In Situ Detection of Novel Bacterial Endosymbionts of Acanthamoeba spp. Phylogenetically Related to Members of the Order Rickettsiales

Acanthamoebae are ubiquitous soil and water bactivores which may serve as amplification vehicles for a variety of pathogenic facultative bacteria and as hosts to other, presently uncultured bacterial endosymbionts. The spectrum of uncultured endosymbionts includes gram-negative rods and gram-variable cocci, the latter recently shown to be members of the Chlamydiales. We report here the isolation from corneal scrapings of two Acanthamoeba strains that harbor gram-negative rod endosymbionts that could not be cultured by standard techniques. These bacteria were phylogenetically characterized following amplification and sequencing of the near-full-length 16S rRNA gene. We used two fluorescently labelled oligonucleotide probes targeting signature regions within the retrieved sequences to detect these organisms in situ. Phylogenetic analyses demonstrated that they displayed 99.6% sequence similarity and formed an independent and well-separated lineage within the Rickettsiales branch of the alpha subdivision of the Proteobacteria. Nearest relatives included members of the genus Rickettsia, with sequence similarities of approximately 85 to 86%, suggesting that these symbionts are representatives of a new genus and, perhaps, family. Distance matrix, parsimony, and maximum-likelihood tree-generating methods all consistently supported deep branching of the 16S rDNA sequences within the Rickettsiales. The oligonucleotide probes displayed at least three mismatches to all other available 16S rDNA sequences, and they both readily permitted the unambiguous detection of rod-shaped bacteria within intact acanthamoebae by confocal laser-scanning microscopy. Considering the long-standing relationship of most Rickettsiales with arthropods, the finding of a related lineage of endosymbionts in protozoan hosts was unexpected and may have implications for the preadaptation and/or recruitment of rickettsia-like bacteria to metazoan hosts.     

Ultrastructure of the rickettsial endosymbiont “MIDORIKO” in the green alga Carteria cerasiformis as revealed by high-pressure freezing and freeze-substitution fixation

Bacterial endosymbionts belonging to the family Rickettsiaceae were recently identified in the unicellular green alga Carteria cerasiformis, providing the first molecular evidence of rickettsial endosymbionts within photosynthetic eukaryotes. However, previous morphological studies using transmission electron microscopy (TEM) with conventional chemical fixation did not demonstrate whether the endosymbionts of C. cerasiformis have the diagnostic characteristics of the family Rickettsiaceae. In this study, we observed the rickettsial endosymbionts “MIDORIKO” within C. cerasiformis cells by TEM with high-pressure freezing and freeze-substitution fixation. The rickettsial endosymbionts resided directly in the C. cerasiformis cytoplasm without engulfing or encompassing membranes or vacuoles. The endosymbionts had a Gram-negative cell envelope composed of outer and inner bilayer membranes. The thicknesses of the outer and inner leaflets of the bacterial cell wall were almost identical. These morphological characteristics are consistent with those of the genus Rickettsia, but the cell wall structure differed from that of the genus Orientia within the family Rickettsiaceae.     

Family- and Genus-Level 16S rRNA-Targeted Oligonucleotide Probes for Ecological Studies of Methanotrophic Bacteria

Methanotrophic bacteria play a major role in the global carbon cycle, degrade xenobiotic pollutants, and have the potential for a variety of biotechnological applications. To facilitate ecological studies of these important organisms, we developed a suite of oligonucleotide probes for quantitative analysis of methanotroph-specific 16S rRNA from environmental samples. Two probes target methanotrophs in the family Methylocystaceae (type II methanotrophs) as a group. No oligonucleotide signatures that distinguish between the two genera in this family, Methylocystis and Methylosinus, were identified. Two other probes target, as a single group, a majority of the known methanotrophs belonging to the family Methylococcaceae (type I/X methanotrophs). The remaining probes target members of individual genera of the Methylococcaceae, including Methylobacter, Methylomonas, Methylomicrobium, Methylococcus, and Methylocaldum. One of the family-level probes also covers all methanotrophic endosymbionts of marine mollusks for which 16S rRNA sequences have been published. The two known species of the newly described genus Methylosarcina gen. nov. are covered by a probe that otherwise targets only members of the closely related genus Methylomicrobium. None of the probes covers strains of the newly proposed genera Methylocella and “Methylothermus,” which are polyphyletic with respect to the recognized methanotrophic families. Empirically determined midpoint dissociation temperatures were 49 to 57°C for all probes. In dot blot screening against RNA from positive- and negative-control strains, the probes were specific to their intended targets. The broad coverage and high degree of specificity of this new suite of probes will provide more detailed, quantitative information about the community structure of methanotrophs in environmental samples than was previously available.     

