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Ecdysteroid titre and metabolism to novel apolar derivatives in adult female Boophilus microplus (Ixodidae).

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Archives of Insect Biochemistry and Physiology 2:39-54 (1985)
Ecdysteroid Titre and Metabolism to Novel
Apolar Derivatives in Adult Female
Boophilus microplus (Ixodidae)
Kim P. Wigglesworth, D a v i d Lewis, a n d Huw H. Rees
Department of Biochemistry, University of Liverpool, Liverpool, England (K.P. W.,
H.H.R.), and
Department of Animal Health Biology, Pfizer Central Research, Sandwich, Kent, England (0.L.)
The free ecdysteroid titre determined by radioimmunoassay in adult female
Boophilus microplus showed a peak just prior t o full engorgement and
detachment of the ticks and decreased subsequently to a very low value. I n
contrast, the titre of polar ecdysteroid conjugates was very low. Ecdysone
was the major ecdysteroid at peak titre and was accompanied by much lower
levels of 20-hydroxyecdysone. I n newly detached ticks, injected [3H]ecdysone
was metabolized primarily (80%) into much less polar compounds, which
could be resolved into at least three groups by reversed-phase h.p.1.c. These
[3H] “apolar” metabolites were transferred to the newly laid eggs, where they
accounted for the vast preponderance of ecdysteroids, the level of free
hormone being low. Hydrolysis of the three groups of compounds with an
esterase preparation from porcine liver yielding [3H]ecdysone, together with
the release of [3H] ecdysteroid and fatty acids upon alkaline saponification of
the compounds, suggests that they are of a fatty acyl ester nature. The
chemical transformation of these “esters” into the corresponding acetonide
derivatives indicates that the 2- and 3-hydroxyls of ecdysone remain
unsubstituted in these compounds. Several tick tissues, including Malpighian
tubules, ovaries, gut, and fat body, metabolized [3H]ecdysone in vitro forming
the ”apolar esters” as major products. The maternal ecdysteroid “esters”
may function as storage forms of hormone (presumably hormonally inactive),
which could be hydrolysed enzymically during embryogenesis releasing free
ecdysteroids. Such enzymic hydrolysis of [3H]ecdysone ”esters” by homogenates from developing eggs of B. microplus has been demonstrated.
Key words: ecdysteroids, fatty acyl esters, ticks, Ixodidae, Soophilus micmphs, h.p.1.c.
Acknowledgments: We thank the Science and Engineering Research Council and Pfizer
Central Research for financial support and a C.A.S.E. Studentship (to K.P.W.), the Royal
Society for an equipment grant, Miss P.A. Duxbury for investigating hydrolysis of the ecdysteroid esters in developing eggs, Dr. j.D. Gee and Dr. K.A.F. Gration for invaluable discussion,
Mr. D. King and Mr. N. Leach-Bing for the supply of ticks, Professor K.-D. Spindler and J.D.
O’Connor for generous gifts of antisera, and Dr. M.E. Rose and Mr. M. Prescott for the mass
spectrometric analyses.
Received April 18,1984; accepted June 20,1984.
Address reprint requests to Dr. H.H. Rees, Department of Biochemistry, The University, P.O.
Box 147, Liverpool, L69 3BX, U.K.
0 1985 Alan R.
Liss,lnc.
40
Wigglesworth, Lewis, and Rees
INTRODUCTION
Moulting in immature stages of insects and other arthropods is controlled
by the steroidal moulting hormones, the ecdysteroids. In many insect species
investigated the major ecdysteroid in larvae and pupae is 20-hydroxyecdysone, which is generally accompanied by smaller quantities of ecdysone [1,2].
However, it has been demonstrated in many insect species that ecdysteroids
occur not only in immature stages, but also in adult females, primarily in the
ovaries [3]. At least in the migratory locust, Locustu migrutoriu, ovarian ecdysteroids are synthesised in the follicle cells [4]. In many insect species, it has
been demonstrated that ovarian ecdysteroids are mainly present as polar
conjugates which are largely passed into the eggs [3,5-81. In the locust,
Schistocercu greguriu, the polar ecdysteroid conjugates in newly laid eggs have
been identified as the 22-phosphate derivatives [9,10], whereas it has been
reported that the major polar conjugates in newly laid eggs of the related
species, L. migrutoriu, are 22-adenosine monophosphate derivatives [11,12].
Although the function of polar ecdysteroid conjugates in developing eggs is
not known, the ecdysteroid 22-phosphates may represent inactive storage
forms of hormone. In fact, embryonic tissues of S . gregariu can enzymically
hydrolyse ecdysteroid 22-phosphates to liberate free ecdysteroids; other
sources, such as de novo synthesis, of free ecdysteroids in embryos are not
precluded [13]. Studies on the developing eggs of a number of insect species
indicate an apparent correlation between peaks of free ecdysteroids and the
formation of embryonic membranelcuticle [for references see 101.
