- Email: [email protected]
Role of the brain in post-diapause adult development in the swallowtail, Papilio xuthus
0022-1910/84 $3.00 + 0.00 Copyright c:: 1984 Pergamon Press Ltd
J. Insect fh.wio/. Vol. 30, No. 2. pp. 165-168. 1984 Printedin Great Britain. All rights reserved.
ROLE OF THE BRAIN IN POST-DIAPAUSE ADULT DEVELOPMENT IN THE SWALLOWTAIL, PAPILIO HIDEHARU NUMATA
XUTHUS and
TOSHITAKA HIDAKA
Department of Zoology, Faculty of Science, Kyoto University, Sakyo, Kyoto 606, Japan (Received 6 July 1983)
Abstract-The role of the brain in adult development was examined by brain removal in unchilled and chilled diapausing pupae of Pupilio xuthus. Chilling was effective in shortening the pupal duration and synchronizing adult emergence, although photoperiod had little effect on diapause development. The brain played a role of shortening the pupal duration and synchronizing adult emergence in both unchilled and
chilled individuals, although it was not essential for post-diapause adult development. The stimulus of low temperature was recorded even in the absence of the brain, because chilling shortened the pupal duration in brainless individuals. The brain showed activity which affected subsequent adult development in chilled pupae within one day after chilling in males. This period was less limited in females. Key Word Index: Pupal diapause. adult development, brain, prothoracicotropic
INTRODUCI’ION
Pupilio xuthus L. (Lepidoptera: Papilionidae), exhibits a facultative pupal diapause, which is induced by a short-day photoperiod during the larval stage (Ishizaki and Kate, 1956; Ishizaki, 1958). The critical daylength for inducing diapause is a little shorter than 13 h at 24°C (Hidaka and Hirai, 1970). Although the cold stimulus is not always necessary for terminating diapause, chilling for about 3 or 4 months is required to synchronize adult emergence in this species, and diapause development is little affected by photoperiod (IchinosC, 1974). Therefore, diapause development in P. xuthus was expected to be the process of restoring the ability of the brain to secrete prothoracicotropic hormone (PTTH) at low temperature, as Williams (1946, 1947, 1952) first showed in Hyalophora cecropia. The brains of mature larvae destined to become non-diapausing pupae initiated adult development, when implanted into diapausing pupae (Ichikawa and Ishizaki, 1958). However, adult development after chilling was not hindered by the decapitation of diapausing pupae immediately after pupation (Ozeki, 1954). Ozeki (1954) concluded that the brain exerts no effect on the prothoracic gland of pupae in P. xuthus. In the present paper, adult development in brainless pupae of P. xuthus was re-examined and the role of the brain in post-diapause adult development is discussed.
The swallowtail,
MATERIALS
AND METHODS
Female adults of P. xuthus were collected in May 1981 or 1982 in the Osaka prefecture. Eggs laid by these females were incubated at 25 + 1.5”C under a
hormone, swallowtail
photoregime of 12-h photophase and 12-h scotophase (12L: 12D). Larvae were reared on fresh leaves of Poncirus trifoliata under the same conditions, which induces pupal diapause in this species (Hidaka and Hirai, 1970). The diapausing pupae thus obtained were used for the experiments conducted in a constant temperature room (25 f l.S”C) except the chilling procedure (4 f 0S”C). Pupae were divided into 2 groups at pupation. One was kept at a constant 25°C (unchilled group), and the other was kept under 16L:8D at 25°C for 12 days and chilled under lOL:14D at 4°C for 100 days, then returned to 25°C (chilled group). Evidence concerning the neurosecretory processes implies that PTTH is released from the corpora cardiaca. However, the release site of cerebral neurosecretory products can be located in organs other than the corpora cardiaca (Raabe, 1982). In vitro assay for PTTH has demonstrated unequivocally that the corpora allata are the neurohaemal organ for PTTH in Mnndwca sexta (Agui et al., 1980). Therefore, operations were carried out as follows: The and brain, suboesophageal ganglion corpus cardiacurn-allatum complex were extirpated altogether from ether-anesthetized pupae, and the wound was sealed with melted paraffin wax. Pupae were inspected daily for 200 days (unchilled group) or 30 days (chilled group) to record adult emergence. Although most of the operated adults failed to emerge from the exuviae by their own efforts, the adult abdomen could be seen to shrink and separate from the pupal cuticle after the adult body was fully formed. The day when the space was first observed between the adult abdomen and the pupal cuticle, was regarded as the day of emergence in those adults. Statistical analyses were carried out by Mann-Whitney U test.
HIDEHARU NUMATAand T~SHITAKA HIDAKA
166
50
100
Days
150 after
200 RD
pupation
Fig. I. Pupal duration in diapausing P. xufhus under 16L:SD and 1OL:14D at 25°C without chilling. Each block represents emergence of an adult (open, male; hatched. female). RD, remain diapausing.
50
100 Days after
150
200 RD
pupation
Fig. 3. Pupal duration in operated diapausing P. xufhtrs under 16L:SD at 25°C without chilling. Operations were carried out 2 days after pupation. Each block represents emergence of an adult (open, male; hatched, female). RD, remain diapausing.
