(ACS Symposium Series 208) Paul A. Hedin (Eds.) - Plant Resistance To Insects-American Chemical Society (1983)

Plant Resistance to Insects
Paul A. Hedin, EDITOR

U.S. Department of Agriculture
Based on a symposium
sponsored by the ACS
Division of Pesticide Chemistry
at the 183rd Meeting of the
American Chemical Society,
Las Vegas, Nevada,
March 28-April 2, 1982
ACS SYMPOSIUM SERIES 208

AMERICAN CHEMICAL SOCIETY
WASHINGTON D.C. 1983
In Plant Resistance to Insects; Hedin, P.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1983.
Library of Congress Cataloging in Publication Data
Plant resistance to insects.
(ACS symposium series, ISSN 0097-6156;208)
"Based on a symposium sponsored by the ACS
Division of Pesticide Chemistr
of the American Chemical Society
March 28-April 2, 1982."
Includes bibliographies and index.
1. Plants—Disease and pest resistance—Congresses.
2. Insects—Host plants—Congresses. 3. Insects, In-
jurious and beneficial—Congresses. I. Hedin, Paul A .
(Paul Arthur), 1926- . II. American Chemical
Society. Division of Pesticide Chemistry. III. Ameri-
can Chemical Society. National Meeting (183rd:
1982:Las Vegas, Nev.) IV. Series.
SB750.P57 1983 632'.7 82-22622
ISBN 0-8412-0756-9 ACSMC8 208 1-375
1983
Copyright © 1983
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FOREWORD
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PREFACE
B IOLOGISTS HAVE LONG RECOGNIZED the specificity of various insects for

plants and the related interactions between plants and herbivorous insects.
A degree of understanding has evolved from the realization that.different
kinds of insects respond differentially to various secondary chemicals occur-
ring in plants. It has also been recognized that plants and insects coevolve,
and the continuing adjustments of one to the other reflect the biosynthesis
of defensive compounds b th plant d th development f detoxificatio
or avoidance mechanism
ship is illustrated by the ability of insects to induce detoxifying mechanisms
within twenty-four hours when challenged by a toxic agent. Plant injury, in
turn, can elicit the biosynthesis of additional quantities of resistance agents.
The resources that plants can muster for defense when attacked by
insects are limited. Plants that stand exposed to insects over long periods
generally develop defense systems that require a relatively large quantity of
the resistance agent(s), and these agents often bind dietary protein or other-
wise interfere with digestion. In this case, the costs of defense are relatively
large and so the yield may be decreased. Plants that are exposed to pests over
a short period generally develop defense systems that involve the biosyn-
thesis of a small amount of a highly toxic agent. Although such defense
systems require that the plant divert less energy from yield to defense, in
evolutionary time the insect may be able to adapt to the plant and defeat
the resistance.
Of the expressions of plant resistance that are chemical, the so-called
secondary plant compounds appear to be dominant. In most cases they
modify or control insect growth, development, and reproduction, but others
such as the antifeedants modify behavior. However, antifeedants may also
be toxicants and toxicants may also be antifeedants; thus their designation
is a function of the bioassay employed. Not all compounds toxic to one
insect are toxic to another. In evolutionary time, some insects develop
mechanisms by adaption to detoxify compounds in plants on which they
must feed whereas others do not. As a result, a compound toxic to one
insect may be a feeding stimulant for a second. To the extent that the bio-
synthesis of the compounds is an expression of genetic information, the
elucidation of the compounds and their roles can provide a guide to selec-
tions by plant breeders. Additionally, as genetic studies become more
sophisticated, the assignments of the role of individual genes in directing
vii

biosynthesis of resistance compounds will be expediated. Eventually,
improved knowledge about genetic engineering will provide the technology
for introducing protective genes into crop plants, thus creating resistant
lines.
The book is divided into four sections: Ecological and Histochemical
Aspects, Biochemical and Physiological Mechanisms, Insect Feeding Me-
chanisms, and Roles of Plant Constituents. It is hoped that this volume will
help to identify unifying themes by which plants express resistance to
insects. With an ever increasing world population exerting greater and
greater pressure on food production systems, every potential breakthrough
is of critical importance.
As the editor, I am grateful to all of the participants for their contribu-
tions to this book. I am also grateful to the U.S Department of Agriculture
for afinancialgrant.
P A U L A. H E D I N
U.S. Department of Agriculture
Mississippi State, MS 39762
September 30,1982
viii

1
Patterns in Defensive Natural Product Chemistry:
Douglas Fir and Western Spruce Budworm
Interactions
REX G. C A T E S , R I C H A R D A . R E D A K , and C O L I N B . H E N D E R S O N
University of New Mexico, Chemical Ecology Laboratory, Department of Biology,
Albuquerque, NM 87131
Studies were undertaken to t e s t various aspects of current

p l a n t - h e r b i v o r e theory P a r t i c u l a r emphasis was given to
i n v e s t i g a t i n g the e f f e c t
success of the western
the most important v a r i a b l e s i n f l u e n c i n g budworm success were the
concentrations of s e v e r a l s p e c i f i c terpenes and changes i n the
distribution of foliar terpenes. A d d i t i o n a l l y , the p r o d u c t i v i t y
of trees was i n v e r s e l y c o r r e l a t e d with budworm success. The
a v a i l a b l e foliar n i t r o g e n did not appear to be an important
f a c t o r determining i n s e c t success. F i n a l l y , escape i n time
appears to be another f a c t o r limiting i n s e c t utilization of newly
emerged D o u g l a s - f i r t i s s u e .
Some of the major o b j e c t i v e s i n coevolutionary b i o l o g y are

to e x p l a i n the patterns of interaction between plants and
h e r b i v o r e s , the s e l e c t i o n pressures maintaining these p a t t e r n s ,
the ways in which these patterns differ among plant communities,
and how they might change over e c o l o g i c a l and e v o l u t i o n a r y time
( 1 ) . The r e c e n t l y developed theory of plant phytophagous i n s e c t
i n t e r a c t i o n s suggests that the population biology of phytophagous
insects i s influenced significantly by the diversity of plant
species w i t h i n a community, the i n t e r t w i n i n g r e l a t i o n s h i p s among
invidual p l a n t s , the predictability of the plant resources i n
space and time to h e r b i v o r e s , the nutritional l e v e l s of plant
t i s s u e s of various growth forms, and the d i v e r s i t y of mechanical
and chemical defense mechanisms of plant t i s s u e s (1-6). This
theory has sparked considerable research e f f o r t w i t h i n s e v e r a l
d i f f e r e n t areas. Appropriate i n v e s t i g a t i o n s , however, of the
ways i n which the above f a c t o r s i n t e r a c t i n molding plant and
h e r b i v o r e l i f e h i s t o r i e s and community s t r u c t u r e are only
beginning to surface i n the l i t e r a t u r e ( 3 _ 7 - 9 ) .
Since we wish to address various aspects of current
p l a n t - h e r b i v o r e theory, a b r i e f d i s c u s s i o n of some of the b a s i c
premises f o l l o w s , emphasizing those that p e r t a i n to woody
0097-6156/83/0208-0003$06.00/0
© 1983 American Chemical Society

4 PLANT RESISTANCE TO INSECTS
p e r e n n i a l s and t h e i r associated h e r b i v o r e s . Rhoades and Cates

(6) suggest that plant resources f o r herbivores vary along a
continuum of i n c r e a s i n g p r e d i c t a b i l i t y . Resources such as
l o n g - l i v e d woody p e r e n n i a l s , or mature, evergreen l e a f t i s s u e s ,
are suggested to be p r e d i c t a b l e , or i n Feeny's (5) terminology,
apparent, resources to h e r b i v o r e s . Examples, on the other hand,
of the unpredictable or unapparent resources that may vary
g r e a t l y i n t h e i r occurrence and a v a i l a b i l i t y to herbivores are
the ephemeral annuals or the q u i c k l y developing young l e a f
t i s s u e s of perennials ( 6 ) .
These d i f f e r e n c e s i n plant and t i s s u e p r e d i c t a b i l i t y and
a v a i l a b i l i t y as a food resource to h e r b i v o r e s , along with various
t i s s u e developmental c o n s t r a i n t s , are suggested to be important
determinants of the defensive system evolved by a plant (_1 6 ) . f
Although p l a n t s produce many secondary products, chemical

defensive compounds ca
as t o x i n s or d i g e s t i b i l i t y - r e d u c i n
t r e a t e d u s u a l l y as small molecular weight compounds that a f f e c t
the metabolic processes w i t h i n herbivores and include cyanogenic
g l y c o s i d e s , c a r d i a c g l y c o s i d e s , terpenes, a l k a l o i d s , and numerous
o t h e r s . D i g e s t i b i l i t y - r e d u c i n g substances, on the other hand,
are o f t e n l a r g e molecular weight substances capable of bonding
w i t h p r o t e i n s and polysaccharides r e s u l t i n g i n a complex that may
be d i f f i c u l t to d i g e s t . These two c l a s s e s are not a b s o l u t e l y
d i s t i n c t but represent a continuum, and i n some cases, compounds
may f u n c t i o n both as a t o x i n as w e l l as a p r o t e i n complexing
substance ( 6 ) .
The a v a i l a b l e data suggest that t o x i n s , or q u a l i t a t i v e
defenses, are c h a r a c t e r i s t i c of ephemeral p l a n t s and plant
t i s s u e s , and that d i g e s t i b i l i t y - r e d u c i n g substances, or
q u a n t i t a t i v e defenses, appear to be c h a r a c t e r i s t i c of p r e d i c t a b l e
p l a n t s and plant t i s s u e s (_5,6). Furthermore, unpredictable plant
resources are thought to escape i n space and/or time from t h e i r
herbivores to a l a r g e r extent than do p r e d i c t a b l e resources ( 6 ) .
This escape i n space and/or time may be more e f f e c t i v e against
s p e c i a l i z e d or monophagous-oligophagous herbivores than against
the g e n e r a l i z e d or polyphagous herbivores since the former l a c k
a l t e r n a t e food resources.
Our o b j e c t i v e i s to examine some aspects of current plant
h e r b i v o r e theory using D o u g l a s - f i r (Pseudotsuga m e n z i e s i i ) and
western spruce budworm (Choristoneura o c c i d e n t a l i s ) . Both plant
and herbivore are widespread i n western North America. N a t u r a l
hosts of the budworm i n c l u d e D o u g l a s - f i r , species of A b i e s , and,
on occasion, other c o n i f e r s ( 9 ) . V a r i a t i o n i n budworm d e n s i t y
occurs on both a geographic and l o c a l s c a l e . We have f r e q u e n t l y
observed d i f f e r e n t i a l d e f o l i a t i o n ' i n trees having overlapping
crowns at s i t e s i n Montana, Idaho, and New Mexico.
I n t e r e s t i n g l y , considerable geographic v a r i a t i o n i s known to
e x i s t i n the terpene chemistry of D o u g l a s - f i r (10,11). Our

1. CATES ET AL. Defensive Natural Product Chemistry 5
p r e l i m i n a r y data show that t h i s v a r i a t i o n a l s o e x i s t s at a l o c a l

l e v e l (Table I ) .
Table I . V a r i a t i o n i n some of the chemical c o n s t i t u e n t s i n the

young needles of D o u g l a s - f i r from s i t e s a t Boulder,
Montana (MT) and Taos, New Mexico (NM).
Value
Constituent Site X SD Min Max
T o t a l Nitrogen MT 1.8 0.5 1.3 3.5

NM 1.3 0.1 1.1 1.4
a-pinene MT 176.5 223.6 0.0 1001.0

NM 105.1 48.4 34.0 221.0
Camphene MT
NM 190.8 84.4 67.0 351.0
0-pinene MT 134.8 93.7 0.0 429.0

NM 75.9 52.5 3.0 221.0
Limonene MT 84.7 69.2 0.0 403.0

NM 81.5 38.0 24.0 193.0
B o r n y l Acetate MT 63.1 38.4 6.0 189.0

NM 92.6 42.1 26.0 190.0
Cadinene Isomer MT 6.9 5.4 0.0 33.2

NM 4.4 8.1 0.0 32.0
T o t a l Terpenes MT 713.6 502.1 147.8 2442.5

NM 616.6 270.2 246.0 1119.0
Polyphenols MT 0.4 0.2 0.1 1.0

NM 16.6 9.1 0.0 40.0
Polyphenols = % f r e s h wt; n i t r o g e n =» % dw; terpenes -

counts/20 mg t i s s u e
Objectives
Our focus was upon n a t u r a l and experimental s t u d i e s that

were designed to I n v e s t i g a t e v a r i o u s aspects of the current
p l a n t - h e r b i v o r e theory using the D o u g l a s - f i r / w e s t e r n spruce
budworm system. S p e c i f i c a l l y , we wished to t e s t whether there
are chemical c h a r a c t e r i s t i c s l a the young needle t i s s u e of
D o u g l a s - f i r that reduce the growth, s u r v i v a l , and adult f e c u n d i t y
of the western spruce budworm. Secondly, we were i n t e r e s t e d i n

e l u c i d a t i n g the r e l a t i o n s h i p between budworm success and p h y s i c a l

a t t r i b u t e s of D o u g l a s - f i r . F i n a l l y , we desired to determine
whether there are any changes i n f o l i a g e q u a l i t y due to increased
water s t r e s s that could account f o r v a r i a b i l i t y i n budworm
success.
Resistance i s defined as the s u i t e of " h e r i t a b l e
c h a r a c t e r i s t i c s by which a plant species, race, clone or
i n d i v i d u a l may reduce the p r o b a b i l i t y of s u c c e s s f u l u t i l i z a t i o n
of that plant as a host by an i n s e c t species, race, biotype, or
i n d i v i d u a l " ( 2 ) . The important aspect of t h i s concept i s that
r e s i s t a n c e c h a r a c t e r i s t i c s are h e r i t a b l e , even though they may be
modified by the p h y s i c a l environment. With regard to the above
c o n s i d e r a t i o n s and questions, emphasis was placed upon
determining the e f f e c t s of v a r i a t i o n i n n u t r i t i o n a l (water,
n i t r o g e n sources) as w e l l as secondary chemical c h a r a c t e r i s t i c s
(terpenes, r e s i n a c i d s
c a p a c i t y ) i n the f o l i a g
growth, s u r v i v a l , d e n s i t y , l e v e l of d e f o l i a t i o n , a d u l t dry
weight, and f e c u n d i t y . Several p h y s i c a l and phenological
measurements of the trees were made at each s i t e i n c l u d i n g age,
h e i g h t , dbh, crown diameter, crown r a t i o , bole r a d i u s , f i v e year
growth increment, average internode l e n g t h , xylem pressure
p o t e n t i a l , and time of budburst.
S i t e S e l e c t i o n and General Methodology
Resistance-Susceptibility Studies
Studies to determine the l e v e l of r e s i s t a n c e or

s u s c e p t i b i l i t y due to f o l i a g e q u a l i t y and tree p h y s i c a l
parameters of D o u g l a s - f i r to the budworm were conducted at the
Boulder, Montana and Barley Canyon, New Mexico s i t e s . The study
a t the Montana s i t e involved trees exposed to moderate budworm
d e n s i t i e s , while the study at Barley Canyon was done using
budworm that were placed on trees that had very low n a t u r a l
d e n s i t i e s of budworm.
Near Boulder, Montana i n the Deer Lodge N a t i o n a l Forest a
s i t e was s e l e c t e d that c o n s i s t e d of a near monoculture of
D o u g l a s - f i r i n which were s c a t t e r e d ponderosa pine, lodgepole
p i n e , and j u n i p e r s . During May and e a r l y June, 1979, 147 trees
were located and tagged at the s i t e which was a t an e l e v a t i o n of
1620 m. The i n i t i a l s e l e c t i o n of these trees was done such that
a l l were of s i m i l a r age, h e i g h t , dbh, and crown diameter. Trees
were selected that were growing i n s i m i l a r microenvironments
( p r o x i m i t y to drainage, angle and a t t i t u d e of slope, and distance
t o neighboring t r e e s ) . A f t e r the i n i t i a l s e l e c t i o n a more exact
determination of the above p h y s i c a l parameters was made, and from
the i n i t i a l 147 t r e e s , 80 were s e l e c t e d f o r t h i s
r e s i s t a n c e - s u s c e p t i b i l i t y study.

N a t u r a l budworm d e n s i t i e s were determined by sampling 6

sprays, each 40 cm long, i n the same quarter of the tree used to
c o l l e c t t i s s u e f o r chemical a n a l y s i s and to c o l l e c t d e f o l i a t i o n
data. D e n s i t i e s were expressed as the average number of budworm
l a r v a e per 100 buds per t r e e . A v i s u a l estimate of the amount of
d e f o l i a t i o n also was made i n the same a r e a of the crown where the
d e n s i t i e s and needle t i s s u e were c o l l e c t e d . Since budworm may
d i s p e r s e from h e a v i l y d e f o l i a t e d t r e e s , (Greenbank, 1963) budworm
d e n s i t i e s from each tree were weighted by the l e v e l of
d e f o l i a t i o n that each tree sustained. This r e s u l t e d i n an
i n f e s t a t i o n i n t e n s i t y measurement (dependent v a r i a b l e ) which was
subjected to m u l t i p l e stepwise c o r r e l a t i o n a n a l y s i s using v a r i o u s
f o l i a g e q u a l i t y and p h y s i c a l tree parameters as the independent
v a r i a b l e s . Thirty-one parameters were used as independent
variables i n this analysis
F i f t e e n to 20 g o
c o l l e c t e d during the tim
5th l a r v a l i n s t a r . The t i s s u e was f r o z e n , and returned to the
l a b o r a t o r y a t the U n i v e r s i t y of New Mexico f o r a n a l y s i s .
Polyphenols and p r o t e i n complexing c a p a c i t y were determined by
p r e c i p i t a t i n g the polyphenols, weighing t h i s f r a c t i o n ,
r e d i s s o l v i n g i n 20% acetone, and f i n a l l y , measuring the p r o t e i n
complexing c a p a c i t y of the e x t r a c t using a 1.5% buffered g e l a t i n
s o l u t i o n . This method, w i t h s l i g h t m o d i f i c a t i o n , i s taken from
Feeny and Bostock (13) and Feeny ( 1 4 ) .
T o t a l n i t r o g e n was determined using standard m i c r o k j e l d a h l
d i g e s t i o n , and terpenes were analyzed using a P e r k i n Elmer 3920
gas chromatograph equipped w i t h a P e r k i n Elmer 0.10 i n x 150 f t
c a p i l l a r y column packed with 85% 0S-138, 14% CO-880, and 1%
V-930. The method foe terpene a n a l y s i s followed Redak (1982)
w i t h only minor m o d i f i c a t i o n s i n column loading time. Dr. D. F.
Z i n k e l (Forest Products Laboratory, USDA, Madison, Wisconsin)
k i n d l y analyzed the young f o l i a g e f o r r e s i n a c i d s . The r e s i n
a c i d s were not detected and no d e t a i l e d a n a l y s i s of the needles
f o r these chemicals was done i n any of our s t u d i e s .
The r e s i s t a n c e - s u s c e p t i b i l i t y study, where budworm larvae
were placed on trees that n a t u r a l l y had few, i f any, budworm, was
conducted In 1980 a t the Barley Canyon s i t e i n the Santa Fe
N a t i o n a l F o r e s t . This s i t e was approximately 3.2 km long and 0.5
km wide at an average e l e v a t i o n of 2440 m. The dominant
v e g e t a t i o n included D o u g l a s - f i r , white f i r , ponderosa pine, and
aspen. Tree s e l e c t i o n was i d e n t i c a l to that described f o r the
Montana s i t e except that i n i t i a l l y 200 t r e e s were s e l e c t e d . From
t h i s group 105 t r e e s e v e n t u a l l y were used f o r the study reported
here. T h i r t y - f o u r of the 105 were trenched i n 1980 to induce
water s t r e s s i n 1981. One-way m u l t i p l e a n a l y s i s of variance
showed no s i g n i f i c a n t d i f f e r e n c e i n the measurements of 34
f o l i a g e q u a l i t y and tree p h y s i c a l c h a r a c t e r i s t i c s between the 34
t r e e s that were trenched i n 1980 and the remaining 71 trees ( 1 5 ) .

Consequently a l l 105 trees were used i n the a n a l y s i s reported

here.
Larvae were c o l l e c t e d from a nearby i n f e s t e d area and
transported i n the cool of the evening to the U n i v e r s i t y of New
Mexico l a b o r a t o r y (15). Approximately 3000, 4th i n s t a r larvae
were c o l l e c t e d from the i n f e s t e d s i t e and placed i n v i a l s
c o n t a i n i n g a small amount of young D o u g l a s - f i r t i s s u e . Each v i a l
contained 25 l a r v a e . V i a l s were then returned to the
environmental chamber to minimize l a r v a l s t r e s s . The f o l l o w i n g
day the experimental larvae were placed on the s e l e c t e d trees at
the Barley Canyon s i t e . Five l a r v a e were placed on each branch
t h a t had at l e a s t 10 new, expanding f o l i a g e buds, and were
contained on the branches using screen s l e e v e s . Seventy percent
of f u l l s u n l i g h t penetrated these s l e e v e s . Five sleeves, each
w i t h f i v e l a r v a e , were placed on each tree to y i e l d a t o t a l of 25
l a r v a e per t r e e . The l a r v a
t r a n s f e r r i n g them to th
sexed. L a r v a l s u r v i v o r s h i p and adult dry weight could then be
determined. In a d d i t i o n , f o r 81 randomly s e l e c t e d females, egg
mass dry weight and numbers of eggs per female were determined to
examine the r e l a t i o n s h i p between f e c u n d i t y and female dry weight
(15) .
During the 5th and 6th i n s t a r , approximately 30 g of
f o l i a g e were c o l l e c t e d from the same side and i n the same midcrown
l e v e l where the larvae were placed. The f o l i a g e samples were put
i n z i p - l o c bags, transported on Ice to the l a b o r a t o r y , and frozen
u n t i l analyzed f o r n i t r o g e n content and p r o t e i n complexing
c a p a c i t y . Nitrogen was analyzed as above. P r o t e i n complexing
c a p a c i t y was determined using the method described by Bate-Smith
(16) w i t h minor m o d i f i c a t i o n s ( 1 5 ) . For the a n a l y s i s of f o l i a g e
terpene content, 30 mg of young f o l i a g e were c o l l e c t e d and weighed
i n the f i e l d , encapsulated i n indium tubing, placed on i c e ,
transported to the l a b o r a t o r y , and frozen u n t i l i t was analyzed
f o r terpene content. Terpenes were analyzed as described by
(15).
A t o t a l of 34 v a r i a b l e s were used i n the m u l t i p l e stepwise
c o r r e l a t i o n a n a l y s i s . Twenty-three were used to determine
f o l i a g e q u a l i t y , while 11 v a r i a b l e s were used to define the
p h y s i c a l and phenological a t t r i b u t e s of the sample t r e e s . The
dependent v a r i a b l e s used were average adult female budworm dry
weight and average adult male budworm dry weight f o r each t r e e .
D e t a i l s are found i n Redak ( 1 5 ) .
Water S t r e s s Studies
Studies to determine the e f f e c t of water s t r e s s on f o l i a g e

q u a l i t y and budworm success were conducted at 2 s i t e s west of
Jemez s p r i n g s , New Mexico, Santa Fe N a t i o n a l F o r e s t . These s i t e s
were chosen such that d i f f e r e n c e s i n water a v a i l a b i l i t y to
D o u g l a s - f i r trees would be maximized. T h i r t y trees were s e l e c t e d

1. CATES ET A L . Defensive Natural Product Chemistry 9
that were growing on a north f a c i n g slope ( n o n - s t r e s s ) , while

another 30 were selected from a south f a c i n g slope ( s t r e s s s i t e ) .
A n a l y s i s of p r e l i m i n a r y data suggested that these trees d i d not
d i f f e r i n i t i a l l y i n foliage quality characteristics.
A d d i t i o n a l l y , a t the s t r e s s s i t e , about a t h i r d of the area
beneath each tree was trenched to cut roots i n an e f f o r t to
f u r t h e r maximize water s t r e s s . Xylem water p o t e n t i a l s were
measured using a Scholander pressure bomb when the larvae were i n
t h e i r 5th i n s t a r . Budworm larvae were placed on each of the
t r e e s at the s i t e s as described above f o r the Barley Canyon
study. Terpenes and n i t r o g e n were analyzed as described above.
Polyphenols and p r o t e i n completing c a p a c i t y were measured as
described f o r the Montana s i t e .
Female adult budworm dry weights and the number of
s u r v i v o r s of budworm were analyzed by m u l t i v a r i a t e a n a l y s i s of
v a r i a n c e to t e s t f o r th
d i s c r i m i n a n t a n a l y s i s wa
p h y s i c a l parameters d i f f e r e d between s i t e s ( 1 7 ) .
Results
Resistance - S u s c e p t i b i l i t y Studies Using N a t u r a l Budworm

D e n s i t i e s and " D e f o l i a t i o n
R e s u l t s of the data gathered a t the Montana s i t e i n d i c a t e

that 50% of the v a r i a t i o n i n the n a t u r a l budworm i n f e s t a t i o n
i n t e n s i t y v a r i a b l e was explained by 9 v a r i a b l e s (Table I I ) .
The acetate f r a c t i o n of the terpenes, myrcene, an u n i d e n t i f i e d
terpene, time of budburst, and bole radius were i n v e r s e l y
c o r r e l a t e d with budworm i n f e s t a t i o n i n t e n s i t y , i n d i c a t i n g that
some aspects of f o l i a g e q u a l i t y may confer r e s i s t a n c e against the
budworm. The evenness i n the q u a n t i t a t i v e d i s t r i b u t i o n of the
terpenes i n the f o l i a g e among the t r e e s , beta-pinene, t o t a l
f o l i a r n i t r o g e n , and tree age were a l l c o r r e l a t e d p o s i t i v e l y with
budworm i n f e s t a t i o n i n t e n s i t y . Examination of the standardized
c o r r e l a t i o n c o e f f i c i e n t s i n d i c a t e d that the acetate f r a c t i o n of
the terpenes, the q u a n t i t a t i v e d i s t r i b u t i o n of the terpenes i n
the f o l i a g e among t r e e s , age, and time of budburst were the most
important of the included v a r i a b l e s i n e x p l a i n i n g the v a r i a t i o n
in infestation intensity.
The evenness measurement, c a l c u l a t e d from the Shannon-
Wiener formula, suggests that trees which have an uneven
d i s t r i b u t i o n of terpenes are more r e s i s t a n t to the budworm. I t
i s l i k e l y that t h i s imbalance i n the terpene d i s t r i b u t i o n i s
represented by the s p e c i f i c terpenes (acetate f r a c t i o n , myrcene,
and the u n i d e n t i f i e d terpene) that were found to be important i n
the a n a l y s i s . The a n a l y s i s a l s o i n d i c a t e d that the polyphenol
and p r o t e i n complexing c a p a c i t y of the e x t r a c t s from the f o l i a g e

were not important i n determining the budworm d e n s i t i e s and

l e v e l s of d e f o l i a t i o n *
Studies Using Experimental Female Budworm L e v e l s on

Non-Infested Trees
R e s u l t s of the data gathered at the Barley Canyon s i t e ,

where budworm were placed on trees that n a t u r a l l y had few l a r v a e ,
i n d i c a t e d that terpenes again were important i n determining
Table I I . M u l t i p l e c o r r e l a t i o n a n a l y s i s using i n f e s t a t i o n
i n t e n s i t y as the dependent v a r i a b l e R^ = 0.50, p <
0.001). Boulder, Montana s i t e , 1979.
Standardized
Independent
Variable
Acetate F r a c t i o n -0.17828 0.0432 -0.547

Evenness i n Terpene
Distribution 45.85419 15.7133 0.389
Tree Age 0.38511 0.1327 0.339
Budburst -3.05608 1.00207 -0.331
Bole Radius -1.74379 0.6490 -0.302
3-pinene 0.02624 0.0138 0.246
T o t a l Nitrogen 5.17278 2.3350 0.239
Myrcene -0.03534 0.0162 -0.227
Unidentified
Terpene No. 10 -0.22199 0.1247 -0.193
N i t r o g e n = % dw; Terpenes • area counts/20 mg dw
budworm success (Table I I I ) . When the u n i d e n t i f i e d terpene,

t o t a l n i t r o g e n , beta-pinene, and myrcene were i n high
c o n c e n t r a t i o n i n the needles of D o u g l a s - f i r t r e e s , the adult
female d r y weights were reduced s i g n i f i c a n t l y . The f i v e year
growth increment a l s o was i n v e r s e l y r e l a t e d with female dry
weight. When b o r n y l a c e t a t e , t e r p i n o l e n e , and geranyl acetate
were higher i n the f o l i a g e , budworm success increased. A l s o ,
budburst, tree age, and twig internode l e n g t h f o r 1980 were
a s s o c i a t e d p o s i t i v e l y with budworm success.
Examination of the standardized c o r r e l a t i o n c o e f f i c i e n t s
i n d i c a t e d that b o r n y l a c e t a t e , the u n i d e n t i f i e d terpene, t o t a l
n i t r o g e n content, and beta-pinene were the most important
v a r i a b l e s i n determining female adult dry weight. I t i s
i n t e r e s t i n g to note that once more p r o t e i n complexing c a p a c i t y of
the e x t r a c t was not important i n determining female dry weight.
I n t e r e s t i n g l y , s e v e r a l more v a r i a b l e s were included i n the
model d e a l i n g w i t h male budworm success on the trees at the
B a r l e y Canyon s i t e (Table I V ) . Terpinolene, c i t r o n e l l y l a c e t a t e ,

1. CATES ET A L . Defensive Natural Product Chemistry 11
alpha-pinene, bornyl acetate, myrcene, an u n i d e n t i f i e d terpene,

and crown r a t i o were i n v e r s e l y r e l a t e d with average adult male
dry weight production per t r e e . P o s i t i v e l y c o r r e l a t e d with male
dry weight were limonene, young needle water content, gamma-
t e r p i n e n e , 3 u n i d e n t i f i e d terpenes, the r a t i o of s o l u b l e to
I n s o l u b l e n i t r o g e n , and the amount of twig internode growth i n
1980. The most important of these 15 v a r i a b l e s i n i n f l u e n c i n g
male adult dry weight production were limonene, t e r p i n o l e n e ,
c i t r o n e l l y l acetate, alpha-pinene, and bornyl acetate. Once
a g a i n , p r o t e i n complexing c a p a c i t y explained none of the
v a r i a t i o n i n average male adult success.
Table I I I . M u l t i p l e c o r r e l a t i o n a n a l y s i s using adult female

dry weight as the dependent v a r i a b l e . (R2 = 0.35;
F - 3.31; p < 0.001). Barley Canyon, New Mexico
(Redak, 1982)
Standardized
Independent Standard Regression
Variable Coefficient Error Coefficient
B o r n y l Acetate 0.00334 0.00122 0.354

Unidentified
Terpene No. 5 -0.04165 0.01862 -0.262
T o t a l Nitrogen -114.75060 51.62314 -0.232
3-pinene -0.00541 0.00290 -0.229
Budburst 0.41324 0.22878 0.193
Terpinolene 0.04044 0.02703 0.172
Myrcene -0.01387 0.000948 -0.165
Tree Age 0.16913 0.12241 0.145
Geranyl Acetate 0.00436 0.00376 0.137
F i v e Yr
Growth Increment -1.59537 1.31204 -0.133
1980 Internode
Length 0.48272 0.37711 0.133
Nitrogen = % dw; Terpenes » Area counts/20 mg dw
Water S t r e s s and Budworm Success
Hypotheses concerning drought s t r e s s and i t s i n f l u e n c e on

budworm success and changes i n f o l i a g e q u a l i t y were tested using
D o u g l a s - f i r trees growing on south f a c i n g ( s t r e s s ) and north
f a c i n g slopes ( n o n - s t r e s s ) . We f i r s t demonstrated that the
degree of water s t r e s s was d i f f e r e n t between the 2 groups of
t r e e s . Xylem pressure p o t e n t i a l s averaged 23% higher f o r the
t r e e s a t the s t r e s s s i t e when compared to the non-stress s i t e
(p < 0.001).

Next, the male and female adult dry weights and the number
of budworm s u r v i v i n g per tree between the 2 s i t e s were compared.
Table V i n d i c a t e s the means f o r each of these v a r i a b l e s
subdivided by s i t e and sex. These data were used i n the
m u l t i v a r i a t e a n a l y s i s of v a r i a n c e , and are presented i n t h i s
t a b l e as a point of reference. Since no dependency was found
between weight and number of s u r v i v o r s ( r = 0.158; p • 0 . 1 2 ) ,
these v a r i a b l e s were subjected to m u l t i v a r i a t e a n a l y s i s of
v a r i a n c e to t e s t f o r the e f f e c t s of s i t e and sex (Table V I ) .
Budworm success, as measured by a d u l t dry weight and number of
s u r v i v o r s , was found to be s i g n i f i c a n t f o r both s i t e and sex,
but there was no i n t e r a c t i o n between these f a c t o r s . The
r e l a t i v e c o n t r i b u t i o n s of weight and numbers of s u r v i v o r s to
the d i f f e r e n c e s between s i t e s and sexes are i l l u s t r a t e d by the
c h a r a c t e r i s t i c vector c o e f f i c i e n t s . Not s u r p r i s i n g l y , they
i n d i c a t e that d i f f e r e n c e
i n weight between male
was that d i f f e r e n c e s between s i t e s were due to d i f f e r e n c e s i n
weight and the number of s u r v i v o r s per t r e e .
Stepwise d i s c r i m i n a n t a n a l y s i s was used to determine how
t r e e chemical, p h e n o l o g i c a l , and p h y s i c a l parameters d i f f e r e d
between s i t e s (Table V I I ) . Only seven of the 18 v a r i a b l e s used
were needed to completely d i f f e r e n t i a t e the trees at the 2
s i t e s (F(7 193} = 210.36; p < 0 . 0 0 1 ) . The magnitudes of the
standardized d i s c r i m i n a n t f u n c t i o n c o e f f i c i e n t s f o r the
i n c l u d e d v a r i a b l e s i n d i c a t e d that the d i f f e r e n c e s between s i t e s
were l a r g e l y due to terpene chemistry (Table V I I I ) . The
d i s c r i m i n a n t f u n c t i o n c o n t r a s t s p r i m a r i l y the r e l a t i v e
c o n c e n t r a t i o n of alpha-pinene versus the c o n c e n t r a t i o n of
s e v e r a l terpenes, p a r t i c u l a r l y bornyl acetate and beta-pinene.
Examination of the d i s c r i m i n a n t scores showed that the stressed
t r e e s loaded n e g a t i v e l y on the f u n c t i o n (x d i s c r i m i n a n t score =
-2.23), w h i l e the non-stressed trees loaded p o s i t i v e l y (x
d i s c r i m i n a n t score = 3.38). In other words, t r e e s from the
s t r e s s e d s i t e were higher i n alpha-pinene while the
non-stressed trees contained more b o r n y l a c e t a t e , beta-pinene,
and other terpenes i n t h e i r young needles.
Discussion
D o u g l a s - f i r F o l i a g e Q u a l i t y and Resistance t o Budworm
Data from a l l three s t u d i e s show t h a t , i n a l l cases, the

terpene chemistry of young f o l i a g e , or q u a l i t a t i v e defenses,
was the most important f a c t o r i n reducing budworm success. The
p r o t e i n complexing c a p a c i t y , or q u a n t i t a t i v e defenses, of t h i s
t i s s u e was not important i n reducing budworm success i n any of
the s t u d i e s .

Table IV. M u l t i p l e c o r r e l a t i o n a n a l y s i s using adult male dry

2
weight as the dependent v a r i a b l e . ( R - 0.35;
F * 2.49; p < 0.005) B a r l e y Canyon, New Mexico
(Redak, 1982).
Standardized
Independent Standard Regression
Variable Coefficient Error Coefficient
Limonene 0.00512 0.00200 0.461

Terpinolene -0.02296 0.00999 -0.300
Citronellyl
Acetate -0.00335 0.00144 -0.256
a-Pinene -0.00079 0.00055 -0.234
B o r n y l Acetate -0.00062 0.00040 -0.202
Myrcene -0.0052
Water Content 6.7411
y-Terpinene 0.00825 0.00543 0.167
Unidentified
Terpene No. 8 -0.02614 0.01863 -0.159
Unidentified
Terpene No. 5 0.00770 0.00598 0.149
Crown Ratio -5.52496 4.97238 -0.124
Unidentified
Terpene No. 9 0.00946 0.00831 0.121
Soluble/
Insoluble
Nitrogen 0.71473 0.68189 0.119
Unidentified
Terpene No. 4 0.00658 0.00686 0.104
1980 Internode
Length 0.11978 0.12284 0.102
N i t r o g e n * % dw; Terpenes = Area Counts/20 mg dw
Table V. Mean adult dry weight and number of budworm s u r v i v i n g

per tree on s t r e s s e d and non-stressed s i t e s . These
data were used i n the m u l t i v a r i a t e a n a l y s i s , the
r e s u l t s of which are given i n Table VI (Cates et
a l . , 1982).
Weight Number
Site Sex N (mg) Survived
Non-stressed Male 22 8.95 3.73

Female 23 18.18 3.04
Stressed Male 27 10.73 4.63

Female 26 23.64 5.15

Table V I . Results of m u l t i v a r i a t e a n a l y s i s of variance f o r

the e f f e c t s of s i t e and sex on adult dry weight and
number of s u r v i v o r s . C h a r a c t e r i s t i c vector co-
e f f i c i e n t s i n d i c a t e the r e l a t i v e c o n t r i b u t i o n of
the dependent v a r i a b l e s to a p a r t i c u l a r e f f e c t
(Cates et a l . , 1982).
C h a r a c t e r i s t i c Vector
Coefficients
F
Source (2 93)
t ? Number Weight
Site 10.78 0.0001 0.022 0.018

Sex 73.97 0.0001 -0.007 0.023
S i t e x Sex 2^42 0.0941 0.018 0.019 _
Table V I I . Chemical, p h e n o l o g l c a l
c h a r a c t e r i s t i c s of D o u g l a s - f i r trees that were
subjected to d i s c r i m i n a n t a n a l y s i s .
Nitrogen Terpenes
Total 13 I n d i v i d u a l Compounds
Soluble Total
P r o t e i n Complexing Tree Age
Capacity
Table V I I I . Standardized d i s c r i m i n a n t f u n c t i o n c o e f f i c i e n t s f o r
the 7 v a r i a b l e s r e s u l t i n g from the a n a l y s i s of
chemical and p h y s i c a l parameters among trees
growing on the stressed and non-stressed s i t e s
(Cates et a l . , 1982).
Standardized D i s c r i m i n a n t
Variable Function C o e f f i c i e n t
a-pinene -5.84
S o l u b l e Nitrogen -0.71
Age 0.34
U n i d e n t i f i e d terpene 1 1.22
U n i d e n t i f i e d terpene 2 1.66
3-pinene 2.57
B o r n y l Acetate 2.81
N i t r o g e n was found to be of l i t t l e importance except f o r

the female model generated from the Barley Canyon Study. In
t h i s case, female success was i n v e r s e l y c o r r e l a t e d with f o l i a r
n i t r o g e n l e v e l s . However, these l e v e l s , based on published
data (18,19), do not appear high enough to be t o x i c to the
budworm. This observation, i n view of the inverse c o r r e l a t i o n
between t o t a l n i t r o g e n and female budworm success, leads us to

suggest that t h i s r e l a t i o n s h i p i s more a r e f l e c t i o n of tree

p r o d u c t i v i t y than i t s r o l e as a primary n u t r i e n t . In other
words, higher l e v e l s of f o l i a r n i t r o g e n i n non-stressed trees
t h a t were growing on good q u a l i t y s i t e s may have been
i n d i c a t i v e of o v e r a l l tree p r o d u c t i v i t y and v i g o r (19,20,21).
These trees would be b e t t e r able, p r o v i d i n g they were
g e n e t i c a l l y predisposed, t o produce e f f e c t i v e defensive
chemistry against the budworm. The inverse r e l a t i o n s h i p
between female budworm success and the 5 year growth increment,
as w e l l as the p o s i t i v e r e l a t i o n s h i p between tree age and
female budworm success, are c o n s i s t e n t with t h i s hypothesis.
Productive trees should be more vigorous, grow b e t t e r , and
hence, possess wider annual r i n g s . The smaller width of annual
r i n g s i n d i c a t e s that older trees may be l e s s productive,
suggesting a reduced a b i l i t y to produce e f f e c t i v e defensive
chemistry.
A l l of our r e s u l t
a s s o c i a t e d with reduced budworm success. In the Montana study
t h i s was evidenced by the r e l a t i o n s h i p between budworm
i n f e s t a t i o n i n t e n s i t y and bole r a d i u s . In the Barley Canyon
study, the r e l a t i o n s h i p between budworm growth and crown r a t i o ,
the 5 year growth increment, and t o t a l n i t r o g e n support t h i s
c o n c l u s i o n as w e l l . Assuming that p r o d u c t i v i t y d e c l i n e s with
age, the p o s i t i v e c o r r e l a t i o n between budworm success and age
a l s o i m p l i e s that a d e c l i n e i n v i g o r increases tree
s u s c e p t i b i l i t y to budworm.
A d d i t i o n a l l y , our data suggest that D o u g l a s - f i r may reduce
the r i s k of damage to new t i s s u e through escape i n time. In
Montana, trees which burst bud l a t e r i n the growing season
s u f f e r e d l e s s damage from budworm. This i s c o n s i s t e n t with
theory (1,5^6) which suggests that u n p r e d i c t a b i l i t y i n space
and time may be an e f f e c t i v e mechanism f o r reducing the adverse
e f f e c t s of monophagous-oligophagous herbivores on young
ephemeral t i s s u e s . On the other hand, trees a t Barley Canyon
show a p o s i t i v e r e l a t i o n s h i p between budburst and budworm s i z e .
This appears to be i n d i r e c t c o n t r a d i c t i o n with the Montana
data and theory, u n t i l the experimental design Is considered.
At Barley Canyon, budworm were placed on trees a f t e r a l l had
b u r s t bud. Consequently, most budworm were subjected to
t i s s u e s that had developed f o r up to 10 days and which
possessed more complete defensive systems. Therefore, the
c o r r e l a t i o n we observed with budburst r e f l e c t e d the length of
time the f o l i a g e was allowed to mature d e f e n s i v e l y before the
budworm feeding occurred. I t d i d not r e f l e c t an escape i n time
component as the Montana study d i d where n a t u r a l populations
were s t u d i e d .

Water Stress and Budworm Success
The study i n v o l v i n g water s t r e s s i n the Jemez mountains

suggests that changes i n f o l i a g e q u a l i t y due to s t r e s s
p o s i t i v e l y i n f l u e n c e s budworm success (17). Female adult
budworm from larvae reared on stressed trees were 30% heavier,
w h i l e the male a d u l t s were 20% h e a v i e r . In a d d i t i o n , a higher
number of both sexes survived on stressed trees (Table V I ) .
The water s t r e s s i n c u r r e d by trees growing on the south s i t e i s
hypothesized to have modified f o l i a g e q u a l i t y such that trees
became h i g h l y s u s c e p t i b l e to the budworm. This appears to have
been p r i m a r i l y due to the decrease i n the r e s i s t a n c e f a c t o r s
beta-pinene and bornyl acetate accompanied by an increase i n
alpha-pinene. I f alpha-pinene c o n s i s t e n t l y Increases i n
s t r e s s e d D o u g l a s - f i r t r e e s , i t i s then p o s s i b l e that budworm
may use i t as an a t t r a c t a n
The primary emphasi
the importance of n i t r o g e n i n f a c i l i t a t i n g outbreaks of
phytophagous i n s e c t s (22). More r e c e n t l y , i t has been
suggested that changes i n defensive chemistry are as important
as the changes i n primary n u t r i e n t s (23,24). In our s t u d i e s ,
n i t r o g e n was not n e a r l y as important as were s e v e r a l terpenes.
Budworm success was most s t r o n g l y a s s o c i a t e d with changes i n
terpene defensive chemistry, p a r t i c u l a r l y i n the r e d u c t i o n of
r e s i s t a n c e f a c t o r s , rather than with changes i n the primary
n u t r i t i o n of the herbivore.
I m p l i c a t i o n s f o r Current Plant-Herbivore Theory
Ephemeral t i s s u e s , such as the young needles of

D o u g l a s - f i r , are suggested to be defended by t o x i n s or
q u a l i t a t i v e defenses (5,6). Because these t i s s u e s are under
strong s e l e c t i o n f o r r a p i d development and growth to a l e v e l
where they are c o n t r i b u t i n g to the net primary p r o d u c t i v i t y of
the p l a n t , s e l e c t i o n i s postulated to favor a defensive system
t h a t does not place a f u r t h e r burden on an already s t r a i n e d
energy budget (1,6). Hence, q u a n t i t a t i v e defenses that r e q u i r e
c o n s i d e r a b l e energy to produce, and must be compartmentalized
so that they do not i n t e r f e r e with metabolic processes, are
p o s t u l a t e d to be a minor or missing defensive system i n
ephemeral t i s s u e s . Q u a l i t a t i v e , or t o x i n defensive systems are
p r e d i c t e d i n these t i s s u e s i n s t e a d . Exceptions to t h i s general
p r e d i c t i o n may p o s s i b l y i n c l u d e the young leaves of evergreen
t r e e s or shrubs which should show slower growth when compared
to s h o r t - l i v e d t i s s u e s of non-evergreen p l a n t s .
While q u a n t i t a t i v e defenses or d i g e s t i b i l i t y reducing
substances are also present i n small q u a n t i t i e s i n the .young
needles, they do not seem to be e f f e c t i v e i n reducing female
dry weight, l a r v a l d e n s i t y , or l e v e l of d e f o l i a t i o n . This i s
c o n s i s t e n t with the reasoning that young t i s s u e development

1. CATES ET AL. Defensive Natural Product Chemistry
c o n s t r a i n t s render d i f f i c u l t the production of a w e l l developed

d i g e s t i b i l i t y reducing system.
As discussed i n the previous s e c t i o n , we have observed
that escape i n time, another p r e d i c t i o n of current
p l a n t - h e r b i v o r e theory, e x i s t s making newly emerged f o l i a g e
l e s s p r e d i c t a b l e to the budworm. Some trees may burst bud as
l a t e as 10-14 days a f t e r the f i r s t trees w i t h i n a stand, and as
our data show, t h i s delay i s a s s o c i a t e d w i t h reduced damage by
the budworm.
Slansky and Feeny (25) and Feeny (_5) suggest that once
q u a l i t a t i v e defenses are overcome they " a f f e c t l a r v a l growth to
a much l e s s e r extent than do the n u t r i e n t c h a r a c t e r i s t i c s of
food p l a n t s , " and that "once overcome by s p e c i f i c adaptation,
they may have l i t t l e a f f e c t on growth o r f i t n e s s . " Our data do
not corroborate these p r e d i c t i o n s Budworm success was reduced
s i g n i f i c a n t l y by the presenc
i n the young needles o
s t u d i e s , n i t r o g e n content was not as important as were the
terpene contents of the f o l i a g e i n i n f l u e n c i n g budworm success.
I n a l l three s t u d i e s , but p a r t i c u l a r l y I n the 2 d e a l i n g
w i t h r e s i s t a n c e - s u s c e p t i b i l i t y c h a r a c t e r i s t i c s of f o l i a g e
q u a l i t y , s p e c i f i c a l l y the higher q u a n t i t i e s of terpenes were
c o r r e l a t e d with reduced budworm success. At any p a r t i c u l a r
s i t e a l l of the trees sampled contained the same b a s i c
complement of terpenes, a l b e i t , some were present i n low
q u a n t i t i e s . The e f f e c t , which we assume i s one of t o x i c i t y , of
the terpenes i n reducing budworm success, then, was due to an
Increase i n t h e i r q u a n t i t a t i v e amounts. I n other words, the
terpenes were a c t i n g i n a q u a n t i t a t i v e o r dosage dependent
fashion.
Q u a l i t a t i v e defenses i n D o u g l a s - f i r a l s o may be s u c c e s s f u l
a g a i n s t the r e l a t i v e l y s p e c i a l i z e d budworm because o f the
d i v e r s i t y of terpenes that are present. I n other words, a
l a r g e number of q u a l i t a t i v e t o x i c combinations are a v a i l a b l e
f o r s e l e c t i o n to a c t upon i n D o u g l a s - f i r . I n t e r e s t i n g l y , we
found that i n the Montana study the greater the Imbalance In
the terpene d i s t r i b u t i o n i n the f o l i a g e , the l e s s w e l l the
budworm performed. This suggests that the more a t r e e ' s
d e f e n s i v e chemistry deviates from a balanced, o r "average"
p a t t e r n , the l e s s l i k e l y i t i s t o be attacked by the c h e m i c a l l y
w e l l adapted budworm. A d d i t i o n a l l y , adaptation by budworm to
the p a r t i c u l a r imbalanced terpene p a t t e r n of one host tree
would confer an advantage to any tree whose terpene p a t t e r n was
predominated by other terpenes.
Thus we f e e l that the e f f e c t of s e l e c t i o n by the budworm
f o r q u a l i t a t i v e terpene defenses would be to produce a number
of "chemical p a t t e r n phenotypes" among I n d i v i d u a l s a t a l o c a l
or i n t r a p o p u l a t i o n a l l e v e l . Chemical d i v e r s i t y should e x i s t
among populations as w e l l , and t h i s has been suggested i n our
work as w e l l as by others (10,11). Such s e l e c t i o n f o r

d i v e r s i t y i n the host plant chemistry should produce races of

budworm that are g e n e r a l l y adapted to the average defensive
chemistry of t h e i r host plants but that may not be w e l l adapted
t o the chemistry of D o u g l a s - f i r from other populations. I f
t h i s scenario i s c o r r e c t , then some very i n t e r e s t i n g
s i l v i c u l t u r a l p r a c t i c e s i n managing f o r e s t s could r e s u l t using
defensive n a t u r a l product chemistry along with other c o n t r o l
measures i n an i n t e g r a t e d pest management system.
I n a sense, many of the data presented from our work are
p r e l i m i n a r y since the above d i s c u s s i o n i s based on c o r r e l a t i o n
a n a l y s i s . Synthetic d i e t s i n c o r p o r a t i n g compounds that are
suggested by these studies to adversely i n f l u e n c e budworm
success, as w e l l as other experimental work with n a t u r a l
populations of D o u g l a s - f i r and budworm, are needed to determine
cause and e f f e c t . A thorough examination of the n a t u r a l
product chemistry i s neede
be i n f l u e n c i n g s i g n i f i c a n t l
Considerable e f f o r t needs to be given to various aspects of
budworm b i o l o g y . But i n the f i n a l a n a l y s i s , these data suggest
t h a t the Douglas-fir-budworm system may be very u s e f u l i n
u n r a v e l i n g various aspects of p l a n t - h e r b i v o r e i n t e r a c t i o n s , and
i n understanding ways i n which we might b e t t e r manage our
f o r e s t s against phytophagous i n s e c t s .
Acknowledgements
We should l i k e to thank T. I. McMurray, H. J . Alexander,

M. Alexander, J . Horner, M. F r e e h l i n g , and numerous
undergraduate students f o r t h e i r help and f o r t h e i r constant
q u e s t i o n i n g and d i s c u s s i o n of various aspects of the p r o j e c t s .
We are g r a t e f u l to C l i f f o r d S. Crawford and F r i t z Taylor f o r
p r o v i d i n g information and d i s c u s s i o n of budworm population
b i o l o g y . Doug Parker and Mike Chavez (USDA FS, Region 3,
Albuquerque), and Jed Dewey, John Hard, and Larry Stipe (USDA
FS, Region 6, Missoula, MT), were h e l p f u l In numerous ways but
p a r t i c u l a r l y i n helping us l o c a t e appropriate s i t e s . We are
extremely g r a t e f u l to Linda L. DeVries f o r typing the
manuscript s e v e r a l times. This research was supported i n part
by NSF grants DEB 7619950 and DEB 7927067 to RGC f o r which we
are g r a t e f u l . Work leading to t h i s p u b l i c a t i o n was funded by
the Canada/United States Spruce Budworms Program, and
A c c e l e r a t e d Research, Development and A p p l i c a t i o n Program,
sponsored by the USDA Forest S e r v i c e .

Literature Cited
1. Cates, Rex G.; Rhoades, D. Biochem. S y s t . E c o l . 1977, 5,

185-93.
2. Cates, Rex G.; Alexander, H. J. In "Bark Beetles in
North American C o n i f e r s : Ecology and E v o l u t i o n " : J . M i t t o n
and K. Sturgeon, (Eds.); U n i v e r s i t y of Texas Press; A u s t i n ,
Texas, in press.
3. Cates, Rex G. Oecologia 1981, 48, 319-26.
4. Cates, Rex G. Oecologia 1980, 46, 22-31.
5. Feeny, P. P. In "Biochemical I n t e r a c t i o n s Between P l a n t s
and I n s e c t s " ; Wallace, J.; M a n s e l l , R., Eds.; Plenum
P r e s s : New York, NY, 1976; pp 1-40.
7. Futuyma, D.; Gould, F. Ecol. Monogr. 1979, 49, 33-50.
8. Rosenthal, G.; Janzen, D., Eds.; "Herbivores: Their
I n t e r a c t i o n w i t h Secondar
P r e s s : New York, NY
9. Johnson, P.; Denton, R. USDA F o r . Ser. Gen. Tech. Rep.
1975, INT-20, 1-144.
10. von R u d l o f f , E. Pure A p p l . Chem. 1973, 34, 401-10.
11. von R u d l o f f , E. Can. J. Bot. 1972, 50, 1025-40.
12. Greenbank, D. Entomol. Soc. Can. Mem. 1963, 31, 174-80.
13. Feeny, P.; Bostock, H. Phytochemistry, 1968, 7, 871-80.
14. Feeny, P. Phytochemistry, 1969, 8, 2119-26.
15. Redak, R. "A Determination of the Resistance-
Susceptibility C h a r a c t e r i s t i c s in D o u g l a s - f i r to the
Western Spruce Budworm"; MS Thesis, Univ. of New Mexico,
Albuquerque, NM, 1982, pp 1-74.
16. Bate-Smith, E. Phytochemistry, 1973, 12, 907-12.
17. Cates, Rex G.; McMurray, T.; Redak, R.; Henderson, C.
Nature, (submitted).
18. Mattson, W. Ann. Rev. E c o l . Syst. 1980, 11, 119-61.
19. Gosz, J . Ecol. Bull, 1981,33,405-26.
20. Miller, H.; Cooper, J.; Miller, J . ; P a u l i n e , O. Can.
J. For. Res. 1979, 9, 19-26.
21. Van den Driessche, R.; D a n g e r f i e l d , J . P l a n t and Soil
1975, 42, 685-02.
22. White, T. Ecology, 1969, 50, 905-9.
23. Rhoades, D. I n ( 8 ) .
24. Mattson, W.; Addy, N. Science, 1975, 190, 515-22.
25. Slansky, F.; Feeny, P. Ecol. Monogr. 1977, 47, 209-28.
RECEIVED September 13, 1982

2
Physiological Constraints on Plant Chemical
Defenses
H . A . M O O N E Y , S. L. G U L M O N , and N. D . J O H N S O N
Stanford University, Department of Biological Sciences, Stanford, C A 94305
Plants allocate their carbon and nitrogen

resources to a variet
constructing and
shoot, reproductive), storage, and defense from
herbivores. There i s a genetically-determined time
schedule of allocation of resources to various
functions which can be modified to a certain
degree by environmental conditions. The
proposition i s examined that this time schedule
represents changing allocation p r i o r i t i e s within
the plant which results i n time "windows" when i t
is more susceptible to herbivore damage than
others. Anti-herbivore "strategies" of plants
must be considered i n r e l a t i o n to changing
patterns of resource a v a i l a b i l i t y to the whole
plant and i n r e l a t i o n to allocation of these
resources to enhance reproductive output.
P l a n t - i n s e c t i n t e r a c t i o n has been considered e x t e n s i v e l y

from the view p o i n t of the chemistry involved i n p l a n t defense
( f o r example reviews i n I), the growth c y c l e of the plant as i t
a f f e c t s i t s "apparency" t o herbivores (2_), and the seasonal
p h y s i c a l and b i o t i c environment of the herbivores ( 3 ) . Here we
consider the i n t e r a c t i o n of the p l a n t ' s p h y s i c a l environment,
that i s , i t s changing resource a v a i l a b i l i t y p a t t e r n , w i t h the
u t i l i z a t i o n of these resources f o r growth, reproduction and
defense against herbivores.The production of p l a n t defensive
chemicals i s , o p t i m a l l y , governed by the cost of the chemicals,
t h e i r e f f e c t i v e n e s s a t d e t e r r i n g h e r b i v o r y , the r i s k o f
h e r b i v o r y , and the cost of herbivory. The e f f e c t i v e n e s s of
d i f f e r e n t chemicals a t d e t e r r i n g h e r b i v o r y has been e x t e n s i v e l y
s t u d i e d , and t h e o r i e s concerning the r i s k of herbivory are w e l l
e s t a b l i s h e d . Much l e s s I s known about the costs of h e r b i v o r y
or the costs of defensive chemicals. The costs of defensive
0097-6156/83/0208-0021$06.00/0

chemicals are a f u n c t i o n of t h e i r b i o s y n t h e t i c maintenance

and turnover c o s t s , but a l s o a f u n c t i o n of the value of the
elements used i n t h e i r c o n s t r u c t i o n . The value of these
resources w i l l be determined by t h e i r importance to other
a c t i v i t i e s such as reproduction, photosynthesis, n u t r i e n t
uptake, or support. Here we assess the consequences, i n terms
of herbivore s u s c e p t i b i l i t y , of simultaneous uses of l i m i t i n g
resources by p l a n t s f o r a v a r i e t y of necessary f u n c t i o n s . We
e a r l i e r discussed c o s t s of defensive compounds i n r e l a t i o n to
h e r b i v o r y (_4).
Carbon and n i t r o g e n are the two primary resources which can
be a l l o c a t e d to defensive s t r u c t u r e s and which may a l s o be
l i m i t i n g i n a p l a n t . They are used d i r e c t l y i n the
c o n s t r u c t i o n of secondary compounds or p r o t e c t i v e s t r u c t u r e s ,
and carbon i s a l s o used as an energy source to c o n s t r u c t these
components. The suppl
the a v a i l a b i l i t y of a l
i n c l u d i n g l i g h t , water, and n u t r i e n t s . Temperature a c t s
i n d i r e c t l y to c o n t r o l resource a v a i l a b i l i t y . V a r i a t i o n i n any
of these r e s u l t s i n f l u c t u a t i o n s i n carbon g a i n and r e c u r r e n t
c y c l e s of storage and u t i l i z a t i o n of carbon i n d i f f e r e n t p a r t s
of the plant ( F i g . 1). L i m i t a t i o n s on the supply of n i t r o g e n
and carbon could r e s u l t i n " p r i o r i t i e s " of use w i t h i n the p l a n t
i n order to maximize f i t n e s s . In the f i r s t part of t h i s paper
we consider general aspects of both n i t r o g e n and carbon
a l l o c a t i o n . Then we examine the way carbon a l l o c a t i o n
p r i o r i t i e s a f f e c t defense from herbivores i n a p a r t i c u l a r
v e g e t a t i o n type, the temperate deciduous f o r e s t .
Nitrogen
N i t r o g e n i s a component of s e v e r a l c l a s s e s of compounds
b e l i e v e d to be important i n p l a n t chemical defense, p r i n c i p a l l y
cyanogenic g l y c o s i d e s , g l u c o s i n o l a t e s , a l k a l o i d s , c e r t a i n
p r o t e i n s , and non-protein amino a c i d s and peptides. The
n i t r o g e n c o n c e n t r a t i o n i n leaves i s o f t e n l i m i t i n g to i n s e c t
l a r v a l growth (see review by Mattson, _5), and i t i s a primary
l i m i t i n g f a c t o r to l e a f carbon-gaining c a p a c i t y (6^ 7_ 9 8).
A l l o c a t i o n of n i t r o g e n to chemical defense must be considered
w i t h i n the context of t h i s complex interdependency.
The q u a n t i t a t i v e commitment of n i t r o g e n to secondary
compounds v a r i e s c o n s i d e r a b l y . At one extreme, the n i c o t i n e
c o n c e n t r a t i o n of tobacco leaves ( N l c o t l a n a tabacum) can range
from 0.17 to 4.93% ( 9 ) . At the upper l e v e l , t h i s i s equivalent
to n e a r l y 1% l e a f n i t r o g e n c o n c e n t r a t i o n , s i n c e n i c o t i n e i s
over 17% n i t r o g e n by weight. The t o t a l n i t r o g e n c o n c e n t r a t i o n
i n leaves r a r e l y exceeds 5%, so n i c o t i n e may comprise up to 20%
of the n i t r o g e n In the l e a f . Not s u p r i s i n g l y , n i c o t i n e
production i s c o r r e l a t e d w i t h f e r t i l i z e r a p p l i c a t i o n ( 9 ) .
In another example, the cyanogenic g l u c o s i d e content i n the

2. MOONEY E T AL. Plant Chemical Defenses 23
Resource
Uptake
Allocation
Stores Growth Protection
Retrieval Leaf
Support
Root
Reproduction
I
t
Loss
Figure 1. Flow scheme for a resource within a plant.

leaves of the evergreen c h a p a r r a l shrub Heteromeles a r b u t i f o l i a

a t t a i n s 7% of t o t a l dry weight (10). Assuming, as the authors
d i d , that a l l of the g l u c o s i d e was prunasin, t h i s i s equivalent
to 0.33% n i t r o g e n by dry weight, or about o n e - t h i r d of the
average t o t a l n i t r o g e n i n the l e a f ( 1 1 ) .
On the other hand, most a l k a l o i d s c o n t a i n much l e s s

n i t r o g e n as a f r a c t i o n of the t o t a l weight than n i c o t i n e (Table
I ) , and t o x i c compounds such as a l k a l o i d s are u s u a l l y found a t
very low concentrations i n the p l a n t (12). Thus, the
a l l o c a t i o n of n i t r o g e n t o nitrogenous secondary compounds can
range from s u b s t a n t i a l t o n e g l i g i b l e , even w i t h comparable
a l l o c a t i o n s of carbon.
Another f a c t o r which i s important i n c o n s i d e r i n g the
commitment of n i t r o g e n to chemical defense i s the high m o b i l i t y
of t h i s element w i t h i n
of the n i t r o g e n i n senescin
i s r e a d i l y moved from senescing leaves to developing f r u i t s
(14), and i n deciduous trees a s u b s t a n t i a l part of s p r i n g
growth i s made u s i n g n i t r o g e n stored i n the r o o t s and stem wood
and bark (15, 16).
The n i t r o g e n i n secondary compounds a l s o appears to be
r e a d i l y mobile and metabolizable. When doubly-labeled n i c o t i n e
( C - N) was f e d to tobacco (]¥. r u s t i c a ) , carbon was
recovered i n a l k a l o i d s , f r e e amino a c i d s , pigments, f r e e
organic acids and f r e e sugars; and n i t r o g e n was recovered i n
p r o t e i n s , a l k a l o i d s , f r e e amino a c i d s and pigments ( 1 7 ) .
C e r t a i n n i t r o g e n - c o n t a i n i n g t o x i c compounds found i n seeds are
completely degraded during germination (18). Other examples o f
a l k a l o i d turnover are given i n Robinson (19) and Digenis ( 2 0 ) .
Examples of t r a n s l o c a t i o n of n i t r o g e n - c o n t a i n i n g secondary
compounds i n c l u d e the cyanogenic g l u c o s i d e , l i n a m a r i n , i n the
w i l d lima bean (Phaseolus l u n a t u s ) , which i s t r a n s l o c a t e d from
seeds to developing seedlings (21). R i c i n i n e i n c a s t o r beans
i s t r a n s l o c a t e d from senescent leaves t o young, developing
t i s s u e and t o seeds. Caffeine i s a l s o t r a n s l o c a t e d among
leaves and i n t o f r u i t s (22). I n the case of Heteromeles
a r b u t i f o l i a , c i t e d e a r l i e r , cyanogenic glucosides appear t o
move from leaves i n t o developing seeds'. McKey (23) gives other
examples of t r a n s l o c a t i o n of t o x i c compounds w i t h i n the
p l a n t . Thus n i t r o g e n i n v e s t e d i n chemical defenses can be
moved throughout the p l a n t to p r o t e c t v u l n e r a b l e organs such as
young leaves or f r u i t s , and can l a t e r be reused f o r growth and
development.
Because nitrogenous compounds are r e a d i l y metabolized or
transported w i t h i n the p l a n t , i t would seem that the major
c o n s t r a i n t on use of n i t r o g e n i n chemical defense i s i t s
e f f e c t i v e n e s s and the p a t t e r n of i t s a v a i l a b i l i t y i n the
h a b i t a t . I f n i t r o g e n i s the major f a c t o r l i m i t i n g to growth,
i t i s u n l i k e l y that l a r g e amounts w i l l be d i v e r t e d to secondary

2. MOONEY E T AL. Plant Chemical Defenses 25
Table 1. Approximate percentages of n i t r o g e n i n some common

plant a l k a l o i d s
Alkaloids
caffeine 28.9
nicotine 17.3
ricinine 17.0
gramine 16.1
lupinin
quinin
mescaline 6.6
colchicine 3.5
tomatine 1 4
Toxic amino a c i d s
3amlno p r o p i o n i t r i l e 40.0
canavanine 31.8
mimosine 14.1
Transport amides
asparagine 21.2
glutamine 19.2
Amino a c i d s
glycine 18.7
alanine 15.7
serine 13.3
proline 12.2
tyrosine 7.7
Cyanogenic g l u c o s i d e s
linamarin 5.7
prunasin 4.7
amygdalin 3.1

compounds, e s p e c i a l l y s i n c e there are many non-nitrogenous

plant products, such as c a r d i a c g l y c o s i d e s , saponins,
f l a v o n o i d s , quinones, e t c , which are a l s o t o x i c .
Although n i t r o g e n i s probably l i m i t i n g to some extent i n
most n a t u r a l systems, i t i s not the major l i m i t i n g n u t r i e n t i n
a l l of them, and i s not l i m i t i n g to the same extent a t a l l
times of the year. In l a r g e areas of A u s t r a l i a f o r example,
phosphorus i s the p r i n c i p l e l i m i t i n g element (24).
Some p l a n t s experience a s h o r t - l i v e d abundance of
n i t r o g e n . Most annuals occupy d i s t u r b e d or semi-disturbed
h a b i t a t s which are c h a r a c t e r i z e d by a sudden r e l e a s e of
n u t r i e n t s (25). Even many herbaceous p e r e n n i a l s occur under
semi-disturbed c o n d i t i o n s (eg. o l d f i e l d s ) , or grow during a
short i n t e r v a l between c l i m a t i c l i m i t a t i o n s and b i o t i c
competition when resources are r e l a t i v e l y abundant, as i s the
case of the f o r e s t understor
P l a n t t o x i n s are widel
as opposed to protein-complexing polyphenols which are more
l i m i t e d to leaves of woody p e r e n n i a l s (12, 23). This p a t t e r n
has been suggested t o r e l a t e to the low p r e d i c t a b i l i t y i n space
and time of herbaceous f o l i a g e i n c o n t r a s t to the f o l i a g e of
woody p l a n t s (2_, 12). However, i t should be noted t h a t , a t
l e a s t i n the case of some annuals (9_, 28), a l l of the n i t r o g e n
i s taken up during the p e r i o d of v e g e t a t i v e growth and simply
r e d i s t r i b u t e d d u r i n g r e p r o d u c t i o n (Figure 2 ) . Although data
are scarce, t h i s p a t t e r n may be widespread among herbaceous
species s i n c e , as already s t a t e d , they o f t e n grow during a
short p e r i o d of resource a v a i l a b i l i t y .
Given a p a t t e r n of i n i t i a l r a p i d n u t r i e n t uptake and
temporal storage i n some cases to high c o n c e n t r a t i o n s , i t i s
c l e a r l y advantageous to maintain any n i t r o g e n not being used
f o r photosynthesis or growth i n a form that i s t o x i c t o
h e r b i v o r e s . Thus, the common occurrence of t o x i c nitrogenous
compounds i n herbaceous p l a n t s may simply r e f l e c t resource
a v a i l a b i l i t y p a t t e r n s and consequent a l l o c a t i o n pathways. This
p a t t e r n may a l s o apply to some woody p e r e n n i a l s . For example,
c h a p a r r a l shrubs have periods of a c t i v e n i t r o g e n uptake i n the
f a l l when growth i s not o c c u r r i n g (29). This s t o r e d n i t r o g e n
i s moved to new growth i n the s p r i n g and a p o r t i o n i s a l l o c a t e d
to nitrogenous defensive compounds (10).
Carbon
L i k e n i t r o g e n a c q u i s i t i o n , the carbon-gaining c a p a c i t y of a
plant i s environmentally l i m i t e d . As described e a r l i e r , t h i s
l i m i t a t i o n r e s u l t s i n apparent " p r i o r i t i e s " f o r a l l o c a t i o n t o
storage, growth of d i f f e r e n t organs, and e l a b o r a t i o n of v a r i o u s
c l a s s e s of m e t a b o l i t e s . These a l l o c a t i o n patterns change
during the seasonal growth of an i n d i v i d u a l . I n t r e e s , f o r
example, root growth g e n e r a l l y takes place a t times d i f f e r e n t

2. MOONEY ET AL. Plant Chemical Defenses 27
from stem e l o n g a t i o n which i n t u r n i s d i s p l a c e d from stem

diameter growth (30, 31) (Figure 3 ) .
Carbon metabolism d i f f e r s fundamentally from n i t r o g e n
metabolism i n that v i r t u a l l y a l l nitrogenous compounds can be
r e c y c l e d w i t h i n the p l a n t , whereas most of the s t r u c t u r a l
components of the p l a n t , p r i n c i p a l l y c e l l u l o s e and l i g n i n , a r e
not reusable. There are a l s o p u t a t i v e defensive compounds i n
t h i s category such as l e a f - e x t e r n a l r e s i n s (32) and p o s s i b l y
some condensed t a n n i n s . Thus the c o n s t r a i n t s on a l l o c a t i o n
have both an immediate and f u t u r e time frame.
The i n t e r a c t i o n s between a v a i l a b i l i t y of resources, carbon
g a i n i n g c a p a c i t y , and the a l l o c a t i o n of carbon t o p l a n t
f u n c t i o n s , i n c l u d i n g chemical defense, w i l l o b v i o u s l y d i f f e r
among environments. We examine a p a r t i c u l a r system, t h e
temperate deciduous f o r e s t t i l l u s t r a t th natur f th
c o n s t r a i n t s on chemica
carbon gain.
The Deciduous Forest
In s p r i n g , 80% of the deciduous f o r e s t canopy i s produced

over a short span of time (31, 33, 34) (Figure 3). I n i t i a l
l e a f expansion, f l o w e r i n g and shoot e x t e n s i o n are h e a v i l y
s u b s i d i z e d from carbohydrate reserves because there i s
i n s u f f i c i e n t p h o t o s y n t h e t i c area t o support these processes
(35, 36) ( F i g . 4 ) . Despite the demand on carbohydrate s t o r e s ,
reserves are not completely depleted and appear t o be renewed
r a p i d l y f o l l o w i n g the growth f l u s h e s (16). I t has been
suggested that maintenance of some storage i s important i n the
event the f i r s t l e a f crop i s destroyed due t o i n t e n s e h e r b i v o r y
(37) o r a l a t e k i l l i n g f r o s t (31), and a second crop must be
produced.
Deciduous t r e e s vary i n the d u r a t i o n of l e a f i n i t i a t i o n and
expansion and new shoot e x t e n s i o n (38-41). This v a r i a t i o n i s
t y p i f i e d by two type examples ( F i g u r e 5): Quercus s p e c i e s
complete a l l l e a f and shoot growth during a s h o r t p e r i o d ,
u s u a l l y l a t e A p r i l t o e a r l y June; B e t u l a , by c o n t r a s t , grows
throughout most o f the summer. Fagus, Carya, and Fraxinus have
growth c y c l e s of the Quercus type, and L l r l o d e n d r o n , Populus,
and Llquldamber have extended growth c y c l e s l i k e B e t u l a . Acer
species are i n t e r m e d i a t e . The r e l a t i o n s h i p between d u r a t i o n of
stem e l o n g a t i o n and dependence on carbohydrate reserves needs
to be e s t a b l i s h e d f o r these t r e e s , o b v i o u s l y though oak species
which form canopies e n t i r e l y from pre-formed l e a f buds have a
heavy d r a i n on carbon reserves f o r a p e r i o d .
The young leaves c o n s t i t u t e an abundant food resource f o r
phytophagous i n s e c t s and they are e s p e c i a l l y v u l n e r a b l e t o
h e r b i v o r e s due to a h i g h n i t r o g e n content and l a c k o f
s c l e r o p h y l l y . These c h a r a c t e r i s t i c s appear t o be u n i v e r s a l i n
new leaves of deciduous f o r e s t s p e c i e s , and t h i s p e r i o d o f

- A
55 -7
CD !c
£ cm
& E
</> ~o 6 -
0
-
1 o"
* E
A
/
/
1 1 1 . 1 . 1 . 1 . 1
o
o
a>
>
0
CD
or
r
19 10 30 19 9 29 18
March April May June July
Figure 3. Seasonal growth activity of a white oak in Missouri. Development of
canopy photosynthetic capacity is shown in A. Relative seasonal growth activity of
various tree components are given in B. Key: F, flower; S, shoot; L, leaf; SC, stem
circumference; RG, root growth; and A, acorn. (Reproduced with permission from
Ref.31.)

4 -
2 -
0 I—i i i i i i i i i i i 1—
A S O N D J F M A M J J
Months
Figure 4. Seasonal changes in available carbohydrates in a Californian evergreen

oak (A), a Vermont sugar maple (B), and a Calif ornian buckeye (C). Maple data for
sapwood are from Ref. 15. Oak and buckeye data are from Ref. 49, for total branch
minus fruits. The evergreen oak has a stable supply of carbon through continuous
photosynthesis. The maple draws upon reserves in the spring to build a new canopy.
The buckeye draws upon reserves to build fruits in the fall as well as new canopy in
late January.

20 30 10
April May
Figure 5. Seasonal height growth of three deciduous species (39). Key: O, white
ash; - • red maple; and gray birch.

2. MOONEY ET A L . Plant Chemical Defenses 31
v u l n e r a b i l i t y t y p i c a l l y l a s t s about one month (42, 43, 44)

(Figure 6 ) .
Apparently, the p o t e n t i a l f o r reducing t h i s window of
v u l n e r a b i l i t y i s f a i r l y l i m i t e d . The p r o t e i n content o f
developing leaves could conceivably be reduced but t h i s would
slow the metabolic processes o f growth and reduce the gross
photosynthetic rate- Both would r e s u l t i n longer development
times, prolonging v u l n e r a b i l i t y , and be a heavier d r a i n on
r e s e r v e s , consequences which would reduce a t r e e ' s competitive
advantage.
A somewhat s i m i l a r c o n s t r a i n t a p p l i e s t o the e a r l y
development of s c l e r o p h y l l y , a p o t e n t i a l h e r b i v o r e d e t e r r i n g
f e a t u r e . C o n s t r u c t i o n of r i g i d c e l l w a l l s and the production
of l i g n i n and other compounds would c e r t a i n l y slow the o v e r a l l
r a t e of l e a f expansion and prolong the p e r i o d during which the
l e a f i s a net importe
We have already mentione
an important competitive advantage and minimizes the time
d u r i n g which young leaves are v u l n e r a b l e to h e r b i v o r e s .
Climate can sometimes be an a d d i t i o n a l c o n s t r a i n t . For
example, i n the white oak f o r e s t i n M i s s o u r i , l e a f development
occurs during an i n t e r v a l o f optimal growing c o n d i t i o n s between
the l a t e s t k i l l i n g f r o s t and the midsummer drought (31).
Deployment o f defensive chemicals during l e a f expansion.
Toxins appear t o be e f f e c t i v e i n s m a l l q u a n t i t i e s (12),

making them an i d e a l low cost defense against g e n e r a l i s t
herbivores even though s p e c i a l i s t s are able t o evolve e f f e c t i v e
d e t o x i f i c a t i o n mechanisms. Studies have shown t h a t t o x i n
c o n c e n t r a t i o n s are g e n e r a l l y higher i n new growth (23, 45), but
there have been few s t u d i e s o f temperate deciduous t r e e s . I t
has been shown that g e n e r a l i s t herbivores are more common on
mature f o l i a g e whereas s p e c i a l i s t h e r b i v o r e s p r e f e r new f o l i a g e
(46), but again, there are few s t u d i e s i n the temperate
deciduous f o r e s t .
Protein-complexing polyphenols are broadly e f f e c t i v e
against h e r b i v o r e s , e s p e c i a l l y those not s p e c i f i c a l l y adapted
to eat p l a n t s c o n t a i n i n g polyphenols (47). They may c o n s t i t u t e
10% o r more o f the l e a f dry weight (12, 37, 44), and t h i s
r e q u i r e s a l l o c a t i o n of s i g n i f i c a n t amounts of carbon and energy
to t h e i r c o n s t r u c t i o n . The use of l i m i t e d reserves f o r such
q u a n t i t a t i v e defenses must be balanced a g a i n s t the requirements
of r a p i d canopy development and new shoot e x t e n s i o n .
P a t t e r n s of polyphenol accumulation i n leaves vary among
the few temperate f o r e s t species which have been s t u d i e d .
1
Feeny s (42) c l a s s i c study on oak leaves (Quercus robur) shows
l e a f t a n n i n l e v e l s r i s i n g s l o w l y u n t i l August, and then
i n c r e a s i n g s h a r p l y . In the cases of sugar maple (Acer
saccharum) and y e l l o w b i r c h ( B e t u l a l u t e a ) (44), r e s p e c t i v e l y ,

Figure 6. Seasonal changes in leaf nitrogen (A) and leaf toughness (B) of a series
of deciduous tree species. (Reproduced with permission from Ref. 43.)

polyphenol concentrations peak i n l a t e June and mid May. In

Betula pubescens (48) the c o n c e n t r a t i o n o f tannins i s s t a b l e
u n t i l l a t e summer and then Increases.
Feeny (2) has coined the term "apparency" t o express t h e
i d e a that some p l a n t s o r p l a n t components are more p r e d i c t a b l e
resources i n space and time t o p o t e n t i a l h e r b i v o r e s . He
suggests that such p r e d i c t a b l e p l a n t s are more a t t r a c t i v e t o
s p e c i a l i s t herbivores ( i n e v o l u t i o n a r y time) because o f t h e i r
p r e d i c t a b i l i t y , and thus should employ q u a n t i t a t i v e defensive
compounds which are l e s s s u s c e p t i b l e t o the e v o l u t i o n o f
d e t o x i f i c a t i o n mechanisms.
We propose that the p a t t e r n s of polyphenol production i n
leaves are r e l a t e d t o the d u r a t i o n of new l e a f p r o d u c t i o n .
Species which accomplish l e a f expansion and shoot extension
w i t h i n a short time p e r i o d e s p e c i a l l y i n c o l d c l i m a t e s should
g e n e r a l l y have low polypheno
compared t o species w i t
There are two reasons f o r the above. F i r s t , the new leaves
of species w i t h short growth periods a r e l e s s "apparent" i n the
sense Feeny (2) has described. They appear e a r l y , when
predator populations may be i n h i b i t e d by c o l d temperatures, and
are not present long enough t o a l l o w build-up of predator
p o p u l a t i o n s . By c o n t r a s t , new leaves o f species w i t h long
growth periods are present over an extended p e r i o d of time,
g e n e r a l l y much o f the summer, and thus c o n s t i t u t e a p r e d i c t a b l e
resource f o r s p e c i a l i z e d h e r b i v o r e s . Second, new l e a f and
shoot growth are a g r e a t e r d r a i n on p l a n t reserves when the
e n t i r e canopy i s produced over a short time p e r i o d (35). In
such s p e c i e s , a l l o c a t i o n of 10-25% o f new l e a f dry weight t o
polyphenol p r o d u c t i o n would f u r t h e r deplete reserves, slow
canopy development, o r both. This would decrease the advantage
that such species have i n terms of escaping h e r b i v o r y i n time,
and would a l s o reduce the p e r i o d o f carbon g a i n which
determines reserves and p o t e n t i a l growth f o r the f o l l o w i n g
season. The p a t t e r n s o f polyphenol accumulation i n Quercus
(42) and Acer and B e t u l a (44) are c o n s i s t e n t w i t h t h i s
hypothesis, but examination of polyphenol concentrations
through time i n many more species i s necessary. Schultz et a l .
(44) suggest t h a t the low polyphenol contents of young leaves
may be due t o the f a c t that p r o t e i n s y n t h e s i s suppresses
phenolic synthesis.
To summarize, s p e c i f i c t o x i n s are e f f e c t i v e d e t e r r e n t s t o
g e n e r a l i s t h e r b i v o r e s , but polyphenols produced i n r e l a t i v e l y
l a r g e q u a n t i t i e s are the only e f f e c t i v e d e t e r r e n t t o
s p e c i a l i s t s . Since l e a f toughness and lower n i t r o g e n content
a l s o deter many h e r b i v o r e s , deciduous f o r e s t t r e e s are
e s p e c i a l l y v u l n e r a b l e t o a t t a c k during the p e r i o d o f canopy
development, which occurs y e a r l y a t a p r e d i c t a b l e time. Canopy
development i s h e a v i l y dependent on a v a i l a b l e reserves o f
carbohydrate and n i t r o g e n , and the production o f q u a n t i t a t i v e

defensive compounds i s expensive i n terms of carbon and

energy. I t t h e r e f o r e appears that the patterns of production
of polyphenols during canopy development are r e l a t e d to the
d u r a t i o n of new growth. Species which produce the e n t i r e
canopy during the s h o r t e s t p e r i o d of time, thus i n c u r r i n g the
greatest d r a i n on r e s e r v e s , may defer maximum polyphenol
production u n t i l a f t e r the canopy i s mature. More i n f o r m a t i o n
i s needed to examine these ideas.
Summary
In order to g a i n an understanding of the options which

p l a n t s can u t i l i z e to defend themselves against herbivores we
need a more comprehensiv vie f th carbo d nitroge
a c q u i s i t i o n and a l l o c a t i o
h a b i t a t s . For example,
a l l o c a t i o n p a t t e r n s between annual versus p e r e n n i a l p l a n t s
which r e l a t e to the resources a v a i l a b l e to them through time.
Further, there are d i f f e r e n c e s i n the r e u t i l i z a t i o n
p o s s i b i l i t i e s of e i t h e r carbon or n i t r o g e n compounds w i t h i n a
plant as i t develops. These d i v e r s e a c q u i s i t i o n and a l l o c a t i o n
p a t t e r n s place c o n s t r a i n t s on the p o s s i b l e defensive s t r a t e g i e s
of p l a n t s and hence on the o p p o r t u n i t i e s f o r h e r b i v o r e a t t a c k .
Acknowledgements. This paper grew out of work supported by NSF

grant DEB810279. We thank B. L i l l e y f o r a s s i s t a n c e and
F.S. Chapin I I I , J.C. S c h u l t z , G. P u t t i c k and J . Glyphis
f o r comments on a v e r s i o n of t h i s paper.
L i t e r a t u r e Cited
1. Rosenthal, G.A.; Janzen D.H., Eds.; "Herbivores. Their

I n t e r a c t i o n w i t h Secondary P l a n t M e t a b o l i t e s " . Academic
Press: N.Y., 1979, p 718.
2. Feeny, P.P. Wallace, J . ; M a n s e l l , R. Eds.;"Biochemical
I n t e r a c t i o n s between P l a n t s and I n s e c t s " . Recent Adv.
Phytochemistry 1979, 10, 1-40.
3. Faeth, S.H.; Mopper, S.; Simberloff D. Oikos 1981, 37, 238-
251.
4. Mooney, H.A.; Gulmon, S.L. BioScience 1982, 32, 198-206.
5. Mattson, W.J. Ann. Rev. E c o l . Syst. 1980, 11, 119-161.
6. Mooney, H.A.; Gulmon, S.L. "Plant P o p u l a t i o n B i o l o g y " .
S o l b r i g , O.T.; J a i n , S. Johnson G.B.; Raven, P.H. eds.;
Columbia U n i v e r s i t y Press N.Y. 1979: p 316.
7. Gulmon, S; Chu, C. Oecologia 1981, 49, 207-212.
8. Mooney, H.A.; F i e l d , C.; Gulmon, S.L.; Bazzaz, F.A.
Oecologia 1981, 50, 109-112.
9. Tso, T.C. "Physiology and Biochemistry of Tobacco
P l a n t s " . Dowden, Hutchinson and Ross, Inc. Pa. p 393.

2. MOONEY E T AL. Plant Chemical Defenses
10. Dement, W.A.; Mooney, H.A. Oecologia 1974, 15, 65-76.

11. Mooney, H.A.; Kummerow, J.; Johnson, A.W.; Parsons, D.J.;
Keeley, S.; Hoffmann, A.; Hays, R.I.; Giliberto J . ; Chu,
C. "Convergent E v o l u t i o n in C h i l e and California. Mooney,
H.A. Ed.; Dowden, Hutchinson and Ross Inc. Stroudsberg, Pa.
1977, p 85.
12. Rhoades, D.; Cates, R.G. "Biochemical I n t e r a c t i o n s Between
P l a n t s and I n s e c t s " Wallace, J . ; M a n s e l l , R.; Eds. Rec.
Adv. Phyto Chem. 10, 168-213. Plenum Press N.Y. London.
13. Ryan, D.F.; Bormann, F.H. BioScience 1982, 32, 29-32.
14. Derman, B.D.; Rupp, D.C.; Nooden, L.D. Amer. J. Bot. 1978,
65, 205-213.
15. T a y l o r , B.K.; May, L.H. Aust. J. Biol. Sci. 1967, 20, 389-
411.
16. Kramer, P.J.; K o z l o w s k i T.T "Physiology of Woody P l a n t s "
Academic Press N.Y.
17. Tso, T.C.; Jeffrey
253-6.
18. S e i g l e r , D.S. Biochemical Systematics and Ecology 1977, 5,
195-199.
19. Robinson, T. Science 1974, 184, 430-435.
20. D i g e n i s , G.A. J. Pharm. Sci. 1969, 58, 39-42.
21. Clegg, D.O.; Conn, E.; Janzen, D. Nature 1979, 278, 343-
344.
22. Waller, G.R.; Nowacki, E.K. " A l k a l o i d Biology and
Metabolism in P l a n t s " . Plenum P r e s s . N.Y., 1978, p 194.
23. McKey, D. Am. Nat. 1974, 108, 305-320.
24. Beadle, N.C.W. Ecology 1953, 34, 426-428.
25. Vitousek, P.M.; R e i n e r s , W.A. B i o S c i e n c e 1975, 25, 376-381.
26. M u l l e r , R.N.; Bormann, F.H. Science 1976, 193, 1126-1128.
27. Mahall B.E.; Bormann, F.H. Bot. Gaz. 1978, 139, 467-481.
28. Herridge, D.F.; Pate, J.S. P l a n t P h y s i o l . 1977, 60, 759-
764.
29. Mooney, H.A.; Rundel, P.W. B o t a n i c a l Gazette 1979, 140,
109-113.
30. Fritts, H.C. "Tree Rings and Climate". Academic Press,
London, 1976, p 567.
31. Dougherty, P.M.; Teskey, R.O.; Phelps, J.E.; H i n c k l e y , T.M.
P l a n t P h y s i o l . 1979, 64, 930-935.
32. L i n c o l n , D.E. Biochem. Syst. and E c o l . 1980, 8, 397-400.
33. Satoo, T. " A n a l y s i s of Temperate Forest Ecosystem".
R e i c h l e , D.E. Ed. Springer V e r l a g , Heidelberg, N. Y.,
1970, p. 55
34. T a y l o r , F.G. Jr. "Phenology and S e a s o n a l i t y Modeling",
Helmut L i e t h (Ed), S p r i n g e r - V e r l a g , N.Y., Heidelberg
Berlin, 1974, p.237.
35. Kozlowski, T.T.; Keller, T. Bot. Rev. 1966, 32, 293-382.
36. Hansen, P. Physiol. P l a n t . 1971, 25, 469-473.
37. Haukioja, E. Holartic Ecology 1979, 2, 272-274.
38. Farnsworth, C.E. Ecology 1955, 36, 285-292.

39. K i e n h o l z , R. E c o l . 1941, 22, 249-258.

40. Kramer, P.J. P l a n t P h y s i o l . 1943, 18, 239-251.
41. Ahlgren, C.E. Ecology 1957, 38, 622-682.
42. Feeny, P.P. Ecology 1970, 51, 565-581.
43. Hough, J.A.; Pimental, D. E n v i r . Entomol. 1978, 7, 97-102.
44. S c h u l t z , J.C.; Nothnagle, P.J.; Baldwin, I.T. Am. J . Bot.
1982, 69, 753-759.
45. S c r i b e r , J.M.; Stansky, F. Jr. Ann. Rev. Entomol. 1981, 26,
183-211.
46. Cates, R.G. Oecologia 1980, 46, 22-31.
47. Bernays, E.A. E c o l . Ent. 1981, 6, 353-360.
48. Haukioya, E.; Niemelä, L.; Iso-Iivari; O j a l a , H.; Aro, E.
Rep Kevo S u b a r c t i c Res. S t a t . 1978, 14, 5-12.
49. Mooney, H.A.; Hays, R.L. F l o r a 1973, 162, 295-304.

3
Impact of Variable Plant Defensive Chemistry
on Susceptibility of Insects to Natural Enemies
JACK C. SCHULTZ
Dartmouth College, Department of Biological Sciences, Hanover, NH 03755
The major role of chemical defenses i n plants i s

hypothesized to be increasing the impact of
insect diseases
of these factor
why e v o l u t i o n a r i l y l a b i l e insects rarely
defoliate t h e i r long-lived hosts. However,
interactions among all of them could increase the
useful evolutionary l i f e t i m e of each and the
effectiveness of all. In p a r t i c u l a r , chemical
v a r i a b i l i t y i s observed to place insects i n
compromise situations which increase t h e i r
exposure and s u s c e p t i b i l i t y to natural enemies.
Forest trees are shown to be highly variable i n
space and time, and the impact of t h i s
variability on c a t e r p i l l a r s i s explored i n
several examples.
Despite the impression made by o c c a s i o n a l widespread p e s t

outbreaks such as those o f the gypsy moth, severe d e f o l i a t i o n o f
f o r e s t e d ecosystems i s q u i t e unusual. Fewer than 10% o f the
species l i s t e d i n the Canadian F o r e s t Survey o f L e p i d o p t e r a (1,
2) e x h i b i t p e r i o d i c or o c c a s i o n a l outbreaks. G e n e r a l l y , d e f o l i -
a t i o n i n f o r e s t s i s l e s s than 7% of primary p r o d u c t i o n p e r year
(_3, b u t see 4) . The v a s t m a j o r i t y o f f o r e s t L e p i d o p t e r a a r e
q u i t e r a r e almost a l l o f the time, and t h e i r numbers do n o t
f l u c t u a t e t o a n o t i c e a b l e degree. These o b s e r v a t i o n s suggest
t h a t some f a c t o r o r f a c t o r s normally r e g u l a t e f o r e s t i n s e c t
p o p u l a t i o n s and keep d e f o l i a t i o n a t low l e v e l s .
A number o f r e g u l a t o r y f a c t o r s have been proposed, and
v a r i o u s p o t e n t i a l r e g u l a t o r s have been shown t o operate i n
c e r t a i n systems (S,6). However, none o f these f a c t o r s can be
shown t o be. g e n e r a l l y e f f e c t i v e i n most o r a l l f o r e s t s . F o r
example, a g i v e n p a r a s i t o i d s p e c i e s may be the most important
i n f l u e n c e on i t s host a t one s i t e b u t not a t another (7). I t i s
d i f f i c u l t t o i d e n t i f y emergent g e n e r a l i z a t i o n s about the
r e l a t i v e importance of v a r i o u s p o t e n t i a l c o n t r o l s .
0097-6156/83/0208-0037$06.00/0

Indeed, e m p i r i c a l o b s e r v a t i o n , e v o l u t i o n a r y theory, and

common sense a l l suggest t h a t s i n g l e - f a c t o r approaches are not
l i k e l y t o i d e n t i f y u n d e r l y i n g c a u s a l r e l a t i o n s h i p s . Consider the
impact of p l a n t defensive chemistry. In recent years i t has
become c l e a r t h a t secondary compounds and the r e l a t i v e concentra-
t i o n s of primary n u t r i e n t s i n p l a n t t i s s u e s may r e s t r i c t f e e d i n g
and growth of herbivorous i n s e c t s (see 8 ) . C l e a r l y the a v a i l -
a b i l i t y of p l a n t t i s s u e s i s a f u n c t i o n of t i s s u e q u a l i t y as w e l l
as q u a n t i t y , h e l p i n g t o e x p l a i n why herbivorous i n s e c t s may
appear f o o d - l i m i t e d before t h e i r host t i s s u e s are exhausted (9).
However, t o maintain f o r e s t i n s e c t p o p u l a t i o n s a t the s t a b l e ,
extremely low l e v e l s we normally observe, p l a n t chemistry would
have t o deter f e e d i n g , poison i n s e c t s , or reduce d i g e s t i o n so
s t r o n g l y t h a t p o p u l a t i o n dynamics of the i n s e c t s are profoundly
depressed. T h i s n e c e s s i t a t e s d r a s t i c a l l y reducing the f i t n e s s of
organisms having enormou
growth r a t e s , and very
Lepidoptera have p o t e n t i a l f e c u n d i t i e s of from 200-1000 eggs/
female, y e t year-to-year p o p u l a t i o n l e v e l s i n d i c a t e s s u r v i v o r -
ship of only 1 or 2 per female (10).
Such a strong impact on s u r v i v o r s h i p or f e c u n d i t y , and on the
f i t n e s s of i n d i v i d u a l s , means e x e r t i n g strong n a t u r a l s e l e c t i o n on
herbivorous i n s e c t s . T h i s should f a v o r the r a p i d e v o l u t i o n of
i n s e c t adaptations which overcome i t . T h i s i s , of course, a
common occurrence i n the a p p l i c a t i o n of p e s t i c i d e s or the develop-
ment of r e s i s t a n t crop p l a n t c u l t i v a r s (11). The s u p p o s i t i o n t h a t
p l a n t defenses s e l e c t f o r d e t o x i c a t i o n adaptations i n i n s e c t s i s
the foundation of the concept of c o e v o l u t i o n (12).
F o r e s t t r e e s represent a p a r t i c u l a r l y v u l n e r a b l e paradox. An
i n d i v i d u a l t r e e may l i v e f o r 300 or more years. During t h i s time
i t does not move, and presumably cannot adapt t o environmental
change. I f t r e e defenses were r e s p o n s i b l e f o r the strong r e g u l a -
t i o n of i n s e c t s and h e r b i v o r y , the l i f e t i m e of a s i n g l e t r e e
ought t o p r o v i d e enough time f o r the e v o l u t i o n of h i g h l y v i r u l e n t
i n s e c t s which c o u l d d e f o l i a t e t h e i r host t r e e s repeatedly. T h i s
does not appear t o happen.
Feeny (13) attempted t o r e s o l v e t h i s dilemma by proposing
t h a t f o r e s t t r e e s may have developed a p a r t i c u l a r l y r e c a l c i t r a n t
defense, one which even i n s e c t s c o u l d not overcome i n hundreds of
generations. H i s suggestion was t h a t protein-complexing p o l y -
phenols, or t a n n i n s , could p r o v i d e such p r o t e c t i o n . However,
there are many i n s e c t s which feed p r e f e r e n t i a l l y on h i g h - t a n n i n
content t i s s u e s (14,L5) , and s p e c i f i c adaptations e x i s t which can
n u l l i f y or reduce the d i g e s t i o n i n h i b i t i o n e f f e c t s of tannins
(16) •
One must conclude t h a t no uniform p h y s i c a l or chemical
defense should be regarded as insurmountable by e v o l v i n g i n s e c t s .
Any uniform chemical p l a n t defense should s e l e c t f o r p e s t s
capable of d e f e a t i n g i t . Obviously, however, there i s a s o l u t i o n

3. SCHULTZ Insect Susceptibility 'to Natural Enemies 39
t o t h i s dilemma, s i n c e , as I have p o i n t e d out, f o r e s t s are not

s t r i p p e d year a f t e r year.
The E v o l u t i o n a r y Importance of Chemical Variability
In f a c t , there are a t l e a s t 3 p o s s i b l e s o l u t i o n s . A l l three

have one t h i n g i n common: they focus on the r a p i d l y - g r o w i n g body
of evidence t h a t t r e e s are not uniform i n defensive chemistry.
Instead, most p l a n t s are h i g h l y complex, dynamic mosaics o f
v a r i a b l e chemistry and n u t r i e n t v a l u e . T h i s o b s e r v a t i o n suggests
ways i n which defensive chemistry may remain e f f e c t i v e over many
i n s e c t generations:
Complex r e s i s t a n c e . Q u a l i t a t i v e and/or q u a n t i t a t i v e
chemical v a r i a t i o n i n p l a n t insect t tha
d e t e r r e n t (or poison) c o n c u r r e n t l y
posed (12^,18,1^) and Pimente (20)
l a b o r a t o r y , t h a t i n s e c t s may be slower t o adapt t o such complex
chemical mixtures. As a r e s u l t , even s u b l e t h a l doses o f t o x i n s
may remain e f f e c t i v e over long p e r i o d s o f time.
Resource r e s t r i c t i o n . I f chemical defenses vary q u a n t i t a -

t i v e l y w i t h i n or between i n d i v i d u a l p l a n t s , then some t i s s u e s may
be defended w h i l e others are not. As a r e s u l t , i n s e c t s have
a v a i l a b l e t o them the e v o l u t i o n a r y o p t i o n o f avoidance; they may
develop the a b i l i t y t o recognize poor q u a l i t y food and a v o i d i t ,
r a t h e r than e v o l v i n g d e t o x i c a t i o n mechanisms (12,18). T h i s should
r e s u l t i n f e e d i n g a c t i v i t y concentrated on a r e s t r i c t e d set o f
t i s s u e s or p l a n t i n d i v i d u a l s . There are two important conse-
quences o f t h i s . F i r s t , contact r a t e s w i t h defenses can be
lowered by a v o i d i n g them. Hence, the e v o l u t i o n of d e t o x i c a t i o n i s
l e s s l i k e l y o r l e s s r a p i d (18). Second, and perhaps more
important, the e f f e c t i v e n e s s o f n a t u r a l enemies may be enhanced
(below).
M u l t i p l e - f a c t o r i n t e r a c t i o n s . Each p o t e n t i a l r e g u l a t o r y
f a c t o r may i n t e r a c t s y n e r g i s t i c a l l y w i t h the others and enhance
t h e i r e f f e c t i v e n e s s . For example, p l a n t chemistry can i n f l u e n c e
the e f f e c t i v e n e s s of p r e d a t o r s , p a r a s i t o i d s and diseases i n a
v a r i e t y of ways (_21,J22,23) . However, the s e l e c t i v e pressure
exerted by uniform chemical defenses should be strengthened by
i n t e r a c t i o n s w i t h n a t u r a l enemies, and t h e i r u s e f u l l i f e w i l l be
shortened.
Consequently, although the ways i n which p l a n t chemistry can
i n f l u e n c e the e f f e c t i v e n e s s o f n a t u r a l enemies are d i z e r s e , they
can remain e f f e c t i v e through e v o l u t i o n a r y time only i f v a r i a b i l i t y
i s p a r t o f the p i c t u r e as w e l l . I n f a c t , although reviews have
tended t o focus on chemical enhancement o f n a t u r a l enemy r e g u l a -
t i o n (23), there are probably as many ways i n which uniform p l a n t
chemistry can i n t e r f e r e w i t h the a c t i o n s o f these enemies as there

are p o s i t i v e e f f e c t s (24,25,26). I t i s not even c l e a r t h a t the

impact o f uniform p l a n t defenses w i l l always be p o s i t i v e from the
p l a n t ' s p o i n t o f view.
I suggest t h a t v a r i a b l e p l a n t chemistry, by r e s t r i c t i n g
resource a v a i l a b i l i t y and f o c u s i n g the a c t i v i t i e s of herbivores
on a few t i s s u e s , promotes compromises between f o o d - f i n d i n g and
r i s k s from n a t u r a l enemies which are n o t r e a d i l y countered by
most i n s e c t s . The s p a t i a l and temporal heterogeneity which
appears t o be common i n f o r e s t t r e e s i s the most important p a r t
of the t r e e ' s defensive system, and i s the only way a p l a n t ' s
chemical defenses can remain e f f e c t i v e over e v o l u t i o n a r y time.
T h i s v a r i a b l e impact on n a t u r a l enemies may be more important i n
r e g u l a t i n g consumption than any s i n g l e f a c t o r can be.
V a r i a b i l i t y i n Tree Defenses
There are many p o s s i b l

v a r i a b i l i t y i n t r e e t i s s u e s (27,28) which r e s u l t i n a wide range
of s p a t i a l a r r a y s o f s u i t a b l e and u n s u i t a b l e food f o r i n s e c t s
(29). Although l a r g e - s c a l e s p a t i a l v a r i a t i o n may i n f l u e n c e
i n s e c t host race formation and have i n t e r e s t i n g consequences f o r
i n s e c t biogeography and host race formation (30), the s c a l e o f
v a r i a t i o n w i t h which the i n d i v i d u a l i n s e c t deals most o f t e n i s
more l o c a l , on the i n d i v i d u a l t r e e or t i s s u e b a s i s .
Most a n t i h e r b i v o r e t r a i t s have been found t o be h i g h l y
v a r i a b l e on t h i s smaller s c a l e ; s i g n i f i c a n t v a r i a t i o n i n n u t r i e n t
content and secondary chemistry i s commonly observed w i t h i n t r e e
canopies, on a branch-to-branch b a s i s (28,29).
On an even f i n e r s c a l e , we have found t h a t adjacent leaves on
s i n g l e sugar maple (Acer saccharum Marsh) and y e l l o w b i r c h
(Betula a l l e g h e n i e n s i s B r i t t . ) may d i f f e r g r e a t l y i n s e v e r a l
t r a i t s important t o herbivorous i n s e c t s (31; F i g u r e 1). Some o f
these d i f f e r e n c e s , e.g., i n tanning c o e f f i c i e n t (32), vary by
f a c t o r s o f 2 o r more from l e a f t o l e a f (Figure 1). The p a t t e r n o f
such v a r i a t i o n appears random i n sugar maple, b u t may be age-
r e l a t e d and hence s p a t i a l l y p r e d i c t a b l e (young leaves occur only
a t c e r t a i n growing p o i n t s ) i n yellow b i r c h (31). I n s e c t s such as
c a t e r p i l l a r s f o r a g i n g along sugar maple branches may have l i t t l e
i n f o r m a t i o n a v a i l a b l e t o them about the s p a t i a l d i s t r i b u t i o n o f
l e a f q u a l i t y , w h i l e those f o r a g i n g i n y e l l o w b i r c h may be able t o
l o c a t e leaves w i t h p a r t i c u l a r t r a i t s by searching i n c e r t a i n
p l a c e s -{e.g., ends o f branches).
The s i g n i f i c a n c e o f such s p a t i a l a r r a y s l i e s i n the
b e h a v i o r a l responses of i n s e c t s f o r a g i n g i n these t r e e s . I f
c e r t a i n l e a f types a r e u n a v a i l a b l e w h i l e others a r e p r e f e r r e d ,
then such s p a t i a l arrays f o r c e i n s e c t s t o move about i n search of
good feeding s i t e s (29). For i n s e c t s which spend much time (or
a l l o f t h e i r l i v e s ) feeding i n one p l a c e ( s e s s i l e s p e c i e s , such as
a p h i d s ) , t h i s search i s performed once; a f t e r a s u i t a b l e s i t e i s
l o c a t e d , these i n s e c t s a r e r e s t r i c t e d t o one p o r t i o n o f t h e i r

SCHULTZ Insect Susceptibility to Natural Enemies
TOUGHNESS
12.0-I g/cm 2
•mm una u Vi^174.75 V ; ^ W / ; # V ; | ^ 97.75

12.0
m ntiii mmm
WATER
J
64.48 70.75
m 'll.JMtMMIk
POLYPHENOLS
V. TAE
0.03
0.177 045
0.03
TANNING
% TAE
0.340
LEAF A B C D E A B C D E
Figure 1. Leaf-to-leaf variation in four traits along a single branch of sugar maple
(left) and yellow birch (right) on 6/23/81. Horizontal axis is mean of each measure
for that branch; hatched area is one standard deviation. Each black bar represents
the actual value for one leaf, plotted as deviation from the mean. Branch terminus
is to right; yellow birch leaves D and E are at least 10 days younger than the others.

h o s t s . More mobile species which r e q u i r e more food t o complete

development (e.g., c a t e r p i l l a r s ) must repeatedly search f o r new
feeding s i t e s throughout t h e i r l i v e s .
Many i n s e c t s do indeed recognize and respond t o t i s s u e
q u a l i t y v a r i a t i o n . There are numerous examples o f l e a f - a g e -
s p e c i f i c preferences among f o l i v o r o u s i n s e c t s , i n c l u d i n g some
which make r i g i d feeding d e c i s i o n s based on t i s s u e ages which
d i f f e r by l e s s than a few weeks (29). The apparent r e s i s t a n c e o f
i n d i v i d u a l t r e e s i n otherwise s u s c e p t i b l e stands has been recog-
n i z e d f o r some time (e.g., 33). The few species o f Lepidoptera
l a r v a e whose f o r a g i n g behavior has been s t u d i e d t r a v e l c o n s i d e r -
able d i s t a n c e s (sometimes s e v e r a l meters) and spend l a r g e
p r o p o r t i o n s o f t h e i r time sampling, r e j e c t i n g and judging the
a c c e p t i b i l i t y o f leaves on i n d i v i d u a l host t r e e s (29). G a l l
forming aphids s e l e c t leaves o f a c e r t a i n s i z e and may engage i n
t e r r i t o r i a l disputes t
clear that individual insect
f o r e s t t r e e leaves.
Temporal v a r i a b i l i t y i n t r e e t i s s u e q u a l i t y i s w e l l known
(see _29r.35 f o r review) . Year-to-year, seasonal, day-to-day, and
d i u r n a l s h i f t s i n n u t r i e n t contents and secondary chemistry have
a l l been observed. P a r t i c u l a r l y i n t r i g u i n g i s the i n c r e a s i n g
body o f evidence t h a t damage by i n s e c t s and pathogens may r e s u l t
i n short-term o r year-to-year changes i n secondary chemistry (36,
32). We have found t h a t ongoing d e f o l i a t i o n by gypsy moth
(Lymantria d i s p a r L.) l a r v a e i s a s s o c i a t e d w i t h profound changes
i n p h e n o l i c chemistry o f red oak (Quercus rubrum) leaves (38).
Over a p e r i o d o f a month, tanning c o e f f i c i e n t s increased
d r a m a t i c a l l y , and seasonal (2 month) i n c r e a s e s i n h y d r o l y z a b l e
tannins were observed i n t r e e s undergoing d e f o l i a t i o n . P r e l i m -
i n a r y s t u d i e s o f y e l l o w b i r c h and sugar maple suggest t h a t day-
to-day responses i n p h e n o l i c p r o d u c t i o n may be generated by
damage t o leaves (39)•
The importance o f seasonal changes i n secondary chemistry
and n u t r i e n t s t o the feeding success and l i f e h i s t o r y p a t t e r n s o f
some f o r e s t Lepidoptera i s w e l l e s t a b l i s h e d (40,41). Year t o
year changes i n chemical phenology may i n f l u e n c e t r e e s u s c e p t i -
b i l i t y and i n s e c t p o p u l a t i o n dynamics (42,43). Hence, a given
t r e e may not present the same d i s t r i b u t i o n o f l e a f q u a l i t y i n
every year. I n s e c t s attempting t o assess host q u a l i t y f o r o f f -
s p r i n g which w i l l feed d u r i n g the next year o r even l a t e r i n the
same season may not have very complete i n f o r m a t i o n a v a i l a b l e f o r
selecting oviposition sites.
Shorter-term temporal v a r i a t i o n i n l e a f q u a l i t y should a c t t o
complicate the s p a t i a l a r r a y s d e s c r i b e d above. Thus, not only may
a f o r a g i n g i n s e c t have d i f f i c u l t y l o c a t i n g s u i t a b l e feeding s i t e s
i n space, but t h e i r l o c a t i o n s may s h i f t from time t o time o r
c o n t i n u o u s l y , as seasonal changes, i n d u c t i o n e f f e c t s , o r even
p l a n t pathogen a t t a c k (44) a l t e r t i s s u e q u a l i t y . A s u i t a b l e
t i s s u e a t one time may not be s u i t a b l e l a t e r i n the day, o r l a t e r
i n the i n s e c t ' s l i f e .

3. SCHULTZ Insect Susceptibility to Natural Enemies 43
The p i c t u r e o f a f o r e s t t r e e t h a t I wish t o p o r t r a y , then, i s

one o f great s p a t i a l h e t e r o g e n e i t y , complicated by ongoing change.
For an i n s e c t capable o f d e a l i n g w i t h some subset o f the great
number o f t i s s u e q u a l i t y f a c t o r s which could i n f l u e n c e f e e d i n g ,
there may be only a l i m i t e d a r r a y o f s u i t a b l e t i s s u e s i n a canopy.
These s u i t a b l e s i t e s may be w i d e l y s c a t t e r e d , f o r c i n g long
searches and much t r a v e l i n g . The p a t t e r n i s complicated f u r t h e r
by c o n s t a n t l y changing t i s s u e q u a l i t i e s . The s i t u a t i o n can be
s a i d t o resemble a " s h e l l game", i n which a v a l u a b l e resource
( s u i t a b l e leaves) i s "hidden" among many other s i m i l a r - a p p e a r i n g
but u n s u i t a b l e resources. The i n s e c t must sample many t i s s u e s t o
i d e n t i f y a good one. The l o c a t i o n o f good t i s s u e s may be
s p a t i a l l y u n p r e d i c t a b l e , and may even change w i t h time. For a
"choosy" o r d i s c r i m i n a t i n g i n s e c t , f i n d i n g s u i t a b l e food i n an
apparently uniform canopy c o u l d be h i g h l y complex.
Impact on N a t u r a l Enemie
Although chemical v a r i a b i l i t y may not a l t e r a l l o f the

p o t e n t i a l e f f e c t s o f p l a n t chemistry on the e f f e c t i v e n e s s o f
n a t u r a l enemies, there are a number o f important q u a l i t a t i v e
d i f f e r e n c e s i n the k i n d s o f i n t e r a c t i o n s p o s s i b l e . I n some cases
the impact v a r i a b l e chemistry may have on an i n s e c t ' s s u s c e p t i -
b i l i t y t o r i s k s i s simply g r e a t e r than i t would be were p l a n t
chemistry uniform. I n other cases w h o l l y d i f f e r e n t r e l a t i o n s h i p s
are p o s s i b l e .
T o x i c substances a c q u i r e d from the host p l a n t may p r o v i d e
r e s i s t a n c e t o p a r a s i t o i d s (24), pathogens (25), and predators
(45). By a v o i d i n g some t o x i n s i n p l a n t m a t e r i a l and s e l e c t i n g
s u p e r i o r food t i s s u e s , i n s e c t s feeding on v a r i a b l e hosts may
become more s u s c e p t i b l e t o some enemies. Of course, other
substances i n p r e f e r r e d t i s s u e s may s t i l l be t o x i c t o c e r t a i n o f
these enemies, but t h i s i s l e s s l i k e l y than i t would be were
p l a n t compounds u n i f o r m l y encountered by the host i n s e c t .
An i n s e c t host's exposure t o p a r a s i t e s and predators may be
increased by v a r i a b l e p l a n t defenses i n three ways. F i r s t , by
r e s t r i c t i n g f e e d i n g a c t i v i t y t o c e r t a i n t i s s u e types o r p o r t i o n s
of the host p l a n t , the p o s i t i o n o f i n s e c t hosts becomes more
p r e d i c t a b l e . P a r a s i t e s (24,46,42) o r predators (48) able t o
recognize p h y s i c a l p l a n t t r a i t s such as t i s s u e c o l o r o r form, o r
those capable o f employing the unique chemistry o f the p r e f e r r e d
t i s s u e s as cues (47,49) would be able t o l o c a t e t h e i r hosts more
r e a d i l y by f o c u s i n g t h e i r search on these t r a i t s .
Second, the increased movement necessary f o r l o c a t i n g w i d e l y
d i s p e r s e d feeding s i t e s should i n c r e a s e contact r a t e s w i t h
enemies. Movement makes i n s e c t s more conspicuous t o p a r a s i t o i d s
or predators s e n s i t i v e t o i t (50,51). Random encounters w i t h
arthropod predators o r p a r a s i t e s should i n c r e a s e w i t h searching
a c t i v i t y , as would r i s k o f dislodgement and f a l l o u t .

