Microbial ecology of sea star wasting disease

从 2013 年 6 月开始一直持续到（2015 年 5 月），超过 20 种海星（Asteroidea、棘皮动物）受到海星消耗病 (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，这可能导致底栖群落结构发生深刻的改变。最近的研究已确定海星相关浓核病毒（SSaDV）是该疾病最可能的病原体。 SSaDV 与栖息在世界各地其他棘皮动物中的浓核病毒有关，并且已在西海岸小行星种群中存在至少 72 年。 因此，我们对 SSaDV 如何实际引发 SSWD 症状的理解仍然存在重大知识差距，特别是棘皮动物宿主、浓核病毒和微生物组成分如何相互作用。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群的组成？2）病毒基因组的变异及其相关毒力是什么？3）幼虫扩散是否会在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、历史和当代病毒株之间的基因组比较，以及通过针对水族馆和自然界中的幼虫和幼年小行星的实验来解决这些假设。这项工作将支持对一名研究生（针对代表性不足的少数群体进行招聘）和几名本科生进行生物海洋学和疾病生态学技术方面的培训。信息将通过两个备受瞩目的太平洋海岸公共水族馆（西雅图和温哥华水族馆）的公共外展活动、专门针对该项目的网站以及公开演讲和媒体曝光来传播。 该项目将解决与海星消耗性疾病（SSWD）相关的三个基本问题：1）SSaDV 如何引起 SSWD 症状以及该疾病如何从原发感染进展到动物死亡； 2) 目前的SSaDV基因型与历史上的基因型有何不同，其毒力与基因组多态性是否相关； 3) 幼虫和幼年小行星是否受到 SSaDV 的不同影响，广播产生的双翅虫是否是 SSaDV 在遥远栖息地之间传播的可行机制。第一个问题将通过用 SSaDV 实验接种幼稚海星，然后对宿主转录进行时程监测（即通过 RNAseq 进行转录组学），通过 16S rRNA 测序和定量 PCR 进行微生物组组成，以及使用定量 PCR 进行病毒载量和流行率来解决。第二个问题将通过扩增 SSaDV 和相关浓核病毒的整个基因组来解决，然后进行基因组间比较，以确定关键蛋白质编码区域的多态性 DNA。第三个问题将通过在产卵小行星种群附近的野外地点从浮游生物中收集双翅虫，并对圈养的幼年海星进行时间过程观察并使用定量 PCR 监测其细菌和病毒载量来解决。这项工作将主要在萨利什海地区进行，使用从阿拉斯加水域收集的 SSaDV（幼稚小行星）。

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.