Effects of salinity on pre- and post-fertilization developmental events in the mangrove oyster Crassostrea rhizophorae (GUILDING, 1828).

The mangrove oyster Crassostrea rhizophorae is identified as a potentially valuable species for tropical aquaculture, however, information on the physiological mechanisms of reproduction under laboratory conditions for this species is limited. This study investigated the effects of salinity at different concentrations (15, 20, 25, 30, 35, and 40 g/L) on the induction of germinal vesicle breakdown (GVBD) of oocytes obtained through stripping, the release of polar bodies (PB1 and PB2), and the larval development of the mangrove oyster. The results revealed a relationship between salinity and the percentage of GVBD, with the most effective range being 30-40 g/L within the hydration time frame between 70 and 120 min. The release of 50 % of PB1 was detected within this salinity range, while for the release of 50 % of PB2, the saline treatments of 35 and 40 g/L showed the best results. Overall, the salinity range of 30-40 g/L is suggested as the most suitable of polyploidy induction methodologies through the retention of PB1 or PB2. Regarding larval hatching, while salinities between 25 and 40 g/L presented similar percentages, at 15 g/L no hatching was observed. This study demonstrated that salinity is a key factor in early pre- and post-fertilization stages for the successful reproduction of mangrove oyster in hatcheries and that the percentages of oocyte maturation and artificial fertilization can be optimized by adjusting salinity.

Effects of salinity on pre- and post-fertilization developmental events in the clam Anomalocardia flexuosa (Linnaeus, 1767).

The knowledge about the effect of salinity on the physiological mechanism of bivalve reproduction is fundamental to improve production strategies in hatcheries. The present work evaluated the influence of different salinity concentrations (15, 20, 25, 30, 35 and 40 g⋅L-1) on pre- and post-fertilization development processes in the clam, Anomalocardia flexuosa, oocytes obtained by stripping. Salinity directly interfered with the germinal vesicle breakdown (GVBD) rate and in the cellular stability of unfertilized oocytes. Salinity concentrations between 30 and 35 g⋅L-1 provided better percentages of stable GVBD within 120 min, and incubation of oocytes in the salinity range of 30-35 g⋅L-1 for a time interval of 80-120 min provided > 80% GVBD. In the post-fertilization analysis, salinity affected the rate of the extrusion of the first and second polar bodies (PB1 and PB2). The release of 50% of the PBs was faster at a salinity of 35 g⋅L-1, with an estimated time of 10 min for PB1 and 30 min for PB2. Thus, chromosome manipulation methodologies aiming triploids should be applied at 35 g⋅L-1 salinity, with application of post-fertilization shock before 10 min for PB1 retention or before 30 min for PB2 retention.

Effect of dosage of orally administered 17α-methyltestosterone on sex reversion of the yellowtail tetra Astyanax lacustris (Lütken, 1875).

The females of yellowtail tetra (Astyanax lacustris), known as the freshwater sardine, are approximately 1.33 times larger than males, and thus, all-female monosex culture would increase production and reduce size variability. The present work aimed to identify the optimal dose of 17α-methyltestosterone (MT) to be used in the masculinization of A. lacustris for indirect sex reversal. Three different concentrations of MT (20, 40, and 60 mg/kg of feed in the diet) were fed to the fry for 30 days. Thirty adult individuals from each treatment, including the control (0 mg MT/kg), were evaluated for gonadal development, morphological and histological sexual identification, zootechnical performance, and the possible genotoxic effect caused by prolonged exposure to MT. MT significantly (P<0.01) affected the differentiation of the gonads, with the presence of possible inhibitory effects in all treatments. Intersex individuals were present in the 20 and 60 mg MT/kg treatments. All treatments were able to masculinize A. lacustris and the treatment with the lowest hormone concentration produced the highest percentage of males 76.7%, while the control had 46.7% males. The presence of erythrocyte nuclear alterations indicated a possible cytotoxic effect of MT in treatments 40 and 60 mg MT/kg, however, the use of the hormone did not affect the growth and the survival of the individuals. Thus, the use of MT is a viable option for obtaining neomales as a first step into the production of all-female progenies.

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 culture's 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.

Sexual reproduction in freshwater fairy shrimp, Dendrocephalus brasiliensis (Pesta, 1921) inferred by Amplified Fragment Length Polymorphism markers / Reprodução sexual do microcrustáceo de água doce, Dendrocephalus brasiliensis (Pesta, 1921), deduzida a partir de marcadores de polimorfismo de comprimento de fragmento amplificado

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.

Há vários anos que a produção de cistos de Artemia sp. apresenta flutuações, o que aumenta consideravelmente o seu valor. Vários organismos têm sido testados para a substituição total ou parcial da Artemia, dentre os quais se destaca o microcrustáceo (Anostraca) Dendrocephalus brasiliensis. O objetivo do presente trabalho foi investigar a estratégia reprodutiva de D. brasiliensis por meio de marcadores de AFLP. A distinção entre uma reprodução sexuada e partenogenética foi feita a partir da genotipagem de sete combinações de primers de AFLP em uma família composta de 13 progênies. Quarenta marcadores polimórficos foram obtidos, dos quais 35% foram de origem paterna e 65%, materna. Os resultados mostraram a segregação mendeliana de todos os marcadores através do teste de qui-quadrado a P≤0.05. Mudanças de sexo foram observadas após 72 horas do agrupamento de indivíduos de mesmo sexo em um recipiente, sugerindo uma possível razão sexual populacional densidade-dependente. A herança paterna mostrou que a reprodução sexual é adotada por essa espécie sob condições experimentais.

