| Neonicotinoids, such as imidacloprid, are selective agonists of insect nicotinic acetylcholine receptors (nAChRs) and display selective toxicity against insects versus insect natural enemy spiders, such as Pardosa pseudoannulata. As the important neurotransmitter receptor and target of insecticides, insect nAChRs are extensively studied. In contrast, little is known on spider nAChRs with agricultural importance. The selectivity of neonicotinoids on insects over spiders might be from the differences in nAChRs, the targets of neonicotinoids, similar to the selectivity on insects versus vertebrates. So, it is interesting to understand well the selectivity mechanisms between insects and spiders.Pardosa pseudoannulata, the target spider in this study, is the predominant spider species in paddy fields and the important natural enemy of some rice insect pests. In this study, several nAChRs subunits are identified in Pardosa pseudoannulata. Comparisons between insects and P. pseudoannulata nAChRs are performed to understand the pharmacological difference between insects and spiders, especially on nAChRsβsubunits. The subunit composition for P. pseudoannulata nAChRs is also under investigation to analyze the selectivity mechanisms of neonicotinoids on insect versus spiders.1. Molecular cloning of nicotinic acetylcholine receptor from Pardosa pseudoannulata.Using RT-PCR with general primers designed on insect nAChRs subunits and RACE techniques, we identified seven nAChRs subunits in P. pseudoannulata. These subunits show high identities to insect nAChRs subunits, in which five subunits display the characteristics of nAChRs a subunits and two withβsubunit properties. These proteins include all typical properties of nAChRs subunits, such as the extracellular N-terminal region with conserved residues within loops A, B, C, D, E and F which are involved in ligand binding, the cys-loop consisting of two disulphide bond-forming cysteines separated by 13 amino acid residues, four well-conserved transmembrane regions (TM1-4) and a variable intracellular region between TM3 and TM4. Based on the identities to insect nAChRs subunits, these proteins are denominated as Ppα1, Ppα, Ppα3, Ppα6, Ppα7, Ppβ1 and Ppβ2.2. Difference in nAChRs (31 subunit confers imidacloprid selectivity between insects and P. pseudoannulata.In the pond wolf spider, P. pseudoannulata, aβ1 subunit Ppβ1 was cloned, with high identity to insectβ1 subunits. Despite this, however, several amino acid differences were found within loops D, E and F between insectβ1 subunits and Ppβ1. In consideration of important roles of insectβsubunit-specific loops on neonicotinoid selectivity, these amino acid differences might be also important in neonicotinoid selectivity against insects over spiders. Hence, the effects of Ppβ1 specific loops and specific residues within these loops on agonist potency were examined using the subunit chimera approach.First, loops D, E and F from Myzus persicae Mpβ1 were introduced into ratβ2 to construct chimeraβ2Mpβ1, which then was co-expressed with insect Nlal. Then loops D, E or F ofβ2Mpβ1 were replaced by that of Ppβ1, singly or together. The results showed these replacements conferred the decreased potency to imidacloprid, but not to ACh. In study of single amino acid changes, R81Q, N137G and F190W, within loops D, E and F respectively, contributed mainly to these sensitivity changes. This study contributes to our understanding of the molecular mechanism underlying selectivity of neonicotinoids between insects and spiders, and suggests the possible target regions on which to focus in the design of new selective insecticides against insects versus spiders.3. Co-assembly of two nicotinic acetylcholine receptorαsubunits and twoβsubunits in P. pseudoannulata.In vertebrates, nAChRs receptors can form "pair type", "riplet type", "quadruplet type" and homomeric pentamer in heterologous expression system. However, little is known on the subunit composition for invertebrate nAChRs, even for insect nAChRs. In the present study, evidence has been obtained for the co-assembly of Ppα1 and Ppα2 subunits with vertebrateβ2 subunit into 'triplet' nAChRs (Ppα1/Ppα2/rβ2). Co-immunoprecipitation studies with native nAChRs preparations, using subunit-selective antisera, have demonstrated the co-assembly of Ppα1, Ppα2, Ppβ1 and Ppβ2 subunits into one receptor complex. A single epibatidine binding site is found in P. pseudoannulata native nAChRs and these found subunits are all involved in forming this unique binding site, which also provide evidence that these four subunits are co-assembled into one receptor complex constituting the single binding site for epibatidine in P. pseudoannulata. This is the first identification for the co-assembly of four subunits (twoαand twoβsubunits) into one nAChR complex in invertebrates. |
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