Research On Breeding And Rapid Detection Of Dichloros Resistance Musca Domestica Linnaeus (Housefly)

Background Musca domestica Linnaeus (House fly), as important vectors of numerous human and animal diseases all of the world, can cause great harm to public health.For a long time,the intensive use of insecticides, in controlling house flies, has made many insects develop the resistance of insecticides.This severely affects the treating effect and impedes the efficient progress of disease prevention. Therefore, based on the understanding of the characteristics and extent of resistance, scientific traetments play important roles in the prevention and control of house fly. There are many factors in the wild house fly which can affect the research of resistance, such as the complex interaction of resistance and stage differences. So resistant house flies directive breeding in the laboratory are used as the appropriate subjects.But it has not confirmed which method can be efficient for screening resistant strains. The reasons of drug resistance are complicated,which might be induced by one or multiple factors.But normally they can be divided into three mechanisms: physiological, biochemical and behavioral.The changes in acetylcholinesterase(AChE),the co-target site of organophosphate and carbamates compounds are one of the major mechanisms of resistance to organophosphates(OPs) and carbamates(CBs) .Currenty it has been confirmed that the AChE of house flies is encoded by a single acetylcholinesterase gene (Ace) and mutation of Ace gene can lead to structural changes in AChE. Altered AChE reduces the affinity and provides insensitivity to different Ops and/or CBs insectticides. Many subsequent studies indicated that there were 15 Ace alleles found and 11 of the alleles coded for a susceptible form of the enzyme. The methods to detect resistance using biochemical and molecular biology have developed rapidly. Compared with the traditional biological methods, these new methods have significant advantages in accuracy, repeatability and early detection of resistance.But considering their limitations on technique,cost and overall evaluation on insects,these new methods are mainly used for genetic research. The aims of this study are to compare breeding methods of resistant house flies and to study characteristics of AChE in resistant house flies, establishing a rapid and easy detection method of resistance to dichlorvos (DDVP) and analyzing Ace gene mutations of resistant houseflies bred in laboratory.Methods In this study, we bred dichlorvos-resistant strains of houseflies with dichlorvos in laboratory and measured the resistant spectrum by topical application. To study methods of breeding and bring insecticide resistance, we selected surface contact method, topical application and larvae phase selection. The toxic effects of DDVP for the larvae was evaluated through the number and the average weight of larvae, pupae and adult, time of pupation and eclosion. The crude enzyme, extracted from the head of different resistant strains, was used to be the enzyme sources of enzymatic reaction. Activity and inhibition of AChE, reflecting the sensitivity of different strains for DDVP, were calculated by measuring the values of absorbance (A). We determined the DDVP concentration, which can complete inhibition AChE activity of susceptible strain, and established a qualitative method to detect resistance of housefly by observing the color reaction. The cDNA of the Ace gene encoding AChE from susceptible strains and dichlorvos-resistant strains had been cloned using RT-PCR and sequenced. DNAstar software was adopted to compare the gene sequence from different strains,and then BLAST Sequence Similarity Searching tools were applied to compare the sequence with those registered in GeneBank,trying to testify the drug resistances of directive breeding house fly on the molecular level.Results After 5 times directive breeding with surface contact method,topical application and larvae phase selection,a strain of house fly with the original resistance ratio of 1.29 exhibited 2.12, 8.91 and 19.35-fole resistance to dichlorvos at the LD50. The toxicity of DDVP in housefly mainly showed the lethal and teratogenic effects. Larval survival rate, pupation rate, emergence rate, pupal and adult average weight were reduced with increasing concentration of DDVP. The distortion number and extent were proportional to the concentration of DDVP. But the DDVP concentration had little effect in emergence and pupation time, and had no relation with the gender of houseflies. The crude enzyme content and AChE activity show no statistically significant difference among resistant strains and susceptible strains. AChE activities of each resistant strain were decreased with increasing concentration of DDVP, however, in a certain range (DDVP concentration <0.6mg/mL), the resistance of housefly AChE showed a certain degree of insensitivity. With the increase of resistance, inhibition of AChE to DDVP was significantly increased.After analysis with the data,0.08mg/mL was chosen as the limit concentration between DDVP sensitive strains and resistant ones,which could be used to rapidly test the housefly's drug resistance. Compared with the susceptible strain, the sequence of Ace gene of resistant strain had 7 point mutations, one insertion and one deficiency.Conclusion1 Compared with surface contact method and topical application methods, larvae phase selection can enhance the level of resistance more quickly.2 Dichlorvos can not delay the pupation and eclision of larvae, but will affect the number and average weight of larvae, pupae and adult.3 Compared with the susceptible strain, the content and activity of the AChE in resistant strains did not increase. The reason of resistance is the decreased sensitivity to organophosphates of AChE.4 Established a qualitative method to detect resistance of housefly rapidly.5 Molecular biology studies have shown that Ace-v15 allele was found in the laboratory resistant strains.