Inactivation and elimination of human enteric viruses by Pacific oysters

Aims: To investigate the comparative elimination of three different human enterically transmitted viruses [i.e. hepatitis A virus (HAV), norovirus (NoV) and poliovirus (PV)] and inactivation of HAV and PV by Pacific oysters. Methods and Results: New Zealand grown Pacific oysters (Crassostrea gigas) were allowed to bioaccumulate HAV, NoV and PV. Samples of oyster gut, faeces and pseudofaeces were then analysed by using real‐time RT‐PCR to determine the amount of viral RNA and cell culture methods to identify changes in the number of plaque forming units. The results suggest that the majority of the PV present in the oyster gut and oyster faeces is noninfectious, while in contrast, most of the HAV detected in the oyster gut are infectious. Depuration experiments identified a large drop in the count of PV in the gut over a 23‐h cleansing period, whereas the levels of HAV and NoV did not significantly decrease. Conclusions: Human enterically transmitted viruses are eliminated and inactivated at different rates by Pacific oysters. Significance and Impact of Study: The research presented in this article has implications for risk management techniques that are used to improve the removal of infectious human enteric viruses from bivalve molluscs.     

Vibrio–bivalve interactions in health and disease

In the marine environment, bivalve mollusks constitute habitats for bacteria of the Vibrionaceae family. Vibrios belong to the microbiota of healthy oysters and mussels, which have the ability to concentrate bacteria in their tissues and body fluids, including the hemolymph. Remarkably, these important aquaculture species respond differently to infectious diseases. While oysters are the subject of recurrent mass mortalities at different life stages, mussels appear rather resistant to infections. Thus, Vibrio species are associated with the main diseases affecting the worldwide oyster production. Here, we review the current knowledge on Vibrio–bivalve interaction in oysters (Crassostrea sp.) and mussels (Mytilus sp.). We discuss the transient versus stable associations of vibrios with their bivalve hosts as well as technical issues limiting the monitoring of these bacteria in bivalve health and disease. Based on the current knowledge of oyster/mussel immunity and their interactions with Vibrio species pathogenic for oyster, we discuss how differences in immune effectors could contribute to the higher resistance of mussels to infections. Finally, we review the multiple strategies evolved by pathogenic vibrios to circumvent the potent immune defences of bivalves and how key virulence mechanisms could have been positively or negatively selected in the marine environment through interactions with predators.     

Cloning of cDNAs and hybridization analysis of lysozymes from two oyster species, Crassostrea gigas and Ostrea edulis

In bivalve molluscs including oysters, lysozymes play an important role in the host defense mechanisms against invading microbes. However, it remains unclear in which sites/cells the lysozyme genes are expressed and which subsequently produced the enzyme. This study cloned lysozyme cDNAs from the digestive organs of Pacific oyster Crassostrea gigas and European flat oyster Ostrea edulis. Both complete sequences of two oysters' lysozymes were composed of 137 amino acids. Two translated proteins present a high content in cysteine residues. Phylogenetic analyses showed that these oysters' lysozymes clustered with the invertebrate-type lysozymes of other bivalve species. In the Pacific oyster, lysozyme mRNA was expressed in all tissues except for those of the adductor muscle. In situ hybridization analyses revealed that lysozyme mRNA was expressed strongly in basophil cells in the digestive gland tubule of C. gigas, but not in digestive cells. Results indicated that the basophil cells of the oyster digestive gland are the sites of lysozyme synthesis.     

Feeding ecology of Xenoturbella bocki (phylum Xenoturbellida) revealed by genetic barcoding

The benthic marine worm Xenoturbella is frequently contaminated with molluscan DNA, which had earlier caused confusion resulting in a suggested bivalve relationship. In order to find the source of the contaminant, we have used molluscan sequences derived from Xenoturbella and compared them to barcodes obtained from several individuals of the nonmicroscopic molluscs sharing the same environment as Xenoturbella. Using cytochrome oxidase 1, we found the contaminating sequences to be 98% similar to the bivalve Ennucula tenuis. Using the highly variable D1-D2 region of the large ribosomal subunit in Xenoturbella, we found three distinct species of contaminating molluscs, one of which is 99% similar to the bivalve Abra nitida, one of the most abundant bivalves in the Gullmarsfjord where Xenoturbella was found, and another 99% similar to the bivalve Nucula sulcata. These data clearly show that Xenoturbella only contains molluscan DNA originating from bivalves living in the same environment, refuting former hypotheses of a bivalve relationship. In addition, these data suggest that Xenoturbella feeds specifically on bivalve prey from multiple species, possibly in the form of eggs and larvae.     

