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Profiles of free and conjugated ecdysteroids and ecdysteroid acids during pupal-adult development of Manduca sexta.

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Archives of Insect Biochemistry and Physiology 12:63-74 (1989)
Profiles of Free and Conjugated Ecdysteroids
and Ecdysteroid Acids During Pupal-Adult
Development of Manduca sexta
R u b e n Lozano, Malcolm J. Thompson, James A. Svoboda, a n d
William R. L u s b y
Insect and Nematode Hormone Laboratoy, Agricultural Research Service, USDA,
Beltsville, Ma yland
The levels of individual free and conjugated ecdysteroids and ecdysteroid acids,
labeled from ['4Clcholesterol, in five different age groups of male Manduca
sexta during pupal-adult development were determined by HPLC. Eight free
ecdysteroids, eight ecdysteroid phosphates, and two ecdysteroid acids were
identified. Newly ecdysed pupae contained predominantly 3-epiecdysteroids
in each of the free, conjugated, and acidic ecdysteroid fractions. The titer of
each ecdysteroid fraction rose sharply by day4, and this was particularly noteworthy with respect to free ecdysone and 3-epi-20-hydroxyecdysonoic acid.
This stage demonstrated high degrees of ecdysone biosynthesis, oxidative
catabolism, and phosphorylation. As development proceeded to day 16, total
ecdysteroid titer remained constant; a decreasing free ecdysteroid titer was
accompanieid by increasing titers of both conjugates and acids resulting from
the metabolic processes of hydroxylation, oxidation, epimerization, and phosphorylation. The predominant metabolites throughout development were
3-epi-20-hydroxyecdysonoicacid and the phosphate conjugates of 3-epi-20hydroxyecdysone and 3-epi-20,26-dihydroxyecdysone. The ultimate inactivation
of the ecdysteroids of M. sexta during pupal-adult development is possibly
mediated by two pairs of metabolically-linked processes, one leading to a
3-epiecdysteroid acid, and the other to 3-epiecdysteroid phosphates.
Key words: ['4C]cholesterol, HPLC, ecdysteroidtiters, ecdysteroid metabolism, epimerization,
hydroxylation, oxidation, epiecdysteroid acids, epiecdysteroid phosphates
Physiological events that occur during pupal-adult metamorphosis of insects
can be correlated with the presence of ecdysteroid hormones whose titer and
Acknowledgments: We gratefully thank Lynda J. Liska and Kenneth R. Wilzer, Jr., for their technical support, Sherry A. Cohen for her secretarial assistance, and Martin L. Weinrich for his
preparation of the manuscript. We also wish to thank John M. Roman of Program Resources,
NCI-FCRS, Frederick, MD, for the FAB-mass spectra.
Received April 21,1989; accepted August 3,1989.
Address reprint requests to Dr. JamesA. Svoboda, USDA, ARS, PSI, Insect Hormone Laboratory, Bldg. 467, BARC-East, Beltsville, MD 20705.
0 1989 Alan R. Liss, Inc.
Lozano et al.
activity during this closed stage are regulated by biosynthesis, enzymatic degradation, and compartmentalization of the hormonally active compounds [l-31.
The pupal-adult development of Munducu sextu is characterizedby quantitative,
qualitative, and temporally specific changes in the ecdysteroid titer [4]. Six of the
free ecdysteroids occurring in M. sextu during this period were originally isolated
and identified by this laboratory using mass spectrometryand NMR spectroscopy
[5]. More recently, a detailed study of the metabolism kinetics and distribution of
ecdysteroids during M. sextu pupal-adult development has been reported by
Warren and Gilbert [6]. They defined a hemolymph ecdysteroid profile resulting
from the synthesis and secretion of ecdysone by the prothoracic glands and the
sequential oxidative metabolism of ecdysone by peripheral tissues. As metamorphosis progressed, ecdysteroids were increasinly localized in the gut.
