Parasitol Res DOI 10.1007/s00436-012-3066-8
ORIGINAL PAPER
Insight into the ultrastructural organisation of sporulated oocysts of Eimeria nieschulzi (Coccidia, Apicomplexa) Eric Seemann & Thomas Kurth & Rolf Entzeroth
Received: 11 July 2012 / Accepted: 20 July 2012 # Springer-Verlag 2012
Abstract Sporulated oocysts of Eimeria contain four sporocysts with two sporozoites each and a sporocyst residuum. The developing sporozoites are protected by the sporocyst wall and the robust double-layered oocyst wall. Because of problems with conventional fixatives, high-pressure freezing, followed by freeze substitution was used to achieve optimal ultrastructural preservation of oocysts, sporocysts and sporozoites. After embedding in Epon®, ultrathin sections were examined by electron microscopy to select specific oocyst regions for further investigation by electron tomography (ET). ET allows high-resolution three-dimensional views of subcellular structures within the oocysts and sporocysts. Analysis of several 300 nm sections by ET revealed a network of small tubular structures with a diameter of 70–120 nm inside the sporocysts which is decribed here for the first time. This network connects the residual body in a sporocyst with the endoplasmic reticulum (ER) of the surrounding sporozoites. The network consists of membrane-bound tubules that contain vesicles but no larger organelles like mitochondria. These tubules, named “sporocord”, may have a function similar to an “umbilical cord” providing the sporozoites with metabolites for long-term survival. Small vesicular structures inside E. Seemann : R. Entzeroth (*) Institute of Zoology, TU Dresden, Zellescher Weg 20B, 01217 Dresden, Germany e-mail:
[email protected] T. Kurth DFG-Center for Regenerative Therapies Dresden, TU Dresden, Fetscherstraße 105, 01307 Dresden, Germany Present Address: E. Seemann Institut für Biochemie I, Universitätsklinikum Jena, Nonnenplan 2, 07743 Jena, Germany
the ER of the sporozoites, multivesicular structures inside the residual bodies and vesicles in the tubules support this hypothesis.
Introduction The rat coccidium Eimeria nieschulzi belongs to the phylum Apicomplexa (Mehlhorn and Armstrong 2001), which is part of the Alveolata (Protista) (Adl et al. 2005). Some Apicomplexa species cause severe parasitic diseases like malaria (Plasmodium falciparum), toxoplasmosis (Toxoplasma gondii) and coccidiosis (Eimeria, Isospora). E. nieschulzi is used as a “model organism” because it can be easily propagated in rats (Rattus norvegicus), has a one-host life cycle and can partially be cultured in vitro. The life cycle consists of an asexual multiplication phase (schizogony/merogony) followed by sexual stages (macrogamonts, microgamonts). After fertilization, zygotes encyst to form oocysts that have at least two layers in their wall. Under influence of oxygen and temperature, oocysts “sporulate” (i.e. become infective) and form sporocysts, each containing two sporozoites. This process needs 2–3 days and results in resistant oocysts capable of surviving for months in the external environment. One of the characteristic features within each sporocyst of E. nieschulzi is a structure called a sporocyst residuum (SR); the SR results from the cell divisions during sporulation (Long 1982), but its precise function is unknown. Ultrastructural analysis of the morphological details of sporozoites is hampered by the protective oocyst wall, which serves as a diffusion barrier for chemical fixatives (Roberts et al. 1970; Birch-Andersen et al. 1976; Ferguson et al. 1978; Marchiondo et al. 1978; Beesly and Latter 1982; Belli et al. 2006). High-pressure freezing (HPF) and freeze substitution (FS) can circumvent this problem and results in samples with decent morphology for EM studies (Kurth et al. 2012).
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Here, we used high-pressure frozen and freeze-substituted samples for 3D-reconstruction of subcellular details of sporozoites by electron tomography (ET). We describe novel structures (termed sporocords) that connect sporozoites with their corresponding sporocyst residuum and might serve an umbilical-cord-like function.
