International Meeting Abstract (2011-)

 
 

2011-01 Characterization of epoxytrienes derived from (3Z,6Z,9Z)-1,3,6,9-tetraenes, sex pheromone components of arctiid moths and related compounds

9,10-Epoxy-(3Z,6Z)-1,3,6-henicosatriene has been identified from female moths of Hyphantria cunea. Since diversity of lepidopteran species suggests that some species utilize the positional isomers for their mating communication, we systematically synthesize and characterized the epoxytrienes with a C19-C21 chain. While 1,2-epoxy-3,6,9-trienes were not yielded, MCPBA oxidation of (3Z,6Z,9Z)-1,3,6,9-tetraenes produced mixtures of three epoxides, 3,4-epoxy-1,6,9-trienes (1), 6,7-epoxy-1,3,9-trienes (2), and 9,10-epoxy-1,3,6-trienes (3), which were separable by GC and LC. Detailed inspection of their mass spectra indicated the following diagnostic ions for determining the chemical structures; m/z 128, 167, M-87, and M-85 for 1, m/z 111, M-125, and M-69 for 2, and m/z M-125 and M-139 for 3. Resolution of two enantiomeres of each epoxytriene was achieved by HPLC equipped with a chiral column. The elution order of the enantiomers was the same as that of the corresponding epoxydiene derived from a (3Z,6Z,9Z)-3,6,9-triene. Chiral HPLC analysis of the pheromone extracted from virgin females of H. cunea revealed 9S,10R configuration of the natural epoxytriene.


 
 

2012-01 GC-FT-IR analyses of sex pheromones secreted by nettle moths

While an IR spectrometer is useful to determine functional groups of an unknown compound, it has been rarely used for pheromone researches because of low sensitivity. Recently, a new type of GC-FT-IR (Discover IR, Spectra Analysis) has been developed by applying a system with a cooled and turned disk, where compounds eluted from a capillary GC column are fixed and irradiated with an IR beam. The FT-IR measurement is carried out sensitively as well as GC-MS.
Several nettle moth species in the family of Limacodidae secrete sex pheromone components with a terminal conjugated dienyl structure. Separation of two geometrical isomers of the terminal dienes is insufficient even on a high polar GC column and it is not easy to determine configuration of the double bond by the chromatographic behaviour. However, GC-FT-IR measurement of a crude pheromone extract of Parasa lepida lepida could confirm Z-configuration of 7,9-decadien-1-ol, which showed no absorption at 960 cm-1. On the other hand, the dienyl component of another nettle moth species, Monema flavescens, showed a band at 960 cm-1 indicating E-configuration.


 
 

2013-01 Enantioselective syntheses of insect pheromones with methyl-branched skeletons verified by chiral HPLC analyses

Many chiral insect pheromones with methyl-branched skeletons have been identified. Synthesis of optically pure stereoisomers is very important because the stereochemistry of natural pheromones can be elucidated by biological activity of the synthetic isomers. In addition to natural occurring chiral substances, commercialized chiral reagents are utilized as starting materials for stereospecific syntheses. Both of the enantiomers of propylene oxide (1) are inexpensive and can be easily converted into various chiral secondary alcohols by a Grignard reaction. Attacks of appropriate nucleophiles upon tosylates (2a and 2b) derived from chiral secondary alcohols are expected to yield chiral building blocks (3a and 3b) with a branched methyl group accompanied by inversion of the stereochemistry. The SN2 reaction, however, has never been utilized for a pheromone synthesis because the complete conversion might be hard to monitor. Recently, we found that enantiomers of some methyl-branched compounds could be separated by chiral HPLC; thus the SN2 reaction accomplished without racemization was verified. By a coupling reaction between 3a and 3b, we successfully synthesized some sex pheromones, such as 5,9-dimethylheptadecane of the leaf mining moth Leucoptera scitella and 6,10,13-trimethyltetradecan-2-one of the stink bug Pallantia macunaima.


