Understanding immune priming in Pacific oysters: A multi-omics exploration of transcriptomic, epigenetic and microbiome regulation: supplemental files for the thesis by Sarah Woodsford

These are the supplemental data files associated with PhD thesis: "Immune priming in Pacific oysters: A multi-omics exploration of transcriptomic, epigenetic and microbiome regulation", by Sarah Woodsford.<b>Thesis abstract</b>Aquaculture has a fundamental role to play in global food security, given the limitations for increase of captures from global fisheries and the pressures they pose on the natural environment. Infectious disease remains an important limitation for the aquaculture industry both because it is financially costly and contributes towards poor animal welfare. For molluscs, widespread mass-mortalities of Pacific oysters (<i>Magallana gigas</i>) have occurred in recent years linked to infection with the bacterial pathogen <i>Vibrio aestuarianus</i>. Disease in farmed oysters is difficult to prevent or to treat and, in this context, immune priming has been proposed as a method to increase resistance to disease, and has been reported to be effective for viral pathogens. However, it is unknown whether an immune response can be primed in oysters to provide protection against <i>Vibrio aestuarianus</i> upon secondary challenge. This thesis aimed to address this knowledge gap and explored the hypothesis that epigenetic and microbiome processes are involved in sustaining long-lasting immune system memory.In order to identify a period of DNA methylation reprogramming, and therefore a candidate window of epigenome malleability, Pacific oyster embryos were exposed to the methylation inhibitor 5-azacytidine at different temporal intervals throughout early development (Chapter 2). A period of methylation inhibitor sensitivity was observed up to approximately 11 hours post fertilisation, which is indicative of a window of methylome reprogramming. Further periods of reprogramming and/or epigenetic sensitivity may exist after this window, but their presence and precise timing was not explored.Pacific oysters were primed with heat-inactivated <i>Vibrio aestuarianus</i> via 24-hour bath exposure at either the larval or young spat stage, then challenged with the non-attenuated form of the pathogen at six months old. A multi-omics approach involving RNA-Seq, 16S amplicon sequencing and whole genome bisulphite sequencing was utilised to interrogate whether immune priming resulted in long-lasting molecular alterations to the oyster transcriptome, microbiome and epigenome (Chapters 3-5). For naïve oysters, transcriptomic analysis evidenced that <i>Vibrio aestuarianus</i> had an immunosuppressant effect on 6-month-old oyster spat, despite high survival rates and no significant alterations in oyster microbiota suggesting spat were not susceptible to disease. Immune priming at either the larval or young spat stage resulted in lasting alterations in the oyster transcriptome, microbiome and epigenome that were sustained for up to five months after the priming took place. Lasting transcriptional alterations were identified in pathways including complement, protein modification and phagocytosis, with upregulation of these pathways suggesting faster pathogen recognition upon challenge. The oyster transcriptome, microbiome and epigenome appeared to remain malleable after metamorphosis, although larval priming appeared to be more beneficial in terms of increasing within-sample microbial diversity.Together, this thesis demonstrates that acquired immunity can be primed in Pacific oysters against <i>Vibrio aestuarianus</i> at either the larval or spat stage and suggests that epigenetics may be responsible for the continued alteration of transcriptional and microbiome regulation after priming. However, it remains unclear how long this acquired pathogen memory may persist for and whether priming during a window of epigenome malleability will enhance adult survival against the virulent pathogen. Overall, this work highlights the potential to utilise sensitive windows during early life to manipulate the microbiome and epigenome in order to induce lasting memory for use in disease control within bivalve molluscs. Future research is required to clarify the precise causative mechanisms of the effects seen, whether transgenerational immunological memory can be primed against <i>Vibrio aestuarianus,</i> and evaluate the feasibility of this approach for building pathogen resistance within invertebrate aquaculture settings.<br>