Fig. 1-2 Life cycle and sample preparation. Fig. 1. Simplified life cycle of E. nieschulzi. Sporogony occurs in oocysts containing four sporocysts (spc) surrounded by a robust oocyst wall (ocw), spo, sporont. For TEM analysis, oocysts suspended in 20 % bovine serum albumin were transferred to a HPF membrane carrier and were high pressure frozen using the Leica EM PACT 2, followed by freeze substitution and embedding into Epon®. Fig. 2. Electron micrograph of a high-pressure-frozen ooycst. The outer and inner oocyst walls (ow, iw) protect the sporozoites (spz) in the sporocysts (spc). Scale bar corresponds to 2 μm
Materials and methods Animals and oocyst isolation Rats (CD, Charles River) were infected orally with 500,000 sporulated oocysts of E. nieschulzi. Faeces were collected 7–10 days post inoculation and incubated in 2.5 % aqueous (w/v) potassium dichromate
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(K2Cr2O7) solution for 5–8 days under constant air supply and agitation (Bürger et al. 1995). The oocysts were ollected as described previously (Hammond et al. 1968; Sheather 1923) and stored in 2.5 % (K2Cr2O7) at 4 °C until fixation. High-pressure freezing, freeze substitution, and Epon® embedding Sample preparation for TEM inspection was performed as described in Kurth et al. (2012). In brief, oocysts were mixed with 20 % BSA and high-pressure frozen using the Leica EM-PACT2 and 100 μm HPF membrane carriers (Leica microsystems, Vienna, Austria). FS was performed in acetone containing 1 % osmium tetroxide (OsO4) and 0.1 % uranyl acetate (5 h at −90 °C) using the Leica AFS2 unit. After FS, the samples were heated to 0 °C at 5 °C/h, washed twice in acetone and heated to room temperature (∼23 °C). Finally, samples were infiltrated and embedded in Epon® 812 and cured at 60 °C. Ultrathin
Figs. 3-6 Electron tomography of E. nieschulzi sporocysts reveals protrusions (arrows) obviously connecting sporozoites and the sporocyst residuum. Fig. 3 Tomographic z-section of a sporocyst (spc), surrounded by a sporocyst wall (spcw). A sporozoite (spz), a sporocyst residuum (sr) and intracellular details such as rhoptries (rh), micronemes (mn) and the inner oocyst wall (iow) are visible. The arrow marks several tubular structures. Fig. 4 Sporozoite (green) and corresponding SR (red). The refractile body (blue), granules (brown) and the ribosomes (pink) of the
sections (80 nm) were stained with lead citrate and aqueous uranyl acetate. Electron tomography Semi-thin (300 nm) serial sections were collected on Formvar-coated copper slot grids, treated with an unspecific gold-labelled antibody (AuroProbe™, Amersham Pharmacia Biotech UK Limited) as a fiducial marker and imaged with a TECNAI F30 intermediatevoltage electron microscope (FEI, The Netherlands) operated at 300 kV. Serial, tilted views were collected over a ±63° range at 1° increment about two orthogonal axes. In total, we recorded six dual tilt and four single tilt data sets of seven different sporocysts. Two of these sporocysts were imaged in two and three serial sections for joining subsequently. ET was supported by the software SerialEM (Mastronarde 2005, 2006). We used IMOD for reconstructing the tilt series,
endoplasmic reticulum are highlighted. Arrows indicate tubules some of which contain electron dense material. Fig. 5 The translucent 3Dreconstruction shows accumulated granula (brown) surrounding the refractile body (blue) and the electron dense structure (seen in B) on the passage from ER to tubules (yellow). Fig. 6 The 3D-reconstruction shows a tubular network and a direct connection (arrow) between the sporozoite (green) and the SR (red). All scale bars 500 nm
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Figs. 7-8 Details of tomographic z-sections of a high-pressure-frozen E. nieschulzi sporocyst. Fig. 7 The tubules which connect the sporozoite (spz) and the SR (sr), contain vesicles (arrows) and filamentous structures (seen in cross-section, arrowhead). Multivesicular bodies (mvb) are found
within the residual body. Fig. 8 Posterior pole of a sporozoite (spz) with the refractile body (rfb) and the endoplasmic reticulum (er) containing globular structures (white arrows). The asterisk marks the connection to the “sporocord”. All scale bars 200 nm
for joining and segmentation of the tomograms, and for measurements (Kremer et al. 1996).
