Fusobacterium, Parvimonas, Veillonella, Prevotella and Peptostreptococcus were enriched in GC, while commensals Bifidobacterium, Bacillus and Blautia were depleted in comparison to SG. The co-occurring correlation strengths of GC-enriched bacteria were increased along disease progression while those of GC-depleted bacteria were decreased. Eight bacterial taxa, including Veillonella, Dialister, Granulicatella, Herbaspirillum, Comamonas, Chryseobacterium, Shewanella and Helicobacter, were newly identified by this study as universal biomarkers for robustly discriminating GC from SG, with an area under the curve (AUC) of 0.85. Moreover, H. pylori-positive samples exhibited reduced microbial diversity, altered microbiota community and weaker interactions among gastric microbes. Our meta-analysis demonstrated comprehensive and generalizable gastric mucosa microbial features associated with histological stages of gastric carcinogenesis, including GC associated bacteria, diagnostic biomarkers, bacterial network alteration and H. pylori influence.The consistency of the associations between gastric mucosal microbiome and gastric cancer across studies remained unexamined. We aimed to identify universal microbial signatures in gastric carcinogenesis through a meta-analysis of gastric microbiome from multiple studies. Compositional and ecological profiles of gastric microbes across stages of gastric carcinogenesis were significantly altered. Meta-analysis revealed that opportunistic pathobionts  H. pylori infection plays a primary role in gastric cancer development [H. pylori is very prevalent, which has been estimated that at least 50% of adults harbour such infection worldwide [H. pylori does not guarantee the prevention of gastric cancer development [H. pylori prefers a healthy stomach environment and that H. pylori colonization decreases at the later stages of carcinogenesis [H. pylori, other factors also contribute to gastric tumorigenesis [Gastric cancer (GC) is the fifth most commonly diagnosed cancer, the fourth leading cause of cancer death, and responsible for 7.7% of all cancer-related deaths worldwide in 2020 . It deveelopment . Infectiorldwide . Howeverelopment , 7. Addiogenesis , 9. Thesigenesis .H. pylori infection causes a decreased secretion of stomach acid, leading to the overgrowth of non-H. pylori microbes in the gastric ecological niche. Furthermore, H. pylori can cause the formation of bacterial biofilms, making it easier for oral bacteria to colonize in the stomach [H. pylori and other microbes in GC development was reported that gastric microbiota after H. pylori eradication could restore to a similar status of negative subjects [H. pylori and the development of GC. Mice harboring a complex microbiota with H. pylori infection developed gastric cancer much faster than germ-free mice monocolonized with H. pylori [H. pylori infection [H. pylori commensals may contribute GC development, together with or independent of H. pylori infection. The potential interactions between H. pylori and gastric microbial communities, which may contribute to gastric carcinogenesis need to be further elucidated. stomach . The asssubjects . Solid e. pylori . Insulinnfection . Thus, nH. pylori on microbial communities.Several independent studies characterised the human gastric microbiota in gastric mucosa tissues from patients with GC and precancerous lesions using next-generation sequencing (NGS) of the bacteria 16 S rRNA gene –18. HoweProteobacteria, Firmicutes, Bacteroidetes, Actinobacteria and Fusobacteria, dominated the gastric microbiota in descending order of overall relative abundance . We first explored the overall microbial compositions along the progression of gastric cancer. Five phyla, nce Fig. . The relcus Fig. . SimilarH. pylori status adjusted using MaAsLin2. Among 52 bacterial genera significantly different between the two stages, 35 genera were enriched in GC compared with SG including Veillonella, Fusobacterium, Prevotella, Stenotrophomonas, Streptococcus and Lactobacillus, whereas 17 were depleted including Shewanella, Halomonas, Helicobacter, Bifidobacterium, Bacillus and Blautia  and two GC-depleted genera (Shewanella and Helicobacter) were identified as potential biomarkers using the backward stepwise selection algorithm. A logistic regression model was built based on the eight biomarkers. To evaluate the performance of the model, receiver operating characteristic (ROC) analysis was conducted, yielding an area under the curve (AUC) of 0.9109 for the training set  among GC-enriched and GC-depleted bacteria were strengthened with disease progression, such as the interactions between Blautia and Parvimonas or Peptostreptococcus.To explore the interaction among diseases-associated bacteria (GC-enriched and depleted genera) along the progression of gastric cancer, we estimated their correlations using SparCC algorithm. We found that the distributions of correlations were significantly altered between GC and all three benign disease stages  was the most depleted pathway in GC  was also significantly depleted in IM compared with SG  was significantly depleted in AG, IM and GC compared with SG  of peptidoglycan biosynthesis. Moreover, the pathways related to purine nucleotide biosynthesis, such as inosine-5’-phosphate biosynthesis and 5-aminoimidazole ribonucleotide biosynthesis, were also enriched in GC. Other GC-enriched pathways are related to carbohydrate degradation and biosynthesis, including starch degradation V, galactose degradation I (Leloir pathway), and glycogen biosynthesis I (from ADP-D-Glucose) Fig. . Interes GC Fig.  as well  GC Fig.  in compa SG Fig. . Additio SG Fig. . We furtSG Fig. S–S6. All H. pylori status on gastric cancer microbiome structure. Overall, the alpha diversity evaluated based on Shannon index revealed that the gastric microbiome in the H. pylori-negative patients had significantly higher microbial diversity  among bacteria were observed in H. pylori-negative group compared with H. pylori-positive group  in distinguishing GC from SG on a multi-cohort dataset, underlying their implications in disease progressive as well as their clinical applications. Besides, we found 9 and 10 bacterial biomarkers capable of classifying AG from SG (AUC of 0.86) and IM from SG (AUC of 0.71). The comparable AUC values obtained by different classification models (logistic regression vs SVM) indicated that the bacterial biomarkers were robust regardless of models used.Stenotrophomonas, Streptococcus, Fusobacterium, Parvimonas Peptostreptococcus and Prevotella. By contrast, for the sub-network generated by GC-depleted genera, there were some putative probiotics, such as Bifidobacterium, Bacillus, and Blautia. We observed increasing strengths of co-occurring correlations among GC-enriched bacteria implied that they could be more active in the later disease stage and therefore contribute to gastric carcinogenesis. We also observed decreasing strengths of co-occurring interactions among GC-depleted bacteria along disease progression, suggesting that they potentially play an essential role in maintaining the balanced composition of gastric microbiota. Moreover, the increase of negative correlation strengths between GC-enriched and GC-depleted bacteria suggested the possibility of reciprocally antagonistic effects between them. The alteration of network structure for the gastric microbial community with GC development was also reported by other researchers. One study found that the correlation strengths of GC-enriched and GC-depleted bacteria increased with disease progression [Helicobacter and Fusobacterium, Neisseria, Prevotella, Veillonella, Rothia were found only in patients with advanced gastric lesions, and were absent in normal/superficial gastritis group [Changes in bacterial correlations could partially explain gastric tumorigenesis and reflect disease-specific microenvironment. We found that the sub-network formed by GC-enriched bacteria included many opportunistic pathogens, including gression . Anotheris group . All theH. pylori infection, allowing the bacterium to persist and induce carcinogenic consequences in the gastric niche [We further addressed the functional features of gastric microbiota across disease stages. Compared with SG, we observed the most significant pathway enriched in GC was related to peptidoglycan biosynthesis. Peptidoglycan plays an important role in modulating host inflammatory response to ic niche . Moreoveic niche , 45, sucic niche .H. pylori on the diversity and interactions of microbial communities for all involved subjects and each disease stage. We observed a decrease in richness of microbiome in H. pylori-positive patients, which is consistent with previous reports [H. pylori status. Additionally, we observed the significant decrease of interactions among bacteria in H. pylori-positive group in all participants and each disease group. These findings were supported by the previous study [H. pylori infection and colonization. During colonization, the adhesins produced and virulence factors delivered by H. pylori may weaken the interactions among other bacteria. All these indicate H. pylori with the capacity to alter gastric microbiota community dramatically.Furthermore, we explored the effects of  reports , 47. Thius study . The dyse.g. Veillonella and Parvimonas) concomitant with the decrease in putative probiotics (e.g. Bifidobacterium) was observed along the stages of disease progressive, as revealed by corresponding ecological and functional shifts in bacterial community. We found that the co-occurring correlation strengths of GC-enriched bacteria were increased while those of GC-depleted bacteria were decreased with GC progression. In the meanwhile, co-excluding correlations among GC-enriched and GC-depleted bacteria were strengthened. The top GC-enriched pathways were related to peptidoglycan biosynthesis and purine nucleotide biosynthesis. Additionally, we showed that H. pylori could modulate gastric microbiota, leading to reduction in microbial diversity and interactions among gastric microbes. Our meta-analysis provides additional insight into the functional involvements and therapeutic targets of the gastric bacteria other than H. pylori in contributing to gastric tumorigenesis.In conclusion, we assessed the gastric microbiome using multi-cohort datasets and identified biomarkers capable of distinguishing patients across disease stages. The increase in the abundance of opportunistic pathogens  . Raw paiH. pylori status adjusted according to clinical details of included subjects. The significance criteria were adjusted p-value < 0.05, mean relative abundance >0.1% and prevalence >20%.The differentially abundant bacteria at genus level among different sample groups were determined by MaAsLin2  with study, age, gender and For each disease stage (i.e. SG), data was randomly split into training and test sets in a ratio of 8:2. The optimal biomarkers for discriminating any two disease stages (i.e. GC vs SG) on training set were iteratively selected from differentially abundant bacteria based on the Akaike Information Criteria (AIC) using backward stepwise selection algorithm from MASS package in R. Logistic regression models were built using the selected bacterial biomarkers with the function “glm” from stats package, and their performance was evaluated on the corresponding test set. In addition to logistic regression model, we evaluated the selected bacteria biomarkers in discriminating disease stages using support vector machine (SVM) model on the same training set and test set as implemented in caret R package. The receiver operating characteristic (ROC) analysis was performed to illustrate the performances of classification models using pROC R package.p-values of correlation coefficients were estimated based on 100 iterations. The correlation coefficients with adjusted p-values < 0.05 were considered significant and visualized with Cytoscape (version 3.7.2).SparCC algorithm was used to estimate the correlations between taxa from sparse compositional data. The empirical p-value < 0.05.Functional prediction was performed using PICRUSt2 . PredictU test). Kruskal-Wallis test was used to compare multiple groups. Fisher’s exact test was performed on categorical variables (gender and H. pylori status). Kolmogorov-Smirnov test was applied to compare the distributions of correlation coefficients between bacteria for different sample groups. Benjamini-Hochberg false discovery rate correction was applied to adjust p-value for multiple tests. All the related statistical analyses were performed using R software (version 4.0.4).Pairwise comparison was performed using two-sided Wilcoxon rank-sum test (Mann-Whitney Figure S1Figure S2Figure S3Figure S4Figure S5Figure S6Figure S7Figure S8Figure S9Figure S10Figure S11Figure S12Table S1Table S2Table S3Table S4Table S5Table S6


