Symbiotic dinoflagellates of the giant clam, tridacna squamosa, express an extracellular Alpha carbonic anhydrase associated with the plasma membrane to promote HCO3− dehydration and CO2 uptake during illumination

Abstract

Giant clams generally harbor phototrophic Symbiodiniaceae dinoflagellates of genera Symbiodinium, Cladocopium, and Durusdinium. The coccoid symbiotic dinoflagellates (zooxanthellae) reside extracellularly inside the lumen of zooxanthellal tubules in the colorful outer mantle. They obtain from the host inorganic carbon (Ci) for photosynthesis and supply photosynthate to the host. The outer mantle has a host-derived carbon concentration mechanism (CCM) to facilitate the transport of Ci from the hemolymph into the luminal fluid. To regulate Ci uptake, the symbionts probably possess their own CCMs that comprise an extracellular alpha carbonic anhydrase (αCA) and a proton transporter. Indeed, we obtained from the outer mantle of the giant clam, Tridacna squamosa, three complete cDNA coding sequences of a membrane-associated αCA derived from Symbiodinium (Symb-αCA), Cladocopium (Clad-αCA), and Durusdinium (Duru-αCA), which consisted of 2808, 2847, and 2829 bp, respectively. The respective encoded proteins had 935 (104.7 kDa), 948 (106.1 kDa), and 942 (105 kDa) amino acids, each containing a transmembrane domain. The outer mantle had the highest level of Duru-αCA transcripts. Phenogramic analyses denoted Duru-αCA as an extracellular CA closely associated with human CA4 and had a dinoflagellate-origin. Using an antibody that could react comprehensively with zooxanthellae-αCAs (Zoox-αCA) derived from all three genera of dinoflagellate, immunofluorescence microscopy revealed immuno-labeling at the dinoflagellate’s plasma membrane. As Symb-αCA, Clad-αCA, and Duru-αCA possessed extracellular catalytic domains, they could catalyze the dehydration of HCO3− to CO2 in the luminal fluid. Importantly, illumination led to significant increases in the gene and protein expression levels of Zoox-αCA/Zoox-αCA in the outer mantle of T. squamosa. Taken together, Zoox-αCA could be part of the symbiont’s light-enhanced external CCM to promote and regulate the acquisition of Ci from the host for photosynthesis. Our results also suggested that the potentials of generating CO2 adjacent to the symbionts’ plasma membrane could differ among different phylotypes of Symbiodinium and Cladocopium.