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Neutral sterols and ecdysteroids of the solitary cactus bee Diadasia rinconis cockerell hymenopteraAnthophoridae.

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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.*
Key words: 20-hydroxyecdysone, makisterone A, 24-methylenecholesterol
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 [3]). 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.
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.
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 [6].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 [7].
The purified extract was fractionated by reversed-phase HPLC. One milliliter
Sterols and Ecdysteroids of Diadasia Bees
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
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.
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.
TABLE 1. Relative Percentages of Neutral Sterols From Adult Female D. rinconis and From
Cactus Pollens as Determined by GLC-Mass Spectrometry
Cactus bee
Prickly peai-”
aFromLusby et al. (unreported data)
%lot detected.
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
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%),
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 [9]. 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.
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
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 [5],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 [15]. 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.
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dealkylation in honey bees. Experientia 39, 1120 (1983).
2. Svoboda JA, Lusby WR: Sterols of phytophagous and omnivorous Hymenoptera. Arch
Insect Biochem Physiol3, 13 (1986).
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and Pharmacology. Kerkut GA, Gilbert LI, eds. Pergamon Press, Oxford, vol10, pp 137-175
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major ecdysteroid from the pupa of the honeybee, Apis mellifera. Insect Biochem 15, 597
5. Feldlaufer MF, Herbert EW Jr, Svoboda JA, Thompson MJ: Biosynthesis of makisterone A
and 20-hydroxyecdysone from labeled sterols by the honey bee, Apis ttielliferu. Arch Insect
Biochem Physiol3, 414 (1986).
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Feldlaufer et al.
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12. Feldlaufer MF, Svoboda JA, Herbert EW Jr: Makisterone A and 24-methylenecholesterol
from the ovaries of the honey bee, Apis rnellifern L. Experientia 42, 200 (1986).
13. Maurer P, Royer C, Mauchamp B, Porcheron P, Debieu D, Riba G: Occurrence of 28- and
27-carbon ecdysteroids and sterols in developing worker pupae of the leaf-cutting ant
Acromyrmex ocfospinosus (Reich) (Hymenoptera, Fwmicidae: Attini). Arch Insect Biochem
PhysioI16, 1(1991).
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rinconis, neutral, hymenopteraanthophoridae, diadasia, cockerell, cactus, ecdysteroids, sterol, bee, solitary
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