STUDIES ON THE MODE OF ACTION OF THE DIAPAUSE HORMONE IN THE SILKWORM, BOMBYX MORI L.

Biol. (1963), 40, 517-52 517 text-figures Printed in Great Britain STUDIES ON THE MODE OF ACTION OF THE DIAPAUSE HORMONE IN THE SILKWORM, BOMBYX MORI L. I. THE ACTION OF DIAPAUSE HORMONE INJECTED INTO PUPAE OF DIFFERENT AGES BYKINSAKU HASEGAWA The SericuUural Laboratory, Faculty of Agriculture, Nagoya University, Anzyo, Aiti-ken, Japan {Received 27 February 1963) INTRODUCTION The effect of temperature on the voltinism of the silkworm, Bombyxrnori,was discovered by Watanabe (1918) over 45 years ago. This followed the rinding of the important role of photoperiod during the incubation period of silkworm eggs (Kogure, 1933). In brief, whether diapause or non-diapause eggs are laid depends on temperature and light experienced by the mother during her embryonic life. In 1950, the present author found that the suboesophageal ganglion is the centre responsible for the induction of diapause eggs (Hasegawa, 1950). This finding was the starting-point or the endocrinological investigation of silkworm diapause by surgical manipulations such as transplantation and extirpation of suboesophageal ganglion, brain, corpus allatum, and so forth (Fukuda, 1951-53; Hasegawa, 1950-52; Morohoshi, 1957, 1959). These extensive works on silkworm diapause have made a great contribution to the understanding of insect diapause. Up to 1950 the phenomenon of insect diapause was believed to be due solely to lack of insect growth hormone. This conception, however, is not applicable to the silkworm where the suboesophageal ganglion lying just behind the brain yields an active principle inducing diapause of silkworm eggs, indicating therefore that the induction of insect diapause comprises two features; one of which is the result of shortage of growth hormone, and the other is the result of positive action of a substance released from the suboesophageal ganglion (diapause hormone). As to the nature of silkworm diapause, different investigators have different conceptions, though it is universally admitted that the suboesophageal ganglion is the actual source of the principle responsible for induction of diapause in silkworm eggs. Surgical procedures such as extirpation and transplantation of organs and ligation experiments, however, will leave some uncertainty about the function of the organs concerned, for such operations cause injuries to the organs to be tested, and, furthermore, the transplants may not show uniform development in the host. The development in hosts with the suboesophageal ganglion alone transplanted seems to be slightly delayed when compared with those in which the suboesophageal ganglion with brain attached is transplanted. If that is so, it suggests that the brain may activate the suboesophageal ganglion by way of the oesophageal connectives. From * This work was aided, in part, by a grant from the Scientific Research Committee of the Department >f Education. 33-a

518 KINSAKU HASEGAWA another point of view, denervated organs such as implants may not behave normally and will therefore fail to reveal the real function of the organs concerned. There is therefore a limit to the use of surgical techniques. These considerations prompted the present author to try extracting diapause hormone from the silkworm, B. mori. In 1956 the first separation of the active part was achieved after 3 years' trial (Hasegawa, 1957). For the past 6 years the purification of the hormone has been carried out in collaboration with Prof. Hirata (Department of Chemistry, Faculty of Science, Nagoya University). At present, the diapause hormone is not in pure form, but it has been purified highly enough to be used for the biological test of the hormone without entailing any detectable changes upon pupal duration, egg production, egg-laying, etc., of the pupae injected with diapause hormone extracts. This first of a series of communications deals with most of the experimental results obtained in 1961 and gives some details of biological tests of the extracts which have brought to light certain points of general interest. MATERIAL AND METHOD Silkworm pupae receiving the diapause hormone extracts are polyvoltine races or their hybrids, their hibernating values (Nagatomo, 1953) being zero to two. In addition, further care was taken to ensure that the female moth laid only non-diapause eggs by exposing her to low temperature and darkness during her embryonic development. Non-operated silkworms and those receiving extracts from which diapause hormone had been removed by chemical procedures or those which received extract containing insufficient amount of diapause hormone deposited only non-diapause eggs, thereby serving as controls in the present investigations. For the injections of diapause hormone extracts dissolved or suspended in water a finely tapered glass capillary calibrated in 5 or 10 /A. units was employed. All injections were made in the dorsum of the pregonadal segment avoiding the dorsal vessel, and most series of experiments involved nine or ten pupae of the same age. The diapause hormone was extracted from the heads of male silkworm moths subjected to high temperature and light during their embryonic life. The active principle was also separated from brain-suboesophageal ganglion complexes of pupae irrespective of sex. The procedure here used to prepare the extracts was modified from the previous paper (Hasegawa, 1957). Pupae were kept at 27-28 0 C. during adult development unless otherwise stated. Excepting female moths deprived of the suboesophageal ganglion, almost all moths injected with diapause hormone extracts behaved and laid eggs normally just like non-injected controls. Occasional moths showing abnormal egg-laying were excluded from consideration, and non-fertilized eggs were not included in the percentage of diapause eggs. EXPERIMENTAL RESULTS I. Effect of extracts from moth heads or brain-suboesophageal ganglion complexes on silkworm pupae deprived of the suboesophageal ganglion Diapause hormone was extracted from male moth heads or brain-suboesophageal ganglion complexes. It is possible that such extracts, even if they are competent to cause polyvoltine races to lay diapause eggs, may stimulate the brain of the recipient

