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Quinone production of some species of Tribolium
J. Insecr Phxsiol., Vol. 24. pp. 785 to 790. c Pergamon Press Ltd. 1978. Printed in Great Britain
QUINONE
PRODUCTION
0022-1910~78,1201-0785
SO2.OOiO
OF SOME SPECIES OF
TRIBOLIUM HAIG MARKARIAN,*GERALDJ. FLORENTINE?and JOHN J. PRATT, JR.:. U.S. Army Natick Research and Development Command. Natick, MA 01760 U.S.A. (Received 8 August 1977; revised 17 April 1978)
Abstract-The quinone secretions of 6 Tribohm species were analyzed qualitatively and quantitatively by gas chromatography. Procedures were developed for collecting and weighing the secretion which prevented its contamination and oxidation of the quinones. Secretions of all species contained 2-methyl- and 2-ethyl1,4-benzoquinones in amounts ranging from 50.5% in T. brevicornis to 73% in T. confusum. Ratios of 2methyl- to 2-ethyl-l ,Cbenzoquinone were 1:I .7 in 2 species and 1:2.6 to 1:2.7 in 4 species. The most prevalent non-quinone in secretions of all species was 1-pentadecene, varying from 15.6% in T. confusum to 19.9% in T. brevicorrzis. Three unidentified compounds, probably long-chain hydrocarbons, were found and designated biosolvents. BS-2 was present in secretions of all species, BS-la in 4 species and traces of BS-lb in 2 species.
The primary function of I-pentadecene, and probably the unidentified biosolvents. is quinone solvency. This is discussed in relation to other previously proposed functions and to the recently discovered activity as a sex pheromone
INTRODUCTION
THE OCCURRENCE of quinones in insect and other arthropod secretions has been reviewed by ROTH and EISNER(I 962) and WEATHERSTON and PERCY(1970). TSCHINKEL(1975a) reported the presence of 2-methyland 2..ethyl-1,4-benzoquinones in secretions of 147 species representing 55 genera of Tenebrionidae. In the genus Tribolium these quinones have been identified in secretions of T. brevicornis (TSCHINKEL, 1975a), T. castaneum (ALEXANDER and BARTON,1943; LOCONTIand ROTH. 1953). T. confusum (HACKMANet al., 1948; ENGLEHARDT et al., 1965), and T. destructor (TSCHINKEL, 1975a). Other compounds have been found associated with the quinones in Tribolium secretions. LOCONTIand ROTH (1953) reported the presence of an “oil” in the secretion of T. castaneum and VON ENDT and WHEELER(1971) identified lpentadecene as a component of T. confusum secretion. TSCHINKEL(1975a) found this alkene in secretions of T. brevicornis and T. destructor and KEVILLE and KANNOWSKI (1975) confirmed its presence in T. confusum. None of the above identifications were reported in terms of the quantity of each compound found in the total secretion. This knowledge was needed for our studies on the effects of insect secretions and excretions on the usability and wholesomeness of military foods. We, therefore, compared qualitatively and quantitatively the composition of quinone secretions from the following 6 Tribolium species: T, audax Hal., T. castaneum (Herbst). T. destructor Myttenb., T. brevicornis (Let.), T. madens (Charp.). T. confusum Duv. (normal and ebony strain).
MATERIALS
AND METHODS
All Tribolium species were reared in glass culture jars containing approximately 2 kg of whole wheat flour with 5% Brewer’s yeast and maintained under laboratory conditions of 24°C and 75 r.h. When samples of quinone secretion were to be obtained, adult beetles were sifted from the culture jars and placed in cylindrical 1.5 1plastic containers containing 5-8 pleated file cards (127 x 76 mm). Since sifting and handling of the beetles caused them to secrete quinones, thus reducing the amount available for collection, no quinone secretions were collected until at least 24 hr after sifting. The pleated file cards provided a large surface area for the beetles to crawl upon and thus facilitated their recovery without stress conducive to quinone secretion. Previous methods of collecting quinones involved air-flow or vacuum sublimation techniques (LADISCHand MCQUE 1953). These methods did not collect whole secretion and in our laboratory, air-flow collection produced oxidation products. The following procedure was developed to obtain uncontaminated and unoxidized secretion. A Petri dish (86-95 mm) was frozen in ice contained in a slightly larger container. When ready for use, the frozen unit was removed from the freezer and the Petri dish washed with acetone and dried. Using extreme care, the pleated file cards containing the resting beetles were removed from the holding container and quickly brushed or tapped onto the cold Petri dish. The transfer and cold stress caused the beetles to expel quinone secretion (ROTH and HOWLAND,1941). The cold dish also served to inactivate the beetles, thus facilitating further handling. When the secretion Present addresses: contacted the cold atmosphere in the Petri dish, it * 5 Ivy Lane, Natick, Massachusetts 01760, U.S.A. t Bureau of Plant Industry, Pennsylvania, Dept. of solidified as small yellow to yellow-brown crystals in the prothoracic and posterior abdominal regions (Fig. Agriculture. 2301 IV. Cameron Street, Harrisburg, 1 and ROTH, 1943; Fig. 4). This method proved Pennsylvania 17120, U.S.A. satisfactory for obtaining good yields from all species 785 I.P. 24,12--r
786
HAIG MARKARIAN, GERALD J. FLORENTINE AND JOHN J. PRATT, JR.