Microbial community of predatory bugs of the genus Macrolophus(Hemiptera: Miridae)

Background

The predatory mirids of the genus Macrolophus are key natural enemies of various economically important agricultural pests. Both M. caliginosus and M. pygmaeus are commercially available for the augmentative biological control of arthropod pests in European greenhouses. The latter species is known to be infected with Wolbachia -inducing cytoplasmic incompatibility in its host- but the presence of other endosymbionts has not been demonstrated. In the present study, the microbial diversity was examined in various populations of M. caliginosus and M. pygmaeus by 16S rRNA sequencing and denaturing gradient gel electrophoresis.

Results

Besides Wolbachia, a co-infection of 2 Rickettsia species was detected in all M. pygmaeus populations. Based on a concatenated alignment of the 16S rRNA gene, the gltA gene and the coxA gene, the first is phylogenetically related to Rickettsia bellii, whereas the other is closely related to Rickettsia limoniae. All M. caliginosus populations were infected with the same Wolbachia and limoniae-like Rickettsia strain as M. pygmaeus, but did not harbour the bellii-like Rickettsia strain. Interestingly, individuals with a single infection were not found. A PCR assay on the ovaries of M. pygmaeus and M. caliginosus indicated that all endosymbionts are vertically transmitted. The presence of Wolbachia and Rickettsia in oocytes was confirmed by a fluorescence in situ hybridisation. A bio-assay comparing an infected and an uninfected M. pygmaeus population suggested that the endosymbionts had minor effects on nymphal development of their insect host and did not influence its fecundity.

Conclusion

Two species of the palaearctic mirid genus Macrolophus are infected with multiple endosymbionts, including Wolbachia and Rickettsia. Independent of the origin, all tested populations of both M. pygmaeus and M. caliginosus were infected with three and two endosymbionts, respectively. There was no indication that infection with endosymbiotic bacteria had a fitness cost in terms of development and fecundity of the predators.

     

Endosymbiotic Bacteroidales Bacteria of the Flagellated Protist Pseudotrichonympha grassii in the Gut of the Termite Coptotermes formosanus

A unique lineage of bacteria belonging to the order Bacteroidales was identified as an intracellular endosymbiont of the protist Pseudotrichonympha grassii (Parabasalia, Hypermastigea) in the gut of the termite Coptotermes formosanus. We identified the 16S rRNA, gyrB, elongation factor Tu, and groEL gene sequences in the endosymbiont and detected a very low level of sequence divergence (<0.9% of the nucleotides) in the endosymbiont population within and among protist cells. The Bacteroidales endosymbiont sequence was affiliated with a cluster comprising only sequences from termite gut bacteria and was not closely related to sequences identified for members of the Bacteroidales attached to the cell surfaces of other gut protists. Transmission electron microscopy showed that there were numerous rod-shaped bacteria in the cytoplasm of the host protist, and we detected the endosymbiont by fluorescence in situ hybridization (FISH) with an oligonucleotide probe specific for the 16S rRNA gene identified. Quantification of the abundance of the Bacteroidales endosymbiont by sequence-specific cleavage of rRNA with RNase H and FISH cell counting revealed, surprisingly, that the endosymbiont accounted for 82% of the total bacterial rRNA and 71% of the total bacterial cells in the gut community. The genetically nearly homogeneous endosymbionts of Pseudotrichonympha were very abundant in the gut symbiotic community of the termite.     