The occurrence of ecdysteroids in nymphs of the hard tick, Amblyommu
hebraeurn [14], and in the soft tick, Ornithodoros moubutu [El, has been reported. However, there have been no reports of the occurrence of ecdysteroids in adult female ticks. We now report the occurrence, titre, metabolism,
and fate of ecdysteroids in adult females of the hard tick, Boophilus microplus.
This work has been presented in preliminary form [16].
MATERIALS AND METHODS
Reagents
H.p.1.c.* grade solvents were purchased from Rathburn Chemicals, Walkerburn, U.K.,Helix pornutiu arylsuphatase preparation (type H-1) and porcine
esterase were from Sigma, and ecdysone was obtained from S h e s , Milan,
Italy. [23,24-3H2]Ecdysone (80 Cilmmole) was purchased from New England
Nuclear, Boston, U.S.A., and N-trimethylsilylimidazole was from Pierce and
Warriner, Chester, U.K.
Ticks
One-host ticks, Boophilus microplus, were reared on calves at 20°C. Under
the conditions used the nymphal-adult moult of B. microplus commenced on
*Abbreviations: fast atom bombardment = FAB; gas chromatography/mass spectrometry (selected ion monitoring) = G U M S (SIM,); high-performance liquid chromatography = h.p.1.c.;
4-morpholine-ethanesulphonic acid = Mes; radioimmunoassay = RIA; relative retention time
= RR,; thin layer chromatography = t.1.c.; N-trimethylsilylimidazole= TMSI.
Ecdysteroids in Ticks
41
the 15th day from initial attachment to the host as larvae. Engorgement was
completed around day 21, when the female ticks detached and began egg
laying at day 24 postinfestation.
Extraction Procedure
Tick material (adult females and eggs) was extracted essentially according
to the method of Dinan and Rees [S]. The adult ticks and eggs were macerated and extracted successively with methanollwater (7:3 vlv; 2 x), methanol
( 2 ~ ) ,and methanolldichloromethane (l:l, vlv; 2 X ) using Polytron and Potter-Elvehjem homogenisers, respectively. The volumes of each solvent used
were: 2 x 15 mllg adult females; 2 x 1ml/lO mg eggs. After evaporation of
solvent under vacuum, the extract was partitioned between methanollwater
(7:3, vlv) and hexane (equal volumes) to remove apolar material, each phase
being back-extracted with the respective counter phase. The residue from the
aqueous methanol phase was evaporated on to Celite and applied to a silicic
acid column. The column was developed sequentially with chloroform, 30%
(vlv) methanol in chloroform* (which eluted free ecdysteroids), and 80%
(vlv) methanol in chloroform (which eluted any highly polar ecdysteroids
including conjugates [S].
During identification of free ecdysteroids at peak titre, further cleanup of
the 30% methanol in chloroform silicic acid column fraction before analysis
by h.p.l.c./RIA (Fig. 2) was effected on a reversed-phase Sep-Pak cartridge
(Waters Associates). The sample dissolved in 10% (vlv) methanol in water
(2 x 2 mi) was applied to the activated cartridge, which was eluted sequentially with 30% (vlv) methanol in water (4 ml), 60% (vlv) methanol in water
(6 ml), and methanol (4 ml). Free ecdysteroids were eluted in the 60%
methanol in water fraction.
Radioimmunoassay
Assay of 30% methanol in chloroform silicic acid column fractions was
performed as described previously [8], using ecdysone as standard. Two
antisera were used, ICT-1, a gift from Professor K. -D. Spindler, University
of Dusseldorf 1181, and DHS-1-15 wk., a gift from Professor J.D. O’Connor,
University of California [19]. The cross-reaction factors of the antisera toward
20-hydroxyecdysone (ratio of the mass of 20-hydroxyecdysone required to
displace 50% of [3H]ecdysone to the mass of ecdysone required) were: ICT-1
= 2.6; DHS-1-15 wk. = 2.8.
Radiochemical Methods
Radioactivity was assayed on a Beckman model LS 9800 liquid scintillation
spectrometer, d.p.m. being computed using the H’ system. For assay of
fractions from h.p.1.c. or t.l.c., Scintran Cocktail T (4 ml; BDH Chemicals
*This fraction will be referred to as the “30% methanol in chloroform silicic acid column
fraction” and was shown in this work to contain highly apolar derivatives of ecdysteroids in
addition to free hormones.
42
Wigglesworth, Lewis, and Rees
Ltd., U.K.) was used, whereas radioimmunoassay samples were assayed in
Aquasol (4 ml; New England Nuclear).
Hydrolysis of Highly Polar Ecdysteroid Conjugates
During estimation of the highly polar ecdysteroid conjugate titre in adult
females, a portion (three-quarters) of the 80% (vlv) methanol in chloroform
silicic acid column fraction was evaporated to dryness, dissolved in 0.2 M Mes buffer pH 5.4 (1ml), and incubated with 0.5 ml of a crude arylsulphatase
preparation from Helix pornutiu (125 units; 1 unit hydrolyses 1.0 pmole of
nitrocatechol sulphate in 1 h at pH 5.0 and 37°C) for 18 h at 37°C. The
reaction was terminated by the addition of ethanol (4 ml) and the protein
precipitate was extracted four times with methanol (4 ml). Chromatography
of the residue after evaporation of the combined alcoholic extracts on a silicic
acid column as before allowed separation of a fraction containing ecdysteroids released from conjugation from any highly polar ecdysteroids refractory
to enzymic hydrolysis.
