Dynamics of Growth in Purebred Pacu (Piaractus mesopotamicus) and Tambaqui (Colossoma macropomum), and Their Reciprocal Hybrids, under Varied Feeding Programs: Insights from Nonlinear Models.

We evaluated the growth performance of pacu and tambaqui and their reciprocal hybrids (tambacu and paqui) under different feeding programs. We raised 30 individuals from each species and their respective crosses, distributing them into three replicate cages with 10 individuals each. Throughout the 5-month experimental period, the fish were weighed six times while exposed to diverse feeding regimens involving three commercial diets with varying combinations of crude protein (CP) levels: 24%, 28%, and 32%. Growth curves were adjusted using nonlinear models. The evaluation period was insufficient for adjusting the logistic model for the tambaqui and paqui treatments with the highest initial protein intake. Pure pacu had a higher (p < 0.05) growth rate (k = 0.0185) than in the tambacu hybrid (k = 0.0134) and proved to have an early performance since animals from this group reached their weight at inflection faster. Despite growing more slowly, tambacu reached a higher (p < 0.05) final weight (A = 1137.12) than in the pacu (A = 889.12). Among the feeding programs, animals that received less CP showed greater growth potential; however, longer evaluation is important to verify if the initial CP intake has no significant positive effect on fish growth.

Genomic selection signatures in farmed Colossoma macropomum from tropical and subtropical regions in South America.

Tambaqui or cachama (Colossoma macropomum) is one of the most important neotropical freshwater fish used for aquaculture in South America, and its production is concentrated at low latitudes (close to the Equator, 0°), where the water temperature is warm. Therefore, understanding how selection shapes genetic variations and structure in farmed populations is of paramount importance in evolutionary biology. High-throughput sequencing to generate genome-wide data for fish species allows for elucidating the genomic basis of adaptation to local or farmed conditions and uncovering genes that control the phenotypes of interest. The present study aimed to detect genomic selection signatures and analyze the genetic variability in farmed populations of tambaqui in South America using single-nucleotide polymorphism (SNP) markers obtained with double-digest restriction site-associated DNA sequencing. Initially, 199 samples of tambaqui farmed populations from different locations (located in Brazil, Colombia, and Peru), a wild population (Amazon River, Brazil), and the base population of a breeding program (Aquaculture Center, CAUNESP, Jaboticabal, SP, Brazil) were genotyped. Observed and expected heterozygosity was 0.231-0.350 and 0.288-0.360, respectively. Significant genetic differentiation was observed using global FST analyses of SNP loci (FST = 0.064, p < 0.050). Farmed populations from Colombia and Peru that differentiated from the Brazilian populations formed distinct groups. Several regions, particularly those harboring the genes of significance to aquaculture, were identified to be under positive selection, suggesting local adaptation to stress under different farming conditions and management practices. Studies aimed at improving the knowledge of genomics of tambaqui farmed populations are essential for aquaculture to gain deeper insights into the evolutionary history of these fish and provide resources for the establishment of breeding programs.

The Satellite DNA Catalogues of Two Serrasalmidae (Teleostei, Characiformes): Conservation of General satDNA Features over 30 Million Years.

Satellite DNAs (satDNAs) are tandemly repeated sequences that are usually located on the heterochromatin, and the entire collection of satDNAs within a genome is called satellitome. Primarily, these sequences are not under selective pressure and evolve by concerted evolution, resulting in elevated rates of divergence between the satDNA profiles of reproductive isolated species/populations. Here, we characterized two additional satellitomes of Characiformes fish (Colossoma macropomum and Piaractus mesopotamicus) that diverged approximately 30 million years ago, while still retaining conserved karyotype features. The results we obtained indicated that several satDNAs (50% of satellite sequences in P. mesopotamicus and 43% in C. macropomum) show levels of conservation between the analyzed species, in the nucleotide and chromosomal levels. We propose that long-life cycles and few genomic changes could slow down rates of satDNA differentiation.

Use of Molecular Genetic Methods to Reduce the Risk of Incorrect Identification of Fish Strains in Brazilian Aquaculture.

