Pheromone biosynthetic pathways: PBAN-regulated rate-limiting steps and differential expression of desaturase genes in moth species.

We combine the use of labeled precursors with enzyme inhibitors to decipher the biosynthetic pathway of pheromone biosynthesis and the rate-limiting step/s that are regulated by pheromone biosynthesis activating neuropeptide (PBAN). We demonstrate that Plodia interpunctella is able to utilize hexadecanoic acid, and to a lesser extent tetradecanoic acid, for the biosynthesis of the main pheromone component (Z,E)-9,12-tetradecadienyl acetate. This indicated that the main pathway involves a Delta11 desaturase, chain shortening, followed by a Delta12 desaturase, but that a functional Delta9 desaturase could also be utilized. Using reverse transcription-quantitative real-time polymerase chain reaction (RT-QPCR) we distinguish two out of nine possible desaturase gene transcripts in P. interpunctella that are expressed at the highest levels. The rate-limiting step for PBAN-stimulation was studied in two moth species so as to compare the biosynthesis of a diene (P. interpunctella) and a monoene (Helicoverpa armigera) main pheromone component. In both species, incorporation of label from the (13)C sodium acetate precursor was activated by PBAN whereas no stimulatory action was observed in the incorporation of the precursors: (13)C malonyl coenzyme A; hexadecanoic 16,16,16-(2)H(3) or tetradecanoic 14,14,14-(2)H(3) acids. The acetyl coenzyme A carboxylase (ACCase) inhibitor, Tralkoxydim, inhibited the PBAN-stimulation of incorporation of stable isotope whereas the fatty-acyl reductase inhibitor, Mevastatin, failed to influence the stimulatory action of PBAN. These results provide irrefutable support to the hypothesis that PBAN affects the production of malonyl coenzyme A from acetate by the action of ACCase in the pheromone glands of these moths.

Desaturases from the spotted fireworm moth (Choristoneura parallela) shed light on the evolutionary origins of novel moth sex pheromone desaturases.

Six acyl-CoA desaturase-encoding cDNAs from mRNA isolated from the spotted fireworm moth, Choristoneura parallela (Lepidoptera: Tortricidae) were characterized and assayed for functionality. The expression levels of these cDNAs were determined in the pheromone gland and fat body by real-time PCR and the resulting patterns are in line with results from published studies on other moth sex pheromone desaturases. The cDNAs were found to correspond to six genes. Using both biochemical and phylogenetic analyses, four of these were found to belong to previously characterized desaturase functional groups [the Delta 10,11, the Delta 9 (16>18) and the Delta 9 (18>16) groups]. A desaturase highly expressed in the pheromone gland was a novel E11 desaturase that was specific to 14-carbon precursor acids. The fifth gene [CpaZ9(14-26)] was found to display a novel Z9 activity indicating that it belongs to a new Delta 9 functional group, whereas the sixth gene was determined to be nonfunctional with respect to desaturase activity. In accordance with previous studies, we find that desaturases of the Delta 10,11 and Delta 14 groups, which are the fastest evolving desaturases and possess the novel pheromone biosynthetic function, are expressed primarily in the pheromone gland whereas all other desaturases, which do not possess the novel reproductive function, evolve more slowly and display the ancestral metabolic function and pattern of gene expression.

Expression and evolution of delta9 and delta11 desaturase genes in the moth Spodoptera littoralis.

Desaturation of fatty acids is a key reaction in the biosynthesis of moth sex pheromones. The main component of Spodoptera littoralis sex pheromone blend is produced by the action of Delta11 and Delta9 desaturases. In this article, we report on the cloning of four desaturase-like genes in this species: one from the fat body (Sls-FL1) and three (Sls-FL2, Sls-FL3 and Sls-FL4) from the pheromone gland. By means of a computational/phylogenetic method, as well as functional assays, the desaturase gene products have been characterized. The fat body gene expressed a Delta9 desaturase that produced (Z)-9-hexadecenoic and (Z)-9-octadecenoic acids in a (1:4.5) ratio, whereas the pheromone gland Sls-FL2 expressed a Delta9 desaturase that produced (Z)-9-hexadecenoic and (Z)-9-octadecenoic acids in a (1.5:1) ratio. Although both Delta9 desaturases produced (Z)-9-tetradecenoic acid from myristic acid, transformed yeast grown in the presence of a mixture of myristic and (E)-11-tetradecenoic acids produced (Z,E)-9,11-tetradecadienoic acid, but not (Z)-9-tetradecenoic acid. The Sls-FL3 gene expressed a protein that produced a mixture of (E)-11-tetradecenoic, (Z)-11-tetradecenoic, (Z)-11-hexadecenoic and (Z)-11-octadecenoic acids in a 5:4:60:31 ratio. Despite having all the characteristics of a desaturase gene, no function could be found for Sls-FL4.

Gene characterized for membrane desaturase that produces (E)-11 isomers of mono- and diunsaturated fatty acids.

Moth species have evolved integral membrane desaturases that exhibit a wide diversity in substrate specificity, as well as in regiospecificity and stereospecificity of the unsaturated products. We report here the cloning and expression of a single desaturase from the sex pheromone gland of the light brown apple moth, Epiphyas postvittana, that makes E11 isomers of monounsaturated (E11-16 and E11-14) fatty acids and a diunsaturated (E9,E11-14) fatty acid. In the pheromone gland, the monoene precursor is made available by beta oxidation of E11-16 acid with a subsequent two-carbon loss to E9-14 acid. A functional assay using a baculovirus expression system required addition of myristic acid and E9-14 acid precursors to demonstrate the unusual regiospecificity and stereospecificity of this desaturase. The amino acid sequence of this desaturase has approximately 61% identity to that of Z11-desaturases from two other insect species, and only approximately 48% identity to the metabolic Z9-desaturases in those species. A pheromone-gland Z9-desaturase gene also was found with the light brown apple moth that differed in its deduced amino acid sequence (66% identity) with the metabolic Z9-desaturase from fat body in this species.