Beneficial Arthropod Behavior Mediated by Airborne Semiochemicals

Journal of Chemical Ecology, Vol. 14, No. 7, 1988

BENEFICIAL ARTHROPOD BEHAVIOR MEDIATED BY AIRBORNE SEMIOCHEMICALS. III. Influence of Age and Experience on Flight Chamber Responses of Microplitis demolitor Wilkinson 1

F. HI~RARD, 2 M.A. KELLER, 3 W.J. LEWIS, 3'4 and J.H. TUMLINSON 3

2European Parasite Laboratory, USDA, ARS 17 rue de la Masse, B~houst 78910, Orgerus, France 31nsect Attractants, Behavior, and Basic Biology Research Laboratory USDA, ARS Gainesville, Florida 32604 4Duty Station, P.O. Box 748 Tifion, Georgia 31793

(Received April 13, 1987; accepted September 24, 1987)

Abstract--Heliothis zea (Boddie) larvae fed cowpea seedlings produced volatile semiochemicals to which Microplitis demolitor Wilkinson responded in a wind tunnel. However, most M. demolitor females reared from H. zea larvae fed an artificial diet were not responsive at emergence to the same volatile semiochemicals. A preflight contact with frass from H. zea fed cowpea was needed to stimulate a response of sustained flight in a wind tunnel. The most consistent flight response was 7-10 days postemergence. Response resulting from both antennal and ovipositor contact with host frass during preflight stimulation was no better than from antennal contact alone. Chilling the parasitoid pupae rendered most of the emerging females unresponsive to volatile semiochemicals.

Key Words--Microplitis demolitor, Hymenoptera, Braconidae, Heliothis zea, Lepidoptera, Noctuidae, Biological control, artificial diet, preflight behavior, wind tunnels, oviposition, age, chemosensory receotors, chilling pupae. t Hymenoptera: Braconidae.

1583 1584 HI~RARD ET AL.

INTRODUCTION

Many authors, of whom we cite only a few, have shown that semiochemicals and other biological communication mediators serve vital roles in the foraging activities of entomophagous insects (Salt, 1935; Monteith, 1955, 1958; Arthur et al., 1964; Carton, 1971; Corbet, 1971; Hendry et al., 1973; Lewis et al., 1975; Lenteren, 1976; Weseloh, 1976, 1977; Prokopy and Webster, 1978; Nordlund et al., 1981a,b; Vet, 1983; Lecomte and Thibout, 1983; Vinson, 1976, 1984a,b; Noldus and Lenteren, 1985). An example is the report of Drost et al. (1986), who conducted wind-tunnel studies with the indigenous Micro- plitis croceipes (Cresson) and showed that responses of the females to volatile semiochemicals required appropriate preflight stimulation. M. demolitor is an important part of the parasitoid community, which attacks Heliothis in several crops, especially soybeans in Australia. It was imported from Australia in 1981 through the Research Quarantine facility in Stoneville, Mississippi. Shepard et al. (1983) found that M. demolitor developed successfully in the cotton bollworm, Heliothis zea (Boddie); the tobacco budworm, Heliothis virescens (F.); the soybean looper, Pseudoplusia includens (Walker); and the cabbage looper, Trichoplusia ni (Hiibner). The studies reported here were designed to determine the response of the exotic Microplitis demolitor Wilkinson reared from Heliothis zea (Boddie) fed on artificial diet. Specific objectives were: to determine the effect of age and prior oviposition activity on flight response of M. demolitor females to odors emanating from the plant-host complex using a wind tunnel; to determine if a specified part of the plant-host complex was more particularly involved in preflight activation of females; to investigate the role of chemosensory receptors of antennae and ovipositor in integration of messages relative to presence of cues from the plant-host complex; and to test the effect of chilling M. demolitor pupae on the quality of females subsequently emerging.

METHODS AND MATERIALS

Culture of M. demolitor in Hosts Reared on Artificial Diet. All tests used M. demolitor that were reared from larvae of H. zea fed on artificial diet. Females prefer first- to third-instar larvae of H. zea for parasitization (Shepard et al., 1983). A detailed description of the rearing techniques for this parasitoid is provided by H6rard et al. (1988). Adult parasitoids were paired (1 male and 1 female) 1-15 hr after emergence. Each pair was placed in a 30-ml cup with honey and water until tested. Preflight Experience. Response to olfaction can be affected by the parasitoid's previous experience (Thorpe and Jones, 1937; Narayanan and Subba Rao, BEHAVIOR OF ARTHROPODS III 1585