Table 1. Ratios of 2-methyl- to 2-etbyi-1,4-benzoquinon~s, per cent total 1.4-~nzoquinones (BS-I) in quinone secretions of 6 Triholium species
Species T. T. T. T. T. T. T.
audax castaneum madens brevicornis destructor ronjiisum conjkum, ebony
Mean ratios of 2-methyl to 2-ethyl 1,Cbenzoquinones (&S.D.) () = N* .-_...-_____~ (7) l:l.7 0.1 (7) (6) (6) (5) (9)
1:1.7 1:2.7 l:2.6 l:2.6 12.7
0.2 0.4 0.3 0.2 0.3
(8) 1:2.7 0.3
Mean % total 1,4benzoquinones by weight (+SD.)N=5* _ 57.5 4.3 58.3 54.1 50.5 61.4 73.0
6.4 6.1 5.5 6.5 2.5
68.6 7.2
and per cent I-pentadecene Mean 7, 1-pentadecene (BS-1) by weight (*SD.) Pi = 5t ~__. 16.7 4.1 15.8 0.9 18.3 2.8 19.9 2.8 IX.3 2.1 17.6. 4.9 15.6 3.9
* Means and variances of replicates within each speciesare equal at the I”/, level. ? Means and variances not equal. at the t*/0level. _ except T. mudens. To increase the yield from this ionization. Approximately 20 pg of secretion (in 5 ~1of species approximately 50-75 adults were placed in a solution) were sufficient for analysis. Standard 100 x 25 mm test tube and shaken briefly. The tube solutions of the above 3 compounds* were was then dipped into a dry ice-acetone bath until the chromato~aphed before and after each analysis to beetles became immobile and produced secretion. The insure its integrity. Quan~tative discrepancies of beetles were then poured onto the iced petri dish to about 50/, were compensated for with a correction keep the solidified secretion from melting. factor. Standard solutions were renewed every 20-30 The beetles. were examined for solidified secretion days because of solvent loss and degradation of the under a binocular dissecting microscope and the solid 1,4-~nzoquinones. secretion removed and collected in the following All analyses were replicated between 5 and 9 times manner. An immobilized beetle was held with forceps as indicated. in Table 2. The means of each set of and the solid secretion picked off the thorax and analyses were tested for equalness by a one-way abdomen with a fine dissecting needle. then placed on analysis of variance and for homogeneity of variance the open end of a OS-O.9 mm i.d. capillary tube by Bartlett’s Test (DIXON and MASSEY,1957). slightly tapered at its end. A warm, pencil soldering iron, its heat regulated by a variable voltage supply, RESULTS was placed near, but not touching, the solid secretion, causing it to melt and be drawn into the capillary tube. Prior to analysis of the quinone secretions. hexane The secretion then remained liquid at room washes of the plastic rearing containers, flour, and all temperature. Pooled samples of about 400 pg of glassware and manipulating tools were analyzed by secretion could be collected in the capillary tube. GC for substances which might interfere with the Capillary tubes for weighing the secretion were drawn .quinone analysis. None were found. out on a micro-pipet puller and formed with a double Pure 2-methyl 1,Cbenzoquinone had a retention taper. The extremely fine portion of the tapered ends time of 20 set after the solvent peak, while that of 2was removed with forceps, the tube weighed, and those ethyl 1,6benzoquinone was 30 sec. To determine approximately 17 mm long and weighing 15 2 0.5 mg possible interference with these rates, retention times were selected for use. Approximately 100-200 pg of for the following related compounds were also secretion were transferred to weighing tubes from the determined: 1,Cbenzoquinone-1 1 set; 2,3-dimethoxy collecting capillaries and weighed on a microbalance. 1,~be~oquinon~35 see; 2-methoxy 1,4-benzoThe secretion was then transferred from the weighing quinone-59 sec. The latter compound, reported capillary into a 100 ~1 volumetric flask with 5 ~1 by LOCONTIand ROTH (1953) as a constituent of 7: graduations, using spectrophotometric grade hexane, castaneum secretion, was not detected in any of the and the volume adjusted to contain approximately 20 species studied. 2-Ethyl- and 2-methyl-l A-benzo,ul of secretion, These solutions could be stored quinones, which were separated from secretions of all overnight at 5°C with only lO-15% loss in volume. The secretions were analyzed quantitatively for 2ethyland 2-methyl- 1,Cbenzoquinone and l- Table 2. Relative quantities of biosolvents (BS) in 18 fig of quinone secretion from each of 6 Tribolium species pentadecene by gas chromatography (GC) (Bendix 2500 with Auto Lab Digital Integrator). BS-lb BS-2 BS-1 BS-la Chromatography columns were glass, 4 mm x 180 Specks cm, Packed with 100/120’mesh Chromosorb W HP ++* T. audax +++* +* supporting 5% OV-1 as the liquid phase. The carrier T. castaneum ++ ++-t+ gas was nitrogen and detection was by hydrogen flame T. madens ++ +++ + tt T. brevicorn~s -l-i+ + * 2-Methyl-1,4-betuoquinone
and I-pentadecene were obtained commercially and purified if required. 2-Ethyl-1,4benzoquinone and all other quinones used in this study were synthesized by Dr. Joseph Bornstein, Boston College, Newton, Mas~chusetts 02167.
