Journal of Chemical Ecology, Vol. 13, No. 4, 1987
ISOLATION A N D IDENTIFICATION OF APOLAR METABOLITES OF INGESTED 20-HYDROXYECDYSONE IN FRASS OF Heliothis virescens LARVAE
ISAO KUBO, S A K A E K O M A T S U , Y U K I H I R O ASAKA, and G E R R I T DE BOER Division of Entomology and Parasitology College of Natural Resources University of California, Berkeley, California 94720 (Received March 17, 1986; accepted May 6, 1986) Abstract--A large amount of 20-hydroxyecdysonewas orally administered to larvae of the tobacco budworm, Heliothis virescens, in order to investigate its detoxification mechanisms. Four major relatively nonpolar metabolites were isolated from their frass. These compounds were identified as the 22linoleate, 22-palmitate, 22-oleate, and 22-stearate of 20-hydroxyecdysone using various forms of spectroscopy, including NMR. This is the first report of this type of metabolite from an insect. Key Wurds--20-Hydroxyecdysone, feeding, insect, metabolites, conjugates, Heliothis virescens, feces. INTRODUCTION Current research in our laboratory is centered around the chemical basis of insect-plant relationships and particularly the physiological effects of phytoecdysteroids. We have isolated several phytoecdysteroids from various tropical plant species which are known to be resistant to insect attack (Kubo et al., 1983a, 1984). One o f the most abundant and widespread phytoecdysteroids is 20-hydroxyecdysone (Bergamasco and Horn, 1983), which is also the moulting hormone in insects themselves. Recent studies in our laboratory demonstrated that small amounts o f this ecdysteroid consumed with diet can inhibit growth and ecdysis in larvae of the lepidopterous species Pectinophora gossypiella (pink bollworm), Spodoptera frugiperda (fall armyworm), and Bombyx mori (silkworm) (Kubo et al., 1983b). The inhibition of ecdysis, which can occur through 785 0098-0331/87/0400-0785505.00/0
9 1987 Plenum Publishing Corporation
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disruption of the normal titer of 20-hydroxecdysone, results in failure to complete ecdysis after normal apolysis and confines the insect in the pharate condition (Kubo and Klocke, 1986). This abnormal pharate condition prevents feeding, excretion, and locomotion and eventually results in death of the affected insect. In contrast, certain insect species are apparently able to survive on plants containing large amounts of phytoecdysteroids, such as the moth Milonia vasalis which feeds on leaves of Podocarpus macrophyllis (Hikino and Takemoto, 1974). Kubo et al. (1981) found that larvae of the bollworm complex, Heliothis zea and H. virescens, are not affected by high concentrations of 20-hydroxyecdysone in their diet. Clearly, these insects must have a defensive mechanism which operates against disruption of the hormonal coordination. Therefore, we examined the physiological basis of this phytoecdysteroid resistance in one such insect, the tobacco budworm, H. virescens, by studying the metabolic fate of the ingested phytoecdysteroid. We describe here the isolation and structural elucidation of several relatively nonpolar metabolites of ingested 20-hydroxyecdysone from frass of tobacco budworm larvae. METHODS AND MATERIALS
Insects. Larvae of the tobacco budworm, Heliothis virescens (Fabricius) (Lepidoptera, Noctuidae) were reared from eggs obtained from a culture maintained at the U.S.D.A. at Stoneville, Mississippi. Upon hatching, larvae were reared individually on an artificial diet (Chanet al., 1978) in plastic containers at 28~ and at 16:8 light-dark cycle. The phytoecdysteroid-containing diet is prepared by adding a solution of a known concentration of ecdysteroid to the cellulose part of the diet. After evaporation of the solvent, the cellulose laced with the ecdysteroid is mixed with the other ingredients of the diet to form the test diet. Control diet is made similarly except for the addition of the ecdysteroid. Newly hatched larvae (N = 50) were reared on an artificial diet containing 1000 ppm 20-hydroxyecdysone through the fifth instar. Fecal material of larvae fed on test and control diet was collected regularly and stored in methanol at 0~ The fecal material of these larvae, as well as the diets themselves, were analyzed for the presence of metabolites of 20-hydroxyecdysone. These samples were homogenized in methanol, and the filtrate was evaporated to dryness and partitioned between n-hexane and water. The aqueous layer was extracted with ethyl ether. The ethyl ether layer was dried over Mg2SO4, filtered, and evaporated to dryness. This fraction was purified by C18 reversed-phase medium-pressure liquid chromatography on a Pharmacia SR10/50 column packed with hand-made 40/zm ODS. The mobile phase used was methanol-water (9 : 1),
20-HYDROXYECDYSONE METABOLITES
787
delivered with a Pharmacia P-3 peristalic pump at a flow rate of t ml/min. Fractions (5 ml) were collected, and an aliquot of each fraction was injected into the HPLC system to check their purity. Chemicals. 20-Hydroxyecdysone had previously been isolated from various tropical medicinal plants (Kubo et al., 1983a, 1984). Solvents used were of reagent or HPLC grade. A Sep-pak Cj8 cartridge (Waters Assoc., Milford, Massachusetts) was used for the preliminary purification of the HPLC samples. Analysis. Analyses were carried out with an Eyela PLC-5 liquid chromatograph system (Tokyo Rikakikai, Tokyo, Japan). A YMC pack. ODS column (15 cm x 6 mm) (Yamamura Chemical, Kyoto, Japan), equipped with Uptight precolumn (2 cm x 2 mm) (Upchurch Scientific, Oak Harbor, Washington) and packed with pellicular ODS (Pell, Whatman Co.), was used. The mobile phase used was methanol-water (9 : 1) at a flow rate of 1.25 ml/min. The effluent was monitored by built-in UV detector at 254 nm. Identification. Isolated compounds were analyzed by several spectroscopic techniques. Ultraviolet (UV) spectra were recorded on a Hitachi 100-80 spectrophotometer. Infrared (IR) spectra were recorded on a Perkin-Elmer 1310 IR spectrophotometer. Secondary ionization mass spectra (SI-MS) were obtained on a Hitachi RMU-6MG apparatus. Proton nuclear magnetic resonance ([tH]NMR) spectra were determined on a Nicolet NT-300 spectrometer. RESULTS AND DISCUSSION
To examine the metabolic fate of ingested phytoecdysteroids in larvae of
H. virescens, fecal material was collected from larvae reared on artificial diet containing 1000 ppm 20-hydroxyecdysone. The following four samples were chromatographically analyzed; I, control diet; II, test diet (containing 1000 ppm of 20-hydroxyecdysone); III, control feces (obtained from insects feeding on the control diet); and IV, test feces (obtained from insects feeding on the test diet). The resulting chromatograms are illustrated in Figure 1. Four compounds appearing in chromatogram IV, M-I, M-2, M-3, and M-4, were not found in the other three samples. 20-Hydroxyecdysone appeared in the chromatogram of II (retention time: 3.0 min). Chromatogram IV also showed 20-hydroxyecdysone. These results suggest that the four major components of sample IV, M-l, M-2, M-3 and M-4, are metabolites of 20-hydroxyecdysone. All four metabolites, M-1 (7.3 mg), M-2 (10.5 mg), M-3 (8.5 rag), and M-4 (3.4 rag), were obtained in large enough quantities to perform various spectroscopic studies. Spectroscopic data of M-l: UV (EtOH) ~kmax 242 nm; IR (CHCI3) 1715, 1654 cm-~; SI-MS m/z 743 (M + 1) § 725 (743-H20), 707 (743-2H20), 689 (743-3H20), 463 (743-fatty acid), 445 (463-H20), 427 (463-2H20), 409 (4633H20), 353 (409-C4H8), and 301 (ecdysone nucleus); [1H]NMR (CDC13) ~ 0.85
788
K U B O ET AL.
