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.

Ammonia transporter 2 as a molecular marker to elucidate the potentials of ammonia transport in phylotypes of Symbiodinium, Cladocopium and Durusdinium in the fluted giant clam, Tridacna squamosa.

Giant clams harbor coccoid Symbiodiniaceae dinoflagellates that are phototrophic. These dinoflagellates generally include multiple phylotypes (species) of Symbiodinium, Cladocopium, and Durusdinium in disparate proportions depending on the environmental conditions. The coccoid symbionts can share photosynthate with the clam host, which in return supply them with nutrients containing inorganic carbon, nitrogen and phosphorus. Symbionts can recycle nitrogen by absorbing and assimilating the endogenous ammonia produced by the host. This study aimed to use the transcript levels of ammonia transporter 2 (AMT2) in Symbiodinium (Symb-AMT2), Cladocopium (Clad-AMT2) and Durusdinium (Duru-AMT2) as molecular indicators to estimate the potential of ammonia transport in these three genera of Symbiodiniaceae dinoflagellates in different organs of the fluted giant clam, Tridacna squamosa, obtained from Vietnam. We also determined the transcript levels of form II ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcII) and nitrate transporter 2 (NRT2) in Symbiodinium (Symb-rbcII; Symb-NRT2), Cladocopium (Clad-rbcII; Clad-NRT2) and Durusdinium (Duru-rbcII; Duru-NRT2), in order to examine the potential of ammonia transport with reference to the potentials of phototrophy or NO3- uptake independent of the quantities and proportion of these Symbiodiniaceae phylotypes. Our results indicated for the first time that phylotypes of Symbiodinium and Cladocopium could have different potentials of ammonia transport, and that phylotypes of Symbiodinium might have higher potential of NO3- transport than ammonia transport. They also suggested that Symbiodiniaceae phylotypes residing in different organs of T. squamosa could have disparate potentials of ammonia transport, alluding to the functional diversity among phylotypes of coccoid Symbiodinium, Cladocopium, and Durusdinium.

Molecular characterization, immunofluorescent localization, and expression levels of two bicarbonate anion transporters in the whitish mantle of the giant clam, Tridacna squamosa, and the implications for light-enhanced shell formation.

Giant clams conduct light-enhanced shell formation, which requires the increased transport of Ca2+ and inorganic carbon (Ci) from the hemolymph through the shell-facing epithelium of the whitish inner mantle to the extrapallial fluid where CaCO3 deposition occurs. The major form of Ci in the hemolymph is HCO3-, but the mechanisms of HCO3- transport through the basolateral and apical membranes of the shell-facing epithelial cells remain unknown. This study aimed to clone from the inner mantle of Tridacna squamosa the complete coding cDNA sequences of electrogenic Na+-HCO3-cotransporter 1 homolog (NBCe1-like-b) and electrogenic Na+-HCO3-cotransporter 2 homolog (NBCe2-like). NBCe1-like-b comprised 3360 bp, encoding a 125.7 kDa protein with 1119 amino acids. NBCe1-like-b was slightly different from NBCe1-like-a of the ctenidium reported elsewhere, as it had a serine residue (Ser1025), which might undergo phosphorylation leading to the transport of Na+: HCO3- at a ratio of 1: 2 into the cell. NBCe1-like-b was localized at the basolateral membrane of the shell-facing epithelial cells, and its gene and protein expression levels increased significantly in the inner mantle during illumination, indicating a role in the light-enhanced uptake of HCO3- from the hemolymph. The sequence of NBCe2-like obtained from the inner mantle was identical to that reported previously for the outer mantle. In the inner mantle, NBCe2-like had an apical localization in the shell-facing epithelial cells, and its protein abundance was upregulated during illumination. Hence, NBCe2-like might take part in the light-enhanced transport of HCO3- through the apical membrane of these cells into the extrapallial fluid.

The colorful mantle of the giant clam Tridacna squamosa expresses a homolog of electrogenic sodium: Bicarbonate cotransporter 2 that mediates the supply of inorganic carbon to photosynthesizing symbionts.

