Fucus vesiculosus, the upper shore species Fucus spiralis, and an intermediate distinctive morphotype of F. spiralis var. platycarpus were morphologically characterized. Two diagnostic microsatellite loci recovered 3 genetic clusters consistent with prior morphological assignment. Phylogenetic analysis based on single nucleotide polymorphisms in 14 protein coding regions unambiguously resolved 3 clades; sympatric F. vesiculosus, F. spiralis, and the allopatric (in southern Iberia) population of F. spiralis var. platycarpus. In contrast, the sympatric F. spiralis var. platycarpus  was distributed across the 3 clades, strongly suggesting hybridization/introgression with both other entities. Common garden experiments showed that physiological resilience following exposure to desiccation/heat stress differed significantly between the 3 sympatric genetic taxa; consistent with their respective vertical distribution on steep environmental clines in exposure time. Phylogenetic analyses indicate that F. spiralis var. platycarpus is a distinct entity in allopatry, but that extensive gene flow occurs with both higher and lower shore species in sympatry. Experimental results suggest that strong selection on physiological traits across steep intertidal exposure gradients acts to maintain the 3 distinct genetic and morphological taxa within their preferred vertical distribution ranges. On the strength of distributional, genetic, physiological and morphological differences, we propose elevation of F. spiralis var. platycarpus from variety to species level, as F. guiryi.Gene flow among hybridizing species with incomplete reproductive barriers blurs species boundaries, while selection under heterogeneous local ecological conditions or along strong gradients may counteract this tendency. Congeneric, externally-fertilizing fucoid brown algae occur as distinct morphotypes along intertidal exposure gradients despite gene flow. Combining analyses of genetic and phenotypic traits, we investigate the potential for physiological resilience to emersion stressors to act as an isolating mechanism in the face of gene flow. Along vertical exposure gradients in the intertidal zone of Northern Portugal and Northwest France, the mid-low shore species  Speciation requires the evolution of reproductive isolation between interbreeding populations, thus preventing the homogenizing effects of gene flow. In addition, following secondary contact, hybridization can blur previously clear species boundaries. Models of speciation based on divergence between geographically isolated populations  have historically dominated the literature Gene flow generally retards speciation by reducing genetic divergence among populations Littorina saxatilisThe intertidal zone, where vertical gradients in emersion time produce strong variation in abiotic  and biotic  conditions over very small (meters) spatial scales Fucus comprises of intertidal brown algae, it is the most species-rich within the family Fucaceae, and appears to have undergone recent radiation that has given rise to several closely related species F. spiralis (L.) individuals are exposed to air at low tide for longer than F. vesiculosus (L.) individuals. Laboratory studies have also shown small, but significant, differences in emersion tolerance between F. spiralis and F. vesiculosusF. vesiculosus can extend its vertical range upshore when F. spiralis is removed F. spiralis is competitively excluded by F. vesiculosus in the midshore region F. spiralis is hermaphroditic whereas F. vesiculosus is unisexual (i.e. dioecious taxon). In both species, planktonic larval phase is absent leading to rapid settlement very near parent algae The genus F. vesiculosus and F. spiralisF. vesiculosus is dioecious (outcrossing), while F. spiralis is a hermaphrodite with high levels of inbreeding Fucus taxa Molecular phylogenies using traditional markers (nuclear ITS and mtDNA) have failed to discriminate between Fucus spiralis morphotypes were described as morphological varieties, currently considered taxonomically equivalent F. spiralis var. platycarpus (Thuret), first described over a century ago F. spiralisF. spiralis Low and F. spiralis High because of their respective vertical distributions in the intertidal both occurring on average higher than F. vesiculosusF. spiralis High and Low morphotypes, a subsequent assessment suggested that F. spiralis High and Low correspond morphologically to F. spiralis var. typicusF. spiralis var. platycarpusAbout a century ago, several F. vesiculosus, F. spiralis, and sympatric/allopatric F. spiralis var. platycarpus. Our data show significant differences in emersion stress resilience between morphotypes, despite extensive asymmetric introgression in F. spiralis var. platycarpus when in sympatry.This study addresses the major question of persistence of genetic divergence in sympatry/parapatry despite gene flow, using as models two species and two varieties within one of those species, which occur along a sharp environmental gradient. We aimed to formally establish morphotype descriptions and the correspondence between morphology and genetic differentiation using previously identified diagnostic microsatellite markers F. vesiculosus and F. spiralis occur in sympatry, with both species present on the open coast and in sheltered habitats . From Northen Portugal southwards towards Morocco the two species have an allopatric distribution, with F. vesiculosus occurring only in estuarine and coastal lagoons and F. spiralis present only on the open coast. Sampling along the sympatric range  was carried out in Viana do Castelo , Portugal, and in Roscoff (48°43′39″N 3°59′20″W), France, both in 2009. Based on previous morphological observations F. vesiculosus, F. spiralis and F. spiralis var. platycarpus co-occur Fucus zone on both shores, which can be divided in five areas  the zone inhabited by F. spiralis and F. spiralis var. platycarpus in sympatry, (C) a mixed area containing all three morphotypes, (D) the zone inhabited by F. vesiculosus and F. spiralis var. platycarpus in sympatry, (E) the lowest zone dominated by F. vesiculosus in parapatry.From Northern Portugal northwards ve areas : (A) theF. spiralis, F. spiralis var. platycarpus, and F. vesiculosus (12 each morphotype) were collected from all the other zones where each morphotype is present, and brought to the laboratory for similar physiological trials.In zone C at both locations, 50 individuals of each morphotype were sampled for genetic analysis using microsatellites, of which 10 were photographed for further characterization of morphological traits. At Viana only, out of the 50 individuals, 12 were brought to the laboratory to test for their physiological resilience to air exposure. In addition, further individuals of F. spiralis individuals from the allopatric, southern range  collected in Vila Nova de Milfontes on the southwest coast of Portugal .Of the 50 individuals of each morphotype collected at Viana, 5 individuals were characterized by sequencing cDNA fragments for 14 protein coding nuclear loci, together with 4 Morphological traits of the southern morphotype were described using a population from Santa Eulália, southern Portugal . At this sheltered location individuals reach a larger size that allows greater confidence in morphological characterization, relative to the typically stunted and wave-damaged individuals encountered on the more exposed southwest coast of Portugal. Therefore only undamaged individuals were collect to avoid morphological biased identification caused by wave brakeage.http://www.shom.fr). Like others Fucoids, these species are perennial algae that do not undergo seasonal shifts in distribution The median tidal height