Biosynthesis and PBAN-regulated transport of pheromone polyenes in the winter moth, Operophtera brumata.

The trienoic and tetraenoic polyenes, (3Z,6Z,9Z)-3,6,9-nonadecatriene, (3Z,6Z,9Z)-3,6,9-henicosatriene, and (3Z,6Z,9Z)-1,3,6,9-henicosatetraene were found in the abdominal cuticle and pheromone gland of the winter moth Operophtera brumata L. (Lepidoptera: Geometridae), in addition to the previously identified single component sex pheromone (3Z,6Z,9Z)-1,3,6,9-nonadecatetraene. The pheromone biosynthesis activating neuropeptide (PBAN) is involved in the regulation of polyene transport from abdominal cuticle to the pheromone gland. In vivo deuterium labeling experiments showed that (11Z,14Z,17Z)-11,14,17-icosatrienoic acid, the malonate elongation product of linolenic acid, (9Z,12Z,15Z)-9,12,15-octadecatrienoic acid, is used to produce (3Z,6Z,9Z)-3,6,9-nonadecatriene and (3Z,6Z,9Z)-1,3,6,9-nonadecatetraene.

Terminal fatty-acyl-CoA desaturase involved in sex pheromone biosynthesis in the winter moth (Operophtera brumata).

The winter moth (Operophtera brumata L., Lepidoptera: Geometridae) utilizes a single hydrocarbon, 1,Z3,Z6,Z9-nonadecatetraene, as its sex pheromone. We tested the hypothesis that a fatty acid precursor, Z11,Z14,Z17,19-nonadecanoic acid, is biosynthesized from α-linolenic acid, through chain elongation by one 2-carbon unit, and subsequent methyl-terminus desaturation. Our results show that labeled α-linolenic acid is indeed incorporated into the pheromone component in vivo. A fatty-acyl-CoA desaturase gene that we found to be expressed in the abdominal epidermal tissue, the presumed site of biosynthesis for type II pheromones, was characterized and expressed heterologously in a yeast system. The transgenic yeast expressing this insect derived gene could convert Z11,Z14,Z17-eicosatrienoic acid into Z11,Z14,Z17,19-eicosatetraenoic acid. These results provide evidence that a terminal desaturation step is involved in the winter moth pheromone biosynthesis, prior to the decarboxylation.

Identification of components of the female sex pheromone of the Simao pine caterpillar moth, Dendrolimus kikuchii Matsumura.

The pine caterpillar moth, Dendrolimus kikuchii Matsumura (Lepidoptera: Lasiocampidae), is a pest of economic importance on pine in southwest China. Three active compounds were detected during analyses of solvent extracts and effluvia sampled by solid phase microextraction (SPME) from virgin female D. kikuchii using gas chromatography (GC) coupled with electroantennographic (EAG) recording with antennae from a male moth. The compounds were identified as (5Z,7E)-5,7-dodecadien-1-yl acetate (Z5,E7-12:OAc), (5Z,7E)-5,7-dodecadien-1-ol (Z5,E7-12:OH), and (5Z)-5-dodecenyl acetate (Z5-12:OAc) by comparison of their GC retention indices, mass spectra, and EAG activities with those of synthetic standards. Microchemical reactions of gland extracts provided further information confirming the identifications of the three components. Solvent extractions and SPME samples of pheromone effluvia from virgin calling females provided 100:18:0.6 and 100:7:1 ratios of Z5,E7-12:OAc:Z5,E7-12:OH:Z5-12:OAc, respectively. Field behavioral assays showed that Z5,E7-12:OAc and Z5,E7-12:OH were essential for attraction of male D. kikuchii moths. However, the most attractive blend contained these three components in a 100:20:25 ratio in a gray rubber septa. Our results demonstrated that the blend of Z5,E7-12:OAc, Z5,E7-12:OH, and Z5-12:OAc comprise the sex pheromone of D. kikuchii. The optimized three-component lure blend is recommended for monitoring D. kikuchii infestations.

