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Effects of fat body on α-ecdysone induced morphogenesis in cultured wing disks of the wax moth, Galleria mellonella
r. Insect Physiol., 1971, Vol. 17, pp. 269 to 276. Pergamon Press. Printed in Great Britain
EFFECTS OF FAT BODY ON a-ECDYSONE INDUCED MORPHOGENESIS IN CULTURED WING DISKS OF THE WAX MOTH, GALLERIA MELLONELLA KENNETH Department
RICHMAN
of Biology,
Brandeis
and HERBERT University,
(Received
13 July
OBERLANDER
Waltham,
Massachusetts
02154
1970)
Abstract-Fat
body promoted ecdysone induced morphogenesis in Galleria Medium preincubated with fat body and cr-ecdysone from any of the first 5 days of the !inal larval instar enhanced tracheal migration and elongation in the disks. Disks from the third, fourth, and fifth days of the
wing disks cultured in vitro.
final larval instar responded equally well to the fat body and cz-ecdysone. We
suggest a physiological r61e for Galleria fat body as an intermediary in the stimulation of wing disk development by ol-ecdysone. INTRODUCTION
CX-ECDYSONE stimulates morphogenesis in wing disks of Galleria mellonella when they are cultured in a defined chemical medium (OBERLANDERand FULCO, 1967; OBERLANDER,1969a). It appears that the disks themselves do not inactivate ecdysone in vitro (OBERLANDER,1969b). S ince it was reported that ecdysone is rapidly inactivated in oivo in Surcophugu peregrinu (OHTAKI et al., 1968), and that the fat body of Calliphora erythrocephala can inactivate ar-ecdysone (KARLSONand BODE,1969), we wished to determine whether fat body inactivated a-ecdysone under in oitro culture conditions. Surprisingly, we found that the fat body of Galleria mellonella larvae had a marked promoting effect on c+ecdysone induced morphogenesis in cultured wing disks. MATERIALS
AND METHODS
Experimental animals The wax moth, Galleria mellonella, was reared at 30°C and 70% r.h. according to the procedures outlined by BECK(1960). Larvae were collected on the day after the final larval ecdysis, and were reared in plastic Petri dishes (100 x 25 mm), ten to a dish, until use. In these cultures the larvae spun cocoons 6 to 7 days after the final larval ecdysis. Larvae were selected for experiments if their weights fell within the central 50 per cent of the weight distribution. All experiments were conducted with disks and fat body from larvae of the final larval instar. In vitro procedures Mesothoracic wing disks were cultured in 1 ml of Graces medium without haemolyrnph (Grand Island Biological Co.) at 25°C as previously reported 10
269
270
KENNETHRICHMANANDHERBERTOBERLANDER
(OBERLANDER, 1969a). The paired mesothoracic wing disks were always divided between experimental and control cultures. This procedure is necessary because disks from larvae of the same age and weight may differ in their response to ecdysone, whereas members of a disk pair behave alike. Each culture dish contained ten disks. The wing disks were cultured in each case for 2 days before the addition of hormone. Fat body was taken from the mesothoracic region of five larvae for each culture dish, and was washed in two changes of sterile medium before use. Typically, the fat body added to each culture weighed 25 mg, though it was not weighed for each experiment. Tracheal migration and elongation (flattening out and extension) of the wing disks were used as criteria of ecdysone induced development in vitro (OBERLANDER, 1969a). These features were also described by SEHNAL (1968) in his study of development of Galleria wing disks in vivo. Unless otherwise stated observations were made daily for two weeks and on alternate days for another 2 weeks. The disks appeared healthy for at least 2 to 3 weeks. Ecdysone ol-Ecdysone (Zoecon corporation) was dissolved in 10% ethanol at a concentration of 100 pg/ml. Unless otherwise indicated 3 pg of Ly-ecdysone was used per ml of culture medium. RESULTS Eflects of various concentrations of wecdysone in the presence of fat body Wing disks from animals on the fifth day of the final larval instar were cultured in Graces medium with and without fat body. After 2 days ol-ecdysone was added to each culture. The results of this experiment are given in Table 1. Increasing the concentration of cY-ecdysone in the absence of fat body increased the percentage of disks with tracheal migration. The percentage of disks exhibiting tracheal migration was uniformly higher in the fat body cultures. Furthermore, the time to achieve the maximum percentage of disks with tracheal migration was less in the cultures which contained fat body in two cases, and equal to the control in the third instance. Increasing the concentration of cll-ecdysone in the absence of fat body also caused an increased percentage of elongated disks. When fat body was present all of the disks elongated, even in the presence of a subthreshold (1 pg) concentration. In each case elongation took less time in the presence of fat body. Effects of fat body and ol-ecdysone on dirks of dzgerent ages Wing disks from the third and fourth days of the final larval instar were cultured with and without fat body. ol-Ecdysone was added 2 days after the beginning of the culture period. Both tracheal migration and elongation occurred in a greater percentage of disks cultured with fat body than without it. In addition the time to
FAT
achieve the maximum cultures (see Table 1).
