Factors Limiting the Spread of the Protective Symbiont Hamiltonella defensa in Aphis craccivora Aphids

Many insects are associated with heritable symbionts that mediate ecological interactions, including host protection against natural enemies. The cowpea aphid, Aphis craccivora, is a polyphagous pest that harbors Hamiltonella defensa, which defends against parasitic wasps. Despite this protective benefit, this symbiont occurs only at intermediate frequencies in field populations. To identify factors constraining H. defensa invasion in Ap. craccivora, we estimated symbiont transmission rates, performed fitness assays, and measured infection dynamics in population cages to evaluate effects of infection. Similar to results with the pea aphid, Acyrthosiphon pisum, we found no consistent costs to infection using component fitness assays, but we did identify clear costs to infection in population cages when no enemies were present. Maternal transmission rates of H. defensa in Ap. craccivora were high (ca. 99%) but not perfect. Transmission failures and infection costs likely limit the spread of protective H. defensa in Ap. craccivora. We also characterized several parameters of H. defensa infection potentially relevant to the protective phenotype. We confirmed the presence of H. defensa in aphid hemolymph, where it potentially interacts with endoparasites, and performed real-time quantitative PCR (qPCR) to estimate symbiont and phage abundance during aphid development. We also examined strain variation of H. defensa and its bacteriophage at multiple loci, and despite our lines being collected in different regions of North America, they were infected with a nearly identical strains of H. defensa and APSE4 phage. The limited strain diversity observed for these defensive elements may result in relatively static protection profile for this defensive symbiosis.     

Impact of environmental stress on aphid clonal resistance to parasitoids: Role of Hamiltonella defensa bacterial symbiosis in association with a new facultative symbiont of the pea aphid

Resistance to endoparasitoids in aphids involves complex interactions between insect and microbial players. It is now generally accepted that the facultative bacterial symbiont Hamiltonella defensa of the pea aphid Acyrthosiphon pisum is implicated in its resistance to the parasitoid Aphidius ervi. It has also been shown that heat negatively affects pea aphid resistance, suggesting the thermosensitivity of its defensive symbiosis. Here we examined the effects of heat and UV-B on the resistance of A. pisum to A. ervi and we relate its stability under heat stress to different facultative bacterial symbionts hosted by the aphid. For six A. pisum clones harboring four different facultative symbiont associations, the impact of heat and UV-B was measured on their ability to resist A. ervi parasitism under controlled conditions. The results revealed that temperature strongly affected resistance, while UV-B did not. As previously shown, highly resistant A. pisum clones singly infected with H. defensa became more susceptible to parasitism after exposure to heat. Interestingly, clones that were superinfected with H. defensa in association with a newly discovered facultative symbiont, referred to as PAXS (pea aphid X-type symbiont), not only remained highly resistant under heat stress, but also expressed previously unknown, very precocious resistance to A. ervi compared to clones with H. defensa alone. The prevalence of dual symbiosis involving PAXS and H. defensa in local aphid populations suggests its importance in protecting aphid immunity to parasitoids under abiotic stress.     

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.     

Cheaper is not always worse: strongly protective isolates of a defensive symbiont are less costly to the aphid host

Defences against parasites are typically associated with costs to the host that contribute to the maintenance of variation in resistance. This also applies to the defence provided by the facultative bacterial endosymbiont Hamiltonella defensa, which protects its aphid hosts against parasitoid wasps while imposing life-history costs. To investigate the cost–benefit relationship within protected hosts, we introduced multiple isolates of H. defensa to the same genetic backgrounds of black bean aphids, Aphis fabae, and we quantified the protection against their parasitoid Lysiphlebus fabarum as well as the costs to the host (reduced lifespan and reproduction) in the absence of parasitoids. Surprisingly, we observed the opposite of a trade-off. Strongly protective isolates of H. defensa reduced lifespan and lifetime reproduction of unparasitized aphids to a lesser extent than weakly protective isolates. This finding has important implications for the evolution of defensive symbiosis and highlights the need for a better understanding of how strain variation in protective symbionts is maintained.     

