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Long-term cholesterol labeling as a convenient means for measuring ecdysteroid production and catabolism in vivoApplication to the last larval instar of Pieris brassicae.

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Archives of Insect Biochemistry and Physiology 5:139-154 (1987)
Long-Term Cholesterol Labeling as a
Convenient Means for Measuring Ecdysteroid
Production and Catabolism In Vivo:
Application to the Last Larval lnstar of
Pieris Brassicae
Philippe Beydon and Rene Lafont
Ecole Nomale Supkrieure, Laboratoire de Zoologie, Paris, France
In vivo biosynthesis of ecdysteroids during t h e last larval instar of Pieris
brassicae was investigated by administering [3H]cholesterol followed byhighperformance liquid chromatography analysis of t h e resulting [3H]
ecdysteroids. The demonstration that t h e specific activity of t h e ecdysteroids
synthesized at a given time is always identical with that of cholesterol indicates
that t h e cholesterol pool is uniformly labeled, and this allows u s to easily
calculate t h e amounts of ecdysteroids produced by animals. The total amount
of ecdysone produced throughout t h e last larval instar was measured as 1.17
nmol/insect. This quantity is more than three-fold t h e maximal level of molting
hormones (ecdysone + 20-hydroxyecdysone) reached during t h e instar (0.37
nmollanimal) because a high catabolic activity occurs at the-beginning of t h e
hormone production period. Larvae thus differ from pupae, where catabolism
is minimal w h e n ecdysone synthesis takes place, resulting in a more
“economical” system.
Key words: development, ecdysone, HPLC, lepidoptera, stools
INTRODUCTION
Ecdysteroids are steroid hormones that control molting and development
of insects. Ecdysteroids are synthesized by insects from sterols originating in
food. Endocrine glands, the molting glands, produce and secrete ecdysone,
Acknowledgments: We are grateful to Dr. J.-M. Ferezou, Ecole Normale Superieure, for GLC
analyses. We acknowledge Dr. C. Hetru, University of Strasbourg, for a gift of [3H]2-deoxyecdysone. We thank Mrs. Morin for mass spectra. We thank Dr. C. Blais for valuable discussions.
Received December 16,1985; accepted December 16,1986.
Address reprint requests to Philippe Beydon, Ecole Norrnale Superieure, Laboratoire d e
Zoologie, CNRS UA 686, 46 rue d’Uhm, 75230 Paris cedex 05, France.
0 1987 Alan R. Liss, Inc.
140
Beydon and Lafont
which is afterward converted by peripheral tissues into various metabolites.
Among them 20E" is considered as the active hormone and is generally the
predominant ecdysteroid in hemolymph. Other metabolites (e.g., 3-epiecdysteroids and ecdysonoic acids) are probably inactive compounds that are
often found in feces [l].
Variations of molting hormone titers (ecdysone + 20E) in hemolymph are
now well documented in many insect species. A large hormone peak occurs
before every molt. During the last larval instar of many holometabolous
species, an additional early small hormone peak is observed, which has been
correlated with the commitment for metamorphosis [2]. Molting hormone
titer changes result from two opposite factors: the ecdysone biosynthetic rate
and the catabolic activity. Both factors have been shown to vary considerably
within each instar [3,4], but their relative contribution to hormone titer
changes is somewhat difficult to determine. In order to answer this question,
several approaches are possible. The first would include in vitro measurements of ecdysone production by molting glands and in vivo catabolic studies
of injected labelled ecdysone or 20-hydroxyecdysone. Another approach
would be the measurement by appropriate radioimmunoassay(s)of ecdysone
and all its subsequent metabolites, as realized by Warren et al. in Manduca
sextu pupae [5] and embryos [6]. Both methods are very time-consuming
experiments and moreover an objection can be made because the first method
may not exactly reflect the true situation. A third type of attempt, previously
applied to Pieris brussicne pupae [7J, uses long-term labeling of the ecdysteroid
pool with [3H]cholesterol.
The aim of this paper is to further assess the validity of this in vivo
cholesterol labeling method and to discuss some limitations in its general
use. The present study was performed to estimate the in situ ecdysone
production and to determine the relative contribution of biosynthesis and
catabolism with changes in the hormone titer that are observed during the
last larval instar of P. brassicae.
