Protection against a fungal pathogen conferred by the aphid facultative endosymbionts Rickettsia and Spiroplasma is expressed in multiple host genotypes and species and is not influenced by co‐infection with another symbiont

Many insects harbour facultative endosymbiotic bacteria, often more than one type at a time. These symbionts can have major effects on their hosts' biology, which may be modulated by the presence of other symbiont species and by the host's genetic background. We investigated these effects by transferring two sets of facultative endosymbionts (one Hamiltonella and Rickettsia, the other Hamiltonella and Spiroplasma) from naturally double‐infected pea aphid hosts into five novel host genotypes of two aphid species. The symbionts were transferred either together or separately. We then measured aphid fecundity and susceptibility to an entomopathogenic fungus. The pathogen‐protective phenotype conferred by the symbionts Rickettsia and Spiroplasma varied among host genotypes, but was not influenced by co‐infection with Hamiltonella. Fecundity varied across single and double infections and between symbiont types, aphid genotypes and species. Some host genotypes benefit from harbouring more than one symbiont type.     

A diversity of endosymbionts across Australian aphids and their persistence in aphid cultures

There is increasing interest in the use of endosymbionts in pest control, which will benefit from the identification of endosymbionts from potential donor species for transfer to pest species. Here, we screened for endosymbionts in 123 Australian aphid samples across 32 species using 16S DNA metabarcoding. We then developed a qPCR method to validate the metabarcoding data set and to monitor endosymbiont persistence in aphid cultures. Pea aphids (Acyrthosiphon pisum) were frequently coinfected with Rickettsiella and Serratia, and glasshouse potato aphids (Aulacorthum solani) were coinfected with Regiella and Spiroplasma; other secondary endosymbionts detected in samples occurred by themselves. Hamiltonella, Rickettsia and Wolbachia were restricted to a single aphid species, whereas Regiella was found in multiple species. Rickettsiella, Hamiltonella and Serratia were stably maintained in laboratory cultures, although others were lost rapidly. The overall incidence of secondary endosymbionts in Australian samples tended to be lower than recorded from aphids overseas. These results indicate that aphid endosymbionts probably exhibit different levels of infectivity and vertical transmission efficiency across hosts, which may contribute to natural infection patterns. The rapid loss of some endosymbionts in cultures raises questions about factors that maintain them under field conditions, while endosymbionts that persisted in laboratory culture provide candidates for interspecific transfers.     

Rickettsia Symbiont in the Pea Aphid Acyrthosiphon pisum: Novel Cellular Tropism, Effect on Host Fitness, and Interaction with the Essential Symbiont Buchnera

In natural populations of the pea aphid Acyrthosiphon pisum, a facultative bacterial symbiont of the genus Rickettsia has been detected at considerable infection frequencies worldwide. We investigated the effects of the Rickettsia symbiont on the host aphid and also on the coexisting essential symbiont Buchnera. In situ hybridization revealed that the Rickettsia symbiont was specifically localized in two types of host cells specialized for endosymbiosis: secondary mycetocytes and sheath cells. Electron microscopy identified bacterial rods, about 2 μm long and 0.5 μm thick, in sheath cells of Rickettsia-infected aphids. Virus-like particles were sometimes observed in association with the bacterial cells. By an antibiotic treatment, we generated Rickettsia-infected and Rickettsia-eliminated aphid strains with an identical genetic background. Comparison of these strains revealed that Rickettsia infection negatively affected some components of the host fitness. Quantitative PCR analysis of the bacterial population dynamics identified a remarkable interaction between the coexisting symbionts: Buchnera population was significantly suppressed in the presence of Rickettsia, particularly at the young adult stage, when the aphid most actively reproduces. On the basis of these results, we discussed the possible mechanisms that enable the prevalence of Rickettsia infection in natural host populations in spite of the negative fitness effects observed in the laboratory.     

