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Progenies from some wild-caught females of Drosophila willistoni and three other sibling species are entirely female. The proclivity for production of unisexual female progeny by these flies was named the sex ratio (SR) trait and was originally thought to be genetic. However, experiments in the laboratory of Donald F. Poulson in the early 1960s demonstrated that this 'trait' was vertically transmitted and infectious, in that it could be artificially transferred by injection from infected females to non-infected females. Motile, helical micro-organisms were observed in females showing the trait. In 1979, the SR organisms were designated as group II in the informal spiroplasma classification system. The organisms proved to be extremely fastidious, but were eventually cultivated in a very complex cell-free medium (H-2) after initial co-cultivation with insect cells. Cultivation in the H-2 medium and the subsequent availability of a triply cloned strain (DW-1T) permitted comparative studies. Cells of strain DW-1T were helical, motile filaments 200-250 nm in diameter and were bound by a single trilaminar membrane. Cells plated on 1.8% Noble agar formed small satellite-free colonies 60-70 microns in diameter with dense centres and uneven edges. The temperature range for growth was 26-30 degrees C; optimum growth occurred at 30 degrees C, with a doubling time in H-2 medium of 15.8 h. The strain passed through filters with 220 nm, but not 100 nm, pores. Reciprocal serological comparisons of strain DW-1T with representatives of other spiroplasma groups showed an extensive pattern of one-way crossing when strain DW-1T was used as antigen. However, variable, usually low-level reciprocal cross-reactions were observed between strain DW-1T and representatives of group I sub-groups. The genome size of strain DW-1T was 2040 kbp, as determined by PFGE. The G + C content was 26 +/- 1 mol%, as determined by buoyant density and melting point methods. The serological and molecular data indicate that strain DW-1T is separated from group I representative strains sufficiently to justify retention of its group status. Continued group designation is also indicated by the ability of SR spiroplasmas to induce male lethality in Drosophila, their vertical transmissibility and their extremely fastidious growth requirements. Group II spiroplasmas, represented by strain DW-1T (ATCC 43153T), are designated Spiroplasma poulsonii.

Significant changes have been made in the systematics of the genus Spiroplasma (class Mollicutes) since it was expanded by revision in 1987 to include 23 groups and eight sub-groups. Since that time, two additional spiroplasmas have been assigned group numbers and species names. More recently, specific epithets have been assigned to nine previously designated groups and three sub-groups. Also, taxonomic descriptions and species names have been published for six previously ungrouped spiroplasmas. These six new organisms are: Spiroplasma alleghenense (strain PLHS-1T) (group XXVI), Spiroplasma lineolae (strain TALS-2T) (group XXVII), Spiroplasma platyhelix (strain PALS-1T) (group XXVIII), Spiroplasma montanense (strain HYOS-1T) (group XXXI), Spiroplasma helicoides (strain TABS-2T) (group XXXII) and Spiroplasma tabanidicola (strain TAUS-1T) (group XXXIII). Also, group XVII, which became vacant when strain DF-1T (Spiroplasma chrysopicola) was transferred to group VIII, has been filled with strain Tab 4c. The discovery of these strains reflects continuing primary search in insect reservoirs, particularly horse flies and deer files (Diptera: Tabanidae). In the current revision, new group designations for 10 spiroplasma strains, including six recently named organisms, are proposed. Three unnamed but newly grouped spiroplasmas are strain TIUS-1 (group XXIX; ATCC 51751) from a typhiid wasp (Hymenoptera: Tiphiidae), strain BIUS-1 (group XXX; ATCC 51750) from floral surfaces of the tickseed sunflower (Bidens sp.) and strain BARC 1901 (group XXXIV; ATCC 700283). Strain BARC 2649 (ATCC 700284) from Tabanus lineola has been proposed as a new sub-group of group VIII. Strains TIUS-1 and BIUS-1 have unusual morphologies, appearing as helices at only certain stages in culture. In this revision, potentially important intergroup serological relationships observed between strain DW-1 (group II) from a neotropical Drosophila species and certain sub-group representatives of group I spiroplasmas are also reported.

