Gram-positive anaerobic (GPA) bacteria inhabit different parts of the human body as commensals but can also cause bacteremia. In this retrospective observational study, we analyzed GPA bacteremia pathogens before (2013–2015) and after (2016–2018) the introduction of the matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS).Parvimonas micra (P. micra) (17.5%) was the second most frequently identified GPA (MALDI-TOF MS); we then retrospectively reviewed electronic medical records for 25 P. micra bacteremia cases at our hospital. We also conducted a literature review of published cases in PubMed from January 1, 1980, until December 31, 2019; 27 cases were retrieved.We conducted a retrospective observational study by searching the microbiology database to identify all positive GPA blood cultures of patients with GPA bacteremia diagnosed using the new technique, MALDI-TOF MS, between January 1, 2016 and December 31, 2018; and using a conventional phenotypic method between January 1, 2013 and December 31, 2015 at a single tertiary center in Japan. P. micra bacteremia were identified after 2015, both, at our institute and from the literature review. They were of mostly elderly patients and had comorbid conditions . In our cases, laryngeal pharynx  and gastrointestinal tract  were identified as the most likely sources of bacteremia; however, the infection source was not identified in 9 cases (36%). P. micra bacteremia were frequently associated with spondylodiscitis (29.6%), oropharyngeal infection (25.9%), intra-abdominal abscess (14.8%), infective endocarditis (11.1%), septic pulmonary emboli (11.1%), and GIT infection (11.1%) in the literature review. Almost all cases were treated successfully with antibiotics and by abscess drainage. The 30-day mortalities were 4 and 3.7% for our cases and the literature cases, respectively.Most cases of P. micra are predominantly associated with GIT, oropharyngeal, vertebral spine, intra-abdominal region, pulmonary, and heart valves. Patients with P. micra bacteremia could have good prognosis following appropriate treatment.Infection sites of  Clostridium spp., Gram-positive anaerobic cocci (GPAC), and Gram-positive non-spore-forming anaerobes (GPNAs) [Clostridium spp., GPAC, and GPNAs account for 20.7, 14.7, and 6% of all anaerobic hematic isolates, respectively [Anaerobic bacteria are major components of the microflora on the mucosal membranes in humans, and several hundred distinct species of anaerobic microorganisms have been identified by classical and molecular methods. Gram-positive anaerobes (GPAs) are found in the commensal microbiota of the digestive tract, with some species colonizing the urogenital tract and skin. GPAs include  (GPNAs) . As repoParvimonas micra (P. micra) is a member of GPAC normally found in the oral cavity, respiratory system, gastrointestinal tract (GIT), and the female genitourinary tract. Originally known as Peptostreptococcus micros, the organism was reclassified as P. micra in 2006 [P. micra is one of the bacterial species most frequently isolated from infected root canals of teeth with chronic apical periodontitis [P. micra has also been implicated in meningitis [P. micra was one of the most commonly isolated GPAC species and characteristics of Parvimonas spp. bacteremia (mostly P. micra) were discussed [P. micra.dontitis . P. micrningitis , cervicaningitis , 6, infeningitis , and sponingitis . Recentliscussed . HoweverA new technique, matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS), was reported to correctly identify 91.2% of anaerobic isolates at the species level compared to 16S rRNA sequencing as the gold standard of diagnosis . MALDI-TP. micra was found to be the second most commonly identified causative organism of GPA bacteremia using MALDI-TOF MS, therefore, we described the clinical features and laboratory diagnosis of P. micra bacteremia. Finally, we conducted a review of literatures on previously reported P. micra bacteremia cases.In this study, we retrospectively analyzed the causative pathogens of GPA bacteremia before (2013–2015) and after (2016–2018) the introduction of MALDI-TOF MS in a tertiary medical center. Blood culture samples were incubated for 7 days in the Bact/Alert 3D instrument  per the manufacturer’s protocol. Bact/Alert FN (anaerobic) blood culture bottles were used at the laboratory throughout 2015; Bact/Alert FN plus (anaerobic) bottles were used thereafter. Subcultures were streaked onto the Centers for Disease Control and Prevention anaerobic blood agar plates  and incubated under anaerobic conditions for 2 days. If bacterial growth was insufficient, the incubation period was extended.Anaerobic bacteria were identified using RapIDTM ANA II  before the introduction of MALDI-TOF MS in our laboratory. Since January 2016, we have used the MALDI Biotyper  for bacterial identification. Bacterial isolates of single colonies were identified using the plate extraction method as previously described . MALDI-TFor each strain of anaerobic bacteria, two preparations of colony/sample material were analyzed. The Biotyper software compares each sample’s mass spectrum to the reference mass spectrum in the database, calculates an arbitrary unit score value between 0 and 3 reflecting the similarity between the sample and reference spectrum, and displays the top 10 matching database records. Log scores were interpreted according to the manufacturer’s instructions. Log scores of > 2.0; > 1.7 but < 2.0; and < 1.7 indicate identification with high confidence, low confidence, and unreliable identification for the specie level, respectively.Antimicrobial susceptibility testing was performed for most isolates using the broth dilution method using ABCM broth . Bacteria were tested against the following antimicrobial agents: penicillin, ampicillin/sulbactam, piperacillin/tazobactam, meropenem, and clindamycin. Minimum inhibitory concentrations (MIC) were routinely reported and interpreted according to Clinical and Laboratory Standards Institute (CLSI) M100 S28 (2018) breakpoints for anaerobes.P. micra, bacteremia was expansively searched from January 2011 to December 2019, in that period, medical records were available for analyzing P. micra bacteremia.This retrospective study was conducted at Japanese Red Cross Nagoya Daini Hospital, an 812-bed tertiary medical center in Nagoya, Japan. By searching our microbiology database, we identified all positive GPA blood cultures from January 2013 through December 2018. GPA bacteremia was identified by conventional phenotypic methods from January 2013 to December 2015 and by MALDI-TOF MS from January 2016 to December 2018. As for Clinical data were collected from medical records. Bacteremia was considered clinically relevant when a patient had one or more positive blood cultures for GPA with clinical evidence consistent with infection: temperature > 38 °C or elevated serum concentrations of C-reactive protein (> 40 mg/L), or the source of infection was definitively identified. Community-onset bacteremia was defined as infection acquired outside of the hospital setting, with a positive blood culture obtained within 48 h of admission. Nosocomial bacteremia was defined as infection acquired while being administered treatment within a healthcare setting, with a positive blood culture obtained more than 48 h after admission. Charlson Comorbidity Score was used to assess comorbidities . IllnessParvimonas micra” or “Peptostreptococcus micros” and “bacteremia” or “Infection.” Articles published from January 1, 1980 to December 31, 2019 were included. Articles were individually assessed for a case of P. micra bacteremia based on the title and/or abstract and/or text. All articles were reviewed to ensure no redundancy of cases, and that adequate information was available for inclusion in the review.We searched the PubMed databases for journal articles, using the search terms “Clostridium perfringens was the most frequently identified causative organism  of GPA bacteremia, followed by P. micra , and Eggerthella lenta  , and 14/25 (56%) patients were male. Most patients  had community-onset disease. Nearly all patients  presented with fever; however, focal symptoms such as sore throat  and abdominal pain  were less frequently observed. The most common comorbidities were cancer  and diabetes . The laryngeal pharynx  and GIT  were identified as the most likely sources of bacteremia. The infection source was not identified in 9 cases . Twelve patients (48%) had polymicrobial bacteremia. The various pathogens associated with concomitant polymicrobial infection included Solobacterium moorei, Staphylococcus capitis, Fusobacterium nucleatum, Pseudomonas citronellolis, Bacillus subtilis, Streptococcus oralis, Streptococcus constellatus, Staphylococcus lugdunensis, Veillonella parvula, Actinomyces meyeri, Prevotella intermedia, Atopobium parvulum, Streptococcus anginosus, and Prevotella oris, Corynebacterium spp. Most P. micra isolates were susceptible to penicillin, ampicillin-sulbactam, piperacillin-tazobactam, and meropenem; however, clindamycin showed slightly elevated MIC  , oropharyngeal infection  intraabdominal abscess , GIT infection , IE , and septic pulmonary emboli . Five patients (18.5%) had polymicrobial bacteremia, and pathogens associated with concomitant polymicrobial infection included Gemella morbillorum, Atopobium rimae, Fusobacterium nucleatum, Prevotella bivia, and Bacteroides fragilis. In the literature cases, P. micra bacteremia also had a favorable outcome .In 27 literature cases, P. micra was a frequently identified causative organism of GPA bacteremia. However, P. micra have rarely been identified as causative pathogens of GPA bacteremia using conventional methods due to difficulties with the laboratory identification. In both ours and the literature cases, P. micra bacteremia cases were increasingly diagnosed after 2015 . In a recent population-based study, GPAC bacteremia, identified using MALDI-TOF MS or 16S rRNA gene sequencing, was reported to be much more common than previously reported with an annual incidence of 3.4 cases per 100,000 persons per year [P. micra was the most commonly isolated in the study. Thus, these new techniques might enable accurate diagnosis of P. micra bacteremia for appropriate treatment.In our cases, using MALDI-TOF MS, per year , while PP. micra bacteremia using MALDI-TOF MS or conventional phenotypic method from 2010 to 2019. We found that oropharyngeal infections  and GIT  were the primary sources of P. micra bacteremia; however, the sources of bacteremia in the remaining 9 patients (36.0%) were not recorded. As the presenting symptoms in these patients were non-specific , thorough searches for the primary source of bacteremia may not have been carefully conducted in these 9 cases. In 27 literature cases, P. micra bacteremia were also frequently associated with oropharyngeal infection  and GIT infection  [P. micra in the literature cases were spondylodiscitis , intra-abdominal abscess , IE , and septic pulmonary emboli  which is inconsistent with our results. Spondylodiscitis caused by P. micra is rare but an important infectious disease, which is sometimes diagnosed from samples of the bone or vertebral disk without positive blood culture [P. micra might be one of the important anaerobic pathogens causing IE. Recently, Badri and coworkers reported clinical features about 100 cases of Parvimonas spp. (96 P. micra and 4 Parvimonas spp.), in which common focal infections were in the abdominal tract, bones and joints, the respiratory tract, and in skin/soft tissue, however, oropharyngeal infection were rarely identified [P. micra bacteremia.We reviewed 25 patients with , 11.1%) –31. Othe culture . IE due  culture , and P. P. micra bacteremia had polymicrobial bacteremia. In the literature cases, 5 patients (18.5%) had polymicrobial bacteremia. Most concomitant pathogens were aerobic and anaerobic members of the oral and gastrointestinal microbiota. Badri et al. showed that 50% of Parvimonas spp. associated bacteremic infections were polymicrobial [P. micra.In our cases, 48% (12/25) of icrobial , which wicrobial , it was icrobial . TherefoP. micra bacteremia in our case series were successfully treated with antibiotics  . In one of the previous cases, a patient died as a consequence of the primary tumor after the completion of antibiotic therapy [Parvimonas spp. was 10% [P. micra can cause complicated infectious diseases including IE and spondylodiscitis, these results suggest that patients with P. micra bacteremia might have good prognosis following appropriate treatment.Most cases of  therapy . In a pr was 10% . In cont was 10% . AlthougP. micra bacteremia was dependent on publication biases, making it impossible to address the real prevalence and incidence of P. micra bacteremia in clinical setting.There were some limitations to this study. First, we may have missed some cases at our institute as well as previously reported cases due to the retrospective design. Second, the cases in our case series were diagnosed in a single tertiary medical center in Japan; therefore, regional and institutional differences were not considered. Third, different blood culture bottles were used in the two study periods. Although a prior study found no difference in the detection rate of anaerobic bacteremia using Bact/Alert FN bottles or Bact/Alert FN plus bottles , we cannP. micra as the second most frequently identified GPA. Our case series and literature review showed that P. micra have been mainly identified in blood culture using MALDI-TOF MS and 16 s rRNA sequencing. Infection sites of P. micra were predominantly associated with GIT, oropharyngeal, vertebral spine, intra-abdominal region, pulmonary, and heart valves. Patients with P. micra bacteremia could have good prognosis following appropriate treatment. This study helps improve our understanding of the clinical characteristics of bloodstream infections by P. micra.We reported the causative pathogens of GPAs bacteremia identified by MALDI-TOF MS, and findings showed Additional file 1: Supplemental Figure 1. Numbers of GPAs identified or not identified at the species level from 2013 to 2018. Anaerobic bacteria were identified by a conventional phenotypic method from 2013 to 2015, and MALDI-TOF MS from 2016 to 2018.Additional file 2: Supplemental Table 1.


