Study On Cloning Of The Sodium Channel Gene And Kdr Mutation In Tetranychus Cinnabarinus (Boisduval)

The carmine spider mite, Tetranychus cinnabarinus (Boisduval), one of the most important pests on cotton and many vegetables, is widely distributed in China. Because of high fecundity and short generation time, the mite can rapidly build a certain number of stocks in a short period, develop resistance easily and thus it is difficult to prevent and control this mite.Resistance of insects and mites is mainly caused by the increase in metabolic capacity of detoxification enzymes and insensitivity of target system. The voltage-gated sodium channel (VGSC) is the target of pyrethroids and DDT. Pyrethroids and DDT are known to exert their insecticidal effects by altering the function of voltage-sensitive sodium channels in nerve membranes, then to kill insects and mites. By changing structure of the sodium channel to reduce in the sensitivity of the insect nervous system, insects and mites develop resistance to pyrethroids and DDT.Based on laboratory maintained susceptible (SS) and fenpropathrin-selected strains (FeR) CMS, the resistance level of different geographical populations to fenpropathrin was monitored. Subsequently, the life table of the experiment population of T. cinnabarinus was studied. Besides, the cDNAs of sodium channel gene from the CSM SS and FeR strains,were cloned. Furthermore, the mRNA expression levels of the sodium channel gene from susceptible and fenpropathrin-resistant CSM were comparatively analyzed. The main results were listed as follows:1. The modified residual contact vial method bioassay results showed that laboratory fenpropathrin-selected strains of T. cinnabarinus has developed a 98.5-fold resistance to fenpropathrin. The resistance ratios of Chongqing. Zhejiang, Hubei and Yunnan populations amounted to 7.6,9.3,11.8 and 16.2-fold, respectively. Resistance of laboratory resistant strains in CSM was about 50%-85% suppressed by three mixed synergists (synergist:fenpropathrin=3:land 9:1).2. Experimental population ecology results showed that compared with the susceptible strain, reproductive disadvantage existed in laboratory resistant strains, meanwhile growth rate accelerated and the average generation time shortened. 3. The full-length cDNAs of the para sodium channel gene were cloned from T. cinnabarinus of both susceptible and resistant strains using the combined techniques of reverse transcriptase-PCR (RT-PCR) with rapid amplification of cDNA ends (RACE). The GenBank accession numbers were GU196305 and GU569881, respectively. The complete cDNAs both consisted of 5664 nucleotides encoding a protein of 1888 amino acids residues. Optional exon (SV1) and alternative exon (SV2) were all found in VGSC genes. A point mutation (F15381) was identified from the sodium channel gene of laboratory fenpropathrin-resistant CSM. The F15381 existed in laboratory resistant strains and different geographical populations by diverse mutation frequency, but the frequency in susceptible strain was 0, indicating that the mutation was the kdr mutation of T. cinnabarinus.4. The expression profiles of the sodium channel gene from fenpropathrin-susceptible and resistant CSM were analyzed. The results revealed that the mRNA abundance in the FeR and AbR strains was significantly lower than that in SS strain, suggesting no direct relationship between the reduction of sodium channels gene mRNA expression and kdr resistance.