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Juvenile hormone esterase activity and ecdysteroid titer in Heliothis virescens larvae injected with Microplitis croceipes teratocytes.

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Archives of Insect Biochemistry and Physiology 20:231-242 (1992)
JuvenileHormone Esterase Activity and
Ecdysteroid Titer in Heliothis virescens Larvae
Injected With Microplitis croceipes
Teratocytes
Deqing Zhang, Douglas L. Dahlman, and Dale B. Gelman
Department of Entomology, University of Kentucky, Lexington (D.Z., D.L.D.); Insect
Neurobiology and Hormone Laboratory, U.S. Department of Agriculture, Agricultural Research
Service, Beltsville, Maryland (D.B.G.)
Juvenile hormone esterase (JHE)activity in the hemolymph of 5th-instar
Heliothis virescens larvae injected with Microplitis croceipes teratocytes was
inversely related to the number of teratocytes injected. JHE activity in the
hemolymph of larvae injected with 750 3-day-old teratocytes (the approximate
number from one parasitoid embryo) was depressed to less than 5% of those
levels found in control larvae. During the latter portion of the digging stage
and in the burrowing-digging (BD) stage JHEactivity in larvae treated with 350
teratocytes was approximately 40% of control values. However, injection of
180 teratocytes did not significantly affect JHEtiters. Two-day-old teratocytes
caused the greatest reduction in JHEtiter with decreasing effects observed
with injections of 3- to 6-day-oldteratocytes. Nevertheless, because 2-day-old
teratocytes were difficult to separate from host hemocytes, 3-day-old teratocytes
were used in most of these studies. Injections of nonparasitired H. virescens
hemolymph plasma, Micrococcus lureus bacterial cell walls, washed M. croceipes eggs, or teratocytes from Coresia congregata did not depress JHEtiters.
Teratocyte injections also significantly reduced growth of host fat body. Ecdysteroid titers in cell formation, day 2 (CF2) larvae injected as new 5th instars
with 350 3-day-old teratocytes failed to increase, as compared to noninjected and
saline-injected controls. An injection of 1 pg/larvaof 20-hydroxyecdysone at the
BD stage permitted normal pupation in 50% of the teratocyte-treated larvae as
compared to 0% pupation for teratocyte-treated control larvae not treated with
20-hydroxyecdysone. Teratocytes seem to be responsible for the inhibition of JHE
release and thus indirectly impact on ecdysteroid titers. o 1992 ~ i ~ e y - L i s Inc.
s,
Key words: tobacco budworm, parasitoid, ecdysteroids, Braconidae, Noctuidae
Acknowledgments: The authors express appreciation for the technical assistance of T.J. Neary
and Becky Wilson for help with insect rearing. The authors gratefully acknowledge support from
USDA Competitive Research Grant 89-37250-4705and a grant from R.J. Reynolds. This is paper
91-07-189of the Kentucky Agriculture Experiment Station, Lexington, KY 40546-0091.
Received October 31,1991; accepted April 9,1992.
Address reprint requeststo Dr. Douglas L. Dahlman, Department of Entomology, 5-225 Agricultural Sciences North, Universityof Kentucky, Lexington, KY 40546-0091.
0 1992 Wiley-Liss, Inc.
232
Zhang et al.
INTRODUCTION
High titers of JH+are responsible for maintaining the larval stages in holometabolous insects [11. In Lepidoptera, JH titer is high immediately following
the last-larval ecdysis but then declines to a very low or even undetectable
level [l]. A short burst of ecdysteroid then permits the reprogramming of the
larval cells to express their pupal form. This is followedby a second large burst
of ecdysteroid which is responsible for the larval-pupal molt. In this sequence
of events the degradation of JH by ester hydrolysis is the major pathway of JH
catabolism. Indeed, THE has been suggested as the primary agent responsible
for the reduction in JH titers, which in turn is necessary for normal insect development [2]. Thus, the decrease in levels of JH is correlated with the level of
JHE which degrades JH in the presence of carrier proteins [3]. The change in
tissue commitment from larvae to pupa is inhibited when the JH titer is maintained at an artificially high level [4-61. Available evidence suggests a very close
relationship between JH titer and JHE level during the last larval stadium [7-91.
