Raw data used to obtain Figs 2–4 and 6–7.

To sustain the gonotrophic cycle, the Aedes aegypti mosquito must acquire a blood meal from a human or other vertebrate host. However, in the process of blood feeding, the mosquito may facilitate the transmission of several bloodborne viral pathogens (e.g., dengue, Zika, and chikungunya). The blood meal is essential as it contains proteins that are digested into polypeptides and amino acid nutrients that are eventually used for egg production. These proteins are digested by several midgut proteolytic enzymes. As such, the female mosquito’s reliance on blood may serve as a potential target for vector and viral transmission control. However, this strategy may prove to be challenging since midgut proteolytic activity is a complex process dependent on several exo- and endo-proteases. Therefore, to understand the complexity of Ae. aegypti blood meal digestion, we used Multiplex Substrate Profiling by Mass Spectrometry (MSP-MS) to generate global proteolytic profiles of sugar- and blood-fed midgut tissue extracts, along with substrate profiles of recombinantly expressed midgut proteases. Our results reveal a shift from high exoproteolytic activity in sugar-fed mosquitoes to an expressive increase in endoproteolytic activity in blood-fed mosquitoes. This approach allowed for the identification of 146 cleaved peptide bonds (by the combined 6 h and 24 h blood-fed samples) in the MSP-MS substrate library, and of these 146, 99 (68%) were cleaved by the five recombinant proteases evaluated. These reveal the individual contribution of each recombinant midgut protease to the overall blood meal digestion process of the Ae. aegypti mosquito. Further, our molecular docking simulations support the substrate specificity of each recombinant protease. Therefore, the present study provides key information of midgut proteases and the blood meal digestion process in mosquitoes, which may be exploited for the development of potential inhibitor targets for vector and viral transmission control strategies.

为维持生殖营养周期（gonotrophic cycle），埃及伊蚊（Aedes aegypti）必须从人类或其他脊椎动物宿主处获取血液餐。然而在吸血过程中，该蚊虫可促成多种血源性病毒病原体（如登革热病毒、寨卡病毒及基孔肯雅病毒）的传播。血液餐是蚊虫生存必需的营养来源，其所含蛋白质可被消化为多肽与氨基酸类营养物质，最终用于虫卵生成。这些蛋白质可由多种中肠蛋白水解酶消化分解。因此，雌性埃及伊蚊对血液的依赖可作为媒介传播与病毒传播防控的潜在靶点。但该防控策略颇具挑战，因为中肠蛋白水解活性是一个复杂过程，依赖多种外肽酶（exoprotease）与内肽酶（endoprotease）的协同调控。为此，为解析埃及伊蚊血液餐消化过程的复杂性，本研究采用质谱多重底物谱分析（Multiplex Substrate Profiling by Mass Spectrometry, MSP-MS），分别获取了喂食糖水与吸血的中肠组织提取物的全局蛋白水解谱，同时获得了重组表达的中肠蛋白酶的底物谱。研究结果显示，喂食糖水的埃及伊蚊以高外蛋白水解活性为主，而吸血个体的内蛋白水解活性出现显著提升。该方法在MSP-MS底物库中共鉴定出146个被裂解的肽键（对应吸血后6小时与24小时的混合样本），其中99个（占比68%）可被本次评估的5种重组蛋白酶裂解。上述结果阐明了每一种重组中肠蛋白酶在埃及伊蚊整体血液餐消化过程中的独立贡献。此外，本研究的分子对接模拟结果验证了各重组蛋白酶的底物特异性。综上，本研究阐明了埃及伊蚊中肠蛋白酶及血液餐消化过程的关键信息，可为开发用于媒介传播与病毒传播防控的潜在抑制剂靶点提供科学依据。