The Physiological and Biochemical Studies on Insect Cuticle Formation and Chitin Synthesis

The tanned pupal cuticle was formed in vitro when the wandering stage larval epidermis of Manduca sexta was cultured in the presence of 5μg/ml of β-ecdysone for 4 days. Since metamorphosis occurs in response to two releases of ecdysone, epidermis was explanted from feeding final-instar larvae (day 3, weighing 6.5-7.0g) before the first release of ecdysone and was cultured in Grace's medium. During the first 24hr of exposure to 1μg/ml of β-ecdysone in vitro, the epidermis changed its cellular commitment to that for pupal cuticle formation. Juvenile hormone inhibited the change of the cellular commitment and the tissue subsequently formed larval cuticle in the presence of JH. The epidermis of the last instar larvae was found to convert JH I into JH-acid. This esterase activity increased as a function of larval age and was considered to be involved in elimination of intracellular JH from the target tissues in preparation for metamorphosis. The nuclei were isolated and assayed for specific JH binding using the competitive nuclear exchange assay. Specific binding was found only for epidermal nuclei from larvae before the change in commitment of the cells. Diflubenzuron was found to inhibit both endocuticle deposition and β-ecdysone-initiated pupal cuticle synthesis by using the in vitro culture technique. Both effects were due to its inhibition of chitin synthesis. When larvae were injected with 14C-glucosamine after treatment of difiubenzuron (DFB), 14C-UDP-N-acetylglucosamine (UDP-AGA) was found to accumulate in vivo. However, chitin synthetase was not inhibited by DFB. Peritrophic membrane in the midgut of Mamestra brassicae final instar larvae was used as a target to study the mode of inhibition of chitin synthesis by DFB. Chitin synthesis was not inhibited only when DFB and UDP-AGA were applied inside the midguts. When 14C-AGA was applied outside the midguts, the amount of the radio-labeled compounds transported across the microvilli membrane was about 1/4 as much in the DFB-treated tissues as in the control tissues. The results lead the conclusion that DFB seems to be an inhibitor of UDP-AGA transport across the microvilli membranes.