P1: IAZ

Journal of Chemical Ecology [joec] pp1129-joec-478533 February 20, 2004 22:23 Style file version June 28th, 2002
Journal of Chemical Ecology, Vol. 30, No. 2, February 2004 (
C
2004)
MALE AGGREGATION PHEROMONE OF DATE PALM FRUIT
STALK BORER Oryctes elegans
DIDIER ROCHAT,
1,∗
KAZEM MOHAMMADPOOR,
2
CHRISTIAN MALOSSE,
1
ARMAN AVAND-FAGHIH,
3
MARTINE LETTERE,
1
JOSIANE BEAUHAIRE,
1
JEAN-PAUL MORIN,
4
ADELINE PEZIER,
1
MICHEL RENOU,
1
and GHOLAM ABBAS ABDOLLAHI
3
1
INRA, Unit´ e de Phytopharmacie et M´ ediateurs Chimiques
Centre de Versailles, Route de Saint-Cyr
78026 Versailles Cedex, France
2
Saravan Plant Pests & Diseases Research Laboratory
P.O. Box 364, 99515 Saravan, Iran
3
Harmful Insects to Plants Research Department
Plant Pests & Diseases Research Institute, P.O. Box 1454
19395 Tehran, Iran
4
CIRAD-CP, TA 80/02, Avenue Agropolis
34398 Montpellier Cedex 5, France
(Received May 26, 2003; accepted September 8, 2003)
Abstract—Laboratory and field investigations were carried out to characterize
the chemical communication system of the date palm fruit stalk borer, Oryctes
elegans, and to develop pheromone-based trapping in Eastern Iran. Adults of
both sexes feeding on date palm pieces attracted conspecifics, whereas date
palm alone was minimally attractive. Males were twice as attractive as females.
More beetles were captured at the palm crown than at ground level. Odors
from adults feeding on sugarcane were sampled and analyzed by gas chro-
matography and mass spectrometry. Whereas females did not emit sex specific
volatiles, males emitted a blend of 4-methyloctanoic acid (1: major component)
and ethyl 4-methyloctanoate (2), occasionally mixed with minor components:
4-methyloctanyl acetate (3), methyl 4-methyloctanoate (4), 4-methyloctanol (5),
and nonanyl acetate (6). Electroantennography and field trapping experiments
demonstrated that compound 1 is an essential component of the male aggregation
pheromone of O. elegans. It was barely attractive by itself but synergistic with
fresh date palm odor. It attracted many more beetles than any of compounds
2–6. The addition of one or several of compounds 2–6 to 1 did not improve
trap captures. During the course of 2 years, we captured 4000 beetles, with a

To whom correspondence should be addressed. E-mail: rochatd@versailles.inra.fr
387
0098-0331/04/0200-0387/0
C
2004 Plenum Publishing Corporation
P1: IAZ
Journal of Chemical Ecology [joec] pp1129-joec-478533 February 20, 2004 22:23 Style file version June 28th, 2002
388 ROCHAT ET AL.
weekly average of 6.3 beetles/trap, and were able to monitor the seasonal flight
of O. elegans. Our results provide the basis for developing mass trapping for
control of this pest.
KeyWords—Coleoptera, Scarabaeidae, Oryctes, aggregationpheromone, EAG,
field trapping, date palm, synergy.
INTRODUCTION
Rhinoceros beetles constitute a group of medium- to large-sized scarabs of the
Dynastinae subfamily whose males are characterized by a large cephalic horn.
Among rhinoceros beetles, the genus Oryctes is specific to the Old World and
includes about 40 species (Lepesme, 1947; Endr¨ odi, 1985). Many rhinoceros bee-
tles are associated with palm trees and are severe pests of coconut, oil, and date
palms (Lepesme, 1947; Bedford, 1980). Adult beetles burrowgalleries in the fresh
tissues of palms for feeding. The damage is particularly severe when the apical
bud is attacked in the young trees or the fruit-stalk in producing trees. Indirect
damage often occurs in areas occupied by palm weevils (Rhynchophorus spp.),
which lay eggs in the galleries made by the dynastine beetles. Palm weevil infes-
tations are often fatal to palms. Larvae have no direct impact on plant production
because most of them feed on dead tissue. Despite insecticides active against most
rhinoceros beetles, efficient and acceptable methods of controlling them are still
lacking in many cases, either because of economic reasons, or more generally,
because adults spend most of their life hidden in galleries, and are excellent flyers,
capable of rapidly colonizing new feeding and breeding sites.
The idea of manipulating adult behavior, especially luring beetles into traps,
was investigated in the 1970s. Some attractants were reported but subsequently
abandoned (Julia and Mariau, 1976; Vander Meer et al., 1979; Vander Meer and
McGovern, 1983). Recently, male aggregation pheromones were reported from
four rhinoceros beetles attacking palmtrees: Oryctes monoceros (Olivier) in Africa
(Gries et al., 1994), Oryctes rhinoceros L. (Hallet et al., 1995; Morin et al., 1996)
in the Asia-Pacific area (further referred to as “the Tropical Oryctes”), Scapanes
australis Boisduval in the New Guinea region, and Strategus aloeus L. in the
New World (Rochat et al., 2000b, 2002). Synthetic pheromone makes it possible
to capture these species in bucket traps. Applications on a large scale have been
reported for O. rhinoceros and Scapanes australis (Hallet et al., 1995; Ho, 1996;
Chung, 1997; Purba et al., 2000) but not for O. monoceros and S. aloeus. For
both O. rhinoceros and S. australis, attraction to the pheromone was enhanced
by, or synergistic with, plant odors from decaying wood in O. rhinoceros (Hallet
et al., 1995; Alfiler, 1999; Sudharto et al., 2001), or from fresh palm tissues in S.
australis (Rochat et al., 2002).
Three Oryctes species develop specifically in date palms and are present in the
Near- and Middle-East and in North Africa: O. elegans Prell [=sinaicus Petrovitz
P1: IAZ
Journal of Chemical Ecology [joec] pp1129-joec-478533 February 20, 2004 22:23 Style file version June 28th, 2002
AGGREGATION PHEROMONE OF Oryctes elegans 389
(non Walker)], O. agamemnon Burmeister (=desertorum Arrow, =persicus
Endr¨ odi), and O. richteri Petrovitz (Endr¨ odi and Petrovitz, 1974). The first two are
common throughout the Near- and Middle-East and seem to be sympatric in many
places. They are univoltine, flying from spring to autumn. Adults cause damage
by mining the stalks of fruit bunches. In the Tropical Oryctes, larvae develop in
the wood of dead trees, and adults feed on living trees. The two feeding habitats
are separate in space. In contrast, larvae and adults of the date palm Oryctes spp.
live in the same living and standing trees. Larvae develop in the crown and at the
periphery of the stem, feeding at the interface of dead and living tissues. Though
not reported as major pests, O. elegans and O. agamemnon inflict chronic dam-
age in date palm groves and cause economic losses in Iran and Iraq (Hurpin
and Fresneau, 1969; Gharib, 1970; Hussain, 1974). Insecticide applications in the
crown against emerging adults are commonly made in Iran in spring and summer.
Winter pruning and burning of the dead fronds and of the dried frond bases just
below the crown is recommended and applied by some growers to eliminate lar-
vae and pupae. Both measures require considerable labor and their impact is not
known.
As ethyl 4-methyloctanoate had been reported as the male aggregation
pheromone of the Tropical Oryctes (Gries et al., 1994; Hallet et al., 1995; Morin
et al., 1996) and also identified in a preliminary analysis of airborne effluvia from
O. elegans males (Rochat and Malosse, unpublished), we tested this compound
as an attractant for trapping O. elegans in Southern and Eastern Iran in 1996–97.
Several weeks of trapping in highly infested areas, without using plant material as
a co-attractant led to the capture of just two adults of O. elegans (Avand-Faghih
and Rochat, unpublished). To understand this apparent contradiction and develop
a trapping strategy against this insect, we undertook the study of the chemical
communication of O. elegans. The work was based on a field approach with
experimental trapping using live insects followed by screening of synthetic pu-
tative pheromone compounds selected from the results of volatile collections, gas
chromatography–mass spectrometry analyses, organic synthesis, and electroan-
tennographic screening.
Here we report the identification and activity of the male aggregation
pheromone of O. elegans and provide practical data to develop an efficient trap-
ping system. The result of a 6-month population monitoring study is presented, and
the differences between the chemical communication systems of the rhinoceros
beetles are discussed.
METHODS AND MATERIAL
Synthetic Chemicals. Chiral molecules were used as racemic mixes.
4-Methyloctanoic acid (1) and ethyl 4-methyloctanoate (2) were purchased from
E.G.N.O.-Chimie (Tancarville, France) and served as starting material to prepare
P1: IAZ
Journal of Chemical Ecology [joec] pp1129-joec-478533 February 20, 2004 22:23 Style file version June 28th, 2002
390 ROCHAT ET AL.
4-methyloctanyl acetate (3), methyl 4-methyloctanoate (4), and 4-methyloctanol
(5). 4 was prepared froma mixture of methanol, 1, and sulfuric acid; 5 was obtained
by reduction of 2 with LiAlH
4
in anhydrous diethyl ether; 3 and nonanyl acetate
(6) were prepared from 5 and nonanol, respectively using acetic anhydride in pyri-
dine. 1–6 were >98% pure by gas chromatography (GC) and 3–5 were free of
detectable traces of 1 and 2. Octanoic (=caprylic; 7) and nonanoic (=pelargonic;
8) acids were purchased from Aldrich (7: UK and 8: Germany) and were 99.5 and
96% pure, respectively.
Insects. O. elegans were collected in date palm groves in the Saravan area,
shipped by air to France, and maintained on sugarcane at 23–28

