Dendroctonus valens LeConte, is an important invasive bark beetle from North America that has caused substantial tree mortality in China. The lack of a high-quality reference genome seriously limits deciphering the extent to which genetic adaptions resulted in a secondary pest becoming so destructive in its invaded area.Biological invasions are responsible for substantial environmental and economic losses. The red turpentine beetle (RTB), Dendroctonus ponderosae, red flour beetle Tribolium castaneum, blister beetle Hycleus cichorii, and Colorado potato beetle Leptinotarsa decemlineata. We identify rapidly expanded gene families and positively selected genes in RTB, which may be responsible for its rapid environmental adaptation. Population genetic structure of RTB was revealed by genome resequencing of geographic populations in native and invaded regions, suggesting substantial divergence of the North American population and illustrates the possible invasion and spread route in China. Selective sweep analysis highlighted the enhanced ability of Chinese populations in environmental adaptation.Here, we present a 322.41 Mb chromosome-scale reference genome of RTB, of which 98% of assembled sequences are anchored onto fourteen linkage groups including the X chromosome with a N50 size of 24.36 Mb, which is significantly greater than other Coleoptera species. Repetitive sequences make up 45.22% of the genome, which is higher than four other Coleoptera species, i.e., Mountain pine beetle Pinus tabuliformis genome.Overall, our high-quality reference genome represents an important resource for genomics study of invasive bark beetles, which will facilitate the functional study and decipher mechanism underlying invasion success of RTB by integrating the The online version contains supplementary material available at 10.1186/s12915-022-01388-y. Anoplophora glabripennis Motsch [Hyphantria cunea Drury [Cydia pomonella Linnaeus [With increasing international trade, biological invasion is a critical concern worldwide, threatening environmental quality and economic security, incurring losses of US$26.8 billion annually worldwide . There hs Motsch ., the faea Drury , and theLinnaeus , to bettTribolium castaneum Herbst (Tenebrionoidea) was first sequenced [Dendroctonus ponderosae Hopkins (Curculionoidea) [A. glabripennis (Cerambycoidea) [Leptinotarsa decemlineata Say (Chrysomeloidea) [Dendroctonus bark beetle in North America and has long recorded history of major outbreaks [Dendroctonus valens LeConte, is a secondary bark beetle that does not commonly cause tree mortality in its native range in North America, but since its introduction into China in the 1980s, it has caused substantial tree death in China [Pinus phloem, providing valuable information for other bark beetles [Coleoptera (beetles) is the most species-rich order of insects with over 400,000 described species , and somequenced , the mouonoidea) , the Asiycoidea) , and Coleloidea)  have beeeloidea) , and conutbreaks . In contin China . KeelingPinus tabuliformis Carr [P. tabuliformis, resulting in multiple outbreaks unlike its native region [exo-brevicomin [In its native range of North America, which extends from Nova Scotia (Canada) west to California (USA) and south to Honduras , RTB is mis Carr .. Yousonmis Carr . Terpenomis Carr , which mmis Carr  and invamis Carr , suggestmis Carr . Howevermis Carr . The mose region , 27. Stue region . Howevere region , 34. More region , 26, whievicomin , 36.To understand how a secondary pest in its native range can become a primary pest in a new region, we investigated the genetic basis of RTB using multiple sequencing technologies. Additionally, we target genomic variations between native and invaded populations by genome resequencing, and explored possible adaptation capacities of RTB to new environments during the course of invasion. We report high quality chromosome-level genome of RTB and describe several highlights including gene family expansion and positively selected genes that might be involved in environmental adaption, the identification of X-chromosome, the origin of RTB population that invaded China, and genes showing signals of selective sweep in Chinese population contributing to evolutionary adaption in its new habitat.A total of 300 × single molecule real-time (SMRT) long reads, 107 × coverage Illumina paired-end reads, and 400 × 10X Genomics reads were generated by PacBio Sequel and Illumina HiSeq X Ten sequencing platform, respectively  (27.93%), long terminal repeat retrotransposons (LTR-RT) 12.19%), and DNA transposons (6.81%) represented the top three most abundant repeat types, followed by tandem repeat and short interspersed nuclear elements (SINE)  were identified in the genome. Furthermore, we manually annotated chemosensory, detoxification, and metabolism-related gene families that are important for RTB adaptation to its environment , the blister beetle (Hycleus Cichorii), MPB (Dendroctonus ponderosae), the Asian longhorned beetle (Anoplophora glabripennis), and the Colorado potato beetle (Leptinotarsa decemlineata) and exhibited the closest distance to MPB  (34 expanded and 29 contracted families)  or contracted (gene loss) families. Results showed all 13,751 genes in RTB were assigned onto 6194 gene families in TreeFam database. The majority of the gene families  were commonly shared by four Coleoptera species, and only a small fraction of gene families (1.04~3.04%) was unique among them , endocytosis (P = 0.012), protein geranylgeranylation (P = 0.015), histone H4-K16 acetylation (P = 0.015), locomotory behavior (P = 0.021), and small GTPase mediated signal transduction (P = 0.040) , methyltransferase activity (P = 0.021), and mevalonate kinase activity (P = 0.030) were closely associated with the PSGs in RTB    Fig. b. LG10 ireported . We alsoTo reveal the genetic variants in RTB across different geographic populations, a total of 107 individual samples were collected from the native region in North America and the invaded country China  based on the phylogeny reconstructed from SNP data, principal component analysis (PCA) and population structure analysis Fig. a–c. The FST) was computed for four populations and three contrast groups, respectively. Notably, WIMN vs. China (CHN) group showed the highest FST value (median: 0.112), followed by AZCO vs. CHN group (median 0.084), and CAMT vs. CHN had the lowest FST value (median 0.042)   and pairwise fixation index  vs. 166 Mb (assembled) in T. castaneum [H. cichorii [Locusta migratoria [Aquatica lateralis [Chromosome-level genome assemblies have been obtained for several Coleoptera through combining PacBio or Nanopore long-read sequencing and Hi-C technology –41. Suchastaneum , 269.87 cichorii , 6.3 Gb gratoria , and 940ateralis . Using tateralis –20, 46, D. valens, which is substantially higher than the percentage of repetitive elements in D. ponderosae (21.12%) [T. castaneum (28.90%) [H. cichorii (31.93%) [L. decemlineata (38.63%) [A. glabripennis (62.1%) [D. valens genome may enhance its environmental adaptation and substantially contribute to its invasiveness. It should be noted that multiple high-quality Coleoptera insect genomes have been reported in recent years, including the firefly Photinus pyralis [Pachyrhynchus sulphureomaculatus [Nebria riversi [Propylea japonica [Harmonia axyridis [The high-quality assembly genome enabled a comprehensive analysis of TEs which play important roles in driving genome evolution in eukaryotes . In this21.12%) , T. cast(28.90%) , H. cich(31.93%) , and L. (38.63%) , and low (62.1%)   , 50. The pyralis , the Easaculatus , the car riversi , and thejaponica  and Harmaxyridis . A systePinus spp have relatively low nitrogen content [Dendroctonus, helping RTB aggregate on host P. tabuliformis [Whether single genes or whole genomes –55, dupl content  and high content , 62, we liformis . Togetheliformis .P. tabuliformis [P. tabuliformis in China.We used genome resequencing data of geographic populations in native and invaded regions to identify the genes that exhibited significant signatures of positive selection in China population. Interestingly, the enrichment of selected genes in terpenoid backbone biosynthesis might indicate a difference in the biosynthesis of hormone and pheromone between China and CAMT subpopulations. In its native range of North America, RTB typically infest weakened pine trees caused by root diseases, fire, and mechanical wounding , 26, andliformis . These tliformis , which mliformis . Besidesliformis , 65–69. Dendroctonus rufipennis Kirby [The invasion and spread pattern of invasive species have long been the focus of attention. Population-based resequencing has been successfully applied to elucidate the origin and spread of populations in a number of species , 71. Ouris Kirby .Dendroctonus is closely linked to its conifer hosts in North America [Pinus spp. underwent a north to south dispersion along the principal mountain ranges of North America, such as the Rocky Mountains [Dendroctonus is in the northern region of North America [The natural history of  America , 76. Hosountains  and prev America . The CenIn contrast to the results from native ranges, 5 Chinese populations  were clustered into one group Fig. a. The ChDaktulosphaira vitifoliae (Fitch), exhibited a higher genetic diversity than did invasive populations, while some introduced populations also showed a higher genetic diversity relative to part of native geographical populations [Strikingly, it was observed that the median genetic diversity of the invasive population of RTB was greater than that of each of three native populations Fig. d, a phenulations . As the P. tabuliformis, particularly interactions with terpenoids. Moreover, as an invasive species, RTB also serves as a model to decipher the genomic basis of how a secondary pest in its original habitat can become highly destructive in invaded ranges. Additionally, gene family expansion and positive selection analysis revealed the significant enhancement of metabolic capacity, signal transduction, and gene expression regulation in RTB, which might be related to adaptation in new environments. The X-chromosome associated linkage group was determined based on female to male coverage ratio analysis, providing genetic basis for studying sex-specific behaviors. Moreover, genome resequencing analysis highlighted the population genetic structure of RTB in its native and invaded regions, suggesting substantial divergence among American subpopulations. Furthermore, selective sweep analysis showed that multiple genes associated with environmental adaptation and stress resistance underwent strong selection in China. Our work provides an important resource for genomic study in forest bark beetles in general. Moreover, it will also facilitate the functional study and decipher how an invasive beetle adapt to a new host pine P. tabuliformis with copious defensive oleoresin in invaded range aided by the recently published genome of P. tabuliformis [A chromosome-level high-quality genome assembly was obtained for the invasive forest pest RTB through integrating multiple sequencing technologies. The genome sequence of RTB provides a new valuable resource in Curculionoidea, many species of which are economically and ecologically important pests in forestry and agriculture. It also provides an important data resource for interaction studies between RTB and its host liformis .P. tabuliformis at Beishe Mountain in Shanxi . Traps were checked daily, and RTB were collected and sexed by stridulation [Field trapping was conducted using eight-funnel Lindgren traps baited with kairomone attractant  in 2016 in a natural stand of dulation . Ten paiFor population resequencing, RTB field population were collected from multiple states in the native habitat of the USA, and several provinces in the invaded region China by field trapping with eight-funnel Lindgren traps baited with the kairomone attractant and sites were tabulated . Three new emerged males/single frozen beetle kept in 100% ethanol were/was homogenized using a cell disrupter  and followed the protocol of the kit to yield genomic DNA. Finally, DNA quantity and quality controls were validated by Qubit, Nanodrop, and Femto Pulse machines.k was set as 17. Subsequently, the genome size, heterozygosity rate, and repeat content were estimated using GenomeScope 2.0 [To evaluate the genome size of RTB, an Illumina high-throughput sequencing library with an insert size of 350 bp was constructed from genomic DNA and paired-end sequenced on the Illumina HiSeq X Ten platform. Raw reads were preprocessed to obtain clean data using Trimmomatic v0.36 software . Generalcope 2.0  based onFor Illumina short-read sequencing, two paired-end sequencing libraries with an insert size of 350 bp were constructed and sequenced on Illumina HiSeq X Ten platform. For Pacific Biosciences (PacBio) long-read sequencing, two SMRT bell sequencing libraries with insert sizes of 40 and 20 kb were constructed and sequenced on the PacBio Sequel sequencing platform, generating 42.3 Gb (40 kb) and 54.16 Gb (20 kb) data separately. The raw sequencing data with adapters, more than 10% of unknown nucleotides (N), and 50% of low quality  bases generated by the Illumina HiSeq X Ten platform were trimmed to obtain high-quality reads. The subreads of PacBio sequencing data were filtered with default parameters.The Chromium library was prepared according to 10X Genomics’ protocols using the Genome Reagent Kit v2 . DNA sample quantity and quality controls were validated by Qubit, Nanodrop, and