Can survival of European flat oysters following experimental infection with Bonamia ostreae be predicted using QTLs?

The present study identifies quantitative trait loci (QTLs) in response to an experimental infection with the parasite responsible for bonamiosis, Bonamia ostreae, in two segregating families of the European flat oyster, Ostrea edulis. We first constructed a genetic-linkage map for each studied family and improved the existing genetic-linkage map for the European flat oyster with a set of SNP markers. This latter map now combines the best accuracy and the best estimate of the genome coverage available for an oyster species. Secondly, by comparing the QTLs detected in this study with those previously published for O. edulis in similar experimental conditions, we identified several potential QTLs that were identical between the different families, and also new specific QTLs. We also detected, within the confidence interval of several QTL regions, some previously predicted candidate genes differentially expressed during an infection with B. ostreae, providing new candidate genome regions which should now be studied more specifically.

OsHV-1 countermeasures to the Pacific oyster’s anti-viral response

The host-pathogen interactions between the Pacific oyster (Crassostrea gigas) and Ostreid herpesvirus type 1 (OsHV-1) are poorly characterised. Herpesviruses are a group of large, DNA viruses that are known to encode gene products that subvert their host’s antiviral response. It is likely that OsHV-1 has also evolved similar strategies as its genome encodes genes with high homology to C. gigas inhibitors of apoptosis (IAPs) and an interferon-stimulated gene (termed CH25H). The first objective of this study was to simultaneously investigate the expression of C. gigas and OsHV-1 genes that share high sequence homology during an acute infection. Comparison of apoptosis-related genes revealed that components of the extrinsic apoptosis pathway (TNF) were induced in response to OsHV-1 infection, but we failed to observe evidence of apoptosis using a combination of biochemical and molecular assays. IAPs encoded by OsHV-1 were highly expressed during the acute stage of infection and may explain why we didn’t observe evidence of apoptosis. However, C. gigas must have an alternative mechanism to apoptosis for clearing OsHV-1 from infected gill cells as we observed a reduction in viral DNA between 27 and 54 h post-infection. The reduction of viral DNA in C. gigas gill cells occurred after the up-regulation of interferon-stimulated genes (viperin, PKR, ADAR). In a second objective, we manipulated the host’s anti-viral response by injecting C. gigas with a small dose of poly I:C at the time of OsHV-1 infection. This small dose of poly I:C was unable to induce transcription of known antiviral effectors (ISGs), but these oysters were still capable of inhibiting OsHV-1 replication. This result suggests dsRNA induces an anti-viral response that is additional to the IFN-like pathway.

Distinct immune responses of juvenile and adult oysters (Crassostrea gigas) to viral and bacterial infections

Since 2008, massive mortality events of Pacific oysters (Crassostrea gigas) have been reported worldwide and these disease events are often associated with Ostreid herpesvirus type 1 (OsHV-1). Epidemiological field studies have also reported oyster age and other pathogens of the Vibrio genus are contributing factors to this syndrome. We undertook a controlled laboratory experiment to simultaneously investigate survival and immunological response of juvenile and adult C. gigas at different time-points post-infection with OsHV-1, Vibrio tasmaniensis LGP32 and V. aestuarianus. Our data corroborates epidemiological studies that juveniles are more susceptible to OsHV-1, whereas adults are more susceptible to Vibrio. We measured the expression of 102 immune-genes by high-throughput RT-qPCR, which revealed oysters have different transcriptional responses to OsHV-1 and Vibrio. The transcriptional response in the early stages of OsHV-1 infection involved genes related to apoptosis and the interferon-pathway. Transcriptional response to Vibrio infection involved antimicrobial peptides, heat shock proteins and galectins. Interestingly, oysters in the later stages of OsHV-1 infection had a transcriptional response that resembled an antibacterial response, which is suggestive of the oyster's microbiome causing secondary infections (dysbiosis-driven pathology). This study provides molecular evidence that oysters can mount distinct immune response to viral and bacterial pathogens and these responses differ depending on the age of the host.

