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Control mechanism of diapause of the pharate first-instar larvae of the silkmoth Antheraea yamamai
J. Insect Physiol. Vol.36, No. 11,PP. 855-860,1990 Printedin Great Britain.All rightsreserved
0022-1910/90 $3.00+ 0.00 Copyright0 1990PergamonPressplc
CONTROL MECHANISM OF DIAPAUSE OF THE PHARATE FIRST-INSTAR LARVAE OF THE SILKMOTH ANTHERAEA YAMAMAI Ko~crn SUZUKI,‘* TSUKASAMINAGAWA,’TAKUYAKUMAGAI,’SHM-ICHINAYA,~ YASUHISAENDO,’MINORUOSANAI’and Encrn K~_J~ANo~ ‘Faculty of Agriculture, Iwate University, Morioka 020, 2Faculty of Textile Science, Kyoto Institute of Technology, Kyoto 606, 3Department of Biology, Tokyo Metropolitan Institute of Gerontology, Tokyo 173 and 4Department of Agriculture Chemistry, Kyushu University, Fukuoka 812, Japan (Received 25 May 1990; revised 3 July 1990)
Abstract-The application of an imidazole derivative (KK-42), body ligation and extirpation of specific ganglia suggest a new endocrine mechanism responsible for the regulation of diapause in pharate first-instar larvae of insects. The present results indicate that a humoral repressive factor originates in the region of the mesothorax and that a humoral maturation factor is released from the region of the 2nd to 5th abdominal segments. Our data suggest that the former inhibits the action of the latter during diapause. Key Word Index: Diapause;
pharate first-instar larva; control mechanism
MATERIALSAND METHODS
INTRODUCTION An arrest of development and differentiation known as diapause can occur in any developmental stage such as the egg, larva, pupa or adult, and is controlled by the central endocrine system in insects (Chippendale and Turunen, 1981; Chippendale, 1983; de Kort, 1981; de Wilde, 1983; Denlinger, 1985; Yamashita and Hasegawa, 1985). Arrested development in the egg has been observed to occur at any of five stages of embryonic development. Development stops at the fully formed, pharate, first-ins& stage in some orthopteran and lepidopteran insects (Umeya, 1946, 1950; Umeya and Osanai, 1953). Although the latter type has been classified as larval diapause (Chippendale, 1977), its hormonal regulation has not been established to date. We have succeeded in terminating diapause in the pharate first-instar of the wild silkmoth, Antheraea yumumai by the application of imidazole derivatives; this resulted in a method for artificial hatching (Suzuki et al., 1989). Following this finding, we have now learned that diapause in the pharate first-instar is regulated by a new endocrine system independent of the central endocrine system which includes the brain (prothoracicotropic hormone), copora allata (juvenile hormone), suboesophageal ganglion (diapause hormone) and prothoracic gland (ecdysone). *To whom all correspondence should be. addressed at: Department of Entomology, College of Agriculture, The University of Arizona, Tucson, AZ 8572I, U.S.A.
Larvae of the wild silkmoth, A. yamamai, were reared on fresh foliage of a Japanese oak, Quercus serruta or artificial diet produced by Nippon Chlorella Co., Ltd (Japan). The oviposited eggs were incubated at 25°C and washed with 0.5% chlorinated lime for IO min to remove the adhesives. Ten days after oviposition the eggs enter diapause as pharate first-instar larvae [Fig. l(A)]. Eggs were held at 25°C but the chorion was removed 1 day before the experiments. The naked, diapausing, pharate, first-instar larvae were held in a Petri dish containing a filter paper immersed in a solution of streptomycin (0.25%), 4hydroxybenzoic acid (1%) and amphotericin (0.25%) as antibiotics. They were used for the experimental procedures including application of the imidazole derivative, body ligation with dental floss, and extirpation of ganglia. The imidazole derivative, l-benzyl-5-[(E)-2,6dimethyl-1,5-heptadienyl] imidazoie (KK-42) synthesized according to the previous method (Kuwano et al., 1985), has been shown to terminate diapause of pharate first-instar larvae (Suzuki et al., 1989). KK-42 was dissolved in acetone and applied to the pharate first-instar larvae and body compartments during diapause. The head-thorax compartment [Fig. l(B)] was produced by a single ligation of the intersegmental membrane between the metathorax and 1st abdominal segment and the posterior region to the ligation being cut-off with fine forceps. The anterior region to this ligation was cut-off and the remnant was used as the 855
KOICHISUZUKIet al.
