Archives of insect Biochemistry and Physiology 14:13-30 (1990)

Qualitative and Quantitative Analyses of

luvenile Hormone and Ecdysteroids from the
Egg to the Pupal Molt in Trichoplusia ni
Christa Grossniklaus-Burgin and Beatrice Lanzrein
Division of Developmental Biology, Zoological Institute, University of Berne, Berne, Switzerland

A method was developed to determine i n the same extract juvenile hormone

and various types o f ecdysteroids i n precisely staged eggs and larvae of
Trichoplusia ni. Ecdysteroids were tentatively identified o n the basis of their
retention time in ion suppression reversed-phaseHPLC and their cross-reactivity
with two relatively non-specific, complimentary antibodies, whereas juvenile
hormone was identified using reversed-phase HPLC combined with Gafleria
bioassay. Freshly laid eggs contained low levels of immunoreactive ecdysteroids.
Mid-polar ecdysteroids increased in the phase of segmentation (14-18 h) and
1st larval cuticle formation (36-44 h), when 20-hydroxyecdysone and 20,26-
dihydroxyecdysone were found to be predominant. Only traces of ecdysone
and 26-hydroxyecdysone were seen. Toward hatching ecdysteroids decreased
and represented mainly compounds more polar than 20,26-dihydroxyecdysone.
In larval development ecdysteroids were low at the beginning of the feeding
phases, increasedtoward cessation of feeding, and reached highest levels 12-15
h before ecdysis. In feeding stages ecdysone and 20-hydroxyecdysone were
predominant, whereas i n molting stages they were seen together w i t h
20,26-dihydroxyecdysone and 20-hydroxyecdysonoic acid. The juvenile hor-
mone titer was very low in freshly laid eggs and was high (approximately 25
ng/g) i n embryos at the stage of 1st larval cuticle formation and eye pigmenta-
tion. I n eggs we tentatively identified juvenile hormones I and II, whereas in
larval stages juvenile hormone I1appeared to be the predominant or exclusive
juvenile hormone. Its titer fluctuated rapidly and was high in early 1st-instar
larvae and again before the molts into the 3rd, 4th, and 5th instar. Highest
titers were reached concomitant with the peak in 20-hydroxyecdysone 12-15
h before ecdysis.

Key words: endocrine parameters, noctuid, embryonic development, larval development

Acknowledgments: We would like to express our thanks to Mrs. A. Tschan and Mrs. A.
Riechsteiner for technical assistance, to Prof. R. Lafont (Ecole Normale Superieure, Paris) for
generous gifts of labeled 2026E and 2OEoic, and to Prof. J.D.O’Connor (UCLA) for the Horn-2
and Horn-22 antibodies. We also thank the agricultural station of Sandoz at Witterswil for their
help in rearing the Trichoplusia ni. Financial support from the Swiss National Science Founda-
tion (grants 3.201-0.82 and 3.061-0.84) is gratefully acknowledged.

Received September 15,1989; accepted December 22,1989.

Address reprint requests to Beatrice Lanzrein, Division of Developmental Biology, Zoological
Institute, University of Berne, Baltzerstrasse4 9, CH-3012 Berne, Switzerland.

0 1990 Wiley-Liss, inc.

14 Grossniklaus-Burginand Lanzrein

INTRODUCTION
Insect growth, development, and metamorphosis are under the endocrine
control of juvenile hormones and ecdysteroids. According to the classical model,
which includes larval-larval, larval-pupal, and pupal-adult molts, the molting
process is induced by ecdysteroids, whereas the concentrationof JH*determines
the nature of the molt [1,2]. This model was primarily based on ligation, trans-
plantation, exstirpation, and parabiosis experiments [2] and was later confirmed
by determining the titer of JHs and ecdysteroids at late larval and pupal stages
in various insect species [2-41. Very little is known with regard to JH and ecdy-
steroid titers in early larval instars and somewhat more in embryos. Analyses
of ecdysteroids in eggs and embryos have revealed marked differences among
species, not only with regard to quantities but also with regard to the type of
ecdysteroid found [5,6]. JHs have been analyzed in eggs and embryos of a few
species only [7-91. These analyses led to the discovery of two new JHs in some
lepidopteran species, namely JH 0 and iso-JH 0 (4-methylJH I) [7] and revealed
the presence of very large quantities of methyl farnesoate and JH I11 in a cock-
roach {8]. The function of JHsand ecdysteroids in embryos appears to be similar
to that in larval stages [6]; for the former, however, additional functions have
been observed [lo]. So far the titers of JH and the two major ecdysteroids,
namely E and 20E, have been measured in embryonic, early larval, and late
larval stages in one hemimetabolousspecies only, namely Nuuphoetu cinereu [ l l ] .
Here we report on qualitative and quantitativeanalyses of JHs and ecdysteroids
from the egg stage to the pupal molt in the holometabolous insect, Trichoplusiu
ni. We developed a method to determine, in the same extract, JH and various
types of ecdysteroidsand used only precisely staged eggs or larvae. We observed
that the ecdysteroid content and composition vary extensively in the course
of embryonic and larval development. Nevertheless the predominant ecdy-
steroids were the same in the embryos at the stage of 1st larval cuticle forma-
tion and in molting larvae, namely E, 20E, and 2026E. The JH titer was high
(approximately25 ng/g) in embryos at the stage of 1st larval cuticle formation
and eye pigmentation, and we tentatively identified the presence of JH I and
I1 at these stages. In larval stages JH I1 seemed to be the predominant or exclu-
sive JH, and its titer fluctuated between 0 and 9 ng/ml, being high in early
1st-instar larvae and before the molt into 3rd-, 4th-, and 5th-instar larvae. Our
data indicate that JH and 20E reach their highest titers simultaneously, approx-
imately 12 h before ecdysis.

