Journal of Chemical Ecology, Vol. 17, No. 4, 1991
DEFENSIVE SECRETION OF Tenebrio molitor (COLEOPTERA: TENEBRIONIDAE) 1
A . B . A T T Y G A L L E , 2 C . L . B L A N K E S P O O R , 3 J. M E I N W A L D , 2 and T. E I S N E R 3'* aDepartment of Chemistry 3Section of Neurobiology and Behavior Cornel[ University Ithaca, New York 14853
(Received December 27, 1990; accepted January 2, t99t) Abstract--The defensive secretion of Tenebrio molitor contains a mixture of 2-methyl-1,4-benzoquinoneand m-cresol. The phenol had not previously been detected in the secretion, although some investigators reported presence of 2-ethyl-l,4-benzoquinone as a second component. We failed to detect the latter quinone in secretion samples from three laboratory populations of 7". molitor. Key Words--2-Methyl-l,4-benzoquinone, m-cresol, Tenebrio molitor, Coteoptera, Tenebrionidae, defensive glands,
INTRODUCTION As part o f a study o f how parasitization encumbers an insect's ability to use its defensive glands, we recently had occasion to look into the chemistry o f the defensive secretion o f Tenebrio molitor, a beetle commonly maintained in laboratory culture and used worldwide in biological research. Unexpectedly, our findings about the composition o f the b e e t l e ' s secretion differed from those previously reported (Schildknecht, 1959; Tschinkel, 1975a; Kanehisa, 1978). W e here present our results. *To whom correspondence should be addressed. ~Paper No. 100 of the series Defense Mechanisms of Arthropods; No. 99 is Storey et al., J. Chem. Ecol., I7,687-693 (1991). 805 0098-033t/9t/0400-0805506.50/0 9 199[ Plenum Publishing Corporation
806
ATTYGALLE ET AL. METHODS AND MATERIALS
Tenebrio molitor. The defensive glands of T. molitor, and the mechanism by which the glands are everted for defensive use, have been described (Tschinkel, 1975b,c). We used beetles from three sources: (1) our Comell culture (replenished periodically with individuals purchased from Rainbow Mealworms, Compton, California); (2) S.C. Johnson & Sons, Inc., Racine, Wisconsin; and (3) Department of Entomology, Kansas State University, Manhattan, Kansas. We "milked" the beetles of secretion by squeezing them gently with forceps so as to cause them to evert the glands, then wiping the glands with small pieces of filter paper, and transferring the papers to vials with hexane for extraction. Beetles were milked without regard to sex. Milkings of beetles from any one source were lumped, providing three samples [beetles milked per sample = 10 (Cornell); 4 (Wisconsin); 6 (Kansas)]. Instrumentation. Gas chromatography [Hewlett-Packard ( = HP) 5890 equipped with flame ionization detector] was performed on a 25-m • 0.22-ram fused-silica capillary column coated with DB-5 (J. & W. Scientific). Temperature program: 40~ for 4 rain, then to 260~ at 8~ Infrared and mass spectra were obtained with a HP 5890 gas chromatograph linked in series to a HP 5965A FTIR detector and a HP 5970 mass selective detector (MSD). Mass spectra were also obtained on a HP 5890 gas chromatograph linked to a Finnigan ion trap detector (ITD 800).
RESULTS
Capillary gas chromatography of the Cornell secretion sample (Figure 1) revealed two primary peaks, accounting for about 99.5% of the volatile components. Mass spectra (ITD and MSD) of the first component were closely similar to those given for 2-methyl-1,4-benzoquinone (McLafferty and Stauffer, 1989) and matched those of an authentic sample of this compound. The natural compound and the authentic sample also had identical gas-phase FTIR spectra. The mass spectrum of the second component showed that it was a cresol [m/z(%), 108(M § 82), 107(100), 90(7), 79(42), 77(40), 63(6), 53(11), 51(11)], but could not unambiguously resolve the isomeric structure of the compound (McLafferty and Stauffer, 1989). However, differences that we determined between the o, m, and p isomers of cresol by FTIR spectrometry (Figure 2) allowed unambiguous identification of the unknown as m-cresol. As further evidence for the nature of the components, peaks 1 and 2 (Figure 1) had chromatographic retention times identical to those of authentic 2-methyl-l,4-benzoquinone and m-cresol, respectively. Of the three cresols,
807
DEFENSIVE SECRETION
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FIo. 1. Gas chromatogram (hexane) of Tenebrio moIitor defensive secretion (Cornell sample). See text for column and temperature program. o-cresol had the shortest retention time; m- and p-cresol eluted closely together, in that order. The secretion samples from the Wisconsin and Kansas beetles proved to contain the same two primary components. There were some differences in the ratio of the components in the three samples (Table 1). DISCUSSION The finding that our three samples contained the same two primary compounds leads us to believe that 2-methyl-l,4-benzoquinone and m-cresol are characteristic components of the T. molitor secretion. Schildknecht (1959) previously had reported the presence of 2-methyl-1,4-benzoquinone in the secretion of this beetle. Tschinkel (1975a) and Kanehisa (1978) also reported presence of this quinone, but claimed the additional presence of 2-ethyl-l,4-benzoquinone. Kanehisa (1978) based his identification of the latter compound on TLC and packed column retention data only. We found no evidence for the presence of this quinone, which we would have detected at the 2-ng level.
