Candidatus Blochmannia Research Articles

Unique antimicrobial activity in honey from the Australian honeypot ant (Camponotus inflatus).

Honey produced by the Australian honeypot ant (Camponotus inflatus) is valued nutritionally and medicinally by Indigenous peoples, but its antimicrobial activity has never been formally studied. Here, we determine the activity of honeypot ant honey (HPAH) against a panel of bacterial and fungal pathogens, investigate its chemical properties, and profile the bacterial and fungal microbiome of the honeypot ant for the first time. We found HPAH to have strong total activity against Staphylococcus aureus but not against other bacteria, and strong non-peroxide activity against Cryptococcus and Aspergillus sp. When compared with therapeutic-grade jarrah and manuka honey produced by honey bees, we found HPAH to have a markedly different antimicrobial activity and chemical properties, suggesting HPAH has a unique mode of antimicrobial action. We found the bacterial microbiome of honeypot ants to be dominated by the known endosymbiont genus Candidatus Blochmannia (99.75%), and the fungal microbiome to be dominated by the plant-associated genus Neocelosporium (92.77%). This study demonstrates that HPAH has unique antimicrobial characteristics that validate its therapeutic use by Indigenous peoples and may provide a lead for the discovery of novel antimicrobial compounds.

Diversity of Bacteria in Ants (Hymenoptera: Formicidae) from Canopy and Understory of Selected Trees at Mount Makiling Forest Reserve, Laguna, Philippines

The Mount Makiling Forest Reserve (MMFR) is a biodiversity hotspot and listed as one of the 170 conservation priority areas established by the Philippine government. Its flora and fauna diversity has been reported, but knowledge gap has been identified concerning the bacterial communities associated with the flora and fauna. This study focused on ants (Hymenoptera: Formicidae), which are dominant in forest canopy and play essential roles in the ecosystem functionality. A metagenomic sequencing approach based on amplified V3–V4 regions of the 16S rRNA was employed to investigate the bacterial communities associated with five arboreal ant species collected from MMFR. The collected ants were identified as Dolichoderus thoracicus, Myrmicaria sp., Colobopsis leonardi, Polyrhachis mindanaensis, and Polyrhachis semiinermis. The sequence analyses revealed that Proteobacteria, Spirochaetes, Firmicutes, Bacteroides, and Actinobacteria were the most abundant phyla. Individual analysis of the bacterial genera associated with the five ant species showed that unclassified members of Rhizobiaceae, Orbaceae, and Burkholderiaceae were dominant in D. thoracicus. Unclassified members of Rhizobiaceae, Spirochaetaceae, and Ruminococcaceae were dominant in Myrmicaria sp. On the other hand, Candidatus Blochmannia, and Wolbachia were abundant in Camponotini ants C. leonardi, P. mindanaensis, and P. semiinermis. Bray-Curtis distance and UPGMA cluster analyses showed that the microbiomes of the Camponotini group clustered together, while D. thoracicus and Myrmicaria sp. exhibited unique bacterial profiles. Predictive gene profile analysis showed that the most functional categories were those associated with metabolism and biosynthesis of amino acids, pathways for metabolism of nucleotide, amino sugars and nitrogen, and utilization of different carbon sources.

Impact of Vitamin D Deficit on the Rat Gut Microbiome.

Inadequate immunologic, metabolic and cardiovascular homeostasis has been related to either an alteration of the gut microbiota or to vitamin D deficiency. We analyzed whether vitamin D deficiency alters rat gut microbiota. Male Wistar rats were fed a standard or a vitamin D-free diet for seven weeks. The microbiome composition was determined in fecal samples by 16S rRNA gene sequencing. The vitamin D-free diet produced mild changes on α- diversity but no effect on β-diversity in the global microbiome. Markers of gut dysbiosis like Firmicutes-to-Bacteroidetes ratio or the short chain fatty acid producing bacterial genera were not significantly affected by vitamin D deficiency. Notably, there was an increase in the relative abundance of the Enterobacteriaceae, with significant rises in its associated genera Escherichia, Candidatus blochmannia and Enterobacter in vitamin D deficient rats. Prevotella and Actinomyces were also increased and Odoribacteraceae and its genus Butyricimonas were decreased in rats with vitamin D-free diet. In conclusion, vitamin D deficit does not induce gut dysbiosis but produces some specific changes in bacterial taxa, which may play a pathophysiological role in the immunologic dysregulation associated with this hypovitaminosis.

