- Email: [email protected]
Hormonal control of diapause in the silkworm
GENERAL
AND
COMPARATIVE
ENDOCRINOLOGY,
ENDOCRINE
Hormonal
REGULATION
Control
DURING
of
Diapause
SOICHI Nishiuzuhashi,
1, 337-340 (1962)
&lpph?meDt
DEVELOPMENT
in the
II
Silkworm
FUKUDA
Matsumoto
INTRODUCTION
The production of the diapause or the nondiapause eggs in the silkworm is a genetical phenomenon. However, in the bivoltine and the quadrivoltine races of the silkworm, environmental factors such as the temperature and the light conditions during t,he period of incubation of the eggs exert an irrevocable effect on the diapausing or the nondiapausing property of the eggs to be laid by the resulting moths, whereas in the univoltine and the multivoltine races, external conditions at any developmental stages including the incubation period scarcely affect the property of the eggs. In our investigations on the mechanism involved in the determination of voltinism, therefore, the hybrids between the strain No. 106 of Japanese bivoltine race and the strain Taizo of Chinese bivoltine race were mainly used as materials for experiments. If the eggs of this hybrid are kept during the incubation period at 26°C under illumination, the resulting moths lay diapause eggs only, whereas, if the eggs are incubated at 15°C in the dark, the moths invariably produce nondiapause eggs only. In the following pages, larvae, pupae, and moths determined to produce diapause eggs only will briefly be referred to as diapause larvae, pupae and moths and those destined to produce nondiapause eggs only as nondiapause larvae, pupae and moths. 337
City,
Japan
ROLE OF PUPAL BRAIN AND SUBESOPHAGEAL GANGLION IN DETERMINATION OF DIAPAUSING AND NONDIAPAUSING PROPERTIES OF EGGS
If the brain of nondiapause pupae was surgically removed shortly after pupation! many of the resultant moths’ laid diapause eggs together with nondiapause eggs or diapause eggs only. The two types of eggs deposited by this series of brainless moths were approximately equal in number. Also, the moths which emerged from diapause pupae deprived of the brain laid nondiapause eggs only or nondiapause eggs together with diapause eggs, the two types of eggs being again about the same in number. ‘Pupae deprived of the brain are capable of transforming into moths. Whereas the normal female moth usually begins to lay eggs only after mating with a male, the brainless moth begins to deposit unfertilized eggs as soon as it finishes shedding the pupal skin. After mating, the brainless moth lays the majority of eggs in one night. The moth from pupa deprived of the subesophageal ganglion is less active than the brainless moth and cannot lay eggs even after mating. After several trials, it was found that the ligation of the body, the transection of the nerve cord, or the removal of any ganglion except for the last induces t.he deposition of eggs in these moth. In our experiments eggs were usually obtained from the moths without the subesophageal ganglion by ligating the body between the thorax and abdomen or by excising the thorax after the ligation
338
SOICHI
From these experiments, it appears evident that in the pupa, the diapausing or the nondiapausing property of the eggs developing in the ovaries is determined by the brain. However, it is unlikely that the effect of the brain on the ovarian eggs is hormonal in nature, since the autotransplantation of the brain into the pupae immediately after its removal failed to prevent the reversal of voltinism in the individuals. On the other hand, the moths which emerged from diapause and nondiapause pupae from which the subesophageal ganglion had been removed, invariably laid nondiapause eggs only. If the subesophageal ganglion was autografted into the pupae, the resultant moths produced both diapause eggs and nondiapause eggs. These results indicate that the subesophageal ganglion releases into the blood the diapause factor which acts on the developing eggs and make them diapause eggs. Furthermore, transplantation of subesophageal ganglion from nondiapause and diapause larvae into nondiapsuse ones demonstrated that there was no difference in the potency of secreting the diapause factor of the subesophageal ganglion between the two types of the silkworm. From these observations, it seems probable that, in the nondiapause pupa, the release of the diapause factor from the subesophageal ganglion is inhibited by the brain by way