Spiroplasma poulsonii sp. nov., a new species associated with male-lethality in Drosophila willistoni, a neotropical species of fruit fly.

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.

Entomoplasma freundtii sp. nov., a new species from a green tiger beetle (Coleoptera: Cicindelidae).

A mollicute (strain BARC 318T) isolated from gut tissue of a green tiger beetle (Coleoptera: Cicindelidae) was found by dark-field microscopy to consist of non-helical, non-motile, pleomorphic coccoid forms of various sizes. In ultrastructural studies, individual cells varied in diameter from 300 to 1200 nm, were surrounded by a cytoplasmic membrane and showed no evidence of cell wall. The organisms were readily filterable through membrane filters with mean pore diameters of 450 and 300 nm, with unusually large numbers of organisms filterable through 200 nm pore membrane filters. Growth occurred over a temperature range of 15-32 degrees C with optimum growth at 30 degrees C. The organism fermented glucose and hydrolysed arginine but did not hydrolyse urea. Strain BARC 318T was insensitive to 500 U penicillin ml-1 and required serum or cholesterol for growth. It was serologically distinct from all currently described sterol-requiring, fermentative Mycoplasma species and from 12 non-sterol-requiring Mesoplasma species, 13 non-sterol-requiring Acholeplasma species and 5 previously described sterol-requiring Entomoplasma species. Strain BARC 318T was shown to have a G + C content of 34 mol% and a genome size of 870 kbp. The 16S rDNA sequence of strain BARC 318T was compared to 16S rDNA sequences of several other Entomoplasma species and to other representative species of the genera Spiroplasma and Mycoplasma, and to other members of the class Mollicutes. These comparisons indicated that strain BARC 318T had close phylogenetic relationships to other Entomoplasma species. On the basis of these findings and other similarities in morphology, growth and temperature requirements and genomic features, the organism was assigned to the genus Entomoplasma. Strain BARC 318T (ATCC 51999T) is designated the type strain of Entomoplasma freundtii sp. nov.

Spiroplasma turonicum sp. nov. from Haematopota horse flies (Diptera: Tabanidae) in France.

Strain Tab4cT, a helical prokaryote that was isolated from the body of a Haematopota sp. fly collected in Champchevrier, Indre-et-Loire, Touraine, France, was found to be a member of the class Mollicutes. The cells of strain Tab4cT were small, motile helices that were devoid of a cell wall. The organism passed through filters with mean pore diameters as small as 0.20 mm. Strain Tab4cT grew rapidly in liquid SP-4 medium at both 30 and 37 degrees C. The organism fermented glucose but did not hydrolyse arginine or urea, and did not require serum for growth. In preliminary electrophoretic analyses, the cell protein patterns of strain Tab4cT were distinct from those of 14 other spiroplasmas found in mosquitoes, deer flies and horse flies from Europe and the Far-East. In reciprocal metabolism inhibition and deformation serological tests, employing antigens and antisera representative of spiroplasma groups I-XXXIII (including all sub-groups), plus ungrouped strains BARC 1901 and BARC 2649, no serological relationship with Tab4cT was found. The G + C content of the DNA of strain Tab4cT was about 25 +/- 1 mol% and its genome size was 1.305 kbp. It is proposed that spiroplasma strain Tab4cT be assigned to group XVII (presently vacant) and that strain (ATCC 700271T) is the type strain of a new species, Spiroplasma turonicum.

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.

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).

Phylogeny of the grassland leafhopper genus Flexamia (Homoptera: Cicadellidae) based on mitochondrial DNA sequences.

Leafhoppers (Cicadellidae) are a highly diverse group of sap-sucking insects, many species of which specialize on grasses. Past attempts to examine the roles of host transfer or host plant coevolution in the diversification of leafhopper species using cladistic methods have been hindered by a paucity of discrete, phylogenetically informative morphological characters. To demonstrate the utility of DNA sequence data for species-level phylogenetic studies of Cicadellidae, we estimated phylogenetic relationships among species in the North American grassland leafhopper genus Flexamia DeLong using partial nucleotide sequences of mitochondrial 16S rDNA and NADH dehydrogenase 1, totaling 1496 base pairs and 810 potentially informative characters. Analyses of the partitioned and combined sequence data using maximum parsimony, neighbor-joining, and maximum likelihood criteria yielded similar estimates of relationships in which most nodes were well-supported by bootstrap and decay indices. These estimates largely agreed with a previously published, intuitive, morphology-based phylogeny for the genus. A parsimony reconstruction of host associations based on these results suggests that the origins of various Flexamia clades coincided with host transfers among grass subfamilies or genera. Nevertheless, associations with certain subfamilies, genera, or species of grasses appear to have been largely conserved in the evolutionary diversification of Flexamia.