Giant clams live in symbiosis with phototrophic dinoflagellates, which reside extracellularly inside zooxanthellal tubules located mainly in the colourful and extensible outer mantle. As symbiotic dinoflagellates have no access to the ambient seawater, they need to obtain inorganic carbon (Ci) from the host for photosynthesis during illumination. The outer mantle has a host-mediated and light-dependent carbon-concentrating mechanism to augment the supply of Ci to the symbionts during illumination. Iridocytes can increase the secretion of H+ through vacuolar H+-ATPase to dehydrate HCO3- present in the hemolymph to CO2. CO2 can permeate the basolateral membrane of the epithelial cells of the zooxanthellal tubules, and rehydrated back to HCO3- in the cytoplasm catalysed by carbonic anhydrase 2. This study aimed to elucidate the molecular mechanism involved in the transport of HCO3- across the apical membrane of these epithelial cells into the luminal fluid surrounding the symbionts. We had obtained the complete cDNA coding sequence of a homolog of electrogenic Na+-HCO3- cotransporter 2 (NBCe2-like gene) from the outer mantle of the fluted giant clam, Tridacna squamosa. NBCe2-like gene comprised 3,399 bp, encoding a protein of 1,132 amino acids of 127.3 kDa. NBCe2-like protein had an apical localization in the epithelial cells of zooxanthellal tubules, denoting that it could transport HCO3- between the epithelial cells and the luminal fluid. Furthermore, illumination augmented the transcript level and protein abundance of NBCe2-like gene/NBCe2-like protein in the outer mantle, indicating that it could mediate the increased transport of HCO3- into the luminal fluid to support photosynthesis in the symbionts.

Basolateral Na+/Ca2+ exchanger 1 and Na+/K+-ATPase, which display light-enhanced gene and protein expression levels, could be involved in the absorption of exogenous Ca2+ through the ctenidium of the giant clam, Tridacna squamosa.

Giant clams perform light-enhanced shell formation (calcification) and therefore need to increase the uptake of exogenous Ca2+ during illumination. The ctenidium of the fluted giant clam, Tridacna squamosa, is involved in light-enhanced Ca2+ uptake. It expresses the pore-forming voltage-gated calcium channel (VGCC) subunit alpha 1 (CACNA1) in the apical membrane of the epithelial cells, and the protein expression level of CACNA1 is upregulated in the ctenidium during illumination. This study aimed to elucidate the mechanism involved in the transport of the absorbed Ca2+ across the basolateral membrane of the ctenidial epithelial cells into the hemolymph. We obtained a homolog of Na+/Ca2+exchanger 1 (NCX1-like) from the ctenidium of T. squamosa, which comprised 2418 bp, encoding a protein of 806 amino acids (88.9 kDa). NCX1-like had a basolateral localization in the epithelial cells of the ctenidial filaments and tertiary water channels. Illumination resulted in significant increases in the transcript and protein levels of NCX1-like/NCX1-like in the ctenidium. Hence, NCX1-like could operate in conjunction with VGCC to increase the transport of Ca2+ from the ambient seawater into the hemolymph during illumination. Illumination also resulted in the upregulation of the gene and protein expression levels of Na+/K+-ATPase (NKA) α-subunit (NKAα/NKAα) in the ctenidium of T. squamosa. As light-enhanced extrusion of Ca2+ into the hemolymph through NCX1-like would lead to a greater influx of extracellular Na+, the increased expression of the basolateral NKA was required to augment the capacity of intracellular Na+ homeostasis.

Using glutamine synthetase 1 to evaluate the symbionts' potential of ammonia assimilation and their responses to illumination in five organs of the giant clam, Tridacna squamosa.

