Journal of Eukaryotic Microbiology最新文献

{"title":"Fine structure and molecular characterization of two new parabasalid species that naturally colonize laboratory mice, Tritrichomonas musculus and Tritrichomonas casperi","authors":"Leila Tuzlak,&nbsp;Eliza V. C. Alves-Ferreira,&nbsp;Cindi L. Schwartz,&nbsp;Andrea Kennard,&nbsp;Jacqueline M. Leung,&nbsp;Christina Shehata,&nbsp;Michael E. Grigg","doi":"10.1111/jeu.12989","DOIUrl":"10.1111/jeu.12989","url":null,"abstract":"<p><i>Tritrichomonas muris</i> is a common flagellated protist isolated from the cecum of wild rodents. This commensal protist has been shown previously to alter immune phenotypes in laboratory mice. Other trichomonads, referred to as <i>Tritrichomonas musculis</i> and <i>Tritrichomonas rainier</i>, also naturally colonize laboratory mice and cause immune alterations. This report formally describes two new trichomonads, <i>Tritrichomonas musculus</i> n. sp., and <i>Tritrichomonas casperi</i> n. sp., at the ultrastructural and molecular level. These two protists were isolated from laboratory mice and were differentiated by their size and the structure of their undulating membrane and posterior flagellum. Analysis at the <i>18S rRNA</i> and trans-<i>ITS</i> genetic loci supported their designation as distinct species, related to <i>T. muris</i>. To assess the true extent of parabasalid diversity infecting laboratory mice, 135 mice bred at the National Institutes of Health (NIH) were screened using pan-parabasalid primers that amplify the trans-<i>ITS</i> region. Forty-four percent of mice were positive for parabasalids, encompassing a total of eight distinct sequence types. <i>Tritrichomonas casperi</i> and <i>Trichomitus-</i>like protists were dominant. <i>T. musculus</i> and <i>T. rainier</i> were also detected, but <i>T. muris</i> was not. Our work establishes a previously underappreciated diversity of commensal trichomonad flagellates that naturally colonize the enteric cavity of laboratory mice.</p>","PeriodicalId":15672,"journal":{"name":"Journal of Eukaryotic Microbiology","volume":"70 6","pages":""},"PeriodicalIF":2.2,"publicationDate":"2023-06-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10261971","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"True molecular phylogenetic position of the cockroach gut commensal Lophomonas blattarum (Lophomonadida, Parabasalia)","authors":"LeAnn Nguyen,&nbsp;Stephen J. Taerum,&nbsp;Daniel E. Jasso-Selles,&nbsp;Claudio H. Slamovits,&nbsp;Jeffrey D. Silberman,&nbsp;Gillian H. Gile","doi":"10.1111/jeu.12988","DOIUrl":"10.1111/jeu.12988","url":null,"abstract":"<p><i>Lophomonas blattarum</i> is a facultative commensal gut dweller of common pest cockroaches. Its cells are roughly spherical in shape with an apical tuft of ~50 flagella. Controversially, it has been implicated in human respiratory infections based on light microscopic observations of similarly shaped cells in sputum or bronchoalveolar lavage fluid. Here, we have sequenced the 18S rRNA gene of <i>L. blattarum</i> and its sole congener, <i>Lophomonas striata</i>, isolated from cockroaches. Both species branch in a fully supported clade with Trichonymphida, consistent with a previous study of <i>L. striata</i>, but not consistent with sequences from human samples attributed to <i>L. blattarum</i>.</p>","PeriodicalId":15672,"journal":{"name":"Journal of Eukaryotic Microbiology","volume":"70 5","pages":""},"PeriodicalIF":2.2,"publicationDate":"2023-06-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10121980","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"New Parabasalia symbionts Snyderella spp. and Daimonympha gen. nov. from South American Rugitermes termites and the parallel evolution of a cell with a rotating “head”","authors":"Elisabeth Hehenberger,&nbsp;Vittorio Boscaro,&nbsp;Erick R. James,&nbsp;Yoshihisa Hirakawa,&nbsp;Morelia Trznadel,&nbsp;Mahara Mtawali,&nbsp;Rebecca Fiorito,&nbsp;Javier del Campo,&nbsp;Anna Karnkowska,&nbsp;Martin Kolisko,&nbsp;Nicholas A. T. Irwin,&nbsp;Varsha Mathur,&nbsp;Rudolf H. Scheffrahn,&nbsp;Patrick