In Prochilodus lineatus B-chromosomes are visualized as reduced size extra elements identified as microchromosomes and are variable in morphology and number. We describe the specific total probe (B-chromosome probe) in P. lineatus obtained by chromosome microdissection and a whole genomic probe (genomic probe) from an individual without B-chromosome. The specific B-chromosome was scraped and processed to obtain DNA with amplification by DOP-PCR, and so did the genomic probe DNA. Fluorescence in situ hybridization using the B-chromosome probe labeled with dUTP-Tetramethyl-rhodamine and the genomic probe labeled with digoxigenin-FITC permitted to establish that in this species supernumerary chromosomes with varying number and morphology had different structure of chromatin when compared to that of the regular chromosomes or A complement, since only these extra elements were labeled in the metaphases. The present findings suggest that modifications in the chromatin structure of B-chromosomes to differentiate them from the A chromosomes could occur along their dispersion in the individuals of the population.

The family Prochilodontidae is considered a group with well conserved chromosomes characterized by their number, morphology and banding patterns. Thence, our study aimed at accomplishing a cytogenetic analysis with conventional methods (Giemsa staining, silver staining of the nucleolus organizer regions-AgNOR, and C-banding) and fluorescence in situ hybridization (FISH) with 18S and 5S ribosomal DNA probes in five species of the Prochilodus genus (Prochilodus argenteus, Prochilodus brevis, Prochilodus costatus, Prochilodus lineatus and Prochilodus nigricans) collected from different Brazilian hydrographic basins. The results revealed conservatism in chromosome number, morphology, AgNORs 18S and 5S rDNAs location and constitutive heterochromatin distribution patterns. The minor differences observed in this work, such as an Ag-NOR on a P. argenteus chromosome and a distinct C-banding pattern in P. lineatus, are not sufficient to question the conservatism described for this group. Future work using repetitive DNA sequences as probes for FISH will be interesting to further test the cytogenetic conservatism in Prochilodus.

Summary Within the genus Prochilodus, the species Prochilodus lineatus is certainly the most studied one from a cytogenetic point of view. In this study, a cytogenetic characterization of specimens of P. lineatus from Mogi-Guaçu River was performed in the period from 2003 to 2007, through utilization of cytogenetic markers, such as Giemsa, Ag-NOR, C-banding and cytogeneticalmolecular markers (FISH) to detect both 5S and 18S ribosomal genes. All analyzed individuals presented 2nϭ54 meta/submetacentric chromosomes, besides bearing up to 7 supernumerary microchromosomes. Polymorphic NORs were detected on a single chromosomal pair. The constitutive heterochromatin was distributed at centromeric region of all chromosomes in the A complement, while the microchromosomes were totally heterochromatic. A syntenic distribution of 5S and 18S ribosomal genes was detected, comprising the NOR-bearing chromosomal pair. No additional ribosomal clusters in other chromosomes were observed. Although the karyotype features are commonly conserved within the genus Prochilodus, the few differences on the distribution of both ribosomal genes and heterochromatin when compared to specimens of P. lineatus from other localities can be associated to the evolutionary changes that these repetitive sequences have undergone through the years.

Cytogenetic studies were developed in Prochilodus lineatus (Valenciennes 1836), describing an interesting system of small supernumerary chromosomes. The purpose of this work is to study the frequency and morphology of B chromosomes in individuals from the parental line and the inheritance patterns of these elements in individuals obtained from controlled crosses in the species P. lineatus. The transmission rate of B chromosomes revealed a k B =0.388 for the acrocentric type, a kB=0.507 for the metacentric type and a k B =0.526 for the submetacentric type. The obtained results raise hypothesis that B-acrocentric chromosomes are involved in an extinction process in this species, while the metacentric and submetacentric supernumerary elements comprises a neutral mechanism and follows a Mendelian transmission rate.

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.

To ascertain the origin of B chromosomes in 2 fish species of the genus Prochilodus, i.e. P. lineatus and P. nigricans, we microdissected them and generated B-specific DNA probes. These probes were used to perform chromosome painting in both species and in 3 further ones belonging to the same genus (P. argenteus, P. brevis and P. costatus). Both probes hybridized with the B chromosomes in P. lineatus and P. nigricans, but with none of the chromosomes in the 5 species. This indicates that the B chromosomes have low similarity with DNAs located in the A chromosomes and suggests the possibility that the B chromosomes in the 2 species have a common origin. The most parsimonious explanation would imply intergeneric hybridization in an ancestor of P. lineatus and P. nigricans yielding the B chromosome as a byproduct, which remained in these 2 species after their phylogenetic origin, but was perhaps lost in other Prochilodus species. This hypothesis predicts that B chromosomes are old genomic elements in this genus, and this could be tested once a species from a relative genus would be found showing homology of its A chromosomes with the B-probes employed here, through a comparison of B chromosome DNA sequences with those in the A chromosomes of this other species.

The genus Prochilodus includes individuals ranging in size from medium to large, being highly relevant for commercial and subsistence fishing. Prochilodus species have a diploid number of 2n = 54 chromosomes and up to seven supernumerary (B) microchromosomes. Previous research has shown that B frequency increased drastically in the Mogi-Guaçu river population of Prochilodus lineatus in the early 1980s, whereas it remained about constant in the 1990s. Here we analyses B frequency in this population during the 2003-2007 period and have found that frequency has not changed significantly since 1987, and that these B chromosomes do not show the intra-individual variation in number that characterized them in the 1980s. This indicates that these B chromosomes have been neutralized, after their invasion, through their mitotic stabilization.

B chromosomes are supernumerary elements present in about 15% of eukaryotic species and are most frequently heterochromatic, behave parasitically, show a transmission rate higher than standard (A) chromosomes, and can provoke harmful effects on carriers. In the current work, Prochilodus lineatus individuals carrying eight and nine B chromosomes were obtained by induced crossing performed involving breeders with different B chromosome numbers in their cells. The high B chromosome numbers found in the offspring were recorded for the first time in this species. The use of cytogenetic techniques applied in the present study revealed that regardless of the increase in number of B chromosomes in the genome of these individuals, those elements did not presented active genes, and showed their normal heterochromatic characteristic.