Fractionation of Different Life Cycle Stages of Microsporidia Nosema grylli from Crickets Gryllus bimaculatus by Centrifugation in Percoll Density Gradient for Biochemical Research

Abstract

ABSTRACT. A new method of fractionation and purification of different life cycle stages of microsporidia Nosema grylli, parasitizing the fat body of cricket Gryllus bimaculatus, by centrifugation in Percoll density gradient is elaborated. The whole procedure can be summarized as: 1) infected fat body preparation, 2) homogenization in buffer and filtration through cotton wad and filter paper, 3) first centrifuging, resulting in the separation of the pellet into three layers containing different life cycle stages, 4) second centrifuging of the chosen layer in Percoll density gradient, 5) washing out the Percoll from the fraction under study. After centrifugation in Percoll density gradient, meronts and early sporonts form a band in the area corresponding to density 1.016 g/ml. Mature spores form the pellet at the bottom of centrifuge tube, while immature spores are distributed throughout the layer of 1.016 g/ml up to the bottom of the centrifuge tube, according to their buoyant densities. The offered technique is simple, it takes about one hour and may become a routine procedure for biochemical studies on microsporidia.

Analogs of the Golgi complex in microsporidia: structure and avesicular mechanisms of function

“…The most plausible candidates to demonstrate ER-to-plasma membrane transport in microsporidia cells are proteins exported out of the cell in the formation of the spore wall. In the P. grylli spore wall, the most prevalent protein has a molecular mass of 40 kDa (p40) (Seleznev et al, 1995). Close connections of the TNs with the forming polar tube, a post-Golgi compartment, are described both elsewhere (Cali and Takvorian, 1999;Vavra and Larsson, 1999) and in the present study.…”

“…Immunoblotting, immunofluorescence analysis and immunoelectron microscopy P. grylli and P. locustae spores and their intracellular stages were purified from the fat bodies of infected Gryllus bimaculatus crickets by Percoll density gradient centrifugation, as described previously (Seleznev et al, 1995). Spores were disrupted by vortexing with glass beads (Bio-Rad, Milan, Italy) in phosphatebuffered saline, for 30 minutes at 4°C.…”

Interaction between SWP9 and Polar Tube Proteins of the Microsporidian Nosema bombycis and Function of SWP9 as a Scaffolding Protein Contribute to Polar Tube Tethering to the Spore Wall

“…Mature spores of N. bombycis isolate CQ1 (CVCC 102059) were purified from infected silkworms and conserved in the China Veterinary Culture Collection Center, Chongqing, China. At the same time, N. bombycis spores at early stages were purified and harvested from laboratory-reared silkworm larvae, as previously described (7,24,27,42,43). The purified spores were washed and stored in sterile phosphate-buffered saline (PBS) with antibiotics (100 g/ml streptomycin, 100 U/ml penicillin) at 4°C for later use.…”

Immunolocalization of an Alternative Respiratory Chain in Antonospora (Paranosema) locustae Spores: Mitosomes Retain Their Role in Microsporidial Energy Metabolism

“…locustae spores and prespore intracellular stages. A. locustae spores and stages of intracellular development were isolated from the fat bodies of experimentally infected Locusta migratoria locusts containing sufficient numbers of spores and merogonial and sporogonial stages, as described previously (19).…”

“…A. locustae spores were broken in TS solution (25 mM Tris-Cl [pH 8.0], 0.3 M sucrose) by vortexing with 2.5-mm glass balls (BDH, United Kingdom) for 30 min, and homogenate was cleared by centrifugation at 100 ϫ g for 10 min. The stages of intracellular development (meronts and sporonts) were purified by centrifugation in Percoll density gradient (19) and ruptured by sonication. For comparative analysis of spores and intracellular stages, both samples were equilibrated in protein concentration by Bradford's method (4).…”