Kairomones Used By Trichogramma Chilonis

Journal of Chemical Ecology, Vol. 26, No. 2, 2000

KAIROMONES USED BY Trichogramma chilonis TO FIND Helicoverpa assulta EGGS

K. S. BOO* and J. P. YANG Division of Applied Biology and Chemistry College of Agriculture and Life Sciences Seoul National University Suwon 441-744, Korea (Received March 13, 1998; accepted September 20, 1999) Abstract—Chemically mediated interactions between an egg parasitoid, Trichogramma chilonis, and its host insect, Helicoverpa assulta, were studied in laboratory experiments. T. chilonis was attracted to the sex pheromone of H. assulta, and, among four components of its sex pheromone, (Z )-11-hexadecenyl acetate seemed to be most attractive. T. chilonis was also highly attracted to (E )-12-tetradecenyl acetate, a component of the sex pheromone of Ostrinia furnacalis, another host. H. assulta eggs were more parasitized by T. chilonis when the eggs were treated with male moth scale extract (MSE) of H. assulta. Parasitism was also affected by the age of the parasitoid, time of day, and MSE concentration. Silica gel chromatography and subsequent argentation chromatography for MSE fractionation indicated the activity was associated with the fraction of saturated hydrocarbons. A linear olfactometer experiment revealed that H. assulta eggs also contain a short-range attractant(s). Key Words—Trichogramma chilonis, kairomones, Helicoverpa assulta sex pheromone, egg odor, scale odor, Ostrinia furnacalis sex pheromone.

INTRODUCTION

Semiochemicals offer good prospects as a tool for managing parasitoid behavior, particularly in a view of possible application to enhance the efficacy of parasitoids in biological control programs (Tumlinson, 1988). Among entomophagous insects, Trichogramma spp. have received considerable attention, due mainly to their potential as biological control agents against lepidopteran * To whom correspondence should be addressed.

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crop pests. Trichogramma pretiosum Riley, for example, is a candidate for inundative release against Heliothis spp. (King et al., 1986; King and Coleman, 1989). Trichogramma species use various semiochemicals including plant synomones and host kairomones in their search for host eggs (Noldus, 1989a,b). Volatile chemicals emanating from female moths mediate increased rates of parasitism (Lewis et al., 1982). Furthermore, the effluvia of virgin female moths attract female wasps (Noldus and van Lenteren, 1985a; Noldus et al., 1991a). The sex pheromone of their host is an important cue used by Trichogramma wasps. This was first noticed in field and greenhouse observations by Lewis et al. (1982) with the noctuid Heliothis zea. Laboratory olfactometer experiments confirmed behavioral response of T. pretiosum to the odor of calling H. zea moths (Noldus, 1988). Similarly, Trichogramma evanescens responded to the sex pheromone of Mamestra brassicae in a four-way airflow olfactometer (Noldus and van Lenteren, 1985a). A wind-tunnel study with T. evanescens and T. pretiosum showed that host sex pheromone elicits orientation behaviors that result in arrestment in the area where the odor is perceived (Noldus et al., 1990, 1991a). In addition to volatile semiochemicals, the searching behavior of Trichogramma spp. is modified by nonvolatile kairomones that increase the possibility of encountering the host. Plants on which adult moths have been present or plant leaves or filter papers treated with extracts of wing scales or eggs are preferred over control leaves by female Trichogramma (Noldus and van Lenteren, 1985b; Zaborski et al., 1987). Jones et al. (1973) isolated docosane, tricosane, tetracosane, and pentacosane from moth scales and demonstrated that these hydrocarbons elicit positive orthokinesis in T. evanescens, tricosane being the most active compound. In contrast, tricosane and various organic acids found in H. zea scales had only minor kairomonal effects on T. pretiosum (Gueldner et al., 1984). Dimethylnonatriacontanes have been identified in the scales of the European corn borer, Ostrinia nubilalis, and proven to effect both klinokinesis and retention of Trichogramma nubilale, which results in an increase in parasitism rates of O. nubilalis eggs and egg masses (Shu and Jones, 1989; Shu et al., 1990). Eggs are the target of oophagous parasitoids and can be a direct source of kairomones. For example, the eggs of several noctuid species are attractive to females of several Trichogramma spp. (Nordlund et al., 1977, 1981; Gross et al., 1981). In the presence of an extract from O. nubilalis or M. brassicae eggs, female Trichogramma brassicae exhibited increased rates of upwind locomotion in the tubes of a linear olfactometer. GC and GC-MS analyses of O. nubilalis or M. brassicae extracts revealed the presence of fatty acids, their ethyl esters, and various hydrocarbons. Exposing the wasps to a mixture of the main saturated hydrocarbons (heneicosane, tricosane, pentacosane, heptacosane, and nonacosane) increased the upwind progression in the olfactometer. Ethyl palmi-

