The chemical and biological insecticides were commonly used in mosquito control with their own shortcomings. New strategies are urgently needed to block the transmission of malaria. For example, malaria transmission can be blocked by interfering with the parasite development in mosquito using transgenic engineering or transfection methods. In this study, we found that HD5 microinjection could effectively inhibit the development of malaria parasites in An. stephensi by enhancing innate immunity of mosquitoes.
HD5 has broad-spectrum antibacterial activity and highly effective antimicrobial activity, which has a promising application prospect (Ericksen et al., 2005; Porter et al., 1997). HD5 can inhibit or kill Gram-positive (G+) bacteria, Gram-negative (G−) bacteria, fungi, spirochete, protozoa, and enveloped virus(Awang and Pongprayoon, 2021; Ericksen et al., 2005; Wanniarachchi et al., 2011). Whether HD5 has the parasiticidal effect on Plasmodium has not been studied so far. The influence of HD5 on the innate immunity of mosquitoes has also not been reported. We found that the oocysts were significantly reduced by microinjection of HD5 into Anopheles mosquitoes, indicating that HD5 can decrease the malaria transmission ability of Anopheles. The oocyst counts varied when HD5 was injected intrathoracically into An. stephensi at different time points prior to or post Plasmodium infection, which may be related to the development process of Plasmodium in the mosquitoes. The female mosquito was infected after ingesting a blood meal containing Plasmodium gametocytes. As zygotes form in the midgut, they develop into motile ookinetes that invade the midgut epithelium 18–26 hours after infection(Volohonsky et al., 2020). As the ookinetes migrate through the midgut epithelium and the oocysts develop on the basal side, the number of parasites is limited. The oocyst development was significantly inhibited when HD5 was given prior to infection or injected at 6 h, 12 h and 24 h but not 72 h post infection, possibly because the up-regulated immune response by HD5 mainly acted on the early stage of oocysts. In addition, we found that HD5 treatment at 12 hpi demonstrated the most significant inhibitory effect on oocysts development. Therefore, the practical application of HD5 in the future needs to consider the administration time.
Besides, the concentration of HD5 also needs to be considered. In this study, we also explored the effects of different concentrations of HD5 on the malaria vector competence of An. stephensi. The mosquitoes were injected with 69 nl HD5 at concentrations of 50 µg/ml, 100 µg/ml and 200 µg/ml. The results showed that administration of HD5 at concentrations of 100 µg/ml and 200 µg/ml led to significantly lower infection intensities of plasmodium than the control group, especially the 200 µg/ml concentration (P = 0.0004 < 0.001). However, no effect was observed when the mosquitoes was injected with HD5 at a concentration of 50 µg/ml (Fig. S1).
The mosquito's innate immune system plays an important role at multiple stages of Plasmodium infection. Plasmodium ookinetes cross the mosquito midgut epithelium and reach the basal lamina, where they were killed by mosquito complement C3-like protein TEP1 (Blandin et al., 2004; Volohonsky et al., 2017). Our study found that the expression of TEP1 was up-regulated at 24 h and 72 h post-infection by HD5 treatment, indicating that TEP1 was involved in the effect of HD5 on restriction of plasmodium. Silence of TEP1 expression via RNAi didn’t reverse the impact of HD5 on the development of Plasmodium, indicating the involvement of other effector molecules. As we mentioned before, numerous studies have demonstrated that the anti-Plasmodium and antibacterial defenses of the mosquito are mainly regulated by the Toll and Imd signaling pathways(Blumberg et al., 2013; Ramirez et al., 2014; Zakovic and Levashina, 2017). Detecting and comparing the expression of key molecules of these two signaling pathways showed that the Toll signaling pathway was up-regulated by HD5 injection, while the Imd pathway was not affected. The role of the Toll signaling pathway in the effect on malaria competence of anopheles was further confirmed by silencing MyD88, a key upstream molecule in the Toll signaling pathway. It is consistent with the study that activation of the Toll signaling pathway in Anopheles gambiae by silencing Cactus promotes the activation of the complement-like system in mosquitoes, leading to elimination of the oocyte-cell malaria parasite(Barletta et al., 2022). HD5 can ameliorate dextran sodium sulfate-induced mice colitis via inhibiting NF-kB pathway(Zeng et al., 2020). The results of present study indicated that HD5 could inhibit malaria parasites development in anopheles via affecting the innate immunity of mosquitoes.
HD5 used in the present study is a polypeptide with 95% purity directly synthesized by chemical methods. The dry powder of HD5 was dissolved and diluted with ddH2O to a final working concentration. Therefore, there were hardly any impurities and other solvents which might interfere the results. Although the purity of HD5 was very high, the impact of the possible impurities on the results cannot be excluded completely. This issue will be considered in our future study.
Defensins play an important role in maintaining the distribution and stability of intestinal flora(Sankaran-Walters et al., 2017). The mosquito’s gut microbiota interferes with Plasmodium infection and affects mosquito vectorial capacity. The composition of microbiome might change with shifts in temperature(de Angeli et al., 2023). This natural barrier could be a new transmission-blocking strategies(Romoli and Gendrin, 2018; Vinayagam S et al., 2023). A gut symbiotic bacterium Serratia strain Y1 was isolated from field-caught female Anopheles sinensis and was found capable of enhancing mosquito resistance to Plasmodium infection through activation of mosquito immune responses(Bai et al., 2019). The effect of HD5 on malaria parasites in Anopheles can be observed more intuitively and conveniently by direct feeding. However, the challenge is to obtain large quantities of HD5 in vitro. It is difficult to extract native HD5 from intestinal fluid and chemical synthesis is expensive. A large amount of HD5 hopes to be obtained to meet the demand through the increasingly mature genetic engineering technology. A Serratia AS1, isolated from Anopheles ovaries, was genetically engineered to a recombinant strain which could inhibit Plasmodium falciparum infection of An. gambiae(Wang et al., 2017). In the future, we hope to construct HD5 into a symbiotic strain to express HD5 in vivo and enhance the activity of Anopheles to defense plasmodium.