A second unveiling: Haplotig masking of the eastern oyster genome improves population-level inference.

Genome assembly can be challenging for species that are characterized by high amounts of polymorphism, heterozygosity, and large effective population sizes. High levels of heterozygosity can result in genome mis-assemblies and a larger than expected genome size due to the haplotig versions of a single locus being assembled as separate loci. Here, we describe the first chromosome-level genome for the eastern oyster, Crassostrea virginica. Publicly released and annotated in 2017, the assembly has a scaffold N50 of 54 mb and is over 97.3% complete based on BUSCO analysis. The genome assembly for the eastern oyster is a critical resource for foundational research into molluscan adaptation to a changing environment and for selective breeding for the aquaculture industry. Subsequent resequencing data suggested the presence of haplotigs in the original assembly, and we developed a post hoc method to break up chimeric contigs and mask haplotigs in published heterozygous genomes and evaluated improvements to the accuracy of downstream analysis. Masking haplotigs had a large impact on SNP discovery and estimates of nucleotide diversity and had more subtle and nuanced effects on estimates of heterozygosity, population structure analysis, and outlier detection. We show that haplotig masking can be a powerful tool for improving genomic inference, and we present an open, reproducible resource for the masking of haplotigs in any published genome.

Draft Genome Sequence of Vibrio parahaemolyticus PSU5579, Isolated during an Outbreak of Acute Hepatopancreatic Necrosis Disease in Thailand.

Here, we announce the draft genome sequence of Vibrio parahaemolyticus strain PSU5579, isolated from a shrimp hatchery in southern Thailand during an outbreak of acute hepatopancreatic necrosis disease (AHPND). The genome contains 44 contigs with a sequence length of 5,229,426 bp, 4,861 coding sequences, and a G+C content of 45.3%.

Development and Evaluation of High-Density SNP Arrays for the Eastern Oyster Crassostrea virginica.

The eastern oyster Crassostrea virginica is a major aquaculture species for the USA. The sustainable development of eastern oyster aquaculture depends upon the continued improvement of cultured stocks through advanced breeding technologies. The Eastern Oyster Breeding Consortium (EOBC) was formed to advance the genetics and breeding of the eastern oyster. To facilitate efficient genotyping needed for genomic studies and selection, the consortium developed two single-nucleotide polymorphism (SNP) arrays for the eastern oyster: one screening array with 566K SNPs and one breeders' array with 66K SNPs. The 566K screening array was developed based on whole-genome resequencing data from 292 oysters from Atlantic and Gulf of Mexico populations; it contains 566,262 SNPs including 47K from protein-coding genes with a marker conversion rate of 48.34%. The 66K array was developed using best-performing SNPs from the screening array, which contained 65,893 oyster SNPs including 22,984 genic markers with a calling rate of 99.34%, a concordance rate of 99.81%, and a much-improved marker conversion rate of 92.04%. Null alleles attributable to large indels were found in 13.1% of the SNPs, suggesting that copy number variation is pervasive. Both arrays provided easy identification and separation of selected stocks from wild progenitor populations. The arrays contain 31 mitochondrial SNPs that allowed unambiguous identification of Gulf mitochondrial genotypes in some Atlantic populations. The arrays also contain 756 probes from 13 oyster and human pathogens for possible detection. Our results show that marker conversion rate is low in high polymorphism species and that the two-step process of array development can greatly improve array performance. The two arrays will advance genomic research and accelerate genetic improvement of the eastern oyster by delineating genetic architecture of production traits and enabling genomic selection. The arrays also may be used to monitor pedigree and inbreeding, identify selected stocks and their introgression into wild populations, and assess the success of oyster restoration.

Development of a multiplex qPCR for the quantification of three protozoan parasites of the eastern oyster Crassostrea virginica.

A multiplex quantitative PCR (qPCR) assay for the simultaneous detection of 3 eastern oyster Crassostrea virginica parasites, Perkinsus marinus, Haplosporidium nelsoni, and H. costale, was developed using 3 different fluorescently labeled hydrolysis probes. The primers and probe from a previously validated singleplex qPCR for P. marinus detection were combined with newly designed primers and probes specific for H. nelsoni and H. costale. The functionality of the multiplex assay was demonstrated on 2 different platforms by the linear relationship of the standard curves and similar cycle threshold (CT) values between parasites. Efficiency of the multiplex qPCR assay on the Roche and BioRad platforms ranged between 93 and 101%. The sensitivity of detection ranged between 10 and 100 copies of plasmid DNA for P. marinus and Haplosporidium spp., respectively. The concordance between the Roche and BioRad platforms in the identification of the parasites P. marinus, H. nelsoni, and H. costale was 91, 97, and 97%, respectively, with a 10-fold increase in the sensitivity of detection of Haplosporidium spp. on the BioRad thermocycler. The concordance between multiplex qPCR and histology for P. marinus, H. nelsoni, and H. costale was 54, 57, and 87%, respectively. Discordances between detection methods were largely related to localized or low levels of infections in oyster tissues, and qPCR was the more sensitive diagnostic. The multiplex qPCR developed here is a sensitive diagnostic tool for the quantification and surveillance of single and mixed infections in the eastern oyster.

