Comparative CompCytogen 6(2): 141–151 (2012) A peer-reviewed open-access journal 141 Cytogenetics Cytogenetic analysis of B chromosomes in one population of the ish... doi: 10.3897/CompCytogen.v6i2.1769 ReSeARCh ARTiCle www.pensoft.net/journals/compcytogen International Journal of Plant & Animal Cytogenetics, Karyosystematics, and Molecular Systematics Cytogenetic analysis of B chromosomes in one population of the fish Moenkhausia sanctaefilomenae (Steindachner, 1907) (Teleostei, Characiformes) Diogo Teruo Hashimoto1, Tatiana Aparecida Voltolin1, Ana Danyelle Noitel Valim de Arruda Paes1, Fausto Foresti2, Jehud Bortolozzi1, Fábio Porto-Foresti1 1 Departamento de Ciências Biológicas, Faculdade de Ciências, Universidade Estadual Paulista (UNESP), Av. Eng. Luiz E. C. Coube, 17033-360, Bauru, SP, Brazil 2 Departamento de Morfologia, Instituto de Biociências, Universidade Estadual Paulista (UNESP), Dist. Rubião Júnior, 18618-000, Botucatu, SP, Brazil Corresponding author: Fábio Porto-Foresti (fpforesti@fc.unesp.br) Academic editor: G. Furgala-Selezniow | Received 2 July 2011 | Accepted 7 March 2012 | Published 9 April 2012 Citation: Hashimoto DT, Voltolin TA, de Arruda Paes ADNV, Foresti F, Bortolozzi J, Porto-Foresti F (2012) Cytogenetic analysis of B chromosomes in one population of the ish Moenkhausia sanctaeilomenae (Steindachner, 1907) (Teleostei, Characiformes). Comparative Cytogenetics 6(2): 141–151. doi: 10.3897/CompCytogen.v6i2.1769 Abstract he aim of this study was to characterize cytogenetically one population of the ish Moenkhausia sanctaeilomenae (Steindachner, 1907), with emphasis on the analysis of B chromosomes. he nucleolar activity in the B microchromosomes was characterized, and an analysis of mitotic instability of these microchromosomes was accomplished. he 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. he 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. he analysis of constitutive heterochromatin patterns showed that the B microchromosomes are heterochromatic or euchromatic, which demonstrates diferentiation of DNA composition between these genomic elements. he calculation of the mitotic instability index implied that B chromosomes in this species might be in a inal stage of instability. Keywords ish cytogenetic, NOR expression, supernumerary chromosomes, mitotic instability Copyright Diogo Teruo Hashimoto et al. This is an open access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC-BY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 142 Diogo Teruo Hashimoto et al. / Comparative Cytogenetics 6(2): 141–151 (2012) 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. sanctaeilomenae (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. sanctaeilomenae, a stable diploid number of 50 chromosomes and few karyotype variations among the diferent populations analyzed have been reported. Furthermore, some populations of M. sanctaeilomenae 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 ish group (Foresti et al. 1989). B chromosome includes a variety of extra chromosomes that display conspicuous heterogeneity in their nature, behavior, and evolutionary dynamics. his deinition 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 diferentiation 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 diferent 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 ish 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). Diferently from other microchromosome-bearing ish species, which exhibit a low frequency and a sporadic occurrence (Hashimoto et al. 2008, Oliveira et al. 2009, 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 ish, it is possible to ind both B macrochromosomes and B microchromosomes (Oliveira et al. 2009), 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, Cytogenetic analysis of B chromosomes in one population of the ish... 143 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. hrough this method, these microchromosomes can be characterized in diferent classes according to the pattern of constitutive heterochromatin; they can be partially and totally heterochromatic, and euchromatic (Foresti et al. 1989). hus, 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 ish 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 he cytogenetic analyses were carried out in chromosomal preparations obtained from 15 specimens (8 males and 7 females) of Moenkhausia sanctaeilomenae. he 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. he voucher specimens were identiied and stored in the ish collection of the Laboratório de Genética de Peixes, UNESP, Bauru, SP, Brazil. Before sacriice, 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). he chromosomal morphology was determined on the basis of arm ratio, as proposed by Levan et al. (1964) and the chromosomes were classiied as metacentric (m), submetacentric (sm), subtelocentric (st), and acrocentric (a). he 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/i)/M.N where ni is the numer of B chromosome in the diferent types of cells that do not coincide with M, and i is the number of cells of each particular type. 144 Diogo Teruo Hashimoto et al. / Comparative Cytogenetics 6(2): 141–151 (2012) Results and discussion In the individuals of Moenkhausia sanctaeilomenae, 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 diference was observed. he diploid chromosome number and the karyotypes composed mainly of metacentric and submetacentric chromosomes seem to be a conserved characteristic observed for diferent M. sanctaeilomenae populations (Foresti et al. 1989, PortelaCastro et al. 2001, Portela-Castro and Júlio Jr. 2002). Extra chromosomes were observed in the genomes of all individuals of M. sanctaeilomenae, 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. he 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 diferences because the presence of 0–2 microchromosomes were reported only in males. hese 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. sanctaeilomenae 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. he supernumerary chromosomes showed diferent 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 diferent DNA composition, mainly of repetitive sequences. his 