856
abdominal compartment [Fig. l(C)]. Wounds were sealed with the powder of streptomycin and phenylthiourea (1: 1) and on the next day the body compartments were treated with acetone or KK-42 (0.1 pg/ 0.5 pi/larva). They were incubated at 25°C under moist conditions and observed every day. Diapause breakdown was determined 4-6 days after the experimental procedures by yellow colour and melanized stripes in the integument, dark legs, reddish cervical shield, standing bristles, and locomotion. When the above features were observed, we determined the insects as diapause-terminated. RESULTS The pharate first-instar larvae exposed by the removal of the chorion sometimes showed a higher mortality compared with normal intact eggs (Suzuki et al., 1989). Seventy to 100% of naked, pharate, firstinstar larvae, however, terminated diapause following the application of KK-42 (Table 1). The optimal concentration was 0.1 pg/larva; about one-two hundredth in comparison with the case of the intact whole egg (Suzuki et al., 1990). To elucidate the action of KK-42 and the mechanism of diapause, intersegmental membranes were ligated with fine dental floss between the main body compartments of pharate first-in&r larva. The results are shown in Fig. l(B, C) and Table 1. The headless compartment of thorax-abdomen broke diapause following the application of KK-42 as well as the intact whole body, but the head-thorax compartment continued an apparent diapause. In addition, KK-42 application was not always indispensable to diapause breakdown on the abdominal compartment which could be induced merely by ligation. We speculate that the head-thorax compartment lacks some maturation factor and consequently this
compartment cannot terminate diapause. On the other hand, it seems that the abdomen compartment may be free from a repressive factor and it is able to mature directly. Therefore, a repressive factor may be present in the head-thorax compartments and a maturation factor must exist in the abdominal compartment. This possibility was examined by moving ligatures and by double ligations of intersegmental membrane from the thorax to the abdomen. As shown in Fig. I@) and Tables 2 and 3, the repressive factor might originate in the region of mesothorax while the maturation factor occurs in the region of the 2nd to 5th abdominal segments. The extirpation of specific ganglia was performed to determine where the two factors originate and whether they function through a humoral or neural system. Although the difficult surgical extirpation of a ganglion with fine opthalmological tweezer caused serious injury to diapausing, pharate, first-instar larvae, several interesting results are shown in Table 4. Following extirpation of the mesothoracic ganglion, treatment with KK-42 terminated diapause successfully. The same results were obtained with the 4th and 5th abdominal ganglia were extirpated and the insect treated with KK-42, although the ratio of diapause breakdown was lower due to the serious injury of the operation. We conclude that a humoral repressive factor originates in the region of the mesothorax and a humoral muturation factor exists in the region of the 2nd to 5th abdominal segments. DJSCUSSION
Yamashita et al. (1987) showed that KK-42 acts directly on the prothoracic gland to inhibit ecdysone synthesis in the silkworm, Bombyx mori. Akai and Mauchamp (1989) reported that this chemical depresses the titres of juvenile hormones I and II in
Table 1. Effect of a single ligation on diapause breakdown in the main body compartments of pharate first-instar larva Nos of compartments Body compartment Whole body-l Whole body-2 Head-thorax* Thorax-abdomen?
Abdomen*
Nos of compartments
Application
Diapausing
Diapause broken
30 30 55 55 65 65 40 40 40 40 40 40
Acetone KK-42 Acetone KK-42 Acetone KK-42 None Acetone KK-42 None Acetone KK-42
30 0 39 11 59 57 33 31 10 19 15 19
0 30 0 22 0 1 0 0 13 13 7 12
Dead$ 0 0
Ratio of diapause breakdown (“/) 0.0 100.0
16
0.0
22 6 7 7 9 17 8 18 9
66.7 0.0 1.7 0.0 0.0 56.5 40.6 31.8 38.7
*See Fig. 1. tThe thorax-abdomen compartment was produced by single ligation at the neck and the rest was cut off. $Body compartments that died during the observation period was excluded from each group. This applied to Tables 2, 3 and 4.
