Gambierdiscus and Fukuyoa genera in order to rapidly screen environmental samples for potentially toxic species. Additionally, a specific assay for F. paulensis was developed as this species is of concern in New Zealand coastal waters. Using the assays we analyzed 31 samples from three locations around New Zealand and the Kingdom of Tonga. Fourteen samples in total were positive for Gambierdiscus/Fukuyoa and two samples were also positive using the F. paulensis assay. Samples from the Kermadec Islands were further characterized using high-throughput sequencing metabarcoding. The majority of reads corresponded to Gambierdiscus species with three species identified at all sites . This is the first confirmed identification of G. polynesiensis, a known ciguatoxin producer, in New Zealand waters. Other known toxin-producing genera were also detected, included Alexandrium, Amphidinium, Azadinium, Dinophysis, Ostreopsis, and Prorocentrum.Ciguatera Fish Poisoning (CFP) is increasing across the Pacific and the distribution of the causative dinoflagellates appears to be expanding. Subtle differences in thecal plate morphology are used to distinguish dinoflagellate species, which are difficult to determine using light microscopy. For these reasons we sought to develop a Quantitative PCR assay that would detect all species from both  Gambierdiscus Adachi and Fukuyo and Fukuyoa Gómez, Qiu, Lopes and Lin are marine benthic dinoflagellates that are widely distributed from tropical to warm-temperate environments [Gambierdiscus species and the closely related Fukuyoa species. Subtle differences in thecal plate morphology are used to distinguish species which are difficult to view without scanning electron microscopy. This has hindered accurate determination of risk associated with potentially toxic taxa. For example, the original species description of G. toxicus [G. toxicus cannot be assigned with any confidence [Gambierdiscus cells can only be identified as Gambierdiscus spp. [Gambierdiscus and Fukuyoa species. Currently, Quantitative Polymerase Chain Reaction (QPCR) is the most utilized molecular monitoring method as it is cost-effective and rapid [The genera ronments ,2. Specironments ,4,5, accronments . CFP is ronments . Advance toxicus , the fir toxicus , includenfidence ,9. This cus spp.  and so end rapid ,12, howeGambierdiscus currently comprises 15 described species  technologies combined with DNA barcoding methodologies, termed metabarcoding has increased [The genus  toxicus ; G. belilizeanus ; G. austesiensis ; G. carirpenteri ; G. excecabrosus ; G. silv. silvae ; G. balebalechii ; G. chelheloniae ; G. honu[G. honu ; and G. lapillus ) and sixbotype 2 ; G. sp. . type 2 ; G. sp. . type 3 ; G. sp.  5 and 6 ). The geasumotoi ; F. ruetuetzleri ; and F. aulensis ). Severaaulensis ,12. Howe species ,25,28. Tncreased ,30,31. Hncreased ,33,34.Gambierdiscus species) appears to be expanding, including into more temperate habitats [Gambierdiscus species and strains also influence the occurrence of CFP and the exact toxin profile most species is unknown [Gambierdiscus and Fukuyoa have been reported from New Zealand’s northern coastal waters [Gambierdiscus and Fukuyoa in New Zealand indicates a need for pro-active and accurate monitoring of benthic harmful communities in this region [Gambierdiscus and Fukuyoa species, we designed a QPCR assay that would detect all species from both genera. Additionally, as F. paulensis is known to occur in New Zealand coastal waters, we designed a specific QPCR assay to detect this species. Environmental samples from New Zealand warm-temperate (Northland) and sub-tropical waters (Kermadec Islands) and the Kingdom of Tonga  were analyzed using both assays. Samples were initially screened using the Gambierdiscus/Fukuyoa assay and positive samples were subsequently analysed using the F. paulensis assay. Samples from the Kermadec Islands were further characterized using dinoflagellate-specific HTS metabarcoding analyses [CFP risk is increasing across the Pacific ,35, and habitats ,36,37. Dl waters . The pres region ,36,39,40analyses .Gambierdiscus and Fukuyoa species and the high diversity of species within the genera, including the likelihood of undescribed cryptic taxa, we aimed to develop a single qPCR assay for the rapid detection of all species from both genera. Once environmental samples have been screened using the QPCR assay, the Gambierdiscus and Fukuyoa community can be further characterized using various molecular tools including species-specific QPCR and HTS metabarcoding. Due to the difficulties in identifying F. paulensis as this species is present in New Zealand waters [Gambierdiscus and Fukuyoa) and species (F. paulensis), as determined via cross-reactivity testing with strains listed in Gambierdiscus and Fukuyoa genera so were unable to comprehensively assess the assay against all described species. However, in silico analyses, using available DNA sequence data for missing species showed the assays are likely to amplify only the specific target genera/species , Kermadec Islands, New Zealand Territory (6 samples), and Tonga (18 samples). Samples were initially analyzed using the Gambierdiscus/Fukuyoa QPCR assay  [Alexandrium and Azadinium, presumably present as cysts in the benthos. The symbiotic dinoflagellate, Symbiodinium, was also present. In order to confirm the identification of the Gambierdiscus species, in particular G. polynesiensis as this species has not been detected in New Zealand water previously, we also sequenced amplicons from the SSU V4 region. The consensus sequences from all reads classified as Gambierdiscus species were aligned with reference sequences. The identification of G. polynesiensis, G. honu and G. australes was supported by the phylogenetic analyses   and the CAWD221)  produce CAWD221) . Planktoanalyses . The con Islands . Overalltrations .Gambierdiscus spp., Ostreopsis spp., and Amphidinium spp.  either because they represent undescribed species or species that are not present in GenBank database.Phylogenetic analyses showed that all taxa classifications using the custom dinoflagellate sequence databases were correct . Some spium spp. . While oes level . AlthougGambierdiscus/Fukuyoa species were detected in six of the eighteen samples collected from Tonga. Unfortunately, these samples were not able to be fully characterized using HTS metabarcoding as DNA quantity was low. Rates of CFP have increased in Tonga over the last four decades [Gambierdiscus/Fukuyoa communities in Tonga, including morphological and molecular descriptions, are currently being undertaken . decades  but littGambierdiscus/Fukuyoa genus and Fukuyoa paulensis assays respectively. Sequences from Gambierdiscus and Fukuyoa species, and other Gonyaulacales dinoflagellates obtained from GenBank (http://www.ncbi.nlm.nih.gov/genbank/). The specificity of the primer sequences was then confirmed using BLAST  at NCBI . The assay was optimized on a Rotor-Gene 6000 , using genomic DNA extracted from an exponentially growing culture of F. paulensis (strain CAWD210) isolated from Northland, New Zealand [® DNA isolation kits  following the manufacturer’s instructions. The optimized assays consisted of a 20 μL reaction containing 10 μL of Platinum® SYBR® Green QPCR SuperMix-UDG , 1 μM of each primer, 0.8 μg non-acetylated bovine serum albumin , and 10 ng of DNA template. Primer sequences for each assay are shown in The target positions for forward and reverse primers were designed using multiple alignments of the large subunit ribosomal RNA (LSU) and the ribosomal internal transcribed spacer (ITS) region   for the  Zealand . GenomicF. paulensis (strain CAWD210) culture and no template controls contained 2 μL of sterile water as a substitute for DNA template. Assays were run in clear 0.2 mL thin-wall PCR tubes . PCR cycling conditions were: 50 °C for 2 min, 95 °C for 2 min and 30 cycles of 95 °C for 15 s and 62 °C for 60 s. The specificity of the assays was verified using DNA from various Gambierdiscus and Fukuyoa species, and other related species , North Gambierdiscus species present in the Kermadec Islands duplicate DNA extractions were also amplified using primer pairs targeting the D1-D2 region of the LSU and the V4 region of the SSU for high-throughput sequencing (HTS) metabarcoding [TM overhang adaptors. DNA extracts were sent to New Zealand Genomics Limited  for further processing. Samples were normalized to five ng·uL−1. Libraries were prepared using the IlluminaTM two-step PCR amplicon library preparation method and sequenced using an IlluminaTM MiSeq sequencer with 2X 250 base paired-end reads. All data generated was quality checked by NZGL using the following processes; FastQC, FastQscreen, and SolexaQA [® using MrBayes 3.1.2 [6 generations, sampling every 1000 trees, discarding a burn-in period of the first 1000 sampling points. After 2 × 106 generations, potential scale reduction factor values were approximately 1.0 and average standard deviation of split frequencies were less than 0.01.In order to fully characterize the arcoding . Samplesarcoding  and the SolexaQA . FurtherSolexaQA  as descrSolexaQA . Bayesiaes 3.1.2 . The evoes 3.1.2 . The conGambierdiscus/Fukuyoa species in environmental samples, enabling select samples to be targeted for further characterization. It is also an advantageous method for monitoring risk at sites where cell concentrations extremely low . HTS metabarcoding (targeting the D1-D2 region of the LSU and the V4 region of the SSU) was used to identify all the co-occurring Gambierdiscus species from the Kermadec samples, as well as the full dinoflagellate community. This is the first confirmed identification of G. polynesiensis, a species associated with CFP, in New Zealand waters. In a more recent sampling expedition to the nearby North Meyer Island (part of the Kermadec archipelago) G. polynesiensis cells were isolated and characterized [G. polynesiensis in New Zealand warrants further investigation as this species can pose a serious risk to human health via the food chain. HTS metabarcoding shows huge potential for characterizing dinoflagellate communities to species-level resolution but turn-around times and costs are still significant, and currently metabarcoding is not suitable for routine monitoring purposes. However, rapid advances in HTS technologies mean these limitations will reduce in the future. At present, the Gambierdiscus/Fukuyoa genus-level QPCR assay is an extremely useful tool for monitoring programmes and taxonomic surveys worldwide. By screening environmental samples for the presence of these potentially toxic taxa, sites with potential risk can be rapidly identified and prioritized for more in-depth species-level characterization.The QPCR assays developed in this study demonstrated high specificity and sensitivity. The assays allowed rapid determination of the presence of cterized . The pre


