Does variation in host plant association and symbiont infection of pea aphid populations induce genetic and behaviour differentiation of its main parasitoid, Aphidius ervi?

The host-associated differentiation (HAD) hypothesis states that higher trophic levels in parasitic associations should exhibit similar divergence in case of host sympatric speciation. We tested HAD on populations of Aphidius ervi the main parasitoid of the pea aphid Acyrthosiphon pisum, emerging from host populations specialized on either alfalfa or red clover. Host and parasitoid populations were assessed for genetic variation and structure, while considering geography, host plant and host aphid protective symbionts Regiella insecticola and Hamiltonella defensa as potential covariables. Cluster and hierarchical analyses were used to assess the contribution of these variables to population structure, based on genotyping pea aphids and associated A. ervi with microsatellites, and host aphid facultative symbionts with 16S rDNA markers. Pea aphid genotypes were clearly distributed in two groups closely corresponding with their plant origins, confirming strong plant associated differentiation of this aphid in North America. Overall parasitism by A. ervi averaged 21.5 % across samples, and many parasitized aphids producing a wasp hosted defensive bacteria, indicating partial or ineffective protective efficacy of these symbionts in the field. The A. ervi population genetic data failed to support differentiation according to the host plant association of their pea aphid host. Potential for parasitoid specialization was also explored in experiments where wasps from alfalfa and clover aphids were reciprocally transplanted on alternate hosts, the hypothesis being that wasp behaviour and parasitic stages should be most adapted to their host of origin. Results revealed higher probability of oviposition on the alfalfa aphids, but higher adult emergence success on red clover aphids, with no interaction as expected under HAD. We conclude that our study provides no support for the HAD in this system. We discuss factors that might impair A. ervi specialization on its divergent aphid hosts on alfalfa and clover.     

Facultative symbionts are associated with host plant specialization in pea aphid populations

The pea aphid, Acyrthosiphon pisum, shows significant reproductive isolation and host plant specialization between populations on alfalfa and clover in New York. We examine whether specialization is seen in pea aphids in California, and whether fitness on alternative host plants is associated with the presence of bacterial symbionts. We measured the fitness of alfalfa- and clover-derived aphids on both types of plants and found no evidence for specialization when all aphid lineages were considered simultaneously. We then screened all aphids for the presence of four facultative bacterial symbionts: PAR, PASS, PABS and PAUS. Aphids with PAUS were host-plant specialized, having twice as many offspring as other aphids on clover, and dying on alfalfa. Other aphids showed no evidence of specialization. Additionally, aphids with PABS had 50% more offspring than aphids with PASS when on alfalfa. Thus, specialist and generalist aphid lineages coexist, and specialization is symbiont associated. Further work will resolve whether PAUS is directly responsible for this variation in fitness or whether PAUS is incidentally associated with host-plant specialized aphid lineages.     

Facultative 'secondary' bacterial symbionts and the nutrition of the pea aphid, Acyrthosiphon pisum

Abstract.  Facultative 'secondary' bacterial symbionts influence various traits of aphids, including plant utilization patterns and resistance to parasitoids. The present study is designed to test the hypothesis that these multiple effects are underlain by symbiont-mediated changes to the aphid requirement for the dominant dietary nutrients, sucrose and amino acids. The performance of pea aphids ( Acyrthosiphon pisum ) on chemically defined diets of systematically altered sucrose and amino acid content varies among eight parthenogenetic clones, with a pattern that does not match the aphid complements of secondary symbionts, Hamiltonella defensa , Regiella insecticola and Serratia symbiotica . Aphid performance is reduced, increased and unaffected by elimination of S. symbiotica , R. insecticola and H. defensa , respectively, but with no significant effect on the range of diets on which aphids performed well. It is concluded that the impact of secondary symbionts on aphid traits is most unlikely to have a purely nutritional basis.     

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.     

