longicaudata (Hymenoptera: Braconidae) in Thailand

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1 1999 The Japan Mendel Society Cytologia 64: , 1999 Metaphase Chromosomes of Parasitic Wasp, Diachasmimorpha longicaudata (Hymenoptera: Braconidae) in Thailand Sangvorn Kitthawee *, Siripong Singhapong and Visut Baimai Department of Biology, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, Thailand Accepted January 5, 1999 Summary Diachasmimorpha longicaudata (Hymenoptera: Braconidae) is a larval endoparasitoid of the fruit fly, Bactrocera correcta (Diptera: Tephritidae) that is important in biological control. We have analysed the metaphase chromosomes of this parasitoid using simple air-drying and squashed air-drying techniques. We examined the karyotypes of reproductive organs of pre-emerged adults and the cerebral ganglia of prepupae. Both show that females are diploid with a chromosome number of (2n)=40, whereas males are haploid with chromosome number of (n)=20. The haploid metaphase karyotype of D. longicaudata comprises 5 metacentric, 13 submetacentric and 2 subtelocentric chromosomes. We could not distinguish the sex chromosomes. These findings are the first record of the metaphase karyotype of this endoparasitoid. The parasitic wasp, Diachasmimorpha longicaudata (Ashmead) (Hymenoptera: Braconidae) is a solitary opiine parasitoid that attacks third instar larvae of tephritid fruit flies (Greany et al. 1976). It is an important endoparasitoid and a potential biological control agent for the Oriental fruit fly, Bactrocera dorsalis (Hendel) (Wong and Ramadan 1987) which is a major crop pest in Thailand and Southeast Asia. From our observations, D. longicaudata is also a natural enemy of B. correcta (Bezzi) which mainly attacks guava in Thailand (Hardy 1973, White and Elson-Harris 1992). In recent years, there have been attempts to suppress or eradicate B. dorsalis by using sterile insect technique (SIT) in northern Thailand (Sutantawong 1991). Such a control measure for B. dorsalis may subsequently lead to an increase in the population density of B. correcta which could become an economically important pest in the managed areas in the future. In such circumstances, D. longicaudata may be useful as a biological control agent for B. correcta in its distribution area. Although D. longicaudata is known to occur in some populations in Thailand, little work has been done on the systematics and genetics of this endoparasitoid despite its potential usefulness in biological control of the related species of fruit fly. Diachasmimorpha longicaudata has an extremely high reproductive potential due to the haplodiploid system which is typical of Hymenoptera. In general, females come from fertilized eggs and are diploid, whereas males develop from unfertilized eggs and are therefore haploid. Diachasmimorpha longicaudata may comprise a number of cryptic species because it shows morphological variations in different geographical populations (Fischer 1966, Wharton and Marsh 1978, Wharton and Gilstrap 1983). Thus it is necessary to undertake genetic investigations of D. longicaudata to elucidate its species status. Like most fruit fly parasitoids, D. longicaudata seems to be an unfavorable insect for cytogenetic study because of its small size, particularly at the larval and pupal stages. This makes it difficult to find cerebral ganglia, ovaries and testes in a suitable stage of its development. We present the first report on the metaphase karyotype of D. longicaudata. Techniques for metaphase chromosome preparations from prepupae and pre-emerged adults of D. longicaudata are described. Corresponding author. grskt@mahidol.ac.th.