20-Hyclroxyecdysone
II c
o
III
20-Hydroxyecdysone
M-I
0
6
12
18
_
24
M-4
30
36
42
48
~n
FIG. 1. HPLC of I, control diet; II, test diet (containing 1000 ppm of 20-hydroxyecdysone); III, control feces (obtained from insects feeding on the control diet); and IV, test feces (obtained from insects feeding on the test diet).
(3H, s, 18-CH3), 0.89 (3H, t, J = 6.7, terminal-CH3), 0.97 (3H, s, 19-CH3), 1.20 (3H, s, 21-CH3), 1.24-1.26 (6H, 2s, 26, 27-CH3), 2.37 (2H, t, J = 7.0, - - O - - C O - - C H 2 - - ) , 2.42 (1H, m, 5/3-H), 2.77 (2H, t, J = 5.8, ll'-CH2), 3.01 (1H, m, 9-H), 3.86 (1H, m, 2-H,x), 4.02 (1H, m, 3-Heq), 4.86 (1H, d, J = 9.4, 22-H), 5.34 (4H, m, 9', 10', 12', 13' olefinic Hs), 5.84 (1H, d, J =
1.6, 7-H). M-2: UV (EtOH) Xm,x 242 nm; IR (CHC13) 1715, 1654 c m - l ; SI-MS m/z 719 (M + 1) § 701 (719-H20), 683 (719-2H20), 665 (719-3H20), 463 (719-fatty acid), 445 (463-H20), 427 (463-2H20), 353 (409-C4H8), and 301 (ecdysone nucleus); [IH]NMR (CDC13) 6 0.86 (3H, s, 18-CH3), 0.88 (3H, t,
789
20-HYDROXYECDYSONE METABOLITES
J = 7.0, terminal-CH3) , 0.98 (3H, s, 19-CH3) 1.24-1.25 (6H, 2s, 26, 27CH3), 2.37 (2H, t, J = 7.1, --O--CO--CH2) , 2.43 (1H, m, 5/3-H), 3.00 (1H, m, 9-H), 3.88 (1H, m, 2-Ha• 4.04 (1H, m, 3-Heq), 4.89 (1H, d, J = 9.4, 22H), 5.85 (1H, d, J = 1.7, 7-H). M-3: UV (EtOH) ~kmax 242 nm; IR (CHC13) 1715, 1654 cm-~; SI-MS m/z 745 (M + 1) § 727 (745-H20), 709 (745-2H20), 691 (745-3H20), 463 (745-fatty acid), 445 (463-H20), 427 (463-2H20), 353 (409-C4H8), and 301 (ecdysone nucleus); [1H]NMR (CDC13) 6 0.86 (3H, s, 18-CH3), 0.88 (3H, t, J = 7.0, terminal-CH3), 1.21 (3H, s, 21-CH3), 1.24 (6H, s, 26, 27-CH3), 2.37 (2H, t, J = 7.1, --O--CO--CH2), 2.43 (1H, m, 5/3-H), 3.00 (1H, m, 9-H), 3.88 (1H, m, 2-Hax), 4.04 (1H, m, 3-Heq), 4.89 (1H, d, J = 9.4, 22-H), 5.34 (2H, m, 9', 10' olefinic Hs), 5.85 (1H, d, J = 1.7, 7-H). M-4: UV (EtOH) Xmax 242 nm; IR (CHC13) 1715, 1654 cm-~; SI-MS m/z 747 (M + 1) +, 729 (747-HZO), 711 (747-2H20), 693 (747-3H20), 463 (747-fatty acid), 445 (463-H20), 427 (463-2H20), 409 (463-3H20), 353 (463C4H8) , and 301 (ecdysone nucleus); [1H]NMR (CDC13) 6 0.86 (3H, s, 18CH3), 0.88 (3H, t, J = 6.7, terminal-CH3), 0.99 (3H, s, 19-CH3), 1.21 (3H, s, 21-CH3), 1.23 (6H, s, 26, 27-CH3), 2.37 (2H, t, J = 7.2, O--CO--CH2--), 2.44 (1H, m, 5t3-H), 3.00 (1H, m, 9-H), 3.89 (1H, m, 2-H,x), 4.05 (1H, m, 3-Heq), 4.88 (1H, d, J = 9.4, 22-H), 5.85 (1H, d, J = 1.6, 7-H). Typical SIMS and [1H]NMR spectra are illustrated in Figures 2 and 3. The UV and IR spectra of all four compounds were quite similar. The presence of ester was suggested by the IR absorption at 1715 cm -~. The SI-
0
100
' x5D 50
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30(1
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I
84
353
/ J,-,
J I ,o, t t
,,. . . . . ,,//, . . . . . . .
