Haemonchus contortus is a haematophagous parasitic nematode of veterinary interest. We have performed a survey of its genome-wide diversity using single-worm whole genome sequencing of 223 individuals sampled from 19 isolates spanning five continents. We find an African origin for the species, together with evidence for parasites spreading during the transatlantic slave trade and colonisation of Australia. Strong selective sweeps surrounding the β-tubulin locus, a target of benzimidazole anthelmintic drug, are identified in independent populations. These sweeps are further supported by signals of diversifying selection enriched in genes involved in response to drugs and other anthelmintic-associated biological functions. We also identify some candidate genes that may play a role in ivermectin resistance. Finally, genetic signatures of climate-driven adaptation are described, revealing a gene acting as an epigenetic regulator and components of the dauer pathway. These results begin to define genetic adaptation to climate in a parasitic nematode. Haemonchus contortus and identify genes that play a role in drug resistance as well as climate-driven adaptations involving an epigenetic regulator.Based on single worm whole genome sequencing, the authors here characterise the global evolution of the gastrointestinal parasite  Their complex life cycles may involve both intermediate hosts, vectors and time spent in the environment, where they face harsh and variable conditions such as frost or drought that they must withstand between infections of their hosts2. Parasitic nematodes have adapted to a wide range of threats, including predation, climate and the immune responses of a great diversity of both plant and animal host species4.Nematodes have evolved to exploit a wide diversity of ecological niches. Although many sustain a free-living lifestyle, parasitic nematodes rely on one or more hosts to complete their life cycle. Many parasitic nematodes undergo a complex series of morphological changes, linked to migration through their hosts to establish a mature infection5, or indirectly via major economic losses in plant3 and livestock production6, and through spending on parasite control. The control of animal parasitic nematodes relies almost exclusively on anthelmintic drugs, administered on a recurrent basis in livestock7 and through large-scale preventative drug administration programmes in humans8. Although the success of such strategies was originally undeniable, the emergence of drug-resistant veterinary parasites6, or the reported lack of efficacy in human-infective species9, threatens ongoing control efforts for many parasitic infections. Vaccines offer an attractive alternate control strategy against these parasites: the extensive genetic diversity and immune-regulatory properties of parasites has, however, greatly hampered vaccine development10, and although two licensed vaccines are currently available for veterinary purposes11, transcriptomic plasticity of the parasite following vaccine challenge may contribute to circumvent the vaccine response of their host11. It is therefore clear that novel, sustainable control strategies are required. The potential of helminths to adapt to and thus escape control measures lies in their underlying genetic diversity. A greater understanding of the extent of this diversity and the processes that shape it throughout their range should provide insight into the mechanisms by which they adapt, and may identify new targets, which may be exploited for control.The evolutionary success of parasitic nematodes comes at a cost to humans, either directly as they significantly impact human health (amounting to a loss of 10 million disability-adjusted life-years)Haemonchus contortus is a gastrointestinal parasite of ruminants in tropical and temperate regions throughout the world, and causes significant economic and animal health burden particularly on sheep husbandry. It is also emerging as a model parasitic nematode system for functional and comparative genomics, largely owing to its rapid ability to acquire drug resistance, the relative tractability of its life cycle under laboratory conditions12, the development of extensive genomic resources14, and its relatively close relationship with other clade V parasitic nematodes of both veterinary and medical importance 15. We use whole genome sequencing of 223 individual H. contortus sampled from 19 isolates spanning five continents to characterise genome- and population-wide genetic diversity throughout its range. This survey of genome-wide diversity reveals old and new genetic connectivity influenced by human history, and signatures of selection in response to anthelmintic exposure and local climatic variation.The trichostrongylid Whole-genome sequencing of 223 individuals from 19 isolates , with only 411,574 SNPs segregating with a minor allele frequency (MAF) > 5% in those individuals. Estimates of nucleotide diversity (π) in isolates with at least five individuals ranged from 0.44% (STA.2) to 1.3%  = 9.49, P = 0.003; Supplementary Note π was estimated for three subsets of isolates with the highest coverage (> 8× on average) from France (FRA.1 and FRA.2), Guadeloupe (GUA), and Namibia (NAM) that yielded slightly higher values that ranged between 0.65% and 1.14%. Overall, these data show equivalent diversity levels to Drosophila melanogaster (ranging between 0.53% and 1.71%)16 but represented ~ 1.6- to 45-fold greater diversity than two filarial nematode species for which similar statistics are available  of genetic variation, which revealed three broad genetic clusters of isolates  of H. contortus to be between 0.60 and 1.05 million. MSMC analysis, which models past recombination events based on heterozygosity patterns along the genome, revealed the historical Ne has remained within a slightly lower range of values for most of the sampled time interval, i.e., from 2.5 to 500 thousand years ago (kya), with extreme estimates falling between 1.5 × 105 and 6.1 × 105 individuals for GUA (633 years ago) and NAM (415 years ago) isolates, respectively , was inconsistent with a simple isolation-by-distance scenario. This observation was supported by, for example, little genetic differentiation (measured by FST) between geographically distant French (FRA) and Oceanian  isolates  and 0.24 (FST range: 0.17–0.33), respectively : windowed mitochondrial nucleotide diversity estimates were 0.63% ± 0.37% (standard deviation), 0.58% ± 0.31%, 0.66 ± 0.32%, 0.6% ± 0.35%, for Mediterranean (n = 360), Oceanian (n = 225), American (n = 92), and south-African (n = 305) isolates, respectively.The greatest genetic dissimilarity was found between subtropical African isolates and the French and Australian isolates, with mean FF = 11.36, P = 0.0009) between STO and other isolates relative to other pairwise comparisons, almost certainly reflects the isolation of an island population relative to continental populations as a result of higher exposure to drift or smaller founding populations  = 581, P < 10–4) to other samples. Inference of the joint history of STO and other African populations supported this view. The best-fitting model , consistent with the initial foundation of the sheep industry in this country. These complex patterns reiterate that human movement has played an important role in shaping the diversity of this livestock parasite throughout the world.The patterns of genetic connectivity support at least three distinct migration events in time and space that we investigated using forward genetic simulations  protein capable of mediating phenotypic resistance. We identified indications of a selective sweep in the region surrounding the Hco-tbb-iso-1 locus in the resistant isolates analysed. Focusing on three isolates with the highest coverage, we found an average 2.31-fold reduction of Tajima’s D coefficient within 1 Mbp  in the beta tubulin isotype 1 , whereas the surrounding region was in favour of topologies congruent with overall population structure  owing to both phenotypically susceptible and resistant individuals being present , 198 (A to T) and 200 (T to A) (Supplementary Table n = 39), and accounted for all samples from the Guadeloupe (n = 14) and the White-River South-African  isolates. Variants at codons 167 and 198 were much less common, i.e., 13 individuals carried mutant alleles at position 198 and only one F167Y mutant was identified in a French isolate. No double homozygous mutants were found, but three individuals from France, Australia and South-Africa were heterozygous at both positions 198 and 200. In each case, inspection of the sequencing reads revealed that the two mutations never appeared together in the same sequencing read, suggesting they cannot co-occur in cis (on the same chromosome copy). Genotype frequencies were consistent with isolate-level benzimidazole efficacies as measured by the percentage of egg excretion after treatment (Supplementary Table n = 20).The F200Y homozygous genotype was the most common and widespread resistant genotype , but is robust to SNP uncertainty25. Within continent pairwise comparisons of African  and Australian isolates  yielded 1740 hotspots of diversification, 48% of which were contained within a gene locus  on chromosome I, and a P-glycoprotein coding gene already involved in ivermectin susceptibility in the equine ascarid Parascaris sp27,  on chromosome V. Although the former showed indication of reduced genetic diversity in its vicinity in resistant isolates relative to others  and response to drug . Additional significant biological process associated terms were related to phenotypes tightly linked to anthelmintic effects. For example, pharyngeal pumping28  and oviposition  genes were enriched, both of which are phenotypes linked to ivermectin and its effect on parasite fecundity29. Neurotransmission is the primary target of anthelmintics such as macrocyclic lactones30 and levamisole31; genes with GO terms related to neurotransmission  were significantly enriched across comparisons. Anthelmintic-associated GO terms including “response to drug”, “regulation of neurotransmitter” and “neurotransmitter transporter activity” were significantly enriched in every comparison involving a South-African multi-resistant field isolates  were found overlapping XP-CLR signals of selection and thus seem candidates for drug resistance loci in H. contortus, as supported by the functions of their C. elegans orthologs . The snf-9 gene encodes a neurotransmitter:sodium symporter belonging to a family of proteins involved in neurotransmitter reuptake, and the latter three genes are expressed in neurons. Unc-24 and B0361.4 are involved in response to lipophilic compounds and aex-3 is critical in synaptic vesicle release32. A reduction in nucleotide diversity surrounding unc-24, B0361.4, aex-3 in the STA.1 isolate in comparison with others  was among the top 10 significant biological process ontologies associated with genes under diversifying selection. Although anthelmintic exposure would be associated with significant stress on susceptible (and perhaps resistant or tolerant) parasites, all free-living stages of H. contortus will be exposed to and must tolerate abiotic factors such as temperature or humidity prior to infection of a new host. To evaluate the impact of such climatic stressors, a genome scan for genetic differentiation between isolates categorised by the climatic conditions prevailing at their sampling locations, i.e., arid (Namibia), temperate (France mainland) and tropical (Guadeloupe), was performed , and a second window (31.345–31.415 Mbp) was common to comparisons between tropical isolates and others (GUA vs. FRA.1 and GUA vs. NAM). Although the two annotated genes in the latter region were not associated with any biological description, the first window overlapped a chromo-domain containing protein  in D. melanogaster, a chromo-domain subunit of the Polycomb PRC1-complex that specifically recognises trimethylated lysine 27 of histone 3 (H3K27me3)33. Nucleotide diversity over these genomic windows revealed reduced genetic diversity in FRA.1 relative to isolates from hotter climates  was shared by all pairwise comparisons. Additional