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tate and palmitic acid were also effective. Single hydrocarbons, however, elicited marginal or no activity. Therefore, it was concluded that various compounds present on the surface of egg masses and eggs of O. nubilalis or M. brassicae may play a role in the orientation of T. brassicae to its host (Renou et al., 1992), and these chemicals appear to act by short-range attraction and/ or by contact (Renou et al., 1989). If the parasitoid has already entered a host habitat, such chemicals may facilitate host location and examination. The Oriental tobacco budworm, Helicoverpa assulta, is widely distributed in Korea, Japan, China, Australia, and Africa and causes serious damage to fruits of the hot pepper (Capsicum annum L.) in Korea. Adverse effects of insecticides and difficulties in H. assulta control led the researchers to study its sex pheromone (Cork et al., 1992) and natural enemies such as the egg parasitoid, Trichogramma chilonis, which is the most important parasitoid of H. assulta in Korea (Choi et al., 1975; Hwang, 1987; Nandihalli, 1994). The current study investigated the sex pheromone, scales, and eggs of H. assulta as potential sources of kairomones used by T. chilonis.

METHODS AND MATERIALS

Insect Rearing. T. chilonis were obtained from the Department of Plant Protection, National Agricultural Science and Technology Institute, Rural Development Administration, Korea, and reared on eggs of H. assulta in 500-ml beakers under a 15L : 9D photoperiod and at 27 ± 18 C. Adult wasps emerged eight days after host eggs were parasitized. They were collected every day, transferred to glass tubes (2.5 cm diam. × 14 cm long), and fed on 20% sugar solution. Only 3-day-old T. chilonis females were used, except when testing age-dependent responses. The females had no previous oviposition experience. H. assulta larvae were reared on an artificial diet (Park, 1991) under a 15L : 9D photoperiod at 25 ± 18 C and 50 ± 10% relative humidity. Some moth eggs laid on gauze were supplied to T. chilonis and others were allowed to develop. After pupation, moths were sexed and segregated. Adults were kept in 30 × 30 × 30-cm acryl cages with 20% sugar solution to obtain eggs. Sex Pheromones of Host Insects. Individual components of synthetic H. assulta sex pheromone, (Z)-11-hexadecenal (Z11–16 : Ald), (Z )-9-hexadecenal (Z9–16 : Ald), (Z )-11-hexadecenyl acetate (Z11–16 : Ac), and (Z )-9-hexadecenyl acetate (Z9–16 : Ac), or a complete mixture at a 50 : 1000 : 15 : 300 ratio (Cork et al., 1992) were tested. In addition, we tested hexadecanal (16 : Ald) because of its significant presence (15.3%) in hexane extract of sex pheromone glands (Cork et al., 1992). Since the response of T. chilonis to H. assulta sex pheromone was high, especially to acetate components, female wasps were examined for their response to sex pheromone components [(Z )-12-tetradecenyl

