The neutral sterols of Megalotomus quinquespinosus say hemipteraAlydidae and identification of makisterone a as the major free ecdysteroid.код для вставкиСкачать
Archives of Insect Biochemistry and Physiology 3:423-430 (1986) The Neutral Sterols of Megalotomus quinquespinosus (Say) (Hemiptera: Alydidae) and Identification of Makisterone A as the Major Free Ecdysteroid M.F. Feldlaufer, J.A. Svoboda, J.R. Aldrich, and W.R. Lusby Insect and Nematode Hormone Laboratoy, USDA, Agricultural Research Service, Beltsville, Maryland Last-stage nymphs of the broad-headed bug, Megalotomus quinquespinosus contain the C28 ecdysteroid makisterone A as their major ecdysteroid. No ecdysone or 20-hydroxyecdysone was detected in whole body extracts analyzed by high performance liquid chromatography and radioimmune assay. Analyses of the neutral sterols of this phytophagous hemipteran revealed that the sterol composition of the nymphs was highly reflective of their dietary sterols. The most abundant nymphal sterols were sitosterol (46.6%), A’-stigmastenol (13.8%) and spinasterol (13.4%). Cholesterol accounted for only 0.2% of the total sterols and indicates that this species is incapable of converting 24-alkyl sterols to cholesterol. Key words: molting hormones, dietary sterols, HPLClRlA INTRODUCTION In the Hemiptera, an interesting relationship exists between dietary sterol metabolism and the chemical nature of the molting hormone [1,2]. Previous studies have shown that most predacious and hematophagous species, which have a preponderance of cholesterol in their diet, produce the C27 ecdysteroid, 20-hydroxyecdysone, whereas plant-feeding species, whose diets contain mostly C-24 alkyl sterols, mainly produce the C28 ecdysteroid, makisterone A [l-41. The one exception, to date, is a secondarily predacious species, that contains high levels of cholesterol [l] yet still produces makisterone A as its molting hormone . The phytophagous species examined Acknowledgments: The technical assistance of Martha Hollenbeck, Owen Duncan, Ill and Susan Wilzer is greatly appreciated. Received October 21, 1985; accepted January 10, 1986. Address reprint requests to Dr. Mark F. Feldlaufer, Insect and Nematode Hormone Laboratory, Bldg. 467; BARC-East, Beltsville, Maryland 20705. 0 1986 Alan R. Liss, Inc. 424 Feldlaufer et al have been shown to lack the ability to convert plant sterols to cholesterol [3,5] and therefore presumably use a C28 plant sterol, unchanged, for ecdysteroid biosynthesis. We have extended our in-depth studies of comparative steroid metabolism in insects by examining the neutral sterol and ecdysteroid content of Megalotomus quinquespinosus, a phytophagous species of true bug belonging to a different taxonomic group (Alydidae) than those hemipterans previously examined. We now report the apparent inability of this species to convert plant sterols to cholesterol and the physico-chemical identification of makisterone A as the major molting hormone. MATERIALS AND METHODS Chemicals Solvents for extraction and purification were reagent grade redistilled. High performance liquid chromatography* grade solvents were obtained from Burdick and Jackson? (American Scientific Products, Columbia, MD). Ecdysteroid standards (Shes, Milano, Italy) were 20-hydroxyecdysone, (20R,22R)-2&3P,14a,20,22,25-hexahydroxy-5~-cholest-7-en-6-one; makisterone A, (20R,22R)-2~,3~-14a,20,22,25-hexahydroxy-24-methyl-5~-cholest-7en-6-one; and ecdysone, (22R)-2/3,3P-14a,22,25-pentahydroxy-5P-cholest-7en-6-one. Insects Colonies of broad-headed bugs, M. quinquespinosus (Say), were maintained on a combination of soybean, sunflower, and alfalfa seeds, with fresh green beans and water, at 27°C under a 16 