Understanding how interspecific interactions mould the molecular basis of adaptations in coevolving species is a long‐sought goal of evolutionary biology. Venom in predators and venom resistance proteins in prey are coevolving molecular phenotypes, and while venoms are highly complex mixtures it is unclear if prey respond with equally complex resistance traits. Here, we use a novel molecular methodology based on protein affinity columns to capture and identify candidate blood serum resistance proteins (“venom interactive proteins” [VIPs]) in California Ground Squirrels (Otospermophilus beecheyi ) that interact with venom proteins from their main predator, Northern Pacific Rattlesnakes (Crotalus o. oreganus ). This assay showed that serum‐based resistance is both population‐ and species‐specific, with serum proteins from ground squirrels showing higher binding affinities for venom proteins of local snakes compared to allopatric individuals. Venom protein specificity assays identified numerous and diverse candidate prey resistance VIPs but also potential targets of venom in prey tissues. Many specific VIPs bind to multiple snake venom proteins and, conversely, single venom proteins bind multiple VIPs, demonstrating that a portion of the squirrel blood serum “resistome” involves broad‐based inhibition of nonself proteins and suggests that resistance involves a toxin scavenging mechanism. Analyses of rates of evolution of VIP protein homologues in related mammals show that most of these proteins evolve under purifying selection possibly due to molecular constraints that limit the evolutionary responses of prey to rapidly evolving snake venom proteins. Our method represents a general approach to identify specific proteins involved in co‐evolutionary interactions between species at the molecular level.

中文翻译：

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响尾蛇-松鼠食肉动物-猎物系统中抗毒液的分子基础。

了解种间相互作用如何塑造共同进化物种的适应性分子基础是进化生物学的长期目标。捕食者中的毒液和猎物中的毒液抗性蛋白是共同进化的分子表型，尽管毒液是高度复杂的混合物，但尚不清楚猎物是否具有同样复杂的抗性特征。在这里，我们使用一种基于蛋白质亲和力列的新颖分子方法来捕获和识别加利福尼亚地松鼠（Otospermophilus beecheyi）中与主要捕食者的毒液蛋白相互作用的候选血清抗药性蛋白（“毒液相互作用蛋白” [VIPs]），北太平洋响尾蛇（响尾蛇o。oreganus）。此测定法表明，基于血清的抗性既具有种群特异性，又具有物种特异性，与松散个体相比，地松鼠的血清蛋白显示出对本地蛇毒蛋白的更高结合亲和力。毒液蛋白特异性测定可鉴定出众多多样的候选猎物抗性VIP，但也鉴定了猎物组织中毒液的潜在靶标。许多特定的VIP结合多种蛇毒蛋白，相反，单个毒液蛋白结合多种VIP，表明部分松鼠血清“抵抗力”涉及广泛的非自身蛋白抑制作用，并表明抗性涉及毒素清除机制。对相关哺乳动物VIP蛋白同源物进化速率的分析表明，这些蛋白中的大多数在纯化选择下进化，可能是由于分子约束限制了猎物对快速进化的蛇毒蛋白的进化反应。我们的方法代表了一种在分子水平上识别物种间共同进化相互作用的特定蛋白质的通用方法。