A Review of Asteroid Biology in the Context of Sea Star Wasting: Possible Causes and Consequences.

AbstractSea star wasting-marked in a variety of sea star species as varying degrees of skin lesions followed by disintegration-recently caused one of the largest marine die-offs ever recorded on the west coast of North America, killing billions of sea stars. Despite the important ramifications this mortality had for coastal benthic ecosystems, such as increased abundance of prey, little is known about the causes of the disease or the mechanisms of its progression. Although there have been studies indicating a range of causal mechanisms, including viruses and environmental effects, the broad spatial and depth range of affected populations leaves many questions remaining about either infectious or non-infectious mechanisms. Wasting appears to start with degradation of mutable connective tissue in the body wall, leading to disintegration of the epidermis. Here, we briefly review basic sea star biology in the context of sea star wasting and present our current knowledge and hypotheses related to the symptoms, the microbiome, the viruses, and the associated environmental stressors. We also highlight throughout the article knowledge gaps and the data needed to better understand sea star wasting mechanistically, its causes, and potential management.

The expanded inhibitor of apoptosis gene family in oysters possesses novel domain architectures and may play diverse roles in apoptosis following immune challenge.

BACKGROUND: Apoptosis plays important roles in a variety of functions, including immunity and response to environmental stress. The Inhibitor of Apoptosis (IAP) gene family of apoptosis regulators is expanded in molluscs, including eastern, Crassostrea virginica, and Pacific, Crassostrea gigas, oysters. The functional importance of IAP expansion in apoptosis and immunity in oysters remains unknown. RESULTS: Phylogenetic analysis of IAP genes in 10 molluscs identified lineage specific gene expansion in bivalve species. Greater IAP gene family expansion was observed in C. virginica than C. gigas (69 vs. 40), resulting mainly from tandem duplications. Functional domain analysis of oyster IAP proteins revealed 3 novel Baculoviral IAP Repeat (BIR) domain types and 14 domain architecture types across gene clusters, 4 of which are not present in model organisms. Phylogenetic analysis of bivalve IAPs suggests a complex history of domain loss and gain. Most IAP genes in oysters (76% of C. virginica and 82% of C. gigas), representing all domain architecture types, were expressed in response to immune challenge (Ostreid Herpesvirus OsHV-1, bacterial probionts Phaeobacter inhibens and Bacillus pumilus, several Vibrio spp., pathogenic Aliiroseovarius crassostreae, and protozoan parasite Perkinsus marinus). Patterns of IAP and apoptosis-related differential gene expression differed between the two oyster species, where C. virginica, in general, differentially expressed a unique set of IAP genes in each challenge, while C. gigas differentially expressed an overlapping set of IAP genes across challenges. Apoptosis gene expression patterns clustered mainly by resistance/susceptibility of the oyster host to immune challenge. Weighted Gene Correlation Network Analysis (WGCNA) revealed unique combinations of transcripts for 1 to 12 IAP domain architecture types, including novel types, were significantly co-expressed in response to immune challenge with transcripts in apoptosis-related pathways. CONCLUSIONS: Unprecedented diversity characterized by novel BIR domains and protein domain architectures was observed in oyster IAPs. Complex patterns of gene expression of novel and conserved IAPs in response to a variety of ecologically-relevant immune challenges, combined with evidence of direct co-expression of IAP genes with apoptosis-related transcripts, suggests IAP expansion facilitates complex and nuanced regulation of apoptosis and other immune responses in oysters.

Perkinsus marinus suppresses in vitro eastern oyster apoptosis via IAP-dependent and caspase-independent pathways involving TNFR, NF-kB, and oxidative pathway crosstalk.

