Differential Expression Of Catalase Gene During Different Phases Of Diapause In The Bivoltine Silkworm, Bombyx Mori

Diapause occurs at the gastrula stage in the silkworm, Bombyx mori. The bivoltine strain becomes the producers of diapause and non-diapause eggs when embryogenesis is incubated at 25℃under continuous illumination and at 15℃under continuous darkness, respectively. The course of diapause in the bivoltine strain can be divided into five different phases: induction, decision, initiation, maintenance and termination. The initiation of diapause can be prevented by the treatment of hydrochloric acid at 20 h or so after oviposition and the diapause state can be effectively terminated by low temperatures around 5℃. However, the molecular mechanism of diapause is not yet clear although much knowledge has been accumulated on the diapause of the silkworm from view points of eco-physiology, physiology, endocrinology, biochemistry and molecular biochemistry.Hydrogen peroxide (H2O2), one kind of reactive oxygen species (ROS) in aerobic cells, can be produced by superoxide dismutase (SOD) and xanthine oxidase (XO) and decomposed by catalase (CAT), respectively. The relationship between the metabolism of H2O2 and different phases of diapause in the silkworm has been investigated in our previous studies. High level of H2O2 coinciding with the induction, decision and termination and low level of H2O2 coinciding with the initiation and maintenance of diapause were observed. In the present study, level of CAT mRNA and activity of catalase in the bivoltine strain were determined using RT-PCR and ultraviolet spectrophotometry to characterize expression patterns of CAT gene coinciding with the induction, decision, initiation, maintenance and termination of diapause. The results showed that expression of CAT gene exhibited different profiles during different phases of diapause in the bivoltine strain. 1 Expression of CAT gene during the induction phaseThe producers of diapause eggs contained higher level of CAT mRNA from stage 20 to stage 29 of embryogenesis compared to the producers of non-diapause eggs. Two peaks of CAT activity were observed at stage 21 and stage 27 in the producers of diapause eggs, which were 2.4 and 2.2 times as high as those of the producers of non-diapause eggs. The results confirmed that enhanced expression of CAT gene coincided with the induction of diapause in the bivoltine strain.2 Expression of CAT gene during the decision phaseThe level of CAT mRNA and activity of CAT both decreased in the producers of diapause and non-diapause eggs from the third day after pupation to eclosion. Similar to the induction of diapause, higher level of CAT mRNA and activity of CAT were observed in the producers of diapause eggs from the third day after pupation to eclosion compared to the producers of non-diapause eggs, revealing enhanced expression of CAT gene coinciding with the decision of diapause in the bivoltine strain.3 Expression of CAT gene during the initiation of phaseA big peak of CAT mRNA occurred at 24 h and disappeared at 72 h after oviposition while activity of CAT generally increased in the diapause eggs. In the case of the non-diapause eggs, level of CAT mRNA increased while CAT activity first decreased before 24 h and increased thereafter at the same time. The results indicated that there were significant differences of CAT gene expression between the diapause and non-diapause eggs during the initiation of diapause phase.4 Effects of hydrochloric acid on expression of CAT geneExpression of CAT gene in the diapause eggs, including level of CAT mRNA and activity of CAT, was significantly inhibited by the treatment of hydrochloric acid at 20 h after oviposition. Furthermore, the developmental profile of CAT gene expression in the diapause eggs treated by hydrochloric acid was similar to that in the non-diapause eggs.5 Expression of CAT gene during the maintenance phaseCAT mRNA decreased to undetected level while activity of CAT kept at low level during the maintenance phase in the diapause eggs. As described above, Level of CAT mRNA and activity of CAT increased in the non-diapause eggs simultaneously. 6 Expression of CAT gene during the termination phaseLevel of CAT mRNA and activity of CAT both increased when diapause eggs were chilled at 5℃, showing enhanced expression of CAT gene coinciding with the termination of diapause. Compared to diapause eggs, diapause eggs treated by hydrochloric acid contained higher level of CAT mRNA and activity of CAT during the chilling of 5℃.Above results confirme that differential expression of CAT gene coincides with the induction, decision, initiation, maintenance and termination of diapause in the bivolotine strain of the silkworm, Bombyx mori. Taken together with variations of H2O2 during different phases of diapause, differential expression of CAT gene may be the key regulatory factor to control level of H2O2 during different phases of diapause.