Streptococcus (S.) pneumoniae, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Legionella pneumophila, Escherichia coli, Middle East respiratory syndrome (MERS) coronavirus, influenza A virus (IAV) and superinfection of IAV-incuced pneumonia with S. pneumoniae. Systematic comparisons of the models revealed striking differences in the distribution of lesions, the characteristics of pneumonia induced, principal inflammatory cell types, lesions in adjacent tissues, and the detectability of the pathogens in histological sections. We therefore identified core criteria for each model suitable for practical semiquantitative scoring systems that take into account the pathogen- and model-specific patterns of pneumonia. Other critical factors that affect experimental pathologies are discussed, including infectious dose, time kinetics, and the genetic background of the mouse strain. The substantial differences between the model-specific pathologies underscore the necessity of pathogen- and model-adapted criteria for the comparative quantification of experimental outcomes. These criteria also allow for the standardized validation and comparison of treatment strategies in preclinical models.Pneumonia may be caused by a wide range of pathogens and is considered the most common infectious cause of death in humans. Murine acute lung infection models mirror human pathologies in many aspects and contribute to our understanding of the disease and the development of novel treatment strategies. Despite progress in other fields of tissue imaging, histopathology remains the most conclusive and practical read out tool for the descriptive and semiquantitative evaluation of mouse pneumonia and therapeutic interventions. Here, we systematically describe and compare the distinctive histopathological features of established models of acute pneumonia in mice induced by  Streptococcus (S.) pneumoniae [S. pneumoniae, resulting in severe progressive pneumonia associated with increased mortality [Legionella (L.) pneumophila is the causative agent of the severe CAP Legionnaires’ disease, and the second most commonly detected pathogen in pneumonia in patients admitted to intensive care units (ICU) in industrialized countries [Staphylococcus (S.) aureus, Klebsiella (K.) pneumoniae, Acinetobacter (A.) baumannii, and Escherichia (E.) coli have been isolated with varying prevalences [K. pneumoniae is a significant opportunistic pathogen causing severe life-threatening hospital-acquired respiratory tract infections [S. aureus, a Gram-positive bacterium, is one of the most prevalent pathogens of community- and hospital-acquired lower respiratory tract infections in humans and accounts for a significant health and economic burden [A. baumannii and E. coli are ubiquitous Gram-negative bacteria which have recently emerged as major causes of community-associated, nosocomial [As one of the most frequent infectious diseases, pneumonia causes a tremendous socioeconomic burden in industrialized countries  and is teumoniae , 8 whicheumoniae , 10. As eumoniae , 11, 12.ortality . In contountries , 15. Howountries  and the valences –19. Morefections –22 whilec burden –25. A. bsocomial , 27 and socomial  as well socomial , 30.In addition, more recently discovered pulmonary pathogens indicate that novel emerging diseases may add to the list of highly relevant pneumonias that may also be of interest to be studied in animal models. For example, the Middle East respiratory syndrome coronavirus (MERS-CoV) which is transmitted by dromedary camels as vectors  has emerThe various forms of pneumonia have been successfully reproduced in specific murine models of experimentally-induced acute pneumonia –39. ThesHistopathology has been a powerful, reliable, and reproducible read-out tool for the evaluation of morphological changes in animal lung infection experiments for many decades , 44. QuaS. pneumoniae and S. aureus-induced pneumonia [Previous studies have revealed first fundamental differences in histopathologic lesions caused by different pathogens in mouse lungs , 41, 42.neumonia , 42. HowS. pneumoniae, S. aureus, K. pneumoniae, A. baumannii, L. pneumophila, E. coli, MERS-CoV, IAV and superinfection with IAV and pneumococci. We provide model-specific criteria that can be used for appropriate histological quantitative comparisons, e.g., when different therapeutic interventions are evaluated within these established models. Whole mouse lung sections were used to obtain complete overviews, particularly of the distributions of lesions and inflammatory patterns. On the basis of the different and oftentimes quite pathogen- and model-specific changes we identified the most suitable evaluation criteria for each model that will allow for more accurate semiquantitative assessments of the severities and distributions of pneumonic lesions.Here, we systematically describe and compare the histopathologies at their peaks of inflammation and injury of nine previously established acute lung infection models induced by A. baumannii and E. coli experiments that will be published elsewhere. All animal procedures and protocols were approved by institutional ethics committees of Charité-University of Berlin, Justus-Liebig University of Giessen, Philipps University of Marburg, University Hospital of Jena and local governmental authorities  Berlin, Regierungspräsidium (RP) Gießen and Darmstadt, Landesamt für Verbraucherschutz (TLV) Thüringen), respectively. Permit numbers were G 0356/10, A-0050/15 (S. pneumoniae), G 0358/11 (S. aureus), G 75/2011, G 110/2014 (K. pneumoniae), A 0299/15 (A. baumannii), G 0175/12 (L. pneumophila), 02-049/12 (E. coli), 114/2012 (MERS-CoV), G 0152/12, and G 0044/11 (IAV and superinfection). All animal studies were conducted in strict accordance with the Federation of European Laboratory Animal Science Associations (FELASA) guidelines and recommendations for the care and use of laboratory animals, and all efforts were made to minimize animal discomfort and suffering.The lung tissues examined here were derived from experiments primarily conducted for purposes other than this study and most have been published elsewhere , 51–53, All mice, except for MERS-CoV infected mice, were monitored at 12 hour intervals throughout the experiment to assess appearance, behavior, grooming, respiration, body weight, and rectal temperature. Humane endpoints were defined  but not reached by any of the mice at the indicated time points of termination of the experiments. MERS-CoV infected mice were monitored once daily and appearance, behavior, grooming, respiration and body weight were protocolled. Here, a single humane endpoint (loss of body weight of >15%) was defined but not reached by any of the mice employed due to their favourable clinical outcome at the infection dose used.K. pneumoniae, female mice (aged 8–12 weeks and weighing 17–22 g) were randomly assigned to groups (n = 2–4) per cage whereas in the K. pneumoniae model female and male mice (aged 23–25 weeks and weighing 22–24 g) were used for model specific reasons [For all experimental infection models, except of  reasons . Further reasons , 54) bacE. coli, mice were anesthetized intraperitoneally with ketamine (80 mg/kg)  and xylazine (25 mg/kg) . For experimental viral infections, mice were anesthetized using inhalation of isoflurane . For lung histology, all mice except of the MERS-CoV model were humanely euthanized by exsanguination via the caudal Vena cava after anesthesia by intraperitoneal injection of premixed ketamine (160 mg/kg) and xylazine (75 mg/kg). MERS-CoV infected mice were humanely euthanized by cervical dislocation after isoflurane anesthesia.For bacterial infections, except for S. pneumoniae , S. aureus Newman (NCTC 10833), K. pneumoniae , A. baumannii (RUH 2037), L. pneumophila  were cultured as described [6 CFU S. pneumoniae (n = 14 mice), 5 x 107 CFU S. aureus (n = 4), 5 x 108 CFU A. baumannii (n = 8), in 20 μl PBS. Mice transnasally infected with L. pneumophila (n = 8) received 1 × 106 CFU in 40 μl PBS. Mice infected with K. pneumoniae (n = 16) received 3.5 x 105 CFU intratracheally in 50 μl NaCl (0.9%) via Microsprayer® Aerosolizer  using intubation-mediated intratracheal instillation through intact airways [ii RUH 20, L. pneuescribed , 55, 56  airways  which ha airways , 57–59.E. coli (ATCC 25922) from -80°C glycerol stock was added to LB broth  and incubated for 12 hours at 200 rpm and 37°C with 5% CO2. Optical density of 0.03 was adjusted in LB broth followed by incubation until midlogarithmic phase for 1.5 hours at 200 rpm and 37°C. After centrifugation, the pellet was resuspended in sterile 0.9% NaCl at 8 x 105 CFU E. coli / 200 μl and administered intraperitoneally (n = 10).8 PFU per inoculum , resulting in hDPP4 expression in the epithelial compartment of the lung [4 TCID50 of MERS-CoV in 20 μl DMEM (n = 17) under isoflurane anesthesia .For initial transduction of human DPP4 for subsequent infection of BALB/c mice with MERS-CoV (hCoV EMC) viruses were cultured and prepared as described , 54, 60.Influenza A/PR/8/34 virus  was grown as described  and miceS. pneumoniae was cultured as described [3 CFU S. pneumoniae in 20 μl PBS (n = 4).For superinfection experiments, the IAV infection procedure was applied as described above with 40 PFU PR8 in 50 μl PBS. 8 days after viral infection, escribed  and resuMice were humanely euthanized at model-specific time points as indicated . BetweenThree evenly distributed whole-organ horizontal sections throughout the entire lungs were microscopically evaluated to assess the distribution and character of pathologic alterations, generating a modified panel of specific lung inflammation parameters adapted to each pathogen used . All exaS. pneumoniae and IAV (H1N1), antigen retrieval was performed with microwave heating (600 W) in 10 mM citric acid  for 12 minutes (min). Lung sections were then incubated with a purified rabbit antibody polyclonal to S. pneumoniae  or with a purified goat antibody polyclonal to IAV H1N1  at 4°C overnight. Incubation with an immuno-purified rabbit or goat antibody at the same dilution served as negative controls. Subsequently, slides were incubated with a secondary, alkaline phosphatase-conjugated goat anti-rabbit  antibody for 30 min at room temperature. The alkaline chromogen triamino-tritolyl-methanechloride (Neufuchsin) was used as phosphatase substrate for color development. All slides were counterstained with hematoxylin, dehydrated through graded ethanols, cleared in xylene and coverslipped.For immunohistochemical detection of S. pneumoniae, serotype 3 resulted in a broad spectrum of tissue lesions and immune cell infiltrations that are typical of aerogenic bacterial pneumonia. Specific for this model, lesions widely expanded down to the periphery of the lung lobes  from S. aureus [L. pneumophila which primarily infects macrophages [H1-response [S. pneumoniae and K. pneumoniae. This massive bacterial spreading throughout the thoracic cavity was exclusively present in these two models and most likely associated with sepsis, responsible for the rapid clinical progression and unfavourable outcome [K. pneumoniae had the tendency of abscess formation which was not seen in pneumococcal pneumonia. Infection with S. aureus and A. baumannii also resulted in similar lesion patterns, except for Acinetobacter-induced lesions widely expanding to the lung periphery while Staphyloccocus-induced pneumonia was restricted to the lung hilus. A second difference between the two was the presence of prominent vascular thrombosis in A. baumannii-induced pneumonia which was absent from Staphylococcus pneumonia. The clinical outcome of mice infected with A. baumannii and S. aureus was more favourable when compared to infection with S. pneumoniae or