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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.

Background Oysters in coastal environments are subject to fluctuating environmental conditions that may impact the ecosystem services they provide. Oyster-associated microbiomes are responsible for some of these services, particularly nutrient cycling in benthic habitats. The effects of climate change on host-associated microbiome composition are well-known, but functional changes and how they may impact host physiology and ecosystem functioning are poorly characterized. We investigated how environmental parameters affect oyster-associated microbial community structure and function along a trophic gradient in Narragansett Bay, Rhode Island, USA. Adult eastern oyster, Crassostrea virginica, gut and seawater samples were collected at 5 sites along this estuarine nutrient gradient in August 2017. Samples were analyzed by 16S rRNA gene sequencing to characterize bacterial community structures and metatranscriptomes were sequenced to determine oyster gut microbiome responses to local environments. Results There were significant differences in bacterial community structure between the eastern oyster gut and water samples, suggesting selection of certain taxa by the oyster host. Increasing salinity, pH, and dissolved oxygen, and decreasing nitrate, nitrite and phosphate concentrations were observed along the North to South gradient. Transcriptionally active bacterial taxa were similar for the different sites, but expression of oyster-associated microbial genes involved in nutrient (nitrogen and phosphorus) cycling varied throughout the Bay, reflecting the local nutrient regimes and prevailing environmental conditions. Conclusions The observed shifts in microbial community composition and function inform how estuarine conditions affect host-associated microbiomes and their ecosystem services. As the effects of estuarine acidification are expected to increase due to the combined effects of eutrophication, coastal pollution, and climate change, it is important to determine relationships between host health, microbial community structure, and environmental conditions in benthic communities.

Larval oysters in hatcheries are susceptible to diseases caused by bacterial pathogens, including Vibrio spp. Previous studies have shown that daily addition of the probiotic Bacillus pumilus RI06-95 to water in rearing tanks increases larval survival when challenged with the pathogen Vibrio coralliilyticus. We propose that the presence of probiotics causes shifts in bacterial community structure in rearing tanks, leading to a net decrease in the relative abundance of potential pathogens. During three trials spanning the 2012-2015 hatchery seasons, larvae, tank biofilm, and rearing water samples were collected from control and probiotic-treated tanks in an oyster hatchery over a 12-day period after spawning. Samples were analyzed by 16S rRNA sequencing of the V4 or V6 regions followed by taxonomic classification, in order to determine bacterial community structures. There were significant differences in bacterial composition over time and between sample types, but no major effect of probiotics on the structure and diversity of bacterial communities (phylum level, Bray-Curtis k = 2, 95% confidence). Probiotic treatment, however, led to a higher relative percent abundance of Oceanospirillales and Bacillus spp. in water and oyster larvae. In the water, an increase in Vibrio spp. diversity in the absence of a net increase in relative read abundance suggests a likely decrease in the abundance of specific pathogenic Vibrio spp., and therefore lower chances of a disease outbreak. Co-occurrence network analysis also suggests that probiotic treatment had a systemic effect on targeted members of the bacterial community, leading to a net decrease in potentially pathogenic species.

Thalassobius sp. I31.1 is a putative pathogen involved in epizootic shell disease in the American lobster (Homarus americanus). We report here the draft genome sequence for Thalassobius sp. I31.1 and provide insight into its metabolism and links to environmental pollutant degradation.

Bacterial pathogens are a major cause of mortality in bivalve hatcheries, and outbreaks can result in shortages of seed supply to the grow-out industry. The use of probiotic bacteria is a potential preventative measure to limit the impact of bacterial diseases. Previous research showed that the marine bacteria Phaeobacter inhibens S4 (S4) and Bacillus pumilus RI06—95 (RI) protect larval eastern oysters (Crassostrea virginica) when challenged with the pathogens Vibrio tubiashii RE22 (now Vibrio coralliilyticus RE22) and Roseovarius crassostreae CV919-312T. In this study, these probiotic bacteria were tested under hatchery conditions. Daily addition of S4 and RI (104 colony forming units (CFU)/ml) to 100-l culture tanks resulted in a significant decrease in the levels of total Vibrios in water and tank surfaces (P < 0.05), but not in oysters. Larval growth and survival was unaffected by the probiotic treatments. Larvae treated with probiotics in the hatchery showed significantly less mortality than...

