Differential effects of silencing crustacean hyperglycemic hormone gene expression on the metabolic profiles of the muscle and hepatopancreas in the crayfish Procambarus clarkii

February Differential effects of silencing crustacean hyperglycemic hormone gene expression on the metabolic profiles of the muscle and hepatopancreas in the crayfish Procambarus clarkii Wenfeng Li 0 1 Kuo-Hsun Chiu 1 Yi-Chun Tien 0 1 Shih-Fu Tsai 0 1 Li-Jane Shih 1 Chien- Hsun Lee 0 1 Jean-Yves Toullec 1 2 Chi-Ying Lee 0 1 0 Department of Biology, National Changhua University of Education, Changhua, Taiwan, 2 Department of Aquaculture, National Kaohsiung Marine University , Kaohsiung, Taiwan , 3 Department of Medical Laboratory, Taoyuan Armed Forces General Hospital , Taoyuan, Taiwan, 4 Sorbonne Universit eÂs , UPMC Universit e Paris 06, UMR 7144 CNRS, Equipe ABICE, Station Biologique de Roscoff , Roscoff , France 1 Editor: Emanuele Buratti, International Centre for Genetic Engineering and Biotechnology , ITALY 2 CNRS, UMR 7144, Adaptation et Diversite en Milieu Marin, Station Biologique de Roscoff , Roscoff , France In order to functionally characterize the metabolic roles of crustacean hyperglycemic hormone (CHH), gene expression of CHH in the crayfish (Procambarus clarkii) was knocked down by in vivo injection of CHH double-stranded RNA (dsRNA), followed by metabolomic analysis of 2 CHH target tissues (the muscle and hepatopancreas) using nuclear magnetic resonance spectroscopy. Compared to the levels in untreated and saline-injected (SAI) animals, levels of CHH transcript, but not those of molt-inhibiting hormone (a CHH-family peptide), in the eyestalk ganglia of CHH dsRNA-injected (DSI) animals were significantly decreased at 24, 48, and 72 hour post injection (hpi), with concomitant changes in levels of CHH peptide in the sinus gland (a neurohemal organ) and hemolymph. Green fluorescence protein (GFP) dsRNA failed to affect levels of CHH transcript in the eyestalk ganglia of GFP DSI animals. Number of metabolites whose levels were significantly changed by CHH dsRNA was 149 and 181 in the muscle and 24 and 12 in the hepatopancreas, at 24 and 48 hpi, respectively. Principal component analysis of these metabolites show that metabolic effects of silencing CHH gene expression were more pronounced in the muscle (with the cluster of CHH DSI group clearly being separated from that of SAI group at 24 hpi) than in the hepatopancreas. Moreover, pathway analysis of the metabolites closely related to carbohydrate and energy metabolism indicate that, for CHH DSI animals at 24 hpi, metabolic profile of the muscle was characterized by reduced synthesis of NAD+ and adenine ribonucleotides, diminished levels of ATP, lower rate of utilization of carbohydrates through glycolysis, and a partially rescued TCA cycle, whereas that of the hepatopancreas by unaffected levels of ATP, lower rate of utilization of carbohydrates, and increased levels of ketone bodies. The combined results of metabolic changes in response to silenced CHH gene - Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: The present study was supported financially by Ministry of Science and Technology, Taiwan (https://www.most.gov.tw/) through grants (102-2311-B-018-002 and 104-2311-B-018-002) to C-YL. WL was supported by a postdoctoral fellowship (104-2811-B-018-001) from Ministry of Science and Technology, Taiwan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. expression reveal that metabolic functions of CHH on the muscle and hepatopancreas are more diverse than previously thought and are differential between the two tissues. Introduction Crustacean eyestalks contain a neuroendocrine tissue, the X-organ/sinus gland complex, which synthesizes and secrets several neurohormones that are implicated in regulating a wide variety of physiological functions, including metabolism, reproduction, growth, ionic balance, and color changes. The wide array of biological processes that are under the hormonal control of the eyestalk factors highlights the importance of the XO/SG complex in crustacean physiology [1±3]. Among these neuropeptides, crustacean hyperglycemic hormone (CHH) is the prototypic member of the CHH family, which also includes molt-inhibiting hormone (MIH), vitellogenesis-inhibiting hormone (VIH), and mandibular organ-inhibiting hormone (MOIH), and insect ion transport peptide (ITP) [ 4, 5 ]. CHH, a 72- or 73-residue polypeptide, is characterized by 3 disulfide bridges formed by 6 highly conserved cysteine residues and Nand C-terminally blocked, with the C-terminal amide being critical for its hyperglycemic activity [6±10]. Molecular characterizations of CHH precursor indicated that the precursor protein consists of a signal peptide, a CHH precursor-related peptide, and a mature CHH peptide [11]. Functionally, the best characterized roles for CHH is its effects on carbohydrate metabolism [ 12 ] and CHH is consid (...truncated)