The Impacts Of Individual And Combined Exposure To Ammonia And Microcystin-LR On Intestinal Immunity And Microbial Community In Female Zebrafish(Danio Rerio)

Due to human activities and industrial wastewater discharge,ammonia nitrogen concentration in water often exceeds the standard,which further aggravates the eutrophication degree of water and lead to the outbreak of cyanobacterial blooms.The frequent occurrence of cyanobacterial blooms has become one of the major environmental problems worldwide.Therefore,ammonia nitrogen and microcystin(MC)may coexist in blooms-contaminated water and cause potential combined toxicity to aquatic organisms.To explore the possible synergistic impacts of ammonia and microcystin-LR(MCLR)on the intestine of fish,the ammonia alone exposure and combined exposure experiments were designed.The experimental results are as follows:In single ammonia exposure experiment,3-month-old female zebrafish were exposed to different concentrations of total ammonia nitrogen(TAN 0,3 and 30 mg/L)for 60 d to explore the impacts of ammonia nitrogen on the intestinal innate system of fish and its mechanism.The results showed that TAN expsoure caused cell vacuolation,lymphocyte infiltration and an increased number of goblet cells in the intestine mucosa.Our further detection showed significant decreases in the contents and gene expression levels of lysozyme(Lys)and β-defensin(def-β)in the intestine of zebrafish,suggesting that chronic ammonia stress decreased intestinal innate immunity of fish.The analysis of TLRs-related genes(tlr4a)indicated that ammonia could activate TLR-mediated myd88-dependent pathway,and eventually induce intestinal inflammation.These results illustrated chronic ammonia exposure can damage intestinal structure,inhibit intestinal immunity and cause intestinal inflammation.In the combined exposure experiment of ammonia and MCLR,adult female zebrafish were exposed to control,ammonia group(30 mg/L TAN),MCLR group(10 μg/L)and mix group(30 mg/L TAN + 10 μg/L)for 45 d,respectively.Histopathological results showed that ammonia alone group decreased intestinal villis area,MCLR alone group cause villus rupture and increased the ratio of vacuolization in intestines.More serious damage was observed in the mix exposure group.Both ammonia nitrogen alone and in combination with MCLR significantly decreased contents and gene expression levels of intestinal lysozyme and β-defensin in all treatment in relative to the control group,suggesting the intestinal immunity of zebrafish was suppressed.Moreover,the mix group increased the expression levels and contents of intestinal inflammatory factors IL-1β and TNF-α by activating nod1/2 and tlr4a/b singals,and caused intestinal inflammation.Intestinal flora analysis results showed that ammonia and MCLR cause changes in the diversity of zebrafish intestinal microbiota.Ammonia nitrogen significantly increased the richness of Firmicutes,while MCLR significantly increased the richness of Actinobacteria and decreased the richness of Proteobacteria.The combined exposure to ammonia nitrogen and MCLR significantly increased the abundance of Pseudomonas and Mycobacterium.Their combined exposure also significantly reduced OTUs of short-chain fatty acids producers and the contents of short-chain fatty acids in the intestine.In conclusion,ammonia combined with MCLR has a synergistic effect on the intestinal immunity and structure of intestinal flora in zebrafish.