Identification And Analysis Of Nicotinic Acetylcholine Receptors In Locusta Migratoria And Pardosa Pseudonnulata

The cholinergic pathway is one of the most important excitatory neurotransmitters in the insect nervous system.Acetylcholine(ACh)is an important neurotransmitter,which is widely distributed in the whole nervous system of cholinergic neurons and plays a key role in the transmission of synaptic nerve signals in the insect.At present,the study found insect had two kinds of acetylcholine receptor:muscarinic type acetylcholine receptor(muscarinic ACh receptors,mAChRs)and nicotinic acetylcholine receptors(nicotinic ACh receptors,nAChRs).In particularly,nicotinic acetylcholine receptors are widely studied as targets for neonicotinoid insecticides.Neonicotinoids insecticides also require specific amino acids to bind to acetylcholine receptors.Depending on the molecular characteristics of each active compound and the types of subunits involved,these residues are different.There are many harmful effects of neonicotinoids insecticides,such as insect resistance,pesticide residues and damage to natural enemies.Although neonicotinoids are selectively toxic to insects and vertebrates,widespread use having side effects on economic insects(eg,honeybees)is gaining attention.In addition,studies have shown that the differences between insect and natural enemies of insecticide targets and detoxification enzymes are important reasons for the selection of pesticide toxicity.Pesticide bioassay found neonicotinoid insecticides on a variety of rice pests have stronger virulence,but virulence is relatively small to Pardosa pseudoannulata.Selective mechanism of pesticides between insects and Pardosa pseudoannulata is worthy of further study.This paper utilized Pardosa pseudoannulata transcriptome sequencing,got a lot of information of nicotinic acetylcholine receptor gene.And the receptor genes have carried on the appraisal and analysis,provided an important information for the pesticide selective research.In addition,we also carried out a study on pesticide targets in the agricultural model pest of Locusta migratoria,hoping to find out the selective mechanism of pesticides on natural enemies-pests.1.Alternative splicing in nicotinic acetylcholine receptor subunits from Locusta migratoria and its influence on acetylcholine potenciesDue to the great abundance within insect central nervous system(CNS),nicotinic acetylcholine receptors(nAChRs)play key roles in insect CNS,which makes it to be the targets of several classes of insecticides,such as neonicotinoids.Insect nAChRs are pentameric complexes consisting of five subunits,and a dozen subunits in one insect species can theoretically comprise diverse nAChRs.The alternative splicing in insect nAChR subunits may increase the diversity of insect nAChRs.In the oriental migratory locust(Locusta migratoria),a model insect species with agricultural importance,the alternative splicing was found in six subunits among nine and two subunits,such as missing conserved residues in Loop D from Loc?1,Loc?6 and Loc?9,a 34-residue insertion in Loc?8 cytoplasmic loop,and truncated transcripts for Loc?4,Loc?7 and Loc?9.Hybrid nAChRs were successfully constructed in Xenopus oocytes through co-expression with rat ?2 and one subunit from L.migratoria,which included Loc?1,Loc?2,Loc?3,Loc?4,Loc?5,Loc?8 and Loc?9.Influences of alternative splicing in Loc1,Loc?8 and Loc?9 on acetylcholine potency were tested on hybrid nAChRs.The alternative splicing in Loc?1 and Loc?9 could increase acetylcholine sensitivities on recombinant receptors,while the splicing in Loc8 showed significant influences on the current amplitudes of oocytes.The results revealed that the alternative splicing at or close to the ligand-binding sites,as well as at cytoplasmic regions away from the ligandbinding sites,in insect nAChR subunits would change the agonist potencies on the receptors,which consequently increased nAChR diversity in functional and pharmacological properties.2.Two distinctive p subunits are separately involved in two binding sites of imidacloprid with different affinities in Locusta migratoria manilensisDue to great diversity of nicotinic acetylcholine receptor(nAChR)subtypes in insects,one ? subunit may be contained in numerous nAChR subtypes.In the locust Locusta migratoria,a model insect species with agricultural importance,the third p subunits(Loc?3)was identified in this study,which reveals at least three p subunits in this insect species.Imidacloprid was found to bind nAChRs in L.migratoria central nervous system at two sites with different affinities,with Kd values of 0.16 and 10.31 nM.The specific antisera(LI-I,L2-I and L3-I)were raised against fusion proteins at the large cytoplasmic loop of Loc?1,Loc?2 and Loc?3 respectively.Specific immunodepletion of Loc?1 with antiserum L1-I resulted in the selective loss of the low affinity binding site for imidacloprid,whereas the immunodepletion of Loc?3 with L3-I caused the selective loss of the high affinity site.Dual immunodepletion with L1-I and L3-I could completely abolish imidacloprid binding.In contrast,the immunodepletion of Loe?2 had no significant effect on the specific[3H]imidacloprid binding.Taken together,these data indicated that Loc?1 and Loc?3 were respectively contained in the low-and high-affinity binding sites for imidacloprid in L.migratoria,which is different to the previous finding in Nilaparvata lugens that Nl?1 was in two binding sites for imidacloprid.The involvement of two ? subunits separately in two binding sites may decrease the risk of imidacloprid resistance due to putative point mutations in ? subunits in L.migratoria.3.Identification and analysis of the acetylcholine receptor of the Pardosa pseudoannulataIt is an important predatory natural enemy in the rice field,and the research on the target of nicotinic insecticides is helpful to the understanding of the selective mechanism of insecticides and the development of selective insecticides.In this chapter,a large number of genes information was obtained by high-throughput sequencing technology,and the target gene of the nAChRs was identified and analyzed.For nine transcriptome All annotation genes for nAChRs have carried on the appraisal analysis,eliminate error annotations and uncertain sequence,Total 63 unigenes were identified as acetylcholine receptors,and found the 5 new sequence.Four of them are a type subunits,and the other is ? type subunits.By Amino acid sequence alignment,c59396_g1 is named as pp?6_B,e42994_g1 and c47708_g1 are respectively named as pp?4 and pp?5,and c29060_g2 is temporarily named as pp?9.Furtherrmore,the sequence of the newly discovered beta subunit(c56827_g1)was named pp?4.The results of phylogenetic analysis showed that the nAChRs gene of the arAChnid was similar to sub-classification of insects.By means of Map and exons prediction of nAChRs subunits with a full open reading frame,a total of 4 genes can completely Map to a scaffold,and each gene has 7 to 8 exons.These results not only provide Pardosa pseudoannulata abundant genetic information,also can be helpful to the research of spider pesticides target.also for pesticides in natural enemy insects and understanding the mechanism of selective and selective pesticide research and development to provide theoretical basis.At the same time,it provides a theoretical basis for the understanding of the selective mechanism between natural enemies and insects and the development of high selective insecticides.