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Journal of the Lepidopterists' Society 52(4), 1998, 356-363

IDENTIFICATION OF MAJOR COMPONENTS OF THE

FEMALE PHEROMONE GLANDS OF EUCHAETES EGLE

DRURY (ARCTIIDAE)

Rebecca B. Simmons

Department of Entomology, University of Minnesota, 1980 Folwell Avenue, Saint Paul, Minnesota 55108, USA

AND

William E. Conner, Robert B. Davidson

Department of Biology, Wake Forest University, 7325 Reynolda Station, Winston-Salem, North Carolina 27109, USA

ABSTRACT. Extracts of the female pheromone glands of Euchaetes egle were analyzed using gas chromatography, infrared spectroscopy and mass spectroscopy. Identified compounds of the pheromone gland extract include a simple alkane (2-methyloctade-cane), one diene (Z,Z-6,9-heneicosadiene), two trienes (Z,Z,Z-3,6,9-eicosatriene, Z,Z,Z-3,6,9-heneicosatriene), and one tetraene (Z,Z,Z-l,3,6,9-heneicosatetraene). The primary compound was found to be Z,Z,Z-3,6,9-heneicosatriene. Male sensitivity to these compounds was determined using an electroantennogram assay, and Z,Z,Z-1,3,6,9-hene-icosatetraene elicited the greatest male response. Trienes are found in pheromone glands of the Arctiidae, Geometridae, Lymantriidae, and Noctuidae, suggesting that the use of these in compounds in pheromone glands is a plesiomorphic character for Arctiidae.

Additional key words: pheromones, mating behavior, Arctiidae.

In Lepidoptera, reproductive isolation among species can be achieved via temporal, chemical, or behavioral mechanisms. In moth systems, temporal and chemical isolation mechanisms are particularly important (Roelofs & Carde 1974, Meyer 1984). Temporal isolation is achieved by variations in flight times. Chemical isolation is achieved by variations in the female pheromone blend, which is emitted from glands located in terminal abdominal segments (Baker 1985, Linn & Roelofs 1989).

Lepidopteran female pheromone glands vary in structure, ranging from a simple band of glandular tissue to eversible sacs or folds (Jefferson et al. 1971, Percy & Weatherston 1974, Percy-Cunningham & Mac-Donald 1987). Female pheromone glands in arctiids usually are paired, air-filled tubular invaginations that open dorsally between the eighth and ninth abdominal segments (Meyer 1984, Conner et al. 1980). The glands have two layers: an outer cellular layer and an inner cuticular layer (Fig. 1). The cuticular layer is then elaborated into internal cuticular spines (Fig. 2) (Conner et al. 1980, Yinn et al. 1991). To release the pheromone, arctiid females rhythmically protrude the eighth and ninth segments (Conner et al. 1985).

Female pheromone blends are typically composed of straight-chained hydrocarbons, 16 to 22 carbons in length. These compounds can contain

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FlG. 1. Portion of the E. egle female pheromone gland, viewed using differential interference contrast (DIC) microscopy (O = outer cellular layer, I = inner cuticular layer, scale bar =100 jLlm).

a variety of functional groups, such as alcohols, aldehydes, acetate esters, epoxides and ketones (Leonhardt 1985, Mayer & McLaughlin 1991). In members of the Arctiidae, the major female pheromone components are trienes, triene derivatives, or 2-methylalkanes (Mayer & McLaughlin 1991). In this study, we examined the pheromone blend of Euchaetes e^e Drury, the milkweed tussock moth, and determined male electroantennogram response to identified major components of the female's pheromone blend.

Materials and Methods

Insects. Adult female E. egle were collected by James Adams in Dal-ton, Georgia. These gravid females were shipped to Wake Forest University, Winston-Salem, North Carolina. Eggs from these females and subsequent generations were also reared on Asclepias syriaca.

Female pheromone identification. Pheromone glands were extracted from nine adult females before the calling cycle, and placed in

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Fig. 2. Lumen of the E. egle female pheromone gland, viewed using DIC microscopy (S = cuticular spines, scale bar = 100 \lm).

100 Ltl of methylene chloride. No internal standard was used. Glands were extracted overnight. This extract was subjected in 1 JLll aliquots to gas chromatography/infrared spectroscopy/mass spectrometry on a Hewlett Packard (HP) 5890 GLC coupled with both an HP 5965A infrared detector and an HP 5970 mass selective detector. Components of the pheromone blend were separated on a DBl fused silica capillary column, measuring 60 m x 0.32 mm x 0.5 mm thickness. The initial temperature was held at 100° C for 2 min and then increased to 240° C at 2.5°C/min.