Leishmania species and transmitted by the bite of sand flies. The genus Lutzomyia and Phlebotomus of sand flies are known to be the responsible vector for transmitting almost all Leishmania species to humans. The detection of Leishmania DNA in species of the genus Sergentomyia, in different regions, suggests their likely role in Leishmania transmission.Leishmaniases are parasitic diseases caused by Sergentomyia minuta, the most dominant Sergentomyia species in Morocco, using ecological niche modeling.Our objective was to determine the potential geographical distribution of S. minuta in Morocco, specifically in northern and central Morocco where visceral and cutaneous leishmaniasis foci occur. There were six abiotic factors affecting the distribution of S. minuta whose annual precipitation, precipitation seasonality and precipitation of driest month as the most important ecological variables of the model.The results showed the widespread geographical distribution of S. minuta abundance was noted suggesting the potential involvement of S. minuta in local Leishmania transmission cycles.A positive statistical correlation between human leishmaniasis cases and  Phlebotomine sand flies (Diptera: Psychodidae) are small nocturnal insects of which only the female is a hematophagous. Currently, about 1000 species of sand flies are distributed in almost all biogeographic regions of the world .Phlebotomus, Lutzomyia and Sergentomyia contain all hematophagous species of sand flies  was investigated in Morocco  and literature using the keyword “species name, Morocco” as a search word to retrieve all data archived in PubMed database. Thus, about 70% of the 190 localities were investigated by the Ecology and Environment Laboratory (L2E) team between 2005 and 2016. The rest of the localities were published observations of the investigations in Morocco (20 publications on PUBMED). The coordinates of all localities were registered with a global positioning system (GPS).http://www.worldclim.org), with a spatial resolution of 30 arc seconds (about 1km2).For ecological niche modeling, 19 climatic variables (Bio1-Bio19) were used with elevation precision (ALT) for each locality . AltitudThe modeling was performed using Maximum entropy (MaxEnt) version 3.3.3 , which uDistribution data for the sand fly species and environmental data were imported into MaxEnt v. 3. 3. 3 , a totalTo evaluate the quality of model produced by MaxEnt, we analyzed the Operating Characteristics Curve Receiver Operating Characteristic (ROC) which assigns a unique value according to the model performance Area Under the Curve (AUC).S. minuta was estimated using the Jackknife Environmental Variables Importance Test , compared to the false positive rate (superfluous forecast error). Also, the ROC analysis evaluates the ability of the model to correctly predict the occurrence of the species. The closer the AUC value is to 1, the closer the correlation of the model  Figs. 2. The impnce Test  (Table 2χ2) was used to analyze the correlation between variation of S. minuta abundance and the presence/absence of human leishmaniasis cases  in all sampling sites.The Chi-squared test  were obtained from official Moroccan Ministry of Health reports .Sergentomyia species were noted: Sergentomyia minuta (N= 150), S. fallax (N= 110), S. dreyfussi (N= 57), S. africana (N= 10) and S. christophersi (N= 15). Phlebotomus species were also recorded, specifically Phlebotomus sergenti (N= 102), P. papatasi (N= 82), P. longicuspis (N= 78), P. perniciosus (N= 76) and P. ariasi (N= 43), while P. alexandri (N= 09), P. kazeruni (N= 03), P. langeroni (N= 02), and P. mariae (N= 02) were noted with very low reporting.In this study, we assembled 190 sampling sites for sand fly species. Five Sergentomyia minuta was the most predominant species and it was collected in the 150/190 sampling sites between 14 and 2123m , which were the most important factors affecting the distribution of S. minuta in the study area.nd 2123m . To idenS. minuta. Warmer colors show areas with better predicted conditions. The white dots indicate the presence locations used for training, while the purple dots show the locations of the tests.Since 1995, leishmaniases are notifiable diseases in Morocco . All human cases are listed in leishmaniasis epidemiological information collection system and published yearly. We used data recorded of CL and VL between 2005 and 2016  correspoS. minuta in Morocco showed a positive correlation between VL by L. infantum and S. minuta abundance (r= 0.211) and between CL by L. tropica and S. minuta abundance (r= 0.166).Statistical analysis of the presence and absence of leishmaniasis cases  and the distribution of χ2) test, S. minuta was more associated to VL distribution  compared to CL .According to Chi-squared test , as competent vector of L. major, L. tropica and L. infantum, respectively  and cutaneous leishmaniasis (r= 0.166) with S. minuta presented an anthropophilic behavior in Portugal (minuta is a ubiquitous species (S. minuta showed abundance from May to September in sub-humid and semi-arid stages in Morocco (S. minuta activity favors the S. minuta-Leishmania temporal overlapping in these areas.Sand fly behaviors can also determine its vector implication. Portugal . In Moro species  which sh species . In addi Morocco . In arid Morocco . This loSergentomyia species, were found highly dependent on environmental conditions; and their abundance and geographical distribution were affected by several ecological variables (S. minuta in Morocco (Leishmania vector species in Mediterranean basin (Sand flies of Morocco, including ariables –62. Acco Morocco . Precipi Morocco . Modelin Morocco , found tan basin .S. minuta is recorded in arid region of Tunisia with an annual precipitation between 88 and 157mm (nd 157mm , also innd 157mm . In the S. minuta with Leishmania species seemed to present the important criteria necessary to be incriminated as a potential vector of the Leishmania species in Morocco. Hence, the present study opened a debate on the potential role of Sergentomyia species, especially S. minuta in the transmission of leishmaniasis in Morocco. Hypothesis deserves further researches including, but not limited to, protozoan isolation from engorged specimens as well as experimental transmission.Effective vector control requires accurate vector identification. The spatial and temporal overlapping of


Gene abundances were statistically assessed with regard to previously determined concentrations of fecal indicator organisms Escherichia coli, intestinal enterococci and somatic coliphages, as well as influential environmental factors. Similar seasonal patterns and strong positive correlations between fecal indicators and blaCTX-M genes indicated identical sources. Accordingly, linear regression analyses showed that blaCTX-M concentrations could largely be explained by fecal pollution. E. coli provided the best estimates (75% explained variance) at the upstream site, where proportions of blaCTX-M genes in relation to fecal indicator organisms were highest. At this site, rainfall proved to be more influential, hinting at surface runoff as an emission source. The level of agricultural impact increased from downstream to upstream, linking increasing blaCTX-M concentrations after rainfall events to the degree of agricultural land use. Exposure assessment revealed that even participants in non-swimming recreational activities were at risk of incidentally ingesting blaCTX-M genes and thus potentially antibiotic resistant bacteria. Considering that blaCTX-M genes are ubiquitous in Lahn River and participants in bathing and non-bathing water sports are at risk of exposure, results highlight the importance of microbial water quality monitoring with an emphasis on antibiotic resistance not only in designated bathing waters. Moreover, E. coli might serve as a suitable estimate for the presence of respective antibiotic resistant strains.Antibiotic-resistant bacteria represent an emerging global health problem and are frequently detected in riverine environments. Analyzing the occurrence of corresponding antibiotic-resistant genes in rivers is of public interest as it contributes towards understanding the origin and dissemination of these emerging microbial contaminants via surface water. This is critical for devising strategies to mitigate the spread of resistances in the environment. Concentrations of  Median infectious doses (N50) and slope parameters  were obtained from Haas et al. )E. coli, ESBL-producing E. coli and diarrheagenic ESBL-producing E. coli was calculated based on prevalence values obtained from the literature [E. coli isolates in several Central European rivers were 32% (Rhine), 34% (New Meuse) and 48% (Meuse) [E. coli isolates from the Seine River in France was as high as 42% [BlaCTX-M abundance in enteric bacteria in some United Kingdom bathing waters was assumed to be 0.1% [rd generation cephalosporin-resistance in E. coli in coastal surface waters in England and Wales of 0.12%. In some Dutch recreational waters ESBL-producing E. coli represented 0.05–1% of the total E. coli population [E. coli harboring ESBL resistance in surface water of Cache La Poudre River, USA. 8.5% of all ESBL-producing E. coli isolates from surface waters in the Netherlands were suspected diarrheagenic variants [As described earlier, theoretical prevalence of antibiotic-resistant terature –48, 54. terature –30. Repo (Meuse) . Antibioh as 42% . BlaCTX- be 0.1% . This is be 0.1%  found a pulation . Higher pulation , who fouE. coli and ARG  [E. coli per children’s swim session  , partici session . However session  is actua session .Nevertheless, Haas et al.  clearly blaCTX-M genes may be ingested per hour of non-swimming water sports resulting in up to 3927 copies theoretically ingested per session , the prudy site , Fig 7.E. coli provided the best estimates for ARG in Lahn River, with three-quarters (75%) of variance explained. Yet, further model validation using an independent reference dataset will be required. ARG and FIO were shown to be similarly distributed and were influenced by environmental factors in a similar way. Thus, multiple linear regression models (MLR) as previously established for the prediction of FIO based on environmental factors [blaCTX-M genes and the actual level of antibiotic resistance, a variety of bacterial isolates should be tested for antibiotic resistances in future studies and correlations between blaCTX-M genes should be analyzed. Results could strengthen the assumptions made on the suitability of blaCTX-M genes as proxy for the total level of antibiotic resistance in river environments.Notably, ARG concentrations can be estimated by FIO with a considerably higher accuracy at site 2 . As expe factors , 54, 79 BlaCTX-M genes were found to be omnipresent in Lahn River surface water. Overall, E. coli and blaCTX-M genes followed a similar trend and their abundances varied according to temporal variations in hydro-meteorological factors. An influence of WWTP discharges on blaCTX-M gene levels was observed under low flow conditions, whereas total concentrations of blaCTX-M genes increased after rainfall events in accordance with the degree of agricultural impact in the surrounding catchment. In general, participants in swimming and non-swimming recreational activities  at Lahn River are at risk of ingesting ARB and ARG. Despite the limitation that the amount of ARG and FIO ingested cannot be linked to an actual risk of infection, results demonstrate potential health risks associated with microbial water quality and water related recreational activities. BlaCTX-M gene abundance was largely explained by fecal pollution, with E. coli providing the best estimates. This information will be helpful in the fields of risk assessment and water management.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.


