Anderson Luis Alves

Cytogenetic analysis in the incertae sedis species Astyanax altiparanae Garutti and Britzki, 2000 and Hyphessobrycon eques Steindachner, 1882 (Characiformes, Characidae) from the upper Paraná river basin.

Abstract Cytogenetic analyses were accomplished in two populations of Astyanax altiparanae Garutti & Britzki, 2000 and one population of Hyphessobrycon eques Steindachner, 1882, considered incertae sedis in Characidae family. Two populations of Astyanax altiparanae (Mogi-Guaçu and Tietê rivers) presented 2n=50, with the same karyotype formula: 6M+12SM+20ST+12A (FN=88). Hyphessobrycon eques from Capivara river presented 2n=52 and karyotype formula 14M+16SM+4ST+18A (FN=86). In each karyotype, the nucleolus organizer regions were detected at the end of the short arm of a single medium-sized subtelocentric chromosome. The Chromomycin A3 (CMA3) marking is coincident for the NORs in chromosomes of the two species and present additionally in two different chromosomes of Astyanax altiparanae thus showinginterpopulation differences in this species. In Hyphessobrycon eques, weak heterochromatic blocks in the position of centromeres and telomeres of most chromosomes and negative C-banding for the NOR bearing chromosome were visualized. The obtained results contribute both to the understanding of karyotype evolution of these species and to the clarifying their phylogenetic relationships.

Chromosomal microstructure diversity in three Astyanax (Characiformes, Characidae) species: comparative analysis of the chromosomal locations of the 18S and 5S rDNAs.

The species of genus Astyanax is widely distributed in freshwater neotropical zones. Astyanax is considered to be taxonomically confused, similar to other genera placed incertae sedis in Characidae. The cytogenetics of this genus is well characterized; species vary widely in diploid number, from 2n=36 chromosomes in Astyanax schubarti to 2n=50 for most species studied. The size, number, and position of different cytological markers vary among species and populations of Astyanax. We analyzed the karyotypes of individuals from three Astyanax species (Astyanax abramis, Astyanax altiparanae, and Astyanax eigenmanniorum) from populations not previously analyzed. We describe variations in several cytogenetic markers and the karyotypic relationships between them, specifically focusing on the characteristics of the conserved and divergent locations of the ribosomal genes. Our data are useful for establishing relationships between species and for investigating the karyotype evolution within the genus.

Phylogeography of Hypostomus strigaticeps (Siluriformes: Loricariidae) inferred by mitochondrial DNA reveals its distribution in the upper Paraná River basin

