Temperate trees locally engineer decomposition and litter-bound microbiomes through differential litter deposits and species-specific soil conditioning.

Leaf decomposition varies widely across temperate forests, shaped by factors like litter quality, climate, soil properties, and decomposers, but forest heterogeneity may mask local tree influences on decomposition and litter-associated microbiomes. We used a 24-yr-old common garden forest to quantify local soil conditioning impacts on decomposition and litter microbiology. We introduced leaf litter bags from 10 tree species (5 arbuscular mycorrhizal; 5 ectomycorrhizal) to soil plots conditioned by all 10 species in a full-factorial design. After 6 months, we assessed litter mass loss, C/N content, and bacterial and fungal composition. We hypothesized that (1) decomposition and litter-associated microbiome composition would be primarily shaped by the mycorrhizal type of litter-producing trees, but (2) modified significantly by underlying soil, based on mycorrhizal type of the conditioning trees. Decomposition and, to a lesser extent, litter-associated microbiome composition, were primarily influenced by the mycorrhizal type of litter-producing trees. Interestingly, however, underlying soils had a significant secondary influence, driven mainly by tree species, not mycorrhizal type. This secondary influence was strongest under trees from the Pinaceae. Temperate trees can locally influence underlying soil to alter decomposition and litter-associated microbiology. Understanding the strength of this effect will help predict biogeochemical responses to forest compositional change.

Leveraging aquatic-terrestrial interfaces to capture putative habitat generalists.

Habitat type is a strong determinant of microbial composition. Habitat interfaces, such as the boundary between aquatic and terrestrial systems, present unique combinations of abiotic factors for microorganisms to contend with. Aside from the spillover of certain harmful microorganisms from agricultural soils into water (e.g. fecal coliform bacteria), we know little about the extent of soil-water habitat switching across microbial taxa. In this study, we developed a proof-of-concept system to facilitate the capture of putatively generalist microorganisms that can colonize and persist in both soil and river water. We aimed to examine the phylogenetic breadth of putative habitat switchers and how this varies across different source environments. Microbial composition was primarily driven by recipient environment type, with the strongest phylogenetic signal seen at the order level for river water colonizers. We also identified more microorganisms colonizing river water when soil was collected from a habitat interface (i.e. soil at the side of an intermittently flooded river, compared to soil collected further from water sources), suggesting that environmental interfaces could be important reservoirs of microbial habitat generalists. Continued development of experimental systems that actively capture microorganisms that thrive in divergent habitats could serve as a powerful tool for identifying and assessing the ecological distribution of microbial generalists.

Functionally discrete fine roots differ in microbial assembly, microbial functional potential, and produced metabolites.

Traditionally, fine roots were grouped using arbitrary size categories, rarely capturing the heterogeneity in physiology, morphology and functionality among different fine root orders. Fine roots with different functional roles are rarely separated in microbiome-focused studies and may result in confounding microbial signals and host-filtering across different root microbiome compartments. Using a 26-year-old common garden, we sampled fine roots from four temperate tree species that varied in root morphology and sorted them into absorptive and transportive fine roots. The rhizoplane and rhizosphere were characterized using 16S rRNA gene and internal transcribed spacer region amplicon sequencing and shotgun metagenomics for the rhizoplane to identify potential microbial functions. Fine roots were subject to metabolomics to spatially characterize resource availability. Both fungi and bacteria differed according to root functional type. We observed additional differences between the bacterial rhizoplane and rhizosphere compartments for absorptive but not transportive fine roots. Rhizoplane bacteria, as well as the root metabolome and potential microbial functions, differed between absorptive and transportive fine roots, but not the rhizosphere bacteria. Functional differences were driven by sugar transport, peptidases and urea transport. Our data highlights the importance of root function when examining root-microbial relationships, emphasizing different host selective pressures imparted on different root microbiome compartments.

Leveraging microbiome rediversification for the ecological rescue of soil function.

