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Diapause termination and metamorphosis in brainless tobacco hornworms (Lepidoptera)
Life Sciences Vol. 11, Part II, pp. 805-811, 1972. Printed in Great Britain
Pergamon Press
DLAPAOSB TßRMINATION AND METAMORPHOSIS IN BRAINLESS TOBACCO HORNiPORMS (LEPIDOPTßRA) 1 Kenneth J. Judy zoecon Corporation, Palo Alto, California 94304 (Received 1 March 1972; in final form 4 May 1972)
SummarY
Diapausinq Manduca sexta pupae differ from certain Fey are able to terminate other Lepidoptera~fFat diapause and initiate adult development even after brain extirpation . A small number of larvae are able to pupate, diapause, and later initiate adult development without brains, indicating that, in this species, the brain is not essential to stimulate molting hormone production . Several possible explanations are discussed . Introduction In the classical scheme of endocrine regulation of growth and development in insects, the "brain hormone", produced by neurosecretory cells in the brain and released through the corpora cardiaca, exerts a tropic influence on the prothoracic glands which produce ecdysona (1-3) .
Scdysone, in tars, stimulates cell
division and brings about molting, metamorphosis and termination of papal diapause .
Ligation or brain extirpation has bean found
to arrest growth, molting and diapause termination in many species of Lepidoptera, and these effects can ba overcome by implantation of "active" brains (1) .
However, McDaniel and Harry (4) have
shown that "'inactive" brains may be activated by injury and in soma exceptional circs~stances injury of brainless pupae may lead to diapause termination.
Robayshi et al .
(5,6) have reported
3'ha initial phase of this study was conducted at the USDA Laboratory, Fargo, North Dakota, and the assistance of Dra . R.A . Hall and S .P . Marks is gratafnlly acknowledged .
806
808
Initiation a~ Metamorphosis
Vol. il, No . 12
diapause termination in Bo®by~ artificially induced to diapause by brain extirpation, further suggesting that the brain may not be absolutely essential . This paper presents evidence which demonstrates that the mechanism of endocrine control over growth and development in the tobacco hornworm, Manduca sexta , is another exception to the generally accepted scheme, specifically, that Manduca larvae and pupae are able to undergo molting and to complete metamorphosis without brains .
Materials and Methods Manduca sexta were laboratory reared for many generations on an artificial diet (7) (plus 0 .49 linseed oil) and papal diapause was induced by rearing larvae under a 12 hour light:l2 hour dark regime (S) .
Diapausinq pupae require no extrinsic signals (i .e . chilling,
increased photoperiod) to terminate diapause, and initiate adult development between one and five months after entering diapause . Papal brains and/or cardiaca-allata complexes were extirpated from C0 2-anesthetiaed animals through a small opening cut between the bases of the antennae, while larval brains were removed through a small opening cut in the front of the head capsule .
A few
crystals of phenylthiourea were placed in the heaoocoel and the woand sealed with melted wax .
Epidermal retraction and subsequent
morphological changes visible through the papal cuticle marked the termination of diapause . Results A total of more than 150 diapausinq pupae, dabrained 20 to 50 days after pupation, have been observed over the past three years . A third of these had received injections of various scdysones (low
Vol. il, No . 12
Iniriarion ad Metamorphosis
807
doses), inactive steroids, or solvents but showed no immediate response .
Virtually all of these brainless pupae eventually ter-
minated diapause and completed or were well along in adult development when they died or were sacrificed .
Only a few were able to
emerge from the papal case without assistance .
~ comparisoa of
the rata of diapause termination in detrained versus normal pupae is shown in Fiq. 1 .
100r
s z W F~
n~ra FIG. 1 Rate of diapause termination in normal (N) versus detrained (0-O) pupae. Days represent time after pupation for normal group and time after brain extirpation (20-50 days after pupation) for the detrained pupae . All of 20 pupae from which only the region of brain containing the median neurosecretory cells was removed, and all 18 pupae from which both the brain and corpus cardiacum-allatum complex was removed also eventually terminated diapause .
