Two O-Methyltransferases from Phylogenetically Unrelated Cow Parsley (Anthriscus sylvestris) and Hinoki-Asunaro (Thujopsis dolabrata var. hondae) as a Signature of Lineage-Specific Evolution in Lignan Biosynthesis.

O-Methyltransferases (OMTs) play important roles in antitumor lignan biosynthesis. To date, six OMTs catalyzing the methylation of dibenzylbutyrolactone lignans as biosynthetic precursors of antitumor lignans have been identified. However, there is still no systematic understanding of the diversity and regularity of the biosynthetic mechanisms among various plant lineages. Herein, we report the characterization of two OMTs from Anthriscus sylvestris and Thujopsis dolabrata var. hondae [designated as AsSecoNorYatein (SNY) OMT and TdSNYOMT] together with the six known OMTs to evaluate their diversity and regularity. Although A. sylvestris 5-O-methylthujaplicatin (SecoNorYatein) and 4-O-demethylyatein (NorYatein) OMT (AsSNYOMT) and TdSNYOMT accept 5-O-methylthujaplicatin and 4-O-demethylyatein as substrates, phylogenetic analysis indicated that these two OMTs shared low amino acid sequence identity, 33.8%, indicating a signature of parallel evolution. The OMTs and the six previously identified OMTs were found to be diverse in terms of their substrate specificity, regioselectivity and amino acid sequence identity, indicating independent evolution in each plant species. Meanwhile, two-entropy analysis detected four amino acid residues as being specifically acquired by dibenzylbutyrolactone lignan OMTs. Site-directed mutation of AsSNYOMT indicated that two of them contributed specifically to 5-O-methylthujaplicatin methylation. The results provide a new example of parallel evolution and the diversity and regularity of OMTs in plant secondary (specialized) metabolism.

Fiber-associated spirochetes are major agents of hemicellulose degradation in the hindgut of wood-feeding higher termites.

Symbiotic digestion of lignocellulose in wood-feeding higher termites (family Termitidae) is a two-step process that involves endogenous host cellulases secreted in the midgut and a dense bacterial community in the hindgut compartment. The genomes of the bacterial gut microbiota encode diverse cellulolytic and hemicellulolytic enzymes, but the contributions of host and bacterial symbionts to lignocellulose degradation remain ambiguous. Our previous studies of Nasutitermes spp. documented that the wood fibers in the hindgut paunch are consistently colonized not only by uncultured members of Fibrobacteres, which have been implicated in cellulose degradation, but also by unique lineages of Spirochaetes. Here, we demonstrate that the degradation of xylan, the major component of hemicellulose, is restricted to the hindgut compartment, where it is preferentially hydrolyzed over cellulose. Metatranscriptomic analysis documented that the majority of glycoside hydrolase (GH) transcripts expressed by the fiber-associated bacterial community belong to family GH11, which consists exclusively of xylanases. The substrate specificity was further confirmed by heterologous expression of the gene encoding the predominant homolog. Although the most abundant transcripts of GH11 in Nasutitermes takasagoensis were phylogenetically placed among their homologs of Firmicutes, immunofluorescence microscopy, compositional binning of metagenomics contigs, and the genomic context of the homologs indicated that they are encoded by Spirochaetes and were most likely obtained by horizontal gene transfer among the intestinal microbiota. The major role of spirochetes in xylan degradation is unprecedented and assigns the fiber-associated Treponema clades in the hindgut of wood-feeding higher termites a prominent part in the breakdown of hemicelluloses.

Recurrent symbiont recruitment from fungal parasites in cicadas.

