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.     

Arthropod cell lines in the isolation and propagation of tickborne spiroplasmas

The ability of several continuous tick cell culture lines to support growth of tickborne spiroplasmas (helical, wall-less prokaryotes in the classMollicutes) was assessed. Seven triturates, prepared from pools ofIxodes pacificus ticks naturally infected with theSpiroplasma sp. (group VI) organism, were retrieved from frozen (–70°C) storage and passaged in three distinct tick cell lines, in antibiotic-free tick cell culture medium alone, or in spiroplasma culture medium (SP-4 formulation). Six spiroplasma strains were recovered in the RML-19 cell line fromDermacentor variabilis, and five isolations were made in another cell line (RML-15) from this tick species. None was recovered in aRhipicephalus sanguineus cell line (RML-23), in tick cell culture medium, or in SP-4 broth medium. One of the spiroplasma isolates (Y43) was maintained through four consecutive weekly refeedings of theD. variabilis cell line and for three feedings ofR. sanguineus cells, where numbers of spiroplasmas in cell supernatants reached levels comparable to those obtained in the SP-4 medium.A laboratory-adapted strain (SMCA) ofSpiroplasma mirum, a second helical mollicute of tick origin (the suckling mouse cataract agent), grew in three tick cell lines (RML-15, RML-23, and RML-16 cells fromD. parumapertus), in three mosquito cell lines (fromAedes albopictus, Ae. aegypti, andCulex quinquefasciatus), and in both cell culture medium alone and in SP-4 medium. The organisms survived for 1–2 weeks, but failed to multiply, in cell lines fromC. tritaeniorhynchus, Antheraea eucalypti, orXenopus laevis. Some evidence of cytopathic effect ofS. mirum on tick cell lines was seen, although growth of the organism in mosquito cell cultures was not associated with cell toxicity. The use of arthropod cell lines appears to have value in the primary isolation of arthropod- or insect-derived mollicutes and for the study of cytopathogenicity of these wall-less prokaryotes.     

Characterization of Spiroplasmas by one- and two-dimensional protein analysis on polyacrylamide slab gels

Differences betweenSpiroplasma citri isolates were detected by one-dimensional electrophoresis of proteins on gradient polyacrylamide slab gels. Two-dimensional protein maps (electrofocusing followed by electrophoresis) showed a highly characteristic pattern for allS. citri isolates examined. Coanalysis of mixed protein samples from pairs ofS. citri strains revealed more than 150 comigrating proteins common to allS. citri isolates, but also a number of noncomigrating proteins. Some noncomigrating proteins were present in one isolate but not in another, while other proteins whose migrational properties were only slightly different from one isolate to the other (homologous proteins), were present in more than one isolate.S. citri isolates had many common and only a few homologous proteins. Comparisons ofS. citri with the corn stunt spiroplasma revealed few common proteins and a large number of homologous proteins. When comparingS. citri and the suckling mouse cataract spiroplasma, few common and homologous proteins were apparent. However, several of these common proteins were also shared by the corn stunt spiroplasma, suggesting that they may well represent genus-specific proteins. The data also offer additional evidence that the suckling mouse cataract spiroplasma differs significantly fromS. citri and corn stunt spiroplasmas and probably deserves a separate species designation.     

Sequencing of the intergenic 16S-23S rRNA spacer (ITS) region of Mollicutes species and their identification using microarray-based assay and DNA sequencing

Abstract We have completed sequencing the 16S-23S rRNA intergenic transcribed spacer (ITS) region of most known Mycoplasma , Acholeplasma , Ureaplasma , Mesoplasma , and Spiroplasma species. Analysis of the sequence data revealed a significant interspecies variability and low intraspecies polymorphism of the ITS region among Mollicutes . This finding enabled the application of a combined polymerase chain reaction–microarray technology for identifying Mollicutes species. The microarray included individual species-specific oligonucleotide probes for characterizing human Mollicutes species and other species known to be common cell line contaminants. Evaluation of the microarray was conducted using multiple, previously characterized, Mollicutes species. The microarray analysis of the samples used demonstrated a highly specific assay, which is capable of rapid and accurate discrimination among Mollicutes species.     