The genus Caedibacter comprises endosymbionts of Paramecium spp. related to the Rickettsiales (Alphaproteobacteria) and to Francisella tularensis (Gammaproteobacteria)

Obligate bacterial endosymbionts of paramecia able to form refractile inclusion bodies (R bodies), thereby conferring a killer trait upon their ciliate hosts, have traditionally been grouped into the genus CAEDIBACTER: Of the six species described to date, only the Paramecium caudatum symbiont Caedibacter caryophilus has been phylogenetically characterized by its 16S rRNA gene sequence, and it was found to be a member of the Alphaproteobacteria related to the RICKETTSIALES: In this study, the Caedibacter taeniospiralis type strain, an R-body-producing cytoplasmatic symbiont of Paramecium tetraurelia strain 51k, was investigated by comparative 16S rRNA sequence analysis and fluorescence in situ hybridization with specific oligonucleotide probes. C. taeniospiralis is not closely related to C. caryophilus (80% 16S rRNA sequence similarity) but forms a novel evolutionary lineage within the Gammaproteobacteria with the family Francisellaceae as a sister group (87% 16S rRNA sequence similarity). These findings demonstrate that the genus Caedibacter is polyphyletic and comprises at least two phylogenetically different bacterial species belonging to two different classes of the PROTEOBACTERIA: Comparative phylogenetic analysis of C. caryophilus, five closely related Acanthamoeba endosymbionts (including one previously uncharacterized amoebal symbiont identified in this study), and their hosts suggests that the progenitor of the alphaproteobacterial C. caryophilus lived within acanthamoebae prior to the infection of paramecia.     

Obligate intracellular bacteria diversity in unfed Leptotrombidium scutellare larvae highlights novel bacterial endosymbionts of mites

It is well known that the mite Leptotrombidium scutellare carries the pathogen of scrub typhus, Orientia tsutsugamushi. However, our understanding of other bacterial endosymbionts of mites is limited. This study investigated the diversity of the obligate intracellular bacteria carried by L. scutellare using 16S rRNA gene amplicon analysis with next-generation sequencing. The results showed that the detected bacteria were classified into the genera Rickettsia, Wolbachia, and Rickettsiella and an unknown genus of the order Rickettsiales. For further classification of the detected bacteria, a representative read that was most closely related to the assigned taxonomic classification was subjected to homology search and phylogenic analysis. The results showed that some bacteria of the genus Rickettsia were identical or very close to the human pathogens Rickettsia akari, Rickettsia aeschlimannii, Rickettsia felis, and Rickettsia australis. The genetic distance between the genus Wolbachia bacteria in the present study and in previous reports is highly indicative that the bacteria in the present study can be classified as a new taxon of Wolbachia. This study detected obligate intracellular bacteria from unfed mites; thus, the mites did not acquire bacteria from infected animals or any other infectious sources. Finally, the present study demonstrated that various and novel bacterial endosymbionts of mites, in addition to O. tsutsugamushi, might uniquely evolve with the host mites throughout overlapping generations of the mite life cycle. The roles of the bacteria in mites and their pathogenicity should be further examined in studies based on bacterial isolation.     

Extremely Acidophilic Protists from Acid Mine Drainage Host Rickettsiales-Lineage Endosymbionts That Have Intervening Sequences in Their 16S rRNA Genes

During a molecular phylogenetic survey of extremely acidic (pH < 1), metal-rich acid mine drainage habitats in the Richmond Mine at Iron Mountain, Calif., we detected 16S rRNA gene sequences of a novel bacterial group belonging to the order Rickettsiales in the Alphaproteobacteria. The closest known relatives of this group (92% 16S rRNA gene sequence identity) are endosymbionts of the protist Acanthamoeba. Oligonucleotide 16S rRNA probes were designed and used to observe members of this group within acidophilic protists. To improve visualization of eukaryotic populations in the acid mine drainage samples, broad-specificity probes for eukaryotes were redesigned and combined to highlight this component of the acid mine drainage community. Approximately 4% of protists in the acid mine drainage samples contained endosymbionts. Measurements of internal pH of the protists showed that their cytosol is close to neutral, indicating that the endosymbionts may be neutrophilic. The endosymbionts had a conserved 273-nucleotide intervening sequence (IVS) in variable region V1 of their 16S rRNA genes. The IVS does not match any sequence in current databases, but the predicted secondary structure forms well-defined stem loops. IVSs are uncommon in rRNA genes and appear to be confined to bacteria living in close association with eukaryotes. Based on the phylogenetic novelty of the endosymbiont sequences and initial culture-independent characterization, we propose the name “Candidatus Captivus acidiprotistae.” To our knowledge, this is the first report of an endosymbiotic relationship in an extremely acidic habitat.     