comparisons between populations with different histories, i.e., Australia, Indonesia, São Tomé, and Zaire also supported this signal . On chromosome I, eight (of the 13) associations with BIO12 fell within a 6 Kbp window , overlapping the region universally differentiated between climatic areas. Chromosome III contained 11 significant associations for both BIO7 (n = 6) and BIO12 (n = 5). The two most significant associations were also found on this chromosome for BIO7 . These two SNPs fell within the HCOI00198200 locus, which codes for a metallopeptidase M1, an ortholog of the C. elegans gene anp-1. Additional BIO7-associated SNPs were found within  and in the vicinity  of a solute carrier coding gene (HCOI00312500) on chromosome IV, whose orthologs in C. elegans are under the control of daf-12, a key player in dauer formation. Of note, two H. contortus orthologs of components of the dauer pathway in C. elegans, namely tax-4 (HCOI00661100) and daf-36 (HCOI00015800), were among genes under diversifying selection  were found with BIO7 and BIO12, respectively Fig. . Consist34. On the contrary, knowledge about their genetic diversity remains limited; a better understanding of population structure and selective pressure applied by environmental factors would yield better predictions of changes in their dispersion35. By exploiting a broad collection of samples from globally distributed isolates, together with chromosome-scale assembly and extensive individual resequencing, we have performed an in-depth characterisation of H. contortus genetic diversity, explored historical contributions to its current population structure and identified important drivers shaping the genome of this parasite.The ecology and epidemiology of gastrointestinal nematodes have been well characterised and exploited to build mathematical models to guide treatment decision in the fieldH. contortus populations displayed high levels of nucleotide diversity, consistent with early estimates based on mitochondrial data37, and recent resequencing experiments of inbred isolates13. These extreme levels of genetic diversity are thought to arise from both a large census population36 and the high fecundity of H. contortus females20.H. contortus population structure at the continent level37. Analyses of these data identified three to four  main phylogenetic clusters as well as evidence for the strong genetic connectivity between Australian, South-African and European isolates37. Our genome-wide data corroborated these early results. However, the use of a chromosome-scale assembly and individual resequencing contributed to our ability to identify genome-wide patterns of genetic diversity in its chromosomal context, and in turn, provided sufficient resolution to identify genes likely associated with selective advantage against drug or climate selection pressures. This had not been possible in previous work with AFLP markers37.An early attempt based on a set of genome-wide AFLP markers obtained for 150 individual worms from 14 countries supported the first exploration of 38, before introduction into Eastern Africa and subsequent spread, likely through cultural diffusion, toward southern Africa ~ 2–2.5 kya39. The timing of the bottleneck identified in our data and occurring between 2.5 and 10 kya is compatible with major migrations of pastoralist populations that ultimately resulted in the import of small ruminants into Southern Africa39. The simultaneous increase in rainfall in central Africa ~ 10.5 kya40 would have supported the population expansion and dispersal of H. contortus. In addition, early radiation towards Asia observed in our data was congruent with evidence obtained from the study of retroviral insertions within the sheep genome that suggested direct migration between Africa and southwest Asia41, and with the timing of Asian sheep expansion between 1.8 and 14 kya42.Major past human migrations and associated sheep movements have contributed to the mixing of parasite populations, which partly accounts for the limited genetic structure and extensive admixture between some of our globally distributed isolates. Our data supported an out-of-Africa expansion derived from ancestral populations from Western Africa, with a bottleneck dated back between 2.5 to 10 kya. Sheep domestication originally took place in the Middle East ~ 10 kya43, before West African breeds more suited to tropical conditions were transported, resulting in an admixed Caribbean sheep population43. The timing of genetic admixture here overlaps with centuries of human and presumably livestock movement during the transport of slaves, most of whom originated in West Africa and were transported to colonies that included the French West Indies21. The Mediterranean ancestry of H. contortus isolates in Guadeloupe and the close relationship with French worm populations suggest additional sheep transport occurred between French mainland and its Guadeloupian overseas territory. Although these movements are difficult to track precisely, live sheep were shipped aboard slave trade vessels departing from French harbours44, and may have introduced European H. contortus to the island. Based on historical data43, a Spanish lineage of H. contortus on Guadeloupe would also be expected to exist, though data are missing to confirm this. It can be speculated that the shared ancestry between Guadeloupian and Moroccan worm isolates might result from the introduction of Spanish Churra sheep, as this breed emerged in Spain while the region was under Arab influence between the 8th and 13th century45. Introduction of H. contortus-infected sheep from the Maghreb may have been associated with the spreading of African worms in Spain, and ultimately, Guadeloupe.The genetic congruence of parasites in Guadeloupe with those from both West Africa and the Mediterranean region is consistent with co-transportation of livestock, including sheep, during the discovery and subsequent colonisation of America. Woolly Churra sheep were originally brought to the Caribbean by Spanish conquistadors46; however, some evidence of shared ancestry between French and Guadeloupe resistant individuals emphasises the risk of spreading resistance without careful monitoring of parasite populations during livestock trade. Furthermore, this highlights the risk of evolution of resistance in other parasitic nematodes, for example, parasites of medical interest that are treated with benzimidazoles in mass-drug administration programmes8.The widespread use of anthelmintic drugs has been a major selective force shaping standing genetic variation in the isolates analysed. Adaptation to drug exposure was clearly illustrated in the genetic diversity surrounding the β-tubulin-coding gene, a target of benzimidazole drugs; strong loss of diversity was observed at this locus in many distinct isolates phenotypically characterised to be resistant to this class of drug. The co-occurrence of the same resistant haplotypes in geographically disconnected isolates is almost certainly owing to the independent evolution of benzimidazole resistance as has been described previously47. Worldwide emergence of ivermectin-resistant veterinary parasitic nematodes6 and evidence of reduced efficacy in human filarial nematodes9 underline the urgency and importance of a better understanding of the mechanisms involved47. Our data identified genes previously associated with ivermectin resistance in parasitic nematodes of either veterinary (pgp-11 in Parascaris sp27.) or medical (aex-3 in Onchocerca volvulus48) interest and uncovered candidates. Additional validation will be needed to determine whether these could serve as useful markers in the field to monitor drug efficacy. Of note, support for a major QTL for ivermectin resistance on chromosome V recently identified in a back-cross experiment in two resistant isolates49 was also present in our genome-wide data and warrants further investigation.Ivermectin is an important anthelmintic for parasite control in both veterinary and medical settingsH. contortus has enabled analyses into climate-driven adaptation in a parasitic nematode. Understanding how climate shapes parasite genetic variation is of primary importance to foresee consequences of climate change on parasite phenology and range dispersion. Parasite dispersal is largely driven by their hosts but H. contortus free-living stages experience climatic conditions that affect their development2 and constrain their spatio-temporal dispersal50. Observations in Northern Europe suggest that climate change has already altered H. contortus winter phenology50. Our analyses identified associations between genetic variation and bioclimatic variables on the one hand, and estimated genome-wide genetic differentiation between populations experiencing contrasting climates. Neither of these two analyses are ideal as bioclimatic variables are correlated and genetic differentiation is influenced by confounding factors such as population ancestry. Nevertheless, the correlation between bioclimatic variables was expected to lead to our analysis underestimating the respective contribution of the most important features51, but BIO12 and BIO7 were consistently ranked as the most important features. Genetic differentiation was conserved across various comparisons, suggesting the reported signal was relatively independent of population ancestry. Our results suggest that adaptation toward annual precipitation was mostly under the control of variation on chromosome I, however, no obvious candidate genes could be identified. In addition, the Hco-tbb-iso-1 locus was close to the region of interest, and linked variation in allele frequency at this locus as a result of benzimidazole selection cannot be ruled out. A second region of chromosome III was associated with both temperature- and precipitation-related variables, within which biologically relevant genes could be identified; first, the strongest genetic associations with annual temperature range were found in a metallopeptidase with zinc ion binding function (anp-1 ortholog), an enzyme linked to drought stress tolerance in Drosophila52, and second, an ortholog of Pc displayed strong genetic differentiation between arid- and wetter temperate or tropical environmental conditions. This gene has been linked to putative epigenetic regulation of xeric adaptation in Drosophila melanogaster, where Pc mutants display lower resistance to desiccation stress53. This finding is also corroborated by observations in plants showing Polycomb-mediated regulation of climate-induced phenotypes54. While in the absence of direct experimental evidence we cannot be definitive, we believe it is possible that this function of this Pc ortholog is conserved in nematodes. Domain prediction from its peptide sequence indicates a chromo- or chromo-shadow domain, supporting a role in chromatin-binding. In addition, the putative C. elegans homologue of this gene55, named cec-1, encodes a protein that recognises the repressive Polycomb-specific H3K27me methylation mark56. Knocking out the expression of this gene through RNA interference should help to support its role in the plastic response to climatic stress. Further links to climatic adaptation include identification members of the dauer pathway;57tax-4 and daf-36 orthologs were under diversifying selection and a daf-12-respondent solute carrier was associated with annual temperature range. Dauer is a developmentally arrested stage in C. elegans that is triggered by environmental stress and mediates tolerance to unfavourable conditions until better conditions are met57, and can occur in parasitic nematodes like H. contortus under semi-arid conditions58. Evidence for climatic adaptation suggests that adaptation in the face of climate change will be constrained by available genetic variability at temperature-selected loci, that may both limit or enable range expansion depending on the region. By a better understanding of the interaction between climatic conditions and phenotypes such as hypobiosis , optimisation of treatment timing may be possible to maximise control efficacy.Our broad sampling throughout the global range of H. contortus, and how this diversity has been shaped by