Fusarium spp. is a destructive disease of wheat. Since the most effective sources of FHB resistance are typically associated with unfavorable agronomic traits, breeding commercial cultivars that combine desired agronomic traits and a high level of FHB resistance remains a considerable challenge. A better understanding of the molecular mechanisms governing FHB resistance will help to design more efficient and precise breeding strategies. Here, multiple molecular tools and assays were deployed to compare the resistant variety Sumai3 with three regionally adapted Canadian cultivars. Macroscopic and microscopic disease evaluation established the relative level of Type II FHB resistance of the four varieties and revealed that the F. graminearum infection process displayed substantial temporal differences among organs. The rachis was found to play a critical role in preventing F. graminearum spread within spikes. Large-scale, organ-specific RNA-seq at different times after F. graminearum infection demonstrated that diverse defense mechanisms were expressed faster and more intensely in the spikelet of resistant varieties. The roles of plant hormones during the interaction of wheat with F. graminearum was inferred based on the transcriptomic data obtained and the quantification of the major plant hormones. Salicylic acid and jasmonic acid were found to play predominantly positive roles in FHB resistance, whereas auxin and ABA were associated with susceptibility, and ethylene appeared to play a dual role during the interaction with F graminearum.Fusarium head blight (FHB or scab) caused by  Fusarium graminearum Schwabe (teleomorph Gibberella zeae (Schweinitz) Petch), is one of the most destructive diseases of wheat (Triticum aestivum L.) [F. graminearum spores attach and germinate onto flowering spikelets. Subcuticular and intercellular growth of F. graminearum has been observed during the first 2 days post infection (dpi), suggesting a biotrophic phase in advance of necrotization of the plant tissue by intracellular hyphae [F. graminearum hyphae pass the rachilla of the inoculated spikelet and enter the cortex of the rachis node [F. graminearum hyphae invade uninoculated spikelets, resulting in severe dark-brown, water-soaked disease symptoms that eventually cause premature bleaching of the wheat spike [Fusarium head blight (FHB), primarily caused by ivum L.) . The funr hyphae . Necrotrr hyphae . At 3 orr hyphae . After 5his node . Severalat spike .F. graminearum [Host resistance is broadly considered as the most economical, ecofriendly and efficient approach to control FHB . Three ominearum . These dminearum . Alteratminearum , reductiminearum  and qualminearum . Severalminearum . To survminearum , phenomeminearum .F. graminearum and wheat will help us to better understand this complex trait and to design more efficient and precise breeding strategies. In this study, the molecular mechanisms of FHB resistance were investigated by comparing the resistant variety Sumai3 with three regionally adapted Canadian cultivars. The extent of FHB resistance in four varieties was determined macroscopically and microscopically through a comprehensive disease evaluation strategy. Large-scale, organ-specific RNA-seq and hormone profiling experiments were performed on the four varieties after F. graminearum infection. Compared with “bulk material”, organ-specific analyses prevent averaged information and diluted signals, and provide more accurate and precise information [F. graminearum infected wheat spikes, which are composed of multiple organs. From this analysis, the role of several plant hormones is proposed during interaction with F. graminearum, including salicylic acid (SA), jasmonic acid (JA), ethylene (ET), auxin and abscisic acid (ABA).Most existing FHB resistance is associated with other unfavorable agronomic traits . In-deptormation . This isAll experiments were conducted in environment-controlled growth chambers. Seeds were sown in peat pots  and maintained in a growth chamber at 21°C/19°C: day/night cycle, with a 16 h photoperiod. Plants were fertilized weekly with 20-20-20 (N-P-K).Four cultivars are used in this study. Sumai3 is among the most resistant cultivars, but displays unacceptably low yield potential and low end-use quality . The CanF. graminearum, 3-ADON chemotype (M9-4-6), originally obtained from Dr. J. Gilbert at the Agriculture and Agri-Food Canada Cereal Research Centre in Winnipeg, MB, was used throughout. Fungal growth and inoculation assays were as described previously [A Western Canadian isolate of eviously . Briefly4 spores ml-1) between the palea and lemma. Inoculated plants were covered with a plastic bag for 2 days. For disease severity tests and microscopic observation, a pair of alternate spikelets in the middle of the head was inoculated. The number of infected rachis internodes and spikelets from the inoculated site was recorded over a three-week period. Two spikes per plant and 20 plants per variety were examined. The experiment was repeated three times with similar results. Data from each time point were analyzed using a one-way Analysis of Variance (ANOVA), General Linear Model , and differences between varieties determined by post-hoc testing (Tukey’s Honest Significant Difference test).At mid-anthesis, single floret inoculation was carried out by pipetting 10 μl of the macroconidial suspension , Alexa Fluor 488 conjugate  and examined by fluorescence stereomicroscopy . Photos were taken with a Zeiss AxioCam HR colored camera.Total RNA from each sample was isolated using the Plant RNeasy Mini Kit , according to the manufacturer’s instructions. All samples were treated with DNase I on column using the Qiagen RNase-Free DNase Set. The yield and RNA purity were determined spectrophotometrically with a Nanodrop 1100 , and the quality of RNA was monitored by determining the RNA Integrity Number (RIN) with an Agilent 2100 bioanalyzer and RNA LabChip . Samples with a RIN value > = 7 were used to prepare cDNA libraries by using the TruSeq RNA Sample Preparation Kit v2 (Illumina). Paired-end sequencing was conducted on the Illumina HiSeq2500, generating 101-nucleotide reads, at the National Research Council, ACRD-Saskatoon, Canada.http://wheat-urgi.versailles.inra.fr/)  and read counts per gene were estimated using STAR (v2.4.2a) [Sequencing adapters were removed and low-quality reads were trimmed following the methods described in . The filv2.4.2a) .Normalization and differential expression analysis was performed with DESeq2 . After cprcomp function from the R base package. Malhalanobis distances and associated p-values were calculated using the pca-utils software tools [Principal component analysis (PCA) was performed using the re tools .Arabidopsis thaliana, Brachypodium distachyon, Oryza sativa Japonica, and Zea mays (EnsemblPlants version 25). GO enrichment analysis was performed by using GOAL software ([http://archive.plants.ensembl.org/Triticum_aestivum/Info/Index). Only GO terms for Biological Process are shown.Gene Ontology (GO) annotations for mRNA transcript sequences were derived from databases containing orthology sequences of oftware . The GOhttps://www.nrc-cnrc.gc.ca/eng/solutions/advisory/plant_hormone.html).Phytohormones were extracted from individual replicate and quantified by UPLC/ESI-MS/MS at the National Research Council, ACRD-Saskatoon, Canada  (F. graminearum had successfully entered the rachis and was able to infect successive rachises and uninoculated spikelets (The on (wpi) . The onlon (wpi)  Fig. Thepikelets .F. graminearum, whereby the fungus predominately uses vertical intercellular hyphae to rapidly colonize the vasculature at early stages, and later deploys lateral intracellular hyphae to penetrate the rachilla of uninoculated spikelets [F. graminearum into uninfected spikelets early in the infection process. At a later stage, F. graminearum switches to its necrotrophic lifestyle to kill host cells and successfully infect uninoculated spikelet [The rachis internodes adjacent to the inoculated spikelets of the three Canadian varieties were already severely bleached or brown at 1 wpi . Milder pikelets . Alternaspikelet .F. graminearum in FL62R1 was slower than in Stettler at all time points and slower than in Muchmore by 3 wpi , while the rachilla and rachis adjacent to inoculated spikelet were harvested together from 2 to 4 dpi (“rachis”). Stettler samples at 4 dpi were not collected due to an unexpected problem. In total, 93 samples (31 sets of triplicates) were collected and submitted for RNA sequencing.To capture the most valuable dynamic transcriptome changes in the four wheat varieties after F. graminearum genomes after quality filtering  per sample were aligned to the wheat and iltering . The perncreased . At 3 dpp to 63% . Furtherp to 63% .F. graminearum infection between the two organs.Principal component analysis PCA,  on diffeF. graminearum infection. Plant hosts may slow down or postpone developmental and photosynthetic processes and reallocate the resources and energy to combat F. graminearum [F. graminearum could disrupt host biological processes when invading a host [A set of differentially expressed genes (DEGs) derived from each variety was identified according to their significance in fold-change expression (p ≤ 0.01) and an additional threshold level cutoff of at least a two-fold change (-1>log2>1) in comparison to the respective mock treatment  Datasetsminearum . Alternag a host .F. graminearum  of SA-related DEGs were up-regulated after F. graminearum infection in all four varieties  is a key defense hormone contributing to defense against biotrophic and hemibiotrophic pathogens . Intercerotrophy , 7. Thisminearum –36. Consarieties . The actarieties  and 5A.NPR1 was only up-regulated in the spikelet  is considered to be a critical hormone for plant defense responses against pathogens with a necrotrophic lifestyle . Previounfection , 39, 40.minearum , 39.F. graminearum infection  is dramatically induced in plant tissues after pathogen challenge ; howeverThe GO enrichment identified three ET-related terms among significantly up-regulated DEGs of the spikelets from Sumai3 and FL62R1 at 1 dpi; “ET biosynthetic processes”, “ET metabolic processes”, and “response to ET” . One dayExtraction of all ET responsive DEGs indicated that most were up-regulated in the spikelet of Sumai3 and FL62R1 from 1dpi, but not until 3 dpi in Stettler and Muchmore . Many ofF. graminearum infection. To our knowledge, this is the first report to combine RNA-seq profiling with ethylene (ACC) quantification. Levels of ACC were dramatically increased, by at least 12-fold, in both organs of all four varieties after F. graminearum infection , the immediate precursor of ET, after nfection . In the F. graminearum has long been characterized as premature senescence [F. graminearum infection. We presume that elevated levels of ET may contribute to disease development through promotion of senescence during F. graminearum infection. Consistent with this hypothesis, silencing a key ET signaling gene, ETHYLENE INSENSITIVE2 (EIN2), reduced F. graminearum -induced FHB symptoms in the moderately susceptible wheat cultivar Bob White [By 4 dpi, levels of ACC in the spikelet and rachis of Sumai3 were lower than in the other varieties . It is knescence . The GO ob White .Based on transcript profiling, there have been conflicting conclusions on the role of ET in FHB resistance. Ding et al.  and Li aF. graminearum infection  were measured after Muchmore . Auxin aarieties . Compareze auxin  and the F. graminearum biomass  has recently