MATERIALS AND METHODS

Insects
T. ni was mass-reared at 28 f 1°C at a photoperiod of 14hand fed an artificial
diet [12]. The larvae were kept in cardboard boxes (150 ml volume) in groups of
50 from the 1st to the 3rd larval instar and in groups of 12 in the 4th and 5th larval
instar. Under these conditions the 1st-to 4th-instar larvae feed for 1day and then

*Abbreviations used: E = ecdysone; 20E = 20-hydroxyecdysone; 2026E = 20,26-dihydroxyec-

dysone; 20Eoic = 20-hydroxyecdysonoic acid; 26E = 26-hydroxyecdysone; eq. = equivalents;
JH = juvenile hormone; IS-RP-HPLC = ion suppression reversed-phase HPLC; NP-HPLC =
normal-phase HPLC.
 JuvenileHormone and Ecdysteroid Titers in T. ni 15

leave the food for 1day until ecdysis to the next instar occurs 10 to 15 h after
lights on ( = day 0). In the 5th ( = last) instar 2 days of feeding are followed by
wandering behavior and subsequent spinning ( = day 3; for details see 1131).
Pupation took place on day 4.Approximately 10 days later the adults emerge,
whereupon they were kept in plastic containers (2.8 1volume) and fed with
diluted sugar water. Eggs were laid onto sterile net or nylon tissue. The majority
of eggs were oviposited at the onset of the scotophase. Hatching of the 1st
instar larvae occurred during the photophase, about 60 to 70 h after oviposition.
Chemicals
Solvents for extraction, purification, and HPLC were of pro analysis or HPLC
grade and were purchased from R o d Chemicals (Shepshed, England) or Merck
(Darmstadt, Germany). [10-3H(N)] JH I11 (specific activity 11-12 Ci/mmol),
[10-3H(N)]JHI (specific activity 13.5CUmmol), [23,24-3H(N)]E(specific activ-
ity 58.8 CYmmol), and [3H(G)]20E (specific activity 2.2 Ci/rnmol) were pur-
chased from New England Nuclear (NEN, Boston). 26E as well as labeled 2026E
and 20Eoic were generous gifts from Dr. R. Lafont. JHs I11 and I were obtained
from Calbiochem (San Diego), JH I1 was from Fluka (Buchs, Switzerland), and
E and 20E were from Simes (Milan, Italy).
Extraction and Determination of JH I1 and Ecdysteroids in Hemolymph of
4th- and 5th-Instar Larvae (Method A)
For the purpose of synchronizationlarvae, which had molted within an interval
of 3 h into the 3rd, 4th, and/or 5th instar, were selected. Hemolymph (from 10 to
50 larvae) was collected into two microcapillaries (one for JH determination, the
other for ecdysteroid determination) by clipping off one of the thoracic prolegs.
For JH determination 70 to 150 p1 was extracted with 1rnl acetonitrile kept
at 4°C.[3H]JHI11 (approximately 30,000 dpm) was added as an internal stan-
dard. Acetonitrile extracts were partitioned between pentane and brine (8%
NaC1) as described by Bergot et al. [14]. The pentane was evaporated under
Nl, and the extract was taken up in 100 pl acetonitrile and purified by HPLC.
We used an RP-18 column (25 cm x 4 mm, 5 pm; Macherey-Nagel, Oensingen,
Switzerland) with a precolumn (6 cm X 4 mm) packed with ODS (30-38 pm;
Whatman, Clifton, NJ). The solvent was 63% acetonitrile in water, and the
flow rate was 650 plhin; fractions were collected with a Helirac fraction col-
lector 2218 (LKB, Lucerne, Switzerland). [3H]JH111was detected by means of
a radioactivity flow monitor (TRACE 7140, Packard Instruments, Zurich, Swit-
zerland) and on the basis of the retention time of [3H]JHI11 a broad fraction
corresponding to JH I1 was collected (this is the only JH found in 4th- and
5th-instar larvae of T. ni by physico-chemical means, Dr. B. Hammock, per-
sonal communication). This fraction was then overlayered with hexane, and
the JH activity in the hexane phase was quantified by the Galleria bioassay
according to the method described by De Wilde et al. [15]. In order to deter-
mine the recovery, (in general between 80 and 95%)the radioactivity in the
fractions containing [3H]JHI11 was quantified by liquid scintillation counting.
For some replicate experiments, extracts were purified by passage in acetoni-
trile over a CISSep-Pak cartridge (Waters, Milford, MA) instead of HPLC. The
acetonitrile was then evaporated in a Speed Vac concentrator; subsequently
extracts were taken up in hexane and prepared for Galleria bioassay. Results
16 Crossniklaus-Biirginand Lanzrein

