Development of duplex TaqMan-based real-time PCR assay for the simultaneous detection of Perkinsus olseni and P. chesapeaki in host Manila clam tissue samples.

The aetiological agent Perkinsus olseni is globally recognised as a major threat for shellfish production considering its wide geographical distribution across Asia, Europe, Australia and South America. Another species, Perkinsus chesapeaki, which has never been known to be associated with significant mortality events, was recently detected along French coasts infecting clam populations sporadically in association with P. olseni. Identifying potential cryptic infections affecting Ruditapes philippinarum is essential to develop appropriate host resource management strategies. Here, we developed a molecular method based on duplex real-time quantitative PCR for the simultaneous detection of these two parasites, P. olseni and P. chesapeaki, in the different clam tissues: gills, digestive gland, foot, mantle, adductor muscle and the rest of the soft body. We firstly checked the presence of possible PCR inhibitors in host tissue samples. The qPCR reactions were inhibited depending on the nature of the host organ. The mantle and the rest of the soft body have a high inhibitory effect from threshold of host gDNA concentration of 2 ng.µL-1, the adductor muscle and the foot have an intermediate inhibition of 5 ng.µL-1, and the gills and digestive gland do not show any inhibition of the qPCR reaction even at the highest host gDNA concentration of 20 ng.µL-1. Then, using the gills as a template, the suitability of the molecular technique was checked in comparison with the Ray's Fluid Thioglycolate Medium methodology recommended by the World Organisation for Animal Health. The duplex qPCR method brought new insights and unveiled cryptic infections as the co-occurrence of P. olseni and P. chesapeaki from in situ tissue samples in contrast to the RFTM diagnosis. The development of this duplex qPCR method is a fundamental work to monitor in situ co-infections that will lead to optimised resource management and conservation strategies to deal with emerging diseases.

Oyster reproduction is affected by exposure to polystyrene microplastics.

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.

Exposure to the toxic dinoflagellate Alexandrium catenella modulates juvenile oyster Crassostrea gigas hemocyte variables subjected to different biotic conditions.

The Pacific oyster Crassostrea gigas is an important commercial species cultured throughout the world. Oyster production practices often include transfers of animals into new environments that can be stressful, especially at young ages. This study was undertaken to determine if a toxic Alexandrium bloom, occurring repeatedly in French oyster beds, could modulate juvenile oyster cellular immune responses (i.e. hemocyte variables). We simulated planting on commercial beds by conducting a cohabitation exposure of juvenile, "specific pathogen-free" (SPF) oysters (naïve from the environment) with previously field-exposed oysters to induce interactions with new microorganisms. Indeed, toxic Alexandrium spp. exposures have been reported to modulate bivalve interaction with specific pathogens, as well as physiological and immunological variables in bivalves. In summary, SPF oysters were subjected to an artificial bloom of Alexandrium catenella, simultaneously with a cohabitation challenge. Exposure to A. catenella, and thus to the paralytic shellfish toxins (PSTs) and extracellular bioactive compounds produced by this alga, induced higher concentration, size, complexity and reactive oxygen species (ROS) production of circulating hemocytes. Challenge by cohabitation with field-exposed oysters also activated these hemocyte responses, suggesting a defense response to new microorganism exposure. These hemocyte responses to cohabitation challenge, however, were partially inhibited by A. catenella exposure, which enhanced hemocyte mortality, suggesting either detrimental effects of the interaction of both stressors on immune capacity, or the implementation of an alternative immune strategy through apoptosis. Indeed, no infection with specific pathogens (herpesvirus OsHV-1 or Vibrio aesturianus) was detected. Additionally, lower PST accumulation in challenged oysters suggests a physiological impairment through alteration of feeding-related processes. Overall, results of this study show that a short-term exposure to A. catenella combined with an exposure to a modified microbial community inhibited some hemocyte responses, and likely compromised physiological condition of the juvenile oysters.

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.

Involvement of Mitochondrial Activity and OXPHOS in ATP Synthesis During the Motility Phase of Spermatozoa in the Pacific Oyster, Crassostrea gigas.

