Effects Of Sublethal Dose Of Bacillus Sphaericus On The Malaria Vector Capacity Of Anopheles Mosquitoes And Its Molecular Mechanisms

Background and ObjectivesMalaria remains a major global public health problem and the malaria incidence has been on the rise for five consecutive years.Although China has reached the elimination standard of malaria,imported malaria still poses a great threat to the health of Chinese people.Moreover,there are a large number of Chinese people who travel to malaria-endemic areas such as Africa and Southeast Asia,and malaria remains a serious threat to these populations.With the emergence and spread of mosquito resistance to chemical insecticides,the use of biolarvicides in mosquito control has become an important strategy to block malaria transmission.Bacillus sphaericus(Bs)is one of the most widely and successfully used biolarvicides.Bs has various advantages such as having strong selectivity for mosquito killing,safe to non-target organisms,easy to be degraded in nature and non-pollution to environment,etc.Meanwhile,it can be recycled in larval carcasses in some cases.It has become the main non-chemical means of mosquito control in Africa and other malaria-endemic areas.However,the killing efficiency of Bs in field use can be affected by several biological and abiotic factors,such as inaccurate measurement of the dosage of Bs or reduced contact between larvae and Bs caused by vegetation growth enveloping in the habitat,as well as p H,turbidity and temperature of water,etc.These factors may degrade B.sphaericus or alter the activity of B.sphaericus in mosquitoes,resulting in a sublethal dose effect and survival of some mosquitoes.The physiological characteristics of surviving Anopheles mosquitoes may change under the selection pressure of biolarvicide and the exposure to B.sphaericus.Changes in the vector capacity(susceptibility to Plasmodium infection/population density)of Anopheles will directly affect the epidemic and transmission of malaria.However,whether the vector capacity of the surviving mosquitoes will change after Bs exposure and its mechanisms have not been clarified.In this study,we firstly used two malaria infection models to test the effects of sublethal dose of Bs on the susceptibility of Anopheles mosquitoes to malaria parasites infection.Then,the immune signaling pathway and key antimalarial molecules of mosquitoes were studied to analyze the innate immune mechanisms involved in the impact of Bs on the susceptibility of Anopheles to Plasmodium infection.Moreover,the effects of sublethal dose of Bs on the fecundity of Anopheles mosquitoes were investigated.Finally,the molecular mechanisms of Bs affecting the reproductive capacity of Anopheles mosquitoes through TOR signaling pathway were analyzed.This study will provide reference for the prediction of possible consequences and the formulation of corresponding countermeasures once sublethal dose effect occurs in the practical application of Bs,guiding the scientific and rational application of Bs.This study also provides new ideas for blocking malaria transmission by mosquito control.Methods1.Effects of sublethal dose of Bs treatment on susceptibility of Anopheles mosquitoes to Plasmodium infectionFirstly,the killing efficiency of Bs on Anopheles stephensi larvae and Anopheles dirus larvae were detected by the method of Probit analysis bioassay.Secondly,the linear relationship between Bs concentration and cumulative mortality of larvae was analyzed to obtain the sub-lethal dose concentrations for the treatment of Bs on An.stephensi and An.dirus larvae.Then,sublethal dose of Bs was used to treat the mosquito larvae,and the surviving adults were collected and the 3 d-adults were infected with Plasmodium parasites;24 h later,the blood-saturated female mosquitoes were isolated and routinely maintained;nine days after infection,the mosquito midguts were dissected and the infection and melanization of oocysts were detected.2.Study on the innate immunity mechanisms of Bs affecting the susceptibility of Anopheles mosquitoes to malaria parasitesFirstly,sublethal dose of Bs was used to treat the mosquito larvae,and the surviving adults were collected and the 3 d-adults were infected with Plasmodium parasites;24 h later,the engorged female mosquitoes were isolated for routine rearing and samples were collected at larvae,pupae,adult mosquito stages and various time points after Plasmodium infection.Then,Real-time quantitative PCR was used to detect the effects of