The identification of fish species using traditional methods is generally based only on morphological characteristics and these methods are currently under review. This kind of identification of hybrid fishes solely based on their morphologies may be unreliable, especially when the samples include juveniles and post-F1 lineage fishes. Therefore, in the present study, we used molecular markers to accurately identify the fish species of economic interest that are used as materials in the projects developed in research institutions. We evaluated six lots of fishes sampled from different research centers, containing a total of 84 specimens acquired from private fish farms that were considered to be the representatives of pure species. Genetic analyses of all the specimens revealed that, globally, 22 samples (26.2%) were interspecific hybrids, while 20 (90.9%) samples were surprisingly characterized as post-F1 hybrids. This result confirms that hybrids are sold in markets without adequate labeling and also indicates the lack of proper control of the commercialization and management of stocks and products in fish farms. In addition, we determined that molecular diagnosis was an extremely effective and necessary method to test the reliability of biological materials currently used in scientific research.

Sex-Dependent Inheritance of B Chromosomes in Psalidodon paranae (Teleostei, Characiformes) Revealed by Directed Crossings.

B chromosomes are additional dispensable elements to the standard chromosomal set of an organism. In most cases, their transmission differs from Mendelian patterns, leading to their accumulation or extinction. The present study aimed to describe, for the first time, the transmission pattern of B chromosome in a population of Psalidodon paranae through directed crosses, as well as to analyze the populational dynamics of B chromosome. Our results revealed the possible elimination of B chromosome in crossings where only females were B-carriers, with a mean transmission rate (kB) of 0.149; however, kB was significantly higher in crossings involving male B-carriers (kB = 0.328-0.450). Moreover, we observed an increase in the frequency of B chromosomes in the natural population of P. paranae in the last two decades. These apparently contradictory results can make sense if the B chromosome provides adaptive advantages to their carriers. Here, we observed a differential transmission of B chromosomes in each sex of parental individuals, with higher transmission rates in crossing involving males B-carriers, in addition to describe the temporal changes of B chromosome frequency in P. paranae.

ETosis in tambaqui Colossoma macropomum: A programmed cell death pathway and approach of leukocytes immune response.

Tambaqui Colossoma macropomum is the most cultivated native fish in South America and Aeromonas hydrophila is one of the main bacteria infecting tropical fish. Despite the economic importance of this round fish, to date, there has been a paucity of investigations into haematological changes in tambaqui. In this study, detailed blood analyses (0 h, 6 h, 24 h, 7 d and 14 d) following intraperitoneal challenge with A. hydrophila were performed. After analysing the results, there was a suspicion of a novel cell death mechanism via extracellular traps (ETosis) in tambaqui. The search for ETosis was based on differential interference contrast (DIC) microscopy and scanning electron microscopy (SEM) assays through application of an adapted protocol applying co-incubation of leukocytes with A. hydrophila. The cells were investigated at: 0 h (control), 4 h and 7 h after incubation. The complete haemogram profile showed an uncommon severe leukopenia in early phases of infection (6 h, p < 0.001 and ≤ 0.05), due to significant decreases in the three main leukocytes: lymphocytes (6 h, p ≤ 0.001), monocytes (6 h, p ≤ 0.05) and neutrophils (6 h and 24 h, p ≤ 0.01 and p ≤ 0.05). Leucocytosis and lymphocytosis (p ≤ 0.01) were ascertained only 7 days post-infection. Through DIC and SEM, we discovered that leukocyte suicide exposed the nuclear contents between 4 and 7 h after stimuli with bacteria. The leukogram profile associated with DIC and SEM analyses suggested that tambaqui leukocytes underwent a programmed death (ETosis) in order to expose chromatin and granule proteins as a trap to bind and then kill bacteria; thus, preventing A. hydrophila from spreading and resulting in leukopenia during the early phase of bacterial infection. In this paper, we presume that ETosis is one of the last resources for tambaqui to contain the infection, and after this leukocyte strategy, a high number of phagocytic cells are produced and released into the peripheral circulation.

Patterns of the innate immune response in tambaqui Colossoma macropomum: Modulation of gene expression in haemorrhagic septicaemia caused by Aeromonas hydrophila.

