Sterols in the termite Nasutitermes rippertii

Insect Biochem., x974, Vol. 4, PP. z8x to z85. Pergamon Press. Printed in Great Britain

z8x

STEROLS IN THE T E R M I T E N A S U T I T E R M E S RIPPERTII KAREL UBIK AND JAN VRKO~ Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Science, 166 xo-Praha 6-Dejvice, Flemingovo nLm. z, Czechoslovakia

(Received z4 ,September I973) ABSTRACT The components of the mixture of free sterols isolated from the termite

Nasutiterraes rippertii have been identified. Androst-5-en-3/3-ol, 5 ~:-androstan3/3-ol, pregn-5-en-3fl-ol, i7~:-isopropylandrost-s-en-3fl-ol, and x~-isopropyl5se-androstan-3/3-ol have been found to constitute the minor components. Furthermore, the proportion of sterols in particular castes of these termites has been determined. IN the course of investigations on substances occurring in the termite Nasutitermes rippertii (Rambur), (Vrko~ et al., I973), a fraction of free sterols has been isolated. As shown by analysis in a combined gas chromatograph and mass spectrometer system ( G L C - M S ) , the fraction of free sterols contains in addition to the usual insect sterols some minor sterols, which have not hitherto been found in nature. T h e side chain of these minor sterols is shortened to an isopropyl and ethyl group or is quite absent. I n the present paper we wish to describe the identification of minor sterols and the composition of sterols isolated from a physogastrique queen, workers, soldiers, and a mixture of all castes and developmental stages. MATERIALS AND METHODS The investigations were performed with the termite Nasutiterraes rippertii, abundantly occurring in Cuba and living in carton nests. Ten kg. of termites (a mixture of all castes and developmental stages imported from Cuba) fixed with ethanol immediately after collecting were finely ground in an atomizer and the resulting slurry filtered. The material on the filter was kept in ethanol overnight and filtered again. The filtrates were combined and concentrated under diminished pressure to the volume of zl. The concentrate was diluted with zl. of water and extracted with four 5oo-ml. portions of methylene chloride. The extracts were dried over sodium sulphate and taken down on a rotary evaporator. The residue (35o g.) was diluted with zSO ml. of benzene and chromatographed on 4 kg. of silica gel (partially deactivated with x5 per cent water). Hydrocarbons and lipids (28o g.) were removed by elution with benzene; elution with ether afforded the more polar compounds along with sterols (3o g.), and elution with ethanol yielded the most polar components (I5 g.). The ethereal eluate was repeatedly rechromatographed on a column of silica gel with the use of benzene containing from x to Io per cent ether as eluant. The sterol-containing (i.8 g.) fractions (as determined by thin-layer chromatography on silica gel in 9 : i benzene/ether) were combined and used for the GLC-MS analysis. The analysis of sterols in particular casts was performed as follows: the insect (I physogastrique queen or zoo workers or zoo soldiers) was frozen in liquid nitrogen, powdered, and the powder extracted with methylene chloride. The extractive substances were separated by preparative thin-layer chromatography on silica gel, in 9 : i benzene-ether. The band corresponding to sterols was removed and eluted with ethyl acetate to yield the free sterols. The free sterols isolated in this way were analysed in a combined GLC-MS system with the use of the following apparatus : m

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1. Finnigan 3000, electron energy 70 eV, source temperature 220 ° C, glass column, 2 mm. diameter and z'5 m. length, 3 per cent OV I, temperature 24 °0 C, isothermal. 2 . PYE I04-AEI M S 902, electron energy 70 eV, source temperature 220 ° C, glass column, 4"2 mm. diameter, 1"5 m. length, 3 per cent OV I, programme 2IO °, I ° C per min up to 25 o° C, and a glass column of the same size, 3 per cent SE 30, programme 2oo ° C, i ° C per rain. For an easier identification of the minor sterols C19 to Ca2 with a short chain or without any chain, the mixture of sterols was repeatedly chromatographed on a column of silica gel under the above conditions. T h e enriched minor sterols were eluted from the column in the last chromatographic fractions. T h e mixture of minor sterols enriched in this manner was then measured in a G L C - M S system (PYE I o 4 - A E I M S 902) under the above conditions, except for the use of an SE 30 column (programme temperature above 18o ° C per minute). In this system the C19 to C,2 sterols were separated in such a manner that the chromatographic record showed an approximately 15 per cent ' valley' between the saturated and unsaturated sterols of the same chain-length. RESULTS AND DISCUSSION Fig. I shows a t y p ic a l r e c o r d f r o m t h e gas c h r o m a t o g r a p h . T h e h e t e r o g e n e o u s n a t u r e o f s o m e c h r o m a t o g r a p h i c peaks was d e m o n s t r a t e d b y r e c o r d i n g a set of mass sp ect r a

9

J 1 0

I 30

I 20

TIME

I 10

I 0

(minutes)

FIC. x . - - A typical G L C record of the mixture of sterols (OV i, programme 21o ° C, 1o C per min).

