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Insects often harbour heritable symbionts that provide defence 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 antifungal and antiparasitoid 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 also housing H. defensa. In contrast, APV titres 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 of 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.

Background Most phages infect free-living bacteria but a few have been identified that infect heritable symbionts of insects or other eukaryotes. Heritable symbionts are usually specialized and isolated from other bacteria with little known about the origins of associated phages. Hamiltonella defensa is a heritable bacterial symbiont of aphids that is usually infected by a tailed, double-stranded DNA phage named APSE. Methods We conducted comparative genomic and phylogenetic studies to determine how APSE is related to other phages and prophages. Results Each APSE genome was organized into four modules and two predicted functional units. Gene content and order were near-fully conserved in modules 1 and 2, which encode predicted DNA metabolism genes, and module 4, which encodes predicted virion assembly genes. Gene content of module 3, which contains predicted toxin, holin and lysozyme genes differed among haplotypes. Comparisons to other sequenced phages suggested APSE genomes are mosaics with modules 1 and 2 sharing similarities with Bordetella-Bcep-Xylostella fastidiosa-like podoviruses, module 4 sharing similarities with P22-like podoviruses, and module 3 sharing no similarities with known phages. Comparisons to other sequenced bacterial genomes identified APSE-like elements in other heritable insect symbionts (Arsenophonus spp.) and enteric bacteria in the family Morganellaceae. Conclusions APSEs are most closely related to phage elements in the genus Arsenophonus and other bacteria in the Morganellaceae.

Facultative, heritable endosymbionts are found at intermediate prevalence within most insect species, playing frequent roles in their hosts' defence 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 frequencies ranged from 23.2% to 68.1% across a six-month longitudinal survey. Rapid spikes and declines were often consistent across fields, and we estimated that selection coefficients for Hamiltonella-infected aphids changed sign within this field season. Prior laboratory research suggested antiparasitoid defence as the major Hamiltonella benefit, and costs under parasitoid absence. While a prior field study suggested these forces can sometimes act as counter-weights in a regime of seasonal balancing selection, our present survey showed no significant relationship between parasitoid wasps and Hamiltonella prevalence. Field cage experiments provided some explanation: parasitoids drove modest ~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 prediction. Instead, our longitudinal survey - and two overwintering studies - showed temperature to be the strongest predictor of Hamiltonella prevalence. Matching some prior lab discoveries, this suggested that thermally sensitive costs and benefits, unrelated to parasitism, can shape Hamiltonella dynamics. These results add to a growing body of evidence for rapid, seasonal adaptation in multivoltine organisms, suggesting that such adaptation can be mediated through the diverse impacts of heritable bacterial endosymbionts.

While the intensification and globalization of agriculture and commerce have provided humanity with numerous benefits, they have simultaneously escalated systemic risk to ecological systems. Global climate change, acting in negative synergy with other anthropogenic stresses, is expected to rapidly increase ongoing losses of biodiversity and profoundly redistribute enduring species. This chapter provides an overview of microbial symbiosis in a rapidly changing world. The ability of many multicellular organisms to relocate, acclimate, or adapt to these changes will depend on their microbial symbionts. But for other groups, specialized symbionts may expose acute vulnerabilities that limit climate resilience unless compensating mechanisms are engaged. We review a number of globally relevant symbioses to examine responses to elevated temperature, drought, ocean acidification, and other climate-driven challenges. While several widespread interactions, including mycorrhizal fungi associated with plants, show the clear potential to mitigate specific threats, others, such as photosymbiotic corals and the obligate nutritional symbioses of arthropods are likely to be among the first mass casualties of climate change.

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.

Like many miniscule phenomena in biology, the importance of intimate, host-associated microbes lay hidden until the timely collaboration between a microscope and a curious mind. Chronicled early in insect groups such as termites, ants, and both sap- and blood-feeders, recent research within the 'omics era has completely transformed our understanding of symbiosis within the hexapods. Bacteria, protists, and fungi shape a range of insect phenotypes, complementing their endogenous abilities for resource acquisition and defence. Other microbes exploit chinks in insect armour, manipulating conserved features of insect cell biology to selfishly spread within host populations. Through the use of ever-improving technology, combined with conceptual and experimental advances, scientists now understand a number of key mechanisms underpinning the function, regulation, and transmission of insect-associated microbes across a range of systems. In this volume of Advances in Insect Physiology we present eight chapters focused on symbiont mechanism, including those involved in resource acquisition among herbivores, detritivores and blood-feeding insects, and those associated with reproductive manipulation and host defence. Within the present introduction, we sandwich summaries of each chapter between a brief historical perspective on insect-microbe symbiosis and a discussion on the implications of growing mechanistic knowledge. We conclude that understanding mechanism brings the potential to manage insects—and their impacts—in a changing world, whether aiding those that are beneficial, or suppressing pest-transmitted diseases. This understanding also promises a powerful, wide-ranging comprehension of complex biological phenomena emerging from symbiosis, spanning the biological spectrum from genes, molecules, cells, organisms, populations, communities, and ecosystems.