808
ATTYGALLE ET AL.
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FIG. 2. Gas-phase FTIR spectra of authentic o-cresol (A), m-cresol (B), and p-cresol (C), and of the second-eluting component (Figure 1) of the Tenebrio molitor defensive secretion (D). Resolution = 8 cm -I.
DEFENSIVE SECRETION
809
TABLE 1. RELATIVE PERCENT OF MAJOR COMPONENTS IN DEFENSIVE SECRETION OF Tenebrio molitor FROM THREE SOURCES
Source
2-Methyl- 1,4-benzoquinone
m-Cresol
Cornell Wisconsin Kansas
87.5 97.0 68.0
12.5 3.0 32.0
Acknowledgments--Supported in part by grants AI-02908 and AI-12020 from NIH, and a NSF predoctoral fellowship to C.L.B. We thank Richard Hoebeke for identification of T. molitor, and Robert Martin from S.C. Johnson and Sons and Nahid Tavakkol from Kansas State University for shipment of beetles.
REFERENCES KANEHISA, K. 1978. Comparative study of the abdominal defensive systems in tenebrionid beetles. Ber. Ohara. Inst. Landwirtsch. Biol. Okayama Univ. 17:47-56. MCLAFFERTY, F.W., and STAUFFER, D.B. 1989. The Wiley/NBS Registry of Mass Spectral Data. John Wiley, New York. TSCn~NKEL, W.R. 1975a. A comparative study of the chemical defensive system of tenebrionid beetles: Chemistry of the secretions. J. Insect Physiol. 21:753-783. TSCmNKEL, W.R. 1975b. A comparative study of the chemical defensive system of tenebrionid beetles. III. Morphology of the glands. J. Morphol. 145:355-370. TSCmNKEL, W.R. 1975c. A comparative study of the chemical defensive system of tenebrionid beetles. Defensive behavior and ancillary features. Ann. Entomol. Soc. Am. 68:439-453. SCHILDKNECHT,H. 1959. Uber das flfichtige Sekret vom gemeinen Mehlk/ifer. Angew. Chem. 71:524.
DEFENSIVE SECRETION OF Tenebrio molitor (COLEOPTERA: TENEBRIONIDAE) 1
A . B . A T T Y G A L L E , 2 C . L . B L A N K E S P O O R , 3 J. M E I N W A L D , 2 and T. E I S N E R 3'* aDepartment of Chemistry 3Section of Neurobiology and Behavior Cornel[ University Ithaca, New York 14853
(Received December 27, 1990; accepted January 2, t99t) Abstract--The defensive secretion of Tenebrio molitor contains a mixture of 2-methyl-1,4-benzoquinoneand m-cresol. The phenol had not previously been detected in the secretion, although some investigators reported presence of 2-ethyl-l,4-benzoquinone as a second component. We failed to detect the latter quinone in secretion samples from three laboratory populations of 7". molitor. Key Words--2-Methyl-l,4-benzoquinone, m-cresol, Tenebrio molitor, Coteoptera, Tenebrionidae, defensive glands,
INTRODUCTION As part o f a study o f how parasitization encumbers an insect's ability to use its defensive glands, we recently had occasion to look into the chemistry o f the defensive secretion o f Tenebrio molitor, a beetle commonly maintained in laboratory culture and used worldwide in biological research. Unexpectedly, our findings about the composition o f the b e e t l e ' s secretion differed from those previously reported (Schildknecht, 1959; Tschinkel, 1975a; Kanehisa, 1978). W e here present our results. *To whom correspondence should be addressed. ~Paper No. 100 of the series Defense Mechanisms of Arthropods; No. 99 is Storey et al., J. Chem. Ecol., I7,687-693 (1991). 805 0098-033t/9t/0400-0805506.50/0 9 199[ Plenum Publishing Corporation
806
ATTYGALLE ET AL. METHODS AND MATERIALS
Tenebrio molitor. The defensive glands of T. molitor, and the mechanism by which the glands are everted for defensive use, have been described (Tschinkel, 1975b,c). We used beetles from three sources: (1) our Comell culture (replenished periodically with individuals purchased from Rainbow Mealworms, Compton, California); (2) S.C. Johnson & Sons, Inc., Racine, Wisconsin; and (3) Department of Entomology, Kansas State University, Manhattan, Kansas. We "milked" the beetles of secretion by squeezing them gently with forceps so as to cause them to evert the glands, then wiping the glands with small pieces of filter paper, and transferring the papers to vials with hexane for extraction. Beetles were milked without regard to sex. Milkings of beetles from any one source were lumped, providing three samples [beetles milked per sample = 10 (Cornell); 4 (Wisconsin); 6 (Kansas)]. Instrumentation. Gas chromatography [Hewlett-Packard ( = HP) 5890 equipped with flame ionization detector] was performed on a 25-m • 0.22-ram fused-silica capillary column coated with DB-5 (J. & W. Scientific). Temperature program: 40~ for 4 rain, then to 260~ at 8~ Infrared and mass spectra were obtained with a HP 5890 gas chromatograph linked in series to a HP 5965A FTIR detector and a HP 5970 mass selective detector (MSD). Mass spectra were also obtained on a HP 5890 gas chromatograph linked to a Finnigan ion trap detector (ITD 800).
