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.     

Comparing constitutive and induced costs of symbiont‐conferred resistance to parasitoids in aphids

Host defenses against parasites do not come for free. The evolution of increased resistance can be constrained by constitutive costs associated with possessing defense mechanisms, and by induced costs of deploying them. These two types of costs are typically considered with respect to resistance as a genetically determined trait, but they may also apply to resistance provided by ‘helpers’ such as bacterial endosymbionts. We investigated the costs of symbiont‐conferred resistance in the black bean aphid, Aphis fabae (Scopoli), which receives strong protection against the parasitoid Lysiphlebus fabarum from the defensive endosymbiont Hamiltonella defensa. Aphids infected with H. defensa were almost ten times more resistant to L. fabarum than genetically identical aphids without this symbiont, but in the absence of parasitoids, they had strongly reduced lifespans, resulting in lower lifetime reproduction. This is evidence for a substantial constitutive cost of harboring H. defensa. We did not observe any induced cost of symbiont‐conferred resistance. On the contrary, symbiont‐protected aphids that resisted a parasitoid attack enjoyed increased longevity and lifetime reproduction compared with unattacked controls, whereas unprotected aphids suffered a reduction of longevity and reproduction after resisting an attack. This surprising result suggests that by focusing exclusively on the protection, we might underestimate the selective advantage of infection with H. defensa in the presence of parasitoids.     

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.     

Strong specificity in the interaction between parasitoids and symbiont‐protected hosts

Coevolution between hosts and parasites may promote the maintenance of genetic variation in both antagonists by negative frequency‐dependence if the host–parasite interaction is genotype‐specific. Here we tested for specificity in the interaction between parasitoids (Lysiphlebus fabarum) and aphid hosts (Aphis fabae) that are protected by a heritable defensive endosymbiont, the γ‐proteobacterium Hamiltonella defensa. Previous studies reported a lack of genotype specificity between unprotected aphids and parasitoids, but suggested that symbiont‐conferred resistance might exhibit a higher degree of specificity. Indeed, in addition to ample variation in host resistance as well as parasitoid infectivity, we found a strong aphid clone‐by‐parasitoid line interaction on the rates of successful parasitism. This genotype specificity appears to be mediated by H. defensa, highlighting the important role that endosymbionts can play in host–parasite coevolution.     

EXPERIMENTAL EVOLUTION OF PARASITOID INFECTIVITY ON SYMBIONT‐PROTECTED HOSTS LEADS TO THE EMERGENCE OF GENOTYPE SPECIFICITY

Host‐parasitoid interactions may lead to strong reciprocal selection for traits involved in host defense and parasitoid counterdefense. In aphids, individuals harboring the facultative bacterial endosymbiont, Hamiltonella defensa, exhibit enhanced resistance to parasitoid wasps. We used an experimental evolution approach to investigate the ability of the parasitoid wasp, Lysiphlebus fabarum, to adapt to the presence of H. defensa in its aphid host Aphis fabae. Sexual populations of the parasitoid were exposed for 11 generations to a single clone of A. fabae, either free of H. defensa or harboring artificial infections with three different isolates of H. defensa. Parasitoids adapted rapidly to the presence of H. defensa in their hosts, but this adaptation was in part specific to the symbiont isolate they were evolving against and did not result in an improved infectivity on all symbiont‐protected hosts. Comparisons of life‐history traits among the evolved lines of parasitoids did not reveal any evidence for costs of adaptation to H. defensa in terms of correlated responses that could constrain such adaptation. These results show that parasitoids readily evolve counter‐adaptations to heritable defensive symbionts of their hosts, but that different symbiont strains impose different evolutionary challenges. The symbionts thus mediate the host‐parasite interaction by inducing line‐by‐line genetic specificity.     

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

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

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

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

A strain of the bacterial symbiont Regiella insecticola protects aphids against parasitoids

Aphids commonly harbour facultative bacterial endosymbionts and may benefit from their presence through increased resistance to parasitoids. This has been demonstrated for Hamiltonella defensa and Serratia symbiotica, while a third common endosymbiont, Regiella insecticola, did not provide such protection. However, this symbiont was recently detected in a highly resistant clone of the peach-potato aphid, Myzus persicae, from Australia. To test if resistance was indeed conferred by the endosymbiont, we eliminated it from this clone with antibiotics, and we transferred it to two other clones of the same and one clone of a different aphid species (Aphis fabae). Exposing these lines to the parasitoid Aphidius colemani showed clearly that unlike other strains of this bacterium, this specific isolate of R. insecticola provides strong protection against parasitic wasps, suggesting that the ability to protect their host against natural enemies may evolve readily in multiple species of endosymbiotic bacteria.     

Genotype‐by‐genotype specificity remains robust to average temperature variation in an aphid/endosymbiont/parasitoid system

Genotype‐by‐genotype interactions demonstrate the existence of variation upon which selection acts in host–parasite systems at respective resistance and infection loci. These interactions can potentially be modified by environmental factors, which would entail that different genotypes are selected under different environmental conditions. In the current study, we checked for a G × G × E interaction in the context of average temperature and the genotypes of asexual lines of the endoparasitoid wasp Lysiphlebus fabarum and isolates of Hamiltonella defensa, a protective secondary endosymbiont of the wasp's host, the black bean aphid Aphis fabae. We exposed genetically identical aphids harbouring different isolates of H. defensa to three asexual lines of the parasitoid and measured parasitism success under three different temperatures (15, 22 and 29 °C). Although there was clear evidence for increased susceptibility to parasitoids at the highest average temperature and a strong G × G interaction between the host's symbionts and the parasitoids, no modifying effect of temperature, that is, no significant G × G × E interaction, was detected. This robustness of the observed specificity suggests that the relative fitness of different parasitoid genotypes on hosts protected by particular symbionts remains uncomplicated by spatial or temporal variation in temperature, which should facilitate biological control strategies.     