of the esophageal connectives, while in the diapause pupa, the brain stimulates the secretion of the factor via the esophageal connectives. This hypothesis is substantiated by the following transplantation experiments using nondiapause female larvae of the fourth instar as recipients. In the first series of experiments, many of the moths which developed from the larvae given transplants of the subesophageal ganglion alone or of the ganglion together with the isolated brain laid both diapause and nondiapause eggs, while all the moths bearing grafts of the brainsubesophageal ganglion complex with esophageal connectives intact laid nondiapause eggs only. In this series, the grafts were
FUKUDA
obtained from nondiapause donor larvae of the same age as the recipient,s. In the second series of experiments, grafts were obtained from diapause larvae of the fourth instar. Results showed that the majority of the moths having transplants of the brain-subesophageal ganglion complex laid diapause eggs, whereas many of the moths bearing grafts of the subesophageal ganglion or the ganglion plus the isolated brain produced both diapause and nondiapause eggs. In similar experiments using grafts from larvae of the univoltine race, many of the moths bearing grafts of the subesophageal ganglion alone laid diapause eggs and nondiapause eggs, while the majority of the moths carrying transplants of the brainsubesophageal ganglion complex diapause eggs only. The results were the same regardless of the incubation conditions to which the donors had been exposed during the embryonic period. On the contrary, none of the moths from larvae which had received grafts of the brain-subesophageal ganglion complex from larvae of the multivoltine race2 deposited diapause eggs, if the donors had been incubated at the temperature below 20°C. Yet, the moths given grafts of the subesophageal ganglion alone deposited both diapause and nondiapause eggs, regardless of the temperature and light conditions ta which the donors had been subjected during the incubation period. These observations seem to indicate that the subesophageal ganglion is capable of secreting the diapause factor irrespective of the genetic constitution of the silkworm and external conditions during the period of incubation. However, it appears likely that the subesophageal ganglion is not ac‘The silkworm of multivoltine race in the strict sense of the word produces nondiapause eggs only, from generation to generation, regardless of environmental conditions to which the silkworm is exposed during its development. In the silkworms used as materials in our experiments, however, if the eggs were exposed to the temperature higher than 26°C during the incubation period, the resulting moths occasionally laid diapause eggs or diapause eggs together with nondiapause eggs.
HORMOXAL
CONTROL
OF
339
DIAPAUSE
tive enough to make all eggs developing in the ovaries diapause ones. It may be that, in the pupa determined to produce diapause eggs only, the activity of the subesophageal ganglion is augmented by the brain by way of the esophageal connectives, while in the pupa destined to produce nondiapause eggs only, the brain inhibits the production or the release of the diapause factor from the subesophageal ganglion via the esophageal connectives. In the absence of the effect of the brain on the subesophageal ganglion, both diapause and nondiapause eggs are produced. Our experiments have also suggested that conditions during the incubation period exert an influence on the brain of the embryo and let it have a potency to stimulate or suppress the secretion of the diapause factor from the subesophageal ganglion during the pupal period.
better developed in comparison with the anterior part. Similar abnormal embryos were also found in the eggs deposited by the moths of the bivoltine race which had been subjected to about 20°C during the incubation period. It is conjectured that the disharmony in the development in these embryos is ascribable to the insufficiency of the diapause factor during the pupal stage. Only those eggs which have been subjected to the sufficient effect of the diapause factor during the course of the pupal period can be in the perfect diapause state after being dcposit,ed. The eggs only insufficiently influenced by the diapause fact,or cannot be perfectly dormant so that development may proceed in some part of the body of the embryos.