High-Performance Liquid Chromatography
A high-performance liquid chromatograph (Waters Associates, Northwich, U.K.) incorporating two M6000A pumps in conjunction with a model
660 solvent programme controller, U6K injector, and a model 441 detector set
at 254 nm, was used. Ecdysteroids were separated on the following systems:
system 1, an Ultrasphere-ODS column (15 cm X 4.6 mm internal diameter;
Anachem, Luton, U.K.) eluted at 1mllmin with methanollwater (9:11, vlv);
system 2, a Partisil ODs-3 column (25 cm x 4.6 mm internal diameter;
Whatman, Maidstone, U.K.) eluted at 2 mllmin with a linear gradient (20
min) of methanol in water changing from (a) 2:3 to 4:l (vlv), or (b) 2:3 to 1:O
(vlv), or (c) 9:l to 1:O (vlv); system 3, an aminopropyl column (APS-Hypersil;
25 cm x 4.5 mm internal diameter; Shandon Southern Products, Runcorn,
U.K.) eluted isocratically at 2 mllmin with either (a) dichloroethanelmethanol
(19:1, vlv) or (b) dichloroethanelmethanol (47:3, vlv) [17 1.
In the case of radioactive samples, fractions were collected at 0.5- or 1-min
intervals for radioassay and scintillation cocktail (4 ml) added directly in the
case of reversed-phase h.p.l.c., but after evaporation of the solvent when
APS-Hypersil columns were used.
Mass Spectrometry
Fast atom bombardment mass spectra. Negative-ion FAB mass spectra were
obtained as described previously [20].
Gas chromatographylmass spectrometry (selected ion monitoring). Ecdysteroids were converted into their pertrimethylsilyl ether derivatives by heating
with TMSI and subjected to GClMS (SIM) as described previously [21].
Makisterone A was used as internal standard and fragment ions at mlz 567
and 561 were monitored.
Administration of [3H]ecdysone
[23,24-3H]ecdysonediluted to 40 Cilmmole was dissolved in insect Ringer
solution and injected into day 22-23 adult female ticks through the dorsal
Ecdysteroids in Ticks
43
wall (5 pl, generally containing 0.10-0.21 pCi 3H per tick). The animals were
then kept at 27°C in a humid atmosphere and then either sacrificed by
freezing 18 h after injection or allowed to lay eggs for 5-6 days, when the
spent females were frozen; the eggs were frozen within 1day of oviposition.
Thin-Layer Chromatography
Samples were chromatographed on aluminium-backed precoated silica gel
plates (0.2 mm thick Kieselgel 60Fm; E. Merck., A.G., Darmstadt, F.R.G.)
developed as described in the appropriate points in the text. Authentic
marker-compounds were located under UV light (253 nm) and the chromatograms were cut into 0.5-cm strips, which were radioassayed directly.
Hydrolysis of Apolar Fractions With an Esterase Enzyme
Fractions containing apolar derivatives of ecdysteroids were subjected to
hydrolysis with an esterase preparation. Samples were dissolved in 0.1 M
borate buffer (0.5 ml), pH 8.4, and incubated with a porcine esterase (10
units; 1unit will hydrolyse 1.0 pmole of ethylbutyrate per min at pH 8.0 and
25°C) for 24 h at 37°C. The reaction was terminated by the addition of
ethanol (2 ml) and the protein precipitate was extracted four times with
methanol (4 ml).
Acetonide Derivative Formation
Acetonide derivatives of ecdysteroids were prepared by the method of
Galbraith and Horn [22].
Incubation In Vitro of Tissues With [3H]ecdysone
Malpighian tubules, ovaries, fat body, and gut were dissected from late
day 20 females in tick saline (NaC1, 200 mh4; KC1, 10 mM; CaCl2, 2 mM; in
50 mh4 Tris-HC1buffer, pH 7.4) and each tissue incubated with [3H]ecdysone
(1pCi; 40 Cilmmole) in 0.5 ml of tick saline pH 7.4 for 6 h at 27°C. The
incubations were terminated by addition of ethanol (1ml) and the reaction
mixtures homogenised and extracted as described earlier.
Hydrolysis of Apolar Ecdysone "Esters" by a Homogenate of Developing
Eggs of B. microplus
Eggs of B. micropEus were collected within 24 h of oviposition and incubated for 15 days at 27°C and approximately 90% relative humidity. Under
these conditions larvae hatch after 21 days. Day 15 developing eggs (500 mg)
were homogenised at 0°C in 0.1 M potassium phosphate buffer pH 6.0 (6.7
ml) with a Potter Elvehjem homogenizer. Duplicate aliquots (2 ml) of the
crude homogenate as well as 2 ml of boiled homogenate (control incubation)
were preincubated in a reciprocating water bath at 50°C (approximate optimal temperature) for 10 min. The reaction was then initiated by addition of
[23,24-3H]ecdysoneesters (58,000 d.p.m. 3H in 10 p1 methanol) purified by
h.p.1.c. from newly laid eggs derived from female B microplus injected with
[3H]ecdy~~ne.
