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

J. Exp. Biol. (4), 4, 55-55 Printed in Great Britain STUDIES ON THE MODE OF ACTION OF THE DIAPAUSE HORMONE IN THE SILKWORM, BOMBYX MORI L. II. CONTENT OF DIAPAUSE HORMONE IN THE SUBOESOPHAGEAL GANGLION BY KINSAKU HASEGAWA The Sericultural Laboratory, Faculty of Agriculture, Nagoya University, Anzyo, Aiti-ken, Japan (Received April 4) INTRODUCTION In 5, 5 and 55 the present author showed that the suboesophageal ganglion (SG) of pupae expected to produce or, in brief, '' pupae, is active enough to produce ; but in the polyvoltine race with 'non-' pupae the action of the ganglion is too weak to produce. These conclusions were confirmed by Morohoshi (5) in transplantation experiments of the SG and by Kobayashi (5) in a histochemical analysis of the SG. As previously reported, extracts of hormone from the heads of male, and from the brain-suboesophageal ganglion complexes of pupae, faithfully reflect the hormone which originates in the SG (Hasegawa, ). It is, therefore, reasonable to expect that an active hormone extract will be isolated from ' ' worms, while weak or no hormone extract will be obtained from polyvoltine or 'non' worms. As long as the same race is used as recipient of the hormone extracts, the occurrence of after hormone injection runs approximately parallel with the amount of the extract injected at a definite pupal stage (Hasegawa, ). By means of this bioassay of hormone extracts, therefore, we can estimate the activity of the SG in different races. The present paper deals with the production of hormone in the SG with special reference to sex, pupal age and polyvoltine race. MATERIALS AND METHODS ' Non-' pupae of polyvoltine and bivoltine races were used as recipients of the hormone extracts. These pupae were determined to produce only non- by low temperature and darkness during their embryonic development. Preparations of hormone extracts and the method of injection are as described previously; the injection of the extracts was carried out on the rd day or 4th day after pupation when the injections are most effective (Hasegawa, ). Non-fertilized laid were not included in the of, and the average was reckoned in. 55-

5 KlNSAKU HASEGAWA Before going further, it must be considered whether 'non-' pupae of any voltinism can be used as recipients for hormone bioassay. To this end, ' non' pupae of iv 4 (polyvoltine race) and Daizo (bivoltine race) were injected with hormone extract (M). The results obtained are tabulated in Table i. Table i. Difference in response of 'non-' pupae of JV 4 {polyvoltine race) and Daizo (bivoltine race) to hormone extract (M) Recipient N, Pupal age at injection (days) Amount of extract injected (mg-) - 4-4 o- -4 non alone o o o o Non no. of laid Diapause 4 5 5 of -5* - Daizo o S 4 4 4 '4 Note. Each series of experiments consists of nine pupae injected (the same applies to the following tables except Table 5). Out of nine, one moth gave a high response to the hormone extract, resulting in - % of, the other range from n-o to -% with an average of - %. 4- As shown in Table, 'non-' pupae of the polyvoltine race, N 4, responded clearly to hormone extract injected especially with pupae days old, while in the bivoltine race, Daizo, the hormone injected is almost ineffective. From this preliminary experiment, it appears that the susceptibility of 'non-' pupae to hormone extracts is different in different silkworm races. Accordingly, the following experiments were carried out by using -day-old pupae of a polyvoltine race (N 4 ) as recipients of the hormone extracts to be tested. EXPERIMENTAL RESULTS () Sex difference in production of hormone in the SG Since female of' ' worms produce, the question arises whether female have a lower content of hormone in the SG than male. To determine this, moth heads of both sexes of '' worms (Ginpaku) were taken. 5 mg. of active extract was obtained from,4 moth heads of females and 44 mg. from, male moth heads of the same race by the modified method of the hormone preparation (Hasegawa, 5). Each extract was injected into 'non-' pupae of N 4 days old (Table ). As shown in Table, injection of o- mg. of the extract from female moth heads was completely ineffective, but the same amount from male moth heads was effective in producing on an average *% of, varying from -5 to -%. Further, i- mg. extract corresponding to the extract of about forty-five female has low activity varying from * to '% with an average of $' %> whereas the