BODY
ECDYSONE
elongation
IN
GALLERIA
WING
DISKS
response was consistently
271
less in the fat body
Eflects of fat body without oz-ecdysone To determine whether the fat body had an effect on the disks in the absence of ol-ecdysone, wing disks from the fifth day of the final larval instar were cultured with and without fat body. Hormone was omitted and the disks were observed for 4 weeks. At the end of this period there was no development in either culture. These observations do not rule out the possibility that the fat body affected the disks directly, but in a way that became manifest only in the presence of a-ecdysone. To test for such an effect disks from 5 day larvae (final instar) were cultured with and without fat body for 2 days. The two sets of disks were then transferred to fresh medium without fat body. ol-Ecdysone was added to both cultures. There was no significant difference between control and experimental disks (Table 2). Effects of medium preconditioned with fat body To discover whether medium preconditioned with fat body enhanced the response of the disks to c+ecdysone the following experiments were conducted. Medium was incubated at 25°C with and without fat body. After 2 days wing disks and ecdysone were added to the two dishes. It is clear from the data in Table 2 that incubation with fat body alone had only a small effect on the response of the disks to ecdysone. In another experiment fat body was incubated with Lu-ecdysone for 2 days and then removed. Disks were then added to that medium and to medium containing cl-ecdysone added 2 days before. The data reported in Table 2 demonstrates an enhanced response of the disks in the fat body-ecdysone conditioned medium. The percentage of disks with tracheal migration and elongation was greater, and the rate of development was faster in the fat body treated culture. Since a-ecdysone was also present for 2 days before the addition of disks in the above experiment it was necessary to compare the development of disks cultured in such medium with the development of disks in medium containing fresh ecdysone. There was no significant difference between these two groups of disks at the end of 2 weeks. Fat body eflects at 5°C If the effects of fat body on the response of cultured disks to ol-ecdysone result from an active interaction it should be sensitive to temperature change. To test this hypothesis fat body was cultured with ecdysone at 5 and 25°C. Two days later the fat body was removed, and wing disks from S-day larvae were added to each dish. Both cultures were placed at 25°C. The data from two such experiments are reported in Table 2. Both elongation and tracheal migration occurred later in disks cultured in medium preincubated with fat body and ecdysone at 5°C. Secondly, tracheal migration occurred in a smaller percentage of disks in the culture preincubated at 5°C.
