The shell matrix and microstructure of the Ram’s Horn squid: Molecular and structural characterization

Abstract

Molluscs are one of the most diversified phyla among metazoans. Most of them produce an external calcified shell, resulting from the secretory activity of a specialized epithelium of the calcifying mantle. This biomineralization process is controlled by a set of extracellular macromolecules, collectively defined as the organic matrix. In spite of several studies, these components are mainly known for bivalve and gastropod classes. In the present study, we investigated the physical and biochemical properties of the internal planispiral shell of the understudied Ram's Horn squid Spirula spirula (Cephalopoda, Decabrachia, Spirulida). Scanning Electron Microscope investigations of the shell reveal a complex microstructural organization, with septa sandwiched into the shell wall, in the form of a bevel. The saccharides constitute a quantitatively important moiety of the matrix, as shown by Fourier-transform infrared spectroscopy. Solid-state nuclear magnetic resonance spectroscopy identified β-chitin and additional polysaccharides, for a total amount of 80% of the insoluble fraction. Proteomics was applied to both soluble and insoluble matrices and in silico searches were performed, first on heterologous metazoans models, and secondly, on an unpublished transcriptome of Spirula spirula. In the first case, several peptides were identified, some of them matching with tyrosinase, chitinase 2, protease inhibitor, or immunoglobulin. In the second case, 38 hits were obtained, including transferrin, a serine protease inhibitor, matrilin, different histone-like, a 2-macroglobulin or a putative heme-binding/calcium-binding protein. The very few similarities with known molluscan shell matrix proteins suggest that Spirula spirula uses a unique set of shell matrix proteins for constructing its internal shell. The absence of similarity with closely related cephalopods such as the cuttlefish Sepia demonstrates that there is no obvious phylogenetic signal in the skeletal matrix of cephalopods.

A Nature’s Curiosity: The Argonaut “Shell” and Its Organic Content

“…The resolved and less crowded aromatic region peaks (e.g., 129 ppm) suggest the occurrence of the aromatic amino acids (e.g., tyrosine) while the broad peak centered at 72 ppm may arise from either hydroxyprolines and/or polysaccharides. Different from A. hians, the bio-organic content of the AIM of the shell of S. spirula (Figure S3c)-another member of the cephalopod class-was shown as being dominated by chitin and possibly other oligosaccharides with only a minor proteinaceous content [28]. For comparison we also show the 13 C CPMAS spectrum of the AIM of Arctica islandica (Figure S3b)-a bivalve whose shell acid insoluble matrix is protein-rich (~90:10 proteins versus saccharides) [29].…”

“…An additional advantage is its ability to identify the structural forms of the calcareous inorganic matrix-crystalline and/or amorphous-and interactions with the bioorganics. [28,29]. 13 C, 31 P Magic Angle Spinning (MAS) NMR measurements were carried out on 300 MHz solid state NMR Bruker AVANCE III spectrometer using a 4 mm triple-resonance MAS NMR probe with zirconia rotors.…”

Independent adoptions of a set of proteins found in the matrix of the mineralized shell-like eggcase of Argonaut octopuses

“…The presence of housekeeping proteins in the shell matrix is not surprising, although it is intriguing because no study has addressed what they are doing there. Housekeeping proteins have also been reported to be present in the shell matrix of Crassostrea angulata and C. hongkongensis (Upadhyay et al 2016), Euhadra quaesita (Shimizu et al 2019), and Spirula spirula (Oudot et al 2020a).…”

“…Because of the absence of EGF-ZP, the eggcase of the Argonauts lacks the nacreous layer, and is composed of only calcite. However, Oudot et al (2020a) analyzed the coiled shell of the Spirulid Spirula spirula, and found that the species shares only a few similarities with known SMPs of the Mollusks.…”

The First In Situ Observation of the Ram’s Horn Squid Spirula spirula Turns “Common Knowledge” Upside Down

“…Although live observations of Spirula are very rare, there have been important advances in our understanding of its shell structure and function [11,12], life history and biogeography [13,14] and phylogeny and systematics [15][16][17]. Still, live observations offer researchers a unique opportunity to directly examine the behavioral responses of organisms about which behavior otherwise has to be merely surmised.…”