J. Keeling","doi":"10.1111/jeu.12987","DOIUrl":"10.1111/jeu.12987","url":null,"abstract":"<p>Most Parabasalia are symbionts in the hindgut of “lower” (non-Termitidae) termites, where they widely vary in morphology and degree of morphological complexity. Large and complex cells in the class Cristamonadea evolved by replicating a fundamental unit, the karyomastigont, in various ways. We describe here four new species of Calonymphidae (Cristamonadea) from <i>Rugitermes</i> hosts, assigned to the genus <i>Snyderella</i> based on diagnostic features (including the karyomastigont pattern) and molecular phylogeny. We also report a new genus of Calonymphidae, <i>Daimonympha</i>, from <i>Rugitermes laticollis</i>. <i>Daimonympha</i>'s morphology does not match that of any known Parabasalia, and its SSU rRNA gene sequence corroborates this distinction. <i>Daimonympha</i> does however share a puzzling feature with a few previously described, but distantly related, Cristamonadea: a rapid, smooth, and continuous rotation of the anterior end of the cell, including the many karyomastigont nuclei. The function of this rotatory movement, the cellular mechanisms enabling it, and the way the cell deals with the consequent cell membrane shear, are all unknown. “Rotating wheel” structures are famously rare in biology, with prokaryotic flagella being the main exception; these mysterious spinning cells found only among Parabasalia are another, far less understood, example.</p>","PeriodicalId":15672,"journal":{"name":"Journal of Eukaryotic Microbiology","volume":"70 5","pages":""},"PeriodicalIF":2.2,"publicationDate":"2023-06-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1111/jeu.12987","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10498254","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Comparison between p-distance and single-locus species delimitation models for delineating reproductively tested strains of pennate diatoms (Bacillariophyceae) using cox1, rbcL and ITS","authors":"Andréa de O. da R. Franco,&nbsp;Matt P. Ashworth,&nbsp;Clarisse Odebrecht","doi":"10.1111/jeu.12986","DOIUrl":"10.1111/jeu.12986","url":null,"abstract":"<p>Several automated molecular methods have emerged for distinguishing eukaryote species based on DNA sequence data. However, there are knowledge gaps around which of these single-locus methods is more accurate for the identification of microalgal species, such as the highly diverse and ecologically relevant diatoms. We applied genetic divergence, Automatic Barcode Gap Discovery for primary species delimitation (ABGD), Assemble Species by Automatic Partitioning (ASAP), Statistical Parsimony Network Analysis (SPNA), Generalized Mixed Yule Coalescent (GMYC) and Poisson Tree Processes (PTP) using partial <i>cox</i>1, <i>rbc</i>L, <i>5.8S + ITS</i>2, <i>ITS</i>1 <i>+ 5.8S + ITS</i>2 markers to delineate species and compare to published polyphasic identification data (morphological features, phylogeny and sexual reproductive isolation) to test the resolution of these methods. ASAP, ABGD, SPNA and PTP models resolved species of <i>Eunotia</i>, <i>Seminavis, Nitzschia, Sellaphora</i> and <i>Pseudo-nitzschia</i> corresponding to previous polyphasic identification, including reproductive isolation studies. In most cases, these models identified diatom species in similar ways, regardless of sequence fragment length. GMYC model presented smallest number of results that agreed with previous published identification. Following the recommendations for proper use of each model presented in the present study, these models can be useful tools to identify cryptic or closely related species of diatoms, even when the datasets have relatively few sequences.