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.


Bancroft, J.D., Gamble, M., 2008. Theory and Practice of Histological Techniques, 6th ed. Churchill Livingstone Elsevier, pp. 249–250.

Becker, A., Ziegler, A., Epple, M., 2005. The mineral phase in the cuticles of two species of Crustacea consists of magnesium calcite, amorphous calcium carbonate and amorphous calcium phosphate. Dalton Trans, 1814–1820. DOI:

Bonucci, E., 2007. Methodology. In: Schreck, S. (ed.): Biological calcification: Normal and Pathological Processes in the Early Stages. Springer – Verlag, Heidelberg, pp. 23–51.

Brečević, L., Nielsen, A.E., 1989. Solubility of amorphous calcium carbonate. J Cryst Growth, 98 (3), 504–510. DOI:

Gal, J.-Y., Bollinger, J.-C., Tolosa, H., Gache, N., 1996. Calcium carbonate solubility: a reappraisal of scale formation and inhibition. Talanta, 43, 1497–1509. DOI:

Hadley, N.F., Hendricks, G.M., 1987. X-ray microanalysis of the cuticle surface of the terrestrial isopod Porcellionides pruinosus. Can J Zool, 65, 1218–1223. DOI:

Hild, S., Marti, O., Ziegler, A., 2008. Spatial distribution of calcite and amorphous calcium carbonate in the cuticle of the terrestrial crustaceans Porcellio scaber and Armadillidium vulgare. Struct Biol, 163 (1), 100–108. DOI:

Hild, S., Neues, F., Žnidaršič, N., Štrus, J., Epple, M., Marti, O., Ziegler, A., 2009. Ultrastructure and mineral distribution in the tergal cuticle of the terrestrial isopod Titanethes albus. Adaptations to a karst cave biotope. J Struct Biol, 168, 426–436. DOI:

Hornung, E., 2011. Evolutionary adaptation of oniscidean isopods to terrestrial life: Structure, physiology and behavior. Terrestrial Arthropod Reviews, 4 (2), 95–130. DOI:

Kiernan, J.A., 2008. Histological and Histochemical Methods: Theory and Practice, 4th ed. Scion Publishing Limited, Bloxham, pp. 338–339.

Lievremont, M., Potus, J., Guillou, B., 1982. Use of alizarin red S for histochemical staining of Ca2+ in the mouse; some parameters of the chemical reaction in vitro. Acta anat, 114, 268–280. DOI:

Luquet, G., 2012. Biomineralization: insights and prospects from crustaceans. Zookeys, 176, 103–121. DOI:

McGee-Russell, S.M., 1958. Histochemical methods for calcium. J Histochem and Cytochem, 6, 22–42. DOI:

Meiron, O.E., Bar-David, E., Aflalo, E.D., Shechter, A., Stepensky, D., Berman, A., Sagi, A., 2011. Solubility and bioavailability of stabilized amorphous calcium carbonate. J Bone Miner Res, 26 (2), 364–372. DOI:

Milatovič, M., Kostanjšek, R., Štrus, J., 2010. Ontogenetic development of Porcellio scaber: Staging based on microscopic anatomy. J Crustacean Biol, 30 (2), 225–235. DOI:

Mrak, P., Žnidaršič, N., Tušek-Žnidarič, M., Klepal, W., Gruber, D., Štrus, J., 2012. Egg envelopes and cuticle renewal in Porcellio embryos and marsupial mancas. Zookeys, 176, 55–72. DOI:

Neues, F., Hild, S., Epple, M., Marti, O., Ziegler, A., 2011. Amorphous and crystalline calcium carbonate distribution in the tergite cuticle of moulting Porcellio scaber (Isopoda, Crustacea). J Struct Biol, 175 (1), 10–20. DOI:

Ouyang, D., Wright, J., 2005. Calcium accumulation in eggs and mancas of Armadillidium vulgare (Isopoda: Oniscidea). J Crustacean Biol, 25 (3), 420–426. DOI:

Presnell, J.K., Schreibmann, M.P., 1997. Humanson's Animal Tissue Techniqes, 5th edition. The Johns Hopkins University Press, Baltimore in London, pp. 223–224.

Puchtler, H., Meloan, S.N., Terry, M.S., 1969. On the history and mechanism of alizarin and alizarin red S stains for calcium. J Histochem and Cytochem, 17 (2), 110–124. DOI:

Seidl, B.H.M., Huemer, K., Neues, F., Hild, S., Epple, M., Ziegler, A., 2011. Ultrastructure and mineral distribution in the tergite cuticle of the beach isopod Tylos europaeus Arcanglei, 1938. J Struct Biol, 174, 512–526. DOI:

Seidl, B.H.M., Ziegler, A., 2012. Electron microscopic and preparative methods for the analysis of isopod cuticle. Zookeys, 176, 73–85. DOI:

Surbida, K.L., Wright, J.C., 2001. Embryo tolerance and maternal control of the marsupial environment in Armadillidium vulgare Brandt (Isopoda: Oniscidea). Physiol Biochem Zool, 74, 894–906. DOI:

Virtanen, P., Isotupa, K., 1980. Staining properties of alizarin red S for growing bone in vitro. Acta anat, 108, 202–207. DOI:

Warburg, M.R., 2011. The oniscid isopod female reproductive system and gestation, with a partial review. Invertebr Reprod Dev, 1–24.

Wolff, C., 2009. The embryonic development of the malacostracan crustacean Porcellio scaber (Isopoda, Oniscidea). Dev Genes Evol, 219, 545–564. DOI:

Ziegler, A., 1994. Ultrastructure and electron spectroscopic diffraction analysis of the sternal calcium deposits of Porcellio scaber Latr. (Isopoda, Crustacea). J Struct Biol, 112, 110–116. DOI:

Ziegler, A., 1997. Ultrastructural changes of the anterior and posterior sternal integument of the terrestrial isopod Porcellio scaber Latr. (Crustacea) during the moult cycle. Tissue Cell, 29 (1), 63–76. DOI:






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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