RESULTS Effect of temperature and photoperiod
Two photoperiodic regimes, a long-day photo(16L: SD) and a short-day photoregime (IOL: 14D), were employed at 25°C in both unchilled and chilled groups. Both male and female adults emerged sporadically S&180 days after pupation in the unchilled group (Fig. 1). In the chilled group, adult emergence was more synchronous than in the unchilled group: Most adults emerged between 10 and 30 days after transfer to 25°C except for a few females. Males emerged significantly earlier (P < 0.01 under each photoregime) and more synchronously than females in this group (Fig. 2). No significant difference was observed in pupal duration between pupae kept under a long-day photoregime and those under a short-day photoregime. Chilling was effective in shortening the pupal duration and synchronizing adult emergence, although the photoregime had little effect on diapause development. Therefore, only a long-day photoregime was employed at 25°C in following experiments. regime
E#ect of brain removal
Brain removal was carried out two days after pupation in the unchilled and chilled groups. In the unchilled group, pupal duration of brainless individuals was significantly longer than that of shamoperated individuals (P < 0.01 in males, P < 0.05 in females), and adult development did not occur in 3 males and 4 females for 200 days (Fig. 3). In the chilled group, the emergence of brainless adults was
a little delayed and not synchronous as compared with that of sham-operated adults, although the difference between these groups was not statistically significant (P = 0.11 in males, P = 0.07 in females). However, almost all of the brainless individuals emerged within 30 days from transfer to 25°C (Fig. 4). Thus, the brain is considered to play a role in shortening the pupal duration and synchronizing adult emergence in unchilled and chilled pupae, although it was not essential for post-diapause adult development. No significant difference was observed in pupal duration between untreated and sham-operated individuals in the unchilled group and chilled females. In chilled males, untreated adults emerged significantly earlier than sham-operated individuals (P < 0.05) (Figs 14). Active period of the brain
In order to clarify the period when the brain affects adult development, decapitation was carried out in the chilled group, immediately (Group A), one day (Group B) or two days (group C) after transfer to 25°C from 4°C. The pupal duration of group A was almost equal to that of individuals with brains removed two days after pupation (P = 0.60 in males, P = 0.40 in females) (Figs 4, 5). Adult males of group A emerged significantly earlier than those of group B (P < O.Ol),
m-El-
-K 0
20
10 Days
after
30 RD
-
0
10
Days after
20
30 RD
chrlllng
chilling
Fig. 2. Pupal duration in diapausing P. xurhus under 16L:8D and 1OL: 14D at 25°C after chilling. Each block represents emergence of an adult (open, male; hatched, female). RD, remain diapausing.
Fig. 4. Pupal duration in operated diapausing P. xurhus under 16L:SD at 25°C after chilling. Operations were carried out 2 days after pupation (before chilling). Each block represents emergence of an adult (open, male; hatched, female). RD, remain diapausing.
Brain and pupal diapause in Papilio
0
20
10 Days
after
30 RD
chllllng
Fig. 5. Pupal duration in decapitated diapausing P. xurhus under 16L:8D at 25°C after chilling. Decapitation was carried out 0, 1 or 2 days after chilling (arrows). Each block represents emergence of an adult (open, male; hatched, female). RD, remain diapausing.
although there was no significant difference in pupal duration of females between group A and Group B (P = 0.62). No significant difference was observed in pupal duration between group B and group C (P = 0.45 in males, P = 0.83 in females) (Fig. 5). In males, the pupal duration of group B or group C was almost equal to that of sham-operated individuals (P = 0.41, P = 0.85 respectively). In females, the pupal duration of group B or group C was even a little longer than that of sham-operated individuals, although the difference was not statistically significant (P = 0.18, P = 0.26 respectively) (Figs 4, 5). Thus, the period when the brain shows activity which affects the subsequent adult development, perhaps release of PT’TH, is within 1 day after chilling in males, and less limited in females. DISCUSSION
In the classical scheme of endocrine control of moulting and metamorphosis in insects, neurosecretory cells in the brain produce PTTH which stimulates the secretion of ecdysone from the prothoracic glands (Williams, 1952; Gilbert and King, 1973). Diapause in the pupa is considered to result from an ecdysone deficiency due to the temporary failure of the brain in secreting PTTH (Gilbert and King, 1973; Novak, 1975). Brainless H. cecropia failed to initiate adult development regardless of whether the operation was carried out in unchilled pupae (Williams, 1946) or promptly after being chilled (Williams, 1956). In P. xuthus, however, Ozeki (1954) concluded that the brain exerts no effects on the prothoracic glands of pupae, because adult development was completed in both diapausing and non-diapausing pupae which were decapitated immediately after pupation. In nondiapausing pupae of this species, the critical period for PTTH secretion may be before pupation as in the
167
