Christian Mingant

Oyster reproduction is affected by exposure to polystyrene microplastics

Significance Plastics are a contaminant of emerging concern accumulating in marine ecosystems. Plastics tend to break down into small particles, called microplastics, which also enter the marine environment directly as fragments from a variety of sources, including cosmetics, clothing, and industrial processes. Given their ubiquitous nature and small dimensions, the ingestion and impact of microplastics on marine life are a cause for concern, notably for filter feeders. Oysters were exposed to polystyrene microparticles, which were shown to interfere with energy uptake and allocation, reproduction, and offspring performance. A drop in energy allocation played a major role in this reproductive impairment. This study provides ground-breaking data on microplastic impacts in an invertebrate model, helping to predict ecological impact in marine ecosystems. Plastics are persistent synthetic polymers that accumulate as waste in the marine environment. Microplastic (MP) particles are derived from the breakdown of larger debris or can enter the environment as microscopic fragments. Because filter-feeder organisms ingest MP while feeding, they are likely to be impacted by MP pollution. To assess the impact of polystyrene microspheres (micro-PS) on the physiology of the Pacific oyster, adult oysters were experimentally exposed to virgin micro-PS (2 and 6 µm in diameter; 0.023 mg·L−1) for 2 mo during a reproductive cycle. Effects were investigated on ecophysiological parameters; cellular, transcriptomic, and proteomic responses; fecundity; and offspring development. Oysters preferentially ingested the 6-µm micro-PS over the 2-µm-diameter particles. Consumption of microalgae and absorption efficiency were significantly higher in exposed oysters, suggesting compensatory and physical effects on both digestive parameters. After 2 mo, exposed oysters had significant decreases in oocyte number (−38%), diameter (−5%), and sperm velocity (−23%). The D-larval yield and larval development of offspring derived from exposed parents decreased by 41% and 18%, respectively, compared with control offspring. Dynamic energy budget modeling, supported by transcriptomic profiles, suggested a significant shift of energy allocation from reproduction to structural growth, and elevated maintenance costs in exposed oysters, which is thought to be caused by interference with energy uptake. Molecular signatures of endocrine disruption were also revealed, but no endocrine disruptors were found in the biological samples. This study provides evidence that micro-PS cause feeding modifications and reproductive disruption in oysters, with significant impacts on offspring.

Autumn conditioning of the oyster Crassostrea gigas: a new approach

Gametogenesis, emitted gametes and larval yield (D larvae) of the oyster, Crassostrea gigas (Thunberg), were simultaneously examined in laboratory under two different conditions of temperature and photoperiod. One batch (“natural” cycle, NC) followed the equivalent cycle of average conditions of these parameters measured in La Tremblade (Charente-Maritime, France) during the last 15 years. The second batch (“accelerated” cycle, AC) was maintained under a variation two times faster of these parameters. Three conditioning experiences were performed during October, November and December 1998 with oysters from both NC and AC conditions. “Accelerated individuals” produced oocytes in growing stage from October and mature oocytes were noticed from December. In contrast, oysters from NC produced growing oocytes until January. Animals in AC produced 42% (October), 56% (November) and 96% (December) of mature oocytes after conditioning. Only the “natural” lot conditioned in December showed growing (23%) and mature oocytes (8%). Higher Walne–Mann index (WMI) values were recorded for “accelerated oysters” and significant differences (P<0.05) between treatments were noticed during October and November, suggesting nutrient accumulation before conditioning in the “accelerated” condition. Gamete emission and D larval yield results were equivalent to those reported in literature during early spring for the same species. The effect of temperature and photoperiod is discussed in order to understand their relationship with gametogenesis under these particular experimental conditions. It was demonstrated that internal clocks regulating gametogenesis can be change if stimulating factors (environmental cues) also change.

A preliminary study of the behaviour and vitality of reseeded juvenile great scallops, of three sizes in three seasons

In order to have a better understanding of recessing in great scallop, Pecten maximus and consequently the causes of mortality at reseeding, this study has monitored, at different seasons, the dispersion and recessing of different sizes of juveniles (about 15, 30 and 45 mm, called ‘small’, ‘medium’ and ‘large’) after seeding. Moreover, the aim was to see when small spat (15 mm) could be seeded, and thus reduce the costs of intermediate culture.Three monitoring approaches were used together: (1) continual observations by remote video camera, of a defined area (less than 1 m2) containing 10 scallops from each size group; (2) daily monitoring of behaviour with divers along three bottom lines, with 20 × 1 m2 plots each and nine marked scallops per plot; and (3) the biochemical content of the muscle: adenylic energetic charge and storage of energy reserves (glucides, proteins, lipids).The video monitoring identified but did not quantify predator behaviour, particularly at night. The role and behaviour of spiny crab, Maia squinado, and of small predators has clearly been shown, such as: (a) small crustaceans, Inachus sp., breaking the edges of scallop valves; and (b) small gobies, Pomatoschistus pictus, pecking the tentacles of the scallop mantle.For the monitoring by divers, filtering appeared much too difficult to look at for it was very disturbed by divers, and anyway the resumption of filtering came immediately after seeding. On the other hand, diver monitoring of dispersal and recessing was quite easy to do with a minimum of practice. On the basis of dispersal, the best seasons for seeding appear to be spring or summer. In autumn, two-thirds of ‘small’ and ‘medium’ juveniles are missing 3 days after seeding, but we could not observe whether they had been eaten by predators or had just moved and recessed farther. There was no experiment in winter owing to adverse conditions for scallop seedings.Biochemical analyses confirmed the unsuitability of autumn for scallop seeding, because of very low glucide content in this season.The adenylic energetic charge in the smooth part of the muscle showed that stress before seeding (aerial exposure, handling), and post-seeding behaviour (swimming, recessing) have a high energetic cost for scallops. In summer and autumn, 3 days after seeding, none of the three size batches recovered their initial vitality.

