| Amphibians are an important part of the animal resources in world,and they play an important role in maintaining the natural ecological balance.However,in recent decades,the number of amphibians has fallen sharply.The causes resulted in amphibian decline include climate change,environmental pollution and so on.It is worth noting that a class of environmental pollutants affect amphibians by altering their thyroid hormone levels.A number of previous studies have investigated the effects of changes in temperature and thyroid hormone levels on the growth and development of amphibians.However,little is known about the effects of temperature and thyroid hormone levels on energy metabolism in amphibians.Therefore,the present study explored the effects of temperature and thyroid hormone levels on leptin signaling pathway of Bufo gargarizans and the molecular mechanisms.This study consists of two parts.The first part evaluated the effects of different temperatures on the growth,development,an d leptin signaling pathway of B.gargarizans.The experiment exposed B.gargarizans larvae to different temperature treatments(15-17,22-24,and 29-31?)from Gs30 to Gs42.Firstly,the indexes of the development stage,total length,body length,and weight of B.gargarizans were detected by morphological measurement on the seventh and fourteenth days after different temperature treatments.Secondly,the morphological parameter(body length,weight and liver weight)of B.gargarizans larvae at Gs42 were measured using the same method as above.Then the histopathological analysis was performed to evaluate the morphological changes of liver in B.gargarizans at Gs42 after different temperature treatments.Finally,the effects of different temperature treatments on the expression of genes related to thyroid hormone signaling pathway and leptin signaling pathway in the liver of B.gargarizans were detected by real-time quantitative PCR(RT-qPCR).The second part of the study evaluated the effects of thyroid hormone levels on the growth,development,and leptin signaling pathway of B.gargarizans.B.gargarizans at Gs30 were administered with different thyroid hormone levels influencing compound solutions(control,sodium perchlorate,and exogenous thyroxine).Firstly,the effects of different solution treatments on the growth and development of B.gargarizans were assessed.Subsequently,the body length,body weight and liver weight of B.gargarizans larvae at Gs42 under different solution exposures were measured.Furthermore,the hematoxylin-eosin(HE)staining methodology was used to detect the alterations of liver structure in B.gargarizans at Gs42.Finally,RT-qPCR was used to determine the expression level of leptin signaling pathway-related genes in the liver of B.gargarizans at Gs42.The main findings of this study are as follows:1.Morphological data showed that under higher temperature treatment group,the average development stage of B.gargarizans larvae were significantly higher,while the duration of metamorphosis,body size and weight at the metamorphic climax were significantly reduced.However,in the lower temperature treatment,the average development stage of the tadpoles were significantly lower,while the duration of metamorphosis,body size and weight at the metamorphic climax were significantly increased.Thus,high temperature accelerated development rate of tadpoles,but reduced body size and weight,while low temperature deferred the development of tadpoles,but increased size and weight.2.Histopathological analysis indicated that morphological changes were found in the liver of B.gargarizans larvae exposed to high temperature,including significant decrease in the size of hepatocytes,unclear cell boundaries,dilation of bile canaliculus and significant increase in the number of melanomacrophage centres and red blood cells.In addition,low temperature treatment significantly reduced the hepatocytes size,and made cytoplasm transparent.Therefore,these results showed that both high temperature and low temperature exposure could cause pathological damage of liver in B.gargarizans.3.Real-time quantitative PCR(RT-qPCR)results revealed that exposure to high temperature significantly upregulated the mRNA expression levels of thyroid hormone-related genes,including type 2 deiodinase(D2)and thyroid hormone receptor alpha(TR?),and genes involved in leptin signaling pathway,including leptin,leptin receptor(LepR),members of the Janus kinase family(JAK1,JAK2,and TYK2),members of the signaling and transcription activation factor family(STAT3,STAT3.1,and STAT6),and suppressor of cytokine signaling 3(SOCS3).However,the mRNA levels of thyroid hormone-related genes,including thyroid receptor alpha and beta(TRa,TR?),and leptin signaling pathway-related genes,including leptin receptor(LepR)and members of the Janus kinase family(JAK1,and TYK2)were significantly deceased in tadpoles treated with low temperature.This indicated that high temperature treatment might enhanced the energy metabolism level of B.gargarizans,while low temperature treatment reduced its energy metabolism level.4.By measuring the morphological indicators of tadpoles under different solution treatments,we found that the exposure to sodium perchlorate(NaClO4)solution significantly reduced the average development stage of tadpoles,but significantly increased the total length,body length,weight and duration of metamorphosis.However,the average development stage of tadpoles was significantly increased,while total length,body length,weight,liver weight and duration of metamorphosis were significantly declined or shortened under the exogenous thyroid hormone(T4)treatment.The results showed that NaClO4 treatment delayed the development of tadpoles and made the tadpoles gain a large weight,while T4 treatment accelerated the development of the tadpoles and made the tadpoles have a smaller size than the normal control group.5.Hematoxylin-eosin(HE)staining results indicated that the increase in the number of melanomacrophage centres and the decrease in the size of hepatocytes were detected in liver of B.gargarizans treated with NaClO4 solution.Besides,degeneration of hepatocytes,atrophy of nucleus,disappearance of hepatocyte membranes,and increase in the number of melanomacrophage centres were found in liver of T4 exposure group.This revealed that changes in thyroid hormone levels could lead to liver damage of B.gargarizans larvae.6.RT-qPCR results showed that NaClO4 treatment significantly inhibited the transcript levels of genes related to thyroid hormone,including D2,TRa and TR?,and genes involved in leptin signaling pathway,including LepR,JAK1,JAK2,and TYK2.However,expression levels of D2,TRa,leptin,LepR,JAK2,TYK2,and STAT1,STAT3,STAT3.1,STAT6,and SOCS3 were significantly upregulated in T4 treatment group.This indicated that NaClO4 treatment could cause the disorder of thyroid hormone synthesis and reduced energy metabolism level of B.gargarizans,while T4 treatment could result in the increase of thyroid hormone levels and energy metabolism level of B.gargarizans.In conclusion,temperature and thyroid hormone levels could interfere with the growth and development of the B.gargarizans,cause tissue injury of liver,and result in disruption of energy metabolism.This study provides experimental references for further research on the effects of temperature and thyroid hormone levels on the growth,development and energy metabolism of amphibians.In addition,this study plays roles in promoting the public awareness of environmental protection and ecology,and provides valuable experimental and theoretical basis for the protection of amphibians. |
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