Cohabitation and roommate bias of symbiotic bacteria in insect hosts

Symbiotic interactions between insects and bacteria have long fascinated ecologists. Aphids have emerged as the model system on which to study the effect of endosymbiotic bacteria on their hosts. Aphid‐symbiont interactions are ecologically interesting as aphids host multiple secondary symbionts that can provide broad benefits, such as protection against heat stress or specialist natural enemies (parasitic wasps and entomopathogenic fungi). There are nine common aphid secondary symbionts and individual aphids host on average 1–2 symbionts. A cost‐benefit trade‐off for hosting symbionts is thought to explain why not all aphids host every possible symbiont in a population. Both positive and negative associations between various symbionts occur, and this could happen due to increased costs when cohosting certain combinations or as a consequence of competitive interactions between the symbionts within a host. In this issue of Molecular Ecology, Mathé‐Hubert, Kaech, Hertaeg, Jaenike, and Vorburger (2019) use data on the symbiont status of field‐collected aphids to inform a model on the evolution of symbiont co‐occurrence. They vary the effective female population size as well as the rate of horizontal and maternal transmission to infer the relative impact of symbiont‐symbiont interactions versus random drift. Additional data analysis revisits an association between two symbionts in a fruit fly species using a long‐term data set to highlight that such interactions are not limited to aphids.     

Effects of biodiversity in agricultural landscapes on the protective microbiome of insects – a review

Symbiotic bacteria in herbivorous insects can have strong beneficial impacts on their host's survival, including conferring resistance to natural enemies such as parasitoid wasps or pathogens, while also imposing energetic costs on the host, resulting in cost‐benefit trade‐offs. Whether these trade‐offs favour the hosting of symbionts depends on the growth environment of the herbivore. Long‐term experimental grassland studies have shown that increasing plant species richness leads to an increased diversity of associated herbivores and their natural enemies. Such a change in natural enemy diversity, related to changes in plant diversity, could also drive changes in the community of symbionts hosted by the herbivorous insects. Aphids are one model system for studying symbionts in insects, and effects of host‐plant species and diversity on aphid‐symbiont interactions have been documented. Yet, we still understand little of the mechanisms underlying such effects. We review the current state of knowledge of how biodiversity can impact aphid‐symbiont communities and the underlying drivers. Then, we discuss this in the framework of sustainable agriculture, where increased plant biodiversity, in the form of wildflower strips, is used to recruit natural enemies to crop fields for their pest control services. Although aphid symbionts have the potential to reduce biological control effectiveness through conferring protection for the host insect, we discuss how increasing plant and natural enemy biodiversity can mitigate these effects and identify future research opportunities. Understanding how to promote beneficial interactions in ecological systems can help in the development of more sustainable agricultural management strategies.     

Effects of bacterial secondary symbionts on host plant use in pea aphids

Aphids possess several facultative bacterial symbionts that have important effects on their hosts'' biology. These have been most closely studied in the pea aphid (Acyrthosiphon pisum), a species that feeds on multiple host plants. Whether secondary symbionts influence host plant utilization is unclear. We report the fitness consequences of introducing different strains of the symbiont Hamiltonella defensa into three aphid clones collected on Lathyrus pratensis that naturally lack symbionts, and of removing symbionts from 20 natural aphid–bacterial associations. Infection decreased fitness on Lathyrus but not on Vicia faba, a plant on which most pea aphids readily feed. This may explain the unusually low prevalence of symbionts in aphids collected on Lathyrus. There was no effect of presence of symbiont on performance of the aphids on the host plants of the clones from which the H. defensa strains were isolated. Removing the symbiont from natural aphid–bacterial associations led to an average approximate 20 per cent reduction in fecundity, both on the natural host plant and on V. faba, suggesting general rather than plant-species-specific effects of the symbiont. Throughout, we find significant genetic variation among aphid clones. The results provide no evidence that secondary symbionts have a major direct role in facilitating aphid utilization of particular host plant species.     