of each zone  was measured by recording the slope angle in the middle of each zone and the distance between each middle point and the lowest air exposed point during low tide (five measures each zone). Mean time of emersion during the year was calculated as the duration of emersion, according to the relative height on the shore, over 2008 in VC and RS using the SHOM database  and site  using a MANOVA on the morphological variables. Post-hoc Tuckey tests were performed to assess the difference between the morphotypes (Statistica software version 8).From the 50 individuals collected based on morphology, we haphazardly chose 10 individuals of each morphotype from Viana and from Roscoff. Digital photographs made in field were stored for morphometric analyses. Image analysis  was used to measure a suite of morphometric variables on each thallus : thallusF. spiralis, F. spiralis var. platycarpus and F. vesiculosus in sympatry 2, 0.03 mM of each dNTP, 0.17 µM of each Forward and 0.33 µM of each Reverse primer, 0.5 U GO Taq Polymerase and 5 µL of diluted DNA 1∶10. Amplifications were carried out on a Thermal Cycler 2720 (Applied Biosystems) using the following profile: initial denaturation at 94°C for 2 minutes; 35 cycles of 94°C for 20 seconds, followed by 35 seconds at 54°C for L20 and at 55°C for L78, 72°C for 40 seconds; and a final extension at 72°C for 20 minutes.DNA was isolated from 5–10 mg of dried tissue with the CTAB method, but a silica filter plate  was used instead of the silica fines step. Microsatellite loci L20 and L78 K was used as an indicator of the strength of the signal detected by Structure and considered as the real number of K cluster K) was unknown and the population structure and selfing rates were inferred . Clustering was running in 4 independent chains for the MCMC algorithm .Structure software version 2.3 F. spiralis var. platycarpus were also included. RNA was isolated from fresh tissue of these individuals using the extraction method of Pearson et al. 2 (10 mM), DTT (0.1 M) ,RNase OUT and SuperScript™ III RT (Invitrogen) were added following the suppliers instructions, and the mix was incubated at 55°C for 1–2 hours, before inactivating the reaction at 80°C for 10 minutes.A total of 14 partial coding regions .From the 50 individuals collected in Viana, we haphazardly chose 5 individuals of each morphotype. An additional 4 individuals from the allopatric population of ions see  were selst, 6th, 7th, 10th, 12th and 13th partitions; Kimura 2-parameter nd, 4th, 8th and 9th partitions; Tamura-Nei th and 11th; and General Time Reversible rd and 14th partition. The combined data set was analyzed as one partition using the TrN model. Two phylogenetic reconstractions were performed, one with F. vesiculosus, F. spiralis and populations of F. spiralis var. platycarpus in sympatry and allopatry; the other excluding F. spiralis var. platycarpus in sympatry. Fucus ceranoides was used as outgroup species based on previous information about the phylogenetic relationships among species in Fucus genus Sequences were aligned first by MAFFT version 6 using an iterative refinement method with global homology G-INS-i FST values were calculated using polymorphism between sequences coded as SNPs information in Arlequin software v.3.5.1.2 Heterozygous sites were included and codified following standard nucleotide ambiguity codes. No gaps were detected in the sequences analyzed. To prevent wrong inference of haplotypes, Maximum likelihood bootstrap analysis with 9999 replicates was performed to infer the phylogenetic relationships for the combined data set using PhyML version 3.0.1 6 with a sampling frequency of 100 generations in dual running process with four chains each run Bayesian-based inference using the same alignment was performed with MrBayes version 3.1.2 −2 s−1) supplied by sodium vapour lamps. Additional samples of 12 individuals  from the other zones where each morphotype is found were also collected and acclimated in the same way. Following acclimation, vegetative apical tips were cut and placed in 5 L tanks and kept for an additional 10 days. Half the seawater volume was replaced every 2 days throughout the acclimation period.Haphazard subsets of 12 individuals  from the genotyped samples were acclimated in seawater (17°C) for seven days in 5 L tanks at low photosynthetic photon flux density (LL: PPFD of 30–50 µmol m−2 s−1) at one of the following temperatures: (a) 33°C (±0.5°C), (b) 37°C (±0.5°C). An additional 40°C (±0.5°C) treatment was performed for individuals coming from zone C. Control treatments were kept in seawater at 17°C in LL.From each individual four tips were selected and duplicates were exposed in air at high photosynthetic photon flux density  was measured with a chlorophyll fluorometer . By relating the capacity for photochemical quenching (Fv) to the total fluorescence emission of closed PSII reaction centers (Fm), Fv/Fm is directly proportional to the quantum efficiency of PSII photochemistry F. spiralis, F. spiralis var. platycarpus, F. vesiculosus) as fixed factors and zone  nested in morphotype. Distance-based homogeneity of dispersion tests, tests of main effects and pair-wise tests on significant interactions were performed as recommended using 999 permutations. Therefore the number of times the permuted p-value was equal to or lying outside the 95% confidence interval was divided by the total number of permutations (999) and the resulted number taken as the permuted p-value.The data were analysed using the PERMANOVA module The biologically defined zones (based on morphotype presence) form a gradient of increasing exposure time from E to A in both Viana and Roscoff . AverageF. vesiculosus individuals had bladders (B) while the other algae did not. The receptacle sterile rim (RR) and monopodial branching (M) were uniquely present in all F. spiralis var. platycarpus individuals. Because bladders, monopodial branching and receptacle sterile rim were diagnostic for a given morphotype, they had zero variance, and were thus excluded from the MANOVA  while the effect of site was not (p = 0.96). Results of post-hoc tests are shown in F. spiralis var. platycarpus (Santa Eulália) shared the morphological traits of the sympatric F. spiralis var. platycarpus, namely the presence of a sterile rim around the receptacles, the relative proportions of the receptacles dimensions, and the monopodial branching (data not shown).All e MANOVA . Except F. spiralis (sensu lato) and F. vesiculosusF. spiralis and F. spiralis var. platycarpus morphotypes and F. vesiculosus at both locations. Nevertheless, several individuals morphologically identified as F. spiralis var. platycarpus and F. vesiculosus displayed genetic characteristics of another entity .Bayesian admixture analyses, implemented by both Structure software r entity . Interes Roscoff , and wheF. vesiculosus and F. spiralis sensu lato, as shown by nucleotide differences and FST values that were significant between all Fucus taxa , but after 6 h recovery F. vesiculosus<F. spiralis var. platycarpus. At both recovery times the resilience of F. spiralis was significantly greater than that of the other two morphotypes  temperature stress reduced Fv/Fm below that of controls, which did not differ between taxa. Zones did not have an effect for any of the taxa. However, while recovery occurred in all taxa after exposure at 33°C, with Fv/Fm increasing between 1 and 6 hours post-stress  significF. spiralis>F. spiralis var. platycarpus≥F. vesiculosus.The results show that resilience to emersion stress in the laboratory correlates with the observed distribution on the shore, with F. vesiculosus/F. spiralis species pair, three genetically distinct fucoid morphology