After 1 h, the incubations were terminated by addition of
44
Wigglesworth, Lewis, and Rees
ethanol (5 ml), the mixture cooled, centrifuged (1,OOOg x 5 min), and the
supernatant removed. The pellet was reextracted with ethanol (5 ml) followed by methanol (2 x 5 d)and the combined supernatants evaporated to
dryness under reduced pressure. [3H]Ecdysone (Rf 0.33) produced by enzymic hydrolysis of the apolar [3H]ecdysone esters (Rf 0.56) was separated
from the latter by t.1.c. of the extract (applied in methanol), developing with
methano1:chloroform (1:4,vlv). The chromatograms were cut into 0.5-cm
strips and radioassayed, and the 3H in the band corresponding to authentic
ecdysone was expressed as a percentage of the total 3H recovered in ecdysone and apolar ecdysone esters.
RESULTS
Ecdysteroid Titre During Adult Female Development
Batches of adult female B. microplus ticks (20-200 individuals) were collected at daily intervals from the time of the nymphal-adult moult (day 15)
until commencement of egg laying (day 24) and stored at -20°C until they
were extracted. The 30% methanol in chloroform silicic acid column fractions
(which include the free ecdysteroids) and ecdysteroids released from conjugation with polar moieties by the Helix pomatia enzymes were subjected to
radioimmunoassay using both the ICT-1 and DHS-1-15 antisera. The resulting titre curve (Fig. 1)shows the presence of one major peak of immunoreactive material in the 30% methanol in chloroform fraction at day 20 of
development prior to complete engorgement; the levels of ecdysteroids released by hydrolysis of the polar ecdysteroid conjugate fractions with Helix
enzymes were comparatively low. Similar titre curves were obtained in a
separate experiment.
]
antiserum
erum
Po,ar
conjugated
ecdysterolds
T l m o f r o m i n l t i a l a t t a o h m o n t of t i c k s t o t h e h o a t (day.)
Fig. 1 Titres of free and polar conjugated ecdysteroids (30% and hydrolysed 80% methanol
in chloroform silicic acid column fractions, respectively) in adult female Boophilus microplus
determined by RIA using two antisera.
Ecdysteroids in Ticks
45
Identification of Free Ecdysteroids at Peak Titre
The nature of the free ecdysteroids at the time of peak titre (day 20) was
investigated initially by reversed-phase h.p.1.c. system 1collecting fractions
every 30 s for radioimmunoassay (ICT-1 antiserum; Fig. 2). A major immunoreactive UV-absorbing peak, cochromatographing with authentic ecdysone, was observed, together with smaller peaks, one of which
cochromatographed with 20-hydroxyecdysone. When the peak cochromatographing with ecdysone was collected and analysed on an APS-Hypersil
column (system 3a), a single UV-absorbing peak (retention time, 13 min)
cochromatographing with ecdysone was observed. Further evidence for the
identity of the ecdysone was furnished by its negative-ion FAB mass spectrum, which showed a major peak at mlz 463 as expected.
Analysis of another portion of the 30% methanol in chloroform silicic acid
column fraction from day 20 ticks by GClMS [SIM] (Fig. 3) showed a peak in
the chromatogram for the ion at mlz 567 having an identical retention time
relative to the pertrimethylsilyl derivative of makisterone A (RR, 0.65) as that
of silylated authentic ecdysone (RR, 0.65) [cf 211, as well as a smaller unidentified peak (RRt 0.84). The fact that the latter shows the ion at mlz 567 rather
than at mlz 561 indicates that the compound is a 20-deoxyecdysteroid having
an ecdysonelike nucleus. The chromatogram for the ion at mlz 561 suggested
Ecd rone
PO-Hydroxyecdyione
Fig. 2 Reversed-phase h.p.1.c. fractionation (system I) of the 30% methanol in chloroform
silicic acid column fraction from day 20 5. microplus females (equivalent to 2 ticks; 75 mg
fresh weight) with collection of fractions every 30 sec for radioimrnunoassasy (ICI-I antiserum). The positions of authentic ecdysteroids are shown.
46
Wigglesworth, Lewis, and Rees
' O 0 . Total ion chromatogram
lool
I\
Intom8l
Stmd8rd
i
m/z 567
3:20
1o:oo
20:oo
Time (min:rec)
Fig. 3 CC/MS(SIM) analysis of a silylated portion of the 30% methanol in chloroform silicic
acid column fraction from day 20 B. microplus females. ions at mlz 561 and 567 were
monitored. The positions of authentic ecdysteroids are shown.
the presence of 20-hydroxyecdysone (RR, 0.49). Comparison of the levels
(pglg tick) of free ecdysone and 20-hydroxyecdysone detected at peak titre
by radioimmunoassay (2.8 and 0.08, respectively) and GUMS (SIM) (2.6 and
0.04, respectively) showed good agreement.
