Costs and benefits of a superinfection of facultative symbionts in aphids

Symbiotic associations between animals and inherited micro-organisms are widespread in nature. In many cases, hosts may be superinfected with multiple inherited symbionts. Acyrthosiphon pisum (the pea aphid) may harbour more than one facultative symbiont (called secondary symbionts) in addition to the obligate primary symbiont, Buchnera aphidicola. Previously we demonstrated that, in a controlled genetic background, A. pisum infected with either Serratia symbiotica or Hamiltonella defensa (called R- and T-type in that study) were more resistant to attack by the parasitoid Aphidius ervi. Here, we examined the consequences of A. pisum superinfected with both resistance-conferring symbionts. We found that an A. pisum line co-infected with both S. symbiotica and H. defensa symbionts exhibits even greater resistance to parasitism by A. ervi than either of the singly infected lines. Despite this added benefit to resistance, superinfections of S. symbiotica and H. defensa symbionts appeared rare in our survey of Utah A. pisum symbionts, which is probably attributable to severe fecundity costs. Quantitative polymerase chain reaction estimates indicate that while the density of H. defensa is similar in singly and superinfected hosts, S. symbiotica densities increased dramatically in superinfected hosts. Over-proliferation of symbionts or antagonistic interactions between symbionts may be harmful to the aphid host. Our results indicate that in addition to host-symbiont interactions, interactions among the symbionts themselves probably play a critical role in determining the distributions of symbionts in natural populations.     

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.     

Costs and benefits of symbiont infection in aphids: variation among symbionts and across temperatures

Symbiosis is prevalent throughout the tree of life and has had a significant impact on the ecology and evolution of many bacteria and eukaryotes. The benevolence of symbiotic interactions often varies with the environment, and such variation is expected to play an important role in shaping the prevalence and distributions of symbiosis throughout nature. In this study, we examine how the fitness of aphids is influenced by infection with one of three maternally transmitted bacteria, 'Candidatus Serratia symbiotica', 'Candidatus Hamiltonella defensa' and 'Candidatus Regiella insecticola', addressing how symbiont benevolence varies with temperature. We find that the effects of these 'secondary' symbionts on Acyrthosiphon pisum depend on when and whether aphids are exposed to a brief period of heat shock. We also demonstrate that symbionts--even closely related isolates--vary in their effects on hosts. Our results indicate similar effects of S. symbiotica and H. defensa in conferring tolerance to high temperatures and a liability of R. insecticola under these same conditions. These findings reveal a role for heritable symbionts in the adaptation of aphids to their abiotic environments and add to an expanding body of knowledge on the adaptive significance of symbiosis.     

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 mum long and 0.5 mum 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.     

Symbiont infection affects aphid defensive behaviours

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

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.     

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.     

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

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

Independent origins and horizontal transfer of bacterial symbionts of aphids

Many insect groups have obligate associations with primary endosymbionts: mutualistic bacteria that are maternally transmitted and derived from an ancient infection. Often, the same insects are hosts to 'secondary' bacterial symbionts which are maternally transmitted but relatively labile within host lineages. To explore the dynamics of secondary symbiont associations in aphids, we characterized bacteria infecting 15 species of macrosiphine aphids using DNA sequencing, diagnostic polymerase chain reaction (PCR), diagnostic restriction digests, phylogenetic analyses, and electron microscopy to examine aphids from nature and from laboratory colonies. Three types of bacteria besides Buchnera were found repeatedly; all three fall within the Enterobacteriaceae. The R-type has a 16S rDNA less than 0.1% different from that of the secondary symbiont previously reported from Acyrthosiphon pisum and is related to Serratia species. The T-type includes a symbiont previously reported from a whitefly; the U-type comprises a new cluster near the T-type. The T-type was found in every one of 40 Uroleucon ambrosiae clones collected throughout the United States. In contrast, A. pisum individuals were infected by any combination of the three symbiont types. Secondary symbionts were maternally transmitted for 11 months within laboratory-reared A. pisum clones and were present in sexually produced eggs. PCR screens for a bacteriophage, APSE-1, indicated its presence in both A. pisum and U. ambrosiae containing secondary symbionts. Electron microscopy of R-type and T-type bacteria in A. pisum and in U. ambrosiae revealed rod-shaped organisms that attain extremely high densities within a few bacteriocytes.     

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.     

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.     

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

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

Only helpful when required: a longevity cost of harbouring defensive symbionts

Maternally transmitted symbionts can spread in host populations if they provide a fitness benefit to their hosts. Hamiltonella defensa, a bacterial endosymbiont of aphids, protects hosts against parasitoids but only occurs at moderate frequencies in most aphid populations. This suggests that harbouring this symbiont is also associated with costs, yet the nature of these costs has remained elusive. Here, we demonstrate an important and clearly defined cost: reduced longevity. Experimental infections with six different isolates of H. defensa caused strongly reduced lifespans in two different clones of the black bean aphid, Aphis fabae, resulting in a significantly lower lifetime reproduction. However, the two aphid clones were unequally affected by the presence of H. defensa, and the magnitude of the longevity cost was further determined by genotype × genotype interactions between host and symbiont, which has important consequences for their coevolution.     