DNA methylation is an important feature of plant epigenomes, involved in the formation of heterochromatin and affecting gene expression. Extensive variation of DNA methylation patterns within a species has been uncovered from studies of natural variation. However, the extent to which DNA methylation varies between flowering plant species is still unclear. To understand the variation in genomic patterning of DNA methylation across flowering plant species, we compared single base resolution DNA methylomes of 34 diverse angiosperm species.By analyzing whole-genome bisulfite sequencing data in a phylogenetic context, it becomes clear that there is extensive variation throughout angiosperms in gene body DNA methylation, euchromatic silencing of transposons and repeats, as well as silencing of heterochromatic transposons. The Brassicaceae have reduced CHG methylation levels and also reduced or loss of CG gene body methylation. The Poaceae are characterized by a lack or reduction of heterochromatic CHH methylation and enrichment of CHH methylation in genic regions. Furthermore, low levels of CHH methylation are observed in a number of species, especially in clonally propagated species.These results reveal the extent of variation in DNA methylation in angiosperms and show that DNA methylation patterns are broadly a reflection of the evolutionary and life histories of plant species.The online version of this article (doi:10.1186/s13059-016-1059-0) contains supplementary material, which is available to authorized users. Arabidopsis thaliana, which is tolerant to significant reductions in DNA methylation, a feature that enabled the discovery of many of the underlying mechanisms. However, A. thaliana has a particularly compact genome, when most plant genomes are much larger [Biological diversity is established at multiple levels. Historically this has focused on studying the contribution of genetic variation. However, epigenetic variations manifested in the form of DNA methylation –3, histoh larger , 21. Theh larger .A. thaliana primarily methylate regions of deep heterochromatin, such as transposons bodies [Zea mays are associated with upstream and downstream of more highly expressed genes where they might function to prevent transcription of neighboring transposons [In plants, cytosine methylation occurs in three sequence contexts; CG, CHG, and CHH , and are under control by distinct mechanisms . Methylas bodies . RdDM res bodies , 32, 36 nsposons . The estA. thaliana [Z. mays [Sequencing and array-based methods allow for studying DNA methylation across entire genomes and within species , 15, 38.thaliana , 40 to e[Z. mays , 36, 42.[Z. mays –46. The [Z. mays  but does[Z. mays .Here, we report a comparative epigenomics study of 34 angiosperms (flowering plants). Differences in mCG and mCHG are in part driven by repetitive DNA and genome size, whereas in the Brassicaceae there are lower mCHG levels and lower numbers or even losses of CG gene body methylation (gbM) when compared to other species. The Poaceae are distinct from other lineages, having low mCHH levels and a lineage-specific distribution of mCHH in the genome. Additionally, species that have been clonally propagated often have low levels of mCHH. Although some features, such as mCHH islands, are found in all species, their association with effects on gene expression is not universal. The extensive variation found suggests that both genomic, life history, and mechanistic differences between species contribute to this variation.A. thaliana cmt3 mutants to establish thresholds with which to identify sites as symmetrical or asymmetrical and [We compared single-base resolution methylomes from the leaves of 34 angiosperm species that have genome assemblies –52 Addi. The proical and  quantifyical and .Fig. 1GeA. thaliana to a high of ~92.5 % in Beta vulgaris. Levels of mCHG varied as much as approximately eight-fold between species, from only ~9.3 % in Eutrema salsugineum to ~81.2 % in B. vulgaris  family, which includes ies Fig. . Furtherels Fig. , which isicaceae , 62. MetGlycine max and Phaseolus vulgaris, show considerably lower per-site mCHH levels as compared to Medicago truncatula and Lotus japonicus, even though they have equivalent levels of genome-wide mCHH  family, CHH Fig. . The Poaeen lost , and it V. vinifera, Theobroma cacao, Manihot esculenta, Eucalyptus grandis. No causal factor based on examined genomic features or examined DNA methylation pathways was identified; however, these plants are commonly propagated via clonal methods [M. esculenta using DNA methylation-sensitive amplified polymorphisms have been observed before [Fragaria x ananassa (common garden strawberry) [A. thaliana suggests that mCHH is re-established during reproduction [M. esculenta plant that had previously undergone clonal propagation and a DNA methylome of its offspring that was germinated from seed. Additionally, the original F. vesca plant used for this study had been micro-propagated for four generations. We germinated seeds from these plants, as they would have undergone sexual reproduction and examined these as well. Differences were slight, showing little substantial evidence of genome-wide changes in a single generation of sexual reproduction :  methods . Among nd before , so has awberry) . If repeoduction , 67. To p value = 2.9 × 10–3) and between mCHG and genome size   events, and in the copy number for repetitive elements. The majority of changes in genome size among the species we examined are due to changes in repeat content as the total gene number in these species only varies two-fold, whereas the genome size exhibits ~8.5-fold change. As genomes increase in size due to increased repeat content, it is expected that DNA methylation levels will increase as well. This was tested using phylogenetic generalized least squares (PGLS)  which ta–6) Fig. , but no –6) Fig. . This dap value = 5 × 10–6) and mCHH , but in contrast found no correlation for mCG   Fig. . This prV. vinifera. However, several Poaceae species show no correlation or even positive correlations between gene number and mCHH levels. Only two grass species showed negative correlations, Setaria viridis and Panicum hallii, which fall in the same clade  and mCHG levels   , but CDS mCG did not  , whereas mCG was not    Fig. . This wa Fig. . TE. salsugineum. Similarly, low levels of mCHH were found in most Poaceae. Across the body of the repeat, most species show elevated levels in all three DNA methylation sequence contexts as compared to outside the repeat . However, they also had many gbM genes that had unmethylated A. thaliana orthologs . Although gbM is generally “conserved” between species, this conservation breaks down over evolutionary distance with gains and losses of gbM in different lineages. In terms of total number of gbM genes, M. truncatula and Mimulus guttatus had the greatest number . Using previously identified mCG regions we identified genes with mCG overlapping the TSS, but lacking either mCHG or mCHH regions within or near genes. These genes had suppressed expression , increasing mCG throughout the gene body and a sharp decrease at the transcriptional termination site (TTS) , 76. GbMen found , 76, gbMA. thaliana as the base for pairwise comparison for all species except the Poaceae where O. sativa was used , and have previously been reported to evolve more slowly , 46. We . lyrata  and betwA. thaliana and G. max have shown that some percentage of protein-coding genes do indeed contain non-CG methylation [Z. mays genes is known to occur, especially in intronic sequences due in part to the presence of transposons [Cucumis sativus has an unusual pattern of mCHH in many highly expressed genes, although this pattern was not observed in a second C. sativus sample and will require further study to understand the basis for this difference  to as high as ~32 % of genes (F. vesca)  Fig. . In all A. thaliana mCHH genes across all species. Previous comparisons of A. thaliana, A. lyrata, and C. rubella have shown no conservation of non-CG methylation between orthologs within the Brassicaceae [Unlike gbM genes, there was no conservation of DNA methylation status across orthologs of mCHG and mCHH genes  hints at this possibility [Z. mays, high mCHH is enriched in the upstream and downstream regions of highly expressed genes and are termed mCHH islands [Z. mays also show an association between mCHH islands and gene expression, many showed no such association, indicating no universal causal relationship between the two  , there were numerous outlying species which may explain the lack of correlation between mCHH islands and total repeat content  or other regulatory elements. To date, there is limited in vivo evidence of such effects in plants, although the recent example of sibility , 85. In sibility . Conservsibility , 88. We  islands , 37. We E. salsugineum, that is associated with loss of CMT3 [We present the methylomes of 34 angiosperm species in a phylogenetic framework using comparative epigenomics, which enables the study of DNA methylation in an evolutionary context. Extensive variation was found between species, both in levels of DNA methylation and distribution of DNA methylation, with the greatest variation being observed in non-CG contexts. The Brassicaceae show overall lower mCHG levels and reduced numbers of gbM genes, leading to a complete loss in  of CMT3 . Whereas of CMT3 , and undSelaginella moellendorffii (lycophyte) [Physcomitrella patens (moss) [Marchantia polymorpha (liverwort) [E. salsugineum indicates that it is dispensable over evolutionary time [Z. mays are an exception, with nearly every gene associated with TEs, suggesting that other causes might also exist. Many non-CG genes lack orthologous genes, which could indicate a preferential targeting of de novo genes, as in the case of the qua-quine starch (QQS) gene in A. thaliana [Variation exists within features of the genome. Repeats and transposons show variation in their DNA methylation level and distribution with impacts on DNA methylation within genes and regulatory regions. Although gbM genes do show many conserved features, this breaks down with increasing evolutionary distance and as gbM is gained or lost in some species. GbM is known to be absent in the basal plant species cophyte) , Physcoms (moss) , and Marverwort) . That itary time . Non-CG thaliana . At a hihttp://schmitzlab.genetics.uga.edu/plantmethylomes). Historically, our understanding has come primarily from A. thaliana, which has served as a great model for studying the mechanistic nature of DNA methylation. However, the extent of variation observed previously [This study demonstrates that widespread variation in DNA methylation exists between flowering plant species. For many species, this is the first reported methylome and methylome browsers for each species have been made available to serve as a resource  [L. japonicus genome was downloaded from the Lotus japonicus Sequencing Project (http://www.kazusa.or.jp/lotus/) [B. vulgaris genome was downloaded from the Beta vulgaris Resource (bvseq.molgen.mpg.de/) [C. sativa genome from the C. sativa (Cannabis) Genome Browser Gateway (http://genome.ccbr.utoronto.ca/cgi-bin/hgGateway) [S. viridis were not available, gene models from the closely related S. italica were mapped onto the S. viridis genome using Exonerate [A. thaliana transposable element sequences [In plants, DNA methylation is highly stable between tissues and across generations , 48, shoal.html) . The L. /lotus/) , the B. mpg.de/) , and theGateway) . As genexonerate  and the xonerate  was usedxonerate  and A. tequences .http://schmitzlab.genetics.uga.edu/plantmethylomes.Sequencing data for each species was aligned to their respective genome  using BEDTools  and custB. distachyon, C. sativus, E. grandis, E. salsugineum, M. truncatula, P. hallii, and R. communis were constructed using the TruSeq Small RNA Library Preparation Kit (Illumina Inc). Small RNA-sequencing (RNA-seq) datasets for additional species were downloaded from GEO and the SRA and reanalyzed [Libraries for analyzed , 129. Thanalyzed . For trianalyzed . BedToolanalyzed  and custE. salsugineum, or to high, as in the case of B. vulgaris, which would further lead to misclassifications.Genes were classified as gbM, mCHG, or mCHH by applying a binomial test to the number of methylated sites in a gene   [q-value < 0.05.TEs mapping to within 2000 bp upstream, within, or 2000 bp downstream of a gene were identified using BedTools. GbM, mCHG, and mCHH genes were then tested for enrichment of TEs upstream, within, or downstream using Fisher’s exact test against the background of all the genes in a genome. GO terms for each gene were downloaded from phytozome 10.1  . GO termal.html)  with theFor each species, the general feature format 3 (gff3) file from phytozome 10.1  was usedO. sativa and dicots to A. thaliana. Reciprocal best BLAST with an e-value cutoff of ≤ 1E-08 was used to identify orthologs between dicot-A. thaliana and monocot-O. sativa pairs. Individual CDS pairs were aligned using MUSCLE [yn00 (Yang-Neilsen) [dS, dN, and ω, respectively) [Substitution rates were calculated between CDS pairs of monocots to g MUSCLE , insertiNeilsen)  model inctively) . DiffereB. distachyon and C. sativus RNA-seq libraries were constructed using Illumina TruSeq Stranded mRNA Library Preparation Kit (Illumina Inc.) and sequenced on a NextSeq500 at the Georgia Genomics Facility. Reads were aligned using Tophat v2.0.13 [RNA-seq datasets , 136–140 v2.0.13  supplied v2.0.13  suppliedA. lyrata-A. thaliana, B. distachyon-O. sativa, F. vesca-P. persica, G. raimondii-T. cacao, M. esculenta-P. trichocarpa). As the genomes of some species analyzed here are as yet unpublished, we restricted our analysis to a representative subset of species with published genomes taken from across the phylogeny. CNS regions were defined as 5’ distal, 5’ proximal, intronic, 3’ proximal, and 3’ distal by the CNS Discovery Pipeline 3.0. Coordinates for CNS regions were extracted and methylation levels calculated across 2 kb upstream, across the CNS, and 2 kb downstream. BED files of called CNS regions are available at GitHub .The CNS Discovery Pipeline 3.0  was usedp value < 0.01 [mCHH islands were identified for both upstream and downstream regions as previously described . Brieflye < 0.01 .


