94 Identification of a Sex Pheromone Component of the Geometrid Moth Milionia basalis pryeri.
A single component in extracts of virgin female
Milonia basalis pryeri moths elicited response from male moth antennae. This compound (ca. 7 ng / female) was identified as (
Z,Z)-(3
S,4
R)-3,4-epoxynonadeca-6,9-diene by GC-MS and NMR analyses, microchemical reactions, and comparative chiral HPLC. In a field test, synthetic (
Z,Z)-(3
S,4
R)-3,4-epoxynonadeca-6,9-diene attracted male moths. The opposite enantiomer, the racemic mixture, and virgin female moths held in small cages attracted no more moths than the solvent controls.
95 Sex Pheromones of Five Olethreutine Species (Lepidoptera: Tortricidae) Associated with the Seedlings and Fruits of Mangrove Plants in the Ryukyu Islands, Japan: Identification and Field Evaluation.
The sex pheromones of three Cryptophlebia, one Centroxena, and one Eucosma species (Lepidoptera: Olethreutinae) inhabiting mangroves in the Ryukyu Islands, Japan, were studied with techniques using gas chromatography (GC) equipped with an electroantennographic (EAG) detector and GC-mass spectrometry (GC-MS). The larvae of each Cryptophlebia species are specifically associated with viviparous seedlings from one of three mangrove Rhizophoraceae plants. While three EAG-active alcohol components, (
Z)-8-dodecen-1-ol, (
E)-8-dodecen-1-ol, and dodecan-1-ol, in a ratio of 100:12:4, were identified from the pheromone gland extract of female moths of
C. horii (host:
Bruguiera gymnorrhiza), two other sibling species did not produced them but produced the corresponding acetates, i.e., (
Z)-8-dodecenyl acetate, (
E)-8-dodecenyl acetate, and dodecyl acetate, in a 100:2:3 ratio from
C. palustris (host:
Rhizophora stylosa in Iriomote-jima Island) and in a 100:7:13 ratio from
C. amamiana (host:
Kandelia candel in Amami-oshima Island). The double-bond positions of the monounsaturated components were confirmed by GC-MS analysis of their adducts with dimethyl disulfide. On the other hand, the larvae of
Centroxena sp. feed on fruits of
Sonneratia alba, another mangrove plant in the Sonneratiaceae, and the extract of the female pheromone glands contained (8
E,10
E)-dodecadienyl acetate and dodecyl acetate in a ratio of 100:5. The double-bond position of the diunsaturated compound was confirmed by GC-MS analysis of its adduct with 4-methyl-1,2,4-triazoline-3,5-dione. Furthermore, (
E)-9-dodecenyl acetate was exclusively identified in the pheromone gland extract of
Eucosma coniogramma females which also appeared from the seedlings of
B. gymnorrhiza. Although the roles of minor components have not been revealed by field tests, synthetic lures baited with the main pheromone component of each species successfully attracted the target males, confirming that the sex pheromone is one of the most important factors for their reproductive isolation.
96 Sex Pheromone and Related Compounds in the Ishigaki and Okinawa Strains of the Tussock Moth Orgyia postica (Walker) (Lepidoptera: Lymantriidae).
Two distinct EAG-active components, A and B, and a weakly active component C were found in a solvent extract from virgin females of the Ishigaki strain of the tussock moth,
Orgyia postica (Walker). Components A, B and C were found in the extract of the females at 4.0, 0.5 and 4.0 ng / female, respectively. Components A, B and C were identified as (6
Z,9
Z,11
S,12
S)-11,12-epoxyhenicosa-6,9-diene [(11
S,12
S)-1: posticlure], (6
Z)-henicos-6-en-11-one (2) and (6
Z,9
Z)-henicosa-6,9-diene (3), respectively. Component B was absent in the extract from an Okinawa strain, in which components A and C were present at 2.0 and 1.5 ng / female, respectively. (11
S,12
S)-1 and the racemic mixture showed attractiveness both for Okinawa and Ishigaki strains, whereas (11
R,12
R)-1 did not. Addition of 2 significantly reduced the trap catches with (11
S,12
S)-1 on Okinawa strain that lacked 2, while there was no significant inhibitory effect on the Ishigaki strain. Addition of 3 to (11
S,12
S)-1 did not significantly affect trap catches at Ishigaki or Okinawa. This confirmed that attractant pheromone of
O. postica of the Ishigaki strain is also (11
S,12
S)-1.
97 Mating Communication Systems of Four Plusiinae Species Distributed in Japan: Identification of the Sex Pheromones and Field Evaluation.
