Initial Steps of XY Sex Chromosome Differentiation in the Armored Catfish Hypostomus albopunctatus (Siluriformes: Loricariidae) Revealed by Heterochromatin Accumulation.

In fish species, heterochromatinization is one process that could trigger sex chromosome differentiation. The present article describes a nascent XX/XY sex chromosome system evidenced by heterochromatin accumulation and microsatellite (GATA)8 in Hypostomus albopunctatus from two populations of the Paraná River basin. The specimens of H. albopunctatus from the Campo and Bossi Rivers share the same karyotype. The species exhibits 74 chromosomes (8m+14sm +16st +36a, fundamental number = 112). The C-banding technique suggests male heterogamety in H. albopunctatus, where the Y-chromosome is morphologically like the X-chromosome but differs from it for having long arms that are entirely heterochromatic. Double fluorescence in situ hybridization (FISH) with 18S and 5S rDNA probes confirmed the Ag-nucleolus organizer region sites in a single pair for both populations, and minor rDNA clusters showed interpopulational variation. FISH with the microsatellite (GATA)8 probe showed a dispersed pattern in the karyotype, accumulating these sequences of sex chromosomes of both populations. FISH with microsatellite (CGC)10 probe showed interpopulational variation. The absence of differentiated sex chromosomes in H. albopunctatus is described previously, and a new variant is documented herein where XY chromosomes can be seen in an early stage of differentiation.

Hidden Diversity in Eigenmannia (Gymnotiformes: Sternopygidae) Populations from Upper Paraná and Paraguay Rivers.

Eigenmannia is a highly diverse genus within the Sternopygidae family, comprising 30 species. Due to its complex taxonomy, molecular analyses have been crucial for species delimitation within this group. Therefore, the present study presents a genetic analysis using sequences of the mitochondrial gene cytochrome c oxidase subunit 1 (COI) in specimens previously identified through alpha taxonomy as E. correntes (with unpublished data), E. virescens, and E. trilineata, originating from various locations within the Upper Paraná and Paraguay River basins in Brazil. The molecular data confirm the taxonomic complexity of the genus, as individuals morphologically identified as E. virescens and E. trilineata shared the same haplotype (H52). Furthermore, the results of the species delimitation tests suggest that specimens morphologically identified as E. virescens belong to the species E. trilineata. In addition, samples morphologically identified as E. correntes may correspond to more than one Operational Taxonomic Units (OTUs). Furthermore, the intraspecific Kimura-2-parameter (K2P) distances within the different studied populations are significant. This study has contributed valuable information about genetic diversity in Eigenmannia, emphasizing the importance of using integrative analyses to resolve taxonomic conflicts within the group. It also supports biogeographical studies and assists in biodiversity conservation efforts.

Genotoxicity and cytotoxicity of textile production effluents, before and after Bacillus subitilis bioremediation, in Astyanax lacustris (Pisces, Characidae).

Textile effluents may be highly toxic and mutagenic. Monitoring studies are important for sustaining the aquatic ecosystems contaminated by these materials, which can cause damage to organisms and loss of biodiversity. We have evaluated the cyto- and genotoxicity of textile effluents on erythrocytes of Astyanax lacustris, before and after bioremediation by Bacillus subitilis treatment. We tested 60 fish (five treatment conditions, four fish per condition, in triplicate). Fish were exposed to contaminants for 7 days. The assays used were biomarker analysis, the micronucleus (MN) test, analysis of cellular morphological changes (CMC), and the comet assay. All concentrations of effluent tested, and the bioremediated effluent, showed damage significantly different from the controls. We conclude that water pollution assessment can be accomplished with these biomarkers. Biodegradation of the textile effluent was only partial, indicating the need for more thorough bioremediation to effect complete neutralization of toxicity.

Cytogenetic and Molecular Characterization of Eigenmannia aff. desantanai (Gymnotiformes: Sternopygidae): A First Report of System of Sex Chromosomes ZW1W2/ZZ in Gymnotiformes.

