IMD-mediated innate immune priming increases Drosophila survival and reduces pathogen transmission.

Invertebrates lack the immune machinery underlying vertebrate-like acquired immunity. However, in many insects past infection by the same pathogen can 'prime' the immune response, resulting in improved survival upon reinfection. Here, we investigated the mechanistic basis and epidemiological consequences of innate immune priming in the fruit fly Drosophila melanogaster when infected with the gram-negative bacterial pathogen Providencia rettgeri. We find that priming in response to P. rettgeri infection is a long-lasting and sexually dimorphic response. We further explore the epidemiological consequences of immune priming and find it has the potential to curtail pathogen transmission by reducing pathogen shedding and spread. The enhanced survival of individuals previously exposed to a non-lethal bacterial inoculum coincided with a transient decrease in bacterial loads, and we provide strong evidence that the effect of priming requires the IMD-responsive antimicrobial-peptide Diptericin-B in the fat body. Further, we show that while Diptericin B is the main effector of bacterial clearance, it is not sufficient for immune priming, which requires regulation of IMD by peptidoglycan recognition proteins. This work underscores the plasticity and complexity of invertebrate responses to infection, providing novel experimental evidence for the effects of innate immune priming on population-level epidemiological outcomes.

Alteration of diet microbiota limits the experimentally evolved immune priming response in flour beetles, but not pathogen resistance.

Host-associated microbiota play a fundamental role in the training and induction of different forms of immunity, including inducible as well as constitutive components. However, direct experiments analysing the relative importance of microbiota on diverse forms of evolved immune functions are missing. We addressed this gap by using experimentally evolved lines of Tribolium castaneum that either produced inducible immune memory-like responses (immune priming) or constitutively expressed basal resistance (without priming), as divergent counterstrategies against Bacillus thuringiensis infection. We altered the microbial communities present in the diet (i.e. wheat flour) of these evolved lines using UV irradiation and estimated the impact on the beetle's ability to mount a priming response versus basal resistance. Populations that had evolved immune priming lost the ability to mount a priming response upon alteration of diet microbiota. Microbiota manipulation also caused a drastic reduction in their reproductive output and post-infection longevity. In contrast, in pathogen-resistant beetles, microbiota manipulation did not affect post-infection survival or reproduction. The divergent evolution of immune responses across beetle lines was thus associated with divergent reliance on the microbiome. Whether the latter is a direct outcome of differential pathogen exposure during selection or reflects evolved immune functions remains unclear. We hope that our results will motivate further experiments to understand the mechanistic basis of these complex evolutionary associations between microbiota, host immune strategies and fitness outcomes.

Ageing leads to reduced specificity of antimicrobial peptide responses in Drosophila melanogaster.

Evolutionary theory predicts a late-life decline in the force of natural selection, possibly leading to late-life deregulations of the immune system. A potential outcome of such deregulations is the inability to produce specific immunity against target pathogens. We tested this possibility by infecting multiple Drosophila melanogaster lines (with bacterial pathogens) across age groups, where either individual or different combinations of Imd- and Toll-inducible antimicrobial peptides (AMPs) were deleted using CRISPR gene editing. We show a high degree of non-redundancy and pathogen-specificity of AMPs in young flies: in some cases, even a single AMP could confer complete resistance. However, ageing led to drastic reductions in such specificity to target pathogens, warranting the action of multiple AMPs across Imd and Toll pathways. Moreover, use of diverse AMPs either lacked survival benefits or even accompanied survival costs post-infection. These features were also sexually dimorphic: females required a larger repertoire of AMPs than males but extracted equivalent survival benefits. Finally, age-specific expansion of the AMP-repertoire was accompanied with ageing-induced downregulation of negative-regulators of the Imd pathway and damage to renal function post-infection, as features of poorly regulated immunity. Overall, we could highlight the potentially non-adaptive role of ageing in producing less-specific AMP responses, across sexes and pathogens.

