Entomol. exp. appl.

43: 25-29, 1987 25

9 Dr W. Junk Publishers, Dordrecht - Printed in the Netherlands

The role of a m m o n i a in the attraction of females of the Mediterranean fruit fly to

protein hydrolysate baits

M. Mazor 1, S. Gothilf 1 & R. Galun2

llnstitute of Plant Protection, ARO, The Volcani Center, Bet Dagan, Israel; and 2Department of
Parasitology, Hebrew University of Jerusalem, Hadassah Medical School, Jerusalem, Israel

Keywords: Mediterranean fruit fly, Ceratitis capitata, attractant, ammonia, protein hydrolysates

Abstract

Attraction of female Mediterranean fruitfly (medfly) to ammonia and to protein baits was studied with
an olfactometer. Ammonia bait (1 cc/trap) proved to be an effective lure. The maximum number of females
was caught in traps loaded with 0.01 M ammonia solution with a release rate of 5.28/zg/cc/h.
A positive correlation was found between female catch and the ammonia release rate of various protein
baits, especially from dry protein hydrolysate of which casein hydrolysate was the most effective. However,
casein hydrolysate was less attractive than certain ammonia solutions having a lower rate of ammonia release.
Elevation of the pH of the liquid commercial baits, Buminal and Naziman, increased the latter's efficacy
as medfly baits but the increased stimulation could not be strictly correlated with the increased rate of ammo-
nia release. It is therefore suggested that medfly olfactory response to protein baits is affected not only by
ammonia but by other volatiles as well.

