Expression and applications of recombinant crustacean hyperglycemic hormone from eyestalks of white shrimp (Litopenaeus vannamei) against bacterial infection Sunee Wanlem a, b , Kidchakan Supamattaya a , Chutima Tantikitti a , Poonsuk Prasertsan b , Potchanapond Graidist c, * a Aquatic Animal Health Research Center, Department of Aquatic Science, Faculty of Natural Resources, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand b Department of Industrial Biotechnology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand c Tumor Biology Research Unit, Department of Biomedical Sciences, Faculty of Medicine, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand article info Article history: Received 7 September 2010 Received in revised form 11 January 2011 Accepted 16 January 2011 Available online 25 January 2011 Keywords: Crustacean hyperglycemic hormone, CHH Glucose White shrimp Litopenaeus vannamei Vibrio harveyi abstract Crustacean hyperglycemic hormone (CHH) has many functions to regulate carbohydrate metabolism, ecdysis and reproduction including ion transport in crustaceans. The cDNA encoding CHH peptides containing 369 bp open reading frame encoding 122 amino acids was cloned from eyestalk of white shrimp (Litopenaeus vannamei) and was produced by a bacterial expression system. The biological activity of recombinant L. vannamei crustacean hyperglycemic hormone (rLV-CHH) was tested. The hemolymph glucose level of shrimp increased two-fold at 1 h after the rLV-CHH injection and then returned to normal after 3 h. In addition to the effect of rLV-CHH administration (25 mg/shrimp) on immunological responses of white shrimp against pathogenic bacteria, Vibrio harveyi was studied. Results showed that the blood parameters of shrimp injected with rLV-CHH; the THC, PO activity, serum protein level and clearance ability to V. harveyi, were also higher than those of Neg-protein and PBS- injected shrimp. The survival of shrimp injected with rLV-CHH was significantly higher (66.0%) than shrimp that injected with Neg-protein (33.3%) and PBS (28.9%) after 14 days. It is possible that the administration of rLV-CHH in L. vannamei exhibited a higher immune response related to resistance against V. harveyi infection. Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved. 1. Introduction Physiological processes in crustaceans are mainly regulated by neuroendocrines of natural peptides, of which approximately fifty neuroendocrines have been described, although the functions of some hormones are still not well defined [1]. Crustacean hyper- glycemic hormone (CHH), the most abundant neuroendocrine, is synthesized by X-organ (XO) and stored in the sinus gland (SG) prior to being released directly into the hemolymph [2]. The X-organ and sinus gland (XO-SG) are located in the optic ganglia in the eyestalk which synthesize and control neuropeptide hormones essential for the regulation of the physiology and metabolism of crustaceans. The major hormone family produced from XO-SG is composed of crustacean hyperglycemic hormone (CHH), molt- inhibiting hormone (MIH), gonad-inhibiting hormone (GIH) or vitellogenesis-inhibiting hormone (VIH) and mandibular organ- inhibiting hormone (MOIH) [3e7]. These hormones are known as the CHH-family of peptides on account of the extensive similarities in their sequences between individuals in a species or among different species. The peptides of the CHH-family consist of 72e78 residues and contain six conserved Cys residues that form three disulfide bonds [8]. The sequence of CHH hormone from Carcinus maenus was first reported by Kegel and coworkers [9]. Subse- quently, CHH peptides sequences have been described and char- acterized in lobster [10], crabs [11], crayfishes [12], prawns [13,14] and shrimp [8,15]. CHH functions mainly in the regulation of glucose levels, as well as in the metabolism of carbohydrates and lipids [16]. In addition, it has a significant role in the processes of molting [17], reproduction [18], and osmoregulation [19,20]. However, there is no report about its role on the activation of the immune response in crustacean. The objective of the present study is therefore to elucidate the characterization of the cDNA encoding CHH peptides of white shrimp (Litopenaeus vannamei) and to investigate the effect of the rLV-CHH protein on the induction of shrimp immunity against luminous bacteria, Vibrio harveyi by intramuscular injection. * Corresponding author. Tel.: þ66 74281184; fax: þ66 74429584. E-mail address: gpotchan@medicine.psu.ac.th (P. Graidist). Contents lists available at ScienceDirect Fish & Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi 1050-4648/$ e see front matter Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2011.01.014 Fish & Shellfish Immunology 30 (2011) 877e885