Evidence for the involvement of branchial Vacuolar-type H+-ATPase in the acidification of the external medium by the West African lungfish, Protopterus annectens, exposed to ammonia-loading conditions.

African lungfishes are obligatory air-breathers with exceptionally high environmental ammonia tolerance. They can lower the pH of the external medium during exposure to ammonia-loading conditions. This study aimed to demonstrate the possible involvement of branchial vacuolar-type H+-ATPase (Vha) in the ammonia-induced acidification of the external medium by the West African lungfish, Protopterus annectens, and to examine whether its capacity to acidify the medium could be augmented after exposure to 100 mmol l-1 NH4Cl for six days. Two full coding cDNA sequences of Vha subunit B (atp6v1b), atp6v1b1 and atp6v1b2, were obtained from the internal gills of P. annectens. The sequence of atp6v1b1 comprised 1548 bp, encoding 515 amino acids (57.4 kDa), while that of atp6v1b2 comprised 1536 bp, encoding 511 amino acids (56.6 kDa). Using a custom-made antibody reactive to both isoforms, immunofluorescence microscopy revealed the collective localization of Atp6v1b (atp6v1b1 and atp6v1b2) at the apical or the basolateral membrane of two different types of branchial Na+/K+-ATPase-immunoreactive ionocyte. The ionocytes labelled apically with Atp6v1b presumably expressed Atp6v1b1 containing a PDZ-binding domain, indicating that the apical Vha was positioned to transport H+ to the external medium. The expression of Atp6v1b was regulated post-transcriptionally, as the protein abundance of Atp6v1b and Vha activity increased significantly in the gills of fish exposed to 100 mmol l-1 NH4Cl for six days. Correspondingly, the fish exposed to ammonia had a greater capacity to acidify the external medium, presumably to decrease the ratio of [NH3] to [NH4+] in order to reduce the influx of exogenous NH3.

RNA sequencing, de novo assembly and differential analysis of the gill transcriptome of freshwater climbing perch Anabas testudineus after 6 days of seawater exposure.

To obtain transcriptomic insights into branchial responses to salinity challenge in Anabas testudineus, this study employed RNA sequencing (RNA-Seq) to analyse the gill transcriptome of A. testudineus exposed to seawater (SW) for 6 days compared with the freshwater (FW) control group. A combined FW and SW gill transcriptome was de novo assembled from 169.9 million 101 bp paired-end reads. In silico validation employing 17 A. testudineus Sanger full-length coding sequences showed that 15/17 of them had greater than 80% of their sequences aligned to the de novo assembled contigs where 5/17 had their full-length (100%) aligned and 9/17 had greater than 90% of their sequences aligned. The combined FW and SW gill transcriptome was mapped to 13,780 unique human identifiers at E-value ≤1.0E-20 while 952 and 886 identifiers were determined as up and down-regulated by 1.5 fold, respectively, in the gills of A. testudineus in SW when compared with FW. These genes were found to be associated with at least 23 biological processes. A larger proportion of genes encoding enzymes and transporters associated with molecular transport, energy production, metabolisms were up-regulated, while a larger proportion of genes encoding transmembrane receptors, G-protein coupled receptors, kinases and transcription regulators associated with cell cycle, growth, development, signalling, morphology and gene expression were relatively lower in the gills of A. testudineus in SW when compared with FW. High correlation (R = 0.99) was observed between RNA-Seq data and real-time quantitative PCR validation for 13 selected genes. The transcriptomic sequence information will facilitate development of molecular resources and tools while the findings will provide insights for future studies into branchial iono-osmoregulation and related cellular processes in A. testudineus.

Na+/H+ Exchanger 3 Is Expressed in Two Distinct Types of Ionocyte, and Probably Augments Ammonia Excretion in One of Them, in the Gills of the Climbing Perch Exposed to Seawater.

