Journal of Chemical Ecology, l/ol. 15, No. 1, 1989

ISOLATION, IDENTIFICATION, AND BIOSYNTHESIS OF COMPOUNDS PRODUCED BY MALE HAIRPENCIL GLANDS OF Heliothis virescens (F.) (LEPIDOPTERA: NOCTUIDAE)

P . E . A . T E A L and J . H . TUMLINSON

Insect Attractants, Behavior, and Basic Biology Research Laboratory Agricultural Research Service, U.S. Department of Agriculture Gainesville, Florida 32604 (Received August 25, 1987; accepted December 22, 1987) Abstract--Extracts of the intact hairpencil glands and hairs from the hairpencil glands of males of Heliothis virescens (F.) were analyzed by capillary gas chromatography (GC) and by GC-mass spectroscopy. These analyses indicated that hexadecanyl acetate (212.4 ng/male), hexadecanol (22.3 ng/ male), (Z)-I 1-hexadecenyl acetate (3.5 ng/male), octadecanyl acetate (14.2 ng/male) octadecanol (7.5 ng/male), tetradecanoic acid (2.7 ng/male), hexadecanoic acid (22.3 ng/male), and octadecanoic acid (6.5 ng/male) were present in the extracts. These compounds also were found in volatiles released from the hairpencil glands. In addition, GC analysis using both polar and apolar capillary columns indicated that extracts of the glands and extracts of the hairs from the hairpencil glands contained small amounts of tetradecanyl acetate, (Z)-9-tetradecenyl acetate, tetradecanol, (Z)-7-hexadecenyl acetate, (Z)-9-hexadecenyl acetate, and (Z)-I 1-hexadecenol. No (Z)-9-tetradecenal was present. Studies indicated that titers of the compounds increased rapidly during the 36 hr after adult eclosion and then leveled off, being maintained at high levels until released when the glandular hairs were exposed. During exposure of the hair pencils substantial amounts of the compounds were released. In vivo application of 500 ng of suspensions of (Z)-1 l-hexadecenyl acetate, (E)- 1l-hexadecenyl acetate, (Z)- 11-tetradecenyl acetate, or (E)- 11tetradecenyl acetate in dimethyl sulfoxide to the surface of the denuded hairpencil gland showed that biosynthesis proceeds to the alcohol via the acetate. Key Words--Heliothis virescens, Lepidoptera, Noctuidae, hairpencil glands, male-produced compounds, hexadecanyl acetate, hexadeeanol, (Z)-I 1-hexadecenyl acetate, octadecanyl acetate, octadecanol, tetradecanoic acid, hexadecanoic acid, octadecanoic acid.

