Impact Of Artemisinin On Vector Capacity Of Anopheles Stephensi To Malaria And Its Molecular Mechanisms

Malaria is an infectious disease caused by plasmodium that seriously threats human health via anopheles mosquitoes transmission.Artemisinin?ART?is the preferred choice for falciparum malaria treatment,and often used by the universal coverage in malaria-affected areas to eliminate potential sources of infection.In many malaria-endemic regions of the world,when malaria occurs or worsens,in addition to increased surveillance and early detection of malaria-infected people,population-wide drug delivery strategies in endemic regions have been vigorously promoted.When anopheles mosquitoes bite healthy people who have taken artemisinin,the artemisinin and its metabolites have a chance to enter the anopheles mosquito.If the anopheles mosquito bites an infected person again,it has not been reported whether the artemisinin and its metabolites inhaled by the anopheles mosquito affect its malaria transmission ability.Therefore,it is of practical significance and scientific value to elucidate the effect of artemisinin on the ability of anopheles mosquitoes to transmit malaria.The innate immune system is the main way for Anopheles stephensi to resist exogenous pathogens and harmful stimuli,and is also an important factor affecting its ability to transmit malaria.Toll-like receptors?TLRs?is accounted as the most important pattern recognition receptors,they transmit extracellular stimulation signals to cells through adaptor protein molecules to activate Myeloid differentiation factor 88?MyD88?,and produce antimicrobial peptide?antimicrobial peptide,AMPs?,which inhibit the infection of plasmodium and reduce the ability of anopheles to transmit malaria via promoting Rel 1 nuclear translocation and activating the humoral immunity of Anopheles stephensi.Meanwhile,population and animal based studies have found that malaria infection can change the composition of host gut microflora community,and the remodeling of host intestinal flora can affect the natural immune status of mosquitoes and the development of plasmodium.However,the role and mechanism of innate immune system and intestinal flora in artemisinin's influence on the ability of Anopheles stephensi to transmit malaria remain to be elucidated.This study focuses on this scientific question.Methods:1.Part I:Impact of artemisinin pretreatment on the ability of Anopheles stephensi to transmit malariaUsing an animal model of Anopheles stephensi and Plasmodium yoelii,mice pretreated with artemisinin were fed to 3 to 5 days old Anopheles stephensi,the anopheles mosquitoes were then fed on mice infected with Plasmodium yoelii 3 days later.On the 9^th day after infection,the mosquito midguts were dissected,the development of the plasmodium oocyst was examined and counted,and the differences of infection rates and densities were analyzed and compared between the two groups.2.Part II:Involvement of innate immune Toll signaling pathway in the impact of artemisinin on vector capacity of anopheles to malariaFirstly,bioinformatic analysis was conducted to analyze the gene sequence of MyD88 of Anopheles stephensi.Then,real-time PCR was carried out to compare the transcriptional levels of MyD88 at different stages of Anopheles stephensi and before and after plasmodium infection,so as to investigate the role of MyD88 in the resistance of Anopheles stephensi to Plasmodium yoelii infection.After that,real-time PCR was used to compare and analyze the transcriptional levels of key antimalarial molecules of the Toll signaling pathway such as MyD88,Rel 1 and Cactus,between the artemisinin treatment group and the control group,so as to clarify the role of the Toll signaling pathway of Anopheles stephensi in the effect of artemisinin on the malaria transmission ability of Anopheles stephensi.3.Part III:Role of gut flora in the impact of artemisinin on vector capacity of anopheles to malariaThe gut microflora of Anopheles stephensi were analyzed and compared between the artemisinin-treated group and the control group before and after challenging by Plasmodium yoelii using high-throughput sequencing of 16S rDNA.According to the changes of gut flora,it was analyzed to illuminate if gut flora was involved in the impact of artemisinin on vector capacity of anopheles mosquitoes to malaria.Results:1.Artemisinin pretreatment significantly reduced the ability of Anopheles stephensi mosquitoes to transmit malaria.The infection rate of the artemisinin-treated group?88.15%?was slightly lower than that of the control group?90.4%?without statistically significant difference between the two groups?P=0.558?.However,the infection density in the artemisinin-treated group was significantly lower than that in the control group?P<0.001?.It was suggested that artemisinin pretreatment could reduce the vector capacity of Anopheles stephensi to Plasmodium yoelii.2.Toll signaling pathway of innate immunity plays an important role in the impact of artemisinin on the vector capacity of anopheles mosquitoes to malaria.The results of this part showed that MyD88 was a key upstream molecule of Toll signaling pathway of the Anopheles stephensi,and its expression was up-regulated at multiple time points after plasmodium infection,suggesting that MyD88 played an important role in mosquito resistance to plasmodium infection.At different time points after artemisinin pretreatment and plasmodium infection,transcriptional levels of Toll signaling pathway activating key molecules MyD88 and Rel 1 were both up-regulated in the artemisinin treatment group comparing with the control group,while the inhibitory molecule Cactus has a downward trend.The results suggested that artemisinin pretreatment could enhance the resistance of anopheles mosquitoes to plasmodium by upregulation of the Toll signaling pathway,thus inhibiting the ability of anopheles mosquitoes to transmit malaria.3.Gut microflora was involved in the impact of artemisinin on the vector capacity of anopheles mosquitoes.It was found that that the gut microbiota of anopheles mosquitoes changed significantly after artemisinin treatment,and some of the antimalarial microbiota such as serratia increased significantly.It suggested that artemisinin could probably affect vector capacity of anopheles to malaria by changing the gut microflora.Conclusions:1.This study is the first to demonstrate that artemisinin pretreatment can reduce the vector capacity of anopheles to malaria.2.Toll signaling pathway of innate immunity of mosquitoes was involved in the impact of artemisinin on the vector capacity of anopheles to malaria.3.Artemisinin may enhance the anti-malaria ability of anopheles mosquitoes by changing the gut flora of mosquitoes.The results above will help to clarify the impact of artemisinin application on the ability of anopheles mosquitoes to transmit malaria and its molecular mechanisms,so as to guide the rational promotion and application of artemisinin drugs and formulate corresponding countermeasures,and lay a theoretical foundation for blocking malaria transmission through vectors control.The results will also provide a reference for China's strategy formulation in the action to eliminate malaria to further consolidate China's achievements in malaria control and prevention.