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Initially, strain CUAS-lT (T = type strain), which was isolated from a frozen triturate of Culex annulus mosquitoes collected in Taiwan, was thought to be a member of spiroplasma group VII. This placement was based on the spiroplasma deformation test titer observed when strain CUAS-lT spiroplasmas were tested with Spiroplasma monobiue MQ-lT antiserum. The results of subsequent reciprocal spiroplasma deformation, metabolism inhibition, and growth inhibition tests clearly revealed that strain CUAS-lT is not serologically related to previously described spiroplasma groups (groups I to XXIV) and thus is a representative of a new group, group XXV. Strain CUAS-lT was characterized by using the minimal standards for mollicute species descriptions. During logarithmic-phase growth, strain CUAS-lT cells are characteristically very short helices with 1.5 to 2 helical turns (1 to 2 pm), highly motile, and bounded by a single trilaminar membrane and form granular colonies with satellites when the organism is grown aerobically on MID medium containing 1.6% agar. Growth in M1D broth occurs at temperatures ranging from 10 to 37"C, and the optimum temperature is 30°C. Substrate utilization tests revealed that cholesterol is required for growth, that glucose is hydrolyzed, and that arginine is not hydrolyzed both in the presence and in the absence of glucose. The genome of strain CUAS-lT is 1,080 kbp long, and the guanine-plus-cytosine content is 26 & 1 mol%. On the basis of the results of our studies we propose that strain CUAS-lT (group XXV) should be placed in a new species, Spiropfasmu diminutum. Strain CUAS-1 (= ATCC 49235) is the type strain of S. diminutum.The first mosquito spiroplasma was isolated from a triturate of female salt marsh mosquitoes (Aedes sollicitans) (12). This organism was subsequently characterized and named Spiroplasma culicicola (8). Mosquitoes have proved to be a rich source of spiroplasmas, and the following two additional mosquito-derived spiroplasmas have been characterized: Spiroplasma sabaudiense (1) and Spiroplasma taiwanense (2). In addition, several spiroplasma strains isolated from mosquitoes collected in France and the United States have recently been shown to be related to each other and to be members of three subgroups of group XVI (3). A previously existing collection of frozen mosquito triturates originally collected by one of us (L.R.) for a mosquito-borne virus survey in Southeast Asia (People's Republic of China, Japan, Taiwan) has been found to contain, in addition to S. taiwanense, several spiroplasma strains that cannot be placed in previously described species. One of these triturates (triturate 250657) was derived from 83 Culex annulus adult female mosquitoes collected in Taishan, Taiwan, in 1980. The spiroplasmas found in this triturate (designated strain CUAS-lT [T = type strain]) were studied because of their small size and rapid growth in broth medium, * Corresponding author. Phone: (516) traits shared with the mosquito-derived organism Spiroplasma culicicola AES-lT. The res...

Spirophsmu isolates recovered from female mosquitoes (Culex tritueniorhynchus) collected in Taiwan werefound to be similar in their serological properties. Strain CT-lT (T = type strain) proved to be serologically unrelated to all currently recognized spiroplasma groups and subgroups. Strain CT-lT was studied by using criteria proposed by the International Committee on Systematic Bacteriology Subcommittee on Taxonomy of Mollicutes for the description of new mollicute species. The organisms were shown to belong to the class Mollicutes by the ultrastructure of their limiting membrane, their colonial morphology, and their filtration patterns and to the family Spiroplusmatuceue by their helical morphology and motility. Growth in SP-4, MlA, and M1D media occurred at 22 to 30°C. Cholesterol was required for growth. Glucose was fermented, but arginine was not hydrolyzed. The base composition (guanine-plus-cytosine content) of the deoxyribonucleic acid of strain CT-lT was found to be 25 f 1 mol%. On the basis of these findings, we propose that spiroplasma strains with these characteristics should be recognized as a new species, Spiroplusmu tuiwunense. Strain CT-lT has been deposited in the American Type Culture Collection as strain ATCC 43302T.In July 1981, female mosquitoes were collected in Taiwan in a survey of a natural population of mosquitoes for the presence of Japanese encephalitis virus @a). In 1985, triturates of these mosquitoes, which had been stored at -7O"C, were tested for the possible presence of spiroplasmas by culture in SP-4 medium. Several isolates of helical motile organisms were obtained in these trials. One of the spiroplasma isolates was designated strain CT-lT (T = type strain) to reflect the name of the mosquito species (Culex tritaeniorhynchus) from which it had been isolated. Strain CT-lT was shown to be distinct from all previously described group I through XXI strains by one-way deformation, metabolism inhibition, and growth inhibition tests and was designated the representative strain of a new group, group XXII, according to the classification originally proposed by Junca et al. (17) and subsequent revisions (27,33,34). Two additional spiroplasma strains, CT-2 and CT-3, isolated in the same year from the same species of mosquito at the same site, had titers similar to those of strain CT-lT when they were tested against CT-lT antiserum in one-way deformation tests.In this report, we summarize evidence that strain CT-lT and related strains fulfill the minimal species requirements for Mollicutes as proposed by the International Committee on Systematic Bacteriology Subcommittee on the Taxonomy of Mollicutes (16, 32). Therefore, we formally propose that strain CT-lT and related strains be recognized as a new species in the genus Spiroplasma. MATERIALS AND METHODSOrigin of strain. Female mosquitoes attracted to animal bait near rice fields were collected at Taishan (near Taipei), * Corresponding author.Taiwan, Republic of China, on 2 July 1981. After capture, mosquitoes were held for 4 days to ...

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 ...

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