Molecular characterization and gene expression analysis of the pro-inflammatory cytokines IL-1ß and IL-8 in the South American fish Piaractus mesopotamicus challenged with Aeromonas dhakensis.

In the present study, the complete characterization of cDNA and genomic sequences of IL-1ß and IL-8, as well as the expression profile of these genes in the South American fish pacu (Piaractus mesopotamicus) is provided. The full-length pmIL-1ß cDNA was composed of 1208 nucleotides that would produce a precursor peptide with 273 amino acid residues. A putative caspase-1 cleavage site, similar to what is found in mammalian IL-1ß, was identified producing a mature peptide with a theoretical molecular weight of 17.21 kDa. The pmIL-8 cDNA sequence consisted of 1019 nucleotides which encoded a 95-amino acid protein with a theoretical molecular weight of 10.43 kDa that showed all typical CXC chemokine features, including a 20-residue signal peptide and four conserved cysteine residues. Constitutive mRNA expression was detected for both genes in the liver, head kidney, gill, intestine, skin and spleen. After a bacterial challenge, up-regulation was detected for both pmIL-1ß and pmIL-8 in the spleen and head kidney at 12 h post-infection. At 24 h post-infection there was a decrease in the expression of both genes, with pmIL-8 showing a significant down-regulation in the liver and head kidney when compared to the control groups.

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

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 48 h 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.

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.

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.

Complete mitochondrial genome from South American catfish Pseudoplatystoma reticulatum (Eigenmann & Eigenmann) and its impact in Siluriformes phylogenetic tree.

The cachara (Pseudoplatystoma reticulatum) is a Neotropical freshwater catfish from family Pimelodidae (Siluriformes) native to Brazil. The species is of relative economic importance for local aquaculture production and basic biological information is under development to help boost efforts to domesticate and raise the species in commercial systems. The complete cachara mitochondrial genome was obtained by assembling Illumina RNA-seq data from pooled samples. The full mitogenome was found to be 16,576 bp in length, showing the same basic structure, order, and genetic organization observed in other Pimelodidae, with 13 protein-coding genes, 2 rNA genes, 22 trNAs, and a control region. Observed base composition was 24.63% T, 28.47% C, 31.45% A, and 15.44% G. With the exception of NAD6 and eight tRNAs, all of the observed mitochondrial genes were found to be coded on the H strand. A total of 107 SNPs were identified in P. reticulatum mtDNA, 67 of which were located in coding regions. Of these SNPs, 10 result in amino acid changes. Analysis of the obtained sequence with 94 publicly available full Siluriformes mitogenomes resulted in a phylogenetic tree that generally agreed with available phylogenetic proposals for the order. The first report of the complete Pseudoplatystoma reticulatum mitochondrial genome sequence revealed general gene organization, structure, content, and order similar to most vertebrates. Specific sequence and content features were observed and may have functional attributes which are now available for further investigation.

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.

Effects of biodegradable detergents in the accumulation of lipofuscin (age pigment) in gill and liver of two neotropical fish species / Efectos de detergentes biodegradables en la acumulación de lipofuscina (pigmento de envejecimiento) en branquias e hígado de dos espécies de peces neotropicales

Currently there is a growing concern, in both population and governments, to identify the effects of substances commonly disposed of into rivers and lakes, on aquatic fauna and flora. Thus the objective of the present study was to verify effects of biodegradable detergents and water from an urban lake on gills and liver of two neotropical fish species of great economic importance, Astyanax altiparanae and Prochilodus lineatus. In order to do so, lipofuscin, also called the ageing pigment, was used as bioindicator. After one and five months of experiment both tissues accumulated this pigment. These data are discussed from physiological points of view, related with lipid peroxidation and mitochondrial damage.

Existe una preocupación creciente de la población y los gobiernos para identificar los efectos de substancias comúnmente arrojadas en ríos y lagos, sobre la fauna y flora acuática. El objetivo fue verificar los efectos de detergentes biodegradables y agua de un lago urbano sobre las branquias e hígado de dos especies de peces neo-tropicales de gran importancia económica, Astyanax altiparanae y Prochilodus lineatus. Analizamos los pigmentos de lipofuscina, también llamado pigmento de envejecimiento, el que fue utilizado como biomarcador. Después de uno y cinco meses de experimento, ambos tejidos acumulados con el pigmento fueron analizados. Los datos fueron discutidos desde el punto de vista fisiológico, relacionado con la peroxidación lipídica y daño mitocondrial.