The freshwater climbing perch, Anabas testudineus, is an euryhaline teleost and an obligate air-breather with the ability to actively excrete ammonia. Members of the Na+/H+ exchanger (NHE) family help maintain intracellular pH homeostasis and ionic balance through the electroneutral exchange of Na+ and H+. This study aimed to obtain, from the gills of A. testudineus, the full cDNA coding sequence of nhe3, and to determine the effects of exposure to seawater or 100 mmol l-1 of NH4Cl in fresh water on its mRNA and protein expression levels. Efforts were also made to elucidate the type of ionocyte that Nhe3 was associated with in the branchial epithelium of A. testudineus. The transcript level and protein abundance of nhe3/Nhe3 were very low in the gills of freshwater A. testudineus, but they increased significantly in the gills of fish acclimated to seawater. In the gills of fish exposed to seawater, Nhe3 was expressed in two distinct types of seawater-inducible Na+/K+-ATPase (Nka)-immunoreactive ionocytes. In Nkaα1b-immunoreactive ionocytes, Nhe3 had an apical localization. As these ionocytes also expressed apical Rhcg1 and basolateral Rhcg2, which are known to transport ammonia, they probably participated in proton-facilitated ammonia excretion in A. testudineus during seawater acclimation. In Nkaα1c-immunoreactive ionocytes, Nhe3 was atypically expressed in the basolateral membrane, and its physiological function is uncertain. For A. testudineus exposed to NH4Cl in fresh water, the transcript and protein expression levels of nhe3/Nhe3 remained low. In conclusion, the branchial Nhe3 of A. testudineus plays a greater physiological role in passive ammonia transport and acid-base balance during seawater acclimation than in active ammonia excretion during environmental ammonia exposure.

Molecular characterization of three Rhesus glycoproteins from the gills of the African lungfish, Protopterus annectens, and effects of aestivation on their mRNA expression levels and protein abundance.

African lungfishes are ammonotelic in water. They can aestivate for long periods on land during drought. During aestivation, the gills are covered with dried mucus and ammonia excretion ceases. In fishes, ammonia excretion through the gills involves Rhesus glycoproteins (RhGP/Rhgp). This study aimed to obtain the complete cDNA coding sequences of rhgp from the gills of Protopterus annectens, and to determine their branchial mRNA and protein expression levels during the induction, maintenance and arousal phases of aestivation. Three isoforms of rhgp (rhag, rhbg and rhcg) were obtained in the gills of P. annectens. Their complete cDNA coding sequences ranged between 1311 and 1398 bp, coding for 436 to 465 amino acids with estimated molecular masses between 46.8 and 50.9 kDa. Dendrogramic analyses indicated that Rhag was grouped closer to fishes, while Rhbg and Rhcg were grouped closer to tetrapods. During the induction phase, the protein abundance of Rhag, but not its transcript level, was down-regulated in the gills, suggesting that there could be a decrease in the release of ammonia from the erythrocytes to the plasma. Furthermore, the branchial transcript levels of rhbg and rhcg decreased significantly, in preparation for the subsequent shutdown of gill functions. During the maintenance phase, the branchial expression levels of rhag/Rhag, rhbg/Rhbg and rhcg/Rhcg decreased significantly, indicating that their transcription and translation were down-regulated. This could be part of an overall mechanism to shut down branchial functions and save metabolic energy used for transcription and translation. It could also be regarded as an adaptive response to stop ammonia excretion. During the arousal phase, it is essential for the lungfish to regain the ability to excrete ammonia. Indeed, the protein abundance of Rhag, Rhbg and Rhcg recovered to the corresponding control levels after 1 day or 3 days of recovery from 6 months of aestivation.

Ammonia exposure affects the mRNA and protein expression levels of certain Rhesus glycoproteins in the gills of climbing perch.