adaptation to its environment and to drug exposure. Understanding the mechanism(s) by which parasites adapt to fluctuating environmental conditions both within and outside their hosts will have important implications for field management of parasitic nematodes in both veterinary and medical settings. Further characterisation of these putative strategies, together with genetic covariation of drug resistance genes, should contribute to refining epidemiological models and guide treatment decisions for more sustainable management of worm populations in the face of a changing climate.In summary, our data describe the extensive global and genome-wide diversity of the blood-feeding parasitic nematode H. contortus were obtained from a collection held at INRA17  were sampled to establish the connectivity between worm populations from Europe and these countries. Ivermectin-resistant isolates  were also retained to evaluate how anthelmintics had shaped H. contortus genomic variability (drug efficacy data have been provided in Supplementary Table n = 9) per isolate and proper allele frequency estimation. Following these criteria, 19 isolates from 12 countries were available  following the manufacturer’s instruction. Sequencing libraries were prepared using an amplification-free protocolftp://ngs.sanger.ac.uk/production/pathogens/Haemonchus_contortus) was performed using SMALT  with a median insert size of 500 bp, k-mer length of 13 bp, and a stringency of 90%. For samples that had two or more BAM files (when split across multiple sequencing lanes), the BAM files were merged using samtools v.0.1.19-4442860, and duplicated reads removed using Picard v.2_14_0 (https://github.com/broadinstitute/picard) before performing realignment around indels using Genome Analysis Toolkit (GATK v3.6)61 RealignerTargetCreator. Mean coverage of the mitochondrial and genomic genomes were estimated using GATK DepthOfCoverage, revealing coverage lower than the estimated target coverage  for most samples. Individuals with > 80% of their mitochondrial sequence with at least 15 reads and a mean mitochondrial genome coverage of at least 20× were retained for population genetic inferences .Read mapping to both the mitochondrial and nuclear genomes  and extraction of variants (SelectVariants), resulting in a total of 30,040,159 unfiltered SNPs across the five autosomes. Sex determination in https://gatkforums.broadinstitute.org/gatk/discussion/39/variant-quality-score-recalibration-vqsr), which first uses a reference (truth) SNP set to estimate the covariance between called SNP quality score annotations and SNP probabilities, followed by application of these probabilities to the raw SNPs of interest  using three independent SNP callers as described in the dedicated Ne trajectory through time, cross-population composite likelihood-ratio) that could not be performed under the probabilistic framework that relied on GLs as implemented in ANGSD62 v. 0.919-20-gb988fab . Realignment around indels was applied with GATK61 and SNP were subsequently called using samtools60 mpileup using only reads that achieved a mapping quality of 30 and base quality of 30. To exclude sites prone to heterozygous signals from further phylogenetic inference analysis, a SNP calling procedure was implemented with the HaplotypeCaller tool to apply hard filtering parameters on the raw SNP sets  with a minimum depth of 20 reads. This procedure excluded 1354 putative heterozygous sites, and retained 72% of the putative SNP sites (3052 out of 4234 SNPs). Nucleotide diversity and Tajima’s D were computed by sliding windows of 100 bp using vcftools v.0.1.1563. A PCA was performed on genotypes using the SNPrelate package64 in R version 3.565. A consensus fasta sequence was subsequently generated with GATK FastaAlternateReferenceMaker for each sample using the filtered variant set, which was used for the phylogenetic analyses.The mitochondrial genome exhibited an average coverage depth of 322×  per sample (Supplementary Data π) was computed for each isolate with at least five individuals using ANGSD62. Using GLs from samtools60 (option GL = 1) as an input, variants were included that had a minimal supporting evidence of five reads, and base and mapping quality phred scores of at least 20. As π values were biased by population mean coverage , Guadeloupe  and Namibia . A slight increase in Tajima’s D values was observed in the same samples following resequencing, i.e., increase in depth of coverage. However, only modest and negative correlations were found between window coverage and Tajima’s D, suggesting that coverage is not causing a systematic bias in estimates of Tajima’s D  = − 0.05, P = 0.55; Supplementary Fig. F66, considering the only individuals with a mean coverage above 2.5× as failure to do so yielded biased estimates  below 10–3. Clustering was robust to coverage variation and closely matched the PCA from the VQSR-called SNP genotypes  trajectory through time, and the cross-divergence time between populations, were estimated using MSMC273 as described in the The effective population size  with the following options: −w1 0.0001 500 2000 –p0 0. This fit a grid of putatively selected points every 2 Kbp along the genome, with a sliding window size around grid points of 0.01 cM, interpolating SNP position in Morgans from the average recombination rate for each chromosome13. Downsampling was applied to windows where >  500 SNPs were found to keep SNP numbers comparable between regions, and no LD-based down-weighing of the CLR scores was applied. A selection score was subsequently computed at every position as the root mean square of XP-CLR coefficients, and the highest 0.1% of selection scores were deemed significant.To further characterise the genetic diversity in Hco-btub-iso-1 were determined by blasting the gene coding sequence from WormBase Parasite75 against current genome assembly . The SNP positions associated with codons 167, 198 and 200 was determined to be at 7027535, 7027755 and 7027758 bp, respectively, along chromosome I after aligning the Hco-btub-iso-1 consensus sequence and published sequence (GenBank accession FJ981629.1) using muscle v3.8.3168. Genotype and GLs at these positions were determined with ANGSD62. Genotypes were only considered for GL > 60% (n = 74), as coverage bias occurred otherwise  following the same coalescent scenario as predicted by MSMC2 for chromosome I (considering a recombination rate of 1.83 cM/Mbp13). Suitable ms input parameters were derived from MSMC2 output files using the msmc2ms.py script from msmc-tools. This approach was implemented for isolates with sufficient mean depth of coverage, i.e., three benzimidazole-resistant isolates. In case of susceptible isolates, the lack of significant departure in the Hco-btub-iso-1 vicinity relative to the rest of chromosome I was tested.The genomic coordinates of 60% n = 7, as cove77. This method computes phylogenetic trees between a set of isolates, using genetic information from short sliding windows (50 Kbp) and by sampling with replacement individuals from each isolate. At each window, a weight is subsequently computed for each of the possible tree topology, ultimately providing inference of gene flow events in discrete locations where tree topology connects phylogenetically distant isolates. Analyses were run using individuals with sufficient mean depth of coverage (5×and more) from French  and Guadeloupian  isolates, adding Namibian isolate  as an outgroup. A fourth isolate was chosen for its common ancestry with Guadeloupian isolates: first, an analysis was run with worms from São Tomé , followed by a second analysis using Moroccan samples , to ensure that evidence of gene flow was consistent in both cases.The introduction of resistant haplotypes from France mainland into Guadeloupian worm isolates was tested by a topology weighting analysis implemented with TWISSTST estimates were calculated every 10 Kbp with a 1 Kbp overlap along the genome with ANGSD62, retaining sites with minimal depth of five reads, mapping and base quality phred scores of at least 30, missing rate below 50%, and windows with at least 1000 sites. Genomic coordinates of the top 0.5% most-differentiated windows were extracted and analysed by BLASTN  against the published V1 H. contortus assembly14, and annotated gene identifiers were inferred from the corresponding GFF file from WormBase Parasite75.To investigate the genetic architecture of ivermectin resistance, a differentiation scan was run between ivermectin-resistant isolates and isolates of unknown status sharing same ancestry, i.e., STA.1 and STA.3 vs. NAM and ZAI in Africa, AUS.1 against AUS.2 in Australia. Windowed FST using ANGSD62, of which test values >0.5% quantile in at least two comparisons were analysed further.To identify SNPs putatively influenced by environmental selection, we first defined the climatic conditions of each isolate using the Köppen–Geiger classification inferred from isolate geographical coordinates. Three isolates with the best coverage, i.e., Namibia, France, and Guadeloupe, were used to contrast dry, temperate and tropical conditions, respectively. We performed a genome-wide scan using pairwise comparisons of F78 to quantify changes in genetic variation (fit as population SNP MAF) along environmental gradients. The gradients consisted of 19 bioclimatic variables79, summarising rainfall and temperature information recorded between 1970 and 2000 . SNP MAFs were estimated from genotype likelihoods with ANGSD, and subsequently filtered to ensure a within-isolate MAF > 10%, at least 90% within-isolate call rate, and shared across the eight considered isolates, resulting in 3758 SNPs retained for further analysis.80 on genotype calls from the VQSR pipeline, considering SNPs with call rate of at least 70% across isolates and minor genotype frequency above 5%, using K = 3 for the latent factor accounting for underlying population structure. Analyses were run five times and P values were combined and adjusted as recommended80.Environmental variables were highly correlated  against the published V1 H. contortus genome assembly14 using BLAST v2.2.25. Any genes falling within most probable blast hit coordinates were retrieved from the H. contortus published assembly available from WormBase Parasite75. This database was also used to retrieve C. elegans orthologs of positional candidate genes and H. contortus Gene Ontology (GO) terms. GO term enrichment analysis was run with the R topGO81 package, considering nodes with at least 10 annotated genes. The “weight01” algorithm was used to account for existing topology between GO terms. This framework makes P value of one GO term conditioned on its neighbours, thereby correcting for multiple testing. Enrichment was tested by the Kolmogorov–Smirnov statistic applied to gene selection or FST score accordingly. GO terms with P values below 1% were deemed significant.Annotation of the revised Further information on research design is available in the Supplementary InformationPeer Review FileReporting SummaryDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5