Symbiotic microbial studies in diverse populations of Aphis gossypii,existing on altered host plants in different localities during different times

Complex interactions between symbiotic bacteria and insects ultimately result in equilibrium in all aspects of life in natural insect populations. In this study, abundance of principal symbiotic bacteria was estimated using qPCR in 1553 individuals of aphids, Aphis gossypii. Aphids were sampled from primary and secondary host plants—hibiscus and cotton. Hibiscus aphids were collected from 24 different locations in April, September, and November, whereas cotton aphids were collected between 2015 and 2017 from areas with wide variations in climatic conditions. About 30%–45% aphids were recorded with the most dominant symbiont, Arsenophonus. The other symbionts were in low frequency, and about 7% of aphids were noted with Hamiltonella, Acinetobacter, and Microbacterium, and 3% of aphids were verified with Serratia and Pseudomonas. Aphids infected with Hamiltonella, Arsenophonus, and Serratia can influence Buchnera densities. Hamiltonella has positive interaction with densities of Arsenophonus and Serratia. Almost 100% coinfection of Hamiltonella and Arsenophonus was detected in Xinxiang aphids and 50% coinfection was reported in aphids from North China, while no coinfection was detected in Hainan aphids. These findings describe the prevalence pattern and richness of core community of symbiotic bacteria in naturally occurring populations of A. gossypii and provide new insights for the study of symbiotic bacteria.     

The Combined Effects of Bacterial Symbionts and Aging on Life History Traits in the Pea Aphid,Acyrthosiphon pisum

While many endosymbionts have beneficial effects on hosts under specific ecological conditions, there can also be associated costs. In order to maximize their own fitness, hosts must facilitate symbiont persistence while preventing symbiont exploitation of resources, which may require tight regulation of symbiont populations. As a host ages, the ability to invest in such mechanisms may lessen or be traded off with demands of other life history traits, such as survival and reproduction. Using the pea aphid, Acyrthosiphon pisum, we measured survival, lifetime fecundity, and immune cell counts (hemocytes, a measure of immune capacity) in the presence of facultative secondary symbionts. Additionally, we quantified the densities of the obligate primary bacterial symbiont, Buchnera aphidicola, and secondary symbionts across the host''s lifetime. We found life history costs to harboring some secondary symbiont species. Secondary symbiont populations were found to increase with host age, while Buchnera populations exhibited a more complicated pattern. Immune cell counts peaked at the midreproductive stage before declining in the oldest aphids. The combined effects of immunosenescence and symbiont population growth may have important consequences for symbiont transmission and maintenance within a host population.     

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.     

Selective and stable elimination of endosymbionts from multiple‐infected whitefly Bemisia tabaci by feeding on a cotton plant cultured in antibiotic solutions

The maternally heritable endosymbiont provides many ecosystem functions. Antibiotic elimination of a specific symbiont and establishment of experimental host lines lacking certain symbionts enable the roles of a given symbiont to be explored. The whitefly Bemisia tabaci (Gennadius) in China harbors obligate symbiont Portiera infecting each individual, as well as facultative symbionts, such as Hamiltonella, Rickettsia and Cardinium, with co‐infections occurring relatively frequently. So far no studies have evaluated the selectivity and efficacy of a specific symbiont elimination using antibiotics in whiteflies co‐infected with different symbionts. Furthermore, no success has been achieved in establishing certain symbiont‐free B. tabaci lines. In this study, we treated Hamiltonella‐infected B. tabaci line, Hamiltonella‐Rickettsia‐co‐infected line and Hamiltonella‐Cardinium co‐infected line by feeding B. tabaci adults with cotton plants cultured in water containing rifampicin, ampicillin or a mixture of them, aiming to selectively curing symbiont infections and establishing stable symbiont‐free lines. We found ampicillin selectively eliminated Cardinium without affecting Portiera, Hamiltonella and Rickettsia, although they coexisted in the same host body. Meanwhile, all of the symbionts considered in our study can be removed by rifampicin. The reduction of facultative symbionts occurred at a much quicker pace than obligate symbiont Portiera during rifampicin treatment. Also, we measured the stability of symbiont elimination in whitefly successive generations and established Rickettsia‐infected and Cardinium‐infected lines which are absent in natural populations. Our results provide new protocols for selective elimination of symbionts co‐existing in a host and establishment of different symbiont‐infected host lines.     