Moving long d i s t a n c e s and t a s t i n g many s u r f a c e s (29) may a l s o

g r e a t l y i n c r e a s e contact r a t e s w i t h pathogens. Pathogens are
o f t e n d i s t r i b u t e d on p l a n t t i s s u e s u r f a c e s by other host i n s e c t s
(52), and are t r a n s m i t t e d by subsequent contact w i t h the same
s u r f a c e s . Increased movement should d i s p e r s e pathogens more
w i d e l y and i n c r e a s e the p r o b a b i l i t y of encountering them. More-
over, the chemistry of p l a n t t i s s u e s may i n f l u e n c e the composition
of t h e i r surface f a u n a s / f l o r a s (53). Hence, by f o c u s i n g f e e d i n g
a c t i v i t i e s on t i s s u e s w i t h c e r t a i n chemical t r a i t s , i n s e c t s may
simultaneously f i n d themselves feeding on t i s s u e s which promote
the growth of pathogens. T h i s may be p a r t i c u l a r l y important when
l e a f age i s a c r i t e r i o n f o r c h o i c e . We have observed c o n s i s t e n t l y
e l e v a t e d v i r a l m o r t a l i t y (70% vs 30%, N = 80) among i n d i v i d u a l s of
the n o c t u i d , O r t h o s i a h i b i s c i Guenee, when fed o l d e r (45 days)
y e l l o w b i r c h leaves as compared w i t h those fed young ( l e s s than 10
days) leaves from the sam
due t o d i f f e r e n t i a l chemistr
more parsimonious hypothesis i s t h a t o l d e r leaves have had more
time t o c o l l e c t more pathogens. Hence, t r a v e l l i n g on o l d e r
leaves may be q u i t e r i s k y .
Some i n s e c t species may avoid the r i s k s of cuing v i s u a l
predators (e.g., b i r d s ) w h i l e moving by being a c t i v e only a t
n i g h t (29,54). I n many h a b i t a t s t h i s would mean reducing the
time a v a i l a b l e f o r feeding by one-half or t w o - t h i r d s , r e s u l t i n g
i n a decrease i n growth r a t e of as much as 40 or 50% (_55,56) .
Slowing the growth r a t e by t h i s much adds to the l e n g t h of time
an i n s e c t i s a v a i l a b l e t o a l l r i s k s (57,58); there i s a t r a d e o f f
between r e s t r i c t e d feeding and r i s k s over the l i f e t i m e of the
i n s e c t as w e l l as d u r i n g each feeding bout. T h i s i s the t h i r d
means by which the i n s e c t ' s exposure t o r i s k s i s increased by
v a r i a b l e host q u a l i t y .
These t r a d e o f f s can be d e p i c t e d g r a p h i c a l l y (Figure 2 ) . I
have suggested (29) t h a t the form of expected food y i e l d d u r i n g
an i n s e c t ' s f o r a g i n g among v a r i a b l e resources should be r e p r e -
sented by a r i s i n g , asymptotic curve i f the i n s e c t s e l e c t s some
subset of t i s s u e s from those i t encounters. We can p l o t the
s u r v i v o r s h i p p r o b a b i l i t y of an i n d i v i d u a l as a negative expo-
n e n t i a l f u n c t i o n of the capture r a t e ; such a f u n c t i o n f o r a
constant capture r a t e of 30% i s shown i n F i g u r e 2. A capture
r a t e of 30% represents the median r a t e of removal of c a t e r p i l l a r s
by b i r d s i n a n o r t h temperate f o r e s t as determined by Holmes e t
a l (59), and i s a c o n s e r v a t i v e estimate f o r p a r a s i t i s m r a t e s a t
moderate host d e n s i t i e s (e.g. 60). The more v a r i a b l e the leaves
from which an i n s e c t must s e l e c t a meal, the lower i t s expected
y i e l d over a given time i n t e r v a l (the lower y i e l d curve i n
F i g u r e 2 represents 1/2 the a v a i l a b l e leaves of the upper curve,
or t w i c e the v a r i a b i l i t y ) . As a r e s u l t of the increased time
spent s e a r c h i n g , the contact r a t e w i t h and p r o b a b i l i t y of capture
by a predator o r p a r a s i t e i n c r e a s e s g r e a t l y f o r a given food
y i e l d as t i s s u e s become more v a r i a b l e . There i s a d i r e c t
i n f l u e n c e of v a r i a b i l i t y on r i s k .

\ 30% Mortality
\
N
Less Variable

TIME OR DISTANCE
Figure 2. Graphical model of yield and risk accruing during foraging by a defoliating insect. Mor-
tality from natural enemies is assumed to be constant at 30%, resulting in exponentially decreasing
survivorship curve (dashed). Yield (solid lines) accumulates curvilinearly (see 29) and more rapidly
when leaves are less variable (because most leaves can be consumed) than when they are highly
variable (many do not contribute to the diet). For an expected yield of 1 hypothetical unit, foraging
longer (because leaves are variable) reduces the probability of surviving by an amount labeled

E(Y)=1 on right axis. The reduction in survivorship is much greater by the time E(Y)—2. Leaf
variability decreases the probability of surviving.
An example employing data f o r gypsy moth l a r v a e and t h e i r

t a c h i n i d f l y p a r a s i t e , B l e p h a r i n a p r a t e n s i s Meigen, i s d e p i c t e d i n
F i g u r e 3. The p a r a s i t o i d i n f e c t i o n r a t e i s d e r i v e d from s t u d i e s
done i n Centre County, PA, under moderate gypsy moth d e n s i t i e s
(60). The f l i e s begin t o o v i p o s i t on f o l i a g e when gypsy moth
l a r v a e are i n the 3d i n s t a r , and the microtype eggs are consumed
by l a r v a e . There i s d i f f e r e n t i a l p a r a s i t o i d s u r v i v o r s h i p i n
c a t e r p i l l a r s of d i f f e r e n t i n s t a r s , and the " s u r v i v a l " curve i n
F i g u r e 3 represents s u c c e s s f u l c a t e r p i l l a r k i l l s c o r r e c t e d f o r
p a r a s i t o i d m o r t a l i t y i n the host. C a t e r p i l l a r dry weights are
taken from a study (61) of the e f f e c t s of gypsy moth d e f o l i a t i o n
on host p l a n t food q u a l i t y and l a r v a l growth. The "normal
f o l i a g e " growth curve approximates the growth r a t e of gypsy moth
l a r v a e on normal oak f o l i a g e through the l a s t 4 i n s t a r s . The
"induced f o l i a g e " curve approximates the growth of l a r v a e on
f o l i a g e from d e f o l i a t e
l a r v a e i s about 4 days
f o l i a g e " l a r v a e (61). Most of the r e t a r d a t i o n occurs i n the
f i r s t 3 i n s t a r s ; by" the 5th and 6th i n s t a r s reduced food q u a l i t y
no longer depresses growth r a t e s below c o n t r o l l a r v a e (M.
Montgomery, pers. comm.).
As a consequence of an apparently induced change i n food
q u a l i t y (probably due to increased t a n n i n contents; _38), develop-
ment time i s lengthened. T h i s i n t u r n r e s u l t s i n an increase i n
p a r a s i t i s m r a t e s . The d e p i c t i o n i n F i g u r e 3, although somewhat
schematic, shows a decrease i n s u r v i v o r s h i p of almost 20% r e s u l t -
ing from a growth r a t e r e d u c t i o n of 3%. I n t e r e s t i n g l y , were
growth r a t e s slowed enough, the c a t e r p i l l a r s could escape
p a r a s i t i s m by t h i s f l y . B. p r a t e n s i s eggs l a s t about 2 weeks on
f o l i a g e . Were development of some c a t e r p i l l a r s delayed enough,
they might enter the 3d i n s t a r l a t e enough t o avoid v i a b l e
p a r a s i t o i d eggs. On the other hand, the a d u l t f l i e s apparently
t r a c k c a t e r p i l l a r p o p u l a t i o n development and time o v i p o s i t i o n t o
c o i n c i d e w i t h entry i n t o the 3d c a t e r p i l l a r i n s t a r (60).
Thus a constant m o r t a l i t y or s u s c e p t i b i l i t y , from a complex
of enemies or from g e n e r a l i z e d predators or p a r a s i t e s , r e s u l t s i n
a steep i n c r e a s e i n r i s k w i t h time vFigure 2). The time
necessary t o accumulate m a t e r i a l s f o r growth and the l e v e l of r i s k
w h i l e doing so may be increased g r e a t l y when food p l a n t q u a l i t y i s
v a r i a b l e . Both s p a t i a l v a r i a b i l i t y and temporal v a r i a b i l i t y (e.g.
induction) can have t h i s e f f e c t . Even when the r i s k accumulation
i s slower and growth i s slowed a very small amount (as i n the
f l y - g y p s y moth c a s e ) , host p l a n t v a r i a t i o n can have a major impact
on exposure t o enemies (Figure 3).
F i n a l l y , density-dependent m o r t a l i t y from v a r i o u s enemies may
be enhanced by host p l a n t v a r i a t i o n . Again, f o c u s i n g feeding
a c t i v i t i e s on a r e s t r i c t e d set of s u i t a b l e t i s s u e s should a l s o
focus the a c t i v i t i e s and abundance of pathogens, p a r a s i t o i d s , and
predators. S e s s i l e i n s e c t s , such as g a l l - f o r m i n g aphids (55,62),

Figure 3. Relationship between growth rate of gypsy moth larvae on "normal" and "induced"
foliage and mortality due to the tachinid parasite, Blepharina pratensis. Larvae grow more slowly
on induced foliage and are exposed to parasitism longer. A given larval weight, X, is attained later
on induced foliage, so that infection rates are higher. Hatched area accounts for larval-age specific

mortality of B . pratensis; upper "survival" curve is corrected survival curve for infected larvae. In
this case, a 3% decrease in growth of gypsy moth larvae results in an approximately 20% decrease
in survivorship. See text for details.
experience increased contagion i n terms of m o r t a l i t y from enemies.

By o c c u r r i n g p r e d i c t a b l y on p a r t i c u l a r p l a n t surfaces or i n
p a r t i c u l a r l o c a t i o n s , i n s e c t s may focus the searching a c t i v i t i e s
of predators and p a r a s i t e s i n a density-dependent f a s h i o n (e.g.,
55,62,63).
More complex, second order i n t e r a c t i o n s may be imagined,
i n v o l v i n g more than one n a t u r a l enemy. For example, consider
i n s e c t s t o which tannins are important d e t e r r e n t s and d i g e s t i o n
i n h i b i t o r s . As mentioned above, e l e v a t e d gut pH appears to be a
way of d e a l i n g w i t h t a n n i n s , since t a n n i n - p r o t e i n complexes are
d i s s o c i a t e d or i n h i b i t e d a t a l k a l i n e pH (16,32). Indeed, u s i n g a
model i n v i t r o system i n which hemoglobin i s employed as a p r o t e i n
s u b s t r a t e , we found t h a t s e v e r a l n a t u r a l tannins and p h e n o l i c
e x t r a c t s do not p r e c i p i t a t e t h i s p r o t e i n when the pH exceeds about
8.5 (Figure 3; 32); b i n d i n g i s q u i t e complete from pH 4 through 8.
Although hemoglobin i s
p l a n t p r o t e i n s i n molecula
f o r example) and i s a u s e f u l comparison (32).
I t i s i n t e r e s t i n g t o note t h a t the s o l u b i l i t y of the
c r y s t a l l i n e t o x i n of a common, important c a t e r p i l l a r pathogen,
B a c i l l u s t h u r i n g i e n s i s ( B t ) , runs from j u s t over pH 8 to about
pH 9.5 (64,65,66). Above pH 9.5, there i s some doubt t h a t the
p r o t e i n t o x i n remains e f f e c t i v e (66). Hence, a c a t e r p i l l a r
adapted f o r feeding on h i g h - t a n n i n foods i s i n a p r e c a r i o u s
s i t u a t i o n , caught between i n c r e a s i n g the d i g e s t i b i l i t y of i t s food
and the r i s k of pathogen " s u s c e p t i b i l i t y . The s o l u b i l i t y of the
p r o t e i n coats of s e v e r a l nuclear p o l y h e d r o s i s v i r u s e s (NPV)- and
hence t h e i r v i r u l e n c e i n the i n s e c t gut- ranges from pH 4.5 t o pH
8.5 (67,68). Hence t a n n i n - t o l e r a n t i n s e c t s w i t h e l e v a t e d gut pH's
may be r e l a t i v e l y r e s i s t a n t t o these pathogens. According t o
theory (6£,70), e a r l y s u c c e s s i o n a l p l a n t s should have low t a n n i n
contents and t h e i r herbivores should have lower gut pH values
(16). An emerging hypothesis would be t h a t c a t e r p i l l a r species
feeding on l a t e s u c c e s s i o n a l t r e e s would be more s u s c e p t i b l e t o Bt
and l e s s s u s c e p t i b l e t o NPV than are t h e i r r e l a t i v e s on e a r l i e r -
s u c c e s s i o n a l p l a n t s . T h i s hypothesis i s as yet untested. It
could have great p r a c t i c a l importance, since these pathogens are
c u r r e n t l y being developed and promoted as b i o l o g i c a l c o n t r o l
agents f o r f o r e s t pests on both h i g h - t a n n i n and low-tannin t r e e
species.
M i c r o b i a l c h i t i n a s e has been proposed as a s y n g e r g i s t f o r Bt
(71). I t s r o l e would be t o d i g e s t holes i n the i n s e c t gut w a l l
and f a c i l i t a t e p e n e t r a t i o n of Bt t o x i n . However, unless the
c a t e r p i l l a r ' s gut pH can be manipulated (71), t h i s i s u n l i k e l y t o
be e f f e c t i v e w i t h B t , but might be f e a s i b l e w i t h NPV (Figure 4 ) .
How does chemical v a r i a b i l i t y enter i n t o t h i s pH scenario?
F i r s t , by c o n c e n t r a t i n g on low-tannin t i s s u e s , an i n s e c t may be
able t o feed on a t r e e species w i t h h i g h average t a n n i n values
w h i l e m a i n t a i n i n g a lower gut pH. However, t h i s could i n c r e a s e
pathogen r i s k (Figure 4 ) . Second, gut pH may f l u c t u a t e w i t h the

SCHULTZ Insect Susceptibility to Natural Enemies 49
Figure 4. Binding of a protein (hemoglobin) to several tannin extracts (tannic

acid, sugar maple tannins, yellow birch tannins, quebracho tannins; see 29) at vari-
ous pH values. Ranges of microbial chitinase activity, NPV activity, and Bt toxicity
are given. See text for discussion and references.

food a c t u a l l y i n g e s t e d , and may decrease i n animals starved f o r 12

hours (72). Thus any i n s e c t e x p e r i e n c i n g long p e r i o d s between
meals may experience lowered gut pH and p o s s i b l e d i g e s t i b i l i t y
problems when feeding begins. More i n t e r e s t i n g , a high gut pH may
d e c l i n e i n t o a r e g i o n of maximum p a t h o g e n i c i t y f o r organisms l i k e
Bt.
But why would a c a t e r p i l l a r not feed f o r up t o 12 hours? I f
s u i t a b l e food i s widely s c a t t e r e d and r i s k s of movement among
f e e d i n g s i t e s are h i g h (above), many i n s e c t s may be forced t o feed
only a t n i g h t (29,54). In north temperate f o r e s t s , such an i n s e c t
w i l l " s t a r v e " f o r from 8 t o 14 hours. One consequence of t h i s
t a c t i c may be t h a t the f i r s t meal of the evening may be very
risky.
Some aspects of p l a n t v a r i a t i o n could i n t e r f e r e w i t h the
impact of n a t u r a l enemies. Some enemies may be unable t o a s s o c i -
ate m i c r o h a b i t a t cues (e.g.
w i t h prey or host l o c a t i o n
f e e d i n g on r e s t r i c t e d t i s s u e s w i l l tend t o appear widely spaced
and they may not be r e a d i l y encountered. I t appears t o me t h a t
many, i f not most, p a r a s i t o i d s and predators can be found t o use
one o r more cues. T h i s negative e f f e c t could be counteracted by
i n c r e a s e d encounter r a t e s d u r i n g h e r b i v o r e searching movements.
The metabolic c o s t s of t r a v e l l i n g among feeding s i t e s and
r e s t i n g long p e r i o d s without feeding could be t r a n s l a t e d d i r e c t l y
i n t o reduced i n s e c t f e c u n d i t y (29). Were t h i s e f f e c t strong
enough, i t i s conceivable t h a t i n s e c t d e n s i t i e s might be reduced
d i r e c t l y . A p o s s i b l e consequence of t h i s would be reduced
density-dependent m o r t a l i t y . There are no data a v a i l a b l e f o r the
metabolic c o s t s of 'walking f o r i n s e c t s such as c a t e r p i l l a r s .
Conclusions and Management Prospects
I have argued t h a t uniform chemical defenses cannot be

e v o l u t i o n a r i l y s t a b l e . For t r e e s , t h i s means they should not
remain e f f e c t i v e even f o r a s i n g l e t r e e generation. But t r e e s are
not uniform; they are dynamic, h i g h l y d i v e r s e h a b i t a t s and food
sources f o r i n s e c t s . Although p l a n t chemistry can i n f l u e n c e
n a t u r a l enemies d i r e c t l y , i t may do so i n e i t h e r p o s i t i v e o r
negative f a s h i o n , and t h i s i n f l u e n c e should not remain e f f e c t i v e
over e v o l u t i o n a r y time, e i t h e r . However, chemical v a r i a b i l i t y
i n f l u e n c e s s u s c e p t i b i l i t y of h e r b i v o r e s t o n a t u r a l enemies by
f o r c i n g c o s t l y t r a d e o f f s upon i n s e c t s which i n v o l v e unavoidable
r i s k s and metabolic c o s t s . These d i f f i c u l t i e s are l e s s e a s i l y
overcome by adaptation. I n a d d i t i o n t o these s y n e r g i s t i c e f f e c t s ,
chemical v a r i a b i l i t y could maintain the e f f e c t i v e n e s s of p l a n t
defenses through a general slowing of adaptation due to lowered
contact r a t e s w i t h s p e c i f i c defenses or the d i f f i c u l t y of d e a l i n g
with multiple factors.
These observations are of p r a c t i c a l importance. The k i n d s of
v a r i a b i l i t y I have d e s c r i b e d appear t o e x e r t c o n t r o l on i n s e c t

p o p u l a t i o n s and consumption i n nature. For example, i n d u c t i o n

responses may be c r i t i c a l t o l i m i t i n g o c c a s i o n a l pest outbreaks
(35,36,38). The t i m i n g and type o f human i n t e r v e n t i o n i n such
events c o u l d d i s r u p t such d e l i c a t e c o n t r o l s . E a r l y r e d u c t i o n i n
i n s e c t d e n s i t i e s d u r i n g an outbreak through the use o f p e s t i c i d e s
or a b i o l o g i c a l c o n t r o l agent a c t i n g e a r l y i n the l i f e h i s t o r y o f
a p e s t l i k e the gypsy moth c o u l d reduce the impact o f c a t e r p i l l a r
feeding on host t r e e s and slow o r h a l t t r e e i n d u c t i o n responses.
J u s t such a s i t u a t i o n , although w i t h l i t t l e c o n s i d e r a t i o n o f the
b i o l o g y o f the p a r t i c i p a n t s , has been modelled by s e v e r a l authors
(73,74). As a r e s u l t o f untimely i n t e r v e n t i o n , i n t e r a c t i o n s
which may n a t u r a l l y l i m i t outbreaks c o u l d be f r u s t r a t e d and
a r t i f i c i a l c o n t r o l s may become necessary over extended p e r i o d s .
Some b i o l o g i c a l c o n t r o l e f f o r t s may be f a u l t y or may be
improved when p l a n t chemistry and v a r i a b i l i t y are taken i n t o
account. For example,
may work w e l l on h i g h - t a n n i
such as those f e e d i n g on l a t e s u c c e s s i o n a l o r slow-growing t r e e
s p e c i e s , but i t may be l e s s e f f e c t i v e on e a r l y s u c c e s s i o n a l
species o r e a r l y i n the growth season on h i g h - t a n n i n t r e e s . NPV
may be more e f f e c t i v e i n e a r l y s u c c e s s i o n a l s i t u a t i o n s o r any
s i t u a t i o n where t a n n i n s are not important p l a n t defenses. I n
a d d i t i o n , some p l a n t chemicals may make c e r t a i n b i o l o g i c a l c o n t r o l
agents l e s s e f f e c t i v e . Examples i n c l u d e p l a n t chemicals which are
t o x i c t o p a r a s i t o i d s (24) and those which are a n t i b a c t e r i a l (e.g.,
monoterpenes i n c o n f e r s ; 26). Knowledge o f n a t u r a l v a r i a t i o n i n
p l a n t chemistry c o u l d g r e a t l y a i d i n improving such c o n t r o l
methods.
F i n a l l y , I would suggest t h a t p l a n t v a r i a b i l i t y , genotypic
and/or phenotypic, i s as important t o t r e e s as i t i s t o
herbaceous s p e c i e s such as crop p l a n t s . I t should thus be as
important i n t r e e p l a n t a t i o n s and f o r e s t management as i t has
become i n a g r i c u l t u r e . As f o r e s t management takes on more
c h a r a c t e r i s t i c s o f l a r g e s c a l e a g r i c u l t u r e , perhaps we should take
a l e s s o n from the mad scramble f o r o l d "new" genes i n corn and
other crops and a v o i d the mistakes i n h e r e n t i n l a r g e , uniform
p l a n t a t i o n s (11,75,76). Tree defense v a r i a b i l i t y may be as
important or more important than uniform r e s i s t a n c e per se. I t
seems reasonable t o suggest m a i n t a i n i n g i t o r mimicking i t under
i n t e n s e management c o n d i t i o n s . C e r t a i n l y , there are s u b s t a n t i a l
grounds f o r c o n c e n t r a t i n g r e s e a r c h e f f o r t s on s t u d i e s o f v a r i a n c e
as w e l l as means.
Acknowledgements
Ideas were developed i n c o n v e r s a t i o n w i t h I.T. Baldwin, R.T.

Holmes, and P.J. Nothnagle. I.T. Baldwin c a r r i e d out chemical
analyses o f b i r c h and maple l e a v e s , and M.J. Richards drew the
f i g u r e s . I thank M i c h a e l Montgomery, USDA F o r e s t S e r v i c e , f o r
p e r m i s s i o n t o use unpublished data. Supported by NSF grant

DEB-8022174 t o JCS and RTH as p a r t of c o n t i n u i n g s t u d i e s of

h e r b i v o r y a t the Hubbard Brook Experimental F o r e s t , W. Thornton,
NH.
Literature Cited
1. P r e n t i c e , R. M. "Forest Lepidoptera of Canada, Recorded by

the F o r e s t I n s e c t Survey 2"; Canada Dept. F o r e s t r y Bull. 128.
2. P r e n t i c e , R. M. "Forest Lepidoptera of Canada, Recorded by
the F o r e s t I n s e c t Survey 3"; Canada Dept. F o r e s t r y , P u b l .
1013.
3. Mattson, W. J . ; Addy, N.D. Science 1975, 190, 515-522.
4. Journet, A. R. P. A u s t r a l . J. E c o l . 1981, 6, 135-138.
5. Andrewartha, H. G.; B i r c h , L. C. "The Distribution and
Abundance of Animals"; Univ. Chicago P r e s s , Chicago, 1954.
6. DeBach, P. " B i o l o g i c a
Cambridge Univ. Press
7. H a s s e l l , M. P. J. Animal E c o l . 1980, 49, 603-628.
8. Rosenthal, G. A.; Janzen, D. H. "Herbivores: T h e i r
I n t e r a c t i o n s w i t h P l a n t Secondary Compounds"; Academic
P r e s s , NY.
9. E h r l i c h , P. R.; B i r c h , L. C. Amer. Natur. 1967, 101, 97-107.
10. Lawton, J. H.; M c N e i l l , S. Symp. British E c o l . Soc. 1979,
20, 223-244.
11. Lupton, F. G. H. I n " O r i g i n s o f P e s t , P a r a s i t e , Disease and
Weed Problems"; J. M. Cherritt, G. R. Sagar, eds., B l a c k w e l l ,
London, 1977; pp. 71-83.
12. E h r l i c h , P. R.; Raven, P. H. E v o l u t i o n 1964, 18, 586-608.
13. Feeny, P. Rec. Adv. Phytochem. 1976, 10, 1-40.
14. Feeny, P. Ecology 1970, 51, 562-581.
15. S c h u l t z , J. C.; Holmes, R. T. i n prep.
16. Berenbaum, M. Amer. Nat. 1980, 115, 138-146.
17. D o l i n g e r , P. M.; Ehrlich P. R.; Fitch, W. L.; Breedlove,
D.E. Oecologia 1973, 13, 191-204.
18. Maiorana, V. B i o l . J . L i n n . Soc. 1979, 11, 387-396.
19. A t s a t t , P. R.; O'Dowd, D. J. Science 1976, 193, 24-29.
20. P i m e n t e l , D.; Belloti, A. C. Amer. Nat. 1976, 110, 877-
888.
21. G r e e n b l a t t , J. A. J . Appl. E c o l . 1981, 18, 1-10.
22. G r e e n b l a t t , J. A.; Barbosa, P. J . Appl. E c o l . 1981, 18,
1-10.
23. P r i c e , P. W.; Bouton, C. E.; Gross, P.; McPheron, B. A.;
Thompson, J. N.; Weis, A. E. Ann. Rev. E c o l . Syst. 1980,
11, 41-65.
24. Campbell, B. C.; Duffey, S. S. Science 1979, 205, 700-702.
25. Maksymiuk, B. J . I n v e r t . Path. 1970, 15, 356-371.
26. Smirnoff, W. A.; Hutchison, P, M. J . I n v e r t . Path. 1965, 7,
273-280.
27. Whitham, T. G.; S l o b o d c h i k o f f , C. N. Oecologia 1981, 49,
287-292.

28. Whitham, T. G. I n " I n s e c t Life H i s t o r y P a t t e r n s " , R. F.

Denno, H. D i n g l e eds., S p r i n g e r - V e r l a g , NY, 1981; pp. 9-27.
29. S c h u l t z , J. C. I n "Impact o f V a r i a b l e Host Q u a l i t y on
Herbivorous I n s e c t s " , R. F. Denno, M. S. McClure, eds.,
Academic P r e s s , NY, in p r e s s .
30. Fox, L. R. Morrow, P. A. Science 1981, 211, 887-893.
;
31. S c h u l t z , J. C.; Nothnagle, P. J.; Baldwin, I . T. Amer. J .

Bot. 1982, 69, 753-759.
32. S c h u l t z , J. C.; Baldwin, I . T.; Nothnagle; P. J. J. A g r i c .
Food Chem. 1981, 29, 823-826.
33. P a i n t e r , R. H. I n "Breeding P e s t - R e s i s t a n t Trees", H. D.
Gerhold, E. J. S c h r e i n e r , R. E. McDermott, J. A. W i n i e s k i ,
eds., Pergamon P r e s s , NY, 1966; pp. 349-355.
34. Whitham, T. G. Amer. Nat. 1980, 115, 449-466.
35. Rhoades, D. F. I n "Herbivores: T h e i r I n t e r a c t i o n w i t h P l a n t
Secondary Compounds"
Academic P r e s s , NY
36. Haukioja, E.; Niemelä, P. Ann. Z o o l . Fenn. 1977, 14, 48-52.
37. Benz, G. I n "Eucarpia/IDBC Working Group: Breeding f o r
Resistance t o I n s e c t s and M i t e s " , Bull. SROP, 1977; pp. 155-
159.
38. S c h u l t z , J. C.: Baldwin, I . T. Science 1982, 217, 149-151.
39. Baldwin, I . T.; S c h u l t z , J. C. in prep.
40. Schweitzer, D. F. Oikos 1979, 32, 403-408.
41. Hough, J. A.; P i m e n t e l , D. E n v i r . Entomol. 1978, 7, 97-102.
42. Campbell, I. T. I n "Breeding Pest R e s i s t a n t Trees", M. D.
Gerhold, E. J. S c h r e i n e r , R. E. McDermott, J. A. W i n i e s k i ,
eds., Pergamon P r e s s , NY, 1966; pp. 129-134.
43. Mattson, W. J.; Lorimer, N.; Leary, R. A. Proc. IUFRO Conf.
Genetics o f H o s t / P a r a s i t e I n t e r a c t i o n s , Centre f o r Agric.
P u b l . and Document., Wageningen, Netherlands, 1982; i n
press.
44. Matta, A. P l a n t Disease 1980, 5, 345-361.
45. Brower, L. P.; Van Zandt Brower, J. Z o o l o g i c a 1964, 49,
137-159.
46. A r t h u r , A. P., Can. Entomol. 1962, 94, 337-347; IBID 1966,
98, 213-223; IBID 1967, 99, 877-886.
47. Read, D. P.; Feeny, P. P.; Root, R. B. Can. Entomol. 1970,
102, 1567-1578.
48. H e i n r i c h , B. Oecologia 1979, 42, 325-337.
49. Ryan, R. B. Can. Entomol. 1979, 111, 477-480.
50. d e R u i t e r , L. Behaviour 1952, 4, 222-232.
51. Richerson, J . V.; DeLoach, C. J. Ann. Entomol. Soc. Amer.
1972, 65, 834-839.
52. Aizawa, K. I n "Insect Pathology, an Advanced T r e a t i s e
V o l . 1, E. A. Steinhaus, ed., Academic P r e s s , NY, 1963; pp.
381-412.
53. L o v e t t , J. V.; Duffield A. M. J . Appl. E c o l . 1981, 18,
283-290.

54. Windsor, D. M. I n "Ecology of A r b o r e a l F o l i v o r e s " , G. G.

Montgomery, ed., Smithsonian I n s t . P r e s s , Washington, DC,
1978; pp. 101-113.
55. McGinnis, A. J.; K a s t i n g , R. Can. J. Z o o l . 1959, 37, 259-
266.
56. Muthukrishna, J.; Delvi, M. R. Oecologia 1974, 16, 227-236.
57. Feeny, P. P. I n "Coevolution o f Animals and P l a n t s " , L.
Gilbert and P. R. Raven, eds., Univ. Texas P r e s s , A u s t i n ,
1975; p. 246.
58. S c h u l t z , J . C. E v o l u t i o n 1981, 35, 171-179.
59. Holmes, R. T.; S c h u l t z , J . C.; Nothnagle, P. J . Science
1979, 206, 462-463.
60. Doane, C. C.; McManus, M. L. USDA Tech. Bull. 1584, USDA-FS,
Washington, DC, 1981; p. 389.
61. W a l l n e r , W. E.; Walton G S Ann Entomol Sco Amer 1979
72, 62-67.
62. Whitham, T. G. I
Herbivorous I n s e c t s " , R. F. Denno, M. S. McClure, eds.,
Academic P r e s s , NY, in p r e s s .
63. Solomon, B. P.; McNaughton, S. J. Oecologia 1979, 42, 47-56.
64. Sharp, E. S.; Detroy, R. W. J . I n v e r t . Path. 1979, 34,
90-91.
65. Raun, E. S.; S u t t e r , G. R.; Revelo, M. A., J. I n v e r t . Path.
1966, 8, 365-375.
66. F a s t , P. G.; M i l n e , R. J . I n v e r t . Path. 1979, 34, 319.
67. Gudauskas, R. T.; Canerday, D. J . I n v e r t . Path. 1968, 12,
405-411.
68. I g n o f f o , C. M.; G a r c i a , C. J . I n v e r t . Path. 1966, 8, 426-
427.
69. Rhoades, D. F.; Cates, R. G. Rec. Adv. Phytochem. 1976, 10,
168-213.
70. Cates, R. G.; O r i a n s , G. H. Ecology 1975, 56, 410-418.
71. Daoust, R. A.; Gunner, H. B. J . I n v e r t . Path. 1979, 33,
368-377.
72. Heimpel, A. M. Can. J. Z o o l . 1955, 33, 94-106.
73. Anderson, R. M.; May, R. M. Science 1980, 210, 658-661.
74. Carpenter, S. R. J.Theor. Biol. 1981, 92, 181-184.
75. Walsh, J. Science 1981, 214, 161-164.
76. M a r s h a l l , D. R. Ann. NY Acad. Sci. 1977, 287, 1-20.

4
Responses of Alder and Willow to Attack
by Tent Caterpillars and Webworms:
Evidence for Pheromonal Sensitivity of Willows
D A V I D F. RHOADES
University of Washington, Department of Zoology, Seattle, WA 98195
Red alder (Alnus rubra) and Sitka willow (Salix sit-

chensis) trees subjecte
(Malacosoma californicu
tria cunea), respectively, exhibited a change in foliage
quality such that bioassay insects fed leaves from the
attacked trees grew more slowly than those fed leaves
from unattacked control trees. In contrast, bioassay
of leaf quality of S. sitchensis, subjected to attack
by tent c a t e r p i l l a r s , indicated that altered leaf quality
had been induced not only i n the attacked trees but also
in nearby unattacked control trees. This suggests that
S. sitchensis i s sensitive to and can respond to s i g -
nals generated by attacked trees or the c a t e r p i l l a r s .
Since no evidence was found for root connections bet-
ween attacked and control willows, the message may be
transferred through airborne pheromonal substances.
During the l a s t s e v e r a l years there has been an i n c r e a s i n g

i n t e r e s t i n , and demonstration of, the f a c t that p l a n t s subjected
to a t t a c k by i n s e c t s or other herbivores can decrease the q u a l i t y
of t h e i r t i s s u e s as food by i n c r e a s i n g t h e i r content o f defensive
substances, decreasing t h e i r content of n u t r i e n t s , o r both (1-5).
There i s evidence f o r both short and long term p l a n t responses t o
a t t a c k . Short term responses o c c u r r i n g during the p e r i o d o f
a t t a c k can be expected t o i n f l u e n c e the f i t n e s s o f the a t t a c k i n g
h e r b i v o r e s , whereas long term responses may i n f l u e n c e the f i t n e s s
of subsequent h e r b i v o r e s . These induced p l a n t responses could
have profound e f f e c t s on the p o p u l a t i o n dynamics of h e r b i v o r e s .
The experiments described here were designed t o detect short term
changes i n l e a f q u a l i t y o f red a l d e r (Alnus rubra Bong; B e t u l a -
ceae) and S i t k a w i l l o w ( S a l i x s i t c h e n s i s Sanson; Salicaceae) i n
response t o a t t a c k by two species o f polyphagous, u n i v o l t i n e ,
c o l o n i a l , d e f o l i a t i n g Lepidoptera, western tent c a t e r p i l l a r s
0097-6156/83/0208-0055$06.00/0

(Malacosoma c a l i f o r n i c u m p l u v i a l e Dyar; Lasiocampidae) and f a l l

webworms (Hyphantria ctmea Drury; A r c t i i d a e ) .
If herbivore a t t a c k can l e a d to reduced food q u a l i t y of
p l a n t s , then i t seems reasonable that n a t u r a l l y attacked p l a n t s
should e x h i b i t decreased food q u a l i t y compared to n a t u r a l l y
unattacked ones. On the other hand, there i s considerable e v i -
dence that herbivores p r e f e r e n t i a l l y a t t a c k p l a n t s or t i s s u e s of
h i g h food q u a l i t y (3). In other words, p l a n t food q u a l i t y can
probably act both as a dependent and an independent v a r i a b l e as
f a r as degree of h e r b i v o r y i s concerned. Therefore, conclusions
drawn from comparisons of f o l i a g e p r o p e r t i e s of n a t u r a l l y
attacked versus n a t u r a l l y unattacked p l a n t s are l i k e l y to be
confounded by the i n t e r a c t i o n of these two e f f e c t s . To avoid
t h i s problem the f o l l o w i n g experiments were conducted by
s u b j e c t i n g p l a n t s to a t t a c k by i n s e c t s placed on them by the
investigators.
Red A l d e r Attacked by Tent C a t e r p i l l a r s
In western Washington State tent c a t e r p i l l a r s were abundant

during the springs of 1975, 1976, and 1977, a t t a c k i n g a l d e r s ,
w i l l o w s , and other species of broad-leafed t r e e s . A l d e r s and
w i l l o w s at our study s i t e near Kent, King Co., Washington, were
h e a v i l y attacked i n 1977, some t r e e s s u f f e r i n g almost complete
d e f o l i a t i o n . In 1978 the l o c a l populations crashed, producing
few v i a b l e egg masses. Since the s p r i n g of 1979, no n a t u r a l
c o l o n i e s have been observed w i t h i n a r a d i u s of 10 km of the
Kent s i t e .
I n s p r i n g of 1979 seven p a i r s of 3-year-old a l d e r t r e e s of
3
average height 3.1 ± 0.1 (S.E.) m and volume 3.5 ± 0.5 m were
s e l e c t e d . P a i r s were chosen on the b a s i s of p r o x i m i t y and s i m i -
l a r i t y i n s i z e and exposure to the sun. For each p a i r one member
was randomly assigned as t e s t t r e e , the other as c o n t r o l . D i s -
tance between each c o n t r o l t r e e and the nearest t e s t t r e e averaged
6.1 ± 1.1 m. Two tent c a t e r p i l l a r egg masses, c o l l e c t e d the
previous w i n t e r from a l d e r trees, were attached to each of the t e s t
t r e e s . A sample of the egg masses (n = 55) contained 214 eggs
per egg mass of which 3.2% were p a r a s i t i z e d and 6.6% d i d not hatch
due to unknown causes. The egg masses had a l l hatched by A p r i l
26. M i g r a t i o n of l a r v a e from the t e s t t r e e s and onto c o n t r o l
t r e e s was prevented by t a n g l e f o o t bands over aluminum f o i l on the
trunks of a l l t r e e s . N e t t i n g bags were placed over assay branches
on both t e s t and c o n t r o l t r e e s to provide equivalent l e a v e s ,
protected from the tent c a t e r p i l l a r s , f o r l a t e r bioassay and
chemical a n a l y s i s . This ensured t h a t any d i f f e r e n c e s found bet-
ween leaves from t e s t and c o n t r o l t r e e s were due to changes i n
l e a f q u a l i t y r a t h e r than w i t h i n - p l a n t heterogeneity combined w i t h
p r e f e r e n t i a l consumption of high q u a l i t y leaves by the i n s e c t s .
I t a l s o ensured that any p l a n t responses observed were systemic
r a t h e r than l o c a l i z e d wound responses. In mid-May the c o l o n i e s

4. RHOADES Evidence for Pheromonal Sensitivity 57
were observed t o be growing more slowly than expected, c o n s t r u c t i n g

very small t e n t s and causing l i t t l e l e a f damage t o the t r e e s , so
on May 20 an average of 3.4 t 0.4 (S.E.) a d d i t i o n a l c o l o n i e s
c o l l e c t e d from a l d e r were placed on each of the t e s t t r e e s .
Commencing May 23, t h i r d and f o u r t h i n s t a r tent c a t e r p i l l a r s
c o l l e c t e d from a l d e r were r a i s e d i n the l a b o r a t o r y (one r e p l i c a t e
per t r e e , 25 l a r v a e per r e p l i c a t e ) on the assay branches detached
from the t e s t and c o n t r o l t r e e s . The assay l a r v a e were weighed,
s u r v i v o r s counted, and dead l a r v a e removed p e r i o d i c a l l y (Figure
1A). Assay branches were replaced each time that the l a r v a e were
weighed and censused. Larvae feeding on leaves from the t e s t
t r e e s grew more s l o w l y and had lower s u r v i v o r s h i p than those fed
leaves from the c o n t r o l t r e e s (Figure LA). Biomass per r e p l i c a t e
of l a r v a e , u n t i l the i n i t i a t i o n of pupation, was s i g n i f i c a n t l y
higher f o r l a r v a e fed c o n t r o l leaves than f o r those fed leaves
from attacked t r e e s on
t a i l e d paired t t e s t ) . Survivorshi
s i g n i f i c a n t l y higher f o r the c o n t r o l s . For pupae s u r v i v o r s h i p
averaged 16.0 ± 6.3% (S.E.) f o r c o n t r o l s versus 6.9 ± 3.5% f o r
those produced from l a r v a e feeding on leaves from attacked p l a n t s
Cp < 0.05, o n e - t a i l e d p a i r e d t t e s t ) . Death of l a r v a e i n both
l a b o r a t o r y groups and the i n s e c t s i n the f i e l d was a s s o c i a t e d w i t h
a c o n d i t i o n i n which, s h o r t l y before death, the l a r v a e produced
l i q u i d feces i n s t e a d of the normal f e c a l p e l l e t . Pupal weights
were not s i g n i f i c a n t l y d i f f e r e n t between t e s t and c o n t r o l groups
f o r e i t h e r male o r female pupae, but a d u l t s from pooled t e s t and
pooled c o n t r o l groups produced only one egg mass i n the former
case and e i g h t egg masses i n the l a t t e r (x^ = 5.59, p < 0.025).
By June 11 a l l o f the f i e l d load i n s e c t s had d i e d o r l e f t the
t r e e s . Leaf damage t o the t e s t t r e e s when measured on June 3 was
r e l a t i v e l y l i g h t . Leaves e x h i b i t i n g n o t i c e a b l e damage averaged
27.6 ± 2.1% (S.E.) f o r the c o n t r o l t r e e s and 49.0 ± 4.7% f o r t e s t
t r e e s (p < 0.01, o n e - t a i l e d p a i r e d t t e s t ) . Estimated l e a f area
l o s s averaged 2.5 ± 0.2% f o r c o n t r o l s and 11.3 ± 2.1% f o r t e s t
t r e e s (p < 0.005, o n e - t a i l e d p a i r e d t t e s t ) . Damage t o c o n t r o l
t r e e s was due t o u n i d e n t i f i e d i n s e c t s other than tent c a t e r p i l l a r s .
Samples o f f r e s h leaves from the assay branches of a l l t r e e s
were e x t r a c t e d w i t h 85% aqueous methanol on June 3. These
e x t r a c t s were assayed f o r t o t a l p h e n o l i c content by the F o l i n -
Denis Method (6) and proanthocyanidins ( 7 ) . On a dry weight b a s i s ,
E
the proanthocyanidin s p e c i f i c e x t i n c t i o n c o e f f i c i e n t s I r m ( 5 0 n m ) ]
C
5
i n d i c a t e d 24% higher l e v e l s , on average, i n leaves from attacked

versus c o n t r o l t r e e s (p < 0.05, o n e - t a i l e d p a i r e d t t e s t ) . Folin-
Denis s p e c i f i c e x t i n c t i o n c o e f f i c i e n t s [E}j? (725 nm)] averaged 12%
m
higher, on a dry weight b a s i s , f o r leaves from attacked t r e e s , but

t h i s d i f f e r e n c e was not s i g n i f i c a n t (p < 0.1, o n e - t a i l e d p a i r e d t
t e s t ) . The chemical nature of a l d e r proanthocyanidins has not
been i n v e s t i g a t e d , but proanthocyanidin values have been
commonly used t o i n d i c a t e l e v e l s of condensed tannins i n p l a n t s
(8, 9, 10).

Figure 1. A: Average biomass and survivorship of groups of western tent caterpil-

lar larvae fed leaves in the laboratory from red alder trees under attack by tent
caterpillars in the field ( ) compared to those fed leaves from unattacked con-
trol alders (—). B: Average biomass and survivorship of groups of tent caterpillar
larvae fed leaves in the laboratory from Sitka willow trees under attack by tent cater-
pillars in the field (—) compared to those fed leaves from unattacked control willows
( ). The test trees were reloaded with additional colonies of tent caterpillars
on the indicated date.

Since t e s t and c o n t r o l a l d e r s had been randomly assigned, no

s i g n i f i c a n t pre-treatment d i f f e r e n c e s i n f o l i a g e q u a l i t y were t o
be expected. Thus a t t a c k of a l d e r by tent c a t e r p i l l a r s produced a
change i n f o l i a g e q u a l i t y which caused decreased growth, s u r v i v a l ,
and egg mass production i n tent c a t e r p i l l a r s r a i s e d i n the l a b o r a -
t o r y on detached branches. Furthermore, t h i s change was induced
by r e l a t i v e l y l i g h t g r a z i n g l e v e l s and occurred w i t h i n a time
period o f 27 days, o r s h o r t e r , o f the i n i t i a t i o n o f a t t a c k . The
change i n f o l i a g e q u a l i t y was systemic s i n c e i t occurred i n leaves
on branches protected from a t t a c k , and i t was a s s o c i a t e d w i t h an
increase i n proanthocyanidin content o f the f o l i a g e . Even though
s i g n i f i c a n t d i f f e r e n c e s i n biomass accumulation and s u r v i v o r s h i p
were observed between t e s t and c o n t r o l assay i n s e c t s , the s u r v i -
v o r s h i p s o f both groups were low, and t h i s was r e f l e c t e d i n the
poor performance o f the a t t a c k i n g i n s e c t s i n the f i e l d . This may
have been due to low f o l i a g
repeated a t t a c k from ten
outbreak (3).
S i t k a Willow Attacked by Tent C a t e r p i l l a r s
During the same s p r i n g (1979) that the above experiment was

performed, a s i m i l a r experiment i n which S i t k a w i l l o w was sub-
j e c t e d t o a t t a c k by tent c a t e r p i l l a r s was c a r r i e d out. Seven
p a i r s o f f o u r - y e a r - o l d w i l l o w t r e e s of average height 4.5 ± 0.2
3
(S.E.) m and volume of 6.1 ± 0.7 m were s e l e c t e d a t the Kent s i t e .
Test and c o n t r o l t r e e s were randomly assigned f o r each p a i r .
Distance between each c o n t r o l and the nearest t e s t t r e e averaged
3,5 i 0,4 m, An average o f 7.4 i 0.3 egg masses c o l l e c t e d from
a l d e r and from the same bulk c o l l e c t i o n used i n the previous
experiment were placed on each of the t e s t t r e e s , a l l hatching by
A p r i l 24. M i g r a t i o n o f l a r v a e from the t e s t t r e e s , onto c o n t r o l
t r e e s and onto assay branches, was prevented by t a n g l e f o o t bands
over aluminum f o i l . A l l t e s t and c o n t r o l t r e e s were t r e a t e d
s i m i l a r l y . On May 11 the l a b o r a t o r y feeding experiment was
i n i t i a t e d . Second and t h i r d i n s t a r tent c a t e r p i l l a r l a r v a e ,
r a i s e d on w i l l o w i n the f i e l d from egg masses c o l l e c t e d from
a l d e r , were fed detached leaves i n the l a b o r a t o r y (25 l a r v a e per
r e p l i c a t e , one r e p l i c a t e per t r e e ) . Feed l e a v e s , detached from
the assay branches, were replaced every second day. I n contrast
to the experiment w i t h a l d e r , no d i f f e r e n c e i n growth r a t e s or
s u r v i v o r s h i p between i n s e c t s r a i s e d on leaves from attacked o r
c o n t r o l t r e e s was observed (Figure I B ) .
Due t o the very poor performance of the i n s e c t s i n the f i e l d ,
as had been noted i n the a l d e r experiment above, the t e s t t r e e s
were reloaded w i t h an average of 8.0 ± 1.0 (S.E.) a d d i t i o n a l
c o l o n i e s of tent c a t e r p i l l a r s , obtained from a l d e r , s i n c e none
were a v a i l a b l e from w i l l o w , on May 22, These a d d i t i o n a l c o l o n i e s
were mainly i n the 4th and e a r l y 5th i n s t a r , whereas the o r i g i n a l
l a r v a e hatched on the t r e e s were s t i l l mainly i n the t h i r d i n s t a r .
Thus, a t the time o f r e l o a d , the t r e e s experienced a l a r g e

increase i n biomass of a t t a c k i n g i n s e c t s . Coincident w i t h the

r e l o a d o p e r a t i o n , a l a r g e drop i n average biomass was observed f o r
both t e s t and c o n t r o l groups of assay i n s e c t s . This was due mainly
to an increased r a t e of m o r t a l i t y from May 25 onward (Figure I B ) .
I t should be noted that biomass and s u r v i v o r s h i p data (Figure IB)
represent performance of assay i n s e c t s fed leaves obtained two
days p r e v i o u s l y so the f i r s t post-load s u r v i v o r s h i p i s that
obtained on May 25.
Death of the assay i n s e c t s was a s s o c i a t e d w i t h the production
of l i q u i d feces. Very l i t t l e feeding by the r e l o a d i n s e c t s was
observed. They spent most of t h e i r time r e s t i n g on the t e n t s or
wandering i n apparent attempts to leave the t r e e s . By the end of
May a l l i n s e c t s had e i t h e r died or dropped from the t r e e s . This
behavior s t r o n g l y c o n t r a s t s w i t h that observed when tent c a t e r -
p i l l a r s are t r a n s f e r r e d to w i l l o w p r e v i o u s l y unattacked by tent
c a t e r p i l l a r s during tha
host range. Under thes
s u r v i v e , grow and remain on the t r e e s f o r extended periods ( f o r
example see f o l l o w i n g experiment). So, i n s p i t e of the l a r g e
numbers of egg masses and mature c o l o n i e s placed on the t e s t t r e e s ,
l e a f damage was low when measured on June 1. An average of
37.7 t 5.4% (S.E.) of leaves on the t e s t t r e e s were attacked.
Estimated l e a f area l o s t from the t e s t t r e e s averaged 6.2 ± 2.4%.
The corresponding f i g u r e s f o r the c o n t r o l s were 11.5 t 1.4%
(p < 0.0025, o n e - t a i l e d p a i r e d t t e s t ) leaves attacked and
1.7 ± 0.5% (p < 0.0025) l e a f area l o s t , due to u n i d e n t i f i e d
i n s e c t s other than t e n t c a t e r p i l l a r s .
Thus, i n c o n t r a s t to the r e s u l t s obtained the same season
w i t h a l d e r , no d i f f e r e n c e s were found i n tent c a t e r p i l l a r growth
or s u r v i v a l when fed leaves from attacked versus c o n t r o l w i l l o w s .
I t i s p o s s i b l e that the r a p i d drop i n biomass of the assay i n s e c t s
feeding on leaves from attacked and c o n t r o l t r e e s c o i n c i d e d w i t h
r e l o a d of the t e s t t r e e s by chance, p o s s i b l y due to the r a p i d
spread of a pathogen through the l a b o r a t o r y i n s e c t p o p u l a t i o n (11).
On the other hand, the p o s s i b i l i t y that r e l o a d of the t e s t t r e e s
caused the biomass drop i n both t e s t and c o n t r o l assay i n s e c t s
cannot be discounted. Both attacked and nearby unattacked c o n t r o l
w i l l o w s may have r a p i d l y decreased the food q u a l i t y of t h e i r
leaves i n response to a sudden increase i n biomass of i n s e c t s on
the attacked t r e e s . I f so, t h i s suggests that unattacked w i l l o w s
are s e n s i t i v e to s i g n a l s from the i n s e c t s or nearby attacked
w i l l o w s . This p o s s i b i l i t y was i n v e s t i g a t e d i n the f o l l o w i n g
experiment.
I n the s p r i n g of 1981, 20 s i x - y e a r - o l d w i l l o w s of average
height 5.6 ± 0.1 (S.E.)m and volume 7.5 t 1.0 m were s e l e c t e d
3
from the same stand used i n the p r e l i m i n a r y experiment i n 1979.

None of these t r e e s had been used i n the previous experiment.
Stems and assay branches were banded w i t h t a n g l e f o o t as before,
and 10 t e s t and 10 nearby c o n t r o l t r e e s were randomly assigned
i n p a i r w i s e f a s h i o n . Distance between each c o n t r o l and the

nearest t e s t t r e e averaged 3.3 i 0,6 (S,E.) m. A f a r c o n t r o l

group of 20 s i x - y e a r - o l d w i l l o w s 1,6 km from the t e s t s i t e was
a l s o s e l e c t e d and banded w i t h t a n g l e f o o t . These t r e e s averaged
3
4.8 ± 0.2 m i n height and 8.9 ± 1.7 m i n volume.
In the p r e l i m i n a r y experiment of 1979, egg masses had been
placed on t h e t e s t w i l l o w s . Approximately one month a f t e r the
eggs had hatched the numbers of l a r v a e a t t a c k i n g the t r e e s had
been r e i n f o r c e d w i t h a d d i t i o n a l c o l o n i e s ( r e l o a d ) . I n 1981,
i n s u f f i c i e n t egg masses were a v a i l a b l e t o repeat t h i s procedure,
but as the season progressed we were able t o l o c a t e tent c a t e r -
p i l l a r c o l o n i e s i n the f i e l d . These c o l o n i e s , c o n t a i n i n g l a r v a e
from t h i r d t o e a r l y f i f t h i n s t a r , were c o l l e c t e d from w i l d rose
(Rosa nutkana), b i t t e r cherry (Prunus emarginata), domestic apple
(Pyrus malus), hawthorn (Crataegus monogyna), and Scouler w i l l o w
( S a l i x s c o u l e r i a n a ) . On May 28 the c o l o n i e s were attached t o the
t e s t t r e e s , unattacked
3.9 ± 0.4 (S.E.) c o l o n i e pe correspond approxi
mately 600 l a r v a e per t r e e . The l a r v a e were censused on the t r e e s
p e r i o d i c a l l y u n t i l the end of June (Figure 2 ) . Leaf q u a l i t y
before and a f t e r adding i n s e c t s (load) was bioassayed i n the
l a b o r a t o r y using tent c a t e r p i l l a r l a r v a e r a i s e d from the egg stage
on w i l l o w . I n c o n t r a s t t o the bioassay used i n previous e x p e r i -
ments, i n which l a r v a e had been grown continuously on p e r i o d i c a l l y
replaced leaves over the e n t i r e course o f the experiment, a d i f -
f e r e n t assay method was used. I n t h i s procedure batches o f l a r v a e
(4-14 l a r v a e per batch, depending on a v a i l a b i l i t y a t the time of
assay, one r e p l i c a t e per t r e e ) were r a i s e d on leaves from assay
branches o f t e s t and c o n t r o l t r e e s f o r an average time p e r i o d o f
22.1 hours (range 21.1 t o 23.0) a f t e r which time the l a r v a e were
weighed and discarded. F r e s h l a r v a e were then used i n subsequent
assays. An advantage o f t h i s method i s that i t g r e a t l y f a c i l i -
t a t e s the d e t e c t i o n of r a p i d changes i n f o l i a g e q u a l i t y . On the
other hand, i t produces no i n f o r m a t i o n concerning the cumulative
e f f e c t s o f f o l i a g e q u a l i t y , and changes thereof, on s u r v i v a l o r
e
8 g production. Since the amounts o f f o l i a g e consumed o r f r a s s
produced by the i n s e c t s were not measured, i t i s not known whether
d i f f e r e n c e s i n growth r a t e s were due to d i f f e r e n c e s i n amounts
eaten o r t o d i f f e r e n c e s i n amounts a s s i m i l a t e d o r both. Whatever
the mechanism may be, d i f f e r e n c e s i n l a r v a l growth imply d i f f e r -
ences i n l e a f q u a l i t y .
R e l a t i v e growth r a t e s of l a r v a e fed leaves from t e s t , near
c o n t r o l , and f a r c o n t r o l groups were c a l c u l a t e d as % increase i n
f r e s h weight mass per u n i t time, over and above t h a t of starved i n -
s e c t s , normalized w i t h respect to the f a r c o n t r o l group (Figure 2).
On May 11, p r i o r to l o a d , there were no s i g n i f i c a n t d i f f e r e n c e s i n
growth r a t e s between the t e s t and c o n t r o l groups, nor were there
any s i g n i f i c a n t d i f f e r e n c e s i n growth f o r the f i r s t three assays
subsequent to load (0.1 < p < 0.25, ANOVA, f o r the most s i g n i f i c a n t
case). However, on June 9, 11.5 days a f t e r l o a d , l a r v a e f e d leaves
from the t e s t t r e e s grew s i g n i f i c a n t l y more s l o w l y than those fed

DAYS AFTER LOAD

0 5 10 15 20 25
1 1 i 1 1 r -
LOAD
MAY JUNE
DATE
Figure 2. Top: normalized average relative growth rates of groups of western tent
caterpillar larvae raised in the laboratory of leaves from test, nearby control, and
far control Sitka willow trees. The test trees were loaded with tent caterpillar colon-
ies on the indicated date. Bottom: density of tent caterpillar larvae attacking the
trees on various dates.

leaves from the near o r f a r c o n t r o l s (Figure 2, p < 0.05, Newman-

Keuls m u l t i p l e range t e s t ) . I n the subsequent assay on June 12th,
14.5 days a f t e r l o a d , l a r v a e fed leaves from both t e s t and nearby
c o n t r o l s grew more s l o w l y than those fed leaves from the f a r con-
t r o l s (Figure 2, p < 0.01, Newman-Keuls m u l t i p l e range t e s t ) .
Leaves damaged averaged 44.8 +1.2% (S.E.) f o r the t e s t t r e e s ,
6.4 ± 0.6% f o r the near c o n t r o l s , and 10.4 ± 0.8% f o r the f a r
c o n t r o l s , when measured a t the beginning o f J u l y . A l l these
d i f f e r e n c e s were s i g n i f i c a n t (p < 0.01 f o r the l e a s t s i g n i f i c a n t
case, Newman-Keuls m u l t i p l e range t e s t ) . Estimated l e a f area
l o s s e s were 15.8 t 1.0% f o r the t e s t t r e e s , 1.1 ± 0.1% f o r the
near c o n t r o l s , and 1.6 * 0.2% f o r the f a r c o n t r o l s Leaf area
l o s t by the t e s t t r e e s was s i g n i f i c a n t l y g r e a t e r than that l o s t
by each of the c o n t r o l groups (p < 0.001), but l e a f areas l o s t by
each of the two c o n t r o l groups were not s i g n i f i c a n t l y d i f f e r e n t
(p < 0.5). Damage to leave
other than tent c a t e r p i l l a r s
were estimated a t 2.6 ± 0.4 (S.E.)% f o r the t e s t t r e e s versus
3.3 ± 0.5% f o r the near c o n t r o l s and 2.4 t 0.3% f o r the f a r
c o n t r o l s . The d i f f e r e n c e s i n sampling pressure were not s i g n i -
f i c a n t (p > 0.5, ANOVA).
These r e s u l t s supported the hypothesis that a l t e r e d l e a f food
q u a l i t y can be induced i n both w i l l o w s attacked by t e n t c a t e r -
p i l l a r s and nearby unattacked w i l l o w s . The chemical b a s i s of the
changes i n l e a f food q u a l i t y are unknown. Leaf F o l i n - D e n i s t o t a l
phenolic and proanthocyanidin s p e c i f i c e x t i n c t i o n c o e f f i c i e n t s
showed l i t t l e d i f f e r e n c e between t e s t and c o n t r o l t r e e s throughout
the experiment. Willows commonly form c l o n a l stands and, con-
c e i v a b l y , subterranean r o o t connections could l i n k each nearby
c o n t r o l t r e e t o a t e s t t r e e . I f so, a l t e r e d l e a f q u a l i t y of the
nearby c o n t r o l s could be due t o a long-range systemic response.
However, S a l i x s i t c h e n s i s i s not known to form c l o n a l stands (12),
and excavation o f 20 j>. s i t c h e n s i s t r e e s (1-3 m i n h e i g h t ) a t the
same s i t e and of the same age as the study t r e e s gave no evidence
of c l o n i n g o r r o o t g r a f t i n g . Therefore, a i r b o r n e pheromonal sub-
stances emitted by the tent c a t e r p i l l a r s o r the a t t a c k e d t r e e s
may be involved.-
S i t k a Willows Attacked by F a l l Webworm
F a l l webworms, l i k e t e n t c a t e r p i l l a r s , are polyphagous,

c o l o n i a l Lepidoptera t h a t a t t a c k a wide v a r i e t y of broad-leafed
t r e e s and shrubs. U n l i k e t e n t c a t e r p i l l a r s , o f which the l a r v a l
stage occurs i n the s p r i n g , webworm l a r v a e occur from mid-summer
to e a r l y f a l l i n western Washington. I n J u l y 1980 an experiment
to i n v e s t i g a t e p o s s i b l e changes i n w i l l o w l e a f q u a l i t y induced by
f a l l webworm a t t a c k was i n i t i a t e d The experiment was designed
to take account o f p o s s i b l e changes i n l e a f q u a l i t y o f unattacked
w i l l o w s near a t t a c k e d ones. Two groups of w i l l o w s , 10 w i l l o w s per
group and approximately 60 m between groups, were s e l e c t e d a t the
Kent s i t e , a t e s t group A to be loaded w i t h webworms and a c o n t r o l

group B (Figure 3). These t r e e s were members of f i v e - y e a r - o l d

even-aged stands, and t h e i r average heights and volumes were
3
5.4 ± 0.2 m (S.E.), 7.5 + 1.9 m f o r group A and 4.7 + 0.2 m,
4.1 t 0.5 m f o r group B. Two a d d i t i o n a l groups of w i l l o w s (C and
3
D), 10 w i l l o w s per group and approximately 69 m between groups,

were s e l e c t e d a t a s i t e near Seattle-Tacoma I n t e r n a t i o n a l A i r p o r t ,
approximately 8 km from the Kent s i t e . Groups C and D were
members of a f i v e - and f o u r - y e a r - o l d stand and measured 4.6 t 0.3
3 3
m, 12.0 ± 2.1 m , and 4.3 ± 0.3 m, 11.5 ± 1.8 m r e s p e c t i v e l y .
These t r e e s served as f a r c o n t r o l groups.
Trunks and assay branches of a l l trees were banded w i t h
t a n g l e f o o t as p r e v i o u s l y described. On August 13 an average of
1.9 t 0.2 (S.E.) c o l o n i e s o f webworms, obtained from a l d e r , were
loaded onto each of the t e s t t r e e s (Group A, F i g u r e 3), Webworms
t r a n s f e r r e d from a l d e r to w i l l o w r e a d i l y feed, and a sample o f
the l a r v a e were r a i s e d
w i l l o w . D i f f i c u l t i e s wer
the t r e e s because they remain concealed w i t h i n t h e i r tent when
not f e e d i n g , so on September 1 a l l c o l o n i e s were removed and
the l a r v a e counted. At t h i s time leaves attacked (%) f o r the t e s t
group A and groups B, C, and D averaged: A, 56.6 ± 2 . 7 (S.E.);
B, 8.6 + 0.7; C, 10.4 ± 1.8; D, 14.4 ± 1.6. Estimated l e a f area
l o s s (%) averaged: A, 20.7 ± 2.8; B, 1.1 ± 0.4; C, 1.6 ± 0.3;
D, 2.6 t 0.4. For both l e a f damage measures Newman-Keuls m u l t i p l e
range t e s t s showed t h a t the t e s t group had s u f f e r e d g r e a t e r l e a f
area damage than the other three groups (p < .001) among which
damage was not s i g n i f i c a n t l y d i f f e r e n t .
On September 9 f r e s h c o l o n i e s obtained from a l d e r were r e -
loaded onto the t e s t t r e e s a t approximately the same d e n s i t y as
i n the o r i g i n a l l o a d (Figure 3). A census was obtained on Septem-
ber 15, and the remaining l a r v a e were removed and counted on Sep-
tember 21. At t h i s time leaves attacked (%) averaged: A ( t e s t
group), 61.4 ± 3.1; B, 8.5 ± 1.1; C, 9.4 ± 1.7; D, 14.7 ± 2.1.
Estimated l e a f area l o s s (%) averaged: A, 30.6 ± 2.7; B,
1.3 ± 0.2; C, 1.7 ± 0.4; D, 2.9 ± 0.3. Again, both measures
showed s i g n i f i c a n t l y g r e a t e r l e a f damage (p < .001) f o r the t e s t
group A than the other three groups which were not s i g n i f i c a n t l y
d i f f e r e n t from each other. Leaves removed i n sampling (%)
averaged : A, 8.9 ± 1.0; B, 11.2 ± 1.3; C, 7.9 ± 1.5; D,
9.7 t 1.4. Sampling pressure was not s i g n i f i c a n t l y d i f f e r e n t
among the four groups (p > 0.5, Newman-Keuls m u l t i p l e range t e s t ) .
Damage to leaves of c o n t r o l t r e e s was due to u n i d e n t i f i e d i n s e c t s
other than webworms.
Leaf q u a l i t y of the four groups of t r e e s before and a f t e r
load was compared by measuring the r e l a t i v e growth r a t e s of web-
worm l a r v a e (14-20 l a r v a e per r e p l i c a t e , one r e p l i c a t e per t r e e )
fed leaves from the assay branches over a one to three day p e r i o d
i n the l a b o r a t o r y (Figure 3 ) . These assay l a r v a e were c o l l e c t e d
from a l d e r and f e d w i l l o w f o r two days p r i o r to the feeding exper-
iments. Since mass changes o f starved i n s e c t s were not measured,

DAYS AFTER INITIAL LOAD

O 10 20 30 40
T 1 1 —r r—
SEPTE
DATE
Figure 3. Top: normalized average relative growth rate of fall webworm larvae
raised on leaves from test (A), nearby control (B), and far control (C and D) groups
of Sitka willow trees. The test trees were loaded with webworms and unloaded on
the indicated dates. Connected points represent growth rates of larvae fed detached
leaves in the laboratory. Unconnected points at the right represent growth rates in
the field of larvae on the various groups of willow trees. Bottom: density of web-
worm larvae attacking the test trees on various dates.

the r e s u l t s are expressed as averages of the % increases i n mass

per u n i t time over the i n i t i a l v a l u e s , normalized w i t h respect to
the B group (Figure 3). The webworms grew f a s t e r on the d i s t a n t
c o n t r o l s at the a i r p o r t s i t e (groups C and D) than on leaves from
the t e s t and nearby c o n t r o l s at the Kent s i t e (groups A and B) i n
a l l f o u r l a b o r a t o r y assays. There were no s i g n i f i c a n t d i f f e r e n c e s
i n growth on leaves from groups C and D throughout the experiment.
Growth r a t e s of the l a b o r a t o r y assay i n s e c t s on leaves from the
t e s t group A and nearby c o n t r o l group B were not s i g n i f i c a n t l y
d i f f e r e n t from each other before load and during the f i r s t two
assays subsequent to load. However, f o l l o w i n g the r e l o a d opera-
t i o n , 35 days a f t e r the o r i g i n a l l o a d , growth r a t e s of the l a b o r a -
t o r y i n s e c t s were lower on leaves from the t e s t t r e e s (A) than on
those of the f a r and nearby c o n t r o l s (Figure 3). This was con-
firmed by measuring the growth r a t e s i n the f i e l d of groups of
l a r v a e (14 l a r v a e per group
n e t t i n g bags on the assa
p e r i o d on September 17-20 (Figure 3 ) . R e l a t i v e growth r a t e s i n
the f i e l d feeding experiment showed a p a t t e r n very s i m i l a r to that
of the f i n a l l a b o r a t o r y experiment (Figure 3). Absolute growth
r a t e s i n the l a b o r a t o r y were much higher than i n the f i e l d assays,
however. They ranged between 5.5 times higher (group A) and 4.1
times higher (group C).
P r o b a b i l i t i e s that the observed d i f f e r e n c e s i n l a r v a l growth
between groups C and D, D and B, and B and A i n the f i n a l l a b o r a -
t o r y feed are merely by chance are > 0.5, < 0.05, and < 0.1
r e s p e c t i v e l y (Newman-Keuls m u l t i p l e range t e s t ) . Corresponding
p r o b a b i l i t i e s f o r the f i e l d feeding experiment are > 0.5, < 0.1,
and < 0.1, r e s p e c t i v e l y . Since these two feeding assays c o n s t i -
t u t e independent t e s t s , we can conclude t h a t during the p e r i o d of
f i n a l l a b o r a t o r y and f i e l d growth assays the t e s t t r e e s e x h i b i t e d
an a l t e r e d l e a f q u a l i t y such that the assay l a r v a e grew more
s l o w l y on these leaves than on leaves from the nearby c o n t r o l
t r e e s (p = 0.1 x 0.1 = 0.01). The p l a n t response occurred w i t h i n
a p e r i o d of 35 days from the time of i n i t i a l l o a d , and i t was
systemic.
Higher growth r a t e s of the l a r v a e fed leaves from w i l l o w s at
the a i r p o r t s i t e than those fed leaves from w i l l o w s at the Kent
s i t e , throughout the experiment, show that s i g n i f i c a n t and f a i r l y
constant d i f f e r e n c e s i n l e a f q u a l i t y can e x i s t between t r e e s a t
d i f f e r e n t s i t e s . Normalized r e l a t i v e growth r a t e s of webworms fed
detached leaves i n the l a b o r a t o r y were s i m i l a r to those obtained
by growing webworms on the t r e e s .
The chemical b a s i s f o r the observed d i f f e r e n c e s i n l e a f food
q u a l i t y i s unknown. L i t t l e d i f f e r e n c e s were found i n F o l i n - D e n i s
t o t a l phenolic or proanthocyanidin e x t i n c t i o n c o e f f i c i e n t s of 85%
aqueous methanol l e a f e x t r a c t s from the v a r i o u s p l a n t groups
throughout the experiment. There were no i n d i c a t i o n s of changes
i n l e a f q u a l i t y of c o n t r o l w i l l o w s 60 m d i s t a n t from w i l l o w s
attacked by f a l l webworm.

Summary and D i s c u s s i o n
These r e s u l t s lend support t o previous suggestions (1, 2, 13,

14) that p l a n t s may commonly decrease the q u a l i t y of t h e i r t i s s u e s
as food i n response to herbivore a t t a c k . Red a l d e r t r e e s sub-
j e c t e d t o a t t a c k by western tent c a t e r p i l l a r s e x h i b i t a change i n
f o l i a g e q u a l i t y such that tent c a t e r p i l l a r s fed unattacked leaves
from attacked t r e e s grew more s l o w l y , died a t a f a s t e r r a t e , and
produced fewer egg masses than those fed leaves from unattacked
t r e e s (Figure 1A). This change i n f o l i a g e q u a l i t y was a s s o c i a t e d
w i t h an i n c r e a s e i n proanthocyanidin e x t i n c t i o n c o e f f i c i e n t s o f
l e a f e x t r a c t s . S i m i l a r l y , f a l l webworms fed unattacked leaves
from S i t k a w i l l o w s subjected to a t t a c k by f a l l webworms grew more
s l o w l y than those fed leaves from unattacked w i l l o w s (Figure 3).
On the other hand, no d i f f e r e n c e s were found i n growth or
mortality of tent c a t e r p i l l a r
compared t o those fed leave
p i l l a r s (Figure I B ) . Reload of the attacked t r e e s w i t h a d d i t i o n a l
tent c a t e r p i l l a r s c o i n c i d e d w i t h a r a p i d decrease i n biomass and
an increased m o r t a l i t y o f i n s e c t s fed leaves from both attacked
and unattacked c o n t r o l t r e e s . This could have been by chance, but
i t suggested the p o s s i b i l i t y that both attacked and c o n t r o l t r e e s
e x h i b i t e d a r a p i d decrease i n food q u a l i t y i n response to the
a d d i t i o n o f more i n s e c t s t o the attacked t r e e s . I f so, t h i s
suggested that unattacked w i l l o w s were s e n s i t i v e t o s i g n a l s from
nearby attacked w i l l o w s o r the a t t a c k i n g i n s e c t s . An experiment
to t e s t t h i s hypothesis produced p o s i t i v e r e s u l t s (Figure 2).
Tent c a t e r p i l l a r s fed leaves from w i l l o w s attacked by tent c a t e r -
p i l l a r s grew more s l o w l y than l a r v a e fed leaves from nearby and
f a r c o n t r o l s 11.5 days a f t e r the i n i t i a t i o n o f a t t a c k . Three
days l a t e r , l a r v a e fed leaves from both attacked w i l l o w s and
nearby c o n t r o l s grew more s l o w l y than l a r v a e fed leaves from the
f a r c o n t r o l s . No evidence was found f o r root connections between
w i l l o w s o f the same age as the study t r e e s a t the same s i t e .
This suggests that the r e s u l t s may be due t o a i r b o r n e pheromonal
substances!
The burden o f proof f o r such an unprecedented e f f e c t should
be h i g h , and the foregoing experiments w i t h w i l l o w s and tent
c a t e r p i l l a r s cannot be considered t o c o n s t i t u t e such proof.
However, a t the very l e a s t , they show that the r e s u l t s of e x p e r i -
ments designed t o t e s t f o r changes i n l e a f q u a l i t y of a t t a c k e d
p l a n t s should be i n t e r p r e t e d w i t h c a u t i o n , p a r t i c u l a r l y i f c o n t r o l
p l a n t s are near attacked ones.
Acknowledgement s
I thank G. H. Orians f o r d i s c u s s i o n and A. F. G. Dixon, G.

B a t z l i , and unknown reviewers f o r commenting on the manuscript.
S p e c i a l thanks are due to L. Erckmann who p a r t i c i p a t e d i n most of
the work. N. E. Beckage, C. J . Baron, A. B. Adams, R. Hagen, and
J . C. Bergdahl have a l l c o n t r i b u t e d t o some aspect. I thank Mr.

R. H. DeBoer, Stoneway, Inc., Mr. W. D. Robertson, Mr. R. A. Marr

and the Seattle-Tacoma I n t e r n a t i o n a l A i r p o r t A u t h o r i t i e s f o r use
of t h e i r f a c i l i t i e s , and Dr. J . P. Donahue f o r i d e n t i f y i n g the
i n s e c t s . This research was supported by N a t i o n a l Science Founda-
t i o n grants DEB 77-03238 and DEB 80-05528 to G. H. Orians and D.
F. Rhoades.
Literature Cited
1. Benz, G. Eucarpia/IOBC Working Group Breeding f o r Resistance

to I n s e c t s and M i t e s . B u l l . SROP, 1977/78, pp 155-159.
2. Rhoades, D. F. "Herbivores: T h e i r I n t e r a c t i o n w i t h Secondary
P l a n t M e t a b o l i t e s " ; Rosenthal, G. A.; Janzen, D. H.; Eds;
Academic P r e s s : New York, 1979, Chapter 1.
3. Rhoades, D. F. "Impact of V a r i a b l e Host Q u a l i t y on H e r b i -
vorous I n s e c t s " ; Denno
in press.
4. Ryan, C. A. "Herbivores: T h e i r I n t e r a c t i o n w i t h Secondary
P l a n t M e t a b o l i t e s " ; Rosenthal, G. A.; Janzen, D. H.; Eds.;
Academic P r e s s : New York, 1979, Chapter 17.
5. Haukioja, E. Oikos, 1980, 35, 202-213.
6. Swain, T. S.; G o l d s t e i n , J. L. "Methods in Polyphenol
Chemistry"; Pridham, J. B., Ed.; Macmillan P r e s s : New York,
1964, pp 131-146.
7. Swain, T.; Hillis, W. E. J . Sci. Food Agric., 1959, 10,
63-68.
8. Bate-Smith, E. C. Phytochemistry, 1975, 14, 1107-1113.
9. Bate-Smith, E. C. Phytochemistry, 1977, 16, 1421-1426.
10. McKey, D.; Waterman, P. G.; Mbi, C. N.; G a r t l a n , J. S.;
Struhsaker, T. T. Science, 1978, 202, 61-64.
11. R i d d i f o r d , L. M. Science, 1967, 22, 1451-1452.
12. Hitchcock, C. L.; Cronquist, A. " F l o r a of the Pacific North-
west"; U n i v e r s i t y of Washington P r e s s : S e a t t l e , 1973, p 66.
13. Benz, G. Z. ang. Ent., 1974, 76, 196-228.
14. Haukioja, E.; Hakala, T. Rep. Kevo S u b a r c t i c Res. S t a t . ,
1975, 12, 1-9.

5
Function and Chemistry of Plant Trichomes
and Glands in Insect Resistance
Protective Chemicals in Plant Epidermal
Glands and Appendages
ROBERT D. STIPANOVIC
U.S. Department of Agriculture, Agricultural Research Service, National Cotton
Pathology Research Laboratory, P.O. Drawer JF, College Station, T X 77841
Plants hav
of defense agains
defensive morphological features are trichomes and
glands. Trichomes may be h a i r - l i k e or glandular.
Plant hairs act as physical barriers keeping smaller
insects away from the leaf surface. Glandular trichomes
and plant glands may exude a sticky substance that entraps
and immobilizes small insects, or they may contain toxic
constitutents which spill into the surrounding tissue when
the gland is ruptured, making it unpalatable or t o x i c .
These toxins are generally weak and do not kill the insect
d i r e c t l y , rather they retard insect growth and delay
pupation. As a result, the insects are more vulnerable to
disease, predation, and the environment. The balance
which has evolved between plants and insects could be
seriously disrupted if secondary plant toxin analogs are
synthesized and used as insecticides. Insects developing
resistance to the analogs might develop resistance to the
natural toxin.
P l a n t s have served as a food source f o r f i s h , i n s e c t s , and

mammals s i n c e e a r l y b i o t i c times. I n response p l a n t s have
developed i n t r i c a t e p h y s i c a l as w e l l as chemical p r o t e c t i v e
mechanisms. The two defensive s t r u c t u r e s that are the primary
subject of t h i s chapter are trichomes and glands. Trichomes are
epidermal appendages o f d i v e r s e form and s t r u c t u r e , such as
p r o t e c t i v e and g l a n d u l a r h a i r s and s c a l e s or p e l t a t e h a i r .
H a i r s , whether they are u n i c e l l u l a r , m u l t i c e l l u l a r , o r p e l t a t e ,
may be g l a n d u l a r . Glandular trichomes e l a b o r a t e v a r i o u s
substances, such as v o l a t i l e o i l s , r e s i n s , mucilages, and gums.
Rupture of the c u t i c l e a l l o w s the g l a n d u l a r contents t o escape.
Other types of glands are a l s o found on the s u r f a c e o f the p l a n t .
These i n c l u d e : 1) the g l a n d u l a r epidermis c o v e r i n g the l e a f
t e e t h as found i n Primus; 2) nectary glands which produce a
This chapter not subject to U . S . copyright.

Published 1983 American Chemical Society.

sugary s e c r e t i o n a s s o c i a t e d w i t h flowers and other p l a n t p a r t s ;

3) glandular e x c r e t o r y s t r u c t u r e s which discharge a v a r i e t y of
o i l s and r e s i n s ; 4) o i l glands which c o n t a i n v a r i o u s terpenoid
oils.
The u t i l i t y of secondary plant chemicals as defensive agents
against pest a t t a c k has only r e c e n t l y been widely accepted.
However, t h i s view i s s t i l l not u n i v e r s a l l y accepted as
i l l u s t r a t e d by Luckner's (1) recent comment:
"....secondary metabolism i s c h a r a c t e r i z e d by a high degree
of order, i . e . by the p r e c i s e r e g u l a t i o n of enzyme l e v e l and
a c t i v i t y i n metabolic pathways, the compartmentation and
channeling of enzymes, p r e c u r s o r s , i n t e r m e d i a t e s , and
products as w e l l as the i n t e g r a t i o n of secondary metabolism
i n the programs of c e l l s p e c i a l i z a t i o n of the producer
organism. However, c h a r a c t e r i s t i c s of secondary metabolism
are a l s o the b i z a r r
products, the r e s t r i c t e
secondary compounds w i t h i n groups of l i v i n g beings and t h e i r
usefulness which u s u a l l y i s s m a l l or absent, i . e . ,
p r o p e r t i e s which g i v e secondary metabolism i t s e r r a t i c
features."
However, I b e l i e v e t h i s and other chapters w i l l more than
adequately show that secondary p l a n t chemicals were and s t i l l are
important weapons i n the p l a n t s a r s e n a l of defense.
The f o l l o w i n g pages review the f u n c t i o n and chemistry of
trichomes and glands. The concluding s e c t i o n s address the
i n s e c t ' s response to the p l a n t ' s defensive chemicals, and end
w i t h an appeal to the chemical p e s t i c i d e i n d u s t r y .
E x t r a f l o r a l Nectaries
I t i s g e n e r a l l y conceded that f l o r a l n e c t a r i e s evolved as an

a t t r a c t a n t to p o l l i n a t i n g i n s e c t s . The f u n c t i o n of e x t r a f l o r a l
n e c t a r i e s i s not so obvious. Some suggest that the s e c r e t i o n of
sugars i s a s s o c i a t e d w i t h a s h i f t from a " s i n k " t o a "source" of
carbohydrates during development (2^, 3) • Others propose that
sugars are excreted i n c i d e n t a l to the e x c r e t i o n of water and
s a l t s (4, _5, 6 ) . However, B. L. Bentley c o n v i n c i n g l y argues i n
her review that e x t r a f l o r a l n e c t a r i e s are a t t r a c t a n t s f o r ants
which act as "pugnacious body guards" i n defense of the p l a n t
(7_). She bases t h i s contention on f o u r observations. 1) E x t r a -
f l o r a l n e c t a r i e s f o l l o w no recognizable d i u r n a l p a t t e r n and
produce nectar throughout the day and n i g h t . 2) A c t i v e n e c t a r i e s
are found on younger p o r t i o n s of the p l a n t and are o f t e n
a s s o c i a t e d with developing reproductive organs. The n e c t a r i e s
are f u l l y developed before the reproductive organ, and s e c r e t i o n
ceases soon a f t e r the organ matures. 3) Secretory a c t i v i t y i s
u s u a l l y greatest at the height of the growing season and may
continue throughout the year i n moist t r o p i c a l r e g i o n s . 4)

5. STIPANOVIC Plant Trichomes and Glands 71
Attack by sucking i n s e c t s o f t e n increases the r a t e o f s e c r e t i o n

i n a p o s i t i v e l y c o r r e l a t e d manner w i t h increased i n f e s t a t i o n
l e v e l s . Bentley argues that n e c t a r i e s f r e q u e n t l y remain a carbo-
hydrate source u n t i l m a t u r i t y , w h i l e developing buds, f l o w e r s ,
and f r u i t never become a carbohydrate source; sucking i n s e c t s
reduce the carbohydrate source pressure o f an organ and yet
nectar s e c r e t i o n s increase w i t h i n c r e a s i n g i n f e s t a t i o n s . Thus,
the source/sink hypothesis does not seem to apply.
In Catalpa, E l i a s and Newcombe report that glandular
trichomes on leaves are not only very s i m i l a r t o n e c t a r i e s but
are t h e i r precursors ( 8 ) . These authors propose these m u l t i p l e
n e c t a r i e s i n the lower l e a f surface v e i n a x i e s act as a t t r a c t a n t s
f o r b e n e f i c i a l i n s e c t s t o c o n t r o l o r minimize the e f f e c t s of
herbivorous i n s e c t s . Predatory ants are g e n e r a l l y considered t o
be b e n e f i c i a l i n s e c t s T h e i r presence on p l a n t s c o n t a i n i n g
e x t r a f l o r a l nectaries
these mercenaries are
L e r s t e n report that one o f the most d e v a s t a t i n g i n s e c t s observed
to be feeding on cottonwood (Populus d e l t o i d e s ) was an Aphis
species herded by an u n i d e n t i f i e d species o f l a r g e black ant ( 9 ) .
Thus, t h i s "body guard" may "pugnaciously" defend the p l a n t from
some pests only to usher i n a plant pest o f i t s choosing.
Trichomes as a Form of P h y s i c a l Resistance
In medieval times, c i t i e s commonly erected w a l l s as a

p r o t e c t i v e b a r r i e r t o i n v a s i o n . P l a n t s have evolved s i m i l a r
p r o t e c t i v e mechanisms. Thorns and n e t t l e s which r e p e l most
herbivores are two examples o f p r o t e c t i v e b a r r i e r s . To ward o f f
i n s e c t a t t a c k , p l a n t s have evolved non-glandular trichomes which
act i n a s i m i l a r f a s h i o n . Thus, i n some p l a n t s , the d e n s i t y ,
l e n g t h , o r branching o f trichomes have been n e g a t i v e l y c o r r e l a t e d
w i t h i n s e c t s u r v i v a l (10).
The degree of l e a f pubescence g r e a t l y a f f e c t s the behavior
of c e r e a l l e a f b e e t l e g r a v i d females, Oulema melanopus (11).
Densely pubsecent wheat had only o n e - t h i r d as many eggs as the
glabrous c o n t r o l . On pubescent leaves i t was o f t e n found that
eggs were l a i d i n areas where a d u l t s had d i s r u p t e d the trichome
cover by feeding. F i e l d cage t e s t s showed that the v i a b i l i t y o f
eggs decreased w i t h i n c r e a s i n g pubescence. Eggs l a i d o r
mechanically placed on pubescent wheats were more s u s c e p t i b l e t o
d e s i c c a t i o n . L a r v a l s u r v i v a l and weight g a i n were a l s o minimized
on densely pubescent leaves (12). This was e s p e c i a l l y true i n
f i r s t - i n s t a r l a r v a e , w h i l e t h i r d - i n s t a r and f o u r t h - i n s t a r l a r v a e
were l i t t l e a f f e c t e d by pubescence. However, long o r dense
trichomes d i d not c o r r e l a t e i n a greenhouse t e s t w i t h r e s i s t a n c e
of wheat t o greenbug, Schizaphis graminum (13).
A h a i r y c o t t o n v a r i e t y that had the most trichomes, the
longest trichomes, and the most branched trichomes was the most
r e s i s t a n t t o s p i d e r mites and leafhoppers (14). I n a no-choice

cage t e s t w i t h three cotton i s o l i n e s , Lygus herperus o v i p o s i t i o n

response was h i g h e s t , but growth r a t e slowest on densely
pubescent c o t t o n as compared t o normal and smooth cottons ( 1 5 ) •
Nymphal s u r v i v a l was e s s e n t i a l l y equal on a l l l i n e s , but the
e f f e c t o f predators was not a f a c t o r i n these cage t e s t s .
However, pubescence i s not always an advantage t o p l a n t s , as
i l l u s t r a t e d by the g r e a t e r r e s i s t a n c e of smooth l e a f c o t t o n t o
fleahoppers ( 1 6 ) •
Hooked trichomes on the French bean, Phaseolus v u l g a r i s have
been reported t o capture a d i v e r s e group of i n s e c t species
i n c l u d i n g the potato leafhopper, Empoasca fabae ( 1 7 ) , and the
aphids, Myzus p e r s i c a e ( 1 8 ) , Aphis fabae ( 1 9 ) , and Aphis
c r a c c i v o r a (20). A recent study has shown t h a t the major f a c t o r
a f f e c t i n g potato leafhopper damage on the French bean was the
d e n s i t y of hooked trichomes ( 2 1 ) . Leafhopper nymphs were impaled
by the trichomes, l e a d i n
same p o s i t i v e c o r r e l a t i o
d e n s i t y a l s o has been reported f o r a d u l t leafhoppers on f i e l d
bean c u l t i v a r s , J?, v u l g a r i s and P. lunatus ( 2 2 ) . Hooked
trichomes growing a t angles l e s s than 3 0 ° are r e p o r t e d l y
i n e f f e c t i v e i n c a p t u r i n g leafhoppers.
The i n h e r i t a n c e o f pubescence type ( 2 3 ) and the morphology
of pubescence i n soybean, G l y c i n e max, have been s t u d i e d , and
densely pubescent p l a n t s were found t o be the most vigorous ( 2 4 ) .
Growth d i f f e r e n c e s were a s s o c i a t e d w i t h IS. fabae i n f e s t a t i o n s .
Glabrous soybeans supported a higher p o p u l a t i o n o f IS. fabae and
had a higher i n c i d e n c e of o v i p o s i t i o n than pubescent v a r i e t i e s
( 2 5 , 2 6 ) . This same o b s e r v a t i o n has been made on other pubescent
host p l a n t s ( 1 7 ) . The d i f f e r e n c e s i n populations of E. fabae
were r e l a t e d t o o r i e n t a t i o n , l e n g t h , and the erectness of the
l e a f trichomes ( 2 7 , 2 8 ) . When populations of E. fabae, which i s
1.0 - 4 . 0 mm i n l e n g t h , were compared t o t h a t of a s p r i n g t a i l ,
Deuterosmiathurus yumanensis, which i s 0 . 2 - 0 . 4 mm i n l e n g t h , on
near i s o g e n i c l i n e s o f soybean, i t was found that both species
had the highest populations on the glabrous genotype. On a
deciduous v a r i e t y , w i t h only misshapen and s e v e r e l y appressed
trichomes, the p o p u l a t i o n o f the s m a l l phytophagus species was
depressed, w h i l e t h a t o f the l a r g e r species was not a f f e c t e d .
Populations of E>. fabae decreased w i t h i n c r e a s i n g trichome
l e n g t h ( 0 . 9 - 1 . 6 mm) r e g a r d l e s s o f trichome d e n s i t y , whereas the
f
s p r i n g t a i l s p o p u l a t i o n decreased w i t h i n c r e a s i n g trichome
d e n s i t y . The populations o f t h r i p s , S e r i c o t h r i p s v a r i a b i l i s , and
bandedwing w h i t e f l y , T r i a l e u r o d e s a b u t i l o n e a , showed no
c o n s i s t e n t response t o trichome v a r i a t i o n s ( 2 9 ) .
Trichomes and Glands as a Form o f Chemical Resistance
In a d d i t i o n t o p h y s i c a l forms o f r e s i s t a n c e , p l a n t s a l s o
r e l y on chemicals t o immobilize, r e p e l and poison phytophagus
i n s e c t s . These chemicals may be l o c a t e d i n v a r i o u s types of o i l

or secretory glands or i n g l a n d u l a r trichomes. Glandular h a i r s

or trichomes are widely d i s t r i b u t e d i n v a s c u l a r p l a n t s . A
c a r e f u l h i s t o c h e m i c a l study showed that 39 o f 43 p l a n t species
from 26 d i f f e r e n t p l a n t s f a m i l i e s had stem h a i r s (30). Some o f
these p l a n t s , such as tomato, had s e v e r a l morphologically
d i s t i n c t h a i r s . P l a n t h a i r s are a l s o chemically d i s t i n c t as
evidenced by t h e i r r e a c t i o n w i t h v a r i o u s h i s t o c h e m i c a l reagents.
Reviews on the i n t r a c e l l u l a r compartmentation o f f l a v o n o i d s
and other secondary metabolites have been published (31, 32). A
review on the chemical c o n s t i t u e n t s o f these glands i s
complicated by a number o f f a c t o r s . The d i v e r s i t y and the
complexity o f chemicals produced i n a gland o r trichome a r e two
such f a c t o r s . However, one of the most formidable o b s t a c l e s t o a
review .on g l a n d u l a r c o n s t i t u e n t s i s the d e t a i l s reported on the
e x t r a c t i o n procedure. I t i s common f o r i n v e s t i g a t o r s t o simply
d i v i d e the p l a n t i n t o i t
p a r t s , flowers and buds
l e a v e s , f o r example, there i s o f t e n no attempt t o d i f f e r e n t i a t e
between v a r i o u s morphological e n t i t i e s . The l e a f i s ground o r
e x t r a c t e d whole. A l l evidence i n d i c a t i n g the occurrence of a
p a r t i c u l a r chemical i n a trichome o r gland i s o b l i t e r a t e d .
In the study o f r e s i s t a n c e mechanisms, i t i s recognized t h a t
the l o c a t i o n of a p a r t i c u l a r t o x i c chemical may be as important
as the presence o r absence o f t h a t chemical. For example, a h i g h
c o n c e n t r a t i o n of a t o x i c chemical i n a p l a n t part that the i n s e c t
does not e a t o r eats only i n the l a t e r stages o f l a r v a l growth,
w i l l probably have minimal a f f e c t on the r e s i s t a n c e of that p l a n t
to i t s host. Conversely, a low c o n c e n t r a t i o n o f a t o x i c chemical
i n a s p e c i f i c s i t e a t which the i n s e c t feeds i n an e a r l y l a r v a l
stage may s i g n i f i c a n t l y a f f e c t the p l a n t ' s r e s i s t a n c e .
In h o s t - p l a n t r e s i s t a n c e s t u d i e s , i t i s t h e r e f o r e imperative
f o r i n v e s t i g a t o r s t o r e p o r t not o n l y what chemicals are present,
but a l s o t o r e p o r t as a c c u r a t e l y as p o s s i b l e the p r i n c i p a l
s i t e ( s ) a t which the chemical i s concentrated. This i n f o r m a t i o n
i s g e n e r a l l y not reported, t h e r e f o r e t h i s review w i l l undoubtedly
omit reported chemicals that a r e l o c a t e d i n glands and g l a n d u l a r
trichomes and improperly i n d i c a t e g l a n d u l a r components that are
a c t u a l l y l o c a t e d elsewhere i n the p l a n t . However, i n f o r m a t i o n on
the l o c a t i o n o f chemicals w i l l be given wherever p o s s i b l e . This
s e c t i o n w i l l review the major types o f chemicals that are present
i n glands and g l a n d u l a r trichomes.
Immobilizing Chemicals. Some p l a n t s produce a s t i c k y , gummy

exudate from g l a n d u l a r trichomes. These exudates e f f e c t i v e l y
immobilize s m a l l i n s e c t s .
A number of p l a n t s o f the Solanum and N i c o t i a n a genera are
p a r t i c u l a r l y adept a t producing s t i c k y l e a f exudates. I n some
w i l d potato s p e c i e s , an exudate i s discharged from g l a n d u l a r
h a i r s when aphids mechanically rupture the c e l l w a l l s (33). The
c l e a r , water s o l u b l e exudate i s s t a b l e i n the absence o f 0 , 9

but r a p i d l y darkens and forms a p r e c i p i t a t e on the aphids limbs

when exposed t o a i r . E v e n t u a l l y the aphid becomes immobilized
and d i e s . I n v e s t i g a t o r s found s e v e r a l m o r p h o l o g i c a l l y d i f f e r e n t
types of glands; however, only one was an apparent source of the
exudate. Once t h i s gland was ruptured, the m a t e r i a l was not
r e p l a c e d . The c l e a r m a t e r i a l gave a p o s i t i v e t e s t w i t h the
F o l i n - D e n i s phenol reagent. The formation of the dark
p r e c i p i t a t e was i n h i b i t e d by the copper c h e l a t i n g agent, sodium
d i e t h y l d i t h i o c a r b a m a t e . Polyphenol oxidase enzymes may be
i n v o l v e d i n t h i s r e a c t i o n s i n c e copper i s e s s e n t i a l f o r t h e i r
r e a c t i v i t y . In other research on Empoasca fabae, the percentage
m o r t a l i t y of nymphs, females, and males confined to the glanded
species, polyadenium, was 78, 64, and 94 r e s p e c t i v e l y ,
compared to l e s s than 20% on 2 nonglandular species (34).
Trichome exudates were found on the mouthparts of IS fabae i n 75
67, and 29% of dead nymphs
Scanning e l e c t r o n microscop
was t o t a l l y occluded by the trichome exudate. The s u r v i v i n g
leafhoppers had no observable exudate on t h e i r bodies. The
viscous exudate, which r a p i d l y darkens and hardens, could be
d i s s o l v e d i n 95% e t h a n o l .
Aphids, attempting to feed on the tomato, Solanum p e n n e l l i i ,
are entangled i n a s t i c k y l e a f exudate and soon d i e (35). This
exudate could be removed by washing w i t h cotton s a t u r a t e d w i t h
95% e t h a n o l , but the washed leaves r a p i d l y s e c r e t e d a new exudate
that a l s o was f a t a l . Trichomes were a d e t e r r e n t to o v i p o s i t i o n
by the tobacco w h i t e f l y , Bemisia t a b a c i , on both tomato and
tobacco leaves (36). The s t i c k y exudate may p a r t i a l l y account
f o r t h i s s i n c e w h i t e f l i e s were found glued to the g l a n d u l a r
hairs.
Glandular h a i r s a l s o have been i m p l i c a t e d i n the r e s i s t a n c e
of c e r t a i n annual Medicago species to the a l f a l f a w e e v i l , Hypera
p o s t i c a (37). The annual s p e c i e s , M. d i s c i f o r m i s , and M.
s c u t e l l a t a , possess e r e c t g l a n d u l a r h a i r s , and the exudate from
these glands acts as a glue to immobilize l a r v a e . The p e r e n n i a l
s p e c i e s , M. s a t i v a , which i s s u s c e p t i b l e to the a l f a l f a w e e v i l ,
possesses only procumbert glands. The exudate from these glands
does not prevent l a r v a l movement (38).
Stylosanthes hamata and S^. scabra, which are h i g h l y
productive and n u t r i t i o u s species of t r o p i c a l pasture legumes,
are covered w i t h g l a n d u l a r trichomes. The trichomes secrete a
viscous s e c r e t i o n that immediately immobilize l a r v a e of the
c a t t l e t i c k , Boophilus microplus (39). T i c k s have a n a t u r a l
tendency to climb p l a n t s , and wait f o r a host animal. The
s e c r e t i o n has no r e p e l l a n t p r o p e r t i e s , so that t i c k s do not
attempt to seek a l t e r n a t i v e p l a n t s . In a d d i t i o n to i m m o b i l i z i n g
the l a r v a e , the p l a n t s produce an u n i d e n t i f i e d v o l a t i l e
compound(s) that poison the larvae, w i t h i n 24 hours.
As w i t h many p l a n t defensive systems, g l a n d u l a r trichomes
may a l s o be d e t r i m e n t a l to the p l a n t . I t has been proposed that

5. STiPANOVic Plant Trichomes and Glands 75
the tobacco i n t r o d u c t i o n 1112 ( T . I . 1112) may be r e s i s t a n t t o

some i n s e c t s because i t s l e a v e s , although h a i r y , l a c k g l a n d u l a r
trichomes (40). The glandular trichomes that cover the a e r i a l
p o r t i o n s of most tobacco p l a n t s are a conspicuous source of
odors, as w e l l as a source of the s t i c k y exudate. I t has been
proposed that the trichome exudate serves as an a t t r a c t a n t o r
o v i p o s i t i o n stimulant f o r tobacco budworm moths and as an
a r r e s t a n t f o r a l a t e green peach aphids. The absence of g l a n d u l a r
trichomes on the leaves of T.I. 1112 may account f o r i t s p a r t i a l
r e s i s t a n c e t o these i n s e c t s . However, T.I. 1112 has glandular
trichomes on the calyces which may e x p l a i n the readiness of
tobacco budworm moths t o o v i p o s i t i o n on i t s flowers and flower
buds. The absence of g l a n d u l a r trichomes a l s o increased
Trichogramma p a r a s i t i s m on H e l i o t h i s eggs on T.I. 1112.
H e l i o t h i s egg m o r t a l i t y from t h i s p a r a s i t e on other tobacco i s
n e g l i g i b l e because th
exudate (41).
Toxic and Repellent Allelochemics
L e v i n has proposed t o c l a s s i f y t o x i c p l a n t chemicals

i n v o l v e d i n r e s i s t a n c e i n t o two broad c a t e g o r i e s , those that
confer s p e c i f i c r e s i s t a n c e and those that confer general
r e s i s t a n c e (42). Compounds e x h i b i t i n g s p e c i f i c r e s i s t a n c e a r e
c h a r a c t e r i z e d as extremely t o x i c t o a small group of s p e c i a l i z e d
pathogens or h e r b i v o r e s . Each compound i s present only i n a few
s p e c i e s , and i s o f t e n t i s s u e s p e c i f i c . They tend t o reach t h e i r
highest concentration i n young leaves and f r u i t s and decrease as
they mature. Examples would be s i n i g r i n , tomatine, s o l a n i n e , and
gossypol. Compounds c l a s s i f i e d as showing general r e s i s t a n c e
d e t e r , r e p e l , o r are weakly t o x i c t o most microorganisms and/or
h e r b i v o r e s . These compounds are present i n s e v e r a l plant species
and sometimes i n f a m i l i e s of d i f f e r e n t orders. They are
g e n e r a l l y not t i s s u e s p e c i f i c , and t h e i r concentration increases
as the t i s s u e matures. Examples i n c l u d e chlorogenic a c i d ,
q u e r c e t i n , and tannins.
Although a t o x i c p l a n t chemical may not f i t e i t h e r category
p e r f e c t l y , those chemicals discussed below that are t i s s u e
s p e c i f i c would g e n e r a l l y be considered t o show s p e c i f i c
r e s i s t a n c e . I t i s i n t e r e s t i n g t o note that those phytochemicals
t h a t are e s p e c i a l l y t o x i c t o one group of i n s e c t s are q u i t e o f t e n
e s s e n t i a l d i e t a r y i n g r e d i e n t s or feeding s t i m u l a n t s t o other
i n s e c t s that feed p r i m a r i l y on that p l a n t .
The phytochemicals that are discussed below may be d i v i d e d
i n t o three c l a s s i c a l c a t e g o r i e s : a l k a l o i d s , f l a v o n o i d s , and
terpenes. S t r a i g h t chain carbon compounds, such as hydrocarbons,
waxes, f a t t y a c i d and a l c o h o l s a l s o occur i n glands. The
a l k a l o i d s tend t o react as feeding deterents and as poisons
(43). They are probably the major c l a s s found i n any l i s t s of
p l a n t t o x i n s (44, 45). Flavonoids are w e l l known a n t i b a c t e r i a l ,

a n t i m i c r o b i a l and a n t i v i r a l agents (46-49). Flavonoids a l s o

i n h i b i t the growth of H e l i o t h i s zea l a r v a e (50). They may a l s o
act as e i t h e r feeding d e t e r r e n t s o r s t i m u l a n t s f o r d i f f e r e n t
i n s e c t s (22, 43, 51.* 52, 53). I t was found that v i c i n a l
h y d r o x y l a t i o n was necessary, but not s u f f i c i e n t f o r growth
i n h i b i t i o n (50). Flavanones, f l a v o n e s , and f l a v o n o i d g l y c o s i d e s
i n h i b i t Na-independent passive t r a n s p o r t of sugars by i n t e s t i n a l
e p i t h e l i a l c e l l s (54). The i n h i b i t o r y a c t i o n i s i n f l u e n c e d by
the extent and p o s i t i o n of h y d r o x y l a t i o n of the f l a v o n o i d
nucleus, the degree of p l a n a r i t y of the molecules, the presence
of a carbonyl group, and g l y c o s y l a t i o n . The occurrance of
f l a v o n o i d aglycones throughout the p l a n t kingdom has been
c a r e f u l l y cataloged (55).
Terpenoids are a d i v e r s e group of compounds showing a wide
spectrum of b i o l o g i c a l a c t i v i t i e s Among the most b i o l o g i c a l l y
a c t i v e terpenoids ar
b i o l o g i c a l a c t i v i t y ha
Sesquiterpene lactones show c y t o t o x i c i t y , mammalian t o x i c i t y ,
a l l e r g i c contact d e r m a t i t i s , a l l e l o p a t h y , a n t i b i o t i c a c t i v i t y t o
b a c t e r i a and f u n g i , s c h i s t o s o m i c i d a l a c t i v i t y , a n t i f e e d a n t
a c t i v i t y toward mammals and i n s e c t s , i n s e c t l a r v a l growth
i n h i b i t i o n , and deter i n s e c t o v i p o s i t i o n . T h e i r a c t i v i t y can be
explained i n many cases by Michael a d d i t i o n r e a c t i o n s between a
n u c l e o p h i l e and the e x o c y c l i c a , 6 - u n s a t u r a t e d y-lactone f u n c t i o n .
Other types of terpenoids a r e a n t i f e e d a n t s t o the A f r i c a n
armyworm (59), and the obscure root w e e v i l (60). General
terpenoid a n t i f e e d a n t s from east A f r i c a n t r o p i c a l p l a n t s have
been reviewed (61, 62). Terpenes may a c t as feeding s t i m u l a n t s
or i n h i b i t o r s depending on c o n c e n t r a t i o n (63)• Terpenoids a l s o
e x h i b i t i n s e c t growth r e g u l a t i o n (64), antiecdysone a c t i v i t y
(65), and are a c t i v e against a wide range of b a c t e r i a and f u n g i
(66-71).
A l k a l o i d s and Phenols from Tobacco, Tomato, and Potato.

Tobacco, tomato, and potato p l a n t s c o n t a i n a number of t o x i c
a l k a l o i d s . Probably the most widely s t u d i e d i s n i c o t i n e . The
i n s e c t i c i d a l p r o p e r t i e s of t h i s and other tobacco a l k a l o i d s have
been reviewed (72). A study of N i c o t i a n a showed that a l k a l o i d s
are secreted by trichomes i n the seven species t e s t e d (73).
N i c o t i n e was the major a l k a l o i d i d e n t i f i e d i n the trichome
s e c r e t i o n (74). Anabasine and n o r n i c o t i n e were a l s o i d e n t i f i e d
i n two species ( 7 5 ) . Other ether s o l u a b l e c o n s t i t u t e n t s have
a l s o been i d e n t i f i e d (76). Aphids were k i l l e d by contact w i t h
these s e c r e t i o n s . Trichome s e c r e t i o n s from N i c o t i a n a a l s o were
t o x i c t o the tobacco hornworm and the two spotted spider mite
(77, 78). In a p h y s i o l o g i c a l study of i n s e c t ' s response t o
n i c o t i n e , i t was found that the p e n e t r a t i o n r a t e of n i c o t i n e i n t o
the i s o l a t e d nerve cord was pH dependent (79)• At the pH of
i n s e c t haemolymph the p e n e t r a t i o n of n i c o t i n e i n t o the nerve cord
was higher on a u n i t weight b a s i s i n the n i c o t i n e - s u s c e p t i b l e

silkworm (Bombyx mori) than i n the n i c o t i n e - r e s i s t a n t hornworm,

Manduca s e x t a . This was i n t e r p r e t e d as i n d i c a t i n g a l e s s
e f f i c i e n t ion-impermeable b a r r i e r of the silkworm nervous
system.
The e f f e c t s of the Solanum a l k a l o i d s on the Colorado potato
b e e t l e , L e p t i n o t a r s a decemlineata, have been c a r e f u l l y s t u d i e d .
Solanine, chaconine, l e p t i n e I , l e p t i n i n e I , l e p t i n i n e I I ,
demissine, and tomatine were a l l found t o be feeding d e t e r r e n t s
(74, 80). N i c o t i n e , even i n q u i t e s m a l l c o n c e n t r a t i o n s , was very
t o x i c (74). The Colorado potato b e e t l e was found t o feed more on
young tomato p l a n t s which had a low c o n c e n t r a t i o n of tomatine
than on mature o r f l o w e r i n g p l a n t s that had a higher
c o n c e n t r a t i o n of tomatine (81). Feeding was a l s o i n h i b i t e d
(20-80%) when tomatine was i n f i l t r a t e d i n t o tomato l e a f d i s k s a t
concentrations between 65 and 165 mg/100 g f r e s h weight
Nornicotine, solanine
the two-striped grasshopper
general feeder (82). Trichome s e c r e t i o n s of tomato are a l s o
t o x i c t o the green peach aphid (83). An extensive l i s t of p l a n t
a l k a l o i d s and other phytochemicals that act as i n s e c t feeding
d e t e r r e n t s and r e p e l l e n t s as w e l l as feeding s t i m u l a n t s and
a t t r a c t a n t s has been published (45). I n the case o f the tobacco
hornworm, M. s e x t a , n o r v a l a t i n e and nonpolar or weakly p o l a r
f r a c t i o n s o f tomato leaves s t i m u l a t e f e e d i n g , w h i l e feeding
d e t e r r e n t s were present i n water e x t r a c t s of tomato leaves (84).
Tomato trichome exudates are t o p i c a l l y t o x i c t o the s p i d e r mite,
Tetranychus u r t i c a e (85). The f l a v o n a l g l y c o s i d e , r u t i n , has
been i s o l a t e d from the t e t r a c e l l u l a r g l a n d u l a r trichomes of the
tomato p l a n t , Lycopersicon esculentum (86). This and other
trichome exudates reduce l a r v a l growth o f H e l i o t h i s zea. A
morphologically d i f f e r e n t type of g l a n d u l a r trichome on the w i l d
tomato, L. hirsutum, contains 2-tridecanone, which i s t o x i c t o 11.
zea and M. sexta when a p p l i e d t o p i c a l l y (87)• T o t a l
g l y c o a l k a l o i d s i n f o l i a g e o f w i l d potatoes were s i g n i f i c a n t l y
c o r r e l a t e d w i t h r e s i s t a n c e t o the potato leafhopper (88).
Flavonoids and Terpenoids i n Bud Exudate. The occurrence of

f l a v o n o i d aglycones i n buds has been reviewed by Wollenweber and
D i e t z (55). They note that the f l a v o n o i d aglycones w i t h a low
number of hydroxyl groups and/or a high number of methoxyl
groups, because of t h e i r l i p o p h i l i c nature, do not accumulate i n
the c e l l sap. Thus, these types of compounds are found i n p l a n t s
w i t h g l a n d u l a r trichomes, e x c r e t i o n c e l l s , o r c a v i t i e s . A
v a r i e t y of p l a n t f a m i l i e s produce bud exudates c o n t a i n i n g widely
v a r y i n g f l a v o n o i d s . I t was p r e v i o u s l y mentioned that f l a v o n o i d s
have a wide range of b i o l o g i c a l a c t i v i t i e s . However, the
b i o l o g i c a l a c t i v i t y o f only a small f r a c t i o n o f the f l a v o n o i d s
has been i n v e s t i g a t e d against an Qven smaller f r a c t i o n of p l a n t
p e s t s . For t h i s reason, s p e c i f i c compounds w i l l not be l i s t e d ,
but i t i s c l e a r that many, i f not most, of the f l a v o n o i d s confer
some p r o t e c t i o n on t h e i r parent p l a n t s .

Table I . F l a v o n o i d Aglycones i n Bud Exudates
CO
o cu
a o
o
CJ
CO tH
CD CO cd
a Q) CJ
a
CO
CD o o
c O a u o o
o a cd CJ
o iH
> cd
cd •H ^3
Family U
O
Salicaeae Populus > a b
4 12 5 1 2 1
Betulaceae Alnus > >c d e
6 14 1 1
Betula
Ostrya #
Rubiaceae Elaegia* 1
Asteraceae Acanthospermum ^ 1
Hippo cas t umaceae Aes cuius 11
Rosaceae Prunus ' a m
9 1
Rhamnaceae Rhamnus ' k m
4
Didieraceae Decry a 11
1
n
f R e f . (55) R e f . (91)
b
R e f . (110) ^ e f . (94)
-*Ref. ( I l l ) ^Ref. (116)
d
R e f . (112) *Ref. ( 9 2 j
*Ref. (113) •"•Ref. (117)
f
R e f . (114) "Ref. (118)
n
*Ref. (115) R e f . (119)
The f l a v o n o i d aglycones r e p o r t e d i n buds of A l n u s , B e t u l a ,

Ostrya, E l a e g i a , Acanthospermum, Aescuius, Primus, Rhamnus, and
Decrya are l i s t e d i n Table 1. The b i o s y n t h e s i s of f l a v o n o i d s i n
s u b c e l l u l a r glands has been i n v e s t i g a t e d i n Populus n i g r a ( 8 9 ) ,
and the f l a v o n o i d s i n the l i p o p h i l i c c o a t i n g i n Populus buds have
been thoroughly analyzed ( 9 0 ) . I n a d d i t i o n t o t h i s wide v a r i e t y
of f l a v o n o i d s , mixtures of terpenoids have a l s o been reported i n
bud exudates. I n B e t u l a n i g r a the bud exudate i s reported t o be
mainly u n i d e n t i f i e d terpenoids (91). U n i d e n t i f i e d terpenoids a r e
found i n bud exudates from Aescuius sp. ( 9 2 ) , Alnus sp., B e t u l a
sp., and Ostrya sp. ( 9 3 ) . Some s p e c i f i c terpenoids have been
i d e n t i f i e d . The t r i t e r p e n e s i s o f o u q u i e r o l , dammarenediol-11, and
(20S)-dammar-24-ene-3 , 20, 2 6 - t r i o l have been i s o l a t e d from the
bud exudate of E l a e g i a u t i l i s ( 9 4 ) , and 6-amyrenone i s reported
to be a major c o n s t i t u e n t of the bud exudate from a l d e r trees

(95). Several raelampolide type sesquiterpene lactones have been

i s o l a t e d from the Tanzanian plant Acanthospermum glabratum (96,
97); however, t h e i r occurence i n the bud exudate i s u n c e r t a i n .
Although the c o n s t i t u e n t s o f bud exudates are u s u a l l y
a s c r i b e d t o f l a v o n o i d s and terpenes, other c o n s t i t u e n t s may a l s o
be present. The flower buds of Alnus pendula produce a viscous
m a t e r i a l which i n c l u d e s a wide v a r i e t y of compounds i n c l u d i n g
a c i d s (cinnamic, 3 - p h e n y l p r o p i o n i c , and b e n z o i c ) , an e s t e r ( 3 -
phenylethyl cinnamate), ketones ( t r a n s , t r a n s - 1 , 7 - d i p h e n y l - l , 3 -
heptadiene-5-one and benzylacetone), an a l c o h o l ( 3 - p h e n y l e t h y l
a l c o h o l ) , an aldehyde (cinnamaldehyde), s t i l b e n e s ( p i n o s y l v i n and
p i n o s y l v i n raonomethyl e t h e r ) , and phenols (eugenol and c h a v i c o l ) ,
as w e l l as p a r a f f i n s , f l a v o n o i d s (pinocembrin, p i n o s t r o b i n ,
a l p i n e t i n , and g a l a n g i n ) , and t r i t e r p e n e s (6 -amyrenone and
taraxerone) (98). In A s i e b o l d i a n a two ketones were a l s o
i s o l a t e d (yashabushiketo
Farinose Exudates o f the Polypodiaceae and Primulaceae.