Based on the measurement of JHE in 11 species of Lepidoptera plus two other
species for which data were available, Jones et al. [lo] concluded that most
Lepidoptera exhibit two peaks of JHE activity during the last larval instar, the
relative size of which varies from species to species. The first peak occurs
near the time of maximum weight and the second peak late in the pharate
pupal phase.
Studies on parasitoid regulation of host development emphasized three independent but interactive sources of regulatory substances which affect the host.
They are 1)the adult female parasitoid, which not only deposits eggs but may
also inject glandular secretions and polydnavirus, 2) the developing parasitoid
larva within the host, and 3) the extra-embryonic tissues, usually called
teratocytes.
Teratocytes originate from the serosal membrane surrounding the developing parasitoid embryo. These cells are liberated into the host hemolymph when
the parasitoid larva hatches [ l l ] and have been suggested to play trophic, immunosuppressive, and secretory roles [12,13]. In a previous study [14], we found
that pupation was prevented when nonparasitized Heliothis virescens larvae
were injected with Microplitis croceipes teratocytes. These injected larvae showed
behavioral and morphological characteristics similar to larvae parasitized by a
wasp and containing the parasitoid larva.
We initiated this study to determine if the injected teratocytes were responsible for the reduced levels of ecdysteroids and JHE that were observed previously in parasitized larvae [15]. This experimental design allows a distinction
between the effects of teratocytes and those of the parasitoid larva and/or adult
wasp products such as poison gland secretions or calyx fluids.
*Abbreviations used: BD = burrowing-digging, 5th stadium; CFI = cell formation, day 1;
CF2 = cell formation, day 2 ; D1 = digging, day I ; D2 = digging, day 2; D3 = digging, day 3;
DLM = delayed larval mortality; 20-OH-ecdysone = 20-hydroxyecdysone; ILPE = incomplete
larval pupal ecdysis; JH = juvenile hormone; ]HE = juvenile hormone esterase; N5 = new
5th stadium; P = normal pupa; PP = pharate pupa; PlTH = prothoracicotropic hormone;
S = slender, 5th stadium.
Teratocyte Effects on JHEand Ecdysteroids
233
MATERIALS AND METHODS
Insects
Methods of rearing host larvae and parasitoids, collection and preparation
of teratocytes, and procedures for injection of teratocytes into nonparasitized
hosts have been described previously [14]. Briefly, hemolymph was collected
from appropriate age parasitized larvae, diluted with saline, and the teratocytes
were separated from hemocytes via several low speed centrifugations. The
concentration of teratocytes was determined and the volume was adjusted in
order to deliver a specified number of teratocytes in a fixed volume of saline.
All nonparasitized larvae used in this study were staged according to the
criteria of Webb and Dahlman [16], which consist of size and behavioral characteristics during the 5th instar. The new and slender stages each last approximately 1 day. The digging stage, usually 2 days long, is followed by a 1-day
long burrowing-digging stage. The larva does not feed during the cell formation stage, which lasts 2 days. A l-day long pharate pupal stage precedes pupation, N5 nonparasitized larvae were injected with 10 pl of various doses of
teratocytes suspended in Pringle’s saline [17] using either a Hamilton syringe
or a 0.5-cc U-100 insulin syringe (Becton Dickinson, Rochelle Park, NJ).
JHE Assay
Hemolymph samples for the JHE assay were obtained by clipping a proleg
from a chilled (OOC), flaccid larva. The hemolymph from each individual larva
was collected into an ice-chilled microcentrifuge tube containing a few crystals
of phenylthiourea (to inhibit tyrosinase activity). For each treatment period,
eight samples were taken, each from an individual larva; all experiments were
repeated at least three times for a total of 24 samples per treatment period.
Hemolymph samples were centrifuged for 4 min in a microcentrifugeat 10,OOOg.