ABNORMAL EMBRYONIC DEVELOPMENT IN IMPERFECT DIAPAUSE EGGS
If two pupae were united in parabio,*is attaching the dorsal sides of the thoraxer together, two moths emerged from the pupae on the same day. In diapause pupanondiapause pupa combinat,ions, many of the moths from diapause pupae produced diapause eggs, while many of those from nondiapause pupae laid diapause eggs together with nondiapause eggs. The result seems to show that the diapause factor passes with the blood from diapause par:\biont to nondiapause partner. However, it was also found that, in the same parabiotic combinations, the moths from diapause parabionts occasionally laid a small number of nondiapsuse eggs in addition to diapause eggs. Even in con)binations of two diapause pupae, one or both of the resultant moths often depositrd a few nondiapause eggs together with dinpause eggs. Furt,hermore, a few of the moths which emerged from the unions of two nondiapause pupae laid a few diapaus:c eggs together with nondiapause eggs. In order t.o ascertain whether t.he operation of parabiotic union per se is effective in inducing such a reversal of voltinism, a small disc of skin was excised from the thoracic tergum in a series of pupae and replaced it by applying melted paraffin
As mentioned above, many of the moths which developed from diapause or nondiapause pupae deprived of the brain lay diapause eggs together with nondiapause eggs. Similar phenomenon has also been observed in t,he moths each given a subesophageal ganglion as a transplant during the larval or the pupal period. In several days after the deposition, t.he eggs in the same batch exhibited differences in coloration varying from pale yellowish nondiapause type to dark purplish diapause type. By dissecting the eggs less darkened than normal diapause eggs, it was frequently found that embryos contained in the eggs were deformed to varying degrees. In some embryos, the posterior part, of the digestive tract had been formed but the cephalic lobes or the anterior part of the body remained undeveloped as in the diapausing embryos. In another embryo, the posterior part of the body was well advanced in development, the clearly segmented abdomen being provided with rudiments of the abdominal legs, while the anterior part, was still dormant. A feature in common in these abnormal embryos was that the posterior part of the body was
EFFECT
OF ON
STRESSFUL VOLTINISM
STIMULI
340
SOICHI
around the cut edge. It was found that such a simple operation provoked the change in voltinism like that observed in the parabiotic experiments. These observations led the author to the assumption that the production of diapause or nondiapause eggs is affected by nonspecific stressful stimuli. Therefore, various noxious treatments, such as thermocauterization of small area of the skin on the ventral or the lateral side of t,he abdomen, removal of small pieces of the fat body, removal of one of the abdominal ganglia, transection of the nerve cord between two adjacent abdominal ganglia, injection into the abdominal cavity of adrenaline, thyradin (a thyroid preparation), or air, insertion of a piece of glass or of silkworm cocoon into the body cavity, were applied to diapause as well as nondiapause pupae. Following subjection to any one of these treatments, some of the moths from diapause pupae laid varying number of nondiapause eggs together with diapause eggs and some of those from nondiapause pupae deposited some diapause eggs besides nondiapause eggs. It may be worthy of mention that any treatment applied to nondiapause pupae from which the subesophageal ganglion had previously been removed could no longer induce the production of diapause eggs, showing that nonspecific stimuli stimulate the subesophageal ganglion to secrete the diapause factor. It has not yet been known whether the stimuli act directly on the subesophageal ganglion or through the intermediary of some center
FUKUDA
in the brain or somewhere in the central nervous system. A similar question may also arise in the case of the production of nondiapause eggs in diapause pupae after exposure to stressful stimuli. At any rate, it appears probable that the reversal of voltinism in these cases is caused by a derangement in the secretory activity of the subesophageal ganglion. DISCUSSION HASEGAWA: In 1952 and 1953, I observed that following transplantation of SG from silkworm larvae of polyvoltine, tetravoltine and bivoltine races, respectively, into larvae of tetravoltine race on the second or the third day of the 4th instar, 31.3, 63.1 and 93.8% on the average, respectively, of the eggs laid by resultant moths were diapause eggs. From this, it seems likely that the activity of SG has already been genetically determined at the larval stage so that the SG of larvae of different races are different in activity, the SG of bivoltine race larvae being most active. In some experiments, SG-brain complexes from tetravoltine moths were also used as transplants. It was found that 55.7% of eggs laid by resultant moths were diapause eggs. It seems probable that the SG-brain complex of tetravoltine animals cannot release enough diapause hormone to affect their own eggs, but if the level of the hormone becomes sufficiently high in the recipients it may exert its effects on developing eggs. FUKUDA: I suppose that the moths of the tetravoltine race you used in your experiments should have normally produced some diapause eggs.