Diverse insects are associated with ancient bacterial symbionts, whose genomes have often suffered drastic reduction and degeneration. In extreme cases, such symbiont genomes seem almost unable to sustain the basic cellular functioning, which comprises an open question in the evolution of symbiosis. Here, we report an insect group wherein an ancient symbiont lineage suffering massive genome erosion has experienced recurrent extinction and replacement by host-associated pathogenic microbes. Cicadas are associated with the ancient bacterial co-obligate symbionts Sulcia and Hodgkinia, whose streamlined genomes are specialized for synthesizing essential amino acids, thereby enabling the host to live on plant sap. However, our inspection of 24 Japanese cicada species revealed that while all species possessed Sulcia, only nine species retained Hodgkinia, and their genomes exhibited substantial structural instability. The remaining 15 species lacked Hodgkinia and instead harbored yeast-like fungal symbionts. Detailed phylogenetic analyses uncovered repeated Hodgkinia-fungus and fungus-fungus replacements in cicadas. The fungal symbionts were phylogenetically intermingled with cicada-parasitizing Ophiocordyceps fungi, identifying entomopathogenic origins of the fungal symbionts. Most fungal symbionts of cicadas were uncultivable, but the fungal symbiont of Meimuna opalifera was cultivable, possibly because it is at an early stage of fungal symbiont replacement. Genome sequencing of the fungal symbiont revealed its metabolic versatility, presumably capable of synthesizing almost all amino acids, vitamins, and other metabolites, which is more than sufficient to compensate for the Hodgkinia loss. These findings highlight a straightforward ecological and evolutionary connection between parasitism and symbiosis, which may provide an evolutionary trajectory to renovate deteriorated ancient symbiosis via pathogen domestication.

Repeated evolution of bacteriocytes in lygaeoid stinkbugs.

Host-microbe symbioses often evolved highly complex developmental processes and colonization mechanisms for establishment of stable associations. It has long been recognized that many insects harbour beneficial bacteria inside specific symbiotic cells (bacteriocytes) or organs (bacteriomes). However, the evolutionary origin and mechanisms underlying bacterial colonization in bacteriocyte/bacteriome formation have been poorly understood. In order to uncover the origin of such evolutionary novelties, we studied the development of symbiotic organs in five stinkbug species representing the superfamily Lygaeoidea in which diverse bacteriocyte/bacteriome systems have evolved. We tracked the symbiont movement within the eggs during the embryonic development and determined crucial stages at which symbiont infection and bacteriocyte formation occur, using whole-mount fluorescence in situ hybridization. In summary, three distinct developmental patterns were observed: two different modes of symbiont transfer from initial symbiont cluster (symbiont ball) to presumptive bacteriocytes in the embryonic abdomen, and direct incorporation of the symbiont ball without translocation of bacterial cells. Across the host taxa, only closely related species seemed to have evolved relatively conserved types of bacteriome development, suggesting repeated evolution of host symbiotic cells and organs from multiple independent origins.

One Hundred Mitochondrial Genomes of Cicadas.

Mitochondrial genomes can provide valuable information on the biology and evolutionary histories of their host organisms. Here, we present and characterize the complete coding regions of 107 mitochondrial genomes (mitogenomes) of cicadas (Insecta: Hemiptera: Auchenorrhyncha: Cicadoidea), representing 31 genera, 61 species, and 83 populations. We show that all cicada mitogenomes retain the organization and gene contents thought to be ancestral in insects, with some variability among cicada clades in the length of a region between the genes nad2 and cox1, which encodes 3 tRNAs. Phylogenetic analyses using these mitogenomes recapitulate a recent 5-gene classification of cicadas into families and subfamilies, but also identify a species that falls outside of the established taxonomic framework. While protein-coding genes are under strong purifying selection, tests of relative evolutionary rates reveal significant variation in evolutionary rates across taxa, highlighting the dynamic nature of mitochondrial genome evolution in cicadas. These data will serve as a useful reference for future research into the systematics, ecology, and evolution of the superfamily Cicadoidea.

Ultrabithorax is essential for bacteriocyte development.