Neofunctionalization in an ancestral insect desaturase lineage led to rare Δ6 pheromone signals in the Chinese tussah silkworm.

The Chinese tussah silkworm, Antheraea pernyi (Lepidoptera: Saturniidae) produces a rare dienoic sex pheromone composed of (E,Z)-6,11-hexadecadienal, (E,Z)-6,11-hexadecadienyl acetate and (E,Z)-4,9-tetradecadienyl acetate, and for which the biosynthetic routes are yet unresolved. By means of gland composition analyses and in vivo labeling we evidenced that pheromone biosynthesis towards the immediate dienoic gland precursor, the (E,Z)-6,11-hexadecadienoic acid, involves desaturation steps with Δ(6) and Δ(11) regioselectivity. cDNA cloning of pheromone gland desaturases and heterologous expression in yeast demonstrated that the 6,11-dienoic pheromone is generated from two biosynthetic routes implicating a Δ(6) and Δ(11) desaturase duo albeit with an inverted reaction order. The two desaturases first catalyze the formation of the (E)-6-hexadecenoic acid or (Z)-11-hexadecenoic acid, key mono-unsaturated biosynthetic intermediates. Subsequently, each enzyme is able to produce the (E,Z)-6,11-hexadecadienoic acid by accommodating its non-respective mono-unsaturated product. Besides elucidating an unusually flexible pheromone biosynthetic pathway, our data provide the first identification of a biosynthetic Δ(6) desaturase involved in insect mate communication. The occurrence of this novel Δ(6) desaturase function is consistent with an evolutionary scenario involving neo-functionalization of an ancestral desaturase belonging to a gene lineage different from the Δ(11) desaturases commonly involved in moth pheromone biosynthesis.

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

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.

Elucidation of the sex-pheromone biosynthesis producing 5,7-dodecadienes in Dendrolimus punctatus (Lepidoptera: Lasiocampidae) reveals Delta 11- and Delta 9-desaturases with unusual catalytic properties.

Sex pheromones produced by female moths of the Lasiocampidae family include conjugated 5,7-dodecadiene components with various oxygenated terminal groups. Here we describe the molecular cloning, heterologous expression and functional characterization of desaturases associated with the biosynthesis of these unusual chemicals. By homology-based PCR screening we characterized five cDNAs from the female moth pheromone gland that were related to other moth desaturases, and investigated their role in the production of the (Z)-5-dodecenol and (Z5,E7)-dodecadienol, major pheromone constituents of the pine caterpillar moth, Dendrolimus punctatus. Functional expression of two desaturase cDNAs belonging to the Delta 11-subfamily, Dpu-Delta 11(1)-APSQ and Dpu-Delta 11(2)-LPAE, showed that they catalysed the formation of unsaturated fatty acyls (UFAs) that can be chain-shortened by beta-oxidation and subsequently reduced to the alcohol components. A first (Z)-11-desaturation step is performed by Dpu-Delta 11(2)-LPAE on stearic acid that leads to (Z)-11-octadecenoic acyl, which is subsequently chain shortened to the (Z)-5-dodecenoic acyl precursor. The Dpu-Delta 11(1)-APSQ desaturase had the unusual property of producing Delta 8 mono-UFA of various chain lengths, but not when transformed yeast were grown in presence of (Z)-9-hexadecenoic acyl, in which case the biosynthetic intermediate (Z9,E11)-hexadecadienoic UFA was produced. In addition to a typical Z9 activity, a third transcript, Dpu-Delta 9-KPSE produced E9 mono-UFAs of various chain lengths. When provided with the (Z)-7-tetradecenoic acyl, it formed the (Z7,E9)-tetradecadienoic UFA, another biosynthetic intermediate that can be chain-shortened to (Z5,E7)-dodecadienoic acyl. Both Dpu-Delta 11(1)-APSQ and Dpu-Delta 9-KPSE thus exhibited desaturase activities consistent with the biosynthesis of the dienoic precursor. The combined action of three desaturases in generating a dienoic sex-pheromone component emphasizes the diversity and complexity of chemical reactions that can be catalysed by pheromone biosynthetic fatty-acyl-CoA desaturases in moths.