Introduction the components and volatiles of protein hydroly-

Tephritid fruit flies require a source of protein to
complete egg maturation. This requirement is prob-
ably the main cause for the strong attraction of fe-
males towards decomposing proteinaceous sub-
stances, first observed by McPhail (1939). Based on
this behavioural reaction, hydrolysates of protein
have been included in baits to control fruit flies
(Steiner, 1952). Since ammonia is emitted from
decomposing proteins, it was assumed that attrac-
tion of fruit flies to protein baits is related to the
release of ammonia. Indeed ammonium salts have
long been used as a lure in fruit fly traps but with
conflicting conclusions concerning their effective-
ness. Some workers suggested that ammonia is a
weak attractant (e.g. Ripley & Hepburn, 1929;
McPhail, 1939; Gow, 1954), while others consid-
ered it to be an effective lure (e.g. Hodson, 1948;
Frick, 1952; Prokopy, 1975; Reissig, 1975; Vita et
al., 1980). Recently Morton & Bateman (1981) and
Bateman & Morton (1981) studied the chemistry of
sates but did not identify any particular compound
as being responsible for the attraction of the
Queensland fruit fly-Dacus tryoni. However, they
did find that certain solutions of ammonium bicar-
bonate were much more effective attractants than
the standard protein baits, and that at low pH the
attraction of the fly to the protein baits is caused
by the small amounts of ammonia released from
the baits. At high pH, other attractive volatiles are
apparently released .from these baits. In the present
work, we have tested the attractancy of the female
Mediterranean fruit fly, Ceratitis capitata (Wied.)
(medfly), to ammonia solutions and several protei-
naceous baits. We examined the relationship be-
tween the attractancy of these baits and their rate
of ammonia release.
Materials and methods
Medfly pupae originating from local fruit plan-
26
tations, reared for 5 to 10 generations in the labora-
tory, were supplied by the Israel Cohen Institute for
Biological Control, Rehovot, Israel. Pupae and the
emerging flies were kept in a room with windows,
under a natural day:night regime and a controlled
temperature of 25 + 1 ~ All experiments were
conducted in a separate room under the same con-
ditions. Three- to 10-day-old females were used for
the experiments. Preliminary tests showed no
difference in the olfactory response of flies in this
age range. Flies were maintained on granulated sug-
ar and water absorbed on cotton wool.
The following baits were tested: the enzymatic
hydrolysates of casein, lactalbumin, soy, and yeast
(ICN, Cleveland, OH, U.S.A.), all in dry powder
form; Buminal | (Bayer, Leverkusen, F.D.R.),
Naziman (Nasid Ltd., Tel Aviv, Israel) and PIB 7
(Staley Manufacturing Co., Decatur, IL. U.S.A.), in
liquid form. The ammonia stock solution was a
concentrated volumetric solution (BDH) adjusted
to several concentrations between 0.0001 and
0.1 M.
The response of the medfly females to the baits
was examined in an olfactometer and in traps
described by Gothilf & Galun (1982). All tests were
of the two-choice type. During the test six traps
were suspended from the horizontally rotating
wheel of the olfactometer. Three traps were loaded
with one bait and three with a second bait or un-
baited. One cc of the experimental bait, whether
liquid or dry powder, in a 3-cm-long 1.5-cm i.d.
glass tube (exposure surface of 1.8 cm2), was put
in each baited trap. Traps of different treatments
were placed alternately in the olfactometer. 250 or
500 female flies were placed in the olfactometer be-
fore the start of testing and the same flies were used
for several tests, usually one test per day, but some-
times two. Dead flies were replaced by live females
of the same age before every test. Each test lasted
one hour. At the end of that time the entrance holes
of the traps were plugged with a piece of cotton
wool and the traps were transferred to a refrigerator
for a few minutes. With this procedure the flies be-
came less active and could be counted more easily.
The trapped flies were collected with an aspirator,
counted, and released back into the olfactometer.
For a given bait in a test, the number of flies cap-
tured in the three traps was recorded and the test
was repeated 5 - 1 2 times. Four olfactometers were
used so that four tests could be run simultaneously.
The release rate of ammonia from protein
hydrolysate baits and from ammonia solutions was
determined as follows: 1 cc of an experimental bait
was put in a 3-cm-long x 1.5-cm-i.d. glass tube of
the type used in the trap, two or three such tubes
were prepared when materials of low ammonia re-
lease rate were tested. The tubes were placed in a
100-ml flask. Air was pulled with a pump over the
sample at a rate of 150 ml/min and then into two
consecutive tubes, through cinter glass filters, each
containing 10 ml distilled water. The emitted am-
monia was trapped mainly in the first water trap;
the second trap contained less than 10% of the
released ammonia. The duration of this procedure
ranged from 1 to 4 h, depending on the expected re-
lease rate of ammonia from the experimental mate-
rial. The amount of ammonia trapped in the dis-
tilled water was determined by the phenolchlorite
method (Solorzano, 1969) and was calculated as #g
ammonia released from 1 cc experimental material
per hour. The release rate was checked twice for
each bait and only small differences were found.
Except for Fig. 2, the average of the two checks is
given. All the chemical work was carried out at an
ambient temp. of 25 _+ 1 ~
Results and discussion
Before questioning the role of ammonia in med-
fly attraction to baits, it was important to assess the
relationship between rate of ammonia release from
a bait and the olfactory reaction of the flies. Using
a bait of pure ammonia solution, which released
only ammonia (ignoring water molecules which
had no stimulatory effect in the present experimen-
tal environment), a clear correlation was found be-
tween the concentration of the solution and its
stimulation (Fig. 1) and between the concentration
and rate of ammonia release (Fig. 2). The relation-
ship between concentration of ammonia solution
and the number of trapped females is linear,
characteristic of log stimulus - response relation
up to a concentration of 0.01 M (Fig. 1). The rela-
tive ammonia release rate at maximum stimulation
was 5.28/~g/cc/h. Beyond 0.01 M the correlation
was negative, i.e., at higher concentrations with
higher release rates of ammonia, the solutions be-
came less and less attractive.
The olfactory response of medfly females to vari-
 27