C. elegans is one of the pioneering animals in which numerous CRISPR-Cas strategies have been rapidly established over the past decade. Ironically, the emergence of numerous methods makes the choice of the correct method difficult. Choosing an appropriate selection or screening approach is the first step in planning a genome modification. This report summarizes the key features and applications of CRISPR-Cas methods using C. elegans, illustrating key strategies. Our overview of significant advances in CRISPR-Cas will help readers understand the current advances in genome editing and navigate various methods of CRISPR-Cas genome editing.CRISPR-Cas allows us to introduce desired genome editing, including mutations, epitopes, and deletions, with unprecedented efficiency. The development of CRISPR-Cas has progressed to such an extent that it is now applicable in various fields, with the help of model organisms.  C. elegans, CRISPR-Cas9 technology was first adopted in 2013  is the current choice for genome editing. It is an RNA-guided system where a crRNA and a trRNA, together called a guide RNA (sgRNA), direct a Cas9 nuclease to the target gene of interest  pathway [unc-119 and dpy-13, which enabled screening of the transgenic mutants by screening uncoordinated (unc) or dumpy (dpy) phenotypes. Additionally, it demonstrated that a gene  exhibiting no distinct phenotypes can also be identified by PCR screening methods. Single guide RNA (sgRNA), which combines crRNA and trRNA, can cleave target sites, thus eliminating the use of crRNA and trRNA separately, as demonstrated in S. pyogenes [Since the first publication of the effects of the CRISPR-Cas9 nuclease in vitro in 2012 , paralle pathway . This stpyogenes .C. elegans was neither an efficient nor a major recombination pathway for double-stranded breaks [C. elegans genome via homologous recombination [Previously, homologous recombination in d breaks ,23. Howebination ,8,9. Henben-1 locus, endowing both hygromycin and benomyl resistance [Dpy-3 was also proposed as a new CRISPR-Cas tool, since its homozygous and hemizygous phenotypes are distinct [Whereas CRISPR induces DNA double-strand break (DSB) at the target genome, and the cell’s DSB repair system completes genome editing, the screening of the corrected genome still remains for scientists to complete. PCR has been the breakthrough technique for screening targeted mutations, and it is still helpful for most small-scale screenings . Howeversistance . Dpy-3 wdistinct .C. elegans genome editing entered a new phase by adopting a second endogenous marker. Kim et al. introduced the Co-CRISPR strategy to detect genome editing. In addition to the gene of interest, unc-22 was targeted and selected by identifying twitching worms. The unc-22 indicates active Cas9 expression and a twitcher-based indel frequency via NHEJ mutations  recognizes NGA PAM sequences instead of canonical NGG [Since the ical NGG . VQR recical NGG , thus brical NGG . Thus, tSince guide RNA must designate the unique site of a gene of interest, multiple factors must be considered to achieve a high efficacy and specificity regarding genome editing. Studies have investigated strategies for designing guide RNA to enhance the genome editing frequency. For example, Farboud et al. reported that a GG motif at the 3′ end of the target sequences induced a high frequency of mutagenesis via NHEJ and HR pathways . The medC. elegans as a model to understand the universal rules underlying genome editing [Later, the same group explored the location of DSB repair events in CRISPR-Cas editing. Interestingly, NHEJ induces asymmetric insertions and deletions (indels) preferentially in regions of 5′ of PAM . Notably editing ,29.C. elegans genome (genome.sfu.ca/gexplore (accessed on 4 December 2022), www.crisprscan.org (accessed on 4 December 2022), crispr.dbcls.jp (accessed on 4 December 2022), crispor.tefor.net (accessed on 4 December 2022) [Since variants of Cas nucleases have expanded the range of genome targets, identifying a proper target may now involve additional effort. Currently, a web-based database of guide RNA can help to identify guide RNAs for target genes and minimize off-target sites. Several database services can identify potential guide RNA for the entire er 2022) ,31,32,33While Cas9 nuclease induces a single DSB at a target site, dual or triple DSBs offer advantages for genome editing. For example, deletion mutants can be achieved by adopting two sgRNAs via NHEJ or HR pathways  [Zhang et al. reported that dual cutting of the repair template, rather than genomic DNA, enhanced precise genome editing efficacy . The duactively) . It is wSince linear DNA is prone to degradation by nucleases present in the cells, the conventional way of delivering a repair template is to supply double-stranded DNA as a part of circular DNA. Therefore, plasmid DNA is desirable, especially for large-sized repair templates. However, a growing number of studies have found that single-stranded oligonucleotides can often be good alternatives for HR-mediated genome editing. Specifically, oligonucleotides offer a few advantages over plasmid DNA: they are cloning-free and can be rapidly synthesized via commercially available resources. Therefore, adopting linear DNA reduces the amount of time required for the entire genome editing process.The first demonstration of oligonucleotides as repair templates was performed in 2014, substantiating their simplicity and efficacy . An oligC. elegans for the first time. Although the modified oligonucleotide resulted in the desired genome editing, it was unclear whether its efficacy was enhanced compared that resulting from to non-modification [Previous research investigated whether modification of the donor template improves HR efficacy. Although controversial at first, a growing number of studies have reported that the modification of repair donors enhances genome editing proficiency . In 2014fication . In mammfication . In contfication .C. elegans study demonstrated that 5′ modifications of the donor improved the efficacy of HR frequency roughly twofold [C. elegans, whereas RNA::TEG was superior to TEG in human cells and zebrafish. They demonstrated that these 5′ modifications suppress donor ligation reactions in a similar way to that of biotin and SpC3 5′ modifications [With this controversy, recent studies have explored donors with 5′-end modifications that enhance genome editing efficacy. Gutierrez-Triana et al. showed that adding biotin at the 5′ ends of dsDNA leads to an increase in HR efficiency of up to 60% in the injected generation of medaka fish embryos. Provocatively, the authors demonstrated that biotin and SpC3 5′ modifications prevent donor multimerization/NHEJ of dsDNA, thereby providing optimal conditions for HR-mediated CRISPR-Cas genome editing . Similar twofold . Interesications .C. elegans studies from the Mello Lab reported additional alterations that enhance HR efficiency dramatically by modifying repair template DNA. First, a single-stranded overhang containing dsDNA donors yielded higher integration rates at three loci [It is worth noting that ree loci . Second,ree loci .C. elegans studies. Chiu et al. performed high-throughput sequencing after CRISPR-Cas genome editing and found no distinct evidence of off-target genomic lesions using GATK pipeline or split-read analysis [Given that genome editing relies on the creation of DSB, potential off-target effects have been one of the major concerns. The off-target effects of CRISPR-Cas9 have been addressed a few times in analysis . Second,analysis . These sC. elegans genome editing protocols. Please see the list of major C. elegans CRISP-Cas publications for more information , rapid life cycle (~3 days), self-fertilization, and easy maintenance [C. elegans were successively adopted in other nematode species. The strategies work in C. remanei, C. briggsae, and the parasitic nematodes Strongyloides spp. and A. freiburgensis [C. briggsae can utilize C. elegans promoters to express sgRNAs and Cas9 and, similar to C. elegans, can use the 3′ GG motif to increase genome editing efficacy [C. elegans to other nematodes would provide a powerful tool to investigate the functions of orthologs, thereby leading to a better understanding of the molecular mechanisms. In contrast, there are intrinsic hurdles in C. elegans genome editing. Although some studies developed a quantity drug screening in C. elegans, a high-throughput CRISPR screening approach for identifying genes or pathways is not feasible. This is mainly because genome editing relies on a slow, multi-step microinjection process. Given that a recent study demonstrated the successful delivery of sgRNA [ntenance ,54. Straurgensis ,58,59,60efficacy . Thus, aof sgRNA , feedingStudies over the last decade have demonstrated that improvements in genome editing can unlock the enormous potential that CRISPR offers. However, CRISPR-Cas is still far from perfect. Consequently, we are expecting various aspects of progress. For example, while conventional strategies rely on type II Cas9, the remaining five CRISPR systems have not been well investigated. Studies on the remaining types expand the targets of CRISPR-Cas application. For example, Cas12 of type V induced staggered breaks in dsDNA, unlike blunt breaks created by Cas9 . Cas13 oC. elegans—60–80% of C. elegans genes are conserved between the two groups of organisms [C. elegans may also be applicable to mammalian systems.Advances in new screening methods, secondary endogenous markers, dual DSBs, modified repair templates, computational tools, and Cas variants have extended the efficiency and specificity of genome editing. This further strengthens overall genome editing strategies and incentivizes their use in science, medicine, pharmaceuticals, and industry. Furthermore, given the high level of conservation between mammals and rganisms ,67—the m


Medicago polymorpha L. (bur clover), an invasive plant species of the genus Medicago, has been traditionally used in China as an edible vegetable crop because of its high nutritive value. However, few molecular markers for M. polymorpha have been identified. Using the recently published high-quality reference genome of M. polymorpha, we performed a specific-locus amplified fragment sequencing (SLAF-seq) analysis of 10 M. polymorpha accessions to identify molecular markers and explore genetic diversity. A total of 52,237 high-quality single nucleotide polymorphisms (SNPs) were developed. These SNPs were mostly distributed on pseudochromosome 3, least distributed on pseudochromosome 7, and relatively evenly distributed on five other pseudochromosomes of M. polymorpha. Phenotypic analysis showed that there was a great difference in phenotypic traits among different M. polymorpha accessions. Moreover, clustering all M. polymorpha accessions based on their phenotypic traits revealed three groups. Both phylogenetic analysis and principal component analysis (PCA) of all M. polymorpha accessions based on SNP markers consistently indicated that all M. polymorpha accessions could be divided into three distinct groups . Subsequent genetic diversity analysis for the 10 M. polymorpha accessions validated the effectiveness of the M. polymorpha germplasm molecular markers in China. Additionally, SSR mining analysis was also performed to identify polymorphic SSR motifs, which could provide valuable candidate markers for the further breeding of M. polymorpha. Since M. polymorpha genetics have not been actively studied, the molecular markers generated from our research will be useful for further research on M. polymorpha resource utilization and marker-assisted breeding. Medicago consists of approximately 87 species including the most important forage crop species M. sativa and the legume model species M. truncatula  markers. Additionally, an analysis using five simple sequence repeat (SSR) markers indicated a low diversity level in eight accessions in China  of 10 M. polymorpha accessions from different regions in China was conducted in this study. It should be noted that there are very few available genetic resources for the species, so the development of a massive set of SNPs is of interest. The accessions used in this study were geographically representative and mainly collected from the main producing areas of M. polymorpha. Moreover, their phenotypic traits were notably different. These characteristics were helpful for the identification of genetic variations.To develop large-scale markers for breeding and understand the genetic diversity of M. polymorpha mainly distributes in five major cultivation areas  with very limited distribution elsewhere. For SLAF-seq analysis, a total of 10 representative M. polymorpha individual accessions with distinct phenotypic characteristics were collected from these five different geographical regions. Among them, six accessions came from Jiangsu province , and the other four accessions from the other four areas . For plant growth form, MP-3 and MP-4 grown in lawn were prostrate, MP-13 grown in farmland was semi erect, and the other seven M. polymorpha accessions grown in farmland were erect  for ball planting. After the seeds of each accession were harvested in May, field experiments