Environmental conditions affect insect fitness, with many species constrained by specific temperature ranges. Aphids are limited to temperate climates and it is hypothesized that this is partly due to their heat-susceptible obligate nutritional symbiont Buchnera. Aphids often carry additional facultative symbionts which can increase the host's fitness after heat stress. Here we used the pea aphid (Acyrthosiphon pisum) and three of its facultative endosymbionts (Candidatus Regiella insecticola, Candidatus Fukatsuia symbiotica (X-type; PAXS), and Candidatus Hamiltonella defensa) to investigate how these species respond to heat stress and whether their presence affects the fitness of the host or the obligate symbiont. We exposed aphid lines to a single high temperature event and measured lifetime fecundity and population densities of both obligate and facultative symbionts. Heat shock reduced aphid fecundity, but for aphids infected with two of the facultative symbionts (Regiella or Fukatsuia), this reduction was less than in uninfected aphids. The population density of Buchnera was also reduced after heat shock, and only recovered in aphids infected with Regiella or Fukatsuia but not in uninfected aphids or those with Hamiltonella. Although heat shock initially reduced the densities of two of the facultative symbionts (Hamiltonella and Fukatsuia), all facultative symbiont densities recovered by adulthood. Two of the facultative symbionts tested therefore aided the recovery of the obligate symbiont and the host, and we discuss possible underlying mechanisms. Our work highlights the beneficial effects of protective symbionts on obligate symbiont recovery after heat stress and how facultative symbionts may affect the wider ecological community.

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Highlights d Pea aphids avoid feeding on leaves with pathogenic bacteria on their surface d Pea aphids use vision in the presence of ultraviolet light to avoid pathogenic bacteria d Avoidance is mediated by visual detection of fluorescence from bacterial pyoverdine

Microbial symbionts often alter the phenotype of their host. Benefits and costs to hosts depend on many factors, including host genotype, symbiont species and genotype, and environmental conditions. Here, we present a study demonstrating genotype-by-genotype (G×G) interactions between multiple species of endosymbionts harboured by an insect, and the first to quantify the relative importance of G×G interactions compared with species interactions in such systems. In the most extensive study to date, we microinjected all possible combinations of five Hamiltonella defensa and five Fukatsuia symbiotica (X-type; PAXS) isolates into the pea aphid, Acyrthosiphon pisum. We applied several ecological challenges: a parasitoid wasp, a fungal pathogen, heat shock, and performance on different host plants. Surprisingly, genetic identity and genotype × genotype interactions explained far more of the phenotypic variation (on average 22% and 31% respectively) than species identity or species interactions (on average 12% and 0.4%, respectively). We determined the costs and benefits associated with co-infection, and how these compared to corresponding single infections. All phenotypes were highly reliant on individual isolates or interactions between isolates of the co-infecting partners. Our findings highlight the importance of exploring the eco-evolutionary consequences of these highly specific interactions in communities of co-inherited species.