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Polytene Chromosome Maps of the Melon Fly Bactrocera Cucurbitae (Diptera: Tephritidae)

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Polytene Chromosome Maps of the Melon Fly Bactrocera Cucurbitae (Diptera: Tephritidae) Color profile: Generic CMYK printer profile Composite Default screen 1167 Polytene chromosome maps of the melon fly Bactrocera cucurbitae (Diptera: Tephritidae) Reza M. Shahjahan and Farzana Yesmin Abstract: Standard photographic maps of the polytene chromosomes are presented for the melon fly Bactrocera cucurbitae, a serious pest of fleshy fruits and vegetables. Five larval salivary gland polytene chromosomes (10 polytene arms) were isolated, and their characteristic features and landmarks have been recognized. Banding patterns of each of the polytene arms are presented, where variation in band intensity and puffs appear to reflect fundamental differences in chromosomes. The whole polytene genome has been typically mapped by dividing it into 100 sections and the sub- sections were lettered. The mitotic chromosomes of larval brain ganglia are also examined, five pairs of autosomes and an XX/XY sex chromosome pair. In addition, a heterochromatic mass corresponding to the sex chromosomes are ob- served in the polytene nuclei of salivary gland tissue. This investigation showed that B. cucurbitae has excellent cyto- logical material for polytene chromosome analysis and proved to be very useful for obtaining more detailed genetic information on the pest’s natural populations. Key words: Bactrocera cucurbitae, salivary gland, banding patterns, polytene maps. Résumé : Les auteurs ont produit des cartes photographiques standard des chromosomes polytènes de la mouche du melon, Bactrocera cucurbitae, un insecte nuisible important dans les cultures fruitières et maraîchères. Cinq chromoso- mes polytènes (10 bras chromosomiques) des glandes salivaires chez des larves ont été isolés et leurs caractéristiques distinctives ont été décrites. Les bandes obtenues suite à des colorations sont présentées pour chaque bras et la varia- tion au niveau de l’intensité des bandes ou des boursouflures semble refléter des différences fondamentales entre les chromosomes. Tel que le veut l’usage, le génome polytène complet a été divisé en 100 sections et les sous-sections ont été identifiées à l’aide de lettres. Les chromosomes mitotiques des ganglions cérébraux chez les larves ont aussi été examinés : cinq paires d’autosomes et une paire de chromosomes sexuels XX/XY ont été observés. De plus, une masse hétérochromatique correspondant aux chromosomes sexuels est également observée au sein des noyaux polytènes des glandes salivaires. Cette étude montre que le B. cucurbitae constitue un excellent matériel cytologique pour l’analyse de chromosomes polytènes et s’avère très utile pour acquérir de plus amples informations génétiques sur les popula- tions naturelles d’insectes nuisibles. Mots clés : Bactrocera cucurbitae, glande salivaire, motifs de bandes, cartes de chromosomes polytènes. [Traduit par la Rédaction] Shahjahan and Yesmin 1174 Introduction fruit are produced in the country annually. Usual loss of fruits and vegetables to insects ranges from 10 to 15%. In The melon fly Bactrocera cucurbitae belongs to the fam- some cases, it causes a total loss of the crop (SYBB 1999). ily Tephritidae and is well known as a destructive pest of The status of Bactrocera spp. as insect pests has inspired re- fleshy fruits and vegetables. It is found in Africa, India, search for more than 50 years into adequate methods of con- southeast Asia, Thailand, the Ryukyu Islands of Japan, and trol aimed at the development and testing of genetic control the Pacific Islands including Hawaii. Females habitually de- methods. The development of improved sterile insect tech- posit their eggs in healthy fruits, and when the larvae emerge nique strategies will largely depend on an understanding of about 24 h later, they begin to feed and burrow into the pulp the genetics of the species (Zhao et al. 1998). Currently, ge- of the host, causing considerable damage (Shahjahan et al. 6 6 netic studies are concentrated on the construction of chro- 2000). About 4.5 × 10 t of vegetables and 1.5 × 10 tof mosome rearrangements, either as major components of the strains being developed for release (Foster et al. 1972, 1976; Received 2 January 2002. Accepted 22 August 2002. Whitten et al. 1977; Whitten 1979) or as tools to facilitate Published on the NRC Research Press Web site at genetic manipulations (Whitten and Foster 1975). For this http://genome.nrc.ca on 4 November 2002. purpose, it has been necessary to establish a solid foundation of cytogenetic data in this species. Corresponding Editor: A.J. Hilliker. Dipteran polytene chromosomes have proved to be favour- R. M. Shahjahan and F. Yesmin.1 Institute of Food and able for cytogenetic and genetic studies because of their con- Radiation Biology (IFRB), Atomic Energy Research stant characteristic banding pattern (Zhao et al. 1998). Establishment (AERE), G.P.O. Box 3787, Dhaka 1000, However, the recognition of polytene chromosomes can be