were corrected for recovery and transformed into JH I1 eq. with a standard
curve of JH 11. One Galleria unit corresponds to 1 pg of racemic JH I1 (FIuka,
Buchs, Switzerland).
For ecdysteroid determinations 50 to 150 p1 of hernolymph were extracted
in 70% methanol in water and centrifuged, and the sediment was washed twice
with 100%methanol. The supernatant was concentrated and the residue taken
up in 5 ml Tris/perchloric acid buffer (20 mM; pH 7-51, and loaded onto a C1g
Sep-Pak cartridge (Waters).Ecdysteroids were eluted with 5 111125% methanol
and a further 5 ml60% methanol. Part of the combined 25% and 60% metha-
nol fraction or the 60% methanol fraction was further purified by IS-RP-HPLC
using a gradient system [20] or an isocratic system of 20% acetonitrile in 20
mM Tris/perchloric acid buffer (pH 7.5) and the same precolumn and column
as for JH analyses (see above). Retention times of E and 20E were determined
with small quantities of labeled hormone to avoid "memory effects." Aliquots
of the 25% and the 60% methanol as well as the HPLC-purified fractions were
quantified with radioimmunoassayaccording to the method of Borst and OCon-
nor [17] by the use of two different antibodies, one more specific for the side
chain ([MI; DHS#-1 or Horn-2, 13.5 weeks) and the other for the E nucleus
([19]; Horn-22, 14 weeks); both were generous gifts from Dr. J.D. O'Connor.

Extraction and Determination of JH and Ecdysteroids in Eggs of 1st- to

3rd-Instar Larvae (Method B)
The eggs were collected at various intervals after oviposition and, in addi-
tion, staged according to morphological markers. Groups of 1st- to 3rd-instar
larvae at similar developmental stages were collected by selecting larvae that
had hatched (eggs) or molted into the 2nd or 3rd instar within an interval of
6 h. Between 100 and 1,000 eggs or 50 to 1,000 1st- to 3rd-instar larvae were
immediately homogenized in 70% methanol (4°C) containing t3H]JHHI as an
internal standard. Homogenates were worked up immediately or, in a few cases,
stored at -20°C. Extracts were centrifuged, the sediment was washed twice
with 100% methanol, and the volume of the supernatant was reduced in a
Speed Vac concentrator before partition between equal volumes of hexane and
water. Each phase was extracted twice with the corresponding counterphase.
The hexane phase was used for JH titer determination and, for larvae, pro-
cessed as described in method A; in the case of eggs, extracts were purified
by CISSep-Pak, if not mentioned otherwise. The aqueous phase, containing
the majority of ecdysteroids, was concentrated and purified by CI8 Sep-Pak
and HJPLC, as described in method A in the case of larval extracts. In the case
of eggs, the aqueous phase was partitioned against an equal volume of n-butanol,
each phase being back-extracted twice with an equal volume of fresh
counterphase and dried on a rotary evaporator. The residue of the butanol
phase was taken up in 5 ml water and purified by CI8Sep-Pak and HPLC, as in
method A. The residue of the aqueous phase was taken up in 10 ml 10% meth-
anol and loaded onto a CISSep-Pak cartridge, and highly polar ecdysteroids
were eluted with 15 ml 30% and 5 ml 40% methanol [20]. The solvent was
evaporated on a rotary evaporator, and the ecdysteroid content was measured
by RIA, as described in method A.
 juvenile Hormoneand EcdysteroidTiters in T. ni 17

5
c
1500 U 1 juvenile hormone

ha mid polar ecdi

El polar ecd.
T
v I highly polar ecd.
u
e,
w 1000
0
N
.-C
v)
.-
V
g
+
500
v)
x
U
0
a,

0
0-3 14-18 20-24 36-40 40-44 58-62 62-64
c

.-
0
I
C
.-0
Y
0
.-
C
0
c
.-
c
OT
Y
K
0
.-
v)
-w

-ea,
0
.-a> k
% 0
v)
v-
0
e
a,
c
0
-xm
c c
.-r
'c
4-
ern

Fig. 1. Simultaneous analysis in eggs of JHand mid-polar, polar, and highly polar ecdysteroids
in relation to time and developmental markers. Extracts were purified according to method B
(see "Materials and Methods"). J H activity of the CIBSep-Pak fraction is expressed i n JH II eq.
and corrected for recovery. Following butanol-water partition ecdysteroids were measured in
the water phase (highly polar ecdysteroids) and in the butanol phase after passage through
C18 Sep-Pak, which separated polar (25% methanol fraction) and mid-polar (60% methanol
fraction) free and conjugated ecdysteroids. Ecdysteroid activitywasdetermined with the Horn-22
antibody and is expressed in 20E eq. The number of determinations was between 1 to 5.

