Neutral sterols and ecdysteroids of the solitary cactus bee Diadasia rinconis cockerell hymenopteraAnthophoridae.код для вставкиСкачать
Archives of Insect Biochemistry and Physiology 23:91-98 (1 993) Neutral Sterols and Ecdysteroids of the Solitary Cactus Bee Diadasia rinconis Cockerell (Hymenoptera: Anthophoridae) Mark F. Feldlaufer, Stephen L. Buchmann, William R. Lusbp Gunter F. Wirich, and JamesA. Swboda U S Depnrtment of Agrtcrrlfrrre, Agricultural Research Service, Insect Neurobrohgy t3 Hormone Laboratory [M F F , W R L , G F W , A S j, Bcltsuzlle, Maryland, and Honey Bee 6 Insat Biolopal Control Laboratory (5 L BJ, Turson, Arizona Using high performance liquid chromatography i n conjunction with radioimmunoassay and mass spectrometry, the major ecdysteroid of the solitary cactus hee, Diadasia rinconis, was detcrmined to be 20-hydroxyecdysone, with lesser amounts of rnakisterone A. Another 28-carbon ecdysteroid thought to be the 24-epimer of makisterone A was also detected. The neutral sterols of Diadasia consisted primarily of Z4-me1hylenecholesterol (92.2%) with lesser amounts of other Cz8 and C2g sterols. Cholesterol accounted for less than 0.1 % ' of the total tissue sterols. The occurrence of 20-hydroxyecdysorw in a phytophagous hymenopteran i s discussed i n relation to the luw level of cholesterol encountcred. cz 1993 ~ i ~ e y - ~ iInc.* ss, Key words: 20-hydroxyecdysone, makisterone A, 24-methylenecholesterol INTRODUCTION The solitary cactus bee Diudasza rinconis is an important pollinator of several Sonoran cacti, feeding primarily on the pollen of Engelmann's prickly pear (Oyuntiu phneacantha)and to a lesser extent on saguaro (Cereus gi$pnieus) and cholla (Opuntiu uersicolor). Our interest in these bees stems from the fact that other phytophagous bees, including the honey bee, Apis nzellifera, are unable to convert dietary plant sterols to cholesterol [1,2], a pathway common to many other phytophagous insect species (see ). We have also shown that the honey bee utilizes the 28-carbon sterol campesterol to synthesize makiAcknowledgments: Thetechnical expertise of Kenneth R. Wilzer, Jr., and Dawn J. Harrison i s greatly appreciated. We also thank Patricia Maurer, INRA, Cuyancourt, France, for chromatographic data concerning 24-epi-makisteroneA and Oliver W. Howarth, Department of Biochemistry, University of Liverpool, for NMR analyses of our refwence rnakisterone A. Received August 26, 1992; accepted October 13, 1992. Address reprint rcquests to Mark F. Feldldufer, Insect Neurobiology 5; Hormone Laboratory, Bldg. 467, BARC-East, Beltsville, MD 20705. 01993 Wiley-Liss,Inc. *This article i s a US Government work and, as such, is in the public domain in the United States of America. 92 Feldlaufer et al. sterone A as its major ecdysteroid [4,5]. We now report on the neutral sterol composition of Diadasia bees and provide evidence that these bees cannot deakylate dietary plant sterols to cholesterol, yet synthesize both the C27 ecdysteroid, 20-hydroxyecdysone, and thc c28 ecdysteroid, makisterone A. MATERIALS AND METHODS Biological Material Pollen from saguaro was both hand-collected and collected from bee trdps outside of Tucson, Arizona. Adult female D. vinconzs were collected from the same area. All samples were stored in methanol at -20°C until analysis. Insect Extractions Adult female D. rinconis (26.61 g fresh wt.) were homogenized in methanol (2 x 200 ml), filtered, and rehomogenized in 75% rnethanol/water (1 x 100 ml). After filtering, the combined filtrates were dried in vacuo. The residue was partitioned between 70% methanoliwater and n-hexane (countersaturated) to remove apolar lipids. The hexane phase was set aside for neutral sterol determination, while the dried methanolic residue (1.78 g) was partitioned between n-butanol and water (countersdturated). The butanolic phase yielded approximately 110 mg of dry residue for ecdysteroid analyses. Sterol Purification The hexane phase from the 70% methanollwater partition was used to determine the relative percentages of neutral sterols in the adult bees. In addition, saguaro pollen was examined for sterol content. Both the bee sample and a methanolic extract of the pollen sample were saponified under reflux using 4% KOH in a solution of ethanol/benzene/water (1O:l:l). After several hours, the solutions were allowed to cool, were acidified with 6 N HC1, and were extracted with hexane (3 x ) and diethyl ether (1 X ). The organic phases of both the bees and pollen were dried over anhydrous magnesium sulfate and then the solvent was removed using a rotary evaporator. The dried residues were fractionated on Florisil (60-100 mesh; Fisher Scientific, Fair Lawn, NJ) in a diethyl etherhexane system as previously described .All fractions were monitored by TLC and capillary GLC and sterols were purified prior to mass spectrometry by reversed-phase HPLC. Ecdysteroid