BACKGROUND: Global biodiversity losses threaten ecosystem services and can impact important functional insurance in a changing world. Microbial diversity and function can become depleted in agricultural systems and attempts to rediversify agricultural soils rely on either targeted microbial introductions or retaining natural lands as biodiversity reservoirs. As many soil functions are provided by a combination of microbial taxa, rather than outsized impacts by single taxa, such functions may benefit more from diverse microbiome additions than additions of individual commercial strains. In this study, we measured the impact of soil microbial diversity loss and rediversification (i.e. rescue) on nitrification by quantifying ammonium and nitrate pools. We manipulated microbial assemblages in two distinct soil types, an agricultural and a forest soil, with a dilution-to-extinction approach and performed a microbiome rediversification experiment by re-introducing microorganisms lost from the dilution. A microbiome water control was included to act as a reference point. We assessed disruption and potential restoration of (1) nitrification, (2) bacterial and fungal composition through 16S rRNA gene and fungal ITS amplicon sequencing and (3) functional genes through shotgun metagenomic sequencing on a subset of samples. RESULTS: Disruption of nitrification corresponded with diversity loss, but nitrification was successfully rescued in the rediversification experiment when high diversity inocula were introduced. Bacterial composition clustered into groups based on high and low diversity inocula. Metagenomic data showed that genes responsible for the conversion of nitrite to nitrate and taxa associated with nitrogen metabolism were absent in the low diversity inocula microcosms but were rescued with high diversity introductions. CONCLUSIONS: In contrast to some previous work, our data suggest that soil functions can be rescued by diverse microbiome additions, but that the concentration of the microbial inoculum is important. By understanding how microbial rediversification impacts soil microbiome performance, we can further our toolkit for microbial management in human-controlled systems in order to restore depleted microbial functions.

Shared and unique responses in the microbiome of allopatric lizards reared in a standardized environment.

The gut microbiome can influence host fitness and, consequently, the ecology and evolution of natural populations. Microbiome composition can be driven by environmental exposure but also by the host's genetic background and phenotype. To contrast environmental and genetic effects on the microbiome we leverage preserved specimens of eastern fence lizards from allopatric lineages east and west of the Mississippi River but reared in standardized conditions. Bacterial composition was indistinguishable between lineages but responded significantly to host age-a proxy for environmental exposure. This was accompanied by a continuous decrease in bacterial diversity in both lineages, partially driven by decreasing evenness seen only in western lizards. These findings indicate that longer exposure to a homogeneous habitat may have a depreciating effect on microbiome diversity in eastern fence lizards, a response shared by both lineages. We highlight the importance of such effects when extrapolating patterns from laboratory experiments to the natural world.

Farm-scale differentiation of active microbial colonizers.

Microbial movement is important for replenishing lost soil microbial biodiversity and driving plant root colonization, particularly in managed agricultural soils, where microbial diversity and composition can be disrupted. Despite abundant survey-type microbiome data in soils, which are obscured by legacy DNA and microbial dormancy, we do not know how active microbial pools are shaped by local soil properties, agricultural management, and at differing spatial scales. To determine how active microbial colonizers are shaped by spatial scale and environmental conditions, we deployed microbial traps (i.e. sterile soil enclosed by small pore membranes) containing two distinct soil types (forest; agricultural), in three neighboring locations, assessing colonization through 16S rRNA gene and fungal ITS amplicon sequencing. Location had a greater impact on fungal colonizers (R2 = 0.31 vs. 0.26), while the soil type within the microbial traps influenced bacterial colonizers more (R2 = 0.09 vs. 0.02). Bacterial colonizers showed greater colonization consistency (within-group similarity) among replicate communities. Relative to bacterial colonizers, fungal colonizers shared a greater compositional overlap to sequences from the surrounding local bulk soil (R2 = 0.08 vs. 0.29), suggesting that these groups respond to distinct environmental constraints and that their in-field management may differ. Understanding how environmental constraints and spatial scales impact microbial recolonization dynamics and community assembly are essential for identifying how soil management can be used to shape agricultural microbiomes.