Carcass Scavenging Relaxes Chemical-Driven Female Interference Competition in Flour Beetles.

AbstractFemale-female nonsexual interference competition is a major fitness determinant of biased sex ratio groups with high female density. What strategies can females use to overcome the negative impact of this competition? To answer this question we used flour beetles (Tribolium castaneum) where competing females from female-biased groups were already known to suppress each other's fecundity by secreting toxic quinones from their stink glands, indicating a unique chemical-driven interference competition. Surprisingly, increasing resources did not alleviate these fitness costs. Females also did not disperse more from the site of interference competition. Hence, the competition was influenced by neither the total resource availability nor the lack of opportunity to avoid chemical interference. Instead, protein sequestered via scavenging of nutrient-rich carcasses relaxed female competition by increasing fecundity and reducing the quinone content. Finally, stink gland components themselves triggered carcass scavenging and increased fecundity, indicating the possibility of a novel chemical-driven feedback loop to reduce the competition. In the present work we provide the rare analyses where multiple competing hypotheses were jointly tested to establish carcass scavenging as an important potential strategy to overcome the fitness costs of intrasexual female interference competition.

The costs and benefits of basal infection resistance vs immune priming responses in an insect.

In insects, basal pathogen resistance and immune priming can evolve as mutually exclusive strategies, with distinct infection outcomes. However, the evolutionary drivers of such diverse immune functions remain poorly understood. Here, we addressed this key issue by systematically analyzing the differential fitness costs and benefits of priming vs resistance evolution in Tribolium beetle populations infected with Bacillus thuringiensis. Surprisingly, resistant beetles had increased post-infection reproduction and a longer lifespan under both starving as well as fed conditions, with no other measurable costs. In contrast, priming reduced offspring early survival, development rate and reproduction. Priming did improve post-infection survival of offspring, but this added trans-generational benefit of immune priming might not compensate for its pervasive costs. Resistance was thus consistently more beneficial. Overall, our work demonstrates the evolutionary change in trans-generational priming response, and provides a detailed comparison of the complex fitness consequences of evolved priming vs resistance.

Why do insects evolve immune priming? A search for crossroads.

Until recently, it was assumed that insects lack immune memory since they do not have vertebrate-like specialized memory cells. Therefore, their most well studied evolutionary response against pathogens was increased basal immunity. However, growing evidence suggests that many insects also exhibit a form of immune memory (immune priming), where prior exposure to a low dose of infection confers protection against subsequent infection by the same pathogen that acts both within and across generations. Most strikingly, they can rapidly evolve as a highly parallel and mutually exclusive strategy from basal immunity, under different selective conditions and with divergent evolutionary trade-offs. However, the relative importance of priming as an optimal immune strategy also has contradictions, primarily because supporting mechanisms are still unclear. In this review, we adopt a comparative approach to highlight several emerging evolutionary, ecological and mechanistic features of priming vs basal immune responses that warrant immediate attention for future research.

Evolution of pathogen tolerance and emerging infections: A missing experimental paradigm.

Researchers worldwide are repeatedly warning us against future zoonotic diseases resulting from humankind's insurgence into natural ecosystems. The same zoonotic pathogens that cause severe infections in a human host frequently fail to produce any disease outcome in their natural hosts. What precise features of the immune system enable natural reservoirs to carry these pathogens so efficiently? To understand these effects, we highlight the importance of tracing the evolutionary basis of pathogen tolerance in reservoir hosts, while drawing implications from their diverse physiological and life-history traits, and ecological contexts of host-pathogen interactions. Long-term co-evolution might allow reservoir hosts to modulate immunity and evolve tolerance to zoonotic pathogens, increasing their circulation and infectious period. Such processes can also create a genetically diverse pathogen pool by allowing more mutations and genetic exchanges between circulating strains, thereby harboring rare alive-on-arrival variants with extended infectivity to new hosts (i.e., spillover). Finally, we end by underscoring the indispensability of a large multidisciplinary empirical framework to explore the proposed link between evolved tolerance, pathogen prevalence, and spillover in the wild.

