Physiological Response And Mechanism Of Juvenile Turbot(Scophthalmus Maximus) To Chronic Nitrate Stress

Turbot（Scophthalmus maximus）is an important marine economic fish species in China.In recent years,with the shortage of groundwater resources and the promotion of green ecological culture model,the culture mode of many important economic fish,such as turbot（Scophthalmusmaximus）,is gradually dominated by industrial recirculating aquaculture mode.The recirculating aquaculture model has the advantages of water saving,energy saving,high efficiency and low environmental impact,and always equipped with core processes such as biological filter,which can control the key water quality parameters such as ammonia and nitrite within the safe range of culture.However,with the continuous transformation of ammonia and nitrite,coupled with the lower water exchange in the system,it is bound to lead to the accumulation of higher concentration of nitrate in aquaculture water.In addition,with the intensification of human activities and the rapid development of industry and agriculture,the concentration of nitrate in natural environmental water is also increasing year by year,and the reports on the toxic effects of nitrate on aquatic organisms are also increasing.Therefore,under the recirculating aquaculture mode,does the higher concentration of nitrate in the rearing water affect the culture of turbot?What is the safe nitrate concentration for turbot culture in recirculating aquaculture culture systems?What is the toxic mechanism and influence pathway of nitrate on turbot?These are the urgent problems to be solved in the study of recirculating aquaculture and nitrate toxicology of turbot.Therefore,in order to explore the toxic effects of nitrate on economic fish,this paper took turbot as the research object and different concentrations of nitrate as stress factors to study the effects of nitrate on the growth,health and welfare of juvenile turbot in the recirculating aquaculture system,and then the toxicity mechanism was analyzed by combining a variety of physiological parameters,biochemical and molecular indexes.The main research contents and results are as follows:（1）Effects of nitrate on growth performance,health status,and endocrine function of juvenile turbot（Scophthalmus maximus）This study was carried out in 12 experimental recirculating aquaculture systems（RASs）,which were divided into four groups:control group（CK）,low nitrate group（LN）,medium nitrate group（MN）and high nitrate group（HN）.The experiment was carried out for 60 days.The growth data showed that the cumulative survival rate（CS）,absolute growth rate（AGR）and specific growth rate（SGR）of the HN were significantly lower than those of the CK（P<0.05）,while the feed coefficient（FCR）of the HN was significantly higher than that of the CK（P<0.05）.The histological results showed that high concentration of nitrate exposure could induce histological damage in gills and liver of juvenile turbot,but there were no significant changes in kidney and spleen.ELISA and q RT-PCR analysis showed that the levels of triiodothyronine（T₃）in LN,MN and HN were significantly lower than those in CK（P<0.05）,the levels of tetraiodothyronine（T₄）in HN were significantly lower than those in CK（P<0.05）,and the levels of growth hormone（GH）,growth hormone receptor b（GHRb）and insulin-like growth factor-1（IGF-1）in HN were significantly lower than those in CK（P<0.05）.The levels of corticotropin releasing hormone（CRH）,adreno-cortico-tropic-hormone（ACTH）and cortisol（cortisol）were significantly increased in HN group（P<0.05）.Other results showed that the levels of glutamic pyruvic transaminase（GPT）,glutamic oxaloacetic transaminase（GOT）and heat shock protein HSP70 in the HN group were significantly higher than those in the CK（P<0.05）.The above results suggest that chronic nitrate exposure has negative effects on the growth,health and endocrine function of juvenile turbot.Nitrate may exert an endocrine-disrupting effect by inducing disorders of the GH/IGF-1,HPT,and HPI axes,thereby causing growth inhibition in juvenile turbot.（2）Effects of nitrate on intestinal health of juvenile turbot（Scophthalmus maximus）The effects of nitrate exposure on intestinal health of juvenile turbot were evaluated by studying the intestinal histological changes,barrier function,immune status and intestinal flora composition of juvenile turbot.The histological results showed that exposure to low,medium and high concentrations of nitrate could induce different degrees of histological damage in the intestines of juvenile turbot,such as intestinal microvilli atrophy and lamina propria necrosis.q RT-PCR analysis showed that the expression of key genes of tight junction protein such as occludin,tricellulin and ZO-1 in HN group was significantly lower than that in control group,while the expression of immune-related genes HSP70,HSP90,TLR-3,IL-1βand TNF-αwas significantly increased（P<0.05）,and the expression of TGF-β,LYS and IGF-1 was significantly decreased in HN group（P<0.05）.The results of intestinal flora analysis showed that exposure to higher concentration of nitrate could induce changes in the structure of intestinal flora of turbot,and may change itsα-diversity andβ-diversity.The proportion of pathogens or opportunistic pathogens increased significantly in MN and HN groups（P<0.05）.The above results showed that chronic nitrate exposure had significant negative effects on intestinal health of juvenile turbot.Long-term exposure to nitrate at concentrations higher than 50 mg/L NO₃^--N can induce intestinal flora imbalance in marine bony fish—turbot,and directly increase the risk of invasion of pathogens or opportunistic pathogens.（3）Effects of nitrate on blood physiological parameters,redox status and apoptosis of juvenile turbot（Scophthalmus maximus）The chronic toxic effects of nitrate on juvenile turbot were evaluated by detecting the contents of nitrate and nitrite in plasma,physiological stress index,many kinds of important ions,antioxidant factors and apoptosis-related gene expression in gills at different time points.The analysis of nitrate and nitrite accumulation showed that the higher the environmental nitrate exposure concentration,the higher the plasma nitrate and nitrite accumulation concentration.The analysis of the content of methemoglobin showed that exposure to the environment could induce methemoglobin in turbot,especially in the HN group.The analysis of physiological indexes showed that the concentrations of plasma cortisol,glucose,triglyceride and lactic acid in the moderate and HN group were significantly higher than those in the CK（P<0.05）.Ion concentration analysis showed that medium and high concentrations of nitrate exposure significantly decreased the plasma concentrations of Na⁺and Cl^-（P<0.05）,and significantly increased the plasma K⁺concentration（P<0.05）.The analysis of antioxidant level showed that from the fifth day,the plasma antioxidants SOD,CAT,GSH and GPx in LN,MN and HN groups decreased in varying degrees,and the level of MDA increased accordingly.The analysis of apoptosis in gills showed that the expressions of apoptosis-related genes p53,caspase-3/7 and Bcl-2 were significantly up-regulated/down-regulated（P<0.05）and the process of apoptosis was abnormal after exposure to high concentration of nitrate.The comprehensive toxicity index analysis showed that the toxic effect of nitrate on juvenile turbot was dose-dependent,and the toxicity of nitrate reached the peak after 15 days of exposure.The above results suggest that the toxic effect of chronic nitrate exposure on juvenile turbot is persistent and may cause irreversible damage.Under the industrial recirculating aquaculture mode,the safe nitrate concentration threshold of turbot juvenile culture should not exceed 50 mg/L NO₃^--N.