Largus than life: Ancient and persistent environmental symbiont reacquisition in bordered plant bugs (Pyrrhocoroidea: Largidae)

Obligate symbioses with bacteria are common in nature and symbiotic bacteria are typically vertically inherited through generations. Within Hemiptera, nearly all phytophagous taxa are thought to depend on bacteria for nutritional supplementation of their diet. However, identities and transmission routes of symbionts have not been investigated for all families, particularly for true bugs (Heteroptera). While Pyrrhocoridae have been relatively well studied with respect to bacterial symbioses, their closest relatives, Largidae, are only beginning to be explored using modern molecular methods. We conducted paired-end Illumina and Sanger sequencing of bacterial 16S amplicons of 30 pyrrhocoroid taxa, including 17 species of Largidae, in order to determine bacterial associates and similarity of associated microbial communities among this group. We also constructed a comprehensive phylogeny of this superfamily (4,800 bp; 5 loci; 56 ingroup + 9 outgroup taxa) to understand evolutionary patterns regarding bacterial associates. We found associations of the plant-beneficial-environmental clade Burkholderia within members of the family Largidae and marked differences between predatory and herbivorous members of the Pyrrhocoridae. Additionally, we undertook multiple lines of investigation (i.e., experimental rearing and phylogenetic and co-evolutionary analyses) to understand transmission routes of largid symbionts. We find evidence that suggests, that like other distantly-related Heteroptera, Largidae acquire bacterial symbionts from the environment every generation, a strategy that has seemingly persisted in some true bug lineages since the Cretaceous.