Spiroplasma chrysopicola sp. nov., Spiroplasma gladiatoris sp. nov., Spiroplasma helicoides sp. nov., and Spiroplasma tabanidicola sp. nov., from Tabanid (Diptera: Tabanidae) Flies

Four spiroplasma strains, DF-1T, TG-1T, TABS-2T, and TAUS-1T, all of which were isolated from deerflies or horseflies (Diptera: Tabanidae), were serologically distinct from previously described spiroplasma species, groups, and subgroups. Strain DF-1Toriginated from a Maryland deerfly (Chrysops sp.); strain TG-1Twas isolated from a Maryland horsefly (Tabanus gladiator); strain TAUS-1Toriginated from a member of the Tabanus abdominalis-limbatinevris complex of horseflies collected in Maryland; and strain TABS-2Twas isolated from a horsefly (Tabanus abactor) collected in Oklahoma. Cells of all of the strains appeared to be helical and motile when they were examined by dark-field microscopy. Cells of strain DF-1Tgrowing in M1D medium were short helices with less than six turns; the helical cells of the other strains were long and usually had six or more turns. The short cells of strain DF-1Tpassed through 450- and 300-nm filter pores with no reduction in titer, but the longer cells of the other strains were partially retained by 450-nm-pore-size filters. Electron microscopic examination of all of the strains revealed wall-less cells surrounded only by a single cytoplasmic membrane. All of the strains grew well in SP-4 liquid media and in conventional mycoplasma or M1D media supplemented with horse or fetal bovine serum. Strains TABS-2T, TAUS-1T, and DF-1Trequired serum or sterol for growth, but strain TG-1Twas able to grow in the absence of serum or sterol. The optimum temperatures for growth of the four strains varied from 30 to 32°C, and growth occurred at 10 to 37°C. All of the strains catabolized glucose but did not hydrolyze urea. Only strain DF-1Thydrolyzed arginine. The guanine-plus-cytosine contents of the DNAs of the strains were: DF-1T, 29 ± 1 mol%; TG-1T, 26 ± 1 mol%; TABS-2T, 27 ± 1 mol%; and TAUS-1T, 26 ± mol%. The genome sizes of strains DF-1Tand TAUS-1Twere 1,270 and 1,375 kbp, respectively. Strain DF-1 (= ATCC 43209), the representative of spiroplasma subgroup VIII-2, is designated the type strain of a new species, Spiroplasma chrysopicola. We also propose that strain TG-1T(= ATCC 43525T), the designated representative of group XXIII, should be placed in a new species, Spiroplasma gladiatoris. In addition, group XXXII spiroplasma strain TABS-2 (= ATCC 51746) is designated the type strain of Spiroplasma helicoides sp. nov., and group XXXIII representative strain TAUS-1 (= ATCC 51747) is designated the type strain of another new species, Spiroplasma tabanidicola.