Metabolism of [3H]ecdysone
To obtain information on possible metabolic transformations responsible
for the decrease in immunoreactive ecdysteroids during the period day 20-22
(see Fig. l), [3H]ecdysonewas injected into adult females at day 22-23 and
its metabolism examined.
Metabolism of ecdysone in adult females. Initially the fate of [3H]ecdysone
was analysed in adult females sacrificed 18 h after injection of [3H] substrate.
The females were extracted and the aqueous methanol phase of the methanollwater-hexane partition fractionated on a silicic acid column. Although
the 80% methanol in chloroform fraction, which includes any highly polar
ecdysteroids, contained some radioactivity, the majority (- 94% of the total
recovered from the column) was associated with the 30% methanol in chlo-
Ecdysteroids in Ticks
47
roform fraction. A portion of the latter fraction was analysed by reversedphase h.p.1.c. using a solvent gradient of 40-80% (vlv) methanol in water
(system 2a), which should elute all known free ecdysteroids. The radiochromatogram observed comprised a major peak of residual ecdysone, a minute
amount of radioactivity cochromatographing with 20-hydroxyecdysone, and
a slightly more prominent peak chromatographing with ecdysone-3-acetate.
In view of the very poor recovery of radioactivity from this chromatographic
system, a portion of the original 30% methanol in chloroform silicic acid
column was analysed by thin-layer chromatography with chloroformlmethan01 (4:l vlv) for development. The majority of the radioactivity (86%) chromatographed with ecdysone-3-acetate (Rf 0.56; cf Rf of ecdysone, 0.33),
although a minor peak of 3H cochromatographed with this compound on the
preceding reversed-phase h. p .l.c. system.
Reanalysis of the original 30% methanol in chloroform silicic acid column
fraction by reversed-phase h.p.1.c. eluting with a linear gradient of 40-100%
(vlv) methanol in water (system 2b) revealed the presence of a major peak of
radioactivity of an apolar nature as compared to ecdysone as well as small
peaks cochromatographing with ecdysone and ecdysone-3-acetate, respectively (Fig. 4A). Because of the "apolar"* nature of the majority of this
material, another portion of the original 30% methanol in chloroform silicic
acid column fraction was reanalysed by reversed-phase h.p.l.c., but eluting
with a linear gradient of 90-100% (vlv) methanol in water (system 2c). In this
A
B
m,o
Apolar
ecdysteroids
Apoler
ecdysteroids
m
.--
m
-Peak 2
I
0
b
a
c
m
14
U
a
a
a
-
I?
P
U
0
12
X
c
U
0
Tlmm(mln)
tlmm (mln)
Fig. 4 Reversed-phase h.p.1.c. (A, system 2b; B, system 2c) radiochromatograms of the 30%
methanol in chloroform silicic acid column fraction from ticks following administration of
[3H]ecdysoneto day 20 adult females. The positions of authentic ecdysteroids are shown.
*In this paper the term "apolar" is used with reference to compounds that are appreciably
less polar than ecdysone.
48
Wigglesworth, Lewis, and Rees
system the large ”apolar” radioactive peak is resolved into three distinct
peaks (Fig. 4B).
Passage of [3H]ecdysteroidsinto the eggs. The possible passage of maternal
ecdysteroids into the eggs was investigated by analysis of eggs derived from
females injected with [TIlecdysone. In a typical experiment, ten adult females were injected with [3H]ecdysone (1.54 pCi total), the newly laid eggs
(130 mg) were collected over the first 6 days of oviposition, the eggs extracted, and the aqueous methanol phase (0.70 pCi) of the methanoliwaterhexane partition fractionated on a silicic acid column. The 30% methanol in
chloroform fraction (0.59 pCi) contained the majority of the radioactivity,
with little (0.03 pCi) being present in the 80% methanol in chloroform (highly
polar ecdysteroid) fraction. Analysis of the 30% methanol in chloroform
fraction by reversed-phase h.p.1.c. using a linear gradient of 40-100% (vlv)
methanol in water (system 2b) showed that it consisted almost entirely of
apolar material, which was resolved using a 90-100% methanol in water
gradient (system 2c) into three distinct peaks of the same polarity as the
major radioactive metabolites observed in adult females (Fig. 5). Thus, it
appears that the apolar compounds which are formed in females as major
ecdysone metabolites are principal radioactive components in newly laid
eggs and are probably formed maternally.
Analysis of the Apolar Metabolites
The apolar nature of the major [3H]ecdysone metabolites suggested that
they could be fatty acyl ester derivatives. Therefore, each of the three [3H]
Apolar
ecdyateroida
CI
m
1
0
v-
01
0
.-
X
0
-*
E
L
Q
Q
0
01
v
U
.-c
>
.-c
>
0
a,
m
m
0
m
.-0
L
U
‘0
1
ID
U
c
2’0
15
10
5
0
Time (min)
Fig. 5 Reversed-phase h.p.1.c. (system 2c) radiochromatogram of the 30% methanol in chloroform silicic acid column fraction from newly laid eggs of ticks which had been injected
with [3H]ecdysone at day 20.
