DataSheet5_Understanding Crassostrea virginica tolerance of Perkinsus marinus through global gene expression analysis.docx

Disease tolerance, a host’s ability to limit damage from a given parasite burden, is quantified by the relationship between pathogen load and host survival or reproduction. Dermo disease, caused by the protozoan parasite P. marinus, negatively impacts survival in both wild and cultured eastern oyster (C. virginica) populations. Resistance to P. marinus has been the focus of previous studies, but tolerance also has important consequences for disease management in cultured and wild populations. In this study we measured dermo tolerance and evaluated global expression patterns of two sensitive and two tolerant eastern oyster families experimentally challenged with distinct doses of P. marinus (0, 106, 107, and 108 parasite spores per gram wet weight, n = 3–5 individuals per family per dose). Weighted Gene Correlation Network Analysis (WGCNA) identified several modules correlated with increasing parasite dose/infection intensity, as well as phenotype. Modules positively correlated with dose included transcripts and enriched GO terms related to hemocyte activation and cell cycle activity. Additionally, these modules included G-protein coupled receptor, toll-like receptor, and tumor necrosis factor pathways, which are important for immune effector molecule and apoptosis activation. Increased metabolic activity was also positively correlated with treatment. The module negatively correlated with infection intensity was enriched with GO terms associated with normal cellular activity and growth, indicating a trade-off with increased immune response. The module positively correlated with the tolerant phenotype was enriched for transcripts associated with “programmed cell death” and contained a large number of tripartite motif-containing proteins. Differential expression analysis was also performed on the 108 dosed group using the most sensitive family as the comparison reference. Results were consistent with the network analysis, but signals for “programmed cell death” and serine protease inhibitors were stronger in one tolerant family than the other, suggesting that there are multiple avenues for disease tolerance. These results provide new insight for defining dermo response traits and have important implications for applying selective breeding for disease management.

疾病耐受性（disease tolerance）是指宿主在特定寄生虫载量下限制机体损伤的能力，其量化依据为病原体负荷与宿主存活率或繁殖率之间的关联关系。帕金虫病（dermo disease）由原生动物寄生虫P. marinus引发，会对野生及养殖的美洲牡蛎（C. virginica）种群的存活率造成负面影响。此前的研究多聚焦于宿主抗P. marinus的能力，但疾病耐受性对野生与养殖种群的病害管理同样具有重要意义。本研究中，我们对帕金虫病耐受性进行了量化，并针对两个敏感型和两个耐受型美洲牡蛎家系开展实验，用不同梯度剂量的P. marinus孢子（每克湿重分别为0、10^6、10^7及10^8个寄生虫孢子，每个剂量下每个家系设置3~5个重复个体）侵染这些家系，同时分析其全基因表达谱。加权基因共表达网络分析（Weighted Gene Correlation Network Analysis, WGCNA）筛选出多个与寄生虫剂量升高/感染强度增强以及宿主表型相关的基因模块。与剂量呈正相关的模块中，其富集的基因本体（Gene Ontology, GO）术语及转录本均与血细胞活化及细胞周期活动相关；此外，这类模块还包含G蛋白偶联受体（G-protein coupled receptor）、Toll样受体（toll-like receptor）以及肿瘤坏死因子（tumor necrosis factor）通路，这些通路在免疫效应分子激活与细胞凋亡过程中发挥关键作用。代谢活动增强同样与侵染处理呈正相关。与感染强度呈负相关的模块则富集了与正常细胞活动及生长相关的GO术语，这提示免疫应答增强与细胞正常生理活动之间存在权衡关系。与耐受型表型呈正相关的模块富集了与“程序性细胞死亡”相关的转录本，且包含大量的三基序蛋白（tripartite motif-containing proteins）。本研究还以最敏感的家系作为对照参考，对10^8剂量侵染组开展了差异表达分析。分析结果与共表达网络分析结果一致，但其中一个耐受型家系的“程序性细胞死亡”相关信号及丝氨酸蛋白酶抑制剂信号要强于另一个耐受型家系，这表明疾病耐受性存在多种调控路径。本研究结果为帕金虫病响应性状的界定提供了新的视角，同时也为通过选择育种开展病害管理提供了重要的理论依据。