Covering: up to October 2014 Insect pheromones are some of the most interesting natural products because they are utilized for interspecific communication between various insects, such as beetles, moths, ants, and cockroaches. A large number of compounds of many kinds have been identified as pheromone components, reflecting the diversity of insect species. While this review deals only with chiral methyl-branched pheromones, the chemical structures of more than one hundred non-terpene compounds have been determined by applying excellent analytical techniques. Furthermore, their stereoselective syntheses have been achieved by employing trustworthy chiral sources and ingenious enantioselective reactions. The information has been reviewed here not only to make them available for new research but also to understand the characteristic chemical structures of the chiral pheromones. Since biosynthetic studies are still limited, it might be meaningful to examine whether the structures, particularly the positions and configurations of the branched methyl groups, are correlated with the taxonomy of the pheromone producers and also with the function of the pheromones in communication systems.

While 11 species in the family Saturniidae are found in Japan, no sex pheromones of the native species had been investigated previously. We collected larvae of Rhodinia fugax in Nagano and Tottori Prefecture, and of Loepa sakaei in Okinawa Prefecture, and extracted sex pheromones of these two species from virgin female moths. In gas chromatography-electroantennogram detection (GC-EAD) analyses, male antennae of each species responded to one component in the respective pheromone extracts of conspecific females. Chemical analyses of the extracts by GC/mass spectrometry revealed that the EAD-active compounds of R. fugax and L. sakaei were a hexadecadienal and a tetradecadienyl acetate, respectively. The two species belong to the subfamily Saturniinae, and the mass spectra of both were similar to that of the 6,11-hexadecadienyl acetate identified from Antheraea polyphemus, classified in the same subfamily, suggesting the same 6,11-dienyl structure for the C16 aldehyde and a 4,9-dienyl structure for the C14 acetate. Based on this assumption, four geometrical isomers of each dienyl compound were stereoselectively synthesized via acetylene intermediates, compared to the natural products, and tested in the field. Male catches confirmed the pheromone structures of the two Japanese saturniid species as (6E,11Z)-6,11-hexadecadienal for R. fugax and (4E,9Z)-4,9-tetradecadienyl acetate for L. sakaei. The compounds have a characteristic 1,6-dienyl motif common to the pheromones of Saturniinae species.

Males of the cerambycid beetle Xylotrechus pyrrhoderus release a mixture of (S)-2-hydroxy-3-octanone [(S)-1] and (2S,3S)-2,3-octanediol [(2S,3S)-2] as a sex pheromone that attracts conspecific females. The chemical structures of these pheromone components include a common motif and are assumed to be biosynthetically related. Here, we show that deuterated (S)-1, applied on the cuticle of a pronotal pheromone gland, was converted into (2S,3S)-2, that included deuterium atoms, but a reverse conversion did not take place. These results reveal a carbonyl reductase to be active in the pheromone gland, and that the ketol is a biosynthetic precursor of the diol. Males did not produce (R)-1; however, deuterated (R)-1 was converted into (2R,3R)-2, indicating an attack of the enzyme from the opposite side of the hydroxyl group at the 2-position. Furthermore, to understand the substrate specificity of the enzyme, racemates of 2-hydroxy-3-hexanone and 2-hydroxy-3-decanone were synthesized and applied to the gland. Their conversion into the corresponding diols suggests that the enzyme reduces the carbonyl group at the 3-position, regardless of the chain length.

Females of the lichen moth, Miltochrista calamina (Arctiidae, Lithosiinae), were previously shown to produce 5-methylheptadecan-7-ol (1) as a sex pheromone. In field tests, males were attracted only by the (5R,7R)-isomer of the four stereoisomers that were prepared by separation from two mixtures of diastereoisomers. A new route to (5R,7R)-1 starting from (S)-propylene oxide was developed utilizing the SN2 reaction of an optically active secondary tosylate and the Jacobsen hydrolytic kinetic resolution of an epoxide intermediate as key steps. Enantioselective HPLC analysis of the product and the antipode synthesized from (R)-propylene oxide confirmed their high enantiomeric excess (> 99 %). Using this stereospecific synthesis, six analogues with the same configuration as (5R,7R)-1 but with different alkyl chain(s) connected to the stereogenic centers were prepared in order to obtain GC/MS data and to examine the ability of M. calamina males to discriminate between them. The mass spectra of the synthetic analogues revealed characteristic fragment ions derived by cleavage around the methyl group in addition to that at the hydroxyl group. In field trapping tests, five out of the six compounds were attractive to male M. calamina moths, indicating that the males distinguished the configurations of methyl and hydroxyl groups but were less able to perceive differences in the lengths of the two alkyl chains in the pheromone.