Hatcheries providing seed for bivalve mollusc aquaculture can suffer from disease outbreaks resulting in high losses of larvae. Previous research demonstrated the effectiveness of candidate probiotics Phaeobacter inhibens S4 (S4) and Bacillus pumilus RI06-95 (RI) in protecting the larvae of eastern oysters Crassostrea virginica, against bacterial challenge. In this study, the ability of this probionts in protecting larvae of other bivalve species, including northern quahog Mercenaria mercenaria, bay scallops Argopecten irradians, blue mussels Mytilus edulis, and razor clams Ensis directus, was investigated. Pretreatment of larvae with 106 colony forming units /ml probiotic S4 and a mixture of S4 and RI protected bay scallop larvae [relative percent survival (RPS): 69% ± 4%]. In pilot-scale hatchery trials, daily additions of candidate probiotics to the water of static tanks containing northern quahog or bay scallop larvae had no significant impact on larval growth and survival. Daily treatment of...

As demand for fish meal as a primary protein source in aquaculture feeds has continued to increase, aquaculturists have sought a replacement with similar nutritional profile and more consistent economic value. Two feeding trials were designed to evaluate the effect of replacing fish meal with soybean meal and soy protein concentrate in summer flounder ( Paralychthis dentatus ) diets on fish growth and survival to challenge with the pathogenic bacterium Vibrio harveyi . Fish fed for 12 weeks with a diet in which 60% of the fish meal was replaced with a 1:1 ratio ( w / w ) of soybean meal and soy protein concentrate (SBM/SPC) increased to a significantly greater mass than fish fed either a fish meal (FM) diet or a 60% replacement diet with soybean meal (SBM; p Statement of relevance Sustainable plant-based diets promoting disease resistance.

Several Vibrio spp. cause acute and severe mortality events in hatcheries where larvae of bivalve mollusks are reared, potentially leading to subsequent shortage of bivalve seed for the grow-out industry. In particular, strains of Vibrio coralliilyticus have been identified as a major cause of disease in Pacific, Crassostrea gigas, and eastern, C. virginica, oyster hatcheries in the United States of America. Probiotic bacteria are an inexpensive, practical, and natural method of disease control. Previous research shows that pretreatment of larval oysters with probiotic bacteria Bacillus pumilus RI06&ndash, 95 (RI) and Phaeobacter inhibens S4 (S4) significantly decreases mortality caused by experimental challenge with the bacterial pathogen V. coralliilyticus RE22 (RE22). This study aims to characterize the immune response of 6&ndash, 10-day-old eastern oyster larvae to experimental challenge with pathogen V. coralliilyticus RE22 and probionts RI and S4. Treatments included (a) pathogen and probiont exposure at a concentration of 5 &times, 104 CFU per mL (~2500 bacterial cells per larva) for a duration of 6 h, (b) probiont exposure at the same concentration for a duration of 24 h, and (c) probiont RI daily treatment of larvae in the hatchery for 4, 11, and 15 days. Differential gene expression analysis compared pathogen or probiotic-treated transcriptomes to unexposed controls. Probiotic and pathogen treatment led to upregulation of transcripts coding for several immune pattern recognition receptors (PRRs) involved in environmental sensing and detection of microbes in oyster larvae. Larval oyster responses to pathogen RE22 suggested suppression of expression of genes in immune signaling pathways (myd88, tak1, nkap), failure in upregulation of immune effector genes, high metabolic demand, and oxidative stress that potentially contributed to mortality. On the other hand, the transcriptomic response to probiotic bacteria RI and S4 suggested activation of immune signaling pathways and expression of immune effectors (e.g., Cv-spi2, mucins and perforin-2). These key features of the host immune response to probiotic bacteria were shared despite the length of probiotic exposure, probiotic species, and the type of environment in which exposures were conducted. This study suggests that pre-exposure of eastern oyster larvae to probiotics for 6&ndash, 24 h prior to pathogenic challenge leads to a robust and effective immune response that may contribute to protecting larvae from subsequent challenge with V. coralliilyticus RE22. This research provides new insights into host-microbe interactions in larval oysters that could be applied in the management of vibriosis in bivalve hatcheries.

