Microbial ecology of sea star wasting disease

从2013年6月开始一直持续到现在（2015年5月），20多种海星（星形科、棘皮科）受到海星消瘦病（SSWD）的影响，影响从阿拉斯加中部到下加利福尼亚州的种群。这种疾病导致这些主要捕食者的数量大大减少或消失，这可能导致底栖生物群落结构的深刻改变。最近的工作已经确定海星相关致密病毒（SSaDV）是该疾病最有可能的病原体。SSaDV与居住在世界各地其他棘皮动物中的致密病毒有关，并且已经在西海岸小行星种群中存在至少72年。因此，对于SSaDV如何引发ssd症状，特别是棘皮动物宿主、致密病毒和微生物组成分如何相互作用，我们的理解仍然存在很大的知识空白。该项目将解决三个主要问题：1)病毒感染是否会改变海星微生物组的组成？2)病毒基因组的变异及其相关的毒力是什么？3)幼虫的扩散是否会在不同的栖息地之间传播疾病？该项目将通过对宿主、病原体和相关微生物的时间过程测量，对历史和当代病毒株的基因组-基因组比较，以及对水族箱和自然界中的幼虫和幼年小行星进行实验，来解决这些假设。这项工作将支持培训一名研究生（针对代表性不足的少数民族招聘）和几名本科生学习生物海洋学和疾病生态学技术。信息将通过太平洋沿岸两个知名的公共水族馆（西雅图水族馆和温哥华水族馆）的公众宣传活动，通过一个专门的项目网站，以及通过公众讲座和媒体曝光来传播。该项目将解决与海星消瘦病（SSWD）有关的三个基本问题：1)海星消瘦病如何引起SSWD症状，以及疾病如何从原发性感染发展到动物死亡；2)当前的SSaDV基因型与历史上存在的基因型有何不同，其毒力是否与基因组多态性有关；3) SSaDV对小行星幼虫和幼崽的影响是否存在差异，以及广播产卵的双胞体是否是SSaDV在遥远栖息地之间传播的可行机制。第一个问题将通过实验接种naïve海星SSaDV来解决，然后通过RNAseq对宿主转录进行时间过程监测（即转录组学），通过16S rRNA测序和定量PCR对微生物组组成进行监测，并使用定量PCR对病毒载量和流行进行监测。第二个问题将通过扩增SSaDV和相关致密病毒的全基因组来解决，然后进行基因组-基因组比较，以鉴定关键蛋白质编码区域的多态性DNA。第三个问题将通过在小行星产卵种群附近的野外地点收集浮游生物的双胞体来解决，并通过对圈养的幼年海星进行时间过程观察，并使用定量PCR监测它们的细菌和病毒载量。这项工作将主要在萨利希海地区进行，使用从阿拉斯加水域收集的SSaDV - naïve小行星。

Beginning in June 2013 and continuing to present (May 2015), over 20 species of sea stars (Asteroidea, Echinodermata) have been affected by sea star wasting disease (SSWD), affecting populations from central Alaska to Baja California. The disease has lead to greatly reduced abundance or disappearance of these keystone predators, which may result in profound alteration to benthic community structure. Recent work has identified the sea star associated densovirus (SSaDV) as the most likely causative agent of the disease. SSaDV is related to densoviruses inhabiting other echinoderms worldwide, and has been present in West Coast asteroid populations for at least 72 years. Hence, there remain significant knowledge gaps in our understanding of how SSaDV actually elicits SSWD symptoms, especially how the echinoderm host, densovirus and microbiome constituents interact. This project will address three major questions: 1) does viral infection change the composition of the sea star microbiome?, 2) what is the variation of viral genomes and their associated virulence?, and 3) does larval dispersal spread the disease between habitats? This project will address these hypotheses through time-course measurements of host, pathogen and associated microorganisms, genome-genome comparisons between historical and contemporary viral strains, and through experiments targeting larvae and juvenile asteroids in aquaria and in nature. This work will support training of a graduate student (recruitment targeting an under-represented minority) and several undergraduates in biological oceanographic and disease ecology techniques. Information will be disseminated through public outreach activities at two high-profile Pacific coast public aquariums (the Seattle and Vancouver Aquariums), through a website dedicated to the project, and through public talks and media exposure. This project will address three fundamental questions relating to Sea Star Wasting Disease (SSWD): 1) How does SSaDV causes SSWD symptoms and how does the disease progress from primary infection through animal mortality; 2) How do current genotypes of SSaDV vary from those present historically, and is virulence related to genome polymorphisms; and 3) Are larvae and juvenile asteroids differentially affected by SSaDV, and are broadcast-spawned bipinarria a viable mechanism for SSaDV dispersal between distant habitats. The first question will be addressed by experimental inoculation of naïve sea stars with SSaDV, then time-course monitoring of host transcription (i.e. transcriptomics via RNAseq), microbiome composition via 16S rRNA sequencing and quantitative PCR, and viral load and prevalence using quantitatice PCR. The second question will be addressed by amplifying the entire genome of SSaDV and related densoviruses, then perform genome-genome comparisons to identify polymorphic DNA in key protein-encoding regions. The third question will be addressed by collecting bipinarria from plankton at field locations adjacent to spawning asteroid populations, and by performing time-course observations of captive juvenile sea stars and monitoring their bacterial and viral loads using quantitative PCR. This work will be performed primarily in the Salish Sea region, with SSaDV - naïve asteroids collected from Alaskan waters.