T. destructor T. con&urn T. con&urn,
ebony
+++ +++
+
+
t
t
+ + +
t
t
* Relative increasing quantities from i- to + + + $Just detectable under analytical conditions used.
Fig. 1.Solidified quinone secretion from TriboIium conjiisum placed on a cold Petri dish. Top: from Prothoracic glands. Secretion has flowed across thorax. Bottom: from abdominal glands. Secretion solidified before flowing.
Quinone
production
of some
n b
T. madens
i
d”““““““““” 1 2
3
4
5
Minutes
Fig. 2. Tracing of gas chromatogram of an 18 pg sample of @none secretion from Tribolium madens. Compounds are: (a) 2-methyl-1.4-benzoquinone, (b) 2-ethyl-1,4benzoquinone. (c) I-pentadecene (biosoivent I), (d) biosolvent la, (e) biosolvent 2.
species, were consistent with the above retention times of pure compounds and were the only quinones found in the secretion. Figure 2 shows a chromatogram of secretion from T. madens, which is typical for all species studied, except for biosolvent lb found only in T. destructor and T. confusum. The greater amount of 2-ethyl than 2-methyll,4-benzoquinone is consistent for all species studied, but the ratios vary (Table 1). Approximately 277; (T. confisum) to 49.5% (T. brevicornis) of the secretions consists of substances other than 1,4benzoquinones (Table 1). One of these substances, having a retention time of 146 set in our GC analysis, and found in all species, was I-pentadecene, first identified in T. confusum secretions by VON ENDTand WHEELER(1971) and subsequently in T. brevicornis and T. destructor by TSCHINKEL(1975a). The per cent by weight of secretion varied with species from 15.6-19.9 . (Table I), showing considerably less difference among species (maximum 4.30;,) than did the 1,Cbenzoquinones (maximum 22.5%). Small amounts of 3 other compounds were found at GC retention times of 150, 160 and 213 sec. We have designated these, and 1-pentadecene, as biosolvents (Table 2) on the assumption that one of their functions may be quinone solvency, which we propose as a prime function of 1-pentadecene. A brief, unsuccessful attempt was made to identify the 3 biosolvents by GC using model compdunds.