Genetic variation in the effect of a facultative symbiont on host-plant use by pea aphids

Ecological specialisation on different host plants occurs frequently among phytophagous insects and is normally assumed to have a genetic basis. However, insects often carry microbial symbionts, which may play a role in the evolution of specialisation. The bacterium Regiella insecticola is a facultative symbiont of pea aphids (Acyrthosiphon pisum) where it is found most frequently in aphid clones feeding on Trifolium giving rise to the hypothesis that it may improve aphid performance on this plant. A study in which R. insecticola was eliminated from a single naturally infected aphid clone supported the hypothesis, but a second involving two aphid clones did not find the same effect. We created a series of new pea aphid–R. insecticola associations by injecting different strains of bacteria into five aphid clones uninfected by symbionts. For all aphid clones, the bacteria decreased the rate at which aphids accepted Vicia faba as a food plant and reduced performance on this plant. Their effect on aphids given Trifolium pratense was more complex: R. insecticola negatively affected acceptance by all aphid clones, had no effect on the performance of four aphid clones, but increased performance of a fifth, thus demonstrating genetic variation in the effect of R. insecticola on pea aphid host use. We discuss how these results may explain the distribution and frequency of this symbiont across different aphid populations. Julia Ferrari and Claire L. Scarborough contributed equally to the work.     

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.     

Resistance to a fungal pathogen and host plant specialization in the pea aphid

Herbivores that show host race formation on different plant species have proven to be valuable model systems for studying the evolution of specialization and speciation. Here, we use the pea aphid, Acyrthosiphon pisum, to investigate a possible link between specialization on two host plant species, Lotus uliginosus and Trifolium pratense, and resistance to a natural enemy, the fungal pathogen Erynia neoaphidis. Pea aphids collected on either plant species in the field showed in most cases poor survival on the alternate host plant. Furthermore, pea aphids specialized on T. pratense were very resistant to E. neoaphidis, whereas aphids specialized on L. uliginosus were susceptible. This susceptibility was not influenced by the actual food plant on which the assays were conducted. We discuss how selection from natural enemies may influence the process of specialization and race formation, and how specialization can affect the evolution of resistance.     

Intraspecific variation in immune gene expression and heritable symbiont density

Host genetic variation plays an important role in the structure and function of heritable microbial communities. Recent studies have shown that insects use immune mechanisms to regulate heritable symbionts. Here we test the hypothesis that variation in symbiont density among hosts is linked to intraspecific differences in the immune response to harboring symbionts. We show that pea aphids (Acyrthosiphon pisum) harboring the bacterial endosymbiont Regiella insecticola (but not all other species of symbionts) downregulate expression of key immune genes. We then functionally link immune expression with symbiont density using RNAi. The pea aphid species complex is comprised of multiple reproductively-isolated host plant-adapted populations. These ‘biotypes’ have distinct patterns of symbiont infections: for example, aphids from the Trifolium biotype are strongly associated with Regiella. Using RNAseq, we compare patterns of gene expression in response to Regiella in aphid genotypes from multiple biotypes, and we show that Trifolium aphids experience no downregulation of immune gene expression while hosting Regiella and harbor symbionts at lower densities. Using F1 hybrids between two biotypes, we find that symbiont density and immune gene expression are both intermediate in hybrids. We propose that in this system, Regiella symbionts are suppressing aphid immune mechanisms to increase their density, but that some hosts have adapted to prevent immune suppression in order to control symbiont numbers. This work therefore suggests that antagonistic coevolution can play a role in host-microbe interactions even when symbionts are transmitted vertically and provide a clear benefit to their hosts. The specific immune mechanisms that we find are downregulated in the presence of Regiella have been previously shown to combat pathogens in aphids, and thus this work also highlights the immune system’s complex dual role in interacting with both beneficial and harmful microbes.     