Action of the hormone in the silkworm 5 same amount of about thirty-six male is active in producing on an average - % of with a range of 5--- %. If appropriate correction is made, i.e. in i- mg. injections abnormally low or high responses of pupae to the extract injected are excluded, the average s are i-o in the female extract and 4- in the male extract as denoted in Table. In view of the activity both per weight of the extract and per numbers of moth heads, it is therefore likely that the SG of male is more active than that of females. This is further supported by the following experiments. Sex of donors Table. Sex difference in content of hormone in moth heads (Ginpaku, '' race) Amount of extract injected (mg.) O-(= h.)» i- (= 45 h.) o- (= ih.) i- C=i ( h.) ) non alone no. of laid Non Diapause 4 5 of o-o 5-at - Note. Recipients are -day-old pupae of N 4 (polyvoltine race). The same applies to the following tables. h. in parentheses is abbreviation of heads, i.e. o p (= h.) means that o - mg. of extract corresponds to the extract of about twenty-three heads, and so on (the same applies to Table 4). t Excluding one moth showing high response (- %), the is on an average i-o ranging from - to -. t Excluding showing low response (S'i and '), the is on an average 4' ranging from 4O'O to -. () Changes of hormone content in the SG during larval-pupal-adult development Silkworm pupae transform into adults in - days after pupation at 5 C. During these short days remarkable physiological and morphological changes take place in the pupal body. As adult development proceeds, for example, the ovaries start to grow vigorously and the SG begins to take on the shape of the adult SG which, unlike that of the larva, joins closely to the brain. To trace the secretory cycle of the SG, during adult development, the hormone content of the SG during larval-pupaladult development was surveyed on''silkworms (Jap. no. 4X Chi.no. 4). This race generally transforms into in about or days after pupation at 5 C. As extirpation of the SG of these worms was carried out in August in, the pupae emerged in or days after pupation at room temperature. 5 SG of last instar larvae of mixed sex at the actively feeding stage, and SG of both sexes of pupae and (apart from SG of female ) were dissected out as material for hormone isolation. SG of larvae and fresh pupae were picked out with the first thoracic ganglion, and SG of the other older pupae and were combined with the brain. These ganglia were homogenized in % methanol and used for hormone isolation. The extracts were dissolved in water, unit

5 KlNSAKU HASEGAWA concentration of the extracts being adjusted so as to be a multiple of 5 SG. The results of the extract-injections (per 5 SG) into 'non-' pupae are illustrated in Table. Table. Activity of extracts of suboesophageal ganglia of '' silkworms (Jap. no. 4X Chi. no. 4) during larval-pupal-adult development Stage of donors Larvae Newly pupated days old 5 days old days old Moth Sex of donors Mixed sex? (nonmated) cj (mated) * non alone 4 5 5 Extract injected, per 5 SG; the other extracts injected each, per 5 SG. S of Non 4 ii no. laid Diapause 5 4 4 4 4 of o-o S-o* ' - ' 4-5- As indicated in Table, the extract from male pupal SG on the day of pupation was very active, but it is uncertain whether this is a true feature or not. Anyhow, the activity of the hormone extract from the SG increases in the late pupal stage, especially in males, indicating greater production or storage of hormone in the SG of the male than in the female, which substantiates the results obtained by the hormone extracts of moth heads (Table ). The figures given in Table are the results of injection of extracts per 5 SG. When injected with extracts per 5 SG, or times the amount, the of reached a high level and the difference in the activity of the extracts injected disappeared, indicating that the pupae in each experiment had received enough hormone. As for the larval SG, the extract per 5 SG was completely negative in producing, while twice as much of the extract amount was very active, and the produced were 5-5-5-% with an average of 5-%, which clearly shows that the SG is functional in producing hormone during the larval stage. It is also interesting that the production of hormone in male moth SG becomes weak after mating. IQ-