RESULTS
Ecdysteroids and JH During Embryogenesis
The results of simultaneous determination of JH and various types of
ecdysteroids during embryogenesis and in relation to developmental markers
are presented in Figure 1. The data indicate that freshly laid eggs of Trichoplusid
ni contain some mid-polar and few polar ecdysteroids but almost no highly
polar ecdysteroids and very little JH. Analysis of the mid-polar ecdysteroids
by HPLC (Fig. 2 top; Table 1)revealed that they were of equal or higher polar-
ity than 2026E and that some were recognized by the Horn-22 antibody only.
In the course of development the quantity of polar ecdysteroids (25% metha-
18 Grossniklaus-Biirginand Lanzrein

EGG 0-3h
V-v HORN-2
*-• HORN-22

1
J
EGG 64-66h

100-

v
a
6-
0
W
0
N

0 10 20 30 40 50 60

froction number

Fig. 2. RIA activity profile of mid-polar ecdysteroids at three stages of embryogenesis. Data
are expressed in 20E eq. for both antibodies. The 60% methanol fraction of C18 Sep-Pak was
separated by isocratic IS-RP-HPLC with 20% acetonitrileR0 mM Tris/perchloric acid buffer (pH
7.5). The flow rate was 500-550 FVmin, and fractions were collected every minute. Retention
times for E, 26E, 20E, and 2026E are indicated.
 JuvenileHormone and Ecdysteroid Titers in T. ni 19

TABLE 1. Stage-DependentProportion of Ecdysteroids of Varying Polarity in Relation to the

Developmental Stage of the Embryo*
More polar than 20E 20E Less polar than 20E
Stage nglg % nglg % nglg %

0-3 h 70.6 95.8 2.6 3.5 1.1 1.5

20-24 h 77.8 85.1 12.8 14.0 0.8 0.9
132.2 82.7 21.9 13.7 5.8 3.6
36-40 h 182.6 58.6 89.6 28.7 39.5 12.7
58-62 h 69.9 58.2 37.4 31.1 12.8 10.6
64-66 h 35.6 89.4 4.6 11.6 2.6 6.5
‘Ecdysteroids were processed as described in method B, and the 60% methanol fraction of the
butanol phase was analyzed by IS-RP-HPLC using 20% acetonitrile in 20 mM Tris/HC104at pH
7.5. Fractions were collected every minute. Ecdysteroid activity was determined with the Horn-22
antibody and is expressed in 20E eq.

no1 fraction) remained low, whereas that of highly polar ecdysteroids (water
phase of butanol partition) rose to high levels (approximately 800 ng 20E eq./g)
in 36-44 h old eggs and then dropped to low levels before hatching. The mid-
polar ecdysteroidsincreased for the first time at the stage of segmentation(14-18
h) and again at the stage of 1st larval cuticle formation and eye pigmentation
(36-44 h); thereafter a decrease was seen (Fig. 1). HPLC analysis of the mid-
polar ecdysteroids in the course of embryogenesisrevealed that 20E reaches a
peak in 36-40 h old eggs (Table l),the stage of 1st larval cuticle formation. At
this stage we also observed E, possibly 26E, 2026E, and more polar ecdysteroids
(Fig. 2, middle). Shortly before hatching ecdysteroids of equal and higher polar-
ity than 2026E were predominant (Table 1;Fig. 2).
JH titers increased continuously to maximum levels of 26 ng/g at the stage
of 1st larval cuticle formation and eye pigmentation; thereafter a rapid decrease
was seen, followed by a slight increase shortly before hatching (Fig. 1). In an
attempt to identify the embryonic JH, extracts (36-44 h) together with a small
quantity of [3H]JHI11 as an internal standard were analyzed by RP-HPLC or
NP-HPLC. Small quantities of t3H]JHI (to determine the retention time) were
injected only after the biological sample to avoid a “memory effect.” Fractions
with retention times of JH I11 up to JH 0 as well as the purge were then tested
using the Galleria bioassay. We observed JH activity in the JH I and I1 fractions
and calculated a proportion of JH 1:II of approximately 1.3-131. The other
fractions showed no JH activity.
Ecdysteroids and JH in Whole-Body Extracts of 1st- to 3rd-Instar Larvae
Figure 3 shows the titer fluctuations of the ecdysteroids in the 60% and 25%
methano1 fraction and of JH in 1st- to 3rd-instar larvae. The ecdysteroids in
the 60% methanol fraction appeared as three low, broad peaks. As we sup-
pose, in analogy to the findings made in 4th- and 5th-instar larvae (Fig. 5),
that the highest values would be attained 12-15 h before each ecdysis, a point
when no measurement was made, we have joined the values obtained 19 h
and 7 h before ecdysis by a dashed line only. The ecdysteroids in the 25%
methanol fraction (possibly 20Eoic, see “Discussion”) increased every time
the larvae left the food. The JH titer decreased in feeding 1st-instar larvae,
 -5
K
W
-
0
C
200 g
i?
%N
._
C

