The cotton stainer's gut microbiota suppresses infection of a cotransmitted trypanosomatid parasite.

The evolutionary and ecological success of many insects is attributed to mutualistic partnerships with bacteria that confer hosts with novel traits including food digestion, nutrient supplementation, detoxification of harmful compounds and defence against natural enemies. Dysdercus fasciatus firebugs (Hemiptera: Pyrrhocoridae), commonly known as cotton stainers, possess a simple but distinctive gut bacterial community including B vitamin-supplementing Coriobacteriaceae symbionts. In addition, their guts are often infested with the intestinal trypanosomatid parasite Leptomonas pyrrhocoris (Kinetoplastida: Trypanosomatidae). In this study, using experimental bioassays and fluorescence in situ hybridization (FISH), we report on the protective role of the D. fasciatus gut bacteria against L. pyrrhocoris. We artificially infected 2nd instars of dysbiotic and symbiotic insects with a parasite culture and measured parasite titres, developmental time and survival rates. Our results show that L. pyrrhocoris infection increases developmental time and slightly modifies the quantitative composition of the gut microbiota. More importantly, we found significantly higher parasite titres and a tendency towards lower survival rates in parasite-infected dysbiotic insects compared to symbiotic controls, indicating that the gut bacteria successfully interfere with the establishment or proliferation of L. pyrrhocoris. The colonization of symbiotic bacteria on the peritrophic matrix along the gut wall, as revealed by FISH, likely acts as a barrier blocking parasite attachment or entry into the hemolymph. Our findings show that in addition to being nutritionally important, D. fasciatus' gut bacteria complement the host's immune system in preventing parasite invasions and that a stable gut microbial community is integral for the host's health.

Multi-modal defences in aphids offer redundant protection and increased costs likely impeding a protective mutualism.

The pea aphid, Acyrthosiphon pisum, maintains extreme variation in resistance to its most common parasitoid wasp enemy, Aphidius ervi, which is sourced from two known mechanisms: protective bacterial symbionts, most commonly Hamiltonella defensa, or endogenously encoded defences. We have recently found that individual aphids may employ each defence individually, occasionally both defences together, or neither. In field populations, Hamiltonella-infected aphids are found at low to moderate frequencies and while less is known about the frequency of resistant genotypes, they show up less often than susceptible genotypes in field collections. To better understand these patterns, we sought to compare the strengths and costs of both types of defence, individually and together, in order to elucidate the selective pressures that maintain multi-modal defence mechanisms or that may favour one over the other. We experimentally infected five aphid genotypes (two lowly and three highly resistant), each with two symbiont strains, Hamiltonella-APSE8 (moderate protection) and Hamiltonella-APSE3 (high protection). This resulted in three sublines per genotype: uninfected, +APSE8 and +APSE3. Each of the 15 total sublines was first subjected to a parasitism assay to determine its resistance phenotype and in a second experiment, a subset was chosen to compare fitness (fecundity and survivorship) in the presence and absence of parasitism. In susceptible aphid genotypes, parasitized sublines infected with Hamiltonella generally showed increased protection with direct fitness benefits, but clear infection costs to fitness in the absence of parasitism. In resistant genotypes, Hamiltonella infection rarely conferred additional protection, often further reduced fecundity and survivorship when enemy challenged, and resulted in constitutive fitness costs in the absence of parasitism. We also identified strong aphid genotype × symbiont-strain interactions, such that the best defensive strategy against parasitoids varied for each aphid genotype; one performed best with no protective symbionts, the others with particular strains of Hamiltonella. This surprising variability in outcomes helps explain why Hamiltonella infection frequencies are often intermediate and do not strongly track parasitism frequencies in field populations. We also find that variation in endogenous traits, such as resistance, among host genotypes may offer redundancy and generally limit the invasion potential of mutualistic microbes in insects.

Specificity of Multi-Modal Aphid Defenses against Two Rival Parasitoids.

Insects are often attacked by multiple natural enemies, imposing dynamic selective pressures for the development and maintenance of enemy-specific resistance. Pea aphids (Acyrthosiphon pisum) have emerged as models for the study of variation in resistance against natural enemies, including parasitoid wasps. Internal defenses against their most common parasitoid wasp, Aphidius ervi, are sourced through two known mechanisms- 1) endogenously encoded resistance or 2) infection with the heritable bacterial symbiont, Hamiltonella defensa. Levels of resistance can range from nearly 0-100% against A. ervi but varies based on aphid genotype and the strain of toxin-encoding bacteriophage (called APSE) carried by Hamiltonella. Previously, other parasitoid wasps were found to commonly attack this host, but North American introductions of A. ervi have apparently displaced all other parasitoids except Praon pequodorum, a related aphidiine braconid wasp, which is still found attacking this host in natural populations. To explain P. pequodorum's persistence, multiple studies have compared direct competition between both wasps, but have not examined specificity of host defenses as an indirectly mediating factor. Using an array of experimental aphid lines, we first examined whether aphid defenses varied in effectiveness toward either wasp species. Expectedly, both types of aphid defenses were effective against A. ervi, but unexpectedly, were completely ineffective against P. pequodorum. Further examination showed that P. pequodorum wasps suffered no consistent fitness costs from developing in even highly 'resistant' aphids. Comparison of both wasps' egg-larval development revealed that P. pequodorum's eggs have thicker chorions and hatch two days later than A. ervi's, likely explaining their differing abilities to overcome aphid defenses. Overall, our results indicate that aphids resistant to A. ervi may serve as reservoirs for P. pequodorum, hence contributing to its persistence in field populations. We find that specificity of host defenses and defensive symbiont infections, may have important roles in influencing enemy compositions by indirectly mediating the interactions and abundance of rival natural enemies.

