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)

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 subsections 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 observed in the polytene nuclei of salivary gland tissue. This investigation showed that B. cucurbitae has excellent cytological 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 chromosomes 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 variation 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 populations 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-

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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 excellent cytological material for analysis of polytene chromosomes.

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 described 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 transferred 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 material 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 composite 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 consists 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

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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. In agreement with the findings of Mavragani-Tsipidou tive size of chromosomes. Therefore, this labelling is not et al. (1992), all five autosomes are more or less based on that of earlier studies (Singh and Gupta 1984) and submetacentric. Autosomes 4, 5, and 6 appear to be smaller it does not imply any correlation with the mitotic chromo- and nearly equal in length, while autosomes 2 and 3 are somes. For locating the site of the centromere, two criteria larger and show little differences in their size. The sex chro- were used, reported by Zacharopoulou (1987): (i) in unbro- mosomes are consisting of a dot-like Y chromosome and a ken chromosomes, the centromere presented a point of dis- small submetacentric X chromosome. continuity (as a form of constriction) and (ii) in broken chromosomes, the centromere was the frequent point of Polytene chromosomes fragmentation. According to the position of the centromere, Because of differential stretching and printing procedure, the two arms in each chromosome are of unequal length. In the proportional length of the chromosomes is not accurate each polytene chromosome, the longer arm was designated and some sections appear thinner and more obscure than as the left arm and the shorter as the right arm. The photo- others. The polytene chromosome maps are shown in graphic maps were analysed using the conventional method Figs. 2–5, and they were numbered I–V according to their of Bedo (1977, 1987) and Mavragani-Tsipidou et al. (1992).

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Fig. 3. Map of polytene chromosomes III and IV in Bactrocera cucurbitae. L, left arm; R, right arm; O, centromere.

In this system, the total polytene chromosome complement of faint bands are present at 14 and 15. The left end of this is assigned 100 sections and the chromosomes allocated a chromosome has a broad tip at 1A. The right arm is charac- number of sections and section limits marked along the terized by a group of prominent dark broad bands in 17B, chromosome using, wherever possible, prominent or distinc- 18, and 19. The tip of right arm (at 22) usually has a slightly tive banding features as delimiters. After the section limits triangular shape. were assigned, subsections were marked, again using the more prominent features available within it. Chromosome II (sections 23–44, Figs. 2 and 5A) This polytene element is recognized by the presence of Chromosome I (sections 1–22, Figs. 2 and 5A) two conspicuous puffs, in the left arm at 33 and in the right Chromosome I is divided into 22 sections. Sections 1–15 arm at 39 followed by three clear bands. Some regions of and 16–22 are assigned to the left and right arm, respec- this chromosome have characteristic dark bands at 27, 28, tively. The left arm is characterized by six heavy bands at 2 29, 31, and 32. There are two clear bands at 35 close to the and 3 and a puff in 8. There is a weak point at 13, which centromere. The right arm has a broad band in 44 and a dot- usually breaks during slide preparation. In addition, a group ted band in 40C.

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Chromosome III (sections 45–66, Figs. 3 and 5A) Fig. 4. Map of polytene chromosome V in Bactrocera The III L arm is recognized by a swelling in 45 with one cucurbitae. L, left arm; R, right arm; O, centromere. heavily stained dark band in section 46B. Region 51 con- tains a pair of dotted bands in 51A. Two prominent puffs are present in the 49 and 57. The end point of this arm is recognized by the presence of a heavily stained thick band in 66C. There is a different type of band structure at 59, characteriz- ing only chromosome III.

Chromosome IV (sections 67–88, Figs. 3 and 5B) There are three successive dark bands in 67. The more important diagnostic landmark is the region 79 near the centromere. There is a puff in 70A. The right arm is identified by its rounded tip (80). A weak point in 82 is characteristic of this arm. The IV R arm is easily recognized by the presence of a number of thick bands in 88.

Chromosome V (sections 89–100, Figs. 4 and 5B) Regions 93A and 93B present the most prominent landmarks of the V L arm. The V R arm has two light bands followed by a dark band (at 95A). This polytene element has two characteristic puffs at 97B and 98 and two heavy bands in 100. In all of the polytene nuclei examined, a heterochromatic structure is observed (Fig. 6). This structure differs in size among different animals and different nuclei, depending on the degree of polytenization of each nucleus.

