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.(AU)

Recruitment of oyster in artificial collectors on the Amazon macrotidal mangrove coast / Recrutamento de ostra em coletores artificiais na costa de manguezais de macromaré amazônico

The purpose of this study was to determine the moment of the year for the oyster recruitment and define the type of collector and environmental conditions that maximize recruitment. Collections were conducted, during 12 months, on Amazon Macrotidal Mangrove at two different sites: raft (point I) and mangrove (point II). In each location three types of collectors were used (1) transparent PET bottles, (2) green PET bottles, and (3) PVC sheets, each with three replicates. Spats were counted and measured at 45-day intervals, while the environmental data were measured every two weeks. Identification of oyster species occurred by genetic testing (multiplex PCR) by randomly selecting individuals by sampling. Results indicated spat capturing was significantly influenced by the collector type, location and period of collection (P<0.05, MANOVA) with significantly higher recruitment in the PVC collector (P<0.05, Tukey test). Oyster recruitment occurred throughout the year, suggesting that these individuals reproduce during all months; however, months with less rain and greater salinity were the best for spat collection, while the rainy period with lower salinity proved to be the best for individuals growth. The location in interaction with the environmental variables, mainly salinity, has a significant effect on the recruitment rate of spat and on their size, so that point II (mangrove) had the best results for recruitment and point I (raft) provided the spats of the largest size. Genetic identification verified two native oysters species (Crassostrea gasar and Crassostrea rhizophorae) in both points (I and II).(AU)

O estudo teve como objetivo determinar o período do ano para coletar sementes de ostras, definir o tipo de coletor e as condições ambientais que maximizam o recrutamento das sementes. As coletas foram conduzidas, durante 12 meses, no manguezal amazônico de macromaré em dois locais distintos: balsa (ponto I) e manguezal (ponto II). Em cada ponto de coleta foram utilizados três tipos de coletores (1) garrafas PET transparentes, (2) garrafas PET verdes e (3) forro de PVC, com três réplicas cada. As sementes foram contadas e medidas a cada 45 dias e os dados ambientais mensurados a cada duas semanas. As espécies de ostras foram identificadas por teste genético (PCR multiplex) com indivíduos aleatoriamente selecionados por coleta. Os resultados indicaram que a captura de sementes foi influenciada significativamente pelo tipo de coletor, localização e período de coleta (P<0,05, MANOVA) com recrutamento significativamente maior no coletor de PVC (P<0,05, teste de Tukey). O recrutamento ocorreu durante todo ano, sugerindo reprodução mensal, contudo, os meses de menor pluviosidade e maior salinidade foram melhores para coletar sementes, enquanto o período chuvoso, com menor salinidade, favoreceu um crescimento superior. O local de coleta em interação com variáveis ambientais, principalmente salinidade, apresentou efeito significativo sobre a fixação e tamanho das sementes de maneira que o ponto II obteve melhores resultados de fixação e o ponto I sementes de maior tamanho. A análise genética identificou duas espécies nativas (Crassostrea gasar e Crassostrea rhizophorae) em ambos os pontos (I e II).(AU)

Molecular identification of native oysters on the coast of Maranhão, Brazil / Identificação molecular de ostras nativas na costa do Maranhão, Brasil

Oysters are found along the whole coast of Brazil. The phenotypes of the species vary considerably according to the characteristics of the habitat. The present study investigated the existence of different oyster species of the genus Crassostrea on the coast of Maranhão, using the Multiplex PCR technique and DNA Barcoding. The results of the Multiplex PCR revealed two distinct bands characteristic of the species C. gasar and C. rhizophorae in a total of 135 samples analyzed. The sequencing of the COI gene of 98 samples produced a 695 bp fragment and 15 haplotypes for C. gasar and 640 bp and eight haplotypes for C. rhizophorae. The haplotype tree divided the two species clearly into different clades with 100% bootstrap support. Intraspecific genetic divergence was 0.2% in both species, while interspecific divergence was 23.6%. The similarity between the sequences generated and those available in BoldSystems ranged from 97.01% to 98.37% for C. rhizophorae and from 97.55% to 99.84% for both C. gasar and C. brasiliana, reinforcing the taxonomic problems in this group, which supports the synonymization of these species. The DNA barcoding permitted the reliable identification of the samples and confirmed the existence of two species of oyster in the study area.