sublethal dose Bs treatment on transcriptional levels of key genes in the Toll,Imd,and JAK-STAT signaling pathways and the corresponding antimalarial immune effect molecules of each signaling pathway.Thirdly,RNA interference was carried out to confirm the function of antimalarial immune effector molecules in changing of susceptibility of Anopheles to Plasmodium infection by Bs exposure.3.Impact of sublethal dose of Bs treatment on fecundity of surviving Anopheles mosquitoesFirstly,mosquito larvae were treated with sublethal dose of Bs,and the surviving adults were collected and fed with blood three days after eclosion.Twenty-four hours later,the engorged female mosquitoes were isolated and reared individually to count the eggs laid on the filter paper.On the 10 th day after feeding the mosquitoes with blood,the ovaries of Anopheles were dissected and the retained eggs were counted.Then,the eggs produced by the above single rearing mosquitoes were put into water to observe the changes of the hatching rate,pupation rate and emergence rate.Thirdly,the effects of Bs on the reproductive capacity of Anopheles larvae were observed after they were treated with different sub-lethal concentrations of Bs.Finally,Anopheles larvae were treated with sublethal dose of Bs,and pupae were collected.Male and female Anopheles larvae were separated after emergence,and the effects of sublethal dose of Bs on survival of female and male mosquitoes and engorged female mosquitoes were observed.4.Study on mechanisms of how Bs impacts the fecundtiy of Anopheles mosquitoes through TOR signaling pathway4.1 In the research of An.stephensiFirstly,Anopheles larvae were treated with sublethal dose of Bs,and the surviving adults were collected and fed with blood three days after eclosion.Twenty-four hours later,the engorged female mosquitoes were isolated and collected for RNA-seq.Transcriptome sequencing results were verified by RT-q PCR,and genes affecting the reproductive capacity of Anopheles mosquitoes treated with sublethal dose of Bs were screened and analyzed.Secondly,RT-q PCR and Western blot were used to detect the effects of sublethal dose of Bs treatment on the transcription level and protein level of Vg gene in Anopheles mosquitoes.Meanwhile,RNAi was conducted to silence expressing of Vg to observe the changes in reproductive capacity of surviving adult mosquitoes treated with sublethal dose of Bs.Thirdly,RT-q PCR and Western blot were used to detect the effects of sublethal Bs treatment on the activation level of TOR signaling pathway in Anopheles mosquitoes.At the same time,rapamycin was used to inhibit TOR,and the changes induced by sublethal Bs treatment on reproductive ability were detected.Forthly,the BCA method was used to detect the effects of sublethal Bs treatment on blood protein concentration in the midgut.Fifthly,adult mosquitoes were fed with Bs bacteria solution mixed with sugar water and the effects on the reproductive capacity of Anopheles mosquitoes were observed.RT-q PCR and Western blot were used to detect the effects of Bs feeding on Vg expression and TOR signaling pathway activation.Meanwhile,rapamycin was used to inhibit TOR,and the effect of Bs direct feeding on the reproductive capacity of adult mosquitoes was detected.Sixly,BCA method was used to detect the effects of Bs feeding on blood protein concentration of Anopheles midgut.Finally,adult mosquitoes were fed directly with the supernatant of Bs bacteria solution mixed with sugar water to observe its effect on the reproductive capacity of Anopheles mosquitoes.RT-q PCR and Western blot were used to detect the effect of directly feeding adult mosquitoes with Bs supernatant on Vg expression.4.2 In the research of An.dirusFirstly,the full-length c DNA of Ad Vg gene was amplified by RACE PCR.Secondly,RT-q PCR and Western blot were used to detect the effect of sublethal dose Bs treatment on Vg expression in mosquitoes.Thirdly,Vg recombinant protein was expressed and purified.The Vg protein was injected into An.dirus via microinjection,and the effect of the Vg protein on mosquito oviposition ability was then observed.Forthly,RT-q PCR and Western blot were used to detect the effects of sublethal Bs treatment on the activation level of TOR signaling pathway in An.dirus.Finally,adult mosquitoes were fed with Bs bacteria solution mixed with sugar water and the effects on the reproductive capacity of Anopheles mosquitoes were observed.RT-q PCR and Western blot were used to detect the effects of Bs feeding on Vg