Anopheles Sinensis Mosquito Insecticide Resistance:Comparison Of Three Mosquito Sample Preparation Methods In Resistance Measurements

ObjectiveTo investigate the effects of mosquito preparation methods on the results of resistance detecting, so as to guide choosing the study subject properly and scientifically in the mosquito resistance detecting; at the same time, to explore the perhaps causes of An. sinensis mosquito resistance in the study sites.MethodsWe chose Huainan City, Anhui Province and Liuyang City, Hunan Province as study sites. In each place, the three commonly used sources mosquitoes were collected:field-collected adults, adults reared from field-collected larvae and F1adults from field blood-fed female. According to the WHO standard protocol, the resistance level, P450monooxygenase (P450s) activity, glutathione s-transferase (GST) activity and Kdr mutation in the three sources mosquitoes were tested, respectively., and then were compared to find their differences among the three sources mosquitoes. These variances were also detected and compared between the blood fed and non-blood fed mosquito, and among the Id,3d,6d,9d,12d,15d,20d old mosquitoes, in order to explore the further confounding factors. At the same time, all of the variances in each group were compared with those in the Labs strain, so as to study the perhaps reasons for the An.sinensis resistance in the local sites.Results(1) The An.sinensis in Huainan and Liuyang have become resistant to deltemethrin, with the mortalities from15.1%to47.6%.(2) In the two sites, the mortalities of field-collected adults were higher than the other two, which shared the same mortality (Liuyang, F=4.85, P<0.05&Huainan, F=27.18, P<0.01). On the other hand, the enzymes activities of the three sources mosquitoes were also different:for the P450monooxygenase (P450s) activity, field-collected adults> adults reared from field-collected larvae>F1adults from field blood-fed female(Huainan,F=8.43, P<0.0001&Liuyang,F=19.84, P<0.01); and for the glutathione s-transferase (GST) activity, field-collected adults> adults reared from field-collected larvae=Fl adults from field blood-fed(Huainan,F=68.18, P<0.01&Liuyang, F=19.12, P<0.01).In the two sites, the Kdr mutation proportions in F1adults from field blood-fed female were3.2-5.8%higher than those in the field-collected adults.(3) The blood fed and non-blood fed mosquito had the similar mortalities (χ2=0.4542, P>0.05),and the blood fed mosquitoes had higher P450s and GST activities than the non-blood fed mosquitoes (t=6.53, P<0.01&t=6.82, P<0.01). All the variances, including the resistance level, GST and P450s enzyme activity, were changing with the mosquito age.(4) In the two sites, all of the P450s activities in the three sources mosquito were higher than the p450s activity in the Labs strain, and the GST activity in the field-collected adults was higher than that in the labs strain. The L1014F mutation in the alive (resistant) mosquito was more often than that in the dead (susceptible) mosquito. All of these results induced that the mosquito preparation method had effects on the resistance detecting, and the mosquito age and the blood fed maybe the causes. L1014F mutation and the elevated P450s activity may be responsible to the resistance in the An. Sinensis in Huainan and Liuyang.Conclusion1、Given the results detected using field-caught adult are easily confounded by blood and mosquito age, and obtaining F1generation is more time-consuming, we suggest the field-emergence-mosquitoes as the preferred study subjects, which can minimize these confounding effects, and then reveal the accurate metabolism enzyme levels in the local population.2、The Kdr mutation and the elevated p450monooxygenase activity may be responsible for the deltemethrin resistance in the An. sinensis in Hunan Liuyang and Anhui Huainan.