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Dinoflagellates from the globally distributed genus Alexandrium are known to produce both paralytic shellfish toxins (PST) and uncharacterized bioactive extracellular compounds (BEC) with allelopathic, ichthyotoxic, hemolytic and cytotoxic activities. In France, blooms of Alexandrium minutum appear generally during the spawning period of most bivalves. These blooms could therefore alter gametes and/or larval development of bivalves, causing severe issues for ecologically and economically important species, such as the Pacific oyster Crassostrea (=Magallana) gigas. The aim of this work was to test the effects of three strains of A. minutum producing either only PST, only BEC, or both PST and BEC upon oyster gametes, and potential consequences on fertilization success. Oocytes and spermatozoa were exposed in vitro for 2 hours to a range of environmentally realistic A. minutum concentrations (10 to 2.5 × 104 cells mL-1). Following exposure, gamete viability and reactive oxygen species (ROS) production were assessed by flow cytometry, spermatozoa motility and fertilization capacities of both spermatozoa and oocytes were analysed by microscopy. Viability and fertilization capacity of spermatozoa and oocytes were drastically reduced following exposure to 2.5 × 104 cells mL-1 of A. minutum. The BEC-producing strain was the most potent strain decreasing spermatozoa motility, increasing ROS production of oocytes, and decreasing fertilization, from the concentration of 2.5 × 103 cells mL-1. This study highlights the significant cellular toxicity of the BEC produced by A. minutum on oyster gametes. Physical contact between gametes and motile thecate A. minutum cells may also contribute to alter oyster gamete integrity. These results suggest that oyster gametes exposure to A. minutum blooms could affect oyster fertility and reproduction success.

Early life stages (ELS) of numerous marine invertebrates mustcope with man-made contaminants, including plastic debris, during their pelagic phase. Among the diversity of plastic particles, nano-sized debris, known as nanoplastics, can induce effects with severe outcomes in ELS of various biological models, including the Pacific oyster Crassostrea gigas. Here, we investigated the effects of a sub-lethal dose (0.1 µg mL−1) of 50 nm polystyrene nanobeads (nano-PS) with amine functions on oyster embryos (24 h exposure) and we assessed consequences on larval and adult performances over two generations of oysters. Only a few effects were observed. Lipid analyses revealed that first-generation (G1) embryos exposed to nano-PS displayed a relative increase in cardiolipin content (+9.7%), suggesting a potential modification of mitochondrial functioning. G1-larvae issued from exposed embryos showed decreases in larval growth (−9%) and lipid storage (−20%). No effect was observed at the G1 adult stage in terms of growth, ecophysiological parameters (clearance and respiration rates, absorption efficiency), or reproductive outputs (gonadic development, gamete quality). Second generation (G2) larvae issued from control G1 displayed a significant growth reduction after G2 embryonic exposure to nano-PS (−24%) compared to control (as observed at the first generation), while no intergenerational effect was detected on G2 larvae issued from G1 exposed embryos. Overall, the present experimental study suggests a low incidence of a short embryonic exposure to nano-PS on oyster phenotypes along the entire life cycle until the next larval generation.

Oysters are keystone species that use external fertilization as a sexual mode. The gametes are planktonic and face a wide range of stressors, including plastic litter. Nanoplastics are of increasing concern because their size allows pronounced interactions with biological membranes, making them a potential hazard to marine life. In the present study, oyster spermatozoa were exposed for 1 h to various doses (from 0.1 to 25 µg mL−1) of 50-nm polystyrene beads with amine (50-NH2 beads) or carboxyl (50-COOH beads) functions. Microscopy revealed adhesion of particles to the spermatozoa membranes, but no translocation of either particle type into cells. Nevertheless, the 50-NH2 beads at 10 µg mL−1 induced a high spermiotoxicity, characterized by a decrease in the percentage of motile spermatozoa (−79%) and in the velocity (−62%) compared to control spermatozoa, with an overall drop in embryogenesis success (−59%). This major reproduction failure could be linked to a homeostasis disruption in exposed spermatozoa. The 50-COOH beads hampered spermatozoa motility only when administered at 25 µg mL−1 and caused a decrease in the percentage of motile spermatozoa (−66%) and in the velocity (−38%), but did not affect embryogenesis success. Microscopy analyses indicated these effects were probably due to physical blockages by microscale aggregates formed by the 50-COOH beads in seawater. This toxicological study emphasizes that oyster spermatozoa are a useful and sensitive model for (i) deciphering the fine interactions underpinning nanoplastic toxicity and (ii) evaluating adverse effects of plastic nanoparticles on marine biota while waiting for their concentration to be known in the environment.