exist on some intertidal rocky shores of Portugal and France F. vesiculosus, F. spiralis var. platycarpus and F. spiralis while admixture analysis using microsatellite markers F. spiralis var. platycarpus from southern Portugal, identified 3 monophyletic clades. However, sympatric/parapatric F. spiralis var. platycarpus was not monophyletic, suggesting that extensive asymmetrical introgression has occurred with local F. vesiculosus and F. spiralis. The phylogenetic analysis excluding sympatric F. spiralis var. platycarpus supported these findings, showing three well supported monophyletic clades.The genetic, ecological, and physiological factors that explain how gene flow persists against countervailing selection have seldom been studied F. vesiculosus and F. spiralis has undergone recent divergence; previous phylogenies based on nuclear ITS F. spiralis and F. vesiculosus, much less detect differentiation within F. spiralis. Nevertheless, microsatellite allele frequencies have demonstrated clear genetic differentiation and therefore a significant level of reproductive isolation between F. spiralis and F. vesiculosus across geographical sites F. spiralis var. platycarpus from southern Iberia [which corresponds morphologically with F. spiralis Low as identified by Billard et al. F. spiralis and F. vesiculosus. In sympatry F. spiralis var. platycarpus morphotypes are polyphyletic, which is suggestive of extensive hybridization and introgression with both the other taxa. Although this conclusion remains tentative at present, the current data for the distribution of F. spiralis var. platycarpus support a model of secondary contact in the northern range. Spatially explicit model simulations F. vesiculosus and F. spiralis morphotypes are monophyletic in sympatry, and showed no evidence of introgression. This is supported by F. vesiculosus mitochondrial haplotypes having integrated into other Fucus  during postglacial re-colonization of Northern Europe Fucus remain permeable to gene flow, despite different reproductive strategies and partial habitat segregation Fucus genetic taxa, overcoming the homogenizing effects of gene flow.Based on phylogenetic and phylogeographic evidence, the clade containing Fucus genetic taxa are partially segregated along the intertidal at both sampling sites and that their vertical distribution along the intertidal mirrors the gradient in the percentage of time out of water throughout a year. During low tide, temperature and desiccation interactively stress intertidal algae. An ecological trade-off occurs in which the protective effect of desiccation, that increases thermo-tolerance, comes at the cost of a decrease in photosynthesis Fucus genetic taxa in response to air exposure at 33°C and 37°C, F. spiralis had the greatest resilience followed by F. spiralis var. platycarpus and then F. vesiculosus. Comparative physiological resilience to emersion between the three morphotypes mirrored their vertical distributions, confirming the crucial role of emersion-induced stress in setting upper vertical limits in the intertidal zone. No intraspecific differences in recovery rates of adult algae from different intertidal heights were detected after acclimation in common garden experiments, suggesting that (intraspecific) local adaptation is null or minor compared with interspecific variation Intertidal organisms are regularly covered and uncovered by the movement of tides that subject them to a transition from aquatic to terrestrial conditions. The upper distributional limits of many rocky intertidal organisms are thought to be set by some aspect of thermal and/or desiccation stress related to aerial exposure at low tide F. spiralis (sensu lato) and F. vesiculosus show contrasting, species-diagnostic mating systems: F. spiralis individuals are hermaphroditic with a high degree of selfing F. vesiculosus is dioecious (obligately outcrossing). Thus, selfing in F. spiralis, as well as limited gamete dispersal capacities F. spiralis and F. vesiculosus nuclear genomes into F. spiralis var. platycarpus  was a distinctive feature of F. spiralis var. platycarpus. These two morphological traits are visually immediate and do not require measurement, therefore they are ideal diagnostic features for field morphotyping. Populations of F. vesiculosus without bladders have been recorded on very exposed sites F. vesiculosus, they are diagnostic for the species relative to other congenerics.The distinct morphotypes studied here could be unambiguously assigned to genetically distinct clusters with the two microsatellite loci used in this study. Together with the microsatellite data, morphological variation between entities showed similar pattern in North Portugal and in France indicating consistency across latitudinal scale. Other than receptacle length, which is similar in F. spiralis morphotypes are coherent with our findings. Many marine organisms have morphologies that reflect their physical environment and some of the best examples of morphological variation in marine organisms with respect to physical forces come from studies of benthic macroalgae Fucus genus, thallus morphology can also play a role in the algae's ability to withstand temperature and desiccation stress when exposed to air at low tide Morphological description and drawings in Perez-Ruzafa F. spiralis, F. spiralis var. platycarpus and F. vesiculosus. We found that F. spiralis var platycarpus occurs in allopatry in southern Portugal, and in allopatry it was shown to form a monophyletic group distinct from F. spiralis. We therefore propose elevation of F. spiralis var. platycarpusF. guiryi. In sympatry, F. guiryi showed strong signals of asymmetric introgression with both local species. Ongoing gene flow across permeable species boundaries has not been sufficient to disrupt adaptive physiological traits associated with emersion-stress resilience. We conclude that the steep vertical selective gradient spanning the intertidal zone is sufficiently strong to facilitate small-scale local adaptation and consequently maintain morphological and physiological species traits, even in the face of extensive  gene flow.We identify physiological, morphological, and genetic (at coding regions of multiple loci) differentiation between the genetic taxa Fucus guiryi Zardi, G.I., Nicastro, K.R., Serrão, E.S., Pearson, G.A. sp. nov.Diagnosis (Latin): Species nova Fuci. Fuco vesiculoso L. forma similis sed receptaculis hermaphroditis, ora sterili receptaculi, ramificatione monopodiali, absentia vesicarum; thallo breviore et longitudine frondis inter hapteron et primam dichotomiam breviore; fronde apicali angustiore; receptaculis altioribus latioribusque; proportione majore latitudinis receptaculorum cum longitudine et altitudinis receptaculorum cum longitudine; positione altiore inter accessum et recessum aestuum et tolerantia aeris majore; et transcriptis genum nuclearium ad proteina facienda  et locis microsatellitum distinguenda . Fuco spirali L. quoque similis sed ora sterili receptaculi et ramificatione monopodiali; thallo longiore et longitudine frondis inter hapteron et primam dichotomiam longiore; fronde apicali latiore; receptaculis minus altis, longioribus, et latioribus; proportione minore latitudinis receptaculorum cum longitudine et altitudinis receptaculorum cum longitudine; positione inferiore inter accessum et recessum aestuum et tolerantia aeris minore; et transcriptis genum nuclearium ad proteina facienda  et locis microsatellitorum distinguenda .Diagnosis (English): New species of the genus Fucus. Similar in morphology to Fucus vesiculosus L. but distinguished from it by hermaphroditic receptacles, the presence of a receptacle sterile rim, monopodial branching, and