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The large-scale loach (Paramisgurnus dabryanus) is one of the most commercially important cultured species. Ammonia nitrogen accumulation is one of the key issue which limited production and animal health in aquaculture, but few of information is available on the molecular mechanisms of ammonia detoxification. We performed transcriptomic analyses of the gill and liver of large-scale loach subjected to 48 h of aerial and ammonia exposure. We obtained 47,473,424 to 56,791,496 clean reads from the aerial exposure, ammonia exposure and control groups, assembled and clustered a total of 92,658 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 large-scale loach were significantly enriched, suggesting that large-scale loach responded to high exogenous and endogenous ammonia stress by enhancing amino acid metabolism. Besides, the expression of several ammonia transporters (i.e., Rhesus glycoproteins and Aquaporins) 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. The results provide valuable information on the molecular mechanism of ammonia detoxification of large-scale loach to endogenous and environmental ammonia loading, will facilitate the molecular assisted breeding of ammonia resistant varieties, and will offer beneficial efforts for establishing an environmental-friendly and sustainable aquaculture industry., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The Paramisgurnus dabryanus was exposed to 30 mmol L -1 NH 4 Cl solution and air to assessing the change of body ammonia and urea contents and the activities of alanine aminotransferase (ALT) and aspartate transaminase (AST). After 48 h of ammonia exposure, ammonia concentration in the plasma, brain, liver and muscle were 3.3-fold, 5.6-fold, 3.5-fold and 4.2-fold, respectively, those of the control values. Plasma, brain, liver and muscle ammonia concentrations increased to 2.2-fold, 3.3-fold, 2.5-fold and 2.9-fold, respectively, those of control values in response to 48 h of aerial exposure. Within the given treatment (ammonia or aerial exposure), there was no change in plasma, brain and liver urea concentrations between exposure durations. The plasma ALT activity was significantly affected by exposure time during aerial exposure, while the liver ALT activity was not affected by ammonia or aerial exposure. Exposure to NH 4 Cl or air had no effect on either plasma or liver AST activity. Our results suggested that P. dabryanus could accumulate quite high level of internal ammonia because of the high ammonia tolerance in its cells and tissues, and NH 3 volatilization would be a possible ammonia detoxification strategy in P. dabryanus. Urea synthesis was not an effective mechanism to deal with environmental or internal ammonia problem. The significant increase of ALT activity in plasma during aerial exposure, indicating that alanine synthesis through certain amino acid catabolism may be subsistent in P. dabryanus.

The weatherloach, Misgurnus angulliacaudatus, is an intestinal air-breathing, freshwater fish that has the unique ability to excrete ammonia through gut volatilization when branchial and cutaneous routes are compromised during high environmental ammonia or air exposure. We hypothesized that transepithelial gut NH(4)(+) transport is facilitated by an apical Na(+)/H(+) (NH(4)(+)) exchanger (NHE) and a basolateral Na(+)/K(+)(NH(4)(+))-ATPase, and that gut boundary layer alkalinization (NH(4)(+) → NH(3) + H(+)) is facilitated by apical HCO(3)(-) secretion through a Cl(-)/HCO(3)(-) anion exchanger. This was tested using a pharmacological approach with anterior (digestive) and posterior (respiratory) intestine preparations mounted in pH-stat-equipped Ussing chambers. The anterior intestine had a markedly higher conductance, increased short-circuit current, and greater net base (J(base)) and ammonia excretion rates (J(amm)) than the posterior intestine. In the anterior intestine, HCO(3)(-) accounted for 70% of J(base). In the presence of an imposed serosal-mucosal ammonia gradient, inhibitors of both NHE (EIPA, 0.1 mmol l(-1)) and Na(+)/K(+)-ATPase (ouabain, 0.1 mmol l(-1)) significantly inhibited J(amm) in the anterior intestine, although only EIPA had an effect in the posterior intestine. In addition, the anion exchange inhibitor DIDS significantly reduced J(base) in the anterior intestine although only at a high dose (1 mmol l(-1)). Carbonic anhydrase does not appear to be associated with gut alkalinization under these conditions as ethoxzolamide was without effect on J(base). Membrane fluidity of the posterior intestine was low, suggesting low permeability, which was also reflected in a lower mucosal-serosal J(amm) in the presence of an imposed gradient, in contrast to that in the anterior intestine. To conclude, although the posterior intestine is highly modified for gas exchange, it is the anterior intestine that is the likely site of ammonia excretion and alkalinization leading to ammonia volatilization in the gut.