 
 

2013-02 Enantioselective synthesis and field evaluation of methyl-branched secondary alcohols, a novel pheromone component of a lichen moth and its structure-related compounds

Females of the lichen moth, Miltochrista calamina (Arctiidae, Lithosiinae) produce 5-methylheptadecan-7-ol (1) as a sex pheromone. Of four synthetic stereoisomers prepared by separation from two synthetic diastereomer mixtures, males in fields were specifically attracted only by the (5R,7R)-isomer. To improve the synthesis, a new route for (5R,7R)-1 starting from (S)-propylene oxide was achieved by utilizing the SN2 reaction of an optically active secondary tosylate and the Jacobsen hydrolytic kinetic resolution of an epoxy intermediate as key steps. Chiral HPLC analysis of the target methyl-branched pheromone and the antipode synthesized from (R)-propylene oxide confirmed their high enantiomeric excess (> 99%). Based on the stereospecific synthesis, six homologues with the same configurations as (5R,7R)-1 but a different alkyl chain(s) connected with the stereogenic centers were produced. Interestingly, attractive activity was observed for five compounds out of six, indicating that the males strictly distinguished the configurations of methyl and hydroxyl groups but inaccurately recognized the length of the two alkyl chains in the pheromone structure.


 
 

2013-03 Propionates and acetates of chiral secondary alcohols: novel sex pheromone components produced by a lichen moth Barsine expressa (Arctiidae: Lithosiinae)

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 GC-EAD and GC-MS. 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 enantio-selectively 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.


 
 

2013-04 Characterization of posticlure and the structure-related sex pheromone candidates prepared by epoxidation of (6Z,9Z,11E)-6,9,11-trienes and (3Z,6Z,9Z,11E)-3,6,9,11-tetraenes

Trans-11,12-epoxy-(6Z,9Z)-6,9-henicosadiene (posticlure) has been identified from a pheromone gland of a lymantriid species, Orgyia postica. Since the diversity of Lepidoptera suggests that some species utilize the structure-related epoxy compound as a sex pheromone component, we systematically synthesized epoxydienes and epoxytrienes from (6Z,9Z,11E)-6,9,11-trienes and (3Z,6Z,9Z,11E)-3,6,9,11-tetraenes with a C19-C21 chain and accumulated the chemical data in order to utilize for new pheromone research. Peracid oxidation of each triene and each tetraene produced a mixture of three epoxydienes (cis-6,7-epoxy-9,11-diene, cis-9,10-epoxy-6,11-diene, and trans-11,12-epoxy-6,9-diene) and four epoxytrienes (cis-3,4-epoxy-6,9,11-triene, cis-6,7-epoxy-3,9,11-triene, cis-9,10-epoxy-3,6,11-triene, and trans-11,12-epoxy-3,6,9-triene), respectively. While the 9,10-epoxy compounds were unstable and interestingly converted into 9-ketones after chromatography over SiO2, each positional isomer could be isolated by HPLC equipped with an ODS column and the chemical structure was determined by NMR analyses. On the GC-MS analysis with a polar column, the positional isomers were also eluted separately and proposed characteristic mass spectra. By comparing the spectral data of the epoxy compounds with a different carbon chain, diagnostic fragment ions reflecting the chemical structure were determined as follows: m/z 79, 109, 113, and M-114 for the 6,7-epoxydienes; m/z 69, 97, 111, 139, and M-111 for the 9,10-epoxydienes; m/z 57, 79, 109, 136, M-151, and M-111 for the 11,12-epoxydienes; m/z 79, 91, 105, and 119 for the 3,4-epoxytrienes; m/z 79, 124, M-124, M-96, and M-69 for the 6,7-epoxytrienes; m/z 79, 95, 109, 137, and M-108 for the 9,10-epoxytrienes; m/z 79, 134, M-149, M-109, and M-95 for the 11,12-epoxytrienes.


