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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.

The haplosporidian parasites Bonamia ostreae (BO) and B. exitiosa (BE) are serious oyster pathogens. Two independent laboratories evaluated fluorescence real-time loop-mediated isothermal amplification (LAMP) assays for rapidly detecting these parasites. Specific LAMP assays were designed on the BO actin-1 and BE actin genes. A further generic assay was conceived on a conserved region of the 18S gene to detect both Bonamia species. The optimal reaction temperature varied from 65 to 67 °C depending on the test and instrument. Melting temperatures were 89.8–90.2 °C, 87.0–87.6 °C, and 86.2–86.6 °C for each of the BO, BE, and generic assays. The analytical sensitivity of these assays was 50 copies/µL in a 30 min run. The BO and BE test sensitivity was ~1 log lower than a real-time PCR, while the generic test sensitivity was similar to the real-time PCR. Both the BO and BE assays were shown to be specific; however, the generic assay potentially cross-reacts with Haplosporidium costale. The performance of the LAMP assays evaluated on samples of known status detected positives within 7–20 min with a test accuracy of 100% for the BO and generic tests and a 95.8% accuracy for BE. The ease of use, rapidity and affordability of these tests allow for field deployment. [ABSTRACT FROM AUTHOR]

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]

Vector or reservoir species of five mollusc diseases listed in the Animal Health Law were identified, based on evidence generated through an extensive literature review, to support a possible updating of Regulation (EU) 2018/1882. Mollusc species on or in which Mikrocytos mackini, Perkinsus marinus, Bonamia exitiosa, Bonamia ostreae and Marteilia refringens were detected, in the field or during experiments, were classified as reservoir species with different levels of certainty depending on the diagnostic tests used. Where experimental evidence indicated transmission of the pathogen from a studied species to another known susceptible species, this studied species was classified as a vector species. Although the quantification of the risk of spread of the pathogens by the vectors or reservoir species was not part of the terms of reference, such risks do exist for the vector species, since transmission from infected vector species to susceptible species was proven. Where evidence for transmission from infected molluscs was not found, these were defined as reservoir. Nonetheless, the risk of the spread of the pathogens from infected reservoir species cannot be excluded. Evidence identifying conditions that may prevent transmission by vectors or reservoir mollusc species during transport was collected from scientific literature. It was concluded that it is very likely to almost certain (90–100%) that M. mackini, P. marinus, B. exitiosaB. ostreae and M. refringens will remain infective at any possible transport condition. Therefore, vector or reservoir species that may have been exposed to these pathogens in an affected area in the wild or at aquaculture establishments or through contaminated water supply can possibly transmit these pathogens. For transmission of M. refringens, the presence of an intermediate host, a copepod, is necessary. This publication is linked to the following EFSA Journal articles: http://onlinelibrary.wiley.com/doi/10.2903/j.efsa.2023.8172/full, http://onlinelibrary.wiley.com/doi/10.2903/j.efsa.2023.8174/full This publication is linked to the following EFSA Supporting Publications articles: http://onlinelibrary.wiley.com/doi/10.2903/sp.efsa.2023.EN-8122/full, http://onlinelibrary.wiley.com/doi/10.2903/sp.efsa.2023.EN-8123/full, http://onlinelibrary.wiley.com/doi/10.2903/sp.efsa.2023.EN-8124/full [ABSTRACT FROM AUTHOR]