The sweet potato vine borer moth, Omphisa anastomosalis (Pyraloidea: Crambidae), is a serious pest in tropical and subtropical Asia-Pacific regions. In previous work using a population from Okinawa, Japan, (10E,14E)-10,14-hexadecadienal (E10,E14-16:Ald) was identified as the major pheromone component, with hexadecanal, (E)-10-hexadecenal, and (E)-14-hexadecenal as minor components. However, traps baited with the synthetic compounds were less effective at attracting males in the field than those baited with virgin females. While Pyraloidea females usually produce only Type I pheromone components (unsaturated fatty alcohols and their derivatives), the pheromones of some Pyraloidea species have been shown to involve a combination of both Type I and Type II components (unsaturated hydrocarbons and their epoxides). We examined an extract of the pheromone glands of female O. anastomosalis from Vietnam by gas chromatography coupled to mass spectrometry and detected (3Z,6Z,9Z)-3,6,9-tricosatriene (Z3,Z6,Z9-23:H) in addition to the compounds identified previously. All four isomers of 10,14–16:Ald were synthesized. A mixture of synthetic E10,E14-16:Ald and Z3,Z6,Z9-23:H in a ratio of 1:0.2–1:2 was attractive to male moths in Vietnam, indicating the strong synergistic effect of the Type II compound. Addition of the other minor pheromone components to the binary blend did not increase the number of male moths captured. Combinations of Z3,Z6,Z9-23:H with the other three geometrical isomers of E10,E14-16:Ald attracted no males, further substantiating the 10E,14E configuration of the natural diene component. E10,E14-16:Ald mixed with other polyunsaturated hydrocarbons showed that mixtures that included a C21 triene, a C22 triene, or a C23 pentaene attracted as many males as did the mixture with Z3,Z6,Z9-23:H. The identification of a highly attractive sex pheromone will help in developing efficient strategies for monitoring and control of O. anastomosalis populations in sweet potato fields.

Females of a lichen moth, Barsine expressa (Arctiidae, Lithosiinae), which inhabit Iriomote Island in Japan, were captured by a black-light trap, and the pheromone gland extract was analyzed by gas chromatography (GC) with an electroantennographic (EAG) detector, and by GC coupled with mass spectrometry. The females produced several EAG-active esters, and the mass spectrum of a major component indicated the mixture consists of propionates derived from C17-saturated secondary alcohols, which were inseparable on the capillary GC column. In addition to these main components, the pheromone glands included two acetate derivatives of C17 alcohols, and other propionates of C16 and C15 alcohols. The crude extract was treated with K2CO3, and a 1:1 mixture of C17 alcohols with a C6- or C7-chain moiety was obtained. The two alcohols were uniformly converted into monodeuterated n-heptadecane by mesylation and succeeding LiAlD4 reduction. This result revealed a straight-chain structure of the C17 alcohols with the acyl groups located at the 7- or 8-position. Field tests on Iriomote Island showed that the synthetic esters were behaviorally active. A 1:1 mixture of racemic 7-propioxyheptadecane and 8-propioxyheptadecane, which were prepared from the secondary alcohols synthesized by a Grignard coupling reaction, attracted male moths. Furthermore, propionates of the alcohols synthesized enantioselectively by using a hydrolytic kinetic resolution with Jacobsen’s catalyst were evaluated. Only the traps baited with a mixture of the two esters with the same S-configuration significantly attracted B. expressa males. In the Tokyo area, the propionate mixture attracted a closely related species, Barsine aberrans aberrans.