Nitrogen-deficient symbiotic dinoflagellates (zooxanthellae) living inside the fluted giant clam, Tridacna squamosa, need to obtain nitrogen from the host. Glutamine synthetase 1 (GS1) is a cytosolic enzyme that assimilates ammonia into glutamine. We determined the transcript levels of zooxanthellal GS1 (Zoox-GS1), which represented comprehensively GS1 transcripts of Symbiodinium, Cladocopium and Durusdinium, in five organs of T. squamosa. The outer mantle had significantly higher transcript level of Zoox-GS1 than the inner mantle, foot muscle, hepatopancreas and ctenidium, but the transcript ratios of Zoox-GS1 to zooxanthellal form II ribulose-1,5-bisphosphate carboxylase/oxygenase (Zoox-rbcII), which represented the potential of ammonia assimilation relative to the phototrophic potential, were comparable among these five organs. Based on transcript ratios of Zoox-GS1 to zooxanthellal Urease (Zoox-URE), the outer mantle had the highest potential of urea degradation relative to ammonia assimilation among the five organs, probably because urea degradation could furnish CO2 and NH3 for photosynthesis and amino acid synthesis, respectively, in the symbionts therein. The protein abundance of Zoox-GS1 was upregulated in the outer mantle and the inner mantle during illumination. Zoox-GS1 could catalyze light-enhanced glutamine formation using ammonia absorbed from the host or ammonia released through urea degradation in the cytoplasm. The glutamine produced could be used to synthesize other nitrogenous compounds, including amino acids in the cytoplasm or in the plastid of the dinoflagellates. Some of the amino acids synthesized by the symbionts in the inner mantle and foot muscle could be donated to the host to support shell organic matrix formation and muscle production, respectively.

Effects of seawater acclimation on two Na+/K+-ATPase α-subunit isoforms in the gills of the marble goby, Oxyeleotris marmorata.

The marble goby, Oxyeleotris marmorata, is a freshwater teleost, but can acclimate progressively to survive in seawater (salinity 30). As an obligatory air-breather, it can also survive long periods of emersion. Two isoforms of Na+/K+-ATPase (nka) α-subunit, nkaα1 and nkaα3, but not nkaα2, had been cloned from the gills of O. marmorata. The cDNA sequence of nkaα1 consisted of 3069 nucleotides, coding for 1023 amino acids (112.5 kDa), whereas nkaα3 consisted of 2976 nucleotides, coding for 992 amino acids (109.5 kDa). As only one form of branchial Nkaα1 was identified using molecular cloning in this study, O. marmorata lacks specific freshwater- and seawater-type Nkaα isoforms as demonstrated by some other euryhaline fish species. The nkaα1 transcript level was about 2.5-fold higher than that of nkaα3 in the gills of freshwater O. marmorata. During exposure to seawater, the branchial transcript level of nkaα1 increased significantly on day 1 (~3.3-fold) and day 6 (~2.6-fold). By contrast, the branchial transcript level of nkaα3 increased significantly on day 1 (~2.6-fold), but not on day 6, of seawater exposure. Six days of exposure to seawater also led to significant increases in protein abundances of Nkaα1 (~6.9-fold) and Nkaα3 (~2.8-fold) in the gills of O. marmorata. Hence, the mRNA and protein expressions of both nkaα1/Nkaα1 and nkaα3/Nkaα3 were up-regulated in O. marmorata during seawater acclimation. This could explain why Vmax increases but Km for Na+ and K+ remain unchanged in Nka extracted from the gills of O. marmorata acclimated to seawater as reported previously.

Illumination enhances the protein abundance of sarcoplasmic reticulum Ca2+-ATPases-like transporter in the ctenidium and whitish inner mantle of the giant clam, Tridacna squamosa, to augment exogenous Ca2+ uptake and shell formation, respectively.

