Oyster Microbiome Research Articles

Persistent tissue-specific resident microbiota in oysters across a broad geographical range.

Marine animals often harbour complex microbial communities that influence their physiology. However, strong evidence for resident microbiomes in marine bivalves is lacking, despite their contribution to estuarine habitats and coastal economies. We investigated whether marine bivalves harbour stable, resident microorganisms in specific tissues or if their microbiomes primarily consist of transient members reflecting the environmental microbial pool. Conducting a latitudinal study of wild eastern oysters (Crassostrea virginica) along the East Coast of the United States, we aimed to identify resident microorganisms that persist across a wide geographical range. Our results revealed that microbial communities in seawater and sediment samples followed latitudinal diversity patterns driven by geographic location. In contrast, oyster-associated microbiomes were distinct from their surrounding environments and exhibited tissue-specific compositions. Notably, oyster microbiomes showed greater similarity within the same tissue type across different geographic locations than among different tissue types within the same location. This indicates the presence of tissue-specific resident microbes that persist across large geographical ranges. We identified a persistent set of resident microbiome members for each tissue type, with key microbial members consistent across all locations. These findings underscore the oyster host's role in selecting its microbiome and highlight the importance of tissue-specific microbial communities in understanding bivalve-associated microbiomes.

Analysis of the core bacterial community associated with consumer-ready Eastern oysters (Crassostrea virginica).

Shellfish, such as the Eastern oyster (Crassostrea virginica), are an important agricultural commodity. Previous research has demonstrated the importance of the native microbiome of oysters against exogenous challenges by non-native pathogens. However, the taxonomic makeup of the oyster microbiome and the impact of environmental factors on it are understudied. Research was conducted quarterly over a calendar year (February 2020 through February 2021) to analyze the taxonomic diversity of bacteria present within the microbiome of consumer-ready-to-eat live Eastern oysters. It was hypothesized that a core group of bacterial species would be present in the microbiome regardless of external factors such as the water temperature at the time of harvest or post-harvesting processing. At each time point, 18 Chesapeake Bay (eastern United States) watershed aquacultured oysters were acquired from a local grocery store, genomic DNA was extracted from the homogenized whole oyster tissues, and the bacterial 16S rRNA gene hypervariable V4 region was PCR-amplified using barcoded primers prior to sequencing via Illumina MiSeq and bioinformatic analysis of the data. A core group of bacteria were identified to be consistently associated with the Eastern oyster, including members of the phyla Firmicutes and Spirochaetota, represented by the families Mycoplasmataceae and Spirochaetaceae, respectively. The phyla Cyanobacterota and Campliobacterota became more predominant in relation to warmer or colder water column temperature, respectively, at the time of oyster harvest.

Understanding the Dynamic of POMS Infection and the Role of Microbiota Composition in the Survival of Pacific Oysters, Crassostrea gigas.

For over a decade, Pacific oyster mortality syndrome (POMS), a polymicrobial disease, induced recurring episodes of massive mortality affecting Crassostrea gigas oysters worldwide. Recent studies evidenced a combined infection of the ostreid herpesvirus (OsHV-1 μVar) and opportunistic bacteria in affected oysters. However, the role of the oyster microbiota in POMS is not fully understood. While some bacteria can protect hosts from infection, even minor changes to the microbial communities may also facilitate infection and worsen disease severity. Using a laboratory-based experimental infection model, we challenged juveniles from 10 biparental oyster families with previously established contrasted genetically based ability to survive POMS in the field. Combining molecular analyses and 16S rRNA gene sequencing with histopathological observations, we described the temporal kinetics of POMS and characterized the changes in microbiota during infection. By associating the microbiota composition with oyster mortality rate, viral load, and viral gene expression, we were able to identify both potentially harmful and beneficial bacterial amplicon sequence variants (ASVs). We also observed a delay in viral infection resulting in a later onset of mortality in oysters compared to previous observations and a lack of evidence of fatal dysbiosis in infected oysters. Overall, these results provide new insights into how the oyster microbiome may influence POMS disease outcomes and open new perspectives on the use of microbiome composition as a complementary screening tool to determine shellfish health and potentially predict oyster vulnerability to POMS. IMPORTANCE For more than a decade, Pacific oyster mortality syndrome (POMS) has severely impacted the Crassostrea gigas aquaculture industry, at times killing up to 100% of young farmed Pacific oysters, a key commercial species that is cultivated globally. These disease outbreaks have caused major financial losses for the oyster aquaculture industry. Selective breeding has improved disease resistance in oysters, but some levels of mortality persist, and additional knowledge of the disease progression and pathogenicity is needed to develop complementary mitigation strategies. In this holistic study, we identified some potentially harmful and beneficial bacteria that can influence the outcome of the disease. These results will contribute to advance disease management and aquaculture practices by improving our understanding of the mechanisms behind genetic resistance to POMS and assisting in predicting oyster vulnerability to POMS.

