Study On Knockdown Resistance(kdr) Mechanizan Of Pyrethroids In The Mosquito, Anohpeles Sinensis

Insectcides play a main role in control of major vectors of diseases. However, development of resistance in many insect species represents a significant and increasing thread to their continued effective use in the recent years. It is important and urgent to elucidate the resistance mechanism in order to slow down the spread and development of resistance of vector populations. Most studies on the resistance-related mechanism of Anopneles sinensis were mainly focused on the occurrence, development and biochemistry of the resistance. There are no research based on molecular biology of An.sinensis as it being very important vector species associated with malaria. In the past thirty years, the mainly use of DDT and pyrethroids insecticides for the impregnation of bet nets and indoor residual spraying in China caused the resistance increased rapidly. But there is no kdr resistance reported, as we all know that was associated with pyrethroids and DDT resistance. In this research, 16 resistance strains which had been collected in Jiangsu and Anhui provinces were studied, a biochemistry and three molecular methods have been adopted to find out the resistance-related mechanism and relationship between the para-knockdown frequencies and bioassay outcome. STR on seven locuses were analyzed. Here are the results.1 Bioassay analyses on larval mosquitoes and adult mosquitoes.1.1 Bioassay analyses on larval mosquitoes of An.sinensis.Larval bioassay results on the five strains from XZ, HY, CS, NJ and SZ all showed resistance to beta-cypermethrin. LC50 of the five strains were 0.894 mg/L, 1.264 mg/L, 1.670mg/L, 2.067 mg/L and 0.677 mg/L,respectively. R/S were 894, 1264, 1670, 2067 and 677-folds,respectively.1.2 Bioassay analyses on adult mosquitoes of An.sinensis.Adult mosquitoes bioassay on the 11 strains from Jiangsu and Anhui provinces with beta-cypermethrin, deltapermethrin and permethrin showed the average KT50 were 8.95-47.62 min. KT50 to beta-cypermethrin was 8.95-26.87min. KT50 to deltamethrin was 10.03-37.03 min. KT50 to permethrin was 16.13-47.62 min. R/S of KT50 of the three kinds of pyrethroids to 11 populations ranged from 2.439- to 12.221-folds. The populations of DT and WH showed the higher R/S, they were 8.64-12.221-folds in DT population and 7.322-9.524-folds in WH population. The lower R/S occurred in XN and YL populations.2. The action of knockdown resistance (kdr) in An.sinensis.2.1 Sequence of the voltage-dependent sodium channel in An.sinensis from S strain.A total 358bp of sodium channel gene had been amplified by RT-PCR, nest-PCR and sequenced from Ss strain. It has been demonstrated that 119 amino acid are encoded including four homologous domains and the hydrophilic linking peptides of varying length without 3` UTR. Although it is incomplete in this para-like cDNA sequence in An. sinensis, but it is long enough to find out the kdr mutation in An.sinensis because it contains all of the mutations loci of L1014 and M989 that have been reported to be related with kdr. It was found that the closest sequence homology to the Culex pipes pallens sodium channel with 98.3%, and with 96.7% sequence identity with Drosophila para, and with 94.2% sequence identity with M domestica para-like (Vssc1),and with 98.3% sequence identity with An.gambiae(para), and with 95.8% sequence identity with B germanica para-lik sodium channel.2.2 cDNA sequence analysis of the sodium channel of the beta-pyrethroid-resistance strain of An.sinensis.The cDNA sequence of IIS4-IIS6 in which kdr mutation located and the partial cDNA sequence have been analyzed from the resistance specimen. Resistance-related mutation of L1014F and L1014C had been found.2.3 Method of AS-PCR was adapted to detect the para-knockdown resistance mutations.Method of AS-PCR was adapted to detect the L1014F ,L1014C mutation in sodium channel . 