Supplementary Material for: HMGB1-Like Dorsal Switch Protein 1 Triggers a Damage Signal in Mosquito Gut to Activate Dual Oxidase via Eicosanoids

Several mosquitoes transmit human pathogens by blood feeding, with the gut being the main entrance for the pathogens. Thus, the gut epithelium defends the pathogens by eliciting potent immune responses. However, it was unclear how the mosquito gut discriminates pathogens among various microflora in the lumen. This study proposed a hypothesis that a damage signal might be specifically induced by pathogens in the gut. The Asian tiger mosquito, <i>Aedes albopictus</i>, encodes dorsal switch protein 1 (<i>Aa-DSP1</i>) as a putative damage-associated molecular pattern (DAMP). Aa-DSP1 was localized in the nucleus of the midgut epithelium in naïve larvae. Upon infection by a pathogenic bacterium, <i>Serratia marcescens</i>, Aa-DSP1 was released to hemocoel and activated phospholipase A₂ (PLA₂). The activated PLA₂ increased the level of prostaglandin E₂ (PGE₂) in the gut and subsequently increased Ca²⁺ signal to produce reactive oxygen species (ROS) via dual oxidase (Duox). Inhibition of Aa-DSP1 via RNA interference or specific inhibitor treatment failed to increase PGE₂/Ca²⁺ signal upon the bacterial infection. Thus, the inhibitors specifically targeting eicosanoid biosynthesis significantly prevented the upregulation of ROS production in the gut and enhanced mosquito mortality after the bacterial infection. However, such inhibitory effects were rescued by adding PGE₂. These suggest that Aa-DSP1 plays an important role in immune response of the mosquito gut as a DAMP during pathogen infection by triggering a signaling pathway, DSP1/PLA₂/Ca²⁺/Duox.

多种蚊虫通过吸血行为传播人类病原体，而肠道是病原体侵入宿主的主要门户。因此，肠道上皮通过激活强效免疫应答抵御病原体。然而，此前学界尚未明确蚊虫肠道如何区分肠腔内的各类共生微生物与病原体。本研究提出假说：病原体可在蚊虫肠道中特异性诱导损伤信号。亚洲虎蚊（<i>Aedes albopictus</i>）可编码背侧开关蛋白1（Aa-DSP1），该蛋白属于潜在的损伤相关分子模式（damage-associated molecular pattern, DAMP）。在未感染的幼虫体内，Aa-DSP1定位于中肠上皮细胞的细胞核内。当受到致病性细菌粘质沙雷氏菌（<i>Serratia marcescens</i>）感染时，Aa-DSP1被释放至血腔，并激活磷脂酶A₂（phospholipase A₂, PLA₂）。被激活的PLA₂可提升肠道内前列腺素E₂（prostaglandin E₂, PGE₂）的水平，随后通过双氧化酶（dual oxidase, Duox）激活钙离子信号以产生活性氧（reactive oxygen species, ROS）。通过RNA干扰（RNA interference）或特异性抑制剂处理抑制Aa-DSP1后，细菌感染时无法再上调PGE₂/钙离子信号通路。因此，靶向类二十烷酸生物合成的特异性抑制剂可显著抑制肠道内ROS生成的上调，并在细菌感染后升高蚊虫的死亡率。但外源性添加PGE₂可逆转此类抑制效应。上述结果表明，Aa-DSP1作为DAMP，在病原体感染过程中通过激活DSP1/PLA₂/Ca²⁺/Duox信号通路，在蚊虫肠道免疫应答中发挥关键作用。