| The large-scale loach（Paramisgurus dabryanus）is one of the important economic aquaculture varieties in China.Previous studies have described the excellent ammonia tolerance in P.dabryanus,however,the ammonia defensive mechanisms of this species are still unclear.The present study accessed the physiological response on ammonia accumulation in P.dabryanus by conducting acute ammonia toxicity test,transcriptomic analysis and screening gene regulation,aimed to provide useful information for revealing the mechanisms of ammonia detoxification in this species.1 In this study,the lethal concentration 50%(LC50)of total ammonia nitrogen was determined by the hydrostatic method.The LC50of total ammonia nitrogen was234.13 mg/L and the safety concentration was 23.41 mg/L.2 We performed transcriptomic analyses of the gill and liver of P.dabryanus subjected to 48 h of aerial and ammonia exposure.We obtained 47473424 to56791496 clean reads from the aerial exposure,ammonia exposure and control groups,assembled and clustered a total of 92658 unigenes with an average length of 909 bp and N50 of 1787 bp.Totals of 489/145 and 424/140 differentially expressed genes（DEGs）were detected in gill/liver of large-scale loach after aerial and ammonia exposure through comparative transcriptome analyses,respectively.In addition,totals of 43 gene ontology（GO）terms and 266 Kyoto Encyclopedia of Genes and Genomes（KEGG）pathways were identified.After aerial and ammonia exposure,amino acid metabolism pathways in liver of P.dabryanus were significantly enriched,suggesting that P.dabryanus responded to high exogenous and endogenous ammonia stress by enhancing amino acid metabolism.Besides,the expression of several ammonia transporters（Rhesus（Rh）glycoproteins and Aquaporins（Aqps））in gill of large-scale loach were markedly changed after 48 h of aerial exposure,suggesting that large-scale loach responded to high endogenous ammonia stress by regulating the expression of Rh glycoproteins and Aqps related genes in gill.3 By screening the transcriptomic data,we obtained three Rh glycoprotein sequences with high expression in tissues（Rhag,Rhbg,Rhcg）.Acute（NH4Cl concentrations:low 60 mmol/L,high 80 mmol/L;exposure tiome:0 h,12 h,24 h,36h,48 h and 96 h）and chronic（NH4Cl concentration:low 20 mmol/L,high 40 mmol/L;exposure time:7 d,14 d and 21 d）ammonia exposure were performed in P.dabryanus.After exposure experiment,the gill and skin of P.dabryanus were sampled to determine Rhag,Rhbg and Rhcg mRNA expression using Quantitative real-time PCR（qPCR）to reveal the role of Rh glycoproteins related genes during ammonia loading.（1）Acute experimentIn gill,Rhag mRNA expression increased first and then decreased with exposure time,and the trend in the high concentration group was the same as that in the low concentration group;compared with the control group,the high concentration group（80 mmol/L）was significantly higher than that in the control group（P<0.05）;at 48 h and 96 h,the expression in the high concentration group（80 mmol/L）was significantly lower than that in the control group（P<0.05）.In skin,the expression of Rhag mRNA at each exposure time point was significantly lower than that in the control group（P<0.05）,and there was no significant difference between the high concentration group and the low concentration group（P>0.05）.In gill,the Rhbg mRNA expression in the low concentration group（60 mmol/L） had no significant change compared with the control group at each time point（P<0.05）,and the high concentration group（80 mmol/L）had no significant change before36 h.The Rhbg mRNA expression within 36 h was significantly higher than that in the control group（P<0.05）;from 48 h to 96 h,the Rhbg mRNA expression in the high concentration group（80 mmol/L）was significantly lower than that in the control group（P<0.05）.In skin Rhbg mRNA expression was significantly higher than that in control group at 36 h and 48 h exposure（P<0.05）.In the gill,the Rhcg mRNA expression in the low concentration group（60 mmol/L）showed a trend of first decreasing,then increasing and then decreasing in time,while the expression in the high concentration group（80 mmol/L）showed an increase and then a decrease in time.The expression level of the low concentration group（60mmol/L）was significantly lower than that of the control group before and after 48 h（P<0.05）,and was significantly higher than that of the control group at 48 h（P<0.05）.The expression level of the high concentration group（80 mmol/L）was significantly higher than that of the control group at 36 h（P<0.05）,and was significantly lower than that of the control group at 96 h（P<0.05）.（2）Chronic experimentIn gill,Rhag mRNA expression in high and low concentration groups was significantly lower than that in control group at 7 days（P<0.05）,and there was no significant difference between 14 days and 21 days（P<0.05）.In skin,the expression levels in the high and low concentration groups were significantly lower than those in the control group（P<0.05）.In gill,Rhbg mRNA expression in high and low concentration groups was significantly lower than that in control group from 0 to 14 d（P<0.05）.In skin,there was no significant difference between the expression levels on 7 d and 14 d compared with the control group（P<0.05）,and the expression level on 21 d was significantly lower than that in the control group（P<0.05）.During ammonia loading,the expression level of Rhag mRNA in the gill tissue of P.dabryanus increased and then decreased with the time,and the expression level of Rhbg mRNA in the gill and skin tissue increased and then decreased with time.Rhcg mRNA expression level in gill tissue showed different trends in high concentration group and low concentration group.The results indicated that P.dabryanus responds to ammonia stress by adjusting the gene expression of Rh glycoprotein,and Rh glycoprotein may play an important role in the ammonia metabolism of P.dabryanus. |