Mycoplasma cavipharyngis and Mycoplasma fastidiosum, the closest relatives to Eperythrozoon spp. and Haemobartonella spp.

The 16S rRNA gene sequences of Mycoplasma cavipharyngis and Mycoplasma fastidiosum have been determined. Phylogenetic analysis showed that these species formed a new cluster within the so-called pneumoniae group of the mollicutes (class Mollicutes). This cluster will be referred to as the M. fastidiosum cluster. Interestingly, the M. fastidiosum cluster formed a sister lineage to the haemotrophic bacteria. Eperythrozoon spp. and Haemobartonella spp. The two latter genera, formerly classified as rickettsias, formed a stable phylogenetic entity in the tree as judged from branch lengths, bootstrap values and sequence signatures. Thus, the members of the M. fastidiosum cluster are the closest known relatives to the haemotrophic bacteria. Our data strongly support that the haemotrophic bacteria should be reclassified to reflect their actual phylogenetic affiliation.     

NewLeptographium species from Indonesia and Eastern North America

Leptographium species have predominantly been described from North America, Canada and Europe. These fungi generally occur on conifers and many cause blue-stain of lumber. MostLeptographium species are also associated with insects and in particular, bark beetles (Coleoptera: Scolytidae). Recently, an unknown species ofLeptographium was isolated from pine infested with anIps sp. in Indonesia. In addition, two unknown species have been collected from red spruce (Picea rubra) and balsam fir (Abies balsamea) roots from high elevation sites in Eastern North America. The latter isolates are unusual in that they are associated with the feeding wounds made by the conifer swift mothKorscheltellus gracilus (Lepidoptera: Hepialidae), which is a habitat unique for species ofLeptographium. Comparison with knownLeptographium species has revealed that the isolates from Indonesia and those from Eastern North America represent three previously undescribed taxa. They are, therefore, described in this study asL. pineti sp. nov,L. abieticolens sp. nov. andL. peucophilum sp. nov.     

A physical and genetic map of the Spiroplasma citri genome.

A physical and genetic map of the Spiroplasma citri genome has been constructed using several restriction enzymes and pulsed field gel electrophoresis. A number of genes were subsequently localized on the map by the use of appropriate probes. The genome size of the spiroplasma estimated from restriction fragments is close to 1780 kbp, the largest of all Mollicutes studied so far. It contains multisite insertions of Spiroplasma virus 1 (SpV1) sequences. The physical and genetic map of the S. citri genome shares several features with that of other Mollicutes, especially those in the Mycoplasma mycoides cluster. This supports the finding that S. citri and these Mycoplasma spp. are phylogenetically related.     

Ophiostomatoid fungi isolated from Japanese red pine and their relationships with bark beetles

We isolated ophiostomatoid fungi from bark beetles infesting Pinus densiflora and their galleries at 24 sites in Japan. Twenty-one ophiostomatoid fungi, including species of Ophiostoma, Grosmannia, Ceratocystiopsis, Leptographium, and Pesotum, were identified. Among these, 11 species were either newly recorded in Japan or were previously undescribed species. Some of these fungal species were isolated from several bark beetles, but other species were isolated from only a particular beetle species. Thus, it is suggested that some ophiostomatoid fungi have specific relationships with particular beetle species. In addition, fungus-beetle biplots from redundancy analysis (RDA) summarizing the effects of beetle ecological characteristics suggested that the association patterns between bark beetles and the associated fungi seemed to be related to the niches occupied by the beetles.     

Species-specific PCR for identification of common contaminant mollicutes in cell culture.

