26. The Relationships between Oxygen Consumption and Duration o f Pupal-Adult Development in the Silkworm Bombyx mandarina

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1 134 Proc. Japan Acad., 69, Ser. B (1993) [Vol. 69(B), 26. The Relationships between Oxygen Consumption and Duration o f Pupal-Adult Development in the Silkworm Bombyx mandarina By Weide SHEN and Kunikatsu HAMANO Department of Biological Production, Faculty of Agriculture, Tokyo University of Agriculture and Technology, Fuchu-shi, Tokyo 183 (Communicated by Seijiro MOROHosHI, M. J. A., June 8, 1993) Abstract: Duration of pupal stage of Bombyx mandarins is 2 to 4 times longer than that of Bombyx mori. We attempted to investigate whether this represents a state of diapause or not. Based on the pupal duration and oxygen consumption, developmental pattern of pupae in B. mandarina was mainly classified to three types, A, B and C, though these were dependent on the rearing seasons. In the spring season, typical type-a pupae had the shortest duration the same as in B. mw-i. Type-B pupae were intermediate of type-a and C. Pupae that had the longest duration were classified as type-c. Oxygen consumption decreased quickly soon after pupation and each type showed a typical U-shape respiration curve during pupal stage. Oxygen consumption in type-c animals was lowest of all 3 types. It reached extremely low levels of 5 /21/hr/g, which went on for more than 2 weeks. When the type-a pupae were maintained in low temperature, pupal duration was elongated in proportion to the temperature decrease. However, in this experiment the respiratory activity curve in the lowest temperature of 20 C was clearly different from that of type-c pupae. In another experiment, type-a and B pupae showed increased respiration, proportionally to the temperature increase, while there was no increase in respiration of the C-type animals when subjected to the different temperatures. Finally, we can conclude that the low level of oxygen consumption in the C-type animals is characteristic to diapausing insects and this is in good accordance with the insensitivity of these animals to different temperatures. It was consequently determined that in B. mandarina there is a kind of facultative diapause in pupal stage. Key words: Summer diapause; Bombyx mandarina. Introduction. In the domestic silkworm, Bombyx mori, diapause occurs only during the embryonic stage, while in the pupal stage no diapausing conditions can be witnessed (Morohoshi, 1959). However, in the wild silkworm Bombyx mandarins-which is regarded as the most phylogenetically related insect to B. mori-it is reported that a sydrome of physiological and behavioural traits similar to diapause is exhibited also during the pupal stage (Oba, 1939; Omura, 1950; Taniguchi and Ninaki, 1983). This diapausing pupal state is largely dependent on the voltinism, the race, the population and the climatic conditions, suggesting that it is both an environmentally and genetically influenced physiological state, with variable intensity within a given population. Its duration is ranging, even within one batch, from two weeks to more than 2 months (Omura, 1950; Taniguchi and Ninaki, 1983). The longest duration in Japanese populations was reported to be 136 days (Oba, 1939). In some animals of Japanese origin, pupal duration lasts for about 2 to 3 weeks, during which pupal-adult development occurs in the same way as in B. mori. In other animals of the same origin this development occurs in a rather slow pace or does not occur at all for a

No. 6] Summer Diapause in Bombyx mandarina 135 considerable length of time, so that the question of whether this represents diapause or simple state of quiescence is rather difficult to answer.