Cascading effects of herbivore protective symbionts on hyperparasitoids

1. Microbial symbionts can play an important role in defending their insect hosts against natural enemies. However, researchers have little idea how the presence of such protective symbionts impacts food web interactions and species diversity. 2. This study investigated the effects of a protective symbiont (Hamiltonella defensa) in pea aphids (Acyrthosiphon pisum) on hyperparasitoids, which are a trophic level above the natural enemy target of the symbiont (primary parasitoids). 3. Pea aphids, with and without their natural infections of H. defensa, were exposed first to a primary parasitoid against which the symbiont provides partial protection (either Aphidius ervi or Aphelinus abdominalis), and second to a hyperparasitoid known to attack the primary parasitoid species. 4. It was found that hyperparasitoid hatch rate was substantially affected by the presence of the symbiont. This effect appears to be entirely due to the removal of potential hosts by the action of the symbiont: there was no additional benefit or cost experienced by the hyperparasitoids in response to symbiont presence. The results were similar across the two different aphid–parasitoid–hyperparasitoid interactions we studied. 5. It is concluded that protective symbionts can have an important cascading effect on multiple trophic levels by altering the success of natural enemies, but that there is no evidence for more complex interactions. These findings demonstrate that the potential influence of protective symbionts on the wider community should be considered in future food web studies.     

Defensive insect symbiont leads to cascading extinctions and community collapse

Animals often engage in mutualistic associations with microorganisms that protect them from predation, parasitism or pathogen infection. Studies of these interactions in insects have mostly focussed on the direct effects of symbiont infection on natural enemies without studying community‐wide effects. Here, we explore the effect of a defensive symbiont on population dynamics and species extinctions in an experimental community composed of three aphid species and their associated specialist parasitoids. We found that introducing a bacterial symbiont with a protective (but not a non‐protective) phenotype into one aphid species led to it being able to escape from its natural enemy and increase in density. This changed the relative density of the three aphid species which resulted in the extinction of the two other parasitoid species. Our results show that defensive symbionts can cause extinction cascades in experimental communities and so may play a significant role in the stability of consumer‐herbivore communities in the field.     

Diversity of bacteria associated with Hormaphidinae aphids (Hemiptera: Aphididae)

Bacteria are ubiquitous inhabitants of animals.Hormaphidinae is a particular aphid group exhibiting very diverse life history traits.However,the microbiota in this group is poorly known.In the present study,using high-throughput sequencing of bacterial 16S ribosomal RNA gene amplicons,we surveyed the bacterial flora in hormaphidine aphids and explored whether the aphid tribe,host plant and geographical distribution are associated with the distribution of secondary symbionts.The most dominant bacteria detected in hormaphidine species are heritable symbionts.As expected,the primary endosymbiont Buchnera aphidicola is the most abundant symbiont across all species and has cospeciated with its host aphids.Six secondary symbionts were detected in Hormaphidinae.Arsenophonus is widespread in Hormaphidinae species,suggesting the possibility of ancient acquisition of this symbiont.Ordination analyses and statistical tests show that the symbiont composition does not seem to relate to any of the aphid tribes,host plants or geographical distributions,which indicate that horizontal transfers might occur for these symbionts in Hormaphidinae.Correlation analysis exhibits negative interference between Buchnera and coexisting secondary symbionts,while the interactions between different secondary symbionts are complicated.These findings display a comprehensive picture of the microbiota in Hormaphidinae and may be helpful in understanding the symbiont diversity within a group of aphids.     

Symbiont infection affects aphid defensive behaviours

Aphids harbour both an obligate bacterial symbiont, Buchnera aphidicola, and a wide range of facultative ones. Facultative symbionts can modify morphological, developmental and physiological host traits that favour their spread within aphid populations. We experimentally investigated the idea that symbionts may also modify aphid behavioural traits to enhance their transmission. Aphids exhibit many behavioural defences against enemies. Despite their benefits, these behaviours have some associated costs leading to reduction in aphid reproduction. Some aphid individuals harbour a facultative symbiont Hamiltonella defensa that provides protection against parasitoids. By analysing aphid behaviours in the presence of parasitoids, we showed that aphids infected with H. defensa exhibited reduced aggressiveness and escape reactions compared with uninfected aphids. The aphid and the symbiont have both benefited from these behavioural changes: both partners reduced the fitness decrements associated with the behavioural defences. Such symbiont-induced changes of behavioural defences may have consequences for coevolutionary processes between host organisms and their enemies.     