Members of the Polypodiaceae produce a yellow o r white powdery
deposit on the lower surface of t h e i r fronds. These deposits are
u s u a l l y r e f e r r e d t o as f a r i n o s e exudates. F a r i n a from species of
Pityrogramma, C h e i l a n t h e s , Adiantum, and Notholaena have been
c a r e f u l l y s t u d i e d . The f a r i n o s e c o a t i n g of these p l a n t s i s
formed by the t e r m i n a l c e l l o f s m a l l h a i r s u s u a l l y found on the
lower surface of the f r o n d .
Wollenweber (99) has reviewed the morphology and chemistry
of these ferns which are prodigious producers o f f a r i n a ,
r e p r e s e n t i n g 0.9%-5.0% of the dry weight of the fronds.
Wollenweber r e p o r t s that f l a v o n o i d s are the major c o n s t i t u e n t s o f
the f a r i n a . Chalcones (100-106) dihydrochalcones (102, 103, 107,
108, 109), f l a v o n o l s (102, 106, 108, 120-128), flavones TT02,
108, 121, 123, 125, 128, 12977 flavanonesTT30), and the
c h a l c o n e - l i k e compound, ceroptene (124, 131), have been reported
i n the f a r i n a from f e r n s . The f l a v o n o i d p a t t e r n i n the f a r i n a of
220 samples from 14 species of the goldenback and s i l v e r b a c k
f e r n s has been reviewed (132). Flavanones w i t h methyl
s u b s t i t u e n t s (133) and f l a v o n o l s e s t e r i f i e d a t the 8 p o s i t i o n
w i t h b u t y r i c and a c e t i c a c i d (134) have a l s o been i d e n t i f i e d i n
f a r i n a . The t r i t e r p e n e s d i p l o p t e r o l , fernene, adiantone,
isoadiantone (135, 136), and 6 3 , 22-dihydroxyhopane (136) and
v a r i o u s p h y t o s t e r o l s (137-140) have been i s o l a t e d from f e r n .
However, the morphological source of these compounds i s not
c l e a r . This i s a l s o t r u e f o r the sesquiterpenes (141, 142, 143),
the l a c t o n e , calomenanolactone (144), the ecdysone analogues
(145, 146), and p-hydroxystyryl-3-D-glucoside (147). However,
f
the novel d i h y d r o s t i l b e n e , 5-hydroxy-3,4 -dimethoxy-6-carbbxylic
a c i d b i b e n z y l , i s a f a r i n o s e c o n s t i t u t e n t of Notholaena dealbata
and N_. l i m i t a n e a (148)• Glycosides of f l a v o n o l s have a l s o been
i s o l a t e d from f a r i n a (121). A group o f hydrocarbons, t e r p e n o i d s ,
and f a t t y acids have been detected i n the glandular l i p i d s of

D r y o p t e r i s a s s i m i l i s f e r n s by gas chromatography (149). The

f
dihydrochalcone, 2 ^ ' - d i h y d r o x y - 4,4'-dimethoxydihydrochalcone,
from Pityrogramma calomelanos has shown marked a n t i f u n g a l
p r o p e r t i e s (150)Tbther f l a v o n o i d s from Pityrogramma are reported
f f f
to be a l l e l o p a t h i c agents. 2 ,6 -Dihydroxy-4 -methoxydihydro-
chalcone i n h i b i t e d spore germination and gametophyte development
by calmelanos at a l l concentrations t e s t e d , but 2,6-dihydroxy-
f
4 -methoxychalcone i n h i b i t e d germination at 5 X 10 M and
s t i m u l a t e d germination at 5 X 10 M. I z a l p i n i n showed
s i m i l a r e f f e c t s , i n h i b i t i n g germination at 5 X 10 M and
s t i m u l a t i n g germination at 5 X 10 M. These f l a v o n o i d s
appear to act as germination i n h i b i t o r s around the parent p l a n t
(151) .
Many primroses (Primula) a l s o produce a f a r i n o s e exudate on
t h e i r stems and l e a v e s Eighteen species have been i n v e s t i g a t e d
(152) . The major component
5-hydroxy-and 5,8-dihydroxyflavon
derivatives.
Sesquiterpene Lactones and Other Glandular Trichome

Components from Veronia, Parthenium, H e l i a n t h u s , and Artemesia.
Sesquiterpene lactones are major c o n s t i t u e n t s of Veronia,
Parthenium, Helianthus and Artemesia. As i n d i c a t e d p r e v i o u s l y ,
the sesquiterpene lactones as a group are a c t i v e i n a wide range
of b i o l o g i c a l systems. Mabry, et a l . found that the
sesquiterpene l a c t o n e , g l a u c o l i d e A, found i n the glandular
trichomes of some species of Veronia, p r o t e c t s these p l a n t s
against some i n s e c t s (153). Leaf d i e t s were prepared w i t h and
without g l a u c o l i d e A f o r s i x i n s e c t l a r v a e : 1) the y e l l o w
woolybear, D i a c r i s i a v i r g i n i c a ; 2) cabbage looper, T r i c h o p l u s i a
n i ; 3) y e l l o w s t r i p e d armyworm, Spodoptera o r n i t h o g a l l i ; 4)
saddleback c a t e r p i l l a r , S i b i n e s t i m u l e a ; 5) f a l l armyworm,
Spodoptera f r u g i p e r d a ; 6) southern armyworm, S^. e r i d a n i a (57).
In a f r e e choice s i t u a t i o n , a l l i n s e c t s p r e f e r r e d the d i e t
without the l a c t o n e , and the Veronia species that d i d not c o n t a i n
g l a u c o l i d e A. Larvae of 3,5, and 6 were s i g n i f i c a n t l y smaller
when fed d i e t s c o n t a i n i n g g l a u c o l i d e A compared w i t h a c o n t r o l
d i e t , w h i l e l a r v a e of 1 and 2 were unaffected. Furthermore, the
days to pupation increased f o r a l l l a r v a e except 1. Other work
has shown that p l a n t s c o n t a i n i n g g l a u c o l i d e A deterred o v i p o s t i o n
i n some i n s e c t s (154). Other sesquiterpene lactones have been
i s o l a t e d from Veronia species (155, 156, 157) as w e l l as
t r i t e r p e n e s (158, 159), long chain alkanes (159), and an i r i d o i d
g l u c o s i d e (160). The morphological o r i g i n of these l a t t e r
compounds i s u n c e r t a i n .
Parthenin i s a major component found i n the trichomes of
Parthenium hysterophorus, making up about 8% of the dry weight of
the p l a n t (161). I t causes d e r m a t i t i s i n humans and c a t t l e (162,
163) and acts as an a l l e l o c h e m i c (164). Parthenin acted as an
a n t i f e e d a n t and was t o x i c o r a l l y at 0.01% t o Dysdereus k o e n i g i ,

Aedes a e g y p t i , T r i b o l i u m castaneum, P e r i p l a n e t a americana, and

Phthormea o p e r c u l e l l a (165)• Two other sesquiterpene l a c t o n e s ,
c o r o n o p i l i n and t e t r a n e u r i n - A (166), and 34 flavones (19
g l y c o s i d e s and 15 aglycones) have been i s o l a t e d from Parthenium
species (167).
Sesquiterpene lactones may o f f e r some i n s e c t r e s i s t a n c e i n
sunflower. Glandular trichomes o c c u r r i n g on the l e a v e s ,
p h y l l a n d r i e s , and anthers of Helianthus m a x i m i l i a n i c o n t a i n a
sesquiterpene l a c t o n e , maximilin-C. The f i r s t i n s t a r l a r v a e o f
the sunflower moth, Homeosoma e l e c t e l l u m , s u f f e r e d a high
m o r t a l i t y r a t e when f e d on a wheat germ d i e t c o n t a i n i n g
concentrations of 1.0 and 10.0% o f maximilin-C (168, 169). A
number of other sequiterpene lactones have a l s o been i s o l a t e d
from Helianthus species (170-175). The d i t e r p e n o i d a c i d s ,
trachyloban-19-oic a c i d d (-)-kaur-16-en-19-oi a c i d hav a l s
been i m p l i c a t e d i n r e s i s t a n c
However, as w i t h maximilin-C, y l a r g dosage (0.5-1.0%)
were r e q u i r e d t o reduce growth on s y n t h e t i c d i e t s by one-half, as
compared t o a c o n t r o l . Other d i t e r p e n o i d s (177, 178, 179),
t r i t e r p e n o i d s (180, 181, 182), two a c e t y l i n i c compounds (172,
183), a flavone (175), and v o l a t i l e c o n s t i t u e n t s (184) have a l s o
been reported from Helianthus s p e c i e s . A p r e l i m i n a r y report
i n d i c a t e s that b r i t t l e brush contains a g l a n d u l a r trichome
sesquiterpene that i s a feeding d e t e r r e n t to moth l a r v a e (185).
The sesquiterpene l a c t o n e s i n Artemesia species have been
reviewed (186). H i s t o c h e m i c a l t e s t s have shown t h a t A. nova has,
i n a d d i t i o n t o nonglandular trichomes, g l a n d u l a r trichomes
c o v e r i n g 21-35% o f i t s l e a f surface (187). These glands hold a
c l e a r f l u i d which contains some of the monoterpenes and a l l of
the sesquiterpene lactones present i n the leaves.
Components of Glandular Trichomes i n Populus, Prunus,

Newcastelia, and S a l v i a . I n Populus d e l t o i d e s , the marginal
t e e t h of the f i r s t leaves t o emerge are covered w i t h
non-glandular trichomes. I n successive leaves, the t e e t h have
glands that s e c r e t e a r e s i n as the lamina u n r o l l s . E x t r a f l o r a l
n e c t a r i e s occur proximal t o each g l a n d u l a r t i p . F i e l d
observations and a l a b o r a t o r y feeding experiment i n d i c a t e that
the r e s i n a c t s as an i n s e c t r e p e l l a n t ( 9 ) . I n V. d e l t o i d e s ,
i n s e c t f e e d i n g was g r e a t e r on newly expanded leaves i n which the
r e s i n had d r i e d than on buds covered w i t h f r e s h l i q u i d r e s i n
(188). Larvae o f the cottonwood l e a f b e e t l e , when given leaves
of d e l t o i d e s i n which only the margin was r e s i n o u s , f e d
normally, pupated, and emerged as normal a d u l t s . Larvae given
resin-covered leaves d i d not eat them. The few l a r v a e that
pupated f a i l e d t o complete t h e i r l i f e c y c l e . Crude e x t r a c t s o f
poplar (Populus) t r e e leaves e x h i b i t e d a n t i b a c t e r i a l and
a n t i f u n g a l a c t i v i t y (189); r e s p o n s i b l e agents have not been
identified.
The l e a f t e e t h i n Prunus are covered by g l a n d u l a r trichomes.

A coumarin g l u c o s i d e , tomenin, has been i s o l a t e d from Prunus

tomentosa, but i t s morphological o r i g i n i s not i n d i c a t e d (190).
F i v e d i f f e r e n t types of terpenoid s e c r e t i n g trichomes have
been described i n the Western A u s t r a l i a n shrub Newcastelia
v i s c i d a (191)• A r e s i n i s r e l e a s e d beneath the c u t i c l e of the
g l a n d u l a r h a i r , which expands and e v e n t u a l l y breaks, r e l e a s i n g
the r e s i n . Once terpenoid production has ceased, the gland i s
c l o s e d o f f by l e a f c u t i n i z a t i o n of the w a l l s . As the gland
matures, a drop of r e s i n forms and runs down onto the a d a x i a l
l e a f s u r f a c e . A f t e r r u p t u r e , the glands apparently become
f u n c t i o n l e s s but new glands are formed during l e a f expansion, and
r e s i n i s continuously produced u n t i l the l e a f i s f u l l y expanded.
Terpenes i d e n t i f i e d i n N. v i s c i d a i n c l u d e the t r i t e r p e n o i c a c i d s ,
o l e a n o l i c and b e t u l i c a c i d s (192), and the t r i c y c l i c d i t e r p e n e ,
isoprimara-9(11),15-diene-3,19-diol (193)
A quick d i p of th
e x t r a c t the g l a n d u l a r trichom
(194). The main component was the t r i t e r p e n o l , a-amyrin. Small
amounts of flavones and f l a v o n o l s were a l s o i s o l a t e d .
Leaf Exudates from Didymocarpus, L a r r e a , Hymenaea, and

B e y e r i a . Didymocarpus p e d i c e l l a t a i s a s m a l l herbaceous p l a n t
found i n the western Himalayas. I t produces a r e d d i s h , dusty
l e a f exudate from which 7-hydroxy-5,6,8-trimethoxyflavone (195),
two chalcones, p e d i c i n , and p e d i c e l l i n , and flavanone, and
i s o p e d i c i n , (196) have been i s o l a t e d . Two other chalcones,
pashonone and m e t h y l p e d i c i n , have been i s o l a t e d from the leaves
of I), p e d i c e l l a t a , and the l a t t e r i s reported to be one of the
major components (197). Leaf components are reported to be t o x i c
to f i s h (198, 199). D i t e r p e n o i d a c i d s have been i s o l a t e d from
the leaves of D. oblonga (200, 201).
L a r r e a t r i d e n t a t a and L. d i v a r i c a t a are a r i d and semiarid
p l a n t s found i n North and South America. L a r r e a produces a l e a f
r e s i n that accounts f o r 10-15% of the dry weight of the leaves.
The r e s i n i s composed of about h a l f n o r d i h y d r o q u a i a r e t i c a c i d ,
which i s one of the most powerful a n t i o x i d a n t s known (202). The
other h a l f i s p r i m a r i l y composed of f l a v o n o i d s (202, 203).
V o l a t i l e c o n s t i t u e n t s of L a r r e a have been reported (204) and
t h e i r a n t i h e r b i v o r e chemistry reviewed (205).
The t r o p i c a l legume, Hymenaea c o u r b a r i l , produces a l e a f
r e s i n t h a t was t e s t e d as a defense a g a i n s t the g e n e r a l i s t
h e r b i v o r e , beet armyworm (Spodoptera exigua) (206). Larvae
showed a dose-response i n the decrease of pupal weight and delay
i n pupation. In a p a l a t a b i l i t y t e s t , S^. exigua s t r o n g l y
p r e f e r r e d untreated to t r e a t e d bean l e a f d i s k s . The primary
components i n l e a f r e s i n were found to be the sesquiterpene
hydrocarbons, c a r y o p h y l l e n e , a - and $-selinene, and 3-copaene
(207).
The leaves of Beyeria b r e v i f o l i a , from western A u s t r a l i a ,
have a hard r e s i n c o a t i n g . D i t e r p e n o l s and d i t e r p e n o i c a c i d s

have been i d e n t i f i e d i n t h i s r e s i n (208). Other d i t e r p e n o i d s

have been i s o l a t e d from IS. c a l y c i n a (209, 210).
Cotton Pigment Glands. P l a n t s belonging t o the genera

Gossypium ( c o t t o n ) , C i e n f u e g o s i a , Thespesia, and Kokia c o n t a i n
subepidermal pigment glands from which the phenol, gossypol, has
been i s o l a t e d (211). S t r u c t u r e e l u c i d a t i o n s t u d i e s , chemical
r e a c t i v i t y (212, 213, 214), and b i o s y n t h e s i s (215) o f gossypol
have been reviewed. Because gossypol i s present i n raw
cottonseed meal, i t s t o x i c i t y , e s p e c i a l l y t o monogastric animals,
has been c a r e f u l l y s t u d i e d . The t o x i c o l o g y , p h y s i o l o g i c a l
e f f e c t s and metabolism have r e c e n t l y been reviewed (216, 217).
The Chinese r e p o r t s that gossypol may a c t as an a n t i f e r t i l i t y
agent i n men (218) have renewed i n t e r e s t i n t h i s and r e l a t e d
compounds.
Gossypol has been
Thespesia populnea ([a
and (±) form from cottonseed (219, 220, 221). Glands i n the
f o l i a r p a r t s of £. hirsutum produce, i n a d d i t i o n t o gossypol, the
t e r p e n o i d s , j>-hemigossypolone (222), h e l i o c i d e s H^, (223), H 2
(224), H (225), and H (223). I n a d d i t i o n t o these

3 4
compounds, barbadense a l s o produces the methyl e t h e r

d e r i v a t i v e s , jp- hemigossypolone methyl ether and the h e l i o c i d e s
B p B (226), B , and B^ (227). G. r a i m o n d i i produces
2 3
raimondal (228). The S c h i f f base, g o s s y r u b i l o n e , has been

detected i n the glands o f £. hirsutum (226). Anthocyanins (229,
230) and flavone (230) have been reported i n glands (229) and
c y a n i d i n - 3 - g l u c o s i d e has been i s o l a t e d from £. barbadense glands
(231). Hemigossypol, hemigossypol-6-methyl e t h e r ,
1
6-deoxyhemigossypol (232), gossypol-6-methyl and 6,6 -dimethyl
ether (233), desoxyhemigossypol and desoxyhemigossypol-6-methyl
ether (234) have been i s o l a t e d from glands i n r o o t s , stems, or
seed.
Several o f these compounds have e x h i b i t e d a n t i b i o t i c
a c t i v i t y . Z a k i , e t a l . found that hemigossypol and
desoxyhemigossypol were more a c t i v e than gossypol a g a i n s t the
fungus, V e r t i c i l l i u m d a h l i a e , (235). Hemigossypolone was a l s o
a c t i v e against V. d a h l i a e (236). Terpenoids were found t o be
exuded i n t o the r h i z o s p h e r e by c o t t o n r o o t s (237) and may be
r e s p o n s i b l e f o r r e s i s t a n c e t o root r o t (238). Gossypol has been
shown t o have a n t i b a c t e r i a l (239), a n t i v i r a l (240, 241, 242) and
antitumor (243) a c t i v i t i e s . Apogossypol, which has a lower
mammalian t o x i c i t y , r e t a i n s t h i s a n t i v i r a l a c t i v i t y (244).
The chemical c o n s t i t u e n t s i n the cotton p l a n t have been
e x t e n s i v e l y s t u d i e d . V o l a t i l e o r steam d i s t i l l a b l e compounds
present i n cotton buds (245-253) and leaves (254, 255, 256) have
been e x t e n s i v e l y cataloged. A number of the surface l i p i d s have
been i d e n t i f i e d (257). Two anthocyanins, cyanidin-3-3-glucoside
(231, 258) and p e l a r g o n i d i n (259), and the f l a v o n o l s and f l a v o n o l
g l y c o s i d e s , q u e r c e t i n , kaempferol, i s o q u e r c i t r i n , q u e r c e t i n - 7 -

f
g l y c o s i d e , and q u e r c e t i n - 3 - g l y c o s i d e (260) have a l s o been
i s o l a t e d . I s o q u e r c i t r i n , q u e r c i t r i n , and q u e r c e t i n are t o x i c and
i n h i b i t growth of the bollworm, H. zea, tobacco budworm, H.
v i r e s c e n s , and the pink bollworm, Pectinophora g o s s y p i e l l a , i n
l a b o r a t o r y t e s t s on a r t i f i c i a l d i e t s (261). These compounds were
found to be more t o x i c to the tobacco budworm than to the
bollworm. Some of these compounds, such as cyanidin-3-3-
g l u c o s i d e and the terpene e s s e n t i a l o i l s , are undoubtedly i n , but
not n e c e s s a r i l y confined t o , the pigment glands. Others may be
l o c a t e d i n g l a n d u l a r trichomes.
V a r i a t i o n s i n the c o n c e n t r a t i o n of gossypol and other
c o n s t i t u e n t s , such as tannins and f l a v o n o i d s , have been measured
w i t h respect to such v a r i a b l e s as c u l t i v a r (244, 262, 263), p l a n t
age (264), and plant part (265). In each study, i n s e c t growth
was n e g a t i v e l y c o r r e l a t e d w i t h gossypol or terpenoid aldehyde
content. Hanny, et a l
damage c o r r e l a t i o n s of
t e r m i n a l l e a f , and l e a f t e r p e n o i d s , r e s p e c t i v e l y (262). Yellow
c o t t o n anthers were found to have a higher gossypol content than
cream-colored anthers. This i s b e l i e v e d to be r e s p o n s i b l e f o r
suppressing H e l i o t h i s v i r e s c e n s l a r v a e growth (265).
Cook was among the f i r s t to propose that the pigment glands
of cotton might act as a r e p e l l a n t to the bollwoim (266).
Lukefahr and Houghtaling found that c u l t i v a r s of c o t t o n w i t h a
flowerbud gossypol content of 1.7% s i g n i f i c a n t l y reduced the
populations of tobacco budworm i n l a r g e , r e p l i c a t e d f i e l d t e s t s
(267). I t a l s o was found that t h i s experimental Upland c o t t o n
was u t i l i z e d l e s s e f f i c i e n t l y by the bollworm than a standard
l i n e c o n t a i n i n g 0.5% gossypol (268). Food consumption by the
tobacco budworm l a r v a e decreased w i t h i n c r e a s i n g gossypol
content. Seaman, et a l . observed a s t r o n g c o r r e l a t i v e
c o e f f i c i e n t between a p l a n t ' s r e s i s t a n c e l e v e l and the
c o n c e n t r a t i o n of gossypol and the h e l i o c i d e s (269). This same
study r e p o r t s that i n l i n e s d i f f e r i n g g r e a t l y i n r e s i s t a n c e ,
hemigossypolone and the four h e l i o c i d e s occur i n roughly the same
p r o p o r t i o n i n buds, w i t h gossypol and the h e l i o c i d e s each
comprising about 44% of the terpenoids. This i s i n c o n t r a s t t o a
subsequent study which reported gossypol as the primary terpenoid
(270) i n buds. These d i f f e r e n c e s may be accounted f o r by the age
of the t i s s u e examined. Seaman a l s o reported that the
c o n c e n t r a t i o n of each terpenoid aldehyde i n r e s i s t a n t i n d i v i d u a l
p l a n t s i s approximately twice the amount found i n s u s c e p t i b l e
p l a n t s (269).
The t o x i c i t y of the cotton terpenoid aldehydes to d i f f e r e n t
l a r v a e has been reported by s e v e r a l groups (227, 259, 270-275).
These r e s u l t s are summarized i n Table 2. Although there i s
v a r i a t i o n i n the t e s t r e s u l t s among the d i f f e r e n t l a b o r a t o r i e s ,
i t i s obvious that gossypol and probably the other terpenoid
aldehydes have a wide range of t o x i c i t y to many moth l a r v a e .
Waiss, et a l . report that the f i r s t i n s t a r l a r v a e of

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H e l i o t h i s zea g e n e r a l l y avoided consuming the pigment glands

(276) i n d i c a t i n g t h a t gossypol may act as a feeding d e t e r r e n t .
Gossypol apparently acts as a f e e d i n g d e t e r r e n t to Spodoptera
l i t t o r a l i s , Egyptian c o t t o n leafworm. When polystyrene l a m e l l a e
were painted on one s i d e w i t h sucrose and on the other s i d e w i t h
gossypol, the 1% gossypol l a m e l l a e s t r o n g l y suppressed feeding
over that of a c o n t r o l (277). The feeding r a t e was n e g a t i v e l y
c o r r e l a t e d w i t h the gossypol c o n c e n t r a t i o n . Larvae f e d about
h a l f as much on lamellae painted w i t h a l e a f e x t r a c t of a h i g h -
gossypol c o t t o n as oh l a m e l l a e painted w i t h an e x t r a c t of a
glandless cotton.
Larvae (90-110 and 170-190 mg) of £. l i t t o r a l i s , r a i s e d on
cotton p l a n t s w i t h a mean content of 1.23% gossypol i n dry l e a f
powder, weighed l e s s , had delayed pupation, and a lower pupal
weight compared to l a r v a e r a i s e d on a c o t t o n having an
intermediate gossypol conten
even more pronounced.
d i e t s c o n t a i n i n g 0.5% gossypol acetate throughout t h e i r l i f e
span, l a r v a l m o r t a l i t y was n e a r l y 70% a f t e r the f i r s t 10 days and
only 0.3% of the l a r v a e pupated. At a c o n c e n t r a t i o n of 0.25%
gossypol a c e t a t e , 44% of the l a r v a e d i e d w i t h i n 10 days and 26%
of the l a r v a e pupated (279).
Studies on the spiny bollworm, E a r i a s i n s u l a n a , have given
s i m i l a r r e s u l t s (273). S u r v i v a l and average weights of l a r v a e
r a i s e d on d i e t s c o n t a i n i n g gossypol were lower than the c o n t r o l .
The same was t r u e w i t h regard t o percent pupation and a d u l t
emergence. Seventy percent of l a r v a e r a i s e d on a cotton c u l t i v a r
w i t h a gossypol content of 0.10% i n the b o l l s pupated, w h i l e only
9% of those r a i s e d on a c u l t i v a r w i t h 2.34% gossypol i n the b o l l s
pupated.
The e f f e c t of gossypol on three consecutive generations of
l i t t o r a l i s has been s t u d i e d by El-Sebae, et a l . (280).
Larvae of S_* l i t t o r a l i s were t r e a t e d t o p i c a l l y w i t h d i f f e r e n t
concentrations of gossypol. A f t e r emergence, the moths were
t r e a t e d t o p i c a l l y at the same concentrations of gossypol and
p a i r e d . The number of eggs and percent h a t c h a b i l i t y were
c a l c u l a t e d . F i r s t i n s t a r l a r v a e were reared on an a r t i f i c i a l
d i e t c o n t a i n i n g d i f f e r e n t gossypol c o n c e n t r a t i o n s . The
treatments were repeated i n the next generation. At the lowest
c o n c e n t r a t i o n of gossypol i n the d i e t (0.25%), the number of eggs
l a i d decreased to about 52% of the c o n t r o l i n the f i r s t
generation. In the second and t h i r d generations, the number of
eggs l a i d decreased to about 10% and 2%, r e s p e c t i v e l y , of the
c o n t r o l . H a t c h a b i l i t y dropped i n the f i r s t , second, and t h i r d
generations to 50%, 43%, and 19%, r e s p e c t i v e l y , of the c o n t r o l .
At a c o n c e n t r a t i o n of 0.50% gossypol i n the d i e t , the e f f e c t was
even more dramatic, w i t h the h a t c h a b i l i t y dropping below 1% i n
the t h i r d generation.
Meisner, et a l . reported i n h i b i t i o n of growth and protease
and amylase a c t i v i t y i n S. l i t t o r a l i s l a r v a e a f t e r f e e d i n g on

high gossypol c u l t i v a r s of cotton (281). El-Sebae, e t a l . a l s o

s t u d i e d the midguts of £. l i t t o r a l i s (280). They found that
gossypol: 1) i n h i b i t e d protease a c t i v i t y and l i p i d p e r o x i d a t i o n ,
2) increased microsomal N-demethylation a c t i v i t y , 3) s t i m u l a t e d
m i t o c h o n d r i a l ATPase a c t i v i t y a t low concentrations ( 10 PM), and
4) i n h i b i t e d ATPase a c t i v i t y a t higher c o n c e n t r a t i o n s . The
i n h i b i t i o n of protease and amylase a c t i v i t y and l i p i d
f
p e r o x i d a t i o n agree w i t h g o s s y p o l s a b i l i t y t o r e t a r d growth by
reducing p r o t e i n b i o s y n t h e s i s . The i n h i b i t i o n of ATPase, which
provides energy f o r b i o s y n t h e s i s , i s a l s o compatible w i t h the
observed r e s u l t s . The i m p l i c a t i o n s of increased microsomal
N-demethylation a c t i v i t y i s discussed below.
Larvae of £. l i t t o r a l i s t r e a t e d t o p i c a l l y w i t h gossypol (100
u g/g body weight) 24 hours before being t r e a t e d w i t h v a r i o u s
i n s e c t i c i d e s had a highe L D C Q tha c o n t r o l i n s e c t (272)
The i n c r e a s e i n L D ^ Q wa
i n s e c t i c i d e , e n d r i n (200%), y organophosphat
i n s e c t i c i d e , Cyolane (154%), the phosphorothioate i n s e c t i c i d e
leptophos (123%), and the carbamate i n s e c t i c i d e , Zectran (87%).
IS. l i t t o r a l i s l a r v a e had a lower m o r t a l i t y r a t e when allowed
to feed on leaves t r e a t e d w i t h the i n s e c t i c i d e , phosfolan, from a
c o t t o n c u l t i v a r w i t h a high gossypol content as compared t o a
c u l t i v a r w i t h l i t t l e o r no gossypol (282). The a b i l i t y o f S».
l i t t o r a l i s larvae to rapidly detoxify insecticides after
treatment w i t h gossypol, agrees w i t h the r e s u l t s o f El-Sebae
(280) that microsomal N-demethylation reaches i t s maximum
a c t i v i t y a f t e r 24 hours. An i n c r e a s e i n microsomal oxidases has
a l s o been observed i n the l i v e r of r a t s which were f e d gossypol
(283).
As opposed t o the r e s u l t s i n d i c a t e d above, the e f f e c t of
gossypol on the b o l l w e e v i l i s q u i t e d i f f e r e n t . Gossypol i s a
f e e d i n g s t i m u l a n t t o the b o l l w e e v i l (260). B o l l weevils feeding
on an a r t i f i c i a l d i e t were h e a l t h i e r and had improved egg hatch
when the gossypol f r a c t i o n from c o t t o n seed was used as the
p r i n c i p l e p r o t e i n source.
Insect Resistance t o Secondary P l a n t A l l e l o c h e m i c s
The i n t r o d u c t i o n o f s y n t h e t i c p e s t i c i d e s heralded a new e r a

i n which a g r i c u l t u r a l production f l o u r i s h e d . However, the use of
p e s t i c i d e s introduced unexpected problems. I n the case o f
i n s e c t i c i d e s , b e n e f i c i a l as w e l l as harmful i n s e c t s were k i l l e d .
Insects r a p i d l y developed r e s i s t a n c e t o the i n s e c t i c i d e s ,
r e q u i r i n g the i n t r o d u c t i o n of new and more potent chemicals.
This same s c e n a r i o has not been observed w i t h p l a n t
a l l e l o c h e m i c s . Phytophagous i n s e c t s and p l a n t s have c o e x i s t e d
f o r eons. I n g e n e r a l , p l a n t s , through t h e i r a l l e l o c h e m i c s have
exacted t h e i r t o l l on i n s e c t s , w h i l e f a l l i n g prey t o these same
i n s e c t s . The reasons f o r these d i f f e r e n c e s deserve comment.
Companies manufacturing p e s t i c i d e s , i n a d d i t i o n t o
c o n s i d e r a t i o n s such as cost and s a f e t y , s e l e c t only those

chemicals that k i l l the m a j o r i t y of the t a r g e t organisms w i t h

which they come i n contact. Indeed, the a g r i c u l t u r a l producer
demands such products. Even f e d e r a l l i c e n s i n g agencies might
balk at p e r m i t t i n g the s a l e of a p e s t i c i d e that was only
m a r g i n a l l y e f f e c t i v e . Yet t h i s i s the path p l a n t s commonly
f o l l o w . Experience has shown that given adequate pressure,
n e a r l y every species of i n s e c t i s capable of developing some
t o l e r a n c e to a p a r t i c u l a r i n s e c t i c i d e (285). The development of
r e s i s t a n c e depends on the presence of r e s i s t a n c e genes and
adequate s e l e c t i o n pressure by which these genes are concentrated
and i n t e g r a t e d i n t o the genome of the p o p u l a t i o n . I f the genetic
p o t e n t i a l f o r r e s i s t a n c e i s present, the r a t e of t h i s development
w i l l depend on f a c t o r s such as the frequency of the r e s i s t a n c e
genes, t h e i r dominance, the s e l e c t i o n pressure and previous
exposure to the i n s e c t i c i d e . Assuming a s u f f i c i e n t s u r v i v a l
r a t e , r e s i s t a n c e w i l l develo
s e l e c t i o n pressure (285)
With p l a n t a l l e l o c h e m i c s , the s e l e c t i o n pressure i s
g e n e r a l l y not h i g h enough to concentrate the r e s i s t a n c e genes
i n the genome of the p o p u l a t i o n . The p l a n t a l l e l o c h e m i c s slow
i n s e c t growth and extend the time of pupation. The l a r v a e thus
become more s u s c e p t i b l e to disease, predators and environmental
s t r e s s . However, some i n s e c t s w i t h s u s c e p t i b l e genes do s u r v i v e
and t h e i r genes are c o n t i n u a l l y i n c l u d e d i n the p o o l .
P l a n t s that produce " s p e c i f i c " t o x i n s may be plagued by
i n s e c t s that develop a t o l e r a n c e to these t o x i n s i n much the same
way as i n s e c t s develop t o l e r a n c e to s y n t h e t i c i n s e c t i c i d e s . Two
examples from t h i s chapter are the tobacco hornworm and the b o l l
w e e v i l which have developed a h i g h t o l e r a n c e to n i c o t i n e and
gossypol, r e s p e c t i v e l y . Some occurrence i n the d i s t a n t past may
have placed s u f f i c i e n t l y high s e l e c t i o n pressure on these
i n s e c t s that they developed t o l e r a n c e to these compounds.
A l t e r n a t i v e l y , the same e f f e c t could have occurred by a low
s e l e c t i o n pressure a p p l i e d over a very long p e r i o d time. Other
p l a n t s p r o t e c t themselves by employing "general" t o x i n s .
S e l e c t i o n pressure from t h i s type of t o x i n would be even l e s s .
Thus p l a n t s have evolved which produce chemicals which are
only m a r g i n a l l y t o x i c to i n s e c t s . Production of chemicals that
are extremely t o x i c to i n s e c t s would g i v e a p l a n t only a
temporary adaptive advantage.
Host-Plant Resistance and the P e s t i c i d e Industry
The recent advances i n i d e n t i f y i n g and u t i l i z i n g

a l l e l o c h e m i c s i n v o l v e d i n h o s t - p l a n t r e s i s t a n c e has drawn the
a t t e n t i o n of the p e s t i c i d e i n d u s t r y . A p o t e n t i a l problem that
may not be recognized, i s the e f f e c t on i n s e c t s i f analogs of
p l a n t p r o t e c t i v e chemicals are sprayed on a g r i c u l t u r a l crops.
Insects t r e a t e d w i t h such analogs, could r a p i d l y become t o l e r a n t
not only to the analog, but a l s o to the n a t u r a l a l l e l o c h e m i c .

Insects t r e a t e d w i t h such analogs, could r a p i d l y become t o l e r a n t

not only t o the analog, but a l s o t o the n a t u r a l a l l e l o c h e m i c .
There are examples o f c r o s s - r e s i s t a n c e i n which an i n s e c t t r e a t e d
w i t h one chemical was found t o have r e s i s t a n c e t o a chemical w i t h
which i t had never been t r e a t e d . A s t r a i n o f house-fly which had
developed r e s i s t a n c e t o the s y n t h e t i c i n s e c t i c i d e , DDT, was found
to be r e s i s t a n t t o the n a t u r a l p y r e t h r i n i n s e c t i c i d e s (286)•
These two i n s e c t i c i d e s are c h e m i c a l l y very d i f f e r e n t , and y e t
they may react a t the same s i t e inducing c r o s s - r e s i s t a n c e .
In c o t t o n , f o r example, i t has been observed that some
i n s e c t s that are not normally cotton pests, become a pest on
g l a n d l e s s cottons which do not c o n t a i n gossypol. Such i n s e c t s ,
which are minor pests because o f t h e i r avoidance o f gossypol, i f
sprayed continuously w i t h the proper i n s e c t i c i d e , could develop,
through c r o s s - r e s i s t a n c e a t o l e r a n c e f o r gossypol and appear as
a new major pest on t h i
developing such r e s i s t a n c
i n s e c t i c i d e s that are q u i t e d i s t i n c t from the p l a n t ' s a l l e l o -
chemic. The p o t e n t i a l becomes i n f i n i t e l y greater i f the a l l e l o -
chemic and s y n t h e t i c i n s e c t i c i d e a r e chemically s i m i l a r . Some
may b e l i e v e that an i n s e c t cannot develop a t o l e r a n c e f o n a t u r a l
i n s e c t i c i d e s . On the c o n t r a r y , the Mexican bean b e e t l e has
developed r e s i s t a n c e t o the n a t u r a l r o t e n o i d i n s e c t i c i d e s (287)•
There are already s c a t t e r e d r e p o r t s o f H e l i o t h i s developing
r e s i s t a n c e t o the p y r e t h r o i d s a f t e r use o f only a few years.
With the one exception o f s e l e c t i o n pressure, a l l other
p r e r e q u i s i t e s f o r the development o f r e s i s t a n c e are present i n
the p l a n t - i n s e c t r e l a t i o n s h i p . Therefore, c a r e f u l s t u d i e s are
needed before such chemicals are introduced as a c o n t r o l
measure.
Acknowledgement s
I thank Jan Cornish, T e r r i Mutchler, Susan C h i l e s , and

Glenda Ward f o r t h e i r a s s i s t a n c e i n the p r e p a r a t i o n o f t h i s
manuscript and Howard W i l l i a m s f o r h e l p f u l d i s c u s s i o n s .
Literature Cited
1. Luckner, M. in "Secondary P l a n t Products"; Bell, E. A.;

Charlwood, B. V., Eds.; Springer-Verlag: New York, 1980;
p. 24.
2. Frey-Wyssling, A. Ber. Schweiz. Bot. Ges. 1933, 42,
109.
3. Rhyne, C. L. Advan. Front. P l a n t Sci. 1965,13,121.
4. Dahlgren, K. V. O. Impatiens, Sv. Bot. T i d s k r . 1940, 34,
53.
5. Dahlgren, K. V. O. Impatiens. Ark. Bot. 1945, 32, 53.
6. S t a h l , E. Flora 1920, 113, 1.

7. B e n t l e y , B. L. in "Annual Review of Ecology and

S t y s t e m a t i c s " , V o l . 8; Johnston, R. F., Ed.; Annual Reviews
Inc.: Palo Alto, 1977, p. 407.
8. Elias, T. S.; Newcombe, L. F. A c t a Bot S i n 1979, 21, 215.
9. C u r t i s , J. D.; L e r s t e n , N. R. Amer. J. Bot. 1974, 61,
835.
10. L e v i n , D. A. Quart. Rev. Biol. 1973, 48, 3.
11. S c h i l l i n g e r , J. A.; G a l l u n , R. L. Ann. Entomol. Soc. Am.
1968, 61, 900.
12. Everson, E. H.; Ringlund, K. Crop Sci. 1968, 8, 705.
13. S t a r k s , K. J.; Merkle, O. G. J. Econ. Entomol. 1977, 70,
305.
14. Kamel, S. A.; E l k a s s a b y , F. Y. J. Econ. Entomol. 1965, 58,
209.
15. Benedict, J. H.; L e i g h T F.; Hyer A N E n v i r Entomol
in press.
16. Lukefahr, M. J.;
Houghtaling, J. E. J. Econ. Entomol. 1968, 61, 661.
17. Poos, F. W.; Smith, F. F. J. Econ. Entomol. 1931, 24,
361.
18. McKinney, K. B. J. Econ. Entomol. 1938, 31, 630.
19. Fluiter, H. J. de; Ankersmit, G. W. T i j d s c h r .
P l a n t e n z i e k t e n 1948, 54, 1.
20. Johnson, B. Bull. Entomol. Res. 1953, 44, 779.
21. P i l l e m e r , E. A.; Tingey, W. M. Entomol. Exp. A p p l . 1978,
24, 83.
22. P i l l e m e r , E. A.; Tingey, W. M. Science 1976, 193, 482.
23. Bernard, R. L.; Singh, B. B. Crop S c i . 1969, 9, 192.
24. Singh, B. B.; Hadley, H. H.; Bernard, R. L. Crop Sci. 1971,
11, 13.
25. Wolfenburger, D. A.; Sleesman, J. P. J. Econ. Entomol.
1963, 56, 895.
26. Robbins, J. C.; Daugherty, D. M. Proc. North Cent. Branch
Entomol. Soc. Am. 1969, 24, 35.
27. Broersma, D. B.; Bernard, R. L.; Luckman, W. H. J. Econ.
Entomol. 1972, 65, 78.
28. Turnipseed, S. G.; S u l l i v a n , M. J. in "World Soybean
Research"; Hill, L. D., Ed; I n t e r s t a t e P r i n t e r s : D o n v i l l e ,
1976, p. 549.
29. Turnipseed, S. G. E n v i r o n . Entomol. 1977, 6, 815.
30. Beckman, C. H.; M u e l l e r , W. C.; McHardy, W. E. P h y s i o l .
Plant P a t h o l . 1972, 2, 69.
31. C h a r r i e r e - L a d r e i x , Y. Proc. FEBS Meetings 1979, 55, 101.
32. Luckner, M.; Diettrich, B.; L e r b s , W. in "Progress in
Phytochemistry", V o l . 6; Reinhold, L.; Harborne, J. B.;
Swain, T., Eds.; Pergamon Press: New York, 1979, p. 103.
33. Gibson, R. W. Ann. A p p l . Biol. 1971, 68, 113.
34. Tingey, W. M.; Gibson, R. W. J. Econ. Entomol. 1978, 71,
856.
35. G e n t i l e , A. G.; Stoner, A. K. J. Econ. Entomol. 1968, 61,
1152.

36. Ohnesorge, B.; Sharaf, N.; Allawi, T. Z. Angerv. Entomol.

1980, 90, 226.
37. Shade, R. E.; Thompson, T. E.; Campbell, W. R. J. Econ.
Entomol. 1975, 68, 399.
38. K r e i t n e r , G. L.; Sorenson, E. L. Crop Sci. 1979, 19,
380.
39. Sutherst, R. W.; Jones, R. J.; S c h n i t z e r l i n g , H. G. Nature
1982, 295, 320.
40. E l s e y , K. D.; C h a p l i n , J. F. J. Econ. Entomol. 1978, 71,
723.
41. Rabb, R. L.; Bradley, J. R. J. Econ. Entomol. 1968, 61,
1249.
42. L e v i n , D. A. in "Annual Review o f Ecology and S y s t e m a t i c s " ,
V o l . 7; Johnston, R. F., Ed.; Annual Reviews Inc.: Palo
Alto, 1976, p. 121.
43. N o r r i s , D. M. AC
44. Schoonhoven, L. M
Phytochemistry", V o l . 5; Runeckles, V. C.; Tso, T. C.,
Eds.; Academic Press: New York, 1972, p. 197.
45. Hedin, P. A.; Maxwell, F. G.; J e n k i n s , J. N. i n
"Proceedings o f the Summer Institute on Biological C o n t r o l
of P l a n t Insects and Diseases"; Maxwell, F. G.; Harris, F.
A. Eds.; U n i v e r s i t y Press o f Mississippi: Jackson, 1974,
p. 494.
46. Vibhute, Y. B.; Wadje, S. S. Indian J. Exp. Biol. 1976, 14,
739.
47. H u f f o r d , C. D.; L a s s w e l l , W. L., Jr. L l o y d i a 1978, 41,
156.
48. Wacker, A.; E i l m e s , H. G. Arzneim-Forsch 1978, 28, 347.
49. M i t s c h e r , L. A.; Park, Y. H.; Omoto, S.; C l a r k , G. W., III;
C l a r k , D. H e t e r o c y c l e s 1978, 9, 1533.
50. Elliger, C. A.; Chan, B. C.; Waiss, A. C., Jr.
Naturwissenschaften 1980, 67, 358.
51. Matsuda, K. Tohuko J. A g r i c . Res. 1976, 27, 115.
52. N i e l s e n , J. K. Entomol. Exp. A p p l . 1978, 24, 562.
53. Matsudo, K. A p p l . Entomol. Z o o l . 1978, 13, 228.
54. Kimmich, G. A.; Randles, J. Membr. Biochem. 1978, 1,
221.
55. Wollenweber, E.; Dietz, V. H. Phytochem. 1981, 20, 869.
56. Rodriguez, E.; Towers, G. H. N.; Mitchell, J. C. Phytochem.
1976, 15, 1573.
57. B u r n e t t , W. C., Jr., Jones, S. B., Jr.; Mabry, T. J. in
"Biochemical Aspects o f P l a n t and Animal Coevolution",
Harborne, J. B., Ed.; Academic Press: New York, 1978, p.
233.
58. I v i e , G. W.; W i t z e l , D. A. "Sesquiterpene Lactones:
S t r u c t u r e , Biological A c t i o n , and T o x i c o l o g i c a l
S i g n i f i c a n c e " , in press.
59. Kubo, I . ; Lee, Y.; Pettei, M.; Nakanishi, K.; Pilkiewicz,
F. J. Chem. Soc., Chem. Commun. 1976, 1013.

60. Doss, R.; L u t h i , R.; H r u t f i o r d , B. F. Phytochem. 1980, 19,

2379.
61. Kubo, I . ; Nakanishi, K. Adv. P e s t i c . Sci., Plenary L e c t .
Symp. Pap. I n t . Congr. P e s t i c . Chem., 4th 1979, 2, 284.
62. Kubo, I . in "Konchu no Seiri to Kagaku";Hidaka, T.;
Takahashi, S.; Isoe, Y., Eds.; K i t a m i Shobo: Tokyo, 1979,
p. 153.
63. A l f a r o , R. I . ; P i e r c e , H. D., Jr.; Borden, J. H.;
Oehtschlager, A. C. Can. J . Z o o l . 1980, 58, 626.
64. Nakajima, S.; Kawazu, K. Heterocycles 1978, 10, 117.
65. Slama, K. Acta Entomol. Bohemoslov. 1978, 75, 65.
66. Makarenko, N. G.; Schmidt, E. N.; Raldugin, V. O.;
Dubovenko, Zh. V. M i k r o b i o l . Zh. (Kiev) 1980, 42, 98.
67. Andrews, R. E.; Parks, L. W.; Spence, K. D. A p p l . E n v i r o n .
M i c r o b i o l . 1980, 40, 301.
68. Agarwal, I . ; Mathela
14, 19.
69. M o r r i s , J. A.; K h e t t r y , A.; S e i t z , E. W. J . Am. Oil Chem.
Soc. 1979, 56, 595.
70. McEnro, F. J . ; F e n i c a l , W. Tetrahedron 1978, 34, 1661.
71. Szabentai, M.; V e r z a r - P e t r i , G.; F l o r i a n , E. Parfuem.
Kosmet. 1977, 58, 121.
72. Schmeltz, I . in " N a t u r a l l y Occurring I n s e c t i c i d e s " ;
Jacobson, M.; Crosby, D. G., Eds.; Dekker P u b l i s h i n g : New
York, 1971, p. 99.
73. Thurston, R.; Smith, W. T.; Cooper, B. P. Entomol. Exp.
A p p l . 1966, 9, 428.
74. Buhr, H.; T o b a l l , R.; S c h r e i b e r , K. Entomol. Exp. A p p l .
1958, 1, 209.
75. Harley, K. L. S.; Thorsteinson, A. J . Canad. J. Zoo. 1967,
45, 305.
76. Chakraborty, M. K.; Weybrew, J. A. Tob. S c i . 1963, 7,
122.
77. Thurston, R. J . Econ. Entomol. 1970, 63, 272.
78. P a t t e r s o n , C. G.; Thurston, R.; Rodriguez, J . G. J . Econ.
Entomol. 1974, 67, 341.
79. Yang, R. S. H.; G u t h r i e , F. E. Ann. Entomol. Soc. Am. 1969,
62, 141.
80. Stürckow, B.; Löw, I . Entomol. Exp. A p p l . 1961, 4, 133.
81. Sinden, S. L.; Schalk, J . M.; Stoner, A. K. J . Am. Soc.
H o r t i c . Sci. 1978, 103, 596.
82. Harley, K. L. S.; Thorsteinson, A. J . Can. J . Z o o l . 1967,
45, 305.
83. A i n a , O. J . ; Rodriguez, J . G.; Knavel, D. E. J . Econ.
Entomol. 1972, 65, 641.
84. S t a e d l e r , E.; Hansen, F. E. Symp. Biol. Hung. 1976, 16,
267.
85. A i n a , O. J.; Rodriguez, J . G.; Knavel, D. E. J . Econ.
Entomol. 1972, 65, 641.

86. Duffey, S. S.; Isman, M. B. E x p e r i e n t i a 1981, 37, 574.

87. W i l l i a m s , W. G.; Kennedy, G. G.; Yamamoto, J. D.; Thacker,
J. D.; Bordner, J. Science 1980, 207, 888.
88. Tingey, W. M.; Mackenzie, J. D.; Gregory, P. Am. Potato J.
1978, 55, 577.
89. C h a r r i e r - L a d r e i x , Y. Physiol. Veg. 1977, 15, 619.
90. Wollenweber, E. Biochem. Syst. E c o l . 1975, 3, 35.
91. Wollenweber, E. Phytochem. 1976, 15, 438.
92. Wollenweber, E. Z. P f l a n z e n p h y s i o l . 1974, 73, 277.
93. Wollenweber, E. Biochem. Sys.Ecol.1975,3,47.
94. B i f t u , T.; Stevenson, R. J. Chem. Soc. P e r k i n 1978, 1,
360.
95. Wollenweber, E. Z. N a t u r f o r s c h . , C 1974, 29, 362.
96. L o t t e r , H.; Wagner, H.; Saleh, A. A.; Cordell, G. A.;
Farnsworth, N. R Z N a t u r f o r s c h . C B i o s c i 1979, 34,
677.
97. Saleh, A. A.; Cordell,
Soc., P e r k i n Trans. 1 1980, 5, 1090.
98. Suga, T.; Iwata, N.; Asakawa, Y. Bull. Chem. Soc. Japan
1972, 45, 2058.
99. Wollenweber, E. Am. Fern J. 1978, 68, 13.
100. N i l s s o n , M. Acta Chem. Scand. 1 9 6 1 , 1 5 , 211.
101. Ramakrishnan, G.; B a n e r j i , A.; Chadha, M. S. Phytochem.
1974, 13, 2317.
104. S t a r , A. E.; Mabry, T. J.; Smith, D. M. Phytochem. 1978,
17, 586.
105. S t a r , A. E.; Mabry, T. J.; Smith, D. M. Phytochem. 1978,
17, 586.
106. Wollenweber, E. Flora 1979, 168, 138.
107. N i l s s o n , M. Acta Chem. Scand. 1961, 15, 154.
108. S t a r , A. E., Mabry, T. J. Phytochem. 1971, 10, 2871.
109. Wollenweber, E.; Dietz, V. H.; Smith, D. M.; S e i g l e r , D. S.
Z. N a t u r f o r s c h . , C 1979, 34, 876.
112. Suga, T.; Iwanatu, N.; Asakawa, Y. Bull. Chem. Soc. Japan
1972, 45, 2058.
115. Popravko, S. A.; Kononenko, G. P.; Tikhomirova, V. I . ;
Wulfson, N. S. Bioorganiches Kaya Khimiya 1979, 5, 1662.
116. Saleh, A. A.; Cordell, G. A.; Farnsworth, N. R. L l o y d i a
1976, 39, 456.
117. Wollenweber, E.; Egger, K. Tetrahedron Lett. 1970, 19,
1601.
118. Wollenweber, E.; Lebreton, P.; Chadenson, M. Z.
N a t u r f o r s c h . , B 1972, 27, 567.
119. Rabesa, Z. A. D o c t o r a l T h e s i s , U. o f Lyon, Lyon, France,
1980.

120. Erdtmann, H.; Novotny, L.; Romanik, M. Tetrahedron, Suppl.

8, P t . I 1966, 22, 71.
121. Rangaswami, S.; I y e r , R. T. I n d i a n J. Chem. 1969, 7, 526.
123. Sunder, R.; Ayengar, K. N. N.; Rangaswami, S. Phytochem.
1974, 13, 1610.
124. S t a r , A. E.; Rösler, H.; Mabry, T. J., Smith, D. M.
Phytochem. 1975, 14, 2275.
125. Wollenweber, E. Ber. Deutsch. Bot. Ges. Bd. 1976, 89,
243.
126. J a y , M.; Favre-Bonvin, J.; Wollenweber, E. Can J. Chem.
1979, 57, 1901.
127. J a y , M.; Wollenweber, E.; Favre-Bonvin, J. Phytochem. 1979,
18, 153.
128. D i e t z , V. H.; Wollenweber E.; Favre-Bonvin J.; Smith D
M. Phytochem. 1981
129. Favre-Bonvin, J.;
Phytochem. 1980, 19, 2043.
130. Wollenweber, E.; D i e t z , V. H.; S c h u l l o , D.; Schilling, G.
Z. N a t u r f o r s c h . , C 1980, 35, 685.
131. N i l s s o n , M. Acta Chem. Scand. 1959, 13, 750.
132. Wollenweber, E.; D i e t z , V. H. Biochem. Syst. Ecol. 1980, 8,
21.
133. Wollenweber, E.; D i e t z , V. H.; M a c N e i l l , C. D.; Schilling,
G. Z. P f l a n z e n p h y s i o l . 1979, 94, 241.
134. Wollenweber, E.; Favre-Bonvin, J.; J a y , M. Z. N a t u r f o r s c h . ,
C 1978, 33, 831.
135. Khosa, R. L.; Wahi, A. K.; Mukherjee, A. K. Curr. Sci.
1978, 47, 624.
136. Gonzalez, A.; Betancor, C.; Hernandez, R.; S a l a z a r , J. A.
Phytochem. 1976, 15, 1996.
137. B o t t a r i , F.; Marshill, A.; Morelli, I . ; P a c c h i a n i , M.
Phytochem. 1972, 11, 2519.
138. S e i g l e r , D. S.; Smith, D. M.; Mabry, T. J. Biochem. S y s t .
E c o l . 1975, 3, 5.
139. Jameson, G. R.; R e i d , E. N. Phytochem. 1975, 14, 2229.
140. L y t t l e , T. F.; Lyttle, J. S.; Caruso, A. Phytochem. 1976,
15, 965.
141. Khan, H.; Zaman, A.; Chetty, H. L.; Gupta, A. S.; Dev, S.
Tetrahedron L e t t . 1971, 46, 4443.
142. Ayengar, K. N. N.; I y e r , R. T.; Rangaswamy, S. I n d i a n J.
Chem. 1972, 10, 482.
143. I y e r , R. T.; Ayengar, K. N. N.; Rangaswamy, S. Indian J.
Chem. 1973,11,1336.
144. B a r d o u i l l e , V.; Mootoo, B. S.; H i r o t s u , K.; C l a r d y , J.
Phytochem. 1978, 17, 275.
145. Imal, S.; Toyosata, T.; S a k a i , M.; Sato, Y.; F u j i o k a , S.;
Murata, E.; Goto, M. Chem. Pharm. Bull. 1969,17,335.
146. Faux, A.; G a l b r a i t h , M. N.; Horn, D. M. S.; M i d d l e t o n , E.
J. Chem. Comm. 1970, 243.

5. STiPANOVic Plant Trichomes and Glands
147. Murakami, T.; Kimura, T.; Tanaka, N.; Saiki, Y.; Chen, C.
Phytochem. 1980, 19, 471.
148. Wollenweber, E.; Favre-Bonvin, J. Phytochem. 1979, 18,
1243.
149. Huhtikangas, A.; Huurre, A.; Partanen, A. P l a n t a Med. 1980
38, 62.
150. B a r d o u i l l e , J . V.; Cox, M. Guyana J. Sci. 1977, 5, 61.
151. S t a r , A. E. Bull. Torrey Bot. Club 1980, 107, 16.
152. Wollenweber, E. Biochem. P h y s i o l . P f l a n z . 1974, 166,
419.
153. Mabry, T. J.; Gill, J . E.; Burnett, W. C., Jr.; Jones, S.
B. in "Host P l a n t Resistance t o P e s t s " , ACS Symposium
S e r i e s , No. 62; Hedin, P. A., Ed.; American Chemical
Society: Washington D. C., 1977, p. 179.
154. Burnett, W. C.; Jones, S. B., Jr.; Mabry, T. J. Am. M i d l .
Nat. 1978, 100,
155. Narain, N. K. Spectrosc
156. Drew, M. G. B.; Hitchman, S. P.; Mann, J.; Lopes, J. L. C.
J. Chem. Soc., Chem. Commun. 1980, 802.
157. Bohlmann, F.; Gupta, R. K.; Jakupovic, J.; K i n g , R. M.;
Robinson, H. L i e b i g s Ann. Chem. 1980, 11, 1904.
158. Narain, N. K. Can. J. Pharm. Sci. 1977, 12, 18.
159. Rustaiyan, A.; Niknejad, A; Danieli, B.; Palmisano, G.;
Jones, S. F i t o t e r a p i a 1977, 48, 266.
160. S t i c h e r , O.; Afifi-Yazar, F. U. Helv. Chim. Acta 1979, 62,
530.
161. Rodriguez, E.; Dillon, M.; Mabry, T.; Mitchell, J . C.;
Towers, G. H. N. E x p e r i e n t i a 1976, 32, 236.
162. Narasimhan, T. R.; Anant, M.; Swamy, M. N.; Babu, M. R.;
Mangala, A.; Rao, P. V. S. Curr. Sci. 1977, 46, 15.
163. Towers, G. H. N.; Mitchell, J . L.; Rodriguez, E.; Bennet,
F. D.; Subba Rao, P. V. Biochem. Rev. 1977, 48, 65.
164. Kanchan, S. D. Curr. Sci. 1975, 44, 358.
165. Sharma, R. N.; Joshi, V. N. Biovigyanam 1977, 3, 225.
166. Picman, A. K.; Towers, G. H. N.; Subba Rao, P. V.
Phytochem. 1980, 19, 2206.
167. Mears, J. A. J. Nat. Prod. 1980, 43, 708.
168. Gershenzon, J.; Mabry, T. J.; A b s t r . Ann. Mtg. Phytochem.
Soc. N. Am. 1981, p. 9.
169. Rogers, C. E. "Proceedings, EUCARPIA Symposium, Oil and
Leguminous Crops, Prague, C z e c h o s l o v a k i a " , in press.
170. Morimoto, H.; Sanno, Y.; Oshio, H. Tetrahedron 1966, 22,
3173.
171. Herz, W.; DeGroote, R. Phytochem. 1977,16,1307.
172. Bohlmann, F.; Dutta, L. N. Phytochem. 1979,18,676.
173. Herz, W.; Kumar, N. Phytochem. 1981, 20, 93.
176. Waiss, A. C., Jr.; Chan, B. G.; Ellinger, C. A.; G a r r e t t , V.

R.; C a r l s o n , F. C.; Beard, B. Naturwissenschaften 1977, 64,

341.
177. S t i p a n o v i c , R. D.; D'Brien, D. H.; Rogers, C. E.; Thompson,
T. E. J. Agri. Food Chem. 1979, 27, 458.
178. Panizo, F. M.; Rodriguez, B. Anales De Quimica 1979, 75,
428.
179. Bohlmann, F.; Jakupovic, J.; K i n g , R. M.; Robinson, H.
Phytochem. 1980, 19, 863.
180. Kasprzyk, Z.; Janiszowska, W. Phytochem. 1971, 10,
1946.
181. St. Pyrek, J . Pol. J. Chem. 1979, 53, 1071.
182. St. Pyrek, J. Pol. J. Chem. 1979, 53, 2465.
183. Bohlmann, F.; Jakupovic, J . ; King, R. M.; Robinson, H.
Phytochem. 1980, 19, 863.
184. Popescu, H.; Fagarasan E Clujul Med 1979 52 171
185. Rodriguez, E. Chem
186. H e r t z , W. in " E f f e c t
K e e l e r , R. F.; van Kampen, K. R.; James, L. F., Eds.;
Academic Press: New York, 1978, p. 487.
187. Kelsey, R. G.; Shafizadeh, F. Biochem. Syst. E c o l . 1980, 8,
371.
188. C u r t i s , J. D.; L e r s t e n , N. R. Amer. J. Bot. 1974, 61, 835.
189. Van Hoof, L.; Vanden Berghe, D. A.; Vlientinck, A. J. Biol.
P l a n t 1980, 22, 265.
190. A h l u w a l i a , V. K.; Prakash, C.; Gupta, M. C.; Mehta, S.
Indian J . Chem., Sect. B 1978, 16, 591.
191. D e l l , B.; McComb, A. J. Aust. J. Bot. 1975, 23, 373.
192. Dawson, R. M.; Jarvis, M. W.; Jefferies, P. R.; Payne, T.
G.; R o s i c h , R. S. Aust. J. Chem. 1966, 19, 2133.
193. Jefferies, P. R.; Ratajczak, T. Aust. J. Chem. 1973, 26,
173.
195. Bose, P. C., Adityachaudhury, N. Phytochem. 1978, 17,
587.
196. S i d d i q u i , S. J. Indian Chem. Soc. 1937, 14, 703.
197. Bhaskar, A.; S e s h a d r i , T. R. Indian J. Chem. 1973, 11,
404.
198. Seshadri, T. R. Proc. Ind. Acad. Sci. 1948, 27A, 129.
199. Seshadri, T. R. Proc. Ind. Acad. Sci. 1949, 28A, 106.
200. Das, A. K.; Mitra, S. R.; Aditayachaudhury, N.; P a t r a , A.;
M i t r a , A. K.; Chattejee, Mrs. A. Indian J . Chem., Sect. B
1979, 18B, 550.
201. M i t r a , S. R.; Das, A. K.; Kirtaniya, C. L.;
Adityachaudhury, N.; P a t r a , A.; M i t r a , A. K. Indian J.
Chem., Sect. B 1980, 19B, 79.
202. Sakakibara, M.; DiFeo, D., Jr.; N a k a t i n i , N.; Timmermann,
B.; Mabry, T. J. Phytochem. 1976, 15, 727.
203. Bernhard, H. O.; T h i e l e , K. P l a n t a Med. 1981,41,100.
204. Bohnstedt, C. F.; Mabry, T. J. Res. Latinoam Quim. 1979,
10, 128.

205. Rhoades, D. F. in "Creosote Bush"; Mabry, T. J.; Hunziker,

J . H.; DiFeo, D. R., Jr., Eds.; Dowden, Hutchinson and
Ross, Inc.: Stroudsburg, 1977.
206. Stubblebine, W. H.; Langenheim, J. H. J. Chem. E c o l . 1977,
3, 633.
207. Langenheim, J. H.; Stubblebine, W. H.; L i n c o l n , D. E.;
F o s t e r , C. E. Biochem. Syst. E c o l . 1978,6,299.
208. Chow, P. W.; Jefferies, P. R. Aust. J . Chem. 1968, 21,
2529.
209. E r r i n g t o n , S. G.; Ghisalberti, E. L.; Jefferies, P. R.
Aust. J . Chem. 1976, 29, 1809.
210. G h i s a l b e r t i , E. L.; Jefferies, P. R.; Sefton, M. A.
Phytochem. 1978, 17, 1961.
211. Lukefahr, M. J.; Fryxell, P. A. Econ. Bot. 1967, 21, 128.
212. Adams, R.; Geissman T A Chem Rev 1960 60 555
213. Seshadri, T. R. Proc
37, 411.
214. B e r a r d i , L. C.; G o l d b l a t t , L. A. in "Toxic C o n s t i t u e n t s of
P l a n t F o o d s t u f f " , 2nd ed.; L i e n e r , I . E., Ed.; Academic
Press: New York, 1969, p. 211.
215. H e i n s t e i n , P.; Widmaier, R.; Wegner, P.; Howe, J. in
"Biochemistry o f P l a n t P h e n o l i c s " ; Swain, T.; Harborne, J.
B.; Van Sumere, C. F., Eds.; Plenum Press: New York,
1979, p. 313.
216. Abou-Donia, M. B. Residue Review 1976, 61, 125.
217. B e r a r d i , L. C.; Golblatt, L. A. in "Toxic Contituents o f
Plant Foodstuff"; L i e n e r , I. E., Ed.; Academic Press: New
York, 1980, p. 183.
218. N a t i o n a l Coordinating Group on Male Antifertility Agents
(China) Gynecol. o b s t e t . Invest. 1979, 10, 163.
219. King, T. J.; D e S i l v a , L. B. Tetrahedron L e t t . 1968, 261.
220. Datta, S. C.; Murti, V. V. S.; Seshadri, T. R. Indian J.
Chem. 1972, 10, 263.
221. Seshadri, T. R.; Sharma, N. N. Curr. Sci. 1973, 42, 821.
222. Gray, J. R.; Mabry, T. J.; Bell, A. A.; S t i p a n o v i c , R. D.;
Lukefahr, M. J. J. Chem. Soc., Chem. Comm. 1976, 109.
223. S t i p a n o v i c , R. D.; Bell, A. A.; O'Brien, D. H.; Lukefahr,
M. J. Agric. and Food Chem. 1978, 26, 115.
M. J. Tetrahedron Lett., 1977, 567.
M. J. Phytochem. 1978,17,151.
226. B e l l , A. A.; S t i p a n o v i c , R. D.; O'Brien, D. H.; Fryxell, P.
A. Phytochem. 1978,17,1297.
227. S t i p a n o v i c , R. D.; Bell, A. A.; Lukefahr, M. J. in "Host
P l a n t Resistance t o P e s t s " , ACS Symposium S e r i e s , No. 62;
Hedin, P. A., Ed.; American Chemical S o c i e t y : Washington,
D. C., 1977, p. 197.
228. S t i p a n o v i c , R. D.; Bell, A. A.; O'Brien, D. H. Phytochem.
1980, 19, 1735.

229. Stanford, E. E.; Viehoever, A. J. Agric. Res. 1918, 13,

419.
230. Chan, B. G.; Waiss, A. C., Jr. Beltwide Cotton Prod. Res.
Conf. Proc. 1981, 49.
231. Chan, B. G.; Waiss, A. C.; Jr.; L u r d i n , R. E.; Asen, S.
A b s t r . Ann. Mtg. Phytochem. Soc. N. Am. 1981, p. 9.
232. B e l l , A. A.; S t i p a n o v i c , R. D.; Howell, C. R.; Fryxell, P.
A. Phytochem. 1975, 14, 225.
233. S t i p a n o v i c , R. D.; Bell, A. A.; Mace, M. E.; Howell, C. R.
Phytochem. 1975, 14, 1077.
234. S t i p a n o v i c , R. D.; Bell, A. A.; Howell, C. R. Phytochem.
1975, 14, 1809.
235. Z a k i , A. I . ; Keen, N. T.; Erwin, D. C. Phytopath. 1972, 62,
1402.
236. Abdullaev, Z. S.; Karimdzhanov A K.; Ismailov A I . ;
Islambekov, S. H.
Prir. Soedin. 1977
237. H a l l o i n , J. M.; Veech, J. A.; C a r t e r , W. W. P l a n t S o i l
1978, 50, 237.
238. B e l l , A. A.; S t i p a n o v i c , R. D. Beltwide Cotton Prod. Res.
Conf. Proc. 1977, 244.
239. M a r g a l i t h , P. A p p l . M i c r o b i o l . 1967,15,952.
240. Vichkanova, S. A.; Goryunova, L. V. Antibiotiki 1968, 13,
828.
241. Vichkanova, S. A.; O i f a , A. I.; Goryunova, L. V.
Antibiotiki 1970, 15, 1071.
242. Shaver, T. N.; G a r c i a , J. A. J. Econ. Entomol. 1973, 66,
327.
243. Vermel, E. M. Acta Unio I n t . Cancrum 1964, 20, 211.
244. D o r s e t t , P. H.; K e r s t i n e , E. E. J. Pharm. Sci. 1975, 64(6),
1073.
245. Minyard, J.P.; Tumlinson, J. H.; Hedin, P. A.; Thompson, A.
C. J. Agric. Food Chem. 1965, 13, 599.
246. Minyard, J. P.; Tumlinson, J. H.; Thompson, A. C.; Hedin,
P. A. J. Agric. Food Chem. 1966, 14, 332.
247. Minyard, J. P.; Tumlinson, J. H.; Thompson, A. C.; Hedin,
P. A. J. Agric. Food Chem. 1967, 1 5 , 517.
248. Minyard, J. P.; Thompson, A. C.; Hedin, P. A. J. Org. Chem.
1968, 33, 909.
249. Minyard, J. P.; Hardee, D. D.; Gueldner, R. C.; Thompson,
A. C.; Wiygul, G.; Hedin, P. A. J. Agric. Food Chem. 1969,
17, 1093.
250. Hedin, P. A.; Thompson, A. C.; Gueldner, R. C.; Minyard, J.
P. Phytochem. 1971, 10, 1692.
251. Hedin, P. A.; Thompson, A. C.; Gueldner, R. C.; Minyard, J.
P. Phytochem. 1971,10,3316.
252. Hedin, P. A.; Thompson, A. C.; Gueldner, R. C.; Ruth, J. M.
Phytochem. 1972,11,2119.
253. Hedin, P. A.; Thompson, A. C.; Gueldner, R. C. Phytochem.
1975, 14, 2087.

254. Thompson, A. C.; Hanny, B. W.; Hedin, P. A.; Gueldner, R.

C. Amer. J. Bot. 1971, 58, 803.
255. Hedin, P. A.; Thompson, A. C.; Gueldner, R. C. Phytochem.
1972, 11, 2356.
256. Kumanoto, J.; Waines, J. G.; H o l l e n b e r g , J. L.; Scora, R.
W. J. Agric. Food Chem. 1979, 27, 203.
257. Hanny, B. W.; Gueldner, R. C. J. Agric. Food Chem. 1976,
24(2), 401.
258. Hedin, P. A.; Minyard, J. P.; Thompson, A. C.; Struck, R.
F.; Frye, J. Phytochem. 1967, 6, 1165.
259. Hedin, P. A.; Collum, D. H.; White, W. H.; P a r r o t t , W. L.;
Lane, H. C.; J e n k i n s , J. N. in "Regulation o f Insect
Development and Behaviour"; Sehnal, R.; Zabza, A.; Menn, J .
J . ; Cymborowski, B., Eds; Wroclaw T e c h n i c a l U n i v e r s i t y
Press: Wroclaw, 1981 1071
260. Hedin, P. A.; M i l e s
J. Agric. Food Chem
261. Shaver, T. N.; Lukefahr, M. J. J. Econ. Entomol. 1969, 62,
643.
262. Hanny, B. W.; M e r e d i t h , W. R., Jr.; Bailey, J. C.; Harvey,
A. J. Crop Sci. 1978, 18, 1071.
263. D i l d a y , R. H.; Shaver, T. N. Crop S c i . 1980, 20, 91.
264. Yang, H. C.; D a v i s , D. D. Crop S c i . 1976, 16, 485.
265. Hanny, B. W. J. Agric. Food Chem. 1980, 28, 504.
266. Cook, O. F. U.S. Dept. A g r i . Bur. P l a n t Indus. Bull. 88
1906, 87.
267. Lukefahr, M. J.; Houghtaling, J. E. J. Econ. Entomol. 1969,
62, 588.
268. Shaver, T. N.; Lukefahr, M. J.; G a r c i a , J. A. J. Econ.
Entomol. 1970, 63, 1544.
269. Seaman, F.; Lukefahr, M. J.; Mabry, T. J. Proc. Nat. Cotton
C o u n c i l , A t l a n t a 1977, 102.
270. Elliger, C. A.; Chan, B. G.; Waiss, A. C., Jr. J. Econ.
Entomol. 1978, 71, 161.
271. Lukefahr, M. J.; M a r t i n , D. F. J. Econ. Entomol. 1966, 59,
176.
272. Abou-Donia, M. B.; Taman, F.; Bakery, N. M.; El-Sebab, A.
H. Experentia 1974, 30, 1151.
273. Meisner, J.; Kehat, M.; Zur, M.; Ascher, K. R. S. Entomol.
Exp. A p p l . 1977, 22, 301.
274. Lukefahr, M. J.; S t i p a n o v i c , R. D.; Bell, A. A.; Gray, J.
R. Beltwide Cotton Prod. Res. Conf. Proc. 1977, 97.
275. Chan, B. G.; Waiss, A. C., Jr.; Binder, R. G.; Elliger, C.
A. Entomol. Exp. A p p l . 1978, 24, 94.
276. Waiss, A. C., Jr.; Chan, B. G.; Elliger, C. A.; Binder, R.
G. Beltwide Cotton Prod. Res. Conf. Proc. 1981, 61.
277. Meisner, J.; Ascher, K. R. S.; Zur, M. J. Econ. Entomol.
1977, 70, 149.
278. Meisner, J.; Zur, M.; Kabonci, E.; Ascher, K. R. S. J .
Econ. Entomol.1977, 70, 714.

279. Meisner, J.; Navon, A.; Zur, M.; Ascher, K. R. S. E n v i r o n .

Entomol. 1977, 6, 243.
280. El-Sebae, A. H.; Sherby, S. I.; Mansour, N. A. J. E n v i r o n .
Sci. Health 1981, B16, 167.
281. Meisner, J.; Ishaaya, I.; Ascher, K. R. S.; Zur, M. Ann.
Entomol. Soc. Am. 1978, 71, 5.
282. Meisner, J.; Ascher, K. R. S.; Zur, M.; Kabonci, E. J.
Econ. Entomol. 1977, 70, 717.
283. Abou-Donia, M. B.; D i e c k e r t , J. W. T o x i c o l . A p p l .
Pharmacol. 1971, 18, 507.
284. Chan, B. G.; Waiss, A. C., Jr.; Lukefahr, M. J. J. Insect
P h y s i o l . 1978, 24, 113.
285. Georghiou, G. P. in "Annual Review o f Ecology and
Systematics", V o l . 3; Johnston, R. F., Ed.; Annual Reviews
Inc.: Palo Alto, 1972 p 133
286. Winteringham, F
35, 1512.
287. Harborne, J . B. in "Herbivores - T h e i r I n t e r a c t i o n w i t h
Secondary M e t a b o l i t e s " ; Rosenthal, G. A.; Janzen, D. H.,
Eds.; Academic Press: New York, 1979, p. 619.
RECEIVED August 23,1982

6
Regulation of Synthesis and Accumulation of
Proteinase Inhibitors in Leaves of Wounded
Tomato Plants
C. E. NELSON, M. WALKER-SIMMONS, D. MAKUS, G. ZUROSKE,
J. GRAHAM, and C. A. R Y A N
Washington State University, Institute of Biological Chemistry and Biochemistry
and Biochemistry/Biophysics Program, Pullman, WA 99164
Two proteinase i n h i b i t o r s Inhibitor I d II,

accumulate i n leave
by chewing insect y
accumulation of these two antinutrient proteins is
apparently a defense response and is i n i t i a t e d by
the release of a putative wound hormone called the
proteinase i n h i b i t o r inducing factor (PIIF). The
direction of flow of PIIF out of wounded leaves i s
primarily towards the apex and transport occurs
maximally about 120 min following wounding. After
a single severe wound, the i n v i t r o translatable
tomato leaf mRNA specific for Inhibitors I and II
increases to a maximum within four hours and
remains constant for about five hours when i t
decreases rapidly to about 50% of the maximum.
The rate of i n vivo accumulation of both i n h i b i t o r
proteins steadily increases, reaching a steady
state after nine hours. However, a second wound
at nine hours results i n a tripling of the steady
state rate of i n h i b i t o r accumulation over the next
several hours. The data indicates that the second
wound causes no change i n the apparent transla-
t i o n a l efficiencies of the mRNA for Inhibitors I
and II but causes increased rates of i n h i b i t o r
accumulation by providing more translatable
i n h i b i t o r messages when the plant's translation
system is operating at high efficiency.
A severe mechanical wound on a s i n g l e l e a f of tomato p l a n t s

i n i t i a t e s a complex s e r i e s of e x t r a c e l l u l a r and i n t r a c e l l u l a r
r e a c t i o n s which r e s u l t i n the s y n t h e s i s and accumulation of two
p r o t e i n a s e i n h i b i t o r s , I n h i b i t o r s I and I I , i n l e a f c e l l s (1_,
2). A second wounding, w i t h i n a few hours, r e s u l t s i n a 2-3
f o l d increase i n the r a t e s of accumulation i n i t i a t e d by the
0097-6156/83/0208-0103$06.00/O

f i r s t wound. A p u t a t i v e wound hormone, c a l l e d the p r o t e i n a s e

i n h i b i t o r i n d u c i n g f a c t o r , P I I F , i s r e l e a s e d at the wound s i t e
and t r a v e l s throughout the p l a n t to i n i t i a t e s y n t h e s i s and
accumulation of the two p r o t e i n a s e i n h i b i t o r s , even i n unwounded
leaves s e v e r a l cm from the wound s i t e . We view t h i s process as
a p r i m i t i v e immune-like response i n which the p l a n t i s respond-
ing to pest damage by producing powerful a n t i n u t r i e n t p r o t e i n s ,
the p r o t e i n a s e i n h i b i t o r s , to help the p l a n t discourage p e r s i s -
tent or f u t u r e a t t a c k s by p e s t s .
This wound response has provided a novel system f o r studying
the r e g u l a t i o n of the expression of the two proteinase i n h i b i t o r s
by the f a c t o r P I I F , t r i g g e r e d by severe environmental s t r e s s .
In t h i s chapter we report our recent data concerning the d i r e c -
t i o n and time course of P I I F t r a n s p o r t through the p l a n t f o l l o w -
ing wounding, and our progress i n i n i t i a t i n g a program to study
the molecular b i o l o g y o
D i r e c t i o n and Rate of Flow of the Wound S i g n a l , P I I F
P I I F a c t i v i t y has been i s o l a t e d by v a r i o u s techniques from

tomato leaves to y i e l d a s i n g l e broad peak from Sephadex G-50
that e x h i b i t s a Mw range of about 5000 to 10,000 daltons and i s
p r i m a r i l y carbohydrate i n composition ( 3 ) . P r o p e r t i e s of h i g h l y
p u r i f i e d P I I F p r e p a r a t i o n s , such as l o s s of a c t i v i t y upon e i t h e r
prolonged a c i d h y d r o l y s i s or periodate o x i d a t i o n , and i t s
monosaccharide composition suggested that i t was s i m i l a r to the
p e c t i c polysaccharides found a s s o c i a t e d w i t h the p l a n t c e l l
wall. In c o l l a b o r a t i o n w i t h Dr. Peter Albersheim, of the
U n i v e r s i t y of Colorado, we found that a sycamore c e l l w a l l -
d e r i v e d p o l y s a c c h a r i d e , c a l l e d rhamnogalacturonan I , was as
a c t i v e as tomato P I I F i n inducing p r o t e i n a s e i n h i b i t o r accumula-
t i o n i n young tomato p l a n t s ( 4 ) . This work s u b s t a n t i a t e d that
tomato P I I F was a fragment of the p l a n t c e l l w a l l . In subsequent
experiments we were able to e n z y m i c a l l y degrade P I I F i n t o
o l i g o s a c c h a r i d e s w i t h molecular weights of about 400 to 2000
that r e t a i n e d the c a p a c i t y to induce p r o t e i n a s e i n h i b i t o r s i n
detached tomato leaves ( 3 ) .
A hypothesis was presented (3) i n which P I I F i s r e l e a s e d as
a mixture of p o l y - and o l i g o s a c c h a r i d e s fragmented from the c e l l
w a l l by h y d r o l y t i c enzymes that e i t h e r are a c t i v a t e d during
wounding or are introduced by invading p e s t s . P I I F , or a
product induced by i t s presence, could then be transported
r a p i d l y through the p l a n t v a s c u l a r system to t a r g e t c e l l s where
i t induces the s y n t h e s i s and accumulation of proteinase i n h i b i t o r
proteins.
D i r e c t i o n of f l o w of P I I F . The time-course of I n h i b i t o r I
accumulation i n leaves of young tomato p l a n t s at the f o u r l e a f
stage, wounded at 0 time and at 72 h r , i s shown i n F i g . 1.
Wounding of an upper l e a f l e t ( l e f t ) d i d not cause accumulation

120

TIME (hours)
Figure 1. Time course of accumulation of Inhibitor I in terminal leaflets of young tomato plants.
Leaves were wounded at the uppermost leaf (left) and the lowest leaf (right) by crushing across the

midrib of the terminal leaflet with a hemostat at zero time and again at 72 h. The concentrations
of Inhibitor I in leaves were determined immunologically.
of I n h i b i t o r I i n lower l e a v e s , but a second wound (at 72 hr) on

the same l e a f was weakly r e g i s t e r e d by the lower leaves and
r e s u l t e d i n some accumulation of the i n h i b i t o r . Wounding of the
lowest l e a f ( r i g h t ) d i d cause upper leaves to accumulate I n h i b -
i t o r I and a second wound at 72 hr s i g n i f i c a n t l y r e i n f o r c e d the
response, p a r t i c u l a r l y i n the two uppermost leaves. In Table I
the average l e v e l s of I n h i b i t o r I i n a l l f o u r t e r m i n a l l e a f l e t s
of tomato p l a n t s , s i n g l y wounded at the v a r i o u s l e a f l e t p o s i -
t i o n s , i s shown. I t i s c l e a r that i n h i b i t o r s p r e f e r e n t i a l l y
accumulated i n leaves near the apex of the p l a n t and that the
t r a n s p o r t of P I I F was p r i m a r i l y a c r o p e t a l l y . The lower leaves
were not disposed to accumulate much I n h i b i t o r I , even when the
lowest l e a f l e t s were wounded. These experiments suggest that
not only are lower o l d e r leaves much l e s s responsive to P I I F ,
but are apparently r e c i p i e n t s of only a s m a l l amount of the
hormone that i s release
demonstrated that a l
PIIF when wounded, although the lowest wounded leaves do not
appear to be r e l e a s i n g as much P I I F as upper l e a v e s .
Rate of flow of P I I F . A s i n g l e s l i c e of a sharp r a z o r

blade through the l e a f p e t i o l e apparently does not r e l e a s e
a p p r e c i a b l e PIIF i n t o the p l a n t as evidenced by the l a c k of
accumulation of i n h i b i t o r i n t i s s u e s of the e x c i s e d l e a f ( 5 ) .
Thus, a s i n g l e wound can be i n f l i c t e d i n a t e r m i n a l l e a f l e t and
then the e n t i r e l e a f can be severed at a measured d i s t a n c e from
the wound s i t e near the base of the p e t i o l e at v a r i o u s times to
determine how long i t takes f o r PIIF to t r a v e l past the p o s i t i o n
of the cut and out of the l e a f , as judged by accumulation of
I n h i b i t o r I i n an adjacent upper l e a f l e t 24 hr l a t e r . Our
previous experiments (6) suggested t h a t about 2 hr was r e q u i r e d
to maximally t r a n s p o r t P I I F out of leaves (a d i s t a n c e of about 6
cm). We repeated these experiments h e r e i n w i t h the purpose of
comparing the r a t e of PIIF t r a n s p o r t w i t h that of [ ^ C ] g l u c o s e
that was a p p l i e d to the wound immediately f o l l o w i n g the i n j u r y .
ltf
The time f o r t r a n s p o r t of [ C ] g l u c o s e from i t s a p p l i c a t i o n
at the wound s i t e to the base of the p e t i o l e ( F i g . 2, r i g h t ) was
n e a r l y i d e n t i c a l to that of P I I F that was r e l e a s e d upon wounding.
The amount of glucose that was t r a n s p o r t e d up to the next l e a f ,
however, was s m a l l compared to what passed through the p e t i o l e
of the wounded l e a f . Very l i t t l e r a d i o a c t i v i t y was detected i n
the adjacent lower l e a f . The maximum r a d i o a c t i v i t y was reached
at about 120 min f o l l o w i n g wounding, the same time that P I I F
t r a n s p o r t through t h i s r e g i o n was maximal ( F i g . 2, l e f t ) . Thus,
11+
both P I I F and [ C]glucose were transported out of the wound
s i t e s at approximately the same times and were both p r e f e r e n -
t i a l l y transported a c r o p e t a l l y .
Mode of t r a n s p o r t . A j e t of hot a i r (80°C) focused on a

segment of the p e t i o l e near the main stem completely destroyed

6. NELSON ET AL. Synthesis and Accumulation of Proteinase Inhibitors 107
TABLE I
INHIBITOR I ACCUMULATION IN LEAVES OF YOUNG TOMATO PLANTS

120 HOURS FOLLOWING WOUNDING OF INDIVIDUAL LEAVES AT
DIFFERENT LOCATIONS ON THE PLANTS
Young tomato p l a n t s having four leaves were wounded a t the l e a f

p o s i t i o n shown below w i t h a hemostat a t 0 and 72 h r . A f t e r 120
hr f o l l o w i n g the i n i t i a l wounding the t e r m i n a l l e a f l e t o f each
l e a f was assayed f o r I n h i b i t o r I c o n c e n t r a t i o n .
Position o
Wounded Leaf 1 2 3 4
I n h i b i t o r I Accumulation
(pg/g t i s s u e )

TIME( min)
Figure 2. Left: Inhibitor I accumulation in leaves of wounded young tomato plants. The middle
leaf of young tomato plants at the three leaf stage was wounded at zero time and excised at the times
shown. Inhibitor I in the upper and lower intact leaves was determined immunologically 48 h fol-
14
lowing wounding. Right: 5 /xL of U-[ C]glucose (specific activity, 28 pCi/mM) was applied directly

to the wounds of the middle leaf immediately upon injury. Radioactivity was determined in 0.5-cm
segments at the base of each of the three petioles at the times indicated.
6. NELSON E T AL. Synthesis and Accumulation of Proteinase Inhibitors 109
the phloem but not the xylem, as evidenced by the d r y i n g of the

t r e a t e d area w i t h i n an hour t o form a f i n e strand o f xylem w i t h
the l e a f s t i l l i n t a c t w i t h good t u r g o r . This treatment caused
the l e a f i t s e l f t o accumulate I n h i b i t o r I over the next 24 h r ,
but i t minimally a f f e c t e d the leaves of the r e s t of the p l a n t ,
i n d i c a t i n g that the d e s t r u c t i o n of the phloem d i d not i n i t i a t e
P I I F t r a n s p o r t out of the l e a f but only i n t o the l e a f i t s e l f .
We subjected a segment of the p e t i o l e of the lowest l e a f of
s e v e r a l 3 l e a f stage tomato p l a n t s , t o a hot a i r j e t t o destroy
the phloem. W i t h i n an hour the i n j u r e d p e t i o l e segments had
d r i e d t o form the t h i n strands of xylem. As b e f o r e , t h i s
treatment d i d not r e l e a s e PIIF i n t o the p l a n t (Table I I , t r e a t -
ment 3) w h i l e the l e a f i t s e l f ( l e a f #1) accumulated considerable
I n h i b i t o r I . Subsequent wounding of leaves whose phloem had
been destroyed (Table I I , treatment 4) d i d not r e s u l t i n I n h i b -
i t o r I accumulation i
phloem d e s t r u c t i o n ha
leaf.
While the evidence presented h e r e i n c u m u l a t i v e l y supports
the involvement of the phloem i n t r a n s l o c a t i n g PIIF out o f
wounded tomato p l a n t s , the v e l o c i t y of t r a n s p o r t appeared to be
much slower than expected f o r phloem t r a n s p o r t . When c a r e f u l l y
measured from p o i n t t o p o i n t i n p e t i o l e t i s s u e the v e l o c i t y of
a s s i m i l a t e out of l e a v e s , i n g e n e r a l , i s i n the order of 1-5
cm/min (_7, 8 ) . Our techniques do not a l l o w d i r e c t measurements
w i t h PIIF i t s e l f , but from our i n d i r e c t measurements we c a l c u l a t e
a gross movement of P I I F from the time of wounding t o time of
maximum t r a n s p o r t of P I I F (as w i t h glucose) t o the base of
the p e t i o l e , 6 cm from the wound s i t e , of about 0.05 cm/min.
This i s over twenty times too slow f o r a process i n v o l v i n g j u s t
phloem t r a n s p o r t . Thus, i f phloem t r a n s p o r t i s o c c u r r i n g , then
a time of n e a r l y 2 h r must be r e q u i r e d t o d e l i v e r a maximum
q u a n t i t y of PIIF i n t o the phloem a t o r near the wound s i t e .
This r a t e i s much slower than normal phloem l o a d i n g , f o r example
i n healthy soybeans ( 9 ) .
The data seems t o i n d i c a t e that the o r i g i n s of P I I F and i t s
i n t r o d u c t i o n i n t o the t r a n s p o r t system of the p l a n t i s a complex
system. We can however speculate about the nature of the
process from a v a i l a b l e i n f o r m a t i o n . The 2 h r p e r i o d r e q u i r e d t o
reach maximum P I I F l e v e l s i n the phloem could be a consequence
of enzymic degradation of the c e l l w a l l f o l l o w i n g wounding,
and/or the production and a s s o c i a t i o n of some type of chemical
to the c e l l w a l l fragments, e v e n t u a l l y e n t e r i n g the t r a n s p o r t
system. Thus, the phloem, which i s i n v o l v e d i n t r a n s l o c a t i n g
carbohydrates, would be a l o g i c a l candidate t o t r a n s p o r t s m a l l
p e c t i c fragments, p r e f e r e n t i a l l y t o the upper leaves. This type
of mechanism of PIIF production and r e l e a s e would r e q u i r e some
type of l o a d i n g mechanism that would a l l o w the fragments t o
enter the phloem as, o r a f t e r , they were produced. As a monitor-
ing system f o r i n s e c t o r pathogen damage, t h i s process would be

TABLE I I
EFFECT OF THE HOT AIR-DESTRUCTION OF PETIOLE PHLOEM ON THE

MOVEMENT OF P I I F OUT OF MECHANICALLY WOUNDED LEAVES
A l l treatments of young tomato p l a n t s (having three leaves) were

at the lowest l e a f (#1) shown below. Hot a i r (80°C) was a p p l i e d
to the base of the p e t i o l e through a window i n a t e f l o n s h i e l d
to destroy a segment o
d i c u l a r to the m i d r i b
n a l l e a f l e t of l e a f #1 three days a f t e r hot a i r treatment and
I n h i b i t o r I l e v e l s were assayed i n the leaves 24 h r l a t e r .
3.
Treatment
If
^•W^t Leaf #
2
I n h i b i t o r I Accumulation
(yg/g t i s s u e )
1. No treatment 0 0 10
2. No phloem d e s t r u c t i o n 133 126 86

l e a f / / l mechanically
wounded
3. Leaf #1 phloem destroyed, 173 35 32

l e a f #1 t i s s u e unwounded
4. Leaf #1 phloem destroyed, 165 14 27

l e a f #1 t i s s u e wounded

w e l l s u i t e d , d i r e c t i n g messages from wounded o r damaged t i s s u e s

to the younger healthy t i s s u e s t o i n i t i a t e i n h i b i t o r accumula-
tion.
In V i t r o Synthesis of P r e - P r o t e i n a s e I n h i b i t o r s w i t h mRNA from

Wounded Tomato P l a n t s
The two p r o t e i n a s e i n h i b i t o r s that accumulate i n leaves of

wounded tomato leaves have been i s o l a t e d and c h a r a c t e r i z e d .
I n h i b i t o r I has a molecular weight of 41,000 and i s composed of
subunits w i t h molecular weights of about 8100 (10). It is,
t h e r e f o r e , a pentamer i n i t s n a t i v e s t a t e . Each subunit
possesses an a c t i v e s i t e s p e c i f i c f o r chymotrypsin, and the
9
apparent f o r the i n h i b i t i o n of chymotrypsin i s about 10"" M
(10). I n h i b i t o r I I has a molecular weight o f about 23,000, i s
composed of two s u b u n i t s
chymotrypsin w i t h K. value
(10) .
Messenger RNA has been prepared from leaves o f wounded and
unwounded tomato p l a n t s and only leaves of wounded p l a n t s
c o n t a i n t r a n s l a t a b l e mRNAs s p e c i f i c f o r I n h i b i t o r s I and I I
(11) . Both p r o t e i n s have been shown t o be t r a n s l a t e d i n v i t r o
i n a r e t i c u l o c y t e l y s a t e system as p r e i n h i b i t o r s , 2000-3000
daltons l a r g e r than those synthesized and accumulated i n v i v o
(11). The p r e i n h i b i t o r s may be important i n the compartmentali-
z a t i o n of the i n h i b i t o r s as they are stored i n the c e n t r a l
v a c u o l e , o r p l a n t lysosome, of the plant c e l l s (12). We have
now studied the time course of the increase i n t r a n s l a t a b l e mRNA
i n leaves of wounded p l a n t s u t i l i z i n g poly(A) mRNA i s o l a t e d a t
v a r i o u s times f o l l o w i n g wounding.
When young tomato p l a n t s are wounded, by chewing i n s e c t s or
by a severe crushing of any type, the l e v e l s of t o t a l poly(A)
t r a n s l a t a b l e mRNA f o r both p r o t e i n a s e I n h i b i t o r I and I n h i b i t o r
I I r i s e r a p i d l y during the f i r s t 4 h r a f t e r wounding (13). This
r i s e was measured by q u a n t i f y i n g the immunoprecipitates that can
be recovered s p e c i f i c a l l y from e l e c t r o p h o r e t i c gels a f t e r
t r a n s l a t i o n i n a c e l l f r e e r a b b i t r e t i c u l o c y t e l y s a t e system
(11, 13). An example of such g e l s i s shown i n F i g . 3. I n t h i s
example the newly t r a n s l a t e d I n h i b i t o r s I and I I were i s o l a t e d
as immunoprecipitates w i t h I n h i b i t o r I IgG p l u s I n h i b i t o r I I
IgG and electrophoresed i n 15% acrylamide gels i n the presence
of SDS and mercaptoethanol. The a n a l y s i s of r a d i o a c t i v i t y
35
( [ S ] m e t h i o n i n e ) i n c o r p o r a t e d i n t o each i n h i b i t o r i n the gels
was taken as a measure of the c o n c e n t r a t i o n of i n h i b i t o r mRNAs.
T r a n s l a t a b l e mRNAs f o r I n h i b i t o r s I and I I are present a t near
maximum l e v e l s w i t h i n 4 h r f o l l o w i n g wounding ( F i g . 4A). The
l e v e l s remain h i g h u n t i l 9 hr. A f t e r 9 h r the l e v e l s decrease
to l e s s than h a l f t h e i r o r i g i n a l l e v e l ( F i g . 4A). In the same
l e a v e s , during the same time, the r a t e s of i n v i v o accumulation
of I n h i b i t o r s I and I I ( F i g . 4B) s t e a d i l y increase during the

Figure 3. SDS-urea electrophoresis of proteinase Inhibitors I and IL Key: Lane 1,

proteinase Inhibitor II; Lane 2, proteinase Inhibitor I (both lanes stained with
35
Coomassie blue); and Lane 3, [ S]preproteinase Inhibitors I and II synthesized in
an in vitro rabbit reticulocyte system immunoprecipitated for Inhibitors I and II.

T 1 r
TIME (hours)
Figure 4. Time course analysis of the accumulation of Inhibitors I and II protein,

translatable mRNAs and apparent translational efficiencies in leaves of singly and
doubly wounded tomato plants. Key: —•—, Inhibitor I, single wound; — O — ,
Inhibitor II, single wound; — • —, Inhibitor I, double wound; and — O — ,
Inhibitor II, double wound.
A: In vitro translation of 4-fig quantities of tomato leaf mRNA and subsequent
isolation of specific preinhibitors through the preformed antibody technique (\\).
B: In vivo accumulation of Inhibitors I and II proteins in wounded tomato leaves.
C: Apparent translational efficiencies.

f i r s t 9 h r a f t e r wounding. By 14 h r the r a t e s of accumulation

have reached a steady s t a t e r a t e that remains constant f o r
s e v e r a l hours. This suggests that some s h i f t i n the c e l l i s
o c c u r r i n g a t about 9 h r that i s r e f l e c t e d i n both the i n v i t r o
t r a n s l a t i o n o f mRNA and i n the i n v i v o accumulation of i n h i b -
i t o r s . The cause o f the s h i f t i n l e v e l s of t r a n s l a t a b l e mRNA i s
unknown and could r e s i d e i n e i t h e r some s t r u c t u r a l f e a t u r e of
the mRNAs themselves that change t h e i r t r a n s l a t i o n a l r a t e s , o r
the d i f f e r e n c e s could r e s u l t i n changes i n t h e i r r a t e s of
s y n t h e s i s o r degradation. Nevertheless, the s h i f t r e s u l t s i n
species of mRNA that are r e s p o n s i b l e f o r the more e f f i c i e n t
steady s t a t e r a t e of s y n t h e s i s of the two i n h i b i t o r s a f t e r 9 h r .
A second wounding of the leaves 9 h r a f t e r the i n i t i a l
wounding, t r i p l e d the r a t e o f accumulation of both I n h i b i t o r s I
and I I ( F i g . 4B) over those of once wounded p l a n t s This second
wound a l s o r e s u l t e d i
present a t 9 h r so tha
wounded p l a n t s d i d not occur. The t r a n s l a t a b l e mRNA l e v e l s
remained high through the 18th h r ( F i g . 4A). Thus, high mRNA
l e v e l s a t 18 h r i n doubly wounded p l a n t s i s r e f l e c t e d i n over a
doubling of I n h i b i t o r s I and I I s y n t h e s i s and accumulation. As
shown i n F i g . 4C, a second wounding a f t e r 9 h r d i d not f u r t h e r
increase the t r a n s l a t i o n a l e f f i c i e n c y of I n h i b i t o r s I and I I
mRNA although i t s i g n i f i c a n t l y increases the r a t e s of i n h i b i t o r
s y n t h e s i s and accumulation. The second wound apparently provides
more mRNA when the p l a n t ' s t r a n s l a t i o n a l system i s already
operating a t high e f f i c i e n c y . The apparent increase i n t r a n s l a -
t i o n a l e f f i c i e n c y of mRNA a f t e r 9 h r i s not the r e s u l t of an
i n c r e a s e i n t o t a l poly(A) mRNA. Two separate e x t r a c t i o n s of
t o t a l mRNA from leaves of wounded tomato p l a n t s showed the
opposite; t h a t s u b s t a n t i a l l y l e s s poly(A) RNA was present a t 14
hr (40 ± 10.6 ug/g of l e a f ) and 18 h r (55 ± 3.5 ug/g of l e a f )
a f t e r a s i n g l e wound than a t 9 h r (96 ± 4.2 ug/g o f l e a f t i s s u e ) .
A s i m i l a r time course f o r t r a n s l a t a b l e mRNA has been
reported (14) i n b a r l e y aleurone l a y e r s , i n response t o g i b b e r e l -
+
l i c a c i d . T o t a l p o l y ( A ) RNA was found t o increase d r a m a t i c a l l y
i n the f i r s t 12 h r a f t e r hormone a p p l i c a t i o n , f o l l o w e d by a
r a p i d d e c l i n e t o 25% o f the maximum a t 18 h r . On the other
hand, the accumulation of ovalbumin mRNA i n response t o proges-
terone i n c h i c k o v i d u c t s (15) i s an example that does not appear
to behave i n t h i s manner.
The p o s s i b i l i t y that the d i f f e r e n c e i n apparent t r a n s l a t i o n
e f f i c i e n c i e s described here might be due t o the presence o f a
d i f f e r e n t , perhaps l a r g e r species of mRNA being present a t 9 o r
18 h r , was i n v e s t i g a t e d by a n a l y s i s of the s i z e s o f mRNAs f o r
I n h i b i t o r s I and I I on l i n e a r sucrose g r a d i e n t s . U l t r a c e n t r i f u -
g a t i o n of 200 ug a l i q u o t s of mRNA from leaves of 9- and 18-hr
s i n g l e wounded p l a n t s and 18-hr double wounded p l a n t s demon-
s t r a t e d that a l l three mRNAS migrated i d e n t i c a l l y ( F i g . 5 ) . To
+
l o c a t e the p o s i t i o n of p o l y ( A ) RNAs s p e c i f i c f o r I n h i b i t o r s I

i r
f r a c t i o n number
Figure 5. Size analysis of Inhibitors I and II specific mRNA from levels of 9- and
18-h singly wounded tomato plants and 18-h doubly wounded plants. PolyfA )* RNA
was applied to 15-30% linear sucrose gradients and was spun at 25,000 rpm.
Twenty-five fractions were collected, the absorbency was measured, and the mRNA
was precipitated by cold ethanol. In vitro translations were performed with each
fraction in a rabbit reticulocyte system, and isolation of the preinhibitors with
preformed antibody precipitates located the position of the two inhibitors. The
gradients were calibrated by centrifugation of tomato leaf poly(A)~ RNA on an
identical gradient. The locations of translatable mRNAs for Inhibitors I and II were
identical with RNA obtained from 9- and 18-h singly wounded or 18-h doubly
wounded plants.

and I I the e l u t e d gradient m a t e r i a l was p r e c i p i t a t e d w i t h ethanol

and i n v i t r o t r a n s l a t i o n performed. F r a c t i o n s c o n t a i n i n g p r e -
i n h i b i t o r s I and I I were i d e n t i f i e d by s p e c i f i c immunoprecipita-
t i o n o f the t r a n s l a t i o n products (data not shown). A n a l y s i s of
the gradient (16) w i t h known standards of tomato l e a f RNA that
do not bind t o o l i g o ( d T ) - c e l l u l o s e columns ( p o l y ( A ) ~ m a t e r i a l )
showed that I n h i b i t o r s I and I I mRNAs always migrated w i t h
sedimentation c o e f f i c i e n t s of 13S and 15S, r e s p e c t i v e l y .
In order t o f u r t h e r p u r i f y the s p e c i f i c messengers f o r the
i n h i b i t o r s , a d d i t i o n a l 15-30% g r a d i e n t s were run on mRNA samples
that had been p a r t i a l l y p u r i f i e d by o l i g o ( d T ) - c e l l u l o s e . Frac-
t i o n s corresponding t o the known p o s i t i o n of I n h i b i t o r s I and I I
mRNA were combined and r e c e n t r i f u g e d i n 10-25% l i n e a r sucrose
g r a d i e n t s ( F i g . 6 ) . By comparing the amount of r a d i o l a b e l
s p e c i f i c a l l y immunoprecipitated t o the t o t a l number of counts
incorporated i n the t o t a
r e l a t i v e p u r i t y of the
techniques have thus provided a 1 5 - f o l d p u r i f i c a t i o n of the
s p e c i f i c mRNA f o r I n h i b i t o r I and a 5 - f o l d p u r i f i c a t i o n of
I n h i b i t o r I I mRNA.
The mRNA f o r both I n h i b i t o r s I and I I appeared t o be
t y p i c a l of e u k a r y o t i c messengers that code f o r s m a l l p r o t e i n s of
+
8-12,000 daltons having a p o l y ( A ) t a i l s i n c e both messengers
bind s p e c i f i c a l l y t o o l i g o ( d T ) - c e l l u l o s e . There i s no evidence
of t r a n s l a t a b l e messengers f o r the two i n h i b i t o r s i n the RNA
f r a c t i o n that d i d not bind t o the o l i g o ( d T ) a f f i n i t y r e s i n (data
not shown).
The l e n g t h of poly(A) segments present i n poly(A) RNA from
pooled sucrose d e n s i t y gradient f r a c t i o n s r i c h i n I n h i b i t o r s I
and I I mRNA was determined by s u b j e c t i n g the RNA t o p a n c r e a t i c
and Tj RNase d i g e s t i o n , and l a b e l i n g the 5' t e r m i n a l ends w i t h
32
[ y - P ] A T P and p o l y n u c l e o t i d e k i n a s e . R e s u l t s of the length
determination i n polyacrylamide g e l s i n d i c a t e that poly(A)
fragments are about 100 n u c l e o t i d e s long. The d i s t r i b u t i o n of
lengths i n the experiment represent a v a r i e t y of mRNAs and the
t a i l lengths of the two I n h i b i t o r s are not known but are assumed
to be w i t h i n t h i s d i s t r i b u t i o n .
In a f u r t h e r experiment we assayed f o r the presence of a
cap s t r u c t u r e on the mRNAs f o r both I n h i b i t o r s I and I I by
competitive i n h i b i t i o n by 7-methyl-guanosine 5'-monophosphate
7 5f
( m G p ) of the i n v i t r o t r a n s l a t i o n of these messengers.
7 5?
Concentrations of 40 uM m G p i n h i b i t e d by 50% the i n v i t r o
+
t r a n s l a t i o n of t o t a l tomato l e a f p o l y ( A ) mRNA ( F i g . 7A). This
l e v e l i s 4 0 - f o l d lower than that r e q u i r e d to s i m i l a r l y i n h i b i t
r a b b i t g l o b i n mRNA t r a n s l a t e d i n a r a b b i t r e t i c u l o c y t e l y s a t e
(17) and 4 - f o l d lower than that r e q u i r e d t o i n h i b i t the same
mRNA i n a wheat germ system (18). I t was of i n t e r e s t that the
7 5
t r a n s l a t i o n of I n h i b i t o r I i s i n h i b i t e d t o 50% by 20 uM m G 'p
w h i l e 50% i n h i b i t i o n of I n h i b i t o r I I r e q u i r e s l e s s than 10 uM
( F i g . 7B). The b a s i s of t h i s d i f f e r e n c e i s not understood but

5 10 15 20 25
FRACTION NUMBER
Figure 6. Partial purification of Inhibitors I and II mRNA. Fractions containing
Inhibitors I and II mRNA determined by in vitro translation analyses were recov-
ered from an initial 15-30% linear sucrose gradient, precipitated by cold ethanol,
and applied to a 10-25% linear sucrose gradient. The sample was centrifuged for
36 h at 25,000 rpm. Fractions of the gradient were collected and subjected to in
35
vitro translation analyses. The upper graph represents total [ S]methionine incorpo-
ration assayed with 1 fxL of the translation mixture as described (11). The bottom
figure quantitates the radiolabel incorporated specifically into Inhibitor I (solid bars)
and Inhibitor II (open bars).

PARTIAL PURIFICATION OF INHIBITORS I AND I I mRNAs
% Counts
Purification Total Translatable I n h i b i t o r s

Total Translatable Protein
Inhibitor I Inhibitor I I
Oligo(dT) Chromatography 0.27% 0.22%
F i r s t Sucrose Gradient 0.42% 0.35%
Second Sucrose Gradient 4.1% 1.2%

7-METHYL GUANOSINE 5'-MONOPHOSPHATE (mM)
7 5
Figure 7. Inhibition of in vitro translation of tomato leaf mRNA by m G 'p.
Quantities of 4 fig mRNA from wounded tomato leaves were translated and
analyzed.
A: Total incorporation of [ S]methionine into trichloroacetic acid insoluble pro-
35
tein. Assays were done with 1-fiL fractions of the translation reaction as described
B: Incorporation of [ S]methionine into pre-Inhibitors I (O) and II (Q) as

35
isolated by preformed antibody precipitates.

could be explained i f there i s a d i f f e r e n c e between the a f f i n -

i t i e s of the two proteinase I n h i b i t o r mRNAs f o r the mRNA b i n d i n g
s i t e on the 40S i n i t i a t i o n complex o r between the I n v i v o cap
7 5
s t r u c t u r e s . I n h i b i t i o n of t r a n s l a t i o n by d e r i v a t i v e s of m G 'p
1
has been used as evidence that a s p e c i f i c mRNA contains a 5
t e r m i n a l cap s t r u c t u r e (19), but t h i s i n t e r p r e t a t i o n must be
t r e a t e d w i t h c a u t i o n (20). However, the f a c t that the t r a n s l a -
t i o n of I n h i b i t o r s I and I I mRNA i s s e v e r a l f o l d more s t r o n g l y
7 5,
i n h i b i t e d by m G p than g l o b i n mRNA lends support t o the
1
presence of capped s t r u c t u r e s a t the 5 terminus of the i n h i b i t o r
mRNAs. The unequivocal i d e n t i f i c a t i o n of the cap s t r u c t u r e s
w i l l r e q u i r e p u r i f i c a t i o n of the mRNAs f o r each i n h i b i t o r .
Thus, s t u d i e s of the p r o p e r t i e s of the mRNAs i s o l a t e d a t
d i f f e r e n t times a f t e r wounding do not r e v e a l any d i f f e r e n c e s
that might r e f l e c t the l a r g e changes i n t r a n s l a t i o n a l e f f i c i e n -
c i e s observed i n tomat
t h i s time there may b
and i n t e r n a l methylation. I t i s a l s o p o s s i b l e that some c e l l u l a r
component i n v o l v e d w i t h s y n t h e s i s and t r a n s p o r t of i n h i b i t o r s
i n t o the c e n t r a l vacuole may be i n v o l v e d . The vacuole o r l y s o -
some i s considered t o o r i g i n a t e w i t h the G o l g i apparatus.
C e l l u l a r events during the f i r s t hours f o l l o w i n g wounding may be
i n v o l v e d w i t h the production of a component(s) t o f a c i l i t a t e
ribosome-mRNA b i n d i n g o r some other process i n the G o l g i appara-
tus t o s p e c i f i c a l l y accommodate t r a n s p o r t of I n h i b i t o r s I and I I
i n t o the vacuole.
A l t e r n a t i v e l y , poor e f f i c i e n c i e s of i n h i b i t o r mRNAs may be
due t o t h e i r i n c o r p o r a t i o n i n t o r i b o n u c l e o p r o t e i n p a r t i c l e s
(RNPs) such as found i n sea u r c h i n embryos (21). Newly made
mRNA i n the embryos i s found i n RNPs and they apparently have
"weak" template a c t i v i t i e s w h i l e i n these p a r t i c l e s . The
presence of newly synthesized tomato mRNA i n s i m i l a r p a r t i c l e s
might e x p l a i n the apparently low t r a n s l a t i o n a l e f f i c i e n c i e s
noted h e r e i n . The use of chaotropic b u f f e r s i n the p r e p a r a t i o n
of tomato l e a f mRNA (11) would not d i f f e r e n t i a t e between f r e e o r
polysome-bound mRNAs and those complexed i n RNPs. I f an RNP o r
s i m i l a r p a r t i c l e i s i n v o l v e d , then i t s r o l e must be a temporal
one s i n c e a second wound does not repeat the phenomenon ( F i g . 4 ) .
Cloning of the mRNAs enriched i n I n h i b i t o r s I and I I
messages i s underway t o provide cDNA probes t o more f u l l y
explore the r e g u l a t i o n of the tomato genes coding f o r I n h i b i t o r s
I and I I i n response t o wounding. With such clones not only can
d i r e c t h y b r i d i z a t i o n techniques be employed t o probe the l e v e l s
of PIIF-induced mRNA, but more i m p o r t a n t l y , the genes f o r the
i n h i b i t o r s can h o p e f u l l y be i s o l a t e d , the s t r u c t u r a l features of
the genes be s t u d i e d and the molecular b a s i s f o r the r e g u l a t i o n
of gene expression i n response t o s t r e s s can be explored.

Summary
The wound-induced s y n t h e s i s and accumulation of p r o t e i n a s e

I n h i b i t o r s I and I I i n tomato leaves has provided a model system
to study the r e g u l a t i o n of p r o t e i n a s e i n h i b i t o r genes i n p l a n t s .
The s i m p l i c i t y of the phenomenon has made i t p o s s i b l e t o i s o l a t e
the wound-factor, o r hormone, and t o study i t s r e l e a s e , d i r e c t i o n
and r a t e of t r a n s p o r t i n tomato p l a n t s . Messenger RNA has been
i s o l a t e d from leaves of wounded p l a n t s and c o n t a i n s t r a n s l a t a b l e
mRNAs f o r the two p r o t e i n a s e i n h i b i t o r s . Studies w i t h these
mRNAs have provided a b a s i s f o r the i n i t i a t i o n of a program t o
clone i n h i b i t o r cDNAs f o r s t u d i e s of the molecular b a s i s o f the
wound-induced process of i n h i b i t o r s y n t h e s i s .
Acknowledgements
We would l i k e t o
a s s i s t a n c e and Richard Hamlin f o r growing the p l a n t s .
Literature Cited
1. Green, T.; Ryan, C.A. Science 1972, 175, 776-777.

2. Ryan, C.A. "Current Topics in Cellular R e g u l a t i o n " ; E.
Stadtman; B.L. Horecker, eds., Academic P r e s s : New York,
1980; pp 1-23.
3. Bishop, P.D.; Makus, D.J.; Pearce, G.; Ryan, C.A. Proc.
N a t l . Acad. Sci. (USA) 1981, 78, 3536-3540.
4. Ryan, C.A.; Bishop, P.; Pearce, G.; Darvill, A.; M c N e i l ,
M.; Albersheim, P. P l a n t P h y s i o l . 1982, 68, 616-618.
5. Ryan, C.A. P l a n t P h y s i o l . 1974, 54, 328-332.
6. Green, T.R.; Ryan, C.A. P l a n t P h y s i o l . 1972, 51, 19-21.
7. Canny, M.J. "Phloem T r a n l o c a t i o n " ; Cambridge Univ. P r e s s :
Cambridge, 1973; pp 301-312.
8. M i n c h i n , P.E.H.; Troughton, J.H. Ann. Rev. P l a n t P h y s i o l .
1980, 31, 191-215.
9 F i s h e r , D.B. P l a n t P h y s i o l . 1970, 45, 114-118.
10. P l u n k e t t , G.; Senear, D.F.; Zuroske, G.; Ryan, C.A. Arch.
Biochem. Biophys. 1982, 213, 463-472.
11. Nelson, C.E.; Ryan, C.A. Proc. N a t l . Acad. Sci. (USA) 1980,
77, 1975-1979.
12. Walker-Simmons, M.; Ryan, C.A. P l a n t P h y s i o l . 1977, 60,
61-63.
13. Nelson, C.E.; Ryan, C.A. Biochem. Biophys. Res. Commun.
1980, 94, 355-359.
14. Muthukrishnan, S.; Chanda, G.R.; Maxwell, E.S. Proc. N a t l .
Acad. Sci. (USA) 1979, 76, 6181-6185.
15. Mulvihill, E.R.; P a l m i t e r , R.D. J. Biol. Chem. 1980, 255,
2085-2091.

