1997-01 PBAN, Biochemical Site for Controlling Pheromone Production
Most of female moths produce their sex pheromones in synchronizing with a light-dark cycle, and the pheromone production is controlled by the hormone named pheromone biosynthesis activating neuropeptide (PBAN), which is secreted by the suboesophageal ganglion (SG). To date, the hormone has been chemically characterized from three species, Helicoverpa zea, Bombyx mori and Lymantria dispar. All of these peptides, composed with 33 or 34 amino acids, are C-terminally amidated and share approximately 80 % sequence homology. In order to apply PBAN for pest management, the biosynthetic process regulated by the hormone must be clarified.
The biosynthetic pathways of many lepidopterous sex pheromones comprise the following steps: construction of saturated acyl moiety, carbon chain elongation or shortening, reduction of the acyl group to an alcohol, and acetylation or oxidation into an aldehyde. In Japan, the mode of action of PBAN has been investigated using mainly B. mori. We concluded from in vivo and in vitro experiments with the decapitated females and 14C-labeled precursors that the target step for PBAN is acyl reduction. However, among the other lepidopterous species in which the target step of their PBAN was examined, these results coincide only with those on Spodoptera littoralis. It is interesting that PBAN might not have a universal target step.
After mating, receptivity of the female moth terminates and oviposition begins. Furthermore, sex pheromone-releasing behavior stops and pheromone biosynthesis ceases. The mechanism involved in the mating-induced termination of pheromone production was examined in some experiments with B. mori. The results indicated that the pheromone gland of the mated female maintained its ability to biosynthesize bombykol; however, it should not produce pheromone due to a suppression of PBAN secretion from the SG, and because the suppression was mediated by a neural signal of mating and not by a substance in the spermatophore.
2000-01 Preparation of Optically Active 1'-Acetoxychavicol Analogs and Their Biological Activities
Many plants in the Zingiberaceae are traditionally utilized for spices and medicinal herbs. From rhizomes of Alpinia galanga in this family, 1'-acetoxychavicol acetate [1'-ACA, 1-acetoxy-4-(1-acetoxy-2-propenyl)benzene], has been identified as anti-ulcer and insecticidal materials. 1'-ACA has a chiral center, and information on the biological activities of stereoisomers is still limited. Therefore, we prepared optically active 1'-ACA and its analogs and examined their repellent and lethal effects on the adzuki bean weevil (ABW), Callosobruchus chinensis (L.). We also investigated a mode of insecticidal action of 1'-ACA using a mitochondrial respiration system.
Racemic mixtures of 1'-acetoxychavicol and its ortho- and meta-analogs, which were synthesized via a Grignard reaction, were supplied to chiral HPLC resolution. After determining their stereochemistry by Mosher's method, optically active 1'-ACA and the analogs were obtained by acetylation of the resolved enantiomers. Repellent activity was measured by a protection time counting numbers of ABW on beans treated with and without each chemical. The results indicated that repellent sensitivity of ABW was depend on the sex, and that (S)-1'-ACA showed longest protection time against the females.
While we found that the insecticidal activity of the ortho-analog was stronger than 1'-ACA, the LD50 values of (S)- and (R)-isomers of each 1'-acetoxychavicol analogs were almost same. Stereo chemistry of 1'-position did not affect on the lethal activity. 1'-ACA and the analog chemicals inhibited respiration of mitochondria prepared from mice livers. Experiments with different substrates, which caused sequential isolation of the coupling sites in an electron transport chain, showed that they are inhibitors of the coupling site III in the chain.
2000-02 Convenient preparation of optically active diepoxyheniocsenes (leucomalure), lymantrid sex pheromone
In order to obtain information about sex pheromones of species in the family Geometridae, one of the biggest family in Lepidoptera, C18 - C23 (Z,Z,Z)-3,6,9-trienes and their racemic monoepoxy derivatives were systematically synthesized. Their random screening tests showed new specific male attraction of ca. 20 species including the Japanese giant looper, a serious defoliator of tea gardens. Referring GC-MS data, C19 3,4-epoxydiene was identified as a main pheromone component of the females. Enantiomers of the epoxydiene were successfully resolved using a chiral HPLC column, thus analysis of this natural pheromone showed that the female produced the racemic mixture. Furthermore, epoxidation in the pheromone gland was examined in vivo, and it was revealed that a monooxygenase of the biosynthesis regiospecifically attacked only the (Z)- double bond at 3-position but substrate specificity of this enzyme was rather low.