An ecological cost associated with protective symbionts of aphids

Beneficial symbioses are widespread and diverse in the functions they provide to the host ranging from nutrition to protection. However, these partnerships with symbionts can be costly for the host. Such costs, so called “direct costs”, arise from a trade‐off between allocating resources to symbiosis and other functions such as reproduction or growth. Ecological costs may also exist when symbiosis negatively affects the interactions between the host and other organisms in the environment. Although ecological costs can deeply impact the evolution of symbiosis, they have received little attention. The pea aphid Acyrthosiphon pisum benefits a strong protection against its main parasitoids from protective bacterial symbionts. The ecological cost of symbiont‐mediated resistance to parasitism in aphids was here investigated by analyzing aphid behavior in the presence of predatory ladybirds. We showed that aphids harboring protective symbionts expressed less defensive behaviors, thus suffering a higher predation than symbiont‐free aphids. Consequently, our study indicates that this underlined ecological cost may affect both the coevolutionary processes between symbiotic partners and the prevalence of such beneficial bacterial symbionts in host natural populations.     

Microbiota associated with Mollitrichosiphum aphids (Hemiptera: Aphididae: Greenideinae): diversity,host species specificity and phylosymbiosis

Symbiotic association is universal in nature, and an array of symbionts play a crucial part in host life history. Aphids and their diverse symbionts have become a good model system to study insect-symbiont interactions. Previous symbiotic diversity surveys have mainly focused on a few aphid clades, and the relative importance of different factors regulating microbial community structure is not well understood. In this study, we collected 65 colonies representing eight species of the aphid genus Mollitrichosiphum from different regions and plants in southern China and Nepal and characterized their microbial compositions using Illumina sequencing of the V3 − V4 hypervariable region of the 16S rRNA gene. We evaluated how microbiota varied across aphid species, geography and host plants and the correlation between microbial community structure and host aphid phylogeny. Heritable symbionts dominated the microbiota associated with Mollitrichosiphum, and multiple infections of secondary symbionts were prevalent. Ordination analyses and statistical tests highlighted the contribution of aphid species in shaping the structures of bacterial, symbiont and secondary symbiont communities. Moreover, we observed a significant correlation between Mollitrichosiphum aphid phylogeny and microbial community composition, providing evidence for a pattern of phylosymbiosis between natural aphid populations and their microbial associates.     

Examination of the success rate of secondary symbiont manipulation by microinjection methods in the pea aphid system

Insects harbor a wide range of microbial symbionts, but their influence on host phenotypes is described in a limited number of biological models. One experimental approach to gain knowledge on the effects of symbionts to their hosts is to create insect lines with and without symbionts and examine their phenotypes. However, the success rate of symbiont elimination and introduction methods is dependent on several parameters that are scarcely tested or described. The pea aphid, Acyrthosiphon pisum Harris (Hemiptera: Aphididae), is a model insect of symbiosis studies. It harbors a primary symbiont that supplies the host with essential amino acids, and an array of secondary symbionts whose effects have been assessed by manipulating their presence/absence in the insect. Here, we describe the influence of key parameters on the success rate of symbiont manipulation using the pea aphid–secondary symbiont system. We compared two elimination methods differing in antibiotic treatment using several aphid–symbiont combinations. We also created new aphid host–symbiont combinations by secondary symbiont introduction and examined the effects of larval stage of recipient aphids on introduction success. Our study revealed that the aphid–symbiont combination has strong influence on both symbiont introduction and elimination success rates, and that the type of antibiotics and the larval stage of recipient aphids influence the elimination and introduction success rate, respectively.     

Variation and covariation of life history traits in aphids are related to infection with the facultative bacterial endosymbiont Hamiltonella defensa

Host–symbiont associations play an important role in insects. In aphids, facultative symbionts affect host plant use and increase thermal tolerance and resistance to natural enemies. In spite of these beneficial effects on aphid fitness, the frequency of facultative symbionts in aphids ranges from low to intermediate. Tradeoffs induced by symbionts could prevent the fixation of symbionts in aphid populations. Therefore, we studied the life history traits and correlations between them in 21 clones of the black bean aphid, Aphis fabae, seven of which were infected with the facultative endosymbiont Hamiltonella defensa. We found that clones harbouring H. defensa exhibited significantly higher body mass at maturity and offspring production, and a marginally higher intrinsic rate of increase. However, development time and offspring body size did not differ between symbiont‐free and infected clones. In addition, body mass at maturity was positively correlated with offspring production, offspring body size and intrinsic rate of increase, whereas development time was negatively correlated with body mass at maturity, offspring production and offspring body size. Excluding infected clones had little effect on these correlations; only correlations between body mass at maturity and offspring production, and between development time and offspring body size, became nonsignificant. Therefore, we did not find any evidence for tradeoffs between life history traits induced by symbiont infection. In fact, infected clones had higher overall fitness than symbiont‐free clones under the conditions of our experiment, suggesting that symbionts do not impose costs on aphids harbouring them. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 100 , 237–247.     