been shown to have biological function towards disease susceptibility . We examat 4 dpi . In the F. graminearum-induced plant responses, most likely associated with susceptibility. Conjugated IAA accumulated to high levels after F. graminearum infection in Canadian germplasm, including FL62R1 that possesses intermediate levels of resistance. This illustrates the complexity of auxin metabolism in response to F. graminearum.These data support a role for auxins in F. graminearum, the predominant penetration route for the fungus is either via cracked anthers, to grow down the filament into the host plant, or through active penetration of the epidermal cuticle and cell wall of floret [F. graminearum implies that the ABA-mediated stomatal closure defense has little impact on FHB resistance. In contrast, co-application of exogenous ABA with F. graminearum increases wheat susceptibility [In addition to its well-documented roles in development and abiotic stress responses, abscisic acid (ABA) has also been implicated in modulated responses to various diseases . Dependif floret , 56. Thetibility , 58, sugF. graminearum infection , possess ABA-like biological activity ranging from the regulation of stomata closure to inhibition of seed germination , 60. EveSimilar to the ABA pattern, PA and DPA accumulated to high levels at 4 dpi in the spikelet of the four varieties . At thisF. graminearum infection, which allowed organ-specific analyses to be performed.Building on the existing well-accepted point inoculation assay, a more comprehensive and precise disease evaluation strategy for FHB was developed. To accurately capture disease development on wheat spikes, extra disease symptom parameters were recorded, including the number of brown or bleached rachis internodes, the assessment of disease symptoms above or below the inoculation site, and the combined macroscopic and microscopic observations. This analysis demonstrated that distinct organs in the spike differentially responded to F. graminearum infection in much the same way as observed for other plant-pathogen systems, including faster and more pronounced activation of well-characterized defense responses in resistant germplasm. However, the availability of a reference wheat genome, the use of RNA-seq technology, and organ-specific profiling yielded more precise and meaningful information than many previous studies. Combined with the comprehensive quantification of multiple plant hormones, detailed information on the potential role of these growth regulators could be inferred, including:Overall, transcript profiling results obtained were consistent with previous reports reviewed in  that wheF. graminearum. Although previous transcriptome analyses emphasized a positive role of JA for FHB resistance [SA and JA play predominantly positive roles in FHB resistance, whereas auxin and ABA may be associated with susceptibility to FHB. ET appears to play a dual role during the interaction with sistance , 39, 40,A number of DEGs were commonly involved in more than one hormone pathway  Fig, indF. graminearum challenge or between varieties, and no increase in SA or JA levels were detected in the rachis following F. graminearum infection.Patterns of hormone-related gene expression and levels of hormones differ substantially between the spikelet and the rachis, with the spikelet displaying faster and greater activation of most hormone-related signaling and response genes in resistant varieties, which was not observed in the rachis. Limited differences in hormone-related gene expression were detected in the rachis at different times after F. graminearum challenge differed considerably between phytohormones with three patterns emerging: (i) up-regulation of both biosynthetic/metabolic and signaling/response category pathways, observed for SA and ET; (ii) down-regulation of biosynthetic/metabolic pathways and up-regulation of signaling/response pathways, seen with JA and ABA; (iii) up-regulation of biosynthetic/metabolic pathways and down-regulation of signaling/response pathways, witnessed for auxin. Comparisons with other genome-wide gene expression datasets will be needed to determine the generality of these patterns, while additional analysis will be required to assess their significance. This could include pharmacological tests involving the application of plant hormones or antagonists, and genetic studies to manipulate the expression and function of key genes within target pathways.Overall patterns of pathway regulation following Overwhelmingly, the same DEGs were identified in all four varieties as well as both organs studied; however, dramatic differences were detected in the levels of expression among varieties and between organs. These are likely the key determinants between resistance and susceptibility.S1 FigPhotographs were taken of the entire inoculated spikelet and adjoining rachis (A), and of the individually excised organs of the inoculated spikelet, lemma (B), glume , and rachilla (E).(TIF)Click here for additional data file.S2 FigThe individually excised organs of inoculated spikelet, lemma (A), palea (B), glume (C), and rachilla (D) were examined at different days after inoculation. All organs were stained with WGA; rachillas were additionally stained with the phloroglucinol-HCl solution to indicate lignification. Samples were separately photographed under fluorescence (left-side photo) and light (right-side photo) microscopy. Fungal hyphae appear in green under fluorescence. Lignin appears as red under light microscopy. The scale bar indicates 200 μM. Each row of photographs represents the same time point among four varieties.(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 FigRed bars represent up-regulated DEGs; green bars represent down-regulated DEGs. The Y axis represents the number of DEGs; X axis represents time points.(TIF)Click here for additional data file.S5 Fighttp://bioinformatics.psb.ugent.be/webtools/Venn/. Pathways related to phytohormones salicylic acid (SA), jasmonic acid (JA), ethylene (ET), abscicic acid (ABA), and auxins were considerred.Venn diagrams were made using (TIF)Click here for additional data file.S1 Dataset(XLSX)Click here for additional data file.S2 Dataset(XLSX)Click here for additional data file.S3 Dataset(XLSX)Click here for additional data file.S4 Dataset(XLSX)Click here for additional data file.S5 Dataset(XLSX)Click here for additional data file.S6 Dataset(XLSX)Click here for additional data file.S7 Dataset(XLSX)Click here for additional data file.


Enterocytozoon bieneusi is an obligate intracellular fungus, infecting various invertebrate and vertebrate hosts, it is common in humans and causes diarrhea in the immunocompromised. In the present study, 801 fecal specimens were collected from pigs on seven large-scale pig farms in Xinjiang, China. Nested polymerase chain reaction (PCR) amplification of the internal transcribed spacer (ITS) gene showed that the overall E. bieneusi infection rate was 48.6% (389/801). The E. bieneusi infection rates differed significantly among the collection sites (20.0–73.0%) . Post-weaned pigs had the highest infection rate , followed by fattening pigs  and pre-weaned suckling pigs . Adult pigs had the lowest infection rate . The E. bieneusi infection rates also differed significantly among age groups . Fifteen genotypes were identified, including 13 known genotypes  and two novel genotypes (XJP-II and XJP-III). Among them, six genotypes  have been reported in humans. Phylogenetic analysis showed that all the genotypes belonged to Group 1 of E. bieneusi. These findings suggest that pigs may play an important role in transmitting E. bieneusi infections to humans. Enterocytozoon bieneusi is considered the most common microsporidial species to cause opportunistic infections in humans  nucleotide sequence polymorphism is widely used for r typing . Over 47orldwide . Phylogeorldwide .E. bieneusi genotypes from pigs in China, thus implicating pigs as dispersing agents and a potential source of human infections .Eight hundred one fresh fecal specimens were collected from Duroc and Landrace pigs on seven large-scale intensive pig farms in Xinjiang between September 2017 and June 2018. Each sampled farm contained 10000–80000 pigs. All farms were visited on a single occasion. A veterinarian randomly collected the fecal specimens either from the rectum or from the internal portion of a stool sample on the ground avoid possible contamination from the specimen surface touched the ground. All specimens (approximately 5–30 g) were collected using sterile disposal latex gloves; marked with the date, age, and farm; stored in insulated boxes; and transferred to the laboratory. Collected specimens included 169 fecal specimens from pre-weaned suckling pigs (<20 days old), 281 specimens from post-weaned suckling pigs (21–70 days old), 129 specimens from fattening pigs (71–180 days old), and 222 specimens from sows (>181 days old).® Stool DNA Kit  per the manufacturer’s instructions with minor modifications. The extracted DNA was stored at −20°C prior to polymerase chain reaction (PCR) analysis.Genomic DNA was directly extracted from the fecal specimens (approximately 200 mg) using the E.Z.N.A.Enterocytozoon bieneusi was identified via nested PCR amplification and sequencing of the ITS region of the rRNA gene. The primers and thermal cycle parameters used for the two PCR amplifications have been described previously  were sequenced by GENEWIZ , and all products were sequenced in both directions to ensure accurate sequencing results. ClustalX 2.14. The posterior probability values were calculated by running 1,000,000 generations. A 50% majority-rule consensus tree was constructed from the final 75% of the trees generated via BI. Analyses were run three times to ensure convergence and insensitivity to priors.Bayesian inference (BI) and Monte Carlo Markov chain methods were used to construct phylogenetic trees in MrBayes, version 3.2.6https://www.ibm.com) was used for the statistical analyses, including Fisher’s exact test and 95% confidence intervals. Differences with p < 0.05 were considered significant.The Statistical Package for the Social Sciences   , which w389/801) , Inner M389/801) ,c, Jilin389/801) , Liaonin389/801) , Sichuan389/801) , Tibet (389/801) , Yunnan 389/801) , but low389/801) ; Table 2ed cages .E. bieneusi infection rates significantly differed among the collection sites . The highest infection rate was found on a farm from Shaya  . Post-weaned pigs had the highest infection rate , while adult pigs (sows) had the lowest infection rate  , Jilin , Zhejiang , and Henan Provinces  , Jilin , Tibet  and Yunnan Provinces   . The E.  25/222) . Most pr137/253) . The hig137/253) . However 42/200) . The difE. bieneusi genotypes were identified from nucleotide sequences via ITS-PCR. These included 13 known genotypes  and 2 novel genotypes  were classified in Group 1, and showed six of the 15 identified genotypes have been found in humans, indicating that pigs may be reservoirs for zoonotic transmission of human microsporidiosis. These findings extend the knowledge of the E. bieneusi distribution among pigs in China.This study revealed that MK778892–K778899 and MK778901–MK778907.The datasets generated for this study can be found in GenBank under the accession numbers Permission was obtained from animal owners or managers before collecting specimens, and no specific permits were required for the described field studies. All work involving animals was carried out in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals. The Research Ethics