Metabarcoding of Hepatitis E Virus Genotype 3 and Norovirus GII from Wastewater Samples in England Using Nanopore Sequencing.

Norovirus is one of the largest causes of gastroenteritis worldwide, and Hepatitis E virus (HEV) is an emerging pathogen that has become the most dominant cause of acute viral hepatitis in recent years. The presence of norovirus and HEV has been reported within wastewater in many countries previously. Here we used amplicon deep sequencing (metabarcoding) to identify norovirus and HEV strains in wastewater samples from England collected in 2019 and 2020. For HEV, we sequenced a fragment of the RNA-dependent RNA polymerase (RdRp) gene targeting genotype three strains. For norovirus, we sequenced the 5' portion of the major capsid protein gene (VP1) of genogroup II strains. Sequencing of the wastewater samples revealed eight different genotypes of norovirus GII (GII.2, GII.3, GII.4, GII.6, GII.7, GII.9, GII.13 and GII.17). Genotypes GII.3 and GII.4 were the most commonly found. The HEV metabarcoding assay was able to identify HEV genotype 3 strains in some samples with a very low viral concentration determined by RT-qPCR. Analysis showed that most HEV strains found in influent wastewater were typed as G3c and G3e and were likely to have originated from humans or swine. However, the small size of the HEV nested PCR amplicon could cause issues with typing, and so this method is more appropriate for samples with high CTs where methods targeting longer genomic regions are unlikely to be successful. This is the first report of HEV RNA in wastewater in England. This study demonstrates the utility of wastewater sequencing and the need for wider surveillance of norovirus and HEV within host species and environments.

Combining Nanopore Sequencing with Recombinase Polymerase Amplification Enables Identification of Dinoflagellates from the Alexandrium Genus, Providing a Rapid, Field Deployable Tool.

The armoured dinoflagellate Alexandrium can be found throughout many of the world's temperate and tropical marine environments. The genus has been studied extensively since approximately half of its members produce a family of potent neurotoxins, collectively called saxitoxin. These compounds represent a significant threat to animal and environmental health. Moreover, the consumption of bivalve molluscs contaminated with saxitoxin poses a threat to human health. The identification of Alexandrium cells collected from sea water samples using light microscopy can provide early warnings of a toxic event, giving harvesters and competent authorities time to implement measures that safeguard consumers. However, this method cannot reliably resolve Alexandrium to a species level and, therefore, is unable to differentiate between toxic and non-toxic variants. The assay outlined in this study uses a quick recombinase polymerase amplification and nanopore sequencing method to first target and amplify a 500 bp fragment of the ribosomal RNA large subunit and then sequence the amplicon so that individual species from the Alexandrium genus can be resolved. The analytical sensitivity and specificity of the assay was assessed using seawater samples spiked with different Alexandrium species. When using a 0.22 µm membrane to capture and resuspend cells, the assay was consistently able to identify a single cell of A. minutum in 50 mL of seawater. Phylogenetic analysis showed the assay could identify the A. catenella, A. minutum, A. tamutum, A. tamarense, A. pacificum, and A. ostenfeldii species from environmental samples, with just the alignment of the reads being sufficient to provide accurate, real-time species identification. By using sequencing data to qualify when the toxic A. catenella species was present, it was possible to improve the correlation between cell counts and shellfish toxicity from r = 0.386 to r = 0.769 (p ≤ 0.05). Furthermore, a McNemar's paired test performed on qualitative data highlighted no statistical differences between samples confirmed positive or negative for toxic species of Alexandrium by both phylogenetic analysis and real time alignment with the presence or absence of toxins in shellfish. The assay was designed to be deployed in the field for the purposes of in situ testing, which required the development of custom tools and state-of-the-art automation. The assay is rapid and resilient to matrix inhibition, making it suitable as a potential alternative detection method or a complementary one, especially when applying regulatory controls.

Methodological advances in the detection of biotoxins and pathogens affecting production and consumption of bivalve molluscs in a changing environment.

The production, harvesting and safe consumption of bivalve molluscs can be disrupted by biological hazards that can be divided into three categories: (1) biotoxins produced by naturally occurring phytoplankton that are bioaccumulated by bivalves during filter-feeding, (2) human pathogens also bioaccumulated by bivalves and (3) bivalve pathogens responsible for disease outbreaks. Environmental changes caused by human activities, such as climate change, can further aggravate these challenges. Early detection and accurate quantification of these hazards are key to implementing measures to mitigate their impact on production and safeguard consumers. This review summarises the methods currently used and the technological advances in the detection of biological hazards affecting bivalves, for the screening of known hazards and discovery of new ones.

