2006-01 Diagnostic fragment ions for the GC-MS analyses of lepidopteran sex pheromones
Sex pheromones, which have been identified from female moths of nearly 560 species from around the world, are mainly classified into Type I or Type II according to their chemical structures. Primary alcohols and their derivatives with a C10?C18 unsaturated straight chain (D1-D3) comprise Type I pheromones, and Type II pheromones are composed of polyunsaturated hydrocarbons (D2-D4) and the epoxy derivatives with a C17?C23 straight chain. GC-MS is commonly used for studies of these components, which are present in minute amounts in the pheromone glands, because their informative mass spectra can be measured very precisely. In addition to the chain lengths of the pheromone components, other structural features could be estimated by some diagnostic fragment ions. In order to clarify the possibilities and limitations for determining the positions of double bonds and epoxy rings by GC-MS analysis, we accumulated mass spectral data of a series of synthetic pheromonal compounds and successfully identified a novel Type I pheromone of the persimmon fruit moth. The females produce 4,6-hexadecadienal with a characteristic base peak at m/z 84, which is expected to be formed by a cyclization after cleavage of the bond between the 5- and 6-positions. Furthermore, we identified several Type II pheromones secreted by highly evolved specie such as the geometrid and lymantriid moths, by referring to the diagnostic ions of monoepoxy derivatives systematically synthesized from 6,9-dienes and 3,6,9-trienes, i.e., ions at m/z M-100, M-114, and 99 of 6,7-epoxy-9-enes, m/z M-111, M-125, and 110 of 9,10-epoxy-6-enes, m/z M-58, M-72, and 79 of 3,4-epoxy-6,9-dienes, m/z M-69, M-83, 111, and 79 of 6,7-epoxy-3,9-dienes, and m/z M-109, M-123, 122, 108, and 79 of 9,10-epoxy-3,6-dienes.
2006-02 Sex pheromone of the citrus leafminer, Phyllocnistis citrella: identification, field attraction and mating disruption by the synthetic 7,11-dienyl compounds.
GC-EAD and GC-MS analyses of a pheromone gland extract from virgin females of the citrus leafminer (
Phyllocnistis citrella, Gracillariidae) revealed that (7Z,11Z)-7,11-hexadecadienal (Z7,Z11-16:Ald), is a pheromone component of
P. citrella. While a field test of lures mixed with extra compounds structurally related to Z7,Z11-16:Ald did not inform candidates for a minor component having a synergistic effect, some compounds, particularly monoenyl derivatives, strongly inhibited the activity of the dienyl pheromone component. Furthermore, two experiments were carried out in citrus orchards to develop a method for disrupting the mating of
P. citrella. In order to estimate the disruptive ability of Z7,Z11-16:Ald and some derivatives, pheromone traps surrounded with septa impregnated with these test compounds were used. However, this first trial was fruitless because the number of captured males actually increased due to the effect of the surrounding compounds such as Z7,Z11-16:Ald. On the other hand, another experiment with polyethylene dispensers containing Z7,Z11-16:Ald revealed a good ability to disrupt the male’s orientation, although eggs and larvae of
P. citrella on a citrus tender flush did not show any significant difference in the control and treated fields. Monitoring traps caught 27-127 males in one night in control fields from July to September, but hardly any males were caught in either field treated with the dispensers at a density of 500 and 1300 tubes/ha.
2007-01 Epoxyalkenyl sex pheromones produced by female moths in highly evolved groups: Biosynthesis and its endocrine regulation
In addition to C10-C18 unsaturated fatty alcohols and their derivatives (Type I pheromones such as bombykol), polyunsaturated hydrocarbons with a C17-C23 straight chain and the epoxy derivatives (Type II pheromones, not including a terminal functional group) have been identified from macrolepidopteran female moths. The variation of the chemical structures in both Type I and Type II compounds results from differences in both enzyme systems and their starting material. In reference to our early study on bombykol biosynthesis, we started biochemical experiments of the Type II pheromone with a geometrid species, the Japanese giant looper, a female moth that produces a C19 3,4-epoxy-6,9-diene (main component) and a C19 3,6,9-triene (minor component), and confirmed that the triene was the direct biosynthetic precursor of the epoxy pheromone. While the expoxidation proceeded in a pheromone gland, some experiments revealed that the precursor was produced outside of the gland, probably in oenocytes or in the fat body, and transported to the pheromone gland via hemolymph after binding to lipophorin. Based on the facts, the selectivity of the epoxidation substrates and of the precursor uptake by the lipophorin and gland was examined.
Furthermore, the neuroendocrine regulation of these processes was studied with in vivo and in vitro experiments using decapitated females and a pheromone biosynthesis-activating neuropeptide (PBAN) secreted from the suboesophageal ganglion in the head. PBAN accelerated the precursor uptake but not a biosynthetic step. These results indicates that biosynthesis of Type II pheromones is quite different form that of Type I pheromones. We were interested in this mode of action of PBAN, and recently clarified the neuropeptide of
A. s. cretacea-PBAN (Asse-PBAN) as a first step. A cDNA isolated from brain-SG complexes of
A. s. cretacea encodes 181 amino acids including PBAN, DH and three NPs. The Asse-PBAN sequence consists of 28 amino acids. It is shorter than those of known PBANs, and its homology is less than 50 %.