Spiroplasma strain DU-1T (T = type strain), which was isolated from hemolymph of the corn rootworm Diabrotica undecimpunctata (Coleoptera:Chrysomelidae), was serologically distinct from other spiroplasma species, groups, and subgroups. Cells of strain DU-1T were shown by light microscopy to be helical motile filaments. Electron microscopy revealed cells bounded by a single cytoplasmic membrane, with no evidence of a cell wall. The organism was not sensitive to 500 U of penicillin per ml. Strain DU-1T grew well in SM-1, M1D, and SP-4 liquid media, in broth supplemented with 1% bovine serum fraction or conventional horse serum, and under both aerobic and anaerobic conditions. This organism did not appear to have a sterol requirement for growth, as has been reported for several other Spiroplasma species or strains. Optimal growth occurred at 32 degrees C, with a doubling time of 0.9 h; strain DU-1T multiplied at 10 to 41 degrees C but failed to grow at 5 or 43 degrees C. It produced acid from glucose but hydrolyzed neither arginine nor urea. The results of reciprocal serologic tests in which antigens or antisera to established Spiroplasma species, groups, subgroups, and putative groups were used indicated that strain DU-1T was serologically distinct. This organism has a DNA guanine-plus-cytosine content of 25 +/- 1 mol% and a genome size of 1,350 kbp. Strain DU-1T is a member of a cluster of fast-growing insect-associated spiroplasmas, as determined by sequence analysis of 16S rRNA. On the basis of the results of this study and previously published data, strain DU-1 (= ATCC 43210) is designated the type strain of a new species, Spiroplasma diabroticae.

Spiroplasma strain EC-1T (T = type strain), which was isolated from the gut of a lampyrid beetle (Ellychnia corrusca) in Maryland, was serologically distinct from other spiroplasma species and groups. Similar strains were obtained from other E. corrusca specimens, and, later, numerous isolates of similar or partially related strains were obtained from several species of tabanid files. Cells of strain EC-1T were helical, motile filaments that were bound by a single cytoplasmic membrane, and there was no evidence of a cell wall. The cells were filterable through 220-nm-pore-size membrane filters but not through 100-nm-pore-size membrane filters. The organism was absolutely resistant to penicillin (1,000 U/ml) and required sterol for growth. Strain EC-1T grew well in M1D and SP-4 liquid media and could be cultivated in the Edward formulation of conventional mycoplasma medium and in 1% serum fraction medium. Optimal growth occurred at 32 degrees C (doubling time, 1.5 h). Strain EC-1T multiplied at 10 to 41 degrees C, but not at 5 or 43 degrees C. This organism produced acid from glucose, but did not hydrolyze arginine or utilize urea. The guanine-plus-cytosine content of the DNA was determined to be 26.3 mol% by the melting temperature method and 27.0 mol% by the buoyant density method. As a result of our studies, strain EC-1 (= ATCC 43212) is designated the type strain of a new species, Spiroplasma corruscae.

Spiroplasma sp. strain EA-1(T) (T = type strain) (subgroup VIII-1), which was isolated from the syrphid fly Eristalis arbustorum, was serologically distinct from other spiroplasma species, groups, and subgroups, The cells of this strain, as revealed by dark-field light microscopy, were short, helical, and motile. An electron microscopic examination revealed wall-less cells delimited by a single membrane. The unusually short cells passed through 220-nm filter pores with no reduction in titer. The organisms grew well in SM-1, M1D, and SP-4 liquid media. Growth also occurred in conventional horse serum medium and 1% serum fraction medium. Strain EA-1(T) grew at temperatures between 10 and 41 degrees C, and optimum growth occurred at 32 degrees C. The doubling time at the optimal temperature was 1.0 h. The strain catabolized glucose and hydrolyzed arginine but did not hydrolyze urea. The guanine-plus-cytosine content of the DNA was 30 +/- 1 mol%. The genome size was about 1,230 kbp. Strain Ea-1 (= ATCC 33826), which represents subgroup VIII-1, is designated the type strain of a new species, Spiroplasma syrphidicola.