Oyster sex determination is influenced by temperature - First clues in spat during first gonadic differentiation and gametogenesis

The sex-determining system of Crassostrea gigas is still poorly known, especially regarding the potential influence of temperature. In order to address this question, mRNA expressions of actors of the molecular cascade (Cg-DMl, Cg-SoxE, Cg-β-catenin, Cg-Foxl2/Cg-Foxl2os) and of Oyvlg, a germ cell marker, were investigated by real-time PCR in spat grown at different temperatures (18, 22, 25 and 28°C). In parallel, gonadic differentiation, gametogenesis and sex ratios were assessed by histology at each of these temperatures. Whatever the temperature, Cg-DMl, Cg-SoxE, Cg-β-catenin and Oyvlg expressions peaked at the same developmental stage, always after Cg-Foxl2/Cg-Foxl2os (around 40-44dpf for spat grown at 18°C). Temperatures increased the kinetics of first gonadic differentiation and gametogenesis. At 25°C a significant switch occurred in sex ratio towards males and in the balance of expression between male and female genes, in favor of males. A slight gametogenesis disturbance was also observed. These results strengthen the hypotheses about the sex-determining time window and molecular cascade governing the development of C. gigas, with notably the involvement of Cg-Foxl2/Cg-Foxl2os in the very early steps. They also suggest an influence of temperature on the oysters sex determination which, associated to genetic control, would induce a mixed sex determination system (GSD+TSD).

Survival, growth and reproduction of cryopreserved larvae from a marine invertebrate, the Pacific oyster (Crassostrea gigas).

This study is the first demonstration of successful post-thawing development to reproduction stage of diploid cryopreserved larvae in an aquatic invertebrate. Survival, growth and reproductive performances were studied in juvenile and adult Pacific oysters grown from cryopreserved embryos. Cryopreservation was performed at three early stages: trochophore (13±2 hours post fertilization: hpf), early D-larvae (24±2 hpf) and late D-larvae (43±2 hpf). From the beginning (88 days) at the end of the ongrowing phase (195 days), no mortality was recorded and mean body weights did not differ between the thawed oysters and the control. At the end of the growing-out phase (982 days), survival of the oysters cryopreserved at 13±2 hpf and at 43±2 hpf was significantly higher (P<0.001) than those of the control (non cryopreserved larvae). Only the batches cryopreserved at 24±2 hpf showed lower survival than the control. Reproductive integrity of the mature oysters, formely cryopreserved at 13±2 hpf and 24±2 hpf, was estimated by the sperm movement and the larval development of their offspring in 13 crosses gamete pools (five males and five females in each pool). In all but two crosses out of 13 tested (P<0.001), development rates of the offspring were not significantly different between frozen and unfrozen parents. In all, the growth and reproductive performances of oysters formerly cryopreserved at larval stages are close to those of controls. Furthermore, these performances did not differ between the three initial larval stages of cryopreservation. The utility of larvae cryopreservation is discussed and compared with the cryopreservation of gametes as a technique for selection programs and shellfish cryobanking.

The quality of great scallop (Pecten maximus) sperm after thawing.

Most publications devoted to the cryopreservation of mollusc sperm have focused on the definition of technical protocols, avoiding the description of sperm quality after thawing. The present study investigated the effects of cryopreservation on sperm quality in the great scallop. Wild scallop were fished during the natural spawning period and conditioned in the hatchery before use. Sperm samples were obtained after intragonadal injection of serotonin and cryopreserved using a previously published protocol. Sperm quality was assessed using a panel of four parameters: sperm motility characteristics, using a computer assisted sperm analysis plugin with Image J, intracellular ATP content using an ATP-Lite kit, sperm integrity, using flow cytometry and sperm morphology, using transmission electron microscopy. For each parameter, fresh (control) and thawed spermatozoa were compared. A significant decrease of both the percentage of motile spermatozoa (reduction: 75%) and sperm swimming speed (86%) were observed for thawed sperm compared with fresh sperm. The percentage of living spermatozoa, as assessed using flow cytometry, was significantly lower for thawed sperm (72.4±2.5%) compared with fresh sperm (86.4±1.1). However, no significant difference of intracellular sperm ATP content was observed between fresh and thawed sperm. Post thawing, while some spermatozoa showed little or no morphological differences compared with fresh sperm, others had undergone drastic changes, including swelling of the plasma membrane, structural alterations of the chromatin and damage to mitochondria. In conclusion, the descriptive parameters studied in the present work showed that the quality of thawed great scallop sperm was lower than that of fresh cells but was still sufficient for use in aquaculture programs and sperm cryobanking for this species.