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In vitro activation of neurosecretory brain cells in Mamestra brassicae by β-ecdysone
GENERAL
AND
COMPARATIVE
In Vitro Activation
ENDOCRINOLOGY
of Neurosecretory Brain Cells in Mamestra brassicae by /3-Ecdysone NORIAKI
Laboratory
of Applied
Zoology,
33, 467-472 (1977)
Faculty
AGWI’
AND KIYOSHI I~~IRIJMA
of Agriculfiure, Tokyo Universiiy Tokyo, IS3, Japan
of Education,
Komaba,
Meguro-ku,
Accepted July 15, 1977 The effect of ecdysone or of the prothoracic gland on the neurosecretory cells of brains taken from diapausing pupae of the cabbage armyworm, Mamestra brassicae L., was investigated histologically and functionally in vitro. When the diapausing brain was cultured in a medium containing fl-ecdysone, the neurosecretory cells of the median group I in the pars intercerebralis increased staining intensity more than those of brains cultured in medium alone. Furthermore, when the prothoracotropic activity of a brain treated with /3-ecdysone was compared with that of an untreated brain in vitro, the brain treated with hormone showed higher activity than the untreated one. These results suggest that p-ecdysone acts on the neurosecretory cells of pupal brain in M. brussicae as a positive feedback regulator.
Secretion of prothoracotropic hormone (PTTH) from neurosecretory cells in the median part of brain has been elucidated by several investigators (Plagge, 1938: Wi~leswo~h, 1940: Williams, 1947) who employed the methods of experimental morphology. Furthermore, it has been reported that the prothoracic gland is stimuIated by its own product (Williams, 1952) and that its activity is enhanced and maintained by juvenile hormone (Gilbert and Schneiderman, 1959: Gilbert, 1962). It has also been proposed that the neuroendocrine system in insects is subjected to some kind of feedback regulation during metamorphosis (Williams, 1950: Bodenstein, 1954). In the present paper, we attempt to clarify how the prothoracic gfand hormone, ecdysone, affects the neurosecretory cells of the diapausing pupal brain of the cabbage armyworm, M. hrassicae, in vitro, and dis-
’ Present address (to which reprint requests should be sent): Department of Medical Entomology, The National Institute of Health, Shinagawa-ku, Tokyo, 141, Japan.
cuss feedback regulation results obtained. MATERIALS
on the basis of the
AND METHODS
For the source of brains and prothoracic glands, cabbage armyworms, M. brassicae, were reared on an artificial diet (Agui et al., 1975) under aseptic conditions, and then semiasepticahy during the last instar. Diapausing M. brassicae pupae were obtained with a short-day photoperiod of 12-hr light and 12-hr dark at 22”. In order to assay prothoracotropic hormone (P’TTH) activity of the brain, both the Chile suppressalis integument test and the cocultured system, which were described previously by Agui (1973, 1975), were used. C. suppressatis larvae were reared on an artificial diet (Kamano, 1973) under 8-hr of light/day at 25”. Culture methods and hormone treatment. The diapausing M. brassicae pupae 40 or 70 days after pupation were su~~ce-steriljzed by submersion in 70% ethanol for 30 set and then in 0.1% aqueous HgCI, for 3 min. They were next rinsed with sterilized distilled water, and their brains were dissected out. Five brains were washed in Ringer-Tyrode solution and then in CSM-2F culture medium (Mitsuhashi, 1968). These were then transferred to a sitting drop (0.025 ml) of CSM-2F medium: either alone, or in which Pecdysone (0.5 I.lgiml) (Rhoto Pha~aceuticai Co.) or cu-ecdysone (5 &ml) (provided by Dr. Ohtaki) was dissolved, or in which five active prothoracic glands taken from G-day-old larvae of Experimental
animals.
467 Copyright t@ 1977 by Academic Press. Inc. All rights of reproduction in any form reserved.
ISSN 00166480
468
AGUI
AND
last-instar M. brassicae were cocultured. The explants were cultured for more than 2 days at 25” in an incubator filled with mixed gas (O,:CO,,l:l). Histological preparation and observation of stainability in neurosecretory brain cells. All brains were fixed with Bouin’s fluid for 24 hr, stained with Gomori’s paraldehyde-fuchsin (AF), and then mounted by the method of Dogra and Tandan (1964). The neurosecretory cells in the brain of M. brassicae can be classified into four groups by cell shape and stainability with AF, as previously described (Agui, 1976). The stain intensity of neurosecretory cells in median group I was classified as follows: (1) not stained: (2) slightly stained dark blue-violet: (3) clearly stained dark blue-violet: (4) slightly tearshaped, and strongly stained dark blue-violet: (5) strongly tear-shaped, and strongly stained dark blue-violet, so that the axons near the neurosecretory cells were also strongly stained. The classification of the neurosecretory cells in median group II and posterior and lateral groups is set up as follows: (1) not stained; (2) slightly stained dark blue-violet: (3) clearly stained dark blue-violet.
