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.

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.

Light exposure enhances urea absorption in the fluted giant clam, Tridacna squamosa, and up-regulates the protein abundance of a light-dependent urea active transporter, DUR3-like, in its ctenidium.

Giant clams live in nutrient-poor reef waters of the Indo-Pacific and rely on symbiotic dinoflagellates (Symbiodinium spp., also known as zooxanthellae) for nutrients. As the symbionts are nitrogen deficient, the host clam has to absorb exogenous nitrogen and supply it to them. This study aimed to demonstrate light-enhanced urea absorption in the fluted giant clam, Tridacna squamosa, and to clone and characterize the urea active transporter DUR3-like from its ctenidium (gill). The results indicate that T. squamosa absorbs exogenous urea, and the rate of urea uptake in the light was significantly higher than that in darkness. The DUR3-like coding sequence obtained from its ctenidium comprised 2346 bp, encoding a protein of 782 amino acids and 87.0 kDa. DUR3-like was expressed strongly in the ctenidium, outer mantle and kidney. Twelve hours of exposure to light had no significant effect on the transcript level of ctenidial DUR3-like However, between 3 and 12 h of light exposure, DUR3-like protein abundance increased progressively in the ctenidium, and became significantly greater than that in the control at 12 h. DUR3-like had an apical localization in the epithelia of the ctenidial filaments and tertiary water channels. Taken together, these results indicate that DUR3-like might participate in light-enhanced urea absorption in the ctenidium of T. squamosa When made available to the symbiotic zooxanthellae that are known to possess urease, the absorbed urea can be metabolized to NH3 and CO2 to support amino acid synthesis and photosynthesis, respectively, during insolation.

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.

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.

Carbonic anhydrase 2-like in the giant clam, Tridacna squamosa: characterization, localization, response to light, and possible role in the transport of inorganic carbon from the host to its symbionts.

The fluted giant clam, Tridacna squamosa, lives in symbiosis with zooxanthellae which reside extracellularly inside a tubular system. Zooxanthellae fix inorganic carbon (Ci) during insolation and donate photosynthate to the host. Carbonic anhydrases catalyze the interconversion of CO2 and HCO3-, of which carbonic anhydrase 2 (CA2) is the most ubiquitous and involved in many biological processes. This study aimed to clone a CA2 homolog (CA2-like) from the fleshy and colorful outer mantle as well as the thin and whitish inner mantle of T. squamosa, to determine its cellular and subcellular localization, and to examine the effects of light exposure on its gene and protein expression levels. The cDNA coding sequence of CA2-like from T. squamosa comprised 789 bp, encoding 263 amino acids with an estimated molecular mass of 29.6 kDa. A phenogramic analysis of the deduced CA2-like sequence denoted an animal origin. CA2-like was not detectable in the shell-facing epithelium of the inner mantle adjacent to the extrapallial fluid. Hence, CA2-like is unlikely to participate directly in light-enhanced calcification. By contrast, the outer mantle, which contains the highest density of tertiary tubules and zooxanthellae, displayed high level of CA2-like expression, and CA2-like was localized to the tubule epithelial cells. More importantly, exposure to light induced significant increases in the protein abundance of CA2-like in the outer mantle. Hence, CA2-like could probably take part in the increased supply of inorganic carbon (Ci) from the host clam to the symbiotic zooxanthellae when the latter conduct photosynthesis to fix Ci during light exposure.

The Whitish Inner Mantle of the Giant Clam, Tridacna squamosa, Expresses an Apical Plasma Membrane Ca2+-ATPase (PMCA) Which Displays Light-Dependent Gene and Protein Expressions.