Can dispersal be leveraged to improve microbial inoculant success?

Microorganisms have long been isolated from soils to develop microbial inoculants, with the goal of spiking them into new soils to augment target functions. However, establishment can be sporadic, and we assume that inoculants simply arrive at their destination. Here, we posit a need for integrating dispersal into inoculant development and deployment. We argue that consideration for an inoculant's dispersal ability, whether via active (e.g., chemotaxis) or passive (e.g., attachment to other organisms) means, and including methods of deployment that allow multiple establishment attempts could help increase the predictability of inoculant success. Dispersal can influence many key aspects of in-field survival, including the ability to escape stressors, seek favorable colonization sites, facilitate multiple establishment attempts, and engage in multikingdom interactions.

Soil salinization accelerates microbiome stabilization in iterative selections for plant performance.

Climate change-related soil salinization increases plant stress and decreases productivity. Soil microorganisms are thought to reduce salt stress through multiple mechanisms, so diverse assemblages could improve plant growth under such conditions. Previous studies have shown that microbiome selection can promote desired plant phenotypes, but with high variability. We hypothesized that microbiome selection would be more consistent in saline soils by increasing potential benefits to the plants. In both salt-amended and untreated soils, we transferred forward Brassica rapa root microbiomes (from high-biomass or randomly selected pots) across six planting generations while assessing bacterial (16S rRNA) and fungal (ITS) composition in detail. Uniquely, we included an add-back control (re-adding initial frozen soil microbiome) as a within-generation reference for microbiome and plant phenotype selection. We observed inconsistent effects of microbiome selection on plant biomass across generations, but microbial composition consistently diverged from the add-back control. Although salt amendment strongly impacted microbial composition, it did not increase the predictability of microbiome effects on plant phenotype, but it did increase the rate at which microbiome selection plateaued. These data highlight a disconnect in the trajectories of microbiomes and plant phenotypes during microbiome selection, emphasizing the role of standard controls to explain microbiome selection outcomes.

Phylogenomic diversity of Vibrio species and other Gammaproteobacteria isolated from Pacific oysters (Crassostrea gigas) during a summer mortality outbreak.

The Pacific oyster (PO), Crassostrea gigas, is an important commercial marine species but periodically experiences large stock losses due to disease events known as summer mortality. Summer mortality has been linked to environmental perturbations and numerous viral and bacterial agents, indicating this disease is multifactorial in nature. In 2013 and 2014, several summer mortality events occurred within the Port Stephens estuary (NSW, Australia). Extensive culture and molecular-based investigations were undertaken and several potentially pathogenic Vibrio species were identified. To improve species identification and genomically characterise isolates obtained from this outbreak, whole-genome sequencing (WGS) and subsequent genomic analyses were performed on 48 bacterial isolates, as well as a further nine isolates from other summer mortality studies using the same batch of juveniles. Average nucleotide identity (ANI) identified most isolates to the species level and included members of the Photobacterium, Pseudoalteromonas, Shewanella and Vibrio genera, with Vibrio species making up more than two-thirds of all species identified. Construction of a phylogenomic tree, ANI analysis, and pan-genome analysis of the 57 isolates represents the most comprehensive culture-based phylogenomic survey of Vibrios during a PO summer mortality event in Australian waters and revealed large genomic diversity in many of the identified species. Our analysis revealed limited and inconsistent associations between isolate species and their geographical origins, or host health status. Together with ANI and pan-genome results, these inconsistencies suggest that to determine the role that microbes may have in Pacific oyster summer mortality events, isolate identification must be at the taxonomic level of strain. Our WGS data (specifically, the accessory genomes) differentiated bacterial strains, and coupled with associated metadata, highlight the possibility of predicting a strain's environmental niche and level of pathogenicity.

Pearl Oyster Bacterial Community Structure Is Governed by Location and Tissue-Type, but Vibrio Species Are Shared Among Oyster Tissues.

