Recent advancements in electron backscatter diffraction (EBSD) data evaluation enable the determination of misorientation between crystals below 0.1°, while with conventional EBSD data evaluation, the smallest misorientation precision between crystals scatters between 0.5°-1°. Sea urchin tests and spines are lightweight biomaterials with a serrated microstructure comprising interlinked calcite crystals. We investigated the microstructure and crystallographic texture of Cidaris cidaris and Paracentrotus lividus test and spine calcite with advanced EBSD measurement and data evaluation. In particular, we re-evaluated the widely accepted single-crystallinity of sea urchin calcite. We found that the test and the spines comprise calcite crystals with different fabrics and a significant variation in crystal co-orientation strength. Even the highly co-oriented calcite of C. cidaris and P. lividus is not perfectly single-crystalline. We found test and spine portions that feature significant internal misorientations (1-3°). Test c-axis orientation in C. cidaris is tangential to the outer test surface, while in the spines, it is parallel to the morphological axis of the spine. Primary and secondary spines feature a bimodal crystal texture comprising co-oriented calcite surrounded by a cortex of misoriented crystals. Crystal misorientation in the spine cortex seems to result mainly from competitive growth determinants. Deciphering the degree of crystallinity and mode of crystal organisation of biological hard tissues is vital for understanding their exceptional control of structure, material architecture and material properties. STATEMENT OF SIGNIFICANCE: Echinoids form lightweight biomineralised skeletal elements with outstanding material properties and a complex microstructure formed of interlinked calcite crystals. The degree of crystallinity and the crystallographic organisation of the calcitic tests and spines are still under debate. We investigate and discuss the crystallinity, microstructure, and texture of Cidaris cidaris and Paracentrotus lividus test and spine crystals. Unprecedented and not yet used for biomineralised carbonate tissues, we apply electron backscatter diffraction pattern matching data evaluation, enabling detection of misorientation precision below 0.1°, relative to 0.5°-1° misorientation precision obtained from conventional EBSD data evaluation. We demonstrate that sea urchin test plates and spines are not single crystals. They feature internal small-angle misorientations and poorly co-oriented, polycrystalline regions with intricate microstructures.

Sea urchins are invertebrates that play a crucial role in marine ecosystems by controlling benthic algal communities and whose natural populations are being affected by different biotic and abiotic factors. Triggering physiological processes promotes the activation of certain metabolic pathways, so oxidative status markers could be a suitable tool to asses maturation stage in which natural populations are. Antioxidant status of three species of Mediterranean Sea urchins, A. lixula, P. lividus and S. granularis, was evaluated in gonadal and digestive tissue. Superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione s-transferase (GST), glucose 6-phosphate dehydrogenase (G6PDH), NAD(P)H: quinone oxidoreductase (NQO1) and lipid peroxidation were assayed. Significant differences were found among species, displaying in general higher antioxidant activity in A. lixula and S. granularis compared to P. lividus. A significant effect of sex was observed with females exhibiting a higher gonadosomatic index and higher levels of lipid peroxidation mainly in A. lixula. These results seem to be related to metabolic fluctuations associated with the gonadal maturation stage. Changes in digestive tissue were less evident, but some differences among species could be related to triggered digestive processes for replenishment of energy reserves in gonads. Oxidative status can be a useful complementary tool to evaluate gonadal condition in species of sea urchin from the same habitat. Integrative physiological and biochemical studies will contribute to the knowledge of invertebrate physiology.