cases of species incapable of pupal diapause (see Williams, 1952). In diapausing pupae, the brain plays a role in shortening the pupal duration and synchronizing the adult emergence, perhaps by PTTH secretion, although it is not essential for postdiapause adult development (Figs 3, 4). Adult development following brain removal, which is not consistent with the classical scheme, has been demonstrated in diapausing pupae of Antheraeapolyphernus (McDaniel and Berry, 1967). Pieris brassicae (Maslennikova, 1970), M. sexta (Judy, 1972; Wilson and Larsen, 1974) and Acronycta rumicis (Kind, 1978). Diapause termination in brainless pupae after a period of developmental arrest artificially induced by brain removal was also reported in Bombyx mori, which is incapable of pupal diapause in nature (Kobayashi et al., 1960; Ishizaki, 1972). Hypotheses to explain these results have been proposed by a number of investigators: (1) brain extirpation only temporarily suppresses prothoracic gland activity, and the gland may spontaneously become active after some period of time (Wigglesworth, 1970; Wilson and Larsen, 1974; (2) ventral nerve cord ganglia may contain cells capable of producing PTTH (Judy, 1972; Gilbert and King, 1973; Novak, 1975); (3) injury stimulates the prothoracic gland activity which initiates adult development (McDaniel and Berry, 1967; Wilson and Larsen, 1974); (4) juvenile hormone or PTTH released from the corpora allata stimulates prothoracic gland activity (Kobayashi et al.. 1960; Raabe, 1982). In the present experiments, the corpora allata were also removed with the brain. Therefore, the adult development in brainless pupae can not be attributed to stimulation by the corpora allata. Furthermore, the eXect of injury stimulating diapause termination was not observed in these experiments (Figs 14). It has been examined, whether the functioning of the prothoracic gland is regulated, apart from by the cerebral brain hormone, by nerve impulses and neurosecretory products originating in certain ventral ganglia (see Raabe, 1982 for review). However, the experiments performed on Lepidoptera by certain authors did not substantiate the existence of PTTH activity in the ventral nerve cord (Gibbs and Riddiford, 1977; Ma16 et al., 1977; Srivastava et al., 1977; Safranek and Williams, 1980). Ecdysteroid secretion without PTTH stimulation has been shown by radioimmunoassay. Detectable concentrations in H. cecropia pupae in the absence of brain, corpora cardiaca and allata (McDaniel, 1979) and haemolymph peaks in headless larvae of Calpodes ethlius (Dean and Steel, 1982) were reported. Thus, the first hypothesis appears to be likely for adult development in brainless pupae of P. xuthus, although the second possibility cannot be denied from the present results. Environmental stimuli (temperature or photoperiod) act directly on the brain to modulate PTTH secretion and thereby to control the termination of pupal diapause in H. cecropia (Williams, 1946, 1956) and in Antheraea pernyi (Williams and Adkisson, 1964). In P. xuthus, however, low temperature treatment remarkably shortened pupal duration not only in untreated or sham-operated individuals but also in brainless individuals (Figs 14). It follows that in this species.
the
stimulus
of low
temperature
can
be
HIDEHARUNUMATA and TOSHITAKAHIDAKA
168 perceived
even in the absence of the brain. Low temperature-initiated adult development is also seen in brainless diapausing pupae of A. poiyphemus (McDaniel and Berry, 1967) and A. rumicis (Kind, 1978) and B. mori tmoae artificiallv induced to diauause bv brain remoial’ (Kobayashi and Burdette, 1963). These authors conclude that the stimulus of low temperature activated the prothoracic glands of brainless pupae. However, in these species, a considerable proportion of the brainless pupae remained in diapause after chilling, although in P. xuthus, almost all the brainless individuals emerged within 30 days after chilling (Fig. 4). In P. xuthus, probably the prothoracic gland itself can perceive the stimulus of low temperature and begin to secrete ecdysone. The brain plays a role in synchronizing adult emergence by releasing PTTH after chilling. On the other hand, the brain is not concerned with the reception of environmental stimuli for the termination of pupal diapause in Heliothis zea and Heliothis punctiger. In diapausing pupae of these species. PTTH is released around the time of pupation and potentiates the prothoracic gland to function. which is then activated by exposure to high temperature (Meola and Adkisson, 1977; Browning, 198 1). It is still an open question whether PTTH release around the time of pupation also may potentiate the prothoracic gland in P. xuthus. Acknowledgement-We thank Dr Y. Kono, Pesticide Research Laboratory, Takeda Chemical Industries Ltd, for his kind advice. REFERENCES
Agui N.. Bohenbacher W. E., Granger N. A. and Gilbert L. 1. (1980) Corpus allatum is release site for insect prothoracicotropic hormone. Nature 285, 669-670. Browning T. 0. (1981) Ecdysteroids and diapause in pupae of Heliolhis ounctiaer. J. Insect Phvsiol. 21. 715-719. Dean R. L. and Steel C. G. H. (1982) Haemolymphecdysteroid peaks occur in headless larvae of Culpodes rfhlius J. Insect Physiol. 28, 2299231. Gibbs D. and Riddiford L. M. (1977) Prothoracicotropic hormone in Manduca sexta: localization by a larval assay. J. e.rp. Biol. 66, 255-266. Gilbert L. 1. and King D. S. (1973) Physiology of growth and development: endocrine aspects. In The Physiology of’ Insecra 2nd edn (Ed. by Rockstein M.). Vol. I. pp. 249-370. Academic Press, New York. Hidaka T. and Hirai Y. (1970) Effect of non-24-hour photoperiod and light interruption of the dark phase on diapause determination in Pupilio xuthus L. Proc. Japan Acad. 46, 541-545. lchikawa M. and Ishizaki H. (1958) Further study on the physiological role of brain in the imaginal differentiation of swallowtail, Papilio xuthus. Mem. CON. Sri. Linir. Kyoto B 25, I I-16. Ichinose T. (1974) Pupal diapause in some Japanese papilionid butterflies, with special reference to the difference in photoperiodic response between the diapausing pupae of Papilio maackii tutanus Fenton and P. xuthus Linnaeus. Konryi. Tokyo 42, 439450. Ishizaki H. (1958) Correlation between colour variation and diapause in the pupa of swallowtail, Papilio xuthus. Phwiol. Ecol. Japan 8, 32-35 (in Japanese with English summary). Ishizaki H. (1972) Arrest of adult development in debrained pupae of the silkworm, Bombyx mori. J. Insect Physiol. 18, 1621-1627.