light:8 dark photoperiod regime. Late fifth-stage nymphs were collected for the study and stored in methanol at -20°C. The length of the fifth stage varied from eight to twelve days under these rearing conditions. Insect Extractions Fifth-stage nymphs (29.06 g) of mixed ages were homogenized in methanol (2 x 200 ml), filtered, and rehomogenized in 75% methanollwater (2 x 100 ml), After filtering, the combined extracts were dried in vacuo. The residue (2.06 g) was partitioned between 70% methanol/water and n-hexane (countersaturated; 50 ml each) across four separatory funnels containing upper phase with two lower phase transfers across all funnels. The combined hexane phases were dried and reserved for neutral sterol workup, while the combined methanolic phases were dried and further partitioned between n- *Abbreviations: desorption chemical ionization-mass spectrometry = DCI-MS; gas liquid chromatography = CLC; high performance liquid chromatography = HPLC; radioimmune assay = RIA. +Mention of a company name or proprietary product does not constitute an endorsement by the US. Department of Agriculture. Sterols and Ecdysteroids of Megalotomus 425 butanol and water (countersaturated; 50 ml each) across five separatory funnels containing upper phase with three transfers of lower phase across all funnels. The dried butanolic phases yielded 160 mg. Sterol Purification and Analyses The hexane phase from the initial partitioning was used to determine the relative percentages of neutral sterols in last-stage nymphs. In addition, the seeds of alfalfa, soybean, and sunflower were also examined for sterol content. All extraction and saponification procedures have been previously reported, as well as alumina column chromatography and capillary column GLC conditions [6,51. Isolation and Purification of Ecdysteroids The dried butanolic residue, containing the free ecdysteroids, was fractionated on a Florisil@open column (5 g, 60-100 mesh; Fisher Scientific, Fair Lawn, NJ) eluted with 5% methanollchloroform (75 ml), 25% methanol/ chloroform (50 ml) and 100% methanol (50 ml). The 25% methanollchloroform fraction yielded 1.6 mg upon drying and this residue was eluted on a CI8 SEP-PAK@as previously described by Thompson et al.  and similar to the method of Lafont et al. . Fractions of lo%, 30%, and 60% methanol/ water and 100% methanol were collected. High Performance Liquid Chromatography A Spectra-Physics solvent delivery system was used in conjunction with a Waters Model 481 UV detector, monitoring the eluant at 242 nm. Nymphal ecdysteroids from the 60% SEP-PAK fraction were injected in methanol and fractionated as previously described [lo]. One milliliter fractions were collected for RIA analysis. After determining the immunoreactive fractions, the sample was further purified by silica HPLC [lo]. Radioimmune Assay Antiserum, from rabbits injected with a carboxymethoxime derivative of 20-hydroxyecdysone, was a gift of J. Koolman (Marburg, FRG). [23,243H]Ecydsone (sp. act. 55-60 Cilmmol; Zoecon Corp., Palo Alto, CA) was used as the labeled ligand. All assays were performed in triplicate [lO,ll]. The cross-reactivity factors for 20-hydroxyecdysone and makisterone A were determined in our laboratory to be 4.85 and 8.65, respectively, b comparing the mass of each ecdysteroid required to displace 50% of the [ Hlecdysone relative to the mass of ecdysone required. Y Mass Spectrometry The highly purified nymphal ecdysteroid from silica HPLC was analyzed by DCI-MS using a Finnigan 4510 mass spectrometer. Methane was used as the reagent gas and spectra were collected from 100-650 a.m.u. with a scan cycle time of 0.85 s. Samples of approximately 150 ng were deposited on