2001-01 Mating Disruption of the Japanese Giant Looper in Tea Gardens Permeated with Synthetic Pheromone and Related Compounds
In
Japan, new pest management techniques without insecticides are a much
desired goal, particularly for defoliators in tea gardens. In 1983, a
product named "Hamaki-con", a polyethylene tube containing
(Z)-11-tetradecenyl acetate, was registered for control of the smaller
tea tortrix (
Adoxophyes honmai sp. nov.) and the Oriental tea tortrix
(
Homona magnanima Diakonoff). This compound is a common component of the
sex pheromones of both species. "Hamaki-con" has been used to keep
these species at a low population level mainly in the Shizuoka, Mie, and
Kumamoto prefectures in Japan.
The Japanese giant looper [mugwort
looper (
Ascotis selenaria cretacea Butler), Geometridae: Ennominae], is
another lepidopterous species which seriously damages tea leaves. The
females produce racemic (Z,Z)-6,9-cis-3,4-epoxynona-decadiene
(epo3,Z6,Z9-19:H, main component) and (Z,Z,Z)-3,6,9-nonadecatriene
(Z3,Z6,Z9-19:H, minor component). The orientation of the males to the
synthetic pheromone placed in a trap was strongly disrupted by
Z3,Z6,Z9-19:H or a mixture of its monoepoxy derivatives (epoxydiene
mixture, EDM) impregnated in septa and placed around the trap. Based on
this result, polyethylene tubes containing Z3,Z6,Z9-19:H or EDM were
prepared and effect of these dispensers was examined in a field.
Disruption of male orientation to synthetic pheromone traps was achieved
in orchards permeated with Z3,Z6,Z9-19:H at dispenser density of 3000
and 5000 tubes/ha (release rate: 0.55-0.61 mg/day/tube) and with EDM at
every tested dose, 250-5000 tubes/ha (release rate: 0.25-0.39
mg/day/tube). Furthermore, disruption of mating in tethered females was
observed in these orchards; particularly, the mating was perfectly
inhibited in the areas treated with EDM at 3000 and 5000 tubes/ha. This
is the first formulation for the mating disruption of a geometrid pest.
2001-02 Substrate Specificity of the Epoxidation Reaction in Sex Pheromone Biosynthesis of the Geometrid Moths
Female
moths in the family of Geometridae produce C17 to C23 hydrocarbons
including two or three (Z)-double bonds at the 3-, 6-, or 9-positions
and their monoepoxy derivatives as sex pheromones. Variation in the
epoxy ring position and the carbon chain length makes complexity of the
geometrid pheromones, playing an important role in their reproductive
isolation. These pheromone components were biosynthesized by chain
elongation, decarboxylation, and epoxidation starting from linoleic and
linolenic acids. We examined the substrate specificity of epoxidation in
the pheromone biosynthesis of the Japanese giant looper,
Acotis
selenaria cretacea, and the mulberry looper,
Hemerophila atrilineata.
The former species secretes cis-3,4-eoxy-(Z,Z)-6,7-nonadecadiene
(epo3,Z6,Z9-19:H, other chemicals are abbreviated in this manner), and
the latter species secretes a 10:1 mixture of Z3,Z6,epo9-18:H and
Z6,epo9-18:H.
The GC-MS analysis of the extract of
A. s. cretacea
pheromone grand treated with D3-Z3,Z6,Z9-19:H showed deuterium
incorporation into the epoxy pheromone. Other Z3,Z6,Z9-trienes with a
C17, C18, C20, or C23 chain, which did not exist in the pheromone gland,
were interestingly converted to the corresponding 3,4-epoxides in the
pheromone gland, indicating that the epoxidation enzyme recognized the
reaction site counting it from the 1-positon in the unsaturated straight
chain compounds. Furthermore, Z3,Z6-diene and Z3-monoene were also
converted to their 3,4-epoxy derivatives, but Z6,Z9-diene and Z2-, Z4-,
and E3-monoenes were not oxidized. This result indicates that the enzyme
specifically attacked the Z3-double.