Mammalian brains show extreme aerobic metabolism. Following hypoxia exposure, mammals usually experience irreversible brain damage and can even develop serious diseases, such as hypoxic ischemic encephalopathy and brain edema. To investigate mechanisms underlying the responses of subterranean rodents to severe hypoxia, we performed a cross-species brain transcriptomic analysis using RNA sequencing and identified differentially expressed genes (DEGs) between the subterranean rodent Lasiopodomys mandarinus and its closely related aboveground species L. brandtii under severe hypoxia  and normoxia .Severe hypoxia induces a series of stress responses in mammals; however, subterranean rodents have evolved several adaptation mechanisms of energy metabolisms and OL. mandarinus and 69,360 in L. brandtii. We identified 359 and 515 DEGs by comparing the hypoxic and normoxia groups of L. mandarinus and L. brandtii, respectively. Gene Ontology (GO) analysis showed that upregulated DEGs in both species displayed similar terms in response to severe hypoxia; the main difference is that GO terms of L. brandtii were enriched in the immune system. However, in the downregulated DEGs, GO terms of L. mandarinus were enriched in cell proliferation and protein transport and those of L. brandtii were enriched in nuclease and hydrolase activities, particularly in terms of developmental functions. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that upregulated DEGs in L. mandarinus were associated with DNA repair and damage prevention as well as angiogenesis and metastasis inhibition, whereas downregulated DEGs were associated with neuronal synaptic transmission and tumor-associated metabolic pathways. In L. brandtii, upregulated KEGG pathways were enriched in the immune, endocrine, and cardiovascular systems and particularly in cancer-related pathways, whereas downregulated DEGs were associated with environmental information processing and misregulation in cancers.We obtained 361 million clean reads, including 69,611 unigenes in L. mandarinus has evolved hypoxia adaptation by enhancing DNA repair, damage prevention, and augmenting sensing, whereas L. brandtii showed a higher risk of tumorigenesis and promoted innate immunity toward severe hypoxia. These results reveal the hypoxic mechanisms of L. mandarinus to severe hypoxia, which may provide research clues for hypoxic diseases. Typically, O2 concentrations in the surroundings change with temperature, humidity, atmospheric pressure, and altitude, among other factors. Many hypoxic environments occur naturally, such as the aquatic habitats, high altitudes  , and melanogenesis].Four enriched pathways for upregulated DEGs in g Angpt2 , Edn1  photoperiod in a laboratory suitable for reproduction L. mandart the Nr , and Swit the Nr  databaserization . GO coveytoscape  was usedhttp://bioconductor.org/packages/release/bioc/html /edgeR.html) was used to obtain the base mean value for identifying the DEGs. To correct for multiple testing, FDR was calculated to adjust the P-value threshold. Transcripts with an FDR of ≤0.05 and a minimum two-fold difference in expression (|log2 ratio| ≥1) were considered as thresholds for the significance of gene expression differences between two groups. Additionally, information for DEGs was collected from unigene annotations, and these genes were subjected to GO and KEGG significant enrichment analyses to identify biological functions and metabolic pathways involving these genes.The fragments per kilobase of exon per million mapped fragments (FPKM) method eliminated the influence of different gene lengths and sequencing levels on the calculation of gene expression. FPKM values were directly used to compare gene expression differences between the samples. The edgeR package  to evaluate the concordance between RT-qPCR results and RNA-seq data. Significant t-test differences were defined as P < 0.05 and highly significant as P < 0.01.To validate the reliability of DEGs identified by RNA-seq, mRNA expression levels of six selected genes were measured by RT-qPCR using the same samples. Primers were designed using Primer-BLAST, and their sequences are shown in Table Additional file 1: Table S1. Illumina sequencing data of the analyzed samples. Table S2. Length distribution and quality metrics of unigenes in L. mandarinus and L. brandtii. Table S3. Functional annotation results for L. mandarinus and L. brandtii transcriptomes. Table S4. Annotated DEGs for L. mandarinus and L. brandtii. Table S5. Significantly enriched GO terms for up- and downregulated DEGs in L. mandarinus and L. brandtii under severe hypoxia. Table S6. Enriched KEGG pathways for up- and downregulated DEGs in L. mandarinus and L. brandtii under severe hypoxia. Table S7. Results of RNA extraction from L. mandarinus and L. brandtii brain tissue. Table S8. RT-qPCR primers for the validation of RNA-Seq data. Table S9. Characteristics of L. mandarinus and L.brandtii samples.Additional file 2: Figure S1. Classification of annotated transcriptome in the Eukaryotic Orthologous Group (KOG). A to Z represent the specific entry information of the KOG annotated by unigenes.Additional file 3: Figure S2. DEGs in the brains of L. mandarinus and L. brandtii under severe hypoxia vs. normoxia. FC, fold change; FDR, false discovery rate. Red, blue, and green dots represent up- and downregulated and unchanged genes, respectively.Additional file 4: Figure S3. Gene-pathway networks for DEGs in L. mandarinus (A) and L. brandtii (B) under severe hypoxia determined using the Clue GO tool in Cytoscape. Small and large dots represent DEGs and enriched pathways, respectively. Genes indicated in blue are upregulated. Lines between dots represent connections between the genes and the pathways in the network; genes with more connections are more important in the network.Additional file 5: Figure S4. Protein interaction network for specific DEGs in L. brandtii (A) and L. mandarinus(B) brain under hypoxia. Pink squares represent upregulated proteins, and green squares represent downregulated proteins; size of the squares represents the importance of the protein in the network, with bigger squares indicating a greater importance; the thickness of line between the squares represents the strength of the association between two proteins, with a wider line indicating a stronger correlation.


Background: In recent years, LncRNA acts as a member of competing endogenous RNA (ceRNA), playing an important role in drug resistance of lung cancer. The aim of this study was to identify potential biomarkers about cisplatin resistant lung cancer cells using a comprehensive ceRNA network.Methods: GSE6410 (GPL-201) analyzed gene expression changes about cisplatin resistance in A549 NSCLC cells. GSE43249 (GPL-14613) included noncoding RNA expression profiling derived from the cisplatin resistant A549 lung cells. GEO2R, an online analysis tool, analyzed the differentially expressed mRNAs and miRNAs (DEmRNAs and DEmiRNAs). To explore the functional enrichment implication of differentially expressed mRNAs, we used the GO (Gene ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis. Through miRDB, Targetscan, Starbase and miRWalk, we found targeted miRNAs. The Kaplan-Meier curve method was used to show clinical survival analysis of targeted RNAs (P<0.05). The Starbase database predicted potential lncRNAs mediated targeted miRNAs. Eventually, the novel ceRNA network of lncRNAs, miRNAs, mRNA was constructed by cytoscape3.7.2.Results: 118 differentially expressed mRNAs were the basis of the mediated ceRNA network. DAVID and Kaplan-Meier picked out BAX, an apoptosis regulator. Venn diagram demonstrated 8 miRNAs commonly regulating BAX. Starbase predicted lncRNA XIST mediated miRNAs. Finally, lncRNA XIST may be a useful biomarker regulating cisplatin resistance in lung cancer cells and further, we explored the BAX may effect tumor-infiltrating immune cells.Conclusions: LncRNA XIST competitively bound to miRNA 520 in the regulation of cisplatin resistance by BAX, participating apoptosis in the p53 signaling pathway. Lung cancer is considered to have a significant role in cancer-related deaths worldwide. It is well known that NSCLC comprises approximately 85% of all lung cancers and lung adenocarcinoma (LAC) accounts for 40% of all lung cancers all over the whole world As for NSCLC, many treatment strategies are effective and include surgical operation, chemotherapy and radiotherapy. NSCLC patients, who are treated with cisplatin, also termed cisdiamminedichloroplatinum (CDDP) or diamminedichloro platinum (DDP); may develop chemoresistance LncRNAs have been considered as oncogenes mediating tumorigenesis and chemoresistance. They may have lower expression and exist in the cytoplasm or nucleus P<0.01). In this present novel, the ceRNA network of lncRNA-miRNA-mRNA about cisplatin resistance in NSCLC was created through RNA sequencing data from the GEO database.BAX https://www.ncbi.nlm.nih.gov/gds/?term=). The inclusion criteria were as follows. Firstly, our goal was to find mRNA and noncoding RNA expression changes about cisplatin resistance in A549 NSCLC cells from GEO database. Through some key words, such as mRNA, noncoding RNA, cisplatin, lung cancer, we screened targeted datasets. Next, for better comparison, we used data including A549 cisplatin-sensitive and cisplatin-resistant non-small cell lung cancer cell lines. The exclusion criteria were incomplete data, which didn't have control group. First, DEmRNAs were analyzed by online software GEO2R. There were 8793 DEmRNAs in GSE6410, and |Log 2 FC|>1 and the P <0.05 We used GSE6410 (6 samples mRNAs) and GSE43249 (6 samples