808
Initiarion of Metamorphosis
Vol. 11, No . 12
Brain extirpation was performed on 20 fourth and 30 1-2 day old fifth instar larvae .
Two of the fourth instar larvae survived
long enough to molt once more, one to a fifth instar larva and one directly to a pupa .
Half of the brainless fifth instar larvae
continued to feed for some time, and 6 of these eventually pupated . After more than 30 days in apparent diapause, 2 of these brainless pupae initiated adult development.
Hoth died without emerging, but
1 had completed a nearly normal adult development .
Discussion It is clear that :
1)
Manduca pupae are capable of termi-
nating diapause and developing to adults without a brain; and 2)
last instar larvae can feed, grow, pupate, diapause, and later
initiate adult development without a brain. The brain certainly plays a role in the regulation of endocrine mediated development, but in Manduca it is not essential to the eventual occurrence of several developmental processes . While "injury response" is known to duplicate many effects of ecdysone
(9) and may in some cases stimulate diapause termination
without the mediation of the brain (4), it is not likely to be the cause of diapause termination two or more months after surgery. It is especially significant that a few larvae were able to pupate and later complete metamorphosis without a brain . Although it has been shown that juvenile hormone can stimulate prothoracic glands (10), this is not a likely explanation for the phenomena reported above, since removal of both the brain and corpora allata is still not sufficient to prevent eventual diapause termination. That brain extirpation relieves an inhibition on the prothoracic glands produced by neurosecretory cells in the lateral
Vol. 11, No . 12
Initiation a~f Metamorphosis
part of the brain (as suggested by Carlisle and Sllis)
809
(11), is
not likely, since removal of only the median brain region has the same effect as total brain extirpation . ldhereas the findings of Robayshi et al . (5,6) support my observations on diapause termination in brainless pupae, it must be noted that they obtained imaginal differentiation in normally non-diapausinq ~ pupae, after a period of developmental arrest artifically induced by brain extirpation .
The underlying cause for
the eventual development observed in Bomi~ may differ free that in Planduca . Roped (12) observed the development of adult Lymantria from brainless pupae
however, in his study animals were debrained as
late last-instar larvae (after the critical period) and adult development occurred as a continuous process after pupationf no diapause intervened . It is clear from a number of diverse reports in the literature that much remains to be learned concerning molting hormone production and its regulation in insects .
It has been suggested
(13) that brain extirpation only temporarily supresses prothoracic gland activity, and the glands may spontaneously become active months later.
However, several investigators (14-18) have reported
molting in insects whose prothoracic glands had been removed . Romer (19) recently reported that oenocytes contain molting hormone, and Moriyama et al . (20) have evidence which suggests that ecdysone can be synthesised from cholesterol in isolated Boar abdomens . Thus, if prothoracic glands are strictly dependant on stimulation from the brain to produce molting hormone, it is possible that alternate sources are present which are not dependent on the brain . Another possible explanation for the observations reported here follows from the recent finding (21) that substances with
810
Initiation ad Metamorphosis
Vol. 11, No . 12
"activation" hormone (prothoracotropic hormone) activity can be extracted from the ventral ganglia of Periplaneta .
Marks (22)
recently observed that several late mater Leucophaea maderae nymphs, of a group debrained 24 hours after molting, pare able to survive and molt once more several seeks later . It is therefore possible that, in Manduca , ganglia other than the brain contain cells capable of producing prothoracotropic hormone . Further experiments using surgical extirpation implantation, ligation, or even isolated body regions could prove most informative .
References 1.
C .M . Williams, Biol . Bull . 103, 120 (1952) .
2.
L.i . Gilbert, in Ph siolo~y of Insects, ed . M. Rockstein, Vol . 1, p. 149 . a c~rees~~ork (1964) .
3.
A.D . Lees, The ~Ph siolo~ of Dia University r~ess,~riagé
4.