The fluted giant clam, Tridacna squamosa, can perform light-enhanced shell formation, aided by its symbiotic dinoflagellates (Symbiodinium, Cladocopium, Durusdinium), which are able to donate organic nutrients to the host. During light-enhanced shell formation, increased Ca2+ transport from the hemolymph through the shell-facing epithelium of the inner mantle to the extrapallial fluid, where calcification occurs, is necessary. Additionally, there must be increased absorption of exogenous Ca2+ from the surrounding seawater, across the epithelial cells of the ctenidium (gill) into the hemolymph, to supply sufficient Ca2+ for light-enhanced shell formation. When Ca2+ moves across these epithelial cells, the low intracellular Ca2+ concentration must be maintained. Sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) regulates the intracellular Ca2+ concentration by pumping Ca2+ into the sarcoplasmic/endoplasmic reticulum (SR/ER) and Golgi apparatus. Indeed, the ctenidium and inner mantle of T. squamosa, expressed a homolog of SERCA (SERCA-like transporter) that consists of 3009 bp, encoding 1002 amino acids of 110.6 kDa. SERCA-like-immunolabeling was non-uniform in the cytoplasm of epithelial cells of ctenidial filaments, and that of the shell-facing epithelial cells of the inner mantle. Importantly, the protein abundance of SERCA-like increased significantly in the ctenidium and the inner mantle of T. squamosa after 12 h and 6 h, respectively, of light exposure. This would increase the capacity of pumping Ca2+ into the endoplasmic reticulum and avert a possible surge in the cytosolic Ca2+ concentration in epithelial cells of the ctenidial filaments during light-enhanced Ca2+ absorption, and in cells of the shell-facing epithelium of the inner mantle during light-enhanced shell formation.

The fluted giant clam (Tridacna squamosa) increases the protein abundance of the host's copper-zinc superoxide dismutase in the colorful outer mantle, but not the whitish inner mantle, during light exposure.

The colorful outer mantle of giant clams contains abundance of symbiotic dinoflagellates (zooxanthellae) and iridocytes, and has direct exposure to light. In light, photosynthesizing dinoflagellates produce O2, and the host cells in the outer mantle would be confronted with hyperoxia-related oxidative stress. In comparison, the whitish inner mantle contains few symbiotic dinoflagellates and no iridocytes. It is involved in shell formation, and is shaded from light. CuZnSOD is a cytosolic enzyme that scavenges intracellular O2-. We had obtained from the outer mantle of the fluted giant clam, Tridacna squamosa, the complete cDNA coding sequence of a host-derived copper zinc superoxide dismutase (CuZnSOD), which comprised 462 bp and encoded for 154 amino acids with a calculated MW of 15.6 kDa. CuZnSOD was expressed strongly in the outer mantle, ctenidium, hepatopancreas and kidney. The transcript level of CuZnSOD remained unchanged in the outer mantle during light exposure, but the protein abundance of CuZnSOD increased ~3-fold after exposure to light for 6 or 12 h. By contrast, 12 h of light exposure had no significant effects on the gene and protein expression levels of CuZnSOD/CuZnSOD in the inner mantle. Hence, the increased expression of CuZnSOD in the outer mantle of T. squamosa was probably a host's response to ameliorate oxidative stress related to photosynthesis in the symbionts, and not simply due to increased metabolic rate in the host cells. Evidently, the host clam must possess light- or O2-responsive anti-oxidative defenses in order to align with the light-dependent photosynthetic activity of its symbionts.

Calcium absorption in the fluted giant clam, Tridacna squamosa, may involve a homolog of voltage-gated calcium channel subunit α1 (CACNA1) that has an apical localization and displays light-enhanced protein expression in the ctenidium.

In light, giant clams can increase rates of shell formation and growth due to their symbiotic relationship with phototrophic zooxanthellae residing extracellularly in a tubular system. Light-enhanced shell formation necessitates increase in the uptake of Ca2+ from the ambient seawater and the supply of Ca2+ through the hemolymph to the extrapallial fluid, where calcification occurs. In this study, the complete coding cDNA sequence of a homolog of voltage-gated calcium channel subunit α1 (CACNA1), which is the pore-forming subunit of L-type voltage-gated calcium channels (VGCCs), was obtained from the ctenidium (gill) of the giant clam, Tridacna squamosa. It consisted of 6081 bp and encoded a 223 kDa polypeptide with 2027 amino acids, which was characterized as the α1D subunit of L-type VGCC. Immunofluorescence microscopy demonstrated that CACNA1 had an apical localization in the epithelial cells of filaments and tertiary water channels in the ctenidium of T. squamosa, indicating that it was well positioned to absorb exogenous Ca2+. Additionally, there was a significant increase in the protein abundance of CACNA1 in the ctenidium of individuals exposed to light for 12 h. With more pore-forming CACNA1, there could be an increase in the permeation of exogenous Ca2+ into the ctenidial epithelial cells through the apical membrane. Taken together, these results denote that VGCC could augment exogenous Ca2+ uptake through the ctenidium to support light-enhanced shell formation in T. squamosa. Furthermore, they support the proposition that light-enhanced phenomena in giant clams are attributable primarily to the direct responses of the host's transporters/enzymes to light, in alignment with the symbionts' phototrophic activity.