Diapause hormone in the silkworm. I 519 to release the diapause factor from the suboesophageal ganglion, for Fukuda (1952) postulated that the brain of non-diapause egg producers inhibits suboesophageal ganglion function, through the oesophageal connectives, and so checks the production of the diapause factor. It is therefore necessary first to see whether or not the extracts stimulate the brain to yield diapause factor from the suboesophageal ganglia. To this end, the following experiment was designed. The suboesophageal ganglia were removed from females of the polyvoltine race N 4 within a few hours after pupation and the operated insects divided into two groups. One group was kept as control and the other was injected with 0-4 mg. of extract (M) (separated from male moth heads and suspended in 0-02 ml. of water) on the fourth day after pupation. In addition, non-operated pupae were also injected with the same amount of the extract at the same pupal age. The results are tabulated in Table 1. Table 1. The response of silkworm pupae deprived of suboesophageal ganglion (SG) to diapause hormone extract Operation SG-removal Non-operated controls Injection of the extract _ + + No. of pupae treated 3 3 9 No. of moths laying fertilized eggs 33 18 9 No. of moths laying non-diapause eggs alone 23 9 (each moth laid less than 9 eggs) 0 No. of moths laying diapause eggs 0 9 (all moths laid more than 10 eggs) 9 Average percentage of diapause eggs o-o 39-4 («= 9); 69-2 (average of 3 moths laying more than 50 eggs) 61-9 Note. Silkworms here used, polyvoltine race (N«); SG-removal, within a few hours after pupation; extract (ivf)-injection, 0-4 mg. each on the 4th day after pupation; adult emergence, on the 8th and 9th day after pupation. Non-operated controls receiving the extract deposited on an average 141, 90 and 8 of diapause, non-diapause and non-fertilized eggs respectively, 1 eggs remaining in the bodies. As predicted, all moths from which the suboesophageal ganglion had been removed laid only non-diapause eggs. Nine out of eighteen operated moths injected with the extract deposited exclusively non-diapause eggs, but the eggs laid were only one to nine.* The other nine moths laid more than ten eggs including diapause ones; average percentage of the latter is 39-4. When the calculation is made with three moths laying more than fifty eggs the percentage rises to 69-2, resembling that of non-operated worms with the extract. Thus the injection of the extract reproduced the results obtained by transplantation of suboesophageal ganglion into isolated pupal abdomen; results which led to the finding of the role of suboesophageal ganglion in voltinism (Hasegawa, 1950). From this preliminary experiment and others described below, and from experiments which will be mentioned in a subsequent communication, it is possible to say that the extract from the heads is diapause hormone extract that has originated from the suboesophageal ganglion. The same applies to other extracts with diapause action. Normal or non-operated female moths lay almost all the eggs in their bodies during the night after mating. Moths deprived of the suboesophageal ganglion lay eggs, but only a small number in a few days. Those laying less than nine eggs, in this case, would deposit diapause eggs if more eggs were laid (see discussion).

