The Pacific oyster Crassostrea gigas accounts for a large part of shellfish aquaculture production worldwide. Aspects of morphological and functional characteristics of oyster oocytes remain poorly documented, and traditional techniques, such as microscopic observations of shape or fertilization rate, are time and space consuming. The purpose of this study was to assess for the first time viability and reactive oxygen species (ROS) production of Pacific oyster oocytes using flow cytometry (FCM) and to apply this method to determine oocyte responses to in vitro exposure to the toxic dinoflagellate Alexandrium minutum. A culture of A. minutum caused a significant increase in oocyte ROS production, which gradually increased with the age of the culture, but viability was not affected. Effect of the supernatant of the same A. minutum culture did not cause any significant modifications of oocyte morphology, viability, or ROS level. This study confirmed that some oocyte cellular characteristics can be assessed using FCM techniques. V C 2014 International Society for Advancement of Cytometry Key terms Pacific oyster; gamete quality; ROS production; viability; flow cytometry; ecotoxicology THE Pacific oyster Crassostrea gigas has considerable commercial value worldwide, including in France. Oyster seed production relies upon gamete quality, fertilization success, and larval development. As gamete quality is the first step in the spatproduction sequence, it is necessary to assess the quality of gametes in broodstock oysters first and foremost.To date, bioassays to assess oocyte quality are limited, and those available are tedious and time-consuming. Oocyte quality can be described using morphological characteristics by microscopy (1). Although microscope observations are unavoidable and useful to validate flow cytometry (FCM), assays based on microscope observations often have little predictive value and can be subjective according to each observer. Determinations of biochemical content (proteins, carbohydrates, and lipids) have been used as criteria of oocyte quality (2-4). More recently, proteomic profiles of oocytes were proposed as quality estimators (5,6). All of these biochemical assays, however, are fairly tedious to perform and provide only retrospective information. Fertilization rate, hatching rate, and larval development are used as complementary end points to evaluate oocyte quality (7-10); however, as these processes depend also upon spermatozoa quality, variability and inconsistency from one experiment to another often are encountered.

Feeding strategies and digestive capacities can have important implications for variation in energetic pathways associated with ecological and economically important traits, such as growth or reproduction in bivalve species. Here, we investigated the role of amylase in the digestive processes of Crassostrea gigas, using in vivo RNA interference. This approach also allowed us to investigate the relationship between energy intake by feeding and gametogenesis in oysters. Double-stranded (ds)RNA designed to target the two α-amylase genes A and B was injected in vivo into the visceral mass of oysters at two doses. These treatments caused significant reductions in mean mRNA levels of the amylase genes: −50.7% and −59% mRNA A, and −71.9% and −70.6% mRNA B in 15 and 75 µg dsRNA-injected oysters, respectively, relative to controls. Interestingly, reproductive knock-down phenotypes were observed for both sexes at 48 days post-injection, with a significant reduction of the gonad area (−22.5% relative to controls) and germ cell underproliferation revealed by histology. In response to the higher dose of dsRNA, we also observed reductions in amylase activity (−53%) and absorption efficiency (−5%). Based on these data, dynamic energy budget modeling showed that the limitation of energy intake by feeding that was induced by injection of amylase dsRNA was insufficient to affect gonadic development at the level observed in the present study. This finding suggests that other driving mechanisms, such as endogenous hormonal modulation, might significantly change energy allocation to reproduction, and increase the maintenance rate in oysters in response to dsRNA injection.