Infection dynamics of Marteilia refringens in flat oyster Ostrea edulis and copepod Paracartia grani in a claire pond of Marennes-Oleron Bay

The protozoan parasite Marteilia refringens has been partly responsible for the severe decrease in the production of the European flat oyster Ostrea edulis Linnaeus in France since the 1970s. The calanoid copepod Paracartia grani Sars was recently found to be a host for M refringens in French shallow-water oyster ponds ('claires'). This study reconsidered M refringens transmission dynamics in the light of this finding, taking into account not only oyster infection dynamics and environmental factors but also data concerning the copepod host. P. grani population dynamics in the claire under study revealed that this species is the dominant planktonic copepod in this confined ecosystem. During winter, M refringens overwintered in O. edulis, with P. grani existing only as resting eggs in the sediment. The increase in temperature in spring controlled and synchronized both the release of M refringens sporangia in the oyster feces, and the hatching of the benthic resting eggs of the copepod. Infection of oysters by M refringens was limited to June, July and August, coinciding with (1) the highest temperature recorded in the claire, and (2) the highest abundance of P. grani. PCR detection of M refringens in P. grani during the summer period was linked to the release of parasite sporangia by the oyster. Our results are supported by previous results on the effective transmission of this parasite from the oyster to the copepod.

Ecophysiologie de la maturation sexuelle et de la ponte de l'huître creuse Crassostrea gigas : réponses métaboliques (respiration) et alimentaires (filtration, absorption) en fonction des différents stades de maturation

The study of maturation and spawning of the oyster is part of a research program to investigate the summer mortalities of the oysters, Crassostrea gigas in Marennes-Oléron Bay. Four maturity stages were simultaneously obtained by diet and thermal conditioning (immature, low maturation, mature and post-spawning stages). Measurements of clearance, filtration, absorption and respiration rates allowed a calculation of the scope for growth and hence an estimation of the oyster's energetic budget at various maturity stages. Male and female oysters had similar physiological responses. The filtration rate ranged from 2.4 to 2.6 1.h(-1) at the early stages of maturation and decreased to 1.8 1.h.' during the maturity stage. Growth rate resulting from gonad development did not induce filtration rate changes. Mature 2.5 and 1.5-year-old oysters showed a negative energy budget reaching -15 and -90 J.h(-1) respectively. By contrast, non-ripe oysters had scope for growth in the range 110 to 170 J.h(-1). A negative energy budget during the high maturation stage resulted from a reduced absorption efficiency. A new allometric relationship for the respiration model of C. gigas was defined during vitellogenesis with a 0.574 coefficient value. Based on Our results, the oyster's physiological weakness during vitellogenesis should be considered as a part of explanation for spring and summer mortalities of cultured oysters in Marennes-Oléron Bay.

Ecophysiologie de la maturation sexuelle et de la ponte de l'huître creuse Crassostrea gigas : réponses métaboliques (respiration) et alimentaires (filtration, absorption) en fonction des différents stades de maturation

The study of sexual maturation and spawning in the Pacific oyster (Crassostrea gigas) is part of a vast research programme that endeavours to understand the causes of mortality that occur sporadically during the spring and summer seasons in the Marennes-Oléron Bay. Thermal and diet conditioning were used to obtain oysters at each stage of maturity simultaneously. Using the measured rates of clearance, consumption, absorption and respiration provided estimates of growth potential and gave the energetic budget of oysters at different stages of sexual maturity. Physiological responses were similar for males and females. Filtration decreased from 2.4 to 2.6 l.h (-1) to 1.8 l.h (-1) with increasing maturity. Weight gain was associated with gonad development and did not appear to have an effect on the clearance rate. Oysters 2.5 years old showed a negative energy budget (-15 J h (-1)) at later maturity stages. This deficit was confirmed (90 J.h (-1)) in oysters 1.5 years old at the same stage of maturity. On the contrary, immature oysters, in the early stages of maturity or post-spawning, had a growth potential of 110 to 170 J.h (-1). The energy deficit observed at later stages of maturity was primarily due to absorption, which decreased sharply during peak gametogenesis. Using measured respiration rates, an allometric relationship specific to gonad growth was determined with a coefficient of 0.574. Low physiological performance of oysters, observed at later stages of sexual maturity, must be taken into account in research on the factors responsible for spring and summer mortalities affecting oyster farms in Marennes-Oléron.

