Division of labor 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 that provide the host with essential nutrients such as amino acids or vitamins lacking in the host’s diet. Therefore, 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 hybridization 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. IMPORTANCE: Heritable beneficial bacterial endosymbionts have been crucial for the evolutionary success of numerous insects by enabling the exploitation of nutritionally limited food sources. Herein, we describe a previously unknown dual endosymbiosis in the psyllid genus Cacopsylla, consisting of 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 perfectly complements the tryptophan biosynthesis pathway that is incomplete in the co-occurring Carsonella. Moreover, the genome of Psyllophila is almost as small as Carsonella’s, suggesting an ancient dual endosymbiosis that has now reached a precarious stage where any additional gene loss would make the system collapse. Hence, our results shed light on the dynamic interactions of psyllids and their endosymbionts over evolutionary time.