HCl Treatment for Preventing Diapause Causes Ca2+ Efflux in Bombyx mori Eggs

To elucidate the mechanism for preventing entry into embryonic diapause or breakdown of diapause in Bombyx mori by HCl and dimethyl sulfoxide (DMSO) treatment or a combination of cold and HCl treatment, we performed quantitative analysis of Ca2+ and Mg2+ in the chorion and egg content using inductively coupled plasma atomic emission spectrometry (ICP-AES). When diapause eggs that had been incubated at 25°C for 2 days from oviposition and at 4°C for an additional six days were treated with HCl solution, the amount of Ca2+ in the chorion and egg content after HCl treatment was reduced to one-seventh, as compared with the amount before treatment. In contrast, there was no change in the amount of Mg2+ with HCl treatment. The amount of Ca2+ in the HCl solution after the diapause eggs were treated increased 7.5-fold, as compared with that of eggs treated with water. Even when 17-day-old diapausing eggs were treated with HCl, which did not break diapause, the amount of Ca2+ in the chorion and egg content was reduced to one-fifth, as compared with the control. Meanwhile, changes in Ca2+ and Mg2+ contents were not observed in 12-hr-old diapause-destined eggs before or after treatment with DMSO, which effectively prevents diapause. These data may suggest that Ca2+ efflux from diapause eggs by HCl is not directly associated with preventing entry into diapause or breaking of diapause. In addition, we discovered that the amount of Ca2+ in diapause-destined eggs was more than 2.4-fold larger than in non-diapause-destined eggs.