The nettle moth Monema flavescens (Limacodidae) is a defoliator of fruit trees, such as Chinese plum and persimmon. The larvae of this species have spines containing a poison that causes serious irritation and inflammation in humans. Coupled gas chromatography-electroantennogram detection and gas chromatography/mass spectrometry analyses of a crude pheromone extract, combined with derivatization, indicated that female moths produced 8-decen-1-ol and 7,9-decadien-1-ol at a ratio of approximately 9:1. The E configuration of the double bonds was assigned for both components from infrared spectra, recorded on a gas chromatograph/Fourier transform-infrared spectrophotometer equipped with a zinc selenide disk cooled to −30 °C. The monoenyl and dienyl alcohols had absorptions characteristic of E geometry at 966 and 951 cm−1, respectively. A band chromatogram at 951 cm−1 was useful for distinguishing geometric isomers, because terminal conjugated diene are difficult to resolve, even on high polarity columns. Furthermore, we identified the Z configuration of the same 7,9-dienyl alcohol secreted by another nettle moth, Parasa lepida lepida, through the absence of this absorption. In field trials, lures baited with a 9:1 mixture of (E)-8-decen-1-ol and (E)-7,9-decadien-1-ol attracted M. flavescens males. Furthermore, the field trials indicated that contamination with the (Z)-diene reduced catches to the pheromone mixture more than did contamination with the (Z)-monoene.

All four of the possible stereoisomers of 5,9-dimethylheptadecane, the major sex pheromone component secreted by female moths of the mountain-ash bentwing (Leucoptera scitella), were synthesized by the coupling of two chiral blocks with a methyl branch at the 2- or 3-position. The blocks were prepared by applying the stereospecific inversion of secondary tosylates, which were derived from (R)- and (S)-propylene oxide, and their enantiopurities were confirmed by chiral HPLC analysis.(5S,9R)-5,9-DimethylheptadecaneC19H40[α]D23=+1.1 (c 0.63, CHCl3)Absolute configuration: (5S,9R)(5S,9S)-5,9-DimethylheptadecaneC19H40[α]D23=+2.2 (c 1.3, CHCl3)Absolute configuration: (5S,9S)(R)-2-HexanolC6H14O[α]D23=-8.1 (c 1.5, CHCl3)Absolute configuration: (R)(R)-2-Hexanyl tosylateC13H20O3S[α]D23=-3.7 (c 2.0, CHCl3)Absolute configuration: (R)(S)-Methyl 3-methylheptanoateC9H18O2[α]D23=-3.1 (c 0.82, CHCl3)Absolute configuration: (S)(S)-3-Methylheptan-1-olC8H18O[α]D23=+2.7 (c 1.2, CHCl3)Absolute configuration: (S)(S)-1-Iodo-3-methylheptaneC8H17I[α]D23=-12.6 (c 1.4, CHCl3)Absolute configuration: (S)(S)-2-DecanolC10H22O[α]D23=+8.1 (c 1.1, CHCl3)Absolute configuration: (S)(S)-2-Decanyl tosylateC17H28O3S[α]D23=-3.5 (c 2.0, CHCl3)Absolute configuration: (S)2-Methyl-1-(4-methylphenylsulfonyl)decaneC18H30O2S[α]D23=-1.1 (c 2.5, CHCl3)Absolute configuration: (R)

Cis-9,10-epoxy-(3Z,6Z)-1,3,6-henicosatriene has been identified from a pheromone gland of arctiid species, such as Hyphantria cunea. Since the diversity of lepidopteran species suggests that structurally related compounds of the 9,10-epoxide are also utilized as a sex pheromone components, epoxytrienes derived from (3Z,6Z,9Z)-1,3,6,9-tetraenes with a C19–C21 chain were systematically synthesized and characterized. While 1,2-epoxy-3,6,9-triene was not obtained, peracid oxidation of each tetraene produced a mixture of three cis-epoxides (3,4-epoxy-1,6,9-triene, 6,7-epoxy-1,3,9-triene, and 9,10-epoxy-1,3,6-triene), which were separable by LC as well as GC. Detailed inspection of the mass spectra of the C19–C21 epoxides indicated the following diagnostic ions for determining the chemical structures: m/z 79, M-70, and M-41 for the 3,4-epoxytrienes; m/z 79, 95, 109, and 149 for the 6,7-epoxytrienes; and m/z 79, 106, 120, M-121, and M-107 for the 9,10-epoxytrienes. Resolution of two enantiomers of each C21 epoxytriene was accomplished by HPLC equipped with a chiral column, and analysis of the pheromone extracted from virgin females of H. cunea revealed the 9S,10R configuration of the natural epoxytriene as the same configuration of C21 9,10-epoxydiene, a main pheromone component of this species. GC-EAD analysis of the optically pure epoxides showed that the antennae of male H. cunea were stimulated more strongly (>100 times) by the (9S,10R)-isomers than the antipodes.