In the Pacific oyster, spermatozoa are characterized by a remarkably long movement phase (i.e., over 24 h) sustained by a capacity to maintain intracellular ATP level. To gain information on oxidative phosphorylation (OXPHOS) functionality during the motility phase of Pacific oyster spermatozoa, we studied 1) changes in spermatozoal mitochondrial activity, that is, mitochondrial membrane potential (MMP), and intracellular ATP content in relation to motion parameters and 2) the involvement of OXPHOS for spermatozoal movement using carbonyl cyanide m-chlorophenyl hydrazone (CCCP). The percentage of motile spermatozoa decreased over a 24 h movement period. MMP increased steadily during the first 9 h of the movement phase and was subsequently maintained at a constant level. Conversely, spermatozoal ATP content decreased steadily during the first 9 h postactivation and was maintained at this level during the following hours of the movement phase. When OXPHOS was decoupled by CCCP, the movement of spermatozoa was maintained 2 h and totally stopped after 4 h of incubation, whereas spermatozoa were still motile in the control after 4 h. Our results suggest that the ATP sustaining flagellar movement of spermatozoa may partially originate from glycolysis or from mobilization of stored ATP or from potential phosphagens during the first 2 h of movement as deduced by the decoupling by CCCP of OXPHOS. However, OXPHOS is required to sustain the long motility phase of Pacific oyster spermatozoa. In addition, spermatozoa may hydrolyze intracellular ATP content during the early part of the movement phase, stimulating mitochondrial activity. This stimulation seems to be involved in sustaining a high ATP level until the end of the motility phase.

Flow cytometric assessment of morphology, viability, and production of reactive oxygen species of Crassostrea gigas oocytes. Application to toxic dinoflagellate (Alexandrium minutum) exposure.

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.

On the horns of a dilemma: Evaluation of synthetic and natural textile microfibre effects on the physiology of the pacific oyster Crassostrea gigas.

Fast fashion and our daily use of fibrous materials cause a massive release of microfibres (MF) into the oceans. Although MF pollution is commonly linked to plastics, the vast majority of collected MF are made from natural materials (e.g. cellulose). We investigated the effects of 96-h exposure to natural (wool, cotton, organic cotton) and synthetic (acrylic, nylon, polyester) textile MF and their associated chemical additives on the capacity of Pacific oysters Crassostrea gigas to ingest MF and the effects of MF and their leachates on key molecular and cellular endpoints. Digestive and glycolytic enzyme activities and immune and detoxification responses were determined at cellular (haemocyte viability, ROS production, ABC pump activity) and molecular (Ikb1, Ikb2, caspase 1 and EcSOD expression) levels, considering environmentally relevant (10 MF L-1) and worst-case scenarios (10 000 MF L-1). Ingestion of natural MF perturbed oyster digestive and immune functions, but synthetic MF had few effects, supposedly related with fibers weaving rather than the material itself. No concentration effects were found, suggesting that an environmental dose of MF is sufficient to trigger these responses. Leachate exposure had minimal effects on oyster physiology. These results suggest that the manufacture of the fibres and their characteristics could be the major factors of MF toxicity and stress the need to consider both natural and synthetic particles and their leachates to thoroughly evaluate the impact of anthropogenic debris. Environmental Implication. Microfibres (MF) are omnipresent in the world oceans with around 2 million tons released every year, resulting in their ingestion by a wide array of marine organisms. In the ocean, a domination of natural MF- representing more than 80% of collected fibres-over synthetic ones was observed. Despite MF pervasiveness, research on their impact on marine organisms, is still in its infancy. The current study aims to investigate the effects of environmental concentrations of both synthetic and natural textile MF and their associated leachates on a model filter feeder.

Co-infection of two eukaryotic pathogens within clam populations in Arcachon Bay.

The parasitic species Perkinsus olseni (= atlanticus) (Perkinsea, Alveolata) infects a wide range of mollusc species and is responsible for mortality events and economic losses in the aquaculture industry and fisheries worldwide. Thus far, most studies conducted in this field have approached the problem from a "one parasite-one disease" perspective, notably with regards to commercially relevant clam species, while the impact of other Perkinsus species should also be considered as it could play a key role in the disease phenotype and dynamics. Co-infection of P. olseni and P. chesapeaki has already been sporadically described in Manila clam populations in Europe. Here, we describe for the first time the parasitic distribution of two Perkinsus species, P. olseni and P. chesapeaki, in individual clam organs and in five different locations across Arcachon Bay (France), using simultaneous in situ detection by quantitative PCR (qPCR) duplex methodology. We show that P. olseni single-infection largely dominated prevalence (46-84%) with high intensities of infection (7.2 to 8.5 log-nb of copies. g-1of wet tissue of Manila clam) depending on location, suggesting that infection is driven by the abiotic characteristics of stations and physiological states of the host. Conversely, single P. chesapeaki infections were observed in only two sampling stations, Ile aux Oiseaux and Gujan, with low prevalences 2 and 14%, respectively. Interestingly, the co-infection by both Perkinsus spp., ranging in prevalence from 12 to 34%, was distributed across four stations of Arcachon Bay, and was detected in one or two organs maximum. Within these co-infected organs, P. olseni largely dominated the global parasitic load. Hence, the co-infection dynamics between P. olseni and P. chesapeaki may rely on a facilitating role of P. olseni in developing a primary infection which in turn may help P. chesapeaki infect R. philippinarum as a reservoir for a preferred host. This ecological study demonstrates that the detection and quantification of both parasitic species, P. olseni and P. chesapeaki, is essential and timely in resolving cryptic infections and their consequences on individual hosts and clam populations.