263bp longer fragment amplified by two outer non allele-specific primers CD1 and CD2. The special primers of Cgd3, Cgd4, Cgd5 were used to detect the three alleles of TTG, TTT, TGT in three parallel tubes with the anti-sense primer CD2 to amplify a 169bp fragment. The sensitivity and specificity of this genotype method was showed to exceed 94%.2.4 Methods of PCR-RFLP were adapted to detect the para-knockdown resistance mutations.According to the mutation sites, after the first PCR based on the primers of C→T submission and digestion with HindⅢ, the presence of sequence with a (G/G) was inferred by the appearance of a single 196-bp fragment, a (T/T) was inferred by the appearance of a 154-bp and a 42-bp fragment and a (G/T) was inferred by the three fragments of 196-bp, 154-bp and 42-bp. In the same way, the second (T/G) involving (T/T),(G/G),(G/T) were inferred by the three fragments of 155-bp, 123-bp and 32-bp(Figure 3). Direct sequencing was used for validating the results of cPASA and PCR-FRLP. The sensitivity and specificity of this genotype method was showed to exceed 96%.2.5 Methods of TaqMan-MGB were adapted to detect the para-knockdown resistance mutations.According to the mutation sites of the para-knockdown resistance gene, three Probees were desired with a Report group of FAM and VIC in the 5',a Quencher group and MGB group in the 3', to detect the three sequences with the 1014 loci of TTG, TTT, TGT. Two non-specific primers were desired overstriding the para-kdr mutations sites. The high sensitivity and specificity of this genotype method leaded to the 99% in sensitivity and 100% in specificity.2.6 Assay of the kdr mutation frequencies and genotyping frequencies.3.6.1 Assay of the kdr mutation frequencies and genotyping frequencies on the larval mosquitoes.In the larval group, a total of five resistance populations from XZ, SZ, CS, NJ and HY cities rural and an Ss population, 290 specimens were tested with cPASA. Kdr frequencies ranged from 16.01% to 84.00%. The kdr alleles existed mainly in kdr-F/F and kdr-F/C genotype, the resistant homozygous form, and only a small portion (1.33%-3.57%) of the mosquitoes possessed kdr-C homozygous (RR-C) kdr alleles, 2.63%-4.76% of the kdr-C and kdr-F heterozygous (RS-C and RS-F) kdr genotype on the five populations. Kdr allelic frequency ranged from 73.68% to 84.00% on the kdr-F, and 16.01% to 23.68% on kdr-C. The genotype frequency of RR-F and RR-F/C ranged from 51.02% to 69.33%, 28.57% to 46.94 respectively.2.6.2 Assay of the kdr mutation frequencies and genotyping frequencies on the adult mosquitoes.A total of 689 An.sinensis specimens were genotyped in AS-PCR method. There did also co-exist the co-occurring knockdown resistance mutations in the eleven strains. There were mainly two genotype exist RR-F/F and RR-F/C. No Ss genotype was found. The para-knockdown resistance mutation frequencies of KDR-F and KDR-C ranged from 75.00%-88.54% and 9.36%-23.47%, respectively. No s-allele on 1014 loci was found in XN, DY and BN strains. Genotyping frequency ranged from 14.58% to 79.17%, which including 48.48%-79.17% in RR-F/F and 14.58%-46.15% in RR-F/C. The highest KDR-F and KDR-C frequencies were found in SZ and PZ population. The highest genotype frequency of RR-F/F and RR-F/C were found in SZ and YL populations.2.7 Hardy-Weinberg equilibrium test2.7.1 Hardy-Weinberg equilibrium test of larval mosquito group.Hardy-Weinberg equilibrium test was accepted for kdr alleles in each population using the T-paired test, the unbiased estimate of P-value of the five populations were 0.989(XZ), 0.930(HY), 0.991(NJ), 0.999(CS), 0.900(SZ), respectively. The results showed that there were no significant difference between the expected value and the observed number either in genic or genotypic differentiation. The five populations were all present genetic equilibrium.2.7.2 Hardy-Weinberg equilibrium test of adult mosquito group.Hardy-Weinberg equilibrium test was also accepted for kdr alleles in the adult mosquito population using the T-paired test, the unbiased estimate of P-value of the eleven populations were 