Hosts do not simply outsource pathogen resistance to protective symbionts

Microbial symbionts commonly protect their hosts from natural enemies, but it is unclear how protective symbionts influence the evolution of host immunity to pathogens. One possibility is that ‘extrinsic’ protection provided by symbionts allows hosts to reduce investment in ‘intrinsic’ immunological resistance mechanisms. We tested this idea using pea aphids (Acyrthosiphon pisum) and their facultative bacterial symbionts that increase host resistance to the fungal pathogen Pandora neoaphidis. The pea aphid taxon is composed of multiple host plant associated populations called biotypes, which harbor characteristic communities of symbionts. We found that biotypes that more frequently carry protective symbionts have higher, rather than lower, levels of intrinsic resistance. Within a biotype there was no difference in intrinsic resistance between clones that did and did not carry a protective symbiont. The host plant on which an aphid feeds did not strongly influence intrinsic resistance. We describe a simple conceptual model of the interaction between intrinsic and extrinsic resistance and suggest that our results may be explained by selection favoring both the acquisition of protective symbionts and enhanced intrinsic resistance in habitats with high pathogen pressure. Such combined protection is potentially more robust than intrinsic resistance alone.     

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.     

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.     

Survival to Parasitoids in an Insect Hosting Defensive Symbionts: A Multivariate Approach to Polymorphic Traits Affecting Host Use by Its Natural Enemy

Insect parasitoids and their insect hosts represent a wide range of parasitic trophic relations that can be used to understand the evolution of biotic diversity on earth. Testing theories of coevolution between hosts and parasites is based on factors directly involved in host susceptibility and parasitoid virulence. We used controlled encounters with potential hosts of the Aphidius ervi wasp to elucidate behavioral and other phenotypic traits of host Acyrthosiphon pisum that most contribute to success or failure of parasitism. The host aphid is at an advanced stage of specialization on different crop plants, and exhibits intra-population polymorphism for traits of parasitoid avoidance and resistance based on clonal variation of color morph and anti-parasitoid bacterial symbionts. Randomly selected aphid clones from alfalfa and clover were matched in 5 minute encounters with wasps of two parasitoid lineages deriving from hosts of each plant biotype in a replicated transplant experimental design. In addition to crop plant affiliation (alfalfa, clover), aphid clones were characterized for color morph (green, pink), Hamiltonella defensa and Regiella insecticola symbionts, and frequently used behaviors in encounters with A. ervi wasps. A total of 12 explanatory variables were examined using redundancy analysis (RDA) to predict host survival or failure to A. ervi parasitism. Aphid color was the best univariate predictor, but was poorly predictive in the RDA model. In contrast, aphid host plant and symbionts were not significant univariate predictors, but significant predictors in the multivariate model. Aphid susceptibility to wasp acceptance as reflected in host attacks and oviposition clearly differed from its suitability to parasitism and progeny development. Parasitoid progeny were three times more likely to survive on clover than alfalfa host aphids, which was compensated by behaviorally adjusting eggs invested per host. Strong variation of the predictive power of intrinsic (body color) and extrinsic traits (symbionts, host plant), indicate that host variables considered as key predictors of outcomes strongly interact and cannot be considered in isolation.     

Host-based divergence in populations of the pea aphid: insights from nuclear markers and the prevalence of facultative symbionts

In North America, the pea aphid Acyrthosiphon pisum encompasses ecologically and genetically distinct host races that offer an ideal biological system for studies on sympatric speciation. In addition to its obligate symbiont Buchnera, pea aphids harbour several facultative and phylogenetically distant symbionts. We explored the relationships between host races of A. pisum and their symbiotic microbiota to gain insights into the historical process of ecological specialization and symbiotic acquisition in this aphid. We used allozyme and microsatellite markers to analyse the extent of genetic differentiation between populations of A. pisum on pea, alfalfa and clover in France. In parallel, we examined: (i) the distribution of four facultative symbionts; and (ii) the genetic variation in the Buchnera genome across host-associated populations of A. pisum. Our study clearly demonstrates that populations of A. pisum on pea, clover and alfalfa in France are genetically divergent, which indicates that they constitute distinct host races. We also found a very strong association between host races of A. pisum and their symbiotic microbiota. We stress the need for phylogeographic studies to shed light on the process of host-race formation and acquisition of facultative symbionts in A. pisum. We also question the effects of these symbionts on aphid host fitness, including their role in adaptation to a host plant.     

