Entomol. exp. appl. 46: 291-294, 1988 �9 Kluwer Academic Publishers, Dordrecht - Printed in the Netherlands

Short communication

Induction of non-diapause eggs in Bombyx mori by a trehalase inhibitor

291

Satoshi Takeda 1, Yoshiaki Kono 2 & Yukihiko Kameda 3 ISericultural Experiment Station, Yatabe, Ibaraki, 305 Japan; 2Takedg Chemical Industries, Ltd, Agricultural Chemicals Division, Ichijoji-Takenouchi-cho, Sakyo-ku, Kyoto, 606 Japan, 3Hokuriku University, School o f Pharmacy, Kanagawa-machi, Kanazawa, 920-H Japan

Accepted: November 5, 1987

Key words: Bombyx mori, egg diapause, trehalase, validoxylamine A

Abstract

The effect of validoxylamine A (VAA), a potent and specific trehalase inhibitor, on the induction of non- diapause in Bombyx mori was examined. The VAA induced non-diapause eggs and prevented the glucogen accumulation in the eggs. Trehalase activity of the pupal ovary was effectively inhibited by the VAA injection.

Introduction

The glycogen content of the diapause eggs which is about 35~ higher than that of non-diapause eggs changes appreciably during the diapause (Chino, 1957). The diapause hormone secreted by the suboesophageal ganglion (Hasegawa, 1951; Fukuda, 1951) stimulates the activity of trehalase in the de- veloping ovaries which operates as the rate-limiting enzyme in glycogen accumulation (Yamashita et al., 1972). The trehalase transforms hemolymph treha- lose into glucose as the source of ovarian glycogen (Yamashita et al., 1972). Based on these results, it is possible to consider that the incidence of embryonic diapause in Bombyx may require a sufficient glyco- gen accumulation in the oocyte. The purpose of this report is, therefore, to examine the effect of validox- ylamine A (VAA), a potent and specific trehalase in- hibitor (Asano et al., 1987; Kameda et al., 1987), on the induction of non-diapause eggs.

kept under a 18L:6D light regime at 25 ~ The silk- worms were reared on an artificial diet containing about 22~ mulberry leaf powder (Horie et al., 1973) throughout the larval stages under a 6L:18D light re- gime to obtain only diapause-egg producers.

Ten tzl of Ringer solution (0.75~ NaC1) contain- ing 0.01 - 1.0/zg of VAA, a specific trehalase inhibi- tor (Asano et al., 1987) was injected into the body cavity of the pharate adults 0, 4, 6 and 8 days after pupal ecdysis. Eggs laid by the adults were kept at 25~ for more than 15 days and the number of hatched eggs was counted in each batch. The num- ber of unfertilized eggs was also checked.

Trehalase activity in crude ovarian extract was measured by the rate of glucose released enzymati- cally from trehalose by the method described in Yamashita et al. (1974). The glycogen content of the eggs was determined according to the method of Yamashita & Hasegawa (1965) within one day after oviposition.

Materials and methods

The hybrid race of the silkworm N 137 x C 137, was used for the experiments. The embryonic stage was

Results and discussion

Effects of VAA injection to the pupae on egg production and the diapause nature of the laid eggs

292

600

500

400

300

2OO

100

600

500

4 0 0

300

200

I 0 0

0

O - d a y - o l d

111 0 . 1 0 . ~ I

4 - d a y - o l d

o . I o . ~ I

VAA a m o u n t ( p g / p u p a )

6 - d a y - o l d - - - - 8 - d a y - o l d

0.01 0 . 0 5 0 . 1 0 . S 1

VAA amount , ( o g / p u p a )

~ . ~ - ~ - ~ - = _

O 0 . O 1 0 . 0 5 O . I 0 . 5 " I

100

8o

60

40 ~

2O

o .

#

Fig. 1. Effect of VAA injection to silkworm pupae producing di-

apause eggs on egg production and non-diapause egg occurrence.