2 No. 6] Summer Diapause in Bombyx mandarina 135 considerable length of time, so that the question of whether this represents diapause or simple state of quiescence is rather difficult to answer. As a general rule, U-shape respiration rate curve can be plotted for all insects during their pupal stage. This physiological trait has been used as a determining factor by many authors to decide whether a physiological state is diapause or not in many insects (Waku, 1965; Wigglesworth,1972). The U-shaped respiration rate in pupal stage is also present in the silkworm B. mori (Itaya, 1940; Ito, 1954). However, it is still unknown if this is the case for B. mandarins also. In this article, we attempted to investigate this long pupal duration and decide whether it represents a state of diapause or not. Our results were compared with that for B. mori and it was determined that in B. mandarina there is a diapausing period in pupal stage in some animals while in others pupal-adult development merely progresses slowly. Materials and methods. Insects. Eggs of the silkworm B. mandarins, were collected from mulberry fields in China (Suzhou city, Jiangsu Province) and in Japan (Fuchu-shi, Tokyo) on the Autumn of Eggs of B. mandarina were incubated under natural climatic condition at late April of Majority of larvae in Chinese population hatched at middle of May. The larvae were reared with fresh mulberry leaves under natural climatic conditions in Japan (Fuchu). The larvae were in pasture on the mulberry trees which were covered with nets to prevent the parasitism of Tachina flies. Pupation of these animals occurred during middle of June to beginning of July. These pupae were collected and maintained in an incubator at an appropriate temperature under natural photoconditions. Domestic silkworms B. mori (Asagiri race) were also used as control. Rearing was done by the conventional method in an incubator using fresh mulberry leaves. Measurement of respiration. Oxygen consumption of pupae was determined individually using an Oxygen Up Tester (TAIYO Co. Ltd, Japan). Five replications could be used at the same time and they were kept at a certain constant temperature. Oxygen uptake was calculated as µlfhrlg fresh body weight. For these calculations the body weight of the insects after the experiment was also used. At the end of each experiment, external morphology of all specimens were carefully investigated to distinguish stages of pupal-adult development. Results and discussion. In the silkworm B. mori, the duration of pupal stage is about 8 to 14 days under natural conditions. The pupae of B. mandarina, on the other hand, exhibited large variations in pupal Fig. 1. Typical changes of oxygen consumption of Bombyx mandarina pupae in Japanese and Chinese populations. Symbols indicate as follows: A: initiation of colouration of compound eye; B: darkening of compound eye; C: darkening of antennae; D: pigmentation of wing; -u-, Type-A; ---A---, Type-B; -, Type-C. The mean of five replicates is shown.

3 136 W. SHEN and K. HAMANO [Vol. 69(B), duration with typical specimen following three types of respiratory activity as shown in Fig. 1. These three types were nominated as type-a, B and C judged by their pupal duration and respiratory activity. Typical type-a pupae had the shortest duration of about two weeks, the same as in B. mm-i. Type-B pupae were intermediate of type-a and C, with about 3 weeks duration. Pupae that had the longest duration were classified as type-c, in which adult emergence took more than 30 days to occur. In Chinese populations of B. mandarina pupae, more than 80% of them were classified as type-a, and the rest were classified as type-b. There was no type-c pupae in Chinese populations. Whereas, in Japanese populations, more than 20% of pupae were classified as type-c. These animals oviposited diapause eggs only in this experiment. There was no C-type group in Japanese trivoltine populations. Oxygen consumption decreased quickly soon after the beginning of the pupal stage and each type showed a typical U-shaped respiration curve. Two to four days after larval-pupal ecdysis in type-a pupae, respiratory activity reached its minimal levels of 130 µljhr/g and this was the highest in all 3 types. Afterwards, oxygen consumption increased sharply in concert with pupal-adult development. Two days before adult emergence, maximal respiratory levels were recorded. This type of respiration was very similar to that of the domestic silkworm B. mori and the program of appearance of morphological developmental signs was also the same. Duration of the type-b pupal stage was twice as long as that of type-a. The lowest respiration level was also more extended than that of type-a pupae and lasted for 7 to 10 days. Minimal respiratory activity was somewhat lower than that of type-a pupae and reached a µl/hr/g level. Thereafter, levels slowly increased, but they were on their lowest point from day 4 to day 6 and therefore eye pigmentation was delayed and occurred around day 12 to 14. Oxygen consumption in type-c was lowest of all 3 types, and reached a level of less than 20 µllhr/g at day-5 to 6 after pupation. One week after pupation, respiratory activity reached extremely low levels of 5 µl/hr/g, and this state went on for more than 2 weeks. There were big variations in this period of low respiratory activity lasting over a range of 15 to 30 days. After several weeks, respiratory rate began to increase gradually and about 2 weeks from the point that exceeded 100 µl/hr/g, adult eclosion occurred. However, maximal oxygen consumption was also lower than that of type-a and B pupae. It must be pointed out that there are two phenotypes of pupal colouration of B. mandarina, black and amber coloured. In the case of the black coloured ones, morphological changes could not be distinguished from outside. In our experiments, therefore, we used only the amber type of pupae. Developmental morphological changes of the amber coloured B. mandarina pupae were not synchronous throughout pupal duration. Timing of eye pigmentation was delayed when pupal duration was expanded and only after respiratory activity recovered, eye colour changed as a sign that the dormant period is over. The relationship between temperature and respiratory activity was also investigated. As shown in Fig. 2, oxygen consumption of the type-a pupae of Chinese origin was recorded when the animals were kept under three different temperatures of 20, 25, and 30 C. Pupal duration was elongated in proportion to the temperature decrease. Maximal levels of respiratory rates also decreased in a steady mode in accordance with the decrease in the temperature and the lowest respiration plane was elongated for few days. However, this respiratory activity curve in the lowest temperature of 20 C was clearly different from that of type-c pupae. Oxygen consumption of 4-day-old pupae of Japanese population of the three types

