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.

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.

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.