Characterization of a β-Adrenergic-Like Octopamine Receptor in the Oriental Fruit Fly, Bactrocera dorsalis (Hendel)

Abstract

The biogenic amine octopamine plays a critical role in the regulation of many physiological processes in insects. Octopamine transmits its action through a set of specific G-protein coupled receptors (GPCRs), namely octopamine receptors. Here, we report on a β-adrenergic-like octopamine receptor gene (BdOctβR1) from the oriental fruit fly, Bactrocera dorsalis (Hendel), a destructive agricultural pest that occurs in North America and the Asia-Pacific region. As indicated by RT-qPCR, BdOctβR1 was highly expressed in the central nervous system (CNS) and Malpighian tubules (MT) in the adult flies, suggesting it may undertake important roles in neural signaling in the CNS as well as physiological functions in the MT of this fly. Furthermore, its ligand specificities were tested in a heterologous expression system where BdOctβR1 was expressed in HEK-293 cells. Based on cyclic AMP response assays, we found that BdOctβR1 could be activated by octopamine in a concentration-dependent manner, confirming that this receptor was functional, while tyramine and dopamine had much less potency than octopamine. Naphazoline possessed the highest agonistic activity among the tested agonists. In antagonistic assays, mianserin had the strongest activity and was followed by phentolamine and chlorpromazine. Furthermore, when the flies were kept under starvation, there was a corresponding increase in the transcript level of BdOctβR1, while high or low temperature stress could not induce significant expression changes. The above results suggest that BdOctβR1 may be involved in the regulation of feeding processes in Bactrocera dorsalis and may provide new potential insecticide leads targeting octopamine receptors.

Keywords: Bactrocera dorsalis; agonist; antagonist; biogenic amine; cyclic AMP; functional expression; octopamine receptor; stress.

Figure 1

Amino acid sequence alignment of BdOctβR1 and two β-adrenergic-like receptors from Drosophila melanogaster (DmOctβR1 and DmOctβR2). TM, transmembrane domains. The seven transmembrane domains are numbered as TM1–7. The shaded sequences highlight the identity level of amino acids between the receptors. Identical amino acids are highlighted in dark gray and conserved amino acids are on a light gray, as determined using the 50% majority rules. Dashes are gaps that were introduced for alignment. Potential phosphorylation sites for protein kinase C are labeled with ▲ and Potential N-glycosylation sites indicated by are ●. Amino acids which are labeled with ▼ are predicted to be involved in octopamine binding. The second phenylalanine after the FxxxWxP motif in TM6, which is a unique feature of aminergic receptors, is indicated by ■.

Figure 2

Phylogenetic tree of BdOctβR1 (red frame) and various biogenic amine receptors. Neighbor-joining tree was constructed in MEGA 5 using 1000 bootstrap tests re-sampling. The numbers at the nodes of the branches represent the level of bootstrap support for each branch. Ag: Anopheles gambiae; Am: Apis mellifera; Bd: Bactrocera dorsalis; Bm: Bombyx mori; Cs: Chilo suppressalis; Dm: Drosophila melanogaster; Tc: Tribolium castaneum. The receptor sequences followed by their GenBank accession numbers are listed in the order illustrated: AgOctαR1, EAA06361; AgTyrR1, EAA07468; AmOctαR1, NP_001011565; AmOctβR1, XP_397139; AmOctβR2, XP_396348; AmOctβR3, XP_003249152; AmOctβR4, CCO13925; AmTyrR1, NP_001011594; BdOctβR1, XP_011212557; BdOctβR2, JAC42311; BmOctαR1, BAF33393; BmOctβR2, BAJ06526; BmTyrR1, BAD11157; BmTyrR2, BAI52937; CsOctβR2, AEO89318; DmFMRF, NP_647758; DmOctαR1, NP_732541; DmOctβR1, Q9VCZ3; DmOctβR2, Q4LBB9; DmOctβR3, Q4LBB6; DmTyrR1, BAB71788; DmTyrR2, NP_650652; TcOctβR1, NP_001280514; TcOctβR2, NP_001280501; TcOctβR3, XP_008198078; TcTyrR1, NP_001164312. The FMRFamide receptor of D. melanogaster was used as an outgroup.

Figure 3

Relative expression levels of BdOctβR1 at different developmental stages. Different stages are displayed by E (egg), L (larva), P (pupa), and A (adult). Different numbers 1, 3, 5, 7 represent days 1, 3, 5, and 7 of the developmental stage, respectively. The data shown are mean ± standard error (S.E.) (n = 3), normalized relative to α-tubulin transcript levels.

Figure 4

Relative expression levels of BdOctβR1 in various tissues of adults. The data shown are mean ± S.E. (n = 3). Normalized relative to α-tubulin transcript levels. CNS: central nervous system; FB: fat body; MG: midgut; MT: Malpighian tubules; OV: ovary; TE: Testis.

Figure 5

Agonist concentration-response curves of BdOctβR1 transiently expressed in HEK-293 cells to five tested ligands (octopamine, naphazoline, tyramine, dopamine, and phentolamine). Each spot represents the mean relative luminescence ± S.E. from three independent tests and three biological replications per experiment. Each value was normalized to the cAMP luminescence obtained with octopamine at 1 × 10⁻⁵ M (=100%).

Figure 6

Antagonist concentration-response curves of BdOctβR1. Inhibition of the octopamine-dependent cAMP luminescence was examined on BdOctβR1-expressing HEK-293 cells with mianserin, phentolamine, and chlorpromazine. Each value was normalized to the cAMP luminescence obtained with the positive control, octopamine at 1.00 × 10⁻⁶ M (=100%). Mean values ± S.E. from three independent tests and three biological replications per experiment.

Figure 7

Expression profile of BdOctβR1 when B. dorsalis adults were kept under thermal (A) or starvation stress (B). The data are means ± S.E. of three independent experiments. Asterisk (*) above indicates a statistical difference determined by the independent samples t-test (* p < 0.05).