Discovery of chitin in skeletons of non-verongiid Red Sea demosponges

Abstract

Marine demosponges (Porifera: Demospongiae) are recognized as first metazoans which have developed over millions of years of evolution effective survival strategies based on unique metabolic pathways to produce both biologically active secondary metabolites and biopolymer-based stiff skeletons with 3D architecture. Up to date, among marine demosponges, only representatives of the Verongiida order have been known to synthetize biologically active substances as well as skeletons made of structural polysaccharide chitin. This work, to our knowledge, demonstrates for the first time that chitin is an important structural component within skeletons of non-verongiid demosponges Acarnus wolffgangi and Echinoclathria gibbosa collected in the Red Sea. Calcofluor white staining, FTIR and Raman analysis, ESI-MS, SEM, and fluorescence microscopy as well as a chitinase digestion assay were applied in order to confirm, with strong evidence, the finding of α-chitin in the skeleton of both species. We suggest that, the finding of chitin within these representatives of Poecilosclerida order is a promising step in the evaluation of these sponges as novel renewable sources for both biologically active metabolites and chitin, which are of prospective application for pharmacology and biomedicine.

Fig 1. Current state of the art concerning distribution of chitin in the phylum porifera.

Fig 2

Specimens of A. wolffgangi (a) and E. gibbosa (c) in their natural environments. Washed with deionized water of the freeze-dried skeletons of A. wolffgangi (b) and E. gibbosa (d) which have been used for chitin isolation in this study.

Fig 3. Step-by-step scheme showing procedure for isolation of chitinous fibers from the skeletons of A. wolffgangi (left line) and E. gibbosa (right line).

Fig 4

Spicule-free, colorless 3D scaffold obtained from A. wolffgangii (a) and E. gibbosa (d) according to the isolation procedure represented in Fig 3. Microstructural features of selected skeletal fibers of A. wolffgangii (marked with arrows) (b) and E. gibbosa (e) prior and after HF-treatment (c and f, respectively) are well visible on the corresponding light microscopy images.

Fig 5

SEM imagery of the purified A. wolffgani (a) and E. gibbosa (d) skeleton’s fragments prior (b and e, respectively) and after demineralization procedure (c and f, respectively). Well visible spicules are marked with arrows.

Fig 6

Purified skeletal fibers of A. wolffgangi (a) and E. gibbiosa (c) after CFW staining observed in light microscopy (a, c) and fluorescence microscopy (b and d) modus, respectively.

Fig 7. FT-IR spectra of chitin isolated from A. wolffgangi and E. gibbosa demosponges in comparison with the of α-chitin standard.

Fig 8. Raman spectroscopy of the chitinous scaffolds isolated from A. wolffgangi and E. gibbosa demosponges in comparison with α-chitin standard.

Fig 9. Visualization of the chitinase digestion test using white light microscopy.

Chitinase digestion of purified and completely demineralized selected skeletal fiber isolated from A. wolffgangi and E. gibbosa prior (a and c, respectively) and after 3 h of chitinase treatment (b and d, respectively).

Fig 10

Comparative ESI-MS analyses from the glucosamine standard (a), and of the hydrolysed chitin from the A. wolffgangi (b) and E. gibbosa (c).

Fig 11. Schematic view of the possible uses of Poecilosclerida sponges including A. wolffgangi and E. gibbosa species.