413 0098-033118910100-0413$06.0010 © 1989 Plenum Publishing Coq~or,ttion

414

TEAL AND TUMLINSON INTRODUCTION

Considerable research has been conducted on pheromone chemistry and on the behavior associated with sex pheromones produced by females of Lepidoptera as well as on the morphology of the glands that produce these compounds (Tamaki, 1986). However, substantially fewer studies have been centered on male-produced pheromones. Studies on the chemistry and both intra- and intersexual behavior associated with pheromones produced by males of Lepidoptera are of importance because such information is critical to the elucidation of the chemical communication systems of these insects (Tumlinson and Teal, 1982). Epidermal glands that produce and disseminate the pheromones produced by males are of diverse structure and varied anatomical position. This diversity is exemplified among moth species when considering the eversible tubelike coremata present on the genital segments of male arctiid moths (Nielsen, 1979), the thoracic brush organs of Phlogophora species (Birch, 1970), wing glands of tortricid and phycitid moths (Grant, 1978), and hairpencil glands present on the genital segments of numerous noctuid species. Variability in structure and anatomical position of the pheromone glands of males is paralleled by the types of chemicals released as pheromone components and by the behavioral roles of these compounds in chemical communication (Tamaki, 1986). Chemicals released as pheromone components include such different molecules as benzaldehyde (Clearwater, 1972), methyl-jasmonate (Baker et al., 1981), and (Z,Z,Z)-3,6,9-heneicosatriene (Heath et al., 1987). Functions of pheromones released by males range from the attraction of females (Dahm et al., 1971; Baker et al., 1981), to inducing female quiescence, and inhibition of the approach of other males (Clearwater, 1972; Hirai et al., 1978). Unfortunately, neither the structures nor functions of pheromones released by males of the majority of moth species have been described. Little work has been conducted on the pheromone produced by the hairpencil gland of males of Heliothis virescens (F.), although various functional roles have been ascribed. For example, Hendricks and Shaver (1975) suggested that the pheromone stopped females from calling. Alternatively, laboratory flight tunnel studies of mating behavior caused Teal et al. (1981) to hypothesize that the pheromone caused females to accept courting males. Recently, Jacobson et al. (1984) reported that (Z)-9-tetradecenal (Z9-14:A1) was the pheromone produced by males of H. virescens. The production of milligram amounts of this compound was of interest because the corresponding alcohol was not identified. This alcohol is present in extracts of the pheromone gland of females (Teal et al., 1986) and is converted to Z9-14 : A1, a critical component of the pheromone blend released by females (Teal and Tumlinson, 1986; Vetter and Baker, 1983). This suggested that males and females of H. virescens employ different methods of pheromone biosynthesis.

MALE HAIR-PENCIL COMPOUNDS

415

For the above reasons, we investigated the chemicals produced in the hairpencil glands of males of H. virescens. The following reports the identification of a blend of components from these glands and discusses the results of in vivo studies on biosynthesis of these compounds. We also describe the structure of the hairpencil gland and discuss the possible mechanism of storage of the compounds identified. A subsequent paper will discuss the behavioral role of the pheromone released from the hairpencil gland in the reproductive behavior of H. virescens.

METHODS AND MATERIALS

General. Heliothis virescens used in this study were obtained as pupae from the Bioenvironmental Insect Control Laboratory, USDA, ARS, Stoneville, Mississippi. Pupae were sexed and maintained under a reversed 16:8 light-dark cycle at 25°C and 55% relative humidity. Newly emerged adults were transferred daily to 30 x 30 x 30-cm clear plastic cages and were provided with a 10% sugar solution soaked onto cotton. Anatomical Studies. The terminal abdominal segments, which include the paired hairpencil glands, one at the base of each clasper, were everted by applying pressure to the anterior abdominal segments. Glutaraldehyde fixative (2 % in phosphate buffer pH 7.2) was then injected into the terminal abdominal segments. After 10 min the hairpencil glands were removed from the abdomen and placed in a vial that contained fixative plus two drops of Photoflow to reduce surface tension. The vial was placed under vacuum at 4°C for 14 hr. Material was postfixed in 2 % OsO4 in phosphate buffer (4 hr) prior to dehydration in ethanol and propylene oxide. Some tissue was then embedded in Spurr's resin and sectioned at 0.5-1.5/xm using glass knives. Sections were mounted in series on gelatin-coated slides and stained using methylene blue in 2% borax (Teal and Philogrne, 1980). The remaining tissue was dried in a desiccator that contained granular SiO2 prior to mounting on stubs using silver conductive paint. These samples were then sputter coated with gold. Scanning electron microscopy was performed using a Cambridge Sterioscan Mark IIA operated at 10 kV. Isolation and Identification. In initial studies, rinses of the whole male hairpencil gland complex, including the elongate glandular hairs and cellular portion, were obtained by removing the abdominal segments of males during the 4th to 6th hr of the dark period of the second complete scotophase after emergence. This was accomplished using forceps by applying pressure to the anterior abdominal segments and dipping the exposed gland into 200 /zl of isooctane (Fisher 99 % mole) for ca. 30 sec. Up to 25 rinses were collected in each sample, and the extracts were analyzed without concentrating the isooctane. Extracts of the elongate hairs associated with the hairpencil gland were