_ _ ,,>-
^
50 9 100 14
100 5 100 6 20 8
4
5
100 6
20
Absent
100
Present
3 pglml
4 days
CULTURED
100 9
5
80
-
_”
,
_
__
..__
,” _ ,_ _ _ _
0
11
30
Absent
1 pglml
5 days
ON
Present
BODY
* Refers to the number of days after the donors ecdysed to the final larval instar.
ma1 elongation response
Percentage of disks exhibiting tracheal migration No. of days to achieve maxima1 tracheal migration response Percentage of disks elongated No. of days to achieve maxi-
Absent
3 fl.g/ml
3 days
S-day * fat body: Present
Ecdysone :
Age of wing disk * :
TABLE ~-EFFECTS OF OI-ECDYSONE ANDFAT
_
100 10
6
90
1
30 19
6
30
Absent
2 tLg/ml
5 days
DISKS
Present
WING
100 7
5
80
Present
90 15
9
6o
Absent
3 tLglm1
5 days
P
P %
i
1_. !I
:
5
F! %
;
B
F z
Wing disks
6
90 9
6
89 9
100 (50) 29:
(L
(Z)
(g)
Wing disks and ecdysone added
WINGDISKS
100 (77)
(G
(3%
Wing disks and ecdysone added to dish after removal of fat body
ON
6
;*%I,
(:)
$)
Wing disks added to dish containing ecdysone after removal of fat body
Fat body and ecdysone
CULTURED
:o”,
(‘9)
(E)
Wing disks added to dish containing ecdysone
Ecdysone
Fat body and ecdysone (25°C)
MEDIUM
Fat body and ecdysone (5°C)
(lz)
(Q
G33q+
(“6:)
(1)
(;+
Wing disks added to dish containing ecdysone after removal of fat body (25°C)
IN PRECONDITIONED
* Donors of both wing disks and fat body were in the fifth day of the final larval instar. t Each of these cultures contained nine wing disks. : Numbers in parentheses are results of a repetition of the experiment.
20
lli
pg/ml)
Fat body
OF OI-ECDYSONE(~
Wing disks transferred to new dish with ecdysone
Percentage of wing disks exhibiting tracheal migration No. of days to achieve maximal tracheal migration response Percentage of wing disks elongated No. of days to achieve maximal elongation response
Treatment
Pre-incubation * (2 days)
2-EFFECTS
Fat body and wing disks
TABLE
90
7
18
8
5
100
3
6
60
100
11
55
90
8
90
15 6
14
5
50
90
-
0
90
4
Ecdysone
4-Day fat body and ecdysone
100
5
10
Ecdysone
3-Day fat body and ecdysone
14
90
-
0
Ecdysone
(:)
100 (100)
(5”,
(2:+
S-Day fat body and ecdysone
(“)
(G
Ecdysone
,,
,,,
,, ,,
,,
,,
&,,
,,,
8,
* Wing disks were added to the dishes containing ecdysone after the fat body was removed. t Each of these cultures contained nine wing disks. Numbers in parentheses are results of a repetition of the experiment.
maximal tracheal migration response Percentage of wing disks elongated No. of days to achieve maximal elongation response
Percentage of wing disks exhibiting tracheal migration No. of davs to
Ecdysone
2-Day fat body and ecdysone
OF OI-ECDYSONE (3 pg/ml) ON WING DISKS CULTUREDIN PRECONDITIONED MEDIUM: EFFECTS OF VARYINGTHE AGEOF THE FAT BODY
1 -Day fat Pre-incubation * body and ecdysone (2 days)
TABLE 3-EFFECTS
FAT BODY ECDYSONE IN
GALLERIA
WING
275
DISKS
Effect offat body of different ages Does fat body from the early part of the last larval instar enhance the response of cultured disks to ol-ecdysone ? Fat body from days 1, 2, 3, and 4 of the final larval instar were cultured with a+ecdysone. The fat body was removed after 2 days. Wing disks from S-day final instar larvae were then added to the cultures. The data is reported in Table 3. It is clear that fat body from any of the first 5 days of the final larval instar increased the percentage of disks with tracheal migration, and shortened the response time for both tracheal migration and elongation. DISCUSSION