Vector or reservoir species of five fish diseases listed in the Animal Health Law were identified, based on evidence generated through an extensive literature review (ELR), to support a possible updating of Regulation (EU) 2018/1882. Fish species on or in which highly polymorphic region‐deleted infectious salmon anaemia virus (HPR∆ ISAV), Koi herpes virus (KHV), epizootic haematopoietic necrosis virus (EHNV), infectious haematopoietic necrosis virus (IHNV) or viral haemorrhagic septicaemia virus (VHSV) were detected, in the field or during experiments, were classified as reservoir species with different levels of certainty depending on the diagnostic tests used. Where experimental evidence indicated transmission of the pathogen from a studied species to another known susceptible species, the studied species was classified as a vector species. Although the quantification of the risk of spread of the pathogens by the vectors or reservoir species was not part of the terms or reference, such risks do exist for the vector species, since transmission from infected vector species to susceptible species was proven. Where evidence for transmission from infected fish was not found, these were defined as reservoirs. Nonetheless, the risk of the spread of the pathogens from infected reservoir species cannot be excluded. Evidence identifying conditions that may prevent transmission by vectors or reservoir fish species during transport was collected from scientific literature. For VHSV, IHNV or HPR∆ ISAV, it was concluded that under transport conditions at temperatures below 25°C, it is likely (66–90%) they will remain infective. Therefore, vector or reservoir species that may have been exposed to these pathogens in an affected area in the wild, aquaculture establishments or through water supply can possibly transmit VHSV, IHNV or HPR∆ ISAV into a non‐affected area when transported at a temperature below 25°C. The conclusion was the same for EHN and KHV; however, they are likely to remain infective under all transport temperatures. This publication is linked to the following EFSA Journal articles: http://onlinelibrary.wiley.com/doi/10.2903/j.efsa.2023.8172/full, http://onlinelibrary.wiley.com/doi/10.2903/j.efsa.2023.8173/full This publication is linked to the following EFSA Supporting Publications articles: http://onlinelibrary.wiley.com/doi/10.2903/sp.efsa.2023.EN-8122/full, http://onlinelibrary.wiley.com/doi/10.2903/sp.efsa.2023.EN-8123/full, http://onlinelibrary.wiley.com/doi/10.2903/sp.efsa.2023.EN-8124/full [ABSTRACT FROM AUTHOR]

Vector or reservoir species of three diseases of crustaceans listed in the Animal Health Law were identified based on evidence generated through an extensive literature review, to support a possible updating of Regulation (EU) 2018/1882. Crustacean species on or in which Taura syndrome virus (TSV), Yellow head virus (YHV) or White spot syndrome virus (WSSV) were identified, in the field or during experiments, were classified as reservoir species with different levels of certainty depending on the diagnostic tests used. Where experimental evidence indicated transmission of the pathogen from a studied species to another known susceptible species, the studied species was classified as vector species. Although the quantification of the risk of spread of the pathogens by the vectors or reservoir species was not part of the terms of reference, such risks do exist for the vector species, since transmission from infected vector species to susceptible species was proven. Where evidence for transmission from infected crustaceans was not found, these were defined as reservoirs. Nonetheless, the risk of the spread of the pathogens from infected reservoir species cannot be excluded. Evidence identifying conditions that may prevent transmission by vectors during transport was collected from scientific literature. It was concluded that it is very likely to almost certain (90–100%) that WSSV, TSV and YHV will remain infective at any possible transport condition. Therefore, vector or reservoir species that may have been exposed to these pathogens in an affected area in the wild or aquaculture establishments or by water supply can possibly transmit WSSV, TSV and YHV. This publication is linked to the following EFSA Journal articles: http://onlinelibrary.wiley.com/doi/10.2903/j.efsa.2023.8173/full, http://onlinelibrary.wiley.com/doi/10.2903/j.efsa.2023.8174/full This publication is linked to the following EFSA Supporting Publications articles: http://onlinelibrary.wiley.com/doi/10.2903/sp.efsa.2023.EN-8122/full, http://onlinelibrary.wiley.com/doi/10.2903/sp.efsa.2023.EN-8123/full, http://onlinelibrary.wiley.com/doi/10.2903/sp.efsa.2023.EN-8124/full [ABSTRACT FROM AUTHOR]