Fig. 1. Photographs of pharate first-instar larva and those body compartments diapausing or breaking from diapause. A = Pharate first-instar larva which starts to enter diapause on the 10th day after oviposition and maintains diapause over one month, was removed from the chorion. The integumentary colour is yellowish-green during diapause. B = Diapausing head-thorax compartments (left, acetone application and right, KK-42 application). C = Abdomen compartment breaking from diapause only by ligation. The integument shows yellow colour and melanized stripes. This body compartment also has dark legs and standing bristles and finally wanders. D = Ligation position shifted at each intersegmental membrane (results shown in Table 2). Numerals are equal to those in Table 2. Scale bar = 6mm.
857
Diapause of pharate first-instar
859
Table 2. Effect of varying the position of ligation on diapause breakdown in pharate first-instar larvae Nos of pharate first-instars Region ligated
N
Anterior and posterior to ligation
Diapausing
Ligation between thoraxes: 30 Anterior* 1. ProthoraxPosterior mesothorax 30 Anterior 2. MesothoraxPosterior metathorax Ligation between abdominal segments: 30 Anterior 3. lst-2nd Posterior 30 Anterior 4.2nd-3rd Posterior 30 Anterior 5.3rd4th Posterior 30 Anterior 6.4th-5th Posterior 30 Anterior 7. Sthdth Posterior 30 Anterior 8.6th-7th Posterior 30 Anterior 9.7th-8th Posterior
Diapause broken
Dead (0)
:8 13
0 0 0 17
28 17 20 2 21 1 25 8 13 13 29 29 20 20
0 11 0 18 0 20 0 17 0 0 0 0 0 0
(2)
30
Ratio of diapause breakdown WI 0.0 0.0 0.0 56.7
(0)
0.0 39.3 0.0 90.0 0.0 95.2 0.0 68.0 0.0 0.0 0.0 0.0 0.0 0.0
(10) (9) (5) (17) (1) (10‘) .’
*Regions anterior and posterior to the ligatures were observed for 7 days and diapause breakdown was determined every day.
the haemolymph of Bombyx larvae. However, our results do not indicate that KK-42 functions as anti-ecdysteroid or anti-juvenile hormone since we determined the titre of ecdysteroids and investigated the effect of rescuing with some juvenile hormone analogues (Suzuki et al., 1990). Our present and previous results suggests that KK-42 reduces the humoral action of a repressive factor present in the
region of mesothorax of pharate first-instar larvae during diapause. Osanai and Arai (1962) reported, for the first time, an occurrence of a diapause factor in the 6th abdominal segment of the 4th (penultimate)-instar larva of nymphalid butterfly, Hestina juponica. This humoral factor causes also integumentary colour change from green to brown at the beginning of this
Table 3. Effect of double ligations on diapause breakdown of pharate tirst-instar larvae Region ligated 1st ligation
2nd ligation
N
Prothorax-mesothorax
and A,-A:
30
Mesothorax-metathorax
Mesothorax-metathorax
Mesothorax-metathorax
Metathorax-A,
Metathorax-A,
and A, -A2
and AZ-A,
and A,-A4
and A, -A4
and A,-A,
30
30
30
30
30
Anterior, middle and posterior to ligation
Nos of pharate first-instars Diapausing
Diapause broken
Anterior? Middle Posterior
25 25 20
8 5
Anterior Middle Posterior
29 29 16
Anterior Middle Posterior
Dead
Ratio of diapause breakdown WI
(5)
0.0 0.0 20.0
0 0 13
(1)
0.0 0.0 44.8
30 18 12
0 12 18
(0)
0.0 40.0 60.0
Anterior Middle Posterior
30 13 4
0 17 26
(0)
0.0 56.7 86.7
Anterior Middle Posterior
:x
(0)
5
0 7 25
0.0 23.3 83.3
Anterior Middle Posterior
30 27 12
0 3 18
(0)
0.0 10.0 60.0
*The 1st ligation was performed at the intersegmental region between the prothorax and mesothorax, and the 2nd ligation was between the 1st and 2nd abdominal segments, -/‘Regions anterior to the 1st ligation, between the 1st and 2nd ligations, and posterior to the 2nd ligation, were observed for 7 days and diapause breakdown was determined every day.