The sex pheromones of four Plusiinae species in the family Noctuidae,
Ctenoplusia albostriata (CA),
Macdunnoughia purissima (MP),
Syngrapha ain (SA), and
Diachrysia. stenochrysis (DS), were identified by GC-EAD and GC-MS analyses. These were subsequently evaluated by a series of field trails in different ecological habitats. CA females produced (
Z)-5-decenyl acetate (I), (
Z)-7-dodecenyl acetate (II), and (
Z)-7-dodecen-1-ol (III) in a ratio of 2:100:13. While the antennae of CA males responded to tall three components, neither I nor III was essential to capture males. The MP females produced II, III, and (
Z)-5-dodecenyl acetate (IV) in a ratio of 100:80:20, and this blend captured the most MP males. Compounds II and III were also identified from SA females, and mixtures ranging from 4:1 to 2:3 were equally as attractive to males. DS females only produced one active compound (
Z)-7-decenyl acetate (V) and is the first Plusiinae species identified as using only the 3-compound and none of 5-compounds, such as II and III, that are common components of Plusiinae pheromones. Using previously published chemical and biosynthetic data, together with the finding from this study, we proposed a classification of the Plusiinae sex pheromones.
98 Identification of the Sex Pheromone of the Citrus Leafminer (Phyllocnistis citrella Stainton, Lepidoptera: Gracillariidae) with a Trial of Control by the Communication Disruption Method.
GC-EAD and GC-MS analyses of a pheromone gland extract from virgin females of the citrus leafminer (CLM,
Phyllocnistis citrella Stainton) confirmed that the male attractant previously found by random screening tests in a field, (7
Z,11
Z)-7,11-hexadecadienal (Z7,Z11-16:Ald), is a real pheromone component of CLM. 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 CLM. 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 CLM eggs and larvae 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.
100 Synthesis and Characterization of 3,13- and 2,13-Octadecadienyl Compounds for Identification of the Sex Pheromone Secreted by a Clearwing Moth, Nokona pernix.
Several geometrical isomers of 3,13- and 2,13-octadecadien-1-ols and their acetates were synthesized starting from 1,8-octanediol or 1,9-nonanediol utilizing acetylene coupling reactions. In addition to commercially available compounds, all geometrical isomers of each dienyl compound were analyzed by NMR and GC-MS to accumulate chemical data for studies of sex pheromones secreted from clearwing moths classified into the family Sesiidae of Lepidoptera. Although acetoxy derivatives of the 3,13- and 2,13-dienes showed almost the same mass spectra, the alcohols were distinguished by comparing the relative intensities of [M-18]
+ at
m/z 248, indicating direct differentiation of the two positional isomers without derivatization. Furthermore, each geometrical isomer eluted from a high-polar GC column with a different retention time. Base on these data, a pheromone gland extract of a sesiid moth,
Nokona pernix, was analyzed by GC-EAD and GC-MS, and two EAG-active components were identified,
viz., the (3
E,13
Z)- and (3
Z,13
Z)-isomers of 3,13-octadecadien-1-ol in a ratio of 9:1. In the field, the synthetic compounds mixed in 9:1 ratio attracted
N. pernix males well, while a single component scarcely attracted the males. The number of attracted males peaked in the middle of June, and a small second peak was observed in August.
101 Transport of a Hydrophobic Biosynthetic Precursor by Lipophorin in the Hemolymph of a Geometrid Female Moth Which Secretes an Epoxyalkenyl Sex Pheromone.
Previous experiments with a geometrid species,
Ascotis selenaria cretacea, have suggested that a pheromonal C
19 3,4-epoxy-6,9-diene is biosynthesized from the corresponding 3,6,9-triene produced outside of a pheromone gland and transported to it via hemolymph after association with lipophorin. In order to clarify this transport, high-density lipophorin (HDLp) in the female moths showing two bands (apoLp I with ca. 250 kDa and apoLp II with ca. 80 kDa) on an SDS-PAGE was purified by KBr equilibrium density-gradient ultracentrifugation, and the association of the triene was confirmed by GC-MS analysis of a solvent extract from the isolated protein. Next, the role of HDLp was revealed by a topical application of the deuterated trienyl precursor to the abdomens of the females. The trienyl precursor was associated with HDLp. In their pheromone glands, the triene and the deuterated epoxy pheromone were detected, indicating movement of the triene via the hemolymph. Experiments with male moths of
A. s. cretacea and female moths of
Bombyx mori showed the same association of HDLp with the triene topically applied. This result suggested that the adult females of
A. s. cretacea did not develop HDLp specialized in the triene transport. Furthermore, the topical application of a mixture including the trienyl precursor and two other related hydrocarbons showed equal amounts of association by HDLp but selective delivery of the precursor to pheromone glands in the
A. s. cretacea females.
102 Sex Pheromone of the Soybean Pod Borer, Leguminivora glycinivorella (Lepidoptera: Tortricidae): Identification and Field Evaluation.