Discussion

Mitotic chromosomes Bactrocera cucurbitae has the same number of mitotic chromosomes as Ceratitis capitata (Bedo 1987; Zacharopoulou 1987, 1990) and as Dacus (Bactrocera) oleae (Mavragani-Tsipidou et al. 1992), both species belong- ing to the same family as Bactrocera (Tephritidae). Both sex chromosomes seem to be largely heterochromatic. Singh and Gupta (1984) reported three pairs of metacentric (including the X chromosomes) and three pairs of submetacentric chromosomes; the Y chromosome was dot-like. Gopalan (1972) showed that the mitotic karyotype consists of three pairs of metacentric and two pairs of acrocentric autosomes and a slightly heteromorphic acrocentric pair (sex chromosomes), while Bhatnagar et al. (1980) reported again a different karyotype for Dacus (Bactrocera) cucurbitae, with one pair of submetacentric and five pairs of metacentric chromosomes including the X chromosomes, the Y chromosome being dot-like. However, our results are in good agreement with the mitotic karyotype given by Singh and Gupta (1984) larger, and most of it stains densely, indicating that it, like for D. cucurbitae and Mavragani-Tsipidou et al. (1992) for the Y chromosome, may be mainly heterochromatic. In D. oleae. B. cucurbitae, the rest of the long arm of the X chromosome Hunwattanakul and Baimai (1994) and Baimai et al. also stains more lightly and also exhibits (like B. oleae)a (1995, 1996) reported similar mitotic karyotypes (2n = 12) chromatid separation during metaphase, which differs from in different species of Bactrocera dorsalis complex and five that of the autosomes. Recently, Zhao et al. (1998) reported species of the genus Bactrocera (B. diversa, B. latifrons, six pairs of somatically paired mitotic chromosomes includ- B. modica, B. rubella, and B. scutellaris), consisting of one ing an XX/XY sex chromosome pair for Bactrocera tryoni. pair of sex chromosomes and five pairs of autosomes. They showed the three largest metacentric or submetacentric By Giemsa staining, the distribution of heterochromatin is autosomes and the two smallest acrocentric pairs and that accumulated to the centromeric area of all autosomes. But as the sex chromosomes are metacentric. In the present study, reported by Mavragani-Tsipidou et al. (1992), in the sex we did not observe any acrocentric chromosomes. The chromosomes, the Y chromosome obtains a deep dark dot- B. tryoni X chromosome was larger and densely stained, in- like colour, whereas the X chromosome is considerably dicating mainly heterochromatin. This is similar to the situa-

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Fig. 5. (A) Diagrammatic sketch of polytene chromosomes (A) I, II, and III and (B) IV and V of Bactrocera cucurbitae, L, left arm; R, right arm; O, centromere.

tion for the X chromosome of B. cucurbitae (shown in described in the larval salivary glands. However, these were Fig. 1). drawings of polytene maps and do not facilitate cytogenetic analysis and are also difficult for further analyses. In this pa- Polytene chromosomes per, a detailed map of the larval salivary gland polytene Previous reports on the polytene map of Dacus chromosomes is presented. Most of the cytogenetists, work- (Bactrocera) cucurbitae (Singh and Gupta 1984) have been ing for several important pest species such as C. capitata,

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Fig. 6. Heterochromatic mass in the potytene nuclei of Bactrocera cucurbitae.

Lucilia cuprina, D. oleae, and B. tryoni, reported that the identifiable by their diagnostic landmarks as well as their salivary gland chromosomes were very difficult to spread characteristic tips as described in the Results section. The because of extensive ectopic pairing. In most cases where problem of the correct nomenclature of the polytene chro- the chromosomes spread, they were broken and it was diffi- mosomes in B. cucurbitae still remains because correlations cult to distinguish each chromosome as a separate entity. We with the mitotic karyotype are necessary to establish chro- also observed such difficulties for B. cucurbitae. mosome identity. The numbering of the polytene chromo- In this species, each polytene nucleus had five pairs of somes (I–V) does not imply any correlation with the mitotic long chromosomes (10 polytene arms). There are no differ- karyotype. Such a correlation could only be achieved by ences between preparations from males and females, indicat- studies of genetically characterized translocated chromo- ing that the sex chromosomes are not polytenized. Thus, the somes (Mavragani-Tsipidou et al. 1992). Presently, such five polytene chromosmes must correspond to the five studies are imposible because of the absence of different autosomes. This is also the case in C. capitata and characteristic marker strains of B. cucurbitae and by the al- in L. cuprina (Bedo 1980, 1987; Foster et al. 1980; most total lack of basic genetic knowledge about the species. Zacharopoulou 1987, 1990). The significance of studying the polytene chromosome In this study, within the polytene nuclei of B. cucurbitae, complement of B. cucurbitae, which causes serious damage no banded sex chromosomes were identified. These chromo- to the fleshy fruits and vegetables, is evident. We have tried somes were represented by a heterochromatic structure in this paper to determine the chromosomal characteristics (Fig. 6). A heterochromatic structure, more or less similar to of this species so that these data can be used as reference that of B. cucurbitae, corresponding to underreplicating sex material for further cytogenetic analysis. With the success in chromosomes, was also identified in polytene tissues of finding good cytological material in B. cucurbitae, genetic C. capitata, L. cuprina, and B. oleae (Childress 1969; Bedo studies can now proceed with excellent cytological support. 1982, 1987; Bedo and Zacharopoulou 1988; Zambetaki et al. This work has revealed the fact that this species has a readily 1995). Also, as reported by Rudkin (1972), the workable cytological system of basic interest, which should heterochromatic chromosomes are not polytenized or provide interesting results in the future. underreplicated in polytene nuclei. Banded sex chromosomes have not been observed in polytene nuclei of several Acknowledgements other Diptera, e.g., Calliphora and Sarcophaga (Whitten 1968). Of course, further studies are necessary for this phe- This work was supported by BAEC/IAEA research con- nomenon to be clarified. Any sex chromosomes or sex- tract No. 8864/RB. Encouragement and support from linked segments could not be detected by Hadi et al. (1995). J. Hendrichs, A. Robinson, and G. Franz from IAEA are cor- In this species, we found five long polytene chromosomes dially acknowledged. Suggestions from A. Zacharopoulou, M. and designated them I–V as is commonly done. These are Tsipidou and M. Frommer thankfully acknowledged. Sincere

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