As ostras são encontradas ao longo de toda a costa do Brasil. Os fenótipos das espécies variam consideravelmente de acordo com as características do habitat. O presente estudo investigou a existência de diferentes espécies de ostra do gênero Crassostrea no litoral do Maranhão, utilizando a técnica de PCR Multiplex e DNA barcoding. Os resultados da PCR Multiplex revelaram duas bandas distintas, características das espécies C. gasar e C. rhizophorae num total de 135 amostras analisadas. No sequenciamento do gene COI de 98 amostras obteve-se fragmentos de 695 pb e 15 haplótipos para C. gasar e de 640 pb e oito haplótipos para C. rhizophorae. A árvore haplótipica agrupou fortemente as duas espécies em clados diferentes com 100% de bootstrap. Divergências intraespecíficas foram de 0,2% para ambas, e a interespecífica de 23,6%. A similaridade das sequencias deste estudo com sequências presentes no BoldSystems variou de 97,01 a 98,37 para C. rhizophorae e de 97,55 a 99,84 tanto para C. gasar como para C. brasiliana, reforçando uma problemática na taxonomia do grupo, que apontam para a sinonimia dessas espécies. Portanto, o DNA barcoding permitiu identificar as amostras coletadas e confirmou a existência de duas espécies de ostras.

Molecular identification of native oysters on the coast of Maranhão, Brazil / Identificação molecular de ostras nativas na costa do Maranhão, Brasil

Oysters are found along the whole coast of Brazil. The phenotypes of the species vary considerably according to the characteristics of the habitat. The present study investigated the existence of different oyster species of the genus Crassostrea on the coast of Maranhão, using the Multiplex PCR technique and DNA Barcoding. The results of the Multiplex PCR revealed two distinct bands characteristic of the species C. gasar and C. rhizophorae in a total of 135 samples analyzed. The sequencing of the COI gene of 98 samples produced a 695 bp fragment and 15 haplotypes for C. gasar and 640 bp and eight haplotypes for C. rhizophorae. The haplotype tree divided the two species clearly into different clades with 100% bootstrap support. Intraspecific genetic divergence was 0.2% in both species, while interspecific divergence was 23.6%. The similarity between the sequences generated and those available in BoldSystems ranged from 97.01% to 98.37% for C. rhizophorae and from 97.55% to 99.84% for both C. gasar and C. brasiliana, reinforcing the taxonomic problems in this group, which supports the synonymization of these species. The DNA barcoding permitted the reliable identification of the samples and confirmed the existence of two species of oyster in the study area.(AU)

As ostras são encontradas ao longo de toda a costa do Brasil. Os fenótipos das espécies variam consideravelmente de acordo com as características do habitat. O presente estudo investigou a existência de diferentes espécies de ostra do gênero Crassostrea no litoral do Maranhão, utilizando a técnica de PCR Multiplex e DNA barcoding. Os resultados da PCR Multiplex revelaram duas bandas distintas, características das espécies C. gasar e C. rhizophorae num total de 135 amostras analisadas. No sequenciamento do gene COI de 98 amostras obteve-se fragmentos de 695 pb e 15 haplótipos para C. gasar e de 640 pb e oito haplótipos para C. rhizophorae. A árvore haplótipica agrupou fortemente as duas espécies em clados diferentes com 100% de bootstrap. Divergências intraespecíficas foram de 0,2% para ambas, e a interespecífica de 23,6%. A similaridade das sequencias deste estudo com sequências presentes no BoldSystems variou de 97,01 a 98,37 para C. rhizophorae e de 97,55 a 99,84 tanto para C. gasar como para C. brasiliana, reforçando uma problemática na taxonomia do grupo, que apontam para a sinonimia dessas espécies. Portanto, o DNA barcoding permitiu identificar as amostras coletadas e confirmou a existência de duas espécies de ostras.(AU)