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E. Hollergschwandtner, T. Schwaha, J. Neumüller, U. Kaindl, D. Gruber, M. Eckhard, M. Stöger-Pollach, S. Reipert:
"Novel mesostructured inclusions in the epidermal lining of Artemia franciscana ovisacs show optical activity";
PeerJ, 3923 (2017), S. 1 - 25.

Background. Biomineralization, e.g., in sea urchins or mollusks, includes the assembly
of mesoscopic superstructures from inorganic crystalline components and biopolymers.
The resulting mesocrystals inspire biophysicists and material scientists alike, because
of their extraordinary physical properties. Current efforts to replicate mesocrystal
synthesis in vitro require understanding the principles of their self-assembly in vivo.
One question, not addressed so far, is whether intracellular crystals of proteins can
assemble with biopolymers into functional mesocrystal-like structures. During our
electron microscopy studies into Artemia franciscana (Crustacea: Branchiopoda), we
found initial evidence of such proteinaceous mesostructures.
Results. EM preparations with high-pressure freezing and accelerated freeze substitution
revealed an extraordinary intracellular source of mesostructured inclusions in both
the cyto-and nucleoplasm of the epidermal lining of ovisacs of A. franciscana. Confocal
reflection microscopy not only confirmed our finding; it also revealed reflective, light
dispersing activity of these flake-like structures, their positioning and orientation with
respect to the ovisac inside. Both the striation of alternating electron dense and electronlucent
components and the sharp edges of the flakes indicate self-assembly of material of
yet unknown origin under supposed participation of crystallization. However, selected
area electron diffraction could not verify the status of crystallization. Energy dispersive
X-ray analysis measured a marked increase in nitrogen within the flake-like inclusion,
and the almost complete absence of elements that are typically involved in inorganic
crystallization. This rise in nitrogen could possibility be related to higher package
density of proteins, achieved by mesostructure assembly.
Conclusions. The ovisac lining of A. franciscana is endowed with numerous mesostructured
inclusions that have not been previously reported. We hypothesize that their
self-assembly was from proteinaceous polycrystalline units and carbohydrates. These
mesostructured flakes displayed active optical properties, as an umbrella-like, reflective
cover of the ovisac, which suggests a functional role in the reproduction of
A. franciscana. In turn, studies into ovisac mesostructured inclusions could help to
optimizing rearing Artemia as feed for fish farming. We propose Artemia ovisacs as an
in vivo model system for studying mesostructure formation.

Artemia franciscana, Brine shrimps, Confocal reflection microscopy, Rapid freeze substitution, Ovisac, Reproduction, Cellular inclusions, Mesocrystals, Transmission electron microscopy

http://dx.doi.org/10.7717/peerj.3923