KOICHI SUZUKIet al.
860
Table 4. Effect of extirpation of specific ganglia on diapause breakdown of pharate first-instar larvae Nos of pharate first-instars Diapause broken
Dead
Ratio of diapause breakdown (“h)
30 13
0 10
0 7
0.0 43.5
None KK-42
30 22
08
0
2::;
None KK-42
30 25
0 4
0 1
0.0 13.8
Application
Extirpation
N
Mesothoracic ganglion
30 30
None KK-42*
4th and 5th Abdominal ganglia
30
5th Abdominal ganglion
30 30
Diapausing
*After extirpation, the insects were chilled at 2°C one day aud on the next day they were treated with KK-42, and thereafter they were observed for 7 days and diapause breakdown was determined every day. larval cliapause. Ecdysone-containing extracts had no influence on the colour changes as well as onset of diapause. On the other hand, Sakate (1984) speculated that in A. yamamai some abdominal segments have a development factor, probably our maturation factor, this is induced by chilling for a long period, but he could not develop this speculation because of his untimely death. Thus these reports suggested partially the operation of a new endocrine system controlling insect diapause. The present study, however, establishes that diapause in pharate first-instar larvae is regulated through the action of a humoral repressive factor which inhibits the expression of humoral maturation factor. It also indicates a new sort of the endocrine system that is responsible for the regulation of insect development and differentiation, independent of the central endocrine system composed of the brain, corpora all&a, suboesophageal ganglion and prothoracic gland. Acknowledgement-We
wish to thank Professor B. W. Bowers of Department of Entomology, The University Arizona, Tex., for critical reading of the manuscript.
REFERENCES
Akai H. and Mauchamp B. (1989) Suppressive effects of an imidazole derivative, KK-42 on JH levels in hemolymph of Bombyx larvae. J. Seric. Sci. Jpn 58, 73-74. Chippendale G. M. (1977) Hormonal regulation of larval diapause. A. Rev. Ent. 22, 121-138. Chippendale G. M. (1983) Larval and pupal diapause. In .&docrinology of Insects (Edited by Downer R. G. and Laufer H.). UD. 343-356. Liss. New York. Chippendale G. M. and Turunen S. (1981) Hormonal and metabolic aspects of the larval diapause of the southwestern corn borer, Diatraea grandiosella (Lepidoptera: Pyralidae). Em. Gen. 7, 223-231. Denlinaer D. L. (1985) Hormonal control of diapause. In Comparative‘ Znsect Physiology, Biochemistry and Pharmacology (Edited by Kerkut G. A. and Gilbert L. I.), Vol. 8, pp. 353-412. Pergamon Press, Oxford.
de Kort A. D. (1981) Hormonal and metabolic regulation of adult diapause in the Colorado Beetle, Leptinotarsa decemlineata (Coleopatera: Chrysomelidae). Em. Gen. 7, 261-271. -__ -._.
Kuwano E., Takeya R. and Eto M. (1985) Synthesis and anti-juvenile hormone activity of I-substituted-5-[(E)-2,6dimethyl-1,5-heptadienyl] imidazoles. Agric. biol. Chem. 49, 483-486.