The results reported in this paper demonstrate that larval fat body enhances the response of cultured wing disks to ar-ecdysone. It is of particular interest that the differences in response between disks of different ages to cu-ecdysone is eliminated in the presence of fat body. Thus, younger disks respond to cu-ecdysone as well as mature disks in the presence of fat body. Furthermore, the differences in response of wing disks to different doses of ol-ecdysone was eliminated by the presence of fat body. Clearly, fat body in some way maximized the effect of ol-ecdysone on the wing disks. The role of the fat body in stimulating wing disk development is not yet known. It may be that the fat body produces an ecdysone co-factor or carrier, that it alters the ecdysone molecule itself, or that it releases a growth factor. We conclude from the preincubation experiments that interaction between a-ecdysone and fat body is necessary for marked enhancement of disk development. The failure of medium preincubated with fat body and ecdysone at 5°C to stimulate development compared to that of the controls is consistent with the hypothesis that the interaction requires metabolic activity of the fat body. At the present time we cannot reconcile our findings with the report that the fat body of Calliphora inactivates ecdysone (KARLSON and BODE, 1969). The inactivating ability of the Calliphora fat body varied with the age of the larvae. It could be expected that an inhibitory effect of GaZZeriafat body would be detected during the early days of the larval instar when the ecdysone titre is presumed to be low. But in our experiments fat body removed from larvae at any day of the last instar, up to the day before cocoon spinning promoted development. An attempt at culturing GaZZeria wing disks in the presence of cl-ecdysone and Sarcophaga bullata fat body failed because the disks did not survive in medium containing the foreign fat body. In vitro work on hormonal interactions with various tissues in a variety of species will be necessary before generalizations can be made. This would seem a propitious time for this work since there are a number of reports of tissues responding to ecdysone in vitro (e.g. MARKS and LEOPOLD, 1970; SENGEL and MANDARON, 1969). Furthermore, WILLIAMS and KAMBYSELLIS (1969) have made the important discovery
that
a co-factor
from
the
haemolymph
is necessary
for
the
testis
of
276
KENNETHRICHMANANDHERBERT OBERLANDER
cynthia pupae to develop when cultured in vitro with ecdysone. The present experiments suggest a physiological r6le for the fat body in GaZZeria, in which an interaction between the fat body and ol-ecdysone is necessary for the development of the wing disks. Acknowledgements-We thank Dr. JOHN SIDDALL (Zoecon Corporation) ecdysone. This research was funded by the National Science Foundation, GB-7992.
for the aGrant No.
REFERENCES BECK S. D. (1960) Growth and development of the greater wax moth, Gal&a mellonella (L.) (Lepidoptera: GalIeriidae). Wis. Acad. Sci. Arts Lett. 49, 137-149. KARLSONP. and BODE C. (1969) Die Inaktivierung des Ecdysons bei der Schmeissfliege Calliphora erythrocephala Meigen. J. Insect Physiol. 15, 11 l-l 18. MARKSE. P. and LEOPOLDR. A. (1970) Cockroach leg regeneration: Effects of ecdysterone in vitro. Science, Wash. 167,61-62. OBERLANDER H. (1969a) Effects of ecdysone, ecdysterone, and inokosterone on the in vitro initiation of metamorphosis of wing disks of Galleria mellonella. J. Insect Physiol. 15, 297-304. OBERLANDER H. (1969b) Ecdysone and DNA synthesis in cultured wing disks of the wax moth, Galleria mellonella. J. Insect Physiol. 15, 1803-1806. OBERLANDER H. and F~LCO L. (1967) Growth and partial metamorphosis of imaginal disks of the greater wax moth, Galleria mellonella, in vitro. Nature, Land. 216, 1140-1141. OHTAKIT., MILKMANR. D., and WILLIAMSC. M. (1968) Dynamics of ecdysone secretion and action in the fleshfly Sarcophaga peregrina. Biol. Bull., Woods Hole 135, 322-334. SEHNALF. (1968) Influence of the corpus allatum on the development of internal organs in Gall&a mellonella L. J. Insect Physiol. 14,73-85. SENGSL P. and MANDARONP. (1969) Aspects morphologiques du developpement in vitro des disques imaginaux de la Drosophile. C.R. Acad. Sci., Paris 268,405-407. WILLIAMS C. M. and KAMBYSELLISM. P. (1969) In vitro action of ecdysone. Proc. nat. Acad. Sci., U.S.A. 63,231.
