Advertisement

Meiosis pp 235-249 | Cite as

Methods for Meiotic Chromosome Preparation, Immunofluorescence, and Fluorescence in situ Hybridization in Daphnia pulex

  • Dai Tsuchiya
  • Brian D. Eads
  • Miriam E. Zolan
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 558)

Abstract

The genus Daphnia has an intriguing reproductive mode of cyclical parthenogenesis. This reproductive mode has been studied for centuries, but cytogenetic information is lacking due to technical limitations of classical methods. We have developed methods for the preparation and examination of meiotic chromosomes of Daphnia pulex from oocytes and spermatocytes. Oocyte chromosome preparations are obtained by isolating individual oocytes after the release of yolk granules from the ovary using pressure and capillary action. Spermatocyte chromosomes are prepared using a conventional squash method. Cryosectioning is an easy and fast way to prepare sections. We also illustrate the application of immunofluorescence staining against α tubulin, as well as fluorescence in situ hybridization (FISH) using the intergenic spacer of ribosomal DNA or single-copy cosmid clones.

Key words

Chromosomes meiosis oocyte spermatocyte Daphnia FISH immunofluorescence staining cryosection D. pulex 

Notes

Acknowledgments

We would like to thank Armin Moczek and Teiya Kijimoto for their help with cryosectioning as well as helpful suggestions. This work was supported by National Science Foundation grant FIBR-0328516.

References

  1. 1.
  2. 2.
    Zaffagnini, F. (1987) Reproduction in Daphnia, in Daphnia memorie (Peters, R.H. and de Bernardi, R., eds.) 45, pp. 245–284.Google Scholar
  3. 3.
    Hoskins, R.A., Carlson, J.W., Kennedy, C., Acevedo, D., Evans-Holm, M., Frise, E., Wan, K.H., Park, S., Mendez-Laqo, M., Rossi, F., Villasante, A., Dimitri, P., Karpen, G.H., and Celniker, S.E. (2007) Sequence finishing and mapping of Drosophila melanogaster heterochromatin. Science. 316, 1625–1628.PubMedCrossRefGoogle Scholar
  4. 4.
    Fritsch, C., Ploeger, G., and Arndt-Jovin, D.J. (2006) Drosophila under the lens: imaging from chromosomes to whole embryos. Chromosome Res. 14, 451–464.PubMedCrossRefGoogle Scholar
  5. 5.
    MacQueen, A.J., Phllips, C.M., Bhalla, N., Weiser, P., Villeneuve, A.M., and Dernburg, A.F. (2005) Chromosome sites play dual roles to establish homologous synapsis during meiosis in C. elegans. Cell. 123, 1037–1050.PubMedCrossRefGoogle Scholar
  6. 6.
    Alpi, A., Pasierbek, P., Gartner, A., and Loidl, J. (2003) Genetic and cytological characterization of recombination protein RAD-51 in Caenorhabditis elegans. Chromosoma. 112, 6–16.PubMedCrossRefGoogle Scholar
  7. 7.
    Ojima, Y. (1958) A cytological study on the development and maturation of the parthenogenetic and sexual eggs of Daphnia pulex (Crustacea-Cladocera). Kwansei Gakuin Unic. Ann. Studies. 6, 123–176.Google Scholar
  8. 8.
    Zaffagnini, F. and Sabelli, B. (1972) Karyologic observations on the maturation of the summer and winter eggs of Daphnia pulex and Daphnia middendorffiana. Chromosoma. 36, 193–203.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Dai Tsuchiya
    • 1
  • Brian D. Eads
    • 1
  • Miriam E. Zolan
    • 1
  1. 1.Department of BiologyIndiana UniversityBloomingtonUSA

Personalised recommendations