expression and TOR signaling pathway activation.Results1.Effects of sublethal dose of Bs treatment on susceptibility of Anopheles mosquitoes to Plasmodium infectionWe found that the LC50 of Bs was 16.57 IU/L for An.stephensi larvae and 5.62 IU/L for An.dirus larvae.Furthermore,in the An.stephensi-P.yoelii susceptible animal model,the sublethal dose of Bs treatment increased the infection density of Plasmodium in the surviving Anopheles mosquitoes without impacting the infection rate.However,in the non-susceptible mosquito model of P.yoelii,sublethal dose of Bs treatment decreased the infection rate and density of Plasmodium in the surviving Anopheles mosquitoes.And,there was no significant difference in melanization rate and density of melanized oocysts,indicating that the melanization mechanism was not involved in the effect of Bs on the susceptibility of An.dirus to P.yoelii infection.2.Study on the innate immunity mechanisms of Bs affecting the susceptibility of Anopheles mosquitoes to malaria parasites2.1 In the research of An.stephensiSublethal dose of Bs treatment inhibited the transcription levels of key molecules of Toll,Imd and JAK-STAT signaling pathways compared with the control group.Moreover,An.stephensi Bs exposure significantly inhibited the expression of effector molecules of the above three signaling pathways such as As DEF1,As TEPs and As NOS genes at multiple time points after Plasmodium infection.2.2 In the research of An.dirusAn.dirus Bs exposure in larval stage significantly up-regulated the expression of Ad PGRP-LC and Ad Rel2 genes in the Imd signaling pathway,but there was no significant difference in the expression of key molecules in the Toll and JAK/STAT signaling pathways between the control and Bs groups.In addition,Bs exposure significantly up-regulated the expression of Ad TEP1 gene in surviving An.dirus mosquitoes after Plasmodium infection and the time of high-level expression was longer than that of the control group.After silencing Ad TEP1 gene by RNAi,the phenomenon that Bs exposure significantly inhibited the susceptibility of An.dirus to Plasmodium disappeared.3.Impact of sublethal dose of Bs treatment on fecundity of surviving Anopheles mosquitoesPrevious studies of our group have found that sublethal dose of Bs can reduce the oviposition ability of An.dirus.In this study,only the effects of sublethal dose of Bs on the reproductive ability of An.stephensi were studied.We found that sub-lethal dose of Bs treatment increased the total eggs(the eggs laid out plus the eggs retained in mosquitoes),the eggs laid out,and the oviposition rate in surviving Anopheles mosquitoes,without inducing any significant difference in gravidity rates between Bs and control groups.In a range of concentrations,the increase of total eggs and laid eggs of the surviving Anopheles mosquitoes showed a concentration-dependent pattern.In addition,larval Bs exposure shortened the life span of the surviving non-bloodfed female mosquitoes,but the impact of Bs on life span of adults disappeared after bloodfeeding.Besides,Bs treatment had no effect on life span of male mosquitoes.There was no significant effect of Bs on the development of the surviving Anopheles’ s offsprings either.4.Study on mechanisms of how Bs impacts the fecundtiy of Anopheles mosquitoes through TOR signaling pathway4.1 In the research of An.stephensiBs exposure in larval stage significantly up-regulated the transcription and protein levels of As Vg after a blood meal.After silencing the expression of As Vg gene by RNAi,the phenomenon that Bs exposure in larval stage enhanced the reproductive capacity of the surviving mosquitoes disappeared.It indicated that As Vg played an important role in Bs enhancing the reproductive capacity of An.stephensi.Meanwhile,Bs exposure in larval stage significantly upregulated the activation level of TOR signaling pathway.After rapamycin treatment,the enhancement of fecundity and up-regulation of As Vg expression through activating TOR signaling pathway by Bs disappeared.In conclusion,the results suggested that Bs exposure in larval stage enhanced the reproductive capacity of An.stephensi by up-regulating the expression of As Vg through activating TOR signaling pathway.In addition,there was no significant difference in blood protein content in the midgut of Anopheles mosquitoes between the Bs group and the control group at 24 h,36 h and 48 h after the blood meal,suggesting that Bs exposure in larval stage had no significant effect on the digestion