Publications

4922

ABSTRACTHere we investigated the bacterial endosymbionts of weevils of the genusCurculio. From all four species ofCurculioweevils examined, a novel group of bacterial gene sequences were consistently identified. Molecular phylogenetic analyses demonstrated that the sequences formed a distinct clade in theGammaproteobacteria, which was not related to previously known groups of weevil endosymbionts such asNardonellaspp. andSodalis-allied symbionts. In situ hybridization revealed that the bacterium was intracellularly harbored in a bacteriome associated with larval midgut. In adult females, the bacterium was localized in the germalia at the tip of each overiole, suggesting vertical transmission via ovarial passage. Diagnostic PCR surveys detected high prevalence of the bacterial infection in natural host populations. Electron microscopy identified the reduced cell wall of the bacterial cells, and the bacterial genes exhibited AT-biased nucleotide composition and accelerated molecular evolution, which are suggestive of a long-lasting endosymbiotic association. On the basis of these results, we conclude that the novel endocellular bacteria represent the primary symbiont ofCurculioweevils and proposed the designation “CandidatusCurculioniphilus buchneri.” In addition to “Ca.Curculioniphilus,” we identifiedSodalis-allied gammaproteobacterial endosymbionts from the chestnut weevil,Curculio sikkimensis, which exhibited partial infection frequencies in host insect populations and neither AT-biased nucleotide composition nor accelerated molecular evolution. We suggest that suchSodalis-allied secondary symbionts in weevils might provide a potential source for symbiont replacements, as has occurred in an ancestor ofSitophilusgrain weevils.

Summary Like other plant sap‐sucking insects, planthoppers within the family Cixiidae (Insecta: Hemiptera: Fulgoromorpha) host a diversified microbiota. We report the identification and first molecular characterization of symbiotic bacteria in cixiid planthoppers (tribe: Pentastirini). Using universal eubacterial primers we first screened the eubacterial 16S rRNA sequences in Pentastiridius leporinus (Linnaeus) with PCR amplification, cloning, and restriction fragment analysis. We identified three main 16S rRNA sequences that corresponded to a Wolbachia bacterium, a plant pathogenic bacterium, and a novel gammaproteobacterial symbiont. A fourth bacterial species affiliated with ‘ Candidatus Sulcia muelleri’ was detected in PCR assays using primers specific for the Bacteroidetes . Within females of two selected cixiid planthoppers, P. leporinus and Oliarus filicicola , fluorescence In situ hybridization analysis and transmission electron microscopy observations showed that ‘ Ca. Sulcia muelleri’ and the novel gammaproteobacterial symbiont were housed in separate bacteriomes. Phylogenetic analysis revealed that both of these symbionts occurred in at least four insect genera within the tribe Pentastirini. ‘ Candidatus Purcelliella pentastirinorum’ was proposed as the novel gammaproteobacterial symbiont.

Bat ticks, Carios kelleyi , from Iowa were examined for the presence of relapsing fever group borreliae. A novel spirochete was characterized by DNA sequence analysis of polymerase chain reaction amplicons for the 16S rRNA , flaB , and glpQ genes in either triturated tick pools or single ticks. All loci and the concatenated DNA sequence of 3,289 bases identified the Carios bacterium as a relapsing fever spirochete most closely related to, but distinct from, Borrelia turicatae . Spirochetes reactive with a Borrelia -specific monoclonal antibody were observed microscopically in the coxal fluid and salivary glands from one tick. These data confirm the presence of a novel species of relapsing fever spirochete in bat ticks and the potential for new enzootic foci for endemic relapsing fever that warrants further investigation. The name Borrelia johnsonii is proposed for this novel spirochete in honor of Dr. Russell C. Johnson.

Abstract Background Metagenomic sequence data are proving to be a vast resource for the discovery of biological components. Yet analysis of this data to identify functional RNAs lags behind efforts to characterize protein diversity. The genome of 'Candidatus Pelagibacter ubique' HTCC 1062 is the closest match for approximately 20% of marine metagenomic sequence reads. It is also small, contains little non-coding DNA, and has strikingly low GC content. Results To aid the discovery of RNA motifs within the marine metagenome we exploited the genomic properties of 'Cand. P. ubique' by targeting our search to long intergenic regions (IGRs) with relatively high GC content. Analysis of known RNAs (rRNA, tRNA, riboswitches etc.) shows that structured RNAs are significantly enriched in such IGRs. To identify additional candidate structured RNAs, we examined other IGRs with similar characteristics from 'Cand. P. ubique' using comparative genomics approaches in conjunction with marine metagenomic data. Employing this strategy, we discovered four candidate structured RNAs including a new riboswitch class as well as three additional likely cis-regulatory elements that precede genes encoding ribosomal proteins S2 and S12, and the cytoplasmic protein component of the signal recognition particle. We also describe four additional potential RNA motifs with few or no examples occurring outside the metagenomic data. Conclusion This work begins the process of identifying functional RNA motifs present in the metagenomic data and illustrates how existing completed genomes may be used to aid in this task.