for phenotypic trait measurement were conducted in a randomized complete block design with three biological replications in October at Yangzhou University. There were two rows for each accession. The seeds of each genotype in each replication were sown in drill with a spacing of 0.5 m in rows and 1 m between two accessions. No fertilizer was used, but timely drainage between the plot ditch, artificial weeding, and timely irrigation were utilized.n), stem diameter (cm) and stem-leaf ratio) were evaluated on 10 plants per replication. Branch number is the number of branches at the base (including the main stem), and plant height is the absolute height from the base of the main stem to the growing point of the main stem. Stem diameter refers to the diameter of the base of the main stem, measured with a vernier caliper. Analysis of agronomical data was carried out using analysis of variance (ANOVA) and the general linear model (GLM) of SAS (V.9.4). Cluster analysis was conducted using the Squared Euclidean Distance method with SPSS software (V.19.0).At the beginning of each accession’s flowering time in April of the following year, six different agronomic traits (leaf length (cm), leaf width (cm), plant height (cm), branch number  using prediction software independently developed by Biomarker Technologies Corporation . Based on the result of the preliminary experiment, RsaI and HaeIII restriction enzymes were selected as the best combination. To further evaluate the accuracy of the ‘RsaI + HaeIII’ combination enzyme digestion experiment, Arabidopsis thaliana ecotype Columbia was used as the control for sequencing. Genomic DNA was then digested with restriction enzyme RsaI , ligated to RsaI adapter, and then digested by additional enzyme HaeIII (NEB). Polymerase chain reactions (PCR) were carried out using the diluted restriction-ligation samples, dNTP, Taq DNA polymerase (NEB), and Rsa I-primer containing a barcode. According to the position of Arabidopsis reads in the genome and the length distribution of inserted fragments in Arabidopsis thaliana ecotype Columbia, DNA fragments of 314–414 bp for M. polymorpha were selected using the Blue Pippin automated DNA size selection system. We classified these as SLAF tags and then sequenced them using the Illumina HiSeq 2500 system  with 125 bp pair-end. The sequencing was performed by Biomarker Technologies Corporation .SLAF-seq was performed with slight modifications as described previously . First, M. polymorpha genotypes. SLAFs with two to four tags were considered polymorphic SLAFs and potential markers. Polymorphic SLAF tags showed sequence polymorphisms between different accessions. Before mapping, low-quality reads with quality scores less than 30 were removed, and high-quality reads with terminal 5-bp and barcode sequences were discarded. All high-quality SLAF paired-end sample reads of the 10 accessions were then aligned to the newly assembled reference genomes of M. polymorpha ‘Huaiyang Jinhuacai’ (https://ngdc.cncb.ac.cn/gwh/Assembly/17540/show) using the Burrows-Wheeler alignment tool (BWA) software (n = 10) were joined into a VCF file with the criteria of integrity >90%, minor allele frequency (MAF) >0.05, and max missing 0.5. The filtered high-quality SNPs were further used to reconstruct the phylogenetic tree with the maximum likelihood estimation method in MEGA-X (https://CRAN.R-project.org/package=scatterplot3d).SLAF marker identification and genotyping were performed as described by software  with defsoftware , and then MEGA-X . The prin MEGA-X . A threen MEGA-X  , observed heterozygosity (Ho), expected heterozygosity (He), Nei diversity index (Nei), Shannon’s information index (I), and polymorphic information content (PIC), were calculated through Perl programming.M. polymorpha accessions. As shown in M. polymorpha materials in this study varied from 1.38 to 2.40 cm, with a variation amplitude of 1.02 cm, and the leaf width varied from 1.45 to 2.23 cm, with an amplitude of 0.78 cm. Among all accessions, the leaf length and width of MP-6 from Wenling, Zhejiang province were the largest, and this material is also known as “Wenling big leaf” in the local area. We found that the germplasm characteristics of the MP-6 large leaves were not changed due to alloplanting. The leaf length and width of MP-4 from Danyang, Jiangsu province were the smallest, followed by MP-3 from Anhui, which retained more wild characteristics. The plant height of M. polymorpha accessions showed obvious differentiation. For example, the plant heights of MP-3 and MP-4 were only about 4 cm, and the lateral branches crept close to the ground because the main stem growth was significantly inhibited. However, other materials grew upright, ranging in height from 20 to 25 cm, except for MP-13. Moreover, the lateral branches of MP-3 and MP-4 were very developed and totaled up to 4, which was significantly more than that of the other materials. Stem diameter ranged from 1.71 to 2.08 cm, with an average of 1.89 cm. MP-9 from Chuxiong, Yunnan province had a stem-to-leaf ratio, of 1.33, indicating that it had the most leaves, while MP-4 had the fewest leaves. The variation coefficient showed that the plant height of the M. polymorpha accessions changed the most (43.15%), followed by branch number (33.42%), and stem-leaf ratio and stem diameter changed the least . Additionally, as the correlation analysis in M. polymorpha accessions with thicker stem had greater branch numbers.There were significant differences in the phenotypic traits across the different M. polymorpha accessions based on their phenotypic traits . The selected size for this method was 314–414 bp using the Blue Pippin system. We predicted that there were 117,528 SLAF tags distributed in the genome with 6.19% located in repetitive regions. To further evaluate the efficiency of the restriction enzyme digestion scheme, Arabidopsis thaliana was selected as an example. Through the detection of the remaining restriction enzyme restriction sites in Arabidopsis read inserts, we found that the ratio of Arabidopsis paired-ended reads mapped to their reference genome was 83.41%, and the enzyme digestion efficiency was 97.79%. This indicates that the double-enzyme digestion scheme should be effective for the SLAF library construction of M. polymorpha.Since the genome of M. polymorpha accessions generated 6.26 Gb clean reads, with a Q30 percentage of 84.97% and GC content of 38.29%  >0.05 were identified in this study , least on pseudochromosome 7 (7%), and nearly evenly distributed on the other pseudochromosomes of M. polymorpha, a variety of molecular markers have been developed, including isozyme, random amplified polymorphic DNA (RAPD), and SSR  and the least on pseudochromosome 7 (7.0%). Cluster analysis of all M. polymorpha accessions based on SNP markers  and phenotypic traits both suggested that they could be divided into three groups. Subsequent genetic diversity analysis for these 10 accessions also validated the effectiveness of the molecular markers of M. polymorpha germplasm in China. Additionally, we performed SSR mining analysis to identify polymorphic SSR motifs, which helped to provide valuable candidate markers for the subsequent breeding of M. polymorpha. Our study will provide important information about the molecular markers of M. polymorpha and will be useful for further research on M. polymorpha resource utilization and marker-assisted breeding.In this study, we applied SLAF sequencing technologies to develop molecular markers and explore the genetic diversity of 10.7717/peerj.14698/supp-1Supplemental Information 1Click here for additional data file.


Bacillus licheniformis has been shown to be safe as a green additive in food and feed. This experiment was conducted to investigate the value of Bacillus licheniformis in the diet of piglets. Our results suggested that dietary Bacillus licheniformis supplementation plays an important role in improving the average daily gain, alleviating diarrhea, improving antioxidant capacity, promoting immune function, and regulating the intestinal microflora of weaned piglets.Bacillus licheniformis (B. licheniformis) is a safe probiotic that can promote animal growth and inhibit pathogenic bacteria. This study aimed to assess the effects of B. licheniformis, one green feed additive, on growth performance, diarrhea incidence, immune function, fecal volatile fatty acids, and microflora structure in weaned piglets. Weaned piglets (n = 180) were randomly divided into three treatment groups and fed a basal diet and a basal diet supplemented with 500 mg B. licheniformis per kg and 1000 mg B. licheniformis per kg, respectively. The dietary 500 mg/kg B. licheniformis inclusion improved the average daily gain, reduced diarrhea incidence, and strengthened antioxidant capacity. Piglets supplemented with B. licheniformis presented increased serum immunoglobulins  compared to the CON group. Meanwhile, the expression of anti-inflammation factors was increased, and the levels of pro-inflammation factors were reduced after B. licheniformis administration. Moreover, the levels of volatile fatty acids, including acetic acid, propionic acid, butyric acid, isobutyric acid, and isovaleric acid, in the BL500 and BL1000 groups were increased compared with the CON group, and the concentration of valeric acid was higher in the BL500 group. Furthermore, piglets in the 500 mg/kg B. licheniformis addition group significantly altered fecal microbiota by increasing Clostridium_sensu_stricto_1 and Oscillospira. In conclusion, dietary B. licheniformis relieved diarrhea, enhanced antioxidant capacity, immunity function, and fecal microflora structure in weaned pigs. Early weaning is a stress, and pigs commonly experience diarrhea and a reduced growth rate during postweaning . AntibioB. licheniformis is a spore-forming Gram-positive bacterium that has been used in animal feed for a long time [B. licheniformis can produce bacitracin against pathogenic microorganisms [B. licheniformis has the function of promoting growth and acting as a competitive repellent, stimulating appetite, promoting digestion, and increasing nutrient retention and absorption [B. licheniformis repaired the imbalance of intestinal flora in deoxynivalenol-challenged mice [B. licheniformis strain CK1 had probiotic characters, including acid resistance, adhesion ability, and resistance to pathogenicity [B. licheniformis could increase digestibility and fecal Lactobacillus counts and reduce fecal toxic gas emissions in growing-finishing pigs [B. licheniformis-fermented feed additive, containing 5 × 1011 CFU/g B. licheniformis, to the basal diet in a weaned piglet experiment. Xu et al. [B. licheniformis in broiler chickens, where the B. licheniformis group was fed a basal diet supplemented with 1.5 × 109 CFU/kg B. licheniformis. Based on previous studies, we set the dose of B. licheniformis for weaned piglets.Several studies have demonstrated that probiotics are beneficial for improving growth performance and immune function in animals . Among tong time . B. lichrganisms . In vivosorption . Researcged mice . B. lichgenicity . Suppleming pigs . Lin et ing pigs  added 0.u et al.  exploredB. licheniformis was a promoter for weaned pig growth and health. The current study aimed to prove the hypothesis that the application of B. licheniformis in piglets’ diet could alleviate the diarrhea incidence, promote growth performance, antioxidant capacity, immune function, and alter the fecal microbial composition in weaned piglets. The combined analysis of these results may help to find specific microorganisms, as well as provide insights into the potential microbial mechanisms of piglets supplemented with B. licheniformis.However, few studies have evaluated whether B. licheniformis per kg of basal diet), and BL1000 . The corresponding amounts of B. licheniformis were evenly mixed into the basic diet for feeding. The experiment lasted for 28 days. The Bacillus licheniformis product was used at a concentration of 1 × 109 CFU per gram of B. licheniformis. The strain has been saved in the China General Microbiological Culture Collection Center (CGMCC) and its collection number is HJ0135. The basal diet for piglets was designed to conform to the nutritional necessities of NRC (2002) without antibiotics . A total of 180 four-week-old weaned Duroc × Landrace × Yorkshire piglets were raised in Zhengxing Animal Husbandry Co., Ltd. , and the piglets were randomly divided into three treatment groups  as follows: CON , BL500 . Blood samples were collected and centrifuged . Feed intake was recorded each day in each pen. The average daily feed intake (ADFI) and feed to gain ratio (F:G) were computed using the recorded data. The number of pigs with diarrhea was observed and recorded daily to evaluate the incidence of diarrhea. The diarrhea rate (%) = total number of diarrhea piglets/ × 100.The serum antioxidant indexes, including glutathione peroxidase (GSH-Px), total antioxidant capacity (T-AOC), superoxide dismutase (SOD), and malondialdehyde (MDA), were