Microbial ecology of sea star wasting disease

从2013年6月到现在(2015年5月)，20多种海星(海星总科、棘皮亚目)受到海星衰减症(SSWD)的影响，影响从阿拉斯加中部到下加利福尼亚州的人口。该病已导致这些Keystone捕食者的丰度大幅下降或消失，这可能导致底栖群落结构的深刻变化。最近的研究发现，海星相关登革热病毒(SSaDV)最有可能是这种疾病的病原体。SSADV与世界各地其他棘皮动物中的致病病毒有关，并已在西海岸小行星种群中存在至少72年。因此，在我们对SSADV如何实际引起SSWD症状，特别是棘皮动物宿主、登革热病毒和微生物组分如何相互作用的理解方面，仍然存在重大的知识差距。这个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)病毒基因组及其相关毒力的变异是什么？以及3)幼虫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进程测量、历史和当代病毒株之间的基因组-基因组比较以及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(面向人数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。将通过在两个备受瞩目的太平洋沿岸公共水族馆(西雅图水族馆和温哥华水族馆)的公共外联活动、通过一个专门介绍该项目的网站以及通过公开演讲和媒体曝光来传播信息。这个项目将解决与海星衰弱病(SSWD)有关的三个基本问题：1)SSADV如何引起SSWD症状，以及疾病如何从最初的感染到动物死亡；2)当前SSADV的基因类型与历史上存在的不同，毒力与基因组多态有关；以及3)SSADV对幼虫和幼年小行星的不同影响，以及广播产卵的小行星是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SSaDV幼稚的海星来解决，然后通过时间过程监测宿主转录(即通过RNAseq转录)，通过16S rRNA测序和定量PCR监测微生物组组成，以及利用定量PCR监测病毒载量和流行率。第二个问题将通过扩增SSaDV和相关密度病毒的整个基因组，然后进行基因组-基因组比较来确定关键蛋白质编码区的多态DNA来解决。第三个问题将通过在小行星种群产卵附近的野外地点收集浮游生物中的双胞幼虫来解决，并对圈养的幼年海星进行时间过程观察，并使用定量聚合酶链式反应监测它们的细菌和病毒载量。这项工作将主要在Salish海地区进行，从阿拉斯加水域收集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.