Integrative Microscopy to Explore Physical and Nanomechanical Properties of Eggshells of Diapausing Embryos in Rotifera: a Proof-of-concept Study

Diapausing embryos of invertebrates represent investments in future populations. Thus, they must be capable of withstanding a variety of environmental assaults. Consequently, their eggshells should be adapted to resist injuries from predators, sediments, or excessive shrinkage if desiccated. To date, there have been no direct nanomechanical measurements of the eggshells of most invertebrates that produce diapausing eggs. Here, we used three approaches to understand how the eggshells of two rotifers, a freshwater species (Brachionus calyciflorus) and a brackish-water species (B. plicatilis), tolerate harsh conditions: (1) atomic force microscopy to measure elasticity and hardness; (2) transmission electron microscopy to study ultrastructure; (3) scanning electron microscopy to examine surface features. We compared these values to measurements of brine shrimp (Artemia salina) cysts and mosquito (Aedes aegypti) overwintering eggs. Our results revealed that rotifer eggshells are structurally similar and have comparable nanomechanical values. While rotifer eggshells had lower Young's moduli (ca 13-16 MPa) and hardness values (1.84-1.85 x 10-2 GPa) than eggshells of Artemia and Aedes, eggshells of all species were relatively elastic and not particularly resistant to deformation. Pliancy of shells that form egg banks (ie Artemia, Brachionus) may be an adaptation to resist cracking under the physical forces associated with burial in sediments. Although there are no obvious relationships among eggshell thickness, ultrastructure, ornamentation, or nanomechanical values in rotifer eggshells, we hypothesise that eggshell composition may play an important role in determining elasticity and hardness. Future studies should consider an integrative approach to understand the importance of eggshell structure, chemistry, and mechanics in protecting diapausing embryos.