Transcriptome-wide comparisons and virulence gene polymorphisms of host-associated genotypes of the cnidarian parasite Ceratonova shasta in salmonids

Ceratonova shasta is an important myxozoan pathogen affecting the health of salmonid fishes in the Pacific Northwest of North America. C. shasta exists as a complex of host-specific genotypes, some with low to moderate virulence, and one that causes a profound, lethal infection in susceptible hosts. High throughput sequencing methods are powerful tools for discovering the genetic basis of these host/virulence differences, but deep sequencing of myxozoans has been challenging due to extremely fast molecular evolution of this group, yielding strongly divergent sequences that are difficult to identify, and unavoidable host contamination. We designed and optimized different bioinformatic pipelines to address these challenges. We obtained a unique set of comprehensive, host-free myxozoan RNA-seq data from C. shasta genotypes of varying virulence from different salmonid hosts. Analyses of transcriptome-wide genetic distances and maximum likelihood multigene phylogenies elucidated the evolutionary relationship between lineages and demonstrated the limited resolution of the established Internal Transcribed Spacer marker for C. shasta genotype identification, as this marker fails to differentiate between biologically distinct genotype II lineages from coho salmon and rainbow trout. We further analyzed the datasets based on polymorphisms in two gene groups related to virulence: cell migration and proteolytic enzymes including their inhibitors. The developed SNP-calling pipeline identified polymorphisms between genotypes and demonstrated that variations in both motility and protease genes were associated with different levels of virulence of C. shasta in its salmonid hosts. The prospective use of proteolytic enzymes as promising candidates for targeted interventions against myxozoans in aquaculture is discussed. We developed host-free transcriptomes of a myxozoan model organism from strains that exhibited different degrees of virulence, as a unique source of data that will foster functional gene analyses and serve as a base for the development of potential therapeutics for efficient control of these parasites.

沙斯塔角孢虫（Ceratonova shasta）是一类重要的黏体动物（myxozoan）病原，可危害北美太平洋西北地区的鲑形目鱼类健康。该病原以宿主特异性基因型复合体的形式存在，部分基因型的毒力处于低至中等水平，另有一类基因型可使易感宿主出现严重的致死性感染。高通量测序（high throughput sequencing）技术是揭示这类宿主与毒力差异遗传基础的有力工具，但由于黏体动物类群的分子进化速率极快，会产生高度分化的序列，难以进行识别，且不可避免地存在宿主污染，因此对其进行深度测序颇具挑战。我们设计并优化了多套生物信息学分析流程以应对上述挑战，并从不同鲑形目宿主来源、毒力各异的沙斯塔角孢虫基因型中，获取了一套独特的无宿主污染黏体动物RNA测序（RNA-seq）数据集。通过对转录组水平的遗传距离进行分析，以及构建最大似然法多基因系统发育树，我们阐明了不同谱系间的进化关系，并证实了现有用于沙斯塔角孢虫基因型鉴定的内转录间隔区（Internal Transcribed Spacer）标记分辨率有限——该标记无法区分银大麻哈鱼和虹鳟来源的、生物学特性迥异的基因型II谱系。我们进一步针对两类与毒力相关的基因家族开展了数据集分析：细胞迁移相关基因，以及包含其抑制剂在内的蛋白水解酶类基因。我们开发的单核苷酸多态性（single nucleotide polymorphism, SNP）识别流程成功鉴定出了不同基因型间的多态性，并证实运动相关基因与蛋白酶基因的变异，均与沙斯塔角孢虫在鲑形目宿主中的不同毒力水平存在关联。本文还讨论了将蛋白水解酶作为水产养殖中针对黏体动物的靶向干预候选方案的潜在应用价值。我们以毒力程度各异的沙斯塔角孢虫菌株为材料，构建了黏体动物模式生物的无宿主污染转录组，这一独特的数据资源将助力功能基因研究，并为开发可高效防控这类寄生虫的潜在治疗手段奠定基础。