Diseases of bivalves of aquacultural importance, including the valuable Australian silver-lipped pearl oyster (Pinctada maxima), have been increasing in frequency and severity. The bivalve microbiome is linked to health and disease dynamics, particularly in oysters, with putative pathogens within the Vibrio genus commonly implicated in oyster diseases. Previous studies have been biased toward the Pacific oyster because of its global dominance in oyster aquaculture, while much less is known about the microbiome of P. maxima. We sought to address this knowledge gap by characterizing the P. maxima bacterial community, and we hypothesized that bacterial community composition, and specifically the occurrence of Vibrio, will vary according to the sampled microenvironment. We also predicted that the inside shell swab bacterial composition could represent a source of microbial spillover biofilm into the solid pearl oyster tissues, thus providing a useful predictive sampling environment. We found that there was significant heterogeneity in bacterial composition between different pearl oyster tissues, which is consistent with patterns reported in other bivalve species and supports the hypothesis that each tissue type represents a unique microenvironment for bacterial colonization. We suggest that, based on the strong effect of tissue-type on the pearl oyster bacterial community, future studies should apply caution when attempting to compare microbial patterns from different locations, and when searching for disease agents. The lack of association with water at each farm also supported the unique nature of the microbial communities in oyster tissues. In contrast to the whole bacterial community, there was no significant difference in the Vibrio community among tissue types nor location. These results suggest that Vibrio species are shared among different pearl oyster tissues. In particular, the similarity between the haemolymph, inside shell and solid tissues, suggests that the haemolymph and inside shell environment is a source of microbial spillover into the oyster tissues, and a potentially useful tool for non-destructive routine disease testing and early warning surveillance. These data provide important foundational information for future studies identifying the factors that drive microbial assembly in a valuable aquaculture species.

Occurrence and dynamics of potentially pathogenic vibrios in the wet-dry tropics of northern Australia.

Bacteria from the Vibrio genus are a ubiquitous component of coastal and estuarine ecosystems with several pathogenic Vibrio species displaying preferences for warm tropical waters. We studied the spatial and temporal abundance of three key human potential pathogens V. parahaemolyticus, V. cholerae and V. vulnificus in northern tropical Australia, over the wet and dry seasons, to identify environmental parameters influencing their abundance. Quantitative PCR (qPCR) analysis revealed that V. parahaemolyticus occurred more frequently and in higher abundance than V. cholerae and V. vulnificus across all locations examined. All three species were more abundant during the wet season, with V. parahaemolyticus abundance correlated to temperature and conductivity, whereas nutrient concentrations and turbidity best explained V. vulnificus abundance. In addition to these targeted qPCR analyses, we assessed the composition and dynamics of the entire Vibrio community using hsp60 amplicon sequencing. Using this approach, 42 Vibrio species were identified, including a number of other pathogenic species such as V. alginolyticus, V. mimicus and V. fluvialis. The Vibrio community was more diverse in the wet season, with temperature and dissolved oxygen as the key factors governing community composition. Seasonal differences were primarily driven by a greater abundance of V. parahaemolyticus and V. vulnificus during the wet season, while spatial differences were driven by different abundances of V. harveyi, V. campbellii, V. cholerae and V. navarrensis. When we related the abundance of Vibrio to other bacterial taxa, defined using 16S rRNA gene amplicon sequencing, V. parahaemolyticus was negatively correlated to several taxa, including members of the Rickettsiales and Saccharimonadales, while V. vulnificus was negatively correlated to Rhobacteriaceae and Cyanobiaceae. In contrast, V. alginolyticus, V. harveyi and V. mediterranei were all positively correlated to Cyanobacteria. These observations highlight the dynamic nature of Vibrio communities and expands current understanding of the processes governing the occurrence of potentially pathogenic Vibrio spp. in tropical coastal ecosystems.

Microbiome response differs among selected lines of Sydney rock oysters to ocean warming and acidification.