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acetate (Z12–14 : Ac), (E )-12-tetradecenyl acetate (E12–14 : Ac), and tetradecanyl acetate (14 : Ac)] of Ostrinia furnacalis (Boo and Park, 1998), another host insect of T. chilonis in Korea. Collection of Volatiles from H. assulta Adults. To collect volatiles with Porapak-Q from H. assulta adults, four females or males were placed in an airtight flask connected to a pump with Teflon tubes. Glassware and Teflon tubes were cleaned with detergent water, rinsed in distilled water, and heated at 1208 C for several hours before use. Redistilled hexane was used as solvent and Porapak-Q as an adsorbent material. Each adsorbent was conditioned by washing with approximately 1 ml of hexane, then with 0.5 ml of diethylether, and heated at 1008 C under a stream of nitrogen for at least 2 hr just before use. Air was pumped out at a rate of 0.2 liters/ min for volatiles to be adsorbed unto Porapak-Q. After lights-on or -off, Porapak-Q was removed and eluted with 0.5 ml hexane. Extracts were stored in a freezer for later bioassays. Collection and Fractionation of Scale Extract from H. assulta Male Adults. Moth scales were collected from laboratory-reared, 0- to 1-day-old H. assulta males after holding them at 08 C for immobilization. Scales were obtained from male moths, instead of females, simply because of the high possibility of contamination with sex pheromone in females. The immobilized moths were shaken in a jar to remove scales, which were collected with a vacuum pump and then extracted with hexane. The hexane extract was filtered with Whatman No. 2 filter paper and then concentrated under nitrogen. The concentrated hexane extract was subjected to column chromatography on a 1.0-cm × 4.5-cm column containing silica gel. The column was eluted successively with 8 ml of hexane, mixtures of hexane and diethyl ether at various ratios (97.5 : 2.5, 95 : 5, 92.5 : 7.5, 90 : 10, 75 : 25, and 50 : 50), diethyl ether, acetone, and methanol. These 10 fractions were stored at − 208 C for subsequent fractionation and bioassay. The hexane fraction was further separated through a 20% silver nitrate Florisil column (1.0 × 4.5 cm), which was eluted successively with 8 ml of hexane, diethyl ether–hexane mixture (2.5 : 97.5), and diethyl ether. These three fractions were stored at − 208 C until bioassay. H. assulta Eggs. H. assulta eggs were tested for their short-range attractiveness to wasps. Female moths were allowed to mate and lay eggs on gauze sheets (5 × 30 cm) hung vertically in acryl cages. The sheets were renewed daily, and those with eggs were immediately refrigerated until use. Bioassays. Three types of bioassay setups were used depending on volatility of and T. chilonis response to odors. T. chilonis responses to stimuli were measured in terms of: (1) how many times the female wasps entered into a compartment or arena in the bioassay setup that was treated with stimuli or control (called the number of enterings in the data); and (2) the time duration they stayed at each compartment or arena, regardless of the number of their enterings (called retention time in the data). Percent retention time means a portion of a reten-

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tion time spent at a particular compartment or an arena out of the total retention time spent in all compartments or arenas during the examination period. We gave more emphasis to retention time since we felt retention time is better in representing behavior towards kairomonal stimuli. All bioassays were conducted from the fifth to ninth hour during the photophase, except for responses at different hours during the photophase. Four-Arm Olfactometer. Bioassays of sex pheromones or their individual components and volatiles collected from adults were conducted in a four-arm olfactometer adapted from the device described by Vet et al. (1983). The exposure chamber was connected to four outlets to create four distinct flow fields apart from the central diaper region, and air was sucked out through the central hole at a flow rate of 1.2 liters/ min. One T. chilonis female was introduced into the central part of the chamber and her frequency of entering each of the four outlets and her retention time at each outlet were recorded for 5 min with a computer program. The device was rotated by 908 between each replicate and thoroughly cleaned with methanol and distilled water after every four trials. Petri-Dish Olfactometer. Bioassays with male moth scale extracts (MSE) and chromatographic fractions (SCF) were conducted in a Petri dish setup (Figure 1) because scales or scale extracts did not attract but rather arrested T. chilonis in a preliminary experiment. MSE was prepared with 10 mg of male moth scales in 1 ml of hexane or other solvents, except in an experiment to test T. chilonis response to different MSE concentrations. MSE was applied to each of two opposite quadrants at a rate of 10 mg of moth scale equivalents per microliter. Hexane or other solvents, as a control, was applied to each of the two remaining quadrants. Both MSE and solvents were applied drop by drop, about 5 ml per drop on each of the M and C quadrants (Figure 1). Thus, the chemicals on the filter papers were not continuous but patchily distributed. A treated Petri dish was placed in a 12- × 12-cm paper box with the top removed, and one female was introduced into the center of the Petri dish and removed after 5 min. The Petri dish was covered by a glass plate that was 2.5 cm above the filter paper. To check the parasitism rate of host insect eggs, one H. assulta egg was laid on each dotted circle (M and C regions in Figure 1). In cases of other T. chilonis responses to MSE and SCF without host eggs, we set four middle regions (EMR) of the quadrant that are located outside the innermost circle but inside the middle circle in the Petri dish setup (Figure 1). The frequencies of entering into an EMR treated with MSE or SCE and with solvent by T. chilonis and that of their retention time in EMR were recorded for 5 min with a computer. After each replicate, a new treated disk was introduced into the Petri dish in the box, and the setup was rotated by 908 . Linear Olfactometer. The bioassay for attractiveness of H. assulta eggs was conducted in a linear olfactometer system (Figure 2). Each of four glass columns was evenly divided into 10 arenas. One T. chilonis female was introduced into

