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Identification of the major constituents of the crystalline powder covering the larval cuticle of Samia cynthia ricini (Jones)
J. Ins. Physiol.,1965, Vol. 11, pp. 1003 to 1011. Pergamon Pms Ltd. Printed in Great Britain
IDENTIFICATION OF THE MAJOR CONSTITUENTS OF THE CRYSTALLINE POWDER COVERING THE LARVAL CUTICLE OF SAMIA CYNTHIA RICINI (JONES) WILLIAM
S. BOWERS
and MALCOLM
J. THOMPSON
Insect Physiology Laboratory, Entomology Research Division, U.S. Department of Agriculture, Beltsville, Maryland (Rece-iwed 8 February
1965)
Abstract-A
white crystalline powder was observed to accumulate superficially on the larval cuticle of Sumiu cynthia ricini (Jones). Most of this powder (92.6 per cent) was shown to be a mixture of two straight chain saturated alcohols, 99.4% n-triacontanol (C,,H,,O) and 0.6% n-octacosanol (C,,H,sO). Larval synthesis of these alcohols was demonstrated by radioactive tracer studies.
INTRODUCTION
THE function of cuticular lipids in water conservation in insects has been dealt with extensively (BEAMENT, 1961). However, the chemical composition of these lipids has enjoyed comparatively little examination. The qualitative and quantitative information available on the specific lipid composition of the insects studied permits few generalizations beyond the fact that two major classes of compounds account for the bulk of these lipids. Hydrocarbons have been isolated and chemically identified in the Mormon cricket (Anabrus simplex Haldeman) by BAKER et al. (1960) and the American cockroach (Periplaneta americana (L.)) by BAKER et al. (1963) and GILBY and Cox (1963). Indeed, hydrocarbons have been shown to be the major cuticular lipid constituents in these insects. Esters of long chain saturated alcohols and acids have been shown to constitute the bulk of the cuticular lipids of several species of insects of the genus Ceroplastes (HACKMAN, 1951; GILBY, 1957) and other lac insects (CHIBNALL and PIPER, 1934; CHIBNALL et al., 1934), as well as the tick, Boophilus microplus (GILBY, 1957). The third-, fourth-, and fifth-instar larvae of the eri silkworm, Sumia Cynthia ricini (Jones), as well as those of related subspecies, were observed to accumulate a white crystalline powder on the cuticle. This powder appeared to be secreted during each instar, and it was shed with the exuviae at every ecdysis. The powder was easily removed by wiping or brushing it off (Fig. 1) and therefore readily lent itself to accumulation and subsequent chemical examination. 1003
1004
WILLIAM S. BOWERSANDMALCOLMJ. THOMPSON MATERIALS
AND METHODS
Collection of the cuticular powder Initially the powder was collected by brushing it from the dorsum of the larvae into a beaker with a camel’s hair brush. Later it was found more convenient to extract the larval cast skins with benzene-methanol (1: 1 (v/v)) in a Soxhlet extractor. Extraction by the latter method was complete after 1 hr. Column chromatography of the cuticular powder and cast skin extract The adsorbent used throughout this study was Florisil* prepared and standardized according to CARROLL (1961). All solvents were redistilled over sodium. Six g of Florisil was slurried in hexane, poured into a glass column (1 x 12 cm) fitted with a sintered-glass filter, and the top of the adsorbent covered with a thin layer of clean sand. The cuticular powder (or cast skin extract), dissolved in a small volume (approx. 5 ml) of hexane, was placed on the column and eluted with the following solvents: 20 ml each of hexane, 5 and 15% diethyl ether in hexane, 35 ml each of 25% diethyl ether in hexane and 4% acetic acid in diethyl ether, and 25 ml of methanol. Solvents were removed in vacua and the residues transferred to tarred vessels and weighed to the nearest microgram on a Mettler Model M5 microbalance. Instrumental and spectral analysis of alcohols and their derivatives Infra-red spectra were obtained in Nujol mulls and/or in carbon disulphide or carbon tetrachloride solution with a Perkin-Elmer Model 221 prism-grating doublebeam spectrophotometer. Gas-liquid chromatographic analyses were performed on Barber-Colman Models 10 and 15. The ionization source in the detector cell was radium sulphate and the carrier gas was argon. All-glass chromatographic columns (6 ft, 4 mm I.D.) were employed. The SE-39 and EGS columns were prepared and coated according to the methods of VANDENHEUVEL et al. (1961). For alcohol analyses the SE-30 column was operated at 236°C and 15 lb/in2 and at 204°C and 24 lb/in2 for hydrocarbons. The EGS column was used for the methyl esters and was operated at 187°C and 40 lb/ix?. Relative percentages of alcohols were determined by planimetric measurement of gas-chromatographic peaks. Melting points were determined on the Kofler block. Thin-layer chromatography Glass plates (20 cm2), coated with silica gel G (containing rhodamine 6G for fluorescent detection (AVIGAN et al., 1963)) and activated at 110°C for 30 min, were used for analytical comparisons. Plates were developed with hexane-diethyl ether-glacial acetic acid 90 : 10 : 1. After development the chromatograms were * Mention of a proprietary product or company does not necessarily imply endorsement of the product or the company by the U.S. Department of Agriculture.
Fie,. 1. Fifth-instar larvae of Samia cynthia ricini (Jones). The crystalline powder has been brushed off of the lower larva.