Chemical effects of different types of rubber-based products on early life stages of Pacific oyster, Crassostrea gigas.

Rubber products and debris with specific chemical signatures can release their constitutive compounds into the surrounding environment. We investigated the chemical toxicity of different types of new and used rubber products (tires, crumb rubber granulates, aquaculture rubber bands) on early life stages of a model marine organism, Pacific oyster Crassostrea gigas. Leachates obtained from used products were generally less toxic than those from new ones. Leachates from new products induced embryotoxicity at different concentrations: oyster-farming rubber bands (lowest observed effect concentration, LOEC = 1 g L-1) and crumb rubber granulates (LOEC = 1 g L-1) > tires (LOEC = 10 g L-1). Moreover, new oyster-farming rubber bands induced spermiotoxicity at 10 g L-1 (-29% survival) resulting in decreased oyster reproductive output (-17% fertilization yield). Targeted chemical analyses revealed some compounds (2 mineral contaminants, 15 PAHs, 2 PCBs) in leachates, which may have played a role. Rubber used in marine aquaculture (rubber bands) or present at sea as waste (tire, crumb rubber granulates) therefore release hazardous chemical molecules under realistic conditions, which may affect oyster development. Aquaculture development work is necessary to improve practices for eco-safety, as efforts to limit the contamination of marine environments by terrestrial rubber debris.

Responses of early life stages of European abalone (Haliotis tuberculata) to ocean acidification after parental conditioning: Insights from a transgenerational experiment.

CO2 absorption is leading to ocean acidification (OA), which is a matter of major concern for marine calcifying species. This study investigated the effects of simulated OA on the reproduction of European abalone Haliotis tuberculata and the survival of its offspring. Four-year-old abalone were exposed during reproductive season to two relevant OA scenarios, ambient pH (8.0) and low pH (7.7). After five months of exposure, abalone were induced to spawn. The gametes, larvae and juveniles were then exposed for five months to the same pH conditions as their parents. Several biological parameters involved in adult reproduction as well as in larval, post-larval and juvenile fitness were measured. No effects on gametes, fertilisation or larval oxidative stress response were detected. However, developmental abnormalities and significant decreases in shell length and calcification were observed at veliger stages. The expression profile of a GABA A receptor-like gene appeared to be regulated by pH, depending on larval stage. Larval and post-larval survival was not affected by low pH. However, a lower survival and a reduction of growth were recorded in juveniles at pH 7.7. Our results confirm that OA negatively impacts larval and juvenile fitness and suggest the absence of carry-over effects on abalone offspring. This may compromise the survival of abalone populations in the near future.

A new insight into allelopathic effects of Alexandrium minutum on photosynthesis and respiration of the diatom Chaetoceros neogracile revealed by photosynthetic-performance analysis and flow cytometry.

The allelopathic effects of Alexandrium minutum, a toxic dinoflagellate, on the diatom Chaetoceros neogracile were evaluated using unialgal cultures evaluated by flow cytometry (FCM) and photosynthetic-performance analysis. Using FCM, we demonstrated that red chlorophyll fluorescence, relative cell size (Forward scatter of blue laser light, FSC) and cell complexity (Side scatter, 90°-angle scatter of blue laser light, SSC) significantly and rapidly decreased in C. neogracile cells exposed to A. minutum. Cells of C. neogracile exposed to A. minutum had fewer active photosynthetic reaction centers and sharply decreased photosynthetic efficiency. These effects were intensified with advancing A. minutum batch culture age and cell density. The supernatant of A. minutum contained the majority of the putative allelopathic compounds, and the biological activity of these compounds remained active less than 9 h after release. This paper describes for the first time specific effects of allelochemicals produced by A. minutum on the photosynthetic apparatus of microalgal target cells. The biochemical composition of A. minutum allelopathic agents, however, remains unknown and still needs to be investigated.