Gymnotiformes a monophyletic group of fish endemic to the Neotropics, represent an important component of the freshwater ichthyofauna that presents relevant taxonomic problems. Thus, in view of the morphological complexity involving Eigenmannia (Gymnotiformes) fish species, this study aimed to characterize Eigenmannia aff. desantanai of the upper Paraguay River basin through cytogenetic and molecular analyses, to help in the correct identification and delimitation of species. This study reports a multiple sex system of the type ZW1W2/ZZ, with 2n = 31 for females and 2n = 30 for males. A single pair of chromosomes carrying the nucleolar organizing regions (NORs) was detected. The heterochromatin was colocated in NOR sites and mainly located in the centromeric regions of chromosomes. Besides that, individual sequences COI from the specimens of E. aff. desantanai were obtained, totalizing three haplotypes. The distance p between the haplotypes in E. aff. desantanai, ranged from 0.2% to 7.1%. Species delimitation tests indicated the existence of two possible operational taxonomic units of E. aff. desantanai. Thus, this study reports a new multiple sex system in Gymnotiformes and these specimens previously identified as E. aff. desantanai may belong to two distinct species.

Cytogenetic and Molecular Characterization of Oligosarcus pintoi (Characidae): A New Record of Supernumerary Chromosome in this Species.

The genus Oligosarcus currently comprises 24 valid species distributed in the major river basins of South America. In this group, nine species were cytogenetically investigated, and found to share a diploid number of 50 chromosomes. Despite the conservation of the diploid number, variations in the karyotypic formula, number and position of the nucleolar organizer regions, and longitudinal bands have been described between both species and populations. In this study, we present cytogenetic and molecular data from Oligosarcus pintoi specimens from the Keller River, a tributary of the Ivaí River (Upper Paraná basin), using DNA barcoding and cytogenetic markers (C-band, silver-stained nucleolar organizer regions, and fluorescence in situ hybridization of 18S and 5S rDNA). The genetic inferences reached after analyzing the cytochrome c oxidade subunit 1 gene allowed us to confirm the identity of the individuals with 2n = 50 chromosomes. However, one specimen contained a medium subtelocentric supernumerary chromosome (2n = 51). This is the second record of additional chromosomes in O. pintoi, thereby confirming the existence of a supernumerary chromosome in allopatric populations of this species, a fact that demonstrates an evolutionary path that is divergent from other populations and/or species of Oligosarcus analyzed so far, contributing to the karyotypic diversification of the group.

Evaluation of the genotoxic and cytotoxic effects of exposure to the herbicide 2,4-dichlorophenoxyacetic acid in Astyanax lacustris (Pisces, Characidae) and the potential for its removal from contaminated water using a biosorbent.

The genotoxic and cytotoxic effects of 2,4-dichlorophenoxyacetic acid (2,4-D) on specimens of Astyanax lacustris were evaluated using different biomarkers. Additionally, this study evaluated the efficiency of an activated carbon filter made from the husks green coconut, which was used as a biosorbent to remove 2,4-D dissolved in the water, and the potential effectiveness of this procedure for the reduction of the toxic effects of this compound on A. lacustris. Three sublethal concentrations of 2,4-D (10, 20, and 40 mg L-1) were tested over 24, 48, and 72 h, and their effects on Astyanax lacustris were evaluated using chromosomal aberration test, the mitotic index, the frequency of micronuclei and nuclear alterations, and the comet assay. Exposure to 2,4-D increased the frequency of chromosomal aberrations, reduced the mitotic index, and caused significant levels of nuclear modification in some of the treatments, in comparison with the negative control. The comet assay revealed DNA damage (classes 1-3) at all 2,4-D concentrations, reaching significant levels in the 20 mg L-1 (48 h) and 40 mg L-1 (72 h) treatments. The coconut husk biosorbent was highly effective for the removal of 2,4-D and the fish exposed to the water decontaminated by this filter had low levels of cellular alteration. The findings of the present study demonstrated, for the first time, the genotoxic and cytotoxic effects of 2,4-D in Astyanax lacustris, as well as suggests the potential application of a biosorbent for the effective decontamination of water contaminated with pesticides.

Chromosomal Diversity in Two Allopatric Populations of Farlowella hahni Meinken 1937 (Teleostei: Siluriformes): Cytogenetics and Cytochrome b Analyses.

