Research paper A novel CHH gene from the Pacific white shrimp Litopenaeus vannamei was characterized and found highly expressed in gut and less in eyestalk and other extra-eyestalk tissues Claudia Ventura-López, Gracia Gómez-Anduro, Fabiola G. Arcos, Raúl Llera-Herrera 1 , Ilie S. Racotta, Ana M. Ibarra ⁎ Centro de Investigaciones Biológicas del Noroeste, S.C. (CIBNOR), Av. Instituto Politécnico Nacional No.195, Col. Playa Palo de Santa Rita, C.P. 23096, La Paz, Baja California Sur, Mexico abstract article info Article history: Received 8 August 2015 Received in revised form 7 January 2016 Accepted 4 February 2016 Available online 6 February 2016 The crustacean hyperglycemic hormone (CHH) family is an important group of neuropeptides involved in con- trolling growth, reproduction, and stress response in decapod species. In this study, a new gene containing 4 exons-3 introns flanked by canonical 5′-GT-AG-3′ intron splice-site junctions was isolated from Litopenaeus vannamei. Two full length transcripts of this CHH were isolated from eyestalk and pericardial tissue of males and females using rapid amplification of cDNA ends (RACE). Transcripts sequences were 1578 bp in length in males pericardial tissues and in males and females eyestalk with 100% identity, but the transcript isolated from females pericardial tissues was shorter (974 bp). The differences in transcripts length is a result of two polyadenylation sites present in the 3'UTR resulting in two transcription termination signals. Transcript se- quences encoded one unique protein that can be classified as type I CHH subfamily because of the 4 exons and 3 introns structure, although the CPRP region is not-well conserved and there is no amidation in the C- terminal of the deduced amino acid sequence. Furthermore, there is a glycine inserted in the mature peptide not at position 12 as in type II CHHs but after amino acid 31 and the phylogenetic analysis did not group the pep- tide within type I, but closer to type II CHHs. We demonstrated by endpoint-PCR, qPCR, and in situ hybridization (ISH), that this gene is expressed in neuroendocrine organs known to express CHHs in penaeid shrimp, including X-organ and optic nerve in eyestalk, supraesophageal ganglion (SoG), but it is also expressed in other organs as gill, gut, pericardial cavity, as well as in terminal ampoule or spermatophore and vas deferens of males. © 2016 Elsevier B.V. All rights reserved. Keywords: Crustacean-hyperglycemic-hormone Extra-eyestalk-expression Pericardial-organ Foregut Ductus-deferens Spermatophore Differential expression ISH 1. Introduction The crustacean hyperglycemic hormone superfamily (CHH) is a multi-gene family identified originally in the medulla terminalis of the X-organ sinus gland complex (XOSG) of decapods (Jaros and Keller, 1979). The primary structure of members of this protein family con- serve 6 cysteine residues forming three intramolecular disulfide bonds, and the mature peptides are 72 to 87 amino acid residues in length (Keller, 1992). According to sequence similarities, CHH super- family members have been classified into two major groups: Type I sub- family is characterized by the presence of a CPRP (CHH Precursor Related Peptide) between the signal and mature peptides, and the genes are composed of 4 exons and 3 introns; this subfamily include all CHHs from crustaceans and ion transport peptides (ITP) from insects (Webster et al., 2012). Type II subfamily contain 3 exons and 2 introns, lack the CPRP region, and comprises molt inhibiting hormone (MIH) and mandibular organ inhibiting hormone (MOIH) members; the vitel- logenesis/gonad inhibiting hormone (VIH/GIH) gene structure is not known as yet (Webster et al., 2012). Both type I and II CHH neuropeptides have been associated with different physiological mechanisms as regulation of blood glucose (review: Fanjul-Moles, 2006; Webster et al., 2012), control of the molt cycle (Chen et al., 2007), osmoregulation (Serrano et al., 2003), and re- production (deKleijn et al., 1998; Tsutsui et al., 2007; Treerattrakool Gene 582 (2016) 148–160 Abbreviations: CHH, crustacean hyperglycemic hormone; XOSG, X-organ sinus gland complex; CPRP, CHH precursor related peptide; VIH/GIH, vitellogenesis/gonad inhibiting hormone; MIH, molt inhibiting hormone; MOIH, mandibular organ inhibiting hormone; ITP, ion transport peptide; ETS, expressed sequence tags; cDNA, DNA complementary to RNA; SSH, suppresive substractive hybridation; MEGA, molecular evolutionary genetics analysis; Lvchh, transcript isolated from L. vannamei; LvCHH_PO_ES, deduced protein se- quence; ISH, in situ hybridization; SoG, supraesophageal ganglion; qPCR, quantitative po- lymerase chain reaction; TAE, tris-acetate-EDTA; RACE, rapid amplification of cDNA ends; JTT, Jones-Taylor-Thornton; PO, pericardial organ; ES, eyestalk; ORF, open reading frag- ment; UTR, untraslated region; PBS, phosphate buffer solution; NBT, nitro blue tetrazoli- um; BCIP, 5-bromo-4-chloro-3-indolyl-phosphate Cep, cuticular epithelium (Cep). ⁎ Corresponding author. E-mail addresses: cventura@cibnor.mx (C. Ventura-López), ggomez@cibnor.mx (G. Gómez-Anduro), farcos04@cibnor.mx (F.G. Arcos), raul.llera@gmail.com (R. Llera-Herrera), iracotta@cibnor.mx (I.S. Racotta), aibarra@cibnor.mx (A.M. Ibarra). 1 Present address: CONACyT Research Fellow — Centro de Investigación en Alimentación y Desarrollo A.C. (CIAD) Unidad Mazatlán, Av. Sábalo-Cerritos s/n. Estero del Yugo, Mazatlán, Sinaloa 82000, Mexico. http://dx.doi.org/10.1016/j.gene.2016.02.011 0378-1119/© 2016 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect Gene journal homepage: www.elsevier.com/locate/gene