Indian Journal of Biotechnology Vol 16, January 2017, pp 37-43 Crustacean hyperglycemic hormone family gene silencing in Penaeus monodon mediated through dsRNA synthesized in vitro from genomic and cDNA S Vrinda 1 , C Reshmi 1 , Seena Jose 1 , P Reynold 2 , K K Vijayan 2 , Rosamma Philip 3 and I S Bright Singh 1 * 1 National Centre for Aquatic Animal Health, Cochin University of Science and Technology, Cochin 682 016, India 2 Central Marine Fisheries Research Institute, Cochin 682 018, India 3 Department of Marine Biology, Microbiology and Biochemistry, Cochin University of Science and Technology Cochin 682 016, India Received 8 January 2015; revised 10 September 2015; accepted 18 September 2015 RNA interference (RNAi) is the phenomenon in which long dsRNA is able to silence cognate gene expression. In the present study, 801 bp crustacean hyperglycemic hormone 1 (CHH1) and 795 bp moult inhibiting hormone 1 (MIH1) specific dsRNAs from genomic DNA, and 316 bp gonad inhibiting hormone (GIH) specific dsRNA from cDNA were constructed in vitro. Then the specific dsRNA constructs were administered into adult shrimps (Penaeus monodon). The gene expression was studied by semi quantitative RT-PCR and by monitoring haemolymph glucose concentration, duration of moulting and expression of vitellogenin as measures of specific biological activity. The gene silencing of CHH1, MIH1 and GIH genes could be attained within 24 h of dsRNA application. GIH gene silencing was observed up to 60 th h. However, a complete silencing of MIH1 and MIH2 continued to 108 h post administration. Physiology of the animals injected with dsRNA of CHH1, MIH1 and GIH corroborated with the silencing of the specific genes resulting in the decrease of haemolymph glucose level, reduction in the days of moulting and expression of vitellogenin gene, respectively in adult shrimp. These results suggest the possibility of using dsRNAs of CHH family hormone genes as molecular tools for silencing inhibitory genes in turn affecting induced maturation in P. monodon. Keywords: CHH family hormone, crustacean hyperglycemic hormone, gonad-inhibiting hormone, dsRNA, moult-inhibiting hormone, RNAi Introduction Physiology of crustaceans is partly regulated by diverse neuropeptides synthesized by medulla terminalis of X-organ sinus gland complex located in the eyestalk. The neuropeptide family includes crustacean hyperglycemic hormone (CHH), moult- inhibiting hormone (MIH), gonad-inhibiting hormone (GIH) also called vitellogenesis-inhibiting hormone (VIH), and mandibular organ-inhibiting hormone (MOIH). This unique peptide family in crustaceans is involved in blood sugar regulation, inhibition of ecdysteroid synthesis, regulation of reproduction and growth 1 . CHH/MIH/GIH family, precisely named as CHH family hormones, is multifunctional despite their similarities in structure 2 . CHH is involved mainly in elevating the glucose level in haemolymph by glycogen degradation in hepatopancreas. However, its function has been demonstrated to be pleiotropic, playing remarkable role in reproduction, moulting, digestion, osmoregulation and lipid metabolism in various species 2 . MIH controls moulting by inhibiting the synthesis of ecdysteroids by Y-organ, the process essential for the development and maturity of decapod crustaceans 3 . GIH controls the gonad maturation playing a highly complex role in reproduction and moulting 4 . Traditionally, shrimp maturation is induced by unilateral ablation of eyestalk, which reduces the CHH family hormone by half 5 . However, being an unethical and destructive process, this necessitates the replacement that would help standardize the maturation process with sustainability. It has been proven that RNA interference (RNAi) mediated gene silencing is operative in whole shrimp 6 . The RNAi has been used for experimental manipulation of gene expression and to prove the function of certain genes at genomic level 7 . Consequently, the technique has revolutionized research in ‘reverse genetics’ by introducing dsRNA into organisms or cells as it could ————— *Author for correspondence: Tel: +91-484-2381120; Fax: +91-484-2381120 isbsingh@gmail.com