RESULTS
Effects of a- and /3-Ecdysone and Prothoracic Gland on the Cultured Brain from the Diapausing Pupa in Vitro As shown in Tables 1 and 2, all groups of neurosecretory cells of the cultured brain TABLE EFFECTS
HIRUMA
taken from diapausing pupae at 70 days were more heavily stained with AF than those of brains in situ. When (Y- or /3-ecdysone was added to the culture medium or active prothoracic glands from 6-day-old larvae were cocultured with diapausing brains, the neurosecretory cells of grade 5 in median group I increased more in stainability than those cultured in medium alone. On the other hand, the increasing stainability of type II cells was only observed in the brain treated with /3-ecdysone. The neurosecretory cells of median group II, the posterior group, or the lateral group showed increased stainability when cultured in medium with or without hormone. However, the lateral group, when treated with a-ecdysone, did not exhibit changes in stainability. The axon tracts, which emerged from neurosecretory cells, were obvious in the cultured brain. The Prothoracotropic Activity of Brains Treated with Ecdysone in Vitro Since the neurosecretory cells treated with ecdysone in vitro increased their stain intensity, we attempted to determine I
OF a- AND B-ECDYSONE AND PROTHORACIC GLAND ON THE NEUROSECRETORY BRAIN CELLS OF DIAPAUSING M. brassicae PUPA in vitroa
Median group 1 (%)*
Days after culture
No. of cultured brains
In situ
0
9
11.1 (a)
Plain medium
2
9
P-Ecdysone (0.3 pg/ml) a-Ecdysone (5 dml)
2
9
33.3
2
9
44.4
PC (5)
2
IO
30.0
Treatments
5
4
3
2
I
66.7 44.4 44.5 77.8
88.9 66.7 (b) 33.3 55.6 22.2 22.2
0 22.2 0 0 0 0
0 11.1 0 0 0 0
11.2 44.4 20.0 20.0
44.4 55.6 40.0 50.0
0 0 10.0 30.0
0 0 0 0
Cl Brains taken from the diapausing pupae 70 days after pupation were stained with Gomori’s paraldehydefuchsin 0 or 2 days after cultivation. b Numbers from I to 5 indicate the classification of stain intensity (see Materials and Methods). Median group 1 consists of two neurosecretory cells of type I (a) and two neurosecretory cells of type II (b).
IN ViTRO
ACTIVATION
OF BRAIN
TABLE EFFECTS
Treatments In situ Plain medium j3-Ecdysone (0.3 &ml) a?-Ecdysone (5 t-&ml) PG (3
IN
469
INSECT
2
OF a AND P-ECDYSONE AND PROTHORACK GLAND ON THE NEUROSECRETORY BRAIN CELLS OF DIAPAUS~NG M. lmssicne PUPA in Vitro’”
Days after culture
No. of cultured brains
0 2 2
Median group IID (%‘c) 3
Posterior (%;)
Lateral (%I
2
I
3
2
9 9 9
II.1 33.3
100.0 88.9 66.7
55.6 33.3
22.2 22.2
100.0 22.2 44.5
2
9
44.4
55.6
44.4
55.6
2
10
0
90.0
30.0
70.0
10.0
1
3
2 44.5 66.7
I 100.0 55.5 33.3 100.0
30.0
70.0
a Brains taken from the diapausing pupae at 70 days after pupation were stained with Gomori’s paraldehydefuchsin 0 or 2 days after cultivation. * Numbers from 1 to 3 indicate the classification of stain intensity (see Materials and Methods).
whether or not the brain treated with /3-ecdysone in rifro can release PTTH into the culture medium during cultivation. This was done by means of the cocultured system of the prothoracic gland and integument (Table 3). The expe~mental procedures were set up as follows: Five brains from diapausing 40-day-old pupae were cultured for 2 days in the medium containing
0.3 &ml of j3-ecdysone and then were transferred to a hormone-free medium for another 2 days in order to allow secretion of the PTTH. Then, the brains were removed from the culture and the conditioned medium was collected. For the assay of PTTH, four inactive prothoracic glands from diapausing pupae were cultured for 4 days in the medium, and then the five frag-
TABLE EFFECTS
(1)
(2)
Brai& + /3-ecdysone
Brain
OF @-ECDYSONE
2 Days i 2 Days
ON THE CULTURED
New medium brain
Medium
3 BRAINS
FROM
DIAPAUSING
Medium
2 Days
4 Days
+ D-PG
+D-PC”
Medium
D-PG f integument 4 Days
Apolysis ’
D-PG
1
211.5
t integument (3)
Brain
4 Days
Medium + D-PG
4 Days
PUPAE
Medium D-PG
4 Days
Apolysis >
O/l5
+ integument B As to experimental procedures, see Results. * D-PG = diapausing prothoracic glands. e Double rectangles mean the conditioned medium with brains for 2 or 4 days. d Brains were taken from the diapausing pupae 40 days after pupation.