C, 75% RH, and
under a 13L:11D photoperiod. Sugarcane (commercial source) was used as food
to replace date palm core not available in France, as for other palm rhinoceros
beetles (Morin et al., 1996; Rochat et al., 2000b, 2002).
Collection of the Natural Pheromone. O. elegans pheromone was collected
fromeffluvia bypassinga streamof air (purifiedover activatedcharcoal; 100ml/min)
into 1-l glass flasks containing four to six adult insects. Odors were adsorbed onto
350 mg Supelpak
TM
-2 (SUPELCO, USA) for 4–7 d. Effluvia were trapped from
batches of males, or females, or both sexes placed on two 20 cm halved sugarcane
pieces, and from sugarcane controls. Five series of collections were carried out
between 1996 and 2002, yielding extracts from males (N = 7), females (N = 6),
and males plus females (N = 2) with sugarcane, and sugarcane control (N = 4)
odors. The adsorbent was eluted with 1.5 ml 99.5% dichloromethane (1996 and
1998) or hexane (2000–2002). The extracts were stored at −40

Cand used for GC
and/or GC–MS.
GC–MS analyses were carried out using a Varian 3300 gas chromatograph
coupled to a Nermag R10-10C quadrupole mass spectrometer. Electron impact
spectra were obtained at 70 eV. Additional spectra were obtained in chemical
ionization mode using NH
3
as reactant gas. The chromatograph was typically
equipped with a WCOT fused silica column (30 m × 0.32 mm id, 0.5-µm MDN-
5S phase; Supelco, Bellefonte, PA, USA) operated from 50

C (1 min) to 300

C at
10

C/min. Additional GC analyses were carried out using a Varian 3400 CX GC
with an equivalent apolar column or a polar column with same dimensions (RTX-
Wax phase; Restek, Bellefonte, PA, USA) and operated from 50

C (1 min), then
heated at 15

C/min to 80

C/(9 min), then heated to 240

Cat 6

C/min. Heliumwas
used as carrier gas at 13 psi. Samples were injected using a SPI injector at 250

C.
The amount of each male-specific compound was estimated by the percentage of
its FID (GC) or TIC (GC–MS) response in the total response recorded to all the
male-specific compounds (Table 1).
Electroantennography. Electroantennograms (EAGs) were recorded using
the same equipment as described for O. rhinoceros (Morin et al., 1996), from
dissected antennae. The antennal lamellae were held apart using small insect pins.
The recording electrode was placed on the central lamella, while the reference
P1: IAZ
Journal of Chemical Ecology [joec] pp1129-joec-478533 February 20, 2004 22:23 Style file version June 28th, 2002
AGGREGATION PHEROMONE OF Oryctes elegans 391
T
A
B
L
E
1
.
C
H
A
R
A
C
T
E
R
I
S
T
I
C
S
A
N
D
A
B
U
N
D
A
N
C
E
O
F
T
H
E
M
A
L
E
-
S
P
E
C
I
F
I
C
C
O
M
P
O
U
N
D
S
I
D
E
N
T
I
F
I
E
D
B
Y
G
A
S
C
H
R
O
M
A
T
O
G
R
A
P
H
Y
A
N
D
M
A
S
S
S
P
E
C
T
R
O
M
E
T
R
Y
F
R
O
M
O
r
y
c
t
e
s
e
l
e
g
a
n
s
E
F
F
L
U
V
I
A
T
R
A
P
P
E
D
O
N
S
U
P
E
L
P
A
K
T
M
-
2
G
C
r
e
t
e
n
t
i
o
n
t
i
m
e
s
(
m
i
n
)
M
o
l
e
c
u
l
a
r
A
p
o
l
a
r
P
o
l
a
r
E
I
m
a
s
s
s
p
e
c
t
r
u
m
a
t
7
0
e
V
c
%
r
a
t
i
o
a
n
d
f
r
e
q
u
e
n
c
y
C
o
m
p
o
u
n
d
w
e
i
g
h
t
R
T
X
-
5
M
S
a
R
T
X
-
W
a
x
b
c
h
a
r
a
c
t
e
r
i
s
t
i
c
i
o
n
s
m
/
z
(
%
)
i
n
m
a
l
e
e
f

u
v
i
a
d
1
:
4
-
M
e
t
h
y
l
o
c
t
a
n
o
i
c
a
c
i
d
1
5
8
1
4
.
8
5

1
5
.
3
5
e
2
9
.
2
2
4
1
,
4
3
,
5
5
,
5
7
(
1
0
0
)
,
6
0
,
6
9
,
7
3
,
8
3
,
9
9
,
1
0
1
,
1
2
9
5
3
±
1
1
(
8
)
2
:
E
t
h
y
l
4
-
m
e
t
h
y
l
o
c
t
a
n
o
a
t
e
1
8
6
1
5
.
2
4
1
4
.
7
0
4
1
,
4
3
,
4
5
,
5
5
,
5
7
,
6
0
,
6
1
,
7
0
,
7
3
,
8
3
,
8
8
(
1
0
0
)
,
9
9
,
1
0
1
,
1
2
3
,
1
2
9
,
1
4
1
,
1
5
7
3
7
±
1
0
(
8
)
3
:
4
-
M
e
t
h
y
l
o
c
t
a
n
y
l
a
c
e
t
a
t
e
1
8
6
1
5
.
6
1
1
6
.
3
3
4
1
,
4
3
(
1
0
0
)
,
5
6
,
6
1
,
6
9
,
7
0
,
8
4
,
9
8
,
1
0
1
,
1
2
6
8
±
4
(
4
)
4
:
M
e
t
h
y
l
4
-
m
e
t
h
y
l
o
c
t
a
n
o
a
t
e
1
7
2
1
3
.
1
9
1
2
.
7
9
4
1
,
4
3
(
1
0
0
)
,
5
5
,
5
7
,
5
9
,
6
9
,
7
4
,
8
7
,
9
9
,
1
1
5
,
1
2
3
,
1
4
1
,
1
4
3
1
±
1
(
3
)
5
:
4
-
M
e
t
h
y
l
o
c
t
a
n
o
l
1
4
4
1
1
.
8
5
1
9
.
0
7
4
1
,
4
3
,
4
5
,
5
6
,
6
9
(
1
0
0
)
,
7
0
,
8
4
,
9
8
,
1
2
6
1
±
1
(
3
)
6
:
N
o
n
a
n
y
l
a
c
e
t
a
t
e
1
8
6
1
7
.
0
5
1
7
.
9
8
4
1
,
4
3
(
1
0
0
)
,
5
5
,
5
6
,
6
1
,
7
0
,
8
4
,
9
8
,
1
1
6
,
1
2
6
0
±
1
(
2
)
a
2
5
m
×
0
.
3
2
m
m
i
d
×
0
.
5
µ
m
d
f
(
R
e
s
t
e
k
)
o
p
e
r
a
t
e
d
f
r
o
m
5
0

C
f
o
r
1
m
i
n
,
5
0

1
0
0

C
a
t
1
5

C
/
m
i
n
,
1
m
i
n
a
t
1
0
0

C
,
a
n
d
1
0
0

2
8
0

C
a
t
5

C
/
m
i
n
.
b
3
0
m
×
0
.
3
2
m
m
i
d
×
0
.
5
µ
m
d
f
(
R
e
s
t
e
k
)
o
p
e
r
a
t
e
d
f
r
o
m
5
0