The production of tambaqui Colossoma macropomum has recently reached a milestone, being considered the main native species produced in South American continental waters. Despite the importance of this fish, its immunity is not well understood. In this study we established some patterns of innate immunity for the species via two experiments. Both studies evaluated the fish in the absence (intraperitoneal saline) or presence (intraperitoneal, 3 x 107 CFU/mL of Aeromonas hydrophila at 0.1 mL/10 g of living weight) of infection at 5 points over time-course of 14 days (0 h, 6 h, 24 h, 7 d, 14 d). In the first experiment, the partial gene sequences and gene expression of IL-1ß, IRAK-1, C3, C4, lysozyme, IL-10, HSP70 and ß-actin were determined in the main secondary lymphoid organs of fish: the spleen and head kidney. The second study was performed to analyse the alternative complement pathway ACH50 in serum to support the elucidation of C3 gene expression. Results of the gene expression assays showed a tendency towards up-regulation of immune genes in infected fish in early phases of infection (mostly around 6 h and 24 h) and in the chronic phase (7 d and 14 d), with the exception of HSP70 which showed a down-regulation in infected fish. Our results also suggested that lysozyme was evolved in both pro- and anti-inflammatory activities. For genes of the complement system, it was demonstrated that C4 regulation followed the tendency of pro-inflammatory genes. However, the C3 gene was, surprisingly, not expressed in most fish and this corroborated with the results of the complement system activity in serum that also did not show activity in most fish. The possible reasons for the regulation of gene expression and association with fish disease are addressed in this paper.

Motile Aeromonas septicemia in tambaqui Colossoma macropomum: Pathogenicity, lethality and new insights for control and disinfection in aquaculture.

Tambaqui Colossoma macropomum is the most produced native fish in South America. Besides the lack of knowledge regarding bacteria-stricken diseases, the unappropriated using of off-label therapies are common. In this study, Aeromonas hydrophila pathogenicity for tambaqui was first established by Koch's Postulate. Lethal doses (LD) were settled for investigation of clinical signs and mortality. The antimicrobial activities were investigated by disk-diffusion test against 11 antibiotics and by broth microdilution methods against 3 antibiotics, 7 disinfectants and 11 herbal medicines. LD experiment showed up to 80% of fish mortality, skin darkness, ulcers, hemorrhage, lethargy and hypo/anorexia in all groups, with exception of control. The LD10,50,90 and 99 were established in 4.1 × 107, 8.8 × 107, 1.9 × 108 e 3.6 × 108 CFU/mL, respectively. Ceftriaxone, florfenicol, oxytetracycline and thiamphenicol were considered promising against A. hydrophila. All herbal medicines were classified as bactericides, but clove Eugenia caryophyllata and cinnamon Cinnamomum zeylanicum displayed strongest activities. Among disinfectants, malachite green was the only that did not present acceptable values, discouraging its use. In conclusion, Koch's postulate was fulfilled and tambaqui entered to the vast list of A. hydrophila hosts and promising results of chemical substances were provided, contributing to motile Aeromonas septicemia (MAS) control.

Transcriptome Profiling of Pacu (Piaractus mesopotamicus) Challenged With Pathogenic Aeromonas hydrophila: Inference on Immune Gene Response.

Pacu (Piaractus mesopotamicus) is a Neotropical fish of major importance for South American aquaculture. Septicemia caused by Aeromonas hydrophila bacteria is currently considered a substantial threat for pacu aquaculture that have provoked infectious disease outbreaks with high economic losses. The understanding of molecular aspects on progress of A. hydrophila infection and pacu immune response is scarce, which have limited the development of genomic selection for resistance to this infection. The present study aimed to generate information on transcriptome of pacu in face of A. hydrophila infection, and compare the transcriptomic responses between two groups of time-series belonging to a disease resistance challenge, peak mortality (HM) and mortality plateau (PM) groups of individuals. Nine RNA sequencing (RNA-Seq) libraries were prepared from liver tissue of challenged individuals, generating ∼160 million 150 bp pair-end reads. After quality trimming/cleanup, these reads were assembled de novo generating 211,259 contigs. When the expression of genes from individuals of HM group were compared to individuals from control group, a total of 4,413 differentially expressed transcripts were found (2,000 upregulated and 2,413 downregulated candidate genes). Additionally, 433 transcripts were differentially expressed when individuals from MP group were compared with those in the control group (155 upregulated and 278 downregulated candidate genes). The resulting differentially expressed transcripts were clustered into the following functional categories: cytokines and signaling, epithelial protection, antigen processing and presentation, apoptosis, phagocytosis, complement system cascades and pattern recognition receptors. The proposed results revealing relevant differential gene expression on HM and PM groups which will contribute to a better understanding of the molecular defense mechanisms during A. hydrophila infection.

Cytogenetic markers as a tool for characterization of hybrids of Astyanax Baird & Girard, 1854 and Hyphessobrycon Eigenmann, 1907.