Worldwide Populations of the Aphid Aphis craccivora Are Infected with Diverse Facultative Bacterial Symbionts

Facultative bacterial endosymbionts can play an important role in the evolutionary trajectory of their hosts. Aphids (Hemiptera: Aphididae) are infected with a wide variety of facultative endosymbionts that can confer ecologically relevant traits, which in turn may drive microevolutionary processes in a dynamic selective environment. However, relatively little is known about how symbiont diversity is structured in most aphid species. Here, we investigate facultative symbiont species richness and prevalence among worldwide populations of the cowpea aphid, Aphis craccivora Koch. We surveyed 44 populations of A. craccivora, and detected 11 strains of facultative symbiotic bacteria, representing six genera. There were two significant associations between facultative symbiont and aphid food plant: the symbiont Arsenophonus was found at high prevalence in A. craccivora populations collected from Robinia sp. (locust), whereas the symbiont Hamiltonella was almost exclusively found in A. craccivora populations from Medicago sativa (alfalfa). Aphids collected from these two food plants also had divergent mitochondrial haplotypes, potentially indicating the formation of specialized aphid lineages associated with food plant (host-associated differentiation). The role of facultative symbionts in this process remains to be determined. Overall, observed facultative symbiont prevalence in A. craccivora was lower than that of some other well-studied aphids (e.g., Aphis fabae and Acyrthosiphon pisum), possibly as a consequence of A. craccivora's almost purely parthenogenetic life history. Finally, most (70 %) of the surveyed populations were polymorphic for facultative symbiont infection, indicating that even when symbiont prevalence is relatively low, symbiont-associated phenotypic variation may allow population-level evolutionary responses to local selection.     

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.     

Coinfection of the secondary symbionts,Hamiltonella defensa and Arsenophonus sp.contribute to the performance of the major aphid pest,Aphis gossypii(Hemiptera:Aphididae)

Bacterial endosymbionts play important roles in ecological traits of aphids.In this study,we characterize the bacterial endosymbionts of A.gossypii collected in Karaj,Iran and their role in the performance of the aphid.Our results indicated that beside Buchnera aphidicola,A.gossypii,also harbors both Hamiltonella defensa and Arsenophonus sp.Quantitative PCR(qPCR)results revealed that the populations of the endosymbionts increased throughout nymphal development up to adult emergence;thereafter,populations of Buchnera and Arsenophonus were diminished while the density of H.defensa constantly increased.Buchnera reduction caused prolonged development and no progeny production.Furthermore,secondary symbiont reduction led to reduction of the total life span and intrinsic rate of natural increase as well as appearance of the deformed dead offspring in comparison with the control insects.Reduction of the secondary symbionts did not affect parasitism rate of the aphid by the parasitic wasp Aphidius matricariae.Together these findings showed that H.defensa and Arsenophonus contributed to the fitness of A.gossypii by enhancing its performance,but not through parasitoid resistance.     

Buchnera has changed flatmate but the repeated replacement of co‐obligate symbionts is not associated with the ecological expansions of their aphid hosts

Symbiotic associations with bacteria have facilitated important evolutionary transitions in insects and resulted in long‐term obligate interactions. Recent evidence suggests that these associations are not always evolutionarily stable and that symbiont replacement, and/or supplementation of an obligate symbiosis by an additional bacterium, has occurred during the history of many insect groups. Yet, the factors favouring one symbiont over another in this evolutionary dynamic are not well understood; progress has been hindered by our incomplete understanding of the distribution of symbionts across phylogenetic and ecological contexts. While many aphids are engaged into an obligate symbiosis with a single Gammaproteobacterium, Buchnera aphidicola, in species of the Lachninae subfamily, this relationship has evolved into a ‘ménage à trois’, in which Buchnera is complemented by a cosymbiont, usually Serratia symbiotica. Using deep sequencing of 16S rRNA bacterial genes from 128 species of Cinara (the most diverse Lachninae genus), we reveal a highly dynamic dual symbiotic system in this aphid lineage. Most species host both Serratia and Buchnera but, in several clades, endosymbionts related to Sodalis, Erwinia or an unnamed member of the Enterobacteriaceae have replaced Serratia. Endosymbiont genome sequences from four aphid species confirm that these coresident symbionts fulfil essential metabolic functions not ensured by Buchnera. We further demonstrate through comparative phylogenetic analyses that cosymbiont replacement is not associated with the adaptation of aphids to new ecological conditions. We propose that symbiont succession was driven by factors intrinsic to the phenomenon of endosymbiosis, such as rapid genome deterioration or competitive interactions between bacteria with similar metabolic capabilities.     

