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Comparative sterol composition of adult female Xyleborus ferrugineus and its mutualistic fungal ectosymbionts
Comp. Biochem.Physiol., 1973, VoL 44II, PP. 499 to 505. PergamonPress.Printedin Great Britain
COMPARATIVE STEROL C O M P O S I T I O N S OF A D U L T FEMALE X Y L E B O R U S FERRUGINEUS AND ITS M U T U A L I S T I C F U N G A L ECTOSYMBIONTS* L O K E T U C K K O K t and D A L E M. N O R R I S Department of Entomology, University of Wisconsin, Madison, Wisconsin 53706 (Received 22 May 1972) A b s t r a c t - - 1 . Sterol compositions of adult female Xyleborus ferrugineu s beetle
and its fungal ectosymbionts, Fusarium solani, Cephalosporium sp. and Graphium sp., were analyzed and compared. 2. Ergosterol was the sole sterol in all three fungi and amounted to 0.12 per cent in Graphium sp., 0"16 per cent in Cephalosporium sp. and 0"24 per cent in F. solani (dry weight basis). 3. /g-Sitosterol (64 per cent) and cholesterol (22 per cent) were the principal sterols in S. ferrugineus; sterol content was 0.05 per cent of the fresh weight of the beetle. 4. Ergosterol, naturally provided by the symbiotic ambrosial fungi, is a critical nutrient to the beetle. INTRODUCTION BASED on numerous studies of the roles of sterols in insect nutrition and development, insects are unable to synthesize these chemicals. T h e only known exceptions had been certain insects with microbial symbionts, but when these are rendered aposymbiotic they also require an exogenous sterol source. Sterols serve as precursors for essential steroid metabolites, such as certain hormones, and as structural components of cells. Our recent studies have established unusual sterol requirements in the ambrosia beetle, Xyleborus ferrugineus, and the apparent sterol-providing role of its fungal ectosymbionts. Norris & Baker (1967) showed that a fungus was required for the reproduction of the beetle, and Baker & Norris (1968) found three fungi, Fusarium solani, Cephalosporium sp. and C-raphium sp., mutualistically involved in its nutrition. In the absence of mutalistic fungi, second brood pupation of X . ferrugineus failed except when ergosterol, the major sterol of fungi, was substituted for cholesterol in the diet (Norris et al., 1969). Subsequent tests revealed that ergosterol or 7-dehydrocholesterol was adequate as the sole sterol source for the continued growth and reproduction of the fungus-free beetle (Chu et al., 1970; *This research was supported by the College of Agricultural and Life Sciences, University of Wisconsin, Madison, Wisconsin; and in part by funds from the Schoenleber Foundation and the Wisconsin Department of Natural Resources. tPresent address: Department of Entomology, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061. 499
500
LOKETUCKKOKANDDALEM. NORRIS
Kok et al., 1970). Because only one species of insect, Drosophila pachea, has previously been reported to require a sterol source other than cholesterol (Heed & Kircher, 1965), the demonstrated sterol requirements of X. ferrugineus appear truly unusual. We hypothesized that such unusual sterol requirements were met by the fungal symbiotes. Comparative studies of sterols in scolytid beetles and their fungal symbionts have not been previously reported. This paper reports our findings on similarities and differences in sterols present in the three ectosymbiotic fungi vs. the beetle. MATERIALS AND METHODS The technique for the culture of the ambrosial fungi and the beetle, and their lipid extraction based on Folch et al. (1957) were similar to that described in our phospholipid study (Kok & Norris, 1972). The chloroform effluent containing the neutral lipids was reduced in volume by rotary-evaporation under vacuum and the sterols isolated by precipitation and subsequent splitting of digitonides by the methods of Sperry (1963). The isolated sterols were weighed and further separated by GLC and TLC,* and structurally checked by mass spectrometry.
Chromatographic methods T L C was carried out on plates 200 x 200 mm coated with 0"25 mm Adsorbosil (Applied Science Labs., Inc.) impregnated with 5% AgNO3, and developed with CHC13-acetone (95 : 5). Sterol constituents were detected by 50% H2SO4 spray and also by u.v. light. Solutions of free sterols for GLC were prepared by dissolving 5 mg sterols/ml CHC13, and of trimethylsilyl ethers by reacting 5 mg sterols/ml of Tri-Sil (Pierce Chemical Co.). GLC was carried out with a Packard 5750 gas chromatograph, equipped with dual hydrogen flame detectors and nitrogen as carrier gas. Columns were 6 ft x 2 mm i.d. coiled stainless steel packed with 1% OV-17 coated on 100/120 mesh Gas-Chrom Q (Applied Science Labs., Inc.) or 1% QF-1 (Dow Chem. Co.) on 60/80 mesh Gas-Chrom Q. Identification of samples was by comparison against authentic knowns.