Farlowella is the second richest genus in Loricariinae, broadly distributed in freshwater streams and rivers of South America. In this article, we aimed to expand on the cytogenetic and molecular data available for two allopatric populations of Farlowella hahni. Both populations had diploid chromosome number 58, but with karyotype differences, indicative of chromosomal rearrangements. C-banding showed large heterochromatic blocks at telomeric regions in acrocentric chromosomes in both populations. Fluorescence in situ hybridization (FISH) revealed a single 18S rDNA site in both populations and a single 5S rDNA site for individuals from lower Paraná River basin (native region) and multiple 5S rDNA sites for individuals from upper Paraná River basin (non-native region). Mitochondrial sequence analyses did not separate the two F. hahni populations. The cytogenetic and molecular data obtained are relevant in a preliminary study and suggested the existence of cryptic diversity and the hypothesis that at least two Farlowella lineages may coexist in the Paraná basin.

Cytogenetic characterization of Hypostomus soniae Hollanda-Carvalho & Weber, 2004 from the Teles Pires River, southern Amazon basin: evidence of an early stage of an XX/XY sex chromosome system.

In the present study, we analyzed individuals of Hypostomus soniae (Loricariidae) collected from the Teles Pires River, southern Amazon basin, Brazil. Hypostomus soniae has a diploid chromosome number of 2n = 64 and a karyotype composed of 12 metacentric (m), 22 submetacentric (sm), 14 subtelocentric (st), and 16 acrocentric (a) chromosomes, with a structural difference between the chromosomes of the two sexes: the presence of a block of heterochromatin in sm pair No. 26, which appears to represent a putative initial stage of the differentiation of an XX/XY sex chromosome system. This chromosome, which had a heterochromatin block, and was designated proto-Y (pY), varied in the length of the long arm (q) in comparison with its homolog, resulting from the addition of constitutive heterochromatin. It is further distinguished by the presence of major ribosomal cistrons in a subterminal position of the long arm (q). The Nucleolus Organizer Region (NOR) had different phenotypes among the H. soniae individuals in terms of the number of Ag-NORs and 18S rDNA sites. The origin, distribution and maintenance of the chromosomal polymorphism found in H. soniae reinforced the hypothesis of the existence of a proto-Y chromosome, demonstrating the rise of an XX/XY sex chromosome system.

Constitutive heterochromatin heteromorphism in the Neotropical armored catfish Hypostomusregani (Ihering, 1905) (Loricariidae, Hypostominae) from the Paraguay River basin (Mato Grosso do Sul, Brazil).

A cytogenetic analysis based on the integration of a number of different chromosomal methodologies, including chromosome microdissection was carried out to characterize the chromosomally polymorphic Hypostomusregani population from the Paraguay River basin, state of Mato Grosso do Sul in Brazil. All specimens had 2n=72 (FN=116) but two distinct karyotype formulas: karyomorph A (12m+14sm+18s+28a) and karyomorph B (13m+14sm+17st+28a). Karyomorph A and B differed only for pair 19 that consisted of two subtelocentrics in karyomorph A and a large metacentric and a subtelocentric in karyomorph B. This heteromorphism was due to extensive heterochromatinization of the short arm of the large metacentric, as highlighted by C-banding. The microdissection of the large metacentric of pair 19 allowed the production of a probe, named HrV (Hypostomusregani Variant), that hybridized to the whole p arm of the large metacentric and the pericentromeric region of the short arm of its (subtelocentric) homologue (karyomorph B) and of both homologs of pair 19 in karyomorph A. Additional cytogenetic techniques (FISH with 18S and 5S rDNA probes, CMA3 and DAPI staining) allowed a finer distinction of the two karyomorphs. These results reinforced the hypothesis that the novel large metacentric of H.regani (karyomorph B) was the result of the amplification of heterochromatin segments, which contributed to karyotypic diversification in this species.

Chromosome Spreading of the Retrotransposable Rex-3 Element and Microsatellite Repeats in Karyotypes of the Ancistrus Populations.

