No. 9] Hormonal Control of Butterfly Seasonal Forms 1083

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1 1082 [Vol. 42, 235. Hormonal Control o f the Development o f Seasonal Forms in the Butterfly, Polygonia c aureum L By Soichi FUKUDA and Katsuhiko ENDO Biological Institute, Faculty of Science, Nagoya University (Comm. by Yo K. OKADA, M.J.A., Nov. 12, 1966) The butterfly, Polygonia c-aureum, which produces four or five generations a year, has two distinct seasonal forms. The butterflies appearing from June to about the end of September are the so-called summer forms. They begin to lay eggs a few days after emergence. The autumn form butterflies are produced in late autumn from eggs laid by females of the last generation of the year. They enter the diapause period continuing for at least 6 months, egg laying not occurring until the following spring. The two seasonal forms are markedly different in coloration. The ground color of the upper side of the wings is brownish yellow in summer forms and reddish yellow in autumn forms, although black spots occur in both of them. Difference in color pattern is still more marked on the under side of the wings than on the upper side (Fig. 1, a and b). In summer forms, the wings are dark yellow in ground color while in autumn forms they are brownish black, Fig. 1. Female butterflies (Polygonia c-aureum) photographed from the under side, x about 2/3. a: summer form, b: autumn form, c: autumn form emerging from decerebrated ND pupa, d: autumn form emerging from ND pupa in which the brain had been divided into two hemispheres, e: autumn form emerging from decerebrated D pupa.

2 No. 9] Hormonal Control of Butterfly Seasonal Forms 1083 females being darker colored than males. Moreover, the wings are different in shape between summer and autumn forms, the wing margin being more profoundly notched in autumn forms than in summer forms. Previous workers have suggested that the most important of the factors for determining the seasonal polymorphism and diapause in a variety of insects is photoperiod or photoperiod plus environmental temperature (de Wilde, 1962, Danilevskii, 1965). In P, c-aureum, Hidaka and Aida (1963) reported that larvae subjected to daily illumination of 14 hours or more underwent pupation and metamorphosed into summer form butterflies, regardless of temperature conditions, while those exposed to daylight period of 12 hours or less invariably became autumn form butterflies, irrespective of thermal surroundings. However, our findings which are dealt with in this paper are not in complete agreement with those of Hidaka and Aida. To disclose internal factors involved in the determination of seasonal forms and diapause in P, c-aureum, experiments were carried out in 1965 and The results have indicated that the neuroendocrine system consisting of the pars intercerebralis and corpora cardiaca is responsible for the production of summer form butterflies in this species. Material and methods. Eggs laid by summer form butterflies were collected in the suburbs of Nagoya and larvae from these eggs were reared in the laboratory under controlled light and temperature conditions. On the basis of the results of a preliminary series of experiments, larvae were subjected to 16-hour photophase and 8-hour scotophase at C, and to 8-hour photophase and 16-hour scotophase at 20 C ± 1, *' to obtain summer form and autumn form butterflies, respectively. Control of photoperiod was effected through the use of programmers wired to a 20 watt fluorescent lamp installed in an incubator. Larvae exposed to these light and temperature conditions during the whole larval period invariably metamorphosed into the respective seasonal forms. For the sake of convenience, insects reared under the former conditions are referred to as ND larvae or pupae and those subjected to the latter conditions as D larvae or pupae in the following description. *) Larvae subjected to 8-hour photophase and 16-hour scotophase at 30 C + 1 during the whole larval period invariably developed into summer forms, in disagreement with the results reported by Hidaka and Aida (1963, l.c.).

3 1084 S, FUKUDA and K, ENDO [Vol. 42, Experiments and results. 1. Extirpation of the brain in pupae. The brain was surgically removed from both ND and D pupae about 12 hours after pupation. Emergence of butterlies occurred from operated and the control pupae on the same day or was delayed half a day at most in pupae operated on. Although the wings failed to expand normally in many of those emerging from decerebrated pupae (Fig. 1, c and e), difference in wing coloration clearly showed whether the butterflies were summer forms or autumn forms. Table I summarizing the results indicates that butterflies emerging from decerebrated D pupae were invariably autumn forms, the removal of the brain being without effect on development of the forms (Fig. 1, e). On the contrary, in those appearing from operated ND pupae, the wing coloration was characteristic of the autumn form, as in butterflies from decerebrated D pupae (Fig. 1, c). Table I, Effects of decerebration, brain implantation after decerebration, and division of brain into two hemispheres on development of seasonal forms in D and ND pupae*) 2. Implantation of the brain into decerebrated pupae. ND and D pupae decerebrated at about 12 hours after pupation were given immediately after operation an implant of a brain into the head capsule from ND and D pupae at a similar age respectively, through the incision from which the brain had been removed. Both kinds of pupae operated on invariably produced autumn form butterflies, showing that grafted brains were totally ineffective in producing summer form butterflies from ND pupae (Table I). 3. Separation o f the right and left brain hemispheres. By