In this study, phylogenetic and phylogeographic analyses of populations identified as Hypostomus strigaticeps from the upper Parana River basin were conducted in order to test whether these different populations comprises cryptic species or structured populations and to assess their genetic variability. The sequences of the mitochondrial DNA ATP sintetase (subunits 6/8) of 27 specimens from 10 populations (one from Mogi-Guacu River, five from Paranapanema River, three from Tiete River and one from Peixe River) were analyzed. The phylogeographic analysis showed the existence of eight haplotypes (A-H), and despite the ancestral haplotype includes only individuals from the Tiete River basin, the distribution of H. strigaticeps was not restricted to this basin. Haplotypes A, B and F were the most frequent. Haplotypes D, E, F, G, and H were present in the sub-basin of Paranapanema, two (A and B) were present in the sub-basin of the Tiete River, one (C) was exclusively distributed in the sub-basin of the Peixe River, and one (B) was also present in the sub-basin of the Grande River. The phylogenetic analysis showed that the populations of H. strigaticeps indeed form a monophyletic unit comprising two lineages: TG, with representatives from the Tiete, Mogi-Guacu and Peixe Rivers; and PP, with specimens from the Paranapanema River. The observed degree of genetic divergence within the TG and PP lineages was 0.1% and 0.2%, respectively, whereas the genetic divergence between the two lineages themselves was approximately 1%. The results of the phylogenetic analysis do not support the hypothesis of existence of crypt species and the phylogeographic analysis confirm the presence of H. strigaticeps in other sub-basins of the upper Parana River: Grande, Peixe, and Paranapanema sub-basins. Neste estudo, foram conduzidas analises filogeneticas e filogeograficas de populacoes identificadas como Hypostomus strigaticeps na bacia do alto rio Parana a fim de testar se essas populacoes compreendem especies cripticas ou populacoes estruturadas e avaliar a variabilidade genetica das mesmas. Foram analisadas sequencias do DNA mitocondrial ATP sintetase (subunidades 6/8) de 27 especimes de 10 populacoes (uma do rio Mogi-Guacu, cinco do rio Paranapanema, tres do rio Tiete e uma do rio do Peixe). A analise filogeografica mostrou a existencia de oito haplotipos (A-H), e apesar do haplotipo ancestral incluir apenas individuos da bacia do rio Tiete, a distribuicao de H. strigaticeps nao se restringe a esta bacia. Os haplotipos A, B e F foram os mais frequentes. D, E, F, G e H estao presentes na sub-bacia do rio Paranapanema, dois (A e B) estao presentes na sub-bacia do rio Tiete, um (C) esta exclusivamente distribuido na sub-bacia do rio do Peixe, e um (B) tambem esta presente na sub-bacia do rio Grande. A analise filogenetica mostrou que as populacoes de H. strigaticeps realmente formam uma unidade monofiletica que compreende duas linhagens: TG, com representantes do rio Tiete, rio Mogi-Guacu e rio do Peixe, e PP, com especimes do rio Paranapanema. O grau de divergencia genetica observada nas linhagens de TG e PP foram de 0,1% e 0,2%, respectivamente, enquanto que a divergencia genetica entre as duas linhagens foi de aproximadamente 1%. Os resultados da analise filogenetica nao suportam a hipotese da existencia de especies cripticas e a analise filogeografica confirma a presenca de H. strigaticeps em outras sub-bacias do alto rio Parana: sub-bacias do rio Grande, rio do Peixe e rio Paranapanema.

Genetic structure of the ornamental tetra fish species Piabucus melanostomus Holmberg, 1891 (CHARACIDAE, IGUANODECTINAE) in the Brazilian Pantanal wetlands inferred by mitochondrial DNA sequences

The subfamily Iguanodectinae comprises a group of small Neotropical fishes composed by two genera and 11 nominal species widely distributed in the Atlantic drainages of South America. Piabucus is the only genus of Iguanodectinae found in the Paraguay River basin, especially in the Pantanal of Mato Grosso State, where it is represented by Piabucus melanostomus. Given the wide distribution and the low dispersion capacity of this species, due the ecological constraints, it is possible that many interesting genetic features could be found in different populations. In this way, the aim of his work was to perform the phylogeographic pattern of P. melanostomus populations using mitochondrial DNA sequences. A total of 13 individuals from three rivers belonging the Mato Grosso wetland were sampled. The ATP sintetase (subunits 6 and 8) gene was completely sequenced, the mean of nucleotide base composition in the sequences was 31.2% (T), 30.2% (C), 26.9% (A) and 11.9% (G), with no gene saturation. The population analysis in the TCS program generated a network with six haplotypes (A to F), where the ancestral haplotype (A) has a frequency of 25% and is composed by individuals from Cuiaba and Paraguay Rivers. The phylogenetic analysis showed the occurrence of two mtDNA lineages (1 and 2), the distance observed between the two lineages was 0.6%. The phylogenetic and phylogeographic results as well as the negative values of Fst for some populations, indicate a possible occurrence of gene flow among the analyzed populations. These results highlights the importance of flood pulse existent on wetland as a vehicle that permits a temporary connection among isolated population maintaining the species genetic variability.