K. pneumoniae [E. coli infection was included here as a model for sepsis-associated ALI [S. pneumoniae, A. baumannii and MERS-CoV, however, to a much lesser extent and only within the most strongly affected areas. MERS-CoV and IAV-associated lesions clearly reflected the known cellular tropisms of these viruses with necrosis of alveolar walls or bronchial epithelial cells, respectively, being the most characteristic histopathologic features [S. pneumoniae potentiated the severity of the IAV-induced lesions and aggravated the course of pneumonia. However, it did not alter the principal histological characteristics of IAV-pneumonia. The patterns seen after single infection with S. pneumonia were not repeated in this superinfection model, likely owing to the much lower inoculation dose which is usually rapidly cleared from virus-naive lungs.The model-associated characteristics of tissue lesions and immune cell infiltrates are widely consistent with well-established properties of the different pathogens used. For example, the destructive tissue damage with mostly neutrophilic infiltrations as seen in peroxide  from S. . aureus , 63. On rophages  resultedresponse , 66. How outcome , 67. Howeumoniae  which maated ALI , 30, 68 features –72. Typifeatures , 73. In features , 74. SubK. pneumoniae infection where the periphery of the lobes was more strongly affected than their hilus areas despite a similar aerogenous route of infection may be due to the aerosolic intratracheal application [E. coli induced pneumonia likely followed the diffuse blood borne entry of the pathogen into the lungs after intraperitoneal infection. Again unique among the pathogens tested here, the IAV-associated pattern affected entire but only select lung lobes with almost complete sparing of others. This distribution probably followed a random spread of the virus along major airways but why some lobes remained virtually unaffected after transnasal infection remains hard to explain. Apart from helping to understand differences in pathogen spread, the uneven and often quite asymmetrical distributions have a tremendous impact in practical terms when acute mouse pneumonia is sampled for molecular studies. When quantitative data on mRNA or protein expression levels or other biochemical information are to be compared with one another or with tissue lesions, it is imperative that only identically affected areas are compared. Since this is impossible to predict or recognize on the macroscopical level for most models, the practice of sampling different regions of such lungs for different readout systems appears highly problematic.When the distributions of lesions were compared among the 9 models tested, four distinct patterns could be clearly distinguished. The most common pattern, where lesions were focused around central airways and blood vessels close to the lung hilus with the periphery less or not affected can likely be explained by the aerogenous route of infection and pathogen entry. The opposite pattern characteristic of lication  of theselication , 57–59. Another implication of the distinct lesion characteristics, immune cell reactions and distributions among the different models appears highly relevant for histological scoring systems that aim at first quantitative comparisons . To narrS. pneumoniae, K. pneumoniae and E. coli were easily visible in HE stained tissue sections in areas with low density of inflammatory cells, e.g., in perivascular spaces, they were very difficult to identify in heavily infiltrated and consolidated lung parenchyma. In contrast, S. aureus, A. baumannii, L. pneumophila and both viruses were entirely invisible by HE staining and thus had to be visualized by appropriate histotechnical stains or immunohistochemistry. Both approaches will likely also allow for a rough quantification of pathogen numbers in tissues when appropriate image analysis tools are used.As standard hematoxylin and eosin (HE) staining of tissue sections failed to visualize most pathogens, traditional special stains as well as immunohistochemical techniques were employed, depending on specific staining properties of the pathogens and the availability of appropriate antibodies. While In this first comparative study of its kind, we examined previously established models with their optimized routes of infection, time points, and infection doses and volumes specific for each model to reach peaks of lung injury and inflammation. Variations of such factors can be expected to result in different lesion severities, composition of the cellular infiltrates, and for some models in different expansions of lesions within the lung. Still, the conditions used here are all based on observations that have evolved during extensive previous establishment studies of these models , 77–79. Histopathology of the lungs may be complex and requires fundamental knowledge in species-specific anatomy, physiology, organ-specific immunology, pathology, and histotechnical procedures. Furthermore, various background lesions in mice, including strain specific spontaneous degenerative or inflammatory conditions and the possibility of accidental infections unrelated to the experiment should not be confused with experimental outcome. Thus, despite our efforts to specify and simplify the criteria relevant for model-specific assessment and quantification of lesions, it appears crucial that trained histopathology experts be involved in the microscopical examination of mouse lungs , 85.Clearly, in addition to descriptive or semiquantitative histology, a number of other parameters may be useful for quantitative comparisons between experimental groups to determine the role of specific cell types, molecules, and therapeutic interventions, depending on the strategy and goal of the study . Such paIn conclusion, we have identified a spectrum of pathogen- and model-specific lesion characteristics in mouse models of acute pneumonia. Our findings underscore the necessity of model-specific criteria for the accurate histopathological characterization and quantitative assessments of experimental pneumonia. This comparative landscaping of acute mouse pneumonia histology provides a comprehensive framework for future studies on the role of individual pathogen or host factors, complex disease mechanisms, and novel therapeutic strategies that could help to treat pneumonia in human patients.


Drosophila suzukii (Matsumura), a pest of numerous economically important stone and small fruit crops, by sampling fruit-producing non-crop hosts adjacent to commercial plantings weekly from June through November in central New York over a two-year period. We found D. suzukii infestation rates (number of flies emerged/kg fruit) peaked mid-August through early September, with Rubus allegheniensis Porter and Lonicera morrowii Asa Gray showing the highest average infestation in both years. Interannual infestation patterns were similar despite a lower number of adults caught in monitoring traps the second year, suggesting D. suzukii host use may be density independent.Invasive, polyphagous crop pests subsist on a number of crop and non-crop resources. While knowing the full range of host species is important, a seasonal investigation into the use of non-crop plants adjacent to cropping systems provide key insights into some of the factors determining local population dynamics. This study investigated the infestation of non-crop plants by the invasive  Drosophila suzukii, the spotted wing drosophila, is a prime example of such a polyphagous, mobile pest. D. suzukii is an invasive pest of several major stone  and other soft-skinned fruit  crops worldwide [D. suzukii-related damage can have significant economic impacts [D. suzukii populations may inhabit and exploit multiple host types simultaneously, possibly affecting the effectiveness of on-farm pest management strategies.Many agricultural pests are highly polyphagous and exploit multiple crop and non-crop host plants throughout their life cycle ,2,3,4. Torldwide ,6,7,8. W impacts ,10. This impacts ,12 that  impacts ,14,15 inD. suzukii utilize non-crop resources is not well understood and likely varies on a regional basis. This is an especially pertinent question for the northern range of D. suzukii, as cold winters may kill off a subset of overwintering adults [D. suzukii emerge from overwintering sites before adequate fruit hosts ripen, but see [D. suzukii can potentially exploit locally-available springtime fruiting non-crop hosts in northern temperate regions to increase adult population levels that later infest summer-fruiting cultivated hosts. If these flies begin their annual infestation cycles in non-crop plants first, those plant species could act as sentinels for early season monitoring and predict infestation risk for growers. At the end of the growing season in northern latitudes, most commercial fruit production ends in October. However, D. suzukii may continue to infest available non-host crops before going into reproductive diapause, which is brought on by a number of factors, including cooler temperatures and shortened day length [The timing and extent to which g adults ,17 and s but see ,19. D. sy length ,21,22.D. suzukii arrives in a perennial agroecosystem, growers need responsive management strategies to protect their crops. Understanding how non-crop areas adjacent to commercial fruit crops can affect infestation risk throughout the fruiting season can help to inform management. This current study complements other studies that assessed non-crop host use at snapshot or periodic intervals . ,23. D. sD. suzukii-susceptible crops , while one location—a cornfield—was used as a comparison site. Wild-growing plants producing soft-skinned fruit adjacent to crop hosts were chosen for inclusion in this study. Ideally, chosen plant species were abundant enough to allow for replication within and across sites while individual plants produced a fruit set large enough to allow for multiple sample collections. Up to five samples  of sound, ripe fruit  per plant species were collected from each sampling location weekly depending on availability. For non-Rubus spp., we collected up to 100 individual fruits per sample. For Rubus, we collected 50 fruits/sample, due to their larger size and weight. While D. suzukii can oviposit into under-ripe fruit [The survey was conducted from June to November in 2013 and 2014. Candidate non-crop hosts were sampled weekly at eight commercial farms in the Finger Lakes region of New York, U.S.A., excepting one week in 2013. Seven farms grew pe fruit ,25, we cD. suzukii male or female, or other Drosophila species.Fruit samples were placed into individual plastic bags and brought back to the lab for processing. Fruit number and weight were recorded before being transferred to a 473.2 mL plastic container . Containers were modified with no-see-um mesh , venting the bottom and top to allow airflow and prevent moisture buildup. The ventilated containers were set into a 946.4 mL  container of the same circumference as the sample cup to collect any liquid released from the fruit. Samples were held at room temperature (21 °C) for two weeks, and monitored for fly development three times per week, for a total of six collection points. All visible pupae and flies were removed at each collection point. Pupae were placed in petri dishes lined with a moistened paper towel square and allowed sufficient time to eclose. Using a dissecting microscope, collected flies were identified as Cornus amomum Miller (Cornaceae); Morrow’s honeysuckle, Lonicera morrowii Asa Gray (Caprifoliaceae), and its hybrids; American pokeweed, Phytolacca americana L. (Phytolaccaceae); European buckthorn, Rhamnus cathartica L. (Rhamnaceae); blackberry, Rubus allegheniensis Porter (Rosaceae), and its hybrids; black raspberry, Rubus occidentalis L. (Rosaceae); and bittersweet nightshade, Solanum dulcamara L. (Solanaceae) (Cornus racemosa Lamarck (Cornaceae); black cherry, Prunus serotina Ehrhart (Rosaceae); choke cherry, Prunus virginiana L. (Rosaceae); red raspberry, Rubus idaeus L. (Rosaceae); and European cranberrybush, Viburnum opulus L. (Adoxaceae). These plants were not sampled in 2014 because either they were found in low abundance or were not infested in 2013  . In 2013 in 2013 .D. suzukii presence was monitored using two clear deli cup traps with 12.5 mm diameter entry holes placed approximately 25 mm from the top of each cup. A yeast, sugar, apple cider vinegar, whole-wheat flour, water mixture was placed in a small insert  within the container, covered with mesh, and surrounded by an apple cider vinegar, ethanol and surfactant drowning solution [D. suzukii, approximately 1 m from a known non-crop host, and at least 10 m from each other. In 2013, traps were placed at all sites, while in 2014, trapping was done at six of the eight sites. Trap catch was collected and bait redeployed each week. Contents of the traps were drained from the drowning solution and examined for D. suzukii and other Drosophila spp. using a dissecting scope. A subsampling procedure was implemented for any trap catch weighing more than 2 g after draining, whereby 10% by weight of the trap catch was randomly removed, counted in its entirety, and used to estimate total D. suzukii numbers. For catches greater than 15 g, a 5% subsample was counted. This methodology was verified by comparing subsample estimates to the full count of the entire sample for five trap collections. The subsample procedure has ~5% margin of error.At each site, solution . The twoD. suzukii caught per week was averaged across all sites. An ANOVA to compare trap catches between years was conducted on log + 0.001 transformed D. suzukii counts. For analysis, the total collection period was summarized by week and month and averaged across all sites. The comparison cornfield site was included with all other sites, as no major differences in infestation were observed. To control for differences in fruit size and weight among the different host fruits, a standardized calculation was made using the mean number of flies reared per kilogram of fruit collected. Pupae that did not produce adults were excluded from the data set. An analysis of variance (ANOVA) was conducted using R software (v3.2.2) to look for the effect of month and year on flies per kilogram  . InfestaLonicera morrowii and Rubus allegheniensis were the plants most heavily infested within and between years , and a male:female D. suzukii ratio of 0.88:1.In general, the pattern of infestation between years was similar—no to low infestation in June and July, a steady increase and peak in August, followed by a sharp decline beginning in mid-September through the end of November . Loniceren years . Plants p < 0.001, F4, 358 = 38.69) than between years , with no significant interannual difference in overall infestation rate for the seven plant species sampled . The baits used in these traps consistently captured more males than females, with a male:female ratio of 1.24:1.The first adult capture of ld hosts , a lag cnt rates . This trnt rates ,31. TrapD. suzukii biology and behavior. The principal difference between 2013 and 2014 results were the winter climate that preceded them, the 2012–2013 winter being largely in line with yearly averages. It was unseasonably cold from November 2013 to March 2014, with monthly low temperatures several degrees lower than average and a total snowfall of 1.88 m, as compared to an average annual snowfall of 1.37 m for the Geneva, NY region , ,23, 12,2mericana , oppositmericana . Lee et mericana  found siNew York . SeveralD. suzukii infestation, even though R. occidentalis is a known host and both L. morrowii and R. allegheniensis samples were later heavily infested. The cold winter preceding the 2014 growing season only delayed D. suzukii infestation by about a week and tracked with the later fruiting times of the sampled plant species. D. suzukii infestation was not detected until mid to late July in both years, indicating flies are not using farm-adjacent areas to build populations to later disperse into crop hosts. We were unable to identify a candidate non-crop host to function as an early season indicator of local D. suzukii presence . Although the plants sampled were abundant in our study area, we cannot exclude the possibility that other alternate springtime hosts may support emerging overwintered flies. However, the lack of infestation in known and locally abundant hosts such as Rubus spp. suggest long distance movement from warmer areas may be a more significant factor in establishing yearly D. suzukii populations than local overwintering adults. Ongoing research on overwintering and dispersal capabilities could help answer where springtime adults originate.As this study shows, early fruiting non-crop plants escape D. suzukii trap capture, but had no effect on the abundance of D. suzukii or overall pest pressure. In our study area, the largest effects from non-crop areas materialized in the middle of August, when the highest numbers of D. suzukii-susceptible hosts were available. As suggested by the number of trap captures and emerging adults per fruit sample, population density spiked during this period. Many non-crop plants stop fruiting in early September, leaving female D. suzukii searching for appropriate oviposition sites. This high density of flies may encourage dispersal into later fruiting crops, such as fall raspberries, figs, and day neutral strawberries—all susceptible to D. suzukii—making fruit protection from infestation more difficult.Our results align with those of Pelton et al. , who fouD. suzukii-susceptible crop fields, but was similarly infested as compared to the commercial fruit production sites (data not shown). There is still a large gap in our knowledge of the extent of this fly’s movement and dispersal capabilities. Other Drosophila spp. are known to move kilometers in a matter of days [D. suzukii infesting crop plants may have emerged from distantly located sources. Many of the wild hosts sampled in this study are common in disturbed areas along the edges of crop fields and roadsides. The specific species sampled in New York have congeners throughout the United States [D. suzukii population dynamics in these regions.The importance of these wild-growing plants for growers may also depend on where these plants are located. The cornfield location was not located near any  of days ,39,40; id States  and othed States  and may D. suzukii population source, it is also likely that some plants may function as population sinks. As Poyet et al. (2015) demonstrated, only a small percentage of eggs laid into species such as P. americana and R. cathartica survive to adulthood [D. suzukii density, these crop-adjacent areas provide additional benefits to growers. Wooded areas containing non-crop D. suzukii hosts near crop fields serve as important refuge sites for insecticide resistance management (IRM). D. suzukii is a crepuscular species, with some flies moving between wooded edge and crop habitat during morning and evening hours [D. suzukii management programs in conventional and organic systems rely primarily on a calendar-based insecticide rotation whenever ripe fruit is available, and there is concern resistance to certain classes of insecticides may develop. Left unmanaged, field edges may help delay the development of resistance, a vital component of IRM [D. suzukii [D. suzukii infestation will help inform a benefit/cost analysis as to the relative risk of these unmanaged areas.How our results can apply to on-farm management practices is less clear. Although non-crop host plants can clearly serve as a dulthood . Regardlng hours ,15. Currt of IRM ,43. Fielt of IRM ,46,47,48 suzukii ,50 have


Background: Abalones are large marine snails in the family Haliotidae and the genus Haliotis belonging to the class Gastropoda of the phylum Mollusca. The family Haliotidae contains only one genus, Haliotis, and this single genus is known to contain several species of abalone. With 18 additional subspecies, the most comprehensive treatment of Haliotidae considers 56 species valid [H. discus hannai genome, a total of 580-G base pairs using Illumina and Pacbio platforms were generated with 322-fold coverage based on the 1.8-Gb estimated genome size of H. discus hannai using flow cytometry. The final genome assembly consisted of 1.86 Gb with 35 450 scaffolds (>2 kb). GC content level was 40.51%, and the N50 length of assembled scaffolds was 211 kb. We identified 29 449 genes using Evidence Modeler based on the gene information from ab initio prediction, protein homology with known genes, and transcriptome evidence of RNA-seq. Here we present the first Haliotidae genome, H. discus hannai, with sequencing data, assembly, and gene annotation information. This will be helpful for resolving the lack of genomic information in the Haliotidae family as well as providing more opportunities for understanding gastropod evolution. Haliotis discus hannai is a widely used ingredient in East Asian cuisine and is a valuable food resource due to its richness in protein and other nutrients  was collected from the brood stock at the Genetic and Breeding Research Center of the National Fisheries Research & Development Institute on Geoje Island, Korea for sampling. Hemolymph (10 ml) was withdrawn from the sole side foot muscle using a syringe. For genomic DNA extraction, hemocytes were harvested from fresh hemolymph by centrifugation at 3000 × rpm for 5 min at 4°C. Genomic DNA was extracted using a DNeasy Animal Mini Kit . A total 39.38 μg of DNA was quantified using the standard procedure of Quant-iT PicoGreen dsDNA Assay Kit  with Synergy HTX Multi-Mode Reader . Quality of DNA was also checked using ND-1000 spectrophotometer .A single wild abalone . The estimated genome size of H. discus hannai using 19-mer distribution was about 1.65 Gb. Based on the 19-mer distribution of paired-end reads, there was a second peak located in the half x-axis of the main peak. This result indicates that the H. discus hannai genome had high heterozygous genetic character or probable DNA contamination from other organisms. Therefore, before genome assembly, raw reads from Hiseq2000 and Nextseq500 paired-end and mate pairs were preprocessed by bacterial sequences, duplicates, and ambiguous nucleotides. To remove the contaminant sequence, clean reads without adapter and low quality bases were mapped to bacterial and ocean metagenome databases downloaded from NCBI by applying the default setting run (-s 0.8 –l 0.5) of clc_mapper (https://www.qiagenbioinformatics.com/). After that, duplicates and ambiguous nucleotides were filtered out using clc_remove_duplicates (https://www.qiagenbioinformatics.com/). The resulting high-quality sequences were used in subsequent assembly. Error correction and initial contig assembly was conducted using clc_assembler within the CLC Assembly Cell (https://www.qiagenbioinformatics.com/products/clc-assembly-cell/) software pipeline. Scaffolds were then built using the mate-pairs and Pacbio RS II reads sequentially by SSPACE [For whole genome shotgun sequencing and draft genome assembly, we used multiple sequencing platforms  with seven different libraries. First, two paired-end libraries with insert sizes of 250 and 350 bp were constructed using Illumina TruSeq DNA Sample Prep. Kit . Mate pair libraries with insert sizes of about 3, 5, 8, and 10 k were constructed for scaffolding process using Illumina Nextera mate-pair library construction protocol (Illumina). For high-quality genome assembly, long mate pair library with insert size over 40 kb is essential. We tried to construct a long mate pair library using 40 kb fosmid clone. However, efficiency of fosmid library construction was very low and we could not retain enough amount of clone. Therefore, Pacbio system was employed for final scaffolding process using long read. Pacbio long reads were generated using P6-C4 chemistry of Pacbio RS II system. Detailed information about the constructed library and generated sequencing data is provided in Table y SSPACE  and PBJey SSPACE . After sy SSPACE  using -lH. discus hannai. After custom library construction, RepeatMasker with RMBlast was used for each genome with ‘no_is’ option, using repeat libraries from RepeatModeler and Repbase. Identified mobile elements are summarized in Table H. discus hannai was 1.86 Gb, and this is the biggest genome among known gastropods. It is 5.31 and 2.02 times larger than genomes size of Lottia gigantea (0.35 Gb) and Aplysia californica (0.92 Gb) in the same Gastropoda class. In animals, the increase of genome size is commonly driven by transposable element, and this is a known genetic adaption mechanism to stressful environments [L. gignatea, a similar marine gastropod with large genome size difference from that of H. discus hannai, to identify the reason for this large difference. Fig. H. discus hannai and L. gigantea is 30.76% and 22.25%, respectively. And the total amount of identified repeat elements in the H. discus hannai genome is almost six times larger than that of L. gigantea same as genome size. Such a linear relationship between genome size and the total proportion of repeat elements is consistent with a previous study [H. discus hannai genome than in L. gigantea genome. Especially, the number of a non-LTR retrotransposon called LINE Element was exceptionally high. Fig. H. discus hannai: LINE/I, DNA/TcMar-Tc1, L. gigantea: DNA/RC, DNA/Maverick) in each genome. DNA/RC and DNA/Maverick, two major mobile elements in L. gigantea genome, are observed in H. discus in somewhat similar distribution. On the other hand, the two signature mobile elements of H. discus hannai genome, LINE/I and DNA/TcMar-Tc1, are specifically abundant in H. discus hannai and seems to have expanded recently diverged compared to other elements. In sum, species specificity can be inferred from the distinctive patterns of repeat element expansion between the two species and the increased genome size of H. discus hannai may be associated with the non-LTR elements  contribution, in parallel to the human genome [Before conducting gene prediction using the assembled sequence, repeat elements were identified using RepeatMasker  with Repronments . Therefous study . The pron genome .ab initio, RNA-seq transcript based, and protein homology-based. For RNA-seq transcript based prediction, transcriptome data from six organ tissues  [ab initio gene prediction, Augustus [H. discus hannai. Manual curation was performed based on the genomic DNA mapping position of the RNA-seq sequence and the protein sequence of the related species. To determine the exon-intron edge of the gene, the genome mapping information of the transcriptome sequence was firstly reflected, and if not, the mapping information of the protein sequence of the related species was referred to secondarily to confirm the gene model. Finally, genes that were not translated into protein sequences in the final gene model were removed. A total of 29 449 genes was predicted in the H. discus hannai genome and summary statistics for the consensus gene set is provided in Table H. discus hannai draft genome, we conducted paired-end read remapping and BUSCO  analysis. 94.89% of paired-end reads with a 350-bp insert size were successfully mapped to the assembled genome and assembled genome contains 609 complete and 130 fragmented genes in BUSCO analysis. The detailed information of BUSCO analysis is summarized in Table Genes were predicted through three different algorithms: es Table  were ali ≤ 1E-4) . Next, t ≤ 1E-4)  to prediAugustus  was traiAugustus  and GeneAugustus  were alsAugustus  to buildH. discus hannai based on various genetic evidence. We expect that the H. discus hannai genome presented here, which is the first genome to be sequenced in the family Haliotidae, will provide useful genomic information for many researchers. H. discus hannai is a cold-water abalone breed that has difficulties dealing with the change in their inhabitable latitude, which is due to global warming and the resulting increase in the rate of sudden perishing. Genomic information of abalone is essential information that can be used for genetic breeding to improve productivity and genetic engineering for the heat resistance breed. It can also provide valuable information for future genomic studies, because only limited genome information about marine animals and mollusks is currently available. Evolutionary signatures recorded in the abalone genome can be identified through future comparative genomic studies and we expect our result will provide more insight into Haliotidae and marine mollusk evolution.In summary, here we report the first annotated Haliotidae genome of GigaScience GigaDB [Raw data is available in project accession PRJNA317403 in the NCBI database. Further supporting data can be found in the e GigaDB .EVM - Evidence ModelerBUSCO - Benchmarking Universal Single-Copy OrthologsAll authors report no competing interests.Sampling - Bo-Hye Nam, Young-Ok Kim, Dong-Gyun KimSequencing - Bo-Hye Nam, Hee Jeong Kong, Woo-Jin Kim, Jeong-Ha Kang, Ji-Young Moon, Choul Ji Park, Duk Kyung KimGenome assembly - Bo-Hye Nam, Woori Kwak, Jae Woong Yu, Joon Yoon, SaetByeol Lee, Samsun Sung, Chul Lee, Sojeong Ka, Kelsey Caetano-AnollesRepeat element analysis - Woori Kwak, Minseok Seo, Kwondo KimGene prediction - Woori Kwak, Younhee Shin, Myunghee Jung, Byeong-Chul Kang, Ga-hee ShinFunding and experimental design – Jung Youn Park, Cheul Min An, Seoae Cho, Heebal KimH. discus hannai.Figure S1. Tree map for sum of repeat element for H. discus hannai and L. gigantea.Figure S2. Comparison of SINE element distribution of H. discus hannai and L. gigantea.Figure S3. Comparison of LINE element distribution of H. discus hannai and L. gigantea.Figure S4. Comparison of LTR element distribution of H. discus hannai and L. gigantea.Figure S5. Comparison of DNA transposon element distribution of GIGA-D-16-00149_Original_Submission.pdfClick here for additional data file.GIGA-D-16-00149_Revision_1.pdfClick here for additional data file.Response_to_reviewer_comments_Original_Manuscript.pdfClick here for additional data file.Reviewer_1_Report_.pdfClick here for additional data file.Reviewer_2_Report_.pdfClick here for additional data file.Reviewer_3_Report_.pdfClick here for additional data file.Supplemental materialH. discus hannai.Figure S1. Tree map for sum of repeat element for H. discus hannai and L. gigantea.Figure S2. Comparison of SINE element distribution of H. discus hannai and L. gigantea.Figure S3. Comparison of LINE element distribution of H. discus hannai and L. gigantea.Figure S4. Comparison of LTR element distribution of H. discus hannai and L. gigantea.Figure S5. Comparison of DNA transposon element distribution of Click here for additional data file.


Zymoseptoria tritici cellular ATP prevents WBs from pore sealing in unwounded cells. Thus, cells appear to exert active control over WB closure. Here, we summarize our current understanding of WB-based pore sealing in ascomycete fungi.In ascomycete fungi, hyphal cells are separated by perforate septa, which allow cell-to-cell communication. To protect against extensive wound-induced damage, septal pores are sealed by peroxisome-derived Woronin bodies (WBs). The mechanism underpinning WB movement is unknown, but cytoplasmic bulk flow may “flush” WBs into the pore. However, some studies suggest a controlled and active mechanism of WB movement. Indeed, in the wheat pathogen  Whilst early studies suggested that each septum is flanked by two rings of 6 WBs . This confers rigidity to the WB and thus facilitates pore plugging.N. crassa Hex1 structure  and ImageJ (https://imagej.nih.gov/ij/).Liquid cultures were prepared for electron microscopy as described previously . Section6.2N. crassa , using options for VTFM optimization, thorough MD optimization, and repeated optimization. The best of ten models was selected on the basis of the MODELLER energy function, and Ramachandran plot quality.Structural modeling of ZtHex1 was based on the published structure of Hex1 from


Campylobacter spp. are a common cause of mostly self-limiting enterocolitis. Although rare, pericarditis and myopericarditis have been increasingly documented as complications following campylobacteriosis. Such cases have occurred predominantly in younger males, and involved a single causative species, namely Campylobacter jejuni. We report the first case of myopericarditis following Campylobacter coli enterocolitis, with illness occurring in an immunocompetent middle-aged female.Campylobacter spp. Matrix assisted laser desorption ionization time-of-flight (Bruker) confirmed the cultured isolate as C. coli.A 51-yo female was admitted to a cardiology unit with a 3-days history of chest pain. The woman had no significant medical history or risk factors for cardiac disease, nor did she report any recent overseas travel. Four days prior to the commencement of chest pain the woman had reported onset of an acute gastrointestinal illness, passing 3–4 loose stools daily, a situation that persisted at the time of presentation. Physical examination showed the woman’s vital signs to be essentially stable, although she was noted to be mildly tachycardic. Laboratory testing showed mildly elevated C-reactive protein and a raised troponin I in the absence of elevation of the serum creatinine kinase. Electrocardiography (ECG) demonstrated concave ST segment elevations, and PR elevation in aVR and depression in lead II. Transthoracic echocardiogram (TTE) revealed normal biventricular size and function with no significant valvular abnormalities. There were no left ventricular regional wall motion abnormalities. No pericardial effusion was present but the pericardium appeared echodense. A diagnosis of myopericarditis was made on the basis of chest pain, typical ECG changes and troponin rise. The chest pain resolved and she was discharged from hospital after 2-days of observation, but with ongoing diarrhoea. Following discharge, a faecal sample taken during the admission, cultured Campylobacter coli enterocolitis. Although rare, myopericarditis is becoming increasingly regarded as a complication following campylobacteriosis. Our report highlights potential for pericardial disease beyond that attributed to Campylobacter jejuni. However uncertainty regarding pathogenesis, coupled with a paucity of population level data continues to restrict conclusions regarding the strength of this apparent association.We report the first case of myopericarditis with a suggested link to an antecedent  Campylobacter species are among the most widespread and commonest cause of bacterial enterocolitis worldwide [C. jejuni is recognised as the leading cause of human illness, although C. coli might account for up to 25% of Campylobacter enterocolitis [C. coli enterocolitis and discuss the epidemiology and pathogenesis of this condition.orldwide . C. jejuocolitis . We descA 51-yo female was admitted to the cardiology unit of an Australian public hospital with a 3-day history of continuous, dull, non-radiating, non-pleuritic, left lateral chest pain, with associated diaphoresis, nausea, palpitations and pre-syncope. Some relief of chest pain was achieved by leaning