Flavobacterium columnare is a Gram-negative fish pathogen causing columnaris disease in wild and cultured fish species. Although the pathogen is widespread in aquatic environments and fish worldwide, little is known about biology of F. columnare and mechanisms of columnaris disease pathogenesis. Previously we presented the complete genome sequence of F. columnare strain ATCC 49512. Here we present a comparison of the strain ATCC 49512 genome to four other Flavobacterium genomes. In this analysis, we identified predicted proteins whose functions indicate F. columnare is capable of denitrification, which would enable anaerobic growth in aquatic pond sediments. Anaerobic growth of F. columnare ATCC 49512 with nitrate supplementation was detected experimentally. F. columnare ATCC 49512 had a relatively high number of insertion sequences and genomic islands compared to the other Flavobacterium species, suggesting a larger degree of horizontal gene exchange and genome plasticity. A type VI subtype III secretion system was encoded in F. columnare along with F. johnsoniae and F. branchiophilum. RNA sequencing proved to be a valuable technique to improve annotation quality; 41 novel protein coding regions were identified, 16 of which had a non-traditional start site . Candidate small noncoding RNAs were also identified. Our results improve our understanding of F. columnare ATCC 49512 biology, and our results support the use of RNA sequencing to improve annotation of bacterial genomes, particularly for type strains. Flavobacterium columnare is in the family Flavobacteriaceae, which is one as the main phyletic lines within the Bacteroidetes  forming yellow-pigmented and typically rhizoid colonies . Thirty-six of the newly identified proteins are conserved hypothetical proteins, of which 30 may be involved in virulence based on similarity to proteins in MvirDB  fry in France  broth or agar , yeast extract (0.80 g), MgSO4 7 H2O (1.00 g), CaCl2 2H2O (0.74 g), NaCl (5.00 g), and sodium citrate (1.50 g) per liter] to an OD600 of 1.0. The culture was diluted 1/10 in FCGM and FCGM with 5 mM sodium nitrate broth medium and incubated under anaerobic conditions at 30°C using a GasPak anaerobic system  in a standard anaerobic jar. Bacterial growth was monitored using OD600 for 1 week.The ability of Nitrite is an intermediate product during denitrification and is reduced to nitric oxide by nitrite reductase. To determine nitrite production during anaerobic growth, a diazotization reaction was used as described Griess,  with somFlavobacterium genomes  were used for genome comparisons. For pathway analyses and comparison of gliding motility components, Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway Database (http://www.genome.jp/kegg/) was used  version 3.0 was used to predict protein localization  were computationally predicted using P2TF  and P2CS (Prokaryotic 2-Component System) .MacSyFinder and TXSScan were used to detect protein secretion systems and their appendages in the Flavobacterium genomes using CRISPRfinder  was downloaded from http://mvirdb.llnl.gov/ . To be considered a match, Flavobacterium proteins had to have BLAST result in the MvirDB with an E-value <1*10−10. For comparison with other Flavobacterium strains, the predicted F. columnare ATCC 49512 virulence factor proteins were uploaded to the NCBI website to identify the best BLAST hit results. Distribution of predicted F. columnare ATCC 49512 virulence factors in metabolic pathways was analyzed using Blast2GO  server  and harvested by centrifugation. RNAprotect Bacteria Reagent (Qiagen) was immediately added, and pellets were stored at −80°C. Total RNA was isolated using RNA Qiagen Mini Kit, and rRNA was removed with the Ribo-Zero™ Magnetic rRNA removal kit . Sequencing libraries were prepared using Truseq RNA v2 (Illumina) at Global Biologics  and pooled. Single-end 50 bp reads were sequenced on two lanes using an Illumina HiSeq 2000.For RNA isolation, F. columnare was performed using RNA-seq. Transcriptome profiling of F. columnare using Illumina Hi-Seq 2000 generated a total of 68,164,700 single-end reads across six samples. Reads were processed for adaptor removal and filtered for quality using FastX clipper and FastQC, respectively. Reads were aligned to the F. columnare ATCC 49512 genome (NC_016510.2) using Bowtie 2  and to identify unannotated functional elements, mRNA expression profiling of https://github.com/jsreddy82/birap) was used for analysis of expression and evaluation of annotation. BIRAP is a command-line Perl pipeline that integrates expression data with in silico annotation to identify expressed annotated as well as unannotated regions. The tool capitalizes on the single nucleotide resolution generated by RNA-seq when reads are initially aligned to the genome using tools such as Bowtie and its subsequent representation as reads per base (RPB) in the pileup output generated by SAMtools   and verifies their association with annotated regions. A region of at least 70 bp in length, identified as expressed, and located between two annotated regions is classified as an EIR. If an EIR is located within 10 bp up- or downstream of an annotated region, then the EIR is marked as associated with that annotated region in the result file, which helps evaluate the annotated region's start and stop sites. If EIRs are not in the vicinity of annotated regions, they are marked as unpredicted EIRs. BIRAP evaluates EIRs as potentially being putative non-coding RNAs when promoters and terminators are predicted in appropriate locations. Small non-coding RNAs are known to regulate gene expression and are generally under the influence of a promoter or a rho-independent terminator  . RNA-Seq accession numbers are: 116-SRR1122816, 117-SRR1122817, 118-SRR1122818, 119-SRR1122819, 120-SRR1122820, 121-SRR1122821.The genome sequence of F. columnare ATCC 49512 chromosome features compared against F. johnsoniae UW101, F. psychrophilum JIP02/86, F. branchiophilum FL-15, and F. indicum GPSTA100-9T are listed in Table Flavobacterium strains have low G+C content. Genome size varies from 2,861,988 to 6,096,872 bp, with the F. johnsoniae UW101 genome being twice the size of the other flavobacteria. Only F. psychrophilum JIP/86 and F. branchiophilum FL-15 carry plasmids. Amino acid composition was similar between all the Flavobacterium genomes , F. columnare ATCC49512 , and F. psychrophilum JIP0286 ], is slightly less than the percentage in environmental isolates F. indicum GPTSA100-9  and F. johnsoniae UW101  , a part of the TCs, are present only in the F. branchiophilum FL-15 and F. columnare ATCC49512 genomes. F. branchiophilum FL-15 encodes two putative unorthodox histidine kinases. F. branchiophilum FL-15 encodes more TFs than the other two fish pathogenic species. However, a similar percentage of histidine kinases and response regulators are encoded in all fish pathogenic Flavobacterium genomes.The percentage of TFs  and TCs (two-component systems) in the fish pathogens' genomes [Flavobacterium secretion systems are shown in Table Flavobacterium genomes encode one whole T9SS operon, but the porQ gene is present in all Flavobacterium genomes twice.Gram-negative bacteria have several types of secretion systems  is restricted to the Bacteroidetes phylum , porin (omf) and the inner-membrane-anchored adaptor protein (mfp). The T1SS is encoded multiple times in the five flavobacterial genomes. T1SS is responsible for secretion of toxins and various other proteins ranging in size from 5.8 to 900 kDa -Cas regions and direct repeat sequences. All the identified CRISPR elements and their components are listed in Table Flavobacterium genomes have CRISPR spacers, and such repeat regions could be the result of the horizontal transfer and contribute to flavobacterial genome plasticity  participates in blocking transcription , but genomic islands are still identifiable using other features.Genomic islands are clusters of genes that often are associated with microbial adaptations of medical or environmental interest   , 6.8 kb (7 ORFs), and 7.5 kb (8 ORFs), and they include ORFs encoding chemical-damaging agent resistance protein and toxic ion resistance protein, which are unique to this genome. The incomplete phage region in F. johnsoniae UW101 has 21 ORFs (15.6 kb), and one of its ORFs encodes arsenate reductase. F. columnare, F. psychrophilum, and F. johnsoniae phage regions all include attL gene, and F. psychrophilum and F. johnsoniae also carry the attR gene. AttL and AttR proteins are responsible for creating recombinant junctions with the help of phage integrase gene.The incomplete F. psychrophilum JIP02/86 is classified as bacteriophage type 6H, which is an abundant type of temperate phage that is lysogenized in a large number of F. psychrophilum strains , which is present in Flavobacterium genomes. These include alginate o-acetyltransferase (AlgI), ABC transporter ATP-binding proteins, aspartate alpha-decarboxylase, bacteriophage components, capsular polysaccharide synthesis enzymes, cold shock-like proteins, CTP synthetize, ferric uptake regulators, glutamate dehydrogenase, invasion regulatory proteins, and thioredoxin reductase (TrxB).Several proteins with matches in MvirDB are encoded in all the evaluated F. columnare ATCC 49512 encodes 89 unique potential virulence proteins that are not in the other four flavobacterial genomes. Among these are a catalase and peroxiredoxin, which are both involved in bacterial resistance to toxic peroxides. A TonB-dependent siderophore receptor is also unique to F. columnare, as well as a thiol-activated cytolysin that is similar to listeriolysin O and streptolysin O. Several polysaccharide envelope synthesis enzymes are unique to F. columnare, including an N-acetyl neuramic acid synthase, a UDP-N-acetylglucosamine-2-epimerase, glycosyl transferases, and capsular polysaccharide synthesis enzymes. Several transcriptional regulators and sensor histidine kinases are also unique to F. columnare ATCC 49512.F. psychrophilum have been extensively described  cycle and glycolysis pathway. All of the other Flavobacterium strains in our comparative analysis also have complete TCA and glycolysis pathways. However, some carbohydrate metabolism components are unique to F. johnsoniae such as pentose phosphate pathway and pentose/glucuronate interconversion elements. Moreover, pathway comparison showed that starch and sucrose utilization pathways are present only in F. johnsoniae and F. branchiophilum.Metabolic pathways of F. columnare ATCC 49512 genome has predicted protein functions in oxidative phosphorylation, which is driven by the electron transport chain and TCA cycle  that are missing in the other Flavobacterium species.F. columnare and F. johnsoniae are unique among the sequenced Flavobacterium species in that they encode more proteins in nitrogen metabolism. The other sequenced Flavobacterium strains do not carry 80% of the nitrogen metabolism genes carried by F. columnare and F. johnsoniae. In particular, F. columnare ATCC 49512 encodes protein functions suggesting it is capable of denitrification, which results in a reduction of nitrate and nitrite to molecular nitrogen (N2). It has predicted protein functions indicating that it is capable of nitrous oxide reduction, nitric oxide reduction, nitrite reduction, nitrate reduction, and nitrite/nitrate transport. Nitrate and nitrite are abundant in eutrophic freshwater environments where F. columnare is commonly found, particularly in warm water aquaculture systems where it is a common fish pathogen. Its ability to utilize nitrate and nitrite as electron acceptors for the electron transport system suggest that it is capable of actively metabolizing and multiplying in pond sediments that are commonly oxygen depleted or anaerobic.F. columnare has a complete urea cycle, but it does not encode a urease. Ammonia is typically abundant in eutrophic freshwater environments, but the advantage to F. columnare of converting ammonia to urea is not currently clear. In Helicobacter pylori, it appears the urea cycle is used to maintain intrabacterial nitrogen balance  is encoded in the F. columnare