- Email: [email protected]
Induction of egg diapause by implantation of corpora cardiaca and corpora allata in Bombyx mori
J. Insect Physiol., 1976, Vol. 22, pp. 941 to 944. Pergamon Press. Printed in Great Britain.
INDUCTION OF EGG DIAPAUSE BY IMPLANTATION OF CORPORA CARDIACA AND CORPORA ALLATA IN BOMBIX MORI SATOSHITAKEDA’
and NOBUO OGURA’
‘Sericultural Laboratory and ‘Laboratory of Applied Entomology and Nematology, Faculty of Agriculture, Nagoya University, Chikusa, Nagoya Japan (Received 3 January 1976)
Abstract-Diapause egg production was examined in non-diapause egg producers by implantation of various cephalic organs into pharate adults 4 days after larval-pupal ecdysis. The implantation of five pairs of corpora cardiaca or corpora allata induced a great amount of egg diapause. Implantation of these organs was effective in inducing egg diapause even when the suboesophageal ganglion of the recipients had been removed, although the effect of the corpora allata decreased moderately. The injection of juvenile hormone into 4-day-old pharate adults did not greatly increase production of diapause eggs.
INTRODUCTION IT IS well known that diapause in the silkworm Bombyx mori, is induced by a neurohormone, the
egg. diapause hormone, which is released from the suboesophageal ganglion (SG) during pupal-adult development (HASEGAWA, 1951, 1952, 1957; FUKUDA, 1951). In early endocrinological work only the SG was thought of as the organ secreting the hormone; other organs such as the brain (Br), corpora allata (CA), and prothoracic gland were not supposed to be concerned with production of the hormone. Recently, OGURA and SAITO(1973) showed by implantation experiments that the brain-corpora cardiaca-corpora allata complexes (Br-CC-CA) of the armyworm, kucania separata, can induce egg diapause in silkworms. Furthermore, the brain of pharate adults of silkworms contains a substance with diapause hormone activity (S~NOBE and KEINO, 1975). Consequently, Br. CC, and CA as well as SG may be responsible for the induction of the egg diapause in silkworms. We have re-examined endocrinologically the organs which contributed to the induction of egg diapause in the silkworm and have found, that both the CC and the CA are concerned in part in the induction of egg diapause in Bombyx mori in addition to the SG. MATERIALS
AND METHODS
The silkworms, Bombyx mori, used were the bivoltine race (Kinshu x Showa) and polyvoltine race (NJ. The former was incubated at 25°C and light during embryonic life to obtain diapause egg producers (diapause pupae) and the latter at 15°C and
dark to avoid the occasional occurrence of diapause eggs. Br, CC, CA, SG, and the 1st thoracic ganglion (first TG) were prepared from the female pupae of diapause egg producers on the day of the larval-pupal ecdysis. These organs were implanted into non-diapause type recipients (N4) on day 4 after the larval-pupal ecdysis. In another experiment, pharate adults, of which the SG were removed just after the larval-pupal ecdysis, were also used as the recipients to see the interaction of CC or CA to SG. Synthetic dl-C1 ,-cecropia juvenile hormone (Zoecon Corporation) was dissolved in 2 d of peanut oil at each concentration, then injected into non-diapause pharate adults 4 days of age. RESULTS Diapause egg production cephalic organs
by
implantation
of
some
First, Br, CC, CA, and 1st TG, as well as SG obtained from diapause pupae. were implanted into the 4-day-old non-diapause type pharate adults with intact SG. These animals kept at 25°C emerged as adults 8 days after the larval-pupal ecdysis, and then laid eggs normally. The diapause nature was determined 15 days after oviposition, when larvae had hatched from non-diapause eggs. The results are shown in Table 1. The Br implantation caused four of the seven adults to lay egg masses consisting of diapause and non-diapause eggs (mixed eggs), the percentage of diapause eggs being 6.8. All the adults implanted with five pairs of CC or CA and five SG laid the mixed eggs, whereas the adults implanted with five first TG laid only non-diapause eggs. In the case of CC implantation, the eggs laid were somewhat
941
S. TAKEDAAND N. OGURA
942
Table 1. Diapause egg production in non-diapause egg producers by implantation of some endocrine organs or ganglia Implants
No. of animals treated
Wean of laid eggs
No. of adults laying Non-diap. egg only
Diap. e9g only
Mixed eggs
Non-diap. Diap. Total 241( 6.8)'
or
7c71*
3
0
4
226
15
CC
S(4)
0
0
4
56
130
186c72.4)
CA
S(4)
0
0
4
74
131
205(68.8)
SG
7(S)
0
0
5
105
103
2011(48.5)
7(7)
7
0
0
7.42
1st TG
0
242( 0.01
Fkv Br, SC, 1st TG and 5 pairs of CC, CA were taken from female pupae of diapause egg producers within 1 day after larval-pupal ecdysis. These organs were implanted into 4-day-old pharate adults of N, (a polyvoltine race). *Numerals in the parentheses refer to number of adults laying fertilized eggs; tnumerals in the parentheses refer to average of diapause egg percentages (the same applies to Tables 2 & 3).
