No. 6] Proc. Japan Acad., 69, Ser. B (1993) 139

27. The Relationships between Pupal Development and Ecdysteroid Levels in Summer Diapausing Silkworm, mandarins

By Weide SHEN, *) Kunikatsu HAMANO,*) and Hajime FUGo**) (Communicatedby Seijiro MoRoxosm,M. J. A., June 8, 1993)

Abstract: In the silkworm, Bombyx mandarins, duration of pupal-adult develop- ment was mainly divided into three different types among the Japanese population: the first one was 2 weeks duration, the second was about 3 weeks and in the third type more than 30 to 40 days were necessary for the adult development, although these were dependent on the rearing seasons. However, the average number of days required for adult development was 14 in the majority of the Chinese population. In the third type of pupal-adult development of Japanese population, the development of pupal stage was arrested. Oxygen consumption of the of which adult eclosion occurred at 34 days after pupation was very low during the pupal development. Ovarian development of these animals was also suppressed considerably. The fluctuation patterns of ecdysteroid titer in haemolymph of Japanese population were quite different from that of the Chinese population. Ecdysteroid titer was undetectable at 4 to 13 days after pupation in the Japanese population, but the titer increased by day-15 and reached a maximum level of 4.5 µg at day-20. The reinitiation of ovarian development was coincident to the increase of ecdysteroids in haemolymph of these animals. It was suggested that the delay of pupal-adult development may be due to the low levels of ecdysteroids in the Japanese population of Bombyx mandarina. Key words: Summer diapause; aestivation; ecdysteroid; Bombyx mandarina.

Introduction. Diapause is a widespread phenomenon in the life span of . diapause is regulated by a variety of neuroendocrine mechanisms (Chippendale, 1983). These control mechanisms include the presence of a diapause hormone regulating the embryonic diapause of the silkworm, (Morohoshi, 1959; Yamashita and Hasegawa, 1985), the presence of juvenile hormone (JH) in diapausing larvae of southwestern corn borer, Diatraea grandiosella (Yin and Chippendale, 1973), the absence of prothoracicotropic hormone (PTTH) in diapausing pupae of the American silkworm, Hyalophora cecropia (Williams, 1952) and the absence of JH in diapausing adult of the Colorado potate beetle, Leptinotarsa decemlineata (de Wilde, 1983). Usually, diapause occurs only once in the life history of a particular . However, the Operophtera brumata is said to undergo obligatory diapause as an embryo and as pupa (Pickfort,1953). This phenomenon commonly occurs in insects (Masaki, 1980). In the silkworm, Bombyx mandarina, eggs enter diapause at an early phase of embryonic development (Umeya, 1946) as the same as Bombyx mori. In the case of Bombyx mandarina, pupal-adult development of Chinese populations is not arrested in pupal stage, but many of the Japanese population aestivate in pupal stage (Shen and *) Department of Biological Production , Faculty of Agriculture, Tokyo University of Agriculture and Technology, Fuchu-shi, Tokyo 183, Japan. **) Department of Environmental Science and Resources , Faculty of Agriculture, Tokyo University of Agriculture and Technology, Fuchu-shi, Tokyo 183, Japan. 140 W. SHEN, K. HAMANO, and H. FUGO [Vol. 69(B),

