Cytogenetic characterization of distinct B chromosomes in a population of the fish Astyanax bockmanni (Teleostei, Characiformes)

Abstract

Individuals of Astyanax bockmanni from one population belonging to the Paranapanema River basin, southeastern Brazil, were cytogenetically examined. In the present paper, we report a diploid number of 50 chromosomes, with some individuals bearing additional chromosomes, characterized as different variants of B chromosomes. The B microchromosome of acrocentric morphology (B_a) was the most frequent type in this population (77.7%). Two other variants were identified as metacentric B chromosomes, but of distinct sizes, characterized as macrochromosome (B_M) and microchromosome (B_m). Conspicuous heterochromatic blocks were observed in the terminal regions of some chromosomes of the standard karyotype and partially heterochromatic blocks were detected in all B chromosomes. 18S ribosomal RNA genes were distributed in the terminal region of several chromosomes, but were not located on the B chromosomes.

Keywords:

• chromosome polymorphism
• fish cytogenetic
• in situ hybridization
• isochromosome
• supernumerary chromosomes

Introduction

Astyanax comprises more than 100 valid species and is one of the most specious incertae sedis genera recognized within the family Characidae (Marinho and Lima 2009). Several species of the genus Astyanax have already been genetically studied, mainly with a cytogenetic focus. Astyanax species show a high level of chromosomal polymorphism, with a great variety of diploid and triploid karyotypes, polymorphism of heterochromatin, nucleolus organizer region (NOR) diversity, and location of histone and ribosomal genes (Moreira-Filho and Bertollo 1991; Oliveira et al. 2009; Hashimoto et al. 2011; Machado et al. 2012).

Moreover, Astyanax species show a high variation of B chromosomes, which is the core issue of several studies in this fish group. B chromosomes are characterized by great diversity in Astyanax scabripinnis, with variations in size, morphology, DNA composition, and frequency, which can be related to altitude, sex, or environmental pollution (Porto-Foresti et al. 1997; Mestriner et al. 2000; Néo et al. 2000). Several approaches have been undertaken to make sense of the complex and variable nature of A. scabripinnis, which is characterized as a “species complex” (Moreira-Filho and Bertollo 1991), and is currently composed of 15 valid species (Bertaco and Lucena 2006).

Some studies have shown a stable diploid number of 50 chromosomes for individuals of Astyanax bockmanni. However, some populations of A. bockmanni can demonstrate a high inter- and intra-individual variability of the NOR phenotypes, as well as an extensive polymorphism in the heterochromatin distribution (Kavalco et al. 2009; Fernandes et al. 2010; Hashimoto and Porto-Foresti 2010).

The present study reports the cytogenetic characterization of A. bockmanni individuals bearing B chromosome variants, which yielded data for the inference of the possible origin of these extra chromosomes in the genome of A. bockmanni.

Materials and methods

Cytogenetic analyses were performed on chromosome preparations obtained from 46 individuals (22 females and 24 males) of A. bockmanni belonging to the population from the Alambari River (22°27′ 6.12′′ S, 49°14′ 25.76′′ W), Paranapanema River basin, southeastern Brazil. The fishes were identified according to Vari and Castro (2007), and the voucher specimens were stored in the fish collection of the Laboratório de Genética de Peixes, UNESP, Bauru, SP, Brazil. Chromosome preparations were obtained from gill and kidney tissues using the technique described by Foresti et al. (1981). Silver staining followed the technique of Howell and Black (1980), and C-banding was performed according to Sumner (1972).

Fluorescent in situ hybridization (FISH) was performed using the method described by Yang et al. (1999). Sequences of the 18S ribosomal DNA (rDNA) were isolated according to Hatanaka and Galetti (2004). For the preparation of the probes, PCR products of 18S rDNA were labeled during the secondary PCR by incorporating the nucleotide tetramethyl-rhodamine-5-dUTP (Roche).Chromosomes were counterstained with DAPI(4′,6-diamidino-2-phenylindole, Vector Laboratories). FISH images were captured and processed using the CytoVision Genus system (Applied Imaging, USA) and a Cohu CCD camera mounted on an Olympus BX-60 microscope.

Results and discussion

Our results showed a diploid number equal to 50 chromosomes and a karyotype formula composed of 8m + 8sm + 18st + 16a (fundamental number = 84), in both sexes (Figure 1). These data corroborate previous information about the diploid chromosome number and karyotype characteristics reported by Hashimoto et al. (2011). In contrast to the species complexes Astyanax fasciatus and Astyanax scabripinnis, which show variable diploid chromosome numbers in different individuals living in sympatry (Moreira-Filho and Bertollo 1991; Artoni, Shibatta et al. 2006, Machado et al. 2012), cytogenetic studies in some populations of A. bockmanni demonstrated a stable 2n (Kavalco et al. 2009; Fernandes et al. 2010; Hashimoto and Porto-Foresti 2010).

Figure 1 Giemsa-stained karyotype showing 2n = 50 chromosomes of one individual of Astyanax bockmanni from the Alambari River, southeastern Brazil.

Our data indicate the occurrence of nine individuals bearing extra chromosomes, characterized as B chromosomes, which demonstrated variations in morphology, size, and frequency. Three different B chromosomes were observed (Table 1 and Figure 2). The most frequent B chromosome variant is an acrocentric chromosome (B_a) of small size, similar to the smallest chromosomes belonging to the complement A (Table 1). Two other B chromosome variants were classified as metacentric, but showed distinct sizes, i.e. a macrochromosome (B_M) similar in size to chromosome pair 1 and a microchromosome (B_m) similar in size to B_a (Table 1 and Figure 2).