1955; Arthur, 1971). Drost et al. (1986) conducted wind-tunnel studies with the indigenous Microplitis croceipes (Cresson) and showed that responses of the females to volatile semiochemicals required appropriate preflight stimulation. Nordlund and Lewis (1985) observed initiation of host-seeking behavior when M. demolitor females contacted frass of H. zea and T. ni larvae fed pea cotyledons. They verified that 13-methylhentriacontane, a compound earlier identified from H. zea larval frass and shown to elicit antennation by M. croceipes (Jones et al., 1971), also stimulated the antennation behavior by females of M. demolitor. Prior to these tests in the flight chamber, individual M. demolitor females were exposed to the components of plant-host complex in a 150-mm-diameter Petri dish for 2 min. The complete experience consisted of contact with greenhouse-grown, pink-eye, purple-hull cowpea seedlings freshly damaged by H. zea larvae; frass from the feeding host larvae; and one or two ovipositions. Contact was limited to a specified part of the plant-host complex to determine the effect of various preflight stimuli. Twelve treatments were tested. Treatment 1 was the control, i.e., the female was tested without preflight expo- sures to plant-host-related materials. Treatment 2 was contact with an undamaged plant. Treatment 3 was contact with an artificially damaged cowpea seedling. Treatment 4 was contact with a plant that had been fed upon and then washed with water. Treatment 5 was contact with natural feeding damage and frass. Treatment 6 was contact with frass alone. In treatment 7, the parasitoid was exposed only to the host odor. The experience chamber contained second- and third-instar host larvae isolated in compartments. The female M. demolitor was placed above the host larvae in a chamber with a double bottom of organdy. In this way the parasitoid was able to detect the odors of larvae without con- tacting them. In treatment 8, the female was allowed to oviposit once. Treat- ment 9 was contact with the complete plant-host complex and one oviposition. Treatment 10 was contact with the same material as in treatment 9 and two ovipositions. Treatment 11 was contact with an artificially damaged seedling and frass. In treatment 12, the source of the odors was a 4-liter jar containing five cowpea seedlings and 25 actively feeding third-instar H. zea larvae pro- ducing damage to plants and frass. An M. demolitor female was placed in a 10- cm 3 cage with a double cover of organdy and the cage placed in the 4-liter jar so that the parasitoid could detect odors from the complete plant-host complex but could not contact the source. Test in the Wind Tunnel. The wind tunnel provided laminar air flow at 16 cm/sec. The test section of the tunnel was 2 m long with a 75 x 75-cm cross- section. Overhead lighting was provided by four 80-W fluorescent light bulbs. The source of airborne semiochemicals consisted of a damaged cowpea seedling bearing fresh frass and five second- and third-instar H. zea larvae, which were feeding actively at the time of each test. The source was prepared 1586 H~RARD EW AL by allowing the larvae to feed on the seedling, the stem of which was immersed in water, for 2 hr prior to testing. During most of the tests, the source was placed at the Center of the upwind end of the flight tunnel. Each naive female M. demolitor was transferred to the experience chamber (Petri dish) and then to the tunnel in clear 3.7-ml vials. The vial was placed 1.3 m downwind from the source with the open end up. When the females walked out of the vial and became exposed to the odor plume, they displayed two principle types of responses. One was a non-target-oriented flight that occurred either immediately or after a brief period of walking while drumming on the release vial with the antennae. The other was a sustained flight to the source of odors, which occurred after a period of upwind orientation on the vial, If a female conducted a non-target-oriented flight in the first trial, we placed her back on the vial and allowed a maximum of four other chances of flight tothe source. We recorded which females displayed a sustained flight in the first through fifth trials. We tested the possible role of vision when M. demoIitor females approached the source in the flight chamber with the technique developed by Zanen et al. (1988). The source was placed outside the wind tunnel, and semiochemicals were introduced through a simple glass nozzle. Thus, the plant-host complex was not visible to the parasitoid as she flew. Females did not need visual cues because they oriented and flew to this nozzle. Effect of Parasitoid Age. The level of response to volatile semiochemicals from a plant-host complex varied widely with the ages of M. demolitor females. We investigated the effect of age on flight response to plant-host odors by experienced females. Female M. demolitor 1-14 days old were separated into seven groups, each with an age range of two days. Effect of Oviposition Activity. An M. demolitor female emerges from its cocoon with some mature eggs available for oviposition during the very first day. The females tested in the above-mentioned experiment were not allowed to oviposit prior to the test. In order to determine if the response to odors from the plant-host complex by 7-day-old females was due to their strong need to lay eggs, we allowed 20 females to deposit five eggs per day and another group of 20 to lay 20 eggs per day for seven days. Their flight responses were compared to 20 females that were not allowed to oviposit during the same period. Role of Chemosensory Receptors of Antennae and Ovipositor in Integra- tion of Messages Relative to Presence of Cues from Plant-Host Complex. While exploring an area containing some cues from the plant-host complex, a female frequently shows intense antennation activity followed by probing with the abdomen. Occurrence of a necessary information exchange process based on messages received by the chemosensory receptors of these two organs was suspected in the recognition of contact kairomones and sensitization to volatile semiochemicals. BEHAVIOR OF ARTHROPODS III 1587