FIG. 2. Secondaryionization mass spectrum of M-I
,",. j .....
790
I 7
. . . .
KUBO ET AL.
I 6
. . . .
I 5
. . . .
I
,I
. . . .
I
3
. . . .
I
2
I
. . . .
. . . . .
t
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FIG. 3. 300-MHz [IH]NMR spectrum of M-1 in CDCI3. MS fragment patterns for all four compounds were almost the same below m/z 463. The main fragmentations of 20-hydroxyecdysone usually occur at C-17/ 20 (m/z 301) and C-20/22 (m/z 363)(Nakanishi, 1971). An intense peak at m/z 301 could also be seen in the SI-MS of all four compounds. However, all SI-MS had a peak at m/z 363 of quite low intensity. It appeared to be replaced by an intense peak at m/z 353, which is not found in the SI-MS of 20-hydroxyecdysone. Since the retained m/z 301 peak is believed to result from cleavage of the C-17 to C-20 bond, we believe its presence suggested any ester group present must be on the side chain. The absence of the m/z 363, representing cleavage of the C-20 to C-22 bond, suggests that the ester group could be placed on the hydroxyl at C-22 in all four compounds. Each acyl group: M-l, C~s:2, M-2, C~6, M-3, C18:1 and M-4, C18 fatty acids, could be conjectured due to each (M + 1)+-463. A comparison of the [~H]NMR spectra of M-l, M-2, M-3, and M-4 with the previously assigned [IH]NMR spectrum of 20-hydroxyecdysone showed strong similarities between the spectra (Kubo et al., 1985). However, there was a shift of the H-22 signal from the value in 20-hydroxyecdysone of di 3.32 to a new value of 6 4.86 in all four compounds. The order of magnitude of this shift is consistent with an esterification of the hydroxyl group at C-22. Also, an HPLC analysis of metabolites hydrolyzed with 0.006% NaOH in ethanol coincided with 20-hydroxyecdysone. A long-range coupling between H-7 (6 5.85) and H-9 (6 3.00) was seen in the two-dimensional contour plot.
791
20-HYDROXYECDYSONE METABOLITES
Typical proton signals of fatty acids, ~ 0.88 and 6 2.37, appeared in all four [~H]NMR spectra. These resonances were assigned to terminal methyl and 2'-methylene in their fatty acid ester side chains. The resonances at 6 2.77 and 5.35 in the spectra of M-1 and at (5 5.34 in the spectra of M-2 coincided with previously reported [~H]NMR shifts and coupling patterns of the olefinic proton signals of methyl linoleate and methyl oleate respectively (Hashimoto et al., 1965). Hence the structure of the metabolites were determined to be the 20-hydroxyecdysone-22-fatty acid esters; M-l: linoleate, M-2: palmitate, M-3: oleate, and M-4: stearate (Scheme 1). Much of the metabolism of ecdysteroids in insects is well documented (Koolman, 1982). Several metabolic pathways have been reported in several
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SCHEME1. Structuresof metabolites in the feces of H. virescens.
792
KUBO ET AL.
species of Lepidoptera including: Antheraea polyphemus (Cherbas and Cherbas, 1970), Bombyx mori (Moriyama et al., 1970; Hikino et al., 1975), Hyalophora cecropia (Gorell et al., 1972), Prodenia eridania (Yang and Wilkinson, 1972), Manduca sexta (King, 1972), Choristoneurafumiferana (Lagueux et al., 1976), and Pieris brassicae (Lafont et al., 1983). According to these investigations, the metabolism of ecdysteroids usually occurs by hydroxylation at C-20; epimerization at C-3; dehydration at C-3; acetylation at 3-OH; conjugation to sulfate esters, phosphate esters, glucosides, and glucuronides; and conversion to 26-oic acids. Recently, apolar metabolites of 20-hydroxyecdysone were found using 3H-labeled 20-hydroxyecdysone in feeding experiments with ticks (Connat et al., 1984; Wigglesworth et al., 1985). The structures of apolar metabolites from tick whole-body extracts were identified by hydrolyzed fatty acid analysis, mass spectrometry, and by comparison to a synthetic 20hydroxyecdysone-22-palmitate (Diehl et al., 1985). Apolar metabolites have been investigated from the cockroach Periplaneta americana (Slinger et al., 1986) and other arthropods (Connat and Diehl, 1986). However, their structures remain unidentified at this time. The compounds isolated in the present study were the same as those previously reported in whole-body extracts of ticks (Diehl et al., 1985). However, this is the first report of this type of metabolite from an insect. These metabolites are found in fecal material at relatively high concentrations: approximately 16 % of ingested 20-hydroxyecdysone. Several metabolic studies of ecdysteroid metabolism in lepidopterous larvae have been carried out by injecting 3H-labeled ecdysteroids into the insect body. However, the oral administration of ecdysteroids must be considered when examining phytoecdysteroids in insect-plant relationships. It has been pointed out that the hydroxy function at C-22 in ecdysteroids is essential for hormonal activity (Sorm, 1974). Synthetic 22-deoxy-20-hydroxyecdysone (Galbraith et al., 1969) and 2,22-dideoxy-20-hydroxyecdysone (Ikekawa et al., 1980) are about 10-100 times less active than 20-hydroxyecdysone. The presence of phosphate or adenosinemonophosphate esters on the 22-hydroxy of ecdysteroids also reduces the biological activity (Sall et al., 1983). This suggests the esterification at the 22-hydroxy group with fatty acids to be a detoxification mechanism in H. virescens against ingested 20-hydroxyecdysone. Research is in progress to examine the mode of action of this defensive mechanism. Acknowledgments--We are grateful to Dr. H. Naoki, Suntory Institute for Bioorganic Research, for [1H]NMR and SI-MS measurements and Dr. R. H. Dadd for critical reading of the manuscript. REFERENCES
BERGAMASCO, R., and HORN, D.H. 1983. Distribution and role of insect hormones in plants, pp. 627-654, in R.G.H. Downer and H. Laufer (eds.). Invertebrate Endocrinology, Vol. 1. Alan R. Liss, New York.