Mobile elements create strain-level variation in the services conferred by an aphid symbiont

Heritable, facultative symbionts are common in arthropods, often functioning in host defence. Despite moderately reduced genomes, facultative symbionts retain evolutionary potential through mobile genetic elements (MGEs). MGEs form the primary basis of strain-level variation in genome content and architecture, and often correlate with variability in symbiont-mediated phenotypes. In pea aphids (Acyrthosiphon pisum), strain-level variation in the type of toxin-encoding bacteriophages (APSEs) carried by the bacterium Hamiltonella defensa correlates with strength of defence against parasitoids. However, co-inheritance creates difficulties for partitioning their relative contributions to aphid defence. Here we identified isolates of H. defensa that were nearly identical except for APSE type. When holding H. defensa genotype constant, protection levels corresponded to APSE virulence module type. Results further indicated that APSEs move repeatedly within some H. defensa clades providing a mechanism for rapid evolution in anti-parasitoid defences. Strain variation in H. defensa also correlates with the presence of a second symbiont Fukatsuia symbiotica. Predictions that nutritional interactions structured this coinfection were not supported by comparative genomics, but bacteriocin-containing plasmids unique to co-infecting strains may contribute to their common pairing. In conclusion, strain diversity, and joint capacities for horizontal transfer of MGEs and symbionts, are emergent players in the rapid evolution of arthropods.     

Sources of variation in dietary requirements in an obligate nutritional symbiosis

The nutritional symbiosis between aphids and their obligate symbiont, Buchnera aphidicola, is often characterized as a highly functional partnership in which the symbiont provides the host with essential nutrients. Despite this, some aphid lineages exhibit dietary requirements for nutrients typically synthesized by Buchnera, suggesting that some aspect of the symbiosis is disrupted. To examine this phenomenon in the pea aphid, Acyrthosiphon pisum, populations were assayed using defined artificial diet to determine dietary requirements for essential amino acids (EAAs). Six clones exhibiting dependence on EAAs in their diet were investigated further. In one aphid clone, a mutation in a Buchnera amino acid biosynthesis gene could account for the clone''s requirement for dietary arginine. Analysis of aphid F₁ hybrids allowed separation of effects of the host and symbiont genomes, and revealed that both affect the requirement for dietary EAAs in the clones tested. Amino acid requirements were minimally affected by secondary symbiont infection. Our results indicate that variation among pea aphids in dependence on dietary amino acids can result from Buchnera mutation as well as variation in the host genotype.     

Diverse Phage-Encoded Toxins in a Protective Insect Endosymbiont

The lysogenic bacteriophage APSE infects “Candidatus Hamiltonella defensa,” a facultative endosymbiont of aphids and other sap-feeding insects. This endosymbiont has established a beneficial association with aphids, increasing survivorship following attack by parasitoid wasps. Although APSE and “Ca. Hamiltonella defensa” are effectively maternally transmitted between aphid generations, they can also be horizontally transferred among insect hosts, which results in genetically distinct “Ca. Hamiltonella defensa” strains infecting the same aphid species and sporadic distributions of both APSE and “Ca. Hamiltonella defensa” among hosts. Aphids infected only with “Ca. Hamiltonella defensa” have significantly less protection than those infected with both “Ca. Hamiltonella defensa” and APSE. This protection has been proposed to be connected to eukaryote-targeted toxins previously discovered in the genomes of two characterized APSE strains. In this study, we have sequenced partial genomes from seven additional APSE strains to address the evolution and extent of toxin variation in this phage. The APSE lysis region has been a hot spot for nonhomologous recombination of novel virulence cassettes. We identified four new toxins from three protein families, Shiga-like toxin, cytolethal distending toxin, and YD-repeat toxins. These recombination events have also resulted in reassortment of the downstream lysozyme and holin genes. Analysis of the conserved APSE genes flanking the variable toxin cassettes reveals a close phylogenetic association with phage sequences from two other facultative endosymbionts of insects. Thus, phage may act as a conduit for ongoing gene exchange among heritable endosymbionts.     

Unpredicted impacts of insect endosymbionts on interactions between soil organisms,plants and aphids

Ecologically significant symbiotic associations are frequently studied in isolation, but such studies of two-way interactions cannot always predict the responses of organisms in a community setting. To explore this issue, we adopt a community approach to examine the role of plant–microbial and insect–microbial symbioses in modulating a plant–herbivore interaction. Potato plants were grown under glass in controlled conditions and subjected to feeding from the potato aphid Macrosiphum euphorbiae. By comparing plant growth in sterile, uncultivated and cultivated soils and the performance of M. euphorbiae clones with and without the facultative endosymbiont Hamiltonella defensa, we provide evidence for complex indirect interactions between insect– and plant–microbial systems. Plant biomass responded positively to the live soil treatments, on average increasing by 15% relative to sterile soil, while aphid feeding produced shifts (increases in stem biomass and reductions in stolon biomass) in plant resource allocation irrespective of soil treatment. Aphid fecundity also responded to soil treatment with aphids on sterile soil exhibiting higher fecundities than those in the uncultivated treatment. The relative allocation of biomass to roots was reduced in the presence of aphids harbouring H. defensa compared with plants inoculated with H. defensa-free aphids and aphid-free control plants. This study provides evidence for the potential of plant and insect symbionts to shift the dynamics of plant–herbivore interactions.     