First Detection of Francisella halioticida Infecting a Wild Population of Blue Mussels Mytilus edulis in the United Kingdom.

In the last decade, declines in the population of wild blue mussels Mytilus edulis in the Tamar estuary (United Kingdom) have been noted. In archived samples collected from 2013 to 2019, between 7% (in 2013) and 18% (in 2019) showed large granulocytoma and haemocytic infiltration in the interstitial tissue of the digestive gland. Four samples were selected for 16S rRNA gene Nanopore sequencing. A consensus sequence of 1449 bp showed nucleotide similarities between 99.93-100% with published sequences of Francisella halioticida. In situ hybridisation (ISH) confirmed the presence of F. halioticida DNA within individual granulocytes of granulocytomas and also in prokaryotic-like inclusion bodies within the digestive epithelial cells. The design of diagnostic tests for surveillance of F. halioticida, including more specific ISH probes and sequencing the genome of the isolates infecting mussels, will shed more light on the pathogenicity and spread of this pathogen.

Hematodinium sp. infection does not drive collateral disease contraction in a crustacean host.

Host, pathogen, and environment are determinants of the disease triangle, the latter being a key driver of disease outcomes and persistence within a community. The dinoflagellate genus Hematodinium is detrimental to crustaceans globally - considered to suppress the innate defences of hosts, making them more susceptible to co-infections. Evidence supporting immune suppression is largely anecdotal and sourced from diffuse accounts of compromised decapods. We used a population of shore crabs (Carcinus maenas), where Hematodinium sp. is endemic, to determine the extent of collateral infections across two distinct environments (open-water, semi-closed dock). Using a multi-resource approach (PCR, histology, haematology, population genetics, eDNA), we identified 162 Hematodinium-positive crabs and size/sex-matched these to 162 Hematodinium-free crabs out of 1191 analysed. Crabs were interrogated for known additional disease-causing agents; haplosporidians, microsporidians, mikrocytids, Vibrio spp., fungi, Sacculina, trematodes, and haemolymph bacterial loads. We found no significant differences in occurrence, severity, or composition of collateral infections between Hematodinium-positive and Hematodinium-free crabs at either site, but crucially, we recorded site-restricted blends of pathogens. We found no gross signs of host cell immune reactivity towards Hematodinium in the presence or absence of other pathogens. We contend Hematodinium sp. is not the proximal driver of co-infections in shore crabs, which suggests an evolutionary drive towards latency in this environmentally plastic host.

Whole Genome Sequencing of Hepatitis A Virus Using a PCR-Free Single-Molecule Nanopore Sequencing Approach.

Hepatitis A virus (HAV) is one of the most common causes of acute viral hepatitis in humans. Although HAV has a relatively small genome, there are several factors limiting whole genome sequencing such as PCR amplification artefacts and ambiguities in de novo assembly. The recently developed Oxford Nanopore technologies (ONT) allows single-molecule sequencing of long-size fragments of DNA or RNA using PCR-free strategies. We have sequenced the whole genome of HAV using a PCR-free approach by direct reverse-transcribed sequencing. We were able to sequence HAV cDNA and obtain reads over 7 kilobases in length containing almost the whole genome of the virus. The comparison of these raw long nanopore reads with the HAV reference wild type revealed a nucleotide sequence identity between 81.1 and 96.6%. By de novo assembly of all HAV reads we obtained a consensus sequence of 7362 bases, with a nucleotide sequence identity of 99.0% with the genome of the HAV strain pHM175/18f. When the assembly was performed using as reference the HAV strain pHM175/18f a consensus with a sequence similarity of 99.8 % was obtained. We have also used an ONT amplicon-based assay to sequence two fragments of the VP3 and VP1 regions which showed a sequence similarity of 100% with matching regions of the consensus sequence obtained using the direct cDNA sequencing approach. This study showed the applicability of ONT sequencing technologies to obtain the whole genome of HAV by direct cDNA nanopore sequencing, highlighting the utility of this PCR-free approach for HAV characterization and potentially other viruses of the Picornaviridae family.

