| Insecticide resistance in vector mosquito populations is a major problem in the control of mosquito-borne diseases. Organophosphate(OP), carbamate and pyrethroid insecticides have been the major classes of chemicals used in pest control. Cx. pipiens pallens is a kind of important vector in North China, which is breeding in sewage, ditch, sewer and polluted puddle and so on, and is easy to resistant to OP and carbamate insectcides because of pollution of water with many agrochemical. It is important to elucidate the resistance mechanism in order to slow down the development of resistance of vector populations. In this research, eleven wild mosquito strains which were collected from eleven sites in Beijing were used as the research material. Two kinds of resistance-related genes have been studied: (1) Acel gene encode Acetylcholinesterase (AChE) within the mosquito nervous system; (2) Est α 2 and Est β 2 genes encode esterase responsible for the metabolic detoxifcation.The results are as follows:1. Resistant level of Cx. pipiens pallens wild strain.A field survey was conducted in the summer of 2004 to evaluate resistance to propoxur, phoxim and DDVP on Cx. pipiens pallens from eleven sewage in Beijing. Based on insecticides bioassays, it was showed that wild strains had low levels of resistance to phoxim: 1. 15-to-3. 33-fold resistance was detected based on LC50. Cx. pipiens pallens had relativelyhigher resistance to propoxur and DDVP, ranging from 3. 13-to-7. 24-fold and 2. 84-to-9. 88-fold at the LC50.2. Bioassay and biochemical analysis of R-lab.Metabolic detoxification induced by esterase have a minor role in R-lab by est-a and estP activity detection. The insensitive AChE may be the main factor in related to the propoxur resistance in R-lab. (R-lab is a strain selected by propoxur in the lab for eight generations which had 12. 07-fold resistance to propoxur after selection.)3. Quantitative analysis of gene amplification in wild Cx. pipiens pallens.Real-time PCR was used to measure mRNA copies of Esta 2 and Est P 2 genes in eleven wild Cx. pipiens pallens strains. This accurate quantitative approach was performed to assess the specific expression profiles of two genes. Esta 2 and Est3 2 genes could not be detected in sensitive strain, indicated that the two genes had tiny expression. The difference between maximal and minimal copies was several millions-fold in Esta 2 genes, but it was only approximately 36-fold in Est 3 2 genes. Correlation-regression analysis showed that Esta 2 and EstP 2 were differentially transcribed in the wild Cx. pipiens pallens strains, the EstP 2 gene had greater transcription than Esta 2 in all wild strain, with an average ratio of 18.7:1. In addition, regression analysis revealed a significant correlation between Est a 2 copy number and the LC50 estimates for DDVP.4. Cloning and expression of the acetylcholinesterase gene from Cx. pipiens pallens.Transposition of the Acel open reading frames (ORF) of Cx. pipiens pallens into the baculovirus and transfection of Sf9 cells were conducted using Bac-To-Bac Baculovirus Expression System. The harvested cells weredisrupted to extract AChEl. And the supernatant was used for enzymatic assay directly. AChEl activity was assayed by spectrophotometric method in a 0. 1M phosphate buffer (pH7. 5) with the substrate acetylcholine iodide. The results showed that the AChEl of Cx. pipiens pallens had expressed within the Sf9 cells and also existed in the supernatant.5. Alternative splicing of Acel in Cx. pipiens pallensAn alternative splicing has been found in Acel gene from a sensitive individual mosquito. This is the first report of alternative splicing in an AChE gene from an mosquito. The splicing site is found in 44 51AA of mature protein after cleavage of the leader peptide. Two proteins which differed in size by eight amino acids were expressed by Bac-To-Bac Baculovirus Expression System. The enzyme activity data indicated that the splicing form of AChEl of Cx. pipiens pallens was less activity than the non-splicing form. Some research showed alternative splicing modulated all aspects of enzymatic activity, such as affinity, substrate specificity, catalytic properties, Vmax and activity regulation. A further research will be necessary to be done on the action of the alternative splicing in AChEl of Cx. pipiens pallens.6. An amino acid substitution in Acel of Cx. pipiens pallens.An amino acid substitution has been found in Acel gene from a wild individual collected from Labagoumen(LBGM). Cx. pipiens pallens Acel encodes a putative 702-amino-acid protein, which is 100% identical to its Cx. p. pipiens homologue. Amino acid sequence from the susceptible and an LBGM individual differed at only one position: the GCC(alanine) codon at position 328 (A201 in Torpedo californica), was replaced by a TCC(serine) codon (mutation A201S). This mutation had been found in Musca domestica and Aphis gossypii. From three-dimensional modeling, it could been found that this mutated residue lied within active 'gorge' of the enzyme, close...
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