the probe tip and heated at a current rate of 20 mA/s from 0 to 1000 mA. Source block temperature was 120°C. 426 Feldlaufer et a1 RESULTS Sterol Analyses The relative percentages of neutral sterolsii (Fig. 1)isolated from nymphal M. quinquespinosus, and their diet, are presented in Table 1. Sitosterol was both the predominant nymphal sterol (46.6%), and the major sterol found in soybean seeds (56.3%), sunflower seeds (56.4%) and green beans (48.0%). While no sitosterol was detected in alfalfa seeds, this foodstuff contained large amounts of two other Cz9 sterols, spinasterol (42.5%) and A7-stigmastenol (39.7%). The nymphs sterols contained 13.8% A7-stigmastenol and 13.4% spinasterol. The bulk of the remaining neutral sterols isolated from M. quinquespinosus were also the 24-alkyl sterols, campesterol (9.0%), isofucosterol (5.8%), and stigmasterol (4.1%). Cz7 sterols (cholesterol and desmosterol) together, accounted for less than 1%of the total sterols isolated from HO Cholesterol HO Desmosterol &&& HO Campesterol 4 HO A7-Campeslenol 24-MeIhylenecholesIcfo~ ~ 7 - Isi ~ m saIen01 SlrosIr?tol Splnesletol lsotucoslerol Fig. 1. Structures of neutral sterols isolated from M. quinquespinosus nymphs. ++Thefollowing systematic names represent the neutral sterols depicted in Figure 1: cholesterol: cholest-5-en-3P-ol; desmosterol: cholesta-5,24-dien-3P-ol; 24-methylenecholesterol: 24methylene-cholest-5-en-3P-ol; campesterol: 24a-methylcholest-5-en-3~-ol;stigmasterol: 24aethylcholesta-5,22-dien-3P-ol; A7-campestenol: 24a-methyl-5a-cholest-7-en-3~-ol;spinasterol: 24c~-ethyl-5a-cholesta-7,22-dien-3~-ol; sitosterol: 24a-ethylcholest-5-en-3P-ol; iso-fucosterol: 24aethylidene-cholest-5-en-fl-ol; A7-stigmastenol: 24a-ethyl-5a-cholest-7-en-3~-oI. Sterols and Ecdysteroids of Megalotomus 427 TABLE 1. Relative Percentage of Neutral Sterols From M . quinquespinosus Nymphs and Their Diet* Sterol Cholesterol Desmosterol 24-Methylenecholesterol Campesterol Stigmasterol A7-Campesterol Spinasterol Sitosterol Isofucosterol A7-Stigmastenol Alfalfa Soybean Sunflower Greenbeana Nymphs -b 0.5 - 0.9 0.8 - - 0.5 23.6 16.5 0.4 0.3 10.3 6.7 1.3 - 0.2 0.5 0.2 9.0 4.1 2.8 13.4 46.6 5.8 13.8 0.2 1.2 7.3 42.5 - - 56.3 0.7 39.7 - 1.1 - 56.4 9.3 4.4 6.0 37.4 - 48.0 7.7 - *Determined by GLC on J&W DB-1 fused-silica capillary column (240°C; 15 m x 0.25 pm film). aFrom Svoboda et al. [l]. bNot detected in sample. nymphs and reflected the relatively small amounts of these sterols in their diets. Ecdysteroid Analysis The residue from the 60% SEP-PAK separation was fractionated by reversed-phase HPLC and analyzed by RIA. This analysis revealed two immunoreactive areas, the larger of which (fractions 13 and 14) corresponded to a visible, UV-absorbing peak having a retention time identical to that of makisterone A (Fig. 2). When this immunoreactive compound was dried and subsequently fractionated by silica HPLC, a strong, UV-absorbing peak was again evident that matched the retention time of makisterone A (Fig. 3). The unknown nymphal ecdysteroid from the silica HPLC fractionation was analyzed by DCI-MS. Methane DCI produced ions at 495[(M+H),44%], 477[(M+ H) -H20, 34%], 459[(M +H) -2H20, 74%], 441[(M+H) -3H20, 79%], and 423[(M+H)-4H20, 39%], along with other ions indicative of a polyhydroxylated ecdysteroid having a molecular weight of 494. The DCI spectrum of the nymphal ecdysteroid closely matched the spectrum of authentic makisterone A. Peak areas from HPLC and RIA (after correcting for cross-reactivity) data indicated approximately 43 ng of makisterone A per gram of nymphs calculated on the basis of fresh weight. A smaller amount of immunoreactivity was also detected in fractions 26 and 27 