of blood protein in the midgut of Anopheles mosquitoes.It’s worth noting that direct feeding of Bs to adult mosquitoes would enhance the expression of As Vg by activating TOR signaling pathway and increase the reproductive capacity of Anopheles mosquitoes,with no significant effect on the digestion of blood protein in the midgut.It was consistent with the phenotype that in Bs exposure in larval stage.The results suggest that Bs may persist in the surviving adult mosquitoes after Bs exposure in larval stage and increase the expression of As Vg by activating the TOR signaling pathway to enhance the reproductive capacity of Anopheles mosquitoes.What’s interesting is that directly feeding adult mosquitoes with Bs supernatant led to increases of total eggs laid and laid eggs of Anopheles,significantly up-regulated the transcription and protein levels of As Vg at 24 h after blood meal in consistency with the up-regulation of As Vg transcription and protein levels in Bs exposure in larval stage.The results suggested that after larvae were treated with sublethal doses of Bs,Bs may persist in the surviving adult mosquitoes and activate TOR signaling pathway through its metabolites resulting in upregulatation of the expression of As Vg and enhancement in the fecundity of Anopheles mosquitoes.4.2 In the research of An.dirusThe full-length Ad Vg gene c DNA sequence was successfully amplified and the Vg domain protein was expressed and purified in this study,which can be used for subsequent research.In addition,exposure to Bs in larvae significantly down-regulated the transcriptional and protein levels of Ad Vg after the blood meal.Moreover,Bs exposure in larval stage significantly inhibited the activation of the TOR signaling pathway in the surviving Anopheles mosquitoes.The results suggested that Bs exposure in larval stage could down-regulate the expression of Ad Vg by inhibiting the activation of the TOR signaling pathway,thereby inhibiting the egg-laying ability of Anopheles mosquitoes.Meanwhile,direct feeding of Bs to adult mosquitoes significantly inhibited the expression of Ad Vg and the activation of TOR signaling pathway,and decreased the oviposition ability,which was also consistent with the down-regulation of fecundity in Bs exposure in larval stage.In conclusion,the results suggested that Bs migh persist in the surviving adult mosquitoes after Bs exposure in larval stage,and downregulate the expression of Ad Vg by inhibiting the activation of TOR signaling pathway,then inhibit the egg-laying ability of An.dirus.Conclusions1.Sublethal dose of Bs showed different effects on the susceptibility of An.stephensi and An.dirus to Plasmodium infection.In a susceptible animal model of An.stephensi-P.yoelii,the sublethal dose of Bs treatment increased the susceptibility of the surviving An.stephensi to P.yoelii infection.While in An.dirus-P.yoelii non-susceptible animal model,sublethal dose of Bs treatment decreased the susceptibility of the surviving An.dirus to P.yoelii infection without involvement of the melanization mechanism.2.In a susceptible animal model of An.stephensi-P.yoelii,Bs exposure in larval stage may down-regulate the expression of As DEF1、As TEPs and As NOS by inhibiting the Toll,Imd,and JAK-STAT signaling pathway,respectively.Then,it will reduce the killing of plasmodium by anti-malarial immune effector molecules and enhance the susceptibility of the surviving An.stephensi to P.yoelii infection.In An.dirus-P.yoelii non-susceptible animal model,Bs exposure in larval stage may upregulate the expression of Ad TEP1 and enhance its killing of Plasmodium through the Imd signaling pathway,thus reducing the susceptibility of the surviving An.dirus to P.yoelii.3.Sublethal dose of Bs treatment significantly enhanced the reproductive capacity of the surviving An.stephensi.Although Bs shortened the life span of the surviving non-bloodfed female mosquitoes,the phenomenon of shortening the life span of female mosquitoes disappeared after blood-feeding.4.In An.stephensi,after Bs exposure in larval stage,Bs might persist in the surviving adult mosquitoes and activate the TOR signaling pathway through its metabolites to upregulate the expression of As Vg,thereby enhancing the reproductive capacity of Anopheles.In An.dirus,after Bs exposure in larval stage,Bs might persist in the surviving adult mosquitoes and downregulate the expression of Ad Vg by inhibiting the activation of the TOR signaling pathway,thereby inhibiting the egg-laying ability of An.dirus.