Truman B. Clark

Spiroplasmas: Diversity of Arthropod Reservoirs and Host-Parasite Relationships

Spiroplasmas were found in 11 new insect hosts representing four orders: Hymenoptera, Hemiptera, Diptera, and Coleoptera. Three of the new spiroplasmas were serologically distinct from all existing groups or subgroups. A spiroplasma that infects digestive tracts of Colorado potato beetles may be transmitted to uninfected insects that feed on contaminated plants. This simple type of spiroplasma-insect relationship may explain a growing list of isolations of spiroplasmas and other wall-less prokaryotes from plant surfaces.

Spiroplasma melliferum, a New Species from the Honeybee (Apis mellifera)

Twenty-eight strains of spiroplasma subgroup I-2 isolated from insects and flower surfaces were similar in their serological properties. Strain BC-3T (T = type strain), which was isolated from the honeybee, was chosen as a representative of this cluster and was characterized according to accepted standards. This strain and other strains of the cluster entered the hemocoel of their insect hosts after per os acquisition, caused pathology in various tissues, and reduced adult longevity. Growth in SM-1 or M1D medium occurred at 20 to 37°, with optimum growth at about 32 to 35°. Cholesterol was required for growth. Glucose, fructose, and other carbohydrates were fermented, and arginine was catabolized. Seventeen strains, including strain BC-3T, reacted with considerable homogeneity in deformation tests and were completely separable from strains of subgroup I-1 (Spiroplasma citri) and subgroup I-3 (corn stunt spiroplasma). A group of five subgroup I-2 strains showed homogeneity upon one-dimensional polyacrylamide gel electrophoresis of cell proteins. Strain BC-3T was also serologically distinct from subgroups I-4 through I-8; from Spiroplasma floricola, Spiroplasma apis, and Spiroplasma mirum; and from representative strains of spiroplasma groups II and VI through XI. Previously published studies on strain BC-3T and related strains demonstrated that (i) these organisms comprise a unique subgroup of the S. citri complex (group I); (ii) deoxyribonucleic acid-deoxyribonucleic acid homologies between strain BC-3T and strains of other group I subgroups do not exceed 70%; (iii) the patterns of protein sharing among group I strains revealed by two-dimensional polyacrylamide gel electrophoresis support molecular genetic indications of partial relatedness; (iv) the EcoRI restriction endonuclease patterns of deoxyribonucleic acids from strain BC-3T and serologically related strains show close relatedness; (v) sequencing of 5S ribosomal ribonucleic acid suggests some degree of relatedness with all organisms now classified in the Mollicutes; (vi) strain BC-3T is capable of viscotactic and chemotactic responses; (vii) strain BC-3T possesses fibrils that may mediate various types of motility; and (viii) a lytic virus (SpV4) isolated from Spiroplasma sp. strain B63 (a representative of subgroup I-2) is morphologically and genomically distinct from other spiroplasma viruses and forms plaques only on lawns of subgroup I-2 spiroplasmas. Previous work on strain AS 576, another member of subgroup I-2, demonstrated (i) a viscotactic response, (ii) moderate sensitivity to osmotic environments, (iii) susceptibility to tetracycline and aminoglycoside antibiotics, (iv) growth in a relatively simple, chemically defined medium, (v) nutritional utilization patterns in defined medium, and (vi) a genome molecular weight of 109. On the basis of our new findings and the previously described properties of strain BC-3T and related subgroup I-2 strains, we propose that spiroplasma strains with the characteristics described here be classified as a new species, Spiroplasma melliferum. Strain BC-3, the type strain, has been deposited in the American Type Culture Collection as strain ATCC 33219.

Spiroplasma kunkelii sp. nov.: Characterization of the Etiological Agent of Corn Stunt Disease

Nine strains of spiroplasma subgroup 1-3, which comprise the etiological agent of corn stunt disease, were similar in their serological properties. Strain E275T(T = type strain) was studied by using criteria proposed by the International Committee on Systematic Bacteriology Subcommittee on Taxonomy of Mollicutes for descriptions of new mollicute species. This strain was shown to belong to the class Mollicutes by the ultrastructure of its limiting membrane, its procaryotic organization, its colonial morphology, and its filtration behavior and to the family Spiroplasmataceae by its helical morphology and motility. Although some serological cross-reactions with other group I spiroplasma strains was observed, strain E275Tcould be readily distinguished from representatives of other group I subgroups. Subgroup 1-3 spiroplasmas and other group I strains also differed in their one- and two-dimensional polyacrylamide gel protein patterns, plant and insect host ranges, and pathogenicities. Growth in MIA or MID medium occurred at 20 to 30°C. Cholesterol was required for growth. Glucose was fermented, and arginine was catabolized. Subgroup 1-3 strains, including strain E275T, reacted with considerable homogeneity in deformation tests and were completely separable from strains belonging to subgroup I-1 (Spiroplasma citri) and subgroup I-2 (Spiroplasma melliferum). Strain E275Twas also serologically distinct from subgroups I-4 through I-8, Spiroplasma floricola (group III), Spiroplasma apis (group IV), Spiroplasma mirum (group V), and representative strains of spiroplasma groups II and VI through XI. The deoxyribonucleic acid of strain E275Thybridized with the deoxyribonucleic acid of S. citri at significant levels (33 to 68%, depending on the technique used). These results demonstrate that strain E275Tand similar strains meet the criteria proposed by the International Committee on Systematic Bacteriology Subcommittee for elevation of spiroplasma subgroups to species. We propose that such strains be named Spiroplasma kunkelii. Strain E275Thas been deposited in the American Type Culture Collection as strain ATCC 29320T.