 
 

2013-05 Identification of the sex pheromone secreted by Palpita nigropunctalis (Lepidoptera: Crambidae)

The lilac pyralid, Palpita nigropunctalis Bremer (Crambidae: Pyraustinae), is a common pest of Oleaceae. A crude pheromone extract of the females was examined by GC-EAD and GC-MS. The GC-EAD analysis indicated three EAG-active components (I-III), and a GC trace by a flame ionization detector indicated a mixing ratio of about 5:1 of I and II and a very low content of III. Mass spectra of I and II were successfully recorded. Ions at m/z 238 (M+) and 220 ([M-18]+) detected for I indicated the structure of a monoenyl aldehyde with a 16-carbon chain. While M+ was not detected for II, ions at m/z 222 ([M-60]+) and 61 ([AcOH+1]+) suggested that II was a monoenyl acetate with a 16-carbon chain. GC-MS analysis of the extract treated with DMDS showed mass spectra of the adducts derived from I and II. Ions at m/z 332, 215, and 117 for the adduct of I and ions at m/z 376, 259, and 117 for the adduct of II revealed double bonds at the same 11-position.
Furthermore, the pheromone extract was examined by GC-FT-IR. An IR spectrum of I showed characteristic absorption at 1716 and 966 cm-1, which indicated a formyl group and E configuration of the double bond, respectively. In the case of II, absorption at 1745 and 968 cm-1 indicated an ester carbonyl and E configuration. As facilitated by comparison with authentic standards, we concluded that I and II were (E)-11-hexadecenal and (E)-11-hexadecenyl acetate, respectively, and speculated that III was (E)-11-hexadecen-1-ol.


 
 

2013-06 Structure determination of the sex pheromone secreted by Loepa sakaei, a giant silkworm moth inhabiting in Okinawa Island

The lepidopteran family of Saturniidae includes more than 1,000 species found all over the world, and sex pheromones have been reported for 16 species. While Japan is inhabited by 11 species, no pheromones of the endemic species have been chemically identified. Therefore, we put the study of saturniid pheromones into practice, starting with Loepa sakaei, which is found only in Okinawa and Amami-Ohshima Islnads and is designated as a quasi-endangered species.
A pheromone gland extract was prepared with two virgin female moths. GC-EAD analysis with an aliquot of the extract (0.25 female equivalent, FE) revealed one EAG active component, and the mass spectrum was obtained by GC-MS analysis with another aliquot (0.25 FE). The highest ion was detected at m/z 192, which was estimated to be [M-60]+ of tetradecadienyl acetate. The spectral pattern was similar to that of 6,11-hexadecadienyl acetate (6,11-16:OAc) published for the saturniid species Antheraea polyphemus. Based on the similarity, we estimated the EAG-active component to be 4,9-tetradecadienyl acetate (4,9-14:OAc), which included two double bonds at the same ω5- and ω10-positions as 6,11-16:OAc.
A field test with the synthetic lures was carried out from May to June 2012 in Okinawa Island. Male moths of L. sakaei were attracted only by the (4E,9Z)-isomer, indicating that mating communication of this species was mediated by E4,Z9-14:OAc. It would be interesting to know whether the C14-chain compound is a species-specific pheromone component of Japanese saturniid species. Therefore, we are analyzing pheromone components of other species, such as Aglia japonica and Rhodinia fugax.


 
 

2013-07 Sex pheromone components and field trapping tests of a clearwing moth Glossosphecia romanovi (Lepidoptera: Sesiidae)

