Dynamics and molecular mechanisms aiding symbiont establishment in Lagria villosa beetles

Abstract

Many animals live in a symbiotic relationship with microbial partners. Among them, beneficial host-associated microorganisms may provide nutritional benefits, protect the host from antagonists, or help in detoxifying harmful compounds. To maintain a stable association, animal hosts vertically transmit symbionts to the offspring, acquire them horizontally from an external source, or carry out both transmission modes. In horizontal and mixed transmission modes, host molecular factors are necessary to screen for the right microbial partners from the environment, and microbial machineries help the symbionts gain entry and establish in the host. However, we know little about molecular factors that mediate symbiont establishment in invertebrates harbouring ectosymbionts. To expand our knowledge, I studied the defensive symbiotic association between Lagria villosa beetles and Burkholderia bacteria. Here, the female Lagria beetles smear symbionts on the egg surface during oviposition. The symbionts colonize three specialized cuticular invaginations on the dorsal surface of the larvae. On eggs and in the larvae, the symbionts produce antifungal compounds and protect the beetle from antagonists. Furthermore, the larvae can occasionally acquire symbionts from the external environment; therefore, they maintain a mixed transmission mode. Among the multiple strains of Burkholderia usually found in an individual, Burkholderia Lv-StB is the most dominant strain, but is uncultivable in the lab. However, Burkholderia gladioli Lv-StA that is occasionally found in the beetles, is cultivable. The ability to rear the beetles in the lab and cultivate one of the symbionts in vitro gives us the opportunity to study host and symbiont molecular factors involved in symbiont establishment. In this dissertation, first, I provide details on the dynamics and mechanisms of symbiont colonization. Symbionts colonize the dorsal structures of the larvae during hatching and a colonization time-window may restrict microbial entry into the host. Further, a transposon-insertion directed sequencing (Tn-seq) method was established in the lab using which 271 potential colonization factors in Lv-StA were identified, including motility, biofilm formation, cell wall structures, lipopeptides, oxidative stress response factors and iron scavenging molecules. Additionally, targeted mutagenesis and colonization assays with Lv-StA reveal that non-motile strains can still colonize the host, and this likely explains the loss of flagellar motility genes in Lv-StB, which has a more reduced genome than Lv-StA. Finally, by performing gene expression analysis across multiple developmental time points in symbiotic and aposymbiotic beetles, the host molecular responses in the presence of Lv-StB was investigated. It reveals that the host may not recognise symbiont presence, likely due to compartmentalization, or may have a constitutive response to the symbionts. Thus, combining our understanding of both host and symbiont molecular factors involved in establishment, interesting questions regarding how partner specificity is achieved in ectosymbioses, are revealed. This study gives us the opportunity to understand the mechanistic details of host – symbiont interactions and further to compare emerging patterns across other model and non-model systems.