The protozoan parasite Perkinsus marinus causes Dermo disease in eastern oysters, Crassostrea virginica, and can suppress apoptosis of infected hemocytes using incompletely understood mechanisms. This study challenged hemocytes in vitro with P. marinus for 1 h in the presence or absence of caspase inhibitor Z-VAD-FMK or Inhibitor of Apoptosis protein (IAP) inhibitor GDC-0152. Hemocytes exposure to P. marinus significantly reduced granulocyte apoptosis, and pre-incubation with Z-VAD-FMK did not affect P. marinus-induced apoptosis suppression. Hemocyte pre-incubation with GDC-0152 prior to P. marinus challenge further reduced apoptosis of granulocytes with engulfed parasite, but not mitochondrial permeabilization. This suggests P. marinus-induced apoptosis suppression may be caspase-independent, affect an IAP-involved pathway, and occur downstream of mitochondrial permeabilization. P. marinus challenge stimulated hemocyte differential expression of oxidation-reduction, TNFR, and NF-kB pathways. WGCNA analysis of P. marinus expression in response to hemocyte exposure revealed correlated protease, kinase, and hydrolase expression that could contribute to P. marinus-induced apoptosis suppression.

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

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.

Extensive genome-wide duplications in the eastern oyster (Crassostrea virginica).

Genomic structural variation is an important source of genetic and phenotypic diversity, playing a critical role in evolution. The recent availability of a high-quality reference genome for the eastern oyster, Crassostrea virginica, and whole-genome sequence data of samples from across the species range in the USA, provides an opportunity to explore structural variation across the genome of this species. Our analysis shows significantly greater individual-level duplications of regions across the genome than that of most model vertebrate species. Duplications are widespread across all ten chromosomes with variation in frequency per chromosome. The eastern oyster shows a large interindividual variation in duplications as well as particular chromosomal regions with a higher density of duplications. A high percentage of duplications seen in C. virginica lie completely within genes and exons, suggesting the potential for impacts on gene function. These results support the hypothesis that structural changes may play a significant role in standing genetic variation in C. virginica, and potentially have a role in their adaptive and evolutionary success. Altogether, these results suggest that copy number variation plays an important role in the genomic variation of C. virginica. This article is part of the Theo Murphy meeting issue 'Molluscan genomics: broad insights and future directions for a neglected phylum'.

Functional plasticity in oyster gut microbiomes along a eutrophication gradient in an urbanized estuary.

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.

Contrasting Immunomodulatory Effects of Probiotic and Pathogenic Bacteria on Eastern Oyster, Crassostrea Virginica, Larvae.

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 RI06-95 (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 6-10-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 × 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 6-24 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.

Bacterial Community Dynamics in an Oyster Hatchery in Response to Probiotic Treatment.

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.

Draft Genome Sequence of the Putative Marine Pathogen Thalassobius sp. I31.1.

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.

From the raw bar to the bench: Bivalves as models for human health.

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.

El Niño drives a widespread ulcerative skin disease outbreak in Galapagos marine fishes.

Climate change increases local climatic variation and unpredictability, which can alter ecological interactions and trigger wildlife disease outbreaks. Here we describe an unprecedented multi-species outbreak of wild fish disease driven by a climate perturbation. The 2015-16 El Niño generated a +2.5 °C sea surface temperature anomaly in the Galapagos Islands lasting six months. This coincided with a novel ulcerative skin disease affecting 18 teleost species from 13 different families. Disease signs included scale loss and hemorrhagic ulcerated patches of skin, fin deterioration, lethargy, and erratic behavior. A bacterial culture isolated from skin lesions of two of the affected fish species was identified by sequencing of the 16S rRNA gene as a Rahnella spp. Disease prevalence rates were linearly correlated with density in three fish species. In January 2016, disease prevalence reached 51.1% in the ring-tailed damselfish Stegastes beebei (n = 570) and 18.7% in the king angelfish Holacanthus passer (n = 318), corresponding to 78% and 86% decreases in their populations relative to a 4.5-year baseline, respectively. We hypothesize that this outbreak was precipitated by the persistent warm temperatures and lack of planktonic productivity that characterize extreme El Niño events, which are predicted to increase in frequency with global warming.

Draft Genome Sequence of Loktanella maritima Strain YPC211, a Commensal Bacterium of the American Lobster (Homarus americanus).

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.

Draft Genome Sequence of the Putative Marine Pathogen Aquimarina sp. Strain I32.4.

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.

Draft Genome Sequence of Bowmanella denitrificans JL63, a Bacterium Isolated from Whiteleg Shrimp (Litopenaeus vannamei) That Can Inhibit the Growth of Vibrio parahaemolyticus.

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.

Sea Star Wasting Disease in Asterias forbesi along the Atlantic Coast of North America.

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.

Subtle Microbiome Manipulation Using Probiotics Reduces Antibiotic-Associated Mortality in Fish.

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.