Ecdysteroids in Ticks
49
apolar peaks were collected from reversed-phase h.p.1.c. (system 2c) and
treated with a porcine liver esterase preparation; parallel control incubations
without esterase were also carried out. Analysis of the products of the
reactions by reversed-phase h.p.1.c. using a linear gradient of 40-100% methanol in water (system 2b) indicated that after enzymic hydrolysis in each case
nearly all the radioactivity ( > 95%) cochromatographed with ecdysone,
whereas control incubations yielded negligible radioactivity in that region
(Fig. 6). Confirmation that the radioactivity was associated with ecdysone in
each case was obtained by cochromatography with authentic material on an
APS-Hypersil h.p.1.c. column (system 3b). These results are consistent with
the notion that the apolar metabolites may be esters of ecdysone. The observed release of [3H]ecdysteroid in alkaline hydrolylsis (6% (wlv) sodium
hydroxide in ethanollwater (lO:l, viv) at room temperature for 18 h) of the
apolar compounds, as well as analysis by g.1.c. of the fatty acids released are
also consistent with this view.
To obtain some information on the position(s) substituted in the apolar
ecdysone metabolites, acetonide derivative formation was attempted on two
of these compounds (peaks 1and 2 ex. reversed-phase h.p.1.c.). Analysis of
the products of the reaction by t.1.c. with chloroformlethanol (9:1, vlv) for
development revealed the presence of a major peak of radioactivity (Rf 0.42)
less polar than ecdysone-2,3-acetonide (Rf 0.20; cf Rf of underivatized compounds 1and 2 (0.03) and of ecdysone (0.03). These results suggest that C-2
Apolmr
ecdymtmrold.
1
1
50
26
20
Tlma
16 10
6
0
(mln)
Fig. 6 Reversed-phase h.p.1.c. (system 2b) radiochromatogram of the product of enzymic
hydrolysis of apolar peak 2 (ex. Fig. 5) with a porcine liver esterase preparation. The position
of authentic ecdysone is shown. Control incubation without esterase, - - -; esterasetreated, -. The radiochromatograms for the products of hydrolysis of apolar peaks 1 and 3
(ex. Fig 5) with the esterase preparation showed a similar pattern to that shown.
50
Wigglesworth, Lewis, and Rees
and C-3 are free in the apolar metabolites 1 and 2, and that another position(s) is substituted in the latter compounds.
Sites of formation of apolar ecdysone metabolites. Certain tissues from late
day 20 females were incubated in vitro with [3H]ecdysone, the reaction
mixture extracted, and the metabolites in the 30% methanol in chloroform
silicic acid column fraction were analysed by reversed-phase h.p.1.c. (system
2b; Fig. 7). In the case of each tissue, when the peak corresponding to apolar
ecdysteroids was collected and rechromatographed on reversed-phase
h.p.l.c., eluting with a linear gradient of methanol in water changing from
9:l to 1 : O (v/v; system 2c; eg, Fig. 8), the radioactivity was resolved into three
separate peaks cochromatographing with the apolar metabolites detected in
females and eggs (Figs. 4,5). Thus, it is apparent that formation of the apolar
ecdysone metabolites occurs, at least in vitro, in the Malpighian tubules,
ovaries, fat body, and gut.
3
0
..
!!
1
Time (min)
Fig. 7 Reversed-phase h.p.1.c. (system 2b) radiochromatograms of the 30% methanol in
chloroform silicic actd column fractions derived from incubation of [3H)ecdysone with various tissues from late day 20 female B. microplus. The positions of authentic ecdysteroids are
shown. Malpighian tubules, 0-0; ovaries 0-0; gut, 0--.-0. The results for fat body
were similar to those for Malpighian tubules.
Ecdysteroids in Ticks
51
T i m e (rnin)
Fig. 8 Reversed-phase h.p.1.c. (system 2c) radiochromatograms of the 30% methanol in
chloroform silicic acid column fractions derived from incubation of [3H]ecdysonewith ovaries
(0-0) or gut (0--.-0)tissues from late day 20 female B. microplus. The relative proportions of apolar ecdysteroids formed in Malpighian tubules and fat body in vitro were similar
to those observed in vivo.
Hydrolysis of apolar ecdysone esters by a homogenate of developing eggs of
B. microplus. When a homogenate (equivalent to 150 mg eggs) of B. microplus
eggs, which had developed for 15 days, was incubated with apolar
[3H]ecdysone esters under the conditions described, 20% hydrolysis of the
substrate was observed. Negligible apparent hydrolysis of substrate was
observed in a control incubation with boiled enzyme. In a separate incubation, the [3H]ecdysteroid product of the reaction cochromatographed with
ecdysone on reversed-phases h.p.1.c. (system 2b).