80 Table 1. Attraction of medfly females to protein hydrolysate

1 I I I I
baits and their corresponding ammonia releases.

70 Bait Mean* 9 Ammonia release

catch _+ s . e . tzg/cc/h

60 Casein hydrolysate 192.0_+ 5.8 a 3.2

Lactalbumin hydrolysate 141.0+_ 7.4 b 2.0
Buminal 135.6_+ 6.0 b 0.4
0 50 Soy hydrolysate 109.2+ 10.6 c 1.4
r~ Naziman 92.4_+ 9.7 c 0.1
PIB 7 83.6+ 9.5 c 0.1
t,U
4O Yeast hydrolysate 29.4+_ 6.1 d 0.1
.-I
Control 4.5 0
3O
h * Average of five tests. Average of control was calculated from
catches in unbaited traps in all tests. No. followed by same
20 letter not significantly different at 5~ level.

iO ous baits and their corresponding release rates o f

a m m o n i a are summarized in Table 1. In this table
0 I 1 I I I the various baits were c o m p a r e d to an unbaited
0.0001 0001 0005 0.01 0.05 O I control. Since the control catch was practically nill,
statistical analysis was unwarrented. However,
AMMONIA CONCENTRATION (M)
within the bait series, significant differences were
detected as indicated in the table. The most attrac-
Fig. 1. Attraction of 9 medflies to traps baited with ammonia
solutions of different concentrations. Dots: means of ten tests. tive a m o n g the tested baits was casein hydrolysate;
Bars: +_S.E. next was lactalbumin hydrolysate and Buminal;
and less stimulatory were soy hydrolysate, Nazi-
man, PIB 7 and yeast hydrolysate. A positive corre-
lation was f o u n d between the stimulatory effect o f
the baits and the a m o u n t s o f a m m o n i a released
A 2O ' I ' I I I ' I ' l/
from them (r =0.77). If the dry baits are considered
0
_- V -" 1 5 8 . 8 3 X + 0.79 iq as a separate group, an even stronger relationship
(r=0.96) exists between a m m o n i a release and
o 16
stimulation (Fig. 3).
In spite o f the p r o n o u n c e d relationship between
IM medfly attraction and a m m o n i a release from the
dry baits shown in Fig. 3, it seems that attraction
t'r
to the dry baits is also affected by other volatiles.
I.iJ In Table 2 various concentrations o f a m m o n i a so-
t~O
,,~ 8
IM
lution were c o m p a r e d to casein hydrolysate in pairs.
_J The flies preferred a m m o n i a solutions at concen-
IM
r,r
trations o f 0.01 M and 0.0075 M although their
4
a m m o n i a release, especially o f the latter, is less
0 than that o f the casein. There was no difference be-
tween the reaction o f flies to casein hydrolysate and
0 to a m m o n i a solutions o f 0.005 M and 0.0025 M
i I i I , I i I , l
0 0.02 0.04 0.06 0.08 O. I whose a m m o n i a release rates are m u c h smaller
than that o f the casein. It seems, therefore, that al-
AMMONIA CONCENTRATION (M)
t h o u g h a m m o n i a release can be used as a criterion
Fig. 2. Ammonia release rate from ammonia solutions of for stimulation o f dry protein hydrolysates, it is not
different concentrations. the only c o m p o u n d which affects the behavioral
 28