Osanai M. and Arai Y. (1962) Uber die Umf%rbuug der Raupen von Hestina japonica zu Beginn de tiberwinterung I. Durchschtiruugsversuche an der Umfgrbung der Hestina-Raupe. Gen. camp. Endocr. 2, 311-316. Sakate S. (1984) Hibernation of the eggs of the Japanese oak silkworm. Tech. Bull. Seric. exp. Stat. 123,31-46. In Japanese. Suzuki K., Fujisawa T., Kurihara M., Abe S. and Kuwano E. (1989) Artificial hatching in the silkworm, Antheraea yamamai: application of KK-42 and its analogs. In Wild Silkmoths ‘88-Proceedings of Workshop in XVZZZZnternational Congress of Entomology (Edited by Aaki H. and
Wu Z. S.), pp. 79-84, Business Center for Academic Societies Japan, Tokyo. Suzuki K., Minakawa T., Kumagai T., Naya S., Fujisawa T. and Kuwano E. (1990) The mode of action of KK-42 on diauause breakdown in uharate first instar of the wild silkmoth, Antheraea yamamai. In Wild Silkmoths ‘89 (Edited by Akai H., Kiuchi M. and Tsubouchi K.). In press. Umeya Y. (1946) Embryonic hibernation and diapause in insects from the viewpoint of the hibernating eggs of the silkworm. Bull. Seric. exp. Stat. 12,393-481. In Japanese with English summary. Umeya Y. (1950) Studies on embryonic hibernation and diapause in insects. Proc. Jpn Acad. 26, l-9. Umeya Y. and Osanai M. (1953) On the diapause type of Rhopalocera. Oyo-Dobutsugaku-Zasshi 18, 73-77. In Japanese with English summary. de Wilde J. (1983) Endocrine aspects of diapause in the adult stage. In Endocrinology of Insects (Edited by Downer R. G. and Laufer H.), pp. 357-367. Liss, New York. Yamashita 0. and Hasegawa K. (1985) Embryonic diapause. In Comparative Insect Physiology, Biochemistry and Pharmacology (Edited by Kerkut G. A. and Gilbert L. I.), Vol. 1, pp. 407-434. Pergamon Press, Oxford. Yamashita O., Kadono-Okuda K., Kuwano E. and Eto M. (1987) An imidazole compound as a potent antiecdysteroid in au insect. Agric. biol. Chem. 51, 22952297.
0022-1910/90 $3.00+ 0.00 Copyright0 1990PergamonPressplc
CONTROL MECHANISM OF DIAPAUSE OF THE PHARATE FIRST-INSTAR LARVAE OF THE SILKMOTH ANTHERAEA YAMAMAI Ko~crn SUZUKI,‘* TSUKASAMINAGAWA,’TAKUYAKUMAGAI,’SHM-ICHINAYA,~ YASUHISAENDO,’MINORUOSANAI’and Encrn K~_J~ANo~ ‘Faculty of Agriculture, Iwate University, Morioka 020, 2Faculty of Textile Science, Kyoto Institute of Technology, Kyoto 606, 3Department of Biology, Tokyo Metropolitan Institute of Gerontology, Tokyo 173 and 4Department of Agriculture Chemistry, Kyushu University, Fukuoka 812, Japan (Received 25 May 1990; revised 3 July 1990)
Abstract-The application of an imidazole derivative (KK-42), body ligation and extirpation of specific ganglia suggest a new endocrine mechanism responsible for the regulation of diapause in pharate first-instar larvae of insects. The present results indicate that a humoral repressive factor originates in the region of the mesothorax and that a humoral maturation factor is released from the region of the 2nd to 5th abdominal segments. Our data suggest that the former inhibits the action of the latter during diapause. Key Word Index: Diapause;
pharate first-instar larva; control mechanism
MATERIALSAND METHODS
INTRODUCTION An arrest of development and differentiation known as diapause can occur in any developmental stage such as the egg, larva, pupa or adult, and is controlled by the central endocrine system in insects (Chippendale and Turunen, 1981; Chippendale, 1983; de Kort, 1981; de Wilde, 1983; Denlinger, 1985; Yamashita and Hasegawa, 1985). Arrested development in the egg has been observed to occur at any of five stages of embryonic development. Development stops at the fully formed, pharate, first-ins& stage in some orthopteran and lepidopteran insects (Umeya, 1946, 1950; Umeya and Osanai, 1953). Although the latter type has been classified as larval diapause (Chippendale, 1977), its hormonal regulation has not been established to date. We have succeeded in terminating diapause in the pharate first-instar of the wild silkmoth, Antheraea yumumai by the application of imidazole derivatives; this resulted in a method for artificial hatching (Suzuki et al., 1989). Following this finding, we have now learned that diapause in the pharate first-instar is regulated by a new endocrine system independent of the central endocrine system which includes the brain (prothoracicotropic hormone), copora allata (juvenile hormone), suboesophageal ganglion (diapause hormone) and prothoracic gland (ecdysone). *To whom all correspondence should be. addressed at: Department of Entomology, College of Agriculture, The University of Arizona, Tucson, AZ 8572I, U.S.A.