16. McConkey, E.H. "Methods in Enzymology," V o l . 12, P t . A; L.

Grossman; K. Moldave, eds., Academic P r e s s : New York, 1967;
pp 620-634.
17. Fresno, M.; Vazquez, D. Eur. J. Biochem. 1980, 103,
125-132.
18. Hickey, E.D.; Weber, L.A.; B a g l i o n i , C. Proc. N a t l . Acad.
Sci. (USA) 1976, 73, 19-23.
19. F i l i p o w i c z , W. FEBS L e t t . 1978, 96, 1-11.
20. Takagi, S.; M o r i , T. J . Biochem. 1979, 86, 231-238.
21. Rudensey, L.M.; I n f a n t e , A.A. Biochem. 1979, 18, 3056-3063.
RECEIVED August 23, 1982

7
Plant Polyphenols and Their Association
with Proteins
J O H N M c M A N U S , T E R E N C E H. L I L L E Y , and E D W I N H A S L A M
University of Sheffield, Department of Chemistry, Sheffield S3 7HF, United Kingdom
Plant polyphenols (syn. vegetable tannins) which complex

strongly with protein
types. The first group
an oligomeric flavan-3-ol structure and the second class
is composed of esters of gallic acid, (R) and (S)-hexa-
hydroxydiphenic acid, and their derivatives usually with
D-glucose. Polyphenols associate with proteins princip-
ally by intermolecular hydrogen bonding. A hypothesis
is advanced to explain the propensity of polyphenols,
as their molecular size increases, to precipitate
proteins from solution. An important corollorary of
this theory is that simple phenols (M.Wt. < 200) should
display similar behaviour if they can be maintained in
solution at sufficiently high concentrations.
For a c e n t u r y and more t h e e l u c i d a t i o n o f t h e c h e m i s t r y o f a

n a t u r a l p r o d u c t (J_, s e c o n d a r y m e t a b o l i t e ) has been a d o m i n a n t theme
of organic chemistry. Today t h e e m p h a s i s has c h a n g e d . The
q u e s t i o n - "what i s t h e r o l e o f s e c o n d a r y m e t a b o l i s m i n t h e l i f e
o f p l a n t s and m i c r o - o r g a n i s m s ? " - i s one t o w h i c h i n v e s t i g a t o r s
i n c r e a s i n g l y address themselves. The p r o b l e m r e m a i n s e s s e n t i a l l y
u n r e s o l v e d a l t h o u g h numerous i d e a s have been p u t f o r w a r d . Several
p r o p o s i t i o n s c e n t r e on t h e s u g g e s t i o n t h a t i t i s t h e p r o c e s s o f
secondary metabolism and n o t , i n t h e general c a s e , t h e secondary
m e t a b o l i t e s themselves which a r e o f importance t o t h e organism
(2). T h e s e s u g g e s t i o n s do n o t e x c l u d e t h e p o s s i b i l i t y , indeed
p r o b a b i l i t y , that the d i s t i n c t i v e p r o p e r t i e s o f i n d i v i d u a l second-
a r y m e t a b o l i t e s have, over t h e course o f e v o l u t i o n , secured a
p a r t i c u l a r n i c h e f o r an o r g a n i s m i n t h e l i v i n g w o r l d . Contrasting
w i t h t h i s v i e w p o i n t a r e t h o s e w h i c h s u g g e s t t h a t an o r g a n i s m ' s
c a p a c i t y t o d e f e n d i t s e l f a g a i n s t p r e d a t i o n by some o r g a n i s m s and
t o a t t r a c t o t h e r s i n c l u d e d t h e e v o l u t i o n o f an a b i l i t y t o s y n -
t h e s i s e an a r r a y o f s e c o n d a r y m e t a b o l i t e s w h i c h i n a p p r o p r i a t e
c i r c u m s t a n c e s m i g h t r e p e l o r a t t r a c t o t h e r o r g a n i s m s (3 > ^ 0 •
0097-6156/83/0208-0123$06.00/0

Plant Polyphenols
P o l y p h e n o l s are a d i s t i n c t i v e group o f h i g h e r p l a n t secondary

m e t a b o l i t e s ( 5 ) . Two i m p o r t a n t c l a s s e s - t h e p r o a n t h o c y a n i d i n s
( 1 , F i g u r e 1) and e s t e r s o f g a l l i c a c i d and (R) and ( S ) - h e x a d y d r o -
x y d i p h e n i c a c i d ( F i g u r e 5) a r e u n i q u e and t h e i r mode o f b i o s y n -
t h e s i s i n the p l a n t e n a b l e s m o l e c u l e s o f m o l e c u l a r w e i g h t > 500
and i n many c a s e s o f a t l e a s t 2-3,000 t o be formed. These m o l e c -
u l e s , i n r e l a t i v e l y low c o n c e n t r a t i o n s , m o r e o v e r p o s s e s s the imp-
o r t a n t p r o p e r t y o f p r e c i p i t a t i n g p r o t e i n s , p o l y s a c c h a r i d e s and
some a l k a l o i d s f r o m s o l u t i o n . This association with proteins i s
the b a s i s o f t h e use o f p o l y p h e n o l s ( s y n . t a n n i n s ) as a g e n t s f o r
the c o n v e r s i o n o f raw a n i m a l s k i n s t o d u r a b l e , impermeable l e a t h e r
(6). I t has a l s o been r e c o g n i s e d as u n d e r l y i n g many o t h e r phe-
nomena a s s o c i a t e d w i t h p l a n t t i s s u e s . Thus i t i s h e l d t o be r e s -
p o n s i b l e f o r the a s t r i n g e n c
t e a and w i n e , f o r t h e i m p a i r e
some f o o d s and f o r the i n a c t i v a t i o n o f m i c r o b i a l enzymes and p l a n t
viruses. The c a p a c i t y o f p l a n t s t o m e t a b o l i s e t h e s e two c l a s s e s
o f p o l y p h e n o l i s a p r i m i t i v e c h a r a c t e r i s t i c t h a t has t e n d e d t o
become l o s t w i t h i n c r e a s i n g p h y l o g e n e t i c s p e c i a l i s a t i o n . Accord-
i n g t o B a t e - S m i t h (7) t h e i r i m p o r t a n c e t o t h e p l a n t l i e s i n t h e i r
e f f e c t i v e n e s s as r e p e l 1 a n t s t o p r e d a t o r s b r o u g h t a b o u t by t h e i r
a b i l i t y t o p r e c i p i t a t e p r o t e i n s w h e t h e r t h i s be an e x t r a c e l l u l a r
m i c r o b i a l enzyme o r the s a l i v a r y p r o t e i n o f a b r o w s i n g a n i m a l .
P r o a n t h o c y a n i d i n s and P r o c y a n i d i n s - In a c l a s s i c a l s t u d y B a t e -
S m i t h (8) used t h e p a t t e r n s o f d i s t r i b u t i o n o f the t h r e e p r i n c i -
pal c l a s s e s o f p h e n o l i c m e t a b o l i t e s , which are found i n the
l e a v e s o f p l a n t s , as a b a s i s f o r c l a s s i f i c a t i o n . The b i o s y n t h e -
s i s of these phenols - ( i ) p r o a n t h o c y a n i d i n s ; ( i i ) g l y c o s y l a t e d
f l a v o n o l s and ( i i i ) h y d r o x y c i n n a m o y l e s t e r s - i s b e l i e v e d t o be
a s s o c i a t e d w i t h the d e v e l o p m e n t i n p l a n t s o f t h e c a p a c i t y t o
s y n t h e s f s e t h e s t r u c t u r a l p o l y m e r l i g n i n by the d i v e r s i o n f r o m
p r o t e i n s y n t h e s i s o f the a m i n o - a c i d s L - p h e n y l a l a n i n e and L - t y r o -
sine. V a s c u l a r p l a n t s t h u s e m p l o y one o r more o f t h e p - h y d r o x y -
cinnamyl a l c o h o l s ( 2 , 3 , and 4 ) , w h i c h a r e d e r i v e d by e n z y m i c
r e d u c t i o n (NADH) o f the coenzyme A e s t e r s o f the c o r r e s p o n d i n g
h y d r o x y c i n n a m i c a c i d s , as p r e c u r s o r s t o l i g n i n . The same coenzyme
A e s t e r s a l s o f o r m the p o i n t s o f b i o s y n t h e t i c d e p a r t u r e f o r the
t h r e e g r o u p s o f p h e n o l i c m e t a b o l i t e s ( ? , i i , i i i ) , F i g u r e 1.
The two p r i n c i p a l c l a s s e s o f p r o a n t h o c y a n i d i n s f o u n d (10) i n
p l a n t t i s s u e s a r e t h e p r o c y a n i d i n s ( 1 , R = H) and the p r o d e l p h i n -
i d i n s ( 1 , R = OH). P r o a n t h o c y a n i d i n s o f mixed a n t h o c y a n i d i n
c h a r a c t e r ( 1 , R = H o r OH) have been n o t e d . In any t i s s u e where
p r o a n t h o c y a n i d i n s y n t h e s i s o c c u r s t h e r e i s i n v a r i a b l y found a
range o f m o l e c u l a r s p e c i e s - f r o m the monomeric f l a v a n - 3 - o l s
( c a t e c h i n s , g a l l o c a t e c h i n s ) t o t h e p o l y m e r i c f o r m s (1) and b i o -
s y n t h e t i c work (11) s u g g e s t s a v e r y c l o s e r e l a t i o n s h i p between
t h e m e t a b o l i s m o f t h e p a r e n t f l a v a n - 3 " o l and the s y n t h e s i s o f
p r o a n t h o c y a n i d i n s , F i g u r e 4.

7. MCMANUS ET AL. Plant Polyphenols 125
The m a j o r i t y o f t h e s t r u c t u r a l and b i o s y n t h e t i c work i n t h i s

f i e l d has been d i r e c t e d t o w a r d s a n u n d e r s t a n d i n g o f t h e p r o c y a n i d -
i n s ( 1 , R = H) - t h e m a j o r g r o u p o f n a t u r a l p r o a n t h o c y a n i d i n s ,
( 1 1 ) F r u i t b e a r i n g p l a n t s have p r o v e d t o be p a r t i c u l a r l y r i c h
s o u r c e s o f o l i g o m e r i c condensed p r o c y a n i d i n s ( 1 1 , 12, 13) and
s t u d i e s have c o n c e n t r a t e d upon t h e f o u r m a j o r n a t u r a l l y o c c u r r i n g
d i m e r i c p r o c y a n i d i n s ( B - 1 , B-2, B-3 and B-4, F i g u r e 2) w h i c h have
been i s o l a t e d f r o m f r u i t , f r u i t p o d s , seeds a n d seed s h e l l s ,
l e a v e s and o t h e r t i s s u e s o f a w i d e r a n g e o f p l a n t s . The p r o c y a n -
i d i n s o c c u r f r e e and u n g l y c o s y l a t e d and a l m o s t i n v a r i a b l y t h e y a r e
found w i t h one o r b o t h o f t h e p a r e n t f 1 a v a n - 3 - o l s , ( + ) - c a t e c h i n o r
( - ) - e p i c a t e c h i n , F i g u r e 2. The p a t t e r n s o f o c c u r r e n c e o f t h e p r o -
c y a n i d i n s a r e most e f f e c t i v e l y r e v e a l e d by h p l c o r by two d i m e n -
s i o n a l paper c h r o m a t o g r a p h y and t h e y may be used a s a t a x o n o m i c
g u i d e ( 1 1 ) . The a b s o l u t e s t e r e o c h e m i s t r y i n p r o c y a n i d i n s B-1 and
B-2 was d e t e r m i n e d a s kR
occupies a q u a s i - a x i a l
h e t e r o c y c l i c r i n g (]k). Conversely the absolute stereochemistry
i n p r o c y a n i d i n s B-3 and B-4 was shown t o be and h e r e t h e i n t e r -
f l a v a n bond i s t h u s i n a q u a s i - e q u a t o r i a l p o s i t i o n on t h e h e t e r o -
c y c l i c r i n g ( 1 4 ) . Thus t h e p r i n c i p a l p r o c y a n i d i n d i m e r s ( F i g u r e
2) a l l p o s s e s s a t r a n s o r i e n t a t i o n o f t h e C~3 h y d r o x y l group and
t h e f l a v a n s u b s t i t u e n t a t C-4.
D e s p i t e t h e s u c c e s s f u l a p p l i c a t i o n o f s p e c t r o s c o p i c methods t o
the s t r u c t u r e d e t e r m i n a t i o n o f t h e d i m e r i c p r o c y a n i d i n s anomalous
f e a t u r e s o f t h e s p e c t r a l e d t o s p e c u l a t i o n (_V2, J 5 , J 6 ) t h a t
r e s t r i c t e d r o t a t i o n e x i s t e d a b o u t t h e i n t e r f l a v a n bond. A
d e t a i l e d s t u d y (14) c o n f i r m e d t h i s h y p o t h e s i s and d e f i n e d two
d i f f e r e n t forms o f h i n d e r e d r o t a t i o n a s s o c i a t e d w i t h t h e two
g r o u p s o f p r o c y a n i d i n d i m e r s (B-1 and B-2, kR - a b s o l u t e c o n f i g -
u r a t i o n ) and (B-3 and B-4, 4 S - a b s o l u t e c o n f i g u r a t i o n ) . Examin-
a t i o n o f m o l e c u l a r models shows t h a t , a l t h o u g h s e v e r a l c o n f o r m a t -
i o n s a r e a c c e s s i b l e by i n t e r f l a v a n bond r o t a t i o n , t h e r e e x i s t s one
p r e f e r r e d conformation f o r each o f the p a i r s o f n a t u r a l p r o c y a n i d -
ins. I f t h e s e a r e v i e w e d f r o m d i f f e r e n t a n g l e s t h e y bear an
a l m o s t o b j e c t t o m i r r o r image r e l a t i o n s h i p ; the structures are
q u a s i - e n a n t i o m e r i c ( F i g u r e 3). Elaboration of the oligomeric
p r o c y a n i d i n s (J^, R = H) by t h e a d d i t i o n o f f u r t h e r i d e n t i c a l
f l a v a n - 3 - o l u n i t s , b e a r i n g i n mind t h e c o n f o r m a t i o n a l r e s t r a i n t s
a b o u t t h e i n t e r f l a v a n bond, l e a d s t o two g e n e r a l s t r u c t u r e s w h i c h
may a d o p t h e l i c a l c o n f o r m a t i o n s w i t h o p p o s i t e h e l i c i t i e s ( 1 1 ) .
The c e n t r a l c o r e o f t h e s e p o l y m e r s i s compcse d o f r i n g s A a n d C o f
t h e f l a v a n r e p e a t u n i t and r i n g B - t h e o r t h o - d i h y d r o x y p h e n y l
r i n g - p r o j e c t s l a t e r a l l y from t h i s c o r e .
E a r l i e r p r o p o s a l s (_12^ J_3, 17) and t h e r e s u l t s o f b i o s y n t h e t i c
e x p e r i m e n t s (18) have been adumbrated i n t o a scheme o f b i o s y n t h e s i s
f o r t h e p r o c y a n i d i n s ( F i g u r e k) i n w h i c h i t i s s u g g e s t e d t h a t t h e y
a r e formed a s b y p r o d u c t s d u r i n g t h e f i n a l s t a g e o f t h e s y n t h e s i s
o f t h e p a r e n t f 1 a v a n - 3 ~ o l s t r u c t u r e s , ( + ) - c a t e c h i n and ( - ) -
e p i c a t e c h i n (_[_[, _18) . A two s t e p r e d u c t i o n o f t h e f 1 av-3~en-3-ol

lignin
(1 R = H or OH )
Figure 1. Phenol biosynthesis in higher plants.

to
Principal naturally occurring dimeric procyanidins.
Figure 2.

PLANT RESISTANCE TO INSECTS

B-4 5 6 5
Figure 4. Biosynthesis of procyanidins.

Is p o s t u l a t e d . I f t h e s u p p l y o f NADPH ( s a y ) i s r a t e l i m i t i n g t h e
i n t e r m e d i a t e C-4 c a r b o c a t i o n s (3S o r 3R s t e r e o c h e m i s t r y ) m i g h t
e s c a p e f r o m t h e enzyme a c t i v e s i t e and r e a c t w i t h t h e r e d u c t i o n
p r o d u c t , t h e f l a v a n - 3 - o l , t o p r o d u c e d i m e r s , t r i m e r s ... and
e v e n t u a l l y polymers. The h y p o t h e s i s h a s been e l a b o r a t e d t o
a c c o u n t f o r t h e v a r i o u s s t e r e o c h e m i c a l f e a t u r e s (11) and has been
s u p p o r t e d by i n v i t r o b i o m i m e t i c e x p e r i m e n t s . The b a l a n c e between
t h e m e t a b o l i c f l u x t o t h e f l a v - 3 - e n - 3 - o l and t h e r a t e o f s u p p l y
o f t h e b i o l o g i c a l r e d u c t a n t (NADPH) p r o b a b l y d e t e r m i n e s t h e
b a l a n c e between t h e l o w e r and h i g h e r o l i g o m e r i c forms o f t h e p r o -
c y a n i d i n s i n each p l a n t t i s s u e . Thus t i s s u e s w i t h a h i g h f l u x
and p o o r s u p p l y o f NADPH w i l l c o n t a i n p r e d o m i n a n t l y t h e h i g h e r
o l i g o m e r s - f o r example t h e seed c o a t o f sorghum ( 1 9 ) .
T h i s f a c e t o f p r o a n t h o c y a n i d i n m e t a b o l i s m was n o t e d by e a r l y
w o r k e r s (20) who r e c o g n i s e d t h e p r e s e n c e i n p l a n t s o f l e u c o a n t h o -
cyanins (proanthocyanidins
i n c r e a s i n g degrees o f
more d i f f i c u l t t o s o l u b i l i s e i n aqueous and a l c o h o l i c media and
t h o s e w h i c h may be c o a x e d i n t o s o l u t i o n may have m o l e c u l a r w e i g h t s
up t o 7,000 - c o r r e s p o n d i n g t o s t r u c t u r e s s u c h a s ( 1 ) where
n s 18. R e c e n t work has c o n c e n t r a t e d upon t h e s t r u c t u r a l e x a m i n -
a t i o n o f these polymers. A l t h o u g h B a t e - S m i t h (8) g e n e r a l l y
d i s c o u n t e d a c o n n e c t i o n w i t h l i g n i n , an e x a m i n a t i o n o f t h e p r o a n -
t h o c y a n i d i n p o l y m e r s , t o a s c e r t a i n i f t h e y do p l a y a s t r u c t u r a l
r o l e w o u l d be v e r y v a l u a b l e .
E s t e r s o f G a l l i c and H e x a h y d r o x y d i p h e n i c A c i d s - An a c u t e
o b s e r v a t i o n made by B a t e - S m i t h ( 8 ) i n h i s t a x o n o m i c work was t h e
complete absence from p l a n t t i s s u e s o f 3 , ^ , 5 " t r i h y d r o x y c i n n a m i c
acid. In s i t u a t i o n s where i t s o c c u r r e n c e m i g h t w e l l have been
p r e d i c t e d , he f o u n d h e x a h y d o r x y d i p h e n i c a c i d and i t s d e r i v a t i v e s
( F i g u r e 5 ) . R e c e n t work has d e m o n s t r a t e d t h e u b i q u i t y i n p l a n t s
o f e s t e r s o f R and S h e x a h y d r o x y d i p h e n i c a c i d w i t h D - g l u c o s e and
o f t h e i r presumed b i o g e n e t i c p r e c u r s o r s t h e g a l l o y l - D - g l u c o s e s
(22, 2 3 ) . G a l l i c a c i d ( F i g u r e 5) t h u s o c c u p i e s a d i s t i n c t i v e
p o s i t i o n i n p h e n o l i c metabolism i n p l a n t s . Almost always i t
o c c u r s i n e s t e r form but i n c o n t r a s t t o o t h e r n a t u r a l p h e n o l i c
a c i d s , w h i c h a r e i n v a r i a b l y f o u n d a s mono- and o c c a s i o n a l l y b i s -
e s t e r s w i t h p o l y o l s , g a l l i c a c i d i s e n c o u n t e r e d i n a range o f
e s t e r i f i e d forms f r o m t h e s i m p l e m o n o e s t e r s t o t h e c o m p l e x p o l y -
e s t e r s w i t h D - g l u c o s e whose m o l e c u l a r w e r g h t s e x t e n d t o a t l e a s t
2,000. The s t r u c t u r a l p a t t e r n s d i s c e r n e d amongst g a l l i c a c i d
m e t a b o l i t e s a r e n e v e r t h e l e s s h i g h l y r e m i n i s c e n t o f t h o s e found i n
o t h e r v a r i a n t s o f secondary metabolism i n o t h e r organisms - e.g.
p o l y k e t i d e s i n moulds and f u n g i .
G a l l i c a c i d i s most commonly a s s o c i a t e d w i t h D - g l u c o s e . Many
p l a n t s m e t a b o l i s e s i m p l e e s t e r s and t h e r e a p p e a r s t o be a p r e d i s -
p o s i t i o n f o r c e r t a i n p o s i t i o n s o f e s t e r i f i c a t i o n on t h e D - g l u c o s e
p y r a n o s e c o r e (1 > 6 > 2 > 3 , * 0 . The m e t a b o l i s m o f 3 - p e n t a k i s -
g a l l o y l - D - g l u c o s e (Figure5) r e p r e s e n t s something o f a m e t a b o l i c

7. MCMANUS ET A L . Plant Polyphenols 131
C°2~
Carbohydrate
OH
* C0 H 2
HO^^OH
Glucose 3 OH Gallic acid G
Group 1 mono
its bis
1,2,6 tris
G -Rrntagalloy I-D-glucose
.OG
Group II
OGi
GO i OG V
depside -2H
-1 / ™ I •2H
OG
1 \ ~ Hp A 6
/?Y
0
GO^^h^OG
3
o G
X
0
GO
-2H
G
OG
1.6 -2,4
M
A-Gallotannins' GO . GO
X
° ^ ^ o c | 0 0
0'
I G^GX OG
I G-G
Dimers *>
\ Dimers,Trimers ?
G-G Trimers ?
Ellagitannins - — C
Hexahydroxydiphenic B
acid
Figure 5. Metabolism of gallic acid and hexahydroxydiphenic acid.

w a t e r s h e d and f r o m t h i s p o i n t a t l e a s t t h r e e d i f f e r e n t b i o s y n t h -
e t i c pathways d i v e r g e . From a t a x o n o m i c p o i n t o f v i e w t h e s e
pathways a r e q u i t e d i s t i n c t (2k). One pathway p r o c e e d s t o f o r m
1 1
d e p s i d e m e t a b o l i t e s ( g a l l o t a n n i n s , g r o u p M A ) . Here f u r t h e r
g a l l i c a c i d m o l e c u l e s a r e e s t e r i f i e d as m-depsides t o 3 - p e n t a k i s -
g a l l o y l - D - g l u c o s e , p r e f e r e n t i a l l y a t p o s i t i o n s 3, and 6 . This
s u p p o r t s t h e v i e w o r i g i n a l l y e x p r e s s e d by Emil F i s c h e r (25) t h a t
these e s t e r s a r e not o n l y m i x t u r e s o f isomers but a l s o o f
substances of d i f f e r i n g e m p i r i c a l formulae.
A s e c o n d and w i d e l y o b s e r v e d p a t t e r n o f m e t a b o l i s m o f $-
p e n t a k i s - g a l l o y l - D - g l u c o s e i s t h a t ( M B , F i g u r e 5) i n w h i c h t h e
s u b s t r a t e i s f u r t h e r t r a n s f o r m e d by o x i d a t i v e c o u p l i n g o f p a i r s
o f a d j a c e n t g a l l o y l e s t e r g r o u p s ( 2 , 3 and 4 , 6) on t h e D - g l u c o -
pyranose r i n g t o g i v e hexahydroxydiphenoyl e s t e r s . Further
m e t a b o l i s m o f t h e s e i n t e r m e d i a t e s t h e n a l s o o c c u r s by i n t e r m o l e c -
u l a r C-0 o x i d a t i v e c o u p l i n
i s o l a t e d f r o m Rubus s p e c i e
c o u p l i n g o f g a l l o y l e s t e r groups takes p l a c e w i t h t h e p r e c u r s o r
4
adopting the e n e r g e t i c a l l y preferred ( Ci) conformation o f the
sugar r i n g . Coupled w i t h t h e o b s e r v a t i o n that t h i s i s t h e
p r e d o m i n a n t mode o f f u r t h e r o x i d a t i v e m e t a b o l i s m o f g a l l o y l e s t e r s
i n p l a n t s , i t a l s o seems r e a s o n a b l e t o c o n c l u d e t h a t i t i s t h e
e n e r g e t i c a l l y p r e f e r r e d pathway.
A t h i r d and r e l a t i v e l y m i n o r pathway o f m e t a b o l i s m o f 3 -
p e n t a k i s - g a l l o y l - D - g l u c o s e ( I I C , F i g u r e 5) o c c u r s i n some p l a n t s .
Here o x i d a t i v e m e t a b o l i s m t a k e s p l a c e v i a t h e e n e r g e t i c a l l y
l
u n f a v o u r a b l e Ci+ c o n f o r m a t i o n o f t h e s u b s t r a t e and c o u p l i n g o f t h e
g a l l o y l e s t e r g r o u p s o c c u r s 1, 6 o r 3 , 6 t o g i v e h e x a h y d r o x y -
diphenoyl e s t e r s o r 2, k t o give the dehydrohexahydroxydiphenoyl
ester. The compound g e r a n i i n i s a key f i g u r e i n t h i s pathway o f
m e t a b o l i sm ( 2 6 ) .
Comparative Aspects o f Polyphenol Metabolism - Proanthocyanidins

and t h e c o m p l e x e s t e r s o f g a l l i c and h e x a h y d r o x y d i p h e n i c a c i d
show many s t r u c t u r a l s i m i l a r i t i e s a s p l a n t m e t a b o l i t e s . The shape
and s i z e o f t h e e s t e r (5) i s t h u s v e r y s i m i l a r t o t h a t o f a
p r o a n t h o c y a n i d i n hexamer ( 1 , n = 4 ) . The most s t r i k i n g f e a t u r e
o f b o t h s t r u c t u r e s h o w e v e r ^ i s t h e manner i n w h i c h f r e e p h e n o l i c
groups a r e d i s t r i b u t e d over t h e s u r f a c e o f t h e m o l e c u l e p r o v i d i n g
a s t r u c t u r e w i t h the i n b u i l t c a p a c i t y f o r m u l t i d e n t a t e attachment
t o o t h e r s p e c i e s by h y d r o g e n b o n d i n g .
A c u r i o u s but p e r h a p s s i g n i f i c a n t o b s e r v a t i o n i s t h a t , a l t h o u g h
several plant f a m i l i e s retain the a b i l i t y to biosynthesise
d i f f e r e n t t y p e s o f complex p o l y p h e n o l , r a r e l y a r e both b i o s y n t h e t i c
c a p a b i l i t i e s d i s p l a y e d m a x i m a l l y . More o f t e n one p a r t i c u l a r
s p e c i e s s p e c i a l i s e s i n one p a r t i c u l a r mode. Thus i n t h e E r i c a c e a e
many p l a n t s a r e r i c h s o u r c e s o f p r o a n t h o c y a n i d i n s b u t A r c t o -
s t a p h y l o s u v a - u r s i has o n l y a m i n i m a l p r o a n t h o c y a n i d i n b i o -
s y n t h e t i c c a p a c i t y but c o m b i n e s w i t h t h i s a v e r y h i g h l e v e l o f
g a l l i c a c i d metabolism.


Polyphenol Interactions with Proteins
S t u d i e s o f t h e a s s o c i a t i o n o f p o l y p h e n o l s w i t h p r o t e i n s have a
long h i s t o r y (27). Loomis (28) h a s s u c c i n c t l y summarised t h e
c o n c l u s i o n s o f t h i s e a r l i e r work. The p r i n c i p a l means whereby
p r o t e i n s and p o l y p h e n o l s a r e t h o u g h t t o r e v e r s i b l y c o m p l e x w i t h
one a n o t h e r a r e ( i ) h y d r o g e n b o n d i n g , ( i i ) i o n i c i n t e r a c t i o n s and
( i i i ) hydrophobic i n t e r a c t i o n s . W h i l s t t h e major t h r u s t i n e a r l i e r
work was t o e m p h a s i z e t h e p a r t p l a y e d by i n t e r m o l e c u l a r h y d r o g e n
b o n d i n g i n t h e c o m p l e x a t i o n , H o f f (29) has drawn a t t e n t i o n t o t h e
p o s s i b i l i t y t h a t h y d r o p h o b i c e f f e c t s may d o m i n a t e t h e a s s o c i a t i o n
between t h e two s p e c i e s .
R e c e n t o b s e r v a t i o n s however now p e r m i t a h y p o t h e s i s t o be
advanced t o e x p l a i n t h e p r o p e n s i t y o f c o m p l e x p o l y p h e n o l s t o
p r e c i p i t a t e p r o t e i n s from aqueous s o l u t i o n ( 3 0 ) Two s i t u a t i o n s
may be e n v i s a g e d . At lo
associates - principall
d i h y d r o x y p h e n y l g r o u p s - a t one o r more s i t e s on t h e p r o t e i n
molecule forming a r e l a t i v e l y hydrophobic surface layer (Figure
6a). A g g r e g a t i o n and p r e c i p i t a t i o n t h e n e n s u e . Where t h e i n i t i a l
p r o t e i n c o n c e n t r a t i o n i s high the hydrophobic s u r f a c e layer i s
formed by c r o s s - l i n k i n g o f d i f f e r e n t p r o t e i n m o l e c u l e s by t h e
multidentate polyphenols (Figure 6b). P r e c i p i t a t i o n then f o l l o w s
as o u t l i n e d , v i d e s u p r a . T h i s t e n d e n c y t o c r o s s - l i n k p r o t e i n
molecules e x p l a i n s the changing s t o i c h i o m e t r y o f the aggregates i n
r e l a t i o n t o t h e i n i t i a l p r o t e i n c o n c e n t r a t i o n . An i n t e r e s t i n g
c o r o l l o r a r y o f t h i s h y p o t h e s i s i s t h a t s i m p l e phenols such a s
p y r o g a l l o l and r e s o r c i n o l s h o u l d a l s o be c a p a b l e o f p r e c i p i t a t i n g
p r o t e i n s from s o l u t i o n i f t h e y c a n be m a i n t a i n e d i n s o l u t i o n a t
c o n c e n t r a t i o n s s u f f i c i e n t t o push t h e e q u i l i b r i u m i n f a v o u r o f t h e
p r o t e i n - p h e n o l c o m p l e x and t h u s e s t a b l i s h a h y d r o p h o b i c l a y e r o f
s i m p l e phenol m o l e c u l e s on t h e p r o t e i n s u r f a c e ( F i g u r e 6 c ) . F o r
many s i m p l e p h e n o l s t h e l i m i t i s d e t e r m i n e d by t h e i r own _ 5
s o l u b i l i t y but i t c a n be a c h i e v e d i n w a t e r w i t h BSA (3 x 10
m o l a l ) and p y r o g a l l o l (1 m o l a l ) and r e s o r c i n o l (2 m o l a l ) .
P o s t s c r i pt
Complex p l a n t p o l y p h e n o l s r e a d i l y and r e v e r s i b l y a s s o c i a t e
w i t h p r o t e i n s and t h e y c a n p r e c i p i t a t e them f r o m d i l u t e s o l u t i o n .
T h i s p r o p e r t y i s however a d i r e c t e x t r a p o l a t i o n o f t h e c h a r a c t e r -
i s t i c s o f simple phenols themselves. The s t r u c t u r a l d e v i c e
r e p r e s e n t e d by t h e p l a n t p o l y p h e n o l s t o a g r e a t e x t e n t o b v i a t e s
t h e need f o r a h i g h m o l a l c o n c e n t r a t i o n o f phenol and i t e m b o d i e s
t h e added f e a t u r e t h a t c r o s s - l i n k i n g between d i f f e r e n t m o l e c u l a r
a g g r e g a t e s may be r e a d i l y a c h i e v e d .
The q u e s t i o n s posed i n i t i a l l y r e m a i n . What p u r p o s e do t h e s e
molecules serve i n p l a n t metabolism? Does t h e i r p r e s e n c e p o i n t
f o r e x a m p l e t o t h e need by p l a n t s e i t h e r now o r d u r i n g t h e c o u r s e
of e v o l u t i o n f o r s u c h t a i l o r e d m o l e c u l e s t o r e v e r s i b l y c o a t t h e

(c)
association.
Protein-polyphenol
(b)
Figure 6.
(a)

s u r f a c e o f p r o t e i n s and hence m o d i f y t h e i r a c t i v i t y a t p a r t i c u l a r
p h a s e s o f d e v e l o p m e n t ? Answers t o t h e s e p r o b l e m s w i l l o n l y emerge
as and when a g r e a t d e a l more i s known c o n c e r n i n g t h e e n z y m o l o g y
o f p r o c e s s e s g o v e r n i n g t h e f o r m a t i o n o f t h e s e m e t a b o l i t e s , how
t h e s e p r o c e s s e s a r e r e g u l a t e d and c o n t r o l l e d , and t h e i r r e l a t i o n -
s h i p t o the network o f p r i m a r y metabolism from which they d e v o l v e .
A t r u l y f o r m i d a b l e t a s k b u t a s R o b e r t L o u i s S t e v e n s o n once w r o t e :
"To t r a v e l h o p e f u l l y i s a b e t t e r t h i n g than t o a r r i v e ,
and the true success i s t o labour".
E l Dorado.
Literature Cited
1. Mothes, K. in "Secondar
P l a n t Physiology Ne
Charlwood B.V.; S p r i n g e r - V e r l a g , Berlin, 1980, p . 1
2. Bu'Lock, J . D . in "The B i o s y n t h e s i s of Mycotoxins", Ed. Steyn,
P.S., Academic P r e s s , New York - London, 1980, p. 1
3. Janzen, K . H . in "Herbivores - Their I n t e r a c t i o n s w i t h
Secondary M e t a b o l i t e s " ; Ed. Rosenthal, G.A. and Janzen, D . H . ;
Academic P r e s s , New York - London, 1979, p . 331
4. Rhoades, D.F. in "Herbivores - Their I n t e r a c t i o n s w i t h
Secondary M e t a b o l i t e s " ; Ed. Rosenthal, G.A. and Janzen, D . H . ;
Academic P r e s s , Nre York - London, 1979, p. 3.
5. Harborne, J . B . in "Secondary P l a n t Products" Encylopaedia
of P l a n t P h y s i o l o g y , New S e r i e s , V o l . 8; Ed. Bell, E . A . and
Charlwood, B.V.; S p r i n g e r - V e r l a g , Berlin, 1980, p. 329.
6. Howes, F.N., "Vegetable Tanning M a t e r i a l s " ; Butterworths,
London, 1939.
7. Bate-Smith, E.C., Food, 1954, 2 3 , 124.
8. Bate-Smith, E.C., J. Linnaen Soc. ( B o t . ) , 1962, 58, 95.
9. Zenk, M.H. in "Recent Advances in Phytochemistry" V o l . 12;
Ed. Swain, T . , Harborne, J . B . and van Sumere, C . , Plenum
P r e s s , London and New York, 1978, p. 139.
10. Bate-Smith, E . C . and L e r n e r , N.H., Biochem. J., 1954, 58,126.
11. Haslam, E., Phytochemistry, 1977, 16, 1625.
12. Thompson, R.S., Jacques, D . , Tanner, R . J . N . and Haslam, E.,
J. Chem. Soc. (Perkin 1 ) , 1972, 1387.
13. Weinges, K., Kaltenhauser, W., Marx, H.-D., Nader, E., Nader,
F., Perner, J. and S e i d e r , D . , Annalen, 1968, 711, 184.
14. F l e t c h e r , A.C., Gupta, R.k., P o r t e r , L.J. and Haslam, E .
J. Chem. Soc. (Perkin 1 ) , 1977, 1628.
15. Weinges, K., Marx, H . - D . and Perner, J., Chem. B e r . , 1970
103, 2344.
16. J u r d , L. and Lundin, R . , Tetrahedron, 1968, 24, 2652.
17. Geissmann, T . A . and Yoshimura, N.N., Tetrahedron L e t t e r s ,
1966, 2669.

18. Opie, C.T., Porter, L.J., Jacques, D. and Haslam, E . , J . Chem.

Soc. (Perkin 1), 1977, 1637.
19. Gupta, R.K. and Haslam, E . , J . Chem. Soc. (Perkin 1), 1978,
892.
20. Robinson, R. and Robinson, G.M., J. Chem. Soc., 1935, 744.
21. Porter, L.J., Czochanska, Z . , Foo, L . Y . , Newman, R . H . ,
Thomas, W.A. and Jones, W.T., J . Chem. Soc. Chem. Commun.,
1979, 375.
22. Okuda, T., Yoshida, T., Mori, K. and Hatano, T.,
Heterocycles, 1981, 15, 1323.
23. Haslam, E . , Fortschr. chem. org. Naturst., 1982.
24. Magnolato, D., Gupta, R . K . , Haddock, E . A . , Layden, K. and
Haslam, E . , Phytochemistry, 1982.
25. Fischer, E . , Ber., 1919, 52, 809.
26. Okuda, T., Nayeshiro H d Yoshida T. Chem Pharm Bull.
(Japan) 1977, 25,
27. Van Buren, J . P . and Robinson, W.B., J. Agric. Food Chem.,
1969, 17, 772.
28. Loomis, W.D., Methods. Enzymol., 1974, 31, 528.
29. Oh, H . I . , Hoff, J.E., Armstrong, G.S. and Haff, L . A . ,
J. Agric. Food Chem., 1980, 28, 394.
30. McManus, J., Lilley, T.H. and Haslam, E . , J . Chem. Soc.,
Chem. Commun., 1981, 309.

The Role of Natural Photosensitizers in Plant
Resistance to Insects
T. ARNASON—University of Ottawa, Biology Department,
Ottawa, Ontario K 1N 6N5, Canada
G. H. N. TOWERS and B. J. R. PHILOGÈNE—University of British Columbia,
Botany Department, Vancouver, British Columbia V6T 1W5, Canada
J. D. H. LAMBERT—Carleton University, Biology Department,
Ottawa, Ontario K15 5T6, Canada
Recent work has suggested that certain secondary meta-

bolites from plants are capable of photosensitizing
insects. With the recognition of increasing numbers
of natural photosensitizers including polyacetylenes,
furanocoumarins, β-carbolines and extended quinones,
this unusual mechanism of plant defense now appears
to be present i n a wide variety of plant families.
Polyacetylenes, a group, of over 500 diverse compounds
which are especially widespread in the Asteraceae are
powerful photosensitizers. At least one sulphur
derivative, α-terthienyl i s more toxic to mosquito
larvae than DDT. The t o x i c i t y of some of these
compounds is mediated by the production of singlet
oxygen although free radicals may also be involved.
Furanocoumarins, furanoquinolines and β-carbolines on
the other hand interact with DNA and cause gross
chromosomal abnormalities i n v i v o . The furanocoumarin,
8-methoxypsoralen has been shown to be highly photo-
toxic to herbivorous Spodoptera larvae. I t has also
been suggested that the leaf r o l l i n g habit of certain
microlepidopteran larvae on leaves containing photo
sensitizing furanocoumarins i s related to l i g h t
avoidance. Although only limited information is a v a i l
able about the effects of naturally occurring compounds
on insects, work with synthetic dye photosensitizers
provides a broader basis for understanding the photo-
sensitization of insects.
0097-6156/83/0208-0139$06.00/0

L i g h t i s o f t e n a f o r g o t t e n or underestimated f a c t o r i n t h e
study of i n s e c t s (1) and u n t i l r e c e n t l y l i t t l e a t t e n t i o n has been
paid t o i t s r o l e i n p l a n t i n s e c t r e l a t i o n s . I n p a r t i c u l a r we
wish t o address the question of a c t i v a t i o n of p l a n t secondary
substances by l i g h t and t h e i r subsequent p h o t o s e n s i t i z i n g e f f e c t s
on i n s e c t s . At l e a s t two examples of i n s e c t p h o t o s e n s i t i z i n g
secondary m e t a b o l i t e s , furanocoumarins and polyacetylenes have
now been reported. These s t u d i e s suggest that t h i s mode of p l a n t
defense has p a r t i c u l a r adaptive advantages and may be more
widespread than p r e v i o u s l y imagined. F o r t u n a t e l y the mode of
a c t i o n of p h o t o s e n s i t i z e r s from p l a n t s i s understood from work
w i t h t a r g e t organisms other than i n s e c t s . I n a d d i t i o n some
d e t a i l e d i n f o r m a t i o n on the e f f e c t s of p h o t o s e n s i t i z e r s on
i n s e c t s i s suggested from work w i t h s y n t h e t i c dye s e n s i t i z e r s .
These l a t t e r aspects a r e c r u c i a l t o the understanding of the
e f f e c t s of n a t u r a l p h o t o s e n s i t i z e r
review.
Photosensitization
For p h o t o s e n s i t i z a t i o n t o occur l i g h t must be absorbed by

the p h o t o s e n s i t i z e r and cause a r e a c t i o n t o take p l a c e i n t h e
b i o l o g i c a l system ( 2 ) . Two main types o f p h o t o s e n s i t i z a t i o n a r e
known t o occur. Those i n v o l v i n g O2 a r e c a l l e d photodynamic
s e n s i t i z a t i o n s and are mediated by the bulk of the 4 0 0 s y n t h e t i c
and n a t u r a l p h o t o s e n s i t i z e r s known ( 3 ) . The second type of
p h o t o s e n s i t i z a t i o n does not i n v o l v e O2 and i s a s s o c i a t e d w i t h the
furanocoumarins.
Photodynamic s e n s i t i z a t i o n s are photo-oxidations which
proceed by one of two pathways (3,4_) . Type I p h o t o s e n s i t i z a t i o n s
( l e s s common) y i e l d superoxide r a d i c a l s ( 0 j ) through a s e r i e s of
e l e c t r o n t r a n s f e r processes f o l l o w i n g l i g h t a b s o r p t i o n . Anthra-
quinone dyes and f l a v i n s operate through t h i s mechanism. I n
type I I s e n s i t i z a t i o n s , the s e n s i t i z e r i s e x c i t e d from the ground
s t a t e , S Q , t o the f i r s t e x c i t e d s t a t e , * S by a b s o r p t i o n of a
photon. Crossing over leads t o the formation o f the e x c i t e d
t t i p l e t state, which i n t e r a c t s w i t h ground s t a t e oxygen, ^02-
The t r a n s f e r of e x c i t a t i o n energy leads t o the production of
s i n g l e t oxygen (^02) and the s e n s i t i z e r r e t u r n s t o i t s ground
state:
(1) s Q
hv
3 3
(2)
S + o 2

8. ARNASON ET A L . Role of Natural Photosensitizers 141
Because the ground s t a t e o f t h e s e n s i t i z e r i s regenerated the

r e a c t i o n i s c a t a l y t i c . Once formed the a c t i v a t e d species o f
oxygen, from type I I r e a c t i o n s Oj from type I mechanisms
cause b i o l o g i c a l damage by o x i d a t i o n o f b i o l o g i c a l molecules.
They are a l s o p o t e n t i a l l y i n t e r c o n v e r t i b l e ( 5 ) . Recent advances
have allowed the i d e n t i f i c a t i o n o f ^2 i n photochemical damage
of b i o l o g i c a l systems by the use o f quenchers (6) o r D2O which
enhances the l i f e t i m e o f ^ 2 (7) Superoxide i s detected i n photo-
o x i d a t i o n s by the use o f the scavenging enzyme superoxide
dismutase ( 8 ) . Important t a r g e t molecules are p r o t e i n s , l i p i d s
( e s p e c i a l l y c h o l e s t e r o l ) and n u c l e i c a c i d s (_3, 4_, 9) but the
e f f e c t s i n v i v o a r e l a r g e l y dependant on the s i t e t o which the
p h o t o s e n s i t i z e r b i n d s . Thus rose bengal (compound I ) binds t o
and l y s e s membranes w h i l e a c r i d i n e orange penetrates t o the
nucleus and causes damage t o DNA (6, 1 0 )
The second group o
chemical damage a t th y requiremen
f o r a c t i v i t y . These i n c l u d e the w e l l s t u d i e d furanocoumarins
(11) and more r e c e n t l y the f u r o q u i n o l i n e a l k a l o i d s (12). Work
w i t h the furanocoumarins suggests that the f i r s t step i s
i n t e r c a l a t i o n o f the p h o t o s e n s i t i z e r i n t o DNA i n such a way that
one o r two o f the double bonds o f the r i n g s t r u c t u r e a l i g n w i t h
the p y r i m i d i n e double bonds. The e x c i t e d s t a t e o f the photosen-
s i t i z e r undergoes c y c l o a d d i t i o n t o the p y r i m i d i n e ( u s u a l l y
thymine) forming a photoadduct ( I I ) . Compounds such as the
f u r o q u i n o l i n e , dictamnine (X) and hindered furanocoumarins form
o n l y monofunctional adducts (11, 12) w h i l e the l i n e a r furanocou-
marins such as 8-methoxypsoralen (8-MOP) (IV) form d i f u n c t i o n a l
adducts ( I I I ) l e a d i n g t o i n t e r s t r a n d c r o s s - l i n k a g e of DNA. Both
types o f damage but e s p e c i a l l y the l a t t e r have s e r i o u s conse-
quences f o r DNA t r a n s c r i p t i o n o r d u p l i c a t i o n and can l e a d t o c e l l
death o r mutagenesis ( n ) .
E f f e c t o f P h o t o s e n s i t i z e r s on I n s e c t s
Because of the a v a i l a b i l i t y o f s y n t h e t i c dye s e n s i t i z e r s ,

t h e i r e f f e c t on i n s e c t s i s somewhat b e t t e r understood than photo-
s e n s i t i z i n g p l a n t secondary substances. The f i r s t r e p o r t o f the
e f f e c t of p h o t o s e n s i t i z e r s on i n s e c t s was made by B a r b i e r i i n
1928 (13), 28 years a f t e r the d i s c o v e r y o f p h o t o s e n s i t i z a t i o n by
Raab. A 1972 review by Graham (14) reported o n l y f i v e i n v e s t i -
gations i n t o the p h o t o s e n s i t i z a t i o n o f i n s e c t s , but the number
has more than doubled s i n c e that time, p r i n c i p a l l y due t o the
work a t M i s s i s s i p p i S t a t e . Some o f these r e p o r t s a r e summarized
i n TABLE I . S t u d i e s have concentrated mainly on the t h i a z i n e
dye methylene b l u e ( I I ) and a range o f xanthene dyes o f which
rose bengal ( I ) was the most prominant. Four orders of i n s e c t s
are represented i n c l u d i n g eggs, l a r v a e , pupae and a d u l t s . No
u n u s u a l l y r e s i s t a n t species have been found and the r e s u l t s w i t h
the b o l l w e e v i l and f i r e ant a r e p a r t i c u l a r l y s i g n i f i c a n t s i n c e


TABLE I oo
Studies of the E f f e c t o f Photodynamic Dye S e n s i t i z e r s on Insects >

Mode o f Admini- >
Order Organism Photosensitizer stration Reference to
O
Diptera Culex p i p i e n s q u i n q u e f a s c i a t u s rose bengal water treatment W
(15) H
(mosquito l a r v a e ) >
Aedes a e g y p t i & Anopheles spp. uranin,erythrosin it (16) 1
r
(mosquito l a r v a e ) a c r i d i n e red e o s i n ,
rhodamine, rose
bengal
Anopheles sp. & Aedes sp.
H
(mosquito l a r v a e ) xanthene dyes (13)
Aedes t r i s e r a t u s and Culex 1
p i p i e n s quinquefasciatus rose bengal fl (17) I
(mosquito l a r v a e )
2
Musca domes t i c a xanthene dyes diet (18)
(adult house f l y )
Musca autumnalis xanthene dyes diet (19)
(adult face f l y )
D r o s o p h i l a melanogaster benzpyrene, methyl diet (20) 3
cholanthrene,

dibenzanthrene
Coleoptera Anthonomus grandis
(adult b o l l w e e v i l ) xanthene dyes diet (21)
Tenebrio m o l i t o r
(meal worm l a r v a e ) mythylene blue injection

Hymenoptera Solenopsis r i c h t e r i xanthene dyes diet (22)
( f i r e ant)
Lepdioptera P i e r r i s b r a s s i c a e methylene blue diet (23)
4^
they i n d i c a t e that even hard bodied i n s e c t s are s u s c e p t i b l e to

photodynamic a c t i o n . M o r t a l i t y i n most cases was found to be
d i r e c t l y p r o p o r t i o n a l to the photon fluence and dye concentra-
t i o n . Except f o r the more recent work, t o x i c i t i e s are d i f f i c u l t
to compare from one study to the next because of the range of
c o n d i t i o n s p o s s i b l e and the d i f f i c u l t y i n e s t i m a t i n g the fluence
of l i g h t absorbed w i t h lamps of d i f f e r e n t s p e c t r a l q u a l i t y and
dyes w i t h d i f f e r e n t a b s o r p t i o n maxima. An attempt a t q u a n t i t a -
t i o n of the r e s u l t s has been made by d e r i v a t i o n of second (22)
and t h i r d order (24) r a t e constants f o r p h o t o - o x i d a t i o n . Using
the second order r a t e constant i n s e c t s can be ranked i n order of
t h e i r s u s c e p t i b i l i t y : Aedes t r i s e r a t u s ( l a r v a e ) > Anthonomus
grandis ( a d u l t ) > Musca autumnalis ( a d u l t ) > Solenopsis r i c h t e r i
( a d u l t ) . With mosquito l a r v a e s u s c e p t i b i l i t y f a l l s w i t h the
number of i n s t a r s . The r a t e constant can a l s o be used to rank
dye t o x i c i t y (18). Bot
y i e l d f o r dye phosphorescenc
e o s i n y e l l o w , p h l o x i n B, e r y t h r o s i n B and rose bengal. This
r e s u l t i s explained by the c u r r e n t view that phosphorescence and
the production of I-O2 i n the p h o t o s e n s i t i z a t i o n r e a c t i o n are
dependant on the p r o p o r t i o n of dye molecules i n t h e i r t r i p l e t
excited state (3).
At the p h y s i o l o g i c a l l e v e l i t i s w e l l e s t a b l i s h e d that
v i t a l dyes such as n i l e b l u e , n e u t r a l red and methylene blue
r e t a r d l a r v a l development under normal l i g h t i n g c o n d i t i o n s
(12L/12D w i t h source u n s p e c i f i e d ) (25~27). Female but not male
pupal weights are a l s o reduced. U n f o r t u n a t e l y experiments were
conducted without dark c o n t r o l s so that i t i s d i f f i c u l t to
evaluate the r o l e of p h o t o s e n s i t i z a t i o n i n these e f f e c t s . As
house f l i e s and f i r e ants succumb to p h o t o s e n s i t i z a t i o n , they
l o s e motor c o n t r o l and become more e x c i t a b l e (28). This
suggested a neurotoxic e f f e c t and i n v e s t i g a t i o n of f i r e ant
a c e t y l c h o l i n e s t e r a s e i n v i t r o revealed that t h i s enzyme was
s e n s i t i v e to photo-oxidation. I n v i v o r e s u l t s , however,
revealed no e f f e c t on the enzyme which suggests another mode of
a c t i o n . Epoxidation of c h o l e s t e r o l and membrane l y s i s may be
a l t e r n a t i v e primary s i t e s . I f t h i s were the case ecdysone
metabolism of i n s e c t s would probably a l s o be e f f e c t e d .
Furanocoumarins
Although furanocoumarins are w e l l s t u d i e s f o r t h e i r e f f e c t s

1
on human s k i n , recent work has suggested t h e i r JuLc&on d H&id i n
p l a n t s may be l i n k e d to t h e i r r o l e as p r o t e c t i v e agents that
are e f f e c t i v e against i n s e c t s or pathogenic f u n g i (29, 30). This
group of compounds i s reported i n 8 f a m i l i e s but f i n d t h e i r
g r e a t e s t d i v e r s i t y i n the Apiaceae and Rutaceae. Berenbaum has
demonstrated t h e i r a c t i v i t y a g a i n s t i n s e c t herbivores i n feeding
t r i a l s w i t h a polyphagous h e r b i v o r e , the f a l l armyworm
Spodoptera e r i a n i a which w i l l feed on c a r r o t which does not

8. ARNASON ET AL. Role of Natural Photosensitizers 145
c o n t a i n furanocoumarins but not on parsnip that does c o n t a i n

these compounds. Larvae were administered 0,1% 8-methoxypsoralen
(IV), a furanocoumarin compound which occurs w i d e l y i n the
Aplaceae by treatment of a r t i f i c i a l d i e t . These l e v e l s are compa-
r a b l e t o those i n p l a n t s and caused 100% m o r t a l i t y i n l a r v a e that
were a l s o t r e a t e d w i t h near UV, the a c t i v a t i n g wavelength range
for these s e n s i t i z e r s . Dark c o n t r o l s and i n s e c t s fed untreated
d i e t s but i r r a d i a t e d w i t h near UV showed a s l i g h t r e d u c t i o n i n
s u r v i v o r s h i p . The e f f e c t of near-UV and 8-M0P together was h i g h l y
s i g n i f i c a n t and i s presumably due t o the e f f e c t of the compound
on l a r v a l DNA. Thus t o feeding g e n e r a l i s t s 8-M0P represents a
formidable b a r r i e r t o g r a z i n g on the p l a n t s that c o n t a i n i t .
Since the precursor t o p s o r a l e n s , u m b e l l i f e r o n e , i s not t o x i c t o
armyworms, Berenbaum hypothesizes that these p l a n t s have escaped
t h e i r enemies by the a l t e r a t i o n of t h e i r chemical phenotype.
I n s e c t s have i n some case
For example aphids tha
and bind 8-M0P b u t are u n a f f e c t e d by i t , perhaps due t o some
d e t o x i f i c a t i o n mechanism (29). Yu, S.J., Berry, R.E., and T e r r i e r e ,
L.C.,(1979) showed that phytophagous i n s e c t s possess enzymes that
are induced by p l a n t secondary substances and that these enzymes
are i n v o l v e d i n r e s i s t a n c e t o otherwise t o x i c compounds (31).
M i c r o l e p i d o p t e r a appear t o have adapted t o l i f e on p h o t o t o x i c
Apiaceae by a l e a f r o l l i n g h a b i t that screens out near UV. (30).
C l u s t e r a n a l y s i s of the fauna of the Apiaceae i n d i c a t e s that the
i n s e c t assemblages of p l a n t s c o n t a i n i n g phototoxic furanocoumarins
are s i m i l a r and d i f f e r e n t from p l a n t s i n the f a m i l y t h a t do not
c o n t a i n phototoxic furanocoumarins (32). This lends support t o the
hypothesis that a s p e c i a l i s t group of i n s e c t s e x i s t s that i s
adapted t o p h o t o t o x i c Apiaceae.
Polyacetylenes
P o l y a c e t y l e n e s are a v e r y l a r g e group of secondary compounds

whose p h o t o s e n s i t i z i n g p r o p e r t i e s have r e c e n t l y been e s t a b l i s h e d
by our research team a t U.B.C. (33). These compounds have conju-
gated double and t r i p l e bond systems (e.g. compound V I I and V I I I )
or may be b i o s y n t h e t i c a l l y c y c l i z e d i n t o thiophene compounds such
as alpha t e r t h i e n y l (a-T) (compound V I ) . P o l y a c e t y l e n e s and t h e i r
thiophene d e r i v a t i v e s occur i n s e v e r a l f a m i l i e s but f i n d t h e i r
g r e a t e s t d i v e r s i t y i n the Asteraceae, the l a r g e s t p l a n t f a m i l y
C34). As i n the case of the furanocoumarins, p h o t o s e n s i t i z a t i o n
i s mediated by the near-UV r e g i o n of the spectrum but the mecha-
nism of a c t i o n does not i n v o l v e c r o s s - l i n k i n g of DNA (35). Some
compounds such as a-T are c l e a r l y photodynamic i n t h e i r mode of
a c t i o n (36) but the s i t u a t i o n i s l e s s c l e a r f o r compounds c o n t a i n -
ing t r i p l e bond systems such as phenylheptatriyne (PHT) ( V I I I ) .
P h o t o - o x i d a t i o n of c h o l e s t e r o l and a c e t y l c h o l i n e s t e r a s e has been
observed (37) w i t h t h i s and other polyacetylenes but l y s i s of r e d
blood c e l l s and p h o t o s e n s i t i z a t i o n _E. c o l i were not 0 dependant
?

C38). These r e s u l t s suggest a novel mechanism of a c t i o n and are

an a c t i v e area of research.
P o l y a c e t y l e n e s are t o x i c to a broad range of organisms (39)
but are e s p e c i a l l y t o x i c t o i n s e c t s . At 0.5 ppm, 9 of 14 com-
pounds t e s t e d were t o x i c to f i r s t i n s t a r mosquito l a r v a e (Aedes
aegypti) i n 30 min treatments w i t h sources of near UV (15 W/m^)
(40). The compounds were more a c t i v e i n s u n l i g h t . For example
a-T k i l l e d second i n s t a r l a r v a e i n s t a n t a n e o u s l y at 4 ppm.
Compounds V I I , a-T and PHT were e s p e c i a l l y a c t i v e and were
s e l e c t e d f o r f u r t h e r t e s t i n g i n dose response experiments ( 4 1 ) .
For s i m i l a r near UV treatments, the LC50 f o r a-T was 19 ppb,
79 ppb f o r compound V I I and 1.0 f o r PHT. Alpha T had some a c t i -
v i t y i n the dark (LC50 =0.74 ppm) i n d i c a t i n g t h a t i t may bind to
a s e n s i t i v e c e l l s i t e and d i r e c t p h o t o s e n s i t i z a t i o n to t h i s s i t e .
This compound was so a c t i v e i n the presence of near UV t h a t i t s
p o t e n t i a l as a commercia
pond t r i a l s . For 500 l a r v a
s u n l i g h t , 100% m o r t a l i t y was observed i n 15 min a t 200 ppb and
120 min at 20 ppb, A d e t a i l e d a c t i o n spectrum f o r p h o t o s e n s i t i -
z a t i o n undertaken w i t h narrowband i n t e r f e r e n c e f i l t e r s i n d i c a t e d
that there was c l o s e agreement between a c t i o n and a b s o r p t i o n .
This suggested t h a t the p o l y a c e t y l e n e was the absorbing species
and was not i n t e r f e r i n g w i t h metabolism i n such a way as to cause
a p h o t o s e n s i t i z i n g product to be formed. Such mechanisms do
occur w i t h i c t e r o g e n s produced by the genus T r i b o l u s which are
ingested by range animals ( 4 2 ) . The r e s u l t w i t h p o l y a c e t y l e n e s
a l s o d i f f e r s from t h a t w i t h psoralens where a DNA-psoralen
complex i s thought to r e s u l t i n the d e v i a t i o n of the a c t i o n
spectrum from the a b s o r p t i o n spectrum (43).
We are c u r r e n t l y i n v e s t i g a t i n g the e f f e c t of p o l y a c e t y l e n e s
and near-UV i n s u b l e t h a l doses during feeding t r i a l s w i t h Euxoa
messoria (Lepidoptera, n o c t u i d a e ) . P o t e n t i a l f o r f u r t h e r work
a l s o e x i s t s w i t h the adapted i n s e c t , the s o l d i e r b e e t l e
(Coleoptera, Cantheridae), which apparently uses a p o l y a c e t y l e n e
as a defense compound (44),
Other compounds
Despite the hypothesis t h a t the e v o l u t i o n a r y s i g n i f i c a n c e of

p h o t o t o x i c secondary substances may be l i n k e d to t h e i r a b i l i t y
to discourage i n s e c t h e r b i v o r e s , most research has been d i r e c t e d
toward t h e i r e f f e c t s on human s k i n and range animals (42). I n
an attempt to extend our knowledge of i n s e c t p h o t o s e n s i t i z e r s we
have screened a number of p l a n t secondary substances (TABLE I I )
f o r t h e i r p h o t o s e n s i t i z i n g a c t i v i t y to 4 t h i n s t a r mosquito l a r v a e
Aedes atropalpus under s o l a r s i m u l a t i n g lamps.
H y p e r i c i n (IX) i s an extended quinone from S t . John's wort
(Hypericum spp), a common pasture weed throughout the w o r l d .
T h i s photodynamic compound causes a p h o t o s e n s i t i v e disease c a l l e d
1
'bighead i n sheep t h a t consume i t (42). Related compounds, the

ARNASON ET AL. Role of Natural Photosensitizers
American Chemical
Society Library
1155 16th st. N. w.
WatUngtofi. D. C. Society:
2003SWashington, DC, 1983.
ACS Symposium Series; American Chemical
TABLE I I
P h o t o t o x i c i t y of P l a n t Secondary M e t a b o l i t e s
to Mosquito Larvae
Compound Type Phototoxic
Hypericin Extended quinone + +
Dictamnine Furo q u i n o l i n e a l k a l o i d + +
Harmaline 3-Carboline a l k a l o i d + +
Methoxyharmaline g-Carboline a l k a l o i d + +
Harman (3-Carboline a l k a l o i d
Norharman $-Carboline a l k a l o i d +
Harmalol $-Carboline a l k a l o i d +
Berberine Isoquinoline alkaloid +
Note; Compounds were screened a t s e v e r a l c o n c e n t r a t i o n s

i n 24-hr acute t o x i c i t y t e s t w i t h f o u r t h i n s t a r
Aedes a t r o p a l p u s l a r v a e . Tests were run w i t h
p a r a l l e l t r i a l s i n the dark and under s o l a r
R
s i m u l a t i n g " v i t a l i t e s " w i t h an i n t e n s i t y of
400 w/m^. A c t i v i t i e s were r a t e d as f o l l o w s
(-) no d i f f e r e n c e between l i g h t and dark t r i a l ,
(+) enhancement of l i g h t t o x i c i t y over dark, (+ +)
l a r g e r enhancement of l i g h t t o x i c i t y over dark.

8. ARNASON ET AL. Role of Natural Photosensitizers 149
fagopyrins occur i n buckwheat (fagopyrum s p ) . I n a d d i t i o n we

have included two groups o f a l k a l o i d s , the g-carbolines (of which
hannaline (X) i s an example) and the f u r o q u i n o l i n e a l k a l o i d
dictamnine (V) which were r e c e n t l y discovered by one o f us
(Towers) t o be p h o t o t o x i c t o yeast (45, 46). The a c t i v i t y o f
berberine was suggested by i t s f l u o r e s c e n c e . Obviously many
compounds a r e phototoxic o r have enhanced t o x i c i t y i n l i g h t as
compared t o dark. We b e l i e v e t h i s demonstrates the p o t e n t i a l f o r
f u r t h e r work i n t h i s f i e l d and suggests that t h i s i s an area that
has been overlooked.
One o f the reasons that many p h o t o s e n s i t i z i n g compounds have
been overlooked i s because o f t h e i r apparent l a c k o f c o l o r . F o r
example the p o l y a c e t y l e n e PHT i s a potent i n s e c t p h o t o s e n s i t i z e r
i n n a t u r a l s u n l i g h t i s completely c o l o r l e s s i n s o l u t i o n . I t s
spectrum r e v e a l s (Figur 1) stron a b s o r p t i o band i th
UV which are w e l l beyon
w i t h i n the range o f wavelength y atmospher
( g e n e r a l l y > 300 nm) (47). I n a d d i t i o n i t should be noted that
the energy absorbed a t these wavelengths i s c o n s i d e r a b l y higher
than i n the v i s i b l e range and may be a f a c t o r i n the h i g h
t o x i c i t y of compounds l i k e alpha T.
I n c o n c l u s i o n i t i s evident that many research o p p o r t u n i t i e s
e x i s t i n the i d e n t i f i c a t i o n and c h a r a c t e r i z a t i o n of new
substances and e v a l u a t i o n o f t h e i r e c o l o g i c a l e v o l u t i o n a r y and
p h y s i o l o g i c a l s i g n i f i c a n c e . I n a p r a c t i c a l sense i t can be
hoped that some o f these new compounds because o f t h e i r n o v e l
mode o f a c t i o n may be u s e f u l f o r the c o n t r o l o f phytophagous
i n s e c t s as p a r t o f i n t e g r a t e d pest management programs.
2.0-
•
o
e i.o-
•»
<
0.0-
I I 300 350
200
250
Wavelength (nm)
Figure 1. Absorption spectrum of 1 ppm of phenylheptatriyne in ethanol.

ACKNOWLEDGEMENT
This work was supported by NSERC and an A g r i c u l t u r e Canada

E.M.R. g r a n t t o one o f us (Arnasoh).
Literature Cited
1. Philogène, B.R. Am. Nat. 1982, 118, i n p r e s s .

2. T u r r o , N.J.; Lamola, A.A. in "The Science o f P h o t o b i o l o g y "
K.S. Smith (ed) Plenum: New York. 1977, 63-86.
3. S p i k e s , J.D. in "The Science o f P h o t o b i o l o g y " K.C. Smith (ed)
Plenum: New York. 1977, 87-112.
4. Grossweiner, L.I. Topics in Rad. Res. Quart. 1976, 11,
141-199.
5. Singh, A. Photochem
6. I t o , K. Photochem. P h o t o b i o l . 1978, 28, 493-508.
7. N i l s s o n , R.; Kearns, D.R. Photochem. P h o t o b i o l . 1973, 17,
65-68.
8. Jahnke, L.S.; F r e n k e l , A.W. Photochem. P h o t o b i o l . 1978, 28
517-523.
9. K u l i g , M.J.; Smith, L.L. J. Org. Chem. P 973, 38, 3639-3642.
10. Lamola, A.A.; Yamane, T.; T r o z z o l o , A.M. Sci. 1973, 179,

1131-1133.
11. Song, P.S.; Tapely, K . J . Photochem. P h o t o b i o l . 1979, 29,
1177-1197.
12. P f y f f e r , G.E.; Panfil, I.; Towers, G.H.N. Photochem.
P h o t o b i o l . 1982, 35, 63-68.
13. B a r b i e r i , A. R i v . Malariol, 1928,7,456-463.
14. Graham, K. Can. J. Z o o l . 1972, 50, 1631-1636.
15. Carpenter, T.L.; Heitz, J.R. E n v i r . Entomol. 1980, 9, 533-7.
16. Schildmacher, H. Biol. Z e n t r a l b l . 1950, 69, 468-477.
17. P i m p r i k a r , G.D.; Norment, B.R.; Heitz, J.R. E n v i r o n .
Entomol 1979, 8, 856-9.
18. Fondren, J.E.; Norment, B.R.; Heitz, J.R. E n v i r o n . Entomol.
1978, 7, 205-208.
19. Fondren, J.E.; H e i t z , J.R. E n v i r o n . Entomol. 1978, 7, 843-6.
20. M a l t o l t s y , G.; Fabian, G. Nat. 1946, 158, 877-8.
21. Callaham, J.J.; Broome, J.R.; Lindig, O.H.; H e i t z , J.R.
E n v i r o n . Entomol. 1975, 4, 837-841.
22. Broome, J.R.; Callaham, M.F.; Heitz, J.R. E n v i r o n . Entomol.
1975, 4, 833-6.
23. Lavialle, M.; Dumortier, B. C.R. Acad. Sc. Paris. 1978, 287,
875-8.
24. Fondren, J.E.; H e i t z , J.R. E n v i r o n . Entomol. 1978, 7, 891-4.
25. Barbarosa, P.; P e t e r s , M. Ent. Exp. & App. 1970, 13, 293-9.
26. Barbarosa, P.; P e t e r s , M. J. Med. Ent. 1970, 7, 693-6.
27. Barbarosa, P.; P e t e r s , M. Histochem. J. 1971,3,71-93.

8. ARNASON E T AL. Role of Natural Photosensitizers 151
28. Callaham, M.F.; Lewis, L.A.; Holloman, M.E.; Broome, J.R.;

H e i t z , J.R. Comp. Biochem. Physiol. 1975, 51C, 123-8.
29. Camm, E.L.; Wat, C.K.; Towers, G.H.N. Can. J. Bot. 1977,
54, 25-
30. Berenbaum, M. Sci. 1978, 201, 532-3.
31. Yu, S.J.; B e r r y , R.E.; Terriere, L.C. P e s t i c i d e Biochem.
P h y s i o l . 1979, 12, 280-4.
32. Berenbaum, M. Ecology. 1980, 62, 1254-66.
33. Camm. E.L.; Towers, G.H.N.; Mitchell, J.C. Phytochem. 1975,
94, 2007-11.
34. Bohlmann, F.; Burkhard, T.; Zdero, C. " N a t u r a l l y Occurring
A c e t y l e n e s " Academic P r e s s , New York, 1973.
35. Wat, C.K.; Biswas, R.K.; Graham, E.A.; Bohm, L.; Towers,
G.H.N.; Waygood, E.R. J . Nat. Prod. 1977, 42, 103-111.
36. Arnason, T.; Chan
G.H.N. Photochem
37. Wat, C.K.; MacRae, W.D.; Yamamoto, E.; Towers, G.H.N.;
Lam, J. Photochem. P h o t o b i o l . 1981. in p r e s s .
38. Arnason, T.; Wat, C.K.; Downum, K.; Yamamoto, E.; Graham,
E.; Towers, G.H.N. Can. J. Microbiol. 1980, 26, 698-705.
39. Towers, G.H.N.; Wat, C.K. Rev. Latinoamer Quim. 1978, 9,
162,170.
40. Wat, C.K.; Prasad, S.K.; Graham, E.A.; P a r t i n g t o n , S.;
Arnason, T.; Towers, G.H.N.; Lam, J. Biochem. S y s t . E c o l .
1981, 9, 59-62.
41. Arnason, T.; Swain, T.; Wat, C.K.; Graham, E.A.; P a r t i n g t o n ,
S.; Towers, G.H.N.; Lam, J. Biochem. S y s t . and Ecol. 1981,
9, 63-68.
42. Giese, A. Photophysiology, 1971, 6, 77.
43. Nakayama, Y.; Morihaw, F.; Fukuda, M.; Hamano, M.; Toda, K.;
Patnak, M. in " S u n l i g h t and Man", Fitzpatrick, T.B. ( e d ) ,
U n i v e r s i t y o f Tokyo P r e s s . 1974.
44. Meinwald, J.; Meinwald, Y.C.; Chalmers, K.M.; E i s n e r , J.
Sci. 1978, 160, 890.
45. McKenna, D.J.; Towers, G.H.N. Phytochem. 1981, 20, 1001-4.
46. Towers, G.H.N.; Graham, E.A.; Spencer, I.D.; Abramowski, Z.
P l a n t a Medica. 1981, 41, 136-142.
47. Jagger, J.J. in "The Science o f Photobiology", Smith, K.D.
(ed), Plenum: New York. 1977.

9
Natural Inducers of Plant Resistance to Insects
MARCOS KOGAN
University of Illinois at Urbana-Champaign, Section of Economic Entomology,
Illinois Natural History Survey and Office of Agricultural Entomology, College of
Agriculture, Urbana, I L 61801
JACK PAXTON
University of Illinois at Urbana-Champaign, Department of Plant Pathology,
Urbana, I L 61801
Many environmental factors may affect herbivore/

plant interaction
tance or s u s c e p t i b i l i t y of the plant to the herbivore.
Among the factors with a demonstrated capability to
induce changes in levels of resistance are tempera-
ture, solar radiation, water stress, s o i l fertility,
insecticides, herbicides, fungicides, growth regula-
tors, pathogen infection, weed competition, and pre-
vious or concurrent herbivore attack. Most chemical
factors, responsible for the resistance of plants to
arthropods studied to date, have involved the geneti-
c a l l y controlled, injury-independent accumulation of
metabolites with allomonal a c t i v i t y . A completely
different plant defense strategy has been demonstrated
for many pathogens for which phytoalexin accumulation
is the result of a post-challenge response. The
aforementioned environmental factors, including pest-
related injury, can induce de novo synthesis and ac-
cumulation of compounds with allomonal properties
(phytoalexins) or change the relative concentration of
both nutrient and non-nutrient compounds. The focus
in this paper is on the post-infestation induction of
resistance by arthropods, in a manner up u n t i l
recently known to result only from pathogen infection.
The i n s e c t - r e s i s t a n c e r o l e of phytoalexins is
discussed and reference is made to other possible
natural sources of inducers of resistance.
Antiherbivory in plants has been ascribed mainly to the

presence of physical defenses or to the injury-independent
accumulation of secondary metabolites that have allomonal
0097-6156/83/0208-0153$06.00/0

properties. These metabolites are compounds that act as

repellents, feeding deterrents, or toxins and are already present
in the plant before herbivore attack. Another defense mechanism,
probably equally important but more d i f f i c u l t to demonstrate,
results from the absence of some required nutrients or compounds
with kairomonal action, such as attractants and feeding excitants
(1).
Antimicrobial defenses in most plants, on the other hand,
have been shown to result from a post-challenge response of the
plant e l i c i t e d by the pathogen through products associated with
the invading organism. Such products are extra-cellular polysac-
charides of bacteria, or the 3-glucans of Phytophthora mycellial
walls ( 2 ) .
Up u n t i l recently, i t has been generally assumed that the two
defense mechanisms have evolved under the specific selection
pressure of animal herbivor
Such mechanisms were studied independently by entomologists and
plant pathologists with l i t t l e or no effort invested to determine
the commonalities that one might have expected to exist between
the two systems. In theory, however, there would be great
advantage for a plant to mobilize i t s defenses against herbivores
only under the reasonable r i s e in risk of injury, such as at the
i n i t i a l stages of an insect infestation. The injury independent
concentration of allomonal compounds may require a considerable
amount of metabolic energy that needs to be diverted from other
v i t a l functions (3) (see also Mooney in this volume). If the
herbivore attack does not occur at a l l , that energy is obviously
wasted.
The post-challenge or injury-dependent response of plants is
a manifestation of induced resistance. For the lack of previous
definitions the following is proposed:
Induced resistance is the qualitative or quantitative
enhancement of a plant's defense mechanisms against pests
in response to extrinsic physical or chemical s t i m u l i .
These extrinsic stimuli are known as inducers or e l i c i t o r s .
Inducers of Plant Resistance or Susceptibility Against Insects
Both physical and biological environmental factors have been

shown to influence resistance or s u s c e p t i b i l i t y of a plant to
insects. Among these factors are: temperature, l i g h t , relative
humidity, s o i l f e r t i l i t y and s o i l moisture, air pollutants (such
as ozone, sulfur dioxide and others) ( 4 ) . These are components of
the plant's physical environment that occur independently of a
man's intrusions. In addition, products made and intentionally
applied by man may influence the quality of plants to insects;
foremost among these are insecticides and fungicides, growth
regulators and herbicides. F i n a l l y , plant pathogens and previous
herbivory also influence the quality of the plant to insects.

9. KOGAN AND PAXTON Natural Inducers 155
Growth regulators, insofar as they are related to plant

hormones, and plant pathogens and herbivores may be perceived as
natural inducing factors. We considered herbicides together with
growth regulators, because of the s i m i l a r i t i e s in their chemistry
and modes of action. We w i l l concentrate our discussion on these
types of inducers of plant defenses against insects.
Induction of Resistance by Growth Regulators and Herbicides
There have been many studies testing the effect of growth

regulators on plants, and attempts have been made to measure this
e f f e c t on i n s e c t s . Table I provides a summary of some
representative studies.
Table I. Examples of Insect Responses to Applied Growth

Regulators.
Inducer Plant Insect Effect Source

Maleic Broad Bean Pea Aphid >Mortality (5)
Hydrazide <Fecundity
Chlormequat Broad Bean Bean Aphid >Mortality (6)

Chloride Cabbage
(=CCC, Aphid
=Cycocel)
Brussels Pea Aphid <Fecundity (8)
Sprout Green Peach
Aphid
Black Hyperomyzus <Fecundity (7)

Currant
Oleander Oleander >Mortality (9)

Aphid
Daminozide Pear Pear Psylla Population (10)
SADH Petunia Whiteflies >Mortality (11)

Gibberi11 in Common Mites <Fecundity (12)
Bean
Apple Mites <Fecundity (13)
Broad Bean Pea Aphid No-effect (5)
Bean Aphid <Fecundity (6)

Application of maleic hydrazide to broadbean plants increases

mortality and reduces the fecundity of bean aphids (5). The
growth retardant CCC (chlormequat) has been tested on several
plant/insect systems, a l l with appreciable increases in resistance
either due to reduced fecundity of females, or increased mortality
of o f f s p r i n g ; both r e s u l t i n g in an o v e r a l l reduction in
populations (6, 7, 8, 9). Occasionally, as i s the case with many
secondary metabolites, certain insect species are affected
detrimentally by the application of a compound, but others are
not. Gibberellin applied to broadbean reduces fecundity in the
bean aphid (6), but has no effect on the pea aphid (5).
Herbicides also have been tested in their effect on insects
(Table II). Results are even more ambiguous than those with
growth regulators. For instance, 2-4D on barley reduced fecundity
in two species of grain aphids, Rhopalosiphum padi and Macrosiphum
avenae (14), but applie
aphids, Rhopalosiphum maydis (15), suggesting an improvement in
the quality of food for the insect. Other herbicides have been
tested with fewer insects, and results vary. Amitrol and Zitron
applied to broadbean reduced fecundity and increased mortality in
the pea aphid Acyrthosiphon pi si (16), but Banvel D, Barban or MCP
applied to barley increased fecundity of the grain aphids M.
avenae, R. padi, and Schizaphis graminum (17).
Table II. Insect Responses to Herbicides Applied to the Food

Plant. (In the "Effect" column, R refers to resistance and _S to
susceptibility.)
Inducer Plant Insect Effect Source

(18)
2,4-D Wheat Stem Sawfly R>Mortality (14)
Barley Grain Aphids R<Fecundity (15)
Corn Corn Leaf S>Fecundity
Aphid (19)
Broad Pea Aphid S>Fecundity
Bean
Rice Rice Stem S>Growth (20)
Borer >Survival
Amitrole Broad Pea Aphid R<Fecundity (16)
Bean >Mortality
Zytron
Banvel D Barley Grain Aphids S>Fecundity (17)
Barban
MCPA

Induction of Resistance by Previous Pathogen Attack
Maclntyre, Dodds and Hare (21), and Hare (22) reported that
some tobacco varieties hypersensitive to tobacco mosaic virus
(TMV) can be protected against attack by other organisms. Local
virus infections induced a systemic protection against the fungi
Phytophthora parasitica var. nicotiana and Peronospora tabacina as
well as the bacterium Pseudomonas tabaci (21). In addition, the
reproductive rate of the green peach aphid, Myzus persicae, was
reduced about 11% when females were allowed to feed and reproduce
parthenogenetically on leaves at plant apices at least 6 nodes
above the s i t e of virus infection (21). Furthermore, i t was found
by Hare and coworkers (22) that growth rates of fourth instar
tobacco hornworms, Manduca sexta, were reduced 27% when reared on
tobacco leaves with local TMV lesions and 16% when reared on
neighboring leaves withou
leaves were on plants tha
The whole plant was systemically induced to higher levels of
resistance.
Induction of Resistance by Previous Herbivory
Feeding a c t i v i t i e s of herbivorous insects often result in

physiological and morphological changes in the host plant. If
these changes are in the direction of the accumulation of
compounds with resistance properties, the herbivores themselves
act as inducers. In Table III examples were drawn from several
insect/ plant systems representing a whole spectrum of annual and
perennial crops as well as vegetable crops, evergreen and
deciduous leaf trees. In a l l these examples the induced
resistance affects the inducing species itself. Thus, for
instance, feeding by the pea aphid on a l f a l f a induces an increase
in coumestrol that may affect additional feeding by other pea
aphids (23). Changes may also occur in the levels of primary
metabolites thus affecting the nutritional value of the leaves.
For example, feeding by gypsy moth, Lymantria dispar, larvae on
gray birch or black oak leaves produces physiological changes in
the plants that may reduce the survival and growth rates of other
larvae feeding on leaves several days after the i n i t i a l attack
(26). It is noteworthy that many changes that occur following
herbivory on trees r e s u l t from accumulations of phenolic
compounds.

Table III. Induction of Resistance by Previous Herbivory.

Inducing Induced
Herbivore Plant Reaction Source
Pea Aphid Alfalfa >Coumestrol (23)
European Pine Pine Polyphenol Synt. (24)
Sawfly
Sweet Potato Sweet Potato Ipomeamarone (25)

Weevi1 (Furanoterpenoid
Phytoalexin)
Gypsy Moth Gray Birch <Nutritional (26)

Black Oak Value
Lep. Larvae Birch >Phenolics (27)
Lygus disponsi Sugar Beet >Quinones (28)

Chinese >Phenolics (29)
Cabbage
Cotton Bollworm Cotton >Phenolics (30)
Striped Cucumber Squash >Cucurbitacins (31)

Beetle
A different type of induction occurs with the striped

cucumber beetle, Acalymma v i t t a t a , feeding on squash (31).
Feeding by this beetle results in an accumulation of cucurbitacins
which are strong feeding excitants for the cucumber beetle i t s e l f .
However, cucurbitacins are deterrent at high concentrations to the
squash beetle, Epilachna tredecimnotata. In f a c t , the squash
beetle seems to have evolved a behavioral adaptation to prevent
accumulation of cucurbitacins in their feeding s i t e s . Before
starting the feeding process, they cut a c i r c u l a r trench
encircling the leaf section upon which they w i l l later feed. The
trench prevents circulation of sap into the enclosed area that
remains free of the allomonal effect of the cucurbitacins, and
suitable to feeding by the beetles (31).
From the above examples i t is thus apparent that a plant
challenged by herbivores, by plant pathogens, or otherwise
stressed by specific chemicals does not remain biochemically
indifferent. The plant responds with more or less drastic
metabolic changes, some of which have a profound effect on
additional herbivores or other plant pathogens. The chemical
nature of these changes is now beginning to be elucidated.

Mechanisms of Induced Resistance
There seems to be a variety of mechanisms involved in

resistance induced by the chemical and biological factors
mentioned above. The mechanisms that we w i l l discuss are the
following: 1) changes in phenological synchronization between
the plant and i t s complement of herbivores; 2) changes in the
physiological state of the plant; 3) changes in nutrient con-
centration; 4) stimulation of compensatory mechanisms; 5) changes
in concentrations of allelochemics—either increases in the
concentration of allomones or reduction in the concentration of
required kairomones; and 6) de novo synthesis of phytoalexins.
This latter is perhaps the most exciting aspect of induced
resistance and one upon which we w i l l dwell in greater length.
Phenological Synchronization
balsam f i r reduce feedin
and desquaring in cotton depress overwintering success of several
major cotton pests. Table IV presents a summary of the work on
these two systems.
TABLE IV. Growth Regulators Used in Induced Phenological

Asynchronization.
Plant Inducer Insect Effect Source

Balsam Abscisic Acid Spruce De1ayed (32)
Fir Budworm budbreak
Maleic
Hydrazide
Chlorflurenol
Cotton 2,4-D (Amine) Pink Bollworm Defoliation (33,34)
Chlorflurenol Heliothis spp. New square (35,36)

shedding
Chlorquemat Boll Weevil Elimination (37)
(CCC) of diapausing
sites
When abscisic acid is applied to balsam f i r , the break of

buds in the spring is delayed and the emerging spruce budworm,
Choristoneura fumiferana, larvae are forced to feed on old
needles, which are a less desirable food. Attempts have been
made, rather unsuccessfully, under natural forest situations, to
manipulate budbreak with growth retardants for the control of the
spruce budworm. However, this system seems to operate under
controlled greenhouse conditions, and with some adjustments, i t
may have potential for practical applications in the f i e l d (32).

Practical results have been obtained in the control of

several cotton pests by the timely application of defoliators or
certain herbicides, such as 2-4D (amine), Chlorflurenol or
Chlorquemat (CCC) (33, 34, 35, 37). Figure 1 shows the phenology
of the cotton plant and the annual cycle of two major cotton
pests: the boll weevil, Anthonomus grandis, and the boll worm,
Heliothis zea. The majority of the bolls are formed and opened by
the middle of September. Those that remain on the plant after
that date do not contribute much to the f i n a l y i e l d , but they do
serve as food and reservoir for diapausing boll weevils,
bollworms, and pink bollworms, Pectinophora gossypiella. It i s
therefore recommended that applications of defoliants be made
starting in the middle of September to eliminate the overwintering
sites or diapausing shelter for these pests. If this i s done on a
wide area, results are indeed outstanding. Even better results
have been obtained fo
interruption of i r r i g a t i o
of defoliants.
Changes in Physiological State of the Plants. Resistance to

f r u i t f l i e s has been obtained with the proper application of
growth retardants. Greany and coworkers in Florida reported that
immature citrus f r u i t s are resistant to f r u i t f l y larvae (39).
Resistance is apparently due to citrus peel o i l s which are mostly
composed of monoterpenes and other terpenoids. Resistance is lost
as the f r u i t s senesce and change color. With timely applications
of g i b b e r l l i c acid, senescence of the peel is retarded, but the
internal maturation processes are not affected. Thus the f r u i t
concentrate sugars while the peel i s s t i l l green and resistant to
the f r u i t f l i e s . Application of these growth regulators
influences a whole range of post harvest pests, not only the f r u i t
f l i e s (39).
Changes in Nutrient Concentration. It has been reported

since the 1960's that g i b b e r e l l i n , CCC and other growth regulators
induce changes in total nitrogen and sugar levels in several
plants. Rodriguez and Campbell (13), working with mites on
apples, and Honeyborne (6), working with aphids on broadbeans and
Brussels sprouts, recorded changes in pest populations following
fluctuations in the concentrations of those nutrients.
Figure 2 (adapted after 13) shows that the increased
concentration of gibberellin solutions applied at weekly intervals
to apples produced a reduction in the concentration of total
nitrogen and sugars with a consequent d e c l i n e in mite
populations.
Stimulation (or Inhibition) of Compensatory Mechanisms. An

interesting effect of herbivore feeding on plants has been
observed by several authors comparing hand defoliation with
herbivory in grasses. One such report shows that the regrowth of

KOGAN AND PAXTON Natural Inducers 161
ACTIVITY Defoliation
Rep.-Diapause
Squares / \ Bolls
/ \ Open
J^^"^ Bolls
PESTS
No./Unit area
Boll Weevil
/ • \
/ \ . " "> -. Bol 1 worm
/ /
L MAY u JUN u JUL w AUG u SEP OCT t NOV
Figure 1. Phenological synchronization of cotton boll opening and diapause of boll

weevils and bollworms (38).
10 50 100 250 500

PPM GIBBERELLIN IN SOLUTION
Figure 2. Effect of weekly sprays of gibberellin solutions on nitrogen (%) and total
sugar (Q) concentration in apple leaves and consequent effect on mite progeny pro-
duction (M) (13).

grasses is stimulated by growth-regulator-type compounds in the

saliva of ruminants (40). However, regrowth seems to be inhibited
in grasses by grasshopper salivary gland and gut extracts at high
defoliation levels, but i t was apparently stimulated at low levels
as is shown in Figure 3 (41). When 1/3 defoliation was
implemented by actual feeding by grasshoppers, there was a sub-
stantial increase in the number of t i l l e r s . T i l l e r i n g was much
less in hand defoliated plants. However, when 100% defoliation
was implemented, hand defoliation produced a greater number of
t i l l e r s than grasshopper induced defoliation. The authors argued
that wheat has evolved a mechanism whereby, at high defoliation
levels induced by grasshoppers, t i l l e r i n g was inhibited. Such
d i f f e r e n t i a l , density-dependent response by the grasses may be
related to a feedback regulatory mechanism of both grass and
grasshopper populations. Such mechanisms are, however, specula-
t i v e , and there is s t i l
Changes in Allelochemic Concentration. Many allelochemic

effects are related to concentrations of diphenolics and the
a c t i v i t y of enzymes involved in their oxidation to qui nones. Hori
and Atalay (29) have shown that the small bug, Lygus disponsi,
feeding on sugar beet leaves induced i n c r e a s e s i n the
concentration of phenolics, and peroxidase and polyphenol oxidase
activities. Similar increases were also observed immediately
after feeding by Lygus disponsi on Chinese cabbage, but levels
returned to near normal after three days. Leaf phenolics that
remain at high concentrations after the i n i t i a l feeding may
influence the amount of herbivory and also pathogen attacks,
although evidence to this effect was not available. Figure 4
(adapted after 29) shows the changes in polyphenol oxidase
a c t i v i t y and in concentration in phenolic compounds following
injury by Lygus disponsi on Chinese cabbage leaves. The a c t i v i t y
and concentration are highest one day after injury, but f a l l and
level off subsequently until 21 days.
De Novo Synthesis of Phytoalexins. Phytoalexins have been

studied in great depth by plant pathologists. Excellent review
papers are available in Hedin's ACS symposium volume, Host Plant
Resistance to Plants (42), and more recently in the book edited by
Horsfall and Cowling, Plant Disease (43). The antiherbivory
effect of phytoalexins, however, is only now beginning to be f u l l y
appreciated. It i s apparent that pathogen induced phytoalexins do
have a definite effect on insect herbivores. There is mounting
evidence that herbivore-inflicted injury may also result in the
induction of phytoalexin production and accumulation.
The antiherbivory effects of phytoalexins have been studied
in our laboratory using the Mexican bean beetle and the soybean
looper (44). The Mexican bean beetle, Epilachna v a r i v e s t i s , i s an
oligophagous species that feeds preferentially "on legume hosts.

1 10r
LEVEL OF DEFOLIATION
Figure 3. Tiller production by wheat plants following defoliation by hand (M) or

by grasshopper grazing (%) (41).
§ soo r
o J 1 I I I I
1 3 6 9 21
DAYS AFTER INJURY
Figure 4. Changes in polyphenol oxidase activity (- • -) and phenolic compounds
(%) in Chinese cabbage tissue after injury by Luygus disponsi (29).

We tested the effect of phytoalexins on the feeding preferences of

both adults and larvae. Soybean seedlings were germinated in the
greenhouse and, when cotyledons were f u l l y opened, small discs
were cut and irradiated with UV-light to e l i c i t phytoalexin
production. Another batch of discs obtained from the second
cotyledonary pair of each plant was not irradiated and was used as
a control. Discs were allowed to incubate for 48 hours, and then
they were exposed in pairs of treated and untreated discs to both
adults and larvae of the Mexican bean beetle. Results of the test
were analyzed using a preference index that gives a value of one
i f there is no preference for either disc in the pair, and a
result smaller than one i f the untreated or control disc i s
preferred. Feeding preferences were measured by counting the
number of scars clearly v i s i b l e on the cotyledon discs. It was
observed that the phytoalexin rich discs were probed, as shown by
fine mandibular markings
was done on the control discs. Feeding preferences on cotyledons
were tested also with the soybean looper, Pseudoplusia includens.
Results showed no feeding deterrency by the phytoalexins. The
effect of pure phytoalexins was tested also using the soybean
looper larvae feeding on a r t i f i c a l media. Due to the small amount
of pure phytoalexins available for the studies, we developed a
miniature system to test the effect of the diet on the growth
rates of newly born larvae up to about the 8th day of l i f e . In
this case, results were not conclusive. When coumestrol was
tested at . 1 , . 5 , and 1% concentration in the d i e t , the percent
survival decreased but the weight gain at the 1% concentration did
not d i f f e r from the control and was twice as high as the weight
gain on the .1% diet (Table V). This is d i f f i c u l t to explain on
toxicological grounds. The survival of larvae on the 1%
glyceollin diet was d r a s t i c a l l y reduced (Table VI), but those
larvae that did survive gained on the average more weight than
those feeding on the control diet.
Table V. Soybean Looper Survival and Weight Gain to

8 Days on A r t i f i c i a l Media + Coumestrol (44).
Coumestrol % Survival % Wgt. Gain

Cone.
0 96.0 100.0
0.1% 64.0 44.1
0.5% 68.0 54.1
1.0% 68.0 96.5

Table VI. Soybean Looper Survival and Weight Gain to

8 days on A r t i f i c i a l Medium + Glyceollin (44).
Glyceollin % Survival % Wgt. Gain

cone.
0 83.2 100.0
0.1% 74.0 76.4
0.5% 75.5 139.2
1.0% 58.0 127.7
To test the v a l i d i t y of the bioassay i t s e l f we prepared a diet

containing increasing amounts of rotenone, a compound derived from
isoflavones and thus chemically not far removed from the soybean
phytoalexins. Results
dose response curve (Table VII). Both survival and weight gain of
larvae were d r a s t i c a l l y affected by increasing concentrations of
rotenone. This experiment showed that the bioassay would be
capable of detecting toxic effects of the phytoalexins on the
soybean looper larvae, i f such effects were acute. It showed also
that the detoxification mechanisms in the soybean looper, a rather
polyphagous insect, may permit i t to adequately overcome the
antibiotic effect of the isoflavonoid phytoalexins, but not that
of the isoflavone rotenone.
Antiherbivory a c t i v i t y of phytoalexins has been demonstrated
also in the f i e l d in studies by Sutherland and his coworkers (45,
46) with scarabaeid grubs feeding on the roots of several forage
legumes. The phytoalexin v e s t i t o l was extracted from the roots of
the forage legume Lotus pedunculatus, and i t was shown to have a
strong feeding deterrent effect on the grubs of Costelytra
zealandica (46). Feeding deterrency was also demonstrated in our
studies with the Mexican bean beetle and phytoalexin r i c h soybean
cotyledons (44).
Table VII. Soybean Looper Survival and Weight Gain to

8 Days on A r t i f i c i a l Medium + Rotenone (Our
Unpublished Data).
Rotenone % Survival % Wgt. Gain

cone.
0 100.0 100.0
0.005% 87.5 46.1
0.05 79.2 16.0
0.5 70.8 9.0
5.0 29.2 3.9

Xn v i t r o studies of the effect of pure phytoalexins on the

feeding of several phytophagous insects, on the other hand, have
provided a diverse picture (Table VIII). For instance, v e s t i t o l
and phaseollin have antifungal a c t i v i t y and also reduce feeding by
Costelytra zealandica and Heperonychus arator. However, various
other phytoalexins such as p i s a t i n , genistein, and coumestrol may
affect one species but not the other. Neither coumestrol nor
genistein seem to i n h i b i t soybean looper feeding or growth.
Table VIII. Effect of Legume Isoflavonoid Phytoalexins

on Feeding by Insects.
Compound Antifungal C. H. P.
Activity zealandica arator includens
in feeding) growth rate)
(-)-Vestitol + + +
(-)-Phaseolin + + +
(+)-Pisatin + +
Genistein - +
Coumestrol - +
Glyceollin + +
One may conclude from these studies that phytoalexins have a

selective effect on herbivores. They i n h i b i t feeding and growth
on some, but seem to be innocuous to others. Given the
revolutionary origin of plant defenses, i t is conceivable that
some phytoalexins may even be kairomones for some insects. There
is ample precedence to this dual role of several allelochemics
(1). The example of cucurbitacins mentioned above is one of
them.
Potential Uses of Induced Resistance in IPM
A better understanding of the role of phytoalexins in plant

defenses and of the mechanisms of induced resistance may
potentially open a powerful new approach to the control of insect
pests of cultivated plants. If indeed, in light of the hypothesis
of optimal defense strategies ( 3 ) , a post-attack response is a
more e f f i c i e n t line of defense than the attack-independent
accumulation of allelochemics, the exploitation of phytoalexin-
producing mechanisms may represent a f e r t i l e f i e l d for future
investigations. Several uses of induced resistance may be
conceived. Four of these approaches are b r i e f l y discussed.

Enhanced Response of Plants to Previous Herbivory by Means of

Classical Breeding Methods. Since plants within the same species
may d i f f e r in t h e i r a b i l i t y to produce and concentrate
phytoalexins in response to s t i m u l i , the idea is to identify such
plants and use their regulating genes in c l a s s i c a l breeding
programs.
Immunization of Plants by Attenuated Forms of Pathogens. As

has been argued by Kuc and Caruso (47), plants can be immunized to
achieve higher levels of resistance to pathogens. Similar
mechanisms may conceivably provide a line of defense against
phytophagous insects without the challenge-independent
accumulation of defensive compounds.
Specific Control of Phytoalexin Accumulation by "Metabolite

Shunting" of Biosyntheti
(personal communication), at the Monsanto Laboratories, St. Louis,
have developed techniques to s e l e c t i v e l y shunt defensive
metabolites, particularly of the shikimic acid cycle. Through
various techniques, certain compounds are applied to plant aerial
or root parts, and these compounds have the property of inducing
specific accumulations of secondary metabolites. The directions
of these accumulations are under known enzymic control (48), and
the regulation of these enzymes is achieved by selecting
appropriate inducers. Such inducers seem to provide a novel
approach to the control of insects by magnifying the a b i l i t y of
plants to produce and concentrate antiherbivory compounds.
Improved Economic I n j u r y L e v e l s Through a B e t t e r

Understanding of the Effects of Low Levels of Herbivory and
Subeconomic Pathogen Infections. One of the c r i t i c a l factors in
IPM is a r e a l i s t i c definition of economic injury levels for single
pests and pest complexes. The understanding of the effect of low
levels of herbivory and subeconomic infections by pathogens may
help reassess the definition of economic injury levels, due to the
evidence that these attacks may actually contribute to an increase
in the a b i l i t y of the plant to withstand future attacks.
Induced Resistance and IPM-Concluding Remarks
Seen from the perspective of a global pest control strategy,

induced r e s i s t a n c e represents a new dimension in c o n t r o l
methodology. As one aspect of the overall f i e l d of plant
resistance, induced resistance is parallel to genetic resistance
and i t may operate through the various modalities that are
c l a s s i c a l l y identified in genetic resistance. Thus, the escape
mechanisms that were discussed as phenological asynchronies may be
obtained or induced by cultural methods or by the timely
application of growth regulators. Antibiosis, either through

PLANT RESISTANCE
I
GENETIC INDUCED
I
•PSEUDORESISTANC
(ESCAPE) BY GROWTH REGULATORS
I
-ANTIBIOSIS —
BY HERBICIDES
NUTRIENT DEFICITS
BY GROWTH REGULATORS
TOXIC ALLOMONES
BY SPECIFIC INDUCERS
-ANTIXENOSIS —
PRESENCE OF ALLOMONES. BY GROWTH REGULATORS
ABSENCE OF KAIROMONES BY SPECIFIC INDUCERS
PHYSICAL CHANGES
•TOLERANCE
REGROWTH
-BY GROWTH REGULATORS
TILLERING
Figure 5. Operation of induced resistance through classical modalities of resistance.

nutrient d e f i c i t s or the accumulation of toxic metabolites, may be

achieved by the application of herbicides, growth regulators or
specific inducers, such as those mentioned under the concept of
metabolite shunting. Antixenosis, or effects on the behavioral
responses of insects, may be the result of accumulation of
allomones, elimination of kairomones or promotion of physical
changes in the plants, a l l of which can be effected by growth
regulators or by specific inducers. F i n a l l y , tolerance may be
obtained through the induction of regrowth or recovery of injured
tissue or the t i l l e r i n g of grasses by growth regulators or by
herbivory i t s e l f .
The increased evidence of the antiherbivory role of
phytoalexins, and improvements in our a b i l i t y to manipulate
phytoalexin and other antiherbivory metabolite accumulations in
plants by the use of growth regulators, specific inducers, or
attenuated forms of pathogen
exciting developments in the fight against insects since the
advent of the modern organo-synthetic i n s e c t i c i d e s . The
difference is that we are enhancing the a b i l i t y of the plants to
produce their own defenses; therefore, we are not tampering with
potentially hazardous chemicals, and we are not grossly impinging
on the integrity of the environment. We are, however, bypassing
the inherently contradictory objective of producing both high
yielding plants and highly resistant plants. From the standpoint
of optimal defense strategies, these two goals are often
impossible to reconcile. Induced resistance represents an
alternative. We may indeed be witnessing the birth of a fourth
generation of insecticides.
Acknowledgements
This publication i s a contribution of the I l l i n o i s Natural

History Survey and I l l i n o i s Agricultural Experiment Station,
College of Agriculture, University of I l l i n o i s at Urbana-
Champaign. Supported in part by USDA Competitive Grant 59-2171-0-
1-452-0, "Role of Phytoalexins in Soybean Resistance to Insect
Pests"; the Regional Project S-157, and the US EPA (through
Texas A&M University; CR-806277-02-0). The opinions expressed
herein are those of the authors and not necessarily those of the
funding i n s t i t u t i o n s .
Literature Cited
1. Norris, D. M.; Kogan, M. in "Breeding Plants Resistant to

Insects" (F. G. Maxwell and P. R. Jennings, ed.), Wiley: New
York, 1980, pp. 23-61.
2. Cruickshank, I.A.M. in "Plant Disease: An Advanced Treatise"
(J. G. Horsfall and E. B. Cowling, ed.), Academic Press: New
York, 1980, pp. 247-67.
3. Rhoades, D. F. in "Herbivores: Their Interaction with

Secondary Plant Metabolites" (G. A. Rosenthal and D. H.

Janzen, ed.), Academic Press: New York 1979, pp. 3-54.
4. Tingey, W. M.; Singh. S. R. in "Breeding Plants Resistant to
Insects" (F. G. Maxwell and P. R. Jennings, ed.), Wiley:
New York, 1980, pp. 87-113.
5. Robinson, A. G. Can. Entomologist 1960, 92, 494-9.
6. Honeyborne, C.H.B. J . S c i . Fd. Agric. 1969, 20, 388-90.
7. Singer, M. C.; Smith, B. D. Ann. Appl. B i o l . 1976, 82, 407-
14.
8. Van Emden, H. F. J . S c i . Fd. Agric. 1969, 20, 385-7.
9. Tahori, A. S.; Halevy, A. H.; Zeidler, G. J . S c i . Fd. Agric.
1965, 16, 568-9.
10. Westigaar, P. H.; Lombard, P. B.; A l l e n , R. B.; Strang, J . G.
Environ. Entomol. 1980, 9, 275-7.
11. Tauber, M. J.; Shalucha, B.; Langhans, R. W. Hort S c i . 1971,
6, 458.
12. Eichmeier, J.; Guyer
13. Rodriguez, J . G.; Campbell, J . M. J . Econ. Entomol. 1961,
54, 984-7.
14. Adams, J.B.; Drew, M. E. Can. J . Zool. 1969, 47, 423-6.
15. Oka, I. N.; Pimental, D. Environ. Entomol. 1979, 3, 911-5.
16. Robinson, A. G. Can. J . Plant S c i . 1961, 41, 413-7.
17. Hintz, S. D.; Schulz, J . T. Proc. N. Cent. Branch Ent. Soc.
Amer. 1969, 24, 114-7.
18. G a l l , A.; Dogger, J . R. J . Econ. Entomol. 1967, 60, 75-7.
19. Maxwell, R. C.; Harwood, R. F. J . Econ. Entomol. 1960, 53,
199-205.
20. I s h i i , S.; Hirano, C. Entomol. Exp. Appl. 1963, 6, 257-62.
21. MacIntyre, J . L.; Dodds, J . A.; Hare, J . D. Phytopathology
1981, 71, 297-301.
22. Hare, J . D. in "Impact of Variable Host Quality on
Herbivorous Insects" (R. F. Denno and M. S. McClure, ed.),
in press.
23. Loper, G. M. Crop S c i . 1968, 8, 104-6.
24. Tejelges, B. A. Can. J . Bot. 1968, 46, 724-5.
25. Akazawa, T.; U r i t a n i , I.; Kubota, H. Arch. Biochem. Biophys.
1960, 88, 150-6.
26. Wallner, W. E.; Walton, G. S. Ann. Ent. Soc. Am. 1979, 72,
62-7.
27. Haukioja, E; Niemela, P. Ann. Univ. Turku (Finland) Ser. A,
1977, 59, 44-7.
28. Hori, K. Appl. Ent. Zool. 1973, 8, 103-12.
29. Hori, K.; Atalay, R. Appl. Ent. Zool. 1980, 15, 234-41.
30. Guerra, D. J . "Natural and Insect-Induced Levels of
Specific Phenolic Compounds in Gossypium hirsutum L." M.S.
Thesis, U. of Arkansas.
31. C a r r o l l , C. R.; Hoffman, C. A. Science 1980, 209, 414-6.
32. E i d t , D. C.; Little, C.H.A. Can. Ent. 1968, 100, 1278-9.
33. Adkisson, P. L. J . Econ. Ent. 1962, 55, 949-51.

34. Kittock, D. L.; Mauney, J . R.; Arle, H. F.; Bariola, L. A.

J. Environ. Qual. 1973, 2, 405-8.
35. Thomas, R. O.; Cleveland, T. C.; Cathey, G. W. Crop S c i .
1979, 19, 861-3.
36. Kittock, D. L.; A r l e , H. F.; Henneberry, T. J.; Bariola,
L. A.; Walhood, V. T. Crop S c i . 1980, 20, 330-3.
37. Bariola, L. A.; Henneberry, T. J.; Kittock, D. L. J . Econ.
Entomol. 1981, 74, 106-9.
38. Hamer, J., ed. "Cotton Pest Management Scouting Handbook"
Missisippi Coop. Extension Service (no date), 48 p.
39. Greany, P. D.; Hutton, T. T.; Rasmussen, G. K.; Shaw, P. E.;
Smoot, J . J . J . Econ. Entomol. in press.
40. Detling, J . K.; Dyer, M. J . Ecology 1981, 62, 485-8.
41. Capinera, J . L.; Roltsch, W. J . J . Econ. Entomol. 1980, 73,
258-61.
42. Hedin, P. A., ed.
American Chemical Society: Washington, D.C., 1979.
43. H o r s f a l l , J . G.; Cowling, E. B., ed; "Plant Disease: An
Advanced Treatise"; Academic Press: New York, 1980.
44. Hart, S. V.; Kogan, M.; Paxton, J . D. J . Chem. Ecol.
(submitted).
45. Russell, G. B.; Sutherland, O. R. W.; Hutchins, R. F. N.;
Christmas, P. E. J . Chem Ecol. 1978, 4, 571-9.
46. Sutherland, O. R. W.; Russell, G. B.; Biggs, D. R.; Lane,
G. A. Biochem. Systematics and Ecol. 1980, 8, 73-75.
47. Kuc, J.; Caruso, F. L. "Host Plant Resistance to Pests,"
American Chemical Society: Washington, D.C., 1979, pp. 78-
89.
48. Hahlbrook, K.; Grisebach, H. Annu. Rev. Plant Physiol.
1979, 30, 105-30.

10
Cytochrome P-450 Involvement in the Interactions
Between Plant Terpenes and Insect Herbivores
LENA B. BRATTSTEN
University of Tennessee, Department of Biochemistry and Graduate Program

in Ecology, Knoxville, TN 37996
The cytochrom
-oxygenase system i s important i n several ways i n
insect herbivores that feed on terpene-containing
plants. Cytochrome P-450 metabolises many terpenes
to polar products that can be excreted, often after
further conjugation reactions, or that may be more
toxic to the insect. Many terpenes induce insect
cytochrome P-450 to higher a c t i v i t y . Changes i n
cytochrome P-450 a c t i v i t y may influence hormone
balance or pheromone production i n the insect,
implicating the plant allelochemicals as factors i n
the regulation of reproductive success of insect
populations. Cytochrome P-450 i s therefore an impor-
tant factor i n insect host-plant specialisations.
Efforts at improving plant resistance to insect
herbivory must take this enzyme system into considera-
tion.
One or more o f the l a r g e v a r i e t y of terpenes, b i o s y n t h e t i c -

a l l y r e l a t e d t o each other as o u t l i n e d i n Figure 1, are present i n
almost a l l higher p l a n t s . The terpenes are a l l f a i r l y to h i g h l y
l i p o p h i l i c compounds depending upon t h e i r s t a t e o f o x i d a t i o n and
g l y c o s y l a t i o n . They t h e r e f o r e have a high p o t e n t i a l f o r t o x i c
i n t e r f e r e n c e w i t h the b a s i c biochemical and p h y s i o l o g i c a l func-
t i o n s of i n s e c t h e r b i v o r e s .
Although the r o l e of the terpenes i n the p l a n t s that produce
them i s s t i l l a matter of debate, evidence of an a n t i - h e r b i v o r e
f u n c t i o n f o r many of them i s accumulating. Modern DNA technology,
gene s p l i c i n g , and c l o n i n g techniques w i l l undoubtedly make i t
p o s s i b l e to i n c o r p o r a t e s u i t a b l e defensive a l l e l o c h e m i c a l s i n t o
s e l e c t e d crop p l a n t s i n order to minimise crop d e v a s t a t i o n by i n -
sect h e r b i v o r e s . However, i t i s necessary to understand the fund-
0097-6156/83/0208-0173$07.00/0

Pheromones
Geranyl-PP (CIO) > Monoterpenes •> Pyrethrum
Gossypol
Farnesyl-PP (CIS) > Sesquiterpenes > Pheromones
Steroids (C27) Triterpenes A

* o d
« r o c n t
' n
Cucurbitacins
Geranylgeranyl-PP (C20) > Oiterpenes (C20)
Graya no toxin
y
Carotenoids (C40)
Figure 1. Outline of the biosynthetic relationships of plant terpenes.

10. BRATTSTEN Cytochrome P-450 Involvement 175
amental mechanisms by which p l a n t populations and i n s e c t popula-

t i o n s c o e x i s t i n p r i s t i n e ecosystems i n order t o take f u l l advan-
tage of t h i s p o t e n t i a l . I t i s c l e a r that the i n s e c t herbivores
have developed many e f f e c t i v e and h i g h l y s p e c i a l i s e d adaptations
f o r d e a l i n g w i t h the unavoidable presence o f these p o t e n t i a l l y
t o x i c chemicals i n t h e i r food supply. Due t o the great d i v e r s i t y
of s p e c i f i c s o l u t i o n s f o r s u r v i v a l that e x i s t i n nature, meaning-
f u l e f f o r t s a t mathematical modeling t o e s t a b l i s h p r e d i c t i v e
models cannot be based simply on the gross behavior of experiment-
a l l y manipulated microcosms. Such models w i l l a t best be v a l i d
only f o r the p a r t i c u l a r s i t u a t i o n on which they are based. Model-
i n g techniques a r e p r e s e n t l y f a r ahead o f the a v a i l a b l e b a s i c
b i o l o g i c a l and e c o l o g i c a l i n f o r m a t i o n upon which t r u l y p r e d i c t i v e
models should be based, i n p a r t i c u l a r when t o x i c a n t s o f any k i n d
are i n v o l v e d .
I n undisturbed ecosystems
ulations coexist i n a
b i o l o g i c a l and p h y s i c a l f a c t o r s ( p a r a s i t e s , predators, p r e c i p i t a -
t i o n , temperature, s o i l q u a l i t y , e t c . ) . But t h i s balance i s a l s o
regulated by myriads of very fundamental i n t e r a c t i o n s , many o r
even most of which are yet unknown, between the p l a n t a l l e l o c h e m i -
c a l s and the b i o c h e m i c a l , p h y s i o l o g i c a l , and b e h a v i o r a l f u n c t i o n s
of the i n s e c t herbivores ( 1 ) .
The complex i n t e r a c t i o n s of terpenes w i t h i n s e c t growth and
reproduction and w i t h the i n s e c t s ' a b i l i t y to metabolise p o t e n t i -
a l l y t o x i c , l i p o p h i l i c f o r e i g n compounds w i l l be discussed b r i e f l y
i n the f o l l o w i n g .
T o x i c i t y of terpenes
As i s the case w i t h p l a n t a l l e l o c h e m i c a l s i n g e n e r a l , the

terpenes are not u s u a l l y a c u t e l y t o x i c . Table I provides a few
examples o f terpene t o x i c i t i e s to mammals and shows that some are,
indeed, h i g h l y t o x i c , e.g. grayanotoxin and e r g o s t e r o l . However,
the v a s t m a j o r i t y of the terpenes show acute mammalian t o x i c i t i e s
only i n the order o f s e v e r a l g/kg, i . e . they a r e not a c u t e l y t o x i c
More data are a v a i l a b l e on p h y t o t o x i n t o x i c i t i e s to mammals than
to i n s e c t s . I t i s not n e c e s s a r i l y true that a compound which i s
t o x i c to a mammal has a s i m i l a r t o x i c e f f e c t i n an i n s e c t . I t i s ,
f o r i n s t a n c e , w e l l known that the p y r e t h r i n s a r e h i g h l y t o x i c t o
i n s e c t s and are used w i t h s a f e t y as s e l e c t i v e i n s e c t i c i d e s .
Limonene, although not t o x i c t o mammals, i s a c u t e l y t o x i c t o
Dendroctonus pine b e e t l e s (2, 3). On the other hand, chemicals
that a r e h i g h l y t o x i c t o mammals may not be t o x i c t o i n s e c t s . An
example i s e r g o s t e r o l which i s h i g h l y t o x i c t o s e v e r a l mammals i n
a d d i t i o n t o the dog, but which l a c k s acute t o x i c i t y t o l a r v a e of
the southern armyworm, Spodoptera e r i d a n i a ( B r a t t s t e n , unpub-
l i s h e d ) . This compound i s probably used as a precursor i n the
s y n t h e s i s o f c h o l e s t e r o l by a t l e a s t some i n s e c t s (4).