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 Paraná 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-Guaçu River, five from Paranapanema River, three from Tietê 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 Tietê 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 Tietê 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 Tietê, Mogi-Guaçu 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 Paraná River: Grande, Peixe, and Paranapanema sub-basins.

Neste estudo, foram conduzidas análises filogenéticas e filogeográficas de populações identificadas como Hypostomus strigaticeps na bacia do alto rio Paraná a fim de testar se essas populações compreendem espécies crípticas ou populações estruturadas e avaliar a variabilidade genética das mesmas. Foram analisadas sequências do DNA mitocondrial ATP sintetase (subunidades 6/8) de 27 espécimes de 10 populações (uma do rio Mogi-Guaçu, cinco do rio Paranapanema, três do rio Tietê e uma do rio do Peixe). A análise filogeográfica mostrou a existência de oito haplótipos (A-H), e apesar do haplótipo ancestral incluir apenas indivíduos da bacia do rio Tietê, a distribuição de H. strigaticeps não se restringe a esta bacia. Os haplótipos A, B e F foram os mais frequentes. D, E, F, G e H estão presentes na sub-bacia do rio Paranapanema, dois (A e B) estão presentes na sub-bacia do rio Tietê, um (C) está exclusivamente distribuído na sub-bacia do rio do Peixe, e um (B) também está presente na sub-bacia do rio Grande. A análise filogenética mostrou que as populações de H. strigaticeps realmente formam uma unidade monofilética que compreende duas linhagens: TG, com representantes do rio Tietê, rio Mogi-Guaçu e rio do Peixe, e PP, com espécimes do rio Paranapanema. O grau de divergência genética observada nas linhagens de TG e PP foram de 0,1% e 0,2%, respectivamente, enquanto que a divergência genética entre as duas linhagens foi de aproximadamente 1%. Os resultados da análise filogenética não suportam a hipótese da existência de espécies crípticas e a análise filogeográfica confirma a presença de H. strigaticeps em outras sub-bacias do alto rio Paraná: 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 / Estrutura genética da espécie de peixe ornamental tetra Piabucus melanostomus Holmberg, 1891 (CHARACIDAE, IGUANODECTINAE) do Pantanal brasileiro inferida por sequências de DNA mitocondrial

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 Cuiabá 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.

A subfamília Iguanodectinae compreende um grupo de pequenos peixes neotropicais composta de dois gêneros e 11 espécies nominais amplamente distribuídas nas drenagens do Atlântico da América do Sul. Piabucus é o único gênero de Iguanodectinae encontrado na bacia do rio Paraguai, especialmente no Pantanal de Mato Grosso, onde é representada por Piabucus melanostomus. Dada a ampla distribuição e a baixa capacidade de dispersão desta espécie, devido às limitações ecológicas, é possível que características genéticas interessantes possam ser encontradas em diferentes populações. Deste modo, o objetivo deste trabalho foi estabelecer os padrões filogeográficos de populações de P. melanostomus utilizando sequências de DNA mitocondrial. Foram amostrados 13 indivíduos de três rios pertencentes ao Pantanal do Mato Grosso. O gene ATP sintetase (subunidades 6 e 8) foi completamente sequenciado, a média da composição de base de nucleotídeos nas sequências foi de 31,2% (T), 30,2% (C), 26,9% (A) e 11,9% (G), não havendo saturação. A análise populacional no programa TCS gerou uma rede com seis haplótipos (A a F), onde o haplótipo ancestral (A) tem uma freqüência de 25% e é composto por indivíduos dos rios Cuiabá e Paraguai. A análise filogenética mostrou a ocorrência de duas linhagens de DNA (1 e 2), a distância observada entre as duas linhagens foi de 0,6%. Os resultados filogenéticos e filogeográficos, bem como os valores negativos de FST para algumas populações, indicam uma possível ocorrência de fluxo de genes entre as populações analisadas. Estes resultados destacam a importância do pulso de inundação existente em zonas úmidas como um veículo que permite uma conexão temporária entre a população isolada, mantendo a variabilidade genética das espécies.

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

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.

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

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.

