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

Authors

  • Polona Mrak
  • Nada Žnidaršič
  • Jasna Štrus

DOI:

https://doi.org/10.14720/abs.56.2.16125

Keywords:

calcification, histochemistry, larval development, terrestrial isopods, Crustacea

Abstract

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.

References

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: https://doi.org/10.1039/b412062k

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: https://doi.org/10.1016/0022-0248(89)90168-1

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: https://doi.org/10.1016/0039-9140(96)01925-X

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: https://doi.org/10.1139/z87-189

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: https://doi.org/10.1016/j.jsb.2008.04.010

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: https://doi.org/10.1016/j.jsb.2009.07.017

Hornung, E., 2011. Evolutionary adaptation of oniscidean isopods to terrestrial life: Structure, physiology and behavior. Terrestrial Arthropod Reviews, 4 (2), 95–130. DOI: https://doi.org/10.1163/187498311X576262

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: https://doi.org/10.1159/000145596

Luquet, G., 2012. Biomineralization: insights and prospects from crustaceans. Zookeys, 176, 103–121. DOI: https://doi.org/10.3897/zookeys.176.2318

McGee-Russell, S.M., 1958. Histochemical methods for calcium. J Histochem and Cytochem, 6, 22–42. DOI: https://doi.org/10.1177/6.1.22

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: https://doi.org/10.1002/jbmr.196

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: https://doi.org/10.1651/09-3189.1

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: https://doi.org/10.3897/zookeys.176.2418

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: https://doi.org/10.1016/j.jsb.2011.03.019

Ouyang, D., Wright, J., 2005. Calcium accumulation in eggs and mancas of Armadillidium vulgare (Isopoda: Oniscidea). J Crustacean Biol, 25 (3), 420–426. DOI: https://doi.org/10.1651/C-2564

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: https://doi.org/10.1177/17.2.110

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: https://doi.org/10.1016/j.jsb.2011.03.005

Seidl, B.H.M., Ziegler, A., 2012. Electron microscopic and preparative methods for the analysis of isopod cuticle. Zookeys, 176, 73–85. DOI: https://doi.org/10.3897/zookeys.176.2294

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: https://doi.org/10.1086/324474

Virtanen, P., Isotupa, K., 1980. Staining properties of alizarin red S for growing bone in vitro. Acta anat, 108, 202–207. DOI: https://doi.org/10.1159/000145301

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: https://doi.org/10.1007/s00427-010-0316-6

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: https://doi.org/10.1006/jsbi.1994.1012

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: https://doi.org/10.1016/S0040-8166(97)80073-0

Downloads

Published

01.12.2013

Issue

Section

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. https://doi.org/10.14720/abs.56.2.16125

Similar Articles

1-10 of 52

You may also start an advanced similarity search for this article.

Most read articles by the same author(s)