DISCUSSION Thoracic and abdominal quinone-secreting glands have some cellular differences in T. rastaneum (ROTH,
species of Triholi~~
789
1943) and in T. destructor (PALM, 1946). Thus qualitative and quantitative differences in secretions could occur: However, LOCONTIand ROTH (1953) concluded that the 1.4-benzoquinone secretions of the thoracic and abdominal glands of T. castaneum were qualitatively the same. In our experiments we collected and pooled secretions indiscriminately between thoracic and abdominal glands. If there were significant qualitative or quantitative differences between glands, variations among replicates for total 1,4-benzoquinones and ratios of 2-methyl-to 2-ethyl1,4-benzoquinones would have shown higher standard deviations and would not have been significantly equal by Bartlett’s Test for homogeneity. Because of the importance of I-pentadecene as a quinone solvent, it seems unlikely that there would be quantitative differences between glands. The possibility, however, remains open because replicate variations in our data did not meet Bartlett’s Test. Qualitative differences among biosolvents la, 1band 2 would not have been resolved by our experimental procedures. The greater amount of 2-ethyl- than 2-methyl-l, 4benzoquinone found in all species agrees with the finding of TSCHINKEL(1975a) that this occurred in 113 of 127 species of Tenebrionidae. Compounds other than quinones constitute 27% (T. corzfusum) to 49.5% (7’. brevicornis) of the secretions. The most prevalent compound was I-pentadccene, but this plus unidentified biosolvents la, 1b and 2, water and procedural losses cannot account for these amounts, except possibly in T. corzjksum. Other compounds are undoubtedly present and might be resolved by different analytical techniques. I-Pentadecene is the prime quinone solvent in the Tribolium species studied, as it and other hydrocarbons are in quinone-secreting arthropods (WEATHERSTONand PERCY, 1970). In the several Tenebrionids in which TSCHINKEL(1975a) found only quinones, his analytical technique probably did not detect existing hydrocarbon solvents, for practically all arthropod-secreted quinones are solids at ambient temperatures of the animals’ habitats. VONENDT and WHEELER (1971) proposed that I-pentadecene functions as a spreading agent in Tribohm in which secretions are known to spread over much of the body (Fig. 1). TSCHINKEL(1975b) has illustrated channels on the integument for this purpose. This would mitigate against a function of increasing permeability of predator cuticle to quinones (WEATHERSTON and PERCY, 1970), for Tribolium would need novel protective substances against quinone absorption in order to prevent self poisoning. KEVILLE and KANNOWSKI (1975) isolated. nhexadecane, I-heptadecene and n-heptadecadiene from T. confusum secretion after our experiments were completed. These may be our unidentified biosolvents. With the exception of n-heptadecadiene which was not tested, they found 1-pentadecene, n-hexadecane and lheptadecene to function as sex pheromones. This novel and probably secondary function of a presumed defensive secretion emphasizes the need to conduct behavioral experiments on secretions whose presumed original function now has limited, if any, biological utility. TSCHINKEL(1975a) states that the usefulness to
7Yll
taxonomy secretions va~ability accurate variances
HAIG MARKARIAN, C~ERALDJ. ~LOREWTINEAND
JOHNJ. PRATT,JR.
of data on analysis of Tenebrionid is limited by its variablility at all levels. This might well be reduced through precise and experimental procedures resulting in low as in our 1,Cbenzoquinone data. The high
LOCONTIJ. D. and ROTHL. M. (1953) Composition of the odorous secretion of Tribolium tzlstuneum. Ann. ent. Sot. Am. 46,281-289. PALMN. (196) Structure and physiology of the stink glands in Tribolium destructor Uytt. (Col.). Opusc. Em. II,
percentages of i,4-benzoquinones in secretions of T. confusum and T. castaneum are of concern to food wholesomeness, for these species are ubiquitous
RUTH L. M. (1943) Studies on the gaseous secretion of Tribolium conji~um Duval-II. The odoriferous glands of Tribolium confiisum. Ann. ent. Sot. Am. 36, 397-424. ROTH L. M. and EISNERT. (1962) Chemical defense of arthropods. Ann. Rev. Ent. 7, I‘O7-136. ROTH L. M. and HOWLAND R. B. (1941) Studies on the gaseous secretion of Tr~bo/i~n~ conf~sum Duvat--I. Abnormalities produced in Tribokum concern Duval by exposure to a secretion given off by the adults. Ann. Em. sot. 34, 115-171. TSCHINKELW. R. (1975a) A comparative study of the chemical defensive system of Tenebrionid beetle& Chemistry of the secretions. J. Insect PItmioi. 21,753-783. TSCHINKELW. R. (1975b) A comparative study of the chemical defensive system of Tenebrionid beettes-II. Defensive behavior and ancillary structures. Ann. em. Sot. Am. t&439-453. VONENDTD. W. and WH~ZZLER J. W. (1971) I-Pentadecene production in Tribolium conjiisum. Science 172, 60-61. WEATHERSTON J. and PERCYJ. E. (1970) Arthropod defense secretions. In Chetiicals Controlling insect Behavior. pp. 95-144. Academic Press, New York.
infestors of many human foods.
and &W-ON D. H. R. (1943) The excretion of ethylquinone by the flour beetle. Biochem. J. 37,463-465. DIXON V. J. and MASSEYF. J. (1957) In Introduction zo Statistical Analysis, pp. 145-152. McGraw-Hill, New York. ENGELHARDT M., RAP~PORTH. and SOKOLOFFA. (1965) Odorous secretion of normal and mutant Triboliwn eonfus~. Science 150,632-633. HACKMANR. H., PRYORM. G. M. and TODDA. R. (1948) The occurrence of phenolic substances in arthropods. Bioehem. .I. 43,474-477. KEVILLER. and KANNOWSKI P. B. (1975) Sexual excitation by pheromones of the confused flour beetle. J. Znsecr Physiol. 21, 8 l-84. LADISCHR. K. and McQus B. (1953) Methods of obtaining quinones from flour beetles. Science 118, 324-325. ALEXANDER P.