The supernatant was diluted (usually 1:1,000) in 0.2 M phosphate buffer (pH
7.4) and used as the enzyme source for the JHE assay. Substrates were prepared
according to Hammock and Roe [18]. [3H]-JuveniIehormone 111 (17.4pCi/mmol)
was purchased from New England Research Products, Boston, MA. Unlabeled
JH was obtained from Fluka Chemical Corp., Ronkonkoma, NY,The assay
[18] was based on the fact that JHE hydrolyzes JH to JH acid. The reaction was
terminated after a specified time and the JH and JH acid were separated by
their different solubilities in immiscible organic and aqueous solvents. The
amount of hydrolyzed JH is determined by the amount of radioactivity associated with each fraction when compared to appropriate controls, The assay
is designed so that the amount of diluted enzyme used produced a linear
increase in product (JH acid). Under most conditions, 100 pl of a 1:1,000 dilution of hemolymph ( = 0.1 pl hemolymph) is incubated with the substrate for
15 rnin. JHE activity is expressed as nmol JH hydrolyzed per min per ml
hemolymph.
To test the effects of other biological preparations on JHE titers, N5 larvae
were injected with either 10 PI of the biological preparations or with 10 p1 of a
suspension of the material made up in Pringle’s saline. The test materials
included: hemolymph plasma from N5 H. virescens (prepared by a 2-min centrifugation at 10,OOOg); 20 Fg of finely ground lyophilized cells of Micrococcus
234
Zhang et al.
luteus; three or four M. croceipes eggs dissected from the ovary of a wasp and
then washed in Pringle’s saline four times before injection; and 500-800 teratocytes from Cotesia congvegufa (collected from the hemolymph of parasitized
Munducu sextu larvae in a manner similar to the collection of M . croceipes
teratocytes). Noninjected and Pringle’s saline-injected larvae served as controls. Hemolymph for the JHE assay was drawn at D2. For each treatment six
to eight larvae were injected. For the JHE assay hemolymph samples from
individual larvae were collected as described above.
Ecdysteroid Titers
To determine the effect of teratocytes on hemolymph ecdysteroid levels, N5
larvae were injected with 350 3-day-old teratocytes. Samples were taken at 24-h
intervals between BD, CF1 and CF2 and then at two 12-h intervals subsequent
to CF2. Because teratocyte-injected larvae seldom demonstrate CF2 characteristics, sample collection was based on chronological age rather than on morphological characters. Each sample contained 50 ~1.1
of hemolymph (10 pl from
each of five larvae) placed in 250 pl of ice-cold 70% methanol. The samples
were stored at - 75°C. At least three replicates were taken at each time period.
Ecdysteroid titers were determined by RIA following the methods of Borst and
OConnor [19] and Bollenbacher et al. [20] as described by Gelman and Woods
[21] and Gelman et al. [22]. The antiecdysone, a gift from W.W. Bollenbacher,
was prepared from a hemisuccinate derivative of ecdysone (at the C-22 hydroxyl
group) that had been coupled to thyrogloblin. The antibody had a high affinity for ecdysone and 20-OH-ecdysone [21].
Rescue Studies
For the rescue study, N5 N.vivescens larvae were injected with 350 3-day-old
teratocytes. When these teratocyte-treated larvae reached BD, they were injected
with 1 p1 of various concentrations of 20-OH-ecdysone (Simes, Milano, Italy)
in 95% ethanol. Larvae injected with 1 ~1.1of 95% ethanol served as solvent
controls. Teratocyte-injected larvae that did not receive the 20-OH-ecdysone
injection and larvae that received neither teratocytes nor 20-OH-ecdysone also
served as controls. In each experiment six or eight larvae were used for each
treatment, and the entire experiment was repeated three times for a total of
22 larvae per treatment. All larvae were weighed daily after the initial injection of teratocytes and records were kept on developmental progress. The final
status attained by treated larvae was either successful pupation, incomplete
larval-pupal ecdysis, or an extended larval instar followed by death. These
parameters have been more fully described by Zhang and Dahlman [14].
Teratocyte Effect on Host Fat Body
Effect of teratocytes on nonparasitized host fat body mass was evaluated by
treating N5 larvae with either 10 p1 of Pringle’s saline or 10 pl of saline containing 350 3-day-old teratocytes. Larvae were dissected 3 days later when the
controls reached the D2 stage. The larvae were chilled on crushed ice, an incision was made along the dorsal midline, and the digestive tract was removed.