C .N . McDaniel and S .J . Berry, Nature 214, 1032 (1967) .
5.
M. Robayshi, M. Fukaya and J . Mitsuhashi, J. Saricul . Sci . J~ 29, 337 (1960) .
6.
M. Robayshi and S . Nakasone, Bull . Sericul . sxp. Sta . .16, i00 (1960) .
7.
R.T . Yamaoto, J. Econ . Entomol . 62, 1427 (1969) .
8.
R.A . Bell, Am . Zool . 8, 791 (1968) .
9.
H .A . Schneiderman and C .M . Williams, Biol . Hull . 105, 320 (1953) .
use in Arthropods , p . 101 .
10 .
L.I . Gilbert and H .A . 3chneiderman, Nature (1959) .
11 .
D .B . Carlisle and P .E . Ellis, Nature (London
12 .
S.
13 .
V .B . Wigglesworth, Insect Hormones , p. 23 . San Francisco (1970~-
14 .
H . Piepho, Naturwiss . 35, 94 (1948) .
Rope,
London
184, 171
220, 706 (1968) .
Biol . Bull . 42, 323 (1922) . W.F . Freeman,
Yol. 11, No . 12
Initiaüon ad Metamorphosis
811
15 .
D. Bodenstein, J. Ex~tl . Zool . 129, 209 (1955) .
16 .
L.E, Chaäpick, Science 121, 435 (1955) .
17 .
L .E, Chadpick, J. Exptl . Zool . 131, 291 (1956) .
18 .
W.L, Nuttinq, Science 122, 30 (1955) .
19 .
F. Rainer, Naturpiss . 58, 324 (1971) .
20 .
H . Moriyama, 1C . Nakanishi and T . Okauchi (unpublished observation) cited in, H . Moriyama, K . Nakanishi, D .S . Rinq, T . Okauchi, J .B . Siddall and W . Aafferl, Gen . C~. Endocrinol . 15, 80 (1970) .
21 .
M. Gersch and J . 3tàrzebecher, J . Insect Physiol . 14, 87 (1968) . -
22 .
E .P . Marks, unpublished observation.
Pergamon Press
DLAPAOSB TßRMINATION AND METAMORPHOSIS IN BRAINLESS TOBACCO HORNiPORMS (LEPIDOPTßRA) 1 Kenneth J. Judy zoecon Corporation, Palo Alto, California 94304 (Received 1 March 1972; in final form 4 May 1972)
SummarY
Diapausinq Manduca sexta pupae differ from certain Fey are able to terminate other Lepidoptera~fFat diapause and initiate adult development even after brain extirpation . A small number of larvae are able to pupate, diapause, and later initiate adult development without brains, indicating that, in this species, the brain is not essential to stimulate molting hormone production . Several possible explanations are discussed . Introduction In the classical scheme of endocrine regulation of growth and development in insects, the "brain hormone", produced by neurosecretory cells in the brain and released through the corpora cardiaca, exerts a tropic influence on the prothoracic glands which produce ecdysona (1-3) .
Scdysone, in tars, stimulates cell
division and brings about molting, metamorphosis and termination of papal diapause .
Ligation or brain extirpation has bean found
to arrest growth, molting and diapause termination in many species of Lepidoptera, and these effects can ba overcome by implantation of "active" brains (1) .
However, McDaniel and Harry (4) have
shown that "'inactive" brains may be activated by injury and in soma exceptional circs~stances injury of brainless pupae may lead to diapause termination.
Robayshi et al .
(5,6) have reported
3'ha initial phase of this study was conducted at the USDA Laboratory, Fargo, North Dakota, and the assistance of Dra . R.A . Hall and S .P . Marks is gratafnlly acknowledged .
806
808
Initiation a~ Metamorphosis
Vol. il, No . 12
diapause termination in Bo®by~ artificially induced to diapause by brain extirpation, further suggesting that the brain may not be absolutely essential . This paper presents evidence which demonstrates that the mechanism of endocrine control over growth and development in the tobacco hornworm, Manduca sexta , is another exception to the generally accepted scheme, specifically, that Manduca larvae and pupae are able to undergo molting and to complete metamorphosis without brains .