Spiroplasma platyhelix sp. nov., a new mollicute with unusual morphology and genome size from the dragonfly Pachydiplax longipennis.

Spiroplasma strain PALS-1T from the gut of the dragonfly Pachydiplax longipennis was shown to be distinct from other species, groups, and subgroups of the genus Spiroplasma as determined by reciprocal serological metabolism inhibition and deformation tests. However, this strain cross-reacted extensively with representatives of other groups when it was used as an antigen. Electron microscopy of cells of strain PALS-1T revealed cells surrounded by a single cytoplasmic membrane. Light microscopy revealed helical cells that exhibited twisting motility rather than rotatory or flexing motility. Variations in the tightness of coiling were transmitted from one end of the helix to the other. The strain was resistant to penicillin, which confirmed that no cell wall was present. The organism grew well in M1D and SP-4 liquid media under either aerobic or anaerobic conditions. Growth also occurred in 1% serum fraction medium and in conventional horse serum medium. The optimum temperature for growth was 30 degrees C, at which the doubling time was 6.4 h. Multiplication occurred at temperatures from 10 to 32 degrees C. Strain PALS-1T catabolized glucose and hydrolyzed arginine but not urea. The guanine-plus-cytosine content of the DNA was 29 +/- 1 mol%. The genome size was 780 kbp, the smallest genome size in the genus Spiroplasma. Strain PALS-1 (= ATCC 51748) is designated the type strain of a new species, Spiroplasma platyhelix.

Spiroplasma diabroticae sp. nov., from the southern corn rootworm beetle, Diabrotica undecimpunctata (Coleoptera:Chrysomelidae).

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 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.

Improved cultivation systems for isolation of the colorado potato beetle spiroplasma.

In North America, the Colorado potato beetle, Leptinotarsa decemlineata, is often infected with the host-specific, gut-inhabiting Colorado potato beetle spiroplasma (CPBS). CPBS is apparently a commensal, but it may be useful in biocontrol if it can be transformed to express an insect-lethal gene. Difficulty in cultivating the organism, however, has hindered the development of a suitable transformation system. In this study, we eliminated the need for coculturing CPBS with insect cells. CPBS was reliably isolated with the BBL Anaerobic GasPak Jar system (low redox, enhanced CO(inf2)), which was easier to use and less expensive than insect cell coculture methods. A further advantage is a reduction in contaminating insect cell components. Use of anaerobiosis should facilitate early-passage screening of isolates for extrachromosomal elements, for use in gene vector constructs. The unique spiral (decreasing amplitude of coils) morphology of CPBS was preserved by anaerobiosis. The use of low-pH (6.0 to 6.5) media allowed aerobic adaptation of CPBS to M1D and SP-4 broth media. These formulations permitted the first cultivation of CPBS on solid media, an accomplishment that will simplify the selection of molecular transformants. Potato beetles collected at four sites in Poland yielded CPBS strains similar to those previously obtained from populations in North America.

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.

Spiroplasma diminutum sp. nov., from Culex annulus mosquitoes collected in Taiwan.

Initially, strain CUAS-1T (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-1T spiroplasmas were tested with Spiroplasma monobiae MQ-1T antiserum. The results of subsequent reciprocal spiroplasma deformation, metabolism inhibition, and growth inhibition tests clearly revealed that strain CUAS-1T 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-1T was characterized by using the minimal standards for mollicute species descriptions. During logarithmic-phase growth, strain CUAS-1T cells are characteristically very short helices with 1.5 to 2 helical turns (1 to 2 microns), 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 MID broth occurs at temperatures ranging from 10 to 37 degrees C, and the optimum temperature is 30 degrees 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-1T 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-1T (group XXV) should be placed in a new species, Spiroplasma diminutum. Strain CUAS-1 (= ATCC 49235) is the type strain of S. diminutum.