416

TEAL AND TUMLINSON

prepared by cutting the hairs carefully from the surface of the gland, leaving a short portion attached and placing the hairs in a 0.5-ml conical microvial. The hairs were then rinsed with 10 p.l of iso-octane that contained the appropriate internal standards per male equivalent (ME). Two methods were used to collect the compounds volatilized from the hairpencil glands. In the first method, individual males were placed in an enlarged holding apparatus similar to that described by Teal et al. (1986). This apparatus was connected to another smaller chamber that contained a rubber septum impregnated with 1 mg of the six-component pheromone blend released by females (Teal et al., 1986). Purified air was passed over the lure and into the chamber holding the males. Volatiles released during hairpencil displays were collected on charcoal microentrainment filters and were recovered in a small amount of dichloromethane and iso-octane as described elsewhere (Teal et al., 1986). In the second method of volatile collection, the hairpencils were everted using forceps, as described earlier, under a stream of dried N2 (250 ml/min) and above a conical microvial that contained 250/A of isooctane for 30 sec so that the N 2 would carry volatiles into the solvent. The volatiles from the hairpencil scales of up to 50 insects were collected in the same sample, and the solvent was never concentrated to less than 25 ~1 under the N2 stream. Prior to methanolysis, the iso-octane was evaporated completely under N2 from extracts of the glandular hairs of groups of five males or volatile collections obtained from groups of 10 males. Methyl esters of acids present in the hairpencil pheromone were formed by acid methanolysis (Bjostad and Roelofs, 1984) and by methanolysis using boron trifluoride in methanol (Morrison and Smith, 1964). Biosynthesis. Studies on age-related production of the identified compounds were conducted using pharate adults that had been removed from the pupal case manually; adult males sampled after emergence but prior to wing expansion; insects that had just completed expansion of the wings; insects sampled 2, 4, and 6 hr after wing expansion; and adult insects 12, 24, 36, and 48 hr old. Studies on the effect of time of day on pheromone production were conducted using insects sampled during the peak of reproductive activity on the second scotophase after adult emergence and on insects sampled 12 hr later during the next light cycle. Studies on depletion of the compounds from the gland were conducted by flying males in a laboratory wind tunnel. The sixcomponent aldehyde pheromone blend identified as the sex pheromone released by females (Teal et al., 1986) was used as a lure. Males were allowed to land and fully expose their hairpencils during a 5-min test period. Only males that fully exposed the hairpencils and attempted copulation numerous times at the Source of the female sex pheromone were sampled. In all of these cases the whole hairpencil gland complex of individual males was everted, removed, and extracted for 30 sec in 30 tzl of iso-octane containing 10 ng of the appropriate

