Mass mortality of pearl oyster (Pinctada fucata (Gould)) in Japan in 2019 and 2020 is caused by an unidentified infectious agent

Mass mortality of 0-year-old pearl oysters, Pinctada fucata (Gould), and anomalies in adults were observed in Japan’s major pearl farming areas in the summer of 2019 and 2020. Although adult oyster mortality was low, both adult and juvenile oysters underwent atrophy of the soft body, detachment of the mantle from nacre (the shiny inner surface of the valves), deposition of brownish material on the nacre, and loss of nacre luster. Infection trials were conducted to verify the involvement of pathogens in this phenomenon. Healthy adult pearl oysters were obtained from areas where this disease had not occurred to use as the recipients. The sources of infection were either affected adult oysters with atrophied soft bodies or batches of juveniles in which mortality had reached conspicuous levels. Transmission of the disease to the healthy oysters were tested either by cohabitation with affected oysters or by injections of the hemolymph of affected animals. The injection infection test examined the effects of filtration and chloroform exposure on the pathogen. Occurrence of the disease was confirmed by the appearance of brown deposits on the nacre and loss of nacre luster. The abnormalities of nacre were clearly reproduced in recipient shells in three out of four cohabitation trials with affected oysters. The disease was also reproduced in six out of six injection trails either with hemolymph filtered through 100 nm filter or with hemolymph treated with chloroform. In a serial passage with hemolymph injections, the disease was successfully transmitted through eight passages. These results suggest that the etiology of the disease is a non-enveloped virus with a diameter ≤100 nm.

Microbiome Analysis Reveals Diversity and Function of Mollicutes Associated with the Eastern Oyster, Crassostrea virginica

ABSTRACT Marine invertebrate microbiomes play important roles in diverse host and ecological processes. However, a mechanistic understanding of host-microbe interactions is currently available for a small number of model organisms. Here, an integrated taxonomic and functional analysis of the microbiome of the eastern oyster, Crassostrea virginica, was performed using 16S rRNA gene-based amplicon profiling, shotgun metagenomics, and genome-scale metabolic reconstruction. Relatively high variability of the microbiome was observed across individual oysters and among different tissue types. Specifically, a significantly higher alpha diversity was observed in the inner shell than in the gut, gill, mantle, and pallial fluid samples, and a distinct microbiome composition was revealed in the gut compared to other tissues examined in this study. Targeted metagenomic sequencing of the gut microbiota led to further characterization of a dominant bacterial taxon, the class Mollicutes, which was captured by the reconstruction of a metagenome-assembled genome (MAG). Genome-scale metabolic reconstruction of the oyster Mollicutes MAG revealed a reduced set of metabolic functions and a high reliance on the uptake of host-derived nutrients. A chitin degradation and an arginine deiminase pathway were unique to the MAG compared to closely related genomes of Mollicutes isolates, indicating distinct mechanisms of carbon and energy acquisition by the oyster-associated Mollicutes. A systematic reanalysis of public eastern oyster-derived microbiome data revealed a high prevalence of the Mollicutes among adult oyster guts and a significantly lower relative abundance of the Mollicutes in oyster larvae and adult oyster biodeposits. IMPORTANCE Despite their biological and ecological significance, a mechanistic characterization of microbiome function is frequently missing from many nonmodel marine invertebrates. As an initial step toward filling this gap for the eastern oyster, Crassostrea virginica, this study provides an integrated taxonomic and functional analysis of the oyster microbiome using samples from a coastal salt pond in August 2017. The study identified high variability of the microbiome across tissue types and among individual oysters, with some dominant taxa showing higher relative abundance in specific tissues. A high prevalence of Mollicutes in the adult oyster gut was revealed by comparative analysis of the gut, biodeposit, and larva microbiomes. Phylogenomic analysis and metabolic reconstruction suggested the oyster-associated Mollicutes is closely related but functionally distinct from Mollicutes isolated from other marine invertebrates. To the best of our knowledge, this study represents the first metagenomics-derived functional inference of Mollicutes in the eastern oyster microbiome.