CONSTITUENTS
OF POWDER COVERING LARVAL CUTICLE OF SAMIA
CYNTHIA
RICINI
1005
exposed briefly to iodine vapour and viewed under U.V. light. Finally, each plate was sprayed with concentrated sulphuric acid and charred overnight in an oven at 110°C. Examination of the cuticular alcohols for unsaturation was made on thin-layer plates impregnated with silver nitrate, prepared according to MORRIS (1963), and developed in hexane-diethyl ether 4 : 1. Detection was accomplished by spraying with sulphuric acid and charring with heat. Appropriate standards were employed with each chromatographic run. Injection of larvae with 1-C14-sodium acetate Five 2-day-old fifth-instar larvae were each injected with 3 million counts of 1-Cl*-sodium acetate (specific activity 23-6 x lo8 cpm/mg) and held without food for 24 hr, after which they were chilled slowly so they would not regurgitate and contaminate themselves externally, and finally they were quickly frozen solid in a freezer. The exterior lipids of the larval cuticle were removed by dipping the frozen larvae successively in each of three separate beakers of benzene. The benzene extracts were pooled and the solvent removed in vacua. The lipids were chromatographed on Florisil by a method previously standardized to yield an alcohol fraction. The lipid dissolved in 2 ml of 10% benzene-hexane solution was placed on a 6 g Florisil column (prepared as described previously in the Methods section). The following fractions were collected: 40 ml of 10% benzene in hexane, 150 ml benzene, and 50 ml of 50% benzene-ether. The solvent was removed in vacua and the fractions weighed on a microbalance. Radiometric determinations were made in triplicate with windowless gas-flow counters and samples were counted for sufficient time to yield a standard error of f 5%. In order to associate the radioactivity with the alcohols, the alcohol fraction was analysed by GLC and several fractions were collected in glass capillary tubes starting from the moment of injection until 1 hr after the emergence of the main alcohol peak, The trapped material was rinsed from the capillary tubes and radioassayed as above. Preparation of derivatives Acid. To a stirred solution of 10 mg of the silkworm alcohol in 10 ml of acetone at room temperature was added dropwise an 8 N solution of chromic acid in dilute sulphuric acid (ca. 40%) until a persistent orange-brown coloration indicated oxidation was completed (BOWERS et al., 1953). The mixture was diluted with water and the white crystalline precipitate collected, washed with water, and dried. Methyl ester. The methyl ester was prepared by treating the carboxylic acid dissolved in ether with an ethereal solution of diazomethane overnight at room temperature. Hydrocarbon. A mixture of 21 mg of the alcohol, 4 ml of pyridine, and 80 mg of p-toluenesulfonyl chloride was allowed to stand overnight at room temperature.
1006
WILLIAM S. BOWERSAND MALCOLM J. THOMPSON
The mixture was poured into ice-cold 2% bicarbonate solution and the precipitate collected and dried. The dried precipitate was dissolved in 75 ml of ether-benzene (1 :l) and 200 mg of solid lithium aluminium hydride was added. The mixture was refluxed overnight, cooled, treated with a few drops of ethyl acetate, water, and 20 ml of 4 N hydrochloric acid. The two layers were separated and the ethereal layer washed with water, dried over sodium sulphate, and concentrated to dryness in vacua. The residue was chromatographed over hexane-washed Florisil. The fraction eluted with hexane gave 10 mg of hydrocarbon. Further elution of the column with benzene gave some unreacted alcohol. RESULTS
Properties
and preliminary
identification
of the crude powder
The crude crystalline powder brushed from the cuticles of eri silkworm larvae (m.p. 86°C) was shown to be a saturated alcohol by i.r. spectroscopy, thin-layer and gas-liquid chromatography, and by the formation of several derivatives. The i.r. spectrum in Nujol revealed broad hydroxyl absorption in the 3200-3340 cm-l region and strong absorption at 1050 cm-l (-OH deformation for primary alcohol). The major component of the crude powder was observed to migrate on thin layers of silica gel in a manner identical with that of several straight chain saturated alcohols (i.e. n-tetradecanol, n-hexacosanol). However, trace quantities of hydrocarbon, ester, sterol, and acid could be detected. After chromatography over 92.6 per cent of Florisil, the alcohol fraction (IS?/, ether in hexane) contained the mass, which was found to be free of contamination by TLC examination. Analysis by GLC (SE-30) of this alcoholic material revealed a minor and a major peak. When chromatographed over Florisil, the material obtained from the cast skin extract yielded 25 mg of crystalline material (70 per cent of the original charge) in A recrystallization from acetone the fraction eluted with 15% ether in hexane. gave 24 mg of spears (m.p. 86.2~86~5°C). Gas-liquid chromatographic analysis of this material showed a trace amount of an impurity preceding the major peak. Comparison of the retention times of this material with a log plot of known standard saturated straight chain alcohols (Table 1) indicated that the two peaks were, in the order of increasing retention time, 0.6% C-28 (n-octacosanol) and 99.4!/, C-30 (n-triacontanol, Lit. m.p. 86~3°C FRANCIS et al., 1937). The saturated nature of these compounds was further demonstrated by a negative tetranitromethane test. Comparison of the movement of these compounds with saturated and unsaturated alcohol standards on a silica gel thin-layer plate impregnated with silver nitrate also failed to show any unsaturated compounds. Elemental analysis for C,,H,,O: calculated: C, 82.11; H, 14.24. Found: C, 81-86; H, 14-55. The remaining 30 per cent of the cast skin extract was found to be composed of 5% hydrocarbon (hexane fraction) and 16% ester (5% ether in hexane fraction). Trace quantities of sterol and acid were seen by TLC analysis of the polar fractions.