Femto Pulse machines. Briefly, ~10 ng of high molecular weight (HMW) gDNA (mean fragment length > 65 kb) was used for each library. A total of four 10X Genomics linked-read libraries were constructed and sequenced on an Illumina HiSeq X Ten platform.One Dovetail Genomics Hi-C library was prepared using the whole body of a single RTB larva which was dealt with 75% alcohol as described previously with certain modifications . Hi-C liThe draft genome was assembled using the raw reads generated by the PacBio and Illumina sequencing platform. Regarding the characteristics of reads generated by two platforms, PacBio long reads were employed for the assembly of the genome framework and Illumina short reads were utilized for improving the genome assembly. First, the assembly of the genome framework was conducted using the FALCON assembler v1.2.4  with defTo generate a chromosome-level genome assembly, Hi-C technology was used to anchor the scaffolds onto chromosomes. First, the high-quality paired-end Hi-C sequencing reads were mapped to RTB draft genome and filtered using HiCUP v0.7.4 . BrieflyTo assess the quality and completeness of the genome assembly, benchmarking universal single-copy orthologs (BUSCO)  was perfhttp://www.repeatmasker.org/RepeatModeler/), RepeatScout [http://www.repeatmasker.org/). Tandem repeats were also predicted using Tandem Repeat Finder . Based on the above procedures, repeat sequences of RTB genome were finally annotated.To identify the repetitive elements in RTB, both homology alignment and ab initio prediction methods were used to annotate transposable elements (TEs) in the RTB genome. First, RepeatModeler , transfer RNAs (tRNAs), small nuclear RNAs (snRNAs), and small nucleolar RNAs (snoRNAs), were identified in RTB genome. Firstly, rRNAs were analyzed by searching against the invertebrate rRNA database using BLAST with an E-value of 1e−10. tRNAs were identified using the tRNAscan-SE software . MeanwhiTo annotate protein-coding genes in the RTB genome, three types of gene annotation methods, including homology-based annotation, transcriptome-based annotation, and ab initio prediction, were simultaneously employed for obtaining a better gene annotation result. For homology-based annotation, reference protein sequences for nine species downloaded from National Center for Biotechnology Information (NCBI) database were aligned against the RTB genome using TBLASTN v2.2.29+ with an E-value cutoff of 1e−5. All BLAST hits were concatenated by the Solar software (v0.9.6). The genomic region corresponding to the 1000 bp upstream and downstream of each candidate gene was extracted for predicting the exact gene structure using GeneWise v2.4.1 . The resTo consider potential functions of protein-coding genes in RTB, we annotated the official gene set against several commonly used database, including NCBI non-redundant protein database (NR), SwissProt, Pfam, InterPro, and Kyoto Encyclopedia of Genes and Genomes (KEGG). Notably, Pfam domain and gene ontology (GO) information were predicted using the InterProScan tool  search aTetranychus urticae) were chosen for finding orthologs. For these 15 species (14 insect species and 1 spider mite), the genome assembly and annotation data were downloaded from NCBI RefSeq assembly or Ensembl invertebrate genome database. For genes with multiple alternative isoforms, only the longest transcript was retained for analysis of orthologs. OrthoMCL was performed with the following parameters: match percentage cutoff = 50%, and E-value cutoff = 1e−5. Markov clustering was performed using mcl with an inflation value of 1.5. After OrthoMCL analysis, single-copy orthologs, universal orthologs, and other types of genes were extracted from the clustering result using an in-house Perl script.For comparison analysis between different genomes, the identification of orthologous genes was rather important for subsequent analysis. To identify orthologs and paralogs across different insects, OrthoMCL v2.0.9 software  was emplDrosophila melanogaster , Ceratitis capitata , Bombyx mori , Spodoptera litura , Apis mellifera , Nasonia vitripennis , Zootermopsis nevadensis , Anoplophora glabripennis , Tribolium castaneum , Leptinotarsa decemlineata , Hycleus cichorii , Dendroctonus ponderosae , Nilaparvata lugens , and Pediculus humanus . Additionally, the two-spotted spider mite  was chosen as an outgroup during the inference of the phylogenetic species tree. First, multiple protein sequence alignment was separately performed for each single-copy gene of all these arthropod species using MUSCLE [To reveal the phylogenetic relationship between RTB and other insect species, a phylogenetic species tree was reconstructed from RTB and 14 other insects, specifically g MUSCLE  with theg MUSCLE  with theg MUSCLE  with theg MUSCLE  with theD. melanogaster and P. humanus, 175–185 Mya for A. mellifera and N. vitripennis, 291–359 Mya for D. melanogaster and A. mellifera, 275–345 Mya for D. melanogaster and T. castaneum, 79–155 Mya for D. melanogaster and C. capitata, 162–254 Mya for A. glabripennis and T. castaneum, and 560–642 Mya for P. humanus and T. urticae. The calibration time points were collected from the fossil records and the TimeTree website (http://www.timetree.org). The parameters of mcmctree were set as follows: burn-in=5000, sample-number=20000, sample-frequency=50.To infer the divergence times of different species in the phylogeny, MCMCtree program within the Phylogenetic Analysis by Maximum Likelihood (PAML) package  was emplP-value < 0.05 were defined as rapidly evolving families.First, all genes were assigned to corresponding gene families through search against the TreeFam v9 protein database for each species using the tool provided by TreeFam database . Then, tD. valens, D. ponderosae, L. decemlineata, H. cichorii, A. glabripennis, and T. castaneum) were separately aligned using MUSCLE [P-value was adjusted using the FDR-based multiple-comparison testing. The genes with FDR < 0.05 were defined as candidate positively selected genes in RTB.The protein sequences encoded by single-copy orthologous genes in six Coleoptera species (g MUSCLE  with theg MUSCLE . For eacg MUSCLE  was perfT. castaneum and H. axyridis), genome-wide synteny analysis was performed using the MCScanX pipeline within JCVI utility libraries [2(F:M coverage ratio) value approximately 0 was defined as autosomes, and a value approximately 1 as X chromosome, and a value equal to or less than −1 as Y chromosome. An in-house script was employed to identify sex-linked scaffolds based on the F:M coverage ratio.To reveal the collinearity relationship between RTB and two Coleoptera species  and five provinces and regions in China . Genomic DNA was extracted from each individual. Subsequently, genomic DNA samples were used for library preparation with TruSeq Nano DNA HT Sample Preparation Kit following the manufacturer’s instructions. The libraries were analyzed for size distribution using Agilent 2100 Bioanalyzer. Finally, these libraries were 150 bp paired-end sequenced on Illumina HiSeq X Ten platform.D. valens reference genome using Burrows-Wheeler Aligner [http://broadinstitute.github.io/picard/) was used to mark and remove PCR duplicates in each sample. To identify SNP sites in resequencing individuals from different locations, the Genome Analysis Toolkit (GATK) v4 [Raw reads were preprocessed to remove adaptors and low-quality sequences using Trimmomatic v0.36 . For eac Aligner  with theGATK) v4  was emplK), ADMIXTURE was implemented with K ranging from 2 to 7, and the best value for K was determined based on the cross-validation error. The format conversion of genomic variants was conducted using plink v1.9 [https://evolution.genetics.washington.edu/phylip). Furthermore, principal component analysis (PCA) of genome-wide SNP data across all resequencing samples was also conducted to determine the clustering status of subpopulations using SMARTPCA program within the EIGENSOFT software (https://github.com/chrchang/eigensoft). Prior to PCA, SNPs were filtered to retain only genotypes with quality ≥ 30 using a Python script vcf2smartpca.py (https://github.com/DeWitP/SFG/blob/master/scripts/vcf2smartpca.py).To investigate the characteristics of resequencing individuals collected from different geographic locations, population structure was determined using ADMIXTURE v1.0  with theink v1.9  prior toFST) and nucleotide diversity (Pi) were computed for detecting the signatures of selective sweeps based on genome resequencing data of RTB from different geographic populations. To calculate the genetic differentiation index, pairwise FST and Pi were calculated using VCFtools with a slide window of 5 kb and a step size of 2.5 kb [FST values and Pi ratios, genomic regions with FST and Pi ratios that are located at upper 5% quantile were considered as candidate genomic regions undergoing strong selection.To identify the genomic regions potentially undergoing strong selective sweeps during the adaption process, fixation index  correction. KEGG pathways with an FDR-corrected P-value of <0.05 were considered statistically significantly enriched.The relevant functions of the protein-coding genes overlapping with selective sweeps were characterized by searching for over-represented GO (gene ontology) terms and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways . DrosophOBAS 3.0 . The P-vAdditional file 1: Table S1. Summary statistics of genome sequencing data of Dendroctonus valens. Table S2. Summary statistics of genome assembly of Dendroctonus valens. Table S3. BUSCO evaluation result for genome assembly of Dendroctonus valens. Table S4. Summary statistics of transposable elements in Dendroctonus valens genome. Table S5. Summary of gene families manually curated in Dendroctonus valens genome. Table S6. Summary statistics of genome annotation in Dendroctonus valens genome. Table S7. List of gene families that are unique in Dendroctonus valens compared to other three Coleoptera species. Table S8. Gene families that are rapidly expanded in Dendroctonus valens revealed by CAFE analysis. Table S9. Gene families that are rapidly contracted in Dendroctonus valens revealed by CAFE analysis. Table S10. List of genes that are positively selected in Dendroctonus valens revealed by codeml analysis. Table S11. Gene ontology enrichment result of positively selected genes in Dendroctonus valens. Table S12. Sampling site information for genome resequencing of geographical populations. Table S13. Summary statistics of genome resequencing data in different populations. Table S14. List of genes that undergo selective sweep in the China population compared to CAMT population.Additional file 2: Figure S1. Genome survey result based on k-mer frequency analysis. K-mer frequency analysis was performed using Jellyfish (k-mer = 17) based on Illumina paired-end sequencing reads of genomic DNA. Genome size, repeat sequence content, and heterozygosity ratio were estimated based on k-mer frequency distribution using GenomeScope 2.0. The estimated genome size was 372.97 Mb. Figure S2. Linkage group contact map informed by Hi-C sequencing data in the red turpentine beetle genome. Fourteen linkage groups were generated after the clustering of contact map. The color bar indicates the frequency of Hi-C interaction intensity from low (yellow) to high (red) in the plot. Figure S3. Venn diagram showing the common and unique gene families across four Coleoptera species. Gene families were assigned by TreeFam database in four Coleoptera species, the red turpentine beetle Dendroctonus valens, the mountain pine beetle Dendroctonus ponderosae, the red flour beetle Tribolium castaneum, and the Asian long-horned beetle Anoplophora glabripennis. Figure S4. Synteny analysis between Dendroctonus valens and two closely related species. Dot plot representation of the syntenic relationship between D. valens and the species in the same genus, Dendroctonus ponderosae. Notably, D. valens linkage groups (LGs) showed strong syntenic relationship with D. ponderosae pseudo-chromosomes. Additionally, many fission and fusion events were observed between D. valens and D. ponderosae. (b) Genome-wide synteny relationship between D. valens and two Coleoptera insects, D. ponderosae and Tribolium castaneum. As shown in the figure, Dpochr1 was formed by the fusion of four complete LGs in D. valens . By contrast, Dpochr9 fused with Dpochr12 to generate LG13 of D. valens. Genome-wide synteny analysis was performed using the MCScan pipeline of JCVI utility libraries. Figure S5. Global representation of sampling sites of different geographical populations and RTB movement. The putative invasion and spread routes was also indicated in the map based on data collected from literature . Resequencing samples were collected from six states (red dots) in the original country North America  and five provinces (blue dots ) in the invaded country China . Figure S6. Number of genomic regions showing signals of selective sweep. Selective sweep analysis was conducted based on genetic differentiation index (FST) and nucleotide diversity (Pi) ratio in three contrast groups, including AZCO vs. CHN (a), CAMT vs. CHN (b), and WIMN vs. CHN (c). The left panel represents the selection status in North American subpopulations, and the right panel stands for the selection status in China populations. The genomic regions that were under selection were determined by the intersection set of FST outliers (top 5% quantile) and Pi ratio outliers (top 5% quantile), which corresponds to the overlapping part in the Venn diagram.