EFFECTS OF FAT BODY ON a-ECDYSONE INDUCED MORPHOGENESIS IN CULTURED WING DISKS OF THE WAX MOTH, GALLERIA MELLONELLA KENNETH Department
RICHMAN
of Biology,
Brandeis
and HERBERT University,
(Received
13 July
OBERLANDER
Waltham,
Massachusetts
02154
1970)
Abstract-Fat
body promoted ecdysone induced morphogenesis in Galleria Medium preincubated with fat body and cr-ecdysone from any of the first 5 days of the !inal larval instar enhanced tracheal migration and elongation in the disks. Disks from the third, fourth, and fifth days of the
wing disks cultured in vitro.
final larval instar responded equally well to the fat body and cz-ecdysone. We
suggest a physiological r61e for Galleria fat body as an intermediary in the stimulation of wing disk development by ol-ecdysone. INTRODUCTION
CX-ECDYSONE stimulates morphogenesis in wing disks of Galleria mellonella when they are cultured in a defined chemical medium (OBERLANDERand FULCO, 1967; OBERLANDER,1969a). It appears that the disks themselves do not inactivate ecdysone in vitro (OBERLANDER,1969b). S ince it was reported that ecdysone is rapidly inactivated in oivo in Surcophugu peregrinu (OHTAKI et al., 1968), and that the fat body of Calliphora erythrocephala can inactivate ar-ecdysone (KARLSONand BODE,1969), we wished to determine whether fat body inactivated a-ecdysone under in oitro culture conditions. Surprisingly, we found that the fat body of Galleria mellonella larvae had a marked promoting effect on c+ecdysone induced morphogenesis in cultured wing disks. MATERIALS
AND METHODS
Experimental animals The wax moth, Galleria mellonella, was reared at 30°C and 70% r.h. according to the procedures outlined by BECK(1960). Larvae were collected on the day after the final larval ecdysis, and were reared in plastic Petri dishes (100 x 25 mm), ten to a dish, until use. In these cultures the larvae spun cocoons 6 to 7 days after the final larval ecdysis. Larvae were selected for experiments if their weights fell within the central 50 per cent of the weight distribution. All experiments were conducted with disks and fat body from larvae of the final larval instar. In vitro procedures Mesothoracic wing disks were cultured in 1 ml of Graces medium without haemolyrnph (Grand Island Biological Co.) at 25°C as previously reported 10
269
270
KENNETHRICHMANANDHERBERTOBERLANDER
(OBERLANDER, 1969a). The paired mesothoracic wing disks were always divided between experimental and control cultures. This procedure is necessary because disks from larvae of the same age and weight may differ in their response to ecdysone, whereas members of a disk pair behave alike. Each culture dish contained ten disks. The wing disks were cultured in each case for 2 days before the addition of hormone. Fat body was taken from the mesothoracic region of five larvae for each culture dish, and was washed in two changes of sterile medium before use. Typically, the fat body added to each culture weighed 25 mg, though it was not weighed for each experiment. Tracheal migration and elongation (flattening out and extension) of the wing disks were used as criteria of ecdysone induced development in vitro (OBERLANDER, 1969a). These features were also described by SEHNAL (1968) in his study of development of Galleria wing disks in vivo. Unless otherwise stated observations were made daily for two weeks and on alternate days for another 2 weeks. The disks appeared healthy for at least 2 to 3 weeks. Ecdysone ol-Ecdysone (Zoecon corporation) was dissolved in 10% ethanol at a concentration of 100 pg/ml. Unless otherwise indicated 3 pg of Ly-ecdysone was used per ml of culture medium. RESULTS Eflects of various concentrations of wecdysone in the presence of fat body Wing disks from animals on the fifth day of the final larval instar were cultured in Graces medium with and without fat body. After 2 days ol-ecdysone was added to each culture. The results of this experiment are given in Table 1. Increasing the concentration of cY-ecdysone in the absence of fat body increased the percentage of disks with tracheal migration. The percentage of disks exhibiting tracheal migration was uniformly higher in the fat body cultures. Furthermore, the time to achieve the maximum percentage of disks with tracheal migration was less in the cultures which contained fat body in two cases, and equal to the control in the third instance. Increasing the concentration of cll-ecdysone in the absence of fat body also caused an increased percentage of elongated disks. When fat body was present all of the disks elongated, even in the presence of a subthreshold (1 pg) concentration. In each case elongation took less time in the presence of fat body. Effects of fat body and ol-ecdysone on dirks of dzgerent ages Wing disks from the third and fourth days of the final larval instar were cultured with and without fat body. ol-Ecdysone was added 2 days after the beginning of the culture period. Both tracheal migration and elongation occurred in a greater percentage of disks cultured with fat body than without it. In addition the time to
FAT
achieve the maximum cultures (see Table 1).
