Waterborne Zinc (⁶⁷Zn) Exposure Influencing Embryos Glucose Metabolism In Grouper （Epinephelus Coioides）

Epinephelus coioides,commonly known as grouper,is a very representative breeding species of grouper（Epinephelus）,and it is a world-famous marine economic fish.In recent years,due to the aggravation of offshore heavy metal pollution,the natural resources of Epinephelus coioides in China are increasingly depleted,and the survival rate of artificial breeding is low.However,there is a lack of research on the effects of heavy metals on the early development of grouper.Zinc is a common heavy metal pollutant in the coastal waters of China,which can lead to high accumulation of heavy metals in grouper through the food chain,and then bring negative effects,even toxic effects.At the same time,zinc is also a necessary trace element for animals,which has the role of insulin-like,which affects the process of glucose metabolism by activating insulin signal pathway.Glycolysis and gluconeogenesis is one of the two main pathways of glucose metabolism.Akt signal pathway is activated by zinc mimicking insulin action.In recent years,studies in mammals have found that zinc can affect the expression of genes related to the above three pathways,but it has not been reported in fish.In this paper,the effects of zinc on glycolysis,gluconeogenesis and Akt signal pathway gene expression in Epinephelus coioides embryos and the accumulation of heavy metal zinc in fish embryos were studied.The main results are as follows:1 Epinephelus coioides embryos in different waterborne zinc(⁶⁷Zn)concentration groups（control,0.05,0.10,0.20,0.70,2.00,4.00 and 8.00 mg/L）.The bioaccumulation of heavy metal zinc was measured after exposure for 5,9,14,20,24 and 28 hours.The results showed that the bioaccumulation of ⁶⁷Zn in embryos was positively correlated with the concentration of waterborne ⁶⁷Zn,and the absorption rate of ⁶⁷Zn decreased with the development of embryos.The results showed that the accumulation of heavy metals in embryos was related to the concentration of waterborne zinc and waterborne exposure zinc time.2 After exposure to different waterborne zinc concentrations（control,0.05,0.10,0.20,0.70,2.00,4.00 and 8.00 mg/L）,the m RNA expression levels of glycolysis,gluconeogenesis and Akt signaling pathway in Epinephelus coioides embryos were measured.⁶⁷Zn exposure up-regulated the m RNA expression of PFK m RNA was in morula stage,blastocyst stage,last gastrula stage,neural stage and heart beat stage,while it were down-regulated in hatching complete stage.⁶⁷Zn exposure decreased the expression of PK gene in blastocyst and last gastrula stage,but increased it in neural stage.Waterborne ⁶⁷Zn concentration groups（2.00,4.00 and 8.00 mg/L）significantly regulated the expression of PC gene（P<0.05）.From morula stage to blastocyst stage,the m RNA expression of PCK in high concentration group（4.00 and 8.00 mg/L）was significantly de-regulated,while that in incubation stage was up-regulated（P<0.05）.Waterborne zinc exposure in water can obviously regulate the expression of FBP gene（P<0.05）.The m RNA expression level of G6PC gene was significantly de-regulated by zinc exposure from heart beat to hatching complete stage（P<0.05）.The m RNA expression of IR gene was significantly up-regulated in waterborne ⁶⁷Zn exposure groups.In the blastocyst stage and late gastrula stage,0.05 mg/L Zn concentration group significantly up-regulated the m RNA expression of AKT2 gene（P<0.05）.In morula stage,the ⁶⁷Zn concentration of 4.00 mg/L could significantly up-regulate the m RNA expression of AKT2 gene（P<0.05）.In morula stage and blastocyst stage,the expression of GSK-3beta in waterborne ⁶⁷Zn exposure group was significantly de-regulated（P<0.05）.However,the low concentration group also significantly up-regulated the expression of GSK-3beta gene in the later stage of development（P<0.05）.In this study,waterborne ⁶⁷Zn significantly down-regulated the expression of GLUT4 gene in morula stage and upregulated the expression of GLUT4 gene in late gastrula stage（P<0.05）.This study proved for the first time that waterborne zinc exposure can affect the expression of key enzymes and related genes of glucose metabolism in fish embryos through time-and concentration-dependent effects,which are fish embryo metal toxicology and glucose metabolism provides a new perspective for research.