119-132.
QUINONE
PRODUCTION
0022-1910~78,1201-0785
SO2.OOiO
OF SOME SPECIES OF
TRIBOLIUM HAIG MARKARIAN,*GERALDJ. FLORENTINE?and JOHN J. PRATT, JR.:. U.S. Army Natick Research and Development Command. Natick, MA 01760 U.S.A. (Received 8 August 1977; revised 17 April 1978)
Abstract-The quinone secretions of 6 Tribohm species were analyzed qualitatively and quantitatively by gas chromatography. Procedures were developed for collecting and weighing the secretion which prevented its contamination and oxidation of the quinones. Secretions of all species contained 2-methyl- and 2-ethyl1,4-benzoquinones in amounts ranging from 50.5% in T. brevicornis to 73% in T. confusum. Ratios of 2methyl- to 2-ethyl-l ,Cbenzoquinone were 1:I .7 in 2 species and 1:2.6 to 1:2.7 in 4 species. The most prevalent non-quinone in secretions of all species was 1-pentadecene, varying from 15.6% in T. confusum to 19.9% in T. brevicorrzis. Three unidentified compounds, probably long-chain hydrocarbons, were found and designated biosolvents. BS-2 was present in secretions of all species, BS-la in 4 species and traces of BS-lb in 2 species.
The primary function of I-pentadecene, and probably the unidentified biosolvents. is quinone solvency. This is discussed in relation to other previously proposed functions and to the recently discovered activity as a sex pheromone
INTRODUCTION
THE OCCURRENCE of quinones in insect and other arthropod secretions has been reviewed by ROTH and EISNER(I 962) and WEATHERSTON and PERCY(1970). TSCHINKEL(1975a) reported the presence of 2-methyland 2..ethyl-1,4-benzoquinones in secretions of 147 species representing 55 genera of Tenebrionidae. In the genus Tribolium these quinones have been identified in secretions of T. brevicornis (TSCHINKEL, 1975a), T. castaneum (ALEXANDER and BARTON,1943; LOCONTIand ROTH. 1953). T. confusum (HACKMANet al., 1948; ENGLEHARDT et al., 1965), and T. destructor (TSCHINKEL, 1975a). Other compounds have been found associated with the quinones in Tribolium secretions. LOCONTIand ROTH (1953) reported the presence of an “oil” in the secretion of T. castaneum and VON ENDT and WHEELER(1971) identified lpentadecene as a component of T. confusum secretion. TSCHINKEL(1975a) found this alkene in secretions of T. brevicornis and T. destructor and KEVILLE and KANNOWSKI (1975) confirmed its presence in T. confusum. None of the above identifications were reported in terms of the quantity of each compound found in the total secretion. This knowledge was needed for our studies on the effects of insect secretions and excretions on the usability and wholesomeness of military foods. We, therefore, compared qualitatively and quantitatively the composition of quinone secretions from the following 6 Tribolium species: T, audax Hal., T. castaneum (Herbst). T. destructor Myttenb., T. brevicornis (Let.), T. madens (Charp.). T. confusum Duv. (normal and ebony strain).
MATERIALS
AND METHODS
All Tribolium species were reared in glass culture jars containing approximately 2 kg of whole wheat flour with 5% Brewer’s yeast and maintained under laboratory conditions of 24°C and 75 r.h. When samples of quinone secretion were to be obtained, adult beetles were sifted from the culture jars and placed in cylindrical 1.5 1plastic containers containing 5-8 pleated file cards (127 x 76 mm). Since sifting and handling of the beetles caused them to secrete quinones, thus reducing the amount available for collection, no quinone secretions were collected until at least 24 hr after sifting. The pleated file cards provided a large surface area for the beetles to crawl upon and thus facilitated their recovery without stress conducive to quinone secretion. Previous methods of collecting quinones involved air-flow or vacuum sublimation techniques (LADISCHand MCQUE 1953). These methods did not collect whole secretion and in our laboratory, air-flow collection produced oxidation products. The following procedure was developed to obtain uncontaminated and unoxidized secretion. A Petri dish (86-95 mm) was frozen in ice contained in a slightly larger container. When ready for use, the frozen unit was removed from the freezer and the Petri dish washed with acetone and dried. Using extreme care, the pleated file cards containing the resting beetles were removed from the holding container and quickly brushed or tapped onto the cold Petri dish. The transfer and cold stress caused the beetles to expel quinone secretion (ROTH and HOWLAND,1941). The cold dish also served to inactivate the beetles, thus facilitating further handling. When the secretion Present addresses: contacted the cold atmosphere in the Petri dish, it * 5 Ivy Lane, Natick, Massachusetts 01760, U.S.A. t Bureau of Plant Industry, Pennsylvania, Dept. of solidified as small yellow to yellow-brown crystals in the prothoracic and posterior abdominal regions (Fig. Agriculture. 2301 IV. Cameron Street, Harrisburg, 1 and ROTH, 1943; Fig. 4). This method proved Pennsylvania 17120, U.S.A. satisfactory for obtaining good yields from all species 785 I.P. 24,12--r
786
HAIG MARKARIAN, GERALD J. FLORENTINE AND JOHN J. PRATT, JR.