determined in accordance with the manufacturer’s instructions using relevant diagnostic kits purchased from the Angle Gene Bioengineering Co., Ltd. . The Lot number of SOD was 202204011C, the Lot number of GSH-Px was 202204033C, the Lot number of MDA was 202204052D, and the Lot number of T-AOC was 202204024B.Concentrations of immunoglobulin  and inflammatory cytokines (interleukin (IL)-6, IL-1β, IL-10, and tumor necrosis factor (TNF)-α) were determined using enzyme-linked immunosorbent assay (ELISA) kits. The kits above were bought from Angle Gene Biotechnology Co. Ltd. . The specific test steps followed the manufacturer’s instructions. The Lot number of IgG was 202111062B, the Lot number of IgG was 202111051C, the Lot number of IgM was 202111032C, the Lot number of IL-1β was 202111063A, the Lot number of IL-10 was 202111041B, the Lot number of TNF-α was 202111063D, and the Lot number of IL-6 was 202111044A.g for 10 min at 4 °C) to gain the supernatant. Then, the supernatant was mixed with 25% metaphosphoric acid (w/v) at a ratio of 5:1, and then centrifuged  to obtain the supernatant. After centrifugation, approximately 500 μL of the supernatant was filtered into sample bottles for further gas chromatography analysis Fecal VFAs were assayed by a Gas Chromatography System  according to previous studies . In brieThe fecal samples from different piglets were used for the microbiome analysis. The genomic DNA of fecal microbial was extracted using the DNeasy Power Soil Kit  following the manufacturer’s instructions. The specific primer sequences 515F (5′-GTGCCAGCMGCCGCGGTAA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) were used for the analysis of V3-V4 region of 16S rRNA gene. The sequencing was conducted on the Illumina Miseq platform , and the data were analyzed on the Majorbio Cloud Platform, an online platform provided by Majorbio Bio-Pharm Technology Co., Ltd. . The amplicon sequence variant (ASV) is a set composed of concerned species. These concerned species can be comprised of unique or common species information in different groups obtained through a Venn diagram. ASV can be used for composition analysis and correlation analysis of specific species. Alpha diversity analyses were used to describe the degree of fecal microflora diversity between each group. p < 0.1 was considered a trend and p < 0.05 was considered a significant difference.All data were checked by the equality of variance with normality distribution, then analyzed with SPSS 25.0 software  using a one-way ANOVA to analyze whether the species diversity was significant between three independent experiments with at least six replicates per independent experiments. The histograms were created using GraphPad Prism 8 software . 0.05 < B. licheniformis on growth performance in weaned piglets are indicated in B. licheniformis supplementation significantly enhanced the ADG in piglets throughout the entire experimental period (p < 0.05). Dietary supplementation with 500 and 1000 mg/kg of B. licheniformis could significantly reduce the diarrhea rate of piglets (p < 0.05).The effects of B. licheniformis on the serum antioxidant parameters in weaned piglets are shown in B. licheniformis significantly increased the serum GSH-Px activity and decreased the MDA level of serum in weaned piglets (p < 0.05). Moreover, the supplementation of 500 mg/kg of B. licheniformis significantly improved the serum levels of T-AOC and SOD (p < 0.05). In addition, dietary 500 mg/kg B. licheniformis markedly improved the serum GSH-Px activity and reduced the MDA level of serum in weaned piglets compared to the BL1000 group (p < 0.05).The effects of B. licheniformis-treated piglets exhibited significantly higher serum IgA and IgM concentrations and lower serum IL-1β and IL-6 concentrations (p < 0.05). The level of serum anti-inflammatory factor IL-10 in the BL500 and BL1000 groups was higher than that in the CON groups (p < 0.05).As demonstrated in p < 0.05). Moreover, the levels of acetic acid, propionic acid, isobutyric acid, butyric acid, and isovaleric acid markedly increased in the BL1000 group compared to the CON group (p < 0.05). Compared with the BL1000 group, the contents of acetic acid, isobutyric acid, and isovaleric acid were significantly higher in the BL500 group (p < 0.05).The results of the VFA analysis are shown in B. licheniformis gave lower Chao index and higher Simpson index readings for fecal microbiota in piglets and 1000 mg/kg B. licheniformis addition showed a lower Chao index . What is more, heatmaps of Spearman’s correlation analysis between fecal microbiota and volatile fatty acids are shown in Oscillospira had an observable positive association with acetic acid, propionic acid, isobutyric acid, and isovaleric acid; meanwhile, Clostridium_sensu_stricto_1 was positively correlated with valeric acid (p < 0.05). However, Megasphaera and Selenomonas had significant negative relations with acetic acid, propionic acid, isobutyric acid, isovaleric acid, and valeric acid (p < 0.05).The results of Spearman’s correlation analysis between fecal microbiota and serum immune cytokines are shown in Bacillus strains could improve infectious enteritis or diarrhea in weaned piglets [B. licheniformis significantly reduced the incidence of diarrhea. According to Lin et al. [B. licheniformis-fermented feed additive not only reduced the incidence of diarrhea, but also tended to increase body weight and the average daily gain of weaned piglets. In this study, supplementation of the diet with 500 mg/kg of B. licheniformis markedly enhanced the ADG compared with the control group, while a diet with B. licheniformis could increase ADFI and reduce F:G but the effects were not significant. What is more, there was no significant difference in growth performance between the BL500 group and the BL1000 group. The improved growth performance of piglets fed B. licheniformis may be related to beneficial metabolites produced by B. licheniformis, such as extracellular digestive enzymes and lysozymes [B. licheniformis could improve the immunity of piglets [B. licheniformis reduced the pathogens in the gut through competitive rejection, while increasing the uptake of nutrients in the intestine, as it was beneficial to improving the growth performance of animals [B. licheniformis was better for modulating the gut health of piglets, but the mechanism needs to be further explored. Piglets fed a diet with B. licheniformis showed reduced damage to the villus height and increased the villus height: crypt depth in the duodenum, jejunum, and ileum [B. licheniformis had positive effects on controlling Salmonella Infantis infection via alleviating inflammation and maintaining the intestinal mucosal barrier integrity in pigs.Pathogen invasion and environmental stress affected the incidence of diarrhea by changing the composition of intestinal flora . Weaning piglets . Similarn et al. , a B. liysozymes . It is a piglets . B. lich animals . The ADGnd ileum . Liu et nd ileum  found thB. licheniformis and Saccharomyces cerevisiae (S. cerevisiae) increased the activities of SOD and GSH-Px. In this study, compared with the CON group, B. licheniformis improved the antioxidant capacities in the serum of piglets by increasing T-AOC, GSH-Px, and SOD activities and by decreasing MDA. Similarly, B. licheniformis enhanced muscle antioxidant capacity and improved meat quality in late-finishing pigs [Researchers hypothesize that oxidative stress affects growth performance in animals . The anting pigs . B. licheniformis could elevate IgA, IgM, and IL-10 levels and reduce the contents of IL-6 and IL-1β compared with control piglets. The results indicated that the lower dose of B. licheniformis could be more beneficial to enhance immune function, possibly because it could better regulate the balance of intestinal flora in piglets, while the exact mechanism needs to be further studied. Kwak et al. [norank_f__Prevotellaceae and Coprococcus had a significant positive association with IL-6, while Selenomonas and Megasphaera had a significant negative relationship with the anti-inflammatory factor IL-10. However, Huang et al. [Coprococcus was negatively correlated with IL-17A, suggesting that it may be involved in pemphigus vulgaris by regulating T cell differentiation and the related cytokines. Indeed, previous data showed that the Megasphaera sp. XA511 promoted the secretion of pro-inflammatory cytokines of peripheral blood mononuclear cells, suggesting that the Megasphaera might enhance the immune response [Epsilonproteobacteria, Erysipelotrichales, Prevotellaceae, Flavobacteriaceae, and other pathogenic bacteria [Serum immunoglobulins and inflammatory cytokines are typical parameters for estimating the immune status of livestock and poultry . Excess k et al.  found thg et al.  showed Cresponse . The actbacteria .B. licheniformis. Adding probiotics to the diet resulted in a higher proportion of VFAs, mainly acetic acid, which may be beneficial to the intestinal environment and may reduce post-weaning diarrhea in piglets [B. licheniformis supplementation induced an incremental increase in fecal VFAs. This result is consistent with the previous research where higher VFAs concentrations were reported because of B. licheniformis supplementation [Oscillospira was positively associated with acetic acid, propionic acid, isobutyric acid, and isovaleric acid, and Clostridium sensu stricto 1 was positively related to valeric acid. Yang et al. [Oscillospira could produce various VFAs, mainly butyric acid. Moreover, Clostridium_sensu_stricto_1 and Clostridium_sensu_stricto_4 were responsible for the change in butyric and propionic acid contents in the colon of anemic animals [The intestinal VFAs play an active role in nutrient metabolism and immune function, while also regulating the intestinal microbiota at the same time , obtaini piglets . These fentation . Spearmag et al.  found th animals . Clostridium_sensu_stricto_1 in the BL500 group compared with the control group. Clostridium sensu stricto has been associated with necrotizing enterocolitis, growth performance, and inflammation in pig models [Clostridium_sensu_stricto_1 and Clostridium_sensu_stricto_2 were two major groups in the Clostridium genus. Compared with healthy piglets, the content of Clostridium_sensu_stricto_2 was higher in piglets with diarrhea, whereas Clostridium_sensu_stricto_1 exhibited contrary results, indicating B. licheniformis could reduce the diarrhea incidence by increasing the level of Clostridium_sensu_stricto_1 [Clostridium_sensu_stricto_1 was reported to produce VFAs using mucus-derived sugars as an energy source and promote intestinal mucus barrier adhesion to pathogens [Clostridium_sensu_stricto_1 significantly increased in the resveratrol group. Through correlation analysis, Zhou et al. [Clostridium_sensu_stricto_1 in cecum contents were negatively correlated with the level of inflammatory factors IL-12, IL-6, and IFN-γ. Various research had interpreted a close link between fecal microbes, VFAs, and immune function ,45. Richg models . Clostritricto_1 . In addiathogens  found thu et al.  showed tB. licheniformis in the diet of weaning piglets could increase the average daily gain, relieve diarrhea, enhance antioxidant capacity, improve immune function, and regulate the intestinal flora in weaned piglets. B. licheniformis may increase the genera Oscillospira and Clostridium_sensu_stricto_1 to promote growth performance. These findings provide novel insight into B. licheniformis enhancing beneficial effects in weaned piglets.In summary, the supplementation of 500 mg/kg


