Journal o f Chemical Ecology Vol. 8. No. 4, 1982
DEFENSIVE AND PHEROMONAL SECRETION OF THE T E R G A L G L A N D OF Aleochara curtula 1
I. The Chemical Composition
K. P E S C H K E 2 a n d M. M E T Z L E R 3 2Zoologisehes Institut 111 der Universitiit Warzburg, ROntgenring 10, D-8700 Wiirzburg ~Institut f u r Pharmakologie und Toxikologie, Versbacher Str. 9, D-8700 Wiirzburg West German),
(Received April 16, 1981; revised August 27, 1981) Abstract--The defensive secretion from the tergal gland of the staphylinid beetle, Aleochara curtula, acting as a supplementary mating stimulant, was investigated by gas-liquid chromatography, mass spectrometry, and IR spectroscopy. The reservoir contains a complex mixture of hydrocarbons, aliphatic aldehydes, and substituted 1,4-benzoquinones. n-Undecane, I-undecene, and (Z)-4-tridecene were identified as the major hydrocarbon components. The main aldehydes are n-dodecanal and (Z)-5-tetradecenal, and the chief quinones are toluquinone and 2-methoxy-3-methyl-l,4benzoquinone, the latter being established structurally by comparison of the mass spectra of the three synthesized isomers. Quantitative GLC analyses revealed no sex specificity of the relative concentrations of the compounds. Key Words--Aleochara curtula (Goeze), Coleoptera, Staphylinidae, tergal gland secretion, hydrocarbons, aliphatic aldehydes, substituted 1,4-benzoquinones, n-undecane, 1-undecene, (Z)-4-tridecene, n-dodecane, (Z)-5tetradeeenal, toluquinone, 2-methoxy-3-methyl-l,4-benzoquinone.
INTRODUCTION W i t h i n t h e s t a p h y l i n i d beetles, a t e r g a l g l a n d s u p p o r t e d w i t h a l a r g e r e s e r v o i r is r e s t r i c t e d to the s u b f a m i l y A l e o c h a r i n a e ( A r a u j o , 1978). H i s t o l o g i c a l i n v e s t i g a t i o n s o n the e p i d e r m a l g l a n d s w e r e m a d e to i n t e r p r e t t h e b e h a v i o r a l a d a p t a t i o n s o f m y r m e c o p h i l o u s a n d t e r m i t o p h i l o u s A l e o c h a r i n a e in c o r n ~Coleoptera: Staphylinidae (Aleocharinae) 773 0098-0331/ 82/0400-0773503.00/09 1982PlenumPublishingCorporation
774
PESCHKE AND METZLER
parison to the free-living species (e.g., Pasteels, 1968; Kistner and Pasteels, 1969). The secretion of the tergal gland of some species has been proved to have defensive properties (Jordan, 1913; Pasteels, 1968; H611dobler, 1970). In Aleochara curtula (Goeze), which is a most abundant species found with carcasses, intraspecific communication by components of the tergal gland secretion was studied and their role as additional mating stimulants established (Peschke, 1982). These effects support the female sex pheromone from epicuticular lipids (Peschke, 1978a). The chemical composition of the tergal gland secretion of two closely related Aleocharinae has also been investigated in Lomechusa strumosa (Blum et al., 1971) and Drusilla canaliculata (Brand et al., 1973), but much more comparative data on tergal gland chemistry are needed in order to make safe conclusions concerning the chemical taxonomy, the function of each compound, and the evolution of this versatile group of beetles. In the present paper we report our chemical investigations on the tergal gland secretion ofA. curtula as a basis for subsequent behavioral investigations. METHODS AND MATERIALS
