Insect Molecular Biology (2005) © 2005 The Royal Entomological Society 1 Blackwell Publishing, Ltd. Characterization of a blood activated chitinolytic system in the midgut of the sand fly vectors Lutzomyia longipalpis and Phlebotomus papatasi J. M. Ramalho-Ortigão*‡§, S. Kamhawi*, M. B. Joshi†, D. Reynoso‡, P. G. Lawyer*, D. M. Dwyer†, D. L. Sacks* and J. G. Valenzuela‡ * Intracellular Parasite Biology Section, † Cell Biology Section, Laboratory of Parasitic Diseases, and ‡ Vector Molecular Biology Unit, Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA Abstract We characterized a cDNA from Phlebotomus papatasi , PpChit1 , which encodes a midgut specific chitinase and show the presence of a functional, blood-induced chitinolytic system in sand flies. PpChit1 is detected only in the midgut and is regulated by blood feeding. A recombinant protein (rPpChit1) produced in HEK 293- F cells exhibited a similar activity profile to that found in the native protein against several specific substrates, including an oligomeric glycol chitin and synthetic 4-methyl-umbelliferone labelled substrates. Western blotting showed that the native protein is recognized by mouse polyclonal antibodies against rPpChit1. Additionally, the rPpChit1 and the native chitinase dis- played similar retention times in a HPLC size fractionation column. When added to rPpChit1 or to midgut lysates, PpChit1 sera reduced chitinolytic activity by 65–70%. Keywords: sand fly, peritrophic matrix, chitinolytic activity, Leishmania . Introduction Upon taking a blood meal, some haematophagous insects secrete a type 1 peritrophic matrix (PM1) that is detectable within a few hours and completely surrounds the blood bolus in the midgut. Several roles have been ascribed for this PM1 including protection of the gut lining and com- partmentalization of the blood meal (Jacobs-Lorena & Oo, 1996; Shao et al ., 2001), acting as a barrier to pathogens (Shahabuddin et al ., 1993 and 1996; Walters et al ., 1992) and as a binding substrate for haeme detoxification (Pascoa et al ., 2002). Recently, Villalon et al . (2003) reported that in Anopheles stephensi and Aedes aegypti , the PM1 may actually limit the rate of blood digestion, presumably by limiting the diffusion of digestive enzymes. In the PM1, chitin is an important component for structure maintenance (Shao et al ., 2001), where it can associate with various proteins and proteoglycans for the development of a fully mature PM (Jacobs-Lorena & Oo, 1996; Tellam et al ., 1999). Moreover, the synthesis of a mature PM1 is thought to occur by a synchronous mechanism that involves chitin deposition and chitin degradation (Shahabuddin et al ., 1993; Pimenta et al ., 1997; Filho et al ., 2002). Chitin degradation is catalysed by the action of chitinases (EC 3.2.1.14), and other glycosylhydrolases, through the hydrolysis of β 1– 4 glycosidic bonds of chitin polymers and oligomers (as reviewed by Merzendorfer & Zimoch, 2003). Leishmania sp. are digenetic parasites transmitted to a vertebrate host by the bite of an infected sand fly vector. In humans, these parasites cause a broad spectrum of dis- eases ranging from mild cutaneous ulcerations to severe and typically fatal visceral disease. All Leishmania have a life cycle that includes two major developmental stages: an obligate intracellular, amastigote form which resides and multiplies within the phago-lysosome of infected mamma- lian macrophages and an extracellular, flagellated promas- tigote form present in the midgut of the sand fly vector (Sacks & Kamhawi, 2001). During Leishmania development in the sand fly midgut, escape from the PM is a crucial event in which chitinases probably play a fundamental role. Schlein et al . (1991) doi: 10.1111/j.1365-2583.2005.00601.x Received 5 February 2005; accepted after revision 28 July 2005. Corre- spondence: J. Marcelo Ramalho-Ortigão, Vector Molecular Biology Unit, Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 12735 Twinbrook Park- way, Room 2E-22, Rockville MD 20852–8132 USA. Tel.: +1 301 4511 082; fax: +1 301 4022 201; e-mail:mortigao@niaid.nih.gov Current address: §Department of Biological Sciences, University of Notre Dame, Galvin Life Sciences, Notre Dame, IN, 46556. Tel.: +1 574 631-0491; m_ortigao@nd.edu