Mass mortality in Pacific oysters is associated with a specific gene expression signature

Mass mortality events occur in natural and cultured communities of bivalve molluscs. The Pacific oyster, Crassostrea gigas, is a dominant species in many intertidal locations as well as an important aquacultured bivalve species, and for the last 50 years, adult oysters have suffered frequent and extreme mass mortality events during summer months. To investigate the molecular changes that precede these mortality events, we employed a novel nonlethal sampling approach to collect haemolymph samples from individual oysters during the period that preceded a mortality event. Microarray-based gene expression screening of the collected haemolymph was used to identify a mortality gene expression signature that distinguished oysters that survived the mortality event from those individuals that died during the event. The signature was cross-validated by comparing two separate episodes of mortality. Here, we report that near-mortality oysters can be distinguished from longer-lived oysters by the elevated expression of genes associated with cell death, lysosomal proteolysis, and cellular assembly and organization. These results show the potential utility of nonlethal sampling approaches for investigating the environmental causes of mortality in natural populations in the field, and for predicting when such events could occur and which individuals will be affected.     

Physiological components of growth differences between individual oysters (Crassostrea gigas) and a comparison with Saccostrea commercialis

Pacific oysters (Crassostrea gigas) of identical age from two genetically distinct lines, one fast growing and the other slow growing, were held at three levels of ration and analysed for physiological traits to explain differences in their rates of growth. The data supported three hypotheses; faster growth was associated with faster rates of consumption of food, reduced metabolic rate at maintenance (i.e., at zero growth), and reduced metabolic costs of growth. A comparison with the Sydney rock oyster, Saccostrea commercialis, based on similar experiments on the two species, indicated that faster growth of Pacific oysters depended on similar physiological differences; the mean metabolic costs of growth, however, were similar in the two species. It is suggested that a general model for genetically linked differences in the growth rate of bivalve molluscs will need to include the processes of metabolic control rather than relying solely on an analysis of the individual components of the energetics of growth.     

In vitro effects of cadmium and mercury on Pacific oyster, Crassostrea gigas (Thunberg), haemocytes

In the past decades, shellfish culture has developed in a significant way around the world. However, culture areas are often subject to recurring anthropic pollution. The recrudescent presence of industrial wastes is a source of heavy metals and results in pollutant transfer towards the aquatic environment in estuarine areas. Because of their mode of life, bivalves, including mussels and oysters, are suggested as ideal indicator organisms. The development of techniques allowing the analysis of the effects of pollutants on bivalve biology may lead to the monitoring of pollutant transfer in estuarine areas. In this context, the effects of cadmium and mercury on defence mechanisms were analysed in Pacific oysters, Crassostrea gigas. Pollutant effects were tested in vitro on oyster haemocytes. Cell viability and enzymatic activities (esterase, peroxidase, aminopeptidase, phagocytosis activities) were monitored by flow cytometry. Enzymatic phenoloxidase-like activity was also evaluated by spectrophotometry. High pollutant concentrations were used in order to detect the acute effect and to approach real pollutant concentrations existing in animal tissues. Cadmium induced no effect on oyster haemocytes under the tested conditions. On the contrary, mercury caused a significant haemocyte mortality after a 24 h in vitro incubation. Aminopeptidase positive cell percentage was enhanced by the pollutant, and phenoloxidase-like activity was inhibited. These in vitro results show that mercury may be expected to have an impact on bivalve immune functions in contaminated areas.     