miRNAs) https://david.ncifcrf.gov/) http://metascape.org) P value <0.05, 5 counts  were most significant in participating DNA damage response, p53 signaling pathway. To GO-CC (Cellular component), calcium channel complex was numerous. Steroid binding accounted most in the GO-MF (molecular function). All genes in the genome have been used as the enrichment background. Through the Kaplan Meier-plotter software (https://kmplot.com) The DAVID database 6.8 (http://www.mirdb.org), Targetscan http://www.targetscan.org), Starbase https://starbase.sysu.edu.cn/), miRWalk  P<0.05) by the Kaplan Meier plotter software (https://kmplot.com) According to the targeted molecule BAX, we found miRNAs, which commonly regulated BAX from miRDB https://starbase.sysu.edu.cn/) http://bioinformatics.psb.ugent.be/webtools/Venn/) Identification of potential lncRNA about 4 miRNAs  was the critical step. The Starbase database (http://www.cytoscape.org/) Figure 1).PPI networks can provide information on the molecular mechanism. The same to us, lncRNA-miRNAs-BAX network were established by cytoscape3.7.2 software obviously (http://www.mircode.org), we predicted potential miRNA by lncRNA XIST. An miRNA family including miRNA 520 and miRNA 214 were also expressed in the ceRNA network. To recognize the differentially expressed miRNAs, GEO2R, an online software was used to conclude the primary data in GSE43249 . Besides, according to the differentially expressed miRNAs in the GSE43249 (GPL14613), we found out miRNA520 in NSCLC. This further proved the importance of miRNA520 in the cisplatin resistant NSCLC cells. Thus lncRNA XIST, miRNA520, BAX were considered to be the key molecules, mediating the apoptosis in the P53 signaling pathway.Contrarily, through the database miRcode p-value <0.05). There were 118 DEmRNAs, including 80 up-regulated genes, 38 down-regulated genes. In order to show DEmRNAs, we used the volcano maps and heat maps . We chose top 25 down regulated and up regulated mRNAs (Table 1).Through comparison and screening, we chose GSE6410 (GPL201) including mRNAs about cisplatin resistant cancer cells. GEO2R, an online software analyzed 8793 DEmRNAs from the samples (absolute Log Fold change>1 and http://metascape.org), an online software, to figure out the functions and pathways of 118 DEmRNAs. The most significant term was shown to represent the cluster . The p53 signaling pathway had numerous counts, including the targeted biomarker BAX. To show pathways of DEmRNAs, the enriched terms were constructed as a network. The different colors were classified by cluster ID, and the common cluster ID was closely related to each other .We used the Metascape (Table 2). Through the GO and KEGG analysis, the terms of P53 signaling pathway were typical. DEmRNAs may take part in this pathway to enhance cisplatin resistance in NSCLC cells. We selected the terms having the best p-values from each of the 20 clusters to show the relationships between the terms. Through the Cytoscape, we constructed the network where each node was an enriched term. Firstly, the network was colored by cluster ID  and then by p-value . We can identify that DEmRNAs were interrelate with each other. PPI networks can provide information on the molecular mechanism underlying cellular activity. In our study, a PPI network of differentially expressed mRNAs in NSCLC provided 4 mRNAs , which were associated with each other tightly . They were all key biomarkers in the P53 signaling pathway.At the same time, the top 5 terms of enrichments were listed as follows  by SPSS17.0 Figure ). In the NSCLC, the expression of BAX was highly showed in resistant lung cancer cells . The Kaplan Meier-plotter software (https://kmplot.com) was used to find the survival analysis of BAX (P<0.01) . By the GEPIA database (http://gepia.cancer-pku.cn/index.html), BAX's expression Profiling was higher in the LAC and LUSC cancer cells . As for cancers, different stages represent people's prognosis. Through our study, we proved that BAX had little impact on patients' staging . Some genes which were similarly with BAX, maybe up-regulated or down-regulated on chromosome . From the graph, some genes were considered to be over-expressed typically on the chromosome 14.Through the function enrichment analysis and pathway, we found that the counts in the P53 signaling pathway were most typical. Four mRNAs, CDKN1A, BAX, MDM2, and GADD45A; attracted our interest deeply. BAX may regulate the cisplatin resistance in NSCLC. We utilized a http://www.mirdb.org); Targetscan (http://www.targ-etscan.org); Starbase (https://starbase.sysu.edu.cn/); and, miRWalk . 8 miRNAs, miR-3681-3p, miR-766-5p, miR-525-5p, miR-4640-3p, miR-128-3p, miR-216a-3p, miR-520a-5p, and miR-214-3p were expressed commonly by the Dram-Venn-Diagram (http://bioinformatics.psb.ugent.be/webtools/Venn/) . The ceRNA network of lncRNA XIST-8 miRNAs-BAX were showed in the map  and we used the cytoHubba to choose the Hub gene. In the NSCLC, we predicted the survival of 8 miRNAs by Kaplan-Meier-Plotter, but only 4 miRNAs significantly affected quality of people's life and longevity . Using the starbase, we found lncRNAs of these 4 miRNAs and selected out together expressed lncRNA . LncRNA XIST and MIR29B2CHG may be the targeted ceRNAs. Combining with some articles reported in recent years, lncRNA XIST has some researches in cisplatin sensitive-resistant NSCLC cells. We considered that lncRNA XIST may act as a miRNA sponge to lead cisplatin resistance. At the same time, we combined the GSE43249's differentially expressed miRNAs, 2 miRNAs were included, such as miR-520, miR-525. miRcode database was used to verify miRNAs from lncRNA XIST. MiR-520, miR-525 were targeted miRNAs of lncRNA XIST. In the present study, lncRNA XIST acted as a member of ceRNA, competitively bound miRNA520 to regulate the BAX, apoptosis associated X, in the P53 signaling pathway.As is shown above, the BAX may be a useful molecule to cisplatin resistance. We chose the BAX as the important molecule to find miRNAs through different databases, including miRDB (http://lce.biohpc.swmed.edu/lungcancer/index.php#page-top). The expression of BAX was higher in 517 tumor samples than 59 normal samples (P=0.0037) . In different types of lung cancer, we found BAX's expression was significant through meta-analysis . At the moment, BAX expression level was commonly higher in all tumors through the TIMER database (https://cistrome.shinyapps.io/timer/) . Details were given in Table  In recent years, the tumor immune infiltration has taken important roles in cancer. The mutation of TP53 and BAX all influence immune cells; such as the B cell, CD8+ T cell, CD4+ T cell, macrophage, neutrophil, and dendritic cell (P<0.05) . Our results revealed positive correlations between BAX and immune cells in LAC and LUSC . LncRNA XIST and targeted molecule BAX's expressions were positive correlation  .In order to verify the accuracy of the data, we used the The Cancer Genome Atlas (TCGA) database through Lung cancer explore (LCE) database  is the most numerous type, with high diversity DDP is a significant chemotherapeutic drug in lung cancer. Thus, the potential mechanism of chemical resistance remains necessary to determine In recent years, lncRNAs have been explored through functional analysis Our study explored the role of potential lncRNA XIST, acting as a ceRNA of miR520 by regulating BAX involving in the P53 signaling pathway. Through the enrichment analysis of 118 DEmRNAs, we found that the targeted molecule BAX had higher expression in DDP. Four online databases were used to identify potential miRNAs mediated BAX. LncRNA XIST regulated four valuable miRNAs by starbase database.Figure ).The present findings clearly demonstrate that the expression of long non-coding RNA (lncRNA XIST) is up-regulated in lung cancer cells, and it maybe involve in the cell proliferation and TGF-β1-mediated apoptosis The implications of the research were providing some new treatment ideas. We found that molecules BAX, miRNA520, lncRNA XIST have been regulated closely each other. In addition, cisplatin can induce cell death by engaging endogenous apoptotic signaling that activates mitochondrial apoptosis P<0.05). Combining with the GSE43249 and miRcode database, miR520 was a potential molecule by regulating BAX in cisplatin sensitive-resistant NSCLC cells. Through the TIMER database For different tumors, the relations of long noncoding RNA (lncRNA) and microRNA influence cell process, cell growth, and drug resistance P<0.05). The interaction between these RNAs may have an important regulatory role in the immune infiltration in NSCLC, thereby affecting the patient's prognosis and immunotherapy efficacy.Immunotherapy has achieved unprecedented success in the treatment of cancer Through our study, potential molecules maybe provided for DDP. In some way, this can reduce cisplatin resistance to chemotherapy and be benefit of clinical NSCLC patients. Of course, there are certain limitations in present study. These associated molecules were predicted only in theoretical aspects. At present, the corresponding experimental verification is lacking. At the same time, the research of molecular mechanism was not deep enough. More importantly, the present novel indicated that lncRNAXIST-miRNA 520-BAX influence cisplatin resistance in NSCLC cells.In conclusion, BAX and LncRNA XIST were up-regulated in NSCLC patients. LncRNA XIST may act as a miRNA520 sponge by regulating BAX, associated apoptosis X, through activating in the P53 signaling pathway to affect cisplatin resistance. To summary, based on our data, this may enrich the effective therapeutic methods for cisplatin resistant NSCLC patients.