Committee of Henan Agricultural University reviewed and approved our study .MQ and L-XZ designed the study. YZ, Y-XJ, J-MX, D-YT, A-YZ, and BJ collected and analyzed the specimens. D-FL and Z-HC analyzed the data. D-FL, MQ, and L-XZ wrote the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Schistosoma mansoni and Paracoccidioides brasiliensis share common geographic areas, determining infectious diseases with high mortality rates worldwide. Histopathological and immunological changes induced by each pathogen are well understood; however, the host responses to S. mansoni and P. brasiliensis coinfection are still unknown. Thus, we investigated liver damage and cytokines production in a murine model acutely and chronically coinfected with these pathogens. Fourty male Swiss mice were infected with S. mansoni and P. brasiliensis alone or coinfected. The animals were euthanized with 50 (acute infection) and 120 (chronic infection) days of infection. All infected animals exhibited liver inflammation. Intense granulomatous inflammation was detected in animals infected with S. mansoni alone and those coinfected. Productive and involutive granulomas were clearly observed in acute and chronic infections, respectively. Granuloma size was reduced in the acute phase and increased in the chronic phase of S. mansoni and P. brasiliensis coinfection, compared with animals infected only with S. mansoni. In the chronic phase of infection, the granulomatous inflammation in coinfected animals was characterized by intense neutrophils accumulation and reduced eosinophils number. IFN-γ, IL-2, IL-4, and IL-5 circulating levels were increased in all infected groups. Coinfected animals presented attenuated IFN-γ and IL-4 production in the acute and chronic infections. Taken together, our findings indicate that coinfected animals exhibited a differential modulation of granulomatous inflammation during the acute and chronic phases of infection, which was potentially associated with a divergent profile of cytokines production and migration of neutrophils and eosinophils in response to S. mansoni and P. brasiliensis antigenic stimulation.The pathogens Th2 vs. Th17) and unbalanced immunological phenotypes simultaneously are required to combat two or more parasite species can compromise the infection resolution [ Schistosoma mansoni that causes schistosomiasis in Latin America [ Paracoccidioides brasiliensis, the etiological agent of paracoccidioidomycosis [The development of infectious diseases is deeply influenced by the interaction between pathogen phenotype  and host conditions, such as immunological health and presence of comorbidities, including coinfections –3. Althosolution . In genesolution , such Sc America  and the omycosis . P. brasiliensis infects about 10 million people in Latin America [ Schistosoma mansoni and P. brasiliensis share common endemic areas [Schistosomiasis is a neglected parasitic disease responsible for more than 230 million people infected in more than 74 countries , 12. In  America , 14. Aft America , 15, 16. America , 17. Altic areas , coinfec Schistosoma oviposition, the formation of schistosomiasis granulomas is mainly mediated by the recruitment of monocytes and neutrophils, which are modulated by Th1 cytokines such as IL-12, INF-γ, and TNF-α [Granulomatous inflammation is the main pathological event associated with severe organ damage in Schistosoma-infected hosts , 19. Grand TNF-α . Howevernd TNF-α , 22. Schistosoma infections [α, and specially IFN-γ) are essential to increase host resistance against P. brasiliensis [ S. mansoni and P. brasiliensis change the host resistance against both pathogens. Thus, in the course of Schistosoma-induced granulomatous inflammation, the attenuation of host defense may favor the opportunistic infection by P. brasiliensis. Conversely, it is also possible that P. brasiliensis infection change the granulomatous inflammation and organ damage in Schistosoma-infected hosts. Considering these hypothesis, by using an experiment model of S. mansoni and P. brasiliensis coinfection, we investigated if these infections might interact to change cytokines production and organ damage in mice.While Th2 phenotype is protective againstfections , 23, Th1iliensis . As comp S. mansoni, Pb: infected with P. brasiliensis, and Sm+Pb: infected with S. mansoni and P. brasiliensis. The animals were maintained in a controlled environment with 60-70% humidity, temperature at 21±2°C, and 12/12h light/dark cycles. Rodent chow and water were provided ad libitum. All experimental protocols were approved by the institutional Ethics Committee for Animals Use in Research (protocol 543/2013).Forty male Swiss mice were equally randomized in four groups with ten animals in each group: control (CG): uninfected, Sm: infected with S. mansoni and the fungus P. brasiliensis. To induce schistosomiasis, mice were subcutaneously infected with 25 cercariae of the LE strain. This strain was obtained from human patients and maintained in multiples passages in Biomphalaria snail and Swiss mice [ P. brasilienses (virulent Pb18 strain) [6 yeast cells/mL, based on the hemocytometer count. Cell viability was determined by staining of Janus Green B vital dye, and only visualization greater than or equal to 80% of viable cells was used [ S. mansoni and P. brasiliensis.The animals attributed to infected groups were inoculated with the infective forms of the helminthiss mice . The fun strain)  at 35°C was used . The ani S. mansoni infection was confirmed through histopathological observation of parasite eggs and schistosomiasis granulomas in liver tissue sections stained with hematoxylin and eosin [ P. brasiliensis infection was also confirmed through microscopic observation of mesenteric fungi deposits, which were revealed by the Grocott-Gomori methenamine silver method for detecting fungi [The confirmation of infection was based on the direct microscopic visualization of the etiological agent in the target organs.nd eosin . P. brasng fungi .μm thick in a rotary microtome and the histological sections were stained with hematoxylin and eosin [Liver samples (median lobe) were collected and fixed in fresh 4% paraformaldehyde solution in sodium phosphate buffer . Organ fragments were dehydrated in ethanol, clarified in xylene, and embedded in paraffin. The paraffin blocks were cut in semi-series at 5 nd eosin . In the  S. mansoni and P. brasiliensis on cytokines production was investigated from an Enzyme-Linked Immunosorbent Assay (ELISA) sandwich method [g, 15 min) in the absence of anticoagulant and used to quantify circulating cytokines levels. For the assay procedure, commercial kits were used to detect the cytokines interferon gamma (IFN-γ), interleukin-2 (IL-2), interleukin-4 (IL-4), and interleukin-5 (IL-5) according to manufacturer instructions . Briefly, 96-wells polystyrene plates were incubated with capture antibodies, serum of control, and infected animals. The reaction was developed with a streptavidin-peroxidase conjugated detection antibody  followed by incubation with the chromogen 3,3′,5,5′ tetramethylbenzidine . The optical densities (OD) of the samples were detected at 450 nm , and cytokine concentrations were calculated by extrapolating the OD obtained from a standard curve for each recombinant cytokine .The impact of the isolated or combined infection withh method . Blood s post-hoc test for multiple comparisons. Nonparametric data were compared using the Kruskal-Wallis test. The results with P value <0.05 were considered statistically significant.The results were expressed as mean and standard deviation (mean ± SD) or median and interquartile range. Normality in the data distribution was assessed using the Kolmogorov-Smirnov test. The data variance was measured by one-way ANOVA. Parametric data were submitted to the Student-Newman-Keuls S. mansoni and P. brasiliensis infections were confirmed. In S. mansoni-infected mice, parasite eggs and hepatic granulomas were clearly observed in bright field microscopy. Similarly, in P. brasiliensis-infected animals, yeast-like forms were observed in the mesentery, marked in a dark-brown color by the Grocott-Gomori methenamine silver method. The fungi were clearly defined in the mesentery after hematoxylin counterstaining . Animals infected only withs sheath . S. mansoni alone compared with animals coinfected with P. brasiliensis (P<0.05). Conversely, in chronic infection, granulomas area and volume were higher in animals coinfected with S. mansoni and P. brasiliensis compared with mice inoculated with S. mansoni alone (P<0.05)  . S. mansoni and those coinfected with P. brasiliensis (P>0.05)  . P. brasiliensis presented increased number of neutrophils in liver tissue compared with S. mansoni-infected mice (P<0.05), which was similar to coinfected animals (P>0.05). Eosinophils infiltrate was similar in animals infected only with S. mansoni and those coinfected (P>0.05). These cells were not identified in liver tissue from control animals and those infected only with P. brasiliensis. In the chronic infection, animals infected with only S. mansoni and only P. brasiliensis exhibited similar neutrophils infiltrate, which was markedly increased in coinfected animals (P<0.05). Eosinophils distribution was reduced in coinfected animals compared with mice only infected with S. mansoni (P<0.05)  . P. brasiliensis presented higher IFN-γ levels compared with mice infected with only either S. mansoni or P. brasiliensis (P<0.05), which exhibited similar results (P>0.05). IL-2 and IL-5 serum levels were similar in all infected groups (P>0.05). IL-4 levels were higher in animals infected only with S. mansoni compared with mice infected only with P. brasiliensis or coinfected (P<0.05), which were similar between them (P>0.05) . Animals infected only with(P>0.05) . S. mansoni and only P. brasiliensis presented similar IFN-γ and IL-2 levels (P>0.05). Coinfected mice had reduced IFN-γ levels compared with S. mansoni-infected animals (P<0.05). IL-2 levels were reduced in coinfected animals compared with mice infected with P. brasiliensis alone (P<0.05). IL-4 levels were similarly reduced in animals infected only with P. brasiliensis and coinfected compared with mice infected only with S. mansoni (P<0.05) . Animals infected with only(P<0.05) . IL-4 se(P<0.05) . S. mansoni and P. brasiliensis coinfection. Thus, we clearly observed the coexistence of S. mansoni eggs in liver and yeast-like forms of P. brasilienses in the mesentery of inoculated animals.Studies on the interaction between pathogens in neglected infectious diseases are still scarce –33. Howe S. mansoni-infected mice represent the most striking histopathological feature in the course of infection. In addition, granuloma organization can be classified in different evolutionary stages such as pregranulomatous , granulomatous (exudative-productive or productive), or involutive (with dissociation of collagen fibers) [ S. mansoni eggs were the main characteristic of productive granulomas. In the chronic infection, modulation/involutive granulomas were typically characterized by a granulomatous sheath with reduced cellularity, marked deposition of eosinophilic collagenous matrix, and dissociated collagen fibers.According to Lenzi et al.  and to H fibers) . From a  fibers)  and Hams fibers) . In the  S. mansoni-induced granulomas in acute infections, Amaral et al. [ S. mansoni infection in mice, indicating an exacerbated inflammatory response induced by antigens of this trematode. Apparently, in our study this response pattern was not impaired when the animals were coinfected