scholarly journals Evidence of Environmental and Vertical Transmission of Burkholderia Symbionts in the Oriental Chinch Bug, Cavelerius saccharivorus (Heteroptera: Blissidae)

2014 ◽  
Vol 80 (19) ◽  
pp. 5974-5983 ◽  
Author(s):  
Hideomi Itoh ◽  
Manabu Aita ◽  
Atsushi Nagayama ◽  
Xian-Ying Meng ◽  
Yoichi Kamagata ◽  
...  
Keyword(s):  
16S Rrna ◽  
Vertical Transmission ◽  
Burkholderia Cepacia ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Diagnostic Pcr ◽  
Content Type ◽  
Chinch Bug ◽  
Cavelerius Saccharivorus

ABSTRACTThe 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 genusBurkholderiaof theBetaproteobacteria. TheBurkholderiasequences were distributed into three distinct clades: theBurkholderia cepaciacomplex (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 ofBurkholderiawas present in ∼89% of the chinch bug field populations tested, while infections with multipleBurkholderiagroups within one insect were observed in only ∼10%. Deep sequencing of the 16S rRNA gene confirmed that theBurkholderiabacteria specifically colonized the crypts and were dominated by one of threeBurkholderiagroups. 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 symbioticBurkholderiavia 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.

scholarly journals Mucilaginibacter koreensis sp. nov., isolated from leaf mould

2014 ◽  
Vol 64 (Pt_7) ◽  
pp. 2274-2279 ◽  
Author(s):  
Cheol Su Park ◽  
Kyudong Han ◽  
Tae-Young Ahn
Keyword(s):  
16S Rrna ◽  
Type Species ◽  
Sequence Similarity ◽  
Optimal Growth ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Strictly Aerobic ◽  
Content Type ◽  
Link Type

A Gram-staining-negative, strictly aerobic, rod-shaped, pale-pink pigmented bacterial strain, designated TF8T, was isolated from leaf mould in Cheonan, Republic of Korea. Its taxonomic position was determined through a polyphasic approach. Optimal growth occurred on R2A agar without NaCl supplementation, at 25–28 °C and at pH 6.0–7.0. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain TF8T belongs to the genus Mucilaginibacter in the family Sphingobacteriaceae . The sequence similarity between 16S rRNA genes of strain TF8T and the type strains of other species of the genus Mucilaginibacter ranged from 92.1 to 94.7 %. The closest relatives of strain TF8T were Mucilaginibacter lutimaris BR-3T (94.7 %), M. soli R9-65T (94.5 %), M. litoreus BR-18T (94.5 %), M. rigui WPCB133T (94.0 %) and M. daejeonensis Jip 10T (93.8 %). The major isoprenoid quinone was MK-7 and the major cellular fatty acids were iso-C15 : 0 (33.0 %), summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c; 24.8 %) and summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c; 13.0 %). The major polar lipids of TF8T were phosphatidylethanolamine and three unidentified aminophospholipids. The G+C content of the genomic DNA was 46.2 mol%. On the basis of the data presented here, strain TF8T is considered to represent a novel species of the genus Mucilaginibacter , for which the name Mucilaginibacter koreensis sp. nov. is proposed. The type strain is TF8T ( = KACC 17468T = JCM 19323T).

scholarly journals Detection of Putatively Thermophilic Anaerobic Methanotrophs in Diffuse Hydrothermal Vent Fluids

2012 ◽  
Vol 79 (3) ◽  
pp. 915-923 ◽  
Author(s):  
Alexander Y. Merkel ◽  
Julie A. Huber ◽  
Nikolay A. Chernyh ◽  
Elizaveta A. Bonch-Osmolovskaya ◽  
Alexander V. Lebedinsky
Keyword(s):  
16S Rrna ◽  
Deep Sea ◽  
Hydrothermal Vents ◽  
Optimal Growth ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Anaerobic Oxidation Of Methane ◽  
Phylogenetic Groups ◽  
Content Type