Ishizaki H. and Kato M. (1956) Environmental factors affecting the formation of orange pupa in Papilio xurhus. Mem. COB. Sci. Unity. Kyoto B 23, 1I-18. Judy K. J. (1972) Diapause termination and metamorphosis in brainless tobacco homworms (Lepidoptera). Life Sci. II, 6055611. Kind T. V. (1978) A study of the reactivation of diapausing pupae of Acronyctn rumicis L. (Lepidoptera, Noctuidae). I. Cold activation of the prothoracic glands in brainless pupae. Em. Reo. 56, 13-16. Kobavashi M. and Burdette W. J. (1963) Effect of low temperature on the induction of imaginal differentiation of dauer pupa of the silkworm. Nature 199, 826827. Kobayashi M., Fukaya M. and Mitsuhashi 3. (1960) Imaginal differentiation of “Dauer-pupae” in the silkworm, Bombyx mori. J. seric. Sci. Japan 29,337-340 (in Japanese with English summary). Mali J.. Granger N. A. and Sehnal F. (1977) Control of prothoracic gland activity in larvae of Galleria mellonella. J. Insect Phjsiol. 23, 309-316. Maslennikova V. A. (1970) On the nroblem of hormonal regulation of diapause in. Pieris brassicae L. Dokl. Akad. Nuuk SSSR 192, 942-945 (in Russian). McDaniel C. N. (1979) Haemolymph ecdysone concentrations in Hyafophora cecropia pupae. dauer pupae and adults. J. Insect Physiol. 25, 143-145. McDaniel C. N. and Berry S. J. (1967) Activation of the prothoracic glands of Antheraea polyphemus. Nature 214, 1032-1034. Meola R. W. and Adkisson P. L. (1977) Release of prothoracicotropic hormone and potentiation of developmental ability during diapause in the bollworm. Heliothis zea. J. Insect Physiol. 23, 683-688. Novak V. J. A. (1975) Insect Hormones 2nd English edn. Chapman and Hall, London. Ozeki K. (1954) Experiments on the formation of imaginal structures in the pupae of the swallowtail, Papilio xuthus Linnaeus. Sri. Pap. Coll. Gen. Educ. Lrnh~. Tokyo 4, 47-56. Raabe M. (I 982) Insecf Neurohormones. Plenum Press, New York. Safranek L. and Williams C. M. (1980) Studies on the prothoracicotropic hormone in the tobacco hornworm. Manduca sexta. Biol. Bull. 158, 141-153. Srivastava K. P., Tiwari R. K. and Kumar P. (1977) Effect of sectioning the prothoracic gland nerves in the larva of the lemon-butterfly, Papilio demoleus L. Experientia 33, 98-99. Wigglesworth V. B. (1970) Insect Hormones. Oliver and Boyd, Edinburgh. Wilson G. R. and Larsen J. R. (1974) Debraining and diapause development inManduca sevta pupae. J. Insect Physiol. 20, 2459-2413. Williams C. M. (1946) Physiology of insect diapause: the role of the brain in the production and termination of pupal dormancy in the giant silkworm, Plutysamia cecropia. Biol. Bull. 90, 234243. Williams C. M. (1947) Physiology of insect diapause. II. Interaction between the pupal brain and prothoracic glands in the metamorphosis of the giant silkworm, Platysamia cecropia. Biol. Bull. 93, 89-98. Williams C. M. (1952) Physiology of insect diapause. IV. The brain and prothoracic glands as an endocrine system in the cecropia silkworm. Biol. Bull. 103, 12&138. Williams C. M. (1956) Physiology of insect diapause. X. An endocrine mechanism for the influence of temperature on the diapausing pupa of the cecropia silkworm. Biol. Bull. l10,201-218. Williams C. M. and Adkisson P. L. (1964) Physiology of insect diapause. XIV. An endocrine mechanism for the photoperiodic control of pupal diapause in the oak silkworm. Aniheraea pernyi. Biol. Bull. 127, 51 l-525.