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FIG. 1. Schematic representation of the Petri dish setup seen from the top. Whatman No. 2 filter paper was trimmed to fit into a 9-cm-diameter Petri dish and then divided into quadrants by dash-lining with a pencil. MSE (male scale extract) or SCF (chromatographic fraction of MSE) was applied to each of the two opposite quadrants (M) and hexane to the two remaining ones (C). One T. chilonis female was introduced into the center of the circle (radius of circles: innermost, 1.5 cm; middle, 3 cm; EMR: middle region of each quadrant).

arena 0 and then her position inside the glass column, in terms of arena number, was recorded every 30 sec for 12.5 min after application of 0.1 mg of H. assulta egg mass. Air was pushed out through each glass column at the flow rate of 0.5 liters/ min. After each replicate, the glass columns and Teflon tubes were washed with a detergent, rinsed with methanol and distilled water, and dried in an oven. In addition, the positions of the control and the egg mass were changed each time. Statistical Analysis. Twenty or 30 replications were run for each experiment. A preliminary analysis of all data with the Shapiro-Wilk test indicated that data obtained in this study depart significantly from normality, and, therefore, arcsin [( y/ 100)(1/ 2) ] × 57.3 transformation and loge ( y + 1) transformation were applied to normalize the retention time data and entering number, respectively. Significance was examined with Duncan’s multiple range test. In the cases of T. chilonis responses to eggs and MSE or its fractions, statistical analyses were performed on the difference in the number of enterings into and their retention time at treated or control EMR. Significance was examined with Wilcoxon rank-sum test.

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FIG. 2. Schematic representation of the linear olfactometer system. Dashed lines and numbers indicate arenas of the glass column and solid and dashed arrows show the directions of the airflow and introduction sites of the parasitoids, respectively. AC: activated charcoal; AFM: airflow meter; GC: glass column (about 30 cm long and 0.5 cm wide); OS: odor source (H. assulta eggs); M: moisture supply; T.c.: T. chilonis female; TT: Teflon tube; VP: vacuum pump.

RESULTS

Response of T. chilonis to Sex Pheromones of Host Insects. T. chilonis was attracted to odors collected from H. assulta females at night (Table 1), but female odor obtained during the daytime or male odor obtained at any time did not elicit any response from female wasps. This response was apparently due to the sex pheromone of H. assulta (Table 2), but there was no difference between the response to the complete mixture of synthetic sex pheromone or its partial mixTABLE 1. RESPONSE OF T. chilonis FEMALE ADULTS TO H. assulta ADULT ODORS COLLECTED WITH PORAPAK-Q IN A FOUR-ARM OLFACTOMETERa Odor source 4 females during nighttime 4 females during daytime 4 males Hexane aFor

Retention time (%) 26.2 15.7 10.1 6.6

± 26.9a ± 16.9b ± 7.7b ± 6.3b

Number of enterings 2.5 1.9 2.4 2.1

± ± ± ±

1.6a 1.7a 1.3a 1.8a

statistical analysis, retention time and the number of enterings were normalized with arcsine square-root and log transformations, respectively. Their significance was examined with Duncan’s multiple-range test at the level of 5% (N c 30, mean ± SD). Significant differences are indicated by letters.

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TABLE 2. RESPONSE OF T. chilonis FEMALE ADULTS TO H. assulta SYNTHETIC SEX PHEROMONE BLEND OR ITS ACETATE OR ALDEHYDE MIXTURE AT DIFFERENT CONCENTRATIONSa Response

Chemicalsb

1 ng

Retention time (%)

Mixture Acetates Aldehydes Hexane Mixture Acetates Aldehydes Hexane

17.9 ± 12.3a 16.9 ± 15.4a 11.0 ± 8.8b 10.1 ± 7.7b 3.5 ± 1.7a 2.7 ± 1.1ab 2.5 ± 1.7b 2.4 ± 1.3b

