Journal of Chemical Ecology, Vol. 17, No. 1, 1991

IDENTIFICATION MALE

OF HAIRPENCIL

SECRETION

FROM

M a m e s t r a b r a s s i c a e (L.) ( L E P I D O P T E R A :

NOCTUIDAE)

AND

ELECTROANTENNOGRAM STUDIES

E. JACQUIN,* P. NAGNAN, and B. FREROT Laboratoire des MOdiateurs chimiques Domaine de Brouessy INRA Magny les Hameaux. F-78114, France (Received May 23, 1990, accepted September 10, 1990)

Abstract--Extracts of male Mamestra brassicae (L.) hairpencils were analyzed by capillary gas chromatography (GC) and by GC-mass spectrometry (GC-MS). The extracts were found to consist of six components. Benzaldehyde, 2-methylpropanoic acid, 2-methylbutanoic acid, and phenol were present in the extracts as well as the previously identified benzyl alcohol and phenyl ethanol. The two major components were 2-phenylethanol and 2-methylbutanoic acid. They represented, respectively, 74% and 12.5% of the total blend in 3-day-old male extracts. Electroantennograms were recorded on male and female antennae in response to stimulation by hairpencil compounds. Male and female antennae responded to each chemical but the female responses were significantly higher than those of the males. Key Words--Mamestra brassicae, Lepidoptera, Noctuidae, hairpencils, male-produced compounds, identification, benzaldehyde, 2-methylpropanoic acid, 2-methylbutanoic acid, benzyl alcohol, 2-phenylethanol, phenol, electroantennography.

INTRODUCTION

Like many noctuid moths, male Mamestra brassicae (L.) possess evertable scent organs known as hairpencils, located in abdominal pockets situated on the first and second abdominal sternites. These structures are associated with two inter*To whom correspondence should be addressed. 239 0098-0331/91/0100-0239506.50/0 9 199l Plenum Publishing Corporation

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JACQUIN ET AL.

nal glands, described by Stobbe (1912), and are everted during courtship behavior. In the vicinity of the female, they release an odor detectable by the human nose. In an attempt to elucidate the effect of male hairpencils and their associated odors on calling females, we compared in a previous work the courtship behavior of males with and without hairpencils (Jacquin, 1989). The number of attempted copulations, followed by either the rejection or the acceptation of the male by the female, was quantified. Our results, according to Birch et al. (1989), suggest that the presence of male hairpencils increases female acceptance. Observed behaviors suggested that hairpencil secretions could affect the recognition of the sexual partners and could be an important factor in sexual selection. In order to provide further evidence for putative functions of the hairpencil secretions, biological tests using pheromonal blends could be used. A reinvestigation of the chemical composition of M. brassicae hairpencil extracts is therefore necessary since no recent study has been reported. Bestmann et al. (1977) identified two compounds in hairpencil extracts of males M. brassicae: 2-phenylethanol (96%) and benzyl alcohol (4%). This paper will discuss the identification of new components and their electrophysiological significance.

METHODS AND MATERIALS

Insects. The insects were reared on a cabbage semisynthetic diet (Poitout and Bues, 1974) and kept under a reversed light-dark 16 : 8 photoperiod at 20 _+ 2~ at every life stage. The insects were sexed as pupae, and the adults were kept in hermetic plastic containers and fed with a sugar solution (10%). Only naive 3-day-old adults were used in this experiment. Hairpencil Extracts. Four hours after the onset of the scotophase, hairpencils were carefully excised out of individual males that had previously been anesthetized with carbon dioxide. The third day of insect life corresponded to the optimum of male sexual activity under our experimental conditions. All dissections were carried out on males that presented a complete retraction of the hairpencils in their abdominal pockets. Each pair of individual male hairpencils was quickly immersed into a glass vial containing 200/xl of pentane (SDS, Vitry, France) and was left to extract at room temperature for one hour. This was repeated with 10 different males. Solutions were then condensed to 100 #1 under a stream of nitrogen before analysis. Chemical Analysis. Identifications were performed by capillary gas chromatography (GC) and by gas chromatography-mass spectrometry coupling (GCMS). GC analyses were performed on a Hewlett-Packard 5890 gas chromatograph equipped with a flame ionization detector (FID) and a FFAP column 25 m x 0.32 mm (ID) (Chrompack) using helium as the carrier gas (Pne = 2.76