CONSTITUENTS OF POWDERCOVERING LARVALCUTICLEOF SAMIA
CYNTHIA
FUCINI
1007
TABLE I-COMPARISON OF GAS-LIQUIDCHROMATOGRAPHIC RETENTIONTIMESOF SILKWORM ALCOHOLS ANDDERIVATIVES WITH N-ALKYLSTANDARDS Retention time (min) Compounds
n-Alkyl standards c-20 c-22 C-23 C-24 C-26 C-28 C-30 (calculated)$ Silkworm alcohol (major peak) Hydrocarbon derivative Methyl ester derivative
Alcohols SE-30*
Hydrocarbons SE-307
Methyl esters EGS$
1.90 -
2.50
3.93 6.65 -
5.65
6.68
11.30 -
9.90 17.00 17.05
13.15 25-20 25.30
33.68 59.10 59.50
* Column 6 ft x 4 mm I.D., 0.75%, SE-30 on loo-140 mesh, Gas Chrom P, 15 lb/in2, 236°C. f Same as SE-30 (1) except operated at 24 lb/in2, 204°C. $ Column 6 ft x 4 mm I.D., 15.0% EGS on 100-140 mesh, Gas Chrom P, 40 lb/in2, 187°C. § Retention times of C-30 compounds calculated from log plots.
Derivatives Oxidation of the alcohol gave triacontanoic acid (m.p. 91-92°C Lit. m.p. 93*6”C, FRANCIS et al., 1937). The i.r. spectrum exhibited strong absorption at 1709 cm-l and the typical broad absorption of a carboxylic groupat 2500-2700 cm-l. The spectrum of the methyl ester (m.p. 71-72°C Lit. m.p. 71~5”C!, ROBINSON, 1934) gave a sharp band at 1735 cm-l (ester, carbonyl). After chromatography of the lithium aluminium hydride reduced material on Florisil, the hydrocarbon fraction gave absorption typical of saturated straight chain hydrocarbons with no free hydroxyl absorption (m.p. 66”C, Lit. m.p. 66”C, FRANCIS et al., 1937). The straight chain nature of the compound also was established by having the material adsorbed by 5 A molecular sieve from a cyclohexane solution. Adsorption was followed by GLC analysis (SE-30). Advantage was taken of the linear relationship which exists in a semilogarithmic plot of the retention time against the carbon chain length of a homologous series of compounds. Thus, each of the compounds examined by GLC (free alcohol, methyl ester, and hydrocarbon) was found to display retention times corresponding to the C,, and C,, members of each homologous series (Fig. 2). The major and
1008
WILLIAM S. BOWERS AND MALCOLM
minor peaks corresponded derivatives, respectively.
to the
triacontanol
Carbon chain
J.THOMPSON
(C-30)
and
octacosanol
(C-28)
length
FIG. 2. Semi-log plot of retention times of the silkworm alcohol, the derived 0, methyl esters; @, hydromethyl ester and hydrocarbon, and n-alkyl standards. carbons; 0, alcohols; S, silkworm alcohol or derivatives.
Incorporation of 1-Cl*-sodium acetate into cuticular alcohols The extract obtained from the cuticles of the frozen larvae gave 7.2 mg of Radioassay of this material gave crystalline residue after removal of the benzene. 14,600 cpm/mg or a total of 105,200 cpm. Thus, of the 15 million counts originally administered, approximately 0.75 per cent was incorporated into the cuticular lipids. The alcohol fraction (benzene fraction) resulting from chromatography on Florisil yielded 3.6 mg of crystalline material, which gave 28,000 cpm/mg, or 96 per cent of the activity extracted from the surface of the larvae. GLC analysis of this crystalline material gave two peaks corresponding to n-triacontanol and n-octacosanol. Trapping of the effluent from the SE-30 column resulted in about a 50 per cent recovery of radioactivity, which is in agreement with similar recoveries of sterols Of the recovered activity, 85.7 per cent from this column in this laboratory. emerged with the n-triacontanol peak.
CONSTITUENTS
OF POWDER COVERING LARVAL CUTICLE OF SAMIA
CYNTHIA
RICINI
1009
DISCUSSION
The straight chain saturated alcohol triacontanol is therefore the major constituent (cu. 93 per cent) of the cuticular lipids of Samia Cynthia ricini larvae. This high percentage of free alcohol presents considerable contrast to the studies of cuticular lipids in the American cockroach (GILBY and Cox, 1963), in which the major cuticular lipids are hydrocarbons and the only free alcohols are sterols. On the other hand, the cuticular lipids of several species of insects of the genus Ceroplastes were reported to contain large amounts of long chain acids and alcohols in esterified form, although HACKMAN (1951) reported the presence of 27% free alcohol (a mixture of hexacosanol and octacosanol) in Ceroplastes destructor Newstead. The tracer studies revealed a fair amount of incorporation of acetate into the cuticular alcohols, indicating significant capacity for the synthesis of alcohols, which It is would tend to discount an exogenous source of the alcohols (e.g. food plant). unknown whether alcohol synthesis is continuous or initiated by some specific stimulus. When the functional role of these unusual cuticular lipids is discerned, the regulation of synthesis will be more easily studied. Synthesis of an alcohol has previously been demonstrated by CLARK and BLOCH (1959) in the hide beetle, Dermestes maculatus De Geer (= vulpinus), by feeding I-Ci4-sodium acetate and recovering from the unsaponifiable fraction a labelled material with the properties of a primary aliphatic alcohol of high molecular weight (27 + 1 carbon atoms). The cuticular alcohols of Samia Cynthia ricini do not form a contiguous sheet or coating similar to the ‘waxes’ of many other insects, but appear to exist on the larval cuticle as a fine dusting or powder. Therefore, the