Effect of acclimatization on hemocyte functional characteristics of the Pacific oyster (Crassostrea gigas) and carpet shell clam (Ruditapes decussatus).

Most experimental procedures on molluscs are done after acclimatization of wild animals to lab conditions. Similarly, short-term acclimation is often unavoidable in a field survey when biological analysis cannot be done within the day of sample collection. However, acclimatization can affect the general physiological condition and particularly the immune cell responses of molluscs. Our aim was to study the changes in the hemocyte characteristics of the Pacific oyster Crassostrea gigas and the carpet shell clam Ruditapes decussatus acclimated 1 or 2 days under emersed conditions at 14 ± 1 °C and for 1, 2, 7, or 10 days to flowing seawater conditions (submerged) at 9 ± 1 °C, when compared to hemolymph withdrawn from organisms sampled in the field and immediately analyzed in the laboratory (unacclimated). The hemocyte characteristics assessed by flow cytometry were the total (THC) and differential hemocyte count, percentage of dead cells, phagocytosis, and reactive oxygen species (ROS) production. Dead hemocytes were lower in oysters acclimated both in emersed and submerged conditions (1%-5%) compared to those sampled in the field (7%). Compared to oysters, the percentage of dead hemocytes was lower in clams (0.4% vs. 1.1%) and showed a tendency to decrease during acclimatization in both emersed and submerged conditions. In comparison to organisms not acclimated, the phagocytosis of hemocytes decreased in both oysters and clams acclimated under submerged conditions, but was similar in those acclimated in emersed conditions. The ROS production remained stable in both oysters and clams acclimated in emersed conditions, whereas in submerged conditions ROS production did not change in both the hyalinocytes and granulocytes of oysters, but increased in clams. In oysters, the THC decreased when they were acclimated 1 and 2 days in submerged conditions and was mainly caused by a decrease in granulocytes, but the decrease in THC in oysters acclimated 2 days in emersed conditions was caused by a decrease in hyalinocytes and small agranular cells. In clams, the THC was significantly lower in comparison to those not acclimated, regardless of the conditions of the acclimatization. These findings demonstrate that hemocyte characteristics were differentially affected in both species by the tested conditions of acclimatization. The phagocytosis and ROS production in clams and phagocytosis in oysters were not different in those acclimated for 1 day under both conditions, i.e. emersed and submerged, and those sampled in the field (unacclimated). The THC was significantly affected by acclimatization conditions, so the differences between clams and oysters should be considered in studies where important concentrations of hemocytes are required. The difference in the immune response between both species could be related to their habitat (epifaunal vs. infaunal) and their ability of resilience to manipulation and adaptation to captivity. Our results suggest that functional characteristics of hemocytes should be analyzed in both oysters and clams during the first 1 or 2 days, preferably acclimated under emersed rather than submerged conditions.

Immune parameters of QX-resistant and wild caught Saccostrea glomerata hemocytes in relation to Marteilia sydneyi infection.

Sydney rock oysters (SRO) Saccostrea glomerata suffer mass mortalities during summer and autumn as a result of infection by a protozoan parasite Marteilia sydneyi (QX disease). Mass selected disease resistant (QXR) lines have been used with some success in affected estuaries in recent years, with resistance attributed to oxidative defense systems. However, the role of hemocytes in resistance to QX by SRO has not been fully explored. In the present study, fifty QXR and fifty wild caught (WC) oysters were collected from a lease at Pimpama River during a QX outbreak in January 2011. Hemocytes characteristics (type, morphology) and functions (mortality, phagocytosis and oxidative activity) from both oyster lines were analyzed by flow cytometry in the context of infection intensity and parasite viability (determined histologically). Amongst the QXR oysters, 20% were diseased containing viable parasite, 74% had killed M. sydneyi and 6% were uninfected. In contrast, 86% of WC oysters were diseased, 2% had killed M. sydneyi and 12% were healthy. Significant differences in hemocyte number and physiology between the two oyster lines were found (ANOVA). Phagocytosis rate and the mean oxidative activity per cell were similar between both oyster lines. Higher numbers of infiltrating and circulating hemocytes, higher percentage of circulating granulocytes, their higher size and complexity in QXR oysters, and the production of reactive oxygen species were associated with the ability to kill the parasite. High abundance of M. sydneyi in the digestive tubule epithelium of both oyster lines implied inability to kill the parasite at the beginning of the infection. However, QXR oysters had the ability to kill M. sydneyi at the stage of sporangiosorae in the epithelium of digestive tubules. The similar phagocytic ability of hemocytes from both oyster lines, the size of the parasite at this infection stage, and its localization suggested that encapsulation is likely to be the main process involved in the eradication of M. sydneyi by QXR oysters.