DISCUSSION
The exact significance of the peak of immunoreactive ecdysteroids in the
30% methanol in chloroform silicic acid column fraction just before complete
engorgement of adult female B. microplus is uncertain. This immunoreactivity
reflects the titre of free ecdysteroids since the apolar ecdysteroid "esters,"
which also occur in the same silicic acid column fraction, are only marginally
immunoreactive towards the two antisera employed. The apolar ecdysteroid
"esters" have not been detected at peak titre, but apparently occur after day
22 of development. The location and source of the ecdysteroids have not yet
been determined. The somewhat lower immunoreactivity detected with the
ICT-1 antiserum than with the DHS-1-15 antiserum may reflect the possible
presence of ecdysteroids having a modified ring structure, since, of the two
52
Wigglesworth, Lewis, and Rees
antisera, the former shows greater specificity for that part of the ecdysteroid
structure [18,19,23].
The occurrence predominantly of ecdysone with very much less 20hydroxyecdysone at peak titre in B. microplus adult females is reminiscent of
the situation in the femalesleggs of most insect species investigated, where
ecdysone, albeit often as a polar conjugate, predominates [see 31.
In comparison with the titre of free ecdysteroids, that of polar conjugates
hydrolysable with the Helix enzymes was much lower. The unlikely possibility still exists that polar conjugates which are not hydrolysed with such
enzymes are present.
Exogenous [3H]ecdysone was metabolised in adult female B. microplus
primarily to appreciably less polar metabolites, which were readily resolved
into three groups by reversed-phase h.p.1.c. Although these metabolites
were hydrolysed upon treatment with an esterase enzyme preparation from
porcine liver, it is always dangerous to draw conclusions regarding the
structure of compounds solely from results of their hydrolyses with enzyme
preparations which may not be pure. However, this observation taken together with the release of [3H]ecdysteroid and fatty acids upon alkaline
saponification of the compounds is consistent with the notion that they are
of an ester nature. It is noteworthy that ecdysone was the only ecdysteroid
moiety in each of the three groups of apolar compounds resolved by reversed-phase h.p.1.c. The demonstration that the ecdysone "esters" (of
peaks 1and 2; Fig. 4B)can be transformed chemically into the corresponding
acetonide derivatives suggests that the 2- and 3-hydroxyls of ecdysone remain unsubstituted in the compounds. Of the remaining hydroxyls, the
secondary 22-hydroxy group would probably be the most likely to be substituted. In fact, preliminary 'H n.m.r. evidence suggests that the C-22 hydroxyl
group of ecdysone is substituted in at least two groups (peaks 1and 2; Fig.
4B)of these apolar derivatives.
The possibility always exists that metabolism of exogenous [3H]ecdysone
in adult female B. microplus may not accurately reflect that of endogenous
hormone. The formation by several tissues of apolar ecdysone metabolites
analogous to those formed in vivo suggests a wide distribution within the
organism of the appropriate enzyme(s). However, the possible physiological
signhcance of this is unclear at present.
The observed metabolism of [3H]ecdysone into the "esters" in adult females, the passage of radioactivity into the eggs, together with the presence
of a vast preponderance of apolar ecdysteroid "esters" compared with free
hormone in the newly laid eggs (Fig. 5) suggest that the "esters" in the latter
are of maternal origin. Although the ecdysteroids in newly laid eggs are
apparently present predominantly as "esters," distinct peaks of free ecdysteroids have been detected during embryogenesis (K.P. Wigglesworth, unpublished results). The observed hydrolysis of the ecdysteroid esters by a
homogenate of developing eggs of B. microplus suggests that such maternal
"esters" may function as storage forms (presumably hormonally inactive),
being enzymically hydrolysed at certain times during embryogenesis releasing free hormones. Of course, other sources of free ecdysteroids (eg, de novo
synthesis) are also tenable. This enzymic hydrolysis of ecdysone "esters" by
Ecdysteroids in licks
53
B microplus embryos is analogous to the release of free ecdysteroids from the
corresponding 22-phosphates by a phosphatase enzyme preparation from
developing embryos of the locust, Schistocercu greguriu [13]. Formation of
apolar metabolites of ecdysteroids, which are probably analogous to those
formed in B. microplus, has been detected recently in nymphs [24] and adult
females of the soft tick, Ornithodoros moubutu [25].
LITERATURE CITED
1. Richards G: Insect hormones in development. Biol Rev 56, 501 (1981).
2. Rees HH: Ecdysteroids. In: Biosynthesis of Isoprenoid Compounds. Porter JW, Spurgeon
SL, eds. Wiley, New York, Vol. 2, pp 463-505 (1983).
3. Hoffmann JA, Lagueux, M, Hetru C, Charlet M, Goltzen6 F: Ecdysone in reproductively
competent female adults and in embryos of insects. In: Progress in Ecdysone Research.
Hoffman JA, ed. ElsevierlNorth-Holland, Amsterdam, pp 431-465 (1980).
4. GoltzenC F, Lagueux M, Charlet M, Hoffmann JA: The follicle cell epithelium of maturing
ovaries of Locustu migrutoriu: A new biosynthetic tissue for ecdysone. Hoppe-Seyler's Z
Physiol Chem 359, 1427 (1978).
