CCHamide-2 Signaling Regulates Food Intake and Metabolism in Gryllus bimaculatus

Abstract

Neuropeptides play vital roles in energy homeostasis in insects and other animals. Although the importance of the regulatory network of neuropeptides in feeding and metabolism has been illuminated, a complete understanding of the mechanisms has not been addressed as many factors are involved in the regulation. CCHamide-2 is a newly identified brain-gut neuropeptide that regulates feeding behavior in several insect species including Drosophila melanogaster. However, little is known about the mechanisms controlling the feeding-related behavior and metabolic functions modulated by CCHamide-2 in other insects. In this study, we addressed the functions of CCHamide-2 in the two-spotted cricket, Gryllus bimaculatus, which was used as the experimental material to research the mechanisms of feeding and metabolism in this omnivorous insect species. Knockdown crickets by RNA interference against GbCCHamide-2R increased the amount of food intake, while injection of chemically synthetic GbCCHamide-2 peptide reduced the amount of food intake. Further, knockdown and peptide injection experiments revealed that GbCCHamide-2 signaling increased the concentrations of circulating lipids and carbohydrates, and the carbohydrate-rich diet increased the transcript levels of GbCCHa-2R. Moreover, GbCCHa-2 injection decreased the transcript level of Gbilp. By contrast, GbCCHamide-2 signaling did not affect nymphal growth or the transcript level of GbAKH, as well as feeding preferences. Taken together, CCHamide-2 signaling in G. bimaculatus regulates food intake associated with alterations in lipid and carbohydrate levels in hemolymph.

Keywords: CCHamide-2 signaling; Gryllus bimaculatus; feeding; metabolism.

Figure 1

RT-PCR analyses of the tissue distributions of GbCCHa-2 and -2R in male eighth-instar nymphs. Elongation factor (EF) was used as the internal control gene. No-RT negative control and no-template control yielded no amplification products (Supplementary Materials, Figure S1). Br, brain; CA, corpora allata; CC, corpora cardiaca; SG, subesophageal ganglion; TG, thoracic ganglia; AG, abdominal ganglia; TAG, terminal abdominal ganglion; Fg, foregut; Mg, midgut; Hg, hindgut; FB, fat body; Mus, muscle; Tes, testis; MT, Malpighian tubules.

Figure 2

RNAi efficiency. The relative mRNA levels of GbCCHa-2R in CNS (a) and gut (b) on the third day after dsRNA injection. ß-actin was used as the reference gene. Values are shown as mean + S.D., n = 5–9, * p < 0.05, ** p < 0.01, unpaired t-test.

Figure 3

Effects of GbCCHa-2 and dsGbCCHa-2R on number of feces and weight gain: (a) Effects of GbCCHa-2R knockdown on the numbers of fecal pellets representing the amount of food intake for 24 h of the third day after dsRNA injection and 12 h after peptide application; (b) Effects of GbCCHa-2R knockdown on the weight gain for 24 h of the third day after dsRNA injection and 12 h after peptide application. In RNAi experiments, values are shown as mean + S.D., n = 15–16, *** p < 0.001, unpaired t-test. In peptide-injection experiments, values are shown as mean ± S.D., * p < 0.05, *** p < 0.001, n = 9, one-way ANOVA with post hoc Dunnett’s test.

Figure 4

Effects of GbCCHa-2 signaling on the levels of lipids and carbohydrates in the hemolymph and fat body. (a,b) The levels of lipid (a) and free carbohydrates (b) in the hemolymph afterGbCCHa-2R knockdown and GbCCHa-2 administration. (c,d) The levels of lipid (c) and glycogen (d) in fat body. Values are showed as mean + S.D., n = 5–9, * p < 0.05, ** p < 0.01, unpaired t-test.

Figure 5

Effects of dietary nutrients on GbCCHa-2R and effects of GbCCHa-2 signaling on Gbilp and GbAKH: (a) The transcript levels of GbCCHa-2R in the crickets fed on normal diet or lipid-, carbohydrate-, and protein-rich diet (L-, C-, and P-rich); (b) The transcript levels of Gbilp in CNS after GbCCHa-2R knockdown and GbCCHa-2 administration; (c) The transcript levels of GbAKH in CNS. Values are showed as mean + S.D., n = 6–9, * p < 0.05, ** p < 0.01, one-way ANOVA with post hoc Dunnett’s test for (a), unpaired t-test for (b,c).

Figure 6

Effects of GbCCHa-2 signaling on food choices: (a–c) Effects of GbCCHa-2R knockdown and peptide injection on the evaluation of the contents of lipids (a), carbohydrates (b), and proteins (c) in diets. Preference index = [W _{(rich nutrient)} − W _{(poor nutrient)}]/[W _{(rich nutrient)} + W _{(poor nutrient)}]. W, weight of diet eaten; (d) Effects of GbCCHa-2R knockdown and GbCCHa-2 application on the differentiation of three macronutrient types. Ratio = W _{(one nutrient)}/W _(total). Values are shown as mean ± S.D., n = 7–11, unpaired t-test.