Though bivalve mollusks are keystone species and major species groups in aquaculture production worldwide, gamete biology is still largely unknown. This review aims to provide a synthesis of current knowledge in the field of sperm biology, including spermatozoa motility, flagellar beating, and energy metabolism; and to illustrate cellular signaling controlling spermatozoa motility initiation in bivalves. Serotonin (5-HT) induces hyper-motility in spermatozoa via a 5-HT receptor, suggesting a serotoninergic system in the male reproductive tract that might regulate sperm physiology. Acidic pH and high concentration of K+ are inhibitory factors of spermatozoa motility in the testis. Motility is initiated at spawning by a Na+-dependent alkalization of intracellular pH mediated by a Na+/H+ exchanger. Increase of 5-HT in the testis and decrease of extracellular K+ when sperm is released in seawater induce hyperpolarization of spermatozoa membrane potential mediated by K+ efflux and associated with an increase in intracellular Ca2+ via opening of voltage-dependent Ca2+ channels under alkaline conditions. These events activate dynein ATPases and Ca2+/calmodulin-dependent proteins resulting in flagellar beating. It may be possible that 5-HT is also involved in intracellular cAMP rise controlling cAMP-dependent protein kinase phosphorylation in the flagellum. Once motility is triggered, flagellum beats in asymmetric wave pattern leading to circular trajectories of spermatozoa. Three different flagellar wave characteristics are reported, including “full”, “twitching”, and “declining” propagation of wave, which are described and illustrated in the present review. Mitochondrial respiration, ATP content, and metabolic pathways producing ATP in bivalve spermatozoa are discussed. Energy metabolism of Pacific oyster spermatozoa differs from previously studied marine species since oxidative phosphorylation synthetizes a stable level of ATP throughout 24-h motility period and the end

Artificial reproduction involves collection and handling of gametes in a way that secures their quality and maximizes the fertilization outcome. In addition to initial sperm quality, numerous steps can affect the final result of fertilization, from the sperm collection process until gamete mixing (or co-incubation) when spermatozoon enters or fuses with the oocyte. In this review, we summarize the whole process of sperm handling, from collection until fertilization for fish, penaeid shrimp, bivalve mollusks and marine mammals. To obtain sperm from captive animals, techniques vary widely across taxa, and include stripping by abdominal massage or testis surgical removal in fish, spermatophore collection in penaeid shrimps, gonadal scarification or temperature shock in bivalve mollusks, and voluntary collection via positive reinforcement in mammals. In most cases, special care is needed to avoid contamination by mucus, seawater, urine, or feces that can either activate spermatozoa motility and/or decrease its quality. We also review techniques and extender solutions used for refrigerated storage of sperm across the aforementioned taxa. Finally, we give an overview of the different protocols for in vivo and in vitro fertilization including activation of spermatozoa motility and methods for gamete co-incubation. The present study provides valuable information regarding breeder management either for animal production or species conservation.

[EN] Artificial reproduction involves collection and handling of gametes in a way that secures their quality and maximizes the fertilization outcome. In addition to initial sperm quality, numerous steps can affect the final result of fertilization, from the sperm collection process until gamete mixing (or co-incubation) when the spermatozoon enters or fuses with the oocyte. In this review, we summarize the whole process of sperm handling, from collection until fertilization for fish, penaeid shrimp, bivalve mollusks and marine mammals. To obtain sperm from captive animals, techniques vary widely across taxa, and include stripping by abdominal massage or testis surgical removal in fish, spermatophore collection in penaeid shrimps, gonadal scarification or temperature shock in bivalve mollusks, and voluntary collection via positive reinforcement in mammals. In most cases, special care is needed to avoid contamination by mucus, seawater, urine, or feces that can either activate sperm motility and/or decrease its quality. We also review techniques and extender solutions used for refrigerated storage of sperm across the afore-mentioned taxa. Finally, we give an overview of the different protocols for in vivo and in vitro fertilization including activation of sperm motility and methods for gamete co-incubation. The present study provides valuable information regarding breeder management either for animal production or species conservation. Crown Copyright (C) 2019 Published by Elsevier Inc. All rights reserved.