4 No. 6] Summer Diapause in Bombyx mandarina 137 Fig. 2. Correlation between respiration and temperature condition in Bombyx mandarina pupae of Chinese population. Symbols indicate as follows: --, 20 C; - -, 25 C; -A-, 30 C, The mean of five replicates is shown. Fig. 3. Effect of temperature variations on the respiratory performance of Bombyx mandarina pupae of Japanese populations. Symbols indicate as follows: -A-, type-a; -i-, type-b; --, type-c. 4-day-old pupae were used. The mean of five replicates is shown. Fig. 4. Sexual differences in oxygen consumption of Bombyx mandarina pupae of Japanese populations. Symbols indicate as follows: solid line -, female; dotted line ---A---, male; A, Type-A; B, Type-B; C, Type-C. The mean of five replicates is shown.

5 138 W. SHEN and K. HAMANO [Vol. 69(B), were investigated. These animals were kept under the same conditions (25 C) before exposed to 3 different temperatures (Fig. 3). In this experiment, there were two kinds of reactions towards the increasing temperature. Type-A and B pupae showed the increased respiration proportionally to the temperature increase. However, there was no reaction of the C-type animals when subjected to the different temperatures. There were also sex differences in the A, B, and C types as those described in B. mori by Ito (1954). In all samples, oxygen consumption of the males was higher until it reached the lowest point of activity and then the increase was more pronounced in females than in males, probably due to the growing ovaries (Fig. 4). Finally, we can conclude that low respiratory levels of 5,cclfhrlg such as these recorded for the C-type animals are characteristic to diapausing insects. A natural developmental program can not be expressed and maintained under such low respiratory conditions. Therefore such conditions represent a diapausing state as described by Masaki (1980). Our conclusion is in good accordance with the insensitivity of those animals to different temperatures as can be seen in Fig. 3. On the other hand, as described by Wigglesworth (1972) respiratory levels are strongly dependent and affected by temperature in ordinary developing insects. Acknowledgements. The authors express their appreciation to Dr. Seijiro Morohoshi, M. J. A., for his kind advice, and Drs. Tosihiko Hukuhara, Toshikazu Oshiki, and Yosimitu Iwashita, Professors of United Graduate School of our Univ. who support most of these experiments. References Itaya, K.: J. Seric. Sci. Jpn., 11, (1940). Ito, T.: Bull. Seric. Exp. Sta., 14, (1954). Masaki, S.: Ann. Rev. Entomol., 25, 1-25 (1980). Morohoshi, S.: J. Insect Physiol., 3, (1959). Oba, H.: Seric. Rep. Kinugasa, 396, (1939). Omura, S.: Bull. Sericul. Exp. Sta., 13, (1950). Taniguchi, Y., and Ninaki, 0.: Acta Sericologica, 126, (1983). Waku, Y.: Res. Rep. Kyoto Ins., 4, (1965). Wigglesworth, V. B.: The Principles of Insect Physiology, London (1972).

163. The Control o f Growth and Development in Bombyx mori. II Genic Balance in Molting Characteristics

No. 8] Proc. Japan Acad., 45 (1969) 733 163. The Control o f Growth and Development in Bombyx mori. II Genic Balance in Molting Characteristics By Seijiro MOROHOSHI Faculty of Agriculture, Tokyo University