On request of the EU Commission, EFSA carried out an Extensive Literature Review (ELR) to provide a list of vector species or reservoirs species of pathogens of crustaceans, listed in Annex II to the AHL, aiming to update the Annex of Implementing Regulation (EU) 2018/1882. In this Technical Report, the detailed review protocol of the ELR and assessment of potential vector and reservoir species is described of the crustacean pathogens listed in Annex II to the AHL: Taura syndrome virus (TSV), Yellow head virus (YHV) or White spot syndrome virus (WSSV). In total 2,530 research publications were collected for abstract screening and from these, 110 were selected for further full text analysis. In the final data collection and assessment 34 relevant research publications were used for extracting information on vector and reservoir species of the above crustacean pathogens. The results for crustacean species for which scientific evidence indicates that a role as vector species or reservoir species is likely are presented as tables in the supplementary material of this report. This publication is linked to the following EFSA Journal articles: http://onlinelibrary.wiley.com/doi/10.2903/j.efsa.2023.8172/full, http://onlinelibrary.wiley.com/doi/10.2903/j.efsa.2023.8173/full, http://onlinelibrary.wiley.com/doi/10.2903/j.efsa.2023.8174/full This publication is linked to the following EFSA Supporting Publications articles: http://onlinelibrary.wiley.com/doi/10.2903/sp.efsa.2023.EN-8122/full, http://onlinelibrary.wiley.com/doi/10.2903/sp.efsa.2023.EN-8124/full [ABSTRACT FROM AUTHOR]

On request of the EU Commission, EFSA carried out an Extensive Literature Review (ELR) to provide a list of vector species or reservoirs species of pathogens of crustaceans, listed in Annex II to the AHL, aiming to update the Annex of Implementing Regulation (EU) 2018/1882. In this Technical Report, the detailed review protocol of the ELR and assessment of potential vector and reservoir species is described of the crustacean pathogens listed in Annex II to the AHL: Taura syndrome virus (TSV), Yellow head virus (YHV) or White spot syndrome virus (WSSV). In total 2,530 research publications were collected for abstract screening and from these, 110 were selected for further full text analysis. In the final data collection and assessment 34 relevant research publications were used for extracting information on vector and reservoir species of the above crustacean pathogens. The results for crustacean species for which scientific evidence indicates that a role as vector species or reservoir species is likely are presented as tables in the supplementary material of this report. This publication is linked to the following EFSA Journal articles: http://onlinelibrary.wiley.com/doi/10.2903/j.efsa.2023.8172/full, http://onlinelibrary.wiley.com/doi/10.2903/j.efsa.2023.8173/full, http://onlinelibrary.wiley.com/doi/10.2903/j.efsa.2023.8174/full This publication is linked to the following EFSA Supporting Publications articles: http://onlinelibrary.wiley.com/doi/10.2903/sp.efsa.2023.EN-8123/full, http://onlinelibrary.wiley.com/doi/10.2903/sp.efsa.2023.EN-8124/full [ABSTRACT FROM AUTHOR]

ABSTRACT The yellow clam Amarilladesma mactroides (Deshayes, 1854) is considered a vulnerable species since the mid-1990s. Populations have experienced mass mortalities throughout their entire range of distribution (23-41°S) along exposed [...]

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 lifecycle 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. [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]

The Pacific cupped oyster Crassostreagigas is one of the most 'globalized' marine invertebrates and its production is predominant in many parts of the world including Europe. However, it is threatened by mortality events associated with pathogenic microorganisms such as the virus OsHV‐1 and the bacteria Vibrio aestuarianus. C. gigas is also a host for protozoan parasites including haplosporidians. In contrast with Haplosporidium nelsoni previously detected in Europe, H. costale was considered exotic although its presence in French oysters was suggested in the 1980s based on ultrastructural examination. Here, a combination of light and transmission electron microscopy, PCR and sequencing allowed characterizing the presence of the parasite in the context of low mortality events which occurred in 2019 in France. Histological observation revealed the presence of uninucleated, plasmodial and spore stages within the connective tissues of some oysters. Ultrastructural features were similar to H. costale ones in particular the presence of axe‐shaped haplosporosomes in spore cytoplasms. Three fragments of the genome including partial small subunit rRNA gene, the ITS‐1, 5.8S and ITS‐2 array and part of the actin gene were successfully sequenced and grouped with H. costale homologous sequences. This is the first time that the presence of H. costale was confirmed in C. gigas in France. Furthermore, a TaqMan real‐time PCR assay was developed and validated [DSe = 92.6% (78.2–99.8) and DSp = 95.5% (92.3–98.6)] to enable the rapid and specific detection of the parasite. The application of the PCR assay on archived samples revealed that the parasite has been present in French oyster populations at least since 2008. Considering the little information available on this parasite, the newly developed TaqMan assay will be very helpful to investigate the temporal and geographic distribution and the life cycle of the parasite in France and more generally in C. gigas geographic range. [ABSTRACT FROM AUTHOR]