forward. Administration of combined paracetamol and codeine phosphate also relieved the pain. Four days prior to the commencement of chest pain the woman had reported onset of an acute gastrointestinal illness, passing 3–4 loose stools daily. This diarrhoea was persistent at the time of presentation. The woman had no significant medical history or risk factors for cardiac disease, nor did she report any recent overseas travel.Physical examination showed the woman to be afebrile but mildly tachycardic, with a heart rate of 106 bpm. Blood pressure was 120/76 mm Hg. Examination of the chest revealed normal heart sounds, without any murmur or rub. Jugular venous pressure was normal. Pulmonary examination was normal with no signs of respiratory distress (RR 18), although mild tachypnea was noted during the course of her admission. The abdomen was soft and non-tender.Laboratory testing showed a mildly elevated C-reactive protein  and a raised troponin I  in the absence of elevation of the serum creatinine kinase. Liver enzymes were mildly elevated with an ALT 65 U/L  and ALKP 142 U/L . Full blood count and serum electrolytes were unremarkable.Serial electrocardiographs demonstrated concave ST segment elevations involving leads I, II, III, aVF and V6, PR elevation in aVR and PR depression in lead II Fig. . DiffereCampylobacter spp. in isolation. Matrix assisted laser desorption ionization time-of-flight (Bruker) confirmed the cultured isolate as C. coli.Clexane and clopidogrel were ceased and colchicine 500mcg bd commenced. The chest pain resolved and she was discharged from hospital after 2 days, but with ongoing diarrhoea. Following hospital discharge, a faecal sample taken during the admission, cultured Acute pericarditis is a not uncommon, frequently idiopathic disorder that mostly follows a benign clinical course. Some myocardial involvement often accompanies acute pericarditis, with the term myopericarditis indicating the pericardium as the primary focus . VirusesCampylobacter spp. have an uncommon but increasingly recognised link with myopericarditis, as supported by a growing body of case reports and reviews of literature [C. coli, a species well recognised as a cause of enterocolitis, but not previously reported in association with myopericarditis. Secondly, the patient was a healthy, middle-aged female, in contrast to previous patients where most were younger immunocompetent males [Campylobacter does show tendency to cause higher rates of disease in men compared to women, the apparent predilection towards men in myopericarditis is striking. However despite this observation, epidemiological evidence of a causal association between campylobacteriosis and myopericarditis is lacking. We identified a single study that showed a greater incidence of myocarditis in Campylobacter positive cases than controls at 16.1 cases per 100,000 person-years (95% CI 2.3–114.4) compared to 1.6 cases per 100,000 person-years (95% CI 0.2–11.4), but the result was not significant and absolute case numbers were small [terature –6. Our rnt males –6. Althore small .C. jejuni or C. coli and that with C. fetus. The former are recognised causes of enteric disease in humans and likely represent archetypal causes of Campylobacter myopericarditis. Conversely C. fetus is an atypical, invasive species, capable of evading the host immune system by means of complement resistant surface proteins [C. jejuni (and C. coli) associated myopericarditis, the putative mechanisms include direct infection of the pericardium or myocardium, an immune hypersensitivity reaction, or the effect of bacterial toxins [C. jejuni detection in pericardial fluid [C. jejuni and development of other immune-mediated disease  [Campylobacter-associated cardiotoxin [C. coli myopericarditis as highlighted by our case might possibly be explained by the dominance of C. jejuni at the species level, along with the lower incidence of cytotoxin and enterotoxin production by C. coli compared to C. jejuni [It is important to make distinction between myopericarditis associated with proteins . As suchproteins . For C. l toxins . Currental fluid , 12. A dthritis) . Althougdiotoxin , the shodiotoxin , 6. Furt. jejuni .Campylobacter spp. myopericarditis, however a recent examination of C. jejuni-associated myopericarditis cases showed treatment with either macrolide or fluoroquinolone antibiotics to be a common practice although without any apparent consensus on dosage and treatment duration [Campylobacter spp. in stool occurred following hospital discharge, with a mechanism for myopericarditis such as direct infection or toxin-mediated response not able to be readily determined. As such antimicrobial therapy was not commenced. Treatment for the myopericarditis was conservative, using nonsteroidal anti-inflammatory drugs (NSAIDS) and colchicine. Among case reports describing Campylobacter-associated myopericarditis we identified a single episode where colchicine, as an adjunctive to aspirin, was used in treating myopericarditis, with the case reported as fully recovered at a 3-month review [C. jejuni (and C. coli) associated myopericarditis appear favourable [It is unclear whether antibiotic treatment influences the outcome of duration . Howeverduration . For ourh review . Anecdotvourable  with lowvourable .C. coli enterocolitis, with the illness running a benign clinical course. While increasingly recognised as a complication of campylobacteriosis, the paucity of population level data restricts further conclusions on the strength of any association between Campylobacter infection and myopericarditis. Further, the mechanism(s) of pathogenesis also remain uncertain, although anecdotally may favour a toxin-mediated response.We report the first case of myopericarditis linked to a


Angiostoma norvegicum n. sp. (Angiostomatidae) is described from the oesophagus, crop and the buccal mass of five species of slugs of the family Arionidae, Arion vulgaris (Moquin-Tandon), Arion ater (L.), Arion fasciatus (Nilsson), Arion fuscus (Müller) and Arion rufus/Arion ater hybrid), collected throughout Norway. Angiostoma norvegicum n. sp. was found parasitising arionids at seven of the 30 sample sites examined (23.3%), and 9.9% of all Arion spp. were infected with this nematode. The new species is characterised by its large size (4.0–8.6 mm long) and in having: lateral alae; 6 + 6 papillae at the cephalic end; a large circular mouth aperture; a spacious stoma; a pharyngeal basal bulb without valvular apparatus; an excretory pore near the base of bulb; a distal part of posterior ovary always outstretched; an anterior ovary distally nearly always outstretched; a vulva situated anterior to mid-body; long, nearly straight spicules and a small gubernaculum; three circumcloacal papillae and caudal genital papillae (GP) arranged in a pattern 1+2/3+3 with GP 5 and GP 8 opened on dorsal side of narrow bursa not reaching tail tip; short conical tails in both sexes with tips supplied by 4 short, unequal denticles. Morphologically, A. norvegicum n. sp. is similar to Angiostoma limacis Dujardin, 1845, which diagnostic characteristics are given based on examination of specimens from Norway and the UK. Conversely, the phylogenetic analyses based on D2D3 large subunit (LSU) rRNA gene sequences performed in the present study did not support the morphological affinity of these two species. Phylogenetic analyses demonstrated that although Angiostoma spp. cluster together, A. norvegicum n. sp. forms a tight monophyletic clade with the milacid nematode parasites Angiostoma margaretae Ross, Malan & Ivanova, 2011 and Angiostoma milacis Ivanova & Wilson, 2009. The association between nematodes and terrestrial molluscs is poorly understood; however their host specificity, diversity of specialisations and mechanisms of infection, indicate that their affiliation is both ancient and widespread  Andrássy, 1983, Agfa flexilis , Alloionema appendiculatum Schneider, 1859 and another species of the same genus, Angiostoma limacis Dujardin, 1845. The new species is described and illustrated below. To demonstrate fine differences between Angiostoma norvegicum n. sp. and the morphologically closest A. limacis, original illustrations of certain morphological structures of A. limacis are also provided.The family Angiostomatidae is comprised of two genera: Slug collectionArion spp. were collected from 30 sample sites in Norway. Habitats included roadside verges, domestic gardens and agricultural land. Localities were found in cooperation with local advisory services and coordinates were recorded. Slugs were collected during late August, September and October 2011, and identified through morphological examination, dissection of genitalia and analysis of mitochondrial DNA  gene  and submitted to the GenBank database, at the National Centre for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).Ethanol-preserved nematodes were transferred to a Chelex/Proteinase K mix for DNA extraction , A. limacis (GQ167725), A. dentiferum  (GQ167726), Angiostoma milacis Ivanova & Wilson, 2009 (FJ949063), Angiostoma margaretae Ross, Malan & Ivanova, 2011 (KU712562), along with Caenorhabditis remanei  (AY602174), Rhabditella axei  (AY602177) and Oscheius myriophilus  (AY602176) which were used as the outgroup . A total of nine nematode D2D3 LSU rRNA gene sequences were compiled and aligned manually using BioEdit Sequence Alignment Editor .Other hosts: Arion ater (Linnaeus), A. fasciatus (Nilsson), A. fuscus (Müller) and A. rufus/A. ater hybrid.Type-locality: Bolsøya , Norway .Other localities: See Table Type-material. Holotype female (19 299), paratype female (11 296), paratype male (21 299) and paratype male on a slide with a paratype female (22 294) deposited in USDA (US Department of Agriculture) Nematode Collection, ARS, Nematology Laboratory, Beltsville, MD, USA.Site in host: Isolated from the oesophagus, crop and the buccal mass of slug hosts.Representative DNA sequences: The sequences obtained for A. norvegicum n. sp. were deposited in NCBI GenBank under accession numbers KU712560 (SSU rRNA gene), KU712561 (D2D3 LSU rRNA gene) and KU710221 (cox1) gene. Sequences of A. norvegicum n. sp. were identical across each gene, so only one representative sequence per gene was submitted to GenBank.Etymology. The species name refers to locality of the gastropod host.General. Body long, cylindrical, slightly tapering to anterior . Proximally, ovaries making two bends followed by short oviducts evidently serving as spermathecae containing large sperm cells c.7–8 in diameter. Oviducts leading to spacious, divergent, equally long (c.800) uteri. Each uterus containing 40–50 eggs , the presence (vs absence) of lateral alae, the presence (vs absence) of a prominent precloacal flap in males, distally outstretched ovaries  (vs reflexed ovaries). Slight differences between these species include thinner stoma walls (mean 3 µm in A. norvegicum n. sp. vs 5 µm in A. limacis) and different proportions of stoma  and pharynx   Fig. A. Also, Clb Fig. Angiostoma norvegicum n. sp. also resembles Angiostoma asamati Spiridonov, 1985, A. milacis and A. margaretae, by having a very similar stoma shape and the presence of lateral alae. However, it can be easily distinguished by the different shape of the tail  , not off-set lip region (vs off-set lip region), tail tip with denticles (vs without denticles), enlarged rectal glands  and different arrangement of genital papillae , longer spicules (mean 119 vs 80 µm) and a much larger stoma , leaving A. glandicola as a sister group to the other Angiostoma spp. Within this group, A. norvegicum n. sp. formed a monophyletic clade with the two milacid parasites, A. margaretae and A. milacis, under strong bootstrap support (96/96/95).Identical tree topologies