RESULTS TLC and GLC retention times of isolated samples and authentic stero]s are summarized in Tables I and 2, respectively. Only one stero], ergosterol, was detected in the three ambrosial fungi. F. solani contained significantly (at the 0.01 level) larger ergosterol content than the other two species. Yields of the three fungi, on dry-weight basis, are shown in Table 3. T h e sterol content after 15 days of incubation was 0.12 per cent in Graphium sp., 0.16 per cent in Cephalosporium sp. and 0.24 per cent in F. solani. Besides quantitative differences between species, the sterol content in F. solani also increased with the age of mycelia. Five-day-old mycelia yielded 0.17 per cent, increasing to 0.20 per cent and 0.24 per cent at 10 and 15 days, respectively. T h e amount in 15-day-old fungus was significantly greater than that in 5-day-old fungus; but differences between 5- and 10-day-old, and between 10- and 15-day-old fungus were not significant at the 0.05 per cent level. *Abbreviations: TLC, thin-layer chromatography; GLC, gas-liquid chromatography; r.h., relative humidity; CHCla, chloroform; H2SO4, sulphuric acid; RI, retention time.
STEROL COMPOSITIONS OF FEMALE X Y L E B O R U S
FERRUGINEUS
501
T A B L E 1 - - O N E - D I M E N S I O N A L THIN-LAYER CHROMATOGRAPHY* OF STEROLS OF AMBROSIAL FUNGI AND THE FEMALE ADULT
Jyleborus ferrugineus BEETLE SHOWING R.,. VALUE AS COMPARED W I T H AUTHENTIC STANDARDS
Sterol Authentic standards Cholesterol 7-Dehydrocholesterol Di-hydrocholesterol Ergosterol Lanosterol fl-Sitosterol Stigmasterol Ambrosial fungal sterols F. solani sterol Cephalosporium sp. sterol Graphium sp. sterol Ambrosia beetle (female adults) sterols X. f errugineus sterols
R/value 0"44 0"14 0"52 0.12 0"67 0"44 0"42 0"12 0"12 0"12 0"32 0"37 0-44
*Adsorbent, Adsorbosil-5% AgNO3 (Applied Science Labs., Inc.); solvent system, chloroform-acetone (95 : 5) ; and glass plates, 200 x 200 mm coated with 0-25 mm adsorbent. In X. ferrugineus, the sterol fraction was 15 mg/30 g of beetle (0.05 per cent of fresh weight). T L C indicated three major spots in the beetle fraction; the largest corresponding to that of fl-sitosterol or cholesterol, with R 1 x 100 of 44 Two minor spots with smaller/71 values, 37 and 32, did not correspond to any of the authentic sterols used (Table 1). Subsequent G L C revealed four sterol peaks from the OV-17 column, but only three from the QF-1 column (Table 2). The principal beetle sterol was fl-sitosterol (64 per cent) and a second one was cholesterol (22 per cent); these together accounted for 86 per cent of the sterol fraction. The remaining 14 per cent consisted of two unidentified steroidal compounds, one 2 per cent and the other 12 per cent. The latter might possibly be desmosterol although this was not definitely identified. DISCUSSION In this study, ergosterol was the only detectable sterol in the three ambrosial fungi. The same sole sterol was also found in three species of Fusarium (Fiore, 1948), fourteen species of Basidiomycetes (Milazzo, 1965) and Ceratocystis fagacearum (Collins & Kalnins, 1969). However, more than one sterol was found in several species of Saprolegniales, Leptomitales and Mucorales (McCorkindale et al., 1969); in the fungal symbiont of the attine ant (Martin et al., 1969); and in the fungus gardens of the termite (Cmelik & Douglas, 1970).
502
LOKB TUCK KOK AND DALE M. Nom~is
T A B L E 2 - - G A S - - L I Q U I D CHROMATOGRAPHY* OF TRIMETHYLSILYL ETHER DERIVATIVES OF STEROLS OF AMBROSIAL FUNGI AND TH.E FEMALE ADULT X. feYrugineus BEETLE AS COMPARED W I T H AUTHENTIC STANDARDS (ALL RETENTION TIMES RELATIVE TO CHOLESTANE, INTERNAL STANDARD, 1.00)
Column Sterol Authentic standards Cholesterol 7-Dehydrocholesterol Dihydrocholesterol Ergosterol fl-Sitosterol Stigmasterol Ambrosial fungal sterols
F. solanisterol Cephalosporium sp. sterol Graphium sp. sterol Ambrosia beetle (female adults) sterols X. ferrugineus sterols
OV-17t
QF-I$
2.75 3"29 2.69 3.71 4.84 3.82
2.86 3"50 2.80 4.14 5.00 4.46
3.71 3"70 3"71
4"14 4' 13 4"13
2"75 3'25 3"87 4"84
2"86 3"87 5"00
*Packard gas chromatograph Series 5750 with dual hydrogen-flame detectors. ~'Stainless steel column 6 f t x 2 m m i.d., 1% OV-17 on 100-120 mesh GasChrom Q, 50 Ib/in 2 nitrogen, 40 ml/min and 230°C temperature. Detector, 310°C; injection port, 285°C; and attenuation, l0 s x 4. Cholestane retention time, 3"2 min. :~Stainless steel column 6 f t x 2 m m i.d., 1% QF-1 on 60-80 mesh Gas-Chrom Q, 50 lb/in 2 nitrogen, 60 ml/min and 200°C temperature. Detector, 290°C; injection port, 275°C; and attenuation, 102 x 4. Cholestane retention time, 1-4 min. T A B L E 3 - - E R G O S T E R O L CONTENT* OF AMBROSIAL FUNGI CULTURED IN 100-ml NEUTRAL-DOXYEAST MEDIUM t PER 5 0 0 - m l FLASK INCUBATED UP TO 1 5 days AT 2 8 ° C AND 70% r . h . IN DARKNESS~ EXPRESSED ON DRY-WEIGHT BASIS
Species and age
Mg/15 g
Percentage
25-7+_2.4 29-9 +_4.8 35"6+-5"7
0.17+_0"013 0.20 _+0"03 0.24_+0"04
23"3+_4"3
0"16+_0"03
18"2 +_1"4
0"12 _+0"007
F. solani 5 days old 10 days old 15 daysold
Cephalosporium sp. 15 days old
Graphium sp. 15 days old
* Mean of three replications + standard error. J- See Norris & Baker (1969).
