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 fast evolving in a
coevolutionary arms race with pathogens. In contrast, previous work
demonstrated that one AMP, Metchikowin (Mtk), has a single residue that
segregates as either proline (P) or arginine (R) in populations of four
different Drosophila species, some of which diverged more than 10 million
years ago. The recurrent finding of this polymorphism regardless of
geography or host species, coupled with evidence of balancing selection in
Drosophila AMPs, suggest there is a distinct functional importance to each
allele. The most likely hypotheses involve alleles having specificity to
different pathogens or the more potent allele conferring a cost on the
host. 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. Here, we report results from experiments assessing the two
hypotheses using these lines. 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. With some pathogens, however, females show results in contrast
with males where Mtk alleles either do not contribute to survival or where
the P allele outperforms 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 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.

抗菌肽（Antimicrobial peptides, AMPs）处于宿主与微生物相互作用的界面，因此被认为在与病原体的协同进化军备竞赛中处于快速进化状态。与之相反，此前的研究表明，一种名为梅奇尼科夫肽（Metchikowin, Mtk）的抗菌肽仅存在一个氨基酸残基多态性：在4个不同果蝇物种（部分物种的分化时间已超过1000万年）的种群中，该位点的氨基酸可为脯氨酸（proline, P）或精氨酸（arginine, R）。无论地理环境或宿主物种如何，该多态性均被反复观测到，加之果蝇抗菌肽存在平衡选择（balancing selection）的相关证据，提示两种等位基因（allele）各自具有独特的功能重要性。最合理的假说包括两类：一是不同等位基因对不同病原体具有特异性，二是功能更强的等位基因会给宿主带来适应性代价。为评估二者的功能差异，我们利用CRISPR/Cas9基因组编辑（CRISPR/Cas9 genome editing）技术构建了携带脯氨酸等位基因、精氨酸等位基因或梅奇尼科夫肽无效突变（null mutation）的黑腹果蝇（Drosophila melanogaster）品系。本文报道了利用这些品系验证上述两类假说的实验结果。在雄性果蝇中，针对一系列细菌与真菌的系统性免疫应答（systemic immune responses）实验显示，精氨酸等位基因在多数感染模型中的表现优于或等同于脯氨酸等位基因与无效等位基因。但在部分病原体感染实验中，雌性果蝇的结果与雄性相反：此时梅奇尼科夫肽等位基因对存活无显著影响，或脯氨酸等位基因的存活表现优于精氨酸等位基因。此外，生活史性状（life history traits）检测结果表明，在无病原体感染的情况下，精氨酸等位基因对雌雄两性均会带来更高的适合度（fitness）代价。上述结果为免疫基因多态性（immune gene polymorphisms）的维持机制提供了强有力的体内实验证据：与“快速进化”的预期相反，等位基因在感染与未感染状态下的适合度差异，以及基于性别的功能差异，可能是维持免疫基因多态性的适应性机制。因此，包括病原体种类与宿主性别在内的多重复杂因素的相互作用，可能会驱动免疫基因型的平衡选择。值得注意的是，这种选择甚至可以作用于抗菌肽中单个氨基酸的多态性位点。