J. Insect fh.wio/. Vol. 30, No. 2. pp. 165-168. 1984 Printedin Great Britain. All rights reserved.
ROLE OF THE BRAIN IN POST-DIAPAUSE ADULT DEVELOPMENT IN THE SWALLOWTAIL, PAPILIO HIDEHARU NUMATA
XUTHUS and
TOSHITAKA HIDAKA
Department of Zoology, Faculty of Science, Kyoto University, Sakyo, Kyoto 606, Japan (Received 6 July 1983)
Abstract-The role of the brain in adult development was examined by brain removal in unchilled and chilled diapausing pupae of Pupilio xuthus. Chilling was effective in shortening the pupal duration and synchronizing adult emergence, although photoperiod had little effect on diapause development. The brain played a role of shortening the pupal duration and synchronizing adult emergence in both unchilled and
chilled individuals, although it was not essential for post-diapause adult development. The stimulus of low temperature was recorded even in the absence of the brain, because chilling shortened the pupal duration in brainless individuals. The brain showed activity which affected subsequent adult development in chilled pupae within one day after chilling in males. This period was less limited in females. Key Word Index: Pupal diapause. adult development, brain, prothoracicotropic
INTRODUCI’ION
Pupilio xuthus L. (Lepidoptera: Papilionidae), exhibits a facultative pupal diapause, which is induced by a short-day photoperiod during the larval stage (Ishizaki and Kate, 1956; Ishizaki, 1958). The critical daylength for inducing diapause is a little shorter than 13 h at 24°C (Hidaka and Hirai, 1970). Although the cold stimulus is not always necessary for terminating diapause, chilling for about 3 or 4 months is required to synchronize adult emergence in this species, and diapause development is little affected by photoperiod (IchinosC, 1974). Therefore, diapause development in P. xuthus was expected to be the process of restoring the ability of the brain to secrete prothoracicotropic hormone (PTTH) at low temperature, as Williams (1946, 1947, 1952) first showed in Hyalophora cecropia. The brains of mature larvae destined to become non-diapausing pupae initiated adult development, when implanted into diapausing pupae (Ichikawa and Ishizaki, 1958). However, adult development after chilling was not hindered by the decapitation of diapausing pupae immediately after pupation (Ozeki, 1954). Ozeki (1954) concluded that the brain exerts no effect on the prothoracic gland of pupae in P. xuthus. In the present paper, adult development in brainless pupae of P. xuthus was re-examined and the role of the brain in post-diapause adult development is discussed.
The swallowtail,
MATERIALS
AND METHODS
Female adults of P. xuthus were collected in May 1981 or 1982 in the Osaka prefecture. Eggs laid by these females were incubated at 25 + 1.5”C under a
hormone, swallowtail
photoregime of 12-h photophase and 12-h scotophase (12L: 12D). Larvae were reared on fresh leaves of Poncirus trifoliata under the same conditions, which induces pupal diapause in this species (Hidaka and Hirai, 1970). The diapausing pupae thus obtained were used for the experiments conducted in a constant temperature room (25 f l.S”C) except the chilling procedure (4 f 0S”C). Pupae were divided into 2 groups at pupation. One was kept at a constant 25°C (unchilled group), and the other was kept under 16L:8D at 25°C for 12 days and chilled under lOL:14D at 4°C for 100 days, then returned to 25°C (chilled group). Evidence concerning the neurosecretory processes implies that PTTH is released from the corpora cardiaca. However, the release site of cerebral neurosecretory products can be located in organs other than the corpora cardiaca (Raabe, 1982). In vitro assay for PTTH has demonstrated unequivocally that the corpora allata are the neurohaemal organ for PTTH in Mnndwca sexta (Agui et al., 1980). Therefore, operations were carried out as follows: The and brain, suboesophageal ganglion corpus cardiacurn-allatum complex were extirpated altogether from ether-anesthetized pupae, and the wound was sealed with melted paraffin wax. Pupae were inspected daily for 200 days (unchilled group) or 30 days (chilled group) to record adult emergence. Although most of the operated adults failed to emerge from the exuviae by their own efforts, the adult abdomen could be seen to shrink and separate from the pupal cuticle after the adult body was fully formed. The day when the space was first observed between the adult abdomen and the pupal cuticle, was regarded as the day of emergence in those adults. Statistical analyses were carried out by Mann-Whitney U test.
HIDEHARU NUMATAand T~SHITAKA HIDAKA
166
50
100
Days
150 after
200 RD
pupation
Fig. I. Pupal duration in diapausing P. xufhus under 16L:SD and 1OL:14D at 25°C without chilling. Each block represents emergence of an adult (open, male; hatched. female). RD, remain diapausing.
50
100 Days after
150
200 RD
pupation
Fig. 3. Pupal duration in operated diapausing P. xufhtrs under 16L:SD at 25°C without chilling. Operations were carried out 2 days after pupation. Each block represents emergence of an adult (open, male; hatched, female). RD, remain diapausing.
