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

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 plant-

natural 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

___________

*Correspondent author; present address:

Defence Research & Development Establishment

Jhansi Road, Gwalior 474 002, India

Telephone: +91-751-2231862; Fax: +91-751-2341148

E-mail: seenivasagan@yahoo.com

INDIAN J EXP BIOL, MAY 2011

376

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

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.

ppm)(1000nhydrocarbosaturated

standardofionConcentrat

nhydrocarbosaturatedstandardofareaPeak

nhydrocarbosaturatedidentifiedofareaPeak

nhydrocarbosaturated

identifiedofQuantity×=

…(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 (10-3

g)

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

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.

INDIAN J EXP BIOL, MAY 2011

378

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

379

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

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

Host plant leaf extracts Host larval body extracts Saturated

straight chain

hydrocarbons Cabbage Cauli-

flower

Broccoli Knol khol Brussels

sprout

Cabbage Cauli-

flower

Broccoli Knol khol Brussels

sprout

Decane (C10) - 1.10 - - - - - - - -

Undecane

(C11) - - - - - - - - - -

Dodecane

(C12) - 2.62 - - - - - - - -

Tridecane

(C13) - - - - - - - - - -

Tetradecane

(C14) - 3.47 0.26 - - 0.21 - 0.13 0.13 -

Pentadecane

(C15) - - - - - - 1.46 - - -

Hexadecane

(C16) 0.14 2.09 0.28 - - - 3.87 0.26 - -

Heptadecane

(C17) - - - - - - 3.30 - - -

Octadecane

(C18) 0.08 0.58 - - - - 4.01 0.13 0.53 0.10

Nonadecane

(C19) - - - - - - 9.66 - - -

Eicosane

(C20) 0.15 0.81 - - - 0.22 22.89 0.22 2.13 0.28

Heneicosane

(C21) - - - - - - 17.34 - - 0.17

Docosane

(C22) - 0.20 - - - - 10.44 - 0.68 0.14

Tricosane

(C23) - - - - - - 6.08 - - 0.14

Tetracosane

(C24) - - - - - - 3.00 - - -

Pentacosane

(C25) - 0.11 - - - 0.16 5.58 - 3.10 0.34

Hexacosane

(C26) - - - 0.43 - - 6.46 - 1.12 0.16

Heptacosane

(C27) 0.62 1.71 0.24 0.57 0.67 0.24 8.00 0.20 4.53 0.36

Octacosane

(C28) 0.09 0.38 2.02 0.22 0.24 0.05 1.40 0.06 0.80 0.07

Nonacosane

(C29) 23.84 81.80 13.70 3.45 66.12 3.79 20.69 21.63 65.41 3.07

Triacontane

(C30) 0.18 0.57 1.24 0.30 1.22 - 0.32 0.21 1.55 0.63

INDIAN J EXP BIOL, MAY 2011

380

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.

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

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].

INDIAN J EXP BIOL, MAY 2011

382

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

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].

SEENIVASAGAN & PAUL: GC AND EAG ANALYSIS OF CRUCIFER SATURATED HYDROCARBONS

383

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

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].

INDIAN J EXP BIOL, MAY 2011

384

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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