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.

Genetics of two marine shrimp hatcheries of the Pacific white shrimp Litopenaeus vannamei (Boone, 1931) in Pernambuco, Brazil / Genética de duas larviculturas de camarão branco do Pacífico Litopenaeus vannamei (Boone, 1931) em Pernambuco, Brasil

The shrimp industry has grown significantly over the past 10 years in Brazil, especially the farmed production of the exotic Pacific white shrimp, Litopenaeus vannamei. In 2004, this industry was marked by a productivity crisis, which stirred interest towards genetic improvement of shrimp stocks. Shrimp breeders importation was banned in Brazil by a govern Normative Instruction in 1997, as a sanitary precaution. Since then, broodstock replacement in hatcheries has been based on domestic stocks, raising concerns on the decline of genetic diversity and if the existing diversity would allow effective genetic improvement programs. In the present research, genetic parameters such as number of alleles, effective allele number, expected and observed heterozygosities, inbreeding coefficient, genetic differentiation index and deviation from Hardy-Weinberg equilibrium have estimated of two important commercial hatcheries in Northeast Brazil, genotyping 5 microsatellite loci. Effective allele number (3 to 10.5) and average observed and expected heterozygosities (0.480 and 0.680) were consistent with those reported for cultured and wild Penaeid populations. However, F IS positive values (0.381 for hatchery A and 0.249 for hatchery B) reflected a significant heterozygous deficiency within hatcheries (P<0.01). Nevertheless, we concluded that even after ten years of limited genetic input, it has been possible to maintain a high level of genetic variability, possibly due to the wide diverse origin of the founder broodstocks and the constant breeders exchange among hatcheries.

A carcinicultura cresceu significativamente no Brasil ao longo dos últimos 10 anos, especialmente a produção do camarão branco do Pacífico, o exótico Litopenaeus vannamei. Em 2004, a atividade foi marcada por uma crise na produção, que despertou interesse na implantação de programas de melhoramento dos estoques de camarão. A importação de crustáceos foi banida do Brasil por uma Instrução Normativa de 1997, como uma medida de precaução sanitária. Desde então, a reposição de matrizes nas larviculturas passou a ser conduzida com estoques domesticados, gerando preocupações sobre o possível declínio da diversidade genética e sobre a possibilidade de que a diversidade genética existente pudesse garantir ganhos efetivos em programas de melhoramento. No presente trabalho, parâmetros genéticos, tais como número de alelos, número de alelos efetivos, heterozigosidade esperada e observada, coeficiente de consanguinidade, coeficiente de diferenciação genética e desvio do equilíbrio de Hardy-Weinberg, foram estimados para duas importantes larviculturas comerciais do Nordeste do Brasil, por meio da genotipagem de cinco marcadores microssatélites. O número de alelos efetivos (3 a 10,5) e as heterozigosidades médias observada e esperada (0,480 e 0,680) foram consistentes com aqueles relatados para populações de peneídeos de cativeiro e selvagens. Entretanto, valores positivos de F IS (0,381 para a larvicultura A e de 0,249 para a larvicultura B) mostraram uma deficiência significativa de heterozigotos (P<0,01). Apesar disso, é possível concluir que, mesmo após 10 anos de proibição na importação de crustáceos, tem sido possível manter um alto nível de variabilidade genética, possivelmente devido a origens múltiplas do estoque de fundadores dessa espécie no Brasil e da constante troca de reprodutores entre larviculturas.

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

O fungo Fusarium solani (teleomorfo Haematonectria haematococca) apresenta uma expressiva importância na agricultura por ser considerado patógeno para várias culturas de interesse econômico causando doença conhecida por podridão das raízes, além de ser patógeno aos animais e ao homem, provocando nestes últimos, micoses superficiais e sistêmicas. A complexidade associada a sua identificação correta através de métodos tradicionais justifica os esforços de usar marcadores moleculares para caracterização dos isolados. Neste trabalho, três métodos baseados na tecnologia da PCR (um por ribotipagem por PCR e dois por impressão genética por PCR) foram utilizados para investigar a variabilidade molecular de dezoito isolados de F. solani de quatro Estados brasileiros, coletados de diferentes substratos. A análise genética revelou a variabilidade intraespecífica dos isolados de F. solani, sem qualquer correlação para a origem geográfica e substrato. Seu polimorfismo foi observado até mesmo na seqüência conservada do locus do rDNA, e o marcador SPAR (GTG)5 mostrou o mais alto polimorfismo. Em conjunto, estes resultados poderão auxiliar nos estudos da relação entre variabilidade do perfil genético de isolados e os fenótipos de resistência de determinados cultivares às doenças provocadas pelo fungo, orientando programas de melhoramento vegetal.