Alizarin red S staining of the crustacean cuticle: implementation in the study of Porcellio scaber larvae


  • Polona Mrak
  • Nada Žnidaršič
  • Jasna Štrus



calcification, histochemistry, larval development, terrestrial isopods, Crustacea


Exoskeletal cuticle of crustaceans is a chitinous matrix, produced apically by epidermis and stiffened by sclerotization and calcification. Embryos of terrestrial isopod crustacean Porcellio scaber develop within the female brood pouch, marsupium, and after hatching larvae mancae continue their development in the marsupium for another week. This study was performed to reveal at the histochemical level whether the exoskeletal cuticle of marsupial mancae is already calcified. Fifteen different procedures of histochemical staining with alizarin red S (ARS), established for calcified tissue localization primarily in vertebrate histology, were evaluated on mancae and adult P. scaber specimens. The best differential staining of the the exoskeletal cuticle was obtained by neutral buffered formaldehyde fixation, followed by paraffin sections staining with ARS 1 (pH 9) or ARS 2 (pH 6.4) or ARS 3 (pH 4.8)
solution. Clear differential staining was achieved also in cryosections of formaldehyde fixed samples, stained with ARS 1 solution (pH 9). Our results suggests that prominent calcification of exoskeletal cuticle is present during postembryonic development of P. scaber mancae in the marsupium. Exoskeleton hardening is likely important also for body movements, that we observed in mancae before they are released from marsupium. The proposed procedures of ARS method are presumed to be applicable for histochemical studies of other calcified chitinous matrices.


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Original Research Paper

How to Cite

Mrak, P., Žnidaršič, N., & Štrus, J. (2013). Alizarin red S staining of the crustacean cuticle: implementation in the study of Porcellio scaber larvae. Acta Biologica Slovenica, 56(2), 51-61.

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