</p>","PeriodicalId":15672,"journal":{"name":"Journal of Eukaryotic Microbiology","volume":"70 5","pages":""},"PeriodicalIF":2.2,"publicationDate":"2023-05-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10125645","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"On stomatocysts of Paraphysomonas caelifrica (Stramenopiles, Paraphysomonadida)","authors":"Dmitry Kapustin,&nbsp;Marina Ignatenko,&nbsp;Tatyana Yatsenko-Stepanova","doi":"10.1111/jeu.12979","DOIUrl":"10.1111/jeu.12979","url":null,"abstract":"<p>Stomatocysts of the rare heterotrophic chrysophyte, <i>Paraphysomonas caelifrica</i>, were discovered from a shallow ephemeral pond Tavolgasai (“Orenburgskiy” State Nature Reserve, Orenburg Region, Russia). Morphology of stomatocysts was studied using scanning electron microscopy. Stomatocysts of <i>P. caelifrica</i> are spherical and smooth with a cylindrical collar surrounding the regular pore. So, they do not belong to the stomatocyst 1 Duff and Smol as previously thought. The description of a new stomatocyst morphotype is provided.</p>","PeriodicalId":15672,"journal":{"name":"Journal of Eukaryotic Microbiology","volume":"70 5","pages":""},"PeriodicalIF":2.2,"publicationDate":"2023-05-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10125164","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Identification and validation of novel Blastocystis subtype ST41 in a Colombian patient undergoing colorectal cancer screening","authors":"Carolina Hernández-Castro,&nbsp;Jenny G. Maloney,&nbsp;Sonia P. Agudelo-López,&nbsp;Miguel A. Toro-Londoño,&nbsp;Jorge H. Botero-Garcés,&nbsp;María C. Orozco,&nbsp;Yulieth C. Quintero-Quinchia,&nbsp;Juan C. Correa-Cote,&nbsp;Alejandro Múnera-Duque,&nbsp;Juan C. Ricaurte-Ciro,&nbsp;Luis I. Londoño-Álvarez,&nbsp;René M. Escobar,&nbsp;Pamela C. Köster,&nbsp;Sergio Sánchez,&nbsp;David Carmena,&nbsp;Mónica Santín","doi":"10.1111/jeu.12978","DOIUrl":"10.1111/jeu.12978","url":null,"abstract":"<p><i>Blastocystis</i> sp. is among the most frequent intestinal protists identified in humans globally. However, characterization of <i>Blastocystis</i> subtype diversity in humans is ongoing. We report here the identification of novel <i>Blastocystis</i> subtype ST41 in a Colombian patient undergoing colorectal cancer screening involving colonoscopy and fecal testing (microscopy, culture, PCR). The full-length <i>ssu</i> rRNA gene sequence of the protist was generated using MinION long-read sequencing technology. The validity of the novel subtype was confirmed via phylogenetic and pairwise distance analyses of the full-length ST41 sequence and all other valid subtypes. The study provides reference material essential for conducting subsequent experimental studies.</p>","PeriodicalId":15672,"journal":{"name":"Journal of Eukaryotic Microbiology","volume":"70 5","pages":""},"PeriodicalIF":2.2,"publicationDate":"2023-05-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10121973","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Vegetative cell fusion and a new stage in the life cycle of the Aphelida (Opisthosporidia)","authors":"Victoria S. Tcvetkova,&nbsp;Igor R. Pozdnyakov,&nbsp;Alexei O. Seliuk,&nbsp;Natalia A. Zorina,&nbsp;Sergey A. Karpov","doi":"10.1111/jeu.12977","DOIUrl":"10.1111/jeu.12977","url":null,"abstract":"<p>The aphelids, intracellular parasitoids of algae, represent a large cluster of species sister to Fungi in molecular phylogenetic trees. Sharing a common ancestor with Fungi, they are very important in terms of evolution of these groups of Holomycota. Aphelid life cycle being superficially similar to that of Chytridiomycetes is understudied. We have found in the aphelids a new stage—big multiflagellar and amoeboid cells, formed from a plasmodium that has two sorts of nuclei after trophic stage fusion. The families of protein-coding genes involved in the vegetative cell fusion in Opisthokonta were also discussed.</p>","PeriodicalId":15672,"journal":{"name":"Journal of Eukaryotic Microbiology","volume":"70 5","pages":""},"PeriodicalIF":2.2,"publicationDate":"2023-04-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10127161","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The photic-aphotic divide is a strong ecological and evolutionary force determining the distribution of ciliates (Alveolata, Ciliophora) in the ocean","authors":"Luciana F. Santoferrara,&nbsp;Aleena Qureshi,&nbsp;Amina Sher,&nbsp;Leocadio Blanco-Bercial","doi":"10.1111/jeu.12976","DOIUrl":"10.1111/jeu.12976","url":null,"abstract":"<p>The bulk of knowledge on marine ciliates is from shallow and/or sunlit waters. We studied ciliate diversity and distribution across epi- and mesopelagic oceanic waters, using DNA metabarcoding and phylogeny-based metrics. We analyzed sequences of the 18S rRNA gene (V4 region) from 369 samples collected at 12 depths (0–1000 m) at the Bermuda Atlantic Time-series Study site of the Sargasso Sea (North Atlantic) monthly for 3 years. The comprehensive depth and temporal resolutions analyzed led to three main findings. First, there was a gradual but significant decrease in alpha-diversity (based on Faith's phylogenetic diversity index) from surface to 1000-m waters. Second, multivariate analyses of beta-diversity (based on UniFrac distances) indicate that ciliate assemblages change significantly from photic to aphotic waters, with a switch from Oligotrichea to Oligohymenophorea prevalence. Third, phylogenetic placement of sequence variants and clade-level correlations (EPA-ng and GAPPA algorithms) show Oligotrichea, Litostomatea, Prostomatea, and Phyllopharyngea as anti-correlated with depth, while Oligohymenophorea (especially Apostomatia) have a direct relationship with depth. Two enigmatic environmental clades include either prevalent variants widely distributed in aphotic layers (the Oligohymenophorea OLIGO5) or subclades differentially distributed in photic versus aphotic waters (the Discotrichidae NASSO1). These results settle contradictory relationships between ciliate alpha-diversity and depth reported before, suggest functional changes in ciliate assemblages from photic to aphotic waters (with the prevalence of algivory and mixotrophy vs. omnivory and parasitism, respectively), and indicate that contemporary taxon distributions in the vertical profile have been strongly influenced by evolutionary processes. Integration of DNA sequences with organismal data (microscopy, functional experiments) and development of databases that link these sources of information remain as major tasks to better understand ciliate diversity, ecological roles, and evolution in the ocean.</p>","PeriodicalId":15672,"journal":{"name":"Journal of Eukaryotic Microbiology","volume":"70 5","pages":""},"PeriodicalIF":2.2,"publicationDate":"2023-04-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1111/jeu.12976","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10123793","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Through the eDNA looking glass: Responses of fjord benthic foraminiferal communities to contrasting environmental conditions","authors":"Inda Brinkmann,&nbsp;Magali Schweizer,&nbsp;David Singer,&nbsp;Sophie Quinchard,&nbsp;Christine Barras,&nbsp;Joan M. Bernhard,&nbsp;Helena L. Filipsson","doi":"10.1111/jeu.12975","DOIUrl":"10.1111/jeu.12975","url":null,"abstract":"<p>The health of coastal marine environments is severely declining with global changes. Proxies, such as those based on microeukaryote communities, can record biodiversity and ecosystem responses. However, conventional studies rely on microscopic observations of limited taxonomic range and size fraction, missing putatively ecologically informative community components. Here, we tested molecular tools to survey foraminiferal biodiversity in a fjord system (Sweden) on spatial and temporal scales: Alpha and beta diversity responses to natural and anthropogenic environmental trends were assessed and variability of foraminiferal environmental DNA (eDNA) compared to morphology-based data. The identification of eDNA-obtained taxonomic units was aided by single-cell barcoding. Our study revealed wide diversity, including typical morphospecies recognized in the fjords, and so-far unrecognized taxa. DNA extraction method impacted community composition outputs significantly. DNA extractions of 10 g sediment more reliably represented present diversity than of 0.5-g samples and, thus, are preferred for environmental assessments in this region. Alpha- and beta diversity of 10-g extracts correlated with bottom-water salinity similar to morpho-assemblage diversity changes. Sub-annual environmental variability resolved only partially, indicating damped sensitivity of foraminiferal communities on short timescales using established metabarcoding techniques. Systematically addressing the current limitations of morphology-based and metabarcoding studies may strongly improve future biodiversity and environmental assessments.