An experiment was set up to observe the flight response of female M. demolitor to volatile semiochemicals following contact with host frass, either by the antennae only or by the antennae and ovipositor. These responses were compared with those of inexperienced females. To allow contact only by the antennae, a pellet of frass maintained on the tip of a pin was presented above the substrate to the female until she palpated it with her antennae. Normal behavior of the female was to catch the pellet with her fore tarsae, climb on the pin, and probe the pellet with her ovipositor. To avoid this contact, we moved the pellet back while the female moved forward still palpating the pellet with her antennae. We stopped the contact after 15-20 sec and then tested the female in the wind tunnel. Effect of Chilling Pupae on Quality of Subsequently Emerging Females. Initially, it was observed from experiments designed for other purposes that M. demolitor and M. croceipes females emerging from chilled pupae were particularly unsuitable for behavioral bioassays. These females had a strong pro- pensity to show only non-target-oriented flights. Since chilling of pupae is commonly practiced in many entomological laboratories, we suspected a gen- eral practical interest in this observation. We tested effect of chilling pupae at 13~ for 4, 7, and 13 days on behavioral and reproductive performance of M. demolitor. Statistical Analysis. The numbers of females tested in each treatment was 20, except for 40 in the seven groups used to study the effect of age, and 30 in the study of the effect of chilling pupae. Incidences of sustained flight in the first trial were compared in the different treatments by using repeated G tests, or replicated goodness-of-fit tests, based on use of the log-likelihood ratio (Sokal and Rohlf, 1969; Zar, 1974). The 0.05 probability level was used for rejection of all null hypotheses.

RESULTS AND DISCUSSION

Description of Response to Contact and Airborne Semiochemicals. Females ofM. demolitor that contacted material from the plant-host complex in an experience chamber exhibited intense antennation activity, drummed the plant material or frass with their antennae while walking or standing still, and probed at the material with the tips of their ovipositors. Flight initiation and orientation sequences of M. demolitor females to odors from the plant-host complex in the wind tunnel were essentially the same as those described by Drost et al. (1986) for M. croceipes: a period of preflight orientation characterized by a sequence of walking while drumming, followed by standing still oriented upwind while holding the antennae in a vertical plane perpendicular to the wind direction, and followed by initiation of the flight pose. 1588 HE,RARD ET AL.

During the flight pose, the female stood on her meta- and mesothoracic legs while making "walking" movements with her prothoracic legs. Takeoff usually was followed by a sequence of wide zigzagging, straight flight, recurrent zigzagging, another straight flight, hovering at about 10 cm downwind from the target, and quick darts toward the target, ending with landing on the target. This characterized sustained flight. Effect of Age. Figure 1 shows that the response to semiochemicals by 1- to 6-day-old females was low and variable. In the youngest females, only a few responded in the first trial, and some did not fly in five trials. However, the percentage of females responding increased from 1 to 6 days of age. Response to semiochemicals in 7- to 10-day-old females was much more consistent, with 55-75 % of them conducting sustained flights in the first trial. In 11- to 14-day-old females, response to semiochemicals decreased and individual variability increased. Forty-five percent of females showed a sustained flight in the first trial, and 20% did not respond in five trials. As no statistical difference was observed between the 7- to 8- and 9- to 10- day-old groups, we considered the 7- to 10-day-old group sufficiently homo- geneous to be used in experiments where a high responsiveness to semiochem-

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PREFLIGHT TREATMENTS FIG. 2. Effect of preliminary egg deposition on flight response to odors from cowpea- Heliothis zea complex by 7- to 8-day-old experienced Microplitis demolitor females. C: no egg laid, no preflight experience; NDF-0: no egg laid, natural damage + frass; NDF- 35:35 eggs laid, natural damage + frass; NDF-140:140 eggs laid, natural damage + frass. The black portion of the bars represents the percentage of females flying in the first trial, and the white plus black bars those flying at least once in five trials. Bars topped by the same letter do not differ significantly; P = 0.05; repeated G tests. 1590 HERARD ET AL.

