Arrangement of spines
Figure 1 shows an optical microscope image and a microfocus X-ray CT image of a whole skeleton of Acanthometra cf. multispina. There are 20 radial spines (“spine” hereafter) radiating from the skeletal center, as illustrated in Fig. 1c. The arrangement of four equatorial spines (e), four diametric (= 8) tropical spines (t), and four diametric (= 8) polar spines (p) agrees with Müller’s law5 (Fig. 1b). Given a unit sphere (Fig. 1d) whose origin is defined at the skeletal center and whose x- and y-axes are designed to the equatorial spines, the deviation angles of tropical and polar spines from the equatorial plane are 30° and 60°, respectively. The equatorial plane is defined by the plane with x- and y-axes.
A more detailed spine arrangement is displayed in the enlarged microfocus X-ray CT images (Figure 2). On the polar-view images (Figure 2b), four equatorial spines and four diametric (= 8) polar spines are at the same positions with fourfold rotational symmetry, and four diametric (= 8) tropical spines are located at intermediate positions. All the spines have four blades around their root and connected through the blades (Figure 2b-ii). From the top views of the spines, we characterized the connecting modes of the spines around the skeletal center. A polar spine is connected to two other polar and two tropical spines (Figure 2c). A tropical spine is linked to two polar and two equatorial spines (Figure 2d). An equatorial spine is directly attached to four tropical spines (Figure 2e). The connecting angles around the equatorial and polar spines are almost the same with a twofold rotational symmetry (Figure 2c, e). The arrangement of the four blades agrees with that reported in a previous study7. On the other hand, the blades of a tropical spine are arranged in a mirror symmetry (Figure 2d).
Micrometric morphology and structure of spines
We revealed the morphology of a spine from several cross-sectional images produced by microfocus X-ray CT. Figure 3 shows cross sections of the polar and tropical spines of small and large specimens. Basically, the shape of the cross section of a spine is rectangular around the root but ellipsoidal in the remaining parts, including the distal end. As mentioned above, four blades are attached around their root. By comparing the fletching root of a small specimen with a large specimen, the bladed parts are roughly equal in size regardless of the different total lengths of the spines. This suggests that the spines elongate from the distal end with ontogenetic growth. The equatorial spines are 1.1–1.3 times longer than the tropical and polar spines (Table S1 in the Supporting Information (SI)).
Figure 4 exhibits SEM images of the partly broken central part, showing the connection among the Fletching-roots of the spines. The spines with blades are found to be separated at the skeletal center. This indicates that the spines are indirectly connected through the blades. The junction planes of the blades are teardrop-shaped.
Figure 5 shows the specimens before and after ethylenediaminetetraacetic acid (EDTA) treatment. By using EDTA, skeletons collapsed upon immersion. The removal of the solid skeleton by this treatment was confirmed by elemental analysis using an energy-dispersive X-ray spectrometer (EDS) (Figure S2 in the SI). This indicates the dissolution of celestite in the skeleton. After dissolution of the solid skeleton, we confirmed an organic membrane covering the spines. From an SEM image of a cross section of a broken spine (Figure 5e), the thickness of the membrane is estimated to be approximately 100 nm. We characterized the organic membranes with a deposition of silver particles to observe surface-plasmon-enhanced Raman spectra (Figure 5f). According to the spectra, the membranes are deduced to be mainly composed of chitin ((C8H13O5N)n). Thus, the spines are enveloped by a chitin-based organic membrane.
Nanometric and crystallographic structures of spines
Figure 6 shows SEM and TEM images of spines with a typical SAED pattern. From the diffraction spots, the spines are assigned to celestite that is elongated in the a-axis direction and that has a single-crystal nature. The blades are suggested to expose the {110} planes. These facts about the crystallographic structure are in agreement with the assignment in a previous work24.
The spines were also assigned to celestite using Raman spectroscopy (Figure 7a). Interestingly, however, we observed a slight shift in the signal due to the S-O asymmetric stretching vibration to a lower wavenumber. This suggests the presence of the lattice strain of a celestite crystal in the skeleton. The strain was recovered after calcination at 600°C for 20 h in air. Figure 7b and 7c shows SEM images of spines after removal of the organic matter by calcination at 600°C for 4 h in air and subsequent etching with pure water for 2 h. Although the associated organic matter was removed with mild calcination for 4 h, the strain remained in the crystalline lattice. We observed fibrous units ∼100 nm wide on the spine surface. Tilted faces are assignable to the (210) plane by comparing the shape of artificially produced celestite crystals25. These results suggest that the spines are not a homogeneous single crystal but a bundle of fibrous units elongated in the a-axis direction. Finally, we conclude that the acantharian skeleton is composed of a celestite mesocrystal consisting of nanoscale units that are arranged in the same crystallographic orientation.
As shown in Figure S3 in the SI, we succeeded in producing celestite mesocrystals consisting of fibrous units. The bundled structure was formed through precipitation in a supersaturated solution containing poly(acrylic acid). The fibrous units that are elongated in the a-axis direction are arranged in the same orientation. The lattice strain of the artificial celestite mesocrystal is similar to that of the acantharian spines. Since the organic content of the products was estimated to be ca. 3wt%, almost the same amounts of organic molecules are deduced to be included in the biological celestite.