RESULTS Effect of temperature and photoperiod
Two photoperiodic regimes, a long-day photo(16L: SD) and a short-day photoregime (IOL: 14D), were employed at 25°C in both unchilled and chilled groups. Both male and female adults emerged sporadically S&180 days after pupation in the unchilled group (Fig. 1). In the chilled group, adult emergence was more synchronous than in the unchilled group: Most adults emerged between 10 and 30 days after transfer to 25°C except for a few females. Males emerged significantly earlier (P < 0.01 under each photoregime) and more synchronously than females in this group (Fig. 2). No significant difference was observed in pupal duration between pupae kept under a long-day photoregime and those under a short-day photoregime. Chilling was effective in shortening the pupal duration and synchronizing adult emergence, although the photoregime had little effect on diapause development. Therefore, only a long-day photoregime was employed at 25°C in following experiments. regime
E#ect of brain removal
Brain removal was carried out two days after pupation in the unchilled and chilled groups. In the unchilled group, pupal duration of brainless individuals was significantly longer than that of shamoperated individuals (P < 0.01 in males, P < 0.05 in females), and adult development did not occur in 3 males and 4 females for 200 days (Fig. 3). In the chilled group, the emergence of brainless adults was
a little delayed and not synchronous as compared with that of sham-operated adults, although the difference between these groups was not statistically significant (P = 0.11 in males, P = 0.07 in females). However, almost all of the brainless individuals emerged within 30 days from transfer to 25°C (Fig. 4). Thus, the brain is considered to play a role in shortening the pupal duration and synchronizing adult emergence in unchilled and chilled pupae, although it was not essential for post-diapause adult development. No significant difference was observed in pupal duration between untreated and sham-operated individuals in the unchilled group and chilled females. In chilled males, untreated adults emerged significantly earlier than sham-operated individuals (P < 0.05) (Figs 14). Active period of the brain
In order to clarify the period when the brain affects adult development, decapitation was carried out in the chilled group, immediately (Group A), one day (Group B) or two days (group C) after transfer to 25°C from 4°C. The pupal duration of group A was almost equal to that of individuals with brains removed two days after pupation (P = 0.60 in males, P = 0.40 in females) (Figs 4, 5). Adult males of group A emerged significantly earlier than those of group B (P < O.Ol),
m-El-
-K 0
20
10 Days
after
30 RD
-
0
10
Days after
20
30 RD
chrlllng
chilling
Fig. 2. Pupal duration in diapausing P. xurhus under 16L:8D and 1OL: 14D at 25°C after chilling. Each block represents emergence of an adult (open, male; hatched, female). RD, remain diapausing.
Fig. 4. Pupal duration in operated diapausing P. xurhus under 16L:SD at 25°C after chilling. Operations were carried out 2 days after pupation (before chilling). Each block represents emergence of an adult (open, male; hatched, female). RD, remain diapausing.
Brain and pupal diapause in Papilio
0
20
10 Days
after
30 RD
chllllng
Fig. 5. Pupal duration in decapitated diapausing P. xurhus under 16L:8D at 25°C after chilling. Decapitation was carried out 0, 1 or 2 days after chilling (arrows). Each block represents emergence of an adult (open, male; hatched, female). RD, remain diapausing.
although there was no significant difference in pupal duration of females between group A and Group B (P = 0.62). No significant difference was observed in pupal duration between group B and group C (P = 0.45 in males, P = 0.83 in females) (Fig. 5). In males, the pupal duration of group B or group C was almost equal to that of sham-operated individuals (P = 0.41, P = 0.85 respectively). In females, the pupal duration of group B or group C was even a little longer than that of sham-operated individuals, although the difference was not statistically significant (P = 0.18, P = 0.26 respectively) (Figs 4, 5). Thus, the period when the brain shows activity which affects the subsequent adult development, perhaps release of PT’TH, is within 1 day after chilling in males, and less limited in females. DISCUSSION
In the classical scheme of endocrine control of moulting and metamorphosis in insects, neurosecretory cells in the brain produce PTTH which stimulates the secretion of ecdysone from the prothoracic glands (Williams, 1952; Gilbert and King, 1973). Diapause in the pupa is considered to result from an ecdysone deficiency due to the temporary failure of the brain in secreting PTTH (Gilbert and King, 1973; Novak, 1975). Brainless H. cecropia failed to initiate adult development regardless of whether the operation was carried out in unchilled pupae (Williams, 1946) or promptly after being chilled (Williams, 1956). In P. xuthus, however, Ozeki (1954) concluded that the brain exerts no effects on the prothoracic glands of pupae, because adult development was completed in both diapausing and non-diapausing pupae which were decapitated immediately after pupation. In nondiapausing pupae of this species, the critical period for PTTH secretion may be before pupation as in the
167