Changes in bovine milk bacterial microbiome from healthy and subclinical mastitis affected animals of the Girolando,Gyr, Guzera,and Holstein breeds

Raw milk samples were collected from 200 dairy cows belonging to Girolando 1/2, Gyr, Guzera, and Holstein breeds, and the bacterial diversity was explored using 16S rRNA amplicon sequencing. SCC analysis showed that 69 animals were classified as affected with subclinical mastitis. The milk bacterial microbiome was dominated by Firmicutes, Proteobacteria, and Actinobacteria, with an increase of Firmicutes in animals with subclinical mastitis and Proteobacteria in healthy animals. At the family and genus level, the milk bacterial microbiome was dominated by Staphylococcus, Acinetobacter, Pseudomonas, members of the family Enterobacteriaceae, Lactococcus, Aerococcus, members of the family Rhizobiaceae, Anaerobacillus, Streptococcus, members of the family Intrasporangiaceae, members of the family Planococcaceae, Corynebacterium, Nocardioides, and Chryseobacterium. Significant differences in alpha and beta diversity analysis suggest an effect of udder health status and breed on the composition of raw bovine milk microbiota. LEfSe analysis showed 45 and 51 discriminative taxonomic biomarkers associated with udder health status and with one of the four breeds respectively, suggesting an effect of subclinical mastitis and breed on the microbiota of milk in cattle.

     

‘Candidatus Megaira polyxenophila’ gen. nov., sp. nov.: Considerations on Evolutionary History,Host Range and Shift of Early Divergent Rickettsiae

“Neglected Rickettsiaceae” (i.e. those harboured by non-hematophagous eukaryotic hosts) display greater phylogenetic variability and more widespread dispersal than pathogenic ones; yet, the knowledge about their actual host range and host shift mechanism is scarce. The present work reports the characterization following the full-cycle rRNA approach (SSU rRNA sequence, specific in situ hybridization, and ultrastructure) of a novel rickettsial bacterium, herewith proposed as ''Candidatus Megaira polyxenophila'' gen. nov., sp. nov. We found it in association with four different free-living ciliates (Diophrys oligothrix, Euplotes octocarinatus, Paramecium caudatum, and Spirostomum sp., all belonging to Alveolata, Ciliophora); furthermore it was recently observed as intracellular occurring in Carteria cerasiformis and Pleodorina japonica (Chlorophyceae, Chlorophyta). Phylogenetic analyses demonstrated the belonging of the candidate new genus to the family Rickettsiaceae (Alphaproteobacteria, Rickettsiales) as a sister group of the genus Rickettsia. In situ observations revealed the ability of the candidate new species to colonize either nuclear or cytoplasmic compartments, depending on the host organism. The presence of the same bacterial species within different, evolutionary distant, hosts indicates that ''Candidatus Megaira polyxenophila'' recently underwent several distinct host shifts, thus suggesting the existence of horizontal transmission pathways. We consider these findings as indicative of an unexpected spread of rickettsial infections in aquatic communities, possibly by means of trophic interactions, and hence propose a new interpretation of the origin and phylogenetic diversification of rickettsial bacteria.     

Molecular characterization of the obligate endosymbiont "Caedibacter macronucleorum"Fokin and Görtz, 1993 and of its host Paramecium duboscqui strain Ku4-8

ABSTRACT. Bacterial endosymbionts of protozoa were often described as new species by protozoologists mainly on the basis of few morphological characters and partly by host specificity. Many of these species have never been validated by prokaryotic microbiologists whose taxonomic rules are quite different from those of protozoologists, who use the Zoological Code of Nomenclature. “Caedibacter macronucleorum” Fokin and Görtz 1993 , an endosymbiont of Paramecium duboscqui, belongs to this category. Here we provide the molecular characterization of this organism and of its host P. duboscqui strain Ku4‐8. Bacterial 16S rRNA gene sequence analysis proved that “C. macronucleorum” belongs to the Alphaproteobacteria. It is closely related to Caedibacter caryophilus but not to Caedibacter taeniospiralis, which belongs to the Gammaproteobacteria. “Caedibacter macronucleorum” and C. caryophilus 16S rRNA genes show a similarity value of 99%. This high 16S rRNA sequence similarity and the lack of a specific oligonucleotide probe for distinguishing the two endosymbionts do not allow validating “C. macronucleorum” as a provisional taxon (Candidatus). Nevertheless, “C. macronucleorum” and C. caryophilus can be easily discriminated on the basis of a highly variable stretch of nucleotides that interrupts the 16S rRNA genes of both organisms.     

