Heritable genetic variation is required for evolution, and while typically encoded within nuclear and organellar genomes, several groups of invertebrates harbour heritable microbes serving as additional sources of genetic variation. Hailing from the symbiont-rich insect order Hemiptera, pea aphids (Acyrthosiphon pisum) possess several heritable symbionts with roles in host plant utilization, thermotolerance and protection against natural enemies. As pea aphids vary in the numbers and types of harboured symbionts, these bacteria provide heritable and functionally important variation within field populations. In this study, we quantified the cytoplasmically inherited genetic variation contributed by symbionts within North American pea aphids. Through the use of Denaturing Gradient Gel Electrophoresis (DGGE) and 454 amplicon pyrosequencing of 16S rRNA genes, we explored the diversity of bacteria harboured by pea aphids from five populations, spanning three locations and three host plants. We also characterized strain variation by analysing 16S rRNA, housekeeping and symbiont-associated bacteriophage genes. Our results identified eight species of facultative symbionts, which often varied in frequency between locations and host plants. We detected 28 cytoplasmic genotypes across 318 surveyed aphids, considering only the various combinations of secondary symbiont species infecting single hosts. Yet the detection of multiple Regiella insecticola, Hamiltonella defensa and Rickettsia strains, and diverse bacteriophage genotypes from H. defensa, suggest even greater diversity. Combined, these findings reveal that heritable bacteria contribute substantially to genetic variation in A. pisum. Given the costs and benefits of these symbionts, it is likely that fluctuating selective forces play a role in the maintenance of this diversity.

Summary Intraspecific variation in dietary breadth can influence important ecological and evolutionary processes, yet the mechanisms generating this variation are usually unknown. Maternally transmitted bacterial symbionts frequently infect insect herbivores, and many have been shown to mediate key ecological interactions. For polyphagous herbivores, infection with particular symbionts is often strongly correlated with feeding on particular plant species, suggesting that facultative symbionts might directly determine herbivore food plant specificity. However, previous tests of this hypothesis have returned inconsistent results, providing little empirical support for a causal relationship between facultative symbiont infection and dietary breadth. Here, we investigate whether heritable facultative symbionts mediate dietary breadth in the polyphagous aphid, Aphis craccivora. We first determined that asexual clones of the aphid differ dramatically in performance across two leguminous food plants, locust and alfalfa, and could be considered biotypes with distinct ecological characteristics. The heritable symbiont Arsenophonus is strongly associated with locust‐origin aphids. We created experimental lines that share aphid genotypes but differed with respect to Arsenophonus infection status, and compared performance across three food plant species. Naturally Arsenophonus‐infected clones performed 2–4× better on locust and up to 75% worse on two alternate plant species than uninfected controls, clearly demonstrating that Arsenophonus promotes specialization on locust. In both laboratory and field experiments, uninfected locust‐ and alfalfa‐origin clones exhibited similar and modest performance on locust, indicating that the ‘locust‐associated biotype’ would not exist without Arsenophonus. We also hypothesized that moving Arsenophonus, via transinfection, to an alfalfa‐origin lineage would improve performance on locust and serve to expand dietary breadth. Indeed, transinfection doubled aphid performance on locust and halved aphid performance on alfalfa. However, because this aphid lineage naturally performs better on alfalfa, the transinfected symbiont functionally equalized aphid performance between locust and alfalfa, making the alfalfa biotype more generalized. Thus, the same symbiont can either reduce or expand dietary breadth, depending on host genotype. Our results unequivocally demonstrate that symbiont gain or loss can instantaneously and substantially change the topology of food plant use in a polyphagous insect, modifying diet in ways that potentially influence the insect's ecological niche, evolutionary trajectory and pest status.

Environmental factors, including temperature, can have large effects on species interactions, including mutualisms and antagonisms. Most insect species are infected with heritable bacterial symbionts with many protecting their hosts from natural enemies. However, many symbionts or their products are thermally sensitive; hence, their effectiveness may vary across a range of temperatures. In the pea aphid, Acyrthosiphon pisum, the bacterial symbiont Hamiltonella defensa and its associated APSE bacteriophages confer resistance to this aphid's dominant parasitoid, Aphidius ervi. Here, we investigate the effects of temperature on both endogenous and symbiont-based protection against this parasitoid. We also explored the defensive properties of the X-type symbiont, a bacterium hypothesized to shape aphid defence when co-occurring with H. defensa. We show that H. defensa protection fails at higher temperatures, although some aphid genotype and H. defensa strain combinations are more robust than others at moderately warmer temperatures. We also found that a single X-type strain neither defended against parasitism by A. ervi nor rescued lost H. defensa protection at higher temperatures. In contrast, endogenous aphid resistance was effective across temperatures, revealing that these distinct defensive modes are not equally robust to changing environments. Through a survey of field-collected pea aphids, we found a negative correlation between H. defensa frequencies and average daily temperatures across North American locales, fitting expectations for reduced symbiont benefits under warm climates. Based on these findings, we propose that rising global temperatures could promote the widespread breakdown of defensive mutualisms, a prospect with implications for both human and ecosystem health.