Clostridium perfringens epsilon toxin (Etx) is categorized as the third most lethal bioterrorism agent by the Centers for Disease Control and Prevention (CDC), with no therapeutic counter measures available for humans. Here, we have developed a high-affinity inhibitory compound by synthesizing and evaluating the structure activity relationship (SAR) of a library of diverse glycosides (numbered 1-12). SAR of glycoside-Etx heptamers revealed exceptionally strong H-bond interactions of glycoside-4 with a druggable pocket in the oligomerization and β-hairpin region of Etx. Analysis of its structure suggested that glycoside-4 might self-aggregate to form a robust micelle-like supra-molecular complex due to its linear side-chain architecture, which was authenticated by fluorescence spectroscopy. Further, this micelle hinders the Etx monomer-monomer interaction required for oligomerization, validated by both surface plasmon resonance (SPR) and immunoblotting. This phenomenon in turn leads to blockage of pore formation. Downstream evaluation revealed that glycoside-4 effectively blocked cell death of Etx-treated cultured primary cells and maintained cellular homeostasis via disrupting oligomerization, blocking pore formation, restoring calcium homeostasis, stabilizing the mitochondrial membrane and impairing high mobility group box 1 (HMGB1) translocation from nucleus to cytoplasm. Furthermore, a single dosage of glycoside-4 protected the Etx-challenged mice and restored normal function to multiple organs. This work reports for the first time a potent, nontoxic glycoside with strong ability to occlude toxin lethality, representing it as a bio-arm therapeutic against Etx-based biological threat. Summary: The authors developed a potent, glycoside-based inhibitor with strong ability to occlude epsilon toxin lethality both in vitro and in vivo. Clostridium perfringens epsilon toxin (Etx) has been categorized as the third most potent toxin after botulinum neurotoxin (BoNT) and anthrax, and is a classified type B agent. Out of five strains (A-E) of C. perfringens, epsilon toxin is secreted by toxinotypes B and D. It causes fatal enterotoxaemia, also called the kidney pulp disease, in domestic ruminants, resulting in heavy economic losses  , Clostric losses . Apart fc losses . If an Ec losses . This sic losses . The sugll death . Additioenotypes . As per enotypes , domain enotypes , formalienotypes , have bein vitro and in vivo reports glycoside-4 as a potential antidote against Etx and can be further developed as a first line of defence against bio-terrorizing agents in humans.To address this issue, using a green synthesis route we have synthesized a library of D-glucose- and D-glucosamine-derived glycosides with a hydrophilic head group and different chain lengths of the tail (lipophilic group). These molecules were found to be completely non-toxic in mammalian cells as well as in animal models. Screening for anti-Etx activity both + IR-120, which gave us the desired product glycoside-1 as an anomeric mixture in good yield as described in the scheme for synthesis . This synthesis followed a green route to prepare desired glycoside as it does not involve any further purification and amberlite resin can be reused by activating it with 1 N HCl in MeOH. Similarly, other compounds in this series (glycoside-2 to glycoside-6) were prepared by adopting similar reaction protocol , which resulted in moderate to good yields . The alkyl glycosides of D-glucosamine glycoside-7 to glycoside-12 were prepared by refluxing D-glucosamine-2 with corresponding alkyl alcohols for 24 h in the presence of amberlite-H+ IR-120 resin, which gave the desired products as an anomeric mixture in good yield as shown . This synthesis was simple: no further workup was required and the used amberlite resin was reusable after activating it with 1 N HCl in MeOH. Further characterizations of all 12 compounds (Table S1) were done using nuclear magnetic resonance (NMR) (supplementary text).The glycoside-derived compounds were designed and synthesized using the commercially available D-glucose-1 and D-glucosamine-2 molecules, which were coupled along with various short- to long-chain alcohols under acidic conditions. The ethyl glycoside D-glucose glycoside-1 was prepared by refluxing in ethanol for 24 h in the presence of amberlite-HC. perfringens Etx was obtained from the Protein Data Bank (PDB) database . Since the crystal structure of the Etx pre-pore was unavailable, we constructed the heptameric pre-pore form, exactly mimicking its active and pre-pore-forming state. Since the inactive protoxin has to be activated by proteolytic removal of the 11-13 and 22-29 residues from the N- and C-terminus, respectively (−1) was calculated. The root-mean-square distance (RMSD) and root-mean-square fluctuation (RMSF) curves were found to be devoid of any severe fluctuations, representing a stable heptamer . The intra-molecular bonding and hydrophobicity analysis of this Etx heptamer revealed predominant existence of amphipathic residues constituting a druggable pocket within domain II (in silico docking of 12 glycoside derivatives with the Etx monomer (PDB: 1UYJ), to understand whether these compounds could bind to the monomeric form as well. The result demonstrated relatively weaker binding  within the pocket . Following this, we performed in silico docking of 12 glycoside derivatives with the Etx heptamer. The docked heptameric Etx-ligand complexes revealed stronger binding energies  localized within this pocket  as compared to the monomeric Etx-ligand .The crystal structure of ectively , we haveectively . To elucomain II C, which Fig. S2D,E). Since MDCK is highly sensitive to Etx, we have evaluated the impact of all 12 glycoside derivatives for anti-toxin activity in Etx-challenged MDCK cells by using the mitochondrial membrane potential (ΨΔm) indicator MTT   showed greater than 50% survival compared to Etx . Further, all three glycosides showed concentration-dependent protection of Etx-treated MDCK cells. Glycoside-4 was found to be the lead molecule among them, with highest survival efficiency  concentrations . The results demonstrated a total of ten strong intermolecular H-bonds between the pocket residues and glycoside-4. The threonine residues at 138 and 193 could make three H-bonds each, while Glu191, Cys139, Asn195 and Val196 made a total of four H-bonds with glycoside-4 (one each) . Upon optimization of the 3D grid parameters, the lead compound, glycoside-4, showed higher binding affinity  and its best docked conformation was further analyzed for polar contacts, including salt bridges and H-bonding (Tables S2 and S3). The results demonstrated ten intermolecular H-bonds (2.6-3.1 Å) and few intramolecular salt bridges within the defined pocket of ligand-bound complex (Tables S2 and S3). In contrast, we obtained only one H-bonding when we docked the Etx monomer to glycoside-4 . We assume that strong binding of glycoside-4 within the domain II region of Etx monomers in the heptamer might introduce steric hindrance and might block the process of the oligomerization, leading to destabilization of β-barrel insertion.To understand the structure-activity relationship of glycoside-4, we performed docking of glycoside-4 to the annotated amphipathic pocket present in domain II of Etx (ne each) C. This cFig. S4A). These morphological changes are often associated with alterations in the cell architecture such as cell-cell and cell–extracellular-matrix (ECM) interactions that determine the barrier integrity, measured by transepithelial electric resistance (TEER) . Further, to observe the membrane changes in real time, we performed live-cell video microscopy. The cells displayed prominent swellings followed by membrane blebbing. Subsequently, the blebs grew bigger , validating the cellular intoxication imposed by purified Etx used in our study. Since structural insights indicated strong binding of glycoside-4 to the annotated region for oligomerization and membrane insertion in the druggable pocket of Etx, we hypothesized that glycoside-4 might block the oligomerization and/or insertion in vitro.Since the heptameric pre-pore complex of Etx employs the β-barrel hairpin structure to make pores in the plasma membrane , we hypoe (TEER) . To assein silico observation, which suggested that glycoside-4 has weaker binding affinity for Etx monomers . Next, to evaluate whether glycoside-4 can also impact oligomerization, we incubated the Etx along with MDCK in the presence or absence of glycoside-4 at 37°C for 1 h, as oligomerization occurs at the same temperature in vitro  to MDCK cells. To prove this, we treated MDCK cells with Etx alone or in combination with glycoside-4 for 1 h at 4°C, followed by western blotting of the crude MDCK pellet. The results indicated that glycoside-4 had no effect on the binding of Etx monomer to the cell membrane  value demonstrates the onset of the formation of micellar assemblies . The data showed that Etx could induce Ca2+ similar to ionomycin, a positive control. Treatment with EGTA, an extracellular Ca2+ chelator, blocked the Etx-invoked influx  staining in both pre-treatment (incubation of Etx with glycoside-4) and post-treatment (without incubation of Etx and glycoside-4) conditions. The results show an intact cellular architecture  residues on the outer cell surface . Furtherc stages A. Upon tI+ cells A, nullifI+ cells B.Fig. 4Another feature of necrosis is the disruption of ΨΔm, which is often correlated with reactive oxygen species (ROS) production and subsequent cell death . Hence, Similarly, glycoside-4-treated cells exhibited decreased levels of ROS, as shown by diminished green staining compared to Etx treatment E,F. ROS 2+ levels, followed by mitochondrial depolarization, ROS generation and HMGB1 release from the nucleus to cytoplasm. In contrast, glycoside-4 rescues Etx-treated MDCK cells by inhibiting all the characteristic features of cell death.Taken together, our data are in line with the previous studies  that Etx50), of purified Etx intraperitoneally (i.p.). Initially, to evaluate the toxicity of glycoside-4 in mice (n=5), doses ranging from 50 to 250 mg/kg body weight were administered (i.p.) and monitored for 48 h. The control group received only PBS (n=5). No mortality or clinical signs  were detected in both the groups during this period. The protective efficacy of the compound was determined by treating the mice with glycoside-4 (50 mg/kg body weight) and 4×LD50 dose of the toxin (n=15). The Etx mice received the same toxin dose  and the animals were observed until 48 h post-injection. All the Etx mice died between 15 and 20 h , whereas a 40% (n=6/15) death rate was seen in mice treated with Etx and glycoside-4 (n=15/15), which mainly affected the kidney, causing enterotoxaemia, followed by brain and liver damage. The changes in the brain included necrosis of neurons, focal gliosis, axonal degeneration . Swelling of brain was detected in Etx-treated mice, whereas the glycoside-4-treated mice showed intact brain structure, comparable to healthy brains. Similar improvements were observed in glycoside-4-treated liver . Only minimal changes, such as a slight increase in glomerular volume and narrowing of the Bowmen's capsule, were seen in glycogen-4 treated kidneys , with the entire tissue structure remaining largely intact. These findings indicate that glycoside-4 can protect multiple affected organs of C57BL/6 mice from Etx-induced lethality.To evaluate the effect of glycoside-4 in Etx-challenged C57BL/6 mice, we injected 150 ng/kg body weight, a median lethal dose  and perfringolysin O (PFO) toxin, responsible for gas gangrene (3 (Gb3-PEDP) and galabiosyl (Gb2)-PEDP neutralize Stx cytotoxicity. This neutralization mechanism involves formation of liposomes and clustering of sugar units . It is noteworthy that the β-barrel structure of domain II plays a major role in Etx-mediated pore formation; thus, we hypothesized that targeting domain II might lead to effective blocking of Etx activity.Structural insights have revealed three crucial domains in Etx: (1) membrane binding, (2) oligomerization and pore formation, and (3) monomer-monomer interaction . Previougangrene . Other ggangrene . Anotherar units . Taking Fig. S2A). During the revision of this paper, the Etx pore structure was published , screening for anti-Etx activity revealed glycoside-4 as the lead molecule . Thus, micelle of glycoside-4 might enforce strong steric hindrance on the heptameric form of Etx following binding to the residues in the β-hairpin, leading to abrogated oligomerization. Further, analysis of oligomerization in vitro corroborated this in silico docking analysis, as the formation of heptamers was substantially impaired in Etx-challenged MDCK cells in the presence of glycoside-4, as evidenced by reduced intensity of the heptameric complex in immune blot .Next, to evaluate whether blocking of oligomerization by glycoside-4 can also abolish pore formation, we added Etx treatment with glycoside-4 to liposomes and analyzed pore formation using TEM B. Intere2+ influx is often correlated with the induction of apoptosis . Along with this, the model also displayed other features of cell death, including membrane blebbing , increased annexin V+/PI+ staining, destabilization of ΨΔm and translocation of HMGB1 from the nucleus to the cytoplasm. Interestingly, upon treatment with glycoside-4, all these features of cell death got reversed, leading to healthy cellular homeostasis  of the mice challenged with 4×LD50 of Etx with a single dosage  exposure (within 30 min), which was approximately ten times higher than in mice exposed to Etx at the same concentration via i.p. The LDted LD50 . This detrations . Treatmee dosage A. Moreove dosage B. To our+ resin (400 mg) was added to a pre-stirred solution of glucose 1  in neat alcohol (5-10 ml). The resulting mixture was stirred at 100°C for 24 h. After completion of the reaction, reaction mixture was cooled down to room temperature, and filtered to remove the resin. The filtrate was evaporated under reduced pressure to obtain compound glycoside-1 to glycoside-6 as white solid in an acceptable to good yield. Amberlite IR 120-H+ resin (400 mg) was added to a pre-stirred solution of N-acetyl glucosamine-2  in neat alcohol (5-10 ml). The resulting mixture was stirred at 100°C for 24 h. After completion of the reaction, reaction mixture was cooled down to room temperature, and filtered to remove the resin. The filtrate was evaporated under reduced pressure to obtain compound glycoside-7 to glycoside-12 as white solid in an acceptable to good yield. 