2 112 Sangvorn Kitthawee, Siripong Singhapong and Visut Baimai Cytologia 64 Materials and methods Parasitoid colony Specimens of D. longicaudata were obtained from collections of fruit that had been parasitized by fruit flies in Nakornpathom Province, central Thailand. Diachasmimorpha longicaudata and its natural host, B. correcta, were maintained in an insectary at Department of Biology, Faculty of Science, Mahidol University, at about 27 Ž with 70 } 10% relative humidity and photoperiod of 12 h light and 12 h dark. A high humidity was maintained by placing damp cloth on the cages. Adults of D. longicaudata were kept in transparent plastic cages (12 ~ 33 ~ 18 cm) and provided with cotton wool soaked in 10% honey and cotton wool soaked in 10% multi-vitamin syrup. Honey (100%) was also streaked on the net cloth at the top of the cages to serve as a food source. The laboratory colony of D. longicaudata was maintained by placing adult females (mated and unmated) in a cage containing third instar larvae of B. correcta which served as the parasitoids' host for ovipositing and developing. The developmental process of this parasitoid from egg to adult required days. The suitable organs for chromosome study We analysed several organs at different stages of development to search for the best source of metaphase chromosomes. We found that the cerebral ganglia of prepupal stage of D. longicaudata provided good quality of metaphase chromosomes. Freshly removed ovaries or testes from the preemerged adults also gave good metaphase chromosomes. Preparations of metaphase chromosomes by air-drying technique Metaphase chromosomes of cerebral ganglia were prepared from prepupae of D. longicaudata using a modified method described by Baimai (1977). The prepupae were treated with 2% colchicine (Sigma) solution for 10 min at room temperature. The cerebral ganglia were then dissected in 1% sodium citrate hypotonic solution and left there for 10 min. The ganglia were transferred to a few drops of fixative (acetic acid : ethanol, 3 : 5) for 5 min then transferred to a drop of 70% acetic acid for 2-3 min. The ganglia were made into a cell suspension by gentle pumping with a pasteur pipette several times. The cell suspension was dropped onto a clean slide which had been pre-warmed to approximately 45 Ž. The air-dried chromosomes were kept for a few days before staining. Metaphase chromosomes from testes and ovaries of pre-emerged adults were prepared in a similar manner after they were pre-treated with demecolcine solution (Sigma) for 4 h in the refrigerator (5 Ž). Dried slides were stained with 8% Giemsa (A. J. P. Scientific Inc.) in phosphate buffer solution (1 part of 0.01 M sodium dihydrogen orthophosphate was adjusted with 3 parts of 0.01 M disodium hydrogen phosphate to ph 7.0 by adding 0.15 M sodium chloride or 0.03 M potassium chloride) for 1 h. Preparations of metaphase chromosomes by the squashed air-drying technique Metaphase chromosomes of D. longicaudata were also prepared from the testes and ovaries using a modified technique of squashed air-drying as described by Imai and Kubota (1972). The testes or ovaries of pre-emerged adults were dissected in a colchicine-hypotonic solution (0.01% colchicine in 0.50% sodium citrate solution) before they were transferred to demecolcine solution (Sigma) for 10 min. They were returned to a few drops of fresh colchicine-hypotonic solution and left for 30 min. The testes or ovaries were then transferred to a drop of fixative (1 : 3, glacial acetic acid : absolute ethanol) on a clean slide and covered with cover slip. The fixed tissues were then squashed gently. The squashed preparations were placed in the freezer (about - 20 Ž) for 2-3 min before flicking out the cover slip. The slide was then quickly rinsed with glacial acetic acid and air-

3 1999 Metaphase Chromosomes of Parasitic Wasp in Thailand 113 dried on a slide warmer plate at approximately 45 Ž. Metaphase chromosomes were stained with 2% lacto-aceto-orcein for 5 min. Chromosome classification Chromosome numbers were determined from observations of at least 100 well-spread metaphase plates. Metaphase chromosomes were selected and measured from 20 cells. Chromosomes were classified and analysed from photomicrographs based on the centromeric position as described by Levan et al. (1964). Results and discussion Both of air-drying and squashed air-drying modified techniques yielded satisfactory metaphase chromosome spreads. However, the air-drying method used for the cerebral ganglia gave a better quality of metaphase chromosomes than the squashed technique. Prepupal cerebral ganglia are very small and therefore difficult to handle. Hence, the alternative method of squashed air-drying of testes or ovaries is more accessible from a practical point of view. Figs Photomicrographs of metaphase chromosome of Diachasmimorpha longicaudata. Diploid chromosome numbers 2n=40: 1) cerebral ganglia, 2) ovary. Haploid chromosome numbers n=20: 3) cerebral ganglia, 4) testes. Figs. 1-3, chromosomes were prepared by air-drying technique and Fig. 4 by squashed air-drying technique.