100 -$
a,
a

a
l
0

DAY
STADlU M

Fig. 3. Simultaneous analysis of ecdysteroids in 60% methanol and 25% methanol fraction of
Sep-Pak (top) and JH II (boitom) in whole-body extracts of 1st- to 3rd-instar larvae. Extracts
were purified according to method 8. JH activity in the HPLC fraction corresponding to JH II
(solid circles) or of C,8 Sep-Pak fraction (open circles), is expressed in JHI I eq. and corrected
for recovery. Ecdysteroids in the 60% and 25% methanol fraction are expressed in 20E eq. of
the Horn-22 antibody. Symbols represent values of single determinations, whereas curves show
the corresponding mean values. Dashed lines indicate the interval, when the molting peak
maximashould occur, and dotted lines indicate the transition to the4th instar. Asterisks denote
stages from which RIA activity profiles given in Figure 4 were obtained. Black bars indicate the
scotophase, and hatched bars indicate the feeding phase.
 JuvenileHormone and Ecdysteroid Titers in T. ni 21

was low during the molt to the 2nd instar, increased during the molt to the
3rd instar, and varied strongly in feeding 3rd-instarlarvae. The similaritybetween
the levels obtained with CI8Sep-Pak or HPLC-purified (JH I1 fraction) extracts
suggests that JH I1 is the predominant or only JH at these stages.
The RIA activity profile observed approximately 6 h before ecdysis into the
3rd instar (asterisk Fig. 3), when the amount of ecdysteroids in the 60% meth-
anol fraction fell, is shown in the upper panel of Figure 4.It reveals the pres-
ence of E, a product slightly less polar than 20E that is not recognized by the
Horn-2 antibody, 20E, and 2026E. The RIA activity profile obtained immedi-
ately before the increase in ecdysteroids in the 60% methanol fraction (aster-
isk Fig, 3) is given in the lower panel of Figure 4 and shows the presence of
mostly 20E, possibly its epimeric form (shoulder seen with the Horn-2 anti-
body only), some E, and ecdysteroids less polar than E.
Ecdysteroids and JH in Hemolymph of Penultimate and Last-Instar Larvae
Figure 5 shows a typical example (out of 3 independent experiments) of a
simultaneous analysis of the ecdysteroids in the 25%and 60% methanol frac-
tion of HPLC-purified E and 20E and of JH I1 in the hemolymph of precisely
staged 4th- and 5th-instar larvae. The titers of ecdysteroids in the 60% metha-
nol fraction displayed rather broad peaks, reaching similar levels in the larval
and pupal molting phase. The amount of ecdysteroids in the 25% methanol
fraction (possibly20Eoic, see "Discussion") was very low during feeding, some-
what higher during larval molting, and increased in the prepupa. The titer of
E was always rather low and reached its highest levels (130 ng E eq./ml) in the
prepupa. The titer of 20E was low during the feeding stages and rose rapidly
to high levels approximately 12 h before ecdysis into the 5th instar and again
12 to 15 h before pupation. In the scotophase of day 2 in the 5th instar, i.e.,
before the onset of wandering, a very small peak of 20E was observed.
The titer of JH I1 fluctuated rapidly between 1 and 4 ng/ml in the feeding
4th-instar larvae and reached its highest level (9 ng/ml) in the late scotophase,
concomitant with the peak in 20E, 12h before ecdysis into the 5th instar. There-
after the JH titer dropped rapidly and then rose again during the scotophase
after ecdysis. In the following days JH was not detectable until the time of
cocoon spinning (day 3), when a slight increase was noted. HPLC analyses
(using a gradient system) of the combined 25% and 60% methanol fraction of
L4D1 and L5DO (stages indicated by asterisks in Fig. 5) are given in Figure 6 .
The top panel (L4D1) shows the presence of 20E and E, and possibly their
epimeric forms (shoulders only recognized by the Horn-2 antibody) in feed-
ing stage 4th instar larvae and of ecdysteroids less polar than E. The lower
panel (L5DO)shows the presence of 20E, E, 2026E, and 20Eoic in larvae shortly
before ecdysis into the 5th instar.

DISCUSSION
Qualitative and Quantitative Variations in Ecdysteroids From the Egg to the
Pupal Molt and Biological Significance
In our analyses ecdysteroids were tentatively identified on the basis of their
retention time in IS-RP-HPLC and their cross-reactivity with two relatively
22 Crossniklaus-Burginand Lanzrein

150

V-- V HORN-2 L3DO

0-• HORN-22
n

.-$0t 100
2 W

s
W
a
0-
Q, 50
w
0
N

300

L3D 1
W
0
N

200 -
V W
w
1
u3
/i
N i!
w
e,
W
0
N

0 10 20 30 40 50

fraction number
Fig. 4. RIA activity profile of molting (L3DO) and feeding (L3D1) 3rd-instar larvae. Data are
expressed in 20E eq. for both antibodies. The 60% methanot fraction of CISSep-Pak was sepa-
rated by isocratic IS-RP-HPLC with 20% acetonitrileR0 m M Trisiperchloric acid buffer (pH 7.5).
The flow rate was 550 plirnin, and fractions were collected every minute. Retention times for E,
20E, and 2026E are indicated.

