Stress Responses And Tolerance Mechanisms Of Ammonia And Nitrite In Litopenaeus Vannamei

Ammonia and nitrite are the major limiting environment factors in aquaculture,which could occur unusual high concentration in aquaculture systems due to the promotion of intensified aquaculture.The increasing accumulation level of ammonia and nitrite in water disrupts multiple physiological functions,including growth,molting,metabolism and excretion,which further lead to the increase of susceptibility to pathogens and microorganisms and the outbreak of diseases.Although the accumulation of ammonia and nitrite in the aquatic environment poses negative effects on shrimp,some shrimps still can withstand somewhat ammonia and nitrite.However,the potential mechanisms underlying this tolerance to ammonia and nitrite are still poorly understood.In this study,the shrimps,Litopenaeus vannamei,from ammonia-tolerant family(LV-AT),ammonia-sensitive family(LV-AS),nitrite-tolerant family(LV-NT)and nitrite-sensitive family(LV-NS)were used for comparative transcriptomic and metabolomic analysis to compare the difference after ammonia and nitrite exposure at m RNA and metabolite levels,respectively.Also,the histopathology observation and the physiological and biochemical indexes were also performed to discover the physiological responses.Collectively,the combination of transcriptomics,metabolomics and physiological results will provide new sights on the potential tolerant molecular mechanisms and the breeding of new varieties of ammonia and nitrite tolerant shrimp.In addition,the multi-omics analysis was also performed between untreated shrimps(LV-C)and ammonia-or nitrite-treated shrimps to investigate the ammonia and nitrite stress responses.All these results will greatly contribute to incrementally understand the ammonia and nitrite stress responses and supply molecular level support for evaluating the environmental effects of ammonia and nitrite on aquatic organisms.The major results are as follows:1.The combination of transcriptomic and metabolomic analysis was performed to reveal the ammonia stress responses of L.vannamei.The results showed that multiple immune,antioxidant and cytoskeleton-related genes were significantly changed after ammonia stress.Also,KEGG pathway enrichment analysis showed that most of DEGs were enriched in Lysosome,Phagosome and Protein processing in endoplasmic reticulum pathways that are associated with immune system and oxidative stress.The metabolomic results showed that most of amino acids,nucleotides and fatty acids were significantly changed after ammonia stress.In addition,the genes related to amino acid metabolism,nucleotide metabolism and fatty acid metabolism were also abnormal alteration.All these results revealed involvement of immune defence,cytoskeleton remodeling,antioxidative system and metabolic pathway in ammonia stress response of L.vannamei.2.The comparison of metabolite and transcript profiles between LV-AT and LV-AS indicated these metabolites involved in energy metabolism such as glycolysis and tricarboxylic acid(TCA)cycle were less accumulated in LV-AT,which may be related to enhanced energy metabolism to enhance the energy supply to adapt highconcentration ammonia stress for LV-AT.Moreover,the ammonia excretion and detoxification-related genes in LV-AT,such as GST,UGTs,CYP450 s,Rhg and NHE,showed higher expression than LV-AS.All these results showed that the higher ability of ammonia excretion and detoxification and the accelerated energy metabolism for energy supplies might be the adaptive strategies for LV-AT relative to LV-AS after ammonia stress.3.The determination of hemocyanin contents after 0 h,4 h,12 h,24 h,48 h,72 h and 96 h of nitrite exposure showed that nitrite stress caused a significant decrease of hemocyanin contents,which further lead to hypoxia.The antioxidant parameter analysis showed that nitrite could induce the oxidative stress and further caused the antioxidative enzyme alterations(T-SOD,T-AOC and CAT).Likewise,nitrite stress caused severe damages of the hepatopancreas and gill structure after histopathology observation.A variety of genes showing the largest magnitude of expression changes including immune system,apoptosis and cytoskeleton-associated genes were detected in hepatopancreas after nitrite exposure according to RNA-seq.In addition,some genes involved in metabolism regulation and the metabolites,including ammonia acids,nucleotides and fatty acids,showed abnormal changes in hepatopancreas after nitrite stress.4.The qPCR results revealed that the LV-NT showed higher expression of HIF-1? in hepatopancreas,gill and muscle than LV-NS after 48 h and 96 h of nitrite stress,which implicated that higher nitrite tolerance of LV-NT than LV-NS may be attributed to enhanced HIF-1? expression to regulate energy supply and gaseous exchange.In addition,most of fatty acids,including monounsaturated fatty acidsand saturated fatty acids,were significantly increased in LV-NT relative to LV-NS,which may activate the defense system to strengthen its tolerance to nitrite.However,the mechanisms how fatty acids deal with nitrite are unknown,which is still worthy of further investigations.5.The comparative gill transcriptomic analysis between LV-NT and LV-NS showed that nitrite stress remarkably enhanced the energy metabolism in LV-NT by activating the expression of genes encoding key enzymes in oxidative phosphorylation(OXPHOS)pathway and TCA cycle,and enhanced extra energy supply in NT for dealing with nitrite stress.Shrimps in LV-NT showed higher nitrite tolerance than LV-NS might be also attributed to the enhancement of gene expressions involved in innate immunity related-genes and pathways,and endoplasmic reticulum stress responses.In addition,we also identified some specific DEGs,such as GILT,KPI,Ferritin,CPB,FLP1,TXNDC11,SOD and COX,that may be involved in protecting shrimp during nitrite stress.