MALE H A I R - P E N C I L C O M P O U N D S

4t7

internal standard immediately after testing in the flight tunnel. The volume of the extract was then reduced to ca. 5/~1 and analyzed. In vivo studies on biosynthesis of the compounds were conducted using glands that were clamped in a fully exposed position with a smooth jawed alligator clip. The glandular hairs of some preparations then were removed carefully with forceps thus exposing the actual gland. The hairs that had been removed were placed in a conical microvial. Other preparations were left intact. Test alcohols or acetates were then applied, with a l-/zl syringe at a concentration of 500 ng in 1 #1 of dimethyl sulfoxide (DMSO), to denuded gland surfaces, the removed hairs, or the intact preparations. Treated and DMSO control preparations were then incubated for 1 hr at 25 °C prior to extraction as described for the individual glands and gas chromatographic analysis. Experiments were conducted during the reproductive period (ca. 4 hr after dark) and 12 hr later during the light period. Chemical Analysis. Chromatographic analyses were conducted using Hewlett Packard (Avondale, Pennsylvania) 5792 and 5890 gas chromatographs (GC) equipped with splitless and cool on-column capillary injectors and flame-ionization detectors. The detectors of the 5792 GC were interfaced to a Hewlett Packard 3390 reporting integrator. Data from the 5890 GC were acquired at a rate of 20 points/sec through a Chromadapt interface and Adalab data acquisition system (Interactive Microware, Inc., State College, Pennsylvania) and processed using an Apple IIe computer with Chromatochart software. Fused silica capillary columns used routinely for GC analsyis included a 15-m x 0.25 mm (ID) DB 225 (J & W Scientific, Folsom, California), a 30-m x 0.25-mm (ID) SPB1 (Supelco), and a 30-m x 0.25-m (id) Supelcowax 10 (Supelco, Bellefonte, Pennsylvania). Only the DB 225 column was used in conjunction with the cool on-column injector. In this case the initial temperature of 80°C was maintained for 0.5 min and then increased at 15°C/min to a final temperature of 135°C. Conditions of chromatography when using the splitless injectors were as follows: initial temperature, 80°C for 1 min; splitless purge at 0.5 min; temperature increase at 25°C/min to 165°C (Supelcowax 10) or 180°C (SPB1). Hydrogen was used as the carrier gas at a linear flow velocity of 38 cm/sec. The primary saturated acetates of tridecanol and pentadecanol (10 ng each) were used as internal standards for both synthetic and natural product samples and were used to calculate relative retention indices and to quantitate the amounts of compounds present in natural-product samples. Further GC confirmation was obtained by cochromatography of the natural product samples with the individual synthetic compounds on all three columns. Electron impact (EI) and chemical ionization (CI) mass spectra (MS) were obtained using VG1212F (VG Instruments, Toronto, Ontario) and Nermag R1010 (Delsi Instruments, Fairfield, New Jersey) instruments interfaced to Hewlett Packard 5792 GCs equipped with cool on-column capillary injectors. Helium

418

TEAL AND TUMLINSON

was used as the carder gas at a linear flow velocity of 18 cm/sec. Both isobutane and methane were used as ionization gases. In addition to positive ion CI-MS we also obtained negative ion (methane) spectra for extracts of the hairpencil scales. Samples were chromatographed on both the SPB1 column used in GC studies and a 50-m × 0.25-mm (id) DB5 (J & W) column under the following conditions: initial temperature, 80°C; temperature program, 20°C/min after 1 min; final temperature, 225°C. The retention times and fragmentation patterns of both the synthetic compounds and those present in natural products were compared. All synthetic chemicals were obtained from Sigma Chemical Company (St. Louis, Missouri). RESULTS

Structure of Hairpencil Gland. The modified hairs that make up the cuticular structures of the hairpencil gland are elongate and distinct from the flattened structure of nonspecialized body scales (Figure 1A and B). The glandular hairs have rows of circular pores separated by cuticular ridges extending their length (Figure 1C and D). Pores communicate with a duct in the interior of the structure (Figure 1E and F). The density of the pores diminishes toward the distal tip (Figure 1D). The cellular units in direct communication with the hairs are trichogen cells having closely associated tormogen and unmodified epidermal cells (Figure 1F and G). Nuclei of the trichogen cells are situated in the basal cell area, are well defined, and of lobulate structure. The central and apical areas of these cells are packed with vacuoles and osmiophylic droplets of similar size and structure to the vacuoles. The vacuoles are assumed to have contained lipid that was extracted during fixation and dehydration because traces of lipid were detected in some vacuoles. The apical cell membrane is involuted, leaving a large space that communicated with the duct of the hair associated with each cell (Figure 1G). Isolation and Identification. Initial GC analyses obtained on three different capillary columns of one male equivalent (ME) (N = 25) of the whole gland extracts indicated the presence of three peaks that were present in a consistent ratio. These peaks had retention indices corresponding to hexadecanol (16 : OH), hexadecanyl acetate (16 : Ac) and (Z)-I 1-hexadecenyl acetate (Z11-16: Ac). In addition, peaks having retention times corresponding to octadecanol (18:OH) and octadecanyl acetate (18 : Ac) were present, but the amounts varied substantially. Three other peaks also were present in chromatograms obtained using the SPB1 column. This column was capable of chromatographing free fatty acids, although peak shape was poor. These peaks had both relative retention indices and peak shapes that suggested that they were tetradecanoic (14 : Acid), hexadecanoic (16: Acid), and octadecanoic (18 :Acid) acids. Methanolysis of the extracts and subsequent chromatography on the three capillary columns indi-