MATERIALS AND METHODS
Animals
P. brussicae larvae were reared on cabbage leaves at constant temperature
(23°C) with a long-day photoperiod. Under these conditions, the last larval
instar lasts 5 days. Four successive developmental stages were distinguished
during this instar: a feeding stage, a wandering stage, a spinning stage, and
then a pharate pupal stage that precedes the pupal ecdysis. Until the wan-
'Abbreviations: 3DE = 3-dehydroecdysone; 3D20E = 3-dehydro-20-hydroxyecdysone; E =
ecdysone; E' = 3-epiecdysone; Eoic = ecdysone-26-oic acid; E'oic = 3-epi-ecdysone-26-oic
acid; E'3P = 3-epiecdysone-3-phosphate; 20E = 20-hydroxyecdysone; 20E' = 3-epi-20-hydroxy-ecdysone; 20Eoic = 20-hydroxyecdysone-26-oic acid; 20E'oic = 3-epi-20-hydroxyecdysone-26-oic acid; 20E'3P = 3-epi-20-hydroxyecdysone-3-phosphate; FW = fresh weight; CLC
= gas-liquid chromatography; HPLC = high-performance liquid chromatography; NP-HPLC
= normal phase HPLC; POPOP = 1,4-di-[2-(phenyloxazoyl)]-benzene; PPO = 2,5-diphenyloxazole; RP-HPLC = reversed phase HPLC; Tris = Tris (hydroxymethy1)-aminomethane.
Ecdysteroid Production in Pieris
141
dering stage, larvae excrete green feces; then they excrete red feces (colored
by ommochromes) at the end of the wandering stage and during the spinning stage.
Chemicals
[la,2c~(N)-~H]
Cholesterol (60 Cilmmol) and [4--14] cholesterol (50 mCil
mmol) were purchased from CEA (Saclay, France). Labeled ecdysone was
prepared in vitro by incubation of [23,24-3H]2-deoxyecdysone (108 Cilmmol,
a gift from Dr Charles Hbtru, Strasbourg, France) with adult male locust
Malpighian tubules [S]. Unlabeled cholesterol was obtained from Sigma (St.
Louis, MO, USA). Unlabeled ecdysteroids-ecdysone, 20-hydroxyecdysone,
and muristerone A-were from Simes (Milano, Italy). Helix digestive juice
was from Merck (E. Merck A. -G., Darmstadt, Federal Republic of Germany).
Thin-Layer Chromatography
TLC of sterols was carried out on silica gel plates (Kieselgel60 F254;Merck)
with hexanelethyl acetate (70:30) for development. After development, sterols were detected under UV light (350 nm) by spraying 2',7'-dichlorofluorescein 0.1% for visualization (Merck); the corresponding band was scraped and
eluted with chloroform.
TLC of ecdysteroids was carried out on silica gel with chloroformlmethano1 (80:20). After development, ecdysteroids were detected under UV light
(254 nm), and eluted from the gel with chloroformlmethanol (1:l).
Gas-Liquid Chromatography
Before GLC analysis, sterols were silylated by heating with pyridinel
hexamethyldisilazaneltrimethylchlorosilane(10:4:2) (250 pl, 70"C, 20 min). A
Girdel gas chromatograph (Girdel, Puteaux, France) was used, fitted with a
flame ionization detector and a 50 m x 0.32 mm capillary column (CPtm Sil
8 CB from Chrompack (Middelburg, Netherlands). The conditions were:
column temperature 290°C; helium carrier gas, 1.2 bar.
High-Performance Liquid Chromatography
HPLC of sterols was performed with a Kratos instrument (Ramsey, USA)
including a Spectroflow 400 pump in conjunction with a Spectroflow 430
gradient former, and a Spectroflow 757 absorbance detector set at 210 nm.
Sterols were separated on a Hypersil-ODS column (25 cm x 4.6 mm, particle
size 5 pm, Chroma-science S.A. Le Blanc Mesnil, France) eluted at 2 mllmin
with acetonitrilelpropanol-2 (85:15) [9].
HPLC of ecdysteroids was carried out with a Kratos instrument (see above)
or a Waters instrument (Milford, MA), incorporating two M6000A pumps in
conjunction with a M720 system controller, a Wisp automatic injector, and a
M440 detector set at 254 nm. Ecdysteroids were separated using either: 1)
RP-HPLC on a Spherisorb-ODScolumn (25 cm x 4.6 mm, particle size 5 pm,
Prolabo, Paris, France) eluted at 1mllmin with a linear gradient of acetonitrile
in 20 mM TrislHC104buffer (pH = 7.5) or in 20 mM potassium citrate buffer
142
Beydon and Lafont
at pH 6.6 or pH 3.3; or 2) NP-HPLC (Zorbax-SIL column, 25 cm x 4.6 mm,
particle size 5 pm, DuPont de Nemours) eluted with 1mllmin dichloromethanelpropanoL2lwater (125:25:2) [lo]. Fractions were collected by a LKB
Redirac fraction collector (LKB, Bromma, Sweden). A Delsi Enica 10 integrator (Delsi Instrument, Suresnes, France) was used for quantification of the
UV peaks.