The result of an incorporation
experiment with
H. atrilineata coincided with that of
A. s. cretacea.
In the pheromone gland of
H. atrilineata, D
3-Z3,Z6,Z9-18:H and
D
3-Z6,Z9-18:H were converted to the pheromone components depending on
amounts of their treatments. Not only Z3,Z6,Z9-trienes with a longer
chain but also Z9-monoene were also successfully changed to 9,10-epoxy
derivatives. H. atrilineata might have only one epoxidation enzyme for
the oxidation of both intermediates, which specifically recognized the
Z9-double bond but did not the chain length. Species-specific pheromone
components of geometrid females might be produced by the strict
construction of unsaturated carbon chain(s) and the following oxidation
at the specific position by an epoxidation enzyme with low substrate
specificity.
2001-03 Biosynthetic Pathway for Production of a Conjugated Dienyl Sex Pheromone of a Plusiinae Moth, Thysanoplusia intermixta
Virgin females of
Thysanoplusia intermixta (Lepidoptera; Noctuidae; Plusiinae) produce (5E,7Z)-5,7-dodecadienyl acetate as a main sex pheromone component. Although pheromones with a 5,7-dodecadienyl chain have been identified from some species in the family of Lasiocampidae, the construction of this distinctive structure has not been elucidated yet. Researches of other dienyl pheromones indicated that the dienes are biosynthesized via a monoenyl intermediate by two different routes (Routes A and B). In Route A proposed for the pheromones such as a 9,11-diene of
Spodoptera littoralis, a-monoene is converted to a,g-diene by one more desaturation keeping the original position of the first double bond. In Route B for the pheromones such as bombykol with a 10,12-dienyl structure, b-monoene is converted to a,g-diene after migration of the first double bond. In order to understand the pathway and substrate specificity of enzymes in the biosynthesis of the
T. intermixta pheromone, we synthesized several deuterated monoenyl fatty acids and examined their fates in the pheromone glands of the females.
GC-MS analysis of the pheromone glands, which were treated with deuterated hexadecanoic, (Z)-11-hexadecenoic, and (Z)-7-docecenoic acids, successfully showed incorporation of the label into the 5,7-diene, while any other deuterated fatty acids tested in this experiments were not incorporated into it. Their incorporation ratios confirmed that the diene structure is produced in Route A, and its biosynthesis proceeds in the following order; D11-desaturation of a C16 acyl intermediate, chain shortening to a C12 compound by b-oxidation, D5-desaturation to produce a 5,7-dienyl system, reduction of the acyl group, and acetylation. These deuterated precursors also converted into a minor pheromone component, (Z)-7-docecenyl acetate, which might be prepared by the same pathway except for the step of D5-desaturation. The labeled dodecenoic acids with (E)-5-, (Z)-6-, and (E)-7-double bonds, which were not incorporated into the dienyl acetate, were changed to the corresponding dodecenyl acetates. This result showed low substrate specificity of the enzymes for reduction and acetylation. Labeled (Z)-10-hexadecenoic acid was not converted into a dodecenyl acetate, indicating the high substrate specificity of the enzyme for b-oxidation.
2002-01 Epoxyalkenyl Sex Pheromones of Lepidoptera: Synthesis, Identification, and Application
Lepidopteran sex pheromones have been identified from more than 500 species. Females in the families of Geometridae, Noctuidae, Arctiidae, and Lymantridae produce (3Z,6Z,9Z)-trienes, (6Z,9Z)-dienes, and their monoepoxides with a C17-C23 straight chain. These compounds constitute an interesting second group of lepidopteran pheromones, different from the main group of unsaturated C10-C18 alcohols and derivatives. We systematically synthesized the epoxyalkenes, utilized them for the identification of natural pheromones, and examined their disruption activity against a mating communication.
Synthesis. The C18-C23 trienes and dienes were synthesized from linolenic and linoleic acids via a Grignard coupling reaction and oxidized by a peracid to convert to a mixture of racemic monoepoxides, 3,4-, 6,7-, and 9,10-epoxides. After separation by MPLC, the chemical structure of each epoxyide was confirmed by 2D NMR. Using a chiral HPLC column, the enantiomers of each racemate were resolved, and the stereochemistry of the separated enantiomers was then determined by a modified Mosher's method after methanolysis of the epoxy ring.