The flat oyster Ostrea edulis is an oyster species native to Europe. It has declined to functional extinction in many areas of the NE Atlantic for several decades. Factors explaining this decline include over-exploitation of natural populations and diseases like bonamiosis, regulated across both the EU and the wider world and caused by the intracellular protozoan parasite Bonamia ostreae. To date, very limited sequence data are available for this Haplosporidian species. We present here the first transcriptome of B. ostreae. As this protozoan is not yet culturable, it remains extremely challenging to obtain high-quality -omic data. Thanks to a specific parasite isolation protocol and a dedicated bioinformatic pipeline, we were able to obtain a high-quality transcriptome for an intracellular marine micro-eukaryote, which will be very helpful to better understand its biology and to consider the development of new relevant diagnostic tools. [ABSTRACT FROM AUTHOR]

Mortality outbreaks of young Pacific oysters, Crassostrea gigas , have seriously affected the oyster-farming economy in several countries around the world. Although the causes of these mortality outbreaks appear complex, a viral agent has been identified as the main factor: a herpesvirus called ostreid herpesvirus 1 (OsHV-1). Autophagy is an important degradation pathway involved in the response to several pathologies including viral diseases. In C. gigas , recent studies indicate that this pathway is conserved and functional in at least haemocytes and the mantle. Furthermore, an experimental infection in combination with compounds known to inhibit or induce autophagy in mammals revealed that autophagy is involved in the response to OsHV-1 infection. In light of these results, the aim of this study was to determine the role of autophagy in the response of the Pacific oyster to infection by virus OsHV-1. For this purpose, an experimental infection in combination with a modulator of autophagy was performed on Pacific oysters known to have intermediate susceptibility to OsHV-1 infection. In haemolymph and the mantle, the autophagy response was monitored by flow cytometry, western blotting, and real-time PCR. At the same time, viral infection was evaluated by quantifying viral DNA and RNA amounts by real-time PCR. Although the results showed activation of autophagy in haemolymph and the mantle 14 hours post infection (after viral replication was initiated), they were also indicative of different regulatory mechanisms of autophagy in the two tissues, thus supporting an important function of autophagy in the response to virus OsHV-1. [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]

Abstract 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.

Summary: 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. [ABSTRACT FROM AUTHOR]