Spiroplasma strain MQ-4T (T = type strain), which was isolated from the hemolymph of the vespid wasp Monobia quadridens, was serologically distinct from other spiroplasma species, groups, putative groups, and subgroups. Each strain MQ-4T cell was helical and motile and was surrounded by a single cytoplasmic membrane; there was no evidence of a cell wall. The strain grew well in 1% serum fraction medium, as well as in SM-1, M1D, and SP-4 liquid media, under both aerobic and anaerobic conditions. Strain MQ-4T grew at temperatures ranging from 10 to 41 degrees C but did not grow at 43 degrees C. The strain grew optimally at 37 degrees C with a doubling time of 0.6 h, the shortest doubling time recorded for any spiroplasma. Strain MQ-4T catabolized glucose and arginine but did not hydrolyze urea. The guanine-plus-cytosine content of the DNA was about 27.5 +/- 1 mol%. The genome size was 1,480 kbp (940 MDa). Strain MQ-4 (= ATCC 35262) is designated the type strain of a new species, Spiroplasma velocicrescens.

Characterization of an extrachromosomal element from an organism in the genus Spiroplasma is likely to be an essential step in the development of cloning vectors which replicate in these organisms. A restriction map for an 11-kb element, designated pCT-1, isolated from Spiroplasma taiwanese strain CT-1 (ATCC 43302) has been constructed using the restriction enzymes Bg/II, EcoRI, HincII, HindIII, HpaI, PstI, and XbaI. This element is distinct from any previously characterized spiroplasma virus or plasmid. pCT-1 has been cloned into the Escherichia coli vector pBR322 as a step in the development of a biphasic shuttle vector system.

Seven methods for the transfection of bacteria were compared and optimized for use in Spiroplasma citri strain HP using the spiroplasma virus SpV1 R8A2 B replicative form (RF). These methods included both chemical-mediated protocols [CaCl2, RbCl/CaCl2, polyethylene glycol (PEG)], liposome-mediated transfection, electroporation, freeze/thaw cycling, and natural competence. The best protocols were those which utilized PEG or electroporation, yielding transfection frequencies of 1.4 x 10(-4) and 9.1 x 10(-4) transfectants/colony-forming unit (CFU), respectively. For both of these protocols, transfection frequencies were higher using CsCl-purified, covalently closed, circular DNA. In the PEG-mediated protocol, Sigma 8000 brand PEG at a final concentration of 44%, and the presence of carrier DNA proved to be optimal with a PEG exposure time of 2 min. Using electroporation, a 1-2 ms pulse of a 6.5 kV/cm electric field was best; washing the host cell pellet prior to electroporation enhanced efficiencies by 50%. Linearization of the DNA resulted in lower transfection efficiencies by either method.

Several spiroplasmas (helical, motile mollicutes) were previously shown to contain extrachromosomal DNA (E-DNA) elements in the form of viruses (double-stranded viruses or the replicative form of single-stranded viruses) or plasmids. These elements are now being investigated as potential vectors for use in spiroplasma transformation systems. Described herein is the first extensive survey of spiroplasma E-DNA in 23 spiroplasma groups (30 strains), a study facilitated by improvements in protocols for E-DNA extraction. E-DNA elements were found in spiroplasmas associated with leafhoppers/plants (spiroplasma subgroups I-1, I-3, and I-8), other insects (subgroups I-2, I-5, I-6, and I-7 and groups IV and XXII), and ticks (subgroup I-4 and groups V and VI). Elements, maintained by passage with their host spiroplasmas, were often lost after extended passage. Whether the current distribution of E-DNA elements is indicative of historical or proximate factors is not known. Many elements (about 75%) from group I spiroplasmas hybridized with Spiroplasma citri viruses SpV1 or SpV3. Of the elements associated with other spiroplasma groups, none hybridized with either virus. These include Spiroplasma apis strains B31 (18 kb) and L89 (18 and 20 kb), S. mirum strains SMCA (20 kb) and Anderson (16 and 20 kb), group VI strain Y32 (7, 9, 10, and 16 kb), and group XXII strain CT-1 (8 kb). Several of these elements will be characterized and examined for their suitability as spiroplasma cloning vectors.