Lysobacterales  is a taxonomically complex group of a large number of gamma-proteobacteria classified in two different families, namely Lysobacteraceae and Rhodanobacteraceae. Current taxonomy is largely based on classical approaches and is devoid of whole-genome information-based analysis. In the present study, we have taken all classified and poorly described species belonging to the order Lysobacterales to perform a phylogenetic analysis based on the 16 S rRNA sequence. Moreover, to obtain robust phylogeny, we have generated whole-genome sequencing data of six type species namely Metallibacterium scheffleri, Panacagrimonas perspica, Thermomonas haemolytica, Fulvimonas soli, Pseudofulvimonas gallinarii and Rhodanobacter lindaniclasticus of the families Lysobacteraceae and Rhodanobacteraceae. Interestingly, whole-genome-based phylogenetic analysis revealed unusual positioning of the type species Pseudofulvimonas, Panacagrimonas, Metallibacterium and Aquimonas at family level. Whole-genome-based phylogeny involving 92 type strains resolved the taxonomic positioning by reshuffling the genus across families Lysobacteraceae and Rhodanobacteraceae. The present study reveals the need and scope for genome-based phylogenetic and comparative studies in order to address relationships of genera and species of order Lysobacterales.Order  Lysobacterales have undergone several reclassifications, until today the taxonomy position of species within the order is largely devoid of whole-genome information. The order Lysobacterales includes more than 200 species with great biotechnological potential. Current phylogeny is based on 16S rRNA gene phylogeny, conserved signature indels (CSIs) and classical approaches, such as morphological, biochemical or low-resolution molecular biology methods. Major limitations of these are not including all species of the order and are based on single or few conserved gene sequences. With the revolution in genome sequencing, whole genome can be utilized in a more profound way to investigate the phylogeny. Hence, this study attempts to achieve robust taxonomy and phylogeny of the order by sequencing type strains and including them up to the whole-genome level resolution. Our basic phylogenomic analysis revealed the existence of novel families within the order. In addition, reshuffling of the genera within the order across families is evident based on whole-genome sequence data. Whole-genome information of the type strain of the genus with unary species can serve as a reference and standard to compare later identified species of the respective genera.Species of order Lysobacterales have been deposited to GenBank and the accession number of each has been provided in Draft genome assemblies of six type species of order Lysobacterales .Phylogenetic tree file in Newick format (.nwk), constructed using the maximum-likelihood method of the 16S rRNA gene sequence of the 206 type strains of order Phylogenetic tree file in Newick file (.nwk), constructed using the 400 most conserved genes from the whole-genome data using PhyloPhlAn.Data file (G-I_OrthoANI.xlxs) used to generate the heat map of orthoANI values of all subgroups (G-I) found to have an unusual grouping in the phylogenetic tree construction using the >400 most conserved genes.Data file (G-II-OrthoANI.xlsx) used to generate the heat map of orthoANI values of all subgroups (G-II) found to have an unusual grouping in the phylogenetic tree construction using the >400 most conserved genes.Data file (G-I_POCP.xlsx) used to generate the heat map of the percentage of conserved protein (POCP) values of the unusual grouping (G-I) obtained from the phylogenetic construction.Data file (G2-POCP.xlxs) used to generate the heat map of the percentage of conserved protein (POCP) values of the unusual grouping (G-I) obtained from the phylogenetic construction.Xanthomonadales [Lysobacterales [Xanthomonadales. Key conserved proteins such as DNA ligase NAD-Dependent, MutS, RecA and DNA polymerase III alpha sub-unit with some amino acid insertion [Lysobacterales [In the advanced era of whole-genome sequencing, taxonomy is also advancing by providing us simpler and highly accurate alternatives to the cumbersome traditional methods. Taxonomy of the order nadales  is highlterales  based onterales . Prior snsertion  were useterales .www.bacterio.net/index.html) the family name Xanthomonadaceae [Lysobacter [Lysobacteraceae [Lysobacterales is sub-divided into two major families Lysobacteraceae and Rhodanobacteraceae [Xanthomonadales genera were out of the order Lysobacterales and were kept as unassigned family and order. Lysobacterales are considered early divergent of bacteria across class gamma-proteobacteria [Lysobacter are Gram-negative, non-flagellated, gliding, flexing and aerobic [Lysobacterales includes bacteria phenotypically, metabolically and ecologically diverse photoheterotrophs and chemoorganotrophs [Lysobacterales is based on phylogenomic and molecular markers to differentiate genera of previously known order Xanthomonadales. Genera such as Metallibacterium [Panacagrimonas [Pseudoxanthomonas [Thermomonas [Fulvimonas [Pseudofulvimonas [Rhodanobacter [According to Rule 51b [obacter , which ieraceae . In accoeraceae . Therefobacteria . The gen aerobic . The ordnotrophs . These anotrophs . Membersnotrophs , biocontnotrophs  and highnotrophs . The curcterium , Panacarimonas , Pseudohomonas , Thermomomonas , Fulvimvimonas , Pseudovimonas , Rhodanobacter  were notLysobacterales. We have generated high-quality draft genomes of six type species of family Lysobacteraceae and Rhodanobacteraceae, which could be an invaluable resource for the future phylogenomic and comparative genomics study of the order.In the current study, we have included genomes of the type strains available along with the six sequenced in the present study. Since this set of genomes represents the whole-genome sequence of the species in the order, this can depict the robust phylogenetic relationship across the genus of order Bacterial cultures were procured from The Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures GmbH and from The Belgian Co-ordinated Collections of Micro-organisms/Laboratory of Microbiology Gent Bacteria Collection (BCCM/LMG). All the isolates were grown as per media and conditions recommended by the respective culture collection centres.www.ncbi.nlm.nih.gov/genome/annotation_prok).Genomic DNA extraction was performed using ZR Fungal/Bacterial DNA MiniPrep Kit  and quantified using Qubit 2.0 Fluorometer (Life Technologies). An Illumina sequencing library of genomic DNA was prepared using Nextera XT sample preparation kit  with dual indexing adapters. The Illumina sequencing library was sequenced using an in-house Illumina MiSeq  platform using 250*2 v2 paired-end sequencing kits in accordance to the manufacturer’s recommendations. Raw reads were automatically de-multiplexing by using the internal control software of Illumina. Assembly of the reads was performed using CLC Genomics Workbench v9.4.2  with default parameters except minimum contig length set at 500 base pairs. Genome assembly quality in terms of completeness and contamination was accessed for all the sequenced genomes using CheckM v1.0.12  with defLysobacterales were fetched from respective International Nucleotide Sequence Database Collaboration (INSDC) number provided in LPSN. The phylogenetic tree based on the maximum-likelihood method was constructed from the multiple sequence alignment (MSA) [Xanthomonadales, including type strains from genus Ignatzschineria [Wohlfahrtiimonas [Vulcaniibacterium [Pseudomonas aeruginosa DSM 50071(T) [mega v7.0.18 [muscle v3.8.31 [Lysobacterales with species of genus Ignatzschineria and Wohlfahrtiimonas (Table S2)P. aeruginosa.  DSM 50071(T), a member of class Gammaproteobacteria, was used as an outgroup in the phylogenetic construction.In total, 16S rRNA gene sequences of all the representative type strains of all the species belonging to the order nt (MSA)  of 206 tiimonas  and genucterium  (Table Scterium  with def50071(T)  was used v7.0.18  using a  v7.0.18 . PhyloPh v7.0.18  to fetch v3.8.31  to perfo v3.8.31 . It inclLysobacterales, we have identified two unusual groupings from phylogenetic trees. Species of genus Rhodanobacter was sandwiched with unusual presence of species of genera Dyella, Frateuria, Luteibacter and Fulvimonas forming group one (G-I). Unusual group two (G-II) includes species from Lysobacter, Luteimonas and Thermomonas. We calculated the average nucleotide identity with OrthoANI v1.2 [https://software.broadinstitute.org/GENE-E/).The whole-genome-based comparative study at order level is a cumbersome task due to the lack of computational tools for genus and family delineation. In order to investigate the taxonomy of the order ANI v1.2 . OrthoANANI v1.2 . POCP usDyella japonica DSM 16301(T) , Lysobacter enzymogenes ATCC 29487(T) and Metallibacterium scheffleri DSM 24874(T) all genomes were more than 98% complete and less than 2% contamination. The whole-genome sequence of Dyella japonica DSM 16301(T) (NZ_JPLA01000000) [Dyella, we have dropped this genome out of our investigation.Raw Illumina sequencing reads were assembled to the high-quality draft genome using CLC Genomics Workbench v9.4.2. Assembled genome size remains in the range of 2.54 to 5.39 Mb, coverage in the range of 158x to 318x, N50 ranges from 38 253 to 222 295 bp. Percentage completeness and percentage contamination for each type species of the representative genus of the family has been summarized in detail with genome features and assembly statistics in Lysobacterales are from a very diverse range of ecology and habitat. The majority of the type species are environmental in nature except for type species of Stenotrophomonas maltophilia ATCC 13637(T), which is a human opportunistic pathogen [Species belonging to different genera of the order pathogen . SpeciesLysobacteraceae and another clade of type strains of the family Rhodanobacteraceae  [Lysobacter and has been kept as new genus Vulcaniibacterium [Lysobacterales. Genus Vulcaniibacterium with two species falls under family Lysobacteraceae. Overall, from the 16S rRNA phylogenetic tree, taxonomic positioning of species of order is shuffling at the family level needs to be genomically investigated.The maximum-likelihood phylogenetic tree of 16S rRNA gene sequences of all 206 type strains covering all genera of the order. Here, one clade consists of type strains belonging to the family eraceae . Further77875(T)  has prevcterium  out of tAquimonas and Pseudofulvimonas from family Rhodanobacteraceae to Lysobacteraceae. Further, the genus Metallibacterium falls in the family Rhodanobacteraceae and not in Lysobacteraceae. Genus Panacagrimonas remains in a separate clade  and 16 (G-II) species each. G-I holds species from genera Rhodanobacter, Dyella, Frateuria, Fulvimonas and Luteibacter. While G-II holds Lysobacter dokdonensis, Thermomonas and Luteimonas. Both the groups with an unusual grouping have been marked in orange and light blue in both 16S rRNA  between the two species. Similarly, in G-II taxonomic position of species Luteimonas mephitis and Luteimonoas tolerans remains separate from the rest of the clade of genus Luteimonas in the phylogenetic tree construction obtained was confirmed with the POCP value  are different species and possibly there is the existence of a minor unexplored genera different from rest of the Luteimonas clade.OrthoANI (cut-off for species boundary 96%) and POCP (cut-off for genus boundary 65%) values are in accordance with each other, reaffirming the misclassifications as obtained by phylogenetic analysis. POCP values suggest the species are diverse in nature and belong to different genera. In G-I, CP value . LuteimLysobacterales. The genus Aquimonas and Pseudofulvimonas need to be removed from the family Rhodanobacteraceae and placed in the family Lysobacteraceae. The genus Metallibacterium and Chiayiivirga need to be removed from the family Lysobacteraceae and placed in the family Rhodanobacteraceae. The genus Panacagrimonas needs to be removed from the family Lysobacteraceae and kept in as an unassigned family of the order Lysobacterales. The genus Vulcaniibacterium (www.bacterio.net/vulcaniibacterium.html), which is an unassigned genus needs to be a part of family Lysobacteraceae. The current study is an attempt to look at the complete order Lysobacterales in light of whole-genome sequence information including the type species of genera belonging to the order. Such a type of whole-genome-based phylogenomics study is a prerequisite for most robust phylogeny.Based on our findings, we suggest the reclassification of the order Lysobacterales is taken from the species definition page of the LPSN List of prokaryotic names with standing in nomenclature (www.bacterio.net/-classifphyla.html) of each genera.Definition of all valid species of the order The Genome (Type species) used in the phylogenomic analysis (detailed list of accession number in the Table S2).Arenimonas donghaensis DSM 18148 (T); AVCJ01000000;1. https://www.ncbi.nlm.nih.gov/nuccore/AVCJ00000000.1Luteimonas mephitis DSM 12574 (T); AULN01000000;2. https://www.ncbi.nlm.nih.gov/nuccore/AULN00000000.1Lysobacter enzymogenes ATCC 29487 (T); FNOG01000000;3. https://www.ncbi.nlm.nih.gov/nuccore/FNOG00000000.1Silanimonas lenta DSM 16282 (T); AUBD01000000;4. https://www.ncbi.nlm.nih.gov/nuccore/AUBD00000000.1Stenotrophomonas maltophilia ATCC13637 ; AE008922;6. https://www.ncbi.nlm.nih.gov/nuccore/AE008922.1Xylella fastidiosa ATCC 35871 (T); AUAJ01000000;7. https://www.ncbi.nlm.nih.gov/nuccore/AUAJ00000000.1Aquimonas voraii DSM 16957 (T); FNAG01000000;8. https://www.ncbi.nlm.nih.gov/nuccore/FNAG00000000.1Dokdonella koreensis DS-123 (T); NZ_CP015249;9. https://www.ncbi.nlm.nih.gov/nuccore/CP015249.1Dyella japonica DSM 16301 (T); JPLA01000000;10. https://www.ncbi.nlm.nih.gov/nuccore/JPLA00000000.1Frateuria aurantia DSM 6220 (T); CP003350;11. https://www.ncbi.nlm.nih.gov/nuccore/CP003350.1Luteibacter rhizovicinus DSM 16549 (T); CP017480;12. https://www.ncbi.nlm.nih.gov/nuccore/CP017480.1Rudaea cellulosilytica DSM 22992 (T); ARJQ01000000;13. https://www.ncbi.nlm.nih.gov/nuccore/ARJQ00000000.1Click here for additional data file.