the absence of bladders; by shorter thallus and shorter frond length between the holdfast and the first dichotomy; by less wide apical frond; by higher and wider receptacles; by higher ratio of receptacle width∶length and receptacle height∶length; by higher intertidal zonation and physiological resilience to air exposure; by differences in the nuclear protein coding gene transcripts  and microsatellite loci . Also similar in morphology to Fucus spiralis L. but distinguished from it by the presence of a receptacle sterile rim and monopodial branching; by longer thallus and frond between the holdfast and the first dichotomy; by wider apical frond; by shorter receptacle height; longer receptacle; wider receptacle; by smaller ratio of receptacle width∶length and receptacle height∶length; by lower intertidal zonation and physiological resilience to air exposure; by differences in the nuclear protein coding gene transcripts  and microsatellite loci .Holotype: Praia da Amoreira, Aljezur, SW Portugal; March 20th, 2011; Gareth Pearson GALW15586.Isotypes: GALW15587, GALW15588, GALW15589, GALW15590, GALW15591.Etymology: Named in honor of Michael Guiry, in recognition to his great contribution to phycology by creating AlgaeBase.Habitat: Marine, intertidal. Where it co-occurs with Fucus spiralis and Fucus vesiculosus on the same shore, average distributional shore height typically in between these two species.Distribution: Morocco, Canary Islands, Atlantic Spain, Portugal, France, north to Britain and Ireland and probably Scandinavia.Synonymy: Fucus platycarpus Thuret, Annales des Sciences Naturelles, Troisième série, Botanique, 16: 9, pl. II (1851), nom. illeg. non F. platycarpus Turner Fuci vol. 3: 23, pl. 144 (1809–11) = Botryoglossum platycarpum (Turner) Kützing.Fucus spiralis var. platycarpus Batters Journal of Botany, British and Foreign 40 (Suppl.): 50, treated in Pérez-Ruzafa Valid publication: The electronic version of this document in itself does not represent a published work according to the International Code of Botanical Nomenclature http://sweetgum.nybg.org/ih/ for herbarium acronyms). A separate print-only edition of the article is available on request from PLoS (Public Library of Science) by sending a request to PLoS ONE, Public Library of Science, 1160 Battery Street, Suite 100, San Francisco, CA 94111, USA along with a check for $10 (to cover printing and postage) payable to “Public Library of Science”. No Unique Digital Identifier was available at the time of writing.Supporting Information S1Nuclear protein coding gene transcript descriptions. Annotations of coding region transcripts used in this study. P indicates partition number for each region used in mixed analyses. Total and used length expressed in base pairs (bp) and aminoacids (aa), as well as primer sequences and accession numbers, are shown.(PDF)Click here for additional data file.


Omentin is an adipocytokine that is abundantly expressed in visceral fat tissue. We investigated the association of omentin with the number of metabolic risk factors.The study population comprised 201 Japanese men who underwent annual health checkups. Plasma omentin levels were determined by enzyme-linked immunosorbent assay. We divided the subjects into 4 groups according to omentin levels. A reduction of plasma omentin levels significantly correlated with an increase in the mean number of metabolic risk factors such as increased waist circumference, dyslipidemia, high blood pressure and glucose intolerance.Circulating omentin levels negatively correlated with the multiplicity of metabolic risk factors, suggesting that omentin acts as a biomarker of metabolic disorders. Obesity has become a major health problem in industrial countries with increasing prevalence in adults and children . ObesityAccumulating evidence indicates that adipose tissue is an active endocrine organ that produces various bioactive substances, also known as adipocytokines or adipokines ,5. ExcesOmentin/intelectin-1 is an adipocytokine that exists in human blood stream -11. WhilThis study included 201 Japanese men who visited Chunichi Hospital in Nagoya for an annual health checkup. All subjects had no history of cardiovascular disease and took no medication. All subjects enrolled in this study provided written informed consent. This study was approved by the ethics committee of the Nagoya University School of Medicine and the Chunichi Hospital.We assessed the presence or absence of four metabolic risk factors: increased waist circumference, dyslipidemia, elevated blood pressure (BP), and dysglycemia/impaired glucose tolerance. An increase in waist circumference was defined as ≥85 cm. Dyslipidemia represented elevated triglycerides (>150 mg/dl) and/or low HDL cholesterol (<40 mg/dl). Elevated BP was defined as systolic BP of ≥ 130 mmHg or diastolic BP of ≥ 85 mmHg on repeated measurements. After an appropriate rest of 10minutes, sitting BP was measured. Dysglycemia/impaired glucose tolerance represented hyperglycemia (fasting glucose level of ≥110 mg/dl).Venous blood samples were obtained for chemical analysis after an overnight fast. Plasma omentin levels were determined with omentin enzyme-linked immunosorbent assay (ELISA) kit . Heparin was used for plasma sampling for measurement of omentin. The intra-assay and interassay coefficients of variation of this kit were 4.1% and 4.8%, respectively. Standard assays were used to measure glucose, hemoglobin A1c, insulin, total cholesterol, HDL cholesterol, LDL cholesterol, triglycerides, creatinine and high-sensitive C-reactive protein (hsCRP) levels.Summary statistics are presented as mean ± standard error (S.E.) for continuous variables. Non-parametric Kruskal-Wallis analysis of variance test and multiple logistic regression analyses were used to analyze the relationship between the number of metabolic risk factors and omentin levels. A value of p <0.05 was considered significant. All analyses were performed using JMP .The study population compromised 201 Japanese men who underwent annual health checkups. Clinical characteristics are shown in Table We categorized the subjects into 4 groups according to plasma omentin levels as follows: category 1, <2.59 ng/ml; category 2, ≥2.59 ng/ml, <2.66 ng/ml; category 3, ≥2.66 ng/ml, <2.72 ng/ml; and category 4, ≥2.72 ng/ml. The number of subjects in the 4 categories were 50, 51, 49 and 51, respectively. The average number of metabolic risk factors significantly increased with the decline in the quartiles of plasma omentin levels  (P < 0.001) (Figure In the present study, we, for the first time, demonstrate that circulating levels of omentin inversely correlate with the number of metabolic risk factors. Individuals with excess of visceral fat accumulation have a high risk of the development of metabolic syndrome . Among vPlasma concentration of omentin is associated with endothelium-dependent vasodilation . CirculaThe role of omentin in regulating metabolic function is poorly defined. A previous study showed that omentin stimulates glucose uptake in response to insulin in cultured adipocytes , suggestIn conclusion, the present study indicates that lower concentration of circulating omentin is linked with an increase in multiplicity of metabolic risk factors, suggesting that omentin serves as a biomarker for assessment of metabolic risk factors.BMI, body mass index; BP, blood pressure; ELISA, enzyme-linked immunosorbent assay; hsCRP, high-sensitive C-reactive protein.The authors declare that they have no competing interestsR.S., N.O., R.T., K.O., and T.M. designed and carried out the studies. N.I., A.H., T.Y., H. T., and S.K. analyzed the data. N.O. and R.S. wrote the paper. All authors read and approved the final manuscript.


