Sex Chromosomes in Stephanolepis Hispidus (Monacanthidae, Tetraodontiformes)
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© 2004 The Japan Mendel Society Cytologia 69(4): 447–452, 2004 Sex Chromosomes in Stephanolepis hispidus (Monacanthidae, Tetraodontiformes) Luiz Gustavo Sá-Gabriel* and Wagner Franco Molina Departamento de Genética e Biologia Celular, Universidade Federal do Rio Grande do Norte, Campus Universitario, s/n, Lagoa Nova, CEP 59078-970, Natal, RN, Brazil Received August 23, 2004; accepted September 21, 2004 Summary Several systems of sex chromosomes were described in fishes. Some of them are char- acterized by male heterogamety (XY/XX, X1X1X2X2/X1X2Y, XX/XY1Y2), others by female heterogamety (ZZ/ZW, ZZ/ZW1W2). In Tetraodontiformes, few multiple sex systems were reported. In the present work, specimens of Stephanolepis hispidus collected along the Bahia and Rio de Janeiro shore were cytogenetically analyzed. All individuals presented a diploid number of 2nϭ33/34 (NFϭ34), with 32 acrocentric chromosomes and a large submetacentric unpaired element in males and 34 acrocentric in females. An odder diploid number and the presence of 3 heteromor- phic unpaired chromosome observed in the males, were characterized as a multiple sex chromosomal system of the type X1X1X2X2/X1X2Y. Ag-NOR marks were identified on a single chromosomal pair at interstitial position. Heterochromatic blocks were visualized at pericentromeric and centromeric regions. The presence of large chromosomes and a reduced diploid number indicates a chromosomal evolutionary pattern determined by centric fusions, similar to that observed in Balistidae species. Key words Sex Chromosome, Stephanolepis hispidus. Sexual differentiation in fish is extremely flexible and favors the establishment of different evo- lutionary strategies in this group (Molina 1995). Cytogenetic analyses in phyllogenetically diverse groups suggest that sex chromosomes functionally differentiated, but cytologically criptic, are a general characteristic in the Teleostei karyotype (Wiberg 1983, Ewulonu et al. 1985). Much is already known about sexual determination in fish but there is still much to be eluci- dated. The data provided by classic studies with sex-linked genes (for revision see Yamamoto 1969, Ohno 1974, Kalmann 1984, Trombka and Avtalion 1993), gains a new dimension with the advent of the more sophisticated cytogenetic and molecular techniques. These expedients for chromosome structural mapping have given a new emphasis to information obtained by the methodologies used in the first studies on the subject. The fact that species are found within a same family showing het- erogamety in the two sexes (Andreata et al. 1993), seems to indicate that the sex determination is not as rigid as in other animal groups, and may be interchangeable during the evolutional history of the species, as Olmo et al. (1987) proposed for lizards. Several sex chromosome systems have al- ready been described in fish. Some show male heterogamety (XY/XX, X1X1X2X2/X1X2Y, XX/XY1Y2) (Bertollo et al. 1983), others, female (ZZ/ZW and ZZ/ZW1W2) (Molina et al. 1998, Moreira-Filho et al. 1993). Although there are a large number of marine fishes, the presence of sex chromosomes seems to be a rare event, and to date sex systems XX/YY, ZZ/ZW (Galetti et al. 2000), and X1X1X2X2/X1X2Y (Ueno et al. 2001) have been identified. Sex chromosome systems have been described for 7 Tetraodontiformes species to date (Table 1). The Tetraodontiformes is represented by about 428 species, of which 107 belong to the Monacanthidae (Nelson 1994). Their representatives occur more commonly in rocky depths and coral reefs. The larger sized species are used commercially (Santos 1992). Stephanolepis hispidus is * Corresponding author, e-mail: [email protected] 448 Luiz Gustavo Sá-Gabriel et al. Cytologia 69(4) Table1.Review of sex chromosomes systems in Tetraodontiformes species (adapted from Devlin and Nagahama, 2002) 2n Families Species sexual Systems References /? Triacanthidae Triacanthus brevirostris 48 47 XX XO Choudhury et al. 1982 Balistidae Rhinecanthus verrucosus 44 44 XX XY Ojima 1985 Rhinecanthus aculeatus 44 44 XX XY Ojima 1985 Odonus niger 42 42 XX XY Ojima 1985 Stephanolepis hispidus 34 33 X1X1X2X2 X1X2YPauls 1993 Stephanolepis cirrhifer 34 33 X1X1X2X2 X1X2Y Murofushi et al. 1980 Tetraodontidae Arothron nigropunctatus 38 37 X1X1X2X2 X1X2Y Ojima 1985 widely distributed in the Western Atlantic, and is found from New Scotia to Uruguay, and frequent- ly all along the Brazilian coast (Figueiredo and Menezes 2000). The objective of this study was to analyze cytogenetically, by chromosome banding (C band- ing, Ag-RONs, EcoRI and AluI restriction enzyme patterns) Stephanolepis hispidus (Monacanthi- dae) with special attention to the characterization of its sex chromosomes. Materials