A clearwing moth, Glossosphecia (formally Toleria) romanovi (Lepidoptera: Sesiidae), is a harmful pest of vine trees in Japan. The larvae of this species bore into the trunk of vine trees and cause serious damage. This species had not attracted much attention from the viticulturist; however, complaints about its damage were increasing in recent 10 years.
Analysis of pheromone gland extracts using GC-EAD and GC-MS showed that the virgin females produced several compounds; such as (3Z,13Z)-3,13-octadecadien-1-ol (Z3,Z13-18:OH, I), the acetate derivative (Z3,Z13-18:OAc, II), and the isomer (E3,Z13-18:OH, III). The mixing ratio of I:II:III was about 100:5:1. In the field, the males were effectively attracted by lures baited with various mixtures of major two components (I and II). However, the lure baited component III with the major two components significantly reduced the number of trapped males. When the traps were placed at 0.5 - 4.0 m high from the ground, the males were effectively caught by the traps at the higher positions.
The number of attracted males showed the bimodal type of occurrence, while this species is considered to be univoltine. From the end of May to the middle of July, small numbers of males were attracted to traps set at various environments include inside or perimeter of vineyard. On the contrary, large numbers of males were attracted to traps set at the ridges of coppices. These results suggest that the season of emergence in vineyard may be from the end of May to the middle of July, and the origin of almost captured males may be coppices near vineyards.


 
 

2013-08 Possible biosynthetic pathway for C15 sex pheromone components of Dolbina tancrei

We surveyed the sex pheromones of fifteen hawk moths and found a novel sex pheromone component 9,11-pentadecadienal from Dolbina tancrei. Pheromone components with functional groups are made from even-carbon chain fatty acids such as palmitic (C16) or stearic (C18) acids. Therefore, odd-carbon chain pheromones may be synthesized by a unique biosynthetic pathway. Here we investigated the sex pheromone precursors of D. tancrei and propose a possible pathway.
To determine sex pheromone precursors, we analyzed fatty acids in female pheromone glands. Pheromone glands of calling females were excised and extracted with dichloromethane and methanol (2:1) solvent. Glycerolipids and free fatty acids from the crude extracts were converted to the corresponding methyl esters by base methanolysis and diazomethane treatment. Chemical structures of the methyl esters were determined by GC-MS and DMDS or MTAD derivatization. As a result, methyl hexadecanoate and methyl octadecanoate were the major components detected. In addition, unsaturated C16 methyl esters, methyl 7-hexadecenoate, methyl 9-hexadecenoate, methyl 11-hexadecenoate, methyl hexadecadienoate, methyl pentadecanoate, methyl 5-pendatecenoate and methyl pendadecadienoate were also detected.
Although C15 fatty acids appear to exist in the pheromone glands, monoenyl C15 intermediates that can be pheromone precursors were not detected in the gland extracts. Assuming a biosynthetic pathway from palmitic acid as known in other hawk moths, the existence of 11-hexadecenoic and 10, 12-hexadecadienoic acids can be explained. These results suggest that 9,11-pentadecadienal is biosynthesized from palmitic acid via 11-hexadecenoic and 10,12-hecadecadienoic acids rather than from C15 fatty acids, and predict a specific enzyme for 1C chain shortening.


 
 

2014-01 Sex pheromone of the Japanese owl moth, Brahmaea japonica (Bombycoidea: Brahmaeidae)

Bombycoidea is divided into 10 families, such as Saturniidae, Sphigidae, and Brahmaeidae. Brahmaeidae consists of seven genera and about 40 species in the world, but only one species, Brahmaea japonica, inhabits Japan. The larva is a defoliator of Oleaceae trees and the large adult (about 10 cm) with huge eyespots in wings is called the Japanese owl moth. We were interested in speciation of this species, and compared the sex pheromone with those of other Bombycoidea females.
A pheromone gland extract was prepared with three virgin females. GC-EAD analysis with an aliquot of the extract (0.5 female equivalent, FE) found three EAG-active components, I - III, approximately in a ratio of 6:1:3. GC-MS analysis with the extract (0.5 FE) showed characteristic ions of I at m/z 238 (M+) and 220 ([M-18]+), indicating a C16 monoenyl aldehyde. The mass spectra of II and III showed diagnostic ions of C16 11,13-dienyl aldehydes at m/z 236 (M+), 95, and 82. With reference to GC-MS data of synthetic compounds, we determined their structures as follows; I = (Z)-11-hexadecenal, II = (11Z,13E)-11,13-hexadecadienal, III = (11Z,13Z)-isomer (Z11,Z13-16:Ald).
Synthetic lures were evaluated in the field during late spring in 2013 and 2014. Interestingly, traps baited with only Z11,Z13-16:Ald, one of the minor components, could successfully attract the males. Other two components showed no synergistic effect on the attraction and their roles in matting communication were unclear. Z11,Z13-16:Ald has been identified from some species in Pyraloidea and Noctuoidea but from no species in Bombycoidea, while the (11E,13E)-isomer is a pheromone component of Agrius convolvuli in Sphigidae.


