Molecular phylogeny of Spirotrichonymphea (Parabasalia) with emphasis on Spironympha, Spirotrichonympha, and three new genera Pseudospironympha, Nanospironympha, and Brugerollina.

Spirotrichonymphea, one of the six classes of phylum Parabasalia, are characterized by bearing many flagella in spiral rows, and they occur exclusively in the guts of termites. Phylogenetic relationships among the 13 described genera are not well understood due to complex morphological evolution and a paucity of molecular data. One such understudied genus is Spironympha. It has been variously considered a valid genus, a subgenus of Spirotrichonympha, or an "immature" life cycle stage of Spirotrichonympha. To clarify this, we sequenced the small subunit rRNA gene sequences of Spironympha and Spirotrichonympha cells isolated from the hindguts of Reticulitermes species and Hodotermopsis sjostedti and confirmed the molecular identity of H. sjostedti symbionts using fluorescence in situ hybridization. Spironympha as currently circumscribed is polyphyletic, with both H. sjostedti symbiont species branching separately from the "true" Spironympha from Reticulitermes. Similarly, the Spirotrichonympha symbiont of H. sjostedti branches separately from the "true" Spirotrichonympha found in Reticulitermes. Our data support Spironympha from Reticulitermes as a valid genus most closely related to Spirotrichonympha, though its monophyly and interspecific relationships are not resolved in our molecular phylogenetic analysis. We propose three new genera to accommodate the H. sjostedti symbionts and two new species of Spirotrichonympha from Reticulitermes.

Lactococcus insecticola sp. nov. and Lactococcus hodotermopsidis sp. nov., isolated from the gut of the wood-feeding lower termite Hodotermopsis sjostedti.

Two strains of lactic acid bacteria, designated Hs20B0-1T and Hs30E4-3T, were isolated from the gut of the damp-wood termite Hodotermopsis sjostedti. These strains were characterized genetically and phenotypically. Strain Hs20B0-1T was related to Lactococcus piscium DSM 6634T showing 96.3 and 84.2 % sequence similarity in 16S rRNA gene and rpoB gene sequences, respectively. Strain Hs30E4-3T was related to Lactococcus plantarum DSM 20686T showing 94.8 and 82.2 % sequence similarity in 16S rRNA gene and rpoB gene sequences, respectively. The 16S rRNA gene sequence similarity between strains Hs20B0-1T and Hs30E4-3T was 95.7 %. Furthermore, genomic comparisons using pairwise average nucleotide identity (ANI) and digital DNA-DNA hybridization (DDH) analyses between strain Hs20B0-1T and L. piscium DSM 6634T resulted in values of 73.5 and 20.1 %, respectively. Strain Hs30E4-3T had 72.8 % ANI similarity and 21.3 % DDH similarity to L. plantarum DSM 20686T. Strains Hs20B0-1T and Hs30E4-3T had 75.4 % ANI similarity and 21.1 % DDH similarity to each other. The cell-wall peptidoglycan types of strains Hs20B0-1T and Hs30E4-3T were A4α, Lys-Asp and A3α, Lys-Thr-Ala, respectively. The two strains, Hs20B0-1T and Hs30E4-3T, are distinguishable from each other and other established Lactococcus species phylogenetically and phenotypically. In conclusion, two novel species of the genus Lactococcus are proposed, namely Lactococcus insecticola Hs20B0-1T (=JCM 33485T=DSM 110147T) and Lactococcus hodotermopsidis Hs30E4-3T (=JCM 33486T=DSM 110148T), respectively.

Lactococcus termiticola sp. nov., isolated from the gut of the wood-feeding higher termite Nasutitermes takasagoensis.

A strain of lactic acid bacteria, designated NtB2T, isolated from the gut of the wood-feeding higher termite Nasutitermes takasagoensis, was characterized genetically and phenotypically. Strain NtB2T was related to Lactococcus lacti subsp. tructae JCM 31125T isolated from brown trout, showing 93.2 and 81.0 % similarity in 16S rRNA gene and rpoB gene sequences, respectively. Furthermore, genomic comparisons using pairwise average nucleotide identity analysis and the Genome-to-Genome Distance Calculator between strain NtB2T and L. lacti subsp. tructae JCM 31125T gave values of 81.0 and 23.2 %, respectively. Major cellular fatty acids produced by strain NtB2T were C18 : 1ω9c and C16 : 0. The cell-wall peptidoglycan type of strain NtB2T was A3α, Lys-Gly-Ser-Ala2. Based on the data presented, the isolate represents a novel species of the genus Lactococcus, for which the name Lactococcus termiticola sp. nov. is proposed. The type strain is NtB2T (=JCM 32569T=DSM 107259T).