Rapid evolution of parasitoids when faced with the symbiont-mediated resistance of their hosts

Insects harbour a wild diversity of symbionts that can spread and persist within populations by providing benefits to their host. The pea aphid Acyrthosiphon pisum maintains a facultative symbiosis with the bacterium Hamiltonella defensa, which provides enhanced resistance against the aphid parasitoid Aphidius ervi. Although the mechanisms associated with this symbiotic‐mediated protection have been investigated thoroughly, little is known about its evolutionary effects on parasitoid populations. We used an experimental evolution procedure in which parasitoids were exposed either to highly resistant aphids harbouring the symbiont or to low innate resistant hosts free of H. defensa. Parasitoids exposed to H. defensa gained virulence over time, reaching the same parasitism rate as those exposed to low aphid innate resistance only. A fitness reduction was associated with this adaptation as the size of parasitoids exposed to H. defensa decreased through generations. This study highlighted the considerable role of symbionts in host–parasite co‐evolutionary dynamics.     

Uncovering symbiont‐driven genetic diversity across North American pea aphids

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.     

Evidence for specificity in symbiont-conferred protection against parasitoids

Many insects harbour facultative symbiotic bacteria, some of which have been shown to provide resistance against natural enemies. One of the best-known protective symbionts is Hamiltonella defensa, which in pea aphid (Acyrthosiphon pisum) confers resistance against attack by parasitoid wasps in the genus Aphidius (Braconidae). We asked (i) whether this symbiont also confers protection against a phylogenetically distant group of parasitoids (Aphelinidae) and (ii) whether there are consistent differences in the effects of bacteria found in pea aphid biotypes adapted to different host plants. We found that some H. defensa strains do provide protection against an aphelinid parasitoid Aphelinus abdominalis. Hamiltonella defensa from the Lotus biotype provided high resistance to A. abdominalis and moderate to low resistance to Aphidius ervi, while the reverse was seen from Medicago biotype isolates. Aphids from Ononis showed no evidence of symbiont-mediated protection against either wasp species and were relatively vulnerable to both. Our results may reflect the different selection pressures exerted by the parasitoid community on aphids feeding on different host plants, and could help explain the maintenance of genetic diversity in bacterial symbionts.     

Cryptic Virulence and Avirulence Alleles Revealed by Controlled Sexual Recombination in Pea Aphids

Although aphids are worldwide crop pests, little is known about aphid effector genes underlying virulence and avirulence. Here we show that controlling the genetics of both aphid and host can reveal novel recombinant genotypes with previously undetected allelic variation in both virulence and avirulence functions. Clonal F₁ progeny populations were derived from reciprocal crosses and self-matings between two parental genotypes of pea aphid (Acyrthosiphon pisum) differing in virulence on a Medicago truncatula host carrying the RAP1 and RAP2 resistance genes. These populations showed Mendelian segregation consistent with aphid performance being controlled largely by a dominant virulence allele derived from only one parent. Altered segregation ratios on near-isogenic host genotypes differing in the region carrying RAP1 were indicative of additional heritable functions likely related to avirulence genes originating from both parents. Unexpectedly, some virulent F₁ progeny were recovered from selfing of an avirulent parent, suggesting a reservoir of cryptic alleles. Host chlorosis was associated with virulence, whereas necrotic hypersensitive-like response was not. No maternal inheritance was found for any of these characteristics, ruling out sex-linked, cytoplasmic, and endosymbiotic factors. Our results demonstrate the tractability of dissecting the genetic basis of pest-host resistance mechanisms and indicate that the annual sexual cycle in aphids may lead to frequent novel genotypes with both increased and decreased virulence. Availability of genomes for both pest and host can facilitate definition of cognate gene-for-gene relationships, potentially leading to selection of crop genotypes with multiple resistance traits.     