All organisms have evolved a wide range of strategies to protect against natural enemies, from complex avoidance behaviours to sophisticated immune systems, and insects are no exception. A more recently recognized, and likely widespread, mode of protection for insects is through the use of microbial symbionts. Many of the best studied examples of defensive symbiosis in insects involve infections with maternally inherited symbionts. While inherited symbionts can protect their insect hosts by priming innate immune systems, or competing with enemies for scarce recourses, they have also been found to synthesize a wide diversity of toxins that effectively kill, weaken, or deter parasitic wasps, parasitic nematodes, pathogenic fungi, and predatory spiders. In this chapter, we provide an overview of defensive symbioses in insects, focusing in particular on toxin-based protection.

Heritable symbionts are common in terrestrial arthropods and often provide beneficial services to hosts. Unlike obligate, nutritional symbionts that largely persist under strict host control within specialized host cells, heritable facultative symbionts exhibit large variation in within-host lifestyles and services rendered with many retaining the capacity to transition among roles. One enigmatic symbiont, Candidatus Fukatsuia symbiotica, frequently infects aphids with reported roles ranging from pathogen, defensive symbiont, mutualism exploiter and nutritional co-obligate symbiont. Here we used an in vitro culture-assisted protocol to sequence the genome of a facultative strain of Fukatsuia from pea aphids (Acyrthosiphon pisum). Phylogenetic and genomic comparisons indicate that Fukatsuia is an aerobic heterotroph, which together with Regiella insecticola and Hamiltonella defensa form a clade of heritable facultative symbionts within the Yersiniaceae (Enterobacteriales). These three heritable facultative symbionts largely share overlapping inventories of genes associated with housekeeping functions, metabolism, and nutrient acquisition, while varying in complements of mobile DNA. One unusual feature of Fukatsuia is its strong tendency to occur as a co-infection with H. defensa. However, the overall similarity of gene inventories among aphid heritable facultative symbionts suggest that metabolic complementarity is not the basis for co-infection, unless playing out on a H. defensa strain-specific basis. We also compared the pea aphid Fukatsuia with a strain from the aphid Cinara confinis (Lachninae) where it is reported to have transitioned to co-obligate status to support decaying Buchnera function. Overall the two genomes are very similar with no clear genomic signatures consistent with such a transition, which suggests co-obligate status in C. confinis was a recent event.

Many insects host facultative, bacterial symbionts that confer conditional fitness benefits to their hosts. Hamiltonella defensa is a common facultative symbiont of aphids that provides protection against parasitoid wasps. Protection levels vary among strains of H. defensa that are also differentially infected by bacteriophages named APSEs. However, little is known about trait variation among strains because only one isolate has been fully sequenced. Generating complete genomes for facultative symbionts is hindered by relatively large genome sizes but low abundances in hosts like aphids that are very small. Here, we took advantage of methods for culturing H. defensa outside of aphids to generate complete genomes and transcriptome data for four strains of H. defensa from the pea aphid Acyrthosiphon pisum. Chosen strains also spanned the breadth of the H. defensa phylogeny and differed in strength of protection conferred against parasitoids. Results indicated that strains shared most genes with roles in nutrient acquisition, metabolism, and essential housekeeping functions. In contrast, the inventory of mobile genetic elements varied substantially, which generated strain specific differences in gene content and genome architecture. In some cases, specific traits correlated with differences in protection against parasitoids, but in others high variation between strains obscured identification of traits with likely roles in defense. Transcriptome data generated continuous distributions to genome assemblies with some genes that were highly expressed and others that were not. Single molecule real-time sequencing further identified differences in DNA methylation patterns and restriction modification systems that provide defense against phage infection.

International audience; Facultative bacterial symbionts can provide their host insects with protection from natural enemies. These symbionts are often found at low to intermediate frequencies among hosts in native populations, suggesting that fewer symbiont taxa (and their corresponding defensive properties) may be present in exotic populations, due to founder effects and drift in newly established populations. We tested this hypothesis by collecting aphid species from their exotic and native regions, and conducting diagnostic surveys for four facultative symbionts: Hamiltonella defensa, Regiella insecticola, Serratia symbiotica, and Arsenophonus nasoniae. We did not find fewer symbiont taxa in exotic host populations, but did find substantial intra-and interpopulation variation in symbiont infection. When we incorporated additional records from the literature, we found moderate support for the hypothesis, although few aphid species were sampled sufficiently to be conclusive. Finally, we tested whether laboratory colonies are prone to losing symbiont infection over time. We established four colonies of the cowpea aphid, Aphis craccivora, each initiated with a single aphid clone infected with H. defensa. Through repeated sampling, we found that all four colonies became uninfected over the course of one year. We suggest that symbiont surveys could aid importation biological control introductions by (1) establishing whether recently established exotic pest populations might have reduced symbiont complements and be particularly vulnerable to natural enemies, (2) providing clues on pest provenance, and (3) determining which native pest populations include the same defensive symbionts as the exotic target populations, as these may be the best sources for prospective agents. We also suggest that laboratory cultures of target and nontarget organisms be routinely monitored for symbiont composition, to ensure that laboratory trials produce field-relevant results.

