Studies On The Expression Of DsEcR And DsMet By Gut Symbiotic Microbiota Against Mosquitoes

Mosquito is a kind of blood-sucking insect,which could transmit a variety of pathogens such as Plasmodium,dengue virus,Zika virus,chikungunya virus,etc.These mosquito-borne infectious diseases have caused many deaths all over the world,and bring a huge burden on public health and economy development.The history of the human struggle with mosquitoes lasts for thousands of years,but there is no effective ways to curb the spread.In recent decades,the development of chemical insecticides such as DDT has brought hope of eliminating mosquitoes.However,with the wide use of chemical pesticides,mosquito resistance is increasing,and chemical agents have caused serious damage to the ecological environment.These problems have increased the urgency of developing new environment-friendly control technologies.RNAi(RNA interference)is a post-transcriptionally inhibit gene expression method that is easy to operate and highly specific to target genes and has been rapidly developed once it was discovered.Therefore,dsRNA(double-stranded RNA)mediated RNAi has been adopted as a novel biological strategy for mosquito control or disease prevention.In previous research,microorganisms such as Escherichia coli,yeast and microalgae have been used to produce dsRNA targeting chitinase or ATPase genes,which achieved good effects.But these microorganisms could not survive and colonize in the host for a long time,which greatly limited the sustainability of RNAi effects and increased the cost of control.In this study,Ecdysone receptor(Ec R)and Methoprene-tolerant(Met)were used as target genes to intervene the growth and development of Anopheles stephensi larvae by RNAi technology.We further isolated a symbiotic bacterium,Serratia fonticola,from the gut of A.stephensi,which was modified to produce and accumulate dsRNA efficiently,and then to play the role of RNAi by colonizing the gut of A.stephensi.The results are as follows:1.dsMet or dsEcR affect the growth and development of A.stephensiAs key receptor genes in juvenile hormone and molting hormone,Ec R and Met genes play important roles in different developmental stages of insects.Block Ec R and Met expression makes the dysregulation of JH and 20 E may lead to growth retardation,molting or pupation failure,or even death.In this study,dsRNAs corresponding to Ec R and Met genes were designed,and their RNAi efficiency was evaluated in adult insects by microinjection.Afterwards,dsRNA was applied to larvae by immersion method,and on the one hand,the morphological development process of larvae was observed,the growth and development of larvae were abnormally retarded,with delayed pupation time and difficulty in pupation;on the other hand,the survival rate of larvae were recorded,which was greatly reduced compared to ds GFP-treated larvae.2.Engineered bacteria affect the growth and development of A.stephensiThe symbiotic bacterium was isolated and identified in the gut of A.stephensi,and a bacterium,Serratia fonticola,which can spread rapidly horizontally or vertically in mosquito populations was selected for further study.The RNase III gene was knockout in S.fonticola to suit for dsRNA expression and accumulation.In addition,the inducible expressed T7 promoter in dsRNA-expressing plasmid was substituted by the constitutively expressed T5 promoter,which could produce dsRNA without exogenous induction.The engineered bacteria fed to the larvae could colonize in larvae intestine and secrete dsRNA.Consequently,the growth of the larvae was gradually retarded after ingestion of the engineered bacteria,and the body size was confined at the average length of the second or third instar,the survival rate significantly decreased at the later stage,and the few surviving larvae had difficulty in pupating or had poor pupal development.This suggests that the genetically modified engineered bacteria can significantly disrupt A.stephensi larvae development as expected.In addition,the survival rate of the mosquitoes and the reproduction-related indexes such as blood meal and egg laying of female mosquitoes were recorded by feeding the engineered bacteria in adult mosquitoes.It was found that the engineered bacteria had no effect on the normal survival of mosquitoes in the short term,and the female mosquitoes were able to suck blood and complete egg laying normally.In summary,this study used RNAi technology to interfere A.stephensi developmental pathway related genes,Ec R and Met,which achieved good lethal effects on the larvae.We also genetically engineered a mosquito symbiotic bacterium to persistently express dsRNA in the mosquito intestine,which has a repressive effect on the larval growth and development.This study provides an intestinal symbiotic microbial-mediated RNAi strategy to knockdown gene expression in host mosquitoes,which provides new ideas and orientation for vector-borne disease control.