MALE HAIR-PENCIL COMPOUNDS

419

FIG. 1. Scanning electron micrographs and micrographs of hairpencil glands and scales: (A) unmodified scales of male genitalia adjacent to hairpencil gland (S); (B) elongate hairs of the hairpencil gland modified for dissemination of chemicals; (C) pores (P) and ridges along surface of the hair; (D) tip of the hair showing reduction in pore size and number (P); (E) section through tip of hair showing pores in cuticle (P) and iniemal ducts (D); (F) section through hairpencil gland complex; H = hair, D = duct of hair, P = pores in scale, SO = socket; (G) section through trichogen gland cell (Tr), N = nucleus, L = lipid droplet, I = infolds in apical cell membrane, To = tormogen cell.

420

TEAL AND TUMLINSON

cated that the acid assignments were correct because the methyl esters of the acids were detected at concentrations of 2.7 ( + 0 . 9 ) ng 14:Acid, 22.2 ( + 7 . 3 ) ng 16:Acid, and 6.3 ( + 1 . 2 ) ng 18:Acid. Gas chromatographic analyses of extracts of the glandular hairs that disseminate the pheromone indicated that all of the above components were present in the same ratio found in whole gland extracts. Extracts of groups of five ME analyzed on all GC columns also contained compounds that had relative retention indices corresponding to tetradecanol ( 1 4 : O H ) , tetradecanyl acetate (14 : Ac), (Z)-9-tetradecenyl acetate (Z9-14 : Ac), (Z)-I 1-hexadecenol (Z1116 :OH), (Z)-7-hexadecenyl acetate (Z7-16 :Ac), and (Z)-9-hexadecenyl acetate ( Z 9 - 1 6 : A c ) (Figure 2). The amount of 1 8 : A c was highly variable, and some extracts did not contain this compound. The ratio and concentrations of these compounds are given in Table 1. Mass spectra (60-400 amu) and relative retention indices of the compounds present in the hair extract of 15-25 ME using both capillary columns coupled with El, positive ion CI (methane), and CI (isobutane) MS confirmed the presence of 1 6 : O H , Z l l - 1 6 : A c , 1 6 : A c , 18 :OH, 18 : Ac, 14:Acid, 16 : Acid, and 18 : Acid. Adequate spectra of the other compounds were not obtained. The use of negative ion CI-MS decreased substantially the amount of each acetate required to obtain spectral data. However, only a single ion, corresponding to M - l , was detected. Alcohols were not detected. When 15 ME of the glandular hair extract was analyzed in this manner, ions representing M-1 (m/z = 281) were detected at retention times coin-

6

10

1

2 3

5

7

9

12

13

14

L I I FIG. 2. Chromatogram obtained from analysis of five male equivalents of the extracts of glandular hairs using the SPBI capillary column. 1 = 14:OH, 2 = 14:Acid, 3 = Z9-14:Ac, 4 = 14:Ac, 5 = Z I I - 1 6 : O H , 6 = 16:OH, 7 = 16:Acid, 8 = Z7-16:Ac, 9 = Z9-16:Ac, 10 = Z I I - 1 6 : A c , 11 = 16:Ac, 12 = 18:OH, 13 = 18:Acid, 14 = 18:Ac.

MALEHAIR-PENCILCOMPOUNDS

421

TABLE 1. COMPOUNDSPRESENTIN HAIRPENCILGLANDEXTRACTSOF MALESOF Heliothis virescens BASEDON GAS CHROMATOGRAPHICANALYSIS(N = 20).