ABSTRACTThe anaerobic oxidation of methane (AOM) is carried out by a globally distributed group of uncultivatedEuryarchaeota, the anaerobic methanotrophic arachaea (ANME). In this work, we used G+C analysis of 16S rRNA genes to identify a putatively thermophilic ANME group and applied newly designed primers to study its distribution in low-temperature diffuse vent fluids from deep-sea hydrothermal vents. We found that the G+C content of the 16S rRNA genes (PGC) is significantly higher in the ANME-1GBa group than in other ANME groups. Based on the positive correlation between thePGCand optimal growth temperatures (Topt) of archaea, we hypothesize that the ANME-1GBa group is adapted to thrive at high temperatures. We designed specific 16S rRNA gene-targeted primers for the ANME-1 cluster to detect all phylogenetic groups within this cluster, including the deeply branching ANME-1GBa group. The primers were successfully tested bothin silicoand in experiments with sediment samples where ANME-1 phylotypes had previously been detected. The primers were further used to screen for the ANME-1 microorganisms in diffuse vent fluid samples from deep-sea hydrothermal vents in the Pacific Ocean, and sequences belonging to the ANME-1 cluster were detected in four individual vents. Phylotypes belonging to the ANME-1GBa group dominated in clone libraries from three of these vents. Our findings provide evidence of existence of a putatively extremely thermophilic group of methanotrophic archaea that occur in geographically and geologically distinct marine hydrothermal habitats.

scholarly journals Mycoplasma anserisalpingitidis sp. nov., isolated from European domestic geese (Anser anser domesticus) with reproductive pathology

2020 ◽  
Vol 70 (4) ◽  
pp. 2369-2381 ◽  
Author(s):  
Dmitriy V. Volokhov ◽  
Dénes Grózner ◽  
Miklós Gyuranecz ◽  
Naola Ferguson-Noel ◽  
Yamei Gao ◽  
...  
Keyword(s):  
16S Rrna ◽  
Type Strain ◽  
Type Species ◽  
Novel Species ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
23S Rrna ◽  
Rrna Gene ◽  
Content Type ◽  
Link Type

In 1983, Mycoplasma sp. strain 1220 was isolated in Hungary from the phallus lymph of a gander with phallus inflammation. Between 1983 and 2017, Mycoplasma sp. 1220 was also identified and isolated from the respiratory tract, liver, ovary, testis, peritoneum and cloaca of diseased geese in several countries. Seventeen studied strains produced acid from glucose and fructose but did not hydrolyse arginine or urea, and all grew under aerobic, microaerophilic and anaerobic conditions at 35 to 37 ˚C in either SP4 or pleuropneumonia-like organism medium supplemented with glucose and serum. Colonies on agar showed a typical fried-egg appearance and transmission electron microscopy revealed a typical mycoplasma cellular morphology. Molecular characterization included analysis of the following genetic loci: 16S rRNA, 23S rRNA, 16S–23S rRNA ITS, rpoB, rpoC, rpoD, uvrA, parC, topA, dnaE, fusA and pyk. The genome was sequenced for type strain 1220T. The 16S rRNA gene sequences of studied strains of Mycoplasma sp. 1220 shared 99.02–99.19 % nucleotide similarity with M. anatis strains but demonstrated ≤95.00–96.70 % nucleotide similarity to the 16S rRNA genes of other species of the genus Mycoplasma . Phylogenetic, average nucleotide and amino acid identity analyses revealed that the novel species was most closely related to Mycoplasma anatis . Based on the genetic data, we propose a novel species of the genus Mycoplasma , for which the name Mycoplasma anserisalpingitidis sp. nov. is proposed with the type strain 1220T (=ATCC BAA-2147T=NCTC 13513T=DSM 23982T). The G+C content is 26.70 mol%, genome size is 959110 bp.

scholarly journals Halomonas icarae sp. nov., a moderately halophilic bacterium isolated from beach soil in India

2020 ◽  
Author(s):  
Kuppusamy Pandiyan ◽  
Prity Kushwaha ◽  
Samadhan Y. Bagul ◽  
Hillol Chakdar ◽  
Munusamy Madhaiyan ◽  
...  
Keyword(s):  
16S Rrna ◽  
Type Species ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Aerobic Bacterium ◽  
Content Type ◽  
Moderately Halophilic ◽  
Link Type ◽  
Moderately Halophilic Bacterium