Genetic effect of domestication selective pressures on Pacific oyster at larval stage

Among bivalve species, the Pacific oyster, Crassostrea gigas, is the most economically important bivalve production over the world. Today, C. gigas is subject to an important production effort that leads to an intensive artificial selection. Larval stage is relatively unknown, specifically in a domestication context. Genetic consequence of artificial selection is still at a preliminary study. We aimed to tackle the consequence of inconscient domestication on the variance reproductive success focusing on larval stage, keystone of the life cycle. We studied two kinds of specific selective processes that common hatchery rearing practices exert : the effect of discarding the smallest larvae on genetic diversity and the artificial environment rearing effect via the temperature providing a contrast resembling wild versus hatchery conditions (20 and 26°C). In order to monitor the effect of the selection of fast growing larvae by sieving, growth variability and genetic diversity in a larval population descended from a factorial breeding was studied. We used a mixed-family approach to reduce potentially confounding environmental biais. The retrospective assignment of individuals to family groups has been performed using a three microsatellite markers set. Two different rearing were carried out in parallel. For three (replicates) 50-l tanks, the smallest larvae were progressively discarded by selective sieving, whereas for the three others no selective sieving was performed. The intensity of selective sieving was adjusted so as to discard 50% of the larvae over the whole rearing period in a progressive manner. As soon as the larvae reached the pediveliger stage, ready to settle larvae were sampled for genetic analysis. Regarding the artificial environment rearing effect via the temperature, we used a similar mixed-family approach. The progeny from a factorial breeding design was divided as follows: three (replicates) 50-l tanks were dedicaced to a rearing at 26°C versus 20°C for three others 50-l tanks. The whole size variability was preserved for this experiment. Individual growth measurements for larvae genetically identified have been performed at days 22 and 30 after fertilization for both conditions. In a same way, we collected individual measurements for genotyped juvenile oysters (80 days after fertilization). At a phenotypic scale, relative survival and settlement success for larvae with sieving were higher. Sieving appears as a time-saving process associated with a better relative survival ratio. But in the same time, our results confirm that a significant genetic variability exist for early developmental traits in the Pacific oyster. This is congruent with the results already obtained that investigated genetic variability and genetic correlations in early life-history traits of Crassostrea gigas. Discarding around 50% of the smallest larvae can lead to significant selection at the larval stage.

Apport d'un programme de génétique à une filière de production aquacole : l'exemple de l'ostréiculture

Two oyster species are currently present along the French coasts : the indigenous European flat oyster (Ostrea edulis), and the Pacific cupped oyster (Crassostrea gigas), that has been introduced from Japan since the beginning of the 70ies. The flat oyster successively suffered from two protozoan diseases during the 60ies and its production decreased from 20 000 tons/year by that time to 1 500 tons/year nowadays. Consequently, the oyster production is principally (99%) based upon the Pacific oyster species with approximately 150 000 tons/year among which 90% are grown from the natural spat. However, the hatchery production of this species is developing and was estimated to 400 to 800 millions spat in 2002. Moreover, strengthened relationships between IFREMER and the 5 commercial hatcheries, that all joined the SYSAAF (Union of the French poultry, shellfish and fish farming selectors), allow to plan for new genetic breeding programs. At the end of the 80ies, IFREMER initiated a genetic breeding program for the resistance of the European flat oyster to the bonamiosis, and obtained strains more tolerant to this disease. After two generations of massal selection, molecular markers had identified a reduced genetic basis in this program. It was then reoriented to an intra-familial selection. However, we were confronted to a zootechnic problem to manage such a scheme and we compromised by an intra-cohorts of families selection scheme managed using molecular markers. The program has now reached the transfer level with experimentation at a professional scale. Concerning the Pacific cupped oyster, and in parallel with the obtaining and the study of polyploids, performance of different Asian cupped oyster strains were compared to the one introduced in France thirty years ago and currently suffering from summer mortalities. The local strain exhibited better performance, certainly based upon a good local adaptation. In other respects, although early growth is a relevant criteria for selection for growth to commercial stage, it is not to be privileged in the context of an oyster producing region with a limited food availability. Contrary, the spat summer mortality became a priority for numerous teams (genetic, physiology, pathology, ecology,...) joined in the MOREST program. The first results showed important survival differences between fullsib and halsib families. They indicate a genetic determinism to this character "survival" and promote for its selection.

