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ABSTRACTThe 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. gigasautophagy 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. gigasautophagy 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.

ABSTRACTMacroautophagy 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. gigasand 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.AbbreviationsMAP1LC3: microtubule associated protein 1 light chain 3; MCA: multiple correspondence analysis; NH4Cl: ammonium chloride; PI: propidium iodide; TEM: transmission electron microscopy

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 sandy beaches of Atlantic South America. Detrimental anthropogenic impacts have further contributed to failure of populations to make a recovery. To determine the factors involved in these events, density prior to a mortality event was calculated and live yellow clams encompassingmost of its geographic range distribution were analyzed histologically to describe parasites and pathologies. Moreover, moribund specimens were analyzed by molecular techniques to test for the presence of the virus OsHV-1. A mortality event was recorded after a maximum density of 127 clams/m2 was attained. No clear pattern was found between the prevalence and intensity of infection and localities, mortality events, or sampling season. Although OsHV-1 was not observed in any of the yellow clams tested, the possibility that another viral agent was implicated cannot be ruled out. The presence of bacteria of the genus Vibrio in combination with stress caused by a relatively high population density is suggested as the likely cause of these episodic mass mortalities. [ABSTRACT FROM AUTHOR]

Diseases represent a major threat for the bivalve production industry. Their control relies on biosecurity measures to prevent their introduction and limit their spread. When maintained in hatcheries, nurseries and depuration centers, bivalves can become infected from the surrounding water and might release pathogens through wastewater effluents. A major effort was done in controlling the safety of bivalves for human consumption, but, on the other hand, information regarding the resistance of mollusc pathogens to water treatment is scarce. The effect of ultraviolet exposure was tested on two protozoan parasites of marine bivalves, the non-culturable parasite Bonamia ostreaeand Perkinsus olseniculturable in DMEM/HAM´s medium. UV exposure experiments were carried out first at the bench scale and then, for P. olseni, at a larger scale mimicking depuration plants, hatcheries and nurseries conditions. At the bench scale, our study indicated that up to 40% of B. ostreaecells and 85% of P. olsenicells died 24 h and 21 days respectively after an exposure to 94 mJ/cm2of UV-C. After 40 mJ/cm2exposure, P. olsenidensity increased between 15 and 21 days of culture suggesting that the parasite is able to recover from low UV intensity exposure. At large scale, no signs of UV recovery were seen in P. olsenicultures, but, at lower intensity (216–244 mJ/cm2), 15% of the parasites remained alive 21 days post exposure. Finally, a minimum dose of 94 mJ/cm2seems required to inhibit proliferation of parasites and 450 mJ/cm2to completely kill all parasites. Taken this into account, a dose higher than 450 mJ/cm2is suggested to properly treat water to avoid dispersion of bivalve protozoan parasites such as P. olseni.

Sporadic high mortalities were reported among larval French scallops (Pecten maximus). Electron microscopy of moribund larvae revealed particles with the characteristics of a herpesvirus in association with cellular lesions. PCR and DNA sequencing showed that the virus is a variant of ostreid herpesvirus-1 that has already been described in clams and oysters. This is the first description of a herpesvirus infection of a scallop species. The virus was transmitted successfully from an extract of infected scallop larvae to uninfected scallop or oyster (Crassostrea gigas) larvae, demonstrating that it is able to infect both species. Detection of viral DNA in asymptomatic adult scallops by in situhybridisation indicates that the herpesvirus may have been transmitted from adults to larvae. It is notable that, unlike most herpesviruses, this virus has a wide host range reflected by its ability to infect several species of marine bivalve.