60
,-” - 60 f 3 4 4o B 20
planted.
We next examined the relationship between implanted number of CC, CA, and SG and rate of diapause eggs in the resultant eggs. As shown in Fig. 1, diapause eggs were produced when one or two pairs of CC were implanted. Furthermore, three and five pairs are highly effective in inducing egg diapause; the adults implanted with three pairs of CC laid about 65yi diapause eggs and with 5 pairs about 754; of CC. When one or two pairs of CA were implanted. no diapause eggs were found. Although three pairs of CA could cause all the adults to lay mixed eggs the percentages of diapause eggs were very low. Five pairs of CA caused all the adults to lay mixed batches of eggs about 704, of eggs produced were diapause eggs. The activity of SG is rather less than that of CC, but a closer relationship was observed between
0
lOor
reduced in number. However, the percentage of diapause eggs was notably high compared with that of CA or SC ~mpl~tation. These results clearly show that CC and CA are effective in inducing egg diapause in non-diapause egg producers when they are im-
‘
I
0
1
2 3 No. of implants
Fig. 1. Relationship between No. of SG or No. of pairs of CC or CA implanted and diapause egg production. CC (A). CA (0) and SC (a) taken from female pupae of diapause egg producers 1 day after larval-pupal ecdysis and then implanted into 4-day-old pharate adults of Nti Each line is drawn through the mean value of each series of experiments on 3 to 5 adults.
Table 2. Diapause egg production by implantation of CC and CA in non-diapause egg producers with the removed SG Inplants
NO. of animals treated
no. of adults laying Diap. Non-diap. egg Only egg only
CC
s(3)*
0
CA
8 (7)
2
1st TG
7(6)
6
15(9)
9
0
None
Mixed eggs
Mean of laid eggs Won-diap.
Diap.
Total
3
24
89
11?(77.3)+
0
5
113
22
135115.5)
0
0
I.15
0
115( 0.0)
0
142
0
142( 0.0)
0
5
The removal of SC of the recipients, within I day after larval-pupal ecdysis. Five first TG and five pairs of CC or CA were taken from female pupae of diapause egg producers f day after larval-pupal ecdysis and then implanted into N, 4-day-old pharate adults.
Induction of egg diapause by CC and CA in Bombyx mori
943
Table 3. Effect of JH injection on diapause egg production in non-diapause egg producers Injection
No.