Bivalves, from raw oysters to steamed clams, are popular choices among seafood lovers and once limited to the coastal areas. The rapid growth of the aquaculture industry and improvement in the preservation and transport of seafood have enabled them to be readily available anywhere in the world. Over the years, oysters, mussels, scallops, and clams have been the focus of research for improving the production, managing resources, and investigating basic biological and ecological questions. During this decade, an impressive amount of information using high-throughput genomic, transcriptomic and proteomic technologies has been produced in various classes of the Mollusca group, and it is anticipated that basic and applied research will significantly benefit from this resource. One aspect that is also taking momentum is the use of bivalves as a model system for human health. In this review, we highlight some of the aspects of the biology of bivalves that have direct implications in human health including the shell formation, stem cells and cell differentiation, the ability to fight opportunistic and specific pathogens in the absence of adaptive immunity, as source of alternative drugs, mucosal immunity and, microbiome turnover, toxicology, and cancer research. There is still a long way to go; however, the next time you order a dozen oysters at your favorite raw bar, think about a tasty model organism that will not only please your palate but also help unlock multiple aspects of molluscan biology and improve human health.

Loktanella maritima strain YPC211 was isolated from the American lobster ( Homarus americanus ). We report here the draft genome sequence for L. maritima YPC211 and identify genes of potential importance to its role within the microbial community.

Aquimarina sp. strain I32.4 (formerly Aquimarina sp. ‘ homaria ’) is a putative pathogen involved in epizootic shell disease in the American lobster ( Homarus americanus ). We report here the draft genome sequence for Aquimarina sp. strain I32.4 and describe virulence factors that may provide insight into its mechanism of pathogenicity.

Bowmanella denitrificans strain JL63 was isolated from a whiteleg shrimp ( Litopenaeus vannamei ) and was determined to have antibacterial activity against an acute hepatopancreatic necrosis disease (AHPND) strain of Vibrio parahaemolyticus . Here, we report the draft genome sequence of this strain and identify genes that are potentially involved in its antibacterial activity.

116 pages, 20 figures, The study of molluscan immune systems, in particular those of bivalve molluscs (e.g., clams, oysters, scallops, mussels), has experienced great growth in recent decades, mainly due to the needs of a rapidly growing aquaculture industry to manage the impacts of disease and the wider application of -omic tools to this diverse group of invertebrate organisms. Several unique aspects of molluscan immune systems highlighted in this chapter include the importance of feeding behavior and mucosal immunity, the discovery of unique levels of diversity in immune genes, and experimental indication of transgenerational immune priming. The development of comparative functional studies using natural and selectively bred disease-resistant strains, together with the potential but yet to be fully developed application of gene-editing technologies, should provide exciting insights into the functional relevance of immune gene family expansion and molecular diversification in bivalves. Other areas of bivalve immunity that deserve further study include elucidation of the process of hematopoiesis, the full characterization of hemocyte subpopulations, and the genetic and molecular mechanisms underlying immune priming. While the most important aspects of the immune system of the largest group of molluscs, gastropods (e.g., snails and slugs), are discussed in detail in Chap. 12, we also briefly outline the most distinctive features of the immune system of another fascinating group of marine molluscs, cephalopods, which include invertebrate animals with extraordinary morphological and behavioral complexity, AF, BN, and RM acknowledge support from the projects AGL2015-65705-R (Ministerio de Economía y Competitividad, Spain) and IN607B 2016/12 (Consellería de Economía, Emprego e Industria (GAIN), Xunta de Galicia). AF, BN, RM, MG, PV, and AP acknowledge support from the project VIVALDI (678589) (EU H2020). MG and AP acknowledge support from the FRA2015 funding program from the University of Trieste. GRV acknowledges support from grants IOS-1656720, IOS-1050518, IOB-0618409, MCB-0077928, and IOS-0822257 from the National Science Foundation, and grant R01GM070589 from the National Institutes of Health, USA. MGC acknowledges support from USDA AFRI grants 2015-67016-22942 and 2016-67016-24905. KR is supported through a scholarship of the Italian Ministry of Foreign Affairs (MAECI), “Entity and diversity of parasite load and his effects on the reproductive status and growth in cephalopod mollusks.” GP is supported by a RITMARE Flagship project (MIUR and Stazione Zoologica Anton Dohrn – SZN)