AND
COMPARATIVE
ENDOCRINOLOGY,
ENDOCRINE
Hormonal
REGULATION
Control
DURING
of
Diapause
SOICHI Nishiuzuhashi,
1, 337-340 (1962)
&lpph?meDt
DEVELOPMENT
in the
II
Silkworm
FUKUDA
Matsumoto
INTRODUCTION
The production of the diapause or the nondiapause eggs in the silkworm is a genetical phenomenon. However, in the bivoltine and the quadrivoltine races of the silkworm, environmental factors such as the temperature and the light conditions during t,he period of incubation of the eggs exert an irrevocable effect on the diapausing or the nondiapausing property of the eggs to be laid by the resulting moths, whereas in the univoltine and the multivoltine races, external conditions at any developmental stages including the incubation period scarcely affect the property of the eggs. In our investigations on the mechanism involved in the determination of voltinism, therefore, the hybrids between the strain No. 106 of Japanese bivoltine race and the strain Taizo of Chinese bivoltine race were mainly used as materials for experiments. If the eggs of this hybrid are kept during the incubation period at 26°C under illumination, the resulting moths lay diapause eggs only, whereas, if the eggs are incubated at 15°C in the dark, the moths invariably produce nondiapause eggs only. In the following pages, larvae, pupae, and moths determined to produce diapause eggs only will briefly be referred to as diapause larvae, pupae and moths and those destined to produce nondiapause eggs only as nondiapause larvae, pupae and moths. 337
City,
Japan
ROLE OF PUPAL BRAIN AND SUBESOPHAGEAL GANGLION IN DETERMINATION OF DIAPAUSING AND NONDIAPAUSING PROPERTIES OF EGGS
If the brain of nondiapause pupae was surgically removed shortly after pupation! many of the resultant moths’ laid diapause eggs together with nondiapause eggs or diapause eggs only. The two types of eggs deposited by this series of brainless moths were approximately equal in number. Also, the moths which emerged from diapause pupae deprived of the brain laid nondiapause eggs only or nondiapause eggs together with diapause eggs, the two types of eggs being again about the same in number. ‘Pupae deprived of the brain are capable of transforming into moths. Whereas the normal female moth usually begins to lay eggs only after mating with a male, the brainless moth begins to deposit unfertilized eggs as soon as it finishes shedding the pupal skin. After mating, the brainless moth lays the majority of eggs in one night. The moth from pupa deprived of the subesophageal ganglion is less active than the brainless moth and cannot lay eggs even after mating. After several trials, it was found that the ligation of the body, the transection of the nerve cord, or the removal of any ganglion except for the last induces t.he deposition of eggs in these moth. In our experiments eggs were usually obtained from the moths without the subesophageal ganglion by ligating the body between the thorax and abdomen or by excising the thorax after the ligation
338
SOICHI
From these experiments, it appears evident that in the pupa, the diapausing or the nondiapausing property of the eggs developing in the ovaries is determined by the brain. However, it is unlikely that the effect of the brain on the ovarian eggs is hormonal in nature, since the autotransplantation of the brain into the pupae immediately after its removal failed to prevent the reversal of voltinism in the individuals. On the other hand, the moths which emerged from diapause and nondiapause pupae from which the subesophageal ganglion had been removed, invariably laid nondiapause eggs only. If the subesophageal ganglion was autografted into the pupae, the resultant moths produced both diapause eggs and nondiapause eggs. These results indicate that the subesophageal ganglion releases into the blood the diapause factor which acts on the developing eggs and make them diapause eggs. Furthermore, transplantation of subesophageal ganglion from nondiapause and diapause larvae into nondiapsuse ones demonstrated that there was no difference in the potency of secreting the diapause factor of the subesophageal ganglion between the two types of the silkworm. From these observations, it seems probable that, in the nondiapause pupa, the release of the diapause factor from the subesophageal ganglion is inhibited by the brain by way of the esophageal connectives, while in the diapause pupa, the brain stimulates the secretion of the factor via the esophageal connectives. This hypothesis is substantiated by the following transplantation experiments using nondiapause female larvae of the fourth instar as recipients. In the first series of experiments, many of the moths which developed from the larvae given transplants of the subesophageal ganglion alone or of the ganglion together with the isolated brain laid both diapause and nondiapause eggs, while all the moths bearing grafts of the brainsubesophageal ganglion complex with esophageal connectives intact laid nondiapause eggs only. In this series, the grafts were
