Effects Of Lead Exposure On The Expression Level Of Antioxidant Genes And The Reproductive Development Related Genes And Activity Of Antioxidant Enzymes In Tissues Of Bombyx Mori

Bombyx mori is the model insect of lepioloptera and it is a widely used experimental animals. Research on heavy metals like lead on mamals (such as human and mouse )and aquatic organisms is common, but relatively little in invertebrates. Research on the insect was less. In our experiment, we used Pb(NO3)2 as the resource of Pb²⁺ to treat Bombyx mori during the whole laval stage. We investgested the accumulation of Pb²⁺ in tissues of silkworm, the basic physical and reproductive development-related indicators and the effects of lead exposure on the expression level of antioxidant genes and the reproductive development related genes and activity of antioxidant enzymes in tissues of Bombyx mori. The major results are as follows:1.When forced by lead(Pb), a heavy metal, the accumulations of Pb²⁺ differs apparently in different tissues of silkworms. A different rule appeared between males and females.In the midgut, gonad and malpighian tube of Bombyx mori, the content of Pb²⁺ increased with improved concentration of Pb(NO3)2 in food which was measured by GFAAS, showing an obvious Pb²⁺ accumulation effect. The content of Pb²⁺ in excrements of silkworms also increased apparently with an effect of concentration gradient. This showed that,the larva of Bombyx mori had a strong excreting ability to detoxification and there were accumulations of Pb²⁺ in gonads.The content of Pb²⁺ in midgut of both sexes obviously increased with the improved exposion concentration of Pb²⁺ , presenting an accordance trend. However, when in the same exposion concentration of Pb²⁺ , the content of Pb²⁺ in midgut of females was higher than males.The content of Pb²⁺ in malpighian tube was close to that in alimentary canal. Meanwhile, with the improved exposion concentration of Pb²⁺, the content of Pb²⁺ in both sexes showed a thend of rising at first and then decreasing. Under the condition of 40 mg/kg exposion concentration, the malpighian of male larvas had a higher excretion ability of Pb²⁺ than that of females.The accumulated amount of Pb²⁺ in gonad was obviously lower than that in alimentary canal and malpighian tube. When the concentration of Pb²⁺ in forage was 40-160 mg/kg, the content of Pb²⁺ in female gonad was higher than males, suggesting that female gonads were influenced greater by Pb²⁺ probably.2.The antioxidant detoxifying enzymes activity in fat body, midgut and gonad of Bombyx mori and expressions of relative genes were sensitivity to Pb²⁺ forcing.After forced by Pb²⁺ ,a heavy metal , silkworms would clear the radical created by Pb²⁺ by koinonia of antioxidant detoxifying enzymes like SOD,CAT,GST and GSH-Px in order to reduce the harm to organism, which was also a flexible response to overcoming harmful environment and preventing toxicosis.When dealed with heavy metals, SOD and CAT in Bombyx mori's fat body and midgut have poison excited and poison inhibit effect, but the ranges were different. When exposed in 10-160 mg/kg of lead, the activity of SOD in both fat body and midgut of Bombyx mori's 5 instar larvaes would significantly rise, however, they were inhibited in gonades. Moreover, the active of CAT in fat body would significantly rise in fatbody under 10-160 mg/kg lead exposure,while the active was inhibited in midgut and gonades.Detected the mRNA expression level of Sod and Cat, we found that the Sod expression levels in fatbody,midgut and gonads of Bombyx mori's 5 instar larvaes were all decreasing. Differently, the expression of Cat were up-regulated in the three tissues showing that rised of enzyme active were not completely rely on the gene transcription and the synthesis of proteins. There were asynchronism between the changes of enzyme activities of CAT and SOD and mRNA expression level changes of relative genes. It suggested that after lead stress the fatbody of silkworm could rise the enzyme active to antidote the stress; But the enzyme active was depressed in gonads suggesting that it was easier to be harmed.Glutathione S-transferase (GST) and glutathione peroxidase (GSH-Px) are important detoxifying enzymes in vivo. In order to study the influencing mechanism of heavy metal lead on lepidopterous insect, we fed the Bombyx mori with artificial diet added with Pb(NO3)2 for Pb²⁺ exposure. Enzyme activity assay and real-time PCR were employed to investigate the activity of enzymes and the mRNA transcription level of related genes in tissues of day 6 larvae of the 5th instar. In normal day 6 larvae of the 5th instar, GST enzyme activity in gonad was 20.0 and 34.6 times of that in fat body and midgut respectively, and GSH-Px enzyme activity was 46.6 and 