We have been performing in vivo studies of ecdysteroid metabolism in M.
sextu using alternate methodology, namely, long-term ['4C]cholesterol labeling of ecdysteroids. The collection and isolation of minute quantities of radiolabeled steroids from various ages of M. sextu ovaries, eggs, and larvae have
allowed us to trace the metabolic fate of maternally acquired ecdysteroids
throughout embryogenesis [7]. Our studies have now extended to the longterm labeling of ecdysteroids during the pupal-adult development of male M.
sextu. Of particular significance, two ecdysteroid acids, 20-hydroxyecdysonoic
acid and its 3a-epimer, were isolated and identified from insects 8 days following pupation as metabolites of ['4C]cholesterol administered to M. sextu as
fifth-instar larvae [8,9]. Ecdysteroid acids and 3-epiecdysteroids are generally
considered as inactivation products of the active molting hormone, 20-hydroxyecdysone [lo], resulting from C-26 oxidation and C-3 epimerization. These
metabolic processes, as well as the formation and hydrolysis of conjugates,
are thus critical factors in the regulation of ecdysteroid hormone titer during
insect development. This report follaws the metabolic fate of radiolabeled free,
acidic, and conjugated ecdysteroids from ['4C]cholesterol during pupal-adult
development of M. sextu, with particular emphasis on the kinetics of the formation of ecdysteroid acids and C-26 oxidized 3-epiecdysteroids.
As described previously [8], each newly molted fifth-instar larva, reared on
an artificial diet, was injected with one microcurie of purified commercial
[4-'4C]cholesterol (Amersham Corporation,' Arlington Heights, IL; specific
activity 53.7 mCi/mmol). Male pupae, staged from ecdysis (day 0), were collected at various ages (0, 4, 8, 12, and 16 days) for analysis in groups of 16-65
in number. Adult eclosion of control insects occurred approximately on day
18. Whole animals were extracted with CHC13/methanol,methanol, and 70%
aqueous methanol [8]. Ecdysteroids were fractionated according to class (free,
acid, or conjugate) and were then analyzed by HPLC.* Separation and purification procedures using Florisil, XAD-16, XAD-2, DEAE-Sephadex A-50, and
C18 Sep-Pak column chromatography have been detailed elsewhere [8,9].
'Mention of a company name or proprietary product does not necessarily constitute endorsement by the U.S. Department of Agriculture.
*Abbreviations used: E = ecdysone; 20E = 20-hydroxyecdysone; 20EA = 20-hydroxyecdysonoic
acid; 26E = 26-hydroxyecdysone; 2026E = 20,26-dihydroxyecdysone; EP, 26EP, etc. = ecdysone phosphate, 26-hydroxyecdysone phosphate, etc.; E', E'A, E'P, etc. = 3-epiecdysone,
3-epiecdysonoicacid, 3-epiecdysone phosphate, etc; X-P = unknown ecdysteroid phosphates.
Ecdysteroid Profiles in Manduca
Free ecdysteroids were analyzed by reverse-phase HPLC on an ODs-Hypersil
x 25 cm, 5 pm particles; Shandon, Sewickley, PA) eluted
with 38%aqueous methanol. Ecdysteroid acids and conjugates were analyzed
by ion-suppression reverse-phase HPLC, also on an ODs-Hypersil c18 column (that provided better resolution than c8 columns used previously) and
were eluted with 28% methanol in 0.03 M aqueous NaH2P04solution (pH 5).
Effluent absorbance was detected at 254 nm. Analyses were performed at 33°C
and a flow rate of 1.0 ml/min. For radioassay, fractions (0.5 ml) were collected
throughout the chromatogram and mixed with scintillation fluid, and their
radioactivity was measured in a liquid scintillation counter (Beckman LS 5801).
Radiolabeled metabolites were identified by comparison with ecdysteroid standards and with ecdysteroid metabolites from M. sexta that had been characterized previously by NMR and mass spectral analyses [9]. Ecdysteroid content
was quantitated by HPLC. Concentrations of free ecdysteroids and ecdysteroid
acids were determined by comparison to standards of each metabolite. Concentrations of ecdysteroid phosphates were determined by comparison to
26-hydroxyecdysone 26-phosphate.