Purification The butanolic residue (110 mg) of the Diadasia extract was fractionated on a column (10 mm i.d.) of silica gel (5 g; 70-230 mesh; EM Science, Gibbstown, NJ) packed in a chloroform slurry and eluted with 50 ml portions of increasing strengths of ethanol in chloroform (5%, 15%, 25% , 40%, and 100%).Ecdysteroids were present in the 15% and 25% fractions (determined by RIA), so these fractions were combined, dried, and then eluted from a c18 SEP-PAK (Waters, Milford, MA) in a methanoliwater system previously described . The purified extract was fractionated by reversed-phase HPLC. One milliliter Sterols and Ecdysteroids of Diadasia Bees 93 fractions were collected and analyzed by RIA, and immunoreactive peaks were further purified by silica HPLC prior to mass spectrometry. Chemicals and Instrumentation All solvents for extraction and purification were reagent grade, redistilled. Solvents for HPLC were from Burdick & Jackson(Baxtcr Scientific, Columbia, MD). Ecdysteroid standards were obtained from Simes (Milan, Italy). For neutral sterol purification and identification, samples wcre analyzed by TLC on high performance silica gel 60 F234 plates (Merck, Darmstadt, Germany) developed in hexaneldiethyletheriaceticacid (60:40:1). Reversed-phase HPLC was performed on a Shandon ODS Hypersil column (250 x 4.6 mm; 5 Fmparticle size;Pittsburgh, PA) eluted with987~rnethanoliwater at 1mllmin. The effluent was monitored at 200 and 215 nm with a Waters 990 photodiode array detector. CapiIIary GLC was performed isothermaiIy at 245°C on a GC-9A gas chromatograph (Shimadzu, Columbia, MD) equipped with a DB-1 fused silica column (15 m x 0.25 mm; 0.25 pm film; J&W Scientific, Folsom, CA). Mass spectra were obtained on a Finnigan 4500 fitted with a J&W DB-1 fused silica column (30 m x 0.32 mm; 0.25 p m film) and temperature programmed from 230°C to 255°C (increased at 5"C/min). Electron impact spectra were collected at 70 eV and a source block temperature of 150°C. For ecdysteroid analyses, reversed-phase HPLC was performed on an IBM octyl column (150 mm x 4.6 mm; 5wm particle size; Danbury, CT) eluted with 37" aqueous methanol at 1ml/min. The effluent was monitored at 215 and 248 nm using a photodiode array detector. Peaks that were immunoreactive (as determined by RIA) and had a UV spectrum similar to an ecdysteroid were subsequently fractionated on a NOVA-PAK silica column (150mm x 4.6 mm; Waters) eluted with methylene chloride:2-propanol:water(125:25:2) at 1 mumin. Putative ecdysteroids were then identified by methane and ammonia desorption chemical ionization mass spectrometry as previously described [4,81. RIA The RIA utilized in this study has been prcviously described [8,9], except that the radioligand ([23,24-'H] ecdysone; specific activity 83 Ci/mmol) was obtained from Dupont (Wilmington, DE) The cross reactivity factors for 20-hydroxyecdysone and makisterone A wcre 4 and 6.65, respectively. RESULTS Sterol Analyses The relative percentages of neutral sterols isolated from adult female D. rinconis a n d saguaro pollen are presented in Table 1. The pollen sterols of Engelmann's prickly pear cactus and of cholla have also been included, since D. rinconis frequent these cacti in addition to saguaro. The predominant sterol in female bees was 24-methylenecholesterol, accounting for over 92% of the total sterols. The remaining sterols cunsisted of small percentages of C2s and C29sterols. Saguaro pollen also Feldlaufer et at. 94 TABLE 1. Relative Percentages of Neutral Sterols From Adult Female D. rinconis and From Cactus Pollens as Determined by GLC-Mass Spectrometry Sterol Cactus bee Saguaro Prickly peai-” Cholla“ (0.1 - 92.2 1.7 0.8 80.3 0.4 - 2.3 91.2 - 1.2 0.6 1.7 0.7 1.8 - - - 1.0 1.3 2.3 7.3 - 5.6 6.6 - 2.2 7.2 2.0 Cholesterol Pollinastanol 24-Dchydropollindstdnol 24-Methylenecholesterol Campesterol 31-Norcycloartenol 24-Methylertepolli~~as tan01 Sitosterol Fucosterol Cycloeucalenol Cycloartenol b - 2.4 - 0.7 81.2 1.1 aFromLusby et al. (unreported data) %lot detected. 2 J. 120 1 0-J e 0 4 8 12 16 20 Time (min.) Fig. 1 . Reversed-phase HPLC trace (248 nm) and RIA analyses of an extract containing the free ecdysteroidsfrom adult D. rinconis females (1 96 of sample). Shaded areas represent RIA activity of the injected sample and are not corrected for cross-reactivity. Elution volumes of knawn standdrds are indicated by the arrows as: 1 = 20-hydroxyecdysone;2 = rnakisterone A; 3 = ecdysone. Fractions indicated by “a” and ” b were collected and rechromatographed under normal phase conditions. Sterols and Ecdysteroids of Diadasia Bees 95 if A 1 I I I I 0 4 8 12 t6 Time (min.) Fig, 2. Uortrial (silica) phase HPLC trace (240 nm) of the putative frec ccdystcroids collected from reversed-phase HPLC. A: The putative 20-hydroxyecdysone peak ("a" from Fig. 1). 