52O KlNSAKU HASEGAWA II. Pupal weight and diapause hormone Egg production in silkworm pupae depends upon pupal weight in general, the heavier the pupae the more numerous the eggs. Nutrients for growth of ovaries amount to about 30% of the weight of young pupae (Hasegawa, 1943). In testing the action of the diapause hormone extracts, pupal weight must be taken into consideration. After measuring the weight of pupae 2 or 3 days old, diapause hormone extracts (0-5 mg. of comp. B and o-6 mg. of 107 B, derived from male moth heads, suspended in o-oi and c-02 ml. of water respectively) were injected into pupae divided into three weight groups in one series of experiments, each group consisting of nine pupae. 50 r - 100 a. 40 80 30 60 8> 2. 20 3 10 o o o 00 6> "o V DO 20 I 0 500 600 700 800 900 Pupal weight (mg.) at the time of hormone Injection Fig. 1. The effect of pupal weight on the response of silkworm pupae (Daizo x NJ to diapause hormone extract. O or #, A pupa injected with 0-5 mg. or i-o mg. of the extract (comp. B) respectively at the a-day-old pupal stage;, a pupa injected with o-6 mg. of the extract (107 B) at the 3-day-old pupal stage. The light-weight group, for example, comprised those up to 700 mg., the mediumweight group those between 700 and 800 mg., and the heavy-weight group those above 800 mg. As shown in Fig. 1, the ratio of diapause eggs to the total number of eggs laid (except non-fertilized ones) seems to be nearly the same for the same amounts of the extracts injected, irrespective of pupal weight. One out of twenty-three moths receiving 0-5 mg. of the extract (comp. B), however, laid an extremely high percentage of diapause eggs. Anomalous high or low response of silkworm pupae to diapause hormone extracts was occasionally observed in other experiments, which are discussed below. In any case, it is highly probable that the percentage of diapause eggs induced by injecting diapause hormone extracts is not connected with pupal weight, indicating that in interpreting experiments of this type pupal weight need not be taken into account.

Diapause hormone in the silkworm. I 521 III. Effect of pupal age and temperature on the response of silkworm pupae to diapause hormone At the outset of diapause hormone injection, pupal age and subsequent treatment of the pupae must be considered. A preliminary experiment on pupal age was performed as follows: nine silkworm pupae (Daizo x N4) in each series ranging from shortly after pupation to pupae 3 days old were injected with 0-5 mg. or o-i mg. of hormone extract 50 - n-9,m=.s07%» 2. I 20 10 0* 0 1 1 Pupal age In d»ys after pupation Fig. a. The effect of pupal age on the response of silkworm pupae (Daizo x NJ to diapause hormone extract (comp. B). O» 0-5 mg. injection;, i-o mg. injection. (comp. B) and kept at 27-28 0 C. throughout adult development. The result, recorded in Fig. a, is of great interest in two points: (1) the older the pupae the greater the response to diapause hormone extract, (2) the extract capable of converting about half a batch of eggs into diapause ones when injected into pupae 3 days old is completely masked or inactivated on the day of pupation. Another preliminary experiment on the effect of temperature on the response of silkworm pupae to diapause hormone was undertaken: polyvoltine silkworm pupae, N 4, having received i-o mg. diapause hormone extract (comp. B) at 2 and 3 days of age, were divided into two groups; one group was kept at 27-28 0 C. throughout adult development as well as before the injection, and the other group was transferred to the same conditions after 5 days' exposure to 15 C. The results, indicated in Fig. 3, show that low temperature favours the action of the diapause hormone.