STEROL C O M P O S I T I O N S OF FEMALE X Y L E B O R U S
FERRUGINEUS
503
Sterol yields of 0.12-0.24 per cent of dry fungal weight in the three ambrosial fungi were at the lower end of the 0.1-1-0 per cent range (also obtained by the digitonide method) of sterol contents reported by Pruess et al. (1931) for a number of yeasts, molds and mushrooms. They were more comparable to values found in the Saprolegniales (0.01-0.25 per cent), but were higher than those of the Leptomitales (0.01-0-05 per cent) and Mucorales (0.005-0.025 per cent) (McCorkindale et al., 1969). The ergosterol content of 14 Basidiomycetes ranged from 0-0170.42 per cent (Milazzo, 1965), but dermatophytes had a wider range of 0.016-1-5 per cent (Blank et al., 1962). Other sterol contents determined by colorimetric method included 0-56 per cent in F. lycopersici and up to 1 per cent in F. lini (Fiore, 1948); 0.18 per cent in F. rasinfectum, 0.22 per cent in Cephalosporium sp. and 0.07-0.84 per cent in a number of yeasts (Appleton et al., 1955). The mean levels of ergosterol in F. solani (0.17-0.24 per cent) and the other two mutualistic ectosymbiotic fungi, 0.12 per cent in Graphium sp. and 0.16 per cent in Cephalosporium sp., were above those generally required (0.01-0.1 per cent) in the diets of insects (Clayton, 1964). Thus, in all three ambrosial fungi, ergosterol was present in an amount which would appear adequate for the growth and development of the beetle. The beetle naturally feeds on young hyphal tips and spores of the ambrosial fungi. Direct comparison of sterol content between X. ferrugineus and other Scolytidae cannot be made because of the absence of previous records of sterols in scolytid beetles. The mean 0.63/~g of sterol per X.ferrugineus female adult was within the range (0.15-1.0/zg/mg of fresh weight) detected in tissues of Periplaneta americana (Casida et al., 1957), but lower than the 1 mg/g ofwetweight found inthe larvae of Neodiprion pratti (Schaefer et al., 1965) and the 3.25 /~g/mg reported from the nerve of Eurycotis floridana (Clayton & Edwards, 1961 ; Clayton, 1964). /~-Sitosterol was the major sterol in X. ferrugineus. This was similarly reported in Leptinotarsa decemlineata (Schreiber et al., 1961) and "Cantharides Russian" (Marker & Shabica, 1940). Among other coleopterans studied, cholesterol and 7-dehydrocholesterol were found to be major sterols in Tribolium confusum (Beck & Kapadia, 1957), although Levinson (1962) reported cholesterol and/~-sitosterol in the same beetle, and also in Phaedon cochleariae and Tenebrio molitor. The differences in sterols isolated from T. confusum apparently can be attributed to the dietary sterol fed to the insect. The large proportion of ~-sitosterol in X. ferrugineus could be at least partially attributed to an accumulation from the diet. Three of the ingredients (elm sawdust, wheat germ and corn-oil) in the oligidic diet contained ~-sitosterol. Air-dried sawdust, a major component of the diet, has a 0.018 per cent ~-sitosterol content (22 mg/120 g). The presence of cholesterol in X. ferrugineus also is probably, at least partially, the result of de-ethylation of the fl-sitosterol side-chain at C24. However, the capacity to dehydrogenate fl-sitosterol to its corresponding 5,7' diene appears limited because of the high fl-sitosterol content remaining in the beetle tissue. The only other major sterol available to the beetle, ergosterol, was derived from the mutualistic fungi growing in the oligidic diet. The absence of ergosterol
504
LOKE TUCK KOK AND DALE M. NORRIS
in the beetle tissues indicates that it is efficiently utilized and rapidly converted to cholesterol and/or other metabolites. Nutritional studies have shown that fungus-free beetles utilized either ergosterol or 7-dehydrocholesterol for pupation (Chu et al., 1970; Kok et al., 1970). In the presence of mutualistic ectosymbiotic fungi, X. ferrugineus developed rapidly without any other exogenous sterol source. Thus, it is highly significant that only ergosterol was detected in all three ambrosial fungi. This strongly indicates that ergosterol which is provided by the ambrosial fungi is a critical nutrient to the beetle, and this dependence establishes a major basis for the existing mutualistic ectosymbiosis. Acknowledgements--The authors would like to thank Professor Barry M. Trost, Department of Chemistry, University of Wisconsin, Madison, for his co-operation in performing the mass spectrometry. REFERENCES APPLETON G. S., KIEBER R. J. & PAYNE W. J. (1955) T h e sterol content of f u n g i - - I I . Screening of representative yeasts and molds for sterol content. Appl. Microbiol. 