Radiochemical Method
Aqueous samples were mixed with three volumes of a scintillation cocktail
(5.5 g PPO, 0.2 g POPOP, 330 mg Triton X l O O and 670 ml toluene), and
counted in a Kontron scintillation spectrometer MR 300 (Kontron, Basle,
Switzerland). Organic samples were evaporated under reduced pressure or
a nitrogen flow, then dissolved in the scintillation cocktail.
Mass Spectrometric Method
Sterol mass spectra were determined with a Riber 10-1OB (Nermag S.A.,
Rueil, France) apparatus equipped with a direct inlet probe. Spectra were
obtained with the electron impact mode.
Administration of Labeled Cholesterol
Labeled cholesterol was administered either by injection or per 0s. For
injection, [3H]cholesterol(50 pcilanimal, 5 pllanimal) was emulsified in water
with Tween 80 (Sigma), 10 times the weight of sterol [ll]. For administration
per os, labeled cholesterol [3H] (50 pCi) andlor [I4C] (5 pCi) was deposited
on cabbage leaves in diethyl ether.
Ecdysone Labeling
[3H]Ecdysone (0.5 pcilanimal) was injected in water (5 pllanimal). To
obtain the ecdysone metabolites from young pupae, [3H]ecdysone was injected into spinning larvae and then the animals were killed after the pupal
ecdysis. For the ecdysone metabolites from red feces (or larval meconium),
ecdysone was injected into wandering larvae; red feces eliminated by the
animals were collected for analysis. The ecdysone metabolites from green
feces were obtained by collecting the feces excreted during the 24 h following
injection of [3H]ecdysoneinto 2-day feeding larvae.
Extraction of Sterols
Samples (0.1-1 g FW) were extracted with 5% potassium hydroxide in
ethanollwater (9:l) (5 ml) and then saponified for 2 h at 60°C. Water (2 ml)
was added and then the nonsaponifiable material was extracted into hexane
(10 ml), evaporated to dryness, and purified by TLC. The resulting free
sterols were analyzed by HPLC. For quantification of cholesterol,
[3H]cholesterol(0.1 pCi) was added before extraction as an internal standard.
The overall recovery (about 60%) was determined during HPLC analysis by
collecting and counting the cholesterol peak.
Ecdysteroid Production in Pieris
143
Extraction of Ecdysteroids
Insects or feces (0.1-1 g FW)were extracted by chloroformlwater (l:l/ 20
ml) [12]. After centrifugation, ecdysteroids were recovered in the aqueous
phase and cholesterol in the chloroform phase. The chloroform phase and
pellet were extracted again with water (10 ml). In cholesterol labeling, the
pellet was further extracted with acetonelethanol (Il, 10 ml). After centrifugation the supernatant was added to the chloroform phase. Nearly all unconverted labeled cholesterol was in this mixture. Ecdysteroids were adsorbed
in a C-18 Sep-pak cartridge (Waters) and then eluted with methanol (5 ml)
and evaporated to dryness. Ecdysteroids were analyzed either directly by
HPLC or by TLC followed by HPLC. Muristerone A (1or 10 p g ) was added
before extraction as internal standard. The muristerone A recovery was
determined during HPLC analysis by uv 254 nm monitoring.
Incorporation Rate Determination
The incorporation rate (R) of labeled cholesterol into ecdysteroids was
calculated according to the formula: R = c I r -T where c = radioactivity
contained in a given ecdysteroid analyzed by HPLC, r = recovery by purification (average 80%), and T = radioactivity of the injected cholesterol (or
radioactivity remaining in the animals at a given time). In practice, T was
equivalent to the radioactivity contained in the chloroform phase and the
acetonelethanol extract, as the incorporation into polar compounds of the
aqueous phase (ecdysteroids) is negligible.
Specific Activity Determinations
The specific activity of cholesterol and of ecdysteroids was determined by
HPLC. Samples were extracted as usual by chloroformlwater; cholesterol
was recovered in the chloroform phase and ecdysteroids in the aqueous
phase. The chloroform phase was evaporated and then the sterols were
saponified (see above). Quantifications of cholesterol were performed by UV
210 nm monitoring. The cholesterol peak was collected and then its radioactivity was measured. Ecdysteroids in water phase were processed on a C-18
Sep-pak cartridge. After TLC of the sample, 20E peak was analyzed twice,
by RP-HPLC and by NP-HPLC. For 20E’oic, the sample was purified by Seppak then by RP-HPLC at pH 6.6. The 2OE’oic peak was collected, desalted
on a Sep-pak cartridge, and then analyzed by RP-HPLC (pH = 3.3) for
calculation of specific activity. For the 20E‘3P specific activity determination,
the sample was purified by Sep-pak and then by RP-HPLC at pH 6.6. The
sample was afterward incubated in Helix juice to hydrolyze 20Er3Pinto 20E’
(10 pl Helix juice, in 1 ml potassium citrate buffer at pH 5.5, overnight, at
37°C). 20E’ was analyzed by NP-HPLC. Ecdysteroids were quantified by UV
254 nm absorbance monitoring, and peaks were collected for radioactivity
determination.