Mycoplasma arginini, M. fermentans, M. hyorhinis, M. orale, and Acholeplasma laidlawii are the members of the class Mollicutes most commonly found in contaminated cell cultures. Previous studies have shown that the published PCR primer pairs designed to detect mollicutes in cell cultures are not entirely specific. The 16S rRNA gene, the 16S-23S rRNA intergenic spacer region, and the 5' end of the 23S rRNA gene, as a whole, are promising targets for design of mollicute species-specific primer pairs. We analyzed the 16S rRNA genes, the 16S-23S rRNA intergenic spacer regions, and the 5' end of the 23S rRNA genes of these mollicutes and developed PCR methods for species identification based on these regions. Using high melting temperatures, we developed a rapid-cycle PCR for detection and identification of contaminant mollicutes. Previously published, putative mollicute-specific primers amplified DNA from 73 contaminated cell lines, but the presence of mollicutes was confirmed by species-specific PCR in only 60. Sequences of the remaining 13 amplicons were identified as those of gram-positive bacterial species. Species-specific PCR primers are needed to confirm the presence of mollicutes in specimens and for identification, if required.     

Characteristic features of embryogenesis and oogenesis in the beetlesAdalia bipunctata L. andHarmonia axyridis Pall. in all-female families

We describe abnormal embryonic development leading to death of 50% of embryos in all-female lines ofAdalia andHarmonia infected with an androcide spiroplasma. The arrest of embryonic development takes place at different stages throughout embryogenesis. Oogenesis of the infected beetles proceeds without any significant morphological changes.     

Pollination ofOpuntia lindheimeri and related species

The large, yellow, bowl-shaped flowers ofOpuntia lindheimeri, O. discata, O. phaeacantha major, andO. compressa in Texas are visited by various species of beetles and bees. The beetles and small bees (Perdita, Dialictus) are pollen thieves. The pollinators are the medium-sized and larger bees (Melissodes, Diadasia, Lithurge, Megachile, Agapostemon, etc.). Different species of theOpuntia lindheimeri group have the same pollination system and there is no evidence of any floral isolation between them. The pollination system of these species ofOpuntia in Texas is essentially the same as that ofEchinocereus fasciculatus andFerocactus wislizeni in Arizona.Pollination of North American Cacti, II.—SeeGrant & Grant (1979).     

Infectivity of beetle spiroplasmas for new host species

Five beetle spiroplasmas, the Colorado potato beetlespiroplasma (CPBS, strain LD-1), the Cantharis carolinusspiroplasma (CCBS, strain CC-1), the Ellychnia corrusca fireflyspiroplasma (FS, strain EC-1), the Diabrotica undecimpunctatacorn rootworm spiroplasma (CRS, strain DU-1), and the Spiroplasmafloricola fall flower spiroplasma (FFS), all associated withbeetles, were fed to beetles (Maladera matrida and Carpophilushumeralis) and mosquitoes (Aedes aegypti and Culex pipiens). CPBSand CCBS were also injected into M. matrida. Attempts to recoverspiroplasmas from regurgitates and hemolymph were conducted 1–10days after their introduction. After day 1, orally administeredspiroplasmas could not be recovered from M. matrida beetles;however, at 2–5 days, four out of five spiroplasmas wererecovered from adult C. humeralis. Injected spiroplasmas survivedin the hemolymph of M. matrida beetles for a relatively longperiod (at least 22 days). All five spiroplasmas were recoveredfrom mosquitoes 1 day post feeding, but only two (CCBS and CRS)survived for five or more days. The results show short andvariable persistence in orally challenged non-host insects, withgeneral failure to pass the gut barrier. Such evidence should beconsidered when attempting to use these microbes in biocontrolprograms.     

Spiroplasma-like organisms closely associated with the gut in five leafhopper species (Hemiptera: Cicadellidae)

Spiroplasmas are bacteria in the Class Mollicutes that are frequently associated with insects and/or plants. Here, we describe the ultrastructure, localization, and occurrence of apparent commensal/symbiotic spiroplasma-like organisms (SLOs) in the midgut and hindgut of five leafhopper species from laboratory-reared colonies. Those found in Dalbulus elimatus, Endria inimica, and Macrosteles quadrilineatus were long and tubular shaped, whereas those in Dalbulus maidis and Graminella nigrifrons were shorter and mostly rod-shaped in their host organisms. These SLOs were found in great numbers in the gut lumen frequently associated with the gut microvilli, but unlike the plant-pathogenic mollicutes, they did not seem to invade the gut epithelium or other tissues in any of these five leafhopper species. Large accumulations of these gut-associated organisms were more commonly found by confocal laser scanning microscopy in males than in females and in crowded than in singly reared leafhoppers. Ultrastructural evidence suggests that these SLOs may be horizontally transmitted between leafhoppers by contamination of the mouth parts with leafhopper excretions.     