IntroductionThe insulin signaling pathway is a highly conserved pathway in animals and is central to nutrient uptake . A key component of the pathway, encoding a substrate of the Insulin Receptor (InR) protein,chicobinds to the InR and thus is also referred to as the Insulin Receptor Substrate (IRS). The IRSchicois essential to the control of cell and organ size , overall growth , including the sex-biased control of cell size and growth , and is involved in lifespan. We propose a gene model for theD. mojavensisortholog of theD. melanogasterchico (chico)gene. The genomic region of the ortholog corresponds to the uncharacterized protein LOC6576893 (RefSeq accession XP_032585220.1) in the dmoj_caf1 Genome Assembly ofD. mojavensis. This model is based on RNA-Seq data fromD. mojavensisand chico inD. melanogaster using FlyBase release FB2022_04 .D.mojavensis is part of themulleri complex in the repletaspecies group within the subgenusDrosophila of the genusDrosophila . It was first described by Patterson .D. mojavensis specializes on rotting cactus as its host and is found in the Mojave and Sonoran Deserts of the southwestern United States and northwestern Mexico including the Baja Peninsula, as well as on the channel-islands off the coast of California (https://www.taxodros.uzh.ch). The Genomics Education Partnership maintains a mirror of the UCSC Genome Browser , which is available athttps://gander.wustl.edu.Syntenychico, occurs onchromosome 2L inD. melanogasterand is flanked upstream byCG31717 and basket (bsk) and downstream by maternal expression at 31B (me31B) and methuselah-like 15 (mthl15). Thetblastnsearch ofD. melanogasterchico-PA (query) against theD. mojavensis(GenBank Accession: GCA_000005175.1) Genome Assembly (database) placed the putative ortholog ofchicowithin scaffold scaffold_6500 (CH933807.1) at locus LOC6576893 (XP_032585220.1)— with an E-value of 0.0 and a percent identity of 44.97%. Furthermore, the putative ortholog is flanked upstream by LOC6576896 (XP_015020894.1) LOC6576895 (XP_002002875.4) and LOC6576894 (XP_015020893.1), which correspond toCG31717, Multi-substrate lipid kinase(Mulk) and trunk(trk)inD. melanogaster. The putative ortholog ofchicois flanked downstream by LOC6576891 (XP_002002871.1) and LOC6576890 (XP_002002870.1), which correspond toCG33303andCG5731inD. melanogaster. The putative ortholog assignment forchico inD. mojavensisis supported by the following evidence: Although the genes surrounding thechico ortholog are not orthologous to the genes at the same locus inD. melanogaster, we conclude that LOC6576893 is the correct ortholog ofchicoinD. mojavensis, supported by strong e-values and percent identities and the lack of other matches while performing theblastpsearch.The target gene,Protein Modelblastpsearch result which shows 44.97% identity betweenD. melanogasterchico-PA and theD. mojavensis gene model as well as the low sensitivity parameters used to generate the dot plot , the dot plot of the two protein sequences contain multiple large gaps along the diagonal.chico in D. mojavensis has 3 identical protein-coding isoforms . Isoform  contains 8 protein-coding exons. Relative to the ortholog inD. melanogaster, the coding-exon number is conserved.The sequence ofchico-PAin D. mojavensishas 44.97% identity  with theprotein-coding isoformchico-PAinD. melanogaster,as determined by blastp . While conducting our research, the presence of a new isoform of chico inD. mojavensiswas discovered. The isoform identified as PA D. mojavensisis orthologous to the PA isoform inD. melanogasterand the PAA isoform is the novel isoform. Lower score Splice Junctions data due to lower levels of RNA-Seq data indicate the chico-PAA isoform is expressed at lower levels, or in fewer cells, than the chico-PA isoform . Coordinates of this curated gene model are stored by NCBI at GenBank/BankIt (accession BK059533). These data are also archived in the CaltechDATA repository (see “Extended Data” section below).Consistent with theSpecial characteristics of the protein modelNovel isoform:A new isoform of chico (chico-PAA) has been discovered based on the available RNA-Seq data and Splice Junctions data . RNA-Seq data drops off in two regions, labeled A and B, and RNA-Seq data increase are displayed in two regions, labeled C and D. Blue and pink data tracks beneath the RNA-Seq data represent Splice Junctions, with pink splice junctions having a higher score than blue splice junctions . The existence of splice junction JUNC00059058 as well as the decrease and sharp increase in RNA-Seq data in exons 6 and 7 which overlap the exons of isoform PA indicates an additional isoform .10.22002/n7ehe-xej65Description: FASTA. Resource Type: Model. DOI:10.22002/26d2v-g8r63Description: GFF. Resource Type: Model. DOI:10.22002/vbfq6-4z544Description: Peptide Sequence. Resource Type: Model. DOI:


Urtica rupestris Guss. (Urticaceae) is a rare endemic plant species occurring on shady cliffs in the southern-eastern part of Sicily. In the last century, the extreme anthropogenic alterations of Hyblaean plateau have caused the continuous and unrestrained fragmentation of natural habitats and consequently the reduction and disappearance of some plant species. A total of 52 vegetation plots, of which 34 are unpublished, were analyzed in order to characterize the floristic composition of the U. rupestris community. All the relevés were classified using classification and ordination methods. The species is mainly linked to shady and wet rock habitats, and only secondarily colonizes the undergrowth shrubs. According to IUCN criteria, we propose a new risk status for this species and the establishment of a new habitat (92/43CEE) for correct long-term conservation. Finally, a new association, Urtico rupestris-Adiantetum capilli-veneris, which falls within the Polysticho setiferi-Phyllitidion scolopendrii alliance (Adiantetea capilli-veneris class), was described. This study can provide useful information for the management and conservation of U. rupestris.The conservation actions of endangered plant species require a clear knowledge of their habitats.  Zelkova sicula Di Pasq., Garfì & Quézel, Trachelium caeruleum L. subsp. lanceolatum (Guss.) Arcang., Anthemis pignattiorum Guarino, Raimondo & Domina, Limonium syracusanum Brullo, L. pachynense Brullo, L. pavonianum Brullo, Ferulago nodosa (L.) Boiss. subsp. geniculata (Guss.) Troìa & Raimondo, Myosotis tineoi C.Brullo & Brullo, Epipactis hyblaea Brullo & Zimmitti, and Solenopsis laurentia (L.) C.Presl. subsp. hyblaea Brullo et al. [Urtica rupestris, which shows some affinities (morifolia-clade) with U. morifolia Poir. (Macaronesian Islands) and a strong similarity with U. fragilis J.Thiébaut  [Urtica rupestris, exclusive to the southern-eastern part of Sicily, is an Urticaceae woody nettle species that grows on the shady cliffs of the Hyblaean district. It was listed in the Red Book of Italian flora [U. rupestris population, in particular, analyzing the floristic composition, identifying the ecological requirements, and re-evaluating the conservation status at a regional level. Moreover, we propose the establishment of a new habitat type according to the European Directive 92/43CEE in order to achieve long-term conservation of the species.Defining effective plant conservation strategies has become a crucial issue in the Mediterranean area due to strong human pressure on the natural landscape, which has led to the loss of habitats and some endemic plant species ,2. Sicilo et al. ,16,17. ILebanon) . Urtica an flora ,20 as a Urtica rupestris, we report below an updated and more detailed description of the species than those available in the literature.Based on our morphological investigations of Urtica rupestris Guss., Cat. Pl. Hort. Boccadifalco: 83(–84). 1821. Type .The species is an erect, perennial, rhizomatous herb 0.3–0.8(−1.0 m) that is woody at the base and forms a perennial root with many unbranched stems. The plants are mostly dioecious and sometimes monoecious. The plant has a very sparse cover of erect stinging hairs that are 0.9–1.2 mm long, with a pluricellular straight base and a ca. 1/3–1/2 of the overall length of the seta. It is subglabrous with scattered, simple trichomes that are 0.1–0.4 mm long. The leaf lamina are 50–100 × 30–50 Mm ovate-acuminate, cuneate, or truncate at the base. The surface is poorly pubescent with short simple trichomes that are 0.1–0.5 mm long and has very few stinging hairs. The margins are coarsely and regularly serrate, with 8–9 teeth on each side. These teeth are 5–7 mm long and are usually undivided. The leaves are opposite and are deciduous with an apex acute to acuminate. The stipules are free (4 per node) and 3–4 mm long. The petioles are 25–45 mm long. The leaves are thin, dark green, and shiny on the upper side and lighter green on the lower leaf page. The racemes are unisexual. The plant has staminate flowers with tepals that are 0.6–0.8 mm long and pistillate flowers with tepals that are 1–1.2 mm long, which are sparsely pubescent. The female inflorescence is 8–18 mm long and is shorter than the subtending petiole, patent, or pendent in fruit. The male inflorescence is 20–40 mm long and is erecto-patent. The female flowers have subglabrous perianth segments. The mature fruit has tepals of 1.2–1.3 mm long and are achenes ovoid, wrinkled, and 1.2–1.3 × 0.8–0.9 mm.Chromosome number: 2n = 26 according to Corsi et al. .Urtica rupestris is a hemicryptophyte scapose, is rhizomatous, and flowers between April and June. It grows on shady cliffs exclusively in the Hyblaean district (south-eastern Sicily), mainly on carbonate rocks and sporadically on volcanic outcrops  [Quercus ilex L., of fluvial tectonic origin, with steep slopes. They are locally named “Cave”. It is a member of the highly specialized shady rupicolous community that is rich in bryophytes and pteridophytes and grows in the water dripping crevices of calcareous rocks. It only secondarily colonizes the Rubus ulmifolius shrubs , and in the presence of rock outcrops, also the Quercus ilex woods , as well as the rare Laurus nobilis communities of the Hedero helicis-Lauretum nobilis Bueno & Prieto 1991 [U. rupestris and the remarkable naturalistic value of its habitat, as well as its vulnerability, we propose the inclusion of this habitat with the name “Shady wet cliffs (Adiantetea capilli-veneris)”, as a new habitat type in Annex I of the Habitat Directive. This shady and wet habitat, with high edaphic humidity, includes the dripping cliffs/walls of the Mediterranean areas that are characterized by chomophytic and chasmophytic vegetation (edaphohygrophylous) related to the Adiantetea capilli-veneris class. In the Mediterranean area, this class includes one order and three alliances [Adiantion capilli-veneris groups plant communities dominated by Adiantum capillus-veneris L., which are particularly rich in bryophytes that grow on siliceous or calcareous dripping cliffs. The second one, Pinguiculion longifoliae, includes a relict herb-rich chomophytic vegetation of