Sexual conflict drives micro- and macroevolution of sexual dimorphism in immunity.

BACKGROUND: Sexual dimorphism in immunity is believed to reflect sex differences in reproductive strategies and trade-offs between competing life history demands. Sexual selection can have major effects on mating rates and sex-specific costs of mating and may thereby influence sex differences in immunity as well as associated host-pathogen dynamics. Yet, experimental evidence linking the mating system to evolved sexual dimorphism in immunity are scarce and the direct effects of mating rate on immunity are not well established. Here, we use transcriptomic analyses, experimental evolution and phylogenetic comparative methods to study the association between the mating system and sexual dimorphism in immunity in seed beetles, where mating causes internal injuries in females. RESULTS: We demonstrate that female phenoloxidase (PO) activity, involved in wound healing and defence against parasitic infections, is elevated relative to males. This difference is accompanied by concomitant sex differences in the expression of genes in the prophenoloxidase activating cascade. We document substantial phenotypic plasticity in female PO activity in response to mating and show that experimental evolution under enforced monogamy (resulting in low remating rates and reduced sexual conflict relative to natural polygamy) rapidly decreases female (but not male) PO activity. Moreover, monogamous females had evolved increased tolerance to bacterial infection unrelated to mating, implying that female responses to costly mating may trade off with other aspects of immune defence, an hypothesis which broadly accords with the documented sex differences in gene expression. Finally, female (but not male) PO activity shows correlated evolution with the perceived harmfulness of male genitalia across 12 species of seed beetles, suggesting that sexual conflict has a significant influence on sexual dimorphisms in immunity in this group of insects. CONCLUSIONS: Our study provides insights into the links between sexual conflict and sexual dimorphism in immunity and suggests that selection pressures moulded by mating interactions can lead to a sex-specific mosaic of immune responses with important implications for host-pathogen dynamics in sexually reproducing organisms.

Pathogen susceptibility and fitness costs explain variation in immune priming across natural populations of flour beetles.

In many insects, individuals primed with low doses of pathogens early in life have higher survival after exposure to the same pathogen later in life. Yet, our understanding of the evolutionary and ecological history of priming of immune response in natural insect populations is limited. Previous work demonstrated population-, sex- and stage-specific variation in the survival benefit of priming response in flour beetles (Tribolium castaneum) infected with their natural pathogen Bacillus thuringiensis. However, the evolutionary forces responsible for this natural variation remained unclear. In the present work, we tested whether the strength of the priming response (measured as the survival benefit after priming and subsequent infection, relative to unprimed controls) was associated with multiple fitness parameters and immune components across 10 flour beetle populations collected from different locations in India. Our results suggest two major selective pressures that may explain the observed inter-population variation in priming: (a) Basal pathogen susceptibility - populations that were more susceptible to infection produced a stronger priming response, and (b) Short-term early reproductive success - populations where primed females produced more offspring early in life (measured over 2 days) had lower survival benefit (measured over 120 days), suggesting a potential trade-off between early reproduction and priming response. However, the negative association between survival and reproduction is limited to priming and infection in adults, but not in larvae. While other components of beetle fitness (starvation resistance and larval development) and immune function (haemolymph antibacterial activity and antimicrobial quinone secretion) also varied widely across populations, none of them was correlated with the variation in priming responses across populations. Our work is the first systematic empirical demonstration of multiple selective pressures that may govern the evolution of immune priming in the wild. We hope that this motivates further experiments to establish the role of pathogen-imposed selection and fitness costs in the evolution of priming in natural insect populations.

Experimental evolution of insect immune memory versus pathogen resistance.

