Effects Of Metamifop On Immunity,Antioxidant,Ammonia Metabolism And Gonadal Axis Of Monopterus Albus

Rice field eel（Monopterus albus）,as an important commercial freshwater species,is popular because of its delicious taste.As a typical kind of air-breathing bony fish,M.albus live in muddy ponds,swamps,canals and rice paddies,and has the habit of burying itself in mud when water is scarce.Therefore,M.albus have a different set of ammonia mechanisms than whole-water fish to cope with the high ammonia concentration in the paddy field during agriculture fertilization and the inability of ammonia to be excreted from the environment during water scarcity.Moreover,M.albus has the biological characteristics of sexual reversal under natural conditions,and it has almost been used as a marker for the study of fish sexual reversal.However,the presence of pollutants may affect the energy metabolism of M.albus,leading to the production of more endogenous ammonia,which subsequently affects the ammonia metabolism of M.albus.In addition,pollutants may also act as endocrine disruptors to interfere with the normal sexual reversal of M.albus.Due to the rise of the"rice-eel farming"model,the use of pesticides in rice fields has made the M.albus easily exposed to various agricultural chemicals.Metamifop（MET）,as a convenient herbicide,is widely used in weeding paddy fields,but there are relatively few studies on the effects of MET on various aspects of biology.Therefore,an acute stress experiment was conducted in this study to investigate whether MET exposure has effects on immunity,antioxidant,ammonia metabolism and gonadal axis of M.albus.Results showed that the semi-lethal concentration of MET exposed for 96 h was0.785 mg L^-1 for M.albus,which was highly toxic.According to the semi-lethal concentration,four MET concentration groups of 0.2-,0.4-,0.6-and 0.8 mg L^-1 were set,and the main results were as follows:（1）Effects of metamifop on immune and antioxidant capacity of Monopterus albus.Low concentrations of metamifop(0.2-and 0.4 mg L^-1)caused a significant increase in leukocyte count,but high concentrations of MET caused a significant decrease in leukocyte count.ROS content was increased with the increase of MET concentration,and ROS content was significantly increased at all concentrations（p<0.01）.The excess ROS resulted in lipid peroxidation,and the content of malondialdehyde（MDA）increased significantly at all MET concentrations.However,ROS did not significantly affect proteins from the perspective of protein carbonylation degree.The activity of antioxidant enzyme SOD decreased significantly after high concentration(0.8 mg L^-1)MET stress（p<0.05）.The activity of GPX decreased with the increase of MET concentration,and was significantly decreased at all MET concentrations（p<0.01）.CAT activity increased significantly at low MET concentration(0.2 mg L^-1),but decreased as MET concentration increased,and was significantly lower than that of the control group when MET concentration was higher than 0.6 mg L^-1（p<0.01）.Immune-related enzymes such as acid phosphatase（ACP）did not change significantly under MET exposure,while alkaline phosphatase（AKP）activity decreased significantly under MET exposure at all concentrations（p<0.01）.Subsequently,the expression levels of inflammation-related genes TNF-α,IL-1βand IL-8 were significantly decreased compared with the control group（p<0.01）.c P53,Bcl-2b,Bax,caspase-3 and caspase-9 were also significantly decreased after all concentrations of MET exposure.This suggests that MET can lead to the production of reactive oxygen species,but the antioxidant capacity is inhibited,lipids are damaged by oxidation after MET exposure,and inflammatory responses and apoptotic pathways are inhibited.MET exposure（even at low levels）inhibits the immune and antioxidant systems of M.albus and prevents them from processing damaged cells through the apoptotic pathway.MET has a strong effect on eel,and it cannot cope with MET stress through autoimmune and antioxidant pathways.