Energy dispersive X-ray spectroscopy (EDX)
For analysis of the elemental composition, three cleaned radulae
(ultrasonic bath for 20 seconds) were attached to glass object slides by
double-sided adhesive tape, following our previous protocol (Krings et
al., 2022a, 2022c, 2023). Then, each radula was surrounded by a small
metallic ring. Afterwrads the ring was filled with epoxy resin (Reckli
Epoxy WST, RECKLI GmbH, Herne, Germany) to cover the readula completely.
After polymerization, lasting for three days at room temperature, glass
object slides and adhesive tape were removed. Samples were polished with
sandpapers of different roughness until teeth were on display and
smoothened with aluminum oxide polishing powder suspension of 0.3 μm
grainsize (PRESI GmbH, Hagen, Germany) on a polishing machine (Minitech
233/333, PRESI GmbH, Hagen, Germany) to receive a plain smooth surface.
The embedding and smoothening prevent artefacts such as electron
scattering during EDX analysis. Embedded samples were subsequently
cleaned in an ultrasonic bath for five minutes, then mounted on SEM
sample holders and sputter-coated with platinum (5 nm layer). Elemental
composition was determined with the SEM Zeiss LEO 1525 equipped with an
Octane Silicon Drift Detector (SDD) (micro analyses system TEAM, EDAX
Inc., New Jersey, USA). For each sample, the same settings were used
(i.e. an acceleration voltage of 20 kV, working distance, lens opening,
etc.). Before analysis, the detector was calibrated with copper.
Small areas (no mapping) were analyzed to receive the data. Following
elements were detected and their proportions measured.: H (hydrogen), C
(carbon), N (nitrogen), O (oxygen), Pt (platinum), Al (aluminum), Ca
(calcium), Cl (chlorine), Cu (copper), Fe (iron), K (potassium), Mg
(magnesium), Na (sodium), P (phosphorus), S (sulphur), Si (silicon), and
Zn (zinc). Some elements were not discussed as they are either the
elemental basis of chitin and proteins (H, C, N, O), the coating (Pt),
or the polishing powder (Al, O). For test purposes, we also performed 10
EDX tests on the epoxy to identify putative pollution due to the
mechanical application, embedding or polishing. We could not detect Si
(which is part of the sandpaper), or any other elements, that we further
discuss as Ae (Ca, Cl, Cu, Fe, K, Mg, P+Pt, S, Si, Zn), in the resin.
Their presence is therefore considered part of the teeth.
The single peak of P overlaps with one of Pt. Due of this, the software
could not discriminate between these two elements and P content could
not be reliably determined. Therefore, P and Pt are discussed together
(P+Pt). We, however, measured 20 areas of pure epoxy to receive values
on their Pt content (mean ± SD; 0.15 ± 0.02 atomic %) to further
estimate the proportions of P in the teeth.
We tested the inner tooth structure by EDX and the thin outer layer
(“surface”) of the teeth (500–1000 nm thickness), which covers the
inner tooth structure. We did not detect high content of elements in the
inner structure and no differences in the distribution there. We thus
decided to summarize the point measurements of the inner structure. With
regard to the surfaces, we could determine variations in the
distribution of elements and thus differentiated between the tooth
basis, the bulges, the basal region of the stylus (stylus, basis), the
terminal region of the stylus (stylus, terminal), the cusps and the
sides (see Figures 1 and 2 for nomenclature). 474 point measurements on
180 mature teeth were conducted: 204 on 70 inner laterals (of which 70
on the inner tooth structure and 134 on the surface); 56 (22 on the
inner tooth structure and 34 on the surface) on 22 outer laterals A, B,
C; 54 (22 on the inner tooth structure and 32 on the surface) on 22
outer laterals D; 48 (22 on the inner tooth structure and 26 on the
surface) on 22 outer laterals E.