title: 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).
abstract: 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.
authors.affiliation: All-Russian Institute for Plant Protection, Russian Academy of Agricultural Sciences, St. Petersburg, Russia. jumacro@yahoo.com
authors.forename: Yu S
authors.initials: YS
authors.lastname: Tokarev
authors.forename: Yu Ya
authors.initials: YY
authors.lastname: Sokolova
authors.forename: R
authors.initials: R
authors.lastname: Entzeroth
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D003044:Coccidia / Q000254:growth & development / Q000502:physiology*; D003048:Coccidiosis / Q000276:immunology / Q000469:parasitology*; D006135:Gryllidae; D006434:Hemocytes / Q000166:cytology*; D008221:Lymphoid Tissue / Q000382:microbiology / Q000469:parasitology; D016814:Microsporidia / Q000254:growth & development / Q000502:physiology*; D016881:Microsporidiosis / Q000276:immunology / Q000382:microbiology*; D010587:Phagocytosis; D013172:Spores, Fungal / Q000254:growth & development; D034101:Sporozoites / Q000254:growth & development