The Toxicity Mechanisms Of Fipronil At Sublethal Concentration To The Pupation Of Leptinotarsa Decemlineata

Leptinotarsa decemlineata is the most destructive pest of potato, and is a major quarantine pest in China. The outbreak of the beetles results in large quantity of yield loss. Nowadays, the control of the pest is mainly relied on traditional chemical insecticides, which has caused serious environmental pollution, and insecticide resistance. Recently, we found that fipronil, butene-fipronil and endosulfan could affect of the pupation of Leptinotarsa decemlineata. In this study, we analyzed the effects of the three noncompetitive y-aminobutyric acid (GABA) inhibitors on the pupation and emergence; we obtained the L. decemlineata transcriptome data; we cloned some full-length cDNAs encoding proteins that involved in nutrition balance and hormone-triggered signaling pathways. We also identified potential functions of proline metabolism-relative genes by RNAi approach and constructed the nutrition balance and hormone-triggered signaling pathways. Finally, we explored the toxicity mechanisms of fipronil at sublethal concentration on larval pupation. The main results were shown as fellows.1. The effects of three noncompetitive GABA inhibitors on pupation and emergence in L. decemlineata and Spodoptera exiguaThe contact and stomach toxicities of fipronil, butene-fipronil and endosulfan were evaluated in L. decemlineata 4th-instar larvae and adults, respectively. The 4th-instar larvae are more susceptible to the three insecticides than the adults. Further bioassay revealed that the pupation and emergence of L. decemlineata 4th-instar larvae were negatively affected by the three noncompetitive GABA inhibitors. Similarly, these inhibitors could also influence the pupation and emergence in S. exigua larvae. Thus, these noncompetitive GABA inhibitors were indicated to disturb insect endocrine systems. In order to elucidate the toxicity mechanisms, three developmental checkpoints for pupation were determined in L. decemlineata 4th-instar larvae:critical weight was 82 mg, the time of reaching critical weight was 24 h after ecdysis, and the timing of the onset of wandering was 80 h after ecdysis.2. The analysis of L. deceamlineata transcripomeSequenced by Illumina platform,39641172 clean short reads were obtained. After assembling and clustering using bioinformatic software, these reads were clustered into 63399 distinct sequences (unigenes). Homology searches revealed that 23633 genes share significant similarity (77% average similarity) with those of nr at amino acid level, of which 33% genes are strong homologous. Beside of those genes,39766 novel genes were identified.23663 nr hits were further categorized into functional groups, classified into 24 COG categories and assigned to 208 KEGG pathways. According to those annotations,423 genes that involved in xenobiotics metabolism, proline metabolism, chemical sense, MH/JH biosynthetic pathways, and PTTH/IIS/TOR signal pathways were retrieved. Moreover, major cyp members and RyR gene were identified. These results demonstrated the correctness of the L. decemlineata transcriptome data.3. Molecular cloning and characterization of genes involved in nutrient balance and hormone-triggered signaling pathways from L. decemlineataBased on L. decemlineata transcriptome data, twenty-four full-length cDNAs were cloned. Among them were 3 glucose transporter genes,1 trehalose transporter gene,5 genes involved in proline metabolism,11 genes involved in insulin-like signaling pathway, and 4 genes involved in ecdysone biosynthesis. The temporal and spatial expression patterns of several representatives were analyzed. The functions of the five proline metabolish-relative genes were characterized by RNA interference (RNAi). Three days of continuous exposure to dsLdProDH decreased 80% of the target gene expression at the mRNA level and increased proline content in hemolymph by nearly 100%, when compared with that from the blank control. The 3-6 days of continuous exposure of adults to bacterially expressed dsLdP5CDh, dsLdALT-2, dsLdPSCR and dsLdP5CS significantly decreased their corresponding mRNA levels (51%-90%), significantly increased the mortality (53.3%-73.3%), and greatly reduced flying speed. Based on the above results, the nutrition balance and hormone-triggered signaling pathways were constructed. Our results provided the fundamental materials of studying the toxicity mechanisms of fipronil to the metamorphosis.4. The toxicity mechanisms of fipronil at sublethal concentration on larval pupation of L. decemlineataFipronil application decreased foliage consumption by 4th-instar larvae, repressed the expression levels of genes encoding proteases and amino acid transporters, decreased the mRNA levels of glucose transporter and trehalose transporter genes, increased the transcript leves of proline metablish-related genes in early stage of 4th-instar larvae. It also delayed the time to reach the critical weight by 8-12 hours. In contrast, the insulin-like signaling pathway was not delayed in larvae that treated with fipronil within 24 hour after ecdysis. In normal feeding L. decemlineata 4th-instar larvae, two expression peaks of either LdAS-B or LdAS-C encoding allatostatins were found at 24 h and 80 h after ecdysis, respectively. At the two timepoints, the two expression peaks were presumed to inactivate corpora allata. After treated by fipronil, however, the first peak was shift to an earlier time, and the second peak was disappeared. The disinhibition of corpora allata by fipronil caused high expression of genes involved in JH biosynthesis such as LdJHAMT even at 80 hours after ecdysis. Moreover, fipronil activate the expression of Halloween genes in the early 4th-instar stage, whereas it depressed their transcription in the late 4th-instar stage. All these transcriptional changes were believed to disturb nutrition balance and hormone-triggered signaling pathways, and eventually caused the failure of pupation and emergence.