Assay of Ecdysone and 20-Hydroxyecdysonein Pieris larvae
Extraction of larvae and partial purification of molting hormones were
done as usual (see above). Levels of ecdysone and 20E were determined by
RP-HPLC [12].
144
Beydon and Lafont
RESULTS
Comparison of Ecdysteroids Obtained After [3H]Cholesterol and
[3H]Ecdysone Labeling
[3H]Cholesterol(50 pcilanimal) was injected into 20 synchronized larvae 2
h after the last larval ecdysis. Injected larvae were reared in individual boxes.
Every 12-h period, the feces produced by each insect were collected and
stored at -20°C until use. Insects were killed by freezing a few hours after
the pupal ecdysis. The different samples (feces and young pupae) were
examined for ecdysteroids by RP-HPLC at pH = 6.6 and pH = 3.3 (Fig. 1).
Retention time of polar ecdysteroids (e.g., ecdysonoic acids or phosphate
conjugates) depends on pH and this provides an easy test for the identification of such compounds. We have distinguished the green feces, and the red
feces, or meconium among the feces samples. Ecdysteroid patterns obtained
with [3H]ecdysone labeling are given in Figure 2. The comparison between
two similar samples indicated that most labeled HPLC peaks originating
from [3H]cholesterol are ecdysteroids, since they are also found after
E
C
P
lo3
Fig. 1. RP-HPLC analyses of [3H]cholesterol metabolites. [3H]Cholesterol was injected into
fieris brassicae larvae after the last larval ecdysis. Operating conditions: column Spherisorb
ODS (25 cm x 4.6mm); flow rate 1 mllmin; linear gradient (in 40 min) from 8% to 30%
acetonitrile in 20 m M potassium citrate buffer at pH = 6.6 (A,C, and E) or pH = 3.3 (B,D, and
F); 0.4-min fractions were collecting. [3H]Cholesterol metabolites in insect before the pupal
ecdysis (A and B), in red feces eliminated during the spinning stage (C and D), and in green
feces eliminated during the feeding stage (E and F). Abscissa units: fraction number. See
abbreviations footnote.
Ecdysteroid Production in Pieris
145
E C
10'
"-
0
50
m
1
w
1w
50
50
F
Y
0
Fig. 2. RP-HPLC analyses of [3H]ecdysone metbolites in Pieris brassicae at p H = 6.6 (A, C,
and E) and pH = 3.3 (B,D, and F). Operating conditions and sampling as in Figure 1. Abscissa
units: fraction number.
[3H]ecdysonelabeling. The chemical nature of these ecdysteroids was previously determined [13]. Moreover, cholesterol labeling confirms the endogenous occurrence of 3-dehydroecdysteroids. The ecdysteroid patterns differ
according to sample. No predominant ecdysteroid is found in green feces;
ecdysonoic acids (20Eoic, 20E'oic, Eoic), phosphate conjugates (20Er3P,E3'P),
3-epiecdysteroids (20E', E'), 3-dehydroecdysteroids (3D20E, 3DE) were detected (Fig. 1E,F). The occurrence of unconverted molting hormones (E +
20E) in green feces has also to be emphasized. In red feces, the predominant
labeled compounds were 20E'3P and 3D20E (Fig. 1C,D). In freshly ecdysed
pupae, the major ecdysteroids were 20E' and 20E'oic.
From these data, we calculated incorporation rates of [3H]cholesterol into
ecdysteroids ( = the sum of all the labeled HPLC peaks, value corrected for
recovery) during the larval-pupal development. The incorporation rates in
feces were calculated by considering the amount of [3H]cholesterol actually
present in animals when feces were produced, since a part of the injected
[3H]cholesterol is eliminated in feces throughout the larval-pupal development (Table 1).Most ecdysteroids recovered in feces were probably synthesized a few hours earlier because the ecdysone half-life (calculated from [3H]ecdysone labeling) in feeding larvae is short (2-3 h). The incorporation rates
in feces increase continuously during the feeding and the wandering stages
(Table 2). The ecdysteroids present at the end of larval-pupal development
146
Beydon and Lafont
TABLE 1. Radioactivity Recovered in Feces of a
Last Instar Larva*
Feces
Hours
0-24
24-36
36-48
48-60
60-72
72-84
In insecta
Radioactivity
x lo6 dpm
x lo6 dpm
x 106dpm
x lo6 dpm
x 106dpm
x lo6 dpm
94. x lo6 dpm
6.0
2.8
2.2
2.2
2.2
9.2
*The insect was injected with [3H]cholesterol after the last
larval ecdysis. Feces produced by the insect were collected.
aRadioactivity recovered in the insect after the pupal ecdysis.