A methyl-branched heptadecanol was found in the pheromone gland extract of a female lichen moth, Miltochrista calamina (Arctiidae, Lithosiinae). GC–MS analyses of the alcohol and a hydrocarbon derived from it by subsequent treatments with methanesulfonyl chloride and LiAlD4 in microscale reactions indicated 5-methylheptadecan-7-ol (1) as one possible structure. The four stereoisomers of 1 in a ratio of 4:4:1:1 were prepared from (S)-β-citronellol with 60% ee, and were separated by a combination of achiral and chiral HPLC columns. The absolute configuration of each isomer was determined by the comparison with the chromatographic behaviors of other samples synthesized by a different scheme, which applied the Jacobsen hydrolytic kinetic resolution of racemic 1,2-epoxydodecane to fix the configuration of the 7-hydroxy group. Only the (5R,7R)-isomer attracted male moths; thus, we concluded that M. calamina females secrete (5R,7R)-1 as a sex pheromone, indicating a new chemical class of lepidopteran female sex pheromones.

The subfamily Geometrinae (Lepidoptera: Geometridae) includes many species called emerald moths. Based on our recent finding of novel polyenyl compounds, including a double bond at the 12-position from two geometrine species, Hemithea tritonaria and Thalassodes immissaria intaminata, (6Z,9Z,12Z)-6,9,12-trienes and (3Z,6Z,9Z,12Z)-3,6,9,12-tetraenes with a C17–C20 straight chain were synthesized and analyzed by GC-MS. The 6,9,12-trienes, which were prepared by a double Wittig reaction between two alkanals and an ylide derived from (Z)-1,6-diiodo-3-hexene, characteristically produced fragment ions at m/z 79, 150, and M–98. The 3,6,9,12-tetraenes, which were prepared by a coupling between (Z)-3-alkenal and an ylide derived from (3Z,6Z)-1-iodo-3,6-nonadiene, showed fragment ions at m/z 79, 148, and M–96. These diagnostic ions were useful to distinguish these compounds from other known polyenyl pheromones, such as 4,6,9- and 6,9,11-trienes and 1,3,6,9-tetraenes. With reference to the GC-MS data, pheromone extracts of other species in Geometrinae inhabiting the Iriomote Islands were analyzed, and the 6,9,12-trienes were identified in the pheromone gland extracts of Pamphlebia rubrolimbraria rubrolimbraria and Maxates versicauda microptera. Furthermore, a field evaluation of the synthetic polyenes in a mixed forest of Tokyo revealed the following new male attractants for emerald moths: Idiochlora ussuriaria by a C17 6,9,12-triene and Jodis lactearia by a C20 3,6,9,12-tetraene, indicating the characteristic chemical structures of Geometrinae pheromones. On the other hand, through reexamination of the pheromone extract of H. tritonaria, (3E,6E)-α-farnesene was identified as an electrophysiologically active component in addition to the C17 6,9,12-triene. The binary mixture attracted more males than the single component lure baited with the triene in the Iriomote Islands.

The pear barkminer moth, Spulerina astaurota Meyrick (Gracillariidae: Gracillariinae), is a harmful pest of the Asian-pear tree. Pheromone components of the female were analyzed by gas chromatography (GC) with an electroantennographic (EAG) detector and GC coupled with mass spectrometry. The analyses of a crude pheromone extract and those of a fractionated extract on a Florisil column indicated three EAG-active components, tetradecadien-1-ol, its acetate, and an aldehyde derivative. Characteristic fragment ions in the mass spectra of the dienyl compounds and derivatives with 4-methyl-1,2,4-triazoline-3,5-dione revealed double bonds at the 9- and 11-positions. By comparing the chromatographic behaviors to those of four authentic geometrical isomers, which were synthesized by three different routes starting from 1,8-octanediol or 1,9-nonanediol, the configuration of each natural component was assigned to be 9Z,11Z; i.e., it was concluded that the S. astaurota females secreted (9Z,11Z)-9,11-tetradecadien-1-ol (Z9,Z11-14:OH) as a main pheromone component, and the acetate and aldehyde derivatives (Z9,Z11-14:OAc and Z9,Z11-14:Ald) as minor components. This identification was confirmed by a field evaluation of the synthetic pheromone. While the male moths could be attracted to a lure baited with Z9,Z11-14:OH alone, Z9,Z11-14:OAc showed a strong synergistic effect on the attraction. Among the lures tested, the mixture of alcohol and acetate in a ratio of 7:3 exhibited the strongest attraction. Addition of Z9,Z11-14:Ald in the mixture did not significantly increase the number of males attracted. Furthermore, the field test indicated that some contamination of a geometrical isomer of the alcohol did not impair the activity of the binary mixture with the 9Z,11Z configuration.