Eight spiroplasma strains from insects and one from spring flowers failed to react with antisera specific for any of the 11 described spiroplasma groups, with sera directed against spiroplasma Group I subgroups, or with sera directed against two unnumbered groups previously reported to occur in tabanid flies. Strains, all from Maryland, were isolated from the hemolymph of the spotted cucumber beetle Diabrotica undecimpunctata and the lampyrid beetle Ellychnia corrusca, and the guts of the cantharid beetles Cantharis bilineatus and C. carolinus. Other strains were obtained from a tabanid fly, Tabanus gladiator and from the firefly Photuris pennsylvanica in Maryland and from the mosquito Culex tritaeniorhynchus in Taiwan. An isolate from pooled Cicadulina bipunctella leafhoppers in Syria apparently represented a unique group. A single isolate from spring flowers in Oklahoma also appeared to be unrelated to existing groups or subgroups. One-way deformation tests using sera prepared against known beetle and tabanid spiroplasmas showed each of the above strains to be unique. Although these results strongly indicate that the nine strains studied are representatives of unique new spiroplasma groups, the formal designation of new groups awaits fulfillment of recently proposed criteria.

A defined medium (H-1) was developed for cultivation of the suckling mouse cataract agent, Spiroplasma mirum, a fastidious member of the class Mollicutes that causes cataracts and chronic brain infection in inoculated neonate mice. The H-1 medium was used to show the importance of sphingomyelin as a growth factor for the culture of the spiroplasma in vitro. The growth of Spiroplasma mirum and the pathology it induces in sphingomyelin-rich tissues in vivo may be related to this dependency.

Half of the spiroplasmas observed microscopically in insects cannot be cultivated and are thus inaccessible to study. Media mixed with cultured insect cells have now been used to isolate two of these spiroplasmas--the sex-ratio organism (SRO) of Drosophila and the Colorado potato beetle spiroplasma (CPBS). Studies described herein indicate that at least one of the cell-supplied factors is involved in redox maintenance. A wide variety of insect cell culture systems were suitable for primary isolation of the CPBS. The SRO and CPBS were found to attach to insect cells in vitro.

Spiroplasmas were observed in seven species of the family Tabanidae (horse flies and deer flies). This is the fifth family of the order Diptera now known to harbor spiroplasmas. Noncultivable spiroplasmas were seen in the hemolymph of three species of the genus Tabanus, and cultivable forms were isolated from the guts of six species in three genera. Isolates from T. calens and T. sulcifrons were serologically similar and closely related to a spiroplasma in the lampyrid beetle, Ellychnia corrusca. These three isolates represent a new serogroup. Isolates from Hybomitra lasiophthalma were related to Group IV strains, while those from T. nigrovittatus and Chrysops sp. both represented new serogroups. At least some tabanids probably acquire spiroplasmas from contaminated flower surfaces. The possibility of vertebrate reservoirs for some tabanid spiroplasmas remains an open question.

The Subgroup I-6 spiroplasma, "Maryland Flower Spiroplasma," originally discovered on fall flowers and subsequently recovered from a syrphid fly and a beetle triungulin, was isolated from two new fall flower hosts and from the guts of nine nectar-imbibing insect species. These data, together with lack of recovery of I-6 spiroplasma from foliage-feeding, plant-sucking, or flightless flower-associated insects, suggest that I-6 spiroplasma may infect and be disseminated by nectar- or pollen-foraging insects, and that the dynamics of maintenance will prove to be complex.