Effects Of Acute Ammonia Stress On Gills And Intestine Of Great Blue-spotted Mudskippers(Boleophthalmus Pectinirostris) And The Preliminary Study On Anti-ammonia Toxicity Effects Of Inositol

The great mudskipper is a large group of amphibious fish in intertidal zone which has the ability to tolerate the drastic changes of daily temperature difference,oxygen content and ammonia nitrogen content.It has important scientific value to study the mechanism of ammonia tolerance for fish.The excessive use of high concentration nitrogen fertilizer to breed benthic diatoms makes the mudskipper which feeds on benthic diatoms on the shoal vulnerable to exogenous ammonia nitrogen stress and thus threatens the survival and yield of mudskippers.Inositol is an important nutrient for fish.It can inhibit the production of oxygen free radicals,improve the antioxidant activity and prevent oxidative damage of fish.Therefore,in this experiment,Boleophthalmus pectinatus was used as the experimental object,a series of experiments were conducted to explore the response mechanism of the gill and intestine of mudskipper under ammonia nitrogen stress.Furthermore,the effect of inositol on alleviating ammonia toxicity was preliminarily studied.1.Effects of acute ammonia stress on gills of mudskippersThe great blue-spotted mudskippers（26.55±0.05 g in weight）were exposed to artificial seawater（15%salinity,control group）or to artificial seawater with high concentration ammonia(15%salinity with 8 mmol·L^-1 NH₄Cl,HEA group).After a 72-hour acute ammonia challenge,the total ammonia content in plasm of mudskipper was significantly increased.The mRNA levels of Rhesus glycoprotein a（Rhag）and Rhesus glycoprotein c1（Rhcg-1）,the non-ionic ammonia transporters in gills were up-regulated.While Rhesus glycoprotein b（Rhbg）and Rhesus glycoprotein c-2（Rhcg-2）in gills were down-regulated,suggesting the decreased inflow of the non-ionic ammonia in plasm and the increased outflow of the non-ionic ammonia through ammonia carriers for the mitochondrial rich cells（MRCs）.The gene expression levels involved in hydrogen ion（H⁺）and ammonium ion（NH₄⁺）transporting,such as carbonic anhydrase-2（CA-2）,carbonic anhydrase-15（CA-15）,H⁺-ATPase,Na⁺/H⁺（NH₄⁺）-exchanger（NHE）and Na⁺/K⁺-ATPase（NKA）,were all significantly up-regulated under ammonia stress,indicating the increased inflow of NH₄⁺from plasma to the MRCs through NKA on the basolateral membrane and increased outflow of NH₄⁺through NHE on the apical membrane.These results show that the great blue-spotted mudskippers which live in amphibious environment adopt the similar ammonia excreting model with the marine fish.The mRNA levels and activities of copper/zinc-superoxide dismutase（Cu/Zn-SOD）,catalase（CAT）and glutathione peroxidase（GPx）were up-regulated under high environmental ammonia（HEA）.Furthermore,the mRNA levels and activities inositol monophosphatase-1（IMPA-1）and inositol-3-phosphate synthase-1（ISYNA-1）,two key enzymes involved in the inositol biosynthesis（MIB）pathway,were up-regulated under ammonia stress,suggesting the acute ammonia stress induced oxidative stress in gills and activated the inositol biosynthesis pathway.The endogenous inositol may respond to oxidative stress by increasing the activity of antioxidant enzymes.2.Effects of acute ammonia stress on intestines of mudskippers.In this study,the two experimental groups were carried out to test the response of the great blue-spotted mudskippers to ammonia toxicity:the fish in the control group were exposed to 15%salinity artificial seawater and perfusion of 0.9%Na Cl（0.3 ml）for 72-h,the fish in the ammonia perfusion group were exposed to 15%salinity artificial seawater and perfusion of NH₄Cl（0.3 ml,60 mg/g fish）for 72-h.The study showed that acute ammonia stress significantly decreased tight junction proteins Claudin-5 gene expression levels from the 6-h to the 72-h,the gene expression level of Occludin（ocln）was decreased from the 3-h to the 6-h and the 48-h to the 72-h,respectively.However,the relative mRNA expression levels of tight junction proteins Claudin-15,Claudin-12 and linker protein in tight junction zonula occludens-1（ZO-1）in ammonia group were significantly increased compared to the control group.In addition,acute ammonia stress up-regulated pro-inflammatory cytokines,such as interleukin1-β（IL1-β）and tumor necrosis factor-α（TNF-α）,from the 3-h to the 72-h,down-regulated anti-inflammatory cytokines,such as transforming growth factor-β（TGF-β）,expression levels from the 6-h to the 72-h（P<0.05）.Additionally,acute ammonia stress increased malondialdehyde（MDA）content while decreased the gene expression levels and enzyme activities of copper/zinc superoxide dismutase（Cu/Zn-SOD）,catalase（CAT）,glutathione peroxidase（GPx）and glutathione-S-transferase（GST）in intestine（P<0.05）at 72-h.Acute ammonia stress also significantly increased apoptosis factors caspase-3 and caspase-9 mRNA expression levels at 72-h（P<0.05）.These results indicated that acute ammonia stress impaired intestinal barrier function via the MLCK signaling pathway by disrupting the tight junction proteins.Oxidative stress occurred in the intestine and induced inflammation and apoptosis,which may be related to the inhibition of antioxidant enzymes.3.Effects of acute ammonia stress on gills and intestines,and the resistance of inositolPrevious experiment found that the expression levels of two key enzymes in the inositol biosynthesis pathway were significantly up-regulated in both gills and intestines.Is this an adverse result of ammonia stress or a self-coping mechanism?For this reason,we performed the experiment of exogenous inositol supplement experiment.In this study,four groups were carried out to test the response of mudskippers to ammonia toxicity and their mitigation through inositol:group 1 was injected with Na Cl and exposed to artificial seawater for 24-h（15%salinity）,group 2was injected with inositol（2.5 mg/g fish）and exposed to artificial seawater for 24-h,group 3 was injected with Na Cl and exposed to high concentration ammonia(15%salinity with 57.025mmol·L^-1 NH₄Cl)for 24-h,and group 4 was injected inositol（2.5 mg/g fish）and exposed to high concentration ammonia for 24-h.Compared to the Na Cl group,ammonia stress disrupted intestinal barrier function（tight junction proteins ZO-1,Occludin,Claudin-5,-12,and-15 mRNA expressions were down-regulated,P<0.05）,increased the gene expression of apoptosis（p53,Bax,caspase-3 and caspase-9）,as well as pro-inflammatory cytokines（TNF-αand IL1-β）,while the genes transcription of anti-inflammatory cytokines TGF-βand IL-10 were depressed.In addition,ammonia poisoning induced oxidative stress（MDA,PC content increased,Cu/Zn-SOD,CAT,GPx,GST activities and T-AOC decreased,also depressed gene expression of the Cu/Zn-SOD,CAT,GPx and GST）.All of the adverse reactions were remarkably reduced by exogenous inositol.Compared to the NH₄Cl group,the concentration of immunoglobulin M（Ig M）and complement 4（C4）in serum were significantly reduced by the exogenous inositol supplement.Therefore,inositol may prevent the disruption of intestinal structural integrity and the inflammation induced by ammonia poisoning.Simultaneously,the acute ammonia stress also induced oxidative stress in gills（MDA content significantly up-regulated,the activity of Cu/Zn-SOD and CAT significantly up-regulated,GPx and GST enzyme activities significantly down-regulated,P<0.05）.Exogenous inositol can also prevent the oxidative stress in the gills of mudskipper under ammonia stress.