Giant clams live in symbiosis with extracellular zooxanthellae and display high rates of growth and shell formation (calcification) in light. Light-enhanced calcification requires an increase in the supply of Ca2+ to, and simultaneously an augmented removal of H+ from, the extrapallial fluid where shell formation occurs. We have obtained the complete coding cDNA sequence of Plasma Membrane Ca2+-ATPase (PMCA) from the thin and whitish inner mantle, which is in touch with the extrapallial fluid, of the giant clam Tridacna squamosa. The deduced PMCA sequence consisted of an apical targeting element. Immunofluorescence microscopy confirmed that PMCA had an apical localization in the shell-facing epithelium of the inner mantle, whereby it can actively secrete Ca2+ in exchange for H+. More importantly, the apical PMCA-immunofluorescence of the shell-facing epithelium of the inner mantle increased significantly after 12 h of exposure to light. The transcript and protein levels of PMCA/PMCA also increased significantly in the inner mantle after 6 or 12 h of light exposure. These results offer insights into a light-dependable mechanism of shell formation in T. squamosa and a novel explanation of light-enhanced calcification in general. As the inner mantle normally lacks light sensitive pigments, our results support a previous proposition that symbiotic zooxanthellae, particularly those in the colorful and extensible outer mantle, may act as light-sensing elements for the host clam.

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.

The inner mantle of the giant clam, Tridacna squamosa, expresses a basolateral Na+/K+-ATPase α-subunit, which displays light-dependent gene and protein expression along the shell-facing epithelium.

Na+/K+-ATPase (NKA) is essential for maintaining the Na+ and K+ gradients, and supporting the secondary active transport of certain ions/molecules, across the plasma membrane of animal cells. This study aimed to clone the NKA α-subunit (NKAα) from the inner mantle adjacent to the extrapallial fluid of Tridacna squamosa, to determine its subcellular localization, and to examine the effects of light exposure on its transcript level and protein abundance. The cDNA coding sequence of NKAα from T. squamosa comprised 3105 bp, encoding 1034 amino acids with an estimated molecular mass of 114 kDa. NKAα had a basolateral localization along the shell-facing epithelium of the inner mantle. Exposure to 12 h of light led to a significantly stronger basolateral NKAα-immunofluorescence at the shell-facing epithelium, indicating that NKA might play a role in light-enhanced calcification in T. squamosa. After 3 h of light exposure, the transcript level of NKAα decreased transiently in the inner mantle, but returned to the control level thereafter. In comparison, the protein abundance of NKAα remained unchanged at hour 3, but became significantly higher than the control after 12 h of light exposure. Hence, the expression of NKAα in the inner mantle of T. squamosa was light-dependent. It is probable that a higher expression level of NKA was needed in the shell-facing epithelial cells of the inner mantle to cope with a rise in Na+ influx, possibly caused by increases in activities of some Na+-dependent ion transporters/channels involved in light-enhanced calcification.

Light-dependent expression of a Na+/H+ exchanger 3-like transporter in the ctenidium of the giant clam, Tridacna squamosa, can be related to increased H+ excretion during light-enhanced calcification.

Na+/H+ exchangers (NHEs) regulate intracellular pH and ionic balance by mediating H+ efflux in exchange for Na+ uptake in a 1:1 stoichiometry. This study aimed to obtain from the ctenidium of the giant clam Tridacna squamosa (TS) the complete cDNA sequence of a NHE3-like transporter (TSNHE3), and to determine the effect of light exposure on its mRNA expression level and protein abundance therein. The coding sequence of TSNHE3 comprised 2886 bp, encoding 961 amino acids with an estimated molecular mass of 105.7 kDa. Immunofluorescence microscopy revealed that TSNHE3 was localized to the apical membrane of epithelial cells of the ctenidial filaments and the tertiary water channels. Particularly, the apical immunofluorescence of the ctenidial filaments was consistently stronger in the ctenidium of clams exposed to 12 h of light than those of the control kept in darkness. Indeed, light induced significant increases in the transcript level and protein abundance of TSNHE3/TSNHE3 in the ctenidium, indicating that the transcription and translation of TSNHE3/TSNHE3 were light-dependent. As light-enhanced calcification generates H+, the increased expression of TSNHE3/TSNHE3 in the ctenidium could be a response to augment H+ excretion in pursuance of whole-body acid-base balance during light exposure. These results signify that shell formation in giant clams requires the collaboration between the ctenidium, which is a respiratory and iono-regulatory organ, and the inner mantle, which is directly involved in the calcification process, and provide new insights into the mechanisms of light-enhanced calcification in giant clams.

