The candidate phylum 'Termite Group 1' of bacteria: phylogenetic diversity, distribution, and endosymbiont members of various gut flagellated protists

The candidate phylum 'Termite Group 1' (TG1) of bacteria, which is abundant in termite guts but has no culturable representative, was investigated with respect to the in situ localization, distribution, and diversity. Based on the 16S rRNA gene sequence analyses and FISH in termite guts, a number of lineages of TG1 members were identified as endosymbionts of a variety of gut flagellated protists from the orders Trichonymphida, Cristamonadida, and Oxymonadida that are mostly unique to termites. However, the survey in various environments using specific PCR primers revealed that TG1 members were also present in termites, a cockroach, and the bovine rumen that typically lack these protist orders. Most of the TG1 members from gut flagellates, termites, cockroaches, and the rumen formed a monophyletic subcluster that showed a shallow branching pattern in the phylogenetic tree, suggesting their recent diversification. Although endosymbionts of the same protist genera tended to be closely related, the endosymbiont lineages were often independent of the higher level classifications of their host protist and were dispersed in the phylogenetic tree. It appears that their cospeciation is not the sole rule for the diversification of TG1 members of endosymbionts.     

The Motility Symbiont of the Termite Gut Flagellate Caduceia versatilis Is a Member of the “Synergistes” Group

The flagellate Caduceia versatilis in the gut of the termite Cryptotermes cavifrons reportedly propels itself not by its own flagella but solely by the flagella of ectosymbiotic bacteria. Previous microscopic observations have revealed that the motility symbionts are flagellated rods partially embedded in the host cell surface and that, together with a fusiform type of ectosymbiotic bacteria without flagella, they cover almost the entire surface. To identify these ectosymbionts, we conducted 16S rRNA clone analyses of bacteria physically associated with the Caduceia cells. Two phylotypes were found to predominate in the clone library and were phylogenetically affiliated with the “Synergistes” phylum and the order Bacteroidales in the Bacteroidetes phylum. Probes specifically targeting 16S rRNAs of the respective phylotypes were designed, and fluorescence in situ hybridization (FISH) was performed. As a result, the “Synergistes” phylotype was identified as the motility symbiont; the Bacteroidales phylotype was the fusiform ectobiont. The “Synergistes” phylotype was a member of a cluster comprising exclusively uncultured clones from the guts of various termite species. Interestingly, four other phylotypes in this cluster, including the one sharing 95% sequence identity with the motility symbiont, were identified as nonectosymbiotic, or free-living, gut bacteria by FISH. We thus suggest that the motility ectosymbiont has evolved from a free-living gut bacterium within this termite-specific cluster. Based on these molecular and previous morphological data, we here propose a novel genus and species, “Candidatus Tammella caduceiae,” for this unique motility ectosymbiont of Caducaia versatilis.     

Phylogenetic diversity, localization, and cell morphologies of members of the candidate phylum TG3 and a subphylum in the phylum Fibrobacteres, recently discovered bacterial groups dominant in termite guts

Recently we discovered two novel, deeply branching lineages in the domain Bacteria from termite guts by PCR-based analyses of 16S rRNA (Y. Hongoh, P. Deevong, T. Inoue, S. Moriya, S. Trakulnaleamsai, M. Ohkuma, C. Vongkaluang, N. Noparatnaraporn, and T. Kudo, Appl. Environ. Microbiol. 71:6590-6599, 2005). Here, we report on the specific detection of these bacteria, the candidate phylum TG3 (Termite Group 3) and a subphylum in the phylum Fibrobacteres, by fluorescence in situ hybridization in the guts of the wood-feeding termites Microcerotermes sp. and Nasutitermes takasagoensis. Both bacterial groups were detected almost exclusively from the luminal fluid of the dilated portion in the hindgut. Each accounted for approximately 10% of the total prokaryotic cells, constituting the second-most dominant groups in the whole-gut microbiota. The detected cells of both groups were in undulate or vibroid forms and apparently resembled small spirochetes. The cell sizes were 0.2 to 0.4 by 1.3 to 6.0 microm and 0.2 to 0.3 by 1.3 to 4.9 microm in the TG3 and Fibrobacteres, respectively. Using PCR screenings with specific primers, we found that both groups are distributed among various termites. The obtained clones formed monophyletic clusters that were delineated by the host genus rather than by the geographic distance, implying a robust association between these bacteria and host termites. TG3 clones were also obtained from a cockroach gut, lake sediment, rice paddy soil, and deep-sea sediments. Our results suggest that the TG3 and Fibrobacteres bacteria are autochthonous gut symbionts of various termites and that the TG3 members are also widely distributed among various other environments.     