Identification. The GC-MS data of a series of epoxides proposed diagnostic fragment ions for the differentiation of these positional isomers. This information was useful to determine the structures of pheromonal epoxides secreted by two geometrid species, as follows: C19 3,4-epoxy-6,9-diene from
Ascotis selenaria cretacea, a serious defoliator of tea gardens, and C19 6,7-epoxy-3,9-diene and 6,7-epoxy-9-ene from
Biston robustum, a polyphagous defoliator unusually reproduced on Hachijo-jima Island. While the natural component of
A. s. cretacea was a racemate, the synthetic (3R,4S)-isomer attracted the males more effectively than the racemate in a field test. The configuration of both natural components of
B. robustum was 6S,7R, and the male moths were successfully attracted to a 9:1 mixture of the two (6S,7R)-isomers with 80 % ee.
Application. Polyethylene dispensers were prepared with a synthetic mixture of epoxydienes derived from C19 triene, and their effect on disruption of the mating communication of
A. s. cretacea was examined in tea gardens. Disruption of male orientation to synthetic pheromone traps was achieved in areas permeated with the mixture at every tested dose, 250-5000 tubes/ha. Furthermore, complete disruption of mating in tethered females was observed in the areas at 3000 and 5000 tubes/ha. This is the first formulation for the mating disruption of a geometrid pest.
2003-01 Bioorganic Chemistry on Sex pheromones Secreted by Lepidopteran Insects and Their Application for Plant Protection
Lepidopteran sex pheromones have been identified from more than 500 species. The pheromones in the most predominant group (Type 1) are composed of unsaturated C10-C18 straight chain compounds with a terminal functional group, such as bombykol produced by the silkworm moth. In addition to them, females in some evolved families produce C17-C23 polyunsaturated hydrocarbons and the epoxy derivatives, constituting a second major group (Type 2). While some synthetic pheromones have been utilized for plant protection on the basis of their strong attractive activities for male moths, many bioorganic chemical studies are currently underway on this exciting topic. This presentation focuses on recent research conducted in the TUAT chemical ecology laboratory and explores the future of pheromone studies and potential applications.
Identification of natural pheromones. Since the pheromone content of female moths is quite low, particularly in small insects, capillary GC-MS data of systematically synthesized pheromone candidates plays an important role in the analysis of natural pheromones. Comparing the accumulating data, new Type 1 compounds were successfully identified from the persimmon fruit moth. A characteristic base peak at m/z 84 of the aldehyde component revealed a 4,6-diene structure. The epoxy components of Type 2 include chiral centers, and it was found that enantiomers were separated by chiral HPLC columns. Stereochemistry was determined for natural pheromones of the Japanese giant looper and the mulberry looper.
Application of synthetic pheromones. To disrupt the sexual communication of several pest insects, synthetic pheromones of Type 1 formulated in polyethylene tubes are commercially available. Application of Type 2 pheromones is limited because of their complicated synthesis, but field evaluation confirmed that mating of the Japanese giant looper was effectively inhibited by a mixture of the epoxy pheromone and its positional isomers. This mixture is synthesized more easily than the pure pheromone and is a promising disruptant.
Pheromone biosynthesis and regulation mechanism. Besides the direct application of synthetic pheromones, it has also been hypothesized that the communication can be disturbed by controlling the pheromone production of female moths. The biosynthetic pathways of Type 1 pheromones have been confirmed by incorporation experiments with labeled precursors, and several biosynthetic inhibitors have been reported. On the other hand, experimental confirmation is still insufficient for the biosynthesis of Type 2 pheromones. Epoxidation of polyunsaturated hydrocarbons, a final step of the biosynthesis, is carried out in the pheromone gland, but the hydrocarbons might be produced outside of the gland, probably in oenocytes. A subesophageal ganglion secretes a hormone named pheromone biosynthesis-activating neuropeptide (PBAN), which suggests that the biosynthetic system is an interesting target for developing a new pest-management technique.
2003-02 Chemical modification and 13C NMR analysis of the fungicidal ambruticin
Ambruticin VS-3 isolated from a myxobacterium in the '70s has a unique chemical structure featuring tetrahydropyran and dihydropyran rings, which are stringed with a long alkenyl chain including a cyclopropane moiety. This compound shows quite strong antifungal activity, particularly against Botrytis cinerea, but its application in agriculture has not been successful because of its photo-instability. In order to prepare new useful derivatives and understand the structure-activity relationship, the methyl ester of Ambruticin VS-3 was analyzed by
13C NMR before further modification experiments. Every
13C signal was undoubtedly assigned using 2D-tequniques such as HMQC, HMBC, and INADEQUATE. We are now in the process of isolating each hydrogenated derivative from a mixture produced by a diimide reduction of the ester and determining the saturated site(s) among four double bonds of the parent compound with reference to its 13C assignment.