Mycosis is a Disease State Encountered Rarely in Shore Crabs, Carcinus maenas.

There is a paucity of knowledge regarding the diversity and impact(s) of disease-causing fungi in marine animals, especially shellfish. In efforts to address this knowledge gap for the shore crab Carcinus maenas, a year-long disease screen was carried out across two sites in Swansea Bay (Wales, UK) with a view to characterising putative fungal infections. Crabs were sampled between November 2017 and October 2018, and screened systematically for disease signatures using haemolymph (blood) preparations, targeted PCR and tissue histopathology. Strikingly, mycosis was confirmed in ~0.4% of total crabs tested (n = 1191) and restricted to one location only (Mumbles Pier). Clinical infections were observed in four out of four infected crabs. In these animals, the gills and hepatopancreas were congested with fungal morphotypes. In addition, some evidence indicates haemocyte (immune cell) reactivity toward the fungi. Phylogenetic placement of the partial internal transcribed spacer (ITS1) gene regions amplified from three mycotic crabs revealed the causative agent to be related to hypocrealean fungi, thereby representing a novel species.

Diagnosis and prevalence of two new species of haplosporidians infecting shore crabs Carcinus maenas: Haplosporidium carcini n. sp., and H. cranc n. sp.

This study provides a morphological and phylogenetic characterization of two novel species of the order Haplosporida (Haplosporidium carcini n. sp., and H. cranc n. sp.) infecting the common shore crab Carcinus maenas collected at one location in Swansea Bay, South Wales, UK. Both parasites were observed in the haemolymph, gills and hepatopancreas. The prevalence of clinical infections (i.e. parasites seen directly in fresh haemolymph preparations) was low, at ~1%, whereas subclinical levels, detected by polymerase chain reaction, were slightly higher at ~2%. Although no spores were found in any of the infected crabs examined histologically (n = 334), the morphology of monokaryotic and dikaryotic unicellular stages of the parasites enabled differentiation between the two new species. Phylogenetic analyses of the new species based on the small subunit (SSU) rDNA gene placed H. cranc in a clade of otherwise uncharacterized environmental sequences from marine samples, and H. carcini in a clade with other crustacean-associated lineages.

The Application of Nanopore Sequencing Technology to the Study of Dinoflagellates: A Proof of Concept Study for Rapid Sequence-Based Discrimination of Potentially Harmful Algae.

Harmful algal blooms (HABs) are a naturally occurring global phenomena that have the potential to impact fisheries, leisure and ecosystems, as well as posing a significant hazard to animal and human health. There is significant interest in the development and application of methodologies to study all aspects of the causative organisms and toxins associated with these events. This paper reports the first application of nanopore sequencing technology for the detection of eukaryotic harmful algal bloom organisms. The MinION sequencing platform from Oxford Nanopore technologies provides long read sequencing capabilities in a compact, low cost, and portable format. In this study we used the MinION to sequence long-range PCR amplicons from multiple dinoflagellate species with a focus on the genus Alexandrium. Primers applicable to a wide range of dinoflagellates were selected, meaning that although the study was primarily focused on Alexandrium the applicability to three additional genera of toxic algae, namely; Gonyaulax, Prorocentrum, and Lingulodinium was also demonstrated. The amplicon generated here spanned approximately 3 kb of the rDNA cassette, including most of the 18S, the complete ITS1, 5.8S, ITS2 and regions D1 and D2 of the 28S. The inclusion of barcode genes as well as highly conserved regions resulted in identification of organisms to the species level. The analysis of reference cultures resulted in over 99% of all sequences being attributed to the correct species with an average identity above 95% from a reference list of over 200 species (see Supplementary Material 1). The use of mock community analysis within environmental samples highlighted that complex matrices did not prevent the ability to distinguish between phylogenetically similar species. Successful identification of causative organisms in environmental samples during natural toxic events further highlighted the potential of the assay. This study proves the suitability of nanopore sequencing technology for taxonomic identification of harmful algal bloom organisms and acquisition of data relevant to the World Health Organisations "one health" approach to marine monitoring.

Prevalence and histopathology of the parasitic barnacle, Sacculina carcini in shore crabs, Carcinus maenas.