Horizontal transfer of bacterial symbionts: heritability and fitness effects in a novel aphid host

Members of several bacterial lineages are known only as symbionts of insects and move among hosts through maternal transmission. Such vertical transfer promotes strong fidelity within these associations, favoring the evolution of microbially mediated effects that improve host fitness. However, phylogenetic evidence indicates occasional horizontal transfer among different insect species, suggesting that some microbial symbionts retain a generalized ability to infect multiple hosts. Here we examine the abilities of three vertically transmitted bacteria from the Gammaproteobacteria to infect and spread within a novel host species, the pea aphid, Acyrthosiphon pisum. Using microinjection, we transferred symbionts from three species of natural aphid hosts into a common host background, comparing transmission efficiencies between novel symbionts and those naturally infecting A. pisum. We also examined the fitness effects of two novel symbionts to determine whether they should persist under natural selection acting at the host level. Our results reveal that these heritable bacteria vary in their capacities to utilize A. pisum as a host. One of three novel symbionts failed to undergo efficient maternal transmission in A. pisum, and one of the two efficiently transmitted bacteria depressed aphid growth rates. Although these findings reveal that negative fitness effects and low transmission efficiency can prevent the establishment of a new infection following horizontal transmission, they also indicate that some symbionts can overcome these obstacles, accounting for their widespread distributions across aphids and related insects.     

Parasitoids as drivers of symbiont diversity in an insect host

Immune systems have repeatedly diversified in response to parasite diversity. Many animals have outsourced part of their immune defence to defensive symbionts, which should be affected by similar evolutionary pressures as the host’s own immune system. Protective symbionts provide efficient and specific protection and respond to changing selection pressure by parasites. Here we use the aphid Aphis fabae, its protective symbiont Hamiltonella defensa, and its parasitoid Lysiphlebus fabarum to test whether parasite diversity can maintain diversity in protective symbionts. We exposed aphid populations with the same initial symbiont composition to parasitoid populations that differed in their diversity. As expected, single parasitoid genotypes mostly favoured a single symbiont that was most protective against that particular parasitoid, while multiple symbionts persisted in aphids exposed to more diverse parasitoid populations, which in turn affected aphid population density and rates of parasitism. Parasite diversity may be crucial to maintaining symbiont diversity in nature.     

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.     

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.     

Parasitoids as vectors of facultative bacterial endosymbionts in aphids

Heritable bacterial endosymbionts play an important role in aphid ecology. Sequence-based evidence suggests that facultative symbionts such as Hamiltonella defensa or Regiella insecticola also undergo horizontal transmission. Other than through male-to-female transfer during the sexual generation in autumn, the routes by which this occurs remain largely unknown. Here, we tested if parasitoids or ectoparasitic mites can act as vectors for horizontal transfer of facultative symbionts. Using symbiont-specific primers for diagnostic PCR, we demonstrate for the first time, to our knowledge, that parasitoids can indeed transfer H. defensa and R. insecticola by sequentially stabbing infected and uninfected individuals of their host, Aphis fabae, establishing new, heritable infections. Thus, a natural route of horizontal symbiont transmission is also available during the many clonal generations of the aphid life cycle. No transmissions by ectoparasitic mites were observed, nor did parasitoids that emerged from symbiont-infected aphids transfer any symbionts in our experiments.     

Complex interactions between a plant pathogen and insect parasitoid via the shared vector-host: consequences for host plant infection

Plant viruses modify the development of their aphid vectors by inducing physiological changes in the shared host plant. The performance of hymenopterous parasitoids exploiting these aphids can also be modified by the presence of the plant pathogen. We used laboratory and glasshouse microcosms containing beans (Vicia faba) as the host plant to examine the interactions between a plant virus (pea enation mosaic virus; PEMV) and a hymenopterous parasitoid (Aphidius ervi) that share the aphid vector/host Acyrthosiphon pisum. Neither PEMV-infection of V. faba, nor the carriage of PEMV virions by A. pisum, affected the growth or morphology of the aphid, or the oviposition behaviour and development of A. ervi. The presence of developing Aphidius ervi larvae within Acyrthosiphon pisum did not affect the ability of the aphids to transmit PEMV. However, by reducing their longevity, parasitism ultimately decreased the time viruliferous aphids were able to inoculate plants. In terms of virus dispersal, parasitized aphids exhibited more movement around experimental arenas than unparasitized controls, causing a slight increase in the proportion of beans infected with PEMV. Exposure to adult Aphidius ervi caused Acyrthosiphon pisum to rapidly drop off bean plants and disperse to new hosts, resulting in considerably higher plant infection rates (70%) than that seen in control arenas (25%). The results of this investigation demonstrate that when parasitoids are added to a plant-pathogen-vector system, benefits to the host plant due to reduced herbivore infestation must be balanced against the consequences of parasitoid-induced aphid dispersal and a subsequent increase in the level of plant infection.