Indian Journal of Experimental Biology
Vol. 49, May 2011, pp. 375-386
Gas-chromatography and electroantennogram analysis of saturated hydrocarbons
of cruciferous host plants and host larval body extracts of Plutella xylostella for
behavioural manipulation of Cotesia plutellae
T Seenivasagan* & A V Navarajan Paul
Division of Entomology, Indian Agricultural Research Institute, New Delhi 110 012, India
Received 12 August 2010; revised 9 December 2010
Saturated hydrocarbons (SHC) of five cruciferous host plants viz., cabbage, cauliflower, broccoli, knol khol and Brussels
sprout and the larvae of diamondback moth (DBM), Plutella xylostella reared on these host plants were identified through
gas-chromatography. The hydrocarbon profile of host plants and larval body extract of DBM reared on respective host
plants revealed a wide variation in quantity as well as quality. Long chain hydrocarbons C26-C30 were detected in all the
extracts. In electroantennogram (EAG) studies, SHCs at 10-3g dose elicited differential EAG response in the antennal
receptors of gravid Cotesia plutellae females. Tricosane (C23) and hexacosane (C26) elicited 10-fold increased EAG response
compared to control stimulus. Long chain hydrocarbons C27, C28 and C29 elicited, 6-7 fold increased responses. The
sensitivity of antenna was 4-5 folds for C25, C14, C24, C15 and C30, while the short chain hydrocarbons elicited 2-3 fold
increased EAG responses. Dual choice flight orientation experiments in a wind tunnel revealed that the gravid C. plutellae
females preferred the odour of C16, C26, C29, C15, C21, C23, C30, C27, C24 and C22 as 60-70% females oriented and landed on
SHC treated substrate compared to control odour, while the odour of eicosane (C20), pentacosane (C25) and octacosane (C28)
were not preferred by the females.
Keywords: Behaviour manipulation, Cotesia plutellae, Electroantennogram, Flight orientation and landing, Gas
chromatography, Hydrocarbons, Plutella xylostella, Wind tunnel
Plant leaf surfaces are coated with a thin layer of
waxy material that has a myriad of functions. This
layer is microcrystalline in structure and form the
outer boundary of the cuticular membrane. It serves
many purposes, for example to limit diffusion of
water and solutes while permitting controlled release
of volatiles that may deter the pests or attract natural
enemies and pollinators1. The surface of the insect is
also covered by a layer of wax and the nature of this
lipid is dependant on species and in general a high
proportion tends to be saturated alkanes (C21 to C31).
It is becoming increasingly clear that a major function
of cuticular hydrocarbons (CHCs) in arthropods is to
serve as recognition signal between individuals2. In
parasitoid wasps, non-volatile host cuticular lipids are
used as very short range signals, while in specialist
parasitoids these lipids serve as chemical recognition
signals to identify host species3-7 or to discriminate
___________
*Correspondent author; present address:
Defence Research & Development Establishment
Jhansi Road, Gwalior 474 002, India
Telephone: +91-751-2231862; Fax: +91-751-2341148
E-mail: [email protected]
suitable individuals for oviposition8. Higher alkanes
are known to be one of the main components of
cuticular waxes of plant leaves and extracts. Among
the various chemical constituents of plant leaf wax
n-alkanes (hydrocarbons) hold a major share. The
other constituents include alkyl esters, fatty acids,
fatty alcohols, aldehydes, ketones and triterpenoids
etc., with different biological functions in the plantnatural enemy interactions in a crop ecosystem.
Several authors have underlined the need to identify
the semiochemicals involved in host location and
recognition to realize their importance in the design of
biological control program9-12. However, the potential
of such approach remains unexplored13.