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CHAN, B.G., WAIss, A.C., STANLEY, W.L., and GOODBAN,A.E. 1978. A rapid diet preparation method for antibiotic phytochemical bioassay. J. Econ. Entomol. 71:366-368. CHERBAS, L., and CHERBAS, P. 1970. Distribution and metabolism of o~-ecdysone in pupae of the silkworm Antheraea polyphemus. Biol. Bull. 138:115-128. CONNAT, J.-L., and DmHL, P.A. 1986. Probable occurrence of ecdysteroid fatty acid esters in different classes of arthropods. Insect Biochem. 16:91-97. CONNAT, J.-L., DmHL, P.A., and MOR~CI, M. 1984. Metabolism of ecdysteroids during the vitel-logenesis of the tick Ornithodoros moubata (Ixodoidea, Argasidae): Accumulation of apolar metabolites in the eggs. Gen. Comp. Endocrinol. 56:100-110. COTTRELL, C.B. 1964. Insect ecdysis with particular emphasis on cuticular hardening and darkening, pp. 175-218, in J.W.L. Beament, J.E. Treheme and V.B. Wigglesworth (eds.). Advances in Insect Physiology, Vol. 2. Academic Press, London. DIEHL, P.A., CONNAT, J.-L., GmAULT, J.P., and LAFONT, R. 1985. A new class of apolar ecdysteroid conjugates: Esters of 20-hydroxyecdysone with long-chain fatty acids in ticks. Int. J. Invert. Reprod. Dev. 8:1-13. GALBRMTH, M.N., HORN, D.H.S., MIDDLETON, E.J., and HACKNEY, R.J. 1969. Moulting hormones of insects and crustaceans: The synthesis of 22-deoxycmstecdysone. Aust. J. Chem. 22:1517-1524. GORELL, T.A., GILBERT, L.I., and TASH, J. 1972. The uptake and conversion of e~-ecdysone by the pupal tissue of Hyalophora cecropia. Insect Biochem. 2:94-106. HASHIMOTO, T., SHIINA, H., and MAMURO, H. 1965. NMR spectra of several fatty acids methyl ester. Kogyo Kagaku Zasshi 68:1434-1437. HTK~NO, H., and TAKEMOTO, T. 1974. Ecdysones of plant origin, pp. 185-203, in W.J. Burdette (ed.). Invertebrate Endocrinology and Hormonal Heterophylly. Springer Verlag, New York. HIK1NO, H., OHIZUMI, Y., and TAKEMOTO, T. 1975. Steroid metabolism in Bombyx mori, I Catabolism of ponasterone A and ecdysterone in Bombyx mori. Hoppe-Seyler's Z. Physiol. Chem. 356:309-314. IKEKAWA, N., IKEDA, T., MIZUNO, T., OHNISHI, E., and SAKURAI, S. 1980. Isolation of new ecdysteroid, 2,22-dideoxy-20-hydroxyecdysone, from the ovaries of the silkworm Bombix mori. Chem. Commun. 119:448-449. I~yc, D.S. 1972. Ecdysone metabolism in insects. Am. Zool. 12:343-345. KOOLMAN, J. 1982. Ecdysone metabolism. Insect Biochem. 12:225-250. KUBO, I., and KLOCKE, J.A. 1986. Insect ecdysis inhibitors, pp. 206-219, in M.B. Green and P.A. Hedin (eds.). Natural Resistance of Plant to Pests. American Chemical Society Symposium 296, Washington, D.C. KUBO, I., KLOCKE, J.A., and ASANO, S. 1981. Insect ecdysis inhibitors from the East African medicinal plant Ajuga remota (Labiatae). Agric. Biol. Chem. 45:1925-1927. KUBO, I., KLOCKE, J.A., GANJIAN, I., ICHIKAWA,N., and MATSUMOTO, W. 1983a. Efficient isolation of phytoecdysones from Ajuga plants by high-performance liquid chromatography and droplet countercurrent chromatography. J. Chromatogr. 257:157-161. KUBO, I., KLOCKE, J.A., and ASANO, S. 1983b. Effects of ingested phytoecdysteroids on the growth and development of two lepidopterous larvae. J. Insect Physiol. 29:307-316. KUBO, I., MATSUMOTO,A., and AYAFOR, J.F. 1984. Efficient isolation of a large amount of 20hydroxyecdysone from Vitex madiensis (Verbenaceae) by droplet countercurrent chromatography. Agric. BioL Chem. 48:1683-1684. KuBo, I., MATSUMOTO,A., and HANKE, F.J. 1985. The [~H]NMR assignment of 20-hydroxyecdysone. Agrie. Biol. Chem. 49:243-244. LAFONT, R., BLAIS, C., BEYDON, P., MODDE, J.-F., ENDERLE, U., and KOOLMAN, J. 1983. Conversion of ecdysone and 20-hydroxyecdysone into 26-oic derivatives is a major pathway in larvae and pupae of species from three insect orders. Arch. Insect Biochem. Physiol. 1:4158. LAGUEU• M., PERRON, J.-M., and HOFFMANN,J.A. 1976. Ecdysone metabolism and endogenous
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moulting hormone titer during larval-pupal development in Choristoneura fumiferana. J. Insect Physiol. 22:57-62. MORIYAMA,H., NAKANISHI,K., KING, D.S., OKAUCHI,T., SIDDALL,J.B., and HAFFERL, W. 1970. On the origin and metabolic fate of c~-ecdysone in insects. Gen. Comp. Endocrinol. 15:8087. NAKANISHI, K. 1971. The ecdysones. Pure Appl. Chem. 25:167-195. SALL, C., TSOUPRAS, G., KAPPLER, C., LAGUEUX,M., ZACHARY, O., LUU, B., and HOFFMANN, J.A. 1983. Fate of maternal conjugated ecdysteroids during embryonic development in Locusta migratoria. J. Insect Physiol. 29:491-507. SLINGER, A.J., DINAN, L.N., and ISAAC, R.E. 1986. Isolation of apolar ecdysteroid conjugates from newly laid oothecae of Periplaneta americana. InsectBiochem. 16:115-119. SORM, F. 1974. Biosynthesis and catabolism of ecdysoids, pp. 318-320, in K. Sl~ima, M. Romanuk, and F. S6rm (eds.). Insect Hormones and Bioanalogues. Springer Verlag, New York. WIGGLESWORTH,K.P., LEWIS, D., and REES, H.H. 1985. Ecdysteroid titer and metabolism to novel apolar derivatives in adult female Boophilus microplus (Ixodidae). Arch. Insect Biochem. Physiol. 2:39-54. YANG, R.S.H., and WILKINSON, C.F. 1972. Enzymic sulphation ofp-nitrophenol and steroids by larval gut tissues of the southern armyworm (Prodenia eridania Cramer). Biochem. J. 130:487493.