were obtained from maximum likelihood (ML), maximum parsimony (MP) and distance analyses. Therefore only the maximum-likelihood tree is presented Fig.  along wiA. norvegicum n. sp., is described from five Arion slug hosts  collected throughout Norway. The nematode was isolated from the oesophagus, crop and the buccal mass of its hosts, thus differing somewhat from other Angiostoma spp. that have been isolated from the intestine , however mean intensity of infection with A. norvegicum n. sp. is higher in all infected host species and particularly in the type-host, A. vulgaris. This species also known as the Iberian or Spanish slug, is highly invasive and its current distribution includes mainland Europe, UK, Ireland and USA  and even shows a greater susceptibility to slug-associated nematodes than native slug species. This fact can point on the limited ability of the host to tolerate nematode invasion in the conditions of the certain area. The presence of the heavy, combined nematode infection  of the invasive slug species on the northern boundary of its distribution, together with its absence closer to its native range  was the highest ever recorded for a species of the Angiostomatidae. However the absence of A. norvegicum n. sp. at certain study sites indicates that the infection of A. norvegicum n. sp. was first based on native larger arionids, i.e. A. ater and A. ater/A. rufus. It can be assumed that A. norvegicum n. sp. was transmitted to A. vulgaris through feeding on other infected Arion spp.The prevalence of Angiostoma are not strictly host-specific, however the infection of certain nematode species tends to be based on related host species , from the families Limacidae and Arionidae, respectively. However further studies demonstrated the presence of A. dentiferum in Limax spp. slugs only .Members of the genus  Mengert  indicate Morand, . MorpholA. norvegicum n. sp. brings the total complement of the genus to 18 species, 14 from terrestrial molluscs and four from amphibian and reptile hosts  and vertebrate (definitive) hosts. In addition, Angiostoma spp. parasitic in both molluscan and vertebrate hosts have been isolated in their adult stages, indicating that they are in their final phase of development. The resolution of the genus can only be confirmed through molecular and phylogenetic analysis of the genus; however, to date insufficient molecular data are available for Angiostoma spp. from vertebrate hosts.The identification of


Deinococcus marmoris strain PAMC 26562 was isolated from Usnea sp., a lichen collected from King George Island, Antarctica. We report here the draft genome sequence of strain PAMC 26562, which has xanthorhodopsin and carbon monoxide dehydrogenase genes in addition to major metabolic pathways presented in deinococcal genomes. Deinococcus-Thermus constitutes one of the major bacterial evolutionary lineages  (The phylum lineages . Species species . The strJ585986) .k-mer values from 5 to 65. The longest N50 and contigs were reached when the k-mer was 27. Bowtie-2 (version 2.2.6) was used to generated a *.sam file (Here, we report the draft genome sequence of the strain PAMC 26562. Strain PAMC 26562 was isolated from a lichen collected from King George Island, Antarctica . The drasam file . BEDToolsam file ; IlluminDeinococcus.There were 5,027 protein-coding sequences in total, and they were annotated by the RAST server , accordiDeinococcus marmoris strain PAMC 26562 has been deposited at DDBJ/ENA/GenBank under the accession no. MSTI00000000. The version described in this paper is version MSTI01000000.This whole-genome shotgun project for


Burkholderia mallei and B. pseudomallei are the causative agents of glanders and melioidosis, respectively, diseases with high morbidity and mortality rates. B. mallei and B. pseudomallei are closely related genetically; B. mallei evolved from an ancestral strain of B. pseudomallei by genome reduction and adaptation to an obligate intracellular lifestyle. Although these two bacteria cause different diseases, they share multiple virulence factors, including bacterial secretion systems, which represent key components of bacterial pathogenicity. Despite recent progress, the secretion system proteins for B. mallei and B. pseudomallei, their pathogenic mechanisms of action, and host factors are not well characterized.B. mallei. Here, we report an expansion of the database with corresponding information about B. pseudomallei. DBSecSys 2.0 contains comprehensive literature-based and computationally derived information about B. mallei ATCC 23344 and literature-based and computationally derived information about B. pseudomallei K96243. The database contains updated information for 163 B. mallei proteins from the previous database and 61 additional B. mallei proteins, and new information for 281 B. pseudomallei proteins associated with 5 secretion systems, their 1,633 human- and murine-interacting targets, and 2,400 host-B. mallei interactions and 2,286 host-B. pseudomallei interactions. The database also includes information about 13 pathogenic mechanisms of action for B. mallei and B. pseudomallei secretion system proteins inferred from the available literature or computationally. Additionally, DBSecSys 2.0 provides details about 82 virulence attenuation experiments for 52 B. mallei secretion system proteins and 98 virulence attenuation experiments for 61 B. pseudomallei secretion system proteins. We updated the Web interface and data access layer to speed-up users’ search of detailed information for orthologous proteins related to secretion systems of the two pathogens.We previously developed a manually curated database, DBSecSys, of bacterial secretion system proteins for B. mallei and B. pseudomallei. They enable studies and comparisons of corresponding proteins of these two closely related pathogens and their host-interacting partners.The updates of DBSecSys 2.0 provide unique capabilities to access comprehensive information about secretion systems of http://dbsecsys.bhsai.org.The database is available at  Burkholderia mallei is the causative agent of glanders, and its phylogenetically closest species, B. pseudomallei, is the causative agent of melioidosis. B. mallei evolved from an ancestral strain of B. pseudomallei by genome reduction and adaptation to an obligate intracellular lifestyle [B. mallei and B. pseudomallei represent an emerging public health threat in their natural environment and as a potential bioterrorism threat. Although the two bacteria cause different diseases, they share multiple virulence factors, including bacterial secretion systems, which represent key components of bacterial pathogenicity [B. mallei and B. pseudomallei pathogenicity [The highly infectious pathogen ifestyle . Given tgenicity , 3. Recegenicity , 5, but Burkholderia proteins [Burkholderia species. In addition, existing databases of secretion systems for multiple pathogens provide general information about secretion system proteins of B. mallei and B. pseudomallei but lack specific information about protein functions and, especially, do not provide information about pathogenic mechanisms of action, experimental results, or host-interacting partners.Several database systems provide general information about proteins –8, whereproteins –12 or muproteins , 14 bactB. mallei and their host factors, we previously developed the Database of Burkholderia mallei Secretion Systems (DBSecSys) [B. pseudomallei. This updated version, DBSecSys 2.0, provides not only annotation for B. mallei and B. pseudomallei proteins associated with secretion systems but also manually curated information about these proteins, their involvement in virulence, their host targets (proteins), and their mechanisms of action inferred from literature and host-pathogen interaction data. These features provide an ability to study and compare secretion systems of the two pathogens not only through characterization of their corresponding pathogen proteins but also through characterization of their host-interacting partners. These data and features are currently not available elsewhere, and they make DBSecSys 2.0 a unique resource for B. mallei and B. pseudomallei secretion system research.To catalogue in-depth information about secretion system proteins of BSecSys) . Since iBurkholderia species and our experimental and computational work on B. mallei [B. mallei ATCC 23344 proteins and 281 B. pseudomallei K96243 proteins and their associated secretion systems, including 200 orthologous proteins. The majority of the proteins are associated with five secretion systems. In the updated database, we also included several recently identified secreted proteins that have been associated with virulence, but whose secretion mechanisms have not yet been determined (denoted as Undetermined Type in the database). We did not include information about contact-dependent interactions, which are involved in bacterial-bacterial interactions for B. pseudomallei [Table . mallei , 17, we domallei , becauseB. mallei secretion system proteins and 98 virulence attenuation experiments for 61 B. pseudomallei secretion system proteins. For a subset of these experiments, we transferred virulence attenuation between orthologous proteins of the two pathogens. In these cases, we provided evidence that the inferred virulence attenuation is based on homology and, thus, needs experimental verification.Published experimental data served as the main source of information for 82 virulence attenuation experiments for 52 B. mallei and human and murine hosts. Thus, we imported PPIs between B. mallei and 1,633 host proteins (795 human and 838 murine) from the previous version. Experimental host-pathogen PPI data for B. pseudomallei are scarce. We found only 4 published PPIs between B. pseudomallei and the human host [B. pseudomallei, we mapped host-pathogen PPIs for B. mallei proteins in the database to their orthologous B. pseudomallei proteins. PPIs between B. mallei protein and host proteins were identified previously in yeast two-hybrid (Y2H) experiments, i.e., they were based on direct protein interactions. Thus, the transfer of PPIs to orthologous B. pseudomallei proteins can be justified. The database now contains 2,400 host-pathogen PPIs between B. mallei and these 2 hosts and 2,286 host-pathogen PPIs between B. pseudomallei and these hosts. As previously described [B. mallei proteins in Y2H experiments [Since the publication of the first version of the database DBSecSys, there were no new published protein-protein interactions (PPIs) between man host . To provescribed , DBSecSyescribed  among 35escribed , 22 amoneriments .B. mallei and B. pseudomallei using literature, experiments, orthology between B. mallei and B. pseudomallei proteins, and computational methods. For all experimental data and observations contained in DBSecSys 2.0, we provided links to their original sources and publications through PubMed.We identified secretion system proteins of B. mallei and murine-B. mallei protein interaction data have been identified in Y2H experiments. As PPI detection experiments did not exhaustively screen for all possible host-B. mallei interactions, we used human-murine orthology information based on the HomoloGene [B. pseudomallei to proteins that have orthologs to B. mallei proteins. We manually curated information about orthologous proteins between the two pathogens from the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways [Human-moloGene  databasemoloGene , 16. Usipathways  and Quarpathways , includiB. mallei and B. pseudomallei proteins, we used information from host PPI networks. For human PPIs, we used data from an experimentally detected PPI network compiled by Yu et al. [To account for the relationship among host proteins