Rodrigo Maggioni

GH overexpression causes muscle hypertrophy independent from local IGF-I in a zebrafish transgenic model

The aim of the present study was to analyse the morphology of white skeletal muscle in males and females from the GH-transgenic zebrafish (Danio rerio) lineage F0104, comparing the expression of genes related to the somatotrophic axis and myogenesis. Histological analysis demonstrated that transgenic fish presented enhanced muscle hypertrophy when compared to non-transgenic fish, with transgenic females being more hypertrophic than transgenic males. The expression of genes related to muscle growth revealed that transgenic hypertrophy is independent from local induction of insulin-like growth factor 1 gene (igf1). In addition, transgenic males exhibited significant induction of myogenin gene (myog) expression, indicating that myog may mediate hypertrophic growth in zebrafish males overexpressing GH. Induction of the α-actin gene (acta1) in males, independently from transgenesis, also was observed. There were no significant differences in total protein content from the muscle. Our results show that muscle hypertrophy is independent from muscle igf1, and is likely to be a direct effect of excess circulating GH and/or IGF1 in this transgenic zebrafish lineage.

First report of Perkinsus beihaiensis in wild clams Anomalocardia brasiliana (Bivalvia: Veneridae) in Brazil

This is the first report of Perkinsus sp. (Bivalvia: Veneridae) infecting wild clams of the species Anomalocardia brasiliana in Brazil. The gill lamellae and rectum of 150 specimens of A. brasiliana collected in the Timonha river estuary (Ceará, Northeastern Brazil) in March 2012 were incubated in Rays fluid thioglycollate medium (RFTM) for detection of Perkinsus sp. In RFTM, the prevalence of Perkinsus sp. was 14.7% (22/150) and the intensity of infection ranged from very light (1-10 cells across the slide) to light (12-100 cells). The presence of Perkinsus sp. was confirmed by PCR in seven (31.8%) out of 22 RFTM-positive specimens. DNA sequencing confirmed the presence of the genus Perkinsus and the phylogenetic analysis strongly indicated Perkinsus beihaiensis as the species responsible for the infection.

Asterionellopsis tropicalis (Bacillariophyceae): a new tropical species found in diatom accumulations.

The diatom Asterionellopsis glacialis sensu lato forms high‐density patches in the surf zone of some sandy beaches worldwide and was until recently considered a cosmopolitan species. With the recent description of four cryptic species, the identity of specimens found in these accumulations remains uncertain. In this study, diatom patches were sampled from two sandy beaches of the Brazilian coast: one tropical (Futuro Beach, 3° S; 38° W) and one subtropical (Cassino Beach, 32° S; 52° W). Fine structure of frustules and the sequencing of three phylogenetic markers revealed the subtropical strains to be A. guyunusae and the tropical strains to be a new species, here described as Asterionellopsis tropicalis sp. nov. A. tropicalis was differentiated morphologically by the number of striae in 10 μm at the foot pole and head (39–44; 38–45, respectively), from A. lenisilicea (46–55; 46–64), A. maritima (46–51; 46–60), and A. thurstonii (42–58; 55–70). The number of striae at the head region of the valvocopula (10 μm) helped to distinguish A. tropicalis (56–62) from A. guyunusae (61–64), but A. tropicalis was morphologically undistinguishable from A. glacialis. The sequence divergence from other identified Asterionellopsis species was 13%–16% (Cox1), 11%–12% (5.8S + ITS2) and 2%–6% (RbcL), and A. tropicalis formed a distinct monophyletic clade with high support in all analyzed phylogenetic trees (single or multi‐locus). This work will aid in the understanding of the ecological and physiological diversity of diatom patches that are key to the trophic webs of sandy beaches.

