Battle-hardening: Combined host and symbiont defenses fortify anti-parasitoid defenses in aphids

Microbial symbionts provide diverse ecological benefits, including defense against natural enemies. The pea aphid, Acyrthosiphon pisum, and its bacterial endosymbiont Hamiltonella defensa represent a well-studied anti-parasitoid defensive symbiosis. However, even highly effective H. defensa strains have key vulnerabilities, including susceptibility to multiple parasitoid ovipositions (superparasitism) and limited effectiveness against certain parasitoid taxa—limitations also shared by the aphid's endogenous defenses. We hypothesized that these respective weaknesses could be mitigated through the collaborative action of phenotypically distinct host and symbiont defensive mechanisms. To test this hypothesis, we created experimental clonal aphid lines with controlled defensive backgrounds and symbiont infection status through microinjection techniques. We then conducted parasitism assays and serial dissections of parasitized insects to evaluate whether aphids could enhance their anti-parasitoid capabilities by combining their endogenous physiological defenses with symbiont-mediated protection. Our results revealed that "doubly-equipped" aphids withstood superparasitism significantly better than aphids relying solely on either symbionts or endogenous defenses. Additionally, these aphids exhibited broader taxonomic resistance by possessing symbiont-mediated and endogenous defenses that targeted different parasitoid genera. These findings provide compelling evidence that host and symbiont anti-parasitoid mechanisms can function additively, creating a more robust defensive system than either component alone. Our study reveals an additional layer of complexity in the defense mechanisms of this significant crop pest that may impact the effectiveness of parasitoid-based biocontrol strategies in agricultural settings.