Localization of 18S ribosomal genes in suckermouth armoured catfishes Loricariidae (Teleostei, Siluriformes) with discussion on the Ag-NOR evolution

Abstract The family Loricariidae with about 690 species divided into six subfamilies, is one of the world’s largest fish families. Cytogenetic studies conducted in the family showed that among 90 species analyzed the diploid number ranges from 2n=38 in Ancistrus sp. to 2n=96 in Hemipsilichthys gobio Luetken, 1874. In the present study, fluorescence in situ hybridization (FISH) was employed to determine the chromosomal localization of the 18S rDNA gene in four suckermouth armoured catfishes: Kronichthys lacerta (Nichols, 1919), Pareiorhaphis splendens (Bizerril, 1995), Liposarcus multiradiatus (Hancock, 1828) and Hypostomus prope plecostomus (Linnaeus, 1758). All species analyzed showed one chromosome pair with 18S rDNA sequences, as observed in the previous Ag-NORs analyses. The presence of size and numerical polymorphism was observed and discussed, with proposing a hypothesis of the Ag-NOR evolution in Loricariidae.

Phylogenetic relationships of Simpsonichthys subgenera (Cyprinodontiformes, Rivulidae), including a proposal for a new genus

The systematic knowledge of Simpsonichthys has changed substantially in recent years, with five subgenera having been elevated to the genus category. In view of these taxonomic changes, the aim of the present study was to identify the phylogenetic relationships among this group in order to test a hypothesis of the division of Simpsonichthys. The ATPase 8 and 6 gene sequences of 53 specimens of Simpsonichthys, Xenurolebias, Ophthalmolebias, Spectrolebias and Hypsolebias genera were assessed. The final topology divided the Simpsonichthys subgenera into five clades: clade I (Hypsolebias antenori group and Ophtalmolebias), clade II (Simpsonichtys and Spectrolebias), clade III (Hypsolebias flammeus and H. magnificus groups), clade IV (H. notatus group) and clade V, which included the Xenurolebias genus as a sister group to all the other Simpsonichthys subgenera. Simpsonichthys, Spectrolebias and Hypsolebias could therefor not be described as monophyletic groups, as has been proposed in some hypotheses. The H. antenori, H. notatus and H. magnificus groups, however, were monophyletic. The molecular results also suggested that H. ocellatus belongs to the Hypsolebias flammeus group. The phylogenetic position of H. fasciatus suggested a new group within Hypsolebias, or possibly even a new genus. The characteristics of the Hypsolebias antenori group and their phylogenetic position suggested that only the species of this group should be included in the Hypsolebias genus. The basal position of the Hypsolebias notatus group and its morphological character may represent a new genus. Although the monophyletism of Simpsonichtys could not be recovered, the branches supported the division of the western and eastern clades, possibly due to allopatric speciation. These facts suggest the division of the genus into at least two major clades. In addition, the study of morphological and molecular data is suggested to obtain a better understanding of such complex organisms, and a major taxonomic review is required.

Effects of Biodegradable Detergents in the Accumulation of Lipofuscin (Age Pigment) in Gill and Liver of Two Neotropical Fish Species

Existe una preocupacion creciente de la poblacion y los gobiernos para identificar los efectos de substancias comunmente arrojadas en rios y lagos, sobre la fauna y flora acuatica. El objetivo fue verificar los efectos de detergentes biodegradables y agua de un lago urbano sobre las branquias e higado de dos especies de peces neo-tropicales de gran importancia economica, Astyanax altiparanae y Prochilodus lineatus. Analizamos los pigmentos de lipofuscina, tambien llamado pigmento de envejecimiento, el que fue utilizado como biomarcador. Despues de uno y cinco meses de experimento, ambos tejidos acumulados con el pigmento fueron analizados. Los datos fueron discutidos desde el punto de vista fisiologico, relacionado con la peroxidacion lipidica y dano mitocondrial.

Karyotypic diversity and evolutionary trends in the Neotropical catfish genus Hypostomus Lacépède, 1803 (Teleostei, Siluriformes, Loricariidae)