High Connectivity among Blue Crab (Callinectes sapidus) Populations in the Western South Atlantic

Population connectivity in the blue crab Callinectes sapidus was evaluated along 740 km of the Western South Atlantic coast. Blue crabs are the most exploited portunid in Brazil. Despite their economic importance, few studies report their ecology or population structure. Here we sampled four estuarine areas in southern Brazil during winter 2013 and summer 2014 in order to evaluate diversity, gene flow and structure of these populations. Nine microsatellite markers were evaluated for 213 adult crabs, with identification of seven polymorphic loci and 183 alleles. Pairwise FST values indicated low population structure ranging from -0.00023 to 0.01755. A Mantel test revealed that the geographic distance does not influence genetic (r = -0.48), and structure/migration rates confirmed this, showing that even the populations located at the opposite extremities of our covered region presented low FST and exchanged migrants. These findings show that there is a significant amount of gene flow between blue crab populations in South Brazil, likely influenced by local current dynamics that allow the transport of a high number of larvae between estuaries. Considering the elevated gene flow, the populations can be considered a single genetic stock. However, further information on population size and dynamics, as well as fishery demands and impacts at different regions, are necessary for harvest management purposes.

RNAi-based inhibition of infectious myonecrosis virus replication in Pacific white shrimp Litopenaeus vannamei

Disease in Pacific white shrimp Litopenaeus vannamei caused by the infectious myonecrosis virus (IMNV) causes significant socioeconomic impacts in infection-prone shrimp aquaculture regions. The use of synthetic dsRNA to activate an RNA interference (RNAi) response is being explored as a means of disease prophylaxis in farmed shrimp. Here, survival was tracked in L. vannamei injected with long synthetic dsRNAs targeted to IMNV open reading frame (ORF) 1a, ORF1b, and ORF2 genome regions prior to injection challenge with IMNV, and real-time RT-PCR was used to track the progress of IMNV infection and mRNA expression levels of the host genes sid1, dicer2, and argonaute2. Injection of dsRNAs targeting the ORF1a and ORF1b genes but not the ORF2 gene strongly inhibited IMNV replication over a 3 wk period following IMNV challenge, and resulted in 90 and 83% shrimp survival, respectively. Host gene mRNA expression data indicated that the Sid1 protein, which forms a transmembrane channel involved in cellular import/export of dsRNA, increased in abundance most significantly in shrimp groups that were most highly protected by virus-specific dsRNA injection. Subclinical IMNV infections present in the experimental L. vannamei used increased markedly in the 2 d between injection of any of the 4 virus-specific or non-specific dsRNAs tested and IMNV challenge. While handling and injection stress are implicated in increasing IMNV replication levels, the underlying molecular factors that may have been involved remain to be elucidated.

Perkinsus sp. infecting the oyster Crassostrea rhizophorae from estuaries of the septentrional Northeast, Brazil

The mangrove oyster Crassostrea rhizophorae is an estuarine resource exploited by riverside communities in Northeast Brazil. Despite its socioeconomic importance, studies on the health status of this bivalve are scanty in this region. The purpose of the present study was to investigate the presence of the protozoan Perkinsus sp. in C. rhizophorae collected in August and September 2011 in three estuaries of the septentrional Northeast, Brazil: Jaguaribe (Ceará), Camurupim (Piauí) and Carnaubeiras (Maranhão) (n= 150 specimens/site). The samples were submitted to Rays fluid thioglycollate medium (RFTM), PCR and histology assays. The RFTM assay revealed spherical, blue or bluish-black hypnospores of the genus Perkinsus in 50 specimens (Jaguaribe= 17.3%, Camurupim= 5.3%, Carnaubeiras= 10.6%). The intensity of the infection ranged from very light (1-10 cells per slide) to severe (more than 40 cells in each of 10 fields of the slide) for Jaguaribe; very light for Camurupim and very light to moderate (at least 40 cells observed in each of 10 fields of the slide) for Carnaubeiras. When submitted to confirmatory PCR analysis, 6 cases were confirmed (Jaguaribe=3, Camurupim=1, Carnaubeiras=2). The histology confirmed 21 cases of infection in specimens from the three estuaries. Although local collectors have reported no mortality in oyster populations that might be attributed to infection by Perkinsus, health surveillance of oyster populations in the septentrional region of Northeast Brazil is advisable.