Number of enterings

10 ng 26.2 21.4 15.7 6.7 2.5 2.6 1.9 2.1

± ± ± ± ± ± ± ±

20.9a 19.9ab 16.9bc 6.3c 1.6a 1.7a 1.7a 1.8a

100 ng 21.5 21.1 20.2 4.0 2.5 2.6 2.1 1.4

± ± ± ± ± ± ± ±

16.5a 14.2a 16.6a 3.5b 1.7a 1.6a 1.4ab 0.8b

1 mg 27.7 30.0 5.8 9.0 2.6 3.1 2.0 2.3

± ± ± ± ± ± ± ±

31.5a 25.0a 5.7b 9.7b 2.3a 2.2a 2.0a 2.9a

aFor

statistical analysis, data for retention time and the number of enterings were normalized with arcsine square-root and log transformations, respectively. Their significance within the same concentration treatment was examined with Duncan’s multiple-range test at the 5% level (N c 30, mean ± SD). Significant differences are indicated by letters. bAcetates: a mixture of Z11–16 : Ac and Z9–16 : Ac (15 : 300); aldehydes: a mixture of Z11–16 : Ald and Z9–16 : Ald (50 : 1000); mixture: a mixture of two acetates and two aldehydes at the same ratio as above.

ture of acetates only. In general, the acetate mixture was more attractive than the aldehyde mixture, which caused a positive response only at the medium range of concentrations. There was no statistical difference between the attractiveness of the two aldehyde components or between the two acetate components (Table 3). Of the sex pheromone components of O. furnacalis, another host insect of T. chilonis, consisting of two acetates only, E 12–14 : Ac was significantly more attractive than Z11–16 : Ac or other acetates in terms of retention time (Table 3). Response of T. chilonis to H. assulta Scale Extract. The average number of H. assulta eggs parasitized by T. chilonis was 1.7 ± 1.3 when treated with MSE, while it was only 0.9 ± 1.0 when treated with hexane only. Wilcoxon rank-sum test showed that this was significantly different at the 5% level. Retention time spent by T. chilonis at an EMR treated with MSE was more pronounced among 3-day-old and older wasp females (Table 4) and also at midday, from 6 to 11 hr after lights-on, when examined during the photoperiod under 15L : 9D regime (Table 5). Such kairomonal activity of MSE was apparent in a rather wide range of its concentrations, but stronger at the concentration of 100 mg to 10 mg of scale per milliliter of hexane (Table 6). MSE fractions obtained with silica gel (Table 7) and successive argentation chromatography (Table 8) indicated the activity to be associated with saturated hydrocarbons. Response of T. chilonis to H. assulta Eggs. The linear olfactometer experiment indicated that H. assulta eggs have their own attractiveness to wasp females. The location of wasp in the glass column connected to H. assulta eggs was significantly different from that of control (Figure 3).

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TABLE 3. RESPONSE OF T. chilonis FEMALE ADULTS TO VARIOUS COMPONENTS OF H. assulta AND Ostrinia furnacalis SEX PHEROMONE AND OTHER ACETATES (1 mg EACH) IN FOUR-ARM OLFACTOMETERa Host insect H. assulta

H. assulta

O. furnacalisc

Chemicalsb

Retention time (%)

Acetates Z9–16 : Ac Z11–16 : Ac Hexane Aldehydes Z9–16 : Ald Z11–16 : Ald 16 : Ald E12–14 : Ac Z12–14 : Ac 14 : Ac Z11–16 : Ac

19.9 16.0 21.7 8.8 23.5 25.9 22.4 10.0 34.6 11.6 8.9 19.9

± ± ± ± ± ± ± ± ± ± ± ±

Number of enterings

25.9a 15.1ab 18.4a 7.9b 22.7a 21.7a 23.0a 7.3b 17.1a 5.5c 6.8c 10.7b

2.1 1.7 2.0 1.3 1.8 3.8 2.6 2.3 3.6 2.2 1.9 3.3

± ± ± ± ± ± ± ± ± ± ± ±

1.6a 1.3ab 1.8ab 0.7b 1.5a 5.5a 2.6a 2.4a 1.1a 1.7b 1.5b 1.3a

aFor

statistical analysis, retention time and the number of enterings were normalized with arcsine square-root and log transformations, respectively. Their significance was examined with Duncan’s multiple-range test at the 5% level (N c 30, mean ± SD). Significant differences are indicated by letters. bAcetates: a mixture consisting of Z11–16 : Ac and Z9–16 : Ac (15 : 300); aldehydes: a mixture consisting of Z11–16 : Ald and Z9–16 : Ald (50 : 1000). cO. furnacalis sex pheromone is composed of E 12–14 : Ac and Z12–14 : Ac (1 : 2) (Boo and Park, 1998).