Acetogenesis from H2 plus CO2 and nitrogen fixation by an endosymbiotic spirochete of a termite-gut cellulolytic protist.

Symbiotic associations of cellulolytic eukaryotic protists and diverse bacteria are common in the gut microbial communities of termites. Besides cellulose degradation by the gut protists, reductive acetogenesis from H2 plus CO2 and nitrogen fixation by gut bacteria play crucial roles in the host termites' nutrition by contributing to the energy demand of termites and supplying nitrogen poor in their diet, respectively. Fractionation of these activities and the identification of key genes from the gut community of the wood-feeding termite Hodotermopsis sjoestedti revealed that substantial activities in the gut--nearly 60% of reductive acetogenesis and almost exclusively for nitrogen fixation--were uniquely attributed to the endosymbiotic bacteria of the cellulolytic protist in the genus Eucomonympha. The rod-shaped endosymbionts were surprisingly identified as a spirochete species in the genus Treponema, which usually exhibits a characteristic spiral morphology. The endosymbionts likely use H2 produced by the protist for these dual functions. Although H2 is known to inhibit nitrogen fixation in some bacteria, it seemed to rather stimulate this important mutualistic process. In addition, the single-cell genome analyses revealed the endosymbiont's potentials of the utilization of sugars for its energy requirement, and of the biosynthesis of valuable nutrients such as amino acids from the fixed nitrogen. These metabolic interactions are suitable for the dual functions of the endosymbiont and reconcile its substantial contributions in the gut.

Evolutionary mechanisms underlying the diversification of nuclear factor of activated T cells across vertebrates.

The mechanisms of immunity linked to biological evolution are crucial for understanding animal morphogenesis, organogenesis, and biodiversity. The nuclear factor of activated T cells (NFAT) family consists of five members (NFATc1-c4, 5) with different functions in the immune system. However, the evolutionary dynamics of NFATs in vertebrates has not been explored. Herein, we investigated the origin and mechanisms underlying the diversification of NFATs by comparing the gene, transcript and protein sequences, and chromosome information. We defined an ancestral origin of NFATs during the bilaterian development, dated approximately 650 million years ago, where NFAT5 and NFATc1-c4 were derived independently. The conserved parallel evolution of NFATs in multiple species was probably attributed to their innate nature. Conversely, frequent gene duplications and chromosomal rearrangements in the recently evolved taxa have suggested their roles in the adaptive immune evolution. A significant correlation was observed between the chromosome rearrangements with gene duplications and the structural fixation changes in vertebrate NFATs, suggesting their role in NFAT diversification. Remarkably, a conserved gene structure around NFAT genes with vertebrate evolutionary-related breaking points indicated the inheritance of NFATs with their neighboring genes as a unit. The close relationship between NFAT diversification and vertebrate immune evolution was suggested.

The features of adolescent irritable bowel syndrome in Japan.

OBJECTIVE AND BACKGROUND: The onset of IBS is in adolescence in many cases. However, the features of adolescent IBS were generally lacking. The objective of this research was to know the features of adolescent IBS in Japan. METHODOLOGY: In 2004 and 2009, we randomly selected Junior high school students in Miyagi prefecture, Japan, according to population of each area. Eight hundred thirty-three boys and 888 girls (age: 15 years old) in 2004 and 256 boys and 335 girls (age: 14 years old) in 2009 participated in this study. They fulfilled self-reported questionnaires those include Rome-II Modular Questionnaire, Self-reported IBS Questionnaire, Generalized Self-Efficacy Scale, Short Form-36 ver.2, other questions on their lives and Toronto Alexithymia Scale-20. RESULTS: The prevalence of adolescent IBS was 14.6% in 2004 and 19% in 2009. Compare with students without abdominal symptoms, IBS showed lower health-related QOL and self-efficacy and complained more sleep disturbance, traumatic episodes and perceived stress in both researches. IBS girls were worse in both physical and psychological aspects. They also have alexithymic tendency and it influenced on severity of IBS symptoms. CONCLUSIONS: Adolescent IBS had almost the same prevalence as adult IBS, however the rate of IBS subtypes was different. They also had psychological problems in addition to physical conditions even though most of them were non-consulters. Improvement of self-efficacy and alxithymia may help to prevent and treat IBS.

