| Amphibian is a key species that undergoes the transition from aquatic to terrestrial and occupies vital position in evolutionary history.In their life history,they change from a completely aquatic larval stage（embryonic stage and early stage of tadpole）to a completely terrestrial frog（adult stage）through a process of complete metamorphosis（metamorphic stage）.Owing to their unique biphasic life history,it has been proved to be a sensitive indicator to assess toxic consequences of aquatic contaminants in environmental monitoring.However,in the recent decades,it has been reported that the amphibians’ populations have sharply declined,and are even on the verge of extinction.The main reasons for this phenomenon can be attributed to climate change,alien species invasion and habitat pollution,etc.Heavy metals become an extremely harmful environmental pollutant to organisms because since they are difficult to degrade naturally and cannot be metabolized in organisms.With the acceleration of urbanization and industrialization,its concentration in the environment continues to increase,and in many heavily polluted areas,the concentration of these ions has exceeded the threshold,which seriously threatens the normal physiological activities of all aquatic organisms.Among the numerous heavy metals,cadmium（Cd）and lead（Pb）are the two main harmful ions that threaten the health of aquatic organisms.The increased levels of Cd and Pb were detected in rivers,estuaries and nearshore waters over the past decades and Cd and Pb always emerge in co-existence form in the environment.However,the current research on the effects of Cd and Pb on amphibians mainly focused on single exposure to Cd or Pb,the research from mixed ions becomes particularly important.Accordingly,Bufo gargarizans（Bufo gargarizans,an amphibian that is widely distributed in China and has a huge number of eggs）were used as the research object in this study.The embryonic stage（Gs16-Gs22 stage）and the pre-metamorphosis（Gs38 stage）.metamorphosis climax（Gs42 stage）and metamorphosis completion（Gs46 stage）were selected to explore the effects of Cd（1μM）,Pb（1μM）and Cd-Pb mixed（1μM Cd+1μM Pb）exposure on the development and intestinal microbiota of Bufo gargarizans.The results were show as follows:1.In the early stage of embryonic development of Bufo gargarizans（Gs16-Gs22）,the effects of Cd/Pb（alone or combined）on Bufo gargarizans were detected through morphological detection,qPCR technology,etc.The results showed that compared to a single Cd or Pb exposure,Cd-Pb mixture caused a more significant gene responses（one-way ANOVA,P<0.05）,such as SOD,GPx,SOCS3,IL-27,etc.In addition,in the Gs19 stage,4.8%,0,65%and 10.3%embryo abnormality rates were observed in the Cd,Pb and Cd-Pb mixed treatment groups,respectively.2.In the Gs38,the host and microorganisms were analyzed separately:1)Paraffin section with H＆E and Masson stained,ultraviolet spectrophotometry,qRT-PCR method and 16S rRNA V3-V4 high-throughput sequencing were used to analyze the intestinal histology,digestive enzymes（lipase,α-amylase,trypsin and pepsin）activity,gene expressions related oxidative stress,apoptosis and immune,and the intestinal microbiota of tadpoles after exposure to Cd/Pb,respectively.The results showed that the growth and development of Bufo gargarizans were delayed after Cd-Pb mixed exposure,and the morphological parameters（body weight,body length,bowel length and bowel weight）were significantly reduced（one-way ANOVA,P<0.05）.Based on the results of H＆E and Masson’s trichrome,the mixture of Cd and Pb exposure caused more severe histological damages of intestines,exhibited as degeneration of enterocyte cells,disappeared palisade arrangements of the enterocyte cells and nuclear pyknosis.The results from digestive enzyme activity showed that the activity of four digestive enzymes in the Cd-Pb mixed treatment group was significantly reduced（one-way ANOVA,P<0.05）.qPCR results showed that exposure to Cd-Pb mixture caused a significant decrease of oxidative stress gene expression,and a significant increase of immune-related genes expression（one-way ANOVA,P<0.05）.2)Exposure to Cd/Pb（both along and mixture）decreased the relative stability and caused significant changes in richness and biodiversity,structure and communities of gut microbiota in B.gargarizans（Bray-Curtis,P<0.05）.Specifically,the phylum Proteobacteria,considered as the front-line responder for an unstable status of microbial structure,dominate the most top 50 OTUs,and meanwhile,increased prevalence of Proteobacteria was observed in the intestine of tadpoles under the Cd/Pb exposure（one-way ANOVA,P<0.05）.Correlation analysis showed that "host factor"was significantly positively correlated with some phyla（such as Fusobacteria）,and significantly negatively correlated with other phyla（Proteobacteria,Firmicutes）（one-way ANOVA,P<0.05）.The prediction results of PICRUSt and Tax4Fun showed that mixed exposure significantly increased the abundance of the KEGG pathways related to metabolism and immune system function in the intestinal microbiota（one-way ANOVA,P<0.05）.3.1)In the Gs42-Gs46 of B.gargarizans,the endochondral ossification of skeleton development,thyroid development and expression of genes related thyroid hormones and ossification,as well as skeleton formation,expression of genes and behavioristics were measured through Masson’s trichrome staining method,alixinlan-alizarin red bone double staining technique and Illumina transcriptome sequencing technology,respectively.The results showed that:long-term Cd-Pb mixed exposure accelerated the metamorphosis of larvae,and significantly shorten the duration（one-way ANOVA,P<0.05）.Thyroid histology results showed that Cd-Pb mixed exposure increased the activity of thyroid gland,exhibited as increased numbers of phagophore per thyroid gland and significant increased thyroid gland area（one-way ANOVA,P<0.05）.However,accelerated metamorphosis caused defects in bone growth and development of B.gargarizans.moreover,the rotio of a variety of bones was significantly reduced,and several were even less than 50%（one-way ANOVA,P<0.05）.The results of whole-body RNAseq showed that the significant up-regulated genes in the exposed group were mainly enriched as the "complement and coagulation cascades",which are involved in the occurrence of a chronic cartilage disease.2)In the Gs46,the results showed that although the metamorphosis rate of the body were accelerated after Cd-Pb mixed exposure,the individual was significantly reduced（one-way ANOVA,P<0.05）.The results of bone staining showed that the ossification rate was still significantly lower than the control group（one-way ANOVA,P<0.05）,and the highest ossification rate was only 85.93%.Finally,this study found that Cd-Pb mixed exposure significantly down-regulated some thyroid hormone-related genes（such as TRα,Dio2,Dio3,etc.）and bone development-related genes（such as MMP9,Runx3）at the mRNA level（one-way ANOVA,P<0.05）.In conclusion,this research evaluated the toxic effects of waterborne Cd and Pb on the development of B.gargarizans by measuring different growth parameters.The results revealed that the exposure at early stage of B.gargarizans might have lasting consequences on the later life history of individuals,including dysbiosis of gut microbiota,inhibition of skeleton development and impaired jumping ability,etc.,which are essential to the later life stage of the amphibian.The results will provide experimental reference for further evaluation of toxicological risks of heavy metal ions on the growth and development of amphibians,as well as an important theoretical basis for assessing the risk of heavy metal pollution in the environment. |
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