2007-02 GC-MS and LC-MS analyses for unraveling diversity of lepidopteran communication systems
As a consequence of the diversity of Lepidoptera, including more than 150,000 species, interesting species-specific mating communication systems are exhibited. The quite varied sex pheromones, which have been identified from female moths of about 600 species from around the world, are mainly classified into two groups (Types I and II) according to their chemical structures. Since the pheromone content in a female moth is very low and each component has many structure-related isomers, chromatographic and spectroscopic data of pheromonal compounds systematically synthesized are informative for a new study of natural pheromones.
In the Type I pheromones composed of primary alcohols and their derivatives with a C10-C18 unsaturated straight chain, a number of conjugated dienyl compounds have been identified. Each diene possessing the double bonds at a side of a terminal methyl group showed the characteristic mass spectrum including diagnostic ions. From
Leguminivora glycinivorella, we could identified (E,E)-8,10-dodecadienyl acetate with reference to the ions at m/z 81 and 68. Among the compounds unsaturated at a side of a functional group, 4,6-dienal showed an unusual base peak at m/z 84. (E,Z)-4,6-Hexadecadienal, a component of
Stathmopoda masinissa pheromone, was successfully identified by the unique mass spectrum. Type II pheromones are composed of polyunsaturated hydrocarbons (6,9-dienes and 3,6,9-trienes) and the epoxy derivatives with a C17-C23 straight chain. In addition to them, some insects produce 1,3,6,9- and 3,6,9,11-tetraenes. These tetraenes are distinguishable with fragment ions at m/z M-54 and 108. (Z,Z,Z)-1,3,6,9-Henicosatetraene was found in the pheromone extract of
Syntomoides imaon.
While HPLC has not been utilized frequently for analyzing natural pheromones, an ODS column is superior in separation of E- and Z-isomers. Two geometrical isomers of a terminal conjugated diene showed almost the same Rts even on a strong polar capillary GC column, but HPLC could completely separate them. The configuration of (Z)-7,9-decadien-1-ol produced secreted by
Parasa lepida was determined by an HPLC analysis. Furthermore, HPLC and LC-MS are useful to analyze heat-unstable components, such as an aldehyde with a 2- or 3-position. Applied them, it was confirmed that
Macroscelesia japona solely secreted (E,Z)-2,13-octadecadienal as an aldehyde component.
2007-03 Synthesis and characterization of 2,13- and 3,13-octadecadienals for the identification of the sex pheromone secreted by a clearwing moth
In addition to 2,13-and 3,13-octadecadien-1-ols and their acetates, an aldehyde analogs has been identified from lepidopteran species in the family Sesiidae. In order 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. Their 1H and 13C NMR data indicated successful preparation with quite 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 though a cool on-column injector was used. In contrast, HPLC analysis with an ODS column was accomplished without isomerization. The 2,13-dienal with a conjugated system was detectable in nanogram amount using a UV detector at 235 nm. Whereas the detection of 3,13-dienals was difficult, 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 had the 2E,13Z configuration.
2007-04 7,11,13-Hexadecatrienal identified from female moths of the citrus leafminer as a new sex pheromone component: Synthesis and field evaluation in Japan and Vietnam
(7Z,11Z)-7,11-Hexadecadienal (Z7,Z11-16:Ald), which has been identified from female moths of the citrus leafminer (
Phyllocnistis citrella), strongly attracts males in Japan. Recently, in addition to the dienyl aldehyde, a trienyl derivative, (7Z,11Z,13E)-7,11,13-hexadecatrienal (Z7,Z11,E13-16:Ald), was found as another sex pheromone component of females collected in California and Brazil. Thus, we synthesized Z7,Z11,E13-16:Ald and its isomer (Z7,E11,E13-16:Ald) to evaluate their effects on the males inhabiting Asia. Starting from 1,7-heptanediol, alcohols with the trienyl structures were prepared by applying the Wittig-coupling reaction twice and oxidized to yield the objective aldehydes after separation by HPLC with a ODS column. At citrus orchards in Ogasawara Islands and Ehime Prefecture of Japan, neither of the trienyl aldehydes attracted males and showed a synergistic effect on the male capture by Z7,Z11-16:Ald. Far from being reinforced, attractant activity of dienyl aldehyde was diminished by mixing of Z7,Z11,E13-16:Ald. At an orchard in Cantho City of Vietnam, males could not be caught by a lure baited only with Z7,Z11-16:Ald, but they were successfully attracted with the 1:3 mixture of Z7,Z11-16:Ald and Z7,Z11,E13-16:Ald. These results indicated that the sex pheromones of the Vietnam and American strains were similar; however, the Japanese strain established a different communication system from those of the foreign strains.