8 column (4.6 mm
Ecdysteroid Conjugate Analysis
Each component of the ecdysteroid conjugate fraction was isolated by HPLC
collection, purified on a
SEP-PAK, and analyzed by negative-ion fast atom
bombardment mass spectrometry. An aliquot of each conjugate was hydrolyzed with an enzyme mixture [8]; after purification, each deconjugated
ecdysteroid was identified by TLC [9] and HPLC.
The results presented are from single experiments with samples of five different groups of male M . sexta taken at 0, 4, 8, 12, and 16 days following
Free Ecdysteroid Analysis
A typical reverse-phase HPLC/radioassay of a free ecdysteroid fraction,
obtained from insects on day 4 following pupation, is shown in Figure 1.Seven
radiolabeled free ecdysteroids were identified in this sample; E was present
in the greatest quantity. From all age groups examined, a total of eight radiolabeled free ecdysteroids were identified: E, 20E, 26E, 2026E, and their four
corresponding 3a-epimers.
The profile of individual free ecdysteroid titers through day 16 of M. sexta
pupal-adult development is shown in Figure 2A. The profile of total free
ecdysteroid titer is shown in Figure 3. The levels of E’ present in all age groups
were very low (20-40 ng/g) and are not shown in Figure 2A. Also not included
(for graphic simplicity) is 26E, which was detected only at day 4 at 0.20 pg/g.
In 0 day pupae, 85% of the total free ecdysteroids were the 3-epiecdysteroids,
20E‘, 26E’, and 2026E’, in relatively small amounts of 0.31, 0.29, and 0.38
pg/g of pupae, respectively. By day 4, there was a 2.5-fold increase in free
ecdysteroid titer, as expressed by the rising concentrations of E (1.86 pg/g),
Lozano et al.
1.005 AU
Fig. 1. Reverse-phase HPLC/radioassay of free ecdysteroids from male M. sexta 4 days postputation. UV absorbance was detected at 254 nm. Shaded areas indicated radioactivity. Component eluting at 20 min was not identified.
20E (0.33pg/g), 26E (0.20 pg/g), and 2026E (0.23 pg/g). The levels of 20E’, 26E’,
and 2026E’ decreased, and free 3-epiecdysteroid content formed only 13%of the
total. At 8 days, the titer of total free ecdysteroids remained the same quantitatively, but it exhibited dramatic qualitative changes. Decreasing levels of E and
26E were accompanied by much increased levels of 20E (1.07 pg/g) and 2026E
(1.21 pg/g). At 12 days following pupation, a decline in free ecdysteroid titer
occurred, particularly that of 20E, whereas 2026E (1.08 pg/g) became predominant and 3-epiecdysteroidcontent increased. By 16 days, the free ecdysteroid titer
decreased again, especially the level of 2026E. However, the titer of 3-epiecdysteroids continued to become more predominant, forming 66% of the total
free ecdysteroids at this stage. 2026E’, which occurred in all age groups, was
the most abundant free ecdysteroid in both 0 and 16 day samples.
Ecdysteroid Conjugate Analysis
A typical ion-suppression reverse-phase HPLC/radioassayof an ecdysteroid
conjugate fraction, obtained from insects 4 days post-pupation, is shown in
Figure 4A. Nine radiolabeled compounds were detected in this sample and
were representative of all of the conjugates found in the other age groups.
Analysis by FAB mass spectrometry of the first conjugate, eluting at 10 min,
produced diagnostic peaks at m/z 79 (PO,)- and 97 (H2P04)-,indicative of a
phosphate ester. The spectrum also showed major peaks in the high mass region
at m/z 575 (M-H)- and 559 (M-OH)-, signifying a phosphate conjugate of a
dihydroxyecdysone. Enzymatic hydrolysis of the conjugate released 2026E as
the ecdysteroid moiety. Therefore, the initial conjugate was identified as 2026EP.
