Sex Chromosomes in Stephanolepis Hispidus (Monacanthidae, Tetraodontiformes)

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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 2n33/34 (NF34), 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 heteromorphic 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 evolutionary 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 heterogamety 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 already 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 banding, 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°58S, 38°31W) and Rio de Janeiro (Angra dos Reis: 23°00S, 44°18W and Niteroi: 22°55S, 43°50W), Northeast and Southeast of Brazil, respectively. 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 restriction endonucleases, EcoRI and AluI, used for genome DNA digestion, were diluted in buffer solution 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 2n33, with 32 acrocentric chromosomes and one large unpaired submetacentric chromosome (Fig. 1a) and the females showed 2n34, all acrocentrics (FN34) (Fig. 1b). The individuals whose sex could not be defined presented 2n33 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. Bar5 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. Bar5 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 performed 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 chromosome study in lower organisms may serve as a base for a better understanding of the sex chromosome evolution in superior vertebrates (Trombka and Avtalion 1993). According to Pauls (1993), who analyzed a Stephanolepis hispidus population on the coast of

Rio de Janeiro, the species would present a multiple X1X1X2X2/X1X2Y system where the females have 34 acrocentric chromosomes, while the males have 2n33 chromosomes. Another species, S. cirrhifer, also showed the same kind of multiple sex system. In this species the males presented 33 chromosomes, one of which was a large unpaired metacentric chromosome, while the females had 2n34 (Murofushi et al. 1980). Some taxonomic groups share a same sex system in different degrees, such as all the representatives of the Triportheus (Characidae, Triportheinae) (Falcão, 1988, Bertollo and Cavallaro 1992, Sánchez and Jorge 1999, Artoni et al. 2001), or some species belonging to the Leporinus (Galetti et al. 2000) that have a ZZ/ZW sex system, indicating respectively in both cases a single origin.

The existence of X1X2Y multiple sex chromosomes among species of the Stephanolepis genus (Monacanthidae) due to the small number of species analyzed, may reﬂect either a sinapomorphic condition where this system would have arisen once before the division of the lines or that originat- ed in independent events by parallelism. Unlike in other sex systems, Stephanolepis hispidus karyotype does not have a large heterochromatin contents, which is limited to centromeric and pericentromeric regions. With this, it can be inferred that heterochromatinization processes were not associated in the origin and differentiation of the system. 2004 Sex Chromosomes in Stephanolepis hispidus 451

Stephanolepis hispidus has 1.25 pg DNA per cell, a value considered small compared with the other representatives of the Tetraodontiformes but a similar condition to the representatives of the Balistidae (Brainerd et al. 2001). This low amount of DNA is mainly evidenced from the small number of chromosomes in the species. This explanation also applies to the Balistidae species, such as Balistes vetula (2n44, FN44) and Melichthys niger (2n40, FN40) (Sá-Gabriel 2004) that, although they present on average a slightly larger genome than Monacanthidae, have either large chromosomes in their complements. The homologies among these two groups are not limited to the aspects of the karyotypic macrostructure but preferentially to similar tendencies in the chromosome evolution, ruled mainly by in tandem or centric fusions, modiﬁed sequentially or not by pericentric inversions.

Acknowledgements The authors especially thank to CAPES for the scholarship granted to LGSG and to Dr. Paulo Roberto Antunes de Mello Affonso for help in collecting samples on the coast of Rio de Janeiro.

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