522 KENSAKU HASEGAWA From these preliminary experiments, it is highly probable that the age of the pup% at the time of injection and its subsequent treatment strongly influence the action of the diapause hormone. Further results relevant to these matters are illustrated in Fig. 4; o-6 mg. or 0-9 mg. of diapause hormone extract (107 B) was injected into nine or ten pupae ranging from those newly pupated to the 6-day-old stage or 1-2 days before adult emergence. As summarized in Fig. 4, production of diapause eggs is greatest when the injection is made at a pupal age of 2-3 days and is less if the pupae are 100 r 80 8 74-5% 60 n = 9 40 20 / y / 27-28 C. throughout adult development n-8 M = 14-4% 8 Pupal age In days after pupation Fig. 3. The effect of temperature on the response of polyvoltine silkworm pupae (N4) to diapause hormone extract (comp. B). Injection, i-omg. of the extract; O, kept at 27-28 0 C. during adult development; #, kept at 15 C. for the first 5 days after injection, then transferred to 27-28 0 C. younger or older. The steep decline in the percentage of diapause eggs after 4 days is not significant, for many of the oocytes in the pupae from these stages onwards would have already started to develop into non-diapause eggs; in other words the older the pupae the lower the percentage of diapause eggs. But it is worthy to note that (1) diapause hormone extracts capable of producing more than 70 % of diapause eggs are almost masked or inactivated when injected on the day of pupation, (2) injections of the extracts are most effective if they are made at a pupal age of about 2-3 days when the ovaries have started to grow vigorously (Hasegawa, 1943), and (3) the masking or inactivation of diapause hormone action is to some extent relieved by low temperature!

Diapause hormone in the silkworm. I 523 ^ the most favourable effect of low temperature for diapause hormone action occurs when the injection is made at a pupal age of 1-2 days and especially at an age of 1 day. As stated above, diapause hormone injected into fresh pupae is masked or inactivated; that is to say, the pupae apparently begin to inactivate the hormone promptly upon injection. Large amounts of active hormone extract, however, can exert the effect even when injected on the day of pupation. For example, nine out of ten pupae of N 4 each having received on the day of pupation i-o mg. of the extract (Po) derived 0 1 2 3 4 5 6 Pupal age In day! after pupation Fig. 4. The effect of pupal age and temperature on the response of silkworm pupae (Daizo x NJ to diapause hormone extract (107 B). O and, Injected with o-6 and 0-9 mg. of the extract respectively; solid line, pupae kept at 27-28 C. during adult development; broken line, pupae kept at 15 C. for 5 days after injection then transferred to 27-28 C. from pupal brain-suboesophageal ganglion complexes were transformed into moths and laid on an average 231, o and 5 of diapause, non-diapause and non-fertilized eggs respectively with no eggs remaining in their bodies; the percentage of diapause eggs ranged from 97-8 to 100, with an average value of 99-7. Similar results were also obtained with pupae of Taiwan No. 8 (polyvoltine race), indicating that the quantity of the hormone injected was sufficient to withstand inactivation. IV. Effect of indian ink on the response of silkworm pupae to diapause hormone From the foregoing results, low temperature (15 0 C.) blocked to some extent the inactivation of diapause hormone, in contrast with the case of juvenile hormone, where the hormone injected into well-chilled Promethea pupa was completely inactivated during prolonged incubation at 15 0 C. (Gilbert & Schneiderman, i960).

524 KlNSAKU HASEGAWA Another attempt to block the inactivation of diapause hormone was made. InjectiSP of indian ink delays the moulting of Rhodnius larvae (Wigglesworth, 1955) and the initiation of adult development in Polyphemus pupae (Schneiderman & Gilbert, 1959), an effect which seems to be due to blockage of haemocytes. Inthecase of theinactivation of diapause hormone the haemocytes also seemed to be concerned. In view of this, indian ink was injected together with diapause hormone extract. Stick ink was made up in distilled water to give a suspension of a density for pen drawing. 100 r.1/3 = 617* n-7 ftm = 61-6% 6 ^ki. 0 1 2 3 4 Pupal age In days after pupation Fig. 5. The effect of indian ink on the response of polyvoltine silkworm pupae (N4) to diapause hormone extract (107 B)., Injection of indian ink alone; O, injection of o-6 mg. of the extract; 9, injection of o-6 mg. of the extract suspended in indian ink. o-6 mg. of diapause hormone extract (107 B) with or without suspension of indian ink was injected into pupae of N 4 from just after pupation to the 4-day-old stage. The results, recorded in Fig. 5, tell us that the inactivation is reduced by indian ink injections when carried out on 1- and 2-day-old stages, though indian ink itself (as here used) had no effect on the pupal duration, occurrence of diapause eggs, etc. Although indian ink injection on the day of pupation relieved slightly the inactivation of hormone, as does low temperature treatment, injection at the 1- and 2-day-old stages is effective in avoiding the inactivation. The reason why indian ink particles injected at the 4-dayold stage were without effect is that the fate of many oocytes can be said to have been determined as non-diapause eggs and only a limited number of oocytes can respond to diapause hormone extract. This phenomenon is in perfect conformity with that of old pupae exposed to low temperature after injections of diapause hormone (see Fig. 4).

