Environmental distribution and seasonal dynamics of Marteilia refringens and Bonamia ostreae, two protozoan parasites of the European flat oyster, Ostrea edulis.

Introduction: Marteilia refringens and Bonamia ostreae are protozoan parasites responsible for mortalities of farmed and wild flat oysters Ostrea edulis in Europe since 1968 and 1979, respectively. Despite almost 40 years of research, the life-cycle of these parasites is still poorly known, especially regarding their environmental distribution. Methods: We carried out an integrated field study to investigate the dynamics of M. refringens and B. ostreae in Rade of Brest, where both parasites are known to be present. We used real-time PCR to monitor seasonally over four years the presence of both parasites in flat oysters. In addition, we used previously developed eDNA based-approaches to detect parasites in planktonic and benthic compartments for the last two years of the survey. Results: M. refringens was detected in flat oysters over the whole sampling period, sometimes with a prevalence exceeding 90%. It was also detected in all the sampled environmental compartments, suggesting their involvement in parasite transmission and overwintering. In contrast, B. ostreae prevalence in flat oysters was low and the parasite was almost never detected in planktonic and benthic compartments. Finally, the analysis of environmental data allowed describing the seasonal dynamics of both parasites in Rade of Brest: M. refringens was more detected in summer and fall than in winter and spring, contrary to B. ostreae which showed higher prevalence in winter and spring. Discussion: The present study emphasizes the difference between M. refringens and B. ostreae ecology, the former presenting a wider environmental distribution than the latter, which seems closely associated to flat oysters. Our findings highlight the key role of planktonic and benthic compartments in M. refringens transmission and storage or potential overwintering, respectively. More generally, we provide here a method that could be useful not only to further investigate non cultivable pathogens life-cycle, but also to support the design of more integrated surveillance programs.

An eDNA/eRNA-based approach to investigate the life cycle of non-cultivable shellfish micro-parasites: the case of Bonamia ostreae, a parasite of the European flat oyster Ostrea edulis.

Environmental DNA approaches are increasingly used to detect microorganisms in environmental compartments, including water. They show considerable advantages to study non-cultivable microorganisms like Bonamia ostreae, a protozoan parasite inducing significant mortality in populations of flat oyster Ostrea edulis. Although B. ostreae development within the host has been well described, questions remain about its behaviour in the environment. As B. ostreae transmission is direct, seawater appears as an interesting target to develop early detection tools and improve our understanding of disease transmission mechanisms. In this context, we have developed an eDNA/eRNA approach allowing detecting and quantifying B. ostreae 18S rDNA/rRNA as well as monitoring its presence in seawater by real-time PCR. B. ostreae DNA could be detected up to 4 days while RNA could be detected up to 30 days, suggesting a higher sensitivity of the eRNA-based tool. Additionally, more than 90% of shed parasites were no longer detected after 2 days outside the oysters. By allowing B. ostreae detection in seawater, this approach would not only be useful to monitor the presence of the parasite in oyster production areas but also to evaluate the effect of changing environmental factors on parasite survival and transmission.

Latitudinal drivers of oyster mortality: deciphering host, pathogen and environmental risk factors.

Diseases pose an ongoing threat to aquaculture, fisheries and conservation of marine species, and determination of risk factors of disease is crucial for management. Our objective was to decipher the effects of host, pathogen and environmental factors on disease-induced mortality of Pacific oysters (Crassostrea gigas) across a latitudinal gradient. We deployed young and adult oysters at 13 sites in France and we monitored survival, pathogens and environmental parameters. The young oysters came from either the wild collection or the hatchery while the adults were from the wild only. We then used Cox regression models to investigate the effect of latitude, site, environmental factors and origin on mortality risk and to extrapolate this mortality risk to the distribution limits of the species in Europe. We found that seawater temperature, food level, sea level atmospheric pressure, rainfall and wind speed were associated with mortality risk. Their effect on hatchery oysters was generally higher than on wild animals, probably reflecting that hatchery oysters were free of Ostreid herpesvirus 1 (OsHV-1) whereas those from the wild were asymptomatic carriers. The risk factors involved in young and adult oyster mortalities were different, reflecting distinct diseases. Mortality risk increases from 0 to 90% with decreasing latitude for young hatchery oysters, but not for young wild oysters or adults. Mortality risk was higher in wild oysters than in hatchery ones at latitude > 47.6°N while this was the opposite at lower latitude. Therefore, latitudinal gradient alters disease-induced mortality risk but interacts with the initial health status of the host and the pathogen involved. Practically, we suggest that mortality can be mitigated by using hatchery oysters in north and wild collected oysters in the south.