Each value is the average o f 8 - 1 0 egg batches. [] Total no. laid

eggs. �9 No. unfertilized eggs, - - Non-diapause egg percentage

to fertilized eggs.

are shown in Fig. 1. All pharate adults which received 10 #1 Ringer solution at 0, 4, 6 and 8 days after pupal ecdysis became adults 10-11 clays after pupal ecdysis and they laid 400- 500 eggs. The eggs in each 8-10 egg batches were exclusively of the di- apause type. On the other hand, the VAA injection induced non-diapause eggs although the total num- ber of eggs laid and the fertility of the eggs were reduced to some extent by the high doses of VAA ad- ministrated. The effect of the injection on each pa- rameter depended on the developmental stage of the pharate adults. VAA injection at the earlier stage (0- and 4-day-old pharate adults) was not effective to in- duce non-diapause eggs but resulted in a large amount of unfertilized eggs at higher doses. The 6- day-old pharate adults responded most positively to the VAA injection in producing non-diapause eggs. Eighteen to 23070 of non-diapause eggs were ob- tained by the injection of 0.05 - 1.0 #g VAA. Howev-

er, when VAA was injected into the 8-day-old pharate adults, only 5~ of non-diapause eggs were produced even at the highest dose (1 #g/insect) of VAA. There was no significant difference in the degree of non-diapause occurrence among the VAA doses (0.05-1 #g) in both the 6- and 8-day-old pharate adults. The number of unfertilized eggs decreased with the pupal age when VAA was inject- ed.

To confirm the induction of non-diapause eggs by VAA, non-diapause egg production was reexamined on a larger experimental scale and the glycogen con- tent of the eggs was determined using 7-day-old pu- pae. As shown in Table 1, a low but significant num- ber of non-diapause eggs was produced in almost all the egg batches of the VAA-injected adults, whereas no control insects laid non-diapause eggs. The glyco- gen content (mg/g eggs) in the VAA-injected adult egg batches was also reduced by about 18070 com- pared with that of the control egg batches. VAA prevented the incidence of embryonic diapause and inhibited the normal accumulation of glycogen in the oocytes.

Then, to clarify the inhibitory mechanism of the glycogen accumulation in the eggs, the trehalase ac- tivity was measured in the ovary of the VAA injected pupae. Trehalase activity in crude ovarian extract of the control pupae was about 4.0 (nmol/min/mg pro- tein) which was almost the same level to that report- ed by Yamashita (1969). No trehalase activity, how- ever, was detected in the extract of VAA-injected pupae as far as the same concentration of the extract to the control was employed (Table 2). VAA has a very strong competitive inhibitory activity against trehalase in various species of organisms, i.e. the Ki value (inhibition constant) of VAA against the treha- lase of the tobacco cutworm larvae was 1.0 x 10 -9 (Kameda et al., 1987). VAA activity is specific to tre- haiase, without any inhibitory activity to other glucoside hydrolases i.e. cellulase, pectinase, chitinase, a-amylase, a- and fl-glucosidases (Asano et al., 1987).

The present results indicated that VAA applica- tion was evidently effective for the inhibition of tre- halase in the ovary of the silkworm pupae at the criti- cal age for the accumulation of glycogen in the eggs.

Takeda & Hasegawa (1975) showed that ouabain

293

Table 1. Effect of VAA-injection on non-diapause egg production and egg glycogen content in silkworms producing diapause eggs. No. pupae treated per injection: 70.

Injection No. egg batches

Diap. egg Mixed Non-diap. only eggs only

No. non-diap. Glycogen eggs in mixed content egg batches* (mg/g eggs)**

Ringer 67 0 0 0 34.3 :t: 3.01 (100) VAA 4 61 0 36.1 + 29.0 28.3 + 1.72 (82.2)

Ten/~g VAA in 10 #1 of 0.75~ NaC1 solution was injected into 7-day-old pupae. *Mean + S.D. of 61 egg batches in which non-diapause eggs were produced. **Mean + S.D. of five egg batches.

Table 2. Effect of VAA-injection on trehalase activity of pupal ovary of the silkworm, Bombyx mori.