Under strong pathogen pressure, insects often evolve resistance to infection. Many insects are also protected via immune memory (immune priming), whereby sublethal exposure to a pathogen enhances survival after secondary infection. Theory predicts that immune memory should evolve when the pathogen is highly virulent, or when pathogen exposure is relatively rare. However, there are no empirical tests of these hypotheses, and the adaptive benefits of immune memory relative to direct resistance against a pathogen are poorly understood. To determine the selective pressures and ecological conditions that shape immune evolution, we imposed strong pathogen selection on flour beetle (Tribolium castaneum) populations, infecting them with Bacillus thuringiensis (Bt) for 11 generations. Populations injected first with heat-killed and then live Bt evolved high basal resistance against multiple Bt strains. By contrast, populations injected only with a high dose of live Bt evolved a less effective but strain-specific priming response. Control populations injected with heat-killed Bt did not evolve priming; and in the ancestor, priming was effective only against a low Bt dose. Intriguingly, one replicate population first evolved priming and subsequently evolved basal resistance, suggesting the potential for dynamic evolution of different immune strategies. Our work is the first report showing that pathogens can select for rapid modulation of insect priming ability, allowing hosts to evolve divergent immune strategies (generalized resistance versus specific immune memory) with potentially distinct mechanisms.

Early-life inflammation, immune response and ageing.

Age-related diseases are often attributed to immunopathology, which results in self-damage caused by an inappropriate inflammatory response. Immunopathology associated with early-life inflammation also appears to cause faster ageing, although we lack direct experimental evidence for this association. To understand the interactions between ageing, inflammation and immunopathology, we used the mealworm beetle Tenebrio molitor as a study organism. We hypothesized that phenoloxidase, an important immune effector in insect defence, may impose substantial immunopathological costs by causing tissue damage to Malpighian tubules (MTs; functionally equivalent to the human kidney), in turn accelerating ageing. In support of this hypothesis, we found that RNAi knockdown of phenoloxidase (PO) transcripts in young adults possibly reduced inflammation-induced autoreactive tissue damage to MTs, and increased adult lifespan. Our work thus suggests a causative link between immunopathological costs of early-life inflammation and faster ageing. We also reasoned that if natural selection weakens with age, older individuals should display increased immunopathological costs associated with an immune response. Indeed, we found that while old infected individuals cleared infection faster than young individuals, possibly they also displayed exacerbated immunopathological costs (larger decline in MT function) and higher post-infection mortality. RNAi-mediated knockdown of PO response partially rescued MTs function in older beetles and resulted in increased lifespan after infection. Taken together, our data are consistent with a direct role of immunopathological consequences of immune response during ageing in insects. Our work is also the first report that highlights the pervasive role of tissue damage under diverse contexts of ageing and immune response.

Immunosenescence and the ability to survive bacterial infection in the red flour beetle Tribolium castaneum.

In most animals, ageing is associated with a decline in immune function (immune senescence). However, different components of the immune system seem to age differentially, and many studies do not measure the ultimate fitness consequences of immune function after infection. Previous work shows that immune function may be traded off with other fitness components such as reproduction. It is possible that age alters the nature of these trade-offs, particularly in conjunction with factors such as gender and mating that can also affect investment in immune function. We tested the impact of age, sex and mating on post-infection survivorship in Tribolium castaneum flour beetles, as well as the components of baseline constitutive innate immunity and external (secreted) immune function in uninfected individuals. We also tested whether the reproductive ability of uninfected females is traded off with immune function (baseline innate and external immunity) and post-infection survivorship across age groups. We found that age, sex and mating significantly affected immune components and infection outcome, although the magnitude and nature of the impact varied in each case. We found that older beetles were more susceptible to infection by the pathogen Bacillus thuringiensis even though major components of the constitutive innate immune defence (antibacterial and phenoloxidase activity) remained unchanged or improved with age. Thus, these aspects of innate immunity cannot explain the observed decline in post-infection survival of older beetles. We did not find trade-offs between the reproductive ability of uninfected females and their immune function. In contrast to innate immunity, external immunity showed an overall decline with age but was also affected by sex and mating. Finally, we show that bacterial infection alters external immunity via complex interactions between age, sex and mating status. Our work uncovers novel interactions between age, sex and mating that can determine the evolution and outcome of immunosenescence by affecting the time course of relative investment in different immune and fitness components.