BODY
ECDYSONE
elongation
IN
GALLERIA
WING
DISKS
response was consistently
271
less in the fat body
Eflects of fat body without oz-ecdysone To determine whether the fat body had an effect on the disks in the absence of ol-ecdysone, wing disks from the fifth day of the final larval instar were cultured with and without fat body. Hormone was omitted and the disks were observed for 4 weeks. At the end of this period there was no development in either culture. These observations do not rule out the possibility that the fat body affected the disks directly, but in a way that became manifest only in the presence of a-ecdysone. To test for such an effect disks from 5 day larvae (final instar) were cultured with and without fat body for 2 days. The two sets of disks were then transferred to fresh medium without fat body. ol-Ecdysone was added to both cultures. There was no significant difference between control and experimental disks (Table 2). Effects of medium preconditioned with fat body To discover whether medium preconditioned with fat body enhanced the response of the disks to c+ecdysone the following experiments were conducted. Medium was incubated at 25°C with and without fat body. After 2 days wing disks and ecdysone were added to the two dishes. It is clear from the data in Table 2 that incubation with fat body alone had only a small effect on the response of the disks to ecdysone. In another experiment fat body was incubated with Lu-ecdysone for 2 days and then removed. Disks were then added to that medium and to medium containing cl-ecdysone added 2 days before. The data reported in Table 2 demonstrates an enhanced response of the disks in the fat body-ecdysone conditioned medium. The percentage of disks with tracheal migration and elongation was greater, and the rate of development was faster in the fat body treated culture. Since a-ecdysone was also present for 2 days before the addition of disks in the above experiment it was necessary to compare the development of disks cultured in such medium with the development of disks in medium containing fresh ecdysone. There was no significant difference between these two groups of disks at the end of 2 weeks. Fat body eflects at 5°C If the effects of fat body on the response of cultured disks to ol-ecdysone result from an active interaction it should be sensitive to temperature change. To test this hypothesis fat body was cultured with ecdysone at 5 and 25°C. Two days later the fat body was removed, and wing disks from S-day larvae were added to each dish. Both cultures were placed at 25°C. The data from two such experiments are reported in Table 2. Both elongation and tracheal migration occurred later in disks cultured in medium preincubated with fat body and ecdysone at 5°C. Secondly, tracheal migration occurred in a smaller percentage of disks in the culture preincubated at 5°C.
_ _ ,,>-
^
50 9 100 14
100 5 100 6 20 8
4
5
100 6
20
Absent
100
Present
3 pglml
4 days
CULTURED
100 9
5
80
-
_”
,
_
__
..__
,” _ ,_ _ _ _
0
11
30
Absent
1 pglml
5 days
ON
Present
BODY
* Refers to the number of days after the donors ecdysed to the final larval instar.