RESULTS
Capillary gas chromatography of the Cornell secretion sample (Figure 1) revealed two primary peaks, accounting for about 99.5% of the volatile components. Mass spectra (ITD and MSD) of the first component were closely similar to those given for 2-methyl-1,4-benzoquinone (McLafferty and Stauffer, 1989) and matched those of an authentic sample of this compound. The natural compound and the authentic sample also had identical gas-phase FTIR spectra. The mass spectrum of the second component showed that it was a cresol [m/z(%), 108(M § 82), 107(100), 90(7), 79(42), 77(40), 63(6), 53(11), 51(11)], but could not unambiguously resolve the isomeric structure of the compound (McLafferty and Stauffer, 1989). However, differences that we determined between the o, m, and p isomers of cresol by FTIR spectrometry (Figure 2) allowed unambiguous identification of the unknown as m-cresol. As further evidence for the nature of the components, peaks 1 and 2 (Figure 1) had chromatographic retention times identical to those of authentic 2-methyl-l,4-benzoquinone and m-cresol, respectively. Of the three cresols,
807
DEFENSIVE SECRETION
o
o
j F
0
0~,,
.
;
2',
min
FIo. 1. Gas chromatogram (hexane) of Tenebrio moIitor defensive secretion (Cornell sample). See text for column and temperature program. o-cresol had the shortest retention time; m- and p-cresol eluted closely together, in that order. The secretion samples from the Wisconsin and Kansas beetles proved to contain the same two primary components. There were some differences in the ratio of the components in the three samples (Table 1). DISCUSSION The finding that our three samples contained the same two primary compounds leads us to believe that 2-methyl-l,4-benzoquinone and m-cresol are characteristic components of the T. molitor secretion. Schildknecht (1959) previously had reported the presence of 2-methyl-1,4-benzoquinone in the secretion of this beetle. Tschinkel (1975a) and Kanehisa (1978) also reported presence of this quinone, but claimed the additional presence of 2-ethyl-l,4-benzoquinone. Kanehisa (1978) based his identification of the latter compound on TLC and packed column retention data only. We found no evidence for the presence of this quinone, which we would have detected at the 2-ng level.
808
ATTYGALLE ET AL.
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FIG. 2. Gas-phase FTIR spectra of authentic o-cresol (A), m-cresol (B), and p-cresol (C), and of the second-eluting component (Figure 1) of the Tenebrio molitor defensive secretion (D). Resolution = 8 cm -I.
DEFENSIVE SECRETION
809
TABLE 1. RELATIVE PERCENT OF MAJOR COMPONENTS IN DEFENSIVE SECRETION OF Tenebrio molitor FROM THREE SOURCES
Source
2-Methyl- 1,4-benzoquinone
m-Cresol
Cornell Wisconsin Kansas
87.5 97.0 68.0
12.5 3.0 32.0
Acknowledgments--Supported in part by grants AI-02908 and AI-12020 from NIH, and a NSF predoctoral fellowship to C.L.B. We thank Richard Hoebeke for identification of T. molitor, and Robert Martin from S.C. Johnson and Sons and Nahid Tavakkol from Kansas State University for shipment of beetles.
REFERENCES KANEHISA, K. 1978. Comparative study of the abdominal defensive systems in tenebrionid beetles. Ber. Ohara. Inst. Landwirtsch. Biol. Okayama Univ. 17:47-56. MCLAFFERTY, F.W., and STAUFFER, D.B. 1989. The Wiley/NBS Registry of Mass Spectral Data. John Wiley, New York. TSCn~NKEL, W.R. 1975a. A comparative study of the chemical defensive system of tenebrionid beetles: Chemistry of the secretions. J. Insect Physiol. 21:753-783. TSCmNKEL, W.R. 1975b. A comparative study of the chemical defensive system of tenebrionid beetles. III. Morphology of the glands. J. Morphol. 145:355-370. TSCmNKEL, W.R. 1975c. A comparative study of the chemical defensive system of tenebrionid beetles. Defensive behavior and ancillary features. Ann. Entomol. Soc. Am. 68:439-453. SCHILDKNECHT,H. 1959. Uber das flfichtige Sekret vom gemeinen Mehlk/ifer. Angew. Chem. 71:524.