1H NMR and 13C NMR spectra in CD3OD and D2O were recorded on a Bruker 400 MHz spectrometer at ambient temperature (1H recorded by 400 MHz and 13C recorded by 100 MHz). Proton chemical shifts are given in ppm relative to the internal standard (tetramethylsilane) or referenced relative to the solvent residual peaks (CD3OD: δ 3.31). Multiplicity was denoted as follows: s (singlet); d (doublet); t (triplet); q (quartet); m (multiplet); dd (doublet of doublet); dt (doublet of triplet); td (triplet of doublet); ddd (doublet of doublet of doublet), etc. Coupling constants (J) were reported in Hz. Column chromatography was performed by using silicagel 100-200 and 230-400 mesh. High resolution mass spectrometry (HRMS) spectra were determined from a quadrapole/Q-TOF mass spectrometer with an ESI source (supplementary text).Amberlite IR 120-HE. coli M15 cells harbouring the pQE60 Etx were induced with 1 mM isopropyl-β-D-thiogalactoside (IPTG) for 5-6 h at 37°C. The soluble fraction containing Etx was purified using (DEAE)-Sepharose anion exchange chromatography . The proto-toxin was further activated to mature toxin (active form) by treatment with trypsin (The purification of Etx was carried out as described elsewhere . In gene trypsin . The proThe Swiss PDB viewer software was utilized for construction of the heptameric unit. Using aerolysin toxin structure as a template (PDB: 5JZT), the monomer units were aligned at similar angles, which were rendered stable after energy minimization. We also used Super-align and TM-align commands on Pymol to structurally align the heptamer of 5JZT, the Cryo-EM structure of Aerolysin pore. 5JZT was aligned to the seven-monomer collection of 1UYJ, thus translating the monomers at similar angles to obtain a heptameric unit. Each heptameric unit of the protein was correctly labelled as chains A-G using in-house scripts. The amino acids were numbered according to UniProt (Q57398_CLOPF). The heptameric molecule was energy-minimized in MOE using CHARMM force field. The lead compound, glycoside-4, was used for further studies. All 12 compounds were drawn through the Sketch module of Molecular Operating Environment (MOE) .+ and Cl−) was added by -neutral option to neutralize the whole protein. The protein and non-protein groups were energy minimized with a tolerance of 1000 kJ mol−1 nm−1 using the steepest descent method for 50,000 steps. All the bonds were constrained using the LINCS algorithm, and the simulation was performed under isothermal-isobaric ensemble (NPT) and canonical ensemble (NVT) conditions, using the v-rescale coupling algorithm and the Parrinello-Rahman coupling algorithm, which stabilized the temperature and pressure . The smooth particles mesh Ewald (PME) method was used with a cut-off of 1.4 nm for electrostatic and van der Waals (vdW) interactions. The electrostatic interactions were calculated with PME using a grid spacing of 0.12 nm. Periodic boundary conditions (PBCs) were employed to eliminate surface effects. The final MD simulations were carried out with a time step of 2 fs and without any position restraints; 17,500,000 steps were performed for a total of 40 ns. The structural deviations were analyzed by computing RMSD and RMSF. The RMSD and RMSF graphs were plotted using XMgrace software .MD simulations of both the energy-minimized monomer and heptameric models of Etx were performed in order to analyze the stability of the modelled oligomeric structure. We used GROMACS version 5.06, which is a versatile collection of programs and libraries for simulating molecular dynamics of large proteins and subsequently analyzing trajectory data. The MD simulation was carried out on a parallel processing internal server (Magus), which is a 60-node, 992-core IBM high performance cluster (HPC). The GROMOS 96 53a6 force field including all hydrogens, along with a simple point-charge (SPC) water model, was used for energy minimization of both monomeric and oligomeric models. The pre-equilibrated SPC water was added to a dodecahedral box, and the individual protein was then placed in the centre of the box. In total, 4,677,864 solvent molecules were externally added into the box in order to solvate the system. Furthermore, an optimal number of ions (NaIn accordance with a hydrophobic pocket within the monomeric structure of Etx (PDB: 1UYJ), a virtual grid was constructed for docking the compounds using the Autogrid module of AutoDockTools. This 3D grid was of 26×28×30 Å measured volume with default spacing and exhaustiveness level of 0.375 Å and 8, respectively. The exhaustiveness level was kept high to increase the time linearly and decrease the probability of not finding the minimum exponentially. The top-ranked poses of the protein with the compound were selected based on the lowest free binding energies. In accordance with proposed active sites lying in domain II of Etx, a virtual grid/space was constructed covering at least two monomeric units for docking the compounds using the Autogrid module of AutoDockTools. This 3D grid was of 40×38×36 Å measured volume with default spacing and exhaustiveness level of 0.375 Å and 8, respectively. We performed molecular docking studies of glycosides 1-12 via Autodock Vina to rationalize its activity against Etx for the monomer as well as heptamer. For all molecular visualization and H-bond analysis of the proteins/ligands and docked protein-ligand complex, PYMOL 1.7.4 software was used.The MDCK cell line was maintained in DMEM (Gibco) supplemented with 10% FBS (Gibco) and 1% penicillin/streptomycin (Gibco).6 cells/well to analyze the cytotoxic effects of purified Etx. Serial dilutions of the Etx mixed in DMEM containing 10% FBS (Gibco) and 1% antibiotic (Gibco) were incubated with MDCK cells in triplicates for 1 h at 37°C. The control wells had an equal volume of media. Cell viability was measured using MTT  (Himedia) and the absorbance was read at A550 using Bio-Rad iMark microplate reader (Bio-Rad). Untreated cells were taken as 100% cell survival, and the viability for Etx-treated cells were calculated accordingly. In the case of pre-treatment, glycoside-4 and Etx were incubated for 30 min before addition onto the cells, whereas, in the case of the post-treatment condition, glycoside-4 and Etx were added to the cells simultaneously.MDCK cells were cultured in 96-well plates at a density of 0.2×102 and 250 µm active gold-plated electrodes. Once the resistance reached a plateau, the cells were washed twice with DMEM containing antibiotics and subsequently the active Etx was added. The data was collected at 4000 Hz using ECIS Zθ (Applied Biophysics) for 24 h post-treatment and analyzed using the Applied Biophysics (abp) software.MDCK cells were grown to confluence in the 8W10E (Applied Biophysics) well arrays with each well having an area of 0.8 cm4 grown on glass-bottom culture dishes (ibidi) were treated with Etx and the morphological changes were observed in real time using a Nikon confocal microscope (Nikon A1R). To access calcium influx and PI positivity, the cells were initially washed twice with PBS followed by addition of Fluo-4 AM  and PI  to the media. The Etx was added, and the Ca2+ and PI changes were observed under a confocal microscope equipped with a temperature-controlled stage . The time-lapse images were captured using a 1.4 numerical aperture lens and analyzed by Nikon ES elements software.MDCK cells with a density of 2×10g for 5 min at 4°C. The crude pellet fraction was boiled along with SDS dye and continued with the western blot using anti-Etx (1:500) as the primary antibody followed by anti-mouse HRP  as the secondary antibody. The blot was developed using Amersham ECL detection reagents . Similarly, to detect the formation of oligomers, the same protocol was used in which MDCK cells were incubated with Etx and/or with glycoside-4 for 1 h at 37°C instead of 4°C followed by western blotting. This protocol has been modified from To detect membrane binding, the cells were grown to confluence in a six-well plate and incubated with Etx and/or with glycoside-4 for 1 h at 4°C. The cells were washed once with PBS and scraped in the same buffer. The harvested cells were lysed by two cycles of freezing and thawing, followed by centrifugation at 8000 All the phospholipids and cholesterol were obtained from Sigma Aldrich. Liposomes were prepared by extrusion method using dipalmitoylphosphatidyl choline (DPPC) and cholesterol at 45:50 mol% as described .A total of 0.5 mg/ml lipid mixture containing DPPC and cholesterol at 50:50 mol% in buffer [20 mM HEPES (pH 7.4) and 40 mM NaCl] was treated with Etx alone or along with glycoside-4 for 30 min at room temperature. After incubation, the liposomes were transferred to coated electron microscope grids and negatively stained with 1% (w/v) urinyl acetate . ElectroTo determine the Etx interactions as well as glycoside-4 interaction with Etx, SPR was performed using Auto Lab Esprit SPR. Etx (7.8 μM) was immobilized on the surface of a nickel-charged NTA SPR chip. Interaction analysis was studied by injecting Etx, glycoside-4 and Etx along with glycoside-4 over the chip surface, with an association and dissociation time of 300 and 150 s, respectively. HEPES buffer was used both as immobilization and binding solutions. The surface of the sensor chip was then regenerated with 50 mM NaOH solution. Data were fit by using Auto Lab SPR Kinetic Evaluation software provided with the instrument.MDCK cells were loaded with Fluo-4 AM and, after an incubation period of 30 min in DMEM medium, cells were washed. Etx was added to MDCK cells, and the increase in fluorescence was analyzed by flow cytometry by FACS Calibur (Becton & Dickinson) using Cell Quest software. Calcium ionophore (Sigma) was used as a positive control. For assessment of extracellular calcium, the assay was performed in the presence and absence of 1 mM EGTA (Sigma). To analyze inhibition by anti-Etx antibody, Etx was incubated with anti-Etx antibody or pre-immune antibody, prior to addition to MDCK cells. Similarly, to check for the inhibition by glycoside-4, Etx and glycoside-4 were incubated prior to addition to MDCK cells and analyzed by FACS Calibur (Becton & Dickinson).MDCK cells were treated with Etx and/or glycoside-4 and incubated for 1 h at 37°C. The cells were washed with PBS and stained with annexin V-FITC  and PI  and imaged. Similarly, staining for the ΨΔm indicator JC-1  was carried out after incubation with Etx in the presence or absence of glycoside-4 for 1 h using the manufacturer's protocol and imaged using FITC and TRITC filters under the Nikon Ti microscope (Nikon).4 MDCK cells were grown on the glass-bottom culture dishes and incubated at 37°C overnight. The cells were treated with Etx or in combination with glycoside-4 for 1 h at 37°C. After incubation, the cells were washed and stained with DCFDA (2-7-dichlorodihydrofluorescein diacetate)  followed by imaging at 485 nm excitation