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-13 (group XXVI), Spiroplasma lineolae (strain TALS-23 (group XXVII), Spiroplasma platyhelix (strain PALS-13 (group XXVlll), Spiroplasma montanense (strain HYOS-13 (group XXXI), Spiroplasma helicoides (strain TABS-23 (group XXXII) and Spiroplasma tabanidicola (strain TAUS-13 (group XXXIII). Also, group XVII, which became vacant when strain DF-IT (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 flies (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-I (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 a t 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.

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-19 permitted comparative studies. Cells of strain DW-lT 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 pm in diameter with dense centres and uneven edges. The temperature range for growth was 26-30 "C; optimum growth occurred a t 30 "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-lT with representatives of other spiroplasma groups showed an extensive pattern of one-way crossing when strain DW-lT was used as antigen. However, variable, usually low-level reciprocal cross-reactions were observed between strain DWlT and representatives of group I sub-groups. The genome size of strain DW-lT was 2040 kbp, as determined by PFGE. The G+C content was 2621 mol%, as determined b y buoyant density and melting point methods. The serological and molecular data indicate that strain DW-lT 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-lT (ATCC 431 533, are designated Spiroplasma poulsonii.

A new method was developed for determination of the doubling times of spiroplasmas. In this procedure, the time required for medium acidification of tubes in tenfold dilution series was recorded. Sixty-four spiroplasma strains, representing 24 groups and 11 subgroups, were studied. Eight strains representing putative new groups were also included in the study. Doubling times at 5, 10, 15, 20, 25, 30, 32, 37, 41, and 43°C were determined. The range of temperatures for spiroplasma growth was 5°-41°C. Twenty-three spiroplasmas had optima of 30°C, 29 had optima of 32°C, and 13 had optima of 37°C. The fastest growing spiroplasma was the MQ-4 strain (group XI), with a doubling time at optimal temperature of 0.6 h. The slowest was the Jamaican corn stunt strain B655 (subgroup I-3), with an optimal doubling time of 36.7 h. Spiroplasma strain B31 (group IV) had the widest range (5°-41°C), while the DW-1 strain and some subgroup I-3 strains had the narrowest, growing only at 25° and 30°C. Some spiroplasmas grew well at 41°C, but none grew at 43°C. The ability of spiroplasmas to withstand a wide range of temperatures may reflect the conditions to which they are exposed in nature, including the temperatures of the insect, tick, and/or plant hosts in which they are carried and the plant surfaces from which they may be acquired by arthropods.

VOL. 47, 1997 FOUR NEW SPIROPLASMA SPECIES FROM TABANID FLIES 719 G. Wagner for assistance with isolation and culture and Mary Fenton for assistance with electron microarauhv.tales ord. nov.), with provision for familial rank to separate species with nonhelical morphology (Entomoplasmataceae fam. nov.) from helical species

Some group XVI spiroplasmas, such as strains CC-1 (Spiroplusma cantharicolu) and CB-1, are associated with cantharid beetles. Fifteen related but heterogeneous strains have been isolated from mosquitoes, other insects, and a flower in France and the United States. In the present study, these seventeen strains have been compared by deformation and metabolism inhibition serological tests, by one-dimensional protein sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and by determination of the guanine-plus-cytosine content of their DNA. Five of the 17 strains were further compared by DNA-DNA hybridization and by restriction enzyme (EcoRI and HindIII) analysis of their DNA. On the basis of the resulting data, we propose that group XVI be subdivided into three subgroups. Subgroup XVI-I is represented by strain CC-1 (ATCC 43207) from a cantharid beetle in the United States, and strain MQ-6 from a wasp; subgroup XVI-2 is represented by strain CB-1 (ATCC 43208) from a cantharid beetle and two strains from mosquitoes, all in the United States; and subgroup XVI-3 is represented by strain Ar-1357 (ATCC 51126) and contains 11 strains from mosquitoes and 1 strain from a flower, all from the Savoy region of France.Since the first isolation of spiroplasmas in 1971 (16), the genus Spiroplasma has continued to grow and currently contains many strains isolated from various insects, ticks, and plants. Serological techniques were first used for spiroplasma classification (26). Genomic and other molecular techniques were subsequently applied to this group of microorganisms (12). These tests include the guanine plus cytosine (G+C) content of DNA, genome size, DNA-DNA hybridization? DNA restriction endonuclease patterns, and polyacrylamide gel electrophoresis (PAGE) patterns of cell proteins (2). The current classification recognizes 24 groups of spiroplasmas (9, 18). A group (12) is a cluster of spiroplasma strains that are serologically related, share 10 to 100% DNA-DNA homology, and possess a significant number of similar proteins by PAGE. Usually, but not always (ll), all strains of groups have an identical G+C content of their DNA (24).Many groups are represented by one strain or by closely related strains (24). However, some groups are heterogeneous. For example, group I has been subdivided into eight subgroups on the basis of serological data, cell protein analysis, and genomic analysis (2, 18). Strains of some other heterogeneous groups (e.g., group IV) (1) are too closely related to warrant a subgroup designation.Group XVI contains strains isolated in the United States (Maryland and Alabama) and in France (Savoy) from various insect species and a flower (4-6, 17, 18). Preliminary serological studies (18) showed that group XVI was heterogeneous. We chose 17 group XVI strains for the present study. To define the taxonomic heterogeneity of these strains, reciprocal deformation (DF) and metabolism inhibition (MI) tests were performed? protein patterns of all strains were analyzed by one-dimensional PAGE, and five strains ...