Relations entre apports terrigènes et conchyliculture dans les Pertuis Charentais

This work presents interactions between quantitative and qualitative river freshwater inputs and the shellfish farming (oyster and mussel) in the Pertuis Charentais. The quantity of freshwater (i.e. salinity) seems to have a weak influence on the shellfish farming contrarily to its quality determined by particulate and dissolved matters contained in the water. In autumn and winter, large precipitations have a "globally positive" effect amending the coastal ecosystem. Associated dissolved nutriments and the organic matter largely determine the quality of the coming spring growth for bred shellfish, itself controlling in turn the annual yield efficiencies. However, in winter their effects are postponed because of strong mineral load, low luminosity and temperature, then limiting the primary production. The spring contributions, directly linked to territorial practices, agriculture and tourism are more variable in quantity and quality from one year to another. They often correspond to high-risk inflows since numerous substances from anthropogenic watersheds can be found diluted in the coastal zone as in the Pertuis Charentais. Their impacts on in situ estuarine ecosystems are still poorly known since these substances are mainly studied and estimated in laboratory in controlled conditions. Several studies showed anthropogenic contaminations (i.e. cadmium, pesticides) could have significant direct or indirect effects on shellfish farming. For instance, the "summer" mortalities between 1990 and 2000 in the South of the Marennes-Oléron bay (MOB), that induced environmental and physiological oyster disorders, could be linked to pesticide effects, measured during consecutive years on the oyster bed of Ronce Perquis in the South of the MOB. The weak results from the spring larval rearing of the IFREMER experimental hatchery in the South of the bay, and chromosomal abnormalities measured on the stocks of wild oysters of the Pertuis could confirm a high-risk spring environment for the shellfish farming. In summer terrestrial inputs are reduced by low precipitations, anthropogenic water removals (drinking water, irrigation) and by plant evapotranspiration. Consequently certain years, a significant salinity increase in water masses of the Pertuis Charentais is observed. However, based on long-term observations, the significant interannual variability noticed in freshwater contributions constitutes one of the most important facts of these last years. When contributions are weak (i.e. 1991 and 2011), the mean annual salinity is 34.5 in the MOB. To the contrary, other years (i.e. 1977, 1981, 1983 and 1988), the mean salinity reduced to 30.5 shows the significant freshwater contributions to the bay. Elsewhere, particularly in the mediterranean region, oyster breeding water conditions characterized by high salinity values show the freshwater does not seem to be necessary for biological functions of the Pacific oyster Crassostrea gigas. Indeed, the oyster embryonic life in particular is well adapted to high salinity values as long as trophic resources are substantial and temperatures remain high. These two factors firstly condition the embryonic survival before the water salinity. Besides, in the Pertuis Charentais, wind conditions and the geographical bloodstock position rather determine the success of the larvae capture than seawater physic-chemical conditions. Finally, a misunderstanding still remains on summer freshwater contributions to the oyster larvae food supply.