functional significance of these alcohols is not immediately clear. Since the alcohols compose so much of the cuticular lipid, it is tempting to speculate about their possible involvement in water conservation, a role traditionally applied to cuticular lipids. If such a possibility is assumed for the moment, how then do the alcohols fulfil this role ? MANSFIELD (1958) demonstrated the capacity of cetyl alcohol to form a monolayer over, and reduce evaporation from, small impoundments of water. The alcohols studied here (triacontanol, octacosanol) would probably function similarly; but, as far as can be observed grossly, the alcohol is not oriented in monolayers unless a monolayer is formed and maintained at the immediate surface of the cuticle to which the alcohol is transported and released, with the readily observed crystalline material accumulating as a result of rapid and continuous synthesis and deposition on the cuticle. How the alcohol could orient as a monolayer on the surface of the cuticle as it does on water is unknown unless one presupposes a highly polar exterior surface of the cuticle which could effect such an orientation of the alcohol. No experimental evidence, however, is available to indicate this possibility. BEAMENT (1961) assumed the presence of alcohols in cockroach ‘grease’ and postulated the presence of a waterproofing monolayer of alcohols in the lower interface of the ‘wax’ layer. However, GILBY and Cox (1963) demonstrated the absence of alcohols, other than sterols, from cockroach ‘grease’. 64
1010
WILLIAM S. BOWERS ANDMALCOLMJ. THOMPSON
Alternatively, it is possible that the eri silkworm cuticular alcohols are not involved in water conservation but perform an entirely different service. KOIDSUMI (1957)
gave experimental
evidence
that the integumental
lipids
of the larvae
of
Bombyx mori L. and Chilo simplex Butler were capable of restricting the growth of pathogenic fungi in vitro and indicated that the antifungal compounds were saturated fatty acids, presumably caprylic and capric acid. However, no certain chemical identification was made. Although these alcohols are present in abundance on the larval cuticle of Samia Cynthia vicini, their functional significance remains unknown. It is anticipated that continued studies, in progress, will clarify their role. Acknowledgement-The authors wish to thank the M. MICHEL and Co., Inc., New York, for their gift of high purity long chain alcohols which were employed as thin-layer and gas-liquid chromatography standards.
REFERENCES AVIGANJ., GOODMAND. S., and STEINBERGD. (1963) Thin-layer chromatography of sterols and steroids. J. Lipid Res. 4, 100-101. BAKER G. L., PEPPER J. H., JOHNSONL. H., and HASTINGS E. (1960) Estimation of the cuticular wax of the Mormon cricket, Anabrus simplex Hald. J. Ins. Physiol. 5, 47-60. BAKER G. L., VROMANH. E., and PADMOREJ. (1963) Hydrocarbons of the American cockroach. Biochem. Biophys. Res. Comm. 13, 360-365. BEAMENTJ. W. L. (1961) The water relations of insect cuticle. Biol. Rev. 36, 281-320. BOWERSA., HALSALLT. G., JONES E. R. H., and LEMIN A. J. (1953) The chemistry of the Elucidation of the structure of Polytriterpenes and related compounds. Part XVIII. porenic acid. J. them. Sot. 2548-2560. CARROLLK. K. (1961) Separation of lipid classes by chromatography on florisil. J. Lipid Res. 2, 135-141. CHIBNALLA. C. and PIPER S. H. (1934) The metabolism of plant and insect waxes. Biochem. J. 28, 2209-2219. CHIBNALL A. C., PIPER S. H., POLLARDA., WILLIAMS E. R., and SAKAI P. N. (1934) The constitution of the primary alcohols, fatty acids, and paraffins present in plant and insect waxes. Biochem. J. 28, 2189-2208. CLARK A. J. and BLOCH K. (1959) The absence of sterol synthesis in insects. J. biol. Chem. 234, 2578-2582. FRANCIS F., COLLINS F. J. E., and PIPER S. H. (1937) N-Fatty acids and certain of their derivatives. Chem. Abstr. 31, 3448. GILBY A. R. (1957) Studies of cuticular lipids of arthropods. II. The chemical composition of the wax from Ceroplastes destructor (Newstead). Arch. Biochem. Biophys. 67, 307-319. III. The chemical composition of the wax from Boophilus microplus. Arch. Biochem. Biophys. 67, 320-324. GILBY A. R. and Cox M. E. (1963) The cuticular lipids of the cockroach, Periplaneta americana (I,.). J. Ins. Physiol. 9, 671-681. HACKMANR. H. (1951) The chemical composition of the wax of the white wax scale, Ceroplastes destructor (Newstead). Arch. Biochem. Biophys. 133, 150-154. KOIDSUMI K. (1957) Antifungal action of cuticular lipids in insects. J. Ins. Physiol. 1, 40-S 1. MANSFIELDW. W. (1958) The influence of monolayers on evaporation from water storages. I. The potential performance of monolayers of cetyl alcohol. Aust. J. appl. Sci. 9, 245-254.
CONSTITUENTSOF POWDERCOVERINGLARVALCUTICLEOF SAMIA
CYNTHIA
RICINI
1011
MORRIS L. J. (1963) Fractionation of cholesterol esters by thin-layer chromatography. J. Lipid Res. 4, 357-359. ROBINSON G. M. (1934) A synthesis of certain higher aliphatic compounds. IV. Synthesis of n-triacontanoic acid from stearic acid. J, them. Sot. 1543-1545. VANDENHEUVELW. J. A., HAAHTI E. 0. A., and HORNINGE. C. (1961) A new liquid phase for gas chromatographic separations of steroids. J. Amer. them. Sot. 83, 1.513-1514.