The freshwater climbing perch, Anabas testudineus, is an obligate air-breathing and euryhaline teleost capable of active ammonia excretion and tolerant of high concentrations of environmental ammonia. As Rhesus glycoproteins (RhGP/Rhgp) are known to transport ammonia, this study aimed to obtain the complete cDNA coding sequences of various rhgp isoforms from the gills of A. testudineus, and to determine their mRNA and protein expression levels during 6 days of exposure to 100 mmol l-1 NH4Cl. The subcellular localization of Rhgp isoforms in the branchial epithelium was also examined in order to elucidate the type of ionocyte involved in active ammonia excretion. Four rhgp (rhag, rhbg, rhcg1 and rhcg2) had been identified from the gills of A. testudineus They had conserved amino acid residues for NH4+ binding, NH4+ deprotonation, channel gating and lining of the vestibules. Despite inwardly directed NH3 and NH4+ gradients, there were significant increases in the mRNA expression levels of the four branchial rhgp in A. testudineus at certain time points during 6 days of ammonia exposure, with significant increases in the protein abundances of Rhag and Rhcg2 on day 6. Immunofluorescence microscopy revealed a type of ammonia-inducible Na+/K+-ATPase α1c-immunoreactive ionocyte with apical Rhag and basolateral Rhcg2 in the gills of fish exposed to ammonia for 6 days. Hence, active ammonia excretion may involve NH4+ entering the ionocyte through the basolateral Rhcg2 and being excreted through the apical Rhag, driven by a transapical membrane electrical potential generated by the apical cystic fibrosis transmembrane conductance regulator Cl- channel, as suggested previously.

Aestivation Induces Changes in the mRNA Expression Levels and Protein Abundance of Two Isoforms of Urea Transporters in the Gills of the African Lungfish, Protopterus annectens.

The African lungfish, Protopterus annectens, is ammonotelic in water despite being ureogenic. When it aestivates in mucus cocoon on land, ammonia is detoxified to urea. During the maintenance phase of aestivation, urea accumulates in the body, which is subsequently excreted upon arousal. Urea excretion involves urea transporters (UT/Ut). This study aimed to clone and sequence the ut isoforms from the gills of P. annectens, and to test the hypothesis that the mRNA and/or protein expression levels of ut/Ut isoforms could vary in the gills of P. annectens during the induction, maintenance, and arousal phases of aestivation. Two isoforms of ut, ut-a2a and ut-a2b, were obtained from the gills of P. annectens. ut-a2a consisted of 1227 bp and coded for 408 amino acids with an estimated molecular mass of 44.7 kDa, while ut-a2b consisted of 1392 bp and coded for 464 amino acids with an estimated molecular mass of 51.2 kDa. Ut-a2a and Ut-a2b of P. annectens had a closer phylogenetic relationship with Ut/UT of tetrapods than Ut of fishes. While the mRNA expression pattern of ut-a2a and ut-a2b across various tissues of P. annectens differed, the transcript levels of ut-a2a and ut-a2b in the gills were comparable, indicating that they might be equally important for branchial urea excretion during the initial arousal phase of aestivation. During the maintenance phase of aestivation, the transcript level of ut-a2a increased significantly, but the protein abundance of Ut-a2a remained unchanged in the gills of P. annectens. This could be an adaptive feature to prepare for an increase in the production of Ut-a2a upon arousal. Indeed, arousal led to a significant increase in the branchial Ut-a2a protein abundance. Although the transcript level of ut-a2b remained unchanged, there were significant increases in the protein abundance of Ut-a2b in the gills of P. annectens throughout the three phases of aestivation. The increase in the protein abundance of Ut-a2b during the maintenance phase could also be an adaptive feature to prepare for efficient urea excretion when water becomes available.

Molecular Characterization of Aquaporin 1 and Aquaporin 3 from the Gills of the African Lungfish, Protopterus annectens, and Changes in Their Branchial mRNA Expression Levels and Protein Abundance during Three Phases of Aestivation.