Similarly, mass spectral analyses of the other isolated conjugates established
that they were all ecdysteroid phosphates of which the position(s) of the phosphate moiety is presently not known. The identities of the ecdysteroid moieties are shown for each conjugate in Figure 4A and were corroborated by
determination of the mass spectra of the intact conjugates.
Ecdysteroid Profiles in Manduca
a 20E’P
0. I
Age of pupae (days after ecdysis)
Fig. 2. Profiles of titers of (A) free ecdysteroids, (B)ecdysteroid conjugates, and (C) ecdysteroid
acids during pupal-adult development of M. sextu males. Titers were quantitated by HPLC/
radioassay analyses. Not shown in 2A are minor quantities of 26E and E’; not shown in 2B is a
minor amount of E‘P (see text).
Lozano et al.
Age of pupae ( d a y s after ecdysis)
Fig. 3. Profiles of titers of total free ecdysteroids, total conjugates, total acids, and total
ecdysteroids (sum of free, conjugates, and acids). Total concentration (&g) is expressed as the
sum of the individual ecdysteroid concentrations (Fig. 2 . )
The two 2026E’Pconjugates, eluting at 12 and 13 min, occurred in an approximate 2:l ratio in every age group. They could possibly be an isomeric pair,
namely, the 25R and 25s isomers, rather than being a pair of 2026E’ conjugates with phosphate groups at different positions. In their previous study,
Warren and Gilbert [6] observed the probable C-25 isomers of free 2026E occurring in a constant 2:l ratio throughout M. sextu pupal development. Under
our HPLC conditions, we could not resolve the C-25 isomers of 2026E, 2026E’,
or 2026EP in our samples, if both isomers were indeed present at all. The radiolabeled peak at 17 min (X-P, Fig. 4A) occurred in significant amounts only on
days 8, 12, and 16 (0.10-0.18 pg/g). The mass spectrum of X-P revealed the
presence of a phosphate group. However, hydrolysis of X-P revealed a mixture of unknown free ecdysteroids. The 26EP conjugate at 24.5 min had the
same retention time as authentic 26-hydroxyecdysone 26-phosphate derived
Ecdysteroid Profiles in Manduca
2 0 2 6 E’ P
2 0 2 6E’P
, 0 0 5 AU
.01 AU
TIME (min)
Fig. 4. Ion-suppression reverse-phase HPLChadioassays of (A) ecdysteroid conjugates from
male M. sexta 4 days post-pupation and (B) ecdysteroid acids from males 16 days post-pupation.
UV absorbance was detected at 254 nm. Shaded areas indicate radioactivity.
from M. sextu eggs [ll]. The 26EP conjugates at 24.5 and 26.5 min might be
the 25R- and 25s-isomeric pair. In summary, we identified phosphate conjugates of E, E‘, 20E’, 26E, 2026E, and 2026E’.
The profile of conjugated ecdysteroids throughout pupal-adult development
is shown in Figures 2B (individual ecdysteroid conjugates) and 3 (total conjugates). For graphic simplicity, E’P is not included in Figure 2B; it was only
detected in 4 day insects at 40 ng/g. A relatively small amount of total conjugates was present in 0 day pupae and consisted mainly of 20E’P (0.13 pg/g)
and 2026E’P (0.10 pg/g). The titer increased fivefold by 4 days, with large
amounts of 20E’P (0.39 Kglg), 26EP (0.40 @g), and 2026E’P (0.44 pg/g) and
Lozano et al.
smaller amounts of EP and E’P. By 8 days, the titer fell, with decreases in the
levels of EP, E’P, and 26EP. From day 8 through day 16, the concentration of
26EP continued to decline, but the total titer gradually rose, with increases in
2026EP and 2026E’P and significant amounts of the unknown mixture, X-P.
20E‘P and 2026E’Pwere the major conjugates in each age group.
Ecdysteroid Acid Analysis
A typical ion-suppression reverse-phase HPLChadioassay of an ecdysteroid
acid fraction, obtained from a 16 day sample, is shown in Figure 4B. 20EA
and 20E’A, characterized by NMR and mass spectral analyses [9], were the
only ecdysteroid acids detected in substantial quantities. They were found in
every age group and accounted for 95-100% of the total radioactivity in each
ecdysteroid acid fraction.
