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...

Drosophila melanogaster cuticular pheromones consist of unsaturated hydrocarbons with at least one double bond in position 7: 7 tricosene (T) in males and 7,11 heptacosadiene (HD) in females. However, in many African populations like the Tai strain, females possess low levels of 7,11 HD and high levels of its positional isomer 5,9 HD. We have previously isolated a desaturase gene, desat1, from the Canton-S strain (CS), a 7,11 HD-2-rich morph of D. melanogaster. This desaturase is located in 87C, a locus that has been involved in the difference between 7,11 HD and 5,9 HD morphs. Therefore, we have searched for different desaturase isoforms in both strains. We first cloned desat1 in the Tai strain and report here functional expression of desat1 in CS and Tai. In both strains, the Desat1 enzymes have the same ⌬9 specificity and preferentially use palmitate as a substrate, leading to the synthesis of 7 fatty acids. Also found was a desaturase sequence, named desat2, with a homologous catalytic domain and a markedly different N-terminal domain compared with desat1. In CS genome, it lies 3.8 kb upstream of desat1 and is not transcribed in either sex. In the Tai strain, it is expressed only in females and acts preferentially on myristate, leading to the synthesis of 5 fatty acids. We suggest, therefore, that desat2 might play a control role in the biosynthesis of 5,9 HD hydrocarbons in Tai females and could explain the dienic hydrocarbon polymorphism in D. melanogaster.acyl-Coa desaturase ͉ pheromone biosynthesis U nsaturated fatty acids are structural components of membrane lipids. In animals, fatty acids are desaturated by a membrane-bound enzyme complex involving cytochrome b5, cytochrome b5 reductase, and a desaturase (1-3). Desaturases catalyze the introduction of a double bond into the hydrocarbon chain of a fatty acyl-CoA, at a position determined by the enzyme specificity. A low number of animal desaturases have been both molecularly and functionally characterized by heterologous expression in yeast or in Arabidopsis: ⌬9 and ⌬6 desaturases from rat (4, 5), ⌬6 desaturase from mouse (6), 3, ⌬6, and ⌬5 desaturases from Caenorhabditis elegans (7-9), and ⌬11 and ⌬9 desaturases from the moth Trichoplusia ni (10, 11).In the Drosophila melanogaster subgroup, flies use double bonds in cuticular hydrocarbons among other structural parameters to signal sex or species (12, 13). Males have high levels of monoenes, whereas females are rich in dienes, for example 7-tricosene (7T) and 7,11 heptacosadiene (7,11 HD) in the Canton-S strain (CS) (14).Studies in D. melanogaster strongly suggest that the biosynthesis of male monoenes and female dienes follows the same elongation-decarboxylation mechanism characterized in Musca domestica (15, 16) and shares early steps up to vaccenic acid, a common precursor (refs. 17 and 18; Fig. 1). The involvement of a desaturase with a ⌬9 specificity to introduce the common double bond has also been hypothesized (18). We have previously isolated a CS D. melanogaster desaturase gene, desat1, expre...

Drosophila melanogaster produces sexually dimorphic cuticular pheromones that are a key component of the courtship behavior leading to copulation. These molecules are hydrocarbons, with lengths of 23 and 25 carbons in males (mainly with one double bond) and 27 and 29 carbons in females (mainly with two double bonds). Here, we describe an elongase gene, eloF, with female-biased expression. The 771-bp ORF encodes a 257-aa protein that shows the highest sequence identity with mouse SSC1 elongase (33%). The activity of the cDNA expressed in yeast was elongation of saturated and unsaturated fatty acids up to C30. RNAi knockdown in Drosophila led to a dramatic modification of female hydrocarbons, with decreased C29 dienes and increased C25 dienes accompanied by a modification of several courtship parameters: an increase in copulation latency and a decrease in both copulation attempts and copulation. Feminization of the hydrocarbon profile in males by using targeted expression of the transformer gene resulted in high expression levels of eloF, suggesting that the gene is under the control of the sex-determination hierarchy. There is no expression of eloF in Drosophila simulans, which synthesize only C23 and C25 hydrocarbons. These results strongly support the hypothesis that eloF is a crucial enzyme for female pheromone biosynthesis and courtship behavior in D. melanogaster.