No.
of
animals treated NO”e
2 ul Of peanut oil
Mean Non-diap.
of
laid Diap.
eggs Total
30
0
0
237
0
237(
O.O+
14
0
0
22R
0
228(
0.0)
9
0
0
255
0
255(
0.0)
9
0
3
213
13
226(
6.8)
8
0
1
237
3
2401
1.41
llg of JH
lO(
pg of JH
15(12)
Of JH
Mixed eggs
30(30)"
2
p9
laying
Diap. egg only
15(14)
0.2
LO
of adults
Non-diap. egg only
10~
9)
9)
JH (synthetic dl-C, *-cecropia juvenile hormone) dissolved in 2 pl of peanut oil at each concentration.
injected into N, 4-day-old pharate adults.
the number of SG implanted and diapause egg production: the percentages of diapause egg increased almost linearly with the increasing number of SG implanted. These results show that implanted CC, CA, as well aS SG, can cause diapause eggs in silkworms, but the effect of the CA is different from that of SG or CC. ~iu~ause egg production by CC and CA implanted into pharate adults with SG removed
The possibility remains that the CC and CA implants may stimulate the SG of recipients to secrete the diapause hormone. In order to clarify this possibility, we implanted five pairs of CC or CA and five first 1% into the pharate adults of which SG were removed on the day of larval-pupal ecdysis and the diapause nature of the eggs laid was examined (Table 2). The SG-removal reduced the number of eggs laid and the reduction was not restored by the implantation of CC, CA. and 1st TG. The adults with only the SG removed or that received five first TG (control) laid non-diapause eggs, as expected. As for CC, all the three adults treated laid mixed eggs and 77.3% of the laid eggs were of the diapause type. There was no difference in diapause egg production between adults with and without intact SG. On the other hand, 5 of the seven adults implanted with five pairs of CA laid mixed eggs, and the rate of diapause eggs was ISSo/, being about one fourth that laid by the adults with intact SG and implanted CA (Table 1). The diapause nature of the eggs remaining in the adults was examined by the Ehrich’s diazo reaction according to HASEGAWA (1952). Sixty to 70”/, of eggs obtained from the adults with CC and 10 to 20% of eggs from the adults with CA showed a positive reaction suggesting they were diapause eggs, whereas none of the eggs from the adults with SG removed alone or that had received five first TG did. From these results, it seems clear that the implanted CC and CA directly induced egg diapause irrespective of the presence or the absence of the intact SG. However, the reduced activity of CA in the absence of SG suggests that the CA is in some way connected with the SG.
Effect ofjuvenile hormone injectiorl into pharate adults of non-diapause egg producers
Synthetic dl-C,,-cecropia JH dissolved in peanut oil was injected into non-diapause egg producers (0.2, 2, and 10 pg in 2 ~1 of peanut oil/animal). The results are shown in Table 3. The injection of JH at 0.2, 2, and 10 pg per animal had no effect on development. None of the adults receiving 2 1.11of peanut oil or 0.2 pg of JH produced diapause eggs. However. 3 of the 12 adults injected with 2 pg of JH and 1 of the 9 adults with 1Opg of JH laid mixed eggs. The percentages of diapause eggs in these adults were 6.8 and 1.4%, respectively. However, these values are too small to suggest that JH injection causes egg diapause in some way. DISCUSSION It has been documented by earlier experiments (HASEGAWA,1952; FUKUDA,1951, 1953) that only SG secretes the hormone responsible for diapause in silkworm eggs. However, present results suggest that, in addition to SG, CC and CA can also induce egg diapause when implanted into non-diapause egg producers. There are clear differences in the developmental stages of both donors and recipients used between Hasegawa’s and Fukuda’s experiments and ours. HASEGAWA(1952) mainly used mature larvae and pharate pupae, and FUKUDA (1951, 1953) used 4th and 5th instar larvae and newly ecdysed pupae. In this experiment, newly ecdysed pupae were used as donors and /t-day-old pharate adults as the recipients. It is therefore probable that the implanted CC and CA are active at a more restricted stage of the recipients than is the SG. Such stage specificity of the recipient was also suggested on the following experiments. The Br-CC-CA complexes from the armyworm larvae, Leucania separata, induced egg diapause in the silkworm when implanted into 4-day-old pharate adults (OGURA and SAITO, 1973), but not when implanted into newly ecdysed pupae (Ogura, unpublished observations. Dependence on number of CC or CA impl~ted (Fig. 1) suggested that the CC or CA secrete a hor-