Increased apical sodium-dependent glucose transporter abundance in the ctenidium of the giant clam Tridacna squamosa upon illumination.

Giant clams contain phototrophic zooxanthellae, and live in nutrient-deficient tropical waters where light is available. We obtained the complete cDNA coding sequence of a homolog of mammalian sodium/glucose cotransporter 1 (SGLT1) - SGLT1-like - from the ctenidium of the fluted giant clam, Tridacna squamosaSGLT1-like had a host origin and was expressed predominantly in the ctenidium. Molecular characterizations reveal that SGLT1-like of T. squamosa could transport urea, in addition to glucose, as other SGLT1s do. It has an apical localization in the epithelium of ctenidial filaments and water channels, and the apical anti-SGLT1-like immunofluorescence was stronger in individuals exposed to light than to darkness. Furthermore, the protein abundance of SGLT1-like increased significantly in the ctenidium of individuals exposed to light for 12 h, although the SGLT1-like transcript level remained unchanged. As expected, T. squamosa could perform light-enhanced glucose absorption, which was impeded by exogenous urea. These results denote the close relationships between light-enhanced glucose absorption and light-enhanced SGLT1-like expression in the ctenidium of T. squamosa Although glucose absorption could be trivial compared with the donation of photosynthates from zooxanthellae in symbiotic adults, SGLT1-like might be essential for the survival of aposymbiotic larvae, leading to its retention in the symbiotic stage. A priori, glucose uptake through SGLT1-like might be augmented by the surface microbiome through nutrient cycling, and the absorbed glucose could partially fulfill the metabolic needs of the ctenidial cells. Additionally, SGLT1-like could partake in urea absorption, as T. squamosa is known to conduct light-enhanced urea uptake to benefit the nitrogen-deficient zooxanthellae.

Molecular characterization, cellular localization, and light-enhanced expression of Beta-Na+/H+ Exchanger-like in the whitish inner mantle of the giant clam, Tridacna squamosa, denote its role in light-enhanced shell formation.

The fluted giant clam, Tridacna squamosa, lives in symbiosis with photosynthetic zooxanthellae, and can engage in light-enhanced growth and shell formation. Light-enhanced shell formation necessitates the elimination of excess H+ from the extrapallial fluid adjacent to the shell. This study aimed to clone Na+/H+Exchanger (NHE) from the whitish inner mantle adjacent to the extrapallial fluid of T. squamosa, to determine its cellular and subcellular localization, and to evaluate the effect of light exposure on its mRNA expression level and protein abundance therein. The complete coding cDNA sequence of NHE obtained was identified as a homolog of beta NHE (ßNHE-like). It consisted of 2925 bp, encoding for a polypeptide of 974 amino acids and 107.1 kDa, and was expressed predominantly in the inner mantle. There, ßNHE-like was localized in the apical membrane of the seawater-facing epithelium by immunofluorescence microscopy. After exposure to light for 12 h, the seawater-facing epithelium of the inner mantle displayed consistently stronger immunostaining than that of the control exposed to 12 h of darkness. Western blotting confirmed that light exposure significantly enhanced the protein abundance of ßNHE-like in the inner mantle. These results denote that some of the excess H+ generated during light-enhanced shell formation can be excreted through the light-dependent ßNHE-like of the seawater-facing epithelium to minimize the impact on the whole-body pH. Importantly, the excreted H+ could dehydrate exogenous HCO3-, and facilitate the absorption of inorganic carbon through the seawater-facing epithelium dedicated for light-enhanced shell formation due to its close proximity with the shell-facing epithelium. NUCLEOTIDE SYMBOL COMBINATIONS: Pairs: R = A/G; W = A/T; Y = C/T. Triples: D = A/G/T.

Light-enhanced expression of Carbonic Anhydrase 4-like supports shell formation in the fluted giant clam Tridacna squamosa.

