Frank E. French

Revised group classification of the genus Spiroplasma

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.

Serologic and Genomic Relatedness of Group VIII and Group XVII Spiroplasmas and Subdivision of Spiroplasma Group VIII into Subgroups

Spiroplasmas are currently classified in a group system. Criteria for separation of the twenty-four currently designated groups include serologic relatedness, polyacrylamide gel electrophoretic patterns of proteins, guanine-plus-cytosine base ratios, and, in some cases, DNA-DNA homology. The analysis of DNA-DNA homology and serologic data from a large array of strains recently discovered in dipteran insects reveals that group VIII strain EA-1 from a syrphid fly, strain TAAS-1 from a horse fly, and group XVII strain DF-1 from a deer fly belong to a large complex (supergroup) of strains with various degrees of interrelatedness. Strains DF-1 and EA-1 share DNA-DNA homology of 33 to 48% (high-stringency conditions), while strain TAAS-1 shares 42 to 67% homology with DF-1 and EA-1. The strains had temperature optima of 30 to 37°C, but the temperature minima and maxima reflected the geographic region of strain origin. These three strains also share G+C values of about 30 mol%, utilize arginine, and tend to grow in culture to very high titers (1011 cells per ml). The helical cells of these strains are smaller than those of other spiroplasmas and readily pass through filter pores of 220 nm. These data support the taxonomic placement of the biotypes represented by strains EA-1, DF-1, and TAAS-1 into one supergroup, group VIII, with subgroups designated as VIII-1, VIII-2, and VIII-3, respectively. It is proposed that group XVII remain vacant.

Spiroplasma Species, Groups, and Subgroups from North American Tabanidae

Abstract. Twenty-one triply cloned spiroplasma strains from the United States east of the Rocky Mountains, all isolated from tabanid (Diptera:Tabanidae) flies or serologically related to strains from tabanids, were compared reciprocally by spiroplasma deformation (DF) and metabolism inhibition (MI) serological tests. Many of the strains were also tested against 28 antisera representing known spiroplasma groups, subgroups, and putative groups isolated from nontabanid hosts. Relationships among strains were indicated by reciprocal cross-reactivity in both DF and MI tests. The strains were found to represent 11 recognized spiroplasma groups or subgroups. On the basis of serological, biochemical, and genomic data, strain BARC 1901 from Tabanus lineola appeared to represent a previously unrecognized candidate group. Strain BARC 2649, also from T. lineola, also appeared to represent a new group, but its morphology, arginine utilization, and some one-way serological crossing patterns suggested that it may be distantly related to group VIII spiroplasmas. Morphological, serological, and genomic data were used to place tabanid spiroplasma strains into three informal clusters. These are (i) groups IV (strain B31) and XXXI (strain HYOS-1); (ii) the three existing subgroups and a new candidate subgroup of group VIII represented by strain BARC 1357 plus ungrouped strain BARC 2649; and (iii) 14 strains, including EC-1 and TATS-1 (group XIV); strains TN-1 and TAAS-2 (group XVIII); strains TG-1, TASS-1, and BARC 4689 (group XXIII), strains TALS-2 (group XXVII), strain TABS-2 (group XXXII), and strains TAUS-1 and TABS-1 (group XXXIII) and ungrouped but closely related strains BARC 1901, BARC 2264 and BARC 2555. Analysis of tabanids from other geographic regions probably will substantially increase the number of known spiroplasma groups from this insect family.

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.

Spiroplasma corruscae sp. nov., from a firefly beetle (Coleoptera: Lampyridae) and tabanid flies (Diptera: Tabanidae).

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 syrphidicola sp. nov., from a Syrphid Fly (Diptera: Syrphidae)

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.

Laboratory Infection and Release of Spiroplasma (Entomoplasmatales: Spiroplasmataceae) from Horse Flies (Diptera: Tabanidae)

Many tabanid flies (Diptera: Tabanidae) are infected with spiroplasmas (Mollicutes: Spiroplasmataceae). Naturally-infected Tabanus gladiator Stone and T. sulcifrons Maquart flies were restrained and fed 10% sucrose to determine the exit points of Spiroplasma from tabanid flies. The flies were allowed to feed for 24 h, and the resulting oral and anal specks were cultured in MID broth. Spiroplasmas were isolated from 21 of 51 oral specks but not from 23 anal specks deposited on plastic. In contrast, when anal specks were deposited in a sucrose solution, 9 of 28 anal specks in sucrose yielded spiroplasma cultures. Tabanus limola F. and T. longiusculus Hine were offered a culture of Spiroplasma strain EC-1 on a stewed raisin or in 5% sucrose in the form of a hanging drop. After 4 d, the minced abdominal viscera of each fly were incubated in MID broth and 25 of 32 tabanids yielded cultures of Spiroplasma.