shaded and water-splashed habitats that are dominated by Pinguicula L. sp. pl., whereas the Polysticho setiferi-Phyllitidion scolopendri groups the fern-rich communities of damp walls and narrow and shady ravines. This shady, wet, and rocky habitat type is characterized by the occurrence of many ferns  Weiss, Pteris vittata L., Pteris cretica L., Osmunda regalis L., Asplenium scolopendrium L. subsp. scolopendrium, and Woodwardia radicans (L.) Sm.), bryophytes (Eucladium verticillatum (Brid.) Bruch & Schimp., Didymodon tophaceus (Brid.) Lisa, Pellia endiviifolia (Dicks.) Dumort., Conocephalum conicum (L.) Dumort., and Palustriella commutata (Hedw.) Ochyra), and vascular plants  Cufod., Hypericum hircinum L. subsp. hircinum, Hypericum androsaemum L., and Samolus valerandi L., etc.). As such, this vegetation type is typically found under the Mediterranean macrobioclimate, and occasionally under the sub-Mediterranean variant of the temperate macrobioclimate [Cratoneurion commutati alliance, including in the Habitat 7220* “Petrifying springs with tufa formation (Cratoneurion)”. The need for a specific habitat type concerning shady dripping cliffs with communities dominated by bryophytes and pteridophytes that belong to the Adiantetea capilli-veneris class (shady dripping cliffs with Woodwardia radicans and other large ferns) has already been highlighted by Spampinato [Woodwardia radicans, Osmunda regalis, Pteris vittata, Pteris cretica, and Asplenium scolopendrium subsp. scolopendrium.outcrops , althougi. Majo) . The speeto 1991 . Considelliances . In partoclimate ,25. In Ioclimate ,28,29,30ampinato , as wellampinato  and Sciaampinato . This prAdiantetea capilli-veneris class (20rel. × 46sp.), whereas the second (cluster B) group (32rel. × 56sp.) includes the thermophilous scrub vegetation of the Scutellario-Urticetum rupestris . In this last association, U. rupestris, together with Scutellaria rubicunda Hornem., was indicated by Brullo et al. [Rhamno-Prunetea class, such as Rubus ulmifolius Schott, Smilax aspera L., Clematis cirrhosa L., C. vitalba L., and Crataegus monogyna Jacq. This phytocoenosis is essentially localized in the thermo-mesophilious woods of the Doronico-Quercetum ilicis, Ostryo-Quercetum ilicis, and Pistacio-Quercetum ilicis. Most species of this phytocoenosis belong to the Mediterranean element (45%), with the dominant life form corresponding to phanerophytes/nanophanerophytes (34%) and hemicryptophytes (31%).The cluster and ordination analysis of all the relevés (52 rel. × 72 sp.) carried out in the Hyblaean territory showed two main groups . The firo et al.  as a chao et al. . It is aQuercus ilex within the Hyblaean caves. The structure of the community is determined by U. rupestris, together with several hygrophilous species of bryophytes and pteridophytes, such as Pellia endiviifolia, Thamnobryum alopecurum (Hedw.) Gangulee, Asplenium scolopendrium subsp. scolopendrium, Dryopteris filix-mas (L.) Schott, and Adiantum capillus-veneris, Asplenium sagittatum (DC.) Bange. Moreover, this vegetation is enriched with several lianose species, such as Hedera helix L., Rubia peregrina L. Dioscorea communis (L.) Caddick & Wilkin, Clematis vitalba, and Aristolochia sempervirens L. Due to its ecological features, U. rupestris has been proposed as a characteristic species of a new association named Urtico rupestris-Adiantetum capilli-veneris ass. nova hoc loco  and second in “Pantani Capo Passero” (Lopriore 1900). In total, U. rupestris falls within 22 cells (2 × 2 km) and eight special areas of conservation (SAC), and outside of four Natura 2000 sites. The area hosting the largest number of individuals was the N2000 “Valle del Fiume Anapo, Cavagrande del Calcinara, Cugni di Sortino” (ITA090009) site. Currently, almost all U. rupestris sites are located in the Syracuse province, with the exception of the locus classicus, which falls within the Catania territory, and one in the Ragusa province. Despite the many records of the distribution of the species, U. rupestris is threatened by many factors that, over time, have altered and reduced its natural habitat. Urtica rupestris was listed in the Red Book of Italian plants [U. rupestris. Considering our current assessments and observations in the field, the species is currently recorded in 22 cells in the Hyblaean territory. According to the reference grid for Italy [2 and an AOO equal to 104 km2. This decline in the original population, because of habitat loss and fragmentation, suggests that this species could be classified as endangered (EN).Our investigations confirmed 13 sites with upestris : 1. Valln plants  as lowerLojacono 04 and seor Italy  and the U. rupestris has been reported were visited. In addition, other sites that were potentially suitable for the species were investigated. In order to analyze the floristic composition of the U. rupestris plant community in the Hyblaean territory, several field activities were carried out in the years between 2015 and 2020. A total of 52 relevés were collected, of which 34 were unpublished and 18 from bibliographic data [p-values for each cluster’s uncertainty using bootstrap resampling. The bootstrap sample size was set to 1000. Detrended correspondence analyses (DCA) were utilized in order to develop a hypothesis about the vegetation/environmental interactions and to establish geographic patterns in the scatter-gram [2) and Extent of Occurrence (EOO) using the Geo-CAT web tool  programme [Data in the literature regarding the distribution of this species were reviewed ,41,42,43hic data ,45. The hic data . All thehic data . Pvclustter-gram . The DCAter-gram . In partter-gram . On the rogramme , which phttp://dryades.units.it/floritaly/ accessed on 15 October 2022) [The identification of vascular plants was carried out according to Pignatti et al. ,52,53,54er 2022) , whereaser 2022) ,57. The er 2022) . The bioer 2022) .The study area, located in the southeast of Sicily, is represented by the Hyblaean plateau which belongs to the African plate. It comprises of a crust of continental types different from that of the rest of Sicily , whereasU. rupestris in the Sicilian territory pointed out the biogeographical importance of this rupicolous species and of the shady dripping cliff habitat that deserves to be included in Annex I of the 93/42 EEC Directive. The re-assessment of the conservation status of this species (EN) highlights the urgent need to primarily preserve the habitats of the “Hyblaean Cave”, which hosts U. rupestris and several other restricted endemic species, such as Zelkova sicula, Trachelium caeruleum subsp. lanceolatum, Anthemis pignattiorum, Limonium pachynense, L. pavonianum, Epipactis hyblaea, etc. Therefore, the outcomes of this research can be included in future conservation and management strategies for this rare endemic taxon. The ecological and phytosociological analyses carried out on


Acanthococcus lagerstroemiae), an invasive polyphagous sap feeder in the United States, has spread across 16 U.S. states in less than two decades, posing potential risks to the Green Industry. Confirming the host range is crucial for effective pest management of invasive insects. However, host range confirmation relying on greenhouse or field trials is often inefficient and time-consuming. In this study, we used the electrical penetration graph (EPG) to monitor the stylet penetration of CMBS in plant tissue in real-time. An R programming-based application was developed to better characterize the insect EPG waveforms recorded by EPG. By analyzing EPG-based EPG parameters, we demonstrated that CMBS has difficulty accessing the phloem tissue  of a resistant plant compared to a susceptible plant. Importantly, we hereby present CMBS typical feeding behaviors on susceptible and non-susceptible plants comparatively, which provides direct evidence for revealing unknown hosts rapidly.Crapemyrtle bark scale , it is imperative to timely identify potential host plants and evaluate plant resistance/susceptibility to pest infestation. In this study, we developed an alternative technology to complement the conventional host confirmation methods. We used electrical penetration graph (EPG) based technology to monitor the A. lagerstroemiae stylet-tip position when it was probing in different plant tissues in real-time. The frequency and relative amplitude of insect EPG waveforms were extracted by an R programming-based software written to generate eleven EPG parameters for comparative analysis between plant species. The results demonstrated that the occurrences of phloem phase and xylem phase offered conclusive evidence for host plant evaluation. Furthermore, parameters including the percentage of insects capable of accessing phloem tissue, time duration spent on initiating phloem phase and ingesting phloem sap, provided insight into why host plant susceptibility differs among similar plant species. In summary, this study developed a novel real-time diagnostic tool for quick A. lagerstroemiae host confirmation, which laid the essential foundation for effective pest management.Host range confirmation of invasive hemipterans relies on the evaluation of plant susceptibility though greenhouse or field trials, which are inefficient and time-consuming. When the green industry faces the fast-spreading threat of invasive pests such as crapemyrtle bark scale ( Acanthococcus lagerstroemiae (Hemiptera: Eriococcidae)] has spread across 16 U.S. states [A. lagerstroemiae from its primary host [crapemyrtle (Lagerstroemia sp.)] to other economically important crops and native species pose a great risk to the Green Industry and ecosystems. Crapemyrtle is the best-selling deciduous flowering tree in the U.S.