Endosymbiont interactions with hosts have important effects on fitness, including the fitness of many pest and beneficial species. Among these interactions, facultative endosymbiotic bacteria can protect aphids from parasitoids. Aphis craccivora and Acyrthosiphon pisum can harbor the symbiotic bacteria Hamiltonella defensa and its bacteriophage APSE. Infection by H. defensa defends these aphids against some but not all parasitoid species in the hymenopteran family Braconidae. Here, we report results on the effect of H. defensa on parasitism of these aphids by species in the other major lineage of aphid parasitoids, Aphelinus species in the family Aphelinidae. Parasitism of aphids infected with H. defensa/APSE by two Aphelinus species did not differ from that of uninfected aphids. While Aphelinus atriplicis showed no difference in fitness components between infected and uninfected aphids, Aphelinus glycinis actually produced more adult progeny and larger female progeny on infected than on uninfected aphids. Aphelinus glycinis may increase host quality for itself by changing the titer of nutritional versus protective bacteria in such a way that aphids infected with H. defensa can be made more suitable for parasitoid development than uninfected aphids. Our results and reasoning suggest that these Aphelinus species may be less prone to harm by H. defensa/APSE that affect eggs because they have anhydropic, heavily chorionated eggs, which may not absorb toxins during embryogenesis.

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.

Reciprocal selection between aphids, their protective endosymbionts, and the parasitoid wasps that prey upon them offers an opportunity to study the basis of their coevolution. We investigated adaptation to symbiont-conferred defense by rearing the parasitoid wasp Lysiphlebus fabarum on aphids (Aphis fabae) possessing different defensive symbiont strains (Hamiltonella defensa). After ten generations of experimental evolution, wasps showed increased abilities to parasitize aphids possessing the H. defensa strain they evolved with, but not aphids possessing the other strain. We show that the two symbiont strains encode different toxins, potentially creating different targets for counter-adaptation. Phenotypic and behavioral comparisons suggest that neither life-history traits nor oviposition behavior differed among evolved parasitoid lineages. In contrast, comparative transcriptomics of adult female wasps identified a suite of differentially expressed genes among lineages, even when reared in a common, symbiont-free, aphid host. In concurrence with the specificity of each parasitoid lineages' infectivity, most differentially expressed parasitoid transcripts were also lineage-specific. These transcripts are enriched with putative venom toxins and contain highly expressed, potentially defensive viral particles. Together, these results suggest that wild populations of L. fabarum employ a complicated offensive arsenal with sufficient genetic variation for wasps to adapt rapidly and specifically to their hosts' microbial defenses.

Evolution lacks foresight, and hence, key adaptations may produce major challenges over the long run. The natural world is rife with examples of long-term ‘side effects’ associated with quick-fix tinkering, including blind spots in vertebrate eyes. An important question is how nature compensates for imperfections once evolution has set a course. The symbioses associated with sap-feeding insects present a fascinating opportunity to address this issue. On one hand, the substantial diversity and biomass of sap-feeding insects are largely due to ancient acquisitions of nutrient-provisioning bacterial symbionts. Yet, the insularity and small population sizes enforced by intracellular life and strict maternal transfer inevitably result in the degradation of symbiont genomes and, often, the beneficial services that symbionts provide. Stabilization through lateral transfer of bacterial genes into the host nucleus (often from exogenous sources) or replacement of the long-standing symbiont with a new partner are potential solutions to this evolutionary dilemma (Bennett & Moran 2015). A third solution is adoption of a cosymbiont that compensates for specific losses in the original resident. Ancient ‘co-obligate’ symbiont pairs in mealybugs, leafhoppers, cicadas and spittlebugs show colocalization, codiversification, metabolite exchange and generally nonredundant nutrient biosynthesis (Bennett & Moran 2015). But in this issue, Meseguer et al. (2017) report on a different flavour of cosymbiosis among conifer-feeding Cinara aphids.

Ecologically relevant symbioses are widespread in terrestrial arthropods but based on recent findings these specialized interactions are likely to be especially vulnerable to climate warming. Importantly, empirical data and climate models indicate that warming is occurring asynchronously, with night-time temperatures increasing faster than daytime temperatures. Daytime (DTW) and night-time warming (NTW) may impact ectothermic animals and their interactions differently as DTW results in greater daily temperature variation and moves organisms nearer to their thermal limits, while NTW avoids thermal limits and may relieve constraints of cooler night-time temperatures; a nuance that has largely been ignored in the literature. In laboratory experiments, we investigated how the timing of warming influences a widespread defensive mutualism involving the pea aphid Acyrthosiphon pisum, and its heritable symbiont, Hamiltonella defensa, which protects against an important natural enemy, the parasitic wasp Aphidius ervi. Three aphid sublines were experimentally created from single aphid genotype susceptible to A. ervi: one line infected with a highly protective H. defensa strain, one infected with a moderately protective strain and one without any facultative symbiont. We examined aphid fitness in the presence and absence of parasitoids and when exposed to an average 2.5°C increase occurring across three warming scenarios (night-time vs. daytime vs. uniform) relative to no-warming controls. An increase of 2.5°C, as predicted to occur by the IPCC before 2100, was sufficient to disable the aphid defensive mutualism regardless of the timing of warming; a surprising result given that the daily maxima for control and NTW scenarios were identical. We also found that warming negatively impacted (a) symbiont-mediated interactions between host and parasitoid more than symbiont-free ones; (b) species interactions (host-parasitoid) more than each participant independently and (c) aphids more than parasitoids even though higher trophic levels are generally predicted to be more affected by warming. Here we show that 2.5°C warming, regardless of timing, negatively impacted a common microbe-mediated defensive mutualism. While this was a laboratory-based study, results suggest that temperature increases predicted in the near-term may disrupt the many ecological symbioses present in terrestrial ecosystems.