Complete genome of the uncultured Termite Group 1 bacteria in a single host protist cell.

Termites harbor a symbiotic gut microbial community that is responsible for their ability to thrive on recalcitrant plant matter. The community comprises diverse microorganisms, most of which are as yet uncultivable; the detailed symbiotic mechanism remains unclear. Here, we present the first complete genome sequence of a termite gut symbiont-an uncultured bacterium named Rs-D17 belonging to the candidate phylum Termite Group 1 (TG1). TG1 is a dominant group in termite guts, found as intracellular symbionts of various cellulolytic protists, without any physiological information. To acquire the complete genome sequence, we collected Rs-D17 cells from only a single host protist cell to minimize their genomic variation and performed isothermal whole-genome amplification. This strategy enabled us to reconstruct a circular chromosome (1,125,857 bp) encoding 761 putative protein-coding genes. The genome additionally contains 121 pseudogenes assigned to categories, such as cell wall biosynthesis, regulators, transporters, and defense mechanisms. Despite its apparent reductive evolution, the ability to synthesize 15 amino acids and various cofactors is retained, some of these genes having been duplicated. Considering that diverse termite-gut protists harbor TG1 bacteria, we suggest that this bacterial group plays a key role in the gut symbiotic system by stably supplying essential nitrogenous compounds deficient in lignocelluloses to their host protists and the termites. Our results provide a breakthrough to clarify the functions of and the interactions among the individual members of this multilayered symbiotic complex.

Molecular Phylogenetic Position of Microjoenia (Parabasalia: Spirotrichonymphea) from Reticulitermes and Hodotermopsis Termite Hosts.

Microjoenia are obligate symbionts of termites. The genus was erected in 1892 for small cells with many flagella that insert near, but not directly from, the cell apex, and an axostyle that can protrude from the cell posterior. Although ultrastructural studies have been carried out on three Microjoenia species to date, no molecular data have been directly attributed to any species. Microjoenia are classified within the parabasalian class Spirotrichonymphea, which is characterized by flagellar bands that emerge near the cell apex and proceed posteriorly in a right-handed helix. In Microjoenia, however, the flagellar bands are very short and proceed longitudinally or with a weakly observable helix. In this study, we have amplified and sequenced the 18S ribosomal RNA gene from individually isolated Microjoenia cells from Reticulitermes and Hodotermopsis hosts as part of an ongoing effort to understand the phylogeny of Spirotrichonymphea and their coevolution with termites. In our 18S rRNA gene phylogeny, Microjoenia forms the sister lineage to Spirotrichonympha, though many other evolutionary relationships within Spirotrichonymphea remain unresolved.

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. RESULTS: Molecular phylogeny of 31 taxa of Bacteroidales symbionts from 17 protist genera in 10 families was examined based on 16S rRNA gene sequences. Their localization, morphology, and specificity were also examined by fluorescent in situ hybridizations. Although a monophyletic grouping of the ectosymbionts occurred in three related protist families, the symbionts of different protist genera were usually dispersed among several phylogenetic clusters unique to termite-gut bacteria. Similar morphologies of the associations occurred in multiple lineages of the symbionts. Nevertheless, the symbionts of congeneric protist species were closely related to one another, and in most cases, each host species harbored a unique Bacteroidales species. The endosymbionts were distantly related to the ectosymbionts examined so far. CONCLUSION: The coevolutionary history of gut protists and their associated Bacteroidales symbionts is complex. We suggest multiple independent acquisitions of the Bacteroidales symbionts by different protist genera from a pool of diverse bacteria in the gut community. In this sense, the gut could serve as a reservoir of diverse bacteria for associations with the protist cells. The similar morphologies are considered a result of evolutionary convergence. Despite the complicated evolutionary history, the host-symbiont relationships are mutually specific, suggesting their cospeciations at the protist genus level with only occasional replacements.

Candidatus Desulfovibrio trichonymphae, a novel intracellular symbiont of the flagellate Trichonympha agilis in termite gut.