—3.03 million plants with a combined value of $69.57 million were sold in 2019 , economically important crops , and invasive plants [Japanese spiraea (Spiraea japonica) and Thunberg’s spiraea (Spiraea thunbergii)]  ,16,17,18bergii)] . TherefoA. lagerstroemiae. The Green Industry is in need of management research of A. lagerstroemiae  at the Department of Biology, Texas A&M University. Crawling adult female of A. lagerstroemiae  obtained from the colony on detached twigs of the plants were used for EPG recordings.Colonies of L. limii  have been characterized for L. limii . Waveford aphids ,44,45,46ealybugs ,32,44. EA. lagerstroemiae started probing and having extracellular stylet pathway activities , representing the phloem phase C③, is ofWaveform G, representing the xylem phase C④, is co2 = 21.56; df = 7, 152.00; p = 0.0030; A. lagerstroemiae (n = 160) started probing on the respective test plants  differed significantly among the plant species  successfully targeted the phloem tissue  than on C. acuminata (476.30 min and 514.84 min) and L. limii (316.23 min and 341.81 min).The phloem phase involves phloem salivation (E1) and phloem ingestion (E2) by m tissue . The timF = 2.36; df = 4, 15.20; p = 0.0769; F = 42.78; df = 4, 17.09; p < 0.0001; A. lagerstroemiae spent a significantly longer time in phloem sap ingestion (L. limii (200.92 min), which were at least three-fold longer than on L. speciosa (64.72 min), F. tikoua (26.49 min), and C. acuminata (24.60 min). No individuals had phloem salivation or phloem ingestion on F. pumila, F. auriculata, or G. max.Among the individuals detected for waveform E, plant species did not affect their total duration of phloem salivation  [A. lagerstroemiae reached the phloem or xylem tissue on the non-hosts F. pumila and F. auriculata [A. lagerstroemiae could not access phloem and xylem tissues should be non-hosts due to the failure to acquire sufficient nutrients.The comparative results of feeding parameters  showed tuminata) . In contriculata . In mostriculata ,67,68. IA. lagerstroemiae in a timesaving manner. As a future research direction, testing other economically important crops and native species using this technology could better estimate this invasive insect’s potential threat to the Green Industry and ecosystems in the U.S.Therefore, our EPG-based monitoring system provided convincing evidence to reveal hosts of L. speciosa was considered more A. lagerstroemiae-resistant than L. limii because the latter had higher number of ovisacs  compared to L. speciosa  in our greenhouse feeding trial. Our feeding parameter results showed that, compared with L. limii, a lower percentage of A. lagerstroemiae successfully reached the phloem phase on L. speciosa. After penetrating the epidermis, A. lagerstroemiae spent a significantly longer time to initiate the phloem salivation/ingestion with a huge decrease in the total duration of phloem ingestion on L. speciosa  potentially resulted from the delays in phloem-locating and difficulties in phloem ingestion. These phenomena could be due to higher plant resistance, involving morphological features and phytochemical adaptions (deterrence and repellents) in the mesophyll and phloem tissue [A. lagerstroemiae [A. lagerstroemiae resistance in crapemyrtles, it is essential to identify and integrate the main plant resistance mechanism by investigating the correlations between the A. lagerstroemiae feeding behavior and nutritive and defensive traits in phloem sap from resistant and susceptible crapemyrtles.Our results indicated that the poor m tissue ,32,69,70troemiae . TherefoA. lagerstroemiae was feeding on the resistant plant L. speciosa, which improved our understanding of the interaction between plant and A. lagerstroemiae. To further decipher the plant resistance mechanisms against A. lagerstroemiae, high-resolution micro-computed tomography would be needed to demonstrate similarities and differences in how A. lagerstroemiae penetrates a resistant plant and a susceptible plant. With the integration of ‘omic’ research approaches , comparative analysis on phloem tissue between L. limii and L. speciosa will provide a holistic understanding of plant resistance to A. lagerstroemiae. Such endeavors will increase the breeding efficiency for desired plant traits, benefiting academia and industry communities.In this study, difficulties in reaching phloem tissue and a decreased duration in phloem ingestion were indicated by feeding parameters when A. lagerstroemiae by the EPG-based monitoring system. Based on the analyses of eleven feeding parameters, the occurrences of waveform E and G could be convincing evidence to reveal host plants of A. lagerstroemiae rapidly. After initiating the first probing, A. lagerstroemiae spent more time locating the phloem tissue and a shorter time for phloem sap salivation and ingestion on resistant plant L. speciosa than on susceptible plant L. limii. This work allows us to clearly distinguish the similarities and differences in feeding features between A. lagerstroemiae and other sap-sucking insects according to the typical EPG waveforms. With the help of EPG technologies, this work improves our understanding of the correlations between plant resistance/susceptibility and feeding parameters of A. lagerstroemiae.In summary, this is the first report to characterize the typical EPG waveforms of


Cardiac arrhythmias are a major cause of morbidity and mortality worldwide. Although recent advances in cell-based models, including human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM), are contributing to our understanding of electrophysiology and arrhythmia mechanisms, preclinical animal studies of cardiovascular disease remain a mainstay. Over the past several decades, animal models of cardiovascular disease have advanced our understanding of pathological remodeling, arrhythmia mechanisms, and drug effects and have led to major improvements in pacing and defibrillation therapies. There exist a variety of methodological approaches for the assessment of cardiac electrophysiology and a plethora of parameters may be assessed with each approach. This guidelines article will provide an overview of the strengths and limitations of several common techniques used to assess electrophysiology and arrhythmia mechanisms at the whole animal, whole heart, and tissue level with a focus on small animal models. We also define key electrophysiological parameters that should be assessed, along with their physiological underpinnings, and the best methods with which to assess these parameters. Cardiac arrhythmias are a major global health burden and a primary cause of morbidity and mortality in a wide spectrum of patients, particularly those with existing cardiovascular disease, including atherosclerosis, coronary heart disease, heart failure, and myocardial infarction , 2. Card1) provide an overview of experimental techniques commonly used for electrophysiological assessments in small animal models, 2) define key electrophysiological measurements and their physiological underpinnings, 3) provide recommendations and key considerations that researchers should take into account when designing studies and using these experimental techniques, and 4) provide an extensive reference list that may serve as a useful guide for those interested in implementing experimental electrophysiological approaches in animal models.These guidelines will examine experimental approaches for the assessment of cardiac electrophysiology and arrhythmia mechanisms at the whole animal, whole heart, and tissue level, with a focus on small animal models. Although experiments in isolated cardiomyocytes or cellular-based tissue constructs are vitally important and often necessary to discern underlying cellular mechanisms, we will not cover those approaches here and refer readers to excellent reviews on these topics –22. The American Journal of Physiology-Heart and Circulatory Physiology. The search included all primary research articles published from January 1, 2019 to May 3, 2022 using the terms “electrophysiology” or “arrhythmia.” This search identified 82 articles that were then evaluated by two authors (C.M.R. and N.G.P.) to only include original research articles that reported one or more electrophysiological measurements from animal models obtained from the intact heart, intact tissue, or in vivo. Articles using only human tissues or cell-based work (primary isolated cardiomyocytes or iPSC-derived cardiomyocytes) were excluded, resulting in 30 articles for further evaluation.To obtain insight into current methods and approaches that are commonly used for measuring cardiac electrophysiology and arrhythmias in animal models, we evaluated recently published articles in the A summary of our search results is shown in In terms of disease models, genetically modified mice , 28–33 oConsidering the heavy reliance on small animals for many recent cardiac electrophysiology studies, these guidelines will focus on best practices for experimental approaches most often used in small animals . However, some of the measurements, experimental techniques, and recommendations can be universally applied to a variety of species.v1.5) is largely preserved across species, the plateau and repolarization phases of the ventricular AP differ significantly across species , ionic currents, and autonomic regulation , 55. For species . Dogs, pm humans , 57–60. m humans , 62, as m humans , 64, whem humans . Althougm humans –68, in mm humans , 70.-tubule structure is an important limitation. These limitations can be partly overcome using genetically altered mice, rats, or rabbits , high conservation of gene function, simplified maintenance, short life span, and straightforward genetic manipulation physiological foundations are the same.+ channel function, cell-cell coupling, structural remodeling), measurements of QRS duration, AP upstroke, activation patterns, conduction velocity, and/or anisotropy should be assessed. On the other hand, if repolarizing factors are suspected, measurements of QT interval, action potential duration (APD), refractory periods, or repolarization heterogeneity should be quantified. Within each subsection below, the citations include many exemplary studies in which these parameters have been used to characterize various disease models. The section Arrhythmogenesis further explains how alternations to specific endpoints may be pro- or antiarrhythmic.It is also worth noting that there are myriad end points that may be assessed to discern underlying mechanisms in both healthy and diseased hearts. Therefore, there is no standard recommendation for which parameters should be measured to characterize a specific disease model. Electrophysiological assessments should be guided a priori by a clear hypothesis. For example, when factors that may influence activation or wave propagation are suspected to be at play  clock”] and intracellular Ca2+ cycling (“the Ca2+ clock”)  via ryanodine receptors (RyR) generate small increments in intracellular Ca2+ concentration. These local calcium release events activate the Na+/Ca2+ exchanger (NCX), which generates an inward INCX current and boosts the diastolic depolarization rate, resulting in the onset of an AP via activation of ICa,L. The function of If, ICa,T, and ICa,L currents can be indirectly estimated from the early (slow) component of diastolic depolarization, whereas the late (and faster) diastolic depolarization component can be used to assess NCX function, as well as the contribution of local Ca2+ release events. Diastolic depolarization can be measured via both electrode-based and optical mapping techniques, but the measurement of local Ca2+ release events requires optical techniques that use Ca2+ sensitive indicators  –79. Pace clock”)  Fig. 3)Vm) clockchannels ] contribdicators .1) spontaneous heart rate (HR) assessed at baseline and in response to autonomic stimulation and/or blockade; 2) HR variability  to charanalyzed –86; 3) i1) sinoatrial delay during spontaneous rhythm, measured as the time between the earliest excitation within the sinus node and first atrial activation ; 2) locnditions , 89; andThe intramural location of the atrioventricular node (AVN) at the posteroinferior region of the interatrial septum  makes it difficult to use electrode-based techniques to directly record AVN activation. Accordingly, AVN