Populus trichocarpa sRNAs was implemented.MicroRNAs (miRNAs) are endogenous small RNAs (sRNAs) with a wide range of regulatory functions in plant development and stress responses. Although miRNAs associated with plant drought stress tolerance have been studied, the use of high-throughput sequencing can provide a much deeper understanding of miRNAs. Drought is a common stress that limits the growth of plants. To obtain more insight into the role of miRNAs in drought stress, Illumina sequencing of P. trichocarpa conserved miRNAs were detected from the two sRNA libraries. In addition, 274 potential candidate miRNAs were found; among them, 65 candidates with star sequences were chosen as novel miRNAs. The expression of nine conserved miRNA and three novel miRNAs showed notable changes in response to drought stress. This was also confirmed by quantitative real time polymerase chain reaction experiments. To confirm the targets of miRNAs experimentally, two degradome libraries from the two treatments were constructed. According to degradome sequencing results, 53 and 19 genes were identified as targets of conserved and new miRNAs, respectively. Functional analysis of these miRNA targets indicated that they are involved in important activities such as the regulation of transcription factors, the stress response, and lipid metabolism.Two sRNA libraries were constructed by sequencing data of control and drought stress treatments of poplar leaves. In total, 207 P. trichocarpa and promote the understanding of miRNA functions during the drought response.We discovered five upregulated miRNAs and seven downregulated miRNAs in response to drought stress. A total of 72 related target genes were detected by degradome sequencing. These findings reveal important information about the regulation mechanism of miRNAs in  Caenorhabditis elegans in 1994 [MicroRNAs (miRNAs) are one of the most abundant classes of small RNAs (sRNAs) in plants and animals. These endogenous sRNAs were first identified in a metazoan called  in 1994  and viru in 1994 . MiRNAs  in 1994 . The mat in 1994 . MiRNAs  in 1994 , growth  in 1994 , develop in 1994 , biotic  in 1994  and abio in 1994 -19 stresArabidopsis, four drought-responsive miRNAs  have been identified by microarray analysis [Medicago truncatula (M. truncatula), Wang et al. (2011) mined drought-responsive miRNAs on a genome-wide scale using the Illumina sequencing technology; 22 members from four miRNA families and 10 members of six miRNA families were identified as up- and downregulated in response to drought, respectively [Populus euphratica (P. euphratica) [An increasing body of evidence indicates that miRNAs are involved in the plant drought stress response ,17,20,21analysis . In tobaanalysis . In riceanalysis . In Medihratica) . FurtherP. trichocarpa was the first model forest species sequenced [P. trichocarpa and identified stress-responsive and novel miRNAs by Sanger sequencing technology [P. trichocarpa miRNAs were further identified by Klevebring et al. (2009) using the 454 sequencing method [P. trichocarpa miRNA in general and of drought-responsive miRNAs in particular.Environmental stressors due to climate change, especially drought stress, could make forests increasingly vulnerable to disease and die-offs . Droughtequenced . Lu et achnology . An addig method . FurtherP. trichocarpa miRNA precursors are annotated in the miRBase (version 18.0) [Oryza sativa and M. truncatula, respectively, two other model organisms. Since the genome size of P. trichocarpa  is similar to that of M. truncatula (approximately 454–526 Mbp) and rice (389 Mbp), the potential for identification of new, specific miRNAs in P. trichocarpa is great. In this context, high-throughput sequencing was used to identify non-conserved miRNAs and drought-responsive miRNAs with the new version of the poplar genome (version 2.0), which has not been used in previous research on P. trichocarpa. The targets of these conserved and novel miRNAs were predicted, and some of them were confirmed by degradome sequencing. We discussed the potential regulatory mechanism between miRNAs and their targets. This may help to unravel the mechanism of drought stress tolerance in P. trichocarpa and other plants.Only 234 on 18.0) , compareP. trichocarpa plants were subjected to control levels  and drought levels . The two libraries were sequenced by an Illumina sequencer, yielding 27,333,282 reads for the control library (CL) and 30,806,496 reads for the drought library (DL)  of water-deficient soil ,28, P. tls RSMC, 5–20%. ThP. trichocarpa sRNA ranged from 16 to 27 nt, and the two major size classes were 24 nt (41.06% in CL and 43.94% in DL) and 21 nt (16.64% in CL and 16.05% in DL). This is in agreement with previous studies on sRNAs of P. trichocarpa [M. truncatula [The size distribution of all unique sRNAs is summarized in Figure chocarpa  and M. tuncatula  using hiP. trichocarpa sRNAs, small interfering RNA (siRNA) and miRNA with various important post-transcription regulating functions were the largest of our acquired sequences. The siRNA is a 22 to 24 nt double-strand RNA, each strand of which is 2 nt longer than the other on the 3’ end [P. trichocarpa. In total, 10,784,410 and 15,674,365 sequencing reads were identified as known poplar miRNAs in the two libraries. Thirty-four families from 207 known miRNAs were found, which accounted for about 87.3% of the total members. The remaining 30 miRNAs were not detected  . For new miRNAs whose targets were not known, we predicted their targets using the plant target prediction pipeline by the P. trichocarpa genome V2.0. The rules used for target prediction were based on those suggested by Allen et al. (2005) [The previously known miRNA targets also identified in this study are available on the PopGenIE site (. (2005)  and Schw. (2005) . We predP. trichocarpa degradome sequencing. In particular, the sRNA-cleaved mRNAs ligated by 5′ RNA adapters used for degradome sequencing acquired 23,326,117 and 34,398,368 reads (longer than 18 nt) in the mRNA libraries of the two treatments after removing redundancy; 108,593 and 234,316 unique reads could be matched to the P. trichocarpa genome (version 2.0) without mismatches  libraries were constructed for the http://www.uniprot.org/), these target transcripts can be grouped into nine categories. The majority of targets fall into the stress-response category, suggesting that these genes are drought-responsive  was identical in P. tomentosa and our research, which might be due to their close genetic relationship.Although miRNAs have been shown to play an important role in the