Prophylactic antibiotics are widespread in the aquaculture industry and are used where vaccination is impossible or overly expensive. If antibiotics impact fish as they do mice and humans, prophylactic administrations in aquaculture and ornamental fish farms may increase downstream disease susceptibility in target hosts, despite short-term pathogen control benefits. Recent research has suggested that their use exacerbates bacterial outbreaks by creating sterile, nutrient-rich environments for invading pathogens to colonize and could help to explain rising economic costs of bacterial outbreaks in aquaculture. Our findings suggest a long-term cost of prophylactic antibiotic use and demonstrate a probiotic-based solution that does not rely on full microbiome community transplantation., Prophylactic antibiotics in the aquaculture and ornamental fish industry are intended to prevent the negative impacts of disease outbreaks. Research in mice and humans suggests that antibiotics may disturb microbiome communities and decrease microbiome-mediated disease resistance, also known as “colonization resistance.” If antibiotics impact fish as they do mice and humans, prophylactic administrations on aquaculture farms may increase downstream disease susceptibility in target hosts, despite short-term pathogen control benefits. We tested the effects of antibiotics on mortality after a pathogen challenge in the Poecilia sphenops black molly and subsequently tested if probiotic inoculations could reverse any antibiotic-induced losses of disease resistance. We found that antibiotic treatment significantly increased fish mortality. We further found that our two candidate probiotic bacterial species, Phaeobacter inhibens S4Sm and Bacillus pumilus RI06-95Sm, were able to colonize black molly microbiomes and reverse the negative impacts of antibiotics. Despite the positive impact on survival, probiotic treatment did not influence overall microbiome community structure or diversity. Our results suggest that subtle manipulations of microbiome composition can have dramatic impacts on host phenotype. The results of this study have implications for how antibiotic-treated microbiomes can be restored and suggest that small-scale additions may be as effective as wholesale transplants. IMPORTANCE Prophylactic antibiotics are widespread in the aquaculture industry and are used where vaccination is impossible or overly expensive. If antibiotics impact fish as they do mice and humans, prophylactic administrations in aquaculture and ornamental fish farms may increase downstream disease susceptibility in target hosts, despite short-term pathogen control benefits. Recent research has suggested that their use exacerbates bacterial outbreaks by creating sterile, nutrient-rich environments for invading pathogens to colonize and could help to explain rising economic costs of bacterial outbreaks in aquaculture. Our findings suggest a long-term cost of prophylactic antibiotic use and demonstrate a probiotic-based solution that does not rely on full microbiome community transplantation.

Advancing the production efficiency and profitability of aquaculture is dependent upon the ability to utilize a diverse array of genetic resources. The ultimate goals of aquaculture genomics, genetics and breeding research are to enhance aquaculture production efficiency, sustainability, product quality, and profitability in support of the commercial sector and for the benefit of consumers. In order to achieve these goals, it is important to understand the genomic structure and organization of aquaculture species, and their genomic and phenomic variations, as well as the genetic basis of traits and their interrelationships. In addition, it is also important to understand the mechanisms of regulation and evolutionary conservation at the levels of genome, transcriptome, proteome, epigenome, and systems biology. With genomic information and information between the genomes and phenomes, technologies for marker/causal mutation-assisted selection, genome selection, and genome editing can be developed for applications in aquaculture. A set of genomic tools and resources must be made available including reference genome sequences and their annotations (including coding and non-coding regulatory elements), genome-wide polymorphic markers, efficient genotyping platforms, high-density and high-resolution linkage maps, and transcriptome resources including non-coding transcripts. Genomic and genetic control of important performance and production traits, such as disease resistance, feed conversion efficiency, growth rate, processing yield, behaviour, reproductive characteristics, and tolerance to environmental stressors like low dissolved oxygen, high or low water temperature and salinity, must be understood. QTL need to be identified, validated across strains, lines and populations, and their mechanisms of control understood. Causal gene(s) need to be identified. Genetic and epigenetic regulation of important aquaculture traits need to be determined, and technologies for marker-assisted selection, causal gene/mutation-assisted selection, genome selection, and genome editing using CRISPR and other technologies must be developed, demonstrated with applicability, and application to aquaculture industries.Major progress has been made in aquaculture genomics for dozens of fish and shellfish species including the development of genetic linkage maps, physical maps, microarrays, single nucleotide polymorphism (SNP) arrays, transcriptome databases and various stages of genome reference sequences. This paper provides a general review of the current status, challenges and future research needs of aquaculture genomics, genetics, and breeding, with a focus on major aquaculture species in the United States: catfish, rainbow trout, Atlantic salmon, tilapia, striped bass, oysters, and shrimp. While the overall research priorities and the practical goals are similar across various aquaculture species, the current status in each species should dictate the next priority areas within the species. This paper is an output of the USDA Workshop for Aquaculture Genomics, Genetics, and Breeding held in late March 2016 in Auburn, Alabama, with participants from all parts of the United States.