In order to reduce variation among samples, only the fat body found between
the first and fifth abdominal segments was collected. Extraneous hemolymph
Teratocyte Effects on JHEand Ecdysteroids
235
was removed from the fat body by capillary action (using absorbent paper),
and the net weight of the fat body from each larva was determined to the
nearest 0.1 mg. A total of 27 larvae were sampled for each treatment.
RESULTS
Teratocyte-injected larvae exhibited abnormally low levels of hemolymph
JHE with the degree of change induced by the teratocytes being dose-dependent
(Table 1, Fig. 1).For each teratocyte treatment (180,350, 750) there was a separate set of controls, the mean values of which, with a few exceptions, were
not significantly different from each other. It should be noted that the trauma
associated with injection resulted in an additional day in the active feeding
stage (D3) and, thus, a delay of 1day to maximum JHE activity, as compared
to noninjected controls. It also resulted in lower JHE values at BD as compared to noninjected controls. There were two JHE peaks for both the noninjected and saline-injected controls. The first peak was large and broad and
associated with the last day of the D phase and with the BD phase. The second peak was small and associated with the PP phase. The timing and titer of
both peaks was as expected, based on information about JHE titers in other
Lepidoptera [lo]. A dose of 750 teratocytes prevented the appearance of
significant amounts of JHE at all times during the 5th stadium. Even a dose of
350 teratocytes (approximately one-half the number derived from a single
parasitoid embryo) kept the JHE titer from reaching more than 40% of the
control level. However, a dose of 180 teratocytes had no apparent effect on
JHE titer.
JHE activity was inversely correlated with the age of teratocytes injected
into the larvae (Table 2). The reduction in JHE activity caused by 2-day-old
teratocytes was significantly greater than that caused by 3-day-old teratocytes.
JHE activity from larvae treated with either 5- or 6-day-old teratocytes was significantly greater than the JHE levels in larvae treated with either 2-or 3-day-old
teratocytes. However, even in larvae treated with 6-day-old teratocytes, JHE
levels were significantly reduced, only being 20% of those found in salinetreated controls.
Results from experiments designed to test the effects of other biological preparations on JHE levels support the hypothesis that M. croceipes teratocytes are
responsible for the low JHE levels observed. No reduction in JHE titer was
observed after any of the treatments other than M . croceipes teratocytes (Table
3). The developmental rate of all larvae treated with these other biological preparations was similar and all pupated. In a separate test, larvae that had received
washed M. croceipes eggs were dissected but teratocytes were not observed
in the host hemolymph.
To determine the effect of teratocyte injection on ecdysteroid titers, larval
hemolymph was sampled daily from BD to .
'
I
P Ecdysteroid titers in noninjected
controls and Pringle's saline-injected controls both demonstrated the expected
large premolt peak at CF2 + 12 h (Table 4).In contrast, the ecdysteroid titers
in teratocyte-treated larvae were significantly higher (approximately 150-200
pg/pl greater) than in either control during the earlier stages (BD through CFI),
but then remained unchanged during the time when ecdysteroid titers surged
in the controls.
81.5 f 9.3
29.4 f 7.7
21.0 f 4.5
77.8 i 8.0
22.8 i 7.9
1.5 +- 1.3
35.2 2 10.5
19.9 2 8.9
3.8 +- 1.0
28.6 % 8.9
15.9 2 3.8
1.4 +- 0.9
21.0 f 3.9
18.8 2 3.5
1.9 2 1.0
18.5 f 8.5
13.8 +- 3.5
0.9 f 0.7
Control
Saline
Teratocytes
Control
Saline
Teratocytes
D3
Phases
BD
CFl
*
-a
0.3 2 0.2
0.4 +- 0.3
1.0 i7 0.9
0.1 % 0.1
1.5 % 0.9
0.6 +- 0.3
1.9 & 0.8
0.1 ? 0.1
1.9 ? 1.0
CF2
*
f 0.7
f 3.0
f 4.2
4.5 1.9
1.9 i 1.4
1.5 2 1.4
6.8
9.3
1.9
7.8 +- 0.9
4.4 2 2.9
8.0f 2.9
PP
S.E.in Heliothis virescens Larvae Injected at N-5With Various Numbers
180 Teratocytes per larva
41.22 4.4
18.5 f 9.6
47.0 f 7.7
20.1 f 5.1
18.0 f 5.9
40.7 f 8.2
19.0 i: 6.4
16.4 f 6.6
350 Teratocytes per larva
65.5 f 6.7
21.2 i 8.8
44.4 f 8.7
18.8 ? 8.6
76.6 +- 9.8
30.1 f 4.0
19.2 f 2.8
2.1 i 1.4
750 Teratocytes per larva
76.4 f 8.5
19.6 f 4.2
10.8 f 4.9
56.8 10.8
49.4 f 9.4
2.5 f 1.4
5.0 f 3.3
1.9 +- 1.1
2
*n = 24 for all samples.