with P. brasiliensis. However, different models of coinfection between S. mansoni and HIV, Plasmodium sp., and Mycobacterium tuberculosis indicated a more intense leucocytes recruitment and inflammatory liver damage in the acute stage of granuloma formation [ S. mansoni and those coinfected indicated modulation/involutive granulomas exhibiting concentric an epithelioid organization of macrophages around S. mansoni eggs. Considering the poor clinical outcome of S. mansoni-infected hosts in combating coinfecting pathogens that induces divergent immunological patterns  [ P. brasiliensis could modify the cellular pattern of the granulomatous reaction. Such modifications were observed by Farah et al. [ S. mansoni and Papio cynocephalus anubis in BALB/c mice. In the coinfection course, these authors reported hepatic fibrosis associated with the development of multinucleated giant cells (MGCs), divergent phenotypic profile of inflammatory cells, intense collagenogenesis, and periportal fibrosis. In our study, the coinfection of schistosomiasis animals with P. brasilienses did not exhibit the same manifestations expected for P. cynocephalus anubis coinfection. According to Almeida et al. [ P. brasiliensis may infect and develop in multiple organs, the disease often presents a self-limited course or remain asymptomatic for years. In this context, our experimental model was consistent with the human infection, in which infected hosts can exhibit continuous antigenic stimulation in a long-term infectious processes, developing low-grade immunological responses previously to the occurrence of clinical manifestation of paracoccidioidomycosis [Considering the dynamism of leucocytes migration and cellular organization inl et al.  describeormation , 10, 38.e., Th1) –41, we eh et al. , who inva et al. , althougomycosis . S. mansoni alone is coherent with the formation of large granulomas. However, in coinfected animals the reduction in granuloma size could reflect an adaptation of the immunological response to simultaneously combat different pathogens. Considering that the target organs of both parasites investigated are different, it is not unrealistic to assume that in coinfection the simultaneous recruitment of leucocytes to different sites can attenuate the immune response directed to a single pathogen [In our investigation, the area and volume of schistosomiasis-elicited granulomas in the acute phase of infection were proportionally larger compared with coinfected animals, suggesting a potential association of this fungus with an attenuated granulomatous inflammation. According to Lenzi , the intpathogen . This prpathogen . S. mansoni alone presented a more pronounced granuloma involution, a response similar to those observed in a previous study of S. mansoni infection [ S. mansoni alone. This finding seems to be potentially aligned with our proposition of a latency period, which would be required for the immune system to recognize and adapt in response to multiples antigenic stimuli, establishing a more pronounced inflammatory response. This proposition is reinforced considering the morphological, molecular, and functional changes in the infective yeast forms of P. brasiliensis after host infection [ P. brasiliensis creates a latency period, in which the immune system activates variable defense mechanisms to improve antimicrobial responses [ S. mansoni female, and the level of egg retention in host tissues [Compared to the acute stage, in the chronic infection the granulomatous response behaved in an opposite way. Thus, animals infected withnfection . Howevernfection , 46. Thenfection , 46. In esponses , 46. Thu tissues , 36. P. brasiliensis as compared to S. mansoni Infection. Conversely, the distribution of these cells in schistosomiasis granulomas was markedly reduced in the chronic infection. The high quantity of these cells in the acute infection was already expected, since the onset of the protective immunological response is notably mediated by neutrophils. However, in chronic infections, the granuloma presented a differential stage of evolution, and these cells become less relevant to combat this pathogen [ P. brasiliensis, in the chronic phase of infection, we observed intense neutrophilia in coinfected animals, a process potentially related to the control of fungi dissemination in hepatic tissue.The size and distribution of schistosomiasis granulomas have been intimately linked to the profile of leucocytes migration . Accordipathogen . Althoug S. mansoni eggs also presented high eosinophilia in the acute and chronic infection. In acute infections, eosinophils constitute approximately 90% of the total cells in granuloma sheath [ P. brasiliensis coinfection contributed to the drastic reduction of these cells in detriment of the neutrophils predominance in granuloma sheath. According to Chuah et al. [In our study, the granulomas induced bya sheath . Accordia sheath , the inca sheath , 51. Inth et al. , in the h et al. .γ, TNF-α, IL1, IL2, and IL-5 stimulated by antigens of schistosomules and adult worms present in liver and mesenteric veins of the infected hosts [γ levels in animals infected with P. brasiliensis compared with S. mansoni alone. Calich et al. [ P. brasiliensis is in fact linked to the predominance of IFN-γ production by Th1 lymphocytes, while the susceptibility is mainly associated with an impaired cellular immune responses and activation of B cells [According to Chuah et al. , the higed hosts , 47, 52.h et al.  showed t B cells .γ levels. Classically, the granuloma evolution is related to a Th2 cytokine profile  and lower IFN-γ expression [γ levels will reflect the cellular reorganization of the schistosomiasis granulomatous as a reflex of the beginning of an involutive granulomatous phase. On the other hand, when coinfected animals are analyzed in both acute and chronic phases, similar IFN-γ levels were observed, indicating the predominance of an immunological pattern associated with the resistance against P. brasiliensis infection [ S. mansoni and other pathogens such as T. cruzi, Fasciola hepatica, and Heligmosomoides polygyrus [ S. mansoni infection increased the host susceptibility to coinfecting pathogens.In the chronic phase, both groups infected with a single pathogen maintained high IFN-pression . It is pnfection . This reolygyrus , 55, whiγ, we observed similar IL-2 and IL-5 levels in the acute and chronic phases in both infected groups. As important proinflammatory cytokines involved in leucocytes recruitment directed to the initial granuloma formation (Stadecker 1999), such high values in the acute phase of S. mansoni infection are expected. Although reduced IL-2 levels are reported in chronic infections in response to Th2 polarization [ S. mansoni infection. In addition, in animals inoculated with P. brasiliensis alone and those coinfected, sustained production of Th1 cytokines  in both phases of infection can also indicate a protective response associated with the resistance of infected hosts against disease progression [Unlike IFN-rization , antigengression , 56. P. brasiliensis alone and those coinfected exhibited higher IL-4 levels than mice infected with S. mansoni alone. This interesting find corroborates the evidence that Th2 cytokines are also produced in infections by pathogens that induces strong Th1 responses, such as P. brasiliensis [ T. cruzi [ S. mansoni infection, being used a complementary marker of disease chronification [ P. brasiliensis predominantly induces a Th1 immunological polarization [ S. mansoni alone. IL-4 also exerts a direct impact on the evolutionary behavior of the granulomatous infection [High IL-4 serum levels in the acute and chronic phases in both infected groups were also observed. However, animals infected withiliensis  and T. cT. cruzi . Since uT. cruzi , 47, an T. cruzi . As obserization , it was γ, IL-2, IL-4, and IL-5, as well as distinct recruitment of neutrophils and eosinophils in response to S. mansoni and P. brasiliensis antigenic stimulation. As we have not identified any studies investigating coinfections by these pathogens, further studies are required and urgent. To investigate new models of coinfection from different animal species and lineages as well as S. mansoni and P. brasiliensis strains with distinct virulence and pathogenicity phenotypes may improve the understanding of the immune response profiles associated with the host resistance or susceptibility to coinfections by these pathogens.Taken together, our findings indicate that coinfected animals exhibited a differential modulation of granulomatous inflammation in the acute and chronic phases of infection, which was potentially associated with a divergent profile of cytokines such as IFN-


Background. Invasive bacterial diseases cause significant disease and death in sub-Saharan Africa. Several are vaccine preventable, although the impact of new vaccines and vaccine policies on disease patterns in these communities is poorly understood owing to limited surveillance data.Methods. We conducted a hospital-based surveillance of invasive bacterial diseases in The Gambia where blood and cerebrospinal fluid (CSF) samples of hospitalized participants were processed. Three surveillance periods were defined in relation to the introduction of pneumococcal conjugate vaccines (PCVs), before (2005- 2009), during (2010–2011) and after (2012–2015) PCV introduction. We determined the prevalences of commonly isolated bacteria and compared them between the different surveillance periods.Results. A total of 14 715 blood and 1103 CSF samples were collected over 11 years; overall, 1045 clinically significant organisms were isolated from 957 patients (972 organisms [6.6%] from blood and 73 [6.6%] from CSF). The most common blood culture isolates were Streptococcus pneumoniae (24.9%), Staphylococcus aureus (22.0%), Escherichia coli (10.9%), and nontyphoidal Salmonella (10.0%). Between the pre-PCV and post-PCV eras, the prevalence of S. pneumoniae bacteremia dropped across all age groups  while S. aureus increased in prevalence, becoming the most prevalent bacteria . Overall, S. pneumoniae (53.4%), Neisseria meningitidis (13.7%), and Haemophilus influenzae (12.3%) were the predominant isolates from CSF. Antimicrobial resistance to common antibiotics was low.Conclusions. Our findings demonstrate that surveillance data on the predominant pathogens associated with invasive disease is necessary to inform vaccine priorities and appropriate management of patients. Staphylococcus aureus has emerged as the leading cause of bacteremia.The etiology of invasive bacterial disease has changed since the introduction of routine pneumococcal vaccines.  