Compound

Mean amount (ng)

SD

Normalized percentage

14: OH 14: Ac Z9-14: Ac 14:Acid 16: OH ZI 1-16 : OH 16: Ac Z7-16 : Ac Z9-16:Ac Z11-16: Ac 16: Acid 18:OH 18: Ac 18 : Acid

0.35 1.2 0.19 2.7 27.3 .27 212.4 0.52 0.18 3.5 22.2 2.5 14.2 6.3

0.22 0.92 0.09 2.1 15.6 .25 78.4 .40 0.15 1.0 7.3 3.6 6.2 1.15

0.16 0.56 0.09 0.13 12.85 0.13 I00.00 0.29 0.08 1.16 10.36 1.18 6.69 2.97

cident with synthetic standards of Z-/-16 : Ac, Z 9 - 1 6 : Ac, and Z 1 1 - 1 6 : Ac. The ratio of ion intensities for m/z = 281 of the peaks corresponding of Z 7 - 1 6 : A c and Z 9 - 1 6 : AC was 2.1 : 1 (mean of three runs), which was similar to the 2.9 : 1 ratio detected in GC analyses. Although this provides good evidence that Z 7 16 : Ac and Z 9 - 1 6 : Ac are present, we cannot be certain of their existence without further and more complete spectral data. Volatiles collected on the charcoal entrainment filter of the pheromone released by males responding to the female sex pheromone contained an 11 : 1 ratio of 1 6 : A c to 1 6 : O H . The greatest amount of 1 6 : A c recovered from a male was 12.2 ng after six hairpencil exposures. Therefore, minor components were not detected due to the small amount of material collected. The 11 : 1 ratio present in these samples is comparable to the 9 : 1 ratio found in glandular hair extracts when considering the recovery efficiencies of the acetates and alcohols from the charcoal filter (Tumlinson et al., 1982). In order to obtain greater quantities of the volatile components, we developed the second technique of volatile collection. GC and GC-mass spectral analysis of volatiles collected in this manner confirmed the presence of 1 6 : A c and 1 6 : O H . GC analyses also indicated the presence of 14 : Ac, Z 1 1 - 1 6 : Ac, and 18 : OH. However, background was too high to obtain useful mass spectra of these compounds. GC and GC-mass spectral analyses of the esterified, volatile samples confirmed the presence of 14:Acid, 16:Acid, and 18:Acid. The ratio of all of the components was consistent with that found in extracts. Biosynthesis. Studies indicated that the glands of pharate adults did not

422

TEAL AND TUMLINSON

contain detectable amounts of either I 6 : A c or I 6 : O H . However, detectable amounts of both compounds were present in extracts of the hairpencil glands of males that had just emerged but had not expanded their wings (Figure 3). The amounts of each compound increased rapidly after adult emergence and peaked at the levels found in isolation identification studies within 36 hr after adult emergence. The titer of these compounds remained high at all times of the light or dark period (Figure 3) provided that the male did not evert the hairpencils. Substantial depletion of both 16:OH and 16:Ac occurred when males exposed their hairpencils numerous times in response to the sex pheromone blend produced by females (Figure 4). The greatest reduction, with respect to control males, was 32-fold for 16:Ac and 27-fold for 16:OH for a male that made eight flights from downwind and exposed his hairpencils several times after each landing. The smallest reduction was fivefold for both 16:Ac and 16:OH, and the majority of insects tested maintained titers of these two compounds that were 8- to 12-fold less than in the control insects. Minor components were not detected in any of the extracts obtained from males that had exposed the hairpencils repeatedly, and the amounts of the acids did not appear to be reduced, although esterified samples were not prepared.

,

100-

'~

I

t.."

75-

0

g. 25-

O

-

~

,;

2',

3'~ Age

,~

o! Male (Hr.)

FIG. 3. Titers of 16:Ac ( O I O ) and 16:OH (+ ... +) in extracts of pheromone glands of males sampled at various ages. Values are normalized to the mean amounts of 16:Ac (212.4 ng) and 16:OH (27.3 ng) present in extracts of males 48 hr old (N = 5 for each point).