function is frequently assessed via ECG by determining its conduction at increasingly rapid rates and its refractory properties with progressively premature stimulation in the right atrium , 91. WenVm because of incomplete repolarization during rapid atrial rhythm  morphology and parameters . These parameters can be correlated with the corresponding ECG parameters, including P wave and QRS duration for atrial and ventricular activation, respectively, and the T wave for ventricular repolarization. Because AP and CaT properties and propagation depend heavily on HR , these parameters should be measured during constant electrical pacing or potentially corrected for HR . Although, HR correction may not be required in mice, as the QT interval is not significantly altered by HR, unless the R-R interval becomes significantly shortened  measured using sharp glass micropipette electrodes represents a gold standard for the analysis of AP characteristics, many AP characteristics can be measured using other electrode-based or optical mapping techniques. For method-specific electrophysiological measurements, see experimental approaches.Although transmembrane potential . Activation time can be measured as the time of maximum derivative during phase 0 , or alternatively, as the time at which the AP reaches 50% of its maximal amplitude (phase 3) to rest (phase 4). Notably, the second derivative is highly sensitive to noise and postprocessing filtering. Therefore, repolarization time is often measured as the time at which the AP has repolarized from its peak by a percentage of the AP amplitude, e.g., repolarized by an amount equal to 30, 50, or 90% of the AP amplitude. APD is measured as the difference between repolarization and activation time (Vm (phase 4) mainly reflects the function of the inwardly rectifying K+ current (IK1), the Na+/K+-ATPase, and other background currents. AP amplitude and upstroke velocity (dVm/dt) reflect the function of fast Na+ channels. APD can be used to indirectly assess the contribution of other ionic currents, including transient outward K+ current Ito (at APD20–30%), ICa,L and INCX (at APD50%), IKs, IKr, IKur, and IK1 (at APD90%), although the relative contribution of these currents throughout the AP are species and location specific . These often-colorful maps have lines or colored bands that indicate areas of tissue that depolarized within the same time interval E. A valu+ channels, the distribution of collagen, and the pattern of excitation (Restitution). CV should be measured along the fastest and slowest directions, which typically align with longitudinal and transverse fiber orientations, respectively  is spatially heterogeneous and depends on multiple factors, including cardiomyocyte size, fiber orientation, the expression level, and spatial distribution of gap junctions and Nacitation –112. CV ectively , 114 . APD and repolarization maps, reconstructed using high-resolution mapping, can be used to identify areas with increased heterogeneity of tissue repolarization, which is a primary cause of reentrant activity. However, such maps do not reveal postrepolarization refractoriness, which may occur during ischemia, wherein tissue repolarization can occur before recovery from refractoriness  activated during the previous beat remain open, resulting in greater peak K+ current than at longer diastolic intervals . APD restitution (at the time of APD90) in the mouse heart is also dependent on the recovery of SR Ca2+ release at short diastolic intervals (30) in rodent hearts has been shown to prolong, rather than shorten, at short diastolic intervals because of incomplete recovery of Ito, which is the main driver of early repolarization in rodents  depend on the preceding diastolic interval (the rest period between repolarization and the next depolarization). In large nonrodent mammals, APD shortens with reductions in the diastolic interval because a significant fraction of Kntervals . Rodent ntervals . Early r rodents  Fig. 6)+ channelArrhythmogenesis. Restitution parameters should be determined using either standard or dynamic restitution pacing protocols  at this pacing cycle length, the pacing is stopped and APD, CV, or CaT duration and/or amplitude of the last paced beat is measured. Pacing is then reinitiated at a progressively shorter cycle length and measurements continue until either 2:1 block or higher-order periodicities occur. Similar to the standard restitution protocol, for each pacing cycle length, the APD, CV, or CaT duration/amplitude are plotted as a function of the preceding diastolic interval.+ channels. Depending on the pacing location, protocols can be implemented to identify atrial, AVN, and ventricular effective refractory periods   , 91, 1292+-sensitive fluorescent indicators must be used along with optical recording methods . CaT rise time . The CaT amplitude is representative of a combination of ICa,L and SR Ca2+ release, which varies by species with ∼70% of total intracellular Ca2+ increase coming from the SR in rabbits and larger mammals and ∼90% coming from the SR in rodents   and Ca2+ efflux from cardiomyocytes (by INCX). The relative contribution of SERCA versus INCX in intracellular Ca2+ decay is species dependent and, at steady state, matches the proportions of Ca2+ released from the SR and Ca2+ carried into the cell via ICa,L, respectively  . The decectively . Rate of2+ channels and RyRs, the AP-to-CaT delay can be measured  or abnormal Ca2+-mediated triggered activity   , 133. Ficitation .2+-dependent triggered activity . At slow heart rates, hearts are more susceptible to ectopic activity  or EAD-based triggered activity, which is promoted by the long APD that occurs at slow heart rates. Posttachycardia pauses tend to promote DAD-based triggered activity (due to loading of the SR with Ca2+ at high heart rates). In pathological conditions, sinus node dysfunction may therefore also play a role in susceptibility to triggered activity, but experimentally, pacing protocols that mimic these rate-dependent phenomena should be used to provoke the suspected arrhythmia mechanism. In some cases, spontaneous arrhythmias that involve abnormal impulse formation may also require pharmacological “stress tests” as discussed in Recording of the optical action potential.Although technical and methodological details on arrhythmia testing protocols are summarized in Recording of the optical action potential, here we provide a brief theoretical foundation behind commonly used protocols for arrhythmia induction. It should be noted that the exact electrophysiological mechanisms of cardiac arrhythmias are not completely understood and remain under intensive investigation. In general, cardiac arrhythmias could be classified into categories of disorders of impulse formation , disorders of impulse conduction , or a combination of both. Because of distinct underlying cellular and molecular mechanisms, different types of arrhythmias require different provocative protocols. For example, for arrhythmias that involve abnormal conduction, fast-pacing protocols (S1-S1) or premature stimuli (S1-S2) are commonly used to assess arrhythmia susceptibility. The initiation of such arrhythmias is associated with the development of local conduction discontinuities and is best described by restitution theory. In contrast, disorders of impulse formation may stem from abnormal automaticity or CaThe restitution properties of myocardial tissue have been linked to its arrhythmogenic potential , 136. Th2+. At fast rates, both refractoriness of SR Ca2+ release (due to refractoriness of RyR) and SERCA Ca2+ reuptake can lead to beat-to-beat alternation in the amplitude of the CaT. CaT alternans, in turn, can result in cellular APD alternans through several electrogenic Ca2+-sensitive sarcolemmal currents. Reduced SERCA expression  or nonstationary (drifting), vortex-like  reentrant activity. Analysis of the dominant frequency of arrhythmic activity can be useful to distinguish between these mechanisms and to characterize the complexity of arrhythmic activity , 166. FoVm(t) at each pixel in an optical mapping data set] are plotted against a time-shifted version of the same signal  to generate a phase portrait , which can trigger an AP  or several (polymorphic) local sources of automaticity. Focal activity can be either ectopic automaticity  or triggered activity . DADs oer an AP .Focal arrhythmias can be characterized via the localization of ectopic foci, their frequency, and spatial-temporal characteristics of myocardial activation. Source-sink interactions are critical determinants of whether cellular-triggered activity can propagate to the surrounding myocardium. Smaller cardiac tissue sizes and ∼one- and ∼two-dimensional structures  are much more susceptible to ectopic activity compared with three-dimensional volumes due to the greater current sink in the larger tissue volumes . NotablyA variety of experimental approaches can be applied to assess the aforementioned parameters.In this section, we discuss the techniques commonly used for in vivo and ex vivo electrophysiological studies and their suitability for the assessment of different electrophysiological parameters. Special considerations and unique challenges in each technique, as well as advantages and limitations, are also discussed see .Acute In Vivo ECG Recording). Another advantage is that data can be continuously collected over days, weeks, or months. Long-term ECG recordings allow for investigations of time as a biological variable, including studies of ECG changes during the light or dark cycle . Signal averaging, if appropriate, will increase the signal-to-noise ratio. Removal of nearby devices that might cause electrical interference and Faraday shielding between cages can also improve ECG signal quality. Commercial or custom-developed software programs can be used to analyze a large amount of data, as well as limit the impact of signal artifacts.In Vivo ECG Telemetry) that motivate use of alternative methods.Although telemetry remains the gold-standard approach for conscious ECG recording in animals, there are a number of limitations  ECG screening.ECGs may also be recorded in conscious animals placed in restraining devices such as plastic tubes or custom-made elastic jackets that fit the thoracic circumference of the animal and hold them in a fixed position –220. TheA tethered ECG system implants wired electrodes that are tunneled under the skin via a midscapular incision and positioned near the four limbs of the animal , 222. ThECG recording in conscious animals is often preferred, as anesthetic agents can depress heart rate, blood pressure, autonomic regulation, and respiration , 223. HoWith all these approaches, the choice of anesthesia needs to be carefully considered. Inhalable isoflurane and injectable ketamine-xylazine cocktail are the most commonly used anesthetic agents for in vivo ECG and programmed electrical stimulation studies. Isoflurane is typically preferred because of its minimal impact on cardiac function and the