The profile of ecdysteroid acid titer is shown in Figures 2C (individual ecdysteroid acids) and 3 (total acids). 20EA (0.23 pg/g) and 20E‘A (0.73 pg/g) were
both present in 0 day pupae. Total acid titer increased as development progressed. Each acid concentration, especially that of 20E’A, had increased by
day 4.The level of 20EA tripled from day 4 to 8, appeared to peak on day 12 (1.77
pglg), and then declined on day 16. The level of 20E’A exhibited little significant
variation from days 4 to 12, but then it rose sharply by day 16 (2.57 pg/g).
Our long-term [14C]cholesterollabeling of ecdysteroids has resulted in the
identification of eight free ecdysteroids, two ecdysteroid acids, and eight
ecdysteroid phosphates in M. sextu during larval-pupal-adult development.
Significant among these compounds are several C-26-oxidized and C-3-epimerized ecdysteroids whose metabolism kinetics during insect metamorphosis have not been examined by HPLC/RIA because of limitations imposed by
the relatively low-binding affinities of the antisera for these ecdysteroids [6].
Our results show an approximatelyconstant titer (6-7 pg/g)of total ecdysteroids
after day 8 (Fig. 3). However, various RIA studies [6,12-141 have shown a declining ecdysteriod titer in the later stages of pupal development, because the
metabolic processes of 26-oxidation and 3-epimerization predominate in the
later stages, and the resulting 26-oxidized 3-epiecdysteroids are not detected
by RIA. Our methods and results thus provide an advantage in that the
declining titer can be accounted for by the isolation and identification of
ecdysteroid metabolites such as 26E’, 2026E’, 2026E’P, and 20E‘A. We also
have identified 20E’P as a major metabolite during M. sextu pupal development. Although this 3-epiecdysteriod is not oxidized at C-26, it had not been
detected previously by H-2 antisera [6], suggesting that the phosphate group
of 20E’P is located in the side chain, possibly at C-22.
However, because of the sensitivity of their HPLC/RIA methodology and
the specificity of their compartmental analyses of hemolymph and gut, Warren and Gilbert [6] have been able to identify certain ecdysteroid metabolites
in M. sexta that we could not identify in this study. These include 2-deoxy-E,
ecdysonoic acid, 20E-conjugates, and three E-conjugates of different polarity.
Our failure to detect these compounds is due partly to their occurrence in
Ecdysteroid Profiles in Manduca
rather low concentrations in whole insects. Also, the relative apolarity of
2-deoxy-E and the E-conjugates may have caused their retention on the HPLC
columns under our elution conditions. Nevertheless, certain observations on
the titers of the major ecdysteroid metabolites can be made from our results.
In newly pupated M. sextu, 3-epiecdysteroidspredominated in the free, conjugate, and acid fractions. Freshly ecdysed pupae of another lepidopteran,
Pieris brussicue, have also been shown to contain mainly 3-epiecdysteroids (20E’
and 20E’A) [15]. The formation of 3-epiecdysteroidsappears to be characteristic
before molting or hatching in these two insects. Newly emerged first-instar
larvae of M. sexta also contained large amounts of 3-epiecdysteroids (26E’,
2026E’, 20E’A) [7].
Between days 0 and 4 of pupal-adult development, the titer of each of the
free, conjugate, and acid fractions rose sharply because of a large synthesis of
E. The 20-monooxygenase and 26-hydroxylation/oxidation enzyme systems
were presumably active at this stage, particularly increasing the level of 20E’A.
Conjugation was very prominent as a variety of ecdysteroid phosphates were
synthesized, especially 20E’P, 26EC and 2026E’P. The major ecdysteroids
on day 4 were E and 20E’A, demonstrating that this early stage of pupal-adult
development and rapid E biosynthesis was also characterized by a variety of
catabolic reactions (oxidation, phosphorylation, epimerization), occurring in
conjunction with each other, that may regulate the titer of free ecdysteroids,
particularly E and 20E. The high catabolic activity decreased the level of free
3-epiecdysteroids as they were metabolized either to 20E’A or to phosphate
As noted previously [6] and verified here, the appearance of maximal levels
of 26E on day 4 particularly in the phosphorylated form indicated that considerable 26-hydroxylation activity occurred early in pupal-adult development.
