Embryonic Responses To Carbonic Anhydrase Inhibition And Exogenous Calcium Supplementation In Eggs Of The American Alligator

RM 216

: During incubation, embryonic archosaurs mobilize calcium from the eggshell, and deposit it in the yolk and the skeleton. Previous experiments on eggs of the American alligator (Alligator mississippiensis) demonstrated that removal of the calcareous eggshell results in embryonic growth retardation. So far, no experiments have ascertained the role of carbonic anhydrase in eggshell calcium mobilization, nor the importance of calcium provenance to embryonic crocodilians. We conducted two experiments to test whether (i) carbonic anhydrase (CA) inhibition has similar effects on the embryo as eggshell removal, and (ii) exogenous calcium supplementation can rescue the original embryonic phenotype. To inhibit CA, we applied topical acetazolamide (150-600 µg/day AZA in DMSO vehicle). As exogenous sources of calcium, we used 0.1M CaCO3 solution and calcite sand. We found AZA reduced embryonic wet mass after five weeks of treatment, with a significant dose-dependent response. Calcium supplementation of eggs with the eggshell removed yielded mixed results. Spraying with CaCO3 solution partially rescued the phenotype, with alligator embryos being intermediate in wet mass between control and non-treated experimental siblings. Egg incubation in calcite sand, however, did not restore normal embryonic growth trajectory. This suggests alligator embryos can potentially obtain calcium from environmental sources, with presence of aqueous calcium especially important.