Bacteria in bivalve shellfish with special reference to the oyster

K ueh , C.S.W. & C han , K-Y. 1985. Bacteria in bivalve shellfish with special reference to the oyster. Journal of Applied Bacteriology , 59 , 41–47.
The bacterial flora of the Pacific oyster Crassostrea gigas , the sea mussel Perna viridis and the arkshell clam Scapharca cornea differed considerably from that of seawater in both numbers and generic composition. The numbers of heterotrophic bacteria in the bivalve shellfish, including the anaerobes and spore-forming bacteria, were greater than that in the surrounding water. Pseudomonas spp. were the dominant organisms, comprising over one third of the 321 strains characterized after isolation from the bivalves and seawater. Other bacteria isolated from the shellfish included Vibrio, Acinetobacter , and Aeromonas spp., whereas the seawater flora consisted mainly of coliform organisms, coryneform bacteria and Flavobacterium/ Cytophaga spp. Bacteria associated with the deposit-feeding clams were higher in density and more distinct in generic composition as compared with those in the suspension-feeding oysters and mussels. Over 90% of the coliform and heterotrophic bacteria in oysters were found in organs associated with the digestive tract. Coliforms were mainly found in the stomach while heterotrophs were present in both stomach and the lower intestine. The results suggest that the stomach flora of oysters are mainly derived from the external environment and, through a process of selection and multiplication, that it may be gradually replaced by a more indigenous population which dominates the lower digestive tract.     

High genetic load in the Pacific oyster Crassostrea gigas

The causes of inbreeding depression and the converse phenomenon of heterosis or hybrid vigor remain poorly understood despite their scientific and agricultural importance. In bivalve molluscs, related phenomena, marker-associated heterosis and distortion of marker segregation ratios, have been widely reported over the past 25 years. A large load of deleterious recessive mutations could explain both phenomena, according to the dominance hypothesis of heterosis. Using inbred lines derived from a natural population of Pacific oysters and classical crossbreeding experiments, we compare the segregation ratios of microsatellite DNA markers at 6 hr and 2-3 months postfertilization in F(2) or F(3) hybrid families. We find evidence for strong and widespread selection against identical-by-descent marker homozygotes. The marker segregation data, when fit to models of selection against linked deleterious recessive mutations and extrapolated to the whole genome, suggest that the wild founders of inbred lines carried a minimum of 8-14 highly deleterious recessive mutations. This evidence for a high genetic load strongly supports the dominance theory of heterosis and inbreeding depression and establishes the oyster as an animal model for understanding the genetic and physiological causes of these economically important phenomena.     

Marine Snails and Slugs: a Great Place To Look for Antiviral Drugs

Molluscs, comprising one of the most successful phyla, lack clear evidence of adaptive immunity and yet thrive in the oceans, which are rich in viruses. There are thought to be nearly 120,000 species of Mollusca, most living in marine habitats. Despite the extraordinary abundance of viruses in oceans, molluscs often have very long life spans (10 to 100 years). Thus, their innate immunity must be highly effective at countering viral infections. Antiviral compounds are a crucial component of molluscan defenses against viruses and have diverse mechanisms of action against a wide variety of viruses, including many that are human pathogens. Antiviral compounds found in abalone, oyster, mussels, and other cultured molluscs are available in large supply, providing good opportunities for future research and development. However, most members of the phylum Mollusca have not been examined for the presence of antiviral compounds. The enormous diversity and adaptations of molluscs imply a potential source of novel antiviral compounds for future drug discovery.     

Molecular and histological identification of Marteilioides infection in Suminoe Oyster Crassostrea ariakensis, Manila Clam Ruditapes philippinarum and Pacific Oyster Crassostrea gigas on the south coast of Korea

The oyster ovarian parasite Marteilioides chungmuensis has been reported from Korea and Japan, damaging the oyster industries. Recently, Marteilioides-like organisms have been identified in other commercially important marine bivalves. In this study, we surveyed Marteilioides infection in the Manila clam Ruditapes philippinarum, Suminoe oyster Crassostrea ariakensis, and Pacific oyster Crassostrea gigas, using histology and Marteilioides-specific small subunit (SSU) rDNA PCR. The SSU rDNA sequence of M. chungmuensis (1716 bp) isolated from C. gigas in Tongyoung bay was 99.9% similar to that of M. chungmuensis reported in Japan. Inclusions of multi-nucleated bodies in the oocytes, typical of Marteilioides infection, were identified for the first time in Suminoe oysters. The SSU rDNA sequence of a Marteilioides-like organism isolated from Suminoe oysters was 99.9% similar to that of M. chungmuensis. Marteilioides sp. was also observed from 7 Manila clams of 1840 individuals examined, and the DNA sequences of which were 98.2% similar to the known sequence of M. chungmuensis. Unlike Marteilioides infection of Pacific oysters, no remarkable pathological symptoms, such as large multiple lumps on the mantle, were observed in infected Suminoe oysters or Manila clams. Distribution of the infected Manila clams, Suminoe oysters and Pacific oysters was limited to small bays on the south coast, suggesting that the southern coast is the enzootic area of Marteilioides infection.     