Collection of Secretion. A. curtula was reared in the laboratory according to Fuldner (1968) and Peschke (1978a). Sexes were separated immediately after emergence. Beetles were freshly killed by freezing, and the tergal gland secretion was collected by inserting a triangle of filter paper between the sixth and seventh abdominal tergites. The triangles from 100 males or females were collected in 1 ml n-hexane. This extract was stored at - 1 7 ~ and could be used for GLC without further preparation. Chemical Isolation and Identification. Gas-liquid chromatographic analyses (GLC) were carried out on a Shimadzu GC-6A instrument with hydrogen flame detectors under standard conditions of hydrogen (25 ml/min), air (250 ml/min), and nitrogen flow (30 ml/min), employing the routine temperature program (70-190 ~ C at 8~ C/rain column temperature, 210 ~ C detector and injector temperature). Comparison of retention times and cochromatographics were run on four liquid phases (3% or 10% Carbowax 20 M, 10% SP 1000, 10% SE 30), using Chromosorb W AW 80/100 mesh as a solid support and made up in 2-m • 3-mm glass columns. Quantitative determination of the compounds was carried out by GLC with a Shimadzu ITX-4 integrator. The peak areas were calibrated to mass by injection of mixtures of weighed standards. For preparative GLC a 2-m X 4-mm steel column supported with 10% Carbowax 20 M on Chromosorb W AW 80/100 mesh was used. Individual substances were trapped by liquid nitrogen. Ozonolysis was carried out according to the procedure of Beroza and
CHEMISTRY OF TERGAL G L A N D SECRETION OF
Aleochara
775
Bierl (1967), and the resulting aldehydes were identified by GLC retention times (3% Carbowax 20 M, 5 min at 70 ~ C, then temperature programmed from 70~ to 190~ at 8~ Gas chromatography-mass spectrometry was carried out on a Varian 2700 gas chromatograph coupled to a Varian CH 7 mass spectrometer through a two-stage Watson Biemann separator using an all-glass system for the connection. Temperatures were 250~ for the separator and the connecting capillary tube. The mass spectra were run at an electron energy of 70 eV. IR spectra of purified compounds dissolved in C6D6 were taken with a Perkin-Elmer 377 Granting infrared spectrophotometer equipped with a 2-~1 microcell. Synthesis of Substituted l, 4-Benzoquinones. 2-Methyl- 1,4-benzoquinone was purchased from Fluka. 2-Methoxy-3-methyl-1,4-benzoquinone, 2-methoxy-5-methyl- 1,4-benzoquinone, and 2-methoxy-6-methyl- 1,4-benzoquinone were synthesized by oxidation with potassium nitrosodisulfonate (Fremy's radical) of 2-methoxy-3-methylaniline, 2-methoxy-5-methylaniline, and 2methoxy-6-methylaniline, respectively (Zimmer et al., 197 l). The amines were obtained from the corresponding nitrocresols by methylation with dimethylsulfate, followed by Raney nickel-catalyzed reduction with hydrazine hydrate. The purity of the synthesized benzoquinones was controlled by GLC and found to be >95%. In the case of 2-methoxy-6-methyl benzoquinone, the structure of a 1,4-benzoquinone was self-evident from the synthesis. For the 2-methoxy-3-methyl isomer and the 2-methoxy-5-methyl isomer, the possibility of a 1,2-benzoquinone structure was ruled out because both compounds failed to give a phenazine derivative with orthophenylenediamine. The synthetic isomers were separated by GLC on a 6-ft X 2-mm ID glass column packed with 3% OV 225 on Gas Chrom Q and operated with a helium flow of 30 ml/min and a temperature program of 100~ with 4~ C/min. Synthetic hydrocarbons and n-dodecanal were purchased from Fluka, Roth, or Sigma. A sample of (Z)-5-tetradecenal was obtained from Dr. O. Vostrowsky, Erlangen.
RESULTS
General Features of Secretion. Like other Aleocharinae (Araujo, 1978; Jordan, 1913; Pasteels, 1968; Kistner and Pasteels, 1969), A. eurtula possesses a tergal gland with a bilobate reservoir formed by an invagination of the intersegmental membrane between the sixth and seventh abdominal tergites (Figure 1). The volume of the reservoir is approximately 0.2 mm 3, as determined by measuring histological preparations. The secretion is immediately sucked from the reservoir by inserting a
776
PESCHKE AND M E T Z L E R
\ FIG. 1. Dorsal view of A. curtula with the reservoir of the tergal gland (dotted area; VI.T, VII.T: sixth and seventh abdominal tergites). No sex specificity was recognized in gross morphology.
