INFLUENCE OF MICROSPORIDIAN SPORES ON PHENOLOXIDASE ACTIVITY IN THE HAEMOCYTES OF GRYLLUS SPP. (INSECTA: ORTHOPTERA) IN VITRO.

After incubation with spores of microsporidia Paranosema spp. during 1­2 h, the quotes of Gryllus spp. haemolymph cells giving positive reaction in histochemical assay for phenoloxidase (PO) activity were significantly lower as compared to the control. Spores of microsporidia P. grylli caused 3- and 5.4-fold decrease of this index in haemocyte monolayers of Gryllus bimaculatus and G. argentinus, respectively. The ability of P. grylli spores to decrease the quote of PO-positive cells in G. bimaculatus haemocyte monolayers was stronger when monolayers and spores had been coincubated for a longer period (2 h) and when the spores used were more fresh (with higher level pf infectivity). Treatment of P. grylli spores with antibiotics resulted in diminishing their ability to decrease the quote of PO-positive cells. The highest level of decrease of this index, being 28.7-fold, was registered when G. bimaculatus haemocytes had been incubated with spores of P. locustae, the highly aggressive parasite which is able to infect orthopteran insect hosts belonging to more than 100 species and to preserve a high level of infectivity for years. Strong correlation has been revealed between infectivity of microsporidian spores and their ability to suppress PO system, which is a key factor in invertebrate immunity.

INTERACTIONS OF MICROSPORIDIA WITH INFECTED HOST CELL.

Microsporidia comprise a group of fungi-related obligate intracellular eukaryotic pathogens with extremely wide host range: from protists to mammals. Adaptation to intracellular parasitism drives these parasites towards significant reduction and modification of the genome and functional apparatus, which causes extreme dependence on the host cell, as well as sophisticated host-parasite relationships. In this review we summarize our results and recent literature data about microsporidian interactions with the host at the cellular level. The impacts of these pathogens to infected cells include induction of hypertrophy, restructuring and modification of the cytoskeleton and the vesicular transport system of the host cells. Microsporidians also able to stimulate the metabolic processes in the infected cells and inhibit their defensive reactions. The main tool of the direct regulatory impact of microsporidia on the host cell apparently is the secretion of the special protein effectors capable to interfere to regulatory and signaling pathways of the host cell.

A primary culture of haemocytes isolated from Gryllus bimaculatus (Orthoptera, Gryllidae) and their interactions with two intracellular parasites--Paranosema grylli (Microsporidia) and Adelina grylli (Coccidia).

Cricket haemocytes were derived from either haemolymph or haemopoietic organs (lymph glands) of insects and introduced to a primary culture. Varied isolation protocols, tissue culture vessels, media compositions and cell densities were tested to determine the optimal conditions for in vitro maintenance of haemocytes, and for subsequent light and electron microscopic analysis of monolayers. Freshly prepared Mitsuhashi and Maramorosh (MM;Sigma, Steinheim, Germany) insect medium (420 mOsm), buffered with sodium bicarbonate (pH 7.2) and supplemented with 10 % FCS, was found to be most appropriate for haemocyte maintenance. All tested tissue culture vessels (FLEXiperm units, multiwell plates and Thermanox slides, with the exception of Melineux agar plates), were suitable for cell attachment and haemocyte monolayers formation. Viability of cultured cells was confirmed by LIVE/DEAD Viability/Cytotoxity Kit for Eukaryotic Cells. Free circulating haemocytes were cultivated up to 27 days and then degraded. Infection with the microsporidian Paranosema grylli or the coccidian Adelina grylli caused noticeable swelling of host lymph glands (haemopoietic tissue) and increase in the number of cells comprising the glands. The cells derived from haemopoietic tissue were maintained for maximum 5 days; thereafter multiplication of bacteria normally inhabiting cricket lymph glands destroyed monolayers and killed the cells. Microsporidian and coccidian invasive stages (spores and sporozoites, respectively) were isolated from infected tissues, resuspended in MM medium and added to haemocyte monolayers in ratios 1 zoite per haemocytes or 10 spores per 1 haemocyte. Actively moving zoites contacted and penetrated the cultured cells. Unlike coccidian zoites, microsporidian spores were phagocytized by haemocytes. Application of fluorescent LIVE/DEAD kit allowed to visualize internalized parasites inside host cells as clearly shaped dark areas. The present study has demonstrated that 1) cricket haemocytes from both circulating haemolymph and lymph glands can be short-term cultivated on tissue culture vessel surfaces which made possible their further light and electron microscopic analysis; 2) short-term haemocyte cultures may be employed to study host-parasite interactions, in particular, to follow the initial steps of parasite internalization inside host cell; 3) Fluorescent assay with Viability/Cytotoxity Kit for Eukaryotic Cells (Molecular Probes, Oregon) allows to observe penetration of these parasites into cultured cells.