Exploring Endosymbiont Biodiversity and Complexity in the Family Cicadidae

所有生物都有有益的微生物伙伴（共生体），有助于它们的健康和生存。其中许多生活在肠道中，被称为微生物群。理论上，昆虫共生体带来了允许物种形成和栖息地入侵的新特征。对于以缺乏重要营养物质的食物为食的昆虫来说，高度特化的微生物栖息在腹部的器官中，提供必需的氨基酸和维生素。本提案的主要目标是研究昆虫和微生物之间的相互作用，以及它们如何在互利关系中共同发挥作用。内共生体在物种形成和生物地理学中的作用在很大程度上尚未被探索，它们的多样性和进化在少数模型系统之外知之甚少。这项研究使用蝉作为研究系统，因为它们的饮食特别缺乏，而且种类繁多，遍布全球。DNA数据将被用来构建一个古老的进化树，以研究蝉和它们的共生伙伴是如何在世界各地传播并分化成新物种的。这一信息将有助于了解微生物如何使其宿主受益，特别是在研究对农业有重要意义的益虫和害虫的入侵方面。蝉及其共生体的生物多样性信息将通过期刊出版物、学会会议和万维网向科学家和公众传播。将设计并提供公民科学项目和生物实验室练习。未来的科学家将在多个实验室接受培训，包括一名博士后和两名研究生；许多本科生参与者将通过网络与国际科学家互动。蝉的原生内共生体已经被证明包括了与其他昆虫惊人不同的进化例子，例如在不同的蝉谱系中猖獗的共生基因组复制/谱系分裂，随后是互补基因的丢失和假基因的形成。为了了解宿主-共生体群落的生物地理和进化，将扩展研究小组先前创建的现有蝉系统发育，并将其作为框架。映射到树上的生态和共生体数据将测试关于蝉栖息地关联和共生体联盟成员关系变化的相对时间的假设。这项工作提出了以下问题：1)次生（兼性）内共生体的获得是否会促进初级（专性）内共生体的分解和/或丧失？或者，2)专性内共生体的破坏或丧失是否允许次级内共生体的入侵？同样，3)肠道菌群的变化是否会影响初级内共生菌（请记住，肠道菌群已知可以为某些宿主合成必需氨基酸）。最后，4)共生体联盟成员是否与外部环境（包括生物和非生物）的变化有关，如不同环境条件的新栖息地和寄主植物的入侵。改进的高通量基因组测序方法和保守的内共生体基因探针将同时扩展蝉科进化树框架，同时生成两种主要的蝉内共生体的遗传数据集。利用宏基因组测序（细菌组和脂肪体用于内共生体）和扩增子测序（肠道内容物用于微生物组研究）对蝉的共生伙伴进行调查，将有助于探索微生物和真菌的共生生物多样性。我们的假设检验将有助于探索生物多样性创新的环境与进化驱动因素。细菌内共生体基因组进化的新信息也将产生。

All organisms possess beneficial microbial partners (symbionts) that contribute to their health and survival. Many of these live in the gut and are known as the microbiome. Insect symbionts are theorized to bring new features that allow species formation and habitat invasions. In the case of insects that feed on food lacking important nutrients, highly specialized microbes inhabit organs in the abdomen and provide essential amino acids and vitamins. The major goal of this proposal is to study the interactions between insects and microbes and how they function together in a mutually beneficial relationship. The roles of endosymbionts in speciation and biogeography are largely unexplored and their diversity and evolution is poorly known outside a handful of model systems. This research uses cicadas as a study system because they have a particularly deficient diet and are diverse and spread across the globe. DNA data will be used to construct a dated evolutionary tree to study how the cicadas and their symbiont partners have spread around the world and diversified into new species. This information will be useful in general understanding how microorganisms benefit their hosts and specifically in the study of invasions of beneficial and pest insects of agricultural importance. Biodiversity information for cicadas and their symbionts will be disseminated to scientists and the general public by journal publications, society meetings, and the World Wide Web. Citizen science projects and biology lab exercises will be designed and made available. Future scientists will be trained in multiple laboratories, including one postdoc and two graduate students; many undergraduate participants will interact with scientists internationally via the web. Cicada primary endosymbionts have already been demonstrated to include examples of evolution that are surprisingly different from those of other insects, such as rampant symbiont genome duplication/lineage splitting in diverse cicada lineages followed by complementary gene loss and pseudogene formation. To understand the biogeography and evolution of host-symbiont consortia, an existing cicada phylogeny created previously by the research team involved in this work will be expanded and used as a framework. Ecological and symbiont data mapped onto the tree will test hypotheses about the relative timing of changes in cicada habitat association and symbiont consortium membership. This work asks: 1) Does the gain of a secondary (facultative) endosymbiont facilitate the breakdown and or loss of primary (obligate) endosymbiont? Or, 2) Does the breakdown or loss of the obligate endosymbiont allow the invasion of a secondary endosymbiont? Similarly, 3) Do changes in the gut microbiota affect primary endosymbionts (keeping in mind that gut microbiota are known to synthesize essential amino acids for some hosts). Finally, 4) Is symbiont consortium membership related to changes in the external environment (both biotic and abiotic) such as the invasion of new habitats with different environmental conditions and host plants. Improved high-throughput genomic sequencing methods and probes for conserved endosymbiont genes will simultaneously expand the framework Cicadidae evolutionary tree while generating genetic datasets for both of the primary cicada endosymbionts. Surveys of cicada symbiotic partners using metagenomic sequencing (of bacteriomes and fat bodies for endosymbionts) and amplicon sequencing (of gut contents for microbiome studies) will allow the exploration of microbial and fungal symbiont biodiversity. Our hypothesis testing will facilitate exploration of environmental versus evolutionary drivers of biodiversity innovation. New information on the evolution of bacterial endosymbiont genomes will also be produced.