drought stress response of ichocarpa, little es Table  [12-15,1P. trichocarpa  were both identified to be cold-responsive miRNAs in chocarpa . TMV-Cg d miR164 . Three ad miR164 . Repressd miR164 . MiR164 d miR164 , and NACd miR164 ,59. ThesP. trichocarpa. Their targets include miR408-targeted plastocyanin-like proteins and miR1444-targeted all plastid polyphenol oxidases [P. trichocarpa, Ptc-miR1444a is reportedly downregulated by dehydration [P. trichocarpa. Drought stress might induce the expression of ERD protein by downregulating the expression of miR408 in P. trichocarpa. This may be one of the mechanisms of regulation of drought-stress tolerance [Two downregulated miRNAs (Ptc-miR408 and Ptc-miR1444) have been reported to be Cu-responsive miRNAs in oxidases ,61. Drouoxidases . Accordiydration , and Ptcydration  and has olerance .Arabidopsis, salt-induced miR394 targets the mRNA of F-box proteins [Other downregulated miRNA is Ptc-miR394, whose predicted targets are annotated as dehydration-responsive protein (POPTR_0002s07760.1) and F-box proteins (POPTR_0001s13770.1 and POPTR_0003s16980.1), which were recently reported to be differentially regulated by stress conditions and to play significant roles in the abiotic stress-response pathway. In proteins ,56.P. trichocarpa. The homolog of TBLP in Arabidopsis is important to the formation of crystalline cellulose in trichomes [From the analysis of predicted targets to downregulated Ptc-miRn6, a CCCH-type zinc finger protein and two trichome birefringence-like proteins (TBLPs) were functionally predicted. Although a cotton CCCH-type zinc finger protein has been identified to enhance abiotic stress tolerance in tobacco , we did richomes . As prevrichomes , and therichomes . This maArabidopsis, miR390 was reported to target TAS genes [P. trichocarpa, no TAS homologs have been found [Arabidopsis, the UDPG is a key regulator of stress adaption through auxin IBA [P. trichocarpa.In AS genes , while ien found . From ouen found . This maen found . As in Auxin IBA  and playuxin IBA . This isP. trichocarpa. Over-expressing or repressing expression of these miRNAs in P. trichocarpa may help to elucidate the regulation mechanism.The degradome sequencing results imply that the miRNAs with no detected targets may silence genes by repressing translation. However, we could not obtain information about translation repression by miRNA through degradome sequencing. Only 19 targets of new miRNAs were identified. The targets of these non-conserved miRNAs are difficult to detect, possibly because of low abundance or a spatial expression pattern. More studies are needed to shed light on the regulation network of these miRNAs in Populus plants.In this study, sRNA libraries and degradome libraries of control and drought treatments were constructed with poplar leaves for high-throughput sequencing. Twelve miRNA members in 11 families were confirmed to be responsive to drought stress, and 65 novel miRNAs with star sequences of 59 families were identified. Through degradome sequencing, 53 and 19 genes were identified as cleavage targets of annotated miRNAs and new miRNAs, respectively. The functions of miRNA targets were analyzed and discussed. This study provides useful information for further analysis of plant miRNAs and drought stress tolerance, particularly in P. trichocarpa seedlings of the same size (~5 cm) from tissue culture were planted in individual pots (15 L) containing loam soil and placed in a greenhouse at Beijing Forestry University. They were well irrigated and grown under control conditions  for three months, the heights of them were about 45 cm. During the period of drought-stress treatment, P. trichocarpa seedlings were sustained at two RSMC levels (70–75% and 15–20%) for 1 month according to a previous publication [lication . The matlication . Then thP. trichocarpa genome (version 2.0) using SOAP software [Illumina sequencing on sRNAs (ranged from 18 nt to 30 nt) was conducted using an Illumina Genome Analyzer, following the Illumina protocol . After rsoftware . tRNA, rsoftware . The remsoftware . The cansoftware . Parametsoftware .p-value for comparison of the miRNA expression between the two libraries was based on previously established methods [2 ratio formula was: log2 ratio = log2 (miRNA reads in drought treatment/miRNA reads in control).The sequence reads of the two libraries were normalized to 1 million by the total number of sRNA reads in each sample. The calculation of the  methods ,78. Specp-value formulae were used:The following 1 is the total number of reads in the sequencing library of the control, N2 is the total number of reads in the sequencing library of the drought treatment, x is the number of reads for an miRNA in the control library, and y is the number of reads for an miRNA in the drought treatment library.where Nhttp://www.genomics.cn/en/navigation/show_navigation?nid=4143). Normalized miRNAs of <1 were filtered in both libraries.All calculations were performed on a BGI Bio-Cloud Computing platform .New d before ,80-82. DP. trichocarpa transcripts database from JGI Phytozome 2.0. At this step, a target plot was also created to distinguish the true miRNA cleavage site from background noise. We ran Cleaveland 2.0 with default parameters using 100 randomized sequencing shuffles. The NCBI database was used to predict functions of targets that were not annotated in JGI Phytozome 2.0. The cleaved target transcripts were categorized into three categories according to the following criteria: I, the abundance of reads in its cleavage site is the maximum on the transcript; II, the abundance of reads in its cleavage site is not the maximum, but is equal to or higher than the median for the transcript; and III, the abundance of reads in its cleavage site is less than the median for the transcript.Degradome sequencing following the PARE protocol was used . Only miPopulus trichocarpa; sRNA: Small RNA; RISC: RNA-induced silencing complex; mRNA: Message RNA; M. truncatula: Medicago truncatula; P. euphratica: Populus euphratica; CL: Control library; DL: Drought library; siRNA: Small interfering RNA; MFE: Minimum free energy; RT-qPCR: Quantitative real time polymerase chain reaction; SOD: Superoxide dismutase; P. beijingensis: Populus beijingensis; MSR: Methionine sulfoxide reductase; VEP1: Vein Patterning 1; ARF: Auxin responsive factor; LOX: Lipoxygenase; UDPG: UDP-glucosyltransferase; RSMC: Relative soil moisture content; PARE: Parallel analysis of RNA ends; TBLP: Trichome birefringence-like protein; ERD: Early responsive dehydration-related.miRNA: MicroRNAs; P. trichocarpa: The authors declare that they have no competing interests.PS DL designed and conducted the experiments, PS DL ZZ analyzed the data, PS DL WY XX drafted the manuscript, WY XX supervised the project. All authors have read and approved the final version of this manuscript.P. trichocarpa small RNAs sequencing.Summary of Click here for file30 undetected miRNAs in this study.Click here for fileP. trichocarpa.Potential miRNA candidates without miRNA*s found only in one library of Click here for fileThe predicted hairpin structures of all the 65 new miRNAs precursors.Click here for fileMiRNAs in response to drought stress.Click here for fileP. trichocarpa miRNAs.Targets prediction of new Click here for fileP. trichocarpa degradome sequencing.Summary of Click here for fileLeaf water potential.Click here for fileLeaf photosynthetic data.Click here for fileThe primers designed for RT-qPCR.Click here for file


