Building a house when supplies are limited: Calcification in ion-poor conditions by the freshwater common pond snail Lymnaea stagnalis

Biological calcification is completed by diverse groupings of organisms in habitats ranging from minimally buffered, low-ion freshwater lakes and streams to relatively well-buffered high ionic strength as in the open ocean. Calcifying organisms must acquire shell-forming ions, calcium and carbonate, to complete calcification while maintaining an environment favorable for continued calcification. The freshwater common pond snail Lymnaea stagnalis produces embryos that complete direct development, hatching as shell-bearing individuals within 10 d despite relatively low ambient calcium and carbonate availability. Using a multi-pronged approach including microscopic developmental evaluations and whole egg mass and microscale ion flux measurements, we observed growth retardation in the absence of ambient calcium. However, embryos did not exhibit dependence upon ambient carbonate availability, developing and hatching in bicarbonate-free water at rates comparable to controls. Post-metamorphic, shell-laying embryos exhibited a significant, saturation-type net calcium uptake as a function of increasing ambient calcium concentration following depletion of maternal stores. However, changes in ambient bicarbonate levels did not influence calcium or apparent titratable alkalinity uptake. Using pharmacological inhibitors in both whole egg mass and microscale measurements, we found that calcium uptake in this species is apparently via calcium channels and electrogenic calcium/proton exchange with an apparent connection to carbonic anhydrase-catalyzed hydration of carbon dioxide. This hydration of endogenous carbon dioxide provides an endogenous carbonate source but also unfavorable acid accumulation. Further experiments using bafilomycin, a V-H⁺ ATPase inhibitor, implicated an outwardly directed H⁺-pump serving the dual function of removing H⁺ to maintain a more alkaline internal environment, which is favorable for continued bicarbonate production and sustaining a membrane potential that electrochemically supports calcium uptake. Apparently calcium requirements for embryonic snail shell formation are met via active uptake but carbonate, which is also necessary for calcification is acquired in part from endogenous sources with no significant correlation to ambient carbonate availability.