genome. F. johnsoniae, F. psychrophilum, F. branchiophilum, and F. indicum have the same iron acquisition elements.Bacteria require iron as an essential nutrient. Iron acquisition is particularly important for pathogenic bacteria because of limited availability inside the host . GldE gene is missing in F. columnare ATCC 49512, F. psychrophilum JIB02/86, and F. indicum GPSTA100-9T. In F. johnsoniae, overexpression of gldE gene partly suppressed the motility defects of a gldB mutant, suggesting that gldB and gldE might have overlapping functions .A total of 63,390,073 reads were aligned to the genome using Bowtie 2 with an average 98% alignment per sample. Genome annotation for A total of 1,132 EIRs of size 70 bp or more were identified by BIRAP and were analyzed. BlastX search against the non-redundant bacterial database identified 41 that had matches with annotated bacterial proteins in other species Table , 16 of wF. columnare ATCC 49512 genome, suggesting they could represent start codon corrections. BlastX searches of these EIRs resulted in updated start sites of 25 annotated protein coding regions . A CRISPR segment consists of three elements: CRISPR array, flanking regions consisting of short direct repeats separated by short variable DNA sequences and cas genes, and short repeat regions  carrying a complete phage region. The other four species only carry remnants or incomplete phage regions, with three incomplete regions in F. branchiophilum FL-15.Despite carrying F. columnare virulence factors have been identified. An initial characterization of F. columnare iron acquisition proteins was described . Also, RNA-seq provided experimental validation for predicted protein-coding ORFs that were identified using automated annotation for the F. columnare ATCC 49512 genome , and it allowed analysis of operon structure within the genome. RNA-seq also enables identification of small non-coding RNAs, which are not annotated using automated pipelines. We identified candidate sRNAs in the F. columnare ATCC 49512 genome, none of which had matches in the Rfam database. This is likely due to a paucity of data on sRNAs in the Bacteroides group, and we consider this to be the first experimental identification of sRNAs in the Flavobacterium genus. Based on our results, we advocate RNA-seq as a relatively low-cost method to improve annotation of reference genomes, particularly in identifying novel proteins and sRNAs that are not identified by automated annotation pipelines.Finally, we used RNA-Seq to improve the F. columnare ATCC 49512 genome revealed several conserved functions the species shares with the other Flavobacterium species. Like the other flavobacteria, F. columnare likely derives energy primarily from carbohydrates and proteins rather than lipids, and it has similar gliding motility functions. However, unlike the other flavobacteria, F. columnare has the genetic capability of utilizing nitrate and other nitrogen molecules for anaerobic respiration. We present experimental evidence to phenotypically substantiate this capability. F. columnare ATCC 49512 also has evidence for more horizontal gene exchange than the other flavobacterial species, as evidenced by a high number of insertion sequences and genomic islands. RNA-seq improved the F. columnare ATCC 49512 annotation and added value by validating automated annotation and adding operon structure. We also report for the first-time candidate non-coding small RNAs in F. columnare.In summary, the HT, AK, JR, and ML conceived and designed the experiments. HT, JR, SK, and SN performed the experiments and analyzed the data. HT wrote and ML and AK edited the manuscript. All authors approve submission of this manuscript to Frontiers in Microbiology.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.


Analyses deriving insights from 23 research sites across the northeastern and east central US reveals that white-tailed deer lower native plant diversity and increase the fraction of the plant community made up of non-native species. Particularly, deer increase the abundance of the invasive plants garlic mustard and Japanese stiltgrass. Deer are known to avoid eating these species in favour of more palatable ones, thereby indirectly facilitating the success of the non-native invasives. Managing deer abundance has implications for forest plant communities. By maintaining lower deer densities, native plants likely will be better sustained and invasions of unpalatable non-native plants limited. Odocoileus virginianus), combined with burgeoning plant invasions raise concerns for native plant diversity and forest regeneration. While individual researchers typically test for the impact of deer on plant invasion at a few sites, the overarching influence of deer on plant invasion across regional scales is unclear. We tested the effects of deer on the abundance and diversity of introduced and native herbaceous and woody plants across 23 white-tailed deer research sites distributed across the east-central and north-eastern USA and representing a wide range of deer densities and invasive plant abundance and identity. Deer access/exclusion or deer population density did not affect introduced plant richness or community-level abundance. Native and total plant species richness, abundance (cover and stem density) and Shannon diversity were lower in deer-access vs. deer-exclusion plots. Among deer-access plots, native species richness, native and total cover, and Shannon diversity (cover) declined as deer density increased. Deer access increased the proportion of introduced species cover (but not of species richness or stem density). As deer density increased, the proportion of introduced species richness, cover and stem density all increased. Because absolute abundance of introduced plants was unaffected by deer, the increase in proportion of introduced plant abundance is likely an indirect effect of deer reducing native cover. Indicator species analysis revealed that deer access favoured three introduced plant species, including Alliaria petiolata and Microstegium vimineum, as well as four native plant species. In contrast, deer exclusion favoured three introduced plant species, including Lonicera japonica and Rosa multiflora, and 15 native plant species. Overall, native deer reduced community diversity, lowering native plant richness and abundance, and benefited certain invasive plants, suggesting pervasive impacts of this keystone herbivore on plant community composition and ecosystem services in native forests across broad swathes of the eastern USA.Herbivores can profoundly influence plant species assembly, including plant invasion, and resulting community composition. Population increases of native herbivores, e.g. white-tailed deer ( Modern plant communities are anthropogenically altered . BecauseCarduus, Centaurea and Cirsium spp., among others, when grazers consume more palatable species , caribou (Rangifer tarandus), Dall’s sheep , elk (Cervus elaphus), moose  and pronghorn (Antilocapra americana), have experienced severe range contractions during the past 200 years  increased steadily following steep population declines in the late 1800s  and unfenced (deer-access) plots gauged deer impacts on forest plant communities e.g. see . Use of Here, we present results of a multisite, regional assessment of white-tailed deer effects on composition, richness and abundance of introduced and native plants in east-central and north-eastern USA. We pool data from 23, paired-plot deer access/exclusion experiments spanning a broad range of invasive plant abundance and deer densities. We predicted that deer access would (i) alter floristic composition and reduce floristic diversity and (ii) increase richness and abundance of introduced plant species and decrease richness and abundance of native plant species.Quercus spp.), maple (Acer spp.), beech (Fagus grandifolia), tulip-poplar (Liriodendron tulipifera) and black cherry (Prunus serotina) (−2 (see Supporting Information—Table S1). The timing and duration of deer exclusion varied across experiments  . We acquerotina) . Deer deeriments . Six siteriments . A receneriments . Fence heriments .We acknowledge that the paired-plot approach has limitations, including fence-line effects, fences providing artificial support for vines and concentrated perch areas for birds, and an unrealistic total absence of deer in fenced plots . Deer alThe pooling approach taken here has the benefit of increasing statistical power and reducing type II error rates . We converted cover data to cover classes  and then used midpoints of cover classes (e.g. the midpoint of a 5–25 % cover class is 15 %) in analyses (hereafter referred to as cover).We analysed plant species presence/absence and two abundance metrics, cover and stem density. Across experiments, plant abundance was quantified in several ways, including stem density (14 sites), per cent cover (8 sites), cover classes  and/or density classes  . We excluded these from analyses that required knowledge of native/introduced status, but otherwise included them in indicator species analyses and when determining total plot species richness and abundance. We statistically tested effects of deer access/exclusion and deer density on richness and abundance of unknown species Database . Taxa wiWe used mixed effects linear regression to test for effects of deer on relationships between native and introduced species richness and abundance. Introduced vegetation  was the response variable, native vegetation and deer access/exclusion or deer population density were the fixed effects, and plot pair and site were the random effects. For the native vegetation effect in these models, native cover was used in the introduced cover analysis, native stem density in the introduced stem density analysis, and native species richness in the introduced species richness analysis. Deer density analyses were utilized only for unfenced, deer-access plot data here and below.H′) (H′ for each abundance metric to determine floristic diversity using the equation:We used the multi-response permutation procedure (MRPP)  to test H′) , a combiH′) , for eacS is the total number of species measured according to each abundance metric and pi is the proportional abundance of species i in the plot.where We used linear mixed effects analysis of variance (ANOVA) to test for effects of deer access/exclusion and linear mixed effects regression to test for effects of deer population density on plant richness and absolute abundance of (i) native species, (ii) introduced species and (iii) total species (native plus introduced plus unknown species) and on Shannon diversity. Deer access/exclusion (fencing treatment) or deer population density were fixed effects and plot pair and site were random effects. We evaluated both absolute and proportion of introduced plant abundance (i.e. relative abundance) and plant species richness because they represent different indices of plant invasion; the former represents actual introduced plant abundance/species richness, while the latter represents the portion of plant community abundance/species richness composed of introduced plants. Absolute introduced plant abundance/species richness was evaluated based on the main effect of deer and proportion introduced plant abundance/species richness was evaluated based on the interaction of the deer effect with total vegetation. A significant interaction indicates that the ratio of introduced to total plant abundance/species richness (i.e. proportion introduced) varies with the deer effect. The ratio of introduced to native vegetation provides another index of plant invasion and was evaluated by testing the interaction of the deer effect with native vegetation. A significant interaction would indicate that the ratio of introduced to native vegetation varies with the deer effect. Total cover could exceed 100 % due to overlapping leaves of different species. We excluded sites lacking introduced plants from community-level mixed model analyses with introduced plants in the response variable.see , is a more accurate sampling approach vs. total population counts, considerable error surrounds single estimates and cannot fully account for season-to-season or year-to-year fluctuations or legacy effects of previous deer populations. Additionally, only deer-access plot data were used in these analyses; thus, the paired-plot baseline provided by fenced-plot data is lacking. Due to these limitations, we exercised caution in interpreting results of regression analyses. All sites were included in deer density analyses except West Point, for which deer population density estimates were unavailable.We acknowledge that accurate deer density estimation is particularly difficult in forests . While dα < 0.05.To follow up the MRPP and determine which species might be driving community-level differences, we used indicator species analysis (ISA) to test for species and genera affinities for deer access or deer exclusion . The ISAWe used linear mixed effects ANOVA to test the main effect of deer access/exclusion on abundance of individual introduced and native plant species. We conducted these species-level abundance analyses for the most frequently occurring introduced plants  and for the 20 most frequent native plant species . Plot pair and site were random effects included in models to control for within- and between-site variability, respectively.