Female moths of Lyclene dharma dharma (Arctiidae, Lithosiinae) produce three sex pheromone components (I–III), for which we assigned the following novel chemical structures; 6-methyl-2-octadecanone (1) for I, 14-methyl-2-octadecanone (2) for II, and 6,14-dimethyl-2-octadecanone (3) for III. In the Iriomote Islands where the insects were collected, a lure including racemic 1 and 2 attracted the male moths without mixing 3. In this study for further confirmation of the plane structures, the positional isomers with a methyl branch at the 4-, 5-, 7-, 13-, or 15-position (4–8, respectively) were synthesized. The GC-MS analyses revealed that natural components I and II were best fitted with those of 1 and 2, respectively, among the methyl-2-octadecanones examined, indicating the usefulness of this analytical instrument and authentic standards for the determination of the positions of methyl branches. In field trapping tests, 4–8 could not substitute for 1 or 2, nor did these compounds inhibit the active binary lure of 1 and 2, indicating that the males strictly recognized the 2-ketones with a methyl branch at the 6- or 14-positions. Next, the absolute configurations of I and II were determined by HPLC with a normal-phased chiral column (Chiralpak AD-H), which could separate the enantiomers of both 1 and 2. The chiral HPLC analysis of a crude pheromone extract indicated that the females exclusively produced (S)-1 and (S)-2. Furthermore, a field evaluation of each enantiomer revealed that (S)-1 and (S)-2 were bioactive but (R)-1 and (R)-2 were not.

Lepidopteran Type II sex pheromones are mainly composed of 6,9-dienes, 3,6,9-trienes, and their epoxy derivatives, which are biosynthesized from linoleic and linolenic acids by the species in some families of higher Lepidoptera. To investigate further structural modifications on this theme, we synthesized polyunsaturated hydrocarbons with a C17–C21 chain, which included an extra double bond. Using the Wittig reaction, (Z,Z,E)-6,9,11-trienes and (Z,Z,Z,E)-3,6,9,11-tetraenes were synthesized from (E)-2-alkenals with appropriate carbon chains, and (Z,Z,Z)-1,3,6,9-tetraenes were synthesized from 3-hexyn-1,6-diol. The gas chromatography-mass spectrometry (GC-MS) analysis of each synthetic polyene, whose chemical structure was confirmed by 1H NMR and 13C NMR, revealed some characteristic fragment ions reflecting the positions of the double bonds, i.e., m/z 79, 110, 163, and M-85 of the 6,9,11-trienes, m/z 79, 108, and M-82 of the 3,6,9,11-tetraenes, and m/z 79, 91, 106, and M-54 of the 1,3,6,9-tetraenes. Because the determination of the unsaturated positions is difficult to accomplish by chemical derivatization with a limited amount of natural pheromones, these diagnostic ions found in authentic samples would help identify the hydrocarbons in a pheromone extract. Furthermore, we carried out field screening tests of these polyenes in forests in Japan, and documented the attraction of four geometrid species in Tokyo and one noctuid species in the Iriomote Islands.