Insects are frequently infected with inherited facultative symbionts known to provide a range of conditionally beneficial services, including host protection. Pea aphids (Acyrthosiphon pisum) often harbour the bacterium Hamiltonella defensa, which together with its associated bacteriophage A. pisum secondary endosymbiont (APSE) confer protection against an important natural enemy, the parasitic wasp Aphidius ervi. Previous studies showed that spontaneous loss of phage APSE resulted in the complete loss of the protective phenotype. Here, we demonstrate that APSEs can be experimentally transferred into phage-free (i.e. non-protecting) Hamiltonella strains. Unexpectedly, trials using injections of phage particles alone failed, with successful transfer occurring only when APSE and Hamiltonella were simultaneously injected. After transfer, stable establishment of APSE fully restored anti-parasitoid defenses. Thus, phages associated with heritable bacterial symbionts can move horizontally among symbiont strains facilitating the rapid transfer of ecologically important traits although natural barriers may preclude regular exchange.

Protective mutualisms are common in nature and include insect infections with cryptic symbionts that defend against pathogens and parasites. An archetypal defensive symbiont, Hamiltonella defensa protects aphids against parasitoids by disabling wasp development. Successful defense requires H. defensa infection with bacteriophages (APSEs), which play other key roles in mutualism maintenance. Genomes of H. defensa strains are highly similar in gene inventories, varying primarily in mobile element content. Protective phenotypes are highly variable across aphid models depending on H. defensa/APSE, aphid and wasp genotypes. Infection frequencies of H. defensa are highly dynamic in field populations, influenced by a variety of selective and non-selective factors confounding biological control implications. Overall, H. defensa infections likely represent a global aphid protection network with effects radiating outward from focal interactions.

The microbiome can significantly impact host phenotypes and serve as an additional source of heritable genetic variation. While patterns across eukaryotes are consistent with a role for symbiotic microbes in host macroevolution, few studies have examined symbiont-driven host evolution or the ecological implications of a dynamic microbiome across temporal, spatial or ecological scales. The pea aphid, Acyrthosiphon pisum, and its eight heritable bacterial endosymbionts have served as a model for studies on symbiosis and its potential contributions to host ecology and evolution. But we know little about the natural dynamics or ecological impacts of the heritable microbiome of this cosmopolitan insect pest. Here we report seasonal shifts in the frequencies of heritable defensive bacteria from natural pea aphid populations across two host races and geographic regions. Microbiome dynamics were consistent with symbiont responses to host-level selection and findings from one population suggested symbiont-driven adaptation to seasonally changing parasitoid pressures. Conversely, symbiont levels were negatively correlated with enemy-driven mortality when measured across host races, suggesting important ecological impacts of host race microbiome divergence. Rapid drops in symbiont frequencies following seasonal peaks suggest microbiome instability in several populations, with potentially large costs of 'superinfection' under certain environmental conditions. In summary, the realization of several laboratory-derived, a priori expectations suggests important natural impacts of defensive symbionts in host-enemy eco-evolutionary feedbacks. Yet negative findings and unanticipated correlations suggest complexities within this system may limit or obscure symbiont-driven contemporary evolution, a finding of broad significance given the widespread nature of defensive microbes across plants and animals.

Summary Maternally inherited bacterial symbionts are present in many, if not most, insect species. While there is rapidly accumulating evidence that facultative, heritable symbionts often protect insect hosts from natural enemies, there have been few clear examples where facultative symbionts mediate herbivore–plant interactions. The phloem-feeding whitefly, Bemisia tabaci, is a major agricultural pest that frequently harbours facultative symbionts, including Hamiltonella defensa. While H. defensa and other facultative symbionts have been shown to improve whitefly performance on particular food plants, no direct and specific roles for symbiont-mediated interactions with food plants have been identified. Here, we conducted a series of assays to determine whether infection with H. defensa improved whitefly performance on tomato (Solanum lycopersicum) and whether this benefit was associated with symbiont effects on induced plant defences. Finally, we tested whether impacts on induced defences involved in the modulation of a salivary factor. The downregulation of plant defences was associated with increased whitefly fecundity and survival. Feeding by H. defensa-infected whiteflies suppressed JA and JA-related anti-herbivore-induced defences in tomato relative to uninfected controls. That saliva-only treatments of damaged tomato leaves from H. defensa-infected whiteflies suppressed induced plant defences compared to saliva from uninfected controls, suggesting that elicitors in saliva were responsible. Characterization of the putative salivary factor(s) revealed they are likely small (< 3-kDa) and nonproteinaceous. Interestingly, suppression of defences was not observed in SA-deficient NahG plants, indicating that suppression of JA-regulated defences depends on the SA signalling pathway. This finding reveals an intriguing example of the crosstalk between SA and JA signalling pathways and suggests that infection with facultative symbionts can result in the manipulation of induced plant defences to the benefit of the insect host and heritable symbiont. Our results show that the bacterial symbiont H. defensa mediates whitefly–plant interactions by suppressing induced plant defences in tomato. This finding is among the first showing a direct role for facultative symbionts in mediating plant–herbivore interactions and demonstrates a novel tactic for insect herbivores to circumvent plant defences. Symbiont-mediated suppression of plant defences may enhance the deleterious effects of insect pests feeding on important crops.