ISOLATION A N D IDENTIFICATION OF APOLAR METABOLITES OF INGESTED 20-HYDROXYECDYSONE IN FRASS OF Heliothis virescens LARVAE
ISAO KUBO, S A K A E K O M A T S U , Y U K I H I R O ASAKA, and G E R R I T DE BOER Division of Entomology and Parasitology College of Natural Resources University of California, Berkeley, California 94720 (Received March 17, 1986; accepted May 6, 1986) Abstract--A large amount of 20-hydroxyecdysonewas orally administered to larvae of the tobacco budworm, Heliothis virescens, in order to investigate its detoxification mechanisms. Four major relatively nonpolar metabolites were isolated from their frass. These compounds were identified as the 22linoleate, 22-palmitate, 22-oleate, and 22-stearate of 20-hydroxyecdysone using various forms of spectroscopy, including NMR. This is the first report of this type of metabolite from an insect. Key Wurds--20-Hydroxyecdysone, feeding, insect, metabolites, conjugates, Heliothis virescens, feces. INTRODUCTION Current research in our laboratory is centered around the chemical basis of insect-plant relationships and particularly the physiological effects of phytoecdysteroids. We have isolated several phytoecdysteroids from various tropical plant species which are known to be resistant to insect attack (Kubo et al., 1983a, 1984). One o f the most abundant and widespread phytoecdysteroids is 20-hydroxyecdysone (Bergamasco and Horn, 1983), which is also the moulting hormone in insects themselves. Recent studies in our laboratory demonstrated that small amounts o f this ecdysteroid consumed with diet can inhibit growth and ecdysis in larvae of the lepidopterous species Pectinophora gossypiella (pink bollworm), Spodoptera frugiperda (fall armyworm), and Bombyx mori (silkworm) (Kubo et al., 1983b). The inhibition of ecdysis, which can occur through 785 0098-0331/87/0400-0785505.00/0
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KUBOET AL.
disruption of the normal titer of 20-hydroxecdysone, results in failure to complete ecdysis after normal apolysis and confines the insect in the pharate condition (Kubo and Klocke, 1986). This abnormal pharate condition prevents feeding, excretion, and locomotion and eventually results in death of the affected insect. In contrast, certain insect species are apparently able to survive on plants containing large amounts of phytoecdysteroids, such as the moth Milonia vasalis which feeds on leaves of Podocarpus macrophyllis (Hikino and Takemoto, 1974). Kubo et al. (1981) found that larvae of the bollworm complex, Heliothis zea and H. virescens, are not affected by high concentrations of 20-hydroxyecdysone in their diet. Clearly, these insects must have a defensive mechanism which operates against disruption of the hormonal coordination. Therefore, we examined the physiological basis of this phytoecdysteroid resistance in one such insect, the tobacco budworm, H. virescens, by studying the metabolic fate of the ingested phytoecdysteroid. We describe here the isolation and structural elucidation of several relatively nonpolar metabolites of ingested 20-hydroxyecdysone from frass of tobacco budworm larvae. METHODS AND MATERIALS
Insects. Larvae of the tobacco budworm, Heliothis virescens (Fabricius) (Lepidoptera, Noctuidae) were reared from eggs obtained from a culture maintained at the U.S.D.A. at Stoneville, Mississippi. Upon hatching, larvae were reared individually on an artificial diet (Chanet al., 1978) in plastic containers at 28~ and at 16:8 light-dark cycle. The phytoecdysteroid-containing diet is prepared by adding a solution of a known concentration of ecdysteroid to the cellulose part of the diet. After evaporation of the solvent, the cellulose laced with the ecdysteroid is mixed with the other ingredients of the diet to form the test diet. Control diet is made similarly except for the addition of the ecdysteroid. Newly hatched larvae (N = 50) were reared on an artificial diet containing 1000 ppm 20-hydroxyecdysone through the fifth instar. Fecal material of larvae fed on test and control diet was collected regularly and stored in methanol at 0~ The fecal material of these larvae, as well as the diets themselves, were analyzed for the presence of metabolites of 20-hydroxyecdysone. These samples were homogenized in methanol, and the filtrate was evaporated to dryness and partitioned between n-hexane and water. The aqueous layer was extracted with ethyl ether. The ethyl ether layer was dried over Mg2SO4, filtered, and evaporated to dryness. This fraction was purified by C18 reversed-phase medium-pressure liquid chromatography on a Pharmacia SR10/50 column packed with hand-made 40/zm ODS. The mobile phase used was methanol-water (9 : 1),
20-HYDROXYECDYSONE METABOLITES
787