Haemophilus influenzae type b (Hib) conjugate vaccine and pneumococcal conjugate vaccines (PCVs) ), S. aureus (22% [214 of 972]), E. coli (11% [106 of 972]), and NTS (10% [97 of 972])  than during the post-PCV period, peaking in 2008 . Of the of 972]) . We alsot season . Howeverevalence . No distS. pneumoniae bacteremia  and increased odds of S. aureus bacteremia  among all age groups in the post-PCV surveillance period, we observed a 9-fold increase in the odds of bacteremia due to N. meningitidis. We observed no change in the E. coli but did note a decrease in NTS. When results were stratified by age, the most significant reduction in S. pneumoniae bacteremia was noted among children aged 2–23 months and adults ≥15 years. Likewise, the increases in S. aureus bacteremia was among children aged 2–23 months. No differences were observed between study periods for other common bacteria. The prevalence of the predominant pathogens by year is summarized in S. aureus bacteremia over the study periods along with a decline in S. pneumoniae.Overall and age-specific prevalences of all blood culture isolates by vaccine surveillance period re summarized in S. pneumoniae (53% [39 of 73]), N. meningitidis (14% [10 of 73]), and H. influenzae (12% [9 of 73]) . However, the prevalence of S. pneumoniae meningitis gradually declined from 2008, before the introduction of PCV. N. meningitidis increased in the post-PCV compared with the pre-PCV period , becoming the most prevalent bacteria in this latter period.Eighty-three (7.5%) of CSF samples cultured were positive, the majority (87.9% [73 of 83]) of which were considered clinically significant, representing 6.6% of the overall samples. Overall, the most common clinically significant isolates were  of 73]) . We obseeumoniae . S. pneu. pneumoniae, 6 N. meningitidis, 5 H. influenzae, and 4 S. aureus). Twenty-three patients had coinfections with >1 pathogen; S. aureus was the most common (n = 12), coinfected with group A or group B streptococci (each n = 3), S. pneumoniae (n = 3), and NTS, H. influenzae, and N. meningitides (each n = 1).The same pathogen was isolated from concomitant blood and CSF samples of 36 patients  were found. In addition, resistance to gentamicin and cephalosporins was low for NTS . Recent data from The Gambia have shown persistence of nasopharyngeal carriage of pneumococci of vaccine serotypes along with an increased carriage of nonvaccine serotypes [Interestingly, the decrease in erotypes . BecauseS. aureus was prevalent over the surveillance period but emerged as the primary cause of bacteremia in the post-PCV era being isolated in 30% of proven bacteremia cases among infants 2–23 months old. Although our study design precludes documenting an increase incidence of S. aureus bacteremia, there is a global increase in incidence with a changing epidemiology attributed to a range of factors such as vaccines, virulence, invasive procedures, antibiotic resistance, and immune suppression [S. aureus bacteremia cases in low- and middle-income countries warrants further investigation, because these may be distinct from the prevalent causes in high-income countries, such as intravenous drug use and nosocomial infections related to intravenous devices and catheters. Other factors, such as skin and soft-tissue infections, bone and joint infections, and endocarditis [S. aureus bacteremia.pression , 33, undcarditis , are impE. coli and NTS were also important causes of bacteremia in our setting. Although the prevalence of E. coli has remained stable, that of NTS significantly declined after PCV introduction, with the overall number of cases relatively small (n = 97). This is consistent with previous reports from rural Gambia [N. meningitidis were observed over the entire surveillance period, and these coincided with an outbreak in the Eastern region. More than 2 decades after the Hib vaccine was introduced, with near-elimination of invasive disease in The Gambia and neighboring Senegal [l Gambia , in whicl Gambia . In addil Gambia , 36. Onl Senegal , 37, spoS. aureus isolates compared with some SSA countries [E. coli. However, overall ciprofloxacin resistance for E. coli may warrant monitoring resistance quinolones for probable emergence of multidrug resistance [Antimicrobial resistance was generally low, including very few methicillin-resistant ountries , 38. Antountries , and ressistance . Althougsistance , becauseThe current study had several limitations intrinsic to the surveillance design. Because our data are hospital based, the observed changes in pathogen prevalence were dependent on sampling and may not reflect changes in disease incidence. It is a retrospective analysis, and any changes in case ascertainment, amount of sample volume collected, or health-seeking behavior in the population may have modified the overall yield of bacteria. Although the case ascertainment for patients seen at the MRCG CSD did not change over the surveillance period, there were several research studies during the period, each with different age inclusion criteria. Although this might not have changed the distribution of pathogens, it probably increased the number of isolates in some years compared with others. The linking of clinical data associated with microbiological findings is an another limitation. In addition, we could not determine what proportion of patients had received the PCV. Finally, we determined antimicrobial sensitivity using disk diffusion without final confirmation by Etest, which may have resulted in an overestimation of the prevalence of antibiotic resistance levels. Still, results show a low prevalence of antibiotic resistance and no trends for an increase.S. aureus and N. meningitis have emerged as the leading causes of bacteremia and meningitis, respectively, in urban Gambia, while S. pneumoniae remains a leading cause of IBD, even after the introduction of PCV13. The changing epidemiology of IBD makes a compelling case for regular microbiological and antimicrobial surveillance data, which is lacking in SSA. Not only are such data necessary for healthcare workers to inform appropriate antibiotic prescribing practices, but they are also vital for prioritizing vaccine development, for emerging pathogens such as S. aureus and optimizing schedules of current vaccines. New vaccines targeting S. aureus should focus on young infants and older children, among whom the prevalence of IBD is highest.Our long surveillance data have shown that Clinical Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at ciz463_suppl_Supplemental-Figure-1Click here for additional data file.


Enterococcus (VRE) are candidates for gauging the degree of AMR bacteria in wastewater. Enterococcus faecalis and Enterococcus faecium are recognized indicators of fecal contamination in water. Comparative genomics of enterococci isolated from conventional activated sludge (CAS) and biological aerated filter (BAF) WWTPs was conducted.Wastewater treatment plants (WWTPs) are considered hotspots for the environmental dissemination of antimicrobial resistance (AMR) determinants. Vancomycin-Resistant E. faecalis (n = 24), E. faecium (n = 11), E. casseliflavus (n = 2) and E. gallinarum (n = 2) were selected for sequencing based on WWTP source, species and AMR phenotype. The pangenomes of E. faecium and E. faecalis were both open. The genomic fraction related to the mobilome was positively correlated with genome size in E. faecium (p < 0.001) and E. faecalis (p < 0.001) and with the number of AMR genes in E. faecium (p = 0.005). Genes conferring vancomycin resistance, including vanA and vanM (E. faecium), vanG , and vanC , were detected in 20 genomes. The most prominent functional AMR genes were efflux pumps and transporters. A minimum of 16, 6, 5 and 3 virulence genes were detected in E. faecium, E. faecalis, E. casseliflavus and E. gallinarum, respectively. Virulence genes were more common in E. faecalis and E. faecium, than E. casseliflavus and E. gallinarum. A number of mobile genetic elements were shared among species. Functional CRISPR/Cas arrays were detected in 13 E. faecalis genomes, with all but one also containing a prophage. The lack of a functional CRISPR/Cas arrays was associated with multi-drug resistance in E. faecium. Phylogenetic analysis demonstrated differential clustering of isolates based on original source but not WWTP. Genes related to phage and CRISPR/Cas arrays could potentially serve as environmental biomarkers.VRE isolates, including E. faecalis and E. faecium have smaller genomes and harbor more virulence, AMR, and mobile genetic elements than other Enterococcus spp.There was no discernible difference between enterococcal genomes from the CAS and BAF WWTPs.  Enterococcus faecalis and Enterococcus faecium, are associated with a variety of clinical infections of the urinary tract, heart, surgical wounds, bloodstream and neonates [Enterococcus spp. is hindered by the development and spread of antimicrobial resistance (AMR) [Enterococci are ubiquitous in nature and can be found in a variety of environments, including soil, plants, surface water, wastewater, food, and the gastrointestinal tract of animals and humans , 60. Entneonates  as well neonates . The abice (AMR) . Resistace (AMR) , 32.Enterococci and antimicrobials are excreted in urine and feces, and in urbanized developed nations, most of this waste is transported to and treated in wastewater treatment plants (WWTPs) prior to discharge into surface waters. WWTPs could be considered points of control for the environmental dissemination of AMR and ideal environments to investigate the epidemiology of AMR from a “One Health” perspective , 44, 57.E. faecalis being the dominant species identified [E. faecalis and E. faecium than other Enterococcus spp. and that the larger genomes in these clinically relevant species would correlate with the number of mobile genetic elements and genes conferring fitness for survival in a broader range of environments.We previously isolated a number of species of enterococci from two WWTPs with different treatment processes, a conventional activated sludge (CAS) and a biological aerated filter (BAF) system, with entified . This woentified . We hypoEnterococcus spp. genomes can be found in Table E. casseliflavus and E. gallinarum  were larger than those of E. faecalis  and E. faecium . The GC content of the genomes ranged from 37.3–37.7%, 37.5–38.1%, and 40.4–42.9% for E. faecalis, E. faecium, and E. casseliflavus/E. gallinarum, respectively.A summary of sequencing statistics for the 39 E. faecium (21–195 contigs) than in other species (11–68 contigs), likely due to the presence of repetitive and insertion genetic elements complicating assembly [E. faecium (3.04 Mbp) than for susceptible strains (2.60 Mbp). The genome size of vancomycin-resistant and multi-drug resistant E. faecalis was similar to their susceptible counterparts.The range in contigs generated during sequencing was greater in assembly . Genome E. faecalis pangenome consisted of 5708 genes with a core of 2054 genes (36%), a soft-core of 91 genes (1.6%), a shell genome of 1193 (20.9%) and a cloud genome of 2370 genes , a shell of 1186 genes (30%) and a cloud genome of 805 genes . Out of the E. faecalis STs identified in this study, ST16 (n = 7) and ST40 (n = 4) were the most common.In the current study, 4 sequence types (STs) for R, TECR, AMPR, ERYR were among the most common resistant phenotypes found in enterococci.Sequenced enterococci exhibited a number of phenotypic antimicrobial resistant profiles, with some isolates being resistant to as many as seven antimicrobials Table . VANR, TE. faecalis isolates were more closely related to livestock and food-derived E. faecalis genomes, while seven wastewater strains  clustered with strains isolated from human infections  isolates clustered together by source, suggesting that agricultural and human clinical strains are phylogenetically distinct. Vancomycin-resistant E. faecalis isolates also did not cluster as they belonged to different STs, unlike vancomycin-resistant E. faecium, which did cluster as all isolates belonged to CC-17  were more closely related to E. faecium F1213D (bovine) and E. faecium NRRL B-2354 (food) than to clinical isolates.Genomes did not cluster based on WWTP, but all species formed separate monophylogenetic groups Fig. . The majEnterococcus spp., but little difference among strains within species, with the exception of the mobilome and genes associated with energy production and conversion  are broad