3, 249-251. BAKER J. M. & NORRIS D. M. (1968) A complex of fungi mutualistically involved in the nutrition of the ambrosia beetle Xyleborus ferrugineus. 07. invert. Pathol. 11, 246-250. BECK S. D. & KAPADIAA. G. (1957) Insect nutrition and metabolism of sterols. Science 126, 258-259. BLANK F., SHORTLANDF. E. & JUST G. (1962) The free sterols of dermatophytes. J. Invest. Dermatol. 39, 91-94. CASIDAJ. E., BECK S. D. & COLE M. J. (1957) Sterol metabolism in the American cockroach. 07. biol. Chem. 224, 365-371. CHU H. M., NORRIS D. M. & KOK L. T. (1970) Pupation requirement of the beetle, Xyleborusferrugineus: sterols other than cholesterol. 07. Insect Physiol. 16, 1379-1387. CLAYTON R. B. (1964) T h e utilization of sterols by insects. 07. Lipid Res. 5, 3-19. CLAYTON R. B. & EDWARDSA. M. (1961) T h e essential cholesterol requirement of the roach, Eurycotis floridana. Biochem. biophys. Res. Commun. 6, 281-284. CMELIK S. H. W. & DOUGLASC. C. (1970) Chemical composition of "fungus gardens" from two species of termites. Comp. Biochem. Physiol. 36, 493-502. COLLINS R. P. & KALNINS K. (1969) T h e occurrence of ergosterol in the fungus Ceratocystis fagacearum. Mycologia LXI, 645-646. FIORE J. V. (1948) On the mechanism of enzyme a c t i o n - - X X X I I . Fat and sterol in Fusarium lini Bolley, Fusarium lycopersici and Fusarium solani D2 Purple. Archs Biochem. 16, 161-168. FOLCH J., LEES M. & SLOANE-STANLEYG. H. (1957) A simple method for the isolation and purification of total lipids from animal tissues. 07. biol. Chem. 226, 497-509. HEED W. B. & KIRCHER H. W. (1965) Unique sterol in the ecology and nutrition of Drosophila pachea. Science 149, 758-761. KOK L. T. & NORRIS D. M. (1972) Comparative phospholipid compositions of adult female Xyleborus ferrugineus and its mutualistic fungal ectosymbionts. Comp. Biochem. Physiol. 42B, 245-254. KOK L. T., NORRIS D. M. & CHU H. M. (1970) Sterol metabolism as a basis for a mutualistic symbiosis. Nature, Lond. 225, 661-662. LEVlNSON Z. H. (1962) The functions of dietary sterols in phytophagous insects..7. Insect Physiol. 8, 191-198.
STEROL COMPOSITIONS OF FEMALE X Y L E B O R U S F E R R U G I N E U S
505
McCoRKINDALE N. J., HUTCHINSON S. A., PURSEYB. A., SCOTT W. T. & WHEELER R. (1969) A comparison of the types of sterol found in species of the Saprolegniales and Leptomitales with those found in some other Phycomycetes. Phytochemistry 8, 861-867. MARKER R. E. ~ SHABICA A. C. (1940) S t e r o l s - - X C I X . Sterols from various sources. 07. Am. Chem. Soc. 62, 2523-2525. MARTIN M. M., CARMAN R. C. & McCoNNELL J. G. (1969) Nutrients derived from the fungus cultured by the fungus-growing ant Atta colombica ton@es. Ann. ent. Soc. Am. 62, 11-13. MILAZZO F. H. (1965) Sterol production by some wood-rotting Basidiomycetes. Can. 07. Bot. 43, 1347-1353. NORRIS D. M. & BAI~R J. M. (1967) Symbiosis: effects of a mutualistic fungus upon the growth and reproduction of Xyleborus ferrugineus. Science 156, 1120-1122. NORRIS D. M. & BAKFJ~J. M. (1969) Nutrition of Xyleborusferrugineus--I. Ethanol in diets as a tunneling (feeding) stimulant. Ann. ent. Soc. Am. 62, 592-594. NORRIS D. M., BAm~RJ. M. & CHU H. M. (1969) Symbiontic inter-relationships between microbes and ambrosia b e e t l e s - - I I I . Ergosterol as the source of sterol to the insects. Arm. ent. Soe. Am. 62, 413-414. PRt~SS L. M., Pm'ERSON W. H., STE~'qBOCKH. & FRED E. B. (1931) Sterol content and antirachitic activatibility of mold mycelia. 07. biol. Chem. 90, 369-384. SCHA~ER C. H., KAPLANISJ. N. & ROBBINS W. E. (1965) The relationship of the sterols of the Virginia pine sawfly, Neodiprion pratti Dyar, to those of two host plants, Pinus virginiana Mill and Pinus rigida Mill. 07. Insect Physiol. 11, 1013-1021. SCHm~IBER K., Ossm~ G. & SEMBDN~R G. (1961) Identifizierung von ~-sitosterin als Hauptsterin des Kartoffelkafers (Leptinotarsa decemlineata Say). Experientia 17, 463-464. SPEm~Y W. M. (1963) Quantitative isolation of sterols. 07. Lipid Res. 4, 221-225.
