Candidatus Symbiodolus in Cryptocephalus beetles: unveiling the nature of this novel insect generalist symbiont

Insects host various bacterial symbionts that can positively or negatively affect their fitness. While many symbionts coevolved within narrow host ranges, only a few generalist symbionts can colonize diverse insect taxa and tissues, with Wolbachia being the only well-studied example. The main aim of this project is to clarify the symbiotic nature of Symbiodolus (Enterobacterales), a newly discovered generalist symbiont infecting insects from six orders and colonizing various tissues. To investigate Symbiodolus' relationship with insects, the genus of phytophagous beetles Cryptocephalus (Coleoptera, Chrysomelidae), including both Symbiodolus-infected and uninfected species, will be used as a model. Specifically, the first project objective is to determine the prevalence and phylogenetic distribution of Symbiodolus within Cryptocephalus to define if it is a phylo-symbiont or if its association with the host is linked to other factors. It will be achieved by characterizing the bacteria hosted by different Cryptocephalus species and testing the phylogenetic signal of Symbiodolus presence/absence across the genus. The second objective is to determine Symbiodolus transmission mode by localizing it in Cryptocephalus body across developmental stages and within its excretions using fluorescence in situ hybridization. The third objective is to assess Symbiodolus impact on Cryptocephalus fitness by analyzing the genomes of strain/s infecting six Cryptocephalus species to determine the presence of genes related to host benefits, pathogenicity, and reproductive manipulation. Symbiosis manipulation experiments will also test the effects of selectively removing or introducing Symbiodolus on the host phenotype. Beyond clarifying Symbiodolus' symbiotic nature, this project will provide insights into the mechanisms governing the establishment and maintenance of associations between insects and generalist bacterial symbionts, as well as the evolutionary adaptations sustaining them.