Indian Journal of Experimental Biology Vol. 42,December 2004, pp. 1230- 1234

Electroantennogram responses of Chrysoperla carne a (Stephens) to volatiles

Ruchira Raina, Mary Joseph & Avalokiteswar Sen*

Laboratory of Entomology, National Chemi cal Laboratory, Dr. Homi Bhabha Road, Pune 411 008

Received 27 April, 2004; revised I September 2004

Electrophysiological responses of adult males and females of C.carnea to commonly occurring plant volatiles were recorded using the electroantennogram technique. Responses to 28 volatiles evaluated indicate that both depolarization and recovery to the baseline were rapid in fe males compared to males. Normalized EAG responses relative to the standard, trans-caryophyllene reveal signifi cant differences between the chemicals as also between the sexes. The response of males to several of the compounds, particularly the terpenoids was higher than females. The pooled averages to the different classes of chemical compounds revea l greater response for fatty acid derivatives and terpenoids, particularly the oxygenated monoterpenes and the sesquiterpenes. These findings are discussed in relation to volati les released in the cotton ecosystem.

Keywords: Cluysoperla carnea, Electroantennograms, Predators, Volati les

The Green lacewing, Chrysoperla carnea (Stephens) (Neuroptera : Chrysopidae) is a cosmopolitan and generalist species commonly found in agricultural ecosystems. The larval stages of chrysopids are voracious feeders of soft bodied arthropods including lepidopteran eggs and young larvae, aphids, spider mites, leaf hoppers, whiteflies etc. 1 In recent years, there is growing evidence suggesting volatiles emanating from both undamaged plan ts2

•3 and

damaged plants4·6 provide the necessary cues to

natural enemies to locate often scarce and well hidden hosts. Despite the apparent importance of these volatile organic compounds (VOC' s) in tritrophic interactions, specific studies aimed at elaborating their function, vis-a-vis, predators is lacking. In our continuing efforts to identify semiochemicals that would possibly serve to manipul ate the behavior of natural enemies, the electrophysiological responses of adult males and females of C. camea have been investigated to major terpenoids, which represent a dominant class of compounds in cotton, to fatty acid derivatives including green leaf volatiles, to ketones, phenoli cs and to other compounds commonly found in a majority of plants, using the electroantennogram (EAU) technique. Such studies will reveal the olfactory sensitivity profiles of C. carnea, besides

• Correspondent author Phone: (020) 2589 3300 ext. 230 1 Telefax: (020) 2589 3153 e-mail: aloksen@yahoo.com

differences between sexes to a senes of chemicals belonging to diverse classes thereby help in identifying compounds that could be used to increase the efficiency of these predators.

Materials and Methods Insects-A nucleus culture of Cluysoperla carnea

(Stephens) was obtained from the Divi sion of Entomology, I.A.R.I. , New Delhi from which subsequent generations have been reared in the laboratory at 25°±2°C and 70% RH. Briefly, 10-15 pairs of newly emerged adults are released in a jar and provided with 20% honey mixed with Proteinex®. The mouth of the jar is secured with black muslin cloth which serves as a substrate for oviposition by the adult females . Eggs are laid within 5-7 days and the larvae on hatching are provided, on alternate days , with UV treated eggs of Corcyra cephalonica for feeding until pupation. The pupal cocoons are separated and kept for emergence.

Chemicals-The chemicals were obtained from commercial suppliers. Twenty-eight test compounds were chosen based on their common distribution in plants fed upon by the host(s) of C. carnea. The test chemicals were divided into different chemical classes such as (a) fatty acid derivatives (hexanal, trans-2-hexanal, cis-3-hexen-1-ol, 1-pentanol, 1-hexanol arid 1-heptanol); (b) hydrocarbon monoterpenes (R(+) limonene, (IS)(-) limonene, (1 S)(-) ~-pinene, a-phellandrene, ~-myrcene and o-3-

RAINA et. al.: ELECTROANTENOGRAM RESPONSE OF CHRYSOPERLA CARNEA TO VOLATILES 1231

carene); (c) oxygenated monoterpenes (linalool, citronella!, citronellol, phytol and nero!); (d) ketones (acetophenone and 2-heptanone); (e) phenolics (eugenol and benzaldehyde); (f) sesquiterpenes (nerolidol, trans-caryophyllene and farnesol) and (g) others (cis-jasmone, phenethyl alcohol, phenyl acetaldehyde and eucalyptol). All of them were more than 95% pure except for two mixtures, phellandrene and phenyl acetaldehyde. Each chemical was dissolved in paraffin oil (Hi-Media) to yield 100 mg/ml solutions.