 
 

2014-02 Reexamination of the female sex pheromone of the sweetpotato vine borer moth: Identification of tricosatriene and its field evaluation

The sweetpotato vine borer moth, Omphisa anastomosalis (Pyraloidea: Crambidae), is a serious pest in tropical and subtropical Asia-Pacific regions. From an Okinawa population in Japan, (10E,14E)-10,14-hexadecadienal (E10,E14-16:Ald) has been identified as a main pheromone component, in addition to 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 virgin females. While Pyraloidea females usually produce only Type I compounds, which are unsaturated fatty alcohols and their derivatives, mating communication of some Pyraloidea species is interestingly mediated by a combination of the Type I compounds and Type II compounds, which are unsaturated hydrocarbons and their epoxides. Based on this knowledge, we examined a pheromone extract of females inhabiting Vietnam by GC-MS and successfully found (3Z,6Z,9Z)-3,6,9-tricosatriene (Z3,Z6,Z9-23:H). A mixture of synthetic E10,E14-16:Ald and Z3,Z6,Z9-23:H could effectively attract the males in a Vietnamese field. In field test with the minor pheromone components mixed to the binary blend did not increase the number of male moths captured. A combination of Z3,Z6,Z9-23:H and another geometrical isomer of E10,E14-16:Ald scarcely attracted the males. Furthermore, E10,E14-16:Ald mixed with other polyunsaturated hydrocarbons showed that the mixtures which included a C21 triene, a C22 triene, or a C23 pentaene interestingly attracted as many males as did the mixture with Z3,Z6,Z9-23:H. The males seemed to have a strict recognition of E10,E14-16:Ald, but a low specificity in perception of Z3,Z6,Z9-23:H. The identification of a highly attractive sex pheromone will help in integrate pest management of O. anastomosalis.


 
 

2015-01 Identification and synthesis of chiral methyl-branched sex pheromones secreted by female moths

A species-specific mating communication plays an important role in reproductive isolation of lepidopteran insects, and sex pheromones have been identified from female moths of about 650 species to date. While most of them are composed of unsaturated fatty alcohols, hydrocarbons, and the derivatives with a straight chain, some species secret methyl-branched compounds. Leafminer moths in the family of Lyonetiidae and hemlock looper moths in the family of Geometridae produce dimethyl hydrocarbons, and we recently identified ketones and a secondary alcohol with a branched skeleton from lichen moths in the family of Arctiidae, indicating structural diversity of the sex pheromones. The identifications were accomplished by analyses of pheromone gland extracts using GC-EAG and GC-MS techniques, and stereochemistry of the natural pheromones was estimated by field evaluation of all enantiomers with a high enantiomeric excess (ee). For the enantioselective synthesis, we achieved a simple method of constructing a chiral methyl-branched skeleton by an SN2 reaction with a tosylate of 2-alkanol, which was derived from commercialized (R)- and (S)-propylene oxide. Perfect configurational inversion was successfully confirmed by enantioselective HPLC analysis, and two kinds of chiral building blocks with a methyl branch at the 3- or 2-position could be obtained by selecting an appropriate carbanion as a nucleophile. In addition to chiral monomethyl pheromones, coupling of these two blocks produced all four enantiomers of a pheromone with a 1,5-dimethyl structure, such as 10,14-dimethyloctadec-1-ene identified from the apple leafminer Lyonetia prunifoliella. To demonstrate the usefulness of the SN2 reaction of secondary tosylates and develop the study about methyl-branched pheromones, we synthesized four stereoisomers of (S)-2-methylpent-3-yl 3,13-dimethylpentadecanoate identified from the bagworm moth Clania variegate.