The Gut Bacteria Associated with Camponotus japonicus Mayr with Culture-Dependent and DGGE Methods

The bacterial composition and distribution in the different gut regions of Camponotus japonicus were investigated using both culture-dependent method and culture-independent method of polymerase chain reaction and denaturing gradient gel electrophoresis (PCR-DGGE). Five different bacterial strains were isolated using culture-dependent method, and they all belong to the phylum Firmicutes, including three genera of bacteria Bacillus, Paenibacillus, and Enterococcus. Bacillus cereus and Enterococcus mundtii were found in the midgut; Paenibacillus sp. was isolated from the hindgut; and the other two Bacillus spp. were isolated from the crop. Twelve distinct DGGE bands were found using PCR-DGGE method, and their sequences blasting analysis shows that they are members of the Proteobacteria and the Firmicutes, respectively, including three genera (Pseudomonas, Candidatus Blochmannia, Fructobacillus) and one uncultured bacterium, in which Pseudomonas was the most dominant bacteria group in all the three gut regions. According to the DGGE profile, the three gut regions had very similar gut communities, and all the DGGE bands were presented in the midgut and hindgut, while just two bands representing Blochmannia were not present in the crop. The results of our study indicate that the gut of C. japonicus harbors several other bacteria besides the obligate endosymbionts Blochmannia, and more work should be carried on to verify if they are common in the guts of other Camponotus ants.

Assessment of bacterial endosymbiont diversity in Otiorhynchus spp. (Coleoptera: Curculionidae) larvae using a multitag 454 pyrosequencing approach

BackgroundWeevils of the genus Otiorhynchus are regarded as devastating pests in a wide variety of horticultural crops worldwide. So far, little is known on the presence of endosymbionts in Otiorhynchus spp.. Investigation of endosymbiosis in this genus may help to understand the evolution of different reproductive strategies in these weevils (parthenogenesis or sexual reproduction), host-symbiont interactions, and may provide a future basis for novel pest management strategy development. Here, we used a multitag 454 pyrosequencing approach to assess the bacterial endosymbiont diversity in larvae of four economically important Otiorhynchus species.ResultsHigh-throughput tag-encoded FLX amplicon pyrosequencing of a bacterial 16S rDNA fragment was used to characterise bacterial communities associated with different Otiorhynchus spp. larvae. By sequencing a total of ~48,000 PCR amplicons, we identified 49 different operational taxonomic units (OTUs) as bacterial endosymbionts in the four studied Otiorhynchus species. More than 90% of all sequence reads belonged either to the genus Rickettsia or showed homology to the phylogenetic group of “Candidatus Blochmannia” and to endosymbionts of the lice Pedicinus obtusus and P. badii. By using specific primers for the genera Rickettsia and “Candidatus Blochmannia”, we identified a new phylogenetic clade of Rickettsia as well as “Candidatus Nardonella” endosymbionts in Otiorhynchus spp. which are closely related to “Candidatus Blochmannia” bacteria.ConclusionsHere, we used multitag 454 pyrosequencing for assessment of insect endosymbiotic communities in weevils. As 454 pyrosequencing generates only quite short sequences, results of such studies can be regarded as a first step towards identifying respective endosymbiotic species in insects. In the second step of our study, we analysed sequences of specific gene regions for a more detailed phylogeny of selected endosymbiont genera. As a result we identified the presence of Rickettsia and “Candidatus Nardonella” endosymbionts in Otiorhynchus spp.. This knowledge is an important step in exploring bacteria-insect associations for potential use in insect pest control.

Bacteria Associated with Gut Lumen of Camponotus japonicus Mayr

Camponotus ants harbor the obligate intracellular endosymbiont Blochmannia in their midgut bacteriocytes, but little is known about intestinal bacteria living in the gut lumen. In this paper we reported the results of a survey of the intestinal microflora of Camponotus japonicus Mayr based on small-subunit rRNA genes (16S rRNAs) polymerase chain reaction (PCR)-restriction fragment-length polymorphism analysis of worker guts. From 107 clones, 11 different restriction fragment-length polymorphism profiles were identified, and sequences blasting analysis found these represent four types of bacteria. Most (91.6%) of the clones were "Candidatus Blochmannia", the obligate endosymbionts of Camponotus ants, and 6.5% of the clones were "Candidatus Serratia symbiotica", a secondary endosymbiont of aphids; the remaining 2% clones were Fructobacillus fructosus and uncultured Burkholderiales bacterium, respectively. These results show that the diversity of gut bacteria in C. japonicus was low. "Candidatus Serratia symbiotica" was identified from Camponotus ants for the first time, an interesting result because Blochmannia's closest bacterial relative is also in the genus Serratia. This discovery supports the scenario that consumption of aphid honeydew or tissue provides an initial step in the evolution of an advanced symbiosis, and suggests that Camponotus ant could acquire other secondary endosymbionts from Hemiptera host through their diet. In addition, Burkholderiales bacterium also was identified from the gut of C. japonicus for the first time, and whether it is a nitrogen-recycling endosymbiont in Camponotus ants needs to be investigated further.