In fact, the peak titers of 26E and 26EP on day 4 coincided with the peak titers
of E and EP. As further development to days 8 and 12 apparently promoted
higher 20-monooxygenase activity, the titers of 26E and 26EP declined in favor
of increased levels of 2026E, 2026EP, and 20EA. The appearance of the peak
titers of 26E and 26EP on day 4 could indicate a specific physiological role for
26E. Male pupal hemolymph of the European corn borer, Ostriniu nubildis,
has been found to exhibit near-coincidental peak titers of E and 26E [14]. We
believe that 26E also is a physiologically active hormone during the embryonic development of M. sextu [71.
Day 8 was characterized by dramatic qualitative changes in the free ecdysteroid titer presumably due to high oxidative metabolism by the 20-monooxygenase and 26-hydroxylase/oxidasesystems. The total ecdysteroid titer rose slightly;
a large increase in the level of 20EA was accompanied by a small decrease in the
conjugate titer. Days 4-8 thus were marked by additional synthesis of E; rapid
oxidation of E to 20E, 2026E, and 20EA; deconjugation of EP, E‘P, and 26EP,
but no changes in 20E’P or 2026E’P; and an inactive 3-epimerase system, indicated by the lack of titer increases for any of the 3-epiecdysteroids. Conjugation
may play a dual role during this period. Because of their deconjugation between days 4 and 8, EP and 26EP could serve as storage or intermediate metabolites to 20E and 2026E, whereas the constant or rising levels of 20E’P, 2026EP,
and 2026E’P suggest their possible role as inactivation products.
Lozano et al.
The total ecdysteroid titer remained unchanged from days 8 to 12 to 16; free
ecdysteroid titer decreased, while acid and conjugate titers increased. These
stages were characterized by more oxidative metabolism, particularly 26-hydroxylation/oxidation,increased 3-epimerization, and phosphorylation. As a
result of these processes, 26-oxidized 3-epiecdysteroids, and a 3-epiecdysteroid
acid especially, predominated in 16 day pupae: 2026E’ in the free fraction,
2026E’P in the conjugate fraction, and 20E’A in the acid fraction. Any significant ecdysone synthesis apparently ceased by day 8.
The present study shows that the ecdysteroid titer in M . sextu, during the
ages of pupal-adult development examined here, is characterized mostly by
the rapid synthesis of E followed by a series of C-20 and C-26 hydroxylations,
C-26 oxidation, C-3 epimerization, and phosphorylation. E, which peaked on
day 4, is metabolized, resulting in a progression of peak titers of oxidized,
epimerized, and phosphorylated ecdysteroid metabolites that produced the
following additional major ecdysteroids: 26E and 26EP peaked on day 4, 20E
on day 8, 2026E between days 8 and 12, and 20EA on day 12; and 20E’A,
2026E‘, 20E’P, 2026EP, and 2026E’P all peaked on day 16. Similar oxidative
sequences have been observed previously in pupae of M . sextu [5,6], Drosophilu melanoguster [12], and 0. nubilutis [14]. The metabolic progression of ecdysone toward a 3-epiecdysteroid acid is probably a general occurrence during
pupal-adult metamorphosis [3].
As noted previously [6], the accumulation and resulting peak titers of E
and 2026E suggests that they, in addition to 20E, may be active hormones during each of their respective intervals of peak titer. The subsequent metabolism of 2026E to the presumably less active 20EA and 2026E’ is in support of a
postulated role for 2026E. 20EA and 20E’A are logical inactivation end-products
of this metabolic sequence because of their anionic nature that facilitates their
excretion via the meconium of the hindgut. On day 16, 20E’A was found in
the largest concentration of any ecdysteroid during development. As the titer
of 20EA increased throughout and appeared to peak on day 12, more 20EA
was probably transported into the gut, where 3-epimerase activity is extremely
high [16], promoting the conversion of 20EA to 20E’A (as well as the conversion of 2026E to 2026E‘ and thereon to 20E’A).