that interact with u et al. , whereasu et al. , 22.B. mallei proteins, we used mechanisms of action based on the literature, Y2H experiments, and additional computations, as previously described [B. pseudomallei proteins, we used the literature as the primary source of information about mechanisms of action. To provide additional information about possible mechanisms of action, we assigned all mechanisms of action bi-directionally between orthologous proteins of B. mallei and B. pseudomallei.We inferred pathogen mechanisms of action for proteins in the database using common terms from the available literature shown in Table escribed . For B. We used the National Center for Biotechnology Information (NCBI)  and Unip1) a backend Oracle database server that stores the data contained in the DBSecSys 2.0 database, 2) the controller, and 3) the presentation tier. We used Java Platform, Enterprise Edition 7, JavaServer Faces 2, and ICEfaces 3 technologies. The presentation tier also includes JBrowse [http://dbsecsys.bhsai.org.In DBSecSys 2.0 database and Web interface, we used a three-tier architecture, which we developed for the previous version : 1) a ba JBrowse , D3.js [ JBrowse , NVD3.js JBrowse , and Cyt JBrowse  JavaScri1) pathogen protein or host protein annotation, 2) pathogen proteins associated with a secretion system, 3) pathogen proteins associated with the pathogen’s mechanism of action, 4) host-pathogen interactions, and 5) experimentally screened virulence factors. We provided details of these functionalities for a single organism in the previous version of the database, which have been updated in the current User’s Guide. Users can download query results in a tab-delimited format and host-pathogen PPIs in the Proteomics Standards Initiative Molecular Interactions (PSI-MI) Tab 2.5 format [Users can browse and query cross-linked data in DBSecSys 2.0 through a Web-based graphical user interface, which supports five functionalities: query for 5 format . DBSecSyImportantly, the new database content and design enable comparative analysis between organisms. We describe two such applications below.B. mallei, to display the protein page with detailed annotation information about this protein, including its Locus Tag BMAA0729, evidence about its association with the type 2 secretion system (T2SS), virulence attenuation experiments, inferred mechanisms of action, and host-pathogen interactions. Selecting the box labeled “Pathogen Ortholog Comparison” and clicking the link for BMAA0729, the database returns information about TssM’s orthologous protein in B. pseudomallei, with Locus Tag BPSS1512, on the lower half of the screen. This enables direct comparison of annotations for the two orthologs. The information about orthologs is very similar because of the database curation strategy to transfer virulence information, mechanisms of action, and host-pathogen interactions between two orthologous proteins. Comparison between secretions systems of these orthologous proteins provides additional insights.Users can study individual (pathogen or host) proteins on the “Proteins” page of the Web interface. Users can search for association of a pathogen protein with a secretion system using standard protein/gene identifiers, such as Locus Tag, Name, or Uniprot ID. When the ortholog of the query protein is present in the database, DBSecSys 2.0 provides a link to it. For example, a search by Name for the virulence factor TssM , 33 retuBurkholderia mallei (strain ATCC 23344) and Burkholderia pseudomallei (strain K96243)” from the drop-down list “Select Pathogen.” The lists of proteins in the corresponding secretion systems of the two pathogens are displayed on the same page and enable direct comparison between individual proteins. In addition, users can open the link to the genome browser, which depicts the positions of these proteins on the genomes of the two organisms, including linear genome maps and schematic circular chromosomes. Positioning the mouse over the marked red bands on the circular chromosomes shows individual proteins or secretion system clusters. Clicking at the positions on chromosome 2 of each organism corresponding to TssM, BMAA0729 in B. mallei and BPSS512 in B. pseudomallei, shows their positions on the corresponding linear genome maps. In addition, by adjusting the coverage of the genomic regions, users can visualize the position of this T2SS protein relative to the adjacent type 6 secretion system (T6SS) proteins in B. mallei and B. pseudomallei, as shown in Fig. B. mallei and BPSS1513 in B. pseudomallei. Previously, we found that both transposon insertion and deletion of this gene in B. mallei fully attenuate virulence [Users can study pathogen proteins associated with a specific secretion system on the “Secretion Systems” page of the Web interface. Continuing the previous example, users can compare secretion systems for two TssM proteins by clicking the link to T2SS and selecting the link “Compare irulence , 34. Becirulence –37, an iB. pseudomallei, which was recently identified as an important virulence factor for B. pseudomallei [B. mallei ortholog for this protein, because the corresponding genomic region was originally annotated as a pseudogene [B. mallei codes for two proteins, designated as BMAA0736A and BMAA0736B [B. pseudomallei, and their protein sequences in B. mallei have the same length and 99 % sequence identity to the corresponding B. pseudomallei orthologs. We included these sequences in DBSecSys 2.0, providing for a more comprehensive comparison of cluster 1 T6SS for the two pathogens.Another interesting protein is TssF, with Locus Tag BPSS1504 in domallei . Neithereudogene . HoweverMAA0736B . Based oThe database content will be expanded with information for additional pathogens and will be updated periodically. Before each update, the current state of the database will be frozen and archived, and can be requested by contacting dbsecsys@bhsai.org.B. mallei secretion system proteins. The database organization provided capability to store and retrieve comprehensive information about pathogen proteins associated with secretion systems, such as their annotation, involvement in virulence, host protein targets, and mechanisms of action inferred from the literature and host-pathogen interaction data. In DBSecSys 2.0, we improved data querying and retrieval, enabling comparison of secretion systems for multiple pathogens. In addition, we expanded the database with new information about B. pseudomallei and updated the information about B. mallei. These updates provide unique capabilities to access comprehensive information about the secretion systems of B. mallei and B. pseudomallei. Importantly, they enable studies and comparisons of corresponding proteins of these two closely related pathogens and their host-interacting partners.We previously developed DBSecSys, a database of


Zizania latifolia (Manchurian rice), Echinochloa colona (a wild grass), and Persicaria sp. (a wild dicot plant). We assembled two new genomes: Ustilago hordei (strain Uhor01) isolated from oats and U. tritici (strain CBS 119.19) isolated from wheat. The smut genomes were of small sizes, ranging from 18.38 to 24.63 Mb. U. hordei species experienced genome expansions due to the proliferation of transposable elements and the amount of these elements varied among the two strains. Phylogenetic analysis confirmed that Ustilago is not a monophyletic genus and, furthermore, detected misclassification of the U. tritici specimen. The comparison between smut pathogens of crop and non-crop hosts did not reveal distinct signatures, suggesting that host domestication did not play a dominant role in shaping the evolution of smuts. We found that host specialization in smut fungi likely has a complex genetic basis: different functional categories were enriched in orphans and lineage-specific selected genes. The diversification and gain/loss of effector genes are probably the most important determinants of host specificity.Host specialization is a key evolutionary process for the diversification and emergence of new pathogens. However, the molecular determinants of host range are poorly understood. Smut fungi are biotrophic pathogens that have distinct and narrow host ranges based on largely unknown genetic determinants. Hence, we aimed to expand comparative genomics analyses of smut fungi by including more species infecting different hosts and to define orphans and positively selected genes to gain further insights into the genetics basis of host specialization. We analyzed nine lineages of smut fungi isolated from eight crop and non-crop hosts: maize, barley, sugarcane, wheat, oats,  Host specialization is commonly found among plant pathogens. Specialist pathogens are favored in ecological contexts of restricted host species diversity, interspecific competition, and due to genetic trade-offs in adaptation to different hosts . MoreoveThe intimate interaction between plants and specialist pathogens suggests that co-speciation should be common. However, host shifts/jumps rather than co-speciation are the main mode of pathogen speciation and a major route for disease emergence . This raMany pathogens show extraordinary genome plasticity enabling the quick response to selection pressures imposed by a new host . AnalysiSmut fungi are a relevant group of host specialized plant pathogens. Despite the growing interest in smut diseases as a threat to agriculture, edible delicacies, and biotechnological applications , the genUstilago maydis, the causal agent of common smut of maize and teosinte, infects all aerial parts of the host plant  and locally induces tumor formation  and two species infecting non-crop hosts . The Persicaria sp. pathogen, Melanopsichum pennsylvanicum, is one of the few Ustilaginaceae smut species known to infect a dicot host  isolated from an oats field in Southern Brazil and the U. tritici from CBS-KNAW Westerdijk Fungal Biodiversity Institute (strain CBS 119.19). Yeast-like cells were obtained from U. hordei teliospores according to CFRVS 40435. For genomic DNA extractions, single colonies from both species were grown in YM liquid medium , at 25°C overnight, in an orbital shaker at 250 rpm. Genomic DNA was extracted using the Genomic-tip 20G kit , according to the manufacturer’s instructions for yeasts. A total of 10 μg of DNA of each sample was sent to the GCB facility at Duke University (United States), where a single large insert library (15–20 kb) was constructed and sequenced in one SMRT cell (P5-C3 chemistry) using the PacBio RS II  sequencing platform. DNA from the same extraction was also used for Illumina paired-end library construction and sequencing using HiSeq2500 platform with 2 × 125 cycles at Center of Functional Genomics . About 10.4 Gb of Illumina and 1.6 Gb of Pacbio data were obtained for U. hordei and about 4.7 Gb of Illumina and 0.5 Gb of Pacbio data were obtained for U. tritici.For genome sequencing, we selected U. maydis, U. hordei, S. reilianum were retrieved from MIPS1. The sequences of U. esculenta, U. trichophora, S. scitamineum were retrieved from NCBI2, and sequences of M. pennsylvanicum from Senckenberg Repository3. Among the genomes of S. scitamineum strains sequenced, we used the best assembly from SSC39B strain in our analyses   using Hmdatabase . The hmmdatabase . Putativdatabase . The secdatabase .5. One-tailed Fisher’s exact test for KOG enrichment were performed for orphan and positively selected gene sets using the KOGMWU R package (Distribution of euKaryotic Orthologous Group (KOG) terms were performed for protein sets using the BLAST search online tool against the eggNOG 4.0 database package .De novo and homology-based identification of repeats were performed using the RepeatModeler