Cuticular chitin synthase and chitinase mRNA of whiteleg shrimp Litopenaeus vannamei during the molting cycle Jorge Rocha a , Fernando L. Garcia-Carreño a , Adriana Muhlia-Almazán b , Alma B. Peregrino-Uriarte b , Gloria Yépiz-Plascencia b , Julio H. Córdova-Murueta a, ⁎ a Centro de Investigaciones Biológicas del Noroeste (CIBNOR), Mar Bermejo 195, Col. Playa Palo de Santa Rita, La Paz, B.C.S. 23096, Mexico b Centro de Investigación en Alimentación y Desarrollo (CIAD), Carretera a La Victoria Km 0.6, Col. Ejido La Victoria, Hermosillo, Sonora 83000, Mexico abstract article info Article history: Received 24 June 2010 Received in revised form 30 November 2011 Accepted 13 December 2011 Available online 29 December 2011 Keywords: Molt cycle Chitinase Chitin synthase Litopenaeus vannamei Chitin metabolism is of high relevance for shrimp growth because it has to be synthesized and cleaved in each molt. We studied chitin synthase and chitinase mRNAs from whiteleg shrimp Litopenaeus vannamei. For this, cDNA coding for cuticular chitin synthase (LvChS) and chitinase isoenzymes (LvChi1, LvChi2 and LvChi3) was amplified, sequenced and identified. In a qualitative analysis, LvChi1 and LvChi3 were detected only in the he- patopancreas and are probably involved in digestion of food chitin. LvChi2 transcript was found in pleopods, uropods, gills, eyestalk, and digestive tube; LvChi2 is likely to be involved in the hydrolysis of chitin from the exoskeleton and peritrophic membrane, but not in food chitin digestion. LvChS was found widely distributed in the organism, including the hepatopancreas. Thus, it seems to be involved in synthesis of chitin to build the exoskeleton and also the peritrophic membrane. Relative expression of LvChS and LvChi2 genes was evaluated by quantitative RT-PCR in the integument. These transcripts had a varying pattern of abundance during the molt cycle, based on the need of shrimp to synthesize or hydrolyze chitin from exoskeleton. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Chitin is a linear homopolymer of β-linked N-acetylglucosamine (NAcGlc) that occurs widely in nature. World annual bioproduction of chitin was estimated at 100 × 10 9 t, being an important source of carbon for heterotrophic bacteria (Cauchie, 2002; Tharanathan and Kittur, 2003). Chitinous structures are found in several taxa as well as the cell wall of fungi (Roncero, 2002), egg shells of nematodes (Brydon et al., 1987) and exoskeleton and peritrophic matrix of insects (Merzendorfer and Zimoch, 2003) and crustaceans (Roer and Dillaman, 1984). Thus, the study of chitin metabolism is of biotechnological, eco- logical, and physiological relevance. The exoskeleton of crustaceans is a four-layered matrix that confers support, rigidity, and impermeability to the body. Chitin is present in the inner three layers, forming a sclerotized complex with structural proteins and calcium salts (O'Brien et al., 1991). For crustaceans to grow, the rigid exoskeleton has to be discarded by hydrolysis of cuticular components and a new exoskeleton has to be synthesized to match the new body size (Promwikorn et al., 2004). The process of synthesizing a new exoskeleton and discarding the old one (ecdysis or molting) is frequently repeated during the life cycle of shrimp. The molt cycle com- prises the time between one molt and another, including five stages: post-molt which includes stages A (subdivided in A 1 and A 2 ) and B, intermolt stage C, pre-molt stage D (which is subdivided in D 0 through D 4 ), and molting or ecdysis (stage E), as described by Smith and Dall (1985). These stages are accompanied by a number of processes induced by hormonal signals, including gene expression, enzyme activity, and physiological and behavioral changes (Mikami, 2005). Chitin is also pre- sent in the peritrophic matrix that is secreted along the length of the di- gestive tract and serves as an impermeable coat for the food bolus, thereby protecting the lumen from digestive enzymes and attack by pathogens (Ceccaldi, 1989). The study of synthesis and hydrolysis of chitin during the molting cycle will contribute to understanding its physiology. Chitin synthase (ChS; E.C. 2.4.1.16, UDP-N-acetyl-D-glucosamine: chi- tin 4-β-N-acetylglucosaminyl transferase) is the enzyme that assembles monomers of NAcGlc into chitin polymers. ChS is a cell membrane- bound enzyme composed of two identical subunits and has been found in the membrane or in cytoplasmic microsomes in insects (Merzendorfer, 2006). After ChS polymerizes chitin, the growing polymer is deposited in the extracellular space (Cohen, 1987). Several sequences of insect ChS cDNA have been reported, including ChS from the sheep fly Lucilia cuprina (Tellam et al., 2000), tobacco hornworm Manduca sexta (Zimoch et al., 2005), mosquito Aedes aegypti (Ibrahim et al., 2000), fruit fly Drosophila melanogaster (Gagou et al., 2002), and red flour beetle Tribolium castaneum (Arakane et al., 2004). Compared to abundant studies of insect ChS, there are no studies about activity or immuno-localization of crustacean ChS, and only one cDNA sequence of Aquaculture 330–333 (2012) 111–115 ⁎ Corresponding author. Tel.: + 52 612 123 8484; fax: + 52 612 125 3625. E-mail address: jcordova@cibnor.mx (J.H. Córdova-Murueta). 0044-8486/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2011.12.024 Contents lists available at SciVerse ScienceDirect Aquaculture journal homepage: www.elsevier.com/locate/aqua-online