Electroantennograms--EAG responses were recorded from 2-3 day old adults. Briefly, the head was mounted on the indifferent electrode containing 0.1 M KCI, while the tip of the antennae was slipped carefully onto the recording electrode containing the same concentration of the electrolyte. The recording electrode was connected through a pre-amplifier (Syntech) to a EAG amplifier (AM-02, Syntech®, Hilversum, The Netherlands). The analog EAG signal was amplified 10 times and digitized through an AID interface (IDAC-02, Syntech®, Hilversum, The Netherlands). With the custom EAG program (Ver. 2.6, 1996, Syntech®, Hilversum, The Netherlands), the resulting EAG was analysed by measuring the maximal millivolt amplitude of depolarization resulting due to a particular stimulus.

An air stimulus controller provided air and odor delivery with a constant flow of 1.8 L/min and 0.6 Llmin respectively. A hexane washed, oven dried filter paper strip (5 mm x 30 mm; Whatman #1) containing the test stimulus was inserted into the Pasteur pipette placed into the side port (3 mm diam.) of a glass tube (10 mm, od) serving as the delivery tube. Stimulus (10 fLl) was applied for 100 msec duration and a duration of 30 sec was kept for antenna! recovery. For control, 10 fLl of paraffin oil was used as a stimulus. Three replicates were conducted for each stimulus and EAGs were recorded from 5 males and 5 females. Following conection with the control stimulus, EAG responses were calculated as per cent amplitude to the standard, trans-caryophyllene. Also, differences in volatility among the test compounds were not considered and as a result, comparisons among responses are relative.

Statistical analysis--Relative values were Log 10

transformed and subjected to a 2-way ANOV A (SPSS, GLM procedure). Subsequently, within each sex, a one-way ANOV A was computed on the relative values and contrasts between chemicals were

examined by the Scheffe's contrast method. For each group of chemical, relative amplitudes to individual compounds were pooled and averaged and differences between sexes were determined by Student's t test.

Results The typical electroantennogram response of

C .camea to plant volatiles is characterized by a fast depolarization followed by a slow return to the baseline. In females, both depolarization and recovery to the baseline were rapid in trans-caryophyllene while they were delayed for aldehydes, alcohols and a majority of the terpenoids. However, the above parameters were relatively delayed in males. The mean response of females to stimulation by trans­caryophyllene (standard) was 5.55±0.32 mV, while it was lower in males, 2.46±0.25 mV. The normalized peak response to the other chemicals varied in females from 11.74±0.42% (&-3-carene) to 66.6±3.53% (trans-2-hexanal) and in males from 8.10±0.14%W-pinene) to 42.28±4.95% (trans-2-hexanal) (Fig. 1).

Of the 28 chemicals tested, a 2-way ANOV A (GLM procedure) of log 10 transformed values indicated strong differences between chemicals (F=24.47, 27df, ?<0.0001), sex (F=27.5,1 df, P<0.0001) and a significant interaction between sex/chemicals (F=22.6, 27df, P<O.OOOI). A one-way ANOVA of relative values on each sex indicated important differences in females (F=72.6, 27df, ?<0.0001) and in males (F=26.02, 27df, ?<0.0001). The analysis of contrasts by the Scheffe's procedure in females indicated significant differences between the standard trans-caryophyllene with the remaining 27 compounds. While differences between trans-2-hexanal, hexanal and nerolidol and between nerolidol, phellandrene and P-pinene were not significant, they differed individually from the remaining 23 compounds. However, differences between the remaining compounds were not significant. In males, the maximal response was obtained with trans-2-hexanal which was higher than the remaining 27 compounds, including the standard, trans­caryophyllene. Although differences between trans­caryophyllene, trans-2-hexanal, linalool, myrcene, cis-3-hexen-1-ol, cis-jasmone and phytol were not significant, they were significantly higher than the remaining compounds. Similarly, differences between myrcene, cis-3-hexen-1-ol, cts-Jasmone, phytol, benzaldehyde, phellandrene, citronellal, hexanal and

1232 INDIAN J EXP BIOL, DECEMBER 2004

8-3-carene, between 8-3-carene, R-limonene, 1-hexanol, 1-pentanol, nerolidol, S-limonene and eucalyptol, between eucalyptol, 1-heptanol, farnesol, 2-heptanone, nerol , phenethyl alcohol, eugenol and acetophenone and between citronellol, phenyl acetaldehyde and P-pinene were not significant. Differences between other compounds were significant at P<O.OOOl.