Complete rpoB gene sequencing as a suitable supplement to DNA-DNA hybridization for bacterial species and genus delineation

DNA-DNA hybridization (DDH), the gold standard for bacterial species delineation, is a laborious method and the alternative, average nucleotide identity (ANI), a genomic sequence-derived parameter, is not applicable to non-sequenced species. A universal cut-off value to delineate bacterial species does not exist, yet a DDH value <70 % and ANI <95+/-0.5 % have proved useful in selected examples. We herein compare published values for DDH and ANI with sequence similarity of rpoB gene sequences retrieved from GenBank for strains of 230 bacterial species representative of 45 genera. Intraspecific rpoB sequence similarity was 98.2-100 %. We observed that an rpoB gene sequence similarity <or=97.7 % significantly correlated with a DDH value <70 % and an ANI value <94.3 %. An rpoB gene sequence similarity <85.5 % correlated with membership of different genera. When applied to fastidious and as-yet-uncultivated organisms lacking experimental DDH values, these cut-off values suggested that 'Candidatus Blochmannia pennsylvanicus' and 'Candidatus Blochmannia floridarius' may belong to different genera, that the different endosymbiotic Buchnera aphidicola organisms may belong to different genera and that, while the tsetse fly enteric symbiont Sodalis glossinidius may belong to the Enterobacteriaceae, the endocellular obligate symbiont Wigglesworthia glossinidia from the same host may belong to the group of as-yet-uncultivated gammaproteobacteria. rpoB gene sequence similarity provides an efficient supplement to DDH and ANI measurements to delineate bacterial species and genera, including delineation of as-yet-uncultivated, non-sequenced organisms.

Small genome of Candidatus Blochmannia, the bacterial endosymbiont of Camponotus, implies irreversible specialization to an intracellular lifestyle.

Blochmannia (Candidatus Blochmannia gen. nov.) is the primary bacterial endosymbiont of the ant genus CAMPONOTUS: Like other obligate endosymbionts of insects, Blochmannia occurs exclusively within eukaryotic cells and has experienced long-term vertical transmission through host lineages. In this study, PFGE was used to estimate the genome size of Blochmannia as approximately 800 kb, which is significantly smaller than its free-living relatives in the enterobacteria. This small genome implies that Blochmannia has deleted most of the genetic machinery of related free-living bacteria. Due to restricted gene exchange in obligate endosymbionts, the substantial gene loss in Blochmannia and other insect mutualists may reflect irreversible specialization to a host cellular environment.

Systematic relationships and cospeciation of bacterial endosymbionts and their carpenter ant host species: proposal of the new taxon Candidatus Blochmannia gen. nov.

The systematic relationships of intracellular bacteria of 13 Camponotus species (carpenter ants) from America and Europe were compared to those of their hosts. Phylogenetic trees of the bacteria and the ants were based on 16S rDNA (rrs) gene sequences and mitochondrial cytochrome oxidase subunit I (COI) gene sequences, respectively. The bacterial endosymbionts of Camponotus spp. form a distinct lineage in the y-subclass of the Proteobacteria. The taxa most closely related to these bacteria are endosymbionts of aphids and the tsetse fly. The bacterial and host phylogenies deduced from the sequence data show a high degree of congruence, providing significant evidence for cospeciation of the bacteria and the ants and a maternal transmission route of the symbionts. The cloned rrs genes of the endosymbionts contain putative intervening sequences (IVSs) with a much lower G+C content than the mean of the respective rrs genes. By in situ hybridization specific 16S rDNA oligonucleotide probes verified the presence of the bacteria within tissues of three of the eukaryotic hosts. It is proposed that the endosymbionts of these three carpenter ants be assigned to a new taxon 'Candidatus Blochmannia gen. nov.' with the symbionts of the individual ants being species named according to their host, 'Candidatus Blochmannia floridanus sp. nov.', 'Candidatus Blochmannia herculeanus sp. nov.' and 'Candidatus Blochmannia rufipes sp. nov.'.