Calcification without Strong Proton Extrusion in the Pearl Oyster Pinctada fucata

Biomineralization in marine calcifying organisms has traditionally been regarded as a process that involves proton release associated with calcium carbonate precipitation, which may lead to localized acidification. In several taxa, including foraminifera, pronounced pH gradients have been reported between calcification sites and the surrounding environment. However, it remains unclear whether strong proton extrusion into the external environment is universally required for shell formation.

In this study, we re-evaluated pH distributions during shell formation in juvenile pearl oysters (Pinctada fucata) using improved HPTS ratiometric fluorescence calibration combined with spatial analysis. We found that regions involved in shell formation, corresponding to extrapallial fluid domains inferred to represent calcification sites, consistently showed relatively higher pH values than internal soft tissues. The pH in these regions was approximately 7.8, which is slightly lower than that of the surrounding seawater (~8.0). At the spatial scale examined, no pronounced acidification was detected in the external environment outside the shell. By contrast, strongly acidic regions reaching pH ~6.0 were observed in internal tissues, which are likely associated with digestive organs. In addition, within or adjacent to the inferred calcification sites, moderately lower-pH regions (approximately pH ~7.0) were observed as ribbon-like distributions composed of small, discrete spots.

These observations indicate that shell formation in P. fucata does not depend on strong proton extrusion into the surrounding seawater, nor on extreme alkalization of the calcification site. Instead, pH regulation in this species appears to occur in a manner that is spatially separated from the surrounding seawater. This suggests that elevation of pH alone may not be the primary factor controlling calcification. Alternative mechanisms may therefore contribute to shell formation, including regulation of calcium concentration, modulation of ionic composition that inhibits calcification (e.g., Mg²⁺ and sulfate ions), and intracellular proton processing mediated by organic components.

Although acidification driven by carbon dioxide production is theoretically expected to accompany calcium carbonate precipitation, such changes could not be directly resolved under the imaging conditions employed in this study. Taken together, our results highlight diversity in proton regulation strategies among marine calcifying organisms and provide a basis for comparative discussions of shell formation mechanisms.