Abies ernestii Rehder is endemic to the montane regions of Southwest China. Till now, phylogenetic relationships between A. ernestii and other closely related species remain unclear. In this study, we first characterized the complete chloroplast (cp) genome of A. ernestii. The whole cp genome was 121,841 bp in size, including one hundred and thirteen genes. Results of comparative cp genome revealed that only ycf1 and ycf2 was characterized by a considerable variation. Our phylogenetic analyses supported the monophyly of the genus Abies and revealed a clear separation between A. ernestii and A. chensiensis Tiegh. This study highlights the significance of using cp genomes to examine species boundaries among closely related fir species. Abies ernestii Rehder is distributed across Southwest China and characterized by high economic and ecological importance , in addition to its distribution in the north and west of the Sichuan province, extending into extreme north-western Yunnan province . The voucher specimen was deposited in the herbarium of the Institute of Botany, CAS (PE)  under the voucher number 5874. The leaf materials were first desiccated in silica gel, and the Ezup plant genomic DNA prep kit was used to extract the total DNA. The relevant cp genome sequence was submitted to the NCBI database (No. MH706707). The associated numbers were PRJNA790664 (BioProject), SRP351570 (SRA), and SAMN24219951 (Bio-Sample), respectively.The specimens and leaf materials of https://chlorobox.mpimp-golm.mpg.de/geseq.html), and tRNAscan-SE v1.3.1 .The extracted DNA was sequenced on the Illumina HiSeq X platform with libraries in 350 bp length. The clean reads were approximately 10.2 million in 150 bp length. We aligned, assembled, and annotated the reads using CLC de novo assembler , GeSeq  , and Abies fargesii Franch. (MH706716)  in the cp genome of Table S1). Juniperus squamata Buchanan–Hamilton ex D. Don in the family Cupressaceae was used as an outgroup.Phylogenetic analysis was performed using the complete cp genomes. We aligned the sequences containing the hotspot mutation regions and chose the maximum-likelihood (ML) analysis as the method in this study. The analysis was performed using a rapid bootstrap analysis and 1000 rapid bootstrap search steps in the RAxML v.7.8  region and short single-copy (SSC) region were 67,157 and 54,156 bp, respectively. It contained 113 genes, including peptide-encoding genes (68), tRNA genes (16), open reading frames (6), and rRNA genes (4), respectively (Table S2). In the LSC region, there were 53 protein-coding genes, 16 tRNA genes, and 3 open reading frames. The SSC region owned all the 4 rRNA. The IR regions were 264 bp and featured trnI-CAU and trnT-GGU. Among the encoded genes, ycf3 and rpl2 had two introns, and 11 genes  had one intron  Silba . Compared with other SSRs, the mononucleotide repeats (62.68%) were the most abundant and they contributed more to the genetic variations. Most mononucleotide repeats were A or T, and three types of dinucleotide SSRs AG/CT/AT were identified. In addition, we detected two types of trinucleotide SSRs (AAT/ATT), twelve types of tetranucleotide SSRs (AAAG/CTTT/AAAT/ATTT/AACC/GGTT/ACCT/AGGT/AGAT/ATCT/ATCC/ATGG), and four types of pentanucleotides SSRs (AACAT/ATGTT/AATCG/ATTCG) . Furthermore, sixty-four long repeats were revealed, and they included 37 forward repeats, 14 tandem repeats, and 13 palindromic repeats .The simple sequence repeats (SSRs) have been widely used in phylogenetic and phylogeographic analyses of highly polymorphic genetic materials   Mill. was distributed in North America and formed a sister lineage with the firs from East Asia (BSML = 100). Among the East Asia firs, Abies beshanzuensis M. H. Wu was nested with others (BSML = 97). Abies ernestii, A. chensiensis, and A. fargesii clustered together as a monophyletic lineage, with an extremely high bootstrap (BSML = 100). Our results further indicated that A. ernestii and A. chensiensis were separated with an extremely high bootstrap (BSML = 100); thus, their current species status was well corroborated. The treatments of A. ernestii as a variety of A. chensiensis, or an identical subspecies of A. chensiensis were rejected  . Abies bAbies. Our results validated the reliability of using cp genome data to delimitate problematic groups at low taxonomic levels. These data provided important genetic resources for these ecologically important Abies species and further cp genome evolution research.This study provides new insights into the species delimitation and phylogenetic relationship in the genus Click here for additional data file.


Bauhinia s.l. (Leguminosae), known as cow’s hoof, unha de boi or pata de vaca, has been used in traditional medicine worldwide. The aim of the present review is to summarize the studies published on the biological activity of the main native medicinal species reported in austral South America. Of the 14 species present in the region, 10 are consumed as leaf infusions to regulate glucose and lipid metabolism, as well as used for their anti-inflammatory and analgesic effects and to treat various diseases. Pharmacological properties have been recorded in seven species. Antioxidant, anticoagulant, antihypertensive, diuretic, antimicrobial and antitumor properties have been reported in B. forficata. Together with B. holophylla, they are important for their antidiabetic properties, since several studies indicate their effectiveness as a hypoglycemic agent. B. bauhinioides is distinguished for its anti-inflammatory and antithrombotic activities and S. microstachya for its analgesic properties. Anti-ulcer and wound healing activities recorded in B. holophylla and B. ungulata, respectively, are of particular interest. Most of the species possess antitumor activity. The antioxidant capacity of flavonoids and other bioactive compounds make these plants good candidates to assist or treat various alterations related with oxidative stress, such as diabetic complications. Thus, these species constitute promising targets for new bioactive substance research and phytotherapy.The genus  Bauhinia s.l. , popularly known as cow’s hoof, cow’s paw, orchid trees, pata de vacca, unha de boi, falsa caoba, pezuña de vaca or pata de vaca, have been traditionally employed by different communities all over the world for medicinal purposes. Bilobed or bifoliolate leaves are consumed in infusions to treat diabetes mellitus, pains, inflammation and several diseases. The genus has promising medicinal potential, since experimental studies have provided evidence of its therapeutic properties pyrene (B[a]P). Aqueous extracts of B. forficata have antimutagenic/protective action on bone marrow cells of Wistar rats; they reduced chromosomal alterations induced by the chemotherapeutic agent cyclophosphamide [B. forficata flavonoid-rich fraction and purified kaempferitrin protected intestinal cells (IEC-6 cells) from cytotoxicity induced by irinotecan [ cancers . More re cancers  found thsphamide . B. forfinotecan . This chB. forficata leaf ethanolic extracts had antimicrobial activity against Candida albicans. Sousa et al. [C. albicans, Escherichia coli or Staphylococcus aureus species, but the extracts increased the effectiveness of norfloxacin against the S. aureus SA1199-B with a concentration-dependent effect. This strain overproduces the NorA efflux pump, a transmembrane protein that extrudes antimicrobial compounds, such as norfloxacin. Thus, B. forficata extract could be potentially used—together with norfloxacin—to treat infections caused by multidrug-resistant S. aureus [B. forficata leaf tincture (20% in a 70% hydroethanolic solution) against oral microorganism strains and mature dental biofilms obtained from salivary samples and formed on membranes or bovine enamel blocks. The tincture is a promising preventive agent of dental caries, with no cytotoxic effect, tested against oral fibroblast cells. Miceli et al. [, E. coli and S. aureus. Aqueous and ethanolic extracts of B. rufa presented antimicrobial activity against Candida spp. [B. ungulata aqueous and esential oils presented antimicrobial activity against various pathogenic microorganisms alone  increased the endothelium-dependent relaxation of phenylephrine-precontracted aortic rings in ALX diabetic rats; this effect was attributed to the antioxidant activity mediated by flavonoids [B. forficata leaves (1–50 μg/mL) were also described in aortic rings of both normotensive and hypertensive rats precontracted with phenylephrine [B. forficata (5–40 mg/kg intravenous administration) presented antihypertensive effects, inducing a dose-dependent transitory hypotension and tachycardia in normotensive rats and reducing mean arterial pressure by 12% in hypertensive rats . These effects seem to involve the release of nitric oxide. Regarding vasorelaxant properties, it has been demonstrated that aqueous–ethanol extracts of leaves of avonoids . Acetylclephrine . The efflephrine  found thBauhinia forficata is popularly consumed for kidney and urinary disorders such as polyuria, cystitis and kidney stones . Plant decoction (250–300 mg/kg/day for 16 weeks) prevented the formation of lipid peroxidation induced by two pro-oxidants tested. Moreover, it partially diminished the vacuous chewing movements induced by haloperidol. Other diverse biological activities have been described in austral South American  species . The chrB. bauhinioides BbCI inhibition of cruzipain—a cysteine proteinase from Trypanosoma cruzi—has shown the potential of this species for the development of anti-Chagas drugs [B. holophylla, enriched in flavonoids, presented a potent activity  against the Dengue virus serotype DENV-2, which is transmitted by mosquitoes. Leaf essential oils of B. cheilantha and B. ungulata showed larvicidal potential against instar III larvae of the Aedes aegypti mosquito  [Bauhinia extracts presented larvicidal activity against A. aegypti  and Culex quinquefasciatus  [The as drugs ,59. Santas drugs  reportedctively) ,117. Oth leaves) ,63,111).B. forficata exhibited some activity against cholinesterases (acetyl- or butyrylcholinesterase) [B. ungulata inhibited acetylcholinesterase [Hydromethanolic extracts of leaves of both subspecies of sterase) , whereasesterase ,120. Theesterase . B. forficata is a promising source of natural inhibitors of the serine proteases involved in blood clotting disturbances induced by snake venoms. The extract neutralized the clotting activity induced by the Bothrops and Crotalus crude venoms and inhibited clotting and fibrinogenolytic activities induced by the isolated thrombin-like enzyme from the Bo. jararacussu venom. It also inhibited the edema induced by C. durissus terrificus venom in mice. On the other side, B. forficata extracts enhanced the Tityus serrulatus scorpion venom’s lethality [Oliveira et al. ) found tethality .B. holophylla. Other proteinase inhibitors (BuXI) and their target proteinases have been characterized in B. ungulata [B. forficata proteome [Finally, the presence of lectins and Kunitz inhibitor activities could have potential uses that still have not been explored ,166,168.ungulata  and two proteome .B. forficata crude extract (2.85 g/kg) injected intraperitoneally caused the death of 50% of the animals, yet oral administration (0.5 to 5.0 g/kg) is not toxic [B. forficata spouted bed-dried extract and B. holophyla aqueous extracts in treated diabetic rats suggests liver injury  and the authors recommended a dilution when preparing formulations, teas and garrafadas [B. forficata aqueous extracts , since they detected alterations in behavior in rat anxiolytic models, such as decreased general activity and increased grooming duration and pentobarbital sleep inducing time; these authors suggest a dosage for central neurotransmitters. Sampaio et al. [B. forficata extracts . Mitochondrial damage was also reported by Ecker et al. [B. forficata aqueous extracts in vitro. They detected a decrease in mitochondrial dehydrogenase activity at high concentrations of extracts (200 and 400 μg/mL) and an induction of swelling (at 25 and 400 μg/mL). Finally, by interviewing 100 Bauhinia spp. consumers in Diadema, Sau Paulo (Brazil), Neto et al. [B. forficata , published by the Brazilian sanitary vigilance agency; they did not find similar reports in the literature searches. Thus, it is important to further investigate the possible adverse effects of the consumption of the Bauhinia species in order to minimize the health risk.Toxic effects were not reported in most methodological approaches e.g., ,141,173),173141,1jury see  [133,140rrafadas . Cavalcarrafadas  warned ao et al.  reportedr et al.  in isolao et al.  reportedBauhinia forficata, B. ungulata, B. bauhinioides, S. microstachya, B. holophylla, B. rufa and B. cheilantha are the austral South American species with records of pharmacological properties that explain their various ethnopharmacological uses. Bauhinia forficata is the most consumed and studied plant, with antidiabetic, antioxidant, anticoagulant, antihypertensive, diuretic, antimicrobial and antitumor properties. Together with B. holophylla, they are important for their antidiabetic properties, since several studies indicate their effectiveness as a hypoglycemic agent. Conflicting results could be explained by differences in extraction methods and preparation, chemical profiles, route of administration and dose, treatment periods, animal models used or plant identification. Clinical studies in B. forficata are still preliminary and deserve further investigation. B. bauhinioides is distinguished for its anti-inflammatory and antithrombotic activities mediated by Kunitz-type inhibitors. S. microstachya is distinguished for its analgesic properties. Anti-ulcer and wound healing activities recorded in B. holophylla and B. ungulata, respectively, are of particular interest. Most of the species possess antitumor activity, mediated by lectins, Kunitz proteinase inhibitors and other compounds. B. forficata extracts alleviate the side effects of chemotherapy, such as intestinal mucositis. The antioxidant capacity of flavonoids and other bioactive compounds present in B. forficata, S. microstachya, B. ungulata and B. holophylla make these plants good candidates for assisting or treating various alterations related with oxidative stress, such as diabetic complications and gastric ulcer, or even for cosmetic use. Thus, these regional species constitute promising targets for new bioactive substance research and phytotherapy.