944
S. TAKEDAANDN. OGURA
monal factor. High activity of CC, even when a small number are impl~ted suggested that their action on the ovaries in inducing egg diapause is direct as shown for the SG. The idea is supported by the fact that the diapause egg production by CC was not reduced in the SG-removed recipients (Table 2). In the case of CA. the dependency was quite different from that of SG or CC. Although five pairs of CA induced a larger number of diapause eggs than that of five SG, three pairs of CA hardly showed an overt effect. From this result, it may be presumed that the action site of CA in the sequence for diapause induc-
tion is different from that of SG or CC: the hormonal substance from CA may act rather indirectly for diapause induction whereas the diapause hormone from SG directly controls the diapause nature of the eggs. Reduced activity of CA implanted into the recipients of which the SG were removed (Table 2) implies that CA affects SC activity in producing diapause eggs. ~OROHOSHI (1972) recently postulated that the activity of the CA in the larval stage influenced the voltinism by controlling the development of the SG. From our present results there is the slight possibility that in the pharate adult JH itself affects diapause egg induction but our results are not clear cut. On the other hand, a higher production of diapause eggs when five pairs of CA were implanted (Table 1) may be in part due to the direct action of CA on the induction of egg diapause. Indeed, this idea was supported by a low but significant induction of egg diapause by the implan~tion of CA into the pharate adults with the SG removed (Table 2). SONOBE and KEINO (1975) reported that substances with diapause hormone activity were extractable from the brain of silkworm pharate adults, which was assayed by 2-hydroxyk~urenine accumulation in ovaries. Their findings and our results indicate the possibility that the substance(s) with diapause hormone activity is (are) produced in neurosecretory cells of the brain as well as in the SG and that they are temporarily stored in CC and CA like some neurohormones produced in the brain in some insects (see
WIGGLESWORTH. 1970). Thus the CC and CA may
release the substance that directly induces egg diapause. Implanted CA also seems to act on SG of recipients to release the diapause hormone. However, it remains to be resolved whether or not the brain CC, and CA in situ induce egg diapause. .-t~i,frltri(~,c!y~~l;rt,/fr,s--The authors ~icknow~edge Prof. S. KA~AS~, S&cultural Laboratory, Faculty of Agriculture, Nagoya University, for his advice and criticism of the manuscript.
REFERENCES FURUIIA S. (1951) The production of the diapause eggs by transplanting the suboesophageai ganglion in the silkworm. Proc. Japan Acud. 27. 672-617. FUKUDA S. (1953) Determination of voltinism in the univoltine silkworm, Proc. Japan Acad. 29, 381-384. HASEGAWAK. (1951) Studies on the voltinism in the silkworm, Bomhyx rnori L., with special reference to the organs concerning determination of voltinism (a preliminary note). Proc. Juputt .Acad. 27. 667-672. HASEGAWAK. (1952) Studies on the voitinism in the silkworm. Bomhjx ttrctri L., with special reference to the organs concerning determination of voltinism. J. Far. Agric. Totcori Unit). 1. 84--1X4. HASEGAWAK. (1957) The diapause hormone of the silkworm Bomhyx mori. Nature, Lond. 179. 1300-1301. MOROHOSHI S. (1973) The control of growth and devetopment in Botnhg.x rtzori-XV. Consideration of the function of the brain on the activity of the corpora allata. Proc. Japan Acad. 48. 433-438.
OGURA N. and SAITOT. (1973) Induction of embryonic diapause in the silkworm, ~u~by~ mori L. (Lepidoptera: 3ombycidae)~ by implantation of ganglia of the common army~orm larvae. Leucania separata Walker (Lepidoptera: Noctuidae). Aool. Eur. Zool. 8. 46-48. SONOBE H. and I&IN~ H. (1975) Diapause factor in the brains, suboesophageal ganglia and prothoracic ganglia of the silkworm. Narurwiss. 62, 348-349. WIGGLESWORTH V. 8. (1970) Insect Hormortes. Oliver & Boyd. Clark, Edinburgh.