cases of species incapable of pupal diapause (see Williams, 1952). In diapausing pupae, the brain plays a role in shortening the pupal duration and synchronizing the adult emergence, perhaps by PTTH secretion, although it is not essential for postdiapause adult development (Figs 3, 4). Adult development following brain removal, which is not consistent with the classical scheme, has been demonstrated in diapausing pupae of Antheraeapolyphernus (McDaniel and Berry, 1967). Pieris brassicae (Maslennikova, 1970), M. sexta (Judy, 1972; Wilson and Larsen, 1974) and Acronycta rumicis (Kind, 1978). Diapause termination in brainless pupae after a period of developmental arrest artificially induced by brain removal was also reported in Bombyx mori, which is incapable of pupal diapause in nature (Kobayashi et al., 1960; Ishizaki, 1972). Hypotheses to explain these results have been proposed by a number of investigators: (1) brain extirpation only temporarily suppresses prothoracic gland activity, and the gland may spontaneously become active after some period of time (Wigglesworth, 1970; Wilson and Larsen, 1974; (2) ventral nerve cord ganglia may contain cells capable of producing PTTH (Judy, 1972; Gilbert and King, 1973; Novak, 1975); (3) injury stimulates the prothoracic gland activity which initiates adult development (McDaniel and Berry, 1967; Wilson and Larsen, 1974); (4) juvenile hormone or PTTH released from the corpora allata stimulates prothoracic gland activity (Kobayashi et al.. 1960; Raabe, 1982). In the present experiments, the corpora allata were also removed with the brain. Therefore, the adult development in brainless pupae can not be attributed to stimulation by the corpora allata. Furthermore, the eXect of injury stimulating diapause termination was not observed in these experiments (Figs 14). It has been examined, whether the functioning of the prothoracic gland is regulated, apart from by the cerebral brain hormone, by nerve impulses and neurosecretory products originating in certain ventral ganglia (see Raabe, 1982 for review). However, the experiments performed on Lepidoptera by certain authors did not substantiate the existence of PTTH activity in the ventral nerve cord (Gibbs and Riddiford, 1977; Ma16 et al., 1977; Srivastava et al., 1977; Safranek and Williams, 1980). Ecdysteroid secretion without PTTH stimulation has been shown by radioimmunoassay. Detectable concentrations in H. cecropia pupae in the absence of brain, corpora cardiaca and allata (McDaniel, 1979) and haemolymph peaks in headless larvae of Calpodes ethlius (Dean and Steel, 1982) were reported. Thus, the first hypothesis appears to be likely for adult development in brainless pupae of P. xuthus, although the second possibility cannot be denied from the present results. Environmental stimuli (temperature or photoperiod) act directly on the brain to modulate PTTH secretion and thereby to control the termination of pupal diapause in H. cecropia (Williams, 1946, 1956) and in Antheraea pernyi (Williams and Adkisson, 1964). In P. xuthus, however, low temperature treatment remarkably shortened pupal duration not only in untreated or sham-operated individuals but also in brainless individuals (Figs 14). It follows that in this species.
the
stimulus
of low
temperature
can
be
HIDEHARUNUMATA and TOSHITAKAHIDAKA
168 perceived
even in the absence of the brain. Low temperature-initiated adult development is also seen in brainless diapausing pupae of A. poiyphemus (McDaniel and Berry, 1967) and A. rumicis (Kind, 1978) and B. mori tmoae artificiallv induced to diauause bv brain remoial’ (Kobayashi and Burdette, 1963). These authors conclude that the stimulus of low temperature activated the prothoracic glands of brainless pupae. However, in these species, a considerable proportion of the brainless pupae remained in diapause after chilling, although in P. xuthus, almost all the brainless individuals emerged within 30 days after chilling (Fig. 4). In P. xuthus, probably the prothoracic gland itself can perceive the stimulus of low temperature and begin to secrete ecdysone. The brain plays a role in synchronizing adult emergence by releasing PTTH after chilling. On the other hand, the brain is not concerned with the reception of environmental stimuli for the termination of pupal diapause in Heliothis zea and Heliothis punctiger. In diapausing pupae of these species. PTTH is released around the time of pupation and potentiates the prothoracic gland to function. which is then activated by exposure to high temperature (Meola and Adkisson, 1977; Browning, 198 1). It is still an open question whether PTTH release around the time of pupation also may potentiate the prothoracic gland in P. xuthus. Acknowledgement-We thank Dr Y. Kono, Pesticide Research Laboratory, Takeda Chemical Industries Ltd, for his kind advice. REFERENCES
Agui N.. Bohenbacher W. E., Granger N. A. and Gilbert L. 1. (1980) Corpus allatum is release site for insect prothoracicotropic hormone. Nature 285, 669-670. Browning T. 0. (1981) Ecdysteroids and diapause in pupae of Heliolhis ounctiaer. J. Insect Phvsiol. 21. 715-719. Dean R. L. and Steel C. G. H. (1982) Haemolymphecdysteroid peaks occur in headless larvae of Culpodes rfhlius J. Insect Physiol. 28, 2299231. Gibbs D. and Riddiford L. M. (1977) Prothoracicotropic hormone in Manduca sexta: localization by a larval assay. J. e.rp. Biol. 66, 255-266. Gilbert L. 1. and King D. S. (1973) Physiology of growth and development: endocrine aspects. In The Physiology of’ Insecra 2nd edn (Ed. by Rockstein M.). Vol. I. pp. 249-370. Academic Press, New York. Hidaka T. and Hirai Y. (1970) Effect of non-24-hour photoperiod and light interruption of the dark phase on diapause determination in Pupilio xuthus L. Proc. Japan Acad. 46, 541-545. lchikawa M. and Ishizaki H. (1958) Further study on the physiological role of brain in the imaginal differentiation of swallowtail, Papilio xuthus. Mem. CON. Sri. Linir. Kyoto B 25, I I-16. Ichinose T. (1974) Pupal diapause in some Japanese papilionid butterflies, with special reference to the difference in photoperiodic response between the diapausing pupae of Papilio maackii tutanus Fenton and P. xuthus Linnaeus. Konryi. Tokyo 42, 439450. Ishizaki H. (1958) Correlation between colour variation and diapause in the pupa of swallowtail, Papilio xuthus. Phwiol. Ecol. Japan 8, 32-35 (in Japanese with English summary). Ishizaki H. (1972) Arrest of adult development in debrained pupae of the silkworm, Bombyx mori. J. Insect Physiol. 18, 1621-1627.