The repetitive DNAs are the expressive substrate to genomic evolution and directly related to chromosomal diversification in eukaryote, including fishes. Ancistrus is an interesting group for studies about interplay between repetitive DNA and karyotype evolution, given its extensive chromosomal variation. In this study, we aimed to understand the evolutionary dynamics in genome of the distinct Ancistrus populations of the Paraná basin to the contribution of three classes of repetitive DNA sequences. Nucleotide sequence was isolated, characterized the nonlong terminal repeat (non-LTR) retrotransposable Rex-3, and evaluated the chromosomal organization in the Ancistrus populations. In addition, we also mapped microsatellite repeats on chromosomes. A high conserved level of the Rex-3 element was presented in Ancistrus genome sequences to record in other fish genomes. We recognized also five domains conserved in the amino acid sequence presumed from nucleotide sequence of the reverse transcriptase fragment, which indicates that it is potentially active in the genome. The physical mapping using the Rex-3 as probe revealed signals scattered throughout the chromosomes of all the Ancistrus specimens, while the microsatellite probes hybridized preferentially in the subterminal and interstitial regions. Physical mapping also reveals interplay between these two classes of repetitive DNA in some chromosome pairs. Besides, the spreading of Rex-3 signals in adjacencies of the 5S recombinant DNA (rDNA) sites could reflect their role in the dispersion of these regions. Our findings provide important insights into the mechanisms of karyotype diversification in the genus Ancistrus, which involve these repetitive sequences.

Comparative Cytogenetics Analysis on Five Genera of Thorny Catfish (Siluriformes, Doradidae): Chromosome Review in the Family and Inferences About Chromosomal Evolution Integrated with Phylogenetic Proposals.

Doradidae has been a target of phylogenetic studies over the last few years, but chromosomal information about the family is still scarce. Therefore, new cytogenetic data are provided herein and they are correlated with phylogenetic proposals to contribute to the knowledge of chromosomal evolution within doradids. Cytogenetic studies were performed on Trachydoras paraguayensis, Anadoras sp. "araguaia," Ossancora eigenmanni, Platydoras armatulus, and Rhinodoras dorbignyi. O. eigenmanni, P. armatulus, and R. dorbignyi had 2n = 58 chromosomes as found for most doradids, but T. paraguayensis and Anadoras sp. "araguaia" had 2n = 56 chromosomes, probably caused by a chromosomal reduction. There is a great maintenance of 2n = 58 verified in doradids, but karyotype formulas are diverse. Moreover, other markers (i.e., nucleolar organizer regions, heterochromatin distribution, and 5S and 18S rDNA) showed a great diversity among the analyzed species. Contrasting the variability in the chromosomal markers with the maintenance of diploid number, it is likely that inversions and translocations played an important role in chromosome differentiation in Doradidae. Herein, we created an integrative discussion linking cytogenetic data to phylogenetic proposals, based on morphological and genetic features, enabling us to identify possible cytogenetic traits, as well as probable chromosomal plesiomorphy and apomorphy of Doradidae species.

Histological and electron microscopy observations on the testis and spermatogenesis of the butterfly Dione juno (Cramer, 1779) and Agraulis vanillae (Linnaeus, 1758) (Lepidoptera: Nymphalidae).

Lepidopteran species present an interesting case of sperm polymorphism and testicular fusion. The study of these features are of great importance in understanding the reproductive biology of these insects, especially in the case of those considered pests. Dione juno and Agraulis vanillae stand out as the most important pests of passion fruit (Passiflora sp.) crops in Brazil. Therefore, the objective of the present study was to characterize the testes and germ cells of Dione juno and Agraulis vanillae at different life stages, using light microscopy and scanning and transmission electron microscopy, to understand the maturation mechanisms of the male gametes in these species. The study showed that the larvae of both species have a pair of brown kidney-shaped testes, covered by epithelial cells which divide the organ into four follicles. The testes are full of spermatogonia which begin to differentiate in the third larval instar. In the fifth larval instar, spermatozoa can be observed. When they enter the prepupal stage the testes begin a fusion process that is completed in the adult insects, where they present as spherical organs divided into eight follicles, containing all the cells of the germ line. Spermatogenesis occurs centripetally, and in both species, sperm dimorphism is observed, where two different types of spermatozoa are formed, eupyrene (nucleated) and apyrene (anucleate), which differ in morphology and function. Apart from contributing to scientific basic research on the reproductive biology of these insects, the present study provides important data that can aid in research on the physiology, systematics, and control of these species.

Chromosomal Diversity of Three Species of Hypostomus Lacépède, 1803 (Siluriformes, Loricariidae), from the Paraná River Basin, Brazil: A Species Complex in Hypostomus ancistroides Reinforced by a ZZ/ZW Sex Chromosome System.

