Relationships Between an Application O F KK-42 and Pupal Developmemt
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144 Proc. Japan Acad., 69, Ser. B (1993) [Vol. 69(B), 28. Relationships between an Application of KK-42 and Pupal Developmemt in the Silkworm, Bombyx mandarina By Weide SHEN,*) Kunikatsu HAMANO,*) and Hajime FUGO**) (Communicatedby Seijiro MoRoxosxl, M.J. A., June 8, 1993) Abstract: Populations of Chinese origin of the silkworm, Bombyx mandarina, do not show arrested pupal stage, however populations of Japanese origin undergo pupal summer diapause. Hormonal control of this pupal summer diapause in Bombyx mandarina of Japanese origin was investigated using KK-42 treated pupae of Chinese origin. When an imidazole compound (KK-42:1-benzyl-5-[(E) 2, 6-dimethyl-1, 5-heptadienyl] imidazole) was injected into the newly ecdysed pupae of Chinese populations, the pupal-adult development was arrested for as long as 40 days. Resumption of pupal-adult development of the KK-42 treated pupae was induced by injection of 20-hydroxyecdysone (20-HE). Oxygen consumption of the KK-42 treated animals was very low comparing to the control but it increased gradually about 35 to 37 days after treatment. Abrupt increase of oxygen consumption was observed about 12 hr after the application of 20-HE in the KK-42 treated animals. These results strongly indicate that the pupal summer diapause in Japanese populations of Bombyx mandarins is due to the low level of ecdysteroids in their body thus preventing them from proceeding to the pupal-adult development. Key words: Summer diapause; imidazole compound; ecdysteroid; Bombyx mandarins; pupae. Introduction. Terpenoid imidazoles had been initially reported as compounds with anti juvenile hormone activity (Kuwano et al., 1984, 1985; Asano et al., 1984, 1986). On the other hand, an inhibitory effect of imidazole compound on the ecdysteroid synthesis in prothoracic glands of the silkworm, Bombyx mori, has also been demonstrated (Kadono-Okuda et al., 1987a, b; Yamashita et al., 1987). When an imidazole compound (KK-42) was applied early in pupal stage, the duration of pupal-adult development was prolonged significantly (Kadono-Okuda et al., 1987a; Fugo and Oshiki, 1988). Furth- ermore, the "dauer pupa" (non-developing pupa) can be induced by the application of KK-42 into the brainless pupa of the silkworm, Bombyx mori (Fugo and Oshiki, 1990). In a previous paper, we demonstrated that the pupal aestivation of the silkworm, Bombyx mandarina, found in Japanese population may be due to the deficiency of ecdysteroids in their body (Shen et al., 1993a). We have also reported that a Chinese population of Bombyx mandanina exhibits no arrested pupal stage, although this population is reared in Japan and is acclimatized in the Japanese environmental conditions (Shen and Hamano, 1993). The present paper deals with the effects of KK-42 injection on the pupal development in Bombyx mandanina, in order to elucidate the role of ecdysteroid on the pupal aestivation in Japanese populations. *) 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. No. 6] Prolongation of Pupal Development by KK-42 145 Materials and methods. Insects. Eggs of the silkworm, Bombyx mandarina, were collected from mulberry fields in China (Suzhou, Jiangsu Province) and in Japan (Fuchu, Tokyo) on the Autumn of 1991. Eggs were incubated under natural conditions from late of April in 1992. Larvae hatched from the eggs were reared with mulberry leaves under natural climatic conditions in Japan (Fuchu); the mulberry trees were surrounded with nets to prevent the parasitism of Tachina flies. In these conditions, pupation occurred during late of May to beginning of June. Pupae were maintained in an incubator at 25±1°C under natural photoperiodic conditions. Estimation of respiration. Oxygen consumption was measured in each individual pupa as the same method described previously (Shen and Hamano, 1993). Chemicals. An imidazole compound (KK-42: 1-benzyl-1, 5-[(E)-2, 6-dimethyl-1, 5-heptadienyl] imidazole) was synthesized by the reaction of citral and benzylamine with tosylmethyl isocyanide (Kuwano et al., 1985). 20-Hydroxyecdysone was purchased from SIGMA Chemical Company (USA). Injection. The synthetic KK-42 was dissolved in 50% aqueous solution of acetone. Twenty ,ug of KK-42 in 2.5µl of 50% acetone were injected into each pupa. As a control, same volume of acetone solution (50%) was injected. 