 
 

2017-01 GC/FT-IR analysis of a novel 2,4,6,9-tetraene occurring in a female pheromone gland of Parasemia plantaginis (Lepidoptera: Arctiidae)

     IR spectrometer is useful to determine functional groups of an unknown compound, but its utilization for pheromone researches is very limited because of its low sensitivity.  Meanwhile, a new type of GC/FT-IR (Discover IR, Spectra Analysis) has been developed by applying a system with a cooled and turned disk, where compounds elute from a capillary GC column are fixed and irradiated with an IR beam.  This FT-IR measurement is carried out sensitively as well as GC-MS.  We had successfully used it to identify a terminal conjugated dienyl pheromone produced by the nettle moth.  As a next challenge, it was tried to apply it for structure determination of a tetraene found in a female pheromone gland of Parasemia plantaginis, the wood tiger moth, in the family Arctiidae.  Noteworthily, 15 subspecies of this species have been recorded in Northern Hemisphere.  We are interested in their mating communication systems and started to investigate the sex pheromone from the females distributed in Japan and Finland.
     GC-EAD analysis of a pheromone extract of the Japanese subspecies showed four EAG-active components (I – IV).  GC-MS analysis utilizing synthetic standards revealed their chemical structures of C₂₁ unsaturated hydrocarbons as follows; 3,6,9-triene for I (most abundant component), 4,6,9-triene for II, and 1,3,6,9-tetraene for III.  The mass spectrum of IV indicated it also to be a C₂₁ tetraene, but its retention time on GC is much longer than that of the 1,3,6,9-tetraene suggesting a longer conjugated system than a conjugated diene.  Since IV is expected to include two double bonds at the 6- and 9-positions in common with other three components, we tentatively assigned a novel 2,4,6,9-tetraenyl structure for it.  Then, GC/FT-IR analysis was conducted in order to determine configuration of the double bonds at the 2- and 4-positions.  While the content of IV is very low, its IR spectrum with absorption at 3019 cm^-1 due to C-H stretching of two (Z)-double bonds at the 6,9-dienyl moiety was successfully recorded.  This spectrum also showed characteristic weak absorptions at 993 and 942 cm^-1 due to C-H bending of one (E)-double bond at the 2,4-dienyl moiety.  Intensities of these absorptions excluded the possibility of the (E,E)-conjugated system, and the (Z,Z)-conjugated system was denied because of no absorption around at 3040 cm^-1.  As a result, we estimated (2Z,4E,6Z,9Z)-configuration for IV.  This GC/FT-IR experiment also confirmed (Z)-double bonds of I – III.
     Similar analytical data was obtained from the extract of a Finnish subspecies.  Furthermore, (2Z,4E,6Z,9Z)-2,4,6,9-henicosatetraene was synthesized utilizing a double Wittig reaction.  It showed a EAG activity and all of its chemical data coincided well with those of the natural component IV.


 
 

2017-02 Unsaturated cuticle hydrocarbons contributed to sex pheromone attraction of the eggplant fruit borer, Leucinodes orbonalis Gueneé (Lepidoptera: Crambidae) inhabiting the Mekong delta of Vietnam

