Chapter 5 : Microsporidian Biochemistry and Physiology

Effect of Ameson michaelis infection on the skeletal muscle composition of the blue crab (Callinectes sapidus). Data are presented as the percentage difference in protein, free amino acid (NPS), and carbohydrate (CHO) levels of thoracic versus cheliped skeletal muscle. Bar direction indicates whether values for thoracic muscle were higher (+) or lower (-) than those for cheliped muscle (mean ± 95% confidence interval).

Lactate concentrations in the hemolymph, thoracic skeletal muscle, and hepatopancreas of normal, lightly infected, and heavily infected blue crabs (mean ± 95% confidence interval).

Glucose utilization, lactate production, and pyruvate production by isolated A. michaelis sporoplasms incubated in medium 199 containing 5.5 mM glucose. Data are presented as (cumulative) micromoles of solute per 1010 sporoplasms (mean ± 95% confidence interval).

Uptake of U-14C-D-glucose by isolated A. michaelis sporoplasms. Data are presented as micromoles of glucose taken up per 1010 sporoplasms. Incubations in low-substrate (minimum essential medium, 0.5 mM glucose) and high-substrate (medium 199, 5.5 mM glucose) media are compared (mean ± 95% confidence interval).

A proposed scheme of energy metabolism in microsporidian spores.

Polarographic measurement of oxygen consumption by mitochondria isolated from a Agrotis segetum (Lepidoptera: Noctuidae) fat body infected with Vairimorpha antheraeae.

Surface topography of the outer envelope surrounding S. lophii spores. (A) Platinum-shadowed spore with the outer envelope partially dispersed after preincubation in urea and a rinse in distilled water. Arrows indicate envelope is compartmentalized. (B) Isolated S. lophii spore envelope stained with phosphotungstic acid. Arrows indicate compartments with internal matrix which probably buffer proteins within these domains. (C) Isolated S. lophii spore envelope stained with uranyl acetate. Visualized proteins (arrows) are weakly stained within the compartments so clearly delineated in panel B. The compartmental channels are 75 to 100 nm in width.

Immunoblot analyses of S. lophii spore envelope proteins specific for G protein (α-subunits). Antisera GC/2 recognizes Goα (lanes 1 and 2); CO/1 recognizes Goα and Giα 3 (lanes 3 and 4) and RM/1 is specific for Gsα (lanes 5 and 6). For controls, rat brain membrane proteins are in lanes 2, 4 and 6, and S. lophii envelope peptides are in lanes 1, 3 and 5.

S. lophii spore envelope isolate. (A) Uranyl acetate-stained proteins partially isolated from spore envelope but still in the native linear arrangement (arrows). (B) Isolate of S. lophii envelope with proteins tested against anticlathrin and reacted with a second antibody coupled to peroxidase. Arrows indicate peroxidase reaction is limited to threadlike elements. Magnification, × 200,000.

S. lophii spore polar aperture area. Arrows indicated precipitate that frequently accumulated near spore aperture after CaCl2 incubation. Since precipitation was confined to this site, one assumption is that the calcium salt accumulates here because of the channel proteins at this aperture (where the Ca2+ influx is believed to occur). Magnification, × 200,000.

Nucleotidase activity in the isolated spore envelope of S. lophii. The envelopes were incubated in nitrophenyl phosphate or ATP (1 mM), and the substrate was incubated in medium with the capture agent cerium. The cytochemical medium contained 0.1 M Tris-maleate (pH 7.4), CeCl3, ATP, or nitrophenyl phosphate (Robinson and Karnovsky, 1983). Reaction was confined to the envelope channels. Magnification, × 35,000.

The presumed anatomy of a Tlielohania sporophorous vesicle with keratin and plaque proteins. The arrow indicates the position of the jacket assemblage (bearing the keratin) that envelops the spore. The evidence for channel continuity with the vesicle exterior is indicated by the permeation of lanthanum and dyes into the area between the jacket and the spores; no probe material penetrated the primary space between the jacket and the vesicle envelope.

Electron micrograph showing spore colony removed from host neuron after 6 h in culture medium. Filament bundles still persist and comprise the matrix between the spores or meronts and hold these cells together. Magnification, ×30,000.

Images of S. lophii sporoplasms in extracellular culture media. (A) Phase light micrograph of sporoplasms after 12 h in medium. (B) Electron micrograph of sporoplasms. Arrows indicate apparent interiorizing on surface of cell. (C and D) DAPI-stained sporoplasms after 24 h in culture. Arrows indicate presumptive divider cells. (E) Sporoplasms after 10-min incubation in rhodamine-albumin with some accumulation of label in small fluorescent domains at the surface (arrows). (F and G) DAPI-stained sporoplasms after 24 h in culture. Arrows show a cell in apparent division. (H and I) Sporoplasms incubated for 20 min in dextran. Arrows point to vacuoles as possible sites of dextran endocytosis. Sporoplasms are 2 μm in diameter.

The periphery of an isolated extracellular S. lophii meront colony after 6 h in culture medium. Cells at the border of the colony (seen here) were apparently in better physical condition than interior cells of the colony, indicating that the dense matrix between meronts may affect nutrient infiltration. Magnification, X25.000.