Effects Of Instant Hydrochloride Acid Soaking And Chilling On The Metabolisms Of NAD(H) And NADP(H) In Diapause Eggs Of The Silkworm, Bombyx Mori

The silkworm, Bombyx mori is a typical insect with embryonic diapause, in wichthe initiation of diapause can be prevented by instant hydrochloric acid soaking (HCl),whereas the status of diapause is terminated by chilling of5oC, respectively. Duringdiapause initiation, the significant decline of oxygen consumption, the largeaccumulation of sorbitol converted from glycogen and the arrest of cell division at theG2phase of cell cycle is observed. However, above phenomena are converted by thetreatment of HCl. During chilling, oxygen consumption retains at a very low level,sorbitol converts into glycogen. When the chilled diapause eggs are transferred into25oC, the significant increase of oxygen consumption and the re-division of cells areobserved. The metabolisms of NAD(H) and NADP(H) are closely involved in oxygenconsumption, biosynthesis and antioxidation. NAD kinase (NADK) can catalyzeNAD(H) into NADP(H). The oxidation of NADH can be catalyzed by malatedehydrogenase (MDH) and lactate dehydrogenase (LDH). On the other hand,glucose-6-phosphate dehydrogenase (G6PDH) and isocitrate dehydrogenase (ICDH) aretwo enzymes catalyzing the reduction of NADP. As oxygen consumption andconvertion between glycogen and sorbitol are involved in the metabolisms of NAD(H)and NADP(H), effects of instant HCl soaking and chilling on NAD(H) and NADP(H)metabolisms were examined in diapause eggs in the present study. The main results arereported as follow:1. Effects of HCl on the metabolisms of NAD(H) and NADP(H)During diapause initiation, the marked declines of NAD(H) level, NADP(H) leveland NADK showed the stronger degradation of NAD and NADP, in which the declineof NAD(H) may result in the decline of oxygen consumption, furthermore, the productsof NAD degradation may be related to the arrest of cell division. NADH/NAD ratio peaked at36h after oviposition, no significant variation in MDH activity and themarked decline of LDH activity were observed, indicaing the weaker oxidation ofNADH. In addition, the slight variation of NADPH/NADP ratio, no significant changein ICDH activity and the significant decline of G6DH activity were observed,suggesting the weaker reduction of NADP.In the HCl-treated diapause eggs, NAD(H) level, NADP(H) level and NADKactivity significantly increased and were significantly higher than those in diapause eggs,which indicated the stronger synthesis in NAD and NADP. The significant increase ofNAD(H) may be the foundation for the rapid increase of oxygen consumption in eggsafer the treatment of HCl. Meanwhile, NADH/NAD ratio fluctuated, MDH activitysignificantly increased, LDH activity significantly declined, which indicated thestronger oxidation of NADH via malate-aspartate shuttle. Except for36h aferoviposition, there was no significant variation of NADH/NAD ratio between diapauseeggs and the HCl-treated diapause eggs. Compared to diapause eggs, the HCl-treateddiapause eggs had higher activities of MDH and LDH, indicating the stronger oxidationof NADH coinciding with the increase of NAD(H). In addition, the marked increase ofNADPH/NADP ratio, no significant variation in G6PDH activity, and the markedincrease of ICDH activity were observed in the HCl-treated diapause eggs, indicatingthe stronger reduction of NADP. There was no significant variation in NADPH/NADPratio between diapause and the HCl-treated diapause eggs, whereas higher activities ofG6PDH and ICDH were observed in the HCl-treated diapause eggs, suggesting thecoinciding of the stronger reduction of NADP and the increase of NADP(H).It showed that the increase of oxygen consumption in the HCl-treated diapauseeggs may be resulted from the enhancement of NAD(H) level rather than NADH/NADratio, whereas it is the enhancement of NADP(H) rather than NADPH/NADP ratio thatleads to the stronger abilities in biosynthesis and antioxidation.2. Effects of chilling on the metabolisms of NAD(H) and NADP(H)From10d to90d after oviposition in diapause eggs incubated at25oC, activitiesof G6PDH and ICDH significantly increased, however, there was no significant variation in the other parameters, showing the metabolisms of NAD(H) and NADP(H)are in the stable status during diapause maintenance.Chilling of5oC resulted in the marked increases of NAD(H) level, NADP(H)level and NADK activity, which were higher than those in diapause eggs at25oC,indicating the stronger synthesis of NAD and NADP. The rapid increase of oxygenconsumption transferred into25oC after chilling may be due to the marked increase ofNAD(H) in the5oC-treated diapause eggs. No marked variation of NADH/NAD ratio,the significant increase of MDH activity and no marked change in LDH activity wereobserved in the5oC-treated diapause eggs, revealing the stronger oxidation of NADHvia the shuttle of malate and aspartate. There was no significant difference inNADH/NAD ratio and LDH activity between diapause eggs and the5oC-treateddiapause eggs, however, higher activity of MDH in the5oC-treated diapause eggs wasobserved, indicating the stronger oxidation of NADH. In addition, there was nomarked variation of NADPH/NADP ratio at the prophase and metaphase of chilling,whereas the marked increase of NADPH/NADP ratio was observed at the anaphase.Meanwhile, G6PDH and ICDH activities markedly increased at the anaphase ofchilling, suggesting the stronger reduction of NADP. There was no significantvariation in NADPH/NADP ratio and ICDH activity at the prophase and metaphasebetween diapause and the5oC-treated diapause eggs, whereas the higher NADP(H)level and G6PDH activity were observed, which indicating the large number ofNADPH may be used in the antioxidation at the prophase and metaphase of chilling.It showed that, in the5oC-treated diapause eggs, it is the enhancement of NAD(H)rather than NADH/NAD ratio that leads to the increase of oxygen consumption,furthermore, the stronger biosynthesis and antioxidation may be due to the increase ofNADP(H) and NADPH/NADP ratio.