Ostreid Herpesvirus 1 Infection among Pacific Oyster (Crassostrea gigas) Spat: Relevance of Water Temperature to Virus Replication and Circulation Prior to the Onset of Mortality

A number of bivalve species worldwide, including the Pacific oyster, Crassostrea gigas, have been affected by mass mortality events associated with herpesviruses, resulting in significant losses. A particular herpesvirus was purified from naturally infected larval Pacific oysters, and its genome was completely sequenced. This virus has been classified as Ostreid herpesvirus 1 (OsHV-1) within the family Malacoherpesviridae. Since 2008, mass mortality outbreaks among C. gigas in Europe have been related to the detection of a variant of OsHV-1 called μVar. Additional data are necessary to better describe mortality events in relation to environmental-parameter fluctuations and OsHV-1 detection. For this purpose, a single batch of Pacific oyster spat was deployed in 4 different locations in the Marennes-Oleron area (France): an oyster pond (“claire”), a shellfish nursery, and two locations in the field. Mortality rates were recorded based on regular observation, and samples were collected to search for and quantify OsHV-1 DNA by real-time PCR. Although similar massive mortality rates were reported at the 4 sites, mortality was detected earlier in the pond and in the nursery than at both field sites. This difference may be related to earlier increases in water temperature. Mass mortality was observed among oysters a few days after increases in the number of PCR-positive oysters and viral-DNA amounts were recorded. An initial increment in the number of PCR-positive oysters was reported at both field sites during the survey in the absence of significant mortality. During this period, the water temperature was below 16°C.     

Metallothioneins (MTs) in marine molluscs.

The presence of MTs in marine molluscs was firstly hypothesized in oyster and in mussel during the seventies, however mussel's and oysters' MTs were completely purified and sequenced rather later. Already from the first studies it was evident that the purification of molluscan MTs was more difficult than in mammals. Mussel's MTs are characterized by the presence of a monomeric and a dimeric form. Several physiological and biochemical parameters can influence the concentration and the isolation of MT from molluscan tissues. Remarkable variations in MT isolation and quantification might depend on the purification and storage protocol. Because of possible artefacts due to the isolation procedure the establishment of a standard protocol for MT quantification in marine mollusc is still an important goal. In a few species the presence of very low molecular weight metal binding ligands has also been reported, in these cases it cannot be excluded that the native MT has been cleaved by the action of proteases. This review aims to report: 1) importance of a standard method for MT purification and quantification in molluscs; 2) distribution of MT among molluscan species; 3) data concerning oyster's and mussel's MTs which are the two more deeply investigated marine molluscs; 4) biotic and abiotic factors influencing MT concentration, and 5) biological role of MT and use of MT as a biochemical marker of heavy metal pollution.     

Shell microstructure of the early bivalve Pojetaia and the independent origin of nacre within the mollusca

Abstract: Pojetaia and Fordilla are the oldest bivalve molluscs, occurring in roughly co‐eval rocks from the Tommotian, and are the only undisputed, well‐known bivalves from the Cambrian. New specimens reveal that Pojetaia had a laminar inner shell microstructure reminiscent of the foliated aragonite of modern monoplacophorans, and the same is true for Fordilla. A similar shell microstructure is seen in Anabarella and Watsonella, providing support for the hypothesis that they are the ancestors of bivalves. Foliated aragonite shares many similarities with nacre, and it may have been the precursor to nacre in bivalves. No cases of undisputed nacre occur in the Cambrian, in spite of much shell microstructure data from molluscs of this time period. Thus, although considered by many to be homologous among molluscs, we conclude that nacre convergently evolved in monoplacophorans, gastropods, bivalves, and cephalopods. This independent origin of nacre appears to have taken place during, or just prior to, the Great Ordovician Biodiversification Event and represents a significant step in the arms race between predators and molluscan prey.     