Oyster microbiomes are integral to healthy function and can be altered by climate change conditions. Genetic variation among oysters is known to influence the response of oysters to climate change and may ameliorate any adverse effects on oyster microbiome; however, this remains unstudied. Nine full-sibling selected breeding lines of the Sydney rock oyster (Saccostrea glomerata) were exposed to predicted warming (ambient = 24°C, elevated = 28°C) and ocean acidification (ambient pCO2 = 400, elevated pCO2 = 1000 µatm) for 4 weeks. The haemolymph bacterial microbiome was characterized using 16S rRNA (V3-V4) gene sequencing and varied among oyster lines in the control (ambient pCO2, 24°C) treatment. Microbiomes were also altered by climate change dependent on oyster lines. Bacterial α-diversity increased in response to elevated pCO2 in two selected lines, while bacterial ß-diversity was significantly altered by combinations of elevated pCO2 and temperature in four selected lines. Climate change treatments caused shifts in the abundance of multiple amplicon sequence variants driving change in the microbiome of some selected lines. We show that oyster genetic background may influence the Sydney rock oyster haemolymph microbiome under climate change and that future assisted evolution breeding programs to enhance resilience should consider the oyster microbiome.

The hierarchy of root branching order determines bacterial composition, microbial carrying capacity and microbial filtering.

Fine roots vary dramatically in their functions, which range from resource absorption to within-plant resource transport. These differences should alter resource availability to root-associated microorganisms, yet most root microbiome studies involve fine root homogenization. We hypothesized that microbial filtering would be greatest in the most distal roots. To test this, we sampled roots of six temperate tree species from a 23-year-old common garden planting, separating by branching order. Rhizoplane bacterial composition was characterized with 16S rRNA gene sequencing, while bacterial abundance was determined on a subset of trees through flow cytometry. Root order strongly impacted composition across tree species, with absorptive lower order roots exerting the greatest selective pressure. Microbial carrying capacity was higher in absorptive roots in two of three tested tree species. This study indicates lower order roots as the main point of microbial interaction with fine roots, suggesting that root homogenization could mask microbial recruitment signatures.

Climate change alters the haemolymph microbiome of oysters.

The wellbeing of marine organisms is connected to their microbiome. Oysters are a vital food source and provide ecological services, yet little is known about how climate change such as ocean acidification and warming will affect their microbiome. We exposed the Sydney rock oyster, Saccostrea glomerata, to orthogonal combinations of temperature (24, 28 °C) and pCO2 (400 and 1000 µatm) for eight weeks and used amplicon sequencing of the 16S rRNA (V3-V4) gene to characterise the bacterial community in haemolymph. Overall, elevated pCO2 and temperature interacted to alter the microbiome of oysters, with a clear partitioning of treatments in CAP ordinations. Elevated pCO2 was the strongest driver of species diversity and richness and elevated temperature also increased species richness. Climate change, both ocean acidification and warming, will alter the microbiome of S. glomerata which may increase the susceptibility of oysters to disease.

Long-lasting effect of mercury contamination on the soil microbiota and its co-selection of antibiotic resistance.

Antibiotic resistance genes (ARGs) in the environment are an exposure risk to humans and animals and is emerging as a global public health concern. In this study, mercury (Hg) driven co-selection of ARGs was investigated under controlled conditions in two Australian non-agricultural soils with differing pH. Soils were spiked with increasing concentrations of inorganic Hg and left to age for 5 years. Both soils contained ARGs conferring resistance to tetracycline (tetA, tetB), sulphonamides (sul1), trimethoprim (dfrA1) and the ARG indicator class 1 integron-integrase gene, intI1, as measured by qPCR. The last resort antibiotic vancomycin resistance gene, vanB and quinolone resistance gene, qnrS were not detected. Hg driven co-selection of several ARGs namely intI1, tetA and tetB were observed in the alkaline soil within the tested Hg concentrations. No co-selection of the experimental ARGs was observed in the neutral pH soil. 16S rRNA sequencing revealed proliferation of Proteobacteria and Bacteriodetes in Hg contaminated neutral and alkaline soils respectively. Multivariate analyses revealed a strong effect of Hg, soil pH and organic carbon content on the co-selection of ARGs in the experimental soils. Additionally, although aging caused a significant reduction in Hg content, agriculturally important bacterial phyla such as Nitrospirae did not regrow in the contaminated soils. The results suggest that mercury can drive co-selection of ARGs in contaminated non-agricultural soils over five years of aging which is linked to soil microbiota shift and metal chemistry in the soil.

