| In intensive aquaculture,especially in factory-type circulating aquaculture systems,due to factors such as excessive culture density,excessive feeding,and unbalanced management operations,ammonia nitrogen will usually gradually accumulate in the water body,which will be detrimental to economic fish.Impact.However,in largemouth bass,there are few reports about ammonia nitrogen stress,and its detoxification mechanism is not clear.Therefore,it is of great significance to study the toxic effect of ammonia on largemouth bass.In this study,largemouth bass was used as the object.First,the effects of ammonia stress on its growth and metabolic status were studied.Second,the effects of ammonia stress on its physiological and biochemical indexes were explored.Finally,its molecular index response mechanism was analyzed from the aspect of immune genes.Results were shown as follows.The effects of ammonia,temperature,and body weight on oxygen consumption rate and asphyxiation point of Micropterus salmoides were studied in a closed-recirculation experiment.Results show that within the range of ammonia concentration from 0 to 8.61 mg/L,the oxygen consumption rate increased first and then decreased with a peak value of 0.338 mg/(g·h).The asphyxiation point increased with the increase of ammonia concentration,and showed significant difference(P<0.05).Within the experimental temperature range of 13-33°C,the oxygen consumption rate increased first,reached a peak of 0.392 mg/(g·h)at 29 °C,and then declined.The oxygen consumption rate decreased with the body weight increase with the lowest value of 0.112mg/(g·h).The oxygen consumption rate showed an obvious change of circadian rhythm with significant difference(P<0.05).The night average and daytime average of the oxygen consumption rate was 0.214 and 0.199mg/(g·h),respectively.The average oxygen consumption rate at night was much higher than that in the daytime,and two peaks appeared at 2:00-6:00 and 18:00-20:00.A study was carried to test the response of largemouth bass(Micropterus salmoides)(30.94±1.71)g for acute ammonia toxicity under three ammonia concentrations(19,28.5,38 mg/L).The tissues of kidney,liver,gill,brain and intestine were taken at 0,6,12,24,48,72,96 h respectively.The total superoxide dismutase(T-SOD),total antioxidant activity(T-AOC)and glutathione peroxidation oxidase(GSH-Px)were studied in different tissues of largemouth bass under acute ammonia exposure.Results: during the experiment,the concentration of ammonia nitrogen was 19 mg/L,and the activity of T-SOD increased gradually with the increase of stress time.The higher the concentration of ammonia nitrogen,the longer the stress time,the stronger the toxic effect,which showed that 38 mg/L ammonia nitrogen treatment group was significantly lower than the normal level after 72 hours or 96 hours.When the concentration of ammonia nitrogen was 19 mg/L,the indexes of T-SOD,T-AOC and GSH-Px were lower than those in the control group(P<0.5),the other were no significant changes(P>0.05).When the concentration of ammonia nitrogen is 28.5mg/L,the content of GSH-Px in kidney,liver,gill,intestine,liver and all tissues were significantly lower than that of the control group(P<0.5).However,the T-SOD,T-AOC and GSH-Px were significantly lower than those of the control group(P<0.5)in 38mg/L.According to the size characteristics of the peak value,under ammonia nitrogen stress,T-SOD of gill filaments and T-AOC and GSH-Px of kidney showed the strongest sensitivity.Under the condition of ammonia stress,the activities of T-SOD,T-AOC and GSH-Px were significantly affected(P<0.5).With the increase of concentration and time,the influence degree increased.It can be seen that ammonia nitrogen in aquaculture water will have a great impact on the enzyme activity of largemouth bass.At the same time,T-SOD is suitable for gill tissue,T-AOC and GSH-Px are suitable for kidney tissue as monitoring indicators of ammonia nitrogen stress.In order to understand the signal transduction mechanism of Toll like receptor 2(TLR2)and explore the immunoprotective effect of TLR2 on largemouth bass(Micropterus salmoides)under ammonia exposure.In this study,TLR2 partial sequence was obtained from the transcriptome of largemouth bass,and TLR2 full-length cDNA was obtained by RT-PCR.The TLR2 gene and amino acid sequence of largemouth bass were analyzed by molecular bioinformatics,and the molecular evolution of TLR2 gene was analyzed according to the phylogenetic tree.qRT-PCR was used to analyze the tissue expression and distribution of TLR2 gene in largemouth bass and the immune response model after ammonia nitrogen stress.The results showed that TLR2 gene was 2626 bp,ORF was 2445 bp,encoding 814 amino acids.The results of sequence homology analysis showed that TLR2 of largemouth bass was similar to other species in TLR and gene structure,with high conservation and homology of 85.66%.Phylogenetic tree analysis showed that the Largemouth bass TLR2 was clustered with Lateolabrax and grouper.qRT-PCR analysis showed that TLR2 was expressed in six healthy tissues(kidney,liver,spleen,brain,gill and intestine),and the expression was high in liver and kidney.The expression pattern of TLR2 gene after ammonia exposure indicated that TLR2 gene played an important role in response to the threat of toxicity.In this pathway,TLR2 mediates NF-?B signal through MyD88 dependent pathway.In order to study the effect of acute ammonia nitrogen stress on the expression of immune response related genes,and to understand the immunoprotective effect of interferon regulatory factor 4(IRF4)on the body of larval sea bass(Micropterus salmoides).In this study,partial sequences of IRF4 were obtained from transcriptome of largemouth bass,and full-length IRF4 cDNA was obtained by RT-PCR.The IRF4 gene sequence and amino acid sequence of largemouth bass were analyzed by molecular bioinformatics,and the molecular evolution of IRF4 gene was analyzed according to the phylogenetic tree constructed.qRT-PCR was used to analyze the distribution of IRF4 gene expression and the immune response pattern after ammonia nitrogen stress.The results showed that the full length of IRF4 gene was 1964 bp,ORFwas 1224 bp,encoding 407 amino acids.Sequence homology analysis showed that the IRF4 gene structure of largemouth bass was similar to that of other species,with high conservation,and the homology was 90.33%.Phylogenetic tree analysis showed that IRF4 and mandarin fish were one.qRT-PCR analysis showed that IRF4 was expressed in six healthy tissues(kidney,liver,spleen,brain,gill and intestine),and the expression was high in kidney.Ammonia exposure could induce the expression of IRF4 gene in six tissues(P<0.05),and the time of peak was different.Type I IFN(IFN ? / ?)induced by IRF4 can participate in the resistance of organism to ammonia exposure.This study showed that the expression pattern of IRF4 gene in largemouth bass under ammonia nitrogen stress showed that IRF4 gene played an important role in dealing with ammonia poisoning. |
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