Helminths, with an estimated 1.5 billion annual global infections, are one of the major health challenges worldwide. The current strategy of the World Health Organization to prevent helminth infection includes increasing hygienic awareness, providing better sanitation and preventative anthelmintic drug therapy in vulnerable populations. Nowadays, anthelmintic drugs are used heavily in livestock, both in case of infection and as a preventative measure. However, this has led to the development of resistance against several of the most common drugs, such as levamisole, ivermectin and thiabendazole. As many as 70% of the livestock in developed countries now has helminths that are drug resistant, and multiple resistance is common. Because of this, novel anthelmintics are urgently needed to help combat large-scale production losses. Prior to this review, no comprehensive review of the anthelmintic effects of essential oils and their components existed. Multiple review articles have been published on the uses of a single plant and its extracts that only briefly touch upon their anthelmintic activity. This review aims to provide a detailed overview of essential oils and their components as anthelmintic treatment against a wider variety of helminths. Parasitic worms or helminths are one of the main health challenges in developing countries in Africa, Asia and South America. Each year, approximately 1.5 billion individuals are infected worldwide, with many being host to multiple parasitic species simultaneously . It is eKnowledge of helminthosis goes back to the Roman Empire and early Arab scholars, but a true understanding of helminths’ biology was only attained starting from the 18th century ,13. TodaThere are an estimated 76,930 species of parasitic helminths, with 43,945 known host species . HoweverSchistosoma mansoni [Prior to this paper, no comprehensive review of the anthelmintic effects of essential oils and their components existed. Multiple review articles discuss the uses of a single plant and its extracts and only briefly touch upon their anthelmintic activity ,31,32. L mansoni . Under tStrongyloides stercoralis can reproduce by parthenogenesis [The most widespread human helminths belong to the phylum Nematoda. They are divided into two groups: the soil-transmitted nematodes  and the filarial nematodes . Several anatomical traits distinguish nematodes from platyhelminths, of which the cylindrical body shape and an exoskeleton (cuticle) are the foremost. Due to this exoskeleton, nematodes must undergo four larval molts before transitioning into adulthood. The parasitic nematodes are typically dioecious, with both male and female forms, although ogenesis ,27,35.Ascaris lumbricoides, Trichuris trichiura (whipworm) and Enterobius vermicularis (pinworm) infect through the ingestion of infective eggs. In contrast, hookworms  and Strongyloides stercoralis infect by means of free-living larval stages that hatch from eggs excreted in fecal matter. E. vermicularis is unique in that females migrate out of the anus to lay eggs on the perianal skin [There are some variations among the life cycles of various soil-transmitted helminths. nal skin ,36. Globnal skin ,3,17. Innal skin .Wuchereria bancrofti and Brugia malayi), onchocerciasis or river blindness (Onchocerca volvulus), loiasis (Loa Loa) and dracunculiasis or Guinea worm disease (Dracunculus medinensis) [D. medinensis, which is not a true filarial nematode [Wuchereria bancrofti is the leading cause of lymphatic filariasis (90% of cases) and has been confirmed to be endemic in 76 countries [W. bancrofti [Filarial nematodes are less widespread. The most common infections are lymphatic filariasis (inensis) . Filarianematode . Lymphatountries . Death iountries ,39,40. Aancrofti . Guinea ancrofti .Clonorchis sinensis, Fasciola hepatica, F. gigantica, Opisthorchis viverrini and O. felineus), lung flukes (Paragonimus spp.), intestinal flukes (Fasciolopsis buski) and blood flukes  [Schistosoma are responsible for the disease schistosomosis (bilharzia). They are not transmitted via food; instead, the cercariae directly penetrate their final host, often via the foot. They develop into adults in the lungs and the hepatic portal system before migrating to the venules of the intestine or the bladder. Schistosomes are dioecious, and the smaller female resides in a tegumental fold on the ventral side of the male [The trematodes that are infectious to humans can be divided into four groups: liver flukes  ,27,42,43calatum) . Annuallcalatum) . The blothe male ,47. In 2the male .Tapeworms have several unique anatomical features. They do not possess an alimentary canal; instead, nutrients diffuse through the integument. In addition, they possess a scolex or head, which is connected via a neck to a series of repeating segments called proglottids. The proglottids at the neck get detached from the posterior end when mature. These terminal segments carry eggs that are passed in the stool upon release. Upon ingestion by the intermediate host, the eggs hatch into oncospheres, which migrate to the muscles and develop into cysticercus larvae . The cysticercoid larvae mature into adults in the intestine of the definitive host .Taenia saginata (beef tapeworm) and T. solium (pork tapeworm) can live in the intestines as adults in a condition called taeniasis. However, infection by the cysticercus stage is also possible after consumption of eggs, leading to cysticercosis [Approximately 40 species of tapeworms are capable of infecting humans, although most of these infections are accidental. cercosis ,49. TaenEchinococcus is another genus of tapeworm capable of infecting humans. They can infect humans as an intermediate host. The disease is called echinococcosis or hydatidosis and is characterized by the formation of hydatid cysts in the liver and lungs. More than 1 million people suffer from echinococcosis worldwide [orldwide ,50.Ostertagia ostertagi (cattle), Haemonchus contortus (sheep and cattle), Teladorsagia circumcincta (sheep and goats), Dictyocaulus viviparus (ruminants), Ascaris suum (pigs) and the trematode Fasciola hepatica (sheep and cattle) [T. saginata and T. solium are transmitted to humans through the ingestion of undercooked meat [F. hepatica and F. gigantica, are able to infect livestock as well as humans [Ascaris suum, the large roundworm of pig, might be the same species as A. lumbricoides, the foremost human roundworm parasite [Helminth infections are not solely limited to humans but are widespread among all vertebrates. Of the estimated 76,930 helminth species, 1555 infect cartilaginous fish, 14,154 infect bony fish, 4225 infect amphibians, 11,486 infect reptilians, 33,849 infect birds, and 11,631 infect mammals . Of spec cattle) ,54. Asidked meat . Some hes humans . Ascarisparasite ,57.Ostertagia larvae can remain on pastures for up to a year, indicating that long-term treatments may be needed to fully eliminate helminths from a herd [Detection of these parasites is often difficult, as many infections are asymptomatic. In the past, preventive treatment with anthelmintics was used, but this led to increasing anthelmintic resistance . Infectim a herd . Many hem a herd . Fasciola spp. of their intermediate hosts [Mucor circinelloides, Pochonia chlamydosporia, Duddingtonia flagrans, Arthrobotrys spp. and Monacrosporium spp., can act as effective biological anthelminthic agents to reduce the presence and survival of free-living stages , thus avoiding infection. The health of the infested animals can also be improved through supplementary feeding. The genetic resistance of livestock can also be increased through selective breeding and crossing. Maasai sheep have increased resistance against nematodes, and trematode resistance has been observed in Javanese thin-tailed sheep. Crossing these breeds with high performance breeds can lead to decreased production loss [H. contortus has been underway for many years, and recent human vaccine trials against hookworms increase the prospect of veterinary vaccines [Haemonchus spp. and Ostertagia spp. [In addition to anthelmintic treatment, there are several strategies to limit infection among livestock. Simple practices, such as grazing management, biological control and supplementary feeding, can be followed without the need for additional research. Grazing management can help limit the exposure of livestock to helminths. Letting livestock sequentially graze on different plots causes the number of infective helminths to decrease due to the absence of their host before the cattle are reintroduced. Biological control involves denying helminths the necessary environment for infection. The most logical procedure is through dung removal, but supporting wildlife can also lead to decreased infection. For example, ducks can feed on snails and deprive te hosts . Additioion loss . Finallyvaccines ,61,62,63gia spp. . Anthelmintics are drugs used for the treatment of parasitic worms in humans, animals or plants. While the vast majority act upon receptors of the neuromuscular system in helminths, the efficacy differs strongly between nematodes, trematodes and cestodes . The antCooperia spp. and Ostertagia spp. [Caenorhabditis elegans. Mutations in the GluCl subunits glc-1, avr-14 and avr-15 can decrease susceptibility. Additionally, increased expression of P-glycoproteins leads to increased resistance against ivermectin, as well as moxidectin, levamisole and pyrantel [Ivermectin, a macrocyclic lactone and derivative of avermectin, is one of the most potent anthelmintics used in both human and veterinary therapies. In humans, it is used as a treatment for filariasis and onchocerciasis, whereas it sees broader antinematodal use in livestock and domestic pets ,67,68. Igia spp. ,70,71. Igia spp. . The genpyrantel ,69,73,74pyrantel . Aside fpyrantel .C. elegans mutants have led to the identification of more than 20 mutant genes that confer resistance, several of which have also been observed in parasites [Nicotinic acetylcholine receptors (nAChRs) are targeted by a variety of anthelmintic drugs with differing chemical structures. Levamisole (an imidazothiazole), pyrantel and morantel (both tetrahydropyrimidines) act as agonists on L-nAChRs (levamisole-sensitive nAchRs), mimicking the action of acetylcholine. They cause prolonged excitation, leading to muscle spasms and paralysis . They ararasites ,69,77.Haemonchus spp. has begun to decline [Derquantel acts as a competitive antagonist of nAChRs and has a preference for bephenium-sensitive B-nAChRs . It has  decline ,78. No mC. elegans through mutations in the acr-23 gene [H. contortus, only four years after its introduction [C. elegans is unclear; recent in vivo selection for resistance could provide insight into the exact mechanisms [Amino-acetonitrile derivatives (AAD) are a relatively new class of