4 No. 9] Hormonal Control of Butterfly Seasonal Forms 1085 operative procedures similar to those described in a previous paper (Fukuda and Takeuchi, 1966), the brain in situ was cut into two hemispheres along the midline in pupae about 12 hours after pupation. A few butterflies emerging from operated pupae had their wings fully expanded. However, butterflies obtained from D pupae operated on were uniformly autumn forms, concordant with the determination during the larval period. On the contrary, all ND pupae subjected to a similar operation transformed into autumn form imagines, instead of becoming summer form butterflies (Table I) and in those having normally expanded wings, the wings were similar in both coloration and shape to those in natural autumn forms (Fig. 1, d). In another series of experiments, the brain was divided into two hemispheres in ND pupae about 36 hours after pupation. From these pupae, summer form and intermediate form butterflies, in addition to autumn form ones, emerged (Table I). In the intermediate forms, the wing coloration was of the type intermediate between the summer and autumn forms. 4. Alaatectomy in larvae of the last instar. The corpora allata were removed from ND and D larvae on the second day of the last instar. Butterflies developed from operated larvae were summer forms and autumn forms, respectively, the determined seasonal forms never being modified by allactectomy (Table II). Table II. Allatectomy and cardiectomy in D and ND larvae of last instar 5. Cardiectomy in larvae o f the last instar. The corpora cardiaca were extirpated from groups of ND and D larvae on the second day of the last instar, their corpora allata being left intact. Butterflies developed from both operated ND and D larvae were autumn forms (Table II). It is evident that cardiectomy brought about change in seasonal form of butterflies developed from ND larvae. Discussion and conclusion. The results of Series 1 of experiments indicated that the brain was essential for ND pupae to metamorphose into summer form butterflies in accordance with the determination by light and temperature conditions during the larval

5 1086 S. FUKUDA and K. ENDO [Vol. 42, period. However, the facts that grafted brains were not effective in this respect in decerebrated pupae (Series 2 of experiments) and that division of the brain in situ into two hemispheres had the effects similar to those of decerebration on seasonal form of resulting butterflies (Series 3) seem to show that the intact brain in situ is necessary for ND pupae to become summer form butterflies. In pupae of Bombyx mori (Fukuda and Takeuchi, 1966), it has been demonstrated that division of the brain into the right and left hemispheres blocks the transport of neurosecretory material from medial neurosecretory cells in the gars intercerebralis to the complex of corpora cardiaca-corpora allata. Accordingly, the results of Series 3 of experiments suggest that in ND pupae determined to become summer form butterflies, some principle involved in the development of the butterflies is secreted from the medial neurosecretory cells and transferred through their axons to the corpora cardiaca, corpora allata or both, from which it is released into the blood. However, in both ND and D larvae of the last instar, allatectomy brought about no modifications in seasonal form of butterflies (Series 4), while cardiectomy in ND larvae of the last instar which had been determined to produce summer form butterflies resulted in the development of autumn forms (Series 5). These findings seem to show that the corpora cardiaca, but not the corpora allata, play an important role in the events. From these results, it may be concluded that in P, c-aureum, external factors determining the development of summer form butterflies, especially daily illumination and temperature during the larval period, act upon medial neurosecretory cells in the pars intercerebralis to render them capable of producing an active principle or principles after pupation. The neurosecretory material in question is transported through axons to the corpora cardiaca of the pupa, from which it is released into the blood to induce the development of the summer form butterfly. It seems likely that pupae metamorphose into autumn form butterflies, if the pars intercerebralis and corpus cardiacum are not activated. References Danilevskii, A. S. (1965): Photoperiodism and Seasonal Development of Insects. Oliver and Boyd, Edinburgh and London. Fukuda, S., and Takeuchi, S. (1966): Difference sexeulle Bans l'activite du systeme pars intercerebralis-corpus cardiacum-corpus allatum chez le Bombyx mori. C. R. Soc. Biol., 160 (in press).

6 No. 9] Hormonal Control of Butterfly Seasonal Forms 1087 Hidaka, T., and Aida, S. (1963): Day length as the main factor of seasonal form determination in Polygonia c-aureum (Lepidoptera, Nymphalidae) (in Japanese). Zool. Mag., 72, Wilde, J, de (1962): Photoperiodism in insects and mites. Ann. Rev. Entom., 7, 1-26.