relative ease of controlling anesthesia depth and duration . However2+ handling abnormalities are suspected because caffeine will sensitize RyR to spontaneous Ca2+ release (quantifying electrophysiology for details) is with the use of in vivo pacing   , 230. Prulation) , 231. Foulation) , 232. Baulation) . The advulation) , 229. ThTypically, transesophageal pacing is performed using a 2-Fr octapolar catheter. Because the induction of certain types of arrhythmias, such as atrial fibrillation (AF), can vary in control and AF-susceptible animals depending on age, sex, strain, and pacing protocol used , it is cTo assess susceptibility to atrial arrhythmias, cumulative pacing-mediated AF duration (or burden) or a binary AF occurrence per animal  can be quantified. Induction of AF episodes [most commonly ≥1 s –240] shoProgrammed electrical stimulation can also be performed to induce ventricular tachyarrhythmias, such as ventricular tachycardia (VT) and ventricular fibrillation (VF). Similarly, the incidence and duration of VT or VF can be quantified upon different pacing protocols . In certain genetic models, such as the catecholaminergic polymorphic ventricular tachycardia mutant mice, injections of epinephrine and caffeine may be needed to mimic β-adrenergic stimulation before pacing –245.Although in vivo electrophysiological recordings provide assessments in the most physiologically relevant conditions, highly detailed measurements of APs, conduction properties, and arrhythmia mechanisms often require excised perfused hearts and tissues. The Langendorff-perfused heart  is the mImportant variables to be considered for ex vivo studies include the type of anesthesia and euthanasia used before tissue excision, the maintenance of adequate coronary flow and pressure during perfusion, and the temperature, composition, pH, and oxygenation of the perfusate –258, as Although more commonly thought of as an approach for in vivo measurement of whole heart electrical activity, the ECG is also often acquired from the isolated heart and tissue preparations. At least two electrodes, an anode, and a cathode are placed at two distinct positions on or near the preparation. A third electrode could be placed in the bath to serve as a common ground to minimize common mode content, such as pacing artifacts. The relative potential difference between the anode and cathode is then acquired. From this, a representation of the overall electrical activity between the electrodes is generated, which is useful for monitoring the electrical features and rhythm of the tissue. This approach is useful in conjunction with localized electrophysiological measurements , to determine their relation to global electrical activity  and to m1) spring-loaded pellet electrodes that are pressed against the tissue (2) needle or hook electrodes that are inserted into the tissue; or 3) when the preparation is in a bath filled with an ionic solution, disk or needle electrodes that are positioned close to, but not in contact with, the tissue will acquire the electrical activity conducted through the solution solution , 268. Insolution , so theiVm of frog muscle fibers. The technique was later improved by Ling and Gerard . The first MAP was recorded in 1882 by Burdon-Sanderson and Page, who placed one electrode on the intact epicardial surface and the other on an injured site of the frog heart and thereby captured the phasic electrical changes of the cardiac cycle . In 1934Vm. Instead, they are useful for assessing relative changes in the AP, as long as a stable baseline recording is obtained before and after an intervention  repolarization, and artificially prolong the later (phase 3) repolarization. Elastic probes with an electrode tip diameter of 0.25 mm are recommended for mouse hearts but should be used carefully as small probes may puncture the myocardium  . Large aArrhythmogenesis) and the lifetimes of individual wavefronts and their fractionations and contact with other wavefronts can be tracked using directed graph  that cannot be accessed by optical mapping . Mappinged graph , 300. Exed graph  as well ed graph , 303, whed graph , 304.Vm and intracellular Ca2+, into an optical signal that is often recorded using a high-speed camera. Fluorescent indicators may be exogenously delivered to cells, where they localize to cell membranes or specific subcellular compartments . Cells can also be genetically encoded to produce voltage or Ca2+-sensitive indicators, which avoid some limitations of exogeneous indicators , are useful for long-term studies, and enable measurements from specific cell types  in response to membrane depolarization, resulting in a change in fluorescence. The fluorescent indicator, optical filters, lens objectives, and mirrors should be selected to maximize both the excitation light that energizes the indicator and the emitted light that carries the signal, while also minimizing the overlap of excitation and emitted light. For example, a broad-spectrum excitation light source may maximally excite an indicator, but excitation light may reflect back through the emission filters to overwhelm the optical signal(s) of interest.All fluorescent indicators absorb photons at one wavelength (excitation spectrum) and emit photons at a longer wavelength (emission spectrum). The optical signal is encoded by changes in the emission spectrum that are caused by a specific physiological phenomenon. For CaWhen simultaneously imaging more than one physiological phenomenon, indicators that have similar excitation spectra are often used together because the same excitation light will energize each indicator, eliminating the need for another light source, as when imaging APs with RH237 and CaTs with Rhod2-AM , 315. ToBecause of the three-dimensional structure of the heart, optical mapping often requires careful positioning of the camera(s) relative to the tissue to provide a relatively “flat” area to be mapped. This can be done by lightly pressing the tissue against a transparent window, carefully pinning tissue to achieve a flat imaging surface, as often done for atrial tissue , or slicOptical mapping is well-suited for studying excised Langendorff-perfused hearts as optical components can be configured to image APs and/or CaTs at high spatial resolution from any epicardial region , includiVm-sensitive indicators include RH237, di-4-ANEPPS, and di-4-ANBDQBS/Di-ANBDQPQ. These may be combined with indicators to measure other parameters, such as intracellular Ca2+ , which appears earlier in the myocardium imaged close to the pacing site and later in the distal myocardium  an important parameter to consider, which should be matched to the expected Ca2+ concentration to be recorded. Given that intracellular Ca2+ often rises from ∼200 nM in diastole to approximately >1 μM in systole (2+-sensitive dyes with a moderate affinity (300–700 Kd) are most frequently used for intracellular Ca2+ recording. Ca2+-sensitive dyes with a lower Kd can saturate before the peak of the CaT and can interfere with calcium buffering , a single wavelength signal must be scaled for every pixel and signal intensity in this case reflects relative, not absolute changes in [Ca2+]i. Dual-wavelength dyes are less affected by some of these artifacts but require a different and more involved calibration method involving the recording of signals at two emission wavelengths  (As the use of Ca point”) .Optical mapping data can be analyzed using various platforms . Often, custom-made analysis routines are developed by research groups, some of which have been published and are publicly available [e.g., Rhythm , ORCA 1, ElectroCardiac arrhythmias are a primary cause of morbidity and mortality, accounting for 10–20% of all deaths worldwide –355. PreIn the presented guidelines, we reviewed current methods and approaches that are most commonly used for measuring cardiac electrophysiology and arrhythmias in small animal models, although recent advances in the field are likely to enhance and evolve these technical approaches in the near future. For example, contactless “all optical” cardiac electrophysiology techniques use genetically encoded voltage and/or calcium indicators  in combination with optogenetic actuation to precisely control cardiac excitation via light-sensitive ion channels . All-optThis work was supported by MAESTRIA Grant 965286 and British Heart Foundation Accelerator Award AA/18/2/34218, DZHK (to L.F.); National Institutes of Health (NIH) Grants R01HL136389, R01HL163277, and R01HL147108 (to N.L); NIH Grants R01HL139472 and R01HD108839 (to N.G.P.); NIH Grants R01HL153042 and R01HL141343 (to B.P.D.); Canadian Institutes of Health Research (CIHR) Grant MOP 342562, Natural Sciences and Engineering Research Council of Canada Grant RGPIN-2022-03150, Government of Canada’s New Frontiers in Research Fund NFRFE-2021-00219, and Heart and Stroke Foundation of Canada Grant G-22-0032127 (to T.A.Q.); CIHR Grants PJT166105 and PJT180474 and Heart and Stroke Foundation of Canada Grant G-22-0032033 (to R.A.R.); NIH Grants R01HL141214, R01HL139738, and R01HL146652 (to A.V.G.); NIH Grants R01HL156652 and R01HL135096 (to T.J.H.); NIH Grants R01HL146169, R01HL147279, and R01HL144157 (to M.W.K.); NIH Grant R01HL133127 and American Heart Association Basic Project Grant 18SFRN34230125 (to K.M.); Netherlands CardioVascular Research Initiative CVON  Grants CVON2018-30 PREDICT2 and CVON2015-12 eDETECT (to C.A.R.); NIH Grant R35HL144980 (to B.C.K.); NIH Grants R01HL138003, R01HL141855, and R01HL102298 (to S.P.); NIH Grants R01HL085727, R01HL085844, and R01HL137228 and VA Merit Review Grants I01 BX000576 and I01 CX001490 (to N.C.); NIH Grants R01HL130212 and R01HL163274 and Burroughs Wellcome Fund CAMS (1009884) (to S.L.R.); and NIH Grants R01HL111600 and OT2OD026580 (to C.M.R.).L.F. has received institutional research grants and nonfinancial support from European Union, DFG, British Heart Foundation, Medical Research Council (UK), NIHR, and several biomedical companies. L.F. is listed as an inventor of two patents . None of the other authors has any conflicts of interest, financial or otherwise, to disclose.American Journal of Physiology-Heart and Circulatory Physiology and was not involved and did not have access to information regarding the peer-review process or final disposition of this article. An alternate editor oversaw the peer-review and decision-making process for this article.Crystal Ripplinger is an editor of C.M.R. and N.G.P. conceived and designed research; C.M.R., A.V.G., M.W.K., N.L., and N.G.P., prepared figures; C.M.R., A.V.G., M.W.K., B.J.B., N.C., B.P.D., L.F., T.J.H., B.C.K. N.L., K.T.M., S.P., T.A.Q., C.A.R., S.L.R., R.A.R., and N.G.P., drafted manuscript; C.M.R., A.V.G., M.W.K., B.J.B., N.C., B.P.D., L.F., T.J.H., B.C.K., N.L., K.T.M., S.P., T.A.Q., C.A.R., S.L.R., R.A.R., and N.G.P., edited and revised manuscript; C.M.R., A.V.G., M.W.K., B.J.B., N.C., B.P.D., L.F., T.J.H., B.C.K., N.L., K.T.M., S.P., T.A.Q., C.A.R., S.L.R., R.A.R., and N.G.P., approved final version of manuscript.