Molecular diversity of bacterial endosymbionts associated with dagger nematodes of the genus Xiphinema (Nematoda: Longidoridae) reveals a high degree of phylogenetic congruence with their host

Bacterial endosymbionts have been detected in some groups of plant‐parasitic nematodes, but few cases have been reported compared to other groups in the phylum Nematoda, such as animal‐parasitic or free‐living nematodes. This study was performed on a wide variety of plant‐parasitic nematode families and species from different host plants and nematode populations. A total of 124 nematode populations (previously identified morphologically and molecularly) were screened for the presence of potential bacterial endosymbionts using the partial 16S rRNA gene and fluorescence in situ hybridization (FISH) and confocal microscopy. Potential bacterial endosymbionts were only detected in nematode species belonging to the genus Xiphinema and specifically in the X. americanum group. Fifty‐seven partial 16S rRNA sequences were obtained from bacterial endosymbionts in this study. One group of sequences was closely related to the genus ‘Candidatus Xiphinematobacter’ (19 bacterial endosymbiont sequences were associated with seven nematode host species, including two that have already been described and three unknown bacterial endosymbionts). The second bacterial endosymbiont group (38 bacterial endosymbiont sequences associated with six nematode species) was related to the family Burkholderiaceae, which includes fungal and soil–plant bacterial endosymbionts. These endosymbionts were reported for the first time in the phylum Nematoda. Our findings suggest that there is a highly specific symbiotic relationship between nematode host and bacterial endosymbionts. Overall, these results were corroborated by a phylogeny of nematode host and bacterial endosymbionts that suggested that there was a high degree of phylogenetic congruence and long‐term evolutionary persistence between hosts and endosymbionts.     

Bacterial endosymbiont infections in ‘living fossils’: a case study of North American vaejovid scorpions

Bacterial endosymbionts are common among arthropods, and maternally inherited forms can affect the reproductive and behavioural traits of their arthropod hosts. The prevalence of bacterial endosymbionts and their role in scorpion evolution have rarely been investigated. In this study, 61 samples from 40 species of scorpion in the family Vaejovidae were screened for the presence of the bacterial endosymbionts Cardinium, Rickettsia, Spiroplasma and Wolbachia. No samples were infected by these bacteria. However, one primer pair specifically designed to amplify Rickettsia amplified nontarget genes of other taxa. Similar off‐target amplification using another endosymbiont‐specific primer was also found during preliminary screenings. Results caution against the overreliance on previously published screening primers to detect bacterial endosymbionts in host taxa and suggest that primer specificity may be higher in primers targeting nuclear rather than mitochondrial genes.     

Rickettsia spp., Ehrlichia sp. and Candidatus Midichloria sp. associated to ticks from a protected urban area in Buenos Aires City (Argentina)

The aim of this study was to determine the infection with Rickettsiales in ticks and birds from the main protected urban area of Buenos Aires City (Argentina). One Amblyomma aureolatum (0.2%) and one Ixodes auritulus (0.1%) were positive by PCR targeting Rickettsia 23S-5S rRNA intergenic spacer. Phylogenetic analysis shows to findings in A. aureolatum are closely to Rickettsia bellii and for I. auritulus are related to ‘Candidatus Rickettsia mendelii’. One I. auritulus (0.1%) and three A. aureolatum (0.6%) were positive by PCR for a fragment of the 16S rRNA gene of the Anaplasmataceae family. The sequences obtained from A. aureolatum were phylogenetically related to Midichloriaceae endosymbionts. The sequence from I. auritulus s.l. had 100% identity with Ehrlichia sp. Magellanica from Chile and two genotypes of Ehrlichia sp. from Uruguay. The results of our study show that Rickettsia and Ehrlichia are present in ticks in the main protected urban area of Buenos Aires City.