Action of the hormone in the silkworm 5 () Content of hormone in the SG of the polyvoltine race From the foregoing experiments, it is possible to say that the s of induced by injections of extracts from moth heads permit one to estimate the activity of the SG of the race. On the basis of transplantation experiments of the SG of the polyvoltine and ' ' races, it was inferred that SG of the polyvoltine race is less active than that of the '' race and that the SG cannot produce by itself without implantation of additional SG (Hasegawa, 5, 5, 55). Whether this inference is wrong or not was examined by means of bioassay of hormone extract from male moth heads of a polyvoltine race. mg. of the hormone extract was isolated from 45 male moth heads of N 4 (polyvoltine race), and, at this time as control experiments, mg. of extract was obtained from, male moth heads of '' worms (Kenpaku) by the same isolation procedure. Both extracts were injected into -day-old pupae of N 4. The results obtained were summarized in Table 4. Table 4. Comparison of activity of hormone extracts from male moth heads of' non-' worms (iv 4 ) and ' ' worms (Kenpaku) Donor of male moth heads Amount of extract injected (mg.) o-(=5h.) i-(= oh.) o-( = 4 h.) i-(s= h.) non alone no. of laid Non Diapause of Kenpaku o o o S 4 4 4 4 -O 4- - o* mg. extract corresponding to about thirty-five male moth heads of N 4 is low in activity, varying from - to *% with an average of *%, while the same amount from '' worms produces on an average -% of (though including a moth with an extremely low response: -4 %). * mg. extract corresponding to about seventy heads from polyvoltine and ' ' worms produced about 5 % and % of respectively. These results reveal that the extract of the polyvoltine race is not so active as that of the ' ' race, but it undoubtedly has the ability to produce. DISCUSSION () Susceptibility of 'non-' pupae to hormone In the previous paper (Hasegawa, ) it was reported that exceptionally high or low responses to the injection of hormone extracts sometimes occurred among pupae receiving the same amount of the extract, and that these responses should be ascribed to individual differences.

KINSAKU HASEGAWA In general, it may be supposed that 'non-' pupae of bivoltine race are more susceptible to hormone than those of polyvoltine race. But this is not so, as shown in the preliminary experiment (Table i), where 'non-' pupae of a bivoltine race unexpectedly did not respond to injected hormone as the polyvoltine race did; i.e. 'non-' pupae of Daizo (bivoltine race) have a lower susceptibility to the hormone, or a stronger hormone-inactivation mechanism (Hasegawa, ), than those of N 4 (polyvoltine race). Accordingly, the susceptibility of 'non-' pupae to hormone, or hormone inactivation, is different in different races, showing that adequate silkworm races have to be selected as test animals for bioassay of hormone. () Sex difference in production of hormone in SG As shown in Table, male moth heads store more hormone than females, which is further substantiated by injection of extracts derived from the SG, where the injections of extracts from the SG of old male pupae and are more effective than those of females (Table ). On the other hand, larval SG themselves of female and male silkworms were reported to show almost the same activity when transplanted into 'non-' worms (Hasegawa, 55, and see below). It is necessary, therefore, to consider the difference of activity observed in hormone extracts of females and males. Although convincing evidence cannot yet be presented, the difference may perhaps come from the presence or absence of ovaries, which are the 'target organ' of the hormone (Hasegawa & Yamashita, unpublished data), resulting in more or less storage of hormone in the SG, which would reflect the activity of hormone extract from heads or SG. () Content of hormone in the SG during larval-pupal-adult development It is worth noting that hormone extract corresponding to 5 SG of 5th-instar larvae is unexpectedly active in producing in high (Table ), indicating that larval SG is producing the hormone during larval life. If larval SG produces hormone without liberation of it into blood, the hormone content in pupal SG would increase thereafter. As shown in Table, however, the extract from pupal SG at an early pupal stage is generally not so effective, thereby indicating that the SG produces hormone while liberating it into the blood. Diapause hormone liberated into the blood at the larval stage may be completely inactivated as observed in fresh pupae (Hasegawa, ) or may have some action. It is conceivable that the hormone during the larval stage may affect larval duration, silk production and so on which are generally observed as differences between '' and 'non-' worms, and these characters were, in fact, analysed by transplantation of SG or brain-sg complex (Nagatomo, 5; Morohoshi, As adult development proceeds, the extracts from the SG become more effective, showing a high content of hormone in the SG at the late pupal stage when the ovaries are approaching maturity. The pupal SG, which is at first of larval type, develops gradually into the adult type and secretes hormone, which in turn