Spiroplasma litorale sp. nov., from Tabanid Flies (Tabanidae: Diptera) in the Southeastern United States

Spiroplasma strain TN-1T (T = type strain), a strain serologically distinct from other spiroplasma species, groups, and subgroups, was isolated from the gut of a horsefly (Tabanus nigrovittatus) from a barrier island off the coast of North Carolina. Related strains were isolated from other Tabanus spp., T. atratus, T. americanus, T. gladiator, T. lineola, T. sulcifrons, and T. zythicolor, from coastal Georgia. Cells of strain TN-1T in culture were helical and motile with an average of 5 to 10 helical turns per cell. Electron microscopic studies determined that the cells of strain TN-1T were surrounded by a single cytoplasmic membrane, and there was no evidence of a cell wall. The spiroplasma grew well in MID and SP-4 liquid media. Serum fraction (1%) medium and conventional horse serum medium also supported growth of strain TN-1T. Fried-egg colonies were not produced; instead, the strain produced small diffuse colonies that were surrounded by satellite growth. The optimum temperature for growth was 32°C, but multiplication was observed at temperatures from 10 to 41°C. The doubling time at the optimum temperature (32°C) was 1.6 h. No growth was observed at 5 or 43°C. Spiroplasma strain TN-1T passed through 220-nm filter pores but failed to pass through 100-nm filter pores. Strain TN-1T catabolized glucose but hydrolyzed neither arginine nor urea. The guanine-plus-cytosine content of the DNA was about 25 ± 1 mol%, and the genome size was 1,370 kbp. Based on results from this study and previously published data, strain TN-1T (= ATCC 43211) (group XVIII) is designated the type strain of a new spiroplasma species, Spiroplasma litorale.

Spiroplasma lineolae sp. nov., from the horsefly Tabanus lineola (diptera : Tabanidae)

Spiroplasma strain TALS-2T from the viscera of the striped horsefly, Tabanus lineola, collected in Georgia was serologically distinct from other Spiroplasma species, groups, putative groups, and subgroups. Light and electron microscopy of cells of strain TALS-2T revealed helical motile cells surrounded only by a single cytoplasmic membrane. The organism grew in M1D and SP-4 liquid media. Growth also occurred in 1% serum fraction medium and in conventional horse serum medium. Growth in liquid media was serum dependent. The strain passed through 220-nm filter pores, but was retained in filters with 100-nm pores. The optimum temperature for growth was 30 degrees C. Multiplication occurred at temperatures from 20 to 37 degrees C, with a doubling time at the optimum temperature of 5.6 h in M1D broth. Strain TALS-2T catabolized glucose but hydrolyzed neither arginine nor urea. The guanine-plus-cytosine content of the DNA was 25 +/- 1 mol%. The genome size was 1,390 kbp. Six isolates serologically similar to strain TALS-2T were obtained from the same host in coastal Georgia. Three strains closely related to strain TALS-2T were isolated from the horsefly Poeciloderas quadripunctatus in Costa Rica. Strain TALS-2T (= ATCC 51749), a representative of group XXVII, is designated the type strain of a new species, Spiroplasma lineolae (Mollicutes: Entomoplasmatales).

Spiroplasma lampyridicola sp. nov., from the Firefly Beetle Photuris pennsylvanicus

Spiroplasma strain PUP-1Twas isolated from the gut fluids of a firefly beetle (Photuris pennsylvanicus) collected in Maryland. Cells of the strain were shown by dark-field microscopy to be helical, motile filaments. Ultrastructural examination by electron microscopy revealed filamentous cells bounded by a single cytoplasmic membrane and no evidence of a cell wall. The cells were not sensitive to 500 U of penicillin per ml and grew under aerobic conditions in M1D, SP-4, and M-2 broth formulations, as well as in conventional mycoplasma medium. The doubling times at 15, 20, 25, and 30°C were 83.1, 32.0, 14.9, and 9.8 h, respectively. Suboptimal growth occurred at 37°C, and no growth was apparent in cultures maintained at 10 or 40°C. The organism required cholesterol for growth and produced acid from glucose, fructose, and trehalose; arginine and urea were not hydrolyzed. The results of previous serological analyses of strain PUP-1Tindicated that the organism was not related to the then currently established Spiroplasma species or group representatives, and the organism was classified as the representative of group XIX. Additional testing of strain PUP-1Tagainst recently recognized Spiroplasma species or group representatives by both metabolism inhibition and deformation tests confirmed the unique serological status of the organism. The guanine-plus-cytosine content of the DNA was 26 ± 1 mol%, and the genome size was 1,375 kbp. These values clearly differentiate strain PUP-1Tfrom group XXI strain W115, with which it cross-reacted reciprocally at a low level in deformation and metabolism inhibition tests. We propose that strain PUP-1 (= ATCC 43206) should be recognized as the type strain of a new species, Spiroplasma lampyridicola.