Biochemical characterization of digestive proteases and carbohydrases of the carob moth, Ectomyelois ceratoniae (Zeller) (Lepidoptera: Pyralidae) Parvin Razavi Tabatabaei, Vahid Hosseininaveh ⁎, Seyed Hossein Goldansaz, Khalil Talebi Department of Plant Protection, College of Agriculture, University of Tehran; Karaj, 31587-77871, Iran abstract article info Article history: Received 22 October 2010 Revised 14 December 2010 Accepted 21 December 2010 Available online 30 December 2010 Keywords: Ectomyelois ceratoniae Digestive Proteases Carbohydrases Enzyme recycling Digestive proteinases and carbohydrases of Ectomyelois ceratoniae (Zeller) larvae were investigated using appropriate substrates and inhibitors. Midgut pH in larvae was determined to be slightly alkaline. Midgut extracts showed optimum activity for proteolysis of hemoglobin at pH 9–12. Midgut proteinases also hydrolyzed the synthetic substrates of trypsin, chymotrypsin, and elastase at pH 8–11. Maximum digestive α- amylase activity was also observed at pH 8–11. However, optimum activity for α- and β-glucosidase occurred at pH 5–8. Alpha- and β-galactosidases optimum activities occurred at pH 5 and pH 6, respectively. Inhibitors of serine proteases were effective on midgut serine proteases (trypsin and chymotrypsin proteases). Zymogram analyses revealed at least five bands of total proteolytic activity in the larval midgut. Protease- specific zymogram analyses revealed at least four, two, and one isozymes for trypsin-, chymotrypsin-, and elastase-like activities respectively. Two α-amylase isozymes were found in the midgut of fifth instar larvae and in the whole bodies of 1st through 5th instar larvae. Zymogram studies also revealed the presence of one and two bands of activity for β- and α-glucosidase, respectively. Recycling of α-amylase and proteases in the larval midgut was not complete. At least one isozyme of trypsin, chymotrypsin, elastase, and α-amylase were not recycled and were observed in the larval hindgut. © Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society, 2010. Published by Elsevier B.V. All rights reserved. Introduction Lepidopteran larvae are one of main crop pests because they can eat each part of host plants, which causes remarkable losses in crop production and affects the product quality (Valencia-Jiménez et al., 2008). The carob moth, Ectomyelois ceratoniae (Zeller) (Lepidoptera: Pyralidae), is a well-known cosmopolitan pest of many crops. It completes larval development on many crops, including almonds and peaches, walnuts, navel oranges, pomegranates, pistachios, apples, acacia, carob, mesquite, castor bean, and many others (Heinrich, 1956; Gothilf, 1970, 1984; Al-Izzi et al., 1985; Warner, 1988; Neunzig, 1990; Gonzalez, 2003). Although the pest can be controlled by chemical pesticides (Warner et al., 1990), the use of insecticide sprays on many stored product crops such as dates and pistachio is considered undesirable by consumers. Therefore, there is an urgent need for the development of alternative pest control strategies and for improvements in the current biological control strategies, including the use of natural enemies. The physiology and biochemistry of the insect midgut have been an important field of study for the development of novel insecticidal strategies. Midgut digestive enzymes can be targeted by plant proteinaceous inhibitors to interfere in normal food digestion and absorption in insect pests (Jongsma and Bolter, 1997; Gatehouse and Gatehouse, 1999). To use this approach, an understanding of the biochemical characteristics of the enzymes active in the insect midgut is necessary (Wilhite et al., 2000). Herbivorous lepidopteran larvae feed voraciously on host crops to derive nutrients for optimum growth and development. The primary constituent of their diet is protein, which is digested into amino acids by proteases. Similarly, complex polysaccharides are broken down into simple sugars by amylases. Any interference in digestion by proteinase inhibitors and amylase inhibitors can lead to developmental malformations. Prote- ase inhibitor-based approaches usually focus on the dominant mechanistic class of digestive protease in Lepidoptera, serine proteases (Srinivasan et al., 2006; Chougule et al., 2008). The lepidopteran larval midgut harbors complex proteolytic enzymes such as trypsins, chymotrypsins, elastases, cathepsin-B like proteases, aminopeptidases, and carboxypeptidases, which are all involved in protein digestion. Serine proteases dominate the larval gut environ- ment and contribute to about 95% of the total digestive activity (Bown et al., 1997; Gatehouse et al., 1997; Patankar et al., 2001; Srinivasan et al., 2006; Chougule et al., 2008). Owing to changes in feeding preferences and to tissue-specific variation of plant proteins, a dynamic modulation of larval digestive proteases seems sensible for achieving the optimal digestion of dietary protein that is required for normal growth and development (Chougule et al. 2005; Srinivasan et Journal of Asia-Pacific Entomology 14 (2011) 187–194 ⁎ Corresponding author. Fax: + 98 261 2238529. E-mail address: vnaveh@ut.ac.ir (V. Hosseininaveh). 1226-8615/$ – see front matter © Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society, 2010. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.aspen.2010.12.010 Contents lists available at ScienceDirect Journal of Asia-Pacific Entomology journal homepage: www.elsevier.com/locate/jape