A high-density linkage map and sex-linked markers for the Amazon Tambaqui Colossoma macropomum.

BACKGROUND: Tambaqui (Colossoma macropomum, Cuvier, 1818) is the most economically important native freshwater fish species in Brazil. It can reach a total length of over 1 m and a weight of over 40 kg. The species displays a clear sex dimorphism in growth performance, with females reaching larger sizes at harvest. In aquaculture, the production of monosex populations in selective breeding programmes has been therefore identified as a key priority. RESULTS: In the present study, a genetic linkage map was generated by double digest restriction-site associated DNA (ddRAD) sequencing from 248 individuals sampled from two F1 families. The map was constructed using 14,805 informative SNPs and spanned 27 linkage groups. From this, the tambaqui draft genome was improved, by ordering the scaffolds into chromosomes, and sex-linked markers were identified. A total of 235 markers on linkage group 26 showed a significant association with the phenotypic sex, supporting an XX/XY sex determination system in the species. The four most informative sex-linked markers were validated on another 206 sexed individuals, demonstrating an accuracy in predicting sex ranging from 90.0 to 96.7%. CONCLUSIONS: The genetic mapping and novel sex-linked DNA markers identified and validated offer new tools for rapid progeny sexing, thus supporting the development of monosex female production in the industry while also supporting breeding programmes of the species.

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.

Genetic diversity and structure in Arapaima gigas populations from Amazon and Araguaia-Tocantins river basins.

BACKGROUND: Arapaima gigas (Schinz, 1822) is the largest freshwater scaled fish in the world, and an emerging species for tropical aquaculture development. Conservation of the species, and the expansion of aquaculture requires the development of genetic tools to study polymorphism, differentiation, and stock structure. This study aimed to investigate genomic polymorphism through ddRAD sequencing, in order to identify a panel of single nucleotide polymorphisms (SNPs) and to simultaneously assess genetic diversity and structure in wild (from rivers Amazon, Solimões, Tocantins and Araguaia) and captive populations. RESULTS: Compared to many other teleosts, the degree of polymorphism in A. gigas was low with only 2.3% of identified RAD-tags (135 bases long) containing SNPs. A panel of 393 informative SNPs was identified and screened across the five populations. Higher genetic diversity indices (number of polymorphic loci and private alleles, Shannon's Index and HO) were found in populations from the Amazon and Solimões, intermediate levels in Tocantins and Captive, and very low levels in the Araguaia population. These results likely reflect larger population sizes from less urbanized environments in the Amazon basin compared to Araguaia. Populations were significantly differentiated with pairwise FST values ranging from 0.086 (Amazon × Solimões) to 0.556 (Amazon × Araguaia). Mean pairwise relatedness among individuals was significant in all populations (P < 0.01), reflecting a degree of inbreeding possibly due to severe depletion of natural stocks, the species sedentary behaviour and possible sampling biases. Although Mantel test was not significant (P = 0.104; R2 = 0.65), Bayesian analysis in STRUCTURE and discriminant analysis of principal components (DAPC) showed populations of Amazon and Solimões to be genetically differentiated from Araguaia, with Tocantins comprising individuals from both identified stocks. CONCLUSIONS: This relatively rapid genotyping by sequencing approach proved to be successful in delineating arapaima stocks. The approach and / or SNP panels identified should prove valuable for more detailed genetic studies of arapaima populations, including the elucidation of the genetic status of described discrete morphotypes and aid in delivery of conservation programs to maintain genetic diversity in reservoirs across the Amazon region.

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.

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.

Molecular identification, phylogeny and geographic distribution of Brazilian mangrove oysters (Crassostrea).

Oysters (Ostreidae) manifest a high degree of phenotypic plasticity, whereby morphology is of limited value for species identification and taxonomy. By using molecular data, the aim was to genetically characterize the species of Crassostrea occurring along the Brazilian coast, and phylogenetically relate these to other Crassostrea from different parts of the world. Sequencing of the partial cytochrome oxidase c subunit I gene (COI), revealed a total of three species of Crassostrea at 16 locations along the Brazilian coast. C. gasar was found from Curuçá (Pará state) to Santos (São Paulo state), and C. rhizophorae from Fortim (Ceará state) to Florianópolis (Santa Catarina state), although small individuals of the latter species were also found at Ajuruteua beach (municipality of Bragança, Pará state). An unidentified Crassostrea species was found only on Canela Island, Bragança. Crassostrea gasar and C. rhizophorae grouped with C. virginica, thereby forming a monophyletic Atlantic group, whereas Crassostrea sp. from Canela Island was shown to be more similar to Indo-Pacific oysters, and either arrived in the Atlantic Ocean before the convergence of the Isthmus of Panama or was accidentally brought to Brazil by ship.

Isolation and characterization of 11 microsatellite loci in the mangrove crab, Ucides cordatus (Ocypodidae: Ucidinae).

Eleven polymorphic microsatellite loci were described for the mangrove crab, Ucides cordatus, an important fishery resource on the Brazilian coast. The number of alleles observed at each locus varied between eight and 23. Observed and expected mean heterozygosities were 0.791 and 0.893 respectively. Amplification of all loci was highly successful, under the same polymerase chain reaction conditions. With the exception of P2D3, all loci adhered to the assumptions of the Hardy-Weinberg equilibrium and did not present deviations reflecting linkage disequilibrium. Given this, these markers will be extremely useful in future management programmes for U. cordatus stocks.

Molecular identification, phylogeny and geographic distribution of Brazilian mangrove oysters (Crassostrea)

Oysters (Ostreidae) manifest a high degree of phenotypic plasticity, whereby morphology is of limited value for species identification and taxonomy. By using molecular data, the aim was to genetically characterize the species of Crassostrea occurring along the Brazilian coast, and phylogenetically relate these to other Crassostrea from different parts of the world. Sequencing of the partial cytochrome oxidase c subunit I gene (COI), revealed a total of three species of Crassostrea at 16 locations along the Brazilian coast. C. gasar was found from Curuçá (Pará state) to Santos (São Paulo state), and C. rhizophorae from Fortim (Ceará state) to Florianópolis (Santa Catarina state), although small individuals of the latter species were also found at Ajuruteua beach (municipality of Bragança, Pará state). An unidentified Crassostrea species was found only on Canela Island, Bragança. Crassostrea gasar and C. rhizophorae grouped with C. virginica, thereby forming a monophyletic Atlantic group, whereas Crassostrea sp. from Canela Island was shown to be more similar to Indo-Pacific oysters, and either arrived in the Atlantic Ocean before the convergence of the Isthmus of Panama or was accidentally brought to Brazil by ship.