Cytogenetic analysis of B chromosomes in one population of the fish Moenkhausia sanctaefilomenae (Steindachner, 1907) (Teleostei, Characiformes)

Abstract The aim of this study was to characterize cytogenetically one population of the fish Moenkhausia sanctaefilomenae (Steindachner, 1907), with emphasis on the analysis of B chromosomes. The nucleolar activity in the B microchromosomes was characterized, and an analysis of mitotic instability of these microchromosomes was accomplished. The results showed a diploid chromosome number of 50 chromosomes. In all individuals, we observed the presence of B microchromosomes with intra- and inter-individual variability. The analysis of the nucleolus organizing regions (NORs) by silver nitrate staining demonstrated multiple NORs. We observed active sites of ribosomal DNA in the B microchromosomes, with a frequency of 20% in the analyzed cells, which shows gene activity in these chromosomal elements. The analysis of constitutive heterochromatin patterns showed that the B microchromosomes are heterochromatic or euchromatic, which demonstrates differentiation of DNA composition between these genomic elements. The calculation of the mitotic instability index implied that B chromosomes in this species might be in a final stage of instability.

introduction Moenkhausia Eigenmann, 1903 is considered as incertae sedis in Characidae and contains 65 valid species widely distributed in the Neotropical river basins (Lima et al. 2003). Although the genus Moenkhausia cannot be characterized as monophyletic, a group consisted of M. oligolepis (Günther, 1864), M. sanctaefilomenae (Steindachner, 1907), M. cotinho Eigenmann, 1908, and M. pyrophthalma Costa, 1994 shares a very similar color pattern (Costa 1994). Moenkhausia systematic is very complex and nowadays several studies have shown that it needs to be more thoroughly addressed (Benine et al. 2009).
Chromosome studies in the genus Moenkhausia are still restricted and cytogenetic data are available only for six species (Portela-Castro et al. 2001). In M. sanctaefilomenae, a stable diploid number of 50 chromosomes and few karyotype variations among the different populations analyzed have been reported. Furthermore, some populations of M. sanctaefilomenae can show a high inter-and intra-individual variability of the NOR (nucleolus organizer region) phenotypes, as well as conspicuous blocks of constitutive heterochromatin in the pericentromeric region of the chromosomes (Foresti et al. 1989, Portela-Castro et al. 2001, Portela-Castro and Júlio Jr. 2002. However, the occurrence of several B microchromosomes in the genome of this species is the most peculiar feature to be studied in this fish group (Foresti et al. 1989).
B chromosome includes a variety of extra chromosomes that display conspicuous heterogeneity in their nature, behavior, and evolutionary dynamics. This definition highlights some of the most universal properties of B chromosomes: their dispensability (that is, they are not necessary for the host to complete a normal life cycle); their origin from chromosomes (either from within the same species or from other species); and their remarkable differentiation relative to A chromosomes, with which they do not recombine (Camacho 2005).
B chromosomes are widely distributed among eukaryotes and their occurrence has been reported in 10 species of the fungi, nearly 1.300 plants (more than 1.400 when different ploidy levels of the same species are considered separately), and over 500 animals (Camacho 2005). In addition, B chromosomes have been described in 61 species of Neotropical fish to date (Carvalho et al. 2008).
In species of Moenkhausia, B chromosomes were documented for M. sanctaefilomenae and M. intermedia Eigenmann, 1908(Portela et al. 1988, Foresti et al. 1989. Differently from other microchromosome-bearing fish species, which exhibit a low frequency and a sporadic occurrence (Hashimoto et al. 2008, Hashimoto et al. 2011, several microchromosomes can be found in the genome of M. sanctaefilomenae and, in certain situations, the frequency can be related to sex (Portela-Castro et al. 2001). In fact, in Neotropical fish, it is possible to find both B macrochromosomes and B microchromosomes ), but in both cases the presence of a large number of B chromosomes in the cells is rare, as was observed in Prochilodus lineatus (Valenciennes, 1836) and M. sanctaefilomenae, which presented up to eight microchromosomes in the cells (Foresti et al. 1989, Voltolin et al. 2011. Another interesting characteristic observed in the B microchromosomes of M. sanctaefilomenae is the polymorphism revealed by C-banding. Through this method, these microchromosomes can be characterized in different classes according to the pattern of constitutive heterochromatin; they can be partially and totally heterochromatic, and euchromatic (Foresti et al. 1989). Thus, such polymorphism indicates a distinct DNA composition between these microchromosomes, especially of repetitive DNA.
In the present study, we carried out cytogenetic analyses in one particular population of the fish M. sanctaefilomenae focusing on two special features concerning the B microchromosomes: the occurrence of nucleolar activity in the B chromosome of this species and a study about the maintenance of microchromosomes in this population through the calculation of the mitotic instability index (MI).