FUKUDA
obtained from nondiapause donor larvae of the same age as the recipient,s. In the second series of experiments, grafts were obtained from diapause larvae of the fourth instar. Results showed that the majority of the moths having transplants of the brain-subesophageal ganglion complex laid diapause eggs, whereas many of the moths bearing grafts of the subesophageal ganglion or the ganglion plus the isolated brain produced both diapause and nondiapause eggs. In similar experiments using grafts from larvae of the univoltine race, many of the moths bearing grafts of the subesophageal ganglion alone laid diapause eggs and nondiapause eggs, while the majority of the moths carrying transplants of the brainsubesophageal ganglion complex diapause eggs only. The results were the same regardless of the incubation conditions to which the donors had been exposed during the embryonic period. On the contrary, none of the moths from larvae which had received grafts of the brain-subesophageal ganglion complex from larvae of the multivoltine race2 deposited diapause eggs, if the donors had been incubated at the temperature below 20°C. Yet, the moths given grafts of the subesophageal ganglion alone deposited both diapause and nondiapause eggs, regardless of the temperature and light conditions ta which the donors had been subjected during the incubation period. These observations seem to indicate that the subesophageal ganglion is capable of secreting the diapause factor irrespective of the genetic constitution of the silkworm and external conditions during the period of incubation. However, it appears likely that the subesophageal ganglion is not ac‘The silkworm of multivoltine race in the strict sense of the word produces nondiapause eggs only, from generation to generation, regardless of environmental conditions to which the silkworm is exposed during its development. In the silkworms used as materials in our experiments, however, if the eggs were exposed to the temperature higher than 26°C during the incubation period, the resulting moths occasionally laid diapause eggs or diapause eggs together with nondiapause eggs.
HORMOXAL
CONTROL
OF
339
DIAPAUSE
tive enough to make all eggs developing in the ovaries diapause ones. It may be that, in the pupa determined to produce diapause eggs only, the activity of the subesophageal ganglion is augmented by the brain by way of the esophageal connectives, while in the pupa destined to produce nondiapause eggs only, the brain inhibits the production or the release of the diapause factor from the subesophageal ganglion via the esophageal connectives. In the absence of the effect of the brain on the subesophageal ganglion, both diapause and nondiapause eggs are produced. Our experiments have also suggested that conditions during the incubation period exert an influence on the brain of the embryo and let it have a potency to stimulate or suppress the secretion of the diapause factor from the subesophageal ganglion during the pupal period.
better developed in comparison with the anterior part. Similar abnormal embryos were also found in the eggs deposited by the moths of the bivoltine race which had been subjected to about 20°C during the incubation period. It is conjectured that the disharmony in the development in these embryos is ascribable to the insufficiency of the diapause factor during the pupal stage. Only those eggs which have been subjected to the sufficient effect of the diapause factor during the course of the pupal period can be in the perfect diapause state after being dcposit,ed. The eggs only insufficiently influenced by the diapause fact,or cannot be perfectly dormant so that development may proceed in some part of the body of the embryos.