Revised serological classification of spiroplasmas, new provisional groups, and recommendations for serotyping of isolates

Five new spiroplasma strains were analyzed in reciprocal growth inhibition, metabolism inhibition (MI), and deformation (DF) serological tests. New provisional groups from the waspMonobia quadridens (VII) and the syrphid flyEristalis arbustorum (VIII) were added to the existing classification. Three serovars—represented by the LB-12 green leaf bug spiroplassignificantly with subgroup I-4 and with each other. These new groups and serovars bring the number of recognized spiroplasma serovars to 16. Serological distinctiveness of a sixth group (VI) fromIxodes ticks was confirmed. Simple DF and/or MI procedures are described for typing new isolates.

A spiroplasma of serogroup IV causes a May-disease-like disorder of honeybees in Southwestern France

Honeybees affected by a disorder resembling the classical “May disease” in southwestern France contained numerous helical, motile organisms in their digestive tracts and hemolymph. Two strains of the organism (B31 and B39) were cultured and triply cloned in the BSR spiroplasma medium. The electrophoretic patterns of spiroplasmal proteins in 1 - and 2-dimensional polyacrylamide gels were similar to those of group IV spiroplasmas F1 and F2, cultured previously from flower surfaces in France. The organism could be introduced into adult bees by injection or food ingestion at various stages after emergence. Agent administered by either route multiplied to high titers in the hemolymph and killed the bees. Both multiplication and the induced lethal effect of the agent could be prevented by tetracycline but not penicillin. Spiroplasmas that were nearly identical to the B31 and B39 strains were also recovered from the surface of flowers collected within the area visited by the bees from the diseased hives.

A filamentous virus of the honey bee

Abstract A common and widespread disease of honey bees, Apis mellifera, is caused by an unoccluded, Feulgen-positive, filamentous nuclear virus. Ovoid viral particles seen in diseased bee hemolymph consisted of a folded nucleocapsid within a viral envelope and were 0.40 by 0.10 μm. Virions with unfolded nucleocapsids were about 3060 by 60 nm. The disease was transmissible to bees both per os and by injection, but efforts to infect oriental cockroaches, Blatta orientalis, and the greater wax moth, Galleria mellonella, failed. The disease is apparently the same as that described as a rickettsial disease of European bees.

Acholeplasmas and similar nonsterol-requiring mollicutes from insects: Missing link in microbial ecology

Wall-less prokaryotes from guts of five insect species (a corn root maggot [Plecia sp.: Diptera: Bibionidae, strain PS-1], a syrphid fly [Diptera: Syrphidae, strain YJS], two tabanid flies [Tabanus catenatus, strain TAC, andChrysops discalis, strain DF-2], and a vespid wasp [Monobia quadridens, strain MQ-3]) were characterized. The strains grew at 23°–32°C in conventional mycoplasma media containing 10% (vol/vol) serum, or in serum-free mycoplasma medium with or without fatty-acid-Tween-80 supplements. No helical forms were noted in dark-field microscopy, and electron micrographs of thin sections of the strains showed a single membrane. Two strains (DF and MQ-3) cross reacted with antiserum againstAcholeplasma florum. Although the other three strains did not react with antisera to establishedAcholeplasma orMycoplasma species, they were otherwise characteristic of acholeplasmas. This study, the first to demonstrate acholeplasmas from Arthropoda, in conjunction with previous isolations from plant surfaces, suggests that insects may constitute an important reservoir for acholeplasmas.

Spiroplasmas from coleopterous insects: New ecological dimensions.

The genusSpiroplasma (helical wall-less prokaryotes) is a recently described group of microorganisms that cause disease in plants, arthropods, and experimentally, in vertebrates. Two spiroplasmas from beetles have now been discovered in a search for microorganisms suitable for biological control of economically important coleopterous insects. Colorado potato beetles (CPB) infected with spiroplasma were commonly found on potato and other solanaceous plants in Maryland. Although this spiroplasma occurred in high concentration in gut fluids and sputum, it could not be cultivated in conventional spiroplasma media. However, another spiroplasma (CN-5 and related strains) reported here to occur commonly in association with larvae and adults of the green June beetle,Cotinus nitida, could be cultivated readily in the SM-1 formulation and several other conventional spiroplasma media. The CN-5 spiroplasma was serologically distinct from representative members of all 8 major groups now recognized. Thus, it represents a ninth major spiroplasma serogroup (IX), and can be considered to be an unnamed species. The CPB spiroplasma is apparently maintained in plant surface-insect gut cycles, but details of maintenance of the CN-5 spiroplasma are incompletely understood. Isolation of CN-5 spiroplasma from soil in which host larvae had fed suggests that transmission of this agent may occur in the soil. Both CN-5 and CPB spiroplasmas exhibited unusually active translational motility in natural fluids, and CN-5 organisms exhibited such motility in culture media. Although we have no evidence that either spiroplasma is pathogenic to its usual host, the pathogenicity of spiroplasmas to many hosts, including the beetle,Melolontha melolontha, suggests possible application for biological control.

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.

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.