The Pacific oyster, Crassostrea gigas, is a mollusk bivalve commercially important as a food source. Pacific oysters are subjected to stress and diseases during culture. The autophagy pathway is involved in numerous cellular processes, including responses to starvation, cell death, and microorganism elimination. Autophagy also exists in C. gigas, and plays a role in the immune response against infections. Although this process is well-documented and conserved in most animals, it is still poorly understood in mollusks. To date, no study has provided a complete overview of the molecular mechanism of autophagy in mollusk bivalves. In this study, human and yeast ATG protein sequences and public databases (Uniprot and NCBI) were used to identify protein members of the C. gigas autophagy pathway. A total of 35 autophagy related proteins were found in the Pacific oyster. RACE-PCR was performed on several genes. Using molecular (real-time PCR) and protein-based (western blot and immunohistochemistry) approaches, the expression and localization of ATG12, ATG9, BECN1, MAP1LC3, MTOR, and SQSTM1, was investigated in different tissues of the Pacific oyster. Comparison with human and yeast counterparts demonstrated a high homology with the human autophagy pathway. The results also demonstrated that the key autophagy genes and their protein products were expressed in all the analyzed tissues of C. gigas. This study allows the characterization of the complete C. gigas autophagy pathway for the first time. Abbreviations: ATG: autophagy related; Atg1/ULK: unc-51 like autophagy activating kinase; ATG7: autophagy related 7; ATG9: autophagy related 9; ATG12: autophagy related 12; BECN1: beclin 1; BSA: bovine serum albumin; cDNA: complementary deoxyribonucleic acid; DNA: deoxyribonucleic acid; GABARAP: GABA type A receptor-associated protein; IHC: immunohistochemistry; MAP1LC3/LC3/Atg8: microtubule associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; NCBI: national center for biotechnology information; ORF: open reading frame; PBS: phosphate-buffered saline; PCR: polymerase chain reaction; PtdIns3K: class III phosphatidylinositol 3-kinase; RACE-PCR: rapid amplification of cDNA-ends by polymerase chain reaction; RNA: ribonucleic acid; SQSTM1: sequestosome 1; Uniprot: universal protein resource; WIPI: WD repeat domain, phosphoinositide interacting. [ABSTRACT FROM AUTHOR]

There is a growing effort throughout Europe to restore populations of native oysters (Ostrea edulis), with the ecological objective of enhancing ecosystem biodiversity and resilience.The introduced parasite, Bonamia ostreae, caused catastrophic mortalities during the 1980s, furthering the decline of this species, and is now present throughout much of the natural range of O. edulis. It is therefore important that restoration attempts avoid further introduction and spread of this parasite, which can cause lethal infections of O. edulis.This article presents a comprehensive overview of the scale and distribution of current infection, transmission pathways, and preventive measure guidelines, focusing on the seas, inlets, and estuaries of north‐west Europe, where most ecological restoration attempts for the native European oyster have occurred so far.This is critical information for restoration project planning in which the risk of Bonamia infection must be taken into account. [ABSTRACT FROM AUTHOR]

Intracellular microcolonies of bacteria (IMC), in some cases developing large extracellular cysts (bacterial aggregates), infecting primarily gill and digestive gland, have been historically reported in a wide diversity of economically important mollusk species worldwide, sometimes associated with severe lesions and mass mortality events. As an effort to characterize those organisms, traditionally named as Rickettsia or Chlamydia -like organisms, 1950 specimens comprising 22 mollusk species were collected over 10 countries and after histology examination, a selection of 99 samples involving 20 species were subjected to 16S rRNA gene amplicon sequencing. Phylogenetic analysis showed Endozoicomonadaceae sequences in all the mollusk species analyzed. Geographical differences in the distribution of Operational Taxonomic Units (OTUs) and a particular OTU associated with pathology in king scallop (OTU_2) were observed. The presence of Endozoicomonadaceae sequences in the IMC was visually confirmed by in situ hybridization (ISH) in eight selected samples. Sequencing data also indicated other symbiotic bacteria. Subsequent phylogenetic analysis of those OTUs revealed a novel microbial diversity associated with molluskan IMC infection distributed among different taxa, including the phylum Spirochetes, the families Anaplasmataceae and Simkaniaceae , the genera Mycoplasma and Francisella , and sulfur-oxidizing endosymbionts. Sequences like Francisella halioticida/philomiragia and Candidatus Brownia rhizoecola were also obtained, however, in the absence of ISH studies, the association between those organisms and the IMCs were not confirmed. The sequences identified in this study will allow for further molecular characterization of the microbial community associated with IMC infection in marine mollusks and their correlation with severity of the lesions to clarify their role as endosymbionts, commensals or true pathogens. [ABSTRACT FROM AUTHOR]