Effect of Varying Preflight Stimuli. Data for the 12 preflight experience designs are shown in Figure 3. Treatments 2, 3, 4, 7, 8, 11, and 12 gave results not significantly different from the control. Contact with the complete plant- host complex (treatment 5) induced a sustained flight in the first trial in 75 % of the females. When components of the plant-host complex were used separately in the experience chamber (treatments 2, 3, 4, 6, and 7), only frass (treatment 6) induced a stimulation (in 70% of the females) which was as good as the complete complex (treatment 5). Three other treatments containing the frass component induced a good sustained flight in the first trial: natural damage + frass (treatment 5), and complete plant-host complex with oviposition (treatments 9 and 10). The additional contact with natural feeding damage and the experience of one or two ovipositions did not significantly increase the degree

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PREFLIGHT TREATMENTS Fro. 3. Effect of varying preflight stimuli on flight response to odors from cowpea- Heliothis zea complex by 7- to 8-day-old experienced Microplitis demolitor females. Treatment 1 (C): no preflight experience; treatment 2 (HP): undamaged host plant; treatment 3 (AD): artificial damage; treatment 4 (ND-w): natural damage, water washed; treatment 5 (NDF): natural damage + frass; treatment 6 (F): frass; treatment 7 (L-od): larval odor; treatment 8 (OV): 1 oviposition; treatment 9 (NDF + 10V): natural damage + frass + I oviposition; treatment 10 (NDF + 20V): natural damage + frass + 2 ovipositions; treatment 11 (ADF): artificial damage + frass; treatment 12 (LNDF- od): odor of larvae, natural damage and frass. The black portion of the bars represents the percentage of females flying in the first trial, and the white plus black bars those flying at least once in five trials. Bars topped by the same letter do not differ significantly; P = 0.05; repeated G tests. BEHAVIOR OF ARTHROPODS III 1591 of response observed with frass alone. Only the combination of preflight contact with frass and artificial damage (treatment 11) gave a response as poor as other treatments lacking frass. This suggests the possibility of a masking or repulsive effect of chemicals emitted by cowpea leaves damaged artificially. A leaf from which the frass was removed was then washed (treatment 4) and did not induce host-seeking behavior in most of the females. Exposure to odors of larvae (treatment 7) or of the complete plant-host complex with no contact with the material (treatment 12) were not valuable preflight experiences. Even a brief contact with a host, limited to a fraction of a second, during which an egg was laid (treatment 8), did not render most of the females tested responsive to volatile semiochemicals. Contact with undamaged or artificially damaged plants (treatments 2 and 3, respectively) had no effect on responses to plant-host odors in most of the M. demolitor females. However, very few females (5%), with no preflight experience at all (treatment 1), showed a sustained flight in the first trial. This indicates that a few females have a sufficiently low inherent threshold of response to volatile semiochemicals such that no prior experience is required. This experiment showed that: (1) most of the M. demolitor females reared from hosts fed artificial diet needed a preflight experience to become responsive to volatile semiochemicals emitted by a target plant-host complex; (2) frass from the host was the most effective source for preflight stimulation and only a brief contact with the source was necessary; and (3) the act of oviposition alone did not increase responses nor did it enhance the effect of contact with the frass. Role of Chemosensory Receptors of Antennae and Ovipositor in Integra- tion of Messages Relative to Presence of Cues from Plant-Host Complex. Fig- ure 4 indicates that there is no significant difference in percentages of females showing a sustained flight in the first trial after experience by contact with frass with only the antennae or with antennae and ovipositor. This indicates that the antennae are the necessary and sufficient olfactory organs involved in: (1) recognition of significant cues from the plant-host complex, (2) detecting host presence in the vicinity and continuation of active search for it, and (3) the threshold level necessary for flight response to volatile semiochemicals. Contact with frass elicits responses of antennation, a short running, and subsequent probing with the abdomen by the female M. demolitor. As soon as the ovipositor touches the frass, running stops and antennation is resumed. Con- sequently, we assume that when the frass is touched with the antennae, it trig- gers a stereotypical behavior of oviposition, which ceases when the chemoreceptors of the ovipositor encounter a substance that is not a suitable host. Very likely, an information exchange process occurs when the stimulation is received alternatively by antennae and ovipositor. Whereas the former would induce activation of search for the host by lowering the threshold of sensitivity I592 HARAR# ET AL.