non-specific, complimentary antibodies. The cross-reactivity of various

ecdysteroids with these antibodies was determined by Warren and Gilbert
[21]. As our data are expressed in 20E eq., the relative affinity of the two
antibodies has to be taken into account in order to roughly calculate absolute
quantities of ecdysteroids in HPLC fractions other than 20E. For the major
ecdysteroids dealt with in this paper this means that, in the case of the Horn-22
 JuvenileHormone and EcdysteroidTiters in T. ni 23

G
1200 \
E
1200
rn
C
-0
v

900 900 g
C

i
600 600
s
N
.-c
u0
P)

300
300 [
0 0

c.
E
-2
C
v
-
-
400 B w
C
0
E
0
-c
.-C
-
0)

200 t 4 2
.-
3

I
I

0 0
1 2 3 4 DAY
LS STADIUM

Fig. 5. Simultaneous analysis of ecdysteroids in the 60% and 25% methanol fraction of Sep-
Pak (top)and of JH II, E and 20 E (bottom) in hemolyrnph of precisely staged 4th- and 5th-instar
larvae. Extracts were purified according to method A. JH activity in the HPLC fraction corres-
ponding to JH I1 is expressed in JH II eq. and corrected for recovery. Ecdysteroids in the 60%
and 25% methanol fraction are expressed in 20E eq., respectively. 20E and E are expressed in
20E and E eq. Solid lines indicate the scotophase and broken lines the feeding phase. Aster-
isks denote the stage from which the RIA activity profile given in Figure 6 was obtained.

antibody, the values given in 20E should be multiplied (according to Warren

and Gilbert [Zl])by a factor of 0.2 for E and 26E and of 1.1for 2026E and 20Eoic.
For the Horn-2 antibody the factors for E, 26E, 2026E, and 20Eoic are 0.3, 5,
20, and 26.7, respectively. In an earlier work we identified the major metabo-
lites formed after injection of [3H]Einto 4th- and 5th-instar larvae as 20E, 2026E,
and 2OEoic [161.
Freshly laid eggs of Trichoplusiu ni contained rather low quantities of immu-
noreactive ecdysteroids, which occurred mainly in the mid-polar fraction (Fig.
24 Crossniklaus-Burgin and Lanzrein

150

L4D 1
w
0
c-4
V -- V HORN-2

100 - HORN-22

50

L5DO
w
400 0
nl

.-00 V
W
0
[\I
200

fraction number
Fig. 6. RIAactivity profile of feeding (L4D1)and molting (L5DO)day4th-instar larvae as expressed
in 20E eq. for both antibodies. Combined 25% and 60% methanol fraction of CISSep-Pak were
injected into IS-RP-HPLC and eluted with a gradient of 840% acetonitrile/20m M Trislperchloric
acid buffer (pH 7.5). The flow rate was 1 ml/min, and fractions were collected every minute.
Retention times for E, 20E, 2026E, and 2OEoic are indicated.

1; Table 1); analysis of the latter by HPLC revealed that these ecdysteroids
were equally polar to or more polar than 2026E. The ecdysteroids of greater
polarity than 2026E were recognized by both antibodies, while those of equal
polarity were recognized mainly by the Horn-22 antibody (Fig. 2). This sug-
gests the presence of 2026E or another side-chain-modified ecdysteroid. In
freshly laid eggs of the phasrnids Clitumnus [22] and Curausius [23], the cricket
Gryllus bimuculatus [24], and the cockroaches Nuuphoeta cinerea [25] and Peeri-
pluneta umericana [26] the quantity of immunoreactive ecdysteroids was also
low. In the latter species they represented mainly apolar ecdysteroid esters
[26,27]. In contrast, newly laid eggs of other species such as Schistocerca greguria,
 juvenile Hormone and Ecdysteroid Titers in T.ni 25