"Stress from injection causes an extra day in the digging state that is not present in the noninjected control larvae.
74.0 f 8.3
44.6 f 9.8
40.1 f 3.9
29.6 i- 3.8
21.5 2 8.7
18.1 2 4.6
D2
23.3 f 3.5
20.8 f 4.5
15.1i 4.4
D1
Control
Saline
Teratocytes
S
TABLE 1. JHE Activity (nmol JH hydrolyzedmidml hernolymph)
of 3-DayOld Microplitis croceipes Teratocytes*
Teratocyte Effects on jHE and Ecdysteroids
-
237
0control
<.-
80
2.-
60
EE3 Pringle's saline
=
K
E
teratocytes
T
N
e
I
-0
2
40
S
D1
D2
03
ED
CF1
CF2
PP
STAGE
Fig. 1. JHEactivity (nmol JHhydrolyzed/min/ml hernolymph) ? S.E. in Hehothis virescens larvae injected at N-5 with 750 3-day-old Microplitis croceipes teratocytes. (n = 24 for each sample time and treatment.)
One-half of the N5 H . virescens larvae treated with 350 teratocytes died as
larvae (DLM) approximately 5 days after the BD stage, well beyond the 4 days
typically needed for pupation (Table 5 ) . The remaining treated larvae expired
as the result of unsuccessful larval-pupalecdyses (ILPE). Treatments with ethanol or 20-OH-ecdysone (0.1 @larva) did not significantly alter this response.
However, a 0.25 pg/larva dose of 20-OH-ecdysone at the BD stage resulted in
13.6% successful pupation. At doses of 1 and 2.5 pg/larvae, pupation rates
TABLE 2. JHE Activity (nmol JH hydrolyzedmidml hemolymph) 2 S.E. in D2 Heliathis
virescens Larvae Injected at N5 With 350 Teratocytes of Various Ages Obtained From
Heliothis virescens Hosts Parasitizedby Microplitis croceipes
Treatment
(Teratocyte age)
2 Days
3 Days
4 Days
5 Days
6 Days
Saline
Noninjected
Parasitizedb
n
JHE Activity"
6
6
6
3.0 1.7
6.7 f 0.8
9.0 f 9.6
12.8 f 6.5
14.5 2 7.7
76.5 f 2.7
83.0 f 3.3
2.9 f 0.3
6
6
4
6
-
*
a
b
bc
C
C
d
d
-
'Means followed by the same letter are not significantly different (a = 0.05; new Duncan's multigle range test).
From Dahlman et al. [15].
238
Zhang et al.
TABLE 3. Effect of Various Biological Preparations on JHE Activity in Heliothis
virescens Lame*
*
Treatment
n
JHE Activity S.E.
(nmoles JH hydrolyzed/min/ml)
N.virescens plasma (10 p1)
M. luteus cell wall (20 pg)
M. croceipes eggs (3 or 4)
C. congreguta teratocytes (500-800)
M . croceipes teratocytes (350)
7
6
6
8
8
6
8
103.0 -+ 42.0
172.7 t 61.3
1078 2 39.1
114.9 19.8
21.0 2 4.5
128.5 26.4
149.6 t 10.1
Saline control (10 pl)
Noninjected control
*
*Larvaewere injected at N5 and JHE was measured at D2.
were increased to 50% or more and delayed larval mortality was either significantly reduced or absent. There was a 2-day delay in pupation of 20-OH-ecdysone-treated larvae as compared to that observed for noninjected controls. The
time required to express ILPE ranged from 5.2 to 7.9 days beyond BD.