Larvae of the wild silkmoth, A. yamamai, were reared on fresh foliage of a Japanese oak, Quercus serruta or artificial diet produced by Nippon Chlorella Co., Ltd (Japan). The oviposited eggs were incubated at 25°C and washed with 0.5% chlorinated lime for IO min to remove the adhesives. Ten days after oviposition the eggs enter diapause as pharate first-instar larvae [Fig. l(A)]. Eggs were held at 25°C but the chorion was removed 1 day before the experiments. The naked, diapausing, pharate, first-instar larvae were held in a Petri dish containing a filter paper immersed in a solution of streptomycin (0.25%), 4hydroxybenzoic acid (1%) and amphotericin (0.25%) as antibiotics. They were used for the experimental procedures including application of the imidazole derivative, body ligation with dental floss, and extirpation of ganglia. The imidazole derivative, l-benzyl-5-[(E)-2,6dimethyl-1,5-heptadienyl] imidazoie (KK-42) synthesized according to the previous method (Kuwano et al., 1985), has been shown to terminate diapause of pharate first-instar larvae (Suzuki et al., 1989). KK-42 was dissolved in acetone and applied to the pharate first-instar larvae and body compartments during diapause. The head-thorax compartment [Fig. l(B)] was produced by a single ligation of the intersegmental membrane between the metathorax and 1st abdominal segment and the posterior region to the ligation being cut-off with fine forceps. The anterior region to this ligation was cut-off and the remnant was used as the 855
KOICHISUZUKIet al.
856
abdominal compartment [Fig. l(C)]. Wounds were sealed with the powder of streptomycin and phenylthiourea (1: 1) and on the next day the body compartments were treated with acetone or KK-42 (0.1 pg/ 0.5 pi/larva). They were incubated at 25°C under moist conditions and observed every day. Diapause breakdown was determined 4-6 days after the experimental procedures by yellow colour and melanized stripes in the integument, dark legs, reddish cervical shield, standing bristles, and locomotion. When the above features were observed, we determined the insects as diapause-terminated. RESULTS The pharate first-instar larvae exposed by the removal of the chorion sometimes showed a higher mortality compared with normal intact eggs (Suzuki et al., 1989). Seventy to 100% of naked, pharate, firstinstar larvae, however, terminated diapause following the application of KK-42 (Table 1). The optimal concentration was 0.1 pg/larva; about one-two hundredth in comparison with the case of the intact whole egg (Suzuki et al., 1990). To elucidate the action of KK-42 and the mechanism of diapause, intersegmental membranes were ligated with fine dental floss between the main body compartments of pharate first-in&r larva. The results are shown in Fig. l(B, C) and Table 1. The headless compartment of thorax-abdomen broke diapause following the application of KK-42 as well as the intact whole body, but the head-thorax compartment continued an apparent diapause. In addition, KK-42 application was not always indispensable to diapause breakdown on the abdominal compartment which could be induced merely by ligation. We speculate that the head-thorax compartment lacks some maturation factor and consequently this