TABLE 2. Incorporation Rates of [3H]Cholesterol Into
Ecdysteroids Recovered in Feces Eliminated During the Last
Larval Instar, and in Insect After the Pupal Ecdysis
Feces
Incorporation
rates
(% f SD)
Hours
0-24
24-36
36-48
48-60
60-72
72-Ma
0.004
0.005
0.007
0.013
f 0.001
f 0.002
& 0.002
f 0.003
0.018 f 0.005
0.057 f 0.010
Insectb
0.077
0.013
aRed feces, larval meconium.
bIncorporation rates in insect after pupal ecdysis.
are stored inside pupal meconium during the whole pupal-adult development and then are eliminated at adult emergence.
Now we may consider how the incorporation rates for cholesterol into
ecdysteroids can be converted into ecdysone biosynthesis rates.
Relationship Between Incorporation Rate and Biosynthetic ActivityChoice of a Method
If the specific activity of ecdysteroids is identical to that of cholesterol, a
simple relation would exist between incorporation rate and biosynthetic
activity. This biosynthetic activity could be calculated as the roduct of the
cholesterol content in the insect by the incorporation rate of [ H]cholesterol
into ecdysteroids. Thus we have 1)to demonstrate that specific activities are
equal and 2) to measure the cholesterol content of Pieris larvae during the
fifth instar. In growing larvae cholesterol pools increase, which results in a
decrease of cholesterol specific activity.
We decided to compare the specific activities of cholesterol and of selected
ecdysteroids at specific times of larval-pupal development, when these ec-
Ecdysteroid Production in Pieris
147
dysteroids are major metabolites (e.g., 20E at the spinning stage when the
hormone titer is high; 20E’oic at pupal ecdysis; 20E’3P in red feces).
As a prerequisite, we analyzed the sterols in P. brassicae larvae by HPLC
(Fig. 3A). Four major peaks (peak 1, 2, 3, and 4) were observed. Peak 2
comigrated with authentic cholesterol. Peaks 3 and 4 were sterols originating
from cabbage (Fig. 3B). Every peak was collected for further analyses. Every
HPLC peak also gave a single peak in GLC (Table 3). Peak 2 gave a single
peak in GLC that comigrated with cholesterol. The sterols were characterized
by mass spectrometry by comparison with reference spectra [14]. Peak 1was
identified as desmosterol, mlz 384 (M +, 18%),369 (22%), 351 (11?40), 300
(26%), 271 (loo%), 253 (22%), 219 (15%), 213 (21%); peak 2 as cholesterol, ml
z 386 (M loo%), 371 (33%), 368 (51%), 353 (37%), 326 (13%), 313 (6%), 301
(70%), 275 (98%), 273 (58%), 255 (8O%), 247 @YO), 213 (96%); peak 3 as
campesterol, mlz 400 (M + 100%), 385 (28%), 382 (40%), 367 (29%), 315
(%YO), 289 (@YO),273 (34%), 255 (46%), 231 (37%), 213 (69%); peak 4 as
sitosterol, mlz 414 (M +, loo%), 399 (24%), 396 (34%), 381 (28%), 329 (@YO),
303 (36%), 273 (32%), 255 (36%), 231 (26%), 213 (52%).
+
A
0
2
5
0
10
time
5
10
(min)
Fig. 3. HPLC analysis of the sterols from Pieris brassicae spinning larvae (A) and from
cabbage on which larvae were reared (B). UV 210 nrn absorbance was monitored. Operating
conditions: column Hypersil ODS (25 cm x 4.6 mrn); flow rate 2 rnllrnin; solvent: acetonitrilel
propanol-2 (85:15). 1, desrnosterol; 2, cholesterol; 3, campesterol; 4, sitosterol.
TABLE 3. The Relative Retention Times* of the Sterols From
Pieris brussicue in HPLC and GLCt
Sterol
HPLC
GLC
Cholesterol
Desmosterol
Campesterol
Sitosterol
1.00
0.66
1.11
1.27
1.00
1.06
1.18
1.35
*Retention time relative to cholesterol.
?After silylation.
148
Beydon and Lafont
Thus, our analytical conditions allow a good separation of cholesterol from
other sterols and cholesterol can be quantified by this method.
The cholesterol content of Pieris larvae throughout the last-larval instar
was determined (Fig. 4); it increased during the feeding stage and then
reached a plateau at the wandering stage.
Comparison Between Specific Activity of Cholesterol and 20E in
Spinning Larvae
Specific activities of cholesterol and of 20E were analyzed in spinning
larvae (six larvae per experiment). Cholesterol labeling of 2-day-old last instar
larvae was done in three different ways: 1) injection of [3H]cholesterol; 2)
administration per 0s of [3H]cholesterol; and 3) administration per 0s of
[14C]cholesterol in admixture with [3H]cholesterol. (Administration per 0s
was preferred because the specific activity of [4-14C]cholesterol is very low.)