HAIRPENCILSECRETIONIN Mamestra brassicae

241

bar). Oven temperature was programmed to increase from 60~ to 140~ at 3~ Samples were injected on a Ross injector after 10 sec of evaporation or on a split/splitless injector. Injector temperature was 220~ and detector temperature was 250~ GC-MS identification was conducted on a Nermag 10-10C quadmpolar mass spectrometer using a Sidar data system (EI, 70 eV, 30-400 ainu). The GC was equipped with a nonpolar capillary column, CPSIL5 CB, 25 m • 0.32 mm (ID) (Chrompack) programmed to increase from 60~ to 140~ at 3~ min. Identifications of the extract components were determined by comparing their retention times on both polar and nonpolar columns and their spectra with those of reference compounds (Aldrich Chimie). Electroantennography. Compounds identified in the crude extracts were tested on both male and female antennae. Electroantennograms (EAG) were recorded according to the technique described by Renou (1979). Chemicals were diluted in hexane and applied on filter papers at a dose of 10 t~g. Stimulations with air alone and hexane alone (1 ~1) were applied as check samples. The stimulation cartridges were only used twice and then renewed to prevent loss of charge due to the volatility of the chemicals. A 1-sec pulse of air, delivered at 60 liters/hr, was allowed to pass through the cartridge. Five male and five female antennae were tested twice each with the set of chemicals.

RESULTS

Identification of Hairpencil Components. GC analyses showed six peaks (Figure 1). The retention times of peaks 4 and 5 matched the retention times of, respectively, benzyl alcohol and 2-phenylethanol, which had previously been identified by Bestmann et al. (1977). GC-MS spectra confirmed these results. The first peak exhibited the same retention time as that of benzaldehyde. This identification was confirmed by the mass spectrum, which matched the reference benzaldehyde spectrum. Peak 6 had the same retention time and mass spectrum as phenol, whereas peaks 2 and 3 showed characteristic ions of 2-methylpropanoic acid and of 2-methylbutanoic acid. The retention times of both acids corresponded with those of the reference samples for the two types of columns. Analyses of individual pairs of hairpencils from 10 individual males confirmed the occurrence of the six previously identified compounds. The percentage composition of the hairpencil extracts is: 1% benzaldehyde, 3.5% 2-methylpropanoic acid, 12.5% 2-methylbutanoic acid, 6.5% benzyl alcohol, 74% 2-phenylethanol, and 2.5% phenol (Table 1). Studies of the correlations (Spearmann test) between the relative percent-

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JACQUIN ET AL.

3

5

,,..~CO0H 4

[~ CH20H

2 '---COOH

OH

CHO

I0

1~5

20

r.t.

(min.)

F~. 1. Capillary column gas chromatogram of hairpencil extract of a virgin Mamestra brassicae male (2/A out of 100/zl extract, other conditions in text). 1: benzaldehyde; 2: 2-methylpropanoic acid; 3: 2-methylbutanoic acid; 4: benzyl alcohol; 5: 2-phenylethanol; 6: phenol.

ages of these six compounds showed there to be negative correlations between phenylethanol and the other compound ratio (P < 0.05). However, phenol did not correlate with any of the five other compounds. The correlations were positive between each of the others (P < 0.05). Electroantennographic Results. Electroantennogram recordings indicated that both male and female antennae responded to the six identified compounds (Figure 2). All six compounds were significantly better perceived by the female antennae than were the controls of hexane or air alone (P < 0.01). However, the responses of male antennae to the 2-methylpropanoic acid did not

H A I R P E N C I L S E C R E T ION IN

TABLE l.

BAG

243

Mamestra brassicae HAIRPENCIL EXTRACT COMPOSITION

Compounds

Percentage composition (means _+ SEM, N = 10)

Benzaldehyde 2-me.prop.ac. 2-me.but.ac. Benzyl OH 2-phenyl ethOH Phenol

1 +_ 0.12 3.5 + 0.6 12.5 _+ I 6.5 _+ 0.6 74 _+ 2.3 2.5 _+ 0.7

~N

male

m

female

responses

-m V +

+ + + +

+_+

o9__ ~4 ~6

l "

4

5

2

3

6

(I) c~ N q3

FIG. 2. Average values of EAG potentials evoked on five antennae of both male and female Mamestra brassicae by 10/xg of each identified compound. Vertical bars indicate the standard error. ( + ) or (+ +) above responses indicates that the responses are significantly different between male and female (Mann-Whitney: + P < 0.05, + + P < 0.01). All the responses to the test compounds are significantly different from controls (air and hexane) (P < 0.05) except for the male response to 2-methylpropanoic acid (2).

significantly differ from the responses to the hexane control. The other compounds elicited significantly higher responses than hexane on male antennae (P < 0.05 for 2-methylbutanoic acid and P < 0.01 for all others). Both males and females showed the same response pattern to the six components of hairpencil extracts. The highest responses were evoked by benzaldehyde, benzyl alcohol, 2-phenylethanol, and phenol for both sexes. The 2-methylpropanoic acid and the 2-methylbutanoic acid showed lower activities. The comparison o f male and female E A G responses to the six compounds

244

JACQUIN ET AL.

showed that female antennae gave significantly higher responses, except for the two acids (Figure 2).

