| Amphibians during the metamorphosis undergo a series of physiological,morphological and behavioral changes,including the resorption of tail,the development of limbs,and remodeling of organs and tissues such as intestine.The intestine is an important organ for lipid absorption,immunity response and health maintenance in the body.Trillions of microbes are inhabited in the intestine,and these microbes play an integral role in maintaining digestive and immune functions.Amphibian metamorphosis is tightly controlled by thyroid hormones(THs).Interference with the normal activity of THs will further affect the growth and development of amphibians.Nitrite,as a part of nitrogen cycle,is one of the most common toxic compounds in aquatic ecosystems.It is therefore necessary to study nitrite toxic effects on amphibians for the monitoring of environmental chemical pollution.This study selected Bufo gargarizans as model organism and set concentrations of 0,10,100,500,and 1000 ?g/L NO2-N to explore the effects of nitrite on tadpole intestinal microbes,thyroid hormones,skeletal development,and locomotion ability during metamorphosis.The study consists of three parts.The first part explored the effects of different concentrations of nitrite on the intestinal tissue parameters,digestive enzyme activities,immune response and intestinal microbial composition of Bufo gargarizans tadpoles at Gs38.In the second part,the effects of different concentrations of nitrite on metamorphosis,thyroid hormone secretion and skeletal development of tadpoles at Gs42 were investigated.In the third part,the responses of nitrite exposure on skeletal development and locomotion ability of tadpoles at Gs46 were investigated.The main results and conclusions are as follows:1.Morphological measurements showed that the body weight of tadpoles at Gs 46 was significantly reduced in 100 ?g/L NO2-N treatment group.The body length and forelimb length of tadpoles at Gs 46 were significantly decreased in 500 ?g/L NO2-N treatment group.The body length,the body weight,forelimb length,and the hind limb length of tadpoles at Gs 46 were significantly reduced in 1000 ?g/L NO2-N treatment group.2.Metamorphosis data showed that the metamorphism rate of tadpoles exposed to 10?g/L NO2-N was significantly faster than that of the control group,and metamorphosis rate of tadpoles exposed to 500 and 1000 ?g/L NO2-N was significantly lower than that of the control group.3.Histopathological studies indicated that nitrite exposure induced histological damage of the intestine and thyroid gland.The results of intestinal histopathology showed that the intestines of control tadpoles showed normal appearance,with close and homogeneous enterocytes,clear cytoplasm,well-delineated cytoplasmic borders and uniform microvilli.However,exposure to 100 ?g/L NO2-N showed indistinct cell boundaries of mucosal epithelial cells.The severely histopathological abnormalities of intestines were found in 500 and 1000 ?g/L NO2-N treatment groups,such as scattered cell nuclei in the lumen of intestine,cytoplasmic damage,unclear cell boundaries,and loss of microvilli.The results of thyroid tissue histopathology showed that the amount of colloid in thyroid follicles remarkably decreased in 10?g/L NO2-N treatment group,and there was almost no colloid in 100,500 and 1000 ?g/LNO2-N treatment groups.In addition,follicular epithelial cell hyperplasia and malformation could be observed in 100,500 and 1000?g/LNO2-N treatment groups.4.The results of intestinal digestive enzymes showed that the activities of trypsase,lipase and pepsin in intestine of tadpoles at Gs 38 were significantly decreased after NO2-N treatment,indicating nitrite exposure might affect digestive process and lead to disorder of normal function of intestine.5.Intestinal microbial ?-diversity results showed that the intestinal microbial richness significantly increased in 1000?g/L NO2-N treatment group,while the microbial diversity significantly decreased in 100 and 500 ?g/L NO2-N treatment groups.The?-diversity results showed that compared with the 10 and 100 ?g/L NO2-N treatment groups,the microbial communities of the 500 and 1000 ?g/L NO2-N treatment groups were farther away from the control group,indicating that the 500 and 1000 ?g/L NO2-N treatment groups had more significant influence on the microbial community structure.Functional prediction of intestinal microbial community showed that the NO2-N exposure changed the abundance of most metabolic pathways,interfered with metabolic function of tadpoles and increased risks of disease of tadpoles.6.The double staining results showed that skeletal development lengths of tibiale,fibulare,pes,antibrachium and phalanges of tadpoles at Gs 42 and Gs 46 in 500 and 1000 ?g/L NO2-N treatment groups were lower than that in control group.This suggests that exposure to high concentration nitrite could inhibit skeletal development and reduce skeletal lengths of tadpoles.7.Jump distance showed that maximum jump distance and mean jump distance of tadpoles at Gs 46 were significantly decreased in 500 and 1000?g/L NO2-N treatment groups,suggesting that exposure to high concentration nitrite could inhibit the locomotion ability of tadpoles.In addition,the correlation analysis between jump distance and morphology showed that jump distance was positively correlated with body weight,body length,hind limb length and forelimb length.8.The results of RT-qPCR showed that in tadpoles at Gs38,the transcription profiles of oxidative stress genes(SOD,GPx and HSP)were significantly decreased in 1000?g/L NO2-N treatment group,and the transcription profiles of inflammation and immunity genes(SOCS3,IL-27,IL-1 and IL-17D)were significantly changed in NO2-N treatment groups.In tadpoles at Gs42 and Gs 46,the transcription levels of Dio2,TRa and TR? were downregulated after NO2-N exposure,while the transcription levels of Dio3,VEGFA,VEGFB,VEGFR1,VEGFR2 and VEGFR3 were remarkably upregulated.These results indicated that nitrite resulted in immune dysfunction,induced disruption of thyroid hormone homeostasis and inhibited the skeletal development of tadpoles.In summary,nitrite exposure can inhibit the normal growth of Bufo gargarizans tadpoles,such as body length,body weight,forelimb length and hind limb length.Nitrite exposure resulted in intestinal tissue damage,immune dysfunction and intestinal microbial changes of tadpoles.Additionally,nitrite caused thyroid tissue damage,affected the metamorphosis of tadpoles,and inhibited the development of bone,further contributing to the poor jumping performance of Bufo gargarizans.The results of this study will provide important new theoretical basis for the effects of nitrite on the immune response and microflora of amphibian tadpole intestine,provide experimental references for studying the effects of nitrite exposure on thyroid damage and skeletal development of amphibian,and provide detailed data for the decline of amphibian population and the protection of water environment caused by excessive nitrogen compounds. |
PDF Full Text Request