The Identification Of Molecular Marker For Kdr Resistance Against Fenpropathrin From Tetranychus Cinnabarinus (Boisduval)

The carmine spider mite (CSM), Tetranychus cinnabarinus (Boisduval), is one of the most important pests in the world agricultural industry. Pyrethroid insecticide has been used to control insects and mites worldwide. However, the intensive use of pyrethroid insecticide resulted in the development of resistance, which had been mainly been induced by a variety of point mutations responsible for voltage-gated sodium channel (VGSC) insensitivity and has became the biggest obstacle to sustain the use of pyrethroid insecticide.Thus, it is necessary for pesticide use and insect resistance management to achieve accurate resistance monitoring.Based on the susceptible strain (SS), fenpropathrin-resisrant strain (FeR) and seven field populations of CSM, we cloned the full legth cDNA of VGSC in T. cinnabarinus and identified the molecular marker of Mr. Besides, the kdr mutation frequencies in seven field populations of CSM were detected.1. Based on the VGSC fragment of T. cinnabarinus, GU196305, we cloned cDNA full length of the para-homologous sodium channel gene from T. cinnabarinus by rapid amplification of cDNA ends (RACE). The complete open reading frame of VGSC of T. cinnabarinus contains6579nucleotides, encodes2193amino acids, contains the domain ⅠS1-ⅣS6, C-terminal and N-terminal (GenBank accession number:JX290514). There is unique mutation, F1538I, was found after comparing the amino acid sequences of FeR strain with SS strain.2. The point mutation, F1538I, was also identified from genome DNA sequences of VGSC in FeR strain after amplification of genome DNA from SS and FeR strains. The result indicated that the F1538kdr mutation in resistant T. cinnabarinus underwent DNA mutation events rather than RNA editing.3. Single nucleotide polymorphisms (SNP) detection of F1538I mutation from SS and FeR strains revealed that this mutation appeared in all FeR strain individuals. Nevertheless, in the detection of the SS strain individuals undergoing the treatment with LC90of fenpropathrin (about10000mg/L), two mutants were found from30survivals and no mutant individual was found from30dead which were randomly selected.These results demonstrated that the higher mutation frequency in the FeR strain resulted from fenpropathrin selection, and the mutant exist in the SS strain with very low frequency (theoretically should be0.67%, i.e.2/30×10%=0.67%).The F1538I mutation in the kdr gene could be used as a molecular marker for fenpropathrin resistance monitoring.4. The F1538I kdr mutation frequency and the resistantant level against fenpropathrin were both detected from seven field populations. The resistant levels ranged from3.8to16.2, and the mutation frequencies ranged from13.6%to45%accordingly, i.e. the higher the resistant level, the larger the mutation frequency in field populations. Significant positive correlation between sodium channel gene F1538I mutation frequency and bioassay-based fenpropathrin resistance phonotype demonstrates that the F1538I mutation could be used as a molecular marker for fenpropathrin resistance monitoring in natural T. cinnabarinus populations.