African lungfishes can undergo long periods of aestivation on land during drought. During aestivation, lungfishes are confronted with desiccation and dehydration, and their gills become non-functional and covered with a thick layer of dried mucus. Aquaporins (Aqps) are a superfamily of integral membrane proteins which generally facilitate the permeation of water through plasma membranes. This study aimed to obtain the complete cDNA coding sequences of aqp1 and aqp3 from the gills of Protopterus annectens, and to determine their branchial mRNA and protein expression levels during the induction, maintenance and arousal phases of aestivation. Dendrogramic analyses of the deduced Aqp1 and Aqp3 amino acid sequences of P. annectens revealed their close relationships with those of Latimeria chalumnae and tetrapods. During the induction phase, there were significant decreases in the transcript levels of aqp1 and aqp3 in the gills of P. annectens, but the branchial Aqp1 and Aqp3 protein abundance remained unchanged. As changes in transcription might precede changes in translation, this could be regarded as an adaptive response to decrease the protein abundance of Aqp1 and Aqp3 in the subsequent maintenance phase of aestivation. As expected, the branchial transcript levels and protein abundance of aqp1/Aqp1 and aqp3/Aqp3 were significantly down-regulated during the maintenance phase, probably attributable to the shutdown of branchial functions and the cessation of volume regulation of branchial epithelial cells. Additionally, these changes could reduce the loss of water through branchial epithelial surfaces, supplementing the anti-desiccating property of the dried mucus. Upon arousal, it was essential for the lungfish to restore branchial functions. Indeed, the protein abundance of Aqp1 recovered partially, with complete recovery of mRNA expression level and protein abundance of Aqp3, in the gills of P. annectens after 3 days of arousal. These results provide insights into how P. annectens regulates branchial Aqp expression to cope with desiccation and rehydration during different phases of aestivation.

Increases in apoptosis, caspase activity and expression of p53 and bax, and the transition between two types of mitochondrion-rich cells, in the gills of the climbing perch, Anabas testudineus, during a progressive acclimation from freshwater to seawater.

This study aimed to test the hypothesis that branchial osmoregulatory acclimation involved increased apoptosis and replacement of mitochdonrion-rich cells (MRCs) in the climbing perch, Anabas testudineus, during a progressive acclimation from freshwater to seawater. A significant increase in branchial caspase-3/-7 activity was observed on day 4 (salinity 20), and an extensive TUNEL-positive apoptosis was detected on day 5 (salinity 25), indicating salinity-induced apoptosis had occurred. This was further supported by an up-regulation of branchial mRNA expression of p53, a key regulator of cell cycle arrest and apoptosis, between day 2 (salinity 10) and day 6 (seawater), and an increase in branchial p53 protein abundance on day 6. Seawater acclimation apparently activated both the extrinsic and intrinsic pathways, as reflected by significant increases in branchial caspase-8 and caspase-9 activities. The involvement of the intrinsic pathway was confirmed by the significant increase in branchial mRNA expression of bax between day 4 (salinity 20) and day 6 (seawater). Western blotting results revealed the presence of a freshwater Na(+)/K(+)-ATPase (Nka) α-isoform, Nka α1a, and a seawater isoform, Nka α1b, the protein abundance of which decreased and increased, respectively, during seawater acclimation. Immunofluorescence microscopy revealed the presence of two types of MRCs distinctly different in sizes, and confirmed that the reduction in Nka α1a expression, and the prominent increases in expression of Nka α1b, Na(+):K(+):2Cl(-) cotransporter 1, and cystic fibrosis transmembrane conductance regulator Cl(-) channel coincided with the salinity-induced apoptotic event. Since modulation of existing MRCs alone could not have led to extensive salinity-induced apoptosis, it is probable that some, if not all, freshwater-type MRCs could have been removed through increased apoptosis and subsequently replaced by seawater-type MRCs in the gills of A. testudineus during seawater acclimation.

Molecular characterization of branchial aquaporin 1aa and effects of seawater acclimation, emersion or ammonia exposure on its mRNA expression in the gills, gut, kidney and skin of the freshwater climbing perch, Anabas testudineus.

