Symbiotic microbes have become increasingly recognized to mediate interactions between natural enemies and their hosts. The ecologies of these symbioses, however, are poorly understood in many ...systems, and a predictive framework is needed to guide future studies. To achieve this, we focus on heritable, defensive microbes of insects. Our review of laboratory‐based studies identifies diverse bacterial species that have independently evolved to protect a range of insects against parasitoids, parasites, predators and pathogens. Although defensive mechanisms are typically unknown, some involve toxins or the upregulation of host immunity. Despite substantial benefits of infection in the presence of natural enemies, the protective symbionts of insects are often found at intermediate levels in natural populations. Using a host‐centred population genetics approach made possible by the host restriction and cytoplasmic inheritance of these microbes, we propose that balancing selection plays a major role in symbiont maintenance, with protective benefits in the presence of enemies and infection costs in their absence. Other mediating factors are likely to be important, including temperature, superinfections and transmission dynamics. While few studies have provided evidence for defence in the field, several studies have shown symbiont infection frequencies to be dynamic, varying across temporal and spatial gradients and food–plant associations. Newly presented data from our pea aphid research reveal that temporal shifts in defensive symbiont prevalence can be quite rapid, with Hamiltonella defensa showing 10–20% shifts around a seasonal average of c. 50%. Such findings contrast with more unidirectional changes seen in laboratory population cages, suggesting temporal changes in the costs and benefits of symbionts in the field. To frame future research on defensive symbiont ecology, we briefly consider a range of studies needed to test laboratory‐ and field‐derived predictions on defensive symbiosis. Included are investigations of defensive mechanisms, symbiont‐driven co‐evolution and community‐level effects. We also consider the need for more thorough and highly resolved molecular diagnostics of natural infections, laboratory studies on functional differences between symbiont strains and species and studies on the relative costs and benefits of defenders in nature. The emerging theme of symbiont‐mediated defence across eukaryotes suggests that knowledge of the functional mechanisms behind protection and natural symbiont dynamics could be key to understanding many of the world's antagonistic species interactions. Thus, the development of insects as a model for such studies holds promise for these organisms and beyond.
Aphids engage in symbiotic associations with a diverse assemblage of heritable bacteria. In addition to their obligate nutrient-provisioning symbiont, Buchnera aphidicola, aphids may also carry one ...or more facultative symbionts. Unlike obligate symbionts, facultative symbionts are not generally required for survival or reproduction and can invade novel hosts, based on both phylogenetic analyses and transfection experiments. Facultative symbionts are mutualistic in the context of various ecological interactions. Experiments on pea aphids (Acyrthosiphon pisum) have demonstrated that facultative symbionts protect against entomopathogenic fungi and parasitoid wasps, ameliorate the detrimental effects of heat, and influence host plant suitability. The protective symbiont, Hamiltonella defensa, has a dynamic genome, exhibiting evidence of recombination, phage-mediated gene uptake, and horizontal gene transfer and containing virulence and toxin-encoding genes. Although transmitted maternally with high fidelity, facultative symbionts occasionally move horizontally within and between species, resulting in the instantaneous acquisition of ecologically important traits, such as parasitoid defense.
Bacteriophages are known to carry key virulence factors for pathogenic bacteria, but their roles in symbiotic bacteria are less well understood. The heritable symbiont Hamiltonella defensa protects ...the aphid Acyrthosiphon pisum from attack by the parasitoid Aphidius ervi by killing developing wasp larvae. In a controlled genetic background, we show that a toxin-encoding bacteriophage is required to produce the protective phenotype. Phage loss occurs repeatedly in laboratory-held H. defensa-infected aphid clonal lines, resulting in increased susceptibility to parasitism in each instance. Our results show that these mobile genetic elements can endow a bacterial symbiont with benefits that extend to the animal host. Thus, phages vector ecologically important traits, such as defense against parasitoids, within and among symbiont and animal host lineages.
