Physiological, Biochemical And Gene Expression Changes In Plutella Xylostella (L.) Under Fipronil Stress

The diamondback moth (DBM), Plutella xylostella (L.) (Lepidoptera: Plutellidae), is an economically important pest of cruciferous crops throughout the world. This insect pest is difficult to control as it can rapidly develop its tolerance to many synthetic insecticides and bioinsecticides. When DBM is exposed to insecticides, it can lead to various behavioral and metabolic responses to insecticides. The responses may invovle multiple physiological changes, which cannot be assigned to only a single gene or protein.In recent years, studies on insceticides have developed at different levels of physiological, biochemical and molecular mechanisms, but information on the biochemical and molecular mechanism of P. xylostella in response to fipronil, a phenylpyrazole insecticide, which blocks the inhibitory GABA-gated chloride channel and is used to protect crops, is still limited.To better understand the DBM's response mechanisms, we observed the various levels of biochemical, proteomic and molecular response in P. xylostella that was exposed to fipronil stress. The proteins that may be involved in the response to fipronil were investigated using differential proteomics. The full-length cDNAs encoding thioredoxin peroxidase, C-type lectin, adenine nucleotide translocase and ATP synthase subunit d were all cloned from P.xylostella and their gene expressions were analyzed at the mRNA level. The major results are summarized in the following sections.1. The biochemical response of DBM to fipronil-induced stressThe short-term biochemical response of P.xylostella to fipronil was studied by examining the activities of detoxifying enzymes. We founded significantly higher activities of the detoxifying enzymes, mixed-function oxidase (MFO), esterase (EST) and glutathione-S-transferases (GSTs), in the DBM larvae that were treated with an LC50 dose of fipronil, compared to the larvae in fipronil-free controls. The activities of these detoxifying enzymes gradually increased over 24h, with MFO being more sensitive to external stimuli and showing higher induced activity than either EST or GSTs. 2. The proteomic response of DBM to fipronil-induced stressThe proteomic response of the DBM to fipronil was examined using two-dimensional electrophoresis (2-DE) and mass spectrometry at 8,16 and 24 h following fipronil treatment. Twenty proteins showed differential expression, including nine down-regulated spots, eleven up-regulated spots, of which five spots expressed only in the treatment group. Seventeen protein spots were successfully identified and include proteins that participate in the immune response and in other metabolic pathways, cytoskeletal proteins, and molecular chaperones. Differences in the expression of these proteins suggest that P. xylostella could create general changes in the cytoskeleton, metabolic level, and many other characteristics in response to the inesticide stress.3. Cloning and expression analysis of genes involved in the DBM response to fipronilThe full-length cDNA encoding thioredoxin peroxidase, C-type lectin, adenine nucleotide translocase and ATP synthase subunit d were cloned from P.xylostella by reverse transcriptase PCR (RT-PCR) and rapid amplification of cDNA ends (RACE). These four gene sequences and their encoding proteins were then analyzed, and a phylogenetic tree were contructed based on the amino acid sequences to reveal the relationship between DBM and other insects. In addition,3D protein structure models were developed using SWISS-MODEL.The mRNA levels of these four genes were measured using real-time PCR. These genes could be detected during the growth and development of DBM and were differentially expressed. However, the expression levels of these genes were also variable in the larvae treated with an LC50 value of fipronil.