Adipokinetic Hormones and Their Receptor Regulate the Locomotor Behavior in Tribolium castaneum

Abstract

The regulation of locomotor behavior is essential for insects to perform their life activities. The central nervous system plays a pivotal role in modulating physiological behaviors, particularly movement, with neuropeptides serving as key modulators of these processes. Among these, adipokinetic hormone (AKH) was originally identified in insects as a neurohormone involved in lipid mobilization. This study investigates the functional role of AKHs (AKH1 and AKH2) and their receptor (AKHR) in regulating locomotion in the red flour beetle, Tribolium castaneum. Using functional calcium reporter assays, we demonstrated that AKHR is activated by two mature AKH peptides from T. castaneum, with half-maximal effective concentrations (EC₅₀) falling within the nanomolar range. Gene expression analysis confirmed the presence of AKH1 and AKH2 transcripts in the brain, while AKHR expression was localized to the fat body and carcass. The silencing of AKHs or AKHR through RNA interference resulted in a significant reduction in both movement distance and duration. Collectively, these findings highlight the regulatory influence of AKH/AKHR signaling in locomotor activity in T. castaneum, thereby advancing our understanding of the molecular mechanisms underlying locomotor control in this economically important insect species.

Keywords: RNAi; locomotor behavior; neuropeptide; red flour beetle.

Figure 1

Gene structures and deduced amino acid sequences of AKHs in T. castaneum. (A) Gene structures of Tc-AKH1 and Tc-AKH2. Exons are represented by boxes and introns by lines. (B) Sequence alignment of mature AKH peptides from T. castaneum, D. melanogaster, B. mori, L. migratoria, A. pisum, S. frugiperda, A. mellifera and L. decemlineata. The calculated sequence logo is shown at the bottom. (C) Deduced amino acid sequences of Tc-AKHs. Putative signal peptides are underlined; mature peptides are shaded in gray, and predicted amidation signals with dibasic cleavage sites (KR) are shown in bold.

Figure 2

Amino acid sequence alignment and cluster analysis of AKHR in T. castaneum. (A) Evolutionary tree analysis of Tc-AKHR (neighbor-joining). The insect AKHR protein sequences of T. castaneum, Z. morio, A. mellifera, A. pisum, N. vitripennis, P. americana, D. pulex, A. aegypti, A. gambiae, B. dorsalis, D. melanogaster, G. morsitans, and B. mori were selected to construct the evolutionary tree. Tc-AKHR was assigned with “▲”. (B) Comparative amino acid sequence alignment of T. castaneum, D. melanogaster, and B. mori AKHR sequences.

Figure 3

(A,B) Tc-AKH1 and Tc-AKH2 docked to Tc-AKHR. The hydrogen bond is represented by a yellow dotted line; the three letters are the abbreviation of the amino acid. and the number is the amino acid number of the receptor. (C) Dose–response curves and EC₅₀ values of Tc-AKH1 and Tc-AKH2, tested on Tc-AKHR expressed in CHO-WTA11 cells.

Figure 4

The relative expression of Tc-AKHR, Tc-AKH1, and Tc-AKH2 in different adult tissues of T. castaneum. Data were obtained from the Beetle Atlas database.

Figure 5

Effect of dsAKHR injected into three-day-old adults on the gene transcript levels and motion situation of Tc-AKHR. The data were analyzed using Student’s t-test or the Mann–Whitney U test. Date are means ± SE. * p < 0.05, ** p < 0.01, and *** p < 0.001. (A) Relative expression levels of Tc-AKHR at 48 h after injection with dsAKHR; dsEGFP was used as a control. (B) Movement trajectory after injection with dsAKHR. (C) Changes in motion distance. (D) Changes in moving time. (E) Changes in resting time. (F) Changes in active time.

Figure 6

Effect of dsAKH1 injected into three-day-old adults on the gene transcript levels and motion situation of Tc-AKH1. Date are means ± SE. * p < 0.05, ** p < 0.01, and *** p < 0.001. (A) Relative expression levels of Tc-AKH1 at 48 h after injection with dsAKH1; dsEGFP was used as a control (n = 5). (B) Movement trajectory after injection with dsAKH1. (C) Changes in motion distance. (D) Changes in moving time. (E) Changes in resting time. (F) Changes in active time. The data in (A,D,E) were analyzed using Student’s t-test. The data in (C,F) were analyzed using the Mann–Whitney U test.

Figure 7

Effect of dsAKH2 injected into three-day-old adults on the gene transcript levels and motion situation of Tc-AKH2. Date are means ± SE. * p < 0.05, and ** p < 0.01. (A) Relative expression levels of Tc-AKH2 at 48 h after injection with dsAKH2; dsEGFP was used as a control (n = 5). The data were analyzed using the Mann–Whitney U test. (B) Movement trajectory after injection with dsAKH2. (C) Changes in motion distance. (D) Changes in moving time. (E) Changes in resting time. (F) Changes in active time. The data in (C–F) were analyzed using Student’s t-test.