C
f
o
r
1
m
i
n
,
5
0

8
0

C
a
t
1
5

C
/
m
i
n
,
9
m
i
n
a
t
8
0

C
,
a
n
d
8
0

2
4
0

C
a
t
6

C
/
m
i
n
.
c
N
e
r
m
a
g
R
1
0
-
1
0
C
q
u
a
d
r
u
p
o
l
e
m
a
s
s
s
p
e
c
t
r
o
m
e
t
e
r
.
d
M
e
a
n
±
S
.
E
.
b
a
s
e
d
o
n
t
o
t
a
l
F
I
D
o
r
T
I
C
r
e
s
p
o
n
s
e
m
e
a
s
u
r
e
d
f
o
r
t
h
e
m
a
l
e
-
s
p
e
c
i

c
p
e
a
k
s
.
T
h
e
n
u
m
b
e
r
o
f
o
c
c
u
r
r
e
n
c
e
s
i
s
g
i
v
e
n
i
n
b
r
a
c
k
e
t
s
(
n
=
9
)
.
e
E
l
u
t
e
s
a
s
a
b
r
o
a
d
a
s
y
m
m
e
t
r
i
c
p
e
a
k
o
v
e
r
l
a
p
p
i
n
g
w
i
t
h
t
h
e
p
e
a
k
s
o
f
2
a
n
d
3
w
h
e
n
l
a
r
g
e
a
m
o
u
n
t
s
a
r
e
i
n
j
e
c
t
e
d
.
P1: IAZ
Journal of Chemical Ecology [joec] pp1129-joec-478533 February 20, 2004 22:23 Style file version June 28th, 2002
392 ROCHAT ET AL.
electrode was pushed into the lumen of the scape. Each antenna was bathed by a
flow of moist air (400 ml/min) and stimulated with compounds 1–4 and 7–8. Each
compound was delivered at four doses, selected randomly from serial dilutions
in hexane (0.1–100 µg; decadic step) The antenna was stimulated by hexane
(solvent) at the beginning, the end, and between each dose series. Compounds
were presented as 1 µl of hexane solutions deposited on filter papers inserted in
Pasteur pipettes just prior to the stimulation (0.5 sec; 200 ml/min). Raw EAG
values for a given antenna were normalized to relative EAGs (percent) by dividing
them by the average response recorded to the complete stimulation series.
Field Trapping Experiments: Chemical Dispensers. Synthetic chemicals
were emitted from heat-sealed sachets made of polyethylene films (ALPLAST,
France), the characteristics and dimensions of which were adapted to the chem-
icals and the targeted release rates after laboratory calibration (e.g., 28 × 40 ×
0.2 mm filled with 0.7 ml to emit 1 at 3 mg/d at 35

C). Calibration was achieved
by preparing dispensers of various sizes and different film thicknesses, filled with
the pure chemicals, and left in a room thermostated at 30, 35, and 40

C for one wk
at each temperature. The dispensers were weighed daily, and the slopes obtained
fromthe linear adjustment of the weight loss versus time furnished the estimations
of the evaporation rates. The dispensers were stored at −10

C and weighed (mg
precision) prior to and at the end of each replicate or assay to determine the daily
release rates of the chemicals (reported in the figures and tables).
Location and General Features. All assays were carried out close to the
Pakistan border in Saravan or Zaboli, a place about 100 km west from Saravan.
Both areas are located in Sistan and Balootchestan province of Iran and constitute
the eastern limit of the area of date palm production in Iran, with a mean elevation
of ca. 1200 mabove sea level and having cold winters. The areas consist of scattered
oases, with a traditional farming system of mixed vegetables, pasture, and fruit
crops, with few or no chemical inputs, either fertilizer or pesticide. Date palms are
mostly Mazafati variety, in highly heterogeneous plots.
Traps consisted of 24-l plastic buckets whose lids were equipped with eight
radial 8 × 5 cm openings. All treatments (except Assay 8) included one piece of
date palm core (ca. 1 kg) freshly cut from a shoot. The palm piece was wrapped
in a punctured plastic bag to limit dehydration (synthetic lures) or put in a 1-l
perforated plastic box with two live O. elegans. In Assay 8, date palm core tested
without beetle was prepared as described above or by chopping the palmpiece and
crushing the tissue with a hammer.
In Assays 1–2, traps were put on the ground at the base of a date palm. From
Assay 3 onwards, traps were hung from the stem at 2.5–3 m above ground level,
generally positioned just below the crown of the palms. Soapy water was poured
into the bottom of the bucket to prevent beetles from escaping. No insecticide was
used. Assays 1–8 were carried out in randomized complete block designs. Traps
were spaced ≥80 m apart. The two blocks were separated by ≥200 m. One assay
P1: IAZ
Journal of Chemical Ecology [joec] pp1129-joec-478533 February 20, 2004 22:23 Style file version June 28th, 2002
AGGREGATION PHEROMONE OF Oryctes elegans 393
consisted of n consecutive periods designated as replicates. During each replicate,
the date palm pieces remained unchanged and the traps were visited at regular
intervals (every 3 or 7 d) to collect trapped beetles, check the health of the O.
elegans used as bait, and replace dead bait insects and empty chemical dispensers
as needed. Date palm tissue was renewed and the treatments were re-randomized
at each replicate (Table 2).
Chronology and Experimental Choices. The experiments are summarized in
Table 2. Host plant was systematically included as bait (except specific Assay 8)
because compound 2 alone had captured almost no beetles. The attraction to con-
specifics was investigated first. Then we searched for the best trap positioning
(Assays 1–3). Synthetic chemicals were further evaluated as pheromone candi-
dates in various situations (Assays 4–8). Finally, Assay 9 evaluated the feasibility
of monitoring O. elegans population over one flight season.
Statistical Analysis. (Minitab, 1998). Assays 4–8: The total captures per trap
per visit were analyzed by a three-way ANOVA (F1: bait; F2: age of date palm
tissue–no. of visit within replicate; F3: replicate) on Ln(x +1) transformed data
using a general linear model (GLM) procedure. Means were compared by multiple
comparison(Tukeytest) or comparisontoa reference (Dunnett test) with P = 0.05.
The data from the four assays were pooled and subjected to another common
ANOVA to evaluate the effect of the age of the date palm tissue. In assays 1–3,
we could not apply ANOVA to the raw data because too many values were zero.
We performed an ANOVA on cumulative captures per trap: the total per replicate
and the mean daily total per assay for Assays 3 and 1–2, respectively. Results of
Assays 1–2 were analyzed together because both had the same structure and gave
similar results. The sex ratio of the captures was analyzed similarly in all assays.
Details of these analyses are not presented because the sex ratio appeared not to
be correlated to the experimental variables and to be variable across the trapping
periods (e.g., Figure 1).
RESULTS
Effluvia Characteristics and Pheromone Structural Identification. Feeding
by beetles of either sex induced the emission of various molecules (f; Figure 1A).
Comparisons of the odors emitted by only sugarcane, and males, females, or both
sexes feeding on sugarcane showed that the presence of males was correlated with
at least six volatile molecules occurring in variable proportions (Figure 1; Table 1).
In contrast, no female-produced chemicals were detected. Based on their mass
spectra and retention times by GC, and comparisons with reference compounds,
the male-specific molecules were identified as 1: 4-methyloctanoic acid, 2: ethyl
4-methyloctanoate, 3: 4-methyloctanyl acetate, 4: methyl 4-methyloctanoate, 5: 4-
methyloctanol, and 6: nonanyl acetate (Table 1). At least one of these compounds
P1: IAZ
Journal of Chemical Ecology [joec] pp1129-joec-478533 February 20, 2004 22:23 Style file version June 28th, 2002
394 ROCHAT ET AL.
T
A
B
L
E
2
.
G
E
N
E
R
A
L
C
H
A
R
A
C
T
E
R
I
S
T
I
C
S
O
F
N
I
N
E
F
I
E
L
D
E
X
P
E
R
I
M
E
N
T
S
C
A
R
R
I
E
D
O
U
T
T
O
T
R
A
P
O
r
y
c
t
e
s
e
l
e
g
a
n
s
U
S
I
N
G
N
A
T
U
R
A
L
O
R
S
Y
N
T
H
E
T
I
C
A
T
T
R
A
C
T
A
N
T
S
I
N
E
A
S
T
E
R
N
I
R
A
N
F
R
O
M
1
9
9
8
T
O
2
0
0
2
W
I
T
H
R
E
F
E
R
E
N
C
E
T
O
T
H
E
F
I
G
U
R
E
S
A
N
D
T
A
B
L
E
S
S
H
O
W
I
N
G
T
H
E
R
E
S
U
L
T
S
C
h
a
r
a
c
t
e
r
i
s
t
i
c
s
:
b
l
o
c
k
s
,
P
u
r
p
o
s
e
(
n
o
.
t
r
e
a
t
m
e
n
t
s
)
n
o
.
×
d
u
r
a
t
i
o
n
o
f
r
e
p
l
i
c
a
t
e
E
x
p
t
.
#
B
a
i
t
s
(
a
d
d
e
d
t
o
d
a
t
e
p
a
l
m
c
o
r
e
e
x
c
e
p
t
A
s
s
a
y
8
)
(
f
r
e
q
u
e
n
c
y
o
f
v
i
s
i
t
s
)
a
D
a
t
e
a
n
d
p
l
a
c
e
1
A
t
t
r
a
c
t
i
o
n
b
y
c
o
n
s
p
e
c
i