We obtained a full cDNA coding sequence of aquaporin 1aa (aqp1aa) from the gills of the freshwater climbing perch, Anabas testudineus, which had the highest expression in the gills and skin, suggesting an important role of Aqp1aa in these organs. Since seawater acclimation had no significant effects on the branchial and intestinal aqp1aa mRNA expression, and since the mRNA expression of aqp1aa in the gut was extremely low, it can be deduced that Aqp1aa, despite being a water channel, did not play a significant osmoregulatory role in A. testudineus. However, terrestrial exposure led to significant increases in the mRNA expression of aqp1aa in the gills and skin of A. testudineus. Since terrestrial exposure would lead to evaporative water loss, these results further support the proposition that Aqp1aa did not function predominantly for the permeation of water through the gills and skin. Rather, increased aqp1aa mRNA expression might be necessary to facilitate increased ammonia excretion during emersion, because A. testudineus is known to utilize amino acids as energy sources for locomotor activity with increased ammonia production on land. Furthermore, ammonia exposure resulted in significant decreases in mRNA expression of aqp1aa in the gills and skin of A. testudineus, presumably to reduce ammonia influx during ammonia loading. This corroborates previous reports on AQP1 being able to facilitate ammonia permeation. However, a molecular characterization of Aqp1aa from A. testudineus revealed that its intrinsic aquapore might not facilitate NH3 transport. Hence, ammonia probably permeated the central fifth pore of the Aqp1aa tetramer as suggested previously. Taken together, our results indicate that Aqp1aa might have a greater physiological role in ammonia excretion than in osmoregulation in A. testudineus.

Roles of three branchial Na(+)-K(+)-ATPase α-subunit isoforms in freshwater adaptation, seawater acclimation, and active ammonia excretion in Anabas testudineus.

Three Na(+)-K(+)-ATPase (nka) α-subunit isoforms, nka α1a, nka α1b, and nka α1c, were identified from gills of the freshwater climbing perch Anabas testudineus. The cDNA sequences of nka α1a and nka α1b consisted of 3,069 bp, coding for 1,023 amino acids, whereas nka α1c was shorter by 22 nucleotides at the 5' end. In freshwater, the quantity of nka α1c mRNA transcripts present in the gills was the highest followed by nka α1a and nka α1b that was almost undetectable. The mRNA expression of nka α1a was downregulated in the gills of fish acclimated to seawater, indicating that it could be involved in branchial Na(+) absorption in a hypoosmotic environment. By contrast, seawater acclimation led to an upregulation of the mRNA expression of nka α1b and to a lesser extent nka α1c, indicating that they could be essential for ion secretion in a hyperosmotic environment. More importantly, ammonia exposure led to a significant upregulation of the mRNA expression of nka α1c, which might be involved in active ammonia excretion. Both seawater acclimation and ammonia exposure led to significant increases in the protein abundance and changes in the kinetic properties of branchial Na(+)-K(+)-ATPase (Nka), but they involved two different types of Nka-immunoreactive cells. Since there was a decrease in the effectiveness of NH(4)(+) to substitute for K(+) to activate branchial Nka from fish exposed to ammonia, Nka probably functioned to remove excess Na(+) and to transport K(+) instead of NH(4)(+) into the cell to maintain intracellular Na(+) and K(+) homeostasis during active ammonia excretion.

Cystic fibrosis transmembrane conductance regulator in the gills of the climbing perch, Anabas testudineus, is involved in both hypoosmotic regulation during seawater acclimation and active ammonia excretion during ammonia exposure.