A moderately halophilic, Gram-stain-negative, aerobic bacterium, strain D1-1T, belonging to the genus Halomonas , was isolated from soil sampled at Pentha beach, Odisha, India. Phylogenetic trees reconstructed based on 16S rRNA genes and multilocus sequence analysis of gyrB and rpoD genes revealed that strain D1-1T belonged to the genus Halomonas and was most closely related to Halomonas alimentaria YKJ-16T (98.1 %) followed by Halomonas ventosae Al12T (97.5 %), Halomonas sediminicola CPS11T (97.5 %), Halomonas fontilapidosi 5CRT (97.4 %) and Halomonas halodenitrificans DSM 735T (97.2 %) on the basis of 16S rRNA gene sequence similarity. Sequence identities with other species within the genus were lower than 97.0 %. The digital DNA–DNA hybridization (dDDH) and average nucleotide identity (ANI) values of 22.4–30 % and 79.5–85.4 % with close relatives of H. halodenitrificans DSM 735T, H . alimentaria YKJ-16T, H. ventosae Al12T and H. fontilapidosi 5CRT were lower than the threshold recommended for species delineation (70 % and 95–96 % for dDDH and ANI, respectively). Further, strain D1-1T formed yellow-coloured colonies; cells were rod-shaped, motile with optimum growth at 30 °C (range, 4–45 °C) and 2–8 % NaCl (w/v; grew up to 24 % NaCl). The major fatty acids were summed feature 8 (C18 : 1  ω7c/C18 : 1  ω6c), summed feature 3 (C16 : 1  ω7c/C16 : 1  ω6c) and C16 : 0 and the main respiratory quinone was ubiquinone Q-9 in line with description of the genus. Based on its chemotaxonomic and phylogenetic characteristics and genome uniqueness, strain D1-1T represents a novel species in the genus Halomonas , for which we propose the name Halomonas icarae sp. nov., within the family Halomonadaceae . The type strain is D1-1T (=JCM 33602T=KACC 21317T=NAIMCC-B-2254T).

Phylogenomic analysis of Anabaenopsis elenkinii (Nostocales, Cyanobacteria)

2021 ◽  
Author(s):  
Endrews Delbaje ◽  
Ana Paula D. Andreote ◽  
Thierry A. Pellegrinetti ◽  
Renata B. Cruz ◽  
Luis H. Z. Branco ◽  
...  
Keyword(s):  
16S Rrna ◽  
Type Species ◽  
Soda Lakes ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Phylogenomic Analysis ◽  
Rrna Gene ◽  
Content Type ◽  
Link Type ◽  
The Family

The saline-alkaline lakes (soda lakes) are the habitat of the haloalkaliphilic cyanobacterium Anabaenopsis elenkinii, the type species of this genus. To obtain robust phylogeny of this type species, we have generated whole-genome sequencing of the bloom-forming Anabaenopsis elenkinii strain CCIBt3563 isolated from a Brazilian soda lake. This strain presents the typical morphology of A. elenkinii with short and curved trichomes with apical heterocytes established after separation of paired intercalary heterocytes and also regarding to cell dimensions. Its genome size is 4 495 068 bp, with a G+C content of 41.98 %, a total of 3932 potential protein coding genes and four 16S rRNA genes. Phylogenomic tree inferred by RAxML based on the alignment of 120 conserved proteins using GTDB-Tk grouped A. elenkinii CCIBt3563 together with other genera of the family Aphanizomenonaceae. However, the only previous available genome of Anabaenopsis circularis NIES-21 was distantly positioned within a clade of Desikacharya strains, a genus from the family Nostocaceae. Furthermore, average nucleotide identity values from 86–98 % were obtained among NIES-21 and Desikacharya genomes, while this value was 76.04 % between NIES-21 and the CCIBt3563 genome. These findings were also corroborated by the phylogenetic tree of 16S rRNA gene sequences, which also showed a strongly supported subcluster of A. elenkinii strains from Brazilian, Mexican and Kenyan soda lakes. This study presents the phylogenomics and genome-scale analyses of an Anabaenopsis elenkinii strain, improving molecular basis for demarcation of this species and framework for the classification of cyanobacteria based on the polyphasic approach.

Hydrogenophaga aromaticivorans sp. nov., isolated from a para-xylene-degrading enrichment culture, capable of degrading benzene, meta- and para-xylene

2021 ◽  
Author(s):  
Sinchan Banerjee ◽  
András Táncsics ◽  
Erika Tóth ◽  
Fruzsina Révész ◽  
Károly Bóka ◽  
...  
Keyword(s):  
16S Rrna ◽  
Type Species ◽  
Enrichment Culture ◽  
Draft Genome ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Content Type ◽  
Link Type ◽  
Para Xylene

A benzene, para- and meta-xylene-degrading Gram-stain-negative, aerobic, yellow-pigmented bacterium, designated as D2P1T, was isolated from a para-xylene-degrading enrichment culture. Phylogenetic analyses based on 16S rRNA genes showed that D2P1T shares a distinct phyletic lineage within the genus Hydrogenophaga and shows highest 16S rRNA gene sequence similarity to Hydrogenophaga taeniospiralis NBRC 102512T (99.2 %) and Hydrogenophaga palleronii NBRC 102513T (98.3 %). The draft genome sequence of D2P1T is 5.63 Mb long and the genomic DNA G+C content is 65.5 %. Orthologous average nucleotide identity (OrthoANI) and digital DNA–DNA hybridization (dDDH) analyses confirmed low genomic relatedness to its closest relatives (OrthoANI <86 %; dDDH <30 %). D2P1T contains ubiquinone 8 (Q-8) as the only respiratory quinone and phospholipid, phosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol as major polar lipids. The main whole-cell fatty acids of D2P1T are summed feature 3 (C16 : 1  ω7c/C16 : 1  ω6c), C16 : 0 and summed feature 8 (C18 : 1  ω7c/C18 : 1  ω6c). The polyphasic taxonomic results indicated that strain D2P1T represents a novel species of the genus Hydrogenophaga , for which the name Hydrogenophaga aromaticivorans sp. nov. is proposed. The type strain is D2P1T (=LMG 31780T=NCAIM B 02655T).