Table 1. Ratios of 2-methyl- to 2-etbyi-1,4-benzoquinon~s, per cent total 1.4-~nzoquinones (BS-I) in quinone secretions of 6 Triholium species
Species T. T. T. T. T. T. T.
audax castaneum madens brevicornis destructor ronjiisum conjkum, ebony
Mean ratios of 2-methyl to 2-ethyl 1,Cbenzoquinones (&S.D.) () = N* .-_...-_____~ (7) l:l.7 0.1 (7) (6) (6) (5) (9)
1:1.7 1:2.7 l:2.6 l:2.6 12.7
0.2 0.4 0.3 0.2 0.3
(8) 1:2.7 0.3
Mean % total 1,4benzoquinones by weight (+SD.)N=5* _ 57.5 4.3 58.3 54.1 50.5 61.4 73.0
6.4 6.1 5.5 6.5 2.5
68.6 7.2
and per cent I-pentadecene Mean 7, 1-pentadecene (BS-1) by weight (*SD.) Pi = 5t ~__. 16.7 4.1 15.8 0.9 18.3 2.8 19.9 2.8 IX.3 2.1 17.6. 4.9 15.6 3.9
* Means and variances of replicates within each speciesare equal at the I”/, level. ? Means and variances not equal. at the t*/0level. _ except T. mudens. To increase the yield from this ionization. Approximately 20 pg of secretion (in 5 ~1of species approximately 50-75 adults were placed in a solution) were sufficient for analysis. Standard 100 x 25 mm test tube and shaken briefly. The tube solutions of the above 3 compounds* were was then dipped into a dry ice-acetone bath until the chromato~aphed before and after each analysis to beetles became immobile and produced secretion. The insure its integrity. Quan~tative discrepancies of beetles were then poured onto the iced petri dish to about 50/, were compensated for with a correction keep the solidified secretion from melting. factor. Standard solutions were renewed every 20-30 The beetles. were examined for solidified secretion days because of solvent loss and degradation of the under a binocular dissecting microscope and the solid 1,4-~nzoquinones. secretion removed and collected in the following All analyses were replicated between 5 and 9 times manner. An immobilized beetle was held with forceps as indicated. in Table 2. The means of each set of and the solid secretion picked off the thorax and analyses were tested for equalness by a one-way abdomen with a fine dissecting needle. then placed on analysis of variance and for homogeneity of variance the open end of a OS-O.9 mm i.d. capillary tube by Bartlett’s Test (DIXON and MASSEY,1957). slightly tapered at its end. A warm, pencil soldering iron, its heat regulated by a variable voltage supply, RESULTS was placed near, but not touching, the solid secretion, causing it to melt and be drawn into the capillary tube. Prior to analysis of the quinone secretions. hexane The secretion then remained liquid at room washes of the plastic rearing containers, flour, and all temperature. Pooled samples of about 400 pg of glassware and manipulating tools were analyzed by secretion could be collected in the capillary tube. GC for substances which might interfere with the Capillary tubes for weighing the secretion were drawn .quinone analysis. None were found. out on a micro-pipet puller and formed with a double Pure 2-methyl 1,Cbenzoquinone had a retention taper. The extremely fine portion of the tapered ends time of 20 set after the solvent peak, while that of 2was removed with forceps, the tube weighed, and those ethyl 1,6benzoquinone was 30 sec. To determine approximately 17 mm long and weighing 15 2 0.5 mg possible interference with these rates, retention times were selected for use. Approximately 100-200 pg of for the following related compounds were also secretion were transferred to weighing tubes from the determined: 1,Cbenzoquinone-1 1 set; 2,3-dimethoxy collecting capillaries and weighed on a microbalance. 1,~be~oquinon~35 see; 2-methoxy 1,4-benzoThe secretion was then transferred from the weighing quinone-59 sec. The latter compound, reported capillary into a 100 ~1 volumetric flask with 5 ~1 by LOCONTIand ROTH (1953) as a constituent of 7: graduations, using spectrophotometric grade hexane, castaneum secretion, was not detected in any of the and the volume adjusted to contain approximately 20 species studied. 2-Ethyl- and 2-methyl-l A-benzo,ul of secretion, These solutions could be stored quinones, which were separated from secretions of all overnight at 5°C with only lO-15% loss in volume. The secretions were analyzed quantitatively for 2ethyland 2-methyl- 1,Cbenzoquinone and l- Table 2. Relative quantities of biosolvents (BS) in 18 fig of quinone secretion from each of 6 Tribolium species pentadecene by gas chromatography (GC) (Bendix 2500 with Auto Lab Digital Integrator). BS-lb BS-2 BS-1 BS-la Chromatography columns were glass, 4 mm x 180 Specks cm, Packed with 100/120’mesh Chromosorb W HP ++* T. audax +++* +* supporting 5% OV-1 as the liquid phase. The carrier T. castaneum ++ ++-t+ gas was nitrogen and detection was by hydrogen flame T. madens ++ +++ + tt T. brevicorn~s -l-i+ + * 2-Methyl-1,4-betuoquinone
and I-pentadecene were obtained commercially and purified if required. 2-Ethyl-1,4benzoquinone and all other quinones used in this study were synthesized by Dr. Joseph Bornstein, Boston College, Newton, Mas~chusetts 02167.
