Maria Raquel Moura Coimbra

A genetic linkage map of the Japanese flounder, Paralichthys olivaceus

Abstract We report the first genetic linkage map of the Japanese flounder ( Paralichthys olivaceus ) constructed with 111 microsatellite markers and 352 AFLP fragments. The parental male linkage map consisted of 25 linkage groups while the female map consisted of 27 groups, with an average resolution of 8 and 6.6 cM, respectively. We have identified linkage among 96% of the markers and the total map length was estimated to be around 1000–1200 cM. This study reports the first low-density linkage map for the Japanese flounder and describes differences in sex recombination. Recombination rates were higher in male flounder compared to the female (7.4:1), a rare condition among vertebrates. This map is a starting point for the mapping of single loci and quantitative traits in flatfish species.

Genetic variability within Fusarium solani specie as revealed by PCR-fingerprinting based on pcr markers

Fusarium solani fungus (teleomorph Haematonectria haematococca) is of relevance for agriculture, producing a disease that causes significant losses for many cultivars. Moreover, F. solani is an opportunistic pathogen to animals and humans. The complexity associated to its correct identification by traditional methods justifies the efforts of using molecular markers for isolates characterization. In this work, three PCR-based methods (one PCR-ribotyping and two PCR-fingerprinting) were used to investigate the molecular variability of eighteen F. solani isolates from four Brazilian States, collected from different substrates. Genetic analysis revealed the intraspecific variability within the F. solani isolates, without any correlation to their geographical origin and substrate. Its polymorphism was observed even in the very conserved sequence of rDNA locus, and the SPAR marker (GTG)5 showed the highest polymorphism. Together, those results may contribute to understand the relation between fungal genetic variability and cultivars resistance phenotypes to fungal-caused diseases, helping plant-breeding programs.

Quantitation of infectious myonecrosis virus in different tissues of naturally infected Pacific white shrimp, Litopenaeus vannamei, using real-time PCR with SYBR Green chemistry

The Pacific white shrimp, Litopenaeus vannamei, is the most important shrimp species in volume in world aquaculture. However, in recent decades, outbreaks of diseases, especially viral diseases, have led to significant economic losses, threatening the sustainability of shrimp farming worldwide. In 2004, Brazilian shrimp farming was seriously affected by a new disease caused by the Infectious myonecrosis virus (IMNV). Thus, disease control based on rapid and sensitive pathogen detection methods has become a priority. In this study, a specific quantitation method for IMNV was developed using real-time PCR with SYBR Green chemistry and viral load of the principal target tissues of chronically infected animals was quantified. The quantitative analysis revealed that mean viral load ranged from 5.08×10(8) to 1.33×10(6)copies/μg of total RNA in the hemolymph, 5.096×10(5) to 1.26×10(3)copies/μg in the pleopods, 6.85×10(8) to 3.09×10(4)copies/μg in muscle and 8.15×10(6) to 3.90×10(3)copies/μg in gills. Different viral loads of IMNV were found with greater values in the hemolymph and muscle, followed by the pleopods and gills.

Genetic Diversity of Captive and Wild Threatened Catfish Pseudoplatystoma corruscans in the São Francisco River

A genetic analysis was conducted of the critically endangered migratory catfish Pseudoplatystoma corruscans from the São Francisco River basin, the basin with the second highest hydropower capacity installed in Brazil. The analysis was conducted using five microsatellite markers previously described in the literature. Genetic diversity was analyzed and compared in three wild populations and a captive broodstock for a restocking program. Catfish populations of this river showed high genetic diversity in terms of heterozygosities; however, only half of the total number of alleles found in other basins were detected. Seven private alleles occurred in this basin and most of them at low frequencies, indicating they are under threat of extinction. Overall FST and assignment test suggested low differentiation among the three wild populations; however, a marked distinction between these populations and the captive broodstock was observed. The number of alleles and low average relatedness estimates found for the captive population suggest that it could be used for restocking in areas where the wild population has collapsed, but should not be used for stock enhancement in areas where the wild population is extant. These results are important as a reference for genetic aspects that should be considered in fish management activities.

