Post-feeding Molecular Responses of Cobia (Rachycentron canadum): RNA-Sequencing as a Tool to Evaluate Postprandial Effects in Hepatic Lipid Metabolism.

We used transcriptome sequencing to investigate the hepatic postprandial responses of Rachycentron canadum (cobia), an important commercial fish species. In total, 150 cobia juveniles (50 per tank, triplicate) were fed ad libitum with a commercial diet for 7 days, fasted for 24 h, and fed for 10 min. The liver was sampled 10 min prior to feeding and 30 min, 1, 2, 4, 8, 12, and 24 h after the feeding event. Each sample was evaluated in terms of liver fatty acid profile and gene expression. Differential gene expressions were evaluated, focusing on fatty acid synthesis and oxidation pathways. In general, the liver fatty acid profile reflected diet composition. Docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and n-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA) levels increased at 8 to 12 h but decreased at 24 h after the feeding event. A high number of differentially expressed genes (DEGs) were observed comparing fish that fasted for 8 h with those fasted for 30 min and 24 h, while a reduced number of DEGs was observed comparing individuals who fasted for 30 min compared with those who fasted for 24 h. Similarly, the main differences in the expression of genes related to the fatty acid biosynthesis and oxidation pathways were noticed in individuals who fasted for 8 h compared with those who fasted for 30 min and 24 h. The results suggested that the adequate time to sample the individuals ranged between 8 and 12 h after the meal since, apparently, after 24 h, differential gene expression was not necessarily influenced by food intake.

Transcriptomic Profiling and Microsatellite Identification in Cobia (Rachycentron canadum), Using High-Throughput RNA Sequencing.

Cobia (Rachycentron canadum) is a marine teleost species with great productive potential worldwide. However, the genomic information currently available for this species in public databases is limited. Such lack of information hinders gene expression assessments that might bring forward novel insights into the physiology, ecology, evolution, and genetics of this potential aquaculture species. In this study, we report the first de novo transcriptome assembly of R. canadum liver, improving the availability of novel gene sequences for this species. Illumina sequencing of liver transcripts generated 1,761,965,794 raw reads, which were filtered into 1,652,319,304 high-quality reads. De novo assembly resulted in 101,789 unigenes and 163,096 isoforms, with an average length of 950.61 and 1,617.34 nt, respectively. Moreover, we found that 126,013 of these transcripts bear potentially coding sequences, and 125,993 of these elements (77.3%) correspond to functionally annotated genes found in six different databases. We also identified 701 putative ncRNA and 35,414 putative lncRNA. Interestingly, homologues for 410 of these putative lncRNAs have already been observed in previous analyses with Danio rerio, Lates calcarifer, Seriola lalandi dorsalis, Seriola dumerili, or Echeneis naucrates. Finally, we identified 7894 microsatellites related to cobia's putative lncRNAs. Thus, the information derived from the transcriptome assembly described herein will likely assist future nutrigenomics and breeding programs involving this important fish farming species.

Variant expression signatures of microRNAs and protein related to growth in a crossbreed between two strains of Nile tilapia (Oreochromis niloticus).

Nile tilapia (Oreochromis niloticus) is a species of worldwide importance for aquaculture. A crossbred lineage was developed through introgressive backcross breeding techniques and combines the high growth performance of the Chitralada (CHIT) lwith attractive reddish color of the Red Stirling (REDS) strains. Since the crossbreed has an unknown genetically improved background, the objective of this work was to characterize expression signatures that portray the advantageous phenotype of the crossbreeds. We characterized the microRNA transcriptome by high throughput sequencing (RNA-seq) and the proteome through mass spectrometry (ESI-Q-TOF-MS) and applied bioinformatics for the comparative analysis of such molecular data on the three strains. Crossbreed expressed a distinct set of miRNAs and proteins compared to the parents. They comprised several microRNAs regulate traits of economic interest. Proteomic profiles revealed differences between parental and crossbreed in expression of proteins associated with glycolisis. Distinctive miRNA and protein signatures contribute to the phenotype of crossbreed.

Use of mucus as a non-invasive sampling method for DNA barcoding of stingrays and skates (batoid elasmobranchs).

