Insect Biochem. Molec. Biol. Vol. 27, No. 7, pp. 693–699, 1997  1997 Elsevier Science Ltd Pergamon PII: S0965-1748(97)00045-3 All rights reserved. Printed in Great Britain 0965-1748/97 $17.00 + 0.00 Molecular Cloning of cDNAs for Two Pro-Phenol Oxidase Subunits from the Malaria Vector, Anopheles gambiae 1 HAOBO JIANG,† YANG WANG,† SVETLANA E. KOROCHKINA,‡ HELEN BENES ˇ ,‡ MICHAEL R. KANOST†* Received 28 May 1997; revised and accepted 20 June 1997 Phenol oxidase exists in insect hemolymph as a zymogen, pro-phenol oxidase (pro-PO), which is activated by specific proteolysis in response to infection or wounding. Phenol oxidase cata- lyses the synthesis of quinones that polymerize to form melanin deposits, which encapsulate parasites and help to seal wounds. Antibodies to pro-PO from Manduca sexta bound to 76, 72, and 71 kDa polypeptide bands from hemolymph of Anopheles gambiae larvae. This anti- serum was used to screen a cDNA library from A. gambiae fourth-instar larvae. Full-length clones were isolated for two different pro-POs, designated A. gambiae proPO-p1 and proPO- p2, which are 67% identical in nucleotide sequence and 66% identical in deduced amino acid sequence. The A. gambiae pro-PO sequences are more similar to pro-PO from Drosophila melanogaster than to lepidopteran or crustacean pro-PO sequences in the GenBank database. Like the other arthropod pro-POs, the A. gambiae pro-PO sequences lack a signal peptide and have two conserved regions predicted to bind two copper atoms in the active site of the enzyme. The availability of these pro-PO cDNAs should be useful in examining the biochemical differences between A. gambiae strains that are refractory or susceptible to Plasmodium infec- tion, and differ in their ability to encapsulate the parasites.  1997 Elsevier Science Ltd Mosquito Hemolymph Encapsulation Antibody INTRODUCTION When a parasite invades an insect’s hemocoel, the insect often responds with an encapsulation reaction in which a layer of melanin is deposited around the parasite. The capsule may also include layers of hemocytes that further sequester the parasite (Nigam et al., 1996). Production of the melanin in such capsules is caused by the action of tyrosinase-type phenol oxidase, which has monophenol monooxygenase (E.C.1.14.18.1) and o-diphenol oxidase (E.C.1.10.3.1) activities. Phenol oxidase produces quin- ones that undergo further enzymatic and non-enzymatic reactions, finally yielding products that polymerize to form melanin (Marmaras et al., 1996; Sugumaran, 1996). *Author for correspondence. Tel: + 1 913 532 6964; Fax: + 1 913 532 7278; E-mail: kanost@ksu.edu. †Department of Biochemistry, Kansas State University, Manhattan, KS 66506, U.S.A. ‡Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, U.S.A. 1 The sequences have been deposited in GenBank under accession num- bers AF004915 and AF004916. 693 The reactive quinone intermediates produced in this pro- cess may be involved in killing the parasite, in addition to their incorporation into melanin. Phenol oxidase exists in hemolymph as a zymogen, pro-phenol oxidase (pro- PO), that can be activated by specific proteolysis. The recognition of certain non-self molecules triggers acti- vation of a serine proteinase that cleaves pro-PO near its amino terminus, to produce active phenol oxidase (So ¨derha ¨ll et al., 1996). The pro-PO activating system is best understood in a crayfish, Pacifasticus leniusculus (So ¨derha ¨ll et al., 1996), and in the silkworm, Bombyx mori (Ashida and Yoshida, 1990; Yasuhara et al., 1995). cDNA clones for pro-PO have recently been isolated from these two species (Aspa ´n et al., 1995; Kawabata et al., 1995) as well as from Manduca sexta (Hall et al., 1995; Jiang et al., 1997) and Drosophila melanogaster (Fujimoto et al., 1995). Activation of pro-PO and the ensuing melanotic encap- sulation are important in the defensive response of mos- quitoes to various parasites (Nigam et al., 1996; Zheng, 1997). The ability of the parasite to avoid or overcome this response may be a key for the successful trans-