Environmental influences on the Pacific oyster (Crassostrea gigas) microbiome and disease associated with Ostreid herpesvirus-1 (OsHV-1)

Pacific oyster mortality syndrome (POMS) is a high mortality disease which has negatively impacted oyster farming. Despite the causal relationship between OsHV-1 and oyster mortality, the incidence and severity of disease is determined by complex interactions between the physiology of oysters, the environmental conditions and secondary pathogens. Understanding the multifactorial nature of the disease is required to develop management strategies. Recently, changes in the microbiome of oysters were associated with POMS. It is important to identify if differences in the microbiome are an outcome of the pathogenesis of the disease or whether the features of the microbiome predispose or contribute to the severity of disease. This thesis focused on the influence of important environmental factors on the composition of the Pacific oyster microbiome and determined how this impacted the outcome of OsHV-1 infections in oysters. Research chapter 1 investigated how the microbiome of genetically related Pacific oysters with a common hatchery origin was different when they were grown in different estuaries. Using the 16S rRNA gene diversity, the different estuarine environments were shown to generate unique microbiomes which were also associated with a differential response to the OsHV-1 infection. The quantitative dynamics of total bacteria and Vibrio spp. during an OsHV-1 infection was assessed using qPCR assays. The microbiome changed with the environment and after an OsHV-1 challenge. A strong correlation was observed between the OsHV-1 and Vibrio quantities in OsHV-1 infected oysters. Different microbiomes responded differently with a differential outcome of OsHV-1 challenge. Optimizing the quality and quantity of bacterial DNA from oyster tissues to facilitate accurate identification of the microbiome proved critical for microbiome studies. Research chapter 2 focused on evaluating methods to sample tissues and extract nucleic acids from Pacific oysters to accurately determine the microbiome. These procedures substantially impacted the quantity and diversity of bacteria identified. The controlled environment of a laboratory experimental system has been utilized to understand infectious diseases of oysters including POMS. A period of acclimation to environment before an experimental study can induce changes to the microbiome that may confound findings attributed to the disease being studied. Research chapter 3 focused on assessing changes in the Pacific oyster microbiome during acclimation to a laboratory environment when oysters are maintained in constant immersion in water and in a simulated tide. The oyster microbiome changed during acclimation irrespective of the management strategy (constant immersion vs. simulated tide). Research chapter 4 discusses the impact of the environment on the microbiome of oysters subsequent to challenge with OsHV-1. Although the microbiome composition changed after viral infection, it was the same for constant and partial immersion and did not explain higher mortality in oysters in simulated tidal conditions. Knowledge of the role of environmental factors on disease expression can help direct advice on oyster farm management strategies. Seawater temperature plays a major role in triggering diseases in the marine environment. Research chapter 5 discusses the impact of temperature and temperature fluctuations in water on the oyster microbiome and how it affects the outcome of an OsHV-1 infection. Unlike with temperature fluctuations the microbiome behaved differently at higher temperatures (26 0C) but did not create any distinct impact on the cumulative survival