Phage toxin variants are linked to protection specificity in a defensive symbiont

Insects often depend on symbiotic bacteria for protection; however, the mechanisms by which these microbes target specific natural enemies remain poorly understood. In aphids, different strains of the facultative symbiont Hamiltonella defensa provide highly specific protection against particular species of parasitoid wasps. To uncover the genetic basis of this specificity, we analyzed 26 Hamiltonella genomes and their toxin-encoding APSE bacteriophages with distinct protective phenotypes. Our analyses revealed that Hamiltonella strains share a conserved core genome but differ significantly in accessory gene content, reflecting their distinct evolutionary origins. Strikingly, we show that variation in toxin types is the key distinguishing feature of APSE phages in Hamiltonella strains that protect against different parasitoid species. These toxin repertoires include several novel candidates, such as variants with MAC/perforin domains and leucine-rich repeat (LRR) proteins previously unreported in insect defensive symbionts. We also reveal cases of multiple cointegrated APSE phages carrying different toxins within a single genomic locus. These findings suggest phage-borne toxins are important determinants of enemy-specific defense and point to phage-driven toxin diversification as a major force shaping the functional evolution of this symbiosis. This work highlights how mobile genetic elements influence the ecological roles and diversification of protective symbionts.