Morphological and Biological Evidence for the Presence of a Male Sex Pheromone of the Diamondback Moth

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1 11 Morphological and Biological Evidence for the Presence of a Male Sex Pheromone of the Diamondback Moth Y. S. Chow, Y. M. Lin, and H. J. Teng Institute of Zoology, Academia Sinica, Nankang, Taipei, Taiwan, ROC Abstract In the present study, besides morphological evidence, two bioassays, antenna-excision and hairpencil-excision, were used to demonstrate the presence of a male sex pheromone of the diamondback moth Plutella xylostella L. Although the antenna-excision bioassay demonstrated that a male aphrodiasic pheromone exists in the diamondback moth, it was proven to be not as important as the female sex pheromone in mating. After the hairpencils of males were excised, the mating success of the females decreased significantly, suggesting that the male hairpencils might play a role in the mating of the female diamondback moth. introduction Hairpencils, highly specialized scales, are present on the integument of the males of various lepidopteran species. They are exposed during the pre-courtship rituals that lead to various species-specific behavior patterns (Grant and Brady 1975, Baker and Carde 1979, Rutowski 1980, Hirai et al 1981). This has been studied in tobacco budworm (Heliothis virescens). The release of an airborne chemical by these organs suppresses the emission of the sex pheromone secreted by the female (Hendricks and Shaver 1975). However, Clearwater (1972) reported that Mythimna separate (Walker) produced a pheromone from hairpencils and suggested that the major component, benzaldehyde, acted as an arrestant that prevented the escape of the female during courtship. On the other hand, Gothilf and Shorey (1976) found that in cabbage looper (Trichoplusia ni) the scent of the male moth was not an essential component in courtship behavior. Recently Baker et al (1981) and Nishida et al (1982) found that a blend of ethyl transcinnamate, methyl 2-epijasmonate, methyl jasmonate, and (R)-(-)-mellein, identified from the hairpencils of male oriental fruit moth, attracts sex pheromone-releasing females several centimeters away. The objectives of the present study were to determine the effect of the hairpencils of the male diamondback moth (DBM), Plutella xylostella L (Lepidoptera: Yponomeutidae) on mating and to demonstrate the presence of a male sex pheromone, if any. Experimental insects Materials and Methods The DBM adults were obtained either from a laboratory where they were reared on kale seedlings, or from field population on cabbage and kale during the pupal or late larval stages. The pupae were kept individually in a 4.5 cm x 1 cm (diam) vial

2 104 Chow, Lin, and Teng maintained at 25 ± 1 C in a 16:8 light-dark photoperiod incubator. Newly emerged presexed adults were maintained individually in the same incubator. A 5% sugar solution was provided as food to the adults. Morphology Hairpencils were exposed by squeezing the base of the male abdomen and removed with fine forceps. They were fixed by immersion in fixative (3% glutaraldehyde), and then transferred to amyl acetate to facilitate critical pointed drying. They were coated with a 50 nm film of gold in a Polaron Sputter Coater and examined and photographed using a Hitachi 450 Scanning Electron Microscope (Chow et al 1976). For light microscopic observation, the male abdomen tip was dissected out and fixed in Zenker s solution for two hours, dehydrated, and embedded in paraffin. Serial sections, 6-10 u thick, were cut and stained with Harris s hematoxylin as described previously (Chow et al 1976). Bioassay Male mating frequency per night One to three day-old adults were used. One male and five female moths were kept in each of the 14, 17 cm x 8 cm (diam) containers. All containers were placed overnight (about 14 h) in a dark room at 22 ± 1 C. The females were anatomized the following day to check for the presence of sphermatophores and the size of bursa copulatrix to judge the success of mating (Yang and Chow 1978, Fujiyoshi et al 1979). The antenna-excision method One half to four day-old adults were used. The insects were anesthetized by ether. Ten males with excised antennae were paired with 10 normal females, 10 females with excised antennae were paired with 10 normal males, and 10 pairs of adults that served as a control were kept in 17 cmx8 cm (diam) containers separately. All were kept in a dark room at 19 ± 3 C overnight. The presence of spermatophores and the size of bursa copulatrix were used to judge mating success on the following day (Yang and Chow 1978, Fujiyoshi et al 1979). The data were analyzed by Duncan s multiple range test. The hairpencil-excision method One half to four day old male moths were separated into four groups. Each group consisted of 19 pairs. In the first group, male moths were anesthetized by ether. Then with the help of forceps covered with a polyethylene tubing the tip of abdomen was pressed tenderly until the hairpencils came out. With another pair of forceps the hairpencils were pulled out. The second group of insects were treated similarly to the first except that the tip of hairpencil tube was sealed with glue to prevent the secretion of sex pheromone from the inside of the tube. This is termed the tube-sealing group. The third group of insects was treated in the same way as the first except that the hairpencils were not touched. The fourth group of insects were normal adult males. Each group of insects was maintained in a 17 cm x 8 cm (diam) container. A 5% sugar solution adsorbed on sponge was placed in each and the containers were covered with cheesecloth to allow ventilation. All four groups were kept in a dark room at 19 ± 2 C overnight. The following day, the insects were dissected to check the progress of mating by the size of bursa copulatrix and the presence of sphermatophores (Yang and Chow 1978, Fujiyoshi et al 1979). The data were analyzed by Duncan s multiple range test.