The E. egle pheromone blend was compared to a set of nine standards, that were subjected to the conditions given above. These standards were: (1) Z,Z-6,9-heneicosadiene (> 99% pure); (2) Z,Z,Z-1,3,6,9-heneicosatetraene (> 95% pure); (3) Z,Z,Z-3,6,9-octadecatriene (> 99% pure); (4) Z,Z,Z-3,6,9-eicosatriene (92% pure); (5) Z,Z,Z-3,6,9-hene-icosatriene (99% pure); (6) Z,Z,Z-3,6,9-docosatriene (94% pure); (7) 2-methyloctadecane (99% pure); (8) 2-methylheneicosane (75% pure);

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and (9) 2-methyldocosane (82% pure). Standards were analyzed as a 1:1:1:1:1:1:1:1:1 blend. Standards were run in concentrations that bracketed all extract concentrations.

Z,Z,Z-l,3,6,9-heneicosatetraene was undetectable using the methods described above due to its instability at high temperatures (Jain et al. 1983). Because of this instability, the pheromone and standard blends were also analyzed using an HP 5790A gas chromatograph with an on-column injection port and a flame ionization detector. An Ultra II (5% phenyl methyl silicone) column was used for this analysis (25 m, 0.32 ID, 0.52 mm film thickness). Carrier flow was set at 5.0 ml/min. The initial temperature was 50°C, which was held for 5 min. and then increased 5°/min until it reached 280°C. Extracts of pheromone glands and the nine-component standard blend were injected as 1 \xl aliquots and compared. The percentage of each sample in the extract was also calculated by dividing the abundance of the individual compound by the total abundance of all compounds.

Electroantennograms. Electroantennograms (EAG's), as described in Rodgers (1991), were used to determine male responsiveness to possible components of the female pheromone blend of E. egle. Antennae were removed from 1 to 2 day old males (n = 7). The proximal end of the antenna was placed in a Petri dish containing EAG saline (NaCl 7.5 g/1, CaCl2 0.21 g/1, KCl 0.35 g/1, NaHCOs 0.2 g/1) (Rodgers 1991). Several segments were removed from the distal end of the antenna. The remaining distal end was placed in contact with a input electrode. The input electrode, via a high impedance electrode (Tektronix model 013-0071-00), was connected to a Tektronix 5115 storage oscilloscope. A continuous charcoal filtered airstream (1800 ml/min) was passed over the antenna. Two ml puffs of air were passed through Pasteur pipettes containing filter paper impregnated with one of nine pheromone standards (10 jag dissolved in 100 jul of methylene chloride) and a negative control (methylene chloride). These puffs were injected into the air stream, through an opening in the air supply tube.

Responses to the following nine standards were measured: (1) Z,Z-6,9-heneicosadiene; (2) Z,Z,Z-3,6,9-octadecatriene; (3) Z,Z,Z-3,6,9-eico-satriene; (4) Z,Z,Z-3,6,9-heneicosatriene; (5) Z,Z,Z-3,6,9-docosatriene; (6) Z,Z,Z-l,3,6,9-heneicosatetraene; (7) 2-methyloctadecane; (8) 2-methylheneicosane; (9) 2-methyldocosane. Standards were presented in random order. Antennal responses were measured twice per antenna for all standards and the control (the order of presentation of the stimuli being reversed for the second set of measurements). These responses were then averaged, and compared across all males using one way analysis of variance (ANOVA) and a Scheffe F-test.

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Table 1. Summary of compounds found in the female pheromone blend of Euchaetes egle. Letters refer to compounds in total ion chromatogram, compound X to subsequent analysis, — = trace amount.

Identification/Comments

Amount in Blend (%)

Mass spectrum indicates an alkane

Parent ion, mass spectrum indicates a

branched alkane

Infrared and mass spectra match

2-methyloctadecane; presence also

confirmed by subsequent gas chromatography.

Infrared and mass spectra indicates an alkane

Mass spectrum is similar to that of

Z,Z?Z-3,6,9- eicosatriene; presence confirmed

by subsequent gas chromatography

Mass spectrum matches

Z,Z-6,9-heneicosadiene; presence confirmed

by subsequent gas chromatography

Infrared and mass spectra match

Z,Z,Z-3,6,9-heneicosatriene; presence

confirmed by subsequent gas chromatography

Mass spectrum indicates an alkane

Mass spectrum corresponds to spectrum of

Z,Z,Z-l,3,6,9-heneicosatetraene, may be a

22-or 23-carbon tetraene

Retention time in subsequent gas chromatography

matches Z,Z?Z-1,3,6,9-heneicosatetraene

23.4

2.7

4.3

37.5

14.8

Results

Female pheromone identification. A total ion chromatogram of the E. egle pheromone blend extract revealed nine compounds. Of these, three (C, F, G) could be matched to standards (Table 1). The retention time for compound E was close to that of Z,Z?Z-3,6,9-eicosatriene, and the compound had a fragmentation pattern indicative of trienes (De-scoins et al. 1986). However, the parent ion of compound E had a mass/ charge ratio of 281, while the parent ion of Z,Z,Z-3,6,9-eicosatriene had a mass/charge ratio of 275. Because compound E was found in trace amounts in the total sample, an infrared spectrum could not be obtained. Compound I did not match any standards tested, but showed a mass spectrum similar to that of Z,Z?Z-l,3,6,9-heneicosatetraene (Jain et al. 1983). The other four unidentified components (A, B, D, H) correspond to alkanes of varying chain lengths (McLafferty 1973).

