Identification of Spiroplasma insolitum symbionts in Anopheles gambiae

Background: Insect symbionts have the potential to block the transmission of vector-borne diseases by their hosts. The advancement of a symbiont-based transmission blocking strategy for malaria requires the identification and study of Anopheles symbionts. Methods: High throughput 16S amplicon sequencing was used to profile the bacteria associated with Anopheles gambiae sensu lato and identify potential symbionts. The polymerase chain reaction (PCR) with specific primers were subsequently used to monitor symbiont prevalence in field populations, as well as symbiont transmission patterns. Results: We report the discovery of the bacterial symbiont, Spiroplasma, in Anopheles gambiae in Kenya. We determine that geographically dispersed Anopheles gambiae populations in Kenya are infected with Spiroplasma at low prevalence levels. Molecular phylogenetics indicates that this Anopheles gambiae associated Spiroplasma is a member of the insolitum clade. We demonstrate that this symbiont is stably maternally transmitted across at least two generations and does not significantly affect the fecundity or egg to adult survival of its host. Conclusions: In diverse insect species, Spiroplasma has been found to render their host resistant to infection by pathogens. The identification of a maternally transmitted strain of Spiroplasma in Anopheles gambiae may therefore open new lines of investigation for the development of symbiont-based strategies for blocking malaria transmission

Spiroplasma Bacteria Enhance Survival of Drosophila hydei Attacked by the Parasitic Wasp Leptopilina heterotoma

Maternally-transmitted associations between endosymbiotic bacteria and insects are ubiquitous. While many of these associations are obligate and mutually beneficial, many are facultative, and the mechanism(s) by which these microbes persist in their host lineages remain elusive. Inherited microbes with imperfect transmission are expected to be lost from their host lineages if no other mechanisms increase their persistence (i.e., host reproductive manipulation and/or fitness benefits to host). Indeed numerous facultative heritable endosymbionts are reproductive manipulators. Nevertheless, many do not manipulate reproduction, so they are expected to confer fitness benefits to their hosts, as has been shown in several studies that report defense against natural enemies, tolerance to environmental stress, and increased fecundity.We examined whether larval to adult survival of Drosophila hydei against attack by a common parasitoid wasp (Leptopilina heterotoma), differed between uninfected flies and flies that were artificially infected with Spiroplasma, a heritable endosymbiont of Drosophila hydei that does not appear to manipulate host reproduction. Survival was significantly greater for Spiroplasma-infected flies, and the effect of Spiroplasma infection was most evident during the host's pupal stage. We examined whether or not increased survival of Spiroplasma-infected flies was due to reduced oviposition by the wasp (i.e., pre-oviposition mechanism). The number of wasp eggs per fly larva did not differ significantly between Spiroplasma-free and Spiroplasma-infected fly larvae, suggesting that differential fly survival is due to a post-oviposition mechanism.Our results suggest that Spiroplasma confers protection to D. hydei against wasp parasitism. This is to our knowledge the first report of a potential defensive mutualism in the genus Spiroplasma. Whether it explains the persistence and high abundance of this strain in natural populations of D. hydei, as well as the widespread distribution of heritable Spiroplasma in Drosophila and other arthropods, remains to be investigated

Challenging the Wigglesworthia, Sodalis, Wolbachia symbiosis dogma in tsetse flies : Spiroplasma is present in both laboratory and natural populations

Profiling of wild and laboratory tsetse populations using 16S rRNA gene amplicon sequencing allowed us to examine whether the “Wigglesworthia-Sodalis-Wolbachia dogma” operates across species and populations. The most abundant taxa, in wild and laboratory populations, were Wigglesworthia (the primary endosymbiont), Sodalis and Wolbachia as previously characterized. The species richness of the microbiota was greater in wild than laboratory populations. Spiroplasma was identified as a new symbiont exclusively in Glossina fuscipes fuscipes and G. tachinoides, members of the palpalis sub-group, and the infection prevalence in several laboratory and natural populations was surveyed. Multi locus sequencing typing (MLST) analysis identified two strains of tsetse-associated Spiroplasma, present in G. f. fuscipes and G. tachinoides. Spiroplasma density in G. f. fuscipes larva guts was significantly higher than in guts from teneral and 15-day old male and female adults. In gonads of teneral and 15-day old insects, Spiroplasma density was higher in testes than ovaries, and was significantly higher density in live versus prematurely deceased females indicating a potentially mutualistic association. Higher Spiroplasma density in testes than in ovaries was also detected by fluorescent in situ hybridization in G. f. fuscipe