Spiroplasma velocicrescens sp. nov., from the vespid wasp Monobia quadridens.

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.

Spiroplasma ixodetis sp. nov., a new species from Ixodes pacificus ticks collected in Oregon.

Eight strains of mollicutes were isolated from pooled suspensions prepared from western black-legged ticks (Ixodes pacificus) collected in Oregon. Morphologic examination by electron and dark-field microscopic techniques showed that each strain consisted of a mixture of motile, tightly coiled helical cells, small coccoid cells with diameters ranging from 300 to 500 nm, and pleomorphic, straight or branched filamentous forms. All cellular forms were surrounded by a single cytoplasmic membrane, and there was no evidence of a cell wall. The organisms were filterable and fastidious in their growth requirements. The optimum temperature for growth was 30 degrees C, but multiplication occurred at temperatures ranging from 23 to 32 degrees C. The strains catabolized glucose but did not hydrolyze arginine or urea. The genome size of strain Y32T (T = type strain) was 2,220 kbp, and the DNA base composition (guanine-plus-cytosine content) of this organism was 25 +/- 1 mol%. The eight isolates were serologically related to each other but were not related to 37 other type or representative strains belonging to the genus Spiroplasma. Strain Y32 (= ATCC 33835) is the type strain of Spiroplasma ixodetis sp. nov.

Taxonomic descriptions of eight new non-sterol-requiring mollicutes assigned to the genus Mesoplasma.

Twenty mollicute strains isolated primarily from insect hosts were characterized and arranged into eight new species in the genus Mesoplasma. Morphological examination of the organisms by electron and dark-field microscopic techniques revealed that the cells of each strain were small, nonhelical, nonmotile, pleomorphic, and coccoid and that each cell was surrounded by a single cytoplasmic membrane with no evidence of a cell wall. Although the new mollicutes grew well in media containing horse or fetal bovine serum, growth in serum-free or cholesterol-free medium occurred only when the medium contained 0.04% polyoxyethylene sorbitan (Tween 80). The optimum temperature for growth was usually 30 degrees C, but multiplication generally occurred over a temperature range of 10 to 32 degrees C. All strains catabolized glucose. Most strains did not hydrolyze arginine or urea, although three related strains isolated from fireflies (the strain PUPA-2T [T = type strain] group) did hydrolyze arginine. The genome sizes ranged from 825 to 930 kbp, and the DNA base compositions (guanine-plus-cytosine contents) ranged from 26.5 to 31.6 mol%. The proposed type strains of the eight new species were not serologically related to the type strains of four other Mesoplasma species, five Entomoplasma species, 11 Acholeplasma species, and 100 Mycoplasma species and subspecies. Strain PS-1 (= ATCC 49582) is the type strain of Mesoplasma pleciae sp. nov., strain PUPA-2 (= ATCC 49581) is the type strain of Mesoplasma photuris sp. nov., strain YJS (= ATCC 51578) [corrected] is the type strain of Mesoplasma syrphidae sp. nov., strain CHPA-2 (= ATCC 49578) is the type strain of Mesoplasma chauliocola sp. nov., strain ELCA-2 (= ATCC 49579) is the type strain of Mesoplasma corruscae sp. nov., strain GRUA-1 (= ATCC 49580) is the type strain of Mesoplasma grammopterae sp. nov., strain BARC 779 (= ATCC 49583) is the type strain of Mesoplasma coleopterae sp. nov., and strain BARC 857 (= ATCC 49584) is the type strain of Mesoplasma tabanidae sp. nov.

Acholeplasma brassicae sp. nov. and Acholeplasma palmae sp. nov., two non-sterol-requiring mollicutes from plant surfaces.