Effects of pea aphid secondary endosymbionts on aphid resistance and development of the aphid parasitoid Aphidius ervi: a correlative study

In order to reduce parasite‐induced mortality, hosts may be involved in mutualistic interactions in which the partner contributes to resistance against the parasite. The pea aphid, Acyrthosiphon pisum Harris (Hemiptera: Aphididae), harbours secondary bacterial endosymbionts, some of which have been reported to confer resistance against aphid parasitoids. Although this resistance often results in death of the developing parasitoid larvae, some parasitoid individuals succeed in developing into adults. Whether these individuals suffer from fitness reduction compared to parasitoids developing in pea aphid clones without symbionts has not been tested so far. Using 30 pea aphid clones that differed in their endosymbiont complement, we studied the effects of these endosymbionts on aphid resistance against the parasitoid Aphidius ervi Haliday (Hymenoptera: Braconidae: Aphidiinae), host–parasitoid physiological interactions, and fitness of emerging adult parasitoids. The number of symbiont species in an aphid clone was positively correlated with a number of resistance measurements but there were also clear symbiont‐specific effects on the host–parasitoid interaction. As in previous studies, pea aphid clones infected with Hamiltonella defensa Moran et al. showed resistance against the parasitoid. In addition, pea aphid clones infected with Regiella insecticola Moran et al. and co‐infections of H. defensa–Spiroplasma, R. insecticola–Spiroplasma, and R. insecticola–H. defensa showed reduced levels of parasitism and mummification. Parasitoids emerging from symbiont‐infected aphid clones often had a longer developmental time and reduced mass. The number of teratocytes was generally lower when parasitoids oviposited in aphid clones with a symbiont complement. Interestingly, unparasitized aphids infected with Serratia symbiotica Moran et al. and R. insecticola had a higher fecundity than unparasitized aphids of uninfected pea aphid clones. We conclude that in addition to conferring resistance, pea aphid symbionts also negatively affect parasitoids that successfully hatch from aphid mummies. Because of the link between aphid resistance and the number of teratocytes, the mechanism underlying resistance by symbiont infection may involve interference with teratocyte development.     

Influence of “protective” symbionts throughout the different steps of an aphid–parasitoid interaction

Microbial associates are widespread in insects, some conferring a protection to their hosts against natural enemies like parasitoids. These protective symbionts may affect the infection success of the parasitoid by modifying behavioral defenses of their hosts, the development success of the parasitoid by conferring a resistance against it or by altering life-history traits of the emerging parasitoids. Here, we assessed the effects of different protective bacterial symbionts on the entire sequence of the host-parasitoid interaction (i.e., from parasitoid attack to offspring emergence) between the pea aphid, Acyrthosiphon pisum, and its main parasitoid, Aphidius ervi and their impacts on the life-history traits of the emerging parasitoids. To test whether symbiont-mediated phenotypes were general or specific to particular aphid–symbiont associations, we considered several aphid lineages, each harboring a different strain of either Hamiltonella defensa or Regiella insecticola, two protective symbionts commonly found in aphids. We found that symbiont species and strains had a weak effect on the ability of aphids to defend themselves against the parasitic wasps during the attack and a strong effect on aphid resistance against parasitoid development. While parasitism resistance was mainly determined by symbionts, their effects on host defensive behaviors varied largely from one aphid–symbiont association to another. Also, the symbiotic status of the aphid individuals had no impact on the attack rate of the parasitic wasps, the parasitoid emergence rate from parasitized aphids nor the life-history traits of the emerging parasitoids. Overall, no correlations between symbiont effects on the different stages of the host–parasitoid interaction was observed, suggesting no trade-offs or positive associations between symbiont-mediated phenotypes. Our study highlights the need to consider various sequences of the host-parasitoid interaction to better assess the outcomes of protective symbioses and understand the ecological and evolutionary dynamics of insect–symbiont associations.     

Insect life history and the evolution of bacterial mutualism

Bacterial symbiosis has played a fundamental role in the evolution of eukaryotes. However, we still know little about how cooperative relationships with bacteria originate, and why they form in some host species but not others. Facultative symbionts that are beneficial, but not essential, provide unique insights into these processes. We use data from over a hundred aphid species to test if host life history is associated with the presence of facultative symbionts. We find that aphid species that have mutualistic associations with ants that protect them from natural enemies are less likely to carry symbionts that provide similar benefits. We also find one symbiont species occurs more frequently in unrelated aphid species that specialise on certain plant genera. In addition, aphid species that attack multiple plants often carry different symbiont complements. Our findings provide evidence of the ecological conditions that facilitate stable, mutually beneficial relationships between microbes and eukaryotic hosts.     