Macroautophagy is a mechanism that is involved in various cellular processes, including cellular homeostasis and innate immunity. This pathway has been described in organisms ranging in complexity from yeasts to mammals, and recent results indicate that it occurs in the mantle of the Pacific oyster, Crassostrea gigas. However, the autophagy pathway has never been explored in the hemocytes of C. gigas, which are the main effectors of its immune system and thus play a key role in the defence of the Pacific oyster against pathogens. To investigate autophagy in oyster hemocytes, tools currently used to monitor this mechanism in mammals, including flow cytometry, fluorescent microscopy and transmission electron microscopy, were adapted and applied to the hemocytes of the Pacific oyster. Oysters were exposed for 24 and 48 h to either an autophagy inducer (carbamazepine, which increases the production of autophagosomes) or an autophagy inhibitor (ammonium chloride, which prevents the degradation of autophagosomes). Autophagy was monitored in fresh hemocytes withdrawn from the adductor muscles of oysters using a combination of the three aforementioned methods. We successfully labelled autophagosomes and observed them by flow cytometry and fluorescence microscopy, and then used electron microscopy to observe ultrastructural modifications related to autophagy, including the presence of double-membrane-bound vacuoles. Our results demonstrated that autophagy occurs in hemocytes of C. gigas and can be modulated by molecules known to modulate autophagy in other organisms. This study describes an integrated approach that can be applied to investigate autophagy in marine bivalves at the cellular level. Abbreviations: MAP1LC3: microtubule associated protein 1 light chain 3; MCA: multiple correspondence analysis; NH4Cl: ammonium chloride; PI: propidium iodide; TEM: transmission electron microscopy [ABSTRACT FROM AUTHOR]

An understanding of husbandry strategies and any associated risk factors is important for designing management control measures that can reduce mortality in Pacific oysters, Crassostrea gigas, caused by ostreid herpesvirus 1 (OsHV‐1). The type of culture facility can be considered in relation to the potential pathways that could lead to the entry of a pathogen and its survival. In addition, the animal host (e.g. age, physiological state, selective breeding programmes), husbandry procedures (e.g. stocking density), the pathogen itself (e.g. pathogenicity, virulence) and environmental effects (e.g. temperature) represent other relevant interconnected factors. However, all these factors provide valuable background information for outlining the mitigation strategies needed by the industry, as well as in the context of surveillance and biosecurity programmes. These control mechanisms for hatchery or nursery areas are related to movement restrictions, water treatment, virus inactivation, the production calendar and practical farm management decisions. This comprehensive literature review compiles information related to such approaches and also includes the different existing guidelines suggested for control of OsHV‐1. Therefore, the review represents a solid foundation for a more critical appraisal currently being developed to support recommendations for disease management strategies. [ABSTRACT FROM AUTHOR]

This is the first study of the metabolic enzyme activity and energy state of the dwarf oyster Ostrea stentina (Payraudeau, 1826) in Tunisia and in the Mediterranean region. The main purpose of this study was to examine the modulation of the physiological status of the oyster O. stentina depending on the season and the presence of parasite Marteilia refringens. The prevalence of bonamiosis and marteiliosis were established by PCR. Bonamia exitiosa was detected only in 2.91% of oysters while the prevalence of M. refringens was 100% (30 Ind./30) in February and 93.93% (28 Ind./30) in March. A 42–87% mortality rate was reported during the study period. Biochemical analyses were carried out to evaluate the management of the energy resources in regard to the biochemical changes of lipids and carbohydrates. The enzyme activity of pyruvate kinase (PK), citrate synthase (CS) and cytochrome c oxidase (CCO) were measured and the mean level of enzyme activity was respectively 20.31 mU mg−1 protein; 12.06 mU mg−1 protein and 3.59 mU mg−1 protein. Carbohydrate contents of O. stentina were very low all year round with an average of 15.18% in dry weight, and lipid contents remain similar (11.77% in dry weight) compared with the values reported for most other temperate bivalves. Enzyme activity significantly decreased over time (P < 0.001). The energy resources of the oyster O. stentina wherein much of the energy was devoted to reproduction seem to affect the defence system. [ABSTRACT FROM PUBLISHER]