423

MALE HAIR-PENCIL COMPOUNDS

S- 16:AC

S-16:OH

Male Hairpencils Not Exposed

S- 16:COOH

Male Hairpencils Exposed

,.L, FIG. 4. Comparison of extract obtained from the hairpencil glands of a virgin male H. virescens with that obtained from a male that had exposed the hairpencil gland numerous times during four flights from downwind in the flight tunnel. SPB1 column; attenuation = 8 × for both chromatograms.

Application of 500 ng of 14 : OH or Z11-16 : OH to intact hairpencil glands, glands denuded o f hairs, and the hairs from denuded glands did not result in changes in the ratios of the corresponding acetates present in extracts. Similarly, other alcohols including (E)- and (Z)-I 1-tetradecenol ( E 1 1 - 1 4 : O H , Z l l 14:OH) or Z9-14 : O H were not converted to the corresponding acetates when applied to the gland surface or to either the removed hairs or intact hairs. Scales incubated with 14: Ac, E 1 1 - 1 4 : Ac, or Z9-14 : Ac did not convert the acetates to the alcohol analogs. However, substantial amounts o f the alcohols were produced when the acetates were applied to the surface of the denuded gland (Figure 5). The conversion showed no specificity for either presence or geometry of double bonds as is indicated by the production o f 4.0 ( + 1.2) ng more 14 : OH

424

TEAL AND TUMLINSON

Helrpancll gland treated with E11-14:AC E11-14:AC 16:AC 10 ng 1 3 : A C ~ (latdl E11-14:OH

16:COOH

16:OH

I

E11-14:OH

Treated with E11-14:OH

10 ng 13:AC

16:COOH 1 161AC

16iOH

7 Untreated gland

10 ng 13:AC

16:COOH

18:OH

I

16:AC I

I

FIG. 5. Comparison of extracts of hairpencil gland (hairs removed) treated with E1114:Ac in DMSO (upper), E11-14:OH in DMSO (middle), or untreated (lower) using the SPB1 capillary column.

425

MALE HAIR-PENCIL COMPOUNDS

than was present in glands of untreated preparations (N = 10) and 3.7 (+0.7) ng (N = 10) of E11-14 : Ac after a 1-hr incubation. Studies conducted 12 hr after the peak of the reproductive period indicated that the temporal periodicity governing male response to the female pheromone had no effect on the biosynthetic capability of the gland because 3.9 (+0.6) ng of E 1 1 - 1 4 : O H was produced by preparations treated with the corresponding acetate during this period. Similar amounts of Z9-14 : OH and Z11-14 : OH were produced when glands were treated with the corresponding acetates.

DISCUSSION

Until recently, the components identified as male pheromones, for example, benzaldehyde and 2-phenylethanol (Tamaki, 1986), have had no structural similarity to the components released by females. However, Heath et al. (1988) have documented that males ofAnticarsia gemmatalis (H~ibner) release (Z,Z,Z)3,6,9-heneicosatriene from abdominal scent scales and have shown that this compound is attractive to other males. This compound is one of the pheromone components also produced by females. The identification of the acetates, alcohols, and acids that correspond to the aldehydic components released by H. virescens females (Teal et al., 1986) shows a close relationship between pheromone biosynthesis by males and females of this species. This close relationship is also demonstrated by the presence of an acetate esterase that functions to produce alcohol precursors in female glands (Teal and Tumlinson, 1987) as well as in the hairpencil glands where it produces the alcohol components. The function of such esterases is common among females of other moth species, for example Choristoneurafumiferana (Clem.) (Morse and Meighen, 1984, 1986), but this is the first report of the function of the esterase in production of pheromone components by males of Lepidoptera. It is of interest to note that the acetates that correspond to all six of the aldehydes, which comprise the pheromone released by females of H. virescens, were found in the hairpencil extracts. However, the ratio of the acetates was very different from that of the aldehydes released by females. For example, the ratio of 16 :Ac to Z l l - 1 6 : Ac present in the hairpencil pheromone gland is approximately 68: 1, while that of hexadecanal to (Z)-I 1-hexadecenal given off by females is 1 : 8 (Teal et al., 1986). This indicates that while both sexes possess the enzymes involved in desaturation (see Bjostad and Roelofs, 1983), the enzyme activities are different. The ratio of the acetates present in greatest amounts to the corresponding alcohols was similar, being about 7 : 1 for 18 : Ac/18 : OH, 8 : 1 for 16 : Ac/ 16:OH, 9:1 forZll-16:Ac/Zll-16:OH, and 6:1 for 14:Ac/14:OH. Therefore, it is probable that the alcohols corresponding to Z7-16:Ac, Z9-16:Ac,