The role of saturated hydrocarbons (SHCs)
functioning as kairomones for egg parasitoids of the
genus Trichogramma14,15, in predators16 and also in a
parasitic wasp of silver leaf whitefly17 have been well
documented in literature. Although, Cotesia plutellae
(Kurdjumov), the solitary larval endoparasitoid of
diamondback moth (DBM), Plutella xylostella (L.),
received lesser attention in this aspect, Roux et al.18
have reported that the females of C. plutellae detect
376
INDIAN J EXP BIOL, MAY 2011
their hosts through a short antennal contact. In another
study, Roux et al.19 using GC-MSD have found that,
the hydrocarbon fraction was more dominant (77%)
than non hydrocarbon fraction in the cuticular lipids
of diamondback moth, which elicited positive
antennal contact by C. plutellae females in laboratory
experiments. To our knowledge there has been limited
work to evaluate systematically the role of
hydrocarbons as orientation cues for C. plutellae,
Hence, the present study was intended to identify
hydrocarbons from the hexane extracts of cruciferous
host plants belonging to genus Brassica as well as the
host larvae reared on these plants using a gas
chromatograph-with flame ionization detector
(GC-FID). Subsequently electroantennogram (EAG)
was employed to evaluate the sensitivity of antennal
receptors of C. plutellae for these identified
hydrocarbons followed by flight orientation studies in
a wind tunnel. In this paper, we give a brief account
of hydrocarbons and their possible semiochemical
functions, which could mediate P. xylostella
(herbivore)- Brassica spp (host plant)-C. plutellae
(natural enemy) interaction in the cruciferous crop
ecosystem.
Materials and Methods
Plant materials and test insects — The cruciferous
host plants of diamondback moth, P. xylostella viz.,
cabbage, cauliflower, broccoli, knol khol and Brussels
sprout were grown in the research farm of the Indian
Agricultural Research Institute (I.A.R.I,) New Delhi
and were used for the present investigation. Both the
host insect P. xylostella and its larval parasitoid
C. plutellae were cultured in the Biological Control
Laboratory, (I.A.R.I) based on the methods described
in detail by Seenivasagan et al.20. P. xylostella was
reared on cabbage and cauliflower leaves at 26°±2°C
in open trays. Briefly, the nucleus culture of larval
parasitoid C. plutellae was obtained from National
Bureau of Agriculturally Important Insects (NBAII),
Bangaluru, and then subsequently reared on the
second and third instar larvae of natural host at
27°±1°C, 60±10% RH, 10L:14D photoperiod. For
electroantennogram and flight orientation bioassays
2-3 day old gravid females were used.
Host plant and host larval body extracts — The
host plant leaf extracts (HPLE) were prepared based
on the method described by Eigenbrode et al.21 with
little modifications to ensure the optimal extraction of
cuticular compounds into the solvent medium.
Seventh or eighth fully expanded leaf was removed
from the uninfested plants of each host plant. Leaves
were gently rinsed in cold tap water and air dried.
Thirty gram (3×10 g fresh wt) of undamaged leaves of
each cruciferous host plant was immersed for 5 min in
300 ml (3×100 ml) of high performance liquid
chromatography (HPLC) grade hexane (Merck Ltd,
Mumbai, MS, India), taking care not to immerse the
cut end of the petiole. The three extracts were
combined for further processing. Similarly, the host
larval body extract (HLBE) of DBM larvae (1 g fresh
wt ~125-150 late 2nd and early 3rd instar) reared on
respective host plants were made by immersing larvae
in 10 ml of hexane as described above. HPLE and
HLBE were filtered using Whatman #1 filter paper
and then dehydrated over anhydrous sodium sulphate
(Na2SO4) for 1h and passed through 120 mesh silica
gel (Qualigens Fine Chemicals, Mumbai, MS, India)
in borosil glass column (Vensil®, Bangalore, KA,
India) with 18mm ID × 45 cm length to remove any
moisture. The eluted extracts were concentrated by
passing N2 to minimize loss of volatiles and stored
at -20°C. Separate columns were used for different
extracts. The concentrated residue of HPLEs and
HLBEs were dissolved separately in a small quantity
of solvent and the volume was made up to 1 ml which
constituted 100% extract and used for gas
chromatography analysis.