T. destructor T. con&urn T. con&urn,
ebony
+++ +++
+
+
t
t
+ + +
t
t
* Relative increasing quantities from i- to + + + $Just detectable under analytical conditions used.
Fig. 1.Solidified quinone secretion from TriboIium conjiisum placed on a cold Petri dish. Top: from Prothoracic glands. Secretion has flowed across thorax. Bottom: from abdominal glands. Secretion solidified before flowing.
Quinone
production
of some
n b
T. madens
i
d”““““““““” 1 2
3
4
5
Minutes
Fig. 2. Tracing of gas chromatogram of an 18 pg sample of @none secretion from Tribolium madens. Compounds are: (a) 2-methyl-1.4-benzoquinone, (b) 2-ethyl-1,4benzoquinone. (c) I-pentadecene (biosoivent I), (d) biosolvent la, (e) biosolvent 2.
species, were consistent with the above retention times of pure compounds and were the only quinones found in the secretion. Figure 2 shows a chromatogram of secretion from T. madens, which is typical for all species studied, except for biosolvent lb found only in T. destructor and T. confusum. The greater amount of 2-ethyl than 2-methyll,4-benzoquinone is consistent for all species studied, but the ratios vary (Table 1). Approximately 277; (T. confisum) to 49.5% (T. brevicornis) of the secretions consists of substances other than 1,4benzoquinones (Table 1). One of these substances, having a retention time of 146 set in our GC analysis, and found in all species, was I-pentadecene, first identified in T. confusum secretions by VON ENDTand WHEELER(1971) and subsequently in T. brevicornis and T. destructor by TSCHINKEL(1975a). The per cent by weight of secretion varied with species from 15.6-19.9 . (Table I), showing considerably less difference among species (maximum 4.30;,) than did the 1,Cbenzoquinones (maximum 22.5%). Small amounts of 3 other compounds were found at GC retention times of 150, 160 and 213 sec. We have designated these, and 1-pentadecene, as biosolvents (Table 2) on the assumption that one of their functions may be quinone solvency, which we propose as a prime function of 1-pentadecene. A brief, unsuccessful attempt was made to identify the 3 biosolvents by GC using model compdunds.