Table I
Mammalian acute t o x i c i t i e s of p l a n t terpenes
Compound ^50 Animal, r o u t e
Grayanotoxin 1.2 mg/kg mouse, i p

Ergosterol 4 mg/kg dog, o r a l
Gossypol 550 mg/kg pig, oral
Pyrethrum 200 mg/kg rat, oral
Pulegone 120 mg/kg rat, ip
Hymenovin 150 mg/kg mouse, o r a l
a-pinene s e v e r a l g/kg rat, oral
Limonene s e v e r a l g/kg rat, oral
Carvone s e v e r a l g/kg rat, oral
Borneol
Cineole
Citral several g/kg rat, oral
Eugenol several g/kg mouse, o r a l
Geraniol several g/kg rat, oral
Menthol several g/kg rat, oral
Terpineol several g/kg rat, oral
Caryophyllene several g/kg rat, oral
Nepetalactone several g/kg rat, oral
A l l i n f o r m a t i o n from (46) except f o r hymenovin ( 4 7 ) .
Terpenes as i n s e c t a t t r a c t a n t s and deterrents
Numerous terpenes are a t t r a c t a n t s f o r i n s e c t s . Table I I

shows some examples. The compounds a r e very o f t e n feeding
a t t r a c t a n t s . They can a l s o be o v i p o s i t i o n s t i m u l i a s , f o r
i n s t a n c e , a-pinene f o r the eastern spruce budworm or methyl i s o -
eugenol f o r the c a r r o t r u s t f l y . The e v o l u t i o n a r y d e t a i l s of
some of these r e l a t i o n s , o f t e n r e v e a l f a s c i n a t i n g cases of i n s e c t
h o s t - p l a n t s p e c i a l i s a t i o n s . A chemical that i s a deterrent to
most i n s e c t s can become an o b l i g a t o r y feeding cue f o r a s p e c i a l i s t ^
e.g. the spotted cucumber b e e t l e ' s dependency on c u c u r b i t a c i n s f o r
food r e c o g n i t i o n . Even more i n t r i c a t e r e l a t i o n s h i p s e x i s t as w i t h
Ips and Dendroctonus bark b e e t l e s using the host t r e e (+)'-a-pinene
as precursor f o r t h e i r own aggregation pheromone component, c i s -
verbenol (_5, _6) .
A study w i t h h o u s e f l i e s (7) shows c l e a r l y that very s m a l l
d i f f e r e n c e s i n the molecular s t r u c t u r e can r e s u l t i n d r a s t i c a l l y
d i f f e r e n t b i o l o g i c a l e f f e c t s as e x e m p l i f i e d i n Table I I I by the
o p t i c a l isomers (-)-limonene, a f l y a t t r a c t a n t , and (+)-limonene,
a f l y d e t e r r e n t . A d i f f e r e n c e i n o x i d a t i o n s t a t e i n the func-
t i o n a l group as i n c i t r o n e l l o l , a f l y a t t r a c t a n t , and c i t r o n e l l a l ,
a f l y d e t e r r e n t , a l s o causes d i f f e r e n t responses. A d i f f e r e n c e i n
the length of the carbon chain as i n f a r n e s o l (C15), a f l y a t t r a c -
t a n t , and g e r a n i o l (C10), a f l y d e t e r r e n t , a l s o confers d i f f e r e n t

Table I I
P l a n t terpenes as a t t r a c t a n t s f o r i n s e c t s
Insect species Compound Referer
Eastern spruce budworm a-pinene 48

Ips bark b e e t l e s
S c o l y t i d bark b e e t l e s
g-pinene
limonene
camphene
geraniol
a-terpineol
Honeybee geraniol 51
Japanese b e e t l e geraniol 52
citronellal
Pine b e e t l e a-terpineol 53
Pales w e e v i l eugenol 54
a-pinene
anethole
citronellal
Oriental f r u i t f l y methyleugenol 55
Carrot r u s t f l y mehyl isoeugenol 56
B o l l weevil a-pinene 57
(3-pinene
limonene
caryophyllene
Lace wing iridodiol 58
Spotted cucumber b e e t l e cucurbitacins 59
Willow b e e t l e salicin 60
S i l k worm t e r p i n y l acetate 61
linalool
hexenol

b i o l o g i c a l p r o p e r t i e s . Table I I I a l s o shows an example of the

c o n c e n t r a t i o n e f f e c t . In t h i s case a low c o n c e n t r a t i o n of carvone
i s an a t t r a c t a n t , whereas a high c o n c e n t r a t i o n i s a d e t e r r e n t .
This i s a very w i d e l y occuring phenomenon, and w e l l known a l s o i n
human s o c i e t y , e.g. i n the contexts of s p i c e s and perfumes.
Table IV gives a few examples of terpenes shown to be i n s e c t
d e t e r r e n t s . As w i t h the previous examples of i n s e c t a t t r a c t a n t s
i n Tables I I and I I I , the s t r u c t u r a l d i v e r s i t y of the d e t e r r e n t
compounds i s remarkable. There are no c l e a r and l o g i c a l s t r u c -
t u r e - a c t i v i t y r e l a t i o n s h i p s among the compounds w i t h these behav-
i o r a l e f f e c t s . S p e c i a l i s e d and unique e f f e c t s and b e h a v i o r a l
adaptations are thus the r u l e i n i n t e r a c t i o n s between species
r a t h e r than the exception.
The Vernonia sesquiterpene l a c t o n e * g l a u c o l i d e A, i s a feed-
ing d e t e r r e n t f o r the southern armyworm, a broadly g e n e r a l i s t
feeder. This compound
worm l a r v a e r e j e c t . Thi
armyworm, J3. f r u g l p e r d a , are capable of feeding on a l a r g e v a r i e t y
of p l a n t s ( 8 ) . Both can a l s o metabolize l i p o p h i l i c f o r e i g n com-
pounds, i n c l u d i n g p l a n t a l l e l o c h e m i c a l s and s y n t h e t i c p e s t i c i d e s ,
to e x c r e t a b l e , p o l a r metabolites by cytochrome P-450-dependent
oxidations.
Southern and f a l l armyworm growth on pulegone-laced d i e t s
The mint monoterpene pulegone i s not only a feeding d e t e r r e n t

f o r the f a l l armyworm but i s a l s o t o x i c to t h i s species ( 9 ) . I t
i s , however, n e i t h e r a feeding d e t e r r e n t nor a c u t e l y t o x i c to the
southern armyworm at s i m i l a r c o n c e n t r a t i o n s . F i g u r e 2a shows that
a c o n c e n t r a t i o n of 0.1% pulegone i n the d i e t (10) i s t o x i c and
k i l l s the f a l l armyworm l a r v a e (11). In c o n t r a s t , the southern
armyworm l a r v a e feed f r e e l y on d i e t s (12) c o n t a i n i n g e i t h e r 0.01%
or 0.1% pulegone. Their weight increases and there i s no delay
i n t h e i r development (Figure 2b). However, f u r t h e r experiments
w i t h the southern armyworm showed that pulegone can be a very
important f a c t o r i n the s u c c e s s f u l coexistence of p l a n t s and her-
b i v o r e s (13).
Pulegone e f f e c t s on the southern armyworm
The data i n Table V (13) show that the southern armyworm

l a r v a e a t t a i n higher maximal f r e s h body weights when pulegone i s
present i n t h e i r d i e t up to 0.1%. But the percentage of non-
water body c o n s t i t u e n t s i s reduced as i s most obvious i n l a r v a e
feeding on a 0.2% pulegone d i e t . The l a t t e r d i e t a l s o prolongs
the time the l a r v a e spend i n the s i x t h i n s t a r and they undergo the
pupal molt 8-10 days l a t e r than l a r v a e fed c o n t r o l d i e t s or d i e t s
w i t h lower pulegone c o n c e n t r a t i o n s . The 0.2% pulegone d i e t a l s o
reduces the pupation success to 57% as shown i n Table VI
(13) , but a d u l t emergence from these pupaeoccursat almost normal
r a t e (85%).

Table I I I
Terpenes as housefl y a t t r a c t a n t s and r e p e l l e n t s ( 7 ) .
Attractants Deterrents
(-)-limonene (+)-limonene
Citronellol Citronellal
Eugenol Citral
Farnesol Geraniol
Carvone (low) Carvone (high)
6 -phellaudrene
Carvacrol
Linalool
Table IV
P l a n t terpenes as d e t e r r e n t s f o r i n s e c t s
Insect species Compound References
Western pine b e e t l e Myrcene 62

Limonene
Gypsymoth l a r v a e Farnesol 63
Geranial
Nerolidol
Many species Nepetalactone 64
Azadirachtin 65
Gossypol 66
Silkworm Terpineol 61
Geranylacetate
Geraniol
A f r i c a n armyworm Warburganal 67
Xylomollin 68
Beet armyworm Caryophyllene 69
F a l l armyworm Pulegone 9
Glaucolide A 70
Southern Armyworm Glaucolide A 70

S. fruoiperdo
V
„4
~« 1 1 1
5 1
Days since molting to 4th instar
5 10 15
Days since molting to 4th instar
Figure 2. Growth from fourth instar to pupation on control or pulegone-containing

diet of fall armyworm larvae (11) (top) and southern armyworm larvae (13) (bot-
torn). Key: control; A, 0.01% pulegone; and A, 0.1% pulegone.

Table V
E f f e c t of d i e t a r y pulegone on body weight and accumulation of non-
water body c o n s t i t u e n t s i n southern armyworm l a r v a e and pupae (13)
Larvae Pupae
Diet mg/fresh l a r v a l %dry mass mg/fresh pupa %dry mass
Control 622.4 111 12 282.8 22 21
0.01% 672.1 24* 10 278.1 20 20
0.1 % 784.8 96* 8* 323.0 53* 20
0.2 % 687.5 88 8* 296.2 36 15*
* S i g n i f i c a n t l y d i f f e r e n t from c o n t r o l a t P< 0.002 ( T - t e s t )

Data are mean ± S.D.
Larvae ate the 0.2% pulegone d i e t during the s i x t h i n s t a r o n l y ;
they ate the other d i e t
pupation.
However, a l l l e v e l s o f d i e t a r y pulegone a f f e c t the egg

p r o d u c t i o n o f the moths. The data i n Table V I i n d i c a t e a reduced
o v i p o s i t i o n r a t e even a t the lowest (0.01%) pulegone c o n c e n t r a t i o n
i n the l a r v a l d i e t . The h i g h e s t c o n c e n t r a t i o n (0.2%) reduces the
egg p r o d u c t i o n to 10% of that i n the c o n t r o l moths and hatching i s
a l s o reduced t o 10% (13).
This case i l l u s t r a t e s a p o s s i b l e , n a t u r a l l y occuring
mechanism whereby even a minute amount o f a b i o a c t i v e p l a n t a l l e -
l o c h e m i c a l may reduce the r e p r o d u c t i v e c a p a c i t y of an i n s e c t her-
b i v o r e p o p u l a t i o n not d r a s t i c a l l y , but conceivably enough so that
continued co-existence i s p o s s i b l e . The i n s e c t p o p u l a t i o n may be
reduced only t o the p o i n t where the p l a n t s can s t i l l grow and r e -
produce s u c c e s s f u l l y , thereby i n s u r i n g a continued food supply f o r
the i n s e c t h e r b i v o r e .
An e n t i r e l y d i f f e r e n t , i n f a c t o p p o s i t e , e f f e c t on i n -
s e c t r e p r o d u c t i o n by terpenes occurs w i t h the desert l o c u s t . I n
t h i s case the monoterpenes a-pinene, $-pinene, limonene, and euge-
n o l evaporating from desert shrubs about to bloom, p r e c i p i t a t e s
synchronised sexual maturation and mating a c t i v i t y i n the l o c u s t s
(14). The spruce budworm i s a l s o s t i m u l a t e d to increased f e r t i l -
i t y l e v e l s by host t r e e monoterpenes (R.G. Cates, personal*commun-
i c a t i o n ) . I t i s p o s s i b l e that even opposite e f f e c t s on reproduc-
t i o n i n i n s e c t s could occur depending on the s p e c i a l i s a t i o n o f the
i n s e c t species t o i t s environment, the d i v e r s i t y o f the b i o l o g i c a l
a c t i v i t i e s o f the compounds, and the high l e v e l o f complexity o f
the r e p r o d u c t i v e processes.

Table VI
E f f e c t s of l a r v a l d i e t a r y pulegone on development and o v i p o s i t i o n
i n the southern armyworm
Diet %Pupation Days to %Emergence Days to Eggs per

emergence oviposition female
Control 96 12 3 96 3.5 1900-2000

0.01% 92 12 3 93 3.5 1500-1800
0.1 % 82 12 3 93 3.0 1000-1200
0.2 % 57 15 3 85 2.5 100-200
Larvae ate the 0.2% pulegone d i e t d u r i n g the s i x t h i n s t a r o n l y ;

they ate the other d i e t s from f o u r t h i n s t a r through to pupation.
1
The "Days to emergence ' dat i n d i c a t that moth durin
6-day period w i t h a pea
Terpene involvement i n i n s e c t r e p r o d u c t i o n
Several d i f f e r e n t molecular mechanisms could be i n v o l v e d i n

the r e p r o d u c t i v e i n h i b i t i o n observed i n the southern armyworm.
For i n s t a n c e , many terpene d e r i v a t i v e s mimic i n s e c t hormone action.
Juvabione (15) i s the c l a s s i c a l example of a j u v e n i l e hormone (JH)
mimic that prevents egg maturation i n P y r r h o c o r i s bugs. Aromatic
terpene ethers (16), methylene dioxyphenyl terpene ethers (17),
and other f a r n e s y l d e r i v a t i v e s a l s o have JH a c t i v i t y and the
l a t t e r ones (18) a l s o cause s t e r i l i t y i n P y r r h o c o r i s . For the most
p a r t JH a c t i v e terpenes are among the sesquiterpenes but s e v e r a l
monoterpenes a l s o have i n s e c t s t e r i l i z i n g e f f e c t s (19, 20). The
a c y c l i c monoterpene c i t r a l reduces the f e r t i l i t y of r a t s by caus-
i n g f o l l i c u l a r degeneration (21).
The precocenes, another c l a s s of terpene d e r i v a t i v e s w i t h JH
a n t a g o n i s t i c e f f e c t s , i n a c t i v a t e JH s y n t h e s i s by s p e c i f i c i n h i b i -
t i o n of corpora a l l a t a (CA) microsomal cytochrome P-450-dependent
mixed-function oxidases (22). These enzymes, of primary impor-
tance i n l i p o p h i l i c f o r e i g n compound metabolism, are e s s e n t i a l i n
the b i o s y n t h e s i s of JH as o u t l i n e d i n F i g u r e 3 (23). They may
a l s o c o n t r i b u t e to the i n a c t i v a t i o n of JH as the metabolic scheme
i n F i g u r e 4 i n d i c a t e s (_24, ^ 5 , 26) although epoxide hydrase and
esterase a c t i v i t i e s dominate here (26, 27). The presence of JH i n
the female a d u l t i s necessary f o r v i t e l l o g e n i n s y n t h e s i s (28, 29)
i n many i n s e c t s . Therefore, s i n c e the cytochrome P-450 oxidase
system i s e s s e n t i a l f o r the maintenance of balanced JH t i t e r s , even
s l i g h t changes i n the P-450 system i n response to e x t e r n a l induc-
ers and i n h i b i t o r s could have profound e f f e c t s on the dynamic
c h a r a c t e r i s t i c s of i n s e c t p o p u l a t i o n s .

BRATTSTEN Cytochrome P-450 Involvement
Farnesyl pyrophosphate
oxidation(s)
Farnesoic acid
^ Methyltransferase, SAM
OCH,
Methylfarnesoate
CA cytochrome P-450
NADPH + H +, 0 2
OCH.
Figure 3. Outline of juvenile hormone biosynthesis based on Ref. 23.

Downloaded by PURDUE UNIVERSITY on September 14, 2010 | http://pubs.acs.org
00
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Η
ce
53
H
>
Ο
W

H
Ο
Figure 4. Outline of juvenile hormone inactivation based on Refs. 25 and 26. 1

w
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H
09
I n t e r a c t i o n of terpenes and cytochrome P-450. Metabolism
The major metabolic f a t e o f higher p l a n t terpenes i n mammals

i s o x i d a t i o n followed by conjugation u s u a l l y to g l u c u r o n i c a c i d
(30). However, s p e c i f i c data on even some of the most common com-
pounds are not r e a d i l y a v a i l a b l e . Limonene metabolism seems to
have been s t u d i e d unusually i n t e n s i v e l y , maybe due to i t s thera-
peutic use t o d i s s o l v e p o s t - o p e r a t i v e l y r e t a i n e d g a l l s t o n e s .
Limonene i s t y p i c a l l y converted to t r a n s d i o l s v i a cytochrome
P-450-mediated epoxidation of e i t h e r one of the two double bonds,
followed by epoxide h y d r a t i o n (31). I t i s a l s o converted t o sev-
e r a l a l c o h o l s (32, 33) which may undergo f u r t h e r o x i d a t i o n by de-
hydrogenases as i n d i c a t e d i n Figure 5. A l l these products are
non-toxic and are excreted, i n some cases a f t e r g l u c u r o n i d a t i o n .
The cases where terpen metabolis ha bee studied i i n
sects are very few indeed
b e e t l e s convert monoterpene a-pinene, 8-pinen myr
cene to o x i d a t i o n products, some of which have pheromonal a c t i v i -
t i e s (_5, ^, J34, 35). A Dendroc tonus bark b e e t l e ' s cytochrome
P-450 converts a-pinene to s e v e r a l o x i d i z e d products a f t e r induc-
t i o n by a-pinene, and to a t l e a s t one o x i d i z e d product without
p r i o r i n d u c t i o n (36). Rat l i v e r cytochrome P-450 a l s o converts
a-pinene t o o x i d a t i o n products (36) and t h i s a c t i v i t y i s induced
by phenobarbitol and 3-naphthoflavone. There i s a l s o the i n t e r -
e s t i n g p o s s i b i l i t y that the b a c t e r i a l f l o r a i n the bark b e e t l e s
may c o n t r i b u t e to the o x i d a t i o n of a-pinene to t r a n s - and c i s -
verbenol. A bacterium, B a c c i l l u s cereus, i s o l a t e d from the hind-
gut of Ips paraconfusus c a t a l y s e s these o x i d a t i o n s (37).
The microsomal cytochrome P-450 system i n the midguts of
southern armyworm l a r v a e o x i d i s e s pulegone in v i t r o . The two
major products, 9-hydroxypulegone and 10-hydroxypulegone are form-
ed by microsomes from l a r v a e induced w i t h e i t h e r pentamethylben-
zene o r a-pinene. Microsomes from c o n t r o l (un-induced) l a r v a e
only o x i d i s e trace amounts o f the compound. The 9-hydroxypulegone
rearranges spontaneously to menthofuran (38).
In both these cases where an i n s e c t cytochrome P-450 system
has been shown to be r e s p o n s i b l e f o r the o x i d a t i o n of a-pinene and
pulegone, the enzyme had to be induced to higher a c t i v i t y to e f -
f e c t i v e l y c a t a l y s e the r e a c t i o n . This leads to the question of
whether i n s e c t P-450-dependent o x i d a t i o n s are s u f f i c i e n t l y a c t i v e
i n n a t u r a l s i t u a t i o n s to produce a s i g n i f i c a n t amount o f the
metabolites. Due to the importance of cytochrome P-450 o x i d a t i o n s
i n p e s t i c i d e metabolism, there are, f o r t u n a t e l y , s e v e r a l s t u d i e s
which show that the i n s e c t oxidase system i s e a s i l y and r a p i d l y
induced i n response to a l a r g e v a r i e t y o f non-nutrient chemicals
i n the food.


I n t e r a c t i o n s of terpenes and cytochrome P-450. Induction
The c l a s s i c a l examples o f cytochrome P-450 inducers among the

terpenes are the hormonal s t e r o i d s . Among the p h y t o s t e r o l s ,
s i t o s t e r o l , s t i g m a s t e r o l (39), and e r g o s t e r o l (Table V I I ) are i n -
ducers of the southern armyworm microsomal oxidases. The i n s e c t
m o l t i n g hormones a-ecdysone and ecdysterone, w i d e l y occuring i n
p l a n t s , a r e very potent inducers of housefly microsomal a l d r i n
epoxidase a c t i v i t y (40). Some of the most a c t i v e inducers of i n -
sect cytochrome P-450 a r e among the monoterpenes, e.g. myrcene,
camphene (39) and other shown i n Table V I I . Menthol, menthone,
a-pinene, and 8-pinene induce a l d r i n epoxidase a c t i v i t y i n micro-
somes from the v a r i e g a t e d cutworm larvae (41). However, i n t h i s
case, limonene was i n h i b i t o r y (41) whereas both (+)- and (-)-
limonene are good inducers of the southern armyworm oxidase system
(_39 and Table V I I ) .
Table V I I
Terpene i n d u c t i o n of midgut microsomal cytochrome P-450 and
pyrethrum-dependent NADPH o x i d a t i o n i n southern armyworm
l a r v a e (42)
Cytochrome P-450 X-max NADPH o x i d a t i o n

Diet (nmole/mg p r o t e i n ) (nm) (nmole/min,mg p r o t e i n )
control 0.350 450.1 10.96

stigmasterol 0.396 450.3 12.13
ergosterol 0.371 449.8 16.57
a-pinene 0.881* 449.9 33.27*
8-pinene 0.741* 449.5 33.89*
(+)-limonene 0.683 449.8 32.55*
(-)-limonene 0.967* 449.8 31.20*
a-terpinene 0.876* 450.0 38.12*
Y-terpinene 1.028* 449.9 41.34*
• S i g n i f i c a n t l y d i f f e r e n t from c o n t r o l a t P < 0.001 ( T - t e s t ) .

S i x t h i n s t a r l a r v a e were fed ad l i b i t u m f o r 3 days on d i e t s con-
t a i n i n g 0.2% o f the terpene.
Table V I I shows the increase i n cytochrome P-450 content i n

microsomes from southern armyworm l a r v a l midguts r e s u l t i n g from
d i e t a r y exposure t o s e v e r a l c y c l i c monoterpenes (42). I t a l s o
shows a c l o s e l y corresponding i n c r e a s e i n the r a t e of NADPH o x i -
d a t i o n when pyrethrum i s the s u b s t r a t e (R) being o x i d i s e d . The
microsomal cytochrome P-450 system i s arranged as o u t l i n e d i n
F i g u r e 6, c o n s i s t i n g of a t e r m i n a l heme-iron p r o t e i n that i n the
o x i d i s e d (Fe3+) s t a t e binds the s u b s t r a t e (R). The complex under-
goes two reductions d u r i n g which bound molecular oxygen i s con-
verted t o f r e e r a d i c a l s p e c i e s , one of which i s i n s e r t e d i n the
s u b s t r a t e molecule, and the other one forms water. The reductions

Figure 6. Outline of the microsomal cytochrome P-450 system.

are s p e c i f i c a l l y dependent upon NADPH and a f l a v o p r o t e i n which

t r a n s p o r t s the reducing e q u i v a l e n t s to the cytochrome. The e n t i r e
system i s deeply embedded i n the endoplasmic r e t i c u l u m membranes
and depends on the membrane p h o s p h o l i p i d f r a c t i o n t o a s s i s t i n the
b i n d i n g o f the l i p o p h i l i c s u b s t r a t e molecule. The NADPH-depen-
dence o f f e r s a convenient spectrophotometric method f o r measuring
the r a t e o f oxygenation o f s u b s t r a t e s i n cases where there i s no
simple means of a n a l y s i n g the r a t e o f product f o r m a t i o n , o r when
s e v e r a l d i f f e r e n t m e t a b o l i t e s a r e formed as i n the cases o f
pyrethrum and pulegone.
The r a t e s o f pulegone-dependent NADPH o x i d a t i o n i n southern
armyworm microsomes are shown i n Table V I I I (38). I n t h i s case
microsomes from c o n t r o l d i e t - f e d l a r v a e show only t r a c e a c t i v i t y
i n agreement w i t h r e s u l t s o f the gas chromatographic m e t a b o l i t e
analyses. The southern armyworm cytochrome P-450 system apparent-
l y more e a s i l y oxygenate
Table V I I I the more poten
i n accordance, the r a t e of pulegone metabolism i s twice as h i g h
w i t h t h i s inducer as when a-pinene i s the inducer. A l l the p l a n t
monoterpenes appear t o induce a molecular form of the cytochrome
that i s i d e n t i c a l t o the c o n s t i t u t i v e form as i n d i c a t e d by the
p o s i t i o n of the a b s o r p t i o n maximum o f the cytochrome-CO d i f f e r e n c e
spectrum. Pentamethylbenzene, on the other hand, appears t o i n -
duce a molecular form w i t h some minute s t r u c t u r a l d i f f e r e n c e ( s )
from the c o n t r o l form as i n d i c a t e d by a s h i f t i n the a b s o r p t i o n
maximum t o 449.0 nm. I n the case o f pulegone o x i d a t i o n , the
M i c h a e l i s constants (Km) i n d i c a t e t h a t both the pentamethylbenzene
and a-pinene-induced cytochrome have s i m i l a r a f f i n i t y f o r the com-
pound. However, the pentamethylbenzene-induced form h y d r o x y l a t e s
pulegone i n the CIO p o s i t i o n three times more o f t e n than the
a-pinene-induced form (38).
The inducing e f f e c t s on the southern armyworm cytochrome
P-450-mediated metabolism shown i n Tables V I I and V I I I r e s u l t e d
1
from 3 days f e e d i n g on d i e t s c o n t a i n i n g 0.2% o f the terpene.
This c o n c e n t r a t i o n may occur i n many p l a n t s p e c i e s . However, a
much s m a l l e r dose of the inducer a l s o e f f e c t s a measureable
d i f f e r e n c e i n the oxygenation r a t e s as shown w i t h a-pinene and
s i n i g r i n i n d u c t i o n o f southern armyworm P-450 (39). The data i n
Table IX show that a s i n g l e dose o f 100 yg/g o f l a r v a l body weight
r e s u l t s i n a c t i v i t i e s h i g h e r than those i n c o n t r o l l a r v a e . Con-
s i d e r i n g the feeding behavior o f the southern armyworm l a r v a e ,
c o n s i s t i n g of feeding bouts o f 10-20 minutes f o l l o w e d by e q u a l l y
long r e s t i n g p e r i o d s ( 4 3 ) , i t seems reasonable t o assume that the
metabolism of some b i o a c t i v e l e a f component may be increased
enough i n a n a t u r a l s i t u a t i o n t o c o n t r i b u t e s i g n i f i c a n t l y t o the
r e v o l u t i o n a r y s t a t u s o f the i n s e c t w i t h i t s host p l a n t ( 3 9 ) .
This would be a v a l i d assumption whether a d e t o x i f i e d o r an
a c t i v a t e d p o l a r product i s formed.

Table V I I I
Pulegone-dependent NADPH o x i d a t i o n i n r e l a t i o n t o cytochrome P-450
content i n midgut microsomes form southern armyworm l a r v a e ( 3 8 ) .
Cytochrome P-450 NADPH o x i d a t i o n

concentration X max V m a x Km
Diet (nmole/mg p r o t e i n ) (nm) (nmole/mi^mg p r o t e i n ) (uM)
control 0.346 450.0 Trace
a-pinene 0.815 449.9 39.86 ± 4.88 10.28 ± 1.94
PMB 1.120 449.0 80.11 ± 11.52 11.64 ± 2.88

Larvae fed ad l i b i t u m f o r 3 days on c o n t r o l d i e t o r d i e t s c o n t a i n i n g 0.2% o f the inducer.

Table IX
Rapid i n d u c t i o n of microsomal oxidase a c t i v i t y

By terpenes i n southern armyworm l a r v a e
Specific activity Percent Significance

Inducer (nmole/mi^mg p r o t e i n ) of c o n t r o l (2-tailed T-test)
control 1.66 ±0.1 (7) 100

d-carvone 2.22 ±0.2 (4) 134 P <0.001
1-carvone 1.92 ±0.2 (4) 115 0.002>P>0.001
caryophyllene 2.07 ±0.2 (4) 125 0.002>P>0.001
carotol 1.95 ±0.2 (4) 117 0.01>P > 0.005

Larvae were i n d i v i d u a l l y f e d a l i m a bean l e a f d i s c loaded w i t h a terpene dose of 100 ± 2 \xg
per gram of t h e i r body weight. They were k i l l e d one hour a f t e r f e e d i n g . A c t i v i t y of p - c h l o r o
N - m e t h y l a n i l i n e N-demethylase was measured i n p o s t - m i t o c h o d r i a l supernatants as d e s c r i b e d e a r l i e r ( 7 1 ) ,
The data a r e mean ±. S.D. of (N) experiments.

Conclusions
I t would be c h a r i t a b l e to say that the cytochrome P-450

system i n i n s e c t h e r b i v o r e s does not e n t i r e l y d i c t a t e t h e i r i n -
t e r a c t i o n s w i t h the host p l a n t s . There i s a very a c t i v e cyto-
chrome P-450 system i n the o l f a c t o r y receptor region of the dog
(44), i n d i c a t i n g the t a n t a l i s i n g p o s s i b i l i t y that such an enzyme
system, i f s u i t a b l y l o c a t ed i n the i n s e c t , i s a l s o c o n t r i b u t i n g
to the e f f e c t s of p l a n t a l l e l o c h e m i c a l s on i n s e c t feeding be-
h a v i o r . The cytochrome P-450 may a l s o be i n v o l v e d i n pheromone
b i o s y n t h e s i s i n more species of i n s e c t s (45) than the bark
b e e t l e s . Cytochrome P-450 i s d i r e c t l y i n v o l v e d i n i n s e c t JH
b i o s y n t h e s i s , of e s s e n t i a l importance i n both development and
reproduction. The microsomal cytochrome P-450 system i s of
c e n t r a l and c r u c i a l importance i n the metabolsim of l i p o p h i l i c
f o r e i g n compounds of a l
biological significanc
p l a n t a l l e l o c h e m i c a l s . I t i s beyond doubt that the cytochrome
P-450 system s t r o n g l y c o n t r i b u t e s to the great v a r i e t y of unique
s o l u t i o n s f o r s u r v i v a l that e x i s t between i n s e c t h e r b i v o r e s and
plants.
Acknowledgement
I thank C.K. Evans, C.A. Gunderson, and J.T. Fleming f o r

e x c e l l e n t a s s i s t a n c e . P r e v i o u s l y unpublished work presented here
was supported by U.S. Department of A g r i c u l t u r e , (SEA-GAMO)
Competitive Research Grant Program grant No. 59-1471-1-1-695-0
and NSF grant PCM 81 00081.
Literature cited
1. C l a y t o n , R.B., i n "Aspects of Terpenoid Chemistry and B i o -

chemistry" (T.W. Goodwin, ed.) Academic P r e s s , New York,
1971, p. 1-27.
2. Smith, R.H. J . Econ. Entomol. 1965, 58, 509-510.
3. Coyne, J.F., L o t t , L.H. Georgia Entomol. Soc. 1976, 11, 297-
301.
4. Thompson, M.J., Svoboda, J.A., K a p l a n i s , J.N., Robbins, W.E.
Proc. Roy. Soc. Lond. 1972, B180, 203-221.
5. Renwick, J.A.A., Hughes, P.R., Ty, T.D. J . Insect P h y s i o l .
1973, 19, 1735-1740.
6. Vité, J.P., Bakke, A., Renwick, J.A.A. Can. Entomol. 1972,
104, 1967-1975.
7. Sharma, R.N., Saxena, K.N. J . Med. Entomol. 1974, 11, 617-
621.
8. T i e t z , H.M.: "An Index to the described L i f e Histories,
e a r l y Stages and Hosts of the Macrolepidoptera of the C o n t i -
n e n t a l United States." A.C. A l l y n P u b l . , Sarasota, 1972.
9. Zalkow, L.H., Gordon, M.M. L a n i r , N. J. Econ. Entomol. 1979,
72, 812-815.

10. Shorey, H.H., Hale, R.L. J. Econ. Entomol. 1965, 58, 522-524.
11. Gunderson, C.A. M.S. T h e s i s , 1982, Univ. o f Tennessee,
Knoxville.
12. B r a t t s t e n , L.B., Price S.L., Gunderson, C.A. Comp. Biochem.
P h y s i o l . 1980, 66C, 231-237.
13. Gunderson, C.A., Samuelian, J.H., Evans, C.K., B r a t t s t e n ,
L.B., Submitted t o Insect P h y s i o l .
14. Carlisle, D.B., Ellis, P.E., B e t t s , E. J. Insect P h y s i o l .
1965, 11, 1541-1558.
15. Bowers, W.S., F a l e s , H.M., Thompson, M.S., Uebel, E.C.
Science, 1966, 154, 1020.
16. Bowers, W.S. Science, 1969, 164, 323-325.
17. Bowers, W.S. Science, 1968, 161, 895-897.
18. Masner, P., Slama, K., Landa, V. Nature, 1968, 219, 395-396.
19. Masner, P. Gen. Comp E n d o c r i n o l 1967 9,472
20. Slama, K. A. Rev
21. T o a f f , M.E., Abramovici, , Sporn, J., , Reprod
F e r t . 1979, 55, 347-352.
22. Soderlund, D.M. F e l d l a u f e r , M.F., Takahashi, S.Y., Bowers,
W.S. in " J u v e n i l e Hormone Biochemistry" (G.E. P r a t t s and G.
T. Brooks, eds) E l s e v i e r , 1981, p. 353-362.
23. Hammock, B.C. Life Sciences, 1975, 17, 323-238.
24. Yu, S.J., Terriere, L.C.J.Insect P h y s i o l . 1974,20,1901-
1912.
25. Yu, S.J., T e r r i e r e , L.C. Pestic. Biochem. P h y s i o l . 1978 9,
237-246.
26. Slade, M., W i l k i n s o n , C.F. Comp. Biochem. P h y s i o l . 1974,
49B, 99-103.
27. Wing, K.D., Sparks, T.C., Lovell, V.M., Levinson, S.O.,
Hammock, B.D. Insect Biochem. 1981,11,473-485.
28. Pan, M.L., Wyatt, G.R. Science, 1971, 174, 503-505.
29. R e i d , P.C., Chen, T.T. Insect Biochem. 1981, 11, 297-305.
30. S c h e l i n e , R.R. "Mammalian Metabolism of P l a n t X e n o b i o t i c s , "
Academic P r e s s , New York, 1978.
31. Watabe, T., H i r a t s u k a , A., Isobe, M., Ogawa, N. Biochem.
Pharmacol. 1980, 29, 1068-1071.
32. Regan, J.W., B j e l d a n e s , L.F. J. Agric. Food Chem. 1976. 24,
377-380.
33. Kodama, R. Yano, T., Furakawa, K., Noda, K., Ide H.
X e n o b i o t i c a , 1976, 6, 377-389.
34. Hughes,P.R. Naturwiss. 1973, 60, 261-262.
35. Hughes, P.R. J. Insect P h y s i o l . 1974, 20, 1271-1275.
36. White, R.A. Franklin, R.T., Agosin, M. Pestic Biochem.
P h y s i o l . 1979, 10, 233-242.
37. Brand, J.M., Bracke, J.W., Markovetz, A . J . , Wood, D.L.
Browne, L.E. Nature, 1975, 254, 136-137.
38. B r a t t s t e n , L.B., Fleming, J.T., Trammell, D.H., Zalkow, L.H.
Submitted t o Nature.
39. B r a t t s t e n , L.B., W i l k i n s o n , C.F., E i s n e r , T. Science, 1977,
196, 1349-1352.
40. T e r r i e r e , L.C., Yu, S.J. Insect Biochem. 1976, 6, 109-114.

41. Yu, S.J., Berry, R.E., Terriere, L.C. Pestic. Biochem.

P h y s i o l . 1979, 12, 380-384.
42. B r a t t s t e n , L.B., Evans, C.K., B o n e t t i , S.J., Zalkow, L.H.
Submitted to Comp. Biochem. P h y s i o l .
43. C r o w e l l , H.H. Ann. Entomol. Soc. Amer. 1943, 36, 243-249.
44. Dahl, A.R., Hadley, W.M. Hahn, F.F., Benson, J.M., M c C l e l l a n ,
R.O. Science, 1981, 216, 57-59.
45. B r a t t s t e n , L.B. Drug Metab. Rev. 1979, 10, 35-58.
46. Duke, J.A. CRC Crit. Rev. T o x i c o l . 1977, 5, 189-237.
47. I v i e , G.W. W i t z e l , D.A. Herz, W., Kannan, R., Norman, J.O.,
Rushing, D.D., Johnson, J.H., Rowe, L.D., Veech, J.A. J .
A g r i c . Food Chem. 1975, 23, 841-845.
48. Städler, E. Ent. exp. a p p l . 1974, 17, 176-188.
49. Renwick, J.A., Hughes, P.R., Krull, I.S. Science, 1976, 191,
199-200.
50. Rudinsky, J.A. Science
51. Free, J.B. J. Agricult.
52. Wilde, J. de, Ghent Landb. Hogesch. Mededel. 1957, 22, 335-
347.
53. Kangas, E. Pertunnen, E. Oksanen, H. Rinne, M. Ann. Entomol.
Fenn. 1965, 31, 61-73.
54. Thomas, H., Hertrell, G. J. Econ. Entomol. 1969, 62, 383-386.
55. Howlett, F.M. Bull. Entomol. Res. 1915, 6, 297-305.
56. Berüter, J., Städler, E. Z e i t s c h r . N a t u r f o r s c h . 1971, 26b,
339-340.
57. Folsom, J.W. J. Econ. Entomol. 1931, 24, 827-833.
58. Sakan, T. I s s a c , S., Hyeon, S. in " C o n t r o l of Insect Be-
h a v i o r by N a t u r a l Products" (D.L. Wood, R.M. Silverstein,
N. Nakajima, eds) Academic P r e s s , New York, 1970, p. 237-247.
59. Benepal, P.S., Hall, C.V. Amer. Soc. Hort. Sci. 1967, 91,
353-359.
60. Kerns, H.G.H. Ann. Rep. A g r i c . Horticult. S t n . , Long Ashton,
1931, p. 199.
61. Hammamura, Y. in " C o n t r o l of Insect Behavior by N a t u r a l
Products," (D.L. Wood, R.M. Silverstein, M. Nakajima, eds)
Academic P r e s s , New York, 1970, p. 55-80.
62. Smith, R.H. For. Sci. 1966, 12, 63-68.
63. Doskotch, R.W., Cheng, H.Y., O d e l l , T.M. G i r a r d , L. J. Chem.
E c o l . 1980, 6, 845-851
64. E i s n e r , T. Science, 1969, 146, 1318-1320.
65. Zanno, P.R., M i u r a , I., N a k a n i s h i , K., E l d e r , D.L. J. Amer.
Chem. Soc. 1975, 97, 1975.
66. B o t t g e r , G.T., Patana, R. J. Econ. Entomol. 1966, 59, 1166-
1168.
67. Kubo, L., Lee, Y.W., Pettei, M., Pilkiewicz, F., N a k a n i s h i ,
K. J.C.S. Chem. Comm. 1976, 1013-1014.
68. Meinwald, J., P r e s t w i c k , B.D., N a k a n i s h i , K., Kubo, L.
Science, 1978, 199, 1167-1173.
69. Langenheim, J.H., F o s t e r , C.E., McGinley, R.B. Biochem. Syst.
E c o l . 1980, 8, 385-396.

70. B u r n e t t , W.C., Jr., Jones, S.B.,Jr.,Mabry, T. J. Amer.

M i d l . Nat. 1978, 100, 242-296.
71. B r a t t s t e n , L.B., W i l k i n s o n , C.F. Pestic, Biochem. P h y s i o l .
1973, 3, 393-407.

11
Nonpreference Mechanisms:
Plant Characteristics Influencing Insect Behavior
J. A . A. R E N W I C K
Boyce Thompson Institute at Cornell University, Ithaca, NY 14853
The selection or avoidance of potential host

plants by phytophagou
complex combinatio
s t i m u l i . Color, shape and olfactory cues may
play a role in the initial orientation, whereas
acceptance or rejection of a plant depends on
texture as well as chemical stimulants or deter-
rents. I n i t i a t i o n of feeding is stimulated or
deterred by the presence or absence of specific
chemicals or groups of chemicals, many of which
have been i d e n t i f i e d . The selection of a suitable
plant for oviposition i s also c r u c i a l for sur-
v i v a l of the progeny of most herbivorous insects,
but the chemical factors involved are known i n
r e l a t i v e l y few cases. Oviposition stimulants and
deterrents often appear to be quite different from
the chemicals that elicit or i n h i b i t feeding
responses of larvae.
The importance of developing crop p l a n t s that a r e

r e s i s t a n t t o major i n s e c t pests has created a need f o r
d e t a i l e d examination o f the mechanisms i n v o l v e d i n r e s i s t a n c e .
The widely recognized c l a s s i f i c a t i o n proposed by P a i n t e r (1)
appears t o provide an acceptable break-down of the p o s s i b l e
bases of r e s i s t a n c e f o r most purposes. However, some
m o d i f i c a t i o n of the terminology may be d e s i r a b l e before
beginning t o analyze the i n d i v i d u a l mechanisms i n v o l v e d . The
term "nonpreference" r e f e r s t o a b e h a v i o r a l response of the
i n s e c t t o a p l a n t , whereas " a n t i b i o s i s " and " t o l e r a n c e " r e f e r
to p l a n t c h a r a c t e r i s t i c s . This anomaly has been addressed by
Kogan and Ortman ( 2 ) , who suggested the term " a n t i x e n o s i s " t o
describe the p l a n t p r o p e r t i e s r e s p o n s i b l e f o r nonpreference.
0097-6156/83/0208-0199$06.00/0

ANTIXENOSIS NONPREFERENCE
Characteristic B e h a v i o r a l response
of p l a n t of i n s e c t
(undesirable host) (avoidance)
A problem may a l s o a r i s e i n the separation of the

mechanisms s i n c e some overlap can occur between a n t i x e n o s i s
and a n t i b i o s i s . These forms of r e s i s t a n c e may be roughly
compared i n the f o l l o w i n g manner:
ANTIXENOSIS ANTIBIOSIS
Undesirability Unsuitability
Avoidance by i n s e c t Adverse e f f e c t s
or Prevention of i n s e c t
a c t i v i t y (e.g. feeding)
I f a p l a n t deters feeding by an i n s e c t , the mechanism of

r e s i s t a n c e may be c l a s s i f i e d as a n t i x e n o s i s or a n t i b i o s i s .
The c r i t i c a l question i s whether the i n s e c t i s completely
prevented from f e e d i n g , thus s t a r v i n g to death ( a n t i b i o s i s ) ,
or would e v e n t u a l l y feed on that p l a n t when given no choice
( a n t i x e n o s i s ) . Since the answers to such questions are not
always known, t h i s d i s c u s s i o n w i l l d e a l w i t h the f a c t o r s
a f f e c t i n g an i n s e c t ' s behavior i n the s e l e c t i o n of a host
p l a n t , w i t h emphasis on behavior.
The choice of host p l a n t s i s a f f e c t e d by a vast array
of p o s i t i v e and negative f a c t o r s . These opposing forces
g e n e r a l l y f a l l i n t o one of the c a t e g o r i e s l i s t e d below:
Positive factors Negative f a c t o r s

Physical stimuli Physical barriers
attractants repellents
feeding s t i m u l a n t s feeding d e t e r r e n t s
o v i p o s i t i o n stimulants o v i p o s i t i o n deterrents
Whether a p o t e n t i a l host p l a n t i s s e l e c t e d by an i n s e c t o f t e n
depends on a d e l i c a t e balance which may be tipped i n e i t h e r
d i r e c t i o n by the presence or absence of one of these f a c t o r s .
The subject of host s e l e c t i o n has been reviewed i n depth
by s e v e r a l authors (3, 4^, 5) , and the chemical f a c t o r s
i n v o l v e d have been h i g h l i g h t e d (6, 7). Reviews by Kogan (8)
and Hedin et a l . (9) have provided comprehensive l i s t s of
chemicals i n v o l v e d i n the i n t e r a c t i o n s between p l a n t s and
i n s e c t s . Despite the vast number of p l a n t - i n s e c t systems that

11. RENWICK Nonpreference Mechanisms 201
have been s t u d i e d , i t i s s u r p r i s i n g how few of the s p e c i f i c

chemicals r e s p o n s i b l e f o r e l i c i t i n g p a r t i c u l a r b e h a v i o r a l
responses have a c t u a l l y been i d e n t i f i e d . No attempt w i l l be
made here t o f u r t h e r review the s u b j e c t . Instead, a few
examples of the v a r i o u s types of i n t e r a c t i o n s that could
c o n t r i b u t e t o p l a n t r e s i s t a n c e w i l l be presented. S p e c i a l
emphasis w i l l be given t o aspects that may have been
overlooked i n the p a s t , and one p l a n t - i n s e c t system w i l l be
examined i n d e t a i l t o demonstrate the i n t e r p l a y between the
v a r i o u s s t i m u l i a f f e c t i n g i n s e c t behavior.
Physical Factors
Although we are p r i m a r i l y concerned here w i t h p l a n t

chemicals t h a t i n f l u e n c e i n s e c t behavior i t i s important t o
recognize the c r i t i c a
host s e l e c t i o n process
v i s u a l and t a c t i l e s t i m u l i . Among the v i s u a l cues a f f e c t i n g
o r i e n t a t i o n towards a host p l a n t , c o l o r and shape appear t o be
most important. A good example i s provided by the apple
maggot f l y , R h a g o l e t i s pomonella, which i s a t t r a c t e d t o the
y e l l o w hue of f o l i a g e f o r feeding and r e s t i n g and t o the form
of the f r u i t f o r mating and o v i p o s i t i o n (10). The spruce
budworm l a y s i t s eggs on the needles of v a r i o u s c o n i f e r s , and
the maximum number of eggs i s found on twigs having a high
d e n s i t y of needles (11). The primary stimulus f o r o v i p o s i t i o n
appears t o be shape. Female moths r e a d i l y l a y eggs on paper
models of c o n i f e r twigs (12). I n choice b i o a s s a y s , we have
found no d i f f e r e n c e i n the number of eggs l a i d on white
spruce, the p r e f e r r e d host, and E n g l i s h yew, a t r e e on which
the l a r v a e cannot s u r v i v e (13).
Morphological c h a r a c t e r i s t i c s of p o t e n t i a l host p l a n t s
may present b a r r i e r s t o i n s e c t feeding and o v i p o s i t i o n .
Glandular h a i r s on p l a n t leaves s e v e r e l y hamper the a c t i v i t i e s
of l e a f hoppers and aphids (14, 15). The potato leafhopper,
f o r example, i s r e s t r i c t e d from feeding by glandular trichomes
of Solanum b e r t h a u l t i i and S^. polyadenium, and m o b i l i t y of the
i n s e c t i s impaired by a s t i c k y exudate (16). Some i n s e c t s
a l s o e x h i b i t a preference f o r e i t h e r smooth o r rough s u r f a c e s .
The cowpea w e e v i l , Callosobruchus maculatus, p r e f e r s smooth-
coated and w e l l - f i l l e d seeds t o rough and w r i n k l e d v a r i e t i e s
f o r o v i p o s i t i o n (17). However, the tobacco budworm moth,
H e l i o t h i s v i r e s c e n s p r e f e r s pubescent over smooth l e a f cotton
p l a n t s f o r o v i p o s i t i o n (18), and even i n a no choice
s i t u a t i o n , more eggs a r e l a i d on the smooth l e a f c u l t i v a r s
(19).

Chemical F a c t o r s
O r i e n t a t i o n . Chemical cues are i n v o l v e d i n a l l three

phases of host s e l e c t i o n behavior, i . e . o r i e n t a t i o n ,
o v i p o s i t i o n and f e e d i n g . Long range o r i e n t a t i o n of many
i n s e c t s to t h e i r host p l a n t s i s known to be guided by chemical
a t t r a c t a n t s , and reviews of the subject have provided l i s t s of
i n s e c t s and the sources of chemicals i n v o l v e d ( 9 ) . But the
s p e c i f i c chemicals r e s p o n s i b l e f o r a t t r a c t i o n are known i n
very few cases. However, some of the best s t u d i e d systems
appear to be found i n the D i p t e r a . E a r l y work on f r u i t f l i e s
revealed the involvement of e s s e n t i a l o i l s i n the host f i n d i n g
process (20), and methyl eugenol was i d e n t i f i e d as an
a t t r a c t a n t f o r the o r i e n t a l f r u i t f l y (21). R e c e n t l y , the
combined e f f e c t of o p t i c a l , chemical and t a c t i l e s t i m u l i has
been demonstrated f o
r e s u l t s have been obtaine
a n t i q u a . The a t t r a c t i v e p r o p e r t i e s of s p e c i f i c s u l f u r
compounds present i n onions has long been recognized (23).
n-Propyl mercaptan and d i p r o p y l d i s u l f i d e are both a t t r a c t a n t s
and o v i p o s i t i o n s t i m u l a n t s f o r the onion f l y (24). However,
recent s t u d i e s have p o i n t e d out the need f o r a d d i t i o n a l
compounds f o r maximum response (25). In a d d i t i o n , synergism
of v i s u a l and chemical cues occurs i n the s e l e c t i o n of
o v i p o s i t i o n s i t e s . A v e r t i c a l image and y e l l o w c o l o r
resembling an onion stem g r e a t l y enhance the e f f e c t of
v o l a t i l e s i n e l i c i t i n g o v i p o s i t i o n (26).
The long range o r i e n t a t i o n of most other i n s e c t s i n
response to chemical a t t r a c t a n t s i s not n e a r l y as c l e a r . The
o l f a c t o r y o r i e n t a t i o n of the Colorado potato b e e t l e has been
s t u d i e d i n c o n s i d e r a b l e d e t a i l by V i s s e r and coworkers.
B e e t l e s are a t t r a c t e d by v o l a t i l e s of s e v e r a l solanaceous
p l a n t s (27). The c o l l e c t i o n and c h a r a c t e r i z a t i o n of compounds
emanating from potato p l a n t s r e s u l t e d i n the i d e n t i f i c a t i o n of
general green l e a f v o l a t i l e s such as hexanol, hexenols and
hexenal, which e l i c i t a p o s i t i v e response (28). But the
s p e c i f i c compounds t h a t enable the i n s e c t s to recognize t h e i r
solanaceous hosts s t i l l remain a mystery.
Feeding. The feeding behavior of phytophagous i n s e c t s

has been s t u d i e d much more w i d e l y than other aspects of the
i n s e c t / p l a n t r e l a t i o n s h i p . The reason f o r t h i s probably l i e s
i n the r e l a t i v e ease w i t h which bioassays can be performed and
the r e s u l t s i n t e r p r e t e d . Many i n s e c t s can be reared on
a r t i f i c i a l d i e t s , and the e f f e c t s of added p l a n t c o n s t i t u e n t s
can r e a d i l y be determined. Some e a r l y s t u d i e s by D e t h i e r (29)
demonstrated a c o r r e l a t i o n between l a r v a l food choice and the
presence of s p e c i f i c chemicals i n the u m b e l l i f e r o u s host
p l a n t s of P a p i l i o polyxenes. However, many of the compounds
t y p i c a l l y found i n the U m b e l l i f e r a e are a l s o present i n other

p l a n t f a m i l i e s , and the P a p i l i o l a r v a e d i d i n f a c t feed on

some of these s p e c i e s . Since t h a t time, compounds o r groups
of compounds that are t y p i c a l of other p l a n t f a m i l i e s have
been i d e n t i f i e d as feeding s t i m u l a n t s f o r i n s e c t s s p e c i a l i z i n g
i n these f a m i l i e s ( 9 ) . Secondary p l a n t substances are
g e n e r a l l y i n v o l v e d i n such cases of s p e c i a l i z a t i o n , even
though the primary f u n c t i o n of these compounds i n the
e v o l u t i o n of the p l a n t i s b e l i e v e d to be i n defense against
herbivores (30). Many secondary p l a n t substances are i n f a c t
powerful feeding d e t e r r e n t s o r a n t i b i o t i c agents, thus
p r o v i d i n g p r o t e c t i o n from g e n e r a l i s t i n s e c t s . The idea of
u t i l i z i n g feeding d e t e r r e n t s as a means of p r o t e c t i n g crops
from i n s e c t damage has r e c e i v e d a l o t of a t t e n t i o n i n the l a s t
few years (31), and l a r g e screening programs have uncovered
s e v e r a l promising compounds (32) However a balance o f t e n
e x i s t s between the r e p u l s i o
a t t r a c t i o n of s p e c i a l i s t s
substances of the c u c u r b i t s o f f e r p r o t e c t i o n from mites and
other herbivores (33) , but these c u c u r b i t a c i n s act as
kairomones f o r a group o f d i a b r o t i c i t e b e e t l e s (34). A l s o ,
s i n i g r i n , a g l y c o s i d e found i n most c r u c i f e r o u s p l a n t s , i s
t o x i c to P a p i l i o polyxenes l a r v a e , which do not normally
a t t a c k c r u c i f e r s . The southern armyworm, a g e n e r a l i s t , i s
i n h i b i t e d by high concentrations of s i n i g r i n , but feeding by
the imported cabbageworm i s a c t u a l l y s t i m u l a t e d by t h i s
compound (35).
The i n t r o d u c t i o n of feeding d e t e r r e n t s i n t o crop p l a n t s
through breeding programs would appear to be an i d e a l s o l u t i o n
to many pest problems. Some progress has been made i n t h i s
d i r e c t i o n through the a n a l y s i s of r e s i s t a n t and s u s c e p t i b l e
v a r i e t i e s . Feeding d e t e r r e n t s have been i s o l a t e d from sorghum
l i n e s r e s i s t a n t t o the greenbug, Schizaphis graminum, and
i d e n t i f i e d as p-hydroxybenzaldehyde, d h u r r i n and p r o c y a n i d i n
(36). Resistance t o the European corn b o r e r , O s t r i n i a
n u b i l a l i s i n maize has been a t t r i b u t e d t o 2,4-dihydroxy-
7-methoxy-benzoxazin-3-one (DIMBOA) (37). This compound
i n h i b i t s normal development and increases m o r t a l i t y of the
l a r v a e . However, recent s t u d i e s have shown that other p l a n t
f a c t o r s are i n v o l v e d i n r e s i s t a n c e , and feeding deterrency may
play an important r o l e (38). Breeding programs f o r i n c o r p o r -
a t i o n of t h i s r e s i s t a n c e i n t o commercially d e s i r a b l e v a r i e t i e s
appear t o o f f e r p a r t i c u l a r promise.
O v i p o s i t i o n . Although most of the research on host

s e l e c t i o n has focused on feeding s t i m u l i , most s c i e n t i s t s
i n v o l v e d i n t h i s work acknowledge the f a c t that the choice of
food i s l a r g e l y predetermined by the g r a v i d female a t the time
of egg l a y i n g . The t i n y h a t c h l i n g l a r v a e are u s u a l l y
incapable of moving any d i s t a n c e t o sample p o t e n t i a l food
p l a n t s . Thus t h e i r s u r v i v a l depends on the j u d i c i o u s

s e l e c t i o n of an o v i p o s i t i o n s i t e by the a d u l t female. The

p a u c i t y of research i n t h i s area i s probably due to the
problems i n v o l v e d i n conducting b e h a v i o r a l experiments that
provide the i n s e c t s w i t h n a t u r a l c o n d i t i o n s f o r f l i g h t ,
l a n d i n g and sensory r e c e p t i o n of the v a r i o u s s t i m u l i .
I t would seem l o g i c a l to assume that s i m i l a r chemical
s t i m u l i are i n v o l v e d i n l a r v a l feeding and a d u l t o v i p o s i t i o n .
This has i n f a c t been demonstrated f o r P i e r i s b r a s s i c a e , which
l a i d i t s eggs on green paper t r e a t e d w i t h s i n i g r i n , a feeding
stimulant f o r the l a r v a e (39). However, few other cases e x i s t
where such a r e l a t i o n s h i p can be d e f i n i t e l y confirmed. A
d i s t i n c t d i f f e r e n c e i n the f a c t o r s a f f e c t i n g o v i p o s i t i o n by
s p e c i a l i s t and g e n e r a l i s t i n s e c t s has been noted. The
polyphagous species may be s t i m u l a t e d to l a y eggs by non-
s p e c i f i c cues such as moisture, sugars and amino a c i d s . The
s p e c i a l i s t s (oligophagou
u s u a l l y respond to s p e c i f i
However, the g e n e r a l i s t s may d i f f e r markedly i n t h e i r
preference f o r p l a n t s c o n t a i n i n g p a r t i c u l a r a l l e l o c h e m i c a l s .
In a comparison of cabbage looper and armyworm o v i p o s i t i o n on
three species of Vernonia (Compositae) the cabbage looper
showed a d i s t i n c t preference f o r V. gigantea and V. glauca,
which c o n t a i n the sesquiterpene l a c t o n e , g l a u c o l i d e A. The
armyworms l a i d more eggs on V. f l a c c i d i f o l i a , which l a c k s t h i s
b i t t e r compound (40).
The r o l e of i n h i b i t o r y s t i m u l i i n the choice of o v i -
p o s i t i o n s i t e by phytophagous i n s e c t s has been emphasized by
Jenny and S z e n t e s i (41). The acceptance or r e j e c t i o n of a
p l a n t u s u a l l y depends on contact w i t h the p l a n t surface or
through probing a f t e r l a n d i n g . S p e c i a l i s t s w i l l o v i p o s i t i f
the r i g h t stimulant i s present, whereas acceptance by
g e n e r a l i s t s i s governed to a l a r g e extent by the absence of
d e t e r r e n t s . S p e c i a l i s t s may a l s o be deterred by non-host
components which can i n t e r f e r e w i t h the response to p o s i t i v e
s i g n a l s . Three species of cabbage b u t t e r f l y were deterred
from o v i p o s i t i n g on cabbage that was t r e a t e d w i t h e x t r a c t s of
tomato and other non-host p l a n t s (42). On the other hand,
P i e r i s b r a s s i c a e has been s t i m u l a t e d to o v i p o s i t on bean
p l a n t s (a non-host) by c u l t u r i n g these p l a n t s i n a s o l u t i o n of
g l u c o s i n o l a t e s (43). So i t appears that the balance between
i n h i b i t o r s and s t i m u l a n t s i s c r i t i c a l .
The o v i p o s i t i o n behavior of P a p i l i o b u t t e r f l i e s has been
s t u d i e d i n some d e t a i l . Gravid females of the c i t r u s
b u t t e r f l y , P a p i l i o demoleus, are a t t r a c t e d to host and
non-host p l a n t s almost e q u a l l y by c o l o r . But the s p e c i f i c
a t t r a c t a n t emitted from c i t r u s p l a n t s increases the chances of
l a n d i n g on these p l a n t s . Then contact chemical s t i m u l i e l i c i t
the o v i p o s i t i o n a l response of the b u t t e r f l i e s (44).
Examination of another s p e c i e s , P a p i l i o protenor demetrius, i n
Japan has i n d i c a t e d that o v i p o s i t i o n occurs i n response to

contact s t i m u l i r e l e a s e d by the drumming a c t i o n of the

f o r e l e g s on the l e a f surface. V o l a t i l e components of the
p l a n t do not appear t o be i n v o l v e d i n t h i s case (45).
V a r i a t i o n i n the behavior of i n d i v i d u a l females of P a p i l i o
machaon has been noted. D i f f e r e n t thresholds f o r acceptance
of a l t e r n a t i v e p l a n t s appear t o occur, so that females
e x h i b i t i n g a g e n e r a l i s t s t r a t e g y may a c t u a l l y l a y eggs on
p l a n t s which are u n s u i t a b l e as food f o r the l a r v a e (46).
B u t t e r f l i e s i n general seem t o r e l y on a combination o f
v i s u a l and chemotactile s t i m u l i f o r o v i p o s i t i o n . F i e l d
observations coupled w i t h l a b o r a t o r y experiments on C o l i a s
b u t t e r f l i e s have shown that chemical preferences f o r v a r i o u s
legume food p l a n t s are under genetic c o n t r o l . But i n some
cases, chemical cues alone a r e not s u f f i c i e n t f o r females t o
d i s c r i m i n a t e between s p e c i e s Lupinus a legume which i s not
u s u a l l y u t i l i z e d by C o l i a s
laboratory, indicatin
p h y s i c a l o r environmental f a c t o r s must p l a y a r o l e i n nature.
Wiklund (48) has a l s o concluded that a d u l t and l a r v a l
preferences of P a p i l i o machaon a r e determined by separate gene
complexes. Thus the p o s s i b i l i t y of o v i p o s i t i o n on u n s u i t a b l e
p l a n t s always e x i s t s .
The involvement of v o l a t i l e s i n the s e l e c t i o n of o v i -
p o s i t i o n s i t e s appears t o be p a r t i c u l a r l y important i n the
D i p t e r a . O r i e n t a t i o n and o v i p o s i t i o n are c l o s e l y t i e d t o
a t t r a c t a n t chemicals f o r the f r u i t f l i e s and the onion f l y ,
which were discussed e a r l i e r . Recent work on the c a r r o t f l y ,
P s i l a rosae, has r e s u l t e d i n the i d e n t i f i c a t i o n of both
v o l a t i l e and n o n - v o l a t i l e components of the r e c o g n i t i o n s i g n a l
(49, 50). The propenylbenzenes trans-methylisoeugenol and
trans-asarone, along w i t h hexanal are s u f f i c i e n t t o e l i c i t
o v i p o s i t i o n . I n a d d i t i o n , a polyacetylene, f a l c a r i n d i o l , a t
the l e a f surface i s h i g h l y s t i m u l a t o r y .
The importance of understanding the f a c t o r s a f f e c t i n g
o v i p o s i t i o n by phytophagous i n s e c t s cannot be overemphasized.
This step i n the l i f e c y c l e i s a key t o s u r v i v a l of most
i n s e c t p o p u l a t i o n s . Despite the problems a s s o c i a t e d w i t h
b e h a v i o r a l s t u d i e s on a d u l t i n s e c t s , considerable progress has
been made, and the p o t e n t i a l f o r breeding p l a n t s that
discourage o v i p o s i t i o n i s g a i n i n g widespread r e c o g n i t i o n .
Environmental Factors
Host p l a n t preferences of i n s e c t pests are o f t e n

i n f l u e n c e d by environmental c o n d i t i o n s . The e f f e c t s of p l a n t
s t r e s s on s u s c e p t i b i l i t y t o i n s e c t a t t a c k have been observed
i n many crop p l a n t s . S t r e s s f a c t o r s a f f e c t i n g the
p h y s i o l o g i c a l s t a t e of the p l a n t i n c l u d e drought, disease,
chemical p o l l u t a n t s , and h i g h s a l t concentrations. Profound

d i f f e r e n c e s i n p h y s i o l o g i c a l c o n d i t i o n s of the p l a n t a l s o
occur w i t h i n c r e a s i n g age of the t i s s u e .
The r e s i s t a n c e of sorghum to grasshoppers has been
r e l a t e d to the content of p h e n o l i c s i n the p l a n t (51), and
those c u l t i v a r s w i t h a high p h e n o l i c content s u f f e r l e s s
damage from leaf-chewing i n s e c t s i n general (52). The l e v e l s
of p h e n o l i c s i n healthy sorghum p l a n t s decrease as the p l a n t
matures, and environmental f a c t o r s such as l i g h t i n t e n s i t y
i n f l u e n c e the c o n c e n t r a t i o n of p h e n o l i c s (53). Thus wide
v a r i a t i o n s i n the feeding deterrent a c t i v i t y may be found
w i t h i n a p a r t i c u l a r c u l t i v a r . S i m i l a r l y , host preferences of
H e l i o t h i s zea have been found to change as the v a r i o u s host
p l a n t s matured or entered a more a t t r a c t i v e stage of
development (54).
The s t r e s s produced by f u n g a l pathogens on p l a n t s may
have a d i s t i n c t e f f e c
P l a n t p a t h o l o g i s t s hav
white f l i e s on p l a n t s i n f e c t e d w i t h V e r t i c i l l i u m before the
disease symptoms are apparent (H. M u s s e l l , personal
communication). The host s e l e c t i o n behavior of grasshoppers
feeding on w i l d sunflower i s a l s o a f f e c t e d by the presence of
a pathogen. Leaf t i s s u e i n f e c t e d w i t h r u s t fungus, damaged by
l e p i d o p t e r a n l a r v a e or w i l t e d by g i r d l i n g b e e t l e s was pre-
f e r r e d over h e a l t h y , green leaves (55).
The e f f e c t of p l a n t age on the host s e l e c t i o n process has
been observed by comparison of two aphid species on b r a s s i c a
p l a n t s (56). A s p e c i a l i s t , Brevicoryne b r a s s i c a e p r e f e r s
young l e a v e s , which are higher i n g l u c o s i n o l a t e s , whereas the
g e n e r a l i s t , Myzus p e r s i c a e p r e f e r s o l d e r l e a v e s , where amino
a c i d s are more important i n the s e l e c t i o n process.
Outbreaks of i n s e c t p e s t s , p a r t i c u l a r l y i n f o r e s t eco-
systems, have o f t e n been l i n k e d to a i r p o l l u t a n t s (57).
Recent s t u d i e s have shown t h a t exposure of p l a n t s to s u l f u r
d i o x i d e can i n f a c t a f f e c t t h e i r s u s c e p t i b i l i t y to i n s e c t
a t t a c k . Bean p l a n t s exposed to low l e v e l s of SO^ were
p r e f e r r e d f o r feeding by the Mexican bean b e e t l e (58).
S i m i l a r preferences were found w i t h soybeans (59) both i n the
l a b o r a t o r y and i n the f i e l d (60). Growth, r a t e of development
and f e c u n d i t y of the b e e t l e s were a l s o increased on the
treated plants.
The s u s c e p t i b i l i t y of a p l a n t to i n s e c t damage may a l s o
be a f f e c t e d by a s s o c i a t e d v e g e t a t i o n . The p r a c t i c e of mixed
cropping i s b e l i e v e d to minimize crop damage i n many of the
developing c o u n t r i e s , where i n s e c t i c i d e a p p l i c a t i o n i s
i m p r a c t i c a l . E f f e c t s on the p o p u l a t i o n dynamics of i n s e c t
pests have been demonstrated i n mixed crops of Brassica/tomato
and Phaseolus/weed grass (61). A recent study by Saxena and
B a s i t (62) i n d i c a t e d that c o t t o n can be protected from
leafhoppers by p l a n t i n g non-hosts such as c a s t o r or sponge
gourd. O v i p o s i t i o n on c o t t o n was reduced i n both cases, but

the mechanisms appear to be d i f f e r e n t . V o l a t i l e s from the

c a s t o r p l a n t s reduced the number of leafhoppers landing on
host p l a n t s , whereas sponge gourd was a t t r a c t i v e t o the
i n s e c t s . O v i p o s i t i o n occurred on the gourd, but emerging
nymphs f a i l e d t o develop on them and d i e d .
Aggregation and D i s p e r s i o n
C e r t a i n i n s e c t s must aggregate on t h e i r host p l a n t s i n

order t o s u r v i v e , and others depend on even d i s t r i b u t i o n of
populations t o prevent overcrowding of l i m i t e d resources (63).
The mechanisms i n v o l v e d i n meeting these requirements o f t e n
depend on p h y s i c a l and chemical c h a r a c t e r i s t i c s of the p l a n t .
The aggregation of bark b e e t l e s on pine t r e e s i s mediated
by pheromones produced by the invading b e e t l e s Attack en
masse i s necessary t
entrance h o l e s . Man
g a t i o n are produced by o x i d a t i o n of terpene hydrocarbons
present i n the r e s i n (64, 65). Furthermore, the production of
a p a r t i c u l a r pheromone may depend on the o p t i c a l r o t a t i o n (or
absolute c o n f i g u r a t i o n ) of a precursor. I n the b e e t l e I p s
paraconfusus, (-)-a-pinene i s o x i d i z e d t o c i s - v e r b e n o l , a
component of the pheromone complex f o r t h i s species (66),
whereas (+)-a-pinene i s o x i d i z e d t o t r a n s - v e r b e n o l , which i s a
pheromone f o r another species (67). Thus t r e e s which l a c k the
precursors o r r i g h t c o n f i g u r a t i o n of precursor might escape
i n v a s i o n by these bark b e e t l e s .
D i s p e r s i o n of i n s e c t populations may a l s o depend on p l a n t
c o n s t i t u e n t s . Recent work i n our l a b o r a t o r y has shown that
when cabbage looper l a r v a e are feeding on a p l a n t , the a d u l t
females w i l l avoid t h i s p l a n t f o r o v i p o s i t i o n (68). The
deterrent i s present i n l a r v a l f r a s s , but a l s o i n d i s r u p t e d
p l a n t t i s s u e (69). Homogenized t i s s u e s from s e v e r a l host
p l a n t s of the cabbage looper were e f f e c t i v e i n d e t e r r i n g
o v i p o s i t i o n . Both v o l a t i l e and n o n - v o l a t i l e components of the
p l a n t appear to be i n v o l v e d (70). I n another study, female
moths of the European corn borer were deterred from
o v i p o s i t i n g by the v o l a t i l e emissions from i n j u r e d p l a n t s
(71). A l s o , the o l i v e f r u i t f l y i s deterred from f u r t h e r
o v i p o s i t i o n on o l i v e s which are already attacked. I n t h i s
case, j u i c e from the o v i p o s i t i o n wound i s a c t i v e i n s i g n a l l i n g
occupancy t o the g r a v i d females (72).
These examples serve t o i l l u s t r a t e the i n d i r e c t r o l e
that p l a n t chemicals may play i n the p o p u l a t i o n dynamics of
i n s e c t pests. Work i n t h i s area has been very l i m i t e d , but
the p o t e n t i a l u t i l i z a t i o n o f an i n s e c t ' s own spacing mechanism
may o f f e r a new approach t o pest management, and a good
understanding of the p l a n t / i n s e c t r e l a t i o n s h i p w i l l be
essential.

Insect Pests of C r u c i f e r s
The i n s e c t community feeding on c r u c i f e r o u s crops has

been widely s t u d i e d over a long period of time. The reason
f o r t h i s i n t e r e s t stems not only from the commercial
importance of these crops, but a l s o from the f a c t that much i s
known about the chemistry of the i n s e c t - p l a n t r e l a t i o n s h i p s .
Furthermore, a t l e a s t two of the major chemicals i n v o l v e d are
commercially a v a i l a b l e .
Many i n s e c t s have become s p e c i a l i s t s on c r u c i f e r s and a
few r e l a t e d p l a n t f a m i l i e s . These i n c l u d e f l e a b e e t l e s , l e a f
b e e t l e s , cabbage root f l y , aphids, cabbage b u t t e r f l i e s and the
diamondback moth. At the same time, s e v e r a l polyphagous
i n s e c t s such as the cabbage looper, armyworms and aphids are
major pests of c r u c i f e r s . Comparative s t u d i e s on these
s p e c i a l i s t s and g e n e r a l i s t
on host r e c o g n i t i o n an
The C r u c i f e r a e are c h a r a c t e r i z e d chemically by the
presence of g l u c o s i n o l a t e s , a group of g l y c o s i d e s which give
r i s e t o v o l a t i l e h y d r o l y s i s products known as the mustard
o i l s . The most common g l u c o s i n o l a t e i s s i n i g r i n , o r
a l l y l g l u c o s i n o l a t e , which r e l e a s e s a l l y l i s o t h i o c y a n a t e upon
h y d r o l y s i s . Both these compounds have been shown t o be
i n v o l v e d i n the host s e l e c t i o n behavior of s e v e r a l i n s e c t s
(73). S i n i g r i n s t i m u l a t e s feeding by the imported
cabbageworm, P i e r i s rapae, the l a r g e white cabbage b u t t e r f l y ,
b r a s s i c a e , the diamondback moth, P l u t e l l a maculipennis, the
mustard b e e t l e , Phaedon c o c h l e a r i a e , and f l e a b e e t l e s ,
P h y l l o t r e t a spp.
When the e f f e c t of s i n i g r i n on s p e c i a l i s t s and
g e n e r a l i s t s i s compared, v a r y i n g responses are obtained (35).
Feeding by P i e r i s rapae (a s p e c i a l i s t ) i s s t i m u l a t e d . Larvae
of Spodoptera e r i d a n i a (a g e n e r a l i s t ) are unaffected by low
c o n c e n t r a t i o n s , but feeding i s i n h i b i t e d by high s i n i g r i n
concentrations. P a p i l i o polyxenes does not feed on c r u c i f e r s ,
and a l l concentrations of s i n i g r i n are t o x i c t o l a r v a e of t h i s
species.
An i n t e r e s t i n g study on the host p l a n t s e l e c t i o n of the
horseradish f l e a b e e t l e , P h y l l o t r e t a armoraciae, has shown
that a f l a v o n o l g l y c o s i d e s t i m u l a t e s feeding by t h i s
monophagous species (74). The combination of s i n i g r i n and
t h i s compound, kaempferol 3 - 0 - x y l o s y l g a l a c t o s i d e , was more
s t i m u l a t o r y than e i t h e r chemical alone. This appears t o be
the f i r s t report of a c r u c i f e r feeding i n s e c t being stimulated
to feed by an a l l e l o c h e m i c which i s not a g l u c o s i n o l a t e .
Feeding d e t e r r e n t s i n other p l a n t s may a l s o e x p l a i n i n part
the s p e c i f i c i t y of the horseradish f l e a b e e t l e . A comparison
of monophagous and oligophagous f l e a b e e t l e s demonstrated
s e l e c t i v e feeding deterrence w i t h c u c u r b i t a c i n s , c a r d i a c
g l y c o s i d e s and cardenolides (75). Cardenolides and

c u c u r b i t a c i n s have been suggested as a second generation of

p r o t e c t i v e compounds i n C r u c i f e r a e .
Several i n s e c t s appear t o be a t t r a c t e d by mustard o i l o r
a l l y l i s o t h i o c y a n a t e . These i n c l u d e the cabbage f l y , Phorbia
f l o r a l i s , the vegetable w e e v i l , L i s t r o d e r e s o b l i q u u s , the f l e a
b e e t l e s , P h y l l o t r e t a c r u c i f e r a e and P_. s t r i o l a t a , the
diamondback moth, P l u t e l l a maculipennis, and l a r v a e of the
imported cabbageworm, P i e r i s rapae (73). However, o r i e n t a t i o n
towards host p l a n t s f o r o v i p o s i t i o n by the P i e r i s b u t t e r f l i e s
seems t o be guided p r i m a r i l y by v i s u a l cues (76)•
Despite the i m p l i c a t i o n s and s p e c u l a t i o n that glucos-
i n o l a t e s and/or mustard o i l i s i n v o l v e d i n host s e l e c t i o n by
P i e r i s rapae, the mechanism of o r i e n t a t i o n and host
r e c o g n i t i o n by t h i s i n s e c t i s not yet c l e a r . The b u t t e r f l i e s
are a t t r a c t e d t o green c o l o r s and o f t e n land on non-hosts as
w e l l as h o s t s . Recognitio
seems t o depend on contac
assay t o study the chemistry of the o v i p o s i t i o n s t i m u l a n t .
B u t t e r f l i e s w i l l r e a d i l y land on green index cards, and i f
these are painted w i t h a water e x t r a c t of cabbage, o v i p o s i t i o n
w i l l occur. Good d i s c r i m i n a t i o n between d i f f e r e n t
concentrations of e x t r a c t were obtained, but when s i n i g r i n
s o l u t i o n s were s u b s t i t u t e d f o r cabbage e x t r a c t s , the o v i p o s i -
t i o n a l response was marginal. S i n i g r i n alone i s t h e r e f o r e not
s u f f i c i e n t t o account f o r the s t i m u l a t o r y a c t i v i t y of cabbage.
P r e l i m i n a r y f r a c t i o n a t i o n has i n d i c a t e d that a t l e a s t four
compounds are i n v o l v e d , but the most a c t i v e m a t e r i a l i s
considerably l e s s p o l a r than s i n i g r i n .
The e f f e c t of v o l a t i l e s on o v i p o s i t i o n by _P. rapae was
t e s t e d by p l a c i n g whole cabbage leaves or a container of
macerated cabbage t i s s u e under the bioassay cards. There was
no evidence f o r any increase i n the number of landings on the
cards w i t h the v o l a t i l e s , and i t appears that o v i p o s i t i o n i s
a c t u a l l y deterred t o some extent by the presence of v o l a t i l e s .
The p o s s i b i l i t y s t i l l e x i s t s that very low concentrations
might be a t t r a c t i v e t o the b u t t e r f l i e s , but random landing
does occur i n the f i e l d , and so i t seems reasonable t o
conclude that the n o n - v o l a t i l e stimulant i s the most important
chemical cue f o r o v i p o s i t i o n .
Conclusions
The behavior of i n s e c t s i n s e l e c t i n g a host p l a n t f o r

food and s h e l t e r i s a f f e c t e d by a wide array of p h y s i c a l and
chemical s t i m u l i . Chemicals that play a r o l e i n r e s i s t a n c e
mechanisms may i n t e r f e r e w i t h an i n s e c t ' s o r i e n t a t i o n , i n h i b i t
feeding, or deter o v i p o s i t i o n . Most of the known mechanisms
of r e s i s t a n c e i n v o l v e feeding d e t e r r e n t s , but the most
v u l n e r a b l e phase of the i n s e c t l i f e c y c l e may prove t o be
o v i p o s i t i o n . Environmental f a c t o r s may i n f l u e n c e the a b i l i t y

of a p l a n t to combat i n s e c t a t t a c k , and chemical c o n s t i t u e n t s

of the p l a n t may have i n d i r e c t e f f e c t s on the success or
f a i l u r e of i t s a t t a c k e r s . The presence of s p e c i f i c chemicals
may o f f e r p r o t e c t i o n from one i n s e c t p e s t , but may i n c r e a s e
the r i s k of i n v a s i o n by other i n s e c t s . Many assumptions have
been made i n even the most thoroughly s t u d i e d p l a n t - i n s e c t
systems. But many more p h y s i c a l and chemical c h a r a c t e r i s t i c s
need to be i d e n t i f i e d t o e x p l a i n the complex b e h a v i o r a l events
i n host s e l e c t i o n by i n d i v i d u a l i n s e c t s p e c i e s .
Literature Cited
1. P a i n t e r , R. H. "Insect Resistance in Crop P l a n t s " ;

U n i v e r s i t y Press f Kansas Lawrence/London 1951 520
pp.
2. Kogan, M.; Ortman, ,
24, 175-6.
3. T h o r s t e i n s o n , A. J. Annu. Rev. Entomol. 1960, 5, 193-218.
4. H a r r i s , P. Proc. Symp. IX I n t . Congr. P l a n t P r o t e c t i o n
1979, 1, 105-9.
5. Kennedy, J. S. Ann. A p p l . Biol. 1965, 56, 317-22.
6. Hsiao, T. H. Ent. exp. & a p p l . 1969, 12, 777-88.
7. Hedin, P. A.; Maxwell, F. G.; J e n k i n s , J . N. I n : "Proc.
Summer I n s t , on Biol. C o n t r o l of P l a n t I n s e c t s and
Diseases" (F. G. Maxwell & F. A. H a r r i s , eds.);
U n i v e r s i t y Press: Mississippi, 1974; p 494-527.
8. Kogan, M. Proc. I n t . Congr. Entomol. 1976, 15, 211-27.
9. Hedin, P. A.; J e n k i n s , J. N.; Maxwell, F. G. I n : "Host
P l a n t Resistance to P e s t s " (P. A. Hedin, e d . ) ; Am. Chem.
Soc. Symp. Ser. 1977, 62, 231-75.
10. Prokopy, R. J., Owens, E. D. Ent. exp. & a p p l . 1978, 24,
409-20.
11. W i l s o n , L. F. J. Econ. Entomol. 1963, 56, 285-8.
12. Städler, E. Ent. exp. & a p p l . 1974, 17, 176-88.
13. Renwick, J. A. A.; Radke, C. D. Environ. Entomol. 1982,
11, 503-5.
14. Pearson, E. D. "The Insect Pests of Cotton in T r o p i c a l
Africa"; Eastern Press: London, 1958; 355 pp.
15. Johnson, B. P l a n t P a t h o l . 1956, 5, 131-2.
16. Tingey, W. M.; Gibson, R. W. J . Econ. Entomol. 1978, 71,
856-8.
17. Nwanze, K. F.; Horber, E.; Pitts, C. W. Environ. Entomol.
1975, 4, 409-12.
18. Lukefahr, M. J . ; Houghtading, J. E.; Graham, H. M.
J . Econ. Entomol. 1971, 64, 486-8.
19. Robinson, S. H.; Wolfenbarger, D. A.; D i l d a y , R. H. Crop
Sci. 1980, 20, 646-9.
20. Howlett, F. M. Trans. Entomol. Soc. London 1912, 412-8.
21. Howlett, F. M. Bull. Entomol. Res. 1915, 6, 297-305.

22. Levinson, H. Z. Z. angew. Entomol. 1977, 84, 1.

23. Peterson, A. J. Econ. Entomol. 1924, 17, 87-94.
24. Matsumoto, Y. I n : " C o n t r o l o f Insect Behavior by N a t u r a l
Products" (D. L. Wood, R. M. Silverstein, & M. Nakajima,
eds.); Academic Press: New York, 1970; p 133-60.
25. P i e r c e , H. D., Jr.; Vernon, R. S.; Borden, J. H.;
Oehlschlager, A. C. J. Chem. E c o l . 1978, 4, 65-72.
26. Miller, J. R.; Harris, M. O. Proc. 5 t h Int. Symp.
I n s e c t - P l a n t R e l a t i o n s h i p s ; Pudoc Press: Wageningen,
1982; in press.
27. V i s s e r , J. H.; N i e l s e n , J. K. Ent. exp. & a p p l . 1977, 21,
14-22.
28. V i s s e r , J. H.; van S t r a t e n , S.; Maarse, H. J. Chem. E c o l .
1979, 5, 11-23.
29. D e t h i e r , V. A. Am 1941 75, 61-73
30. F r a e n k e l , G. Scienc
31. Munakata, K. Pure Appl. Chem. 1975, 42, 57-66.
32. Shalk, J. M.; Ratcliffe, R. H. FAO P l a n t P r o t . Bull.
1977, 25, 9-14.
33. DaCosta, C. P.; Jones, C. M. Science 1971, 172, 1145-6.
34. M e t c a l f , R. L.; M e t c a l f , R. A.; Rhodes, A. M. Proc. N a t l .
Acad. Sci. USA 1980, 77, 3769-72.
35. B l a u , P. A.; Feeny, P.; Contardo, L.; Robson, D. S.
Science 1978, 200, 1296-8.
36. Dreyer, D. L.; Reese, J. C.; Jones, K. C. J. Chem. E c o l .
1981, 7, 273-84.
37. K l i m , J. A.; T i p t o n , C. L.; B r i n d l e y , T. A. J. Econ.
Entomol. 1967, 60, 1529-33.
38. S c r i b e r , J. M.; Tingey, W. M.; Gracen, V. E.; Sullivan,
S. L. J. Econ. Entomol. 1975, 68, 823-6.
39. David, W. A. L.; Gardiner, B. O. C. Bull. Entomol. Res.
1962, 52, 91-109.
40. B u r n e t t , W. C.; Jones, S. B. Jr.; Mabry, T. J. Am. Midl.
Nat. 1978, 100, 242-6.
41. Jermy, T.; S z e n t e s i , A. Ent. exp. & a p p l . 1978, 24,
258-71.
42. Lundgren, L. Z o o l . S c r . 1975, 4, 253-8.
43. Ma, W. C.; Schoonhoven, L. M. Ent. exp. & a p p l . 1973, 16,
343-57.
44. Saxena, K. N.; Goyal, S. Ent. exp. & a p p l . 1978, 24,
1-10.
45. Ichinosé, T.; Handa, H. Appl. Ent. Z o o l . 1978, 13,
103-14.
46. Wiklund, C. Oikos 1981, 36, 163-70.
47. Stanton, M. L. Oecologia ( B e r l . ) 1979, 39, 79-91.
48. Wiklund, C. Oecologia ( B e r l . ) 1975, 18, 185-97.
49. Guerin, P. M.; Städler, E.; Buser, H. R. Proc. 5 t h I n t .
Symp. I n s e c t - P l a n t R e l a t i o n s h i p s ; Pudoc Press:
Wageningen, 1982; in press.
50. Städler, E.; Buser, H. R. Proc. 5 t h Int. Symp.


1982; in press.
51. Woodhead, S.; Bernays, E. A. Ent. exp. & a p p l . 1978, 24,
123-44.
52. Woodhead, S.; Padgham, D. E.; Bernays, E. A. Ann. A p p l .
Biol. 1980, 95, 151-7.
53. Woodhead, S. J. Chem. E c o l . 1981, 7, 1035-47.
54. Stadelbucher, E. A. Environ. Entomol. 1980, 9, 542-5.
55. Lewis, A. C. Proc. 5th I n t . Symp. I n s e c t - P l a n t R e l a t i o n -
s h i p s ; Pudoc Press: Wageningen, 1982; in press.
56. van Emden, H. F. I n : "Phytochemical Ecology" ( J . B.
Harborne, ed.); Academic Press: New York, 1972; pp 25-43.
57. Wenzel, K. F.; Ohnesorge, B. F o r s t a r c h i v 1961, 32, 177.
58. Hughes, P. R.; P o t t e r , J. E.; Weinstein, L. H. Environ.
Entomol. 1981, 10, 741-4.
59. Hughes, P. R.; P o t t e r
Entomol. 1982, 11
60. Hughes, P. R.; Weinstein, L. H.; Laurence, J. A.; Sacher,
R. F.; D i c k i e , A.; Johnson, L. Proc. 5 t h I n t . Symp.
1982; in press.
61. P e r r i n , R. M.; Phillips, M. L. Ent. exp. & a p p l . 1978,
24, 385-93.
62. Saxena, K. N.; B a s i t , A. Proc. 5 t h I n t . Symp.
1982; in press.
63. Prokopy, R. J. I n : "Semiochemicals. T h e i r Role in Pest
C o n t r o l " (D. A. Nordlund, R. L. Jones, W. J . Lewis,
eds.); W i l e y - I n t e r s c i e n c e : New York, 1981; pp 181-213.
64. Hughes, P. R. Z. angew. Entomol. 1973, 73, 294-312.
65. Hughes, P. R. J . Insect P h y s i o l . 1974, 20, 1271-5.
66. Silverstein, R. M.; Rodin, J. O.; Wood, D. L. Science
1966, 154, 509-10.
67. Pitman, G. B.; Vité, J. P. Can. Entomol. 1969, 101,
143-9.
68. Renwick, J. A. A.; Radke, C. D. Environ. Entomol. 1980,
9, 318-20.
69. Renwick, J. A. A.; Radke, C. D. Ent. exp. & a p p l . 1981,
30, 201-204.
70. Renwick, J. A. A.; Radke, C. D. Proc. 5th I n t . Symp.
I n s e c t - P l a n t R e l a t i o n s h i p s ; Pudoc: Wageningen, 1982; in
press.
71. Schurr, K; Holdaway, F. G. Ent. exp. & a p p l . 1970, 13,
455-61.
72. C i r i o , U. Redia 1971, 52, 577-600.
73. Schoonhoven, L. M. Recent Adv. Phytochem. 1972, 5,
197-224.

74. N i e l s e n , J . K.; Larsen, L. M.; Sørensen, H.; Ent. exp. &

a p p l . 1979, 26, 40-8.
75. N i e l s e n , J. K. Ent. exp. & a p p l . 1978, 24, 41-54.
76. Hawkes, C.; P a t t o n , S.; Coaker, T. H. Ent. exp. & a p p l .
1978, 24, 219-27.
RECEIVED August 2 3 , 1 9 8 2

12
Differential Sensory Perceptions of Plant
Compounds by Insects
J. H. VISSER
Agricultural University, Department of Entomology, Binnenhaven 7, 6709 PD
Wageningen, The Netherlands
The insect's perception of plant characters i s

directed toward
on which feeding
reproduction. By means of i t s gustatory receptors,
an insect i s informed about the n u t r i t i o n a l quality
of a plant. Plant odours are the chemical messengers
for the insect's orientation. The olfactory
orientation of the Colorado beetle in response to
plant odours, and the s p e c i f i c i t y and s e n s i t i v i t y
of i t s olfactory receptors are outlined. The
d i f f e r e n t i a l perception of green odour , being
composed of C6 alcohols, aldehydes and the derivative
acetate, i s a common feature in phytophagous insects.
The s e n s i t i v i t y of olfactory receptors for the
individual components of this complex vary in and
between phytophagous insect species. Although leaf
odours are characterized by the particular composition
of their green odour, other v o l a t i l e compounds are
involved as w e l l .
Most phytophagous i n s e c t s e x h i b i t s p e c i a l i z e d f e e d i n g h a b i t s ;
they feed on a r e s t r i c t e d range of taxonomically r e l a t e d p l a n t
s p e c i e s , and are even s p e c i a l i z e d t o feed on p a r t i c u l a r p a r t s of
these p l a n t s l i k e l e a v e s , stems, f l o w e r s , f r u i t s o r r o o t s (J_).
The d i v e r s i t y i n i n s e c t - p l a n t i n t e r a c t i o n s i s overwhelming as
each i n s e c t species shows a s e r i e s of adaptations t o i t s host
p l a n t s . These adaptations i n v o l v e morphological f e a t u r e s l i k e the
i n s e c t ' s mouthparts, as w e l l as b e h a v i o u r a l and metabolic changes
i n order t o cope w i t h the p h y s i c a l and chemical c h a r a c t e r i s t i c s
of the p l a n t s t o which phytophagous i n s e c t s became adapted i n
e v o l u t i o n a r y time. I t i s beyond the scope of the present paper t o
l i s t a l l the adaptations of i n s e c t s t o p l a n t s , o r even a l l the
counter-adaptations of p l a n t s t o i n s e c t s . At the r i s k t o
g e n e r a l i z e t o an extent which o v e r - s i m p l i f i e s the d i v e r s i t y i n
0097-615 6 / 83/0208-0215 $ 0 6 . 0 0 / 0

i n s e c t - p l a n t i n t e r a c t i o n s , one might s t a t e that the chemical

d i v e r s i t y among p l a n t s i s the p r i n c i p a l f a c t o r u n d e r l y i n g host
s p e c i f i c i t y of phytophagous i n s e c t s (2,3).
The n u t r i t i o n a l requirements of i n s e c t species e x h i b i t i n g
d i f f e r e n t feeding h a b i t s l i k e scavengers, p a r a s i t e s , predators
and phytophagous i n s e c t s , are s i m i l a r i n a q u a l i t a t i v e sense ( 4 ) .
Each i n s e c t species needs, however, a p a r t i c u l a r q u a n t i t a t i v e
composition of n u t r i e n t s i n i t s d i e t t o complete development ( 5 ) .
The presence of t o x i c substances i n p l a n t s , secondary p l a n t
substances as they were formerly c a l l e d by phytochemists, forms
a b a r r i e r which phytophagous i n s e c t s have overcome by
s p e c i a l i z a t i o n . Thus, an i n s e c t can t o l e r a t e o r d e t o x i f y the
secondary p l a n t substances present i n i t s host p l a n t s , w h i l e the
m a j o r i t y of these substances being present i n other p l a n t s s t i l l
acts as t o x i n s ( 1 ) . I n t h i s way phytophagous i n s e c t s are adapted
to the metabolic q u a l i t i e
p a r t i c u l a r chemical compositio
substances.
At the same time i n s e c t s are able t o d i s c r i m i n a t e between
host and non-host p l a n t species as they s e l e c t p l a n t s on which
feeding u l t i m a t e l y r e s u l t s i n growth and r e p r o d u c t i o n , and on the
other hand avoid p o i s o n i n g o r m a l n u t r i t i o n on non-host p l a n t s . By
means of chemosensory s e n s i l l a , i n s e c t s are able t o perform the
d i f f i c u l t t a s k , being w e l l equipped a n a l y t i c a l chemists, of
i d e n t i f y i n g the chemical composition of p l a n t s that i n s e c t s meet
i n t h e i r environment ( 6 ) .
Gustation
Lepidopterous l a r v a e bear on t h e i r mouthparts two p a i r of

s t y l o c o n i c s e n s i l l a (see F i g u r e 1 ) . The p a p i l l a of each s e n s i l l u m
possesses one t e r m i n a l pore which gives entrance t o the d e n d r i t i c
regions of f o u r gustatory receptor c e l l s . B e s i d e s , a f i f t h c e l l
i n each s e n s i l l u m acts as a mechanoreceptor i n d e t e c t i n g
p o s i t i o n a l changes of the p a p i l l a (_7,80 .
The response s p e c t r a of the i n d i v i d u a l gustatory receptor
c e l l s can be recorded by making use of e l e c t r o p h y s i o l o g i c a l
techniques ( 7 , 8 ) . A c a p i l l a r y c o n t a i n i n g the t e s t compound
d i s s o l v e d i n a s a l i n e s o l u t i o n i s placed i n contact w i t h the
t e r m i n a l opening of the s e n s i l l u m and the responses of the
gustatory receptor c e l l s t o t h i s compound a r e recorded by the %
same e l e c t r o d e being connected t o an a m p l i f i e r , an o s c i l l o s c o p e

and r e c o r d e r . I n case the t e s t chemical evokes a response, a
t r a i n of a c t i o n p o t e n t i a l s i s recorded. The response s p e c t r a of
the i n d i v i d u a l gustatory receptor c e l l s i n the s e n s i l l a
s t y l o c o n i c a of P i e r i s b r a s s i c a e l a r v a e are shown i n Table I .

12. VISSER Differential Sensory Perceptions 217
Table I . Response s p e c t r a of gustatory receptor cells

i n P i e r i s brassicae larvae (7,8).
medial cell sugars

sensillum styloconicum 2
i
feeding i n h i b i t o r s
3 glucosinolates
4 salts
j
cell 5 sugars
lateral 6 glucosinolates
sensillum styloconicum 7 amino a c i d s
8 anthocyanins
epipharyngeal s e n s i l l u
11 salts
This set o f contact chemoreceptors enables the larvae t o

perceive n u t r i e n t s l i k e sugars, s a l t s and amino a c i d s , as w e l l as
secondary p l a n t substances. The feeding i n h i b i t o r s e n s i t i v e c e l l
i n the medial s e n s i l l u m styloconicum responds t o a v a r i e t y of
a l k a l o i d s and s t e r o i d s , these are compounds which possess strong
feeding i n h i b i t o r a c t i o n l i k e s t r y c h n i n e , conessine and
a z a d i r a c h t i n . On the other hand the gustatory s e n s i l l a r e a c t t o
g l u c o s i n o l a t e s , a c l a s s o f secondary p l a n t substances d i s t r i b u t e d
i n the host p l a n t s of t h i s i n s e c t i . e . , B r a s s i c a s p e c i e s . Though
the m e d i a l , as w e l l as the l a t e r a l s e n s i l l u m styloconicum c o n t a i n
a sugar s e n s i t i v e and a g l u c o s i n o l a t e s e n s i t i v e c e l l , the s e n s i l l a
show d i f f e r e n t s p e c i f i c i t i e s . The sugar s e n s i t i v e c e l l i n the
medial s e n s i l l u m responds t o a number o f carbohydrates, whereas the
receptor c e l l i n the l a t e r a l s e n s i l l u m responds r e s t r i c t i v e l y t o
sucrose and glucose. Aromatic g l u c o s i n o l a t e s are detected both
i n the medial as w e l l i n the l a t e r a l s e n s i l l u m ; the responses t o
a l i p h a t i c g l u c o s i n o l a t e s are r e s t r i c t e d t o the l a t e r a l s e n s i l l u m .
I t i s remarkable that p l a n t pigments l i k e anthocyanins a r e
perceived as a t a s t e by brassicae larvae.
In a d d i t i o n t o the s e n s i l l a s t y l o c o n i c a , lepidopterous
l a r v a e possess gustatory s e n s i l l a on the m a x i l l a r y p a l p s . Eight
b a s i c o n i c s e n s i l l a are l o c a t e d on top o f each palpus (see Figure
1 ) . F i v e of them possess a t e r m i n a l pore, and f o r that reason
these s e n s i l l a might be considered as contact chemoreceptors. The
remaining three show numerous small p e r f o r a t i o n s a l l over the
c u t i c l e , which i n d i c a t e s an o l f a c t o r y f u n c t i o n ( 8 ) . The response
spectra o f these s e n s i l l a a r e , however, s t i l l obscure.
One p a i r of epipharyngeal s e n s i l l a l o c a t e d i n the buccal
c a v i t y completes the set of contact chemoreceptors i n
lepidopterous l a r v a e . In jP. b r a s s i c a e l a r v a e , each o f these

p a p i l l a - s h a p e d s e n s i l l a c o n t a i n three sensory c e l l s : a) one c e l l

responds to sucrose and g l u c o s e , b) one f e e d i n g i n h i b i t o r
s e n s i t i v e c e l l , and c) one s a l t s e n s i t i v e c e l l (see Table I ) .
In b i t i n g t h e i r food and by means of a r e l a t i v e l y small
number of gustatory r e c e p t o r c e l l s , the l a r v a e are informed about
the composition of n u t r i e n t s and secondary p l a n t substances. Taste
p e r c e p t i o n , the i n t e g r a t i o n of sensory i n f o r m a t i o n i n the i n s e c t ' s
c e n t r a l nervous system, i s not merely a process of summation.
S y n e r g i s t i c as w e l l as a n t a g o n i s t i c e f f e c t s between i n d i v i d u a l
compounds can be observed i n the food uptake of l a r v a e on
a r t i f i c i a l diets.
Sucrose i n c o r p o r a t e d i n these d i e t s enhances the food uptake
by I \ b r a s s i c a e l a r v a e already a t 0.002 Molar (see F i g u r e 2 ) . In
t h i s respect D-glucose i s l e s s e f f e c t i v e than sucrose.
G l u c o s i n o l a t e s , amino a c i d s and s a l t s do not induce food uptake
by themselves. In the presenc
c o n c e n t r a t i o n s of sucrose
food uptake (Figure 2 ) . The s y n e r g i s t i c e f f e c t of s i n a l b i n
already occurs below 10"^ Molar. In t h i s way the i n t e r a c t i o n of
i n d i v i d u a l t a s t e substances as a r e s u l t of the c e n t r a l
i n t e g r a t i o n of sensory i n f o r m a t i o n , d e f i n e s the motor p a t t e r n s
l e a d i n g to f e e d i n g behaviour. The i n d i v i d u a l t a s t e substances
can be d e s c r i b e d i n terms of t h e i r a c t i o n on food uptake and
t h e i r consecutive s i g n a l f u n c t i o n . For JP. b r a s s i c a e l a r v a e , t a s t e
substances can be c a l l e d e i t h e r f e e d i n g s t i m u l a n t s (sucrose, D-
glucose), feeding i n c i t a n t s (glucosinolates), feeding co-factors
(amino a c i d s , s a l t s ) , or f e e d i n g i n h i b i t o r s ( s t r y c h n i n e ,
conessine, a z a d i r a c h t i n ) .
D i f f e r e n t i n s e c t species posses d i f f e r e n t gustatory r e c e p t o r
c e l l s , t h e i r response s p e c t r a being adapted to the p e r c e p t i o n
of chemical components d i s t r i b u t e d i n t h e i r host p l a n t species
(9). Taste p e r c e p t i o n i n P. b r a s s i c a e l a r v a e forms a
r e p r e s e n t a t i v e example f o r phytophagous i n s e c t s , which are able
to d i s c r i m i n a t e a number of compounds l i k e sugars, amino a c i d s ,
s a l t s , and secondary p l a n t substances a c t i n g as f e e d i n g
i n h i b i t o r s or f e e d i n g i n c i t a n t s (3,6,8,10).
Olfaction
Green odour. Food chemists are w e l l aware of the complexity

of food odour blends being composed of numerous v o l a t i l e s . The
chemical complexity of food odour blends i s somewhat confusing
since analyses are f r e q u e n t l y c a r r i e d out on processed foods,
which i n c l u d e s canning and cooking of the o r i g i n a l p l a n t m a t e r i a l
(11). During the h e a t i n g p r o c e s s , a v a r i e t y of v o l a t i l e
substances i s formed from n o n - v o l a t i l e p r e c u r s o r s . Therefore, one
might s t a t e that the present knowledge concerning the chemical
composition of odour blends which o r i g i n a t e from l i v i n g p l a n t s ,
i s but l i t t l e . In order to t r a p the v o l a t i l e s from the a i r over
p l a n t s , analyses can be c a r r i e d out by making use of adsorbents
l i k e carbon or Porapak Q (12).

Figure 1. The head of a Pieris brassicae larva. The chemosensory sensilla are
shown in detail (7,8).
Log Molar Concentration Log Molar Concentration
Figure 2. Feces production by Pieris brassicae larvae on artificial diets containing

different amounts of sucrose and different amounts of sinalbin in the presence of
sucrose (7).

To study the odour over potato p l a n t l e a v e s , i n 20 minutes

30 l i t r e s of a i r were drawn through a sample f l a s k (volume: 5
l i t r e ) c o n t a i n i n g cut potato leaves (930 g ) , and the a i r b o r n e
components were trapped onto carbon (1-2 mg, see 13). The carbon
t r a p was e x t r a c t e d w i t h CS2, and the e x t r a c t was subjected to
gas chromatography (Figure 3 ) . Components were i d e n t i f i e d by
u s i n g the gas chromatograph-mass spectrometer-computer system
of the C e n t r a l I n s t i t u t e f o r N u t r i t i o n and Food Research TNO (14).
In the a i r space above leaves of f u l l y grown potato p l a n t s , the
f o l l o w i n g v o l a t i l e s were detected: trans-2-hexenal., c i s - 3 - h e x e n y l
a c e t a t e , cis-3-hexen-1-ol and trans-2-hexen-1-ol. Besides,
sesquiterpenes were o b v i o u s l y present at r e t e n t i o n times of
23-29 minutes; t h e i r i d e n t i t i e s were not worked out i n d e t a i l .
The c l a s s of s t r a i g h t c h a i n C( >9 s a t u r a t e d and unsaturated
a l c o h o l s , aldehydes and the d e r i v a t i v e a c e t a t e forms a
s i g n i f i c a n t part of a l
components o r i g i n a t e fro
a c i d s i n leaves i . e . , l i n o l e i c and l i n o l e n i c a c i d . D i f f e r e n t
p l a n t species may show d i f f e r e n t compositions of t h e i r l e a f odour
blends i n the p r o p o r t i o n s of the i n d i v i d u a l components of t h i s
green odour complex (15). In the a i r over c a u l i f l o w e r l e a v e s ,
c i s - 3 - h e x e n y l acetate i s the predominant component (16) , whereas
i n the a i r over leaves of f u l l y grown potato p l a n t s cis-3-hexen-
1-ol i s the main component, f o l l o w e d by c i s - 3 - h e x e n y l a c e t a t e ,
trans-2-hexenal and trans-2-hexen-1-ol (see F i g u r e 3 ) .
D i f f e r e n t i a l sensory s e n s i t i v i t y . The i n s e c t ' s p e r c e p t i o n

of p l a n t odours d i f f e r s e s s e n t i a l l y from t h e i r d i s c r i m i n a t i o n
of n o n - v o l a t i l e t a s t e substances, as phytophagous i n s e c t s may
already p e r c e i v e the odour at some d i s t a n c e from the p l a n t . In
a d u l t phytophagous i n s e c t s the antennae bear a l a r g e number of
o l f a c t o r y s e n s i l l a i n order to detect the minute c o n c e n t r a t i o n s
of the l e a f odour components i n the a i r downwind from a p l a n t . The
o v e r a l l s e n s i t i v i t y of the antennal o l f a c t o r y r e c e p t o r system can
be measured by making use of the electroantennogram technique
(17). An electroantennogram (EAG) i s the change i n p o t e n t i a l
between the t i p of an antenna and i t s base, i n response to
s t i m u l a t i o n by an odour component. Such an EAG r e f l e c t s the
r e c e p t o r p o t e n t i a l s of the o l f a c t o r y r e c e p t o r c e l l p o p u l a t i o n i n
the antenna.
The antennal o l f a c t o r y r e c e p t o r system i n s e v e r a l
phytophagous i n s e c t s i s very s e n s i t i v e i n the d e t e c t i o n of the
green odour components. In the Colorado b e e t l e L e p t i n o t a r s a
decemlineata, the t h r e s h o l d of response f o r trans-2-hexen-1-ol
i s c i r c a 108 molecules per ml of a i r (17) . In comparison, at 760
mm Hg and 20°C, 1 ml of a i r contains about 10^9 molecules. The
i n s e c t s t e s t e d i . e . , the migratory l o c u s t Locusta m i g r a t o r i a , the
c a r r o t f l y P s i l a rosae (18), the c e r e a l aphid S i t o b i o n avenae
(19) , the Colorado b e e t l e L. decemlineata (17) , L e p t i n o t a r s a

haldemani, the oak f l e a w e e v i l Rhynchaenus quercus (20) , t h e

summer f r u i t t o r t r i x moth Adoxophyes orana, and the l a r g e white
b u t t e r f l y P. b r a s s i c a e , show d i f f e r e n t i a l sensory s e n s i t i v i t i e s
f o r the i n d i v i d u a l green odour components (see F i g u r e 4 ) . F o r
example, the antennal r e c e p t o r system of the Colorado b e e t l e i s
more s e n s i t i v e f o r the a l c o h o l s than the corresponding aldehydes.
Whereas i n the c a r r o t f l y and i n a l a t e v i r g i n o p a r a e of the c e r e a l
aphid the aldehydes cause h i g h e r responses than t h e corresponding
a l c o h o l s . Each o f the i n s e c t species shows c e r t a i n t r a i t s i n the
c h a r a c t e r of t h e i r s e n s i t i v i t y s p e c t r a f o r the green odour
components. This d i f f e r e n t i a l sensory s e n s i t i v i t y might represent
a s p e c i e s s p e c i f i c a d a p t a t i o n of the s e t o f o l f a c t o r y r e c e p t o r s
to the p a r t i c u l a r green odour composition of the host p l a n t s .
The s e n s i t i v i t y of t h e antennal o l f a c t o r y r e c e p t o r system
d i f f e r s even between Colorado b e e t l e p o p u l a t i o n s (see F i g u r e 5 )
The b e e t l e s of t h e f i e l
more s e n s i t i v e f o r c i s - 3 - h e x e n y
of t h e l a b o r a t o r y stock c u l t u r e . B e e t l e s of the Utah p o p u l a t i o n
are r e l a t i v e l y l e s s s e n s i t i v e f o r trans-2-hexen-1-ol and
trans-2-hexenal than the i n d i v i d u a l s of the f i e l d p o p u l a t i o n i n
Wageningen, and those i n s e c t s obtained from the l a b o r a t o r y stock
c u l t u r e . The f u n c t i o n a l s i g n i f i c a n c e of these d i f f e r e n c e s f o r the
geographic v a r i a t i o n i n host p l a n t range of t h i s i n s e c t s p e c i e s
needs f u r t h e r e l u c i d a t i o n (21,22).
D i f f e r e n t i a l sensory p e r c e p t i o n . Phytophagous i n s e c t s may

use the p a r t i c u l a r green odour blend of t h e i r host p l a n t s t o
l o c a t e these s u i t a b l e f e e d i n g and/or o v i p o s i t i o n s i t e s . The
p a r t i c u l a r composition of the green odour a f f e c t s the long-range
o l f a c t o r y o r i e n t a t i o n of Colorado b e e t l e s . When s t a r v e d , these
i n s e c t s respond t o the odour of t h e i r h o s t p l a n t potato by w a l k i n g
upwind (odour-conditioned p o s i t i v e anemotaxis), and by i n c r e a s i n g
t h e i r speed o f locomotion ( d i r e c t chemo-orthokinesis), as shown
i n F i g u r e 6 (23,14). The odours of s e v e r a l other solanaceous p l a n t
species induce these b e h a v i o u r a l responses i n Colorado b e e t l e s
a l s o (24). The group o f solanaceous p l a n t species t e s t e d ,
comprises host p l a n t s l i k e Solanum c a r o l i n e n s e and Solanum
dulcamara, as w e l l as non-host p l a n t s l i k e N i c o t i a n a tabacum,
P e t u n i a "hybrida and Solanum nigrum. I n g e n e r a l , the vapours of
non-solanaceous p l a n t s p e c i e s do not induce upwind locomotory
responses i n Colorado b e e t l e s , and can be regarded as "non-
a t t r a c t i v e " o r n e u t r a l i n t h i s r e s p e c t . At some d i s t a n c e from
p l a n t s , l e a f odour blends a r e the chemical cues which enable the
Colorado b e e t l e t o d i s c r i m i n a t e between solanaceous and non-
solanaceous p l a n t s p e c i e s . I n t h i s way through o l f a c t o r y
o r i e n t a t i o n , the b e e t l e ' s e x p l o r a t i o n i s t o some extent c o n f i n e d
to a r e l e v a n t p a r t of the v e g e t a t i o n i n which host p l a n t s occur.
The p r o p o r t i o n s o f the i n d i v i d u a l green odour components
i n the l e a f odour c o n s t i t u t e a chemical message which, when
p e r c e i v e d , d i r e c t s the motor p a t t e r n s of t h i s i n s e c t . The

30 20 10
Time in minutes
Figure 3. Components trapped
were extracted with 60 fiL of CS»; IfiL was used for GC (detector FID). GC condi-
tions: WCOT Carbowax 20M column, 50 m long; temperature programmed 70-
150 °C. Key: a, trans-2-hexenal; b, cis-3-hexenyl acetate; c, cis-3-hexen-l-ol; and
d, trzns-2-hexen-l-ol (14).
T T T
Locusta migratoria 9
a
Psila rosae <$ 9
b
Sitobion avenae
c
Leptinotarsa decemlineata a V
Leptinotarsa haldemani 6* ?
d
Rhynchaenus quercus 6* 9
Adoxophyes orana 6* 9
Pieris brassicae 9
Figure 4. Sensitivity spectrum of the antennal olfactory receptor system in several
phytophagous insect species to the green odor components. EAG amplitudes in re-
sponse to the individual components are visualized in the areas of circles. Data were
derived from Refs. 18 (a), 19 (b), 17 (c), and 20 (d).

VISSER Differential Sensory Perceptions 223
< <
VV V
o o O
ill
I*; rii
i
e X I 3 |
5 ± ? j ?
O (it o <? -m
^j^*f<°£:
F i e w c 5. Mean responses of three male and three female Colorado beetles
from different populations to ums-2-hexen-l-ol(t-2-H-l-ol),
H-l-ol), cis-3-hexenyl acetate (c-3-H-ace), and Kms-2-hexenal (l-2-H-al) at two
3 s t o c k
dilutions in paraffin oil, 10' and 10> (v/v). Key: A laboratory .
field population Wageningen; C, field population Utah; vertical lines indicate 95%
confidence intervals; and *, significant atP < 0.01 (Mann-Whitney V test).

n = 687
Chemo-orthokinesis
Odour-conditioned anemotaxis
n
100 reaching the upwind or downwind edge
100-1 1-5: categories of response
-° 50H
50-
0 1
•1:2:3:A:5: : i '2\r.u\5\ wind wind* potato
wind wind* potato
time in minutes

Figure 6. Long-range orientation responses of female Colorado beetles to wind and to wind plus
potato odor; 687 individuals were tested. Left: tracks divided into five categories—1 and 2, straight
and indirectly downwind; 3, indifferent; 4 and 5 , indirectly and straight upwind. Right: time periods
required to reach upwind or downwind edge (14).

chemical message i s d i s t o r t e d as the c o n c e n t r a t i o n r a t i o s o f the

green odour components i n the l e a f odour blend are changed
a r t i f i c i a l l y (25). On i n c r e a s i n g the p r o p o r t i o n o f trans-2-hexen-
1-ol o r trans-2-hexenal i n the potato l e a f odour, the b e e t l e ' s
upwind locomotory response i s "turned o f f " . Thus, a change i n
the composition o f the green odour impairs the a t t r a c t i v e n e s s of
a p a r t i c u l a r l e a f odour blend (25).
The responses of s i n g l e o l f a c t o r y receptor c e l l s i n the
antenna o f the Colorado b e e t l e t o the green odour components and
t h e i r isomers have been analysed (26). For the present purpose,
these data are reduced t o the elements a c t i n g i n long-range
o l f a c t o r y o r i e n t a t i o n o f t h i s i n s e c t towards i t s host p l a n t
potato. The r e s u l t s from receptors responding w i t h i n h i b i t i o n are
d i s c a r d e d , as from t h e i r l e v e l s of spontaneous a c t i v i t y , these
responses are suspected t o represent a r t i f a c t s . Moreover, the
response thresholds of thes
d o u b t f u l . The green odou
considered t o be the adequate s t i m u l i f o r a number o f o l f a c t o r y
receptor c e l l s i n the Colorado b e e t l e antenna. The r e l a t i v e
i n t e n s i t i e s of response i n the r e c e p t o r c e l l s , which increase
t h e i r n e u r a l a c t i v i t y upon s t i m u l a t i o n by the i n d i v i d u a l green
odour components of potato l e a f , are shown i n F i g u r e 7 (26,14).
Since not a l l s i n g l e c e l l s were t e s t e d f o r t h e i r responses t o
c i s - 3 - h e x e n y l a c e t a t e , t h i s compound i s not discussed here.
The array o f o l f a c t o r y receptor c e l l s r e a c t s d i f f e r e n t i a l l y
to the green odour component s t i m u l i of potato l e a f , which i s
manifest by the continuum of t h e i r response s p e c t r a (see F i g u r e
7). The observed i n t e r f e r e n c e w i t h potato l e a f odour caused by
i n c r e a s i n g the c o n c e n t r a t i o n o f one green odour component occurs
at concentrations near o r even below the thresholds f o r an
electroantennogram response (25). At these extremely low
c o n c e n t r a t i o n s , the l e v e l of spontaneous a c t i v i t y i n one s i n g l e
o l f a c t o r y receptor c e l l i s h a r d l y changed. Therefore, i n l o n g -
range o l f a c t o r y o r i e n t a t i o n , the p e r c e p t i o n of green odour
i n v o l v e s a concerted change of n e u r a l a c t i v i t y i n numbers o f
o l f a c t o r y receptor c e l l s . An increase of the p r o p o r t i o n o f
trans-2-hexen-1-ol o r trans-2-hexenal heightens the n e u r a l
a c t i v i t y i n l i n e s 1-11 more than i n l i n e s 12-23 (see F i g u r e 7 ) .
This d i f f e r e n t i a l increment a l t e r s the c o n t r a s t i n the a c r o s s -
f i b r e p a t t e r n and, a t the l e v e l of the c e n t r a l nervous system,
modulates the b e e t l e ' s o r i e n t a t i o n responses. Thus, green odour
p e r c e p t i o n i n v o l v e s a d i f f e r e n t i a l response i n the a r r a y o f
o l f a c t o r y receptor c e l l s (14).
Leaf odours. The t o t a l essence which emanates from growing

leaves i s not s o l e l y c o n s t i t u t e d o f s t r a i g h t c h a i n a l c o h o l s and
aldehydes. I n the i n s e c t ' s s e l e c t i o n o f a host p l a n t , s p e c i e s -
s p e c i f i c components might be i n v o l v e d . The leek m o t h A c r o l e p i o p s i s
a s s e c t e l l a i s a t t r a c t e d by t h i o s u l f i n a t e s , compounds i s o l a t e d
from leek. Cis-3-hexen-1-ol was a l s o shown t o be a t t r a c t i v e (27).