INDUCTION OF EGG DIAPAUSE BY IMPLANTATION OF CORPORA CARDIACA AND CORPORA ALLATA IN BOMBIX MORI SATOSHITAKEDA’
and NOBUO OGURA’
‘Sericultural Laboratory and ‘Laboratory of Applied Entomology and Nematology, Faculty of Agriculture, Nagoya University, Chikusa, Nagoya Japan (Received 3 January 1976)
Abstract-Diapause egg production was examined in non-diapause egg producers by implantation of various cephalic organs into pharate adults 4 days after larval-pupal ecdysis. The implantation of five pairs of corpora cardiaca or corpora allata induced a great amount of egg diapause. Implantation of these organs was effective in inducing egg diapause even when the suboesophageal ganglion of the recipients had been removed, although the effect of the corpora allata decreased moderately. The injection of juvenile hormone into 4-day-old pharate adults did not greatly increase production of diapause eggs.
INTRODUCTION IT IS well known that diapause in the silkworm Bombyx mori, is induced by a neurohormone, the
egg. diapause hormone, which is released from the suboesophageal ganglion (SG) during pupal-adult development (HASEGAWA, 1951, 1952, 1957; FUKUDA, 1951). In early endocrinological work only the SG was thought of as the organ secreting the hormone; other organs such as the brain (Br), corpora allata (CA), and prothoracic gland were not supposed to be concerned with production of the hormone. Recently, OGURA and SAITO(1973) showed by implantation experiments that the brain-corpora cardiaca-corpora allata complexes (Br-CC-CA) of the armyworm, kucania separata, can induce egg diapause in silkworms. Furthermore, the brain of pharate adults of silkworms contains a substance with diapause hormone activity (S~NOBE and KEINO, 1975). Consequently, Br. CC, and CA as well as SG may be responsible for the induction of the egg diapause in silkworms. We have re-examined endocrinologically the organs which contributed to the induction of egg diapause in the silkworm and have found, that both the CC and the CA are concerned in part in the induction of egg diapause in Bombyx mori in addition to the SG. MATERIALS
AND METHODS
The silkworms, Bombyx mori, used were the bivoltine race (Kinshu x Showa) and polyvoltine race (NJ. The former was incubated at 25°C and light during embryonic life to obtain diapause egg producers (diapause pupae) and the latter at 15°C and
dark to avoid the occasional occurrence of diapause eggs. Br, CC, CA, SG, and the 1st thoracic ganglion (first TG) were prepared from the female pupae of diapause egg producers on the day of the larval-pupal ecdysis. These organs were implanted into non-diapause type recipients (N4) on day 4 after the larval-pupal ecdysis. In another experiment, pharate adults, of which the SG were removed just after the larval-pupal ecdysis, were also used as the recipients to see the interaction of CC or CA to SG. Synthetic dl-C1 ,-cecropia juvenile hormone (Zoecon Corporation) was dissolved in 2 d of peanut oil at each concentration, then injected into non-diapause pharate adults 4 days of age. RESULTS Diapause egg production cephalic organs
by
implantation
of
some
First, Br, CC, CA, and 1st TG, as well as SG obtained from diapause pupae. were implanted into the 4-day-old non-diapause type pharate adults with intact SG. These animals kept at 25°C emerged as adults 8 days after the larval-pupal ecdysis, and then laid eggs normally. The diapause nature was determined 15 days after oviposition, when larvae had hatched from non-diapause eggs. The results are shown in Table 1. The Br implantation caused four of the seven adults to lay egg masses consisting of diapause and non-diapause eggs (mixed eggs), the percentage of diapause eggs being 6.8. All the adults implanted with five pairs of CC or CA and five SG laid the mixed eggs, whereas the adults implanted with five first TG laid only non-diapause eggs. In the case of CC implantation, the eggs laid were somewhat
941
S. TAKEDAAND N. OGURA
942
Table 1. Diapause egg production in non-diapause egg producers by implantation of some endocrine organs or ganglia Implants
No. of animals treated
Wean of laid eggs
No. of adults laying Non-diap. egg only
Diap. e9g only
Mixed eggs
Non-diap. Diap. Total 241( 6.8)'
or
7c71*
3
0
4
226
15
CC
S(4)
0
0
4
56
130
186c72.4)
CA
S(4)
0
0
4
74
131
205(68.8)
SG
7(S)
0
0
5
105
103
2011(48.5)
7(7)
7
0
0
7.42
1st TG
0
242( 0.01
Fkv Br, SC, 1st TG and 5 pairs of CC, CA were taken from female pupae of diapause egg producers within 1 day after larval-pupal ecdysis. These organs were implanted into 4-day-old pharate adults of N, (a polyvoltine race). *Numerals in the parentheses refer to number of adults laying fertilized eggs; tnumerals in the parentheses refer to average of diapause egg percentages (the same applies to Tables 2 & 3).