The Increased Expression of an Engrailed to Sustain Shell Formation in Response to Ocean Acidification

Engrailed is a transcription factor required in numerous species for important developmental steps such as neurogenesis, segment formation, preblastoderm organization, and compartment formation. Recent study has proved that engrailed is also a key gene related to shell formation in marine bivalves. In the present study, the expression pattern of an engrailed gene (Cgengrailed-1) in Pacific oyster Crassostrea gigas under CO2-driven acidification was investigated to understand its possible role in the regulation of shell formation and adaptation to ocean acidification (OA). The open reading frame (ORF) of Cgengrailed-1 was obtained, which was of 690 bp encoding a polypeptide of 229 amino acids with a HOX domain. Phylogenetic analysis indicated that the deduced amino acid sequence of Cgengrailed-1 shared high homology with other engraileds from Drosophila melanogaster, Mizuhopecten yessoensi, and Crassostrea virginica. The mRNA transcripts of Cgengrailed-1 were constitutively expressed in various tissues with the highest expression levels detected in labial palp and mantle, which were 86.83-fold (p < 0.05) and 75.87-fold (p < 0.05) higher than that in hepatopancreas. The mRNA expression of Cgengrailed-1 in mantle decreased dramatically after moderate (pH 7.8) and severe (pH 7.4) acidification treatment (0.75- and 0.15-fold of that in control group, p < 0.05). The results of immunofluorescence assay demonstrated that the expression level of Cgengrailed-1 in the middle fold of mantle increased significantly upon moderate and severe acidification treatment. Moreover, after the oyster larvae received acidification treatment at trochophore stage, the mRNA expression levels of Cgengrailed-1 increased significantly in D-shape larvae stages, which was 3.11- (pH 7.8) and 4.39-fold (pH 7.4) of that in control group (p < 0.05). The whole-mount immunofluorescence assay showed that Cgengrailed-1 was mainly expressed on the margin of shell gland, and the periostracum in trochophore, early D-shape larvae and D-shape larvae in both control and acidification treatment groups, and the intensity of positive signals in early D-shape larvae and D-shape larvae increased dramatically under acidification treatment. These results collectively suggested that the expression of Cgengrailed-1 could be triggered by CO2-driven acidification treatment, which might contribute to induce the initial shell formation in oyster larvae and the formation of periostracum in adult oyster to adapt to the acidifying marine environment.

Spatial epidemiological modelling of infection by Vibrio aestuarianus shows that connectivity and temperature control oyster mortality

Vibrio aestuarianus infection in oyster populations causes massive mortality, resulting in losses for oyster farmers. Such dynamics result from host-pathogen interactions and contagion through water-borne transmission. To assess the spatiotemporal spread of V. aestuarianus infection and associated oyster mortality at a bay scale, we built a mathematical model informed by experimental infection data at 2 temperatures and spatially dependent marine connectivity of oyster farms. We applied the model to a real system and tested the importance of each factor using a number of modelling scenarios. Results suggest that introducing V. aestuarianus in a fully susceptible adult oyster population in the bay would lead to the mortality of all farmed oysters over 6 to 12 mo, depending on the location in which infection was initiated. The effect of temperature was captured by the basic reproduction number (R0), which was >1 at high seawater temperatures, as opposed to values <1 at low temperatures. At the ecosystem scale, simulations showed the existence of long-distance dispersal of free-living bacteria. The western part of the bay could be reached by bacteria originating from the eastern side, though the spread time was greatly increased. Further developments of the model, including the consideration of the anthropogenic movements of oysters and oyster-specific sensitivity factors, would allow the development of accurate maps of epidemiological risks and help define aquaculture zoning.

A comparative study on the nursery culture of hatchery-reared sub-adult cupped oyster, Crassostrea iredalei (Faustino, 1932), in an earthen pond and a mangrove canal

A comparative study on the nursery culture of the spat of the tropical oyster, Crassostrea iredalei, in an earthen pond and a mangrove canal was conducted over two months. The results revealed no differences in the absolute growth rate determined by shell width between the two culture sites (P < 0.05). Sub-adult oysters cultured in the mangrove canal showed a higher absolute growth rate in shell length and a higher daily growth rate than the oysters cultured in the earthen pond (P < 0.05). The mean survival rate of sub-adult oysters cultured in the earthen pond (99.8 ± 0.2%) was significantly higher than for those cultured in the mangrove canal (66.7 ± 31.4%). Decreased density from the loss of sub-adult oyster nursery culture in the mangrove canal led to higher growth performance than in the earthen pond. However, no difference was found for the fraction of oysters larger or smaller than 5 cm for the two culture sites (P < 0.05). A significant difference was noted in the Condition Index (CI) between the two culture sites (P < 0.05). The high primary productivity in mangroves is a major supporter of higher CI in sub-adult oysters cultured in mangrove canals versus in earthen ponds. Water exchange in the earthen pond to maintain calcium and magnesium concentrations resulted in no differences in the shell compressibility of sub-adult oysters compared with those cultured in the mangrove canal.