Variation in Survival and Gut Microbiome Composition of Hatchery-Grown Native Oysters at Various Locations within the Puget Sound.

ABSTRACTThe Olympia oyster (Ostrea lurida) of the Puget Sound suffered a dramatic population crash, but restoration efforts hope to revive this native species. One overlooked variable in the process of assessing ecosystem health is association of bacteria with marine organisms and the environments they occupy. Oyster microbiomes are known to differ significantly between species, tissue type, and the habitat in which they are found. The goals of this study were to determine the impact of field site and habitat on the oyster microbiome and to identify core oyster-associated bacteria in the Puget Sound. Olympia oysters from one parental family were deployed at four sites in the Puget Sound both inside and outside of eelgrass (Zostera marina) beds. Using 16S rRNA gene amplicon sequencing of the oyster gut, shell, and surrounding seawater and sediment, we demonstrate that gut-associated bacteria are distinct from the surrounding environment and vary by field site. Furthermore, regional differences in the gut microbiota are associated with the survival rates of oysters at each site after 2 months of field exposure. However, habitat type had no influence on microbiome diversity. Further work is needed to identify the specific bacterial dynamics that are associated with oyster physiology and survival rates.IMPORTANCE This is the first exploration of the microbial colonizers of the Olympia oyster, a native oyster species to the West Coast, which is a focus of restoration efforts. The patterns of differential microbial colonization by location reveal microscale characteristics of potential restoration sites which are not typically considered. These microbial dynamics can provide a more holistic perspective on the factors that may influence oyster performance.

Can seagrass modify the effects of ocean acidification on oysters?

Solutions are being sought to ameliorate the impacts of anthropogenic climate change. Seagrass may be a solution to provide refugia from climate change for marine organisms. This study aimed to determine if the seagrass Zostera muelleri sub spp. capricorni benefits the Sydney rock oyster Saccostrea glomerata, and if these benefits can modify any anticipated negative impacts of ocean acidification. Future and ambient ocean acidification conditions were simulated in 52 L mesocosms at control (381 μatm) and elevated (848 μatm) CO2 with and without Z. muelleri. Oyster growth, physiology and microbiomes of oysters and seagrass were measured. Seagrass was beneficial to oyster growth at ambient pCO2, but did not positively modify the impacts of ocean acidification on oysters at elevated pCO2. Oyster microbiomes were altered by the presence of seagrass but not by elevated pCO2. Our results indicate seagrasses may not be a panacea for the impacts of climate change.

Determining the Composition of Resident and Transient Members of the Oyster Microbiome.

To better understand how complex microbial communities become assembled on eukaryotic hosts, it is essential to disentangle the balance between stochastic and deterministic processes that drive their assembly. Deterministic processes can create consistent patterns of microbiome membership that result in persistent resident communities, while stochastic processes can result in random fluctuation of microbiome members that are transient with regard to their association to the host. We sampled oyster reefs from six different populations across the east coast of the United States. At each site we collected gill tissues for microbial community analysis and additionally collected and shipped live oysters to Northeastern University where they were held in a common garden experiment. We then examined the microbiome shifts in gill tissues weekly for 6 weeks using 16S rRNA gene amplicon sequencing. We found a strong population-specific signal in the microbial community composition of field-sampled oysters. Surprisingly, the oysters sampled during the common garden experiment maintained compositionally distinct gill-associated microbial communities that reflected their wild population of origin, even after rearing them in a common garden for several weeks. This indicates that oyster gill-associated microbiota are predominantly composed of resident microbes specific to host population, rather than being a reflection of their immediate biotic and abiotic surroundings. However, certain bacterial taxa tended to appear more frequently on individuals from different populations than on individuals from the same population, indicating that there is a small portion of the gill microbiome that is transient and is readily exchanged with the environmental pool of microbes. Regardless, the majority of gill-associated microbes were resident members that were specific to each oyster population, suggesting that there are strong deterministic factors that govern a large portion of the gill microbiome. A small portion of the microbial communities, however, was transient and moved among oyster populations, indicating that stochastic assembly also contributes to the oyster gill microbiome. Our results are relevant to the oyster aquaculture industry and oyster conservation efforts because resident members of the oyster microbiome may represent microbes that are important to oyster health and some of these key members vary depending on oyster population.