ma1 elongation response
Percentage of disks exhibiting tracheal migration No. of days to achieve maxima1 tracheal migration response Percentage of disks elongated No. of days to achieve maxi-
Absent
3 fl.g/ml
3 days
S-day * fat body: Present
Ecdysone :
Age of wing disk * :
TABLE ~-EFFECTS OF OI-ECDYSONE ANDFAT
_
100 10
6
90
1
30 19
6
30
Absent
2 tLg/ml
5 days
DISKS
Present
WING
100 7
5
80
Present
90 15
9
6o
Absent
3 tLglm1
5 days
P
P %
i
1_. !I
:
5
F! %
;
B
F z
Wing disks
6
90 9
6
89 9
100 (50) 29:
(L
(Z)
(g)
Wing disks and ecdysone added
WINGDISKS
100 (77)
(G
(3%
Wing disks and ecdysone added to dish after removal of fat body
ON
6
;*%I,
(:)
$)
Wing disks added to dish containing ecdysone after removal of fat body
Fat body and ecdysone
CULTURED
:o”,
(‘9)
(E)
Wing disks added to dish containing ecdysone
Ecdysone
Fat body and ecdysone (25°C)
MEDIUM
Fat body and ecdysone (5°C)
(lz)
(Q
G33q+
(“6:)
(1)
(;+
Wing disks added to dish containing ecdysone after removal of fat body (25°C)
IN PRECONDITIONED
* Donors of both wing disks and fat body were in the fifth day of the final larval instar. t Each of these cultures contained nine wing disks. : Numbers in parentheses are results of a repetition of the experiment.
20
lli
pg/ml)
Fat body
OF OI-ECDYSONE(~
Wing disks transferred to new dish with ecdysone
Percentage of wing disks exhibiting tracheal migration No. of days to achieve maximal tracheal migration response Percentage of wing disks elongated No. of days to achieve maximal elongation response
Treatment
Pre-incubation * (2 days)
2-EFFECTS
Fat body and wing disks
TABLE
90
7
18
8
5
100
3
6
60
100
11
55
90
8
90
15 6
14
5
50
90
-
0
90
4
Ecdysone
4-Day fat body and ecdysone
100
5
10
Ecdysone
3-Day fat body and ecdysone
14
90
-
0
Ecdysone
(:)
100 (100)
(5”,
(2:+
S-Day fat body and ecdysone
(“)
(G
Ecdysone
,,
,,,
,, ,,
,,
,,
&,,
,,,
8,
* Wing disks were added to the dishes containing ecdysone after the fat body was removed. t Each of these cultures contained nine wing disks. Numbers in parentheses are results of a repetition of the experiment.
maximal tracheal migration response Percentage of wing disks elongated No. of days to achieve maximal elongation response
Percentage of wing disks exhibiting tracheal migration No. of davs to
Ecdysone
2-Day fat body and ecdysone
OF OI-ECDYSONE (3 pg/ml) ON WING DISKS CULTUREDIN PRECONDITIONED MEDIUM: EFFECTS OF VARYINGTHE AGEOF THE FAT BODY
1 -Day fat Pre-incubation * body and ecdysone (2 days)
TABLE 3-EFFECTS
FAT BODY ECDYSONE IN
GALLERIA
WING
275
DISKS
Effect offat body of different ages Does fat body from the early part of the last larval instar enhance the response of cultured disks to ol-ecdysone ? Fat body from days 1, 2, 3, and 4 of the final larval instar were cultured with a+ecdysone. The fat body was removed after 2 days. Wing disks from S-day final instar larvae were then added to the cultures. The data is reported in Table 3. It is clear that fat body from any of the first 5 days of the final larval instar increased the percentage of disks with tracheal migration, and shortened the response time for both tracheal migration and elongation. DISCUSSION
The results reported in this paper demonstrate that larval fat body enhances the response of cultured wing disks to ar-ecdysone. It is of particular interest that the differences in response between disks of different ages to cu-ecdysone is eliminated in the presence of fat body. Thus, younger disks respond to cu-ecdysone as well as mature disks in the presence of fat body. Furthermore, the differences in response of wing disks to different doses of ol-ecdysone was eliminated by the presence of fat body. Clearly, fat body in some way maximized the effect of ol-ecdysone on the wing disks. The role of the fat body in