Zizania latifolia shoots, referred to as gall formation, depends on infection with the fungal endophyte Ustilago esculenta. The swollen and juicy shoots are a popular vegetable in Asia. A key role for cytokinin action in this process was postulated. Here, trans-zeatin stimulated swelling in fungi-infected Z. latifolia. A two-component system (TCS) linked cytokinin binding to receptors with transcriptional regulation in the nucleus and played important roles in diverse biological processes. We characterized 69 TCS genes encoding for 25 histidine kinase/histidine-kinase-like (HK(L)) (21 HKs and 4 HKLs), 8 histidine phosphotransfer proteins (HP) (5 authentic and 3 pseudo), and 36 response regulators  in the genome of Z. latifolia. These TCS genes have a close phylogenetic relationship with their rice counterparts. Nineteen duplicated TCS gene pairs were found and the ratio of nonsynonymous to synonymous mutations indicated that a strong purifying selection acted on these duplicated genes, leading to few mutations during evolution. Finally, ZlCHK1, ZlRRA5, ZIRRA9, ZlRRA10, ZlPRR1, and ZlPHYA expression was associated with gall formation. Among them, ARR5, ARR9, and ZlPHYA are quickly induced by trans-zeatin, suggesting a role for cytokinin signaling in shoot swelling of Z. latifolia.The thickening of  Zizania latifolia belongs to the wild rice genus Zizania and was an important grain crop in ancient China. Around 2000 years ago, Z. latifolia was infected by the fungal endophyte Ustilago esculenta which causes swelling in shoot apical tissues, resulting in juicy galls. Nowadays, the swollen shoots of Z. latifolia are a popular vegetable in China , East Asian countries, and Japan [nd Japan ,2.Agrobacterium tumefaciens carries the Ti plasmid, which can integrate a DNA segment called T-DNA into the host’s genome [In nature, plant tumors or “galls” can be induced by different pathogens such as bacteria, viruses, fungi, protists, and insects. Despite different pathogenic mechanism to induce galls, most of these plant pathogens demonstrate the ability to change the phytohormone level in the host plants, especially cytokinins. For example, the well-studied gall-forming s genome . The T-Ds genome .Ustilago esculenta liquid culture, suggesting this fungi may also produce cytokinin in vivo. Another study revealed that the content of cis-zeatin riboside and cytokinin activity in terms of kinetin equivalents from fungi-infected gall tissue was elevated compared to non-fungi-infected shoots in Z. latifolia [Z. latifolia shoots.Increased production of cytokinins in the infected plant leads to uncontrolled cell proliferation and gall formation ,6. Chan atifolia . These rArabidopsis thaliana model have been identified and functionally characterized. Three histidine kinase proteins of AHK2, AHK3, and AHK4 have been characterized as cytokinin receptors [Cytokinins are a class of key phytohormones that mediate several processes including apical dominance, lateral branching, germination, flower and fruit development, chloroplast differentiation, plant–pathogen interactions, senescence, cell division in shoots, and spatial control of differentiation in roots ,11,12,13eceptors . All cyteceptors . Upon peeceptors ,17,18. Teceptors ,28,29,30Z. latifolia shoots swelling was investigated. Exogenous cytokinin stimulated the gall formation in fungi-infected shoots, in line with previous reports. As a first step towards understanding this response to cytokinins, genome-wide identification of TCS genes in Z. latifolia was conducted, which included sequences and functional domains analysis, phylogenetic relationship prediction, genes and protein structure analysis, and the analysis of the evolution of these members. Secondly, the expression of the TCS genes during shoot swelling was investigated by transcriptomics. Thirdly, the response of a subset of TCS genes to exogenous cytokinin was analyzed by quantitative real-time PCR (qRT-PCR). The obtained results establish a base for future functional analysis of TCS genes in cytokinin signal transduction in Z. latifolia, especially during gall formation.Capitalizing on the progress in genome sequencing, TCS genes have been identified and characterized in many other different plant species such as rice , maize , soybeanZ. latifolia shoots, Ustilago esculenta-infected plants develop galls which become up to 20 cm long over two weeks (p < 0.05) (p < 0.05)  B,C. Furt < 0.05) D,E. Chaned shoot . Furthered shoot . CollectZ. latifolia, HMM searches and BLASTP analysis were performed by employing 415 TCS protein sequences from Arabidopsis [Z. latifolia genome. All TCS members in Z. latifolia were named according to the homologous genes in Arabidopsis [Z. latifolia (n = 69) is similar to that in tomato (n = 65) but larger than that of all reported species except soybean (n = 98) and Chinese cabbage  and 4 related proteins (ZlHKLs), based on whether their HK domains were conserved or divergent  were 61% and 63% similar, respectively, to Arabidopsis AHK3. Another four AHK4-like cytokinin receptor genes  were highly similar (55%–72%) to Arabidopsis CRE1/AHK4/WOL  domains . Althougbidopsis . The HATbidopsis .Z. latifolia HK genes, we analyzed the exon–intron organization and conserved protein motifs  contained long C-terminal extensions with a Myb-like DNA binding domain  all had a conserved receiver domain  contained a receiver domain  trees [Z. latifolia usually have much closer relationships to those of rice and maize than other species, as they are all members of the monocotyledon. This indicates that the HK(L)s gene expansion occurred after the divergence of monocot and dicot plants. In addition, HK(L)s genes of monocots and eudicots showed an alternative distribution pattern in each subfamily, but no monocots occurred in the AHK1-like subgroup.To investigate the evolutionary relationships of TCS genes, we used all amino acid sequences of 124 HK(L)s, 53 HPs, and 275 RRs from bidopsis , rice [2bidopsis , maize [bidopsis , soybeanbidopsis , tomato L) trees . On the L) trees ,17,21. TAll HP proteins from the seven species were divided into three clades—clades I, II, and III . Clade IZ. latifolia were phylogenetically closer to rice OsRRs in all these groups. Phylogenetic analyses indicated that all type A RRs shared a close evolutionary relationship and an alternating distribution between monocots and dicots plants, suggesting that type A RR genes might already have expanded before the monocot–dicot divergence. Type B RRs from these species could be divided into three subgroups , consistent with previous studies [Arabidopsis, Chinese cabbage, and tomato; the RRs from monocots might have been lost during evolution. All type B ZlRRs from Z. latifolia except for ZlRRB3 and ZlRRB14 were classified into the type B I subfamily. A similar phenomenon was also viewed in soybean and cucumber [The 275 RR proteins from the seven plant species were classified into type A, type B, type C, and pseudo-RR groups . General studies ,26. In dcucumber ,28. All Z. latifolia expanded during evolution, the gene duplication events were investigated. Nineteen duplicated gene pairs in Z. latifolia TCS genes were identified including 10 ZlHK pairs, 3 ZlHP pairs, and 6 ZlRR pairs  were compared to samples from young but infected unswollen shoots (collected from fungi-infected plant with eight leaves) and samples from developing galls  transiently accumulated during gall formation and the activation of ZIPDK2 in young infected shoots , 2, 4, 8, and 24 h. The mock-sprayed samples displayed a similar trend for all genes measured. In these mock samples, we observed a transient decline in transcript levels followed by an increase after 8 h. In contrast, transcript levels of ZIRRA5, ZIRRA9, and ZIPHYA were elevated by zeatin treatment after two to four hours . To investigate the morphological changes of Z. latifolia in response to cytokinin, a 150 mg/L trans-zeatin  solution in 2% EtOH was sprayed onto the leaves of fungi-infected plants two weeks before their shoot swelling began. Two percent EtOH was used as mock treatment. The frequency of swelling was recorded daily over a period of 18 days. As the swelling ceased, shoots were harvested, and weight and size were recorded.To examine the expression profiles of TCS genes responding to cytokinin, 150 mg/L trans-zeatin solution was sprayed until the fully expanded leaves were all covered with the solution. The shoots’ apical tissues were then collected at 0, 1, 2, 4, 8, and 24 h after treatments. All samples were collected in three biological replicates with 3 shoots each and then stored at −80 °C until RNA extraction.Z. latifolia were downloaded from the Ricerelativesb GD (http://ibi.zju.edu.cn/ricerelativesgd/download.php). The protein sequences of other TCS genes from Arabidopsis, rice, maize, tomato, soybean, and Chinese cabbage were downloaded from Phytozome  or NCBI (www.ncbi.nlm.nih.gov). All TCS sequences were used to build a local blast database and then used as queries to perform BLASTP  searches with an E-value of 1e−5 as the threshold [Z. latifolia were searched with the hidden Markov model (HMM) of TCS characteristic domains from Pfam (http://pfam.janelia.org/). On the basis of the above two independent methods, the redundancy removal sequences were obtained. To confirm the reliability of the searched results, all candidate protein sequences were then confirmed with SMART (http://smart.embl-heidelberg.de/) [http://pfam.xfam.org/) [https://www.ncbi.nlm.nih.gov/cdd/) [The genome sequences of hreshold ,60. Howeerg.de/) , Pfam (ham.org/) ,63, and ov/cdd/)  accordinhttp://gsds.cbi.pku.edu.cn/) [PI) and protein molecular weight in kDa were obtained using ExPASy proteomics server (http://www.expasy.org/tools/) [http://www.cbs.dtu.dk/services/TargetP/). Multiple Expectation Maximization for Motif Elicitation  [Analysis of exons and introns were conducted using Gene Structure Display Server 2.04 (edu.cn/)  by compa/tools/) . Proteinro.html)  was usedro.html) . Next, pro.html) .Z. latifolia were subjected to a manual BLAST search, and two genes with a similarity of >75% were considered paralogous gene pairs. However, the tandem or segmental duplication genes could not be determined due to the lack of chromosome information. The selection pressure of paralogs was then determined by analyzing the synonymous and nonsynonymous nucleotide substitutions rates (Ka/Ks ratio) of each paralogous gene pair. In theory, Ka/Ks < 1 indicates purifying or negative selection, Ka/Ks = 1 indicates neutral selection, and Ka/Ks > 1 indicates positive selection [The TCS paralogous genes in election . The Ka/election .Z. latifolia. Samples from shoots apical tissue (0.5 cm in length) were collected from wild-type Z. latifolia (no fungi infected and never swollen), and galls in different size including non-swollen, 5, 10, and 20 cm, respectively. All samples were collected in three biological replicates with 3 shoots each, frozen immediately in liquid nitrogen, and then stored at −80 °C until used. RNA was extracted and the cDNA library was constructed using Illumina TruseqTM RNA sample prep Kit. The Illumina Novaseq 6000 platform was used for transcriptome sequencing, which generated a total of 254.5 Gb of sequence data. Upon filtering, the sequencing data to eliminate poor quality sequences, the sequences were mapped to the Z. latifolia genome using TopHat2  [To obtain a better understanding of the functions of the TCS genes, we investigated their transcriptional levels in the different gall formation stages in md.edu/) . The Tra® Green Realtime PCR Master Mix . The ACTIN gene was used as the housekeeping loading reference. The relative gene expression was calculated using the 2−ΔΔCt method [Total RNA was extracted using an RNAprep Pure Plant Plus Kit . The first cDNA strand was synthesized with 2 μg of total RNA by using a FastKing RT Kit (with gDNase) , as per the manufacturer’s instructions. qRT-PCR was performed with the CFX96 Touch C1000 Real-Time PCR System  by using specific primers designed by Primer 5 Software . Three bt method .Z. latifolia in line with earlier reports [Z. latifolia genome; the classification was supported by conserved motifs and domains, exon and intron structure, and phylogeny. Our phylogenetic analysis revealed the closest relationships between rice and Z. latifolia in seven species comparisons. Gene duplication events contributed to the TCS gene expansion in the Z. latifolia genome, and Ka/Ks analysis suggested that these duplicated gene pairs experienced strong positive selection during evolution.A role for cytokinin in gall formation was postulated. Here we observed a positive effect of cytokinin treatment on the gall formation in  reports ,8, whichWe observed the activation of the expression of several of these TCS genes during gall formation, either in the very early stages or during the development of the gall. Among these, two genes encoding for RRA-type response regulators were also rapidly induced by cytokinin treatment. This transient upregulation of these RRA type was in line with RRA genes being a direct target of cytokinin signaling in other plant systems. Furthermore, the far-red light receptor phytochrome A was revealed to be a putative target.Ustilago esculenta and its host Z. latifolia.In conclusion, these observations suggest an important role for cytokinin signaling in the process of gall formation and they highlight an important role for cytokinins in the ancient relationship between the endophyte