As keystone species, sea stars serve to maintain biodiversity and species distribution through trophic level interactions in marine ecosystems. Recently, Sea Star Wasting Disease (SSWD) has caused widespread mass mortality in several sea star species from the Pacific Coast of the United States of America (USA) and Asterias forbesi on the Atlantic Coast. A densovirus, named Sea Star associated Densovirus (SSaDV), has been associated with the wasting disease in Pacific Coast sea stars, and limited samples of A. forbesi. The goal of this research is to examine the pathogenesis of SSWD in A. forbesi on the Atlantic Coast of the USA and to determine if SSaDV is associated with the wasting disease in this species. Histological examination of A. forbesi tissues affected with SSWD showed cuticle loss, vacuolation and necrosis of epidermal cells, and oedema of the dermis, but no consistent evidence indicating the cause of the lesions. Challenge experiments by cohabitation and immersion in infected water suggest that the cause of SSWD is viral in nature, as filtration (0.22 μm) of water from tanks with sea stars exhibiting SSWD did not prevent the transmission and progression of the disease. Death of challenged sea stars occurred 7-10 d after exposure to infected water or sea stars, and the infectivity crossed species (A. forbesi and Pateria miniata) with equal penetrance. Of the 48 stars tested by quantitative real time PCR, 29 (60%) were positive for the SSaDV VP1 gene. These stars represent field-collected sea stars from all geographical regions (South Carolina to Maine) in 2012-2015, as well as stars exposed to infected stars or water from affected tanks. However, a clear association between the presence of SSaDV and SSWD signs in experimental and field-collected A. forbesi was not found in this study.

Vibrio tubiashii subsp. europaeus is a bivalve pathogen isolated during episodes of mortality affecting larval cultures in different shellfish hatcheries. Here, we announce the draft genome sequence of the type strain PP-638 and describe potential virulence factors, which may provide insight into the mechanism of pathogenicity.

Aliiroseovarius crassostreae CV919-312 is a marine alphaproteobacterium and the causative agent of Roseovarius oyster disease. We announce here the draft genome sequence of A. crassostreae CV919-312 and identify potential virulence genes involved in pathogenicity.

Vibrio bivalvicida is a novel pathogen of bivalve larvae responsible for recent vibriosis outbreaks affecting shellfish hatcheries. Here, we announce the draft genome sequence of V. bivalvicida 605 T and describe potential virulence factors.