DISCUSSION Thoracic and abdominal quinone-secreting glands have some cellular differences in T. rastaneum (ROTH,
species of Triholi~~
789
1943) and in T. destructor (PALM, 1946). Thus qualitative and quantitative differences in secretions could occur: However, LOCONTIand ROTH (1953) concluded that the 1.4-benzoquinone secretions of the thoracic and abdominal glands of T. castaneum were qualitatively the same. In our experiments we collected and pooled secretions indiscriminately between thoracic and abdominal glands. If there were significant qualitative or quantitative differences between glands, variations among replicates for total 1,4-benzoquinones and ratios of 2-methyl-to 2-ethyl1,4-benzoquinones would have shown higher standard deviations and would not have been significantly equal by Bartlett’s Test for homogeneity. Because of the importance of I-pentadecene as a quinone solvent, it seems unlikely that there would be quantitative differences between glands. The possibility, however, remains open because replicate variations in our data did not meet Bartlett’s Test. Qualitative differences among biosolvents la, 1band 2 would not have been resolved by our experimental procedures. The greater amount of 2-ethyl- than 2-methyl-l, 4benzoquinone found in all species agrees with the finding of TSCHINKEL(1975a) that this occurred in 113 of 127 species of Tenebrionidae. Compounds other than quinones constitute 27% (T. corzfusum) to 49.5% (7’. brevicornis) of the secretions. The most prevalent compound was I-pentadccene, but this plus unidentified biosolvents la, 1b and 2, water and procedural losses cannot account for these amounts, except possibly in T. corzjksum. Other compounds are undoubtedly present and might be resolved by different analytical techniques. I-Pentadecene is the prime quinone solvent in the Tribolium species studied, as it and other hydrocarbons are in quinone-secreting arthropods (WEATHERSTONand PERCY, 1970). In the several Tenebrionids in which TSCHINKEL(1975a) found only quinones, his analytical technique probably did not detect existing hydrocarbon solvents, for practically all arthropod-secreted quinones are solids at ambient temperatures of the animals’ habitats. VONENDT and WHEELER (1971) proposed that I-pentadecene functions as a spreading agent in Tribohm in which secretions are known to spread over much of the body (Fig. 1). TSCHINKEL(1975b) has illustrated channels on the integument for this purpose. This would mitigate against a function of increasing permeability of predator cuticle to quinones (WEATHERSTON and PERCY, 1970), for Tribolium would need novel protective substances against quinone absorption in order to prevent self poisoning. KEVILLE and KANNOWSKI (1975) isolated. nhexadecane, I-heptadecene and n-heptadecadiene from T. confusum secretion after our experiments were completed. These may be our unidentified biosolvents. With the exception of n-heptadecadiene which was not tested, they found 1-pentadecene, n-hexadecane and lheptadecene to function as sex pheromones. This novel and probably secondary function of a presumed defensive secretion emphasizes the need to conduct behavioral experiments on secretions whose presumed original function now has limited, if any, biological utility. TSCHINKEL(1975a) states that the usefulness to
7Yll
taxonomy secretions va~ability accurate variances
HAIG MARKARIAN, C~ERALDJ. ~LOREWTINEAND
JOHNJ. PRATT,JR.
of data on analysis of Tenebrionid is limited by its variablility at all levels. This might well be reduced through precise and experimental procedures resulting in low as in our 1,Cbenzoquinone data. The high
LOCONTIJ. D. and ROTHL. M. (1953) Composition of the odorous secretion of Tribolium tzlstuneum. Ann. ent. Sot. Am. 46,281-289. PALMN. (196) Structure and physiology of the stink glands in Tribolium destructor Uytt. (Col.). Opusc. Em. II,
percentages of i,4-benzoquinones in secretions of T. confusum and T. castaneum are of concern to food wholesomeness, for these species are ubiquitous
RUTH L. M. (1943) Studies on the gaseous secretion of Tribolium conji~um Duval-II. The odoriferous glands of Tribolium confiisum. Ann. ent. Sot. Am. 36, 397-424. ROTH L. M. and EISNERT. (1962) Chemical defense of arthropods. Ann. Rev. Ent. 7, I‘O7-136. ROTH L. M. and HOWLAND R. B. (1941) Studies on the gaseous secretion of Tr~bo/i~n~ conf~sum Duvat--I. Abnormalities produced in Tribokum concern Duval by exposure to a secretion given off by the adults. Ann. Em. sot. 34, 115-171. TSCHINKELW. R. (1975a) A comparative study of the chemical defensive system of Tenebrionid beetle& Chemistry of the secretions. J. Insect PItmioi. 21,753-783. TSCHINKELW. R. (1975b) A comparative study of the chemical defensive system of Tenebrionid beettes-II. Defensive behavior and ancillary structures. Ann. em. Sot. Am. t&439-453. VONENDTD. W. and WH~ZZLER J. W. (1971) I-Pentadecene production in Tribolium conjiisum. Science 172, 60-61. WEATHERSTON J. and PERCYJ. E. (1970) Arthropod defense secretions. In Chetiicals Controlling insect Behavior. pp. 95-144. Academic Press, New York.
infestors of many human foods.
and &W-ON D. H. R. (1943) The excretion of ethylquinone by the flour beetle. Biochem. J. 37,463-465. DIXON V. J. and MASSEYF. J. (1957) In Introduction zo Statistical Analysis, pp. 145-152. McGraw-Hill, New York. ENGELHARDT M., RAP~PORTH. and SOKOLOFFA. (1965) Odorous secretion of normal and mutant Triboliwn eonfus~. Science 150,632-633. HACKMANR. H., PRYORM. G. M. and TODDA. R. (1948) The occurrence of phenolic substances in arthropods. Bioehem. .I. 43,474-477. KEVILLER. and KANNOWSKI P. B. (1975) Sexual excitation by pheromones of the confused flour beetle. J. Znsecr Physiol. 21, 8 l-84. LADISCHR. K. and McQus B. (1953) Methods of obtaining quinones from flour beetles. Science 118, 324-325. ALEXANDER P.
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