Brachionus calyciflorus, Pallas, 1776

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, Journal of Natural History 57, pp. 1984-2005 : 1991

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 ).

Figures

Figure 1. Characterisation of diapausing embryos of Brachionus calyciflorus. a. Scanning Electron Microscope image of a single egg. b. Close-up of the surface of the egg. c. Ultrastructure of the shell viewed with Transmission Electron Microscopy. d. Close-up of shell ultrastructure. e. Topographic image of the external surface of an egg generated with Atomic Force Microscopy. f. Force–distance (FD) curve generated from a single location on an egg. Parameters for the position of the location on the eggshell are indicated by the cursor positions (software screenshot). Maximum force (MF, green circle) and 20% maximum force (20% MF, orange circle) are indicated on the FD curve. Abbreviations: al, alveolar layer; ar, amorphous layer around embryo; em, embryo; s1, sublayer. Scale bars:A = 25 µm; B = 5 µm; C = 3.5 µm; D = 1.7 µm.