Insects often harbor heritable symbionts that provide defense against specialized natural enemies, yet little is known about symbiont protection when hosts face simultaneous threats. In pea aphids (Acyrthosiphon pisum), the facultative endosymbiont Hamiltonella defensa confers protection against the parasitoid, Aphidius ervi, and Regiella insecticola protects against aphid-specific fungal pathogens, including Pandora neoaphidis. Here we investigated whether these two common aphid symbionts protect against a specialized virus A. pisum virus (APV), and whether their anti-fungal and anti-parasitoid services are impacted by APV infection. We found that APV imposed large fitness costs on symbiont-free aphids and these costs were elevated in aphids housing H. defensa. In contrast, APV titers were significantly reduced and costs to APV infection were largely eliminated in aphids with R. insecticola. To our knowledge, R. insecticola is the first aphid symbiont shown to protect against a viral pathogen, and only the second arthropod symbiont reported to do so. In contrast, APV infection did not impact the protective services either R. insecticola or H. defensa. To better understand APV biology, we produced five genomes and examined transmission routes. We found that moderate rates of vertical transmission, combined with horizontal transfer through food plants, were the major route of APV spread, although lateral transfer by parasitoids also occurred. Transmission was unaffected by facultative symbionts. In summary, the presence and species identity of facultative symbionts resulted in highly divergent outcomes for aphids infected with APV, while not impacting defensive services that target other enemies. These findings add to the diverse phenotypes conferred by aphid symbionts, and to the growing body of work highlighting extensive variation in symbiont-mediated interactions.

Facultative, heritable endosymbionts are found at intermediate prevalence within most insect species, playing frequent roles in their hosts’ defense against environmental pressures. Focusing on Hamiltonella defensa, a common bacterial endosymbiont of aphids, we tested the hypothesis that such pressures impose seasonal balancing selection, shaping a widespread infection polymorphism. In our studied pea aphid (Acyrthosiphon pisum) population, Hamiltonella infection frequencies ranged from 23.2% to 68.1% across a six-month longitudinal survey. Rapid spikes and declines were consistent across fields, and we estimated that selection coefficients, for Hamiltonella-infected aphids, changed sign within this single season. Prior laboratory research suggested anti-parasitoid defense as the major Hamiltonella benefit, and costs under parasitoid absence. While a prior field study supported these forces as counter-weights in a regime of seasonal balancing selection, our present survey showed no significant relationship between parasitoid wasps and Hamiltonella. Field cage experiments provided some explanation: parasitoids drove ~10% boosts to Hamiltonella frequencies that would be hard to detect under less controlled conditions. They also showed that Hamiltonella was not always costly under parasitoid exclusion, contradicting another long-held prediction. Instead, our longitudinal survey – and two overwintering studies - showed temperature to be the strongest predictor of Hamiltonella infection, matching some lab discoveries, and suggesting thermally sensitive costs and benefits, unrelated to parasitism, can shape this symbiont’s prevalence. These results add to a growing body of evidence arguing for rapid, seasonal adaptation in multivoltine organisms. For many insects, such adaptation may be mediated through the diverse impacts of heritable symbionts on host phenotypes.