4
peak
identity
t nonane 2 decane 3 undecone 9
DEFENSIVE AND PHEROMONAL SECRETION OF THE T E R G A L G L A N D OF Aleochara curtula 1
I. The Chemical Composition
K. P E S C H K E 2 a n d M. M E T Z L E R 3 2Zoologisehes Institut 111 der Universitiit Warzburg, ROntgenring 10, D-8700 Wiirzburg ~Institut f u r Pharmakologie und Toxikologie, Versbacher Str. 9, D-8700 Wiirzburg West German),
(Received April 16, 1981; revised August 27, 1981) Abstract--The defensive secretion from the tergal gland of the staphylinid beetle, Aleochara curtula, acting as a supplementary mating stimulant, was investigated by gas-liquid chromatography, mass spectrometry, and IR spectroscopy. The reservoir contains a complex mixture of hydrocarbons, aliphatic aldehydes, and substituted 1,4-benzoquinones. n-Undecane, I-undecene, and (Z)-4-tridecene were identified as the major hydrocarbon components. The main aldehydes are n-dodecanal and (Z)-5-tetradecenal, and the chief quinones are toluquinone and 2-methoxy-3-methyl-l,4benzoquinone, the latter being established structurally by comparison of the mass spectra of the three synthesized isomers. Quantitative GLC analyses revealed no sex specificity of the relative concentrations of the compounds. Key Words--Aleochara curtula (Goeze), Coleoptera, Staphylinidae, tergal gland secretion, hydrocarbons, aliphatic aldehydes, substituted 1,4-benzoquinones, n-undecane, 1-undecene, (Z)-4-tridecene, n-dodecane, (Z)-5tetradeeenal, toluquinone, 2-methoxy-3-methyl-l,4-benzoquinone.
INTRODUCTION W i t h i n t h e s t a p h y l i n i d beetles, a t e r g a l g l a n d s u p p o r t e d w i t h a l a r g e r e s e r v o i r is r e s t r i c t e d to the s u b f a m i l y A l e o c h a r i n a e ( A r a u j o , 1978). H i s t o l o g i c a l i n v e s t i g a t i o n s o n the e p i d e r m a l g l a n d s w e r e m a d e to i n t e r p r e t t h e b e h a v i o r a l a d a p t a t i o n s o f m y r m e c o p h i l o u s a n d t e r m i t o p h i l o u s A l e o c h a r i n a e in c o r n ~Coleoptera: Staphylinidae (Aleocharinae) 773 0098-0331/ 82/0400-0773503.00/09 1982PlenumPublishingCorporation
774
PESCHKE AND METZLER
parison to the free-living species (e.g., Pasteels, 1968; Kistner and Pasteels, 1969). The secretion of the tergal gland of some species has been proved to have defensive properties (Jordan, 1913; Pasteels, 1968; H611dobler, 1970). In Aleochara curtula (Goeze), which is a most abundant species found with carcasses, intraspecific communication by components of the tergal gland secretion was studied and their role as additional mating stimulants established (Peschke, 1982). These effects support the female sex pheromone from epicuticular lipids (Peschke, 1978a). The chemical composition of the tergal gland secretion of two closely related Aleocharinae has also been investigated in Lomechusa strumosa (Blum et al., 1971) and Drusilla canaliculata (Brand et al., 1973), but much more comparative data on tergal gland chemistry are needed in order to make safe conclusions concerning the chemical taxonomy, the function of each compound, and the evolution of this versatile group of beetles. In the present paper we report our chemical investigations on the tergal gland secretion ofA. curtula as a basis for subsequent behavioral investigations. METHODS AND MATERIALS
