The genetic basis of phylosymbiosis and microbe-mediated hybrid lethality

动物的肠道中有一些最密集的微生物共生体群落，过去十年的研究阐明了它们在宿主生物学中令人震惊的作用。在一些情况下，宿主-微生物区系联合的保真度反映在这些肠道细菌群落及其元基因组的宿主特有的组成上。此外，肠道细菌群落关系可以概括宿主物种的进化关联性--这种模式被称为“系统共生”。值得注意的是，肠道细菌群落系统共生的破坏可能与杂交宿主的严重疾病有关，并可能推动宿主物种形成。调节系统共生的寄主遗传因素和机制是什么？这些基因如何影响与肠道微生物区系相关的杂交条件？这一提议的中心假设是，系统共生和依赖微生物的杂交致死是(1)肠道细菌异常迁移到血淋巴中，(2)杂交中特定免疫基因的错误表达，否则将控制纯物种中的驻留细菌种群。Nasonia黄蜂模型系统是检验这些假说的理想工具，因为已观察到肠道微生物区系对种间杂交幼虫的严重致死作用。使用这个模型系统，我将解决以下两个目标：i：跟踪肠道细菌定植，并识别在杂交种中高表达的宿主基因，以响应肠道定植。奇异变形杆菌(Proteus Mirabilis)是纳氏菌肠道微生物区系中分布广泛的一员，它会导致纳氏菌杂交种的黑化过多和死亡。这个目标将使用绿色荧光蛋白标记的奇异P.mirabilis来检验这样的假设，即在纯物种中，细菌以良性的“游泳者”形态存在，但在杂交物种中分化为更具毒性的“游泳者”形态，从而导致血淋巴中的黑化，继而死亡。将使用荧光显微镜研究奇异支原体的增殖速度和位置。此外，对无菌黄蜂和被微生物区系定植的黄蜂的RNA-seq分析和qPCR将检验这一假说，即杂交种中特定的候选免疫基因参与了宿主-微生物区系相互作用的致死性破坏。II：从功能上询问杂交致死性和系统共生的候选基因。负责杂交致死和系统共生的候选基因将根据目标1中从杂交和非杂交幼虫获得的表达谱入围。这一目标将检验这样一种假设，即当杂交致死的重要候选基因被RNAi或CRISPR抑制时，杂交活性将得到恢复。此外，物种内相同基因的抑制将扰乱系统共生群落的组装，从而探索了影响杂交致死的相同基因也影响系统共生的假设。

The intestinal tracts of animals house some of the densest communities of microbial symbionts, and studies over the last decade have elucidated their astounding roles in host biology. In several cases, the fidelity of host-microbiota associations is reflected by host-specific composition of these gut bacterial communities and their metagenomes. Moreover, gut bacterial community relationships can recapitulate the evolutionary relatedness of the host species – a pattern known as “phylosymbiosis”. Notably, disruptions of phylosymbiosis in gut bacterial communities can be linked to severe maladies in hybrid hosts and could be driving host speciation. What are the host genetic factors and mechanisms that regulate phylosymbiosis? How do these genes influence hybrid conditions associated with the gut microbiota? The central hypothesis of this proposal is that phylosymbiosis and microbe-dependent hybrid lethality result from (1) an abnormal migration of gut bacteria into the hemolymph and (2) a misexpression of particular immune genes in hybrids that would otherwise control the resident bacterial population in pure species. The Nasonia wasp model system is ideal to test these hypotheses, because gut microbiota have been observed to contribute to severe larval lethality in interspecific hybrids. Using this model system, I will address the following two aims:I: Track bacterial colonization of the gut and identify the host genes that are hyperexpressed in hybrids in response to gut colonization. Proteus mirabilis is a widespread member of the Nasonia gut microbiota and contributes to hypermelanization and death of gnotobiotic Nasonia hybrids. This aim will use GFP-tagged P. mirabilis to test the hypothesis that, in the pure species, the bacterium exists in the benign, “swimmer” morphotype, but differentiates into the more virulent, “swarmer” morphotype in the hybrid to cause melanization in the hemolymph followed by death. The proliferation rate and sites of P. mirabilis will be studied using fluorescence microscopy. Additionally, RNA-seq analysis and qPCR in germ-free and wasps colonized by microbiota will test the hypothesis that specific candidate immune genes in the hybrid are involved in lethal breakdown in host-microbiota interactions.II: Functionally interrogate candidate genes underlying hybrid lethality and phylosymbiosis. Candidate genes responsible for hybrid lethality and phylosymbiosis will be shortlisted based on expression profiles obtained in Aim 1 from hybrid and non-hybrid larvae. This aim will test the hypothesis that when an important candidate gene for hybrid lethality is repressed by RNAi or CRISPR, hybrid viability will be restored. Moreover, repression of the same genes within species will disrupt a phylosymbiotic community assembly, thus probing the hypothesis that the same genes that influence hybrid lethality also influence phylosymbiosis.