IDENTIFICATION OF THE MAJOR CONSTITUENTS OF THE CRYSTALLINE POWDER COVERING THE LARVAL CUTICLE OF SAMIA CYNTHIA RICINI (JONES) WILLIAM
S. BOWERS
and MALCOLM
J. THOMPSON
Insect Physiology Laboratory, Entomology Research Division, U.S. Department of Agriculture, Beltsville, Maryland (Rece-iwed 8 February
1965)
Abstract-A
white crystalline powder was observed to accumulate superficially on the larval cuticle of Sumiu cynthia ricini (Jones). Most of this powder (92.6 per cent) was shown to be a mixture of two straight chain saturated alcohols, 99.4% n-triacontanol (C,,H,,O) and 0.6% n-octacosanol (C,,H,sO). Larval synthesis of these alcohols was demonstrated by radioactive tracer studies.
INTRODUCTION
THE function of cuticular lipids in water conservation in insects has been dealt with extensively (BEAMENT, 1961). However, the chemical composition of these lipids has enjoyed comparatively little examination. The qualitative and quantitative information available on the specific lipid composition of the insects studied permits few generalizations beyond the fact that two major classes of compounds account for the bulk of these lipids. Hydrocarbons have been isolated and chemically identified in the Mormon cricket (Anabrus simplex Haldeman) by BAKER et al. (1960) and the American cockroach (Periplaneta americana (L.)) by BAKER et al. (1963) and GILBY and Cox (1963). Indeed, hydrocarbons have been shown to be the major cuticular lipid constituents in these insects. Esters of long chain saturated alcohols and acids have been shown to constitute the bulk of the cuticular lipids of several species of insects of the genus Ceroplastes (HACKMAN, 1951; GILBY, 1957) and other lac insects (CHIBNALL and PIPER, 1934; CHIBNALL et al., 1934), as well as the tick, Boophilus microplus (GILBY, 1957). The third-, fourth-, and fifth-instar larvae of the eri silkworm, Sumia Cynthia ricini (Jones), as well as those of related subspecies, were observed to accumulate a white crystalline powder on the cuticle. This powder appeared to be secreted during each instar, and it was shed with the exuviae at every ecdysis. The powder was easily removed by wiping or brushing it off (Fig. 1) and therefore readily lent itself to accumulation and subsequent chemical examination. 1003
1004
WILLIAM S. BOWERSANDMALCOLMJ. THOMPSON MATERIALS
AND METHODS
Collection of the cuticular powder Initially the powder was collected by brushing it from the dorsum of the larvae into a beaker with a camel’s hair brush. Later it was found more convenient to extract the larval cast skins with benzene-methanol (1: 1 (v/v)) in a Soxhlet extractor. Extraction by the latter method was complete after 1 hr. Column chromatography of the cuticular powder and cast skin extract The adsorbent used throughout this study was Florisil* prepared and standardized according to CARROLL (1961). All solvents were redistilled over sodium. Six g of Florisil was slurried in hexane, poured into a glass column (1 x 12 cm) fitted with a sintered-glass filter, and the top of the adsorbent covered with a thin layer of clean sand. The cuticular powder (or cast skin extract), dissolved in a small volume (approx. 5 ml) of hexane, was placed on the column and eluted with the following solvents: 20 ml each of hexane, 5 and 15% diethyl ether in hexane, 35 ml each of 25% diethyl ether in hexane and 4% acetic acid in diethyl ether, and 25 ml of methanol. Solvents were removed in vacua and the residues transferred to tarred vessels and weighed to the nearest microgram on a Mettler Model M5 microbalance. Instrumental and spectral analysis of alcohols and their derivatives Infra-red spectra were obtained in Nujol mulls and/or in carbon disulphide or carbon tetrachloride solution with a Perkin-Elmer Model 221 prism-grating doublebeam spectrophotometer. Gas-liquid chromatographic analyses were performed on Barber-Colman Models 10 and 15. The ionization source in the detector cell was radium sulphate and the carrier gas was argon. All-glass chromatographic columns (6 ft, 4 mm I.D.) were employed. The SE-39 and EGS columns were prepared and coated according to the methods of VANDENHEUVEL et al. (1961). For alcohol analyses the SE-30 column was operated at 236°C and 15 lb/in2 and at 204°C and 24 lb/in2 for hydrocarbons. The EGS column was used for the methyl esters and was operated at 187°C and 40 lb/ix?. Relative percentages of alcohols were determined by planimetric measurement of gas-chromatographic peaks. Melting points were determined on the Kofler block. Thin-layer chromatography Glass plates (20 cm2), coated with silica gel G (containing rhodamine 6G for fluorescent detection (AVIGAN et al., 1963)) and activated at 110°C for 30 min, were used for analytical comparisons. Plates were developed with hexane-diethyl ether-glacial acetic acid 90 : 10 : 1. After development the chromatograms were * Mention of a proprietary product or company does not necessarily imply endorsement of the product or the company by the U.S. Department of Agriculture.
Fie,. 1. Fifth-instar larvae of Samia cynthia ricini (Jones). The crystalline powder has been brushed off of the lower larva.