DISCUSSION

Low-molecular-weight compounds have been isolated previously from hairpencils of various noctuid males (reviewed by Zagatti, 1981; Boppr6, 1984; Fitzpatrick and McNeil, 1988; Birch et al., 1990). All are simple terpenoids, aromatics, and carboxylic acids. The six compounds isolated in Mamestra brassicae hairpencil extracts are structurally similar and have been reported previously in other noctuids as hairpencil components (Aplin and Birch, 1970). However, phenol has never been identified in male hairpencils of any other lepidoptera. Nevertheless, phenols are described as defensive allomones of millipedes (Blum, 1987) and as a sex attractant for the grass grab beetle Costelytra zealandica (Henzell and Lowe, 1970). Phenols also are known to be produced by wounded trees and to act as a repellent or a toxic agent to bark beetles (Raffa and Berryman, 1983). Fitzpatrick et al. (1989) relate that many no ctuids are attracted to exudates of tree wounds. It is curious to find phenol as a hairpencil constituent, and its biological function could differ from the other component functions. Further behavioral studies will be conducted to test the different functions of the hairpencil components. Benzaldehyde is an extremely common hairpencil component and is found to be a major component of male scent brushes of many Leucania and Mythimna species, and a minor component in Polia nebulosa (Hufn.) and Mamestra persicariae (L.) (Aplin and Birch, 1970). Up to now, it has not been detected in M. brassicae. This was probably due to the low quantity present in hairpencil extracts (1%). In our experiment, benzyl alcohol and 2-phenylethanol have been described as hairpencil components of M. brassicae with approximately the same relative ratios as those published by Bestmann et al. (1977). However, 2-methylpropanoic acid and 2-methylbutanoic acid have been newly identified in this study as components of M. brassicae hairpencil extracts. The 2-methylpropanoic acid has been found at a level of about 20 % in hairpencil extracts of several Hadeninae [Leucania impura (Hueb.), L. conigera (Schiff.), L. pallens (L.)] (Aplin and Birch, 1970). 2-Methylbutanoic acid is also produced by some noctuid species (Amphipyrinae and Cucullinae) but has never been reported in Hadeninae. This compound appeared in our experiment to be one of the main compounds of M. brassicae male extracts (12.5 %). The 10 analyses of individual male hairpencil extracts reflected a low vari-

HAIRPENCIL SECRETION IN Mamestra

brassicae

245

ability of the component percentages between individuals. However, the quantities detected seemed to be very variable from one male to another. EAG recordings indicated that female antennae responded to the six compounds identified in male hairpencil extracts. It is of interest that male M. brassicae also responded to the majority of their own hairpencil components, except to 2-methylpropanoic acid. This suggests that the male scent could affect male behavior. Although Toth (1982) does not find any evidence for male-to-male inhibition, other effects should be considered. Male and female antennae responded in the same way to the different hairpencil components. This is in accordance with the results on Trichoplusia ni (Htibn.) (Grant, 1970) and suggests that the antennae of both sexes possess similar receptors for detecting male scents. Nevertheless, nothing is known on the specificity of those receptors for the hairpencil components. The female responses were higher than the male responses. As EAG is thought to be the summed potential of the activated receptor cells of the antennae (Boeckh, 1969), our results suggest that female antennae possess more acceptor sites for all of the compounds than do the male antennae. The fact that the phenol ratio did not correlate with the ratios of the other identified compounds appears to be consistent with the hypothesis that there is a different metabolic origin between this chemical and the other aromatic compounds. Phenol is known to originate in tyrosine (Blum, 1987) while phenylethanol and benzaldehyde originate in L-phenylalanine (Clearwater, 1975). The two acid precursors are not known and could be valine and isoleucine for, respectively, 2-methylpropanoic acid and 2-methylbutanoic acid as described in other insects (Blum, 1987). Identification of new components in Mamestra brassicae hairpencils may provide some clues for the understanding of the specificity of chemical communication between males and females. Many noctuid males within Hadeninae are considered to produce closely related scent blends, but it may be worthwhile to reinvestigate hairpencil secretions by GC on capillary columns and by GCMS. Minor unidentified components could act to promote species isolation. To date, we do not know whether the female can discriminate variable blends of the same components or whether the quality of the blend can interact with sexual behavior and act on sexual selection. Males seem to produce variable quantities of the same blend of compounds. Does this fact interact with the female choice or acceptance? Further studies on this subject will be conducted on Mamestra brassicae. Acknowledgments--The authors are pleased to thank Dr. P. Zagatti and Dr. M. Renou for their helpful comments, Dr. C. Descoins for reviewing the manuscript and C. Malosse for GC and GC-MS advice.

