The functions of Ctenidia in light-enhanced calcification in Tridacna squamosa  

The giant clam Tridacna squamosa harbours symbiotic zooxanthellae which are distributed mainly in the extensible outer mantle. Like corals, it can perform light-enhanced calcification. During calcification, calcium is deposited as calcium carbonate according to the reaction: Ca²⁺ + HCO₃⁻ ó CaCO₃ + H⁺. Hence, the rate of calcification can be enhanced by the removal of H⁺ from the calcification site. However, what happens to the excess H⁺ removed from the extrapallial fluid by the inner mantle during light enhanced calcification in T. squamosa is unclear at present.  Furthermore, an increased rate of CaCO₃ deposition in light demands an increase in the rate of Ca²⁺ supply by the inner mantle to the extrapallial fluid. However, little is known of the mechanisms involved in Ca²⁺ absorption from the external medium in giant clams, and in molluscs in general. As zooxanthellae symbionts are known to be nitrogen-limited, it would be advantageous for the clam host to supply the symbionts with ammonia during photosynthesis. Indeed, T. squamosa can actively absorb ammonia from the environment, but there is a dearth of information on the site and mechanisms involved. Therefore, we aimed to elucidate the functions of ctenidia (gills) in Ca²⁺ and NH₄⁺ absorption and H⁺ excretion in T. squamosa. Unlike corals, giant clams possess ctenidia as respiratory organs. A ctenidium is shaped like a comb, with many filaments to increase surface area for gas exchange. There are indications that ctenidia can also function in ionoregulation and acid-base balance in molluscs.  Therefore, we made an effort to obtain the complete coding cDNA sequences of several genes related to the transport of Ca²⁺, H⁺ or NH₃/NH₄⁺ from the ctenidia of T. squamosa, and to determine the effects of light exposure on their mRNA expression levels using quantitative real-time PCR. We confirmed that light increased the mRNA expression levels of Plasma Membrane Ca²⁺-ATPase (PCMA), Na⁺/H⁺ Exchanger isoforms (NHEs) and Na⁺:K⁺(NH₄⁺):2Cl^- cotransporter 2 (NKCC2), and decreased those of Ammonia transporter (AMT) in ctenidia of T. squamosa. These results indicate that (1) ctenidia can be the site of active Ca²⁺ absorption for light-enhanced calcification, (2) light-enhanced calcification may perturb acid-base balance in the hemolymph and require an increase H⁺ excretion through the ctenidia, and (3) active ammonia absorption from the external medium may be increased during light-enhanced calcification to support photosynthesis in the symbiotic zooxanthellae. As ctenidia are non-pigmented and positioned inside the mantle cavity, it is logical to deduce that they respond to light indirectly through some signaling molecules produced and released by the zooxanthellae which act as light sensors in the extensible outer mantle. Our results indicate for the first time that light-enhanced calcification in giant clams involves the collaboration between ctenidia and the inner mantle, coordinated possibly by some signaling molecules produced by the zooxanthellae in response to light.