With reference to the data of synthetic standards, GC-EAD and GC-MS analyses of a pheromone gland extract of the soybean pod borer,
Leguminivora glycinivorella (Lepidoptera: Tortricidae), resulted in three acetates [dodecyl acetate, (8
E,10
E)-8,10-dodecadienyl acetate (E8,E10-12:OAc), and its (8
E,10
Z)-isomer] at a ratio of 10:100:2.5 as a candidate of the pheromone. While the contents in the glands were low and even the titer of the major pheromonal component was ca. 0.5 ng/female, the positions of the two double bonds were confirmed by a mass spectrum of the adduct with 4-methyl-1,2,4-triazoline-3,5-dione. In a soybean field, synthetic E8,E10-12:OAc successfully attracted male moths of
L. glycinivorella, and highly selective attraction was observed for E8,E10-12:OAc among the geometrical isomers of the 8,10-diene. Neither of the two minor components showed a synergistic effect on field attraction by E8,E10-12:OAc, and their roles were unclear. A binary lure of E8,E10-12:OAc and (
E)-8-dodecenyl acetate (E8-12:OAc) was necessary to attract the male moths of
Matsumuraeses falcana, the soybean pod worm, but E8-12:OAc strongly inhibited the attraction of
L. glycinivorella by E8,E10-12:OAc, indicating that E8-12:OAc secreted by
M. falcana is one of the most important factors in the reproductive isolation of these two species.
103 Sex Pheromones of Two Melittini Species, Macroscelesia japona and M. longipes (Lepidoptera: Sesiidae): Identification and Field Attraction.
Two Melittini species,
Macroscelesia japona and
M. longipes (Lepidoptera: Sesiidae) are native to Japan, but occupy different localities since their host plants never grow together. Using pheromone gland extracts of adult females that were obtained after rearing larvae collected in the field, the sex pheromones of the two species were investigated by GC-EAD and GC-MS analyses. Two EAG-active components were found in a crude extract of
M. japona female pheromone gland, and assigned to (2
E,13
Z)-2,13-octadecadien-1-ol (E2,Z13-18:OH) and (2
E,13
Z)-2,13-octadecadienal (E2,Z13-18:Ald). The average ratio of these two components analyzed with twenty females individually was about 1:10. In the field, while
M. japona males could be attracted by traps baited with a single component lure of E2,Z13-18:Ald, the strongest attraction was observed with a 1:100 mixture of E2,Z13-18:OH and E2,Z13-18:Ald. Interestingly, the corresponding acetate, which was not detected in the extract, boosted the strength of the attractant. In an extract of
M. longipes, the same two components were found but in a markedly different mixing ratio. Mating behavior in
M. longipes males was most strongly induced by a 20:1 mixture of E2,Z13-18:OH and E2,Z13-18:Ald, while a lure of unmixed E2,Z13-18:OH could also catch males, indicating that reproductive isolation of these two species is ensured by the difference in composition ratios of the pheromone components.
104 Determination of the Pheromone Producing Region That Has Epoxidation Activity in the Abdominal Tip of the Japanese Giant Looper, Ascotis selenaria cretacea (Lepidoptera: Geometridae).
The epoxydienyl sex pheromone of
A. s. cretacea can be detected only within a rod-like abdominal tip (RAT) of the female. To clarify which part of the RAT is the sex pheromone producing region, the RAT was morphologically divided into three sections, defined positionally from the abdomen as sections A, B, and C. GC-MS measurements clearly showed that the sex pheromone compound levels in section B were four times greater than those of the other sections. Microscopic dissection analysis revealed that section B consists of four tissues: rectum, oviduct, musculature, and intersegmental membrane. GC-MS analysis of the individual tissues revealed that ~90% of the sex pheromone in section B is localized in the intersegmental membrane. A cell layer was found in the intersegmental membrane after staining with propidium iodide. Furthermore, incubation of tissues dissected from section B with a deuterated trienyl pheromone precursor revealed that the labeled epoxy pheromonal component was detected exclusively in the intersegmental membrane. We have determined that the sex pheromone producing region of
A. s. cretacea is on the terminal side of the intersegmental membrane located between the 8th and 9th abdominal segments.