functional categories used to assign proteins to their specific function . FunctioE. casseliflavus and E. gallinarum compared to enterococci more frequently associated with clinical infections.Given the variation in genome size between and within species, the relationships between genome size and the number of genes associated with specific functional categories was determined Fig. , Sheet 6Enterococcus genomes were pooled, there was a strong negative correlation (p < 0.001) between genome size and nucleotide transport and metabolism, lipid metabolism and translation, ribosomal structure and biogenesis and a strong positive correlation (p < 0.001) between genome size and cell motility  with genome size. This is reflective of the greater genome size of environmental species compared to E. faecium and E. faecalis. The total number of genes related to cell division and chromosome partitioning, cell envelope biogenesis, outer membrane and post translational modification, protein turnover, and transcription were negatively correlated (p < 0.001) with genome size.When all of the wastewater  p < 0.00 between E. faecalis and E. faecium, a larger genome was strongly correlated with the mobilome (p < 0.001)  between the number of unique AMR genes and genome size of E. faecium, suggesting the acquisition of AMR genes occurs through horizontal gene transfer. For example, E. faecium R337 had a genome of 3.02 kbp, 58 genes associated with the mobilome and 23 AMR genes; while E. faecium C329 had a genome of 2.48kbp and 15 genes associated with the mobilome and 3 AMR genes.The species-specific patterns in genomic proportions for each functional category differ from the pooled genomes for the genus. In both 01) Fig.  b and c,p < 0.001), DNA replication, recombination, and repair (p < 0.001), extracellular structures (p < 0.001), and mobilome (p < 0.001) was positively correlated with genome size in E. faecium. The number of AMR genes also showed a positive correlation (p = 0.002) with the amount of genes related to the mobilome in this species , while E. faecalis genomes were more diverse.The total number of genes related to cell motility  associated with multi-drug efflux pumps and other transporters were detected. These efflux proteins may confer intermediate resistance to a variety of antimicrobials.In this study, we screened 39 multi-antimicrobial resistant enterococci genomes against the CARD database for antimicrobial resistance genes (ARGs)  resistance were detected in 20 of the genomes. In ErmB confers resistance to macrolide-lincosamide-streptogramin B (MLSB) antimicrobials and was found in E. faecium (n = 7) and E. faecalis (n = 4). Other erm genes  were detected in one E. faecium genome. msrC, which codes for a macrolide efflux pump, was only detected in E. faecium (n = 11). The most common macrolide resistance gene detected in enterococcal genomes was ermB (n = 15).ant(9′)-Ia, aad(6′), aph(3′)-IIIa, SAT-4, ant(6′)-Ia, and aac(6′)-Ie-aph(2″)-Ia) were detected in the genomes of up to 5 E. faecalis and 7 E. faecium aminoglycoside resistant isolates. Gentamicin resistance arises as the result of the acquisition of aac(6′)-Ie-aph(2″)-Ia, which was detected in 7 genomes  and confers resistance to all aminoglycosides except streptomycin [E. faecium and gentamicin resistance more common in E. faecalis.Thirteen of the enterococci isolates were resistant to high concentrations of gentamicin and streptomycin. In our study, cross-resistance to levofloxacin and the aminoglycosides (gentamicin and streptomycin) occurred in 5 isolates with 3 additional isolates exhibiting intermediate resistance to one or more of these antimicrobials. In our study, additional aminoglycoside genes  were also detected in E. faecium and E. faecalis. Two E. faecalis genomes also had genes associated with lincosamide resistance (InuB and InuG).Genes encoding tetracycline resistance were detected in 26 of the genomes, including E. faecium, E. casseliflavus, E. faecalis, and E. gallinarum, respectively , cell wall biosynthesis , cellular defense (lisR), biofilm formation and surface proteins . Other functions including bile salt degradation (bsh), proteases (tip/ropA), biofilm formation (bopD), enolase (eno), and antiphagocytosis and capsule formation (rfbA-1) were also identified. All of the E. faecalis genomes contained genes for cell adhesion (tuf), carbohydrate metabolism (hyl), endocarditic and biofilm association (ebp) pili (ebpA), Type III secretion proteins (bopD) and fibrinogen-binding proteins (fss1). All of the E. casseliflavus genomes contained the same five virulence genes with functions of: capsule biosynthesis (capE), enolase (eno), leucine aminopeptidase (lap), heat shock protein (hsp60), and protein modification (lplA1). All of the E. gallinarum genomes had an enolase (eno), a flagellar biosynthesis protein (flhA) and a bile salt hydrolase (bsh). One of the E. gallinarum genomes also contained genes related to capsule proteins and another isolated from effluent possessed 2 genes associated with metal transporter (ssaB and psaA) as well as those associated with the CAS system. Hyaluronidase (hyl) genes were detected in all the E. faecalis genomes.The number of shared virulence genes among genomes of the same species were 16, 6, 5 and 3 for E. faecalis genomes. One transposon, Tn6098 was present in all genomes. A multidrug resistance transposon, Tn5385 was also found in all E. faecalis genomes. Other Tn5801 and Tn6013-like ICE elements of unknown function were also present in all E. faecium isolates, in addition to a cadmium and arsenic resistance ICE, ICESde3396. All of the E. gallinarum and E. casseliflavus isolates had Tn916-type transposons . Seven out of the unique 27 ICE were present in genomes of more than one Enterococcus species.ICE and transposons present in the genomes were identified and described using the ICEberg database  genome.Bacteriocins were identified in 8 tivities . LassopeE. faecalis from wastewater that clustered with agricultural and animal sources revealed a biomarker associated with CRISPR-associated genes that differentiated (score = 0.8043) these isolates from E. faecalis from wastewater that clustered with human sources. A comparison of E. faecium from clinical (inclusion) and wastewater (exclusion) sources yielded 7 biomarkers with scores greater than 0.80. These were associated with phage (n = 6) and hypothetical proteins (n = 1). A search for potential biomarkers that distinguished among species in this study resulted in 98 signatures for E. faecalis, 130 signatures for E. faecium, and 3 signatures E. casseliflavus and E. gallinarum. These signatures include genes related to various types of nucleotide and carbohydrate metabolism, as well as other functions.The small number of genomes limited the identification of biomarkers, particularly for searches within the same species isolated from different sources  exhibited high level gentamicin resistance. The association of these sequence types and gentamicin resistance may differ between studies because of geographical location, as gentamicin resistance is transferable, and because it may not be present in all ST16 and ST40 E. faecalis isolates.In tewater,  suggestirom pigs , 59. HowgroESL locus [E. faecalis than E. faecium. The distinct clustering between clinical and wastewater strains of E. faecium may be due to the large accessory genome and characterization of these genes may provide insight into the mechanisms whereby enterococci adapt to specific environments.The genomes forming monophylogenetic groups support our previous results of speciation of enterococci based on the SL locus , 79. TheE. faecalis and E. faecium was completed to determine if some of these COGs could be increasing the fitness of multi-drug resistant enterococci. This could ultimately increase the risk of infection with these strains and the transfer of virulence and AMR determinants to other bacteria.A disproportionate increase in genes associated with energy conversion, regulatory function, transport and secondary metabolism has been noted with expansion in genome size in previous comparative bacterial genomic studies , 34, 66.E. casseliflavus and E. gallinarum some COGs were over represented . These functional categories could allow for higher fitness in aquatic environments where more diverse substrates are typically available at much lower concentrations than in the digestive tract. The increase in cell motility related genes may also enable these species to undertake chemotaxis in aquatic environments where nutrients may be scarce [E. faecalis and E. faecium, these genomes also contained more genes encoding for secondary metabolites including antimicrobial agents. Although these genes are not required for growth, they can confer competitiveness in diverse environments [E. casseliflavus and E. gallinarum are known to be more environmentally fit than E. faecalis and E. faecium as a result of a variety of mechanisms. For instance, the yellow pigment of E. casseliflavus can protect this species from photo-inactivation in aquatic environments [E. faecium and E. faecalis are still the predominant species in wastewater, likely due to the continuous input of fecal waste into these systems.In e scarce . Compareronments . E. cassronments . HoweverE. faecium and E. faecalis and this would suggest that the mobilome is a significant factor in the evolution of these bacteria within wastewater, contributing to genomic expansion and diversity. However, there was a lack of diversity in E. faecium isolates compared to E. faecalis, suggesting that E. faecium isolates may be more specifically adapted to clinical environments.The number of genes related to the mobilome increased with genome size in vanA, vanB and vanC being the most common in wastewater enterococci. The most common determinant for teicoplanin resistance is vanZ, which can be integrated into the van operon, although it is absent in the vanB operon, and confers resistance to both vancomycin and teicoplanin [Vancomycin-resistant enterococci have been known to exhibit resistance to a number of antimicrobials , 74. Entcoplanin . As a recoplanin , 21, 35.ermB, can also confer resistance to streptogramins [Resistance to erythromycin and other macrolides can arise as a result of mutations in the 23S rRNA gene or by efflux pumps . Macroliogramins .aac(6′)-Ii. Gentamicin and streptomycin are clinically-important as they are not inactivated by aac(6′)-Ii; and E. faecium are typically sensitive to these antimicrobials [Enterococci exhibit intrinsic resistance to low concentrations of aminoglycosides as a result of the presence of crobials . Aside fcrobials . Streptocrobials .Tet(L) encodes an efflux protein and tet(M) and tet(S) encode for ribosomal protection proteins. Disk susceptibility testing revealed that these isolates were resistant to doxycycline, whilst those containing tet(L) were susceptible, suggesting specificity for the tet(L) efflux protein. In general, bacteria that are resistant to doxycycline are also resistant to tetracycline and oxytetracycline [There are multiple tetracycline resistance genes. acycline , 56. Tetacycline , 40.ace, acm, agg, bop, ccf, cob, cpd, ebpABC, ecbA, efaA, esp, fsrABC, gelE, pil, scm, sgrA, sprE, and srt). These genes are ubiquitous as they likely play a role in the fitness of enterococci in both the human digestive tract and WWTPs. A number of capsule protein genes were also common among the genomes and not only confer resistance to phagocytosis in humans and animals [hyl) genes have been associated with increased vancomycin resistance