Divergent immune priming responses across flour beetle life stages and populations.

Growing evidence shows that low doses of pathogens may prime the immune response in many insects, conferring subsequent protection against infection in the same developmental stage (within-life stage priming), across life stages (ontogenic priming), or to offspring (transgenerational priming). Recent work also suggests that immune priming is a costly response. Thus, depending on host and pathogen ecology and evolutionary history, tradeoffs with other fitness components may constrain the evolution of priming. However, the relative impacts of priming at different life stages and across natural populations remain unknown. We quantified immune priming responses of 10 natural populations of the red flour beetle Tribolium castaneum, primed and infected with the natural insect pathogen Bacillus thuringiensis. We found that priming responses were highly variable both across life stages and populations, ranging from no detectable response to a 13-fold survival benefit. Comparing across stages, we found that ontogenic immune priming at the larval stage conferred maximum protection against infection. Finally, we found that various forms of priming showed sex-specific associations that may represent tradeoffs or shared mechanisms. These results indicate the importance of sex-, life stage-, and population-specific selective pressures that can cause substantial divergence in priming responses even within a species. Our work highlights the necessity of further work to understand the mechanistic basis of this variability.

Changing bacterial profile of Sundarbans, the world heritage mangrove: impact of anthropogenic interventions.

Mangrove microbial communities and their associated activities have profound impact on biogeochemical cycles. Although microbial composition and structure are known to be influenced by biotic and abiotic factors in the mangrove sediments, finding direct correlations between them remains a challenge. In this study we have explored sediment bacterial diversity of the Sundarbans, a world heritage site using a culture-independent molecular approach. Bacterial diversity was analyzed from three different locations with a history of exposure to differential anthropogenic activities. 16S rRNA gene libraries were constructed and partial sequencing of the clones was performed to identify the microbial strains. We identified bacterial strains known to be involved in a variety of biodegradation/biotransformation processes including hydrocarbon degradation, and heavy metal resistance. Canonical Correspondence Analysis of the environmental and exploratory datasets revealed correlations between the ecological indices associated with pollutant levels and bacterial diversity across the sites. Our results indicate that sites with similar exposure of anthropogenic intervention reflect similar patterns of microbial diversity besides spatial commonalities.

Male Drosophila melanogaster show adaptive mating bias in response to female infection status.

Given the non-trivial cost of reproduction for males and substantial variation in female quality, males have been predicted to show mating bias as an evolved strategy. Using a large outbred population of Drosophila melanogaster, we test this prediction and show that males may adaptively bias their mating effort in response to the infection status of females. Given a simultaneous choice between females infected with pathogenic bacteria and sham infected females, males preferentially mated with the latter, who had a higher reproductive output compared to infected females. This may provide evidence for pre-copulatory male mate choice. Assessment of the reproductive behaviour ensured that the observed pattern of mating bias was not due to differences in receptivity between females infected with pathogenic bacteria and sham infected females. Further, there was no evidence for post-copulatory male mate choice measured in terms of copulation duration.

The aging of the immune response in Drosophila melanogaster.

Senescence of functional immunity in invertebrates has been a topic of recent interest. Results from previous studies have been inconsistent with older adults exhibiting wide variation in response to infection. In the present study, we assayed the senescence of functional immune response using a large outbred population of Drososphila melanogaster as the model host and Serratia marcescens as the model pathogen. We assessed the effect of an individual's age, parental age, sex, and mating status on overall antibacterial immunity. We found an improvement of immunity with the progression of age with 13-day-old flies exhibiting lower bacterial load compared with 3-day-old flies. Parental age did not show consistent effects on the antibacterial immunity of the offspring. Neither mating status nor the sex of an individual had any significant effect on immune response.