of the reversed-phase HPLC fractionation (Fig. 2), although no clear, UV-absorbing peak was seen and an insufficient quantity of material precluded further investigation. There was no immunological evidence supporting the presence of the C27ecdysteroids, ecdysone or 20-hydroxyecdysoner two ecdysteroids that have a greater affinity for our antisera than does makisterone A. In addition, there was neither immunological nor chromatographic evidence for the presence of makisterone C, a C29 phytoecdysteroid. Makisterone C, which has not, to date, been isolated from an insect, elutes at approximately 39.7 minutes in our HPLC system, well after makisterone A. 428 Feldlaufer et al n 0 Q. U 1000 >- - I- > - I- 0 a a - 500 K 6 12 18 24 30 T I M E Cminl Fig. 2. Reversed-phase HPLC trace (242 nm) and RIA analyses of an extract containing the free ecdysteroids from nymphal M. quinquespinosus. Column: C8 (150 mm x 4.6 mm i.d.); mobile phase: 35% methanol/water; flow rate: 1 ml/min; column temp: 33OC. Shaded areas represent RIA activity expressed as ecdysone equivalents per gram of fresh weight and is not corrected for cross-reactivity. Solid bar indicates absorption units. The elution volumes of ecdysteroid standards are indicated by the arrows as: 1 = 20-hydroxyecdysone; 2 = makisterone A; 3 = ecdysone. DISCUSSION The neutral sterols isolated from M. quinquespinosus nymphs were closely reflective of their dietary sterols. Of the ten sterols detected in nymphs, five were C29sterols, accounting for almost 84% of the sterol composition, three were C28 sterols (12%), and two were C27sterols (0.7%). The preponderance of 28 and 29 carbon sterols in M. quinquespinosus nymphs indicates that this species is incapable of converting 24-alkyl phytosterols to cholesterol . Other species of plant-feeding hemipterans incapable of this conversion belong to the Pyrrhocoridae , the Lygaeidae , or the Pentatomidae [l]. The apparent inability of M. quinquespinosus to dealkylate dietary sterols shows that this phenomenon is not limited to only these families of true bugs. Outside the Hemiptera, several species of phytophagous Hymenoptera have also been shown to contain mostly C28and C29sterols. These include the honey bee, Apis mellifera [13-151, the alfalfa leafcutter bee, Megachile rotundata [q and a leaf-cutting cutting ant, Atta cephalotes isthmicola . Five species of Hemiptera have previously been shown to contain makisterone A as their major free ecdysteroid (see [lqfor orig. references). In most cases, these bugs-like M. quinquespinosus-are phytophagous and contain a preponderance of 24-alkyl sterols, with little or no detectable levels of cholesterol [l].The one exception is the pentatomid Podisus maculiventris, a secondarily predacious species that has very high levels of cholesterol, yet produces makisterone A as its molting hormone , apparently retaining the biosyn- Sterols and Ecdysteroids of Megalotomus 3 2 v 429 1 v v E cv * cv c I .005AU I 6 I I 12 I I 18 I I 24 I I 30 Fig. 3. Silica HPLC trace (242 nm) of the putative rnakisterone A fraction (13 and 14) from Figure 2. Column: Rainin Microsorb (150 mm x 44.6 rnrn i.d.); mobile phase: methylenechloridel2-propanoVwater (125/25/2);flow rate: 1 m l h i n ; column temp: 33OC. Standards are as in Figure 2. thetic capabilities of phytophagous ancestors. Interestingly, this species contains only low levels of campesterol(O.6%),the proposed sterol precursor of makisterone A [3,18]. A report that the adult milkweed bug, Ovlcopeltus fusciutus, can convert radiolabeled cholesterol to both C27and C28ecdysteroids  should probably be reinvestigated. The honey bee, for instance, has recently been shown to convert radiolabeled campesterol to makisterone A and radiolabeled cholesterol to 20-hydroxyecdysoner although no interconversion of either c 2 8 sterol to C27ecdysteroid, or C27sterol to c28 ecdysteroid was observed . LITERATURE CITED 1. Svoboda JA, Lusby WR, Aldrich JR: Neutral sterols of representatives of two groups of Hemiptera and their correlation to ecdysteroid content. Arch Insect Biochem Physiol I, 139 (1984). 