（2）The effects of metamifop on energy and ammonia metabolism of Monopterus albus.Under MET exposure,the ammonia discharge rate was decreased at all time periods.When MET concentration was higher than 0.4 mg L^-1,the ammonia discharge rate at all time periods was significantly decreased compared with the control group（p<0.01）.Ammonia content in hemolymph increased significantly after MET exposure（p<0.05）,but urea content decreased after MET exposure,and urea content decreased significantly after MET concentration was higher than 0.6 mg L^-1（p<0.01）.When MET concentration increased,the protein content in liver and brain decreased and increased,respectively.The protein content in liver and brain changed significantly when MET reached 0.6 mg L^-1.Amino acid content in liver increased after MET exposure,and amino acid content in brain decreased significantly at all MET concentrations（p<0.01）.After MET exposure,the activity of acid protease（CP）in liver did not change significantly,the activity of neutral protease（NP）was significantly increased（p<0.01）,and the activity of alkaline protease（KP）was significantly decreased（p<0.01）.In the brain,the activity of CP,NP and KP decreased,with the neutral protease significantly decreased at 0.2 mg L^-1 MET concentration（p<0.01）,while the CP and KP significantly decreased at 0.6 mg L^-1 and0.4 mg L^-1 and higher concentration of MET,respectively.ATP content in liver was significantly increased after MET exposure,while ATP content in brain was decreased after MET exposure,especially when the concentration reached 0.6 mg L^-1（p<0.01）.Aminotransferase,such as glutamic-pyruvic transaminase（GPT）,was not significantly changed in liver,but significantly decreased in brain（p<0.01）.The activity of glutamine synthetase（GS）in both liver and brain was increased with the increase of MET concentration,while the increased level in liver was significant（p<0.01）,and no significant changes were found in glutamine synthetase in brain after exposure to MET concentrations（p>0.05）.GLS activity in liver was significantly decreased after MET exposure at all concentrations（p<0.05）,while GLS activity in brain was significantly increased after MET exposure at 0.4 mg L^-1 or higher concentrations（p<0.05）.The results showed that MET stress enhanced the energy metabolism in the liver of M,albus,which led to the degradation of more proteins and amino acids,and the organisms did not take corresponding measures to reduce the production of ammonia,which resulted in the production of more ammonia.However,MET inhibits the ammonia excretion system of M,albus,and the ammonia produced by the body cannot be excreted.But the liver can reduce the effects of ammonia by converting some of it to glutamine for preservation.The brain responds by synthesizing proteins in response to MET stress,but this mechanism may lead to a decrease in brain energy synthesis,which prevents brain tissue from making or reducing energy-intensive glutamine synthesis in response to elevated endogenous ammonia.These results indicate that MET can increase the production of endogenous ammonia and inhibit the excretion of ammonia,so that the M.albus is in a higher level of ammonia stress.（3）Effects of metamifop on hypothalamic-pituitary-gonadal axis of Monopterus albus.No significant effects on measured plasma E2 and T levels were observed at all concentrations of MET（p>0.05）,and the ratio of estrogen to androgen（E2/T）remained unchanged after MET exposure.Plasma VTG content of female eel treated with 0.8 mg L^-1 was significantly increased（p<0.05）.In liver,VTG levels in 0.6-and 0.8 mg L^-1MET treatment groups were significantly increased compared with the control group（p<0.05）.In the brain,the expressions of CYP19A1b,CYP17 and LHβwere significantly increased in the MET exposed group compared with the control group（p<0.01）,but FSHβwas down-regulated in all exposed groups.The expression levels of CYP19A1a and CYP17 in ovary were significantly decreased in all MET exposed groups（p<0.05）,the m RNA levels of FSHR and LHCGR were significantly decreased at all MET concentrations,and the expressions of HSD11b2 and 3β-HSD were not significantly changed at low MET concentration(0.2 mg L^-1).The expression levels of HSD11b2 and3β-HSD decreased with increased MET concentration.The expression levels of Dmrt1and Foxl3 were down-regulated in all exposed groups,while the expression levels of JNK1 were slightly increased.In liver,esr1,esr2a,and esr2b were down-regulated in all MET exposed groups.The results showed that MET had perturbative estrogen effect on female eel.MET directly affects the expression of genes on the HPG axis,but does not affect sex hormone levels.MET exposure increased the expression of CYP19A1b and CYP17,which regulate sex hormone production in the brain,but all genes associated with ovarian sex hormone production were suppressed.In addition,sex-related genes did not show a trend of sex reversal after MET.