Sacculina carcini is a common parasite of the European shore crab, Carcinus maenas. Following successful penetration of the host, numerous rootlets are formed that permeate through the hosts' tissues. Ultimately, these form an externa that houses the developing nauplii larvae of the parasite. Most studies have quantified levels of infection by counting the presence of reproductive externae and their breakdown structures, called scars. However, the diagnosis of the disease based only on external features may lead to underreporting the prevalence of the parasite. In the current study, we examined the presence and severity of S. carcini in C. maenas (n = 221) in the Prince of Wales Dock, South Wales, U.K. using a range of diagnostic approaches to give an accurate representation of temporal dynamics of infection. Parasitized crabs were found with a mean prevalence of 24% as determined by histological examination of the hepatopancreas. However, the prevalence of S. carcini based on the presence of externae and scars was only 6.3% and 1.8%, respectively. Overall, parasitism was associated with smaller crabs, crabs later in the moulting cycle that were orange in colour (as opposed to green or yellow), and those with a higher number of bacteria in the haemolymph. Interestingly, only 7.5% of infected crabs showed evidence of distinct host (cellular) response to the presence of rootlets in the hepatopancreas.

Spatial and temporal disease dynamics of the parasite Hematodinium sp. in shore crabs, Carcinus maenas.

BACKGROUND: The parasitic dinoflagellates of the genus Hematodinium represent the causative agent of so-called bitter or pink crab disease in a broad range of shellfish taxa. Outbreaks of Hematodinium-associated disease can devastate local fishing and aquaculture efforts. The goal of our study was to examine the potential role of the common shore (green) crab Carcinus maenas as a reservoir for Hematodinium. Carcinus maenas is native to all shores of the UK and Ireland and the North East Atlantic but has been introduced to, and subsequently invaded waters of, the USA, South Africa and Australia. This species is notable for its capacity to harbour a range of micro- and macro-parasites, and therefore may act as a vector for disease transfer. METHODS: Over a 12-month period, we interrogated 1191 crabs across two distinct locations (intertidal pier, semi-closed dock) in Swansea Bay (Wales, UK) for the presence and severity of Hematodinium in the haemolymph, gills, hepatopancreas and surrounding waters (eDNA) using PCR-based methods, haemolymph preparations and histopathology. RESULTS: Overall, 13.6% were Hematodinium-positive via PCR and confirmed via tissue examination. Only a small difference was observed between locations with 14.4% and 12.8% infected crabs in the Dock and Pier, respectively. Binomial logistic regression models revealed seasonality (P < 0.002) and sex (P < 0.001) to be significant factors in Hematodinium detection with peak infection recorded in spring (March to May). Male crabs overall were more likely to be infected. Phylogenetic analyses of the partial ITS and 18S rRNA gene regions of Hematodinium amplified from crabs determined the causative agent to be the host generalist Hematodinium sp., which blights several valuable crustaceans in the UK alone, including edible crabs (Cancer pagurus) and langoustines (Nephrops norvegicus). CONCLUSIONS: Shore crabs were infected with the host generalist parasite Hematodinium sp. in each location tested, thereby enabling the parasite to persist in an environment shared with commercially important shellfish.

Evolution and diversity of alpha-carbonic anhydrases in the mantle of the Mediterranean mussel (Mytilus galloprovincialis).

The α-carbonic anhydrases (α-CAs) are a large and ancient group of metazoan-specific enzymes. They generate bicarbonate from metabolic carbon dioxide and through calcium carbonate crystal formation play a key role in the regulation of mineralized structures. To better understand how α-CAs contribute to shell mineralization in the marine Mediterranean mussel (Mytilus galloprovincialis) we characterized them in the mantle. Phylogenetic analysis revealed that mollusc α-CA evolution was affected by lineage and species-specific events. Ten α-CAs were found in the Mediterranean mussel mantle and the most abundant form was named, MgNACR, as it grouped with oyster nacreins (NACR). Exposure of the Mediterranean mussel to reduced water salinity (18 vs 37 ppt), caused a significant reduction (p < 0.05) in mantle esterase activity and MgNACR transcript abundance (p < 0.05). Protonograms revealed multiple proteins in the mantle with α-CA hydratase activity and mapped to a protein with a similar size to that deduced for monomeric MgNACR. Our data indicate that MgNACR is a major α-CA enzyme in mantle and that by homology with oyster nacreins likely regulates mussel shell production. We propose that species-dependent α-CA evolution may contribute to explain the diversity of bivalve shell structures and their vulnerability to environmental changes.

