| The diamondback moth (DBM), Plutella xylostella (L.) is one of important cosmopolitan pests of cruciferous crops all around the world. Now, it has received much attention in the past mainly due to its seemingly unlimited capacity to become resistance to almost all kinds of insecticides. Chlorantraniliprole, as an alternative to traditional insecticides, is a reduced-risk insecticide being introduced for global control of DBM and has been shown to be a highly efficacious insecticide in pest control in recent years. However, resistance to chlorantraniliprole has been observed in China and Philoppine Island since 2010.In order to study the resistance of DBM to chlorantraniliprole, a series experiments were carried out in this paper, including resistance monitoring, biochemical mechanism study and influence study of chlorantraniliprole on DBM by adult feeding. The major research results are as following:1. Resistance monitoring:In order to determine if resistance to chlorantraniliprole had evolved in the field,4 field populations of DBM, that were collected since 2009 from Guangdong Huizhou, Guangzhou Shijing, Guangzhou Dongsheng and Yuebei Lianzhou, were tested with laboratory bioassays. The results showed that although the planting patterns can affect the resistance of DBM to some extent, but all 4 field populations of DBM developed resistance to chlorantraniliprole at the same time and appered suddenly in 2011, the highest resistance ratio (RR) was 1749.96 (Guangdong Huizhou). The early stage of resistance was instability and was significantly decreased after a period of time to stop using the insecticide. Seasonal variation was investigated in different fields and autumn resistance is higher than spring resistance in generally, especially in Yuebei Lianzhou. The development of resistance has gradually expanded from a point area to a larger area. And now, it has a stable high level of resistance (RR>100) to chlorantraniliprole in field populations of DBM in Guangdong province.2. Detoxification mechanism study of chlorantraniliprole resistance:2.1 Biochemical mechanism study:In this work, a resistance strain (RR=82.18) of DBM was selected firstly. And then the effect of metabolic synergists on toxicity, the detoxification enzymes assays and the effect of short-term exposure to several insecticides on biochemical response were carried out. The results showed that selected resistant strain and susceptible strain could be synergized by known metabolic inhibitors such as piperonyl butoxide (PBO), triphenyl phosphate (TPP) and diethyl-maleate (DEM) at different levels (1.68-5.50-fold and 2.20-2.89 fold, respectively), and DEM showed the maximum synergism in both strains. In enzymes assays, a high level of glutathione-S-transferase (GSTs) was observed in the resistant strain,3.34-fold to susceptible strain. The findings of short-term exposure of chlorantraniliprole on biochemical response showed that all the three detoxification enzymes in susceptible DBM larvae could be induced significantly in short treatment time, especially P450s. But in resistant DBM larvae, all the three detoxification enzymes needed more time to induce, GSTs was the best. However, all the results above showed that there is a strong correlation between the enzymes activity and resistance.2.2. Molecular cloning and analysis of detoxifying enzyme genes:Based on our previous studies, the full-length cDNA sequence encoding the PxCYP, PxCCE and PxGST (1960 bp,2422bp and 833 bp), were cloned by RT-PCR and RACE tecnology, respectively. Sequences analysis showed that the full length cDNA sequence of PxCYP was 1960bp long with a 1503 bp ORF, which encoded a protein of 500 amino acid residues, preidted mass and theoretical PI value of this protein was 56.62 kDa and 8.35, respectively; the full length cDNA sequence of PxCCE was 1773bp with a 1629bp ORF, which encoded a protein of 542 amino acids, and this cDNA predicted mass of 61.561 kDa and a theoretical isoelectric point value of 5.78; the full length cDNA sequence of PxGST was 833bp with a 651bp ORF, which encoded a protein of 216 amino acid residues, preidted mass and theoretical PI value of this protein was 24.08 kDa and 8.5. Among them, PxCYP and PxCCE might be reported to be related with chlorantraniliprole resistant at the first time in DBM. Then the PxCYP was named as CYP321E1 by the P450 Nomenclature Committee and has been deposited to GenBank under accession no. KC626090. The pxCCE was name as PxCCE016b here temporary and also has been deposited to GenBank under accession no. KM008609. The PxGST gene shared 100% identity similarity with PxGSTel which was reported several years ago (accession no. AAC35245). In this paper, the expression profiles of 3 detoxifying enzyme genes of DBM was examined by using RT-qPCR. β-actin was used as an internal reference gene. RT-qPCR analyses indicated that all 3 detoxifying enzyme genes here were expressed at all developmental stages but were highest in 4th instars larvae. Furthermore, the relatively high expression was observed in the midgut of the 4th instars larvae, followed by fat bodies and epidermis. The expression patterns of these genes are consistent with their detoxifying functions.2.3. CYP321E1 gene RNAi functional study:The insecticides exposure experiment was carried out, and the result showed that the expression of CZP321E1 in DBM was differentially affected by alphamethrin, abamectin and chlorantraniliprole at sublethal dose, the exposure to chlorantraniliprole resulted in the largest transcript level of CYP321EI gene (P<0.05). The findings suggested potential involvement of CYP321E1 in chlorantraniliprole resistance of DBM. To further assess the functional link of CYP321E1 to chlorantraniliprole resistance, RNAi by dsRNA injecting was used. Results revealed that injection delivery of dsRNA can greatly reduce gene expression after 24h. As a consequence of RNAi, a significant increment in mortality of larvae injected CYP321EI dsRNA was observed. These results support our notion that CYP321E1 gene may play some role in chlorantraniliprole detoxification in the DBM and is partly responsible for its resistance.3. Influence of chlorantraniliprole on DBM adult:Here, using of suplement nutrition characteristic of DBM adult,10% honey solution was prepared and compounded with chlorantraniliprole, and then investigated influence on DBM adult and their offspring. The results showed that high concentration of chlorantraniliprole (final concentration was 250 mg L"’) could directly kill adult life, reduced concentration of chlorantraniliprole (final concentration was 5-50mg L-1) in poisonous diets could notable lower adult mating and fecundity, and could inhibite progeny population growth in a short term. Index of interference of population control (IIPC) was decreased with increasing concentration of chlorantraniliprole. |
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