In this study we tested the use of mucus from five species of Neotropical marine batoid elasmobranchs to extract genomic DNA for barcoding and phylogenetic analysis. The DNA from all individuals sampled was successfully amplified and sequenced for molecular barcode, allowing 99-100% accuracy to the species level. This method proved to provide reliable and good-quality DNA for barcoding and phylogenetic analysis of Neotropical elasmobranchs, through rapid handling and with low disturbance to animals.

Phylogeography and Historical Biogeography of the Astyanax bimaculatus Species Complex (Teleostei: Characidae) in Coastal Southeastern South America.

Astyanax bimaculatus, a ubiquitous species in many Neotropical basins, is characterized by a complex taxonomy and are currently considered a species complex. The goal of this study was to analyze 31 populations (N = 136) of this species from southeastern Brazil using cytogenetic techniques: conventional staining, nucleolar organizer region (Ag-NOR), C-banding, and 18S and 5S recombinant DNA fluorescent in situ hybridization (FISH) probes; and molecular techniques: S72, RAG2, and COI. All populations were 2n = 50 (6m + 20sm +18st +6a); Ag-NORs were predominantly simple, C-banding revealed high variation levels within and among basins, and the FISH probes 18S and 5S were restricted to chromosome pairs 14 and 7, respectively. The S72 was uninformative for phylogenetic analyses, and RAG2 showed no variation among populations. The COI gene revealed three haplogroups. The most basal was composed of Pandeiros population (São Francisco Basin) that diverged in the Middle Miocene. The second was composed of A. altiparanae from the Upper Paraná Basin and Espírito Santo Stream (Paraíba do Sul Basin), whereas the third was composed of Astyanax lacustris from São Francisco and coastal basins. The second and third haplogroups diverged in the Pleistocene, indicating that diversification of the bimaculatus complex was driven by tectonic activity and sea-level fluctuations.

Genome-wide microRNA screening in Nile tilapia reveals pervasive isomiRs' transcription, sex-biased arm switching and increasing complexity of expression throughout development.

MicroRNAs (miRNAs) are key regulators of gene expression in multicellular organisms. The elucidation of miRNA function and evolution depends on the identification and characterization of miRNA repertoire of strategic organisms, as the fast-evolving cichlid fishes. Using RNA-seq and comparative genomics we carried out an in-depth report of miRNAs in Nile tilapia (Oreochromis niloticus), an emergent model organism to investigate evo-devo mechanisms. Five hundred known miRNAs and almost one hundred putative novel vertebrate miRNAs have been identified, many of which seem to be teleost-specific, cichlid-specific or tilapia-specific. Abundant miRNA isoforms (isomiRs) were identified with modifications in both 5p and 3p miRNA transcripts. Changes in arm usage (arm switching) of nine miRNAs were detected in early development, adult stage and even between male and female samples. We found an increasing complexity of miRNA expression during ontogenetic development, revealing a remarkable synchronism between the rate of new miRNAs recruitment and morphological changes. Overall, our results enlarge vertebrate miRNA collection and reveal a notable differential ratio of miRNA arms and isoforms influenced by sex and developmental life stage, providing a better picture of the evolutionary and spatiotemporal dynamics of miRNAs.

Fishing Into the MicroRNA Transcriptome.

In the last decade, several studies have been focused on revealing the microRNA (miRNA) repertoire and determining their functions in farm animals such as poultry, pigs, cattle, and fish. These small non-protein coding RNA molecules (18-25 nucleotides) are capable of controlling gene expression by binding to messenger RNA (mRNA) targets, thus interfering in the final protein output. MiRNAs have been recognized as the main regulators of biological features of economic interest, including body growth, muscle development, fat deposition, and immunology, among other highly valuable traits, in aquatic livestock. Currently, the miRNA repertoire of some farmed fish species has been identified and characterized, bringing insights about miRNA functions, and novel perspectives for improving health and productivity. In this review, we summarize the current advances in miRNA research by examining available data on Neotropical and other key species exploited by fisheries and in aquaculture worldwide and discuss how future studies on Neotropical fish could benefit from this knowledge. We also make a horizontal comparison of major results and discuss forefront strategies for miRNA manipulation in aquaculture focusing on forward-looking ideas for forthcoming research.

Genetic Diversity of an Imperiled Neotropical Catfish and Recommendations for Its Restoration.