2003-03 Lepidopteran Epoxyalkenyl Pheromones: Synthesis and Identification
Lepidopteran sex pheromones have been identified from more than 500 species. Females in the families of Geometridae, Noctuidae, Lymantriidae, and Arctiidae produce (3Z,6Z,9Z)-trienes, (6Z,9Z)-dienes, and their epoxy derivatives with a C17-C23 straight chain. These compounds constitute an interesting second group of lepidopteran pheromones, different from the main group of unsaturated C10-C18 chain compounds with a terminal functional group. The research here began with a systematic synthesis of monoepoxides.
Synthesis and field evaluation. The C18-C23 trienes and dienes were synthesized from linolenic and linoleic acids via a Grignard coupling reaction and oxidized by a peracid to convert to a mixture of racemic cis-monoepoxides. After separation by MPLC, the activity of each epoxide was tested in a field. New male attraction of about 20 geometrid and noctuid species was found in random screening tests in Japan.
Optical resolution. Each racemate was successfully resolved using chiral HPLC columns operated under a normal-phase condition (Chiralpak AS and AD) or a reversed-phase condition (Chiralcel OJ-R). The stereochemistry of the separated enantiomers was determined by a modified Mosher's method after methanolysis of the epoxy ring.
Identification of geometrid pheromones. The GC-MS data of a series of epodixes proposed diagnostic fragment ions for the differentiation of these positional isomers. This information was useful to determine the structures of natural pheromones secreted by two geometrid species; i.e., C19 3,4-epoxy-6,9-diene from
Ascotis selenaria cretacea, a serious defoliator of tea gardens, and C19 6,7-epoxy-3,9-diene and 6,7-epoxy-9-ene from
Biston robustum, a polyphagous defoliator in forests. While the pheromonal epoxide of
A. s. cretacea was a racemate, the synthetic (3R,4S)-isomer attracted more effectively than the racemate in a field test. The configuration of both natural components of
B. robustum was 6S,7R, and the male moths were successfully attracted to a 9:1 mixture of the two (6S,7R)-isomers with 80 % ee.
Identification of novel lymantriid pheromones. In spite of the great number of species in the above families, the structural diversity known to date for the epoxy pheromones is limited. It is assumed that some females secrete further modified compounds as their sex pheromones, and, recently, Dr. Wakamura of the National Institute of Agrobiological Sciences in Japan and our team successfully identified novel components from two lymantriid species,
Orgyia postica and
Perina nuda. Females of the former tussock moth produced C21 (11S,12S)-11,12-epoxy-6,9-diene (posticlure) with a trans-epoxy ring, and females of the species produced C21 (3R,4S,6S,7R)- and (3S,4R,,6S,7R)- 3,4-6,7-diepoxy-9-enes with two cis-epoxy rings.
2003-04 Biosynthesis of epoxyalkenyl sex pheromones: biosynthetic pathway, substrate specificity, and endocrine control
Epoxyalkenyl
compounds with a C17-C23 straight chain make a second major class (Type
II) of lepidopteran sex pheromones. Their chemical structures indicate
the biosynthesis from linolenic or linoleic acids in a diet, but studies
with deuterated precursors have confirmed only the final epoxidation
step in a pheromone gland. Recently, polyunsaturated hydrocarbons
corresponding to the epoxy pheromones have been detected in the female
hemolympha of Geometridae and Arctiidae species that secrete Type II
pheromones. The polyenyl precursors are most likely produced in
oenocytes and transported to the pheromone gland via the hemolympha as a
monoenyl precursor of disparlure, a methyl-branched epoxy pheromone of
the gipsy moth. Referring to a pioneer work with the gipsy moth, we are
going to analyze an oenocyte extract and conduct incorporation
experiments with incubated oenocytes to identify a biosynthetic site and
a pathway to the polyenes.
The pathways predicted for polyenes with
odd-numbered and even-numbered chains are different. While alkanes with
an odd-numbered chain dominantly exist in hemolympha, alkanes with an
even-numbered chain have been also detected in it. The production
mechanism for polyenyl precursors with a specific chain length is an
interesting research target. The mechanism of the precursor uptake by
the pheromone gland is another challenge. Injection of various polyenes
into female abdomens will reveal the selectivity of the uptake.