‘Candidatus Phytoplasma’

Phy.to.plas-ma.Gr.masc.n.phytosaplant;N.L.neut.n. Phytoplasma a form from a plant. Phytoplasmas are wall-less, nutritionally fastidi- ous, and phytopathogenic prokaryotes 0.2–0.8μm in diameter that morphologically resemble nonhelical members of the class Mollicutes. Sequencing of nearly full-length PCR-amplified 16S rRNA genes, combined with earlier studies, provided the first comprehensive phylogeny of the organisms and showed that they constitute a unique, monophyletic clade within the Mollicutes. These organisms are most closely related to members of the genus Acholeplasma within the Anaero- plasma clade as defined by Weisburg et al. Despite decades of efforts and recent progress, sustained pure culture in cell-free media has not yet been demon- strated for any phytoplasma. Their genome sizes have been estimated to range from 530 to 1,350kb, and the DNA G+C content is about 23–30mol%. The presence of a characteristic oligonucleotide sequence in the 16S rRNA gene, CAA GAY BAT KAT GTK TAG CYG GDC T, and standard codon usage indicate thaphytoplasmasrepresentadistinctgenus-leveltaxonfor which the name ‘Candidatus Phytoplasma’ has been adopted. At present, for any subtaxa within the pro- visional genus, the designation ‘Candidatus’ must still be used

Serologic and genomic relatedness of group XIV spiroplasma isolates from a lampyrid beetle and tabanid flies: an ecologic paradox

Spiroplasma group XIV strain EC-1T and other isolates from the lampyrid beetle Ellychnia corrusca form a serogroup with tabanid spiroplasma strains (TC-1 and TS-1). It was hypothesized that similarities among these strains reflect a transmission cycle in which lampyrid beetles serve as overwintering hosts and tabanid adults become infected and transmit a homogeneous population of spiroplasma strains during spring, summer and autumn. In the present study, variations in restriction fragment length patterns suggest the presence of multiple genovars. Genotypic analysis may therefore be a companion to serology in elucidating spiroplasma diversity, and may provide clues to strain host range

The cpcE and cpcF genes of Synechococcus sp. PCC 7002. Construction and phenotypic characterization of interposon mutants

The 3\u27 region of the cpc operon of Synechococcus sp. PCC 7002 has been sequenced, transcriptionally characterized, and analyzed by interposon mutagenesis. The cpc operon contains six genes, 5\u27 cpcB-cpcA-cpcC-cpcD-cpcE-cpcF 3\u27, and gives rise to at least eight (more likely ten) discrete mRNA transcripts. The steady-state levels of transcripts for the cpcE and cpcF genes are very low and are estimated to represent only about 1-2% of the total transcripts arising from the cpc locus. The cpcE gene predicts a protein of 268 amino acid residues, whereas the cpcF gene predicts a protein of 205 amino acid residues. The deduced amino acid sequences of these proteins are about 50% identical and 70% similar to the predicted products of homologous genes which have been identified in other cyanobacterial cpc operons. Interposon insertion mutations were constructed in the cpcE and cpcF genes, and an interposon deletion mutation affecting both genes was constructed. The phenotypes of all mutant strains were similar. These strains were yellow-green in color, had doubling times approximately twice that of the wild-type strain, and failed to accumulate normal levels of phycocyanin. Further analyses indicated that these strains contained substantial amounts of apparently normal phycocyanin beta subunits; however the majority of the phycocyanin alpha subunit (about 90%) did not carry a phycocyanobilin chromophore. During serial subculturing of the mutant strains, suppressor mutations, which allowed cells to regain the ability to synthesize phycocyanin, arose at significant frequency. Based upon the results reported here, as well as those presented in the accompanying paper (Swanson, R. V., Zhou, J., Leary, J. A., Williams, T., de Lorimier, R., Bryant, D. A., and Glazer, A. N. (1992) J. Biol. Chem. 267, 16146-16154), we propose that the CpcE and CpcF polypeptides are the two subunits of a phycocyanobilin lyase specifically required for chromophorylation of the phycocyanin alpha subunit

Nucleotide sequence and further characterization of the Synechococcus sp. strain PCC 7002 recA gene: complementation of a cyanobacterial recA mutation by the Escherichia coli recA gene.

