Primary crustacean cell culture was introduced in the 1960s, but to date limited cell lines have been established. Skogsbergia lerneri is a myodocopid ostracod, which has a body enclosed within a thin, durable, transparent bivalved carapace, through which the eye can see. The epidermal layer lines the inner surface of the carapace and is responsible for carapace synthesis. The purpose of the present study was to develop an in vitro epidermal tissue and cell culture method for S. lerneri. First, an optimal environment for the viability of this epidermal tissue was ascertained, while maintaining its cell proliferative capacity. Next, a microdissection technique to remove the epidermal layer for explant culture was established and finally, a cell dissociation method for epidermal cell culture was determined. Maintenance of sterility, cell viability and proliferation were key throughout these processes. This novel approach for viable S. lerneri epidermal tissue and cell culture augments our understanding of crustacean cell biology and the complex biosynthesis of the ostracod carapace. In addition, these techniques have great potential in the fields of biomaterial manufacture, the military and fisheries, for example, in vitro toxicity testing.
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.
The Skogsbergia lerneri is a marine ostracod which possesses a carapace that is both protective and transparent. Since development of this carapace and how it is maintained in the adult is not known, the aim of this investigation was to carry out an in-depth ultrastructural study of the ostracod carapace at different developmental stages. Standard transmission electron microscopy and novel serial block face scanning electron microscopy (SBF-SEM) were undertaken to discern carapace ultrastructure in both two and three dimensions. Analysis revealed a carapace consisting of the same basic layer structure as other myodocopid ostracods, namely an epicuticle, exocuticle, endocuticle and membranous layer, but with a thinner adult carapace of mean thickness of 19.2 ± 1.78 µm, n = 5. The carapace layers, except for instar 1 ostracods, had similar relative proportions throughout development. The endocuticle and membranous layer thickened through advancing developmental stages due to an increase in calcified crystalline polyhedrons and a greater number of chitinous lamellae in the membranous layer. Crystalline polyhedron dimensions were significantly smaller near the boundary with the membranous layer. The borders between the carapace layers were indistinct; SBF-SEM revealed an abundance of epicuticle projections into the exocuticle and apparent gradual merging at the boundary of the exocuticle and the endocuticle. Here, we discuss how the S. lerneri carapace layer structure has evolved to serve a specific mechanical function, allowing surface protection and rigidity. In addition, we suggest that the lack of pigment and graduated layer boundaries contribute to the transparency of the carapace.
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