20-Hydroxyecdysone was dissolved in distilled water and each pupa received 5 µg of this compound in 5 µl of distilled water. Five ul of distilled water was injected into each pupa as control in the same way as with the chemical injection. Results and discussion. Diapause is known as a specific developmental stage characterized by reduced metabolic activity and arrested morphogenesis (Chippendale, 1983; Tauber et al.,1986). In general, diapause can occur during any of the life span of the insect but usually occurs only once in the life history of a particular species (Masaki,1980; Beherens, 1984). In the silkworm, Bombyx mandarina, egg enters diapause at an early phase, the dumbbell-shaped embryo, of embryonic development (Umeya, 1946) in the same way as Bombyx mori (Morohoshi,1959; Yamashita and Hasegawa,1985). In a previous paper, we reported that Bombyx mandarin in Japanese populations aestivates in pupal stage and this may be caused by the deficiency of ecdysteroids in the pupal stage (Shen et al. ,1993a, b). In order to clarify the induction mechanism of pupal aestivation in this Japanese population of Bombyx mandarina, effects of KK-42 injection on the pupal-adult development in Chinese populations, which do not aestivate in pupal stage, was investigated. Since insect diapause or aestivation, once it occurs, is characterized by a more or less reduced oxidative metabolism and since oxygen consumption is the commonly used way to quantify oxidative rates (Masaki,1980; Chippendale, 1983), we measured the oxygen consumption of the KK-42 treated animals as one of the criteria for the pupal-adult development. In the control animals (typical type-A: Shen and Hamano, 1993), the duration of pupal-adult development was 14 days; eye pigmentation completed by day-7, wing pigmentation occurred on day-12 and adult eclosion was observed on day-14. Oxygen consumption after pupal ecdysis was about 430 µl O2/hr/g fresh weight. During day-2 to day-4, levels dropped significantly, and thereafter increased sharply. The maximum rate of oxygen consumption was found at day-11. Twenty µg of KK-42 in 2.5 µl of 50% acetone were injected into a pupa of type-A within 3 hr after pupation. In the KK-42 treated animals, oxygen consumption suddenly decreased with age and a steady phase continued for about one month and meanwhile pupal-adult development was quite suppressed. The reinitiation of respiration and pupal-adult development was observed by day-34 and thereafter oxygen consumption increased and adult development proceeded with age. Differentiation of pupal-adult 146 W. SHEN, K. HAMANO, and H. FUGO [Vol. 69(B), Fig. 1. Effect of KK-42 injection on the oxygen consumption in the silkworm, Bombyx mandarina. At least 10 animals were used for each experiment. Symbols indicate as follows: P. pupation; EP, eye pigmenta- tion; WP, wing pigmentation; E, eclosion; --• , control; --o--, 20 ug of KK-42. The mean and standard deviation (vertical bars) of four to five replicates are shown. Fig. 2. Restoration of oxygen consumption in KK-42 treated pupae by injection of 20-hydroxyecdysone. Twenty µg of KK-42 were injected into a pupa within 3 hr after pupal ecdysis. KK-42 treated animals were kept in 25°C. Indicated time (arrows), 5µg of 20-hydroxyecdysone were injected into a KK-42 treated animal. Ten animals were used for each run of assay. The mean and standard deviation (vertical bars) of four to five replicates are shown. --• : 20-hydroxyecdysone; --0--: control. development seemed to be coincident with the increase of oxygen uptake (Fig. 1). As mentioned above, the induction of prolonged pupal-adult development in the Chinese population of Bombyx mandarins is caused by an application of KK-42. In Japanese populations, aestivation occurs frequently in summer season, and the duration of pupal-adult development is 30 to 40 days (Shen and Hamano, 1993). This prolongation of pupal-adult development may be due to the deficiency of ecdysteroids (Shen et at., 1993a, b). Therefore if the delay of the development is caused by the low level of ecdysteroids, it is expected that exogeneous ecdysteroid can shorten the duration of pupal-adult develop- ment. Resumption of oxygen consumption in KK-42 treated pupae was induced by an injection of 20-HE (Fig. 2). When 20 µg of KK-42 were injected into the newly ecdysed pupae, oxygen uptake of these animals decreased suddenly as shown in Figs. 1 and 2. However, respiratory activity began to increased spontaneously in these animals by 32nd No. 6] Prolongation of Pupal Development by KK-42 147 Table I. Elevation of oxygen consumption in KK-42 treated pupae by injection of 20-hydroxyecdysone to 34th day after KK-42 treatment. Injection of 20-HE caused a rapid resumption of oxygen consumption at 10-day-old and 29-day-old KK-42 treated animals. Fluctuation patterns of oxygen consumption were almost similar to that of the control (Fig. 2). The restoration of respiratory activity was observed when 20-HE was injected into the KK-42 treated animals. This indicates that the reinitiation of arrested development was induced by this insect hormone. Elevation of oxygen consumption after treatment with 20-HE was clearly demonstrated. About 4.5 times increase of oxygen uptake was recorded on 12 hr after injection (Table I) and by ten times and more increase was observed by 36th to 48th hr after treatment; this indicates rapid metabolic reinitiation occurred after this application (Table I).