anthelmintic drugs . Monepan-23 gene . There aoduction ,78,81. Wchanisms . Benzimidazoles include thiabendazole and albendazole among others and are used to treat a wide range of flukes, tapeworms and roundworms. They act by interfering with the cytoskeletal structure through the binding of β-tubulin monomers. This results in decreased intracellular transport with far-ranging effects, ending in paralysis and death ,69,83. DC. elegans, it is thought to act intracellularly on the calcium-activated phosphate channel SLO-1 located in the body wall muscle. The activation results in increased opening of the channels, and as a result, a decrease in locomotion and feeding behavior [Emodepside was developed as a novel anthelmintic effective against levamisole-, ivermectin- and benzimidazole-resistant helminths . It is abehavior . It has behavior ,91.The discovery of novel anthelmintic drugs is advancing slowly, despite the severity of human helminthoses. The main drive behind anthelmintic research are veterinary applications, and many drugs used in humans today were originally developed for livestock .Anthelmintic resistance is a widespread and growing phenomenon, especially in small ruminants and, to a lesser but increasing degree, in cattle ,92. ResiWhether anthelmintic resistance is present in human helminths is unclear, but the possibility should be considered seriously, and efforts should be undertaken to limit it ,98,99.Aside from possible methods to limit anthelmintic resistance, there is an urgent need for novel anthelmintics . SeveralC. elegans, to measure anthelmintic activity [C. elegans [The most straightforward method to test anthelmintic activity is the fecal egg count reduction test—an in vivo assay that compares the number of helminth eggs in the feces between treated and untreated groups . Large-sactivity . Such asactivity . Variousactivity ,105. Eggactivity ,107, as  elegans ,106. How elegans . Similar elegans ,109.C. elegans in order to assess motility. Two sequential scans are performed, which allows the identification of immobile or dead worms [C. elegans in a microtiter plate. These fluctuations in the signal intensity are used as a measure of the locomotor activity [Adult (and L4) motility and mortality assays can take several forms . The simad worms ,111. Recad worms . A thirdactivity ,114.Positive hits from the aforementioned assays can be further tested in egg-hatching assays of parasitic nematodes as an additional test of relevance . The assEssential oils (EOs) are mixtures of volatile hydrophobic secondary plant metabolites of low molecular weight. They are usually extracted from plants through steam distillation and more rarely by cold pressing. Chemically, they are blends of up to hundreds of different plant metabolites  and inclH. contortus (24 articles) as the most utilized helminth species. Other Nematoda are unspecified gastro-intestinal nematodes in in vivo research (nine articles) and Anisakis simplex in research on fish parasites (four articles). The anthelminthic effects on Trematoda are studied in 13 articles: 12 on Schistosoma mansoni and 1 on Schistosoma haematobium. Echinococcus spp. belonging to the phylum Cestoda are studied in 10 articles. Monogenean Gyrodactylus spp. and the Annelid Pheritima posthuma are studied in one article each. The range of assays used in both in vitro and in vivo studies is shown in Echinococcus spp. (10 articles). In vivo studies depend heavily on fecal egg count reduction assays (10 articles) and measurements of the animal’s worm burden (8 articles).Due to their promising biological effects, EOs and/or their derived components have gained much attention nowadays ,125,126.Eucalyptus staigeriana, Citrus reticulata, C. limonia and C. aurantifolia [50 against H. contortus, a pathogenic nematode of ruminants, as determined by egg hatch assays and larval development assays [Schistosoma mansoni, causing full inhibition of motion at 43.75 µg/mL and with an LC50 of 81.7 µg/mL [Limonene is a major terpene (28% to 73%) in EOs from ntifolia ,145,152.t assays . These at assays . Its eff.7 µg/mL .Zanthoxylum zanthoxyloides, Melaleuca alternifolia and Bunium persicum EOs [Echinococcus spp. (3.125 to 50 µL/mL) [H. contortus, determined via the egg hatch assay (LC50 = 430 µg/mL) [Another monoterpene, gamma-terpenene, has been isolated as a major component (10 to 35%) of icum EOs ,151,162.0 µg/mL) .p-Cymene (synonym: p-cymol or p-isopropyltoluene) is an alkyl-substituted aromatic compound. Although not a major constituent, it is present in EOs of many plants, such as Origanum compactum, Alpinia zerumbet, Thymus vulgaris, Trachyspermum ammi and Nigella sativa [Echinococcus spp. and H. contortus. This compound is well known for its health benefits and antiparasitic properties [a sativa ,163,166.operties .Myrtus communis and Pinus nigra) is active against hydatid cysts of Echinococcus spp. assessed through protoscolex viability assays, where 6.3 ± 0.3% and 100% reduction occurred after, respectively, with 5 to 30 min of exposure [Artemisia campestris (36.4%) and Ferula gummosa (57.0%), and is effective against Heligmosomoides polygyrus [Echinococcus spp. [Citrus aurantifolia EO, along with others (limonene (56.37%), β-pinene (11.86%) and γ-terpinene (11.42%)), and was effective against H. contortus [α-Pinene . In several animal studies, β-myrcene has shown promising health benefits. Along with other constituents, it was active against Trichostrongylus spp. and H. contortus when tested in a larval development assay [Monoterpene myrcene (β-myrcene) is a minor constituent in the EOs of nt assay .Bidens sulphurea (10.23%) [Newbouldia laevis (36.08%) [Schistosoma mansoni. In addition, it is found in the EO of Plectranthus neochilus, which is effective against Strongyloides ratti [Pinus nigra Arn. subsp. pallasiana (active against Echinococcus spp.) assessed through a larval development assay [Baccharis trimera, Mentha x villosa etc. [Schistosoma mansoni. (E)-Caryophyllene, present in variable amounts in several EOs, such as those from Ageratum conyzoides and Lippias idoides, is effective against several helminths, such as Trichostrongylus spp., Haemonchus spp., Syphacia obvelata and Aspiculuris tetraptera [Lantana camara are also effective in an egg-hatching assay with H. contortus [Sesquiterpenes are terpenoids (C15) containing three isoprene units and are the second most important group of compounds in plant EOs. Several sesquiterpenes have been reported as antiprotozoal since the discovery of artemisinin . β-Caryo(10.23%)  and Newb(36.08%) , and is es ratti , as wellnt assay . Trans-Cosa etc. ,160, is traptera ,139. Carontortus .Eucalyptus citriodora and Pelargonium roseum (37.7%), is a natural acyclic monoterpenoid. It is effective against hydatid cysts of Echinococcus spp. assessed through protoscolices viability assays measured after 60 min of exposure [Citronellol or dihydrogeraniol, present in the EOs of exposure .Cymbopogon. Its concentration varied from 53 to 81% in the EO of lemon grasses, such as Cymbopogon citratus, C. schoenathus and C. martinii. The in vivo efficacy has been tested by Katiki et al. [H. contortus in sheep through fecal egg count reduction, egg hatch and larval development assays. The in vitro effects were tested through fecal egg count reduction, egg hatch and larval development assays against Trichostrongylus spp. (CL50s = 40–160 µg/mL) [Pheritima posthuma (20 mg/mL) [Mentha piperita EO were found to be effective against Trichostrongylus spp.  [Schistosoma mansoni at 10 µg/mL EC50 using Baccharis dracunculifolia EO, which contains (E)-nerolidol (33.51%) and spathulenol (16.24%).Geraniol is the principal component in many EOs of the genus i et al.  with the0 µg/mL)  and Pher0 mg/mL) . Gerania0 mg/mL) . Menthol0 µg/mL) . Parreir0 µg/mL)  observedAnisakis simplex  [H.contortus  [Terpinen-4-ol is the main bioactive component of tea-tree oil. It is active against y assay)  and H.coh assay) .Coriandrum sativum EO, is active against H. contortus at 630–2890 µg/mL based on egg hatch and larval development assays [β-Linalool, the major component (73.21%) of t assays .10H14O. It is abundantly (50–60%) found in the oils of plants, such as Melaleuca alternifolia, Trachyspermum ammi, Thymus vulgaris and Lippia sidoides, has a pleasant aromatic odor and strong antiseptic properties. It was the treatment of choice for hookworm infection in the United States after 1910 [50s , IC50s  and LD50s , it is highly effective against H. contortus [Anisakis simplex [Syphacia obvelata and Aspiculuris tetraptera [Haemonchus spp., and Trichostrongylus spp., gastro-intestinal nematodes [Echinococcus spp. [Thymol, also called 2-isopropyl-5-methylphenol (IPMP), is a natural colorless crystalline mono-terpenoid phenol derivative of cymene, Cter 1910 ,184. Basontortus , Anisaki simplex , Syphacitraptera , Haemoncematodes  and hydacus spp. . Worm buZanthoxylum simulans, Satureja montana (40–64%), S. khuzistanica (94%), Origanum dictamnus (44–50%), O. compactum (50–59%), Thymus caespititius (54%), Thymbra capitata (68%), Origanum vulgare (68.5%), Mentha spicata (64.5%) and O. syriacum (82%), etc. This compound is active against H. contortus  [Anisakis simplex  [Echinococcus spp. and protoscolices  [Schistosoma haematobium  [Carvacrol, isomeric to thymol, is a major monoterpene phenol in the EO of several plants, especially h assay) , Anisakiy assay) , hydatidxposure)  and Schitituent) .Ocimum basilicum and other plants, such as clove. Its efficacy was assessed through mortality and egg hatch assays, and it has been proven with H. contortus (662.5 to 5.300 µg/mL). According to El-Kady et al. [Foeniculum vulgare and is effective (10 to 100 µg/mL) against Schistosoma mansoni (a water-borne blood fluke of humans) based on mortality tests [Croton zehntneri and effective against H. contortus [Ocimum basilicum. It is mostly active against GI nematodes  [Eugenol, an allyl chain-substituted guaiacol, is a significant component (43.7 to 53%) of the EO of y et al. , there ay et al. . Anetholty tests . It is aontortus . Estrago assays) .Haemonchus contorts parasites at concentrations of 130 to 1370 µg/mL  [Citral  is the second most effective drug after carvacrol against  assays) .H. contortus is 410 to 1700 µg/mL  [Citronellal is a monoterpenoid aldehyde, which is the main component (around 67%) in the mixture of terpenoids that give citronella EO its distinctive lemon scent. It’s in vitro efficacy against y assay) . When tey assay) , it is eLavandula officinalis EO was effective in an egg-hatching assay against H. contortus (IC50 = 316 µg/mL) [Tetradenia riparia EO is also effective against Anisakis simplex [Cymbopogon martini EO, is effective against Trichostrongylus spp. when tested in egg hatch and larval development assays [Anthemis nobilis EO are effective against H. contortus in egg hatch and larval development assays [Pelargonium roseum EO, is effective against Echinococcus spp. [Esters are also common in EOs due to their widespread occurrence in nature. Linalool acetate (35.97%), along with trans-sabinene hydrate (29.17%), in 6 µg/mL) . Geranyl simplex . Isomentt assays . Isobutyt assays . Citronecus spp. .Anethum graveolens EO, dihydrocarvone (39.1%), along with carvone (22.24%) and D-limonene (16.84%), was effective against H. contortus at IC50 = 6 µg/mL, ascertained through egg hatch and larval development assays [Monoterpenoid dihydrocarvone is a key building block for synthesizing sesquiterpenes. In t assays .Tanacetum vulgare EO, is effective against Schistosoma mansoni in a mortality assay (100–200 µg/mL) [Salvia and Artemisia and is known to be toxic.