c
s
a
n
d
s
e
x
e
f
f
e
c
t
(
4
)
4
b
l
o
c
k
s
,
4
×
1
4
d
M
a
y
2
1

J
u
l
2
0
,
1
9
9
8
,
Z
a
b
o
l
i
B
a
i
t
s
:
2
,
2
,
2
+
2
,
n
o
b
e
e
t
l
e
s
(
c
)
(
7
d
)
Z
a
b
o
l
i
2
A
t
t
r
a
c
t
i
o
n
b
y
c
o
n
s
p
e
c
i

c
s
a
n
d
s
e
x
e
f
f
e
c
t
(
4
)
4
b
l
o
c
k
s
,
1
0
×
9
d
M
a
y
5

A
u
g
3
,
1
9
9
9
,
S
a
r
a
v
a
n
B
a
i
t
s
:
2
,
2
,
2
+
2
,
n
o
b
e
e
t
l
e
s
(
c
)
(
3
d
)
3
E
f
f
e
c
t
o
f
t
r
a
p
h
e
i
g
h
t
f
r
o
m
g
r
o
u
n
d
(
9
)
4
b
l
o
c
k
s
,
2
×
9
d
J
u
l
9

S
e
p
1
0
,
2
0
0
0
,
S
a
r
a
v
a
n
B
a
i
t
s
:
2
a
t
0
,
2
,
5
m
,
2
a
t
0
,
2
,
5
m
,
n
o
b
e
e
t
l
e
s
a
t
0
,
2
,
5
m
(
3
d
)
S
y
n
t
h
e
t
i
c
c
o
m
p
o
u
n
d
s
(
m
a
l
e
-
s
p
e
c
i

c
:
1

6
o
r
a
n
a
l
o
g
s
:
7

8
)
4
T
e
s
t
o
f
c
o
m
p
o
u
n
d
s
1

3
e
m
i
t
t
e
d
b
y
s
a
m
e
d
i
s
p
e
n
s
e
r
m
o
d
e
l
(
6
)
5
b
l
o
c
k
s
,
4
×
9
d
J
u
n
1
4

J
u
l
2
0
,
2
0
0
1
,
S
a
r
a
v
a
n
B
a
i
t
s
:
1
,
2
,
3
,
1
:
2
(
1
:
1
v
/
v
)
,
1
:
2
:
3
(
1
:
1
:
1
)
,
2
(
c
)
(
3
d
)
5
T
e
s
t
o
f
c
o
m
p
o
u
n
d
s
1
-
8
a
t
s
a
m
e
t
a
r
g
e
t
d
o
s
e
o
f
5
m
g
/
d
a
y
(
9
)
4
b
l
o
c
k
s
,
2
×
9
d
J
u
n
2
0
-
J
u
l
8
,
2
0
0
2
,
S
a
r
a
v
a
n
B
a
i
t
s
:
1
,
2
,
3
,
4
,
5
,
6
,
7
,
8
,
2
(
c
)
(
3
d
)
6
E
f
f
e
c
t
o
f
a
d
d
i
n
g
c
o
m
p
o
u
n
d
s
2

6
t
o
c
o
m
p
o
u
n
d
1
:
t
e
s
t
o
f

v
e
5
b
l
o
c
k
s
,
2
×
9
d
J
u
l
2
6
-
A
u
g
1
3
,
2
0
0
2
,
S
a
r
a
v
a
n
1

6
m
i
x
e
s
(
B
1

B
5
)
a
t
t
a
r
g
e
t
d
o
s
e
o
f
5
m
g
/
d
a
y
v
s
.
o
n
l
y
1
(
9
)
(
3
d
)
B
a
i
t
s
:
B
1
,
B
2
,
B
3
,
B
4
,
B
5
,
1
(
3
a
n
d
6
m
g
/
d
a
y
t
a
r
g
e
t
d
o
s
e
s
)
,
2
(
c
)
7
D
o
s
e
-
r
e
s
p
o
n
s
e
t
o
c
o
m
p
o
u
n
d
1
(
5
)
6
b
l
o
c
k
s
,
2
×
9
d
J
u
l
8

2
6
,
2
0
0
2
,
S
a
r
a
v
a
n
B
a
i
t
s
:
f
o
u
r
d
o
s
e
s
o
f
1
,
2
(
c
)
(
3
d
)
8
S
y
n
e
r
g
y
b
e
t
w
e
e
n
c
o
m
p
o
u
n
d
1
a
n
d
d
a
t
e
p
a
l
m
c
o
r
e
-
D
P
-
(
5
)
5
b
l
o
c
k
s
,
2
×
9
d
A
u
g
1
3

3
1
,
2
0
0
2
,
S
a
r
a
v
a
n
B
a
i
t
s
:
1
,
D
P
,
c
r
u
s
h
e
d
D
P
,
1
+
D
P
,
1
+
c
r
u
s
h
e
d
D
P
,
2
+
D
P
(
c
)
(
3
d
)
9
S
e
a
s
o
n
a
l
p
o
p
u
l
a
t
i
o
n
m
o
n
i
t
o
r
i
n
g
(
1
)
1
0
t
r
a
p
s
,
2
5
×
7
d
A
p
r
7