Phenotypic Effect of “Candidatus Rickettsiella viridis,” a Facultative Symbiont of the Pea Aphid (Acyrthosiphon pisum), and Its Interaction with a Coexisting Symbiont

A gammaproteobacterial facultative symbiont of the genus Rickettsiella was recently identified in the pea aphid, Acyrthosiphon pisum. Infection with this symbiont altered the color of the aphid body from red to green, potentially affecting the host''s ecological characteristics, such as attractiveness to different natural enemies. In European populations of A. pisum, the majority of Rickettsiella-infected aphids also harbor another facultative symbiont, of the genus Hamiltonella. We investigated this Rickettsiella symbiont for its interactions with the coinfecting Hamiltonella symbiont, its phenotypic effects on A. pisum with and without Hamiltonella coinfection, and its infection prevalence in A. pisum populations. Histological analyses revealed that coinfecting Rickettsiella and Hamiltonella exhibited overlapping localizations in secondary bacteriocytes, sheath cells, and hemolymph, while Rickettsiella-specific localization was found in oenocytes. Rickettsiella infections consistently altered hosts'' body color from red to green, where the greenish hue was affected by both host and symbiont genotypes. Rickettsiella-Hamiltonella coinfections also changed red aphids to green; this greenish hue tended to be enhanced by Hamiltonella coinfection. With different host genotypes, Rickettsiella infection exhibited either weakly beneficial or nearly neutral effects on host fitness, whereas Hamiltonella infection and Rickettsiella-Hamiltonella coinfection had negative effects. Despite considerable frequencies of Rickettsiella infection in European and North American A. pisum populations, no Rickettsiella infection was detected among 1,093 insects collected from 14 sites in Japan. On the basis of these results, we discuss possible mechanisms for the interaction of Rickettsiella with other facultative symbionts, their effects on their hosts'' phenotypes, and their persistence in natural host populations. We propose the designation “Candidatus Rickettsiella viridis” for the symbiont.     

Unrelated facultative endosymbionts protect aphids against a fungal pathogen

The importance of microbial facultative endosymbionts to insects is increasingly being recognized, but our understanding of how the fitness effects of infection are distributed across symbiont taxa is limited. In the pea aphid, some of the seven known species of facultative symbionts influence their host's resistance to natural enemies, including parasitoid wasps and a pathogenic fungus. Here we show that protection against this entomopathogen, Pandora neoaphidis, can be conferred by strains of four distantly related symbionts (in the genera Regiella, Rickettsia, Rickettsiella and Spiroplasma). They reduce mortality and also decrease fungal sporulation on dead aphids which may help protect nearby genetically identical insects. Pea aphids thus obtain protection from natural enemies through association with a wider range of microbial associates than has previously been thought. Providing resistance against natural enemies appears to be a particularly common way for facultative endosymbionts to increase in frequency within host populations.     

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.     

Stress‐induced changes in abundance differ among obligate and facultative endosymbionts of the soybean aphid

Bacterial endosymbionts can drive evolutionary novelty by conferring adaptive benefits under adverse environmental conditions. Among aphid species there is growing evidence that symbionts influence tolerance to various forms of stress. However, the extent to which stress inflicted on the aphid host has cascading effects on symbiont community dynamics remains poorly understood. Here we simultaneously quantified the effect of host‐plant induced and xenobiotic stress on soybean aphid (Aphis glycines) fitness and relative abundance of its three bacterial symbionts. Exposure to soybean defensive stress (Rag1 gene) and a neurotoxic insecticide (thiamethoxam) substantially reduced aphid composite fitness (survival × reproduction) by 74 ± 10% and 92 ± 2%, respectively, which in turn induced distinctive changes in the endosymbiont microbiota. When challenged by host‐plant defenses a 1.4‐fold reduction in abundance of the obligate symbiont Buchnera was observed across four aphid clonal lines. Among facultative symbionts of Rag1‐stressed aphids, Wolbachia abundance increased twofold and Arsenophonus decreased 1.5‐fold. A similar pattern was observed under xenobiotic stress, with Buchnera and Arsenophonus titers decreasing (1.3‐fold) and Wolbachia increasing (1.5‐fold). Furthermore, variation in aphid virulence to Rag1 was positively correlated with changes in Arsenophonus titers, but not Wolbachia or Buchnera. A single Arsenophonus multi‐locus genotype was found among aphid clonal lines, indicating strain diversity is not primarily responsible for correlated host‐symbiont stress levels. Overall, our results demonstrate the nature of aphid symbioses can significantly affect the outcome of interactions under stress and suggests general changes in the microbiome can occur across multiple stress types.     

