INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Jan. 1988, p. 103-107 Vol. 38, No. I 0020-7713/88/010103-05$02.00/0 Copyright 0 1988, International Union of Microbiological Societies Spiroplasma taiwanense sp. nov. from Culex tritaeniorhynchus Mosquitoes Collected in Taiwan M. L. ABALAIN-COLLOC,l* L. ROSEN,* J. G. TULLY,3 J. M. BOVE,4 C. CHASTEL,l AND D. L. WILLIAMSON' Laboratoire de Bact&riologie,Faculte' de Me'decine, 22, Avenue Camille Desmoulins, 29285 Brest, France'; University of Hawaii at Manoa, Pacijic Biomedical Research Center, Honolulu, Hawaii, 968222; Mycoplasma Section, Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, Frederick Cancer Research Center, Frederick, Maryland 21 7013; Laboratoire de Biologie Cellulaire et Mole'culaire, Institut National de la Recherche Agronomique et Universite' de Bordeaux 11, Domaine de la Grande Ferrade, 33140 Pont de la Maye, France4; and Department of Anatomical Sciences, Health Sciences Center, State University of New York, Stony Brook, New York I I 794' Spirophsmu isolates recovered from female mosquitoes (Culex tritueniorhynchus) collected in Taiwan were found 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-lTwas 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 Taiwan, Republic of China, on 2 July 1981. After capture, in a survey of a natural population of mosquitoes for the mosquitoes were held for 4 days to allow any blood present presence of Japanese encephalitis virus @a). In 1985, tritu- to be digested. They were then separated by species and rates of these mosquitoes, which had been stored at -7O"C, stored at -70°C until thawed for processing. were tested for the possible presence of spiroplasmas by Culture media and cultivation procedures. A triturate was culture in SP-4 medium. Several isolates of helical motile prepared for viral assay by grinding a pool of 100 adult organisms were obtained in these trials. One of the spiro- female Culex tritaeniorhynchus mosquitoes in 1 to 2 ml of plasma isolates was designated strain CT-lT (T = type phosphate-buffered saline containing 30% calf serum, peni- strain) to reflect the name of the mosquito species (Culex cillin (750 U/ml), and streptomycin (750 pg/ml). After viral tritaeniorhynchus) from which it had been isolated. assay, the triturate was refrozen and stored at -70°C. In Strain CT-lT was shown to be distinct from all previously 1985, the frozen triturate was thawed and filtered through a described group I through XXI strains by one-way deforma- 450-nm membrane filter, and 0.1 ml was inoculated into 1.5 tion, metabolism inhibition, and growth inhibition tests and ml of SP-4 medium (29, 30). The culture was incubated at was designated the representative strain of a new group, 28°C. Color change was noted 12 days later, and motile group XXII, according to the classification originally pro- helical filaments were observed in samples of the cultures posed by Junca et al. (17) and subsequent revisions (27, 33, examined by dark-field microscopy. Tenfold dilutions of an 34). Two additional spiroplasma strains, CT-2 and CT-3, early pass (P3) were sent to D. L. Williamson, State Univer- isolated in the same year from the same species of mosquito sity of New York, Stony Brook. The isolate was first passed at the same site, had titers similar to those of strain CT-lT into M1D broth medium (35) and triply filter-cloned on M1D when they were tested against CT-lT antiserum in one-way agar medium, and then 1 to 2-ml samples of a batch culture deformation tests. were lyophilized. Strain C-intaiffeB in M1D In this report, we summarize evidence that strain CT-lT medium by passing a 1OYGnocu~umevery day into fresh and related strains fulfill the minimal species requirements media at 30°C. Agar colonies were obtained by plating for Mollicutes as proposed by the International Committee suspensions on 1.6% Noble agar (Difco Laboratories) in on Systematic Bacteriology Subcommittee on the Taxonomy M1D medium. To provide a uniform source of spiroplaSmas, of Mollicutes (16, 32). Therefore, we formally propose that a 25-ml batch culture was prepared and separated in mid- strain CT-lT and related strains be recognized as a new logarithmic phase into 0.5- and 1-ml volumes in microtubes, species in the genus Spiroplasma. which were stored at -70°C. Other spiroplasmas