Hamano, 1993). It is well known that pupal diapause occurring in winter is regulated by neuroendocrine control from brain (Williams,1952; Bollenbacher and Granger, 1985). It is also speculated that summer diapause or aestivation may be controlled by the brain. In the present paper, we demonstrate the possible induction mechanism of the summer diapause (aestivation) in Bombyx mandarina. Materials and methods. Insects. Eggs of the silkworm, Bombyx mandarina were collected from mulberry fields in China (Suzhou city, Jiangsu Province) and in Japan (Fuchu-shi, Tokyo) on the Autumn of 1991. Eggs of Bombyx mandarins were incubated under natural climatic conditions at late of April in 1992. Majority of the larvae hatched at middle of May. The larvae were reared with fresh mulberry under natural climatic conditions in Japan (Fuchu). Larvae were in pasture on the mulberry trees which were surrounded with nets to prevent the parasitism of Tachina flies. At the middle of June to beginning of July, the pupae were collected and maintained in an incubator at 25±1°C under natural photoconditions. Estimation of respiration. Oxygen consumption of each individual pupa was measured by an Oxygen Up Tester (TAIYO Co. Ltd. Japan) for 1 hr at 25°C and was expressed as µl 02/hr/g fresh weight (Shen and Hamano, 1993). Ovarian development. Ovarian development was determined by measuring the weight of ovaries in the animals. The dissected ovaries were rinsed with distilled water and blotted on filter paper to remove excess water, and immediately weighed gravimetri- cally. Estimation of ecdysteroid in haemolymph. Estimation of ecdysteroid in haemo- lymph of pupae was performed by radioimmunoassay (RIA) method as descrived by Takeda et al. (1986). Since 20-hydroxyecdysone (20-HE) was used as a standard, the RIA activity was expressed as µg of 20-HE equivalent per ml of haemolymph. The antiserum against 20-HE was a kind gift from Dr. Satoshi Takeda, National Institute of Sericulture and Entomological Science, Tsukuba, Japan. Results and discussion. It is accepted that in Bombyx mandarins diapause occurs at embryonic stage. However, there is a summer diapause (aestivation) among the populations of Bombyx mandarina (Shen and Hamano, 1993). Summer diapause is not only common among insects but also widespread throughout invertebrate (Masaki,1980). Insect diapause is under hormonal control. Ecdysteroids and juvenile hormones as well as a diapause hormone are engaged in its control (Behrens, 1984). The endocrine control of summer diapause conforms to the basic system governing winter diapause, according to a few endocrinological studies (Masaki, 1980). There are three different types of pupal duration among the populations of Bombyx mandarina; one is 2 weeks pupal duration (type-A), about 3 weeks duration are the intermediate (type-B) and more than 30 to 40 days are necessary for adult eclosion on the third type (type-C) (Shen and Hamano, 1993). In Chinese populations of Bombyx mandarins, pupal duration is about 2 weeks and there is no summer diapause (aestivation) when the same population was reared in Japan. In this experiment, we used the animals of three typical different types of pupal development. About 2 weeks duration of pupal-adult development was most frequently observed in Chinese populations; such a pupal-adult development is referred in our experiment as type-A. In this type, eye pigmentation was completed by day-7, the wing pigmentation occurred on day-12 and adult eclosion on day-14 as was observed for Chinese population (Shen and Hamano, 1993; Shen et al., 1993). During the progress of pupal development, oxygen consumption fluctuated: the rate at pupation was 380 µl 02fhr/g of body weight but during day-2 to day-4 rates dropped obviously. Afterwards, oxygen consumption increased sharply with age (Fig. 1). Typical U-shaped oxygen consumption No. 6] Ecdysteroid Levels in Bombyx mandarins 141

Fig. 1. Typical fluctuation patterns of oxygen consumption during the pupal-adult development in the silkworm, Bombyx mandarina. Ten to fifteen animals were used for each experiment. The mean of four to five replicates is shown. The standard deviations are less than 10% of each mean. was seen in the Chinese population of Bombyx mandarina and this type of respiration is similar to the silkworm, Bombyx moni. The fluctuation of ecdysteroids in haemolymph is shown in Fig. 2. Ecdysteroid level at pupation was about 500 ng/ml haemolymph. The levels abruptly increased with age and reached a maximum level of about 4µg/ml haemolymph on day-6. Afterwards, the level decreased gradually and during 8th to 14th ecdysteroid level was below the limitation of measurement of this system. In accordance with the decrease of ecdysteroid level in haemolymph, the differentiation of ovaries occurred sharply with age. The ecdysteroid titer during the pupal-adult development has been reported in many insects, and it has been revealed that the peak of ecdysteroid titer existed in the first half of the pupal stage, as shown in Fig. 2. Although pupal duration between the three types of the Japanese populations ranged significantly, almost same pupal duration as that shown for the Chinese population was observed also for the Japanese population of type-A (data not shown). However, the durations of development for type-B and type-C are prolonged in Summer. The percentage of this delayed development was about 80 in the Japanese population of Bombyx mandarina (Shen and Hamano, 1993). In the intermediate type (type-B) of pupal-adult duration in the Japanese population, changes of oxygen consumption were relatively slow; oxygen consumption of an just after pupation was 310 ,ul/hr/g animal and it dropped about 1/6 at day-2 (Fig. 1-B). The 142 W. SHEN, K. HAMANO, and H. FUGO [Vol. 69(B),