filter and 535 nm emission filter under the Nikon Ti microscope (Nikon).A total of 2×10MDCK cells were grown on the sterile coverslips (Borosil) and incubated for 12 h at 37°C. The cells were treated as described in the culture section, and the coverslips were fixed with 2.5% paraffin. HMGB1  was used as primary antibody followed by addition of anti-rabbit Alexa Fluor 594  as the secondary antibody. The coverslips were mounted on a glass slide using DAPI antifade  and imaged under the Nikon Ti microscope (Nikon).ad libitum access to food and water, and were kept under standard laboratory conditions . The Animal Ethics Committee of the institute (NII) approved the animal usage and procedures (IAEC Code #288/11). The control mice received only PBS and all the dilutions were prepared in PBS. The route for injection was intraperitoneal (i.p.) in all the cases.BALB/c mice (4-5 weeks) were injected with the activated Etx (10 μg/mouse) and the anti-sera was collected. C57BL/6 mice weighing about 20-25 g (5-7 weeks) of both sexes were used for the Etx and glycoside study. All the animals were obtained from the animal house of the National Institute of Immunology (NII), Aruna Asaf Ali Marg, New Delhi, India. The animals had 50 of the toxin preparation, we used serial dilutions of toxin ranging from 1 µg/mouse to 0.01 µg/mouse. A total of eight mice received 200 µl of toxin (i.p.) of each dilution in PBS. Negative control mice were treated with PBS only. All the animals were monitored for 72 h post-injection. For the challenge experiments, the Etx+glycoside-4 group (n=15) received a single dose of 2×LD50, 4×LD50 and 6×LD50 of the toxin (i.p.) along with the 50 mg/kg body weight drug. The Etx control and PBS control were kept in all the cases. All the animals were monitored for 48 h post-injection. The mice were sacrificed at the end of the duration by cervical dislocation, and the brain, kidney and liver were removed for H&E staining.In order to determine the LDThe brain, kidney and liver from all the groups  were removed rapidly and stored in freshly prepared 10% neutral buffered formalin (Sigma), processed and embedded in paraffin. The sections were 3 µm thick and cut on the sagittal plane. These were stained with H&E for histopathological alterations. All the images were taken using the Nikon light microscope and processed using the Nikon software.t-test was performed wherever applicable. P-values of <0.05, <0.01 and <0.005 were considered significant . Results represent mean±s.d. of at least three independent experiments.One-way analysis of variance (ANOVA) followed by Bonferroni test was used for comparison. When appropriate, Student's


Hypera postica) is a well‐known example of a worldwide‐distributed pest with high genetic variation. Based on the mitochondrial genes, the alfalfa weevil clusters into two main mitochondrial lineages. However, there is no clear picture of the global diversity and distribution of these lineages; neither the drivers of its diversification are known. However, it appears likely that historic demographic events including founder effects played a role. In addition, Wolbachia, a widespread intracellular parasite/symbiont, likely played an important role in the evolution of the species. Wolbachia infection so far was only detected in the Western lineage of H. postica with no information on the infecting strain, its frequency, and its consequences on the genetic diversity of the host. We here used a combination of mitochondrial and nuclear sequences of the host and sequence information on Wolbachia to document the distribution of strains and the degree of infection. The Eastern lineage has a higher genetic diversity and is found in the Mediterranean, the Middle East, Eastern Europe, and eastern America, whereas the less diverse Western lineage is found in Central Europe and the western America. Both lineages are infected with the same common strain of Wolbachia belonging to Supergroup B. Based on neutrality tests, selection tests, and the current distribution and diversification of Wolbachia in H. postica, we suggested the Wolbachia infection did not shape genetic diversity of the host. The introduced populations in the United States are generally genetically less diverse, which is in line with founder effects.The alfalfa weevil ( For the first time, we mapped the current distribution of the two lineages of alfalfa weevil in its native and invasive distribution range covering North America, Europe, the Middle East, and Eastern Asia. We further report the degrees of intra‐and interpopulation divergence and conducted neutrality analyses to test for signs of selection potentially caused by Wolbachia. Finally, we report the degree of infection for all populations and discuss its potential role for the genetic diversification of the host species.In order to fill this knowledge gap, we generated a large molecular dataset  and Chinese milk vetch , respectively; Lozitsa (Bulgaria) samples were collected from Bird vetch (Vicia cracca L.). Samples were preserved in pure ethanol and stored in a freezer at −20°C until further processing.Specimens of H. postica. Further, one specimen of Hypera mele  , three specimens of Hypera viciae  , and one specimen of Brachypera zoilus    and ClustalX . Two mitochondrial (COI and CytB) and two nuclear genes (EF1a and CAD) were amplified with specifically designed primers  or by demography events . Three groups were defined for these tests: (a) Western lineage versus Eastern lineage (b) Eastern endemic infected (Bulgaria) versus Eastern endemic uninfected , (c) Eastern infected (Bulgaria) versus Eastern uninfected .Estimates of genetic diversity the number of segregating sites (S), number of haplotypes (H), haplotype diversity (Hd), the average number of nucleotide differences (K), nucleotide diversity (pi), singleton variable sites (V), and parsimony informative sites (P) were assessed using DnaSP v. 4.0  for Eastern and Western lineages observed on the phylogenetic tree was measured with CodeML implemented in the PAML package Yang,  of PamlX2.4p value >.05). We ran MrBayes for 10 million generations, sampling every 1,000 generations yielding a total of 10,000 trees; the first 25% of trees were discarded as burn‐in. Convergence was checked using the average standard deviations of split frequencies, which were below 0.005 and effective sample sizes (above 200). Trees were visualized and edited with FigTree v. 1.4.2 . Automatic barcode gap discovery  and the wsp locus  , the Wolbachia strain type (profile) of each positively infected weevil was determined. We obtained an additional 26 Wolbachia sequences for phylogenetic comparison from GenBank and the MLST database in order to confirm the Supergroup of the alfalfa weevil Wolbachia strains.  revealed a deep divergence between the two lineages  (Table ST = .041), and higher between the Japanese and Koran Western lineage (ΦST = .244). Low genetic differentiation was also detected among American Western lineage populations. This, however, may at least partially be an artifact due to insufficient sample size; we did not find evidence of genetic differentiation between Western lineage populations from Europe and American Western lineage populations.In Iran, genetic differentiation was low (.21 > Φ5) Table ; the samST estimates and showed that <2% of the genetic variance was found among populations (see among population comparison in Table AMOVA supported the results of ΦWolbachia infected populations of the Eastern lineage (Bulgaria) .Tajima's D, Fu's F, and Li's D tests on the mitochondrial genes Table A rejectea) Table . Similar3.5p‐value = .17) and were used jointly to construct a phylogenetic tree. In addition to the high support of each lineage, Western lineage sample of Lozitsa (Bulgaria) formed a single supportive clade against other Western lineage samples  were the Western and Eastern lineages with a deep gap   and among them, coxA, fbpA, and wsp were successfully amplified for 35 specimens. Analyses of the sequence data suggested that three different Wolbachia strains of Supergroup B infected the samples. The first one (subsequently named WHypera1) was the predominant one and appeared in 57 samples. WHypera2 was detected in one sample from California differs only by one base pair from WHypera1 (gatB gene). WHypera3 was detected in a sample of the America‐Montana population (HP6033) and has 5.6% and 6.9% difference for coxA, and fbpA and only one base per difference for gatB.PCR products of ARM primers showed strong infection in positive controls, but not in our samples, indicating a wHypera1 and wHypera3 in one sample. However, based on the limitation of our methodology, we can only suggest the possibility of double infection. The most frequent Wolbachia types wHypera1 and wHypera2 have not been previously reported (based on blast in both GenBank and MLST database in 01.05.2019). The single gatB sequence of wHypera1 we were able to generate is compatible with MLST allele number 9. The Bayesian tree based on three MLST genes, supported by ClonalFrameML approach  resulting from recent host shifts in a cosmopolitan host species, rather than a uniform infection of a certain strain across the host's range  were infected with the same Wolbachia strain. WHypera1 might be found more widely within Hypera and other related genera. However, it is also possible that Wolbachia is horizontally transmitted to H. vicieae as they both can coexist in the same fields.4.3Wolbachia drives or demographic events.The low genetic diversity of the Western lineage can be an intrinsic character of this lineage. The recent evolved clades usually are observed with lower genetic diversity compare to the elder (ancestor) clade Tilmon, . AlthougWolbachia and the beetle hosts may have an unstable relationship leading to the lack of a clear effect of Wolbachia infection on the host's ecology and phylogeny , but also, such with high genetic diversity  may be infected with Wolbachia. Further, we did not detect any signs of selection on mitochondrial DNA in most populations regardless of their genetic diversity and lineage  rather than that of Wolbachia infection.The question remains whether ge Table . FinallyWolbachia may have had a negative effect on genetic diversity. Among populations with sufficient individuals, the two Montana populations have one of the highest infection rates (Table Wolbachia may have caused linkage disequilibrium between mitochondrial and nuclear genes. Such Wolbachia effects may well explain the low genetic diversity in the Montana population. While the two mentioned populations may have suffered genetic reduction by Wolbachia, other populations with Wolbachia infection did not show reduced genetic diversity and infection rates were similar independent of the host haplotype (Figure However, in a few populations, our data suggest that es Table . Neutrales Table . Therefoe Figure .5Wolbachia endosymbiont diversity. In addition, we found that regardless of lineage and population, most of the infection of alfalfa weevils are caused by a common strain of Wolbachia named wHypera1. However, high mitochondrial genetic diversity of the Eastern lineage and the low diversity in the Western lineage may be explained better by demographic events rather than Wolbachia infection. The data generated in this study may provide a useful basis for future studies on alfalfa weevil evolution, but may also provide some information for pest management.The comprehensive molecular data, which is generated in this study, provided a better picture of the global distribution of both alfalfa weevil and its The authors declare that they have no competing interests.ES, IK, and MH conceived the idea. ES and MJK performed molecular analyses including DNA extractions, PCRs, and the preparation of the raw data. ES, MS, MR, MT, TBT, and DA performed fieldwork and collecting materials. ES, IK, MH, and MJK performed phylogenetic, population genetic, and neutrality test analysis. ES, MH, MS, MR, MT, and TBT contributed to writing of the manuscript, and the final version was prepared by ES. All authors read and approved the final version of the manuscript. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.