Enterococcus (E.) faecalis in evaluating patients with CLD, including those treated with rifaximin , and in patients with alcohol-associated liver disease (ALD).The gut-liver axis contributes to disease progression, a rise in infection rate, organ failure and a poor overall outcome in chronic liver diseases (CLD). Monitoring of the gut-liver axis is critical in understanding disease status, but biomarkers have not been elucidated. The aim of this study is to determine the level of serum antibodies against E. faecalis translocation, we developed an assay for the detection of a serum antibody against E. faecalis capsular polysaccharide (E.CPS).We enrolled 109 CLD patients (cohort 1), 30 hepatic encephalopathy patients treated with rifaximin (cohort 2), 53 inpatients with ALD undergoing alcohol cessation (cohort 3) and 33 healthy subjects. To assess the consequences of E.CPS antibody titer was elevated only in those patients with advanced CLD and ALD. The E.CPS antibody titer was an independent prognostic factor (p < 0.05), while Mac-2 binding protein and albumin-bilirubin score were not independent predictors of survival. The improvement of predictive model in integrated factors was significant [continuous net reclassification index  and integrated discrimination improvement ]. Furthermore, rifaximin treatment led to a decrease of serum E.CPS antibody titer resulting in a significantly longer overall rate of survival.Serum E.CPS antibody titer appears to be a strong predictor of survival in CLD patients. Serum E.CPS levels decrease in CLD patients receiving rifaximin, and may be associated with an overall improvement in rate of survival.The  Fecal dysbiosis, small intestinal bacterial overgrowth, intestinal epithelial barrier dysfunction and increased permeability have been described as key events in patients with advanced chronic liver diseases (CLD) . Gut dysThe most common tools used to predict the overall outcome of CLD patients include the following: assessing severity of portal hypertension, scoring systems such as the model of end-stage liver disease (MELD) and Child-Pugh score, and blood biomarkers related to complications and/or survival rate . RecentlEnterococcus (E.) faecalis is a critical contributor to the progression of alcoholic hepatitis  in patients with chronic pancreatitis and pancreatic cancer , Mac-2, as a cell-associated macrophage antigen, binding protein   during a period of alcohol cessation.In this study, we investigate the utility of monitoring serum antibody levels against  marker) , Aleuria marker)  and albuThe study protocol was approved by the ethics committee of Mie and Osaka Universities, Mie Prefectural Mental Care Center, and Omiya City Clinic. This study was performed retrospectively on stored samples, and subjects were allowed to opt out of their data being used. Written informed consent was obtained from all subjects at the time of blood sampling. A total of 109 patients were recruited by their stage of CLD for this study (cohort 1). Patients positive for hepatitis B surface antigen were diagnosed with hepatitis B virus (HBV) infection, whereas those positive for hepatitis C virus (HCV) RNA were diagnosed with HCV infection. ALD was defined as the presence of alcohol consumption >60 g/day. The diagnosis of liver cirrhosis and hepatocellular carcinoma (HCC) were based on clinical history, serologic testing, and radiologic imaging and HCC patients were grouped by the Barcelona Clinic Liver Cancer (BCLC) staging classification . PatientE.CPS antibody levels and mortality (cohort 2). A separate 53 inpatients with ALD who had been treated with alcohol cessation intervention were enrolled as cohort 3. In cohort 3, alcohol abstinence was confirmed by the percent carbohydrate deficient transferrin (%CDT) using the N Latex CDT direct immuno-nephelometric assay   . Thirty-E.CPS antibody, LBP, Mac-2 bp and AAL-Hp measurements could be performed. The presence of infection with E. faecalis was evaluated using a CPS-specific ELISA , aspartate aminotransferase (AST), albumin (ALB), glutamyl-transferase (GGT), total bilirubin (T-bil), sodium (Na), blood urea nitrogen (BUN) and creatinine (Cr) were retrospectively evaluated. Blood samples were kept at −80 degrees until ic ELISA . A lectiic ELISA . The seric ELISA  accordinU-test in two groups and one-way analysis of variance for comparison of continuous variables. The categorical data were compared using the Chi-squared test. The relationship between the serum E.CPS antibody titer and clinical data were examined using Spearman’s rank correlation coefficient. Receiver operator characteristic (ROC) curves and the corresponding area under the curve (AUC) were used to obtain cut-offs for the outcomes. The Youden index was applied to calculate the optimal cut-off point. We also conducted time-dependent ROC analysis for the prediction of CLD patient survival or death based on duration after CLD diagnosis. Overall survival (OS) was measured using the Kaplan-Meier method and compared using the log-rank test. Associations between predictor variables and OS were determined by the hazard ratio (HR) and 95% confidence interval (CI) calculated using Cox proportional hazards regression. The improvements on the basic model were assessed by the continuous net reclassification index (NRI) and integrated discrimination improvement (IDI) of which values above 0 were regarded as significant. We further conducted time-dependent concordance index (C-index) analysis for the prediction of CLD patient survival, or death, based on the duration of time post CLD diagnosis. For cohort 2 in post-rifaximin, we combined patients who had E.CPS at 3 and 6 months available to do the analysis and defined rifaximin effective patients with below 0.018 E.CPS antibody levels at 3 or 6 months or at both 3 and 6 months in the same patients. The statistical analyses were performed using the JMP software program  for univariate and multivariate logistic regression analyses. Differences were considered to be significant at p < 0.05.Continuous variables are presented as mean ± standard deviation or median (25th and 75th percentiles), and categorical variables are shown as numbers of patients. Data were analyzed using the Mann-Whitney Clinical features of the 109 CLD patients (cohort 1) are shown in E.CPS antibody, Mac-2 bp or AAL-Hp and clinical parameters in CLD patients are shown in E.CPS antibody titer was significantly correlated with AST, hepatic function (ALB and T-bil) and fibrosis markers (FIB-4) and Cr values. Mac-2 bp was significantly correlated with transaminases, hepatic function, fibrosis markers, and Cr values.The correlations between LBP, p < 0.001, p < 0.01, respectively; E.CPS antibody titer was significantly elevated in liver cirrhosis patients compared with chronic hepatitis patients (p < 0.01). Moreover, E.CPS antibody titer was gradually increased in liver cirrhosis progression . ALBI score was significantly increased in liver cirrhosis compared to healthy controls or chronic hepatitis . Serum E.CPS antibody titer and Mac-2 bp levels, as well as ALBI score, were significantly increased in the deceased group when compared with the survival group . In contrast, serum AAL-Hp levels showed no difference between the deceased and survival groups (p = 0.0588) for LBP, 0.74 (p < 0.05) for E.CPS, 0.77 (p < 0.01) for Mac-2 bp and 0.84 (p < 0.001) for the ALBI score (E.CPS at 0.1 (sensitivity 44.4% and specificity 95.9%), Mac-2 bp at 1,500 μg/mL (sensitivity 100% and specificity 44.4%) and the ALBI score at −2.55 (sensitivity 88.9% and specificity 73.7%) (E.CPS antibody titer (<0.1), low serum Mac-2 bp levels (<1500) and a high ALBI score (≥−2.55) showed significantly better OS than patients with high serum LBP levels, high serum Mac-2 bp levels, high E.CPS antibody titer and a low ALBI score, respectively  died in the average follow-up period of 1652 ± 623 days during our study period. The ultimate cause of death for all patients was considered liver-related. There is no patient received a liver transplant in this study. An increasing trend in serum LBP levels was observed in the deceased group (l groups . ROC anaBI score . From ouy 73.7%) . From Kaong-term .E.CPS antibody titer and ALBI score were evaluated by univariate analysis using a Cox proportional hazard model (E.CPS antibody titer (p < 0.0001), as well as serum LBP (p < 0.001), Mac-2 bp levels (p < 0.01) and ALBI score (p < 0.001). Using multivariate analysis, the E.CPS antibody titer and LBP levels were independent prognostic factors , while serum Mac-2 bp levels and ALBI score were not independent predictors of survival , continuous NRI , and IDI   . C-indexong-term .E.CPS antibody titer. Twelve out of 30 patients (40%) died in the average follow-up period of 977.5 ± 578.5 days during our study period. E.CPS antibody titer was significantly elevated in the deceased group  for E.CPS (E.CPS antibody titer at 0.018 (sensitivity 75.0% and specificity 77.8%) from our ROC analysis of survival curves. Patients with low E.CPS antibody titer (<0.018) showed significantly better OS than patients with high E.CPS antibody titer , at 3 or 6 months post-rifaximin treatment or at both 3 and 6 months post-rifaximin treatment in the same patients, showed significantly better OS than patients with high E.CPS antibody titer (≥0.018) at 3 or 6 months , while serum LBP, Mac-2 bp and AAL-Hp levels are elevated not only between investigatory groups (chronic hepatitis and liver cirrhosis) and healthy controls, but between the investigatory groups themselves. Notably, we verified that serum E.CPS antibody titer can be used as a prognostic factor in CLD patients independent of serum Mac-2 bp levels or ALBI score. Furthermore, we revealed that serum E.CPS antibody titer may indicate the overall condition of the liver-gut axis; rifaximin treatment-induced titer level changes altered OS in liver cirrhosis patients, while titer levels were unchanged by 1–2 months of abstinence from alcohol in ALD patients.Our study demonstrates that serum via two routes (via the lymphatic system to the systemic vasculature. The gut-associated lymphoid tissue, including Peyer’s patches and mesenteric lymph nodes, confine the bacteria thus preventing unwanted systemic exposure and subsequent immune response. Previous findings in experimental ALD mice, and in patients with ALD, showed that translocation of intestinal E. faecalis to the liver induces ethanol-induced liver inflammation and hepatocyte damage (E. faecalis increases in parallel with severity of cirrhosis and MELD score (E. faecalis and CLD progression has not been well studied clinically, and no clinical study has investigated the usefulness of serum antibody titer against E. faecalis as a predictive marker for survival in cases of CLD. To assess the consequences of E. faecalis translocation on immunity, we developed an assay for the detection of serum antibody titer against E.CPS according to previous reports (E.CPS antibody titer is elevated only in advanced CLD patients. The elevation of E.CPS antibody titer may be associated with the consequences of gut-associated lymphoid tissue inflammation or E. bacteremia. Further study is required using a larger cohort, as well as using multiple samples per subject, in order to establish reproducibility.The gut-liver axis affects the progression of CLD and its complications, which leads to enhanced systemic inflammation o routes . The fire damage . A recenLD score . However reports . We founE.CPS antibody titer greater than 0.1. In addition, serum LBP and Mac-2 bp levels, as well as ALBI score, were also prognostic factors in CLD patients, which is consistent with previous reports (E.CPS antibody titer can be used as an independent predictor of survival, suggesting that E.CPS antibody titer may be a more accurate prognostic factor.The ALBI score is a well-known predictive measure for patient prognosis in those who have CLD following liver failure . In this reports , 22. We E.CPS, to infiltrate the portal circulation, lymphoid tissue and systemic circulation. In this study, E.CPS antibody titer at baseline, or 3 and 6 months post-rifaximin treatment, were associated with increased OS in liver cirrhosis patients with hepatic encephalopathy. Rifaximin treatment provides potent activity against several species of E. bacteremia and a E.CPS antibody may be useful in evaluating and monitoring the reactivity to E. faecalis product translocation.A recent investigation has reported that rifaximin significantly decreases the occurrence of overall complications in patients with advanced stages of cirrhosis, leading to prolonged survival . RifaximE.CPS antibody titer in patients with ALD was relatively higher than the levels observed in liver cirrhosis patients. This evidence may suggest an interaction between the progression of ALD and the consequences of gut-associated lymphoid tissue inflammation or E. bacteremia.There is strong evidence that the gut-liver axis is causatively linked to the progression of ALD and systemic inflammation, both in patients and experimental animal models . Indeed,E.CPS antibody titer in patients with CLD, should be conducted using a randomized control study. In addition, a comprehensive evaluation of E.CPS antibody behavior in ALD patients is also necessary.Despite our important findings, there are limitations to the present study. This study was retrospective and was conducted within two medical centers using a small number of patients in each cohort. It will be important to examine a multicenter validation study in the future. The effectiveness of rifaximin for prolonging survival, accompanied by an improvement in serum E.CPS antibody titer appears to be a strong predictor of survival in CLD patients. Serum E.CPS antibody titer decreases in liver cirrhosis patients receiving rifaximin, and may be associated with an overall improvement in rate of survival.In conclusion, the present study illustrated that The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Mie and Osaka Universities, Mie Prefectural Mental Care Center, and Omiya City Clinic. The patients/participants provided their written informed consent to participate in this study.AE and MI contributed to study concept and design, interpretation of data and drafting of the manuscript. YT, RS, RN, HH, JK, MM, and EM contributed to the acquisition of data. EM and HN contributed to study supervision. All authors have read and agreed to the published version of the manuscript.