| Anopheles sinensis is an important vector of malaria and filariasis.It is wide distributed in China and is the dominant mosquito species in plain water network and rice growing areas.Vector control plays an extremely important role in the control and elimination of malaria,and most important measures are insecticide indoor residue spray(IRS)and insecticide treated bed nets(ITN)have been used.Due to its high efficiency and low toxicity,deltamethrin have been widely used for a long time,which has played an important role in controlling malaria in China.In recent years,it has shown that Anopheles sinensis has developed resistance to deltamethrin insecticides in varying degrees.It has become an important threat to current vector control activities.In this study,we compared the expression level of deltamethrin resistance related gene family cytochrome P450 genes(CYP genes)in Anopheles sinensis with different levels of resistance,to identify the CYP genes that may play a major role in the mechanism to deltamethrin resistance in Anopheles sinensis,to provide foundation for further understanding of the molecular mechanism and resistance control measures.This study is consisted of three parts: Part I Analysis of the deltamethrin resistance in Anopheles sinensisIn order to detect the deltamethrin resistance of Anopheles sinensis from different regions,the bottle bioassay recommended by the US Disease Control and Prevention Centers was used.The deltamethrin resistance in Anopheles sinensis that collect in different sites in Jiangsu Province(Yancheng,Changzhou,and Taizhou)was evaluated,and differentiated the degree of deltamethrin resistance of Anopheles sinensis in different areas.The results showed that the mortality of laboratory mosquitoes was 100% within the diagnosis time(30 min)at the diagnostic dose of deltamethrin(12.5 ?g/bottle),indicate that the laboratory mosquito is deltamethrin sensitive mosquito.Combined with mortality of mosquitoes at different concentration and data fitting showed that the median lethal concentration(LC50)of deltamethrin was 3.849 ?g/bottle in laboratory mosquito.The mortalities of Anopheles sinensis from Yancheng were 11.11%,46.67%,and 100% at concentrations of 125,250 and 375 ?g/bottle respectively,the mortalities of Anopheles sinensis from Changzhou were 34.48%,46.43%,and 100% at concentrations of 375,500,and 625 ?g/bottle respectively,the mortalities of Anopheles sinensis from Taizhou at concentrations of 375,500,and 625 ?g/bottle were 21.88%,40.83%,and 84.21% respectively.In this part of the study showed that the laboratory mosquitos were deltamethrin sensitive mosquito,while the field mosquitos were deltamethrin resistance mosquitoes,and the field mosquitos deltamethrin resistance level was highly resistant.However,the levels of deltamethrin resistance in Anopheles sinensis in different areas were significantly different,from high to low were Taizhou,Changzhou and Yancheng.Part II Analysis of deltamethrin resistance related genes in Anopheles sinensisTo analyze the molecular mechanism of deltamethrin resistance in Anopheles sinensis,the Allglo probe was used to detect the mutation of kdr(knock down resistance)gene in three Anopheles sinensis using Real-time fluorescence quantitative PCR.The results showed that there were two kdr mutation types,L1014 F and L1014 C,the genotypes including TTT/TTT,TGT/TGT,TTG/TTT,TTT/TGT,no wild type TTG/TTG and heterozygous TTG/TGT,statistical analysis showed that the frequencies of different kdr alleles and L1014 F and L1014 C mutation in Anopheles sinensis were not statistically different.It is suggested that the deltamethrin resistance of the Anopheles sinensis in the three sites is related to the kdr mutation,but the difference in the resistance level among them may not be due to the mutation of the kdr gene.Secondly,RT-qPCR was used to detect the expression level of six related CYP genes(CYP6M3,CYP6Y1,CYP6P5,CYP4H14,CYP4G17,and CYP12F16)in Anopheles sinensis samples of three sites.In this part of the study,the kdr genotypes of the laboratory Anopheles sinensis samples were found to be homozygous,while field Anopheles sinensis samples were all mutant,and there was no significant difference in kdr gene mutations of Anopheles sinensis in different regions.Therefore,we believe that Anopheles sinensis deltamethrin resistance is related to the kdr mutation,but the difference in resistance is not caused by the kdr mutation.In the detection of resistance-related CYP gene expression,it was found that the expression level of CYP6M3 gene was significantly correlated with the level of resistance,presumably it may be a major gene of deltamethrin resistance.Part III Analysis of expression characteristic of deltamethrin resistance related cytochrome P450 genes in Anopheles sinensisIn order to further study the expression of the CYP genes in Anopheles sinensis,we detected the expression level of the CYP genes of laboratory samples in the different development stages of Anopheles sinensis : egg,larvae,pupa,adult(male and female);different tissues : salivary gland,malpighian tubule,midgut,ovaries and fat body;different deltamethrin exposure dose : 0,1.25,3.75,6.25,12.5 ?g/bottle and different deltamethrin exposure time : 0,5,15,30,60 minutes.The study showed that CYP6M3 and CYP6Y1 were expressed in the highest level in male mosquitoes,the expression of CYP6M3 in male mosquitoes was 35.1 times than that in female mosquitoes,and the expression of CYP6Y1 in male mosquitoes was 61.4 times than that in female mosquitoes;CYP4H14 had the lowest expression level in larval stage,the expression of CYP4H14 in females was 22.5 times than that in fourth instar larvae.The related CYP genes expression was significantly different in different tissues of Anopheles sinensis and showed significant enrichment,the expression of CYP6M3 and CYP12F16 were 38.9 times and 4.4 times higher than that in the ovary,the expression of CYP6Y1 and CYP4G17 in the fat body were 9.1 times and 4.6 times higher than that in the ovary,the expression of CYP6P5 in the midgut was 30.3 times higher than that in the ovary.Exposure to different deltamethrin doses and time showed the induction effect on the expression level of related CYP genes.In the process of exposure to different deltamethrin concentration,the expression levels of CYP6P5,CYPH14,and CYP12F16 increased with increasing contact dose,the expression level of CYP6Y1 increased first and then decreased with increasing exposure dose,the expression level of CYP4G17 decreased first and then increased with increasing exposure dose.During exposure to deltamethrin for different periods of time,changes in the expression level of resistant related CYP genes showed a tendency of increasing first and then decreasing with time(5-60 min).For example,CYP6M3,CYP6P5,CYP4G17 and CYP12F16 all showed this trend,and the expression levels of CYP6M3,CYP6P5,CYP4G17 and CYP12F16 reached the maximum at 30 min.In this part of the study,it was found that the resistance-associated CYP genes showed significant differences in different developmental stages and in different tissues of Anopheles sinensis,and its expression level showed significant enrichment in some tissues.The expression level of resistance-associated CYP genes showed a certain inducing effect with insecticide exposure,which further suggested that CYP family members play an important role in the deltamethrin resistance of Anopheles sinensis. |
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