Ammonia Stress Influences Intestinal Barrier,Microbiota And Glucolipid Metabolism Of Chinese Striped Neck Turtle (Mauremys Sinensis)

Ammonia is one of the major pollutants in aquaculture that induces severe stress to organisms when it exceeds its suitable concentration.During intensive culture farming,the use of high protein diet is excreted as ammonia（the end product of protein metabolism）which leads to ammonia elevation and causes stress.Ammonia exists in two forms（NH₄⁺and NH₃）in water bodies,and NH₃easily crosses cytomembrane and causes toxicity.A high concentration of ammonia could disrupt aqueous life including turtles.Chinese striped neck turtle（Mauremys sinensis）,a freshwater species,is well known for traditional medicine,food,decoration,research activities and plays a key role in economic development.It has been found that the increased concentration of NH₃in the body fluids of aquatic animal could adversely affect survival,development,growth,immunity and energy metabolism.The main goal of our research was to investigate the effects of ammonia exposure on M.sinensis;thus,we examined the effect of ammonia stress on intestinal histomorphology,intestinal immune status,intestinal microbiota and hepatic glucolipid metabolism.Therefore,the turtles were divided into three groups i-e control(total ammonia<13 mg L^-1),A100(NH₃:1.53 mg L^-1)and A200(NH₃:2.98 mg L^-1)for24 h and 48 h and 30 days.The main findings are following:1.Ammonia affected intestinal histomorphology and immune status of M.sinensisOur results showed that ammonia reduced the villus length in ammonia exposed groups as compared to control but it did not vary significantly（P>0.05）.Moreover,ammonia exposure for 30 days induced multiplication of heterotopic granulocytes and inflammatory cells.In addition,the epithelial tight junction genes,oclaudin and zonola occludin remarkably downregulated in ammonia exposed groups while claudin1 expression decreased only at A100 as compared to control group（P<0.05）.Similarly,the mRNA transcription profile of the MUC-2gene also noticeably decreased in ammonia treated groups.However,the expression of intestinal immune related genes such as IL-10,IL-12,TGF-β1,TNF-αand IFN-γmarkedly increased except IL-6.Therefore,it confirmed that ammonia could disrupt intestinal structure in M.sinensis by disturbing paracellular permeability,impairment of mucosal structure and triggering inflammatory reactions.2.The effect of ammonia on intestinal microbiota of M.sinensisIn current study,we found total 1,878,149 raw reads from 27 samples of M.sinensis intestinal contents,with an average of 69,561 reads per sample.The average length of each sequence was 480 bp.Out of 680 OTUs（53.62%,71.28%and 62.04%of all sequences in control,A100 and A200,respectively）were common.56 OTUs in A100 and 74 OTUs in A200were unique correspondingly,and 127 OTUs were common between them.Furthermore,ammonia changed gut microbial diversity variedly.At the phylum levels,Firmicutes increased,while Bacteroidota,Desulfobacterota and Synergistota decreased greatly.Whereas,at genus levels,Lachnospiraceae,Bacteroides,Eubacteriaceae,Desulfovibrio,Muribaculaceae,Bilophila,Cloacibacillus,Christensenellaceae,Ruminococcus and Parabacteroides decreased while Romboutsia and Turicibacter increased in ammonia exposed groups.Consequently,it clarified that ammonia could affect the composition and ability of intestinal bacteria,increase the chance of opportunistic pathogenic bacteria which rupture the intestinal epithelium,alter metabolic processes and disturb the functioning of intestinal barriers.3.Effect of Ammonia stress on carbohydrate and lipid metabolism in M.sinensisIn this study,M.sinensis were subjected to two different ammonia groups（A100 and A200）for 24 h and 48 h,and the effects of ammonia on carbohydrates and lipid metabolism in Chinese striped neck turtles were explored.Results showed that the mRNA expression of AMPKα1 significantly increased only in A100 at 24 h,while its activity increased at 24 h and 48h in all ammonia treated groups.Furthermore,the two AMPK-regulated transcription factors supervising carbohydrate metabolism,HNF4αelevated whereas Fox O1 decreased in ammonia treated groups.Subsequently,PFK and G-6-PAS expression downregulated.Additionally,the expression profiles of transcription factors Ch REBP and SREBP-1c responsible for lipogenesis were reduced and their downstream genes including（ACC,FAS and SCD-1）were also suppressed.Besides,the glucose contents significantly decreased only in A200,while triglyceride content uplifted in A200 at 24 h.The results indicated that ammonia stress activated AMPK signaling to inhibit gluconeogenesis and lipogenesis,by influencing HNF4α,Fox O1,Ch REBP and SREBP-1c,and promote glycolysis in M.sinensis to accomplish energy demand and survive in stressful environment.However,detail study is needed to explore complex pathways that might involve in energy regulation in M.sinensis and enable it to confer ammonia stress.Our study provides basis for introducing healthy environment for aquatic animals.