60
,-” - 60 f 3 4 4o B 20
planted.
We next examined the relationship between implanted number of CC, CA, and SG and rate of diapause eggs in the resultant eggs. As shown in Fig. 1, diapause eggs were produced when one or two pairs of CC were implanted. Furthermore, three and five pairs are highly effective in inducing egg diapause; the adults implanted with three pairs of CC laid about 65yi diapause eggs and with 5 pairs about 754; of CC. When one or two pairs of CA were implanted. no diapause eggs were found. Although three pairs of CA could cause all the adults to lay mixed eggs the percentages of diapause eggs were very low. Five pairs of CA caused all the adults to lay mixed batches of eggs about 704, of eggs produced were diapause eggs. The activity of SG is rather less than that of CC, but a closer relationship was observed between
0
lOor
reduced in number. However, the percentage of diapause eggs was notably high compared with that of CA or SC ~mpl~tation. These results clearly show that CC and CA are effective in inducing egg diapause in non-diapause egg producers when they are im-
‘
I
0
1
2 3 No. of implants
Fig. 1. Relationship between No. of SG or No. of pairs of CC or CA implanted and diapause egg production. CC (A). CA (0) and SC (a) taken from female pupae of diapause egg producers 1 day after larval-pupal ecdysis and then implanted into 4-day-old pharate adults of Nti Each line is drawn through the mean value of each series of experiments on 3 to 5 adults.
Table 2. Diapause egg production by implantation of CC and CA in non-diapause egg producers with the removed SG Inplants
NO. of animals treated
no. of adults laying Diap. Non-diap. egg Only egg only
CC
s(3)*
0
CA
8 (7)
2
1st TG
7(6)
6
15(9)
9
0
None
Mixed eggs
Mean of laid eggs Won-diap.
Diap.
Total
3
24
89
11?(77.3)+
0
5
113
22
135115.5)
0
0
I.15
0
115( 0.0)
0
142
0
142( 0.0)
0
5
The removal of SC of the recipients, within I day after larval-pupal ecdysis. Five first TG and five pairs of CC or CA were taken from female pupae of diapause egg producers f day after larval-pupal ecdysis and then implanted into N, 4-day-old pharate adults.
Induction of egg diapause by CC and CA in Bombyx mori
943
Table 3. Effect of JH injection on diapause egg production in non-diapause egg producers Injection
No.