57.5 times of that in fat body and midgut respectively. After the larvae fed on artificial diet containing 10 mg/kg or higher Pb²⁺, activity of the two enzymes in gonad decreased by many times. When the artificial diet contained 20-80 mg/kg Pb²⁺, mRNA transcriptional levels of Gstd1 gene and Gsh-Px gene were also significantly down-regulated, with more significant down-regulation in males over females. After the larvae fed on artificial diet containing 10-160 mg/kg Pb²⁺, the activity of GST and GSH-Px in fat body increased significantly. Effective Pb²⁺ concentrations to these two enzymes could be divided into two ranges: 10-160 mg/kg and 20-80 mg/kg, respectively. However, within the range of 20-80 mg/kg Pb²⁺, mRNA transcriptions of Gsh-Px and Gstd1 were inhibited. Moreover, GSH-Px activity and mRNA level of Gsh-Px in fat body of female larvae, and GST activity and mRNA level of Gstd1 in fat body of male larvae were more sensitive to Pb²⁺ exposure. After 20 mg/kg or more Pb²⁺ exposure, GST activity in the midgut increased dramatically, but GSH-Px activity only changed slightly and there was no significant difference between male and female larvae. mRNA transcriptional level of Gstd1 gene had a trend of up-regulation, while that of Gsh-Px was significantly down-regulated. The results suggested that, although silkworm larvae have higher enzyme activity of GST and GSH-Px in gonads than in midgut and fat body, they are sensitive to toxicity of Pb²⁺. Nevertheless, male larvae are more sensitive than female ones, and the antioxidant defense system in gonads is quite weak in resisting toxicity from Pb²⁺.3.Lead stress had a significant impact on the expression of Bombyx mori vitellogenin gene and egg specific protein gene.Vitellogenin gene and egg specific protein gene were two key genes of Bombyx mori's reproductive development. EST electric expression profiles showed that Vg gene only expressed in fat body and ovary on both spinning stage and pupa stage of silkworm, and specific protein gene Esp only expressed in testis on pupa stage.In order to investgate if lead have the enviromental estrogen effect, the paper study the specific expression profiles of Vg and Esp. The results indicated that expression of Vg gene trended to decrease than normal with disposals of Pb²⁺ concentrations within 20-160 mg/kg in the fat body of 5L female silkworm. Meanwhile, the expression of all disposed groups dropped dramatically except that of 20 mg/kg concentration group in male silkworm which turned out to be up significantly and it was as 1.15 times as that of control group in male. It can be driven that faint induction of Vg gene expression took place in male fat body in the concentration of 20 mg/kg, though it was still in a relatively low level compared with that of female fat body. When the lead exposure concentration rised to 40-160 mg/kg, expression of Vg gene in fatbody of males trended to decrease remarkably than normal. In gonads of female and male Bombyx mori there were little expression of Vg, but higer in females than males. In testis of silkworms under 20 mg/kg lead exposure, the Vg expression level was rised absolutely, which was coincident with the result of fatbody of male larves suggesting that the concentration of lead had the enviromental estrogen effect of induction expression of Vg in males.The expression of Vg gene in fat body on pupa stage of normal silkworm was tremendously higher than that of larva fat body and had a sex difference. The expression of Vg gene in all disposed groups rose drastic (P<0.01) in comparison to the control except the group exposed to the concentration of 20 mg/kg which decreased drastic (P<0.01). The expression of Vg gene in male fat body on pupa stage of normal silkworm was also lower than that of female fat body, which was tantamount to 3.59% of the female control. The mRNA expression level of Vg gene in all disposed male fat body on larva stage dropped dramatically within the Pb²⁺ concentration of 20 mg/kg-160 mg/kg compared with that of the control. Therefore, a disparity existed in mRNA expression of Vg gene in fat body between pupal and larva stages. The difference between sexes of pupa stage was less than the stage of larva; After lead exposure, Vg expression in ovary of 3d pupa depressed significantly while fluctuation at low levels intesis. Therefore, a disparity existed in influence pattern of Vg gene between pupal and larva stages.Investigation on the mRNA expression level of Esp gene in genital gland on pupal stage showed that Pb²⁺ concentrations used in this study made expression of Esp gene down in females, while Esp gene expression wasn't induced in males. Therefore, Esp gene is different to Vg gene, thus Esp gene can not be used as the indicators of environmentally endocrine disruptors.