Two toll-like receptors identified in the mantle of Mytilus coruscus are abundant in haemocytes.

Toll-like receptors (TLRs) are a large family of pattern recognition receptors (PRRs) that play a critical role in innate immunity. TLRs are activated when they recognize microbial associated molecular patterns (MAMPs) of bacteria, viruses, or fungus. In the present study, two TLRs were isolated from the mantle of the hard-shelled mussel (Mytilus coruscus) and designated McTLR2 and McTLR3 based on their sequence similarity and phylogenetic clustering with Crassostrea gigas, CgiTLR2 and CgiTLR3, respectively. Quantitative RT-PCR analysis demonstrated that McTLR2 and McTLR3 were constitutively expressed in many tissues but at low abundance.

Elevated Seawater Temperatures Decrease Microbial Diversity in the Gut of Mytilus coruscus.

The gut microbial community is critical for the host immune system, and in recent years, it has been extensively studied in vertebrates using 'omic' technologies. In contrast, knowledge about how the interactions between water temperature and diet affect the gut microbiota of marine invertebrates that do not thermoregulate is much less studied. In the present study, the effect of elevated seawater temperature and diet (Isochrysis zhanjiangensis and Platymonas helgolandica var. tsingtaoensis) on the gut microbial community of the commercial mussel, Mytilus coruscus, was investigated. The 16S rRNA gene sequencing was used to characterize the microbial community in M. coruscus gut. The mortality of M. coruscus exposed to a high water temperature (31°C) increased after 3 days and the diversity of the bacterial community in the gut of live M. coruscus was significantly reduced. For example, the abundance of Bacteroides (Bacteroidetes) and norank_Marinilabiaceae (Bacteroidetes) increased in the gut of M. coruscus fed I. zhanjiangensis. In M. coruscus fed P. helgolandica, the abundance of Arcobacter (Proteobacteria) and norank_Marinilabiaceae increased and the abundance of unclassified_Flavobacteriaceae (Bacteroidetes) decreased. The results obtained in the present study suggest that high temperatures favored the proliferation of opportunistic bacteria, including Bacteroides and Arcobacter, which may increase host susceptibility to disease. Microbial community composition of the gut in live M. coruscus was not impacted by the microalgal diet but it was modified in the group of mussels that died. The present study provides insight into the potential effects on the gut microbiome and mussel-bacteria interactions of rising seawater temperatures.

Evidence of vertical transmission of ostreid herpesvirus 1 in the Portuguese oyster Crassostrea angulata.

In order to determine if ostreid herpesvirus 1 (OsHV-1) can be vertically transmitted, 9 full-sib families of the Portuguese oyster Crassostrea angulata were produced using a factorial mating design with 3 males and 3 females. The parents were survivors from an OsHV-1 mortality outbreak. OsHV-1 DNA was not detected by conventional PCR in the mantle of parents, gametes or 3day-old larvae. However, viral DNA was detected by real-time PCR in all gametes and larvae samples. These results show that C. angulata that have survived an OsHV-1 mortality outbreak can carry the virus and vertically transmit it to their offspring.

Experimental infection of European flat oyster Ostrea edulis with ostreid herpesvirus 1 microvar (OsHV-1µvar): Mortality, viral load and detection of viral transcripts by in situ hybridization.

Ostreid herpesvirus 1 (OsHV-1) infections have been reported in several bivalve species. Mortality of Pacific oyster Crassostrea gigas spat has increased considerably in Europe since 2008 linked to the spread of a variant of OsHV-1 called µvar. In the present study we demonstrated that O. edulis juveniles can be infected by OsHV-1µvar when administered as an intramuscular injection. Mortality in the oysters injected with OsHV-1µvar was first detected 4 days after injection and reached 25% mortality at day 10. Moreover, the high viral load observed and the detection of viral transcripts by in situ hybridization in several tissues of dying oysters suggested that OsHV-1µvar was the cause of mortality in the O. edulis juveniles. This is therefore the first study to provide evidence about the pathogenicity of OsHV-1µvar in a species that does not belong to the Crassostrea genus. Additionally, we present a novel method to detect OsHV-1 transcripts in infected individuals' using in situ hybridization.

Perkinsosis in the clams Ruditapes decussatus and R. philippinarum in the Northeastern Atlantic and Mediterranean Sea: A review.