Cytogenetic markers as diagnoses in the identification of the hybrid between Piauçu (Leporinus macrocephalus) and Piapara (Leporinus elongatus)

The genetic monitoring of interspecific hybrids involves the application of methodologies able to provide an easy and indubitable genetic characterization of both parental and hybrid individuals. In the present work, cytogenetic techniques were used to identify a hybrid lineage of "Piaupara" in order to caracterize them in relation to the parental species, Leporinus macrocephalus (piauçu) and L. elongatus (piapara). The cytogenetic analysis revealed that L. macrocephalus presented 2n = 54 chromosomes and a nucleolar organizer regions (NOR) at the telomere of the long arm of the submetacentric chromosome pair 2. Analysis of constitutive heterochromatin (C-banding) revealed a conspicuous block at the pericentromeric region on the long arm of a submetacentric chromosome pair. L. elongatus presented the same diploid number, 2n = 54, and a karyotypic formula similar to that of L. macrocephalus. The NORs were also at the telomere of the long arm of the submetacentric pair 2, which was morphologically different from that of L. macrocephalus. Heterochromatic blocks were observed at both telomeres of a submetacentric chromosome pair. The hybrid "Piaupara" presented the same diploid number (2n = 54) and karyotypic formula as the parental species and there were no visible differences between parental and hybrid individuals. Differently from the Giemsa staining, NOR- and C-banding analysis showed marked differences which allowed the identification of the hybrids by the different morphology and/or size of the chromosomes carrying the NORs and patterns of heterochromatin distribution in their chromosomes. Such genetic studies are important for fish culture since they can provide tools for monitoring natural and artificial hybridization. They are also useful in biological conservation programmes and in the proper management of natural and reared fish stocks.

Supernumerary chromosomes in the pufferfish Sphoeroides spengleri: first occurrence in marine Teleostean Tetraodontiformes fish

Cytogenetic analyses carried out in eight specimens of Sphoeroides spengleri revealed the presence of 2n = 46 chromosomes (20 M/SM and 26 ST/A). Besides the standard karyotypical set, the presence of B microchromosomes was observed in two individuals, ranging from 0 to 2 microchromosomes per cell. A karyotype composed by 2n = 46 chromosomes with occurrence of M and SM chromosomes is considered basal for the species from the clade comprising the families Tetraodontidae, Balistidae, and Diodontidae, although it represents a derived condition for the order Tetraodontiformes, whose basal karyotype would be composed by 2n = 48 acrocentric chromosomes. The occurrence of B microchromosomes in marine Tetraodontiformes fish was not known, and this represents the first report of such a chromosomal type.

Karyotypic relationships among the tribes of Hypostominae (Siluriformes: Loricariidae) with description of XO sex chromosome system in a Neotropical fish species.

The family Loricariidae, with about 683 species, is one the largest fish families in the world. The subfamily Hypostominae was recently reviewed and is now divided in five tribes. With the main objective of contributing to a better understanding of the relationships of the members of the subfamily Hypostominae, cytogenetic analyses were conducted in seven species (three Hypostomini, three Pterygoplichthini and two Ancistrini) from Brazil and Venezuela. In Pterygoplichthini, all species show 2n = 52 chromosomes. In Hypostomini Hypostomus ancistroides has 2n = 68, H. regani 2n = 72 and Hypostomus goyazensis 2n = 72 chromosomes. In Ancistrini Ancistrus n. sp. 1 has 2n = 39/40 with a sex chromosome system of the type XX/X0, which is a novelty for neotropical fishes, and Ancistrus n. sp. 2 has 2n = 52 chromosomes. Six species have single Ag-NORs and two multiple Ag-NORs. The possible cytogenetic relationships among the species of Hypostominae are discussed.

Comparative cytogenetic analysis of eleven species of subfamilies Neoplecostominae and Hypostominae (Siluriformes: Loricariidae).

The family Loricariidae with about 690 species divided into six subfamilies, is one of the world's largest fish families. Recent studies have shown the existence of several problems in the definition of natural groups in the family, which has made the characterization of the subfamilies and even of some genera quite difficult. With the main objective of contributing for a better understanding of the relationships between loricariids, cytogenetic analysis were conducted with two species of Neoplecostominae and nine species of Hypostominae that, according to morphological and molecular data, may belong to a new monophyletic unit. The results obtained showed a marked chromosomal conservation with the presence of 2n = 54 chromosomes and single interstitial Ag-NORs in all species analyzed. Considering that Neoplecostominae is the primitive sister-group of all other loricariids, with exception of Lithogeneinae, this karyotypic structure may represent the primitive condition for the family Loricariidae. The cytogenetic characteristics partaken by the species of Neoplecostominae and Hypostominae analyzed in the present study reinforce the hypothesis that the species of both these subfamilies might belong to a natural group.