Material and methods
The cytogenetic analyses were carried out in chromosomal preparations obtained from 15 specimens (8 males and 7 females) of Moenkhausia sanctaefilomenae. The individuals were collected from a population of the Batalha River (22°7.02'S, 49°16.01'W), belonging to Tietê River basin, São Paulo State, southeastern Brazil. The voucher specimens were identified and stored in the fish collection of the Laboratório de Genética de Peixes, UNESP, Bauru, SP, Brazil.
Before sacrifice, the animals were inoculated with yeast cell suspension to increase the number of metaphase cells (Oliveira et al. 1988). Chromosomal preparations were obtained from gill and kidney tissues using the technique described by Foresti et al. (1993). Silver staining (Ag-staining) of the nucleolus organizer regions followed the technique of Howell and Black (1980), and C-banding was performed according to Sumner (1972). The chromosomal morphology was determined on the basis of arm ratio, as proposed by Levan et al. (1964) and the chromosomes were classified as metacentric (m), submetacentric (sm), subtelocentric (st), and acrocentric (a).
The index to quantify the mitotic instability of B chromosome, MI, which was calculated as the sum of the absolute values of every deviation in B number with respect to the median (M), and normalized by dividing the median and the number of cells analyzed (N) so that the index is independent of the number of B and the sample size were performed by means of one-way ANOVA.

MI = (M-ni/fi)/M.N
where ni is the numer of B chromosome in the different types of cells that do not coincide with M, and fi is the number of cells of each particular type.

Results and discussion
In the individuals of Moenkhausia sanctaefilomenae, our results showed a diploid chromosome number of 50 chromosomes, with karyotypes composed of 6 m, 16 sm and 28 st (fundamental number FN = 100) (Fig. 1). No sex-related karyotype difference was observed. The diploid chromosome number and the karyotypes composed mainly of metacentric and submetacentric chromosomes seem to be a conserved characteristic observed for different M. sanctaefilomenae populations (Foresti et al. 1989, Portela-Castro et al. 2001, Portela-Castro and Júlio Jr. 2002.
Extra chromosomes were observed in the genomes of all individuals of M. sanctaefilomenae, which were characterized as B microchromosomes (Fig. 1). We detected inter-and intra-individual variation in relation to the number of B chromosomes in the cells, with specimens bearing up to eight microchromosomes. Metaphase counts for 13 individuals showing the variation in supernumerary chromosome numbers are presented in Table 1. The modal numbers were of 2 and 3 microchromosomes. Such variation is in accordance with the pioneer study of Foresti et al. (1989), who also analyzed a population from the Tietê River basin. On the other hand, the specimens from the Paraná River analyzed by Portela-Castro et al. (2001), showed differences because the presence of 0-2 microchromosomes were reported only in males. These polymorphisms concerning the distribution of B chromosomes indicate a process of genetic divergence in distinct populations that likely occurs in some species restricted to small tributaries and streams, as reported for species of Astyanax (Moreira-Filho and Bertollo 1991, Vicari et al. 2008, Hashimoto et al. 2011. Analysis of the constitutive heterochromatin patterns by C-banding showed heterochromatic blocks in the centromeric and pericentromeric regions in the majority of the chromosomes (Fig. 2a, b). Such general heterochromatin pattern was also observed in previous analyses for other M. sanctaefilomenae populations (Foresti et al. 1989, Portela-Castro et al. 2001, Portela-Castro and Júlio Jr. 2002, demonstrating that these chromosomal regions present a highly conservative distribution in this species. The supernumerary chromosomes showed different C-banding patterns. We observed euchromatic (Fig. 2a) as well as partially or totally heterochromatic microchromosomes (Fig. 2a, b), evidencing that these B chromosomes can have a different DNA composition, mainly of repetitive sequences. This is a common feature also reported for B chromosomes in other characid species (Néo et al. 2000, Jesus et al. 2003, Moreira-Filho et al. 2004).
The Ag-impregnation revealed intra- (Fig. 3a, b) and inter-individual (Fig. 3c, d) variability for the NOR phenotypes in metaphases of M. sanctaefilomenae, ranging from two to five Ag-positive sites, distributed in the interstitial and terminal regions of distinct chromosomes (Fig. 3e). However, the chromosomes 6 always presented active Ag-NORs, and consequently, were considered the major NOR-bearing chromosomes. The minor NORs showed a very variable pattern of activity. Such NOR features were previously reported by Foresti et al. (1989).