246

JACQUIN ET AL. REFERENCES

APLIN, R.T., and BIRCH, M.C. 1970. Identification of odorous compounds from male Lepidoptera. Experientia 26:1193 - 1194. BESTMANN, H.J., VOSTROWSKY, O., and PLATZ, H. 1977. M~innchenduftstoffc von Noctuiden (Lepidoptera). Experientia 33:874-875. BIRCH, M.C., LUCAS, D., and WHITE,P.R. 1989. The courtship behavior of the cabbage moth, Mamestra brassicae (Lepidoptera: Noctuidae), and the role of hairpencils. J. Insect. Behav. 2:227-239. BIRCH, M.C., POPPY, G.M. and BACKER,T.L. 1990. Scents and eversible scent structures of male moths. Annu. Rev. Entomol. 35:25-58. BLUM, M.S. 1987. Biosynthesis of arthropod exocrine compounds. Annu. Rev. Entomol. 32:381413. BOECKH, J. 1969. Electrical activity in olfactory receptor cells, pp. 34-51, in C. Pfaffman (ed.). Olfaction and Taste, Vol III. Rockefeller University Press, New York. BOPPR~, M. 1984. Chemically mediated interactions between butterflies, pp. 261-275, in R.I. Vane-Wright and P.R. Ackery (eds.). The Biology of Butterflies. Academic Press, London. CLEARWATER,J.R. 1975. Synthesis of a pheromone precursor in the male noctuid moth, Mamestra configurata. J. Insect Biochem. 5:737-746. FITZPATRICK, S.M. and MCNEIL, J.N. 1988. Male scent in Lepidopteran communication: The role of male pheromone in mating behaviour of Pseudaletia unipuncta (Haw.) (Lepidoptera: Noctuidae). Mere. Entomol. Soc. Can. 146:131-151. FITZPATRICK, S.M., MCNEIL, J.N., and MILLER, D. 1989. Age-specific titer and antennal perception of acetic acid, a component of male Pseudaletia unipuncta (Haw.) hairpcncil secretion. J. Chem. Ecol. 15:641-648. GRANT, G.G. 1970. Evidence for a male sex pheromone in the noctuid, Trichoplusia ni. Nature 227:1345-1346. HENZELL, R.F., and LOWE, M.D. 1970. Sex attractant of the grass grub beetle. Science 168:10051006. JACQUIN, E. 1989. Etude comportementale et 61ectrophysiologique des ph6romones mfiles de la noctuelle du chou Mamestra brassicae (L.). M6moire de DAA Protection des Cultures, INAPG Paris. 24 pp. POITOUT, S., and BUES, R. 1974. Elevage de 28 esp~ces de 16pidopt~res Noctuidae et de 2 espbces d'Arctiidae sur milieu artificiel simplifi6. Particularit6s selon les espbces. Ann. Zool. Ecol. Anita. 6:431-441. RAPFA, K.F., and BERRYMAN,A.A. 1983. Physiological aspects of lodgepole pine wound responses to a fungal symbiont of the mountain pine beetle, Dendroctonus ponderosae (Coleoptera: Scolytiidae). Can. EntomoL 115:723-734. RENOU, M. 1979. Contribution ~ l'etude de la communication ph6romonale chez trois Tinbides ravageurs des cultures: Acrolepiopsis assectella (Z.), Scobipalpa ocellatella (Boyd.), Prays oleae (Bem.), Thbse de 3~me cycle, Paris VI. 161 pp. STOBBE, R. 1912. Die abdominalen Duftorgane der M~nnlichen Sphingiden und Noctuiden. Zool. Jb. 32:493-532. TOTH, M. 1982. Male produced pheromone of Mamestra brassicae L. (Lepidoptera: Noctnidae): Its possible role in courtship behaviour and study of other effects. Acta Phytopathol. Acad. Sci. Hung. 17:123-132. ZAGATTI, P. 1981. Micro-comportements induits par les phdromones sexuelles chez quelques ldpidoptbres ravageurs des cultures en milieu sah61ien. Thbse de 3bme cycle, Paris VI. 161 pp.