Symbiosis often entails the emergence of novel adaptive traits in organisms. Microbial symbionts are indispensable for diverse insects via provisioning of essential nutrients, wherein novel host cells and organs for harboring the microbes, called bacteriocytes and bacteriomes, have evolved repeatedly. Molecular and developmental mechanisms underpinning the emergence of novel symbiotic cells and organs comprise an unsolved question in evolutionary developmental biology. Here, we report that a conserved homeotic gene, Ultrabithorax, plays a pivotal role in the bacteriocyte differentiation in a hemipteran insect Nysius plebeius. During embryonic development, six pairs of aggregated presumptive bacteriocytes appear on both sides of six abdominal segments, incorporate the symbiotic bacteria at the stage of germband retraction, and fuse into a pair of lateral bacteriomes at the stage of germband flip, where bacteriocyte-associated Ultrabithorax expression coincides with the symbiont infection process. Suppression of Ultrabithorax expression by maternal RNA interference results in disappearance of the bacteriocytes and the symbiont localization therein, suggesting that Ultrabithorax is involved in differentiation of the host cells for symbiosis. Suppression of other homeotic genes abdominal-A and Antennapedia disturbs integrity and positioning of the bacteriomes, affecting the configuration of the host organs for symbiosis. Our findings unveil the molecular and developmental mechanisms underlying the bacteriocyte differentiation, which may have evolved either via cooption of the transcription factors for inducing the novel symbiotic cells, or via revival of the developmental pathway for the bacteriocytes that had existed in the ancestral hemipterans.

Intrasperm vertical symbiont transmission.

Symbiotic bacteria are commonly associated with cells and tissues of diverse animals and other organisms, which affect hosts' biology in a variety of ways. Most of these symbionts are present in the cytoplasm of host cells and maternally transmitted through host generations. The paucity of paternal symbiont transmission is likely relevant to the extremely streamlined sperm structure: the head consisting of condensed nucleus and the tail made of microtubule bundles, without the symbiont-harboring cytoplasm that is discarded in the process of spermatogenesis. Here, we report a previously unknown mechanism of paternal symbiont transmission via an intrasperm passage. In the leafhopper Nephotettix cincticeps, a facultative Rickettsia symbiont was found not only in the cytoplasm but also in the nucleus of host cells. In male insects, strikingly, most sperm heads contained multiple intranuclear Rickettsia cells. The Rickettsia infection scarcely affected the host fitness including normal sperm functioning. Mating experiments revealed both maternal and paternal transmission of the Rickettsia symbiont through host generations. When cultured with mosquito and silkworm cell lines, the Rickettsia symbiont was preferentially localized within the insect cell nuclei, indicating that the Rickettsia symbiont itself must have a mechanism for targeting nucleus. The mechanisms underlying the sperm head infection without disturbing sperm functioning are, although currently unknown, of both basic and applied interest.

Bacterial symbionts of a devastating coffee plant pest, the stinkbug Antestiopsis thunbergii (Hemiptera: Pentatomidae).

Stinkbugs of the genus Antestiopsis, so-called antestia bugs or variegated coffee bugs, are notorious pests of coffee plants in Africa. We investigated the symbiotic bacteria associated with Antestiopsis thunbergii, a major coffee plant pest in Rwanda. PCR, cloning, sequencing, and phylogenetic analysis of bacterial genes identified four distinct bacterial lineages associated with A. thunbergii: a gammaproteobacterial gut symbiont and symbionts representing the genera Sodalis, Spiroplasma, and Rickettsia. In situ hybridization showed that the gut symbiont densely occupied the lumen of midgut crypts, whereas the Sodalis symbiont, the Spiroplasma symbiont, and the Rickettsia symbiont sparsely and sporadically infected various cells and tissues. Diagnostic PCR survey of 154 A. thunbergii individuals collected at 8 localities in Rwanda revealed high infection frequencies (100% for the gut symbiont, 51.3% for the Sodalis symbiont, 52.6% for the Spiroplasma symbiont, and 24.0% for the Rickettsia symbiont). These results suggest that the gut symbiont is the primary symbiotic associate of obligate nature for A. thunbergii, whereas the Sodalis symbiont, the Spiroplasma symbiont, and the Rickettsia symbiont are the secondary symbiotic associates of facultative nature. We observed high coinfection frequencies, i.e., 7.8% of individuals with quadruple infection with all the symbionts, 32.5% with triple infections with the gut symbiont and two of the secondary symbionts, and 39.6% with double infections with the gut symbiont and any of the three secondary symbionts, which were statistically not different from the expected coinfection frequencies and probably reflected random associations. The knowledge of symbiotic microbiota in A. thunbergii will provide useful background information for controlling this devastating coffee plant pest.

Intracellularity, extracellularity, and squeezing in the symbiotic organ underpin nurturing and functioning of bacterial symbiont in leaf beetles.

Cassidine leaf beetles are associated with genome-reduced symbiotic bacteria Stammera involved in pectin digestion. Stammera cells appear to be harbored in paired symbiotic organs located at the foregut-midgut junction either intracellularly or extracellularly, whereas the symbiont is extracellular in the ovary-accessory glands of adult females and during caplet transmission in eggs. However, using fluorescence and electron microscopy, an intracellular symbiotic configuration of Stammera was observed in Notosacantha species. Detailed inspection of other cassidine species revealed fragmented cell membrane and cytoplasm of the symbiotic organs, wherein Stammera cells are in an intermediate status between intracellularity and extracellularity. We also identified a mitochondria-rich region adjacent to the symbiont-filled region and well-developed muscle fibers surrounding the whole symbiotic organ. Based on these observations, we discuss why the Stammera genome has been reduced so drastically and how symbiont-derived pectinases are produced and supplied to the host's alimentary tract for plant cell wall digestion.

Paleocene origin of a streamlined digestive symbiosis in leaf beetles.

Timing the acquisition of a beneficial microbe relative to the evolutionary history of its host can shed light on the adaptive impact of a partnership. Here, we investigated the onset and molecular evolution of an obligate symbiosis between Cassidinae leaf beetles and Candidatus Stammera capleta, a γ-proteobacterium. Residing extracellularly within foregut symbiotic organs, Stammera upgrades the digestive physiology of its host by supplementing plant cell wall-degrading enzymes. We observe that Stammera is a shared symbiont across tortoise and hispine beetles that collectively comprise the Cassidinae subfamily, despite differences in their folivorous habits. In contrast to its transcriptional profile during vertical transmission, Stammera elevates the expression of genes encoding digestive enzymes while in the foregut symbiotic organs, matching the nutritional requirements of its host. Despite the widespread distribution of Stammera across Cassidinae beetles, symbiont acquisition during the Paleocene (∼62 mya) did not coincide with the origin of the subfamily. Early diverging lineages lack the symbiont and the specialized organs that house it. Reconstructing the ancestral state of host-beneficial factors revealed that Stammera encoded three digestive enzymes at the onset of symbiosis, including polygalacturonase-a pectinase that is universally shared. Although non-symbiotic cassidines encode polygalacturonase endogenously, their repertoire of plant cell wall-degrading enzymes is more limited compared with symbiotic beetles supplemented with digestive enzymes from Stammera. Highlighting the potential impact of a symbiotic condition and an upgraded metabolic potential, Stammera-harboring beetles exploit a greater variety of plants and are more speciose compared with non-symbiotic members of the Cassidinae.

Diversity of bacterial endosymbionts associated with Macrosteles leafhoppers vectoring phytopathogenic phytoplasmas.

Here, we investigate the endosymbiotic microbiota of the Macrosteles leafhoppers M. striifrons and M. sexnotatus, known as vectors of phytopathogenic phytoplasmas. PCR, cloning, sequencing, and phylogenetic analyses of bacterial 16S rRNA genes identified two obligate endosymbionts, "Candidatus Sulcia muelleri" and "Candidatus Nasuia deltocephalinicola," and five facultative endosymbionts, Wolbachia, Rickettsia, Burkholderia, Diplorickettsia, and a novel bacterium belonging to the Rickettsiaceae, from the leafhoppers. "Ca. Sulcia muelleri" and "Ca. Nasuia deltocephalinicola" exhibited 100% infection frequencies in the host species and populations and were separately harbored within different bacteriocytes that constituted a pair of coherent bacteriomes in the abdomen of the host insects, as in other deltocephaline leafhoppers. Wolbachia, Rickettsia, Burkholderia, Diplorickettsia, and the novel Rickettsiaceae bacterium exhibited infection frequencies at 7%, 31%, 12%, 0%, and 24% in M. striifrons and at 20%, 0%, 0%, 20%, and 0% in M. sexnotatus, respectively. Although undetected in the above analyses, phytoplasma infections were detected in 16% of M. striifrons and 60% of M. sexnotatus insects by nested PCR of 16S rRNA genes. Two genetically distinct phytoplasmas, namely, "Candidatus Phytoplasma asteris," associated with aster yellows and related plant diseases, and "Candidatus Phytoplasma oryzae," associated with rice yellow dwarf disease, were identified from the leafhoppers. These results highlight strikingly complex endosymbiotic microbiota of the Macrosteles leafhoppers and suggest ecological interactions between the obligate endosymbionts, the facultative endosymbionts, and the phytopathogenic phytoplasmas within the same host insects, which may affect vector competence of the leafhoppers.

Novel clade of alphaproteobacterial endosymbionts associated with stinkbugs and other arthropods.

Here we report a novel clade of secondary endosymbionts associated with insects and other arthropods. Seed bugs of the genus Nysius (Hemiptera: Lygaeidae) harbor the primary gammaproteobacterial symbiont Schneideria nysicola within a pair of bacteriomes in the abdomen. Our survey of Nysius species for their facultative bacterial associates consistently yielded a novel type of alphaproteobacterial 16S rRNA gene sequence in addition to those of Wolbachia. Diagnostic PCR survey of 343 individuals representing 24 populations of four Nysius species revealed overall detection rates of the alphaproteobacteria at 77.6% in Nysius plebeius, 87.7% in Nysius sp. 1, 81.0% in Nysius sp. 2, and 100% in Nysius expressus. Further survey of diverse stinkbugs representing 24 families, 191 species, and 582 individuals detected the alphaproteobacteria from an additional 12 species representing six families. Molecular phylogenetic analysis showed that the alphaproteobacteria from the stinkbugs form a distinct and coherent monophyletic group in the order Rickettsiales together with several uncharacterized endosymbionts from fleas and ticks. The alphaproteobacterial symbiont clade was allied to bacterial clades such as the endosymbionts of acanthamoebae, the endosymbionts of cnidarians, and Midichloria spp., the mitochondrion-associated endosymbionts of ticks. In situ hybridization and electron microscopy identified small filamentous bacterial cells in various tissues of N. plebeius, including the bacteriome and ovary. The concentrated localization of the symbiont cells at the anterior pole of oocytes indicated its vertical transmission route through host insect generations. The designation "Candidatus Lariskella arthropodarum" is proposed for the endosymbiont clade.

RNAi of the circadian clock gene period disrupts the circadian rhythm but not the circatidal rhythm in the mangrove cricket.

The clock mechanism for circatidal rhythm has long been controversial, and its molecular basis is completely unknown. The mangrove cricket, Apteronemobius asahinai, shows two rhythms simultaneously in its locomotor activity: a circatidal rhythm producing active and inactive phases as well as a circadian rhythm modifying the activity intensity of circatidal active phases. The role of the clock gene period (per), one of the key components of the circadian clock in insects, was investigated in the circadian and circatidal rhythms of A. asahinai using RNAi. After injection of double-stranded RNA of per, most crickets did not show the circadian modulation of activity but the circatidal rhythm persisted without a significant difference in the period from controls. Thus, per is functionally involved in the circadian rhythm but plays no role, or a less important role, in the circatidal rhythm. We conclude that the circatidal rhythm in A. asahinai is controlled by a circatidal clock whose molecular mechanism is different from that of the circadian clock.

Symbiont coordinates stem cell proliferation, apoptosis, and morphogenesis of gut symbiotic organ in the stinkbug-Caballeronia symbiosis.

The bean bug Riptortus pedestris obtains a specific bacterial symbiont, Caballeronia insecticola (Burkholderia insecticola), from the environmental soil and harbors it in the posterior midgut region that is composed of hundreds of crypts. While newly hatched aposymbiotic insects possess primordial midgut crypts with little or no lumen, colonization of C. insecticola triggers swift development of the symbiotic organ, forming enlarged and opened crypts, and the symbiont subsequently fills the luminal cavities of those mature crypts. The cellular processes of crypt development triggered by C. insecticola colonization are poorly understood. Here we identified a fundamental mechanism of the symbiont-mediated midgut development by investigating cell cycles of intestinal epithelial cells. Intestinal stem cells of the bean bug are located and proliferate at the crypt base. Differentiated enterocytes migrate upward along the epithelial cell layer of the crypt as the midgut develops, induction of apoptosis in enterocytes primarily occurred on the tip side of the crypts, and apoptotic cells then eventually were shed from the crypts into the hemolymph. The proliferation rate of the stem cells at the base of the crypts was low while a high apoptotic rate was observed at the crypt tip in aposymbiotic insects, resulting in undeveloped short crypts. On the contrary, the gut-colonizing C. insecticola promoted the proliferation of the stem cells at the base of crypts and simultaneously inhibited apoptosis at the tip of crypts, resulting in a net growth of the crypts and the generation of a crypt lumen that becomes colonized by the bacterial symbiont. These results demonstrated that the Caballeronia symbiont colonization induces the development of the midgut crypts via finely regulating the enterocyte cell cycles, enabling it to stably and abundantly colonize the generated spacious crypts of the bean bug host.

Displaced humeral head after intramedullary nailing for proximal humeral fracture is associated with worse short-term outcomes-a multicenter TRON study.

Background: In recent years, complex and unstable proximal humeral fractures (PHFs) are treated with intramedullary nails (IMNs) in the elderly; however, the postoperative radiographic findings related to the clinical outcome are not clear. This study evaluated the association of clinical outcomes with the radiographic findings of PHFs treated with IMNs. Methods: We collected data of patients aged >60 years with PHFs treated with IMNs from 2015 to 2019 in 13 associated centers' database (named TRON). We excluded patients lost to follow-up of <6 months postoperatively (PO6M). We evaluated clinical outcomes with the University of California at Los Angeles (UCLA) score at PO6M and defined a score of <27 as poor. We assessed the radiographic findings on the anteroposterior view of the humeral head postoperatively, and each radiographic finding such as humeral head height (HHH), head shaft angle, and cranialization of the greater tuberosity was divided into two groups: poor and good. Factors associated with poor UCLA at PO6M were extracted by logistic regression analysis, and the factors were divided into two groups (poor and good) and matched for age, sex, and fracture type. The UCLA score at PO6M between the groups was examined by the Mann-Whitney U test, and the significance level was set at 0.05. The minimal clinical important difference in the UCLA score was set 2 points. Results: The study included 243 patients (mean age, 76 years; range, 60-95 years). The mean follow-up period was 12 months (range, 6-56 months). The correlation coefficients indicated that there was either no or only a weak correlation between HHH, head shaft angle, and cranialization of the greater tuberosity. A poor HHH (HHH <0 or >10 mm) was extracted as a factor associated with a poor UCLA score at PO6M by logistic regression analysis (odds ratio: 5.78, 95% confidence interval = 1.2-27.7, P = .0287). In matched pair analysis, the UCLA score at PO6M was significantly lower in the poor HHH group (26 [range: 9-33] vs. 24 [range: 10-35], P = .0458). Conclusion: We revealed that the HHH was an independent risk factor for poor short-term outcomes. There was a significant difference in the UCLA score between groups divided by the HHH in cases treated with IMNs. The HHH can be used intraoperatively or postoperatively as a reliable parameter to predict clinical outcomes in PHFs treated with IMNs.

Ophiocordyceps salganeicola, a parasite of social cockroaches in Japan and insights into the evolution of other closely-related Blattodea-associated lineages.