Reciprocal selection between aphids, their protective endosymbionts, and the parasitoid wasps that prey upon them offers an opportunity to study the basis of their coevolution. We investigated ...adaptation to symbiont-conferred defense by rearing the parasitoid wasp Lysiphlebus fabarum on aphids (Aphis fabae) possessing different defensive symbiont strains (Hamiltonella defensa). After ten generations of experimental evolution, wasps showed increased abilities to parasitize aphids possessing the H. defensa strain they evolved with, but not aphids possessing the other strain. We show that the two symbiont strains encode different toxins, potentially creating different targets for counter-adaptation. Phenotypic and behavioral comparisons suggest that neither life-history traits nor oviposition behavior differed among evolved parasitoid lineages. In contrast, comparative transcriptomics of adult female wasps identified a suite of differentially expressed genes among lineages, even when reared in a common, symbiont-free, aphid host. In concurrence with the specificity of each parasitoid lineages’ infectivity, most differentially expressed parasitoid transcripts were also lineage-specific. These transcripts are enriched with putative venom toxins and contain highly expressed, potentially defensive viral particles. Together, these results suggest that wild populations of L. fabarum employ a complicated offensive arsenal with sufficient genetic variation for wasps to adapt rapidly and specifically to their host’ microbial defenses.
The cadherin (cdh) superfamily of adhesion molecules carry O-linked mannose (O-Man) glycans at highly conserved sites localized to specific β-strands of their extracellular cdh (EC) domains. These ...O-Man glycans do not appear to be elongated like O-Man glycans found on α-dystroglycan (α-DG), and we recently demonstrated that initiation of cdh/protocadherin (pcdh) O-Man glycosylation is not dependent on the evolutionary conserved POMT1/POMT2 enzymes that initiate O-Man glycosylation on α-DG. Here, we used a CRISPR/Cas9 genetic dissection strategy combined with sensitive and quantitative O-Man glycoproteomics to identify a homologous family of four putative protein O-mannosyltransferases encoded by the TMTC1–4 genes, which were found to be imperative for cdh and pcdh O-Man glycosylation. KO of all four TMTC genes in HEK293 cells resulted in specific loss of cdh and pcdh O-Man glycosylation, whereas combined KO of TMTC1 and TMTC3 resulted in selective loss of O-Man glycans on specific β-strands of EC domains, suggesting that each isoenzyme serves a different function. In addition, O-Man glycosylation of IPT/TIG domains of plexins and hepatocyte growth factor receptor was not affected in TMTC KO cells, suggesting the existence of yet another O-Man glycosylation machinery. Our study demonstrates that regulation of O-mannosylation in higher eukaryotes is more complex than envisioned, and the discovery of the functions of TMTCs provide insight into cobblestone lissencephaly caused by deficiency in TMTC3.
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.
Insects and other animals commonly form symbioses with heritable bacteria, which can exert large influences on host biology and ecology. The pea aphid,
, is a model for studying effects of infection ...with heritable facultative symbionts (HFS), and each of its seven common HFS species has been reported to provide resistance to biotic or abiotic stresses. However, one common HFS, called X-type, rarely occurs as a single infection in field populations and instead typically superinfects individual aphids with
, another HFS that protects aphids against attack by parasitic wasps. Using experimental aphid lines comprised of all possible infection combinations in a uniform aphid genotype, we investigated whether the most common strain of X-type provides any of the established benefits associated with aphid HFS as a single infection or superinfection with
We found that X-type does not confer protection to any tested threats, including parasitoid wasps, fungal pathogens, or thermal stress. Instead, component fitness assays identified large costs associated with X-type infection, costs which were ameliorated in superinfected aphids. Together these findings suggest that X-type exploits the aphid/
mutualism and is maintained primarily as a superinfection by "hitchhiking" via the mutualistic benefits provided by another HFS. Exploitative symbionts potentially restrict the functions and distributions of mutualistic symbioses with effects that extend to other community members.
Maternally transmitted bacterial symbionts are widespread and can have major impacts on the biology of arthropods, including insects of medical and agricultural importance. Given that host fitness and symbiont fitness are tightly linked, inherited symbionts can spread within host populations by providing beneficial services. Many insects, however, are frequently infected with multiple heritable symbiont species, providing potential alternative routes of symbiont maintenance. Here we show that a common pea aphid symbiont called X-type likely employs an exploitative strategy of hitchhiking off the benefits of a protective symbiont,
Infection with X-type provides none of the benefits conferred by other aphid symbionts and instead results in large fitness costs, costs lessened by superinfection with
These findings are corroborated by natural infections in field populations, where X-type is mostly found superinfecting aphids with
Exploitative symbionts may be common in hosts with communities of heritable symbionts and serve to hasten the breakdown of mutualisms.