Daktulosphaira vitifoliae across the globe altered the course of grape cultivation. For the past 150 years, viticulture relied on grafting-resistant North American Vitis species as rootstocks, thereby limiting genetic stocks tolerant to other stressors such as pathogens and climate change. Limited understanding of the insect genetics resulted in successive outbreaks across the globe when rootstocks failed. Here we report the 294-Mb genome of D. vitifoliae as a basic tool to understand host plant manipulation, nutritional endosymbiosis, and enhance global viticulture.Although native to North America, the invasion of the aphid-like grape phylloxera Using a combination of genome, RNA, and population resequencing, we found grape phylloxera showed high duplication rates since its common ancestor with aphids, but similarity in most metabolic genes, despite lacking obligate nutritional symbioses and feeding from parenchyma. Similarly, no enrichment occurred in development genes in relation to viviparity. However, phylloxera evolved > 2700 unique genes that resemble putative effectors and are active during feeding. Population sequencing revealed the global invasion began from the upper Mississippi River in North America, spread to Europe and from there to the rest of the world.The grape phylloxera genome reveals genetic architecture relative to the evolution of nutritional endosymbiosis, viviparity, and herbivory. The extraordinary expansion in effector genes also suggests novel adaptations to plant feeding and how insects induce complex plant phenotypes, for instance galls. Finally, our understanding of the origin of this invasive species and its genome provide genetics resources to alleviate rootstock bottlenecks restricting the advancement of viticulture. Daktulosphaira vitifoliae (Fitch), did on the European grape, Vitis vinifera . Curated genes were merged with the automatic annotation using a custom script [https://github.com/abretaud/ogs-tools/tree/master/ogs_merge]. Putative functions of predicted proteins by the above pipeline were identified with blastp (v2.6.0) against Genbank NR , and interproscan v5.13-52.0 against Interpro. Associated GO terms were collected from blast NR and interproscan results with blast2GO (v2.5). Transmembrane domain signal peptides were identified by tmhmm v2.0c and signalP (euk v4.1), respectively. All genome resources and the Apollo server were made available online on BIPAA, within the AphidBase section [http://bipaa.genouest.org/is/aphidbase/daktulosphaira_vitifoliae/] [https://galaxy-genome-annotation.github.io/] [Gene predictions were generated using MAKER2 . Within n Apollo  server wfoliae/] . This syfoliae/] , Chado [foliae/] , Tripal foliae/] ) and devhub.io/] .A screening of contaminants was performed on scaffolds (blastp of the predicted proteins to nr), which allowed to eliminate 3 scaffolds identified as bacterial. We also used BLOB  which scnad5 sequence. Closer inspection showed a possible insertion of 45 T nucleotides within nad5. PacBio reads were mapped to this region, revealing that the insertion was likely due to a sequencing or assembly error. This insertion was removed, resulting in an intact nad5 gene sequence. The final assembled scaffold is 15,568 bp in length and has a GC content of 15.6%. A gene prediction analysis was performed on this scaffold using MITOS [The mitochondrial genome sequence from the grape phylloxera was found during genome assembly. The initial mitochondrial scaffold was 15,613 bp in length, and inspection of the predicted gene sequences revealed a frameshift within the ng MITOS  and ARWEng MITOS .A. pisum as query sequences for blastp searches on the predicted proteins of the phylloxera genome. The A. pisum genome also contains genes from bacterial sources [A. pisum sequences as queries, we performed blastp searches on protein databases for the genomes of D. vitifoliae, A. pisum, Myzus persicae, Diuraphis noxia, Aphis glycines, Rhopalosiphum padi, Diaphorina citri (Psyllidae), Pachypsylla venusta (Psyllidae), and Bemisia tabaci (Aleyrodidae). A blastp search was also conducted in the NCBI non-redundant protein sequence database, in order to identify other species where these genes might be present. The alignments were made using MAFFT v7.313 using default parameters [To determine if genes for carotenoid biosynthesis were present in the phylloxera genome, we used genes of this pathway previously characterized in  sources , 18, andrameters . PhylogeTransposable elements (TEs) were identified and annotated using the REPET package v2.2 , 117. MaA. pisum  , M. persicae  , D. noxia  , C. cedri  , A. glycines Matsumara, 1917 , R. padi  , D. citri Kuwayama, 1908 , B. tabaci  , and the true bug Rhodnius prolixus  . As outgroups, we selected four insect taxa: D. melanogaster Meigen, 1830 , Nasonia vitripennis  , Frankliniella occidentalis  , and Pediculus humanus  . Genome versions are indicated in Additional File http://bipaa.genouest.org/sp/daktulosphaira_vitifoliae/), we performed a blastp search against the custom proteome database built from the genomes listed above, which included a total of 252,530 proteins. Results were filtered using an e value of 1e−05 and a minimum of 50% overlap between the query and the hit sequences. Multiple sequence alignments were reconstructed in forward and in reverse [http://phylomedb.org), with the phylomeID 196. A species-overlap algorithm, as implemented in ETE v3.0 [The phylome  of grape phylloxera was reconstructed to obtain a dynamic view of gene family expansion within this genome. We included nine other fully sequenced genomes of Hemiptera based on their phylogenetic position:  Harris, 776  and of other factors, we removed all genes annotated as proteins encoded by TEs, prior to the inference of gene expansions, GO term enrichment and gene gains, losses, and duplications.For each gene tree, we selected the nodes that contained only phylloxera sequences with ETE v3.0 . Nodes whttp://bipaa.genouest.org/sp/daktulosphaira_vitifoliae/), all genes in the phylloxera genome were functionally annotated with InterProScan v.5.19 [In addition to the automatic and manual annotation performed on the phylloxera Official Gene Set (OGS) . To evaluate synonymous distances (dS) among paralogs, we used a Reciprocal Best Hit approach (RBH) by blasting gene collections against themselves, determining pairs of genes that matched the RBH criteria. Doing this in just one step would lead us to focus on terminal branches in expanded gene families, neglecting deeper nodes and thus missing the ancient dynamics in the history of duplications. To account for this, we applied an approach similar to that used in [A. pisum genome  and for D. melanogaster .To remove potentially spurious gene models from the official gene set, we first used a filtering step, eliminating genes which had very weak support: these were defined as genes with no manual annotation, no hit to the nr database of GenBank, and very low RNA-Seq support . This left us with  used in : after a used in ; this st used in ). For cohttp://arthropodacyc.cycadsys.org/).We used the Cyc Annotation Database System (CycADS ), an autD. vitifoliae, A. pisum, and M. persicae (clone G006) were exported in the SBML format and imported into the PSAMM software [gapcheck” function, which reports a list of all metabolites not produced in the metabolic network. Then, the objective function was defined for each non-producing metabolite, and a gap-filling procedure was performed for each objective function through individual rounds of simulations using the PSAMM implementation of the fastgapfill algorithm [A. pisum and M. persicae were used as candidates for identifying potentially missing annotations. Following the gap-filling predictions, candidate missing genes were identified through the identification of homologs to annotated genes in A. pisum and M. persicae. This was achieved with manual curations using evidence from blast alignments, Pfam protein domain identifications [https://www.brenda-enzymes.org/), InterPro [https://www.genome.jp/kegg/), PhylomeDB [Metabolic reconstructions from the ArthropodaCyc databases for software . First, lgorithm . In the ications , phylomeications , transcrInterPro , KEGG orhylomeDB , and crohylomeDB  genome bD. melanogaster reference proteins. This approach was then extended to a complete collection of D. melanogaster immune genes. For reciprocal best hits (RBH) between phylloxera and D. melanogaster, the D. melanogaster annotation was directly transferred to phylloxera. In other cases (non-RBH relationship), a manual curation was performed, using the genomic information for arthropods with well-annotated immune pathways  or for other aphid genomes (A. pisum and M. persicae) available in Genbank, ArthropodaCyc, and ImmunoDB [Immune genes were annotated using bidirectional blastp analyses. We first used the phylloxera gene set to identify proteins with similarity to genes of the IMD and TOLL pathways. These putative phylloxera proteins were then blasted against ImmunoDB  databasehttp://aias.biol.uoa.gr/CutProtFam-Pred/), with standard settings. Candidate genes were then fully manually curated on AphidBase through Apollo. Phylogenetic analyses were performed using the updated protein sequences of sets of RR-1 or RR-2 genes of M. persicae [A. pisum [D. noxia [http://www.expasy.org/tools/) and removed; then phylogenetic analyses were conducted on the mature sequences. For RR-2 proteins, only the extended 69 amino acids RR domain (pfam00379) was used for phylogenetic analysis, the rest of the sequences being too divergent to align. Alignments were made with Clustal Omega [To determine the full set of genes coding for cuticular proteins (CPs) (including cuticular proteins with R&R motif defined as CPR proteins ), we seapersicae , A. pisuA. pisum , and D. D. noxia . RR-1 anal Omega , and phyal Omega  where alSelenoproteins contain the non-canonical amino acid selenocysteine (Sec), known as the 21st amino acid. Sec is encoded by a