These results suggest that activity of the 3-epimerase was significant only
at the beginning and end of pupal-adult development. Conversely, minimal
activity occurred during the interval from days 4 to 8, coincident with the stage
of peak titers of 20E and 2026E. This perhaps reflects the critical role played
by this gut enzyme in M . sextu during those late stages of development, i.e.,
late fifth-instar, pre-adult-eclosion, and late embryogenesis [7], when ecdysteroids are prepared for excretion by becoming more concentrated in the gut.
Conjugation of ecdysteroids occurred via phosphorylation. Phosphate esters
of E and 26E were mentioned above as possible storage products that occur during a period of high E biosynthesis (day 4), thus providing for the release of
free E and 26E to be metabolized by the 20-monooxygenase when high titers of
20E and 2026E are required to fill their postulated roles (at day 8). However, two
other conjugates, 20E ‘P and 2026E‘C predominated during development. Their
titers, and that of 2026EE either increased or remained fairly constant throughout, suggesting that conjugation, linked with 3-epimerization, served to inac-
Ecdysteroid Profiles in Manduca
tivate the free ecdysteroids, 20E and 2026E. The primary role of conjugation in
M . sextu as an inactivation process during pupal-adult development is in contrast to its primary role as a storage mechanism in embryonic development
during which 26EP was synthesized in adult ovaries and then hydrolyzed during
embryogenesis to release the presumably active hormone, free 26E [7].
In M . sextu pupal-adult development, conjugation and epimerization enzymes
played relatively minor roles during the time (days 8 to 12) of peak titer of the
active hormone 20E and possibly active 2026E, hormones whose titers were
more critically regulated by inactivation via the oxidative sequence leading
to 20EA. Conversely, epimerization and conjugation attained more significant
inactivation roles during intervals of low ecdysone biosynthesis and presumably low 20-monooxygenase activity (days 0, 12, 16). Perhaps such intervals
initiate the induction of the epimerase and phosphorylase systems, or else
they merely reflect the increased transport of metabolized ecdysteroids into
the gut, a site where the two enzymes may be highly active [6].
Inactivation of the ecdysteroid molting hormones in M. sextu near the end
of pupal-adult development is therefore characterized by two pairs of metabolically linked processes: l) oxidation and epimerization leading to a 3-epiecdysteroid acid (20E'A); and 2) epimerization and phosphorylation leading
to 3-epiecdysteroid phosphates (20E'P and 2026E'P).
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7, pp 295-341 (1985).
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5. KaplanisJN, Thompson MJ, Dutky SR, Robbins WE: The ecdysteroids from the tobacco hornworm during pupal-adult development five days after peak titer of molting hormone activity. Steroids 34,333 (1979).
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9. Lozano R, Thompson MJ, Lusby WR, Svoboda JA, Rees HH: Metabolism of ['4C]cholesterol
in Munducu sextu pupae: Isolation and identification of sterol sulfates, free ecdysteroids, and
ecdysteroid acids. Arch Insect Biochem Physiol7,249 (1988).
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of Invertebrate Hormones. Hoffman J, Porchet M, eds. Springer-Verlag, Berlin, pp 196-226
11. Thompson MJ, Weirich GF, Rees HH, Svoboda JA, Feldlaufer MF, Wilzer KR: New ecdysteroid
conjugate: Isolation and identification of 26-hydroxyecdysone 26-phosphate from eggs of
the tobacco hornworm, Munduca sextu (L.). Arch Insect Biochem Physiol2,227 (1985).
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12. Pak MD, Gilbert LI A developmental analysis of ecdysteroids during the metamorphosis of
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development, adults, acid, profiler, free, conjugate, pupal, ecdysteroids, sexta, manduca
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