TABLE 4. DIFFERENCES IN RESPONSES OF T. chilonis FEMALE ADULTS OF DIFFERENT AGES TO QUADRANTS TREATED WITH MSE OR HEXANEa Differencesb Age (days) 0 1 2 3 4 5 6 7 aBioassay

Retention time (sec) 1.4 2.1 3.7 8.8 6.3 7.1 6.2 5.1

± ± ± ± ± ± ± ±

3.1 5.9 4.7* 12.4* 8.43** 10.2* 7.8** 6.3*

Number of enterings 0.3 0.2 0.4 0.4 0.7 1.0 1.1 1.1

± ± ± ± ± ± ± ±

0.6 1.7 0.9 1.0* 1.5* 2.1* 2.1* 1.5*

was performed from 5th to 9th hour during the photophase (Wilcoxon rank-sum test, 20 replicates, mean ± SD). Significant differences are indicated by asterisks [5%(*) or 1%(**)]. bThe difference between retention times in or the numbers of entering by the wasps into quadrants treated with MSE or hexane.

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TABLE 5. DIFFERENCES IN RESPONSES OF 3-DAY-OLD T. chilonis FEMALE ADULTS TO QUADRANTS TREATED WITH MSE OR HEXANE DURING PHOTOPHASEa Differencesb Hours after lights-on 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Retention time (sec) − 0.4 1.0 2.4 2.1 3.3 7.1 7.8 5.4 6.5 5.2 4.8 3.2 2.6 3.7 3.0

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

Number of enterings − 0.4 0.5 1.1 0.9 1.0 2.7 2.2 1.3 2.2 1.5 1.1 1.3 0.9 1.1 0.9

5.5 3.7 3.8* 3.6* 5.3* 8.0** 8.8** 7.2* 7.6** 6.3* 7.2* 2.3* 5.0* 7.8* 6.7*

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.8 1.3 1.3* 1.5* 1.7* 2.7* 2.3* 1.9* 2.3* 1.7* 1.9* 0.9* 1.8* 2.2* 1.7*

aBioassay

was performed every hour during the photophase (Wilcoxon rank-sum test, 20 replicates, mean ± SD). Significant differences [5% (*) or 1% (**)] are indicated by asterisks. bThe difference between retention times in or the numbers of entering by the wasps into quadrants treated with MSE or hexane.

TABLE 6. DIFFERENCES IN RESPONSES OF 3-DAY-OLD T. chilonis FEMALE ADULTS TO QUADRANTS TREATED WITH HEXANE OR MSE OF DIFFERENT CONCENTRATIONSa Differencesb MSE concentration (per ml hexane) 1 mg 10 mg 100 mg 1 mg 10 mg 100 mg 1g

Retention time (sec) 3.4 2.3 7.6 11.4 8.4 5.1 − 1.5

± ± ± ± ± ± ±

5.2* 2.5* 13.2* 15.0* 10.8* 6.8* 2.6

Number of enterings 0.3 0.4 0.6 0.8 0.3 0.3 − 0.2

± ± ± ± ± ± ±

0.8 0.5* 1.3* 1.1* 0.6 0.7 0.6

rank-sum test (20 replicates, mean ± SD). Significant differences [5% (*)] are indicated by asterisk. bThe difference between retention times in or the numbers of entering by the wasps into quadrants treated with MSE or hexane. aWilcoxon

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TABLE 7. DIFFERENCES IN RESPONSES OF T. chilonis FEMALE ADULTS TO H. assulta MSE FRACTIONS OBTAINED IN SILICA GEL CHROMATOGRAPHY WITH DIFFERENT SOLVENTS Differencesb Fraction S1 S2 S3 S4 S5 S6 S7 S8 S9 S10

Retention time (sec) 12.7 − 0.9 1.8 0.6 − 0.2 0.9 0.9 − 0.2 − 0.9 − 0.8

± ± ± ± ± ± ± ± ± ±

Number of enterings

19.9** 6.8 5.6 3.3 4.6 5.3 6.2 4.5 5.3 2.5

0.3 − 0.3 − 0.2 − 0.2 − 0.3 0.1 0.1 − 0.2 − 0.3 − 0.3

± ± ± ± ± ± ± ± ± ±

1.4* 0.7 0.8 0.8 1.3 1.5 1.1 0.7 1.5 1.2

aOnly

hexane fraction (S1) elicited T. chilonis response significantly different by Wilcoxon rank-sum test. Solvents used were hexane (S1); hexane and diethyl ether mixtures at 97.5 : 2.5 (S2), 95 : 5 (S3), 92.5 : 7.5 (S4), 90 : 10 (S5), 75 : 25 (S6) and 50 : 50 (S7); diethyl ether (S8); acetone (S9); and methanol (S10) (30 replicates, mean ± SD). Significant differences [5% (*) or 1% (**)] are indicated by asterisks. bThe difference between retention times in or the numbers of entering by the wasps into quadrants treated with MSE or hexane.

