No. 6] Proc. Japan Acad., 46 (1970) 541 127. Effect o f Non 24 Hour Photo period and Light Interruption o f the Dark Phase on Diapause Determination in Papilio xuthus L. By Toshitaka HIDAKA and Yoshio HIRAI*> Laboratory of Biology, Tokyo University of Agriculture and Technology, Fuchu, Tokyo (Comm. by Yo K. OKADA, M. J. A., June 12, 1970) The mechanism of the photoperiodic reaction in insects is now subj ect to active discussions. Although many contradictions exist, its understanding by coincidence of an endogenous circadian oscillation, containing a strictly fixed "light-sensitive point", with a given external photoperiod seems to be generally accepted. Our experiments utilizing the Swallowtail butterfly, Papilio xuthus L., in which the pupal diapause is photoperiodically controlled (Kato, 1963), appear to show, however, a fact which escapes this classic interpretation. Material and methods. Eggs and young larvae of Papilio xuthus were collected in the suburbs of Tokyo. The larvae were reared under various photoperiodic conditions from hatching until pupation, with leaves of rutaceous plants, Citrus natsudaidai and/or Poncirus tri f oliata as the diet. Rearing chambers for various photoperiods were constructed from light-proof wooden boxes (50 x 35 x 40 cm) lined with tin-plate. As the light source, a 10-watt "Daylight" type fluorescent tube (Toshiba) was attached on the lid of the box. Photoperiods of the 24-hour cycle were controlled by time switches. Non-24-hour-cycle photoperiods were regulated manually. The temperature inside the boxes was kept at 24±1 C by water flowing through polyethylene tubing. Discrimination of diapausing and non-diapausing pupae was made morphologically. Two or three days after pupation, the chromatic type of the pupae was examined, and the pupae of Types Green and Brown were regarded as non-diapausing while those of Types Green-D and Orange were classified as diapausing (Hidaka, 1961). This discrimination was re-checked by keeping the pupae at 25 C during successive 20 days. When pupal period lasted more than 20 days instead of 10 to 14 days as in non-diapausing pupae, those pupae were finally confirmed as diapausing. The morphological criterion proved always highly reliable. The problem of the "inten- *) Institute of Forest Zoology, Faculty of Agriculture, University of Tokyo.
542 T. HIDAKA and Y. HIRAI [Vol. 46, sity" of diapause is not discussed here. Experiments and results. When larvae were subjected to a 24-h-cycle photoperiod with 0-, 10-, 12-, 13-, 14- or 24-h photophase, pupae entered diapause after larvae were exposed to photophases of less than 12 h, while those pupae that had developed under a photoperiod of 14-h light emerged within 12 days without entering diapause. At a 13-h-light photoperiod, 16.7% of pupae were diapausing (Fig. 1). It will follow therefore that the critical "day length" for inducing pupal diapause in this species is a little shorter than 13-h under this temperature condition. Larvae were then subj ected to various non-24-h-cycle photoperiods all containing the light and the dark period in the same ratio Fig. 1. Effect of 24-h-cycle photoperiods with various photophase on the rate of pupal diapause in Papilio xuthus. Fig. 2. Effect of various non-24-h-cycle photoperiods on the rate of pupal diapause in Papilio xuthus, as compared with that of 24-h-cycle ones.
No. 6] Diapause Determination in Papilio xuthus L. 543 of 1 :1. Almost equal rates of diapause were obtained when the photoperiods contained dark phases of the same length (Fig. 2). The same duration of photophase failed to induce diapause to the same degree. This fact strongly suggests that it is the length of the dark period, rather than of the light period, which is responsible for the induction of diapause, as it is now widely believed. Here, the maximum length of the dark period for complete inhibition of diapause seems to be 10-h. But in such non-24-h cycles, the circadian clock would have been largely disordered. The light interruption experiments carried out with the scheme Fig. 3. Effect of 1-h light interruption during the scotophase of an 8-h-light-16-h-dark substrate photoperiod. All photoperiods contained therefore 9 h light period and 15 h of dark period.
544 T. HIDAKA and Y. HIRAI [Vol. 46, as shown in Fig. 3 gave interesting results. The substrate cycle consisted of an 8-h photophase and a 16-h scotophase scanned by a 1-h light interruption. The diapause-inhibiting effect of the light interruption was observed only when it started 3-4 h after the beginning of the scotophase. In disagreement with the results reported by Adkisson (1964) in Pectinophora gossypiella, a second diapause-inhibiting light-sensitive point in the later part of the scotophase seemed to be absent. Even after 10 h from the beginning of the scotophase, 1-h light interruption gave practically no influence on the rate of diapause (Fig. 3). What is more curious is the location of the light-sensitive point. The strongest inhibition of diapause occurred when the interrupting light fell on the 12th hour from the "dawn". In normal, uninterrupted photo-scoto-cycles, however, presence of light at the 12th hour from the dawn is never sufficient for inhibiting diapause, because 12-h-light-12-h-dark photoperiod induces very high rate of diapause (Fig. 1). 4n the other hand, light interruption at the 14th hour failed to prevent diapause in disagreement with what is expected from the effect of 14-h uninterrupted-light photoperiod. Discussion. These results can not be explained by the length of the dark period remaining after the light interruption. Light interruption at the 12th hour leaves a continuous dark period naturally longer than the critical "night length" of 10-11 hours, and yet it is diapause-preventing. Light interruption at the 14th hour, on the contrary, leaves only 10-h dark period which should normally assure the development of non-diapausing pupae, and yet it acts as highly diapause-inducing. Nor this shift of light-sensitive point can be attributed to the lowering of temperature. The temperature, although really lower than in the light period, still remained at 23 C during the dark period. A possible interpretation of the event will be the hypothesis that the light-sensitive point on which the light acts as inducer (Pittendrigh and Minis, 1964) is not fixed. Although many works since Buunning's hypothesis (Bunning, 1960) appear to have repeatedly confirmed the accurate fixedness of the diapause-preventing light-sensitive point parallel to the accuracy of the circadian clock, and although this is believed to give a reasonable explanation of photoperiodism in insects, there are, on the other hand, so many facts speaking of the "shift" of critical daylength by temperature and food conditions as well as by influence of the maternal generation (Hidaka and Takahashi, 1967). These facts are more simply explained by supposing that the position of the light-sensitive point within a circadian cycle is essentially flexible than by thinking about
No. 6] Diapause Determination in Papilio xuthus L. 545 the "temperature compensation" of circadian rhythms. The fixedness of the circadian clock will not necessarily impose the fixedness of the location of the light-sensitive point. As our present results seem to suggest, the light-sensitive point will be, at least within a definite range, subject to influence of various factors, independently of the rigidity of the circadian clock system itself. References Adkisson, P. L. (1964): Amer. Naturalist, 98, 357-374. Bunning, E. (1960): Cold Spring Harbor Symp. Quant. Biol., 25, 249-256. Hidaka, T. (1961) : J. Fac. Sci., Univ. Tokyo, Sect. IV, 9, 223-261. Hidaka, T., and H. Takahashi (1967): Annot. Zool. Japon., 40, 200-204. Kato, M. (1963): Kagaku, 33, 485-488. Pittendrigh, C. S., and D. H. Minis (1964): Amer. Naturalist, 98, 261-294.