Labeling was stopped by freezing the insects 2 days after the treatment; at
this time, the larvae were in the spinning stage and their 20E titer was high.
The data summarized in Table 4 show the following.
1. Cholesterol was converted into 20E with low incorporation rates (Table
4A). The differences between the incorporation rates in the three experiments
originated probably in the absence of a perfect synchronism among insects.
2. The specific activities of cholesterol and 20E were approximately equal
in each ex eriment (Table 4B).
3. The 3pH/I4Cradioactivity ratios of cholesterol and 20E were similar (Table
4C), suggesting that the 2 a hydrogen of cholesterol was retained during
hydroxylation at C-2. This result is in agreement with that previously obtained with adult females of Schistocercu greqaria [15].
0
I
1
2
3
I
4
Is1
5
pp
days
I
Fig. 4. Changes in cholesterol titer during the last larval instar of Pieris brassicae (O),
in
pmollanimal. e, ecdysis; F, feeding stage; W, wandering; S, spinning stage; PP, prepupal stage.
Ecdysteroid Production in Pieris
149
TABLE 4. Comparison Between Specific Activities of Cholesterol and
Ecdysteroids
TABLE 4A. Incorporation Rates of [3H]CholesterolInto 20E at the
Spinning Stage*
Incorporation rate
("/.I
Experiment
[3H]Cholesterolinjected
[3H]Cholesterolper 0s
[3H] and [14C]Cholesterol
in admixture per 0 s
0.025
0.015
0.031
L3H1
*Cholesterol was administered to 2-day fifth instar larvae. The insects were
examined for cholesterol and 20E at the spinning stage.
TABLE 4B. Comparison Between the Specific Activities of 20E and
Cholesterol in Each Experiment
Experiment
[3H]Cholesterolinjected
[3H]Cholesterolper 0 s
['4C]Cholesterol in admixture
with [3H]cholesterolper 0 s
Specific activity
(dpminmol)
Cholesterol
95,000"
97,000
44,000"
46,000
12,500"
12,000
[3~1
20E
105,O0Ob
110,000'
37,O0Ob
41,000'
10,000b
9,500'
pH1
"The sample was analyzed twice.
bSpecific activity calculated from RP-HPLC analysis (see text).
'Specific activity calculated from NP-HPLC analysis (see text).
TABLE 4C. 3H/14CRadioactivity Ratios in 20E and Cholesterol After
Administration of [414C]Cholesterolin Admixture With
ria. 2~~-~HlCholesterol
Per 0 s
3H/'4C radioactivity ratio
Cholesterol
20E
3.84"
3.81
3.85b
3.85'
"The sterol sample was analyzed twice.
bRadioactivityratio calculated from RP-HPLC analysis.
'Radioactivity ratio calculated from NP-HPLC analysis.
Comparison Between Specific Activity of Cholesterol and of Some
Ecdysteroids Recovered in Feces or in Young Pupae
Twelve 2-day last instar larvae were injected with [3H]cholesterol. The red
feces produced by the insects during wandering and spinning were collected
and then examined for specific activity of 20E'3P. The insects were killed by
freezing after the pupal ecdysis and examined for the specific activity of
cholesterol and 20E'oic.
Table 5 shows that the specific activity of cholesterol, of 20E'oic, and of
20E'3P were nearly identical. Thus, in all the above experiments the specific
activities of cholesterol and of some predominant ecdysteroids are equal.
150
Beydon and Lafont
TABLE 5. Comparison Between the Specific Activities of Cholesterol and
Some Ecdysteroids*
Specific activity
(dpmlnmol)
Steroid
99,000
Cholesterol
20E’oica
20EJ3Pb
100,000
108,000
*[3H]Cholesterol was injected into 2-day-old fifth instar larvae. After the pupal
ecdysis, the insects were analyzed for 2OE’oic. Their red feces were collected
for ZOE’3P.
a20E’oicrecovered in the insects after the pupal ecdysis.
b20E’3Precovered in the red feces of the insects.
TABLE 6. Ecdysteroid Content in the Feces Produced by an Insect and
Ecdysteroid Content in the Same Insect After Pupal Ecdysis
Incorporation
ratea
Feces
Cholesterol
contentb
(nmolllarva)
Ecdysteroid
content‘
(nmolllarva)
0.004
0.005
0.007
0.013
0.018
0.057
125
200
0.005
0.01
330
0.077
730
0.025
0.06
0.11
0.40
0.56
(”/I
Hours
0-24
24-36
36-48
48-60
60-72
72-84
In insect
after pupal
ecdysis
Total
ecdysteroid
production
460
610
700
1.17
aFrom Table 2.
bFrom Figure 4.