The weatherloach, Misgurnus anguillicaudatus, is a freshwater, facultative air-breathing fish that lives in streams and rice paddy fields, where it may experience drought and/or high environmental ammonia (HEA) conditions. The aim of this study was to determine what roles branchial Na(+)/K(+)-ATPase, H(+)-ATPase, and Rhcg have in ammonia tolerance and how the weatherloach copes with ammonia loading conditions. The loach's high ammonia tolerance was confirmed as was evident from its high 96 h LC(50) value and high tissue tolerance to ammonia. The weatherloach does not appear to make use of Na(+)/NH(4)(+)-ATPase facilitated transport to excrete ammonia when exposed to HEA or to high environmental pH since no changes in activity were observed. Using immunofluorescence microscopy, distinct populations of vacuolar (V)-type H(+)-ATPase and Na(+)/K(+)-ATPase immunoreactive cells were identified in branchial epithelia, with apical and basolateral staining patterns, respectively. Rhesus C glycoprotein (Rhcg1), an ammonia transport protein, immunoreactivity was also found in a similar pattern as H(+)-ATPase. Rhcg1 (Slc42a3) mRNA expression also increased significantly during aerial exposure, although not significantly under ammonia loading conditions. The colocalization of H(+)-ATPase and Rhcg1 to the similar non-Na(+)/K(+)-ATPase immunoreactive cell type would support a role for H(+)-ATPase in ammonia excretion via Rhcg by NH(4)(+) trapping. The importance of gill boundary layer acidification in net ammonia excretion was confirmed in this fish; however, it was not associated with an increase in H(+)-ATPase expression, since tissue activity and protein levels did not increase with high environmental pH and/or HEA. However the V-ATPase inhibitor, bafilomycin, did decrease net ammonia flux whereas other ion transport inhibitors (amiloride, SITS) had no effect. H(+)-ATPase inhibition also resulted in a consequent elevation in plasma ammonia levels and a decrease in the net acid flux. In gill, aerial exposure was also associated with a significant increase in membrane fluidity (or increase in permeability) which would presumably enhance NH(3) permeation through the plasma membrane. Taken together, these results indicate the gill of the weatherloach is responsive to aerial conditions that would aid ammonia excretion.

The oriental weatherloach Misgurnus anguillicaudatus is an extremely ammonia-tolerant fish. Many ammonia-protection mechanisms have been reported in this fish. Six strategies used by this fish to deal with the problem of excess ammonia are described. The fish can (1) reduce ammonia production through reduction in protein and/or amino acid catabolism; (2) reduce ammonia production and obtain energy through partial amino acid catabolism leading to alanine formation; (3) detoxify ammonia to glutamine; (4) tolerate very high ammonia levels in its tissues; (5) get rid of ammonia as NH(3) gas and, probably, (6) possesses background K(+) channels that are impermeable to NH(4)(+). The effects of extracellular ammonia on the contraction performance of the heart from this fish were found to be the same as in rainbow trout, an ammonia-sensitive fish. It suggests that the hearts of most, if not all, fish species are protected against ammonia. MK-801, an NMDA receptor blocker, was found to have a protective effect against ammonia intoxication in the oriental weatherloach, which suggests that the NMDA receptor, as in mammals, is involved in ammonia toxicity.

The effect of endogenous ammonia produced in dead fish or ammonia in ammonium bicarbonate solutions was evaluated on the viability of Clonorchis sinensis metacercariae. Viability was determined by worm recovery in rats infected with metacercariae exposed to ammonia in decaying fish or in vitro solutions. The recovery rates were 58%, 48%, 44% and 2% in fish that had been allowed to decay for 5, 10, 20 and 30 days at 4 degrees C, respectively, and this rate ( Y) was inversely correlated with the ammonia concentration ( X) in fish muscle extract ( Y=80.62-0.0667 X, r(2)=0.905, P=0.049). C. sinensis metacercariae barely survived in ammonia solutions with concentrations exceeding 1 g N/l. Our results indicate that the ammonia produced in dead fish is lethal to the viability of C. sinensis metacercariae.