3 Male Sex Pheromone in DBM 105 Morphology Results and Discussion The photomicrograph of artificially extruded hairpencils and accompanying scent brush glands of the male DBM are shown in Figure 1 and the normal non-exposed hairpencils in Figure 2. Morphologically speaking, these hairpencils were identical to those of cabbage looper moth described by Gothilf and Shorey (1976) and of the flour moth described by Corbet and Fook (1977). In Figure 2, small droplets within the hairpencils are easily seen. It is suspected that they secrete the pheromone. Whether the droplets observed by us are identical to small vesicles in noctuid male moths observed by Clearwater (1975) still needs further study. Figure 1. a. A scanning electronmicrograph of the exposed hairpencils (arrow) of the male DBM showing the position on the abdomen, and the genital clasper (C) (100x). b. Enlarged view of the exposed hairpencils (500x). c. and d. Enlarged view of the exposed scent brush gland (3000x and 15000x) Bioassay Mating frequency A male moth mated with only one female per night. This was confirmed by the observation of the size of bursa copulatrix and the presence of spermatophores within bursa copulatrix in females.

4 106 Chow, Lin, and Teng Figure 2. a. A scanning electronmicrograph of the non- exposed hairpencils (arrow) and the infold scent brush gland. b. Photomicrograph of the hairpencils and their scent brush gland (100x). c. and d. Same as b, but with greater magnification (200x and 400x) showing the small droplets within the hairpencils The antenna-excision method The effect of antenna-excision on mating success is presented in Table 1. The average number of females with spermatophores in the control group was significantly greater than in antenna-excision groups. The number of moths with spermatophores in the female antenna-excision group was significantly greater than in the male excision group. Since electroantennograms (EAG) have been used extensively to identify the female or male sex pheromones (Grant et al 1972, Jacobson et al 1976, Chow et al 1980), the antenna is believed to be the only olfactory receptor of sex pheromone. It has also been established that female sex pheromone in many lepidopteran species, including DBM, plays an important role in mating success. In the present study, our results confirmed this. When the antennae of male moths were excised, the mating success decreased to almost nil. When the antennae of females were excised, the mating success decreased significantly but not as drastically as when antennae of males were excised. This indicates that a male sex pheromone was involved in mating success but it was not as important as the female sex pheromone. Table 1. The effect of antenna-excision on mating of DBM Treatment Control Female without antenna Male without antenna Mean number of females/10 with spermatophores 8.6a 5.8b 0.4c Means followed by the same letter are not significantly different at 5% level according to Duncan's multiple range test. The hairpencil-excision method The results of the effect of hairpencil-excision on mating success are presented in Table 2. The average number of females with spermatophores in the normal group was not significantly different from that in the control group. Both groups were significantly different from the hairpencil-excision