After
decapitation of the females, epoxyalkenyl pheromones disappeared from
the pheromone glands, such as Type I pheromones with a terminal
functional group, indicating that the biosynthesis of Type II pheromones
is also controlled by a hormone, probably a PBAN-like neuropeptide. It
is interesting to determine which site is activated by this hormone, the
pheromone gland or the oenocyte.
Type II pheromones are produced by
species in highly evolved insect groups in quite a different manner
from Type I pheromones. The evolution of biosynthetic systems is beyond
our grasp at this point. Hybridized chemicals with Types I and II, such
as epoxy derivatives with a terminal functional group, have not been
identified from any female moths.
2003-05 Diversity of Sex Pheromones Produced by Plusiinae Moths Reflecting Their Evolution
Plusiinae
is a sub-family of Noctuidae, the largest family in Lepidoptera, and
includes about 400 species recorded mainly in tropical and temperature
zones throughout the world. Sex pheromones of 14 Plusiinae species had
already been identified chemically when this research started, with 9
species that used (Z)-7-dodecenyl acetate (Z7-12:OAc) as a major
component indicating their common ancestor. To understand the evolution
of Plusiinae more precisely, sexual communication in additional species
has been defined.
A total of 55 species are distributed in Japan,
and some of them are defoliators of cultivated plants. GC-MS analysis of
pheromone gland extracts from
Anadevidia peponis (a pest of cucumber
plants) and
Macduunoughia confusa (a pest of edible burdock plants)
revealed that the females produced Z7-12:OAc as a major component, but
the types and mixing ratios of some minor components were different,
indicating one of the main factors for the reproductive isolation of
these species. Seven extra minor components were identified form the
A.
peponis extract, and (Z)-5-decenyl acetate (Z5-10:OAc) was an
indispensable component for male attraction. On the contrary, the
M.
confusa extract included four minor components, and (Z)-9-tetradecenyl
acetate (Z9-14:OAc) was necessary for male attraction. In the field
tests for
A. peponis, the sex attractants of two other Plusiinae
species,
Macduunoughia purissima and
Ctenoplusia albosteriata, were
found. With the use of this information, their real sex pheromones were
also successfully defined.
Furthermore, the sex pheromones of other
Plusiinae species,
Thysanoplusia intermixta,
T. orchalcea (a pest of
carrot plants), and Plusia festucae (a pest of rice plants), were
studied. In addition to Z7-12:OAc, (E,Z)-5,7-dodecadienyl acetate
(E5,Z7-12:OAc) was identified from the former two species. The main
component of the last species is (Z)-5-dodecenyl acetate (Z5-12:OAc),
and, interestingly, Z7-12:OAc is completely excluded from the pheromone
blend.
Z7-12:OAc, Z5-10:OAc, and Z9-14:OAc are biosynthesized via
D11-desaturation of C16 acid (Route I), but the double bond of Z5-12:OAc
is introduced by D11-desaturation of C18 acid or D9-desaturation of C16
acid (Route II). Some Plusiinae species utilize Z9-12:OAc, which is
biosynthesized via D11-desaturation of C14 acid or D5-desaturation of
C16 acid (Route III). It has been reported that D5-desaturation of a C12
intermediate acts on the formation of E5,Z7-12:OAc (Route IV). Based on
the synthetic pathways (Routes I-IV) for key components clarified in
our field tests and other past trials, the pheromone blends of the
Plusiinae species were rationally classified into five groups according
to their chemotaxonomy.
2003-06 Polyunsaturated Hydrocarbons in the Hemolymph: Biosynthetic Precursors of Epoxy Pheromones of Geometrid and Arctiid Moths
Female moths of many species in Geometridae, Arctiidae and some other macrolepidopteran families produce epoxy pheromones, which are probably derived from polyunsaturated hydrocarbons. The final biosynthetic step has been verified by our previous experiment with the deuterated C19 (Z,Z,Z)-3,6,9-triene, which was used to treat the pheromone gland of a geometrid species,
Ascotis selenaria cretacea (Japanese giant looper). The double bond at the 3-position was specifically oxidized to make the pheromonal C19 3,4-epoxy-6,9-diene. This C19 triene is probably produced by decarboxylation of C20 fatty acid, which was derived from linolenic acid by a C2 elongation reaction. However, the C20 acid has not been found in lipids extracted from the pheromone gland of
A. s. cretacea, and the labeled C20 acid applied to the pheromone gland was not converted into the epoxy pheromone or even into the C19 triene.