Impact of seawater temperature on the Pacific oyster (

Context Intertidal estuarine environments expose oysters to wide temperature variations. This can shift their microbiome composition towards pathogen-dominated communities. Understanding the impact of temperature on the microbiome will facilitate oyster health management. Aims The present study was conducted to (1) assess the Pacific oyster microbiome at different temperatures (21°C, 22°C, 26°C and diurnal fluctuation between 22°C and 26°C) and (2) investigate microbiome changes in response to exposure to the pathogen Ostreid herpesvirus-1 (OsHV-1) at different temperatures. Methods Pacific oysters (15 months of age; n = 480) were acclimated to different temperatures in laboratory aquaria. Samples were obtained before and after acclimation and after OsHV-1 exposure for quantification of OsHV-1, total bacteria and Vibrio, by quantitative PCR. Bacterial 16S rRNA gene (V1–V3) sequences were used to characterise the gill bacterial community. Key results The alpha diversity (number of observed amplicon sequence variants) and total number of bacteria associated with the gills of oysters did not change with acclimation to different water temperature profiles, but there was variation in beta diversity. The highest mortality after OsHV-1 exposure occurred at 26°C and these, together with oysters at 22/26°C, had a higher concentration of OsHV-1 DNA compared with to the ones at the lower constant temperatures (P < 0.05). The total bacterial quantity increased after the OsHV-1 challenge in oysters at 22/26°C. The alpha diversity of microbiota increased after the OsHV-1 challenge in oysters at 21°C and 22/26°C. The beta diversity changed both after acclimation and OsHV-1 challenge. The highest abundance of Vibrio and higher OsHV-1 loads were seen in OsHV-1-challenged oysters at 26°C (P < 0.05). Conclusions The gill microbiome altered with seawater temperature and OsHV-1 challenge. Higher mortality following OsHV-1 exposure was associated with a higher water temperature and greater abundance of Vibrio spp. arising from the microbiome. Implications Higher seawater temperature can be considered a key risk factor influencing oyster health by altering the microbiome, increasing susceptibility to OsHV-1 and increasing the Vibrio fraction in the oyster microbiome.

Ocean acidification alters the diversity and structure of oyster associated microbial communities

AbstractHost‐associated microbial communities are fundamental to host physiology, yet it is unclear how these communities will respond to environmental disturbances. Here, we disentangle the environment‐linked and host‐linked effects of ocean acidification on oyster‐associated microbial communities. We exposed adult oysters (Crassostrea virginica) to CO2‐induced ocean acidification (400 vs. 2800 ppm) for 80 d. We measured the oyster extrapallial fluid pH and sampled the gills for microbial analysis at six time points. We found that different subsets of microbes were linked to acidification (n = 34 amplicon sequence variants [ASVs]) and to host response (n = 20 ASVs) with little overlap (n = 8 ASVs), suggesting that some members of the oyster microbiome were more responsive to environmental conditions while others were more tightly linked to host condition. Our results provide insight into which members of the oyster microbiome may contribute to the health and resistance of their host, and which members are the most vulnerable to changing environmental conditions.

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 β-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.

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

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.

Microbiome of the Black-Lipped Pearl Oyster Pinctada margaritifera, a Multi-Tissue Description With Functional Profiling.