Curvivirga aplysinae gen. nov., sp. nov., a marine bacterium isolated from the sea sponge Aplysina fistularis

2021 ◽  
Vol 71 (7) ◽  
Author(s):  
Stacey R. Goldberg ◽  
Brad A. Haltli ◽  
Hebelin Correa ◽  
Russell G. Kerr
Keyword(s):  
16S Rrna ◽  
Type Species ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Strictly Aerobic ◽  
Content Type ◽  
San Salvador ◽  
Link Type ◽  
Aplysina Fistularis

A Gram-stain-negative, strictly aerobic, motile bacterium, designated strain RKSG073T, was isolated from the sea sponge Aplysina fistularis, collected off the west coast of San Salvador, The Bahamas. Cells were curved-to-spiral rods with single, bipolar (amphitrichous) flagella, oxidase- and catalase-positive, non-nitrate-reducing and required salt for growth. RKSG073T grew optimally at 30–37 °C, pH 6–7, and with 2–3 % (w/v) NaCl. The predominant fatty acids of RKSG073T were summed feature 8 (C18 : 1ω6c and/or C18 : 1ω7c) and C16 : 0. Major isoprenoid quinones were identified as Q-10 and Q-9. Phylogenetic analyses of nearly complete 16S rRNA genes and genome sequences positioned strain RKSG073T in a clade with its closest relative Aestuariispira insulae AH-MY2T (92.1 % 16S rRNA gene sequence similarity), which subsequently clustered with Hwanghaeella grinnelliae Gri0909T, Marivibrio halodurans ZC80T and type species of the genera Kiloniella , Thalassospira and Terasakiella . The DNA G+C content calculated from the genome of RKSG073T was 42.2 mol%. On the basis of phylogenetic distinctiveness and polyphasic analysis, here we propose that RKSG073T (culture deposit numbers: ATCC collection = TSD-74T, BCCM collection = LMG 29869T) represents the type strain of a novel genus and species within the family Kiloniellaceae , order Rhodospirillales and class Alphaproteobacteria , for which the name Curvivirga aplysinae gen. nov., sp. nov. is proposed.

scholarly journals Aceticlastic and NaCl-Requiring Methanogen “Methanosaeta pelagica” sp. nov., Isolated from Marine Tidal Flat Sediment

2012 ◽  
Vol 78 (9) ◽  
pp. 3416-3423 ◽  
Author(s):  
Koji Mori ◽  
Takao Iino ◽  
Ken-Ichiro Suzuki ◽  
Kaoru Yamaguchi ◽  
Yoichi Kamagata
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Tidal Flat ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Acetate Metabolism ◽  
Rrna Gene ◽  
Marine Environments ◽  
Content Type ◽  
Tidal Flat Sediment

ABSTRACTAmong methanogens, only 2 genera,MethanosaetaandMethanosarcina, are known to contribute to methanogenesis from acetate, andMethanosaetais a specialist that uses acetate specifically. However,Methanosaetastrains so far have mainly been isolated from anaerobic digesters, despite the fact that it is widespread, not only in anaerobic methanogenic reactors and freshwater environments, but also in marine environments, based upon extensive 16S rRNA gene-cloning analyses. In this study, we isolated an aceticlastic methanogen, designated strain 03d30qT, from a tidal flat sediment. Phylogenetic analyses based on 16S rRNA andmcrAgenes revealed that the isolate belongs to the genusMethanosaeta. Unlike the other knownMethanosaetaspecies, this isolate grows at Na+concentrations of 0.20 to 0.80 M, with an optimum concentration of 0.28 M. Quantitative estimation using real-time PCR detected the 16S rRNA gene of the genusMethanosaetain the marine sediment, and relative abundance ranged from 3.9% to 11.8% of the total archaeal 16S rRNA genes. In addition, the number ofMethanosaetaorganisms increased with increasing depth and was much higher than that ofMethanosarcinaorganisms, suggesting that aceticlastic methanogens contribute to acetate metabolism to a greater extent than previously thought in marine environments, where sulfate-reducing acetate oxidation prevails. This is the first report on marineMethanosaetaspecies, and based on phylogenetic and characteristic studies, the name “Methanosaeta pelagica” sp. nov. is proposed for this novel species, with type strain 03d30q.

Halosimplex pelagicum sp. nov. and Halosimplex rubrum sp. nov., isolated from salted brown alga Laminaria, and emended description of the genus Halosimplex

2014 ◽  
Vol 64 (Pt_1) ◽  
pp. 169-173 ◽  
Author(s):  
Dong Han ◽  
Heng-Lin Cui
Keyword(s):  
16S Rrna ◽  
Brown Alga ◽  
Type Species ◽  
Liaoning Province ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Content Type ◽  
Link Type ◽  
Emended Description

Two halophilic archaeal strains, R2T and R27T, were isolated from the brown alga Laminaria produced at Dalian, Liaoning Province, China. Both had pleomorphic cells that lysed in distilled water, stained Gram-negative and formed red-pigmented colonies. They grew optimally at 42 °C, pH 7.0 and in the presence of 3.1–3.4 M NaCl and 0.03–0.5 M Mg2+. The major polar lipids of the two strains were phosphatidylglycerol (PG), phosphatidylglycerol phosphate methyl ester (PGP-Me) and four major glycolipids chromatographically identical to those of Halosimplex carlsbadense JCM 11222T. 16S rRNA gene analysis revealed that each strain had two dissimilar 16S rRNA genes and both strains were phylogenetically related to Halosimplex carlsbadense JCM 11222T (92.7–98.8 % similarities). The rpoB′ gene similarities between strains R2T and R27T and between these strains and Halosimplex carlsbadense JCM 11222T were 95.7 %, 96.1 % and 95.8 %, respectively. The DNA G+C contents of strains R2T and R27T were 62.5 mol% and 64.0 mol%, respectively. The DNA–DNA hybridization values between strains R2T and R27T and between the two strains and Halosimplex carlsbadense JCM 11222T were 43 %, 52 % and 47 %, respectively. It was concluded that strain R2T ( = CGMCC 1.10586T = JCM 17263T) and strain R27T ( = CGMCC 1.10591T = JCM 17268T) represent two novel species of the genus Halosimplex , for which the names Halosimplex pelagicum sp. nov. and Halosimplex rubrum sp. nov. are proposed. An emended description of the genus Halosimplex is also presented.

Natronoarchaeum rubrum sp. nov., isolated from a marine solar saltern, and emended description of the genus Natronoarchaeum

2014 ◽  
Vol 64 (Pt_3) ◽  
pp. 952-956 ◽  
Author(s):  
Xing-Xing Qiu ◽  
Mei-Lin Zhao ◽  
Heng-Lin Cui
Keyword(s):  
16S Rrna ◽  
Type Species ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Solar Saltern ◽  
Marine Solar Saltern ◽  
Content Type ◽  
Link Type ◽  
Emended Description

A halophilic archaeal strain, GX48T, was isolated from the Gangxi marine solar saltern near Weihai city in Shandong Province, China. Cells of the strain were rod-shaped, stained Gram-negative and formed red-pigmented colonies. Strain GX48T was able to grow at 25–50 °C (optimum 37 °C), in the presence of 1.4–4.8 M NaCl (optimum 2.6 M NaCl), with 0–1.0 M MgCl2 (optimum 0.05 M MgCl2) and at pH 5.5–9.5 (optimum pH 7.0). Cells lysed in distilled water and the minimal NaCl concentration to prevent cell lysis was 8 % (w/v). The major polar lipids of the strain were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and two major glycolipids chromatographically identical to those of Natronoarchaeum mannanilyticum YSM-123T and Natronoarchaeum philippinense 294-194-5T. 16S rRNA gene analysis revealed that strain GX48T had two dissimilar 16S rRNA genes and both of them were phylogenetically related to those of the two current members of the genus Natronoarchaeum (96.2–98.3 % similarities). The rpoB′ gene sequence similarities between strain GX48T and Natronoarchaeum mannanilyticum YSM-123T and Natronoarchaeum philippinense 294-194-5T were 96.0 % and 94.7 %, respectively. The DNA G+C content of strain GX48T was 66.2 mol%. Strain GX48T showed low DNA–DNA relatedness with the two members of the genus Natronoarchaeum . It was concluded that strain GX48T ( = CGMCC 1.10388T = JCM 17119T) represents a novel species of the genus Natronoarchaeum , for which the name Natronoarchaeum rubrum sp. nov. is proposed. An emended description of the genus Natronoarchaeum is also presented.

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