Induction of apoptosis by UV in the flat oyster, Ostrea edulis

Apoptosis is a fundamental feature in the development of many organisms and tissue systems. It is also a mechanism of host defense against environmental stress factors or pathogens by contributing to the elimination of infected cells. Hemocytes play a key role in defense mechanisms in invertebrates and previous studies have shown that physical or chemical stress can increase apoptosis in hemocytes in mollusks. However this phenomenon has rarely been investigated in bivalves especially in the flat oyster Ostrea edulis. The apoptotic response of hemocytes from flat oysters, O. edulis, was investigated after exposure to UV and dexamethasone, two agents known to induce apoptosis in vertebrates. Flow cytometry and microscopy were combined to demonstrate that apoptosis occurs in flat oyster hemocytes. Investigated parameters like intracytoplasmic calcium activity, mitochondrial membrane potential and phosphatidyl-serine externalization were significantly modulated in cells exposed to UV whereas dexamethasone only induced an increase of DNA fragmentation. Morphological changes were also observed on UV-treated cells using fluorescence microscopy and transmission electron microscopy. Our results confirm the apoptotic effect of UV on hemocytes of O. edulis and suggest that apoptosis is an important mechanism developed by the flat oyster against stress factors.

Disruption of amylase genes by RNA interference affects reproduction in the Pacific oyster Crassostrea gigas

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 under-proliferation 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.

Exposure to toxic Alexandrium minutum activates the detoxifying and antioxidant systems in gills of the oyster Crassostrea gigas

Harmful algal blooms of Alexandrium spp. dinoflagellates regularly occur in French coastal waters contaminating shellfish. Studies have demonstrated that toxic Alexandrium spp. disrupt behavioural and physiological processes in marine filter-feeders, but molecular modifications triggered by phycotoxins are less well understood. This study analyzed the mRNA levels of 7 genes encoding antioxidant/detoxifying enzymes in gills of Pacific oysters (Crassostrea gigas) exposed to a cultured, toxic strain of A. minutum, a producer of paralytic shellfish toxins (PST) or fed Tisochrysis lutea (T. lutea, formerly Isochrysis sp., clone Tahitian (T. iso)), a non-toxic control diet, in four repeated experiments. Transcript levels of sigma-class glutathione S-transferase (GST), glutathione reductase (GR) and ferritin (Fer) were significantly higher in oysters exposed to A. minutum compared to oysters fed T. lutea. The detoxification pathway based upon glutathione (GSH)-conjugation of toxic compounds (phase II) is likely activated, and catalyzed by GST. This system appeared to be activated in gills probably for the detoxification of PST and/or extra-cellular compounds, produced by A. minutum. GST, GR and Fer can also contribute to antioxidant functions to prevent cellular damage from increased reactive oxygen species (ROS) originating either from A. minutum cells directly, from oyster hemocytes during immune response, or from other gill cells as by-products of detoxification.

Macro-geographical differences influenced by family-based expression on cultured pearl grade, shape and colour in the black-lip "pearl oyster" Pinctada margaritifera: a preliminary bi-local case study in French Polynesia

In French Polynesia, the aquaculture of P. margaritifera is carried out in numerous grow-out sites, located over three archipelagos (Gambier, Society and Tuamotu). To evaluate the impact of macro-geographical effects of these growing sites on pearl quality traits, five hatcheries produced families were used as homogeneous donor oysters in an experimental graft. The molluscs were then reared in two commercial locations: Tahaa island (Society) and Rangiroa atoll (Tuamotu). At harvest, eight pearl quality traits were recorded and compared: surface defects, lustre, grade, circles, shape categories, darkness level, body and secondary colour and visual colour categories. Overall inter-site comparison revealed that: 1) all traits were affected by grow-out location except for lustre and round shape, and 2) a higher mean rate of valuable pearls was produced in Rangiroa. Indeed, for pearl grade, Rangiroa showed twice as many A-B and less reject samples than Tahaa. This was related to the number of surface defects (grade component): in Rangiroa, twice as many pearls had no defects and less pearls had up to 10 defects. Concerning pearl shape, more circled and baroque pearls were found in Tahaa (+10%). For colour variation, 10% more pearls have an attractive green overtone in Rangiroa than in Tahaa, where more grey bodycolor were harvested. Lustre does not seem to be affected by these two culture site (except at a family scale). This is the first time P. margaritifera donor family have been shown to vary in the quality of pearls they produce depending on their grow-out location.