Locusta migraforia, Galleria mellonella, Bombyx mori,and Munduca sexfu were shown
to contain very high quantities of immunoreactive ecdysteroids, which were
mainly highly polar conjugates [6,20,28,29].
In the course of embryonic development content and composition of
ecdysteroids varied extensively (Figs. 1,2; Table 1).The highly polar products
reached high levels concomitant with the mid-polar ecdysteroids (Fig. 1)and
may represent ecdysteroid acids or conjugates. The mid-polar ecdysteroids
increased in the phases of segmentation (14-18 h) and 1st larval cuticle forma-
tion (36-44 h), and analysis by HPLC in 36-to-40-h old eggs revealed the pres-
ence of E, 20E, 2026E, possibly 26E, and several unidentified ecdysteroids (Fig.
2). Calculation of absolute quantities, taking into account the different cross-
reactivity, showed 2026E and 20E to be most abundant, namely 121 ng/g and
105 ng/g, respectively. In comparison, E and 26E were present at concentra-
tions of only 8.2 ng/g and 2.5 nglg, respectively. In Munducu sextu 26E and 2026E
are the predominant free embryonic ecdysteroids [20,28,30], and 20E increases
only in 48-to-64-h oId eggs [30], which are at the stage of 1st larval cuticle
formation. At this stage the free ecdysteroids reach peak levels in many spe-
cies and consist mainly of E, 20E, and 2026E in Nuuphoefa cinereu [31]and in
Loctrsta migraforia [32], of E and 20E in Schistucerca gregariu [33,34], Perzplanefa
[27], and Gryllus 124,351, and of 20E, 2026E, and 2-deoxy-20E in C U ~ R U[23]. S~~S
Among the diversity in quality and composition of the ecdysteroids seen dur-
ing embryonic development in the various species, one common characteris-
tic is the increase of 20E at the stage of 1st larval cuticle formation.
Toward hatching all ecdysteroids dropped to low levels (Fig. 1)and HPLC
analysis of the mid-polar ecdysteroids revealed that E, 26E, and 20E had almost
disappeared and that ecdysteroids equally polar to and more polar than 2026E
were predominant (Fig. 2; Table 1).
In larval development the content and composition of ecdysteroids changed
in relation to the molting cycles (Figs. 3, 5). At the beginning of the feeding
phases the ecdysteroid titer was always low. Toward cessation of feeding it
increased in 1st- to 5th-instar larvae, E and mainly 20E being predominant (Figs.
4, 6). At that stage two ecdysteroids less polar than E were also noted (Figs.
4, 6); a labeled compound of similar polarity was seen also after injection of
[3H]E into feeding 4th- and 5th-instar larvae [16]. In addition, the presence of
the epimeric forms of E and 20E is suggested by the fact that the E and 20E
peaks were broader when analyzed with Horn-2 than with the Horn-22 anti-
body (Figs. 4, 6). [3H]Emetabolism experiments also indicated epimerization
of E and 20E in feeding larvae [16]. In the molting phases the ecdysteroids in
the 60% methanol fraction showed broad peaks (Figs. 3,5). At the end of the
molting phase the predominant ecdysteroids were E, 20E, 2026E, and 20Eoic
(Figs. 4, 6). The fact that the peak with a retention time of 2026E was very
broad (Figs. 4,6) suggests the presence of an additional ecdysteroid that has a
polarity between that of 20E and 2026E. A metabolite of similar polarity was
also seen after injection of [3H]Einto molting larvae [16]. As this additional
ecdysteroid was recognized almost exclusively by the Horn-22 antibody (Figs.
4,6), it would appear to be a side-chain modified ecdysteroid.
The ecdysteroids in the 25% methanol fraction represent mostly 20Eoic
(unpublished results) and increased in phases when the larvae were away from
the food (Figs. 3,5).
26 Crossniklaus-Burginand Lanzrein

The most detailed analyses of titer changes were carried out with 4th- and
5th-instar larvae (Fig. 5). The data obtained revealed broad and high peaks
of ecdysteroids in the 60% methanol fraction, while the peaks of 20E were
sharper and lower. This indicates that changes in ecdysteroids in the 60% meth-
anol fraction only partially reflect changes in 20E. Peak levels of 20E were attained
in the late scotophase 12-15 h before ecdysis. In the 5th instar we observed a
slight increase in 20E during the scotophase before the onset of wandering
(Fig. 5). A small increase in total ecdysteroidsat this stage has also been reported
by Newitt and Hammock [36] and Jones et al. [37]. Presumably this minor
peak in the absence of JH induces wandering behavior and pupal commit-
ment as shown for Munduca sextu [38,39]. The increase in 20E seen in the early
prepupa (Fig, 5) may stimulate epidermal mitoses [40] and/or the production
of JH [41] as proposed in Manduca sexta.
Qualitative and Quantitative Variations in JH From the Egg to the Pupal Molt
and Relation to 20E
In our analyses JH activity was measured using the Galleria wax test after
the extracts had been purified through HPLC or CISSep-Pak. In earlier stud-
ies we found that this assay produced reliable data that, in comparative mea-
surements, corresponded well with data obtained using physico-chemical
methods [11]. Based on lack of activity of 10s JH I11 in the Galleria wax test
[42], it is assumed that only one enantiomer (10R) of JHs I and I1 is also active
in this test. In this case, the titers of JHs I and I1 measured, based on racemic
material, are overestimated by a factor of 2.
With regard to the identity of JH in Trichoplusia ni physico-chemical analy-
ses had indicated the presence of only JH 11 in hemolymph of 4th- and 5th-instar
larvae (B. Hammock, personal communication),and our analyses of these stages
confirmed this finding (data not shown), On the other hand, in 36 to 44 h old
eggs we tentatively identified JHs I and I1 in the ratio of 1.3-1.8:1, using HPLC
analysis. In 1st- to 3rd-instar larvae JH I1 seemed to be the predominant or
only JH, since values obtained in the HPLC fraction corresponding to JH I1
were very similar to those obtained in extracts purified by Sep-Pak only (Fig.
3). These observations indicate a change in the composition of JHs from the
embryonic to the larval stages. Similar although more complex changes have
been observed in Manduca, where iso-JH 0, JH 0, and JH I were predominant
in the egg [7], JHs I1 and I in early 4th-instar [43], JH I1 in late 4th-instar [44],
and JHs I and I1 plus their acids in 5th-instar larvae [MI.
The quantity of JH in eggs of T. ni varied extensively in the course of embry-
onic development: in freshly laid eggs the titer was very low (220 pg JH I1
eq./g) and subsequently increased, together with the increase in 20E, to reach
highest levels (26 ng JH I1 eq./g) at the stage of eye pigmentation. Thereafter it
decreased rapidly to low levels and increased again slightly shortly before hatch-
ing at the stage of head capsule pigmentation (Fig. 1).In Manducu the quan-
tity of JH varied between 0 ng/g in freshly laid eggs and 5.5 ng/g (iso-JH0, JH
0 and JH I) in the middle of embryonic development [7]. In Bombyx JH was
absent in newly laid eggs [45], but later appeared and rose twice to very high
levels, namely up to 1,000 ng/g, according to RIA measurements [46]. In two
other lepidopteran species, namely Hyulophoru cecropia and Heliothis virescens,
 juvenile Hormone and Ecdysteroid Titers in T, ni 27