‘Calculated as the product of the cholesterol content in the insect by the
incorporation rate of [3H]cholesterol into ecdysteroid.
Thus we conclude that we are allowed to calculate the amounts of ecdysone
from incorporation rates and cholesterol content.
Estimation of Ecdysone Production
The quantities of ecdysteroids produced (Table 6) were calculated by
considering the mean incorporation rate and the mean cholesterol content
(in nmol) of the insect at the time of incorporation (Fig. 4). The biosynthetic
activity was calculated as the product of cholesterol content by incorporation
rate.
In Figure 5, the same data are used to represent the cumulative ecdysteroid
synthesis during the last larval instar. Thus the total amount of ecdysteroids
synthesized during the last larval instar is 1.17 nmollinsect). In Figure 5, we
have also plotted the levels of molting hormones (E + 20E) during larvalpupal development of P. brussicue. We can observe that, due to catabolism,
Ecdysteroid Production in Pieris
I
I
I
I
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1
2
3
4
151
I....
days
Fig. 5. Time-course of ecdysteroid production during the larval-pupal development (-).
Dotted line: levels of molting hormones (E + 20E) in whole animals measured by HPLC.
the whole amount of ecdysteroids synthesized during the instar is much
higher than the maximal content of E + 20E in insects. This peak occurs
during the spinning stage, after the red feces have been excreted. The
comparison between the ecdysteroid content at this time (0.51 nmol/insectwith ecdysone and 20E accounting for 75% of this value-data not shown),
and the ecdysteroid content before pupal ecdysis (0.54 nmollinsect-essentially 2OE’oic) indicates that ecdysone production stops after the molting
hormone peak.
DISCUSSION
Injected (or ingested) labeled cholesterol is converted into ecdysteroids by
P. bvassicae larvae. The incorporation rates are rather low (in the range of
0.1% for the whole ecdysteroids during the last larval instar), due to the
respective sizes of the cholesterol and ecdysteroid pools in the insects, but
they can nevertheless be measured accurately, provided that sufficient radioactivity was injected. Cholesterol labeling can probably be applied to any
biological model; in our laboratory, the technique was used successfully with
the fruit fly, Drosophila melanogaster [16] and the shore crab, Carcinus maenas
[In.
Cholesterol labeling provides both qualitative and quantitative information.
The labeled ecdysteroids found in the insects after cholesterol administration
are produced biosynthetically, and we may expect that their fate actually
152
Beydon and Lafont
reflects that of unlabeled hormones. Therefore, they provide a physiological
picture; by contrast, it cannot be ascertained that the fate of injected labeled
ecdysteroids is the same as that of endogenously synthesized molecules (due
to differences in the tissue distribution or binding to proteins). Thus some of
the qualitative findings of the present study seem of general interest for
insect physiologists; for example, they concern 1)the evidence for 3-dehydroecdysteroids as true endogenous ecdysteroids (except in Locustu eggs,
they have never been isolated from insects and they have been characterized
only as exogenous hormone metabolites [l]; and 2) the presence of unconverted ecdysone and 20-hydroxyecdysone in green feces (the presence of free
E and 20E had previously been reported in S. greguriu larvae feces [18].
In P. brussicue, the metabolic reactions affect the ecdysone molecule at
carbons C-3, C-20, and C-26. At C-20, hydroxylation probably leads to more
active compounds. At C-26, hydroxylation leads to 26-hydroxyecdysteroid
and then by oxidation to ecdysonoic acids. At C-3, the reactions consist of an
oxidation that can be followed by epimerization and then possibly by phosphate conjugation. The reactions at C-3 and C-26 produce ecdysteroids that
are generally considered inactive compounds [l].
We note that most ecdysteroids recovered in feces are affected at a single
position at C-3 or C-26 while the predominant ecdysteroid in young pupae is
20E’oic modified at both positions.
Cholesterol labeling may also deserve quantitative analyses. The data
concerning the specific activities of cholesterol and of some ecdysteroids are
consistent with the hypothesis of a uniform labeling of cholesterol and
ecdysteroid pools. Therefore, there is a close connection between the radioactivity and the quantity of a given ecdysteroid.
The total amount of ecdysone produced throughout the last larval instar
of P. brussicue was estimated. In agreement with other data [19-221, ecdysone
biosynthesis mainly occurs when the levels of molting hormones are increasing. The results of this study indicate that the total amount of ecdysone
produced is in fact much higher than the maximal level of molting hormones
in insects, due to a high catabolic activity during the first part of the ecdysone
production phase. In contrast, after the wandering stage, ecdysone biosynthesis occurs during reduced catabolic activity; as a consequence, the levels
of 20E increase at first and then 20E is converted into 20E’oic while the
ecdysone biosynthesis is off. Therefore, the E + 20E level at the molting
hormone peak and the amount of 20E’oic at the pupal ecdysis are almost the
same. Similar data were observed in P. brussicue pupae [18] with successive
accumulations of ecdysone, 20E, and 20Eoic during the pupal-adult development. A noticeable difference, however, is that the entire biosynthesis
period occurred during low catabolic activity and this resulted in a better
economy’’ for the insect.