CONSTITUENTS
OF POWDER COVERING LARVAL CUTICLE OF SAMIA
CYNTHIA
RICINI
1005
exposed briefly to iodine vapour and viewed under U.V. light. Finally, each plate was sprayed with concentrated sulphuric acid and charred overnight in an oven at 110°C. Examination of the cuticular alcohols for unsaturation was made on thin-layer plates impregnated with silver nitrate, prepared according to MORRIS (1963), and developed in hexane-diethyl ether 4 : 1. Detection was accomplished by spraying with sulphuric acid and charring with heat. Appropriate standards were employed with each chromatographic run. Injection of larvae with 1-C14-sodium acetate Five 2-day-old fifth-instar larvae were each injected with 3 million counts of 1-Cl*-sodium acetate (specific activity 23-6 x lo8 cpm/mg) and held without food for 24 hr, after which they were chilled slowly so they would not regurgitate and contaminate themselves externally, and finally they were quickly frozen solid in a freezer. The exterior lipids of the larval cuticle were removed by dipping the frozen larvae successively in each of three separate beakers of benzene. The benzene extracts were pooled and the solvent removed in vacua. The lipids were chromatographed on Florisil by a method previously standardized to yield an alcohol fraction. The lipid dissolved in 2 ml of 10% benzene-hexane solution was placed on a 6 g Florisil column (prepared as described previously in the Methods section). The following fractions were collected: 40 ml of 10% benzene in hexane, 150 ml benzene, and 50 ml of 50% benzene-ether. The solvent was removed in vacua and the fractions weighed on a microbalance. Radiometric determinations were made in triplicate with windowless gas-flow counters and samples were counted for sufficient time to yield a standard error of f 5%. In order to associate the radioactivity with the alcohols, the alcohol fraction was analysed by GLC and several fractions were collected in glass capillary tubes starting from the moment of injection until 1 hr after the emergence of the main alcohol peak, The trapped material was rinsed from the capillary tubes and radioassayed as above. Preparation of derivatives Acid. To a stirred solution of 10 mg of the silkworm alcohol in 10 ml of acetone at room temperature was added dropwise an 8 N solution of chromic acid in dilute sulphuric acid (ca. 40%) until a persistent orange-brown coloration indicated oxidation was completed (BOWERS et al., 1953). The mixture was diluted with water and the white crystalline precipitate collected, washed with water, and dried. Methyl ester. The methyl ester was prepared by treating the carboxylic acid dissolved in ether with an ethereal solution of diazomethane overnight at room temperature. Hydrocarbon. A mixture of 21 mg of the alcohol, 4 ml of pyridine, and 80 mg of p-toluenesulfonyl chloride was allowed to stand overnight at room temperature.
1006
WILLIAM S. BOWERSAND MALCOLM J. THOMPSON
The mixture was poured into ice-cold 2% bicarbonate solution and the precipitate collected and dried. The dried precipitate was dissolved in 75 ml of ether-benzene (1 :l) and 200 mg of solid lithium aluminium hydride was added. The mixture was refluxed overnight, cooled, treated with a few drops of ethyl acetate, water, and 20 ml of 4 N hydrochloric acid. The two layers were separated and the ethereal layer washed with water, dried over sodium sulphate, and concentrated to dryness in vacua. The residue was chromatographed over hexane-washed Florisil. The fraction eluted with hexane gave 10 mg of hydrocarbon. Further elution of the column with benzene gave some unreacted alcohol. RESULTS
Properties
and preliminary
identification
of the crude powder
The crude crystalline powder brushed from the cuticles of eri silkworm larvae (m.p. 86°C) was shown to be a saturated alcohol by i.r. spectroscopy, thin-layer and gas-liquid chromatography, and by the formation of several derivatives. The i.r. spectrum in Nujol revealed broad hydroxyl absorption in the 3200-3340 cm-l region and strong absorption at 1050 cm-l (-OH deformation for primary alcohol). The major component of the crude powder was observed to migrate on thin layers of silica gel in a manner identical with that of several straight chain saturated alcohols (i.e. n-tetradecanol, n-hexacosanol). However, trace quantities of hydrocarbon, ester, sterol, and acid could be detected. After chromatography over 92.6 per cent of Florisil, the alcohol fraction (IS?/, ether in hexane) contained the mass, which was found to be free of contamination by TLC examination. Analysis by GLC (SE-30) of this alcoholic material revealed a minor and a major peak. When chromatographed over Florisil, the material obtained from the cast skin extract yielded 25 mg of crystalline material (70 per cent of the original charge) in A recrystallization from acetone the fraction eluted with 15% ether in hexane. gave 24 mg of spears (m.p. 86.2~86~5°C). Gas-liquid chromatographic analysis of this material showed a trace amount of an impurity preceding the major peak. Comparison of the retention times of this material with a log plot of known standard saturated straight chain alcohols (Table 1) indicated that the two peaks were, in the order of increasing retention time, 0.6% C-28 (n-octacosanol) and 99.4!/, C-30 (n-triacontanol, Lit. m.p. 86~3°C FRANCIS et al., 1937). The saturated nature of these compounds was further demonstrated by a negative tetranitromethane test. Comparison of the movement of these compounds with saturated and unsaturated alcohol standards on a silica gel thin-layer plate impregnated with silver nitrate also failed to show any unsaturated compounds. Elemental analysis for C,,H,,O: calculated: C, 82.11; H, 14.24. Found: C, 81-86; H, 14-55. The remaining 30 per cent of the cast skin extract was found to be composed of 5% hydrocarbon (hexane fraction) and 16% ester (5% ether in hexane fraction). Trace quantities of sterol and acid were seen by TLC analysis of the polar fractions.