Diversity,frequency, and geographic distribution of facultative bacterial endosymbionts in introduced aphid pests

Facultative bacterial endosymbionts in insects have been under intense study during the last years. Endosymbionts can modify the insect's phenotype, conferring adaptive advantages under environmental stress. This seems particularly relevant for a group of worldwide agricultural aphid pests, because endosymbionts modify key fitness‐related traits, including host plant use, protection against natural enemies and heat tolerance. Aimed to understand the role of facultative endosymbionts on the success of introduced aphid pests, the distribution and abundance of 5 facultative endosymbionts (Hamiltonella defensa, Regiella insecticola, Serratia symbiotica, Rickettsia and Spiroplasma) were studied and compared in 4 cereal aphids (Sitobion avenae, Diuraphis noxia, Metopolophium dirhodum and Schizaphis graminium) and in the pea aphid Acyrthosiphon pisum complex from 2 agroclimatic zones in Chile. Overall, infections with facultative endosymbionts exhibited a highly variable and characteristic pattern depending on the aphid species/host race and geographic zone, which could explain the success of aphid pest populations after their introduction. While S. symbiotica and H. defensa were the most frequent endosymbionts carried by the A. pisum pea‐race and A. pisum alfalfa‐race aphids, respectively, the most frequent facultative endosymbiont carried by all cereal aphids was R. insecticola. Interestingly, a highly variable composition of endosymbionts carried by S. avenae was also observed between agroclimatic zones, suggesting that endosymbionts are responding differentially to abiotic variables (temperature and precipitations). In addition, our findings constitute the first report of bacterial endosymbionts in cereal aphid species not screened before, and also the first report of aphid endosymbionts in Chile.     

Identification of Critical Conditions for Immunostaining in the Pea Aphid Embryos: Increasing Tissue Permeability and Decreasing Background Staining

The pea aphid Acyrthosiphon pisum, with a sequenced genome and abundant phenotypic plasticity, has become an emerging model for genomic and developmental studies. Like other aphids, A. pisum propagate rapidly via parthenogenetic viviparous reproduction, where the embryos develop within egg chambers in an assembly-line fashion in the ovariole. Previously we have established a robust platform of whole-mount in situ hybridization allowing detection of mRNA expression in the aphid embryos. For analyzing the expression of protein, though, established protocols for immunostaining the ovarioles of asexual viviparous aphids did not produce satisfactory results. Here we report conditions optimized for increasing tissue permeability and decreasing background staining, both of which were problems when applying established approaches. Optimizations include: (1) incubation of proteinase K (1 µg/ml, 10 min), which was found essential for antibody penetration in mid- and late-stage aphid embryos; (2) replacement of normal goat serum/bovine serum albumin with a blocking reagent supplied by a Digoxigenin (DIG)-based buffer set and (3) application of methanol rather hydrogen peroxide (H₂O₂) for bleaching endogenous peroxidase; which significantly reduced the background staining in the aphid tissues. These critical conditions optimized for immunostaining will allow effective detection of gene products in the embryos of A. pisum and other aphids.     

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.     

Symbiont‐conferred protection against Hymenopteran parasitoids in aphids: how general is it?

1. Hosts are often targeted by multiple species of parasites, leading to a confluence of selective pressures on them. In response, hosts may either evolve defences that act very generally, or specific defences against particular parasites. Aphids are attacked by multiple species of endoparasitoid wasps, and there is clear evidence that heritable endosymbionts can confer resistance against some of these wasps. Less clear is how symbiont‐conferred resistance in a single host acts against multiple parasitoid species. 2. This question was addressed in the black bean aphid, Aphis fabae (Scopoli). Unprotected aphids and aphids protected by three different strains of the defensive endosymbiont Hamiltonella defensa were exposed to four species of parasitic wasps: the parthenogenetic species Lysiphlebus fabarum (Marshall), which was represented by three different asexual lines, and the sexual species Aphidius colemani (Viereck), Binodoxys angelicae (Halliday), and Aphelinus chaonia (Walker). 3. Hamiltonella defensa provided strong protection against L. fabarum and Aphidius colemani, but there was no evidence that H. defensa‐infected aphids were more resistant to the other parasitoid species. While Aphidius colemani was virtually unable to parasitise any aphids harbouring H. defensa, there was variation among the three asexual lines of L. fabarum in how susceptible they were to the defence provided by the different symbiont strains, resulting in a significant genotype‐by‐genotype interaction. 4. The present results suggest that symbiosis with H. defensa does not provide aphids with a general defence against parasitoid wasps, possibly because some species have evolved specific counter adaptations or because biological differences preclude the symbiont's effectiveness against these species.     

