| Background:The Asian tiger mosquito,Aedes albopictus,is the vector for several arboviruses,including dengue,Zika and chikungunya fever viruses,bringing with it a significant burden to public health.Vector control,which relies mainly on chemical insecticides,is the main strategy of mosquito-borne disease prevention and control.However,wide and improper application of insecticides have led to ever-increasing insecticide resistance,especially against the most commonly-used pyrethroid insecticides,and this has compromised the effectiveness of many control programs.At present,the research on insecticide resistance of mosquitoes is mostly a retrospective analysis of multiple factors in the field studies,which makes it difficult to clarify the evolution of resistance,fitness cost and vectorial capacity of specific mosquitoes to a single insecticide.Objective:1.Deltamethrin,the representative insecticide of pyrethroids,was selected as a single variable factor to study the change rule and molecular mechanism of resistance evolution of Ae.albopictus to deltamethrin,and to screen the molecular markers of generation of insecticide resistance.2.Functional verification of molecular targets of deltamethrin resistance in Ae.albopictus was conducted to elucidate the molecular mechanism of resistance generation and develop a reliable molecular detection method for population resistance monitoring.3.To study the contribution of resistance mechanisms to fitness cost in Ae.albopictus,and to evaluate the comprehensive impact of resistance evolution and fitness cost on vectorial capacity of dengue virus.Methods:In this study,deltamethrin was used to screen the resistance of laboratory susceptible strain of Ae.albopictus.The change rule and molecular mechanism of the evolution of deltamethrin resistance in Ae.albopictus were analyzed by monitoring the resistance level of different screening generations and detecting the target-site resistance and metabolic resistance during the process of resistance screening.CRISPR/Cas9 technology and genetic backcross experiment were used to verify the in vivo function of molecular target generated by resistance,and the specific mechanism of resistance induced by molecular target was elucidated by computer modeling of target protein and transcription detection of target gene.The contribution of different resistance mechanisms to fitness cost was quantitatively analyzed,and the comprehensive impact of resistance evolution of Ae.albopictus to deltamethrin and fitness cost on transmission of DENV-2 were systematically investigated using vectorial capacity formula.Results:Deltamethrin was pressurised to select susceptible strain to the 30th generation.It was observed that the development of deltamethrin resistance in Ae.albopictus was observed to be slow at first and then fast.It was verified that the introduction of voltage-gated sodium channel(VGSC)F1534S mutation in Ae.albopictus could conferred deltamethrin resistance on a previously susceptible strain,and removed this mutation restored the susceptible phenotype.The resistance phenotype caused by F1534S is associated with decreased sodium channel binding ability to insecticide and decreased VGSC transcription level.Furthermore,F1534S mutation was detected in deltamethrin-resistant Ae.albopictus populations collected in the field.Based on the above results,a molecular tool for detecting F1534S mutation was developed as one of the criteria for monitoring the resistance of Ae.albopictus population to deltamethrin.Laboratory strains representing different resistance mechanisms were isolated and identified.By comparing the resistance level and fitness cost of several strains,it was found that the F1534S mutation heterozygote of VGSC gene in the early stage of resistance generation not only confer high level of resistance,but also had no significant fitness cost,leading to the rapid spread of resistance in Ae.albopictus population.Because of the significant fitness cost of F1534S homozygote,the resistant population evolved an I1532T mutation with low fitness cost to adapt to the screening pressure;With the evolution of resistance,metabolic resistance that has no significant fitness cost and mediates high resistance phenotype may play its dominant role.In addition,we found that the parameters related to fitness cost in the vectorial capacity formula of resistant strains decreased their VC value.However,the high vector density after insecticide exposure caused by resistance reversed the disadvantage of fitness cost,making the vectorial capacity of the resistant strain to DENV-2 higher than that of the susceptible strain.Among them,the resistant strain dominated by metabolic resistance has the highest vectorial capacity,mainly due to its high resistance level and the lowest fitness cost.Conclusion:We used deltamethrin to screen important vectors of Ae.albopictus,and established an evolutionary model of resistance in mosquito.The initial stage of deltamethrin resistance in Ae.albopictus is the key to vector control,at which point the F1534S mutation of VGSC gene plays an important role.Combined with the functional verification of F1534S mutation in vivo and the detection results of field resistant populations,a molecular detection method based on F1534S mutation was developed for the resistance monitoring of Ae.albopictus population.The molecular mechanism of deltamethrin resistance evolution in Ae.albopictus was revealed by comparing and analyzing the fitness costs of different strains representing different resistance mechanisms.Using vectorial capacity formula to comprehensively analyze the resistance level,fitness cost and susceptibility to DENV-2.It was found that the evolution of deltamethrin resistance of Ae.albopictus will increase the transmission risk of dengue virus,and the control of vector density is still the primary goal of vectorborne diseases.The results of this study will provide scientific guidance for the use of insecticides,resistance management and prevention and control of mosquito-borne diseases. |
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