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1 Louisiana State University, Baton Rouge, LA
2 University of Louisiana at Monroe, Monroe, LA
The noted parasitologist Horace W. Stunkard characterized microsporidians as among the most widespread of parasites, possessing significant survival adaptations, a consequence of their long-term host associations (1). One of the more obvious adaptations within this group is the extrusion apparatus of the infective spore stage. This apparatus consists of an aperture, a polaroplast membrane, a polar filament, and a posterior vacuole, or swelling organelle. The energy source for the firing of this apparatus is thought to reside in the posterior vacuole (2). In an earlier report (3), we indicated that the posterior vacuole had properties of peroxisomes, which function primarily to process very long chain fatty acids (VLCFA) with the assistance of key enzymes, acyl-CoA oxidase (ACOX) and catalase. Although peroxisomes are characteristically found in aerobic cells with mitochondria, there is evidence that peroxisomal enzymes occur in the amitochondriate microsporidian Spraguea lophii. In particular, biochemical assays and western blot analyses indicate that ACOX and catalase are discharged from the spores of S. lophii (Findley, unpubl. data). Here, we report the localization of catalase within the spore cell.
We used a cytochemical protocol described by Angermuller and Fahimi (4). In brief, spores were prefixed in 2.5% glutaraldehyde (to eliminate resident peroxiase activity) and were subsequently incubated in Tris buffer (pH 10.5) in the presence of diaminobenzidine (DAB) and hydrogen peroxide. The cells were then post-fixed in 2% osmium tetroxide and processed for transmission electron microscopy.
Electron micrographs revealed DAB reaction product in the posterior vacuole of the spores before and during its discharge (Fig. 1A). As the invasion tube emerges from the firing spore, the DAB reaction is associated with the inner surface of the tube (Fig. 1B) and also along the outer surface of the discharged tube. In the control preparations without DAB and hydrogen peroxide, there was no catalase-induced reaction product in the spore posterior vacuole (Fig. 1C). In addition to the peroxisomal enzymes, spores of S. lophii extrude the VLCFA nervonic acid into the external medium. Nervonic acid is a characteristic peroxisomal component, and the data indicate that it is discharged from the spores along with the catalase and ACOX enzymes. Of particular interest is the sizeable drop in nervonic acid levels that occurs during and after discharge of S. lophii spores (Findley, unpubl. data).
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If the Lom and Vavra model is correct, the membrane of the extruded sporoplasm may be derived from the extrusion apparatus and may therefore be reversed. Indeed, studies with cytoplasmic protein probes indicate that in discharged sporoplasms, the membrane orientation is reversed as proposed (DeGiorgis and Weidner, unpubl. obs.).
Literature Cited
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