| Cadmium(Cd)is a highly toxic heavy metal contaminant,and it has the properties of teratogenic,mutagenic,and carcinogenic.Several anthropogenic activities(mining activities,fertilizer use and industrial discharge et.)are responsible for the pronounced increase in the concentration of Cd in the aquatic environment.The studies of our laboratory have shown that Bufo gargarizans larvae is ideal study spicies to evaluate negative consequences of toxic effects of aquatic contaminants due to its high-sensitivity to water pollution.In this study,we have been evaluated the chronic toxic effects of Cd exposure on B.gargarizans larvae,the main findings were as follows:1.In order to evaluate the effects of chronic Cd exposure to histological changes,fatty acid synthesis,fatty acid β-oxidation and bile secretion in liver of B.gargarizans tadpoles.B.gargarizans tadpoles were treated in different Cd concentrations of 0,5,10,50,100 and 200 μg L-1 from Gs 26 to Gs 42.We examined the liver histological alterations on B.gargarizans tadpoles at Gs 42.We chose the liver of B.gargarizans of control,5μg Cd L-1(as low concentration exposure)and 200 μg Cd L-1(as high concentration exposure groups)for RNA-seq analysis.We selected 21 differential expression genes relate to fatty acid synthesis,fatty acid β-oxidation and bile secretion in livers based on the annotation results of the RNA-Seq data,and assessed the expressions of 12 genes by performing RT-qPCR to evaluate the effects of Cd exposure to these metabolic processes.Our results showed that histological changes characterized by disappearance of cell boundaries,nuclear pyknosis and increase in the number of melanomacrophage centres were observed in liver at 200 μg Cd L-1.RT-qPCR results showed that mRNA expression of ELOVL1 HSD17B12,MECR and TER(genes relate to fatty acid synthesis)was significantly up-regulated in all Cd treatment groups.Also,in the genes related to fatty acid p-oxidation,mRNA expression of ACAA2 and ECHS1 genes were significantly up-regulation in larvae treated with 5,10,50,100 and 200 μg Cd L-1,but mRNA expression ACOT1,ACAA1,HADH and PPT were significantly downregulated at 100 and 200 μg Cd L-1,and HADHA only at 200 μg Cd L-1.The results of in situ hybridization shown that the location of ABCB11 mRNA expression was in the hepatocyte nucleus and plasma membrane,and positive signals was weaker only in hepatocyte nucleus of 200 μg Cd L-1 treatment group than that of control group.The location of HSD17B12 mRN A expression was in the plasma membrane,and positive signal was weaker at 200 μg Cd L-1 treatment group than that of control group.2.In order to evaluate the effects of chronic Cd exposure to gut hispathological alterations and microbiota alterations of B.gargarizans tadpoles.B.gargarizans tadpoles were treated in different Cd concentrations of 0,5,100 and 200 μg L-1 from Gs 26 to Gs 38.We examined gut histological and microbiota alterations on B.gargarizans tadpoles at Gs 38 by using histological analysis and next generation sequencing(NGS)base high throughput sequencing.Our results showed that histological changes characterized by degeneration of epithelial cells and damage in normal palisade arrangements of epithelial cells,unclear cell boundaries and nuclear pyknosis were observed in gut at 100 and 200μg Cd L-1.Based on the analysis of MiSeq sequencing date of 16S rRNA genes.Chao and Ace index gradually declined with the increase of Cd concentration,which indicated that species richness of gut microbiota was decreased under Cd exposure conditions.Moreover,the Shannon and Simpson index showed that the diversity of gut microbiota was significantly decreased in 100 μg Cd L-1 exposure groups.The results of principal component analysis(PCA),hierarchical clustering tree illuminated and non-metric nultidimensional scaling(NMDS)shown gut microbial communities differed distinctly in control and three Cd exposure groups.The result of taxonomic analysis shown that the major bacterial phyla were Proteobacteria,Bacteroidetes,and Firmicutes in B.gargarizans tadpoles at Gs 38,Proteobacteria had significant increase in 5 and 100 μg Cd L-1 exposure groups compared with control.Also Klebsiella and Aeromonas are two dominant genera in gut microbiota of B.gargarizans tadpoles,the abundance of Klebsiella in 5 μg Cd L-1 exposure group and Aeromonas in 100 μg Cd L-1 exposure group was significantly increased compared to control group.The result of PICRUSt approach analysis shown that,compare with the controls,the relative abundance of carbohydrate metabolism,amino acid metabolism,energy metabolism,metabolism of cofactors and vitamins,nucleotide metabolism,lipid metabolism,xenobiotics biodegradation and metabolism were significantly increased in the 5 μg Cd L-1 exposure groups,and significantly reduced in 200 μg Cd L-1 exposure groups. |
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