2007-05 Novel sex pheromone components from a Lithosiinae moth, Lyclene dharma dharma, in the family of Arctiidae
The family of Arctiidae is mainly divided into two subfamilies, Arctiinae and Lithosiinae. From some species in Arctiinae, Type II sex pheromones composed of polyunsaturated hydrocarbons and/or the epoxy derivatives have been identified. In Lithosiinae, however, neither the pheromones nor the male attractants have been reported, which suggests that their mating communication systems depend on different types of chemicals. In order to understand the diversity of lepidopteran pheromones, we caught several Lithosiinae species in the Iriomote Islands using a black-light trap and found novel compounds in a pheromone extract of Lyclene dharma dharma females. The GC-EAD analysis showed that the extract includes three EAG-active components (Comps. I-III) at a ratio of about 4:3:2. On a column chromatography with Florisil, these EAG-active components were eluted together with 10 % ether in hexane, indicating that these components were more polar than a Type II epoxy pheromone and less polar than a Type I alcohol pheromone. The GC-MS analysis revealed the structure of Comps. I-III as ketones without a C=C bond; i.e., methyl-2-octadecanones for Comps. I and II, and diemethyl-2-octadecanone for Comp. III. We estimated the positions of the methyl groups by Wolff-Kishner reduction of the natural pheromone and are going to synthesize candidates to confirm the structures.
2007-06 Determination of the PBAN receptor (PBANR) in the Japanese giant looper, Ascotis selenaria cretacea, which produces an epoxyalkenyl sex pheromone
The biosynthesis of lepidopteran sex pheromones is regulated by a pheromone biosynthesis-activating neuropeptide (PBAN). Receptors of the peptide (PBANR) have been identified from insects secreting Type I pheromones such as
Bombyx mori and
Helicoverpa zea. The PBAN of a geometrid moth (
Ascotis selenaria cretacea) secreting the Type II pheromone has a unique structure and mode of action. It has double FXPRL motifs and activates the pheromone gland to incorporate a biosynthetic precursor in hemolymph. In order to clarify the details of the activation process, we attempted to identify the PBANR of
A. s. cretacea. A partial sequence of the putative Assc-PBANR was amplified from cDNA of the pheromone gland with degenerate primers designed for the other PBANRs. Using a PCR-based cloning strategy, a cDNA clone encoding the predicted seven transmembrane domains was isolated; however, full-length cDNA was not identified at the 3-prime. An RT-PCR experiment with the mRNA of A. s. cretacea females confirmed the clone expression at the pheromone gland. Interestingly, the external-membrane region from the N-terminus to the first transmembrane domain has 64 amino acids. This sequence is longer than those of
B. mori and
H. zea by 26 and 28 amino acids, respectively, suggesting that it is a candidate for the binding site of Assc-PBAN with a different structure from that of the PBANs of other species.
2007-07 Identification of 11,14,17-icosatrienoic and 13,16,19-docosatrienoic acids, biosynthetic intermediates of lepidopteran sex pheromones derived from linolenic acid
Polyunsaturated hydrocarbons and their epoxy derivatives with a C17-C23 straight chain compose the second major group of lepidopteran sex pheromones. These Type II pheromones commonly include double bonds at the 3- and/or 6- and/or 9-positions, indicating their biosynthesis from dietary linoleic or linolenic acids via decarboxylation after chain elongation. Our previous studies with a geometrid species,
Ascotis selenaria cretacea, revealed that the pheromonal hydrocarbon (C19 3,6,9-triene) was produced outside of a pheromone gland, transported to the pheromone gland after associating with lipophorin, and oxidized to the epoxide. In order to confirm the biosynthetic pathway, we analyzed fatty acids in an abdominal integument including oenocyte cells, where hydrocarbons in an epicuticle are produced. After purification using HPLC with an ODS column and methylation, the GC-MS analysis showed the occurrence of methyl 11,14,17-icosatrienoate in a lipid extraction of the
A. s. cretacea females. In another experiment with an arctiid species,
Syntomoides imaon, which secreted C21 3,6,9-trienes, methyl 13,16,19-docosatrienoate, in addition to the icosatrienoate, was detected. This constitures first identification of novel acyl intermediates in Type II pheromone biosynthesis, and lack of the docosatrienoate in
A. s. cretacea indicates that the biosynthesis is strictly regulated by a species-specific system of the chain elongation.
2007-08 Analysis of odorant-binding proteins in antennae of the geometrid species, which produces lepidopteran Type II sex pheromone components
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, 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 PBP1 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.