stimulating wing disk development is not yet known. It may be that the fat body produces an ecdysone co-factor or carrier, that it alters the ecdysone molecule itself, or that it releases a growth factor. We conclude from the preincubation experiments that interaction between a-ecdysone and fat body is necessary for marked enhancement of disk development. The failure of medium preincubated with fat body and ecdysone at 5°C to stimulate development compared to that of the controls is consistent with the hypothesis that the interaction requires metabolic activity of the fat body. At the present time we cannot reconcile our findings with the report that the fat body of Calliphora inactivates ecdysone (KARLSON and BODE, 1969). The inactivating ability of the Calliphora fat body varied with the age of the larvae. It could be expected that an inhibitory effect of GaZZeriafat body would be detected during the early days of the larval instar when the ecdysone titre is presumed to be low. But in our experiments fat body removed from larvae at any day of the last instar, up to the day before cocoon spinning promoted development. An attempt at culturing GaZZeria wing disks in the presence of cl-ecdysone and Sarcophaga bullata fat body failed because the disks did not survive in medium containing the foreign fat body. In vitro work on hormonal interactions with various tissues in a variety of species will be necessary before generalizations can be made. This would seem a propitious time for this work since there are a number of reports of tissues responding to ecdysone in vitro (e.g. MARKS and LEOPOLD, 1970; SENGEL and MANDARON, 1969). Furthermore, WILLIAMS and KAMBYSELLIS (1969) have made the important discovery
that
a co-factor
from
the
haemolymph
is necessary
for
the
testis
of
276
KENNETHRICHMANANDHERBERT OBERLANDER
cynthia pupae to develop when cultured in vitro with ecdysone. The present experiments suggest a physiological r6le for the fat body in GaZZeria, in which an interaction between the fat body and ol-ecdysone is necessary for the development of the wing disks. Acknowledgements-We thank Dr. JOHN SIDDALL (Zoecon Corporation) ecdysone. This research was funded by the National Science Foundation, GB-7992.
for the aGrant No.
REFERENCES BECK S. D. (1960) Growth and development of the greater wax moth, Gal&a mellonella (L.) (Lepidoptera: GalIeriidae). Wis. Acad. Sci. Arts Lett. 49, 137-149. KARLSONP. and BODE C. (1969) Die Inaktivierung des Ecdysons bei der Schmeissfliege Calliphora erythrocephala Meigen. J. Insect Physiol. 15, 11 l-l 18. MARKSE. P. and LEOPOLDR. A. (1970) Cockroach leg regeneration: Effects of ecdysterone in vitro. Science, Wash. 167,61-62. OBERLANDER H. (1969a) Effects of ecdysone, ecdysterone, and inokosterone on the in vitro initiation of metamorphosis of wing disks of Galleria mellonella. J. Insect Physiol. 15, 297-304. OBERLANDER H. (1969b) Ecdysone and DNA synthesis in cultured wing disks of the wax moth, Galleria mellonella. J. Insect Physiol. 15, 1803-1806. OBERLANDER H. and F~LCO L. (1967) Growth and partial metamorphosis of imaginal disks of the greater wax moth, Galleria mellonella, in vitro. Nature, Land. 216, 1140-1141. OHTAKIT., MILKMANR. D., and WILLIAMSC. M. (1968) Dynamics of ecdysone secretion and action in the fleshfly Sarcophaga peregrina. Biol. Bull., Woods Hole 135, 322-334. SEHNALF. (1968) Influence of the corpus allatum on the development of internal organs in Gall&a mellonella L. J. Insect Physiol. 14,73-85. SENGSL P. and MANDARONP. (1969) Aspects morphologiques du developpement in vitro des disques imaginaux de la Drosophile. C.R. Acad. Sci., Paris 268,405-407. WILLIAMS C. M. and KAMBYSELLISM. P. (1969) In vitro action of ecdysone. Proc. nat. Acad. Sci., U.S.A. 63,231.