426

TEAL AND TUMLINSON

and Z 9 - 1 4 : A c are present in the hairpencil gland, but were not identified because o f the small amounts present. This hypothesis is supported by our studies on biosynthesis, which have indicated that the acetates are converted to the corresponding alcohols. There also may be a similar correlation between the unsaturated acetates and their corresponding acids, given that the saturated acids that are analogous to 1 4 : A c , 1 6 : A c , and 18"Ac were identified. However, this hypothesis cannot be supported at present because, as yet, none o f the unsaturated acid analogs have been identified. The rise in amounts o f the alcohol and acetate components present in the hairpencil gland after adult emergence correlates well with reproductive maturity and parallels the increase in production o f the sex pheromone produced by females (Shorey et al., 1968). However, while titers of pheromone components drop dramatically after the calling period in the glands o f virgin female moths (Pope et al., 1982), the concentration in males remains constant. Therefore, it appears that biosynthesis by males proceeds until a specific titer is reached and then stops with the products being stored, perhaps in the extracellular pocket subtending the hairs. Our data indicate that after depletion o f these compounds, during hairpencil exposure, the alcohols and acetates are regenerated at a rate that would ensure that males would have a full titer o f these compounds during the next reproductive period. Given that the components released from the hairpencils are important for reproductive success in that they cause females to begin to call and to become receptive to male courtship advances (Teal et al., in preparation), depletion o f the compounds during unsuccessful mating attempts would explain why these males are less likely to mate on subsequent attempts during the same reproductive period than are males that have not exposed their hairpencils (Teal et al., 1981).

REFERENCES BAKER,T.C., NISHIDA,R., and ROELOFS,W.L. 1981. Close-range attraction of female Oriental

fruit moths to male hairpencils herbal essence. Science 214:1359-1361. BIRCH, M.C. 1970. Structure and function of the pheromone-producing brash-organs in males of Phologophora metriculosa (L.) (Lepidoptera: Noctuidae). Trans. R. Entomol. Soc. London

122:277-292. BJOSTAD,L.B., and ROELOFS,W.L. 1983. Sex pheromone biosynthesis in Trichoplusia hi: Key

steps involve delta-I l-desaturation and chain shortening. Science 220: 1387-1389. BJOSTAD,L.B., and ROELOFS,W.L. 1984. Sex pheromone biosynthetic precursors in Bombyx mori. bisect Biochem. 14:275-278.

CLEARWATER,J.R. 1972. Chemistry and function of pheromone produced by the male of the south-

ern armyworm, Pseudaletia separata. J. Insect Physiol. 18:781-789. DAHM, K.H., MEYER,D., FINN, W.E., REINHOLD,V., and ROLLER,H. 1971. The olfactory and auditory mediated sex attraction in Achroia grisella (Fabr.). Naturwissenschaften 58:265-266. GRANT, G.G. 1978. Morphology of the presumed male pheromone glands in the forewings of tortricid and phycitid moths. Ann. Entomol. Soc. Am. 71:423-431.

MALE HAIR-PENCIL COMPOUNDS

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Isolation, identification, and biosynthesis of compounds produced by male hairpencil glands ofHeliothis virescens (F.) (Lepidoptera: Noctuidae).

Extracts of the intact hairpencil glands and hairs from the hairpencil glands of males ofHeliothis virescens (F.) were analyzed by capillary gas chrom...
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