Fish may display variations in ploidy, including three sets of chromosomes, known as triploidy. A recent study revealed a frequency of ~ 2% spontaneous  triploidy in domesticated Atlantic salmon produced in Norwegian aquaculture in the period 2007–2014. In contrast, the frequency of triploidy in wild salmon populations has not been studied thus far, and in wild populations of other organisms, it has been very rarely studied. In population genetic data sets, individuals that potentially display chromosome abnormalities, such as triploids with three alleles, are typically excluded on the premise that they may reflect polluted or otherwise compromised samples. Here, we critically re-investigated the microsatellite genetic profile of ~ 6000 wild Atlantic salmon sampled from 80 rivers in Norway and Russia, to investigate the frequency of triploid individuals in wild salmon populations for the first time.We detected a single triploid salmon, and five individuals displaying three alleles at one of the loci, thus regarded as putatively trisomic. This gave an overall frequency of triploid and putatively trisomic individuals in the data set of 0.017 and 0.083% respectively. The triploid salmon was an adult female, and had spent 2 years in freshwater and 2 years in the sea.We conclude that the frequency of naturally-occurring triploid Atlantic salmon in wild Norwegian and Russian populations is very low, and many-fold lower than the frequency of spontaneous triploids observed in aquaculture. Our results suggest that aquaculture rearing conditions substantially increase the probability of triploidy to develop, and/or permits greater survival of triploid individuals, in comparison to the wild.The online version of this article (10.1186/s12863-018-0676-x) contains supplementary material, which is available to authorized users. Polyploidy, i.e., the development of multiple copies of chromosomes within an organism, occurs naturally and is thought to play a role in the evolution of species –3. A triFrom the wild, there is a lack of datasets from different species, which makes rigorous testing of how and why polyploidy develops in the natural environment a relatively unsolved challenge . Fish anSalmo salar L.) is an anadromous salmonid that inhabits temperate rivers on both sides of the North-Atlantic. This species shows highly significant population genetic structuring throughout its native range [Atlantic salmon , consisted of triploid fish that had occurred spontaneously  . FurtherAtlantic salmon is a species where a large number of population genetic data sets, often based upon multiple polymorphic microsatellites, exist –31. In sThe present study is based upon an extensive re-examination of microsatellite genetic profiles of 5994 Atlantic salmon from 56 Norwegian and 24 Russian rivers Fig. . The samAll microsatellite genotyping was performed at the molecular genetics laboratory the Institute of Marine Research, Norway, and included the analysis of 18 loci . The exaThe microsatellite genetic profiles for the 5994 salmon included in this study were carefully re-analysed in the program Genemapper to identify potential triploids. The implemented protocol for this re-analysis of existing data was very similar to that used to identify triploid salmon in other data sets generated in this laboratory , 32. In Salmon trutta L.), which is known to occur in the wild, was excluded. This was achieved by two methods. First, a number of brown trout and trout salmon hybrids have been inadvertently genotyped in this laboratory, and excluded based upon their microsatellite DNA profiles. Especially loci SsaD486 and SSp3016 display non-overlapping alleles between these two species, and thus such individuals are easily identified. However, in order to fully exclude this possibility, all of the triploid and putatively trisomic salmon identified here were genotyped at the diagnostic 5S rDNA locus to for identification of hybrids between these species [The remote possibility that any of the triploid or putative trisomic salmon identified here represented spurious results caused through hybridization between salmon and wild brown trout  that had their genotypes confirmed in all three analyses, one out of the 18 microsatellites displayed three alleles. This was either revealed as three distinct alleles for individuals UMB09–27, AL05–6-8 and BO07-St1–30 at the loci SSsp3016, SsaD157 or SSsp2201 respectively, or alternatively, by the longer allele amplifying greater than the shorter allele for individuals ET06–9 and AD09-St1–23, at the loci SSspG7 and SsOSL85 respectively , and five individuals that were classified as putatively trisomic . The frequency of natural triploidy observed here (1 in 5994: 0.017%) was thus observed to be ~ 10 times lower than in escaped farmed salmon recaptured in Norwegian rivers in the period 2007–2014 , and ~ 1It has been suggested that in commercial aquaculture or supportive breeding hatcheries, spontaneous triploidy may arise due to over-aging of the eggs prior to fertilization, possibly in combination with increased temperatures of the eggs , 41, andCold shock can be used to deliberately induce triploidy in fish farming , and hasPelophylax sp.) have triploid subpopulations that reproduce by mechanisms that are not known in fish [Cypriniformes, Gymnotiformes, Siluriformes, Characiformes) that are known to have variable ploidy populations also utilize multiple modes of both sexual and asexual reproduction [Rates of natural triploidy vary greatly among species, however, the reasons for this are unknown. In humans, where triploidy is lethal, the rate, determined from genotyping of spontaneous abortions, is less than 1% . In amph in fish . Many otoduction , 45, 46.oduction . It is tLittle direct evidence is available regarding survival rates of triploid salmonids in the wild. However, triploid salmon are known to display reduced swimming endurance, increased temperature sensitivity, poorer disease resistance and impaired stress responses in comparison with diploid salmon , 47. It 18 = 3.8e-6). We therefore conclude that our triploid identification method has accurately estimated the number of triploid fish in the samples used here.This study used microsatellite DNA analysis for identifying triploid individuals, a method that has been tested before by us against flow-cytometry , 32, as Trisomy is also caused by a failure of chromosome separation, but affecting only a single chromosome . TrisomyIn conclusion, we have investigated for the first time, the frequency of triploidy and putative trisomy in wild salmon populations. The observed very low frequency of triploidy revealed in the wild here, demonstrates that the rates of up to 28% seen in some fish farms, is highly likely to be due to the specific conditions of the breeding in that environment, although a role of additional mortality of triploids in the wild cannot be ruled out. Further investigation in the wild, for fertilized eggs could reveal further insights into this.Additional file 1:Summary of salmon ploidy per river. (XLSX 21 kb)Additional file 2:Microsatellite genotyping results for all salmon identified as possibly triploid or trisomic. (PDF 874 kb)


Results showed that BRA generally presents moderate levels of genetic diversity when compared with the major canine breeds. The estimated effective population size (recent Ne = 51) shows a similar declining pattern over generations as all other dog breeds, pointing at a common demographic history of modern canine breeds, clearly different from the demography of feral wolves. Multidimensional scaling (MDS), Bayesian clustering and Neighbor Joining tree were used to visualize and explore the genetic relationships among breeds, and revealed that BRA was highly differentiated and presented only low levels of admixture with other breeds. Brittany Spaniel, English Setter, Gordon Setter and Weimaraner dogs are the closest breeds to BRA. The exact reason for BRA being so divergent from other dog breeds, based on these results, is not yet clear. Further studies including additional ≪braccoid≫ breeds will be needed to refine the results presented here and to investigate the origin of the BRA breed. Nonetheless, the genome-wide characterization reported here provides a comprehensive insight into the genome diversity and population structure of the Braque Français, type Pyrénées breed.The evaluation of genetic variability is a useful research tool for the correct management of selection and conservation strategies in dog breeds. In addition to pedigree genealogies, genomic data allow a deeper knowledge of the variability and genetic structure of populations. To date, many dog breeds, such as small regional breeds, still remain uncharacterized. Braque Français type Pyrénées (BRA) is a dog breed originating from a very old type of gun-dog used for pointing the location of game birds to hunters. Despite the ancient background, the knowledge about levels of genetic diversity, degree of inbreeding and population structure is scarce. This may raise concerns on the possibility that few inbred bloodlines may dominate the breed, and on its future health. The aim of this work was therefore to provide a high-resolution representation of the genome-wide diversity and population structure of BRA dogs, using the 170K genome-wide SNP array. Genome-wide polymorphisms in BRA were compared with those of other worldwide dog breeds. Between-dog relationships estimated from genomic data were very similar to pedigree relationships (Pearson correlation  Canis lupus), and through domestication and artificial selection have diversified into a large collection of sub-populations, or breeds, with well-defined morphological and physiological traits   . The R pNe), iii) runs of homozygosity (ROH), and iv) molecular inbreeding. LD between syntenic pairs of SNPs was estimated as r2 [For the genetic characterization of the BRA dog breed, the following molecular parameters were estimated: i) linkage disequilibrium (LD), ii) effective population size , Ne and recombination rate, as illustrated by Sved [Ne was estimated separately for each breed, and recent vs historical Ne were differentiated based on inter-locus distance.Recent and historical  by Sved . Ne was FROH) was estimated per animal by the ratio between the total sum of ROH lengths and the total size of the autosomal genome [Runs of homozygosity (ROH) were detected in each animal based on a sliding-window scan of the genome . No expll genome .Within-breed genomic relationships based on SNP genotypes were estimated as described in Yang et al : brieflyK) was estimated through 5-fold cross-validation of the accuracy to assign samples to the correct cluster: the K value that minimizes the cross-validation prediction error is then assumed as the most likely. An unweighted neighbor-joining (NJ) tree was constructed using a shared allele index based on the genetic distance matrix estimated from the SNP data.The degree of population substructure was evaluated via the model-based clustering algorithm implemented in ADMIXTURE . The moseP tool