S
e
p
1
,
2
0
0
2
,
S
a
r
a
v
a
n
B
a
i
t
:
c
o
m
p
o
u
n
d
1
(
t
a
r
g
e
t
d
o
s
e
o
f
3
m
g
/
d
)
(
7
d
)
S
a
r
a
v
a
n
N
o
t
e
.
(
c
)
:
c
o
n
t
r
o
l
.
a
F
o
r
e
x
a
m
p
l
e
:
A
s
s
a
y
3
(
f
o
u
r
c
o
m
p
l
e
t
e
b
l
o
c
k
s
)
l
a
s
t
e
d
4
×
9
d
=
3
6
d
.
T
h
e
n
u
m
b
e
r
o
f
v
i
s
i
t
s
(
b
a
s
i
c
d
a
t
a
f
o
r
A
N
O
V
A
)
w
a
s
4
×
(
9
d
/
3
d
)
=
1
2
.
I
n
A
s
s
a
y
8
,
t
h
e
1
0
t
r
a
p
s
(
s
i
n
g
l
e
t
r
e
a
t
m
e
n
t
)
w
e
r
e
v
i
s
i
t
e
d
w
e
e
k
l
y
f
o
r
2
5
w
k
.
P1: IAZ
Journal of Chemical Ecology [joec] pp1129-joec-478533 February 20, 2004 22:23 Style file version June 28th, 2002
AGGREGATION PHEROMONE OF Oryctes elegans 395
FIG. 1. Overlaid chromatograms of Supelpak
TM
-2-trapped volatiles emitted by sugarcane,
and Oryctes elegans males or females feeding on sugarcane, showing two profiles of male-
associated compounds. A: GC analysis on a polar RTX-Wax column showing male effluvia
characterized by 1 and 2 (55:45 ratio; 2001 collection) and food volatiles induced by the
beetle feeding (f: white arrow heads); and B: GC–MS analyses using an apolar MDN-
5MS column showing compounds 1– 6 (21:37:34:3:4:1 ratio) associated with males (2000
collection).
P1: IAZ
Journal of Chemical Ecology [joec] pp1129-joec-478533 February 20, 2004 22:23 Style file version June 28th, 2002
396 ROCHAT ET AL.
FIG. 2. Relative electroantennogram (EAG) responses by Oryctes elegans males and fe-
males (mean +S.E.; N = 4 of each sex) to six synthetic molecules (four compounds isolated
from the male effluvia: 1–4; and two related molecules: 7–8) at four doses. The horizontal
lines within the graphs indicate the mean responses to solvent.
was detected from eight of the nine batches with males. 1 and 2 were the most
abundant and regular components (respectively, 53 ±11 and 37 ±10%total male-
specific volatiles; mean ±S.E.). 1 was emitted at a daily rate estimated between 0.1
and 5 µg/male. In six batches, 1 was more abundant than 2 (80 to 50%) followed
by 2. In two cases, 2 was the most abundant male-specific molecule (88 and 37%)
either mixed with only 2 (12%) or 3 (35%) and other minor components. 3–6 were
detected in less than half the samples. 3 was the third most abundant component
(8 ± 4%) whereas 4–6 appeared as sporadic trace components, accounting for
<1% of the male-specific volatiles.
Electroantennography. (Figure 2). EAG responses recorded to the six tested
molecules differed between sexes. In females, most of the responses to the chem-
icals were higher than to the solvent. The most stimulatory compound was the
P1: IAZ
Journal of Chemical Ecology [joec] pp1129-joec-478533 February 20, 2004 22:23 Style file version June 28th, 2002
AGGREGATION PHEROMONE OF Oryctes elegans 397
FIG. 3. Captures of Oryctes elegans by bucket traps baited with a piece of date palm core
with or without caged live conspecifics in two experiments in Eastern Iran [Right dotted
bars: Experiment 1, total captures = 44 (1 :0.5 ); left plain bars: Experiment 2, total
captures = 108 (1 :0.7 )]. Captures surmounted by thy same letter are not statistically
different in both experiments (Tukey multiple comparison test, P < 0.05).
acid 1, which triggered responses that increased with dose. Compound 8 elicited
negligible responses, and compound 7 elicited only small responses. The other
male-specific compounds (2–4) did not elicit clear EAGs. In males, the responses
were less strong and only responses to 1 were greater than to the solvent. The
highest dosage of most of the molecules appeared to inhibit responses.
Trapping with Live Beetles. In experiments 1–2, using live beetles as bait,
few beetles were caught. Nevertheless the effect of the bait was significant on the
captures (F(3, 31) = 7.91, P < 0.001; Figure 3). The traps with male O. elegans
bait (with or without females) captured more conspecifics than the traps without
beetles, (Tukeytest; P < 0.05; Figure 3). The traps withfemales as bait capturedan
intermediarynumber of beetles, about half of those containingonlytwomales (with
or without females). The difference was suggestive but not significant (P = 0.07
and P = 0.18 for female vs. male and male + female, respectively) due to high
variability and a small number of replicates (4).
In experiment 3, the height of the traps influenced the captures whatever the
bait (F(2, 215) = 4.14, P < 0.017) with higher traps being more effective. Traps
placed about 5 m above ground captured more beetles than traps on the ground.
P1: IAZ
Journal of Chemical Ecology [joec] pp1129-joec-478533 February 20, 2004 22:23 Style file version June 28th, 2002
398 ROCHAT ET AL.
FIG. 4. Effect of the position of the trap on captures of Oryctes elegans using a piece
of date palm core with or without caged live conspecifics as bait (Eastern Iran Assay 3).
Whatever the bait, captures associated with the same letter are not statistically different.
Catches by males and females were statistically equivalent and greater than by no beetles
(Tukey multiple comparison test, P < 0.05).
At about 2 m high, traps captured an intermediate number of insects. Attraction
to odors of males and females was equivalent and greater than to traps with no
beetles whatever the trap position (Tukey test, P < 0.05; Figure 4).
Evaluation of Synthetic Chemicals. Whatever the synthetic bait and the repli-
cate, trap catches decreasedas the date palmtissue aged(F(2, 1115) = 67.23, P <
0.001; Figure 5) in experiments 4–8. The mean captures were significantly lower at
a given visit than at the previous one. They decreased by 20 and 66% respectively
at the second (day 6) or third (day 9) visit as compared to the first (day 3).
The effect of the replicate (time) was significant in experiments 4, 5, and
7, (F(3, 354) = 40.23, F(1, 208) = 43.32, and F(1, 179) = 5.23, respectively;
P < 0.001), indicating that the level of catches changed with time whatever the
baits. In experiments 6 and 8, the level of capture was equivalent in the two
replicates.
In experiment 4 (Table 3), the effect of the bait was significant (F(5, 354) =
7.56, P < 0.001). Combinedwithdate palmcore, compound1was most attractive,
significantly more than compounds 2, 3, and the 2:3 blend (1:1). The 1:2:3 mix
P1: IAZ
Journal of Chemical Ecology [joec] pp1129-joec-478533 February 20, 2004 22:23 Style file version June 28th, 2002
AGGREGATION PHEROMONE OF Oryctes elegans 399
FIG. 5. Effect of date palm core aging (chronology of the visits) on the mean captures
of Oryctes elegans by traps baited with various synthetic male-produced compounds or
related compounds, or two live conspecific males, plus a piece of date palm core in Eastern
Iran; common analysis on Experiments 4–8. Captures associated with different letters are
statistically different (Tukey multiple comparison test, P < 0.05).
(1:1:1) and two live males were intermediate in attractiveness (Tukey test; P <
0.05). The synthetic baits were emitted within a broad range of release rates, from
2.8 (1) to 16.5 mg/d (2).
In experiment 5 (Table 4), combined with date palm core, 1 attracted larger
numbers of beetles than any other treatment, including two live males. The syn-
thetic chemicals were emitted in a narrower range than in experiment 4, from 4.3
(1) to 12.5 mg/d (7), especially five of the six compounds produced by males
(4.3–6.2 mg/d).
Inexperiment 6(Table 5), combinedwithdate palmcore, 1emittedat 5.1mg/d
(reference) or 7.6 mg/d was equally attractive. The synthetic baits were emitted
between 5.1 mg/d (1) and 8.6 mg/d (B1). There was no evidence of synergism or
additive effects from the minor components. In fact, several of the blends were
less attractive than 1 as a single component.
In a dose response trial (experiment 7) 1 emitted at 2.2, 3.2, or 9.2 mg/d,
combined with date palm core was equally attractive. A release rate of 1 mg/d was
less attractive, as was the trap baited with two male beetles (+date palm core).
(Dunnett test; P < 0.05.)
P1: IAZ
Journal of Chemical Ecology [joec] pp1129-joec-478533 February 20, 2004 22:23 Style file version June 28th, 2002
400 ROCHAT ET AL.
TABLE 3. COMPARATIVE CAPTURES OF Oryctes elegans BY THREE SYNTHETIC
COMPOUNDS (1–3: THE MAJOR COMPONENTS OF THE MALE EFFLUVIA), ALONE OR
MIXED, AND EMITTED BY THE SAME TYPE OF DISPENSER, AND TWO LIVE CONSPECIFIC
MALES IN BUCKET TRAPS CONTAINING A PIECE OF DATE PALM CORE (EASTERN IRAN,
EXPERIMENT 4)
Mean ± S.E. release rate
Bait added to date palm core
a
of the chemical bait (mg/day)
b
Mean ± S.E. captures/trap/3 d
c
1: 4-Methyloctanoic acid 2.8 ± 0.4 4.0 ± 0.6
b
2: Ethyl 4-methyloctanoate 16.5 ± 1.3 1.4 ± 0.2
a
3: 4-Methyloctanyl acetate 13.6 ± 0.3 2.2 ± 0.5
a
2:3 (1:1) 15.6 ± 1.0 1.5 ± 0.3
a
1:2:3 (1:1:1) 8.5 ± 0.4 2.6 ± 0.5
ab
Two males (control) — 2.5 ± 0.5
ab
Notes. Total catches = 844 (1 : 1.4 ). S.E.: standard error of the mean.
a
Values in brackets indicate the relative proportion of the components within mixes.
b
Actual rates during the experiment (n = 25). Values for each component of the mixes undetermined.
c
Captures followed by the same letter are not significantly different (Tukey multiple comparison test,
P < 0.05).
In the 8th experiment (Table 6), captures by all the baits were significantly
different from zero. Crushed date palm core tended to attract more beetles, either
alone (×2.6) or combined with 1 (×1.4), than a standard date palm core piece. As
before, 1 combined with date palm core was more attractive than two live males
on date palm core, alone, or date palm core alone (Dunnett test; P < 0.05.)
TABLE 4. COMPARATIVE CAPTURES OF Oryctes elegans BY EIGHT SYNTHETIC
MOLECULES AT THE SAME TARGET DOSE OF 5 Mg/Day (SIX COMPOUNDS ISOLATED
FROM THE MALE EFFLUVIA: 1–6 AND TWO RELATED MOLECULES: 7–8), AND TWO LIVE
CONSPECIFIC MALES IN BUCKET TRAPS CONTAINING A PIECE OF DATE PALM CORE
(EASTERN IRAN, EXPERIMENT 5)
Mean ± S.E. release rate
Bait added to date palm core of the chemical bait (mg/day)
a
Mean ± S.E. captures/trap/3 d
b
1: 4-Methyloctanoic acid 4.3 ± 0.2 9.6 ± 2.5
a
2: Ethyl 4-methyloctanoate 5.5 ± 0.2 2.3 ± 1.0
b
3: 4-Methyloctanyl acetate 8.2 ± 0.1 2.7 ± 0.3
b
4: Methyl 4-methyloctanoate 4.8 ± 0.2 0.9 ± 0.9
b
5: 4-Methyloctanol 6.2 ± 0.4 2.7 ± 0.8
b
6: Nonanyl acetate 5.4 ± 0.2 0.5 ± 0.7
b
7: Octanoic acid 12.7 ± 2.0 1.0 ± 0.2
b
8: Nonanoic acid 9.5 ± 0.5 1.7 ± 0.6
b
Two males (control) — 1.8 ± 0.6
b
Notes: Total catches = 653 (1 : 0.5 ). S.E.: standard error of the mean.
a
Actual rates during the experiment (6 ≤ n ≤ 8).
b
Captures followed by the same letter are not significantly different (Tukey multiple comparison test,
P < 0.05).
P1: IAZ
Journal of Chemical Ecology [joec] pp1129-joec-478533 February 20, 2004 22:23 Style file version June 28th, 2002
AGGREGATION PHEROMONE OF Oryctes elegans 401
TABLE 5. COMPARATIVE CAPTURES OF Oryctes elegans BY FIVE SYNTHETIC MIXES
(B1-B5) OF THE SIX COMPOUNDS ISOLATED FROM THE MALE EFFLUVIA 1–6, TWO DOSES
OF 1, THE MAJOR COMPOUND OF THE MALE EFFLUVIA, AND TWO LIVE CONSPECIFIC
MALES IN BUCKET TRAPS CONTAINING A PIECE OF DATE PALM CORE IN EASTERN IRAN
(EXPERIMENT 6)
Mean ± S.E. release rate Mean ± S.E. captures/
Bait added to date palm core
a
of the chemical bait (mg/day)
b
trap/3 d
c
B1: 1:2:3 (1:1:1) 8.6 ± 1.2 1.8 ± 0.4

