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

Abstract

SUMMARY 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 Ca2+ 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 CO2 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 Ca2+via voltage-dependent Ca2+ channels and possibly an electrogenic Ca2+/1H+ exchanger. Additionally, CO2 hydration provides an endogenous source of HCO –3. Thus, hydration of endogenous CO2 forms HCO –3 for calcification while hydrogen ions are excreted, contributing to continued Ca2+ uptake, as well as creating favorable alkaline internal conditions for calcification. The connections between Ca2+ and HCO –3 acquisition mechanisms that we describe here provide new insight into this efficient, embryonic calcification in freshwater.