A single amino acid polymorphism in natural Metchnikowin alleles of Drosophila results in systemic immunity and life history tradeoffs.

Antimicrobial peptides (AMPs) are at the interface of interactions between hosts and microbes and are therefore expected to be rapidly evolving in a coevolutionary arms race with pathogens. In contrast, previous work demonstrated that insect AMPs tend to evolve more slowly than the genome average. Metchikowin (Mtk) is a Drosophila AMP that has a single amino acid residue that segregates as either proline (P) or arginine (R) in populations of four different species, some of which diverged more than 10 million years ago. These results suggest that there is a distinct functional importance to each allele. The most likely hypotheses are driven by two main questions: does each allele have a different efficacy against different specific pathogens (specificity hypothesis)? Or, is one allele a more potent antimicrobial, but with a host fitness cost (autoimmune hypothesis)? To assess their functional differences, we created D. melanogaster lines with the P allele, R allele, or Mtk null mutation using CRISPR/Cas9 genome editing and performed a series of life history and infection assays to assess them. In males, testing of systemic immune responses to a repertoire of bacteria and fungi demonstrated that the R allele performs as well or better than the P and null alleles with most infections. Females show some results that contrast with males, with Mtk alleles either not contributing to survival or with the P allele outperforming the R allele. In addition, measurements of life history traits demonstrate that the R allele is more costly in the absence of infection for both sexes. These results are consistent with both the specificity hypothesis (either allele can perform better against certain pathogens depending on context), and the autoimmune hypothesis (the R allele is generally the more potent antimicrobial in males, and carries a fitness cost). These results provide strong in vivo evidence that differential fitness with or without infection and sex-based functional differences in alleles may be adaptive mechanisms of maintaining immune gene polymorphisms in contrast with expectations of rapid evolution. Therefore, a complex interplay of forces including pathogen species and host sex may lead to balancing selection for immune genotypes. Strikingly, this selection may act on even a single amino acid polymorphism in an AMP. Author summary: Antimicrobial peptides (AMPs) are small peptides produced by many living organisms as a frontline immune response to a variety of microbial pathogens. These peptides directly interface with pathogens to kill them shortly after infection. Importantly, multiple AMP alleles can be balanced within a population, but little is known about how this immune variation among individuals is maintained over time. Multiple species of Drosophila balance two specific alleles of the AMP, Metchnikowin (Mtk), a canonically antifungal and antibacterial peptide. Here, various life-history and infection assays assess the mechanism of Metchnikowin balancing selection using CRISPR-generated D. melanogaster with a null mutation or either of the two alleles, Mtk^R or Mtk^P. Findings demonstrate that the Mtk^R allele usually confers greater protection than the Mtk^P allele in males, but carries a fitness cost without infection. However, this pattern flips in females with certain infections, with the Mtk^P allele conferring greater protection than Mtk^R. This suggests that the two alleles are repeatedly maintained in fly populations due to differential fitness of hosts in the context of many variables including host genotype, sex, infection status, and pathogen species. It also suggests that maintenance of immune variation in a population can be based on just a single amino acid difference between individuals. [ABSTRACT FROM AUTHOR]