Diapause hormone in the silkworm. I 525 DISCUSSION I. Anomalous response of silkworm pupae to diapause hormone. Among those having received 0-5 mg. of Comp. B, recorded in Fig. 1, one moth laid n o diapause eggs and 174 non-diapause eggs, no eggs remaining in her body; the percentage of diapause eggs was 38-7, whereas the other twenty-two moths with the same amount of the hormone extract laid 4-1-14-2% of diapause eggs, the average being 9-8 %. As it has been shown that the pupal age at the time of hormone injection is closely connected with the response of pupae to diapause hormone extracts (Figs. 2-5), it is conceivable that the pupa showing high response was older than the others. But this explanation is also disproved by the pupal duration, for this pupa transformed into moth in 5 days after the injection as well as the others did, i.e. apparently identical pupae receiving the same amount of the hormone extract responded quite differently to the extract. Such instances of exceptionally high or low responses to diapause hormone extracts occurred from time to time, and some examples were picked out in Table 2. Pupal duration of moths showing anomalous response to diapause hormone extracts is the same as for the others in each series of experiments. Although pupae in Table 2 experienced low-temperature treatment, untreated silkworms also happened to show abnormal responses in another experiment. Nothing could be said as to how these anomalous responses were brought about, but this state of affairs will be attributable to individual difference in the response to diapause hormone or diapause hormone inactivation of pupae after all. II. Oocyte development and diapause hormone At present, it is highly probable that the oocyte is the sole target organ of diapause hormone extract as used here. It is clear from the foregoing results that the diapause hormone function has a close connexion with the pattern of ovarian development. Apart from the hormone inactivation in fresh pupae, where ovaries are of larval type, the injection of diapause hormone is most effective at 2- and 3-day-old stages when the ovaries have started to grow rapidly and the ovarioles are floating in the abdomen. Injection of the hormone thereafter is not so efficient, for oocytes located in the lower part of the ovarioles are acquiring characters of non-diapause eggs, and are laid earlier (as non-diapause eggs) than other eggs, which demonstrates why nine moths laying less than nine eggs showed no response to injection of diapause hormone, and the other nine moths laying more than ten eggs deposited diapause eggs (Table 1). That the older the pupae the less the effect of low-temperature treatment and indian ink injection (Figs. 4, 5) is reasonable in the light of the mechanism of ovarian development discussed here. The same sequence has been encountered in the case of uranium nitrate injection which switches the course of oocyte development in diapause egg producers to that of non-diapause eggs, proceeding in the reverse direction from diapause hormone action (Hasegawa, 1947).

