Characterisation of carapace composition in developing and adult ostracods (Skogsbergia lerneri) and its potential for biomaterials

Abstract

The protective carapace of Skogsbergia lerneri, a marine ostracod, is scratch-resistant and transparent. The compositional and structural organisation of the carapace that underlies these properties is unknown. In this study, we aimed to quantify and determine the distribution of chemical elements and chitin within the carapace of adult ostracods, as well as at different stages of ostracod development, to gain insight into its composition. Elemental analyses included X-ray absorption near-edge structure, X-ray fluorescence and X-ray diffraction. Nonlinear microscopy and spectral imaging were performed to determine chitin distribution within the carapace. High levels of calcium (20.3%) and substantial levels of magnesium (1.89%) were identified throughout development. Amorphous calcium carbonate (ACC) was detected in carapaces of all developmental stages, with the polymorph, aragonite, identified in A-1 and adult carapaces. Novel chitin-derived second harmonic generation signals (430/5 nm) were detected. Quantification of relative chitin content within the developing and adult carapaces identified negligible differences in chitin content between developmental stages and adult carapaces, except for the lower chitin contribution in A-2 (66.8 ± 7.6%) compared to A-5 (85.5 ± 10%) (p = 0.03). Skogsbergia lerneri carapace calcium carbonate composition was distinct to other myodocopid ostracods. These calcium polymorphs and ACC are described in other biological transparent materials, and with the consistent chitin distribution throughout S. lerneri development, may imply a biological adaptation to preserve carapace physical properties. Realisation of S. lerneri carapace synthesis and structural organisation will enable exploitation to manufacture biomaterials and biomimetics with huge potential in industrial and military applications.

Keywords: Development; Energy-dispersive X-ray spectroscopy; Nonlinear microscopy; Ostracod; Second harmonic generation; Skogsbergia lerneri; Two-photon excited fluorescence; X-ray analysis near-edge structure; X-ray fluorescence.

Fig. 1

a Light microscope image of an adult S. lerneri ostracod showing the transparent carapace through which body parts can be observed. Representative cartoons showing b the area of the valve for energy-dispersive X-ray spectroscopy (EDS) and nonlinear imaging. Ostracod orientation is shown (anterior–posterior and ventral-dorsal) with the red dotted line indicating position from which tissue sections were cut. c Diagram of S. lerneri outer lamella ultra-structure identifying the distinct layers including the chitin (the exocuticle and the membranous layer), the crystalline endocuticle and the epidermal cell layers

Fig. 2

Ca K-edge offset XANES spectra showing internal absorption energy generated from 4–6 scan points on the long axis of S. lerneri valves of a A-5, b A-3, c A-1 and d adult ostracods. Each spectrum is labelled to denote the prominent form of CaCO₃; identified as ACC (amorphous calcium carbonate) or aragonite. Asterisks indicate the curve features that distinguish aragonite from ACC. e Contribution by either ACC or aragonite differed during development, with ACC present in all samples and aragonite only detected in A-1 and adult valves, n = 3

Fig. 3

Comparison of element levels in ostracod valves at different developmental stages in a magnesium, b phosphorous, c sulphur and d chlorine. *Represents p < 0.05. Error bars denote standard error, n = 3 except A-3 n = 2

Fig. 4

SEM images of a transverse section through the centre of an a adult S. lerneri and g A-5 valve at 3280 × magnification; ML membranous layer, En endocuticle, Ex exocuticle, EP epicuticle. Corresponding elemental maps are overlaid for adult b–f and A-5 h–l valves. An intense calcium signal was observed throughout the carapace depth. Phosphorus expression was highest within the epicuticle, the outermost layer of the carapace. m Element percentage weight through developmental stages. The band of higher signal expression is indicated with arrows, all scale bars = 25 µm. Asterisks indicate significant differences in percentage weight of elements (*p < 0.05 and ***p < 0.001), n = 4

Fig. 5

Development of a chitin-derived nonlinear signal. a Emission spectra derived from purified chitin flakes excited at 920 nm. A sharp peak can be seen at half the wavelength for SHG and a broad peak for TPEF. b SHG signals (emission wavelengths 455–465 nm) from purified chitin flakes, excited at 920 nm. c TPEF signals (emission wavelengths 480–670 nm) from purified chitin flakes, excited at 920 nm. d Emission spectra of an adult carapace section at a range of excitation wavelengths. All curves show a broad TPEF signal, however the sharpest peak i.e., the SHG signal, was observed at 840 nm, n = 3. e Emission spectra derived from the valves at developmental stages, A-5 to adult, using 840 nm excitation, n = 4. The area between the dashed lines represent the areas selected for SHG (red) and TPEF (black) expression

Fig. 6

Grayscale binary images created from SHG image datasets showing representative regions used to calculate percentage chitin-attributable SHG pixels in valves of a A-5, b A-4, c A-3, d A-2, e A-1 and f adult ostracods. All white pixels represent chitin-derived SHG signals. Scale bars: 10 µm, Ex represents the exocuticle, ML represents the membranous layer g Percentage chitin contribution to carapace at different developmental stages. Percentage chitin was similar at all stages, except for the lower values identified at A-2. Error bars represent ± SD and the asterisk denotes p < 0.05, n = 5