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from the same peak. The successive determinations of mass spectra made it possible to identify the minor sterols which would otherwise remain undetermined. Peak I consists of a mixture of androst-5-en-3fl-ol, M + 274; m/e 259 (M-IS); m/e 256 (M-I8); m/e 241 (M-Is-IS), base peak; m/e i45 , x63, I89; and 5~-androstan-3fl-ol, M + 276, base peak; m/e 261 (M-Is); m/e 258 (M-IS); m/e 243 (M-Is-IS); m/e 95, 2o4. The combined spectrum of the two compounds is in accordance with spectra of authentic androst-5-en-3fl-ol and 5a-androstan-3fl-ol, but it is not possible to exclude the simultaneous presence of 5fl-androstan-3fl-ol. Peak 2 contains pregn-5-en-3fl-ol, M + 3o2; m/e 287 (M-IS); m/e 284 (M-IS); m/e I97, base peak, 213 ; the spectrum is in accordance with that of an authentic specimen. Peak 3 consists of a mixture of I7¢-isopropylandrost-5-en-3fl-ol, M + 316; m/e 3Ol (M-15); m/e 298 (M-18); m/e 231 (M-side chain-42); m/e 2i 3 (23 I-iS); m/e 43(C3H7), base peak; and i7~-isopropy1-5¢-androstan-3fl-o1 , M + 3 I8; m/e 3o3(M -I 5); m/e 300 (M-18); m/e 233 (M-side chain-42); m/e 2i 5 (233-i8); m/e 43 (CaH~), base peak. The fragmentation of these two compounds is in accordance with that reported for sterols carrying a side chain (Friedland et al., i959; Knights, 1967). To be sure that the reported minor components (Peaks 1-3) are actually formed by the termites and that they are not artifacts formed from the decomposition of sterol hydroperoxides (van Lier and Smith, i97o), a small sample was provided using the same procedure as above, except that all steps were carried out in an argon atmosphere. The remaining peaks exhibit fragmentations of As- and A¢-3fl-hydroxysterols and their saturated derivatives; the side chain is of varying length and contains a double bond in some cases. Table i shows compounds identified in the mixture of all castes and developmental stages, on comparison of mass spectra obtained from particular peaks with fragmentations of known compounds (Friedland et aL, i959; Clark-Lewis and Dainis, I967; Galli and Maroni, i967; Knights, i967; Wyllie and Djerassi, i968; Hutchins et al., 197o). Table

I . - - T H E COMPOSITION OF FREE STEROLS IN THE MIXTURE OF ALL CASTES AND DEVELOP. MENTAL STAGES

PEAK

RELATIVE ABUNDANCE

I

O'7

2 3

o.8 I'I

4 5

7"i 55"6

6 7

x2"9 5"5

8

3"7

9

8"9

xo

3'7

STRUCTURE

Androst-5-en-3~-ol 5 ~:-Androstan-3~-ol Pregn-5-en-3fl-ol x7 ~-Isopropylandrost-s-en-3~-ol x7~-Isopropyl-5 ~:-androstan-3~-ol Cholest-5,zz-dien-3~-ol Cholesterol Cholestanol 24-Methylcholestan-5,zz-dien-3~-ol 24-Methylcholest-5-en-3]~-ol 24-Methylcholestan-3fl-ol

24-Ethylcholest-7,X-dien-3fl-o1 24-Ethylcholest-7-en-3~-o1 24-Ethylcholest-5-en-3]~-ol 24-Ethylcholestan-3]~-ol Unidentified sterol

RETENTIONTIME MOL.WT. OF (w.r.t. choleTHE STEROL sterol) o'x7 ~. f "~ f "~. I

o'23 o'39 o'92 z'o I'O7 1"2o i .28 i "39 1'46

274 276 3o2 316 318 384 386 388 398 4o0 4o2 412 414 414 416 4x4

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284

Table 2.--PROPORTION OF STEROLS IN PARTICULARCASTES RELATIVE ABUNDANCE STRUCTURE

Cholesta-5,22-dien-3fl-ol Cholesterol Cholestanol 24-Methylcholesta-5,22-dien-3fl-ol 24-Methylcholest-5-en-3fl-ol 24-Methylcholestan-3fl-ol 24-Ethylcholesta-7,X-dien- 3fl-ol 24-Ethylcholest-7-en-3fl-ol 24-Ethylcholest-5-en-3fl-ol 24-Ethylcholestan-3fl-ol Unidentified sterol

"~

~.

