Evolutionary theory predicts late-life decline in the force of natural selection, which could lead to late-life deregulation of immune pathways with increased immunopathological effects. A potential outcome of such ageing-induced immune deregulation is the inability to produce specific immune responses against target pathogens. Instead, non-specific responses would produce an extended set of immune repertoires with little or no fitness benefits, or even increasing fitness costs. We tested this possibility by using two entomopathogens Providencia rettgeri and Pseudomonas entomophila to infect multiple Drosophila melanogaster lines with CRISPR/Cas9-induced knockout of either individual or different combinations of Imd and Toll-inducible antimicrobial peptides (AMPs). As expected, in young flies, AMPs showed a high degree of non-redundancy and pathogen-specificity such that in some cases even a single AMP could confer complete resistance. In contrast, ageing led to a complete loss of specificity, producing complex interactions between multiple AMPs across Toll and Imd pathways. Moreover, nonspecific responses using diverse AMPs with ageing either had no survival benefits, or imposed survival costs against P. rettgeri and P. entomophila. These features of immune senescence were also sexually dimorphic: females expressed a larger repertoire of AMPs compared to males but extracted equivalent survival benefits. Finally, age-specific expansion of the AMP pool was associated with several potential features of a poorly regulated immune system, such as downregulation of negative regulators of the Imd-pathway (e.g., caudal & pirk) and a trend of reduced renal function (i.e., Malpighian tubule activity), following infection, indicating the risk of increased immunopathological damage. Taken together, we demonstrate age-dependent changes in AMP specificity, and how this is associated with variation in immune senescence across sexes and pathogens.

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.

Invertebrates lack the specialized immune-memory cells responsible for vertebrate-like acquired immunity. However, there is increasing evidence that past infection by the same pathogen can "prime" the insect immune response, resulting in improved survival upon reinfection. The mechanisms underlying these phenomenological accounts of priming are diverse, and often not completely clear. Here, we investigated the generality, specificity and mechanistic basis of immune priming in the fruit flyDrosophila melanogasterwhen infected with the gram-negative bacterial pathogenProvidencia rettgeri. We further explore the epidemiological consequences of immune priming and find it has the potential to curtail pathogen transmission by reducing pathogen shedding. We find that priming inDrosophilais a long-lasting, pathogen-specific response, occurring in several fly genetic backgrounds and is particularly stronger in male flies. Mechanistically, we find that the enhanced survival of individuals primed with an initial non-lethal bacterial inoculum coincides with a transient decrease in bacterial loads, and that this is likely driven by the IMD-responsive antimicrobial-peptide Diptericin-B in the fat body. Further, we show that while Diptericins are required as the effector of bacterial clearance, it is not solely sufficient for immune priming, and requires regulation by the peptidoglycan recognition proteins PGRP-LB, PGRP-LC and PGRP-LE. We discuss potential explanations for the observed sex differences in priming, and discuss the epidemiological consequences of innate immune priming in invertebrates.

In many insects, individuals primed with low doses of pathogens live longer after being exposed 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 pathogenBacillus thuringiensis. However, the evolutionary forces responsible for this natural variation remained unclear. Here, 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 across 10 flour beetle populations. 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) Reproductive success – populations where primed females produced more offspring had lower survival benefit, suggesting a trade-off between priming response and reproduction. Our work is the first empirical demonstration of multiple selective pressures that may govern the adaptive 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.

2Under strong pathogen pressure, insects often evolve resistance to infection. Many insects are also 3 protected via immune memory ('immune priming'), whereby sub-lethal exposure to a pathogen 4 enhances survival after secondary infection. To understand the evolution and consequences of these 5 immune responses, we imposed strong pathogen selection on flour beetles (Tribolium castaneum), 6infecting them with Bacillus thuringiensis (Bt) for 11 generations. Populations injected first with heat-