in Vitroa 4 Days ’
Apolysis 8115
470
AGUI AND HIRUMA
ments of Chifo integument were cocultured with prothoracic glands for 4 days in order to assay ecdysone activity. As a control [(2) and (3) in Table 31, the brains were cultured for 2 or 4 days in medium alone, and then the conditioned medium was collected and the prothoracotropic activity in the medium was assayed by the same methods. Table 3 shows that about 55% of the integument fragments underwent apolysis when the brain taken from diapausing pupa was treated with ecdysone. However, when the brains were cultured in medium alone for 2 or 4 days, only about 13% of the epidermal fragments underwent apolysis. Tables 4 and 5 show the differences in stain intensity of neurosecretory cells in the brain treated under various conditions. The stain intensity of median group I neurosecretory cells in the cultured brains treated with or without ecdysone was greater than that in intact brains. When the brains were treated with hormone for 2 days or treated with hormone for the same period and then left an additional 2 days without hormone, there appeared to be no differences between the
two treatments. However, about 70% of the axon tracts from neurosecretory cells in median group were stained in the latter treatment. The nuerosecretory cells in median group II or in the posterior group, which were treated with hormone, showed increased stain intensity over those in the intact brain or in brains in the medium alone. In the lateral group, there was no increase in stain intensity. DISCUSSION
Generally, an increasing stain intensity of neurosecretory cells or axon tracts with AF suggests an increasing accumulation of neurosecretory materials. Agui (1975) showed that the neurosecretory cells or axon tracts from neurosecretory cells in the brain taken from either diapausing or nondiapausing M. brassicae pupae increased in stain intensity at 2-3 days after cultivation. Furthermore, Hiruma and Agui (1977) showed, by implantation experiments using neurosecretory cell-ablated brains or neurosecretory cell clusters of brain in M.
TABLE 4 EFFECTS OF&ECDYSONE ON THE STAIN INTENSITY OF NEUROSECRETORY BRAIN CELLS in Vitro” Days after culture 0
2
No. of cultured brains IO
14
4
13
W-J3
13
2(/3-E) + 2
15
Median group I (%‘c)~ 5 20.0 (a) 10.0 (b) 42.9 14.3 38.5 38.5 69.2 46.1 66.6 60.0
4
3
2
I
50.0 40.0 35.7 50.0 30.8 23.1 23.1 30.8 26.1 20.0
30.0 40.0 14.3 21.4 23.1 30.8 1.1 23. I 6.7 20.0
0
10.0 7.1 14.3 7.6 7.6 0 0 0 0
0 0 0
0 0 0 0 0 0 0
Stained axon (%) 10.0
7.1 38.5 53.9 70.3
D Brains taken from the diapausing pupae 40 days after pupation were cultured in medium with or without P-ecdysone (0.3 pg/ml, P-E), and then were stained with Gomori’s paraldehyde-fuchsin 0, 2, or 4 days after cultivation. b Numbers from I to 5 indicate the classification of stain intensity (see Materials and Methods). Median group 1 consists of two neurosecretory cells of type 1 (a) and two neurosecretory cells of type II (b).
IN
ACTIVATION
VITRO
TABLE EFFECTS
Days after culture 0 2 4
OF ~ECDYSONE
No. of cultured brains IO 14 13
2(/3-E)
13
2(/3-E) + 2
15
471
OF BRAIN IN INSECT 5
ON THE STAIN INTENSITY BRAIN CELLS in Vi/m”
Median group II (%J)~
OF NEUROSECRETOR\
Posterior (‘?r)
Lateral (%)
3
2
I
3
2
I
3
2
I
40.0 35.7 38.5 23.1 53.3
60.0 50.0 46. I 38.4 26.7
10.0
14.3 15.4 38.5 20.0
0 21.4 53.9 30.8 40.0
90.0 78.6 46.1 38.4 33.3
40.0 14.3
60.0 85.7 92.3 76.9 73.3
0 0 7.7 15.4 0
30.8 26.7
7.7 26.7
a Brains taken from the diapausing pupae 40 days after pupation were cultured in medium with or without /3-ecdysone (0.3 &ml, /3-E). and then were stained with Gomori’s paraldehyde-fuchsin 0. 2, or 4 days after cultivation. * Numbers from I to 3 indicate the classification of stain intensity (see Materials and Methods).