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 myostatin from the skeletal muscle of the African lungfish, Protopterus annectens, and changes in its mRNA and protein expression levels during three phases of aestivation.

African lungfishes can aestivate and remain torpid without food and water for years, but disuse muscle atrophy is not prominent during aestivation. This study aimed to clone myostatin (mstn/Mstn), a factor associated with disuse muscle atrophy in mammals, from the skeletal muscle of the African lungfish Protopterus annectens, and to determine its mRNA expression level and protein abundance therein during the induction, maintenance, and arousal phases of aestivation. The complete coding cDNA sequence of mstn comprised 1128 bp, encoding for 376 amino acids with an estimated molecular mass of 42.9 kDa. It was grouped together with Mstn/MSTN of coelacanth and tetrapods in a clade separated from teleost Mstn. After 6 days (the induction phase) of aestivation, the mstn transcript level in the muscle increased significantly, while the protein abundance of Mstn remained comparable to the control. Following that, a significant increase in the expression levels of mstn/Mstn occurred on day 12 (the early maintenance phase) of aestivation. After 6 months of aestivation (the prolonged maintenance phase), the expression levels of mstn/Mstn returned to control levels, indicating the possible impediment of a drastic increase in muscle degradation to prevent muscle atrophy. During 1-3 days of arousal from aestivation, the expression levels of mstn/Mstn in the muscle remained comparable to the control. Hence, tissue reconstruction/regeneration of certain organs might not involve the mobilization of amino acids from the muscle during the early arousal. These results provide insights into how aestivating P. annectens regulates the expression of mstn/Mstn possibly to ameliorate disuse muscle atrophy.

Voltage-Gated Na+ Channel Isoforms and Their mRNA Expression Levels and Protein Abundance in Three Electric Organs and the Skeletal Muscle of the Electric Eel Electrophorus electricus.

This study aimed to obtain the coding cDNA sequences of voltage-gated Na+ channel (scn) α-subunit (scna) and ß-subunit (scnb) isoforms from, and to quantify their transcript levels in, the main electric organ (EO), Hunter's EO, Sach's EO and the skeletal muscle (SM) of the electric eel, Electrophorus electricus, which can generate both high and low voltage electric organ discharges (EODs). The full coding sequences of two scna (scn4aa and scn4ab) and three scnb (scn1b, scn2b and scn4b) were identified for the first time (except scn4aa) in E. electricus. In adult fish, the scn4aa transcript level was the highest in the main EO and the lowest in the Sach's EO, indicating that it might play an important role in generating high voltage EODs. For scn4ab/Scn4ab, the transcript and protein levels were unexpectedly high in the EOs, with expression levels in the main EO and the Hunter's EO comparable to those of scn4aa. As the key domains affecting the properties of the channel were mostly conserved between Scn4aa and Scn4ab, Scn4ab might play a role in electrogenesis. Concerning scnb, the transcript level of scn4b was much higher than those of scn1b and scn2b in the EOs and the SM. While the transcript level of scn4b was the highest in the main EO, protein abundance of Scn4b was the highest in the SM. Taken together, it is unlikely that Scna could function independently to generate EODs in the EOs as previously suggested. It is probable that different combinations of Scn4aa/Scn4ab and various Scnb isoforms in the three EOs account for the differences in EODs produced in E. electricus. In general, the transcript levels of various scn isoforms in the EOs and the SM were much higher in adult than in juvenile, and the three EOs of the juvenile fish could be functionally indistinct.

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.