Astyanax Baird et Girard, 1854, is one of the largest genera in the family Characidae and comprises 177 valid species. This genus has been the focus of cytogenetic studies primarily owing to the presence of B chromosomes and high karyotypic diversity among different populations. The intense genetic variability in Astyanax is one of the factors responsible for the occurrence of species complexes, which are groups (1) with certain difficulties in establishing common genetic pools or (2) belonging to different cryptic species. To evaluate cytogenetic marker inheritance and the possibility of the identification of these hybrids, this study aimed to describe cytogenetic hybrids from three strains of species of the genera Astyanax and Hyphessobrycon Eigenmann, 1908. A. lacustris Lütken, 1875, A. schubarti Britski, 1964, A. fasciatus Cuvier, 1819, and H. anisitsi Eigenmann, 1907 were used to generate three hybrid lineages. The diploid number, heterochromatin sites, and ribosomal genes (18S and 5S rDNA) of the parental strains and the hybrids were analyzed. The results indicated that the three hybrid lineages had cytogenetic markers of both parents, presenting Mendelian inheritance. However, differences in distribution of heterochromatic blocks were observed between the hybrids and the parent strains. Our results allowed the identification of the hybrid strains based on the cytogenetic markers applied, reinforcing the efficiency of cytogenetic markers as tools for identification and indicating that such events may increase the karyotypic diversity in the genera Astyanax and Hyphessobrycon.

Publisher Correction: High-throughput analysis unveils a highly shared satellite DNA library among three species of fish genus Astyanax.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Corrigenda: Cytogenetic markers as a tool for characterization of hybrids of Astyanax Baird & Girard, 1854 and Hyphessobrycon Eigenmann, 1907. Comparative Cytogenetics 14(2): 231-242. https://doi.org/10.3897/CompCytogen.v14i2.49513.

[This corrects the article DOI: 10.3897/CompCytogen.v14i2.49513.].

Assessing Genetic Diversity for a Pre-Breeding Program in Piaractus mesopotamicus by SNPs and SSRs.

The pacu (Piaractus mesopotamicus) is a Neotropical fish with remarkable productive performance for aquaculture. Knowledge of genetic resources in Neotropical fish is essential for their applications in breeding programs. The aim of this study was to characterize the genetic diversity of seven farmed populations of pacu which will constitute the basis for a broodstock foundation for coming breeding programs in Brazil. Analysis of one wild population (Paraná River) was used as a reference to compare genetic parameters in the farmed populations. The analyses were performed using 32 single-nucleotide polymorphisms (SNP) and 8 simple sequence repeat (SSR) markers. No significant differences in genetic diversity between populations estimated through the number of alleles and allelic richness, observed heterozygosity, expected heterozygosity, and minimum allele frequency were detected (p > 0.05). Low genetic diversity was observed in all farmed stocks and the wild population. Moreover, we detected low genetic structure when comparing farmed and wild populations for SNPs (FST = 0.07; K = 3) and SSRs (FST = 0.08; K = 2). Analysis of molecular variance (AMOVA) demonstrated that genetic variation was mostly within populations. Kinship analysis showed that most fish farms included related individuals at a proportion of at least 25%. Our results suggest that the basal broodstock for pacu breeding programs should be founded with individuals from different fish farms for higher genetic diversity and to avoid inbreeding risks.

Satellitome landscape analysis of Megaleporinus macrocephalus (Teleostei, Anostomidae) reveals intense accumulation of satellite sequences on the heteromorphic sex chromosome.

The accumulation of repetitive DNA sequences on the sex-limited W or Y chromosomes is a well-known process that is likely triggered by the suppression of recombination between the sex chromosomes, which leads to major differences in their sizes and genetic content. Here, we report an analysis conducted on the satellitome of Megaleporinus macrocephalus that focuses specifically on the satDNAs that have been shown to have higher abundances in females and are putatively located on the W chromosome in this species. We characterized 164 satellite families in M. macrocephalus, which is, by far, the most satellite-rich species discovered to date. Subsequently, we mapped 30 satellites, 22 of which were located on the W chromosome, and 14 were shown to exist only on the W chromosome. Finally, we report two simple, quick and reliable methods that can be used for sex identification in M. macrocephalus individuals using fin clips or scales, which could be applicable to future studies conducted in the field of aquaculture.

High-throughput analysis unveils a highly shared satellite DNA library among three species of fish genus Astyanax.