Genetic basis of sex pheromone blend difference between Helicoverpa armigera (Hübner) and Helicoverpa assulta (Guenée) (Lepidoptera: Noctuidae).

The two closely related moth species, Helicoverpa armigera and H. assulta, are sympatric in China. Both species use a mixture of (Z)-11-hexadecenal (Z11-16:Ald) and (Z)-9-hexadecenal (Z9-16:Ald) as their sex pheromones but in widely different ratios. Hybridization and backcrossing experiments between H. armigera and H. assulta were conducted and sex pheromone compositions of the parent species, their F(1) hybrids and backcrosses were compared to study the genetic basis of the production of their sex pheromone blend composition. Results show that the difference in sex pheromone blend ratios of these Helicoverpa species is mainly controlled by an autosomal locus with two alleles, with the allele from H. armigera being almost completely dominant over that derived from H. assulta.

Female sex pheromone of the Yunnan pine caterpillar moth Dendrolimus houi: first (E,Z)-isomers in pheromone components of Dendrolimus spp.

The Yunnan pine caterpillar Dendrolimus houi Lajonquière is a serious defoliator of coniferous forests in southwestern China. Gas chromatography-electroantennography (GC-EAG) analyses of extracts of female sex pheromone glands of D. houi moths revealed the presence of three compounds eliciting antennal responses. These were identified as (5E,7Z)-5,7-dodecadien-1-ol (E5,Z7-12:OH), (5E,7Z)-5,7-dodecadien-1-yl acetate (E5,Z7-12:OAc), and (5E,7Z)-5,7-dodecadienal (E5,Z7-12:Ald) by comparison of their GC retention indices, mass spectra, and EAG activities with those of synthetic standards. Average amounts of E5,Z7-12:OH, E5,Z7-12:OAc, and E5,Z7-12:Ald per calling virgin D. houi female were 14.7 +/- 12.9 ng (+/- SD), 5.8 +/- 5.4 ng, and 0.8+1.4 ng, respectively, in a ratio of 100:39.7:5.6. These three components were also collected from the headspace of calling virgin female moths by solid-phase microextraction (SPME). In addition, trace quantities of (Z)-5-dodecen-1-ol (Z5-12:OH), (5Z,7E)-5,7-dodecadien-1-ol (Z5,E7-12:OH), (5E,7E)-5, 7-dodecadien-1-ol (E5,E7-12:OH), (5Z,7E)-5,7-dodecadien-1-yl acetate (Z5,E7-12:OAc), (5Z,7Z)-5,7-dodecadien-1-yl acetate (Z5,Z7-12:OAc), and (5E,7E)-5,7-dodecadien-1-yl acetate (E5,E7-12:OAc) were tentatively identified in female pheromone gland extracts by selected ion monitoring GC-MS. Field trapping experiments showed that E5,Z7-12:OH, E5,Z7-12:OAc, and E5,Z7-12:Ald were essential for attraction of male D. houi moths. Traps baited with a 20:1:1 blend (alcohol/acetate/aldehyde) loaded on gray rubber septa were as effective as traps baited with virgin female moths. The optimum ratio of acetate to aldehyde was 1:1, and this ratio was more critical than the ratio of either compound to the alcohol. This represents the first example of (E,Z)-isomers in pheromone blends of Dendrolimus species.

Putative chemical signals about sex, individuality, and genetic background in the preputial gland and urine of the house mouse (Mus musculus).

