Application of the nitroblue tetrazolium-reduction method for studies on the production of reactive oxygen species in insect haemocytes.

The in vitro generation of reactive oxygen species (ROS) in haemocytes of Galleria mellonella, Aporia crataegi, Dendrolimus sibiricus, Aglais urticae (Lepidoptera) and Gryllus bimaculatus (Orthoptera), was studied by the method of nitroblue tetrazolium (NBT) reduction. Formazan formation (product of NBT reduction) was observed in haemocytes of all the insects examined, except A. urticae. Lypopolysaccharide and zymosan reduced the number of NBT-positive cells after 1 h incubation and an increase was registered after 4 h incubation. The inhibitors of the respiratory chain enzyme (sodium azide) and melanogenesis (phenylthiourea) reduced formazan formation in nonactivated insect blood cells. No influence of sodium azide and phenylthiourea was found on the activated haemocytes. The results suggest that the generation of ROS in insect haemocytes occured as a result of processes such as respiration and melanization during phagocytosis and encapsulation.

Analysis of antibodies raised against soluble and membrane bound proteins of Nosema grylli (Microspora) spores.

Microsporidia (M), representatives of the phylum Microspora, make a world-wide distributed group of intracellular protists, parasitic in the vast number of hosts, from Protozoa to Primates. In their morpho-functional organization, both very primitive and extremely specialized features are seen definitely combined. Data available on RNA and DNA sequences suggest that M may be the most ancient eukaryotes. By the present, as many as 13 microsporidian species have been recognized as opportunistic pathogens in AIDS and transplant patients. Information about structural, transport and regulatory proteins of M, as well as on their enzymes is scarce, though it could serve as a basis for understanding pathogenicity of M and indicate some possible sites of relevant suppressive therapy. The present study persuaded two main goals: 1) to examine two ways of antigen preparation (from the infected organ and from the purified spores) and to evaluate their relation to the yield of the resulting antibodies; 2) to identify and localize new proteins with the help of the obtained antibodies by means of IFA, IEM and WB. Mice were immunized: 1) with dissolved proteins of heavily loaded with parasites fat bodies isolated from crickets Gryllus bimaculatus infected with Nosema grylli, and 2) with proteins of the purified spores of N. grylli. As a result two antisera were obtained. Antiserum 1 reacted predominantly with spore walls on IFA slides and ultrathin sections (IEM). It also reacted with a broad spectrum of parasite and host cell proteins on WB. Antiserum 2 recognized polar filaments and walls of discharged spores in IFA and IEM tests. It did not react with undischarged spores or fat bodies of uninfected crickets and gave a comparatively weak reaction with those of the infected hosts. Hybridization of spleen cells of immune mice with murine myeloma cells resulted in several hybridoma clones. They produced Mabs, 5 of which were tested by IFA, IEM and WB. Mab 1BF3 recognized 55 kDa protein connected with polar filaments as it was clearly suggested by IEM and IFA. Mab 1BD9 recognized 25, 34, 43 kDa proteins from the fraction of membrane bound proteins of spore walls, the sites of their interaction with antigens being marked with uneven fluorescence (IFA) and by gold precipitates on spore walls (IEM). Mab 1BB9 reacted with 36, 45, 65 and 75 kDa proteins, which belong mainly to the fraction of membrane-bound spore proteins, and gave a weak fluorescence associated with spores. Mab 2AB3 recognized 44 and 60 kDa proteins from the fraction of soluble spore proteins, and Mab 2AD4 acknowledged a single protein of 55 kDa from the same fraction. The obtained antibodies add to the existing microsporidian antibody bank and can be used for further work of isolation, description and sequencing the microsporidian proteins in order to understand eventually their functions.