Fig. 2. Typical fluctuation patterns of ecdysteroids in haemolymph and ovarian weight during the pupal-adult development in the silkworm, Bombyx mandarina. Ten to fifteen animals were used for each experiment. The mean of four to five replicates is shown. The standard deviations are less than 10% of each mean. low level of respiration was sustained until day-10 and then level of oxygen consumption gradually increased by day-22. The ovarian development in pupae of this type of oxygen consumption was not proceeded obviously until day-10. However, abrupt increase of ovarian weight was observed from day-12 to day-20 and the eggs were matured in their body by day-23. Adult eclosion occurred on day-27. The fluctuation of ecdysteroid titer in haemolymph was observed, but the dramatic change of ecdysteroid titer as that found in the Chinese populations was not shown (Fig. 2-B). Ecdysteroid level was between 2 to 2.5 ug/ml haemolymph and such a level continued for several days. But the level dropped suddenly on day-14. RIA activity in haemolymph of day-20 to day-27 was not detectable by this assay system. In the case of the third type of pupal-adult duration (pupal-adult development completed in 34 days in this experiment), the value of oxygen consumption was similar to former types of duration just after pupation. However, the level dropped to 5 to 10 ,ul 02Ihr/g at day-4 and this situation remained for about 2 weeks (Fig. 1-C). Reinitiation or restoration of oxygen consumption was observed from day-15 to day-16 and the levels gradually increased with age thereafter. Ovarian development was suppressed during the period in which extremely low levels oxygen consumption continued. On day-17 to day-18, the differentiation of ovaries was initiated and almost all of the eggs were matured by day-30. The necessary days for adult eclosion were 34 to 40 days in this Japanese No. 6] Ecdysteroid Levels in Bombyx mandarina 143 population (Shen and Hamano, 1993). At this moment, type-C can not be distinguished from the other types of pupal duration at the beginning of pupation. Consequently, measurement of ecdysteroid titer began from day-4. The ecdysteroid level in these animals was quite low and this situation continued for at least 2 weeks. By day-14, the level began to increase and reached a maximum of almost 5 µg by day-20. Afterwards the decreasing tendency was similar to those of type-A and type-B animals (Fig. 2-C). It is well recognized that juvenile hormones seem to be less important in pupal diapause regulation in contrast to the endocrine regulation in larvae (Chippendale, 1983). Molting hormone (ecdysteroid) is necessary for adult development and differentiation. Ecdysteroids are found only in trace levels in all pupal diapausing insects studied so far. In this study, ecdysteroid titer and oxygen consumption were observed to be very low in the Japanese population in which the animals are considered to be in a state of summer diapause. Because insect diapause is characterized by a more or less reduced oxidative metabolism and the great majority of diapausing pupae exhibit reduced oxygen consumption, the Japanese population which has a prolongation of pupal-adult develop- ment is considered to be the summer diapause state. Application of KK 42, which has a potency of inhibiting ecdysteroids synthesis in prothoracic glands, causes a delay of pupal-adult development and also suppresses the oxygen consumption in these animals (Shen et al., 1993). In conclusion, prolongation of development in Japanese population seems to be due to lack or deficiency of ecdysteroids which are necessary for adult development. Acknowledgements. We are grateful to Dr. Seijiro Morohoshi, M.J. A., for his kind advice and suggestions, and Drs. Tosihiko Hukuhara, Toshikazu Oshiki and Yoshimitsu Iwashita, Professors of United Graduate School of our University, who support most of these experiments.

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