brassicae, that the neurosecretory cells of the type II cells in the pars intercerebralis are probably able to secrete PTTH. In the present experiments, the stain intensity of type II cells in group I was also increased by P-ecdysone application in vitro. When the PTTH activity of neurosecretory cells of brains treated with /3-ecdysone was assayed, a higher prothoracotropic activity was obtained than with cells from untreated brains. These results indicate that the brains taken from diapausing M. brassicae pupae were activated by in vitro treatment with @-ecydsone. By in vivo experiments, Agui and Hiruma (1977) similarly showed that prothoracic gland removal in M. brassicae larva promoted the release of neurosecretory materials from the neurosecretory cells. Injection of P-ecdysone into prothoracic glandablated larvae increased both the synthesis and transport of neurosecretory materials in the neurosecretory cells. The in vivo and in vitro experiments clearly suggest that the release and synthesis of neurohormone in the brain of M. brassicae are regulated by the prothoracic glands via a positive feedback mechanism. In Rhodnius prolixus, when fifth-instar 1 day after feeding, were wmphs, parabiosed to decapitated older nymphs or injected with P-ecdysone, the median
nuerosecretory cells of the former insects were induced to switch their normal sequence of cytological changes to that characteristic of the older insect (Steel, 1973; 1975). It was concluded from these results that the behavior of median neurosecretory cells is a negative feedback with regard to the release of neurosecretory materials, but with a stimulation of synthesis. Marks er al. (1972) presented somewhat similar results to those obtained with M. brussicae, i.e., that P-ecdysone facilitated only the release of accumulated neurosecretory materials from neurosecretory cells of the cultured brains of the cockroach, Leucophaea maderae. Since then, evidence has appeared for negative feedback regulation in the brain of M. brassicae in vivo and in vitro. When juvenile hormone analog (JHA, ZR-515) was applied to larval brains, both in vivo and in vitro, the release of neurosecretory materials from neurosecretory cells into the axon tract was disturbed (Hiruma et al., unpublished). Consequently, the rapid increase of ecdysone titer in the hemolymph may be induced by these sequential processes: (1) Brain elicits prothoracic gland activation by the prothoracotropic neurohormone; (2) the activated prothoracic glands secrete (Yecdysone; (3) a-ecdysone is quickly converted to /3-ecdysone: (41 P-ecdysone itself
472
AGUI
AND
acts on both the prothoracic glands and brain as a positive feedback regulator. ACKNOWLEDGMENTS We wish to thank Dr. S. Yagi, of our laboratory, for his valuable suggestions. Thanks are also due to Dr. E. P. Marks, Metabolism and Radiation Research Laboratory, USDA, and Prof. L. I. Gilbert, Department of Biological Sciences, Northwestern University, for their critical reading of the manuscript. This work was supported by Grant No. 111908 from the Ministry of Education, Japan.
REFERENCES Agui, N. (1973). Quantitative bioassay of moulting hormone in vitro. Appl. Entomol. Zool. 8, 236240. Agui, N. (1975). Activation of prothoracic glands by brain in vitro. J. Insect Physiol. 21, 903-913. Agui, N. (1976). Studies on insect endocrinology by means of organ culture. Mem. Fat. Agr. Tokyo Unvi. Educ. 22, 173-235. (Japanese with English summary). Agui, N., and Hiruma, K. (1977). Ecdysone as a feedback regulator for the neurosecretory brain cells in the cabbage armyworm, Mamestra brassicae L. J. Insect Physiol. in press. Agui, N., Ogura, N., and Okawara, M. (1975). Rearing of the cabbage armyworm, Mamestra brassicae L. (Lepidoptera:Noctuidae) and some lepidopterous larvae on artificial diets. Japan. J. Appl. Entomol. Zoo/. 19, 91-96. (Japanese with English abstract). Bodenstein, D. (1954). Endocrine mechanisms in the life of insects. Rec. Progr. Hormones Res. 10, 157-182. Dogra, G. S., and Tandan, B. K. (1964). Adaptation of certain histological techniques for in situ demonstration of the neuroendocrine system of insects and other animals. Quart. J. Microsc. Sci. 105, 455-466. Gilbert, L. I. (1962). Maintenance of prothoracic gland by the juvenile hormone in insects. Nature (London)
Gilbert,
193, 1205-1206.
L. I., and Schneiderman,
H. A. (1959).
HIRUMA
Prothoracic gland stimulation by juvenile hormone extracts of insects. Nature (London) 184, 171-173. Hiruma, K., and Agui, N. (1977). Relationship between histological changes and functions of the neurosecretory cells in the brains of the cabbage armyworm, Mamestra brassicae L. Appl. Entomol.
Zool.
12, 42-49.