Implication of the Type IV Secretion System in the Pathogenicity of Vibrio tapetis, the Etiological Agent of Brown Ring Disease Affecting the Manila Clam Ruditapes philippinarum.

Vibrio tapetis is a Gram-negative bacterium that causes infections of mollusk bivalves and fish. The Brown Ring Disease (BRD) is an infection caused by V. tapetis that primarily affects the Manila clam Ruditapes philippinarum. Recent studies have shown that a type IV secretion system (T4SS) gene cluster is exclusively found in strains of V. tapetis pathogenic to clams. However, whether the T4SS is implicated or not during the infection process remains unknown. The aim of this study was to create and characterize a V. tapetis T4SS null mutant, obtained by a near-complete deletion of the virB4 gene, in order to determine the role of T4SS in the development of BRD. This study demonstrated that the T4SS is neither responsible for the loss of hemocyte adhesion capacities, nor for the decrease of the lysosomal activity during BRD. Nevertheless, we observed a 50% decrease of the BRD prevalence and a decrease of mortality dynamics with the ΔvirB4 mutant. This work demonstrates that the T4SS of V. tapetis plays an important role in the development of BRD in the Manila clam.

The toxic dinoflagellate Alexandrium minutum affects oyster gamete health and fertilization potential.

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 h to a range of environmentally realistic A. minutum concentrations (10-2.5 × 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.

Cultures of Dinophysis sacculus, D. acuminata and pectenotoxin 2 affect gametes and fertilization success of the Pacific oyster, Crassostrea gigas.

Harmful algal blooms (HABs) of toxic species of the dinoflagellate genus Dinophysis are a threat to human health as they are mainly responsible for diarrheic shellfish poisoning (DSP) in the consumers of contaminated shellfish. Such contamination leads to shellfish farm closures causing major economic and social issues. The direct effects of numerous HAB species have been demonstrated on adult bivalves, whereas the effects on critical early life stages remain relatively unexplored. The present study aimed to determine the in vitro effects of either cultivated strains of D. sacculus and D. acuminata isolated from France or their associated toxins (i.e. okadaic acid (OA) and pectenotoxin 2 (PTX2)) on the quality of the gametes of the Pacific oyster Crassostrea gigas. This was performed by assessing the ROS production and viability of the gametes using flow cytometry, and fertilization success using microscopic counts. Oocytes were more affected than spermatozoa and their mortality and ROS production increased in the presence of D. sacculus and PTX2, respectively. A decrease in fertilization success was observed at concentrations as low as 0.5 cell mL-1 of Dinophysis spp. and 5 nM of PTX2, whereas no effect of OA could be observed. The effect on fertilization success was higher when both gamete types were concomitantly exposed compared to separate exposures, suggesting a synergistic effect. Our results also suggest that the effects could be due to cell-to-cell contact. These results highlight a potential effect of Dinophysis spp. and PTX2 on reproduction and recruitment of the Pacific oyster.

Sex-, gametogenesis, and tidal height-related differences in levels of HSP70 and metallothioneins in the Pacific oyster Crassostrea gigas.

Pacific oysters, Crassostrea gigas, living at a range of tidal heights, routinely encounter large fluctuations in temperature. We demonstrate that levels of heat shock proteins (HSP) and other stress proteins (metallothioneins, MTs) quantified by ELISA, remained similar in gills, mantle and digestive gland between oysters inhabiting low and high tidal heights. In contrast, endogenous HSPs and MTs levels in gonad changed significantly during gametogenesis. In female gonads, the constitutive form of HSP70 and the MTs increased from immature (or resting) to mature stages (about more than 3-fold) and decreased after spawning. In male gonads, the same expression patterns were observed, whereas proteins levels decreased once fully mature. Females presented higher concentration of HSP70 and MTs than males during the spawning period. No significant difference in HSPs and MTs patterns was found among oysters sampled at low and high tidal heights. We hypothesize that the high level of stress proteins in eggs may increase survival of oyster progeny.