α < 0.05) and, for mixed effect models with a significant deer treatment effect (α < 0.05), we determined least square means using t-tests (based on the Satterthwaite approximation for denominator degrees of freedom). To determine significance of random factors, we used log-likelihood ratio tests . We used the lme4 . We detected no effect of deer on the ratio of introduced to native vegetation (non-significant interactions between deer effect and native species vegetation)  than of introduced species (56 %). Introduced and native species richness and abundance were significantly positively correlated (etation) . At fiveH′) than deer-exclusion plots (H′ (cover but not density) was negatively correlated with deer density  of deer-access plots  is consistent with research showing that introduced plant species invade ‘hot spots’ of diversity at large spatial scales , A. petiolata (garlic mustard) and M. vimineum (Japanese stilt-grass)   and 7. Pltissima . The facalatable , yet anealatable  could oualatable , i.e. naL. japonica (Japanese honeysuckle) and R. multiflora (multiflora rose) . Tangentially, palatable invasive shrubs, such as L. maackii, which offers a leafy source of protein in early spring when native species are still leafless, might serve to boost deer populations . (ii) Increased propagule pressure via bird-dispersal could account for the higher abundance of fleshy-fruited species observed in fenced plots. Birds are attracted to the additional habitat  and fences occurring where deer are excluded  site were in deer-exclusion plots see , implicating deer exclusion as causal in increasing these vines’ abundance.While deer facilitated an increase in the abundance of several unpalatable invaders in unfenced plots, deer exclusion in fenced plots resulted in higher abundance of several other invaders, including ra rose) . Loniceron plots , reinforon plots . Indeed,excluded  and theyexcluded . (iii) Vol plots , yet theA. rubrum, Carya spp., F. americana and Quercus spp., appear to benefit from deer exclusion  also report negative impacts of abundant deer on native tree species, implying that forest regeneration could be at risk. Shrubs, including L. benzoin, R. periclymenoides and V. acerifolium, also appear negatively influenced by deer  than the average across sites   . At Fernsee  and S5. While sites were not selected randomly, most were not established to study invasive plants and spanned a wide deer abundance gradient , plant native status, deer access/deer exclusion and site.Table S6. Taxa frequency by deer access/deer exclusion and plant native status, with sites of occurrence.Figure S1. Vegetation cover class categories used to estimate plant abundance across 15 sites.Figure S2. Relationships between introduced and native species richness and abundance.This work was funded by: the United States Department of Agriculture National Needs Program (K.M.A. and D.A.M.), Penn State College of Agricultural Sciences (K.M.A.), National Science Foundation (NSF) awards DEB 1457531 and DEB 0958676 (S.K.), the NSF award DBI 0851303 and DBI 1156799 (J.D.P.), the Cooperative Agreement H399206006 from the National Park Service (J.D.P.), the U.S. Forest Service award RWU NE-4557 (through agreement JV-11242328-121 with Hood College) (D.H.B. and K.L.C.), the U.S. Department of Energy (DOE) Fermilab National Environmental Research Park  (V.A.N.) and the Strategic Environmental Research and Development Program (SERDP) of the U.S. Department of Defense (Grant RC-1542) (B.B.).K.M.A., D.A.M., E.A.H.S., S.K., W.J.M., N.A.B. and J.D.P. conceived of the project. A.A.R. established Long Run and Marienville deer exclusion experimental sites, which were re-sampled by K.M.A. and D.A.M. K.M.A., D.A.M., S.K., W.J.M., N.A.B., J.D.P., M.D.A., D.K.A., B.B., D.H.B., K.L.C., S.E.J., R.M. and V.A.N. established experiments and/or collected data. S.E.J. created Fig. 1. K.M.A. analysed the data and wrote the manuscript. All authors contributed to revisions.None declared.Supporting InformationClick here for additional data file.


Tetradonia (Staphylinidae: Aleocharinae: Athetini), and specialist ant-mimics in the genera Ecitomorpha and Ecitophya (Staphylinidae: Aleocharinae: Ecitocharini). Similar to a previous study of Tetradonia beetles, we combined DNA barcoding with morphological studies to define species boundaries in ant-mimicking beetles. This approach found four ant-mimicking species at our study site at La Selva Biological Station in Costa Rica. Community sampling of Eciton army ant parasites revealed that ant-mimicking beetles were perfect host specialists, each beetle species being associated with a single Eciton species. These specialists were seamlessly integrated into the host colony, while generalists avoided physical contact to host ants in behavioral assays. Analysis of the ants’ nestmate recognition cues, i.e. cuticular hydrocarbons (CHCs), showed close similarity in CHC composition and CHC concentration between specialists and Eciton burchellii foreli host ants. On the contrary, the chemical profiles of generalists matched host profiles less well, indicating that high accuracy in chemical host resemblance is only accomplished by socially integrated species. Considering the interplay between behavior, morphology, and cuticular chemistry, specialists but not generalists have cracked the ants’ social code with respect to various sensory modalities. Our results support the long-standing idea that the evolution of host-specialization in parasites is a trade-off between the range of potential host species and the level of specialization on any particular host.Host-symbiont interactions are embedded in ecological communities and range from unspecific to highly specific relationships. Army ants and their arthropod guests represent a fascinating example of species-rich host-symbiont associations where host specificity ranges across the entire generalist - specialist continuum. In the present study, we compared the behavioral and chemical integration mechanisms of two extremes of the generalist - specialist continuum: generalist ant-predators in the genus The online version of this article (10.1186/s12983-018-0249-x) contains supplementary material, which is available to authorized users. Wem das gelingt, dem ist eben durch die Gesellschaft seiner furchtbarsten Feinde ein mächtiger Schutz und eine reichgedeckte Tafel gesichert.“ Erich Wasmann (1895)It is the most successful adaptive strategy to join the superior opponent as a friend and to follow the principle: To run with the pack. For those who succeed, the companionship with their most terrifying enemies guarantees powerful protection and richly laden tables.In 1894, the Austrian entomologist Erich Wasmann provided a first inventory of ant-associated arthropods, in which he listed more than 1000 species   or between Tetradonia species . Specimens of the different species were pooled into the categories specialists and generalists, respectively.The amounts of CHCs [in ng] were calculated based on the internal standard hexadecane as described in . To cons/3] (see ). The la/3] (see ). The fralt odor , 79. OwiTetradonia beetles  in the Eciton-myrmecophile community at La Selva . Similarly, there was no sequence overlap between genetic clusters in CAD or in Wg . The distribution of intraspecific- and interspecific genetic p-distances in pairwise comparisons of COI sequences revealed a gap between maximum intraspecific- and minimum interspecific genetic distances of mitochondrial sequences  and five candidate species of tles see ). The NJEcitophya simulans Wasmann 1889, Ecitophya gracillima Mann 1925, Ecitomorpha cf. nevermanni Reichensperger 1935 and Ecitomorpha cf. breviceps Reichensperger 1933.Guided by the molecular work, we were able to reliably distinguish the four candidate species of beetle specialists on the basis of several anatomical characters. We identified them as the following species: Ecitophya simulans and E. gracillima are similar to each other externally, but they are clearly distinguished in the head capsule and antennal shapes. In E. simulans the head length is less than twice as long as wide (head length/ head width = 1.91–1.93), and the antennal segment II is about half as long as the segment X, while in E. gracillima the head length is more than twice as long as wide (head length/ head width = 2.26–2.30), and the segment II is less than 1/3 of the segment X. The two species can also be distinguished by their color. The abdominal tergite VI of Ecitophya simulans is almost uniformly reddish brown, but that of E. gracillima is clearly dark with brownish tinge around middle area. The two Ecitomorpha species are easily distinguishable by the shape of the pronotal incision, the absolute and relative length of antennal segments, and the shape of the head  [E. arachnoides, E. melanotica Mann 1926, E. breviceps Reichensperger 1933, and E. nevermanni Reichensperger 1935 [Ecitomorpha species, which matched well to the species descriptions of E. breviceps and E. nevermanni .nn 1889) . In the ger 1935 . HoweverEcitophya gracillima was exclusively associated with E. hamatum . Note that another Wasmannian mimic, the beetle Ecitophya rettenmeyeri Kistner & Jacobson 1990, was previously collected at La Selva from E. lucanoides but was not detected in the current samples. Figure Tetradonia beetles. These data have been published previously [Each species of Wasmannian mimic was specifically associated with a single host Fig. . Ecitophtum Fig. . It was eviously .Tetradonia beetles avoided host contact and mostly hid in gypsum cavities. Congeneric E. hamatum workers also avoided contact but were regularly seized by E. burchellii foreli workers. Accordingly, we found differences among groups in host contact frequency  . In contrast, Additional file 4: Interactions between Wasmannian mimics and army ant host workers. Video credit: Philipp O. Hönle. (MP4 34 mb)2 = 68.47, df = 2, P < 0.001; Fig. E. hamatum workers (N = 10) were aggressed by E. burchellii foreli workers (mean ± SD: 6 ± 4 aggressive events per minute), while we detected no aggression towards Wasmannian mimics and Tetradonia beetles  (Table Workers of ds Table : heneicos) Table . No addis) Table .Table 1RE. hamatum', 'E. burchellii major', 'E. burchellii intermediate', 'E. burchellii minor', and 'E. burchellii larva' . In PERMANOVA pairwise comparisons all groups differed from each other  except for Wasmannian mimics  and E. burchellii larvae . We also detected differences in the within-group variance  with CHC compositional data being more dispersed in Tetradonia beetles than in ants and Wasmannian mimics  had distinct chemical profiles  and Tetradonia beetles [Communities of army ant myrmecophiles are species-rich , 31. To ear loci –23. Distsee also ), which see also , 23, inc beetles , 21.Eciton-associated Ecitophya and Ecitomorpha beetles. Genetic and morphological inspection agreed on the presence of four distinct species in the La Selva