Abstract
Marine teleost fish produce CaCO3 in their intestine as part of their osmoregulatory strategy. This precipitation is critical for rehydration and survival of the largest vertebrate group on earth, yet the molecular mechanisms that regulate this reaction are unknown. Here, we isolate and characterize an organic matrix associated with the intestinal precipitates produced by Gulf toadfish (Opsanus beta). Toadfish precipitates were purified using two different methods, and the associated organic matrix was extracted. Greater than 150 proteins were identified in the isolated matrix by mass spectrometry and subsequent database searching using an O. beta transcriptomic sequence library produced here. Many of the identified proteins were enriched in the matrix compared to the intestinal fluid, and three showed no substantial homology to any previously characterized protein in the NCBI database. To test the functionality of the isolated matrix, a micro-modified in vitro calcification assay was designed, which revealed that low concentrations of isolated matrix substantially promoted CaCO3 production, where high concentrations showed an inhibitory effect. High concentrations of matrix also decreased the incorporation of magnesium into the forming mineral, potentially providing an explanation for the variability in magnesium content observed in precipitates produced by different fish species.
Highlights
Excreted to the environment as a waste product
In order to determine if the intestinal precipitates formed in marine fish contain an organic matrix, the CaCO3 precipitates were purified by two different methods in order to remove intestinal contaminants that could be associated with the mineral
Precipitates prepared using each of the procedures, as well as unpurified precipitates, were imaged by scanning electron microscopy (SEM) to observe if the mucus layer and other large contaminants had been removed from the mineral
Summary
Excreted to the environment as a waste product. Without the reduced osmolality in the intestinal lumen resulting from this precipitation reaction, teleosts would be unable to inhabit marine environments, as they would be unable to sufficiently rehydrate[8]. Due to the large biomass, as well as high individual rates of carbonate production and excretion, a conservatively estimated 40 to 110 × 106 metric tons of CaCO3 is produced by marine teleosts and excreted into the environment each year, which accounts for approximately 3 to 15% of the total oceanic CaCO3 production[6]. In most instances, these carbonates contain high amounts of Mg, which make them more soluble than other marine derived carbonate minerals such as aragonite[9,10]. We demonstrate that these isolated proteins modulate the rate of CaCO3 production in vitro, as well as the Mg incorporation into the resulting mineral, in a highly dose-dependent, but not straightforward manner
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