Giant clams represent symbiotic associations between a host clam and its extracellular zooxanthellae. They are able to grow in nutrient-deficient tropical marine environments and conduct light-enhanced shell formation (calcification) with the aid of photosynthates donated by the symbiotic zooxanthellae. In light, there is a high demand for inorganic carbon (Ci) to support photosynthesis in the symbionts and light-enhanced calcification in the host. In this study, we cloned and characterized a host Carbonic Anhydrase 4 homolog (CA4-like) from the whitish inner mantle of the giant clam Tridacna squamosa. The full cDNA coding sequence of CA4-like consisted of 1002 bp, encoding for 334 amino acids of 38.5 kDa. The host CA4-like was phenogramically distinct from algal CAs. The transcript level of CA4-like in the inner mantle was ~3-fold higher than those in the colorful outer mantle and the ctenidium. In the inner mantle, CA4-like was immunolocalized in the apical membrane of the seawater-facing epithelial cells, but absent from the shell-facing epithelium. Hence, CA4-like was positioned to catalyze the conversion of HCO3- to CO2 in the ambient seawater which would facilitate CO2 uptake. The absorbed CO2 could be converted back to HCO3- by the cytoplasmic CA2-like. As the protein abundance of CA4-like increased in the inner mantle after 6 or 12 h of light exposure, there could be an augmentation of the total CA4-like activity to increase Ci uptake in light. It is plausible that the absorbed Ci was allocated preferentially for shell formation due to the close proximity of the seawater-facing epithelium to the shell-facing epithelium in the inner mantle that contains only few zooxanthellae.

The Non-ureogenic Stinging Catfish, Heteropneustes fossilis, Actively Excretes Ammonia With the Help of Na+/K+-ATPase When Exposed to Environmental Ammonia.

The stinging catfish, Heteropneustes fossilis, can tolerate high concentrations of environmental ammonia. Previously, it was regarded as ureogenic, having a functional ornithine-urea cycle (OUC) that could be up-regulated during ammonia-loading. However, contradictory results indicated that increased urea synthesis and switching to ureotelism could not explain its high ammonia tolerance. Hence, we re-examined the effects of exposure to 30 mmol l-1 NH4Cl on its ammonia and urea excretion rates, and its tissue ammonia and urea concentrations. Our results confirmed that H. fossilis did not increase urea excretion or accumulation during 6 days of ammonia exposure, and lacked detectable carbamoyl phosphate synthetase I or III activity in its liver. However, we discovered that it could actively excrete ammonia during exposure to 8 mmol l-1 NH4Cl. As active ammonia excretion is known to involve Na+/K+-ATPase (Nka) indirectly in several ammonia-tolerant fishes, we also cloned various nkaα-subunit isoforms from the gills of H. fossilis, and determined the effects of ammonia exposure on their branchial transcripts levels and protein abundances. Results obtained revealed the presence of five nkaα-subunit isoforms, with nkaα1b having the highest transcript level. Exposure to 30 mmol l-1 NH4Cl led to significant increases in the transcript levels of nkaα1b (on day 6) and nkaα1c1 (on day 1 and 3) as compared with the control. In addition, the protein abundances of Nkaα1c1, Nkaα1c2, and total NKAα increased significantly on day 6. Therefore, the high environmental ammonia tolerance of H. fossilis is attributable partly to its ability to actively excrete ammonia with the aid of Nka.

Air-breathing and excretory nitrogen metabolism in fishes.