B2: 1:2:3 (10:1:1) 5.2 ± 0.1 2.0 ± 0.3

B3: 1:2:3:4:5:6 (100:10:10:1:10:1) 5.3 ± 0.3 3.3 ± 0.6
B4: 1:2:3:4:5:6 (10:10:10:1:10:1) 7.3 ± 0.8 3.3 ± 0.6
B5: 1:2:3:4:5:6 (10:10:10:1:1:1) 7.6 ± 0.8 1.5 ± 0.3

1: 4-Methyloctanoic acid 5.1 ± 0.2 →3.9 ± 0.6
1: 4-Methyloctanoic acid 7.6 ± 0.5 4.5 ± 0.7
Two males (control) — 0.7 ± 0.2

Notes. Total catches = 623 (1 : 1.3 ). S.E.: standard error of the mean.
a
2: Ethyl 4-methyloctanoate, 3: 4-methyloctanyl acetate, 4: methyl 4-methyloctanoate, 5: 4-
methyloctanol, and 6: nonanyl acetate). Values in brackets indicate the relative proportion of the
compounds within the mixes B1–B5.
b
Actual rates during the experiment (9 ≤ n ≤ 10). Values for each component of the mixes undeter-
mined.
c
Captures asterisked are significantly lower than compound 1 at 5.1 mg/d (Dunnett test, P < 0.05).
Population Monitoring over 25 wk (experiment 9: Figure 6). A total of 841
beetles (55% female) was captured in 10 traps. Weekly captures per trap, which
were nil in mid-April, increased regularly until the beginning of June (5.3 ± 1.4
beetles). Then, they oscillated around that level until mid-September with a sharp
TABLE 6. SYNERGISTIC CAPTURES OF Oryctes elegans BY COMBINED DATE PALM CORE
(CRUSHED OR NOT) AND SYNTHETIC 1, THE MAJOR COMPONENT OF THE MALE
AGGREGATION PHEROMONE AS COMPARED TO TWO LIVE MALE CONSPECIFICS ON DATE
PALM CORE IN BUCKET TRAPS (EASTERN IRAN EXPERIMENT 8)
Mean ± S.E. release rate Mean ± S.E. captures/
Bait of the chemical bait (mg/day)
a
trap/3 d
b
1: 4-Methyloctanoic acid 3.0 ± 0.1 0.3 ± 0.1
b
Date palm core — 0.2 ± 0.1
b
Crushed date palm core — 0.6 ± 0.3
b
1 + Date palm core 3.5 ± 0.1 5.2 ± 0.7
a
1 + Crushed date palm core 3.3 ± 0.1 7.4 ± 0.9
a
Two males + date palm core — 1.0 ± 0.3
b
Notes. Total catches = 435 (1 : 1.5 ). S.E.: standard error of the mean.
a
Actual rates during the experiment (n = 8).
b
Captures followed by the same letter are not significantly different (Tukey multiple comparison test,
P < 0.05).
P1: IAZ
Journal of Chemical Ecology [joec] pp1129-joec-478533 February 20, 2004 22:23 Style file version June 28th, 2002
402 ROCHAT ET AL.
FIG. 6. Weekly captures of Oryctes elegans per trap—bottom, left scale—with correspond-
ing sex-ratios—top, right scale—from 10 traps baited with a piece of date palm core and 1
(4-methyloctanoic acid, the major component of the male aggregation pheromone) emitted
at a rate of 2.2 ± 0.1 mg/d (mean ± S.E.) for 25 wk between April and September 2002 in
Eastern Iran (Experiment 9). Marked points indicate that the represented values are averages
due to a single visit on July 7 with captures from 2 wk accumulated in traps.
peak of capture at the beginning of August (11.5 ± 1.9 beetles) and decreased
during the two final weeks of the trial. Considering the mean captures per trap
calculated on a monthly basis, the population reached a maximum in summer
(mid-July–mid-August: 5.9 ± 0.8 beetles). The sex ratio of the catches fluctuated
throughout the trial, being approximately balanced in spring and then showing an
excess of females through summer, especially in August.
DISCUSSION
Our data demonstrate that male O. elegans produce anaggregationpheromone
like all the dynastid species studied to date. Its active component is 4-
methyloctanoic acid, a molecule previously reported from the Tropical Oryctes,
O. monoceros and O. rhinoceros (Gries et al., 1994; Hallet et al., 1995; Morin et
al., 1996). However, besides these common features, the O. elegans pheromone
communication system differs from the pheromone communication system of the
Tropical Oryctes in two major aspects: (1) O. elegans’ most active pheromone
compound is not active in the Tropical Oryctes, and (2) O. elegans pheromone is
weakly attractive by itself but highly synergistic with the odor emitted by freshly
cut and macerated palmtissue. This is not the case for the aggregation pheromones
of the Tropical Oryctes, which are highly attractive without plant chemicals.
P1: IAZ
Journal of Chemical Ecology [joec] pp1129-joec-478533 February 20, 2004 22:23 Style file version June 28th, 2002
AGGREGATION PHEROMONE OF Oryctes elegans 403
Attraction to Conspecifics in Natural Conditions. Both sexes of O. elegans
attracted conspecifics when placed on date palm core, suggesting the emission
of (a) pheromone(s) and/or plant volatiles induced by beetle feeding. The latter
hypothesis is in agreement with the catches by either sex feeding on palm tissue
recorded in the initial assays. GC analyses also showed that feeding of beetles of
either sex on sugarcane resulted in the emission of several compounds.
Trap captures in traps baited with males were never significantly greater than
in traps baited with females and the initial field assays did not conclusively support
the emission of a male aggregation pheromone. Evidence for a male-produced
pheromone was provided by the analyses of the beetles’ effluvia and subsequent
assays of synthetic chemicals. Conversely, the existence of a female aggregation
pheromone seems unlikely because no female-specific compounds were detected
from the female effluvia. Thus, attraction of beetles to females feeding on date
palm may only reflect a response to palm volatiles induced by beetle feeding.
In addition, our first assays were performed in harsh environmental conditions
(>50