Kamano, S. (1973). Studies on artificial diet and laboratory rearing method suitable for successive generations of the rice stem borer, Chilo suppressalis Walker. Bull. Nat. Inst. Agr. Sci. Ser. C 27, l-51. (Japanese with English summary). Marks, E. P., Ittycheriah, P. I., and Leloup, A. M. (1972). The effect of P-ecdysone on insect neurosecretion in vitro. J. Insect Physiol. 18, 847-850. Mitsuhashi, J. (1968). Tissue culture of the rice stem borer Chile suppressalis Walker (Lepidoptera: Pyralidae). III. Effects of temperatures and cold-stage on the multiplication of the cell line from larval hemocytes. Appl. Entomol. Zool. 3, l-4. Plagge, E. (1938). Weitere Untersuchungen iiber des Verpuppungshormone bei Schmetterlingen. Biol. Zentralbl. SE, l-12. Steel, C. G. H. (1973). Humoral regulation of the cerebral neurosecretory system of Rhodnius prolixus (Stal) during growth and moulting. J. Exp. Biol. 58, 177-187. Steel, C. G. H. (1975). A neuroendocrine feedback mechanism in insect moulting cycle. Nature (London)
253, 267-269.
Wigglesworth, V. B. (1940). The determination of characters at metamorphosis in Rhodnius prolixus.
.I. Exp.
Biol.
17, 201-222.
Williams, C. M. (1947). Physiology of insect diapause. II. Interaction between the pupal brain and prothoracic glands in the metamorphosis in the giant silkworm, Platysamia cecropia. Biol. Bull. 93, 89-98. Williams, C. M. (1950). The metamorphosis of insects. Sci. Amer.
182, 24-28.
Williams, C. M. (1952). Physiology of insect diapause. IV. The brain and prothoracic glands as an endocrine system in the Cecropia silkworm. Biol. Bull. 121, 572-585.
AND
COMPARATIVE
In Vitro Activation
ENDOCRINOLOGY
of Neurosecretory Brain Cells in Mamestra brassicae by /3-Ecdysone NORIAKI
Laboratory
of Applied
Zoology,
33, 467-472 (1977)
Faculty
AGWI’
AND KIYOSHI I~~IRIJMA
of Agriculfiure, Tokyo Universiiy Tokyo, IS3, Japan
of Education,
Komaba,
Meguro-ku,
Accepted July 15, 1977 The effect of ecdysone or of the prothoracic gland on the neurosecretory cells of brains taken from diapausing pupae of the cabbage armyworm, Mamestra brassicae L., was investigated histologically and functionally in vitro. When the diapausing brain was cultured in a medium containing fl-ecdysone, the neurosecretory cells of the median group I in the pars intercerebralis increased staining intensity more than those of brains cultured in medium alone. Furthermore, when the prothoracotropic activity of a brain treated with /3-ecdysone was compared with that of an untreated brain in vitro, the brain treated with hormone showed higher activity than the untreated one. These results suggest that p-ecdysone acts on the neurosecretory cells of pupal brain in M. brussicae as a positive feedback regulator.
Secretion of prothoracotropic hormone (PTTH) from neurosecretory cells in the median part of brain has been elucidated by several investigators (Plagge, 1938: Wi~leswo~h, 1940: Williams, 1947) who employed the methods of experimental morphology. Furthermore, it has been reported that the prothoracic gland is stimuIated by its own product (Williams, 1952) and that its activity is enhanced and maintained by juvenile hormone (Gilbert and Schneiderman, 1959: Gilbert, 1962). It has also been proposed that the neuroendocrine system in insects is subjected to some kind of feedback regulation during metamorphosis (Williams, 1950: Bodenstein, 1954). In the present paper, we attempt to clarify how the prothoracic gfand hormone, ecdysone, affects the neurosecretory cells of the diapausing pupal brain of the cabbage armyworm, M. hrassicae, in vitro, and dis-
’ Present address (to which reprint requests should be sent): Department of Medical Entomology, The National Institute of Health, Shinagawa-ku, Tokyo, 141, Japan.
cuss feedback regulation results obtained. MATERIALS
on the basis of the
AND METHODS
For the source of brains and prothoracic glands, cabbage armyworms, M. brassicae, were reared on an artificial diet (Agui et al., 1975) under aseptic conditions, and then semiasepticahy during the last instar. Diapausing M. brassicae pupae were obtained with a short-day photoperiod of 12-hr light and 12-hr dark at 22”. In order to assay prothoracotropic hormone (P’TTH) activity of the brain, both the Chile suppressalis integument test and the cocultured system, which were described previously by Agui (1973, 1975), were used. C. suppressatis larvae were reared on an artificial diet (Kamano, 1973) under 8-hr of light/day at 25”. Culture methods and hormone treatment. The diapausing M. brassicae pupae 40 or 70 days after pupation were su~~ce-steriljzed by submersion in 70% ethanol for 30 set and then in 0.1% aqueous HgCI, for 3 min. They were next rinsed with sterilized distilled water, and their brains were dissected out. Five brains were washed in Ringer-Tyrode solution and then in CSM-2F culture medium (Mitsuhashi, 1968). These were then transferred to a sitting drop (0.025 ml) of CSM-2F medium: either alone, or in which Pecdysone (0.5 I.lgiml) (Rhoto Pha~aceuticai Co.) or cu-ecdysone (5 &ml) (provided by Dr. Ohtaki) was dissolved, or in which five active prothoracic glands taken from G-day-old larvae of Experimental
animals.
