| Octopamine(OA),the vertebrate counterpart of noradrenaline,is an important biogenic amine in invertebrates.OA modulates many physiological and behavioral processes including growth,development,feeding and metabolism in insects,which make insects to respond correctly to external stimuli.Because OA is the biogenic amines whose physiological is presumably restricted to invertebrates,pharmacologists have focused their attention on the octopamine receptors(OARs),which are believed to represent promising targets for new insecticides.In this study.We have cloned six OA receptor genes from the brown planthopper,Nilaparvata lugens,which one of the main pests that cause serious damage to rice production.We have investigated expression patterns of the OA receptor genes in different developmental stages and in various tissues for N.lugens by qPCR.Moreover,the pharmacological properties of N10A2B2 receptor,including agonists and antagonists,were researched by in vitro eukaryotic expression.The effects of N10A2B2 receptor on oviposition behavior of N.lugens were studied by pharmacology and RNAi technique.1.Gene clone and sequence analyses of octopamine receptors from N.lugensWe identified and cloned six OA receptor genes from N.lugens by the TBLASTN comparison between the genome data of Drosophila melanogaster and the genome data or the transcriptome data of N.lugens.Those OA receptor genes were named NlOA1,NlOA2B1,NlOA2B2,N10A2B3,NlOA3(N10A3-S and N10A3-L),respectively.The receptor gene(NlOA3)encodes two molecularly distinct transcripts,N10A3-S and NlOA3-L.N10A3-L differs from N10A3-S on account of the presence of an additional 8 amino acids within the third intracellular loop.The results of transmembrane prediction showed that these OA receptors display typical properties of GPCRs that contains seven transmembrane domains(TM1 to TM7),three extra-and intracellular loop,the C-terminal region within the cytosol and the N-terminus region located the outside of the membrane.Bioinformatics analysis showed that those conserved domains were predicted and confirmed in other insect octopamine receptors,such as phosphorylation sites,glycosylation sites,palmitoylation sites,and ligand binding sites are also present in OA receptors of N.lugens.Homology analysis showed that the homology between the OA receptor genes of N.lugens and other insect homologous genes was very high,the homology was 50%-70%.Gene structure analysis showed that the CDS region of the NlOA3 gene had the largest exon/intron structure except the NlOAl gene and the NlOA2B3 gene because the genomic data was lost and the full length was not cloned,respectively.2.Expression pattern of OA receptor genes in N.lugensWe had studies the expression patterns of six OA receptor genes which were cloned in N.lugens by qPCR method.we found all OA receptors could be detected in the whole developmental stage of N.lugens from the experimental results.The N10A1,N10A2B1,N10A2B3,N10A3-L and N10A3-S were highly expressed in eggs,first and second instar larvaes,and then the expression levels were gradually decreased with the development of the age.In the adult stage,the expression level of the males was higher than the females.However,the N10A2B2 was lower expressed in eggs and young nymphs than adults,the expression level was the highest in male.In various tissues,we found all OA receptors were highly expressed in brain of N.lugens,and the expression levels were different in gut,fat body,malpighian tubule,haemolymph.All OA receptors also were expressed in female reproductive organs and male reproductive organs except NlOA2B2,the expression level of N10A2B2 in leg was just lower than brain for N.lugens.In addition,we also studied the expression pattern of N10A2B2 gene in the ovary,oviduct,copulatory pouch and spermatheca of N.lugens,the N10A2B2 was lowly expressed in ovary,but there was no difference between oviduct,copulatory pouch and spermatheca.3.Pharmacological characterization and physiological functions of a β-like-adrenergic OARs in N.lugens.The HEK293 cell line stably expressing the N10A2B2 gene was used to determine the intracellular cAMP under the stimulation of octopamine(OA),tyramine(TA),dopamine(DA)and serotonin(5-HT)by ELISA method.OA and TA significantly induced the increase of cAMP production in N10A2B2-expressing cells,but the biogenic amines DA and 5-HT did not induce detectable responses at the same concentration.The EC50 values for OA and TA were 1.14 × 10-7 M and 1.36 × 10-6 M,respectively.It can be seen that OA was eight-fold of magnitude more potent than TA.The NlOA2B2-expressing cell line was tested for Ca2+ signals after stimulation with OA.None of the NlOA2B2-expressing cell line showed a Ca2+ response.Therefore,we determined that NlOA2B2 was a typical OA receptor mediating its action via increases[cAMP]i.In addition,we also determined agonists naphazoline and clonidine with EC50 values of 1.22 x 10-7 M and 6.03 x 10-7 M,respectively.For assays with antagonists,the elevation of[cAMP];caused by OA was significantly reduced by epinastine,mianserin,phentolamine,methiothepin,butaclamol and methysergide.However,prazosin,chlorpromazine and ketanserin did not significantly antagonize the action of octopamine.We performed pharmacological injection experiments to determine whether NlOA2B2 is involved in ovulation or egg-laying behavior of N.lugens.The results showed that injection of two antagonists,epinastine and phentolamine,have significantly reduced the number of eggs laid and spawning marks.However,the survival rate was not affected by these two antagonists.Knocked down NlOA2B2 gene using RNAi significantly decreased the expression of NlOA2B2 gene in whole body and oviduct,respectively.The NlOA2B2 immunoreactivity in oviduct was significantly reduced by RNAi effects.Whereas the dsgfp-injected N.lugens showed a high level of eggs laid and spawning marks,N.lugens injected dsNloa2b2 did not show obvious difference in survival rate,but eggs laid and spawning marks wer significantly declined.When the ovaries of N.lugens after ovulation for 5 days was dissected,we found that the ovaries became larger and a large number of eggs remained in the ovary when the NlOA2B2 gene was silenced.It was proved that the NlOA2B2 gene plays an important role in the ovulation behavior of the female N.lugens. |