Fertilization of the oocyte is a critical process of sexual reproduction depending among other factors on spermatozoa functionality. Mitochondria participate in many crucial processes for spermatozoa motility and fertilizing ability. The Pacific oyster, Crassostrea gigas, is characterized by a high inter-individual variability of its reproductive success which has been shown to be partially due to the variability of gamete quality. The present study explored spermatozoa characteristics such as: i) relationships among mitochondrial functionality (ATP content, mitochondrial membrane potential (MMP), reactive oxygen species production); sperm viability (percentages of live, dying, and dead); and motility; and ii) their involvement in inter-oyster variability of fertilizing ability demonstrated using simple and multiple regressions. Our results showed that ATP content depends on both MMP and viability of spermatozoa. Using multiple regressions, 61% of the variability of the trochophore-larval yield was explained by a model combining the ATP content and the percentage of dying spermatozoa (P < 0.001). Our results reveal that capacity of spermatozoa to maintain a high level of ATP via OXPHOS partly explains the inter-individual variability of fertilization success in the Pacific oyster. Sperm ATP content and viability assays will provide valuable tools for assessing sperm quality of this species in aquaculture production, cryopreservation, and bioassays.

Pacific oyster hatchery production remains highly variable, partially due to inconsistent gamete quality. However, little attention has been paid to oocyte quality in Pacific oyster compared to important vertebrate aquaculture species such as fishes. The aims of the present study were to identify criteria of oocyte and trochophore quality used as predictive tools for embryo developmental success at the D-larval stage. Twenty-six potential proxies of oocyte and trochophore quality were studied on 25 mature females, as determined using histology. Some morphological and biochemical characteristics of mature oocytes were described. Protein appeared to be the main constituent of fully mature Pacific oyster oocytes. Neutral lipids accounted for 61% of the total lipids and triglycerides for 85% of the neutral lipids. Oocyte fertilization capacity was assessed by determining the D-larval yield. High inter-individual variation of D-larval yield was observed (ranging from 1 to 58%). D-larval yield was positively correlated with the percentage of motile trochophores (R2 = 0.237) and with trochophore Velocity of the Average Path (VAP; R2 = 0.255) and negatively correlated with oocyte total steryl esters (R2 = 0.244). The use of a multivariate model, including D-larval yield, trochophore VAP and total steryl esters, led to a higher coefficient of determination (R2 = 0.352), highlighting that the prediction of embryo development in molluscs is a complex process requiring sophisticated multi-factor models.

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.

This work assesses the present knowledge on Pacific oyster sperm biology in comparison to two marine fish species (turbot and seabass) whose sperm characteristics are well described. Sperm morphology mainly differs by the presence of an acrosome in Pacific oyster which is absent in both fish species. In turbot as in Pacific oyster, a sperm maturation process along the genital tract is observed. Sperm motility is triggered by changes in osmolality for seabass and turbot and in pH for Pacific oyster. However, complementary factors are involved to maintain sperm immotile in the genital tract. Sperm movement duration is very long in Pacific oyster (2024 h), compared to turbot (35 min) and seabass (4050 s). A high capacity of ATP regeneration is observed in Pacific oyster sperm, sustained by the limited changes in its morphology observed at the end of the swimming phase. Then, the total distance covered by spermatozoa is very different among the studied species (seabass: 2 mm, turbot: 12 mm, Pacific oyster: 1 m). Considering the main characteristics of sperm movement, the three studied species can be separated in two groups: the sprint racer group (seabass: high velocity and short distance covered) and the marathonian racer one (Pacific oyster: low velocity but covering long distances). To an intermediate extent, turbot sperm belongs to the sprint racer group. Then, the two different sperm movement strategies observed in the three species, are compensated by the behaviour of the breeders.