Phylum Rotifera: Brachionus calyciflorus SEM The ovoid, diapausing embryos measured ca 139 µm in diameter (̄x = 138.4 ± 5.8 μm). Shells appeared largely devoid of ornamentation at lower magnifications (<1000×), but showed a more rugose surface at higher magnifications (Figure 1a,b). Shells often appeared pitted with small pores and craters (0.07–2.96 µm diameter) and occasionally some surface bumps of various textures. TEM The shell consisted of two layers – a thick apical layer and a thin basal layer – that together averaged 7.20 ± 1.45 µm (SD) thick (Figure 1c). The surface of the shell had a wavy appearance and consisted of high ridges and numerous small bumps and valleys. Immediately beneath this was a thick zone of empty pockets that were interconnected through a framework of electron-dense bridges; this layer is described by Wurdak et al. (1978) as the alveolar layer (al: Figure 1d). Immediately beneath the alveolar layer was a zone [s1: Wurdak et al. (1978)] of less opaque electron density that consisted of two subzones, each containing many fine laminae, and separated by an electron-dense line. An extraembryonic space of variable size separated the physical shell from an amorphous region (ar) that was proximal to the plasma membrane of the embryo. AFM Topography of the outer shell layer was similar to that viewed with SEM, but pores were less visible (̄x = 0.23 µm diameter) and surface bumps were less recognisable (Figure 1e). An example force–distance curve is provided (Figure 1f) that displays how Young̾s modulus values were collected for a single spot on one egg. The Young̾s modulus for two diapausing embryos ranged from 9.73 to 17.21 MPa with an average of 13.62 ± 2.43 MPa (SD) (Table 1). Hardness values ranged from 1.62 × 10−2 to 2.18 × 10−2 GPa with an average hardness of 1.80 × 10−2 GPa ± 1.80 × 10−3 GPa (Table 2).

Published as part of Meyer, Stephanie, Araujo, Thiago Q., Walsh, Elizabeth J., Wallace, Robert L. & Hochberg, Rick, 2023, Integrative microscopy to explore physical and nanomechanical properties of eggshells of diapausing embryos in Rotifera: a proof-of-concept study, pp. 1984-2005 in Journal of Natural History 57 on page 1991, DOI: 10.1080/00222933.2023.2279255, http://zenodo.org/record/10479029