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.

Aphid-encoded variability in susceptibility to a parasitoid.

BACKGROUND: Many animals exhibit variation in resistance to specific natural enemies. Such variation may be encoded in their genomes or derived from infection with protective symbionts. The pea aphid, Acyrthosiphon pisum, for example, exhibits tremendous variation in susceptibility to a common natural enemy, the parasitic wasp Aphidius ervi. Pea aphids are often infected with the heritable bacterial symbiont, Hamiltonella defensa, which confers partial to complete resistance against this parasitoid depending on bacterial strain and associated bacteriophages. That previous studies found that pea aphids without H. defensa (or other symbionts) were generally susceptible to parasitism, together with observations of a limited encapsulation response, suggested that pea aphids largely rely on infection with H. defensa for protection against parasitoids. However, the limited number of uninfected clones previously examined, and our recent report of two symbiont-free resistant clones, led us to explicitly examine aphid-encoded variability in resistance to parasitoids. RESULTS: After rigorous screening for known and unknown symbionts, and microsatellite genotyping to confirm clonal identity, we conducted parasitism assays using fifteen clonal pea aphid lines. We recovered significant variability in aphid-encoded resistance, with variation levels comparable to that contributed by H. defensa. Because resistance can be costly, we also measured aphid longevity and cumulative fecundity of the most and least resistant aphid lines under permissive conditions, but found no trade-offs between higher resistance and these fitness parameters. CONCLUSIONS: These results indicate that pea aphid resistance to A. ervi is more complex than previously appreciated, and that aphids employ multiple tactics to aid in their defense. While we did not detect a tradeoff, these may become apparent under stressful conditions or when resistant and susceptible aphids are in direct competition. Understanding sources and amounts of variation in resistance to natural enemies is necessary to understand the ecological and evolutionary dynamics of antagonistic interactions, such as the potential for coevolution, but also for the successful management of pest populations through biological control.

Effects of parasitism on aphid nutritional and protective symbioses.

Insects often carry heritable symbionts that negotiate interactions with food plants or natural enemies. All pea aphids, Acyrthosiphon pisum, require infection with the nutritional symbiont Buchnera, and many are also infected with Hamiltonella, which protects against the parasitoid Aphidius ervi. Hamiltonella-based protection requires bacteriophages called APSEs with protection levels varying by strain and associated APSE. Endoparasitoids, including A. ervi, may benefit from protecting the nutritional symbiosis and suppressing the protective one, while the aphid and its heritable symbionts have aligned interests when attacked by the wasp. We investigated the effects of parasitism on the abundance of aphid nutritional and protective symbionts. First, we determined strength of protection associated with multiple symbiont strains and aphid genotypes as these likely impact symbiont responses. Unexpectedly, some A. pisum genotypes cured of facultative symbionts were resistant to parasitism and resistant aphid lines carried Hamiltonella strains that conferred no additional protection. Susceptible aphid clones carried protective strains. qPCR estimates show that parasitism significantly influenced both Buchnera and Hamiltonella titres, with multiple factors contributing to variation. In susceptible lines, parasitism led to increases in Buchnera near the time of larval wasp emergence consistent with parasite manipulation, but effects were variable in resistant lines. Parasitism also resulted in increases in APSE and subsequent decreases in Hamiltonella, and we discuss how this response may relate to the protective phenotype. In summary, we show that parasitism alters the within-host ecology of both nutritional and protective symbioses with effects likely significant for all players in this antagonistic interaction.

How resident microbes modulate ecologically-important traits of insects.

The microbiota inhabiting insects influence a wide range of ecologically-important traits. In addition to their better-known roles in nutrient provisioning and degrading plant polymers, there is emerging evidence that microorganisms also aid herbivores in countering plant defenses. The latter can be mediated by enzymes that degrade plant allelochemicals or via the modulation of plant signaling pathways. Symbionts are also increasingly recognized to protect insects from attack by a wide range of natural enemies. Underlying mechanisms are poorly understood, but some microbes produce antimicrobials or toxins, while others modulate insect immune responses. Ecologically-relevant symbioses can exhibit dynamic variation in strength and specificity of conferred phenotypes, transfer key traits among unrelated insects, and have effects that extend to interacting players and beyond.