Evolutionary genetics of a defensive facultative symbiont of insects: exchange of toxin-encoding bacteriophage

The facultative endosymbiont of aphids, Hamiltonella defensa , kills parasitoid wasp larvae, allowing aphid hosts to survive and reproduce. This protection may depend on toxins that are encoded by the genomes of H. defensa and of its bacteriophage (APSE). Strains of H. defensa vary in degree of protection conferred upon Acyrthosiphon pisum (pea aphid). Although H. defensa is known to undergo some horizontal transmission among aphid maternal lineages, divergence, recombination, and population structure in H. defensa and APSE have not been characterized. We performed a multilocus sequence analysis of 10 bacterial and five phage loci for strains isolated from A. pisum and other aphid species. The H. defensa chromosome was found to be largely clonal, allowing us to generate a well-resolved H. defensa strain phylogeny. In contrast, APSE chromosomes undergo recombination and numerous H. defensa strains have probably lost the phage. Within a set of H. defensa strains that are indistinguishable on the basis of chromosomal genes or restriction digests of chromosomal fragments, loss of APSE is associated with decreased protection, strongly suggesting that APSE-encoded genes contribute to the defensive phenotype. Thus, homologous recombination of APSE genes and sexual transmission of symbionts and phage are likely factors influencing the exchange of ecologically important genes among symbionts. Although H. defensa has been lost, transferred and gained within A. pisum , one subclade of H. defensa appears to be universal within a subclade of the aphid genus Uroleucon , suggesting a transition from facultative, horizontal transmission to strictly vertical inheritance.     

The price of protection:a defensive endosymbiont impairs nymph growth in the bird cherry-oat aphid,Rhopalosiphum padi

Bacterial endosymbionts have enabled aphids to adapt to a range of stressors,but their effects in many aphid species remain to be established.The bird cherry-oat aphid,Rhopalosiphum padi(Linnaeus),is an important pest of cereals worldwide and has been reported to form symbiotic associations with Serratia symbiotica and Sitobion miscanthi L-type symbiont endobacteria,although the resulting aphid phenotype has not been described.This study presents the first report of R.padi infection with the facultative bacterial endosymbiont Hamiltonella defensa.Individuals of R.padi were sampled from populations in Eastern Scotland,UK,and shown to represent seven R.padi genotypes based on the size of polymorphic microsatellite markers;two of these genotypes harbored H.defensa.In parasitism assays,survival of H.defensa-infected nymphs following attack by the parasitoid wasp Aphidius colemani(Viereck)was 5 fold higher than for uninfected nymphs.Aphid genotype was a major determinant of aphid performance on two Hordeum species,a modern cultivar of barley H.vulgare and a wild relative H.spontaneum,although aphids infected with H.defensa showed 16%lower nymph mass gain on the partially resistant wild relative compared with uninfected individuals.These findings suggest that deploying resistance traits in barley will favor the fittest R.padi genotypes,but symbiontinfected individuals will be favored when parasitoids are abundant,although these aphids will not achieve optimal performance on a poor quality host plant.     

Bacterial Communities Associated with Host-Adapted Populations of Pea Aphids Revealed by Deep Sequencing of 16S Ribosomal DNA

Associations between microbes and animals are ubiquitous and hosts may benefit from harbouring microbial communities through improved resource exploitation or resistance to environmental stress. The pea aphid, Acyrthosiphon pisum, is the host of heritable bacterial symbionts, including the obligate endosymbiont Buchnera aphidicola and several facultative symbionts. While obligate symbionts supply aphids with key nutrients, facultative symbionts influence their hosts in many ways such as protection against natural enemies, heat tolerance, color change and reproduction alteration. The pea aphid also encompasses multiple plant-specialized biotypes, each adapted to one or a few legume species. Facultative symbiont communities differ strongly between biotypes, although bacterial involvement in plant specialization is uncertain. Here, we analyse the diversity of bacterial communities associated with nine biotypes of the pea aphid complex using amplicon pyrosequencing of 16S rRNA genes. Combined clustering and phylogenetic analyses of 16S sequences allowed identifying 21 bacterial OTUs (Operational Taxonomic Unit). More than 98% of the sequencing reads were assigned to known pea aphid symbionts. The presence of Wolbachia was confirmed in A. pisum while Erwinia and Pantoea, two gut associates, were detected in multiple samples. The diversity of bacterial communities harboured by pea aphid biotypes was very low, ranging from 3 to 11 OTUs across samples. Bacterial communities differed more between than within biotypes but this difference did not correlate with the genetic divergence between biotypes. Altogether, these results confirm that the aphid microbiota is dominated by a few heritable symbionts and that plant specialization is an important structuring factor of bacterial communities associated with the pea aphid complex. However, since we examined the microbiota of aphid samples kept a few generations in controlled conditions, it may be that bacterial diversity was underestimated due to the possible loss of environmental or transient taxa.