The long-whiskered catfish Steindachneridion parahybae (Family Pimelodidae) is endemic to the Paraíba do Sul River basin in southeastern Brazil. This species was heavily exploited by artisanal fisheries and faces challenges posed by dams, introduced species, and deterioration of critical habitat. The remaining populations are small and extirpated from some locales, and the species is listed as critically endangered in Brazil. Screening variation at a partial mitochondrial control region sequence (mtCR) and 20 microsatellite loci, we: (i) describe the patterns of genetic diversity along its current distributional range; (ii) test the null hypothesis of panmixia; (iii) investigate the main factors driving its current population structure, and (iv) propose management of broodstock for fostering recovery of wild populations through genetically cognizant restocking. Our microsatellite data for 70 individuals from five collections indicate moderate levels of heterozygosity (HO = 0.45) and low levels of inbreeding (FIS = 0.016). Individual-based cluster analyses showed clear genetic structure, with three clusters of individuals over the collection area with no mis-assigned individuals, suggesting no recent migration among the three clusters. Pairwise DEST values showed moderate and significant genetic differentiation among all populations so identified. The MUR population may have suffered a recent demographic reduction. mtCRs for 70 individuals exhibited 36 haplotypes resulting from 38 polymorphic sites. Overall, mitochondrial haplotype diversity was 0.930 (±0.023) and nucleotide diversity was 0.011 (±0.002). Significant population structure was observed, with ϕST = 0.226. Genetic markers could be used in a hatchery-based restoration program emphasizing breeding of pairs with low kinship values in order to promote retention of genetic diversity and avoid inbreeding. Individual average kinship relationships showed 87.3% advised matings, 11.0% marginal matings, and 1.7% advised against. While these results comprise a contribution toward planning better breeding management and monitoring, parallel actions to be undertaken include surveying healthy riverine habits for reintroduction and continued searching for wild individuals to introduce new variation into the captive broodstock to avoid adaptation to captivity and to minimize inbreeding.

Influence of spawning procedure on gametes fertilization success in Salminus hilarii Valenciennes, 1850 (Teleostei: Characidae): Implications for the conservation of this species

Artificial reproduction and gamete fertilization were evaluated in Salminus hilarii wild and domesticated broodstocks. Wild and domesticated broodstocks were artificially induced to reproduction using a carp pituitary treatment. Four groups were considered: Group 1 (G1), fish caught in the wild maintained for three years in the same conditions as the domesticated broodstocks and spawned naturally; Group 2 (G2), broodstock born and raised in captivity and spawned naturally; Group 3 (G3), wild broodstocks, which were manually stripped for gamete collection and dry fertilization; and Group 4 (G4), domesticated males and females, also manually stripped. Oocytes, eggs, and larvae were sampled at different time intervals throughout embryonic development. Yolk sac absorption occurred approximately 24-29 h after hatching. Twenty-six h after hatching, the larvae mouths opened. Cannibalism was identified just 28-30 h after hatching. There was no morphological difference in embryonic development among all groups. The number of released eggs per gram of female was: G1: 83.3 ± 24.5 and G2: 103.8 ± 37.4; however, the fertilization success was lower in G2 (42.0 ± 6.37 percent) compared with G1 (54.7 ± 3.02 percent) (P = 0.011). Hand-stripping of oocytes was not successful and the fertilization rate was zero. The reproduction of this species in captivity is viable, but it is necessary to improve broodstock management to enhance fertilization rates and obtain better fingerling production for restocking programs.