During water-land transition, ancient fishes acquired the ability to breathe air, but air-breathing engendered problems in nitrogenous waste excretion. Nitrogen is a fundamental component of amino acids, proteins, and nucleic acids, and the degradation of these nitrogen-containing compounds releases ammonia. Ammonia is toxic and must be removed. Fishes in water excrete ammonia as the major nitrogenous waste through gills, but gills of air-breathing fishes are modified for air-breathing or largely replaced by air-breathing organs. Notably, fishes emerged from water can no longer excrete ammonia effectively because of a lack of water to flush the gills. Hence, ancient fishes that participated in water-land transition must have developed means to deal with ammonia toxicity. Extant air-breathing fishes, particularly amphibious ones, can serve as models to examine adaptations which might have facilitated the emergence of ancient fishes from water. Some of these fishes can actively emerge from water and display complex behaviors on land, while a few can burrow into mud and survive for years during drought. Many of them are equipped with mechanisms to ameliorate ammonia toxicity during emersion. In this review, the mechanisms adopted by air-breathing fishes to deal with ammonia toxicity during emersion were organized into seven disparate strategies. In addition, eight extant air-breathing fishes with distinctive terrestrial behaviors and peculiar natural habitats were selected to describe in detail how these seven strategies could be adopted in disparate combinations to ameliorate ammonia toxicity during emersion.

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.

Molecular Characterization of a Dual Domain Carbonic Anhydrase From the Ctenidium of the Giant Clam, Tridacna squamosa, and Its Expression Levels After Light Exposure, Cellular Localization, and Possible Role in the Uptake of Exogenous Inorganic Carbon.

A Dual-Domain Carbonic Anhydrase (DDCA) had been sequenced and characterized from the ctenidia (gills) of the giant clam, Tridacna squamosa, which lives in symbiosis with zooxanthellae. DDCA was expressed predominantly in the ctenidium. The complete cDNA coding sequence of DDCA from T. squamosa comprised 1,803 bp, encoding a protein of 601 amino acids and 66.7 kDa. The deduced DDCA sequence contained two distinct α-CA domains, each with a specific catalytic site. It had a high sequence similarity with tgCA from Tridacna gigas. In T. squamosa, the DDCA was localized apically in certain epithelial cells near the base of the ctenidial filament and the epithelial cells surrounding the tertiary water channels. Due to the presence of two transmembrane regions in the DDCA, one of the Zn2+-containing active sites could be located externally and the other one inside the cell. These results denote that the ctenidial DDCA was positioned to dehydrate [Formula: see text] to CO2 in seawater, and to hydrate the CO2 that had permeated the apical membrane back to [Formula: see text] in the cytoplasm. During insolation, the host clam needs to increase the uptake of inorganic carbon from the ambient seawater to benefit the symbiotic zooxanthellae; only then, can the symbionts conduct photosynthesis and share the photosynthates with the host. Indeed, the transcript and protein levels of DDCA/DDCA in the ctenidium of T. squamosa increased significantly after 6 and 12 h of exposure to light, respectively, denoting that DDCA could participate in the light-enhanced uptake and assimilation of exogenous inorganic carbon.

The ctenidium of the giant clam, Tridacna squamosa, expresses an ammonium transporter 1 that displays light-suppressed gene and protein expression and may be involved in ammonia excretion.

Ammonium transporters (AMTs) can participate in ammonia uptake or excretion across the plasma membrane of prokaryotic, plant and invertebrate cells. The giant clam, Tridacna squamosa, harbors nitrogen-deficient symbiotic zooxanthellae, and normally conducts light-enhanced ammonia absorption to benefit the symbionts. Nonetheless, it can excrete ammonia when there is a supply of exogenous nitrogen or exposed to continuous darkness. This study aimed to elucidate the role of AMT1 in the ctenidium of T. squamosa by cloning and characterizing the AMT1/AMT1, determining its subcellular localization, and examining changes in its transcript and protein expression levels in response to light exposure. The cDNA coding sequence of AMT1 from T. squamosa consisted of 1527 bp and encoded 508 amino acids of 54.6 kDa. AMT1-immunofluorescence was detected mainly at the apical epithelium of ctenidial filaments, and it decreased significantly after 12 h of exposure to light. By contrast, the epithelial cells surrounding the tertiary water channels in the ctentidium, which are known to exhibit light-enhanced glutamine synthetase expression and take part in the assimilation of exogenous ammonia in light, did not display any AMT1-immunolabelling. Furthermore, the transcript level and protein abundance of ctenidial AMT1/AMT1 decreased significantly at the 6th and 12th h of light exposure. Taken together, these results indicate that AMT1 might participate in ammonia excretion instead of ammonia absorption and assimilation in T. squamosa. It is probable that the expression levels of AMT1/AMT1 need to be down-regulated during light exposure to achieve light-enhanced ammonia uptake.