The catches of c a r r o t f l i e s i n y e l l o w s t i c k y t r a p s are enhanced

by the r e l e a s e of hexanal and compounds i s o l a t e d from the surface
wax of c a r r o t leaves i . e . , trans-methyliso-eugenol and t r a n s -
asarone (28,29). The propenylbenzenes a l s o s t i m u l a t e o v i p o s i t i o n
by t h i s i n s e c t (29).
E l e c t r o p h y s i o l o g i c a l r e s u l t s i n d i c a t e that green odour
components are not the s o l e .compounds involved i n the p e r c e p t i o n
of l e a f odour blends. EAGs of S. avenae to benzaldehyde ( 1 9 ) ,
and EAGs of A. orana t o benzaldehyde, l i n a l o o l , 1-octen-3-ol,
a-phellandrene and a - t e r p i n e o l show c i r c a the same s i z e as EAGs
i n response to green odour components. Whereas i n P. b r a s s i c a e
EAGs to a l l y l i s o t h i o c y a n a t e are small compared w i t h the responses
to trans-2-hexenal (30). In P. b r a s s i c a e l a r v a e , the m a j o r i t y of
c e l l s i n the two l a r g e s e n s i l l a b a s i c o n i c a on the antenna (see
Figure 1) responds d i f f e r e n t i a l l y to green odour components. Two
c e l l types react to a l l y l i s o t h i o c y a n a t e
of n e u r a l discharges i
the second c e l l .
Most of the present-day i n f o r m a t i o n concerning the r o l e of
l e a f odour blends i n host s e l e c t i o n by phytophagous i n s e c t s does
not proceed beyond the suggestion that some v o l a t i l e compounds
might be c a l l e d " a t t r a c t a n t s " and other " r e p e l l e n t s " (31,32) . In
case a v o l a t i l e compound was shown to s t i m u l a t e o v i p o s i t i o n , i t i s
suggestive to d e s c r i b e the component as an " a t t r a c t a n t " : "although
a p o s i t i v e response i s judged by the number of eggs l a i d , the
f l i e s must be a t t r a c t e d to the odor before o v i p o s i t i o n " (33) . In
t h i s l i n e of thought, t e s t s on long-range o l f a c t o r y o r i e n t a t i o n
are not e s s e n t i a l f o r a s s e s s i n g a t t r a c t i v e f e a t u r e s . J.S. Kennedy
has summarized the disadvantages of making use of the concept of
o l f a c t o r y " a t t r a c t i o n " , and s t a t e d " t h i s term ( a t t r a c t a n t ) remains
no more than a b l a n k e t t e l e o l o g i c a l term s i g n i f y i n g and e n d - r e s u l t ,
conveying n o t h i n g about the component s t i m u l i or r e a c t i o n s " (34).
In the same way, one might s t a t e that the term " r e p e l l e n t " i s
misleading.
Phytophagous i n s e c t s might be " a t t r a c t e d " over a long-range
i n response to l e a f odours, the i n s e c t s showing an odour-
c o n d i t i o n e d p o s i t i v e anemotaxis. The p r i n c i p a l b e h a v i o u r a l
response i s d i r e c t e d to the wind, and i s induced by the i n s e c t ' s
p e r c e p t i o n of the minute c o n c e n t r a t i o n of odour downwind from the
source. In the s t r i c t sense of the d e f i n i t i o n s an i n s e c t should
respond to an " a t t r a c t a n t " by moving towards the source, which
c o i n c i d e s by moving upwind, and respond to a " r e p e l l e n t " by moving
away from the source. Thus, the response to a " r e p e l l e n t " should
r e s u l t i n the i n s e c t moving downwind, which makes no sense as
the i n s e c t does not escape from the " r e p e l l e n t " odour plume.
Moreover, i t i s h a r d l y f e a s i b l e phytophagous i n s e c t s are equipped
w i t h large numbers of o l f a c t o r y receptors tuned to " r e p e l l e n t s "
i n order to detect the minute concentrations of these components
at long d i s t a n c e s from the p l a n t . For these reasons the e f f e c t s
of " r e p e l l e n t s " might expected to be r e s t r i c t e d to c l o s e range,

11} • T • T T V ? M :
I : T f
j i ?
T ] r; f
l-t . • • • • • • • • • • • • • • • •
: I I I i I !I! ! ; I i . . I ! ! ! !
1 2 3 ^ 5 6 7
Single Cells - •
Figure 7. Intensity of neural activity in 23 olfactory receptor cells in the antenna

of the Colorado beetle, in response to trans-2-hexen-1-ol (t-2-H-l-ol), cis-3-hexen-
l-ol (c-3-H-l-ol), and lrans-2-hexenal (t-2-H-al). The increase of neural activity in
response to individual green odor components is visualized in the areas of circles
(14, 26).
Figure 8. Host selection by phytophagous insects illustrated in a model of marbles.

Row 6 is the host plant; rows 1-5 and 7-10 are non-host plants.

and d i f f e r e n t from d e t e r r e n t s as f a r as the v o l a t i l e nature allows

these compounds to be detected j u s t before an i n s e c t makes contact
w i t h the p l a n t .
Conclusions
The chemical f a c t o r s u n d e r l y i n g host s e l e c t i o n by

phytophagous i n s e c t s are o u t l i n e d i n F i g u r e 8. P l a n t s are
p r o t e c t e d against herbivores mainly through secondary p l a n t
substances. Phytophagous i n s e c t s have to spend p a r t of t h e i r
metabolic energy on d e t o x i c a t i n g these noxious p l a n t components,
and i n e v o l u t i o n a r y time developed a degree of t o l e r a n c e to the
c l a s s of secondary compounds present i n t h e i r host p l a n t range.
Besides secondary p l a n t substances, the p r o p o r t i o n s of i n d i v i d u a l
n u t r i e n t s d e f i n e the metabolic q u a l i t y of a given p l a n t which
i s manifested i n i n s e c
In the c o - e v o l u t i o
chemosensory receptors i n phytophagous i n s e c t s adapted as to
perceive the metabolic q u a l i t i e s of p l a n t s . Thus, t a s t e informs
an i n s e c t of the presence and concentrations of n u t r i e n t s ,
f e e d i n g i n c i t a n t s and f e e d i n g i n h i b i t o r s . An i n s e c t depends
completely on the perceived t a s t e q u a l i t y i n d e c i d i n g f o r "take
i t or leave i t " . The p e r c e p t i o n of p l a n t odour enables an i n s e c t
to d i r e c t i t s motor p a t t e r n s i n order to explore e f f i c i e n t l y the
surrounding v e g e t a t i o n . In t h i s way host f i n d i n g i s not a matter
of " t r i a l and e r r o r " . Host p l a n t s meet the p h y s i o l o g i c a l
requirements of a given i n s e c t s p e c i e s , i n the sense that an
appropriate sensory q u a l i t y c o i n c i d e s w i t h an a p p r o p r i a t e
metabolic q u a l i t y . Non-host and r e s i s t a n t p l a n t s are d e f i c i e n t
i n at l e a s t one of the q u a l i t y f a c t o r s .
Host s e l e c t i o n behaviour of phytophagous i n s e c t s i s a
catenary process. In the succession of the elements i . e . ,
o l f a c t o r y o r i e n t a t i o n , f o l l o w e d by contact and b i t i n g responses,
one observes an increment i n s p e c i f i c i t y of both the chemical
p l a n t s i g n a l s as w e l l as the chemoreceptors i n v o l v e d : (a) i n s e c t
o l f a c t o r y receptor c e l l s responding d i f f e r e n t i a l l y t o general
green odour components - the q u a l i t y of an odour blend i s coded
i n an a c r o s s - f i b r e p a t t e r n -, and (b) gustatory receptor c e l l s
responding to p l a n t s p e c i e s - s p e c i f i c f e e d i n g i n c i t a n t s and
i n h i b i t o r s - the n e u r a l code i s processed i n l a b e l e d l i n e s -.
Acknowledgement s
The f i n a l drawings and glossy p r i n t s were prepared by W.C.T.

M i d d e l p l a a t s and J.W. Brangert r e s p e c t i v e l y .
Literature Cited
1. Beck, S.D.; Schoonhoven, L.M. "Breeding P l a n t s R e s i s t a n t to

I n s e c t s " ; Maxwell, F . G . , J e n n i n g s , P.R., Eds.;John Wiley:
New York, 1980; p. 115-35.

2. Swain, T. Ann. Rev. P l a n t P h y s i o l . 1977, 28, 479-501.

3. Schoonhoven, L.M. Ent. exp. & a p p l . 1982, 31, 57-69.
4. Southwood, T.R.E. " I n s e c t / P l a n t R e l a t i o n s h i p s " ; van Emden,
H.F., Ed.; Symp. Roy. ent. Soc. London, 1972, 6; B l a c k w e l l :
Oxford; p. 3-30.
5. House, H.L. Ent. exp. & a p p l . 1969, 12, 651-69.
6. S t a e d l e r , E. Proc. XV I n t . Congr. Ent. Washington D.C., 1976,
p. 228-48.
7. Ma, W.C. Meded. Landbouwhogeschool Wageningen, 1972, 72-11.
8. Schoonhoven, L.M. " I n s e c t / P l a n t R e l a t i o n s h i p s " ; van Emden,
H.F., Ed.; Symp. Roy. e n t . Soc. London, 1972, 6, B l a c k w e l l :
Oxford; p. 87-99.
9. Drongelen, W. van J. comp. P h y s i o l . 1979, 134, 265-79.
10. Schoonhoven, L.M. "Semiochemicals: their Role in Pest
C o n t r o l " ; Nordlund D.A. Ed.; John Wiley: New York 1981;
p. 31-50.
11. S t r a t e n , S. van "Volatile
C e n t r a l Institute f o r Nutrition and Food Research TNO: Zeist,
1979.
12. P i e r c e , H.D., Jr.; Vernon, R.S.; Borden, J.H.; Oehlschlager,
A.C. J. chem. E c o l . 1978, 4, 65-72.
13. Grob, K.; Zürcher, F. J. Chromat. 1976, 117, 285-94.
14. V i s s e r , J.H. " O l f a c t i o n in the Colorado B e e t l e a t the Onset
of Host P l a n t S e l e c t i o n " ; T h e s i s , Agricultural University,
Wageningen, 1979.
15. V i s s e r , J.H.; S t r a t e n , S. van; Maarse, H. J. chem. E c o l . 1979,
5, 13-25.
16. Wallbank, B.E.; Wheatley, G.A. Phytochemistry, 1976, 15,
763-6.
17. Visser, J.H. Ent. exp. & a p p l . 1979, 25, 86-97.
18. Guerin, P.M.; Visser, J.H. P h y s i o l o g i c a l Entomology 1980,
5, 111-9.
19. Yan, Fu-shun; V i s s e r , J.H. Proc. 5 t hInt.Symp. I n s e c t - P l a n t
R e l a t i o n s h i p s Wageningen; V i s s e r , J.H., Minks, A.K., Eds.;
Pudoc: Wageningen, 1982, in p r e s s .
20. K o z l o w s k i , M.W.; V i s s e r , J.H. Ent. exp. & a p p l . 1981, 30,
169-75.
21. Hsiao, T.H. Ent. exp. & a p p l . 1978, 24, 437-47.
22. Hsiao, T.H. Proc. 5 t hInt.Symp. I n s e c t - P l a n t R e l a t i o n s h i p s
Wageningen; V i s s e r , J.H., Minks, A.K., Eds.; Pudoc:
Wageningen, 1982, in p r e s s .
23. V i s s e r , J.H. Ent. exp. & a p p l . 1976, 20, 275-88.
24. V i s s e r , J.H.; N i e l s e n , J.K. Ent. exp. & a p p l . 1977, 21, 14-22.
25. V i s s e r , J.H.; Avé, D.A. Ent. exp. & a p p l . 1978, 24, 738-49.
26. Ma, W.C.; V i s s e r , J.H. Ent. exp. & a p p l . 1978, 24, 520-33.
27. Thibout, E.; Auger, J.; Lecomte, C. Proc. 5 t hInt.Symp.
I n s e c t - P l a n t R e l a t i o n s h i p s Wageningen; V i s s e r , J.H., Minks,
A.K., Eds.; Pudoc: Wageningen, 1982, in press.
28. Guerin, P.M.; S t a e d l e r , E. Proc. 5 t h I n t . Symp. I n s e c t - P l a n t


Pudoc: Wageningen, 1982, in p r e s s .
29. S t a e d l e r , E.; Buser, H.R. Proc. 5 t h Int. Symp. Insect-Plant
Pudoc: Wageningen, 1982, in press.
30. Behan, M.; Schoonhoven, L.M. Ent. exp. & a p p l . 1978, 24,
163-79.
31. D e t h i e r , V.G. "Chemical Insect A t t r a c t a n t s and R e p e l l e n t s " ;
B l a k i s t o n : P h i l a d e l p h i a , 1947.
32. Kogan, M. Proc. XV I n t . Congr. Ent. Washington D.C., 1976,
p. 211-27.
33. Vernon, R.S.; P i e r c e , H.D., Jr.; Borden, J.H.; Oehlschlager,
A.C. Environm. Entomology 1978, 7, 728-31.
34. Kennedy, J.S. P h y s i o l o g i c a l Entomology 1978, 3, 91-8.

13
Nutrient-Allelochemical Interactions in Host
Plant Resistance
1
JOHN C. REESE
University of Delaware, Department of Entomology and Applied Ecology,
Newark, DE 19711
Once an i n s e c t has l o c a t e d a p l a n t , the p l a n t must

be capable o f supportin
r e p r o d u c t i o n , if
The s u r v i v a l o f plants, however, is due in part
to t h e i r defensive s t r a t e g i e s . Evidence is pre-
sented supporting the hypothesis that even sus-
c e p t i b l e p l a n t s are well-defended. Black c u t -
worm larvae fed Pioneer 3368A corn seedlings
grew t o a weight o f only 8.1% that of l a r v a e
fed an artificial d i e t . Some o f the ability
to inhibit growth can be e x t r a c t e d out of the
p l a n t and incorporated i n t o the artificial diet
demonstrating a chemical component, but p h y s i c a l
and morphological f a c t o r s appear t o play an im-
portant r o l e too. Data is presented indicating
that neonate l a r v a e are f a r more s e n s i t i v e t o the
d e l e t e r i o u s e f f e c t s o f the seedlings than l a r v a e
a few days o l d . Neonate l a r v a e are a l s o s e n s i t i v e
to h a n d l i n g , and using eggs i n s t e a d of l a r v a e t o
i n f e s t p l a n t s or i n o c u l a t e d i e t seems a d v i s a b l e .
"As greater understanding of i n s e c t and p l a n t b i o l o g y ,

chemistry, and ecology i s a t t a i n e d , we w i l l be able to approach
the goal of developing economic p l a n t s that are d e l i b e r a t e l y and
f o r e s i g h t e d l y designed to be i n s e c t r e s i s t a n t " ( 1 ) . One of the
more i n t e r e s t i n g , and perhaps more poorly understood, areas o f
"— i n s e c t and p l a n t b i o l o g y , chemistry, and ecology — , " i s
the study of the i n t e r a c t i o n s between a l l e l o c h e m i c s and n u t r i e n t s .
A s u i t a b l e host p l a n t must be capable o f supporting growth,
development, and r e p r o d u c t i o n , i f the p l a n t i s t o serve as a host.
The feeding i n s e c t must i n g e s t food " . . . t h a t not only meets i t s
n u t r i t i o n a l requirements, but i s a l s o capable o f being a s s i m i l a t e d
and converted i n t o the energy and s t r u c t u r a l substances f o r normal
1
Current address, Department of Entomology, Kansas State University, Manhattan,
KS 66506
0097-6156/83/0208-0231$06.00/0

11
a c t i v i t y and development (2_ ,3) . Thus, the concept of i n s e c t
d i e t e t i c s i n c l u d e s a good d e a l more than n u t r i t i o n i n the narrow
sense; feeding behavior, n u t r i t i o n , and p o s t - i n g e s t i v e e f f e c t s
are a l s o p a r t of i n s e c t d i e t e t i c s . The i n t e r a c t i o n s between
n u t r i e n t s and a l l e l o c h e m i c s a t the b e h a v i o r a l , n u t r i t i o n a l , and
p o s t - i n g e s t i v e l e v e l s may be very important parts o f the mecha-
nisms u n d e r l y i n g the d e l e t e r i o u s e f f e c t s of p l a n t s on i n s e c t
growth and development. I propose that these d e l e t e r i o u s e f f e c t s
are probably not confined j u s t t o non-hosts o r t o h i g h l y r e s i s t a n t
crop v a r i e t i e s , but may be the r u l e ; i f a p l a n t were t r u l y unde-
fended against h e r b i v o r e and pathogen a t t a c k , i t would i n a l l
l i k e l i h o o d not s u r v i v e long enough t o reproduce o r produce a crop.
Defense of S u s c e p t i b l e P l a n t s
The very s u r v i v a l
"... due l a r g e l y t o t h e i
Most researchers probably accept t h i s concept, and yet the pre-
f e r r e d host p l a n t o f an i n s e c t s p e c i e s , o r a crop that i s decimat-
ed by a p e s t , i s u s u a l l y thought of as being q u i t e s u s c e p t i b l e
and r a t h e r poorly defended against i n s e c t a t t a c k .
I have s t a r t e d t e s t i n g the hypothesis that even the most
s u s c e p t i b l e p l a n t s are i n f a c t remarkably w e l l defended against
i n s e c t a t t a c k when compared t o an a r t i f i c i a l d i e t c o n t a i n i n g low
concentrations of defensive compounds, and having no morphological
means of defense. A few species of i n s e c t s , i n f a c t , have already
been observed t o have greater f e c u n d i t y and growth on a r t i f i c i a l
d i e t s , than on p r e f e r r e d host p l a n t s 05, 6^, 7 ) .
While working a t the USDA Western Regional Research Center
i n C a l i f o r n i a w i t h A. C. Waiss, J r . , I placed H e l i o t h i s zea l a r v a e
on a glanded c o t t o n v a r i e t y , a glandless v a r i e t y (presumably sus-
c e p t i b l e t o bollworm l a r v a e ) , and on a r t i f i c i a l d i e t . Even the
weight of the glandless cotton group of l a r v a e was only 25.1% o f
the c o n t r o l s on a r t i f i c i a l d i e t , i n d i c a t i n g that even the suppos-
edly s u s c e p t i b l e p l a n t was chemically and/or m o r p h o l o g i c a l l y q u i t e
capable o f reducing i n s e c t growth.
Black cutworms ( A g r o t i s i p s i l o n ) were fed Pioneer 3368A corn
seedlings (not known t o be r e s i s t a n t ) and a r t i f i c i a l d i e t (8)
(Table I ) . By the s i x t h day the weight g a i n of l a r v a e fed seed-
l i n g s was a s m a l l f r a c t i o n of that f o r l a r v a e reared on d i e t .
Even though b l a c k cutworms can be severe pests of corn s e e d l i n g s ,
t h e i r growth i s d r a m a t i c a l l y reduced by them. The a r t i f i c i a l d i e t
may have been a r i c h e r source o f n u t r i e n t s than corn s e e d l i n g s .
Therefore, I had samples o f d i e t and s e e d l i n g s analyzed f o r n i t r o -
gen content (as one o v e r a l l measure of n u t r i t i o n a l q u a l i t y ) a t the
U n i v e r s i t y of Delaware S o i l Chemistry Laboratory. The a r t i f i c i a l
d i e t was a c t u a l l y much lower i n n i t r o g e n than the s e e d l i n g s .
Amino a c i d a n a l y s i s showed no s i g n i f i c a n t d i f f e r e n c e s between
corn s e e d l i n g s and the a r t i f i c i a l d i e t .

13. REESE Nutrient-Allelochemical Interactions 233
Table I . E f f e c t s of Pioneer 3368A corn seedlings and a r t i f i c i a l

d i e t (8) on mean weights of black cutworm l a r v a e .
Mean L a r v a l Weights (mg) + SD
L a r v a l Age Reared on P l a n t s Reared on D i e t % of C o n t r o l

(Days) (Diet)
2 0.3 + 0.1 0.8 + 0.2 37.5% ***
3 0.4 + 0.2 2.4 + 1.2 16.7% ***
4 0.6 + 0.2 4.2 + 2.6 14.3% ***
5 1.0 + 0.5 11.7 + 5.3 8.5% ***
6 1.7 + 1.2 21.1 + 4.6 8.1% ***
*** i n d i c a t e s s t a t i s t i c a
reared l a r v a e a t P<0.00
The reduced growth of b l a c k cutworm larvae fed corn seedlings

may be due t o chemical and/or p h y s i c a l f a c t o r s . I n terms of
chemical f a c t o r s , the p l a n t may be low i n some e s s e n t i a l n u t r i e n t
and/or have growth i n h i b i t i n g compounds present. These compounds
may i n t e r a c t with n u t r i e n t s d i r e c t l y o r i n f l u e n c e the o v e r a l l
n u t r i t i o n a l physiology of the i n s e c t i n some way. A bioassay
searching f o r the absence o f something i s e m p i r i c a l at best.
Assessing the importance o f p h y s i c a l d i f f e r e n c e s between a p l a n t
and an a r t i f i c i a l d i e t i s no simple matter e i t h e r . Therefore, I
have s t a r t e d t o f r a c t i o n a t e the p l a n t i n search of the presence
of g r o w t h - i n h i b i t i n g f a c t o r s to see how important chemical f a c t o r s
are before t r y i n g t o assess p h y s i c a l f a c t o r s . E x t r a c t s were ad-
sorbed on the a l p h a c e l p o r t i o n o f the d i e t i n a way s i m i l a r to the
techniques of Chan e t a l (9) and using a m o d i f i c a t i o n of the black
cutworm d i e t ( 8 ) . F r e e z e - d r i e d , powdered, corn s e e d l i n g t i s s u e
was e x t r a c t e d i n a s o x h l e t e x t r a c t o r w i t h i n c r e a s i n g l y p o l a r s o l -
vents. The residue was t e s t e d by s u b s t i t u t i n g i t f o r the a l p h a c e l
p o r t i o n of the d i e t . The greatest b i o l o g i c a l a c t i v i t y was found
i n the acetone f r a c t i o n (Table I I ) . When the e f f e c t s of v a r i o u s
concentrations (20 l a r v a e per treatment) were bioassayed, there
was a s i g n i f i c a n t c o r r e l a t i o n between concentration and weight
gain o f the l a r v a e i n terms of percent of the c o n t r o l s ( r = 0.85,
6 d f ) . Methanol and benzene had s i m i l a r dosage response curves
(r = 0.69, 15 df and 0.75, 7 d f , r e s p e c t i v e l y ) .
Using s i n g l e s o l v e n t e x t r a c t s a t 100% p l a n t equivalency does
not show enough a c t i v i t y t o account e n t i r e l y f o r the growth-in-
h i b i t i n g a b i l i t i e s of the i n t a c t s e e d l i n g s . The recombining of
d i f f e r e n t f r a c t i o n s may show a d d i t i v e o r s y n e r g i s t i c e f f e c t s ; i t
may a l s o be that the p h y s i c a l c h a r a c t e r i s t i c s o f the i n t a c t and
s t r u c t u r a l l y complex p l a n t t i s s u e form a major defense.

Table I I . E f f e c t s of e x t r a c t s of Pioneer 3368A corn seedlings

on 10 day weights of b l a c k cutworm l a r v a e . For
weights, each e x t r a c t bioassayed at 100% p l a n t equiv-
alency (PE) (same c o n c e n t r a t i o n as i n i n t a c t p l a n t ) .
Residue bioassay at 90% of PE due to d i f f i c u l t y of i n -
corporating i n t o d i e t . Correlation c o e f f i c i e n t i s
given f o r experiments performed over a range of con-
c e n t r a t i o n s (25 - 100% PE).
Extract Weight (% of Controls ) _ SD r (degrees of freedom)

Benzene 77.7 + 21.0 0.75 (7)*
Acetone 67.5+10.3 0.85 (6)**
Methanol 75.2+20.5 0.69 (15)**
Water 90.7
Residue 95.0 + 2.8
* i n d i c a t e s s t a t i s t i c a l s i g n i f i c a n c e at P< 0.05 l e v e l .
** i n d i c a t e s s t a t i s t i c a l s i g n i f i c a n c e at P < 0.01 l e v e l .
Neonate S e n s i t i v i t y
In developing s e n s i t i v e bioassays to i n v e s t i g a t e e f f e c t s
of p l a n t s on i n s e c t growth and development, black cutworm l a r v a e
were placed on a r t i f i c i a l d i e t and on corn s e e d l i n g s . Neonate
l a r v a e placed on corn seedlings weighed 1.7 mg 7 days l a t e r ,
w h i l e neonate l a r v a e put on a r t i f i c i a l d i e t f o r 2 days and then
fed corn seedlings f o r 5 days weighed 10.2 mg. Thus, even though
the ones s t a r t e d on d i e t spent 71% of the experimental p e r i o d on
p l a n t s , they weighed 7 times as much as the ones on p l a n t s from
the s t a r t . To see i f t h i s r e a l l y suggests neonate s e n s i t i v i t y ,
a 48 hour feeding p e r i o d on a r t i f i c i a l d i e t was i n s e r t e d at
various p o i n t s during the experiment. A l l of the treatments
y i e l d e d mean l a r v a l weights that were h i g h l y s i g n i f i c a n t l y
d i f f e r e n t from the c o n t r o l s (Table I I I ) , but what i s p a r t i c u l a r l y
important i s that l a r v a e given a boost on a r t i f i c i a l d i e t during
the f i r s t 48 h r s . of the experiment were 3.3 times heavier at the
end of 8 days than those given d i e t during the second 48 h r s .
( s i g n i f i c a n t at P<.001 l e v e l ) . This c e r t a i n l y suggests that neo-
nate l a r v a e are f a r more s e n s i t i v e to p l a n t a l l e l o c h e m i c s and/or
morphological defenses than o l d e r l a r v a e . Judging by the magni-
tude of the d i f f e r e n c e s i n s e n s i t i v i t y , I suspect that the ob-
served growth i n h i b i t i o n i s due to a combination of both chemical
and morphological f a c t o r s . Changes i n s e n s i t i v i t y to a l l e l o -
chemics may revolve around the i n d u c t i o n of d e t o x i f i c a t i o n s y s -
tems (10), w h i l e morphological aspects may i n v o l v e such aspects
as s c l e r o t i z a t i o n of mandibles and thus the greater a b i l i t y to
deal w i t h such f a c t o r s as s i l i c a . The magnitude of the observed
s e n s i t i v i t y may i n d i c a t e that both chemical and morphological

f a c t o r s c o n t r i b u t e to growth r e d u c t i o n . Evidence suggesting

neonate s e n s i t i v i t y has been found i n a few other species (11,
12, 13), but has never been e x p l o i t e d very e f f e c t i v e l y i n a b i o -
assay system. Such s e n s i t i v i t y to d i f f e r e n t experimental regimes
may help e x p l a i n d i f f i c u l t i e s sometimes encountered by d i f f e r e n t
i n v e s t i g a t o r s t r y i n g t o reproduce each others r e s u l t s .
Table I I I . E f f e c t s of being f e d a r t i f i c i a l d i e t a t v a r i o u s times

d u r i n g experimental p e r i o d . Black cutworm l a r v a e f e d
on Pioneer 3368A corn s e e d l i n g s when they were not on
diet.
Mean Weight (mg) a t 8 Days ~ SD % o f Controls

P l a n t s Throughout 1.8 + 1.4 1.9% ***
D i e t 1st 48 h r s
Diet 2nd 48 h r s 4.4 + 3.0 4.5% *
Diet 3rd 48 h r s 2.6 + 1.7 2.7% ***
Diet l a s t 48 h r s 2.4 + 1.9 2.4% ***
Diet Throughout 97.6 + 29.4 100.0%
*** i n d i c a t e s s t a t i s t i c a l l y d i f f e r e n t from l a r v a e fed d i e t

throughout experiment, a t P<.001 l e v e l . N = 17.
Neonate b l a c k cutworm l a r v a e are not only p a r t i c u l a r l y sen-

s i t i v e t o the growth i n h i b i t i n g e f f e c t s o f p l a n t s , but are a l s o
remarkably s e n s i t i v e t o v a r i o u s types o f h a n d l i n g . This i s a
f a c t that i s r a r e l y taken i n t o account i n designing bioassays
(most workers p l a c e l a r v a e on d i e t o r p l a n t s w i t h a brush o r w i t h
an i n o c u l a t o r u s i n g l a r v a e mixed w i t h corn cob g r i t s ) . I t i s
a l s o an aspect o f i n s e c t physiology about which very l i t t l e i s
known. I n an attempt t o see i f the hours between e c l o s i o n and
being placed on d i e t o r on p l a n t s are s t r e s s f u l t o l a r v a e , eggs
were taken every 4 h r s from a s i n g l e c r y s t a l l i z i n g d i s h of
t r y p s i n i z e d eggs (eggs are r o u t i n e l y t r y p s i n i z e d o f f of the
cheesecloth s u b s t r a t e and f i l t e r e d onto a piece o f f i l t e r paper
i n a Buchner f u n n e l ; the egg-covered f i l t e r paper i s placed i n a
c r y s t a l l i z i n g d i s h that contains a l a y e r of agar t o maintain
uniform humidity without excessive condensation) and placed onto
d i e t . A f t e r enough l a r v a e had eclosed i n the c r y s t a l l i z i n g d i s h ,
l a r v a e were placed on d i e t . Weight and s u r v i v a l data taken a t
the end of the experimental p e r i o d i n d i c a t e not only a s t r i k i n g
r e d u c t i o n i n both s u r v i v a l and weight ( e s p e c i a l l y weight) a f t e r
e c l o s i o n , but a l s o a very i n t r i g u i n g r e l a t i o n s h i p between age of
the eggs a t the time they were placed on d i e t and growth of r e -
s u l t i n g l a r v a e (Table I V ) .

Table IV. Weights of black cutworm l a r v a e which were placed on

d i e t as eggs or l a r v a e at v a r i o u s times a f t e r t r y p -
sinizing.
Hours A f t e r Stage When Placed Mean
Trypsinizing on D i e t Weight (mg) ± SD
8 Egg 29.6 + 10.4
**
28 Egg 47.6 + 14.9
***
52 Egg 70.6 + 16.1
60 Egg 64.0 + 34.0
64
***
68 Larval 2.2 + 2.7
A s t e r i s k s i n d i c a t e s t a t i s t i c a l s i g n i f i c a n c e between two adjacent
means. ** i n d i c a t e s s t a t i s t i c a l d i f f e r e n c e at P<0.01 l e v e l . ***
i n d i c a t e s s t a t i s t i c a l d i f f e r e n c e at P<0.001 l e v e l . Larvae were
weighed 7 days a f t e r e c l o s i o n . N = 15.
One f a c t o r i n the l a r g e r e d u c t i o n i n weight of l a r v a e placed

on d i e t as l a r v a e i n s t e a d of as eggs may be the p h y s i c a l movement
of the l a r v a e . An experiment was performed i n which l a r v a e were
t r a n s f e r r e d from one plug of d i e t to a f r e s h one at v a r i o u s times
during the experiment, beginning 6 hrs a f t e r e c l o s i o n (Table V).
C l e a r l y , there was much more s e n s i t i v i t y to being t r a n s f e r r e d
during the f i r s t few hours a f t e r e c l o s i o n . Although t h i s appears
to be due to h a n d l i n g , the p o s s i b i l i t y that the d i e t i s being
conditioned i n some way by the l a r v a e can not be ignored. The
experiment has been repeated s e v e r a l times w i t h both f r e s h d i e t
and w i t h the l a r v a e being t r a n s f e r r e d back to the same piece of
d i e t w i t h i d e n t i c a l r e s u l t s i n each case. Thus, the observed
weight r e d u c t i o n i s apparently due to being handled.
Insect D i e t e t i c s
As a l l u d e d to above, even a crop that s u s t a i n s l o s s e s from

an i n s e c t pest may a c t u a l l y be capable of i n h i b i t i n g i n s e c t
growth. A number of aspects of the i n s e c t d i e t e t i c s may be
i n t e r a c t i n g to produce such a s i t u a t i o n . The feeding i n s e c t must
i n g e s t food "that not only meets i t s n u t r i t i o n a l requirements,
but i s a l s o capable of being a s s i m i l a t e d and converted i n t o the
energy and s t r u c t u r a l substances r e q u i r e d f o r normal a c t i v i t y and
development" 02, 3) .

Table V. Weight a t 10 days o f b l a c k cutworm l a r v a e t r a n s f e r r e d

from one p l u g of d i e t t o another a t v a r i o u s times a f t e r
eclosion.
Time o f T r a n s f e r Mean Weight

(Hours a f t e r E c l o s i o n ) (mg) ± SD % of Control
6 110.7 + 54.4 58.7 ***
12 123.2 + 63.1 65.3 **
24 127.1 + 51.0 67.4 **
48 140.8 + 53.4 74.6
96 158.8 + 61.9 84.2
144 167.1 + 49.6 88.6
192
Control 188.6 + 65.2 100.0
*** i n d i c a t e s s t a t i s t i c a l d i f f e r e n c e from c o n t r o l s a t P<0.001
l e v e l . ** i n d i c a t e s s i g n i f i c a n c e a t P<0.005 l e v e l . * i n d i c a t e s
s i g n i f i c a n c e a t P<0.01 l e v e l . N = 20.
The r o l e o f a l l e l o c h e m i c - n u t r i e n t i n t e r a c t i o n s i n i n s e c t
d i e t e t i c s has been i n v e s t i g a t e d only r a r e l y . Examples o f such
i n t e r a c t i o n s abound i n v e r t e b r a t e l i t e r a t u r e (14, 15) and may
supply u s e f u l leads f o r researchers working w i t h i n s e c t s . Many
of the d e l e t e r i o u s p h y s i o l o g i c a l e f f e c t s o f p l a n t a l l e l o c h e m i c s
may be due p r i m a r i l y t o v a r i o u s i n t e r a c t i o n s between these
a l l e l o c h e m i c s and e s s e n t i a l n u t r i e n t s . I n other words, i t i s
important t o not only consider the presence of n u t r i e n t s , but
a l s o the " b i o - a v a i l a b i l i t y " of these n u t r i e n t s t o the phytopha-
gous i n s e c t .
Some o f the more i n t e r e s t i n g examples o f n u t r i e n t - non-
n u t r i e n t i n t e r a c t i o n s i n c l u d e some o f the compounds that are
analogs of n u t r i e n t s . Mattson e t a l (16) found that c h o l e s t e r o l
a b s o r p t i o n decreased when v a r i o u s p l a n t s t e r o l s were added t o the
d i e t s of r a t s . A number o f p l a n t amino acids are not o r d i n a r i l y
r e q u i r e d by h e r b i v o r e s and are u s u a l l y not incorporated i n t o
proteins. For example, the s t r u c t u r e o f 3,4-dihydroxyphenyl-
alanine (L-dopa) i s s i m i l a r t o that o f t y r o s i n e . L-Dopa may
play a r o l e i n favism ( 1 7 ) , as w e l l as having a number o f other
d e l e t e r i o u s e f f e c t s (18, 19, 20). E s s e n t i a l amino acids them-
selves can be d e l e t e r i o u s i f they a r e ingested i n excessive
q u a n t i t i e s o r i f they are not i n balance w i t h other amino acids
(21, 2 0 ) .
Many more examples o f n u t r i e n t a l l e l o c h e m i c i n t e r a c t i o n s
could be c i t e d from the v e r t e b r a t e l i t e r a t u r e (see L i e n e r (15)
f o r a recent update o f t h i s t o p i c ) ; these i n t e r a c t i o n s i n i n s e c t s

have been i n v e s t i g a t e d at the molecular l e v e l i n only a few

cases. However, the i n h i b i t i o n of a s s i m i l a t i o n or the e f f i c i e n c y
of conversion of a s s i m i l a t e d food demonstrates an i n t e r a c t i o n
w i t h the o v e r a l l n u t r i t i o n a l s t a t u s of the organism. These i n -
dices have been employed to q u a n t i f y host s u i t a b i l i t y and to
assess the i n t e r a c t i o n s between p l a n t compounds i n c o r p o r a t e d
i n t o a r t i f i c i a l d i e t s and the n u t r i t i o n a l physiology of the
i n s e c t . Such techniques have shown, f o r example that a phytoph-
agous i n s e c t does not grow e q u a l l y w e l l on v a r i o u s p l a n t s , even
i f no apparent b e h a v i o r a l b a r r i e r s to feeding occur.
Three of the most u s e f u l n u t r i t i o n a l i n d i c e s are a s s i m i l a -
t i o n (AD), e f f i c i e n c y of conversion of a s s i m i l a t e d food (ECD),
and e f f i c i e n c y of conversion of ingested food (ECI). These and
a number of other i n d i c e s were r e c e n t l y discussed by S c r i b e r and
Slansky (22). I f growth i s i n h i b i t e d , then i t must e i t h e r be
r e f l e c t e d i n the amoun
or both.
As w i t h any experimental technique, there are c e r t a i n i n -
herent sources of e r r o r i n the n u t r i t i o n a l index technique. Some
of these e r r o r s may be g r e a t l y magnified due to the c a l c u l a t i o n s
i n v o l v e d and due to the exact ways i n which the techniques are
employed. In my l a b o r a t o r y , Schmidt (unpublished data) has
succeeded i n i d e n t i f y i n g and q u a n t i f y i n g three major sources of
e r r o r . The f i r s t i s the s e p a r a t i o n of uneaten d i e t from f e c a l
m a t e r i a l . I f the l a r v a masticates or bores i n t o the d i e t , t h i s
becomes very d i f f i c u l t . Secondly, even i f great care i s used,
there i s probably always some e r r o r i n the t a k i n g of weight, i n -
c l u d i n g those used to c a l c u l a t e the percent dry matter of the
d i e t . These e r r o r s r e l a t e to the dry weight eaten by each
l a r v a , which i n t u r n a f f e c t s the three i n d i c e s . Most important-
l y , he found that very s l i g h t e r r o r s can be magnified tremen-
dously through the mathematics i n v o l v e d i n c a l c u l a t i n g the i n -
d i c e s . I f the amount of the d i e t eaten by a l a r v a i s s m a l l
r e l a t i v e to the amount given to i t , then s m a l l e r r o r s i n such
things as percent dry matter of the d i e t are magnified to an un-
acceptable l e v e l . I f the l a r v a eats 80% or more of the d i e t
given to i t , t h i s e r r o r m a g n i f i c a t i o n i s reduced tremendously.
Thus, the s i n g l e most important e r r o r - r e d u c i n g technique i s to
ensure that l a r v a e are given an amount of d i e t such that they
w i l l consume most of i t during the experiment.
The e f f e c t s on n u t r i t i o n a l i n d i c e s can demonstrate an i n t e r -
a c t i o n between an a l l e l o c h e m i c and the n u t r i t i o n a l s t a t u s of the
i n s e c t , but the number of a c t u a l compound-to-compound i n t e r -
a c t i o n s that have been e l u c i d a t e d i n i n s e c t s has been s m a l l . One
of the most prominent examples i s the s t r u c t u r a l analog of
L - a r g i n i n e , L-canavanine (see paper by G. A. Rosenthal i n t h i s
symposium). B r i e f l y , canavanine i s s i m i l a r enough to a r g i n i n e to
be i n c o r p o r a t e d i n t o the p r o t e i n s of most i n s e c t s , but these
canavanyl p r o t e i n s do not f u n c t i o n p r o p e r l y (23). Thus, i t can
act as a competitive i n h i b i t o r of a r g i n i n e metabolism (24, 25),

as an i n h i b i t o r o f i n s e c t s u r v i v a l (26, 27, 20), reproduction

(28), and metamorphosis (29) .
Tannins are u s u a l l y c i t e d as examples o f substances that can
block the a v a i l a b i l i t y o f p r o t e i n s by forming complexes. Thus,
when Feeny (30) found that oak l e a f tannin reduced the growth o f
w i n t e r moth l a r v a e (Operophtera brumata (L.)) and subsequently
showed that oak l e a f t a n n i n forms a h y d r o l y s i s - r e s i s t a n t complex
w i t h casein i n v i t r o (31), i t was w i d e l y assumed that the growth-
i n h i b i t i n g e f f e c t s of tannins i n i n s e c t s are due t o the formation
i n the gut t r a c t o f t a n n i n - d i e t a r y p r o t e i n complexes that are not
r e a d i l y d i g e s t e d. I t i s a l s o widely assumed that many d i g e s t i v e
enzymes may be complexed, f u r t h e r reducing the r a t e o f a s s i m i l a -
t i o n across the gut w a l l . Contrary t o these assumptions, Fox and
Macauley (32) found that tannins do not a p p r e c i a b l y reduce the
a v a i l a b i l i t y o f n i t r o g e n i n Paropsis atomaria O l i v . l a r v a e when
fed Eucalyptus spp. havin
c e n t r a t i o n s . Bernays (33
ments using some r a t h e r high l e v e l s of t a n n i n , but found l i t t l e
evidence f o r a r e d u c t i o n i n d i g e s t i o n . Chan e t a l (36) have
i s o l a t e d a condensed t a n n i n from c o t t o n w i t h a molecular weight
of about 4850. Although t h i s tannin i n h i b i t s the growth o f
H e l i o t h i s v i r e s c e n s , experiments w i t h condensed t a n n i n - c a s e i n o r
condensed tannin-polyamide complexes showed no r e d u c t i o n i n b i o -
l o g i c a l a c t i v i t y (36). This suggests that the a b i l i t y o f t a n n i n
to i n h i b i t growth i n v o l v e s something other than a r e d u c t i o n i n
a s s i m i l a t i o n due t o complexing w i t h gut t r a c t p r o t e i n s . I n
experiments w i t h H. zea (37), cotton condensed t a n n i n was found
to be a r e l a t i v e l y potent growth i n h i b i t o r , but no evidence was
found f o r a r e d u c t i o n i n a s s i m i l a t i o n . Instead, the primary
growth i n h i b i t i n g mechanism was an i n h i b i t i o n o f i n g e s t i o n .
Thus, b e h a v i o r a l , as w e l l as p o s t - i n g e s t i v e processes may a f f e c t
the a v a i l a b i l i t y of n u t r i e n t s .
In the case o f the w i n t e r moth, tannins may very w e l l reduce
a s s i m i l a t i o n by complexing w i t h d i e t a r y p r o t e i n s . However, the
assumption that t h i s i s how tannins i n h i b i t growth i n species
other than the w i n t e r moth needs t o be c a r e f u l l y re-examined. I n
the experiments c i t e d above, mechanisms other than a r e d u c t i o n i n
a s s i m i l a t i o n appear t o be o p e r a t i n g .
There are mechanisms other than the complexing o f p r o t e i n s
that may prevent n u t r i e n t s from passing across the gut w a l l .
Protease i n h i b i t o r s decrease the a v a i l a b i l i t y o f n u t r i e n t s pre-
v e n t i n g the break-down of p r o t e i n s i n t o t h e i r component amino
a c i d s . The e f f e c t s of protease i n h i b i t o r s on i n s e c t s have been
reviewed (.38, 39, 40; paper by C. A. Ryan i n t h i s symposium).
B i r k and Applebaum (41) have s t u d i e d the adverse e f f e c t s of soy-
bean t r y p s i n i n h i b i t o r s on development and protease a c t i v i t y i n
T r i b o H u m castaneum. I n S i t o p h i l u s oryzae, h i g h doses o f soybean
t r y p s i n i n h i b i t o r caused a d u l t m o r t a l i t y (42). The wound-induced
accumulation o f these i n h i b i t o r s i s discussed by C. A. Ryan e l s e -
where i n t h i s symposium.

Other enzymes may be i n h i b i t e d , too. L i e n e r (43) r e c e n t l y

summarized the l i t e r a t u r e on amylase i n h i b i t o r s i n wheat and
t h e i r e f f e c t i v e n e s s against s t o r e d product pests*
While not o f t e n l i s t e d as an e s s e n t i a l n u t r i e n t , water i s

c r i t i c a l to l i f e and i n t e r a c t s w i t h a l l other n u t r i e n t s . For
i n s e c t s , the moisture l e v e l of the host may have profound e f f e c t s
on the n u t r i t i o n a l physiology of the i n s e c t . Although s t o r e d
products i n s e c t s may have remarkable a b i l i t i e s to conserve water,
many i n s e c t s which l i v e on growing p l a n t t i s s u e r e q u i r e r e l a t i v e -
l y high moisture l e v e l s (44, 45, 46). On the other hand, water
d i l u t e s the n u t r i e n t s of the d i e t . For example, C e l e r i o
euphorbiae l a r v a e tend to eat more as the n u t r i e n t s become more
d i l u t e (47). D i l u t i o n of the d i e t of Prodenia e r i d a n i a caused
an i n c r e a s e i n e f f i c i e n c y of conversion (48, 49) Similarly,
Feeny (4) found that th
food decreased w i t h decreasin
of v a r i o u s lepidopterous l a r v a e . In Hyalophora c e c r o p i a l a r v a e ,
moisture l e v e l and e f f i c i e n c y of conversion of both the n i t r o -
genous and the c a l o r i c contents of the food were d i r e c t l y r e l a t e d
(50). The same decreasing e f f i c i e n c y of conversion w i t h de^
c r e a s i n g moisture l e v e l was found f o r b l a c k cutworm l a r v a e (51).
The optimal moisture l e v e l f o r growth was q u i t e d i f f e r e n t from
that f o r e f f i c i e n c y of conversion, due to the i n t e r a c t i o n be-
tween e f f i c i e n c y and the a c t u a l amount of dry m a t e r i a l the l a r v a e
ingested. This k i n d of i n f o r m a t i o n has important i m p l i c a t i o n s
f o r the i n t e r a c t i o n s of n u t r i e n t s and water which can help ex-
p l a i n many of our observations of seasonal t r e n d s , herbivore
success on h o s t s , e t c . (22).
Concluding Remarks
Many aspects of n u t r i e n t - a l l e l o c h e m i c a l i n t e r a c t i o n s are

probably key f a c t o r s i n the s u i t a b i l i t y of a given p l a n t species
as a host f o r a p a r t i c u l a r i n s e c t . At b e s t , t h i s may be a l e s s
than o p t i m a l s i t u a t i o n f o r the i n s e c t , s i n c e even what appears
to be a s u s c e p t i b l e p l a n t i s l i k e l y to be f a i r l y w e l l defended
against i n s e c t and pathogen a t t a c k . I f the p l a n t contains the
e s s e n t i a l n u t r i e n t s f o r the i n s e c t , but the u t i l i z a t i o n of these
n u t r i e n t s i s blocked i n some way by a l l e l o c h e m i c s or by too much
or too l i t t l e water, then growth may be slowed. I f , due to be-
h a v i o r a l m o d i f i e r s , the i n s e c t w i l l not feed on the p l a n t , then
p l a n t n u t r i e n t s are not a v a i l a b l e to the i n s e c t .
Acknowledgement
P u b l i s h e d w i t h the approval of the D i r e c t o r of the Delaware

A g r i c u l t u r a l Experiment S t a t i o n as Miscellaneous Paper No. 988 ,
C o n t r i b u t i o n No. 519 of the Department of Entomology and
A p p l i e d Ecology, U n i v e r s i t y of Delaware, Newark, Delaware.

Research supported by U n i v e r s i t y of Delaware Research Foundation

grant, P l a n t Defense t o Insect A t t a c k and Hatch P r o j e c t 215, Host
P l a n t Resistance t o B l a c k Cutworms i n Corn.
I thank Meredith D. F i e l d and Robert E. Johnson f o r conduct-
i n g many of the experiments reported here.
Literature Cited
1. Beck, S. D. Ann. Rev. Entomol. 1965, 10, 207-232.

2. Beck, S. D.; Reese, J. C. Rec. Adv. Phytochem. 1976, 10, 41-
92.
3. Beck, S. D. "Insect and M i t e Nutrition," J. G. Rodriquez, Ed.
North-Holland P u b l . , Amsterdam, 1972; pp. 1-6.
4. Feeny, P. P. " C o e v o l u t i o n o f Animals and P l a n t s , " Gilbert,
L. E.; Raven, P. H Eds U n i v e r s i t y o f Texas P r e s s , A u s t i n ,
1975; pp. 3-19.
5. Beck, S. D. "Proceeding
ical C o n t r o l o f P l a n t I n s e c t s and Diseases," Maxwell, F. G.;
H a r r i s , F. A. Eds. U n i v e r s i t y Press of Mississippi, Jackson,
1974; pp. 290-311.
6. Whitworth, R. J.; Poston, F. L. Ann. Entomol. Soc. Am. 1979,
72:253-255.
7. Whitworth, R. J.; Poston, F. L. Proc. N. Centr. B r . -
Entomol. Soc. Am. 1979, 33:22.
8. Reese, J. C.; E n g l i s h , L. M.; Yonke, T. R.; Fairchild, M. L.
J. Econ. Entomol. 1972, 65:1047-1050.
9. Chan, B. G.; Waiss, A. C., Jr.; S t a n l e y , W. L.; Goodban, A.
E. J. Econ. Entomol. 1978, 71:366-368.
10. B r a t t s t e n , L. B. "Herbivores: T h e i r I n t e r a c t i o n w i t h
Secondary P l a n t M e t a b o l i t e s , " Rosenthal, G. A.; Janzen, D.
H. Eds. Academic P r e s s , New York, 1979; pp. 199-270.
11. Freedman, B.; Nowak, L. J.; Kwolek, W. F.; B e r r y , E. C.;
G u t h r i e , W. D. J. Econ. Entomol. 1981, 72:541-545.
12. Reese, J. C. "Current Topics in Insect Endocrinology and
Nutrition," Rodriquez, J. G., Ed. Plunum P r e s s , New York,
1981; pp. 317-336.
13. Shaver, T. N.; Parrott, W. L. J. Econ. Entomol. 1970, 63:
1802-1804.
14. Reese, J. C. "Herbivores: T h e i r I n t e r a c t i o n w i t h Secondary
P l a n t M e t a b o l i t i e s , " Rosenthal, G. A.; Janzen, D. H. Eds.
Academic P r e s s , New York, 1979; pp. 309-330.
15. L i e n e r , I . E., ed.; "Toxic C o n s t i t u e n t s o f P l a n t F o o d s t u f f s , "
Academic P r e s s , New York, 1980; p 502.
16. Mattson, F. H.; Volpenhein, R. A.; E r i c k s o n , B. A. J. Nutr.
1977, 107:1139-1146.
17. Bell, E. A. "Toxicants O c c u r r i n g N a t u r a l l y in Foods," 2nd
Ed., Nat. Acad. Sci., Washington, D. C., 1973; pp. 153-169.
18. Reese, J. C.; Beck, S. D. Ann. Entomol. Soc. Am. 1976, 69
68-72.

19. Rehr, S. S.; Janzen, D. H.; Feeny, P. P. Science 1973, 181:

81-82.
20. Janzen, D. H.; J u s t e r , H. B.; Bell, E. A. Phytochem. 1977,
16:223-227.
21. Strong, F. M. "Toxicants Occurring N a t u r a l l y in Foods," 2nd
Ed., Nat. Acad. Sci., Washington, D. C., 1973; pp. 1-5.
22. S c r i b e r , J. M.; Slansky, F., Jr. Ann. Rev. Entomol. 1981; 26:
183-211.
23. Rosenthal, G. A. Q. Rev. Biol. 1977, 52:155-178.
24. Vanderzant, E. S.; Chremos, J. H. Ann. Entomol. Soc. Am.
1971, 64:480-485.
25. Dahlman, D. L.; Rosenthal, G. A. J. Insect P h y s i o l . 1976, 22:
265-271.
26. I s o g a i , A.; Chang, C.; Murakoshi, S.; S u z u k i , A.; Tamura, S.
J . Agr. Chem. Soc. Japan 1973, 47:443-447.
27. Harry, P.; Dror, Y.
6:273-279.
28. Hegdeker, D. M. J. Econ. Entomol. 1970, 63:1950-1956.
29. Isoga, A.; Murakoshi, S.; S u z u k i , A.; Tamura, S. J. Agr.
Chem. Soc. Japan 1973, 47:449-453.
30. Feeny, P. P. J. Insect P h y s i o l . 1968, 14:805-817.
31. Feeny, P. P. Phytochem. 1969, 8:2119-2126.
32. Fox, L. R.; Macauley, B. J. Oecologia 1977, 29:146-162.
33. Bernays, E. A. Entomol. Exp. App. 1978, 24:244-253.
34. Bernays, E. A. E c o l . Entomol. 1981, 6:353-360.
35. Bernays, E. A.; Chamberlain, D. J.; Leather, E. M. J. Chem.
E c o l . 1981, 7:247-256.
36. Chan, B. G.; Waiss, A. C., Jr.; Lukefahr, M. J. Insect
P h y s i o l . 1978, 24:113-118.
37. Reese, J. C.; Chan, B. G.; Waiss, A. C., Jr. J. Chem. E c o l .
1982, I n P r e s s .
38. Ryan, C. A. Ann. Rev. P l a n t P h y s i o l . 1973, 24:173-196.
39. Ryan, C. A. "Herbivores: T h e i r I n t e r a c t i o n w i t h Secondary
P l a n t M e t a b o l i t e s , " Rosenthal, G. A.; Janzen, D. H. Eds.;
Academic P r e s s , 1979; pp. 599-618.
40. Ryan, C. A.; Green, T. R. Recent Adv. Phytochem. 1974, 8:
123-140.
41. B i r k , Y.; Applebaum, S. W. Enzymologia 1960, 22:318-326.
42. Su, H. C. F.; S p e i r s , R. D.; Mahany, P. G. J. Georgia
Entomol. Soc. 1974, 9:86-87.
43. L i e n e r , I. E. "Toxic C o n s t i t u e n t s of P l a n t F o o d s t u f f s , "
2nd ed., L i e n e r , I. E. Ed., Academic P r e s s , New York, 1980;
pp. 429-467.
44. Waldbauer, G. P. Entomol. Exp. Appl. 1962, 5:147-158.
45. Waldbauer, G. P. Entomol. Exp. A p p l . 1964, 7:257-269.
46. Waldbauer, G. P. Adv. Insect P h y s i o l . 1968, 5:229-288.
47. House, H. L. Canad. Entomol. 1965, 97:62-68.
48. Soo Hoo, C. F.; F r a e n k e l , G. J. Insect P h y s i o l . 1966a, 12:
693-709.

49. Soo Hoo, C. F.; F r a e n k e l , G. J. Insect P h y s i o l . 1966b, 12:

711-730.
50. S c r i b e r , J . M. Oecologia ( B e r l . ) 1977, 28:269-287.
51. Reese, J. C.; Beck, S. C. J. Insect P h y s i o l . 1978, 24:473-
479.

14
Chemical Basis for Host Plant Selection
JON BORDNER, DAVID A. DANEHOWER, and J. D. THACKER

North Carolina State University, Department of Chemistry, Raleigh, NC 27650
GEORGE G. KENNEDY, R. E. STINNER, and K A R E N G. WILSON
North Carolina State University, Department of Entomology, Raleigh, NC 27650
The host range of the tobacco hornworm (Manduca

sexta) is limited t
Solanaceae. I n a
ical b a s i s f o r the host p l a n t s e l e c t i o n process, we have
undertaken an examination of both hornworm p r e f e r r e d and
non-preferred members o f the Solanaceae. Our i n v e s t i g a -
t i o n s have shown this t c be a complex system i n v o l v i n g
the s u b t l e i n t e r a c t i o n between such b e h a v i o r a l modula-
t o r s as: (1) O v i p o s i t i o n a l s t i m u l a n t s ; (2) Feeding
s t i m u l a n t s and i m p r i n t e r s ; (3) A n t i - f e e d a n t s ; (4) Repel-
l a n t s ; (5) I n s e c t i c i d e s . The results o f these investi-
gations will be discussed.
In a d d i t i o n t o examining secondary p l a n t substances
and t h e i r r o l e in modulating i n s e c t behavior towards
host p l a n t s e l e c t i o n , we have been concerned w i t h the
r o l e of the major p l a n t metabolites i n i n s e c t physiology
after the s e l e c t i o n process has been completed. I t has
been observed that the Mexican bean b e e t l e s (Epilachna
varivestis) feeding upon soybean (Glycine max) i n the
southeastern regions o f the U.S. are l e s s well able t o
withstand the s t r e s s of d e s i c c a t i o n than do b e e t l e
l a r v a e feeding upon lima beans (Phaseolus lunatum). We
are p r e s e n t l y i n v e s t i g a t i n g the r o l e that d i e t a r y lipids
may play i n t h i s phenomena.
Through e v o l u t i o n a r y time, phytophagous i n s e c t s have tended

towards s p e c i a l i z a t i o n i n t h e i r food s e l e c t i o n process. The a b i l -
i t y o f i n s e c t s t o s e l e c t a s u i t a b l e h o s t p l a n t amid numerous
species of nonhosts i s widely b e l i e v e d t o be a t t r i b u t e d t o the
i n s e c t ' s a b i l i t y t o d i s c e r n the chemical d i f f e r e n c e between host
and non-host. However, the host p l a n t r e c o g n i t i o n process i s not
simply a matter of an i n s e c t r e c o g n i z i n g a p a r t i c u l a r kairomone
or avoiding a p a r t i c u l a r allomone. Rather the process i s the
r e s u l t of the i n t e g r a t i o n of numerous chemical and non-chemical
f a c t o r s , the complexity of which we are j u s t beginning t o appre-
0097-6156/83/0208-0245$06.25/0

c i a t e . Schoonhoven s u c c i n c t l y summarized contemporary thought on

t h i s subject i n t o 5 separate theorems (1) .
Numerous attempts to i s o l a t e and i d e n t i f y host s p e c i f i c
kairomones and allomones f o r a v a r i e t y of p l a n t and i n s e c t species
have been reported (2). However, only i n a few cases, p a r t i c u l a r -
l y where the i n s e c t predator i s monophagous, has a comprehensive
p i c t u r e of the chemical b a s i s f o r p l a n t - i n s e c t i n t e r a c t i o n begun
to emerge. I n the more general case of the oligophagous i n s e c t -
host p l a n t system there has been a great d e a l of research e f f o r t
expended to u n r a v e l the chemical r a t i o n a l e f o r host p l a n t s p e c i f i -
c i t y , but the r e s u l t s have been fragmentary and/or incomplete.
Although there remains l i t t l e doubt that indeed i n s e c t s respond to
chemical cues produced by hosts and non-hosts, the r e l a t i v e
p a u c i t y of hard chemical evidence c o n t r i b u t i n g to a comprehensive
p i c t u r e of i n s e c t - p l a n t i n t e r a c t i o n makes any g e n e r a l i z a t i o n s
tenuous and s p e c u l a t i v
to be made may be that
which i n s e c t s s e l e c t t h e i r r e s p e c t i v e host p l a n t s . As M. S. Blum
(3) pointed out i n h i s eloquent d i s c u s s i o n of the mechanisms
i n s e c t s use to d e t o x i f y , avoid or t o l e r a t e allomones, each i n s e c t
species and allomone must be regarded as a unique e n t i t y , i . e . i t
appears that the a v a i l a b l e methods f o r d e a l i n g w i t h t o x i n s are as
numerous as the i n s e c t species themselves. Indeed, the mechanisms
of host p l a n t r e c o g n i t i o n may be e q u a l l y unique and d i v e r s e .
I f the chemical r a t i o n a l e f o r i n s e c t - h o s t p l a n t i n t e r a c t i o n s
i s to be understood, then i t i s imperative t h a t a d d i t i o n a l i n s e c t -
host p l a n t systems be comprehensively examined w i t h the assurance
that any chemical inferences represent b i o l o g i c a l f a c t . Implicit-
l y , any chemical methodologies that r e s u l t from such i n v e s t i g a -
t i o n s should serve as g u i d e l i n e s f o r i n v e s t i g a t i o n s i n t o other
systems as w e l l . With these precepts i n mind, we have undertaken
a thorough i n v e s t i g a t i o n i n t o the chemical b a s i s f o r h o s t - p l a n t
s e l e c t i o n process of Manduca sexta which confines i t s host range
to s e l e c t e d members of the p l a n t f a m i l y Solanaceae.
The scope of our i n v e s t i g a t i o n s i n t o the M. sexta/Solanaceae
model system are best summarized i n Table I . With three excep-
t i o n s noted i n Table I , we are c u r r e n t l y i n v e s t i g a t i n g the
chemical f a c t o r s r e s p o n s i b l e f o r the observed behaviors i n both
hornworm p r e f e r r e d and non-preferred members of Solanaceae.
However, f o r the sake of b r e v i t y , we w i l l d i s c u s s only the a l l o -
mone i s o l a t e d from the w i l d tomato, Lycopersicon hirsutum f. g l a -
bratum and the kairomonal f a c t o r s found i n h o r s e n e t t l e , Solanum
c a r o l i n e n s e . (See Table I)
The i s o l a t i o n of the allomonal f a c t o r of L. hirsutum was
r e l a t i v e l y s t r a i g h t f o r w a r d and unremarkable as t h i s f a c t o r was
t y p i c a l of many organic molecules i n i t s s o l u b i l i t y p r o p e r t i e s
and was not apparently prone to thermal, p h o t o l y t i c or o x i d a t i v e
degradation. The b i o l o g i c a l l y a c t i v e p r i n c i p l e could be removed
from the l e a f surface w i t h a chloroform wash and could be s e l e c -
t i v e l y removed from unwanted pigments and waxes by r e d i s s o l v i n g

14. BORDNER ET A L . Chemical Basis for Host Plant Selection 247
Table I . The M. s e x t a - Solanaceae Model
Genus/Species Preference Behavior
Solanum c a r o l i n e n s e
(horsenettle)
Ii- hirsutum f. glabratum Non-preferred Repellant

( w i l d tomato)
L. esculentum Preferred Feeding, O v i p o s i t i o n

(domestic tomato) Orientation
Nicandra physaloides Non-preferred R e p e l l a n t (4)
N. physaloides v a r . Preferred
Albiflora Feeding
Datura stromonium Preferred

(Jimson weed) O r i e n t a t i o n (5)
J5. tuberosum Preferred

(potato) Feeding, O v i p o s i t i o n
N i c o t i a n a spp. Preferred
(tobacco) Feeding, O v i p o s i t i o n
P e t u n i a spp. Non-preferred
(petunia) R e p e l l a n t (6)
American Chemical
Society Library
1155 16th St. N.W.
ACS Symposium Series;Washington, D. C.
American Chemical 20036
Society: Washington, DC, 1983.
the d r i e d e x t r a c t i n e t h a n o l . C l a s s i c column chromatography using

a s t a t i o n a r y support of s i l i c i c a c i d and e l u t i o n w i t h a heptane to
chloroform g r a d i e n t a f f o r d e d a b i o l o g i c a l l y a c t i v e f r a c t i o n t h a t
was 85% pure by GLC a n a l y s i s . F i n a l p u r i f i c a t i o n was e f f e c t e d by
HPLC using a PAC bonded phase column (Whatman, Inc.) and e l u t i o n
w i t h a 5% gradient from heptane to methylene c h l o r i d e .
The s t r u c t u r e of t h i s allomonal f a c t o r was e s t a b l i s h e d as
2-tridecanone using c o n v e n t i o n a l s p e c t r a l data. Having i d e n t i f i e d
the compound apparently r e s p o n s i b l e f o r the observed t o x i c i t y , i t
then became necessary to q u a n t i t a t e the n a t u r a l l e v e l s of 2 - t r i d e -
canone and i d e a l l y to use a s i n g l e l e a f of JL. hirsutum i n the
assay procedure. F o r t u n a t e l y , 2-tridecanone was amenable to GC
methodologies. Using t h i s a n a l y s i s on r e s i s t a n t (L. hirsutum)
and s u s c e p t i b l e (_L. esculentum) species of tomato, i t was d e t e r -
mined t h a t f o l i a g e of L^. hirsutum contained an average of 44.6 yg
2
2-tridecanone per cm o
the c u l t i v a t e d tomato,
2
decanone per cm . This i n f o r m a t i o n , together w i t h the r e s u l t s of
i n v i t r o s t u d i e s showing the L C 5 0 value 2-tridecanone f o r 1st
2
i n s t a r M. s e x t a l a r v a e to be 17.1 yg/cm of t r e a t e d s u r f a c e , sug-
gests that 2-tridecanone i s the allomonal f a c t o r r e s p o n s i b l e f o r
the m o r t a l i t y of hornworm l a r v a e on L. hirsutum. A more compre-
hensive d i s c u s s i o n of t h i s research appears on references 1_ and JB.
Turning our a t t e n t i o n to kairomonal f a c t o r s , i t has been
observed that hornworm l a r v a e show a feeding preference f o r the
host p l a n t to which they were exposed during the 1st i n s t a r ( 9 ) .
Yamamoto developed a bioassay u t i l i z i n g a n e u t r a l s u b s t r a t e
(dandelion, Taraxacum o f f i c i n a l e , l e a f d i s k s ) which could be i n -
fused w i t h e x t r a c t s of the p r e f e r r e d host p l a n t i n order to
detect a feeding preference i n hornworm l a r v a e . A f t e r screening
numerous members of the Solanaceae, we s e l e c t e d Solanum
c a r o l i n e n s e ( h o r s e n e t t l e ) as i t appeared to i n i t i a t e the s t r o n g e s t
preference and have higher l e v e l s of the kairomonal f a c t o r i n l e a f
tissues.
I s o l a t i o n of the feeding f a c t o r f o r M. s e x t a was a f a r more
d i f f i c u l t task. Whereas 2-tridecanone i s a simple, s t a b l e mole-
c u l e s o l u b l e i n o r g a n i c s o l v e n t s , the feeding f a c t o r i s water
s o l u b l e , occurs at t r a c e l e v e l s i n p l a n t t i s s u e , and i s e a s i l y
hydrolyzed under m i l d a l k a l i n e or a c i d i c c o n d i t i o n s w i t h subse-
quent l o s s of b i o l o g i c a l a c t i v i t y . The i s o l a t i o n of such a com-
pound was a formidable o b s t a c l e r e q u i r i n g a departure from the more
c l a s s i c a l approach of h y d r o l y s i s or chemical d e r i v a t i z a t i o n f o l l o w -
ed by i s o l a t i o n of the l i p o p h i l i c product. The n e c e s s i t y that pure
substance be i s o l a t e d w i t h r e t e n t i o n of b i o l o g i c a l a c t i v i t y r e -
q u i r e d some b a s i c research i n modern s e p a r a t i o n techniques to
develop a s u i t a b l y m i l d i s o l a t i o n s t r a t e g y .
The i s o l a t i o n procedure t h a t we have developed i n v o l v e s ex-
t r a c t i n g the l e a f t i s s u e w i t h b o i l i n g , d e i o n i z e d water, p r e c i p i -
t a t i o n of p h e n o l i c components w i t h l e a d acetate (PbOAc2'3^0) and

14. BORDNER ET AL. Chemical Basis for Host Plant Selection 249
e x t r a c t i o n of the c l a r i f i e d aqueous l a y e r w i t h n-butanol. The

n-butanol i s then evaporated t o dryness and the r e s i d u e r e d i s -
solved i n d e i o n i z e d H 0 and the sample d e i o n i z e d through a c a t i o n
2
4-
exchange column (Dowex SOW X-8, H ) u s i n g d e i o n i z e d water as the
eluent. The column e f f l u e n t i s then reduced i n volume and passed
through a column packed w i t h Sephadex G-15 (Pharmacia, Inc.)
and e l u t e d w i t h d e i o n i z e d water. S e v e r a l f r a c t i o n s are obtained
w i t h the a c t i v i t y confined t o the f r a c t i o n e l u t i n g a t the v o i d
volume. HPLC a n a l y s i s of t h i s f r a c t i o n r e v e a l e d s e v e r a l compo-
nents (Figure 1) so a p r e p a r a t i v e s c a l e medium r e s o l u t i o n reversed
phase column was employed to f r a c t i o n a t e the mixture f u r t h e r . Two
a c t i v e f e e d i n g f r a c t i o n s were obtained and each was analyzed by
HPLC. The f i r s t f r a c t i o n corresponded t o the peak a t 9.3 minutes
and the second f r a c t i o n corresponded t o the peaks between 9.7 and
10.6 minutes. F i n a l p u r i f i c a t i o n of the peak e l u t i n g a t 9.3
minutes was accomplishe
No attempts have been mad
e l u t i n g between 9.7 and 10.6 minutes.
1
The 400.13 MHz H-nmr (10) was obtained and i s presented i n
Figure 2. Although t h i s spectrum i s complex, c e r t a i n s t r u c t u r a l
f e a t u r e s a r e d i s c e r n a b l e . The l o s s of s i g n a l s i n the r e g i o n of
40-55 ppm upon the a d d i t i o n of D2O suggests the presence of a
g l y c o s i d e . Other s t r u c t u r a l f e a t u r e s i n c l u d e a r o m a t i c i t y (<10%
of the t o t a l i n t e g r a t e d proton a r e a ) , o l e f i n i c protons and a
methylene, methyl p a t t e r n t y p i c a l of a h i g h l y s u b s t i t u t e d fused
3
r i n g system. The C-nmr (100.58 MHz) (10) i s somewhat more
i n f o r m a t i v e and the s a l i e n t features are presented i n Table I I .
F u r t h e r , i t should be noted that molecular weight e s t i m a t i o n from
the 54 observed carbon s i g n a l s suggests a molecular weight of
1,000-1,200 a.m.u.
13
Table I I . C-nmr of Feeding Stimulant (100.58 MHz)
Absorbance(ppm) S t r u c t u r a l moiety
197 b e n z y l i c ketone
164 enol ether
130 (7 s i g n a l s ) a r o m a t i c i t y ( 6 ) , e n o l ether (1)
100 (3 s i g n a l s ) anomeric carbons of sugar
residues
80-60 carbanols of a carbohydrate
<60 methylene and methyl of a
steroid
We had e a r l i e r observed that the b i o l o g i c a l l y a c t i v e f r a c -

t i o n s a f t e r HPLC r e a d i l y hydrolyzed t o g i v e a chloroform s o l u b l e
product. 100 mg of the combined f r a c t i o n s (HPLC peaks 9.3-10.6)

| • 1 1
* • * * * * ' * * A
• |
0 10 20 30
MINUTES
Figure 1. Analytical HPLC of the phagostimulants. Conditions: Partisil 10 ODS-2

(4.6 x 250 mm) column; mobile phase, 100% H O to 100% CH CN, 2 mL/min;
g S
sample, 10 pL 10 mg/mL; ambient temperature; detector, SP8310 (Spectra-

Physics), 254 nm, 0.16 A UFS, 10 MVFS.

14. BORDNER ET AL. Chemical Basis for Host Plant Selection
„ CD

were hydrolyzed under a c i d i c c o n d i t i o n s according to the procedure

described by M. E. W a l l (11) i n an attempt to see i f the aglycones
of the s e v e r a l b i o l o g i c a l l y a c t i v e components were the same and to
i s o l a t e a r e a d i l y i d e n t i f i a b l e product. A s i n g l e product a f t e r
h y d r o l y s i s was obtained! The p e r t i n e n t IR and UV data before and
a f t e r h y d r o l y s i s are presented i n Table I I I .
Table I I I . IR and UV Data Before and A f t e r

Acid Hydrolysis
Feeding F a c t o r s Before H y d r o l y s i s
UV(EtOH) IR(Thin Film)
Amax 245nm
280nm 3400 cm ( b r )X
H y d r o l y s i s Product
UV(EtOH) IR(CHCl^)
Xmax 245nm Vmax 1690, 1715 &

1
280nm 1725 cm"
-1
340nm 2820 c m
1
3400 cm- (br)
Upon examination of the 400.13 MHz ^-nmr c e r t a i n s p e c t r a l s i m i l a r -

i t i e s w i t h nicandrenone, a w i t h a n o l i d e i s o l a t e d from Nicandra
physaloides ( 4 ) , became obvious. A comparison of these two s p e c t r a
i s presented i n F i g u r e 3. The dotted l i n e s are drawn to l i n e up
regions of equivalent chemical s h i f t s i n the two s p e c t r a . Numerous
s i m i l a r i t i e s are seen, however two major d i f f e r e n c e s should be
d i s c u s s e d . The f i r s t of these i s that the protons a t C-16 and
C-18 a r e s h i f t e d 0.5 ppm downfield w h i l e the C-15 proton remains
unchanged as does the s p l i t t i n g p a t t e r n of the aromatic m u l t i p l e t .
This o b s e r v a t i o n suggests t h a t C-12 i s a c a r b o n y l . The other
major d i f f e r e n c e i s the l o s s of the l a r g e m u l t i p l e t centered
around 3 ppm i n nicandrenone. This m u l t i p l e t c o n s i s t s p r i m a r i l y
of h y d r o x y l and a l l y l i c protons. Deuterium exchange has removed
the h y d r o x y l s i g n a l s i n the h y d r o l y s i s product and the broad hump
remaining may be due t o no more than 2 a l l y l i c proton. To account
f o r these o b s e r v a t i o n s , we propose the m o d i f i c a t i o n s of nicandre-
none shown i n F i g u r e 4. Beginning w i t h r i n g A, the enol-ether
allows f o r the h y d r o l y t i c l a b i l i t y of the feeding s t i m u l a n t , the
13
assignment of C - l a t 164.5 ppm i n the C-nmr, assignment of C-2
13
as the seventh "aromatic" carbon i n the 130 ppm r e g i o n of C-nmr,
-1
the appearance of a ketone a f t e r h y d r o l y s i s (IR, 1715 c m ) , and a
convenient p o i n t of attachment f o r the g l y c o s y l r e s i d u e s . I n the

14. BORDNER E T AL. Chemical Basis for Host Plant Selection 253
Si
s: s
y *
is
tes
g 2υ
Η
Ρ
^ ^»
e§

Figure 4. Comparison of nicandrenone structure (top) to the proposed structure

of the feeding stimulant (bottom). R = glycosyl.

13
C r i n g , C-12 i s modified to a b e n z y l i c ketone as suggested by C -
1
nmr (197 ppm) and IR (1690 cm" ). The s u b s t i t u t i o n p a t t e r n of the
aromatic D r i n g i s unchanged, however the presence of the 12-oxo
carbon w i l l s h i f t the protons a t C-18 and C-16 p a t t e r n downfield
i n the ^-nmr. The C-21 methyl of the feeding s t i m u l a n t i s
assigned on the b a s i s of the C-nmr (13.8 ppm) and the ^^nmr
(1.5 ppm, d) which compares f a v o r a b l y t o that of nicandrenone (14
ppm and 1.25 ppm, d r e s p e c t i v e l y ) . The s t r u c t u r e of the E r i n g i s
proposed p r i m a r i l y t o account f o r the appearance of an aldehyde
upon a c i d h y d r o l y s i s of the feeding s t i m u l a n t . To be sure, the
s t r u c t u r e proposed i n F i g u r e 4 i s only t e n t a t i v e and experiments
i n v o l v i n g 2 dimensional J spectroscopy, double i r r a d i a t i o n , and
f a s t atom bombardment mass spectroscopy are c u r r e n t l y underway to
confirm t h i s s t r u c t u r e .
The i s o l a t i o n of the o v i p o s i t i o n a l f a c t o r f o r a d u l t g r a v i d
female hornworms (M. sexta
as f a r along as the tw
s e v e r a l i n t e r e s t i n g chemical and b i o l o g i c a l p o i n t s have been r e -
vealed and a r e worthy of d i s c u s s i o n .
The i s o l a t i o n procedure that we have developed i n v o l v e s an
i n i t i a l hot water e x t r a c t i o n of the d r i e d leaves of e i t h e r horse-
n e t t l e (Solanum c a r o l i n e n s e ) , tomato (L.esculentum), potato (S.
tuberosum) or tobacco ( N i c o t i a n a spp.). D i f f e r e n t i a l s o l u b i l i t y
u t i l i z i n g a n-butanol-water p a r t i t i o n followed by decreasing the
p o l a r i t y of the n-butanol l a y e r w i t h the a d d i t i o n of d i e t h y l ether
to a 50% ether-butanol mix and back e x t r a c t i o n w i t h water i s an
e f f e c t i v e cleanup procedure. The b i o l o g i c a l l y a c t i v e aqueous
l a y e r has been analyzed by m u l t i p l e t h i n l a y e r chromatographic
procedures and a r e l a t i v e l y c l e a n o v i p o s i t i o n a l l y a c t i v e f r a c t i o n
has been obtained. The l a r v a l feeding s t i m u l a n t i s a l s o r e s o l v e d
from the o v i p o s i t i o n a l s t i m u l a n t at t h i s stage and t h e r e f o r e
proves that the o v i p o s i t i o n and feeding f a c t o r s are two d i f f e r e n t
compounds. HPLC a n a l y s i s of the o v i p o s i t i o n a l f r a c t i o n s obtained
from the v a r i o u s members of the Solanaceae shows t h a t each of
these f r a c t i o n s a r e 90% pure. However, each have s l i g h t l y d i f -
f e r e n t r e t e n t i o n times and are t h e r e f o r e d i f f e r e n t . Chemical
a n a l y s i s of the o v i p o s i t i o n a l f a c t o r s obtained from these
d i f f e r e n t sources suggests that i t i s a n i t r o g e n c o n t a i n i n g pheno-
l i c g l y c o s i d e (12, 13). The r e s u l t s of the chemical and s p e c t r a l
evidences are summarized i n Table IV.
F u r t h e r , the phagostimulative f r a c t i o n was analyzed v i a HPLC
and were chromatographically s i m i l a r to the phagostimulative sub-
stances described e a r l i e r . The chemical and s p e c t r a l data ob-
t a i n e d from the phagostimulative f r a c t i o n s of these v a r i o u s mem-
bers of the Solanaceae a l s o suggests a g l y c o s i d i c s t e r o i d .
Although the o v i p o s i t i o n a l s t i m u l a n t and the phagostimulant
are necessary t o i l l i c i t t h e i r r e s p e c t i v e responses, they may not
be s u f f i c i e n t to account f o r h o s t - p l a n t s e l e c t i o n . I n the case of
Nicandra p h y s a l o i d e s , a member of the Solanaceae not p r e f e r r e d as
a host p l a n t by M. s e x t a , we have shown that aqueous e x t r a c t s

c o n t a i n the f e e d i n g f a c t o r and hornworm l a r v a e w i l l feed on these

e x t r a c t s once nicandrenone has been removed. F u r t h e r , we have
Table IV. Oviposition Factor - Preliminary Characterization
Functionality Chemical Tests S p e c t r a l Data
Phenol (Yes) Positive FeCl & 3

IR: 3400-3200 cm_^,
K Fe(CN) , V a n i l l i n ,
3 6 1650-1590 cm
Ammonia Vapor, UV: Xmax 320, 255 nm
Bromine water, Xmax(base) 328,
FeCl3, P r e c i p i t a t i o n 260 nm
w i t h Pb(0Ac) *3H 0
2 2
X
NMR: H 6.9-8.0 ppm
Carbohydrate (Yes) Positive Molisch, NMR: *H 3.4-4.2 ppm

13
Phenol/H S0.o C 55-77 ppm
2 4
Amine (Yes) Positive Ninhydrin IR: 3400-3200 cm" 1
P o s i t i v e Dragendorff
Carbonyl (No) Negative 2,4 DNPH IR: No peak 1800-

1
1700 c n f
13
NMR: C No peak
150-200 ppm
r e c e n t l y r e c e i v e d a v a r i e t y of N. physaloides (var. a l b i f l o r a )
which has been shown to l a c k nicandrenone (14) and hornworm l a r v a e
w i l l feed upon t h i s v a r i e t y . Obviously, nicandrenone i s a phago-
d e t e r r e n t and i t s presence deters hornworm l a r v a e i n s p i t e of the
presence of the feeding s t i m u l a n t .
Jermy (15) has emphasized the importance of allomones i n the
host p l a n t s e l e c t i o n process. Although the o v i p o s i t i o n a l and
phagostimulative kairomones do not appear to be s u f f i c i e n t to
account f o r host s p e c i f i c i t y by M. sexta i n the host p l a n t s e l e c -
t i o n process, the mere avoidance of allomones does not appear to
be s u f f i c i e n t e i t h e r . Rather, the presence of a d e t e c t a b l e
allomone i s s u f f i c i e n t to account f o r n o n - s e l e c t i o n of a p o t e n t i a l
host p l a n t . For example, g i v e n a choice between L. esculentum
and any other s u i t a b l e host p l a n t M. s e x t a moths s e l e c t L. excu-
1enturn (16). No allomones are i n v o l v e d ! To account f o r t h i s
preference, the presence of v o l a t i l e o r i e n t a t i o n f a c t o r ( s ) may be
i n v o l v e d . I n f a c t , Morgan and Lyon (5) i s o l a t e d amyl s a l i c y l a t e
from the host p l a n t Datura stromonium as an o r i e n t a t i o n f a c t o r f o r
g r a v i d female moths. We have a l s o shown that an o r i e n t a t i o n
f a c t o r i s present i n the steam d i s t i l l a t e of L,. esculentum l e a v e s .
I t i s i n t e r e s t i n g to note that the feeding s t i m u l a n t i s the
g l y c o s i d e of a w i t h a n o l i d e , a known c l a s s of allomones unique to

14. BORDNER E T AL. Chemical Basis for Host Plant Selection 257
the Solanaceae (17). This o b s e r v a t i o n tends t o support the view

that s p e c i a l i z a t i o n i n host p l a n t s e l e c t i o n i s the r e s u l t of the
evolved a b i l i t y of an i n s e c t species t o t o l e r a t e o r d e t o x i f y t o x i c
f a c t o r s and i n some cases u t i l i z e these compounds as kairomones.
T
Although the o v i p o s i t i o n a l f a c t o r i s u b i q u i t o u s w i t h i n M. s e x t a s
host range, i t has not been determined whether o r not these com-
pounds are unique t o the Solanaceae, i f they occur i n non-prefer-
red members of the Solanaceae as w e l l , o r i f they represent
another example of an allomone turned kairomone.
Another i n s e c t - h o s t p l a n t system under i n v e s t i g a t i o n i n our
l a b o r a t o r i e s i s that of the oligophagous Mexican bean b e e t l e
(Epilachna v a r i v e s t i s , Mulsant) and i t s predominant hosts i n the
c o n t i n e n t a l United States - snap beans (Phaseolus v u l g a r i s ) , l i m a
beans (P. lunatum), and soybeans (Glycine max) - a l l of which are
members of the f a m i l y Leguminosae> Our i n t e r e s t i n the bean
b e e t l e focuses on a f r e q u e n t l
i t s s e n s i t i v i t y to climati
goes beyond the r o l e of secondary p l a n t m e t a b o l i t e s i n modulating
i n s e c t behavior during the host p l a n t s e l e c t i o n process. Rather,
we a r e examining the r o l e of major p l a n t m e t a b o l i t e s i n i n s e c t
physiology once the s e l e c t i o n process has been completed.
The supposed area of o r i g i n f o r the Mexican bean b e e t l e (MBB)
i s the h i g h p l a t e a u r e g i o n of C e n t r a l America (18) - an area
c h a r a c t e r i z e d by d a i l y r a i n s and moderate temperatures d u r i n g the
growing season. This i s presumably the c l i m a t e to which t h i s
i n s e c t adapted i n i t s e v o l u t i o n a r y past. E a r l y i n v e s t i g a t o r s
noted that p o p u l a t i o n s of t h i s b e e t l e were l i m i t e d t o i r r i g a t e d
Phaseolus f i e l d s i n the southwestern United States (19).
As the MBB spread i n t o the southern United S t a t e s , f i e l d ob-
s e r v a t i o n s confirmed the s e n s i t i v i t y of t h i s i n s e c t t o h i g h
temperatures and low h u m i d i t i e s (20, 21). Q u a l i t a t i v e l a b o r a t o r y
s t u d i e s s u c c e s s f u l l y s u b s t a n t i a t e d the c o n c l u s i o n s of the e a r l i e r
f i e l d i n v e s t i g a t o r s (22).
A l l of t h i s e a r l y work was concerned w i t h s u r v i v a l on pre-
f e r r e d hosts i n the genus Phaseolus. The i n t r o d u c t i o n and r a p i d
expansion of soybean p r o d u c t i o n c o i n c i d e d w i t h the MBB's spread
to the southeastern United S t a t e s , and t h i s important a g r i c u l t u r a l
crop u n f o r t u n a t e l y became a secondary host f o r t h i s i n s e c t .
Although soybeans are only a secondary host ( i . e . given a c h o i c e ,
the i n s e c t p r e f e r s Phaseolus s p e c i e s ) , the p o t e n t i a l f o r adapta-
t i o n of the MBB when presented l a r g e monocultures of G l y c i n e
( e s s e n t i a l l y a ho-choice s i t u a t i o n ) has not been overlooked by
entomologists. S e v e r a l s t u d i e s i n the Department of "Entomology
at NCSU have demonstrated an exaggerated s e n s i t i v i t y of t h i s
i n s e c t t o hot, dry c o n d i t i o n s when f e e d i n g on soybean h o s t s . (23,
24, 25) Of p a r t i c u l a r p e r t i n e n c e t o our work i n the Department of
Chemistry were K. G. Wilson's f i n d i n g s (26) that t r a n s p i r a t i o n a l
water l o s s i n MBB l a r v a e i s dependent on both r e a r i n g temperature
and h o s t p l a n t . Those l a r v a e reared on soy hosts d i s p l a y e d i n -
creased t r a n s p i r a t i o n , e s p e c i a l l y a t e l e v a t e d temperatures. Our

l a b o r a t o r i e s are c u r r e n t l y i n v o l v e d i n t e s t i n g the hypothesis that

these d i f f e r e n c e s are r e l a t e d to the c u t i c l e chemistry of t h i s
insect.
C u t i c u l a r p e r m e a b i l i t y i n i n s e c t s i s c o r r e l a t e d w i t h a number
of f a c t o r s i n c l u d i n g growth stage of the i n s e c t , surface to volume
r a t i o s , the degree of tanning i n the c u t i c l e , c o n d i t i o n i n g
temperatures, and c u t i c u l a r l i p i d s (27, 28, 29). The c u t i c l e of
an i n s e c t i s a complex m a t r i x of c h i t i n (a N - a c e t y l glucose amine
polymer i n the form of overlapping f i b r i l s ) , a c h i t i n - p r o t e i n
complex known as the e x o c u t i c l e , a t h i n l i p o p r o t e i n m a t r i x
( c u t i c u l i n ) and an outermost l a y e r of wax - the e p i c u t i c u l a r l i p i d
(Figure 5). The p r o d u c t i o n and composition of t h i s s t r u c t u r e i s
regulated by the epidermal c e l l s . Entomologists and chemists
concerned w i t h the s t r u c t u r e and f u n c t i o n of i n s e c t c u t i c l e have
demonstrated that the e p i c u t i c u l a r wax l a y e r of an i n s e c t i s of
p r i n c i p l e importance i
i n s e c t s . (30, 31, 32) I
i n s e c t l i p i d s to r e a r i n g temperature (28, 33 9 34) i n d i c a t e d that
greater s a t u r a t i o n of the f a t t y acids e i t h e r i n t h e i r f r e e form
or as part of a l a r g e r molecule (wax e s t e r s , g l y c e r i d e s , etc.)
was c o r r e l a t e d w i t h those i n s e c t s reared at higher temperatures.
Few s t u d i e s have attempted to c o r r e l a t e d i e t a r y l i p i d composition
w i t h the i n s e c t ' s c u t i c u l a r l i p i d s however. This r e l a t i o n s h i p was
one which i n t r i g u e d us, e s p e c i a l l y s i n c e R. F. Wilson (35) of the
Department of Crop Science had performed analyses of soy l e a f
l i p i d s which showed that a high p r o p o r t i o n of these f a t s were
unsaturated. The c u l m i n a t i o n of our i n t e r e s t s i n bean b e e t l e
clim at e s t r e s s , the r o l e of c u t i c u l a r l i p i d s i n i n s e c t water
balance, and the p o s s i b i l i t y that t h i s i n s e c t ' s s e n s i t i v i t y to
such s t r e s s e s might be r e l a t e d to the l i p i d composition of i t s
host l e d us to undertake a d e t a i l e d a n a l y s i s of Mexican bean
b e e t l e c u t i c u l a r l i p i d s as they r e l a t e to h o s t p l a n t , l i f e stage,
and r e a r i n g temperature. In a d d i t i o n , analyses of p l a n t f o l i a r
l i p i d s have a l s o been c a r r i e d out.
Studies of changes as they depended on l i f e stage ( l a r v a e ,
pupae, and a d u l t s ) are being c a r r i e d out on f i e l d i n s e c t s reared
on l i m a , snap, and soybean hosts. The experiments i n v o l v i n g
r e a r i n g temperature dependent changes are underway using l a r v a e
reared on limas or soybeans. Temperatures of r e a r i n g were 23, 27,
and 32°C, which correspond to the values used i n Wilson's water
l o s s experiments. G r a v i m e t r i c , t h i n l a y e r , and gas chromatograph-
i c methodologies are employed i n these analyses. Thin l a y e r
densitometry (36, 37, 38) of the whole l i p i d e x t r a c t has given us
composition by l i p i d c l a s s e s . These analyses i n d i c a t e d that a
m a j o r i t y of the l i p i d found i n l a r v a l c u t i c l e (65-70%) i s composed
of f a t t y a c i d c o n t a i n i n g substances. Major c l a s s e s i n c l u d e hydro-
carbon (the major f r a c t i o n of a d u l t c u t i c u l a r l i p i d ) , wax e s t e r s
(predominant c l a s s of l a r v a e ) , f r e e f a t t y a c i d s , t r i g l y c e r i d e s ,
and s t e r o l s . Gas chromatographic a n a l y s i s by i n d i v i d u a l c l a s s was
deemed impossible due to time and q u a n t i t y c o n s i d e r a t i o n s , there-

Figure 5. The cuticle of an insect.

f o r e the a n a l y s i s was c a r r i e d out on hydrolyzed samples which gave

hydrocarbon, f a t t y a c i d , and a l c o h o l f r a c t i o n s . These analyses
are based i n part on methodologies employed i n previous c u t i c u l a r
l i p i d analyses (39, 40 and references t h e r e i n ) .
Although these analyses are s t i l l underway i n our l a b o r a t o r -
i e s , some p r e l i m i n a r y i n f o r m a t i o n i s a v a i l a b l e . Gas chromato-
graphy of the l i m a bean l e a f l i p i d s , i n comparison w i t h the soy
a n a l y s i s c a r r i e d out by Dr. R. F. Wilson shows that both c o n t a i n
a high p r o p o r t i o n of unsaturated f a t s (Table V ) . Both of these
analyses were c a r r i e d out using packed column GC methodologies.
C a p i l l a r y column a n a l y s i s of the l a r v a l l i p i d s , which provided
considerably more r e s o l u t i o n than i n the p l a n t l i p i d s t u d i e s ,
i n d i c a t e a r a t h e r complicated composition (Table V I ) . The degree
of u n s a t u r a t i o n i s higher i n soy reared l a r v a e , a p o t e n t i a l l y
p o s i t i v e c o r r e l a t i o n w i t h the d i f f e r e n c e s i n c u t i c u l a r permeabil-
ity. In p a r t i c u l a r , th
higher p r o p o r t i o n of s t e a r i
b e e t l e s . S t e a r i c a c i d i s the major f r a c t i o n i n both cases.
Comparison w i t h the l e a f l i p i d data shows that s t e a r i c a c i d i s a
minor component i n both p l a n t s , but i t nevertheless occurs i n
approximately the same r a t i o as i n the i n s e c t ' s c u t i c l e . Any
c o r r e l a t i o n of p l a n t l i p i d and i n s e c t l i p i d composition must,
however, await completion of a l l analyses. Of pertinence w i l l be
comparisons of the l i p i d composition of the c u t i c l e from those
i n s e c t s reared under the c o n t r o l l e d temperature c o n d i t i o n s as w e l l
as a r e - a n a l y s i s of p l a n t l i p i d using the r e c e n t l y acquired c a p i l -
l a r y system w i t h i t s much g r e a t e r r e s o l v i n g c a p a b i l i t i e s .
I t has become i n c r e a s i n g l y obvious to us that r e g u l a t i o n of
water balance i n i n s e c t s i s one of the keys to the success of
these animals i n t h e i r adaptation to such a wide v a r i e t y of h a b i -
t a t s . The c o m p l e x i t i e s i n v o l v e d i n undertaking s t u d i e s of such
systems may r e q u i r e expansion of our i n t e r d i s c i p l i n a r y approach
so as to i n c l u d e such d i v e r s e f i e l d s as b i o p h y s i c s , ecology, and
c e l l b i o l o g y . The subject i s a f a s c i n a t i n g one, perhaps a l l the
more so due to i t s complexity, and promises to open up new areas
i n which i n t e r d i s c i p l i n a r y research w i l l p l a y a r o l e .
I t i s beginning to appear that the host p l a n t s e l e c t i o n pro-
cess i s indeed h i g h l y complex and i n v o l v e s the s u b t l e i n t e r p l a y
of o r i e n t a t i o n f a c t o r s , o v i p o s i t i o n a l f a c t o r s , feeding f a c t o r s and
allomonal f a c t o r s as w e l l as v i s u a l and t a c t i l e s t i m u l i , e n v i r o n -
mental f a c t o r s and plant n u t r i e n t s . I t has been the goal of our
research e f f o r t s to provide a c l e a r e r understanding of the dynamic
e q u i l i b r i u m that e x i s t s between an i n s e c t and i t s host p l a n t s .
F o r t u n a t e l y , the l a s t decade has brought f o r t h the technologies
necessary to i s o l a t e and i d e n t i f y complex bio-molecules and sub-
sequently analyze b i o l o g i c a l systems i n a manner that was p r e v i o u s -
l y i m p o s s i b l e . Such h o l i s t i c i n v e s t i g a t i o n s promise to y i e l d
methods of c o n t r o l l i n g i n s e c t pests on v a l u a b l e crop p l a n t s . These
methods may then be i n t e g r a t e d i n t o a t r u l y b i o - r a t i o n a l approach
to pest management.

Table V. A Comparison of P l a n t Leaf F a t t y Acids
Fatty Acid G l y c i n e max (35) Phaseolus lunatum

(Soybeans - Bragg) (Lima beans-Henderson)
12:0 lauric
14:0 m y r i s t i c 1.6
16:0 p a l m i t i c 14.5 ± 0.7 11.0 ± 0.2
16:1 p a l m i t o l e i c 2.8 ± 0.3 3.1 ± 0.3
16:2 hexadecadienoic trace 0.4 ± 0.2
18:0 stearic 3.4 ± 0.5 6.4 ± 0.3
18:1 oleic 2.3 ± 0.3 2.3 ± 0.1
18:2 l i n o l e i c 13.1 ± 1.1 10.1 ± 0.4
18:3 linolenic 63.9 ± 1.6 63.7 ± 1.1
20:0 a r a c h i d i c 0.4 ± 0.1
22:0 behenic 0.6 ± 0.1
22:1 e r u c i c 0.3 ± 0.1
T o t a l Unsaturated 82.1% 79.9%
A n a l y s i s done on a Carbowax 20M column,

2 meters x 1/4" O.D., 30 ml/min, N , T . 2 cQ
120 + 220 a t 5°/min. P e r k i n Elmer Sigma

3B w i t h FID.

Table V I . Comparison of Larvae C u t i c l e F a t t y Acids
Fatty Acid max _P. lunatum

Reared Larvae Reared Larvae
12: 0 l a u r i c trace trace

14: 0 m y r i s t i c 5.97 + 0.20 1.44 ± 0.00
16: 0 p a l m i t i c 1.40 ± 0.03 0.58 + 0.09
16: 1 p a l m i t o l e i c trace trace
16: 2 hexadecadienoic
18: 0 s t e a r i c
18: 1 o l e i c
18: 2 l i n o l e i c 10.71 ± 0.16 19.57 ± 1.44
18: 3 l i n o l e n i c 8.70 ± 0.30 8.36 + 0.19
20: 0 a r a c h i d i c 8.56 ± 0.71 4.63 + 0.31
20 1 e i c o s e n o i c 3.07 + 0.10 0.67 + 0.11
20: 2 e i c o s a d i e n o i c 1.80 + 0.21 0.47 + 0.03
22 0 behenic 1.91 ± 0.28 1.90 ± 0.27
22: 1 erucate 2.66 + 0.35 0.31 + 0.04
22 2 docosodienoic 2.83 + 0.28 1.15 + 0.24
24 0 l i g n o c e r i c 0.65 + 0.08 0.99 + 0.09
24 1 nervonic 1.91 + 0.54 0.26 + 0.00
24 2 tetradodecadionoic 2.57 + 0.05 1.26 ± 0.01
26 0 hexadodecanoic 1.52 ± 0.26 0.82 ± 0.00
% Unsaturated 56. 53 48.16
Analyses performed on a 10 meter SP2100

c a p i l l a r y column, 50:1 s p l i t r a t i o , l i n e a r
gas v e l o c i t y 28.5 cm/sec, T 120
c q 1 220°C
at 5°/min, P e r k i n Elmer Sigma 3B w/FID
i n t e r f a c e d to Spectra Physics SP4000 Data
System.

Acknowledgements
The authors wish t o g r a t e f u l l y acknowledge the f i n a n c i a l

support of t h i s p r o j e c t by the Frasch Foundation and the United
States Department of A g r i c u l t u r e (SEA/CRGO), NSF Grant //DEB
7822738.
Literature Cited
1. Schoonhoven, Louis M. " O f f p r i n t s from Semiochemicals: T h e i r

Role in Pest C o n t r o l " ; Nordlund, Donald A., Ed.; John Wiley
and Sons, Inc.:New York, 1981; p. 31.
2. Hedin, P a u l A.; Maxwell, Fowden G.; J e n k i n s , Johnie N.
"Proceedings of the Summer Institute on Biological C o n t r o l
of P l a n t I n s e c t s and Diseases" Maxwell Fowde Harris
F.A., Ed.; U n i v e r s i t
p. 494.
3. Blum, M.S., Chapter _ in this book.
4. Bates, R.B.; E c k e r t , D.J. J. Amer. Chem. Soc., 1972, 94,
8258.
5. Morgan, A.C.; Lyon, S.C. J. Econ. Ent. 1928, 21, 189-191.
6. Yamamoto, R.T.; F r a e n k e l , G. Ann. Ent. Soc. 1960a, 53,
499-503.
7. W i l l i a m s , W.G.; Kennedy, George G.; Yamamoto, Robert T.;
Thacker, J.D.; Bordner, Jon Science 1980, 207, 888-889.
8. Kennedy, G.G.; Yamamoto, R.T.; Dimock, M.B.; W i l l i a m s , W.G.;
Bordner, J. J. Chem. E c o l . 1981, 7, 707-716.
9. Yamamoto, R.T. J. Insect P h y s i o l . 1974, 20, 641-650.
10. Ellis, P.D.; Inners, R.R. South C a r o l i n a Magnetic Resonance
Laboratory, Department of Chemistry, U n i v e r s i t y of South
C a r o l i n a , Columbia, S.C. NSF Grant No. CHE 78-18723.
11. W a l l , Monroe E.; K r i d e r , Merle M.; Rothman, Edward S.;
Eddy, C.Roland J . Biol. Chem. 1952, 198, 533-543.
12. Coco, Wm. J. M.S. T h e s i s , North C a r o l i n a State U n i v e r s i t y ,
R a l e i g h , N.C. 1981.
13. F r a e n k e l , G.; Nayer, J.; Nalbandov, O.; Yamamoto, R.T.
Proc. I n t e r n . Congr. Entomol. 11th Vienna 3, 1960, p. 122.
14. Tishbee, Arye; K i r s o n , Isaac J. Chromatog. 1980, 195,
425-430.
15. Jermy, T. Entomol. Exp. A p p l . 1966, 9, 1-12.
16. Yamamoto, R.T.; F r a e n k e l , G. Proc. I n t e r n . Congr. Entomol.
11th Vienna 3. 1960, p. 127.
17. K i r s o n , Isaac; G l o t t e r , Erwin J. Nat. Prod. 1981, 44, 633-647.
18. L a n d i s , B.J.; Plummer, C.C. J. Agric. Res. 1935, 50, 989-1001.
19. Sweetman, H.L. Ecology 1929, 10, 228-244.
20. Howard, N.J. Florida S t . Pl. Bd. Quart. Bull. 1921, 6, 15-24.
21. Eddy, C.O.; M c A l i s t e r , L.C., Jr. S.C. A g r i c . Exp. S t a . Bull.
1927, #236. 33 pp.
22. Sweetman, H.L.; Fermald, H.T. Mass. A g r i c . S t a . Report 1980,
#261. 25 pp.

23. Kitayama, K.; Stinner, R . E . ; Rabb, R.L. Environ. Ent. 1979, 8,

458-64.
24. Sprenkel, R.K.; Rabb, R.L. Environ. Ent., in press.
25. Wilson, K.G.; Stinner, R . E . ; Rabb, R.L. Environ. Ent. 1982,
11, 121-26.
26. Wilson, K.G. PhD Thesis, North Carolina State University,
Raleigh, N.C., 1982, 86 pp.
27. Baker, John F.; Baker, Antje Ecol. Ent. 1980, 5, 309-14.
28. Munson, Sam C. Econ. Ent. 1953, 46, 657-66.
29. Gilby, A.R. "Advances in Insect Physiology" Vol. 15
Academic Press:New York, 1980; pp 1-30.
30. Wigglesworth, V.B. Ann. Rev. Entomol. 1957, 2, 37-54.
31. Armold, M.T.; Regnier, F.Ε. J. Insect Physiol. 1975, 21,
1827-33.
32. Jackson, L . L . ; Baker
33. Cherry, Lois M. Ent
34. Hadley, Neil F. Insect Biochem. 1977, 7, 277-283.
35. Wilson, R . F . ; personal communication.
36. Downing, Donald T. "TLC:Quantitative Environmental and
Clinical Applications"; Touchstone, J . C . ; and Rogers,
Dexter, eds.; Wiley-Interscience:New York, 1980, pp 495-516.
37. Getz, Melvin. Ibid, pp 99-113.
38. Privett, Orville S.; Dougherty, Kathryn Α.; Erdahl, Warren L.
"Quantitative Thin Layer Chromatography"; Touchstone, Joseph
C. ed.; Wiley-Interscience:New York, 1973; pp 57-78.
39. Baker, J . E . Insect Biochem. 1978, 8, 287-92.
40. Warthen, J . D . ; Uebel, E . C . ; Lusby, W.R.; Adler, V . E . Insect
Biochem. 1981, 11, 467-72.

15
Detoxication, Deactivation, and Utilization
of Plant Compounds by Insects
M U R R A Y S. B L U M
University of Georgia, Department of Entomology, Athens, GA 30602
While the r o l e s o f most p l a n t n a t u r a l products are p o o r l y

understood, it is a t least well
compounds can f u n c t i o
e i t h e r unpalatable o r produce d e l e t e r i o u s e f f e c t s f o r a variety
of animals. These t o x i c p l a n t s , endowed w i t h such compounds,
g e n e r a l l y c o n s t i t u t e a considerable challenge t o animals that
i n g e s t t h e i r t i s s u e s , and a r e , in a real sense, "forbidden fruits"
f o r the host o f herbivores w i t h which they share t h e i r living
space. However, notwithstanding the pronounced toxicological
a t t r i b u t e s o f these n a t u r a l products, a m u l t i t u d e o f herbivorous
i n s e c t s utilize t o x i c p l a n t sources as food, u s u a l l y selecting
these p l a n t s as p r e f e r r e d hosts. These i n v e r t e b r a t e s demonstrate
that the term t o x i c is relative a t b e s t , s i n c e these p l a n t s
provide them w i t h both a home and a resource that can be readily
utilized to support both growth and development. Their
predilection f o r a t t a c k i n g "so called" t o x i c p l a n t s has eventuated
in a lifestyle that has enabled these i n s e c t s to exploit a
multitude of p l a n t species that are "off limits" f o r most
herbivores.
Some i n s e c t herbivores s t o r e these t o x i c compounds in their
bodies during d i f f e r e n t developmental stages. Insects in a t least
six orders sequester a v a r i e t y o f p l a n t n a t u r a l products (1)
i n c l u d i n g a l k a l o i d s ( 2 ) , cardenolides ( 3 ) , nitrophenanthrenes
( 4 ) , mustard oils ( 5 ) , and cannabinoids ( 6 ) . On the other hand,
various i n s e c t species feeding on the same species of p l a n t s do
not appear to sequester these compounds ( 1 ) . This d i v e r s e
behavior emphasizes how inadequately we understand the
p h y s i o l o g i c a l bases f o r s e q u e s t r a t i o n (7) or the e v o l u t i o n a r y
f a c t o r s that promote the r e t e n t i o n o f these compounds i n the
bodies o f s e l e c t e d h e r b i v o r e s . I n s h o r t , w h i l e i t i s one t h i n g
to demonstrate that a c e r t a i n species o f phytophagous i n s e c t may
s t o r e s p e c i f i c n a t u r a l products i n i t s body t i s s u e s , i t i s q u i t e
something e l s e to i n t e r p r e t t h i s s e q u e s t r a t i v e a b i l i t y i n e i t h e r
d e t o x i c a t i v e o r metabolic terms. Indeed, i t i s now c l e a r that
the s e q u e s t r a t i o n of a compound can represent an end product o f a
0097-6156/83/0208-0265$06.00/0

concatenation of biochemical and p h y s i o l o g i c a l events that

r e f l e c t s a s p e c i e s ' i d i o s y n c r a t i c p r o c e s s i n g o f the a l l e l o c h e m i c .
Analyses of the i n t e r p l a y o f f a c t o r s that c h a r a c t e r i z e the f a t e s
of n a t u r a l products ingested by s e l e c t e d i n s e c t s i n d i c a t e that
p h y s i o l o g i c a l v a r i e t y i s the s p i c e of herbivorous l i f e .
The demonstration that e c l e c t i c mechanisms have been evolved
by i n s e c t s f o r coping w i t h the p o t e n t i a l l y t o x i c concomitants of
t h e i r ingested n u t r i e n t s n e c e s s i t a t e s c a r e f u l analyses of the
processing of each of these compounds by each adapted h e r b i v o r e .
Furthermore, i t i s important to r e a l i z e that i n i t s e l f ,
s e q u e s t r a t i o n i s nothing more than an end product of a s e r i e s of
r e a c t i o n s that may r e f l e c t s e l e c t i v e a b s o r p t i o n , metabolism of
s p e c i f i c compounds, and e x c r e t i o n of s e l e c t e d a l l e l o c h e m i c s ( 8 ) .
In the present review, these d i v e r s e processing s t r a t e g i e s w i l l
be explored i n order to i l l u s t r a t e the v a r i o u s ways i n which a
multitude o f herbivores
lepidopterous species w i l
w i l l emphasize both the elegance and complexity i d e n t i f i e d w i t h
i n s e c t s as processors of p l a n t - d e r i v e d compounds.
Processing o f P l a n t N a t u r a l Products by Herbivores
The i n t r u s i o n of a p l a n t a l l e l o c h e m i c i n the gut o f an

adapted herbivore t r i g g e r s a s e r i e s of r e a c t i o n s that may r e s u l t
i n the compound being excreted o r absorbed and sequestered, w i t h
or without being metabolized. The metabolism of an absorbed
compound may c o n s t i t u t e d e t o x i c a t i o n o r i t may represent a means
of producing a compound that can be more r e a d i l y sequestered than
the nonderived a l l e l o c h e m i c , t o x i c i t y notwithstanding. Since
l i t t l e i n f o r m a t i o n i s a v a i l a b l e on the t o x i c i t i e s of p l a n t
a l l e l o c h e m i c s o r t h e i r metabolites to adapted h e r b i v o r e s , i t i s
v i r t u a l l y impossible to i n t e r p r e t the d e t o x i c a t i v e s i g n i f i c a n c e
of these metabolic transformations. Superimposed on t h i s
i n f o r m a t i o n a l h i a t u s i s a l a c k of data on the e c o l o g i c a l
c o r r e l a t e s o f s t o r i n g these a l l e l o c h e m i c s v i s - a - v i s pathogens
or predators. Thus, w h i l e the f a t e s of s p e c i f i c p l a n t n a t u r a l
products can be explored a f t e r t h e i r i n g e s t i o n and metabolism by
h e r b i v o r e s , t h e i r p o t e n t i a l r o l e s , as f o r example, p o s s i b l e
emetics o r t o x i n s f o r p r e d a t o r s , i s a t best t e r r a i n c o g n i t a .
E x c r e t i o n . The l a c k of d e t e c t a b l e p l a n t a l l e l o c h e m i c s i n the
bodies of a host of i n s e c t species that had developed on p l a n t s
c o n t a i n i n g p o t e n t i a l l y t o x i c n a t u r a l products (1) may r e f l e c t the
r a p i d e x c r e t i o n of these compounds subsequent to i n g e s t i o n .
However, the demonstration that s e l e c t e d herbivores e l i m i n a t e ,
during pupal o r a d u l t development, compounds sequestered by l a r v a l
stages ( 9 ) , m i l i t a t e s against drawing conclusions about the
s e q u e s t r a t i v e a b i l i t i e s of l a r v a e based s o l e l y on analyses of
a d u l t s . Thus, w h i l e i t seems reasonable to assume that
the l a c k o f ingested a l l e l o c h e m i c s i n the body of a h e r b i v o r e

15. BLUM Detoxification, Deactivation & Utilization 267
probably r e f l e c t s e x c r e t i o n (or metabolism) o f these compounds,

without s u p p o r t i v e experimental data i t i s best t o be cautious
i n interpreting this information.
N i c o t i n e i s r a p i d l y e x c r e t e d by three i n s e c t species t h a t
normally feed on N i c o t i a n a tabacum (10,11). Manduca s e x t a ,
T r i c h o p l u s i a n i , and H e l i o t h i s v i r e s c e n s e l i m i n a t e the unchanged
a l k a l o i d a f t e r i n g e s t i o n , and no evidence was obtained t o i n d i c a t e
that n i c o t i n e was s u b j e c t e d t o any metabolic t r a n s f o r m a t i o n s .
M. s e x t a absorbed s m a l l amounts o f n i c o t i n e , b u t t h i s compound
could only be detected i n the blood f o r a r e l a t i v e l y s h o r t p e r i o d
of time.
Larvae o f the l y m a n t r i i d , E l o r i a n o y e s i , an o b l i g a t o r y
feeder on Erythoxylum coca, show very l i t t l e tendency t o absorb
cocaine, the major a l k a l o i d present i n the l e a v e s . Almost a l l
of the i n g e s t e d a l k a l o i d i s excreted unchanged only t r a c e s being
d e t e c t a b l e i n the bloo
excretes v i r t u a l l y a l
gut, traces o f t h i s tropane a l k a l o i d are present i n a d u l t s ,
i n d i c a t i n g t h a t s m a l l amounts o f t h i s compound, absorbed by
l a r v a e , are r e t a i n e d during development.
The presence o f h i g h l e v e l s of a l k a l o i d s such as n i c o t i n e and
cocaine i n the guts o f l a r v a l i n s e c t s may be h i g h l y adaptive f o r
these i n v e r t e b r a t e s i n terms o f p o t e n t i a l p r e d a t o r s . Molested
l a r v a e , such as those o f E. n o y e s i , discharge e n t e r i c f l u i d s
from the mouth when d i s t u r b e d (12), an a c t which can expose
predators t o high l e v e l s o f a l k a l o i d s . Since these compounds
are e x c e l l e n t r e p e l l e n t s f o r a v a r i e t y o f i n v e r t e b r a t e s , t h e i r
value as d e t e r r e n t s f o r predatory animals may be c o n s i d e r a b l e .
Metabolism. P l a n t a l l e l o c h e m i c s i n g e s t e d by h e r b i v o r e s
may be m e t a b o l i c a l l y a l t e r e d i n the d i g e s t i v e t r a c t o r a f t e r
a b s o r p t i o n across the gut i n t o the hemolymph. I n some cases
metabolism o f these compounds r e s u l t s i n compounds known to be
c o n s i d e r a b l y l e s s t o x i c than the o r i g i n a l n a t u r a l products and
thus c o n s t i t u t e s true d e t o x i c a t i o n . On the other hand, the
t o x i c o l o g i c a l s i g n i f i c a n c e o f many o f these m e t a b o l i t e s i s unknown
f o r e i t h e r t h e i r producers o r f o r p o t e n t i a l p r e d a t o r s . While
these a l l e l o c h e m i c a l d e r i v a t i v e s may be much more amenable t o
s e q u e s t r a t i o n than t h e i r parent compounds, i t w i l l not prove
s u r p r i s i n g i f they are sometimes true d e t o x i c a t i o n products.
N i c o t i n e (I) i s metabolized t o c o n t i n i n e ( I I ) by v a r i o u s
i n s e c t s , i n c l u d i n g species adapted t o feed on a l k a l o i d - c o n t a i n i n g
leaves and those t h a t are not. C o n t i n i n e , which i s v i r t u a l l y
n o n t o x i c t o i n s e c t s , i s the primary n i c o t i n e m e t a b o l i t e produced
by some coleopterous and orthopterous species t h a t feed on
tobacco; other minor m e t a b o l i t e s are produced as w e l l (11). Two
species o f cockroaches and the h o u s e f l y , Musca domestica, a l s o
convert n i c o t i n e t o c o n t i n i n e , although these i n s e c t s do not
normally feed on n i c o t i n e - f o r t i f i e d p l a n t s (11).

I II
S i n i g r i n ( I I I ) , a g l y c o s i d e produced by many species of

c r u c i f e r o u s p l a n t s , can be converted to a h i g h l y t o x i c aglycone,
a l l y l i s o t h i o c y a n a t e ( I V ) , a f t e r h y d r o l y s i s by adapted and
nonadapted i n s e c t s (_5, 13, 14). On the other hand, the mustard
o i l i s the p r e f e r r e d storage form of l a r v a e of two P i e r i s
s p e c i e s , P. b r a s s i c a e an
plants containing s i n i g r i
must be assumed that the mustard o i l can be sequestered more
e f f i c i e n t l y than s i n i g r i n . The P i e r i s species obviously have
evolved mechanisms f o r a v o i d i n g the w e l l - e s t a b l i s h e d t o x i c i t y of
the former.
CH OH
2
III IV
Larvae of an a r c t i i d , S e i r a r c t i a echo, have evolved a novel

s t r a t e g y f o r coping w i t h the t o x i c e f f e c t s of methylazoxymethanol
(V), the aglycone of c y c a s i n ( V I ) , a c o n s t i t u e n t i n the cycad
leaves upon which they feed. When l a r v a e of S. echo are fed the
aglycone, they convert i t to the 8-glycoside c y c a s i n which i s
then sequestered (15). When cycad leaves c o n t a i n i n g an
azoxyglucoside d i f f e r e n t from c y c a s i n were i n g e s t e d , the " f o r e i g n "
g l y c o s i d e was f i r s t converted to c y c a s i n i n the gut, f o l l o w i n g
which i t was sequestered. The a b i l i t y of the l a r v a e to s e l e c t i v e l y
absorb c y c a s i n , which i s much l e s s t o x i c than i t s aglycone, i s
c o r r e l a t e d w i t h the l i m i t e d d i s t r i b u t i o n of 8-glucosidase. This
enzyme, which can e i t h e r s y n t h e s i z e c y c a s i n from methylazoxy-
methanol or hydrolyze i t to the l a t t e r , i s l i m i t e d i n i t s
d i s t r i b u t i o n to the gut, thus i n s u r i n g that absorbed g l y c o s i d e
w i l l not be converted to the t o x i c aglycone.