Collection of Secretion. A. curtula was reared in the laboratory according to Fuldner (1968) and Peschke (1978a). Sexes were separated immediately after emergence. Beetles were freshly killed by freezing, and the tergal gland secretion was collected by inserting a triangle of filter paper between the sixth and seventh abdominal tergites. The triangles from 100 males or females were collected in 1 ml n-hexane. This extract was stored at - 1 7 ~ and could be used for GLC without further preparation. Chemical Isolation and Identification. Gas-liquid chromatographic analyses (GLC) were carried out on a Shimadzu GC-6A instrument with hydrogen flame detectors under standard conditions of hydrogen (25 ml/min), air (250 ml/min), and nitrogen flow (30 ml/min), employing the routine temperature program (70-190 ~ C at 8~ C/rain column temperature, 210 ~ C detector and injector temperature). Comparison of retention times and cochromatographics were run on four liquid phases (3% or 10% Carbowax 20 M, 10% SP 1000, 10% SE 30), using Chromosorb W AW 80/100 mesh as a solid support and made up in 2-m • 3-mm glass columns. Quantitative determination of the compounds was carried out by GLC with a Shimadzu ITX-4 integrator. The peak areas were calibrated to mass by injection of mixtures of weighed standards. For preparative GLC a 2-m X 4-mm steel column supported with 10% Carbowax 20 M on Chromosorb W AW 80/100 mesh was used. Individual substances were trapped by liquid nitrogen. Ozonolysis was carried out according to the procedure of Beroza and
CHEMISTRY OF TERGAL G L A N D SECRETION OF
Aleochara
775
Bierl (1967), and the resulting aldehydes were identified by GLC retention times (3% Carbowax 20 M, 5 min at 70 ~ C, then temperature programmed from 70~ to 190~ at 8~ Gas chromatography-mass spectrometry was carried out on a Varian 2700 gas chromatograph coupled to a Varian CH 7 mass spectrometer through a two-stage Watson Biemann separator using an all-glass system for the connection. Temperatures were 250~ for the separator and the connecting capillary tube. The mass spectra were run at an electron energy of 70 eV. IR spectra of purified compounds dissolved in C6D6 were taken with a Perkin-Elmer 377 Granting infrared spectrophotometer equipped with a 2-~1 microcell. Synthesis of Substituted l, 4-Benzoquinones. 2-Methyl- 1,4-benzoquinone was purchased from Fluka. 2-Methoxy-3-methyl-1,4-benzoquinone, 2-methoxy-5-methyl- 1,4-benzoquinone, and 2-methoxy-6-methyl- 1,4-benzoquinone were synthesized by oxidation with potassium nitrosodisulfonate (Fremy's radical) of 2-methoxy-3-methylaniline, 2-methoxy-5-methylaniline, and 2methoxy-6-methylaniline, respectively (Zimmer et al., 197 l). The amines were obtained from the corresponding nitrocresols by methylation with dimethylsulfate, followed by Raney nickel-catalyzed reduction with hydrazine hydrate. The purity of the synthesized benzoquinones was controlled by GLC and found to be >95%. In the case of 2-methoxy-6-methyl benzoquinone, the structure of a 1,4-benzoquinone was self-evident from the synthesis. For the 2-methoxy-3-methyl isomer and the 2-methoxy-5-methyl isomer, the possibility of a 1,2-benzoquinone structure was ruled out because both compounds failed to give a phenazine derivative with orthophenylenediamine. The synthetic isomers were separated by GLC on a 6-ft X 2-mm ID glass column packed with 3% OV 225 on Gas Chrom Q and operated with a helium flow of 30 ml/min and a temperature program of 100~ with 4~ C/min. Synthetic hydrocarbons and n-dodecanal were purchased from Fluka, Roth, or Sigma. A sample of (Z)-5-tetradecenal was obtained from Dr. O. Vostrowsky, Erlangen.
RESULTS
General Features of Secretion. Like other Aleocharinae (Araujo, 1978; Jordan, 1913; Pasteels, 1968; Kistner and Pasteels, 1969), A. eurtula possesses a tergal gland with a bilobate reservoir formed by an invagination of the intersegmental membrane between the sixth and seventh abdominal tergites (Figure 1). The volume of the reservoir is approximately 0.2 mm 3, as determined by measuring histological preparations. The secretion is immediately sucked from the reservoir by inserting a
776
PESCHKE AND M E T Z L E R
\ FIG. 1. Dorsal view of A. curtula with the reservoir of the tergal gland (dotted area; VI.T, VII.T: sixth and seventh abdominal tergites). No sex specificity was recognized in gross morphology.
4
peak
identity
t nonane 2 decane 3 undecone 9