2007-09 Hydrocarbons with a 1,3,6,9-, 3,6,9,11-, or 6,9,11-polyene system: sex pheromone candidates of lepidopteran insects in highly evolved groups
Lepidopteran Type II sex pheromones composed of 3,6,9-trienes, 6,9-dienes, and their epoxy derivatives were biosynthesized from linolenic and linoleic acids in the species of highly evolved families. Since diversity of an insect species indicates further structural modification, such as the introduction of an extra double bond, we synthesized 1,3,6,9- and 3,6,9,11-tetraenes and 6,9,11-trienes with C17-C21 chains to accumulate new information for the pheromone research. Namely, using the Wittig reaction, (Z,Z,Z)-1,3,6,9-tetraenes were synthesized from 3-hexyn-1,6-diol, and (Z,Z,Z,E)-3,6,9,11-tetraenes and (Z,Z,E)-6,9,11-trienes were synthesized from (E)-2-alkenals with appropriate carbon chains. GC-MS analysis of each synthetic polyene, whose chemical structure was confirmed by
1H- and
13C-NMR, revealed some characteristic fragment ions reflecting the positions of the double bonds; i.e., m/z 79, 106, and M-54 of 1,3,6,9-tetraenes, m/z 79, M-56, and M-82 of 3,6,9,11-tetraenes, and m/z 57, 79, and 163 of 6,9,11-trienes. 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 to discover new sex attractants of lepidopteran insects in forests in Tokyo and Iriomote Island in Japan. To date, the attraction of four geometrid species in Tokyo and one noctuid species in Iriomote Island was observed.
2007-10 Attractiveness of the synthetic sex pheromone to the citrus flower moth (Prays citri Milliere) in the Mekong Delta of Vietnam
The citrus flower moth,
Prays citri (Yponomeutidae), is a widespread pest of citrus. Meanwhile the
P. citri larvae attack citrus flowers, especially lemon flowers, in many countries, this species mostly attacks primarily pomelo fruits in the Mekong delta of Vietnam. From a strain cultured in Israel, the sex pheromone composed of one component, (Z)-7-tetradecenal (Z7-14:Ald), has already been characterized [Nesbitt et al., 1977 (Insect Biochem., 7: 355-3599)]. Therefore, we synthesized Z7-14:Ald and the related compounds and examined the attractiveness at a pomelo orchard in Vinh Long province, which is located near the center of the Mekong delta. A trap baited with a rubber septum including Z7-14:Ald (0.5 mg) captured more
P. citri males than one baited with three virgin females. In the dose-response examination, the one mg-dose of Z7-14:Ald attracted the
P. citri males the most strongly among five tested doses (0, 0.1, 0.3, 0.5, and 1 mg). These results showed that the synthetic lure was useful as a monitoring tool. (Z)-7-Tetradecen-1-ol strongly inhibited the attractiveness of Z7-14:Ald, but the addition of (Z)-7-tetradecenyl acetate slightly increased the number of males captured by Z7-14:Ald, indicating that the acetate was a minor pheromone component. In a future study, we will analyze pheromone components of
P. citri distributed in Vietnam.
2007-11 Study on an epoxygenase working on the Type II pheromone biosynthesis
Many genes encoding a desaturase implicated in the sex pheromone biosynthesis of female moths are identified, whereas no gene encoding epoxygenase is reported. Virgin female of Japanese giant looper,
Ascotis selenaria cretacea, produces epoxyalkene [(Z,Z)-6,9-cis-3,4-epoxynonadecadiene] as a main pheromone component from a triene precursor [(Z,Z,Z)-3,6,9-nonadecatrien] via enzymatic epoxidation in a pheromone gland. Decade ago, an epoxygenase from the plant had been characterized as a membrane protein containing non-heme iron. The plant epoxygenase shows sequence similarity to acetylenase and desaturase that have a histidine-rich motif (his-box). Based on this finding, we assumed that the insect epoxygenase for the sex pheromone biosynthesis might contain non-heme iron similar to the insect desaturase. As a result of PCR using degenerate primers, five genes (epoc1-5) were isolated. RT-PCR expression analysis demonstrated that epoc1 is specifically expressed in the pheromone gland. Although in vitro functional assay using insect cells (Sf9) failed to demonstrate that Epoc1 was epoxygenase, down-regulation of endogenous epoc1 by RNAi reduced the pheromone titer to 40% of the control. Furthermore, when the deuterium labeled triene was injected into the abdomen of the female moths, the corresponding labeled epoxy pheromone was not detected in epoc1 knock down individuals. These results indicate that epoc1 plays some role in the pheromone gland.
2007-12 Mating disruption of the persimmon fruit moth, Stathmopoda masinissa, by the synthetic sex pheromone
The persimmon fruit moth,
Stathmopoda masinissa (Oecophoridae), is a well-known harmful pest of persimmon fruits in Japan. We identified the sex pheromone of
S. masinissa females and developed a method for the mating disruption. GC-EAD and GC-MS analyses found three EAG active components with a 4,6-diene system in a C16 chain; i.e. (4E,6Z)-4,6-hexadecadienyl acetate (E4,Z6-16:OAc) and the corresponding derivatives, aldehyde (E4,Z6-16:Ald) and alcohol (E4,Z6-16:OH). Meanwhile the role of E4,Z6-16:OH in the mating communication has not been clarified, lures baited with a mixture of E4,Z6-16:OAc and E4,Z6-16:Ald (10:1) effectively attracted
S. masinissa males in a field. The number of males attracted to both artificial pheromone lures and tethered virgin females were strongly decreased in the fields with mass-application of the sex pheromone, comparing to the control fields. Consequently, we successfully decreased the damage of the fruits using the sex pheromone.
