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.Thesis abstractAquaculture 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 (Magallana gigas) have occurred in recent years linked to infection with the bacterial pathogen Vibrio aestuarianus. 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 Vibrio aestuarianus 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 Vibrio aestuarianus 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 Vibrio aestuarianus 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 Vibrio aestuarianus 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 Vibrio aestuarianus, and evaluate the feasibility of this approach for building pathogen resistance within invertebrate aquaculture settings.

本部分为萨拉·伍兹福德（Sarah Woodsford）博士论文《太平洋牡蛎（Pacific oyster, Magallana gigas）的免疫预刺激：转录组、表观基因组与微生物组调控的多组学探索》的配套补充数据文件。

论文摘要：
鉴于全球渔业捕捞量增长受限且对自然环境造成压力，水产养殖对于保障全球粮食安全具有核心作用。传染性疾病仍是水产养殖业的重要制约因素，不仅造成经济损失，还会加剧动物福利问题。近年来，太平洋牡蛎（Magallana gigas）发生大规模集体死亡事件，其诱因与病原菌鳗弧菌（Vibrio aestuarianus）感染密切相关。养殖牡蛎的病害难以预防和治疗，在此背景下，免疫预刺激（immune priming）被提议作为提升疾病抗性的手段，且已被证实对病毒性病原体有效。然而，目前尚不清楚能否在牡蛎中预激免疫应答，使其在二次感染时获得抗鳗弧菌的保护作用。

本论文旨在填补这一知识空白，提出并验证了“表观遗传与微生物组过程参与维持长期免疫记忆”的假说。

为确定DNA甲基化重编程周期，进而筛选表观基因组可塑性的候选窗口，研究团队在太平洋牡蛎胚胎早期发育的不同时间节点，将其暴露于甲基化抑制剂5-氮胞苷（5-azacytidine）中（第2章）。观察到受精后约11小时内均对甲基化抑制剂敏感，这一结果提示存在甲基化组重编程窗口。该窗口之后可能还存在其他重编程或表观遗传敏感周期，但本研究未对其存在性及精确时序展开探究。

研究团队分别在幼虫期或稚贝早期，通过24小时浸浴方式用热灭活鳗弧菌对太平洋牡蛎进行免疫预刺激，随后在养殖6个月后用强毒株病原菌进行二次感染。本研究采用RNA测序（RNA-Seq）、16S扩增子测序（16S amplicon sequencing）及全基因组亚硫酸氢盐测序（whole genome bisulphite sequencing）的多组学策略，分析免疫预刺激是否会对牡蛎的转录组、微生物组及表观基因组产生长期分子层面的改变（第3-5章）。

针对未预刺激的牡蛎，转录组分析显示，尽管养殖6个月的稚贝存活率较高且微生物组未发生显著改变，表明其未感染病害，但鳗弧菌对其具有免疫抑制效应。无论是在幼虫期还是稚贝早期进行免疫预刺激，均会使牡蛎的转录组、微生物组及表观基因组产生长期改变，且该改变在预刺激后可维持长达5个月。研究在补体系统、蛋白质修饰及吞噬作用等通路中发现了持久的转录调控改变，这些通路的上调提示二次感染时病原体识别速度更快。尽管幼虫期预刺激在提升样本内微生物多样性方面效果更显著，但牡蛎的转录组、微生物组及表观基因组在变态后仍具有可塑性。

综上，本论文证实可在太平洋牡蛎的幼虫期或稚贝早期预激获得性免疫，使其获得抗鳗弧菌的保护能力，并提示表观遗传机制可能参与预刺激后转录组与微生物组调控的持续改变。不过目前仍不清楚该获得性病原菌记忆的持续时长，以及在表观基因组可塑性窗口内进行预刺激是否能提升成体对抗强毒病原菌的存活率。总体而言，本研究揭示了可利用早期生命中的敏感窗口调控微生物组与表观基因组，从而诱导双壳类软体动物产生长期免疫记忆，用于病害防控。未来仍需进一步明确上述效应的确切致病机制，探究能否通过预刺激产生跨代免疫记忆以对抗鳗弧菌，并评估该方法在无脊椎动物水产养殖场景中构建病原菌抗性的可行性。