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Astyanax is one of the most specious fish groups in the Neotropical region, with many cryptic species, which represents a challenge for correct identification through traditional taxonomic methods. Psalidodon is a recently resurrected genus group of species previously belonging to Astyanax , specifically those with extensive chromosomal variation of the A. scabripinnis and fasciatu s complexes. In the present study, the mitochondrial genes cytochrome c oxidase subunit 1 (COI), mitochondrial ATP synthase 6 and 8 (ATPase 6/8), and NADH dehydrogenase subunit 2 (ND2) were used in conjunction with chromosomal data to characterize molecularly and cytogenetically populations of Astyanax and Psalidodon from rivers and streams of the Ivaí River Basin (Paraná Basin). The results demonstrated the effectiveness of the integrative use of molecular and cytogenetic techniques, with the confirmation of at least three species for the sampled sites: A. lacustris , P. paranae , and P. fasciatus , which showed inter- and intrapopulation karyotype variations. In addition, extensive haplotypic variation can be observed for these species within the Ivaí River Basin and throughout the Paraná River Basin. The data demonstrate a hidden diversity among the species analyzed, enrich the ichthyofaunistic knowledge of small rivers and streams, and contribute to future conservation projects in these areas.

The herbicide glyphosate (N-(phosphonomethyl)glycine) efficiently eliminates weeds, is frequently present in surface waters, and may damage the health of various non-target organisms. The main objective of this study was to investigate cytotoxic and genotoxic effects in erythrocytes, DNA, and chromosomes of native South American fish Astyanax lacustris exposed to a glyphosate-based commercial herbicide Templo®. The presenty study evaluated the presence of micronuclei (MN), chromosomal aberrations (CA), DNA damage revealed by comet assay, and cellular morphological changes (CMC) as biomarkers. The A. lacustris specimens were exposed to Templo® for 96 h at concentrations below the permitted Brazilian legislation for freshwater environments. The glyphosate-based herbicide caused MN formation, an increased incidence of CA, DNA damage, and several types of CMC in all tested concentrations on A. lacustris. Notably, analyses were significant (p<0.05) for all concentrations, except in the frequency mean of MN at 3.7 µg/L. Thus, considering the intensive use of commercial glyphosate formulations in crops, the herbicide Templo® represents a potential risk of genotoxicity and cytotoxicity for aquatic organisms. Therefore, environmental protection agencies must review regulations for glyphosate-based herbicides in freshwater environments., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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.

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.

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 ZW 1 W 2 /ZZ, with 2 n  = 31 for females and 2 n  = 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.

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., Competing Interests: Conflict of interest The authors declare that there are no conflicts of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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 2 n  = 50 chromosomes. However, one specimen contained a medium subtelocentric supernumerary chromosome (2 n  = 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.

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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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.

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., (Luciene Castuera de Oliveira, Marcos Otávio Ribeiro, Gerlane de Medeiros Costa, Cláudio Henrique Zawadzki, Ana Camila Prizon-Nakajima, Luciana Andreia Borin-Carvalho, Isabel Martins-Santos, Ana Luiza de Brito Portela-Castro.)

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, CMA 3 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.

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.

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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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.

Only one species of armored catfish, Ancistrus cirrhosus Valenciennes 1836, has been historically described in the basin of the Paraná River, from Misiones (Argentina). However, the ample variation found in the morphology and coloration of the populations sampled in the tributaries of the Brazilian state of Paraná makes it difficult to establish the real taxonomic status and evolutionary history of the Ancistrus specimens, suggesting that A. cirrhosus is not the only species found in this basin. By combining data on mitochondrial DNA (COI gene) and chromosomal markers from different Ancistrus populations, totaling 144 specimens, in the tributaries of the Paraná, and specimens from Misiones (type-locality of A. cirrhosus ), we detected five distinct evolutionary lineages. All the specimens were 2n = 50, but had four distinct karyotype formulae. The results of the Generalized Mixed Yule Coalescent (GYMC) and the genetic distances (uncorrected P -values) between lineages ranged from 3 to 5%. Clusters of 18S rDNA were observed in a single chromosome pair in seven populations of Ancistrus , but at different positions, in some cases, in synteny with the 5S rDNA sites. Multiple 5S sites were observed in all populations. Overall, the cytogenetic data reinforce the genetic evidence of the diversification of lineages, and indicate the existence of candidate species in the study region. The evidence indicates that at least four candidate species of the Ancistrus may coexist in the Paraná basin besides A. cirrhosus . Overall, our results provide a comprehensive scenario for the genetic variation among Ancistrus populations and reinforce the conclusion that the true diversity of the freshwater fish of the Neotropical regions has been underestimated.

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.