Bacterial symbionts in insects or the story of communities affecting communities

Bacterial symbionts are widespread in insects and other animals. Most of them are predominantly vertically transmitted, along with their hosts' genes, and thus extend the heritable genetic variation present in one species. These passengers have a variety of repercussions on the host's phenotypes: besides the cost imposed on the host for maintaining the symbiont population, they can provide fitness advantages to the host or manipulate the host's reproduction. We argue that insect symbioses are ideal model systems for community genetics. First, bacterial symbionts directly or indirectly affect the interactions with other species within a community. Examples include their involvement in modifying the use of host plants by phytophagous insects, in providing resistance to natural enemies, but also in reducing the global genetic diversity or gene flow between populations within some species. Second, one emerging picture in insect symbioses is that many species are simultaneously infected with more than one symbiont, which permits studying the factors that shape bacterial communities; for example, horizontal transmission, interactions between host genotype, symbiont genotype and the environment and interactions among symbionts. One conclusion is that insects' symbiotic complements are dynamic communities that affect and are affected by the communities in which they are embedded.     

The evolutionary ecology of symbiont‐conferred resistance to parasitoids in aphids

Aphids may harbor a wide variety of facultative bacterial endosymbionts. These symbionts are transmitted maternally with high fidelity and they show horizontal transmission as well, albeit at rates too low to enable infectious spread. Such symbionts need to provide a net fitness benefit to their hosts to persist and spread. Several symbionts have achieved this by evolving the ability to protect their hosts against parasitoids. Reviewing empirical work and some models, I explore the evolutionary ecology of symbiont‐conferred resistance to parasitoids in order to understand how defensive symbiont frequencies are maintained at the intermediate levels observed in aphid populations. I further show that defensive symbionts alter the reciprocal selection between aphids and parasitoids by augmenting the heritable variation for resistance, by increasing the genetic specificity of the host–parasitoid interaction, and by inducing environment‐dependent trade‐offs. These effects are conducive to very dynamic, symbiont‐mediated coevolution that is driven by frequency‐dependent selection. Finally I argue that defensive symbionts represent a problem for biological control of pest aphids, and I propose to mitigate this problem by exploiting the parasitoids’ demonstrated ability to rapidly evolve counteradaptations to symbiont‐conferred resistance.     

Diversity of the Most Commonly Reported Facultative Symbionts in Two Closely-Related Aphids with Different Host Ranges

Richness and abundance of facultative symbionts vary strongly with aphid species and genotype, symbiont strain, host plant, biogeography, and a number of abiotic factors. Despite indications that aphids in the same ecological niche show similar levels of facultative symbiont richness, existing reports do not consider the potential role of host plants on aphid microbial community. Little is known about how oligophagy and polyphagy may be influenced by secondary symbiont distribution, mainly because studies on secondary symbiont diversity are biased towards polyphagous aphids from the Northern Hemisphere. Here, we demonstrate the richness and abundance of the most common aphid-associated facultative symbionts in two tropical aphid species, the oligophagous Aphis (Toxoptera) citricidus (Kirkaldy) (Hemiptera: Aphididae) and the polyphagous Aphis aurantii (Boyer de Fonscolombe) (Hemiptera: Aphididae). Aphis citricidus is restricted to Citrus sp. host plants and closely related genera, whereas A. aurantii successfully exploits a wide variety of host plants from different families. Both were collected in the same ecological niche and our data basically indicated the same richness of secondary symbionts, but the abundance at which secondary symbionts occurred was very distinct between the two species. Spiroplasma was the most abundant facultative symbiont associated with A. citricidus and A. aurantii in the ecological niche studied. Single and multiple secondary symbiont infections were observed, but diversity of multiple infections was particularly high in A. citricidus. We discuss our findings and suggest hypotheses to explain causes and consequences of the differences in secondary symbiont diversity observed between these two aphid species.     