ABNORMAL EMBRYONIC DEVELOPMENT IN IMPERFECT DIAPAUSE EGGS
If two pupae were united in parabio,*is attaching the dorsal sides of the thoraxer together, two moths emerged from the pupae on the same day. In diapause pupanondiapause pupa combinat,ions, many of the moths from diapause pupae produced diapause eggs, while many of those from nondiapause pupae laid diapause eggs together with nondiapause eggs. The result seems to show that the diapause factor passes with the blood from diapause par:\biont to nondiapause partner. However, it was also found that, in the same parabiotic combinations, the moths from diapause parabionts occasionally laid a small number of nondiapsuse eggs in addition to diapause eggs. Even in con)binations of two diapause pupae, one or both of the resultant moths often depositrd a few nondiapause eggs together with dinpause eggs. Furt,hermore, a few of the moths which emerged from the unions of two nondiapause pupae laid a few diapaus:c eggs together with nondiapause eggs. In order t.o ascertain whether t.he operation of parabiotic union per se is effective in inducing such a reversal of voltinism, a small disc of skin was excised from the thoracic tergum in a series of pupae and replaced it by applying melted paraffin
As mentioned above, many of the moths which developed from diapause or nondiapause pupae deprived of the brain lay diapause eggs together with nondiapause eggs. Similar phenomenon has also been observed in t,he moths each given a subesophageal ganglion as a transplant during the larval or the pupal period. In several days after the deposition, t.he eggs in the same batch exhibited differences in coloration varying from pale yellowish nondiapause type to dark purplish diapause type. By dissecting the eggs less darkened than normal diapause eggs, it was frequently found that embryos contained in the eggs were deformed to varying degrees. In some embryos, the posterior part, of the digestive tract had been formed but the cephalic lobes or the anterior part of the body remained undeveloped as in the diapausing embryos. In another embryo, the posterior part of the body was well advanced in development, the clearly segmented abdomen being provided with rudiments of the abdominal legs, while the anterior part, was still dormant. A feature in common in these abnormal embryos was that the posterior part of the body was
EFFECT
OF ON
STRESSFUL VOLTINISM
STIMULI
340
SOICHI
around the cut edge. It was found that such a simple operation provoked the change in voltinism like that observed in the parabiotic experiments. These observations led the author to the assumption that the production of diapause or nondiapause eggs is affected by nonspecific stressful stimuli. Therefore, various noxious treatments, such as thermocauterization of small area of the skin on the ventral or the lateral side of t,he abdomen, removal of small pieces of the fat body, removal of one of the abdominal ganglia, transection of the nerve cord between two adjacent abdominal ganglia, injection into the abdominal cavity of adrenaline, thyradin (a thyroid preparation), or air, insertion of a piece of glass or of silkworm cocoon into the body cavity, were applied to diapause as well as nondiapause pupae. Following subjection to any one of these treatments, some of the moths from diapause pupae laid varying number of nondiapause eggs together with diapause eggs and some of those from nondiapause pupae deposited some diapause eggs besides nondiapause eggs. It may be worthy of mention that any treatment applied to nondiapause pupae from which the subesophageal ganglion had previously been removed could no longer induce the production of diapause eggs, showing that nonspecific stimuli stimulate the subesophageal ganglion to secrete the diapause factor. It has not yet been known whether the stimuli act directly on the subesophageal ganglion or through the intermediary of some center
FUKUDA
in the brain or somewhere in the central nervous system. A similar question may also arise in the case of the production of nondiapause eggs in diapause pupae after exposure to stressful stimuli. At any rate, it appears probable that the reversal of voltinism in these cases is caused by a derangement in the secretory activity of the subesophageal ganglion. DISCUSSION HASEGAWA: In 1952 and 1953, I observed that following transplantation of SG from silkworm larvae of polyvoltine, tetravoltine and bivoltine races, respectively, into larvae of tetravoltine race on the second or the third day of the 4th instar, 31.3, 63.1 and 93.8% on the average, respectively, of the eggs laid by resultant moths were diapause eggs. From this, it seems likely that the activity of SG has already been genetically determined at the larval stage so that the SG of larvae of different races are different in activity, the SG of bivoltine race larvae being most active. In some experiments, SG-brain complexes from tetravoltine moths were also used as transplants. It was found that 55.7% of eggs laid by resultant moths were diapause eggs. It seems probable that the SG-brain complex of tetravoltine animals cannot release enough diapause hormone to affect their own eggs, but if the level of the hormone becomes sufficiently high in the recipients it may exert its effects on developing eggs. FUKUDA: I suppose that the moths of the tetravoltine race you used in your experiments should have normally produced some diapause eggs.