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) *)
Yu. S. Tokarev,1 Yu. Ya. Sokolova,2 R. Entzeroth 3
1 All-Russian Institute for Plant Protection, Russian Academy of Agricultural
Sciences, 2 Institute of Cytology, RAS, St. Petersburg, Russia, and 3 Technical University of Dresden,
Germany;
e-mail: jumacro@yahoo.com
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 % PCS, 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 giylli 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, resuspendcd 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.
Key words: Microsporidia, Orthoptera, haemocyte, cell culture, Paranosema grylli, Adelina grylli,
Gryllus bimaculatus
*) Paper in English
|