Do transparent exopolymeric particles (TEP) affect the toxicity of nanoplastics on Chaetoceros neogracile?

The potential presence of nanoplastics (NP) in aquatic environments represents a growing concern regarding their possible effects on aquatic organisms. The objective of this study was to assess the impact of polystyrene (PS) amino-modified particles (50  nm PSNH2) on the cellular and metabolic responses of the diatom Chaetoceros neogracile cultures at two essential phases of the growth cycle, i.e. exponential (division) and stationary (storage) phases. Both cultures were exposed for 4 days to low (0.05 µg mL-1) and high (5 µg mL-1) concentrations of PS-NH2. Exposure to NP impaired more drastically the major cellular and physiological parameters during exponential phase than during the stationary phase. Only an increase in ROS production was observed at both culture phases following NP exposures. In exponential phase cultures, large decreases in chlorophyll content, esterase activity, cellular growth and photosynthetic efficiency were recorded upon NP exposure, which could have consequences on the diatoms life cycle and higher food-web levels. The observed differential responses to NP exposure according to culture phase could reflect i) the higher concentration of Transparent Exopolymer Particles (TEP) at stationary phase leading to NP aggregation and thus, probably minimizing NP effects, and/or ii) the fact that dividing cells during exponential phase may be intrinsically more sensitive to stress. This work evidenced the importance of algae physiological state for assessing the NP impacts with interactions between NP and TEP being one key factor affecting the fate of NP in algal media and their impact to algal' cells.

The dinoflagellate Alexandrium minutum affects development of the oyster Crassostrea gigas, through parental or direct exposure.

Harmful algal blooms are a threat to aquatic organisms and coastal ecosystems. Among harmful species, the widespread distributed genus Alexandrium is of global importance. This genus is well-known for the synthesis of paralytic shellfish toxins which are toxic for humans through the consumption of contaminated shellfish. While the effects of Alexandrium species upon the physiology of bivalves are now well documented, consequences on reproduction remain poorly studied. In France, Alexandrium minutum blooms have been recurrent for the last decades, generally appearing during the reproduction season of most bivalves including the oyster Crassostrea gigas. These blooms could not only affect gametogenesis but also spawning, larval development or juvenile recruitment. This study assesses the effect of toxic A. minutum blooms on C. gigas reproduction. Adult oysters were experimentally exposed to A. minutum, at environmentally realistic concentrations (102 to 103 cells mL-1) for two months during their gametogenesis and a control group, not exposed to A. minutum was fed with a non-toxic dinoflagellate. To determine both consequences to next generation and direct effects of A. minutum exposure on larvae, the embryo-larval development of subsequent offspring was conducted with and without A. minutum exposure at 102 cells mL-1. Effects at each stage of the reproduction were investigated on ecophysiological parameters, cellular responses, and offspring development. Broodstock exposed to A. minutum produced spermatozoa with decreased motility and larvae of smaller size which showed higher mortalities during settlement. Embryo-larval exposure to A. minutum significantly reduced growth and settlement of larvae compared to non-exposed offspring. This detrimental consequence on larval growth was stronger in larvae derived from control parents compared to offspring from exposed parents. This study provides evidence that A. minutum blooms, whether they occur during gametogenesis, spawning or larval development, can either affect gamete quality and/or larval development of C. gigas, thus potentially impacting oyster recruitment.

DjlA, a membrane-anchored DnaJ-like protein, is required for cytotoxicity of clam pathogen Vibrio tapetis to hemocytes.

DjlA is an inner membrane cochaperone belonging to the DnaJ family, which has been shown to be involved in Legionella sp. pathogenesis. In this study, we explored the role of this protein in the physiology and virulence of Vibrio tapetis, the etiological agent of brown ring disease (BRD) in Manila clam (Ruditapes philippinarum). Analysis of the djlA locus in V. tapetis revealed a putative organization in an operon with a downstream gene that we designated duf924(Vt), which encodes a conserved protein with an unknown function and has homologues in bacteria and eukaryotes. djlA mutants displayed a reduced growth rate and showed an important loss of cytotoxic activity against R. philippinarum hemocytes in vitro, which could be restored by extrachromosomal expression of wild-type djlA(Vt) but not duf924(Vt). These results are in keeping with the potential importance of DjlA for bacterial pathogenicity and open new perspectives for understanding the mechanism of action of this protein in the novel V. tapetis-R. philippinarum interaction model.