A gut-specific chitinase gene essential for regulation of chitin content of peritrophic matrix and growth of Ostrinia nubilalis larvae Chitvan Khajuria a , Lawrent L. Buschman a , Ming-Shun Chen a, b , Subbaratnam Muthukrishnan c , Kun Yan Zhu a, * a Department of Entomology, 123 Waters Hall, Kansas State University, Manhattan, KS 66506, USA b Plant Science and Entomology Research Unit, USDA-ARS, Manhattan, KS 66506, USA c Department of Biochemistry,141 Chalmers Hall, Kansas State University, Manhattan, KS 66506, USA article info Article history: Received 16 April 2010 Received in revised form 4 June 2010 Accepted 7 June 2010 Keywords: Chitinase Chitin synthase European corn borer Ostrinia nubilalis Peritrophic matrix RNA interference abstract Chitinases belong to a large and diverse family of hydrolytic enzymes that break down glycosidic bonds of chitin. However, very little is known about the function of chitinase genes in regulating the chitin content in peritrophic matrix (PM) of the midgut in insects. We identified a cDNA putatively encoding a chitinase (OnCht) in European corn borer (ECB; Ostrinia nubilalis). The OnCht transcript was predom- inately found in larval midgut but undetectable in eggs, pupae, or adults. When the larvae were fed on an artificial diet, the OnCht transcript level increased by 4.4-fold but the transcript level of a gut-specific chitin synthase (OnCHS2) gene decreased by 2.5-fold as compared with those of unfed larvae. In contrast, when the larvae were fed with the food and then starved for 24 h, the OnCht transcript level decreased by 1.8-fold but the transcript level of OnCHS2 increased by 1.8-fold. Furthermore, there was a negative relationship between OnCht transcript level and chitin content in the midgut. By using a feeding-based RNAi technique, we were able to reduce the OnCht transcript level by 63-64% in the larval midgut. Consequently, these larvae showed significantly increased chitin content (26%) in the PM but decreased larval body weight (54%) as compared with the control larvae fed on the diet containing GFP dsRNA. Therefore, for the first time, we provide strong evidence that OnCht plays an important role in regulating chitin content of the PM and subsequently affecting the growth and development of the ECB larvae. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction Chitin, a linear polymer of b-(1,4)-N-acetyl-D-glucosamine (GlcNAc), is an important structural component of the insect cuticle, cuticular lining of the foregut, hindgut, trachea, and peri- trophic matrix (PM) that lines the lumen of the midgut (Lehane, 1997; Kramer and Muthukrishnan, 2005). For insects to grow and develop from one stage to another, a part of chitin in the old cuticle needs to be digested followed by the synthesis of chitin for the formation of new cuticle during molting (Nation, 2008). To date, two chitin synthase genes (CHS1 and CHS2, also known as CHS-A and CHS-B, respectively) have been identified in various insect species (Tellam et al., 2000; Arakane et al., 2005; Bolognesi et al., 2005). CHS1 is responsible for biosynthesis of the chitin found in the cuticular exoskeleton and other tissues that are ectodermal in origin, such as the foregut, hindgut and trachea, whereas CHS2 is responsible for biosynthesis of the chitin associated with the PM in epithelial cells of the midgut (Merzendorfer, 2006). In contrast, insect chitinases, which belong to the family 18 glycosyl hydro- lases, are responsible for chitin degradation in the cuticle (Nation, 2008; Zhu et al., 2008b) and presumably in the PM (Shen and Jacobs-Lorena, 1997; Bolognesi et al., 2005) during the insect molting. It was previously thought that there was only one chiti- nase-like gene in most insects (Choi et al., 1997). But now several fully annotated insect genomes are available and it has become clear that chitinases are encoded by a rather diverse family of genes and can be classified into at least eight groups (Arakane and Muthukrishnan, 2010). The genes encoding chitinase and chitinase-like proteins have been characterized in several insect species, including Manduca sexta (Kramer et al., 1993), Anopheles gambiae (Shen and Jacobs- Lorena, 1997), Bombyx mori, Hyphantria cunea (Kim et al., 1998), Aedes aegypti (Vega et al., 1998), Phaedon cochleariae (Girard and Jouanin, 1999), Chironomus tentans (Feix et al., 2000), Spodoptera litura (Shinoda et al., 2001), Tenebrio molitor (Royer et al., 2002; * Corresponding author. Tel.: þ1 785 532 4721; fax: þ1 785 532 6232. E-mail address: kzhu@ksu.edu (K.Y. Zhu). Contents lists available at ScienceDirect Insect Biochemistry and Molecular Biology journal homepage: www.elsevier.com/locate/ibmb 0965-1748/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibmb.2010.06.003 Insect Biochemistry and Molecular Biology 40 (2010) 621e629