5 Male Sex Pheromone in DBM 107 group and the tube-sealing group. The tube-sealing group was not significantly different from the hairpencil-excision group. Table 2. Effect of hairpencil-excision and tube-sealing on mating of DBM Treatment Normal Control Without hairpencils Mean number of females/10 with spermatophores 8.18a 6.18a 3.36b Tube-sealing 3.09b Means followed by the same letter are not significantly different at 5% level according to Duncan's multiple range test. In this part of the study, when the hairpencils were excised, the average matings decreased considerably compared with the normal and control groups. This showed that the hairpencils of DBM do play a very important part in mating success. The success of mating of some pairs suggests that either the hairpencils were not pulled out completely or that other scent glands present in other parts of body, such as small scent glands and wing glands which are present in male cabbage looper (Grant 1971), are involved in mating. Further histological studies are necessary to define their involvement. Mating success of moths in the tube-sealing group did not differ significantly from those without hairpencils. This could be for the following two reasons. Firstly, the quantity of male sex pheromone did not reach the behavior response threshold. Naturally when the hairpencils are displayed before a female moth it secretes a large quantity of male sex pheromone. But when the hairpencils were pulled out, no such action has been involved. Therefore, there is no difference. Secondly, the glue probably cannot completely prevent the escape of volatile sex pheromone chemicals of the male moth. Both antenna-excision and hairpencil-excision methods proved the presence of a male sex pheromone in DBM. Further studies involving electroantennogram and isolation and identification of the pheromone structure are needed to reach an understanding of the vital function of hairpencils. Acknowledgement This study was supported by the National Science Council, Taiwan, ROC (NSC B001-27). Literature Cited Baker, T. C. and R. T. Carde Courtship behavior of the oriental fruit moth (Grapholitha molesta): experimental analysis and consideration of the role of sexual secretion in the evolution of courtship pheromones in the Lepidoptera. Ann. Entomol. Soc. Amer. 72: Baker, T. C., R. Nishii, and W. L. Roelofs Close-range attraction of female oriental fruit moths to herbal scent of male hairpencils. Sciene 214: Chow, Y. S., J. Chen, and S. H. Lin-Chow Anatomy of the female sex pheromone gland of the diamondback moth. Int. J. Insect Morphol. Embryol. 5: Chow, Y. S., M. S. Mayer, and J. H. Tumlinson Electroantennogram response of Plodia interpunctella to its sex pheromone and wing gland extracts. Bull. Inst. Zool., Acad. Sin. 19: Clearwater, J. R Chemistry and function of a pheromone produced by the male of the southern armyworm Pseudaletia separata. J. Insect Physiol. 18:

6 108 Chow, Lin, and Teng Clearwater, J. R Structure development and evolution of the male pheromone system in some noctuidae (Lepidoptera). J. Morphol. 146: Corbet, S. A. and J. L. Fook The hairpencils of the flour moth Ephestia kuehniella J. Zool. 181: Fujiyoshi, N., K. Miyashita, and K. Kawasaki Mating inhibition in the diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae) with its synthetic sex pheromone. Jpn. J. Appl. Entomol. Zool. 23: Grant, G. G Scent apparatus of the male cabbage looper, Trichophsia ni. Ann. Entomol. SOC. Amer. 64: Grant, G. G. and U. E. Brady Courtship behavior of Phycitid moths. I. Comparison of Plodia interpunctella and Cadra cautella and role of male scent glands. Can. J. Zool. 53: Grant, G. G., U. E. Brady, and J. M. Brand Male armyworm scent brush secretion: identification and electroantennogram study of major components. Ann. Entomol. SOC. Amer. 65: Gothilf, S. and H. H. Shorey Sex pheromone of lepidoptera: Examination of the role of male scent brushes in courtship behavior of Trichoplusia ni. Envirn. Entomol. 5: Hendricks, D. E. and T. N. Shaver Tabaco budworm: Male pheromone suppressed emission of sex pheromone by the female. Environ. Entomol. 4: Hirai, K., T. Okada, T. Kadosawa, and H. Santa Heterocera (moths) in eastern part of Hiroshima Pref. and their brush organs. Bull. Chugoku National Agricultural Experiment Station Series E (Environment division) No. 19: Jacobson, M., V. E. Adler, A. N. Kishaba, and E. Priesner phenylethanol, a presumed sexual stimulant produced by the male cabbage looper moth, Trichoplusia ni. Experientia 32: Nishida, R., T. C. Baker, and W. 0. Roelofs Hairpencil pheromone components of male oriental fruit moths, Grapholitha molesta. J. Chem. Ecol. 8: Rutowski, R. L Male scent-producing structures in Colias butterflies. J. Chem. Ecol. 6: Yang, L. C., and Y. C. Chow Spermatophore formation and the morphology of the reproductive system of the diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae). Bull. Inst. Zool. Acad. Sin. 17: