The Mechanism Of Oxidative Stress In Drosophila Melanogaster Induced By Azadirachtin Stress

Azadirachtin is one of the most widely used botanical pesticides with great potential value of research and development. It has higher biological activity and its insecticidal effects could work via variety ways. Serval researches at home and abroad focus on the complex mechanisms and some specific activities of azadirachtin, such as regulation of growth and development and antifeedant effect have been elucidated. But many more aspects, including its effect on the oxidative stress system as well as the physiological effect remain unclear.We took D. melanogaster as the tested insect to studied the effect of azadirachtin on the antioxidation system and lifespan of D. melanogaster. The lifespan of D. melanogaster affected by azadirachtin was determinated through the survival test. According to the measurement of catalytic activity of enzymes, catalytic activity of the total superoxide dismutase(T-SOD), copper/zinc SOD, manganese SOD, catalase(CAT),and glutathione-s-transferases(GSTs) activity of the third instar larvae stage, pupal stage and 0d age and 15 d age adult stage of D. melanogaster was measured. Meanwhile,the gene expression of Cu/Zn sod, Mn sod, catalase, prx gene family, mth, sir2 and d FOXO of the different development period of D. melanogaster was measured through the quantitative real-time PCR. In addition,the effect of azadirachtin on the reactive oxygen species(ROS),malondialdehyde(MDA) content and the genome DNA of the different development period of D. melanogaster was studied. The main results were as follows:The D. melanogaster was dealt with the feed mixed with 1mg/kg and 4mg/kg azadirachtin.The newly eclosion of F2 generation of both the treatment group and control group were collected to do the survival experiment.The result indicated that azadirachtin could significantly shorten the maximum lifespan,50% survival time and mean lifespan,and reduce the survival rate of D. melanogaster.According to the measurement of catalytic activity of enzymes,the change of the activity of antioxidant enzymes in D. melanogaster treated with azadirachtin was assayed.The result showed that azadirachtin could significantly inhibit the activity of the total SOD,Cu/Zn SOD, Mn SOD, catalase(CAT),and glutathione-s-transferases(GSTs) in D.melanogaster. However,at the third instar larvae stage,the activity of total SOD and Cu/Zn SOD in D. melanogaster in the treatment groups was significantly improved. The exceptions may be associated with the oxidative stress adjustment in D. melanogaster itself.By using the quantitative real-time PCR, it was found that azadirachtin could significantly down-regulate the gene expression of Cu/Zn sod, Mn sod, catalase and prx gene family of the different development period of D. melanogaster.Meanwhile, the effect of azadirachtin on the gene expression of antioxidant enzymes gene was consistent with that of the antioxidant enzymes activity. At the third instar larvae stage,the gene expression of Cu/Zn sod in D.melanogaster treated with azadirachtin was significantly up-regulated.The exceptions may be associated with the oxidative stress adjustment in D. melanogaster itself.In addition,the research on the prx gene family of D. melanogaster treated with azadirachtin showed that azadirachtin could interfere with the gene expression of prx gene family,which was related to azadirachtin dose in the treatment and the different development stages. The research on the gene mth,sir2 and d FOXO indicated that azadirachtin could significantly inhibit the sir2 expression,down-regulate the mth expression of adult fruit flies and mainly promote the d FOXO expression.The change of the ROS and MDA content in D. melanogaster treated by azadirachtin was determined.Compared to the control group,the ROS and MDA content in D.melanogaster in the treated groups were significantly increased.The results indicated that azadirachtin potentially lead to the accumulation of ROS and aggravation of the lipid peroxidation in the body of D. melanogaster.As a result, the biological macromolecules may be damaged.However, through agarose gel electrophoresis to check the integrity of the genome DNA of the different development period of D. melanogaster,the result showed that azadirachtin could induce no obvious damage of genome DNA within the test dose.According to experimental results,the research indicated that azadirachtin could inducethe oxidative stress damage of D.melanogaste,inhibit the activity of antioxidant enzymes,down-regulate the gene expression of antioxidant enzyme, disturb the dynamic balance of the free radicals, aggravate the lipid peroxidation and harm the biological macromolecules in the body of D. melanogaster, which furtuer accelerated the aging process and shortened the lifespan of fruit flies.However, whether azadirachtin could damage the genome DNA of D. melanogaster remained to be further confirmed.The results of the research laid a good foundation of theory and experiment for illuminating the effect on the oxidative stress of the insects, the physiological effect and the mechanism of action of azadirachtin.The research deserved further exploring.