Insects use hydrocarbons as cuticular waterproofing agents and as contact pheromones. Although their biosynthesis from fatty acyl precursors is well established, the last step of hydrocarbon biosynthesis from long-chain fatty aldehydes has remained mysterious. We show here that insects use a P450 enzyme of the CYP4G family to oxidatively produce hydrocarbons from aldehydes. Oenocyte-directed RNAi knock-down of Drosophila CYP4G1 or NADPH-cytochrome P450 reductase results in flies deficient in cuticular hydrocarbons, highly susceptible to desiccation, and with reduced viability upon adult emergence. The heterologously expressed enzyme converts C 18 -trideuterated octadecanal to C 17 -trideuterated heptadecane, showing that the insect enzyme is an oxidative decarbonylase that catalyzes the cleavage of long-chain aldehydes to hydrocarbons with the release of carbon dioxide. This process is unlike cyanobacteria that use a nonheme diiron decarbonylase to make alkanes from aldehydes with the release of formate. The unique and highly conserved insect CYP4G enzymes are a key evolutionary innovation that allowed their colonization of land. Insects are the largest group of extant terrestrial organisms. As their crustacean ancestors moved from aquatic to terrestrial environments, insects were confronted with a new, dry frontier. Insects solved the ecophysiological problem of how to restrict water loss to prevent dessication by depositing long-chain hydrocarbons as essential waterproofing components on their epicuticle (1). These diverse chemicals now serve many additional functions, particularly in defense, reproduction, and communication (2). In flies, cuticular hydrocarbons (CHs) are a complex blend of long-chain (∼C21-C37+) alkanes and alkenes that serve as species-and sex-specific semiochemicals, and some components are sex pheromones. CHs also serve in nest mate recognition by social insects and as trail pheromones in ants; the complexity of their blend can be useful in taxonomic discrimination of mosquitoes. Much is known about the biosynthesis of CHs from fatty acids in insects, involving a complex network of fatty acid synthases, elongases, and desaturases, leading to very long-chain acyl-CoA thioesters. These are converted by acyl-CoA reductases to aldehydes that serve as substrates for the last oxidative decarbonylation step (2) (Fig. 1). The single carbon chain-shortening conversion of precursor aldehydes to hydrocarbons is catalyzed by a P450 enzyme, P450hyd, with release of CO 2 (3), but this enzyme has not yet been identified. The only known decarbonylase that has recently been described occurs in cyanobacteria that use a nonheme diiron enzyme (4-8) to catalyze the last step (Fig. 1). Insect CH are synthesized in large ectodermally derived cells (9) called oenocytes (10-12), and are then shuttled by hemolymph lipophorin (13,14) to the cuticle, where they are deposited on the outer epicuticular layer. Here we identify P450hyd as CYP4G1 in Drosophila melanogaster, and show that the enzyme is massively coexpresse...
The role of cuticle changes in insecticide resistance in the major malaria vector Anopheles gambiae was assessed. The rate of internalization of 14 C deltamethrin was significantly slower in a resistant strain than in a susceptible strain. Topical application of an acetone insecticide formulation to circumvent lipid-based uptake barriers decreased the resistance ratio by ∼50%. Cuticle analysis by electron microscopy and characterization of lipid extracts indicated that resistant mosquitoes had a thicker epicuticular layer and a significant increase in cuticular hydrocarbon (CHC) content (∼29%). However, the CHC profile and relative distribution were similar in resistant and susceptible insects. The cellular localization and in vitro activity of two P450 enzymes, CYP4G16 and CYP4G17, whose genes are frequently overexpressed in resistant Anopheles mosquitoes, were analyzed. These enzymes are potential orthologs of the CYP4G1/2 enzymes that catalyze the final step of CHC biosynthesis in Drosophila and Musca domestica, respectively. Immunostaining indicated that both CYP4G16 and CYP4G17 are highly abundant in oenocytes, the insect cell type thought to secrete hydrocarbons. However, an intriguing difference was indicated; CYP4G17 occurs throughout the cell, as expected for a microsomal P450, but CYP4G16 localizes to the periphery of the cell and lies on the cytoplasmic side of the cell membrane, a unique position for a P450 enzyme. CYP4G16 and CYP4G17 were functionally expressed in insect cells. CYP4G16 produced hydrocarbons from a C18 aldehyde substrate and thus has bona fide decarbonylase activity similar to that of dmCYP4G1/2. The data support the hypothesis that the coevolution of multiple mechanisms, including cuticular barriers, has occurred in highly pyrethroid-resistant An. gambiae. malaria | insecticide resistance | hydrocarbons | mosquito cuticle | cytochrome P450
Geranyl diphosphate synthase (GPPS) catalyzes the condensation of dimethylallyl diphosphate and isopentenyl diphosphate to form geranyl diphosphate. Geranyl diphosphate is the precursor of monoterpenes, a large family of natural occurring C 10 compounds predominately found in plants. Similar to plants but unique to animals, some bark beetle genera (Coleoptera: Scolytidae) produce monoterpenes that function in intraspecific chemical communication as aggregation and dispersion pheromones. The release of monoterpene aggregation pheromone mediates host colonization and mating. It has been debated whether these monoterpene pheromone components are derived de novo through the mevalonate pathway or result from simple modifications of dietary precursors. The data reported here provide conclusive evidence for de novo biosynthesis of monoterpene pheromone components from bark beetles. We describe GPPS in the midgut tissue of pheromone-producing male Ips pini. GPPS expression levels are regulated by juvenile hormone III, similar to other mevalonate pathway genes involved in pheromone biosynthesis. In addition, GPPS transcript is almost exclusively expressed in the anterior midgut of male I. pini, the site of aggregation pheromone biosynthesis. The recombinant enzyme was functionally expressed and produced geranyl diphosphate as its major product. The threedimensional model structure of GPPS shows that the insect enzyme has the sequence structural motifs common to E-isoprenyl diphosphate synthases.isoprenyl diphosphate synthase ͉ monoterpene biosynthesis ͉ ipsdienol
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