and virulence in mouse peritonitis models [The virulence genes detected have additional functions for improved environmental fitness. For instance, the majority of the virulence genes detected in the genomes from this study were also associated with biofilm formation or adherence to surfaces  was found to be associated with Tn1545 and Tn917 [E. faecalis with these isolates exhibiting erythromycin and doxycycline resistance as this transposon commonly carries these resistance genes [E. casseliflavus and E. gallinarum can carry genes coding for tetracycline, minocycline and erythromycin resistance [E. casseliflavus and E. gallinarum, they did not exhibit erythromycin resistance and no associated AMR genes were detected in their genomes.Mobile genetic elements (MGEs) play an important role in horizontal gene transfer and the spread of AMR among isolates in the environment, humans and animal hosts. MGEs include plasmids, transposable elements, prophages and various genomic islands such as integrative conjugative elements (ICE) . The trace genes . Tn916-tsistance , 55. WhiType II CRISPR-Cas systems are typically described in enterococci. Multiple CRISPR arrays can often be detected in bacterial genomes, but not all arrays are accompanied by Cas genes. The absence of CRISPR/Cas systems may compromise genome defence, increasing the likelihood of acquisition of AMR determinants from bacteriophage and plasmids . When a Enterococcus spp. belonging to Podoviridae, Inoviridae, Leviridae, Guttaviridae and Fuselloviridae have also been described [Phages found in the genomes were members of the Siphoviridae and Myoviridae. Other prophages in escribed , 41. Proescribed .Enterococcus spp [Enterococcus spp. is unclear. Aryl polyene biosynthetic clusters produce a pigment that protects the organism from reactive oxygen species [Bacteriocins are ribosomally synthesized antimicrobial peptides produced by Gram-positive and Gram-negative bacteria that have antimicrobial activity against closely related bacteria. They could provide a competitive advantage to the survival of bacteria in ecological niches that exhibit poor nutrient concentrations, heat and extreme pH . Lantipeccus spp . Terpeneccus spp , but the species .E. casseliflavus and E. gallinarum include genes related to various types of nucleotide and carbohydrate metabolism, and genes with other functions which can improve environmental fitness, including a variety of transporters and housekeeping genes related to DNA replication, transcription and translation.Biomarkers are genes or gene fragments only present in one group of genomes and not others making them possible identifiers of the origin of collected isolates. For instance, Weigand et al.  conducteE. faecalis and E. faecium have smaller genomes and may be less equipped to survive outside of their target niche than E. casseliflavus and E. gallinarum. However, they do harbor more virulence, AMR, and mobile genetic elements than other Enterococcus spp. A larger genome size in E. faecalis and E. faecium was positively correlated with an expansion in the mobilome. In E. faecium, there was a positive correlation between the number of AMR genes and the mobilome. So, while the larger genome size of E. casseliflavus and E. gallinarum is accompanied by more genes related to metabolism and secondary functions, possibly increasing their fitness in the environment, this was not the case for E. faecium and E. faecalis. This study suggests that the key to understanding the impact of WWTPs on AMR dissemination is likely understanding the mobilome and discerning linkages between enterococci in wastewater and other environmental and clinical sources.In this study, enterococci did not cluster phylogenetically based on point of isolation during wastewater treatment or on the type of WWTPs. Despite being the dominant species in wastewater, Enterococcus spp., including E. faecalis (n = 24), E. faecium (n = 11), E. casseliflavus (n = 2) and E. gallinarum (n = 2), isolated from wastewater were selected for whole genome sequencing. These were selected from a collection of 308 isolates from the primary and final effluents of two WWTPs in Kingston, Ontario, Canada, a BAF and a CAS system between 2014 and 2016. Isolates were speciated and subsequently underwent disc susceptibility testing for a panel of 12 antimicrobial agents. Nine to ten Enterococcus isolates were chosen from each of the primary and final effluent of the two WWTPs to represent the most prominent species isolated from the samples and the most prominent unique antimicrobial resistance phenotypic profiles. While all of these isolates grew in Todd-Hewitt broth supplemented with vancomycin (≥ 4 mg/L), not all met the requirements for vancomycin resistance using disc susceptibility testing following CLSI and EUCAST guidelines. This procedure used reference strains E. faecium ATCC 700221 (MIC ≥32 mg/L), E. faecalis ATCC 51299 (MIC ≥4 mg/L) and E. faecalis ATCC 29212 (susceptible) and Staphylococcus aureus ATCC 25923. The final isolates selected included 21 vancomycin-susceptible, multi-drug resistant enterococci and 18 enterococci with either intermediate resistance or resistance to vancomycin based on disc susceptibility testing. The AMR phenotypic profiles of the selected isolates are available in Table Thirty-nine alis n = 4, E. faeEnterococcus spp. were grown on Brain Heart Infusion (BHI) agar  overnight at 37 °C. Colonies from a freshly grown culture plate were suspended in TE buffer to achieve an OD600 of 2 in order to harvest 2 × 109 cells, and 1 mL was transferred to a microcentrifuge tube and centrifuged for 2 min at 14000 x g. Genomic DNA was extracted using a modified DNeasy Blood & Tissue Kit  with the addition of an enzymatic lysis step. Bacterial cells were incubated at 37 °C with shaking (150 rpm) in lysis buffer consisting of 20 mM Tris-Cl (pH 8.0), 2 mM sodium EDTA, 1.2% Triton X-100 and 40 mg/mL lysozyme . Proteinase K and 5 μL of 100 mg/mL RNase A were added , and the mixture was incubated at room temperature for 10 min before proceeding to the next step. The quality of the genomic DNA was determined using a Nanodrop One UV-Vis Spectrophotometer  and a Qubit fluorometer (Thermo Scientific). Genomic library construction was performed using the Illumina Nextera XT DNA sample preparation kit  following the manufacturer’s instructions. The library was sequenced on an Illumina MiSeq platform . FASTA data was filtered for quality and high-quality reads were de novo assembled using SPAdes genome assembler 3.6.0 [er 3.6.0 .Enterococcus faecalis MLST  and Enterococcus faecium MLST (https://pubmlst.org/ efaecium/) based at the University of Oxford [Enterococcus spp. genomes were aligned to the appropriate reference genome to generate read pileups . The presence and absence matrices were generated using Phandango [E. faecalis and E. faecium . Assignment of proteins into clusters of orthologous groups (COGs) was performed using the compare genomes function of DOE Joint Genome Institute Integrated Microbial Genomes & Microbiomes platform [Pangenomic analysis was completed using the contigs extracted from the Genbank file which were re-annotated using Prokka 1.13.3 . This generated GFF files that were used as input to Roary 3.12 . Multi-lf Oxford  and fundf Oxford  using unhandango . Whole gplatform . Correlaplatform ) and figplatform  and corrplatform .Enterococcus spp. were investigated for the presence of putative virulence and AMR genes, mobile gene elements, bacteriophage, and CRISPR/Cas arrays. The contigs of each draft genome were ordered based on alignment against a reference genome using progressive Mauve [Draft genome sequences of the 39 ve Mauve . Virulenve Mauve  and CARDve Mauve , respectve Mauve . The genve Mauve . CRISPR-ve Mauve . Secondave Mauve .E. faecalis V583 (NC_004668), E. faecium DO (NC_017960), and E. casseliflavus B72 (this study).A biomarker search was carried out with the 39 genomes from this study and an additional 59 genomes retrieved from NCBI using Neptune  and a GaAdditional file 1: Supplementary Details on Genomic Comparisons. Sheet 1: Complete List of Genomes Used; Sheet 2: Genome Characteristics of Sequenced Genomes; Sheet 3: COG Analysis; Sheet 4: COG Pivot Chart; Sheet 5: COG graphs; Sheet 6: COG correlation and p values; Sheet 7: AMR phenotypic profiles and breakpoints; Sheet 8: AMR genes detected; Sheet 9: Virulence Factors Raw Output; Sheet 10: Virulence Factors by Species; Sheet 11: Virulence Factors Summary; Sheet 12: Phage Detected; Sheet 13: CRISPRfinder Output; Sheet 14: CRISPRfinder Confirmed Systems; Sheet 15: CRISPRdb BLAST Results; Sheet 16: CRISPR Summary Table; Sheet 17: ICEberg Raw Results; Sheet 18: antiSMASH results; Sheet 19: Neptune Biomarker Combinations


Acer and Quercus would have more herbivorous pests than exotic congeners and different communities of arthropod natural enemies. The density of scale insects, common urban tree pests, was greatest on a native Acer and a native Quercus than exotic congeners in both years of our research (2012 and 2016) and sometimes reached damaging levels. However, differences in predator and parasitoid abundance, diversity, and communities were not consistent between native and exotic species in either genus and were generally similar. For example, in 2012 neither predator nor parasitoid abundance differed among native and exotic Acer congeners but in 2016 a native species, A. saccharum, had the least of both groups. A native, Q. phellos, had significantly more predators and parasitoids in 2012 than its native and exotic congeners but no differences in 2016. Parasitoid communities were significantly different among Acer species and Quercus species due in each case to greater abundance of a single family on one native tree species. These native and exotic tree species could help conserve arthropod natural enemies and achieve pest management goals.Urban trees serve a critical conservation function by supporting arthropod and vertebrate communities but are often subject to arthropod pest infestations. Native trees are thought to support richer arthropod communities than exotic trees but may also be more susceptible to herbivorous pests. Exotic trees may be less susceptible to herbivores but provide less conservation value as a consequence. We tested the hypotheses that native species in  Urban trees serve a critical conservation function by supporting herbivorous arthropod communities that, in turn, support vertebrate and invertebrate communities at higher trophic levels . HoweverCities contain many native tree species that share an evolutionary history with local herbivores and many exotic tree species that do not . The evoThe composition and effects of herbivore communities supported by exotic tree species in cities are difficult to predict. Cities are relatively recent human constructions and have many unnatural characteristics to which few animal species have evolved . For exaQuercus and Acer. These genera contain some of the most common trees in urban landscapes  are among the most common and abundant herbivores on urban trees . Scales ndscapes , and natndscapes . Our firAcer palmatum (Japanese maple), A. platanoides (Norway maple), A. rubrum (red maple), A. saccharum (sugar maple), Quercus acutissima (sawtooth oak), Q. alba (white oak), and Q. phellos (willow oak). Acer palmatum is native to Japan, South Korea, and eastern China. The other exotic maple, A. platanoides, is native to eastern Europe and western Russia from Sweden south to Greece. It is also an invasive species that has become established in parts of