community. Two of these species were members of the genus Ecitomorpha, which formally contains only a single species . It seevermanni  and Ecitreviceps  and werereviceps . HoweverEcitophya and Ecitomorpha. The study revealed strong intrageneric genetic divergence in beetles associated with two distinct host subspecies, Eciton burchellii parvispinum and Eciton burchellii foreli, which were studied in West and Central Panama, respectively. However, given the lack of morphological data in that study, it remains to be determined whether the detected genetic differences are in fact continuous and derive from limited gene flow due to the geographic distance separating the two study areas, or whether the two populations indeed represent reproductively isolated populations and, therefore, different species . Eitherssion in  and discssion in ).Eciton colonies within a single community with the genetic and morphological assessment of species boundaries allowed us to reliably determine host specificities, confirming a single host association in four species of Wasmannian mimics.The level of host specificity plays a fundamental role in symbiotic interactions , 81, 82.Almost 100 years after Wasmann hypothesized that ant-mimicking beetles resemble ants chemically  we can cThe same line of argument applies to beetle generalists. Compared to Wasmannian mimics, these generalists resembled the host’s CHC profile less well and showed a higher profile variation and lower CHC concentrations, which might partly explain their lower level of social integration. Imperfect CHC mimicry in myrmecophile-ant systems is common, but the ultimate and proximate mechanisms behind a weak chemical host resemblance mostly remain speculative. Carrying only small quantities of CHCs compared to host ants has often been suggested to hamper recognition due to an inability of host ants to detect these cues , mrmy ants , 91. We rmy ants ). This mrmy ants ).Tetradonia beetles. Wasmannian mimics engaged in intensive and constant host contact , which see also , 58. Tetton ants , 26 and ton ants , 41, 58)Tetradonia beetles, but carrying them out with Wasmannian mimics is difficult for two reasons. First, all adults have been collected from host colonies and immatures have not been found yet, so CHC profiles of individuals without previous host contact cannot be studied. Second, separating the beetles from host ants is impossible as they die after a few days of isolation  and in ace area , 77. Ideace area , 77, butace area ) and in ace area ).It is important to mention that integration mechanisms of myrmecophiles are multifaceted. Chemical mimicry of host CHCs is one among many possible mechanisms to cope with ants, also including morphological and behavioral adaptations , 31, 110The evolution of host-specialization in parasites and parasitoids is considered to be a trade-off . SpecialAdditional file 1:Table S1. Colony collection data . (XLSX 13 kb)Additional file 2:Table S2. Specimen and collection information including GenBank accession numbers . (XLSX 36 kb)Additional file 3:Figure S1. Assessment of species boundaries in Eciton-associated Wasmannian mimics via nuclear loci. Figure S2. Dry weight measurements of ants and parasites. Figure S3. Relationship of dry weight and CHC amount. Figure S4. Distribution of pairwise p-distances. Figure S5. Morphological distinction of studied Ecitomorpha species. Table S3. Re-assignment of samples to groups using a DAPC analysis. (PDF 4155 kb)


Anopheles arabiensis from 140 clusters in four malaria-endemic areas of Sudan from 2011 to 2014. All clusters received LLINs, while half (n = 70), distributed across the four regions, had additional IRS campaigns.Long-lasting insecticidal nets (LLINs) (with pyrethroids) and indoor residual spraying (IRS) are the cornerstones of the Sudanese malaria control program. Insecticide resistance to the principal insecticides in LLINs and IRS is a major concern. This study was designed to monitor insecticide resistance in Anopheles gambiae (s.l.) mosquitoes were identified to species level using PCR techniques. Standard WHO insecticide susceptibility bioassays were carried out to detect resistance to deltamethrin (0.05%), DDT (4%) and bendiocarb (0.1%). TaqMan assays were performed on random samples of deltamethrin-resistant phenotyped and pyrethrum spray collected individuals to determine Vgsc-1014 knockdown resistance mutations.Anopheles arabiensis accounted for 99.9% of any anopheline species collected across all sites. Bioassay screening indicated that mosquitoes remained susceptible to bendiocarb but were resistance to deltamethrin and DDT in all areas. There were significant increases in deltamethrin resistance over the four years, with overall mean percent mortality to deltamethrin declining from 81.0% (95% CI: 77.6–84.3%) in 2011 to 47.7% (95% CI: 43.5–51.8%) in 2014. The rate of increase in phenotypic deltamethrin-resistance was significantly slower in the LLIN + IRS arm than in the LLIN-only arm . The frequency of Vgsc-1014F mutation varied spatiotemporally with highest frequencies in Galabat (range 0.375–0.616) and New Halfa (range 0.241–0.447). Deltamethrin phenotypic-resistance correlated with Vgsc-1014F frequency.Combining LLIN and IRS, with different classes of insecticide, may delay pyrethroid resistance development, but the speed at which resistance develops may be area-specific. Continued monitoring is vital to ensure optimal management and control.The online version of this article (10.1186/s13071-018-2732-9) contains supplementary material, which is available to authorized users. Plasmodium falciparum . Anopheles arabiensis is the principal malaria vector in all parts of the country   was restricted to the southern part of the country [d Sennar , easternd Sennar , 29, 39,d Sennar  which sh country .An. arabiensis populations from all sentinel sites across the four study areas were susceptible to bendiocarb. In Sudan, all studies undertaken to date indicated that An. arabiensis populations showed no evidence of resistance to bendiocarb insecticide [An. arabiensis and An. gambiae (s.s.) were found fully susceptible to bendiocarb [An. arabiensis was found to be susceptible in the South but resistant in the North [This is the most comprehensive insecticide resistance monitoring programme that has been conducted in Sudan and to our knowledge is the largest known dataset on insecticide resistance. Overall, ecticide , 18, 20.ndiocarb , 41. In he North , whilst he North .An. gambiae (s.l.), from Cote d’Ivoire [Anopheles arabiensis populations from all sentinel sites exhibited high levels of resistance to deltamethrin and DDT. Previous studies have reported An. arabiensis resistant to pyrethoids and DDT in Gezira and Sennar state [Conversely, in the West African continent, resistance to carbamate insecticides was reported in d’Ivoire , Benin [d’Ivoire , Burkinad’Ivoire , 47, Mald’Ivoire  and Soutd’Ivoire . Anophelar state , White Nar state , Khartouar state  and in tar state . In factAn. gambiae (s.s.) and An. arabiensis is correlated with insecticide use for control of cotton and rice pests [An. arabiensis, from neighbouring states  or bordering countries such as Ethiopia [Recent studies have suggested that the rapid increase in pyrethroid resistance in ce pests , 51–53. Ethiopia , 54.Vgsc-1014F resistance mutation was more geographically widespread than previously documented. The Vgsc-1014S was detected only in three study areas during 2010 and at very low allele frequencies. However, after the intervention (2011–2014) this mutation was not detected in any An. arabiensis population screened. In Sudan Vgsc-1014S has previously been reported in population from Kassala (frequency = 0.16) [An. arabiensis populations exhibited variable levels of Vgsc-1014F frequency [Results of this study revealed that the  = 0.16)  and Khar = 0.16)  suggestirequency –23.Vgsc-1014F frequency declining over time in all study areas and in both arms. This decline in Vgsc-1014F frequency, which has not been reported elsewhere, is in contrast to the increasing levels of phenotypic resistance and suggests that alternative resistance mechanisms are playing a more important role in the study areas. Recent studies have documented that the over-expression of detoxification enzymes in An. arabiensis is correlated with pyrethroid resistance [Results from the current study show the sistance , 56.vs LLIN + IRS (carbamate) in Tanzania showed a broadly similar pattern [Insecticide resistance management (IRM) has received much attention, and various approaches have been suggested to delay the emergence of resistance . However pattern .An. arabiensis kdr (L1014F) is a strong predictor of pyrethroid resistance at the individual level [Results from the current study (2012) demonstrate a significant negative association between genotypic and deltamethrin phenotypic mortality at the population level, but not for the three other years of data. There are numerous studies showing that in al level –68. HoweAn. arabiensis. Importantly for efforts to delay resistance emergence we provide valuable proof-of-principle data that demonstrates the possibility of delaying pyrethroid phenotypic resistance development by combining pyrethroid-LLINs with a non-pyrethroid IRS (bendiocarb) compared to LLIN alone.The present study documented that deltamethrin and DDT resistance is geographically widespread in Sudanese Additional file 1:Table S1. Mean % mortality (95% CI) of female An. arabiensis populations from the four study areas exposed to standard WHO discriminating concentration of bendiocarb in Sudan 2011–2014. (DOC 37 kb)Additional file 2:Table S2. Mean % mortality (95% CI) of An. arabiensis populations from four study areas exposed to standard WHO discriminating concentration of DDT in Sudan 2011–2014. (DOC 39 kb)Additional file 3:Table S3. Mean % mortality (95% CI) of An. arabiensis populations from four study areas exposed to standard WHO discriminating concentration of deltamethrin in Sudan 2011–2014. (DOC 37 kb)


Borrelia burgdorferi sensu lato in Ixodes ricinus tick vector by MALDI-TOF MS. To this aim, experimental infection model of I. ricinus ticks by B. afzelii was carried out and specimens collected in the field were also included in the study. Borrelia infectious status of I. ricinus ticks was molecularly controlled using half-idiosome to classify specimens. Among the 39 ticks engorged on infected mice, 14 were confirmed to be infected by B. afzelii. For field collection, 14.8% (n = 12/81) I. ricinus ticks were validated molecularly as infected by B. burgdorferi sl. To determine the body part allowing the detection of MS protein profile changes between non-infected and B. afzelii infected specimens, ticks were dissected in three compartments  prior to MS analysis. Highly reproducible MS spectra were obtained for I. ricinus ticks according to the compartment tested and their infectious status. However, no MS profile change was found when paired body part comparison between non-infected and B. afzelii infected specimens was made. Statistical analyses did not succeed to discover, per body part, specific MS peaks distinguishing Borrelia-infected from non-infected ticks whatever their origins, laboratory reared or field collected. Despite the unsuccessful of MALDI-TOF MS to classify tick specimens according to their B. afzelii infectious status, this proteomic tool remains a promising method for rapid, economic and accurate identification of tick species. Moreover, the singularity of MS spectra between legs and half-idiosome of I. ricinus could be used to reinforce this proteomic identification by submission of both these compartments to MS.Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) has been demonstrated to be useful for tick identification at the species level. More recently, this tool has been successfully applied for the detection of bacterial pathogens directly in tick vectors. The present work has assessed the detection of  Borrelia burgdorferi sensu lato (sl). B. burgdorferi sl complex includes, up to now, 21 known bacteria species [B. afzelii is the most prevalent bacterium in Europe [Ixodes genus. In western Europe, I. ricinus is the most common vector [Lyme borreliosis is the most prevalent vector borne disease in the northern hemisphere . This muBorrelia infectious status. Tick species identification could be carried out morphologically and the detection of B. burgdorferi sl. is mainly achieved using molecular biology methods [Borrelia infectious status.Until now, there is no human licensed vaccine available, prevention and vector controls remain the main protective measures. To inform populations and to establish efficient protective devices, realization of vector epidemiological studies is required to map tick species and to determine their  methods . The expRecently, Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) has been successfully applied for the identification of several arthropod families, including ticks . DiffereBorrelia crocidurae detection in soft ticks, Ornithodoros sonrai, by submitting leg protein extracts to MS [Rickettsia spp. pathogens in infected ticks using MALDI-TOF MS, reported the dual identification of tick species and Rickettsia spp. infectious status using tick legs [Rickettsia-free and–infected conspecific specimens.Moreover, this last decade, MALDI-TOF MS has been introduced in clinical microbiology laboratories for the identification and classification of micro-organisms, including bacteria and yeast . The samts to MS . More reick legs  or tick ick legs . Indeed,B. afzelii in I. ricinus tick vector which could be useful for entomological diagnosis. I. ricinus ticks collected in the field or infected experimentally by B. afzelii NE4049 were used for this demonstration. The development of a rapid, economic and reliable method for dual identification; the determination of tick species and their Borrelia infectious status, is becoming more and more essential in the framework of Lyme disease vectors monitoring and pathogen circulation.The aim of the present study was to assess MALDI-TOF MS ability to detect Borrelia afzelii NE4049 [i NE4049  was cultI. ricinus ticks collected in the field (n = 81) or laboratory-reared (n = 68) were used for this study. Field collection of ticks was done in the Murbach area  in June 2016, by dragging a white flannel flag (1x1 m) over low vegetation. Ticks species were determined by morphological identification under a binocular loupe at a magnification of ×56  using taxonomic keys [I. ricinus ticks were included in this work. The laboratory rearing of I. ricinus ticks was performed as previously described [Borrelia experimental infections of ticks, I. ricinus specimens from the larva stage were fed on C3H/HeN mice infected (n = 39) by B. afzelii strain NE4049 or not infected (n = 29) as previously described [A total of 149 escribed ,16. For escribed . At the ad libitum. At the end of the experiment, mice were killed by isoflurane gas overdose.Mice used in tick experiments were three to four-week-old C3H/HeN pathogen-free. They were obtained from our breeding colony and provided food and water Ticks were processed as previously described . BrieflyDNA of each half-idiosome with legs was individually extracted with ammonium hydroxide (Sigma-Aldrich) as previously described ,19. PuriI. ricinus specimen collected in the field were validated by sequencing a PCR fragment of 480 bp from cytochrome oxidase I (COI) gene as previously described [® Corporate, Barcelona, Spain), and were then blasted against GenBank (http://blast.ncbi.nlm.nih.gov).DNA from 14 morphologically identified escribed . The seqB. burgdorferi sl in ticks was determined by a real-time PCR assay targeting a conserved region of the flagellin gene. Species genotyping was achieved by melting curve analysis as previously described [B. afzelii, B garinii and B. burgdorferi ss were added or not to the PCR mix as positive and negative controls, respectively.The presence of escribed . DNA froet al. [Pseudomonas aeruginosa ATCC 27853) respectively as a positive or negative control. Spectra were acquired on a Microflex LT MALDI-TOF Mass Spectrometer  as previously described [Each compartment dissected was homogenized individually using a FastPrep-24 device  and glass beads  in a mix (50/50) of 15 μL 70% (v/v) formic acid (Sigma) and 15 μL 50% (v/v) acetonitrile  for protein extraction according to the standardized automated setting described by Nebbak et al. . A quickescribed .B. afzelii were suspended in water to obtain 1.107 bacteria/μL. Serial dilution in water were done to add 106, 105 or 104 bacteria to half-idiosome protein extract from Borrelia-free I. ricinus. One microliter of this mix was spotted in quadruplicate on the MALDI-TOF target plate. Half-idiosome protein extract from Borrelia-free I. ricinus and B. afzelii bacteria protein extract were loaded on the MS target plate as controls.Pelleted Borrelia infectious status were objectified using clustering analyses and Composite Correlation Index (CCI) tool. Cluster analyses (MS dendrogram) were performed based on comparison of the MSP given by MALDI-Biotyper v3.0. software and clustered according to protein mass profile . The CCI tool from MALDI-Biotyper v3.0. software was also used, to assess the spectral variations within and between each sample group, according to the body part and Borrelia infectious status, as previously described [Borrelia-infectious status). To visualize MS spectra distribution from ticks collected in the field, according to body part and/or Borrelia-infectious status, principal component analysis (PCA) from ClinProTools v2.2 software was performed. To list discriminating peaks between compartments and/or infectious status, MS spectra were analysed using the genetic algorithm (GA) model from ClinProTools v 2.2 software. The maximum number of generations was set to 250 and the number of neighbours was three for K nearest neighbour (KNN) classification. A manual inspection and validation of the selected peaks gave recognition capability (RC) value together with the highest cross-validation (CV) value to assess the reliability and accuracy of the model. The discriminating peak masses generated by the model were searched in the peak report created for each compartment and condition.MS spectra profiles were firstly controlled visually with flexAnalysis v3.3 software . MS spectra were then exported to ClinProTools v2.2 and MALDI-Biotyper v3.0.  for data processing . MS spectra reproducibility was assessed by the comparison of the average spectral profiles  obtained from the four spots for each specimen according to body part and infectious status with MALDI-Biotyper v3.0 software . MS spectra reproducibility and specificity taking into account tick body part and escribed ,24. HighI. ricinus ticks at the nymphal stage using MALDI-Biotyper software v3.0.  [The reference MS spectra were created using spectra from legs and half-idiosomes of five ltonics) . MS specltonics) ,23, a LSBorrelia infected mice (N°APAFIS 885–2015062209113374) were approved by the Comité Régional d’Ethique en Matière d’Expérimentation Animale de Strasbourg . These protocols follow the European directive 2010/63/EU. The authority who issued the permission to collect ticks for each location was the ONF . The field studies did not involve endangered or protected species.The protocols to maintain tick colony (N°APAFIS 886–2015062209279407) and to infect ticks on I. ricinus larva, laboratory-reared and experimentally exposed to mice infected by B. afzelii, 36% of the ticks (14/39) were found infected by B. afzelii at the nymphal stage, according to real-time PCR results. The absence of B. afzelii was confirmed by RT-PCR in the 29 I. ricinus nymphs, laboratory-reared and engorged on pathogen-free mice at the larva stage.Among the 39 I. ricinus by morphological identification. Among them, the DNA extracted from half-idiosome of 13 specimens, randomly selected, were submitted to tick species identification by molecular tool. BLAST analyses corroborated morphological identification showing 99% sequence similarities with I. ricinus COI sequence from GenBank (Accession number: KF197134.1). Concerning their Borrelia infectious status, 12 ticks (14.8%) were found infected, including six, three and two specimens by B. afzelii, B. burgdorferi ss and B. garinii, respectively, and one co-infected by B. garinii and B. burgdorferi ss. This rate of infection is commonly found in this area [B. afzelii is the most prevalent species in Europe [A total of 81 ticks at the nymphal stage were collected by flagging in the Murbach area (France). All these specimens were classified as n Europe .I. ricinus infectious status was done in three body compartments, the four legs, the capitulum and the half-idiosome legs less. The legs were chosen because all anterior descriptions of distinctive MS spectra in ticks between pathogen-free and bacteria-infected specimens were achieved using legs [B. crocidurae in the legs of soft ticks and some Rickettsia species in the legs and hemolymph of hard ticks underlined that these bacteria disseminate in the ticks at sufficiently elevated concentration to modify MS spectra. The capitulum was tested to comfort the specificity of leg MS profile changes following Borrelia infection. Indeed, if MS profile changes were detected at the leg level, it appeared essential to submit to MS another body part of the same tick specimen to assess whether MS pattern modifications were also detected in this second compartment and whether some of these MS peak changes were shared between legs and capitulum. Finally, as the presence of B. burgdorferi sl was repeatedly reported in the gut of infected I. ricinus ticks [The research of MS spectra changes according to ing legs ,11 or heing legs . The detus ticks ,28, the Borrelia-infectious status, reproducible MS spectra had to be obtained for I. ricinus for each compartment and Borrelia-infectious status. The MS spectra comparison from five laboratory reared specimens infected  and capitula  were closely related. Conversely, MS spectra from half-idiosomes  appeared more distinct compared to the MS spectra from the two other body parts. Nevertheless, for each body part, no visible MS peak clearly distinguished Borrelia-infected (ii) from pathogen-free specimens (i).Before comparing MS spectra from the same tick compartment according to the infected  –ii or ninfected  –i by BoBorrelia-infected and pathogen-free specimens was observed for each compartment on the dendrogram. These results indicated that MS protein profiles were weakly affected by Borrelia infection and comforted the low specificity of MS spectra to distinguish tick legs and capitula. These data were confirmed by CCI matrix highlighting a low correlation of MS spectra between idiosomes and legs or capitula , did not reveal differences in peak position. The detection of few peaks of low intensity modifications could explain the decrease of MS spectra correlation for paired comparison of the idiosome profiles according to Borrelia-infectious status. These intensity MS peak changes could be attributed to tick response to bacterial infection. Indeed, previous works reported transcriptome [I. ricinus infected by Borrelia. The appearance and/or disappearance of at least a few MS peaks following tick infection by a specific Borrelia seems necessary to sustain the detection of a specific MS profile.To assess whether specific MS profiles could be associated to tick infectious status per body part, five MS profiles per condition were used to perform clustering and correlation analyses. The dendrogram showed that half-idiosome MS spectra clustered on distinct branches from legs and capitula . Moreovecriptome  or protecriptome  changes Borrelia species identified in I. ricinus ticks was performed. To visualize their distribution according to their Borrelia-infectious status, PCA was performed. Twelve I. ricinus ticks (co)-infected by a B. burgdorferi sl species, plus five Borrelia-free specimens and five laboratory reared I. ricinus ticks were included in this analysis. An intertwining of the dot-reflecting MS spectra distribution from half-idiosomes .A comparison of MS spectra distribution from specimens collected in the field taking into account diversity of diosomes  and legsdiosomes  was obsepartment . Then, tI. ricinus specimens  and, onrochetes . The bloy glands . Then, ty glands .B. burgdorferi sl-infectious status could be to analyze peptide MS profiles instead of intact proteins. This approach was successfully applied for Culicoides species identification [B. burgdorferi sl-infection by peptide sequencing using tandem MS (MS/MS). However, the resort to more sophisticated mass spectrometry apparatus is required such as MALDI-TOF MS/MS or liquid chromatography electrospray ionization (LC/ESI) MS/MS machines [Borrelia burgdorferi sl-infectious status. Nevertheless, proteomic methods present the advantage to detect pathogenic agent protein products which is more convincing to claim that bacteria are alive than DNA amplification which could correspond to trace of death bacteria. It also participates in the demonstration of vector competence of an arthropod vector [An alternative proteomic strategy for elucidation tick fication . With thmachines . The MS machines ,10 to sed vector .Borrelia detection by MS. To evaluate sensitivity of MALDI-TOF MS for this dual detection, serial dilutions of B. afzelii were added to half-idiosome I. ricinus Borrelia-free protein extract. The comparison of the resulting MS profiles of these mix samples with unmixed ones, revealed that MS peaks were shared with B. afzelii MS spectra, only for the tick sample added with the highest bacteria concentration  of the MS spectra queried against the database, obtained LSVs over 1.8, the threshold established for relevant identification [The MS spectra from legs, capitula and idiosomes of the 5 specimens, laboratory-reared and non-exposed, used for clustering analysis, were loaded into MALDI-Biotyper v.3.0.  