No.
of
animals treated NO”e
2 ul Of peanut oil
Mean Non-diap.
of
laid Diap.
eggs Total
30
0
0
237
0
237(
O.O+
14
0
0
22R
0
228(
0.0)
9
0
0
255
0
255(
0.0)
9
0
3
213
13
226(
6.8)
8
0
1
237
3
2401
1.41
llg of JH
lO(
pg of JH
15(12)
Of JH
Mixed eggs
30(30)"
2
p9
laying
Diap. egg only
15(14)
0.2
LO
of adults
Non-diap. egg only
10~
9)
9)
JH (synthetic dl-C, *-cecropia juvenile hormone) dissolved in 2 pl of peanut oil at each concentration.
injected into N, 4-day-old pharate adults.
the number of SG implanted and diapause egg production: the percentages of diapause egg increased almost linearly with the increasing number of SG implanted. These results show that implanted CC, CA, as well aS SG, can cause diapause eggs in silkworms, but the effect of the CA is different from that of SG or CC. ~iu~ause egg production by CC and CA implanted into pharate adults with SG removed
The possibility remains that the CC and CA implants may stimulate the SG of recipients to secrete the diapause hormone. In order to clarify this possibility, we implanted five pairs of CC or CA and five first 1% into the pharate adults of which SG were removed on the day of larval-pupal ecdysis and the diapause nature of the eggs laid was examined (Table 2). The SG-removal reduced the number of eggs laid and the reduction was not restored by the implantation of CC, CA. and 1st TG. The adults with only the SG removed or that received five first TG (control) laid non-diapause eggs, as expected. As for CC, all the three adults treated laid mixed eggs and 77.3% of the laid eggs were of the diapause type. There was no difference in diapause egg production between adults with and without intact SG. On the other hand, 5 of the seven adults implanted with five pairs of CA laid mixed eggs, and the rate of diapause eggs was ISSo/, being about one fourth that laid by the adults with intact SG and implanted CA (Table 1). The diapause nature of the eggs remaining in the adults was examined by the Ehrich’s diazo reaction according to HASEGAWA (1952). Sixty to 70”/, of eggs obtained from the adults with CC and 10 to 20% of eggs from the adults with CA showed a positive reaction suggesting they were diapause eggs, whereas none of the eggs from the adults with SG removed alone or that had received five first TG did. From these results, it seems clear that the implanted CC and CA directly induced egg diapause irrespective of the presence or the absence of the intact SG. However, the reduced activity of CA in the absence of SG suggests that the CA is in some way connected with the SG.
Effect ofjuvenile hormone injectiorl into pharate adults of non-diapause egg producers
Synthetic dl-C,,-cecropia JH dissolved in peanut oil was injected into non-diapause egg producers (0.2, 2, and 10 pg in 2 ~1 of peanut oil/animal). The results are shown in Table 3. The injection of JH at 0.2, 2, and 10 pg per animal had no effect on development. None of the adults receiving 2 1.11of peanut oil or 0.2 pg of JH produced diapause eggs. However. 3 of the 12 adults injected with 2 pg of JH and 1 of the 9 adults with 1Opg of JH laid mixed eggs. The percentages of diapause eggs in these adults were 6.8 and 1.4%, respectively. However, these values are too small to suggest that JH injection causes egg diapause in some way. DISCUSSION It has been documented by earlier experiments (HASEGAWA,1952; FUKUDA,1951, 1953) that only SG secretes the hormone responsible for diapause in silkworm eggs. However, present results suggest that, in addition to SG, CC and CA can also induce egg diapause when implanted into non-diapause egg producers. There are clear differences in the developmental stages of both donors and recipients used between Hasegawa’s and Fukuda’s experiments and ours. HASEGAWA(1952) mainly used mature larvae and pharate pupae, and FUKUDA (1951, 1953) used 4th and 5th instar larvae and newly ecdysed pupae. In this experiment, newly ecdysed pupae were used as donors and /t-day-old pharate adults as the recipients. It is therefore probable that the implanted CC and CA are active at a more restricted stage of the recipients than is the SG. Such stage specificity of the recipient was also suggested on the following experiments. The Br-CC-CA complexes from the armyworm larvae, Leucania separata, induced egg diapause in the silkworm when implanted into 4-day-old pharate adults (OGURA and SAITO, 1973), but not when implanted into newly ecdysed pupae (Ogura, unpublished observations. Dependence on number of CC or CA impl~ted (Fig. 1) suggested that the CC or CA secrete a hor-
