More Is Not Always Better: Coinfections with Defensive Symbionts Generate Highly Variable Outcomes.

Animal-associated microbes are highly variable, contributing to a diverse set of symbiont-mediated phenotypes. Given that host and symbiont genotypes, and their interactions, can impact symbiont-based phenotypes across environments, there is potential for extensive variation in fitness outcomes. Pea aphids, Acyrthosiphon pisum, host a diverse assemblage of heritable facultative symbionts (HFS) with characterized roles in host defense. Protective phenotypes have been largely studied as single infections, but pea aphids often carry multiple HFS species, and particular combinations may be enriched or depleted compared to expectations based on chance. Here, we examined the consequences of single infection versus coinfection with two common HFS exhibiting variable enrichment, the antiparasitoid Hamiltonella defensa and the antipathogen Regiella insecticola, across three host genotypes and environments. As expected, single infections with either H. defensa or R. insecticola raised defenses against their respective targets. Single infections with protective H. defensa lowered aphid fitness in the absence of enemy challenge, while R. insecticola was comparatively benign. However, as a coinfection, R. insecticola ameliorated H. defensa infection costs. Coinfected aphids continued to receive antiparasitoid protection from H. defensa, but protection was weakened by R. insecticola in two clones. Notably, H. defensa eliminated survival benefits conferred after pathogen exposure by coinfecting R. insecticola Since pathogen sporulation was suppressed by R. insecticola in coinfected aphids, the poor performance likely stemmed from H. defensa-imposed costs rather than weakened defenses. Our results reveal a complex set of coinfection outcomes which may partially explain natural infection patterns and suggest that symbiont-based phenotypes may not be easily predicted based solely on infection status.IMPORTANCE The hyperdiverse arthropods often harbor maternally transmitted bacteria that protect against natural enemies. In many species, low-diversity communities of heritable symbionts are common, providing opportunities for cooperation and conflict among symbionts, which can impact the defensive services rendered. Using the pea aphid, a model for defensive symbiosis, we show that coinfections with two common defensive symbionts, the antipathogen Regiella and the antiparasite Hamiltonella, produce outcomes that are highly variable compared to single infections, which consistently protect against designated enemies. Compared to single infections, coinfections often reduced defensive services during enemy challenge yet improved aphid fitness in the absence of enemies. Thus, infection with multiple symbionts does not necessarily create generalist aphids with "Swiss army knife" defenses against numerous enemies. Instead, particular combinations of symbionts may be favored for a variety of reasons, including their abilities to lessen the costs of other defensive symbionts when enemies are not present.

Phage loss and the breakdown of a defensive symbiosis in aphids.

Terrestrial arthropods are often infected with heritable bacterial symbionts, which may themselves be infected by bacteriophages. However, what role, if any, bacteriophages play in the regulation and maintenance of insect-bacteria symbioses is largely unknown. Infection of the aphid Acyrthosiphon pisum by the bacterial symbiont Hamiltonella defensa confers protection against parasitoid wasps, but only when H. defensa is itself infected by the phage A. pisum secondary endosymbiont (APSE). Here, we use a controlled genetic background and correlation-based assays to show that loss of APSE is associated with up to sevenfold increases in the intra-aphid abundance of H. defensa. APSE loss is also associated with severe deleterious effects on aphid fitness: aphids infected with H. defensa lacking APSE have a significantly delayed onset of reproduction, lower weight at adulthood and half as many total offspring as aphids infected with phage-harbouring H. defensa, indicating that phage loss can rapidly lead to the breakdown of the defensive symbiosis. Our results overall indicate that bacteriophages play critical roles in both aphid defence and the maintenance of heritable symbiosis.