The high-throughput analysis of satellite DNA (satDNA) content, by means of Illumina sequencing, unveiled 45 satDNA families in the genome of Astyanax paranae, with repeat unit length (RUL) ranging from 6 to 365 bp and marked predominance of short satellites (median length = 59 bp). The analysis of chromosomal location of 35 satDNAs in A. paranae, A. fasciatus and A. bockmanni revealed that most satellites are shared between the three species and show highly similar patterns of chromosome distribution. The high similarity in satellite DNA content between these species is most likely due to their recent common descent. Among the few differences found, the ApaSat44-21 satellite was present only on the B chromosome of A. paranae, but not on the A or B chromosomes of the two other species. Likewise, the ApaSat20-18 satellite was B-specific in A. paranae but was however present on A and B chromosomes of A. fasciatus and A. bockmanni. The isochromosome nature of B chromosomes in these species was evidenced by the symmetric location of many satDNAs on both B chromosome arms, and the lower symmetry observed in the A. fasciatus BfMa chromosome suggests that it is older than those analyzed in A. paranae and A. bockmanni.

Uncovering the Ancestry of B Chromosomes in Moenkhausia sanctaefilomenae (Teleostei, Characidae).

B chromosomes constitute a heterogeneous mixture of genomic parasites that are sometimes derived intraspecifically from the standard genome of the host species, but result from interspecific hybridization in other cases. The mode of origin determines the DNA content, with the B chromosomes showing high similarity with the A genome in the first case, but presenting higher similarity with a different species in the second. The characid fish Moenkhausia sanctaefilomenae harbours highly invasive B chromosomes, which are present in all populations analyzed to date in the Parana and Tietê rivers. To investigate the origin of these B chromosomes, we analyzed two natural populations: one carrying B chromosomes and the other lacking them, using a combination of molecular cytogenetic techniques, nucleotide sequence analysis and high-throughput sequencing (Illumina HiSeq2000). Our results showed that i) B chromosomes have not yet reached the Paranapanema River basin; ii) B chromosomes are mitotically unstable; iii) there are two types of B chromosomes, the most frequent of which is lightly C-banded (similar to euchromatin in A chromosomes) (B1), while the other is darkly C-banded (heterochromatin-like) (B2); iv) the two B types contain the same tandem repeat DNA sequences (18S ribosomal DNA, H3 histone genes, MS3 and MS7 satellite DNA), with a higher content of 18S rDNA in the heterochromatic variant; v) all of these repetitive DNAs are present together only in the paracentromeric region of autosome pair no. 6, suggesting that the B chromosomes are derived from this A chromosome; vi) the two B chromosome variants show MS3 sequences that are highly divergent from each other and from the 0B genome, although the B2-derived sequences exhibit higher similarity with the 0B genome (this suggests an independent origin of the two B variants, with the less frequent, B2 type presumably being younger); and vii) the dN/dS ratio for the H3.2 histone gene is almost 4-6 times higher for B chromosomes than for A chromosome sequences, suggesting that purifying selection is relaxed for the DNA sequences located on the B chromosomes, presumably because they are mostly inactive.

Origin of B chromosomes in the genus Astyanax (Characiformes, Characidae) and the limits of chromosome painting.

Eukaryote genomes are frequently burdened with the presence of supernumerary (B) chromosomes. Their origin is frequently investigated by chromosome painting, under the hypothesis that sharing the repetitive DNA sequences contained in the painting probes is a sign of common descent. However, the intragenomic mobility of many anonymous DNA sequences contained in these probes (e.g., transposable elements) adds high uncertainty to this conclusion. Here we test the validity of chromosome painting to investigate B chromosome origin by comparing its results for seven B chromosome types in two fish species genus Astyanax, with those obtained (1) by means of the physical mapping of 18S ribosomal DNA (rDNA), H1 histone genes, the As51 satellite DNA and the (AC)15 microsatellite, and (2) by comparing the nucleotide sequence of one of these families (ITS regions from ribosomal DNA) between genomic DNA from B-lacking individuals in both species and the microdissected DNA from two metacentric B chromosomes found in these same species. Intra- and inter-specific painting suggested that all B chromosomes that were assayed shared homologous DNA sequences among them, as well as with a variable number of A chromosomes in each species. This finding would be consistent with a common origin for all seven B chromosomes analyzed. By contrast, the physical mapping of repetitive DNA sequences failed to give support to this hypothesis, as no more than two B-types shared a given repetitive DNA. Finally, sequence analysis of the ITS regions suggested that at least some of the B chromosomes could have had a common origin.

First Description of supernumerary Chromosomes in Ictaluruspunctatus Rafinesque 1818 Reveals Active Ribosomal Genes in the B Complement.