467 Copyright t@ 1977 by Academic Press. Inc. All rights of reproduction in any form reserved.
ISSN 00166480
468
AGUI
AND
last-instar M. brassicae were cocultured. The explants were cultured for more than 2 days at 25” in an incubator filled with mixed gas (O,:CO,,l:l). Histological preparation and observation of stainability in neurosecretory brain cells. All brains were fixed with Bouin’s fluid for 24 hr, stained with Gomori’s paraldehyde-fuchsin (AF), and then mounted by the method of Dogra and Tandan (1964). The neurosecretory cells in the brain of M. brassicae can be classified into four groups by cell shape and stainability with AF, as previously described (Agui, 1976). The stain intensity of neurosecretory cells in median group I was classified as follows: (1) not stained: (2) slightly stained dark blue-violet: (3) clearly stained dark blue-violet: (4) slightly tearshaped, and strongly stained dark blue-violet: (5) strongly tear-shaped, and strongly stained dark blue-violet, so that the axons near the neurosecretory cells were also strongly stained. The classification of the neurosecretory cells in median group II and posterior and lateral groups is set up as follows: (1) not stained; (2) slightly stained dark blue-violet: (3) clearly stained dark blue-violet.
RESULTS
Effects of a- and /3-Ecdysone and Prothoracic Gland on the Cultured Brain from the Diapausing Pupa in Vitro As shown in Tables 1 and 2, all groups of neurosecretory cells of the cultured brain TABLE EFFECTS
HIRUMA
taken from diapausing pupae at 70 days were more heavily stained with AF than those of brains in situ. When (Y- or /3-ecdysone was added to the culture medium or active prothoracic glands from 6-day-old larvae were cocultured with diapausing brains, the neurosecretory cells of grade 5 in median group I increased more in stainability than those cultured in medium alone. On the other hand, the increasing stainability of type II cells was only observed in the brain treated with /3-ecdysone. The neurosecretory cells of median group II, the posterior group, or the lateral group showed increased stainability when cultured in medium with or without hormone. However, the lateral group, when treated with a-ecdysone, did not exhibit changes in stainability. The axon tracts, which emerged from neurosecretory cells, were obvious in the cultured brain. The Prothoracotropic Activity of Brains Treated with Ecdysone in Vitro Since the neurosecretory cells treated with ecdysone in vitro increased their stain intensity, we attempted to determine I
OF a- AND B-ECDYSONE AND PROTHORACIC GLAND ON THE NEUROSECRETORY BRAIN CELLS OF DIAPAUSING M. brassicae PUPA in vitroa
Median group 1 (%)*
Days after culture
No. of cultured brains
In situ
0
9
11.1 (a)
Plain medium
2
9
P-Ecdysone (0.3 pg/ml) a-Ecdysone (5 dml)
2
9
33.3
2
9
44.4
PC (5)
2
IO
30.0
Treatments
5
4
3
2
I
66.7 44.4 44.5 77.8
88.9 66.7 (b) 33.3 55.6 22.2 22.2
0 22.2 0 0 0 0
0 11.1 0 0 0 0
11.2 44.4 20.0 20.0
44.4 55.6 40.0 50.0
0 0 10.0 30.0
0 0 0 0
Cl Brains taken from the diapausing pupae 70 days after pupation were stained with Gomori’s paraldehydefuchsin 0 or 2 days after cultivation. b Numbers from I to 5 indicate the classification of stain intensity (see Materials and Methods). Median group 1 consists of two neurosecretory cells of type I (a) and two neurosecretory cells of type II (b).