C, very low humidity). These conditions were fatal to many bait insects as
shown by the high mortality recorded on the weekly visits (experiments 1–2), and
males probably did not emit pheromone during the major part of the assays.
Male Pheromone Composition. The male pheromone secretion of O. elegans
was more complex and variable than reported in the Tropical Oryctes. In addition
to the major compounds 1 and 2 that were reported from them, O. elegans males
produced the new compounds 3–5, which have obvious structural relationship
with 1 and 2, and with ethyl 3-methylheptanoate, reported from O. rhinoceros
(Hallet et al., 1995). These molecules share a methyl-branched carbon skeleton,
suggesting a common biogenetic pathway. The trace amounts of 3–6 detected from
certain batches of male O. elegans could be artifacts of our sampling procedure
due to reactions between fermentation products from sugarcane with compounds
1–2. However, SPME sampling showed that two males placed in silanized glass
vials without food could emit 0.1–1 µg of 1, 2, and 5 simultaneously (Rochat and
P´ ezier, unpublished). The noticeable amounts of the acetate 3 detected in at least
two batches of male effluvia also support production of 3 and 5 by the insect itself.
O. elegans male-specific effluvia were variable both in the quantities emitted
and in their qualitative composition. This is possibly related to our sampling con-
ditions because we used wild beetles, of unknown age and physiological status,
at different seasons, and grouped in different numbers with only males or mixed
sexes. Pheromone collection from more standardized samples, coupled to an ap-
propriate method for GCquantification will be required to determine the biological
factors which modulate pheromone emission.
Pheromone Activity. Insects responded to racemic 1, indicating that chirality
was not a critical point inthe species or suggestingthat it uses a racemic pheromone.
Hallet et al. (1995) reported 4S-2 to be as attractive as (±)-2 in O. rhinoceros and
concluded that this species produces either the S enantiomer or a racemic mix. We
P1: IAZ
Journal of Chemical Ecology [joec] pp1129-joec-478533 February 20, 2004 22:23 Style file version June 28th, 2002
404 ROCHAT ET AL.
did not investigate the enantiomeric composition of the molecules produced by O.
elegans.
A strong response to the acid 1 was the constant feature of the comparative
trials performed, in accordance with the prevalence of this molecule in the male
pheromone secretion. The specificity of the response was correlated to the methyl-
branched carbon skeleton because responses to the acid analogs 7 and 8 were
negligible. In the field, 1–10 mg/d release rates of 1 were active. Higher doses
were not evaluated because release rates of 2–3 mg/d were as good as the higher
dose of 10 mg/d.
All the other male-specific chemicals were less active than 1, either as sin-
gle compounds or blends (experiment 3), and even tested at higher doses. These
observations are in agreement with the strong EAG responses recorded to 1, in
contrast to those recorded to the other molecules. The presence of large amounts
of 2 (ethyl ester) or 3 (acetate) with 1 (experiments 4–5) seemed to reduce the
trapping efficiency with the exception of the mix 1:2:3:4:5:6 (10:10:10:1: 1:1) in
experiment 5. The good activity of this mix may be due to the large amount of
5, a compound that was not tested by EAG. However, because no clear effect, ei-
ther inhibitory or enhancing, was observed to the blends, and because most minor
male-specific chemicals appeared as erratic components in the male effluvia, the
responses to the molecules other than 1–2 in the field may be due to their structural
relationship with 1. Alternately, these compounds might be involved in reproduc-
tive isolation with O. agamemnon, which is sympatric with O. elegans in lowland
areas of the Middle East; in that case it would not be expected that their presence
would improve the efficiency of the traps.
The strong activity of 1 in O. elegans and the weak activity of 2 is opposite to
what happens in the Tropical Oryctes: 2 proved to be the only or essential active
pheromone compound in these species while 1 resulted in negligible captures of
O. rhinoceros at 30 mg/d (Hallet et al., 1995). 1, inactive when evaluated by GC-
EAD of natural male effluvia, was not evaluated in the field with O. monoceros
(Gries et al., 1994). In O. rhinoceros, a third molecule, ethyl 3-methylheptanoate
was also reported from males and attracted about four times fewer beetles than 2
at 30 mg/d (Hallet et al., 1995), indicating the species was moderately responsive
to another molecule produced by males, with a close structural relationship to the
major pheromone compound.
The sex ratio of the beetles captured was not correlated to the pheromone
bait but varied during the flight period of the beetle. An equal ratio of males and
females was captured over the whole set of experiments with some variations,
the origin of which is unknown. Both sexes seemed to emerge simultaneously in
spring according to the 2002 monitoring. The female excess recorded in summer
may reflect a shorter life span of the males or a greater mobility of females.
Plant Co-Attraction. Although 1 as a single component and date palm odor
were attractive alone catches dramatically increased when both odors were
P1: IAZ
Journal of Chemical Ecology [joec] pp1129-joec-478533 February 20, 2004 22:23 Style file version June 28th, 2002
AGGREGATION PHEROMONE OF Oryctes elegans 405
combined. The effect was strong with fresh palmmaterial, becoming weaker as the
tissue aged, and the effect tended to be enhanced when the palmtissue was crushed.
Whereas this phenomenon is classical in palm weevils (Giblin-Davis et al., 1996;
Rochat et al., 2000a), it has not been reported in other dynastid beetles, or, if so,
to a much lesser extent. It may be correlated to the ecology of O. elegans, which
colonizes live trees for mating and egg laying in the same way as the palmweevils.
In the Tropical Oryctes, mating sites are separate fromfeeding sites. Males are
always found singly in feeding galleries, an observation fully in agreement with
the absence of pheromone emission from galleries (Morin et al., unpublished).
Their aggregation pheromone is attractive by itself (Hallet et al. 1995; Morin and
Allou, unpublished). An enhanced or synergistic response, not as dramatic as in
O. elegans, can be recorded by adding decaying woody material to the pheromone
of O. rhinoceros, an egg laying substrate where the beetles hide and the males
emit their pheromone (Hallet et al., 1995; Alfiler, 1999; Sudharto, 2001).
The dynast S. australis presents an intermediate case between O. elegans
and the Tropical Oryctes. Rochat et al. (2000b) showed that the male aggregation
pheromone was quite attractive by itself but also synergistic with odors emitted by
freshly cut coconut or sugarcane tissues. Male S. australis emit their aggregation
pheromone from the feeding galleries burrowed in such fresh tissue, attracting
females and mating with them, but also attracting other males which they get rid
of by fighting (Prior et al., 2000). The egg-laying sites are separate fromthe feeding
sites, as in the Tropical Oryctes and contrary to O. elegans. Therefore, a synergy
between the male pheromone and plant odors appears particularly intense when it
involves fresh tissues where the males are emitting pheromone. The mechanism
of the plant–insect interaction, especially in the synergistic response and in the
pheromone emission, remains unknown.
Applied Perspectives. More than 4000 O. elegans were caught over the
two trapping seasons during which the synthetic pheromone was evaluated. The
captures averaged 6.3 beetles/trap/wk considering only the traps baited with 1 and