Gas-chromatography — A gas chromatograph
(Varian 3900 XL) equipped with flame ionization
detector (FID) and a WCOT fused silica CP-SIL
24 LB/MS (#CP5860), Varian Chrompack capillary
column (30 m × 0.32 mm ID) was used for analysis of
extracts. The oven temperature was programmed
between 100°-260°C for 56 min in four ramps
initially at 100°C for 5 min, then increased at 10°C
/min to 150°C and held for 10 min, then further
increased to 200°C at 10°C/min and held for 10 min,
and finally the oven temperature was increased to
260°C at 10°C /min and held for 15 min to ensure
complete and orderly elution hydrocarbon standards
(Fig. 1) as well as the components in the extract. Both
the injector and detector temperature was set at
300°C. Nitrogen was used as carrier gas with a flow
rate of 300 ml/min. The flow rate at 30 and 29 ml/min
respectively, was maintained for hydrogen and zero
air. Extract samples (3 µl) were injected using a
Hamilton precision syringe into split/split less injector
with 80:20 split ratio. To generate the basic data on
the retention time of standard hydrocarbons (C10-C30-
SEENIVASAGAN & PAUL: GC AND EAG ANALYSIS OF CRUCIFER SATURATED HYDROCARBONS
377
Fig. 1—Standard chromatogram of saturated straight hydrocarbon mixture dissolved in hexane at 1µg/µl concentration injected into a
split/splitless injector (80:20 split ratio) in a Varian 39 XL gas chromatograph. Peaks are labeled with chain length of respective
hydrocarbons. Value within the parenthesis is retention time of the eluting peak.
Sigma Aldrich, St. Louis, MO, USA), each
hydrocarbon was prepared in 1000 ppm concentration
(ie. 1 µg/µl) in hexane (HPLC grade, Merck Ltd,
Mumbai, MS, India). A mixture of hydrocarbon
standards was prepared by dissolving all the 21 SHCs
in hexane at required quantity to obtain 1000 ppm.
For identification by retention time matching and
quantification of hydrocarbons in the extract samples,
SHC mixture was injected first into the column and
their peak areas were noted and subsequently, the
extract samples were run in a day. The
chromatograms were analyzed with the help of
interactive
graphics
software
(Varian
Star
chromatography workstation, version 6.0) for peak
integration, qualitative identification and quantification
of SHCs of host plant leaves as well as the host larvae
reared on various cruciferous host plants.
Peak area of identified
Quantity of identified saturated hydrocarbon
Concentration of standard
=
×
saturated hydrocarbon Peak area of standard saturated hydrocarbon (1000 ppm)
saturated hydrocarbon
…(1)
After the identification of SHCs by matching their
retention time with that of standards the quantity of
hydrocarbons in the extracts were determined by
following the above mentioned formula.
Electroantennography —- The electroantennogram
responses were recorded from 2-3 days old gravid
female wasps of C. plutellae with at least seven
different excised antennas constituting seven
replicates
using
electroantennogram
(EAG)
instrument
(M/s
Syntech,
Hilversum,
The
Netherlands). Briefly, neck of the female was clipped
off through the foremen magnum of the head from a
cold immobilized adult female wasp, similarly the tip
of the antenna was clipped off using a fine micro
scissor under KCl (0.1 M) solution. The base of the
antenna was mounted onto indifferent electrode using
Electrode gel (Spectra 360; Parker Laboratories Inc,
Fairfield, NJ, USA) and the tip of the antenna was
connected to recording electrode. A stable base line
with minimum fluctuations in the oscillograph of
EAG program indicated an ideal electrical contact of
antenna between the electrodes. The charcoal filtered
and humidified air (500 ml/min) was delivered
continuously through borosil glass tube with 0.5 cm
ID over the antennal preparation from a distance of
1.5 to 2 cm using a stimulus controller (Syntech,
Hilversum, The Netherlands). Test chemicals were
adsorbed onto a piece of hexane washed filter paper
(3 × 1 cm) folded in zig-zag pattern. Ten micro litres
(10 µl) of test stimuli at 1000 ppm (10-3g)
concentration was applied on the filter paper and was
kept for 10 sec that resulted in evaporation of solvent.