4 114 Sangvorn Kitthawee, Siripong Singhapong and Visut Baimai Cytologia 64 Fig. 5. Karyogram of the haploid chromosome of D. longicaudata arranged from the largest no. 1 to the smallest no. 20 prepared from cerebral ganglia using the air-drying method. Fig. 6. Diagrammatic representation of haploid idiogram of D. longicaudata. Analysis of metaphase karyotypes obtained from cerebral ganglia, testes and ovaries of D. longicaudata has revealed 2n=40 in diploid females (Figs. 1, 2) and n=20 in haploid males (Figs. 3, 4). The metaphase chromosomes were numbered in order of size with 1 being the largest and 20 being the smallest (Figs. 5, 6). According chromosomal classification by Levan et al. (1964), these chromosomes have 2 subtelocentric (nos. 4, 9), 13 submetacentric (nos. 1-3, nos. 5-8, nos ) and 5 metacentric (nos ). It was not possible to distinguish the sex chromosomes in this study. There have been few reports of metaphase karyotypes and chromosome numbers from other species of Braconidae. Makino (1951) listed chromosome numbers of some species of parasitoid studied by various authors: Apanteles glomeratus, a larval parasitoid of lepidopteran cabbageworm (Pieris rapae) has a haploid chromosome number of n=12, Bracon brevicornis, B. hebetor (Crozier 1975), and B. pectinophorae which are larval parasitoids of the lepidopteran pink bollworm, sugarcane borer and stalk borer all have haploid chromosome numbers of n=10. Surprisingly, there was no list of chromosome numbers of fruit fly parasitoids mentioned. Thus, cytological study of the fruit fly parasitoids is lacking in spite of their potential use as natural enemy of the important insect pests of the genus Bactrocera (Clancy 1950), partly due to their small size and difficulty of handling the materials. Our attempt to develop cytological techniques provides the first record of metaphase karyotypes of D. longicaudata showing a large number of chromosomes (n=20) compared with other known species of the family Braconidae. This basic knowledge on the metaphase karyotypes may be a useful tool in the cytotaxonomic study of this interesting group of fruit fly parasitoids. Further study on the population cytogenetics of D. longicaudata may shed some light on the species status and karyotypic evolution of this important fruit fly parasitoid in Thailand and Southeast Asia. Acknowledgements We wish to express our appreciation to Dr. R. A. Wharton for identification of the specimens and to Dr. R. A. Sharpe for reading the manuscript. This work was supported by the Thailand Research Fund grants RSA and RTA and the TRF/BIOTEC Special Programme for Biodiversity Research and Training grant BRT

5 1999 Metaphase Chromosomes of Parasitic Wasp in Thailand 115 Baimai, V Chromosomal polymorphism of constitutive heterochromatin and inversions in Drosophila. Genetics 85: Clancy, D. W Notes on parasites of tephritid flies. Proc. Hawaii. Entomol. Soc. 14: Crozier, R. H Hymenoptera. In: John, B. (ed.) Animal Cytogenetics. Vol. 3. Insecta 7. Gebriider Bortraeger, Berlin and Stuttgart. Fischer, M Revision der Indo-Australischen Opiinae. W Junk, The Hague. Greany, P D., Ashley, T. R., Baranowski, R. M. and Chambers, D. L Rearing and life history studies on Biosteres longicaudatus (Hym: Braconidae). Entomophaga 21: Hardy, D. E The fruit flies (Tephritidae-Diptera) of Thailand and bordering countries. Pac. Insects Monogr. 31: Imai, H. T. and Kubota, M Karyological studies of Japanese ants (Hymenoptera, Formicidae). III. Karyotypes of nine species in Ponerinae, Formicinae, and Myrmicinae. Chromosoma 37: Levan, A., Fredga, K. and Sandberg, A. A Nomenclature for centromeric position on chromosomes. Hereditas 52: Makino, S An Atlas of the Chromosome Numbers in Animals. Iowa State Univ. Press, Ames. Sutantawong, M Fruit Flies and Their Control by the Sterile Insect Technique in Thailand. In: Kawasaki, K., Iwahashi, O. and Kaneshiro, K. Y. (eds.) Proceeding of the International Symposium on the Biology and Control of Fruit Flies, Ginowan, Okinawa, Japan. Wharton, R. A. and Gilstrap, F. E Key to and status of opiine braconid (Hymenoptera) parasitoids used in biological control of Ceratitis and Dacus s.l. (Diptera: Tephritidae). Ann. Entomol. Soc. Am. 76: Marsh, P. M New World Opiinae (Hymenoptera: Braconidae) parasitic on Tephritidae (Diptera). J. Wash. Acad. Sci. 68: White, I. M. and Elson-Harris, M. M Fruit Flies of Economic Significance: Their Identification and Bionomics. Centre for Agriculture and Biosciences International, Wallingford Oxon, UK. Wong, T. T. Y. and Ramadan, M. M Parasitization of the Mediterranean and Oriental fruit flies (Diptera: Tephritidae) in Kula area of Maui, Hawaii. J. Econ. Entomol. 80: