Extracellular vesicles of Weizmannia coagulans lilac-01 reduced cell death of primary microglia and increased mitochondrial content in dermal fibroblasts in vitro.

We investigated the properties of extracellular vesicles from the probiotic Weizmannia coagulans lilac-01 (Lilac-01EVs). The phospholipids in the Lilac-01EV membrane were phosphatidylglycerol and mitochondria-specific cardiolipin. We found that applying Lilac-01EVs to primary rat microglia in vitro resulted in a reduction in primary microglial cell death (P < .05). Lilac-01EVs, which contain cardiolipin and phosphatidylglycerol, may have the potential to inhibit cell death in primary microglia. The addition of Lilac-01EVs to senescent human dermal fibroblasts suggested that Lilac-01 EVs increase the mitochondrial content without affecting their membrane potential in these cells.

Host's guardian protein counters degenerative symbiont evolution.

Microbial symbioses significantly contribute to diverse organisms, where long-lasting associations tend to result in symbiont genome erosion, uncultivability, extinction, and replacement. How such inherently deteriorating symbiosis can be harnessed to stable partnership is of general evolutionary interest. Here, we report the discovery of a host protein essential for sustaining symbiosis. Plataspid stinkbugs obligatorily host an uncultivable and genome-reduced gut symbiont, Ishikawaella Upon oviposition, females deposit "capsules" for symbiont delivery to offspring. Within the capsules, the fragile symbiotic bacteria survive the harsh conditions outside the host until acquired by newborn nymphs to establish vertical transmission. We identified a single protein dominating the capsule content, which is massively secreted by female-specific intestinal organs, embedding the symbiont cells, and packaged into the capsules. Knockdown of the protein resulted in symbiont degeneration, arrested capsule production, symbiont transmission failure, and retarded nymphal growth, unveiling its essential function for ensuring symbiont survival and vertical transmission. The protein originated from a lineage of odorant-binding protein-like multigene family, shedding light on the origin of evolutionary novelty regarding symbiosis. Experimental suppression of capsule production extended the female's lifespan, uncovering a substantial cost for maintaining symbiosis. In addition to the host's guardian protein, the symbiont's molecular chaperone, GroEL, was overproduced in the capsules, highlighting that the symbiont's eroding functionality is compensated for by stabilizer molecules of host and symbiont origins. Our finding provides insight into how intimate host-symbiont associations can be maintained over evolutionary time despite the symbiont's potential vulnerability to degeneration and malfunctioning.

Granulimonas faecalis gen. nov., sp. nov., and Leptogranulimonas caecicola gen. nov., sp. nov., novel lactate-producing Atopobiaceae bacteria isolated from mouse intestines, and an emended description of the family Atopobiaceae.

Two strictly anaerobic, Gram-stain-positive, non-motile bacteria (strains OPF53T and TOC12T) were isolated from mouse intestines. Strains OPF53T and TOC12T grew at pH 5.5-9.0 and 5.0-9.0, respectively, and at temperatures of 30-45 °C. The cell morphologies of these strains were short rods and rods, respectively, and the cells possessed intracellular granules. The major cellular fatty acids of OPF53T were C18  :  1 cis 9 and C18  :  1 cis 9 dimethyl acetal, whereas those of TOC12T were C18  :  0 and C18  :  1 cis 9. In OPF53T, the main end-products of modified peptone-yeast extract-glucose (PYG) fermentation were lactate, formate and butyrate, whereas, in addition to these acids, TOC12T also produced hydrogen. The genomes of OPF53T and TOC12T were respectively 2.2 and 2.0 Mbp in size with a DNA G+C contents of 69.1 and 58.7 %. The 16S rRNA gene sequences of OPF53T and TOC12T showed the highest similarity to members of the family Atopobiaceae, namely, Olsenella phocaeensis Marseille-P2936T (94.3 %) and Olsenella umbonata KCTC 15140T (93.2 %), respectively. Phylogenetic analyses revealed that both isolates formed distinct lineages from other genera of the family Atopobiaceae. In addition, the two strains were characterized by relatively low 16S rRNA gene sequence similarity (93.4 %) and can be distinguished by their distinctive traits (including cell shape, DNA G+C content, and major fatty acids profiles). On the basis of their polyphasic taxonomic properties, these isolates represent two noel species of two novel genera within the family Atopobiaceae, for which the names Granulimonas faecalis gen. nov., sp. nov. (OPF53T=JCM 35015T=KCTC 25474T) and Leptogranulimonas caecicola gen. nov., sp. nov. (TOC12T=JCM 35017T=KCTC 25472T) are proposed.

Host-symbiont specificity determined by microbe-microbe competition in an insect gut.

Despite the omnipresence of specific host-symbiont associations with acquisition of the microbial symbiont from the environment, little is known about how the specificity of the interaction evolved and is maintained. The bean bug Riptortus pedestris acquires a specific bacterial symbiont of the genus Burkholderia from environmental soil and harbors it in midgut crypts. The genus Burkholderia consists of over 100 species, showing ecologically diverse lifestyles, and including serious human pathogens, plant pathogens, and nodule-forming plant mutualists, as well as insect mutualists. Through infection tests of 34 Burkholderia species and 18 taxonomically diverse bacterial species, we demonstrate here that nonsymbiotic Burkholderia and even its outgroup Pandoraea could stably colonize the gut symbiotic organ and provide beneficial effects to the bean bug when inoculated on aposymbiotic hosts. However, coinoculation revealed that the native symbiont always outcompeted the nonnative bacteria inside the gut symbiotic organ, explaining the predominance of the native Burkholderia symbiont in natural bean bug populations. Hence, the abilities for colonization and cooperation, usually thought of as specific traits of mutualists, are not unique to the native Burkholderia symbiont but, to the contrary, competitiveness inside the gut is a derived trait of the native symbiont lineage only and was thus critical in the evolution of the insect gut symbiont.

Exaggeration and cooption of innate immunity for social defense.

Social insects often exhibit striking altruistic behaviors, of which the most spectacular ones may be self-destructive defensive behaviors called autothysis, "self-explosion," or "suicidal bombing." In the social aphid Nipponaphis monzeni, when enemies damage their plant-made nest called the gall, soldier nymphs erupt to discharge a large amount of body fluid, mix the secretion with their legs, and skillfully plaster it over the plant injury. Dozens of soldiers come out, erupt, mix, and plaster, and the gall breach is promptly sealed with the coagulated body fluid. What molecular and cellular mechanisms underlie the self-sacrificing nest repair with body fluid for the insect society? Here we demonstrate that the body cavity of soldier nymphs is full of highly differentiated large hemocytes that contain huge amounts of lipid droplets and phenoloxidase (PO), whereas their hemolymph accumulates huge amounts of tyrosine and a unique repeat-containing protein (RCP). Upon breakage of the gall, soldiers gather around the breach and massively discharge the body fluid. The large hemocytes rupture and release lipid droplets, which promptly form a lipidic clot, and, concurrently, activated PO converts tyrosine to reactive quinones, which cross-link RCP and other macromolecules to physically reinforce the clot to seal the gall breach. Here, soldiers' humoral and cellular immune mechanisms for wound sealing are extremely up-regulated and utilized for colony defense, which provides a striking case of direct evolutionary connection between individual immunity and social immunity and highlights the importance of exaggeration and cooption of preexisting traits to create evolutionary novelties.

Environmental Acquisition of Gut Symbiotic Bacteria in the Saw-Toothed Stinkbug, Megymenum gracilicorne (Hemiptera: Pentatomoidea: Dinidoridae).

Many plant-sucking stinkbugs possess a specialized symbiotic organ with numerous crypts in a posterior region of the midgut. In stinkbugs of the superfamily Pentatomoidea, specific γ-proteobacteria are hosted in the crypt cavities, which are vertically transmitted through host generations and essential for normal growth and survival of the host insects. Here we report the discovery of an exceptional gut symbiotic association in the saw-toothed stinkbug, Megymenum gracilicorne (Hemiptera: Pentatomoidea: Dinidoridae), in which specific γ-proteobacterial symbionts are not transmitted vertically but acquired environmentally. Histological inspection identified a very thin and long midgut symbiotic organ with two rows of tiny crypts whose cavities harbor rod-shaped bacterial cells. Molecular phylogenetic analyses of bacterial 16S rRNA gene sequences from the symbiotic organs of field-collected insects revealed that (i) M. gracilicorne is stably associated with Pantoea-allied γ-proteobacteria within the midgut crypts, (ii) the symbiotic bacteria exhibit a considerable level of diversity across host individuals and populations, (iii) the major symbiotic bacteria represent an environmental bacterial lineage that was reported to be capable of symbiosis with the stinkbug Plautia stali, and (iv) the minor symbiotic bacteria also represent several bacterial lineages that were reported as cultivable symbionts of P. stali and other stinkbugs. The symbiotic bacteria were shown to be generally cultivable. Microbial inspection of ovipositing adult females and their eggs and nymphs uncovered the absence of stable vertical transmission of the symbiotic bacteria. Rearing experiments showed that symbiont-supplemented newborn nymphs exhibit improved survival, suggesting the beneficial nature of the symbiotic association.

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.

Bacteriocytes and Blattabacterium Endosymbionts of the German Cockroach Blattella germanica, the Forest Cockroach Blattella nipponica, and Other Cockroach Species.

Cockroaches are commonly found in human residences and notorious as hygienic and nuisance pests. Notably, however, no more than 30 cockroach species are regarded as pests, while the majority of 4,500 cockroaches in the world are living in forest environments with little relevance to human life. Why some cockroaches have exceptionally adapted to anthropic environments and established pest status is of interest. Here we investigated the German cockroach Blattella germanica, which is a cosmopolitan pest species, and the forest cockroach Blattella nipponica, which is a wild species closely related to B. germanica. In contrast to easy rearing of B. germanica, laboratory rearing of B. nipponica was challenging-several trials enabled us to keep the insects for up to three months. We particularly focused on the distribution patterns of specialized cells, bacteriocytes, for harboring endosymbiotic Blattabacterium, which has been suggested to contribute to host's nitrogen metabolism and recycling, during the postembryonic development of the insects. The bacteriocytes were consistently localized to visceral fat bodies filling the abdominal body cavity, where a number of single bacteriocytes were scattered among the adipocytes, throughout the developmental stages in both females and males. The distribution patterns of the bacteriocytes were quite similar between B. germanica and B. nipponica, and also among other diverse cockroach species, plausibly reflecting the highly conserved cockroach-Blattabacterium symbiotic association over evolutionary time. Our study lays a foundation to experimentally investigate the origin and the processes of urban pest evolution, on account of possible involvement of microbial associates.

Small genome symbiont underlies cuticle hardness in beetles.

Beetles, representing the majority of the insect species diversity, are characterized by thick and hard cuticle, which plays important roles for their environmental adaptation and underpins their inordinate diversity and prosperity. Here, we report a bacterial endosymbiont extremely specialized for sustaining beetle's cuticle formation. Many weevils are associated with a γ-proteobacterial endosymbiont lineage Nardonella, whose evolutionary origin is estimated as older than 100 million years, but its functional aspect has been elusive. Sequencing of Nardonella genomes from diverse weevils unveiled drastic size reduction to 0.2 Mb, in which minimal complete gene sets for bacterial replication, transcription, and translation were present but almost all of the other metabolic pathway genes were missing. Notably, the only metabolic pathway retained in the Nardonella genomes was the tyrosine synthesis pathway, identifying tyrosine provisioning as Nardonella's sole biological role. Weevils are armored with hard cuticle, tyrosine is the principal precursor for cuticle formation, and experimental suppression of Nardonella resulted in emergence of reddish and soft weevils with low tyrosine titer, confirming the importance of Nardonella-mediated tyrosine production for host's cuticle formation and hardening. Notably, Nardonella's tyrosine synthesis pathway was incomplete, lacking the final step transaminase gene. RNA sequencing identified host's aminotransferase genes up-regulated in the bacteriome. RNA interference targeting the aminotransferase genes induced reddish and soft weevils with low tyrosine titer, verifying host's final step regulation of the tyrosine synthesis pathway. Our finding highlights an impressively intimate and focused aspect of the host-symbiont metabolic integrity via streamlined evolution for a single biological function of ecological relevance.

Burkholderia insecticola sp. nov., a gut symbiotic bacterium of the bean bug Riptortus pedestris.

A Gram-negative, aerobic, rod-shaped, non-spore-forming, motile bacterium, designated strain RPE64T, was isolated from the gut symbiotic organ of the bean bug Riptortus pedestris, collected in Tsukuba, Japan, in 2007. 16S rRNA gene sequencing showed that this strain belongs to the Burkholderia glathei clade, exhibiting the highest sequence similarity to Burkholderia peredens LMG 29314T (100 %), Burkholderia turbans LMG 29316T (99.52 %) and Burkholderia ptereochthonis LMG 29326T (99.04 %). Phylogenomic analyses based on 107 single-copy core genes and Genome blast Distance Phylogeny confirmed B. peredens LMG 29314T, B. ptereochthonis LMG 29326T and several uncultivated, endophytic Burkholderia species as its nearest phylogenetic neighbours. Digital DNA-DNA hybridization experiments unambiguously demonstrated that strain RPE64T represents a novel species in this lineage. The G+C content of its genome was 63.2 mol%. The isoprenoid quinone was ubiquinone 8 and the predominant fatty acid components were C16 : 0, C18 : 1ω7c and C17 : 0 cyclo. The absence of nitrate reduction and the capacity to grow at pH 8 clearly differentiated strain RPE64T from related Burkholderia species. Based on these genotypic and phenotypic characteristics, strain RPE64T is classified as representing a novel species of the genus Burkholderia, for which the name Burkholderia insecticola sp. nov. is proposed. The type strain is RPE64T (=NCIMB 15023T=JCM 31142T).

Insect's intestinal organ for symbiont sorting.

Symbiosis has significantly contributed to organismal adaptation and diversification. For establishment and maintenance of such host-symbiont associations, host organisms must have evolved mechanisms for selective incorporation, accommodation, and maintenance of their specific microbial partners. Here we report the discovery of a previously unrecognized type of animal organ for symbiont sorting. In the bean bug Riptortus pedestris, the posterior midgut is morphologically differentiated for harboring specific symbiotic bacteria of a beneficial nature. The sorting organ lies in the middle of the intestine as a constricted region, which partitions the midgut into an anterior nonsymbiotic region and a posterior symbiotic region. Oral administration of GFP-labeled Burkholderia symbionts to nymphal stinkbugs showed that the symbionts pass through the constricted region and colonize the posterior midgut. However, administration of food colorings revealed that food fluid enters neither the constricted region nor the posterior midgut, indicating selective symbiont passage at the constricted region and functional isolation of the posterior midgut for symbiosis. Coadministration of the GFP-labeled symbiont and red fluorescent protein-labeled Escherichia coli unveiled selective passage of the symbiont and blockage of E. coli at the constricted region, demonstrating the organ's ability to discriminate the specific bacterial symbiont from nonsymbiotic bacteria. Transposon mutagenesis and screening revealed that symbiont mutants in flagella-related genes fail to pass through the constricted region, highlighting that both host's control and symbiont's motility are involved in the sorting process. The blocking of food flow at the constricted region is conserved among diverse stinkbug groups, suggesting the evolutionary origin of the intestinal organ in their common ancestor.

A Novel Symbiotic Ciliate (Ciliophora: Peritrichia) in the Hindgut of a Stag Beetle (Coleoptera: Lucanidae).

Bell-shaped ciliates of the subclass Peritrichia, such as Vorticella, Carchesium and Epistylis, are commonly found in freshwater and other aquatic environments, either solitary or colonial. Peritrichs attach to a substratum via a contractile or non-motile stalk, and collect food particles by water current using ciliary rows around the edge of the bell, called the peristome. Some peritrichs are epibiotic and ectocommensalistic associates of aquatic insects and other animals, settling on the surface of their specific hosts. Only a few peritrichs are known to establish a more internal association with their hosts, locating within the preoral cavity or esophagus of water beetles and presumably subsisting on food materials chewed and ingested by the insects. To date, no endoparasitic or endocommensalistic peritrichs have been reported from insects. Host insects reported to date have all been aquatic, and given the aquatic lifestyle of peritrichs, terrestrial hosts have been considered unlikely. In the present study, we report a dense population of bizarre microbes within the gut of a terrestrial insect, and histological, ultrastructural and molecular phylogenetic analyses identified it as a peritrich ciliate. The highly-developed hindgut of the stag beetle Aegus currani contained oval colonial peritrichs connected by branched stalks resembling grape clusters. Each zooid exhibited a reduced peristome without disc, a vestibulum with active ciliary movement inside, and an elongated macronucleus. These features are morphologically reminiscent of but distinct in some respects from those in Operculariella parasitica, known from the esophagus of dysticid diving beetles. Taxonomic, ecological and functional aspects of this gut-dwelling peritrich warrant future study.

Isolation and characterization of Flexilinea flocculi gen. nov., sp. nov., a filamentous, anaerobic bacterium belonging to the class Anaerolineae in the phylum Chloroflexi.

A novel obligately anaerobic bacterium, designated strain TC1T, was isolated from methanogenic granular sludge in a full-scale mesophilic upflow anaerobic sludge blanket reactor treating high-strength starch-based wastewater. Cells had a multicellular filamentous morphology, stained Gram-negative and were non-motile. The filaments were flexible, generally >100 µm long and 0.3-0.4 µm wide. Growth of the isolate was observed at 25-43 °C (optimum 37 °C) and pH 6.0-8.5 (optimum pH 7.0). Strain TC1T grew chemo-organotrophically on a range of carbohydrates under anaerobic conditions. Yeast extract was required for growth. The major fermentative end products of glucose, supplemented with yeast extract, were acetate, lactate, succinate, propionate, formate and hydrogen. Co-cultivation with the hydrogenotrophic methanogen Methanospirillum hungatei DSM 864T enhanced growth of the isolate. The DNA G+C content was determined experimentally to be 42.1 mol%. The major cellular fatty acids were anteiso-C15 : 0, iso-C15 : 0 and iso-C17 : 0 3-OH. Based on 16S rRNA gene sequence analysis, strain TC1T belonged to the class Anaerolineae in the phylum Chloroflexi, in which Ornatilinea apprima P3M-1T was its closest phylogenetic relative (88.3 % nucleotide identity). Phylogenomic analyses using 38 and 83 single-copy marker genes also supported the novelty of strain TC1T at least at the genus level. Based on phylogenetic, genomic and phenotypic characteristics, we propose that strain TC1T represents a novel species of a new genus, for which we suggest the name Flexilinea flocculi gen. nov., sp. nov. The type strain of Flexilinea flocculi is strain TC1T ( = JCM 30897T = CGMCC 1.5202T).

Lentimicrobium saccharophilum gen. nov., sp. nov., a strictly anaerobic bacterium representing a new family in the phylum Bacteroidetes, and proposal of Lentimicrobiaceae fam. nov.

A novel, strictly anaerobic, short rod-shaped bacterium, designated strain TBC1T, was isolated from methanogenic granular sludge in a full-scale mesophilic upflow anaerobic sludge blanket reactor treating high-strength starch-based organic wastewater. Cells of this strain were 2-4 µm long and 0.4-0.6 µm wide. They were non-motile and Gram-stain-negative. The optimum growth temperature was 30-37 °C, with a range of 20-40 °C. The optimum pH for growth was around pH 7.0, while growth occurred in a range of pH 6.5-9.0. Strain TBC1T grew chemo-organotrophically on a narrow range of carbohydrates under anaerobic conditions. Yeast extract was required for its growth. The major fermentative end products from glucose, supplemented with yeast extract, were acetate, malate, propionate, formate and hydrogen. Doubling time under optimal growth conditions was estimated to be 1 day. The DNA G+C content of strain TBC1T was 49.2 mol% as determined by HPLC. Major cellular fatty acids were C16 : 0, C18 : 0, C16 : 1ω9c and C18 : 1ω9c. Based on its 16S rRNA gene sequence, strain TBC1T was shown to represent a distinct lineage at the family level in the phylum Bacteroidetes. Among previously described species of this phylum, Mucilaginibacter boryungensis BDR-9T (Sphingobacteriaceae) displayed the highest sequence similarity (85.9 %) with strain TBC1T. Phylogenomic analyses using 38-83 single copy marker genes also supported the novelty of strain TBC1T at the family level. Based on its characteristics, strain TBC1T (=JCM 30898T=DSM 100618T) is considered to be the type strain of a novel species of a new genus, Lentimicrobium saccharophilum gen. nov., sp. nov. A new family, Lentimicrobiaceae fam. nov., is also proposed encompassing the strain and related environmental 16S rRNA gene clone sequences.

Clinical Analysis of Familial Nonmedullary Thyroid Carcinoma.

OBJECTIVE: To analyze the clinical characteristics of familial nonmedullary thyroid carcinoma (FNMTC), in order to provide evidence for early diagnosis and treatment. METHODS: We retrospectively investigated the inpatients between September 2006 and September 2013 in the First Bethune Hospital of Jilin University, in which 78 patients with FNMTC from 31 families were analyzed by a comparison with 3445 control cases from the patients with sporadic nonmedullary thyroid carcinoma (SNMTC). RESULTS: There was no significant difference in gender, age, and tumor size between FNMTC and SNMTC patients. However, the characteristics of disease in multifoci, neck lymph node metastasis, invasion to the surrounding tissues, and coexistence with Hashimoto disease in two types of cancer patients show significant difference. They are: multifoci: 71.8% (56/78) in FNMTC versus 46.3% (1595/3445) in SNMTC; neck lymph node metastasis: 52.6% (41/78) in FNMTC versus 33.3% (1148/3445) in SNMTC; surrounding tissue invasion: 64.1% (50/78) in FNMTC versus 48.5% (1670/3445) in SNMTC; coexistence with Hashimoto disease: 30.8% (24/78) in FNMTC versus 20.0% (689/3445) in SNMTC. CONCLUSION: Lymph node metastasis, multifoci, invasion to the surrounding tissues, and combination with chronic lymphocytic thyroiditis are the main features of FNMTC, which suggests the extent of the operation for FNMTC patients should be amplified properly.

Female-specific specialization of a posterior end region of the midgut symbiotic organ in Plautia splendens and allied stinkbugs.

Many stinkbugs (Insecta: Hemiptera: Heteroptera) are associated with bacterial symbionts in a posterior region of the midgut. In these stinkbugs, adult females excrete symbiont-containing materials from the anus for transmission of the beneficial symbionts to their offspring. For ensuring the vertical symbiont transmission, a variety of female-specific elaborate traits at the cellular, morphological, developmental, and behavioral levels have been reported from diverse stinkbugs of the families Plataspidae, Urostylididae, Parastrachiidae, etc. Meanwhile, such elaborate female-specific traits for vertical symbiont transmission have been poorly characterized for the largest and economically important stinkbug family Pentatomidae. Here, we investigated the midgut symbiotic system of a pentatomid stinkbug, Plautia splendens. A specific gammaproteobacterial symbiont was consistently present extracellularly in the cavity of numerous crypts arranged in four rows on the midgut fourth section. The symbiont was smeared on the egg surface upon oviposition by adult females, orally acquired by newborn nymphs, and thereby transmitted vertically to the next generation and important for growth and survival of the host insects. We found that, specifically in adult females, several rows of crypts at the posterior end region of the symbiotic midgut were morphologically differentiated and conspicuously enlarged, often discharging the symbiotic bacteria from the crypt cavity to the main tract of the symbiotic midgut. The female-specific enlarged end crypts were also found in other pentatomid stinkbugs Plautia stali and Carbula crassiventris. These results suggest that the enlarged end crypts represent a female-specific specialized morphological trait for vertical symbiont transmission commonly found among stinkbugs of the family Pentatomidae.

Cellular mechanism for selective vertical transmission of an obligate insect symbiont at the bacteriocyte-embryo interface.

Many insects are associated with obligate symbiotic bacteria, which are localized in specialized cells called bacteriocytes, vertically transmitted through host generations via ovarial passage, and essential for growth and reproduction of their hosts. Although vertical transmission is pivotal for maintenance of such intimate host-symbiont associations, molecular and cellular mechanisms underlying the process are largely unknown. Here we report a cellular mechanism for vertical transmission of the obligate symbiont Buchnera in the pea aphid Acyrthosiphon pisum. In the aphid body, Buchnera cells are transmitted from maternal bacteriocytes to adjacent blastulae at the ovariole tips in a highly coordinated manner. By making use of symbiont-manipulated strains of A. pisum, we demonstrated that the facultative symbiont Serratia is, unlike Buchnera, not transmitted from maternal bacteriocytes to blastulae, suggesting a specific mechanism for Buchnera transmission. EM observations revealed a series of exo-/endocytotic processes operating at the bacteriocyte-blastula interface: Buchnera cells are exocytosed from the maternal bacteriocyte, temporarily released to the extracellular space, and endocytosed by the posterior syncytial cytoplasm of the blastula. These results suggest that the selective Buchnera transmission is likely attributable to Buchnera-specific exocytosis by the maternal bacteriocyte, whereas both Buchnera and Serratia are nonselectively incorporated by the endocytotic activity of the posterior region of the blastula. The sophisticated cellular mechanism for vertical transmission of Buchnera must have evolved to ensure the obligate host-symbiont association, whereas facultative symbionts like Serratia may coopt the endocytotic component of the mechanism for their entry into the host embryos.

Evidence of environmental and vertical transmission of Burkholderia symbionts in the oriental chinch bug, Cavelerius saccharivorus (Heteroptera: Blissidae).

The vertical transmission of symbiotic microorganisms is omnipresent in insects, while the evolutionary process remains totally unclear. The oriental chinch bug, Cavelerius saccharivorus (Heteroptera: Blissidae), is a serious sugarcane pest, in which symbiotic bacteria densely populate the lumen of the numerous tubule-like midgut crypts that the chinch bug develops. Cloning and sequence analyses of the 16S rRNA genes revealed that the crypts were dominated by a specific group of bacteria belonging to the genus Burkholderia of the Betaproteobacteria. The Burkholderia sequences were distributed into three distinct clades: the Burkholderia cepacia complex (BCC), the plant-associated beneficial and environmental (PBE) group, and the stinkbug-associated beneficial and environmental group (SBE). Diagnostic PCR revealed that only one of the three groups of Burkholderia was present in ∼89% of the chinch bug field populations tested, while infections with multiple Burkholderia groups within one insect were observed in only ∼10%. Deep sequencing of the 16S rRNA gene confirmed that the Burkholderia bacteria specifically colonized the crypts and were dominated by one of three Burkholderia groups. The lack of phylogenetic congruence between the symbiont and the host population strongly suggested host-symbiont promiscuity, which is probably caused by environmental acquisition of the symbionts by some hosts. Meanwhile, inspections of eggs and hatchlings by diagnostic PCR and egg surface sterilization demonstrated that almost 30% of the hatchlings vertically acquire symbiotic Burkholderia via symbiont-contaminated egg surfaces. The mixed strategy of symbiont transmission found in the oriental chinch bug might be an intermediate stage in evolution from environmental acquisition to strict vertical transmission in insects.

Granulicella cerasi sp. nov., an acidophilic bacterium isolated from cherry bark.

A novel acidobacterial strain, Sakura1(T), was isolated from pieces of cherry bark. Cells of strain Sakura1(T) were non-motile, rod-shaped and stained Gram-negative. This strictly aerobic isolate was mesophilic but was able to grow at temperatures as low as 10 °C. Colonies were pink due to production of carotenoids, and its pigmentation was more pronounced in cells grown at lower temperature. This strain had endoglucanase activity. The main respiratory quinone was menaquinone-8, and major cellular fatty acids were iso-C(15 : 0), C(16 : 1)ω7c and C(16 : 0). Phylogenetic analysis of 16S rRNA gene sequences revealed that the strain was closely related to species of the genus Granulicella in subdivision 1 of the phylum Acidobacteria. The closest relative was Granulicella paludicola OB1010(T) (97.1% 16S rRNA gene sequence similarity). While common characteristics were found among the isolate and species of the genus Granulicella, there were obvious differences between them such as their cell morphology, cellulolytic activity, and tolerance to low temperature and NaCl concentration. Based on phylogenetic and phenotypic findings, a novel species is proposed in the genus Granulicella with the name Granulicella cerasi sp. nov. The type strain is Sakura1(T) ( = NBRC 107139(T) = DSM 23641(T)).

Methanohalophilus levihalophilus sp. nov., a slightly halophilic, methylotrophic methanogen isolated from natural gas-bearing deep aquifers, and emended description of the genus Methanohalophilus.

A mesophilic, slightly halophilic, obligately methylotrophic, methanogenic archaeon, designated strain GTA13(T), was isolated from natural gas-bearing confined aquifers in the Minami-Kanto gas field, Japan. The cells were non-motile, slightly irregular cocci, 0.7-1.0 µm in diameter and occurred singly, in pairs or as small aggregates. The cells grew with tri- or dimethylamine but not with H2/CO2, formate, acetate, methanol or dimethyl sulphide. Vitamins, sodium and magnesium were required for growth. Optimal growth occurred at pH 7.0-7.5, 35 °C, 0.35-0.40 M NaCl and 15-50 mM MgCl2. The NaCl range for growth was 0.2-1.3 M. The DNA G+C content was 43.7 mol%. Strain GTA13(T) showed highest levels of 16S rRNA gene sequence similarity with Methanohalophilus portucalensis FDF-1(T) (96.4% sequence similarity) and Methanohalophilus halophilus DSM 3094(T) (96.0%). On the basis of physiological and phylogenetic features, strain GTA13(T) is considered to represent a novel species of the genus Methanohalophilus, for which the name Methanohalophilus levihalophilus sp. nov. is proposed. The type strain is GTA13(T) ( = NBRC 110099(T) = DSM 28452(T)). An emended description of the genus Methanohalophilus is also proposed.