The North American channel catfish Ictalurus punctatus Rafinesque 1818 is cultivated in the United States, Asia and Brazilian fish farms, and also utilized as a model species in aquaculture and genetic studies. In this work, cytogenetic analysis of . punctatus from Brazilian aquaculture revealed for the first time the presence of extra elements (supernumerary or B chromosomes) in this species. These elements were characterized as dot-like micro B chromosomes and were found in three individuals (varying from 0 to 1) and in one individual with higher incidence per cell (varying from 0 to 5; mean number of Bs per cell = 2.01). More specific cytogenetic techniques in this individual revealed 58 A chromosomes (standard complement) containing heterochromatic bands in the centromeric regions, a single Ag-NOR in a subtelocentric pair (also positive for 18S rDNA using the FISH technique) and multiple 5S rDNA clusters in three different subtelocentric chromosomes. Four B chromosomes were entirely Ag-NOR positive (also fully heterochromatic) and three presented 18S rDNA clusters by FISH. The occurrence of Ag-NOR and 18S ribosomal genes in both A and B chromosome complements may indicate an intraspecific origin for these extra chromosomes. Additionally, the terminal location of 18S ribosomal clusters in the Ag-NOR-bearing chromosomes and the presence of active NOR in the B chromosomes suggested that breakage events may be related to a possible recent origin of these extra elements. We suggest this data may be useful as cytogenetic information for future elucidation of the composition, origin and evolution of extra chromosomes in fishes.

New insights into karyotypic relationships among populations of Astyanax bockmanni (Teleostei, Characiformes) of different watersheds.

The fish constitute about 50% of all vertebrates, including a wide morphological and biological diversity, where the genus Astyanax is the most common and diverse, as described in virtually all freshwater environments. By occupying a basal position in the phylogeny of vertebrates, fish are an extremely favorable group for cytogenetic and evolutionary studies. The karyotype found in genus Astyanax diversity may involve a number of polymorphisms, which may be related to ploidy and karyotypic macrostructure, presence of B chromosomes, heterochromatin polymorphisms, and location of ribosomal genes. Nevertheless, the relationship between populations of this species is still poorly studied. Thus, the present work aimed to investigate karyotype variation, chromosomal relationships, and the behavior of 5S and 18S ribosomal genes in six populations of Astyanax bockmanni. The results confirmed the diploid number of 50 chromosomes in all the populations sampled, with the occurrence of one supernumerary chromosome in just one of them. In addition, all populations showed divergent patterns of constitutive heterochromatin and repetitive nucleolar sites. The fluorescence in situ hybridization (FISH) technique using 5S and 18S rDNA probes revealed distinct patterns of distribution for these conserved genes, while 5S rDNA genes were found located in two chromosome pairs, the 18S genes showed multiple marks dispersed in the genome characterizing an inter and intraindividual polymorphic behavior, as previously reported to occur with the utilization of the Ag-NOR technique. Thus, besides minor modifications observed in chromosome morphology, the populations of A. bockmanni analyzed revealed a preserved macrostructural feature, especially concerning to the diploid number; on the other hand, differences in microstructural characteristics indicated by the nucleolus organizer region (NOR) location, constitutive heterochromatin patterns, and distribution of ribosomal genes along the genome were clearly evident in the populations from different river basins, even located at short distances.

Delimiting the origin of a B chromosome by FISH mapping, chromosome painting and DNA sequence analysis in Astyanax paranae (Teleostei, Characiformes).

Supernumerary (B) chromosomes have been shown to contain a wide variety of repetitive sequences. For this reason, fluorescent in situ hybridisation (FISH) is a useful tool for ascertaining the origin of these genomic elements, especially when combined with painting from microdissected B chromosomes. In order to investigate the origin of B chromosomes in the fish species Astyanax paranae, these two approaches were used along with PCR amplification of specific DNA sequences obtained from the B chromosomes and its comparison with those residing in the A chromosomes. Remarkably, chromosome painting with the one-arm metacentric B chromosome probe showed hybridization signals on entire B chromosome, while FISH mapping revealed the presence of H1 histone and 18S rDNA genes symmetrically placed in both arms of the B chromosome. These results support the hypothesis that the B chromosome of A. paranae is an isochromosome. Additionally, the chromosome pairs Nos. 2 or 23 are considered the possible B chromosome ancestors since both contain syntenic H1 and 18S rRNA sequences. The analysis of DNA sequence fragments of the histone and rRNA genes obtained from the microdissected B chromosomes showed high similarity with those obtained from 0B individuals, which supports the intraspecific origin of B chromosomes in A. paranae. Finally, the population hereby analysed showed a female-biased B chromosome presence suggesting that B chromosomes in this species could influence sex determinism.