This study aimed to clone and sequence the cystic fibrosis transmembrane conductance regulator (cftr) from, and to determine the effects of seawater acclimation or exposure to 100 mmol l⁻¹ NH4Cl in freshwater on its mRNA and protein expressions in, the gills of Anabas testudineus. There were 4,530 bp coding for 1,510 amino acids in the cftr cDNA sequence from A. testudineus. The branchial mRNA expression of cftr in fish kept in freshwater was low (<50 copies of transcript per ng cDNA), but significant increases were observed in fish acclimated to seawater for 1 day (92-fold) or 6 days (219-fold). Branchial Cftr expression was detected in fish acclimated to seawater but not in the freshwater control, indicating that Cl⁻ excretion through the apical Cftr of the branchial epithelium was essential to seawater acclimation. More importantly, fish exposed to ammonia also exhibited a significant increase (12-fold) in branchial mRNA expression of cftr, with Cftr being expressed in a type of Na⁺/K⁺-ATPase-immunoreactive cells that was apparently different from the type involved in seawater acclimation. It is probable that Cl⁻ excretion through Cftr generated a favorable electrical potential across the apical membrane to drive the excretion of NH4⁺ against a concentration gradient through a yet to be determined transporter, but it led to a slight loss of endogenous Cl⁻. Since ammonia exposure also resulted in significant decreases in blood pH, [HCO3⁻] and [total CO2] in A. testudineus, it can be deduced that active NH4⁺ excretion could also be driven by the exit of HCO3⁻ through the apical Cftr. Furthermore, A. testudineus uniquely responded to ammonia exposure by increasing the ambient pH and decreasing the branchial bafilomycin-sensitive V-type H⁺-ATPase activity, which suggests that its gills might have low NH3 permeability.

Both seawater acclimation and environmental ammonia exposure lead to increases in mRNA expression and protein abundance of Na⁺:K⁺:2Cl⁻ cotransporter in the gills of the climbing perch, Anabas testudineus.

The freshwater climbing perch, Anabas testudineus, is an obligatory air-breathing teleost which can acclimate to seawater, survive long period of emersion, and actively excrete ammonia against high concentrations of environmental ammonia. This study aimed to clone and sequence the Na⁺:K⁺:2Cl⁻ cotransporter (nkcc) from the gills of A. testudineus, and to determine the effects of seawater acclimation or exposure to 100 mmol l⁻¹ NH4Cl in freshwater on its branchial mRNA expression. The complete coding cDNA sequence of nkcc from the gills of A. testudineus consisted of 3,495 bp, which was translated into a protein with 1,165 amino acid residues and an estimated molecular mass of 127.4 kDa. A phylogenetic analysis revealed that the translated Nkcc of A. testudineus was closer to fish Nkcc1a than to fish Nkcc1b or Nkcc2. After a progressive increase in salinity, there were significant increases in the mRNA expression and protein abundance of nkcc1a in the gills of fish acclimated to seawater as compared with that of the freshwater control. Hence, it can be concluded that similar to marine teleosts, Cl⁻ excretion through basolateral Nkcc1 of mitochondrion-rich cells (MRCs) was essential to seawater acclimation in A. testudineus. Exposure of A. testudineus to 100 mmol l⁻¹ NH4Cl for 1 or 6 days also resulted in significant increases in the mRNA expression of nkcc1a in the gills, indicating a functional role of Nkcc1a in active ammonia excretion. It is probable that NH4⁺ enter MRCs through basolateral Nkcc1a before being actively transported across the apical membrane. Since the operation of Nkcc1a would lead to an increase in the intracellular Na⁺ concentration, it can be deduced that an upregulation of basolateral Na⁺/K⁺-ATPase (Nka) activity would be necessary to compensate for the increased influx of Na⁺ into MRCs during active NH4⁺ excretion. This would imply that the main function of Nka in active NH4⁺ excretion is to maintain intracellular Na⁺ and K⁺ homeostasis instead of transporting NH4⁺ directly into MRCs as proposed previously. In conclusion, active salt secretion during seawater acclimation and active NH4⁺ excretion during exposure to ammonia in freshwater could involve similar transport mechanisms in the gills of A. testudineus.