| ObjectiveTo investigate the resistance level to deltamethrin of Anopheles sinensis in Yunnan, Jiangsu and He’nan Province, so as to know the Kdr mutation in these three places. We detected the effect of sample size and exposed period on the estimated insecticide resistance level of Anopheles sinensis. We studied on insect cuticle proteins genes(ICPG) at the same time. We tried to find the relationship between the resistant mosquitoes and the susceptible ones, and the changes in different growth stages.Methods(1) We made Yingjiang, Suining and Kaifeng as the investigated places, the sample collection and feeding were carried out in the fields. Resistant mosquitoes and susceptible ones were collected by resistant tests.1～2L larvae,3～4 L larvae, pupae,0 d,5 d,10 d,15 d old mosquitoes were also collected.(2) This study used the WHO tube bioassays with varied sample sizes when the exposed period is 100 min, and varied exposed periods when the sample size is 20. Chi-square test was carried out on the corrected mortality rate in the three places with the SPSS19.0.(3) Kdr gene was amplified by PCR and sequenced to detect the mutation frequency in the investigated places, We used chi-square or Fisher test to compare the Kdr mutation frequencies of alive mosquitoes and dead mosquitoes.(4) We found all ICPG related to resistance and screened the genes of this study by bioinformatics. ICPG expression was determinded by RT-qPCR.2-△△Ct was used to calculate the data. ICPG expressions of resistant mosquitoes, susceptible mosquitoes and laboratory mosquitoes were compared by Kruskal Wallis test. ICPG expressions of mosquitoes in different stages were analyzed and compared by Graphpad prism 5, tested by ANOVA.Results(1) Mosquitoes in Yingjiang, Suining and Kaifeng were all resistant ones (R),24 h corrected mortality rates were 67.42%,54.33% and 47.40% (χ2=12.240, P<0.05). The frequency of Kdr mutation in the three places was 4.0%,96.0% and 100%, respectively. In Yingjiang and Suining, the difference of kdr mutation frequency between survival and death mosquitoes has no statistical significance (P=0.190,0.628, P> 0.05), while has statistically significant difference in Kaifeng County (χ2=75.81, P< 0.05).(2) 60 min exposure test cannot accurately estimate the population resistance for the mosquitoes with high resistance level, the test can reach the knockdown plateau after 80 min, with rKD 66.78% in Kaifeng and 71.30% in Yingjiang. Less sample size can lead to later knockdown time.(3) Results of RT-qPCR showed that CPF3, CPLCG3 expression in resistant mosquitoes was significantly higher than that of susceptible ones(P<0.01). CPR30 only express in resistant mosquitoes.(4) The expressions of ICPG in different growth stages (1-2 L,3-4 L, pupae,0 d,5 d,10 d, 15 d) were different. CPLCG3 was increased with the increasing of age, CPF3/4 was expressed specifically in adult mosquitoes, and CPR78 was expressed specifically in the pupa stage.Conclusion(1) The resistance levels of mosquitoes in these places are high. Kdr mutations are not the most essential mechanism for the generation of resistance.(2) Sample size and exposed period can make great effect on the estimated insecticide resistance level of Anopheles sinensis. High level resistant mosquitoes should be appropriately extended the exposed period. To obtain more accurate resistance level with less sample size, we should make corresponding mathematical statistics models and more practice.(3) The expressions of ICPG in the field resistant and susceptible mosquitoes were different, while change with age. |
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