A reprodução artificial e fertilização dos gametas foram avaliados em reprodutores selvagens e de cativeiro de Salminus hilarii. Reprodutores selvagens e de cativeiro foram induzidos artificialmente à reprodução utilizando hipófise de carpa. Quatros grupos foram considerados: Grupo 1 (G1), peixes capturados na natureza, mantidos por três anos nas mesmas condições de reprodutores de cativeiro e desovados naturalmente; Grupo 2 (G2), reprodutores nascidos e criados em cativeiro e desovados naturalmente; Grupo 3 (G3), reprodutores selvagens que foram extrusados manualmente para a coleta de gametas e fertilização a seco; e Grupo 4 (G4), com machos e fêmeas domesticadas, também extrusados manualmente. Oócitos, ovos e larvas foram amostrados em diferentes intervalos de tempo ao longo do desenvolvimento embrionário. A absorção do saco vitelínico ocorreu aproximadamente 24-29 h após a eclosão. Vinte e seis h após a eclosão, as larvas abriram a boca. O canibalismo foi identificado apenas 28-30 h após a eclosão. Não houve diferença morfológica no desenvolvimento embrionário entre todos os grupos. O número de ovos liberados por grama de fêmea foi: G1: 83,3 ± 24,5 e G2: 103,8 ± 37,4; embora, o sucesso na fertilização tenha sido menor no G2 (42,0 ± 6,37 por cento) em comparação com G1 (54,7 ± 3,02 por cento) (P = 0,011). A extrusão manual dos oócitos não foi bem sucedida e a taxa de fertilização foi zero. A reprodução em cativeiro desta espécie é viável, mas é necessário um melhor manejo dos reprodutores para aumentar as taxas de fertilização, visando a obtenção de uma melhor produção de alevinos para os programas de repovoamento.

Color pattern variation in Trichomycterus iheringi (Eigenmann, 1917) (Siluriformes: Trichomycteridae) from rio Itatinga and rio Claro, São Paulo, Brazil

Color pattern is recognized as an important characteristic for diagnosing Trichomycterus species and for elucidating their relationships. An analysis based on morphological and molecular data confirms the existence of a single species of Trichomycterus in the rio Itatinga, a costal river drainage on the escarpment of the Serra do Mar and the rio Claro on the upper course of the rio Tietê. The only species found, Trichomycterus iheringi, shows two clearly distinct patterns of body pigmentation and intermediate color patterns related to body size and microhabitat preference.

O padrão de coloração é reconhecido como uma característica importante para a diagnose das espécies do gênero Trichomycterus e no reconhecimento de suas relações de parentesco. A análise baseada em dados morfológicos e moleculares confirma a existência de uma única espécie de Trichomycterus no rio Itatinga, drenagem litorânea da Serra do Mar e no rio Claro, curso superior do rio Tietê. A única espécie do gênero encontrada, Trichomycterus iheringi, apresenta dois padrões de pigmentação do corpo bastante distintos e padrões de coloração intermediários relacionados ao tamanho corporal e ao micro-habitat.

Induced spawning of the endangered Neotropical species Steindachneridion parahybae (Siluriformes: Pimelodidae)

The "surubim do Paraíba" (Steindachneridion parahybae) is a freshwater catfish endemic to the Paraíba do Sul River basin, Brazil. This species has been seriously threatened by environmental disturbances in the last several decades. Wild Steindachneridion parahybae males and females were collected in 2003 and taken to the hatchery of a power plant of the Companhia Energética de São Paulo (CESP). Steindachneridion parahybae broodstocks were artificially induced to reproduce in December 2003 using a combination of carp pituitary extract (CPE) and human chorionic gonadotropin (hCG). Oocytes and milt were stripped; the fertilized eggs were transferred to 60-liter conical incubators and hatched larvae distributed in nine horizontal trays. Exogenous feed was started just after yolk sac absorption. A high rate of cannibalism and photophobia were observed during the larval period, resulting in a 26 percent survival rate from larvae to fingerlings.(AU)

O "surubim do Paraíba" (Steindachneridion parahybae) é um bagre de água doce, endêmico da bacia do rio Paraíba do Sul, Brasil. Esta espécie foi seriamente ameaçada por distúrbios ambientais nas últimas décadas. Machos e fêmeas selvagens de Steindachneridion parahybae foram coletados em 2003 e transferidos para a piscicultura da CESP (Companhia Energética de São Paulo). Reprodutores de S. parahybae foram induzidos à reprodução artificial em dezembro de 2003 usando uma combinação de extrato hipofisário de carpa (CPE) e gonadotropina coriônica humana (hCG). Após a extrusão dos óvulos e do sêmen, os ovos fertilizados foram transferidos para incubadoras cônicas de 60 litros e, em seguida, as larvas eclodidas distribuídas em nove incubadoras horizontais. Após a absorção do saco vitelino, a alimentação exógena foi iniciada. Uma alta taxa de canibalismo e fotofobia foram observados durante o período larval, resultando em uma taxa de sobrevivência de 26 por cento de larvas para os alevinos(AU)