Rs-N31, a 16S rRNA phylotype affiliated with the genus Desulfovibrio, has frequently been detected from the gut of the wood-feeding termite Reticulitermes speratus. In this study, we designed a probe specifically targeting phylotype Rs-N31 and performed fluorescence in situ hybridization to identify the corresponding cells. The signals were detected exclusively inside the cells of the flagellate Trichonympha agilis, which simultaneously harbours another intracellular bacterium belonging to the candidate phylum Termite Group 1 (TG1). The detected cells were coccoid or short rods and specifically localized in the cortical layer of mainly, the anterior part of the flagellate cell. Approximately 1800 cells were contained in a single host cell, accounting for, in total, 2% of the whole prokaryotic gut microbiota. The genes dsrAB and apsA for sulfate reduction and a gene-encoding H(2)-uptake hydrogenase, both possessing a high sequence identity with those of known desulfovibrios, were obtained by polymerase chain reaction (PCR) from the host cells isolated using a micromanipulator, and their expression was verified by reverse-transcription PCR. Thus, we suggest that this endosymbiont acts as a sink for the hydrogen generated by both the flagellates and possibly TG1 symbionts. For this uncultured bacterium, we propose a novel species, 'Candidatus Desulfovibrio trichonymphae'.

Inheritance and diversification of symbiotic trichonymphid flagellates from a common ancestor of termites and the cockroach Cryptocercus.

Cryptocercus cockroaches and lower termites harbour obligate, diverse and unique symbiotic cellulolytic flagellates in their hindgut that are considered critical in the development of social behaviour in their hosts. However, there has been controversy concerning the origin of these symbiotic flagellates. Here, molecular sequences encoding small subunit rRNA and glyceraldehyde-3-phosphate dehydrogenase were identified in the symbiotic flagellates of the order Trichonymphida (phylum Parabasalia) in the gut of Cryptocercus punctulatus and compared phylogenetically to the corresponding species in termites. In each of the monophyletic lineages that represent family-level groups in Trichonymphida, the symbionts of Cryptocercus were robustly sister to those of termites. Together with the recent evidence for the sister-group relationship of the host insects, this first comprehensive study comparing symbiont molecular phylogeny strongly suggests that a set of symbiotic flagellates representative of extant diversity was already established in an ancestor common to Cryptocercus and termites, was vertically transmitted to their offspring, and subsequently became diversified to distinct levels, depending on both the host and the symbiont lineages.

Molecular phylogeny of parabasalids with emphasis on the order Cristamonadida and its complex morphological evolution.

Parabasalia represents a complex assemblage of species, which recently received extensive reorganization. The newly created order Cristamonadida unites complex hypermastigids belonging to the Lophomonadida like the joeniids, the multinucleate polymonad Calonymphidae, and well-developed trichomonads in the Devescovinidae. All these protists exclusively occur in the guts of termites and related insects. In this study, small subunit rRNA and glyceraldehyde-3-phosphate dehydrogenase genes were identified without cultivation from 14 species in Cristamonadida including previously unstudied genera such as Joenina, Joenia, Joenoides, Macrotrichomonas, Gigantomonas, and Foaina. Despite the great morphological diversity of Cristamonadida, our phylogenetic analyses supported the monophyly of this order. However, almost all the families and subfamilies composing this order are polyphyletic suggesting a complicated morphological evolution. Our analyses also showed that Cristamonadida descends from one lineage of rudimentary trichomonads and that joeniids was basal in this order. Several successive and independent morphological transitions such as the development and reduction of flagellar apparatus and associated cytoskeleton and transition to multinucleated status have likely led to the diversity and complexity of cristamonad lineages.

Molecular identification and phylogenetic relationships of trichomonad isolates of galliform birds inferred from nuclear small subunit rRNA gene sequences.

Histomonas meleagridis is the etiological agent of histomonosis or blackhead disease. Recently, genotyping, based on polymerase chain reaction and sequencing of internal transcribed spacer-1 sequences was applied to various isolates originating from fowl. Three genotypes were described: types I and II isolates were associated with clinical disease and probably derived from H. meleagridis, whereas, type III isolates were not disease-associated and likely corresponded to Parahistomonas wenrichi according to morphological observations. However, this latter species has never been characterized at the molecular level and its phylogenetic relationships with other parabasalids remained hypothetical. To confirm the identification of these isolates, small subunit rRNA gene sequences were obtained from representatives of types I, II, and III and analyzed in a broad phylogeny including 64 other parabasalid sequences. From our phylogenetic trees, we confirmed that types I and II isolates were closely related, if not identical, to H. meleagridis, while type III isolates represented P. wenrichi. Both species clustered together with high support. This grouping suggested that speciation leading to these two species inhabiting the same hosts and ecological niche occurred recently in birds. In addition, speciation was likely followed by loss of pathogenicity in P. wenrichi.