The natural occurrence of secondary bacterial symbionts in aphids

1. Many insects host secondary bacterial symbionts that are known to have wide‐ranging effects on their hosts, from host‐plant use to resistance against natural enemies. This has been most widely studied in aphids, which have become a model system to study insect–bacteria interactions. 2. While there is an increasing understanding of the role of symbionts in aphids from controlled laboratory studies, we are only beginning to explore the impact of hosting these symbionts on eco‐evolutionary dynamics in natural systems. To date, many research groups have identified bacterial symbionts from various aphid species, providing us with a bank of literature on aphid–symbiont associations in natural populations. 3. The role of secondary symbionts in aphids is discussed, and the taxonomic and geographical distribution of symbionts among aphids are summarised, and the potential reasons for the patterns observed. The need to test for multiple symbiont species (and co‐infections) across many individuals and the whole distribution range of an aphid is highlighted, including sampling on all known host‐plant species. 4. It is further important also to consider variation within the symbiont, the aphid‐host and the surrounding community, e.g. host‐plants or the natural enemies, to understand how these have the potential to mediate aphid–symbiont interactions. 5. Finally, the knowledge gained from experimental work should now be used to understand the role of aphid secondary symbionts in field systems, to fully understand the potentially far‐reaching consequences of aphid endosymbionts on community and ecosystem processes.     

刺吸式昆虫次生内共生菌的研究进展

蚜虫作为典型的刺吸式昆虫,需要以取食植物韧皮部汁液来补充营养,几乎所有蚜虫均带有一种能为其提供植物韧皮部缺失营养物质的初生共生菌Buchnera aphidicola。此外,蚜虫还可携带一种或多种次生内共生菌。在众多共生菌—寄主系统中,蚜虫与其所带内共生菌间的互作研究最为透彻。虽然次生内共生菌对寄主的存活和生殖影响并不显著,但其在寄主对环境耐受力、天敌防御能力等方面作用明显。本文在查阅大量蚜虫次生内共生菌相关文献的基础上,着重对蚜虫次生内共生菌的种类及传播规律、次生内共生菌对蚜虫表型的影响、蚜虫次生内共生菌基因组学等方面的研究现状进行综述,以求为刺吸式昆虫次生内共生菌的研究提供参考。     

Impact of plant nutrients on the relationship between a herbivorous insect and its symbiotic bacteria

The interactions between herbivorous insects and their symbiotic micro-organisms can be influenced by the plant species on which the insects are reared, but the underlying mechanisms are not understood. Here, we identify plant nutrients, specifically amino acids, as a candidate factor affecting the impact of symbiotic bacteria on the performance of the phloem-feeding aphid Aphis fabae. Aphis fabae grew more slowly on the labiate plant Lamium purpureum than on an alternative host plant Vicia faba, and the negative effect of L. purpureum on aphid growth was consistently exacerbated by the bacterial secondary symbionts Regiella insecticola and Hamiltonella defensa, which attained high densities in L. purpureum-reared aphids. The amino acid content of the phloem sap of L. purpureum was very low; and A. fabae on chemically defined diets of low amino acid content also grew slowly and had elevated secondary symbiont densities. It is suggested that the phloem nutrient profile of L. purpureum promotes deleterious traits in the secondary symbionts and disturbs insect controls over bacterial abundance.     

