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Aquaculture including shellfish production is an important food resource worldwide which is particularly vulnerable to infectious diseases. Marteilia refringens, Bonamia ostreae and Bonamia exitiosa are regulated protozoan parasites infecting flat oysters Ostrea edulis that are endemic in Europe. Although some PCR assays have been already developed for their detection, a formal validation to assess the performances of those tools is often lacking. In order to facilitate the diagnosis of flat oyster regulated diseases, we have developed and evaluated a new multiplex Taqman® PCR allowing the detection of both M. refringens and Bonamia sp. parasites in one step. First part of this work consisted in assessing analytical sensitivity and specificity of the new PCR assay. Then, diagnostic performances were assessed by testing a panel of field samples with the new real-time PCR and currently recommended conventional PCR methods for the detection of M. refringens and Bonamia sp. Samples were collected from the main flat oyster production sites in France (N = 386 for M. refringens and N = 349 for B. ostreae). In the absence of gold standard, diagnostic sensitivity and specificity of the new PCR were estimated through Bayesian latent class analysis (DSe 87,2% and DSp 98,4% for the detection M. refringens, DSe 77,5% and DSp 98,4% for the detection of Bonamia sp.). Those results suggest equivalent performances for the detection of Bonamia sp. and an improved sensitivity for the detection of M. refringens compared to commonly used conventional protocols. Finally, the new PCR was evaluated in the context of an inter-laboratory comparison study including 17 European laboratories. Results revealed a very good reproducibility with a global accordance (intra-laboratory precision) >96% and a global concordance (inter-laboratory precision) >93% for both targets, demonstrating that this new tool is easily transferable to different laboratory settings. This is the first assay designed to detect both Marteilia refringens and Bonamia sp. in a single step and it should allow reducing the number of analysis to monitor both diseases, and where relevant to demonstrate freedom from infection., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The Gulf of La Spezia (northern Tyrrhenian Sea, Italy) is a commercially important area both as a shipping port and for mussel farming. Recently, there has been increased concern over environmental disturbances caused by anthropogenic activities such as ship traffic and dredging and the effects they have on the health of farmed mussels. This paper reports the results of microbiological and histological analyses, as well as of measurement of several biomarkers which were performed to assess the health status of mussels (Mytilus galloprovincialis) from four rearing sites in the Gulf of La Spezia. Mussels were collected between October 2015 and September 2016 and histological analyses (including gonadal maturation stage), as well as the presence of pathogenic bacteria (Vibrio splendidus clade, V. aestuarianus and V. harveyi), viruses (Herpes virus and ostreid Herpes virus 1) and protozoa (Marteilia spp., in the summer season only) were carried out on a monthly basis. Conversely, biomarker responses in haemocyte/haemolymph (total haemocyte count, haemocyte diameter and volume, lysozyme and lactate dehydrogenase activities in cell-free haemolymph, and micronuclei frequency) and in gills and digestive gland (cortisol-like steroids and lipid peroxidation levels), were evaluated bimonthly. Microbiological data indicated that mussels contain a reservoir of potentially pathogenic bacteria, viruses and protozoa that in certain environmental conditions may cause a weakening of the immune system of animals leading to mortality episodes. The percentage of parasites detected in the mussels was generally low (9.6% for Steinhausia mytilovum, that is 17 samples out of 177 examined females; 3.4% for Proctoeces maculatus; 0.9% for Mytilicola intestinalis and 2% for ciliated protozoa), while symbiont loads were higher (31% for Eugymnanthea inquilina and Urastoma cyprinae). Interestingly, a previously undescribed haplosporidian was detected in a single mussel sample (0.2%) and was confirmed by in situ hybridization. Cells morphologically similar to Perkinsus sp. trophozoites were observed in 0.7% of the mussels analysed; however, infection with Perkinsus spp. could neither be confirmed by ISH nor by PCR. Different pathological aspects, such as host defence responses and regressive/progressive changes were detected in the gills, digestive glands, gonads and mantle. Only one single case of disseminated neoplasia (0.2%) was observed. As for the biomarker evaluation, the MANOVA analysis revealed the statistically significant effect that the variable "sampling site" had on the biological parameter measured, thus suggesting that the multibiomarker approach was able to differentiate the rearing sites., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Since its molecular characterisation, Ostreid herpesvirus 1 (OsHV-1) has been regularly detected in Crassostrea gigas in France. Although its pathogenicity was demonstrated on larval stages, its involvement during mortality outbreaks at the juvenile stage was highly suspected but not evidenced. To investigate mortality outbreaks, the French National Network for Surveillance and Monitoring of Mollusc Health (REPAMO) carried out two surveys in juvenile C. gigas. The first survey lasted from 1998 to 2006 and was an epidemiological inquiry occurring when oyster farmers reported mortality outbreaks. The second survey, a longitudinal one, was set up in 1998 to complete the network observations on OsHV-1. Data analysis showed a specific pattern of mortality outbreaks associated with OsHV-1 detection. Ostreid herpesvirus 1 detection mainly appeared during the summer, suggesting the influence of the seawater temperature on its occurrence. It mostly presented a patchy distribution in the field in contrast to the nursery. Significant relationship between OsHV-1 detection and spat mortality was found, preferentially in sheltered and closed environments. The longitudinal survey confirmed most of the network observations. Although subsequent works particularly epidemiological surveys would be useful to confirm the causal link between the detection of OsHV-1 and the mortality outbreaks in juvenile C. gigas, the role of OsHV-1 in oyster mortality is progressing.