I I I I their attractiveness resulted only from their ammo-

200
nia release which was low compared with the re-
Y = 52.17X 3 0 . 8 0
lease rate of dry baits of similar attractiveness (Ta-
ble 1). That such low rates of ammonia release are
not enough for the recorded females catches, can
150 also be deduced from the tests with pure ammonia
1- which were mentioned before (Figs. 1 and 2).
Following the results of Bateman & Morton
(1981) who succeeded in improving the attractancy
IJJ I00 of yeast hydrolyate to Dacus tryoni by elevating its
_1 pH, we examined this process with Buminal and
:E Naziman which are commonly used in bait sprays
IJJ
M. of fruit trees. The pH of the commercial prepara-
tions is 5.8 for Buminal and 4.5 for Naziman. At
50
these pHs their ammonia release rate is relatively
low (Table 1). Using 5N NaOH, the pH of both
YH
baits was adjusted also to 7.5, 8, 8.5 and 9. The
catch corresponding to a given pH was compared
o I I I 1 to that of an unbaited control. Again the control
0 0.8 1.6 2.4 3.2
catch was virtually nill and results were analyzed as
AMMONIA RELEASE RATE (IJg/ec/hour) in Table 1. Table 3 illustrates the differences detect-
ed among the various formulations. Buminal at pH
Fig. 3. Relationship between rate of a m m o n i a release and 9
of 7.5, 8 and 8.5 was more attractive to the females
catch in traps baited with dry protein hydrolysates. (CH) casein
hydrolysate; (LH) laetaibumin hydrolysate; (SH) soy hydroly-
than at pH of 5.8 and 9, with no significant differ-
sate; (YH) yeast hydrolysate. Dots: See Fig. 1, but 5 tests. Bars: ence in number of females trapped between pHs of
see Fig. 1. 7.5, 8 and 8.5. Naziman showed a slightly different
pattern of relationship between attractancy and
Table 2. Medfly attraction to a m m o n i a solutions and to casein
pH: at pH 8 it was more attractive than at lower or
hydrolysate. higher pHs. (Table 3). It is obvious from these
results that the attractiveness of these liquid baits
Test Bait Mean* 9 A m m o n i a release**
catch_+ s.c. tzg/cc/h
Table 3. Female catch and a m m o n i a release of Buminal and
Naziman at different pHs.
1 Ammonia0.001M 23.7+_2.0b 0.1
Casein hydrolysate 40.4+ 3.3 a 3.1
pH Mean* 9 A m m o n i a release
2 Ammonia0.0025 M 21.2+1.4a 1.2
catch _+ s.e. #g/cc/h
Casein hydrolysate 24.0_+ 2.3 a 3.1
3 A m m o n i a 0.005 M 27.7+2.5 a 1.6
Naziman
Casein hydrolysate 24.1 4.0 a 3.1
4.5 22.2+_2.9 c 0.1
4 A m m o n i a 0.0075 M 46.4 +- 3.1 a 2.0
7.5 42.1+_2.9 b 2.5
Casein hydrolysate 20.0_+ 1.8 b 3.1
8.0 75.3 +_5.3 a 6.4
5 A m m o n i a 0.01 M 56.8 a 2.4
8.5 53.4+_5.6 b 15.8
Casein hydrolysate 11.5 +_ 1.7 b 3.1
9.0 25.2 5.4 c 26.8
Control 0.3
* Average of ten tests. Significancy, see Table 1.
** Values of release from a m m o n i a solutions calculated from
Buminal
regression line of Fig. 2.
5.8 29.8+2.5 b 0.4
7.5 45.7+2.1 a 4.6
8.0 49.4+6.1 a 7.4
reaction of the flies to these baits, and that other 8.5 49.9 _+4.4 a 11.5
9.0 36.9+4.2 ab 23.6
volatiles, apparently repellents, are involved in the
Control 0.2
fly-bait relationship.
As for the three liquid baits, it is improbable that * Average of twelve tests. Significancy, see Table 1.

