Composition and structural organization of tardigrades feeding apparatus focusing on chitin and other autofluorescent molecules

Chitin is a structural sugar broadly distributed among all kingdoms, and it is present in Tardigrada as & alpha;-chitin. Its presence in the buccopharyngeal apparatus of tardigrades was confirmed in previous studies and associated with autofluorescence emission, and within this emission three components were identified. This study aimed to localize the chitin in the buccopharyngeal apparatus and to understand the nature of the other autofluorescent components. Calcofluor white staining of three tardigrade species (Echiniscus sp., Milnesium sp., Macrobiotus sp.) allowed the chitin localization in the feeding structures and the separation of its signal from the whole autofluorescence emission via confocal laser scanning microscopy. Comparing different fluorochromes found in tardigrades and in the nematode Panagrellus pycnus, lambda and square lambda analyses showed that part of the autofluorescent signal is imputable to a collagen-like compound. The images acquired were analysed for morphological, functional, and evolutionary purposes. Chitin and the other autofluorescent compound were localized in the structures of the buccopharyngeal apparatus. Their distribution highlighted the presence of different levels of 'flexibility/strength' of the structures providing better understanding of their mechanical properties and relating them to the characteristics of the three major limnoterrestrial clades of the phylum Tardigrada.