Perkinsosis is a disease of gastropod and bivalve molluscs caused by protozoan parasites of the Perkinsus genus. These parasites have been responsible for mass mortalities worldwide, with a significant impact in both fisheries and aquaculture, and resulting in severe economic losses. This review focuses mainly on current knowledge of diagnostic methods and on the distribution of Perkinsus spp. in the Northeastern Atlantic and Mediterranean Sea, which infect the grooved carpet shell clam Ruditapes decussatus and the Japanese carpet shell clam Ruditapes philippinarum. The association between perkinsosis and high mortality rates of R. decussatus and R. philippinarum in southern European countries such as Portugal and Italy is discussed as is the role of environmental factors in those mortality outbreaks. The putative introduction of Perkinsus olseni into the Northeastern Atlantic and Mediterranean Sea is also discussed, as are management strategies that could be used to mitigate the impact of perkinsosis in production of R. decussatus and R. philippinarum.

Detection of Marteilia refringens infecting the European flat oyster Ostrea edulis and the dwarf oyster Ostrea stentina in southern Portugal and Spain.

In the present study, Marteilia sp. was detected by histological examination and in situ hybridisation in Ostrea edulis and Ostrea stentina collected in southern Iberian Peninsula. Marteilia refringens DNA was detected by PCR in O. edulis (collected in southern Portugal) and O. stentina (collected in southern Spain and Portugal). Sequencing analysis revealed the presence of M. refringens type O in O. edulis, and type O and M in O. stentina. This is the first confirmed detection of M. refringens in Portugal and the first report on the occurrence of M. refringens infecting O. stentina in Europe.

Acidification effects on biofouling communities: winners and losers.

How ocean acidification affects marine life is a major concern for science and society. However, its impacts on encrusting biofouling communities, that are both the initial colonizers of hard substrata and of great economic importance, are almost unknown. We showed that community composition changed significantly, from 92% spirorbids, 3% ascidians and 4% sponges initially to 47% spirorbids, 23% ascidians and 29% sponges after 100 days in acidified conditions (pH 7.7). In low pH, numbers of the spirorbid Neodexiospira pseudocorrugata were reduced ×5 compared to controls. The two ascidians present behaved differently with Aplidium sp. decreasing ×10 in pH 7.7, whereas Molgula sp. numbers were ×4 higher in low pH than controls. Calcareous sponge (Leucosolenia sp.) numbers increased ×2.5 in pH 7.7 over controls. The diatom and filamentous algal community was also more poorly developed in the low pH treatments compared to controls. Colonization of new surfaces likewise showed large decreases in spirorbid numbers, but numbers of sponges and Molgula sp. increased. Spirorbid losses appeared due to both recruitment failure and loss of existing tubes. Spirorbid tubes are comprised of a loose prismatic fabric of calcite crystals. Loss of tube materials appeared due to changes in the binding matrix and not crystal dissolution, as SEM analyses showed crystal surfaces were not pitted or dissolved in low pH conditions. Biofouling communities face dramatic future changes with reductions in groups with hard exposed exoskeletons and domination by soft-bodied ascidians and sponges.

Persistence, seasonal dynamics and pathogenic potential of Vibrio communities from Pacific oyster hemolymph.

Bacteria of the genus Vibrio occur at a continuum from free-living to symbiotic life forms, including opportunists and pathogens, that can contribute to severe diseases, for instance summer mortality events of Pacific oysters Crassostrea gigas. While most studies focused on Vibrio isolated from moribund oysters during mortality outbreaks, investigations of the Vibrio community in healthy oysters are rare. Therefore, we characterized the persistence, diversity, seasonal dynamics, and pathogenicity of the Vibrio community isolated from healthy Pacific oysters. In a reciprocal transplant experiment we repeatedly sampled hemolymph from adult Pacific oysters to differentiate population from site-specific effects during six months of in situ incubation in the field. We characterized virulence phenotypes and genomic diversity based on multilocus sequence typing in a total of 70 Vibrio strains. Based on controlled infection experiments we could show that strains with the ability to colonize healthy adult oysters can also have the potential to induce high mortality rates on larvae. Diversity and abundance of Vibrio varied significantly over time with highest values during and after spawning season. Vibrio communities from transplanted and stationary oysters converged over time, indicating that communities were not population specific, but rather assemble from the surrounding environment forming communities, some of which can persist over longer periods.