Abstract: 【Objective】Octopaminergic signaling system plays a crucial role in the reregulation of behavioral and physiological processes in insects. The red flour beetle (Tribolium castaneum) is a model insect which has been widely used in the study of insect growth, development and physiology. The objective of this study is to utilize T. castaneum as the research insect and elucidate the vital functions of octopamine receptors involved in the physiology and behavior.【Method】Based on the sequence information in GenBank (XP_008198078), the cDNA of an octopamine receptor (TcOctβR3) was cloned by RT-PCR. The open reading frame (ORF), deduced amino acid sequence and the membrane structure domains were predicted by using online service, and phylogenetic tree associated with OctβR3 from other insects was constructed by using neighbor-joining method to clarity its phylogenetic relationship. In addition, the RNA was extracted from different developmental stages (egg, larva, pupa, and adult), different tissues (central nervous system, fat body, midgut, hindgut, malpighian tubule, testis and ovary), as well as the larvae under stress of starvation, respectively. Ribosomal protein S3 gene (TcRPS3) was used as an internal reference. qRT-PCR (real-time quantitative PCR) was employed to determine its expression patterns in different developmental stages, different tissues as well as the induced expression profiles under the stress of starvation. TcOctβR3 was transiently expressed in human embryonic renal cell (HEK293) by using heterologous expression system, and cAMP measuring method was performed to determine the activity of its ability to combine with ligands. Finally, the double stranded RNA of TcOctβR3 was synthesized in vitro, and the physiological functions were verified by track ball behavior analysis as well as RNA interference (RNAi) technology. 【Result】 A complete sequence of TcOctβR3 was cloned with open reading frame (ORF) of 1 305 bp, encoding 434 amino acids, with a signature of 7 transmembrane domains which belongs to the superfamily of G-protein coupled receptors. This gene exhibited a close relationship with the AtOctβR3 from Aethina tumida based on neighbor-joining phylogenetic tree. qRT-PCR results indicated that TcOctβR3 was expressed at all tested developmental stages, particularly high at the early larval stage. However, the expression of TcOctβR3 was not significantly different from other developmental stages. Besides, the expression of TcOctβR3 was remarkably higher in the central nervous system (CNS) than that in other tissues. In the process of starvation for 24 h, the expression of TcOctβR3 significantly fluctuated and then returned to the normal level. The lowest expression of TcOctβR3 was 0.47 fold at 6 h and the highest expression was 1.80 folds at 16 h compared with control, respectively. Based on cyclic AMP response assay, it was found that TcOctβR3 could be activated by octopamine (OA) in a dose-dependent manner with a median effective concentration (EC₅₀) of 8.68×10^-7 mol·L^-1 after heterogenous expression in HEK293 cells. Naphazoline (NA) strongly activated this receptor with EC₅₀ of 8.56×10^-8 mol·L^-1 compared with other ligands. Generally, the rank order for the potency of 4 tested ligands was naphazoline＞tyramine (TA)＞octopamine＞dopamine (DA). RNAi results indicated that the transcript level of TcOctβR3 was significantly knocked down 61.5% by injected the targeted dsRNA. However, no effect on the walking speed and fecundity of T. castaneum adults was observed. 【Conclusion】TcOctβR3 plays an important role in the central nervous system, which modulates the response of the beetle larvae upon starvation. The molecular characterizations were determined based on a cAMP assay, which will provide a solid basis for the future screening of its high efficiency agonists and inhibitors.

Key words: Tribolium castaneum, octopamine receptor, expression profile, biological activity, physiological function