Materials and Methods Manduca sexta were laboratory reared for many generations on an artificial diet (7) (plus 0 .49 linseed oil) and papal diapause was induced by rearing larvae under a 12 hour light:l2 hour dark regime (S) .
Diapausinq pupae require no extrinsic signals (i .e . chilling,
increased photoperiod) to terminate diapause, and initiate adult development between one and five months after entering diapause . Papal brains and/or cardiaca-allata complexes were extirpated from C0 2-anesthetiaed animals through a small opening cut between the bases of the antennae, while larval brains were removed through a small opening cut in the front of the head capsule .
A few
crystals of phenylthiourea were placed in the heaoocoel and the woand sealed with melted wax .
Epidermal retraction and subsequent
morphological changes visible through the papal cuticle marked the termination of diapause . Results A total of more than 150 diapausinq pupae, dabrained 20 to 50 days after pupation, have been observed over the past three years . A third of these had received injections of various scdysones (low
Vol. il, No . 12
Iniriarion ad Metamorphosis
807
doses), inactive steroids, or solvents but showed no immediate response .
Virtually all of these brainless pupae eventually ter-
minated diapause and completed or were well along in adult development when they died or were sacrificed .
Only a few were able to
emerge from the papal case without assistance .
~ comparisoa of
the rata of diapause termination in detrained versus normal pupae is shown in Fiq. 1 .
100r
s z W F~
n~ra FIG. 1 Rate of diapause termination in normal (N) versus detrained (0-O) pupae. Days represent time after pupation for normal group and time after brain extirpation (20-50 days after pupation) for the detrained pupae . All of 20 pupae from which only the region of brain containing the median neurosecretory cells was removed, and all 18 pupae from which both the brain and corpus cardiacum-allatum complex was removed also eventually terminated diapause .
808
Initiarion of Metamorphosis
Vol. 11, No . 12
Brain extirpation was performed on 20 fourth and 30 1-2 day old fifth instar larvae .
Two of the fourth instar larvae survived
long enough to molt once more, one to a fifth instar larva and one directly to a pupa .
Half of the brainless fifth instar larvae
continued to feed for some time, and 6 of these eventually pupated . After more than 30 days in apparent diapause, 2 of these brainless pupae initiated adult development.
Hoth died without emerging, but
1 had completed a nearly normal adult development .
Discussion It is clear that :
1)
Manduca pupae are capable of termi-
nating diapause and developing to adults without a brain; and 2)
last instar larvae can feed, grow, pupate, diapause, and later
initiate adult development without a brain. The brain certainly plays a role in the regulation of endocrine mediated development, but in Manduca it is not essential to the eventual occurrence of several developmental processes . While "injury response" is known to duplicate many effects of ecdysone
(9) and may in some cases stimulate diapause termination
without the mediation of the brain (4), it is not likely to be the cause of diapause termination two or more months after surgery. It is especially significant that a few larvae were able to pupate and later complete metamorphosis without a brain . Although it has been shown that juvenile hormone can stimulate prothoracic glands (10), this is not a likely explanation for the phenomena reported above, since removal of both the brain and corpora allata is still not sufficient to prevent eventual diapause termination. That brain extirpation relieves an inhibition on the prothoracic glands produced by neurosecretory cells in the lateral
Vol. 11, No . 12
Initiation a~f Metamorphosis
part of the brain (as suggested by Carlisle and Sllis)
809
(11), is
not likely, since removal of only the median brain region has the same effect as total brain extirpation . ldhereas the findings of Robayshi et al . (5,6) support my observations on diapause termination in brainless pupae, it must be noted that they obtained imaginal differentiation in normally non-diapausinq ~ pupae, after a period of developmental arrest artifically induced by brain extirpation .