Comparative genomics research in non-model species has highlighted how invertebrate hosts possess complex diversified repertoires of immune molecules. The levels of diversification in particular immune gene families appear to differ between invertebrate lineages and even between species within lineages, reflecting differences not only in evolutionary histories, but also in life histories, environmental niches, and pathogen exposures. The goal of this research was to identify immune-related gene families experiencing high levels of diversification in eastern oysters, Crassostrea virginica. Families containing 1) transcripts differentially expressed in eastern oysters in response to bacterial challenge and 2) a larger number of transcripts compared to other species included those coding for the C1q and C-type lectin domain containing proteins (C1qDC and CTLDC), GTPase of the immune-associated proteins (GIMAP), scavenger receptors (SR), fibrinogen-C domain containing proteins (also known as FREPs), dopamine beta-hydrolase (DBH), interferon-inducible 44 (IFI44), serine protease inhibitors, apextrin, and dermatopontin. Phylogenetic analysis of two of the families significantly expanded in bivalves, IFI44 and GIMAP, showed a patchy distribution within both protostomes and deuterostomes, suggesting multiple independent losses and lineage-specific expansions. Increased availability of genomic information for a broader range of non-model species broadly distributed through vertebrate and invertebrate phyla will likely lead to improved knowledge on mechanisms of immune-gene diversification.

Background The probiotic bacterium Phaeobacter inhibens strain S4Sm, isolated from the inner shell surface of a healthy oyster, secretes the antibiotic tropodithietic acid (TDA), is an excellent biofilm former, and increases oyster larvae survival when challenged with bacterial pathogens. In this study, we investigated the specific roles of TDA secretion and biofilm formation in the probiotic activity of S4Sm. Results Mutations in clpX (ATP-dependent ATPase) and exoP (an exopolysaccharide biosynthesis gene) were created by insertional mutagenesis using homologous recombination. Mutation of clpX resulted in the loss of TDA production, no decline in biofilm formation, and loss of the ability to inhibit the growth of Vibrio tubiashii and Vibrio anguillarum in co-colonization experiments. Mutation of exoP resulted in a ~60 % decline in biofilm formation, no decline in TDA production, and delayed inhibitory activity towards Vibrio pathogens in co-colonization experiments. Both clpX and exoP mutants exhibited reduced ability to protect oyster larvae from death when challenged by Vibrio tubiashii. Complementation of the clpX and exoP mutations restored the wild type phenotype. We also found that pre-colonization of surfaces by S4Sm was critical for this bacterium to inhibit pathogen colonization and growth. Conclusions Our observations demonstrate that probiotic activity by P. inhibens S4Sm involves contributions from both biofilm formation and the production of the antibiotic TDA. Further, probiotic activity also requires colonization of surfaces by S4Sm prior to the introduction of the pathogen. Electronic supplementary material The online version of this article (doi:10.1186/s12866-015-0617-z) contains supplementary material, which is available to authorized users.

Vibriosis represents the main bottleneck for the larval production process in shellfish aquaculture. While the signs of this disease in bivalve larvae are well known, the infection process by pathogenic Vibrio spp. during episodes of vibriosis has not been elucidated. To investigate the infection process in bivalves, the pathogens of larvae as V. tubiashii subsp. europaensis, V. neptunius and V. bivalvicida were tagged with green fluorescent protein (GFP). Larvae of Manila clam (Ruditapes philippinarum) were inoculated with the GFP-labeled pathogens in different infection assays and monitored by microscopy. Manila clam larvae infected by distinct GFP-tagged Vibrio spp. in different challenges showed the same progression in the infection process, defining three infection stages. GFP-tagged Vibrio spp. were filtered by the larvae through the vellum and entered in the digestive system through the esophagus and stomach and colonized the digestive gland and particularly the intestine, where they proliferated during the first 2h of contact (Stage I), suggesting a chemotactic response. Then, GFP-tagged Vibrio spp. expanded rapidly to the surrounding organs in the body cavity from the dorsal to ventral region (Stage II; 6-8h), colonizing the larvae completely at the peak of infection (Stage III) (14-24h). Results demonstrated for the first time that the vibriosis is asymptomatic in Manila clam larvae during the early infection stages. Thus, the early colonization and the rapid proliferation of Vibrio pathogens within the body cavity supported the sudden and fatal effect of the vibriosis, since the larvae exhibited the first signs of disease when the infection process is advanced. As a first step in the elucidation of the potential mechanisms of bacterial pathogenesis in bivalve larvae the enzymatic activities of the extracellular products released from the wild type V. neptunius, V. tubiashii subsp. europaensis and V. bivalvicida were determined and their cytotoxicity was demonstrated in fish and homeothermic cell lines for the first time. That activity was lost after heat treatment.