TABLE 8. DIFFERENCES IN RESPONSES OF T. chilonis FEMALE ADULTS TO H. assulta MSE FRACTIONS ELUTED WITH DIFFERENT SOLVENTS THROUGH ARGENTATION CHROMATOGRAPHYa Differencesb Fractions

Retention time (sec)

Number of enterings

Hexane 2.5% ether in hexane Ethyl ether

6.3 ± 8.2* 0.5 ± 8.5 − 0.1 ± 8.5

1.2 ± 1.5* − 0.4 ± 1.3 − 0.5 ± 1.2

aOnly

hexane fraction elicited significantly different response from T. chilonis, when examined with Wilcoxon rank-sum test (30 replicates, mean ± SD). Significant differences are indicated by asterisk [5% (*)]. bThe difference between retention times in or the numbers of entering by the wasps into quadrants treated with MSE or hexane. DISCUSSION

Our studies suggests that T. chilonis uses some components of its hosts sex pheromone and also odors of the plant (Boo and Yang, 1998) to locate sites

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FIG. 3. Upwind displacement of T. chilonis female adults toward H. assulta eggs in the linear olfactometer. Odor (H. assulta eggs) and control tests were simultaneously run in four parallel tubes with one female each. Location of each wasp in the glass column was recorded every 30 sec for 12.5 min. Wasps’ response in tubes connected to H. assulta eggs was significantly different in comparison to that of the control (Wilcoxon rank-sum test, N c 20). The letter “a” indicates a significant difference at the level of P c 0.05 beginning 2 min after the start of the test and later, but not “b” during the first 90 sec.

that may carry hosts. T. chilonis responded to volatiles released from H. assulta females during nights, but not to those from females during daytime or from males. This is probably due to the sex pheromone of the moth, since the wasps positively responded to the sex pheromone. This is not surprising since many parasitoids and predators have already been reported to use host pheromones as kairomones (Lewis et al., 1982 and references therein; Boo et al., 1998 and references therein). Acetate components in the sex pheromone of H. assulta seem to be responsible for the attraction of T. chilonis. The wasp’s response to the complete mixture of synthetic H. assulta sex pheromone was not better than that to the mixture of the two acetate components only. Among the aldehyde components, Z9–16 : Ald and Z11–16 : Ald were statistically better than 16 : Ald in retaining T. chilonis, but this response was not regarded as important since the mixture of these two aldehydes was statistically inferior to the acetate mixture in eliciting a response. It may be argued that these responses are due to the attractiveness of some components and/ or due to repulsion of some other components. This study does not distinguish between these two possibilities. In the case of the two acetate components in the O. furnacalis sex pheromone, T. chilonis responded

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more positively to a single component, E12–14 : Ac. It is known that not all components of sex pheromones are active in attracting parasitoids. For example, Telenomus busseolae responds to three of four different components of the sex pheromone of its host Sesamia nonagriodes (Colazza et al., 1997). Tricogramma evanescens does not respond to the major component, Z11–16 : Ac, of the sex pheromone of Mamestra brassicae, even though the wasp is attracted by volatiles released by calling virgin female M. brassicae (Noldus and van Lenteren, 1985a). Z11–16 : Ac is not the major component of H. assulta sex pheromone in terms of quantity or activity, even among geographical populations of the species (Boo, 1998 and references therein). This chemical, however, is one of the sex pheromone components that is widely used among insect species. Arn et al. (1992) listed it as a component in sex attractants and/ or pheromones in 168 insect species of 92 genera of 13 lepidopteran families. On the other hand, E12–14 : Ac is used (Arn et al., 1992) by two Yponomeuta species only, besides O. furnacalis. T. chilonis apparently employs chemicals, Z11–16 : Ac and E12–14 : Ac, which are either widely or rarely used for sexual communication among insects. Trichogramma spp. are considered generalists in their choice of hosts (Sheehan, 1986; Thomson and Stinner, 1989). Our studis have suggested, however, that T. chilonis is not simply a generalist employing general chemical cues in searching for hosts. This claim is supported by the fact that T. chilonis uses odor(s) from the host plant (Capsicum annuum) of its host insect (H. assulta), but not from other host plants such as tobacco (Nicotiana tobacum) or other plant species such as eggplant (Solanum melongena) or carrot (Daucus carota var. sativa) (Boo and Yang, 1998). Volatiles released by virgin females do not necessarily have a direct temporal or spatial correlation with the moment or site of oviposition. Therefore, it is not expected that the egg parasite is guided directly towards the place of release. The kairomone might rather lead the parasitoid to an area where mating is in progress and where oviposition is likely to take place or to have taken place. If the mating site is also used as an oviposition site, at the moment a single female host is calling, eggs of conspecifics may already be present in the surroundings. Therefore, responding to the sex pheromone of the host seems adaptively favorable for an egg parasitoid. There is another problem, a time gap of the daytime response of T. chilonis to the sex pheromone released at night from its host insects. Other studies have shown that sex pheromones can be adsorbed onto plant surfaces and subsequently be rereleased. For example, cotton leaves treated with a female Pectinophora gossypiella (Saunders) attracted male moths (Colwell et al., 1978). Further studies with the pea moth, Cydia nigricana (F.), showed that wheat plants adsorbed the synthetic sex attractant and subsequently attracted male moths (Wall et al., 1981; Wall and Perry, 1983). In these studies, the

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adsorption–rerelease effect was obtained with rather high dosages of pheromone. During scent marking, P. gossypiella deposit a considerable quantity on the substrate. In their experiments with C. nigricana, Wall et al. (1981) used a synthetic sex attractant at a dosage that may have been higher than what is released by calling females in natural conditions. Noldus et al. (1991b), however, showed that the airborne sex pheromone originating from a single female M. brassicae moth can adsorb to a Brussels sprout leaf and subsequently elicit behavioral responses in conspecific male moths, as well as in Trichogramma parasitoids. This phenomenon may also apply to the sex pheromone of H. assulta, which is released at night. It may be detectable to T. chilonis during the day. Prolonged searching in an area where host pheromone is perceived will increase the probability that the wasp contacts other cues with a closer spatial correlation with oviposition sites. Actual location of the host eggs may require the detection of other kairomones; for example, scale or egg chemicals left by ovipositing hosts. The kairomones in the moth scales work as sign stimuli, releasing and reinforcing an intensified searching behavior, rather than as a guiding cue, attracting and directing the parasitoid to the host. Naturally occurring eggs of H. assulta are often accompanied by a patch of scales left by the ovipositing moth. This could explain the behavioral response exhibited by T. chilonis upon encountering kairomone patches with scale extract. The localized nature of the response would allow the parasitoid to concentrate its search in a small area likely to harbor hosts. At the same time, the tendency for the intensity of the response to wane allows the parasitoid to quickly confine and search the area of the patch, and ultimately allows it to abandon unrewarding patches. Response of T. chilonis to egg odors indicates that some volatile components from the eggs influence the searching behavior of T. chilonis at a distance. Chemicals that are part of the cement coating of the eggs are characterized by a high molecular weight and low volatility and, thus, may act mainly as contact kairomones (Nordlund et al., 1981; Strand and Vinson, 1983; Zaborski et al., 1987). This kairomonal activity from H. assulta eggs might be due to saturated hydrocarbons, as reported for other insect eggs (Jones et al., 1973; Renou et al., 1992; Strand and Vinson, 1983). However, their role under natural conditions is still to be demonstrated. In conclusion, our studies demonstrate that T. chilonis utilizes some components of the sex pheromone of its host insects (this study) and of the host plant of its host insect (Boo and Yang, 1998) as a relatively long-distance guide for locating prey. Over a short distance within its host habitat, however, the wasp seems to use low volatility or contact type chemicals from the eggs and/ or scales of its host insects. Acknowledgments—This study was financially supported with grants from the Korean Ministry

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of Education and Korea Science and Engineering Foundation through the Research Center for New Bio-Materials in Agriculture, Seoul National University. The authors would also like to thank an anonymous reviewer and editors for greatly improving the original manuscript.

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