Consequences of coinfection with protective symbionts on the host phenotype and symbiont titres in the pea aphid system

Symbiotic associations between microbes and insects are widespread, and it is frequent that several symbionts share the same host individual. Hence, interactions can occur between these symbionts, influencing their respective abundance within the host with consequences on its phenotype. Here, we investigate the effects of multiple infections in the pea aphid, Acyrthosiphon pisum, which is the host of an obligatory and several facultative symbionts. In particular, we study the influence of a coinfection with 2 protective symbionts: Hamiltonella defensa, which confers protection against parasitoids, and Rickettsiella viridis, which provides protection against fungal pathogens and predators. The effects of Hamiltonella‐Rickettsiella coinfection on the respective abundance of the symbionts, host fitness and efficacy of enemy protection were studied. Asymmetrical interactions between the 2 protective symbionts have been found: when they coinfect the same aphid individuals, the Rickettsiella infection affected Hamiltonella abundance within hosts but not the Hamiltonella‐mediated protective phenotype while the Hamiltonella infection negatively influences the Rickettsiella‐mediated protective phenotype but not its abundance. Harboring the 2 protective symbionts also reduced the survival and fecundity of host individuals. Overall, this work highlights the effects of multiple infections on symbiont abundances and host traits that are likely to impact the maintenance of the symbiotic associations in natural habitats.     

Evolution and Diversity of Facultative Symbionts from the Aphid Subfamily Lachninae

Many aphids harbor a variety of endosymbiotic bacteria. The functions of these symbionts can range from an obligate nutritional role to a facultative role in protecting their hosts against environmental stresses. One such symbiont is “Candidatus Serratia symbiotica,” which is involved in defense against heat and potentially also in aphid nutrition. Lachnid aphids have been the focus of several recent studies investigating the transition of this symbiont from a facultative symbiont to an obligate symbiont. In a phylogenetic analysis of Serratia symbionts from 51 lachnid hosts, we found that diversity in symbiont morphology, distribution, and function is due to multiple independent origins of symbiosis from ancestors belonging to Serratia and possibly also to evolution within distinct symbiont clades. Our results do not support cocladogenesis of “Ca. Serratia symbiotica” with Cinara subgenus Cinara species and weigh against an obligate nutritional role. Finally, we show that species belonging to the subfamily Lachninae have a high incidence of facultative symbiont infection.Many insect species harbor heritable endosymbiotic bacteria. Among the best studied of these species are aphids. Almost all aphids are infected with the obligate nutritional symbiont Buchnera aphidicola, which is generally required for the survival of aphids and provides essential amino acids that are rare in their phloem sap diet (32). Many aphids also possess additional symbionts that may be facultative from the host''s perspective and that coexist with Buchnera (20).Three lineages of facultative symbionts that are prevalent in aphids belong to the Enterobacteriaceae. Two of these lineages (“Candidatus Hamiltonella defensa” and “Candidatus Regiella insecticola”) form well-defined clades distinct from free-living bacterial species (4, 20) and confer clear advantages to their hosts by protecting them against natural enemies. “Ca. Hamiltonella defensa” prevents wasp parasitism by arresting development of wasp larvae in pea aphids, and “Ca. Regiella insecticola” provides resistance against the fungal pathogen Pandora neoaphidis (24, 31). The third lineage, “Candidatus Serratia symbiotica,” is closely related to free-living members of the genus Serratia. This symbiont is distributed sporadically among aphid species and has been proposed to have a variety of effects on hosts. In pea aphids (Acyrthosiphon pisum; Macrosiphini), “Ca. Serratia symbiotica” ameliorates the deleterious fitness effects of heat shock by protecting symbiont-harboring bacteriocyte cells (2, 19, 29). Additionally, a strain of “Ca. Serratia symbiotica” provided some resistance to parasitoid wasp attack (24). “Ca. Serratia symbiotica” has been proposed to play a role in nutrition by producing amino acids for its aphid host and by decreasing its host''s reliance on Buchnera (10, 15, 16, 26). In contrast to most Buchnera strains, Buchnera strains from Cinara cedri (Lachnini) have lost the genes for biosynthesis of the essential amino acid tryptophan, while “Ca. Serratia symbiotica” in the same host possesses at least part of the pathway, suggesting that it has a mutualistic role in the nutrition of aphids (26).In A. pisum, “Ca. Serratia symbiotica” cells are rod-shaped bacteria that are present in the sheath cells, hemolymph, and bacteriocytes of some individuals. In contrast, in C. cedri “Ca. Serratia symbiotica” occurs in all individuals, and its cells are large, round, and pleomorphic, similar to the cells of many obligate bacterial aphid endosymbionts, including Buchnera (10, 26). Furthermore, “Ca. Serratia symbiotica” has consistently been present in other Cinara species sampled (28). Both the rod-shaped and pleomorphic forms are assigned to “Ca. Serratia symbiotica” based on phylogenetic analyses of several gene sequences, but they fall into two distinct sister clades of symbiont lineages that seem to coincide with bacterial morphology (17, 20).This diversity in “Ca. Serratia symbiotica” morphology, distribution, and functions may represent evolution of different features within lineages of a single symbiont clade. If “Ca. Serratia symbiotica” is an obligate nutritional symbiont in Cinara hosts, it is expected that Cinara-associated symbionts would form a clade in which the intraclade relationships mirror those of the hosts (cocladogenesis), as observed for Buchnera and other obligate nutritional symbionts of insects (13, 21, 38). Indeed, Lamelas et al. postulated that, based on their similar phylogenies, Serratia symbionts from aphids belonging to the subgenus Cinara have had a long-term relationship with their hosts (17).In addition to the three most common facultative symbiont types found in aphids described above, several other symbiont lineages with unknown functions have been identified by amplification of bacterial 16S rRNA gene sequences from various aphid species (10, 28, 39). Here we examine the diversity of Serratia and other facultative symbionts in aphids belonging to the subfamily Lachninae. We investigated the distribution of symbionts in aphid species and geographic locations and looked for coevolutionary patterns that may correspond to the functions of facultative symbionts within their hosts.     