Dictyocaulus species are strongylid nematodes of major veterinary significance in ruminants, such as cattle and cervids, and cause serious bronchitis or pneumonia (dictyocaulosis or “husk”). There has been ongoing controversy surrounding the validity of some Dictyocaulus species and their host specificity. Here, we sequenced and characterized the mitochondrial (mt) genomes of Dictyocaulus viviparus (from Bos taurus) with Dictyocaulus sp. cf. eckerti from red deer (Cervus elaphus), used mt datasets to assess the genetic relationship between these and related parasites, and predicted markers for future population genetic or molecular epidemiological studies.D. viviparus and Dictyocaulus sp. cf. eckerti (from red deer) by long-PCR, sequenced using 454-technology and annotated using bioinformatic tools. Amino acid sequences inferred from individual genes of each of the two mt genomes were compared, concatenated and subjected to phylogenetic analysis using Bayesian inference (BI), also employing data for other strongylids for comparative purposes.The mt genomes were amplified from single adult males of D. viviparus) and 13,296 bp (Dictyocaulus sp. cf. eckerti) in size, and each contained 12 protein-encoding, 22 transfer RNA and 2 ribosomal RNA genes, consistent with other strongylid nematodes sequenced to date. Sliding window analysis identified genes with high or low levels of nucleotide diversity between the mt genomes. At the predicted mt proteomic level, there was an overall sequence difference of 34.5% between D. viviparus and Dictyocaulus sp. cf. eckerti, and amino acid sequence variation within each species was usually much lower than differences between species. Phylogenetic analysis of the concatenated amino acid sequence data for all 12 mt proteins showed that both D. viviparus and Dictyocaulus sp. cf. eckerti were closely related, and grouped to the exclusion of selected members of the superfamilies Metastrongyloidea, Trichostrongyloidea, Ancylostomatoidea and Strongyloidea.The circular mt genomes were 13,310 bp (Dictyocaulus sp. cf. eckerti (red deer) and D. viviparus are separate species. Barcodes in the two mt genomes and proteomes should serve as markers for future studies of the population genetics and/or epidemiology of these and related species of Dictyocaulus.Consistent with previous findings for nuclear ribosomal DNA sequence data, the present analyses indicate that  Dictyocaulus (Strongylida: Dictyocaulidae) are economically important parasitic nematodes of the lungs of various ungulate animals, including domestic and wild ruminants  [Species of nd deer) , and arend deer) . All memnd deer) . Adult nnd deer) .Dictyocaulus was first erected [Dictyocaulus species and populations is not only important from a taxonomic perspective, but also has implications for studying the host and geographical distributions of the parasites, the cross transmissibility of Dictyocaulus between or among host species (particularly between bovine and cervid hosts) and also for the control of dictyocaulosis. Although molecular tools, employing genetic markers in ribosomal DNA, have found utility for systematic and/or epidemiological studies of some species [Dictyocaulus populations in different ruminant host species and countries around the world.Since  erected , there h erected , because species -13, therDictyocaulus viviparus[D. viviparus studied to date [Dictyocaulus species from a range of ungulate hosts. Here, as a first step, we used a massively parallel sequencing method and semi-automated bioinformatic pipeline for the characterisation of the mt genomes of D. viviparus (from Bos taurus) and Dictyocaulus sp. cf. eckerti (from Cervus elaphus)  genomes provide markers for systematic, genetic and epidemiological investigations of species of strongylid nematodes, with a perspective on discovering population variants and cryptic species and exploring transmission patterns linked to particular genotypes of a species -16. For viviparus,17, conc to date , the inf to date . Importa to date . High th to date  have undus) (cf. ), which us)  or red deer  in previous studies [Adult specimens of  studies ,20,22,23D. viviparus from B. taurus and of Dictyocaulus sp. cf. eckerti from C. elaphus. Amplicons were consistently produced from the positive control samples ; in no case was a product detected for any of the negative (no-template) controls. Amplicons were then treated with shrimp alkaline phosphatase and exonuclease I, and quantified in a spectrophotometer . Following an electrophoretic analysis of quality, the two amplicons (2.5 μg of each) from each of the two worms from each host species were pooled and then sequenced using the 454 Genome Sequencer FLX (Roche) [Using the protocol described by Hu et al. , the com (Roche) . The mt  (Roche) .Metastrongylus pudendotectus; accession no. GQ888714; [rrn  genes  were identified by local alignment of nucleotide sequence data. The trn (transfer RNA = tRNA) genes were predicted (from both strands) according to their structure, using scalable models, based on the standard nematode mt tRNAs [trn genes were then grouped, based on their anti-codon sequence, and identified based on the amino acid encoded by this anti-codon. Two separate trn gene groups were predicted each for serine  and leucine , because these trn genes are duplicated in many invertebrate mt genomes [viahttp://www.ncbi.nlm.nih.gov).The genes and features of each mt genome were annotated using an established computational pipeline . In brieQ888714; ). The lamt tRNAs . All pre genomes . All pretrn genes of each mt genome were then identified and annotated based on maximum sequence identity to known nematode tRNAs. Annotated sequence data were imported