Abstract The family Loricariidae with 813 nominal species is one of the largest fish families of the world. Hypostominae, its more complex subfamily, was recently divided into five tribes. The tribe Hypostomini is composed of a single genus, Hypostomus Lacépède, 1803, which exhibits the largest karyotypic diversity in the family Loricariidae. With the main objective of contributing to a better understanding of the relationship and the patterns of evolution among the karyotypes of Hypostomus species, cytogenetic studies were conducted in six species of the genus from Brazil and Venezuela. The results show a great chromosome variety with diploid numbers ranging from 2n=68 to 2n=76, with a clear predominance of acrocentric chromosomes. The Ag-NORs are located in terminal position in all species analyzed. Three species have single Ag-NORs (Hypostomus albopunctatus (Regan, 1908), Hypostomus prope plecostomus (Linnaeus, 1758), and Hypostomus prope paulinus (Ihering, 1905)) and three have multiple Ag-NORs (Hypostomus ancistroides (Ihering, 1911), Hypostomus prope iheringi (Regan, 1908), and Hypostomus strigaticeps (Regan, 1908)). In the process of karyotype evolution of the group, the main type of chromosome rearrangements was possibly centric fissions, which may have been facilitated by the putative tetraploid origin of Hypostomus species. The relationship between the karyotype changes and the evolution in the genus is discussed.

Quantifying structural modifications of gills of two fish species Astyanax altiparanae (Lambari) and Prochilodus lineatus (Curimbatá) after exposure to biodegradable detergents in urban lake water

ABSTRACT Anthropic actions in rivers and urban lakes are a cause for concern to our ecosystem. The effects on fauna and flora of substances discharged into waterways have become a focus for investigations globally. Biodegradable detergents are widely used in residences and small industries, but little is known regarding the consequences on fish fauna. The objective of the present study was to identify modifications in gill structure in two fish species, Astyanax altiparanae and Prochilodus lineatus, after treatment with water obtained from an urban lake and an exposure to 1 ppm diluted biodegradable detergents (linear alkylbenzene sulfonate). Data demonstrated exposure to urban lake produced various alterations in gill functions such as lamellar fusions, aneurysms, mucous, and chlorine cell proliferation, which may be attributed to the presence of detergents in the water but may also be a consequence of synergetic actions of detergents with other pollutants. Results showed that the levels of NO−2, Na, F–, Cl–, and Fe were significantly higher in urban lake water but in the presence of detergents Ni was also detected. Evidence indicates that biodegradable detergents produce damage to gill functions, which subsequently alters the fish physiology and reduces the ability to cope with stress and survival.

Characterization and gene expression analysis of pacu (Piaractus mesopotamicus) inducible nitric oxide synthase (iNOS) following Aeromonas dhakensis infection

ABSTRACT Nitric oxide (NO) is an important effector molecule which is involved in a myriad of biological processes, including immune responses against pathogens such as parasites, virus and bacteria. During the inflammatory processes in vertebrates, NO is produced by the inducible nitric oxide synthase (iNOS) enzyme in practically all nucleated cells to suppress or kill intracellular pathogens. The aim of the present study was to characterize the full coding region of the iNOS gene of pacu (Piaractus mesopotamicus), an economically and ecologically important South American fish species, and to analyze mRNA expression levels following intraperitoneal infection with the pathogenic bacterium Aeromonas dhakensis by means of quantitative real time PCR (qPCR). The results showed that the pacu iNOS transcript is 3237 bp in length, encoding a putative protein composed of 1078 amino acid residues. The amino acid sequence showed similarities ranging from 69.03% to 94.34% with other teleost fish and 57.70% with the human iNOS, with all characteristic domains and cofactor binding sites of the enzyme detected. Phylogenetic analysis showed that the iNOS from the red‐bellied piranha, another South American characiform, was the closest related sequence to the pacu iNOS. iNOS transcripts were constitutively detected in the liver, spleen and head kidney, and there was a significant upregulation in the liver and spleen at 12, 24 and 48h after infection with A. dhakensis. No significant variations were observed in the head kidney during the periods analyzed. These results show that iNOS expression was induced by A. dhakensis infection and suggest that this enzyme may be involved in the response to this bacterium in pacu. HIGHLIGHTSThe complete iNOS mRNA coding sequence from pacu is characterized.The pacu iNOS domain and motif structure is provided.The phylogenetic relationship of pacu iNOS is compared to other vertebrates.iNOS transcription is significantly up‐regulated in the liver, spleen and head kidney of pacu after A. dhakensis infection.