Odour laden filter paper was then placed inside the
Pasteur pipette (Sigma-Aldrich, St. Louis, MO, USA)
for saturation of air space and subsequently puffed
onto every stabilized antenna for a pulse duration of
0.3 sec which delivered 2.5 ml of odour stimulus
378
INDIAN J EXP BIOL, MAY 2011
laden air to the antenna. Between subsequent
stimulations at least 1 min interval was given for the
recovery of antenna. To ensure complete mixing of
stimulus odour with continuous air flow, the stimulus
was injected into the mixing tube through a side port
located at 10 cm distance from the antennal
preparation. Each recording session was initiated by
application of air, hexane (solvent control) followed
by 10-3 g dose of different SHCs and terminated with
reverse order of first two stimulations. At least seven
replicates were performed with different antenna from
the test insects. The stimulation sequence of
hydrocarbons was randomly shuffled in each
replicate. The resulting signals were amplified 10×
and directly imported via an Intelligent Data
Acquisition Controller (IDAC) interface box and an
analog to digital (A/D) converter into an Intel based
personal computer. Recordings were analyzed by
means of EAG software (EAG 2000 Version 2.7c,
Syntech, Hilversum, The Netherlands). Freshly
prepared 10% honey was used as standard and was
puffed over the antenna at starting and finishing of
each recording session to test the responsiveness of
antenna. The EAG amplitude of air was subtracted
from other responses to nullify any mechanical
stimulation of antennal receptors, and the responses
elicited by hydrocarbons were compared with that of
solvent control for analysis.
Flight orientation — The flight orientation response
of gravid C. plutellae females was studied in a
Plexiglas wind tunnel (100×30×30 cm) as per the
methodology described by Potting et al.22 with few
modifications. A suction fan with a regulator drew the
charcoal filtered and humidified air into the wind
tunnel at a speed of 25 cm/sec. The experimental
arena was covered by nylon wire mesh on both sides
to prevent escape of released wasps. In the lower side
of wind tunnel, there was a motor to continuously roll
a muslin cloth marked with green paint in cross
linking pattern to simulate natural environment inside
the arena. At the upwind end of the wind tunnel two
perforated platforms on a Plexiglas stand of ca. 15 cm
in height were used for holding the treatment and
control stimuli on a piece of sterilized absorbent
cotton. At the downwind end the test insects were
held in a release tube made of Plexiglas with wire
mesh on one side and a sliding plate with wire mesh
on the other side. The sliding plate contained a
separate circular hole to release the insects into the
arena. The insect holding tube was held in a stand of
ca.15 cm height. The distance between insect holding
stand and the stimulus stands was ca. 70-75 cm.
A group of ten cold anesthetized/immobilized
gravid females were transferred into the release tube
using an aspirator and held for a minute, and
simultaneously the air flow was switched on into the
experimental arena for the recovery of test insects.
About 100 µl of each hydrocarbon stimulus
(100 ppm) and equal amount of solvent control was
loaded onto the cotton. After the evaporation of
solvent both the stimuli were kept inside the
experimental arena 15 cm apart at the upwind end of
the wind tunnel. The air flow was switched on to
release the odour of test and control stimulus into the
tunnel and simultaneously the release tube was
opened to release the test insects. The experimental
duration was 5 min to observe the orientation
behaviour and response of the C. plutellae females.
After 5 min the air flow was stopped and the number
of females landed on treatment and control stimulus
substrate were counted to assess the preference of the
test insects. At least seven replicates were performed
with new set of C. plutellae females for each
hydrocarbon. With every new replicate the location of
control and treatment stimulus holding stands were
alternated. Between every experimental trial the wind
tunnel was wiped with moist cotton and subsequently
hot air was blown for 3-5 min to remove the odour of
previous trials.
Statistical analysis
EAG amplitude (-mVolt) values obtained by
stimulating the antennal receptors of C. plutellae for
each hydrocarbon at 10 µg dose was subjected to one
way analysis of variance (SPSS Inc, Chicago, IL,
USA). The difference between the mean of two
treatments were separated by Tukey honestly
significant difference (HSD) test using SPSS 10.0
statistical software. A 2×2 contingency table on the
total number of insects responded to treatment and
control stimulus in a dual choice flight orientation
experiment was analyzed by chi-square test assuming
50:50 distribution in the response of test insects.