Key Word Index--Ectosymbiosis, lipids, ambrosial fungi, ambrosia beetle; mutualistic fungi; sterol composition in scolytid beetle and fungal symbionts.
COMPARATIVE STEROL C O M P O S I T I O N S OF A D U L T FEMALE X Y L E B O R U S FERRUGINEUS AND ITS M U T U A L I S T I C F U N G A L ECTOSYMBIONTS* L O K E T U C K K O K t and D A L E M. N O R R I S Department of Entomology, University of Wisconsin, Madison, Wisconsin 53706 (Received 22 May 1972) A b s t r a c t - - 1 . Sterol compositions of adult female Xyleborus ferrugineu s beetle
and its fungal ectosymbionts, Fusarium solani, Cephalosporium sp. and Graphium sp., were analyzed and compared. 2. Ergosterol was the sole sterol in all three fungi and amounted to 0.12 per cent in Graphium sp., 0"16 per cent in Cephalosporium sp. and 0"24 per cent in F. solani (dry weight basis). 3. /g-Sitosterol (64 per cent) and cholesterol (22 per cent) were the principal sterols in S. ferrugineus; sterol content was 0.05 per cent of the fresh weight of the beetle. 4. Ergosterol, naturally provided by the symbiotic ambrosial fungi, is a critical nutrient to the beetle. INTRODUCTION BASED on numerous studies of the roles of sterols in insect nutrition and development, insects are unable to synthesize these chemicals. T h e only known exceptions had been certain insects with microbial symbionts, but when these are rendered aposymbiotic they also require an exogenous sterol source. Sterols serve as precursors for essential steroid metabolites, such as certain hormones, and as structural components of cells. Our recent studies have established unusual sterol requirements in the ambrosia beetle, Xyleborus ferrugineus, and the apparent sterol-providing role of its fungal ectosymbionts. Norris & Baker (1967) showed that a fungus was required for the reproduction of the beetle, and Baker & Norris (1968) found three fungi, Fusarium solani, Cephalosporium sp. and C-raphium sp., mutualistically involved in its nutrition. In the absence of mutalistic fungi, second brood pupation of X . ferrugineus failed except when ergosterol, the major sterol of fungi, was substituted for cholesterol in the diet (Norris et al., 1969). Subsequent tests revealed that ergosterol or 7-dehydrocholesterol was adequate as the sole sterol source for the continued growth and reproduction of the fungus-free beetle (Chu et al., 1970; *This research was supported by the College of Agricultural and Life Sciences, University of Wisconsin, Madison, Wisconsin; and in part by funds from the Schoenleber Foundation and the Wisconsin Department of Natural Resources. tPresent address: Department of Entomology, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061. 499
500
LOKETUCKKOKANDDALEM. NORRIS
Kok et al., 1970). Because only one species of insect, Drosophila pachea, has previously been reported to require a sterol source other than cholesterol (Heed & Kircher, 1965), the demonstrated sterol requirements of X. ferrugineus appear truly unusual. We hypothesized that such unusual sterol requirements were met by the fungal symbiotes. Comparative studies of sterols in scolytid beetles and their fungal symbionts have not been previously reported. This paper reports our findings on similarities and differences in sterols present in the three ectosymbiotic fungi vs. the beetle. MATERIALS AND METHODS The technique for the culture of the ambrosial fungi and the beetle, and their lipid extraction based on Folch et al. (1957) were similar to that described in our phospholipid study (Kok & Norris, 1972). The chloroform effluent containing the neutral lipids was reduced in volume by rotary-evaporation under vacuum and the sterols isolated by precipitation and subsequent splitting of digitonides by the methods of Sperry (1963). The isolated sterols were weighed and further separated by GLC and TLC,* and structurally checked by mass spectrometry.
Chromatographic methods T L C was carried out on plates 200 x 200 mm coated with 0"25 mm Adsorbosil (Applied Science Labs., Inc.) impregnated with 5% AgNO3, and developed with CHC13-acetone (95 : 5). Sterol constituents were detected by 50% H2SO4 spray and also by u.v. light. Solutions of free sterols for GLC were prepared by dissolving 5 mg sterols/ml CHC13, and of trimethylsilyl ethers by reacting 5 mg sterols/ml of Tri-Sil (Pierce Chemical Co.). GLC was carried out with a Packard 5750 gas chromatograph, equipped with dual hydrogen flame detectors and nitrogen as carrier gas. Columns were 6 ft x 2 mm i.d. coiled stainless steel packed with 1% OV-17 coated on 100/120 mesh Gas-Chrom Q (Applied Science Labs., Inc.) or 1% QF-1 (Dow Chem. Co.) on 60/80 mesh Gas-Chrom Q. Identification of samples was by comparison against authentic knowns.
RESULTS TLC and GLC retention times of isolated samples and authentic stero]s are summarized in Tables I and 2, respectively. Only one stero], ergosterol, was detected in the three ambrosial fungi. F. solani contained significantly (at the 0.01 level) larger ergosterol content than the other two species. Yields of the three fungi, on dry-weight basis, are shown in Table 3. T h e sterol content after 15 days of incubation was 0.12 per cent in Graphium sp., 0.16 per cent in Cephalosporium sp. and 0.24 per cent in F. solani. Besides quantitative differences between species, the sterol content in F. solani also increased with the age of mycelia. Five-day-old mycelia yielded 0.17 per cent, increasing to 0.20 per cent and 0.24 per cent at 10 and 15 days, respectively. T h e amount in 15-day-old fungus was significantly greater than that in 5-day-old fungus; but differences between 5- and 10-day-old, and between 10- and 15-day-old fungus were not significant at the 0.05 per cent level. *Abbreviations: TLC, thin-layer chromatography; GLC, gas-liquid chromatography; r.h., relative humidity; CHCla, chloroform; H2SO4, sulphuric acid; RI, retention time.