parasites and demonstrated that they met the characteristics of nanotubes (Gerdes et al. 2007; Rupp et al. 2011). The function of tubular structures in malaria parasite gametes seems to be different from sporocords in the sporulated oocyst, which is a “dormant” infective “Dauerstadium.” In E. nieschulzi, these structures are in connection with sporozoites and are formed during the divisions of the sporoblasts into two sporozoites each. Tomographic z-section of a sporocyst (spc) showed an intensive nanotubular network with extensions from the sporozoites towards the SR within the sporocysts (Figs. 3–5). The SR may be membrane-bound and contains vesicles of protein and lipids but no mitochondria. The SR differs in size in the Eimeria species that possess it and its function is unknown. It had been suggested by Pérard (1924) that the SR contains nutritive material and decreases gradually in size once the oocyst reaches the external environment. Sporulated oocysts can survive in a moist environment for several months and be
Results and discussion Typical E. nieschulzi oocysts contain four sporocysts each containing two sporozoites and a SR of unknown function. Sporocysts of E. nieschulzi are limited by a wall of 20– 25 nm and contain a Stieda body, a preformed opening at their more pointed end through which the sporozoites excyst (Long 1982). The oocysts are surrounded by a protective, robust wall, which renders good chemical fixation difficult. Improvement of fixation by HPF followed by FS leads to better preservation of the oocyst and sporocyst structures, which allows a more detailed analysis of sporozoite morphology (Fig. 1; Kurth et al. 2012). Using this technique combined with ET revealed tubular structures within E. nieschulzi sporocysts. These tubules are 70–120 nm wide and several micrometers long and seemed to be budding from the sporozoite’s surface. (Figs. 3–7). These structures are hitherto undescribed in Eimeria and other coccidians as Cryptosporidium probably due to the limited information on the ultrastructure of oocysts and sporozoites (e.g. Birch-Andersen et al. 1976; Ferguson et al. 1978; Marchiondo et al. 1978; Beesly and Latter, 1982; Uni et al. 1987; Pakandl et al. 2001; Azevedo 2001). However, similar tubules have been recently described from malaria parasites during gamogony in the mosquito midgut and were reported as nanotubes or FIGS0filaments of gametes (Rupp et al. 2011). In Plasmodium falciparum the filaments originate from the parasite plasma membrane, are close ended, express adhesion proteins like Pfs230, Pfs48/45 or Pfs25 and contain F-actin. It was claimed that the tubular structures represent long distance cell-to-cell connections between sexual stages
Fig. 9 3D Model of a sporulated oocyst showing sporocysts with sporozoites connected via “sporocords” to the SR
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infective for their specific host (rat) up to 1 year. Electron micrographs reveal that there are vesicles within the “nanotubes” (Fig. 7). These vesicles may be transported within these tubes. However, actin filaments like in P. falciparum have not been observed. The tubular structures are observed in several cases to be connected to the posterior end of sporozoites (Fig. 8). In the region the inner membrane complex of the pellicle forms a posterior ring-shaped structure (Fig. 8, asterisk) and leaves access to the cytoplasm of the parasite via the endoplasmic reticulum. This leads us to the conclusion that this nanotube-connection may serve as a long-term supply line between sporozoite and residual body during the resting stage (Figs. 7, 8, and 9). This may explain why coccidian oocysts can survive in moist environment for months up to years (Williams et al. 2010).
Conclusion The 3D-reconstruction of the sporocysts of a rat coccidium, E. nieschulzi, revealed a tubular network surrounding the two sporozoites. Some of these tubules form direct connections between the sporozoites and the residual body. We further observed vesicles and fibrous material inside the tubules and multivesicular bodies and also within the residual body. In some areas the peripheral endoplasmic reticulum of the sporozoite appears to be continuous with the tubules. These facts lead to our hypothesis of the presence of an “umbilicord-like” structure of the connecting tubules named “sporocord” which allows long-term survival of a resting stage until uptake by an appropriate host. A 3D-reconstruction of a whole sporocyst using serial section, ET, and TEM may confirm our hypothesis of a functional “sporocord” in Eimeria sporocysts. Acknowledgements The authors wish to thank the MPI-Facility of the MPI-CBG Dresden, especially Jean-Marc Verbavatz, Daniela Vorkel, Kimberley Gibson and Quentin de Robillard for their support with the tomography, and Donald W. Duszynski for helpful comments on the manuscript.
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