Ecological relevance and molecular regulation of antibiotic-producing bacterial symbionts in Lagriinae beetles

与微生物的共生相互作用是真核生物进化的驱动力。除了营养共生外，具有防御基础的共生生物在自然界中也很常见，但人们对它们的生态和进化知之甚少，特别是在自然条件下。草兔亚科甲虫与多种伯克霍尔德氏菌细菌共生，这些细菌从母亲传给后代，可以保护宿主卵免受拮抗真菌的侵害。最近的发现表明，共存的共生菌每个都可以产生一套不同的生物活性次生代谢物；总共至少有五种。利用这个模型系统，我们的目的是更好地理解这种防御性共生的生态、进化和分子基础。该提案涉及两个项目，第一个项目侧重于共生体介导的防御在宿主发育过程中和跨兔尾蛾亚科物种之间的生态相关性。利用抗生素生物合成基因表达谱、分析化学、质谱学成像、荧光原位杂交(FISH)和操纵性分析相结合的方法，我们将表征抗生素生产的动态，并评估其在昆虫脆弱的蜕皮阶段的重要性。此外，评估来自不同兔亚科物种的共生体产生这些化合物的情况将有助于了解它们对共生进化的重要性。第二个项目将研究防御性共生的分子调控，考虑来自宿主和共生体的因素，这些因素可能是建立和维持这种联系的决定性因素。来自共生体寄主组织定植的关键时间点的转录数据将在共生体和无共生体的甲虫之间进行比较，以确定对共生体调控重要的候选免疫基因的子集。这些基因将通过在甲虫身上进行RNAi来消除，并将使用qPCR和FISH来评估其对共生建立的影响。为了研究与昆虫相关的共生体特征，将使用随机转座子突变来创建共生体突变体文库，并重新感染没有共生体的甲虫。在寄主中成功建立的突变体群落将与原始文库进行对比，以确定可能对在甲虫中定居和生存至关重要的基因。重要的是，恢复的突变体库中的遗传图谱将与自然产生的菌株进行比较，评估某些基因的存在或缺失是否反映并因此可能解释特定菌株的流行。这两个项目应该有助于我们在宿主保护的背景下对动物-微生物相互作用的一般理解，并将为多菌株共生相互作用的进化和稳定性提供有价值的见解。

Symbiotic interactions with microorganisms are a driving force in the evolution of eukaryotes. Along with nutritional symbioses, those with a defensive basis are frequent in nature, yet less is known about their ecology and evolution, especially under natural conditions. Lagriinae beetles engage in a symbiosis with multiple strains of Burkholderia gladioli bacteria, which are transmitted from mother to offspring and can protect the host eggs from antagonistic fungi. Recent findings show that co-existing symbiont strains can each produce a distinct set of bioactive secondary metabolites; at least five in total. Using this model system, our aim is to better understand the ecology, evolution and molecular basis of this defensive symbiosis. The proposal involves two projects, the first one focuses on the ecological relevance of symbiont-mediated defense during host development and across Lagriinae species. Using a combination of antibiotic-biosynthesis gene expression profiling, analytical chemistry, mass-spectrometry imaging, fluorescence in-situ hybridization (FISH) and manipulative assays, we will characterize the dynamics of antibiotic production and assess its importance during the vulnerable molting stages of the insect. Additionally, evaluating the production of these compounds by symbionts from different Lagriinae species will inform on their importance for the evolution of the symbiosis. A second project will investigate the molecular regulation of the defensive symbiosis, considering factors from both host and symbiont that might be determinant for the establishment and maintenance of the association. Transcriptomic data from key time points for host tissue colonization by the symbionts will be compared between symbiotic and symbiont-free beetles, in order to identify a subset of candidate immune genes that are important for symbiont regulation. These will be knocked-down using RNAi on the beetles, and the effect on symbiosis establishment will be assessed using qPCR and FISH. To investigate symbiont traits that are relevant for associating to the insect, random transposon mutagenesis will be used to create a library of symbiont mutants and re-infect symbiont-free beetles. The community of mutants that successfully establishes in the host will be contrasted to the original library to identify genes that are likely crucial for colonizing and surviving in the beetle. Importantly, the genetic repertoire in the recovered mutant library will be compared to the naturally occurring strains, assessing whether the presence or absence of certain genes reflects, and might therefore explain, the prevalence of specific strains. Both projects should contribute to our general understanding of animal-microbe interactions in the context of host protection, and will provide valuable insights on the evolution and stability of multi-strain symbiotic interactions.