Gas chromatography–mass spectrometry (GC–MS) and GC–electroantennographic detection (EAD) analyses of the sex pheromone extract from a wasp moth, Syntomoides imaon (Lepidoptera: Arctiidae: Syntominae), showed that virgin females produced (Z,Z,Z)-3,6,9-henicosatriene and (Z,Z,Z)-1,3,6,9-henicosatetraene with a trace amount of their C20 analogs. Identification of the chemical structures was facilitated by comparison with authentic standards and the double-bond positions were confirmed by dimethyl disulfide derivatization of monoenes produced by a diimide reduction. In a field test in the Yonaguni-jima Islands, males of the diurnal species were captured in traps baited with a 1:2 mixture of the above-described synthetic C21 polyenes. Lipids were extracted from the abdominal integument and its associated oenocytes and peripheral fat bodies. Following derivatization, fatty acid methyl esters (FAMEs) were fractionated by HPLC equipped with an ODS column, and methyl (Z,Z,Z)-11,14,17-icosatrienoate and (Z,Z,Z)-13,16,19-docosatrienoate were identified by GC–MS. These novel C20 and C22 acid moieties are longer-chain analogs of linolenic acid, (Z,Z,Z)-9,12,15-octadecatrienoic acid. They are presumed to be biosynthetic precursors of the S. imaon pheromone because the C21 trienyl component might be formed by decarboxylation of the C22 acid. On the other hand, the C20 acid, but not the C22 acid, was found in FAMEs of Ascotis selenaria cretacea (Lepidoptera: Geometridae), which secretes C19 pheromone components, (Z,Z,Z)-3,6,9-nonadecatriene and the monoepoxy derivative, indicating that different systems of the chain elongation might play an important role in developing species-specific communication systems mediated with polyunsaturated hydrocarbons and/or epoxy derivatives, components of Type II lepidopteran sex pheromones.

In addition to 2,13- and 3,13-octadecadien-1-ols and their acetates, aldehyde analogs have been identified from lepidopteran species in the family Sesiidae. To establish a reliable analytical method for determining the positions and configurations of the two double bonds in natural pheromone components, all geometric isomers of the 2,13- and 3,13-octadecadienals were synthesized by Dess-Martin oxidation of the corresponding alcohols with limited isomerization of the double bond at the 2- or 3-position. GC-MS analysis of these aldehydes showed isomerization of (Z)-2-, (Z)-3-, and (E)-3-double bonds to an (E)-2-double bond, even with a cool on-column injection. In contrast, HPLC analysis with an ODS column was accomplished without isomerization. The geometric isomers of each dienal eluted in the order ZZ → EZ → ZE → EE. The conjugated 2,13-dienals were detectable in nanogram amounts with a UV detector at 235 nm. Whereas the detection of 3,13-dienals was difficult because of the lack of a chromophore, a highly sensitive analysis was achieved after derivatization with 2,4-dinitrophenylhydrazine. LC-MS with atmospheric pressure chemical ionization showed a strong [M-1]- at m/z 443 for the derivatives. Based on these analytical data, a pheromone extract of a sesiid moth, Macroscelesia japona, was examined by HPLC and LC-MS, and it was confirmed that the octadecadienal tentatively identified by a previous GC-MS analysis did indeed have the 2E,13Z configuration. Furthermore, field evaluation of four synthetic geometric isomers of the 2,13-dienal revealed specific attraction to a lure with the (2E,13Z)-isomer as a main component.

Information on the olfactory system in antennae of Geometridae moths is very limited, and odorant-binding proteins (OBPs) working as transporters of lipophilic odors have not been identified. In the first investigation on this family of insects, we examined antennal OBPs of the Japanese giant looper, Ascotis selenaria cretacea. RT-PCR experiments using several pairs of degenerate primers designed from known cDNA sequences encoding lepidopteran OBPs successfully amplified partial sequences of two pheromone-binding proteins (PBPs), named AscrPBP1 and AscrPBP2 in reference to their corresponding nucleotide sequence homologies with other PBPs. Using 5′- and 3′-rapid amplification of cDNA end strategies, a cDNA clone for AscrPBP1 encoding a protein of 141 amino acids was isolated. Western blotting with the antiserum against recombinant AscrPBP1 overexpressed in Escherichia coli showed that the AscrPBP1 gene was more strongly expressed in male antennae than in female antennae. Furthermore, natural AscrPBP1was isolated by immunoprecipitation with the antiserum, and its binding ability was evaluated by using synthetic sex pheromonal compounds with a C19 chain. The result indicated that AscrPBP1 bound not only the pheromone components, 3,6,9-nonadecatriene and its 3,4-epoxy derivative, but also unnatural 6,7- and 9,10-epoxy derivatives. While no general odorant-binding proteins (GOBPs) were amplified in the RT-PCR experiments, two antisera prepared from GOBP1 and GOBP2 of Bombyx mori suggested the occurrence of at least two GOBPs in the A. s. cretacea antennae.