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). he Ag-impregnation revealed intra- (Fig. 3a, b) and inter-individual (Fig. 3c, d) variability for the NOR phenotypes in metaphases of M. sanctaeilomenae, ranging from two to ive 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. he minor NORs showed a very variable pattern of activity. Such NOR features were previously reported by Foresti et al. (1989). Cytogenetic analysis of B chromosomes in one population of the ish... 145 Figure 1. Giemsa-stained karyotype showing 2n = 50 chromosomes of one individual of Moenkhausia sanctaeilomenae. In evidence, eight B microchromosomes. Bars = 10 µm. Table 1. Metaphase counts for 13 specimens of Moenkhausia sanctaeilomenae demonstrating the variation in B microchromosome numbers. Specimen identiication 849 852 853 857 887 888 889 1233 1235 1240 1241 1242 1246 0 6 2 9 3 1 8 9 24 5 Number of B microchromosomes per cell 1 2 3 4 5 6 7 12 22 32 36 10 70 3 6 10 9 9 2 3 12 22 4 2 7 13 6 6 41 5 10 3 12 63 11 3 4 10 24 26 29 13 4 6 31 79 15 8 31 33 26 3 137 175 4 85 27 25 4 5 4 1 - 8 2 - Number of cells counted 40 80 79 42 65 68 92 110 140 101 325 136 44 Indeed, NOR expression was detected in a B chromosome of one individual, which carried only this microchromosome (Fig. 3a, b). We analyzed 60 cells by Ag-staining and observed that about 20% had active ribosomal DNA sites in the B chromosome of this individual. Moreover, this supernumerary chromosome showed to be euchromatic by C-banding. he nucleolar region is a dynamic cell compartment involved in the control of numerous cellular functions that can be visualized after Ag coloration, 146 Diogo Teruo Hashimoto et al. / Comparative Cytogenetics 6(2): 141–151 (2012) Figure 2. Metaphases from specimens of Moenkhausia sanctaeilomenae after C-banding technique. In (a), metaphase shows euchromatic (eB), partially heterochromatic (phB) and totally heterochromatic (hB) microchromosomes. In (b), metaphase demonstrates only heterochromatic B chromosomes. he boxes show enlarged B chromosomes. Major and minor arrows indicate totally and partially heterochromatic B microchromosomes, respectively. Arrowheads exhibit euchromatic B microchromosomes. Bars = 10 µm. when the genes present activities in the interphase that anticipates the mitosis (Roussel et al. 1996, Caperta et al. 2007, Hiscox 2007). herefore, Ag-staining provides a simple and reliable method to detect ribosomal RNA (rRNA) gene transcription (Bakkali et al. 2001, Teruel et al. 2009). B chromosomes in several species carry rRNA genes (Camacho 2005), including ish species (Baroni et al. 2009, Poletto et al. 2010), and in most of the cases, rRNA has been detected by Ag-staining evidencing the presence of active genes, as demonstrated in the present study. However, further analysis using FISH technique will be necessary to detect positions of additional rDNA genes not only during their activity. he fact that the NORs located in the chromosomes 6 were always active can suggest that a process of nucleolar dominance can inluence the rRNA gene transcription in order to provide the proper amount of rRNA for ribosome assembly. Nucleolar dominance is an epigenetic phenomenon common in interspeciic hybrids, in which ribosomal RNA genes set inherited from one parental are rather transcribed in relation to the other (Hashimoto et al. 2009). Nucleolar dominance can also be a consequence of the regulatory process that controls the efective dosage of rRNA genes in pure species (nonhybrid) (Pikaard 2000). Nowadays, the mechanisms by which whole NORs or rRNA genes subsets are selected for inactivation still remains unclear (Preuss and Pikaard 2007). he chromosome context appears to be important for NOR activity, as deduced from changes in the on/of activity status following chromosome rearrangements moving NORs to new locations (Pikaard 2000). he present indings show that the B chromosome plays an important role in the genome organization of M. sanctaeilom- Cytogenetic analysis of B chromosomes in one population of the ish... 147 Figure 3. Metaphases from specimens of Moenkhausia sanctaeilomenae submitted to the silver coloration. In (a) and (b), metaphases of one individual show intra-individual variability of active NORs. he boxes exhibit enlarged B chromosomes with nucleolar activity. In (c) and (d), metaphases of diferent samples demonstrate inter-individual variability for the NORs. In (e), schematic representation shows the NORbearing chromosomes (4, 6, 8, 11 and B). Major arrows indicate major NOR-bearing chromosomes (chromosomes 6). Minor arrows show nucleolar activity in the B microchromosomes (a) and (b). Arrowheads exhibit minor NORs demonstrating a variable pattern of activity in diferent chromosomes. Bars = 10 µm. 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 inluence A chromosome NOR activity. In relation to the mitotic instability and maintenance of B chromosomes in M. sanctaeilomenae, 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- 148 Diogo Teruo Hashimoto et al. / Comparative Cytogenetics 6(2): 141–151 (2012) lyzed individuals was observed. he analysis of the standard maintenance of these B chromosomes by calculating the mitotic instability index (MI) revealed that the M. sanctaeilomenae population analyzed by Foresti et al. (1989) showed a MI = 0.6; however, the M. sanctaeilomenae 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. sanctaeilomenae 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 ish, 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 ish, most of the studies about B chromosomes are still descriptive, because many species have not yet been cytogenetically analyzed. hus, studies focusing B chromosomes in Neotropical ish 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, 2011, Hashimoto et al. 2011), towards which eforts are more thoroughly addressed. hus, our results show that B chromosomes of M. sanctaeilomenae are excellent models and also that extensive studies in this species are essential to improve the knowledge of the diversiication of B chromosomes. Acknowledgements his work was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Cientíico e Tecnológico (CNPq). 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