compartment cannot terminate diapause. On the other hand, it seems that the abdomen compartment may be free from a repressive factor and it is able to mature directly. Therefore, a repressive factor may be present in the head-thorax compartments and a maturation factor must exist in the abdominal compartment. This possibility was examined by moving ligatures and by double ligations of intersegmental membrane from the thorax to the abdomen. As shown in Fig. I@) and Tables 2 and 3, the repressive factor might originate in the region of mesothorax while the maturation factor occurs in the region of the 2nd to 5th abdominal segments. The extirpation of specific ganglia was performed to determine where the two factors originate and whether they function through a humoral or neural system. Although the difficult surgical extirpation of a ganglion with fine opthalmological tweezer caused serious injury to diapausing, pharate, first-instar larvae, several interesting results are shown in Table 4. Following extirpation of the mesothoracic ganglion, treatment with KK-42 terminated diapause successfully. The same results were obtained with the 4th and 5th abdominal ganglia were extirpated and the insect treated with KK-42, although the ratio of diapause breakdown was lower due to the serious injury of the operation. We conclude that a humoral repressive factor originates in the region of the mesothorax and a humoral muturation factor exists in the region of the 2nd to 5th abdominal segments. DJSCUSSION
Yamashita et al. (1987) showed that KK-42 acts directly on the prothoracic gland to inhibit ecdysone synthesis in the silkworm, Bombyx mori. Akai and Mauchamp (1989) reported that this chemical depresses the titres of juvenile hormones I and II in
Table 1. Effect of a single ligation on diapause breakdown in the main body compartments of pharate first-instar larva Nos of compartments Body compartment Whole body-l Whole body-2 Head-thorax* Thorax-abdomen?
Abdomen*
Nos of compartments
Application
Diapausing
Diapause broken
30 30 55 55 65 65 40 40 40 40 40 40
Acetone KK-42 Acetone KK-42 Acetone KK-42 None Acetone KK-42 None Acetone KK-42
30 0 39 11 59 57 33 31 10 19 15 19
0 30 0 22 0 1 0 0 13 13 7 12
Dead$ 0 0
Ratio of diapause breakdown (“/) 0.0 100.0
16
0.0
22 6 7 7 9 17 8 18 9
66.7 0.0 1.7 0.0 0.0 56.5 40.6 31.8 38.7
*See Fig. 1. tThe thorax-abdomen compartment was produced by single ligation at the neck and the rest was cut off. $Body compartments that died during the observation period was excluded from each group. This applied to Tables 2, 3 and 4.
Fig. 1. Photographs of pharate first-instar larva and those body compartments diapausing or breaking from diapause. A = Pharate first-instar larva which starts to enter diapause on the 10th day after oviposition and maintains diapause over one month, was removed from the chorion. The integumentary colour is yellowish-green during diapause. B = Diapausing head-thorax compartments (left, acetone application and right, KK-42 application). C = Abdomen compartment breaking from diapause only by ligation. The integument shows yellow colour and melanized stripes. This body compartment also has dark legs and standing bristles and finally wanders. D = Ligation position shifted at each intersegmental membrane (results shown in Table 2). Numerals are equal to those in Table 2. Scale bar = 6mm.
857
Diapause of pharate first-instar
859
Table 2. Effect of varying the position of ligation on diapause breakdown in pharate first-instar larvae Nos of pharate first-instars Region ligated
N
Anterior and posterior to ligation
Diapausing
Ligation between thoraxes: 30 Anterior* 1. ProthoraxPosterior mesothorax 30 Anterior 2. MesothoraxPosterior metathorax Ligation between abdominal segments: 30 Anterior 3. lst-2nd Posterior 30 Anterior 4.2nd-3rd Posterior 30 Anterior 5.3rd4th Posterior 30 Anterior 6.4th-5th Posterior 30 Anterior 7. Sthdth Posterior 30 Anterior 8.6th-7th Posterior 30 Anterior 9.7th-8th Posterior
Diapause broken
Dead (0)
:8 13
0 0 0 17
28 17 20 2 21 1 25 8 13 13 29 29 20 20
0 11 0 18 0 20 0 17 0 0 0 0 0 0
(2)
30
Ratio of diapause breakdown WI 0.0 0.0 0.0 56.7
(0)
0.0 39.3 0.0 90.0 0.0 95.2 0.0 68.0 0.0 0.0 0.0 0.0 0.0 0.0
(10) (9) (5) (17) (1) (10‘) .’