Our results indicate that molting hormone titers are regulated in P. brussicue by two different mechanisms according to developmental stage. In pupae
or at the end of the last larval instar, there is a mechanism by which molting
hormone peaks rise when the hormone inactivation processes are low. In
such a mechanism, the biosynthetic and catabolic steps are separated. In the
second mechanism, at the beginning of the last larval instar, the hormone
I ,
Ecdysteroid Production in Pieris
153
production and inactivation occur almost at the same time, and production
is counterbalanced by inactivation. Consequently, hormone titers can fluctuate rapidly. We think that this latter mechanism could be related to the
pupal commitment that occurs in P. brussicue during this period. In fact, a
low molting hormone peak in hemolymph has been observed before the
pupal commitment [23]. In P. brussicue, this fluctuation is rather rudimentary,
but more complex oscillations have been reported in Surcophuga bullaka at a
similar stage [24] and such rapid variations can only take place if hormone
half-life is very short at the same time.
In conclusion, cholesterol labeling seems to be a powerful tool for investigating ecdysteroid metabolism for the following reasons.
1. It is easy to separate ecdysteroids from unconverted cholesterol during
extraction, and in RP-HPLC, the labeled peaks are solely ecdysteroids; this
may, however, not be valid in at least two cases: a) when ecdysteroid
metabolism leads to apolar metabolites like fatty acid esters, which would be
found in the chloroform phase with cholesterol [25]; and b) in adults of some
species that synthesize noticeable amounts of glucoside derivatives(s) of
vertebrate-type steroids whose polarity is similar to that of ecdysteroids [26].
2. We have a better physiological picture of steroid metabolism than that
obtained with ecdysone labeling (although we must admit that in the present
study the differences are not great-compare Figures 1and 2.
3. All ecdysteroids are observed and quantified easily at the same time,
assuming they have the same specific activity, as was found in the present
study; it is sufficient just to measure the specific activity of one major
compound, e.g., 20-hydroxyecdysone, and the radioactivity in each ecdysteroid. The present method would thus represent an alternative to HPLC
coupled with RIA detection, a method recently applied to M. sextu pupae [5]
and embryos [6].
LITERATURE CITED
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Action of Invertebrate Hormones. Hoffman JA and Porchet M, eds. Springer-Verlag,
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Oxford, Vol8, p 37 (1985).
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154
Beydon and Lafont
8. Modde J-F, Lafont R, Hoffmann JA: Ecdysone metabolism in Locusta migrutoria larvae and
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(1979).
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Schistocerca gregaria. J Insect Physiol23, 1387 (1977).
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feeding larval stage. Arch Insect Biochem Physiol, 4, 139 (1987).
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19. Bollenbacher WE, Vedeckis WV, Gilbert LI, O'Connor JD: Ecdysone titers and prothoracic
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20. Hirn M, H6tru C, Lagueux M, Hoffmann JA: Prothoracic gland activity and blood titres of
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25, 255 (1979).
21. Schaller F, Charlet M: Neuroendocrine control and rate of ecdysone biosynthesis in larvae
of paleopteran insect: Aeshna cyuna Miiller. In: Progress in Ecdysone Research. Hoffmann
JA, ed. ElsevieriNorth Holland, Amsterdam, Dev Endocrinol Vol. 7, p 99 (1980).
22. Okuda M, Sakurai S, Ohtaki T: Activity of the prothoracic gland and its sensitivity to
prothoracicotropic hormone in the penultimate and last-larval instar of Bombyx rnon'. J
Insect Physiol32, 455 (1985).
23. Beydon P, Mauchamp B, Lhonor6 J, Bouthier A, Lafont R: The epidermal cell cycle during
the last larval instar of Pieris brassicae (Lepidoptera). 11. Endocrine control of xanthommatin
content. J Comp Physiol 236, 21 (1980).
24. Roberts B: Photoperiodic regulation of prothoracicotropic hormone release in late larval,
prepupal and pupal stages of Sarcophugu bullata. In: Photoperiodism Regulation of Insect
and Molluscan Hormones. Ciba Foundation Symposium 104. Pitman, London, p 170
(1984).
25. Connat J-L, Diehl PA: Probable occurrence of ecdysteroid fatty acid esters in different
classes of arthropods. Insect Biochem 16, 91 (1986).
26. Thompson MJ, Svoboda JA, Lusby WR, Rees HH, Oliver JE, Weirich GF, Wilzer KR:
Biosynthesis of a C2, steroid conjugate in an insect. J Biol Chem 260, 15410 (1985).
.
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