Key words: persimmon fruit moth, sex pheromone, mating disruption, disruptant, pest control.
2007-13 Clearwing moths use visual cues with olfactory cues on their mating behavior
Many lepidopterans recognize mating partners with visual and/or chemical cues. Mainly nocturnal moths use volatile sex pheromone, and contrary diurnal butterflies use visual cues to recognize their mating partners. On the other hand, information about the mating behavior of diurnal moths is very limited. It si expected that no little diurnal moths possibly use visual cue on their mating in a diurnal condition, but both usage of the visual cue and female pheromone have been reported only in a few Zygaenidae species (Zagatti & Renou, 1984; Koshio & Hidaka, 1995).
Since the clearwing moth (Sesiidae), which flies only daytime and has a specific colorful wasp-mimicry body, seems to be one of the best insects to study the relationships of visual cues and sex pheromones on the mating behavior, we started identification of the sex pheromone of two sesiid species,
Nokona pernix and
Synanthedon hector. GC-MS and GC-EAD analyses identified (E,Z)-3,13-octadecadien-1-ol and the (Z,Z)-isomer in a pheromone extract of the former species and two acetate derivatives in that o f the corresponding males in a field, indicating that each species secreted the pheromone blend with high species-specificity. However, while the synthetic pheromone baited to rubber septa showed strong attraction activities, a copulatory behavior to the lures was not observed. Interestingly, visual female models (black or wasp-mimic pattern) were necessary for the male moths to recognize their partners and try copulation.
2008-01 Identification of C21 Type II sex pheromone components and novel C20 and C22 trienyl biosynthetic precursors from a wasp moth, Syntomoides imaon (Arctiidae: Syntominae)
GC-MS and GC-EAD analyses of the pheromone extract from a wasp moth,
Syntomoides imaon (Arctiidae: Syntominae), showed that the virgin females produced (Z,Z,Z)-3,6,9-henicosatriene (Z3,Z6,Z9-21:H) and (Z,Z,Z)-1,3,6,9-henicosatetraene with a trace amount of their C20 analogues. The chemical structures were elucidated by comparing with authentic standards, and, furthermore, the positions of the double bonds were confirmed by chemical reactions, i.e., dimethyl disulfide derivatization of monoens produced by a diimide reduction. In a field in the Yonaguni-jima Islands, males of the diurnal species were captured by traps baited with a 1:2 mixture of the synthetic C21 polyenes. In addition to the pheromone components, lipids extracted from abdominal integuments, which were associated with oenocytes and peripheral fat bodies, were examined. After fractionation of fatty acid methyl esters (FAMEs) derived from the lipids by HPLC equipped with an ODS column, GC-MS analysis identified methyl esters of (Z,Z,Z)-11,14,17-icosatrienoic acid (Z11,Z14,Z17-20:Acid) and (Z,Z,Z)-13,16,19-docosatrienoic acid (Z13,Z16,Z19-22:Acid). These novel C20 and C22 acids are longer-chain analogues of linolenic acid, (Z,Z,Z)-9,12,15-octadecatrienoic acid (Z9,Z12,Z15-18:Acid). The C22 acid is presumed to be a direct biosynthetic precursor of the
S. imaon pheromone because the C21 trienyl component can be formed by decarboxydation of the C22 acid. Furthermore, the C20 acid, but not the C22 acid, was found in FAMEs of
Ascotis selenaria cretacea (Geometridae), which secreted C19 pheromone components, (Z,Z,Z)-3,6,9-nonadecatriene (Z3,Z6,Z9-19:H) and the monoepoxy derivative. These results indicate that difference of their chain elongation systems might play an important role in producing species-specific Type II lepidopteran sex pheromones composed of polyunsaturated hydrocarbons and/or epoxy derivatives.
2009-01 A novel lepidopteran sex pheromone produced by females of a lichen moth, Lyclene dharma dharma (Lithosiinae: Arctiidae)
Lithosiinae is one of the subfamilies in Arctiidae. Larvae of the Lithosiinae species, which mainly feed on lichen in a forest, are not pest insects but important organisms contributing to the ecosystem of the forest. While many pheromones have been identified from species in the two other subfamilies, Arctiinae and Synthominae, there has been no information about the pheromones of the Lithosiinae species. The analysis of a pheromone gland extract by GC-EAD showed that the L. d. dharma females, which were collected in a subtropical island in Japan, produced three active components (I-III) in a ratio of 2:1:1. GC-MS analyses of the extracts before and after Wollf-Kishner reduction enabled their structures to be estimated as follows: 6-methyl-2-octadecanone (I), 14-methyl-2-octadecanone (II), and 6,14-dimethyl-2-octadecanone (III). These methyl-branched ketones have not been identified as a natural product, indicating that they constitute a new chemical group of lepidopteran female sex pheromones.