944
S. TAKEDAANDN. OGURA
monal factor. High activity of CC, even when a small number are impl~ted suggested that their action on the ovaries in inducing egg diapause is direct as shown for the SG. The idea is supported by the fact that the diapause egg production by CC was not reduced in the SG-removed recipients (Table 2). In the case of CA. the dependency was quite different from that of SG or CC. Although five pairs of CA induced a larger number of diapause eggs than that of five SG, three pairs of CA hardly showed an overt effect. From this result, it may be presumed that the action site of CA in the sequence for diapause induc-
tion is different from that of SG or CC: the hormonal substance from CA may act rather indirectly for diapause induction whereas the diapause hormone from SG directly controls the diapause nature of the eggs. Reduced activity of CA implanted into the recipients of which the SG were removed (Table 2) implies that CA affects SC activity in producing diapause eggs. ~OROHOSHI (1972) recently postulated that the activity of the CA in the larval stage influenced the voltinism by controlling the development of the SG. From our present results there is the slight possibility that in the pharate adult JH itself affects diapause egg induction but our results are not clear cut. On the other hand, a higher production of diapause eggs when five pairs of CA were implanted (Table 1) may be in part due to the direct action of CA on the induction of egg diapause. Indeed, this idea was supported by a low but significant induction of egg diapause by the implan~tion of CA into the pharate adults with the SG removed (Table 2). SONOBE and KEINO (1975) reported that substances with diapause hormone activity were extractable from the brain of silkworm pharate adults, which was assayed by 2-hydroxyk~urenine accumulation in ovaries. Their findings and our results indicate the possibility that the substance(s) with diapause hormone activity is (are) produced in neurosecretory cells of the brain as well as in the SG and that they are temporarily stored in CC and CA like some neurohormones produced in the brain in some insects (see
WIGGLESWORTH. 1970). Thus the CC and CA may
release the substance that directly induces egg diapause. Implanted CA also seems to act on SG of recipients to release the diapause hormone. However, it remains to be resolved whether or not the brain CC, and CA in situ induce egg diapause. .-t~i,frltri(~,c!y~~l;rt,/fr,s--The authors ~icknow~edge Prof. S. KA~AS~, S&cultural Laboratory, Faculty of Agriculture, Nagoya University, for his advice and criticism of the manuscript.
REFERENCES FURUIIA S. (1951) The production of the diapause eggs by transplanting the suboesophageai ganglion in the silkworm. Proc. Japan Acud. 27. 672-617. FUKUDA S. (1953) Determination of voltinism in the univoltine silkworm, Proc. Japan Acad. 29, 381-384. HASEGAWAK. (1951) Studies on the voltinism in the silkworm, Bomhyx rnori L., with special reference to the organs concerning determination of voltinism (a preliminary note). Proc. Juputt .Acad. 27. 667-672. HASEGAWAK. (1952) Studies on the voitinism in the silkworm. Bomhjx ttrctri L., with special reference to the organs concerning determination of voltinism. J. Far. Agric. Totcori Unit). 1. 84--1X4. HASEGAWAK. (1957) The diapause hormone of the silkworm Bomhyx mori. Nature, Lond. 179. 1300-1301. MOROHOSHI S. (1973) The control of growth and devetopment in Botnhg.x rtzori-XV. Consideration of the function of the brain on the activity of the corpora allata. Proc. Japan Acad. 48. 433-438.
OGURA N. and SAITOT. (1973) Induction of embryonic diapause in the silkworm, ~u~by~ mori L. (Lepidoptera: 3ombycidae)~ by implantation of ganglia of the common army~orm larvae. Leucania separata Walker (Lepidoptera: Noctuidae). Aool. Eur. Zool. 8. 46-48. SONOBE H. and I&IN~ H. (1975) Diapause factor in the brains, suboesophageal ganglia and prothoracic ganglia of the silkworm. Narurwiss. 62, 348-349. WIGGLESWORTH V. 8. (1970) Insect Hormortes. Oliver & Boyd. Clark, Edinburgh.