the eastern and northwestern US and Canada . In 2012, we located 10 or 15 trees  of each d Canada . Acer rurida, US . Acer saer south . Quercusthern US . Quercusexas, US . Quercusw Jersey . North CIn October 2012, we pruned five 30 cm branches from each tree and used a dissecting microscope to identify and count scale insects on the branches. Many scale insects, especially those in the family Diaspididae, leave behind waxy covers that can persist on trees after the soft-bodied scale insect has died. In 2012, we counted all scales insects, live or dead, on the branches and identified armored scales (Hemiptera: Diaspididae), soft scales (Hemiptera: Coccidae), and pit scales (Hemiptera: Astrolecaniidae) to family. In May 2012, we used 7.6 × 12.7 cm yellow sticky cards  to capture scale insect natural enemies in the canopy of each tree. Sticky cards were placed in the interior of the canopy, above the first lateral branches, to avoid attracting insects from other areas and incidental capture of insects flying in the vicinity of the trees. Arthropods were sampled for 7–13 days in each tree. We identified common predators to family or order and counted parasitoid wasps without identifying them. One sticky card was lost during sampling.A. platanoides, which was no longer present in sufficient numbers on campus, with another exotic, A. buergerianum (trident maple) which is native to eastern Asia. Trees of each species were at least 100 m apart, with the exception of three white oaks  which, out of necessity, were at least 50 m apart. From late February through early May 2016, we collected two terminal branches that were at least three m high from each tree to count scale insect abundance. We used a dissecting microscope to identify and count scale insects on the terminal 30 cm of each branch. We used metal probes to determine which scale insects were alive and included only live scale insects in 2016 data analysis. We counted all live scales and identified live armored scales (Hemiptera: Diaspididae), soft scales (Hemiptera: Coccidae), and pit scales (Hemiptera: Astrolecaniidae) to family. We used 7.6 × 12.7 cm yellow sticky cards  to survey natural enemy abundance. We placed a single card in the canopy of each tree for two weeks in June 2016, and we repeated this in July and August 2016. Four sticky cards were lost. We used a dissecting microscope to identify parasitoid wasps to family and to identify common scale insect predators  to randomly select 10 study trees of each species from a geo-referenced inventory of trees maintained by the North Carolina State University Facilities Division. We included the same tree species as in 2012 except we replaced the exotic species redators . Here, wagricolae .We used the R package vegan  to calcu2 = 15.11, p < 0.002), with the native A. rubrum having the highest abundance and the exotic A. platanoides having the lowest abundance , with highest abundance on the native Q. alba and lowest abundance on the exotic Q. acutissima  of 21.68 (± 83.74) scale insects per 30 cm of branch per tree. Scale insect abundance varied by tree species for maples  and oaks ; both native species of each genus had significantly more scale insects than their exotic congener(s)  of 12.51 (± 43.25) live scale insects per 30 cm of branch per tree. Scale insect abundance was less than in 2012 since we only counted live scales in 2016. Scale abundance varied by tree species for maples (χgener(s) . On mapl2 = 2.08, df = 3, p = 0.555) or oak species  , with lower total predator abundance within the exotic A. palmatum than in the other maple species   . In 2016 species . There w= 0.134) .p = 0.134) or oaks . In 2016, the predator community differed among maple species ; the exotic A. palmatum had a different community than all other Acer species   . The exoual taxa .2 = 4.64, df = 3, p = 0.200) but differed among the oak species , with highest abundance within a native, Q. phellos, and lowest abundance within the exotic, Q. acutissima , and did not differ among oak species   of 52.5 (± 47.2) parasitoid wasps per 7-day sample. The number of parasitoid wasps per 7-day sample was not different among maple species (χutissima . In 2016= 0.431) .F3,36 = 2.168, p = 0.109) or oak species . Parasitoid community compositions differed among maple species , driven primarily by high Signiphoridae abundance within A. rubrum and high Aphelinidae abundance within A. palmatum .The Shannon diversity index did not differ among maple species  carried out by trees, the densities of scale insects observed on A. rubrum, Q. phellos, and other urban trees are potentially detrimental. At such densities, scale insects can reduce photosynthesis  (Corythucha arcuata) and oak treehopper (Platycotis vittata) and many generalists  . Oaks sueralists , 2005. Teralists . Coccophr region . A total phellos . Most Pl phellos . All spe phellos .A. saccharum, had the lowest abundance of predators and parasitoid wasps. The native species A. rubrum, which often has severe infestations of gloomy scales (Melanaspis tenebricosa) in cities (Signiphora spp. (Signiphoridae), Encarsia spp. (Aphelinidae), Ablerus spp. (Aphelinidae), and Coccidoctonus (Encyrtidae) have been reared from gloomy scales , lacewings , ants (Hymenptera: Formicidae), 17 families of spiders, and many others  or crape myrtle (Lagerstroemia spp.) are likely more distinct than comparisons between congeners .Pairwise comparisons of predator communities (for five taxa) on maples in 2016. p values for overall pairwise tests were adjusted using the Benjamini-Hochberg method (BH). Univariate p values were adjusted using the standard step-down resampling procedure in Click here for additional data file.10.7717/peerj.6531/supp-2Supplemental Information 2mvabund. Acronyms identifying exotic tree species are bolded. .Pairwise comparisons of predator communities (for five taxa) on oaks in 2016. p values for overall pairwise tests were adjusted using the Benjamini-Hochberg method (BH). Univariate p values were adjusted using the standard step-down resampling procedure in Click here for additional data file.10.7717/peerj.6531/supp-3Supplemental Information 3mvabund. A dash indicates that parasitoid abundance was not high enough for a particular pairwise comparison. Acronyms identifying exotic tree species are bolded. .Pairwise comparisons of parasitoid communities on maples in 2016. p values for overall tests were adjusted using the Benjamini-Hochberg method (BH). Univariate p values were adjusted using the standard step-down resampling procedure in Click here for additional data file.10.7717/peerj.6531/supp-4Supplemental Information 4mvabund. A dash indicates that parasitoid abundance was not high enough for a particular pairwise comparison. In the global univariate test, Eulophidae did not show a significant difference across tree species  and thus is not marked with an asterisk on Q. acutissima, QUAL: Q. alba, QUPH: Q. phellos).Pairwise comparisons of parasitoid communities on oaks in 2016. p values for overall tests were adjusted using the Benjamini-Hochberg method (BH). Univariate p values were adjusted using the standard step-down resampling procedure in Click here for additional data file.


Multiple sclerosis (MS) is an autoimmune disease of the central nervous system characterized by loss of coordination, weakness, dysfunctions in bladder capacity, bowel movement, and cognitive impairment. Thus, the disease leads to a significant socioeconomic burden. In the pathophysiology of the disease, both genetic and environmental risk factors are involved. Gene x environment interaction is modulated by epigenetic mechanisms. Epigenetics refers to a sophisticated system that regulates gene expression with no changes in the DNA sequence. The most studied epigenetic mechanism is the DNA methylation. In this review, we summarize the data available from the current literature by grouping sets of differentially methylated genes in distinct biological categories: the immune system including innate and adaptive response, the DNA damage, and the central nervous system. Multiple sclerosis (MS) is a potentially disabling central nervous system (CNS) disease characterized by inflammation, demyelination, and axonal degeneration. The pathophysiological mechanisms involved in this autoimmune disease differ between patients. In MS pathogenesis, antigen-presenting cells (APCs) stimulate CD4+ T cells in the periphery, favoring their differentiation into CD4+ T helper (Th) cells. Upon their activation, these immune cells cross the blood–brain barrier (BBB) and migrate into the brain, where they are reactivated by resident APCs. Proinflammatory cytokines and chemokines are released by reactivated CD4+ T cells, while infiltrated cytotoxic CD8+ T cells impair neuronal integrity. In parallel, plasma B cells release antibodies against self myelin epitopes, contributing to demyelination  . AHR activity is negatively regulated by the encoded protein for the aryl hydrocarbon receptor repressor (AHRR). MS patients showed lower expression levels of circulating AHR than their matched controls . In lineIn DCs, high levels of cyclic adenosine monophosphate (cAMP) activate Th17 response through the stimulation of guanine nucleotide-binding protein, alpha stimulating (GNAS). High levels of activated Th17 cells are associated with autoimmunity . In linevia enhanced interleukin 4 synthesis  cells is associated with an upregulation of RAS p21 protein activator 3 (RASA3). In contrast, reduced expression of RASA3 suppresses pTh17 cell generation ynthesis . An elevynthesis  and PBMCynthesis , presumaynthesis . The actynthesis . High mRynthesis  and haveynthesis . A lowerynthesis . The proynthesis . A hypomynthesis . Thus, hignaling . The addignaling  might prtivation . Cytokintivation . In agretivation .2+ homeostasis. ER Ca2+ depletion potentiates the inflammatory response, ER stress and, lastly, DNA damage (2+ influx in T cells. Deficiency of protein junctions containing MORN motifs aggravates the store-operated Ca2+ entry in T cells and causes DNA damage (The endoplasmic reticulum (ER), along with the plasma membrane (PM) junctions, is essential for CaA damage . Upon acA damage . An elevA damage  and mighA damage . On the A damage , as wellA damage . The SecA damage . High leA damage . In MS, A damage  and demyA damage . This miA damage .De novo telomere addition of long interspersed nuclear elements (LINE-1) into genomic DNA causes genetic defects, alters the regulatory mechanisms and the structural properties of the genome at their insertion place and might lead to genomic remodeling  during ongoing remyelination . In NAWMeceptors . An overeceptors . Thus, ieceptors . Similareceptors . When reeceptors . In lineeceptors . Additioeceptors . Cadherieceptors . Recentleceptors  probablyeceptors .Multiple sclerosis is a chronic inflammatory CNS disease originating from a complex interaction between genes and the environment. As far as we know, genetics accounts for almost 30% of MS prevalence worldwide, while the remaining percentage corresponds to epigenetic modifications due to exposure to distinct environmental factors . This reNC and JT-R researched the literature and drafted the manuscript. JT-R critically reviewed and edited the work. Both authors read and approved the final manuscript.The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This review was funded by the Deutsche Forschungsgemeinschaft to Dr. Jordi Tomas Roig (ref. TO 977/1-1) and the University of Girona to Mrs Naiara Celarain Sanz (ref. IFUdG2017).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.