     Leucinodes orbonalis is one of the most serious pest insects of the eggplant in the Mekong delta of Vietnam.  While (E)-11-hexadecenyl acetate (E11-16:OAc) and its alcohol (E11-16:OH, a minor component) have been identified as the sex pheromone, the activity of male attraction is weak in the Vietnamese fields. In order to utilize its pheromone as a monitoring and mass trapping tools for sustainable pest management programs, development of a lure comparable to the virgin female is necessary.  We focused on extra pheromone components in another chemical group such as unsaturated hydrocarbons and conducted instrumental analyses of some extracts prepared newly from the L. orbonolis females.  Then, roles of the identified hydrocarbons were investigated by field evaluation of the corresponding synthetic compounds. 
     GC-EAD and GC-MS analyses of the abdominal tip extracts revealed a trace of (3Z,6Z,9Z)-3,6,9-tricosatriene (Z3,Z6,Z9-23:H) as an EAG-active component in addition to E11-16:OAc. The ratio of E11-16:OAc and Z3,Z6,Z9-23:H is about 100:2. On the other hand, extracts of the whole body with a pheromone gland showed an abundant of Z3,Z6,Z9-23:H and one more component, (3Z,6Z,9Z)-3,6,9-docosatriene (Z3,Z6,Z9-22:H).  The ratio of E11-16:OAc, Z3,Z6,Z9-22:H and Z3,Z6,Z9-23:H is about 100:1:45.  Quantitative analysis showed that amounts of E11-16:OAc and Z3,Z6,Z9-23:H were ca. 33.7 and 18.5 ng/female, respectively. Z3,Z6,Z9-23:H was also detected in the wing extract.  These results indicate that the two EAG-active trienes are mainly released from the body surface, not from the pheromone gland.
     In the fields, traps baited with E11-16:OAc alone could captured significantly more males than those of control.  However, its activity was weaker than the virgin female and addition of E11-16:OH to the lure as a minor component did not improve the attraction.  The addition of E11-16:OH at a higher ratio than 10%, the attraction by E11-16:OAc was inhibited.  On the contrary, addition of Z3,Z6,Z9-22:H or Z3,Z6,Z9-23:H significantly increased the number of captured males, confirming that these C₂₂ and C₂₃ trienes effectively contributed as the sex pheromone components to attract the males synergistically.  Interestingly, in the field test of some other synthetic unsaturated hydrocarbons, unnatural C₂₃ (3Z,6Z,9Z,12Z,15Z)-pentaene also showed a good synergistic effect, but C₂₁ and C₂₅ trienes and C₂₅ pentaene didn’t.  The male moths seem to recognize more strictly chain length of the hydrocarbons than unsaturated degrees.


 
 

2017-03 Application of the sex pheromone and plant volatiles for mass trapping of the diamond back moth, Plutella xylostella L. (Lepidoptera: Plutellidae), in the Southern Vietnam

     The diamond back moth, Plutella xylostella, is one of the most serious pest insects of cabbage in the Southern Vietnam.  Since effective control of P. xylostella by spraying insecticides is difficult due to its high resistance against the chemicals, mass trapping by its sex pheromone and other volatile compounds plant volatiles is expected to be an alternative effective method.  As a first step, we examined effective synthetic lures for attraction of the moth.  The pheromone lure composed of three components (Z11-16:Ald, Z11-16:OAc, and Z11-16:OH) at a ratio of 5:5:1 could attracted P. xylostella males as strong as the virgin female did in our field tests, while one- or two-component lures attracted significantly lower numbers of the males than those by the three-component lure.  Another test of the three component lures at some different doses from 0.01 to 1.0 mg/lure showed that 0.01 mg/lure was an optimal dose for attraction of the males (averagely 158 males/trap/week).  On the other hand, lures prepared with (Z)-3-hexenyl acetate (0.01–1.0 mg/lure), allyl isothiocyanate (AITC, 0.01–1.0 mg/lure), or a brassica leaf grinded solution (1.0 ml) showed high attraction of the moths of both sexes.  Interestingly, the males and females were not equally attracted and the ratio of the captured females was 5–10%.  Furthermore, our field examination confirmed that AITC caused the number of captured moths to increase when it was added to the pheromone lure.  Next, based on these results, a mass trapping experiment was conducted.  The traps, which were bated with a lure composed of the sex pheromone and AITC and placed at a density of 120 traps/ha, showed the same effectiveness as application of conventional insecticides (5-time sprays) for the control of P. xylostella in a cabbage field.