pipeline. A combined repeat library was constructed concatenating the RepBase library (release of August 2015) with the de novo repeat family predictions. The combined repeat library was used as input for RepeatMasker6.e-value cutoff of 1e-5, percent match cutoff of 50, and inflation index of 1.5 .Orthologous and paralogous groups among the nine genomes were determined using OrthoMCL with default parameters: BLASTp x of 1.5 . The outn) and blocks with length smaller than 5 (-b4 = 5) in each alignment. After Gblocks filtering, protein alignments smaller than 100 amino acids were excluded. A total of 1,637 protein alignments were retained and concatenated for a total 624,996 amino acid positions. The best-fit amino acid substitution model for the data was obtained using ProtTest v.3.4.2  were aligned using MUSCLE v.3.6 . Gblocks v.3.4.2 . The mod v.3.4.2  with 100 v.3.4.2  was used v.3.4.2 .U. hordei lineages, we also consider the species branch (named UhoOB) as the foreground branch. LRTs was performed between the models bsA  and bsA1 (positive selection) and BEB was used for detect significant sites (≥0.95), according to A total of 4,374 protein-coding sequences with one-to-one orthologs among the nine smut species were aligned with the codon-aware aligner pal2nal v.14  and gapsU. hordei strain isolated from oats were aligned to the reference genome of U. hordei strain isolated from barley using the NUCmer module from MUMmer v3.0 (The scaffolds from the genome assembly of the mer v3.0 . We usedU. hordei from oats and U. tritici from wheat) using a combination of Illumina and PacBio reads. The de novo assemblies resulted in a genome size of 18.63 Mb assembled in 73 contigs ( = 500 bp) for U. tritici and 24.63 Mb assembled in 2,200 contigs ( = 500 bp) for U. hordei. The assembly of U. tritici had a higher degree of contiguity (N50 610 kb) than U. hordei (N50 40 kb). A total of 7,892 and 6,776 protein-coding genes were predicted in the U. hordei and U. tritici genomes, respectively (Supplementary File a and b) were each located on different scaffolds .We assembled the genomes of two smut species infecting important crops .Ustilago was not monophyletic, clustering with members of the Sporisorium Melanopsichum, Pseudozyma, and Kalmanozyma genera . The dicot-infecting species, M. pennsylvanicum, was closely related to the monocot-infecting pathogens in the phylogenetic tree. The phylogeny of the smut fungi also did not separate pathogens according to the wild or domesticated status of their hosts. U. hordei was placed as the earliest diverging species among the analyzed smut fungi.The phylogenomic tree showed that the genus U. tritici (former Tilletia tritici (Bjerk.) G. Winter, 1874) acquired from the CBS-KNAW culture collection under the accession CBS 119.19 . The CBS-KNAW U. tritici strain was placed close to Sporisorium species and clustered apart from U. tritici and Tilletia species in the ITS-based tree using NCBI accessions .A total of 17,454,837 bp (70.83%) of the S. reilianum – U. maydis – M. pennsylvanicum . More extensive chromosomal rearrangements were observed between closely related species, such as S. reilianum – S. scitamineum – CBS119.19, suggesting that these events occurred after the species diverged from the last common ancestor. Translocations and inversions also occurred at mating-type harboring scaffolds .Pairwise dotplot sequence comparisons showed more evident syntenic relationship among high quality assembled genomes, since fragmented genomes result in many tiny syntenic blocks. Interestingly, conservation of long-range synteny was observed between more distantly related species, such as Ustilago hordei species showed the largest genomes among the smut fungi, which ranged from 18.38 Mb in S. reilianum to 24.63 Mb in U. hordei (oats) . The larger genome size in U. hordei species was also accompanied by an increase in the repetitive elements content. In particular, the content in transposable elements ranged from 0.61% in the genome of CBS119.19 strain to 23.93% in U. hordei (oats). The predicted gene repertoire varied from 6,280 genes in M. pennsylvanicum to 7,892 in U. hordei (oats). M. pennsylvanicum also encoded the smallest number of secreted protein (291) and predicted effectors (55), while S. reilianum had the largest secretome (443) and effector content (127). The total number and the diversity of sub-categories of CAZyme, protease, and lipase domains were similar among smut species . U. hordei species were an exception, because there was an expansion of the peptidase family A11A (Copia transposon peptidase) compared to the other species (Supplementary File Table 2). All species have at least one cluster encoding for putative terpene synthase (TS), non-ribosomal peptide synthase (NRPS), and type 1 polyketide synthases (t1PKS). Only U. trichophora presented a hybrid cluster of NRPS-Indole-t1PKS.U. hordei and U. maydis to 98% among orthologs of the two U. hordei strains .We compared the predicted proteome of nine smut species and found 7,187 orthologous clusters (orthogroups) using OrthoMCL  and U. hordei (oats) the largest  . Most of the species-specific encoded proteins were uncharacterized (lacking a conserved domain). Transposases and reverse transcriptases were frequent among orphan proteins, mainly in U. hordei proteomes. Some predicted effectors (ranging from 7 in U. hordei (oats) to 32 in U. trichophora) were also species-specific. Among the orphan proteins with a conserved domain, we found enzymes acting in primary and secondary metabolic pathways, proteins associated with transcriptional regulation, signaling, cell cycle control, morphogenesis, and stress response .U. maydis, S. reilianum, and U. hordei (found on barley) were screened for orthologs in the other species. We identified some effectors that were present in all proteomes, including Cmu1 (Chorismate mutase 1), Stp1 (Stop after penetration 1), ApB73 (Apathogenic in B73), and members of the Eff1 family. Homology searches by tblastn identified putative orthologs of some effectors , See1 (Seedling efficient effector1), and members of the Mig1 (Maize-induced gene 1) family were also detected in all genomes. Additional effectors were only found in a subset of species . The leaf-specific effector candidates, um06223 and um12217, were present only in U. maydis. The effector Sad1  was specific to S. reilianum using the automated annotation procedure, but then recovered in the genomes of S. scitamineum, CBS119.19, U. maydis, and U. esculenta. The genomic region coding for Pit2 (Protein involved in tumors 2) was also identified in U. trichophora. Although the 14 highly conserved residues of Pit2 protein sequence were detected in U. trichophora sequence, the signal peptide is missing. It remains to be established whether Pit2 is secreted using a non-conventional pathway and its functional role if any in U. trichophora. Phylogenetic trees and protein identity matrices of effectors are provided in Supplementary File Effectors characterized in S. scitamineum both in vitro and in planta, and Sad1 only in planta. Genes encoding Pep1 and Sad1 were expressed in U. esculenta also in in vitro and in planta, and the gene encoding UhAvr1 in S. reilianum was expressed in vitro . Among the positively selected genes, there were also genes encoding enzymes acting on primary and secondary metabolism, proteins associated with regulation of cell cycle and morphogenesis, signaling, response to stress .Branch-site models were also used to identify sites evolving under episodic selection. For U. hordei (Uhor01 strain) and a new specimen also belonging to Ustilaginaceae isolated from wheat (CBS119.19 strain). The similarities in cell biology and lifestyle among the nine smut species was reflected in the fact that more than 65% of orthologs groups were shared among species. Most of the orthologous genes were detected as one-to-one orthologs. The species shared also a similar content of KOG functional categories. However, among those genes we found significant evidence of episodic positive selection. Moreover, sets of orphan genes were detected for each species. Hence, each genome offered a particular repertoire of genes that can be related to host-specialization. We are aware that it is difficult to distinguish the genetic changes that directly contribute to the host specialization from those that were a consequence of the divergence after host specialization, but some insights are discussed.In this study, we extended the comparative genomic analyses of Ustilaginaceae smut fungi by using seven genomes available. In order to increase the host range survey, we additionally sequenced and assembled two genomes: Ustilago remained polyphyletic, despite of many taxonomic revisions into to the Ustilaginaceae family have been recently proposed  and, therefore, requires revision.The phylogenomic tree based on a distance super-matrix approach showed that the genus proposed . Anotherle genes . The U. The decomposition of gene trees in quartets showed some additional phylogenetic conflicts. U. hordei is an exception and clearly experienced a genome expansion by containing more protein-coding genes and repetitive elements. U. hordei genome is under less stringent control and that active transposons have translocated some candidate effector genes. The sequencing of a second U. hordei strain herein supports the hypothesis of active transposable elements by showing an even greater content of repetitive DNA than the previously sequenced strain.The smut pathogens sequenced so far have compact genomes depleted of paralogs and repetitive DNA. U. hordei, where virulent and avirulent isolates on Hannchen barley cultivar differed for an insertion of a transposon-derived sequences in the promoter region of the UhAvr1 effector gene    were speSAD1 of S. reilianum, whose orthologs in the S. scitamineum and U. esculenta genomes were identified in this work. The S. reilianum SAD1 effector alters the inflorescence branching architecture of maize plants by inducing loss of apical dominance , siderophores, protection against biotic and abiotic factors  synthesis. TG is a storage lipid which serves as energy reservoir, source of signaling molecules, and substrate for membrane biogenesis , we also brought new knowledge to less studied smut species . We identified lineage-specific sets of orphans and positively selected genes enriched for different functional categories, highlighting genes that have a potential role in host–pathogen interaction. The presence of distinct effector repertoires, with some being detected exclusively in each genome, is emphasized as the most likely important determinants of host specificity. Therefore, we provided good candidate genes for further functional characterization in different smut species. A comparative transcriptomic profile will also achieve additional insights, since differences in host specificity can also be due to distinct expression pattern of orthologs. Moreover, the comparison of U. hordei isolates herein showed the ongoing activity of transposable elements, with variable amounts predicted between the two strains. A population genomic study in U. hordei is also promising to reveal the extent of the divergence among barley and oat isolates.In summary, our comparative genomic study provided further insights on smut host-specificity and symptoms development. In addition to sequencing and characterize two new genomes (from CBS 119.19 strain and in vitro growth of the isolates and commented on and edited the manuscript. CM-V and DC supervised the research.JB analyzed the data. JB, DC, and CM-V designed the analyses and wrote the manuscript. EK and NT-S contributed to the purchase and The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.