Mechanisms of HCO3/-uptake as a source of dissolved inorganic carbon (DIC) for photosynthesis by the intracellular symbiont, Symbiodinium sp. were studied using microcolonies of the coral Galaxea fascicularis, freshly isolated zooxanthellae (FIZ) and cultured zooxanthellae (CZ). For this purpose we used specific inhibitors of anion transport 4-acetamido- 4'- isothiocyanatostilbene-2,2'-disulfonic acid - SITS -, 4,4'- diisothiocyanato- stilbene-2,2'-disulfonic acid - DIDS -, carbonic anhydrase (acetazolamide, ethoxyzolamide), H+-ATPase (N,N'-dicyclohexylcarbodiimide - DCCD -, diethylstilbestrol - DES -, vanadate) or Ca2+channels (verapamil). We also tested the effect of ions known to play a role in HCO3/-transport, like Na+and Ca2+. Chloride uptake experiments were also performed to determine whether Cl-and HCO3/-fluxes were coupled in CZ. Furthermore, the presence of carbonic anhydrase was tested using indirect immunofluorescence. Our results suggest that bicarbonate uptake by the animal symbiont is likely to be achieved by two types of DIDS- sensitive HCO3/-carriers, each sharing 50% of the total uptake. The first is Na+- dependent, while the second is Na+-independent. We suggest the presence of a Na+independent Cl/HCO3/+exchange and either a Na+-dependent Cl/HCO3/-exchange or a Na+/HCO3/-symport. Pharmacological data suggest that the enzyme carbonic anhydrase plays an important role in maintaining the photosynthetic rate. In the intact symbiosis, the major fraction of carbonic anhydrase activity is located in the zooxanthellae. Striking differences in DIC absorption mechanisms were found for FIZ and CZ. In FIZ, H+-ATPase and carbonic anhydrase participate in the carbon supply while in CZ the mechanism of HCO3/-uptake appears to be strictly Na+- dependent and could be the result of Na+/HCO3/-symport activity. We hypothesize that stimulation of HCO3/-uptake by the animal host is a consequence of intracellular pH alkalization by zooxanthellae photosynthesis. These results were summarized in a synthetic scheme of DIC absorption by both host cell and isolated zooxanthellae.
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