Results
Identification and quantification of SHCs in host
plant leaf extracts—Gas chromatographic analysis of
selected cruciferous host plants of diamondback moth
revealed the presence of saturated hydrocarbons in
the leaf extracts (Table 1). Cauliflower leaf extract
SEENIVASAGAN & PAUL: GC AND EAG ANALYSIS OF CRUCIFER SATURATED HYDROCARBONS
contained 12 hydrocarbons with carbon number
ranging from C10-C30, in which C29 was detected in
highest quantity (82 µg). In cauliflower extract
exclusively C10 and C12 hydrocarbons were identified
which were not detected in other host plant extracts.
The hydrocarbon, C14 was detected only in
cauliflower and broccoli extracts, whereas C16 was
detected only in cabbage, cauliflower, and broccoli
379
extracts. C18 and C20 were detected in cabbage and
cauliflower extracts. C22 and C25 were detected only in
cauliflower; while, C26 was found only in knol khol
leaf extract. The HPLEs from knol khol and Brussels
sprout (66 µg) contained only long chain SHCs
ranging from C26-C30, in which C29 was present in
higher quantity followed by cabbage (24 µg),
broccoli (14 µg) and knol khol (3.5 µg). The
Table 1—Quantity of identified saturated hydrocarbons detected in extracts by Varian 39XL Gas chromatograph
Quantity of saturated hydrocarbons (µg/µl) detected in extracts by Varian 39XL Gas chromatograph
Saturated
straight chain
hydrocarbons
Decane (C10)
Undecane
(C11)
Dodecane
(C12)
Tridecane
(C13)
Tetradecane
(C14)
Pentadecane
(C15)
Hexadecane
(C16)
Heptadecane
(C17)
Octadecane
(C18)
Nonadecane
(C19)
Eicosane
(C20)
Heneicosane
(C21)
Docosane
(C22)
Tricosane
(C23)
Tetracosane
(C24)
Pentacosane
(C25)
Hexacosane
(C26)
Heptacosane
(C27)
Octacosane
(C28)
Nonacosane
(C29)
Triacontane
(C30)
Host plant leaf extracts
Host larval body extracts
Cabbage
Cauliflower
Broccoli
Knol khol
Brussels
sprout
Cabbage
Cauliflower
Broccoli
Knol khol
Brussels
sprout
-
1.10
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
2.62
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
3.47
0.26
-
-
0.21
-
0.13
0.13
-
-
-
-
-
-
-
1.46
-
-
-
0.14
2.09
0.28
-
-
-
3.87
0.26
-
-
-
-
-
-
-
-
3.30
-
-
-
0.08
0.58
-
-
-
-
4.01
0.13
0.53
0.10
-
-
-
-
-
-
9.66
-
-
-
0.15
0.81
-
-
-
0.22
22.89
0.22
2.13
0.28
-
-
-
-
-
-
17.34
-
-
0.17
-
0.20
-
-
-
-
10.44
-
0.68
0.14
-
-
-
-
-
-
6.08
-
-
0.14
-
-
-
-
-
-
3.00
-
-
-
-
0.11
-
-
-
0.16
5.58
-
3.10
0.34
-
-
-
0.43
-
-
6.46
-
1.12
0.16
0.62
1.71
0.24
0.57
0.67
0.24
8.00
0.20
4.53
0.36
0.09
0.38
2.02
0.22
0.24
0.05
1.40
0.06
0.80
0.07
23.84
81.80
13.70
3.45
66.12
3.79
20.69
21.63
65.41
3.07
0.18
0.57
1.24
0.30
1.22
-
0.32
0.21
1.55
0.63
380
INDIAN J EXP BIOL, MAY 2011
following hydrocarbons C11, C13, C15, C17, C19, C21,
C23, and C24 were not detected from any of the HPLEs
subjected for GC analysis. The long chain SHCs like
C27, C28, C29 and C30 were detected in all the host
plant leaf extracts (Fig. 2).
Identification and quantification of SHCs in host
larval body extracts—Gas chromatographic analysis
of HLBEs obtained by rearing DBM larvae on
respective host plants revealed that the HLBE from
cauliflower and knol khol contained maximum
number of hydrocarbons ranging from C15-C29 and
C14-C30 respectively in relatively larger amount
compared to other larval body extracts [LBEs]
(Table 1). C29 was found in highest quantity (65-3 µg)
in almost all the LBE of DBM reared on different
cruciferous host plants. In cauliflower, C20 was
detected in higher quantity (23 µg) followed by C29
(21 µg), C21 (17 µg) and C22 (10 µg). Whereas, C15
(1.5 µg), C17 (3.3 µg), C19 (9.6 µg) and C24 (3 µg) was
detected only in HLBE obtained from cauliflower.