The planar structures of I-III were confirmed by achiral syntheses started from diols. Their activities were evaluated in a field, and effective male attraction was observed for the 2:1:1 mixture of I-III. This result indicates that the females do not produce only one stereoisomer for each component or that the response of the males is not disturbed by the other stereoisomers of natural isomers produced by the females. The field test also revealed that the two-component lure of I and II captured as many males as the mixture of I-III, while lures baited with two components in other combinations and with only one component scarcely exhibited any male attraction ability.
2009-02 Synthesis of deuterated sex pheromone components of the grape borer, Xylotrechus pyrrohoders
Adult males of the grape borer,
Xylotrechus pyrrohoders, secrete (S)-2-hydroxy-3-octanone [(S)-1] and (2S,3S)-2,3-octanediol [(2S,3S)-2] from their nota of prothoraces as sex pheromone components. Their structural similarity suggests that one of them is a biosynthetic precursor of another component. In order to confirm the biochemical conversion, deuterated derivatives of both components were synthesized starting from a Wittig reaction between hexanal and an ylide derived from D5-iodoethane and ending with enantiomeric resolution by chiral HPLC. Molecular ions of 1 and 2 were scarcely detected using GC-MS analysis, and the labeled compounds showed similar mass spectra as unlabeled pheromone components. However, several fragment ions, including four deuterium atoms, were observed in the mass spectra of their acetate derivatives, indicating that the conversion could be confirmed by examining a compound with the diagnostic ions after the acetylation of volatiles collected from the insects treated with the labeled precursors.
2009-03 (6Z,9Z,12Z)-6,9,12-Octadecatriene and (3Z,6Z,9Z,12Z)-3,6,9,12-icosatetraene, the novel sex pheromones produced by emerald moths
(6Z,9Z,12Z)-6,9,12-Octadecatriene and (3Z,6Z,9Z,12Z)-3,6,9,12-icosatetraene, hydrocarbons unsaturated more highly than usual lepidopteran Type II pheromones, were identified from geometrid females of
Hemithea tritonaria and
Thalassodes immissaria intaminata, respectively. The triene was synthesized by a double Wittig reaction between hexanal and an ylide derived from (Z)-1,6-diiodo-3-hexene, and the tetraene was synthesized by a coupling between (Z)-3-undecenal and an ylide derived from (3Z,6Z)-1-iodo-3,6-nonadiene. Each synthetic compound attracted males of the corresponding emerald moths in a field. In order to obtain further information for the pheromones, we are synthesizing (6Z,9Z,12Z)-trienes and (3Z,6Z,9Z,12Z)-tetraenes with a C17-C23 chain and carrying out their random screening tests to find new lepidopteran attraction in a field.
2009-04 Synthesis and field evaluation of methyl-branched ketones, sex pheromone components produced by female lichen moths in the family of Arctiidae
Female moths of
Lyclene dharma dharma (Arctiidae, Lithosiinae) produce a novel sex pheromone composed of three methyl-branched ketones (I-III) in a ratio of 2:1:1. In order to confirm the structure of III (6,14-dimethyl-2-octadecanone), a mixture of its four stereoisomers was synthesized via chain elongation by two Wittig reactions, starting from 1,7-hexanediol. GC-MS data of the synthetic III were satisfactorily coincident with those of the natural component. In addition to the racemic mixtures of I (6-methyl-2-octadecanone) and II (14-methyl-2-octadecanone), previously synthesized, the activity of III was evaluated in the Iriomote Islands, and effective male attraction was observed for the 2:1:1 mixture of I-III. This result indicates that the females do not produce only one stereoisomer for each component or that the response of the males is not disturbed by the other stereoisomers of natural isomers produced by the females. The field test also revealed that the two-component lure of I and II captured as many males as the mixture of I-III, while lures baited with two components in other combinations and with only one component scarcely exhibited any male attraction ability.
2009-05 Monitoring, mass trapping, and mating disruption of the citrus fruit moth, Prays sp., by the synthetic sex pheromone in the Mekong Delta of Vietnam
GC-EAD and GC-MS analyses showed that (Z)-7-tetradecenal (Z7-14:Ald) is a major sex pheromone component of the citrus fruit moth (
Prays sp., Yponomeutidae; Praydinae), which is a harmful pest of pomelos in Vietnam and different species from the citrus flower moth,
Prays citri. Therefore, synthetic Z7-14:Ald was applied to control this species at pomelo orchards in Vinh Long province. Monitoring with sticky traps baited with the lure (rubber septum with 0.5 mg of Z7-14:Ald) revealed that the adult flights were observed throughout the year with two remarkable peaks; one was in January and another was in April. In a trial of mass trapping, damage of the fruits was more effectively suppressed by 20 traps/0.1 ha (the lure were renewed every month) than by traditional spraying of an insecticide. Furthermore, mating disruption with the pheromone (polyethylene tube with 80 mg of Z7-14:Ald) successfully control the insect infection. The damaged fruits were about 7% of the total harvesting in a disrupted area (0.75 ha, 400 dispensers/ha) and 10% in another disrupted area (0.6 ha, 200 dispensers/ha), meanwhile more than 50% of fruits were infected by the larva in an untreated area.
