Biosynthesis of unusual moth pheromone components involves two different pathways in the navel orangeworm, Amyelois transitella

Abstract

The sex pheromone of the navel orangeworm, Amyelois transitella (Walker) (Lepidoptera: Pyralidae), consists of two different types of components, one type including (11Z,13Z)-11,13-hexadecadienal (11Z,13Z-16:Ald) with a terminal functional group containing oxygen, similar to the majority of moth pheromones reported, and another type including the unusual long-chain pentaenes, (3Z,6Z,9Z,12Z,15Z)-3,6,9,12,15-tricosapentaene (3Z,6Z,9Z,12Z,15Z-23:H) and (3Z,6Z,9Z,12Z,15Z)- 3,6,9,12,15-pentacosapentaene (3Z,6Z,9Z,12Z,15Z-25:H). After decapitation of females, the titer of 11Z,13Z-16:Ald in the pheromone gland decreased significantly, whereas the titer of the pentaenes remained unchanged. Injection of a pheromone biosynthesis activating peptide (PBAN) into the abdomens of decapitated females restored the titer of 11Z,13Z-16:Ald and even increased it above that in intact females, whereas the titer of the pentaenes in the pheromone gland was not affected by PBAN injection. In addition to common fatty acids, two likely precursors of 11Z,13Z-16:Ald, i.e., (Z)-11-hexadecenoic and (11Z,13Z)-11,13-hexadecadienoic acid, as well as traces of (Z)-6-hexadecenoic acid, were found in gland extracts. In addition, pheromone gland lipids contained (5Z,8Z,11Z,14Z,17Z)-5,8,11,14,17-icosapentaenoic acid, which also was found in extracts of the rest of the abdomen. Deuterium-labeled fatty acids, (16,16,16-D(3))-hexadecanoic acid and (Z)-[13,13,14,14,15,15,16,16,16-D(9)]-11-hexadecenoic acid, were incorporated into 11Z,13Z-16:Ald after topical application to the sex pheromone gland coupled with abdominal injection of PBAN. Deuterium label was incorporated into the C(23) and C(25) pentaenes after injection of (9Z,12Z,15Z)- [17,17,18,18,18-D(5)]-9,12,15-octadecatrienoic acid into 1-2 d old female pupae. These labeling results, in conjunction with the composition of fatty acid intermediates found in pheromone gland extracts, support different pathways leading to the two pheromone components. 11Z,13Z-16:Ald is probably produced in the pheromone gland by Delta11 desaturation of palmitic acid to 11Z-16:Acid followed by a second desaturation to form 11Z,13Z-16:Acid and subsequent reduction and oxidation. The production of 3Z,6Z,9Z,12Z,15Z-23:H and 3Z,6Z,9Z,12Z,15Z-25:H may take place outside the pheromone gland, and appears to start from linolenic acid, which is elongated and desaturated to form (5Z,8Z,11Z,14Z,17Z)-5,8,11,14,17-icosapentaenoic acid, followed by two or three further elongation steps and finally reductive decarboxylation.

Fig. 1

Titers of 11Z,13Z-16:Ald, 3Z,6Z,9Z,12Z,15Z-23:H, and3Z,6Z,9Z,12Z,15Z-25:H in pheromone gland extracts from Amyelois transitella intact females, decapitated females injected with saline, and decapitated females injected with 10 pmol PBAN in saline (PBAN). For each column color, significant differences between means were determined by Student’s t-tests (“*”: P < 0.05; “**”: P < 0.01)

Fig. 2

Total ion chromatograms of the fatty acid compositions (as the methyl esters) of representative pheromone gland (a) and abdominal tissue (b) extracts (HP-1MS capillary column). Fatty acid methyl esters are coded with a number indicating chain length, followed by a number indicating the number of double bonds, with the positions and geometries given if known. Peak numbers correspond to those listed in Table 2. The omega ion at m/z 108, diagnostic for a 3,6,9-triene motif, and the alpha ion at m/z 180, diagnostic for a 5,8,11-triene motif counting from the other end of the chain are indicated in the mass spectrum of the C20:5 acid methyl ester (c). The weak molecular ion at m/z 316 was not detected in full scan mode, but it was detected in the more sensitive selected ion monitoring mode, at a same retention time as the two diagnostic ions at m/z 79 and 108