delivered with a Pharmacia P-3 peristalic pump at a flow rate of t ml/min. Fractions (5 ml) were collected, and an aliquot of each fraction was injected into the HPLC system to check their purity. Chemicals. 20-Hydroxyecdysone had previously been isolated from various tropical medicinal plants (Kubo et al., 1983a, 1984). Solvents used were of reagent or HPLC grade. A Sep-pak Cj8 cartridge (Waters Assoc., Milford, Massachusetts) was used for the preliminary purification of the HPLC samples. Analysis. Analyses were carried out with an Eyela PLC-5 liquid chromatograph system (Tokyo Rikakikai, Tokyo, Japan). A YMC pack. ODS column (15 cm x 6 mm) (Yamamura Chemical, Kyoto, Japan), equipped with Uptight precolumn (2 cm x 2 mm) (Upchurch Scientific, Oak Harbor, Washington) and packed with pellicular ODS (Pell, Whatman Co.), was used. The mobile phase used was methanol-water (9 : 1) at a flow rate of 1.25 ml/min. The effluent was monitored by built-in UV detector at 254 nm. Identification. Isolated compounds were analyzed by several spectroscopic techniques. Ultraviolet (UV) spectra were recorded on a Hitachi 100-80 spectrophotometer. Infrared (IR) spectra were recorded on a Perkin-Elmer 1310 IR spectrophotometer. Secondary ionization mass spectra (SI-MS) were obtained on a Hitachi RMU-6MG apparatus. Proton nuclear magnetic resonance ([tH]NMR) spectra were determined on a Nicolet NT-300 spectrometer. RESULTS AND DISCUSSION
To examine the metabolic fate of ingested phytoecdysteroids in larvae of
H. virescens, fecal material was collected from larvae reared on artificial diet containing 1000 ppm 20-hydroxyecdysone. The following four samples were chromatographically analyzed; I, control diet; II, test diet (containing 1000 ppm of 20-hydroxyecdysone); III, control feces (obtained from insects feeding on the control diet); and IV, test feces (obtained from insects feeding on the test diet). The resulting chromatograms are illustrated in Figure 1. Four compounds appearing in chromatogram IV, M-I, M-2, M-3, and M-4, were not found in the other three samples. 20-Hydroxyecdysone appeared in the chromatogram of II (retention time: 3.0 min). Chromatogram IV also showed 20-hydroxyecdysone. These results suggest that the four major components of sample IV, M-l, M-2, M-3 and M-4, are metabolites of 20-hydroxyecdysone. All four metabolites, M-1 (7.3 mg), M-2 (10.5 mg), M-3 (8.5 rag), and M-4 (3.4 rag), were obtained in large enough quantities to perform various spectroscopic studies. Spectroscopic data of M-l: UV (EtOH) ~kmax 242 nm; IR (CHCI3) 1715, 1654 cm-~; SI-MS m/z 743 (M + 1) § 725 (743-H20), 707 (743-2H20), 689 (743-3H20), 463 (743-fatty acid), 445 (463-H20), 427 (463-2H20), 409 (4633H20), 353 (409-C4H8), and 301 (ecdysone nucleus); [1H]NMR (CDC13) ~ 0.85
788
K U B O ET AL.
20-Hyclroxyecdysone
II c
o
III
20-Hydroxyecdysone
M-I
0
6
12
18
_
24
M-4
30
36
42
48
~n
FIG. 1. HPLC of I, control diet; II, test diet (containing 1000 ppm of 20-hydroxyecdysone); III, control feces (obtained from insects feeding on the control diet); and IV, test feces (obtained from insects feeding on the test diet).
(3H, s, 18-CH3), 0.89 (3H, t, J = 6.7, terminal-CH3), 0.97 (3H, s, 19-CH3), 1.20 (3H, s, 21-CH3), 1.24-1.26 (6H, 2s, 26, 27-CH3), 2.37 (2H, t, J = 7.0, - - O - - C O - - C H 2 - - ) , 2.42 (1H, m, 5/3-H), 2.77 (2H, t, J = 5.8, ll'-CH2), 3.01 (1H, m, 9-H), 3.86 (1H, m, 2-H,x), 4.02 (1H, m, 3-Heq), 4.86 (1H, d, J = 9.4, 22-H), 5.34 (4H, m, 9', 10', 12', 13' olefinic Hs), 5.84 (1H, d, J =
1.6, 7-H). M-2: UV (EtOH) Xm,x 242 nm; IR (CHC13) 1715, 1654 c m - l ; SI-MS m/z 719 (M + 1) § 701 (719-H20), 683 (719-2H20), 665 (719-3H20), 463 (719-fatty acid), 445 (463-H20), 427 (463-2H20), 353 (409-C4H8), and 301 (ecdysone nucleus); [IH]NMR (CDC13) 6 0.86 (3H, s, 18-CH3), 0.88 (3H, t,
789
20-HYDROXYECDYSONE METABOLITES
J = 7.0, terminal-CH3) , 0.98 (3H, s, 19-CH3) 1.24-1.25 (6H, 2s, 26, 27CH3), 2.37 (2H, t, J = 7.1, --O--CO--CH2) , 2.43 (1H, m, 5/3-H), 3.00 (1H, m, 9-H), 3.88 (1H, m, 2-Ha• 4.04 (1H, m, 3-Heq), 4.89 (1H, d, J = 9.4, 22H), 5.85 (1H, d, J = 1.7, 7-H). M-3: UV (EtOH) ~kmax 242 nm; IR (CHC13) 1715, 1654 cm-~; SI-MS m/z 745 (M + 1) § 727 (745-H20), 709 (745-2H20), 691 (745-3H20), 463 (745-fatty acid), 445 (463-H20), 427 (463-2H20), 353 (409-C4H8), and 301 (ecdysone nucleus); [1H]NMR (CDC13) 6 0.86 (3H, s, 18-CH3), 0.88 (3H, t, J = 7.0, terminal-CH3), 1.21 (3H, s, 21-CH3), 1.24 (6H, s, 26, 27-CH3), 2.37 (2H, t, J = 7.1, --O--CO--CH2), 2.43 (1H, m, 5/3-H), 3.00 (1H, m, 9-H), 3.88 (1H, m, 2-Hax), 4.04 (1H, m, 3-Heq), 4.89 (1H, d, J = 9.4, 22-H), 5.34 (2H, m, 9', 10' olefinic Hs), 5.85 (1H, d, J = 1.7, 7-H). M-4: UV (EtOH) Xmax 242 nm; IR (CHC13) 1715, 1654 cm-~; SI-MS m/z 747 (M + 1) +, 729 (747-HZO), 711 (747-2H20), 693 (747-3H20), 463 (747-fatty acid), 445 (463-H20), 427 (463-2H20), 409 (463-3H20), 353 (463C4H8) , and 301 (ecdysone nucleus); [1H]NMR (CDC13) 6 0.86 (3H, s, 18CH3), 0.88 (3H, t, J = 6.7, terminal-CH3), 0.99 (3H, s, 19-CH3), 1.21 (3H, s, 21-CH3), 1.23 (6H, s, 26, 27-CH3), 2.37 (2H, t, J = 7.2, O--CO--CH2--), 2.44 (1H, m, 5t3-H), 3.00 (1H, m, 9-H), 3.89 (1H, m, 2-H,x), 4.05 (1H, m, 3-Heq), 4.88 (1H, d, J = 9.4, 22-H), 5.85 (1H, d, J = 1.6, 7-H). Typical SIMS and [1H]NMR spectra are illustrated in Figures 2 and 3. The UV and IR spectra of all four compounds were quite similar. The presence of ester was suggested by the IR absorption at 1715 cm -~. The SI-
0
100
' x5D 50
301 h
30(1
725 743
I
84
353
/ J,-,
J I ,o, t t
,,. . . . . ,,//, . . . . . . .