Regional and oyster microenvironmental scale heterogeneity in the Pacific oyster bacterial community.

Different organs of a host represent distinct microenvironments resulting in the establishment of multiple discrete bacterial communities within a host. These discrete bacterial communities can also vary according to geographical location. For the Pacific oyster, Crassostrea gigas, the factors governing bacterial diversity and abundance of different oyster microenvironments are poorly understood. In this study, the factors shaping bacterial abundance, diversity and composition associated with the C. gigas mantle, gill, adductor muscle and digestive gland were characterised using 16S (V3-V4) rRNA amplicon sequencing across six discrete estuaries. Both location and tissue-type, with tissue-type being the stronger determinant, were factors driving bacterial community composition. Bacterial communities from wave-dominated estuaries had similar compositions and higher bacterial abundance despite being geographically distant from one another, possibly indicating that functional estuarine morphology characteristics are a factor shaping the oyster bacterial community. Despite the bacterial community heterogeneity, examinations of the core bacterial community identified Spirochaetaceae bacteria as conserved across all sites and samples. Whereas members of the Vulcaniibacterium, Spirochaetaceae and Margulisbacteria, and Polynucleobacter were regionally conserved members of the digestive gland, gill and mantle bacterial communities, respectively. This indicates that baseline bacterial community profiles for specific locations are necessary when investigating bacterial communities in oyster health.

Variability in the Composition of Pacific Oyster Microbiomes Across Oyster Families Exhibiting Different Levels of Susceptibility to OsHV-1 µvar Disease.

Oyster diseases are a major impediment to the profitability and growth of the oyster aquaculture industry. In recent years, geographically widespread outbreaks of disease caused by ostreid herpesvirus-1 microvariant (OsHV-1 µvar) have led to mass mortalities among Crassostrea gigas, the Pacific Oyster. Attempts to minimize the impact of this disease have been largely focused on breeding programs, and although these have shown some success in producing oyster families with reduced mortality, the mechanism(s) behind this protection is poorly understood. One possible factor is modification of the C. gigas microbiome. To explore how breeding for resistance to OsHV-1 µvar affects the oyster microbiome, we used 16S rRNA amplicon sequencing to characterize the bacterial communities associated with 35 C. gigas families, incorporating oysters with different levels of susceptibility to OsHV-1 µvar disease. The microbiomes of disease-susceptible families were significantly different to the microbiomes of disease-resistant families. OTUs assigned to the Photobacterium, Vibrio, Aliivibrio, Streptococcus, and Roseovarius genera were associated with low disease resistance. In partial support of this finding, qPCR identified a statistically significant increase of Vibrio-specific 16S rRNA gene copies in the low disease resistance families, possibly indicative of a reduced host immune response to these pathogens. In addition to these results, examination of the core microbiome revealed that each family possessed a small core community, with OTUs assigned to the Winogradskyella genus and the Bradyrhizobiaceae family consistent members across most disease-resistant families. This study examines patterns in the microbiome of oyster families exhibiting differing levels of OsHV-1 µvar disease resistance and reveals some key bacterial taxa that may provide a protective or detrimental role in OsHV-1 µvar disease outbreaks.

A New High Throughput Sequencing Assay for Characterizing the Diversity of Natural Vibrio Communities and Its Application to a Pacific Oyster Mortality Event.

