Mechanism Of Toxic Response Of Yunlong Grouper(Epinephelus Moara ♀× Epinephelus Lanceolatus ♂) To Manganese Stress

Manganese is an essential trace element in living organisms and is commonly found in the natural environment.With the accelerated industrialization and urbanization,improper discharge of waste can cause water environment pollution,resulting in the accumulation of large amounts of manganese in aquatic ecosystems,which can be harmful to the health of aquatic organisms,especially during the early growth of fish.Salt groundwater is an important water source for industrialized culture,and for a period of time,the manganese content in the Salt groundwater in the northern coast of China has been generally high,which may have a negative impact on the growth of fish in the industrialized culture.Yunlong grouper（Epinephelus moara ♀× Epinephelus lanceolatus ♂）has the advantages of being both the sire and dam,with high-quality meat,nutritional value,and fast growth rate,it has become an important aquaculture variety in our country.There are few reports on the toxic effects of Mn²⁺ exposure on fish at present,and the toxic mechanism is still unclear.Therefore,in order to better understand the toxic effects of Mn²⁺ exposure on mariculture fish,this study took a suitable species for industrialized culture,Yunlong grouper,as the research object and used MnCl2·4H2O as the exposure source to simulate the Mn²⁺ exposure environment in the factory culture process to evaluate the effects on the growth and digestion of Yunlong grouper.And systematically analyze the response mechanism of grouper in response to Mn²⁺ stress in terms of oxidative stress,immune response,liver damage,apoptosis and neuroendocrine aspects.The main research contents and results are as follows:1.Bioaccumulation of manganese and its effects on growth and digestion of Yunlong GrouperThis study focuses on the accumulation characteristics of manganese in different tissues（gill,liver,intestinal tract,muscle）of juvenile Yunlong grouper exposed to Mn²⁺（0-4 mg/L;30 days）,and its effects on the growth status and digestive enzyme activities.The results showed that the bioaccumulation of Mn in fish was related to dose and time,and has differences in tissue.The accumulation of Mn in each tissue increases with the increase of Mn²⁺ exposure concentration,the accumulation sequence of Mn in different tissues is as follows:liver>gill>intestine>muscle.The growth status of juvenile grouper also varied with exposure concentration.Low exposure concentration（0.5 and 1 mg/L）had no significant difference in the growth parameters of juvenile grouper（P≥0.05）.When exposure concentration reached 4 mg/L,the growth of juvenile grouper was inhibited after exposure for 30 days.The body length（BL）,body weight（BW）,weight gain rate（WGR）and specific growth rate（SGR）were significantly decreased,while the feeding rate（FR）was significantly decreased,and the feed coefficient（FCR）was significantly increased（P<0.05）.In addition,the activities of pepsin,trypsin,αamylase and lipase decreased with the extension of exposure time,which also reflects the inhibitory effect of Mn²⁺ on digestive enzymes,which is not conducive to the digestion and utilization of nutrients in fish.2.Effects of manganese exposure on oxidative stress and immune response of Yunlong GrouperIn this study,the effects of Mn²⁺ on oxidative stress and immune ability of Yunlong grouper were evaluated by detecting enzyme activity,protein content and gene expression related to antioxidant capacity and immune function in liver.The results showed as follows:Compared with the control group,no significant changes in antioxidant-related parameters were observed in the liver of Yunlong grouper after exposure to 0.5 mg/L Mn²⁺ for 30 days（P≥0.05）;SOD and CAT activities were significantly increased after 10 and 20 days of exposure in the 2 and 4 mg/L concentration groups,but were substantially decreased in the 4 mg/L exposure group after 30 days（P<0.05）.Similarly,the activity of GSH and GPx in the liver increased significantly after 10 days of exposure in the 4 mg/L concentration group.However,it decreased significantly after 20 and 30 days（P<0.05）.The corresponding antioxidant-related genes（sod,cat,gr and gpx）also showed a similar expression trend.At the same time,MDA levels were significantly increased over time when exposed to 2 and 4 mg/L Mn²⁺（P<0.05）.In addition,Mn²⁺exposure increased the levels of HSP 70 and HSP 90,C3 and C4,on the contrary,significantly decreased the levels of LZM and IgM（P<0.05）.Mn²⁺ also significantly upregulated the expression of immune-related genes（tlr3,tnf-α,il-1β and il-6）（P<0.05）.The results indicate that Mn²⁺ can induce oxidative stress and immunotoxicity through bioaccumulation in fish.3.Effects of long-term manganese exposure on liver injury and cell apoptosis of grouperIn this study,the effects of Mn²⁺stress on liver damage and cell apoptosis of juvenile Yunlong grouper were evaluated.The results showed that after exposure to high concentrations of Mn²⁺（2 and 4 mg/L）for 30 days,the liver tissues of the Yunlong grouper showed some liver tissue pathological changes,such as hepatic sinuses congestion,dilatation,cellular vacuolation and necrosis.Compared with the control group,the activities of alanine aminotransferase（ALT）and aspartate aminotransferase（AST）and the level of 8-hydroxydeoxyguanosine（8-OHdG）in liver of Yunlong grouper after 30 days of Mn²⁺ exposure were significantly increased in 1,2,and 4 mg/L groups（P<0.05）.In addition,long-term Mn²⁺ exposure altered the expression patterns of key apoptosis-related genes,such as significantly up-regulated the expressions of p53,bax,apaf-1,caspase-9,and caspase-3 and significantly down-regulated the expression of bcl2（P<0.05）,indicating that Mn²⁺ exposure induced apoptosis through mitochondrial pathway.4.Regulation mechanism of inhibiting growth of grouper under manganese stressIn this study,the effects of Mn²⁺ stress on GH/IGF axis and HPT axis related hormone and gene expression and monoaminergic neurotransmitter levels of juvenile Yunlong grouper after 30 days of exposure were evaluated.The results showed as follows:after 30 days of exposure,the levels of growth related hormone（GH and IGF-1）in 2 and 4 mg/L Mn²⁺ groups were significantly decreased compared with the control group,thyroid hormone（T4）levels were significantly decreased in 1,2 and 4 mg/L Mn²⁺ groups,while T3 and thyroid stimulating hormone（TSH）levels were significantly increased at the same time and concentration（P<0.05）.At the same time,1,2 and 4 mg/L Mn²⁺inhibited the expressions of ghra and ghrb,while the expressions of igflra and igfbpla were not significantly changed in all concentration groups（P≥0.05）.The expressions of HPT axis related genes diol and dio2 were significantly increased in 2 and 4 mg/L Mn²⁺ groups.Finally,the levels of dopamine（DA）and serotonin（5-HT）were also significantly decreased after exposure to high concentrations of Mn²⁺（2 and 4 mg/L）for 30 days Their metabolite DOPAC did not change significantly in each concentration group,and 5-HIAA levels decreased significantly at 2 and 4 mg/L Mn²⁺ groups.These results indicate that high Mn²⁺ exposure can interfere with GH/IGF axis and HPT axis function,affect the synthesis and metabolism of DA and 5-HT,and thus interfere with endocrine disorder of the body.