The estuarine geochemical reactivity of Zn isotopes and its relevance for the biomonitoring of anthropogenic Zn and Cd contaminations from metallurgical activities: Example of the Gironde fluvial-estuarine system, France

Zinc stable isotopes measurements by MC-ICP-MS, validated by laboratory intercalibrations, were performed on wild oysters, suspended particles and filtered river/estuarine water samples to provide new constraints for the use of Zn isotopes as environmental tracers. The samples selected were representative of the long range (400 km) transport of metal (Zn, Cd, etc.) contamination from former Zn-refining activities at Decazeville (i.e. δ66Zn > 1 ‰) and its phasing out, recorded during 30 years in wild oysters from the Gironde Estuary mouth (RNO/ROCCH sample bank). The study also addresses additional anthropogenic sources (urban and viticulture) and focuses on geochemical reactivity of Zn in the turbidity gradient and the maximum turbidity zone (MTZ) of the fluvial Gironde Estuary. In this area, dissolved Zn showed a strong removal onto suspended particulate matter (SPM) and progressive enrichment in heavy isotopes with increasing SPM concentrations varying from δ66Zn = -0.02 ‰ at 2 mg/L to +0.90 ‰ at 1310 mg/L. These signatures were attributed to kinetically driven adsorption due to strongly increasing sorption sites in the turbidity gradient and MTZ of the estuary. Oysters from the estuary mouth, contaminated sediments from the Lot River and SPM entering the estuary showed parallel historical evolutions (1979-2010) for Zn/Cd ratios but not for δ66Zn values. Oysters had signatures varying from δ66Zn = 1.43 ‰ in 1983 to 1.18 ‰ in 2010 and were offset by δ66Zn = 0.6 - 0.7 ‰ compared to past (1988) and present SPM from the salinity gradient. Isotopic signatures in river-borne particles entering the Gironde Estuary under contrasting freshwater discharge regimes during 2003-2011 showed similar values (δ66Zn ≈ 0.35 ± 0.03 ‰; 1SD, n=15), i.e. they were neither related to former metal refining activities at least for the past decade nor clearly affected by other anthropogenic sources. Therefore, the Zn isotopic signatures in Gironde oysters reflect the geochemical reactivity of Zn in the estuary rather than signatures of past metallurgical contaminations in the watershed as recorded in contaminated river sediments. The study also shows that the isotopic composition of Zn is strongly fractionated by its geochemical reactivity in the Gironde Estuary, representative of meso-macrotidal estuarine systems.

Impacts of Deepwater Horizon oil and associated dispersant on early development of the Eastern oyster Crassostrea virginica

The explosion of the Deepwater Horizon (DWH) oil platform resulted in large amounts of crude oil and dispersant Corexit 9500A® released into the Gulf of Mexico and coincided with the spawning season of the oyster, Crassostrea virginica. The effects of exposing gametes and embryos of C. virginica to dispersant alone (Corexit), mechanically (HEWAF) and chemically dispersed (CEWAF) DWH oil were evaluated. Fertilization success and the morphological development, growth, and survival of larvae were assessed. Gamete exposure reduced fertilization (HEWAF: EC201 h = 1650 μg tPAH50 L− 1; CEWAF: EC201 h = 19.4 μg tPAH50 L− 1; Corexit: EC201 h = 6.9 mg L− 1). CEWAF and Corexit showed a similar toxicity on early life stages at equivalent nominal concentrations. Oysters exposed from gametes to CEWAF and Corexit experienced more deleterious effects than oysters exposed from embryos. Results suggest the presence of oil and dispersant during oyster spawning season may interfere with larval development and subsequent recruitment.