A sea urchin Na+K+2Cl- cotransporter is involved in the maintenance of calcification-relevant cytoplasmic cords in Strongylocentrotus droebachiensis larvae

The cellular mechanisms of calcification in sea urchin larvae are still not well understood. Primary mesenchyme cells within the larval body cavity form a syncytium to secrete CaCO3 spicules from intracellular amorphous CaCO3 (ACC) stores. We studied the role of Na+K+2Cl- cotransporter (NKCC) in intracellular ACC accumulation and larval spicule formation of Strongylocentrotus droebachiensis. First, we incubated growing larvae with three different loop diuretics (azosemide, bumetanide, and furosemide) and established concentration-response curves. All loop diuretics were able to inhibit calcification already at concentrations that specifically inhibit NKCC. Calcification was most effectively inhibited by azosemide (IC50 = 6.5 µM), while larval mortality and swimming ability were not negatively impacted by the treatment. The inhibition by bumetanide (IC50 = 26.4 µM) and furosemide (IC50 = 315.4 µM) resembled the pharmacological fingerprint of the mammalian NKCC1 isoform. We further examined the effect of azosemide on the maintenance of cytoplasmic cords and on the occurrence of calcification vesicles using fluorescent dyes (calcein, FM1-43). Fifty micromolars of azosemide inhibited the maintenance of cytoplasmic cords and resulted in increased calcein fluorescence within calcification vesicles. The expression of NKCC in S. droebachiensis was verified by PCR and Western blot with a specific NKCC antibody. In summary, the pharmacological profile of loop diuretics and their specific effects on calcification in sea urchin larvae suggest that they act by inhibition of NKCC via repression of cytoplasmic cord formation and maintenance.

Negative effects of ocean acidification on calcification vary within the coccolithophore genus Calcidiscus

A large percentage of CO2 emitted into the atmosphere is absorbed by the oceans, causing chemical changes in surface waters known as ocean acidification (OA). Despite the high interest and increased pace of OA research to understand the effects of OA on marine organisms, many ecologically important organisms remain unstudied. Calcidiscus is a heavily calcified coccolithophore genus that is widespread and genetically and morphologically diverse. It contributes substantially to global calcium carbonate production, organic carbon production, oceanic carbon burial, and ocean-atmosphere CO2 exchange. Despite the importance of this genus, relatively little work has examined its responses to OA. We examined changes in growth, morphology, and carbon allocation in multiple strains of Calcidiscus leptoporus in response to ocean acidification. We also, for the first time, examined the OA response of Calcidiscus quadriperforatus, a larger and more heavily calcified Calcidiscus congener. All Calcidiscus coccolithophores responded negatively to OA with impaired coccolith morphology and a decreased ratio of particulate inorganic to organic carbon (PIC:POC). However, strains responded variably; C. quadriperforatus showed the most sensitivity, while the most lightly calcified strain of C. leptoporus showed little response to OA. Our findings suggest that calcium carbonate production relative to organic carbon production by Calcidiscus coccolithophores may decrease in future oceans and that Calcidiscus distributions may shift if more resilient strains and species become dominant in assemblages. This study demonstrates that variable responses to OA may be strain or species specific in a way that is closely linked to physiological traits, such as cellular calcite quota.