Ishizaki H. and Kato M. (1956) Environmental factors affecting the formation of orange pupa in Papilio xurhus. Mem. COB. Sci. Unity. Kyoto B 23, 1I-18. Judy K. J. (1972) Diapause termination and metamorphosis in brainless tobacco homworms (Lepidoptera). Life Sci. II, 6055611. Kind T. V. (1978) A study of the reactivation of diapausing pupae of Acronyctn rumicis L. (Lepidoptera, Noctuidae). I. Cold activation of the prothoracic glands in brainless pupae. Em. Reo. 56, 13-16. Kobavashi M. and Burdette W. J. (1963) Effect of low temperature on the induction of imaginal differentiation of dauer pupa of the silkworm. Nature 199, 826827. Kobayashi M., Fukaya M. and Mitsuhashi 3. (1960) Imaginal differentiation of “Dauer-pupae” in the silkworm, Bombyx mori. J. seric. Sci. Japan 29,337-340 (in Japanese with English summary). Mali J.. Granger N. A. and Sehnal F. (1977) Control of prothoracic gland activity in larvae of Galleria mellonella. J. Insect Phjsiol. 23, 309-316. Maslennikova V. A. (1970) On the nroblem of hormonal regulation of diapause in. Pieris brassicae L. Dokl. Akad. Nuuk SSSR 192, 942-945 (in Russian). McDaniel C. N. (1979) Haemolymph ecdysone concentrations in Hyafophora cecropia pupae. dauer pupae and adults. J. Insect Physiol. 25, 143-145. McDaniel C. N. and Berry S. J. (1967) Activation of the prothoracic glands of Antheraea polyphemus. Nature 214, 1032-1034. Meola R. W. and Adkisson P. L. (1977) Release of prothoracicotropic hormone and potentiation of developmental ability during diapause in the bollworm. Heliothis zea. J. Insect Physiol. 23, 683-688. Novak V. J. A. (1975) Insect Hormones 2nd English edn. Chapman and Hall, London. Ozeki K. (1954) Experiments on the formation of imaginal structures in the pupae of the swallowtail, Papilio xuthus Linnaeus. Sri. Pap. Coll. Gen. Educ. Lrnh~. Tokyo 4, 47-56. Raabe M. (I 982) Insecf Neurohormones. Plenum Press, New York. Safranek L. and Williams C. M. (1980) Studies on the prothoracicotropic hormone in the tobacco hornworm. Manduca sexta. Biol. Bull. 158, 141-153. Srivastava K. P., Tiwari R. K. and Kumar P. (1977) Effect of sectioning the prothoracic gland nerves in the larva of the lemon-butterfly, Papilio demoleus L. Experientia 33, 98-99. Wigglesworth V. B. (1970) Insect Hormones. Oliver and Boyd, Edinburgh. Wilson G. R. and Larsen J. R. (1974) Debraining and diapause development inManduca sevta pupae. J. Insect Physiol. 20, 2459-2413. Williams C. M. (1946) Physiology of insect diapause: the role of the brain in the production and termination of pupal dormancy in the giant silkworm, Plutysamia cecropia. Biol. Bull. 90, 234243. Williams C. M. (1947) Physiology of insect diapause. II. Interaction between the pupal brain and prothoracic glands in the metamorphosis of the giant silkworm, Platysamia cecropia. Biol. Bull. 93, 89-98. Williams C. M. (1952) Physiology of insect diapause. IV. The brain and prothoracic glands as an endocrine system in the cecropia silkworm. Biol. Bull. 103, 12&138. Williams C. M. (1956) Physiology of insect diapause. X. An endocrine mechanism for the influence of temperature on the diapausing pupa of the cecropia silkworm. Biol. Bull. l10,201-218. Williams C. M. and Adkisson P. L. (1964) Physiology of insect diapause. XIV. An endocrine mechanism for the photoperiodic control of pupal diapause in the oak silkworm. Aniheraea pernyi. Biol. Bull. 127, 51 l-525.