In situ distribution and characterization of the organic content of the oyster shell Crassostrea gigas (Mollusca, Bivalvia).

Cultivation of commercial oysters is now facing the possible influence of global change in sea water composition, commonly referred to as "ocean acidification". In order to test the potential consequence of the predicted environmental changes, a cultivation experiment was carried out. The left and right valves of the oyster shell Crassostrea gigas differ in their structure; moreover, lenses of non compact layers are irregular. The shell layers of juvenile C. gigas are studied using a variety of highly spatially resolved techniques to establish their composition and structure. Our results confirm the presence of three different calcitic structural types. The role of the lenses of chalky layers is not yet deciplered. Despite a common mineralogy, the elemental composition of the layers differs. The sulphur aminoacids and sulphated polysaccharide contents of the intracrystalline and intercrystalline matrices differ, as well as those of the structural types. The possible different sensitivity of these structures to environmental changes is still unknown.

Influence of plankton concentration on gametogenesis and spawning of the black lip pearl oyster Pinctada margaritifera in Ahe atoll lagoon (Tuamotu Archipelago, French polynesia).

Pearl culture industry represents one of the dominant business sector of French Polynesia. However, it still entirely relies on unpredictable spat collection success. Our aim was to assess the influence of natural plankton concentration fluctuations on maturation and spawning of the black lip pearl oyster Pinctada margaritifera, during a 4 months survey conducted in Ahe atoll lagoon. Plankton concentration was assessed by chlorophyll a extraction and by microscope counts while gonadic index, gonado-visceral dry weights and histology were used to measure pearl oysters reproduction activity. We found that (i) plankton concentration fluctuations were mainly related to wind regime, (ii) gametogenesis rate was mainly related to plankton concentration, (iii) spawning occurred when maximal gonad storage was reached, (iv) plankton concentration was the main spawning synchronizing factor. These results contribute explaining P. margaritifera spat collection variability in French Polynesian atoll lagoon.

Pearl oysters Pinctada margaritifera grazing on natural plankton in Ahe atoll lagoon (Tuamotu Archipelago, French Polynesia).

In atoll lagoons of French Polynesia, growth and reproduction of pearl oysters are mainly driven by plankton concentration. However, the actual diet of black-lip pearl oysters Pinctada margaritifera in these lagoons is poorly known. To fill this gap, we used the flow through chamber method to measure clearance rates of P. margaritifera in Ahe atoll lagoon (Tuamotu Archipelago, French Polynesia). We found: (i) that pearl oysters cleared plankton at a rate that was positively related to plankton biovolume, (ii) that nanoflagellates were the main source of carbon for the pearl oysters, and (iii) that the quantity and origin of carbon filtrated by pearl oysters was highly dependent on the concentration and composition of plankton. These results provide essential elements for the comprehension of growth and reproduction variability of pearl oysters in atoll lagoons of French Polynesia.

Transcriptional regulation of pyruvate kinase and phosphoenolpyruvate carboxykinase in the adductor muscle of the oyster Crassostrea gigas during prolonged hypoxia.