Animal-associated microbiomes are often comprised of structured, multispecies communities, with particular microbes showing trends of co-occurrence or exclusion. Such structure suggests variable community stability, or variable costs and benefits-possibilities with implications for symbiont-driven host adaptation. In this study, we performed systematic screening for maternally transmitted, facultative endosymbionts of the pea aphid, Acyrthosiphon pisum. Sampling across six locales, with up to 5 years of collection in each, netted significant and consistent trends of community structure. Co-infections between Serratia symbiotica and Rickettsiella viridis were more common than expected, while Rickettsia and X-type symbionts colonized aphids with Hamiltonella defensa more often than expected. Spiroplasma co-infected with other endosymbionts quite rarely, showing tendencies to colonize as a single species monoculture. Field estimates of maternal transmission rates help to explain our findings: while Serratia and Rickettsiella improved each other's transmission, Spiroplasma reduced transmission rates of co-infecting endosymbionts. In summary, our findings show that North American pea aphids harbour recurring combinations of facultative endosymbionts. Common symbiont partners play distinct roles in pea aphid biology, suggesting the creation of "generalist" aphids receiving symbiont-based defence against multiple ecological stressors. Multimodal selection, at the host level, may thus partially explain our results. But more conclusively, our findings show that within-host microbe interactions, and their resulting impacts on transmission rates, are an important determinant of community structure. Widespread distributions of heritable symbionts across plants and invertebrates hint at the far-reaching implications for these findings, and our work further shows the benefits of symbiosis research within a natural context.

Since 2014, populations of the kudzu bug, Megacopta cribraria (F.) (Hemiptera: Plataspidae), have declined in the southeastern United States and seldom require treatment. This decline follows the discovery of Paratelenomus saccharalis (Dodd; Hymenoptera: Platygastridae), a non-native egg parasitoid. The objective of this project was to observe the temporal and spatial dynamics of P. saccharalis parasitism of kudzu bug egg masses in commercial soybean fields. Four fields were sampled weekly for kudzu bugs and egg masses at a density of one sample per 0.6 ha. Sampling commenced when soybean reached the R2 maturity stage and continued until no more egg masses were present. Responses including kudzu bugs, egg masses, and parasitism rates were analyzed using ANOVA, Spatial Analysis by Distance Indices (SADIE), and SaTScan spatial analysis software. Egg masses were collected from the field, held in the lab and monitored for emergence of kudzu bug nymphs or P. saccharalis. Kudzu bug populations were generally lower than previously reported in the literature and spatial aggregation was not consistently observed. Egg parasitism was first detected in early July and increased to nearly 40% in mid-August. Significant spatial patterns in parasitism were observed with spatio-temporal clusters being loosely associated with clusters of egg masses. There were no significant differences in parasitism rates between field margins and interiors, suggesting that P. saccharalis is an effective parasitoid of kudzu bug egg masses on a whole-field scale.

Reciprocal selection between aphids, their protective endosymbionts, and the parasitoid wasps that prey upon them offers an opportunity to study the basis of their coevolution. We investigated adaptation to symbiont-conferred defense by rearing the parasitoid wasp Lysiphlebus fabarum on aphids (Aphis fabae) possessing different defensive symbiont strains (Hamiltonella defensa). After ten generations of experimental evolution, wasps showed increased abilities to parasitize aphids possessing the H. defensa strain they evolved with, but not aphids possessing the other strain. We show that the two symbiont strains encode different toxins, potentially creating different targets for counter-adaptation. Phenotypic and behavioral comparisons suggest that neither life-history traits nor oviposition behavior differed among evolved parasitoid lineages. In contrast, comparative transcriptomics of adult female wasps identified a suite of differentially expressed genes among lineages, even when reared in a common, symbiont-free, aphid host. In concurrence with the specificity of each parasitoid lineages' infectivity, most differentially expressed parasitoid transcripts were also lineage-specific. These transcripts are enriched with putative venom toxins and contain highly expressed, potentially defensive viral particles. Together, these results suggest that wild populations of L. fabarum employ a complicated offensive arsenal with sufficient genetic variation for wasps to adapt rapidly and specifically to their hosts' microbial defenses.