From the HLBE of DBM reared on cauliflower, C21
(17 µg) and C22 (10 µg) were detected in higher
quantity. C25 was absent in HLBEs derived from
broccoli, whereas C26 was not detected in HLBEs
obtained from rearing DBM on cabbage and broccoli.
HLBE of DBM reared on cabbage leaves doesn’t
contain C30, however, it was detected in higher
quantity (1.5 µg) in the HLBE derived from knol khol
followed by Brussels sprout (0.6 µg), cauliflower
(0.3 µg) and broccoli extract (0.2 µg). The long chain
hydrocarbons C27, C28 and C29 were detected in
all the kairomonal/host larva extracts, whereas C10,
C11, C12 and C13 were not detected in any of the
extracts (Fig. 3).
Electroantennogram and flight orientation response
of Cotesia plutellae—The electroantennogram (EAG)
response of C. plutellae to the identified SHCs
revealed a differential sensitivity of antennal receptors
(Fig. 4). Our initial dose response studies of most
commonly detected hydrocarbons at a dose regime of
10-6 g to 10-2 g revealed that 10-3 g dose elicited
significant EAG responses from antenna of
C. plutellae females compared to control as well as
lower doses of SHCs. At higher dose (i.e 10-2 g) the
sensitivity of antenna was reduced or inhibited. Hence
10-3 g was selected as the optimal dose to stimulate
the antenna and to compare the EAG response profile
of all the 21 SHCs used in this investigation.
Interestingly, gravid females of C. plutellae exhibited
very high level of sensitivity to C23 and C26 to an
Fig. 2—Chromatographic profiles of saturated straight chain
hydrocarbons identified in the waxy layer of cruciferous host
plant leaves of diamondback moth [ (a) cabbage; (b) cauliflower;
(c) broccoli; (d) knoll khol; and (e) Brussels sprout]
SEENIVASAGAN & PAUL: GC AND EAG ANALYSIS OF CRUCIFER SATURATED HYDROCARBONS
381
extent of 10 fold increased EAG amplitude compared
to control stimulus. The long chain hydrocarbons C27,
C28 and C29 elicited 6-7 fold increased EAG
responses. The sensitivity of antenna was 4-5 folds for
C25, C14, C24, C15 and C30; while the other
hydrocarbons elicited 2-3 fold increased EAG
responses. The response to C13 was lowest and at par
with C12, C19 and C11, however, it was slightly higher
and significantly different from that of solvent
control.
In dual choice wind tunnel experiments, the odour
plume of C16, C26, C29 and C15 attracted 70% of gravid
C. plutellae females compared to hexane controls.
The attractancy of hydrocarbons varied from 69 to
47%. The decreasing order of attractancy viz.,
C21>C23>C30>C27>C24>C22>C17>C19 was presented by
SHCs to C. plutellae females. Although C. plutellae
females preferentially oriented toward many long
chain hydrocarbons; they were significantly repelled
by the odour of C20, C25 and C28 as evidenced by only
few number/proportion (18-20%) of females landing
on odour laden substrates compared to control
stimulus (Fig. 5). The short chain hydrocarbons
attracted significantly fewer number of gravid
C. plutellae wasps, however, the percentage of
non-responsive females were higher for C13, C12, C10,
C11 and C14 which indicates the unfavorable nature of
these hydrocarbons to the responding C. plutellae
females.
Fig. 3—Chromatographic profiles of saturated straight chain
hydrocarbons identified in the cuticle of larval body extracts of
diamondback moth reared on various host plants [ (a) cabbage; (b)
cauliflower; (c) broccoli; (d) knoll khol; and (e) Brussels sprout].
Discussion
Behaviour of a natural enemy can be manipulated
by selecting appropriate plant variety through
breeding for certain characters which could
potentially enhance the foraging ability of a parasitoid
in an ecosystem against the target pest9. Under natural
situations, the interface where tri-trophic interaction
takes place is often the cuticle of a plant23. The
epicuticular wax layer of plants has been shown to
influence the foraging success of natural enemies24.