Molecular characterization, light-dependent expression, and cellular localization of a host vacuolar-type H+-ATPase (VHA) subunit A in the giant clam, Tridacna squamosa, indicate the involvement of the host VHA in the uptake of inorganic carbon and its supply to the symbiotic zooxanthellae.

The giant clam, Tridacna squamosa, represents a clam-zooxanthellae association. In light, the host clam and the symbiotic zooxanthellae conduct light-enhanced calcification and photosynthesis, respectively. We had cloned the cDNA coding sequence of a Vacuolar-type Proton ATPase (VHA) subunit A, ATP6V1A, from T. squamosa, whereby the VHA is an electrogenic transporter that actively 'pumps' H+ out of the cell. The ATP6V1A of T. squamosa comprised 1866 bp, encoding a protein of 622 amino acids and 69.9 kDa, and had a host-origin. Its gene expression was strong in the ctenidium and the colorful outer mantle, but weak in the whitish inner mantle, corroborating a previous proposition that VHA might have a trivial role in light-enhanced calcification. Light exposure led to significant increases in the gene and protein expression levels of ATP6V1A/ATP6V1A in the ctenidium and the outer mantle. In the ctenidium, the ATP6V1A was localized in the apical epithelia of the filaments and tertiary water channels, indicating that the VHA could participate in the increased excretion of H+ produced during light-enhanced calcification. Additionally, the excreted H+ would augment HCO3- dehydration in the external medium and facilitate the uptake of CO2 by the ctenidium during insolation. In the outer mantle, the ATP6V1A was detected in intracellular vesicles in a type of cells, presumably iridocytes, surrounding the zooxanthellal tubules, and in the apical epithelium of zooxanthellal tubules. Hence, the host VHA could participate in the transfer of inorganic carbon from the hemolymph to the luminal fluid of the tubules by increasing the supply of H+ for the dehydration of HCO3- to CO2 during insolation to benefit the photosynthesizing zooxanthellae.

Molecular characterization of a novel algal glutamine synthetase (GS) and an algal glutamate synthase (GOGAT) from the colorful outer mantle of the giant clam, Tridacna squamosa, and the putative GS-GOGAT cycle in its symbiotic zooxanthellae.

Giant clams harbor symbiotic zooxanthellae (Symbiodinium), which are nitrogen-deficient, mainly in the fleshy and colorful outer mantle. This study aimed to sequence and characterize the algal Glutamine Synthetase (GS) and Glutamate Synthase (GLT), which constitute the glutamate synthase cycle (or GS-GOGAT cycle, whereby GOGAT is the protein acronym of GLT) of nitrogen assimilation, from the outer mantle of the fluted giant clam, Tridacna squamosa. We had identified a novel GS-like cDNA coding sequence of 2325 bp, and named it as T. squamosa Symbiodinium GS1 (TSSGS1). The deduced TSSGS1 sequence had 774 amino acids with a molecular mass of 85 kDa, and displayed the characteristics of GS1 and Nucleotide Diphosphate Kinase. The cDNA coding sequence of the algal GLT, named as T. squamosa Symbiodinium GLT (TSSGLT), comprised 6399 bp, encoding a protein of 2133 amino acids and 232.4 kDa. The zooxanthellal origin of TSSGS1 and TSSGOGAT was confirmed by sequence comparison and phylogenetic analyses. Indeed, TSSGS1 and TSSGOGAT were expressed predominately in the outer mantle, which contained the majority of the zooxanthellae. Immunofluorescence microscopy confirmed the expression of TSSGS1 and TSSGOGAT in the cytoplasm and the plastids, respectively, of the zooxanthellae in the outer mantle. It can be concluded that the symbiotic zooxanthellae of T. squamosa possesses a glutamate synthase (TSSGS1-TSSGOGAT) cycle that can assimilate endogenous ammonia produced by the host clam into glutamate, which can act as a substrate for amino acid syntheses. Thus, our results provide insights into why intact giant clam-zooxanthellae associations do not excrete ammonia under normal circumstances.