To explore whether preputial gland secretions and/or urine from the house mouse (Mus musculus) can be used for coding information about sex, individuality, and/or the genetic background of strain [ICR/albino, Kunming (KM), and C57BL/6], we compared the volatile compositions of mouse preputial glands and urine using a combination of dichloromethane extraction and gas chromatography coupled with mass spectrometry (GC-MS). Of the 40 identified compounds in preputial gland secretions, 31 were esters, 2 sesquiterpens, and 7 alcohols. We failed to find any compound unique to a specific sex, individual, or strain. However, many low molecular weight compounds between the sexes, most compounds among individuals, and several compounds among the 3 strains varied significantly in relative ratios. These quantitative differences in preputial gland volatiles (analog coding) are likely to convey information about sex, individual, and the genetic background of mouse strain. We identified 2 new main and male-elevated compounds, 1-hexadecanol (Z=3.676, P=0.000, N=19 in ICR; Z=3.576, P=0.000, N=18) and 1-hexadecanol acetate (Z=3.429, P=0.000, N=19 in ICR; Z=3.225, P=0.001, N=18), which were eluted in GC chromatogram after the 2 sesquiterpens. They might also be potential male pheromones, in addition to the well-known E-beta-farnesene and E,E-alpha-farnesene. Additionally, a few compounds including 1-hexadecanol also varied with strains and might also code for genetic information. Of the 9 identified volatile compounds in male urine, (s)-2-sec-butyl-4,5-dihydrothiazole and R,R-3,4-dehydro-exo-brevicomin are known urine-originated male pheromones from previous studies. We also detected 6-hydroxy-6-methyl-3-heptanone, a male urinary pheromonal compound, which had not been directly detected by GC-MS previously. Chemical analysis shows that the genetically more closely related ICR and KM strains had a higher similarity in the volatile compositions of preputial glands and urine than that between ICR or KM and C57BL/6. R,R-3,4-dehydro-exo-brevicomin, in particular, was sensitive to genetic shifts and differed in relative abundance among the 3 strains, whereas (s)-2-sec-butyl-4,5-dihydrothiazole differed between ICR or Km and C57BL/6. Hence, these 2 compounds might code for information about their genetic background.

Comparative study of sex pheromone composition and biosynthesis in Helicoverpa armigera, H. assulta and their hybrid.

Two Helicoverpa species, H. armigera and H. assulta use (Z)-11-hexadecenal and (Z)-9-hexadecenal as their sex attractant pheromone components but in opposite ratios. Since both female and male interspecific hybrids produced by female H. assulta and male H. armigera have been obtained in our laboratory, we can make a comparative study of sex pheromone composition and biosynthesis in the two species and their hybrid. With GC and GC-MS analyses using single gland extracts, the ratio of (Z)-9-hexadecenal to (Z)-11-hexadecenal was determined as 2.1:100 in H. armigera, and 1739:100 in H. assulta. The hybrid has a ratio of 4.0: 100, which is closer to that of H. armigera, but significantly different from H. armigera. We investigated pheromone biosynthesis with labeling experiments, using various fatty acid precursors in H. armigera, H. assulta and the hybrid. In H. armigera, (Z)-11-hexadecenal is produced by delta11 desaturation of palmitic acid, followed by reduction and terminal oxidation; (Z)-9-hexadecenal results from delta11 desaturation of stearic acid, followed by one cycle of chain shortening, reduction and terminal oxidation. delta11 desaturase is the unique desaturase for the production of the two pheromone components. In our Chinese strain of H. assulta, palmitic acid is used as the substrate to form both the major pheromone component, (Z)-9-hexadecenal and the minor one, (Z)-11-hexadecenal. Our data suggest that delta9 desaturase is the major desaturase, and delta11 desaturase is responsible for the minor component in H. assulta, which is consistent with previous work. However, the weak chain shortening acting on (Z)-9 and (Z)-11-octadecenoic acid, which is present in the pheromone glands, does occur in this species to produce (Z)-7 and (Z)-9-hexadecenoic acid. In the hybrid, the major pheromone component, (Z)-11-hexadecenal is produced by delta11 desaturation of palmitic acid, followed by reduction and terminal oxidation. The direct fatty acid precursor of the minor component, (Z)-9-hexadecenoic acid is mainly produced by delta9 desaturation of palmitic acid, but also by delta11 desaturation of stearic acid and one cycle of chain shortening. The greater relative amounts of (Z)-9-hexadecenal in the hybrid are due to the fact that both palmitic and stearic acids are used as substrates, whereas only stearic acid is used as substrate in H. armigera. The evolutionary relationships between the desaturases in several Helicoverpa species are also discussed in this paper.