*Regions anterior and posterior to the ligatures were observed for 7 days and diapause breakdown was determined every day.
the haemolymph of Bombyx larvae. However, our results do not indicate that KK-42 functions as anti-ecdysteroid or anti-juvenile hormone since we determined the titre of ecdysteroids and investigated the effect of rescuing with some juvenile hormone analogues (Suzuki et al., 1990). Our present and previous results suggests that KK-42 reduces the humoral action of a repressive factor present in the
region of mesothorax of pharate first-instar larvae during diapause. Osanai and Arai (1962) reported, for the first time, an occurrence of a diapause factor in the 6th abdominal segment of the 4th (penultimate)-instar larva of nymphalid butterfly, Hestina juponica. This humoral factor causes also integumentary colour change from green to brown at the beginning of this
Table 3. Effect of double ligations on diapause breakdown of pharate tirst-instar larvae Region ligated 1st ligation
2nd ligation
N
Prothorax-mesothorax
and A,-A:
30
Mesothorax-metathorax
Mesothorax-metathorax
Mesothorax-metathorax
Metathorax-A,
Metathorax-A,
and A, -A2
and AZ-A,
and A,-A4
and A, -A4
and A,-A,
30
30
30
30
30
Anterior, middle and posterior to ligation
Nos of pharate first-instars Diapausing
Diapause broken
Anterior? Middle Posterior
25 25 20
8 5
Anterior Middle Posterior
29 29 16
Anterior Middle Posterior
Dead
Ratio of diapause breakdown WI
(5)
0.0 0.0 20.0
0 0 13
(1)
0.0 0.0 44.8
30 18 12
0 12 18
(0)
0.0 40.0 60.0
Anterior Middle Posterior
30 13 4
0 17 26
(0)
0.0 56.7 86.7
Anterior Middle Posterior
:x
(0)
5
0 7 25
0.0 23.3 83.3
Anterior Middle Posterior
30 27 12
0 3 18
(0)
0.0 10.0 60.0
*The 1st ligation was performed at the intersegmental region between the prothorax and mesothorax, and the 2nd ligation was between the 1st and 2nd abdominal segments, -/‘Regions anterior to the 1st ligation, between the 1st and 2nd ligations, and posterior to the 2nd ligation, were observed for 7 days and diapause breakdown was determined every day.
KOICHI SUZUKIet al.
860
Table 4. Effect of extirpation of specific ganglia on diapause breakdown of pharate first-instar larvae Nos of pharate first-instars Diapause broken
Dead
Ratio of diapause breakdown (“h)
30 13
0 10
0 7
0.0 43.5
None KK-42
30 22
08
0
2::;
None KK-42
30 25
0 4
0 1
0.0 13.8
Application
Extirpation
N
Mesothoracic ganglion
30 30
None KK-42*
4th and 5th Abdominal ganglia
30
5th Abdominal ganglion
30 30
Diapausing
*After extirpation, the insects were chilled at 2°C one day aud on the next day they were treated with KK-42, and thereafter they were observed for 7 days and diapause breakdown was determined every day. larval cliapause. Ecdysone-containing extracts had no influence on the colour changes as well as onset of diapause. On the other hand, Sakate (1984) speculated that in A. yamamai some abdominal segments have a development factor, probably our maturation factor, this is induced by chilling for a long period, but he could not develop this speculation because of his untimely death. Thus these reports suggested partially the operation of a new endocrine system controlling insect diapause. The present study, however, establishes that diapause in pharate first-instar larvae is regulated through the action of a humoral repressive factor which inhibits the expression of humoral maturation factor. It also indicates a new sort of the endocrine system that is responsible for the regulation of insect development and differentiation, independent of the central endocrine system composed of the brain, corpora all&a, suboesophageal ganglion and prothoracic gland. Acknowledgement-We
wish to thank Professor B. W. Bowers of Department of Entomology, The University Arizona, Tex., for critical reading of the manuscript.
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