5. Mizuno T, Ohnishi E: Conjugated ecdysone in the eggs of the silkworm, Bombyx mori.
Dev Growth Diff 27, 219 (1975).
6. Hsiao TH, Hsiao C: Ecdysteroids in the ovary and the egg of the greater wax moth. J.
Insect Physiol25, 45 (1979).
7. Gande AR, Morgan ED, Wilson ID: Ecdysteroid levels throughout the life cycle of the
desert locust, Schistoceru greguria. J Insect Physiol25, 669 (1979).
8. Dinan LN, Rees HH: The identification and titres of coniugated and free ecdvsteroids in
developing ovaries and newly laid eggs of Schistocercu greguriu. J. Insect Physiol 27, 51
(1981).
9. Issac RE, Rose ME, Rees HH, Goodwin TW: Identification of ecdysone-22-phosphate and
2-deoxyecdysone-22-phosphate in eggs of the desert locust, Schistocercu greguriu, by fast
atom bombardment mass spectrometry and n.m.r. spectroscopy. J Chem SOCChem
Commun 249 (1982).
10. Isaac RE, Rose ME, Rees HH, Goodwin TW: Identification of the 22-phosphate esters of
ecdysone, 2-deoxyecdysone, 20-hydroxyecdysone and 2-deoxy-20-hydroxyecdysone from
newly laid eggs of the desert locust, Schistocercu greguriu. Biochem J 273, 533 (1983).
11. Tsoupras G, Hetru C, Lu B, Constantin E, Lagueux M, Hoffmann J: Identification and
metabolic fate of ovarian 22-adenosine monophosphoric ester of 2-deoxyecdysone in
ovaries and eggs of an insect, Locustu migrutoriu. Tetrahedron 39, 1789 (1983).
12. Tsoupras G, Luu B, Hoffman JA: A cytokinin (isopentenyl-adenosyl-mononucleotide)
linked to ecdysone in newly laid eggs of Locustu migrutoriu. Science 220, 507 (1983).
13. Isaac RE, Sweeney FP, Rees HH: Enzymic hydrolysis of ecdysteroid phosphate during
embryogenesis in the desert locust (Schistocercu gieguriu). Biochem SOCTrans / I , 379 (1983).
14. Delbecque JP, Diehl PA, O'Connor JD: Presence of ecdysone and ecdysterone in the tick
Amblyommu hebrueum Koch. Experientia 34, 1379 (1978).
15. Germond JE, Diehl PA, Morici M: Correlations between integument structure and ecdysteroid titers in fifth-stage nymphs of the tick, Ornithodoros moubutu. Gen Comp Endocrinol
46, 255 (1982).
16. Wigglesworth KP, Rees HH: Ecdysteroid titre and metabolism in adult females of the tick
Boophilus microplus. Presented at the CNRS Symposium on Invertebrate Hormones, Strasbourg (1983).
17. Dinan LN, Donnahey PL, Rees HH, Goodwin TW:High-performance liquid chromatography of ecdysteroids and their 3-epi, 3-dehydro and 26-hydroxy derivatives. J Chromatogr 205, 139 (1981).
18. Spindler KD, Beckers C, Groschel-Stewart U, Emmerich H: A radioimmunoassay for
arthropod moulting hormones, introducing a novel method of immunogen coupling.
Hoppe-Seyler's Z Physiol Chem 359, 1269 (1978).
I
"
54
Wigglesworth, Lewis, and Rees
19. Soumoff C, Horn DHS, O’Connor JD: Production of a new antiserum to arthropod
molting hormone and comparison with two other antisera. J Steroid Biochem 14, 429
(1981).
20. Gibson JM, Majumder MSI, Mendis AHW, Rees HH: Absence of phytosterol dealkylation
and identification of the major ecdysteroid as makisterone A in Dysdercus fusciutus (Heteroptera, Pyrrhocoridae). Arch Insect Biochem Physiol I, 105 (1983).
21. Mendis AHW, Rose ME, Rees, HH, Goodwin TW: Ecdysteroids in adults of the nematode,
Dirofiluria irnrnifis. Mol Biochem Parasitol 9, 209 (1983).
22. Galbraith MN, Horn DHS: Insect moulting hormones: Crustecdysone (20-hydroxyecdysone) from Podocurpus elatus. Aust J Chem 22, 1045 (1969).
23. Reum L, Koolman J: Analysis of ecdysteroids by radioimmunoassay: Comparison of three
different antisera. Insect Biochem 9, 135 (1979).
24. Bouvier J, Diehl PA, Morici M: Ecdysone metabolism in the tick Ornithodoros moubufu
(Argasidae, Ixodoidea). Rev Suisse Zoo1 89, 967 (1982).
25. Diehl P, Connat J-L, Vuilleme P, Morici M, Bouvier J: Metabolism of ecdysteroids in the
tick Ornithodoros rnoubufu (Argasidae, Ixodoidea). Presented at the CNRS Symposium on
Invertebrate Hormones, Strasbourg (1983).
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