Sex pheromone components of the sandthorn carpenterworm, Holcocerus hippophaecolus.

Extracts of female sex pheromone gland of the carpenterworm moth, Holcocerus hippophaecolus Hua, a pest of sandthorn, Hippophae rhamnoides L. were found to contain (E)-3-tetradecenyl acetate (E3-14:Ac), (Z)-3-tetradecenyl acetate (Z3-14:Ac), (Z)-7-tetradecenyl acetate (Z7-14:Ac), the corresponding alcohols, E3-14:OH, Z3-14:OH, Z7-14:OH, and (E)-9-tetradecenyl acetate (E9-14:Ac). Electroantennographic (EAG) analysis of these chemicals and their analogs demonstrated that Z7-14:Ac elicited the largest male EAG response, followed by E3-14:Ac. In field trials, traps baited with either Z7-14:Ac or E3-14:Ac alone caught no male moths, whereas a combination of these two components in a 1:1 ratio caught more males than control traps. Addition of Z7-14:OH and Z3-14:OH or the alcohols plus E9-14:Ac did not enhance trap catches. We conclude that the sex pheromone of H. hippophaecolus is composed of Z7-14:Ac and E3-14:Ac. Optimal ratios and doses of these two components, and the possible role of other minor components, remain to be determined.

Sex pheromone biosynthesis in the pine caterpillar moth, Dendrolimus punctatus (Lepidoptera: Lasiocampidae): pathways leading to Z5-monoene and 5,7-conjugated diene components.

Biosynthesis of the sex pheromone components (Z)-5-dodecenol and (Z,E)-5,7-dodecadienol in Dendrolimus punctatus was studied by topical application of deuterium-labeled fatty acids to pheromone glands and subsequent analysis of fatty acyl groups and pheromone components by gas chromatography-mass spectrometry. Our studies suggest that both (Z)-5-dodecenol and (Z,E)-5,7-dodecadienol can be biosynthetically derived from chain elongation of palmitate to stearate in the gland, and its subsequent Delta11 desaturation to produce (Z)-11-octadecenoate. After three cycles of 2-carbon chain-shortening, the pheromone glands produce (Z)-5-dodecenoate, which is then converted to (Z)-5-dodecenol by reduction. A second Delta11 desaturation of (Z)-9-hexadecenoate produces (Z,E)-9,11-hexadecadienoate, which is then chain shortened in two cycles of beta-oxidation and finally converted to (Z,E)-5,7-dodecadienol by reduction.

Stimulation of sex pheromone production by PBAN-like substance in the pine caterpillar moth, Dendrolimus punctatus (Lepidoptera: Lasiocampidae).

Sex pheromone production in the female pine caterpillar moth, Dendrolimus punctatus is controlled by a PBAN-like substance located in the head of female moth. Pheromone titer was significantly decreased by decapitation of female moth, and restored by injection of either Hez-PBAN or head extract prepared from male or female moth. Stimulation of pheromone production by head extract followed a dose-dependent pattern from 0.5 to at least 4 head equivalent. A gland in vitro assay was used to study the relationship between gland incubation time and pheromone production as well as calcium involvement in the stimulation of pheromone production by head extract. Maximum pheromone production was occurred at 60 min after pheromone gland was incubated with two equivalents of head extracts. In vitro experiments showed that the presence of calcium in the incubation medium was necessary for stimulation of pheromone production. The calcium ionophore, A 23187, alone stimulated pheromone production. The pheromone components (Z,E)-5,7-dodecadienol and its acetate and propionate were produced in these experiments but in addition to the aldehyde, (Z,E)-5,7-dodecadienal was also found. This indicates that females are capable of producing four oxygenated functional groups. The PBAN-like substance control of the pheromone biosynthetic pathway was investigated by monitoring the incorporation of the labeled precursor into both pheromone and pheromone intermediates.