Phylogenetic Position and In Situ Identification of Ectosymbiotic Spirochetes on Protists in the Termite Gut

Phylogenetic relationships, diversity, and in situ identification of spirochetes in the gut of the termite Neotermes koshunensis were examined without cultivation, with an emphasis on ectosymbionts attached to flagellated protists. Spirochetes in the gut microbial community investigated so far are related to the genus Treponema and divided into two phylogenetic clusters. In situ hybridizations with a 16S rRNA-targeting consensus oligonucleotide probe for one cluster (known as termite Treponema cluster I) detected both the ectosymbiotic spirochetes on gut protists and the free-swimming spirochetes in the gut fluid of N. koshunensis. The probe for the other cluster (cluster II), which has been identified as ectosymbionts on gut protists of two other termite species, Reticulitermes speratus and Hodotermopsis sjoestedti, failed to detect any spirochete population. The absence of cluster II spirochetes in N. koshunensis was confirmed by intensive 16S ribosomal DNA (rDNA) clone analysis, in which remarkably diverse spirochetes of 45 phylotypes were identified, almost all belonging to cluster I. Ectosymbiotic spirochetes of the three gut protist species Devescovina sp., Stephanonympha sp., and Oxymonas sp. in N. koshunensis were identified by their 16S rDNA and by in situ hybridizations using specific probes. The probes specific for these ectosymbionts did not receive a signal from the free-swimming spirochetes. The ectosymbionts were dispersed in cluster I of the phylogeny, and they formed distinct phylogenetic lineages, suggesting multiple origins of the spirochete attachment. Each single protist cell harbored multiple spirochete species, and some of the spirochetes were common among protist species. The results indicate complex relationships of the ectosymbiotic spirochetes with the gut protists.     

Complex coevolutionary history of symbiotic Bacteroidales bacteria of various protists in the gut of termites

Background  

The microbial community in the gut of termites is responsible for the efficient decomposition of recalcitrant lignocellulose. Prominent features of this community are its complexity and the associations of prokaryotes with the cells of cellulolytic flagellated protists. Bacteria in the order Bacteroidales are involved in associations with a wide variety of gut protist species as either intracellular endosymbionts or surface-attached ectosymbionts. In particular, ectosymbionts exhibit distinct morphological patterns of the associations. Therefore, these Bacteroidales symbionts provide an opportunity to investigate not only the coevolutionary relationships with the host protists and their morphological evolution but also how symbiotic associations between prokaryotes and eukaryotes occur and evolve within a complex symbiotic community.     

Tunicamycin-Induced Cell Death in the Trigeminal Ganglion is Suppressed by Nerve Growth Factor in the Mouse Embryo

