Resistance Monitoring And CDNA Cloning Of Cytochrome P450 In Panonychus Citri (McGregor) (Acari: Tetranychidae)

The citrus red mite, Panonychus citri McGregor (Acari:Tetranychidae), is an important pest mite in the world that devastates citrus trees and thus seriously affects yield and quality of citrus. P. citri has developed severe resistance to most commonly used acaricides, which has resulted in extensive attentions of researchers and producers of citrus.To clarify the resistance level and mechanism of P. citri to commonly used acaricides in the field, as well as the effects of citrus germplasm on their susceptibilities, the biossays of four acaricides against P. citri were conducted, and the synergism of three synergists (TPP, DEM, and PBO) on acaricides were also applied with the slide-dip method. The objective of this study is to develop an effective strategy for management of resistance. In addition, a fragment of cytochrome P450 was cloned by using the technique of transcriptase-polymerase chain reaction (RT-PCR), and one full-length cDNA of cytochrome P450 were cloned by using the technique of rapid amplification of cDNA ends (RACE). This will provide the first step toward the further study about the molecular analysis of resistance mechanism of cytochrome P450. The main results are summarized as follows:1. Monitoring on acaricides susceptibility of P. citri field populations1.1 Resistance monitoring on commonly used acaricides against P. citriThe results of the two years'monitoring showed that the citrus red mite expressed the highest increase of insensitivity (5.1-folds) to fenpropathrin, and the LC_50^S in 2008 and 2009 amounted to 4.93 mg·L^-1 and 25.17 mg·L^-1, respectively. There was also a decrease of susceptibility (2.2-folds) to pyridaben, the LC_50^S in 2008 and 2009 were 8.38 mg·L^-1 and 3.78 mg·L^-1, respectively. The susceptibility of the citrus red mite to abamectin was almost the same between two years, while the susceptibility to emamectin benzoate increased by 40%.1.2 Effects of different citruses germplasm on susceptibility of P. citri to acaricidesThe citrus red mite fed on three different citrus germplasm showed different susceptibility to fenpropathrin and pyridaben. For fenpropathrin, The Pomelo population expressed the highest susceptibility and followed by the Tangerine population, while the Trifoliate orange population expressed the lowest sensitivity, with corresponding LC_50^S of 2.34-folds to Pomelo population. The susceptibility of the three populations to pyridaben from the highest to lowest were the Pomelo population, the Trifoliate orange population and the Tangerine population. Thus, the Tangerine population revealed the least susceptibility with a relative resistance ratio of 3.9 folds higher than the Pomelo population. However, no significant difference was observed in the three populations to abamectin.2. Synergism of three synergists to acaricides against P. citriThe synergism experiments were performed to explore the mechanisms of the decrease of susceptibility in P. citri. The results indicated that TPP had the synergism effect (3.2,2.0, and 3.8-folds, respectively) to fenpropathrin, abamectin and emamectin benzoate, but had no synergism effect to pyridaben. Synergism of DEM to pyridaben and emamectin benzoate increased by 3.3 and 2.6-folds, respectively, while had no synergism effect to fenpropathrin and abamectin. PBO also had extremely synergism to fenpropathrin, pyridaben, emamectin benzoate, by 2.7,2.4 and 7.1-folds, respectively, and some synergism to abamectin, by-1.7 folds.3. cDNA cloning of cytochrome P450 in P. citriA 486bp and one 470 bp cDNA fragments of P450 gene were isolated from citrus red mite, P. citri. They were named as N1 and N2 and deposited in GenBank under the accession numbers FJ480403 and FJ480404, respectively. The complete cDNA of N2 were cloned by using the technique of rapid amplification of cDNA ends (RACE), and named as CYP4CF1. The full length of CYP4CF1 cDNA was 1880 bp, with a single open reading frame (ORF) of 1527 bp that encoded a protein of 508 amino acids, included a 5'untranslated region (UTR) of 87 nucleotides, a 3' untranslated region (UTR) of 286 nucleotides, start codon (ATG), stop codon (TAA) and the poly (A) tail with a consensus signal sequence for polyadenylation (AATAAA). The CYP signature motif, F××G×××C×G, in which the cysteine residue is crucial for heme binding, and E××R were fond both in the deduced amino acid sequence of N1 and N2. The sequence EVDTFMFEGHDTT, which is characteristic of the CYP4 family, is present in N2 (CYP4CF1).