Pharmacological Properties Of Two Biogenic Amine Receptors In Plutella Xylostella

The metabolites of tyrosine are octopamine（OA）and tyramine（TA）in insects.OA and TA regulate intracellular Ca²⁺concentration and cyclic adenosine monophosphate（cAMP）concentration by binding and activating specific octopamine receptors（OARs）and tyramine receptors（TARs）,effect the physiological behavior of the insect.Therefore,OARs and TARs may become effective targets for pesticide insecticides.In this study,Plutella xylostella,an important pest of the cruciferous family,was used as a research object.The typeβ2 octopamine receptor（OA2B2）and the type 1 tyramine receptor（TAR1）genes were cloned by molecular biology and other methods.The pharmacological properties of OA2B2 and TAR1 were studied.1.Pharmacological characterization of the typeβ2 octopamine receptor in Plutella xylostellaThe putative open reading frame sequence of the Pxoa2b2 gene in P.xylostella was cloned.Orthologous sequence alignment,phylogenetic tree analysis,and protein sequence analysis all showed that the cloned receptor belongs to the OA2B2 protein family.PxOA2B2 was transiently expressed in HEK-293 cells.It was found that PxOA2B2 could be activated by both octopamine and tyramine,resulting in increased intracellular cyclic AMP（cAMP）levels.Further studies with series of PxOA2B2agonists and antagonists showed that all four tested agonists（e.g.,naphazoline,clonidine,2-phenylethylamine,and amitraz）could activate the PxOA2B2 receptor,and two of tested antagonists（e.g.,phentolamine and mianserin）had significant antagonistic effects.However,antagonist of yohimbine had no effects.Quantitative real-time polymerase chain reaction analysis showed that Pxoa2b2 gene was expressed in all developmental stages of P.xylostella and that the highest expression occurred in male adults.Further analysis with fourth-instar P.xylostella larvae showed that the Pxoa2b2gene was mainly expressed inMalpighian tubule,epidermal,and head tissues.2.Pharmacological properties of the type 1 tyramine receptor in the diamondback moth,Plutella xylostellaPxtar1,a candidate tyramine receptor gene in P.xylostella,was successfully cloned.Homologous sequence alignment and phylogenetic tree analysis showed that PxTAR1 belongs to the insect tyramine receptor 1（TAR1）family.PxTAR1,stably expressed in the HEK-293 cell line（TAR1/293T）,was used to test the biological activity of putative agonists and antagonists on the TAR1 receptor.Results showed that TA could activate the PxTAR1 receptor,increasing of intracellular Ca²⁺concentration([Ca²⁺]i)at 10μM.Tyramine could also reduce forskolin（10μM）-stimulated intracellular cAMP concentration（[cAMP]i）,with IC₅₀ of 446 nM.OA could weakly activate the PxTAR1([cAMP]i,IC₅₀=13.7μM),but did not increase intracellular calcium at a concentration of 10μM.Pharmacological analysis showed that DPMF（a metabolite of amitraz）could act as a PxTAR1 receptor agonist,linking to the both endogenous calcium pathway and cAMP pathway.L（-）-carvone also acted as a PxTAR1agonist,strongly inducing Ca²⁺response.Yohimbine and mianserin had significant antagonistic effects on PxTAR1.Yohimbine completely inhibited TA-induced[cAMP]i at a concentration of 10μM.To further explore the biological function of TAR1,we analyzed the expression profile of Pxtar1.The mRNA expression of tar1 in male adults was higher than in any other stage.In both in larvae and adults,tar1 had the highest expression in the head capsule.Furthermore,expression of tar1 in male reproductive organs（MRO）was higher than in female reproductive organs（FRO）.In this study,theβ2 octopamine receptor（PxOA2B2）and type 1 tyramine receptor（PxTAR1）genes were cloned and the agonists and antagonists were tested to screen for PxOA2B2 and PxTAR1.Our study provides a reference platform for the screening of chemical molecules with insecticidal activity.Furthermore,spatial-temporal expression differences of oa2b2 and tar1 gene can provide a starting basis for the further understanding of the physiological function of OA2B2 and TAR1 in P.xylostella.