CONSTITUENTS OF POWDERCOVERING LARVALCUTICLEOF SAMIA
CYNTHIA
FUCINI
1007
TABLE I-COMPARISON OF GAS-LIQUIDCHROMATOGRAPHIC RETENTIONTIMESOF SILKWORM ALCOHOLS ANDDERIVATIVES WITH N-ALKYLSTANDARDS Retention time (min) Compounds
n-Alkyl standards c-20 c-22 C-23 C-24 C-26 C-28 C-30 (calculated)$ Silkworm alcohol (major peak) Hydrocarbon derivative Methyl ester derivative
Alcohols SE-30*
Hydrocarbons SE-307
Methyl esters EGS$
1.90 -
2.50
3.93 6.65 -
5.65
6.68
11.30 -
9.90 17.00 17.05
13.15 25-20 25.30
33.68 59.10 59.50
* Column 6 ft x 4 mm I.D., 0.75%, SE-30 on loo-140 mesh, Gas Chrom P, 15 lb/in2, 236°C. f Same as SE-30 (1) except operated at 24 lb/in2, 204°C. $ Column 6 ft x 4 mm I.D., 15.0% EGS on 100-140 mesh, Gas Chrom P, 40 lb/in2, 187°C. § Retention times of C-30 compounds calculated from log plots.
Derivatives Oxidation of the alcohol gave triacontanoic acid (m.p. 91-92°C Lit. m.p. 93*6”C, FRANCIS et al., 1937). The i.r. spectrum exhibited strong absorption at 1709 cm-l and the typical broad absorption of a carboxylic groupat 2500-2700 cm-l. The spectrum of the methyl ester (m.p. 71-72°C Lit. m.p. 71~5”C!, ROBINSON, 1934) gave a sharp band at 1735 cm-l (ester, carbonyl). After chromatography of the lithium aluminium hydride reduced material on Florisil, the hydrocarbon fraction gave absorption typical of saturated straight chain hydrocarbons with no free hydroxyl absorption (m.p. 66”C, Lit. m.p. 66”C, FRANCIS et al., 1937). The straight chain nature of the compound also was established by having the material adsorbed by 5 A molecular sieve from a cyclohexane solution. Adsorption was followed by GLC analysis (SE-30). Advantage was taken of the linear relationship which exists in a semilogarithmic plot of the retention time against the carbon chain length of a homologous series of compounds. Thus, each of the compounds examined by GLC (free alcohol, methyl ester, and hydrocarbon) was found to display retention times corresponding to the C,, and C,, members of each homologous series (Fig. 2). The major and
1008
WILLIAM S. BOWERS AND MALCOLM
minor peaks corresponded derivatives, respectively.
to the
triacontanol
Carbon chain
J.THOMPSON
(C-30)
and
octacosanol
(C-28)
length
FIG. 2. Semi-log plot of retention times of the silkworm alcohol, the derived 0, methyl esters; @, hydromethyl ester and hydrocarbon, and n-alkyl standards. carbons; 0, alcohols; S, silkworm alcohol or derivatives.
Incorporation of 1-Cl*-sodium acetate into cuticular alcohols The extract obtained from the cuticles of the frozen larvae gave 7.2 mg of Radioassay of this material gave crystalline residue after removal of the benzene. 14,600 cpm/mg or a total of 105,200 cpm. Thus, of the 15 million counts originally administered, approximately 0.75 per cent was incorporated into the cuticular lipids. The alcohol fraction (benzene fraction) resulting from chromatography on Florisil yielded 3.6 mg of crystalline material, which gave 28,000 cpm/mg, or 96 per cent of the activity extracted from the surface of the larvae. GLC analysis of this crystalline material gave two peaks corresponding to n-triacontanol and n-octacosanol. Trapping of the effluent from the SE-30 column resulted in about a 50 per cent recovery of radioactivity, which is in agreement with similar recoveries of sterols Of the recovered activity, 85.7 per cent from this column in this laboratory. emerged with the n-triacontanol peak.
CONSTITUENTS
OF POWDER COVERING LARVAL CUTICLE OF SAMIA
CYNTHIA
RICINI
1009
DISCUSSION
The straight chain saturated alcohol triacontanol is therefore the major constituent (cu. 93 per cent) of the cuticular lipids of Samia Cynthia ricini larvae. This high percentage of free alcohol presents considerable contrast to the studies of cuticular lipids in the American cockroach (GILBY and Cox, 1963), in which the major cuticular lipids are hydrocarbons and the only free alcohols are sterols. On the other hand, the cuticular lipids of several species of insects of the genus Ceroplastes were reported to contain large amounts of long chain acids and alcohols in esterified form, although HACKMAN (1951) reported the presence of 27% free alcohol (a mixture of hexacosanol and octacosanol) in Ceroplastes destructor Newstead. The tracer studies revealed a fair amount of incorporation of acetate into the cuticular alcohols, indicating significant capacity for the synthesis of alcohols, which It is would tend to discount an exogenous source of the alcohols (e.g. food plant). unknown whether alcohol synthesis is continuous or initiated by some specific stimulus. When the functional role of these unusual cuticular lipids is discerned, the regulation of synthesis will be more easily studied. Synthesis of an alcohol has previously been demonstrated by CLARK and BLOCH (1959) in the hide beetle, Dermestes maculatus De Geer (= vulpinus), by feeding I-Ci4-sodium acetate and recovering from the unsaponifiable fraction a labelled material with the properties of a primary aliphatic alcohol of high molecular weight (27 + 1 carbon atoms). The cuticular alcohols of Samia Cynthia ricini do not form a contiguous sheet or coating similar to the ‘waxes’ of many other insects, but appear to exist on the larval cuticle as a fine dusting or