date palm, which emerged as the operational bait for further trapping. The 25-wk
trapping campaign showed that seasonal monitoring of the pest can be carried out
efficiently by this means. The trap catch data agreed with data available in the
literature and obtained fromfarmers: O. elegans adults appear in spring and can be
found until the temperature drops in late fall. The maximumabundance is recorded
in summer, based on the individuals observed at lights.
Trapping could be used as a monitoring and decision tool to improve the
timingof insecticide application, whichis generallyperformedwhenfarmers prune
leaves aroundfruit bunches andplace themindriedleaf nets inorder toprotect them
fromwind and predators. Insecticide is applied as a prophylatic measure, and there
is no simple way to evaluate the population level. Trapping could also be integrated
with insecticide applications to directly control the pest, with a double benefit: a
more efficient and safer elimination of the beetles, which can be done using soapy
P1: IAZ
Journal of Chemical Ecology [joec] pp1129-joec-478533 February 20, 2004 22:23 Style file version June 28th, 2002
406 ROCHAT ET AL.
water without insecticide in traps as reported for S. australis (Rochat et al., 2002).
This method is particularly attractive considering the low economic level of the
farmer, the human health risks from insecticides, and the fragile environment of
the oases.
Besides the cost of the pheromone, one critical point of this trapping strategy
remains, i.e., the necessity to use fresh date palmtissue to synergize the pheromone.
Date palm tissue must be renewed weekly in order to maintain a satisfactory level
of captures. However, such material is precious for farmers because shoots are sold
for the multiplication of the palms. We need to find cheap substitutes for the date
palm shoots, either natural or synthetic, before trapping can be fully developed.
Finally, the development of a common trapping of O. elegans and the red
palm weevil, Rhynchophorus ferrugineus Oliv., now sympatric with the dynast in
many places, is also planned for a better and more economic general control of
date palm beetle pests.
Acknowledgments—We thank Hooshang Boroomand from the Insect Taxonomy Research De-
partment, Coleoptera Laboratory, in Tehran for help and visits to the Oryctes collections from the
Hayk Mirzayans Museum, all the farmers and technicians from Saravan, especially Reza Erfanian and
Faridoddin Yousefi without whom the field work could not have been possible, Fr´ ed´ eric Marion-Poll
for English revision, and two anonymous reviewers for helpful comments.
REFERENCES
ALFILER, A. R. R. 1999. Increased attraction of Oryctes rhinoceros aggregation pheromone, ethyl
4-methyloctanoate, with coconut wood. Coconut Res. Dev. 15:131–149.
BEDFORD, G. O. 1980. Biology, ecology, and control of palm Rhinoceros beetles. Annu. Rev. Entomol.
25:309–339.
CHUNG, G. F. 1997. The bioefficacy of the aggregation pheromone in mass trapping of rhinoceros
beetles (Oryctes rhinoceros L.) in Malaysia. The Planter (Kuala Lumpur) 73:119–127.
ENDR
¨
ODI, S., 1985. The Dynastinae of the World. Dr. W. Junk Publishers, the Hague, The Netherlands,
ISBN 90-6193-138-X, Series Entomologica 28, 800 pp.
ENDR
¨
ODI, S. and PETROVITZ, R. 1974. Die Arten und Rassen der Gattung Oryctes Illiger in Iran.
Entomol. Phytopathol. Appl. (Iran) 36:4–19.
GHARIB, A. 1970. Oryctes elegans Prell. (Coleoptera—Dynastidae). Entomol. Phytopathol. Appl. (Iran)
29:10–12.
GIBLIN-DAVIS, R. M., OEHLSCHLAGER, A. C., PEREZ, A., GRIES, G., GRIES, R., WEISSLING, T. J.,
CHINCHILA, C. M., PENA, J. E., HALLET, R. H., PIERCE, H. D. JR., and GONZALEZ, L. M., 1996.
Chemical and behavioral ecology of palm weevils (Curculionidae: Rhynchophorinae). Fla. En-
tomol. 79:153–167.
GRIES, G., GRIES, R., PEREZ, A. L., OEHLSCHLAGER, A. C., GONZALEZ, L. M., PIERCE, H. D., JR.,
ZEBEYOU, M., and KOUAME, B. 1994. Aggregation pheromone of the African rhinoceros beetles,
Oryctes monoceros (Olivier) (Coleoptera: Scarabaeidae). Z. Naturforsch. 49c:363–366.
HALLET, R. H., PEREZ, A. L., GRIES, G., GRIES, R., PIERCE, H. D., JR., YUE, J., OEHLSCHLAGER,
A. C., GONZALEZ, L. M., and BORDEN, J. H. 1995. Aggregation pheromone of the coconut
rhinoceros beetles, Oryctes rhinoceros (L.) (Coleoptera: Scarabaeidae). J. Chem. Ecol. 21:1549–
1570.
P1: IAZ
Journal of Chemical Ecology [joec] pp1129-joec-478533 February 20, 2004 22:23 Style file version June 28th, 2002
AGGREGATION PHEROMONE OF Oryctes elegans 407
HO, C. T. 1996. The integrated management of Oryctes rhinoceros (L.) populations in the zero burning
environment, in Proceedings of the PORIM International Oil Palm Congress, Kuala Lumpur,
Malaysia. pp. 336–368.
HURPIN, B. and FRESNEAU, M. 1969. Contribution ` a l’´ etude de Oryctes elegans [Col. Dynastinae].
Ann. Soc. Entomol. Fr. (N.S.) 5:595–612.
HUSSAIN, A. A., 1974. Date Palms and Dates with Their Pests. University of Baghdad, Baghdad, Iraq,
pp. 93–95.
JULIA, J.-F. and MARIAU, D. 1976. Research on Oryctes monoceros Ol. in Ivory Coast. III. Olfactory
trapping with ethyl chrysanthemate. Ol´ eagineux 31:263–272.
LEPESME, P. 1947. Les insectes des palmiers. Paul Lechevalier, Paris, pp. 447–473.
MINITAB, 1998. Statistical software, version 12.2 in French, Minitab Inc., France.
MORIN, J.-P., ROCHAT, D., MALOSSE, C., LETTERE, M., DESMIER DE CHENON, R., WIBWO, H.,
and DESCOINS, C. 1996. Ethyl-4-methyloctanoate, major component of Oryctes rhinoceros (L.)
(Coleoptera, Dynastidae) pheromone. C.R. Acad. Sci. Paris, Life Sci. 319:595–602
PRIOR, R., MORIN, J.-P., ROCHAT, D., BEAUDOIN-OLLIVIER, L., STATHERS, T., KAKUL, K., EMBUPA,
S., and NANGUAI, R. 2000. New aspects of the biology of the Melanesian rhinoceros beetle
Scapanes australis (Col., Dynastidae) and evidence for field attraction to males. J. Appl. Entomol.
124:41–50.
PURBA, R., PRAWIROSUKARTO, S., DESMIER DE CHENON, R., MORIN, J.-P., and ROCHAT, D. 2000.
Effect of Oryctes rhinoceros pheromone (ethyl 4-methyloctanoate) diffusion rate on the size of
pest catches, in Proceedings of the XXIth International Congress of Entomology. Foz do Iguaçu,
Brasil, August 20–26, 2000, Vol. I, p. 181.
ROCHAT, D., MORIN, J.-P., KAKUL, T., BEAUDOIN-OLLIVIER, L., PRIOR, R., RENOU, M., MALOSSE, I.,
STATHERS, T., EMBUPA, S., and LAUP, S. 2002. Activity of male pheromone of the Melanesian
rhinoceros beetle Scapanes australis. J. Chem. Ecol. 28:479–500.
ROCHAT, D., NAGNAN-LE MEILLOUR, P., ESTEBAN-DURAN, J. R., MALOSSE, C., PERTHUIS, B., MORIN,
J.-P., and DESCOINS, C. 2000a. Identification of pheromone synergists in American palm weevil,
Rhynchophorus palmarum, and attraction of related Dynamis borassi (Coleoptera, Curculionidae).
J. Chem. Ecol. 26:155–187.
ROCHAT, D., RAMIREZ-LUCAS, P., MALOSSE, C., ALDANA, R., KAKUL, T., and MORIN, J.-P. 2000b. Role
of solid-phase microextraction in identification of highly volatile pheromones of two rhinoceros
beetles Scapanes australis andStrategus aloeus (Coleoptera, Scarabaeidae, Dynastinae). J. Chrom.
A 885:433–444.
SUDHARTO, P. S., PURBA, R. Y., ROCHAT, D., and MORIN, J.-P. 2001. Synergy between empty oil palm
fruit bunch and synthetic aggregation pheromone (ethyl 4-methyloctanoate) for mass trapping of
Oryctes rhinoceros beetles in the oil palm plantation in Indonesia, in Proceedings of the PORIM
International Oil Palm Congress, Kuala Lumpur, Malaysia, pp. 661–664.
VANDER MEER, R. K. and MCGOVERN, T. P. 1983. Structure-activity correlations for derivatives of
siglure: Attractants for Oryctes rhinoceros L. (Coleoptera: Scarabaeidae). J. Econ. Entomol.
76:723–727.
VANDER MEER, R. K., GHATAK, U. R., ALAM, S. K., and CHAKRABORTI, P. C. 1979. (Plus or minus)-
Des-N-morphinan: A unique bridged hydrocarbon attractant for the rhinoceros beetle, Oryctes
rhinoceros, and development of an olfactometer. Environ. Entomol. 8:16–10.