UGA codon, normally a stop codon, and is inserted through a recording mechanism that requires a dedicated set of factors known as the Sec machinery . SelenopVitis host plants, we modified a bioinformatics pipeline from [Mayetiola destructor), a plant manipulating herbivore [e value < 1e−5) the salivary gland-derived Hessian fly effector genes. We therefore screened the 24,585 automated gene models (OGS3.0_20161223_proteins) and predicted a first set of 354 effector genes that classified (using OrthoMCL) into 78 clusters according to sequence similarity. We then performed manual annotation on each of these clusters to (1) correct gene models based on the transcript data from gallicole, radicicole, and egg samples [D. vitifoliae. While conducting manual annotation on the genome, we detected a number of putative genes which had particular characteristics: (1) absence of automatic annotation , (2) presence of an ORF usually encoding more than 200 amino acids and corresponding to a monoexonic structure, and (3) clear RNA-Seq support, in particular in the radicicole samples. The two former points suggested that these ORFs represented bona fide genes, with a particular intron-less structure. Such pattern is usually penalized in gene prediction tools for Eukaryotes, which could explain the absence from the automatic gene model prediction. Additional traits of these genes suggested that they encoded effectors because of (1) the presence of a secretory signal peptide in the N-terminus; (2) clusters of similar gene copies, indicative of tandem gene duplication; and (3) some sequence similarity to the putative effectors predicted using the AGMB approach described above. To generalize the search of similar genes, we performed tblastn searches to the genome and annotated matching regions which shared the above patterns. Because we usually found different hits in each search, but with a relatively low amino acid identity (as low as 20%), it appeared that the grape phylloxera genome encodes highly expanded gene families characterized by high evolutionary rates. To ensure that we collected the most complete collection of genes, the tblastn searches were performed iteratively, each time using the collection of manually annotated monoexonic effector candidates as a query data set, then annotating the new hits, and repeating this process until no new hits were detected. Some of the effector candidates identified using this non-automated gene model-based (NAGMB) approach overlapped with those identified through the AGMB pipeline and therefore were combined with the latter resulting into a total number of 2741 manually annotated predicted effectors (PREFs) in the phylloxera genome. Genes were clustered using SiLiX. Because numerous PREFs appeared unique, lacking sequence homology to other PREFs, and comparisons were based within species rather than among species, the final clusters were determined through an iterative process in SiLiX. As overlap among sequences increased to 60%, very few new clusters were formed. Similarly, as identity decreased down to 20%, the number of clusters reached a minimum. Thus, 60% overlap and 20% identity were designated as conservative thresholds per parameters defined in SiLiX. As PREF function is validated through further study, these thresholds may change to best organize clusters without breaking up families of known function predicted from sequence motifs. Phylogenetic analyses of the largest orthogroup  were performed following the protocol described by [To identify genes underlying effector proteins active when grape phylloxera interacts with ine from . This piine from , 158. Teerbivore , showed  samples  integratribed by  with modribed by  with “auVitis species: Vitis arizonica (Arizona), Vitis labrusca (Massachusetts), Vitis aestivalis (West Virginia), Vitis vulpina , Vitis riparia , interspecific hybrid Chambourcin and Concord , rootstocks 1103P . Populations from introduced areas  were collected from galls on leaves of Vitis vinifera cv. Details on this sampling are presented in Additional File https://github.com/broadinstitute/picard). Each pileup file was then subsampled with Popoolation2/subsample-pileup.pl [D) were calculated with Popoolation2/Variancesliding.pl. The counts of major alleles for each population and for each position were calculated from the subsamples and used as entry for the PCA (FactomineR). We used popoolation2/FST_sliding to estimate pairwise FST after the synchronization of the pileup files with Popoolation2/mpileup2sync.jar, extraction of polymorphic sites  and subsampling (as above). The average of FST pairwise were computed and used for generating a distance matrix distance for the MDS plot (done with R/ggplot2).Phylloxera individuals were collected from both native and introduced areas. All samples consisted of gall-feeding adult insects except for two American populations  that were sampled as root-feeding insects. Insects collected in the same geographical area  and on the same host plant species or cultivar were pooled and considered to represent one population for genome resequencing. In the native area, samples were collected either on cultivated grapevines or on wild ileup.pl  in orderIn order to test various demographic scenarios for the introduction of phylloxera in Europe, we used diyABC, and abcRF . We randAdditional file 1: Figures. S1-S22, Table S1-S20, Methods and Results. Figure S1. Mitochondrial genome view of grape phylloxera. Figure S2. Proportion of transposable elements (TE) in the genome. Figure S3. GO terms of phylloxera-specific genes. Figure S4. Enriched GO terms in the phylloxera genome with and without TEs. Figure S5. Gene gain/loss at different nodes or branches. Figure S6. Species phylogenetic tree based on insect genomes and the transcriptomes of Planoccoccus citri and Adelges tsugae. Figure S7. Diagram of the gap-filling and annotation process. Figure S8. Urea cycle in D. vitifoliae and A. pisum. Figure S9. IMD immune pathway in D. vitifoliae.Figure S10. Phylogenetic tree of RR-1 cuticular proteins.Figure S11. Phylogenetic tree of RR-2 cuticular proteins.Figure S12. Comparison of miRNAs in D. vitifoliae and other insect genomes. Figure S13. Phylogenetic tree of aquaporin protein sequences. Figure S14. Comparison of the phylloxera PER protein with other insects. Figure S15. Amino acid alignment of PTTH amino acid sequences. Figure S16. Phylogeny of hemipteran ORs. Figure S17. Phylogeny of hemipteran GRs. Figure S18. Phylogenetic analysis of OBPs. Figure S19. Phylogenetic analysis of CSPs. Figure S20. Phylogenetic analysis of NPC2s. Figure S21. Distribution of cluster sizes of putative effectors. Figure S22. Physical distribution of the three largest clusters of effectors. Table S1. Genes of bacterial and fungal origin. Table S2. Statistics on TEs. Table S3. GO enrichment of genes duplicated at different ancestral nodes. Table S4. Metabolic gaps in the D. vitifoliae reaction network. Table S5. Functional annotation of metabolic genes. Table S6. Genes of the TOLL pathway. Table S7. Genes of the IMD pathway. Table S8. Statistics on cuticular proteins. Table S9. Developmental genes in D. vitifoliae and A. pisum. Table S10. miRNAs. Table S11. Clock-related genes. Table S12. List of ORs and GRs. Table S13. Number of OBPs, CSPs and NPC2s. Table S14. List of Cytochromes P450. Table S15. List of genes involved in detoxification. Table S16. Effector genes with predicted domains and their corresponding functions. Table S17. Statistics on sequence reads and SRA accessions used for the reference genome. Table S18. List of species used to study gene expansions. Table S19. Sampling sites and SRA used for population genomics analyses. Table S20. Prior distribution of parameters used for ABC modeling of invasion routes.


Leishmania donovani parasites. L. donovani variants causing cutaneous manifestations in this region are different from those causing visceral leishmaniasis in northeastern India.We conducted a molecular study of parasite sequences from a cohort of cutaneous leishmaniasis patients in Himachal Pradesh, India. Results revealed atypical cutaneous disease caused by  Leishmania species generally associated with VL can cause CL and vice versa. Leishmaniasis is a complex disease with cutaneous, mucocutaneous, or visceral manifestations depending on the parasite species and host immunity. Despite continued elimination efforts, leishmaniasis continues to afflict known and newer endemic regions, where 0.5–0.9 million new cases of visceral leishmaniasis (VL) and 0.6–1.0 million new cases of cutaneous leishmaniasis (CL) occur every year  in Kinnaur, Shimla, and Kullu and the previously nonendemic districts of Mandi and Solan  analysis of parasite species–specific internal transcribed spacer 1 (ITS1) sequences by using appropriate standard controls. We detected the expected ≈320-bp product with a nfection  Figure 4https://blast.ncbi.nlm.nih.gov/Blast.cgi) of 44 ITS1 test sequences showed all the samples to be closest to L. donovani, having maximum identity to L. donovani isolates from Bhutan (GenBank accession nos. JQ730001–2) and possibly L. infantum. None of the CL cases were consistent with L. tropica infection, unlike in a previous report  stretch  exhibit a high degree of polymorphism and have been used to identify atedness , panel AL. donovani isolate from Sri Lanka and distinct from the VL-causing L. donovani isolates from India and Bangladesh and CL-causing isolates from Kerala and Sri Lanka (L. infantum strain, although ITS1 analysis using BLAST and microsatellite repeat sequences showed regions of similarity with L. donovani. ITS1 and 6PGDH sequence analysis suggest that Himachal Pradesh isolates from CL patients consist of heterogenous L. donovani variants and possibly represent hybrid genotypes.Phylogenetic analysis of 6PGDH amino acid sequences of CL isolates grouped them into a heterogeneous cluster; variants were closer to a viserotropic ri Lanka , panel BL. donovani–specific ITS1 (L. donovani infection in a fraction of CL patients.None of the CL patients had VL-specific symptoms or VL history. Ten of 43 patient blood samples tested positive for rK39 antibody, and 37 of 51 samples were positive for the circulating parasite DNA with L. donovani variants distinct from the viscerotropic L. donovani strain from northeast India. The CL isolates in Himachal Pradesh exhibit considerable heterogeneity and indicate the possible existence of genetic hybrids. The scenario appears somewhat similar to Sri Lanka and Kerala, where L. donovani parasites cause cutaneous disease, albeit with differences in the region-specific L. donovani variants. In lieu of the coexistence of VL and CL in Himachal Pradesh, parasite isolates from VL patients also need to be characterized. To understand the biology of atypical L. donovani variants with cutaneous manifestations and to genetically differentiate the dermotropic versus viscerotropic potential of L. donovani variants, comparison of CL- and VL-causing isolates in Himachal Pradesh using whole-genome sequence analysis is required.The presence of leishmaniasis in Himachal Pradesh is not yet well known in India and globally  Figure 1L. donovani parasites in the blood of some CL patients represent human reservoirs similar to asymptomatic VL carriers, and the parasite variants have the potential to cause full-blown VL manifestations. An elaborate surveillance program dedicated to the Himachal Pradesh region is urgently required for better diagnosis, treatment, prediction of parasite variants in different afflicted pockets, and prevention of transmission of the disease to other regions.Leishmania donovani infection with atypical cutaneous manifestations, Himachal Pradesh, India, 2014–2018.Additional information about