Population genetic structure and secondary symbionts in host-associated populations of the pea aphid complex

Polyphagous insect herbivores experience different selection pressures on their various host plant species. How this affects population divergence and speciation may be influenced by the bacterial endosymbionts that many harbor. Here, we study the population structure and symbiont community of the pea aphid (Acyrthosiphon pisum), which feeds on a range of legume species and is known to form genetically differentiated host-adapted populations. Aphids were collected from eight legume genera in England and Germany. Extensive host plant associated differentiation was observed with this collection of pea aphids comprising nine genetic clusters, each of which could be associated with a specific food plant. Compared to host plant, geography contributed little to genetic differentiation. The genetic clusters were differentiated to varying degrees, but this did not correlate with their degree of divergence in host use. We surveyed the pea aphid clones for the presence of six facultative (secondary) bacterial endosymbionts and found they were nonrandomly distributed across the aphid genetic clusters and this distribution was similar in the two countries. Aphid clones on average carried 1.4 species of secondary symbiont with those associated with Lathyrus having significantly fewer. The results are interpreted in the light of the evolution of specialization and ecological speciation.     

Aphid clonal resistance to a parasitoid fails under heat stress

Parasitoid virulence and host resistance are complex interactions depending on metabolic rate and cellular activity, which in aphids additionally implicate heritable secondary symbionts among the Enterobacteriaceae. As performance of the parasitoid, the aphid host and its symbionts may differentially respond to temperature, the success or failure of aphid parasitism is difficult to predict when temperature varies. We tested the hypothesis that resistance of the pea aphid Acyrthosiphon pisum to the parasitoid Aphidius ervi, which is linked to aphid secondary symbionts, may depend on temperature in several resistant and non-resistant aphid clonal lineages of different geographic origin and of known bacterial symbiosis, using experiments in controlled environments. Complete immunity to A. ervi at 20 degrees C in three different aphid clones whose symbiosis is characterized by the possession of Hamiltonella defensa reversed to high susceptibility at 25 degrees C and especially 30 degrees C, suggesting that the aphid's immune responses to the establishment and early development of the parasitoid is strongly reduced at moderately high temperatures. There was no evidence that a pea aphid control genotype that was susceptible to A. ervi at 20 degrees C could become more resistant as temperature increases, as has been suggested for insect fungal pathogens. By contrast, our results suggest that aphid clonal resistance to A. ervi and related parasitoids is characteristic of cool temperature conditions, similar to various other fitness attributes of aphids. Based on evidence that H. defensa symbionts characterized all three A. ervi resistant pea aphid clones studied, but was absent in control aphids that remained susceptible at all temperatures, we suggest that secondary symbiosis plays a key role in the heat sensitivity of aphid clonal resistance. Our study may also indicate that aphid natural control of variably susceptible host populations by aphid parasitoids is more likely at moderate to high temperatures.     

Interspecific symbiont transfection confers a novel ecological trait to the recipient insect

In Japan, pea aphids Acyrthosiphon pisum mainly feed on vetch and clover, and many aphid clones produce more progeny on vetch than on clover. In this context, particular genotypes of the facultative symbiont Regiella insecticola enhance reproduction of infected pea aphids specifically on clover, thereby broadening the suitable food plant range of the insect. A species that is sympatric to A. pisum, vetch aphids Megoura crassicauda, are commonly found on vetch but not on clover. Laboratory rearing of M. crassicauda strains revealed active reproduction on vetch but substantially no reproduction on clover. Experimental transfection of Regiella from A. pisum to M. crassicauda by haemolymph injection established stable and heritable infection in the recipients, although no Regiella infection has been detected in natural populations of M. crassicauda. Different strains of Regiella-transfected M. crassicauda grew and reproduced on vetch, but exhibited lower fitness in comparison with corresponding uninfected aphid strains. Strikingly, the Regiella-transfected M. crassicauda exhibited improved survival and some reproduction on clover. These results suggest that Regiella has the potential to confer an ecological trait, adaptation to clover, on novel insect hosts, and also account for why Regiella is able to infect M. crassicauda but is scarcely found in these aphid populations.