Insects and other animals commonly form symbioses with heritable bacteria, which can exert large influences on host biology and ecology. The pea aphid, Acyrthosiphon pisum , is a model for studying effects of infection with heritable facultative symbionts (HFS), and each of its seven common HFS species has been reported to provide resistance to biotic or abiotic stresses. However, one common HFS, called X-type, rarely occurs as a single infection in field populations and instead typically superinfects individual aphids with Hamiltonella defensa , another HFS that protects aphids against attack by parasitic wasps. Using experimental aphid lines comprised of all possible infection combinations in a uniform aphid genotype, we investigated whether the most common strain of X-type provides any of the established benefits associated with aphid HFS as a single infection or superinfection with H. defensa . We found that X-type does not confer protection to any tested threats, including parasitoid wasps, fungal pathogens, or thermal stress. Instead, component fitness assays identified large costs associated with X-type infection, costs which were ameliorated in superinfected aphids. Together these findings suggest that X-type exploits the aphid/ H. defensa mutualism and is maintained primarily as a superinfection by “hitchhiking” via the mutualistic benefits provided by another HFS. Exploitative symbionts potentially restrict the functions and distributions of mutualistic symbioses with effects that extend to other community members. IMPORTANCE Maternally transmitted bacterial symbionts are widespread and can have major impacts on the biology of arthropods, including insects of medical and agricultural importance. Given that host fitness and symbiont fitness are tightly linked, inherited symbionts can spread within host populations by providing beneficial services. Many insects, however, are frequently infected with multiple heritable symbiont species, providing potential alternative routes of symbiont maintenance. Here we show that a common pea aphid symbiont called X-type likely employs an exploitative strategy of hitchhiking off the benefits of a protective symbiont, Hamiltonella . Infection with X-type provides none of the benefits conferred by other aphid symbionts and instead results in large fitness costs, costs lessened by superinfection with Hamiltonella . These findings are corroborated by natural infections in field populations, where X-type is mostly found superinfecting aphids with Hamiltonella . Exploitative symbionts may be common in hosts with communities of heritable symbionts and serve to hasten the breakdown of mutualisms.

Insects use endogenous mechanisms and infection with protective symbionts to thwart attacks from natural enemies. Defenses that target specific enemies, however, potentially mediate competition between rivals and thereby impact community composition. Following its introduction to North America to control pea aphids (Acyrthosiphon pisum), the parasitoid Aphidius ervi competitively displaced other parasitoids, except for the native Praon pequodorum. The pea aphid exhibits tremendous clonal variation in resistance to A. ervi, primarily through infection with the heritable bacterial symbiont Hamiltonella defensa, although some symbiont-free aphid genotypes encode endogenous resistance. Interestingly, H. defensa strains and aphid genotypes that protect against A. ervi, provide no protection against the closely related, P. pequodorum. Given the specificity of aphid defenses, we hypothesized that aphid resistance traits may contribute to the continued persistence of P. pequodorum. We conducted multiparasitism assays to determine whether aphid resistance traits mediate internal competition between these two solitary parasitoid species, but found this was not the case; P. pequodorum was the successful internal competitor across lines varying in susceptibility to A. ervi. Next, to determine whether resistance traits influence competitive interactions resulting in the stable persistence of P. pequodorum, we established replicated cages varying in the proportion of resistant aphids and recorded successful parasitism for each wasp species over time. As expected, A. ervi outcompeted P. pequodorum in cages containing only susceptible aphids. However, P. pequodorum not only persisted, but was the superior competitor in populations containing any proportion (20-100%) of resistant aphids (20-100%). Smaller scale, better replicated competition cage studies corroborated this finding, and no-competition and behavioral assays provide insight into the processes mediating competition. Genetic variation, including that acquired via infection with protective symbionts, may provide a supply of hosts susceptible only to particular enemies, mediating competition with effects on community richness and stability.

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.

The microbiota inhabiting insects influence a wide range of ecologically-important traits. In addition to their better-known roles in nutrient provisioning and degrading plant polymers, there is emerging evidence that microorganisms also aid herbivores in countering plant defenses. The latter can be mediated by enzymes that degrade plant allelochemicals or via the modulation of plant signaling pathways. Symbionts are also increasingly recognized to protect insects from attack by a wide range of natural enemies. Underlying mechanisms are poorly understood, but some microbes produce antimicrobials or toxins, while others modulate insect immune responses. Ecologically-relevant symbioses can exhibit dynamic variation in strength and specificity of conferred phenotypes, transfer key traits among unrelated insects, and have effects that extend to interacting players and beyond.

1. Infection by the bacterial endosymbiont Hamiltonella defensa is capable of protecting the pea aphid from parasitism by Aphidius ervi and the black bean aphid from parasitism by Lysiphlebus fabarum. Here we investigate protection of a third aphid species, the cowpea aphid, Aphis craccivora, from four parasitoid species: Binodoxys communis, B. koreanus, Lysiphlebus orientalis, and Aphidius colemani. 2. We compared parasitism of A. craccivora lines that were either infected with, or cured of H. defensa separately for the four parasitoid species. Infection by H. defensa almost completely eliminated parasitism of A. craccivora by B. communis and B. koreanus, but had no effect on parasitism by L. orientalis and A. colemani. 3. This indicates at least genus-level specificity of protective effects by H. defensa and we discuss implications of our findings on the known world-wide distribution of this host/symbiont interaction.