     

Diversity of secondary endosymbionts among different putative species of the whitefly Bemisia tabaci

Abstract Endosymbionts are important components of arthropod biology. The whitefly Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) is a cryptic species complex composed of ≥ 28 putative species. In addition to the primary endosymbiont Portiera aleyrodidarum, six secondary endosymbionts (S‐endosymbionts), Hamiltonella, Rickettsia, Wolbachia, Cardinium, Arsenophonus and Fritschea, have been identified in B. tabaci thus far. Here, we tested five of the six S‐endosymbiont lineages (excluding Fritschea) from 340 whitely individuals representing six putative species from China. Hamiltonella was detected only in the two exotic invaders, Middle East‐Asia Minor 1 (MEAM1) and Mediterranean (MED). Rickettsia was absent in Asia II 1 and MED, scarce in Asia II 3 (13%), but abundant in Asia II 7 (63.2%), China 1 (84.7%) and MEAM1 (100%). Wolbachia, Cardinium and Arsenophonus were absent in the invasive MEAM1 and MED but mostly abundant in the native putative species. Furthermore, phylogenetic analyses revealed that some S‐endosymbionts have several clades and different B. tabaci putative species can harbor different clades of a given S‐endosymbiont, demonstrating further the complexity of S‐endosymbionts in B. tabaci. All together, our results demonstrate the variation and diversity of S‐endosymbionts in different putative species of B. tabaci, especially between invasive and native whiteflies.     

Diversity and phylogenetic analysis of endosymbionts from Trioza erytreae (Del Guercio) and its parasitoids in Kenya

The African citrus triozid (ACT), Trioza erytreae (Del Guercio), is the primary vector of Candidatus Liberibacter africanus (CLaf), the causative agent of Africa citrus greening disease (ACGD). This study evaluates the diversity of ACT parasitoids and further characterizes endosymbionts associated with both T. erytreae and its parasitoids that could be used as biological control agents of T. erytreae and management of ACGD. Mitochondrial cytochrome oxidase I gene was used to reconstruct T. erytreae and its parasitoids phylogeny, while 16S rRNA gene was used for the bacterial phylogeny. One well-supported clade of ACT was detected within the Triozidae phylogeny, while the parasitoid species clustered into four groups within eulophid and encyrtid phylogeny. The phylogenetic result of parasitoids was supported by morphological identification where five different parasitoid species could be identified, that is Tamarixia dryi, Psyllaephagus pulvinatus, Tetrastichus sp., Aphidencyrtus cassatus and Charipine species. Moreover, four eubacterial symbionts (Wolbachia,Rickettsia,Arsenophonus and Candidatus Liberibacter sp.) were detected in T. erytreae and three symbionts (Wolbachia,Rickettsia and Cardinuim) in the parasitoid specimens. Maximum likelihood phylogenetic inferences clustered the identified eubacterial symbionts within α and γ proteobacteria subdivisions. Phylogenetic inferences of 16S rRNA gene sequences indicated that Wolbachia strains from ACT and the parasitoids did not form a single monophyletic clade; however, both clustered within Supergroup B. The impacts of these parasitoid species and endosymbionts on ACT are still unknown, but their occurrence and broad distribution indicate the possibility of future use for control of T. erytreae.     

“Endomicrobia”: Cytoplasmic Symbionts of Termite Gut Protozoa Form a Separate Phylum of Prokaryotes

Lignocellulose digestion by wood-feeding termites depends on the mutualistic interaction of unusual, flagellate protists located in their hindgut. Most of the flagellates harbor numerous prokaryotic endosymbionts of so-far-unknown identity and function. Using a full-cycle molecular approach, we show here that the endosymbionts of the larger gut flagellates of Reticulitermes santonensis belong to the so-called termite group 1 (TG-1) bacteria, a group of clones previously obtained exclusively from gut homogenates of Reticulitermes speratus that are only distantly related to other bacteria and are considered a novel bacterial phylum based on their 16S rRNA gene sequences. Fluorescence in situ hybridization with specifically designed oligonucleotide probes confirmed that TG-1 bacteria are indeed located within the flagellate cells and demonstrated that Trichonympha agilis (Hypermastigida) and Pyrsonympha vertens (Oxymonadida) harbor phylogenetically distinct populations of symbionts (<95% sequence similarity). Transmission electron microscopy revealed that the symbionts are small, spindle-shaped cells (0.6 μm in length and 0.3 μm in diameter) surrounded by two membranes and located within the cytoplasm of their hosts. The symbionts of the two flagellates are described as candidate species in the candidate genus “Endomicrobium.” Moreover, we provide evidence that the members of the TG-1 phylum, for which we propose the candidate name “Endomicrobia,” are phylogenetically extremely diverse and are present in and also restricted to the guts of all lower termites and wood-feeding cockroaches of the genus Cryptocercus, the only insects that are in an exclusive, obligately mutualistic association with such unique cellulose-fermenting protists.     