DETECTION OF VIRULENCE GENES IN ENVIRONMENTAL STRAINS OF Vibrio cholerae FROM ESTUARIES IN NORTHEASTERN BRAZIL

The objectives of this study were to detect the presence of Vibrio cholerae in tropical estuaries (Northeastern Brazil) and to search for virulence factors in the environmental isolates. Water and sediment samples were inoculated onto a vibrio-selective medium (TCBS), and colonies with morphological resemblance to V. cholerae were isolated. The cultures were identified phenotypically using a dichotomous key based on biochemical characteristics. The total DNA extracted was amplified by PCR to detect ompW and by multiplex PCR to detect the virulence genes ctx, tcp, zot and rfbO1. The results of the phenotypic and genotypic identification were compared. Nine strains of V. cholerae were identified phenotypically, five of which were confirmed by detection of the species-specific gene ompW. The dichotomous key was efficient at differentiating environmental strains of V. cholerae. Strains of V. cholerae were found in all four estuaries, but none possessed virulence genes.

Genetic variability of marine shrimp in the Brazilian industry

The objective of this work was to estimate the genetic variability level and distribution in Brazilian broodstocks of marine shrimp (Litopenaeus vannamei). Nine of the countrys largest hatcheries were evaluated using codominant and highly polymorphic microsatellite markers. The results obtained from genotyping of ten microsatellite loci are indicative of genetic variability that is compatible with that found in wild populations of L. vannamei in Mexico and Central America. A possible explanation is the highly diversified and relatively recent origin of the available broodstocks. Bayesian analysis detected a signal for five founding populations. The distribution of genetic distances partially reflects geographical location, and this information will be useful for the creation of new broodstocks. Therefore, L. vannamei genetic variability among nine of the largest national hatcheries can be considered high.

First record of Perkinsus chesapeaki infecting Crassostrea rhizophorae in South America

This study investigated Perkinsus spp. infecting Crassostrea rhizophorae from the Jaguaribe River estuary, Ceará, Brazil. Fragments of gills and rectum of the oysters (n=150) were incubated in Rays fluid thioglycollate medium (RFTM). Genus Perkinsus-specific PerkITS85/750 PCR assays were performed and their amplicons were sequenced by the Sanger method. The RFTM assays confirmed Perkinsus spp. The sequencing of the amplified fragments from the rDNA internal transcribed spacers (ITS) of Perkinsus spp. confirmed Perkinsus chesapeaki. Neighbor-Joining analyzes place P. chesapeaki identified in this study in a well-supported clade with other isolates of the same species. This is the first record of P. chesapeaki infecting C. rhizophorae in South America.

Genetics of two marine shrimp hatcheries of the Pacific white shrimp Litopenaeus vannamei (Boone, 1931) in Pernambuco, Brazil.

A carcinicultura cresceu significativamente no Brasil ao longo dos ultimos 10 anos, especialmente a producao do camarao branco do Pacifico, o exotico Litopenaeus vannamei. Em 2004, a atividade foi marcada por uma crise na producao, que despertou interesse na implantacao de programas de melhoramento dos estoques de camarao. A importacao de crustaceos foi banida do Brasil por uma Instrucao Normativa de 1997, como uma medida de precaucao sanitaria. Desde entao, a reposicao de matrizes nas larviculturas passou a ser conduzida com estoques domesticados, gerando preocupacoes sobre o possivel declinio da diversidade genetica e sobre a possibilidade de que a diversidade genetica existente pudesse garantir ganhos efetivos em programas de melhoramento. No presente trabalho, parâmetros geneticos, tais como numero de alelos, numero de alelos efetivos, heterozigosidade esperada e observada, coeficiente de consanguinidade, coeficiente de diferenciacao genetica e desvio do equilibrio de Hardy-Weinberg, foram estimados para duas importantes larviculturas comerciais do Nordeste do Brasil, por meio da genotipagem de cinco marcadores microssatelites. O numero de alelos efetivos (3 a 10,5) e as heterozigosidades medias observada e esperada (0,480 e 0,680) foram consistentes com aqueles relatados para populacoes de peneideos de cativeiro e selvagens. Entretanto, valores positivos de FIS (0,381 para a larvicultura A e de 0,249 para a larvicultura B) mostraram uma deficiencia significativa de heterozigotos (P<0,01). Apesar disso, e possivel concluir que, mesmo apos 10 anos de proibicao na importacao de crustaceos, tem sido possivel manter um alto nivel de variabilidade genetica, possivelmente devido a origens multiplas do estoque de fundadores dessa especie no Brasil e da constante troca de reprodutores entre larviculturas.