Injection Ovary fresh Trehalase activity weight (mg)

(nmole/min/g (nmole/min/mg ovary) protein)

Ringer 423.5 + 70.9 254.0 + 25.8 4.09 + 0.59 VAA 455.8 + 30.5 -46.2 + 10.3" -1.25 +_ 0.21"

Each value indicates Mean + S.D. of 6 pupae. Injection as in Table 1, but then the ovary was obtained 3 h after injection. Crude ovarian extract in 0.02M phosphate buffer (pH 6.0) used as enzyme preparation. *Apparent negative activity caused by reduction of glucose content in enzyme reaction mixture for 30 min incubation.

prevents the diapause incidence of the s i lkworm eggs

by suppressing the release of the diapause h o r m o n e

from the suboesophageal gangl ion wi thout directly

affecting oocytes. However, this effect was observed

in the early to middle stage of the development of

the adults and the degree of non-d iapause egg occur-

rence was very high compared with VAA.

On the other hand, based on these results, it is

highly probable tha t VAA inject ion led pr imar i ly to

the inh ib i t ion of ovar ian trehalase and consequent ly

the reduct ion of the glycogen accumula t ion in the

eggs and prevented the diapause incidence. In addi-

t ion it is suggested that the trehalase activity deter-

min ing the diapause of the eggs was specifically in-

creased dur ing a short per iod a round the 7th day of

pupa l age.

VAA, thus, was revealed to be a useful tool for the

ana lyza t ion not only of the relat ionship between

glycogen accumula t ion and diapause incidence in

B o m b y x eggs bu t also of the critical role of trehalase

in the sugar metabol i sm in insects.

R e f e r e n c e s

Asano, N., T. Yamaguchi, Y. Kameda & Matsui, 1987. Effect of validamycins on glycohydrolases of Rhizoctonia solani. J. An- tibiotics 40: 526- 532.

Chino, H., 1957. Carbohydrate metabolism in diapanse eggs of the silkworm, Bombyx mori. I. Diapause and the change of glycogen content. Embryologia 3: 295-316.

Fukuda, S., 1951. The production of the diapause eggs by trans- planting the suboesophageal ganglion in the silkworm. Proc. Japan Acad. 27: 672-677.

Hasegawa, K., 1951. Studies on the voltinism in the silkworm, Bombyx mori L., with special reference to the organs concern- ing the determination of voltinism (A preliminary note). Proc. Japan Acad. 27: 667-672.

Horie, Y., T. Inokuchi, K. Watanabe, A. Nakasone & K. Yanaga- wa, 1973. A few ~periments on the improvement of the silk- worm artificial diet. Bull Seric, Exp. Station, (96) 7 - 20 (In Jap- anese).

Kameda, Y., N. Asano, T. Yamaguchi & K. Matsui, 1987. Validox- ylamines as trehalase inhibitors. J. Antibiotics 40: 563-565.

Takeda, S. & K. Hasegawa, 1975. Alteration of egg diapause in Bombyx moriby ouabain injected into diapause egg producers. J. Insect Physiol. 21: 1995-2003.

294

Yamashita, O., 1969. Studies on the glycogen metabolism in pupal ovaries of the silkworm, Bombyx mori L. J. seric. Sci. Japan 38: 329-339.

Yamashita, O. & K. Hasegawa, 1965. Studies on the mode of ac- tion of the diapause hormone in the silkworm, Bombyx mori L. (V) Effect of diapause hormone on the carbohydrate metab- olism during the adult development. (In Japanese with English summary) J. seric, Sci. Japan 34: 235-243.

Yamashita, O. & K. Hasegawa, 1970. Oocyte age sensitive to the

diapause hormone from the stand point of glycogen synthesis in the silkworm, Bombyx mori. J. Insect Physiol. 16:

2377-2383. Yamashita, O., K. Hasegawa & M. Seki, 1972. Effect of the di-

apause hormone on trehalase activity in pupal ovaries of the silkworm, Bombyx mori L. Gen comp. Endocr. 18: 515-523.

Yamashita, O., M. Sumida & K. Hasegawa, 1974. Developmental changes in midgut trehalase activity and its localization in the silkworm, Bombyx mori. J. Insect Physiol. 20: 1079-1086.