Bangladesh. more difficult because of the presence of weak points that 1Corresponding author (e-mail: [email protected]). may break during slide preparation. Unfortunately, no quan- Genome 45: 1167–1174 (2002) DOI: 10.1139/G02-081 © 2002 NRC Canada I:\gen\gen4506\G02-081.vp Wednesday, October 30, 2002 8:26:58 AM Color profile: Generic CMYK printer profile Composite Default screen 1168 Genome Vol. 45, 2002 titative data are currently available to describe the polytene Fig. 1. Mitotic karyotype from a neural ganglion of a male larva complement of B. cucurbitae. Thus, it is not certain whether of Bactrocera cucurbitae after the air-drying technique. (A) all elements can in fact be reliably recognized. Scanned photograph; (B) diagrammatic representation. The In this paper, banding pattern and photographic maps of centromeres are indicated by constrictions of each chromosome. salivary gland polytene chromosomes of B. cucurbitae are Black areas represent heterochromatic portions. Chromosome presented. The mitotic karyotypes have also been analysed lengths, arm ratios, and heterochromatic portions are shown in to make correspondence between them and the polytene proportion. chromosomes. The results indicate that this species has ex- cellent cytological material for analysis of polytene chromo- somes. Materials and methods Fly stocks Cultures of B. cucurbitae were maintained in the labora- tory of the Pest Control and Management Division, Institute of Food and Radiation Biology, Atomic Energy Research Establishment, Savar, Dhaka, at a temperature of 28 ± 2°C with 70–80% relative humidity and a 12 h light : 12 h dark cycle. Adult flies were usually supplied with yeast, casein hydrolysate, and sugar (1:1:2, w/w) and water in soaked cot- ton or 1% agar gel cubes. Larvae were grown on sweet gourd. This population has been maintained in our labora- tory for more than 6 years. Mitotic chromosome preparation The preparations were made following the method de- scribed by Zacharopoulou (1987) and Willhoeft and Franz (1996). Brain tissue from third-instar larvae (5–6 days old) was used for mitotic chromosome spreads. After dissection of the tissue in Ringer’s solution (0.9% NaCl, 0.02% CaCl2, 0.02% KCl, 0.01% NaHCO3 (pH 6.9)), it was pretreated in 1% sodium citrate for 10–15 min and prefixed in methanol – acetic acid (3:1) for 1 min. The fixed ganglia were trans- ferred to a well slide containing a small drop of 60% acetic acid. The tissue was disrupted with forceps and then macer- ated, using a micropipette, by drawing it several times into the pipette, forming a cell suspension. After that, the mate- rial was laid on clean slides, placed on a worm hotplate (45– 50°C), and dried. Finally, the slides were stained in 5% Giemsa in 0.01 M phosphate buffer. mosomal region were used for the construction of the com- posite chromosome maps. These maps were scanned at 1200 Polytene chromosome preparation dpi with Adobe Photoshop 5.5 (Scanner, Hewlett Packard Third-instar larvae (5–6 days old) were used for the sali- 1125). Finally, the maps were printed on a Hewlett Packard vary gland polytene chromosome preparations following the 1175 printer. The polytene chromosomes of this species are method described by Zacharopoulou (1987) and Mavragani- difficult material for high-quality photographs, and as we do Tsipidou et al. (1992). Larvae were dissected in 45% glacial not have any trained microphotoprinting laboratories in Ban- acetic acid and the glands were fixed in 3 N HCl for 3– gladesh, we have been unable to provide photographs of op- 5 min. They were then transferred to a drop of lactoacetic timal quality. However, we have minimized the problem this acid (80% lactic acid – 60% acetic acid, 1:2) for about creates by providing diagrammatic sketches of the polytene 5 min. The glands were then stained in lactoacetic orcein for chromosomes. about 30–60 min. Excess stain was removed by washing the glands two or three times in a drop of lactoacetic acid before Results squashing. A phase-contrast microscope (Nikon Optiphot 2) was used to examine the polytene chromosome preparations Mitotic chromosomes using a 100× objective. The mitotic metaphase complement of B. cucurbitae con- sists of six pairs of somatically paired chromosomes, includ- Construction of photographic maps ing an XX/XY sex chromosome pair (Fig. 1). The labelling Well-spread chromosomes with clear banding patterns system followed in the figure was proposed by Radu et al. were photographed under 100× magnification using Fuji 100 (1975) and also by Mavragani-Tsipidou et al. (1992) for black and white film. Selected photographs from each chro- Dacus (Bactrocera) oleae. This system labels the sex pair as © 2002 NRC Canada I:\gen\gen4506\G02-081.vp Wednesday, October 30, 2002 8:27:04 AM Color profile: Generic CMYK printer profile Composite Default screen Shahjahan and Yesmin 1169 Fig. 2. Map of polytene chromosomes I and II in Bactrocera cucurbitae. L, left arm; R, right arm; O, centromere. the first and autosomes from 2 to 6 in order of descending size. This labelling is arbitrary and indicates only the rela- size.
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