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PREFLIGHT TREATMENTS FIG. 4. Role of chemosensory receptors of antennae and ovipositor in establishing experience of frass, and effect on flight response to odors from cowpea-Heliothis zea complex by 7- to 8-day-old Microplitis demolitor females. C: no preflight experience; F-a: experience with frass by the antennae only; F-a + o: experience with frass with both the antennae and ovipositor. The black portion of the bars represents the percentage of females flying in the first trial, and the white plus black bars those flying at least once in five trials. Bars topped by the same letter do not differ significantly; P = 0.05; repeated G tests. to airborne semiochemicals and by initiating the reflex of oviposition, the latter would have a regulatory effect on this reflex and would act mainly in host recognition and host acceptance. Many parasitoids are guided to the host by volatile odors and are stimulated to accept it or reject it by chemotactile stimuli on the cuticle or in the body, perceived through receptors on the antennae, tarsi, or ovipositor (Herrebout, 1969; Vinson and Lewis, 1965; Weseloh and Bartlett, 1971; Corbet, 1971; Wilson et al., 1974; Weseloh, 1974; Prokopy and Web- ster, 1978). Effect of Chilling Pupae on Quality of Subsequently Emerging Females. The percentage of experienced females that performed a sustained flight to the odor source in the first trial in each of the three chilled groups was significantly lower than in the nonchilled group (Figure 5). Even a four-day period of chilling led to a decrease in the response of subsequently emerging adults. Experienced BEHAVIOR OF ARTHROPODS III 1593

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PREFLIGHT TREATMENTS Fro. 5. Effect of chilling pupae on flight response to odors from cowpea-Heliothis zea complex by 7- to 8-day-old experienced Microplitis demolitor females, subsequently emerged from this pupae. CH-0: not chilled; CH-4: 4-day chilling; CH-7: 7-day chilling; CH-13: 13-day chilling. All the females were given a preflight exposure to natural damage + frass. The black portion of the bars represents the percentage of females flying in the first trial, and the white plus black bars those flying at least once in five trials. Bars topped by the same letter do not differ significantly; P = 0.05; repeated G tests.

females did not respond to volatile semiochemicals in most cases. It may be that such parasites, released in the field, would be ineffective for biological control. In addition, reproductive performance of the seven- and 13-day chilled individuals was so altered that 94% of their progeny were males. These data suggest that females were not inseminated because of a misfunctioning of the pheromonal system or because the sperm transfer was altered by the cold. In order to answer this question, we interbred females emerging from seven-day- chilled cocoons with normal males, and seven-day-chilled males with normal females. Observation of the males' response to the female pheromone suggested that chilled females produced the pheromone normally. Both chilled and unchilled males responded positively and chilled males were sensitive to the 1594 HERARD ET AL.

female pheromone. Matings were observed in the two groups. The percentage of male progeny in both groups was higher than in typical rearing (60 % instead of 40-50 %). The results indicate that chilling of pupae altered the reproductive performance of both the females and males. The combination of these effects, when chilled males and females were interbred, led to production of a particularly high percentage of males among the progeny. These studies demonstrated the importance of airborne semiochemicals in the host-searching behavior of M. demolitor and the importance of preflight handling on behavioral performance of the parasitoid. Understanding the process of preflight stimulation has a high practical value. It appears that conditioning of beneficial insects by contact with particular cues from the target plant- host complex before their release results in a much more effective host-searching behavior by them and their retention in the release area. Gross et al. (1975) demonstrated in greenhouse and field studies that stimulation with host-seeking stimuli just prior to release resulted in higher parasitization rates with M. croceipes, Trichogramma pretiosum (Riley), and T. achaeae Nagaraji. Lewis et al. (1975) showed that retention of Trichogramma spp. increased when a kairomone (Tricosane) was applied to plant foliage.

Acknowledgments--We are most grateful to Lisa Hill and Joe Spurlin, who maintained insect and plant cultures; to Deryck Perkins and Margaret Wood, who generously provided host larvae and artificial diet; and to Ted Turlings, Fred Eller, Lucas Noldus, Yvonne Drost, and Olivier Zanen for helpful discussions and suggestions. Raymond Moore, Walker Jones, David Perkins, John McLaughlin, John Hamm, and Genevieve Prdvost gave valuable suggestions for manuscript revi- sions.

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