</p>","PeriodicalId":15672,"journal":{"name":"Journal of Eukaryotic Microbiology","volume":"70 4","pages":""},"PeriodicalIF":2.2,"publicationDate":"2023-03-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1111/jeu.12975","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9754709","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"In Memoriam: Richard Dean Allen","authors":"Klaus Hausmann,&nbsp;Helmut Plattner","doi":"10.1111/jeu.12974","DOIUrl":"10.1111/jeu.12974","url":null,"abstract":"Richard Dean Allen passed away on February 10, 2023, in Honolulu, Oahu, Hawaii, the United States. The protozoological and cell biological community sadly lost one of its best and highly esteemed colleagues with international reputation. He was born on September 20, 1935, in Dallas Center, Iowa. Being raised on a farm likely sparked his lifelong interest in biology and his passion for the natural world. After graduating from Dallas Center High School in 1953, Richard left his hometown to obtain a B.A. degree in Biology from Greenville College, Illinois, in 1957, then, in 1960, from the University of Illinois a M.S. degree in Botany. Later, in 1964, he received a Ph.D. in Cell Biology from Iowa State University, Ames, Iowa, followed by work as a Postdoc in Cell Biology at Harvard University, Cambridge, Massachusetts, 1964– 1965, where he was working with one of the first pioneers in electron microscopy, Prof. Keith Roberts Porter. After stations as an assistant professor of biology, Messiah College, Grantham, Pennsylvania (1965– 1968), and as a Director of an EM Service Laboratory, Harvard University, Cambridge, Massachusetts (1968– 1969), he moved with his family to Oahu, Hawaii, in 1969 to accept a position as an Associate Professor of Microbiology in the Pacific Biomedical Research Center at the University of Hawaii in Honolulu, Oahu. In 1975, he became a professor of microbiology. Starting in 1985, he was Director of the Biological EM Facility of the University of Hawaii (Figure 1) until his retirement in December 2006. The scientific topic of his Ph.D.thesis originated from the field of botany. Richard studied ultrastructurally the mitotic cell division in sporogenous cells of the fernlike vascular plant Psilotum nudum, using transmission electron microscopy (Allen & Bowen, 1966). During his time in Messiah College, and Harvard University, Richard started ultrastructural studies about the cortex and associated structures in Tetrahymena and Paramecium. Later, being settled in Hawaii, his further research concentrated preferably on protozoa, predominantly on ciliates, eventually almost exclusively on Paramecium. Over a long period of time, his research was carried out under the heading Membrane Dynamics in Intracellular Digestion (1979– 1992) followed by Endosome System and Membrane Trafficking (1992– 1995). Starting in 1995, he focused his interest on Osmoregulation and Contractile Vacuole Function, working in this field until his retirement. A great deal of the results of all these studies already found entrance in protistological textbooks (Hausmann et al., 2003). Richard Allen has contributed valuable data on the dynamic structure of Paramecium cells. This started with the demonstration of microtubular “rails” as longrange signals for cyclosis in Paramecium (Allen, 1974). Vesicle trafficking was his leitmotif throughout his work. In several regards, ciliates are very complicated cells, but one can much profit from the distinct routes of ves","PeriodicalId":15672,"journal":{"name":"Journal of Eukaryotic Microbiology","volume":"70 4","pages":""},"PeriodicalIF":2.2,"publicationDate":"2023-03-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9742518","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}