Action of the hormone in the silkworm affects the content of -hydroxykynurenine and glycogen (Hasegawa & Yamashita, 4a, b; Yamashita & Hasegawa,, 4) and the ribonucleic acid pattern (Hayashi & Hasegawa, ) of the pupal ovaries. (4) Activity of the SG of the polyvoltine race By transplanting SG of the polyvoltine race into 'non-' worms, the activity of the SG has been shown to be weaker than that of ' ' worms (Hasegawa, 5, 5, 55). This conclusion has been accepted by Morohoshi (5) and supported by Kobayashi (5) from a neurosecretory analysis of the SG. It is of interest to compare the experimental results obtained by injections of hormone extracts (Table 4) with those of SG-transplantation carried out in 5. Some examples of these transplantation experiments have been extracted from the previous paper (Hasegawa, 55) and set out in Table 5. Donor of SG and sex Cambodge? Table 5. Occurrence of after transplanting one SG of \th-instar 'non-' and '' larvae into ^th-instar larva of polyvoltine race (iv 4 ) CO CO non alone i 5, Below % II o classified by of denoting the distribution of -4 % 4-% -% 4 Above % SyS O IS 4- * 4 IS O Note. larvae transplanted, twenty-one except twenty-three of female ' Cambodge ; Cambodge and N 4, polyvoltine races, Sy, '' worms of a bivoltine race. S 4 of - 5-5 - - Although transplantation experiments fail to reveal the real function of the SG itself (see Hasegawa, ), we can form some estimate of its activity. As shown in Table 5, which have received SG from the polyvoltine races (Cambodge and N 4 ) show a wide variation in the numbers of, the average being -5 m Cambodge and - in N 4. The SG of ' ' worms, however, usually induced more than % of in each moth, the average being -5. It is therefore probable that the SG is active in the order '' worms > N 4 > Cambodge. This table also shows that the function of SG is the same in the two sexes. Comparing Table 5 with Table 4, where the recipients in both are of the same race, it is clear that both the hormone extract derived from male moth heads of N 4, and the implanted SG of the same race, are weaker in effect than those of '' worms. i- mg. of extract, corresponding to about moth heads of N 4, approaches the activity of a single implanted SG of this race; while - mg. of extract, corresponding to about sixty-seven male moth heads of the ' ' race, has the same activity as

KINSAKU HASEGAWA a living SG implanted from ' ' worms. It is, therefore, highly probable that the bioassay by injecting hormone extract does reveal the activity of the living SG of silkworms. As stated above, the SG of the polyvoltine race are evidently functional, though a little weaker than those of the ' ' race; nevertheless, the polyvoltine race does not produce. Recalling Table, the larval extract per 5 SG is completely ineffective, while twice this amount is very effective, the average being 5- of with a range from 5-5 to 5-. Pupal extracts from 5 SG at a young pupal stage are also weak in activity, but, though excluded in Table, three times this amount is very effective, Such examples are observed on injecting extracts of N 4 SG, as seen in Table 4, where out of seven pupae receiving o- mg. extract two pupae do not show any response and the other five pupae have only a slight response, but all receiving twice the amount laid ranging in number from 4-5 to 5- with an average of 4-%. From the state of these affairs, it is probable that the SG of the polyvoltine race is functional but not sufficiently so to yield, and that the determination of in the silkworm is not an 'all-or-none' reaction but a 'threshold' reaction towards the hormone. As stated previously, the inactivation mechanism of the hormone is present in pupal bodies irrespective of '' and 'non-' worms (Hasegawa, ) and the susceptibility of silkworm pupae to hormone is different in different races (Table i), which affords further evidence that the mechanism of determination in the silkworm is not a simple matter as commonly believed. Anyhow, a working hypothesis tentatively advanced on the basis of the experiments of SG-transplantation (Hasegawa, 5) has been taken a step further by the isolation of the hormone and the injection of the hormone extract. SUMMARY. The susceptibility of 'non-' pupae to hormone, the sexual difference in production of hormone in the suboesophageal ganglion (SG), the changes in hormone content of the SG during larval-pupal-adult development, and the hormone production in polyvoltine race, were studied by injecting hormone extracts into pupae expected to produce non-.. The susceptibility of 'non-' pupae of a bivoltine race (Daizo) was very low, or the hormone injected was almost inactivated. The susceptibility of 'non' race to hormone is different in different races.. As for sexual differences in content of hormone in moth heads, males are superior to females, which was further substantiated by injecting extracts from the SG of both sexes. 4. From the activity of hormone extracts derived from larval, pupal and adult SG, the hormone production in SG is raised during the late pupal stage, especially in males. It is also observed that larval SG produces the hormone, and the hormone production in the SG of male after mating becomes weak. 5. Male moth heads of 'non-' worms (polyvoltine race), as well as of ' ' worms, contain hormone; though activity is less than in' ' worms, which reflects the occurrence of caused by SG-transplantation.