An oxidative carbon-carbon bond formation system in cycloalkane-based thermomorphic multiphase solution.

A selective anodic oxidation system in which a carbocation intermediate is generated exclusively by use of a temperature-controlled multiphase solution to separate the different stages of the reaction from each other and from the products is described. The formation of a thermomorphic middle layer in an electrolytic solution composed of c-Hex and LPC/MeNO2 results in enhanced interaction between aliphatic alkenes and polar unstable cation.

Draft Genome Sequence of Lactococcus sp. Strain NtB2 (JCM 32569), Isolated from the Gut of the Higher Termite Nasutitermes takasagoensis.

Lactic acid bacteria are widely distributed in the termite gut. Here, we report the draft genome sequence of Lactococcus sp. strain NtB2, which was isolated from the gut of a wood-feeding higher termite.

Host-Symbiont Cospeciation of Termite-Gut Cellulolytic Protists of the Genera Teranympha and Eucomonympha and their Treponema Endosymbionts.

Cellulolytic flagellated protists inhabit the hindgut of termites. They are unique and essential to termites and related wood-feeding cockroaches, enabling host feeding on cellulosic matter. Protists of two genera in the family Teranymphidae (phylum Parabasalia), Eucomonympha and Teranympha, are phylogenetically closely related and harbor intracellular endosymbiotic bacteria from the genus Treponema. In order to obtain a clearer understanding of the evolutionary history of this triplex symbiotic relationship, the molecular phylogenies of the three symbiotic partners, the Teranymphidae protists, their Treponema endosymbionts, and their host termites, were inferred and compared. Strong congruence was observed in the tree topologies of all interacting partners, implying their cospeciating relationships. In contrast, the coevolutionary relationship between the Eucomonympha protists and their endosymbionts was more complex, and evidence of incongruence against cospeciating relationships suggested frequent host switches of the endosymbionts, possibly because multiple Eucomonympha species are present in the same gut community. Similarities in the 16S rRNA and gyrB gene sequences of the endosymbionts were higher among Teranympha spp. (>99.25% and >97.2%, respectively), whereas those between Teranympha and Eucomonympha were lower (<97.1% and <91.9%, respectively). In addition, the endosymbionts of Teranympha spp. formed a phylogenetic clade distinct from those of Eucomonympha spp. Therefore, the endosymbiont species of Teranympha spp., designated here as "Candidatus Treponema teratonymphae", needs to be classified as a species distinct from the endosymbiont species of Eucomonympha spp.

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.

In situ hybridization of termite microbes.

In situ hybridization is one of the most direct and reliable ways to ascertain the origin of the gene from complex mixed cellular systems. This method is essential for studying communities of uncultured microorganism in their natural ecosystem. In this chapter, we introduce our protocols for the in situ hybridization of the messenger RNA of uncultured symbiotic protists of termite hindgut and the ribosomal RNA of the symbiotic bacteria of the protists using nonradioactive labeling protocols. We hope that you will find these protocols useful in your own work to unravel the complex functions and to discover new organisms in the ecosystem.

Distribution and evolution of nitrogen fixation genes in the phylum Bacteroidetes.

Diazotrophs had not previously been identified among bacterial species in the phylum Bacteroidetes until the rapid expansion of bacterial genome sequences, which revealed the presence of nitrogen fixation (nif) genes in this phylum. We herein determined the draft genome sequences of Bacteroides graminisolvens JCM 15093(T) and Geofilum rubicundum JCM 15548(T). In addition to these and previously reported 'Candidatus Azobacteroides pseudotrichonymphae' and Paludibacter propionicigenes, an extensive survey of the genome sequences of diverse Bacteroidetes members revealed the presence of a set of nif genes (nifHDKENB) in strains of Dysgonomonas gadei, Dysgonomonas capnocytophagoides, Saccharicrinis fermentans, and Alkaliflexus imshenetskii. These eight species belonged to and were distributed sporadically within the order Bacteroidales. Acetylene reduction activity was detected in the five species examined, strongly suggesting their diazotrophic nature. Phylogenetic analyses showed monophyletic clustering of the six Nif protein sequences in the eight Bacteroidales species, implying that nitrogen fixation is ancestral to Bacteroidales and has been retained in these species, but lost in many other lineages. The identification of nif genes in Bacteroidales facilitates the prediction of the organismal origins of related sequences directly obtained from various environments.

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.