to create a reference MS database. Then, the remaining 144 legs, 144 idiosomes and 63 capitula of fication .I. ricinus body part. For legs and capitula, cross-recognition occurred due to the proximity of the MS spectra between these two body parts, as reported above. However, for laboratory reared specimens, lower heterogeneity of LSVs from legs and idiosomes were obtained in comparison to capitula  could itrometry . However


Helicobacter pylori (H. pylori) infection is advised to reduce metachronous recurrence. Optimal timing of such therapy (yet to be established) was investigated herein, examining early active and late scarring stages of post-ER iatrogenic ulcers.After endoscopic resection (ER) of gastric tumors, eradication of H. pylori eradication after ER for gastric neoplasms between January 2008 and June 2015. Clinicopathologic characteristics, particularly the timing of triple therapy, were used to compare eradication rates, assigning patients to early- (≤2 weeks), intermediate- (2–8 weeks), and late-phase (≥8 weeks) treatment groups.Analysis included 514 patients who received proton-pump inhibitor (PPI)-based triple therapy for H. pylori eradication rates differed significantly by timing of triple therapy after ER . However, eradication success rates were not significantly affected by age, smoking, alcohol consumption, preexisting comorbidity, method of ER, size and location of iatrogenic ulcer, and duration of therapeutic regimen. Early initiation of H. pylori eradication was also identified as a significant independent predictor of eradication success in multivariate analysis .H. pylori offer the best chance of eradication success.In patients undergoing ER of gastric tumors, early post-ER attempts at eradication of  Helicobacter pylori (H. pylori) may reduce this risk[H. pylori is thus an important therapeutic indication in this setting, dictated by post-ER status[Endoscopic resection (ER) is a widely accepted means of treating gastric adenomas and types of early gastric cancer (EGC), generally through endoscopic submucosal dissection (ESD) or endoscopic mucosal resection (EMR),2. Althothis risk–9. As wiER status,7,9–11.H. pylori, given the gradually declining success rates due primarily to acquired antibiotic resistance[H. pylori include smoking habits, alcohol consumption, age, body mass index, underlying disease, CYP2C19 genotype, existing PUD, patient compliance, regimen duration, and of course antibiotic resistance[H. pylori eradication rates in the aftermath of ER, and none have focused on the optimal timing of therapy to eradicate H. pylori post-ER.Researchers are actively seeking ways to improve eradication rate of esistance–14. Factesistance–17. Resuesistance. HoweverH. pylori.Therefore, this study was conducted to evaluate whether differences in timing of eradication therapy after ER of gastric tumors (early active vs late scarring stages of iatrogenic ulcers) impacts success rates in eradicating H. pylori eradication therapy and use of drugs affecting H. pylori positivity  within the preceding 2 weeks were grounds for exclusion. The Institutional Review Board (IRB) of Gangnam Severance Hospital approved this study. We received a consent exemption from the IRB. Patients records and information was anonymized.Overall, 1637 patients underwent ER of gastric neoplasms between January, 2008 and June, 2015 at Gangnam Severance Hospital. Among them, 514 post-ER recipients of proton-pump inhibitor (PPI)-based triple therapy  were selected for retrospective analysis. The patient selection and grouping flow diagram is shown in H. pylori eradication, specific clinicopathologic factors, such as age, gender, smoking, alcohol consumption, preexisting comorbidity, method of ER , size and location of iatrogenic ulcer, endoscopic finding of background mucosa and histopathologic results, were collected from medical records. We defined the patients who smoke presently as “smoker” and the patient who drink more than 40g a day in men and more than 20g a day in women as as “alcoholics”. And, “preexisting comorbidity” included following disorders; diabetes, hypertension, heart disease, liver disease, kidney disease, pulmonary disease, etc. Atrophy and intestinal metaplasia were evaluated endoscopically at the time of ER. Atrophic gastric mucosa was defined by whitish to yellowish color change, visible submucosal vessels and absence of rugae. Intestinal metaplasia was characterized as metaplastic mucosa with fine or coarse plaques. The size of ulcers created via ER were extrapolated from the excised specimens. In the post-ER period, patients were grouped by timing  of therapy to eradicate H. pylori as follows: 1) early (≤ 2 weeks), 2) intermediate (2–8weeks) or 3) late (≥ 8weeks).In addition to timing and duration of H. pylori infection was determined by histologic sections (hematoxylin & eosin and Wright-Giemsa stains), by rapid urease test , and by serologic assay . Infection was confirmed by either histologic means or rapid urease test positivity, or by positive serologic assay, in the absence of medication to eradicate H. pylori. Eradication of H. pylori was achieved through PPI-based triple therapy, consisting of PPI (standard dosing), amoxicillin (1.0 gm), and clarithromycin (500 mg), given twice daily for 7–14 days after ER. Initiation of therapy varied, depending on physician or patient preference, confirmation of infection (point in time), and follow-up scheduling. H. pylori eradication was established by urea breath test or rapid urease test at least 4 weeks after completion of triple therapy, and also at least 2 weeks after discontinuation of PPI or histamine-2 receptor blocker due to the possibility of false negative result.t-test for non-categorical variables. Factors impacting H. pylori eradication rates were subjected to logistic regression analysis. Statistical significance was set at p<0.05. All computations relied on standard software .Between-group comparisons of clinicopathologic characteristics were conducted using chi-square or Fisher’s exact test, applying Student H. pylori eradication success was 81.9% (421/514). Clinicopathologic characteristics of the study population are shown in H. pylori showed statistically significant associations with H. pylori eradication rates . This subgroup analysis also showed that the early treatment group  also achieved a significantly higher eradication rate than did the intermediate  or the late  treatment group   .p <.001)  achieved a significantly higher eradication rate than did the intermediate  or the late  treatment group  .H. pylori eradication after ER, early initiation of H. pylori eradication and male gender were identified as significant independent predictors of eradication success   .H. pylori is an important therapeutic adjunct[H. pylori eradication success .To prevent metachronous lesions after ER of gastric tumors, especially in instances of EGC, eradication of c adjunct–9 that ic adjunct. HoweverH. pylori have gradually declined in many countries due to acquired microbial resistance to antibiotics. Recently, Shin et al. reported that H. pylori eradication rates with clarithromycin-containing triple therapy in Korea showed a decreasing trend over the past 10 years. The eradication rates of triple therapy ranged 84.9–87.5% from 2001 to 2007, and 80.0–81.4% from 2008 to 2010[Success rates in eradicating 8 to 2010,21,22 stH. pylori eradication rates are higher in patients with PUD than in patients with non-ulcerative dyspepsia[et al reported that active ulcers were likewise found to be independent predictors of successful eradication and concluded that early H.pylori eradication should be considered in patients with peptic ulcer bleeding for higher eradication rate[Many studies have found that post-ER iatrogenic ulcers tend to heal within 8 weeks–25, suppdyspepsia,27,28. Mtion rate. UnfortuMalfertheineret al, correlating with intense (vs mild) neutrophilic infiltration of gastric antrum[H. pylori infections more susceptible to antibiotic therapy, conferring higher eradication rates in the context of active repair.The disparate levels of inflammatory immune reactivity inherent in active and scarred gastric ulcers or defects may offer one explanation. In this regard, a significantly higher eradication rate has been observed by ic antrum. It may ic antrum. Hence, H. pylori eradication success, although this finding appears controversial. Moayyedi et al have reported similar results, citing gender-related differences in gastric physiology[et al provided further confirmation[H. pylori.Our analysis also revealed a significant association between male gender and hysiology, and recfirmation. Howeverfirmation, castingH. pylori eradication in post-ER setting. As noted by Cheon et al., early therapy to eradicate H. pylori may additionally serve to speed the healing of post-ER iatrogenic ulcers[H. pylori eradication for this reason. We nevertheless maintain that early initiation of therapy after ER is a sound rationale, capable of enhancing eradication success. However, early eradication therapy should be reconsidered in patients with acute symptoms after ER  for it may affect not only the patient’s quality of life but also completion rate of eradication therapy.There was no guideline regarding the optimal timing of ic ulcers. Howeveric ulcers, some phH.pylori according to the duration of regimen (p = 0.207), it is also a limitation of this study. Finally, assays of bacterial antibiotic resistance were not feasible. Therefore, further randomized prospective study is needed to validate our result. Nevertheless, our result yields important clues in clinical practice as to when H. pylori eradication should be initiated in patients who underwent ER for gastric neoplasm.Our study has several acknowledged limitations. First, the analysis was retrospective and non-randomized, causing unavoidable potential for selection bias. And for the same reason, we could not evaluate the side effects and completion rates of eradication therapy in each groups. In addition, the duration of regimen showed differences among three groups . Although there was no significant difference in eradication rate of H. pylori, initiated ≤2 weeks after ER of gastric tumors, emerged in this study as an independent predictor of eradication success and is thus encouraged as an important therapeutic adjunct, although further prospective study is needed to validate our result.In conclusion, early treatment to eradicate