Abstract
Some hemipteran insects rely on multiple endosymbionts for essential nutrients. However, the evolution of multi-partner symbiotic systems is not well-established. Here, we report a co-obligate symbiosis in the eusocial aphid, Ceratovacuna japonica. 16S rRNA amplicon sequencing unveiled co-infection with a novel Arsenophonus sp. symbiont and Buchnera aphidicola, a common obligate endosymbiont in aphids. Both symbionts were housed within distinct bacteriocytes and were maternally transmitted. The Buchnera and Arsenophonus symbionts had streamlined genomes of 432,286 bp and 853,149 bp, respectively, and exhibited metabolic complementarity in riboflavin and peptidoglycan synthesis pathways. These anatomical and genomic properties were similar to those of independently evolved multi-partner symbiotic systems, such as Buchnera-Serratia in Lachninae and Periphyllus aphids, representing remarkable parallelism. Furthermore, symbiont populations and bacteriome morphology differed between reproductive and soldier castes. Our study provides the first example of co-obligate symbiosis in Hormaphidinae and gives insight into the evolutionary genetics of this complex system.