Host specificity of mollicutes oriC plasmids: functional analysis of replication origin

Recently, artificial oriC plasmids containing the chromosomal dnaA gene and surrounding DnaA box sequences were obtained for the mollicutes Spiroplasma citri and Mycoplasma pulmonis. In order to study the specificity of these plasmids among mollicutes, a set of similar oriC plasmids was developed for three mycoplasmas belonging to the mycoides cluster, Mycoplasma mycoides subsp. mycoides LC (MmmLC), M.mycoides subsp. mycoides SC (MmmSC) and Mycoplasma capricolum subsp. capricolum. Mycoplasmas from the mycoides cluster, S.citri and M.pulmonis were used as recipients for transformation experiments by homologous and heterologous oriC plasmids. All five mollicutes were successfully transformed by homologous plasmids, suggesting that the dnaA gene region represents the functional replication origin of the mollicute chromosomes. However, the ability of mollicutes to replicate heterologous oriC plasmids was found to vary noticeably with the species. For example, the oriC plasmid from M.capricolum did not replicate in the closely related species MmmSC and MmmLC. In contrast, plasmids harbouring the oriC from MmmSC, MmmLC and the more distant species S.citri were all found to replicate in M.capricolum. Our results suggest that the cis-elements present in oriC sequences are not the only determinants of this host specificity.     

Proposal for classifying human strain navel and related simian mycoplasmas as Mycoplasma primatum sp. n

Mycoplasmas isolated from simian (Cercopithecus aethiops) tissues were shown to have biological, biochemical, and serological properties and electrophoretic cell protein patterns similar to strain Navel isolated from man 15 years ago. The simian and human Navel strains comprised a single serogroup, distinct from the established Mycoplasma and Acholeplasma species of the class Mollicutes. It is proposed that strains with the properties described be named Mycoplasma primatum.     

Rickettsial and mollicute infections in hepatopancreatic cells of cultured Pacific white shrimp (Penaeus vannamei)

Infections by multiple species of bacteria occurred in hepatopancreatic epithelial cells of cultured Pacific white shrimp (Penaeus vannamei). Grossly, hepatopancreases of moribund shrimp were pale white. Light microscopically, hepatopancreatic tubules appeared atrophied and were associated with granulomas. Examination by scanning and transmission electron microscopy revealed heavy cytoplasmic infections by three forms of microorganisms: (1) a rickettsia-like bacterium, (2) a helical form of a mollicute-like bacterium, and (3) a filamentous mollicute-like bacterium. The rod-shaped rickettsia (900 nm long by 300 nm wide) appeared to be free in the cytoplasm and had both a plasma membrane and a cell wall. Neither form of mollicute possessed a cell wall. The helical mollicute was blunt at its wide end (about 260 nm in diameter) where it contained electron-lucent bodies. Helical turns along its tapered axis resembled those of a spiroplasma (the only helical form of mycoplasma in the class Mollicutes) or a spirochete. The helical bacterium did not possess periplasmic flagella characteristic of spirochetes, which lends support to its being a type of spiroplasma. The filamentous mollicute consisted of masses of short, branched filaments 60 nm wide with intermittent spherical dilations and terminal blebs on the branches. The presumed mollicutes have not been reported previously in crustaceans. Each bacterium, or concurrent infections of the bacteria, are pathogenic to cultured shrimp, could impact culture operations and thus deserve more study.     