A dose of 350 3-day-old teratocytes significantly reduced the mass of fat body
produced in a host. At the D2 stage there was only about 60% the amount of
fat body tissue in the insects that received the teratocytes (35.6 1.6 mg fresh
weight) as compared to 56.5 k 1.7 mg for the saline controls and 56.8 & 1.4mg
for noninjected controls. Injection of saline had no effect on fat body development.
*
DISCUSSION
Teratocytes reduced hemolymph JHEina dose-dependent manner. JHE activity remained at very low levels in D2-BD larvae injected with the embryoequivalent of 750 3-day-old teratocytes, in contrast to high levels observed in
normal larvae. Larvae treated with 350 teratocytes had somewhat higher JHE
activity, although levels remained significantlybelow normal. The specific mode
of action of teratocyte inhibition of JHE remains unknown. It is possible that
the teratocytes release an inhibitor which blocks JHE synthesis and/or release.
Since fat body development was retarded by teratocyte injections and fat body
synthesizes JHE [23], inhibition of JHE synthesis, either directly via some prod-
*
TABLE 4. EcdysteroidTiter (pg 20-OH-Ecdysoneequivalentdpl hemolymph S.E.)in Heliothis
virescens Larvae Injected With 350 3-Day-Old Microplitis croceipes Teratocytes at N5 Larvae
Control
Saline
Teratocytes
BD
CFl
47 c 12
(5)a
77 f 18
(6)
363 f 35
(6)
130 f 26
(6)
45 k 18
(6)
300 k 28
(6)
aNumbers in parentheses = n.
Phases
CF2
or
CF1 $. 24h
469
k 114
(3)
405 -t 96
(5)
173 35
*
(6)
CF2 + 12 h
or
CFl + 36h
CF2 + 24 h
or
CF1 + 48 h
1,426 5 463
(6)
2,534 577
(2)
314 f 61
(2)
4,293 905
(3)
4,235 2 682
(3)
254 f 81
(3)
*
*
Teratocyte Effects on JHEand Ecdysteroids
239
TABLE 5. Effects of Rescue With 20-OH-Ecdysone on Heliothis virescens Larvae Treated With
350 3-Day-Old Microplitis croceipes Teratocytes at N5*
Treatment
(CLg per
larva)
2.5
DLM
(”/.I
0
1.0
5.5
0.25
40.9
0.10
63.6
Days to
larval
mortality”
( 2 S.D.)
-
ILPE
(“/.I
45.5
6.0
45.5
6.3
( + 1.5)
6.9
45.5
(-1
36.4
( 2 1.4)
Solvent
59.1
Nonrescued
50.0
5.9
( + 1.2)
40.9
5.2
50.0
(f0.4)
Noninjected
control
0
-
0
Days to
ILPE
( 2 S.D.)
5.2
( * 0.9)
6.0
( * 1.2)
7.9
( 2 1.7)
7.6
( k 0.5)
6.7
( 2 1.7)
6.0
(i0.9)
-
P
(%)
54.5
Days to
P
( k S.D.)
0
5.6
0.9)
6.1
( * 1.1)
6.0
(f1.0)
-
0
-
0
-
100
3.8
( + 0.4)
(k
50.0
13.6
*20-OH-Ecdysone treatment applied at BD, 22 larvae per treatment.
aDaysmeasured from time of 20-OH-ecdysone treatment at BD.
uct released by the teratocytes or indirectly via general inhibition of fat body
growth, may be responsible for the observed results. Alternatively, Hayakawa
[24,25] reported purifying a 4,500-dalton peptide JHE repressor factor from
the hemolymph of Pseuduletia sepuruta parasitized by Apunteles karzyui. Perhaps
this peptide was released by the teratocytes from A . kariyui and teratocytes
from M. croceipes release a similar factor.
