A proteinaceous organic matrix regulates carbonate mineral production in the marine teleost intestine

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.

Amorphous calcium carbonate particles form coral skeletons

“…Reggi et al (26) and Falini et al (27) showed that the organic matrix proteins extracted from coral skeletons stabilize ACC in vitro in the absence of cells. ACC stabilization by proteins was also observed in sea urchin spicules (32) and in fish intestines (49). These proteins, therefore, may be in the same vesicles as ACC.…”

Calcium carbonate production by fish in temperate marine environments

“…However, temperature effects on magnesium uptake are not always clear and can be overridden or distorted by biological controls (so-called "vital effects", sensu Urey et al 1951), solution pH (Lea et al 1999), and the ratio of Mg to Ca in solution (Mewes et al 2014). Within the piscine intestine, proteins capable of regulating magnesium uptake, precipitation rate, and precipitate morphology, at least in vitro, offer a potential organic control (Schauer et al 2016(Schauer et al , 2018Schauer and Grosell 2017), while somewhat variable and often high values of pH, [Mg] 2+ , and Mg/Ca are likely important inorganic controls (Wilson et al 1996;Grosell et al 2001). Intriguingly though, the sole available data point for temperate fish carbonates is consistent with a temperature effect: 10 mol% MgCO 3 in HMC produced at 13 C (Wilson et al 2009) being low compared with typical values of 20-40 mol% from warm-water settings (20-27 C-Walsh et al 1991;Perry et al 2011;Heuer et al 2012;Salter et al 2012Salter et al , 2017Salter et al , 2018.…”

Long-Read De Novo Genome Assembly of Gulf toadfish Opsanus beta.

“…Genome completeness was measured by calculating the number of Actinopterygii single copy orthologs retained in the assembly via BUSCO (91) with the actinopterygii_odb10 database. Completeness was further assessed with read mapping rate by mapping publicly available paired-end Illumina short read RNA (47) and DNA (PRJNA196921) libraries, as well as input HiFi reads to the super-scaffolded assembly.…”

“…Transfer RNAs (tRNAs) were predicted using trnascan v1.4.0 (111). Gene models were then refined and UTRs added with funannotate::update (46) using IsoSeq high quality transcripts and O.beta illumina short reads (PRJNA313355; (47).…”