The effect of nerve growth factor (NGF) on tunicamycin (Tm)-treated neurons in the trigeminal ganglion was investigated by use of caspase-3 immunohistochemistry. In intact embryos at embryonic day 16.5, only a few caspase-3-immunoreactivity were detected in the ganglion neurons. Mean ± SE of the density of the immunoreactivity was 0.22 ± 0.03%. In contrast, the number of the immunoreactive neurons was increased at 24 h after injection of 0.5 μg Tm in 1 μl of 0.05 N NaOH solution into mouse embryos at embryonic day 15.5. The density of immunoreactivity was also increased (mean ± SE = 1.44 ± 0.11%) compared to intact and 0.05 N NaOH-treated embryos (mean ± SE = 0.35 ± 0.03%). The Tm treatment caused increase of the number of trigeminal neurons representing apoptotic profiles (intact, mean ± SE = 79.3 ± 8.5; 0.05 N NaOH, mean ± SE = 132 ± 11.5; 0.5 μg Tm, mean ± SE = 370.2 ± 64.8). In addition, NGF significantly prevented the increase of density of the immunoreactivity (mean ± SE = 0.54 ± 0.16%) and the number of apoptotic cells (mean ± SE = 146.2 ± 11.3). Saline application (without NGF) had no effect on Tm-induced increase of the immunoreactivity (mean ± SE = 1.78 ± 0.23%) or the apoptotic profiles (mean ± SE = 431.9 ± 80.5). These results indicate that Tm-induced cell death in the trigeminal ganglion is suppressed by NGF in the mouse embryo.     

Intranuclear verrucomicrobial symbionts and evidence of lateral gene transfer to the host protist in the termite gut

In 1944, Harold Kirby described microorganisms living within nuclei of the protists Trichonympha in guts of termites; however, their taxonomic assignment remains to be accomplished. Here, we identified intranuclear symbionts of Trichonympha agilis in the gut of the termite Reticulitermes speratus. We isolated single nuclei of T. agilis, performed whole-genome amplification, and obtained bacterial 16S rRNA genes by PCR. Unexpectedly, however, all of the analyzed clones were from pseudogenes of 16S rRNA with large deletions and numerous sequence variations even within a single-nucleus sample. Authentic 16S rRNA gene sequences were finally recovered by digesting the nuclear DNA; these pseudogenes were present on the host Trichonympha genome. The authentic sequences represented two distinct bacterial species belonging to the phylum Verrucomicrobia, and the pseudogenes have originated from each of the two species. Fluorescence in situ hybridization confirmed that both species are specifically localized, and occasionally co-localized, within nuclei of T. agilis. Transmission electron microscopy revealed that they are distorted cocci with characteristic electron-dense and lucent regions, which resemble the intranuclear symbionts illustrated by Kirby. For these symbionts, we propose a novel genus and species, ‘Candidatus Nucleococcus trichonymphae'' and ‘Candidatus Nucleococcus kirbyi''. These formed a termite-specific cluster with database sequences, other members of which were also detected within nuclei of various gut protists, including both parabasalids and oxymonads. We suggest that this group is widely distributed as intranuclear symbionts of diverse protists in termite guts and that they might have affected the evolution of the host genome through lateral gene transfer.     

Adaptation and Evaluation of a Multi-Criteria Decision Analysis Model for Lyme Disease Prevention

Designing preventive programs relevant to vector-borne diseases such as Lyme disease (LD) can be complex given the need to include multiple issues and perspectives into prioritizing public health actions. A multi-criteria decision aid (MCDA) model was previously used to rank interventions for LD prevention in Quebec, Canada, where the disease is emerging. The aim of the current study was to adapt and evaluate the decision model constructed in Quebec under a different epidemiological context, in Switzerland, where LD has been endemic for the last thirty years. The model adaptation was undertaken with a group of Swiss stakeholders using a participatory approach. The PROMETHEE method was used for multi-criteria analysis. Key elements and results of the MCDA model are described and contrasted with the Quebec model. All criteria and most interventions of the MCDA model developed for LD prevention in Quebec were directly transferable to the Swiss context. Four new decision criteria were added, and the list of proposed interventions was modified. Based on the overall group ranking, interventions targeting human populations were prioritized in the Swiss model, with the top ranked action being the implementation of a large communication campaign. The addition of criteria did not significantly alter the intervention rankings, but increased the capacity of the model to discriminate between highest and lowest ranked interventions. The current study suggests that beyond the specificity of the MCDA models developed for Quebec and Switzerland, their general structure captures the fundamental and common issues that characterize the complexity of vector-borne disease prevention. These results should encourage public health organizations to adapt, use and share MCDA models as an effective and functional approach to enable the integration of multiple perspectives and considerations in the prevention and control of complex public health issues such as Lyme disease or other vector-borne and zoonotic diseases.