IN ViTRO
ACTIVATION
OF BRAIN
TABLE EFFECTS
Treatments In situ Plain medium j3-Ecdysone (0.3 &ml) a?-Ecdysone (5 t-&ml) PG (3
IN
469
INSECT
2
OF a AND P-ECDYSONE AND PROTHORACK GLAND ON THE NEUROSECRETORY BRAIN CELLS OF DIAPAUS~NG M. lmssicne PUPA in Vitro’”
Days after culture
No. of cultured brains
0 2 2
Median group IID (%‘c) 3
Posterior (%;)
Lateral (%I
2
I
3
2
9 9 9
II.1 33.3
100.0 88.9 66.7
55.6 33.3
22.2 22.2
100.0 22.2 44.5
2
9
44.4
55.6
44.4
55.6
2
10
0
90.0
30.0
70.0
10.0
1
3
2 44.5 66.7
I 100.0 55.5 33.3 100.0
30.0
70.0
a Brains taken from the diapausing pupae at 70 days after pupation were stained with Gomori’s paraldehydefuchsin 0 or 2 days after cultivation. * Numbers from 1 to 3 indicate the classification of stain intensity (see Materials and Methods).
whether or not the brain treated with /3-ecdysone in rifro can release PTTH into the culture medium during cultivation. This was done by means of the cocultured system of the prothoracic gland and integument (Table 3). The expe~mental procedures were set up as follows: Five brains from diapausing 40-day-old pupae were cultured for 2 days in the medium containing
0.3 &ml of j3-ecdysone and then were transferred to a hormone-free medium for another 2 days in order to allow secretion of the PTTH. Then, the brains were removed from the culture and the conditioned medium was collected. For the assay of PTTH, four inactive prothoracic glands from diapausing pupae were cultured for 4 days in the medium, and then the five frag-
TABLE EFFECTS
(1)
(2)
Brai& + /3-ecdysone
Brain
OF @-ECDYSONE
2 Days i 2 Days
ON THE CULTURED
New medium brain
Medium
3 BRAINS
FROM
DIAPAUSING
Medium
2 Days
4 Days
+ D-PG
+D-PC”
Medium
D-PG f integument 4 Days
Apolysis ’
D-PG
1
211.5
t integument (3)
Brain
4 Days
Medium + D-PG
4 Days
PUPAE
Medium D-PG
4 Days
Apolysis >
O/l5
+ integument B As to experimental procedures, see Results. * D-PG = diapausing prothoracic glands. e Double rectangles mean the conditioned medium with brains for 2 or 4 days. d Brains were taken from the diapausing pupae 40 days after pupation.
in Vitroa 4 Days ’
Apolysis 8115
470
AGUI AND HIRUMA
ments of Chifo integument were cocultured with prothoracic glands for 4 days in order to assay ecdysone activity. As a control [(2) and (3) in Table 31, the brains were cultured for 2 or 4 days in medium alone, and then the conditioned medium was collected and the prothoracotropic activity in the medium was assayed by the same methods. Table 3 shows that about 55% of the integument fragments underwent apolysis when the brain taken from diapausing pupa was treated with ecdysone. However, when the brains were cultured in medium alone for 2 or 4 days, only about 13% of the epidermal fragments underwent apolysis. Tables 4 and 5 show the differences in stain intensity of neurosecretory cells in the brain treated under various conditions. The stain intensity of median group I neurosecretory cells in the cultured brains treated with or without ecdysone was greater than that in intact brains. When the brains were treated with hormone for 2 days or treated with hormone for the same period and then left an additional 2 days without hormone, there appeared to be no differences between the
two treatments. However, about 70% of the axon tracts from neurosecretory cells in median group were stained in the latter treatment. The nuerosecretory cells in median group II or in the posterior group, which were treated with hormone, showed increased stain intensity over those in the intact brain or in brains in the medium alone. In the lateral group, there was no increase in stain intensity. DISCUSSION
Generally, an increasing stain intensity of neurosecretory cells or axon tracts with AF suggests an increasing accumulation of neurosecretory materials. Agui (1975) showed that the neurosecretory cells or axon tracts from neurosecretory cells in the brain taken from either diapausing or nondiapausing M. brassicae pupae increased in stain intensity at 2-3 days after cultivation. Furthermore, Hiruma and Agui (1977) showed, by implantation experiments using neurosecretory cell-ablated brains or neurosecretory cell clusters of brain in M.
TABLE 4 EFFECTS OF&ECDYSONE ON THE STAIN INTENSITY OF NEUROSECRETORY BRAIN CELLS in Vitro” Days after culture 0
2
No. of cultured brains IO
14
4
13
W-J3
13
2(/3-E) + 2
15
Median group I (%‘c)~ 5 20.0 (a) 10.0 (b) 42.9 14.3 38.5 38.5 69.2 46.1 66.6 60.0
4
3
2
I
50.0 40.0 35.7 50.0 30.8 23.1 23.1 30.8 26.1 20.0
30.0 40.0 14.3 21.4 23.1 30.8 1.1 23. I 6.7 20.0
0
10.0 7.1 14.3 7.6 7.6 0 0 0 0
0 0 0
0 0 0 0 0 0 0
Stained axon (%) 10.0
7.1 38.5 53.9 70.3
D Brains taken from the diapausing pupae 40 days after pupation were cultured in medium with or without P-ecdysone (0.3 pg/ml, P-E), and then were stained with Gomori’s paraldehyde-fuchsin 0, 2, or 4 days after cultivation. b Numbers from I to 5 indicate the classification of stain intensity (see Materials and Methods). Median group 1 consists of two neurosecretory cells of type 1 (a) and two neurosecretory cells of type II (b).