to medfly females can be increased by elevating the
pH. Although increase in pH was followed by
higher ammonia release, the reason for the increase
in attractiveness cannot be attributed solely to
elevated ammonia release, because the relation be-
tween pH, ammonia release and female catch was
different and specific for each of the two liquid
baits (Table 2). It can therefore be assumed that
volatiles other than ammonia also play a role in fe-
male medfly attraction to these baits.
We show here in agreement with the findings of
Bateman & Morton (1981) with the Queensland
fruit fly, that ammonia is a very effective attractant
for female medfly. It may be an effective lure also
for many other fruit flies. As for the protein baits,
their stimulation of medfly females could not be
attributed solely to their ammonia volatiles. It is
suggested that other unidentified volatiles have an
important effect on the olfactory reaction of the fly
to protein baits.
Acknowledgement
Contribution from the Agricultural Research Or-
ganization, (ARO), Bet Dagan, Israel. No. 1653-E,
1986 series.
R~sum~
Le role de l'ammoniaque dans l'attraction des
femelles de la mouche mdditerandenne aux attrac-
tifs ?l base d'hydrolysats de proteines
Uattraction de la femelle de la mouche mOditer-
anOenne h l'ammoniaque et aux attractifs protOi-
niques a 6t6 6tudiOe au moyen de l'olfactomOtre
dOcrit par Gothilf & Galun (1982). Uammoniaque
(1 cc/piege) s'avOra ~tre un attractif efficace. Le
nombre maximum de femelles fut attrapp6 dans les
piOges chargOs avec une solution d'ammoniaque
0.01 M ayant une vitesse de diffusion de
5.28/tzg/cc/h.
Une corrOlation positive a &6 trouvde entre les
captures de femelles et les vitesses de diffusion de
divers attractifs protOiniques, particulierement ceux
29
base d'hydrolysats de protOines secs parmi
lesquels le plus effectif a 6t6 l'hydrolysat de casOine.
Toutefois, l'hydrolysat de casOine a 6t6 moins at-
tractif que certaines solutions d'ammoniaque ayant
une vitesse de diffusion plus basse. Une hausse du
pH des attractifs liquides commerciaux, Buminal et
Naziman, a augment6 leur efficacit6 en tant qu'at-
tractifs pour la mouche mOditeranOenne; toutefois
la stimulation plus intense n'a pu 6tre mise en corre-
lation stricte avec une vitesse accrue de la diffusion
de l'ammoniac. C'est pourquoi il est suggOr6 que la
rOaction olfactive de la mouche mOditerranOenne
est rOgie, non seulement par l'ammoniaque mais
aussi par d'autres substances volatiles.
References
Bateman, M.A. & T. C. Morton, 1981. The importance of am-
monia in proteinaceous attractants for fruit flies (Fami-
ly.q'ephritidae). Aust. J. agric. Res. 32: 883-903.
Frick, K. E., 1952. Determining emergence of the cherry fruitfly
with ammonium bicarbonate bait traps. J. econ. Ent. 45:
262-263.
Gothilf, S. & R. Galun, 1982. Olfactometer and trap for evaluat-
ing attractants for the Mediterranean fruit fly, Ceratitis
capitata. Phytoparasitica 10: 79-84.
Gow, P. L., 1954. Proteinaceous bait for the Oriental fruit fly.
J. econ. Ent. 47: 153-160.
Hodson, A. C., 1948. Further studies of lure attractive to the ap-
ple maggot. J. econ. Ent. 41: 61-66.
McPhail, M., 1939. Protein lures for fruit flies. J. econ. Ent. 32:
758-761.
Morton, T. C. & M. A. Bateman, 1981. Chemical studies on pro-
teinaceous attractants for fruit flies, including the identifica-
tion of volatile constituents. Aust. J. agric. Res. 32:905-916.
Prokopy, R. J., 1975. Selective new trap for Rhagoletis cingulata
and R. pomonella flies. Envir. Ent. 4: 420-424.
Reissig, W. H., 1975. Evaluation of traps for apple maggot in
unsprayed and commercial apple orchards. J. econ. Ent. 68:
4 4 5 - 448.
Ripley, L. B. & G. A. Hepburn, 1929. Fruit fly control. Fmg. S.
Aft. 4: 345-346, 357.
Solorzano, L., 1969. Determination of ammonia in natural
waters by the phenolpochlorite method. Limnol. Oceanogr. 5:
799- 801.
Steiner, L. F., 1952. Fruit fly control in Hawaii with poison-bait
sprays containing protein hydrolysates. J. econ. Ent. 45:
838- 843.
Vita, G., B. Rossi & A. Carpita, 1980. Valutazione in campo del-
l'attrattivita esplicata verso il Dacus oleae (Gml.) da parta di
formulati chimici rilascianti ammoniaca. Redia 63: 185-196.
Accepted: July 5, 1986.