Evolution of symbiotic organs and endosymbionts in lygaeid stinkbugs

We investigated seed bugs of the genus Nysius (Insecta: Hemiptera: Lygaeidae) for their symbiotic bacteria. From all the samples representing 4 species, 18 populations and 281 individuals, specific bacterial 16S rRNA gene sequences were consistently identified, which formed a distinct clade in the Gammaproteobacteria. In situ hybridization showed that the bacterium was endocellularly localized in a pair of large bacteriomes that were amorphous in shape, deep red in color, and in association with gonads. In the ovary of adult females, the endosymbiont was also localized in the ‘infection zone'' in the middle of each germarium and in the ‘symbiont ball'' at the anterior pole of each oocyte, indicating vertical transmission of the endosymbiont through the ovarial passage. Phylogenetic analyses based on bacterial 16S rRNA, groEL and gyrB genes consistently supported a coherent monophyly of the Nysius endosymbionts. The possibility of a sister relationship to ‘Candidatus Kleidoceria schneideri'', the bacteriome-associated endosymbiont of a lygaeid bug Kleidocerys resedae, was statistically rejected, indicating independent evolutionary origins of the endosymbionts in the Lygaeidae. The endosymbiont genes consistently exhibited AT-biased nucleotide compositions and accelerated rates of molecular evolution, and the endosymbiont genome was only 0.6 Mb in size. The endosymbiont phylogeny was congruent with the host insect phylogeny, suggesting strict vertical transmission and host–symbiont co-speciation over evolutionary time. Based on these results, we discuss the evolution of bacteriomes and endosymbionts in the Heteroptera, most members of which are associated with gut symbiotic bacteria. The designation ‘Candidatus Schneideria nysicola'' is proposed for the endosymbiont clade.     

The Whole-genome Sequencing of the Obligate Intracellular Bacterium Orientia tsutsugamushi Revealed Massive Gene Amplification During Reductive Genome Evolution

Scrub typhus (‘Tsutsugamushi’ disease in Japanese) is a mite-borne infectious disease. The causative agent is Orientia tsutsugamushi, an obligate intracellular bacterium belonging to the family Rickettsiaceae of the subdivision alpha-Proteobacteria. In this study, we determined the complete genome sequence of O. tsutsugamushi strain Ikeda, which comprises a single chromosome of 2 008 987 bp and contains 1967 protein coding sequences (CDSs). The chromosome is much larger than those of other members of Rickettsiaceae, and 46.7% of the sequence was occupied by repetitive sequences derived from an integrative and conjugative element, 10 types of transposable elements, and seven types of short repeats of unknown origins. The massive amplification and degradation of these elements have generated a huge number of repeated genes (1196 CDSs, categorized into 85 families), many of which are pseudogenes (766 CDSs), and also induced intensive genome shuffling. By comparing the gene content with those of other family members of Rickettsiacea, we identified the core gene set of the family Rickettsiaceae and found that, while much more extensive gene loss has taken place among the housekeeping genes of Orientia than those of Rickettsia, O. tsutsugamushi has acquired a large number of foreign genes. The O. tsutsugamushi genome sequence is thus a prominent example of the high plasticity of bacterial genomes, and provides the genetic basis for a better understanding of the biology of O. tsutsugamushi and the pathogenesis of ‘Tsutsugamushi’ disease.Key words: Orientia tsutsugamushi, genome sequencing, obligate intracellular bacterium, repetitive sequence, IS element, integrative and conjugative element, gene amplification, genome reduction