Elucidating the role of prokaryotic symbionts in mediating host physiology has emerged as an important area of research. Since oysters are the world’s most heavily cultivated bivalve molluscs, numerous studies have applied molecular techniques to understand the taxonomic and functional diversity of their associated bacteria. Here, we expand on this research by assessing the composition and putative functional profiles of prokaryotic communities from different organs/compartments of the black-lipped pearl oyster Pinctada margaritifera, a commercially important shellfish valued for cultured pearl production in the Pacific region. Seven tissues, in addition to mucous secretions, were targeted from P. margaritifera individuals: the gill, gonad, byssus gland, haemolymph, mantle, adductor muscle, mucus, and gut. Richness of bacterial Operational Taxonomic Units (OTUs) and phylogenetic diversity differed between host tissues, with mucous layers displaying the highest richness and diversity. This multi-tissues approach permitted the identification of consistent microbial members, together constituting the core microbiome of P. margaritifera, including Alpha- and Gammaproteobacteria, Flavobacteriia, and Spirochaetes. We also found a high representation of Endozoicimonaceae symbionts, indicating that they may be of particular importance to oyster health, survival and homeostasis, as in many other coral reef animals. Our study demonstrates that the microbial communities and their associated predicted functional profiles are tissue specific. Inferred physiological functions were supported by current physiological data available for the associated bacterial taxa specific to each tissue. This work provides the first baseline of microbial community composition in P. margaritifera, providing a solid foundation for future research into this commercially important species and emphasises the important effects of tissue differentiation in structuring the oyster microbiome.

Influence of environment on the pathogenesis of Ostreid herpesvirus-1 (OsHV-1) infections in Pacific oysters (Crassostrea gigas) through differential microbiome responses

The oyster microbiome is thought to contribute to the pathogenesis of mass mortality disease in Pacific oysters, associated with OsHV-1. As filter-feeders, oysters host a microbiota that can be influenced by the estuarine environment. This may alter susceptibility to OsHV-1 infections, causing variable mortality. This study aimed at: (1) differences in the microbiome of Pacific oysters with a common origin but grown in geographically distinct estuaries; (2) evaluating changes occurring in the microbiota, especially in Vibrio, and (3) differential responses of the oyster microbiome, in response to an OsHV-1 infection. Pacific oysters sourced from a single hatchery but raised separately in Patonga Creek, Shoalhaven River and Clyde River of NSW, Australia, were used and challenged with OsHV-1. The initial microbiome composition was different in the three batches and changed further, post-injection (p < 0.05). The Patonga oysters with the highest mortality also had higher OsHV-1 and Vibrio quantities compared to the other two batches (p < 0.05). The higher initial bacterial diversity in Patonga oysters decreased in moribund oysters which was not observed in the other two batches (p < 0.05). The microbiome of survivors of OsHV-1 infection and negative control oysters of two batches, did not show any changes with the relevant pre-challenged microbiome. A strong correlation was observed between the OsHV-1 and Vibrio quantities in OsHV-1 infected oysters (r = 0.6; p < 0.001). In conclusion, the Pacific oyster microbiome differed in different batches despite a common hatchery origin. Different microbiomes responded differently with a differential outcome of OsHV-1 challenge. The higher Vibrio load in oysters with higher OsHV-1 content and higher mortality, suggests a role in Vibrio in the pathogenesis of this mortality disease. This study provided insights of the potential of different estuarine environments to shape the Pacific oyster microbiome and how different microbiomes are associated with different outcomes of OsHV-1 infection.

The sydney rock oyster microbiome is influenced by local environmental parameters and QX disease resistance

The sydney rock oyster microbiome is influenced by local environmental parameters and QX disease resistance

The role of tissue type, sampling and nucleic acid purification methodology on the inferred composition of Pacific oyster (Crassostrea gigas) microbiome.

This study evaluated methods to sample and extract nucleic acids from Pacific oysters to accurately determine the microbiome associated with different tissues. Samples were collected from haemolymph, gill, gut and adductor muscle, using swabs and homogenates of solid tissues. Nucleic acids were extracted from fresh and frozen samples using three different commercial kits. The bacterial DNA yield varied between methods (P<0·05) and each tissue harboured a unique microbiota, except for gill and muscle. Higher bacterial DNA yields were obtained by swabbing compared to tissue homogenates and from fresh tissues compared to frozen tissues, without impacting the bacterial community composition estimated by 16S rRNA gene (V1-V3 region) sequencing. Despite the higher bacterial DNA yields with QIAamp® DNA Microbiome Kit, the E.Z.N.A.® Mollusc DNA Kit identified twice as many operational taxonomic units (OTUs) and eliminated PCR inhibition from gut tissues. Sampling and nucleic acid purification substantially affected the quantity and diversity of bacteria identified in Pacific oyster microbiome studies and a fit-for-purpose strategy is recommended. Accurate identification of Pacific oyster microbial diversity is instrumental for understanding the polymicrobial aetiology of Pacific oyster mortality diseases which greatly impact oyster production.