Bacterial symbionts infect most insect species, including important pests such as whitefly, Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae), and often exert important effects on host ecology. The facultative symbiont Hamiltonella is found at high frequencies in the B. tabaciMED (type: Mediterranean—MED) in China. The prevalence of this symbiont in natural populations suggests beneficial effects of infection or manipulation of host reproduction. To date, however, no empirical studies on the biological role of Hamiltonella on the host B. tabaci have been reported. Here, we investigated the effects of Hamiltonella infection on the sex ratio and several fitness parameters in B. tabaci MED by comparing Hamiltonella-infected whiteflies with Hamiltonella-free ones. We found that Hamiltonella-infected whiteflies produced significantly more eggs, exhibited significantly higher nymphal survival, faster development times, and larger adult body size in comparison with Hamiltonella-free whiteflies, while ...

Insects often carry heritable symbionts that negotiate interactions with food plants or natural enemies. All pea aphids, Acyrthosiphon pisum, require infection with the nutritional symbiont Buchnera, and many are also infected with Hamiltonella, which protects against the parasitoid Aphidius ervi. Hamiltonella-based protection requires bacteriophages called APSEs with protection levels varying by strain and associated APSE. Endoparasitoids, including A. ervi, may benefit from protecting the nutritional symbiosis and suppressing the protective one, while the aphid and its heritable symbionts have aligned interests when attacked by the wasp. We investigated the effects of parasitism on the abundance of aphid nutritional and protective symbionts. First, we determined strength of protection associated with multiple symbiont strains and aphid genotypes as these likely impact symbiont responses. Unexpectedly, some A. pisum genotypes cured of facultative symbionts were resistant to parasitism and resistant aphid lines carried Hamiltonella strains that conferred no additional protection. Susceptible aphid clones carried protective strains. qPCR estimates show that parasitism significantly influenced both Buchnera and Hamiltonella titres, with multiple factors contributing to variation. In susceptible lines, parasitism led to increases in Buchnera near the time of larval wasp emergence consistent with parasite manipulation, but effects were variable in resistant lines. Parasitism also resulted in increases in APSE and subsequent decreases in Hamiltonella, and we discuss how this response may relate to the protective phenotype. In summary, we show that parasitism alters the within-host ecology of both nutritional and protective symbioses with effects likely significant for all players in this antagonistic interaction.

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.

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.

Symbiosis is the persistent association of two or more dissimilar species. This phenomenon is widespread in nature and has contributed to major transitions in the evolution of life on Earth. Symbiosis can serve as a source of evolutionary innovation. Diverse plant and animal species harbor microbial symbionts that aid their hosts in resource acquisition and defense. Symbioses can originate from diverse interaction types, and can be maintained or broken down through a variety of mechanisms. Understanding and manipulating symbioses may provide direct benefits to human health and agricultural systems.

Despite the well-described importance of bacteriophages to bacterial pathogens, little is known about their influence on the many bacterial species that form beneficial symbioses with eukaryotes. Most insect species, for example, are infected with one or more maternally inherited symbionts, which provide nutritional and defensive services in exchange for housing. The pea aphid, Acyrthosiphon pisum, harbors at least seven common heritable symbionts that mediate a range of ecological interactions and has emerged as a model for studies of beneficial symbionts. One common pea aphid defensive symbiont, Hamiltonella defensa, protects against parasitic wasps, which are important natural enemies. The bacterium is itself infected by temperate bacteriophages, called APSEs (A cyrthosiphon p isum secondary endosymbionts), which are necessary for H. defensa-mediated protection. This represents the first known instance of a bacteria-insect mutualism requiring a viral partner. APSEs play other key roles in the regulation and maintenance of H. defensa: APSE loss results in high titers of the bacterial symbiont, which is correlated with severe fitness costs to the aphid host. These costs to the aphid incurred by phage loss likely lead to phage-free H. defensa-infected aphids being rapidly removed from the population, thus limiting the invasion potential of H. defensa. Below we review the roles of APSEs in the H. defensa-aphid defensive symbiosis and suggest a framework for future studies. We then make comparisons with another well-studied phage-symbiont interaction (Wolbachia-WO) and consider the roles of bacteriophages in the evolution of heritable symbioses and how they may influence other insect-associated bacteria, such as the gut microbiota.

Mutualists that provide redundant services to the same organism have been shown both to coexist and compete for access to the partner. Aphids, for example, are known to receive protection against natural enemies from both heritable bacterial symbionts as well as ants, which tend aphids in exchange for sugary honeydew excretions. Little is known, however, about how ant attendance might influence interactions between aphids and their protective bacteria. Assuming costs to infection, we hypothesized that protective symbiont prevalence would decrease in the presence of ants, because of duplication of services. We surveyed natural aphid populations and found Hamiltonella defensa, a bacterium that provides resistance to aphids against parasitoid wasps in laboratory settings, among several aphid species, including Obtusicauda frigidae (Oestlund 1886). We excluded ants from protecting O. frigidae aggregations and measured the effect of ant absence on the frequency of parasitoid attack and H. defensa infection in aphid groups. Ant presence did not reduce H. defensa frequency in aphids, likely because ants and H. defensa did not significantly lower parasitoid wasp attack rates. Metabolic costs likely control H. defensa prevalence more than the effect of ant-tending.