Validation of a habitat suitability index for oyster restoration

Habitat suitability index (HSI) models provide spatially explicit information on the capacity of a given habitat to support a species of interest, and their prevalence has increased dramatically in recent years. Despite caution that the reliability of HSIs must be validated using independent, quantitative data, most HSIs intended to inform terrestrial and marine species management remain unvalidated. Furthermore, of the eight HSI models developed for eastern oyster (Crassostrea virginica) restoration and fishery production, none has been validated. Consequently, we developed, calibrated, and validated an HSI for the eastern oyster to identify optimal habitat for restoration in a tributary of Chesapeake Bay, the Great Wicomico River (GWR). The GWR harbors an unparalleled, restored oyster population, and therefore serves as an excellent model system for assessing the validity of the HSI. The HSI was derived from GIS layers of bottom type, salinity, and water depth (surrogate for dissolved oxygen), and was tested using live adult oyster density data from a survey of high vertical relief reefs (HRR) and low vertical relief reefs (LRR) in the sanctuary network. Live adult oyster density was a statistically significant sigmoid function of the HSI, which validates the HSI as a robust predictor of suitable oyster reef habitat for rehabilitation or restoration. In addition, HRR had on average 103-116 more adults m-2than LRR at a given level of the HSI. For HRR, HSI values ≥0.3 exceeded the accepted restoration target of 50 live adult oysters m-2. For LRR, the HSI was generally able to predict live adult oyster densities that meet or exceed the target at HSI values ≥0.3. The HSI indicated that there remain large areas of suitable habitat for restoration in the GWR. This study provides a robust framework for HSI model development and validation, which can be refined and applied to other systems and previously developed HSIs to improve the efficacy of native oyster restoration.

Characteristics of the Aragonitic Layer in Adult Oyster Shells,Crassostrea gigas: Structural Study of Myostracum including the Adductor Muscle Scar

Myostracum, which is connected from the umbo to the edge of a scar, is not a single layer composed of prismatic layers, but a hierarchically complex multilayered shape composed of minerals and an organic matrix. Through the analysis of the secondary structure, the results revealed that aβ-antiparallel structure was predominant in the mineral phase interface between the myostracum (aragonite) and bottom folia (calcite). After the complete decalcification and deproteinization, the membrane obtained from the interface between the myostracum buried in upper folia, and the bottom folia was identified as chitin. The transitional zone in the interface between the adductor muscle scar and folia are verified. The myostracum disappeared at the edge of the scar of the posterior side. From this study, the entire structure of the myostracum from the adult oyster shell ofCrassostrea gigascould be proposed.

Functional morphology of the coelomocytes of the larval oysters (Crassostrea virginica and Crassostrea gigas)

The functional morphology of the coelomocytes of larval oysters, Crassostrea virginica and C. gigas, based on observations of live animals and histological and ultrastructural examination of tissues, is presented. Two predominant types of coelomocytes were found in the larval oysters. One, the SER cell, not found in the adult oyster, is large with a spheroidal nucleus, and exhibits basophilic cytoplasm which consists of abundant smooth endoplastic reticulum. This cell appears to participate in metabolic conversion processes. The other predominant cell type, the phagocyte, appears identical to the phagocyte observed in the adult oyster. Both differential cell types were observed from one day post fertilization to metamorphosis, the oldest stage studied.

MORPHOLOGICAL ANOMALIES IN ADULT OYSTER, SCALLOP, AND ATLANTIC SILVERSIDES EXPOSED TO WASTE MOTOR OIL

ABSTRACT Waste motor oil concentrations of 20 ppm and higher induced lesions in the vascular systems of Atlantic silversides and oyster. These lesions were associated with the pseudobranch, the heart, and the arterial system of the Atlantic silversides, and the branchial efferent vein of the oyster. Lesions occurred in the gastro-intestinal tract of oyster, the gill and kidney of scallop, and the mantle of both species. Based on these preliminary exposures, the scallop, of the three species tested, demonstrated the highest sensitivity to the toxic effects of 20 ppm waste motor oil, or higher, for periods up to 60 days. The LC-50 (96 hr) value for waste motor oil and adult Atlantic silversides was 1,7000 ppm. Acute toxicity values were not determined for the oyster or scallop.