The origin of the mechanical properties
In some taxa (i.e., Polyplacophora or Patellogastropoda), high proportions of inorganics, such as iron, silicon, and calcium, are incorporated into some tooth cusps which directly relate to mechanical property differences in the various tooth regions (for in depth reviews, see Brooker & Shaw, 2012; Faivre & Ukmar-Godec, 2015; Joester & Brooker, 2016). In Gastropteron rubrum , each tooth showed mechanical property gradients in its inner structure; but we could not relate these gradients with the inorganic content.
As the radula is composed of an organic matrix of chitin fibers with associated proteins (Runham, 1963; Guralnick & Smith, 1999), the fiber architecture (i.e., fiber density, size, etc.) can promote regional mechanical differences (Runham et al., 1963; van der Wal, 1989; Evans et al., 1990, 1994; van der Wal et al., 1999; Wealthall et al., 2005; Shaw et al., 2010; Gordon & Joester, 2011; Lu & Barber, 2012; Wang et al., 2013; Grunenfelder et al., 2014; Ukmar-Godec, 2016; Ukmar-Godec et al., 2017; Stegbauer et al., 2021; Krings et al., 2020, 2022a). Whether this is the case with Gastropteron rubrum awaits further investigations, e.g., in the form of tooth section investigations in TEM.
Chitin can also show different regional degrees of tanning, which result in different mechanical properties. The degree of tanning can be visualized by applying CLSM, applying the protocol of (Michels & Gorb, 2012). This protocol was developed for insect cuticle, which consists of unmineralized chitin. It allowed previously the identification of cuticle regions with certain dominating material composition: (a) Blue signals (i.e., autofluorescence signal produced by the laser of wavelength 405 nm) in cuticule was induced from regions containing high proportions of resilin or proteins; these regions were relatively soft and flexible. (b) Sclerotized cuticle was associated with a red signal (i.e., autofluorescence signal produced by the laser of wavelengths 555 and 639 nm); this region was relatively hard and stiff. (c) Weakly-sclerotized chitin with a green signal (i.e., autofluorescence signals produced by the laser of wavelength 488 nm) indicated regions which were flexible and relatively tough. When proteins were abundant, those structures appeared brown, yellow, or pink in overlay (i.e., showed strong autofluorescence signals produced by the laser of wavelengths 405 and 488 nm). This protocol (Michels & Gorb 2012) was applied in many studies on arthropod cuticles (e.g., Peisker et al., 2013; Friedrich & Kubiak, 2018; Beutel et al., 2020, Matsumura et al., 2021; Lehnert et al., 2021) and cross-validated by employing nanoindentation in lady beetles and antlions (Peisker et al., 2013; Krings & Gorb, 2023b).
The same protocol was applied for the first time bei Krings et al. (2022d) to the radula of the paludomid Lavigeria grandis , which possesses relatively low mineral content. Here, the autofluorescence signals directly related to the mechanical property values received from nanoindentation technique. We also applied this protocol for the radulae of the nudibranch gastropods Felimare picta and Doris pseudoargus , but detected that the autofluorescence signal was distorted by the content of Ca and Si in the tooth surfaces (Krings et al., 2023). Nudibranch teeth with surfaces full of Ca showed a strong blue signal (i.e., autofluorescence signal produced by the laser of wavelength 405 nm), and teeth containing high Si content in the surfaces a strong green signal (i.e., autofluorescence signal produced by the laser of wavelength 488 nm). This pattern was previously also detected in crustacean feeding structures, the gnathobases and gastric mill teeth, containing either high content of Ca or Si (Michels et al., 2012; Michels & Gorb, 2012; Krings et al., 2022b). In G. rubrum , we detected that the surfaces containing Si appeared green (i.e., showed a strong autofluorescence signal after excitement by the laser of wavelength 488 nm). This shows that the protocol of Michels & Gorb (2012) cannot be directly applied to mineralized structures and that EDX analyses should be included into studies. However, the tooth sides ofG. rubrum did not contain high proportions of Ca and Si, so that the protocol can be applied to these regions. The results indicate, that the teeth are probably sclerotized with a decreasing degree towards the basis. The bases and bulges of G. rubrum teeth were unmineralized and appeared blue (i.e., showed a strong autofluorescence signal after excitement by the laser of wavelength 405 nm), which indicates that less crosslinked organic materials are abundant in these regions, which probably increase both the softness and flexibility of material.