Characterization of mechanisms for Ca2+ and HCO3–/CO32– acquisition for shell formation in embryos of the freshwater common pond snail Lymnaea stagnalis

The freshwater common pond snail Lymnaea stagnalis produces embryos that complete direct development, hatching as shell-bearing individuals within 10 days despite relatively low ambient calcium and carbonate availability. This development is impaired by removal of ambient total calcium but not by removal of bicarbonate and/or carbonate. In this study we utilized pharmacological agents to target possible acquisition pathways for both Ca(2+) and accumulation of carbonate in post-metamorphic, shell-laying embryos. Using whole egg mass flux measurements and ion-specific microelectrode analytical techniques, we have demonstrated that carbonic anhydrase-catalyzed hydration of CO(2) is central in the acquisition of both shell-forming ions because it provides the hydrogen ions for an electrogenic vacuolar-type H(+)-ATPase that fuels the uptake of Ca(2+) via voltage-dependent Ca(2+) channels and possibly an electrogenic Ca(2+)/1H(+) exchanger. Additionally, CO(2) hydration provides an endogenous source of HCO(3)(-). Thus, hydration of endogenous CO(2) forms HCO(3)(-) for calcification while hydrogen ions are excreted, contributing to continued Ca(2+) uptake, as well as creating favorable alkaline internal conditions for calcification. The connections between Ca(2+) and HCO(3)(-) acquisition mechanisms that we describe here provide new insight into this efficient, embryonic calcification in freshwater.