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-1 of 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. [ABSTRACT FROM AUTHOR]

Crassostrea gigas oysters represent a significant global food source with 4.7 million tons harvested per year. In 2001, the bacterium Vibrio aestuarianus subsp. francensis emerged as a pathogen that causes adult oyster mortality in France and Ireland. Its impact on oyster aquaculture has increased in Europe since its re‐emergence in 2012. To better understand the evolutionary mechanisms leading to the emergence and persistence over time of this pathogen, we conducted a survey of mollusc diseases through national reference laboratories across Europe. We analysed 54 new genomes of Vibrio aestuarianus (Va) isolated from multiple environmental compartments since 2001, in areas with and without bivalve mortalities. We used a combination of comparative genomics and population genetics approaches and show that Va has a classical epidemic population structure from which the pathogenic Va francensis subspecies emerged and clonally expanded. Furthermore, we identified a specific cus‐cop‐containing island conferring copper resistance to Va francensis whose acquisition may have favoured the emergence of pathogenic lineages adapted and specialized to oysters. [ABSTRACT FROM AUTHOR]

In the fifty years since the introduction of the Pacific oyster Crassostrea gigas and the first reports of the parasites Marteilia refringens and Bonamia ostreae in European waters, numerous research projects dedicated to the native European flat oyster Ostrea edulis have been conducted, notably in France. Most of these projects have been dedicated to developing controlled reproduction and hatchery technology for seed production, examining pathological aspects to understand and control diseases, and using genetics to develop resistant lines. While the long-term objective of most studies has been to revive the aquaculture production of O. edulis, a smaller number have addressed the ecology of local remnant beds and reefs in France. This article provides an overview of the major outcomes of all these projects, focusing on results obtained in France and prospects for future work there, taking into account the rising interest in increasing aquaculture production and ecological motivation to restore declining populations as part of the framework of the Native Oyster Restoration Alliance (NORA) and in line with UN Decade for Ecosystem Restoration. [ABSTRACT FROM AUTHOR]

The conservation and active restoration of European flat oyster (Ostrea edulis) populations are a major focus of ecological restoration efforts to take advantage of the wide‐ranging ecosystem functions and services this species provides. Accordingly, additional and new demands for seed oysters have arisen. In commercial aquaculture (mariculture), the production of O. edulis is still largely based on natural seed collection. Considering the specific requirements, related to ecological restoration, such as the absence of pathogens and the preservation of high genetic diversity, the current supply is insufficient. Despite the development of breeding and controlled reproduction techniques for this species since the late 1930s, seed production today is mainly based on empirical concepts. Several of the issues that producers still face are already subjects of research; many others are still unanswered or even unaddressed. This review provides a summary of all available knowledge and technologies of O. edulis seed production. Furthermore, it provides a detailed reflection on implications for restoration, future challenges, open questions and it identifies relevant research topics for sustainable seed supply. The study covers the following aspects on (i) biology of the species, (ii) stressors – including pathogens and pollutants, (iii) genetics, (iv) history of production technologies, (v) seed production in polls, (vi) seed production in ponds and (vii) seed production in hatcheries. Future research needs on sex determinism, gametogenesis, cryopreservation, nutrition, selective breeding, pathogens and disease, and the development of reliable protocols for production are highlighted. [ABSTRACT FROM AUTHOR]

Bonamiosis is a parasitic disease (causative agent: Bonamia ostreae) affecting the European flat oyster Ostrea edulis, responsible for a drastic decline in its aquaculture production. Selective breeding programs for resistance to bonamiosis have been undertaken to counter this disease. In the present study, a 6-month cohabitation challenge experiment was performed in order to transmit the disease from wild oysters injected with the parasite to two tested families of oysters (20 and 8-month old at the beginning of the experiment, with different genetic backgrounds) originating from a selective breeding program developed by IFREMER in France. Mortalities were checked daily and ventricular heart smears were performed on dying or moribund oysters to detect the level of infection by B. ostreae. Mortality started after 4 months of cohabitation in the tested oysters. The cumulative mortalities after 6 months were 58% for the wild oysters, 9% for Family 1 (20-month old) and 20% for Family 2 (8-month old). In the dying oysters, the parasite could be detected in 67% of the wild oysters, 68% of Family 1 and 89% of Family 2. It was detected in only 11% of the surviving oysters of Family 2. The mortality and the level of infection by the parasite were significantly higher in Family 2 than in Family 1. Our results demonstrate that prespawning oysters as young as 1 year-old can become infected with the parasite and, most importantly, can die from bonamiosis. This result is inconsistent with the commonly accepted critical age of 2 years-old for the disease development. Additionally, no clear relationship between shell length and level of infection was observed. We also review the different methods for infection of the European flat oyster O. edulis with B. ostreae under experimental conditions and their main results. [ABSTRACT FROM AUTHOR]

[Display omitted] • High-throughput sequencing technologies enabled research on bivalve-microbiota interactions. • Bivalve-microbiota interactions depend on host factors nested within each other. • Microbiota interaction with surrounding environment is important for bivalve health. • Recent studies highlight the involvement of oyster microbiota in disease resistance. • Microbiome studies will provide new strategies to improve bivalve aquaculture. In bivalves, no clear-cut functional role of microbiota has yet been identified, although many publications suggest that they could be involved in nutrition or immunity of their host. In the context of climate change, integrative approaches at the crossroads of disciplines have been developed to explore the environment-host-pathogen-microbiota system. Here, we attempt to synthesize work on (1) the current methodologies to analyse bivalve microbiota, (2) the comparison of microbiota between species, between host compartments and their surrounding habitat, (3) how the bivalve microbiota are governed by environmental factors and host genetics and (4) how host-associated microorganisms act as a buffer against pathogens and/or promote recovery, and could thereby play a role in the prevention of disease or mortalities. [ABSTRACT FROM AUTHOR]