The underlying cause for
the eventual development observed in Bomi~ may differ free that in Planduca . Roped (12) observed the development of adult Lymantria from brainless pupae
however, in his study animals were debrained as
late last-instar larvae (after the critical period) and adult development occurred as a continuous process after pupationf no diapause intervened . It is clear from a number of diverse reports in the literature that much remains to be learned concerning molting hormone production and its regulation in insects .
It has been suggested
(13) that brain extirpation only temporarily supresses prothoracic gland activity, and the glands may spontaneously become active months later.
However, several investigators (14-18) have reported
molting in insects whose prothoracic glands had been removed . Romer (19) recently reported that oenocytes contain molting hormone, and Moriyama et al . (20) have evidence which suggests that ecdysone can be synthesised from cholesterol in isolated Boar abdomens . Thus, if prothoracic glands are strictly dependant on stimulation from the brain to produce molting hormone, it is possible that alternate sources are present which are not dependent on the brain . Another possible explanation for the observations reported here follows from the recent finding (21) that substances with
810
Initiation ad Metamorphosis
Vol. 11, No . 12
"activation" hormone (prothoracotropic hormone) activity can be extracted from the ventral ganglia of Periplaneta .
Marks (22)
recently observed that several late mater Leucophaea maderae nymphs, of a group debrained 24 hours after molting, pare able to survive and molt once more several seeks later . It is therefore possible that, in Manduca , ganglia other than the brain contain cells capable of producing prothoracotropic hormone . Further experiments using surgical extirpation implantation, ligation, or even isolated body regions could prove most informative .
References 1.
C .M . Williams, Biol . Bull . 103, 120 (1952) .
2.
L.i . Gilbert, in Ph siolo~y of Insects, ed . M. Rockstein, Vol . 1, p. 149 . a c~rees~~ork (1964) .
3.
A.D . Lees, The ~Ph siolo~ of Dia University r~ess,~riagé
4.
C .N . McDaniel and S .J . Berry, Nature 214, 1032 (1967) .
5.
M. Robayshi, M. Fukaya and J . Mitsuhashi, J. Saricul . Sci . J~ 29, 337 (1960) .
6.
M. Robayshi and S . Nakasone, Bull . Sericul . sxp. Sta . .16, i00 (1960) .
7.
R.T . Yamaoto, J. Econ . Entomol . 62, 1427 (1969) .
8.
R.A . Bell, Am . Zool . 8, 791 (1968) .
9.
H .A . Schneiderman and C .M . Williams, Biol . Hull . 105, 320 (1953) .
use in Arthropods , p . 101 .
10 .
L.I . Gilbert and H .A . 3chneiderman, Nature (1959) .
11 .
D .B . Carlisle and P .E . Ellis, Nature (London
12 .
S.
13 .
V .B . Wigglesworth, Insect Hormones , p. 23 . San Francisco (1970~-
14 .
H . Piepho, Naturwiss . 35, 94 (1948) .
Rope,
London
184, 171
220, 706 (1968) .
Biol . Bull . 42, 323 (1922) . W.F . Freeman,
Yol. 11, No . 12
Initiaüon ad Metamorphosis
811
15 .
D. Bodenstein, J. Ex~tl . Zool . 129, 209 (1955) .
16 .
L.E, Chaäpick, Science 121, 435 (1955) .
17 .
L .E, Chadpick, J. Exptl . Zool . 131, 291 (1956) .
18 .
W.L, Nuttinq, Science 122, 30 (1955) .
19 .
F. Rainer, Naturpiss . 58, 324 (1971) .
20 .
H . Moriyama, 1C . Nakanishi and T . Okauchi (unpublished observation) cited in, H . Moriyama, K . Nakanishi, D .S . Rinq, T . Okauchi, J .B . Siddall and W . Aafferl, Gen . C~. Endocrinol . 15, 80 (1970) .
21 .
M. Gersch and J . 3tàrzebecher, J . Insect Physiol . 14, 87 (1968) . -
22 .
E .P . Marks, unpublished observation.