Research Article: Correlative and Dynamic Imaging of the Hatching Biology of Schistosoma japonicum from Eggs Prepared by High Pressure Freezing

Date Published: November 11, 2008

Publisher: Public Library of Science

Author(s): Malcolm K. Jones, Sze How Bong, Kathryn M. Green, Philadelphia Holmes, Mary Duke, Alex Loukas, Donald P. McManus, Jennifer Keiser

Abstract: BackgroundSchistosome eggs must traverse tissues of the intestine or bladder to escape the human host and further the life cycle. Escape from host tissues is facilitated by secretion of immuno-reactive molecules by eggs and the formation of an intense strong granulomatous response by the host which acts to exclude the egg into gut or bladder lumens. Schistosome eggs hatch on contact with freshwater, but the mechanisms of activation and hatching are poorly understood. In view of the lack of knowledge of the behaviour of egg hatching in schistosomes, we undertook a detailed dynamic and correlative study of the hatching biology of Schistosoma japonicum.Methodology/Principal FindingsHatching eggs of S. japonicum were studied using correlative light and electron microscopy (EM). The hatching behaviour was recorded by video microscopy. EM preparative methods incorporating high pressure freezing and cryo-substitution were used to investigate ultrastructural features of the miracidium and extra-embryonic envelopes in pre-activated and activated eggs, and immediately after eggshell rupture. Lectin cytochemistry was performed on egg tissues to investigate subcellular location of specific carbohydrate groups.Conclusions/SignificanceThe hatching of S. japonicum eggs is a striking phenomenon, whereby the larva is liberated explosively while still encapsulated within its sub-shell envelopes. The major alterations that occur in the egg during activation are scission of the outer envelope-eggshell boundary, autolysis of the cellular inner envelope, and likely hydration of abundant complex and simple polysaccharides in the lacunal space between the miracidial larva and surrounding envelopes. These observations on hatching provide insight into the dynamic activity of the eggs and the biology of schistosomes within the host.

Partial Text: The pathology associated with chronic schistosomiasis is related to host responsiveness to antigens released by schistosome eggs entrapped in tissues [1],[2]. The primary source of the secreted antigens in developing eggs is a distinct extra-embryonal layer that surrounds the differentiating embryo (miracidium) [3]. This layer has been variously called the Reynold’s layer or the outer envelope (OE), and is derived in early development from cells that delaminate from the embryo [3]–[5]. Secreted antigens, released through preformed pores in the shell [3],[4], consist of a range of peptides and glycans, particularly those containing core mucins and fucose [3],[6],[7].

This study, the first detailed study of the ultrastructure of S. japonicum eggs, shows that the use of HPF as a means of immobilizing the eggs and egg contents provided a superior method of specimen preparation compared with other methods used previously for studies of S. mansoni eggs [3]–[5]. The structure of the eggshell and its sub-shell compartments were preserved with high quality and without freezing artifacts. Despite the use of osmium tetroxide as fixative, some cytochemical labelling using biotinylated lectins in conjunction with colloidal gold probes was possible, as has been demonstrated for studies on insects and nematodes [24]. The ability of the technique to immobilize eggs during hatching, allowing morphological and cytochemical observations, helps overcome problems brought about by the impervious shell [25]. The method provides a means of adding valuable data on the biological roles of drugs, such as praziquantel.



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