2009-06 Identification and field evaluation of the sex pheromone components secreted by the Japanese cherry treeborer, Synanthedon hector (Lepidoptera: Sesiidae)
The Japanese cherry treeborer,
Synanthedon hector (Lepidoptera: Sesiidae), is a harmful pest of Prunus trees. After discovery of the male attraction by a 50:50 mixture of (3Z,13Z)- and (3E,13Z)-3,13-octadecadienyl acetates (I and II) (Yaginuma et al., 1976), a mating disruptant (cherrylure, Sukashiba-conR) formulated with the two 3,13-dienes was developed and commercialized; however, the sex pheromone secreted by the females has not been characterized. Analyses of pheromone gland extracts using gas chromatography (GC) equipped with an electroantennographic detector and GC combined with mass spectrometry showed that the virgin females produced I and II, in addition to (2E,13Z)-2,13-octadecadienyl acetate (III). The ratio of the three components (I:II:III) was about 60:36:4. In the field, the S. hector males were effectively attracted by a lure baited with the mixture of I and II at ratios of 70:30 - 30:70, while III showed neither synergistic nor inhibitory effects on the attraction. Further experiments with other geometrical isomers of the 3,13-dienes indicated that male moths strictly discriminated the configurations of the two double bonds. Mixtures combining one insect-produced isomer (I or II) and another unnatural isomer attracted few males in the field. When the traps were placed at 0.5 - 4.5 m high from the ground, the males were effectively caught by the traps at the higher positions. The color of traps did not affect the male attraction.
2010-01 Chiral HPLC analysis of lepidopteran sex pheromones
Since the pheromone content in insects is very low, the absolute configuration of natural pheromones has been estimated by the biological activity of the synthetic stereoisomers, such as field attraction. However, it is important to understand the stereochemistry of the pheromones actually produced by the insects. In the case of lepidopteran female sex pheromones, epoxy compounds include chiral centers. A chiral GC column seems to have the possibility to separate enantiomers, but the applications are still very limited. We examined the resolution abilities of commercialized chiral HPLC columns in detail and observed desirable separation under a normal-phase condition. By applying the chiral columns, we successfully determined the absolute configuration of several epoxy pheromones. Recently, we identified methyl-branched ketones from a lichen moth as sex pheromone components; 6-methyl-2-octadecanone (1) and 14-methyl-2-octadecanone (2). These components include chiral centers. Male moths were attracted by a mixture of (S)-1 and (S)-2 and also by a mixture of racemic 1 and 2. While the chiral GC did not achieve the resolution of the methyl-branched compounds, a normal-phase chiral HPLC column interestingly succeeded in the enantiometric separation of 1 and 2. The chiral HPLC analysis of the pheromone extract revealed that the females did not produce optically pure compounds but dominantly (S)-1 and (S)-2. This study is the first successful application of chiral HPLC to methyl-branched pheromones, and it is noteworthy that enantiomers of methyl-branched compounds showed different chromatographic behaviors on a chiral HPLC column.
2010-02 Novel sex pheromones produced by emerald moths
Lepidopteran Type II sex pheromones, which are biosynthesized from dietary linolenic and linolic acids, are mainly composed of 6,9-dienes, 3,6,9-trienes, and their epoxides and identified from the species in highly evolved families, such as Geometridae. This group is classified into several subfamilies, such as Larentiinae, Ennominae, and Geometrinae, and sex pheromones have been exclusively studied in species in the first two subfamilies. Geometrinae includes many species called emerald moths with green wings; however, the pheromones from these species have not been identified, indicating a high probability of finding new pheromone compounds in the emerald moths. In order to understand Type II pheromones in depth, we collected male and females in several emerald moths using a light trap and analyzed their pheromone extracts using GC-EAD and GC-MS. Consequently, in addition to known unsaturated hydrocarbons, we could find novel Type II pheromones, (Z,Z,Z)-6,9,12-octadecatriene from
Hemithea tritonaria and (Z,Z,Z,Z)-3,6,9,12-icosatetraene from
Thalassodes immissaria intaminata. The triene was synthesized by a double Wittig reaction between hexanal and an ylide derived from (Z)-1,6-diiodo-3-hexene. The tetraene was synthesized by a coupling between (Z)-3-undecenal and an ylide derived from (Z,Z)-1-iodo-3,6-nonadiene. Furthermore, pheromone gland extracts of both species interestingly contained (E,E)-α-farnesene as an EAG-active component. A field evaluation of the synthetic compounds showed that the Type II components were essential for male attraction and (E,E)-α-farnesene acted as a synergist. This is the first mating communication system of lepidopteran species mediated by farnesene, a representative sesquiterpene.