STEROL COMPOSITIONS OF FEMALE X Y L E B O R U S
FERRUGINEUS
501
T A B L E 1 - - O N E - D I M E N S I O N A L THIN-LAYER CHROMATOGRAPHY* OF STEROLS OF AMBROSIAL FUNGI AND THE FEMALE ADULT
Jyleborus ferrugineus BEETLE SHOWING R.,. VALUE AS COMPARED W I T H AUTHENTIC STANDARDS
Sterol Authentic standards Cholesterol 7-Dehydrocholesterol Di-hydrocholesterol Ergosterol Lanosterol fl-Sitosterol Stigmasterol Ambrosial fungal sterols F. solani sterol Cephalosporium sp. sterol Graphium sp. sterol Ambrosia beetle (female adults) sterols X. f errugineus sterols
R/value 0"44 0"14 0"52 0.12 0"67 0"44 0"42 0"12 0"12 0"12 0"32 0"37 0-44
*Adsorbent, Adsorbosil-5% AgNO3 (Applied Science Labs., Inc.); solvent system, chloroform-acetone (95 : 5) ; and glass plates, 200 x 200 mm coated with 0-25 mm adsorbent. In X. ferrugineus, the sterol fraction was 15 mg/30 g of beetle (0.05 per cent of fresh weight). T L C indicated three major spots in the beetle fraction; the largest corresponding to that of fl-sitosterol or cholesterol, with R 1 x 100 of 44 Two minor spots with smaller/71 values, 37 and 32, did not correspond to any of the authentic sterols used (Table 1). Subsequent G L C revealed four sterol peaks from the OV-17 column, but only three from the QF-1 column (Table 2). The principal beetle sterol was fl-sitosterol (64 per cent) and a second one was cholesterol (22 per cent); these together accounted for 86 per cent of the sterol fraction. The remaining 14 per cent consisted of two unidentified steroidal compounds, one 2 per cent and the other 12 per cent. The latter might possibly be desmosterol although this was not definitely identified. DISCUSSION In this study, ergosterol was the only detectable sterol in the three ambrosial fungi. The same sole sterol was also found in three species of Fusarium (Fiore, 1948), fourteen species of Basidiomycetes (Milazzo, 1965) and Ceratocystis fagacearum (Collins & Kalnins, 1969). However, more than one sterol was found in several species of Saprolegniales, Leptomitales and Mucorales (McCorkindale et al., 1969); in the fungal symbiont of the attine ant (Martin et al., 1969); and in the fungus gardens of the termite (Cmelik & Douglas, 1970).
502
LOKB TUCK KOK AND DALE M. Nom~is
T A B L E 2 - - G A S - - L I Q U I D CHROMATOGRAPHY* OF TRIMETHYLSILYL ETHER DERIVATIVES OF STEROLS OF AMBROSIAL FUNGI AND TH.E FEMALE ADULT X. feYrugineus BEETLE AS COMPARED W I T H AUTHENTIC STANDARDS (ALL RETENTION TIMES RELATIVE TO CHOLESTANE, INTERNAL STANDARD, 1.00)
Column Sterol Authentic standards Cholesterol 7-Dehydrocholesterol Dihydrocholesterol Ergosterol fl-Sitosterol Stigmasterol Ambrosial fungal sterols
F. solanisterol Cephalosporium sp. sterol Graphium sp. sterol Ambrosia beetle (female adults) sterols X. ferrugineus sterols
OV-17t
QF-I$
2.75 3"29 2.69 3.71 4.84 3.82
2.86 3"50 2.80 4.14 5.00 4.46
3.71 3"70 3"71
4"14 4' 13 4"13
2"75 3'25 3"87 4"84
2"86 3"87 5"00
*Packard gas chromatograph Series 5750 with dual hydrogen-flame detectors. ~'Stainless steel column 6 f t x 2 m m i.d., 1% OV-17 on 100-120 mesh GasChrom Q, 50 Ib/in 2 nitrogen, 40 ml/min and 230°C temperature. Detector, 310°C; injection port, 285°C; and attenuation, l0 s x 4. Cholestane retention time, 3"2 min. :~Stainless steel column 6 f t x 2 m m i.d., 1% QF-1 on 60-80 mesh Gas-Chrom Q, 50 lb/in 2 nitrogen, 60 ml/min and 200°C temperature. Detector, 290°C; injection port, 275°C; and attenuation, 102 x 4. Cholestane retention time, 1-4 min. T A B L E 3 - - E R G O S T E R O L CONTENT* OF AMBROSIAL FUNGI CULTURED IN 100-ml NEUTRAL-DOXYEAST MEDIUM t PER 5 0 0 - m l FLASK INCUBATED UP TO 1 5 days AT 2 8 ° C AND 70% r . h . IN DARKNESS~ EXPRESSED ON DRY-WEIGHT BASIS
Species and age
Mg/15 g
Percentage
25-7+_2.4 29-9 +_4.8 35"6+-5"7
0.17+_0"013 0.20 _+0"03 0.24_+0"04
23"3+_4"3
0"16+_0"03
18"2 +_1"4
0"12 _+0"007
F. solani 5 days old 10 days old 15 daysold
Cephalosporium sp. 15 days old
Graphium sp. 15 days old
* Mean of three replications + standard error. J- See Norris & Baker (1969).