Summary
Intraspecific variation in dietary breadth can influence important ecological and evolutionary processes, yet the mechanisms generating this variation are usually unknown. Maternally ...transmitted bacterial symbionts frequently infect insect herbivores, and many have been shown to mediate key ecological interactions. For polyphagous herbivores, infection with particular symbionts is often strongly correlated with feeding on particular plant species, suggesting that facultative symbionts might directly determine herbivore food plant specificity. However, previous tests of this hypothesis have returned inconsistent results, providing little empirical support for a causal relationship between facultative symbiont infection and dietary breadth.
Here, we investigate whether heritable facultative symbionts mediate dietary breadth in the polyphagous aphid, Aphis craccivora. We first determined that asexual clones of the aphid differ dramatically in performance across two leguminous food plants, locust and alfalfa, and could be considered biotypes with distinct ecological characteristics. The heritable symbiont Arsenophonus is strongly associated with locust‐origin aphids.
We created experimental lines that share aphid genotypes but differed with respect to Arsenophonus infection status, and compared performance across three food plant species. Naturally Arsenophonus‐infected clones performed 2–4× better on locust and up to 75% worse on two alternate plant species than uninfected controls, clearly demonstrating that Arsenophonus promotes specialization on locust. In both laboratory and field experiments, uninfected locust‐ and alfalfa‐origin clones exhibited similar and modest performance on locust, indicating that the ‘locust‐associated biotype’ would not exist without Arsenophonus.
We also hypothesized that moving Arsenophonus, via transinfection, to an alfalfa‐origin lineage would improve performance on locust and serve to expand dietary breadth. Indeed, transinfection doubled aphid performance on locust and halved aphid performance on alfalfa. However, because this aphid lineage naturally performs better on alfalfa, the transinfected symbiont functionally equalized aphid performance between locust and alfalfa, making the alfalfa biotype more generalized. Thus, the same symbiont can either reduce or expand dietary breadth, depending on host genotype.
Our results unequivocally demonstrate that symbiont gain or loss can instantaneously and substantially change the topology of food plant use in a polyphagous insect, modifying diet in ways that potentially influence the insect's ecological niche, evolutionary trajectory and pest status.
Lay Summary
Insects often harbour heritable symbionts that provide defence against specialized natural enemies, yet little is known about symbiont protection when hosts face simultaneous threats. In pea aphids ...(Acyrthosiphon pisum), the facultative endosymbiont Hamiltonella defensa confers protection against the parasitoid, Aphidius ervi, and Regiella insecticola protects against aphid‐specific fungal pathogens, including Pandora neoaphidis. Here, we investigated whether these two common aphid symbionts protect against a specialized virus A. pisum virus (APV), and whether their antifungal and antiparasitoid services are impacted by APV infection. We found that APV imposed large fitness costs on symbiont‐free aphids and these costs were elevated in aphids also housing H. defensa. In contrast, APV titres were significantly reduced and costs to APV infection were largely eliminated in aphids with R. insecticola. To our knowledge, R. insecticola is the first aphid symbiont shown to protect against a viral pathogen, and only the second arthropod symbiont reported to do so. In contrast, APV infection did not impact the protective services of either R. insecticola or H. defensa. To better understand APV biology, we produced five genomes and examined transmission routes. We found that moderate rates of vertical transmission, combined with horizontal transfer through food plants, were the major route of APV spread, although lateral transfer by parasitoids also occurred. Transmission was unaffected by facultative symbionts. In summary, the presence and species identity of facultative symbionts resulted in highly divergent outcomes for aphids infected with APV, while not impacting defensive services that target other enemies. These findings add to the diverse phenotypes conferred by aphid symbionts, and to the growing body of work highlighting extensive variation in symbiont‐mediated interactions.
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