How Tyramine β-Hydroxylase Controls the Production of Octopamine, Modulating the Mobility of Beetles

Abstract

Biogenic amines perform many kinds of important physiological functions in the central nervous system (CNS) of insects, acting as neuromodulators, neurotransmitters, and neurohormones. The five most abundant types of biogenic amines in invertebrates are dopamine, histamine, serotonin, tyramine, and octopamine (OA). However, in beetles, an important group of model and pest insects, the role of tyramine β-hydroxylase (TβH) in the OA biosynthesis pathway and the regulation of behavior remains unknown so far. We therefore investigated the molecular characterization and spatiotemporal expression profiles of TβH in red flour beetles (Triboliun castaneum). Most importantly, we detected the production of OA and measured the crawling speed of beetles after dsTcTβH injection. We concluded that TcTβH controls the biosynthesis amount of OA in the CNS, and this in turn modulates the mobility of the beetles. Our new results provided basic information about the key genes in the OA biosynthesis pathway of the beetles, and expanded our knowledge on the physiological functions of OA in insects.

Keywords: Tribolium castaneum; mobility; octopamine; tyramine-β-hydroxylase.

Figure 1

Multiple sequence alignment of TcTβH from four insect species: Apis mellifera (AmTβH), Drosophila melanogaster (DmTβH), Bombyx mori (BmTβH) and Periplaneta americana (PaTβH), by DNAMAN. The three different color lines represent the conserved domains, the black line is the DOMON domain, the red line the Cu2-monooxygen domain and the yellow line the Cu2-monoox-C domain. The information on TβH in these species is listed in Table S2.

Figure 2

The phylogenetic tree of TcTβH (marked by black square) and various amino acids of the TβH neighbor-joining tree were 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. The information on TβH in these species is listed in Table S2.

Figure 3

The expression profiling of TcTβH. (A) Relative expression levels of TcTβH at different developmental stages. The first letters, E and O represent early and old. Different stages are displayed by capitalized letters: E (egg), L (larva), P (pupa), and A (adult). The expression of EA served as the calibrator; (B) Relative expression levels of TcTβH in various tissues of adults. CNS: central nervous system; FB: fat body; MG: midgut; MT: Malpighian tubules; OV: ovary; TE: testis. The data shown are the mean of the relative expression ± standard error (S.E.) (n = 3), normalized to RPS3 transcript levels. Different letters (a, ab, b) above the bar represent a significant difference after ANOVA (least significant difference, LSD, p < 0.05).

Figure 4

The prominent peak retention time of samples by HPLC with a C30 column. The black arrows are octopamine and tyramine.

Figure 5

The RNA interference (RNAi) of TcTβH. (A) The RNAi efficiency was tested by qRT-PCR (the result comes from software of qbase). Data were obtained by analyzing three independent groups of four individuals (two females; two males) per group; (B) The confirmation of RNAi efficiency by RT-PCR. Double asterisks represent a significant difference by an independent t test (n = 3, **, p < 0.01), the target gene of TcTβH was carried out with 32 cycles and the reference gene TcrpS was carried out with 27 cycles.

Figure 6

The level of octopamine (OA) and tyramine (TA) by HPLC. (A) The detection of the OA level by HPLC after RNAi; (B) The detection of the TA level by HPLC after RNAi. The asterisk represents a significant difference by an independent t test (n = 3, *, p < 0.05).

Figure 7

Effect of RNAi of TcTβH on the crawling speed (in millimeters per second) of T. castaneum. The red box plot shows the moving speed of double-stranded RNA (dsRNA)-treated adults, while the blue box plot shows the controls. Double asterisks represent a significant difference by an independent t test (n = 100, **, p < 0.01).