STEROL C O M P O S I T I O N S OF FEMALE X Y L E B O R U S
FERRUGINEUS
503
Sterol yields of 0.12-0.24 per cent of dry fungal weight in the three ambrosial fungi were at the lower end of the 0.1-1-0 per cent range (also obtained by the digitonide method) of sterol contents reported by Pruess et al. (1931) for a number of yeasts, molds and mushrooms. They were more comparable to values found in the Saprolegniales (0.01-0.25 per cent), but were higher than those of the Leptomitales (0.01-0-05 per cent) and Mucorales (0.005-0.025 per cent) (McCorkindale et al., 1969). The ergosterol content of 14 Basidiomycetes ranged from 0-0170.42 per cent (Milazzo, 1965), but dermatophytes had a wider range of 0.016-1-5 per cent (Blank et al., 1962). Other sterol contents determined by colorimetric method included 0-56 per cent in F. lycopersici and up to 1 per cent in F. lini (Fiore, 1948); 0.18 per cent in F. rasinfectum, 0.22 per cent in Cephalosporium sp. and 0.07-0.84 per cent in a number of yeasts (Appleton et al., 1955). The mean levels of ergosterol in F. solani (0.17-0.24 per cent) and the other two mutualistic ectosymbiotic fungi, 0.12 per cent in Graphium sp. and 0.16 per cent in Cephalosporium sp., were above those generally required (0.01-0.1 per cent) in the diets of insects (Clayton, 1964). Thus, in all three ambrosial fungi, ergosterol was present in an amount which would appear adequate for the growth and development of the beetle. The beetle naturally feeds on young hyphal tips and spores of the ambrosial fungi. Direct comparison of sterol content between X. ferrugineus and other Scolytidae cannot be made because of the absence of previous records of sterols in scolytid beetles. The mean 0.63/~g of sterol per X.ferrugineus female adult was within the range (0.15-1.0/zg/mg of fresh weight) detected in tissues of Periplaneta americana (Casida et al., 1957), but lower than the 1 mg/g ofwetweight found inthe larvae of Neodiprion pratti (Schaefer et al., 1965) and the 3.25 /~g/mg reported from the nerve of Eurycotis floridana (Clayton & Edwards, 1961 ; Clayton, 1964). /~-Sitosterol was the major sterol in X. ferrugineus. This was similarly reported in Leptinotarsa decemlineata (Schreiber et al., 1961) and "Cantharides Russian" (Marker & Shabica, 1940). Among other coleopterans studied, cholesterol and 7-dehydrocholesterol were found to be major sterols in Tribolium confusum (Beck & Kapadia, 1957), although Levinson (1962) reported cholesterol and/~-sitosterol in the same beetle, and also in Phaedon cochleariae and Tenebrio molitor. The differences in sterols isolated from T. confusum apparently can be attributed to the dietary sterol fed to the insect. The large proportion of ~-sitosterol in X. ferrugineus could be at least partially attributed to an accumulation from the diet. Three of the ingredients (elm sawdust, wheat germ and corn-oil) in the oligidic diet contained ~-sitosterol. Air-dried sawdust, a major component of the diet, has a 0.018 per cent ~-sitosterol content (22 mg/120 g). The presence of cholesterol in X. ferrugineus also is probably, at least partially, the result of de-ethylation of the fl-sitosterol side-chain at C24. However, the capacity to dehydrogenate fl-sitosterol to its corresponding 5,7' diene appears limited because of the high fl-sitosterol content remaining in the beetle tissue. The only other major sterol available to the beetle, ergosterol, was derived from the mutualistic fungi growing in the oligidic diet. The absence of ergosterol
504
LOKE TUCK KOK AND DALE M. NORRIS
in the beetle tissues indicates that it is efficiently utilized and rapidly converted to cholesterol and/or other metabolites. Nutritional studies have shown that fungus-free beetles utilized either ergosterol or 7-dehydrocholesterol for pupation (Chu et al., 1970; Kok et al., 1970). In the presence of mutualistic ectosymbiotic fungi, X. ferrugineus developed rapidly without any other exogenous sterol source. Thus, it is highly significant that only ergosterol was detected in all three ambrosial fungi. This strongly indicates that ergosterol which is provided by the ambrosial fungi is a critical nutrient to the beetle, and this dependence establishes a major basis for the existing mutualistic ectosymbiosis. Acknowledgements--The authors would like to thank Professor Barry M. Trost, Department of Chemistry, University of Wisconsin, Madison, for his co-operation in performing the mass spectrometry. REFERENCES APPLETON G. S., KIEBER R. J. & PAYNE W. J. (1955) T h e sterol content of f u n g i - - I I . Screening of representative yeasts and molds for sterol content. Appl. Microbiol. 