CHCs are also known from other herbivore–parasitoid
associations to serve as kairomones25,26. Further,
Takabayashi et al27 and Turlings et al28 stated that the
plant is more important in affecting the composition
of volatile blend than the herbivore. Comparison of
the hydrocarbon profiles of both HPLEs and HLBEs
in the present study supports this view. We have used
the split ratio of 80:20 to analyze the extracts with an
aim to identify the hydrocarbons occurring in minimal
quantity. Because extracting the leaf and larval
382
INDIAN J EXP BIOL, MAY 2011
Fig. 4—Electroantennogram response of Cotesia plutellae gravid females to saturated straight chain hydrocarbons.
[Values are mean ± SE (n=7). Mean EAG amplitude connected by cross lines with different letters are significantly different
(F(21,132)= 23.15, P<0.001].
materials for a duration of 5 min in hexane, although
extracts maximum cuticular lipids, some of the
compound which may be behaviourally very
significant for the responding natural enemy may
likely to be undetected if smaller fraction of injected
sample is placed into the column during the GC
analysis. Further, the chemical composition and
amount of plant cuticular waxes may vary greatly
depending on species, genotype or even within plant
parts. In turn, this variation can modulate the outcome
of many interactions between plants, herbivores and
their natural enemies29.
Although, there are many published reports on
Brassica plant-P. xylostella-natural enemy interaction
for the host location behaviour by the parasitoids
associated with cruciferous crop ecosystem22,30-35, to
our knowledge the role of hydrocarbons has not been
given much attention for mediating behavioural
responses in C. plutellae. The GC analysis revealed a
wide variation in quality and quantity of SHCs in each
host plant as well as HLBEs. In general, the
quantity of hydrocarbons detected in LBEs was
higher than leaf extracts. Roux et al19 have detected
forty compounds by GC analysis ranging from 23 to
29 carbon atoms, in which C29 (21.4%),
15-nonacosanone (18.2%), 11-MeC27 (13.1%) and
7,16-diMeC27 (6.8%) dominated and represented
more than 59% of the total cuticular lipid extract.
Further, the hydrocarbon fraction represented 77% of
the amount of total cuticular lipids while the
non-hydrocarbon fraction contributed 22.5% of
cuticular lipids. In the present study, the LBEs of
P. xylostella reared on various host plants contained
C27, C28 and C29 in varying quantity. The DBM larva
reared on cauliflower contained maximum number of
hydrocarbons, which would be due to the use of
highly palatable and undamaged leaves of the plants
at active vegetative stage for rearing the larvae.
Because, at this stage the acquisition of nutrients and
release of volatiles from such leaves are higher due to
larger leaf area, which invites both the pest as well as
its natural enemy i.e., C. plutellae in the natural
environment. This could have contributed to increased
number and higher concentration of hydrocarbons
detected in HLBE of DBM which might have
acquired/ingested more chemical constituents while
feeding on cauliflower leaves compared to other
plants.
Smid et al.36 identified 20 compounds in Brussels
sprout through gas chromatography coupled
electroantennogram detection (GC-EAD) that elicited
responses in Cotesia glomerata and Cotesia rubecula,
however they have not reported any hydrocarbons
from the head space analysis. It might be due to the
variety of the plant, which was different from the one
we have used for extraction in this study. Our results
on electroantennogram and flight orientation response
of gravid C. plutellae suggest that amongst the
21 SHCs evaluated, 8 hydrocarbons (C15, C16, C21,
C23, C26, C27, C29 and C30) were more attractive to the
SEENIVASAGAN & PAUL: GC AND EAG ANALYSIS OF CRUCIFER SATURATED HYDROCARBONS
383
Fig. 5—Flight orientation response of Cotesia plutellae gravid females to saturated straight chain hydrocarbons in a dual choice
experiment in a wind tunnel. [Values are mean ± SE (n=7). Significant differences at P<*0.05, **0.01 and ***0.001, respectively
in χ2 test for 50:50 distribution in a 2×2 contingency table compared to control stimulus].
foraging females with more than 63-73% of females
landing on SHC treated substrate, while 50-60%
positive orientation and landing was observed for C17,
C19, C22 and C24. In spite of eliciting good EAG
response, three hydrocarbons C20, C25 and C28 elicited
negative orientation as 60-64% of gravid females
oriented to control odour laden substrate (Fig. 5).