 
 

2017-04 Identification of female sex pheromones of rare moth species: Perspective for conservation biology

     Assessing the conservation status of rare/threatened insect species is important for keeping species diversity.  Pheromone-based monitoring is a useful method especially moth species which possesses a long-range communication system.  We are interested in the studies of rare/threatened moth species and carrying out now their potential application for assessments of biodiversity, conservation of the species in population declines, and environmental protection.
     Prismosticta hyalinata (Bombycoidea: Prismostictidae) has been thought to be a rare species because female and male adults of this species are not phototactic.  By a happy coincidence, we could collect a pair of newly mated adults, and the female oviposited fertilized eggs onto cuttings of Symplocos sawafutagi (Ericales: Symplocaceae, formally S. chinensis) in laboratory conditions.  We successfully bred them using fresh leaves of S. coreana, and could perform the observations of circadian periodicity.  In laboratory observation, adults flew only during the daytime and showed mating behavior mainly in the afternoon. GC-EAD analysis of the crude extract of female glands indicated only one EAG-active component, and GC/MS analysis assigned (E,E)-bombykal to a chemical structure of the pheromone candidate.
     Rubber septa impregnated with the synthetic (E,E)-bombykal and/or related compounds were prepared as pheromone lures.  Sticky traps baited with the lures were placed on 1.5 m above at coppices in Tottori Prefecture, Western region of Honshu, Japan.  Surprisingly, many males flew around the traps baited with (E,E)-bombykal just after hanging on a twig in the afternoon.  A large number of males could be captured specifically by (E,E)-bombykal suggesting that population level of P. hyalinata is not low.
     The field test revealed three novel findings.  First, P. hyalinata is a common species and may be distributed in various environments where Symplocos trees are distributed.  Second, the adults are diurnal but their catching using a butterfly net seems to be difficult because of their unexpected quick flight.  Third, this species is multivoltine; the males were captured by traps in late May, June, and early September in Tottori Prefecture.
     Furthermore, we are conducting to identify female sex pheromones of some so-called rare moth species; e.g., a lappet moth Pyrosis idiota and a winter geometrid moth Sebastosema bubonarium.  Various biological aspects of this species, e.g., distribution, habitat, rarity/risk of extinction, daily rhythms and seasonal prevalence, will be understand.

 
 

2018-01 Lepidopteran sex pheromones: Wonderland for a natural product chemist

     Lepidopteran sex pheromones have been identified from more than 680 species by chemical analysis, and the sex attractants of 1280 other species have been found in field tests of synthetic pheromones and related compounds.  Our laboratory studies based on organic synthesis contribute to the research of the pheromones of 64 species in 16 moth families and the attractants of 250 species.  While our information is still rudimentary considering the species diversity, other natural products have not been investigated for as many different species as the lepidopteran sex pheromone.
     By analyzing many mass spectra of Type I and II pheromones, we clarified the diagnostic fragment ions reflecting the positions of double bonds and an epoxy ring in order to utilize them for studies on new natural pheromones.  In the case of the epoxy pheromones, their stereochemistry was successfully determined by applying enantioselective HPLC analysis.  Furthermore, we found novel methyl-branched pheromones and developed a widely applicable synthetic route via the S_N2 reaction of a secondary tosylate derived from optically active propylene oxide. 
     Our laboratory is also interested in insect physiology and has investigated pheromone biosynthesis.  Experiments with handmade ¹⁴C- or D-labeled precursors proposed the biosynthetic pathways of some pheromones, and molecular biology experiments successfully cloned key enzymes of the biosynthesis, such as a desaturase for Type I pheromones and an epoxidase for Type II pheromones.  In addition to the pheromones, pheromone-biosynthesis-activating neuropeptides were identified, and biosynthetic steps regulated by the hormone were revealed.  Our understanding increased rapidly, but the diversity of lepidopteran insects still provides many interesting research topics, such as evolution.