Artemia franciscana as a vector for infectious myonecrosis virus (IMNV) to Litopenaeus vannamei juvenile.

In 2004, the infectious myonecrosis virus (IMNV) was recognized as the main cause of Litopenaeusvannamei shrimp cultures drop in Brazil. In health animal control programs, in order to reduce virus prevalence in production units it is necessary to screen live feed used. Among live diets used in aquaculture, the brine shrimp Artemia sp. is essential in crustacean larviculture and maturation. The aim of the present study was to investigate the susceptibility of Artemiafranciscana to IMNV through an immersion challenge and virus-phytoplankton adhesion route and to elucidate its role as a vector for IMNV transmission to L.vannamei. A. franciscana adults were infected with IMNV through both routes, as demonstrated by PCR-positive reactions. However, infected A. franciscana showed no signs of infection. More than 40% of L. vannamei juveniles fed with IMNV-infected A. franciscana by virus-phytoplankton adhesion route were positive by real-time PCR, whereas only a 10% infection rate was found among shrimp fed with IMNV-infected brine shrimp using the immersion challenge. Significant differences were found in mean viral load between immersion and virus-phytoplankton adhesion shrimp treatments (p ⩽ 0.05). Moreover, the mean viral loads were 1.34 × 10(2) and 1.48 × 10(4) copies/μg(-1) of total RNA for virus-phytoplankton adhesion and IMNV-infected tissue treatments, respectively, and the difference was not significant (p ⩾ 0.05). The results indicated that A. franciscana act as a vector for IMNV transmission under the experimental conditions examined. Although no mass mortalities were detected in L. vannamei fed with IMNV-infected brine shrimp, these infected shrimp should not be disregarded as a source of IMNV in grow-out units.

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.

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.

Meiosis Maturation in the Marine Clam Anomalocardia brasiliana (Veneridae)

ABSTRACT Advances in oyster farming have been achieved with biotechnological applications, such as chromosome manipulations aimed at polyploidy. The bivalve Anomalocardia brasiliana is of considerable importance to artisanal fishing activities and is a potential organism for aquaculture in Brazil. The cytogenetic behavior of polar bodies during meiosis provides essential information for chromosome manipulation directed at the production of triploid organisms. The objective of the present study was to identify postfertilization times in which the polar bodies are expelled and determine the most frequent number of chromosomes in A. brasiliana. Individuals were caught on the coast of the state of Pernambuco, in northeast Brazil, and subsequently induced to release the gametes. Samples of the oocyte solution were taken before and every 2 min after fertilization. The material was fixed in Carnoys solution, stained with 4′,6-diamidino-2-phenylindole and photographed under an epifluorescence microscope. Among the 50 oocytes analyzed in metaphase I, 19 bivalents were found. The release of the first polar body was detected 10 min after fertilization among 70% of the eggs, whereas the second polar body was released at 16 min among 62% of the eggs. This work provides relevant information on the time of initiation of shock treatments for chromosome set manipulation, aiming triploids of this important marine fishery resource in Brazil.

Sexual reproduction in freshwater fairy shrimp, Dendrocephalus brasiliensis (Pesta, 1921) inferred by Amplified Fragment Length Polymorphism markers

Fluctuations in world harvests of Artemia sp cysts have increased prices sharply in the past years. Several organisms have been tested as alternative sources for a total or partial replacement of Artemia sp, among which the microcrustacean (Anostraca) Dendrocephalus brasiliensis stands out. The objective of this study was to investigate the reproductive strategy of D. brasiliensis by means of AFLP markers. The distinction between sexual and parthenogenetic reproduction was conducted using 7 EcoRI and MseI primer combinations in a full-sib family composed of 13 progenies. Forty polymorphic markers were obtained, of these, 35% were from paternal origin and 65% were maternally inherited. Results showed mendelian segregation in all markers through the chi-square at P≤0.05. Sex changes were observed after 72 hours of grouping individuals of the same sex in a container, suggesting a possible sex density-dependent behavior. Paternal inheritance revealed that this species adopts sexual reproduction under experimental conditions.