2. Kelly TJ, Aldrich JR, Woods CW, Borkovec AB: Makisterone A: Its distribution and physiological role as the moulting hormone of true bugs. Experientia 40, 996 (1984). 3. Gibson JM, Majumder MSI, Mendis AHW, Rees HH: Absence of phytosterol deakylation and identification of the major ecdysteroid as makisterone A in Dysdercus fusciufus (HeteropteraPyrrhocoridae). Arch Insect Biochem Physiol I, 105 (1983). 4. Aldrich JR, Kelly TJ, Woods CW: Larval moulting hormone of trichophoran HemipteraHeteroptera: Makisterone A, not 20-hydroxyecdysone. J Insect Physiol28, 857 (1982). 5. Svoboda JA, Dutky SR, Robbins WE, Kaplanis JN: Sterol composition and phytosterol utilization and metabolism in the milkweed bug. Lipids 12, 318 (1977). 6. Svoboda JA, Herbert EW Jr, Lusby WR, Thompson MJ: Comparison of sterols of pollens, honeybee workers, and prepupae from field sites. Arch Insect Biochem Physiol 1, 25 (1983). 430 Feldlaufer et al 7. Svoboda JA, Lusby WR: Sterols of phytophagous and omnivorous species of Hymenoptera. Arch Insect Biochem Physiol3, 13 (1986). 8. Thompson MJ, Weirich GF, Svoboda JA: Ecdysone 3-epimerase. In: Methods in Enzymology. Law JH, Rilling HC, eds. Academic Press, New York, pp 437-442 (1985). 9. Lafont R, Pennetier J-L, Andrianjafintrimo M, Claret J, Modde J-F, Blais C: Sample processing for high-performance liquid chromatography of ecdysteroids. J Chromatogr 236, 137 (1982). 10. Feldlaufer MF, Herbert EW Jr,.Svoboda JA, Thompson MJ, Lusby WR: Makisterone A: The major ecdysteroid from the pupa of the honeybee, Apis mellifera. Insect Biochem 25, 597 (1985). 11. Feldlaufer MF, Lusby WR, Svoboda JA, Thompson MJ: Identification of 20-hydroxyecdysone and ecdysone from the pupae of the gypsy moth, Lymantria dispar. Arch Insect Biochem Physiol 2, 323 (1984). 12. Svoboda JA, Thompson MJ: Steroids. In: Comprehensive Insect Physiology, Biochemistry and Pharmacology. Kerkut GA, Gilbert LI, eds. Pergamon Press, Oxford, Vol. 10, pp 137175 (1985). 13. Barbier M, Schindler 0: Isolierung von 24-methylencholestenn aus Koniginnin und Arbeiterinnen dor Honigbiene (Apis mellifera L.). Helv Chim Acta 42, 1998 (1959). 14. Svoboda JA, Herbert EW Jr, Thompson MJ: Definitive evidence for lack of phytosterol dealkylation in honey bees. Experientia 39, 1120 (1983). 15. Feldlaufer MF, Svoboda JA, Herbert EW Jr: Makisterone A and 24-methylenecholesterol from the ovaries of the honey bee, Apis mellifera L. Experientia, (1986) 42, 200. 16. Ritter KS, Weiss BA, Norrbom AL, Nes WR: Identification of A5r7-24-methylene- and methylsterols in the brain and whole body of Attu cephalofes isthmicola. Comp Biochem Physiol 72B, 345 (1982). 17. Feldlaufer MF, Svoboda JA: Makisterone A: A 28-carbon insect ecdysteroid. Insect Biochem 26, 45 (1986). 18. Svoboda JA, Thompson MJ: Comparative sterol metabolism in insects. In: Metabolic Aspects of Lipid Nutrition in Insects. Mittler TE, Dadd RH, eds. Westview Press, Boulder CO, pp 1-16 (1983). 19. Dorn A: Ecdysone synthesis in castrated imagines of Oncopeltus fasciatus Dallas (Insecta, Heteroptera). Gen Comp Endocrinol34, 106 (1978). 20. Feldlaufer MF, Herbert EW Jr, Svoboda JA, Thompson MJ: Biosynthesis of makisterone A and 20-hydroxyecdysone from labeled sterols by the honey bee, Apis rnellifera. Arch Insect Biochem Physiol, (1986) 3, 415-421.