Infection of Three Insect Cell Cultures by Spiroplasma citri and Other Spiroplasmas

Spiroplasma citri R8A2, S. floricola BNR-1, S. apis PPS-1 and the spiroplasma strains B88, CSS, 277F were inoculated into AS-2 (Aceratagallia sanguinolenta) , AC-20 (Agallia constricta) and Dm-1 (Drosophila melanogaster) cell cultures and the resulting interactions were studied. Cytopathogenic effects leading to culture death were observed with all strains except CSS. Strains B88, 277F, BNR-1 and PPS-1 grew as well in AS-2 and AC-20 cultures as in fresh LB medium. Strain R8A2 multiplied faster and reached higher titers in AS-2 and AC-20 cultures than in fresh LB medium (on day 3 the titers in AS-2 and fresh LB medium were 5.0 x 108 and 8.1 x 106 CFU/ml, respectively). This S. citri growth stimulating factor was studied by means of conditioned medium. All 6 strains cytadsorbed to the cell. Strains R8A2, CSS, BNR-1 and PPS-1 adsorbed sliehtlv. and strains B88 and 277B adsorbed heavily. Fluorescent DNA staining revealed " * the presence of non-helical forms within the AS-2 -and AC-20 cells. Interactions take place between mycoplasmas and cells, tissues and organs of the insect vectors of mycoplasmal plant diseases. Such interactions can be studied in vitro using insect tissue systems. We selected two leafhopper cell lines, AS-2 (Aceratagallia sanguinolenta) and AC-20 (Agallia constricta) and the Dm-1 Drosophila melanogaster line for this purpose. Spiroplasmas as well as mammalian mycoplasmas and Acholeplasma laidlawii multiplied to high titers in Dm-1 cultures (9, 10). Spiroplasma dependent cytopathogenic effects (CPE) and culture death was observed. Attachment of spiroplasmas to the cells but no fusion of membranes was seen by transmission electron microscopy. The 277F spiroplasma was occasionally located intracellularly within vacuoles (11). Intracellular membrane bound spiroplasmas were also found in another insect cell line (7) . The spiroplasmas seemed to be filamentous or helical within the vacuoles. In leafhopper tissues, Spiroplasma citri and the corn stunt spiroplasma (CSS) seem to be pleomorphic, not helical, with a morphology comparable to mycoplasma-like-organisms (5, 8) . S. citri was located intracellularly in clusters which were not surrounded by membranes in the natural vector, Circulifer tenellus (5) . Our present studies add some new features to these observations and describe some interesting interactions between leafhopper cells and several spiroplasmas which hopefully will contribute to a better understanding of the hostparasite relationship. MATERIALS AND METHODS The agallian cell lines AS-2 and AC-20 were grown in the LB medium (6) a t 28°C and passaged every 3 to 5 days. The Drosophila melanogaster Dm-1 cell line was maintained a t 25°C in Schneider's medium and passaged a t weekly intervals. The spiroplasma species and strains used were: S. citri R8A2 (group 1-1 of Junca et al. (4) ) ,S. floricola BNR-1 (111), S. apis PPS-1 (IV) and the unspeciated strains B88 (1-2, from honey bees), the corn stunt spiroplasma CSS E-275 (1-3) and the tick strain 277F (1-4). They were cultured a t 30°C in BSR medium with the exception of CSS which was grown in MIA medium. Culture flasks (T 25) containing 0.4x106 cells/ml were infected with approximately lo6 CFU/ml Ninth IOCV Conference of spiroplasmas a few hours after passage. Infected agallian cell cultures were observed daily with an inverted microscope for gross CPE. At selected days the spiroplasma titers were determined by agar plating and the cells were counted with a hemocytometer using the Trypan blue exclusion method. Conditioned medium was prepared in the following way. Confluent AS-2 or AC-20 cultures were passaged and incubated a t 28°C. The supernatant was collected and filtered through 0.22 or 0.45 pm membrane filters to remove cellular materials after a growth period of normally 3 days. This medium, conditioned by the growth of cells, was then used to study the S. citri stimulating factor. Cells were seeded in Leighton tubes with glass coverslips for fluorescent microscopy. Cultures were infected by approximately lo6 CFU spiroplasmas after cell attachment, were incubated for varying periods, and processed. Fluorescent DNA staining with Hoechst 33258 was as described elsewhere (9). Coverslips were fixed in acetone, washed 3 times in water and incubated a t room temperature for 30 minutes with the rabbit anti RSA2 antiserum for indirect immunofluorescence. Fluorescein conjugated goat anti-rabbit antiserum was added after additional washings and the coverslips were rewashed 30 minutes later and mounted.