Insects use endogenous mechanisms and infection with protective symbionts to thwart attacks from natural enemies. Defenses that target specific enemies, however, potentially mediate competition between rivals and thereby impact community composition. Following its introduction to North America to control pea aphids (Acyrthosiphon pisum), the parasitoid Aphidius ervi competitively displaced other parasitoids, except for the native Praon pequodorum. The pea aphid exhibits tremendous clonal variation in resistance to A. ervi, primarily through infection with the heritable bacterial symbiont Hamiltonella defensa, although some symbiont-free aphid genotypes encode endogenous resistance. Interestingly, H. defensa strains and aphid genotypes that protect against A. ervi, provide no protection against the closely related, P. pequodorum. Given the specificity of aphid defenses, we hypothesized that aphid resistance traits may contribute to the continued persistence of P. pequodorum. We conducted multiparasitism assays to determine whether aphid resistance traits mediate internal competition between these two solitary parasitoid species, but found this was not the case; P. pequodorum was the successful internal competitor across lines varying in susceptibility to A. ervi. Next, to determine whether resistance traits influence competitive interactions resulting in the stable persistence of P. pequodorum, we established replicated cages varying in the proportion of resistant aphids and recorded successful parasitism for each wasp species over time. As expected, A. ervi outcompeted P. pequodorum in cages containing only susceptible aphids. However, P. pequodorum not only persisted, but was the superior competitor in populations containing any proportion (20–100%) of resistant aphids (20–100%). Smaller scale, better replicated competition cage studies corroborated this finding, and no-competition and behavioral assays provide insight into the processes mediating competition. Genetic variation, including that acquired via infection with protective symbionts, may provide a supply of hosts susceptible only to particular enemies, mediating competition with effects on community richness and stability.

Aphids engage in symbiotic associations with a diverse assemblage of heritable bacteria. In addition to their obligate nutrient-provisioning symbiont, Buchnera aphidicola, aphids may also carry one or more facultative symbionts. Unlike obligate symbionts, facultative symbionts are not generally required for survival or reproduction and can invade novel hosts, based on both phylogenetic analyses and transfection experiments. Facultative symbionts are mutualistic in the context of various ecological interactions. Experiments on pea aphids (Acyrthosiphon pisum) have demonstrated that facultative symbionts protect against entomopathogenic fungi and parasitoid wasps, ameliorate the detrimental effects of heat, and influence host plant suitability. The protective symbiont, Hamiltonella defensa, has a dynamic genome, exhibiting evidence of recombination, phage-mediated gene uptake, and horizontal gene transfer and containing virulence and toxin-encoding genes. Although transmitted maternally with high fidelity, facultative symbionts occasionally move horizontally within and between species, resulting in the instantaneous acquisition of ecologically important traits, such as parasitoid defense.

Heritable symbionts are common in insects with many contributing to host defence. Hamiltonella defensa is a facultative, bacterial symbiont of the pea aphid, Acyrthosiphon pisum that provides protection against the endoparasitoid wasp Aphidius ervi . Protection levels vary among strains of H. defensa that are differentially infected by bacteriophages named APSEs. By contrast, little is known about mechanism(s) of resistance owing to the intractability of host-restricted microbes for functional study. Here, we developed methods for culturing strains of H. defensa that varied in the presence and type of APSE. Most H. defensa strains proliferated at 27°C in co-cultures with the TN5 cell line or as pure cultures with no insect cells. The strain infected by APSE3, which provides high levels of protection in vivo , produced a soluble factor(s) that disabled development of A. ervi embryos independent of any aphid factors. Experimental transfer of APSE3 also conferred the ability to disable A. ervi development to a phage-free strain of H. defensa . Altogether, these results provide a critical foundation for characterizing symbiont-derived factor(s) involved in host protection and other functions. Our results also demonstrate that phage-mediated transfer of traits provides a mechanism for innovation in host restricted symbionts.

Interactions between multiple anthropogenic environmental changes can drive non-additive effects in ecological systems, and the non-additive effects can in turn be amplified or dampened by spatial covariation among environmental changes. We investigated the combined effects of night-time warming and light pollution on pea aphids and two predatory ladybeetle species. As expected, neither night-time warming nor light pollution changed the suppression of aphids by the ladybeetle species that forages effectively in darkness. However, for the more-visual predator, warming and light had non-additive effects in which together they caused much lower aphid abundances. These results are particularly relevant for agriculture near urban areas that experience both light pollution and warming from urban heat islands. Because warming and light pollution can have non-additive effects, predicting their possible combined consequences over broad spatial scales requires knowing how they co-occur. We found that night-time temperature change since 1949 covaried positively with light pollution, which has the potential to increase their non-additive effects on pea aphid control by 70% in US alfalfa. Our results highlight the importance of non-additive effects of multiple environmental factors on species and food webs, especially when these factors co-occur.