Recently, Jurenka et al. identified the alkenyl precursor of disparlure, the methyl-branched epoxy pheromone of
Limantria dispar (gypsy moth), from the female hemolymph, and showed that the precursor is most likely transported to the pheromone gland after the production in oenocytes. In order to understand a biosynthetic site for the precursors of epoxyalkenyl pheromones, hemolymph from both sexes of two geometrid species,
A. s. cretacea and
Hemerophila artilineata (mulberry looper), and one arctiid species,
Spilosoma imparilis (mulberry tiger moth), was shaken with
n-hexane and the solvent extracts were analyzed by GC-MS. In addition to many saturated hydrocarbons, polyunsaturated hydrocarbons corresponding to the pheromonal epoxy components were sex-specifically identified from the extracts of the female hemolymph; i.e., C19 3,6,9-triene from that of
A. s. cretacea secreting the 3,4-epoxide, C18 3,6,9-triene and 6,9-diene from that of
H. artilineata secreting their 9,10-epoxides, and C21 1,3,6,9-tetraene, C21 3,6,9-triene, C23 3,6,9-triene, and C23 6,9-diene from that of
S. imparilis secreting their 9,10-epoxides. No epoxy compounds were detected in the hemolymph.
Based on this analysis, deuterated polyunsaturated hydrocarbons were injected into the abdomens of two geometrid females, and the labeled epoxy components were successfully yielded from the pheromone glands. This result indicated that the polyunsaturated hydrocarbons occurring in the female hemolymph were direct pheromone precursors, which might be produced outside of the pheromone gland, probably in oenocytes associated with abdominal epidermal cells or in the fat body, and transported to the pheromone gland via the hemolymph for their epoxydation and emission into an atmosphere in a similar manner proposed for dispalure.
2003-07 Chemical characterization of cytoplasmic lipid droplets in the pheromone-producing cells of the silkmoth, Bombyx mori
Accumulation of lipid droplets within the cytoplasm is a common feature of the pheromone gland cells of many Lepidopteran species. The cytoplasmic lipid droplets in the pheromone-producing cells of the silkmoth,
Bombyx mori, were effectively extracted by dipping the trimmed glands in acetone for 10 min. In order to analyze the components originating from the lipid droplets, we separated the acetone extracts prepared before and after adult eclosion using HPLC, and specified the peaks showing a similar pattern of stage-dependence to that in the morphological change of the lipid droplets. Finally, we specified the peaks separated by reversed-phase HPLC as lipid droplet contents. Structure elucidation using FAB-MS / MS-MS, GC-EI-MS, and LC-APCI-MS confirmed that they were triacylglycerols (TGs). Fatty acyl groups contained in these TGs were limited to 5 unsaturated C16 and C18 fatty acyl groups (Δ11-hexadecenoate, Δ10,12-hexadecadienoate, Δ9-octadecenoate, Δ9,12-ocatadecadienoates, and Δ9,12,15-ocatadecatrienoates) and 2 saturated C16 and C18 fatty acyls, with the bombykol precursor Δ10,12-hexadecadienoate as a major component. Digestion with porcine pancreatic lipase confirmed that 3 major TGs eluted in the peaks #3-5 all contained C18 fatty acyl groups at the sn-2 position, indicating that the bombykol precursor is sequestered preferentially at the position of sn-1 and/or sn-3. The ratio of fatty acyls at sn-1 and sn-3 of the TGs were also analyzed by LC-MS using a chiral column. Following hydrolysis of TGs with the Grignard reagent, the result out 1,2(2,3)-DGs were converted to DNPU derivatives, which were further separated and analyzed with LC-MS using a chiral column. There were no difference between the fatty acyls at the position of sn-1 and -3.
2005-01 Biosynthesis of epoxyalkenyl sex pheromones produced by female moths in highly evolved groups
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. Referring 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 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 from that of Type I pheromones.
2005-02 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.
