Table_1_A zone-of-inhibition assay to screen for humoral antimicrobial activity in mosquito hemolymph.pdf

In insects, antibacterial immunity largely depends on the activation of downstream signaling and effector responses, leading to the synthesis and secretion of soluble effector molecules, such as antimicrobial peptides (AMPs). AMPs are acute infection response peptides secreted into the hemolymph upon bacterial stimulation. The transcription of innate immunity genes encoding for AMPs is highly dependent on several signaling cascade pathways, such as the Toll pathway. In the African malaria mosquito, Anopheles gambiae, AMPs hold a special interest as their upregulation have been shown to limit the growth of malaria parasites, bacteria, and fungi. Most of the current knowledge on the regulation of insect AMPs in microbial infection have been obtained from Drosophila. However, largely due to the lack of convenient assays, the regulation of antimicrobial activity in mosquito hemolymph is still not completely understood. In this study, we report a zone of inhibition assay to identify the contribution of AMPs and components of the Toll pathway to the antimicrobial activity of A. gambiae hemolymph. As a proof of principle, we demonstrate that Micrococcus luteus challenge induces antimicrobial activity in the adult female mosquito hemolymph, which is largely dependent on defensin 1. Moreover, by using RNAi to silence Cactus, REL1, and MyD88, we showed that Cactus kd induces antimicrobial activity in the mosquito hemolymph, whereas the antimicrobial activity in REL1 kd and MyD88 kd is reduced after challenge. Finally, while injection itself is not sufficient to induce antimicrobial activity, our results show that it primes the response to bacterial challenge. Our study provides information that increases our knowledge of the regulation of antimicrobial activity in response to microbial infections in mosquitoes. Furthermore, this assay represents an ex vivo medium throughput assay that can be used to determine the upstream regulatory elements of antimicrobial activity in A. gambiae hemolymph.

在昆虫体内，抗菌免疫主要依赖于下游信号转导与效应应答的激活，进而促成可溶性效应分子（如抗菌肽（antimicrobial peptides））的合成与分泌。抗菌肽是一类受细菌刺激后分泌至血淋巴的急性感染应答肽类。编码抗菌肽的先天免疫基因的转录，高度依赖于包括Toll通路在内的多种信号级联通路。在作为非洲疟疾传播媒介的冈比亚按蚊（*Anopheles gambiae*）体内，抗菌肽因其上调表达可抑制疟原虫、细菌与真菌的增殖而受到广泛关注。目前学界关于昆虫抗菌肽在微生物感染中的调控机制的认知，大多来自对果蝇（*Drosophila*）的研究。然而，由于缺乏便捷的实验检测方法，学界对蚊类血淋巴中抗菌活性的调控机制仍未完全明晰。本研究建立了一种抑菌圈实验法，用以解析抗菌肽与Toll通路组分对冈比亚按蚊血淋巴抗菌活性的调控作用。作为原理验证实验，我们证实：藤黄微球菌（*Micrococcus luteus*）侵染可诱导成年雌蚊血淋巴产生抗菌活性，且该活性主要依赖于防御素1（defensin 1）。此外，通过RNA干扰（RNA interference）技术沉默Cactus、REL1与MyD88的表达，我们发现：Cactus基因敲低可诱导蚊类血淋巴产生抗菌活性，而REL1基因敲低与MyD88基因敲低的蚊个体在受侵染后其血淋巴抗菌活性则显著降低。最后，尽管单纯注射操作本身不足以诱导抗菌活性，但本研究结果显示，注射操作可预激活机体对细菌侵染的应答反应。本研究的结果丰富了学界对蚊类在微生物感染过程中抗菌活性调控机制的认知。此外，本研究所建立的抑菌圈实验法属于一种离体中等通量检测体系，可用于解析冈比亚按蚊血淋巴抗菌活性的上游调控元件。