Hypostomus shows wide morphological patterns, corroborated by great chromosomal diversity that has suggested the existence of new species, especially from small tributaries. Cytogenetic analysis has contributed to estimate a cryptic diversity providing important data for taxonomic and evolutionary studies. Cytogenetic techniques were carried out on species from a small tributary of Ivaí River, Keller River (upper Paraná River basin): Hypostomus aff. ancistroides, Hypostomus topavae, and Hypostomus aff. hermanni that presented 2n = 68, 80, and 72 chromosomes, respectively. Each species showed the same diploid number from previous descriptions for other populations but different karyotype formulas, and Hypostomus aff. ancistroides had a ZZ/ZW sex chromosome system. Multiple NORs (nucleolar organizer regions) and pericentromeric heterochromatin blocks were found in the three species. Moreover, each of them showed species-specific heterochromatins. Multiple 5S rDNA sites were detected in Hypostomus aff. ancistroides and H. topavae, whereas Hypostomus aff. hermanni had only one pair bearing these sites. In addition to the divergence in the karyotype formulas, chromosomal markers used showed karyotype differences in the three species related to other respective populations studied. Furthermore, the first description of a ZZ/ZW system for Hypostomus aff. ancistroides reinforces the hypothesis that it may correspond to a species complex and yet, confirming an unknown cryptic diversity existent in small rivers.

Activity of the endophytic fungi Phlebia sp. and Paecilomyces formosus in decolourisation and the reduction of reactive dyes' cytotoxicity in fish erythrocytes.

The current study investigates the potential for discolouration and degradation of Reactive Blue 19 and Reactive Black 5 textile dyes by endophytic fungi Phlebia sp. and Paecilomyces formosus as well as the potential cytotoxicity of products or by-products generated by the treatments in fish erythrocytes. It was observed at 30 days that both endophytes showed biodegradation activity with 0.1 g mL-1 of dyes. P. formosus showed highest extracellular and intracellular protein content levels after the 15th day, and Phlebia sp. stands out for production of extracellular laccase, indicating that this enzyme may be associated with the decolouration capacity. The dyes showed toxic effects in fishes at 0.01 g mL-1 concentration, resulting in the appearance of micronuclei in erythrocyte cells. When degraded dyes treated by endophytes were tested, the frequency of micronuclei reduced approximately 20%, indicating the effectiveness of these endophytic in the treatment of textile dyes with less environmental impact, thus indicating a potential for application of these fungi in bioremediation process.

Cytogenetic data on Ancistrus sp. (Siluriformes, Loricariidae) of the Paraguay River basin (MS) sheds light on intrageneric karyotype diversification.

Ancistrus Kner, 1854 is a diverse catfish genus, currently comprising 66 valid species, but karyotype data were recorded for 33 species, although only ten have their taxonomic status defined. Considerable karyotype diversity has been found within this genus, with 2n varying from 34 to 54 and structural variability including heteromorphic sex chromosomes. In many cases, uncertainty on the taxonomic status of the study populations hampers reliable interpretation of the complex chromosomal evolutionary history of the group. This study aims to present the first karyotype data for a population of the Ancistrus sp. collected in Criminoso stream (tributary of the Paraguay River Basin, Mato Grosso do Sul, Brazil) in which a combination of different chromosomal markers was used and results integrated in broad discussion on karyotype evolution in the genus. The specimens presented 2n=42 with 18m+16sm+8st and a single NOR revealed by silver nitrate and fluorescence in situ hybridization (FISH) with 18S rDNA probe, located in pair No. 10. Clusters of 5S rDNA were located in the pericentromeric region of three chromosomes: pair No. 1 (metacentric) and one of the homologues of the nucleolar pair No. 10. Heterogeneity in the molecular composition of the heterochromatin was confirmed by the association of C-banding and fluorochrome CMA3/DAPI-staining. Exploring the differential composition of constitutive heterochromatin in Ancistrus may provide an important perspective to understand genome organization and evolution within this group. Our data reinforce the chromosomal diversity present in Ancistrus genus and we discuss the potential sources these variation. The karyotype structure of Ancistrus sp. "Criminoso stream" appears to be consistent with the existence of a new candidate species.