The pea aphid, Acyrthosiphon pisum, maintains extreme variation in resistance to its most common parasitoid wasp enemy, Aphidius ervi, which is sourced from two known mechanisms: protective bacterial symbionts, most commonly Hamiltonella defensa, or endogenously encoded defences. We have recently found that individual aphids may employ each defence individually, occasionally both defences together, or neither. In field populations, Hamiltonella-infected aphids are found at low to moderate frequencies and while less is known about the frequency of resistant genotypes, they show up less often than susceptible genotypes in field collections. To better understand these patterns, we sought to compare the strengths and costs of both types of defence, individually and together, in order to elucidate the selective pressures that maintain multi-modal defence mechanisms or that may favour one over the other. We experimentally infected five aphid genotypes (two lowly and three highly resistant), each with two symbiont strains, Hamiltonella-APSE8 (moderate protection) and Hamiltonella-APSE3 (high protection). This resulted in three sublines per genotype: uninfected, +APSE8 and +APSE3. Each of the 15 total sublines was first subjected to a parasitism assay to determine its resistance phenotype and in a second experiment, a subset was chosen to compare fitness (fecundity and survivorship) in the presence and absence of parasitism. In susceptible aphid genotypes, parasitized sublines infected with Hamiltonella generally showed increased protection with direct fitness benefits, but clear infection costs to fitness in the absence of parasitism. In resistant genotypes, Hamiltonella infection rarely conferred additional protection, often further reduced fecundity and survivorship when enemy challenged, and resulted in constitutive fitness costs in the absence of parasitism. We also identified strong aphid genotype × symbiont-strain interactions, such that the best defensive strategy against parasitoids varied for each aphid genotype; one performed best with no protective symbionts, the others with particular strains of Hamiltonella. This surprising variability in outcomes helps explain why Hamiltonella infection frequencies are often intermediate and do not strongly track parasitism frequencies in field populations. We also find that variation in endogenous traits, such as resistance, among host genotypes may offer redundancy and generally limit the invasion potential of mutualistic microbes in insects.

Insects are often attacked by multiple natural enemies, imposing dynamic selective pressures for the development and maintenance of enemy-specific resistance. Pea aphids (Acyrthosiphon pisum) have emerged as models for the study of variation in resistance against natural enemies, including parasitoid wasps. Internal defenses against their most common parasitoid wasp, Aphidius ervi, are sourced through two known mechanisms– 1) endogenously encoded resistance or 2) infection with the heritable bacterial symbiont, Hamiltonella defensa. Levels of resistance can range from nearly 0–100% against A. ervi but varies based on aphid genotype and the strain of toxin-encoding bacteriophage (called APSE) carried by Hamiltonella. Previously, other parasitoid wasps were found to commonly attack this host, but North American introductions of A. ervi have apparently displaced all other parasitoids except Praon pequodorum, a related aphidiine braconid wasp, which is still found attacking this host in natural populations. To explain P. pequodorum’s persistence, multiple studies have compared direct competition between both wasps, but have not examined specificity of host defenses as an indirectly mediating factor. Using an array of experimental aphid lines, we first examined whether aphid defenses varied in effectiveness toward either wasp species. Expectedly, both types of aphid defenses were effective against A. ervi, but unexpectedly, were completely ineffective against P. pequodorum. Further examination showed that P. pequodorum wasps suffered no consistent fitness costs from developing in even highly ‘resistant’ aphids. Comparison of both wasps’ egg-larval development revealed that P. pequodorum’s eggs have thicker chorions and hatch two days later than A. ervi’s, likely explaining their differing abilities to overcome aphid defenses. Overall, our results indicate that aphids resistant to A. ervi may serve as reservoirs for P. pequodorum, hence contributing to its persistence in field populations. We find that specificity of host defenses and defensive symbiont infections, may have important roles in influencing enemy compositions by indirectly mediating the interactions and abundance of rival natural enemies.