N V C H 2 O H
v VI
S e v e r a l p y r r o l i z i d i n e a l k a l o i d s are sequestered by
lepidopterous and orthopterous species a f t e r metabolic conversion
from i n g e s t e d a l k a l o i d s . Larvae o f the a r c t i i d T y r i a jacobaeae
p r i m a r i l y sequester th
t h i s compound i s found
jacobaeae, as the N-oxide ( V I I I ) ( 2 ) . I n the case o f T.
jacobaeae, the l i p h o p h i l i c f r e e compound, s e n e c i p h y l l i n e , i s the
p r e f e r r e d s e q u e s t r a t i o n form o f the a l k a l o i d , the w a t e r - s o l u b l e
N-oxide apparently being unsuited f o r ready storage. Conversely,
VII
the pyrgomorphid grasshopper Zonocerus v a r i e g a t u s appears t o

convert a l a r g e p r o p o r t i o n o f an i n g e s t e d p y r r o l i z i d i n e a l k a l o i d ,
monocrotaline ( I X ) , to i t s N-oxide (X) before sequestering the
a l k a l o i d s (16). Whereas the f r e e a l k a l o i d and the N-oxide a r e
present i n r e l a t i v e l y s i m i l a r amounts i n the p l a n t ( C r o t a l a r i a
s p . ) , Z. variegatus sequesters the a l k a l o i d and N-oxide i n a 1 : 3
r a t i o . Assuming that t h i s s e q u e s t r a t i o n p a t t e r n does not simply
r e f l e c t s e l e c t i v e s e q u e s t r a t i o n of the N-oxide, i t appears that
t h i s pyrgomorphid emphasizes the storage o f p o l a r a l k a l o i d s , some
of which are o x i d a t i v e l y d e r i v e d .

o
IX x
Notwithstanding th a b i l i t f larva f A r c t i caj
develop on p l a n t s c o n t a i n i n
a l k a l o i d s and cardenolides ( 1 ) , they cannot mature on marijuana
(Cannabis s a t i v a ) p l a n t s r i c h i n A-^-tetrahydrocannibinol (THC)
( X I ) . On the other hand, these a r c t i i d s can develop on
marijuana s t r a i n s r i c h i n a l e s s t o x i c cannabinoid, c a n n a b i d i o l
(CBD) ( X I I ) , although developmental time i s somewhat prolonged
(6). Although l a r v a e reared on the CBD-rich s t r a i n of C. s a t i v a
c o n t a i n only t r a c e amounts of t h i s cannabinoid, c o n s i d e r a b l y
more i s sequestered i f they are t r a n s f e r r e d to a THC-rich
s t r a i n of marijuana. These l a r v a e , which s u c c e s s f u l l y
completed development, sequestered s u b s t a n t i a l amounts of both
THC and CBD, demonstrating a we11-developed a b i l i t y t o metabolize
cannabinoids.
XI XEI
Cardenolides appear to be metabolized by a v a r i e t y of

s p e c i e s , p o s s i b l y as a mechanism f o r c o n v e r t i n g these s t e r o i d s
i n t o compounds t h a t can be e f f i c i e n t l y sequestered. The
milkweed bug, Qncopeltus f a s c i a t u s , metabolizes ( h y d r o x y l a t e s ? )
the nonpolar c a r d e n o l i d e d i g i t o x i n to more p o l a r compounds t h a t
are subsequently sequestered i n the d o r s o l a t e r a l space f l u i d
(17, 18). Larvae of another cardenolide-adapted i n s e c t , the
monarch b u t t e r f l y , Danaus p l e x i p p u s , a l s o convert these s t e r o i d s
i n t o compounds t h a t are r e a d i l y sequestered. For example,
1
u s c h a r i d i n , which contains a carbonyl group a t C-3 (*) of the

sugar moiety, i s converted t o c a l a c t i n and c a l o t r o p i n (hydroxyl

1
group a t C-3 ) ( X I I I ) by l a r v a e a f t e r which these s t e r o i d s are
s t o r e d i n s p e c i f i c t i s s u e s (19). The propensity o f i n s e c t s
feeding on c a r d e n o l i d e - r i c h p l a n t s t o s e l e c t i v e l y sequester these
compounds probably r e f l e c t s the e v o l u t i o n of s p e c i f i c metabolic
pathways f o r producing cardenolide d e r i v a t i v e s t h a t are amenable
to f a c i l e s e q u e s t r a t i o n .
H
XIII
S e l e c t i v e Sequestration. Sequestration o f p l a n t n a t u r a l
products i s an i d i o s y n c r a t i c phenomenon, and there i s l i t t l e
i n d i c a t i o n that the p r o f i l e s o f i n s e c t - s t o r e d a l l e l o c h e m i c s i n
any way m i r r o r those o f t h e i r p l a n t food sources. I n general,
each i n s e c t species t r e a t s ingested p l a n t a l l e l o c h e m i c s
d i s t i n c t i v e l y , so that a compound excreted i n t o t o by one species
may c o n s t i t u t e the main s e q u e s t r a t i o n product o f another.
U n f o r t u n a t e l y , w h i l e these s e q u e s t r a t i v e i d i o s y n c r a s i e s a r e
q u i t e e v i d e n t , t h e i r s i g n i f i c a n c e i s not.
The pyrgomorphid grasshopper P o e k i l o c e r u s bufonius sequesters
c a l a c t i n and c a l o t r o p i n , two o f the s i x cardenolides present
i n i t s ascelepiadaceous food source, and these s t e r o i d s a r e
present i n the eggs as w e l l (20). Another i n s e c t that feeds on
a c a r d e n o l i d e - r i c h food source, the ctenuchid moth Syntomeida
e p i l a i s , sequesters o l e a n d r i n , the main s t e r o i d present i n
leaves o f i t s host p l a n t , oleander (21). On the other hand,
a v a r i e t y o f aphids and coccids feeding on oleander do not
sequester oleandrin(1_).
The s e q u e s t r a t i v e p r o p e n s i t i e s o f i n s e c t s feeding on
p l a n t s f o r t i f i e d w i t h p y r r o l i z i d i n e a l k a l o i d s are e q u a l l y
unpredictable. For example, l a r v a e o f the a r c t i i d
A m p h i c a l l i a b e l l a t r i x sequester c r i s p a t i n e and trichodesmine,
whereas the main a l k a l o i d present i n the leaves i s crosemperine
(22). Another a r c t i i d , T y r i a jacobaeae, concentrates senecionine
i n i t s t i s s u e s i n s p i t e o f the f a c t that t h i s compound i s a t r a c e
c o n s t i t u e n t i n the leaves o f i t s food p l a n t , Senecio sp. (23).
On the other hand, Jacobine, j a c o z i n e , and j a c o l i n e , three
major a l k a l o i d s i n the leaves o f the p l a n t , c o n s t i t u t e minor
storage c o n s t i t u e n t s i n a d u l t s o f T\ jacobaeae.

The a b i l i t y of species to s y n t h e s i z e t h e i r own defensive

allomones may have important e c o l o g i c a l c o r r e l a t e s v i s - a - v i s
t h e i r tendency to sequester p l a n t a l l e l o c h e m i c s . Chrysomelid
b e e t l e s i n the genus C h r y s o l i n a sequester h y p e r i c i n , a
naphthodianthrone present i n t h e i r food p l a n t s , Hypericum
spp. (24). On the other hand, many species i n t h i s genus
s y n t h e s i z e cardenolides de novo, but those that feed on
Hypericum do not (25). I t appears that there has been no
s e l e c t i o n pressure f o r the b i o s y n t h e s i s of cardenolides i n
C h r y s o l i n a species that sequester h y p e r i c i n , a known mammalian
t o x i n , i n t h e i r body t i s s u e s .
Insect Sequestrators and P l a n t A l l e l o c h e m i c s — Two Selected

Case Studies
While i t i s f i r m l
products ingested by l a r v a
u l t i m a t e l y sequestered i n a d u l t t i s s u e s , almost no i n f o r m a t i o n
i s a v a i l a b l e on the d e t a i l e d f a t e s of these compounds during
development. What happens to these a l l e l o c h e m i c s during l a r v a l ,
pupal, and a d u l t molts, e s p e c i a l l y when extensive t i s s u e
r e o r g a n i z a t i o n occurs? Are these compounds i r r e v o c a b l y
sequestered i n a d u l t t i s s u e s or are they exchanged between
d i f f e r e n t t i s s u e s , or for: that matter excreted? Is s e q u e s t r a t i o n
sometimes based on a nonenergy-intensive p h y s i c a l process, as has
been demonstrated f o r the cardenolides t h a t are s t o r e d i n the
d o r s o l a t e r a l space f l u i d of the milkweed bug, Qncopeltus
f a s c i a t u s (18)? These questions are i l l u s t r a t i v e of the lacunae
i n our knowledge of how adapted i n s e c t s process the p o t e n t i a l
t o x i c compounds that are i n t h e i r p r e f e r r e d food p l a n t s .
Some i n s i g h t s i n t o how i n s e c t s p h y s i o l o g i c a l l y manipulate
absorbed a l l e l o c h e m i c s have been provided r e c e n t l y by studying
the f a t e s of cardenolides i n two u n r e l a t e d lepidopterous species
that develop on milkweeds f o r t i f i e d w i t h these s t e r o i d s .
Analyses of cardenolide p r o c e s s i n g i n the a r c t i i d , Cycnia
i n o p i n a t u s , and the monarch, Danaus p l e x i p p u s , demonstrate that
each species has evolved d i s t i n c t i v e p h y s i o l o g i c a l mechanisms
f o r manipulating the medley of a l l e l o c h e m i c s that are i n t e r n a l l y
omnipresent (26).
Cycnia i n o p i n a t u s . Larvae of t h i s a r c t i i d develop on

A s c l e p i a s h u m i s t r a t a , a milkweed species that contains very high
l e v e l s of cardenolides. The hemolymph of the l a r v a e sequesters
and maintains these s t e r o i d s at very h i g h l e v e l s , thus i n s u r i n g
that i n t e r n a l concentrations of a l l e l o c h e m i c s w i l l always be
s u b s t a n t i a l . S i g n i f i c a n t l y , the blood s t o r e s a predominance
of p o l a r cardenolides which appear to be r e t a i n e d i n t h i s
f l u i d medium throughout metamorphic development (26).
E l i m i n a t i o n of cardenolides during l a r v a l l i f e occurs p r i m a r i l y by

means o f the exuviae, which are shed a t each l a r v a l molt. About

25% o f the t o t a l cardenolide l o a d o f the l a r v a e are excreted
as e x u v i a l c o n s t i t u e n t s . Indeed, cardenolide l o s s as l a r v a l
e x u v i a l c o n s t i t u e n t s corresponds t o a c o n c e n t r a t i o n two t o three
times h i g h e r than those i n the d i f f e r e n t body regions of a d u l t s
of jC. i n o p i n a t u s . Pupal exuviae c o n t a i n l e s s cardenolides than
those o f the l a r v a e , the c o n c e n t r a t i o n corresponding t o about
50% o f t h a t i n the d i f f e r e n t a d u l t body regions.
The cardenolides i n the l a r v a l hemolymph are about a t an
e q u i v a l e n t c o n c e n t r a t i o n w i t h those d i s t r i b u t e d i n the other
l a r v a l t i s s u e s , n e g l i g i b l e amounts o f these s t e r o i d s being
present i n the gut (26). I n the a d u l t , cardenolides are
e q u a l l y d i s t r i b u t e d between the three main body regions and
the wings, w i t h females c o n t a i n i n g about twice as much o f these
s t e r o i d s as males. This female b i a s i n c a r d e n o l i d e c o n c e n t r a t i o n
appears to be d i r e c t l y
s u b s t a n t i a l amounts o
Larvae o f C. i n o p i n a t u s emerge as major sequestrators of
cardenolides p r i m a r i l y because t h e i r hemolymph, which i s present
i n a r e l a t i v e l y l a r g e volume, e f f e c t i v e l y sequesters high
concentrations o f p o l a r cardenolides (26). Cardenolide e x c r e t i o n
l a r g e l y r e f l e c t s l o s s o f these s t e r o i d s as components o f the
l a r v a l exuviae, the c o n c e n t r a t i o n o f these compounds becoming
r e l a t i v e l y s t a b l e a f t e r pupal e c d y s i s . These s t e r o i d s a r e
u b i q u i t o u s l y d i s t r i b u t e d i n the a d u l t moth, having been derived
p r i m a r i l y from the r i c h cardenolide p o o l i n the l a r v a l blood.
Danaus p l e x i p p u s . The mechanism o f c a r d e n o l i d e p r o c e s s i n g

by the monarch, D. p l e x i p p u s , shares some common denominators
w i t h C. i n o p i n a t u s but i s mainly c h a r a c t e r i z e d by d i f f e r e n c e s .
U n l i k e l a r v a e o f C. i n o p i n a t u s , those o f the monarch have a l a r g e
volume o f gut f l u i d which contains p o l a r cardenolides (26).
Monarch l a r v a e sequester f a r more e f f i c i e n t l y from p l a n t s w i t h
low cardenolide concentrations than from those w i t h high
c o n c e n t r a t i o n s ; i n some cases t h e i r s e q u e s t r a t i v e e f f i c i e n c y i s
not great enough t o s t o r e the high c a r d e n o l i d e concentrations i n
some p l a n t s p e c i e s . I n c o n t r a s t , C. i n o p i n a t u s l a r v a e are
e f f e c t i v e sequestrators even when cardenolide concentrations i n
the host p l a n t are i n o r d i n a t e l y h i g h , such as are found i n
A s c l e p i a s humistrata,a milkweed species upon which both l e p i d o p -
terans develop.
For monarch l a r v a e , the l a r g e volume o f gut f l u i d appears t o
c o n s t i t u t e the e v o l u t i o n a r y breakthrough that made a sequestra-
t i v e l i f e s t y l e p o s s i b l e on c a r d e n o l i d e - r i c h milkweeds. This
c a r d e n o l i d e - f o r t i f i e d f l u i d , which may exceed o n e - t h i r d o f i t s
t o t a l l i q u i d volume, i s withdrawn a t pupation to become p a r t o f
the hemolymph p o o l , being s t o r e d p r i m a r i l y under the wings (26).
Subsequently, the wing s c a l e s , along w i t h the hemolymph, become
the r i c h e s t source o f cardenolides i n the body, the s t e r o i d s from
the gut f l u i d having been e x p l o i t e d as an a l l e l o c h e m i c source f o r

the wings and blood. Gut f l u i d , which diminishes d r a s t i c a l l y

during the prepupal p e r i o d , reappears when the m o l t i n g f l u i d i s
s e c r e t e d , and increases i n volume as e c d y s i s approaches. During
pupal development the gut f l u i d again diminishes as i t i s
converted to hemolymph only to emerge again i n the pharate
a d u l t . This c a r d e n o l i d e - r i c h l i q u i d i s then again converted
to hemolymph during a d u l t development, so that very l i t t l e i s
l o s t a t emergence when the a d u l t evacuates f l u i d s from the gut.
Cardenolides i n the gut f l u i d c o n s t i t u t e the major s t e r o i d a l
pool which i s manipulated e c l e c t i c a l l y i n order to channel the
cardenolides to d i f f e r e n t s i t e s during development. L a r v a l
exuviae are r i c h i n cardenolides and these discarded t i s s u e s
c o n s t i t u t e an excretory form f o r these compounds (26), as i s the
case f o r jC. inopinatus l a r v a e . Cardenolides are a l s o concen-
t r a t e d i n the prepupal m o l t i n g f l u i d , and presumably p a r t of t h i s
s t e r o i d a l pool i s e l i m i n a t e
pupal e c d y s i s . In a two-da
i n low concentrations i n the gut f l u i d but are much more
concentrated i n the hemolymph. However, before wing expansion
the cardenolide c o n c e n t r a t i o n i n the t e n e r a l a d u l t i s at i t s
lowest i m a g i n a l l e v e l , r a p i d l y c l i m b i n g a f t e r t h i s time so
that i t reaches the highest l e v e l encountered i n any l i f e stage.
The presence of h i g h concentrations of cardenolides i n the
blood of a d u l t s demonstrates that a l a r g e percentage of these
s t e r o i d s i s not locked i n t i s s u e s i n k s but i s f r e e l y
c i r c u l a t i n g i n the body. Whether these blood-borne s t e r o i d s
exchange w i t h those sequestered i n t i s s u e s i s unknown, but t h e i r
presence as major c o n s t i t u e n t s i n a d u l t hemolymph demonstrates
that they are i n a dynamic s t a t e , even i n a stage which no longer
i n g e s t s them (26). These r e s u l t s c o n t r a s t w i t h those obtained
f o r C. i n o p i n a t u s , and emphasize the importance of regarding each
species and i t s a l l e l o c h e m i c - f o r t i f l e d host p l a n t as a unique
e v o l u t i o n a r y case.
Literature Cited
1. R o t h s c h i l d , M. Symp. Roy. Ent. Soc. London 1972, 6, 59.
2. A p l i n , R. T.; R o t h s c h i l d , M. in "Toxins of P l a n t and Animal
O r i g i n " ; de V r i e s , A.; Kochva, K., Eds., Gordon and Breach:
New York, 1972; pp 579-595.
3. R e i c h s t e i n , T.; von Euw, J . ; Parsons, J . A.; R o t h s c h i l d , M.
Science 1968, 161, 861.
4. von Euw, J . ; R e i c h s t e i n , T.; R o t h s c h i l d , M.; I s r a e l , J .
Chem. 1968, 6, 659.
5. A p l i n , R. T.; Ward, R. d'Arcy; R o t h s c h i l d , M. J. Ent., Ser.
A. 1975, 50, 73.
6. R o t h s c h i l d , M.; Rowan, M. G.; F a i r b u r n , J . W. Nature 1977,
266, 650.
7. Duffey, S. S. Ann. Rev. Ent. 1980, 25, 447.
8. Blum, M. S. "Chemical Defenses of Arthropods"; Academic
Press: New York, 1981.

9. R o t h s c h i l d , M.; von Euw, J.; R e i c h s t e i n , T.; Smith, D. A. S.;

Pierre, J . Proc. Roy. Soc. London, Ser. B. 1975, 190, 1.
10. S e l f , L. S.; G u t h r i e , F. E.; Hodgson, E. Nature 1964, 204,
300.
11. S e l f , L. S.; G u t h r i e , F. E.; Hodgson, E. J . Insect P h y s i o l .
1964, 10, 907.
12. Blum, M. S.; Rivier, L.; Plowman, T. Phytochem. 1981, 20,
2499.
13. E r i c k s o n , J. M.; Feeney, P. Ecology 1974, 55, 103.
14. B l a u , P. A.; Feeney, P.; Contardo, L.; Robson, D. S. Science
1978, 200, 1296.
15. Teas, H. J. Biochem. Biophys. Res. Comm. 1967, 26, 686.
16. Bernays, E.; Edgar, J. A.; R o t h s c h i l d , M. J. Z o o l . 1977,
182, 85.
17. Duffey, S. S.; Scudder G G E J. Insect P h y s i o l 1972 18
63.
18. Duffey, S. S.; Blum, , ,
J. Insect P h y s i o l . 1978, 24, 639.
19. Seiber, J. N.; Tuskes, P. M.; Brower, L. P.; Nelson, C. J .
J . Chem. E c o l . 1980, 6, 321.
20. von Euw, J.; F i s h e l s o n , L.; Parsons, J. A.; R e i c h s t e i n , T.;
R o t h s c h i l d , M. Nature 1967, 214, 35.
21. R o t h s c h i l d , M.; von Euw, J.; R e i c h s t e i n , T. Proc. Roy. Soc.
London, Ser. B. 1973, 183, 227.
22. R o t h s c h i l d , M.; Aplin, R. T. in "Chemical Releasers in
I n s e c t s " ; T a h o r i , A. S., Ed., Gordon and Breach: New York,
1971, pp 177-182.
23. A p l i n , R. T.; Benn, M. H.; R o t h s c h i l d , M. Nature, 1968, 219,
747.
24. Rees, C. J. C. Ent. Exp. Appl. 1969, 12, 565.
25. P a s t e e l s , J. M.; Daloze, D. Science 1977, 197, 70.
26. N i s h i o , S. "The Fates and Adaptive S i g n f i c a n c e o f
Cardenolides Sequestered by Larvae of Danaus plexippus (L.)
and Cycnia inopinatus (Hy. Edwards)"; Ph. D. Thesis,
U n i v e r s i t y o f Georgia, 1980.

16
L-Canavanine and L-Canaline: Protective
Allelochemicals of Certain Leguminous Plants
GERALD A. ROSENTHAL
University of Kentucky, T. H. Morgan School of Biological Sciences and the
Graduate Center for Toxicology, Lexington, KY 40506
L-Canavanine and L-canaline are non-protein

amino acids of certain leguminous plants that
function as protectiv
nine i s incorporate synthesize
proteins in place of arginine; there i s sug-
gestive evidence that formation of such anomalous
proteins figures s i g n i f i c a n t l y in canavanine's
adverse biological effects. Canavanine, however,
does not appear to i n h i b i t overall protein
synthesis. Thus, an important basis for canava-
nine's antimetabolic properties resides in the
sustained production of b i o l o g i c a l l y aberrant
proteins.
L-Canaline i s an ineffective antimetabolite
of L-ornithine since it has little ability to
antagonize ornithine-dependent reactions. On the
other hand, it forms a covalently bound Schiff-base
complex with the pyridoxal phosphate moiety of
B -containing enzymes. As such it i s a potent
6
i n h i b i t o r of many decarboxylases and aminotrans-

ferases that utilize this vitamin.
L-Canavanine, 2-amino-4-guanidinooxybutyric acid, i s a

structural analogue of L-arginine in which the terminal methylene
group i s replaced with oxygen. This creates the guanidinooxy
group having a pK value of 7.01 as compared to 12.48 for a r g i -
nine' s guanidino group Q ) .
Arginase (EC 3.5.3.1) mediates the hydrolytic cleavage of L-
canavanine to produce L-canaline and urea. L-Canaline, 2-amino-
4-aminooxybutyric acid, bears the same structural analogy to L-
ornithine as canavanine does to arginine. The aminooxy group
of canaline with i t s pK value of 3.96 differs markedly from the
6-amino function of ornithine (pK = 10.76).
These secondary plant metabolites are two of nearly 250
non-protein amino acids currently known to be proliferated by
0097-6156/83/0208-0279$06.00/O

higher plants {2). Canavanine and canaline are constituents of

a much more limited assemblage of non-protein amino acids, d i s -
t i n c t i v e by virtue of their i n t r i n s i c t o x i c i t y and potent a n t i -
metabolic properties (2-j>). They are markedly growth-inhibitory
to insects, and can be insecticidal as well (4,_7,8,j)).
Canavanine Toxicity and Aberrant Protein Formation
Canavanine i s a potent arginine antagonist that functions

in v i r t u a l l y a l l of the reactions for which arginine i s the
preferred substrate (10). It is readily activated and amino-
acylated by arginyl-tRNA synthetase and subsequently incorporated
into the nascent polypeptide chain. This property i s not unique
to canavanine but rather i s observed with many non-protein amino
acids bearing structural analogy to a protein amino acid counter-
part (11). Increasingly
important, perhaps eve
properties of non-protein amino acids structurally akin to a
protein amino acid, resides in the formation of anomalous
proteins.
Isolation of alkaline phosphatase from Escherichia c o l i in
which 85% of the proline residues were replaced by 3,4-dehydro-
proline affected the heat l a b i l i t y and ultraviolet spectrum of
the protein but the important c r i t e r i a of c a t a l y t i c function
such as the K and V
m m a x were unaltered Q 2 ) . Massive replacement
of methionine by selenomethionine in the 3-galactosidase of E,.
c o l i also failed to influence the c a t a l y t i c a c t i v i t y . Canavanine
f a c i l e l y replaced arginine in the alkaline phosphatase of this
bacterium; at least 13 and perhaps 20 to 22 arginyl residues were
substituted. This replacement by canavanine caused subunit
accumulation since the altered subunits did not dimerize to yield
the active enzyme (13). Nevertheless, these workers stated:
"There was also formed, however, a significant amount of enzy-
matically active protein in which most arginine residues had been
replaced by canavanine." An e a r l i e r study in which either 7-
azatryptophan or tryptazan replaced tryptophan resulted in active
protein comparable to the native enzyme (14).
More recently, canavanine was incorporated into the pro-
collagen of cultured embryonic tendon c e l l s of the chicken (15).
Canavanyl procollagen was secreted less rapidly and accumulated
i n t r a c e l l u l a r ^ as compared to the native molecule. It i s very
significant that no change in such c r i t i c a l processes as hydroxy-
lation of proline to hydroxyproline, t r i p l e helix formation in
collagen, basic glycosylation, or the functional parameters of
the resulting collagen was reported (J_5). Canavanine i s also
placed into pro-opiomelanocortin, a glycoprotein progenitor of
adrenocorticotropic and 3-lipotropin. While canavanine affected
prohormone conversion to biologically active components, the
question of impaired biological function of the assembled macro-
molecule was not addressed (16).

ROSENTHAL L-Canavanine and L-Canaline
CPK space-filling model of L-arginine (right) and L-canavanine (left).

As mentioned e a r l i e r , canavanine i s less basic than argin-

ine, a property that can affect R-group interactions and disrupt
t e r t i a r y and/or quaternary interactions essential for the uni-
quely correct three dimensional configuration of a particular
macromolecule. Canavanine i s incorporated into the proteins of
organisms such as insects that do not normally synthesize this
compound. A l l female insects produce vitellogenin—an extra-
ovarial protein secreted by the fat body into the hemolymph and
used in the manufacture of v i t e l l i n , a major storage protein of
the oocyte (V7). Excised fat bodies of the migratory locust,
Locusta migratoria migratorioides maintained by in vitro culture
techniques retain their a b i l i t y to produce and secrete v i t e l l o -
genin at a rate comparable to in vivo a c t i v i t y (18). This system
established the formation of canavanyl vitellogenin and permitted
demonstration of i t s altered physicochemical but not immuno-
logical properties. Thus, canavanine can be incorporated into
de novo synthesized protei
on the resulting physicochemical properties. The germane
question of the biochemical and biological ramifications of
formation of an aberrant canavanyl protein has not been properly
addressed nor resolved.
Canavanine and Protein Synthesis
Experiments conducted with the tobacco hornworm, Manduca

sexta and the aquatic plant, Lemna minor are consistent in
finding that canavanine does not affect whole organism a b i l i t y
to incorporate [ H]leucine into trichloroacetic acid-insoluble
materials (see Table I ) . Such determinations evaluate the
balance between reactions fostering protein synthesis and those
responsible for the turnover and degradation of proteins. If
the treatment time i s reduced to 30 min, so as to accentuate
synthetic reactions over those of catabolism, the result i s the
same.
Similar evaluations employing an in vitro reticulocyte
assay system f a i l e d to provide evidence of diminished polypep-
tide formation (unpub. obser.). Thus, a consistent pattern
emerges in these three systems: canavanine does not impede
protein synthesis, including aberrant, canavanyl proteins.
Canavanine and Nucleic Acid Metabolism
Studies of canavanine interaction with the tobacco hornworm

and I. minor also revealed the marked a b i l i t y of canavanine^to
i n h i b i t whole organism incorporation of [ H]thymidine and [ H]-
uridine into trichloroacetic acid-precipitated materials. When
canavanine is provided simultaneously with the appropriate radio-
labeled precursor, ample evidence for curtailed nucleic acid
metabolism emerges but protein synthesis i s unaffected (Table I,
exp. I ) . In experiment II, canavanine i s allowed to assimilate

16. ROSENTHAL L-Canavanine and L-Canaline 283
into the metabolic reactions of the rapidly growing larvae for

24 hrs (at t h i s time, at least 3.5% of the administered canavanine
can be recovered from de novo-synthesized proteins). In experi-
ment II r e l a t i v e to I, there i s approximately a 3-fold increase
in canavanine-mediated i n h i b i t i o n of both DNA and RNA metabolism;
a result possibly reflecting the formation of errant proteins
(Table I ) . Even under this extremely deleterious s i t u a t i o n , how-
ever, protein synthesis i s not altered.
Table I
The Effect of L-Canavanine on Macromolecular Synthesis
In experiment A, newly ecdysed f i f t h stadium tobacco

hornworm larvae received 5 uCi of labeled compound and 1 mg
canavanine/g fresh body weight. Three hrs l a t e r , the treated
larvae were collected an
nine was injected f i r s t and 24 hrs l a t e r , the labeled compound
was administered. A l l larvae were collected and processed 3
hrs l a t e r (from the work of Rosenthal and Dahlman).
Radioactivity in the t r i c h l o r a c e t i c - a c i d
insoluble fraction^
Injected compound
Experiment A Experiment B
(% of the control)
[ H]leucine 96 ± 1 103 ± 4
[ H]thymidine 85 ± 6 57 ± 1
[ H]uridine 76 ± 4 34 ± 5
Each value i s the mean of 3 determinations involving 3

larvae per determination ± SE.
Canavanine and Vitellogenin Production
The previously described test system involving in vitro

analysis of fat body vitellogenin production and secretion i s
apparently d i s t i n c t i v e in providing evidence for canavanine-
mediated curtailment of protein synthesis. At l e a s t , canavanine
attenuates the amount of [ H]leucine-labeled protein secreted
by the fat body (Fig. 1). As shown in figure 1, the effect of
arginine depletion i t s e l f i s d e b i l i t a t i n g and i t becomes i n -
creasingly deleterious with time—quite apart from any effect
canavanine may e l i c i t . With increased treatment time, canavanine-

150 r-
E 100
a
TJ
a CAV
LU
h- -ARG
LU
u
LU
en
z
50-
o
a:
CL
3 6 9 3 6 9 3 6 9
HOURS
Figure 1. Influence of arginine depletion and canavanine treatment on protein

secretion by cultured fat bodies of Locusta migratoria migratoroides. The fat body
3
samples were maintained in standard incubation medium containing 5 fxCi of [ H]
leucine and supplemented with 5 mM arginine or 10 mM canavanine or lacking
arginine. After 3 and 6 h the fat bodies were transferred to appropriate fresh radio-
active medium. The time on the abscissa denotes the hour at the beginning of which
the samples were removed for determining protein production. The data presented
are typical of several determinations that gave essentially the same results. (Repro-
duced, with permission, from Ref. 19. Copyright 1981, National Academy of
Sciences.)

exposed fat bodies exhibit greater i n h i b i t i o n in the secretion

of radioactive proteins than their arginine-denied counterpart.
It i s possible that what is actually occurring i s the a b i l i t y of
canavanine to block the uptake and u t i l i z a t i o n of arginine
c r i t i c a l to protein synthesis. Consistent with this assertion,
addition of 5 mM arginine and 10 mM canavanine to the arginine-
depleted fat body reduced the extent of canavanine-mediated
i n h i b i t i o n s i g n i f i c a n t l y since protein secretion f e l l to only
55% of the control level (J9). At lower canavanine concentrations,
supplemental arginine was even more effective in blocking the
adverse effect of canavanine. This point is being evaluated
further at this time.
Insectan Canavanyl Protein Production
The female bruchid

her eggs as a cluster on the outside of the f r u i t of the Central
American legume, Dioclea megacarpa. The seed of this plant i s
d i s t i n c t i v e in i t s containment of sufficient canavanine to
account for as much as 13% of the seed dry weight (20) and 95%
of the nitrogen allocated to free amino acid formation (21_). If
canavanyl protein production lacks adverse biological effect, i t
i s reasonable to expect that this seed predator would produce
canavanine-containing proteins. In contrast, i f an adaptive
advantage accrues to the developing larva by avoiding the pro-
duction of canavanyl proteins, then one would expect acquisition
of the a b i l i t y to avoid errant protein formation.
Examination of this question with the tobacco hornworm, an
insect known to be canavanine-sensitive (this insect normally
feeds on canavanine-free plants) revealed that i t readily incorpor-
ates [ C]canavanine into i t s newly synthesized proteins. Caryedes
b r a s i l i e n s i s , however, very effectively avoids the production of
such radiolabeled proteins. When the arginyl-t_RNA synthetase
a c t i v i t y of these insects was compared, tobacco hornworm larvae
readily activated canavanine while the larvae of the bruchid
beetle possess an arginyl-tRNA synthetase with a marked a b i l i t y
to discriminate between arginine and i t s structural analogue (22).
Recent study of the arginine activating system of the bru-
chid beetle disclosed that injection of 7,500 nCi of L-[guani-
dinooxy- C]canavanine resulted in the incorporation of only
0.95 nCi of radioactive canavanine into newly produced proteins.
This compares with 347 nCi of incorporated L-[guanidino- C]
arginine from 2,850 nCi of injected material (the difference in
the amount of injected radioactive amino acid reflects the
internal pool size of each compound. In this way, the i n i -
t i a l specific a c t i v i t y of both compounds was i d e n t i c a l ) . Nearly
300 times more radioactive canavanine was incorporated into the
proteins of the tobacco hornworm, a canavanine-sensitive organism.
I am presently examining the question of whether bruchid beetle
acquisition of an activating system able to discriminate between

canavanine and arginine, provides broad spectrum resistance

to other arginine analogues capable of being incorporated
into proteins.
Elucidation of whether or not canavanine affects protein
synthesis i s a point of considerable significance in our under-
standing of non-protein amino acid t o x i c i t y . A l l of the a v a i l -
able evidence support the a b i l i t y of arginyl-jtRNA synthetase to
activate canavanine. This observation i s reported consistently
for organisms in which t h i s arginine analogue i s a foreign
compound. On the other hand, canavanine-producing organisms
have.an arginyl-tRNA synthetase that does not form canavanyl-
jtRNA 9
and thus maintains a high f i d e l i t y in the manufacture
of essential proteins. While this point proves nothing, i t i s
consistent with a link between canavanyl protein production and
its intrinsic toxicity.
If canavanine placemen
basis for i t s t o x i c i t y an
protein synthesis—assertions which are both reasonable and con-
sistent with the available evidence; then these properties would
be mutually complementary and reinforcing. It would foster a
marked antimetabolic effect in species naive to canavanine.
Higher plant allelochemical production of a compound that pro-
duces anomalous proteins while scrupulously avoiding diminution
in the formation of these aberrant macromolecules certainly repre-
sents a subtly deceitful and highly effective form of chemical
defense.
Canaline Toxicity
L-Canaline shares with canavanine an appreciable potential

for e l i c i t i n g adverse biological effects. Evaluations with L..
minor of some 55 naturally occurring and synthetic amino acids
indicated that canavanine and canaline were amongst the most
t o x i c ; canaline exhibited s l i g h t l y greater growth-inhibition than
canavanine (23). These toxic non-protein amino acids interact to
curtail additively the proliferation of this aquatic plant (24).
L i t t l e i s known of canaline t o x i c i t y in whole animals or
plants. Canaline-fed tobacco hornworm larvae grew poorly, ex-
hibited much more deformity, and succumbed in larger numbers than
the controls (8). This ornithine analogue is neurotoxic to the
adult moth where i t induces almost continuous motor a c t i v i t y .
Axonal conduction of action potentials was unaffected but the
postsyntaptic potential of f l i g h t muscle fibers was prolonged.
Central nervous system functions were affected but i t s exact mode
of action remains unknown (_7).
Canaline and Pyridoxal Phosphate Interaction
Canaline reacts f a c i l e l y with the aldehyde group of free pyri-

doxal phosphate to y i e l d a covalently linked complex having the

following postulated structure at pH 3.0. NMR data of the complex

are consistent with t h i s structure and assert to a Schiff-base
linkage between canaline and pyridoxal phosphate (25).
H C = N - 0 - C H - C H - CH(NH+)COO "
2 2
I
C OH
// \ I
HO-C C-CH -0-P = 0
2
I II I
H C-C
3 C
\ /
N
+
H
L-Canaline-pyridoxal phosphate complex
The canaline-pyridoxal phosphate complex lacks discernible

t o x i c i t y as evaluated by the JL. minor bioassay, even at a concen-
tration of 10 M where canaline v i r t u a l l y stops plant growth.
Canaline reacts with the pyridoxal phosphate moiety of
enzymes possessing this vitamin; BV-containing decarboxylases and
transaminases t y p i c a l l y are inhibited strongly by this non-protein
amino acid (25, 26, 27). When canaline reacts with free pyridoxal
phosphate, d i s t i n c t i v e changes in the absorbance spectrum of
t h i s vitamin result; similar spectral shifts also occur with B^-
containing enzymes treated with canaline {Z7_9 28). Such a l t e r -
ations in absorbance correlate closely with canaline-mediated i n -
h i b i t i o n of enzyme a c t i v i t y (Table I I ) .

Table II
L-Tyrosine Decarboxylase A c t i v i t y Determinations
L-Tyrosine decarboxylase (2 mg/ml, 0.8 ml) in 100 mM sodium
acetate buffer (pH 5.5) was treated with canaline as indicated
for 30 min. A unit of tyrosine decarboxylase a c t i v i t y i s that
amount of enzyme forming 1 umol C0 /min. See original for
2
additional d e t a i l s .
Enzyme form Treatment Activity
(units/mg)
Holoenzyme none 0.221
Holoenzyme 0.1 mM canaline 0.178
0.5 mM canaline 0.071

1.0 mM canaline 0.023
Apoenzyme none 0.017
Apoenzyme 5.0 mM pyridoxal 0.218
phosphate
Apoenzyme 1.0 mM canaline 0.022
Source: Reproduced with permission from Ref. 25.
Canaline as an Ornithine Antagonist
Several lines of investigation assert to the i n a b i l i t y of

canaline to function as an effective ornithine antagonist. Or-
nithine interaction with canaline has been evaluated with the
ornithine carbamoyl transferase (EC 2.1.3.3) of human l i v e r .
Neither canaline nor ornithine inhibited this enzyme when the
other member of this set served as the carbamoyl group recipient
(29). The ornithine antagonist, 2,4-diaminobutyric acid dras-
t i c a l l y reduced urea production in the r a t ; t h i s reflected cur-
tailment of the ornithine carbamoyltransferase-mediated conver-
sion of ornithine to c i t r u l l i n e . Yet, canaline had no such
effect on urea formation in this mammal (30).
Canaline i s a potent inhibitor of a l l seven pyridoxal phos-
phate-containing enzymes studied by Rahiala et al_. (27_) but i t
lacks adverse effects on three o r n i t h i n e - u t i l i z i n g enzymes lack-
ing a BV cofactor. F i n a l l y , in jack bean, Canavalia ensiformis,
ornithine carbamoyl transferase can form 0-ureido-L-homoserine
from canaline and carbamoyl phosphate as i t does c i t r u l l i n e
from ornithine and carbamoyl phosphate. Nevertheless, neither
compound inhibited formation of the reaction products (31).
It i s evident, therefore, that i t i s the binding of canaline
to the pyridoxal phosphate moiety of the enzyme rather than
effective competition with ornithine for the active site that i s
responsible for the antimetabolic properties of canaline.

16. ROSENTHAL h-Canavanine and L-Canaline 289
Acknowledgment
The author gratefully acknowledges the support of the

National Institutes of Health (AM-17322), the National Science
Foundation (PCM-78-20167) and the Kentucky Research Foundation
for studies by the author described in this communication.
Literature Cited
1. Boyar, Α., Marsh, R.E. J . Am. Chem. Soc. 1982, 50, 1995.
2. Rosenthal, G.A. "Plant Nonprotein Amino and Imino Acids.
Their B i o l o g i c a l , Biochemical, and Toxicological Properties";
Academic Press, New York, 1982.
3. Fowden, L.; Lea, P.J.; and Bell, E.A Adv Enzymol 1979
50, 117.
4. Rosenthal, G . A . ; Bell,
Interaction with Secondary Plant Metabolites"; (G.A.
Rosenthal and D.H. Janzen, eds.), Academic Press, New York,
1979; p. 353.
5. B e l l , E.A. Prog. Phytochem. 1980, 7, 171.
6. Fowden, L. Perspect. Exptl. B i o l . 1976, 2, 263.
7. Kammer, A.E.; Dahlman, D . L . ; Rosenthal, G.A. J. Exptl.
Biol. 1978, 75, 123.
8. Rosenthal, G . A . ; Dahlman, D.L. Comp. Biochem. Physiol.
1975, 52A, 105.
9. Rehr, S.S.; Bell, E . A . ; Janzen, D . H . ; Feeny, P.P. Biochem.
Syst. Ecol. 1973, 1, 63.
10. Rosenthal, G.A. Quant. Rev. B i o l . 1977, 52, 155.
11. Fowden, L; Lea, P . J . In: "Herbivores: Their Interaction
with Secondary Plant Metabolites"; (G.A. Rosenthal; D.H.
Janzen, eds.), Academic Press, New York, 1979, p. 135.
12. Fowden, L; Lewis, D; Tristram, H. Adv. Enzymol. 1967, 29,
89.
13. A t t i a s , J; Schlesinger, M . J . ; Schlesinger, S. J. B i o l .
Chem. 1969, 244, 3810.
14. Schlesinger, S. J . B i o l . Chem. 1968, 243, 3877.
15. Crine, P . ; Lemieux, E. J . B i o l . Chem. 1982, 257, 832.
16. Schein, J.; Harsch, M . ; Cywinski, Α.; Rosenbloom, J. Arch.
Biochem. Biophys. 1982, 203, 572.
17. Hagedorn, H . H . ; Kunkel, J . G . Annu. Rev. Entomol. 1979, 24,
475.
18. Harry, P; Pines, M; Applebaum, S.W. Comp. Biochem. Physiol.
1979, 63B, 287.
19. Pines, M . ; Rosenthal, G . A . ; Applebaum, S.W. Proc. Natl.
Acad. S c i . 1981, 78, 5480.
20. Rosenthal, G . A . ; Janzen, D.H.; Dahlman, D.L. Science
1977, 196, 658.
21. Rosenthal, G.A. Biochem. Syst. Ecol. 1977, 5, 219.
22. Rosenthal, G . A . ; Dahlman, D . L . ; Janzen, D.H. Science 1976,
192, 256.

23. G u l a t i , D . K . ; Chambers, C . L . ; Rosenthal, G . A . ; Sabharwal,

P.S. Envir. Exptl. Bot. 1981, 21, 225.
24. Rosenthal, G . A . ; G u l a t i , D . K . ; Sabharwal, P.S. Plant
Physiol. 1976, 57, 493.
25. Rosenthal, G.A. Eur. J. Biochem. 1981, 114, 301.
26. Rahiala, E.L.; Kekomäki, M . ; Janne, J.; Räinä, Α.; Raiha,
N.C.R. Biochim. Biophys. Acta 1971, 227, 337.
27. Katunuma, N . ; Okada, M . ; Matsuzawa, T.; Otsuka, Y. J . Bio
chem. (Tokyo) 1965, 57, 445.
28. Beeler, T.; Churchich, J.E. J. B i o l . Chem. 1976, 251, 5267.
29. Natelson, S.; K o l l e r , Α.; Tseng, H . - Y . ; Dods, R.F. C l i n .
Chem. 1977, 23, 960.
30. Kekomäki, M . ; Rahiala, E.-L.; Raiha, N.C.R. Am. Med.
Exp. Fenn. 1969, 47, 33.
31. O'Neal, T.D. Plan

17
Cytotoxic and Insecticidal Chemicals of Desert
Plants
ELOY RODRIGUEZ
University of California, Phytochemical Laboratory, Department of Ecology and
Biology, Irvine, CA 92717
A d i v e r s e group of n a t u r a l chemicals are produced

by a r i d land p l a n t s
which are store
i n c l u d e t e r p e n o i d s , alkaloids, phenolics and
amines. Some of these compounds coat the l e a f and
stem surfaces and prevent water l o s s through the
cuticle and probably p r o t e c t the p l a n t from
excessive damage by radiation. Another important
e c o l o g i c a l r o l e of secondary metabolites is that
of defense against phytophagous i n s e c t s and
pathogens. Recent phytochemical i n v e s t i g a t i o n s
in our l a b o r a t o r y i n d i c a t e t h a t chromenes,
prenylated quinones and sesquiterpenes e s t e r i f i e d
w i t h phenolic a c i d s , are e x c e l l e n t r e p e l l e n t s and
in some cases are c y t o t o x i c and inhibit larval
growth and development. The chemistry and r o l e of
these c y t o t o x i n s and insecticides in desert p l a n t s
of Baja California and Chihuahua is reviewed.
Deserts cover approximately one-seventh of the earth's land

s u r f a c e , w i t h the North American deserts populated by a d i v e r s i t y
of b i z a r r e plant l i f e forms ranging from c a c t i to creosote bushes
to the boojum t r e e s of Baja C a l i f o r n i a , Mexico. An outstanding
c h a r a c t e r i s t i c of a m a j o r i t y of p l a n t s that dominate the desert
landscape i s t h e i r enormous photosynthetic c a p a c i t y to produce
an array of secondary metabolites • Many of these n a t u r a l products
are e s s e n t i a l t o the everyday s u r v i v a l o f p l a n t s exposed t o the
harsh, hot and d r y environment of the d e s e r t . These n a t u r a l
products, which i n c l u d e t e r p e n o i d s , a l k a l o i d s , p h e n o l i c s , amines
and tannins are produced i n l a r g e q u a n t i t i e s by s p e c i a l i z e d
glandular h a i r s c a l l e d trichomes and coat l e a f , stem and flower
surfaces • This t h i n l a y e r of secondary chemicals are b e l i e v e d t o
prevent water l o s s ( a n t i d e s s i c a n t s ) through the c u t i c l e and i n
some cases prevent excessive c e l l damage by b l o c k i n g u l t r a v i o l e t
0097-6156/ 83/0208-0291 $ 0 6 . 0 0 / 0

r a d i a t i o n . More important, these b i o l o g i c a l l y a c t i v e c o n s t i t u e n t s

a l s o r e p e l and i n some cases k i l l phytophagous i n s e c t s and plant
pathogens. I t i s the desert a l l e l o c h e m i c a l s ( c o n s t i t u e n t s which
have a d e l e t e r i o u s e f f e c t on herbivorous i n s e c t s and pathogens)
which are of considerable i n t e r e s t to phytochemists concerned
w i t h c o n t r o l l i n g i n s e c t pests of crop p l a n t s . With the current
a g r i c u l t u r a l development of desert hydrocarbon crop plants,such
as guayule (Parthenium argentatum) and j o j o b a (Simmondsia
c h i n e n s i s ) , i t i s important that a l i n e of n a t u r a l i n s e c t i c i d e s
and r e p e l l e n t s be developed against i n s e c t s e s p e c i a l l y adapted
to p l a n t s c u l t i v a t e d i n marginal a r i d lands (1_) •
In t h i s communication, we present our l a t e s t phytochemical
f i n d i n g s on prenylated quinones and chromenes that are c y t o t o x i c
and i n h i b i t l a r v a l growth and development. Many of these
b i o a c t i v e chemicals are unique to desert p l a n t s and have probably
been s e l e c t e d against deser
Desert P l a n t s and Their Secondary M e t a b o l i t e s
As p r e v i o u s l y noted, desert p l a n t s are no d i f f e r e n t from

temperate p l a n t s i n producing a wide-range of secondary
metabolites (2)« Dominant p l a n t species such as Larrea t r i d e n t a t a
(Zygophyllaceae) may contain up to 15% of the dry weight i n
l i g n a n s , f l a v o n o i d s , chromenes, t r i t e r p e n e s and v o l a t i l e
terpenes ( 3 ) . Important desert f a m i l i e s i n c l u d e the Asteraceae,
Fabaceae, Agavaceae, Burseraceae, Euphorbiaceae and Fonquieriaceae
which synthesize d i v e r s e products such as sesquiterpene l a c t o n e s ,
methylated f l a v o n o i d s , p o l y a c e t y l e n e s , s t e r o i d s , saponins,
a l k a l o i d s , cyanogenic g l y c o s i d e s , aromatic terpenes and amines
( r e f e r to Figure 1 and 2 f o r r e p r e s e n t a t i v e s t r u c t u r e s ) . These
n a t u r a l products are not only used by n a t i v e peoples f o r
medicines, food and n a t u r a l a n t i b i o t i c s , but many of these
compounds are very e f f e c t i v e against a host of i n s e c t s and
pathogens ( 4 ) . In our s t u d i e s we have concentrated on prenylated
quinones and chromenes that are present i n desert species of the
Asteraceae and Hydrophyllaceae. In the ensuing paragraphs we
summarize the chemistry and b i o l o g i c a l e f f e c t s of a s e l e c t e d group
of quinones and chromenes that are c y t o t o x i c and i n s e c t i c i d a l
to milkweed bugs (Oncopeltus) and mealworm beetles (Tenebrio)•
Prenylated Quinones
Quinonoid compounds are q u i t e common throughout the plant

kingdom, but only i n a few cases have they been reported i n
glandular h a i r s (trichomes) of desert herbaceous p l a n t s . Many of
these quinonoid compounds are extremely a c t i v e and are the major
cause of a l l e r g i c r e a c t i o n s i n humans• For example, Primula
obconica (Primulaceae), an ornamental p l a n t common to Europe,
produces a quinone which i s stored i n small non-capitate
trichomes which r e l e a s e t h e i r contents when touched. These

RODRIGUEZ Cytotoxic and Insecticidal Chemicals
Flourensia Acid
Sesquiterpene Farinosin
Flourensia cernu
Encelia forinosa
Figure 1. Terpenoid constituents of desert plants.

Nordihydroguairetic Acid
Lignan
Phenylethylamines
Cactaceae
Figure 2. Phenolic and alkaloid constituents of desert plants.

17. RODRIGUEZ Cytotoxic and Insecticidal Chemicals 295
secreted chemicals are very e f f e c t i v e a g a i n s t aphids that c r a w l

on the l e a f . These chemicals, l i k e the s e c r e t i o n of Solanum
tuberosum (Solanaceae), hardens on mouth parts and t a r s i of
i n s e c t s and i n h i b i t s movement and f e e d i n g (5-6)•
Recently we have i s o l a t e d a s e r i e s of p r e n y l a t e d quinones
and quinonoid compounds from the v i s c i d c a p i t a t e - g l a n d u l a r
trichomes of P h a c e l i a (Hydrophyllaceae) that are c y t o t o x i c ,
a l l e r g e n i c and i n s e c t i c i d a l . Most o f the species noted t o have
the prenylated quinones are r e s t r i c t e d t o the Sonoran desert o f
Baja C a l i f o r n i a , Mexico and the Mojave desert o f the Southwest
(7-10). P h a c e l i a c r e n u l a t a , an annual which i s common along
desert roadsides i n the southwestern United States and northern
Mexico, i s r e s p o n s i b l e f o r a d e r m a t i t i s s i m i l a r t o that of
poison oak, but i s i s a l s o been demonstrated t o i n h i b i t l a r v a l
f o r m a t i o n . The e f f e c t s are s i m i l a r t o those noted f o r milkbugs
(Oncopeltus) t r e a t e d w i t
for their anti-juvenil
c o n s t i t u e n t s are geranylhydroquinone ( I ) and i n l e s s e r amounts
geranylbenzoquinone ( H ) ( 8 ) . Both compounds are new n a t u r a l
products t o higher p l a n t s , but have been reported i n marine
urochordates ( 1 2 ) . Compound ( I ) has been synthesized as a
drug and found t o have cancer p r e v e n t i v e p r o p e r t i e s i n
experimental animals ( 1 3 ) . Another s p e c i e s , V_. Ixodes from Baja
C a l i f o r n i a , Mexico c o n t a i n s numerous quinones i n the trichomes
(10)• The compounds have been i d e n t i f i e d as geranylhydroquine
( I ) , 3-geranyl-2,5-dihydroxyphenyl a c e t a t e ( I I I ) ,
geranylbenzoquinone ( I I ) , 2-geranyl-6-hydroxy-4-methoxyphenyl
acetate ( I V ) , 2-geranyl-4-hydroxyphenyl a c e t a t e (V) and
6-hydroxy-2-methyl-2,4-methyl-3-pentenyl)-chromene ( V I ) .
Compounds ( I ) through (IV) were assayed f o r t h e i r c y t o t o x i c
and a l l e r g e n i c p o t e n t i a l on guinea p i g s , which are e f f e c t i v e
i n d i c a t o r s of contact a l l e r g e n i c i t y i n humans ( 1 0 ) . The same
compounds were a l s o tested on Tenebrio sp. (mealworm b e e t l e s ) ,
an experimental i n s e c t used t o t e s t the p o t e n t i a l of i n s e c t i c i d e s •
Geranyl-benzoquinone proved t o be a very potent e l i c i t o r of
a l l e r g i c s k i n r e a c t i o n s as w e l l as a potent i n s e c t i c i d e ( 1 0 ) •
A t o p i c a l a p p l i c a t i o n of 100>*g of geranylbenzoquinone on pupae
of Tenebrio caused severe a b n o r m a l i t i e s and death. Although
the exact mechanism of t o x i c i t y f o r ( I I ) i s not known, i t s
s i m i l a r i t y to the j u v e n i l e hormones suggests t h a t ( I I ) might
be a powerful a l k y l a t o r of enzymes r e g u l a t i n g j u v e n i l e hormone
s y n t h e s i s . Compound (VI) was not as potent as ( I I ) , but previous
r e p o r t s of (VI) i n C o r d i a a l l i o d o r a (Boraginaceae) i n d i c a t e that
(VI) i s a l s o an e f f e c t i v e i n s e c t i c i d e ( 1 4 ) .
Two species of P h a c e l i a , P. minor and p a r r y i , widespread
throughout the semi-arid mountains o f southern C a l i f o r n i a
contained as the major c o n s t i t u e n t geranylgeranylhydroquinone
( V I I ) . A minor c o n s t i t u e n t , 2-(-l-oxofarnesyl)-hydroquinone
( V I I I ) was a l s o present and p r e v i o u s l y reported i n a brown a l g a
(15) and Wigardia k u n t h i i o f the f a m i l y Hydrophyllaceae ( 1 6 ) .


Geranylgeranylhydroquinone ( V I I ) was a p p l i e d ( l O O x g ) to f i f t h
i n s t a r s of Oncopeltus (milkbugs) and found t o stunt growth and
wing development.
It i s apparent from our i n v e s t i g a t i o n s of prenylated
quinones from desert annuals, that these compounds are not only
potent s k i n s e n s i t i z e r s but a l s o p o t e n t i a l i n s e c t i c i d e s . The
t o x i c i t y of the prenylated quinones i s i n part due to t h e i r
l i p o p h i l i c i t y which permits the compounds t o penetrate the
c u t i c l e and a l k y l a t e important metabolic enzymes. Another
p o s s i b l e e x p l a n a t i o n f o r t h e i r a n t i j u v e n i l e a c t i o n i s that the
prenylated quinones are transformed i n t o a quinone methide
species which mimic the j u v e n i l e hormones (JH) and p o s s i b l y
i n t e r f e r e w i t h the p r o d u c t i o n of JH o r d e a c t i v a t e the hormones•
Chromenes i n the Asteraceae
In a recent l i t e r a t u r
i n f l o w e r i n g p l a n t s , we have documented that approximately 90%
of the 200 compounds i s o l a t e d are present i n the sunflower
f a m i l y (Asteraceae). These n a t u r a l l y o c c u r r i n g chemicals have
r e c e n t l y received considerable a t t e n t i o n because of t h e i r potent
c y t o t o x i c and i n s e c t i c i d a l a c t i v i t y . Chemotaxonomically, not a l l
t r i b e s of the Asteraceae seem to produce chromenes or benzofurans,
w i t h the major t r i b e s capable o f s y n t h e s i z i n g chromenes i d e n t i f i e d
as the E u p a t o r i e a e , H e l i a n t h e a e , I n u l e a e , Senecioneae and the
Astereae (17)•
The chromenes and benzofurans are r a t h e r simple compounds
b u i l t from acetate and isoprene m e t a b o l i t e s . H e t e r o c y c l i c r i n g
formation gives r i s e to 2,2-dimethyl chromene or 2-isoprophenyl
benzofurans. The m a j o r i t y of known chromenes and benzofurans
e x h i b i t a methyl ketone moiety a t a p o s i t i o n para to the oxygen
of the h e t e r o c y c l i c r i n g . C o n s t i t u e n t s e s t e r i f i e d w i t h phenolic
a c i d s or l a c k i n g methyl ketones are r a r e .
The chromenes are well-known f o r t h e i r c y t o t o x i c and
a n t i j u v e n i l e a c t i v i t y i n i n s e c t s (18-19). The prococenes, simple
chromenes f i r s t i s o l a t e d from Ageratum (Asteraceae), have been
shown to act d i r e c t l y on the corpus a l l a t u m by d i r e c t c y t o t o x i c
d e s t r u c t i o n o f the parenchymal c e l l s ( 2 0 ) . The precocenes, when
a p p l i e d e x t e r n a l l y t o the second i n s t a r l a r v a e of the milkweed
bug (Oncopeltus f a s c i a t u s ) cause the nymphs t o molt to normal
t h i r d and f o u r t h i n s t a r l a r v a e , and then to precocious a d u l t s •
Recent biochemical s t u d i e s have suggested that the precocenes
undergo o x i d a t i v e a c t i v a t i o n w i t h the corpus alluatum and form
r e a c t i v e epoxides that a l k y l a t e n u c l e o p h i l i c s u b s t r a t e s . The
r e a c t i v e precocene intermediate i s suggested to be the quinone
methide ( 2 0 ) , a s i m i l a r r e a c t i v e species that we have proposed
for the prenylated quinones o f P h a c e l i a .
Although considerable i n f o r m a t i o n has been gathered on
c y t o t o x i c and a n t i j u v e n i l e a c t i o n of the precocenes, l i t t l e
information i s a v a i l a b l e on the d i s t r i b u t i o n of chromenes and

benzofurans i n desert p l a n t s and t h e i r p o s s i b l e r o l e as

phototoxic agents and feeding d e t e r r e n t s . With t h i s i n mind, we
have begun a d e t a i l e d phytochemical i n v e s t i g a t i o n of chromenes
and benzofurans i n dominant desert shrubs of the Sonoran and
Chihuahuan deserts that are c y t o t o x i c , phototoxic and
insecticidal.
Chromenes and Benzofurans i n Desert Shrubs
Using HPLC, we have surveyed i n d e t a i l taxa of E n c e l i a ,

F l o u r e n s i a , and Geraea. A l l of these genera were found to
produce l a r g e q u a n t i t i e s of chromenes and benzofurans (21)• I n
the genus E n c e l i a , chromenes can comprise up to 10% of the d r y
weight m a t e r i a l . The compounds are more common i n l e a v e s , but
have been detected i n the stem, f l o w e r i n g heads and i n some cases
i n seeds •
A detailed quantitativ
conducted on E n c e l i a f a r i n o s a ( b r i t t l e b u s h ) , a desert shrub common
throughout Baja C a l i f o r n i a , Sonora and southern C a l i f o r n i a . The
major chromene was i d e n t i f i e d as e n c e c a l i n ( I X ) , but a l s o present
was the benzofuran euparin (X) which i s p r i m a r i l y produced i n the
stems (22). A p p l i c a t i o n of e n c e c a l i n t o the f i r s t i n s t a r of
l a r v a e of the milkweed bug proved t o be moderately i n s e c t i c i d a l .
E n c e c a l i n , euparin, and 7-hydroxyencecalin (XI) were d i s s o l v e d
i n methanol and were a p p l i e d a t concentrations 5 mg-lOOj^g. Each
p e t r i d i s h contained 25-30 f i r s t i n s t a r l a r v a e of (). f a s c u l a t u s .
Concentrations of 1.2 mg/petri d i s h (or higher) of e n c e c a l i n
were l e t h a l to the l a r v a e w i t h i n a p e r i o d of three days. Lesser
concentrations of e n c e c a l i n showed no e f f e c t s , w h i l e euparin and
7-hydroxyencecalin was not t o x i c (23)• E n c e c a l i n does not
compare i n t o x i c i t y w i t h the precocenes, s i n c e 44ju-g of precocene
I I have been reported t o induce precocious metamorphosis i n
milkweed bug l a r v a e . The presence of a methylketone moiety i n
e n c e c a l i n i n s t e a d of a methoxy s u b s t i t u e n t probably r e s u l t s i n a
l o s s of a n t i j u v e n i l e a c t i v i t y noted f o r the precocenes. On the
other hand, 7-hydroxyencecalin was l e s s a c t i v e than e n c e c a l i n and
t h i s could be due to a more r a p i d d e t o x i f i c a t i o n of phenolic
compounds e x h i b i t i n g f r e e hydroxyl groups r a t h e r than methoxyl
groups (23).
As a feeding d e t e r r e n t , e n c e c a l i n was more a c t i v e . F i f t h
i n s t a r s of H e l i o t h i s zea (Lepidoptera) were exposed t o a r t i f i c i a l
d i e t s c o n t a i n i n g v a r y i n g amounts of e n c e c a l i n . At concentrations
of 0.35%, H. zea starved t o death (24). I t should be noted that
e n c e c a l i n i s present i n higher amounts i n the l e a v e s , t h e r e f o r e
suggesting that e n c e c a l i n and other l e s s c y t o t o x i c chromenes and
benzofurans are feeding d e t e r r e n t s . I t should be added that most
desert phytophagous i n s e c t s e i t h e r chew or suck p l a n t parts and
therefore are l i k e l y t o be r e p e l l e d before they consume the
chromenes. T o p i c a l a p p l i c a t i o n s are l e s s l i k e l y , but one could
speculate that the a c c i d e n t a l r u p t u r i n g of glandular h a i r s could


r e s u l t i n the d e p o s i t i o n of a c t i v e c y t o t o x i n s on an i n s e c t
cuticle•
A n a l y s i s of the stems of E n c e l i a ventorum from Baja
C a l i f o r n i a showed the presence of a number of benzopyran
and benzofuran d e r i v a t i v e s and two stereo isomers of a
novel e u p a r i n - e n c e c a l i n ( X I I ) dimer (25). A closely related
euparin-encecalin dimer ( X I I I ) , was p r e v i o u s l y i s o l a t e d from E.
f a r i n o s a (26)* Although the dimer i s not photoactive,
p r e l i m i n a r y s t u d i e s i n d i c a t e that i t i s a feeding deterrent to
H e l i o t h i s zea.
The benzofuran 6-methoxyeuparin (XIV) and the two chromenes
e n c e c a l i n and 7-hydroxyencecalin from species of E n c e l i a
(Asteraceae) from Baja C a l i f o r n i a have been shown to be phototoxic
to s e v e r a l b a c t e r i a and yeast i n long wave UV l i g h t (23).
Compound (XIV) was the most a c t i v i t y against Pseudomonas
f l u o r e s c e n s , an organis
p h o t o s e n s i t i z e r 8-methoxypsorale
a c t i v e against Saccharomyces c e r e v i s i a e and Candida a l b i c a n s .
P r e l i m i n a r y experiments w i t h human erythrocytes w i t h t h i s
new c l a s s of p h o t o s e n s i t i z e r s r u l e s out the membrane as a t a r g e t ,
since the chromenes seem to behave l i k e the p h o t o s e n s i t i z i n g
furanocoumarins by i n t e r a c t i n g w i t h n u c l e i c acids or i n t r a c e l l u l a r
molecules i n l i g h t • Further experimentation i s needed to c l e a r l y
understand t h e i r mode of a c t i o n .
Numerous chromenes and benzofurans have a l s o been i s o l a t e d
from F l o u r e n s i a , a genus that i s dominant i n the desert of
Chihuahua, Mexico. A benzofuran (XV) w i t h a cinnamic a c i d moiety
from F^. dentata and i l i c i f o l i a has r e c e n t l y been shown to be
extremely t o x i c to m i l k weed bugs and h i g h l y phototoxic (28).
Approximately 30 chromenes and benzofurans have been i s o l a t e d
from other desert sunflowers and we are c u r r e n t l y screening them
f o r i n s e c t i c i d a l and phototoxic a c t i v i t y .
Concluding Remarks
Desert p l a n t s are remarkable phytochemical f a c t o r i e s • In

t h i s chapter, we have covered only two c l a s s e s of compounds;
chromenes and prenylated quinones that are a l l e r g e n i c , c y t o t o x i c
and i n s e c t i c i d a l . Many other desert p l a n t s produce r e s i n s that
are complex mixtures of s e s q u i t e r p e n o i d s , chromenes, f l a v o n o i d s
and quinones. These mixtures might not be of i n t e r e s t to
phytochemists, but to the p l a n t , secondary metabolites are
e s s e n t i a l f o r s u r v i v a l and r e p r o d u c t i o n . L i k e the chromenes and
quinones, many n a t u r a l c o n s t i t u e n t s of a r i d land plants play a
dual r o l e . In some cases, the n a t u r a l products excreted on the
l e a f or secreted by trichomes are f u n c t i o n i n g as a n t i d e s s i c a n t s
(prevent water l o s s through the c u t i c l e ) , p r o t e c t i n g the l e a f
from harmful r a d i a t i o n and, most important, keeping the plant
healthy against phytophagous i n s e c t s and pathogens. The chromenes
and benzofurans are chemicals that are antifeedants and e x h i b i t

a n t i j u v e n i l e a c t i v i t y when a p p l i e d t o p i c a l l y . The chromenes are

p r i m a r i l y r e s t r i c t e d t o members o f the Asteraceae and are more
widespread i n desert sunflower species than p r e v i o u s l y thought.
In combination w i t h sesquiterpene l a c t o n e s , the chromenes and
benzofurans are another group of defensive compounds that desert
i n v e r t e b r a t e s have to d e t o x i f y . Indeed, the success of many
desert sunflower species i s i n part due t o t h e i r d i v e r s e secondary
chemistry. P r e n y l a t e d quinones, on the other hand seem t o be
r e s t r i c t e d to the Hydrophyllaceae and t r o p i c a l t r e e s . The
quinones are extremely a c t i v e chemicals that e x h i b i t i n s e c t i c i d a l
a c t i v i t y a t c o n c e n t r a t i o n s lower than many chromenes• The
prenylated quinones of P h a c e l i a are a l s o potent c y t o t o x i n s and
allergens•
Acknowledgement s
This research has

PCM-8209100 and the Focused Research Program a t UCI. I am g r e a t l y
indebted to my research a s s o c i a t e s and colleagues c i t e d i n the
r e f e r e n c e s , namely, Dr. G.H.N. Towers (UBC, Canada), Dr. Gary
Reynolds (LSU), Dr. Peter Proksch (UCI), Charles Wisdom (UCI),
Manuel A r e g u l l i n (UCI) and Margareta Proksch (UCI).
Literature Cited
1. Rodriguez, E. "Aspects o f American H i s p a n i c and Indian

Involvement in Biomedical Research." M a r t i n e z , J.V. and
Marinez, D.I. Eds. SACNAS, Bethesda, Maryland. 1981, p. 244.
2. Campos-Lopez, E.; Roman-Alemany, A. J. Agric. Food Chem.
1980, 28, 171-183.
3. Mabry, T.J. and U l u b e l e n , A. J. Agric. Food Chem. 1980, 28,
188-196.
4. Rosenthal, G.A.; Janzen, D.H. "Herbivores: Their I n t e r a c t i o n
w i t h Secondary Plant M e t a b o l i t e s . " ; Academic Press: New York,
1979, p. 604.
5. K e l s e y , R.; Reynolds, G.; Rodriguez, E. "Biology and
Chemistry of Plant Trichomes." Healey, P., Mabry, T. and
Rodriguez, E. Eds. Plenum P r e s s : New York, in p r e s s .
6. Gibson, R.W. Ann. A p p l . Biol. 1976, 68, 113-117.
7. Rodriguez, E. Rev. Latinoamer. de Quimica 1978, 9, 125-131.
8. Reynolds, G.: Rodriguez, E. Phytochem. 1979, 18, 1567-68.
9. Reynolds, G.; E p s t e i n , W.L.; T e r r y , D.; Rodriguez, E. J.
Contact D e r m a t i t i s 1980, 6, 272-274.
10. Reynolds, G.; Rodriguez, E. P l a n t a Medica 1981, 43, 187-193.
11. Bowers, W.S. "The J u v e n i l e Hormones" G i l b e r t , L . I . Ed.
Plenum: New York, 1976, pp. 394-408.
12. F e n i c a l , W. "4th Proceeding of Food Drugs from the Sea."
Marine T e c h n o l o g i c a l S o c i e t y : Washington D.C., 1974.
13. R u d a l i , P.G.; M e n e t r i e r , L. Therapie 1967, 22, 895-899.

14. J u r d , L.; Manners, G.D. J. Agric. Food Chem. 1980, 28,

183-188.
15. Ochi, M.; K o t s u k i , H.; Inoue, S.; T a n i g u c h i , M.;
Tokoroyama, T. Chem. Lett. 1979, 831-833.
16. Gomez, F.; Quijano, L.; Calderon, J.S.; R i o s , T.
Phytochem. 1980, 19, 2202-2203.
17. Proksch, P.; Rodriguez, E. Phytochem. 1982, in p r e s s .
18. Bowers, W.S.; Ohta, T.; C l e e r e , J.S.; M a r s e l l a , P.A.
Science 1979, 193, 542-548.
19. Bowers, W.S. Am. Z o o l . 1981,21,737.
20. Bowers, W.S. Science 1982, 217, 647-648.
21. Proksch, P.; Rodriguez, E. J. of Chroma. 1982, 240,
543-546.
22. Wisdom, C.; Rodriguez, E. Biochem. System. and Ecology,
1982, 10, 43-48.
23. Proksch, P.; Proksch
J. of N a t u r a l Products
24. Wisdom, C. Ph.D. T h e s i s , U n i v e r s i t y of California, I r v i n e ,
CA, 1982.
25. Proksch, P., Aregullin, M. and Rodriguez, E. P l a n t a Med.,
1982, in p r e s s .
26. S t e e l i n k , C.; M a r s h a l l , G.P. J. Org. Chem.: 1979, 44,
1429-1433.
27. Towers, G.H.N.; Graham, E.A.; Spenser, I.D.; Abramowski,
Z. P l a n t a Med. 1981, 41, 136-138.
28. Aregullin, M.; personal communication.

18
Role of Lipids in Plant Resistance to Insects
D A V I D S. S E I G L E R
University of Illinois, Department of Botany, Urbana, IL 61801
In the coevolutionary interactions of plants and animals

l i p i d s play a major r o l e
allomones and kairomones
t i o n . Host plant resistance i s p a r t i a l l y dependent on these
chemical constituents. Lipids may be subdivided into two types.
V o l a t i l e l i p i d s are generally involved i n long distance inter-
actions whereas non-volatile l i p i d s are generally involved after
the insect has contacted the host plant. Several examples of
each are reviewed. U t i l i z a t i o n of these compounds to promote
increased host plant resistance could be accomplished by selection
of plants rich i n allomones, lacking kairomones for a particular
pest or those with inducible systems of defense. Another approach
is to isolate the defensive compounds of one plant and apply them
to crop plants. Trap crops could also be used to lure insects
away from other crops.
Despite the fact that a majority of insects are phytophagous
(i.e. they eat plants), the world around us is still green (1,2).
Of approximately 1,000,000 known insect species, only a few thou-
sand are "pests" and of these only about 500 cause appreciable
damage (3). Although many factors are involved i n maintaining
this balance between plants and insects, plant secondary compounds
are generally conceded to play a major r o l e .
Coevolution of Plants and Insects
In order to understand f u l l y the importance of these chemical

factors, i t 1s necessary to consider the processes which are
probably responsible for their d i v e r s i f i c a t i o n . A mechanism of
coevolution of insects and plants was set forth eloquently by
Erlich and Raven (4). According to their hypothesis, angiosperms
produced a series of chemical compounds which were not d i r e c t l y
related to their basic (or primary) metabolic pathways, but which
were otherwise not harmful to the plants growth and development.
In practice, these compounds may play other roles such as interac-
tions within plants as primary compounds (5^8) or i n interactions
0097-6156/83/0208-0303$07.25/0

with other organisms (plant-plant, plant-fungus, plant-bacterium,

e t c . ) - Functions such as these almost certainly predated the
roles of these secondary compounds in pi ant-insect interactions,
and i t i s clear that repellancy and attraction cannot be the sole
raison d'etre of these substances (9,10).
Fortuitously, some of the compounds may have reduced the p a l -
a t a b i l i t y of the plants in which they were produced and/or in some
manner reduced the fitness of the insects which normally ate the
plant, and then these plants entered a new adaptive zone. Evolu-
tionary radiation of the plants might then have followed and
eventually what began as a chance mutation, might ultimately have
characterized an entire family or group of families (4).
In a similar fashion, i f a recombinant or mutation occurred
1n a population of insects that enabled Individuals to feed on
some previously protected group of plants, selection would carry
that l i n e into a new adaptiv
be able to diversify wit
phagous insects. A l l in a l l , the diversity of plants would tend
to augment the diversity of Insects and the diversity of phyto-
phagous insects would tend to enhance the diversity of plants.
The situation i s more complex than that proposed as no insect
nor any plant evolves with regard to only one other organism (11,
12). Any change which occurs must not modify unfavorably the
organism's overall fitness with regard to other organisms,
physical and environmental factors and the organisms own develop-
mental sequence and physiological processes. The success or
f a i l u r e of an organism i s rarely ascribable to any single factor.
As an insect became more highly adapted to a particular food
source, recognition of the food plant by v i s u a l , chemical or other
means would be strongly favored.
The secondary compounds involved in these coevolutionary
processes have been called "allelochemics" (V3) and "ecomones"
14,15). Three major classes have been defined: pheromones,
which are i n t r a - s p e c i f i c chemical messengers, and allomones (com-
pounds which are deleterious to the receiving organism) and
kairomones (compounds which are beneficial to the receiving
organism) (13,16,17). Blum (lj3) suggests that the term "ecomone"
be used for a l l external chemical messengers as was proposed by
Florkin (14) and that terms other than pheromone ( i n t r a s p e d f i c
messengers! and allomone (Interspecific messengers) be abandoned
as many Individual compounds may at the same time serve 1n d i f -
ferent roles among various organisms. By e x p l i c i t l y stating the
function of the allomone 1n the particular case analyzed, i t s
idiosyncratic role can be stressed without obscuring any other
functions that i t may possess. The potential use of pheromones
for control of insect pests has recently been reviewed (18).
Allomones are those substances involved in i n t e r s p e c i f i c communi-
cation which give an adaptive advantage to the producing organism
and kairomones are compounds which give an adaptive advantage to
the receiving organism. Kairomones are often allomones to which

18. SEIGLER Role of Lipids 305
insects have become coadapted (19), but may have other origins
(20). —
For example, gossypol, a terpenoid substance, i s an allomone
that l i m i t s herbivory by several lepidopteran species on cotton
(Gossypium spp., Malvaceae), whereas i t i s a kairomone for the
boll weevil, Anthonomus grandis and acts as a feeding stimulant
for this Insect (3,21,22).
The means by which many insects select a suitable host plant
1s by being attracted by plant secondary compounds that also serve
as allomones. In other cases the Insect may avoid the presence of
a toxic compound by the correlated presence of other materials
which may be repellant.
Some compounds such as gymnemic acid from Gymnema sylvestre
(Asclepiadaceae) (which depresses the precelved sweetness of
sugars) are known to d i s t o r t the taste of others (23) and may play
a role in "disguising" th
substances.
It has been suggested that insects were primitively poly-
phagous herbivores (24,25) although others (9) have suggested that
many primitive insects were saprophagus feeders. In either case,
in the process of coevolution, there has been a tendency toward
specialization (2,26) and today, most insects are monophagous or
oligophagous. Many apparently polyphagous groups are comprised of
closely related subtaxa which are each monophagous (1_). Factors
other than plant chemistry are also sometimes Important in the
evolution of biochemically specialized insects (27).
The process of host selection by an insect i s complex and
involves five major steps: host habitat finding, host finding,
host recognition, host acceptance, and host s u i t a b i l i t y (25).
Each step in the process of host-plant selection may be mediated
by plant components. Both the secondary chemistry and nutritional
value play a major role in the s u i t a b i l i t y of the host.
About 85% of a l l Insects are holometabolous, and the food
plants of larval and adult stages often d i f f e r (28). In most holo-
metabolous insects host recognition may have been predetermined
for the larva by the ovipositing female (1_). In some cases i t has
been demonstrated that the larva prefers to eat the food upon
which they are i n i t i a l l y fed (Induction), even i f It's not the
appropriate host (29).
Mechanisms of Host Plant Resistance
For an Insect to successfully u t i l i z e i t s host plant, i t must

complete each of the f i v e aforementioned steps. Any circumstances
which preclude this w i l l convey an advantage to the host plant.
A l l components must be present at the proper time and in adequate
amounts. Plant resistance may result from disruption of the
normal sequence of events or reduced presence of kairomones or
enhancement of allomones (25). This resistance is under genetic
control but may vary with environmental conditions; factors
involved in ecological resistance have been reviewed (25).

Three principal types of genetic resistance have been pro-

posed: preference or non-preference, a n t i b i o s i s , and tolerance
(30).
A number of resistance factors influence behavioral processes
and hence determine an insect's preference or non-preference for a
particular plant. Hosts which do not contain the proper kairo-
monal compounds are often t o t a l l y rejected as food plants and by
ovipositing females. Dethier (29) noted, however, that plants are
almost never neutral, but are almost always either attractive or
repellant. As previously observed, the ovipositional choice of
the female imago and the food choice of the larvae usually
coincide (25).
The presence of kairomones i s usually involved in the selec-
tion of a food plant. Resistant plants usually lack or have too
l i t t l e of the normal kairomones, the kairomones are inhibited or
blocked by antagonisti
(25,31).
"Adverse physiological effects (antibiosis) resulting from
ingestion of a plant by an insect may range from mild to l e t h a l ;
the principal symptoms are death of the larvae in the f i r s t few
stadia, abnormal growth rates, abnormal conversion of ingested
and/or digested food, f a i l u r e to pupate, f a i l u r e of adult emer-
gence from the pupae, malformed or subsized adults, f a i l u r e to
concentrate food reserves followed by unsuccessful hibernation,
decreased fecundity, reduced f e r t i l i t y , and restlessness or other
irregular behavior (25). These a n t i b i o t i c effects may be caused
by several factors, one of which i s the presence of toxic metab-
o l i t e s (see reference 25 for a more complete l i s t ) . Antibiosis
is often the most evident mechanism of resistance.
Morphological or structural plant features which impair
normal feeding or oviposition by insects or contribute to the
action of these mortality factors are often grouped as "phenetic
resistance" (25).
Certain plants can repair injury or produce an adequate y i e l d
despite supporting an insect population at a level capable of
damaging a more susceptible host. Several factors are important
in tolerance (see reference 25).
The role of other plants in the community and of other
trophic levels in resistance has been emphasized (11_,12).
Insect Resistance in Crop Plants
Resistance to insects has been successfully selected and

introduced into important cultivars of a number of plant species
including potatoes, wheat, corn, grapes, a l f a l f a , barley, beans
sorghum, rice and sugar cane (25). This resistance i s usually due
to a combination of factors and only in a few cases is a single
chemical factor i d e n t i f i a b l e as responsible for resistance (32).
In general, decreased pest resistance has occurred in the

domestication of crop plants. This factor and the fact that many
crops are "apparent" or "predictable" has increased the levels of
herbivory on them (33).
There are a number of p o s s i b i l i t i e s for using plant secondary
chemistry to control herbivory in crop plants. One p o s s i b i l i t y 1s
to select for insect resistant lines and though 1t has been done
in only a few cases, select for s p e c i f i c allomones. There are,
however, some potential problems with this approach. There i s a
cost for the production of the secondary compounds which may be
useful for defense (33). Insect resistant soybean cultivars pro-
duce lower yields of seeds and accumulate nitrogen at a slower
rate than insect susceptible varieties 1n the absence of herbivores
(34). Conversely, varieties of crop plants selected for high y i e l d
are often more susceptible to insects, pathogens, and weeds (35).
Several trophic levels must be considered. Breeding plants
with greater allomone conten
herbivores to accumulate higher levels of these compounds and
discourages parasites that normally control herbivore levels (36).
The presence of secondary compounds may also a l t e r the usefulness
of the crop plant to man or his domestic animals. Lines of cotton
with high gossypol content have Increased insect resistance with
regard to a number of insects, but have reduced value as food
materials for livestock.
In the plant, part of the metabolic cost of producing and
maintaining pools of secondary compounds may be reduced by using
compounds which contain only carbon, hydrogen and oxygen (which
are rarely l i m i t i n g ) , by recycling the compounds or by use of
Inducible systems of defense.
Inducible systems of defense (phytoalexins) are widespread 1n
plants and are effective against many types of fungi and bacteria
(37,38). Similar systems have been demonstrated in a few cases
with insects and are probably common 1n nature (see for example
reference 39). Although Inducible systems of insect resistance
would seem to be e f f i c i e n t and e f f e c t i v e , no system 1s foolproof.
The larvae of Epilachna tredecimnotata cut a c i r c u l a r trench in
Cucurbita leaves and prevent mobilization of the deterrent sub-
stances to the area which i s then consumed (40).
It should be possible to create "trap crops", preferentially
attract the insects (via kairomones), destroy them and thus
protect the desired crop (25).
Another approach may be to Isolate the allomones from Qjie
plant and apply them to the surface of another plant and thus
protect 1t (25,31). The p r a c t i c a l i t y of this approach depends on
many factorsHTncTuding residual t o x i c i t y , cost, and s t a b i l i t y of
the compounds involved.
Several l i s t s of plant secondary compounds which are involved
1n plant-insect interactions have been compiled (1,9,13,17,31,41-
461. I n
addition, l i s t s of plants with insecticidal properties
have been published (47,48). Many of these compounds are physi-
c a l l y located on the outside of the plant, either in the epidermal

layer, in trichomes or glands or on the outer surface. Others are

located in flowers, seeds and f r u i t s (1,33,44,49-51).
Table 1. Principal Classes of Chemical Plant Factors ( A l l e l o -

chemics) and the Corresponding Behavioral or Physiological Effect
on Insects.
Allelochemic Factors Behavioral or Physiological Effects
Al1omones Give adaptive advantage to the producing

organism
Repellents Orient Insects away from plant
Locomotor excitants Start or speed movement
Suppressants
Deterrents Prevent maintenance of feeding or o v i -
position
Antibiotics Disrupt normal growth and development of
larvae; reduce longevity and fecundity
of adults
Antixenotics Disrupt normal host reduction behavior
Kairomones Give adaptive advantage to the receiving
organism
Attractants Orient Insects toward host plant
Arrestants Slow or stop movement
Feeding or o v i - E l i c i t b i t i n g , piercing, or oviposition;
position excitants promote continuation of feeding
Source: Reproduced with permission from Ref. 134.
A system of c l a s s i f i c a t i o n (based largely on the previously

proposed systems of Dethier et a l - (52), and Beck (53J, and
Whlttaker and Feeney (16)) of the major types of chemical factors
Involved in plant-insect interactions has been proposed (25)
(Table 1). Unfortunately, i t 1s often d i f f i c u l t to judge from
l i t e r a t u r e data, Into which class of allomone or kalromone a
particular compound should be placed (see also reference 15).
Compounds which are referred to as "antifeedants" in the l i t e r a -
ture are Involved 1n the Inhibition of b i t i n g and piercing and
inhibition maintenance of feeding. These compounds generally do
not k i l l the insect but cause starvation because they prevent
feeding (54).
Many of the plant secondary compounds Involved in plant-
insect relations are l i p i d s . This group is not of a single bio-
synthetic origin but is comprised generally of compounds soluble
in non-polar solvents. Among the common groups of l i p i d s found in
plants are fatty acids and t h e i r derivatives (hydrocarbons, alde-
hydes, alcohols, esters, glycerides, acetylenic compounds, waxes,
e t c . ) , phenylpropanoids (flavonoid aglycones, lignans, coumarins

e t c . ) , and terpenoid derived compounds (monoterpenes, sesquiter-

penes, diterpenes, triterpenes, steroids, tetraterpenoids or
carotenoids). Although many alkaloids are l i p i d s , they are not
discussed in this review. Lipids are often localized on the sur-
face of the paint or produced by special glands or trichomes (50).
Other compounds of this type serve as energy reserves within tfie"
plant and are especially important to both plants and herbivores.
Lipids may be subdivided into two general groups: v o l a t i l e and
non-volatile l i p i d s . There i s considerable overlap between the
two groups.
In the following sections, recent examples from the l i t e r a t u r e
have been cited. They are by no means exhaustive. Many previously
described examples are cited in e a r l i e r works, especially that of
Hedin (41).
V o l a t i l e Lipids
V o l a t i l e compounds are often involved in long distance

attraction and are especially important as attractants and repel-
lents (as defined by Kogan, 2!5). One major class of v o l a t i l e
materials, essential o i l s , i s comprised of complex mixtures of
terpenes, phenylpropanoid derived compounds and a number of esters,
alcohols, aldehydes, ketones, acids, and hydrocarbons. The con-
stituent compounds are mostly of low to medium molecular weight
and generally not highly oxygenated. Some of the biological
properties of these compounds have been reviewed (17,41,46,55,56).
Many of the monoterpenes found in essential o i l s of plants
also occur as pheromonal substances in insects (45,57-60) and are
often involved in plant-insect interactions. Some compounds found
both in plants and insects are the monoterpenes c i t r o n e l l a l , c i t -
r o n e l l o l , geraniol, myrcene, c i t r a l , B-phellandrene, limonene,
2-terpinolene, a-pinene, 6-pinene, 1,8-cineole, and verbenone.
Oil of c i t r o n e l l a (the essential o i l of Andropogon nardus,
Poaceae or Gramineae) has long been used as a mosquito repellent.
This o i l i s mostly composed of geraniol with lesser amounts of
c i t r o n e l l o l , c i t r o n e l l a l , and borneol. Other essential o i l s have
also been used to repel insects (49J. A number of monoterpenes
and methyl esters of fatty acids were evaluated for their repel-
lent and attractant properties toward Ips, Dentroctonus, and
Hylurgops species. Although a c t i v i t y was observed in the labora-
tory s none of the compounds tested appeared active in f i e l d tests
(61), Bay leaves (Laurus n o b i l i s , Lauraceae) and cineole,
geraniol, and piperidine (which occur in bay leaves) possess
repellent properties toward cockroaches (62). Essential o i l s are
involved in the feeding response of several Papilio species to
members of the Apiaceae (Umbelliferae) (9). The aphid Cavariella
aeciopodii which lives on members of the Apiaceae in summer, i s
attracted to the monoterpene carvone but less so i n the presence
of l i n a l o o l which has a repellent effect (63). (+)-3-Thujone and
(-)-3-isothujone, which make up most (80-90%) of the leaf essential

3-phellandrene limonene 2-terpinolene a-pinene
6-pinene 1,8-cineole verbenone
o i l of Thuja p l i c a t a , western red cedar, are feeding deterrents to

the white pine weevil (Pissodes strobi) (64),
Essential o i l s are known to i n h i b i t microbial a c t i v i t y in
ruminants and disrupt digestive processes (65).
Some v o l a t i l e i r i d o i d monoterpenes with biological a c t i v i t y
are also found in essential o i l s and in insect pheromonal and
defensive substances. Eisner (66) found that 17 species of insects
were repelled by the i r i d o i d monoterpene nepetalactone. Lacewings
(Chrysopa septempunctata) are attracted by the leaves and f r u i t s
of Actinidia polygama (Actinidiaceae) which contain a series of
v o l a t i l e i r i d o i d monoterpenes (67).
Many sesquiterpenes which are not highly oxygenated are also
found in essential o i l s . Several of these are reported to possess
a c t i v i t y . a-Farnesene from apples i s an attractant and oviposition
stimulant for the codling moth (68) and farnesol has been demon-
strated to be an active feeding deterrent to gypsy moth larvae
(Lymontria dispar) (69).
A number of v o l a t i l e phenylpropanoid compounds have pro-
nounced biological properties and are also found in essential

farnesol
o i l s (70J. The t o x i c i t y of phenyl propanoic! compounds in seed o i l s

has been reviewed (55). Several simple, v o l a t i l e phenylpropanoid
compounds such as eugenol and myristicin are known to be a l l e l o -
chemics (17). M y r i s t i c i n , isolated from parsnip, Pastinaca sativa
(ApiaceaeJT has been shown to be an antifeedant compound (71JT
The essential o i l of Acoru
This compound causes depression of development of the gonads and
ovaries of the insect Dysdercus koenigii (72). Although t h i r d and
fourth instar larvae moulted normally to the next i n s t a r s , 5th

instar larvae moulted normal to adults but the ovaries were

i r r e v e r s i b i l y affected. In such adults, the ovaries remained
permanently immature.
Several phenylpropanoid compounds have pronounced antifeeding
a c t i v i t y . Isoasarone (from Piper futokadzura, Piperaceae) and
piperenone were highly active against Spodoptera l i t u r a (54).
Many short chain fatty acids, aldehydes, alcohols, esters,
ketones and hydrocarbons are produced by metabolism of fatty acids
( C i 6 - C i ) . These compounds are common in essential o i l s and are
8
also found in insects.

Acetaldehyde, ethyl alcohol and ethyl acetate, which are
present in ripening figs are attractive to several Carpophilus
species (Coleoptera: Nitidulidae) (73). Several short chain
alcohols including ethanol, n-propanol, 2-propanol, isobutanol and
n-butanol serve as oviposition stimulants for the moth Ectomyelois
ceratoniae. This moth onl
infested with a fungus of the genus Phomopsis which apparently i s
responsible for producing the alcohols (74). Compounds such as
trans-2-hexen-l-ol, 1-hexanol, cis-3-hexen-T-ol and trans-2-hexenol
(and l i n a l o o l ) are involved in the olfactory orientation of the
Colorado beetle, Leptinotarsa decemlineata to the foliage of
potato (Solanum tuberosum, Solanaceae) (75). A similar complement
of compounds seem to be involved in the attraction of the a l f a l f a
seed chalcid (Bruchophaqus roddi) to a l f a l f a (Medicago s a t i v a ,
Fabaceae) (7(6). Similar groups of compounds are probably involved
in many problems of attraction of insects over r e l a t i v e l y long
distances.
Mustard o i l s which are found in some essential o i l s and are
probably the hydrolysis or breakdown products of glucosinolates,
are involved in host plant location of a number of groups of
insects (9J e.£. P i e r i s braccicae and P_. rapae on plants in the
Brassicaceae (Cruciferae)
Essential o i l s are especially important in mutualistic r e l a -
tionships between plants and insects such as p o l l i n a t i o n and seed
and f r u i t dissemination (37,45,77-79). In these instances essen-
t i a l o i l components serve as attractive substances for the plant.
These compounds vary widely in composition but contain most of the
chemical types described above. In some cases even v o l a t i l e
amines and skatole are involved, as in the pollination of
Sauronatum guttatum (Araceae) (80).
Non-Volatile Lipids
While v o l a t i l e l i p i d s are often involved in attraction of the

insect to the plant from a distance, non-volatile l i p i d s are
frequently involved in b i t i n g s t i m u l i , continued feeding or pre-
vention of feeding and the disruption of normal growth (25). Many
types of compounds that are l i p o p h i l i c in nature grade into more
polar groups, especially in series which are extensively oxygenated.
Among these are oxygenated sesquiterpenes, diterpenes, triterpenes,

tetraterpenes (carotenoids), phenylpropanoid compounds (simple,

and oxygenated phenylpropanoids, coumarins, flavonoids, lignans,
e t c . ) , but also glycerides, fatty acids and their derivatives.
A number of l i p i d s are pigments in plants. The most impor-
tant group i s probably carotenoids. The role of carotenoids in
plant-insect interactions has been reviewed (81,82).
Several terpenoid compounds serve as juvenile hormones (or
mimics) and ecydsones respectively (83) in insects.
The biological a c t i v i t y and t o x i c i t y of terpenoids to herbi-
vores has been discussed (56^,84) and representatives of each major
type of terpene are known to be active. Well known examples are
sesquiterpene lactones, pyrethrins, and several classes of d i t e r -
penes and triterpenes.
Several non-volatile i r i d o i d monoterpenes occur as glycosides
and have been observed to have biological a c t i v i t y . For example,
xylomolin, from the unrip
(Meliaceae) has antifeedant a c t i v i t y against Spodoptera exempta an
African armyworm at 100 ppm (85) and crotepoxide from Croton
macrostachys (Euphorbiaceae) possesses antifeedant a c t i v i t y
against Spodoptera exempta (85).
A number of sesquiterpenes have been demonstrated to have
pronounced biological a c t i v i t y (84); among the non-volatile com-
pounds the sesquiterpene lactones are best known (86) but other
oxygenated sesquiterpenes are also known to be active. For
example, the role of gossypol, a dimeric sesquiterpene and struc-
t u r a l l y related compounds has been investigated (21,22). The
oxygenated sesquiterpenes, shiromodiol monoacetate and diacetate,
from Parabenzoin trilobum (=Lindera t r i l o b a Blume) possess potent
antifeeding a c t i v i t y toward Spodoptera l i t u r a larvae (85).
l a s i d i o l angelate

Leaf cutter ants, abundant from Texas to Argentina are polyphagous

herbivores, but w i l l not attack several plants. The ant, Atta
cephalotes, for example, does not feed on Lasianthaea fruticosa
(Asteraceae). The active repellent substance has been demonstrated
to be l a s i d i o l angelate (87).
Polygodial, ungandensidial, and warburganal from the bark of
Warburgia stuhlmanii and W. uqandensis (Cannellaceae) have also
proven to be highly active against Spodoptera exempta. Warburganal
is a highly active antifeedant (0.1 ppm against S.. exempta) (85).
warburganal polygodi al
The i r i d o i d monoterpenes, catalpal and catalposide occur in

nectar in flowers of Catalpa speciosa (Bignoniaceae) and are toxic
to many non-coadapted insects which attempt to rob nectar. The
bees which normally pollinate this plant are r e l a t i v e l y insensitive
to the effects of these compounds (88).
A number of diterpenes are known to be active against herbi-
vores (84). The diterpenes a b i e t i c , dehydroabietic, 12-methoxy-
a b i e t i c , sandaracopimaric, and isopimaric acid serve as feeding
deterrents for the larch sawfly, Pristiphora erichsonii in single
needles from new shoots of tamarack (Larix l a r i c i n a ) (133). The
larvae of this insect do eat tufted needles on short shoots of the
same trees.
Varieties of sunflower (Helianthus annums, Asteraceae) that
are resistant to attack by larvae of the sunflower moth (Homeosoma
electellum) contain high concentrations of trachyloban-19-oic acid
and (-)-16-kauren-19-oic acid in their florets (84,132).
Ryania speciosa (Flacourtiaceae) and several related species
are unique in that their insecticidal a c t i v i t y was discovered as
a part of a search for new insecticides (89). The toxic principle
is a diterpene e s t e r i f i e d to pyrrole-2-carboxylic acid. This
insecticide proved to be somewhat selective in i t s a c t i v i t y (89).
A series of complex oxygenated diterpenes i s known to be
antifeedant. Several of these occur in the Lamiaceae (Labiatae)
and Verbenaceae, but are found in other families as well ( e . £ . in

trachyloban-19-oic acid (-)-16-kauren-19-oic acid
the Asteraceae). Clerodendrin A & B from verbenaceous plants

possess potent antifeeding a c t i v i t y against some insects (e.c[.
Spodoptera l i t u r a ) , but less against others (Calospilos miranda).
Several genera of plants from this family contain similar com-
pounds: for example Clerodendron, Caryopteris, Callicarpa (54).
Ajugarins, from Ajuga remota (Lamiaceae or Labiatae) have ent-
clerodane diterpenoid structure. They have antifeedant a c t i v i t y
against Spodoptera exempta at 100 ppm (85J, Ent kaurenoid d i t e r -
penes such as i n f l e x i n from Isodon inflexus (Lamiaceae) and i s o -
domedin from L shikokianus var. intermedius had antifeedant
a c t i v i t y against the African armyworm (Spodoptera exempta). They
are also highly cytotoxic (LD 5.4 and 4.0 yg/ml respectively)
50
(85), Two diterpenes, cinnzeylanine and cinnzeylanol from

Cinnamomum zeylanicum (Lauraceae) have been demonstrated to possess
insecticidal a c t i v i t y (90). At 2 to 4 ppm these compounds inhib-
ited larval ecdysis in Bombyx mori.
Many triterpenes also have antiherbivore a c t i v i t y . In
general, those which are highly oxygenated seem to be more
active in this regard (84). The role of cardiac glycosides,
insects and their predators has been reviewed (91-94). A number
of metabolically altered triterpenes from the Rutaceae, Meliaceae
and Simaroubaceae are antifeedants. Extracts of neem tree seeds
(Azadirachta indica, Meliaceae) were shown to be repellent to a
number of insects when applied to various crop plants at low
concentrations. The probable active compound i s t e t r a n o r t r i t e r -
pene, azadirachtin (95). This compound from the leaves and f r u i t s

of A. indica and Melia azedarach (Meliaceae) gives 100% feeding

inhibition at 40 yg/liter against Schistocerca gregaria (desert
locust) (85). Harrisonin from Harrisonia abyssinica (Simaroubaceae)
had antifeedant a c t i v i t y against Spodoptera exempta at 20 ppm (85).
Both harrisonin and azadarichtin are limonoid compounds.
Several simple non-volatile phenylpropanoids are known to be
allelochemics. Among these are p-coumaric acid, cis and trans-
caffeic acid (17), chlorogenic acid, and a caffeyl derivative of
an aldaric acid (97).
Coumarin (0.1%), f e r u l i c acid (0.1%) and p-coumaric acid (5%)
were shown to be toxic to the larvae of the bruchid beetle,
Callosobruchus maculatus (96).
Furocoumarins such as isopimpinellin, bergapten, and kokusagin
have antifeedant a c t i v i t y against Spodoptera l i t u r a (54). A number
of similar compounds from umbelliferous plants have been demon-
strated to be active antifeedant
Periplaneta americana, Musc
Stylopyga rhombifolia (98).
Xanthotoxin, a linear furocoumarin occurs in many plants of
the Apiaceae. This compound is not appreciably toxic to the
larvae of Papilio polyxenes which normally feed on umbelliferous
plants. Angelicin, an angular furanocoumarin, which i s found in
only a few r e l a t i v e l y advanced tribes of the Apiaceae, reduces
growth rate and fecundity in this insect (100). A few insects can
u t i l i z e plants with angular furocoumarins, however, and these
authors suggest that the pathway leading to angular attachment of
the furan ring may have been favored in the Apiaceae by specialized
herbivores that had adapted to feeding on linear furocoumarins.
The t o x i c i t y of furocoumarins to mammals has been reviewed
(99).
xanthotoxi n angelicin
Flavonoid aglycones and especially highly methylated ones are

often found as resinous exudates on plants (101).
Many flavonoids are known to be phytoalexins, a n t i v i r a l
agents, and to serve as antiinflammatory and antitumor compounds.
Several isoflavones have estrogenic a c t i v i t y in mammals (70).
(-)-Vestitol and sativan, isoflavans from Lotus species, are
phytoalexins. 3R(-)-Vestitol from the resistant pasture legume
Lotus pedunculatus, has been demonstrated to be a feeding deterrent

to larvae of Costelytra zealandica (Coleoptera: Scarabaeidae), a

serious agricultural pest in New Zealand (102). Pastures con-
taining perennial rye grass (Lolium perenneT"and as l i t t l e as 20%
Lotus pedunculatus were r e l a t i v e l y resistant to attack by this
insect.
The l i p o p h i l i c material found on the surface of Larrea
species (Zygophyllaceae) i s comprised of several methylated f l a v -
onoid aglycones and lignans such as nordihydroguaiaretic acid.
This resinous material was shown to act as an antiherbivore sub-
stance and appeared to reduce d i g e s t i b i l i t y of the plant for
several herbivores (103).
Although somewhat less known than other groups of phenylpro-
panoid compounds, lignans are widely distributed among higher
plants (70). Several lignans are known to have antitumor a c t i v i t y
and many are cytotoxic. Lignans are also known to be active in
plant-insect relationships
Sesamum indicum (Pedaliaceae) seed o i l as well as in other plants
was isolated from Magnolia kobus (Magnoliaceae) and demonstrated
to be a growth i n h i b i t i n g substance for Bombyx mori (104).
sesamin
2-Methyl, 2-hydroxymethyl-, and 2-formylanthraquinones in the

heart wood of teak (Tectona grandis, Verbenaceae) are effective in
inhibiting termite a c t i v i t y (105). Several naphthoquinone deriva-
tives including lapachol are thought to impart marine borer
resistance to woods, e.c[. that of Tabebuia guayacan (Bignoniaceae)
(108).
Often related insect species are not a l l sensitive to a
particular allomone. For example, juglone i s a feeding deterrent
to the smaller European elm bark beetle (Scolytus multistriatus)
but not to the closely related hickory bark beet1eH[Scolytus
quadrispinosus) (106,107).
Most insects have a dietary requirement for polyunsaturated
fatty acids, usually l i n o l e n i c acids, but the exact requirements
appear to d i f f e r from species to species and few have been studied
thoroughly (109). Moreover, the presence of certain fatty acids

o o
HO O
lapachol juglone
in the diet may i n h i b i t growth and cause mortality in insects that

do not require them.
General physiological roles for fatty acids in c e l l u l a r l i p i d s
are c a l o r i c storage, membran
cursors. The f i r s t of these mainly involved the formation and
hydrolysis of t r i a c y l g l y c e r o l s , transport and activation of non-
e s t e r i f i e d fatty acids, and other steps leading to energy
conversion (110). The second role primarily involves activation
and incorporation into 1- and 2- positions of different phospho-
l i p i d s which form a major part of membranes. The third role i s
linked to the requirement for certain unsaturated fatty acids in
the diets of most animals (110).
Incorporation of different fatty acids into l i p i d s depends on
the relative abundance of t h e i r CoA derivatives and t h e i r a c y l -
transferase 1^ values. The synthetic enzymes which form membrane
phospholipids may select the acid by molecular features not in
accord with the optimal physiological properties of the products
(110), resulting in the formation of membranes which do not func-
tion adequately.
When trans-fatty acids are fed to rats with adequate amounts
of essential fatty acids, they have l i t t l e effect on growth,
longevity, or reproduction, but when fed as the sole source of
l i p i d s they exaggerate the symptoms of essential fatty acid
deficiency (111). An effect on the metabolism of long chain poly-
unsaturated fatty acids was noted however.
Fatty acids are known to be feeding stimulants for certain
insects. Linolenic acid (as a free fatty acid) in mulberry leaves
stimulates potent feeding a c t i v i t y in Bombyx mori, the silkworm.
Linoleate and laurate also have a c t i v i t y whereas myristate,
palmitate, stearate, elaidate, oleate and vaccenate have none.
Oleate promotes growth but does not promote feeding a c t i v i t y . A
synergistic effect of 3-sitosterol has been observed with l i n o -
lenate, l i n o l e a t e , vaccenate and laurate. Linoleic and l i n o l e n i c
acids are phagostimulants for the f i r e ant Solenopsis saevissima
var. r i c h t e r i (112). Palmitate and stearate produces a positive
feeding response in Dermestes maculata, and valeric acid in Trogo-
derma granarium. Fatty acids ( C - C n ) are repellent to Tribolium
5
castaneum.

The antifungal properties of fatty acids and several of their

derivatives such as amides and methyl esters have been reviewed
(113) as have their antimicrobial a c t i v i t i e s (114). Monolaurin
(tRe ester of 1 auric acid and glycerol) i s the most potent l i p i d
derivative tested to date with regard to a n t i b i o t i c a c t i v i t y .
The effects of selected fatty acid (Ci -Ci ) methyl esters on
0 2
the pink bollworm (Pectinophora gossypiella), bollworm (Heliothis

zea) and tobacco budworm (Heliothis virescens) were determined,
and a number of cyclopropyl, o l e f i n i c and acetylenic methyl esters
were also tested (115). Methyl (Z,Z)-deca-2,8-diene-4,6-diynoate
(matricaria ester) was lethal at low concentrations to a l l three
insects. This l a s t ester was isolated from Conyza canadensis but
is found in vegetative matter of many plants of the Asteraceae.
It was toxic to the pink bollworm at 0.005% and to the bollworm
and the tobacco budworm at 0.15% in a r t i f i c i a l d i e t s . Esters
(C10-C12) were also toxi
ester was also shown to be a potent insect antifeedant compound to
these insects (115).
A number of mammalian and fungal enzyme systems, whole c e l l s
and entire animals have been shown to respond d i f f e r e n t l y to fatty
acids which vary in number and position of unsaturation, geometri-
cal isomers, or cyclopropyl ring positions (110,116).
The salts of fatty acids (not naturally occurring) have long
been known to have i n s e c t i c i d a l properties. The most effective
potassium salts center around oleate in the monounsaturated and
saturated series, although potassium caprate (Ci ) was especially 0
active against Choristoneura occidental i s (Western spruce budworm)

and Aden's gloverana (Western blackheaded budworm) (117).
A number of l i p i d materials were shown to be toxic to the
larvae of the bruchid beetle, Callosobruchus maculatus (96J.
Among these were a cyclopropane fatty acid from Sterculia foetida
(Sterculiaceae) seed o i l (0.1%) and cyanolipids from Koelreuteria
CH (CH ) C = C(CH ) C00H

3 2 7 2 7 s t e r c u l i c acid
paniculata, Sapindus drummondii, and Ungnadia speciosa (Sapinda-

ceae), A"~pentane extract of the seed of tung (Aleurites f o r d i i ,
Euphorbiaceae) contained a compound which was shown to be strongly
repellent to Anthonomis grandis, the boll weevil. Some fractions
were also active to the striped and spotted cucumber beetles, the
codling moth and the redbanded l e a f r o l l e r (95). The active com-
pounds were a-eleostearic acid and erythro-9,10-dihydroxy-1-
octadecanyl acetate (118).
Fatty acids and various esters are also involved in mutual-
i s t i c relationships between certain types of bees and several
plant species (123). These compounds serve as food reserve for
the pollinating species. For example, several species of Krameria

produce free 3-acetoxy-fatty acids which are collected by the

genus Centris.
Seeds of the v i o l e t , Viola odorata are disseminated by the
ant Aphaenogaster rudis. Elaiosomes (appendages attached to the
outside of the seed coat) often contain high concentrations of
l i p i d s and are associated with attraction of the ants. 1,2-
Diolein, a diglyceride, i s largely responsible for this attraction
(124).
C H ( C H ) C H = C H CH=CH CH=CH(CH ) COOH

3 2 3 2 7
a-eleostearic acid
C H ( C H ) C H CH ( C H ) C H O H
3 2 7 2 7 2
HO OH
QAythn.o-9,10-di hydroxy-1 -octadecanoate
Amide derivatives of unsaturated a l i p h a t i c fatty acids ( C i - 0
Ci ) are especially common in members of the Asteraceae, Piper-

8
aceae, and Rutaceae. Among these, the isobutyl amides have

pronounced insecticidal a c t i v i t y (119). Most of these compounds
have not proven useful as commercial insecticides because of their
i r r i t a t i n g properties to mammals and their i n s t a b i l i t y .
A series of structurally similar compounds derived by acetate
extension of phenylpropanoid precursors i s also found in the
Piperaceae. Pipercide from Piper nigrum (Piperaceae) was i n s e c t i -
c i d a l , but the mixture of amides from this plant was s i g n i f i c a n t l y
more toxic and i t appears that co-occurring compounds exert
synergistic effects (120).
Many non-volatile l i p i d s in plants are also deived from fatty
acids. A number of these compounds are known to possess biological
activity.
The wild tomato, Lycopersicon hirsutum f, glabratum i s
covered with trichomes which contain 2-tridecanone, The level of
this compound i s much lower in the domesticated tomato, L_.
esculentum. This exudate proved to be toxic to Manduca sexta
(tobacco hornworm) and to Heliothis zea (121). The density of
glandular trichomes, which secrete 2-tridecanone, was influenced

,CH,
H
CH CH=CH CH=CH CH= CH (CH ) CH=CH C N CH CH
3 2 2 2
echinacein
CH (CH ) CH=CH (CH ) C H = C H C N CH CH

3 2 2 2 4 2
O X
CH 3
neoherculin
pipercide
by an interaction between day length and l i g h t intensity. The

toxic compound was s i g n i f i c a n t l y more abundant on foliage of
plants grown under long day regimes (122), This finding i s of
considerable importance as there i s a p o s s i b i l i t y of introducing
this resistance into acceptable tomato c u l t i v a r s .
Most acetylenic compounds in plants are derived from meta-
b o l i c a l l y altered fatty acids. These often are active in plant-
insect relationships. 8-cis-Dihydromatricaria acid i s also found
in the defensive secretion of the soldier beetle (Chauliognathus
lecontei) (125), and has subsequently been shown to have a n t i -
feedant properties against Phidippus spp. (jumping spiders) (126).
As previously mentioned matricaria ester has antifeedant properties
to the pink bollworm, bollworm and tobacco budworm (115).
Several nematicidal acetylenic compounds have been isolated.
Most are from the Asteraceae (127), Recently isolated are (8R,9R,
10S)-9,10-epoxyheptadec-16-ene-4,6-diyne-8-ol and other compounds
from Cirsium japonicum (128,129). 1-Phenylhepta-1,3,5-triyne and
2-phenyl-5-(l'-propynyl)-thiopene, from Coreopsis lanceolata and
ci s-dehydromatri cari a ester from Soli dago altissima have been
shown to be f l y ovicidal substances (130,131).

C H C H C H C ^ C C = C CH C H - C H ( C H ) C H = C H
3 2 2 2 2
OH
(8R,9R,10S)-9,10-epoxyheptadec-16-ene-4,6-diyne-8-ol
Literature Cited
1. Kogan, M. Proc. XV Int. Cong. Ent. 1976, 211-226.

2. Feeny, P. P. In "Coevolution of Animals and Plants" (L. E.
Gilbert and P. H. Raven, eds.), 3-19. Univ. of Texas Press,
Austin, 1975.
3. Waiss, A. C. Jr., Chan, B. G. and E l l i g e r , C. Α. In "Host
Plant Resistance to Pests" (P. A. Hedin, ed.) 115-128.
Symposium Series No. 62, Amer. Chem. Soc., Washington, 1977.
4. Ehrlich, P. R. and Raven, P. H. Evolution 1964, 18, 568-608.
5. Seigler, D. S. Biochem. Syst & Ecol. 1977, 5, 195-199.
6. Seigler, D. S. and P r i c e , P. W. Amer. Nat. 1976, 110, 101-
104.
7. Jones, D. A. Amer. Nat. 1979, 113, 445-451.
8. Swain, T. Nova Acta Leopoldina, Suppl. 7 1976, 411-421.
9. Jones, O. T. and Coaker, T. H. Ent. Exp. and Appl. 1978,
24, 272-284.
10. Jermy, T. Symp. B i o l . Hung. 1976, 16, 109-113.
11. Atsatt, P. R. and O'Dowd, D. J. Science 1976, 193, 24-29.
12. Price, P. W., Bouton, C. E., Gross, P . , McPheron, Β. Α.,
Thompson, J. N. and Weis, A. E. Ann. Rev. Ecol. Syst. 1980,
11, 41-65.
13. Whittaker, R. H. and Feeny, P. P. Science 1971, 171, 757-
770.
14. Florkin, M. B u l l . C l . S c i . Acad. Roy. Belg. 1965, 51 239-
256.
15. Blum, M. S. In "Animals and Environmental Fitness" (R.
Gilles, ed.). Pergamon, Oxford, 1980, pp. 207-222.
16. Brown, W. L., Jr., Eisner, T. and Whittaker, R. H.
BioScience 1970, 20, 21-22.

17. Beck, S. D. and Reese, J. C. In "Biochemical Interaction

Between Plants and Insects" ( J . W. Wallace and R. L. Mansell,
eds.) 41-92. Plenum, New York, 1976.
18. S i l v e r s t e i n , R. M. Science 1981, 213, 1326-1332.
19. Blum, M. S. In "Chemical Control of Insect Behavior" (H. H.
Shorey and J. J. McKelvy, Jr., eds.) 149-167. New York,
1977.
20. Weldon, P. J. J . Chem. Ecol. 1980, 6, 719-724.
21. Stipanovic, R. D . , Bell, A. A. and Lukefahr, M. J. In "Host
22. Hedin, P. Α., Thompson, A. L. and Gueldner, R. C. In
"Biochemical Interaction Between Plants and Insects" (J. W.
Wallace and R. L. Mansell, eds.) 271-350. Plenum, New York,
1976.
23. Eisner, T. and Halpern
24. Dethier, V. G. Evolutio
25. Kogan, Μ. In "Introduction to Insect Pest Management" (R. L.
Metcalf and W. H. Luckman, eds.) 103-146. Wiley, New York,
1975.
26. G i l b e r t , L. Ε. In "Coevolution of Animals and Plants" (L. E.
Gilbert and P. H. Raven, eds.) 210-240. Univ. of Texas
Press, Austin, 1975.
27. Smiley, J . Science 1978, 201, 745-747.
28. Price, P. W. "Insect Ecology", Wiley, New York, 1975.
29. Dethier, V. G. In "Chemical Ecology" (E. Sondheimer and
J. B. Simeone, eds.) 83-102. Academic Press, New York, 1970.
30. Painter, R. H. "Insect Resistance in Crop Plants",
MacMillan, New York, 1951.
31. Chapman, R. F. B u l l . Ent. Res. 1974, 64, 339-363.
32. Maxwell, F. G. In "Chemical Control of Insect Behavior"
(Η. H. Shorey and J. J. McKelvy, Jr., eds.) 299-304. Wiley,
New York, 1977.
33. Rhoades, D. F. In "Herbovores" (G. A. Rosenthal and D. H.
Janzen, eds.) 3-54. Academic Press, New York, 1979.
34. Tester, C. F. Phytochemistry 1977, 16, 1899-1901.
35. Pimentel, D. B u l l . Ent. Soc. Am. 1976, 22, 20-26.
36. Campbell, B. C. and Duffey, S. S. Science 1979, 205, 700-
702.
37. Harborne, J . B. In "Introduction to Ecological Biochemistry",
2nd Ed. Academic Press, London, 1982.
38. Gross, D. Fort. Chem. Org. Naturst. 1977, 34, 187-247.
39. Haukioja, E. Oikos 1980, 35, 202-213.
40. C a r r o l l , C. R. and Hoffman, C. A. Science 1980, 209, 414-
416.
41. Hedin, P. Α., Maxwell, F. G. and Jenkins, J. N. In "Proc.
of Summer Inst. of Biol. Control of Plant Insects and
Diseases" (F. G. Maxwell and F. A. Harris, eds.) 494-527.
University of Mississippi Press, Jackson, 1974.
42. Hedin, P. Α., Jenkins, J. N. and Maxwell, F. G. In "Host


43. Jacobson, M. and Crosby, D. G. In "Naturally Occurring
Insecticides", Dekker, New York, 1971.
44. McKey, D. In "Herbivores" (G. A. Rosenthal and D. H. Janzen,
eds.) 56-133. Academic Press, New York, 1979.
45. Rodriguez, E. and Levin, D. A. In "Biochemical Interaction
Between Plants and Insects" (J. W. Wallace and R. L. Mansell,
46. Schoonhoven, L. M. Rec. Adv. Phytochem. 1973, 6, 197-224.
47. Jacobson, M. "Insecticides from Plants", Agriculture Hand
book No. 154, USDA, Washington, D . C . , 1958.
48. Jacobson, M. "Insecticides from Plants", Agriculture Hand
book No. 461, USDA, Washington, D . C . , 1975.
49. Levin, D. Quart. Rev. Biol. 1973, 48, 3-15.
50. Levin, D. A. Ann
51. McKey, D. Amer
52. Dethier, V. G . , Brown, L. B. and Smith, C. N. J . Econ.
Entom. 1960, 53, 134-136.
53. Beck. S. D. Ann. Rev. Entomol. 1965, 10, 207-232.
54. Munakata, K. In "Host Plant Resistance to Pests" (P. A.
Hedin, ed.) 185-196. Symposium Series No. 62, Amer. Chem.
Soc., Washington, 1977.
55. Seigler, D. S. In "Herbivores (G. H. Rosenthal and D. H.
56. Herout, V. In "Progress in Phytochemistry", Vol. 2. (L.
Reinhold and Y. L i v s c h i t z , eds.) 143-202. Interscience,
London, 1970.
57. Blum, M. S. In "Biochemistry of Insects" (M. Rockstein, ed.)
466-513. Academic Press, New York, 1978.
58. Nahrstedt, A. Planta Med. 1982, 44, 2-14.
59. Weatherston, J . and Percy, J. E. in "Chemicals Controlling
Insect Behavior" (M. Beroza, ed.) 95-144. Academic Press,
New York, 1970.
60. Duffey, S. S. Proc. XV Int. Cong. Ent. 1976, 15, 323-394.
61. Vasechko, G. I. Z. Angew. Entomol. 1978, 85, 66-76.
62. Verma, M. and Meloan, C. E. Amer. Lab. 1981, 64-69.
63. Chapman, R. F . , Bernays, E. A. and Simpson, S. J. J . Chem.
Ecol. 1981, 7, 881-888.
64. Alfaro, R. I., Pierce, H. D., J r . Borden J. H. and
Oehlschlager, A. C. J. Chem. Ecol. 1981, 7, 39-48.
65. Oh, Η. Κ., Sakai, T . , Jones, M. B. and Longhurst, W. M.
Appl. Microbiol. 1967, 15, 777-784.
66. Eisner, T. Science 1964, 146, 1318-1320.
67. Sakan, T . , Isoe, S. and Hyeon, S. B. In "Control of Insect
Behavior by Natural Products" (D. L. Wood, R. M. Silverstein
and M. Nakajima, eds.) 239-247. Academic Press, New York,
1970.
68. Sutherland, O. R. W., Wearing, C. H. and Hutchins, R. F. N.
J. Chem. Ecol. 1977, 3, 625-631.