were compared with strain CT-lT. MATERIALS AND METHODS These included the following organisms: Spiroplasma citri Maroc R8A2T (= ATCC 27556T) (subgroup I-l), Spiro- Origin of strain. Female mosquitoes attracted to animal plasma melliferum BC-3T (= ATCC 33219T) (subgroup I-2), bait near rice fields were collected at Taishan (near Taipei), Spiroplasma kunkelii E275T (= ATCC 29320T) (subgroup I-3), tick spiroplasma strain 277F (= ATCC 29761) (subgroup * Corresponding author. I-4), strain LB-12 (= ATCC 33649) (subgroup I-5), Maryland 103 104 ABALAIN-COLLOC ET AL. INT.J. SYST.BACTERIOL. flower spiroplasma strain M55 (= ATCC 33502) (subgroup '[-6), Cocos spiroplasma strain N525 (= ATCC 33287) (sub- group I-7), Spiroplasrna phoeniceurn strain P40 (= ATCC 43115) (subgroup I-@, uncultivated strain WSRO of the Dro- sophila sex ratio organism (group 11), Spiroplasrna floricola OBMG (= ATCC 33221) (group 111), Spiroplasrna apis B31T I:= ATCC 33834T) (group IV), Spiroplasrna rnirurn SMUT I:= ATCC 29335T) (group V), Ixodes tick spiroplasma strain Y32 (= ATCC 33835) (group VI), Monobia spiroplasma strain MQ-1 (= ATCC 33825) (group VII), syrphid spiroplasma $strainEA-1 (= ATCC 33826) (group VIII), strain CN-5 (= ATCC 33827) (group IX), Spiroplasrna culicicola AES-1 (= ATCC 35112) (group X), strain MQ-4 (= ATCC 35262) (group XI), strain DU-1 (= ATCC 43210) (group XII), Spiroplasrna sabaudiense Ar-1343T (= ATCC 43303T) (group XIII), strain EC-1 (= ATCC 43212) (group XIV), strain 1-25 (= ATCC 43262) (group XV), strain CC-1 (= ATCC 43207) (group XVI), strain DF-1 (= ATCC 43209) (group XVII), strain TN-1 (= ATCC 43211) (group XVIII), strain PUP-1 (= ATCC 43206) '(group XIX), strain LD-1 (= ATCC 43213) (group XX), strain W115 (= ATCC 43260) (group XXI), and strain TG-1 (= ATCC 43525) (group XXIII). The origins and histories of these strains have been reviewed recently (1, 27). Filtration studies. An M1D broth culture of strain CT-lT FIG. 1. Electron micrograph of spiroplasma strain CT-lT. Thin- that had been incubated for 1day at 22°C was passed through section preparation of a cell pellet obtained from a 24-h broth culture a series of membrane filters with graded pore diameters (450, grown in M1D medium and stained with 2% aqueous uranyl acetate 300, 220, and 100 nm) by using a hypodermic syringe and and Reynold lead citrate. Arrows indicate the unit membrane on the minimum hand pressure. Each filtrate was diluted in a series cells. Bar = 100 nm. of 10-fold dilutions in M1D medium, and all tubes were incubated at 22°C. After 7 days, the tubes were examined for growth by identifying the last tube that showed growth of Genomic analysis. The techniques used for extraction and helical cells and a color change. The results were expressed determination of the guanine-plus-cytosine content (in moles in color-changing units (CCU) per milliliter. percent) of the deoxyribonucleic acid of strain CT-lr have Morphology. Cultures of strain CT-lT were routinely mon- been described elsewhere (3, 5, 6, 17). itored by dark-field microscopy (26). For electron micros- (copy, cells were fixed for 1 h in 2% glutaraldehyde in M1D RESULTS medium, postfixed for 1 h in 1% osmium tetroxide, dehy- Idrated in acetone, and embedded in Epon-Araldite; sections Morphological and cultural properties. A primary culture were stained with 2% aqueous uranyl acetate and Reynold of strain CT-lT was obtained after 12 days in SP-4 medium at lead citrate. 30°C. The subcultures grew rapidly, produced turbidity, and Temperature requirements. Temperature requirements acidified the medium within 24 h. Similar rapid growth was were determined by diluting the batch culture suspension in apparent when the organisms were subcultured in other M1D medium to give about lo6 CCU/ml. Six tubes, each spiroplasma broth media (M1A and M1D media and medium containing 2 ml of this dilution, were incubated at one of six containing 1% serum fraction). The presence of penicillin temperatures (22, 25, 30, 32, 35, and 37°C). At subsequent (500 U/ml) had no influence on growth rates or yields. Strain intervals, growth was assessed by removing 0.1 ml and CT-lT grew at 22,25, and 30"C, but not at 32,35, or 37°C. At preparing a 10-fold dilution series to determine the number 25 and 30"C, titers of lo1' CCUlml were achieved. Growth at of CCU per milliter.