Herpes virus in juvenile Pacific oysters Crassostrea gigas from Tomales Bay, California, coincides with summer mortality episodes

Pacific Crassostrea gigas and eastern C. virginica oysters were examined between June 2002 and April 2003 from 8 locations along the east, west and south USA coasts for oyster herpes virus (OsHV) infections using the A primer set in a previously developed PCR test. Only surviving Pacific oysters from a mortality event in Tomales Bay, California, USA, where annual losses of oysters have occurred each summer since 1993, were infected with a herpes-like virus in 2002. PCR examination using template amounts of both 50 and 500 ng were essential for OsHV detection. Sequence analysis indicated that the Tomales Bay OsHV was similar to that identified in France with the exception of a single base pair substitution in a 917 bp fragment of the viral genome. However, unlike the French OsHV-1, the Tomales Bay OsHV did not amplify with the primer pair of a second OsHV-1 PCR assay, suggesting that further characterization of these viruses is warranted. No evidence of Cowdry type A viral infections characteristic of herpes virus infections or other pathogens were observed in OsHV-infected oysters. Hemocytosis, diapedesis and hemocyte degeneration characterized by nuclear pycnosis and fragmentation were observed in infected oysters, which is consistent with previous observations of OsHV infections in France. Together these data suggest that OsHV may be associated with the annual summer Pacific oyster seed mortality observed in Tomales Bay, but establishment of a causal relationship warrants further investigation.     

Infectious diseases of the Pacific oyster,Crassostrea gigas

The Pacific oyster, Crassostrea gigas, is known for not having been affected by major epizootics of infectious diseases, unlike many other commercially important oysters worldwide. Nonetheless, review of the scientific literature reveals more than ten infectious diseases of this species including those with viral, bacterial, protozoan, and metazoan etiologies. These include diseases of larval, juvenile, and adult oysters. Diseases such as oyster velar virus disease, herpes-like infection, and ligament disease are known because of their importance in intensive husbandry systems of this bivalve. Nocardiosis, Marteilioides infection, haplosporidiosis, Denman Island disease, and others are primarily known from their effect on extensively cultured populations of the Pacific oyster. These diseases are reviewed in terms of their disease manifestations, etilogy, epizootiology and economic importance, prevention, and management and diagnosis.     

A p53-like protein from a freshwater mollusc Lamellidens corrianus

p53 is the most frequently mutated protein in human cancers and the accumulation of its high levels is a potential novel marker for malignancy. Recently, its homologues such as p63 and p73 have been reported in human, mice and fish. Environmentally induced alterations in p53 protein have been reported to contribute to pathogenesis of leukemia in soft-shell clam Mya arenaria inhabiting polluted water, suggesting that p53 proteins can also be used as pollution markers. In the present study, the presence of p53 protein or its homologues was investigated in tissues of bivalve molluscs Lamellidens corrianus that are predominant in the freshwater riverine environment and are well suited to act as test organisms for evaluation of habitat degradation. The molluscs were collected live from the river Ganga at three sampling sites viz., Kanpur, Allahabad and Varanasi and different tissues (foot, gill and mantle) were collected. Proteins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). On immunoblot analysis, a 45 kDa protein (p45) was recognized by the monoclonal anti-p53 antibody in the molluscan tissues. The p45 showed immunoreactivity in all the three tissues of molluscs collected at Kanpur, in foot and gill tissues in those collected at Allahabad, and in foot tissue only, in those collected at Varanasi. Since monoclonal anti-p53 recognizes a denaturation-resistant epitope on the p53 (53 kDa) nuclear protein and does not react with other cellular proteins, the molluscan p45 is a p53-homologue or p53-like protein. Further, the differential expression of p45 in the different organs might serve as a useful biomarker that would help in establishing pollution gradient for environmental monitoring in the large aquatic ecosystems.