69.Doskotch, R. W., Cheng, H. Y., Odell, T. M. and Girard, L.

J. Chem. Ecol. 1980, 6, 845-851.
70. Mabry, T. J. and Ulubelen, A. J . Agr. Fd. Chem. 1980, 28,
188-196.
71. Lichtenstein, E. P. and Casida, J . E. J . Agr. Fd. Chem.
1963, 11, 410-415.
72. Saxena, B. P . , Koul, O., Tikku, K. and A t a l , C. K. Nature
1977, 270, 512-513.
73. Smilanick, J . M . , Ehler, L. E. and Birch, M. C. J . Chem.
Ecol. 1978, 4, 701-707.
74. Gothilf, S., Levy, E. C . , Cooper, R. and Lavie, D. J . Chem.
Ecol. 1975, 1, 457-464.
75. Visser, J . H . , Van Straten, S. and Maarse, H. S. J . Chem.
Ecol. 1979, 5, 13-25.
76. Buttery, R. G. and Kamm J. A J Agr Fd Chem 1980 28
978-981.
77. Yeo, P. F. In Roy Sympos , (H
Emden, Ed.) 51-57. Blackwell, London, 1973.
78. Faegri, K. and van der Pijl, "The Principles of Pollination
Ecology", 3rd Ed. Pergamon, London, 1979.
79. Dodson, C. Η. In "Coevolution of Animals and Plants" (L. E.
Gilbert and P. H. Raven, eds.) 91-99. University of Texas
Press, Austin, 1975.
80. Smith, Β. N. and Meeuse, B. J. D. Plant Phys. 1966, 41,
343-347.
81. Rothschild, M. In "Biochemical Aspects of Plant and Animal
Coevolution", ( J . B. Harborne, ed.) 259-276. Academic Press,
New York, 1978.
82. Rothschild, Μ. In "Coevolution of Animals and Plants",
(L. E. Gilbert and P. H. Raven, eds.) 20-50. University of
Texas Press, Austin, 1975.
83. Sláma, K. In "Herbivores" (G. A. Rosenthal and D. H.
84. Mabry, T. J. and Gill, J . E. In "Herbivores" (G. A.
Rosenthal and D. H. Janzen, eds.) 501-537. Academic Press,
New York, 1979.
85. Kubo, I. and Nakanishi, K. In "Host Plant Resistance to
Pests" (P. A. Hedin, ed.) 165-178. Symposium Series No. 62,
Amer. Chem. Soc., Washington, 1977.
86. Burnett, W. C . , Jr., Jones, S. B. J r . and Mabry, T. J . In
"Biochemical Aspects of Plant and Animal Coevolution, ( J . B.
Harborne, ed.) 233-257. Academic Press, New York, 1978.
87. Wiemer, D. F. and Ales, D. C. J . Org. Chem. 1981, 46,
5449-5450.
88. Stephenson, A. G. J . Chem. Ecol. in press.
89. Crosby, D. G. In "Naturally Occurring Insecticides", (M.
Jacobson and D. G. Crosby, eds.) 177-239. Dekker, New York,
1971.
90. Isogai, Α., Murakoshi, S., Suzuki, A. and Tamura, S. Agr.
B i o l . Chem. 1977, 41, 1770-1784.

91. Roeske, C. N., Seiber, J . N . , Brower, L. P. and Moffitt,

C. M. In "Biochemical Interaction Between Plants and
Insects", ( J . W. Wallace and R. L. Mansell, eds.) 93-163.
Plenum, New York, 1976.
92. Nelson, C. J., Seiber, J . N. and Brower, L. P. J . Chem.
Ecol. 1981, 7, 981-1009.
93. Seiber, J . N., Tuskes, P. M . , Brower, L. P. and Nelson, C. J .
J. Chem. Ecol. 1980, 6, 321-339.
94. Fink, L. S. and Brower, L. P. Nature 1981, 291, 67-70.
95. Jacobson, M . , Reed, D. Κ., C r y s t a l , Μ. Μ., Moreno, D. S. and
Soderstrom, E. L. Ent. Exp. Appl. 1978, 24, 248-257.
96. Janzen, D. H . , Juster, H. B. and Bell, E. A. Phytochemistry
1977, 16, 223-227.
97. E l l i g e r , C. Α., Wong, Y., Chan, B. G. and Waiss, A. C. Jr.,
J. Chem. Ecol. 1981 7 753-758
98. Yajima, T. and Munakata
1701-1706.
99. Ivie, G. W. In "Effects of Poisonous Plants on Livestock"
(R. F. Keeler, K. R. van Kampen and L. F. James, eds.) 475-
485. Academic Press, New York, 1978.
100. Berenbaum, M. and Feeny, P. Science 1981, 212, 927-929.
101. Wollenweber, E. and Dietz, V. H. Phytochemistry 1981, 20,
869-932.
102. Russell, G. B., Sutherland, O. R. W., Hutchins, R. F. N.
and Christmas, P. E. J . Chem. Ecol. 1978, 4, 571-579.
103. Rhoades, D. F. and Cates, R. G. In "Biochemical Interaction
Between Plants and Insects" ( J . W. Wallace and R. L. Mansell,
104. Kamikado, T . , Chang, C . , Murakoshi, S., Sakurai, A. and
Tamura, S. Agr. Biol. Chem. 1975, 39, 833-836.
105. Rudman, R. and Gay, F. J. Holzforschung 1961, 15, 117.
106. G i l b e r t , B. L. and Norris, D. M. J . Insect Physiol. 1968,
14, 1063-1068.
107. G i l b e r t , L. I. and King, D. S. Physiology Insecta 1973, 1,
249-370.
108. Manners, G. D . , Jurd, L., Wong, R. and Palmer, K. Tetra
hedron 1975, 31, 3019-3024.
109, McFarland, J. E. In "The Pharmacological Effects of Lipids"
J. J . Kabara, ed.) 97-104. Amer. Oil Chem. Soc. Monograph
No. 5, Champaign, IL, 1978.
110. Lands, W. Ε. M. In "Geometrical and Positional Fatty Acid
Isomers" (E. A. Emken and H. J. Dutton, eds.) 181-212. Amer.
Oil Chem. Soc. Monograph No. 6, Champaign, I L , 1979.
111. A l f i n - S l a t e r , R. B. and Aftergood, L. In "Geometrical and
Positional Fatty Acid Isomers" (E. A. Emken and H. J. Dutton,
eds.) 53-74. Amer. Oil Chem. Soc. Monograph No. 6,
Champaign, IL, 1979.
112. Vinson, S. B., Thompson, J . L. and Green, H. B. J . Insect
Physiol. 1967, 13, 1729-1736.
113. Gershon, H. and Shanks, L. In "The Pharmacological Effects

of Lipids" ( J . J . Kabara, ed.) 51-62. Amer. Oil Chem. Soc.

Monograph No. 5, Champaign, IL, 1978.
114. Kabara, J. J . In "The Pharmacological Effects of Lipids"
( J . J . Kabara, ed.) 1-14. Amer. Oil Chem. Soc. Monograph
115. Binder, R. G . , Chan, B. G. and E l l i g e r , C. A. Agr. B i o l .
Chem. 1979, 43, 2467-2471.
116. Holman, R. T. In "Geometrical and Positional Fatty Acid
Isomers" (E. A. Emken and H. J. Dutton, eds.) 283-298.
Amer. Oil Chem. Soc. Monograph No. 6, Champaign, I L , 1979.
117. Puritch, G. S. In "The Pharmacological Effect of Lipids"
( J . J . Kabara, ed.) 105-112. Amer. Oil Chem. Soc. Monograph
118. Jacobson, M . , C r y s t a l , M. M. and Warthen, J . D. Jr., J . Agr.
Fd. Chem. 1981, 29 591-593
119. Jacobson, M. In
Jacobson and D. G Crosby, ) , ,
1971.
120. Miyakado, M . , Nakayama, I. and Yoshioka, H. Agr. Biol.
Chem. 1980, 44, 1701-1703.
121. Williams, W. G . , Kennedy, G. G . , Yamamoto, R. T . , Thacker,
J. D. and Bordner, J . Science 1980, 207, 888-889.
122. Kennedy, G. G . , Yamamoto, R. T . , Dimock, M. B., Williams,
W. G. and Bordner, J . J . Chem. Ecol. 1981, 7, 707-716.
123. Simpson, B. B. and Neff, J . L. Ann. Missouri Bot. Gard.
1981, 69, 301-322.
124. Marshall, D. L., Beattie, A. J. and Bollenbacher, W. E.
J. Chem. Ecol. 1979, 335-344.
125. Meinwald, J., Meinwald, Y. C . , Chalmers, A. M. and Eisner, T.
Science 1968, 160, 890-892.
126. Eisner, T . , Hill, D . , Goetz, M . , J a i n , S., Alsop, D . ,
Camazine, S. and Meinwald, J. J . Chem. Ecol. 1981, 7, 1149-
1159.
127. Bohlmann, F . , Burckhardt, T. and Zdero, C. In "Naturally
Occurring Acetylenes", Academic Press, London, 1973.
128. Kawazu, K . , Nishii, Y. and Kakajima, S. Agr. Biol. Chem.
1980, 44, 903-906.
129. Sugiyama, K. and Yamashita, K. Agr. Biol. Chem. 1980, 44,
1983-1984.
130. Nakajima, S. and Kawazu, K. Agr. Biol. Chem. 1980, 44,
1529-1533.
131. Kawazu, K . , Ariwa, M. and Kii, Y. Agr. Biol. Chem. 1977,
41, 223-224.
132. E l l i g e r , C. A., Zinkel, D. F., Chan, G. B. and Waiss, A. C.
Jr., Experientia 1976, 32, 1364-1366.
133. Ohigashi, H . , Wagner, M. R., Matsumura, F. and Benjamin,
D. M. J . Chem. Ecol. 1981, 7, 599-614.
134. Kogan, M. In Introduction to Insect Pest Management" (R. L.
Metcalf and W. H. Luckman, eds.) 2nd Ed. Wiley, New York,
1982 in press.

19
Isolation of Phytoecdysones as Insect Ecdysis
Inhibitors and Feeding Deterrents
ISAO K U B O and J A M E S A . K L O C K E
University of California, Division of Entomology and Parasitology, College
of Natural Resources, Berkeley, CA 94720
Phytoecdysones, due t o their e f f e c t s on the

behavior and th
i n s e c t s , appear
defensive s t r a t e g i e s found in some i n s e c t - r e s i s t a n t
p l a n t species.
Observations in nature obviate the f a c t that c e r t a i n p l a n t

species and cultivars are more r e s i s t a n t t o i n s e c t a t t a c k 1~3
than are others. Ajuga remota (Labiatae) is an example of this.
A survey of a Kenyan savannah f o l l o w i n g a l o c u s t a t t a c k revealed
that the only v e g e t a t i o n t o s u r v i v e the a s s a u l t was A. remota4.
In order t o t e s t f o r chemical f a c t o r s i n v o l v e d i n this ob-
served r e s i s t a n c e , e x t r a c t s of A. remota f o l i a g e were i n c o r p o r a -
ted i n t o a r t i f i c i a l d i e t s optimized f o r s e v e r a l economically im-
portant pest i n s e c t s ( F i g . 1 ) 5 . B r i e f l y , a methanolic e x t r a c t
was d i s s o l v e d i n solvent and added t o a n o n - n u t r i t i v e f i l l e r
( a - c e l l u l o s e ) , evaporated t o dryness, and added t o the components
of a meridic a r t i f i c i a l d i e t , i n c l u d i n g s o l i d n u t r i e n t s ( c a s e i n ,
sucrose, wheat germ, Wesson s a l t s ) , vitamins (C and B-complex),
and 4% agar. Newly-hatched larvae of the pink bollworm, Peetino-
phora gossypiella and of the f a l l armyworm, Spodoptera frugiperda
were placed s i n g l y on p o r t i o n s of the d i e t i n p l a s t i c v i a l s .
A d d i t i o n a l bioassays were conducted w i t h the silkworm, Bombyx
moriy by i n c o r p o r a t i n g d i s s o l v e d A* remota e x t r a c t s d i r e c t l y i n t o
d r i e d mulberry powder (Nihon Nosan), evaporating the solvent t o
dryness, and adding a 2% agar s o l u t i o n .
A n a l y s i s of the t e s t i n s e c t s f e d the A. remota e x t r a c t s r e -
vealed a developmental d i s r u p t i o n i n which the i n s e c t s died i n
the pharate c o n d i t i o n f o l l o w i n g i n i t i a t i o n of molting ( a p o l y s i s ) ,
but before completion of molting ( e c d y s i s ) ( F i g . 2 - 4 ) 6 . Insect
molting c y c l e i s i n i t i a t e d when the c u t i c u l a r e p i t h e l i u m sepa-
r a t e s from the o v e r l y i n g c u t i c l e i n the process of a p o l y s i s . The
molting c y c l e i s terminated, upon the completion of c u t i c l e syn-
t h e s i s , by h y d r o s t a t i c expansion of the new c u t i c l e during the
process of e c d y s i s . The A* remota e x t r a c t apparently upset the
0097-6156/ 83/0208-0329$06.00/0

Sample Solution
<g: —a-Cellulose
Inoculate Larvae (Neonate)
Observation
Weighing
Figure 1. Artificial diet feeding bioassay for lepidopterous larvae. The diet con-
tained (I) Solid nutrients (casein, sugar, salt, wheat germ); (2) Buffered vitamin
solution (B vitamins, vitamin C); and (3) 4% agar.

KUBO AND KLOCKE Isolation of Phytoecdysones 331
Figure 2. A molting cycle failure of the silkworm, Bombyx mori, caused by inges-
tion of the crude methanol extract of Ajuga remota root. The insect underwent nor-
mal apolysis, but failed to complete ecdysis. Thus, it could not remove its head
capsule or its trunk exuviae. Magnification X11.
Figure 3. Electron micrograph of a fall armyworm, Spodoptera frugiperda, after

ingestion of the crude methanol extract of Ajuga remota roots. This insect has three
head capsules that mask its functional mouthparts. The insect eventually starved to
death. Magnification X 38.

Figure 4. Electron micrograph of a pink bollworm, Pectinophora gossypiella, after

ingestion of the crude methanol extract of Ajuga remota roots. This insect has three
head capsules that mask its functional mouthparts. The insect eventually starved to
death. Magnification X 113.
_( ( Ajugasterone C
5 10 MIN
Figure 5. C18 reversed phase HPLC. Conditions: H O-CH CN-MeOH

x 3 (82:18:1.8
v/v); 1.5 mL/min; 254 nm.

19. KUBO AND KLOCKE Isolation of Phytoecdysones 333
temporal p a t t e r n i n g such that the molting c y c l e f a i l e d due t o an

i n h i b i t i o n of e c d y s i s . F i g . 2 e x e m p l i f i e s t h i s as the newly-
molted B. mori l a r v a died while encased by the o l d c u t i c u l a r s k i n
and head capsule (pharate c o n d i t i o n ) . The e f f e c t s of t h i s pha-
rate c o n d i t i o n are t o prevent feeding because of masking of the
mouthparts by the head capsule and a l s o t o prevent locomotory and
e x c r e t o r y f u n c t i o n s because the whole body i s trapped by the
r e t e n t i o n of the e n t i r e c u t i c u l a r s k i n .
F i g s . 3 and 4 are e l e c t r o n micrographs of a f a l l armyworm,
and a pink bollworm, r e s p e c t i v e l y . Both i n s e c t s have three head
capsules because they underwent two f a i l e d molting c y c l e s before
death. That i s , even though feeding became impossible a f t e r the
f i r s t i n h i b i t e d e c d y s i s because the adhering second head capsule
covered the mouthparts, these l a r v a e could synthesize a t h i r d
7
head c a p s u l e .
Chromatographic f r a c t i o n a t i o n
f a i l u r e bioassay r e s u l t e
1
compounds. S p e c t r a l i d e n t i f i c a t i o n (UV, IR, MS and NMR ( H and
l^c)) r e s u l t e d i n B-ecdysone, cyasterone, and ajugasterone C
as the a c t i v e p r i n c i p l e s from the A. remota e x t r a c t ( F i g . 5)**.
We have used the m o l t i n g c y c l e f a i l u r e bioassay t o detect phyto-
ecdysones i n other plant species observed t o be r e s i s t a n t t o i n -
sect a t t a c k , i n c l u d i n g A. reptans , A. ohameaoistus y Podooarpus
gracilior, and o t h e r s . A. reptans was found t o c o n t a i n fl-ecdy-
sone and cyasterone, i n a d d i t i o n t o ajugalactone. P. graoilior
was found t o c o n t a i n ponasterone A as the a c t i v e p r i n c i p l e i n
t h i s bioassay^.
In order t o o b t a i n s u f f i c i e n t q u a n t i t i e s of these phytoecdy-
sones f o r more d e t a i l e d b i o l o g i c a l s t u d i e s , d r o p l e t counter-
10
current chromatography (DCCC) was a d a p t e d . DCCC i s an espe-
c i a l l y e f f i c i e n t method f o r the p r e p a r a t i v e s e p a r a t i o n of p o l a r
compounds l i k e the phytoecdysones. Thus w i t h DCCC, while r e q u i r -
ing only s m a l l volumes of s o l v e n t , more than 50 mg o f each of the
Ajuga phytoecdysones were r a p i d l y and n o n d e s t r u c t i v e l y separated
8
and f u l l y r e c o v e r e d ( F i g . 6) from each 500 mg i n j e c t i o n .
A comparison of the f i v e i s o l a t e d phytoecdysones, t e s t e d i n
the a r t i f i c i a l d i e t feeding bioassay w i t h pink bollworm l a r v a e ,
showed the importance of the phytoecdysone s i d e chain i n the
s t r u c t u r e / a c t i v i t y r e l a t i o n s h i p (Table 1 ) . Thus, ponasterone A
showed the most potent e c d y s i s i n h i b i t o r y (EI95) as w e l l as
growth i n h i b i t o r y (ED50) a c t i v i t i e s , while ajugalactone was
i n a c t i v e as e c d y s i s i n h i b i t o r t o c o n c e n t r a t i o n s as high as 1000
ppm. fi-ecdysone, cyasterone, and ajugasterone C were compara-
t i v e l y a c t i v e i n t h i s bioassay (Table 1 ) .
Ponasterone A was a l s o the most potent of three phytoecdy-
sones o r a l l y i n j e c t e d i n t o f o u r t h i n s t a r mori l a r v a e (Table
2). A l l of the l a r v a e t r e a t e d w i t h > 5 ug ponasterone A were
induced t o i n i t i a t e m o l t i n g ( a p o l y s i s ) , but were unable t o com-
p l e t e t h i s molt due t o an i n h i b i t i o n of e c d y s i s . Thus, a l l of
these ponasterone A-treated l a r v a e d i e d i n the pharate c o n d i t i o n .

Table 1 . E f f e c t s o f 5 phytoecdysones on growth and

development o f pink bollworm l a r v a e .
Amount i n d i e t
Compound (ppm) Effect
fi-ecdysone 35 ED^Q
50 E I 9 5
Cyasterone 2
40 E I 9 5
Ponasterone A 1 E D
5 0
2 EI 9 5
Ajugasterone C 14 E D
5 0
45 EI 9 5
Ajugalactone 430 ED^^

* EI
Values are based on three o r more r e p l i c a t e s , each

of which c o n s i s t e d o f 3 0 o r more neonate pink b o l l -
worm assayed f o r 1 2 days. ED^^ r e f e r s t o the
effective dose f o r 5 0 % growth inhibition, while
EI<^ refers t o the e f f e c t i v e dose f o r 9 5 % k i l l
due t o e c d y s i s i n h i b i t i o n .
* No ecdysis inhibition was observed to

concentrations of 1 0 0 0 ppm.

Table 2. E f f e c t s o f fi-ecdysone as compared t o cyasterone

and ponasterone A when o r a l l y i n j e c t e d t o 4th i n s t a r
5. movi # 25 larvae were used per treatment.
Developmen- Activity
t a l stage yg phyto-
i n 4th i n - ecdysone
s t a r (day) per os fi-ecdysone Cyasterone Ponasterone A
1st 30 P; 74% E I I ( p ) ; 10%EI P; 100% E I

2nd 30 P
20 P ( p )
10 P; 10% E I I ( p ) ; 16%EI P; 100% E I

5 1 P; 100% E I
2.5 NE
3rd 20 P; 46% EI
10 P; 15% E I —
4th 30 P
20 P
10 P
5 NE —
P : Promotion o f 100% o f t r e a t e d larvae t o a p o l y s i s w i t h i n 24-

48 hr p o s t - i n j e c t i o n .
I : Delay o f 100% o f t r e a t e d larvae t o a p o l y s i s f o r 24-72 hr
compared t o c o n t r o l .
I^ ^ p : Delay o f >82% o f treated larvae t o a p o l y s i s f o r > 72 hr
compared t o c o n t r o l . (P) i n d i c a t e s <18% o f treated larvae
were promoted t o a p o l y s i s w i t h i n 24-48 hr p o s t i n j e c t i o n .
A l l o f the l a t t e r group died through e c d y s i s i n h i b i t i o n
(EI).
E I : E c d y s i s i n h i b i t i o n r e s u l t i n g i n death. A l l other larvae
recovered•

34 mg
0-Ecdysone Ajugasterone C Cyasterone
0 50 100 150 200
Fraction Number
Figure 6. DCCC of the ethyl acetate extract of Ajuga remota (500 mg) with
CHCl -MeOH-H,0
3 (13:7:4) by the ascending method.
Maxillolabialhypopharyngeal complex (mc)
Mandible
Figure 7. Schematic depicting (ventral side) dysfunctional mouth-parts in ecdysis-

inhibited Bombyx mori larvae.
Left: Normally ecdysed 5th instar head capsule with fully closed mandibles.
Right: Ecdy sis-inhibited 5 th instar head capsule following artificial removal of 4th
instar head capsule. The adhering 4th instar head capsule prevented full expansion
of the 5th instar head capsule resulting in the forward position of the maxillolabial-
hypopharyngeal complex (mc) such that the mandibles could not fully close.

O r a l i n j e c t i o n s >10 yg of B-ecdysone a l s o r e s u l t e d i n a promoted

a p o l y s i s , but depending upon c o n c e n t r a t i o n and the exact age of
the t r e a t e d f o u r t h i n s t a r l a r v a e , a v a r i a b l e number of the t r e a -
ted l a r v a e were able t o complete m o l t i n g and e s s e n t i a l l y recover.
Cyasterone i n j e c t i o n s >10 pg, i n g e n e r a l , caused a delay ( a n t i -
ecdysone) of m o l t i n g of the t r e a t e d f o u r t h i n s t a r l a r v a e t o t h e
1
f i f t h instar. This 'antiecdysone e f f e c t of cyasterone, at i t s
most severe, r e s u l t e d i n p r o t h e t e l y (precocious development) of
a small % of the t r e a t e d l a r v a e .
In order t o examine a n a t o m i c a l l y the newly-synthesized head
of f o u r t h i n s t a r l a r v a e undergoing m o l t i n g c y c l e f a i l u r e , the
adhering e x u v i a l head capsule was c a r e f u l l y removed w i t h f o r c e p s .
This procedure revealed the morphological d i s r u p t i o n of the f e e d -
ing apparatus ( F i g . 7). The f i f t h i n s t a r head capsule was com-
pressed by the adhering f o u r t h i n s t a r head capsule which r e s u l
ted i n the pushing forwar
complex (mc) such that y
In order t o study how i n j e c t e d (or ingested) phytoecdysone
causes an i n h i b i t i o n i n e c d y s i s , s e v e r a l biochemical parameters
of l a r v a l m o l t i n g f l u i d were analysed, i n c l u d i n g phenoloxidase
a c t i v i t y , t o t a l p r o t e i n , and t o t a l a s c o r b i c a c i d (Table 3 ) .
M o l t i n g f l u i d may be i m p l i c a t e d i n the i n h i b i t i o n of e c d y s i s
since e i t h e r t o t a l removal of the m o l t i n g f l u i d (unpublished) o r
a simple d e l e t i o n of a s c o r b i c a c i d from the m o l t i n g f l u i d of Spo-
11
doptera littoralis (Navon, 1 9 7 8 ) ( f e d w i t h no phytoecdysone)
d u p l i c a t e d the phytoecdysone-induced i n h i b i t i o n of e c d y s i s . The
a n t i o x i d a n t a s c o r b i c a c i d has been hypothesized t o c o n t r o l pheno-
l o x i d a s e hardening of the newly-synthesized c u t i c l e before i t s
h y d r o s t a t i c expansion. I n a d d i t i o n , B-ecdysone i s known t o a c t i -
vate the enzyme which c a t a l y s e s the s y n t h e s i s of a phenoloxidase
1
from i t s proenzyme ^.
Nevertheless, a s c o r b i c a c i d l e v e l s i n both the m o l t i n g f l u i d
and the hemolymph of phytoecdysone-treated l a r v a e are comparable
to those l e v e l s found i n untreated l a r v a e . In a d d i t i o n , phenol-
oxidase a c t i v i t y , as measured w i t h c a t e c h o l s u b s t r a t e , i s a c t u a l -
l y l e s s i n phytoecdysone-induced l a r v a e as compared t o c o n t r o l
larvae.
These negative r e s u l t s do not e l i m i n a t e the involvement i n
ecdysis i n h i b i t i o n o f other biochemical parameters i n the m o l t i n g
f l u i d , i . e . c h i t i n a s e , protease, but do seem t o i n d i c a t e that
premature phenoloxidase-catalysed c u t i c l e hardening i s not the
cause f o r phytoecdysone-induced f a i l e d e c d y s i s .
More d e t a i l e d data w i t h B. mori showed that the e f f e c t s o f
ingested B-ecdysone i n c l u d e d , besides an i n h i b i t i o n of e c d y s i s ,
death without m o l t i n g , death f o l l o w i n g completion of promoted
m o l t i n g , and an i n h i b i t i o n i n growth w i t h no e f f e c t on m o l t i n g .
These v a r i o u s e f f e c t s are dependent upon the c o n c e n t r a t i o n of
exogenous B-ecdysone, the p r e c i s e developmental stage of the
treated l a r v a e , and the d u r a t i o n of exposure.
>
Concentrations of B-ecdysone 5 0 ppm i n a r t i f i c i a l d i e t i n -

Table 3. Comparison of phenoloxidase activity, protein, and

ascorbic acid levels i n the molt fluid and ascorbic
acid levels i n the hemolymph approximately 36 hr
following apolysis to the 5th i n s t a r . B. mori larvae
were i n j e c t e d per os at 4th i n s t a r 2nd day w i t h 10 u l
30% aq/EtOH alone o r w i t h 10 y l 30% aq/EtOH + phyto-
ecdysone. 25 larvae were used/treatment/parameter and
analyzed.
Orally A 4 7 Q /min/ ug A s c o r b i c ug A s c o r b i c ug P r o t e i n /
applied u l molt acid/ul acid/ul u l molt
treatment fluid molt f l u i d hemolymph fluid
Control 0.031
+ 0.05 0.13 16.0
(solvent o n l y )
0.012
30 ug 0.008
3-Ecdysone + 0.06 0.12
(in solvent) 0.005
10 ug 0.006
Ponasterone A
15.0
(in solvent)

duced premature m o l t i n g i n 5. mori and r e s u l t e d i n 100% m o r t a l i -

t y . Much of t h i s m o r t a l i t y , however, occurred not as a r e s u l t
of an i n h i b i t i o n of e c d y s i s , but a c t u a l l y a f t e r molting had been
completed. I n f a c t , w h i l e the lower c o n c e n t r a t i o n s o f B-ecdysone
r e s u l t e d i n <100% m o r t a l i t y , the % of the t o t a l m o r t a l i t y occur-
r i n g during the m o l t i n g process ( i . e . , e c d y s i s i n h i b i t i o n ) i n -
creases as the dose of B-ecdysone i s decreased (Table 4). Thus,
death of B* mori by ingested B-ecdysj>ne cannot be e n t i r e l y ex-
p l a i n e d by an i n h i b i t i o n of e c d y s i s .
At 25 ppm B-ecdysone, m o l t i n g i s delayed (Table 5) so that
88% of the exposed l a r v a e d i e during the second i n s t a r ( t h a t i s ,
before m o l t i n g o c c u r s ) . Doses > 6.25 ppm but <25 ppm a l s o r e -
s u l t i n a delay of m o l t i n g t o the t h i r d i n s t a r , such that 25 ppm
seems t o be the d i e t a r y c o n c e n t r a t i o n above which enhancement,
and below which r e t a r d a t i o n of molting takes p l a c e
T o t a l m o r t a l i t y an
t a l i t y are dependent upo
dysone. I n a d d i t i o n , growth i n h i b i t i o n by d i e t a r y B-ecdysone i s
a l s o a concentration-dependent phenomenon.
H y p o t h e t i c a l l y , the l a r v a e fed high c o n c e n t r a t i o n s o f ecdy-
sone (> 25 ppm) had a high t i t re of ecdysone b u i l t i n the hemo-
lymph, a t i t r e which could not be metabolized o r excreted r a p i d l y
enough t o prevent hormonal imbalance r e s u l t i n g i n molting promo-
t i o n and death. Larvae fed lower c o n c e n t r a t i o n s (< 25 ppm) o f
B-ecdysone grow more slowly than c o n t r o l and molt l a t e r than
control. P o s s i b l y the ecdysone a t these lower c o n c e n t r a t i o n s
induced metabolism of both trie exogenous and the endogenous ecdy-
sone such that there was a delay i n a p o l y s i s .
13
H i k i n o et at. ( 1 9 7 5 ) showed that the c a t a b o l i c a c t i v i t y
on B-ecdysone o f B* mox*i v a r i e d during the course of i t s growth
and development. This i s i l l u s t r a t e d i n Tables 2 and 6 i n which
i t can be seen that the l a r v a e are more s e n s i t i v e t o e i t h e r i n -
gested (Table 6) o r i n j e c t e d (Table 2) 3-ecdysone d u r i n g t h e
e a r l i e r phase o f the f o u r t h i n s t a r .
An a d d i t i o n a l bioassay w i t h a pest aphid species was conduc-
ted t o t e s t f o r a feeding d e t e r r e n t e f f e c t o f d i e t a r y phytoecdy-
1
s o n e s ^ ( F i g . 8 ) . One of s e v e r a l phytoecdysones was d i s s o l v e d
d i r e c t l y i n t o an aqueous d i e t optimized f o r maximal aphid f e e d -
i n g . The c o n t r o l aqueous d i e t c o n s i s t e d of vitamins (C and B-
complex), sucrose, amino a c i d s , t r a c e metals, s a l t s , c h o l e s t e r o l ,
brought t o pH 8.7 w i t h K3PO4. The aqueous d i e t was placed
i n t o poly-ethylene v i a l caps and each o f these caps were f i t t e d
i n t o c i r c u l a r holes punched i n t o p l a s t i c snap-on l i d s f o r p o l y -
styrene catsup cups (1 o z ) . Between 50-100 Biotype C greenbugs,
Schizaphis graminim , an important pest on sorghum (and other
economically important g r a i n s ) i n the midwestern U.S., were
t r a n s f e r r e d from sorghum p l a n t s i n t o each of the 1 oz catsup cups
which were immediately f i t t e d w i t h the d i e t cap-containing snap-
on l i d s . A f t e r 24 h r s a t room temperature the no. of aphids
f e e d i n g / t o t a l no. aphids was determined f o r each treatment.

Table 4. E f f e c t s of fi-ecdysone on the l a r v a l development

a
of second i n s t a r Borribyx mori
Concen- % % of t o t a l mortality
tration Development t o
i n diet 3rd 4th mortality 2nd Ecdysis 3rd
(ppm) instar instar % instar inhibition instar
100 28 0 100 72 0 28
50 28 0 100 56 16 28
25 8 0 100 88 4 8
12-5 63 50 51 41 33 25
6.25 73 69 31 26 61 13
3.125 100 96 4 0 0 100
Control 100 96 4 0 0 100
a) 25 second i n s t a r f i r s t day larvae/treatment

Table 5. E f f e c t s of B-ecdysone on the l a r v a l
developmental p e r i o d of second i n s t a r Bombyx mori
Concentration Average Period (days) t o

in diet Molted Ecdysed Ecdysed
(ppm) 2nd I n s t a 3rd I n s t a 4th I n s t a
100 3.0(7) ' b c

4.1(7) c
Molting
50 3.9(ll) c
4.9(7) c
- Promotion
25 8.0(2) c
9.5(2) c
- Molting
12.5 6.4(19) c
6.9(15) c
14.3(10) Delay
6.25 6.0(24) c
6.0(19) c
14.3(18)
3.125 5.0(25) 6.0(25) 13.7(24)
Control 4.9(25) 5.9(25) 14.0(24)
a) 25 second i n s t a r f i r s t day larvae/treatment.
b) Figures i n p a r e n t h e s i s show the number of l a r v a e

a l i v e out of the o r i g i n a l 25 second i n s t a r f i r s t
day l a r v a e .
c) S i g n i f i c a n t d i f f e r e n c e from control a t P=0.001

by Mann-Whitney U t e s t .

Aqueous
Sample S o l u t i o n
Akey Diet
Amino a c i d s , Sucrose, B-vitamins
Vitamin C S a l t s Trace Metals
350 v l
1.5 cm ID polyethylene v i a l cap
Parafilm
Catshup cup l i d w i t h inserted d i e t cap
50-100 Biotype C aphids i n 1 oz. catshup cup
Observation o f Number Feeding a f t e r 24 h
Figure 8. Antifeedant bioassay for the greenbug, Schizaphis graminum-

Table 6. E f f e c t s of varying no- consecutive days feeding on

d i e t a r y fi-ecdysone by second i n s t a r B. movi larvae
Days of treatment
w i t h fi-ecdysone f o l l o w i n g Concentration of
molt t o 2nd i n s t a r d i e t a r y fi-ecdysone (ppm)
a
of B. moH l a r v a e ) 100 50 25 12.5 6.25
l s t - 5 t h days i n c l u s i v e 0.20 0.22 j 0.30 0.48 0.84
1st day only 1

0.61 0.67 0.66 0.69 0.86
2nd day only I 0.58 0.66 0.67 0.74 0.88
3rd day only 0.68 0.72 0.75 0.80 0.86
4th day only 0.83 0.78 0.86 0.93 0.90 1
a) 25 second i n s t a r f i r s t day larvae/treatment
b) No. i n d i c a t e weight ratio of t r e a t e d l a r v a e / c o n t r o l

l a r v a e on the f i f t h day of second i n s t a r .
i n d i c a t e s treatment a f f e c t i n g i n h i b i t i o n of m o l t i n g t o
the t h i r d i n s t a r .
i n d i c a t e s treatment a f f e c t i n g promotion of molting t o

the t h i r d i n s t a r .

Appropriate c o n t r o l s , which c o n s i s t e n t l y r e s u l t e d i n > 90% feed-

i n g , were then used to compare to each of the treatments i n order
to determine ED50 values f o r each of the t e s t e d phytoecdysones.
ED50 i s the e f f e c t i v e dose f o r 50% feeding compared to c o n t r o l .
From Table 7 i t can be seen that ajugasterone C i s more than
1 0 - f o l d more potent than fi-ecdysone, and more than 3 0 - f o l d more
potent than cyasterone, as a feeding d e t e r r e n t to S. graminum
when i n c o r p o r a t e d i n t o the a r t i f i c a l d i e t of t h i s b e h a v i o r a l b i o -
assay.
Although ingested phytoecdysones do have a potent and unique
hormonal a c t i v i t y a g a i n s t s u s c e p t i b l e species l i k e the silkworm
and the pink bollworm, other i n s e c t species are unaffected by
d i e t a r y phytoecdysones. For example, Heliothis complex fed more
than 3000 ppm phytoecdysones i n a r t i f i c i a l d i e t show no obvious
morphological or developmental changes. However, other chemicals
besides phytoecdysone
remota and P. gracilior
Heliothis and other i n s e c t s p e c i e s . A . remota e x t r a c t s have
y i e l d e d 6 d i t e r p e n e s causing a n t i f e e d a n t and i n s e c t i c i d a l a c t i v i -
•.yl-3,15^ while s e v e r a l i n s e c t i c i d a l n a g i l a c t o n e s coupled to
an a n t i f e e d a n t a c t i v i t y as w e l l as two g r o w t h - i n h i b i t i n g b i s f l a -
vones have been i s o l a t e d from P. gracilior foliage^.
Such multicomponent defensive s t r a t e g i e s , as those e l u c i d a t e d
i n A* remota and i n P. gracilior, may be more the r u l e than the
exception i n r e s i s t a n t p l a n t c u l t i v a r s . The e l u c i d a t i o n of
these s t r a t e g i e s , p a r t i c u l a r l y the chemical aspects of them, i s
important f o r an understanding of e c o l o g i c a l and e v o l u t i o n a r y
aspects of host p l a n t r e s i s t a n c e . In a d d i t i o n , the mechanistic
understanding of host p l a n t r e s i s t a n c e may have economical i m p l i -
c a t i o n s i n that ' r e s i s t a n c e ' chemicals may be bred i n t o crop
p l a n t s , or they may be e x t r a c t e d from one p l a n t species and
a p p l i e d d i r e c t l y to another economically important p l a n t s p e c i e s ,
or they may serve as l e a d i n g structui»2S i n s y n t h e t i c p e s t i c i d e
research.
The r o l e of phytoecdysones i n t h i s scheme i s that of an
important component i n a r a t h e r complex defensive s t r a t e g y of
some p l a n t s . T h e i r presence i n p l a n t s probably serves a l i m i t e d ,
yet important, p r o t e c t i v e r o l e . For example, c o t t o n b o l l s bred
w i t h s e v e r a l ppm of ponasterone A would very l i k e l y be r e s i s t a n t
to a t t a c k by pink bollworm.
In summary, then, whether a c t i n g alone or i n c o n j u n c t i o n w i t h
other chemicals, the unique and potent p h y s i o l o g i c a l , biochemi-
c a l , and morphological e f f e c t s induced by the phytoecdysones con-
f e r s an i n t e g r a l r o l e f o r them i n host p l a n t r e s i s t a n c e .

KUBO AND KLOCKE Isolation of Phytoecdysones
Table 7. Feeding deterrency of three phyto-

ecdysones o greenbug Sohizaphis graminurft
n
Compounds E D
5 0 * diet)**
B-ecdysone 650
Cyasterone 2000
Ajugasterone C 62
a) Biotype C o f 5. graminum from a mixed

p o p u l a t i o n i n a 2 4 h no-choice bioassay.
b) E D ^ Q i s the e f f e c t i v e dose f o r 50% feeding

compared t o c o n t r o l .

Acknowledgement
I n s e c t s were k i n d l y supplied by the agencies of the USDA i n

B r o w n s v i l l e , Tx; Phoenix, Az; and T i f t o n , Ga. The authors thank
J . DeBenedictis f o r h i s help w i t h e l e c t r o n micrographs and D.
Dreyer and K. Jones f o r t h e i r help w i t h the aphid bioassay.
Authentic samples of phytoecdysones were g i f t s from P r o f e s s o r T.
Takemoto ;and P r o f e s s o r K. Nakanishi.
References
1. Kubo, I . ; Lee, Y. W.; Balogh-Nair, V.; N a k a n i s h i , K.; Chapya,

A. J. Chem. Soc. Chem. Commun. 1976, 949.
2. Kubo, I . ; Kido, M.; Fukuyama, Y. J. Chem. Soc. Chem. Commun.,
1980, 897.
3. Kubo, I . ; K l o c k e
Soc. Chem. Commun., 1982, 618.
4. Kubo, I . Science Year 1982, World Book-Childcraft Interna-
tional, Chicago, 1981, 126.
5. Chan, B. G.; Waiss, A. C. Jr.; S t a n l e y , W. L.; Goodban, A.
E. J. Econ. Entomol., 1978, 71, 366.
6. Kubo, I . ; Klocke, J. A.; Asano, S. Agric. Biol. Chem., 1981,
45, 1925.
7. Kubo, I . ; Klocke, J . A.; Asano, S., in p r e p a r a t i o n .
8. Kubo, I . ; Ganjian, I . ; Klocke, J . A., in p r e p a r a t i o n .
9. Kubo, I . ; K l o c k e , J . A., in p r e p a r a t i o n .
10. Hostettmann, K. Planeta Medica, 1980, 39, 1.
11. Navon, A. J. Insect Physiol., 1978, 24, 39.
12. Chapman, R. F. "The I n s e c t s , S t r u c t u r e and Function, American
E l s e v i e r P u b l i s h i n g Company, New York, 1971, 700.
13. H i k i n o , H.; Ohizumi, Y.; Takemoto, T. J. Insect Physiol.,
1975, 21, 1953.
14. Dreyer, D. L.; Reese, J . C.; Jones, K. C.J.Chem.Ecol.,
1981, 21, 273.
15. Kubo, I . ; N a k a n i s h i , K. Host Plant Resistance to Pests, ACS
Symposium S e r i e s 62, American Chemical S o c i e t y , Washington,
D.C., 1977, 165.

20
Multiple Factors in Cotton Contributing
to Resistance to the Tobacco Budworm,
Heliothis virescens F.
P. A. HEDIN, J. N. JENKINS, D. H. COLLUM, W. H. WHITE, and
W. L. PARROTT
U.S. Department of Agriculture, Science and Education Administration,
Agricultural Research Southern Region, Boll Weevil Research Laboratory,
Chemistry Research Unit, Mississippi State,MI39762
Cyanidin-3-ß-glucosid
important f a c t o
hirsutum L. leaves t o the feeding of tobacco buworm
Heliothis v i r e s c e n s (Fab.) in the field. The
reported e f f e c t i v e n e s s of gossypol was confirmed, but
the condensed tannins (proanthocyanidins) i n t e r m i n a l
leaves were not c o r r e l a t e d w i t h r e s i s t a n c e . Para-
doxically, these 3 compounds when incorporated in
l a b o r a t o r y d i e t s are e q u a l l y t o x i c t o l a r v a e . These
f i n d i n g s provide a potential b a s i s f o r a c h i e v i n g
i n s e c t r e s i s t a n c e in non-glanded cotton and other
crops i n f e s t e d by Heliothis.
Since the o r i g i n a l development of glandless cotton by

McMichael ( 1 ) , entomologists and p l a n t breeders have noted that
the experimental g l a n d l e s s l i n e s are g e n e r a l l y s u s c e p t i b l e t o
c e r t a i n phytophagous i n s e c t s . Bottger e t a l . (2), r e p o r t i n g on
the r e l a t i o n s h i p between the gossypol content of cotton p l a n t s
and i n s e c t r e s i s t a n c e , noted that s e v e r a l i n s e c t s f e d on a
g l a n d l e s s l i n e i n preference t o glanded l i n e s . Jenkins e t a l .
(3_) reported increased s u s c e p t i b i l i t y of s e v e r a l g l a n d l e s s l i n e s
t o the bollworm ( H e l i o t h i s zea Boddie). Lukefahr e t a l . (4_)
showed that the growth of bollworm and tobacco budworm ( H e l i o t h i s
v i r e s c e n s F.) l a r v a e increased on d i e t s of g l a n d l e s s cotton
l i n e s compared w i t h that on d i e t s of the corresponding glanded
l i n e . Lukefahr and M a r t i n (5) incorporated 3 cotton pigments,
gossypol, q u e r c e t i n , and r u t i n , i n t o a standard bollworm d i e t
whereupon l a r v a l growth was decreased. They suggested that p l a n t
breeders might s e l e c t f o r cotton p l a n t s w i t h higher pigment
(gossypol and q u e r c e t i n ) content as a mechanism of r e s i s t a n c e .
I t was observed that flower buds from c e r t a i n w i l d and p r i m i -
t i v e cottons showed more i n s e c t i c i d a l a c t i v i t y than could be
accounted f o r by gossypol, and the a d d i t i o n a l a c t i v i t y was
a s c r i b e d t o "X" f a c t o r s ( 6 , 7 ) . The "X" f a c t o r s were i d e n t i f i e d
American Chemical
This c h a p t j ^ ^ J £ « L t a I l S . copyright.
Published l^J^rfcyn'fihlmical Society.
1155 16th St. N. W.
InWashington,
Plant ResistanceD. C. 20036
to Insects; Hedin, P.;
as sesquiterpenoid quinones, hemigossypols, and h e l i o c i d e s i n a

s e r i e s of i n v e s t i g a t i o n s by t h i s College S t a t i o n , Texas group
(8-11). In summary, they i s o l a t e d at l e a s t 15 terpenoid aldehy-
des and r e l a t e d compounds from glanded cotton p l a n t s , present i n
v a r y i n g amounts from d i f f e r e n t l i n e s . They reported that t o x i -
c i t y (ED50; c o n c e n t r a t i o n to reduce l a r v a l growth by 50%)
approached or e q u a l l e d gossypol (0.05%) f o r a number of these
compounds when i n c o r p o r a t e d i n d i e t s fed to tobacco budworms.
Gossypol was g e n e r a l l y found i n considerably higher concentations
than the other terpenoid aldehydes i n the l i n e s i n v e s t i g a t e d .
This group (12) a l s o chromatographically i d e n t i f i e d c a t e c h i n ,
g a l l o c a t e c h i n , q u e r c e t i n , and condensed tannin from V e r t i c i l l i u m
w i l t - r e s i s t a n t young cotton leaves i n higher concentrations than
from s u s c e p t i b l e l a r g e r l e a v e s .
Subsequently, a s e r i e s of i n v e s t i g a t i o n s by a group at the
Western Regional Researc
have shown condensed cotto
f o r the bollworm, tobacco budworm, and pink bollworm (13, 14,
15). I n i t i a l l y , they sought to i n v e s t i g a t e the "X" f a c t o r that
had been described by i t s e x t r a c t i o n from freeze dehydrated
t i s s u e w i t h e t h y l ether or acetone. They found that i f f r e e z e
dehydrated cotton powder was f i r s t e x t r a c t e d w i t h hexane or a
s i m i l a r non-polar solvent system, gossypol and the other t e r -
penoid aldehydes were i s o l a t e d i n the e x t r a c t . This e x t r a c t when
incorporated i n i n s e c t d i e t s decreased l a r v a l growth as pre-
v i o u s l y reported. The r e s i d u a l powder was then e x t r a c t e d w i t h
methanol; t h i s e x t r a c t when i n c o r p o r a t e d i n the l a r v a l d i e t
depressed growth s e v e r e l y . In f a c t , i n a study where c o n t r o l
tobacco budworm l a r v a e weighed 306 mg a f t e r 14 days, l a r v a e fed
the hexane e x t r a c t (from 6 g of powder added to 30 g of d i e t )
weighed 56.6 mg, those fed the acetone e x t r a c t weighed 343 mg,
and those fed the comparable amount of the ethanol e x t r a c t
from the methanolic e x t r a c t weighed 1.1 g. The major a n t i b i o t i c
compound i n the methanol s o l u b l e f r a c t i o n was subsequently
c h a r a c t e r i z e d as condensed t a n n i n , which when hydrolyzed w i t h HC1
i n n-butanol y i e l d e d c y a n i d i n and d e l p h i n i d i n . By osmotic
measurements, the average molecular weight was estimated to be
4850. In r e l a t e d work, Chan et a l . (14) i s o l a t e d the cycloprope-
n o i d f a t t y a c i d s , the terpene aldehydes i n c l u d i n g gossypol, the
f l a v o n o i d s , and the condensed t a n n i n s . When fed to tobacco bud-
worm [ a l s o bollworm and pink bollworm (Pectinophora g o s s y p i e l l a
Saunders)] h a t c h l i n g l a r v a e i n d i e t s , the ED50 values (percent of
d i e t ) were as f o l l o w s : gossypol, 0.12; hemigossypolone, 0.08;
h e l i o c i d e H , 0.12; h e l i o c i d e H , 0.13; c a t e c h i n , 0.13; quer-
x 2
c e t i n , 0.05; condensed t a n n i n , 0.15; methyl s t e r c u l a t e , 0.41; and

methyl malvalate, 0.49. S t i p a n o v i c (16) obtained s i m i l a r ED50
values f o r the tobacco budworm w i t h gossypol, hemigossypolone,
and h e l i o c i d e s and H2.
In another t e s t , Chan et a l . (14) analyzed the condensed t a n -
nin and gossypol ( w i t h analogs) content of 10 cotton p l a n t p a r t s .

20. HEDIN ET AL. Contributing Factors in Cotton 349
I n b r i e f , the t a n n i n content of various l e a f t i s s u e s was f a i r l y

h i g h w h i l e the gossypol content was r e l a t i v e l y low. In the
anthers, c o r o l l a , and c a l y x , the reverse s i t u a t i o n e x i s t e d . When
the p l a n t parts were fed t o budworms, they had comparatively
l i t t l e t o x i c i t y ( E D - 1.7%) d e s p i t e the r e l a t i v e l y high gossy-
5 0
p o l content. Except f o r the high t o x i c i t y of c o r o l l a t i s s u e

(0.15%) which was a t t r i b u t e d to f l a v o n o i d s , the various l e a f
t i s s u e s , ( e a r l y feeding s i t e s i n the f i e l d ) were otherwise the
most t o x i c (0.27-0.50%).
Shaver and P a r r o t t (17) reared bollworm and tobacco budworm
l a r v a e on a standard l a r v a l d i e t , and t r a n s f e r r e d them a t 5 ages
onto media c o n t a i n i n g 0-0.4% gossypol. The i n f l u e n c e on develop-
ment increased w i t h l a r v a l age a t the time of t r a n s f e r . Recently
Waiss and h i s co-workers (18) incorporated condensed t a n n i n i n
d i e t s fed t o the tobacco budworm where the l a r v a e i n i t i a l l y were
of d i f f e r e n t ages. Th
0.10; 3 day, 0.10-0.15
Thus, i t i s evident that l a r v a e a l s o become more t o l e r a n t t o con-
densed tannin w i t h age.
Shaver e t a l . (19) s t u d i e d feeding and l a r v a l growth i n the
l a b o r a t o r y of the tobacco budworm on component parts of cotton
f l o w e r buds. They found that most of the feeding of 2-4 day l a r -
vae occurred on the anthers a f t e r they penetrated i n t o the
i n t e r i o r of the bud through the p e t a l s . For p e r s p e c t i v e , i t
should be noted that females o v i p o s i t on the young t e r m i n a l s and
young l e a v e s , so that the h a t c h l i n g l a r v a e feed on these s i t e s
before m i g r a t i n g t o the bud. When component parts of the flower
bud were incorporated i n t o l a r v a l d i e t , only the p e t a l s ( c o r o l l a )
i n h i b i t e d growth of 3 day l a r v a e . This i n h i b i t i o n occurred both
on glanded and g l a n d l e s s l i n e s , so i t can not be a t t r i b u t e d t o
the gossypol content alone. L a t e r , Shaver et a l . (20) f e d
3-day-old tobacco budworm l a r v a e l a b o r a t o r y d i e t s i n t o which
e t h y l ether e x t r a c t s of flower buds were i n c o r p o r a t e d . They
reported that l i n e s w i t h high gossypol and gossypol r e l a t e d com-
pounds, as determined by the a n i l i n e t e s t , reduced l a r v a l weight
g a i n s . Bud tannins were not analyzed, nor were they e x t r a c t e d
and fed i n t h i s study.
Schuster e t a l . (21) i d e n t i f i e d cotton plant r e s i s t a n c e t o
the two-spotted s p i d e r mite (Tetranycus u r t i c a Koch) by mass
screening s e e d l i n g s . L a t e r Schuster and Lane (22) were able to
show that high t a n n i n l i n e s , p a r t i c u l a r l y TX-1055, showed
r e s i s t a n c e t o t h i s arachnid and the bollworm.
Another t r a i t of cotton a f f e c t i n g l a r v a l growth of tobacco
budworm i s p o l l e n c o l o r which ranges from cream t o yellow t o even
orange. Hanny e t a l . (23) f e d 1st i n s t a r tobacco budworm l a r v a e
whole f r e s h anthers of 5 cream and yellow l i n e s . I n a number of
t e s t s conducted during 1977 and 1978, weight gains a f t e r 7 days
were 13-15% l e s s on the y e l l o w p o l l e n . Hanny (24) reported the
f o l l o w i n g average analyses: gossypol, yellow 0.88%; cream 0.70%;
condensed t a n n i n s , y e l l o w 4.79%; cream 5.34%; and f l a v o n o i d s ,

yellow 0.56%, cream 0.54%. The I s o l a t i o n of 4 gossypetin g l y c o -

s i d e s (but not gossypin), 8 q u e r c e t i n g l y c o s i d e s , and an antho-
cyanin were reported.
In p r e l i m i n a r y work at t h i s l o c a t i o n (25), gossypol and t a n -
n i n were n e g a t i v e l y c o r r e l a t e d with weight gains of tobacco bud-
worm l a r v a e f e d i n the f i e l d on plant terminals. This was based
on seasonal averages f o r a f i v e c u l t i v a r t e s t . In l a b o r a t o r y
t e s t s , the ED50 values f o r s e v e r a l flavonoids and condensed c o t -
ton tannins were determined and found to be s i m i l a r (0.05-0.15%)
to the values reported by Chan et a l . (14) and S t i p a n o v i c ( 1 6 ) .
The ED50 values f o r a number of cotton c u l t i v a r s , h i b i s c u s ,
sorghum, and s i n f o i n , were found to range from 0.03-0.10%, i n d i -
c a t i v e that the tannin could be b i o l o g i c a l l y s i m i l a r . L a r v a l
feeding t e s t s were a l s o performed on a number of chromatographic
f r a c t i o n s obtained from solvent e x t r a c t s of cotton terminal
t i s s u e . Those containin
most t o x i c .
From the preceding, i t appears that chemical r e s i s t a n c e i n
cotton to H e l i o t h i s i n s e c t s i s due to m u l t i p l e f a c t o r s .
D i f f e r e n t l i n e s , each with i n s e c t r e s i s t a n c e , may possess d i f -
f e r e n t r a t i o s of a n t i b i o t i c compounds. Thus, i t may be p o s s i b l e
to increase r e s i s t a n c e by c r o s s i n g l i n e s where each c o n t r i b u t e s
genes f o r b i o s y n t h e s i s of d i f f e r e n t a n t i b i o t i c compounds. The
tobacco budworm was s e l e c t e d f o r study i n preference to the c o t -
ton bollworm because i t i s e a s i e r to rear and use i n the l a b o r a -
t o r y , i s more r e s i s t a n t to i n s e c t i c i d e s i n the f i e l d , and i t i s
approximately as s u s c e p t i b l e to cotton c o n s t i t u e n t s incorporated
i n laboratory d i e t s ( 1 4 ) . This present study was c a r r i e d out to
i d e n t i f y and analyze f o r cotton c o n s t i t u e n t s that were t o x i c i n
l a b o r a t o r y feeding t e s t s , and to determine whether there were
p o s i t i v e c o r r e l a t i o n s of t h e i r content i n leaves and/or other
t i s s u e with f i e l d r e s i s t a n c e . From t h i s information, the genera-
t i o n of l i n e s with m u l t i p l e f a c t o r s f o r r e s i s t a n c e could be i n i -
tiated.
M a t e r i a l s and Methods 1/
Agronomic and entomological p r a c t i c e s . Plants of d i v e r s e

cotton l i n e s were grown i n f i e l d p l o t s during the years
1978-1982 on the Plant Science Farm at M i s s i s s i p p i State
U n i v e r s i t y . Plants were t r e a t e d f o r b o l l weevils with Guthion,
and normal f e r t i l i z e r , h e r b i c i d e , and other c u l t u r a l p r a c t i c e s
were a p p l i e d . The f i e l d design was normally a randomized
complete block with 4 r e p l i c a t i o n s . Plant m a t e r i a l ( t e r m i n a l s ,
If Mention of a trademark, p r o p r i e t a r y product or vendor does

not c o n s t i t u t e a guarantee or warranty of the product by the U.S.
Department of A g r i c u l t u r e and does not imply i t s approval to the
e x c l u s i o n of other products or vendors that may a l s o be s u i t a b l e .

l a r g e r l e a v e s , squares, b r a c t s , f l o w e r s , and b o l l s ) was c o l l e c t e d

a t i n t e r v a l s throughout the season, freeze dehydrated, ground,
and stored at -10°C u n t i l used.
F i r s t i n s t a r tobacco budworm l a r v a e were r e s t r a i n e d i n 15 cm
long d i a l y s i s casings that were s l i p p e d over the t e r m i n a l s , and
c o l l e c t e d and weighed a f t e r f i v e days. Adequate numbers and
r e p l i c a t i o n s were employed f o r s t a t i s t i c a l e v a l u a t i o n .
Laboratory bioassays were performed by p l a c i n g 1st i n s t a r tobacco
budworm l a r v a e on a commercial medium (742-A) prepared by Bioserv
I n c . , Frenchtown, NJ. Organic s o l v e n t - s o l u b l e t e s t compounds
were d i l u t e d i n e i t h e r hexane or e t h a n o l , added to c a s e i n and
evaporated to dryness i n a r o t a r y evaporator. Water s o l u b l e com-
pounds were d i s s o l v e d i n water and added i n place of the
p r e s c r i b e d water content. The c a s e i n was then incorporated w i t h
the remainder of the i n g r e d i e n t s and poured i n t o 10 cm p e t r i p l a -
tes to g e l . D i e t c y l i n d e r
cut w i t h a cork borer an
v i a l s . A neonate l a r v a was added and the v i a l s were incubated at
26°C, 60%RH, 12L:12D f o r 5 days at which time the l a r v a e were
weighed. A randomized complete block design w i t h 8 r e p l i c a t e s of
5 l a r v a e each was used. Compounds were t e s t e d at 5-8 con-
c e n t r a t i o n s ranging from 0.006 to 0.6% of the d i e t on a dry
weight b a s i s .
Chemical analyses and f r a c t i o n a t i o n s . Freeze-dehydrated c o t -

ton t i s s u e was analyzed f o r condensed tannin (heated n-BuOH-HCl),
gossypol ( p h l o r o g l u c i n o l - H C l ) , and anthocyanins-anthocyanidins
( a l e . HC1 at 540 nm). Other analyses performed but not reported
here were f o r c a t e c h i n , t o t a l phenols, E ^ ( t a n n i n ) , and a n i l i n e
r e a c t i v e terpenes ( g o s s y p o l ) .
Freeze dehydrated p l a n t t i s s u e was e x t r a c t e d by Soxhlet f i r s t
w i t h cyclohexane/ethyl a c e t a t e / a c e t i c acid:500/500/1 (CHEA) and
then w i t h acetone/water:7/3. The CHEA e x t r a c t was chromato-
graphed on a 40 cm s i l i c i c a c i d column w i t h hexane and s o l v e n t s
of i n c r e a s i n g p o l a r i t y to y i e l d gossypol and s e v e r a l other com-
ponents, each of which was formulated f o r l a b o r a t o r y bioassay
t e s t i n g . The aqueous acetone f r a c t i o n was chromatographed on a 1
m Sephadex LH-20 column w i t h 70% aqueous methanol, methanol and
dimethyl formamide/methanol:10/90 and 25/75 to y i e l d the f l a v o -
n o i d s , anthocyanin, and condensed t a n n i n . S i m i l a r l y , each
was formulated f o r l a b o r a t o r y bioassay t e s t i n g . The i s o -
l a t e d components were rechromatographed on LH-20, polyamide, and
c e l l u l o s e columns as r e q u i r e d to achieve p u r i t y f o r b i o l o g i c a l
e v a l u a t i o n and i d e n t i f i c a t i o n work.
I d e n t i f i c a t i o n of c o t t o n p l a n t compounds. The i d e n t i t y of
gossypol was confirmed by comparison of the s p e c t r a l (NMR, MS)
and chromatographic p r o p e r t i e s w i t h an a u t h e n t i c sample. The
i d e n t i t y of the cotton l e a f anthocyanin was confirmed by com-
p a r i s o n of the chromatographic and s p e c t r a l p r o p e r t i e s of the

chry8anthemln i s o l a t e d from cotton f l o w e r s , by *H NMR, and by

procedures that we described p r e v i o u s l y (26). The condensed t a n -
nins (polymeric proanthocyanidins) were c h a r a c t e r i z e d w i t h regard
t o t h e i r stereochemistry, s t r u c t u r a l u n i t s , and molecular weight
by the procedures of Czochanska et a l . (27). The condensed tan-
n i n s were found t o c o n s i s t of a mixture of r e l a t e d polymers, the
molecular weight ranging from 1500-6000, the p r o d e l p h i n i d i n :
p r o c y a n i d i n r a t i o from 1.8-3.7, and the stereochemistry of the
monomer u n i t s p r i m a r i l y c i s (81-95%). Figure 1 i s a 13C MNR
spectrum of a h i g h l y p u r i f i e d c u l t i v a r BJA-592 condensed t a n n i n .
The s p e c t r a were obtained w i t h an a c q u i s i t i o n time of 0.2 s e c ,
a pulse width of 13 u sec, and a 45° f l i p angle. The average
molecular weight was deduced from the r a t i o of the s i g n a l s
at 72/67 o t o be 4221.
H i s t o l o g i c a l Examinations
h i s t o c h e m i c a l work, f r e s
c r y o s t a t ( I n t . Equip. Co., Model CTI) , mounted on specimen
h o l d e r s , trimmed, and s l i c e d w i t h wedge shaped knives at -20C t o
produce 20-30 um t i s s u e s l i c e s . For t a n n i n s , small pieces of
l e a f (or other t i s s u e ) were f i x e d f o r 78h i n FeS04-5% formol
s a l i n e , s l i c e d a t 30 ym and picked up on warm (room temp.) s l i -
des. F e r r i c c h l o r i d e (1%) or 10% KOH were f r e q u e n t l y used as
s t a i n i n t e n s i f i e r s . Tannin granules ( c e l l s ) s t a i n e d b l a c k i s h i n
unmodified f i x , b l u i s h w i t h FeCl3 modified f i x , and brown t o red-
brown i n KOH modified f i x . In unfixed t i s s u e s , gossypol s t a i n e d
red w i t h p h l o r o g l u c i n o l - H C l , and l i g n i f i e d elements s t a i n e d l i g h t
v i o l e t . To v i s u a l i z e anthocyanins and gossypol, 30 um f r e s h
f r o z e n s e c t i o n s were f i x e d over formaldehyde vapors f o r 24h, and
t r e a t e d w i t h 5% HC1 o r NaOH t o produce red or green anthocyanin
r e a c t i o n s r e s p e c t i v e l y . Gossypol remained yellow w i t h a c i d or
base treatment, i n c o n t r a s t to the anthocyanins.
Flower p e t a l s were examined under 10-70X m a g n i f i c a t i o n and
photographed i n s i t u a f t e r treatment w i t h 5% KOH which s t a i n e d
anthocyanins green. P h l o r o g l u c i n o l - H C l t r e a t e d gossypol glands
were s t a i n e d red, and f l a v o n o i d s y e l l o w .
R e s u l t s and D i s c u s s i o n
T o x i c i t y of cotton p l a n t compounds i n d i e t s t o TBW l a r v a e .

Table I gives ED5Q values f o r a number of cotton c o n s t i t u e n t s
t e s t e d as i n h i b i t o r s of tobacco budworm l a r v a l growth. For com-
p a r i s o n , values obtained independently by Chan e t a l . (12) and
S t i p a n o v i c (unpublished data) using e s s e n t i a l l y i d e n t i c i a l proce-
dues are i n c l u d e d . The ED50 values were q u i t e r e p r o d u c i b l e by
each l a b o r a t o r y . Further separation of the tannin by Sephadex
LH-20 chromatography gave f r a c t i o n s w i t h average molecular
weights ranging from 1500 t o 6000. When bioassayed, the
ED5Q values v a r i e d no more than from 0.05 to 0.10%, so i t i s
deduced that w i t h i n l i m i t s , the s i z e of the molecule does not

HEDIN ET AL. Contributing Factors in Cotton 353

TABLE I
I n h i b i t i o n of Tobacco Budworm L a r v a l Growth by Cotton C o n s t i t u e n t s , ED50 as Percent of D i e t
Constituent Chan et a l . S t i p a n o v i c M i s s . State Regression equation

Gossypol 0.12 0.05 0.113 y=104.66-504.30x+596.12
r=0.68 P>F=0.01
Hemigossypolone 0.03 0.29
H e l i o c i d e Hj 0.12 0.10 —
Methyl sterculate 0.41 — N.T.
(+)-Catechin 0.13 — 0.052 y=7.06x--562

2
r - 0 . 9 0 P>F=0.001
Condensed tannin 0.15 — 0.063 y=2.07x

2
r =0.87 P>F=0.00
0.05 — 0.042 y=3.29x"-705

Quercetin
2
r =0.90 P>F=.001
Isoquercitrin 0.10 — 0.060 y=4.49x~»888

2
r =0.85 P>F-.001
— 0.166 y=105.66-332.89x
Cyanidin —
2
r =0.71 P>F=.001
Delphinidin — — 0.138 y=124.43-540.36x

2
r =0.89 P>F=.001
Chrysanthemin — 0.070 y=7.91x"*707

2
r =0.81 P>F=.001
a p p r e c i a b l y a f f e c t t o x i c i t y . I n recent years as the chemical

bases f o r r e s i s t a n c e were explored, there was the expectation
that some i n d i v i d u a l compound or group of r e l a t e d compounds would
be found that could account f o r the r e s i s t a n c e of cotton t o t h i s
i n s e c t . This has been shown not t o be the case based on d i e t a r y
i n c o r p o r a t i o n bioassays, because the t o x i c i t i e s are e s s e n t i a l l y
e q u i v a l e n t at the ED50 v a l u e s . However, the growth response
becomes c u r v e l i n e a r a t higher l e v e l s . While i t i s p o s s i b l e t o
reduce growth t o l e s s than 10% of the c o n t r o l w i t h gossypol and
chrysanthemin, i t was impossible t o reduce growth t o l e s s than 20%
w i t h some o t h e r s .
E f f e c t of gossypol, t a n n i n , and chrysanthemin i n terminals on

TBW l a r v a l growth. Figures 2, 3 and 4 give percent con-
c e n t r a t i o n s of gossypol, chrysanthemin, and tannins i n t e r m i n a l
l e a v e s , and a l s o give tobacc
c o t t o n l i n e s , 15 glande
l a r v a e feeding on i n t a c t p l a n t s i n the f i e l d . The percent con-
t e n t s of gossypol and chrysanthemin were n e g a t i v e l y c o r r e l a t e d
w i t h l a r v a l weights ( r » -.38 and -.40) while the tannins were
weakly p o s i t i v e l y c o r r e l a t e d ( r « +.16); i n f a c t , g l a n d l e s s l i n e s
that produced l a r g e l a r v a e were as high i n tannin as most of the
l i n e s that produced s m a l l l a r v a e . Thus, these s t u d i e s c o r r o -
borate the work of Hanny e t a l . (23,24) and suggest that the
absolute c o n c e n t r a t i o n of the tannins i n the t o t a l t i s s u e does
not e x p l a i n the expected t o x i c e f f e c t s of the i n s e c t feeding on
i n t a c t t i s s u e . Female tobacco budworm moths o v i p o s i t mainly on
the t e r m i n a l l e a v e s , although i n mid- and late-season they ovipo-
s i t s i g n i f i c a n t numbers on the square b r a c t s . Consequently, the
i n i t i a l feeding s i t e i s most o f t e n the t e r m i n a l l e a v e s . Our s t u -
d i e s (25) have shown that the l a r v a e migrate down the p l a n t ,
feeding on b r a c t s , meristematic ( o l d e r ) t i s s u e buds, flower
p e t a l s , and b o l l s i n the process where the content of antho-
cyanins, gossypol, and tannins i s mostly s i m i l a r .
Table I I i s a summary of the averages of t a n n i n s , gossypol,
and chrysanthemin i n 18 c u l t i v a r s harvested as 3 r e p l i c a t e s i n
August 1981. The values (somewhat lower than i n 1980) show that
the 3 c o n s t i t u e n t s are present i n comparable q u a n t i t i e s i n a l l
t i s s u e s analyzed except lower i n medium b o l l s . I t i s t o the
medium b o l l s that the l a r v a e e v e n t u a l l y migrate, perhaps t o avoid
h i g h l e v e l s of a l l e l o c h e m i c s . As the season progresses, t a n n i n
i n c r e a s e s i n a l l t i s s u e s sharply while gossypol and chrysanthemin
g r a d u a l l y decrease (Figure 5 ) . During the same p e r i o d , the l a r -
v a l weight gains remained s i m i l a r , however.

Figure 3. Percent chrysanthemin in cotton terminal leaves and ranked tobacco

budworm larval weights for 20 strains (r = 0.3958).

HEDIN ET AL. Contributing Factors in Cotton 357
Figure 4. Percent tannins in cotton terminal leaves and ranked tobacco budworm
larval weights for 20 strains (r — 0.1613).
.10
20 27 I* 11 18 25 1 8 15 22 29
JULY AUGUST
Figure 5. Seasonal trends in chrysanthemin (—), gossypol ( —), and tannin

( ) content during 1981.

TABLE I I
Content of Tannins, Gossypol, and Chrysanthemin i n Cotton
P l a n t T i s s u e s ; Seasonal Averages f o r 3 August 1981 R e p l i c a t e s ,
18 C u l t i v a r s
Tissue Tannins, % Gossypol, % Chrysanthemin,%
Terminals 6.02 0.21 0.14

Leaves 8.10 0.23 0.18
Squares 7.92 0.50*/ 0.10
Square b r a c t s 6.02 0.21 0.14
Small b o l l s 11.71 0.29 0.11
Medium b o l l s 9.3
Medium b o l l 17.0
carpels
a/ Somewhat high; average of 0.28 i n 1980, 0.23 f o r 16 other

l i n e s i n 1981, but 0.47 by E l l i g e r et a l . ( 1 5 ) .
E f f e c t of g o s s y p o l , t a n n i n , and chrysanthemin i n flower p e t a l s

on TBW l a r v a l growth and s u r v i v a l . Our recent observations
demonstrated that i n the l a b o r a t o r y , l a r v a e fed more s u c c e s s f u l l y
on w h i t e , f i r s t day p e t a l s ( i n t a c t ) than on red, second day
p e t a l s , and that they fed more s u c c e s s f u l l y on non-glanded p e t a l s
than on glanded p e t a l s . Table I I I presents t a n n i n , g o s s y p o l , and
chrysanthemin c o n c e n t r a t i o n s of white and red p e t a l s of 2 glanded
and 2 non-glanded ( g l a n d l e s s ) l i n e s . Values f o r tobacco budworm 5
day l a r v a l weights and percent s u r v i v a l are a l s o i n c l u d e d . The
t a n n i n contents of the non-glanded white p e t a l s were higher than
those of the glanded s t r a i n s , but there was l i t t l e d i f f e r e n c e i n
the red p e t a l t a n n i n contents. The gossypol contents of the
p e t a l s of the glanded l i n e s were much higher than those of the
non-glanded l i n e s as could be expected. The chrysanthemin con-
t e n t s of a l l red p e t a l s were a l s o much higher than those of white
p e t a l s as was expected. The weights and s u r v i v a l of larvae
feeding on red p e t a l s were c o n s i d e r a b l y l e s s than those f e e d i n g on
white p e t a l s . Both percents t a n n i n and chyrsanthemin were higher
i n red p e t a l s than i n white p e t a l s (Table I I I ) . Larvae weights
and s u r v i v a l were reduced on red p e t a l s when compared w i t h white
p e t a l s . Based c o l l e c t i v e l y on data i n Table I I I and Figures 3 and
4, we conclude that chrysanthemin i s more important than t a n n i n
f o r the reduced l a r v a l s i z e and s u r v i v a l on g l a n d l e s s
(gossypol-low) red p e t a l s . Gossypol and chrysanthemin c o n t r i b u t e
to the t o x i c i t y of glanded red p e t a l s , and gossypol t o that of
glanded white p e t a l s . We now have p r e l i m i n a r y data that l a r v a e
fed leaves and b r a c t s of red cottons gained 20% l e s s ( s t a t i s -
t i c a l l y s i g n i f i c a n t ) than those fed leaves and b r a c t s from com-

TABLE I I I
R e l a t i v e E f f e c t s of Cotton Flower P e t a l C o n s t i t u e n t s on Tobacco Budworm Growth and S u r v i v a l ^ /
Cultivar Petal color Tannins, % Gossypol, % Chrysanthemin, % L a r v a l Wt, mgf*/ L a r v a l s u r v i v a l , %
ST-7AGN (NG)b/ IS/ 5.79 0.10 0.07 4.72 a 39.5

R 8.68 0.11 0.67 0.46 7.0
DH 66 (NG) W 5.40 0.17 0.07 4.18 a 31.0

R 8.55 0.13 0.73 0.56 16.5
ST-213 (G) W 3.49 0.52 0.13 1.52 b 28.0

R 8.75 0.79 0.59 0.41 8.5
DH 126 (G) W 3.16 1.72 0.18 —e/

R 6.25 2.46 0.65 —

a/ % of dry weight.
b/ NG = Nonglanded, G = glanded.
cJ W = w h i t e ; f i r s t day flower c o l o r , R = red; second day flower c o l o r . Means of l a r v a e f e d on white
p e t a l s not s i g n i f i c a n t l y d i f f e r e n t at .05 l e v e l i f followed by the same l e t t e r .

dj Average tobacco budworm weight a f t e r feeding 5 days on p e t a l s ,
e/ Not f e d .
parable green s t r a i n s . Thus, red c o l o r a t i o n now appears to be a

f a c t o r of considerable importance i n i n s e c t feeding of both p e t a l s
and leaves. There i s s t i l l a r e s i d u a l m o r t a l i t y of i n s e c t s feeding
on white g l a n d l e s s p e t a l s (Table I I I ) . This can be a t t r i b u t e d at
l e a s t i n part to the f l a v o n o i d s , some of which we have p r e v i o u s l y
i d e n t i f i e d (28) and demonstrated to be t o x i c to t h i s i n s e c t (Table
I).
H i s t o c h e m i c a l s t u d i e s on l o c a l i z a t i o n of t a n n i n s , gossypol,
and anthocyannins i n p l a n t t i s s u e s . We were able to observe by
m a g n i f i c a t i o n that tobacco budworm l a r v a e avoided gossypol glands
d u r i n g feeding ( F i g u r e 6 ) . Waiss et a l . (29) had made s i m i l a r
observations w i t h J*, zea. To determine where the t a n n i n s , gossy-
p o l , and anthocyanin are l o c a l i z e d i n the p l a n t , some h i s t o l o g i c a l
s t u d i e s were c a r r i e d out. Figure 7 shows m a g n i f i c a t i o n s of t i s s u e
s l i c e s of a glanded l i n e
and s t a i n e d w i t h p h l o r o g l u c i n o l - H C
m i d r i b i s prominent i n the center of Figure 7, and the tannins
appear to be concentrated near the surface i n granular form. In
g l a n d l e s s l i n e s , t a n n i n i s more d i f f u s e . Figure 8 i s a 5pm
p a r a p l a s t s e c t i o n through a c o t t o n l e a f at the gossypol gland s i t e
which shows the outer anthocyanin-containing envelope (halo)
surrounding the gossypol gland. In f r e s h t i s s u e s e c t i o n s , the
outer halo s t a i n s b r i g h t red i n a c i d . The halo when subsequently
n e u t r a l i z e d w i t h KOH i s converted to green, v e r i f y i n g that i t i s
anthocyanin. I t i s tempting to speculate from t h i s that the
biosyntheses of l e a f gossypol and anthocyanin are p l e i o t r o p i c a l l y
r e l a t e d because the content of each i s much higher i n glanded than
i n non-glanded leaves (Figures 2 and 3 ) . However, the anthocyanin
content can be high and gossypol low as i n flower p e t a l s (Table
I I I ) . Gossypol and r e l a t e d aldehydes are b i o s y n t h e s i z e d v i a the
acetogenic pathway and anthocyanins and other f l a v o n o i d s by the
mixed a c e t a t e / s h i k i m i c pathways. Thus each should be able to
i n c r e a s e independently i n the p l a n t as we have demonstrated i n
glanded and g l a n d l e s s red p e t a l s . This halo e f f e c t may a l s o a l t e r
the i n t e r p r e t a t i o n of Figure 6 that shows the apparent avoidance
of gossypol glands by tobacco budworm l a r v a e . The presumed
feeding deterrence of gossypol may be i n f a c t caused by the antho-
cyanin h a l o . Although both the anthocyanin and gossypol are t o x i c
when ingested, an a n t i f e e d a n t mechanism may be expressed i n t h i s
instance.
F i g u r e 9 shows a Pima (G. barbadense L.) flower p e t a l
t r e a t e d w i t h HC1 to v i s u a l i z e red anthocyanin "granules".
Treatment w i t h a l k a l i converted the granule c o l o r to green. The
halo surrounding the gossypol gland i n s i d e the outer c a r p e l w a l l
of DH-126 buds (Figure 10) i s a l s o anthocyanin. The anthocyanin
(chrysanthemin) i n very young S t o n v i l l e - 2 1 3 b o l l s on the day of
a n t h e s i s was found by d i s s e c t i n g , e x t r a c t i o n , and TLC a n a l y s i s to
be l o c a t e d p r i m a r i l y i n the outer c a r p e l w a l l .

Figure 6. A voidance of gossypol glands by tobacco budworm while feeding.
Figure 7. Cross-section of midrib and adjacent blade tissue of DH-126 leaves

showing tannin cells.

Figure 8. Paraplast section (5 m) of a gossypol gland and the anthocyanin

envelope.

HEDIN ET AL. Contributing Factors in Cotton
Figure 9. Acid treatment of Pima flower petal to visualize anthocyanin.
Figure 10. Acid visualization of anthocyanin halo in Stoneville 213 bud.

I n summary, the contents of chrysanthemin and gossypol were

shown to be n e g a t i v e l y c o r r e l a t e d w i t h tobacco budworm l a r v a l
growth i n the f i e l d w h i l e the tannins were s l i g h t l y p o s i t i v e l y
correlated. The r e c o g n i t i o n of chrysanthemin as a r e s i s t a n c e
f a c t o r provides a b a s i s for developing r e s i s t a n t cotton c u l t i v a r s
that are low or devoid of g o s s y p o l , a long sought o b j e c t i v e . It
a l s o suggests that anthocyanins i n general may be a b a s i s for
s e l e c t i n g f o r r e s i s t a n c e i n other crops to H e l i o t h i s .
Literature Cited
1. McMichael, S. C. Agr. J. 1959. 51, 30.

2. B o t t g e r , G. T., Sheehan, E . T. Lukefahr, M . J. J. Econ.
Entomol. 1964, 57, 283.
3. J e n k i n s , J. N., Maxwell F G., L a f e v e r H N J. Econ
Entomol. 1966. 59,
4. Lukefahr, M . J., Nobel
Entomol. 1966, 59, 817.
5. Lukefahr, M . J., M a r t i n , D. F . J. Econ. Entomol. 1966,
59, 176.
6. Lukefahr, M . J., Shaver, T. N., Cruhm, D. E., Houghtaling,
J. E . P r o c . Beltwide Cotton P r o d . Res. C o n f . , 1974,
Memphis, TN, p . 93.
7. Bell, A . A., S t i p a n o v i c , R. D., H o w e l l , C. R., Mace, M. E.
P r o c . Beltwide Cotton P r o d . Res. Conf. 1974, J a n . 7-9,
D a l l a s , TX, 40.
8. Gray, J. R . , Mabry, R. J., Bell, A . A., S t i p a n o v i c , R. D.,
Lukefahr, M . J. J. C . S. Chem. Comm. 1976. 109.
9. Bell, A . A., S t i p a n o v i c , R. D. H o w e l l , C. R., Fryxell, P .
A . Phytochemistry. 1975, 14
10. Bell, A . A., S t i p a n o v i c , R. D. P r o c . Beltwide Cotton
P r o d u c t i o n Res. Conf. 1977, J a n . 10-12, A t l a n t a , GA, 244.
11. S t i p a n o v i c , R. D., Bell, A . A., O ' B r i e n , D. H., Lukefahr,
M . J. Tetrahedron L e t t e r s . 1977. 6, 567.
12. H o w e l l , C. R., Bell, A . A., S t i p a n o v i c , R. D. P h y s i o l .
P l a n t P a t h o l . 1976. 8, 181.
13. Chan, B . G., Waiss, A . C., Lukefahr, M. J. J. Insect
Physiol. 1978. 24, 113.
14. Chan, B . C., Waiss, A . C., B i n d e r , R. G., Elliger, C. A .
E n t . Exp. & A p p l . 1978. 24, 94.
15. Elliger, C . A., Chan, B . G., Waiss, A . C. J. Econ.
Entomol. 1978. 71, 161.
16. S t i p a n o v i c , R. D . ; personal communication.
17. Shaver, T. W., P a r r o t t , W. L. J. Econ. Entomol. 1970.
63, 1802.
18. Waiss, A . C.; personal communication.
19. Shaver, T. N., G a r c i a , J. A., D i l d a y , R. H . E n v i r o n .
Entomol. 1977. 6, 82.
20. Shaver, T. N., D i l d a y , R. H., W i l s o n , F . D. Crop Sci.
1980. 20, 545.

21. Schuster, M. F., Maxwell, F. G., J e n k i n s , J. N., P a r r o t t ,

W. L. J. Econ. Entomol. 1972. 65, 1104.
22. Schuster, M. F., Lane, H. C. Proc. Beltwide Cotton Prod. Res.
Conf. 1980. Jan. 6-10, S t . L o u i s , MO, p. 83.
23. Hanny, B. W., B a i l e y , J. C., Meredith, W. R. E n v i r o n .
Entomol. 1979. 8, 706.
24. Hanny, B. W. J. Agr. Food Chem. 1980. 28, 504.
25. Hedin, P. A., Collum, D. H., White, W. H., P a r r o t t , W. L.,
Lane, H. C., J e n k i n s , J. N. P r o c . I n t . Conf. on R e g u l a t i o n
of Insect Development and Behavior, Wroclaw Tech. Univ.
P r e s s , Wroclaw, Poland. 1980, 1071.
26. Hedin, P. A., Minyard, J. P., Thompson, A. C., S t r u c k , R.
E., Frye, J. Phytochemistry. 1967. 6, 1165.
27. Czochanska, Z., Foo, L. P., Newman, R. H., P o r t e r , L. J .
J.C.S. P e r k i n I . 1980 2278
28. Hedin, P. A., M i l e s
J. Agr. Food Chem
29. Waiss, A. C., Chan, B. G., Elliger, C. A., and Binder, R.
G. P r o c . Beltwide Cotton Proc. Res. Conf. 1981. Jan. 4-8.
New Orleans, La. p. 61.

INDEX
A Armyworm
fall, deterrent 178
Aedes aegypti, polyacetylene toxicity .. 146 southern, pulegone effects 178-82
Age effects 206 southern, reproductive inhibition .... 182
Aggregation and dispersion 207 Artemesia, glandular trichome
Aglycones, flavonoid 78, 78/ components 80, 81
Agrotis ipsilon—See Black cutworms Assimilated food, conversion
Airborne pheromones efficiency 238
effect on neighboring willow
effect on unattacked willow
Ajuga remota, attack resistanc terpenes 176-78
Alder responses to tent caterpillar thiosulfinates 224
attack 56-59
biomass and survivorship data 58/
B
foliage quality 59
Alders, bud exudate 78 Bacillus thuringiensis 48
Alkaloids 75-77 Bacteria, polysaccharides 154
of plants, nitrogen in 25t Bay leaves, repellent properties 309
in sequestration 271 Behavior, influence of plant
Allelochemic-nutrient interactions in characteristics 199-210
insect dietetics 236-40 Benzofurans 297
Allelochemics 80,304 in desert shrubs 298-300
effect on reproductive capacities .... 181 Betula nigra, bud exudate 78
ingestion 267
Beyeria, leaf exudates 82
neonate sensitivity 234-36
Biomass and survivorship data
and physiological effects 3081
alder responses to tent caterpillar
protective 279-88
attack 58/
repellent 75-87
willow responses to tent caterpillar
and sequestrators 212-1A
attack 58/
toxic 75-87
Allomones 154, 304 Biosynthesis of juvenile hormone 183/
Allylisothiocyanate 226 Biosynthetic relationship, terpenes .... 174
Alnus rubra—See Alder Black cutworms 232
2-Amino-4-aminooxybutyric acid— artificial diet effects 235^
See L-Canaline effects of diet 233/, 234/
2-Amino-4-guanidinooxybutyric sensitivity to handling 235
acid—See L-Canavanine 279 trypsinizing effects 236
Antibiosis 199,306 Blepharina pratensis Meigen 46
vs. antixenosis 200 Boll weevil, gossypol effects 87
Antiherbivory 153 Bollworm, pink 333
Antijuvenile activity 297 electron micrographs 332/
Antitumor activity 317 phytoecdysone effect on growth 334/
Antixenosis 199,200 repellent 84
vs. antibiosis 200 Bud burst and budworm size 15, 17
Ants 71 Budworm densities
Apiaceae and defoliation 9-11
furanocoumarin in 145 natural 7
phototoxic, adaptation of insects .... 145 Budworm resistance and terpene
Apple maggot fly, host selection 201 chemistry 9-11, 12, 13/
367

Budworm success Chromenes

and terpene chemistry 17 in the Asteraceae 297
and tree productivity 15 in desert shrubs 298-300
and water stress 11-12, 13/, 16 Chrysanthemin, effects on tobacco
Budworm, western spruce, interactions budworm growth 355
with Douglas fir 3-18 Chymotrypsin inhibition Ill
1,8-Cineole 310/
C Citronella oil 309
Coevolution of plants and insects 303-5
L-Canaline 279-88 Colorado potato beetle, Solanum
ornithine antagonist 288 alkaloid effects 77
and pyridoxal phosphate Complex resistance 39
interaction 286-88 Condensed tannins, identification 352
structure 279 Continine 267
toxicity 286 Conversion efficiency
L-Canavanine 279-88 of assimilated food 238
aberrant protein formation 280-82 of ingested food 238
effect on macromolecular synthesis 283/ Cost-effectiveness, chemical defensive
incorporation into procollage
and nucleic acid metabolism
protein synthesis 28 chemical analyses and
structure 279 fractionations 351
toxicity 280-82,286 chemical resistance to Heliothis
and vitellogenin production 283-85 insects 350
Canavanyl protein production 285 effects on tobacco budworm growth 354/
proteins 238 flower petals vs. terminals 355-60
Cannabidiol 270 glanded, terpenoid aldehydes 348
Canopy development 33 histochemical studies 360-64
Carbon identification, compounds 351
and chemical defenses 26-34 isolation of cyclopropenoid fatty
and defensive structures 22 acids 348
Carbon uptake 26-34 pigment glands 83-87
Cardenolide sequestration 273 pollen color effects 349
Cardenolides resistance to tobacco budworm ...347-64
in blood 274 tannin content 349
metabolism 270 tobacco budworm feeding 349
Chemical defense(s) Cotton compounds, identification .351, 352
carbon and nitrogen components ... 22 Cotton compounds, toxicity 352-55
and deciduous forest 27-31 Cotton terpenoid aldehydes,
and insect susceptibility 37-51 toxicity to larvae 85/
multiple-factor interactions 39 Crop plants, insect resistance 306
physiological constraints 21-34 Cropping, mixed 206
resource restriction 39 Cross-resistance 89
Chemical defense variability 39-51 Crucifers, pests 208, 209
and evolution 37-51 Cuticle, larvae, comparison of
impact on natural enemies 43-50 fatty acids 262/
Chemical defensive compounds Cuticular permeability 258
cost-effectiveness 21-22 Cyanogenic glucoside, foliar 22, 24
plant 4 Cycnia inopinatus, sequestration and
Chemical resistance allelochemics 272, 21 3
r
in cotton, Heliothus insects 350 Cytochrome P-450

trichomes and glands 72-75 Dendroctonus bark beetle 185
Chemical variability 40 induction, terpene interaction 186
Chemistry of red oak, defoliation interactions, terpenes 185-86
effects 42 metabolism 17 3-92
Chitinase, microbial 48 oxidase, juvenile hormone
Choice of host 200, 201 maintenance 182
See also Host selection rat liver 185

INDEX 369
Cytochrome P-450—Continued E
southern armyworm 189
Ecdysis
system 188/
inhibition, molting fluid 337
Cytotoxic chemicals of desert
inhibitors 329-45
plants 291-301
0-Ecdysone
effect on molting 338/, 339
D effects 335/
Danaus plexippus, sequestration effects on larval development .340/, 341/
1
and allelochemics 273 ingested 337

Deactivation 265-1A Ecomones 304
Deciduous forest and chemical Elaegia utilis, bud exudate 78
defenses 27-31 Electroantennogram 220
Defense mechanisms 232-34 Encecalin 298
Defense, inducible systems, Encelia farinosa, chromenes 298
phytoalexins 307 Environmental effects 205-7
Defensive chemicals and leaf Ephemeral tissues and toxins 16-17
expansion 31-34 Epicuticular wax layer 258
Defensive chemistry, solutions 3 Epidermal glands plant—See Glands
Defensive compounds, chemical
Defensive structures, carbon an
nitrogen components 22 Ergosterol 175
Defensive system, heterogeneity 40 Escape in time 17
Defoliation budworm densities 9-11 Esters
Dendroctonus bark beetle, of gallic acids 124,131,132
cytochrome P-450 185 of hexahydroxydiphenic acids ....131, 132
Density-dependent mortality 46 Evolution and chemical defense
Depside metabolites 132 variability 37-51
Desert plants Excretion 266, 267
cytotoxic chemicals 291-301 Exposure to parasites and predators,
insecticidal chemicals 291-301 variability effects 43, 44
secondary metabolites 292 Extrafloral nectaries 70, 71
trichomes 292
Desert shrubs, benzofurans and F
chromenes 298-300
Destruction of petiole phloem effects, Fagopyrins 149
proteinase inhibitor inducing Fall armyworm
factor 110 deterrent 178
Deterrents, feeding 200 electron micrographs 331/
See also Feeding deterrents pulegone 178
Deterrents, terpenes 179/ Farina constituents 79
Detoxication 265-74 Farinose exudates 79, 80
Development time, food quality 46 Fatty acids
Didymocarpus, leaf exudates 82 larvae cuticle 262/, 318
Dietetics 232 leaf 261/
Differential sensory perceptions 215-28 roles 318
Digestibility-reducing substances ....4, 31, 33 Feeding 202,203
Dimeric procyanidins 125, 127/ prolonged times between 50
Dispersion and aggregation 207 Feeding behavior 202, 203
Diterpenes 314 Feeding deterrent 200
D N A transcription, damage encecalin 298
consequences 141 myristicin 311
Douglas fir, terpene chemistry, phytoecdysones 329-45, 345/
geographic variation 4, 5/ warburganal 314
and western spruce budworm xylomolin 313
interactions 3-18
Feeding inhibitor 217
Droplet countercurrent chroma-
tography 333 Feeding inhibitor sensitive cells 217
Dye phosphorescence 144 Feeding prevention 200
Dye toxicity, ranking 144 Feeding sites 221

1 3
Feeding stimulant, C N M R 249/ Grazing, barriers 145
Flavonoid aglycones 78/ Green odor 218,220
Flavonoids 76-79 differential sensory sensitivity 220
Flavonols 125, 127/ perception 224
Floral nectaries 70 Growth, food quality 46
Flower petals vs. terminals in Growth inhibiting compounds,
cotton 355-60 biological activity 233
Fly parasite oviposits, effects on Growth regulators, resistance
gypsy moth larvae 46 induction 155, 156
Foliage analysis 7, 8 Gustation 216-19
Foliar Gustatory receptor cells 216-19
cyanogenic glucoside 22, 24 Gut p H effects 48
lipids 258 Gypsy moth larvae, effects of fly
nitrogen concentration 22, 24 parasite oviposits 46
polyphenol accumulation 31,33 parasitoid survivorship 47/
Food quality, variability 46
French bean, potato leafhopper H
damage 72 Handling effects 235
Fungal pathogen effects 20
Furanocoumarins 141
Furocoumarins 316 Heliothis insects, chemical resistance
of cotton 350
Heliothis zea larvae 232
Herbicides, resistance induction ... 155, 156
Gallic acid 130/
Herbivores, specialized 31-34
esters 123,124,131,132
High-tannin adapted pests 51
Gallotannins 132
biological control 51
Galloyl esters, oxidative metabolism .. 132
Hooked trichomes 72
General resistance compounds 75
Horseradish flea beetle, host selection 208
Genetic resistance 306
Host-plant resistance 88, 89
Geographical differences, leaf quality 66
Host quality, effect on insect
Geographic variation in Douglas fir
mortality 43,44
terpene chemistry 4, 5/
Host selection
Geraniin 132, 133/
aggregation and dispersion 207
Geranylgeranylhydroquinone,
chemical basis 245-62
growth effects 295,297
chemical factors 202-5, 228
Germination, inhibitors 80
environmental factors 205-7
Glanded cotton, terpenoid aldehydes .. 348
mechanisms 305, 306
Glands, function and chemistry 68-89
morphological characteristics 201
Glandular hairs 69, 292
physical factors 201
Glandular trichomes 69, 201
Houseflies
immobilizing chemicals 73-75
biological effects of terpenes 176
importance of location 73
dye toxicity 144
Glaucolide A 80
Housefly attractants, terpenes 177
D-Glucose 130
Hymenaea, leaf exudates 82
Glucosinolate sensitive cells 217
Hypericin 146
Glucosinolates 208
Hyphantria cunea—See Webworm
Glycine max—See Soybean
Glycoside of withanolide 256-57
I
Gossypol 83,305,348
antibiotic activity 83 Immobilizing chemicals 73
effects on boll weevil 87 Inactivation of juvenile hormone 184/
effects on Spodoptera littoralis 87 Induced resistance
effects on tobacco budworm growth 84 definition 154
feeding deterrent 86 mechanisms 159-66
histochemical studies 360 uses 166, 167
histological examinations 352-55 Inducers
identification 351 natural 153-69
isolation 83, 348 resistance 154-58

INDEX 371
Inducible systems of defense, Leptinotarsa decemlineata—See

phytoalexins 307 Colorado potato beetle
Induction of microsomal oxidase Lignans 317
activity 191/ Limonene 176,310/
Ingested food, conversion efficiency ... 238 metabolism 185
Ingestion 267 Linolenic acid 318
Inhibitor accumulation in wounded Lipids
tomato plant leaves 107/ localization 309
Insect nonvolatile 312-22
dietetics 232,236-40 role 303-22
exposure to parasites and predators 43 volatile 309-12
immobilization 73 Lycopersicon hirsutum, isolation ...246, 248
resistance to crop plants 306-9
resistance to secondary plant M
allelochemics 87, 88
resistant crops, problems 307 Malacosoma californicum pluviale—
resistant soybean cultivars 307 See Tent caterpillar
Insectan canavanyl protein production 285 Manduca sexta
Insecticidal chemicals of desert
plants 291-30
Interflavan bond 125 host selection 246
Isolation procedure, feeding factor Solanaceae model 247/
for Manduca sexta 249 Maximilin-C 81
Mechanisms, nonpreference 199-210
J Messenger R N A I l l , 114
translation inhibition 120
Juvenile hormones 313 Metabolism 267-71
activity 182 Metabolites
biosynthesis 183/ phenolic 124, 126/
inactivation 184/ secondary, phototoxicity to
Juvenile hormone active terpenes 182 mosquito larvae 148
8-Methoxypsoralen 145
K Methyl eugenol 202
Kairomonal factors 248 Methylazoxymethanol, coping with
Kairomones 304 toxic effects 268
Methylene blue 141, 142/
L Mexican bean beetle, origin 257
Microbial chitinase 48
Larrea, leaf exudates 82 Microsomal oxidase activity, induc-
Larvae cuticle fatty acids 262/ tion, terpenes 191/
Larvae sensitivity, neonate 234-36 Mint monoterpene pulegone 178
Laurus nobilis—See Bay leaves 309 Mixed cropping 206
Leaf Mobility of nitrogen in plants 24
age effects 44 Moisture level 240
fatty acids 261/ Molting fluid 337
initiation and expansion 27 Multiple factor interactions 39
odors 224-28 Mustard oil 268,312
pubescence 71 Myristicin 311/
quality, geographical differences .... 66
Leaf damage, tent caterpillar attack N
on willows 63
Leaf expansion Natural inducers 153-69
and defensive chemicals 31-34 Natural photosensitizers 139
and polyphenols 33 Natural products, processing 266-72
Leaf-to-leaf variability 40 Neonate larvae sensitivity 234-36
Leguminous plants, protective New shoot extension 27
allelochemics 279-88 Newcastelia, glandular trichome
Lepidopterous larvae, gustatory components 81, 82
sensilla 216-18 Nicandrenone 252,253/

Nicotine 76, 77 Pesticide industry, problems with

and chemical defenses 21-26 insect tolerance 88
excretion 267 Pesticides, synthetic, problems 87
insects' response 76 Pests, high-tannin adapted 51
metabolism 267 biological control 51
in tobacco leaves 22, 24 Phacelia crenulata, glandular
Nitrogen and chemical defenses 21-26 trichomes 295
Nitrogen Phagodeterrent 256
and defensive structures 22 Phagostimulant, analysis 255
mobility in plants 24 Phaseolus vulgaris—See French bean
in plant alkaloids 25/ Phenolic chemistry of red oak,
Nonpreference, defined 199 defoliation effects 42
Nonpreference mechanisms 199-210 Phenolic metabolism, gallic acid role 130
Nonprotein amino acids 279 Phenolic metabolites 124, 126/
Nonvolatile lipids 312-22 Phenolics 206
derivation from fatty acids 320 Phenological synchronization 159, 160
Notholaena dealbata Pheromones 304
farinose constituent 79 aggregation 207
farinose exudates 7
Nucleic acid metabolism and
canavanine 282, 283 Photosensitization 140, 141
Nutrient-allelochemical interactions furanocoumarins 141, 144-45
in resistance 231-40 polyacetylenes 145, 146
Nutrient concentration changes 160 without oxygen 140
Nutrient-non-nutrient interactions 237 Photosensitizers
Nutrient variability 40 activated species 141
Nutrients and allelochemics 231-40 effect on insects 141-44
Nutritional indices 238 insect susceptibility 144
lack of color 149
O natural 139
role in resistance 139-49
Oil of citronella 309
Phototoxic Apiaceae, adaptation of
Olfaction 218-28
insects 145
Orientation 202
Phyllotreta armoraciae—See Horse-
Ornithine antagonist, 2,4-diamino-
radish flea beetle
butyric acid 288
Physical defenses, trichomes 71,72
Oviposition 203-5
Physiological constraints on chemical
role of inhibitory stimuli 204
defenses 21-34
role of volatiles 205 Physiological effects and allelo-
Oviposition factor, characterization .. 256* chemics 308/
Oviposition sites, differential sensory
Phytoalexins, synthesis 162-66
perception 221, 224
Phytochemicals, categories 75
Oviposition stimulants 312 Phytoecdysone effects on growth 334/
P Phytoecdysones
feeding deterrent 345
Papilio butterflies, oviposition isolation 329-45
behavior 204 Phytophagous insects 303
Parasitoid survivorship in gypsy Pieris brassicae larvae, response of
moth larvae 47/ gustatory receptor cells 217/
Parthenin 80 Pieris butterflies, orientation for
Parthenium glandular trichome oviposition 209
components 80, 81 Pigments 313
Pathogen exposure PIIF—See Proteinase inhibitor
resistance induction 157 inducing factor
variability effects 44 Pink bollworm 333
Pathogen susceptibility and tannins 48 electron micrographs 332/
Pathogens 44 larvae, phytoecdysone effect on
fungal, effects 206 growth 334
/3-Pentakisgalloyl-D-glucose, Plant-herbivore theory 3-18
metabolism 131, 132 Plant alkaloids, nitrogen 25

INDEX 373
Plant allelochemics, secondary, Proteinase inhibitors—Continued

insect resistance 87, 88 synthesis regulation 103-21
Plant epidermal glands—See Glands translation inhibition 120
Plant trichomes, function and Proteinase preinhibitors, synthesis
chemistry 69-89 route HI
Pollen color 349 Proteins
Polyacetylene(s) 145, 146 polyphenol interactions 134
Polyacetylene P H T , spectrum 149/ and polyphenols 123-36
Polyphagous species, oviposition 204 Prunus, glandular trichome
Polyphenol interactions components 81, 82
with proteins 134 Pubescent leaves 71
high protein concentrations 134 Pulegone 178
low protein concentrations 134 Pulegone oxidation 185
Polyphenols 124 southern armyworm 189, 190/
See also Tannins Pyrethrins 175
association with proteins 123-36 Pyridoxal phosphate and L-canaline
biosynthetic pathways 132 interaction 286-88
metabolism 132
structural types 12
toxins 31-3
Polypodiacae farinose exudates 79, 80 Qualitativ
Polysaccharides of bacteria 154 Quantitative defenses 4,31,33
Populus, glandular trichome Quinones, prenylated 292-97
components 81, 82
Potato leafhopper damage to R
French bean 72
Potato plant leaves Rat liver, cytochrome P-450 185
green odor components 224 Red oak, phenolic chemistry of,
odor characterization 220 defoliation effects 42
Precocenes 182,298 Repellent allelochemics 75-87
Prenylated quinones 292-97 to bollworm 84
Pre-proteinase inhibitors, in vitro Repellents 226
synthesis 111-20 Reproduction and terpenes, repro-
Primulaceae, farinose exudates 79, 80 ductive inhibition 182
Proanthocyanidins 123, 124-31 Resistance, complex 39
Resistance-susceptibility studies using
molecular species 124
Douglas fir-budworm 6-8,9-11
synthesis 129/
Resource restriction 39
Processing of natural products 266-72
Rhagoletis pomonella—See Apple
Procollagen 280
maggot fly
Procyanidin Rhamnogalacturonan I 104
biosynthesis 129/ Role of fatty acids 318
dimers 124-31,127/ Role of lipids 303-22
stereochemistry 125 Rose bengal 141, 142/
Protein production, canavanyl 285
Protein synthesis, L-canavanine 282
S
Proteinase inhibitor inducing factor .. 104
destruction of petiole phloem Salix sitchensis—See Willow
effects 110/ Salvia, glandular trichome
direction and flow rate 104-11 components 81, 82
flow rate 106 Secondary metabolites 123,203
means of introduction 109 allomonal properties 153, 154
petiole role 109 and desert plants 292
phloem involvement 109 phototoxicity to mosquito larvae .... 148/
transport mode 106, 109 Secondary plant allelochemics,
Proteinase inhibitors 239 insect resistance 87, 88
accumulation in wounded tomato Selective sequestration 271,272
plants 103-21 Sensillum 216
partial purification 118/ Sensitizations, photodynamic 140
segment length determination 116 Sensory perceptions, differential 215-28

Sequestrators and allelochemics 212-1A Terpene hydrocarbons, oxidation 207

Sesquiterpene lactones 76, 80, 81 Terpene induction of cytochrome
Sesquiterpenes 310 P-450 187*
biological activity 313 Terpenes
Sinigrin 208 as attractants 176-78
structure 268 biosynthetic relationship 174/
Sitka willow—See Willow cytochrome P-450 metabolism ...173-92
Solarium, alkaloids, effects on Colo- as deterrents 176-78, 179*
rado potato beetle 77 effect on houseflies 176
Southern armyworm 178 effect on reproductive capacities ... 181
body weight effects of pulegone ... 181* for houseflies 179*
cytochrome P-450 189 juvenile hormone, active 182
developmental effects of mammalian acute toxicity 176
pulegone 178-82, 182* reproductive inhibition 182
pulegone oxidation 189, 190/ structural effects 176
reproductive inhibition 182 toxicity 175
Soybean(s) 257 Terpenoids 76-79
cultivars, insect resistant 307 a-Terthienyl 145
pubescence 7
Specific resistance compounds 7
Spodoptera littoralis, gossypol effects . 87 Tobacco budworm
Storage, mustard oil 268 and cotton resistance 347-64
Styloconic sensilla 216 feeding on cotton 349
Sucrose 218 gossypol effects 84
Sugar sensitive cells 217 inhibition of growth 354*
Sulfur dioxide effects 206 Tobacco budworm growth
Sunflower, insect resistance 81 chrysanthemin effects 355-60
Susceptibility effects of cotton flower petals 359*
insect, and chemical defenses 37-51 gossypol effects 355-60
to insects 154-58 tannin effects 355-60
Synthetic pesticides, insect tolerance Tobacco hornworm 77
development 88 canavanyl protein production 285
Synthetic pesticides, problems 87 Tobacco leaves, nicotine in 22, 24
Tolerance 199
T Tolerance to synthetic pesticides,
development in insects 88
Tannin(s) Tomato plant leaves, subsequent
See also Polyphenols wounding 114
assimilation 239 Tomato plants
condensed, identification 352 synthesis regulation of proteinase
histological examinations 352, 360 inhibitors 103-21
and pathogen susceptibility 48 wounded, proteinase inhibitors .103-21
Tannin content, cotton 349 Toxic allelochemics 75-87
Taste perception 218 Toxic compounds, storage 265
Temporal variability 42 Toxicity, cotton terpenoid aldehydes .. 85*
Tent caterpillar attack, Alder
Toxicity of terpenes 175
responses 56-59
Tent caterpillar attack, Alder Toxins
responses of foliage quality 59 See also Terpenes
biomass and survivorship data 58/ alkaloids 75-77
Tent caterpillar attack, willow cotton pigment glands 83-87
responses 59-63 and ephemeral tissues 16-17
biomass and survivorship data 58/ flavonoids 75,76,77-79
leaf quality damage 63 plant 4
Terpene chemistry polyphenol 31-34
See also Toxins Translation inhibition,
and budworm resistance 9-12, 13* proteinase inhibitors 120
and budworm success 17 Tree age and productivity 15
geographic variation in Douglas fir.. 4, 5* Tree defenses, variability 40-43

INDEX 375
Trichomes 69,292 Volatile lipids 309-12

and quinones 295 Volatiles in oviposition 205
chemical resistance 72-75
glandular 69 W
hooked 72
of desert plants 292 Warburganal 314/
physical defenses 71 Water balance 260
Triterpenes, antiherbivore activity 315 Water stress and budworm success
Trypsin inhibition Ill 11-12, 13/, 16
L-Tyrosine decarboxylase activity 288/ Water stress studies using Douglas
fir-budworm 8-9,11-12
Webworm, larval stage 63
U Webworms 63
Western spruce budworm, interactions
Unattacked willows
with Douglas fir 3-18
airborne pheromones effect 67
Willow responses to tent caterpillar
leaf food quality 63
attack 59-63
Utilization 265-74
biomass and survivorship data 58/
V
attack 63-67
Variability Willows, unattacked, leaf food
chemical defense 39-51 quality 63
leaf to leaf 40 Withanolide, glycoside 256-57
spatial arrays 40 Wound signal
in tree defenses 40-43 See also Proteinase
Vegetable tannins direction and flow rate 104-11
See Polyphenols Wounding of tomato plant leaves,
Veronica, glandular trichome inhibitor accumulation 107/
components 80, 81
Vitellogenin 282 X
and canavanine production 283-85
Vitellogenin production, arginine Xylem pressure potentials 9, 11
depletion effects 284/ Xylomolin 313


(ACS Symposium Series 208) Paul A. Hedin (Eds.) - Plant Resistance To Insects-American Chemical Society (1983)

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(ACS Symposium Series 208) Paul A. Hedin (Eds.) - Plant Resistance To Insects-American Chemical Society (1983)

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Plant Resistance to Insects

Paul A. Hedin, EDITOR

sponsored by the ACS

Division of Pesticide Chemistry

at the 183rd Meeting of the

American Chemical Society,

Las Vegas, Nevada,

March 28-April 2, 1982

ACS SYMPOSIUM SERIES 208

In Plant Resistance to Insects; Hedin, P.;

In Plant Resistance to Insects; Hedin, P.;

David L. Allara Robert Ory

Robert Baker Geoffrey D. Parfitt

Donald D. Dollberg Theodore Provder

Brian M . Harney Charles N. Satterfield

W. Jeffrey Howe Dennis Schuetzle

Herbert D. Kaesz Davis L. Temple, Jr.

Marvin Margoshes Charles S. Tuesday

Donald E. Moreland C. Grant Willson

In Plant Resistance to Insects; Hedin, P.;

In Plant Resistance to Insects; Hedin, P.;

B IOLOGISTS HAVE LONG RECOGNIZED the specificity of various insects for

In Plant Resistance to Insects; Hedin, P.;

In Plant Resistance to Insects; Hedin, P.;

Studies were undertaken to t e s t various aspects of current

Some of the major o b j e c t i v e s i n coevolutionary b i o l o g y are

In Plant Resistance to Insects; Hedin, P.;

p e r e n n i a l s and t h e i r associated h e r b i v o r e s . Rhoades and Cates

Although p l a n t s produce many secondary products, chemical

In Plant Resistance to Insects; Hedin, P.;

p r e l i m i n a r y data show that t h i s v a r i a t i o n a l s o e x i s t s at a l o c a l

Table I . V a r i a t i o n i n some of the chemical c o n s t i t u e n t s i n the

T o t a l Nitrogen MT 1.8 0.5 1.3 3.5

a-pinene MT 176.5 223.6 0.0 1001.0

0-pinene MT 134.8 93.7 0.0 429.0

Limonene MT 84.7 69.2 0.0 403.0

B o r n y l Acetate MT 63.1 38.4 6.0 189.0

Cadinene Isomer MT 6.9 5.4 0.0 33.2

T o t a l Terpenes MT 713.6 502.1 147.8 2442.5

Polyphenols MT 0.4 0.2 0.1 1.0

Polyphenols = % f r e s h wt; n i t r o g e n =» % dw; terpenes -

Our focus was upon n a t u r a l and experimental s t u d i e s that

In Plant Resistance to Insects; Hedin, P.;

e l u c i d a t i n g the r e l a t i o n s h i p between budworm success and p h y s i c a l

S i t e S e l e c t i o n and General Methodology

Studies to determine the l e v e l of r e s i s t a n c e or

In Plant Resistance to Insects; Hedin, P.;

N a t u r a l budworm d e n s i t i e s were determined by sampling 6

In Plant Resistance to Insects; Hedin, P.;

Consequently a l l 105 trees were used i n the a n a l y s i s reported

Studies to determine the e f f e c t of water s t r e s s on f o l i a g e

In Plant Resistance to Insects; Hedin, P.;

that were growing on a north f a c i n g slope ( n o n - s t r e s s ) , while

Resistance - S u s c e p t i b i l i t y Studies Using N a t u r a l Budworm

R e s u l t s of the data gathered a t the Montana s i t e i n d i c a t e

In Plant Resistance to Insects; Hedin, P.;

were not important i n determining the budworm d e n s i t i e s and

Studies Using Experimental Female Budworm L e v e l s on

R e s u l t s of the data gathered at the Barley Canyon s i t e ,

Acetate F r a c t i o n -0.17828 0.0432 -0.547

N i t r o g e n = % dw; Terpenes • area counts/20 mg dw

budworm success (Table I I I ) . When the u n i d e n t i f i e d terpene,

In Plant Resistance to Insects; Hedin, P.;