the highest values measured were 0.5 ng/g (JHs 0, I and 11) and 1.5 ng/g (JHs
0, I, and 11), respectively [7]. In the cockroach Nuuphoeta JH was absent in newly
formed eggs 1311but later appeared and rose to high levels (960 ng JH III/g) at
the stages of 1st larval cuticle formation and eye pigmentation [ll]; in this spe-
cies we observed very high quantities of methyl farnesoate concomitant with
the increase in JH 111[8].In Locusta, freshly laid eggs contained 1.8 ng/g JH 111
and embryos at the stage of 1st cuticle formation 8 ng/g JH I11 [9]. It would
seem that the composition and content of JH in embryos varies considerably
among different species. Nevertheless one general phenomenon appears to
be an increase in the JH titer concomitant with the increase in 20E at the stage
of 1st larval cuticle formation, a high JH titer at the stage of eye pigmentation
and a decrease toward hatching.
In early larval instars the titer of JH I1 fluctuated between 0.4 and 2.6 ngig,
being high after hatching from the egg and again concomitant with the increase
in ecdysteroids before the molt into the 3rd and 4th instar, but not into the
2nd instar (Fig. 3). As no other data are available on JH and ecdysteroid fluc-
tuations throughout larval development, it is not clear whether a low JH titer
before the molt into the 2nd instar is a general phenomenon or a pecularity of
Trichoplusia ni. Clearly more data will have to be accumulated to verify this. In
4th-instar larvae, where measurements were carried out every 6 h, the JH I1
titer in hemolymph increased twice for a short time from 1 to 4 ng/ml in the
feeding phase (Fig. 5). We propose that the 1st of these peaks induces the
secretion and/or production of prothoracicotropichormone, as allatectomy and
JH-application experiments in penultimate instar larvae of Marnestru brussicae
showed JH to be necessary for the production of prothoracicotropic hormone
[47]. The 2nd peak may act morphogenetically as concluded from ligation and
JH-application experiments in Manduca sexta [48]. Twelve to 16 h before ecdy-
sis the JH titer increased to 10 ng/ml concomitant with the increase in 20E
(Fig. 5 ) . This JH peak may play a role in the coloration and pigmentation of
the cuticle, as indicated by experiments made in M. sextu [49,50]. After ecdy-
sis into the 5th instar the JH I1 titer was high again for several hours, as also
seen in M. sexta [44] and then dropped to undetectable levels until the wan-
dering phase. The absence of JH at this stage is responsible for the onset of
metamorphosis [38] and was seen to be necessary for the prothoracic glands
to gain steroidogeniccompetence [51-531. In the prepupa JH I1 increased slightly;
at this stage an increase in JH and predominantly JH acids was noted in M .
sexta [44]. In T. ni we did not analyze JH acid titers and it is not known whether
their corpora allata also release JH acids at this stage, as do the corpora allata
of M . sexta [54]. According to the results of JH application and deprivation
experiments using T. ni, this peak of JH is necessary for the proper formation
of the pupa and for the induction of JH esterases [36,55] in this species. JH I1
titers measured physico-chemically in 4th- and 5th-instar larvae were in the
range of 0.5 ng/g (B. Hammock, personal communication) and correspond
well with our data. On the other hand, the few values reported by Jones et al.
[37], who analyzed non-purified extracts by the black mutant bioassay, were
10-50 times higher. JH titer determinations performed in penultimate and last-
instar larvae of Mamestra brussicue and Bartha brussicue [56] and Galleria mellonellu
[57,58] showed a similar tendency to those seen in Trichoplusia ni, but mea-
surements were taken at much greater intervals in these cases.
28 Crossniklaus-Burghand Lanzrein

In conclusion, our data reveal great variations in the content and composi-
tion of both JHs and ecdysteroids in the course of embryonic and larval devel-
opment of T. ni. The analyses made at short intervals indicate that titers of JH
and 20E fluctuate very rapidly and revealed the presence of previously unob-
served peaks of JH and 20E. It is clear that much more research needs to be
done in order to determine the biological function of the various ecdysteroids
and JHs as well as the interaction between these two classes of hormones
throughout the development of an insect.

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