Table 1. Relation of different individuals bearing B chromosome variants in Astyanax bockmanni from the population of the Alambari River, southeastern Brazil.

Specimen identification	Ba chromosome	Bm chromosome	BM chromosome	Frequency of B chromosomes in metaphases analyzed^∗
♂ 2030	1	—	—	73.5%
♂ 2028	—	—	1	55.2%
♀ 2034	1	—	1	47.9%
♂ 2763	1	1	—	71.2%
♀ 2762	1	—	—	75.8%
♂ 3237	1	—	—	75.0%
♂ 3238	2	—	—	77.3%
♀ 3597	—	1	—	82.7%
♂ 4145	2	—	—	84.8%
^∗Average number of 30 metaphases analyzed.

Figure 2 Metaphases from different specimens bearing B chromosome variants in Astyanax bockmanni. (a) Individual bearing the B_a chromosome; (b) specimen carrying the B_m chromosome; and (c) individual bearing the B_M chromosome. (¹) Metaphases by conventional Giemsa staining; and (²) metaphases after C-banding technique. Arrows indicate the B chromosomes.

Distinct B chromosomes have been described in several species of the genus Astyanax, and the most frequent variant is the metacentric macrochromosome (B_M) similar to chromosome pair 1, which might be considered the ancestral B chromosome with probably an early origin preceding the species differentiation (Moreira-Filho et al. 2001; Vicari et al. 2011; Machado et al. 2012).

Other variants represent rare occurrences, such as a submetacentric similar in size to the B_M, a medium metacentric and an acrocentric smaller than the B_M, and B microchromosomes (for a review, see Moreira-Filho et al. 2004). The secondary forms of B chromosomes probably originated from chromosome rearrangements, such as pericentric inversion and deletion (Moreira-Filho et al. 2004). In our study, a B_M variant similar to those described for Astyanax species was also observed, but its frequency was lower than that found in the B_a variant. Similar data exhibiting the occasional presence of B chromosomes of different heterochromatin morphologies and patterns were analyzed by Machado et al. (2012).

In A. scabripinnis, structural and functional evidence suggest that B_M corresponds to an isochromosome derived from an acrocentric chromosome of the standard karyotype (Mestriner et al. 2000; Vicari et al. 2011; Machado et al. 2012). However, according to the isochromosomes origin, the centromere misdivision of an acrocentric chromosome ancestor and subsequent chromatid non-disjunction may simultaneously originate B microchromosomes and B_M variants, an event that was not found in any population of A. scabripinnis (Moreira-Filho et al. 2004).

Conversely, our results showed the occurrence of B_M and B microchromosome variants together in the same population, which supports the isochromosome origin of the B chromosomes in A. bockmanni. The distinct morphologies between the microchromosomes (B_a and B_m) found herein may be ascribable to structural rearrangements, as it occurs in the B chromosome variants of the fish Prochilodus lineatus (Artoni, Vicari et al. 2006).

The analysis of constitutive heterochromatin patterns showed heterochromatic blocks in the pericentromeric regions of most chromosomes and evident signals in the terminal regions of some chromosomes (Figure 2), similar to those found in other populations of A. bockmanni (Kavalco et al. 2009; Hashimoto and Porto-Foresti 2010). In relation to the B chromosomes, all three morphological variants (B_M, B_m, and B_a) were identified as heterochromatic (Figure 2), in accordance with a common characteristic reported for B chromosomes in the genus Astyanax (Néo et al. 2000; Moreira-Filho et al. 2001; Hashimoto et al. 2008; Machado et al. 2012).

FISH signals of the 18S rDNA probe were detected in the terminal regions of several chromosomes, while the silver staining showed active NORs in only four chromosomes (Figure 3), according to the polymorphism frequently observed in different species of Astyanax (Kavalco et al. 2009). B chromosomes in several species of plants (Camacho 2005) and animals, including fish species (Poletto et al. 2010), carry rRNA genes. However, in the present study, nucleolar activity or rRNA genes were not found in the B chromosome variants of A. bockmanni.

Figure 3 (Colour online) Metaphases showing the chromosome location of (a) 18S rRNA genes by FISH; and (b) nucleolar regions detected by silver staining. Arrows indicate the B chromosomes. Arrowheads show chromosomes bearing (a) 18S rDNA clusters and (b) NORs.

Several species of Astyanax present populations bearing B chromosomes in the genome of particular individuals, but most of them represent sporadic cases (Moreira-Filho et al. 2004). According to Hashimoto and Porto-Foresti (2010), A. bockmanni shows a high degree of chromosome similarity to the species complex A. scabripinnis. Thus, B chromosomes also appear as an interesting feature that can be fixed in other populations of A. bockmanni, not representing a sporadic event. Although few populations of A. bockmanni have been cytogenetically analyzed, the occurrence of three morphologically variant B chromosomes demonstrates that extensive studies in this species are essential to improve the knowledge of the chromosome diversification in this fish group.

Acknowledgments

The authors wish to thank Fernando C.P. D’Agosta and Ricardo M.C. Castro for the taxonomical identification. This work was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo – FAPESP (Proc. 2010/06919-8, 2011/15007-5) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).