Phylogenetic analysis of heme peroxidases (HPXs) of Culicidae and other insects revealed six highly conserved ancient HPX lineages, each of which originated by gene duplication prior to the most recent common ancestor (MRCA) of Hemimetabola and Holmetabola. In addition, culicid HPX7 and HPX12 arose by gene duplication after the MRCA of Culicidae and Drosophilidae, while HPX2 orthologs were not found in any other order analyzed except Diptera. Within Diptera, HPX2, HPX7, and HPX12 were relatively poorly conserved at the amino acid level in comparison to the six ancient lineages. The genome of Anopheles gambiae included genes ecoding five proteins (HPX10, HPX11, HPX13, HXP14, and HPX15) without ortholgs in other genomes analyzed. Overall, gene expression patterns did not seem to reflect phylogenetic relationships, but genes that evolved rapidly at the amino acid sequence level tended to have divergent expression patterns as well. The uniquely high level of duplication of HPXs in A. gambiae may have played a role in coevolution with malaria parasites. 1. Introduction The production of nitric oxide (NO) is an important immune defense mechanism against cellular microorganisms in insects and other invertebrates [1]. Nitric oxide synthase (NOS), encoded by a single gene in insect genomes sequenced to date [2], is the major enzyme involved in NO production, but the full pathway of NO production is only beginning to be understood. In the mosquito Anopheles gambiae (Diptera: Culicidae) when infected by malaria parasites (Apicomplexa: Plasmodium), a heme peroxidase (HPX2) and NADPH oxidase (NOX5) were found to play a crucial role in potentiating NO in antiparasite defense [3]. NOX5 is represented by a single ortholog in insects, but HPX2 is a member of a multigene heme peroxidase family in A. gambiae and other insects [3–5]. Other mosquito heme peroxidases (HPXs) of known function include those expressed in the salivary glands of female A. gambiae and A. albimanus [6, 7] and one involved in the catalysis of protein-crosslinking in the chorion of Aedes aegypti eggs [8]. In another member of Diptera, the fruit fly Drosophila melanogaster, heme peroxidases have been implicated in chorion assembly and other developmental processes [9, 10]. For nearly 5000 orthologous genes, Waterhouse and colleagues [4] compared the amino acid sequence distance between D. melanogaster and A. gambiae with that between D. melanogaster and Ae. aegypti. On average genes with known immune function showed a greater level of amino acid sequence divergence than other genes, but certain
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