Β-Thujone (84.13%), a monoterpene ketone found in 0 µg/mL) . This coLippia sidoides and Nigella sativa (42%), and is active against hydatid cysts of Echinococcus spp., as has been demonstrated by Mahmoudvand et al. [Thymoquinone is also a monoterpene  found in the EOs of plants, such as d et al.  through Eucalyptus. This compound is active against H. contortus and other GI nematodes (sheep). Camphor (16.65%) and 1,8-cineole (34.56%) are active against H. contortus [Alpinia zerumbet EO [Rosmarinus officinalis EO [Myrtus communis EO are effective against Echinococcus spp. [Ocimum gratissimum EO, is effective against H. contortus in an egg hatch assay [Lantana camara EO are also effective in egg-hatching and larval development assays against H. contortus [1,8-Cineole  is a major component of EOs (17 to 42%) of several plants, including ontortus . 1,8-Cinumbet EO  and 1,8-nalis EO , are effcus spp. . 1,8-Cinch assay . Caryophontortus .Zanthoxylum simulans EO in an egg-hatching assay with H. contortus [Tagetes patula (23.5%), is active against H. contortus  [Piper aduncum) effective against H. contortus . De Melo et al. [Ageratum conyzoides EO, present along with (E)-caryophyllene (14.23%). Rotundifolone (70.96%) is active against Schistosoma mansoni at 10 to 100 µg/mL in a mortality assay [Borneol (18.61%) was more effective than β-elemene (10.87%) in ontortus . Piperith assay) . Gaínza h assay)  found dio et al. , using aty assay .50 or IC50 values even when tested on the same species based on the chemical class of the components (monoterpene or phenylpropanoid) or based on the main functional groups . It seems that the carbon backbone is important, with cyclohexene-containing compounds differing significantly from both the phenylic compounds and the alkenes. The difference between phenylic compounds and those with an alkene structure was also observed among the tests. This suggests that the overall structure of the compound is more important for bioactivity than the specific functional groups or other properties. It is also interesting to note that the effective dose may differ between the isomers, suggesting an interaction with a specific drug target .It appears from the above that the efficacy of EOs is quite variable. There are differences among the strains, the concentrations used and the assay methods. Significant differences have been observed in ECEOs are complex mixtures of substances, which are volatile in nature; they have been used in traditional medicine for millennia. These EOs and their EOC are known to possess many bioactivities, but in the past two decades, researchers have devoted special attention toward their anthelmintic properties. Because of their lower toxicity and frequent use by local practitioners for many parasitic infections in animals and humans, they offer attractive leads for inexpensive and safe drugs.Pinus nigra is frequently used for the treatment of worm infection in cattle [Origanum syriacum is used as vermifuge [Anisakis L3 larvae [Origanum syriacum are proposed as an ecofriendly nematocide and support the folk usages of this plant as an antiparasitic [Citrus aurantifolia, Anthemis nobile and Lavandula officinalis, in folk medicine as anthelmintics. They studied their EOs on different developmental stages of H. contortus and concluded that their study validates the ethnopharmacological importance of three EOs as anthelmintics.In Turkish folk medicine, n cattle . This inn cattle  to studyn cattle . Origanuermifuge . Due to 3 larvae ,167. Accarasitic . Other aarasitic ,145 alsoDysphania ambrosioides (Chenopodiaceae) is widely used as a vermifuge, and its extract, as well as infusion, were used as an anthelmintic [Schistosoma mansoni, finding it 31.8 times more toxic to adult S. mansoni worms compared with GM07492-A cells [Thymus vulgaris (a plant from the mint family) is used in traditional medicine as an anthelmintic agent [H. contortus in vivo model [elmintic . Its EO elmintic . Becauseelmintic  further -A cells . Thymus ic agent , which lvo model . Thymol vo model .Foeniculum vulgare (Apiaceae) is reported in folk medicine as vermifugal [S. mansoni; they concluded that the EO of F. vulgare in combination with praziquantel could be considered as an alternative schistosomosis treatment, which can slow down the development of resistance toward praziquantel [Xanthoxylum bungeanum (Rutaceae) is another frequently used plant in traditional Chinese medicine to kill intestinal parasites [H. contortus and concluded that the available information supported the use of this EO as an alternative means to control gastro-intestinal nematodes [The EO of rmifugal . This usrmifugal  to studyiquantel . Xanthoxarasites . Therefoarasites  studied ematodes .Cymbopogon citratus (Poaceae) is another shrub reported to have anthelmintic activity in ethnoveterinary practice [Cyperus articulatus (Poaceae) is used in traditional medicine by tribes in northwestern Cameroon in various ways, e.g., chopped roots and rhizome, decoction with boiling water to treat onchocerciasis. This led to further study on the activity of its roots and rhizomes against the microfilariae and adult worms of O. ochengi by Metuge et al. [Tanacetum vulgare (Asteraceae) is commonly used in folk medicine as a vermifuge [S. mansoni. The bioactive compounds, as well as its mechanism of action, were elucidated, reinforcing the traditional use of T. vulgare as a vermifuge and an anthelmintic [Cosmos sulphureus (synonym: Bidens sulphurea), which is used to treat malaria in Brazil [B. sulphurea has been evaluated against S. mansoni and considered a promising source of a new schistosomocidal drug [Mentha × villosa; its EO and EOC  were tested against adult S. mansoni. Rotundifolone was considered a potential source for the development of a new drug against S. mansoni [Satureja khuzistanica. The activity they demonstrated supports its use in traditional medicine. Another interesting finding supporting the traditional use of two plants, Zanthoxylum zanthoxyloides and Newbouldia laevis, saw them being selected based on an ethnopharmacology survey, which documented their use by small-scale farmers for treating digestive helminths in small ruminants [Strongyloides ratti, both in egg-hatching and larval migration inhibition assays, at concentrations comparable to established anthelmintics  [Croton zehntneri and Lippia sidoides are used for the treatment of gastro-intestinal diseases [Syphacia obvelata and Aspiculuris tetraptera), as well as in vitro assays on H. contortus. Both EOs proved effective, and the authors concluded that further (in vivo) studies are necessary on target species, such as sheep, after determining the absorption and metabolism of these EOs [practice . Infusiopractice . As the practice . Cyperuse et al. , who cone et al. . Tanacetermifuge , leadingelmintic . Anothern Brazil . The EO dal drug . Another mansoni . Based o mansoni  studied uminants . The EOsamisole) . In Brazdiseases . Camurçadiseases  studied hese EOs .Commiphora molmol resin and 3.5 parts of its volatile oils being tested on seven individuals exhibiting signs of fasciolosis. The administered dosage regimen (12 mg/kg per day) reduced common symptoms, such as abdominal pain, fever and weight loss, and it reduced fecal egg counts to zero three weeks after treatment [Eucalyptus citriodora, Eucalyptus staigeriana, Lippia sidoides and Thymus vulgaris [Complete or ongoing clinical trials of EOs as anthelmintic agents are lacking. A study by Massoud et al. , althougreatment . Althougvulgaris ,139,141 S. mansoni-infected children and increased their hemoglobin level [We found two clinical studies with EO components . Artemether (6 mg/kg every 3 weeks in 5 cycles) was used for schistosomosis . Artesunin level .https://worldwide.Espacenet.com/patent/ (accessed on 31 May 2020)). The search yielded 187 patents, of which only 18 described anthelminthic treatment methods using EOs or their components, and only 6 patents were granted  online platform Espacenet , sesquiterpenes , alcohols , EO-phenol , phenyl methyl ethers (eugenol—43–53%), aldehydes , esters , ketones and EO quinones (thymoquinone—42%), which occur in diverse plant species, have proven effective against a range of helminthic parasites.The current approach to controll helminthic infections is mostly based on preventive pharmacotherapies. However, this has disadvantages, such as drug resistance, adverse effects, undesirable toxicity and recurrence. It is evident plant-derived EOs and some of their components have considerable potential for safe treatment and prevention of helminthic disease. The major EO components, such as monoterpenes  presents a significant hurdle for compounds with poor prospects for patent protection . MoreoveOn the other hand, EOs possess some unusual properties, which may prove advantageous for therapy. The lipophilic nature of EOs allows them to cross the membranes of parasites, as well as the blood–brain barrier. In addition, EOs induce oxidative stress in parasites, increase the levels of nitric oxide in the infected host, reduce parasite resistance to reactive oxygen species and increase lipid peroxidation; all these effects ultimately lead to serious damage to cell membranes.However, this prospect is hindered due to limited in vivo studies. Moreover, the mechanism of action of such components remains largely unknown. More in vitro and in vivo studies on the mechanisms of action are essential to establish the efficacy of EOs. In addition, most of the studies are focused on the major constituents of EOs, but minor constituents and the synergy of action among various components will be an exciting area of future research. Bioassay-guided purification and identification of constituents using LC-MS and development of authentic standards are promising. Application of nanotechnology (nano-emulsion preparation with the EOs) may help solve problems, such as solubility, release and permeation, bio-availability, odor and bio-destruction, which restrict the therapeutic use of EOs in biological systems.