Diapause hormone in the silkworm. I 527 III. Diapause hormone inactivation Putting aside the injection of large amounts of diapause hormone extract, we can observe masking or inactivation of the hormone extracts in the early pupal stages, especially on the day of pupation. The injections of diapause hormone extracts thereafter, however, are effective, indicating that the diapause hormone is promptly inactivated in the silkworm body during 1-2 days after injection. Injections of active extracts of juvenile hormone into Rhodmus larvae have given variable and often negative results (Wigglesworth, 1958). It is said that the injected hormone might be presented in a form which is not readily mobilized in the body of the insect or which is rapidly eliminated by metabolism. Juvenile hormone injected into well-chilled Promethea or Polyphemus pupae is completely inactivated by exposing the pupae at 15 0 C. (Gilbert & Schneiderman, i960) or by the 2 weeks' delay in the initiation of the adult development following indian ink injection (Schneiderman & Gilbert, 1959). In the case of diapause hormone extracts, however, low temperature or indian ink injection seems to favour the hormone action, as the preceding experiments demonstrate. The inactivation mechanism of diapause hormone, therefore, appears to be different from that of juvenile hormone. Table 3. The response of diapause egg producers deprived of suboesophageal ganglion (SG) to diapause hormone extract Average number of egg» laid (and the aver- No, of moths No. of moths No. of moths age of moths Operation SG-removal (control) SG-removal + extractinjection No. of pupae laying ferti- laying non- laving diaoperated lized eggs diapause eggs pause eggs 23 33 17 14 17 14 laying more than 50 eggs) 119, n = 17 (191, n = 10) oa, n = 14 (168, n = 7) Note. Diapause egg producers here used, (Daizo x N«) x Daizo experiencing high temperature and light during their embryonic life; SG-removal, on the day of pupation; extract-injection, o - 6 mg. (107 B) each on the day of pupation after SG-removal. Non-operated worms laid no non-diapause eggs but an average of 365 diapause and 11 non-fertilized eggs. Silkworm moths of bivoltine races subjected to high or low temperature during embryonic development produce only diapause or non-diapause eggs respectively irrespective of conditions experienced during the larval and pupal stages. Those subjected to medium temperature, however, produce more or fewer diapause eggs according to subsequent conditions. That is, low temperature after the mounting stage of mature larvae is favourable for the pupae to yield diapause eggs, and high temperature has just the opposite effect (Watanabe, 1918, 1924). In view of diapause hormone inactivation and its lessening by low-temperature treatment, this observation is explained for the first time in the present investigation. The next step is to see whether the mechanism of diapause hormone inactivation is present in the pupal bodies destined to produce diapause eggs. To this end, the following experiments were designed. Diapause egg producers were deprived of the

528 KlNSAKU HASEGAWA suboesophageal ganglion just after pupation and received o-6 mg. of diapause hormone extract (107 B), the quantity of which is sufficient to induce moths to lay about 40-80 % diapause eggs when injected into 3-day-old pupae (see Figs. 4, 5). As Table 3 indicates, no moths deposited diapause eggs, demonstrating the existence of the hormone inactivation even in the pupae destined to produce diapause eggs. This fact was further substantiated from the following experiment. As has long been known, male pupae of diapause egg producers are also capable of producing diapause eggs if the ovaries of larvae have been implanted into them (Hasegawa, 1952); the blood of male moths would be suitable material for diapause hormone extraction if it were not for diapause hormone inactivation in the males. The result was negative, i.e. 3 mg. of 108 mg. extract, obtained from the blood of 800 male moths by the use of a procedure identical with that of diapause hormone extraction, was not able to produce a single diapause egg. On this account it is highly probable that the mechanism of diapause hormone inactivation is present in silkworm pupae irrespective of their being diapause or non-diapause egg producers, though the site and mode of the hormone inactivation is still uncertain. SUMMARY 1. The action of the diapause hormone has been studied by injecting extracts of the heads of male moths or of the brain-suboesophageal ganglion complexes of pupae into pupae expected to produce non-diapause eggs. 2. The effect of the injection of hormone upon young oocytes is to make them develop into diapause eggs. Older oocytes, which have already acquired non-diapause characters, are not affected. 3. The hormone is almost completely inactivated when injected on the day of pupation. The hormone is most effective when injected into pupae 2-3 days old, at which stage the ovarioles have started to grow vigorously. It is ineffective 1-2 days before adult emergence, by which time all the oocytes have acquired non-diapause characters. 4. The hormone is inactivated in all pupae irrespective of whether they are destined to produce diapause eggs or non-diapause eggs. Inactivation of diapause hormone (in contrast to that of juvenile hormone) is partially relieved by exposure to low temperature or by simultaneous injection of indian ink. 5. The extracts prepared as in (1) above do not serve as a stimulant for the brain causing the suboesophageal ganglion to produce diapause hormone. The action of the extract faithfully reflects the function of the diapause hormone which originates in the suboesophageal ganglion. I am extremely grateful to Dr T. Yokoyama, Director of the Sericultural Experiment Station, for his kindness and his heartiest support for this work. Thanks are due also to Prof. Y. Hirata who has co-operated in diapause hormone isolation and to Prof. V. B. Wigglesworth who read the manuscript.

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