Bonamiosis due to the parasite Bonamia ostreae has been associated with massive mortality outbreaks in European flat oyster stocks in Europe. As eradication and treatment are not possible, the control of the disease mainly relies on transfer restriction. Moreover, selection has been applied to produce resistant flat oyster families, which present better survival and lower prevalence than non-selected oysters. In order to better understand the mechanisms involved in resistance to bonamiosis, cellular and molecular responses of 2 oyster groups (selected oysters and wild-type oysters) were analyzed in the context of experimental injection and cohabitation infections. Cellular responses including non-specific esterases detection, ROS production and phagocytosis activity were analyzed by flow cytometry. Four genes homologous to those shown to be involved in immunity were selected (Inhibitor of apotosis OeIAP, Fas ligand OeFas-ligand, Oe-SOD, and OeEc-SOD) and monitored by quantitative reverse-transcription PCR (qRT-PCR). Infected oysters showed higher phagocytosis activity than controls. Infected selected oyster show a lower phagocytosis activity which might be a protection against the parasite infection. The expression of OeIAP and OeFas-ligand gene was significantly increased in selected oysters at 5 days post-injection. OeIAP gene expression appeared to be significantly increased in wild-type oysters at 8 days post-injection. Our results suggest that resistance to bonamiosis partly relies on the ability of the oysters tomodulate apoptosis. [ABSTRACT FROM AUTHOR]

Although a wide range of viruses have been reported in marine molluscs, most of these reports rely on ultrastructural examination and few of these viruses have been fully characterized. The lack of marine mollusc cell lines restricts virus isolation capacities and subsequent characterization works. Our current knowledge is mostly restricted to viruses affecting farmed species such as oysters Crassostrea gigas , abalone Haliotis diversicolor supertexta or the scallop Chlamys farreri . Molecular approaches which are needed to identify virus affiliation have been carried out for a small number of viruses, most of them belonging to the Herpesviridae and birnaviridae families. These last years, the use of New Generation Sequencing approach has allowed increasing the number of sequenced viral genomes and has improved our capacity to investigate the diversity of viruses infecting marine molluscs. This new information has in turn allowed designing more efficient diagnostic tools. Moreover, the development of experimental infection protocols has answered some questions regarding the pathogenesis of these viruses and their interactions with their hosts. Control and management of viral diseases in molluscs mostly involve active surveillance, implementation of effective bio security measures and development of breeding programs. However factors triggering pathogen development and the life cycle and status of the viruses outside their mollusc hosts still need further investigations. [ABSTRACT FROM AUTHOR]

The SAR group (Stramenopila, Alveolata, Rhizaria) is one of the largest clades in the tree of eukaryotes and includes a great number of parasitic lineages. Rhizarian parasites are obligate and have devastating effects on commercially important plants and animals but despite this fact, our knowledge of their biology and evolution is limited. Here, we present rhizarian transcriptomes from all major parasitic lineages in order to elucidate their evolutionary relationships using a phylogenomic approach. Our results suggest that Ascetosporea, parasites of marine invertebrates, are sister to the novel clade Apofilosa. The phytomyxean plant parasites branch sister to the vampyrellid algal ectoparasites in the novel clade Phytorhiza. They also show that Ascetosporea + Apofilosa + Retaria + Filosa + Phytorhiza form a monophyletic clade, although the branching pattern within this clade is difficult to resolve and appears to be model-dependent. Our study does not support the monophyly of the rhizarian parasitic lineages (Endomyxa), suggesting independent origins for rhizarian animal and plant parasites. [ABSTRACT FROM AUTHOR]

