The Toxicology Characteristic Of Nicotinig Acetylcholine Receptor In Cyrtorhinus Lividipennis, An Important Predatory Enemy Of Rice Planthooper

As the important predatory enemie of rice planthopper, the Cyrtorhinus lividipennis which mainly prey on the eggs and young nymphs of rice planthopper, play an important role in biological control of rice planthopper. However, the irrational use of insecticides has led to resistance of pest and strong lethality to C. lividipennis. The previous studies found that the neonicotinoid insecticides like imidacloprid have no selectivity between C. lividipennis and Nilaparvata lugens, and the sublethal dose of the neonicotinoid insecticides significantly impacted the egg hatchability, predacious number and fecundity of C. lividipennis. However, little research has been done into the nicotinic acetylcholine receptor (nAChR) which is the target site of neonicotinoid insecticides in C. lividipennis. Because of the importance of natural enemy protection, we have to research the toxicology characteristic of nAChR of C. lividipennis to explore the molecular mechanism of high sensitivity to the neonicotinoid insecticides which is helpful for developing the high efficiency and selective insecticide.In this study, we cloned the nAChRs subunits from C. lividipennis by the high-throughput techniques and modern molecular biology methods. Meanwhile we studied the toxicity characteristic of subunit a8 which contain the important difference in amino acids between C. lividipennis and N. lugens we can explore and find the reason of the high sensitivity of C. lividipennis to the neonicotinoid insecticides, which can explore the molecular mechanism of neonicotinoid insecticides selectivity.1. The evaluation of selectivity of novel neonicotinoid insecticides between C. lividipennis and N. lugensData of contact toxicity of 4 neonicotinoid insecticides against C. lividipennis and N. lugens tritonymphs are summarized in this study. The results showed that the acute contact toxicity of neonicotinoid insecticides to C. lividipennis were significantly higher than to N. lugens. The selectivity ratios for thiacloprid, imidacloprid and clothianidin were much less than 1 (0.034,0.058 and 0.131), which indicated that these three insecticides had no selectivity between BPH and the nature enemy, miridbug. By contrast, dinotefuran had the selectivity ratio of 0.510 and showed the comparable toxic to C. lividipennis and N. lugens.2. Transcriptome sequencing of C. lividipennis head and the clone of C. lividipennis nAChRTranscriptome sequencing, RT-PCR and RACE were used to clone the nine C. lividipennis nAChR subunit genes by using the methods in combination. We cloned the four full-length sequence and five almost full length sequence. By the sequences alignment and analysis among the other insects' nAChRs, we found six of them had the typical character of subunit a, and three of them had the typical character of subunit p. The analysis of typical character showed that the nine subunits had the typical character of nAChRs subunits, such as the conservative cysteine, loopA-C and loopD-F which are closely related to the target between genin and the nAChRs, four transmembrane fragments and glycosylation sites. And the nine subunits were denominated as Cl?1,Cla??Cla3, Cla4, Cl?7, Cl?8, Cl?1, Cl?2 and Cl?3.3. The key role of amino acid residues of C. lividipennis nAChR subunit a8 in the selectivity to the neonicotinoid insecticideThe single and double mutants in the key amino acid residues of C. lividipennis nAChR subunit a8 were fused with rat subunit ?2 to construct recombinants of Cl?8/?2 for respective expression in the Xenopus oocytes. Contrasting with the agonist conjugation reaction of wild type Cl?8/r?2, Cl?8/r?2 mutants and Nl?8/r?2, we researched the influence of toxicology characteristic of the key amino acid residues of Cla8 in the Loop B and Loop C. We found that the single mutation (N191F) in loop B reduced sensibility to imidacloprid of Cl?8/r?2, the single mutation (E240T) in loop C did not show influence. But the mutant Cl?8/r?2 (N191F/E240T) showed lower sensibility than the mutant Cl?8/r?2 (N191F). In conclusion the N191 play the key role in the sensibility to imidacloprid and the E240 may have indirect effect on it.