Trematode worms have the neoophoran mode of development in which several specialized vitelline cells surround the zygote. This vitelline cell mass appears just before the zygote passes through the ootype, a thickening of the oviduct, where the egg shell is formed. The great amount of vitelline material blurs the visualization of embryo development in whole egg seen by brightfield microscopy. The eggshell is difficult to cut into thin or ultrathin sections and acts as a barrier to fixation and infiltration with embedding media. The egg shell is also brightly fluorescent when analyzed by fluorescence microscopy. To overcome these technical disadvantages a simple staining protocol widely used in adult helminth morphological analysis was adapted for the study of the embryonic development of two different trematode species. The effects of potassium hydroxide as bleach and ethylene glycol as mounting medium were also evaluated. Confocal microscopy allowed virtual sectioning of whole-mounted eggs and made possible internal morphological detailed analysis of different embryonic stages. This method could contribute to the study of helminth egg embryology.
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The authors thank Pedro Paulo de Abreu Manso from the Laboratório de Patologia, IOC, for his assistance with CLSM analyses; and Dr. Paulo Marcos Zech Coelho from the Laboratório de Esquistossomose, Instituto René Rachou-Fiocruz, MG, and Dr. John Kusel from the Glasgow University for critical review of the manuscript. A.D.J. received a scholarship from the Programa Institucional de Bolsas de Iniciação Científica from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (PIBIC/CNPq). The work was supported by Fundação Oswaldo Cruz (Fiocruz) and CNPq, Brazil.
Communicated by D.A. Weisblat
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Jurberg, A.D., Pascarelli, B.M., Pelajo-Machado, M. et al. Trematode embryology: a new method for whole-egg analysis by confocal microscopy. Dev Genes Evol 218, 267–271 (2008). https://doi.org/10.1007/s00427-008-0209-0
- Schistosoma mansoni
- Echinostoma paraensei
- Carmine staining
- Confocal microscopy