Two mollicutes (strains 0502T [T = type strain] and J233T), which were isolated from the surfaces of broccoli (Brassica oleracea var. italica) plants or the crown tissues of the coconut palm (Cocos nucifera), were capable of sustained growth in serum-free (or cholesterol-free) mycoplasma broth media. Examination by electron and dark-field microscopic techniques revealed that the cells of each strain were small, nonhelical, nonmotile, pleomorphic, and coccoid and that each cell was surrounded by a single cytoplasmic membrane. No evidence of a cell wall was found. The organisms were filterable and grew rapidly in most conventional mycoplasma culture medium formulations containing horse or fetal bovine sera under either aerobic or anaerobic conditions. The optimum temperature for growth of both organisms was 30 degrees C, but multiplication occurred over a temperature range from 18 to 37 degrees C. Both strains catabolized glucose, but did not hydrolyze arbutin, arginine, or urea. The genome size of strain 0502T was 1,215 kbp, and the DNA base composition (guanine-plus-cytosine content) was 35.5 mol%. The genome size of strain J233T was 1,610 kbp, and the DNA base composition was 30.0 mol%. The two isolates were not serologically related to each other or to the type strains of 11 previously described Acholeplasma species. Strain 0502 (= ATCC 49388) is the type strain of Acholeplasma brassicae sp. nov., and strain J233 (= ATCC 49389) is the type strain of Acholeplasma palmae sp. nov.

Molecular taxonomy using single-strand conformation polymorphism (SSCP) analysis of mitochondrial ribosomal DNA genes.

Single-strand conformation polymorphism (SSCP) analysis detects single point mutations in DNA molecules. We demonstrate that SSCP analysis of mitochondrial ribosomal DNA (rDNA) genes is a sensitive taxonomic tool because these genes often differ at numerous sites among closely related species. Using conserved primers, portions of the 12S or 16S rDNA genes were amplified using the polymerase chain reaction (PCR) in congeneric species of ticks, leafhoppers, mosquitoes, and closely related endoparasitic wasps. SSCP was performed and products were visualized with silver staining. Species-specific patterns were observed in all taxa. Intraspecific variation at the level of single nucleotide substitutions was detected. SSCP diagnostics are less expensive and time consuming to develop than PCR with species-specific primers, and, unlike PCR with arbitrary primers, there is minimal concern with DNA contamination from non-target organisms.

A test for measuring growth responses of mollicutes to serum and polyoxyethylene sorbitan.

A test is described that is useful for characterizing mollicutes in terms of the ability to maintain growth in medium containing 15 to 20% fetal bovine serum or in serum-free media with or without 0.04% Tween 80 (polyoxyethylene sorbitan). Representative Acholeplasma species maintained growth in serum-free medium, and about half of the strains tested grew well in Tween 80-supplemented medium. Representative Mycoplasma and Entomoplasma species did not maintain growth in either serum-free medium alone or when Tween 80 was added. Spiroplasma species and group representatives generally failed to sustain growth in serum-free medium with or without Tween 80, but at least four of the spiroplasmas tested maintained growth in serum-free medium. The representative Mesoplasma species grew in serum-free media only when Tween 80 was added, as did Mycoplasma lactucae. Although the test has obvious determinative uses for members of the class Mollicutes, it does not supplant the conventional methodology for assaying the cholesterol requirements of these organisms.

A phylogeny of New World Deltocephalus-like leafhopper genera based on mitochondrial 16S ribosomal DNA sequences.

The Deltocephalus-like leafhoppers are a group of grass-feeding insects that share common forms in the male genitalia, chiefly a fused aedeagal-connective structure. The group contains 27 New World genera. The phylogenetic relationships among these genera and other genera in the tribe are unclear because fusion of the aedeagus and connective, although clearly apomorphic, has occurred in both related and unrelated groups. An independent set of characters is required to test the hypothesis that the Deltocephalus-like genera are monophyletic and to derive relationships among these genera. We examined 562 sites in the 3'-end of the mitochondrial 16S ribosomal DNA from 21 species representing 19 New World genera. Eight species in closely related groups or tribes were also analyzed. The region was sequenced directly from PCR-amplified DNA of field-collected and museum-preserved dried specimens. The transversion rate was twice as high as the transition rate; however, 74% of these were adenine-thymine transversions. Analysis of secondary structure indicated that substitution rates were equal in stem and loop forming regions of the processed rRNA. Using either parsimony or distance analysis, derived phylogenies suggested that, with the exception of the genus Cabrulus, the Deltocephalus-like leafhoppers are genera within a monophyletic group.

Occurrence of extrachromosomal deoxyribonucleic acids in spiroplasmas associated with plants, insects, and ticks.

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.