FIG. 2. Secondaryionization mass spectrum of M-I
,",. j .....
790
I 7
. . . .
KUBO ET AL.
I 6
. . . .
I 5
. . . .
I
,I
. . . .
I
3
. . . .
I
2
I
. . . .
. . . . .
t
t o
FIG. 3. 300-MHz [IH]NMR spectrum of M-1 in CDCI3. MS fragment patterns for all four compounds were almost the same below m/z 463. The main fragmentations of 20-hydroxyecdysone usually occur at C-17/ 20 (m/z 301) and C-20/22 (m/z 363)(Nakanishi, 1971). An intense peak at m/z 301 could also be seen in the SI-MS of all four compounds. However, all SI-MS had a peak at m/z 363 of quite low intensity. It appeared to be replaced by an intense peak at m/z 353, which is not found in the SI-MS of 20-hydroxyecdysone. Since the retained m/z 301 peak is believed to result from cleavage of the C-17 to C-20 bond, we believe its presence suggested any ester group present must be on the side chain. The absence of the m/z 363, representing cleavage of the C-20 to C-22 bond, suggests that the ester group could be placed on the hydroxyl at C-22 in all four compounds. Each acyl group: M-l, C~s:2, M-2, C~6, M-3, C18:1 and M-4, C18 fatty acids, could be conjectured due to each (M + 1)+-463. A comparison of the [~H]NMR spectra of M-l, M-2, M-3, and M-4 with the previously assigned [IH]NMR spectrum of 20-hydroxyecdysone showed strong similarities between the spectra (Kubo et al., 1985). However, there was a shift of the H-22 signal from the value in 20-hydroxyecdysone of di 3.32 to a new value of 6 4.86 in all four compounds. The order of magnitude of this shift is consistent with an esterification of the hydroxyl group at C-22. Also, an HPLC analysis of metabolites hydrolyzed with 0.006% NaOH in ethanol coincided with 20-hydroxyecdysone. A long-range coupling between H-7 (6 5.85) and H-9 (6 3.00) was seen in the two-dimensional contour plot.
791
20-HYDROXYECDYSONE METABOLITES
Typical proton signals of fatty acids, ~ 0.88 and 6 2.37, appeared in all four [~H]NMR spectra. These resonances were assigned to terminal methyl and 2'-methylene in their fatty acid ester side chains. The resonances at 6 2.77 and 5.35 in the spectra of M-1 and at (5 5.34 in the spectra of M-2 coincided with previously reported [~H]NMR shifts and coupling patterns of the olefinic proton signals of methyl linoleate and methyl oleate respectively (Hashimoto et al., 1965). Hence the structure of the metabolites were determined to be the 20-hydroxyecdysone-22-fatty acid esters; M-l: linoleate, M-2: palmitate, M-3: oleate, and M-4: stearate (Scheme 1). Much of the metabolism of ecdysteroids in insects is well documented (Koolman, 1982). Several metabolic pathways have been reported in several
HOo- y HO"
-.~
~
~.-
-./
~
v
~-~,,
Oi"l
HO'~- v - ~
o
M-1
M-2
~.,
o HOg Y
v
v
v
v
v v v
M-3
O
H O.~
- ~ H
l
t
'
M-4
SCHEME1. Structuresof metabolites in the feces of H. virescens.
792
KUBO ET AL.
species of Lepidoptera including: Antheraea polyphemus (Cherbas and Cherbas, 1970), Bombyx mori (Moriyama et al., 1970; Hikino et al., 1975), Hyalophora cecropia (Gorell et al., 1972), Prodenia eridania (Yang and Wilkinson, 1972), Manduca sexta (King, 1972), Choristoneurafumiferana (Lagueux et al., 1976), and Pieris brassicae (Lafont et al., 1983). According to these investigations, the metabolism of ecdysteroids usually occurs by hydroxylation at C-20; epimerization at C-3; dehydration at C-3; acetylation at 3-OH; conjugation to sulfate esters, phosphate esters, glucosides, and glucuronides; and conversion to 26-oic acids. Recently, apolar metabolites of 20-hydroxyecdysone were found using 3H-labeled 20-hydroxyecdysone in feeding experiments with ticks (Connat et al., 1984; Wigglesworth et al., 1985). The structures of apolar metabolites from tick whole-body extracts were identified by hydrolyzed fatty acid analysis, mass spectrometry, and by comparison to a synthetic 20hydroxyecdysone-22-palmitate (Diehl et al., 1985). Apolar metabolites have been investigated from the cockroach Periplaneta americana (Slinger et al., 1986) and other arthropods (Connat and Diehl, 1986). However, their structures remain unidentified at this time. The compounds isolated in the present study were the same as those previously reported in whole-body extracts of ticks (Diehl et al., 1985). However, this is the first report of this type of metabolite from an insect. These metabolites are found in fecal material at relatively high concentrations: approximately 16 % of ingested 20-hydroxyecdysone. Several metabolic studies of ecdysteroid metabolism in lepidopterous larvae have been carried out by injecting 3H-labeled ecdysteroids into the insect body. However, the oral administration of ecdysteroids must be considered when examining phytoecdysteroids in insect-plant relationships. It has been pointed out that the hydroxy function at C-22 in ecdysteroids is essential for hormonal activity (Sorm, 1974). Synthetic 22-deoxy-20-hydroxyecdysone (Galbraith et al., 1969) and 2,22-dideoxy-20-hydroxyecdysone (Ikekawa et al., 1980) are about 10-100 times less active than 20-hydroxyecdysone. The presence of phosphate or adenosinemonophosphate esters on the 22-hydroxy of ecdysteroids also reduces the biological activity (Sall et al., 1983). This suggests the esterification at the 22-hydroxy group with fatty acids to be a detoxification mechanism in H. virescens against ingested 20-hydroxyecdysone. Research is in progress to examine the mode of action of this defensive mechanism. Acknowledgments--We are grateful to Dr. H. Naoki, Suntory Institute for Bioorganic Research, for [1H]NMR and SI-MS measurements and Dr. R. H. Dadd for critical reading of the manuscript. REFERENCES
BERGAMASCO, R., and HORN, D.H. 1983. Distribution and role of insect hormones in plants, pp. 627-654, in R.G.H. Downer and H. Laufer (eds.). Invertebrate Endocrinology, Vol. 1. Alan R. Liss, New York.