was used to estimate effective population sizes (Ne) [R environment for statistical programming [ggplot2 [ape [Quality control and filtering of the genotypic data, LD estimation, ROH detection, estimation of within- and between-breed genomic relationships were performed using PLINK v. 1.9 . The SNezes (Ne) . The R egramming  was used[ggplot2 ) and to ot2 [ape ).The aim of this work was to characterize the genetic variation and population structure of Braque Français, type Pyrénées dogs using genealogical and molecular data: first, to estimate whether the breed still maintains sufficient levels of genetic variability, instrumental in preparing conservation and genetic improvement plans; and second, to place this specific breed in the global context of canine populations, comparing genome-wide polymorphisms in BRA with those of other dog breeds from all over the world.Fi > 0, spanning from a minimum of 0.036 (3.6%) to a maximum of 0.25 (25%). The average pedigree inbreeding per generation was rather erratic, going from 0.06 four generations ago to 0.05 in the last available generation in the past, with a maximum at 0.12 (12%) six generations ago, with no clear trend.From pedigree data, additive genetic relationships between dogs were estimated. The average inbreeding coefficient  was 0.48. Wiener et al. [F and FROH of 0.78. Some studies in livestock species have shown lower correlations between the two inbreeding coefficients; for example, 0.015 in Brazilian Landrace pigs and 0.24 in Brazilian Large White pigs [F and FROH has been reported by several authors –here examples in cattle [Genomic relationships estimated from SNP genotypes were very similar to pedigree relationships, with Pearson correlation of 0.92  (0.371) and English Bulldog (EBD) (0.357). Other breeds generally showed moderate FROH values, like BRA (0.176 ± 0.049), in a range from 0.091 to 0.328. Recently, similar average values (0.17 ± 0.02) were reported for the Czechoslovakian Wolfdog [FROH ranges from 0.061 in Jack Russell Terriers to 0.151 in Bulldogs, while Dreger et al. [FROH = 0.39 in Pharaoh Hounds and minimum FROH = 0.03 in Mastino Abruzzese. The BRA breed shows therefore inbreeding levels well within the range detected in other dog breeds.The ROH coverage of the genome differs considerably among breeds . The hig Wolfdog . The bre Wolfdog  reportedr et al.  reportedNe) of a real population can be defined as the size of a hypothetical ideal population that would result in the same amount of genetic drift as in the  current population. Ne is an important population parameter that helps explain how populations have evolved, and it can be used to improve the understanding and modeling of the genetic architecture underlying complex traits [Ne is promising, especially in situations where the depth of the available pedigree data is low and pedigree-based estimates are thus likely to be inaccurate.As detailed previously , the effx traits . The genNe at each of t ∈ T generations in the past  are shown in Ne over generations was observed. The decreasing trend is likely due to the progressive selection and the within-breed use of top performing dogs. Ne estimates showed that 13 generations ago the effective population size was < 70 for all breeds. Different estimates have been reported by Dreger et al. [Ne values ranging from 71 (Neapolitan Mastiff) to 303 . Ancestors of the contemporary Wolf (WLF) exhibited considerably larger Ne values. In the present study, recent (13 generations ago) and past (995 generations ago) Ne in BRA were estimated to be 51 and ∼ 1530, respectively. The breeds that showed the lowest recent Ne value were Irish Wolfhound (IRW) (Ne = 30) and Eurasian (EUR) (Ne = 31).Estimated r et al.  with recNe decreases, there is an increased risk of mating genetically related individuals. A population with Ne under 50 can be considered at risk from the negative effects of inbreeding depression [Ne = 50, may imply that the viability of the breed is not compromised if the population is well managed. Additionally, while qualitative Ne estimates (shape and pattern of Ne over generations) tend to be rather robust, this is not true for quantitative Ne estimates. Ne point estimates are highly sensitive to parameters like sample size, LD extent and metric, and binning system (inter-locus distances), and their values may change dramatically between studies, especially for more than 4 ⋅ Ne generations in the past [As the pression . This mapression ). Severapression . Therefothe past .r2 statistic was chosen over the D′ estimator. D′ estimates tend to be inflated with small sample sizes or at low haplotype frequencies [r2 is generally thought to be the best suited measure of LD for biallelic markers and to avoid the influence of small sample size [r2—over all 38 autosomes together in each breed is displayed in Ne values. However, for distant markers (650–1000 kb) the LD extent stretched longer in wolves (WLF) than in Gordon Setter (GOS). As a general pattern, breed differences decreased as between-marker distance increased. BRA dogs showed intermediate LD decay, with the average r2 falling below 0.20 after 220 kb. Stern et al. [r2 dropped below 0.25 in three dog breeds: from 100-200 kbps in Newfoundland dogs, to 200-500 kbps in Golden Retrievers, to 0.5–1 Mb in Rottweilers. Average r2 < 0.1 for markers over 820 kbps apart was reported in UK Labrador Retrievers [To predict the extent of LD the quencies . r2 is gple size . The LD trievers . The varAscertainment of population structure and genetic relationships between animals have proven useful in conservation and management practices. Multidimensional scaling (MDS), Bayesian-model based clustering, and Neighbor Joining were used to visualize and explore the genetic relationships among breeds.http://www.fci.be/en/) Group 7 “Pointing Dogs group and Setters” present in the LUPA Database is the Weimaraner (WEI). BRA is thought to be closely related to WEI with which shares structural characteristics and the hunting attitude for wild feathered game. This is supported by the first two dimensions of the MDS plot  revealed three main clusters. In particular, the majority of “modern” European dog breeds were clustered together in the centre of plot and they could not be easily distinguished from each other, suggesting a relatively high gene flow between them. East Asian (Shar-Pei (SHP)), Arctic (Greenland Sledge Dog (GSL), Siberian Husky (HUS) and Samoyed (SAM)) breeds, together with the Czechoslovakian Wolfdog (CWD), Eurasian (EUR), Saarloos (SAR) and Finnish Spitz (FSP) breeds, formed a distinct cluster from most European breeds. Some of these divergent breeds (SHP and HUS) have been pinpointed and collectively termed “ancient breeds” . Fig 4 aK) in a Bayesian clustering algorithm (K = 2) reflected the separation of Wolves (WLF) from domestic dog breeds. At K = 4, we observed a strong genetic differentiation between BRA and the remaining dog breeds and this was also found at higher K-values. At the same K-value, Border Terrier (BOT), English Bull Terrier (EBT) and Greenland Sledge Dog (GSL) showed distinctiveness from the other breeds.All dogs were assigned with the highest probability to the breed from which they were sampled: as reported by Vaysse et al.  this sholgorithm . The firK = 40. At this K value, generally a distinct cluster is observed for each dog breed, although with some differences. As a matter of fact, some breeds (Jack Russell Terrier (JRT), Poodles (PDL)) together with BRA showed less distinct clusters. This result is in line with the historical information available on BRA, suggesting that this breed may be a mixture of European (Spanish) Pointer dogs.The most probable number of populations present in the total sample, as suggested by the ADMIXTURE cross-validation procedure was When K was increased, breeds were progressively assigned to separate clusters. In general, the ADMIXTURE analysis followed the affinities among breeds found in the MDS plot and NJ tree. BRA was highly differentiated and presented only low levels of admixture with other breeds. This could be related to a combination of low population size and isolation from other breeds. Very little is known with certainty about the origin of BRA, as this breed was developed before written records of dog breeding began to be kept. What is known is that the breed was developed in France before the late 1700s, and that it was primarily used for tracking, pointing at and retrieving game. Selection programmes began in the last decades of the 1800s, as was the case for most modern dog breeds, from a population with ancestral morphology (a rather heavy Braque type) with the aim of developing a lighter type. Over time selection led to two different types characterized by different morphological aspects and consequently, different functions.The exact reason for BRA being so divergent is not yet clear. Based on the results here reported, none of the major canine breeds appears to have played a major role in the ancestry of BRA, supporting its origin from old Spanish Pointer dogs which goes far back in time. Further studies including other ≪Braccoid≫ dog breeds, in particular the Braque Français type Gascogne, would be relevant to unravel the origin of BRA. Nonetheless, the genome-wide characterization reported here provides a comprehensive insight into the genomic diversity of Braque Français, type Pyrénées dogs.This is the first study to estimate the population structure of Braque Français type Pyrénées dogs from a genome-wide perspective. From a within-breed perspective, BRA shows lower than average molecular inbreeding and extent of LD, and higher than average effective population size, when compared to the other dog breeds considered in the study. From a between-breeds perspective, BRA displayed clear genetic differentiation, thereby confirming the available historical information which suggests that BRA is a mixture of older European Pointer dogs.Monitoring inbreeding and genetic variability is important to reduce the incidence of heritable diseases and maintain overall fitness of the breed. Therefore, considering that genetic diversity is an intrinsic factor that influences the adaptive capacity and resilience of populations, the outlined genomic approaches represent useful tools for the implementation of carefully planned selection and mating strategies within breeds. These results further suggested that the combined use of genomic and pedigree data may be a valid option for the selection of lineages that minimize the inbreeding rate per generation. Breeders should be aware of the risks related to excessive inbreeding and of the opportunities offered by genomics, so to design breeding systems that foster and maintain genetic variation in the BRA breed.S1 FileA few elements on the history of the Braque Français type Pyrénées canine breed, and the directional selection practiced in Italy.(PDF)Click here for additional data file.S1 FigHeatmap of kinship matrices in BRA estimated from pedigree data or from molecular marker data.(PDF)Click here for additional data file.