The nucleotide sequence and transcript initiation site of the Synechococcus sp. strain PCC 7002 recA gene have been determined. The deduced amino acid sequence of the RecA protein of this cyanobacterium is 56% identical and 73% similar to the Escherichia coli RecA protein. Northern (RNA) blot analysis indicates that the Synechococcus strain PCC 7002 recA gene is transcribed as a monocistronic transcript 1,200 bases in length. The 5' endpoint of the recA mRNA was mapped by primer extension by using synthetic oligonucleotides of 17 and 27 nucleotides as primers. The nucleotide sequence 5' to the mapped endpoint contained sequence motifs bearing a striking resemblance to the heat shock (sigma 32-specific) promoters of E. coli but did not contain sequences similar to the E. coli SOS operator recognized by the LexA repressor. An insertion mutation introduced into the recA locus of Synechococcus strain PCC 7002 via homologous recombination resulted in the formation of diploids carrying both mutant and wild-type recA alleles. A variety of growth regimens and transformation procedures failed to produce a recA Synechococcus strain PCC 7002 mutant. However, introduction into these diploid cells of the E. coli recA gene in trans on a biphasic shuttle vector resulted in segregation of the cyanobacterial recA alleles, indicating that the E. coli recA gene was able to provide a function required for growth of recA Synechococcus strain PCC 7002 cells. This interpretation is supported by the observation that the E. coli recA gene is maintained in these cells when antibiotic selection for the shuttle vector is removed

Absence of Spiroplasma or Other Bacterial 16S rRNA Genes in Brain Tissue of Hamsters with Scrapie

Spiroplasma spp. have been proposed to be the etiological agents of the transmissible spongiform encephalopathies (TSEs). In a blind study, a panel of 20 DNA samples was prepared from the brains of uninfected hamsters or hamsters infected with the 263K strain of scrapie. The brains of the infected hamsters contained ≥10(10) infectious doses/g. The coded panel was searched for bacterial 16S rRNA gene sequences, using primers selective for spiroplasma sequences, primers selective for mollicutes in general, and universal bacterial primers. After 35 PCR cycles, no samples were positive for spiroplasma or any other bacterial DNA, while control Spiroplasma mirum genomic DNA, spiked at 1% of the concentration required to account for the scrapie infectivity present, was readily detected. After 70 PCR cycles, nearly all samples yielded amplified products which were homologous to various bacterial 16S rRNA gene sequences, including those of frequent environmental contaminants. These sequences were seen in uninfected as well as infected samples. Because the concentration of scrapie infectivity was at a known high level, it is very unlikely that a bacterial infection at the same concentration could have escaped detection. We conclude that the infectious agent responsible for TSE disease cannot be a spiroplasma or any other eubacterial species

The Spiroplasma Motility Inhibition Test, a New Method for Determining Intraspecific Variation among Colorado Potato Beetle Spiroplasmas

The Colorado potato beetle, Leptinotarsa decemlineata, is a major holarctic pest of solanaceous crops. Presumably, this insect spread from Solanum species in central America to the Mexican plateau, and this was followed by multiple invasions of North America and Europe. Attempts are being made to control this beetle by using a genetically modified spiroplasma that occurs naturally in its gut. In the current study, spiroplasmas isolated from beetles collected in North America and Poland exhibited serologic (spiroplasma motility inhibition test) and genomic (restriction fragment length polymorphism) profiles that suggest that there were multiple spiroplasma introductions. Two serovars were identified; one is found in northern North America and at high elevations in Poland, and the other is found in southern North America and at low elevations in Poland. The patterns of genovar distribution coincide with the serovar patterns. The existence of such biovars—intraspecific taxal units reflected by serologic and genomic differences—should be taken into consideration when taxonomies are developed and strains are chosen for biocontrol