20-HYDROXYECDYSONE METABOLITES
793
CHAN, B.G., WAIss, A.C., STANLEY, W.L., and GOODBAN,A.E. 1978. A rapid diet preparation method for antibiotic phytochemical bioassay. J. Econ. Entomol. 71:366-368. CHERBAS, L., and CHERBAS, P. 1970. Distribution and metabolism of o~-ecdysone in pupae of the silkworm Antheraea polyphemus. Biol. Bull. 138:115-128. CONNAT, J.-L., and DmHL, P.A. 1986. Probable occurrence of ecdysteroid fatty acid esters in different classes of arthropods. Insect Biochem. 16:91-97. CONNAT, J.-L., DmHL, P.A., and MOR~CI, M. 1984. Metabolism of ecdysteroids during the vitel-logenesis of the tick Ornithodoros moubata (Ixodoidea, Argasidae): Accumulation of apolar metabolites in the eggs. Gen. Comp. Endocrinol. 56:100-110. COTTRELL, C.B. 1964. Insect ecdysis with particular emphasis on cuticular hardening and darkening, pp. 175-218, in J.W.L. Beament, J.E. Treheme and V.B. Wigglesworth (eds.). Advances in Insect Physiology, Vol. 2. Academic Press, London. DIEHL, P.A., CONNAT, J.-L., GmAULT, J.P., and LAFONT, R. 1985. A new class of apolar ecdysteroid conjugates: Esters of 20-hydroxyecdysone with long-chain fatty acids in ticks. Int. J. Invert. Reprod. Dev. 8:1-13. GALBRMTH, M.N., HORN, D.H.S., MIDDLETON, E.J., and HACKNEY, R.J. 1969. Moulting hormones of insects and crustaceans: The synthesis of 22-deoxycmstecdysone. Aust. J. Chem. 22:1517-1524. GORELL, T.A., GILBERT, L.I., and TASH, J. 1972. The uptake and conversion of e~-ecdysone by the pupal tissue of Hyalophora cecropia. Insect Biochem. 2:94-106. HASHIMOTO, T., SHIINA, H., and MAMURO, H. 1965. NMR spectra of several fatty acids methyl ester. Kogyo Kagaku Zasshi 68:1434-1437. HTK~NO, H., and TAKEMOTO, T. 1974. Ecdysones of plant origin, pp. 185-203, in W.J. Burdette (ed.). Invertebrate Endocrinology and Hormonal Heterophylly. Springer Verlag, New York. HIK1NO, H., OHIZUMI, Y., and TAKEMOTO, T. 1975. Steroid metabolism in Bombyx mori, I Catabolism of ponasterone A and ecdysterone in Bombyx mori. Hoppe-Seyler's Z. Physiol. Chem. 356:309-314. IKEKAWA, N., IKEDA, T., MIZUNO, T., OHNISHI, E., and SAKURAI, S. 1980. Isolation of new ecdysteroid, 2,22-dideoxy-20-hydroxyecdysone, from the ovaries of the silkworm Bombix mori. Chem. Commun. 119:448-449. I~yc, D.S. 1972. Ecdysone metabolism in insects. Am. Zool. 12:343-345. KOOLMAN, J. 1982. Ecdysone metabolism. Insect Biochem. 12:225-250. KUBO, I., and KLOCKE, J.A. 1986. Insect ecdysis inhibitors, pp. 206-219, in M.B. Green and P.A. Hedin (eds.). Natural Resistance of Plant to Pests. American Chemical Society Symposium 296, Washington, D.C. KUBO, I., KLOCKE, J.A., and ASANO, S. 1981. Insect ecdysis inhibitors from the East African medicinal plant Ajuga remota (Labiatae). Agric. Biol. Chem. 45:1925-1927. KUBO, I., KLOCKE, J.A., GANJIAN, I., ICHIKAWA,N., and MATSUMOTO, W. 1983a. Efficient isolation of phytoecdysones from Ajuga plants by high-performance liquid chromatography and droplet countercurrent chromatography. J. Chromatogr. 257:157-161. KUBO, I., KLOCKE, J.A., and ASANO, S. 1983b. Effects of ingested phytoecdysteroids on the growth and development of two lepidopterous larvae. J. Insect Physiol. 29:307-316. KUBO, I., MATSUMOTO,A., and AYAFOR, J.F. 1984. Efficient isolation of a large amount of 20hydroxyecdysone from Vitex madiensis (Verbenaceae) by droplet countercurrent chromatography. Agric. BioL Chem. 48:1683-1684. KuBo, I., MATSUMOTO,A., and HANKE, F.J. 1985. The [~H]NMR assignment of 20-hydroxyecdysone. Agrie. Biol. Chem. 49:243-244. LAFONT, R., BLAIS, C., BEYDON, P., MODDE, J.-F., ENDERLE, U., and KOOLMAN, J. 1983. Conversion of ecdysone and 20-hydroxyecdysone into 26-oic derivatives is a major pathway in larvae and pupae of species from three insect orders. Arch. Insect Biochem. Physiol. 1:4158. LAGUEU• M., PERRON, J.-M., and HOFFMANN,J.A. 1976. Ecdysone metabolism and endogenous
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