DataSheet1_Differential responses of selectively bred mussels (Perna canaliculus) to heat stress—survival, immunology, gene expression and microbiome diversity.docx

New Zealand’s green-lipped mussel (Perna canaliculus) is an ecologically and economically important species. Marine heatwaves are increasing in frequency around NZ’s coastline, and these events are correlated with increased stress and mortality of some aquaculture species. This study aimed to identify general biomarkers of heat stress in P. canaliculus and to assess whether responses differed between genetically distinct selectively bred mussels. We exposed three families of selectively bred mussels (families A, B and C) to three seawater temperature regimes in the laboratory: 1) a “control” treatment (ambient 12°C), 2) a 26°C heat challenge with a subsequent recovery period, and 3) a sustained 26°C heat challenge with no recovery. We investigated whether the survival, immune response (hemocyte concentration and viability, oxidative stress and total antioxidant capacity), hemocyte gene expression and gill microbiome differed between the families during the temperature challenges. In the sustained heat-stress treatment, family A had the highest survival rate (42% compared with 25% and 5% for families C and B, respectively). Gene expression levels significantly shifted during thermal stress and differed between families, with family A more dissimilar than families B and C. Family C had substantially more genes impacted by temperature treatment and timepoint than the other families, while family B had very little genes/pathways that responded to thermal stress. Genes related to heat shock proteins and immune responses (e.g., AIF1, CTSC, TOLL8, CASP9, FNTA, AHCY, CRYAB, PPIF) were upregulated in all families during heat stress. Microbiome species-richness differed between families before and during heat-stress, with family A having a distinctly different microbiome flora than the other families. Microbial diversity changed similarly in all families exposed to prolonged heat-stress, with species of Vibrio and Campylobacter increasing in these mussels. Our study highlights the use of non-lethal sampling of hemocytes as a diagnostic tool to explore the immune response and gene expression of selectively bred mussels, to predict their response to ocean warming. This approach can identify potential thermotolerant candidates for further selective breeding, which may increase the resilience of the mussel aquaculture industry in a warming ocean.

新西兰绿唇贻贝（Perna canaliculus）是兼具生态与经济重要性的物种。新西兰周边海域的海洋热浪（marine heatwaves）频率正逐年升高，这类事件与部分水产养殖物种的胁迫压力上升及死亡率增加显著相关。本研究旨在鉴定绿唇贻贝热胁迫的通用生物标志物（biomarker），并评估不同遗传背景的选育贻贝之间的响应差异。我们在实验室中将3个选育贻贝家系（A、B、C家系）置于3种海水温度条件下进行暴露处理：1）"对照组"（环境温度12℃）；2）26℃热胁迫并伴随恢复期的处理组；3）持续26℃热胁迫且无恢复期的处理组。我们探究了在温度胁迫期间，不同家系的存活率、免疫反应（血细胞（hemocyte）浓度与活性、氧化应激（oxidative stress）及总抗氧化能力（total antioxidant capacity））、血细胞基因表达以及鳃微生物组（gill microbiome）是否存在差异。在持续热胁迫处理组中，A家系的存活率最高（42%，C、B家系分别为25%和5%）。热胁迫期间基因表达水平发生显著变化，且不同家系间存在差异，其中A家系的基因表达谱与B、C家系的相似度更低。C家系受温度处理与采样时间点影响的基因数量远多于其他两个家系，而B家系对热胁迫产生响应的基因/通路极少。热胁迫期间，所有家系中与热休克蛋白（heat shock proteins）及免疫反应相关的基因（如AIF1、CTSC、TOLL8、CASP9、FNTA、AHCY、CRYAB、PPIF）均出现上调表达。热胁迫前后不同家系的微生物群落物种丰富度存在差异，其中A家系的微生物菌群组成与其他家系显著不同。所有经历长期热胁迫的家系，其微生物多样性变化趋势相似，弧菌属（Vibrio）和弯曲杆菌属（Campylobacter）的物种在这些贻贝体内丰度上升。本研究证明，可通过非致死性的血细胞采样作为诊断工具，探究选育贻贝的免疫反应与基因表达情况，以此预测其对海洋变暖的响应。该方法可筛选出潜在的耐热候选个体用于后续选育工作，从而提升变暖海洋环境中贻贝养殖业的抗逆能力。