Drosophila melanogaster shows sexually dimorphic cuticular hydrocarbons, with monoenes produced in males and dienes produced in females. Here we describe a female-specific desaturase gene, desatF. RNAi knock-down led to a dramatic decrease in female dienes and increase in monoenes paralleled with an increase in copulation latency and a decrease in courtship index and copulation attempts by the males. The desatF gene was also expressed in females from D. sechellia, rich in dienes, but not D. simulans, which produce only monoenes. When hydrocarbons were feminized in D. melanogaster males by targeted expression of the transformer gene, the expression of desatF occurred. These results strongly suggest that desatF is a crucial enzyme for female pheromone biosynthesis and courtship behaviour in D. melanogaster.

Fatty acid (FA) metabolism plays a central role in body homeostasis and related diseases. Thus, FA metabolic enzymes are attractive targets for drug therapy. Mouse studies on Acetyl-coenzymeA-carboxylase (ACC), the rate-limiting enzyme for FA synthesis, have highlighted its homeostatic role in liver and adipose tissue. We took advantage of the powerful genetics of Drosophila melanogaster to investigate the role of the unique Drosophila ACC homologue in the fat body and the oenocytes. The fat body accomplishes hepatic and storage functions, whereas the oenocytes are proposed to produce the cuticular lipids and to contribute to the hepatic function. RNA–interfering disruption of ACC in the fat body does not affect viability but does result in a dramatic reduction in triglyceride storage and a concurrent increase in glycogen accumulation. These metabolic perturbations further highlight the role of triglyceride and glycogen storage in controlling circulatory sugar levels, thereby validating Drosophila as a relevant model to explore the tissue-specific function of FA metabolic enzymes. In contrast, ACC disruption in the oenocytes through RNA–interference or tissue-targeted mutation induces lethality, as does oenocyte ablation. Surprisingly, this lethality is associated with a failure in the watertightness of the spiracles—the organs controlling the entry of air into the trachea. At the cellular level, we have observed that, in defective spiracles, lipids fail to transfer from the spiracular gland to the point of air entry. This phenotype is caused by disrupted synthesis of a putative very-long-chain-FA (VLCFA) within the oenocytes, which ultimately results in a lethal anoxic issue. Preventing liquid entry into respiratory systems is a universal issue for air-breathing animals. Here, we have shown that, in Drosophila, this process is controlled by a putative VLCFA produced within the oenocytes.

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Drosophila melanogaster is polymorphic for the major cuticular hydrocarbon of females. In most populations this hydrocarbon is 7,11-heptacosadiene, but females from Africa and the Caribbean usually possess low levels of 7,11-heptacosadiene and high quantities of its position isomer 5,9-heptacosadiene. Genetic analysis shows that the difference between these two morphs is due to variation at a single segregating factor located on the right arm of chromosome 3 near map position 51.5 and cytological position 87C-D. This is precisely the position of a desaturase gene previously sequenced using primers derived from yeast and mouse, and localized by in situ hybridization to the polytene chromosomes of D. melanogaster. Alleles of this desaturase gene may therefore be responsible for producing the two hydrocarbon morphs. Mating tests following the transfer of these isomers between females of the two morphs show that, in contrast to previous studies, the hydrocarbon profiles have no detectable effect on mating behaviour or sexual isolation.

D. simulans and D. melanogaster present two types of polymorphism in their cuticular hydrocarbon (HC) composition. Especially both sexes of D. simulans, and D. melanogaster males display 7-tricosene (7T) as the major compound type [7T]s and [7T]m, or 7-pentacosene (7P) [7P]s and [7P]m. D. melanogaster females display 7,11-heptacosadiene (7,11HD) as the major compound: [7,11HD]m, or 5,9-heptacosadiene (5,9HD): [5,9HD]m. The [7P]s, [7P]m and [5,9HD]m are mainly present in central Africa. A significant correlation was found between latitude and the proportion of compounds with 23 and 25 carbon atoms, especially 7T and 7P in both sexes of D. melanogaster. [7P]m type of D. melanogaster, characterized with an excess of C25 compounds, presents a higher resistance against desiccation than [7T]m type, where C23 compounds are more abundant. These differences can be correlated with calculated HC fusion temperatures. Moreover, increasing the breeding temperature from 18 to 29 degrees C induces in D. melanogaster males an increase in 25C compounds and a decrease in 23C compounds, but the opposite effect in D. simulans. A mathematical model of biosynthesis, based on kinetics of elongation and decarboxylation enzymes, suggests that a simple variation of the efficiency of an elongation enzyme may account for the differences observed between the [7T]m and [7P]m types of D. melanogaster and [7T]s and [7P]s types D. simulans. Finally on the basis of the geographical distribution of the HC types of both Drosophila species, an evolutionary dispersal pathway is proposed and discussed in relation to the environment and reproductive behavior.