Number of B microchromosomes per cell
The chromosome context appears to be important for NOR activity, as deduced from changes in the on/off activity status following chromosome rearrangements moving NORs to new locations (Pikaard 2000). The present findings show that the B chromosome plays an important role in the genome organization of M. sanctaefilom- enae, and will be useful for further analyses to determine whether the frequency of B chromosomes expressing their NOR is changing over time and how the B chromosome context can influence A chromosome NOR activity.
In relation to the mitotic instability and maintenance of B chromosomes in M. sanctaefilomenae, we compared the results reported by Foresti et al. (1989) with the data described in this study, because both populations were collected from the Tietê River basin (Brazil). In both populations, a pattern of mitotic instability for all ana- lyzed individuals was observed. The analysis of the standard maintenance of these B chromosomes by calculating the mitotic instability index (MI) revealed that the M. sanctaefilomenae population analyzed by Foresti et al. (1989) showed a MI = 0.6; however, the M. sanctaefilomenae population analyzed in the present study showed a MI = 0.2. Taking account the high variability of B chromosomes in the genomes of these M. sanctaefilomenae specimens, further studies are still necessary to verify if these B chromosomes might be underway towards the neutralization stage, in accordance with the life cycle of B chromosomes described by Camacho et al. (1997).
In fish, the possibility of neutralization through mitotic stabilization of B-chromosomes was also observed in Prochilodus lineatus, in the population from the Mogi-Guaçu River (Brazil) (Oliveira et al. 1997). Afterwards, in this same population, Cavallaro et al. (2000) found a drastic temporal decline in the degree of B mitotic instability; Voltolin et al. (2010) showed that the stabilization process was continuous for over 15 years; and currently, the population of P. lineatus from the Mogi-Guaçu River presents a total mitotic stability index (MI = 0) and the B chromosomes were considered completely neutralized.
In Neotropical fish, most of the studies about B chromosomes are still descriptive, because many species have not yet been cytogenetically analyzed. Thus, studies focusing B chromosomes in Neotropical fish are extremely necessary to better understand this intriguing class of chromosomes, as has been done for some species, such as Prochilodus lineatus and Astyanax species (Moreira-Filho et al. 2004, Voltolin et al. 2010, Hashimoto et al. 2011, towards which efforts are more thoroughly addressed. Thus, our results show that B chromosomes of M. sanctaefilomenae are excellent models and also that extensive studies in this species are essential to improve the knowledge of the diversification of B chromosomes.