The response of Crassostrea gigas to prolonged hypoxia was investigated for the first time by analyzing the metabolic branch point formed by pyruvate kinase (PK) and hosphoenolpyruvate carboxykinase (PEPCK). PK and PEPCK cDNAs were cloned and sequenced. The main functional domains of the PK sequence, such as the binding sites for ADP/ATP and phosphoenolpyruvate (PEP), were identified whereas the PEPCK sequence showed the specific domain to bind PEP in addition to the kinase-1 and kinase-2 motifs to bind guanosine triphosphate (GTP) and Mg(2+), specific for all PEPCKs. A C-terminal extension was detected for the first time in eukaryota PK. Separation of mitochondrial and cytosolic fraction showed that more than 92% of the PEPCK enzyme activity was cytosolic in gills, digestive gland, mantle and muscle. PK and PEPCK mRNAs and enzyme activities have been measured in muscle during prolonged hypoxia for 20 days. Adaptation of PK in hypoxic muscle at transcriptional level occurred lately by decreasing significantly the PK mRNA level at day 20 while PK enzyme activity was inhibited by the high content of alanine. The PEPCK mRNA ratio in hypoxic muscle significantly increased at day 10 simultaneously to the PEPCK enzyme activity. Succinate accumulation observed at day 10 and day 20 confirmed the anaerobic pathway of muscle metabolism in oyster subjected to hypoxia. Regulation of C. gigas PEPCK in muscle occurred at gene transcription level while PK was first regulated at enzyme level with alanine as allosteric inhibitor, and then at molecular level under a fast effect of hypoxia.

Noninvasive characterization of gonad maturation and determination of the sex of Pacific oysters by MRI.

The aim of this study was to test the ability of magnetic resonance imaging (MRI) technique to characterize gonad development and to determine the sex of live Pacific oysters through their shells. A preliminary nuclear magnetic resonance (NMR) relaxometry study was conducted to characterize T1 and T2 NMR relaxation parameters for the main oyster organs. This showed that T1-weighted MRI sequences were most appropriate to optimize contrasts between tissues in images. The results showed that gray levels of gonads in images acquired with gradient-echo sequence were variably affected by T2* weighting effect. However, the ovaries systematically gave a hypersignal in spin-echo T1-weighted images, and stack histograms of female oysters showed a peak well separated from that of male oysters. An automated method is proposed to quantify the development of oysters and their gonad maturation and to identify their sex.

Ovarian and sperm regulatory peptides regulate ovulation in the oyster Crassostrea gigas.

For more than six decades, several studies have shown that genital products to entering the mantle cavity via the incurrent siphon, initiate in oyster, strong and rhythmic contractions of the adductor muscle (AM). In order to characterize the regulatory peptides capable of triggering AM contractions, we focused on the identification of putative myotropic peptides from genital products. Two experimental approaches were developed. The first one, based on a mass spectrometry screening of the male genital products, led to the identification of the tetrapeptide APGWamide. This neuropeptide was also detected in the seminal secretions of the cephalopod Sepia officinalis. In this species, APGWamide is directly involved in the oocyte transport. In Crassostrea, in vitro bioassay demonstrated that APGWamide modulates the AM contractions that insure the release of oocytes in the external medium. Exposure of oysters to a physiological concentration of APGWamide triggered repetitive shell closures. The second experimental approach was based on the monitoring of HPLC purification by a myotropic bioassay using the cuttlefish oviduct contractions as a target. The successive purification steps of the acidic extraction of ovaries from mature female oysters, led to the characterization of the hexapeptide PIESVD. When applied to mature female oysters, this peptide triggered the increase of shell closure frequency. The activity of these two regulatory peptides is the first experimental evidence of a peptidergic control of egg-laying in oyster. APGWamide and PIESVD could be used, in commercial and experimental hatcheries, for the identification of mature females to be selected for in vitro fertilization.

The oyster vasa-like gene: a specific marker of the germline in Crassostrea gigas.

The vasa gene is a key determinant for germline formation in eukaryotes. This gene, highly conserved through evolution, encodes a RNA helicase protein member of the DEAD-box family. To understand the germline formation in oyster, we report here the isolation and the characterization of a vasa orthologue in Crassostrea gigas (Oyvlg). OyVLG contained the eight consensus domains of the DEAD-box including those providing RNA unwinding activity. The expression pattern of Oyvlg was examined in adult oyster tissues at different reproductive stages. Its expression was restricted to germline cells both in males and females, including germinal stem cells and auxiliary cells. The expression of Oyvlg, strongest in early germ cells, decreased as the maturation proceeded. These data and the evolutionary conservation observed suggested the role of Oyvlg in germline development. Oyvlg is the first germ cell specific marker in oyster and will be very useful in studies of oyster germline formation.