The entomopathogenic genus Ophiocordyceps includes a highly diverse group of fungal species, predominantly parasitizing insects in the orders Coleoptera, Hemiptera, Hymenoptera and Lepidoptera. However, other insect orders are also parasitized by these fungi, for example the Blattodea (termites and cockroaches). Despite their ubiquity in nearly all environments insects occur, blattodeans are rarely found infected by filamentous fungi and thus, their ecology and evolutionary history remain obscure. In this study, we propose a new species of Ophiocordyceps infecting the social cockroaches Salganea esakii and S. taiwanensis, based on 16 years of collections and field observations in Japan, especially in the Ryukyu Archipelago. We found a high degree of genetic similarity between specimens from different islands, infecting these two Salganea species and that this relationship is ancient, likely not originating from a recent host jump. Furthermore, we found that Ophiocordyceps lineages infecting cockroaches evolved around the same time, at least twice, one from beetles and the other from termites. We have also investigated the evolutionary relationships between Ophiocordyceps and termites and present the phylogenetic placement of O. cf. blattae. Our analyses also show that O. sinensis could have originated from an ancestor infecting termite, instead of beetle larvae as previously proposed.

Worker-dependent gut symbiosis in an ant.

The hallmark of eusocial insects, honeybees, ants, and termites, is division of labor between reproductive and non-reproductive worker castes. In addition, environmental adaption and ecological dominance are also underpinned by symbiotic associations with beneficial microorganisms. Microbial symbionts are generally considered to be maintained in an insect colony in two alternative ways: shared among all colony members or inherited only by a specific caste. Especially in ants, the reproductive caste plays a crucial role in transmission of the symbionts shared among colony members over generations. Here, we report an exceptional case, the worker-dependent microbiota in an ant, Diacamma cf. indicum from Japan. By collecting almost all the individuals from 22 colonies in the field, we revealed that microbiota of workers is characterized by a single dominant bacterium localized at the hindgut. The bacterium belonging to an unclassified member within the phylum Firmicutes, which is scarce or mostly absent in the reproductive castes. Furthermore, we show that the gut symbiont is acquired at the adult stage. Collectively, our findings strongly suggest that the specific symbiont is maintained by only workers, demonstrating a novel pattern of ant-associated bacterial symbiosis, and thus further our understanding of host-microbe interactions in the light of sociobiology.

A Peptidoglycan Amidase Mutant of Burkholderia insecticola Adapts an L-form-like Shape in the Gut Symbiotic Organ of the Bean Bug Riptortus pedestris.

Bacterial cell shapes may be altered by the cell cycle, nutrient availability, environmental stress, and interactions with other organisms. The bean bug Riptortus pedestris possesses a symbiotic bacterium, Burkholderia insecticola, in its midgut crypts. This symbiont is a typical rod-shaped bacterium under in vitro culture conditions, but changes to a spherical shape inside the gut symbiotic organ of the host insect, suggesting the induction of morphological alterations in B. insecticola by host factors. The present study revealed that a deletion mutant of a peptidoglycan amidase gene (amiC), showing a filamentous chain form in vitro, adapted a swollen L-form-like cell shape in midgut crypts. Spatiotemporal observations of the ΔamiC mutant in midgut crypts revealed the induction of swollen cells, particularly prior to the molting of insects. To elucidate the mechanisms underlying in vivo-specific morphological alterations, the symbiont was cultured under 13 different conditions and its cell shape was examined. Swollen cells, similar to symbiont cells in midgut crypts, were induced when the mutant was treated with fosfomycin, an inhibitor of peptidoglycan precursor biosynthesis. Collectively, these results strongly suggest that the Burkholderia symbiont in midgut crypts is under the control of the host insect via a cell wall-attacking agent.

Huge symbiotic organs in giant scale insects of the genus Drosicha (Coccoidea: Monophlebidae) harbor flavobacterial and enterobacterial endosymbionts.