0.335(YL),0.23(XN),0.853(SZ),0.535(DT),0.998(DY),0.706(CS),0.997(BN),0.403(PZ),0.989(FC),1(CZ),0.995(WH),respectively. The results showed that there were no significant difference between the expected value and the observed number either in genic or genotypic differentiation. The eleven populations were all present genetic equilibrium. The P-value of YL and XN populations showed the two populations were in rim of balance.3. Frequency of L1014F and L1014C mutations in response to pyrethroid .3.1 Frequency of L1014F and L1014C mutations in response to beta-cypermethrin.Spearman's rank correlation analysis based on the correlate analysis showed significant correlations between the LC50 and the kdr allele frequencies of kdr-F, kdr-C (p≤0.01). Regression analysis revealed a significant correlation between LC50 estimates and the frequency of kdr-(F+C) ( R2=0.981), kdr-C (R2=0.9548), kdr-F (R2=0.9513) and F/C genotype (R2=0.8399) . So we conclude that the mutant allele of L1014F and L1014C could be the marker of the resistance to beta-cypermethrin in An.sinensis.3.2 Frequency of L1014F and L1014C mutations in response to KT50 of beta-cypermethrin, deltamethrin and permethrin .Regression analysis revealed a significant correlation between KT50 estimates to beta-cypermethrin and the frequency of kdr-F ( R2=0.6195), RR-F/F (R2=0.0.6166), kdr (F+C) (R2=0.6202). Regression analysis revealed a correlation between KT50 estimates to deltamethrin and permethrin and the frequency of kdr-F ( R2=0.4662), kdr (F+C) (R2=0.4712), so we conclude that the mutant allele of L1014F and L1014C could be the marker of the resistance to beta-cypermethrin, deltamethrin and permethrin in An.sinensis in adult mosquitoes.4. Microsatellite loci of An.sinensis analyses4.1 Results of amplification and sequenced.The 15 paired-primers marked with FAM, HEX and TAMARA fluor groups based on the flanking sequence of microsatellite were used to amplify the microsatellite sequence. Only 7 paired primers amplified and sequenced with capillary tube method successfully. These results from sequencing would be used for analysis. 4.2 Polymorphism on the microsatellite location.Polymorphism on the seven microsatellite locations arranged from 0.21 to 0.75 and average was 0.55. The results showed the seven microsatellite locations being in high genetic information. The five higher polymorphism information content locations exist in AN1,AN2,AN5,AN11,AN15.4.3 Hardy-weinberg equilibrium tests .Hardy-weinberg equilibrium tests of the seven microsatellite locations showed that heterozygote excess on different locations were in equilibrium and parts of the locations out of equilibrium in heterozygote deficit test.4.4 Linkage disequilibrium analysisLinkage disequilibrium analysis was done between the different populations and microsatellite locations. The results showed non linkage relationship between different populations and different microsatellite locations based on the P-values. We further suggested that there was independent assortment ship in the gametes based on the results, No special selection ships were found from one genotype to another.4.5 Gene flow and kdr mutationsThe result showed a low transport ratio in the 10 populations. No direct correlation relationship exist between the frequencies of geneflow and kdr frequencies (R2=0.2251,0.3086,0.3059). The F statistic values also showed Fit, Fst and Fis were 34.29%,11.56% and 25.75%. The paired-comparing results showed the signifcant difference of gene loci existing in the populations form DT and BN(28.63%),the minimal difference existing in the populations from XN and SZ(1.6%)。The difference of kdr frequencies between the different populations weren't accordant to the change of increasing or decreasing of gene flow and Nm estimations. According to the pre-results we suggested that the kdr allele mostly coming from the transporting of inner exchanging between An.sinensis specimens in the same populations. It was concluded that selection played an important role on the population structure.