Workers

Soldiers

Physogastrique Queen

2"0 77"5

x I'5 59"5

9"O 57"5

4"o

13"5

I6"5

2"5 2"5

i.o 1"5

3"5 3"5

io.o

i x.5

8"5

1"5

x'5

1"5

Table 2 shows the composition of sterols in particular castes, but the identification of the C19, C~1, and C2z sterols in the gas-chromatographic region is not quite conclusive, because these sterols are accompanied by some interfering substances, the removal of which by thin-layer chromatography on silica gel alone was not satisfactory. The metabolism of steroids in insects has been described in numerous papers and several reviews (Clayton, 1964; Gilbert, 1967; Ritter and Wientjens, 1967; Thompson et al., i972 ). It has been shown that with insects which are not able to effect the de novo biosyntheses of the steroidal system, an external source of sterols is required for the normal growth, metamorphosis, and reproduction. The sterols act as structural components of cells and tissues and also serve as precursors of the fundamental steroidal metabolites and regulators. In spite of the requirement for specific sterol structures, some insect species are capable of modifying the dietetic sterols to structures required for specific physiological functions; this modification consists of the degradation of carbon atoms C-28 and C-29 of plant sterols and in the subsequent oxidative transformations. The steroidal compounds usually occurring in insects, together with the polyhydroxyketosteroidal moulting hormones, contain a side chain of the cholestane type. In this connexion it is of interest to mention the finding of C19, C~1, and C2~ sterols, containing the system of a 3fl-hydroxy-AS-sterol or the corresponding dihydro system, i.e., compounds the side chain of which is shortened to an isopropyl and ethyl group or is quite absent. The specific requirements of termites for sterols have not been hitherto systematically investigated, but it may be assumed that the fundamental sterol of animals, the cholesterol, is produced in termites by metabolism of phytosterols supplied by food. In spite of the ability of termites to biosynthesise de novo the terpenoids for the purpose of communication and defence (Moore, I969; Vrko6, unpublished results), there is no reason to assume that termites, in contrast to other groups of insects, could be able to effect the biosynthesis of fundamental sterols from acyclic precursors. Additional investigations would be required to make clear whether the sterols with a shortened side chain are of any physiological importance for the termites. It must be born in mind that in the intestinal tract of Nasutitermes rippertii, as in other Nasutitermitinae (Hrd)~, un-

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published results), there exist some symbionts the potential relation of which to sterols deserves elucidation. T h e C19 and C~1 steroids which have been hitherto isolated (Schildknecht, i97o ) from some insects are identical with steroidal hormones of vertebrates and, to the present knowledge, are supposed to serve only as defensive substances against poikilothermic vertebrates. REFERENCES CLARK-LEwis J. W. and DAINISI. (1967) The phytosterols from Acacia species. Aust. ~. Chem. ~o, 1961-1974. CLAYTONR. B. (1964) The utilisation of sterols by insects, ft. Lipid Res. 5, 3-19 • FRIEDLANDS. S., LANEG. H. J., LONGMANR. T., TRAINK. E., and O'NEALM. J., jun. (1959) Mass spectra of steroids. Analyt. Chem. 31, 169-174. GALLI G. and MARONI S. (1967) Mass spectrometric investigation of some unsaturated sterols biosynthetically related to eho]esterol. Steroids Io, 189-197. GILBERT L. I. (1967) Lipid metabolism and function in insects. Adv. Insect Physiol. 4, 69-21 i. HUTCHINS R. F. N., THOMPSONM. J., and SVOBODAJ. A. (197o) The synthesis and the mass and nuclear magnetic resonance spectra of side chain isomers of cholesta-5,22-dien-3]~-oland cholesta-5,22,24-trian-3]~-ol. Steroids I5, 113-13o. KNIGHT B. A. (1967) Identification of plant sterols using combined GLC/mass spectrometry. J. Gas Chromat. 5, 273-281. MooRE B. P. (1969) Biology of Termites (ed. Krishna and Weesner) vol. i, p. 4o7. New York:

Academic Press. RITTER F. J. and WIENTJEN W. H. J. M. (1967) Sterol metabolism in insects. T N O Nieuws 22, 381-392 . SemLDKNEeHTH. (197o) Arthropod protective substances. 4o. Defensive chemistry of land and water beetles. Agnew. Chem. Int. Edn. Engl. 9, 1-9. THOMPSONM. J., SVOBODAJ. A., KAPLANISJ. N., and ROBBINSW. E. (1972) Metabolic pathways of steroids in insects. Proc. R. Soc. (B) I8O, 2o3-221. VAN Lma J. E. and SMITH L. L. (I97O) Autooxidation of cholesterol via hydroperoxide intermediates, ft. org. Chem. 35, 2627-263 I. VRKO~J., UBIK K., DOLEJ§L., and HRD¢/ I. (1973) On the chemical composition of frontal gland secretion in termites of the genus Nasutitermes (N.costalis and N. rippertii; Isoptera). Acta ent. Bohemoslov. 7o, 74-8o. WYLLIE S. G. and DJERASSIC. (1968) Mass spectrometry and stereochemical problems. CXLVI. Mass spectrometric fragmentations typical of sterols with unsaturated side chains. J. org. Chem. 33,

305-313 •

Key Word Index: Nasutitermes rippertii, sterols, GLC/MS.