Marine mollusc production contributes to food and economic security worldwide and provides valuable ecological services, yet diseases threaten these industries and wild populations. Although the infrastructure for mollusc aquaculture health management is well characterized, its foundations are not without flaws. Use of notifiable pathogen lists can leave blind spots with regard to detection of unlisted and emerging pathogens. Increased reliance on molecular tools has come without similar attention to diagnostic validation, raising questions about assay performance, and has been accompanied by a reduced emphasis on microscopic diagnostic expertise that could weaken pathogen detection capabilities. Persistent questions concerning pathogen biology and ecology promote regulatory paralysis that impedes trade and which could weaken biosecurity by driving commerce to surreptitious channels. Solutions that might be pursued to improve shellfish aquaculture health management include the establishment of more broad-based surveillance programmes, wider training and use of general methods like histopathology to ensure alertness to emerging diseases, an increased focus on assay assessment and validation as fundamental to assay development, investment in basic research, and application of risk analyses to improve regulation. A continual sharpening of diagnostic tools and approaches and deepening of scientific knowledge is necessary to manage diseases and promote sustainable molluscan shellfish industries. [ABSTRACT FROM AUTHOR]

The protist phylum Haplosporidia comprises over 40 described species with representatives infecting a range of mollusc hosts, including several ecologically and economically significant pathogens. Continuing exploration of haplosporidian diversity has added ten new species in recent years and brought the phylogenetics of the group into somewhat clearer focus, with monophyletic Bonamia and Minchinia lineages continuing to be supported. However, the addition of new sequences to phylogenetic analyses has left the paraphyletic genus Haplosporidium ’s picture less resolved. It is not clear that even two genera will be enough to accommodate the species presently drawn to the Haplosporidium regions of the haplosporidian tree. In this review, we summarize recent findings in haplosporidian diversity and phylogenetics, and provide a synthesis of our understanding of the life cycles and environmental influences on haplosporidians, with particular emphasis on the important pathogens Haplosporidium nelsoni and Bonamia ostreae . Additionally, we consider the evolution of the “microcell haplosporidian” lifestyle of Bonamia parasites, and suggest that colonization of high-density oyster host populations in relatively stable euhaline marine environments may have been an important development favoring the evolution of the microcell haplosporidian life strategy. [ABSTRACT FROM AUTHOR]

In this study, we described the cytosolic HSP90 of Bonamia ostreae, an intracellular parasite of Ostrea edulis hemocytes. The complete open reading frame was assembled by Rapid Amplification cDNA Ends reactions on cDNA of B. ostreae-infected hemocytes. HSP90 amplification was corroborated in infected oysters and B. ostreae purified cells. The functionality of the HSP90, studied by inhibitory assays with radicicol, suggests that this protein may play a role in hemocyte invasion. Our results inform the molecular basis that governs B. ostreae- O. edulis interactions. [ABSTRACT FROM AUTHOR]

The health status of cultured and wild Mediterranean mussels in the Slovene Sea has so far been unexplored. Initially, 1280 adult Mediterranean mussels (Mytilus galloprovincialis), 960 from a shellfish farm and 320 from natural beds, were collected over a one-year period. Water temperature, oxygenation and salinity were measured at each sampling. Mussels were measured and weighted to calculate the condition index and microscopically examined for the presence of haemocytic neoplasia. Haemocytic neoplasia was detected in 14 mussels (1.1%) with a higher prevalence in cultured mussels. Neoplastic cells singularly infiltrated the connective tissue, in small foci or diffusely. Necrosis and multifocal atrophy of digestive tubules were noticed in mussels with diffuse neoplasia, whereas severe haemocytic infiltration of connective tissue was seen in mussels with single neoplastic cells. Haemocytic neoplasia was more frequently observed in spring and autumn. The average condition index of mussels with haemocytic neoplasia was slightly higher than in healthy ones. This is the first report of haemocytic neoplasia in Mediterranean mussels in the Northern Adriatic Sea. The disease occurs only sporadically and to date no significant impact on the mussel population has been noted. [ABSTRACT FROM AUTHOR]

The article presents information on a study which investigated the effect of temperature and salinity of different seawater media on the survival of Bonamia ostreae, a parasite that infects flat oysters Ostrea edulis. A brief overview is given on the protistan parasite the belongs to the phylum Haplosporidia. It discusses the incubation of purified parasites in various seawater media at various temperatures and then subjected to a range of salinities. It explores the purification of Bonamia ostreae. It also relates of the findings of the analysis of viability of the parasite using flow cytometry protocols.