| The Aedes aegypti mosquito transmits dengue,Zika,yellow fever,chikungunya virus disease and other insect-borne infections,causing hundreds of millions of infections each year and posing a serious public health problem worldwide.Due to the lack of specific drugs for the above infectious diseases and the absence of efficient vaccine protection,chemical control of their vectors has been the main way to prevent and control the spread and prevalence of insect-borne diseases.Due to the long-term use of a large number of insecticides,the resistance of mosquitoes is increasing day by day,in recent years,the effective use of mosquitoes olfactory behavior characteristics,the search for new control methods gradually become a research hot spot.In the life history of Ae.aegypti,life activities such as group dance mating,sugar feeding,blood feeding,finding water and laying eggs are all inseparable from its keen olfactory system.In this study,we firstly investigated the expression of OR genes in the tentacles of male and female Ae.aegypti mosquitoes,post-suckling females and post-oviposition females.qRT-PCR was applied to detect the expression of OR genes in different mosquito stages and perform relative quantification to screen for differentially expressed OR genes that may be associated with host localization,blood-suckling and egg-laying behaviors of Ae.aegypti mosquitoes.Subsequently,CRISPR/Cas9 technology was applied to gene editing in Ae.aegypti embryos for the screened OR110 gene with significant expression differences,and olfactory behavioral and electrophysiological techniques were applied to analyze the behavioral and electrophysiological responses of Ae.aegypti after OR110 gene knockdown for functional validation of the target gene.The main results of this research include:1.qRT-PCR results showed that there were 46 OR genes with lower expression in male Ae.aegypti tentacles than females,including OR2,OR10,OR11 and OR13,among which OR2,OR4,OR6,OR71,OR115 and OR132 contained variable splicing.There were six OR genes with higher expression in male mosquito tentacles than females,namely OR32,OR75,OR76,OR77,OR85,and OR89.2.There were 8 OR genes in the tentacles of Ae.aegypti with statistically different expression before and after blood sampling（p<0.05）.OR115-V2,OR115-V3,OR116 were down-regulated after blood sampling compared to before blood sampling.OR27,OR30,OR62,OR86,OR91,OR117 were up-regulated after blood sampling compared to before blood sampling.3.eight OR genes in Ae.aegypti tentacles were statistically different in expression before and after oviposition（p<0.05）,five OR genes were expressed at lower levels after oviposition than before oviposition,including OR38,OR93,OR110,OR112,OR115-V2 and OR115-V3;three OR genes were expressed at higher levels after oviposition than before oviposition,including OR75,OR77 and OR117.4.Ten OR genes in Ae.aegypti were expressed at lower levels in post-oviposition female tentacles than in blood-sucking females,including OR4-C,OR27,OR30,OR62,OR81,OR86,OR91,OR93,OR110,and OR119;three OR genes were expressed at higher levels in post-oviposition female tentacles than in blood-sucking females,including OR75,OR76,and OR77.5.OR38 and OR112 were found to be differentially expressed between Ae.aegypti females and males,and also differentially expressed before and after egg laying in females.OR30 and OR86 were differentially expressed in female mosquitoes before and after blood-sucking,and also differentially expressed in female mosquitoes after egg-laying and after blood-sucking.OR27,OR62 and OR91 were differentially expressed not only between Ae.aegypti mosquito females and males,but also before and after egg-laying,but also after egg-laying and after blood-sucking.6.The OR110 gene of Ae.aegypti,expressed differently between male and female,before and after oviposition,but also after oviposition and blood sucking,was deleted by using CRISPR/Cas9 gene editing technique,the results of behavioral experiments showed that it had no significant effect on the number of eggs laid and the time needed to search for water.Ae.aegypti has a large family of odor receptors,and knockdown of one of the odor receptors may compensate for other functional close odor receptors.On the other hand,OR110 is not related to the search for water sources,but may be associated with the search for other egg-laying attractants,such as 4-ethylphenol,dodecanic acid,etc.Phylogenetic analysis shows that OR110 is an odor receptor unique to Aedes mosquitoes,and it plans to study the specific behavior of Aedes mosquitoes.This study systematically screened a large number of ORs that may affect the host-seeking,blood-sucking,and egg-laying behaviors of Ae.aegypti mosquitoes,providing a direction for the next research on protein structure and interactions.ORs with significant intergroup differences and high expression,represented by OR93,OR110,OR115,etc.,provide possible targets for the development of mosquito control pathways based on olfactory system. |