Responses to individual groups of compounds were pooled and averaged to allow for comparisons between groups of structural classes. The mean EAG

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amplitude to sesquiterpenes was significantly higher to that of other classes (Student's t test, ?<0.0001). The fatty acid derivatives produced significantly higher amplitudes than hydrocarbon monoterpenes and oxygenated monoterpenes (Student's t test, P<O.OOI), while differences between phenolics, ketones and other compounds were not significant. In females, the relative mean EAG amplitude was higher for sesquiterpenes, while the oxygenated monotergenes elicited maximal responses in males. Among the different classes of compounds,

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RAINA et. al.: ELECTROANTENOGRAM RESPONSE OF CHRYSOPERLA CARNEA TO VOLATILES 1233

differences between males and females were significant in all cases except for the hydrocarbon monoterpenes and oxygenated monoterpenes (Student's t test, ?<0.001). In general, the phenolic compounds produced the least response in both males and females .

Discussion The amplitude of depolarization has been used to

measure the sensitivity of insects to volatile compounds. Differences observed, particularly in males, with respect to relatively longer durations for depolarizations and recovery to baseline is probably due to deactivation processes of the receptor sites7

. It reflects that several of the molecules evaluated, viz.' fatty acid derivatives, ketones and certain terpenoids bind to the receptor sites of olfactory neurons of antenna! sensilla which may have an effect on the predatory behavior of C. carnea.

Using the rationale that peripheral olfactory systems have evolved sensitivity to behaviorally important odors, the present study demonstrates that both males and females of C. carnea respond to the 28 plant volatiles evaluated in electroantennogram studies with differences observed with several chemicals, viz., linalool, myrcene, cis-jasmone and benzaldehyde eliciting higher responses in males than in females. The abi lity of both males and females to detect the odors presented is probably due to their similar habitat requiring the use of the same cues to locate host plants for survival and reproduction. The hierarchy of responses was greater for the fatty acid derivatives, oxygenated monoterpenes and sesquiterpenes. The greater preference of females for fatty acid derivatives, essentially comprising green leaf volatiles could be of adaptive significance in enabling the gravid females to a potential plant for oviposition as has been suggested for Trichogramma chilonis8

. Although the higher response of males of C. carnea for oxygenated monoterpenes has also been reported in some phytophagous insects, their response, in general to plant volatiles is intriguing. Evidence for the role of cotton volatiles in mate location by male Campoletis sonorensis (Cameron) has been documented9 as also the ability of Trichogramma spp. to utilize the sex pheromone or its components of the host has been observed 10

•11

. It is p~ssible that the high responses observed particularly w1th trans-2-hexanal and other saturated aldehydes may be due to their ability to stimulate pheromone receptors.

Olfactometer studies have indicated that C. carnea adult are attracted to volatiles from eggplant, okra and pepper, hosts of the mite, Tetranychus ludeni 12

. Both sexes responded to odors emanating from mite infested eggplants, okra and pepper than uninfested healthy plants. Cotton plants attacked by herbivorous insects emit relatively high amounts of characteristic volatile terpenoids that have been implicated in the attraction of natural enemies of the herbivores 13

.

Thus, the systemic release of myrcene, (Z)-3-hexenyl acetate, (E)-~-ocimene, linalool, (E)-4,8, dimethyl­! ,3, 7 -nonatriene, (E)- ~-farnesene and (E,E)-4,8, 12-trimethyl-1 ,3,7, 11-tridecatetraene are attractive to both the generalist parasitoid, Cotesia marginiventris and the specialist, Microplitis croceipes 14

. In addition 15 ' Flint et al. reported that damaged cotton plants

release the terpenoid, caryophyllene, which attracts C. carnea. The present study demonstrates that several of the compounds, particularly, the terpenoids that are highly responsive to adults of C.carnea are molecules that are released from cotton plants, upon insect herbivory.

In natural ecosystems, herbivore mortality is more commonly mediated by a suite of generalist natural

• 16 Th .d enemies . e WI e spectrum of responses of both males and females of C. carnea to different compounds belonging to diverse chemical classes is due to their wide host range of insects which feed on a variety of host plants. Differences observed in the EAG responses to individual groups of compounds reflects differential tuning of receptors on the antennae thereby suggesting that using plant cues is adaptive for a generalist predator like C. carnea. Efforts are currently underway to study the effect of induced plant volatiles on the predatory efficiency of C. carnea.

Acknowledgement This study was supported by research grants from

the Department of Science and Technology under the OYS Scheme toRR and a grant-in-aid to AS. Thanks are due to SH Kelkar & Co., Mumbai for gift samples of some of the volatiles.

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1234 INDIAN J EXP BIOL, DECEMBER 2004

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