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.

Impact of Phaeobacter inhibens on marine eukaryote-associated microbial communities.

Bacteria-host interactions are universal in nature and have significant effects on host functionality. Bacterial secondary metabolites are believed to play key roles in such interactions as well as in interactions within the host-associated microbial community. Hence, prominent secondary metabolite-producing bacteria may be strong drivers of microbial community composition in natural host-associated microbiomes. This has, however, not been rigorously tested, and the purpose of this study was to investigate how the secondary metabolite producer Phaeobacter inhibens affects the diversity and composition of microbiomes associated with the microalga Emiliania huxleyi and the European flat oyster, Ostrea edulis. Roseobacters were indigenous to both communities exhibiting relative abundances between 2.8% and 7.0%. Addition of P. inhibens caused substantial changes in the overall structure of the low-complexity microbiome of E. huxleyi, but did not shape microbial community structure to the same degree in the more complex oyster microbiomes. Species-specific interactions occurred in both microbiomes and specifically the abundances of other putative secondary metabolite-producers such as vibrios and pseudoalteromonads were reduced. Thus, the impact of a bioactive strain like P. inhibens on host-associated microbiomes depends on the complexity and composition of the existing microbiome.

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.

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.

Potential impacts of hypoxia and a warming ocean on oyster microbiomes

Potential impacts of hypoxia and a warming ocean on oyster microbiomes

Denitrification potential of the eastern oyster microbiome using a 16S rRNA gene based metabolic inference approach.

The eastern oyster (Crassostrea virginica) is a foundation species providing significant ecosystem services. However, the roles of oyster microbiomes have not been integrated into any of the services, particularly nitrogen removal through denitrification. We investigated the composition and denitrification potential of oyster microbiomes with an approach that combined 16S rRNA gene analysis, metabolic inference, qPCR of the nitrous oxide reductase gene (nosZ), and N2 flux measurements. Microbiomes of the oyster digestive gland, the oyster shell, and sediments adjacent to the oyster reef were examined based on next generation sequencing (NGS) of 16S rRNA gene amplicons. Denitrification potentials of the microbiomes were determined by metabolic inferences using a customized denitrification gene and genome database with the paprica (PAthway PRediction by phylogenetIC plAcement) bioinformatics pipeline. Denitrification genes examined included nitrite reductase (nirS and nirK) and nitrous oxide reductase (nosZ), which was further subdivided by genotype into clade I (nosZI) or clade II (nosZII). Continuous flow through experiments measuring N2 fluxes were conducted with the oysters, shells, and sediments to compare denitrification activities. Paprica properly classified the composition of microbiomes, showing similar classification results from Silva, Greengenes and RDP databases. Microbiomes of the oyster digestive glands and shells were quite different from each other and from the sediments. The relative abundance of denitrifying bacteria inferred by paprica was higher in oysters and shells than in sediments suggesting that oysters act as hotspots for denitrification in the marine environment. Similarly, the inferred nosZI gene abundances were also higher in the oyster and shell microbiomes than in the sediment microbiome. Gene abundances for nosZI were verified with qPCR of nosZI genes, which showed a significant positive correlation (F1,7 = 14.7, p = 6.0x10-3, R2 = 0.68). N2 flux rates were significantly higher in the oyster (364.4 ± 23.5 μmol N-N2 m-2 h-1) and oyster shell (355.3 ± 6.4 μmol N-N2 m-2 h-1) compared to the sediment (270.5 ± 20.1 μmol N-N2 m-2 h-1). Thus, bacteria carrying nosZI genes were found to be an important denitrifier, facilitating nitrogen removal in oyster reefs. In addition, this is the first study to validate the use of 16S gene based metabolic inference as a method for determining microbiome function, such as denitrification, by comparing inference results with qPCR gene quantification and rate measurements.