105 Identification of a Unique Phromonotropic Neuropeptide Including Double FXPRL Motifs from a Geometrid Species, Ascotis selenaria cretacea, Which Produces an Epoxyalkenyl Sex Pheromone.
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 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 8 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 SGNP, i.e., SVDFTPRLGRQLVDDVPQRQQIEEDRLGSRTRFFSPRL-NH
2, as the first determination of PBAN from the insects producing an epoxyalkenyl sex pheromone.
107 Analysis of Odorant-binding Proteins in Antennae of a Geometrid Species, Ascotis selenaria cretacea, 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 AscrPBP1 was isolated by immunoprecipitation with the antiserum, and its binding ability was evaluated by using synthetic sex pheromonal compounds with a C
19 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.
108 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 four geometric isomers of each dienal eluted in the following order: ZZ → EZ → ZE → EE. 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 because of the lack of a conjugated system, 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. Furthermore, field evaluation of four synthetic geometric isomers of the 2,13-dienal revealed specific attraction to a lure with the (2
E,13
Z)-isomer as a main component.
109 A Novel Lepidopteran Sex Pheromone Produced by Females of a Lithosiinae Species, Lyclene dharma dharma, in the Family of Arctiidae.
Female moths of
Lyclene dharma dharma (Arctiidae, Lithosiinae) produced three pheromone components (I-III), which strongly stimulated male antennae. Using GC-MS analysis and chemical derivatizations, the following structures were estimated: 6-methyl-2-octadecanone (I), 14-methyl-2-octadecanone (II), and 6,14-dimethyl-2-octadecanone (III). While the stereochemistry of the chiral centers could not be determined because it was difficult to collect a sufficient amount of the natural pheromone, the plain structures of I and II were confirmed by synthesis of the racemic mixtures starting from diols. 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.
110 7,11,13-Hexadecatrienal Identified from Female Moths of the Citrus Leafminer as a New Sex Pheromone Component: Synthesis and Field Evaluation in Vietnam and Japan.
(7
Z,11
Z)-7,11-Hexadecadienal (Z7,Z11-16:Ald), which has been identified from female moths of the citrus leafminer (
Phyllocnistis citrella, Lepidoptera: Gracillariidae), strongly attracts conspecific males in Japan. Recently, in addition to the dienyl aldehyde, a trienyl derivative, (7
Z,11
Z,13
E)-7,11,13-hexadecatrienal (Z7,Z11,E13-16:Ald), was found as another sex pheromone component of females collected in Brazil and California. Thus, we synthesized Z7,Z11,E13-16:Ald and its isomer (Z7,E11,E13-16:Ald) to evaluate their effects on males inhabiting Asia. Starting from 1,7-heptanediol, two corresponding alcohols with trienyl structures were prepared by two applications of the Wittig-coupling reaction and then oxidized to yield objective aldehydes after separation by HPLC with an ODS column. In a citrus orchard in Can-Tho City, Vietnam,
P. citrella males could not be caught by a lure baited only with Z7,Z11-16:Ald, but were successfully attracted with a 1:3 mixture of Z7,Z11-16:Ald and Z7,Z11,E13-16:Ald. On the other hand, in citrus orchards in Ogasawara (Bonin) Islands and Ehime Prefecture, Japan, neither trienals showed a synergistic effect on male capture by the dienal. Far from being reinforced, the attraction activity of the dienal was diminished by mixing in Z7,Z11,E13-16:Ald. These results indicated that the sex pheromone of the Vietnamese strain is similar to that of Brazilian and Californian strains, but the Japanese strain has established a different communication system from those of the foreign strains.
111 Synthesis and Chemical Characterization of Hydrocarbons with a 6,9,11-, 3,6,9,11-, or 1,3,6,9-Polyene System, Pheromone Candidates in Lepidoptera.
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. Since diversity in an insect species is especially associated with further structural modification, we synthesized polyunsaturated hydrocarbons with a C
17-C
21 chain including an extra double bond, in order to accumulate new information for pheromone research. 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 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, 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.