An attachment tip and pili-like structures in insect- and plant-pathogenic spiroplasmas of the class Mollicutes

Ultrastructural studies using scanning electron microscopy (SEM), negative-staining transmission electron microscopy (TEM), and thin-sectioning TEM on four species of Spiroplasma, in vitro and/or in vivo, indicated that their helices commonly possess one tapered end (tip structure) and one blunt or round end. These tip structures appeared morphologically different from the rest of the helix, exhibiting an electron-dense conical or rod-shaped core. In thin sections of the midgut of the leafhopper Dalbulus elimatus, the tip structures of Spiroplasma kunkelii in the midgut lumen were mostly aligned between microvilli, perpendicular to the apical plasma membrane of epithelial cells. These tip structures appeared frequently attached or closely apposed to the plasma membrane, in which cup-shaped invaginations close to the tips were observed. Pleomorphic forms of spiroplasma, enclosed in membranous vesicles, were found in the cytoplasm of the midgut epithelial cells. These findings suggest that the tip structure may be involved in the orientation and attachment of spiroplasma helices in relation to their host cells, and thus may be functionally comparable to the attachment organelle of mycoplasmas. Additionally, pili-like structures were observed by negative-staining TEM on the surface of Spiroplasma melliferum, and in thin sections of S. kunkelii infecting the leafhopper vector Dalbulus gelbus. Abbreviations CSS Corn stunt spiroplasma - SEM Scanning electron microscopy - TBS Tris-buffered saline - TEM Transmission electron microscopy     

Direct isolation in cell-free medium of a spiroplasma fromHaemaphysalis leporispalustris (Acari: Ixodidae) in Maryland

A spiroplasma isolate, was obtained from rabbit ticks (Haemaphysalis leporispalustris) taken from cottontail rabbits in Maryland by inoculation of tick suspensions into SP-4 medium. The isolate was indistinguishable from an experimental vertebrate pathogen (suckling mouse cataract agent spiroplasma) when tested with other plant and tick spiroplasmas in growth inhibition, deformation, and metabolism inhibition tests. The isolated organism had a pathogenic profile for suckling rats and embryonated chicken eggs that differed significantly from that of other suckling mouse cataract agent strains. This is the first report of a direct spiroplasma isolation from ticks in cell-free medium, and confirms the specific association of spiroplasmas of the suckling mouse cataract agent serogroup with rabbit ticks.     

<Emphasis Type="Italic">Geosmithia</Emphasis> Fungi are Highly Diverse and Consistent Bark Beetle Associates: Evidence from their Community Structure in Temperate Europe

Geosmithia spp. (Ascomycota: Hypocreales) are little-studied, dry-spored fungi that occur in galleries built by many phloeophagous bark beetles. This study mapped the distribution and environmental preferences of Geosmithia species occurring in galleries of temperate European bark beetles. One hundred seven host tree samples of 16 tree species infested with 23 subcortical insect species were collected from across Europe during the years 1997–2005. Over 600 Geosmithia isolates from the beetles were sorted into 17 operational taxonomic units (OTUs) based on their phenotype similarity and phylogeny of internal transcribed spacer (ITS) region of rDNA (ITS1-5.8S-ITS2). The OTUs represent six known species and eight undescribed taxa. Ninety-two samples infested with subcortical insects were characterized by the presence/absence of OTUs and the similarity among the samples was evaluated. Geographically distant populations of the same beetle species host relatively uniform Geosmithia communities across large geographic areas (ranging from southern Bulgaria to the Czech Republic). This suggests effective dispersal of Geosmithia spp. by bark beetles. Clustering of similar samples in ordination analysis is correlated predominantly with the isolation source (bark beetles and their respective feeding plant), but not with their geographical origin. The composition of the Geosmithia OTU community of each bark beetle species depends on the degree of isolation of the species’ niches. Thus, Geosmithia communities associated with regularly co-occurring bark beetle species are highly similar. The similarity decreases with decreasing frequency of beetle species’ co-occurrence, a pattern resembling that of entomochoric ophiostomatoid fungi. These findings suggest that: 1) communities of Geosmithia spp. are vector-specific; 2) at least in some cases, the association between Geosmithia OTUs and bark beetles may have been very stable and symbioses are likely to be a fundamental factor in the speciation of Geosmithia fungi; and 3) that even nonsticky spores of Geosmithia are suitable for maintaining an insect–fungus association, contrary to previous hypotheses. An erratum to this article can be found at