2005-03 Sex pheromones of five olethreutine species (Lepidoptera: Tortricidae) associated with the seedlings and fruits of mangrove plants in the Ryukyu Islands, Japan
The sex pheromones of three Cryptophlebia, one Centroxena, and one Eucosma species (Tortricidae: Olethreutinae) inhabiting mangroves in the Ryukyu Islands, Japan, were studied with techniques using GC-EAD and 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, Z8-12:OH, E8-12:OH, and 12:OH 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., Z8-12:OAc, E8-12:OAc, and 12:OAc 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 DMDS. 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 E8,E10-12:OAc and 12:OAc in a ratio of 100:5. The double-bond position of the diunsaturated compound was confirmed by GC-MS analysis of its adduct with MTAD. Furthermore, E9-12:OAc 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.
2005-04 Internal transportation of a sex pheromone precursor by lipophorin in the geometrid female moth, which secretes an epoxyalkenyl pheromone
Monoepoxy derivatives of polyunsaturated hydrocarbons with a C17?C23 straight chain, which have been identified from highly evolved species, such as the geometrid and arctiid moths, comprise a second major group of lepidopteran sex pheromones. Our previous experiments with a geometrid species,
Ascotis selenaria cretacea, revealed the formation of a pheromonal C19 3,4-epoxy-6,9-diene from the corresponding 3,6,9-triene in a pheromone gland. The biosynthesis of the trienyl precursor is not certified, but it is expected to be produced outside of the pheromone glands, probably in oenocytes or in the fat body, and transported to the glands via a hemolymph after binding with lipophorin. In order to confirm this transportation, we analyzed proteins in the hemolymph of
A. s. cretacea females on an SDS-PAGE. Two bands (apoLP I with 250 KDa and apoLP I with 80 KDa) were detected at positions indicating similarity to the lipophorin of
Bombyx mori. Next, the hemolymph of
A. s. cretacea was fractionated by KBr equilibrium density-gradient ultracentrifugation, and compounds bounded with proteins in each fraction were quantitatively analyzed by GC-MS. The triene was found only in the fractions associating with the lipophorin of the females in addition to saturated hydrocarbons with a C23?C27 straight or a methyl-branched chain. Furthermore, the binding selectivity of lipophorin was examined using a deuterated C19 trienyl precursor, a saturated hydrocarbon, and an unlabelled C23 trienyl derivative. When a mixture of the three compounds was topically applied to the abdomens of the female moths, the three hydrocarbons were associated with the lipophorin with approximately equal amounts indicating low binding selectivity. In this experiment, deuterated C19 epoxy pheromone was detected in a pheromone gland extract but an epoxy compound derived from the C23 triene was scarcely produced.
2005-05 Synthesis of 1,3,6,9-tetraene and these monoepoxy derivatives: sex pheromone components of Arctiidae
Introduction
1,3,6,9-Tetraene (1) and these monoepoxy derivatives (2) were identified from Arctiidae and other species, but fall webworm moth (
Hyphantria cunea) is the only identified species in Japan. We synthesized these compounds with C17-C23 carbon chain to search the species used these compounds as pheromone components.
Synthesis and analysis of 1,3,6,9-tetraene and these monoepoxy derivatives
At first, (Z)-1,6-diiodo-3-hexene was synthesized from 3-hexyne-1,6-diol (3) as starting material. After conversion into wittig reagent (4) , 1,3,6,9-tetraene (1) was synthesized from acrolein and n-aldehyde by double coupling reaction in onepot. Mixture of monoepoxides synthesized from 1 by mCPBA oxidation was separated by HPLC with ODS column. Result of NMR analysis of separated monoepoxides indicate that 1-epoxide is not synthesized by this oxidation, and 3-epoxide, 6-epoxide and 9-epoxide indicated the diagnostic fragment ions by GC-MS.
Stereochemistry of 1,Z3,Z6,epo9-21:H and pheromonal component of
Spilosoma imparilis Larva of mulberry tiger moth (
Spilosoma imparilis) is a harmful defoliator of mulberry, cherry and apple in Japan. Pheromone gland extract of this female include Z3,Z6,epo9-21:H as main component and 1,Z3,Z6,epo9-21:H as minor component. The result of chiral HPLC analysis of synthetic compounds and pheromone gland extract, pheromone component was only Fr.2. Compare the saturated epoxide reduced from synthetic 1,Z3,Z6,epo9-21:H and (9R,10S)-Z3,Z6,epo9-21:H by chiral HPLC, it is clearly that 1st fraction is (9R,10S)-epoxide and 2nd fraction is (9S,10R)-epoxide.