A subsequent analysis using on-column injection, Ultra II 5% phenyl methyl silicone column revealed compounds that had matching retention times to the following standards (relative percentage of compound in the total sample): 2-methyloctadecane (23.4%), Z?Z-6,9-heneicosadi-ene (4.3%), Z,Z,Z-3,6,9-eicosatriene (2.7%), and Z,Z,Z-3,6,9-hene-icosatriene (37.5%). Additionally, 2-methyldocosane (1.7%) and Z,Z,Z-

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23456 7 89

Pheromone Standard

FlG. 3. Mean electrical response of male antennae to female pheromone standards and a control (1—10) (n = 7). * = compound found in pheromone blend extract, ♦ = significantly different from control, 1 = Control, 2 = Z,Z-6,9-heneicosadiene, 3 = Z,Z,Z-3,6,9-oc-tadecatriene, 4 = Z,Z,Z-3,6,9-eicosatriene, 5 = Z,Z,Z-3,6,9-heneicosatriene, 6 = Z,Z,Z-3,6,9-docosatriene, 7 = Z,Z,Z-l,3,6,9-heneicosatetraene, 8 = 2-methyloctadecane, 9 = 2-methylheneicosane, 10 = 2-methyldocosane.

3,6,9-docosatriene (1.8%) were also found. This analysis also revealed an additional peak (Compound X; Table 1) at 30.61 minutes, which matches the retention time of Z,Z,Z-l,3,6,9-heneicosatetraene (14.8%). Electroantennograms. Electrical responses of male antennae were significantly higher to Z,Z,Z-l,3,6,9-heneicosatetraene (7) than to the control (1) (P < 0.05). Responses to other standards, including those compounds found in the E. egle pheromone gland extract, were not significantly different than responses to the control (Fig. 3).

Discussion

It should be noted that the compounds identified are components of the extract of a pheromone gland and that this extract may include pheromonal precursors as well as true pheromone components. Compounds found in the pheromone gland extracts of E. egle are similar to pheromonal compounds of other arctiids. Z,Z,Z-3,6,9-heneicosatriene, is found in Arctia villica L.(Einhorn et al. 1984), two species of Cre-atonotos (Bell & Meinwald 1986, Wunderer et al. 1986), Halysidota leda Druce (C. Descions pers. comm.), Paraeuchaetes pseudoinsulata (Walker) (Schneider et al. 1992), Phragmatobia fuliginosa L. (C. De-scions pers. comm.), Syntomeida epilais Walker (C. Descions pers. comm.), and Utetheisa ornatrix L. (Conner et al. 1980, Jain et al. 1983). The triene, Z,Z,Z-3,6,9-eicosatriene, is a component of the pheromone blend of Paraeuchaetes pseudoinsulata (Walker) (Schneider et al. 1992). Trienes are found in the pheromone glands of Geometridae, Lymantri-idae, and Noctuidae (Mayer & McLaughlin 1991). This distribution suggests that triene use is the ancestral condition for Arctiidae, and perhaps much of Noctuoidea.

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The other components found in the E. egle gland extract are also found in other arctiid pheromone blends. A secondary diene component, Z7Z-6,9-heneicosadiene, is present in pheromone blends of members of Creatonotos (Bell & Meinwald 1986, Wunderer et al. 1986), Faraeuchaetes pseudoinsulata (Walker) (Schneider et al. 1992), and Utetheisa ornatrix L. (Jain et al. 1983). Alkanes, such as 2-methylalka-nes, are found in the female pheromones of many species of Holomelina and in Fyrrharctia Isabella (J.E. Sm.) (Roelofs & Carde 1977).

EAG's performed on E. egle males (Fig. 3) show that the compound (7), ZjZ^Z-l^^^-heneicosatetraene, elicits the greatest response. It is also found as a secondary component in the pheromone blends of Arc-tia villica L. (Einhorn et al. 1984), and two species of Utetheisa (C. De-scoins pers. comm., Jain et al. 1983). The tetraene may act as the species specific cue, guiding E. egle males to appropriate mates.

Acknowledgments

We thank James Adams for providing E. egle larvae and adults, and Jerrold Meinwald, E. W. Underbill, George Pomonis, and P. E. Sonnet for providing pheromone standards. Chemical analyses were performed in William Coleman's laboratory at R. J. Reynolds, Winston-Salem, North Carolina. Ruth Boada and Jessica Kent aided with rearing larvae and EAG assays. Dennis Kubinski aided with light microscopy of the pheromone glands. We also thank Susan Weller, William Miller, Lawrence Gall, and two anonymous reviewers for comments on this manuscript. This project was supported in part by a grant-in-aid of research from Sigma Xi, The Scientific Research Society (R. B. Simmons), the Whitehall Foundation (W E. Conner), and the National Science Foundation (DUE-9350763 to W. E. Conner).

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Received for publication 30 October 1997; revised and accepted 30 June 1998.