The fall webworm, Hyphantria cunea Drury (Lepidoptera: Arctiidae), is a harmful polyphagous defoliator. Female moths produce the following four pheromone components in a ratio of about 5:4:10:2; (9Z,12Z)-9,12-octadecadienal (I), (9Z,12Z,15Z)-9,12,15-octadecatrienal (II), cis-9,10-epoxy-(3Z,6Z)-3,6-henicosadiene (III), and cis-9,10-epoxy-(3Z,6Z)-1,3,6-henicosatriene (IV). Although 13C-labeled linolenic acid was not converted into trienal II at the pheromone glands of H. cunea females, GC–MS analysis of an extract of the pheromone gland treated topically with 13C-labeled linolenyl alcohol showed the aldehyde incorporating the isotope. Other C18 and C19 fatty alcohols were also oxidized to the corresponding aldehydes in the pheromone gland, indicating a biosynthetic pathway of II via linolenyl alcohol and low substrate selectivity of the alcohol oxidase in the pheromone gland. On the other hand, epoxydiene III was expected to be produced by specific 9,10-epoxidation of the corresponding C21 trienyl hydrocarbon, which might be biosynthesized from dietary linolenic acid in oenocytes and transported to the pheromone gland. The final biosynthetic step in the pheromone gland was confirmed by an experiment using deuterated C21 triene, which was synthesized by the chain elongation of linolenic acid and LiAlD4 reduction as key reactions. When the labeled triene was administered to the female by topical application at the pheromone gland or injection into the abdomen, deuterated III was detected in a pheromone extract by GC–MS analysis. Furthermore, the substrate selectivity of epoxidase and selective incorporation by the pheromone glands were examined by treatments with mixtures of the deuterated precursor and other hydrocarbons such as C19–C23 trienyl, C21 dienyl, and C21 monoenyl hydrocarbons. The 9,10-epoxy derivative of each alkene was produced, while the epoxidation of the C21 monoene was poorer than those of the trienes and diene. The low selectivity indicated that the species-specific pheromone of the H. cunea female was mainly due to the critical formation of the precursor of each component.Download high-res image (112KB)Download full-size imageHighlights► Biosynthetic pathways were examined by experiments using 13C- or D-labeled precursors. ► Linolenic alcohol was the direct precursors of an aldehyde pheromone component. ► 3,6,9-Henicosatriene was the direct precursor of an expoxydiene pheromone component. ► Oxidation of structure-related compounds revealed low substrate selectivity of the enzymes.

Virgin females of the Japanese giant looper (Ascotis selenaria cretacea, Assc) in the family of Geometridae secrete an epoxyalkenyl sex pheromone to attract males. To regulate its biosynthesis in the pheromone gland, Assc females produce a pheromone biosynthesis-activating neuropeptide (PBAN) in the suboesophageal ganglion (SG), as do females in many lepidopteran species. We have isolated Assc-PBAN cDNA, which encodes 181 amino acids, including a PBAN homologue and four other putative peptides: a diapause hormone (DH) homologue, α-SG neuropeptide (SGNP), β-SGNP, and γ-SGNP, all of which shared an FXPR(K)L motif on their C-termini. Although PBANs with 30–35 amino acids have been characterized from 15 other species, the Assc-PBAN homologue consisted of 28 amino acids and showed low homology (<46%) compared with the others. Assc-β-SGNP with eight amino acids was also shorter than the other β-SGNPs (16–22 amino acids). Furthermore, all of the known PBAN cDNAs have a GRR sequence between β-SGNP and PBAN as a cleavage site, but the Assc-PBAN cDNA showed an unusual GR sequence at the corresponding position, indicating the possibility of non-cleavage between the β-SGNP and PBAN. When the GR sequence was a cleavage site, the question arose of whether or not the glutamine residue at the N-terminus of the Assc-PBAN homologue was cyclized. To identify the sequence of the Assc-PBAN, the brain-SG extract was fractionated by HPLC referring to three synthetic peptides with the predicted sequences. The chromatographic behavior of the natural pheromonotropic peptide revealed the unique structure of Assc-PBAN including β-SGNP, i.e., SVDFTPRLGRQLVDDVPQRQQIEEDRLGSRTRFFSPRL-NH2, as the first determination of PBAN from the insects producing an epoxyalkenyl sex pheromone.