In a study on host parasitoid interaction, Paul
et al.14 have reported some hydrocarbons as
favourable, because they have elicited more activity
in the egg parasitoid Trichogramma spp to parasitize
the host eggs, while the other hydrocarbons which
elicited reduced level of parasitism were grouped as
unfavourable hydrocarbons. Another study by
Ibrahim et al.37 on the response of C. plutellae to
volatile compounds has shown that C. plutellae
preferred DBM damaged plants with limonene over
plants without limonene application. Similarly,
Charlesten et al.38 have found that C. plutellae
females are more attracted to infested cabbage plants
treated with certain botanical pesticides. Further,
Rostas et al.39 have demonstrated that the plant
384
INDIAN J EXP BIOL, MAY 2011
surface wax affects the response of a specialist larval
parasitoid Cotesia marginiventris to the foot print of
its host Spodoptera frugiperda. In the present study,
although different extracts showed variation in
quantity and composition of SHCs, their ability to
elicit significant behavioural response in the flight
orientation was prominent for every SHCs assayed in
wind tunnel. However, under natural conditions these
SHCs mediate the orientation response of foraging
parasitoids in tandem with other constituents of plant
leaves to attract and enhance the activity of
C. plutellae in a cruciferous crop ecosystem.
Since, these hydrocarbons are synthesized through
fatty acid biosynthesis40 the acquisition and
accumulation of dietary constituents of a host plant
should be taken into consideration, because they
might alter the composition of epicuticular waxy layer
of feeding larvae that in turn influence the behaviour
of its natural enemy in the ecosystem. Recently
Fernandes et al.41,42 have reported the acquisition and
fate of dietary constituents in Pieris brassicae fed
with kale leaves. In our earlier study20 we observed,
that the larval body extract of P. xylostella reared on
various host plants were more attractive to gravid
C. plutellae females compared to virgin females.
Subsequently, in a field study, Seenivasagan et al.43
have reported that, the C. plutellae females caused
maximum parasitization of P. xylostella larva on an
artificially infested cauliflower, cabbage and Brussels
spout plants, possibly due the emission of green leaf
volatiles, as well as the release of hydrocarbons
present in the waxy layer of leaves by the feeding of
P. xylostella larvae that could have attracted large
number of gravid females for parasitizing the host
larvae. These findings support our present results that
the SHCs when presented individually can influence
and guide the gravid females to differentially orient
toward and land on a treated substrate in the wind
tunnel.
In conclusion, biological control of insect pests has
become increasingly important in agriculture because
of the need to minimize the amount of toxic chemicals
released into the environment. Using crop varieties
with appropriate wax surfaces may enhance the
efficiency of parasitoids and could thus improve the
biological control of pests. In this study we have
demonstrated that C. plutellae can distinguish and
locate in-flight, to land on the substrate laden with
saturated hydrocarbon compared to control stimulus.
The results of GC study suggest the difference in the
CHC composition of the DBM larvae fed on different
host plants. EAG studies provided evidence that the
antennal receptors of C. plutellae were differentially
sensitive to these hydrocarbons. It would be
interesting to investigate further the role of these
hydrocarbons in combination with other attractive
volatiles for the behavioural manipulation of this
solitary larval endoparasitoid as a component of
integrated pest management for the biological control
of diamondback moth in a cruciferous crop
ecosystem.
Acknowledgement
We are grateful to the Indian Agricultural Research
Institute and Council of Scientific and Industrial
Research (CSIR) for granting Merit Scholarship &
Senior Research Fellowship respectively during the
period of study. We sincerely thank Dr. Alok Sen,
National Chemical Laboratory, Pune for guidance on
Electroantennogram experiments and analysis of the
data. We are thankful to the Director, IARI and Head,
Division of Entomology for the providing the required
facilities during the course of research work.
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