2010-03 Identification of sex pheromone components of the pear barkminer moth (Gracillariidae)
By GC-EAD analysis, three EAG-active components (I-III) were found in a pheromone gland extract of the female moths of the pear barkminer,
Spulerina astaurota. This insect is a harmful pest of pear trees,
Pyrus pyrifolia (Rosaceae) in Japan, and it also habits Korea and India. The larvae mine and feed inside the bark of branches, causing injuries, which might contribute to infection by other pest insects and diseases. On column chromatography with Florisil, the minor components I and II were mainly eluted with 10% ether in hexane and the major component III with 30% ether, indicating differences in the terminal functional groups of Type I lepidopteran pheromones. While the content of I was too small to determine the structure, GC-MS analysis indicated that II and III were C14 dienyl acetate and alcohol, respectively. Their spectra included diagnostic ions for 9,11-dienyl compounds at m/z 82 and 95, and the double bond position was confirmed by an experiment with 4-methyl-1,2,4-triazoline-3,5-dionone. On a GC system with a polar capillary column, the natural pheromone components showed the same retention times as those of synthetic standards with a (Z9,Z11)-configuration among the four geometrical isomers of 9,11-dienes. Therefore, we synthesized the (Z9,Z11)-isomers by using a Sonogashira coupling and hydroboration protonolysis as the key reactions. In this year, we will evaluate the synthetic lures and examine application of the sex pheromone for the IPM programs.
2010-04 Sex pheromone biosynthesis in the fall webworm moth, Hyphantria cunea (Arctiidae)
Part 1: Conversion of linolenyl alcohol into an aldehyde component and substrate specificity of the oxidation
The fall webworm is a harmful polyphagous defoliator. The female moths produce the following four pheromone components in a ratio of about 5:4:10:2; (Z,Z)-9,12-octadecadienal (I), (Z,Z,Z)-9,12,15-octadecatrienal (II), cis-9,10-epoxy-(Z,Z)-3,6-henicosadiene (III), and cis-9,10-epoxy-(Z,Z)-1,3,6-henicosatriene (IV). All of the components are expected to be biosynthesized from linoleic or linolenic acids in the diets, but different pathways are proposed for the aldehydes and expoxides noted above. It is interesting to identify the enzymes involved in biosynthesis as well as the mechanisms of the regulation of the mixing ratio. Before starting these studies, we confirmed the biosynthetic pathways and the substrate specificity of the enzymes.
As the first step of the study on the biosynthesis of the aldehyde II, the pheromone glands of virgin females were treated with 13C18-linolenic acid or 13C18-linolenyl alcohol. Products in the glands were extracted with hexane at 4 or 20 hr after the treatment, and then analyzed by GC-MS. While II including 18 13C showed almost the same Rt as unlabeled endogenous II, the labeled aldehyde with M+ at m/z 280 was found in the pheromone glands treated with alcohol but not with acid. This result showed that II was biosynthesized via the corresponding alcohol by oxidation. Furthermore, long-chain analogs (10,13-nonadecadien-1-ol and 10,13,16-nonadecatrien-1-ol) were converted into the corresponding aldehydes in the pheromone glands, suggesting the low substrate specificity of the alcohol oxidase in the pheromone gland.
2010-05 Sex pheromone biosynthesis in the fall webworm moth, Hyphantria cunea (Arctiidae)
Part 2: Conversion of C21 triene into the epoxydiene component and substrate specificity of the oxidation
The female moths secrete cis-9,10-epoxy-(Z,Z)-3,6-henicosadiene (Z3,Z6,epo9-21:H) as a major pheromone component. This epoxydiene is expected to be produced by epoxidation of the corresponding C21 triene (Z3,Z6,Z9-21:H), which is biosynthesized from dietary linolenic acid in oenocytes and transported to a pheromone gland via the hemolymph with the help of lipophorin. To confirm the final biosynthetic step in the pheromone gland, [19,20,21,21,21]-D5-Z3,Z6,Z9-21:H was synthesized by chain elongation of linolenic acid and LiAlD4 reduction as key reactions. GC-MS analysis of the extract of pheromone glands topically applied with D5-Z3,Z6,Z9-21:H revealed the formation of D5-Z3,Z6,epo9-21:H, indicating specific epoxidation of the double bond at the 9-position in the female glands. Based on this result, the substrate specificity of epoxidase was examined by treatments with mixtures of D5-Z3,Z6,epo9-21:H and some other hydrocarbons, such as C19-C23 trienes, C21 diene (Z6,Z9-21:H), and C21 monoene (Z9-21:H). The 9,10-epoxy derivative of each unsaturated hydrocarbon was detected in the GC-MS analysis, while the yields of the epoxides from the C22 and C23 trienes and the C21 monoene were poorer than those from the other trienes and diene. This experiment indicates that the substrate specificity of the eopxidase in the pheromone gland is low and the species-specific pheromone of
H. cunea females is mainly owed to the critical formation of polyenyl hydrocarbons restricted to the precursors of epoxy components. By injection of the mixed DMSO solutions, we are examining the selectivity of the step for the transportation of the precursors incorporated into the pheromone gland from the hemolymph.