powder. Therefore, the functional significance of these alcohols is not immediately clear. Since the alcohols compose so much of the cuticular lipid, it is tempting to speculate about their possible involvement in water conservation, a role traditionally applied to cuticular lipids. If such a possibility is assumed for the moment, how then do the alcohols fulfil this role ? MANSFIELD (1958) demonstrated the capacity of cetyl alcohol to form a monolayer over, and reduce evaporation from, small impoundments of water. The alcohols studied here (triacontanol, octacosanol) would probably function similarly; but, as far as can be observed grossly, the alcohol is not oriented in monolayers unless a monolayer is formed and maintained at the immediate surface of the cuticle to which the alcohol is transported and released, with the readily observed crystalline material accumulating as a result of rapid and continuous synthesis and deposition on the cuticle. How the alcohol could orient as a monolayer on the surface of the cuticle as it does on water is unknown unless one presupposes a highly polar exterior surface of the cuticle which could effect such an orientation of the alcohol. No experimental evidence, however, is available to indicate this possibility. BEAMENT (1961) assumed the presence of alcohols in cockroach ‘grease’ and postulated the presence of a waterproofing monolayer of alcohols in the lower interface of the ‘wax’ layer. However, GILBY and Cox (1963) demonstrated the absence of alcohols, other than sterols, from cockroach ‘grease’. 64
1010
WILLIAM S. BOWERS ANDMALCOLMJ. THOMPSON
Alternatively, it is possible that the eri silkworm cuticular alcohols are not involved in water conservation but perform an entirely different service. KOIDSUMI (1957)
gave experimental
evidence
that the integumental
lipids
of the larvae
of
Bombyx mori L. and Chilo simplex Butler were capable of restricting the growth of pathogenic fungi in vitro and indicated that the antifungal compounds were saturated fatty acids, presumably caprylic and capric acid. However, no certain chemical identification was made. Although these alcohols are present in abundance on the larval cuticle of Samia Cynthia vicini, their functional significance remains unknown. It is anticipated that continued studies, in progress, will clarify their role. Acknowledgement-The authors wish to thank the M. MICHEL and Co., Inc., New York, for their gift of high purity long chain alcohols which were employed as thin-layer and gas-liquid chromatography standards.
REFERENCES AVIGANJ., GOODMAND. S., and STEINBERGD. (1963) Thin-layer chromatography of sterols and steroids. J. Lipid Res. 4, 100-101. BAKER G. L., PEPPER J. H., JOHNSONL. H., and HASTINGS E. (1960) Estimation of the cuticular wax of the Mormon cricket, Anabrus simplex Hald. J. Ins. Physiol. 5, 47-60. BAKER G. L., VROMANH. E., and PADMOREJ. (1963) Hydrocarbons of the American cockroach. Biochem. Biophys. Res. Comm. 13, 360-365. BEAMENTJ. W. L. (1961) The water relations of insect cuticle. Biol. Rev. 36, 281-320. BOWERSA., HALSALLT. G., JONES E. R. H., and LEMIN A. J. (1953) The chemistry of the Elucidation of the structure of Polytriterpenes and related compounds. Part XVIII. porenic acid. J. them. Sot. 2548-2560. CARROLLK. K. (1961) Separation of lipid classes by chromatography on florisil. J. Lipid Res. 2, 135-141. CHIBNALLA. C. and PIPER S. H. (1934) The metabolism of plant and insect waxes. Biochem. J. 28, 2209-2219. CHIBNALL A. C., PIPER S. H., POLLARDA., WILLIAMS E. R., and SAKAI P. N. (1934) The constitution of the primary alcohols, fatty acids, and paraffins present in plant and insect waxes. Biochem. J. 28, 2189-2208. CLARK A. J. and BLOCH K. (1959) The absence of sterol synthesis in insects. J. biol. Chem. 234, 2578-2582. FRANCIS F., COLLINS F. J. E., and PIPER S. H. (1937) N-Fatty acids and certain of their derivatives. Chem. Abstr. 31, 3448. GILBY A. R. (1957) Studies of cuticular lipids of arthropods. II. The chemical composition of the wax from Ceroplastes destructor (Newstead). Arch. Biochem. Biophys. 67, 307-319. III. The chemical composition of the wax from Boophilus microplus. Arch. Biochem. Biophys. 67, 320-324. GILBY A. R. and Cox M. E. (1963) The cuticular lipids of the cockroach, Periplaneta americana (I,.). J. Ins. Physiol. 9, 671-681. HACKMANR. H. (1951) The chemical composition of the wax of the white wax scale, Ceroplastes destructor (Newstead). Arch. Biochem. Biophys. 133, 150-154. KOIDSUMI K. (1957) Antifungal action of cuticular lipids in insects. J. Ins. Physiol. 1, 40-S 1. MANSFIELDW. W. (1958) The influence of monolayers on evaporation from water storages. I. The potential performance of monolayers of cetyl alcohol. Aust. J. appl. Sci. 9, 245-254.
CONSTITUENTSOF POWDERCOVERINGLARVALCUTICLEOF SAMIA
CYNTHIA
RICINI
1011
MORRIS L. J. (1963) Fractionation of cholesterol esters by thin-layer chromatography. J. Lipid Res. 4, 357-359. ROBINSON G. M. (1934) A synthesis of certain higher aliphatic compounds. IV. Synthesis of n-triacontanoic acid from stearic acid. J, them. Sot. 1543-1545. VANDENHEUVELW. J. A., HAAHTI E. 0. A., and HORNINGE. C. (1961) A new liquid phase for gas chromatographic separations of steroids. J. Amer. them. Sot. 83, 1.513-1514.