Bacillus pumilus RI06-95 is a marine bacterium isolated in Narragansett, Rhode Island, which has shown probiotic activity against marine pathogens in larval shellfish. We report the genome of B. pumilus RI06-95, which provides insight into the microbe's probiotic ability and may be used in future studies of the probiotic mechanism.

The American oyster Crassostrea virginica, an ecologically and economically important estuarine organism, can suffer high mortalities in areas in the Northeast United States due to Roseovarius Oyster Disease (ROD), caused by the gram-negative bacterial pathogen Roseovarius crassostreae. The goals of this research were to provide insights into: 1) the responses of American oysters to R. crassostreae, and 2) potential mechanisms of resistance or susceptibility to ROD. The responses of oysters to bacterial challenge were characterized by exposing oysters from ROD-resistant and susceptible families to R. crassostreae, followed by high-throughput sequencing of cDNA samples from various timepoints after disease challenge. Sequence data was assembled into a reference transcriptome and analyzed through differential gene expression and functional enrichment to uncover genes and processes potentially involved in responses to ROD in the American oyster. While susceptible oysters experienced constant levels of mortality when challenged with R. crassostreae, resistant oysters showed levels of mortality similar to non-challenged oysters. Oysters exposed to R. crassostreae showed differential expression of transcripts involved in immune recognition, signaling, protease inhibition, detoxification, and apoptosis. Transcripts involved in metabolism were enriched in susceptible oysters, suggesting that bacterial infection places a large metabolic demand on these oysters. Transcripts differentially expressed in resistant oysters in response to infection included the immune modulators IL-17 and arginase, as well as several genes involved in extracellular matrix remodeling. The identification of potential genes and processes responsible for defense against R. crassostreae in the American oyster provides insights into potential mechanisms of disease resistance.

Advancing the production efficiency and profitability of aquaculture is dependent upon the ability to utilize a diverse array of genetic resources. The ultimate goals of aquaculture genomics, genetics and breeding research are to enhance aquaculture production efficiency, sustainability, product quality, and profitability in support of the commercial sector and for the benefit of consumers. In order to achieve these goals, it is important to understand the genomic structure and organization of aquaculture species, and their genomic and phenomic variations, as well as the genetic basis of traits and their interrelationships. In addition, it is also important to understand the mechanisms of regulation and evolutionary conservation at the levels of genome, transcriptome, proteome, epigenome, and systems biology. With genomic information and information between the genomes and phenomes, technologies for marker/causal mutation-assisted selection, genome selection, and genome editing can be developed for applications in aquaculture. A set of genomic tools and resources must be made available including reference genome sequences and their annotations (including coding and non-coding regulatory elements), genome-wide polymorphic markers, efficient genotyping platforms, high-density and high-resolution linkage maps, and transcriptome resources including non-coding transcripts. Genomic and genetic control of important performance and production traits, such as disease resistance, feed conversion efficiency, growth rate, processing yield, behaviour, reproductive characteristics, and tolerance to environmental stressors like low dissolved oxygen, high or low water temperature and salinity, must be understood. QTL need to be identified, validated across strains, lines and populations, and their mechanisms of control understood. Causal gene(s) need to be identified. Genetic and epigenetic regulation of important aquaculture traits need to be determined, and technologies for marker-assisted selection, causal gene/mutation-assisted selection, genome selection, and genome editing using CRISPR and other technologies must be developed, demonstrated with applicability, and application to aquaculture industries. Major progress has been made in aquaculture genomics for dozens of fish and shellfish species including the development of genetic linkage maps, physical maps, microarrays, single nucleotide polymorphism (SNP) arrays, transcriptome databases and various stages of genome reference sequences. This paper provides a general review of the current status, challenges and future research needs of aquaculture genomics, genetics, and breeding, with a focus on major aquaculture species in the United States: catfish, rainbow trout, Atlantic salmon, tilapia, striped bass, oysters, and shrimp. While the overall research priorities and the practical goals are similar across various aquaculture species, the current status in each species should dictate the next priority areas within the species. This paper is an output of the USDA Workshop for Aquaculture Genomics, Genetics, and Breeding held in late March 2016 in Auburn, Alabama, with participants from all parts of the United States.