Diversity in symbiont consortia in the pea aphid complex is associated with large phenotypic variation in the insect host

Virtually all eukaryotes host microbial symbionts that influence their phenotype in many ways. In a host population, individuals may differ in their symbiotic complement in terms of symbiont species and strains. Hence, the combined expression of symbiont and host genotypes may generate a range of phenotypic diversity on which selection can operate and influence host population ecology and evolution. Here, we used the pea aphid to examine how the infection with various symbiotic complements contributes to phenotypic diversity of this insect species. The pea aphid hosts an obligate symbiont (Buchnera aphidicola) and several secondary symbionts among which is Hamiltonella defensa. This secondary symbiont confers a protection against parasitoids but can also reduce the host’s longevity and fecundity. These phenotypic effects of H. defensa infection have been described for a small fraction of the pea aphid complex which encompasses multiple plant-specialized biotypes. In this study, we examined phenotypic differences in four pea aphid biotypes where H. defensa occurs at high frequency and sometimes associated with other secondary symbionts. For each biotype, we measured the fecundity, lifespan and level of parasitoid protection in several aphid lineages differing in their symbiotic complement. Our results showed little variation in longevity and fecundity among lineages but strong differences in their protection level. These differences in protective levels largely resulted from the strain type of H. defensa and the symbiotic consortium in the host. This study highlights the important role of symbiotic complement in the emergence of phenotypic divergence among host populations of the same species.     

Cowpea aphid (Aphis craccivora) associated with different host plants has different facultative endosymbionts

Maternally inherited facultative endosymbiotic bacteria are common among insects, including many polyphagous insect herbivores. To investigate whether symbiont infection is structured by host plant in the polyphagous aphid Aphis craccivora Koch, pyrosequencing and diagnostic PCR were performed on 26 populations from two different host plants, alfalfa (Medicago sativa) or black locust (Robinia pseudoacacia). Results indicated that Aphis craccivora harbours distinctly different microbial communities in alfalfa versus locust. The facultative symbiont Hamiltonella was found only in aphids collected from alfalfa, and the facultative symbiont Arsenophonus was found only in aphids from locust. Hamiltonella is known to protect aphids against hymenopteran parasitoids, whereas the phenotypic effects of Arsenophonus in aphids are unknown. Correspondingly, a screen of the aphid samples for hymenopteran DNA indicated that Hamiltonella‐bearing alfalfa populations of A. craccivora experienced lower parasitism than Arsenophonus‐bearing locust populations. This study contributes to the growing body of evidence that correlative associations between bacterial endosymbionts and host plants may be a common phenomenon in polyphagous herbivores, and suggests that microbial symbionts have the potential to act as drivers for observed ecological differences among host‐associated populations of polyphagous insects.     

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.