It is important to recognize that ail ages of teratocyte tested were effective,
even though the same number of 6-day-old teratocytes were less efficient than
a similar number of 2-day-old teratocytes. Although the teratocytes do not
undergo cellular division, the ploidy number increases [26] and they increase
in size [ll];thus, the older teratocytes are larger and perhaps have a greater
capacity to secrete substances that inhibit JHE production. Because we observed
a slight reduction of potency in older teratocytes, their effectiveness per unit
weight may truly be reduced. Under our experimental conditions, the teratocytes in parasitized larvae would be approximately 6 days old at the time of
JHE release (D2-BD) in nonparasitized larvae. However, they were present in
the host as free floating cells all during the 5th stadium and could have been
affecting fat body development during this entire time. The minimum time
between teratocyte injection and JHE inhibition has not been determined but
will be the subject of future studies.
We did not conduct an exhaustive test for other biological preparations that
may cause reduced levels of JHE, but we did show that several foreign materials, including M. croceipes eggs and teratocytes from another braconid species, did not prevent synthesis and release of JHE in H. virescens. Only M.
croceipes teratocytes caused a marked reduction in hemolymph JHE levels.
Hayakawa [24] reported that the peptide he isolated had limited in vivo activity in P. sepurutu. However, he showed that multiple injections (five injections
240
Zhang et al.
maximum) performed at daily intervals kept the JHE titer depressed for as
long as the injection regimen was maintained. Presumably, when the inhibitory effect of the peptide diminished, JHE was released, the JH titer dropped,
and the larval-pupal molt was initiated. We have not determined JH titers in
parasitized or teratocyte-injected larvae but they might be expected to remain
at a level similar to those found in the larval stage prior to the release of JHE.
The actual mechanisms by which JHE activity is suppressed and larval developmental events are mediated are not known.
Prior to pupal commitment, high JH titers inhibit ecdysone production [27,28]
by preventing the release of PTTH from the brain, as well as by influencing
the competency of the prothoracic glands to respond to MTH and a hemolymph
ecdysiotropic factor [29,30]. In teratocyte-injected larvae, high JH titers may
continue to prevent the release of PTTH and/or may keep the prothoracic gland
in an incompetent state. While JH titers prior to JHE release are primarily controlled by synthesis [29], the timely release of JHE is responsible for the rapid
degradation of existing JH in the hemolymph [23], thus setting the stage for
subsequent developmental events. It has been reported that treatment of young
larvae with JH decreases the JHE activity or inhibits its production at the appropriate time [30,31]. Without a drop in JH titer and the resulting return to compe tency of the prothoracic gland, ecdysteroid production should be minimal.
However, there are other reports that JH production initiates JHE synthesis
and/or release [8]. Thus, teratocytes may cause JH titers to be low and the
JH-induced JHE synthesis is never initiated.
During the earlier part of the 5th stadium, ecdysteroid titers in N.virescens
parasitized by M. croceipes were higher (150-200 pg/Fl) than in controls [15].
In this report we observed the same pattern in larvae treated with teratocytes. Presumably this titer is adequate to program larval tissues for pupal
commitment, but larvae do not advance in development beyond CF1 because
the second large peak of ecdysteroid is absent. Parasitized larvae showed
tissue response to injections of 20-OH-ecdysone by forming pupal cuticle and attempting to ecdyse [32], and we have shown here that 20-OHecdysone rescued a significant number of larvae previously injected with
teratocytes.
We suggest that the action of teratocytes, while not specifically known,
impacts the complex interplay of hormone titers and feedback mechanisms to
prevent the synthesis, release, and/or catabolism of adequate amounts of
ecdysteroid. In the case of M . croceipes, the action is independent of any effect
from parasitoid venom or calyx fluid. In contrast, Wani et al. [33] showed that
teratocytes from A . kariyui prevent larval-pupal ecdysis only in the presence
of both venom and calyx fluid. As an alternative, the teratocytes could be synthesizing and releasing JH [34] which could keep ecdysteroid titers depressed
via feedback mechanisms [29]. However, if JH levels were high, the 20-OHecdysone rescue experiments should have caused larval-larval molts rather
than larval-pupal molts unless JH levels dropped for a short time (to allow
tissue commitment) and then increased again. In either case, the parasitized and teratocyte-treated larvae remain in the CF1 stage because of the
inadequate titers of ecdysteroid necessary to drive the cells to the larval-pupal
transformation.
Teratocyte Effects on JHEand Ecdysteroids
241
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