Uniaxial orientation of β-chitin nanofibres used as an organic framework in the scales of a hot vent snail

Abstract

Organisms use various forms and orientations of chitin nanofibres to make structures with a wide range of functions, from insect wings to mussel shells. Lophotrochozoan animals such as snails and annelid worms possess an ancient ‘biomineralization toolkit’, enabling them to flexibly and rapidly evolve unique hard parts. The scaly-foot snail is a gastropod endemic to deep-sea hydrothermal vents, unique in producing dermal sclerites used as sites of sulfur detoxification. Once considered to be strictly proteinaceous, recent data pointed to the presence of chitin in these sclerites, but direct evidence is still lacking. Here, we show that β-chitin fibres (approx. 5% of native weight) are indeed the building framework, through a combination of solid-state nuclear magnetic resonance spectroscopy, wide-angle X-ray diffraction, and electron microscopy. The fibres are uniaxially oriented, likely forming a structural basis for column-like channels into which the scaly-foot snail is known to actively secrete sulfur waste—expanding the known function of chitinous hard parts in animals. Our results add to the existing evidence that animals are capable of modifying and co-opting chitin synthesis pathways flexibly and rapidly, in order to serve novel functions during their evolution.

Identification of Chitin Allomorphs in Poorly Crystalline Samples Based on the Complexation with Ethylenediamine

“…It should be noted that β-chitin treated with 5, 6, 7, and 8 M HCl showed a sharp peak at 2θ = 26.3°, which is indicative of α-chitin (the 0 1 3 diffraction), but this peak is not useful in the determination of chitin allomorphs, since in 2θ = 20°and above the peaks of residual, insoluble inorganic substances appear and blur the diffraction from chitin. 65 In addition to the acid treatment, alkaline treatment was conducted in order to examine the transformation from βchitin to chitosan through deacetylation (Figure 6a). By immersing in 12.5 M aqueous NaOH solution, the β-chitin in siboglinid worm tubes gradually lost the DA and its high crystallinity, and a new peak was observed at 2θ = 8.5°, which corresponded to an unknown hydrate form of chitosan.…”

“…Therefore, the allomorphs were correctly identified with EDA complexation. It should be noted that β-chitin treated with 5, 6, 7, and 8 M HCl showed a sharp peak at 2 θ = 26.3°, which is indicative of α-chitin (the 0 1 3 diffraction), but this peak is not useful in the determination of chitin allomorphs, since in 2 θ = 20° and above the peaks of residual, insoluble inorganic substances appear and blur the diffraction from chitin …”

“…For the Scaly-foot Snail, we used the part of its scales close to the distal tip. As the scales show accretionary growth in the longitudinal direction, the distal tip has been exposed longest to the environment and the β-chitin would be more disordered than freshly secreted parts near the base . In Figure , the peak position of scale samples complexed with EDA was located at the border of the known peak position for β-chitin, identifying it correctly as β-chitin.…”

“…We then extended this EDA complexation method to chitin samples characterized by nearly-amorphous, very poor crystallinity, including the α-chitin planktic copepod exoskeletons ( Pontella fera , Calanoida) 67 and the β-chitin scales of the Scaly-foot Snail ( Chrysomallon squamiferum ). 65 For copepods ( Figure 7 ) , the peak top position in the dry state was between α-chitin and β-chitin monohydrate due to the very poor crystallinity. However, EDA complexation revealed that copepod exoskeletons consisted of poorly crystalline α-chitin or chitosan.…”

New mitogenomes in deep-water endemic Cocculinida and Neomphalida shed light on lineage-specific gene orders in major gastropod clades

“…Within Peltospiridae, the Scaly-foot Snail C. squamiferum was sister to all other peltospirid species included, with its divergent time from other peltospirids dating back to Late Jurassic. The distinctive morphological features of hundreds of chitinous sclerites (Isobe et al, 2022) covering the dorsal foot in Scalyfoot Snail may be linked to its unique phylogeny position. Our mitogenome level molecular phylogeny also lends support to the convergent evolution scenario of endosymbiosis (Chen et al, 2015) in two divergent genera within Peltospiridae, i.e., Chrysomallon and Gigantopelta (Lan et al, 2021), since they are not recovered as sister genera and the other peltospirid snails are asymbiotic (Chen et al, 2015).…”