The Vibrio genus is notable for including several pathogens of marine animals and humans, yet characterization of Vibrio diversity using routine 16S rRNA sequencing methods is often constrained by poor resolution beyond the genus level. Here, a new high throughput sequencing approach targeting the heat shock protein (hsp60) as a phylogenetic marker was developed to more precisely discriminate members of the Vibrio genus in environmental samples. The utility of this new assay was tested using mock communities constructed from known dilutions of Vibrio isolates. Relative to standard and Vibrio-specific 16S rRNA sequencing assays, the hsp60 assay delivered high levels of fidelity with the mock community composition at the species level, including discrimination of species within the Vibrio harveyi clade. This assay was subsequently applied to characterize Vibrio community composition in seawater and delivered substantially improved taxonomic resolution of Vibrio species compared to 16S rRNA analysis. Finally, this assay was applied to examine patterns in the Vibrio community within oysters during a Pacific oyster mortality event. In these oysters, the hsp60 assay identified species-level Vibrio community shifts prior to disease onset, pinpointing V. harveyi as a putative pathogen. Given that shifts in the Vibrio community can precede, cause, and follow disease onset in numerous marine organisms, there is a need for an accurate high throughput assay for defining Vibrio community composition in natural samples. This Vibrio-centric hsp60 sequencing assay offers the potential for precise high throughput characterization of Vibrio diversity, providing an enhanced platform for dissecting Vibrio dynamics in the environment.

Characterisation of the Pacific Oyster Microbiome During a Summer Mortality Event.

The Pacific oyster, Crassostrea gigas, is a key commercial species that is cultivated globally. In recent years, disease outbreaks have heavily impacted C. gigas stocks worldwide, with many losses incurred during summer. A number of infectious agents have been associated with these summer mortality events, including viruses (particularly Ostreid herpesvirus 1, OsHV-1) and bacteria; however, cases where no known aetiological agent can be identified are common. In this study, we examined the microbiome of disease-affected and disease-unaffected C. gigas during a 2013-2014 summer mortality event in Port Stephens (Australia) where known oyster pathogens including OsHV-1 were not detected. The adductor muscle microbiomes of 70 C. gigas samples across 12 study sites in the Port Stephens estuary were characterised using 16S rRNA (V1-V3 region) amplicon sequencing, with the aim of comparing the influence of spatial location and disease state on the oyster microbiome. Spatial location was found to be a significant determinant of the disease-affected oyster microbiome. Furthermore, microbiome comparisons between disease states identified a significant increase in rare operational taxonomic units (OTUs) belonging to Vibrio harveyi and an unidentified member of the Vibrio genus in the disease-affected microbiome. This is indicative of a potential role of Vibrio species in oyster disease and supportive of previous culture-based examination of this mortality event.

Simulated Marine Heat Wave Alters Abundance and Structure of Vibrio Populations Associated with the Pacific Oyster Resulting in a Mass Mortality Event.

Marine heat waves are predicted to become more frequent and intense due to anthropogenically induced climate change, which will impact global production of seafood. Links between rising seawater temperature and disease have been documented for many aquaculture species, including the Pacific oyster Crassostrea gigas. The oyster harbours a diverse microbial community that may act as a source of opportunistic pathogens during temperature stress. We rapidly raised the seawater temperature from 20 °C to 25 °C resulting in an oyster mortality rate of 77.4%. Under the same temperature conditions and with the addition of antibiotics, the mortality rate was only 4.3%, strongly indicating a role for bacteria in temperature-induced mortality. 16S rRNA amplicon sequencing revealed a change in the oyster microbiome when the temperature was increased to 25 °C, with a notable increase in the proportion of Vibrio sequences. This pattern was confirmed by qPCR, which revealed heat stress increased the abundance of Vibrio harveyi and Vibrio fortis by 324-fold and 10-fold, respectively. Our findings indicate that heat stress-induced mortality of C. gigas coincides with an increase in the abundance of putative bacterial pathogens in the oyster microbiome and highlights the negative consequences of marine heat waves on food production from aquaculture.