Inoculation of susceptible and resistant potato plants with the late blight pathogen Phytophthora infestans: effects on an aphid and its parasitoid

Plants are exposed to microbial pathogens as well as herbivorous insects and their natural enemies. Here, we examined the effects of inoculation of potato plants, Solanum tuberosum L. (Solanaceae), with the late blight pathogen Phytophthora infestans (Mont.) de Bary (Peronosporales: Pythiaceae) on an aphid species commonly infesting potato crops and one of the aphid's major parasitoids. We observed the peach‐potato aphid, Myzus persicae Sulzer (Hemiptera: Aphididae), and its natural enemy, the biocontrol agent Aphidius colemani Viereck (Hymenoptera: Braconidae), on potato either inoculated with water or P. infestans. Population growth of the aphid, parasitism rate of its natural enemy, and other insect life‐history traits were compared on several potato genotypes, the susceptible cultivar Désirée and genetically modified (GM) isogenic lines carrying genes conferring resistance to P. infestans. Effects of P. infestans inoculation on the intrinsic rate of aphid population increase and the performance of the parasitoid were only found on the susceptible cultivar. Insect traits were similar when comparing inoculated with non‐inoculated resistant GM genotypes. We also tested how GM‐plant characteristics such as location of gene insertion and number of R genes could influence non‐target insects by comparing insect performance among GM events. Different transformation events leading to different positions of R‐gene insertion in the genome influenced aphids either with or without P. infestans infection, whereas effects of position of R‐gene insertion on the parasitoid A. colemani were evident only in the presence of inoculation with P. infestans. We conclude that it is important to study different transformation events before continuing with further stages of risk assessment of this GM crop. This provides important information on the effects of plant resistance to a phytopathogen on non‐target insects at various trophic levels.     

Evidence for specificity in symbiont-conferred protection against parasitoids

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

Intercropping oilseed rape as a potential relay crop for enhancing the biological control of green peach aphids and aphid‐transmitted virus diseases

Tobacco viruses transmitted by green peach aphids, Myzus persicae (Sulzer) (Hemiptera: Aphididae), cause severe disease in flue‐cured tobacco, Nicotiana tabacum L. (Solanaceae), in China and throughout the world. Field experiments were conducted in 2016 and 2017 in Longyan City, Fujian Province, China, to determine whether M. persicae and aphid‐transmitted virus diseases are affected by intercropping of oilseed rape, Brassica napus L. (Brassicaceae), in tobacco fields. The results showed that, compared with those in monocultured fields, the densities of M. persicae and winged aphids in intercropped fields significantly decreased in both 2016 and 2017. In particular, the appearance of winged aphids was delayed by ca. 7 days. Moreover, the densities of Aphidius gifuensis Ashmead (Hymenoptera: Aphidiidae), a parasitoid of the aphid, significantly increased in 2016 and 2017. Accordingly, the incidence rates of aphid‐transmitted virus diseases (those caused by the cucumber mosaic virus, potato virus Y, and tobacco etch virus) significantly decreased in the intercropped fields in 2016 and 2017. Tobacco yields and monetary value significantly increased in 2016 (by 10–25 and 14–29%, respectively) and 2017 (by 17–22 and 22–34%, respectively). Consequently, our results suggest that intercropping oilseed rape in tobacco fields is a good approach to regulating and controlling aphids and tobacco mosaic viruses, for example potyvirus, and this intercropping can help control aphid‐transmitted virus diseases in tobacco.     

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

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

The NIa‐Pro protein of Turnip mosaic virus improves growth and reproduction of the aphid vector,Myzus persicae (green peach aphid)

Many plant viruses depend on aphids and other phloem‐feeding insects for transmission within and among host plants. Thus, viruses may promote their own transmission by manipulating plant physiology to attract aphids and increase aphid reproduction. Consistent with this hypothesis, Myzus persicae (green peach aphids) prefer to settle on Nicotiana benthamiana infected with Turnip mosaic virus (TuMV) and fecundity on virus‐infected N. benthamiana and Arabidopsis thaliana (Arabidopsis) is higher than on uninfected controls. TuMV infection suppresses callose deposition, an important plant defense, and increases the amount of free amino acids, the major source of nitrogen for aphids. To investigate the underlying molecular mechanisms of this phenomenon, 10 TuMV genes were over‐expressed in plants to determine their effects on aphid reproduction. Production of a single TuMV protein, nuclear inclusion a‐protease domain (NIa‐Pro), increased M. persicae reproduction on both N. benthamiana and Arabidopsis. Similar to the effects that are observed during TuMV infection, NIa‐Pro expression alone increased aphid arrestment, suppressed callose deposition and increased the abundance of free amino acids. Together, these results suggest a function for the TuMV NIa‐Pro protein in manipulating the physiology of host plants. By attracting aphid vectors and promoting their reproduction, TuMV may influence plant–aphid interactions to promote its own transmission.     

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

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

Cascading effects of herbivore protective symbionts on hyperparasitoids

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

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.     

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.