112 Identification of the Sex Pheromone Secreted by Synanthedon hector (Lepidoptera: Sesiidae).
The Japanese cherry treeborer,
Synanthedon hector (Lepidoptera: Sesiidae), is a harmful pest of Prunus trees. After male attraction using a 50:50 mixture of (3
Z,13
Z)- and (3
E,13
Z)-3,13-octadecadienyl acetates (I and II) was determined, a mating disruptant formulated with the two compounds was developed and commercialized, the sex pheromone secreted by the
S. hector females, however, has not been characterized. Analyses of a pheromone gland extract using a gas chromatography (GC) equipped with an electroantennographic detector and GC combined with mass spectrometry showed that the virgin females produced the two 3,13-dienes, I and II, in addition to (2
E,13
Z)-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 a mixture of I and II at a ratio of 70:30 - 30:70, and III showed neither synergistic nor inhibitory effects on the attraction. Further experiments with other geometrical isomers of the 3,13-dienes indicated that the male moths strictly discriminated the configurations of the two double bonds. Mixtures combining one insect-produced isomer (I or II) and other unnatural isomers scarcely attracted the males in the field.
113 Identification of novel C20 and C22 trienoic acids from arctiid and geometrid female moths that produce polyenyl Type II sex pheromone components.
GC-MS and GC-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 C
20 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 by traps baited with a 1:2 mixture of the above-described synthetic C
21 polyenes. In addition to the pheromone components, lipids were extracted from the abdominal integument and its associated oenocytes and peripheral fat bodies. Fatty acid methyl esters (FAMEs) derived from the lipids 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 analysis. These novel C
20 and C
22 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 C
21 trienyl component might be formed by decarboxylation of the C
22 acid. On the other hand, the C
20 acid, but not the C
22 acid, was found in FAMEs of
Ascotis selenaria cretacea (Lepidoptera: Geometridae), which secretes C
19 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.
115 Instrumental analysis of terminal-conjugated dienes for reexamination of the sex pheromone secreted by a nettle moth, Parasa lepida lepida.
Conjugated dienyl compounds make one of the main groups of lepidopteran sex pheromones, and GC has been frequently used to determine the configurations of the double bonds. However, the separation of two geometric isomers of a terminal-conjugated diene, such as 7,9-decadien-1-ol secreted by a nettle moth
Parasa lepida lepida (Limacodidae), is assumed to be difficult. In order to clarify the chromatographic separation of the terminal dienes, 7,9-decadienyl and 9,11-dodecadienyl compounds (alcohols, acetates, and aldehydes) were analyzed by GC and HPLC. On a capillary GC column, the (
E)-isomers flowed out slightly faster than the corresponding (
Z)-isomers, but their peaks almost overlapped. On the other hand, HPLC equipped with an ODS column completely separated the two geometric isomers examined and the (
Z)-isomers eluted from the column faster than the (
E)-isomers without dependence on a functional group. In addition to undergoing direct HPLC analysis without derivatization, the dienyl alcohols were converted into 3,5-dinitrobenzoates and analyzed by LC-ESI-MS operated under the same reversed-phase condition. The two separated geometric isomers were sensitively monitored by negative ions at
m/z 211, M, M+1, M+17, and M+31, which were characteristically derived from the benzoates. Based on these results, a pheromone extract of
P. l. lepida was examined, and it was confirmed that the female moths exclusively produced the (
Z)-isomer of the 7,9-diene. Furthermore, a GC-EAD analysis and a field evaluation with both geometrical isomers indicated that the mating communication of
P. l. lepida is predominantly mediated with the (
Z)-isomer.
116 Identification of a new pheromone-binding protein in the antennae of a geometrid species and preparation of its antibody to analyze the antennal proteins of moths secreting type II sex pheromone components.
The full-length cDNA sequence of a new pheromone-binding protein (AscrPBP2) was determined from a geometrid moth,
Ascotis selenaria cretacea, which secreted a Type II sex pheromone, and an antiserum against its recombinant protein overexpressed in
Escherichia coli was prepared. In addition to this antiserum against AscrPBP2, antibodies against AscrPBP1 and general odorant-binding proteins of
Bombyx mori were used in Western blotting experiments to analyze the proteins in the antennae of several lepidopteran species secreting Type II sex pheromone components.
117 Synthesis and field evaluation of methyl-branched ketones, sex pheromone components produced by Lithosiinae female 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.
118 (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.
(6
Z,9
Z,12
Z)-6,9,12-Octadecatriene and (3
Z,6
Z,9
Z,12
Z)-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 (3
Z,6
Z)-1-iodo-3,6-nonadiene. Each synthetic compound attracted males of the corresponding emerald moths in a field.
119 Synthesis of deuterated sex pheromone components of the grape borer, Xylotrechus pyrrhoderus.
Adult males of the grape borer,
Xylotrechus pyrrhoderus, secrete (
S)-2-hydroxy-3-octanone [(
S)-1] and (2
S,3
S)-2,3-octanediol [(2
S,3
S)-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 D
5-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.