and methods S. hispidus specimens (three males, two females and four individuals without defined sex) were collected on the coast of Bahia (12°58ЈS, 38°31ЈW) and Rio de Janeiro (Angra dos Reis: 23°00ЈS, 44°18ЈW and Niteroi: 22°55ЈS, 43°50ЈW), Northeast and Southeast of Brazil, respective- ly. The individuals were submitted to mitotic stimulation by intraperitoneal injection of glucose yeast solution (Lee and Elder 1980), before the procedures to obtain mitotic chromosomes by the in vitro method (Gold et al. 1990). The sex was established by macro- and microscopic gonad obser- vation. Chromosome preparations were performed from kidney tissue, dissociated in 9.5 ml RPMI 1640 medium with 0.2 ml colchicine for 30 min, followed by hypotonization with KCl 0.075 for 25 min at ambient temperature. The materials were fixed with methanol/acetic acid (3 : 1) and stained with 5% Giemsa solution. The NORs sites were detected using the technique by Howell and Black (1980) and the distribution of heterochromatic regions according Sumner (1972). The restric- tion endonucleases, EcoRI and AluI, used for genome DNA digestion, were diluted in buffer solu- tion according to the manufacturer’s (Amersham Pharmacia) recommendations, using a final con- centration/time ratio of 0.5 U/ml/10 h and 0.3 U/m/4 h, respectively. A volume of 40 ml was added to each previously prepared slide and incubated in a moist chamber at 37°C. The chromosomes were classified according to the pattern established by Levan et al. (1964). Results Two diploid modal values were observed in S. hispidus regarding sex. The males showed 2nϭ33, with 32 acrocentric chromosomes and one large unpaired submetacentric chromosome (Fig. 1a) and the females showed 2nϭ34, all acrocentrics (FNϭ34) (Fig. 1b). The individuals whose sex could not be defined presented 2nϭ33 with karyotypic, structural and numerical pattern similar to the males and were considered as belonging to this sex. Ag-NORs sites were localized in pericentromeric position on the longest arm of the 2nd chromosome pair, based on the comparison of its size with the other chromosomes of the complement (Fig. 1a). The Y chromosome, a large submetacentric, presented few herochromatin that was restricted to the centromeric and telomeric regions. The X1 chromosomes, 5th pair and X2 18th pair, were identified by inference, because they did not present specific patterns that enabled their differentia- 2004 Sex Chromosomes in Stephanolepis hispidus 449 Fig. 1. a) Karyotype of male S. hispidus. In the large box the sex chromosomes. In order, in the small- er box, 2nd pair, carrier of the nucleolar organizer regions. b) Karyotype of female S. hispidus. Barϭ5 mm. tion from the others. The X1 chromosome presented a size equivalent to the 5th largest pair of the karyotype, while X2 showed an inferior size to the smallest autosome pair. Heterochromatic blocks were identified in centromeric and pericentromeric regions (Fig. 2). Banding obtained by RE AluI revealed a pattern similar to C banding, with some intermediate sized chromosomes showing more evident heterochromatic blocks. The treatment with EcoRI restriction endonuclease did not reveal any apparent bands. Discussion The numerous fish group shows a wide variety of colors, shapes and a very flexible pattern of sex determination. This can be shown in their different evolutionary strategies in sex differentiation such as synchronic hermaphroditism, protandria, protoginia and gonocorism. A general characteris- 450 Luiz Gustavo Sá-Gabriel et al. Cytologia 69(4) Fig. 2. Heterochromatin pattern found in the male Stephanolepis hispidus. The sex chromosomes and banding patterns of Y chromosome, by Giemsa, C banding and by the use of EcoRI are highlighted. Barϭ5 mm. tic in the teleosts karyotype is the presence of functionally differentiated sex chromosomes, but cytogenetically cryptic (Molina 1995), as shown in the numerous cytogenetic analyses already per- formed on this group. The multiple sex systems derive from the simple systems, through translocations. In the X1X1X2X2/X1X2Y sex system the diploid value of the males presented one chromosome more than the females. The origin of this system may be linked to the existence of centric fusions (Robertson- ian rearrangements). This type of sex system has been observed in different marine groups, Monacanthidae (Murofushi et al. 1980, Pauls 1993), Ophichthidae (Murofushi and Yosida 1984), Blenniidae (Carbone et al. 1987), Clupeidae (Brum et al. 1992) and five species of the Chan- nichthyidae (Morescalchi et al. 1992), teleosts endemic on the Antarctic coasts (among others). Besides the practical importance of understanding sex determination mechanisms, sex chro- mosome study in lower organisms may serve as a base for a better understanding of the sex chro- mosome evolution in superior vertebrates (Trombka