3, 249-251. BAKER J. M. & NORRIS D. M. (1968) A complex of fungi mutualistically involved in the nutrition of the ambrosia beetle Xyleborus ferrugineus. 07. invert. Pathol. 11, 246-250. BECK S. D. & KAPADIAA. G. (1957) Insect nutrition and metabolism of sterols. Science 126, 258-259. BLANK F., SHORTLANDF. E. & JUST G. (1962) The free sterols of dermatophytes. J. Invest. Dermatol. 39, 91-94. CASIDAJ. E., BECK S. D. & COLE M. J. (1957) Sterol metabolism in the American cockroach. 07. biol. Chem. 224, 365-371. CHU H. M., NORRIS D. M. & KOK L. T. (1970) Pupation requirement of the beetle, Xyleborusferrugineus: sterols other than cholesterol. 07. Insect Physiol. 16, 1379-1387. CLAYTON R. B. (1964) T h e utilization of sterols by insects. 07. Lipid Res. 5, 3-19. CLAYTON R. B. & EDWARDSA. M. (1961) T h e essential cholesterol requirement of the roach, Eurycotis floridana. Biochem. biophys. Res. Commun. 6, 281-284. CMELIK S. H. W. & DOUGLASC. C. (1970) Chemical composition of "fungus gardens" from two species of termites. Comp. Biochem. Physiol. 36, 493-502. COLLINS R. P. & KALNINS K. (1969) T h e occurrence of ergosterol in the fungus Ceratocystis fagacearum. Mycologia LXI, 645-646. FIORE J. V. (1948) On the mechanism of enzyme a c t i o n - - X X X I I . Fat and sterol in Fusarium lini Bolley, Fusarium lycopersici and Fusarium solani D2 Purple. Archs Biochem. 16, 161-168. FOLCH J., LEES M. & SLOANE-STANLEYG. H. (1957) A simple method for the isolation and purification of total lipids from animal tissues. 07. biol. Chem. 226, 497-509. HEED W. B. & KIRCHER H. W. (1965) Unique sterol in the ecology and nutrition of Drosophila pachea. Science 149, 758-761. KOK L. T. & NORRIS D. M. (1972) Comparative phospholipid compositions of adult female Xyleborus ferrugineus and its mutualistic fungal ectosymbionts. Comp. Biochem. Physiol. 42B, 245-254. KOK L. T., NORRIS D. M. & CHU H. M. (1970) Sterol metabolism as a basis for a mutualistic symbiosis. Nature, Lond. 225, 661-662. LEVlNSON Z. H. (1962) The functions of dietary sterols in phytophagous insects..7. Insect Physiol. 8, 191-198.
STEROL COMPOSITIONS OF FEMALE X Y L E B O R U S F E R R U G I N E U S
505
McCoRKINDALE N. J., HUTCHINSON S. A., PURSEYB. A., SCOTT W. T. & WHEELER R. (1969) A comparison of the types of sterol found in species of the Saprolegniales and Leptomitales with those found in some other Phycomycetes. Phytochemistry 8, 861-867. MARKER R. E. ~ SHABICA A. C. (1940) S t e r o l s - - X C I X . Sterols from various sources. 07. Am. Chem. Soc. 62, 2523-2525. MARTIN M. M., CARMAN R. C. & McCoNNELL J. G. (1969) Nutrients derived from the fungus cultured by the fungus-growing ant Atta colombica ton@es. Ann. ent. Soc. Am. 62, 11-13. MILAZZO F. H. (1965) Sterol production by some wood-rotting Basidiomycetes. Can. 07. Bot. 43, 1347-1353. NORRIS D. M. & BAI~R J. M. (1967) Symbiosis: effects of a mutualistic fungus upon the growth and reproduction of Xyleborus ferrugineus. Science 156, 1120-1122. NORRIS D. M. & BAKFJ~J. M. (1969) Nutrition of Xyleborusferrugineus--I. Ethanol in diets as a tunneling (feeding) stimulant. Ann. ent. Soc. Am. 62, 592-594. NORRIS D. M., BAm~RJ. M. & CHU H. M. (1969) Symbiontic inter-relationships between microbes and ambrosia b e e t l e s - - I I I . Ergosterol as the source of sterol to the insects. Arm. ent. Soe. Am. 62, 413-414. PRt~SS L. M., Pm'ERSON W. H., STE~'qBOCKH. & FRED E. B. (1931) Sterol content and antirachitic activatibility of mold mycelia. 07. biol. Chem. 90, 369-384. SCHA~ER C. H., KAPLANISJ. N. & ROBBINS W. E. (1965) The relationship of the sterols of the Virginia pine sawfly, Neodiprion pratti Dyar, to those of two host plants, Pinus virginiana Mill and Pinus rigida Mill. 07. Insect Physiol. 11, 1013-1021. SCHm~IBER K., Ossm~ G. & SEMBDN~R G. (1961) Identifizierung von ~-sitosterin als Hauptsterin des Kartoffelkafers (Leptinotarsa decemlineata Say). Experientia 17, 463-464. SPEm~Y W. M. (1963) Quantitative isolation of sterols. 07. Lipid Res. 4, 221-225.
Key Word Index--Ectosymbiosis, lipids, ambrosial fungi, ambrosia beetle; mutualistic fungi; sterol composition in scolytid beetle and fungal symbionts.