Action of the hormone in the silkworm. It is highly probable that the bioassay by injecting hormone extracts reveals the activity of the SG in the silkworm.. The activity of the SG in the polyvoltine race has been discussed from the standpoint of determination in the silkworm. This work was aided, in part, by a grant from the Scientific Research Committee of the Department of Education. REFERENCES HASEGAWA, K. (5). Studies on the voltinism in the silkworm, Bombyx mori L., with special reference to the organs concerning determination of voltinism (a preliminary note). Proc. Jap. Acad. zy, -. HASEGAWA, K. (5). Studies on the voltinism in the silkworm, Bombyx mori L., with special reference to the organs concerning determination of voltinism. J. Fac. Agric. Tottori Univ., -4. HASEGAWA, K. (55). On the organs concerning determination of voltinism in the silkworm, Bombyx mori L. and the application of the organs to the sericultural science (in Japanese). J. Tokai Branch, Seric. Soci. Japan,, 5-. HASEGAWA, K. (5). The dispause hormone of the silkworm, Bombyx mori. Nature, Lond.,,-. HASEGAWA, K. (). Studies on the mode of action of the hormone in the silkworm, Bombyx mori L. I. The action of hormone injected into pupae of different ages. J. Exp. Biol. 4, 5-. HASEGAWA, K. & YAMASHITA, O. (4a). Effect of hormone on -hydroxykynurenine content in pupal ovaries of the silkworm, Bombyx mori. Nature, Lond. (in the Press). HASEGAWA, K. & YAMASHITA, O. (4). Effect of hormone on the glycogen content in pupal ovaries and the blood sugar level of the silkworm, Bombyx mori. Nature, Lond. (in the Press). HAYASHI, Y. & HASEGAWA, K. (). Effect of hormone on ribonucleic acid pattern in pupal ovaries of the silkworm, Bombyx mori (in Japanese). J. Tokai Branch, Seric. Soci. Japan,,. KOBAYASHI, M. (5). Studies on the neurosecretion in the silkworm, Bombyx mori L. (in Japanese). Bull. Seric. Exp. Sta. 5, -. MOROHOSHI, S. (5). Hormonal studies on the and non- of the silkworm, Bombyx mori L. J. Ins. Physiol., -4. NACATOMO, T. (5). Function of the brain-suboesophageal ganglion complex in the silkworm, Bombyx mori. Proc. Jap. Acad., 5-. YAMASHITA, O. & HASEGAWA, K. (). Further studies on the mode of action of hormone in the silkworm, Bombyx mori. (In Japanese). J. Tokai Branch, Seric. Soci. Japan,,. YAMASHITA, O. & HASEGAWA, K. (4). Studies on the mode of action of the hormone in the silkworm, Bombyx mori L. III. Effect of hormone on -hydroxykynurenine content in pupal ovaries. (In Japanese). J. Seric. Sci. Japan (in the Press).