Fig. 3

Representative sections of the total ion chromatograms (TIC) of fatty acid methyl ester DMDS adducts, analyzed on an InnoWax capillary column. a Section of TIC from pheromone gland fatty acid methyl esters. b Selected ion monitoring GC-MS analyses of monounsaturated C16 fatty acid methyl ester DMDS adducts. Double bond positions were identified from the diagnostic ions from cleavage of the C-C bond between the two carbons bearing methylthiol groups: Δ6-16, 175/187/362; Δ9-16, 217/145/362; Δ11-16, 245/117/362. c Confirmation of the “cis” configuration in the insect-produced Δ6-16 monounsaturated fatty acid methyl ester by comparison of the chromatographic retention times of the DMDS adducts, using the diagnostic ions at m/z 175 and 187. a: Δ6-16:Me DMDS adduct in insect abdomen extract; b: authentic 6Z-16:Me DMDS adduct; c: authentic 6E-16:Me DMDS adduct. d Full scan mass spectrum of the Δ6-16 monounsaturated fatty acid methyl ester DMDS adduct from insect abdomen extract (a) and authentic 6Z-16:Me DMDS adduct (b)

Fig. 4

Selected ion monitoring chromatograms showing incorporation of labeled fatty acid precursors into 11Z,13Z-16:Ald, using ions 239 and 243 to monitor D₃-11Z,13Z-16:Ald and D₇-11Z,13Z-16:Ald, respectively. a Deuterium label incorporation into D₃-11Z,13Z-16:Ald after topical application of D₃-16:Acid in DMSO onto the pheromone gland; b Deuterium label incorporation into D₇-11Z,13Z-16:Ald after topical application of D₉-11Z-16:Acid in DMSO onto the pheromone gland; c diagnostic ions for unlabeled 11Z,13Z-16:Ald, D₃-labeled 11Z,13Z-16:Ald and D₇-labeled 11Z,13Z-16:Ald

Fig. 5

Selected ion monitoring chromatograms showing incorporation of labeled fatty acid precursors into 3Z,6Z,9Z,12Z,15Z-23:H and 3Z,6Z,9Z,12Z,15Z-25:H after abdominal injection of D₅-linolenic acid into 1–2 d old pupae. a Deuterium label incorporation into D₅-3Z,6Z,9Z,12Z,15Z-23:H; b Deuterium label incorporation into D₅-3Z,6Z,9Z,12Z,15Z-25:H; c Ions 108 and 178, and ions 108 and 206 were used to monitor unlabeled 3Z,6Z,9Z,12Z,15Z-23:H and 3Z,6Z,9Z,12Z,15Z-25:H, respectively. Ions 113 and ion 178 were used to monitor D₅-3Z,6Z,9Z,12Z,15Z-23:H, and ions 113 and 206 were used to monitor D₅-3Z,6Z,9Z,12Z,15Z-25:H. Because of the small amounts of both native and labeled 3Z,6Z,9Z,12Z,15Z-25:H, the 206 ion was not detected in labeled D₅-3Z,6Z,9Z,12Z,15Z-25:H

Fig. 6

Proposed biosynthetic pathway for the Type I 11Z,13Z-16:Ald sex pheromone component produced by female Amyelois transitella. “–2H” represents desaturation to form a double bond, “Red” indicates functional group reduction and “Oxi” indicates terminal oxidation

Fig. 7

Proposed biosynthetic pathway for the Type II pentaene sex pheromone components produced by female Amyelois transitella. “–2H” represents desaturation to form a double bond, “+2C” represents one cycle of chain elongation by two carbons, “–COOH” means decarboxylation