Giant scale insects (Drosicha: Coccoldea: Monophlebidae) were investigated for their symbiotic organs and bacterial endosymbionts. Two types of bacterial 16S rRNA gene sequences, flavobacterial and enterobacterial, were consistently detected in D. corpulenta and D. pinicola. The former sequences formed a compact clade in the Bacteroidetes, allied to the symbionts of cushion and armored scales. The latter sequences formed a robust clade in the gamma-Proteobacteria, allied to enteric bacteria like Enterobacter aerogenes and Escherichia coli. Another type of 16S sequence derived from Wolbachia was also detected in D. pinicola. In-situ hybridization demonstrated that the flavobacterial and enterobacterial symbionts were localized in a pair of huge bacteriomes in the abdomen, the former in uninucleated peripheral bacteriocytes and the latter in syncytial central bacteriocytes. Electron microscopy confirmed the endocellular locations of the pleomorphic flavobacterial symbiont and the rod-shaped enterobacterial symbiont, and also revealed the location and fine structure of the Wolbachia symbiont in D. pinicola. Infection frequencies of the flavobacterial and enterobacterial symbionts were consistently 100% in populations of D. corpulenta and D. pinicola, while the Wolbachia symbiont exhibited 0% and 100% infection frequencies in D. corpulente and D. pinicola, respectively. Neither the flavobacterial symbiont nor the enterobacterial symbiont exhibited AT-biased nucleotide composition or accelerated molecular evolution. The huge bacteriomes of Drosicha giant scales would provide a useful system for investigating biochemical, physiological, and genomic aspects of the host-symbiont and symbiont-symbiont interactions.

Impact of initial plasma presepsin level for clinical outcome in hospitalized patients with pneumonia.

BACKGROUND: Presepsin, the soluble CD14 subtype, is known as a sepsis biomarker. However, its clinical significance in pneumonia is unclear. We investigated the effects of plasma presepsin level on clinical outcomes in patients with pneumonia. METHODS: Patients over 18 years old admitted to our hospital due to pneumonia from May 2016 through November 2017 were reviewed using electronic medical records. One hundred and seventy-two patients who underwent measurement of plasma presepsin levels on admission were enrolled. Median age of enrolled patients was 81 years [interquartile range (IQR), 68-86 years]. Pneumonia severity index (PSI) class and A-DROP score on admission were calculated. The receiver operating characteristic (ROC) curve analysis was performed to assess the prognostic value of 30-day mortality and to identify the optimal cut-off value of plasma presepsin level. Correlations between plasma presepsin level and other factors were assessed using the Spearman's test. The Kaplan-Meier survival analysis and the log-rank test were performed to assess the two curves differentiated with the optimal cut-off value of plasma presepsin level. RESULTS: Seventeen patients (9.9%) died within 30 days of admission. The deceased patients had higher value of plasma presepsin on admission (539 pg/mL; IQR, 414-832 pg/mL) compared with the survivors (334 pg/mL; IQR, 223-484 pg/mL) (P=0.001). The areas under ROC curve for predicting 30-day mortality were 0.742 for plasma presepsin, 0.755 for A-DROP score, and 0.774 for PSI class. Plasma presepsin level was not associated with etiology of pneumonia. However, it was moderately correlated with serum creatinine level (rs =0.524, P<0.001). The ROC curve analysis derived 470 pg/mL of plasma presepsin level as the optimal cut-off value for predicting 30-day mortality. The Kaplan-Meier survival analysis showed that patients with plasma presepsin level ≥470 pg/mL on admission had significantly higher 30-day mortality than those with plasma presepsin level <470 pg/mL (P<0.001). Among patients with A-DROP score ≥3, those with plasma presepsin level ≥470 mg on admission had significantly higher 30-day mortality (P=0.013). Similarly, among patients with PSI class ≥4, those with plasma presepsin level ≥470 mg on admission had significantly higher 30-day mortality (P=0.005). CONCLUSIONS: In hospitalized pneumonia patients, plasma presepsin level on admission could be a useful predictor of 30-day mortality and an additional prognostic biomarker on existing severity assessment scales.