Dynamics and functionality of a multipartite defensive symbiosis in immature Lagria beetles
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Abstract
Almost all macroorganisms engage in partnerships with microorganisms, which facilitated not only their origin and evolution but have also great impact on their ecology. Microbial symbionts play important roles in the web of life, by taking over tasks essential for survival or development of their hosts. Of those, defense against natural enemies is a challenge for all animals on Earth, including insects. Although insects have evolved numerous traits to cope with different kinds of natural enemies on their own, their association with symbiotic microbes opened up new opportunities to adapt to challenges occurring with antagonistic encounters.
This dissertation focuses on the defensive symbiosis between darkling beetles of the genus Lagria (Coleoptera: Tenebrionidae) and several defensive symbionts including Burkholderia bacteria (Pseudomonadota: Betaproteobacteria). Lagria beetles accommodate their symbionts throughout development. They begin their journey in accessory glands of the adult female reproductive system, after which they are applied within a secretion onto the egg surface during oviposition and colonize three peculiar dorsal cuticular invaginations in the larvae. On the eggs, symbionts produce antimicrobial compounds to protect the egg stage against fungal infections from the environment. In larvae both sexes carry the symbionts, while male adults do not contain symbionts, indicating that the symbionts fulfill an important function also in post-hatch stages, which might be antifungal defense as well. With this hypothesis, the aim of this thesis was to investigate the symbiosis in the developing life stages of two Lagria species, L. villosa and L. hirta. Therefore, I intended to (i) elucidate the abundance, composition, and localization of several symbiont strains across host development, (ii) investigate the defensive potential of several members of the symbiont community in different life stages, and (iii) examine the morphological modifications of the beetles for symbiont transmission and maintenance throughout molting and metamorphosis. Bacterial community analyses via 16S amplicon sequencing (Illumina) and qPCR of L. villosa and L. hirta revealed that the beetles are consistently associated with their symbionts throughout all life stages. Moreover, they harbor a diverse community, which predominantly consists of Burkholderia strains, while also symbionts of other families are consistently present in lower abundance. In L. villosa, one Burkholderia strain (LvStB) is highly abundant in the community of all life stages. Despite its reduced genome and metabolic capacities, and its putative immotility compared to other Burkholderia strains, it dominates and is often found as the only Burkholderia strain. Its ability to produce the antifungal compound lagriamide is therefore a potential key factor for its stability and leading role in the beetle. Meanwhile in L. hirta, the two strains LhStH and LhStG dominate the community in varying abundances across the different life stages, proving that also closely related symbiont strains can coexist within single individuals, likely through functional complementation of defensive traits. Histological sections, fluorescence in situ hybridizations (FISH), light sheet microscopy, and micro-computed tomography (µCT) revealed that the symbionts are housed in three pouches of the dorsal thorax in larvae, which are needed for colonization and undergo morphological changes in the pupal stage. The symbiotic organs are formed through invaginations of the cuticle but remain open to the outer surface through a channel and are maintained throughout larval development. Through this channel, symbionts can be internalized after hatching from the egg, ensuring a vertical transmission route, but they can also be released to the outer surface. In pupae, a morphological dimorphism of the organs in females and males leads to a decline of symbionts during pupation in males and ultimately to the complete loss in male adults. For female pupae, the localization on the outer surface enables successful vertical symbiont transmission independent of the reorganization process of the internal tissue during metamorphosis. In vitro and in vivo assays, FISH and MALDI-imaging revealed that the symbionts and lagriamide are not only present inside the symbiotic organs, but also on the surface, where they protect young larvae against pathogenic fungi. On the surface, they inhibit fungal infestation on pupae and larvae, leading to higher larval survival probability. Keeping ectosymbionts accessible to the surface illustrates an effective defense strategy for the beetles against infections from fungal enemies. Furthermore, HPLC-MS, in vivo assays, and genome analyses revealed that in addition to its importance and consistent production in L. villosa, a close relative of lagriamide, namely lagriamide II, seems to be similarly responsible for the egg protection in L. hirta. However, lagriamide and their producers are not the only defensive symbionts, but also other Burkholderia strains protect the larval (LvStA) or egg stage (LvStA, LhStG) and have the metabolic capabilities for various protective compounds. Additionally, at least three non-Burkholderia symbionts (AcinetobacterLv1, LuteibacterLv2, VariovoraxLv3) showed protective capabilities on the eggs and through the presence of candidate biosynthetic gene clusters in their genomes their potential for defense.
With this thesis, I explored missing links in the developmental stages of the Lagria host and contributed to the general knowledge of defensive symbioses through several aspects: By shedding light on the structure, development, and function of the symbiotic organs, a unique morphological adaptation for insects to tie and maintain symbionts throughout development and host generations was revealed. The open structure of the cuticular invaginations housing antibiotic-producing ectosymbionts exemplifies an effective defense strategy for insects against fungal infections, especially during vulnerable phases of molting and metamorphosis. Moreover, their open structure allows for the horizontal acquisition of other putative symbionts, keeping the association flexible to potentially respond quickly to changing environments. This flexibility is furthermore reflected by the presence and coexistence of multiple protective symbionts next to dominant strains with different metabolic capabilities. Whether multiple symbiont infections are advantageous for the host and symbiont, and how they can persist in a long-term relationship are evolutionary interesting questions that can be further addressed in the Lagria symbiosis.