Division of labour within psyllids: Metagenomics reveals an ancient dual endosymbiosis with metabolic complementarity in the genus Cacopsylla

Hemipteran insects are well-known for their ancient associations with beneficial bacterial endosymbionts, particularly nutritional symbionts providing the host with essential nutrients such as amino acids or vitamins lacking from the hosts diet. Thereby, these primary endosymbionts enable the exploitation of nutrient-poor food sources such as plant sap or vertebrate blood. In turn, the strictly host-associated lifestyle strongly impacts the genome evolution of the endosymbionts, resulting in small and degraded genomes. Over time, even the essential nutritional functions can be compromised, leading to the complementation or replacement of an ancient endosymbiont by another, more functionally versatile, bacterium. Herein, we provide evidence for a dual primary endosymbiosis in several psyllid species. Using metagenome sequencing, we produced the complete genome sequences of both the primary endosymbiont Candidatus Carsonella ruddii and an as yet uncharacterized Enterobacteriaceae bacterium from four species of the genus Cacopsylla. The latter represents a new psyllid-associated endosymbiont clade for which we propose the name Candidatus Psyllophila symbiotica. Fluorescent in situ hybridisation confirmed the co-localization of both endosymbionts in the bacteriome. The metabolic repertoire of Psyllophila is highly conserved across host species and complements the tryptophan biosynthesis pathway that is incomplete in the co-occurring Carsonella. Unlike co-primary endosymbionts in other insects, the genome of Psyllophila is almost as small as the one of Carsonella, indicating an ancient co-obligate endosymbiosis rather than a recent association to rescue a degrading primary endosymbiont. IMPORTANCEHeritable beneficial bacterial endosymbionts have been crucial for the evolutionary success of numerous insects, enabling the exploitation of nutritionally limited food sources such as vertebrate blood and plant sap. Herein, we describe a previously unknown dual endosymbiosis in the psyllid genus Cacospylla, consisting in the primary endosymbiont Candidatus Carsonella ruddii and a co-occurring Enterobacteriaceae bacterium for which we propose the name Candidatus Psyllophila symbiotica. Its localization within the bacteriome and its small genome size confirm that Psyllophila is a co-primary endosymbiont widespread within the genus Cacopsylla. Despite its highly eroded genome, Psyllophila complements the tryptophan biosynthesis pathway that is incomplete in the co-occurring Carsonella. Moreover, the genome of Psyllophila is almost as small as the one of Carsonella, indicating an ancient dual endosymbiosis rather than a recent acquisition of a new symbiont. Hence, our results shed light on the dynamic interactions of psyllids and their endosymbionts over evolutionary time.