Differential expression of the Bombyx mori diapause-termination timer gene Ea4 in diapause-inducing temperature and photoperiod.

Diapause in Bombyx mori eggs is induced by temperature and photoperiod at the stage of embryonic development in the maternal generation. In those diapause eggs, Esterase-A4 is suggested to serve as a diapause-termination timer (TIME-EA4), because its ATPase activity shows an interval-timer elevation after acid treatment or chilling of eggs to break diapause. To clarify whether the timed ATPase activity of TIME-EA4 is related to its gene (Ea4) expression, we analyzed Ea4 mRNA of eggs in diapause-inducing environmental conditions. Reverse transcription PCR (RT-PCR) analysis showed that the level of Ea4 mRNA was lower in 15DD than in 25LL (P < 0.01) or in 20LD (P < 0.01) but did not oscillate when photoperiod and temperature periodically oscillated (P > 0.05). Furthermore, expressed sequence tag profile and gene microarray analysis demonstrated that Ea4 showed stage-specific and tissue-specific expression during postembryonic stages, high Ea4 mRNA in the spinning and eclosion stages, and in integument and head, but low in gonads of fifth-instar day-3 larvae. Then we analyzed the relationship between TIME-EA4 ATPase activities and Ea4 gene expression. The ATPase activities in diapause eggs laid by the resultant adults transiently elevated after treatments to break diapause, that is, at 1.5 h after common-acid treatment and day 13 after chilling at 5°C. However, these elevations of enzyme activities were not accompanied by any increases in Ea4 mRNA levels. In conclusion, the termination of the Bombyx embryonic diapause is related to TIME-EA4 but not to its gene expression.