IN
ACTIVATION
VITRO
TABLE EFFECTS
Days after culture 0 2 4
OF ~ECDYSONE
No. of cultured brains IO 14 13
2(/3-E)
13
2(/3-E) + 2
15
471
OF BRAIN IN INSECT 5
ON THE STAIN INTENSITY BRAIN CELLS in Vi/m”
Median group II (%J)~
OF NEUROSECRETOR\
Posterior (‘?r)
Lateral (%)
3
2
I
3
2
I
3
2
I
40.0 35.7 38.5 23.1 53.3
60.0 50.0 46. I 38.4 26.7
10.0
14.3 15.4 38.5 20.0
0 21.4 53.9 30.8 40.0
90.0 78.6 46.1 38.4 33.3
40.0 14.3
60.0 85.7 92.3 76.9 73.3
0 0 7.7 15.4 0
30.8 26.7
7.7 26.7
a Brains taken from the diapausing pupae 40 days after pupation were cultured in medium with or without /3-ecdysone (0.3 &ml, /3-E). and then were stained with Gomori’s paraldehyde-fuchsin 0. 2, or 4 days after cultivation. * Numbers from I to 3 indicate the classification of stain intensity (see Materials and Methods).
brassicae, that the neurosecretory cells of the type II cells in the pars intercerebralis are probably able to secrete PTTH. In the present experiments, the stain intensity of type II cells in group I was also increased by P-ecdysone application in vitro. When the PTTH activity of neurosecretory cells of brains treated with /3-ecdysone was assayed, a higher prothoracotropic activity was obtained than with cells from untreated brains. These results indicate that the brains taken from diapausing M. brassicae pupae were activated by in vitro treatment with @-ecydsone. By in vivo experiments, Agui and Hiruma (1977) similarly showed that prothoracic gland removal in M. brassicae larva promoted the release of neurosecretory materials from the neurosecretory cells. Injection of P-ecdysone into prothoracic glandablated larvae increased both the synthesis and transport of neurosecretory materials in the neurosecretory cells. The in vivo and in vitro experiments clearly suggest that the release and synthesis of neurohormone in the brain of M. brassicae are regulated by the prothoracic glands via a positive feedback mechanism. In Rhodnius prolixus, when fifth-instar 1 day after feeding, were wmphs, parabiosed to decapitated older nymphs or injected with P-ecdysone, the median
nuerosecretory cells of the former insects were induced to switch their normal sequence of cytological changes to that characteristic of the older insect (Steel, 1973; 1975). It was concluded from these results that the behavior of median neurosecretory cells is a negative feedback with regard to the release of neurosecretory materials, but with a stimulation of synthesis. Marks er al. (1972) presented somewhat similar results to those obtained with M. brussicae, i.e., that P-ecdysone facilitated only the release of accumulated neurosecretory materials from neurosecretory cells of the cultured brains of the cockroach, Leucophaea maderae. Since then, evidence has appeared for negative feedback regulation in the brain of M. brassicae in vivo and in vitro. When juvenile hormone analog (JHA, ZR-515) was applied to larval brains, both in vivo and in vitro, the release of neurosecretory materials from neurosecretory cells into the axon tract was disturbed (Hiruma et al., unpublished). Consequently, the rapid increase of ecdysone titer in the hemolymph may be induced by these sequential processes: (1) Brain elicits prothoracic gland activation by the prothoracotropic neurohormone; (2) the activated prothoracic glands secrete (Yecdysone; (3) a-ecdysone is quickly converted to /3-ecdysone: (41 P-ecdysone itself
472
AGUI
AND
acts on both the prothoracic glands and brain as a positive feedback regulator. ACKNOWLEDGMENTS We wish to thank Dr. S. Yagi, of our laboratory, for his valuable suggestions. Thanks are also due to Dr. E. P. Marks, Metabolism and Radiation Research Laboratory, USDA, and Prof. L. I. Gilbert, Department of Biological Sciences, Northwestern University, for their critical reading of the manuscript. This work was supported by Grant No. 111908 from the Ministry of Education, Japan.
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H. A. (1959).
HIRUMA
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Williams, C. M. (1952). Physiology of insect diapause. IV. The brain and prothoracic glands as an endocrine system in the Cecropia silkworm. Biol. Bull. 121, 572-585.