8: The putative rnakisterone A peak ("b" from Fig. 1 ) Standards are as in Figure 1. Bar indicating compound " X " was thought to be the 24-epimer of rnakisterone A (see Results). contained mostly 24-methylenecholesterol (91.2%), as did prickly pear (80.3%)and cholla (81.2%).Although cholesterol was not detected in any of the pollen sources, it was detected in the bee sample, though only in trace amounts (less than O.l%), 96 Feldlaufer et al. Ecdysteroid Analyses Analysis of the Uiudusiu ecdysteroids by reversed-phase HPLC in conjunction with RIA revealed two immunoreactive areas (Fig. 1).The first area eluted in fractions 7 and 8 and corresponded to a visible, U’J-absorbing peak (”a”) having a retention time similar to that of 20-hydroxyecdysone. The second area (fractions 12 and 13) corresponded to a smaller peak (“b”) having a retention time similar to a makisterone A standard, In addition to being immunoreactive and matching the retention times of known standards, both peaks had a UV spectrum (from 190 to 400 nm) indicative of an ecdysteroid. The remaining bulk of the sample was fractionated, and peaks “a” and “b” were collected separately. Both peaks were then individually fractionated by silica HPLC (Fig. 2). Peak “a” exhibited a retention time identical to that of 20-hydroxyecdysone (Fig. 2A). Analysis by mass spectrometry indicated a molecular weight of 480, as well as a spectrum matching that of authentic 20-hydroxyecdysone . When the putative makisterone A (”b”)was fractionated by silica HPLC, two peaks were resolved (Fig. 2B). The larger of the two peaks had a retention time and mass spectrum identical to that of our makisterone A standard. Interestingly, the smaller of the two peaks (designated ” x ”) had a UV spectrum indicative of an ecdysteroid (Amax = 248 nm). Analysis of X ” by mass spectrometry yielded a molecular weight of 494 and a spectrum identical to that of rnakisterone A. Enough material did not exist for NMR analysis. However, based on mass spectrometry and the chromatographic behavior of ” x “ relative to makisterone A in two solvent systems, compound ” x ” is thought to be 24-epi-makisterone A (P. Maurer, personal communication). Overall, there was approximately three to four times the amount of 20hydroxyecdysone as makisterone A and about five times the amount of makisterone A as the putative 24-epimer. “ DISCUSSION 24-Methylenecholesterol was the major sterol in adult Diadusiu females. Since this sterol was also the predominant sterol in all dietary (pollen) sources, there is no indication for the existence of a selective sterol transfer mechanism in D. rinconis of the kind shown to exist in the honey bee, where nurse bees selectively transferred 24-methylenecholesterol to the developing brood, regardless of the sterol composition of the adult bees (see [lo]). Additionally, the high percentage (92.2) of 24-methylenecholesterol in the bees could be a result of pollen in the crop or gut, since no effort was made to remove the digestive tract prior to extraction. It is probably fair to assume, though, that Diadasiu, like the honey bee, is incapable of converting plant sterols to cholesterol, since the tissue sterols of Diadasiu contained only trace amounts of cholestcrol. The origin of this small amount of cholesterol in the cactus bees is obscure. Since cholesterol was not detected in any of the dietary sources examined, the possibility exists that D . rinconis visited an additional pollen source that may have contained cholesterol. Sterols and Ecdysteroids of Diadasia Bees 97 Given the small amount of cholesterol present, it is odd that 20-hydroxyecdysonc is the major ecdysteroid in adult females. While A . mellifera pupae were shown to be able to synthesize 20-hydroxyecdysonewhen injected with radiolabeled cholesterol ,makisterone A was the major endogenous ecdysteroid, with little or no 20-hydroxyecdysone found 141. While 20-hydroxyecdysone was detected in larval honey bees, makisterone A was still the predominant molting hormone present [ll]. The ovaries from honey bee queens also contained only makisterone A, though cholesterol accounted for 0.7% of the tissue sterol 1121. Maurer et al. have demonstrated that pupae of the leaf-cutting ant Acrornyrmex octospinosus, whose fungal diet contains little cholesterol, nevertheless contain a mixture of cZ7 and c 2 8 ecdysteroids, though 20-hydroxyecdysone was six times less prevalent than the 28-carbon molting hormones detected [13,14]. The production of 20-hydroxyecdysone by Diadasiu females is reminiscent of the house fly, M u m dornesfica, whose pupae were shown to produce 20-hydroxyecdysoneeven in a low cholesterol environment . Taken together, these studies indicate that it is not prudent to generalize about the utilization of dietary plant sterols for ecdysteroid biosynthesis even among closely related taxonomic groups. LITERATURE CITED 1. 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