Chromosomal polymorphism in Rineloricaria lanceolata Günther, 1868 (Loricariidae: Loricariinae) of the Paraguay basin (Mato Grosso do Sul, Brazil): evidence of fusions and their consequences in the population.

Rineloricaria is the most species-rich genus of the Loricariinae (armored catfish) with 65 valid species. However, the karyotype structure is known only for eight species in this group. This study provides cytogenetic data for Rineloricaria lanceolata collected from the upper Paraguay basin (Mato Grosso do Sul). The specimens revealed extensive chromosomal polymorphism constituting 10 karyotypes, which differed in the diploid number (48 to 45 chromosomes) and fundamental number (FN) between 52 and 55. Three types of chromosome variants were observed: a medium-sized submetacentric, a large submetacentric, and a small acrocentric form. Internal telomere sequences were demonstrated by a telomeric (TTAGGG)n probe in submetacentric chromosome variants, suggesting Robertsonian and tandem fusions. Considering the karyotype 2n=48 (4m+2st+42a, FN=54) as the starting point for this polymorphism, these rearrangements contributed to the reduction in diploid number (48-45). Furthermore, a remarkable polymorphism of 18S rDNA resulted in three nucleolus organizer region phenotypes (I, II, and III) with variable frequencies. Interestingly, this polymorphism has remained in the population through interbreeding between specimens, resulting in different viable combinations. The data obtained confirm that diversification/karyotype evolution in Rineloricaria was marked by numerous chromosomal rearrangements which appear to be well tolerated in the panmitic population.

First record of supernumerary (B) chromosomes in electric fish (Gymnotiformes) and the karyotype structure of three species of the same order from the upper Paraná River basin.

Cytogenetic studies were performed on the species Apteronotus prope albifrons Linnaeus, 1766, Rhamphichthys hahni Meinken, 1937 and Brachyhypopomus gauderio Giora & Malabarba, 2009, collected in the upper Paraná River floodplain, Porto Rico (PR), Brazil. Apteronotus prope albifrons showed a diploid number of 2n=24 chromosomes for both sexes and a karyotype formula of 14m+2sm+2st+6a (FN=42). Besides the standard karyotype, three specimens had one to three extra microchromosomes with inter- and intra-individual variations, which suggested the occurrence of B chromosomes in the species. The chromosomal data of Rhamphichthys hahni, described here for the first time, consists of 50 chromosomes and a formula comprised of 20m+24sm+6a (FN=94). Brachyhypopomus gauderio specimens demonstrated 2n=42 chromosomes infemales, all acrocentric, and 2n=41 chromosomes in males, with 40 acrocentric and 1 medium-sized metacentric chromosome. These differences concern with a multiple system of sexchromosome determination X1X1X2X2/X1X2Y (FN=42) in Brachyhypopomus gauderio. The analysis of nucleolar organizer regions by Ag-NOR and FISH 18S banding revealed a simple NOR system in Apteronotus prope albifrons and Rhamphichthys hahni and a multiple NOR system in Brachyhypopomus gauderio, that is unusual for Gymnotiformes fishes. Constitutive heterochromatin was mainly found in the pericentromere region in most of the chromosomes of the three species, although each species had its own peculiarities. The B chromosomes in Apteronotus prope albifrons demonstrated heterochromatin positioned in the centromeric and telomeric regions whereas Rhamphichthys hahni presented conspicuous blocks of heterochromatin on the long arms in three submetacentric pairs. Brachyhypopomus gauderio showed blocks of heterochromatin on the long arm in the interstitial and telomere positions. The finding of B chromosomes in Apteronotus prope albifrons represents the first description of these elements in the Gymnotiformes order. Although the karyotype of this species is similar with that described for populations in the Amazon basin, the presence of B chromosomes could represent a specific characteristic of this population. A comparative analysis of karyotypes of Rhamphichthys hahni with other species of the genus showed a relatively conservative structure suggesting 2n=50 as a common number in this group. The karyotype of Brachyhypopomus gauderio, a new species, provides an important reference for future chromosome studies of the Brachyhypopmus Mago-Lecia, 1994, and it might be also significant for cytotaxonomy in this group. The cytogenetic data also demonstrate the need of more comparative cytogenetic studies in the families of the highly diversified and taxonomically difficult complex Gymnotiformes.