Molecular characterization, cellular localization and light-dependent expression of dinoflagellate vacuolar-type H+-ATPase (VHA) subunit B in the colourful outer mantle of the giant clam, Tridacna squamosa, indicate the involvement of VHA in CO2 uptake in the photosynthesizing symbionts

Abstract

Giant clams are animal-dinoflagellate associations found in Indo-Pacific reef ecosystems. The clam host obtains organic nutrients from phototrophic dinoflagellates of genera Symbiodinium, Cladocopium, and Durusdinium, which reside extracellularly as symbionts (alias zooxanthellae) in the luminal fluid of zooxanthellal tubules located mainly in the colourful outer mantle. The host also needs to supply the symbionts with inorganic carbon for photosynthesis. Symbiont photosynthesis can be impeded by inhibitors of vacuolar H+-ATPase (VHA) because the host possesses a carbon concentration mechanism consisting of VHA to facilitate the supply of CO2(aq) to the symbionts. Here, we report that VHA was also expressed in dinoflagellates residing in the outer mantle of the fluted giant clam, Tridacna squamosa. Three complete cDNA coding sequences of VHA subunit B (VHA-B), one for each genus of dinoflagellate, had been obtained, and each sequence comprised 1482 bp, encoding a protein of 493 amino acids (~55 kDa). As these three sequences were highly similar, we could only design real-time PCR primers to quantify comprehensively zooxanthellae-VHA-B (Zoox-VHA-B) that represented VHA-B of all three genera of dinoflagellates. The outer mantle had the highest transcript level of Zoox-VHA-B among the three organs studied, and illumination led to a significant increase in the protein abundance of Zoox-VHA-B therein. Zoox-VHA-B was immunolocalized to intracellular vesicles, which could apparently align and fuse with the plasma membrane, in the symbiotic dinoflagellates. Overall, these results indicate that photosynthesizing symbionts could increase the capacity of H+ secretion through VHA-containing vesicles to promote the dehydration of luminal HCO3− and the absorption of CO2(aq) during illumination.