Bottom–up regulates top–down: the effects of hybridization of grass endophytes on an aphid herbivore and its generalist predator

The ecological consequences of hybridization of microbial symbionts are largely unknown. We tested the hypothesis that hybridization of microbial symbionts of plants can negatively affect performance of herbivores and their natural enemies. In addition, we studied the effects of hybridization of these symbionts on feeding preference of herbivores and their natural enemies. We used Arizona fescue as the host‐plant, Neotyphodium endophytes as symbionts, the bird cherry–oat aphid as the herbivore and the pink spotted ladybird beetle as the predator in controlled experiments. Neither endophyte infection (infected or not infected) nor hybrid status (hybrid and non‐hybrid infection) affected aphid reproduction, proportion of winged forms in the aphid populations, aphid host‐plant preference or body mass of the ladybirds. However, development of ladybird larvae was delayed when fed with aphids grown on hybrid (H+) endophyte infected grasses compared to larvae fed with aphids from non‐hybrid (NH+) infected grasses, non‐hybrid, endophyte‐removed grasses (NH?) and hybrid, endophyte‐removed (H?) grasses. Furthermore, adult beetles were more likely to choose all other types of grasses harboring aphids rather than H+ infected grasses. In addition, development of ladybirds was delayed when fed with aphids from naturally uninfected (E?) grasses compared to ladybirds that were fed with aphids from NH+ and NH? grasses. Our results suggest that hybridization of microbial symbionts may negatively affect generalist predators such as the pink spotted ladybird and protect herbivores like the bird cherry–oat aphids from predation even though the direct effects on herbivores are not evident.     

Facultative symbiont infections affect aphid reproduction

Some bacterial symbionts alter their hosts reproduction through various mechanisms that enhance their transmission in the host population. In addition to its obligatory symbiont Buchnera aphidicola, the pea aphid Acyrthosiphon pisum harbors several facultative symbionts influencing several aspects of host ecology. Aphids reproduce by cyclical parthenogenesis whereby clonal and sexual reproduction alternate within the annual life cycle. Many species, including the pea aphid, also show variation in their reproductive mode at the population level, with some lineages reproducing by cyclical parthenogenesis and others by permanent parthenogenesis. While the role of facultative symbionts has been well studied during the parthenogenetic phase of their aphid hosts, very little is known on their possible influence during the sexual phase. Here we investigated whether facultative symbionts modulate the capacity to produce sexual forms in various genetic backgrounds of the pea aphid with controlled symbiont composition and also in different aphid genotypes from natural populations with previously characterized infection status and reproductive mode. We found that most facultative symbionts exhibited detrimental effects on their hosts fitness under sex-inducing conditions in comparison with the reference lines. We also showed that the loss of sexual phase in permanently parthenogenetic lineages of A. pisum was not explained by facultative symbionts. Finally, we demonstrated that Spiroplasma infection annihilated the production of males in the host progeny by inducing a male-killing phenotype, an unexpected result for organisms such as aphids that reproduce primarily through clonal reproduction.     

The Endosymbiont Arsenophonus Is Widespread in Soybean Aphid,Aphis glycines,but Does Not Provide Protection from Parasitoids or a Fungal Pathogen

Aphids commonly harbor bacterial facultative symbionts that have a variety of effects upon their aphid hosts, including defense against hymenopteran parasitoids and fungal pathogens. The soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), is infected with the symbiont Arsenophonus sp., which has an unknown role in its aphid host. Our research goals were to document the infection frequency and diversity of the symbiont in field-collected soybean aphids, and to determine whether Arsenophonus is defending soybean aphid against natural enemies. We performed diagnostic PCR and sequenced four Arsenophonus genes in soybean aphids from their native and introduced range to estimate infection frequency and genetic diversity, and found that Arsenophonus infection is highly prevalent and genetically uniform. To evaluate the defensive role of Arsenophonus, we cured two aphid genotypes of their natural Arsenophonus infection through ampicillin microinjection, resulting in infected and uninfected isolines within the same genetic background. These isolines were subjected to parasitoid assays using a recently introduced biological control agent, Binodoxys communis [Braconidae], a naturally recruited parasitoid, Aphelinus certus [Aphelinidae], and a commercially available biological control agent, Aphidius colemani [Braconidae]. We also assayed the effect of the common aphid fungal pathogen, Pandora neoaphidis (Remaudiere & Hennebert) Humber (Entomophthorales: Entomophthoraceae), on the same aphid isolines. We did not find differences in successful parasitism for any of the parasitoid species, nor did we find differences in P. neoaphidis infection between our treatments. Our conclusion is that Arsenophonus does not defend its soybean aphid host against these major parasitoid and fungal natural enemies.