using the program SEQUIN (available viahttp://www.ncbi.nlm.nih.gov/Sequin/) for the final verification of the mt genome organization/annotation prior to submission to the GenBank database.All Dictyocaulus species using DnaSP v.5 [Sliding window analysis was performed on the aligned, complete mt genome sequences of the two naSP v.5 . The alinaSP v.5 , as implnaSP v.5 . KeepingD. viviparus and Dictyocaulus sp. cf. eckerti) were concatenated. Selected for comparison were concatenated amino acid sequences predicted from published mt genomes from key nematodes representing the order Strongylida, including M. pudendotectus, M. salmi, Angiostrongylus cantonensis and A. costaricensis , Haemonchus contortus , Ancylostoma caninum  and Oesophagostomum dentatum  [Metastrongylus species, the earliest divergent metastrongyloid in Jex et al. [The amino acid sequences conceptually translated from individual genes of each of the two mt genomes (representing yloidea)  and thenD. viviparus and Dictyocaulus sp. cf. eckerti were 13,310 bp and 13,296 bp in size  and M. salmi  [Dictyocaulus mt genomes were similar, being ~ 24.6% for A, 6.3% for C, 17.3% for G and 51.8% for T   . The nucted (cf. ), lackedD. viviparus were compared with those of Dicyocaulus sp. cf. eckerti (Table D. viviparus was ATT and TTG (for three of 12 proteins each), followed by ATA and GTT (two genes), ATC and TTA (one gene each); for Dictyocaulus sp. cf. eckerti, it was ATA (four genes), followed by TTG (three genes), ATG (two genes), ATT, GTT and TTA (one gene each). Eight mt protein genes in D. viviparus and nine in Dictyocaulus sp. cf. eckerti had a TAA or TAG translation termination codon; the other protein genes ended in an abbreviated stop codon, such as TA or T  and GAC (Asp) were not utilized in the mt genome of Dictyocaulus sp. cf. eckerti. The preferred nucleotide usage at the third codon position of mt protein genes of D. viviparus and Dictyocaulus sp. cf. eckerti reflects the overall nucleotide composition of these mt genomes. At this position, T is the most frequently, and C the least frequently used. For D. viviparus and Dictyocaulus sp. cf. eckerti, the codons ending in A have higher frequencies than the codons ending in G, which is similar to, for example, other members of the order Strongylida and Caenorhabditis elegans (Rhabditida), but distinct from Ascaris suum (Ascaridida) and Onchocerca volvulus (Spirurida) . As the nos. in ,31.D. viviparus and Dictyocaulus sp. cf. eckerti, the most frequently used codons are TTT (Phe), TTA (Leu), ATT (Ile), TTG (Leu), TAT (Tyr) and GTT . The least frequently used codons were CTA, CTG (Leu), ATC (Ile), GTC , AGC (Ser), CCC (Pro), GCC (Ala), TAC (Tyr), CAC (His), AAC (Asn), CGA (Arg), TCC (Ser) and GGC (Gly). All four GC-rich only codons are represented here, and every codon had at least one C. When the frequencies of synonymous codons within the AT-rich group, such as Phe , Ile , were compared between both mt genomes, the frequency was always less if the third position was a C.AT bias in the nucleotide composition was also reflected in a bias in the amino acid composition of proteins. The AT-rich codons represent the amino acids Phe, Ile, Met, Tyr, Asn or Lys, and the GC-rich codons represent Pro, Ala, Arg or Gly. In the mt genomes of trn genes were identified in the mt genomes of both D. viviparus and Dictyocaulus sp. cf. eckerti. The trn gene sequences ranged from 50–68 nt in length. The trn structures had a 7 bp amino-acyl arm, a 3–4 bp DHU arm, a 4–5 bp anticodon stem, a 7 base anticodon loop, with a T always preceding an anticodon and a purine always following an anticodon. Twenty of the 22 trn genes (i.e. excluding the two trnS genes) had a predicted secondary structure with a 3–4 bp DHU stem and a DHU loop of 5–8 bases, in which the variable TψC arm and loop were replaced by a “TV-replacement loop” of 6–12 bases, in accordance with other nematodes [trnS of both Dictyocaulus mt genomes had a secondary structure consisting of a DHU replacement loop of 4–6 bases, 3 bp TψC arm, TψC loop of 6–9 bases and a variable loop of 4 bases, consistent with other nematodes of the class Secernentea [Trichinella spiralis and Trichuris suis (class Adenophorea) [Twenty-two ematodes . The mt ernentea ,33, but ophorea) ,35.rrnS and rrnL genes of each of the two Dictyocaulus species were identified by sequence comparisons with homologous sequences of A. cantonensis. The rrnS gene was located between trnE and trnS (UCN), and rrnL was between trnH and nad3. The two genes were separated from one another by the protein genes nad3, nad5, nad6 and nad4L . TheD. viviparus were compared with those of Dictyocaulus sp. cf. eckerti  to the exclusion of Metastrongylus and Angiostrongylus species (Metastrongyloidea) as well as H. contortus (Trichostrongyloidea), An. caninum (Ancylostomatoidea) and O. dentatum (Strongyloidea)  Figure .D. viviparus and Dictyocaulus sp. cf. eckerti, achieved by sliding window analyses, are shown in Figure cox1 (452 bp), and of trnC_M_D_G (320 bp), rrnL (484 bp), nad5 (426 bp) and cox3 (401 bp) used previously as markers to assess genetic diversity within D. viviparus populations [nad6, followed by peaks of variation within nad5, nad1 and atp6. Gene-by-gene nucleotide diversity was highly variable, but, by far, the least variation was recorded within cox1. Of the markers used previously by Hu et al. [cox1) captured the least, and M4 (within nad5) captured the greatest sequence variation. The relatively low variation captured by targeting trn genes is also indicated by a trough for M2. Overall, the full sliding window indicates a wealth of new genes capable of providing high levels of nucleotide variation for population genetic studies.Nucleotide diversities calculated from pairwise comparisons across the mt genomes of ulations ,17. In FnC_M_D_G 20 bp, rru et al.  (i.e. M2D. viviparus (from Bos taurus) and Dictyocaulus sp. cf. eckerti from red deer, and explored the genetic relationships of these two lungworms and selected representatives of the Strongylida. Using sliding window analysis , fallow deer (Dama dama) and moose  [Dictyocaulus sp. cf. eckerti and D. viviparus, supported by previous results for the ITS-2 region [D. viviparus[cox1, cox3 or nad5 regions (375–396 nt) among 72–252 individual worms. These levels of within-species variation are much lower than estimated levels of difference  between Dictyocaulus sp. cf. eckerti and D. viviparus for the same gene regions (this study). At the amino acid level, variation in COX-1 (131 amino acids) within D. viviparus was ≤1.5%, but there was no amino acid difference between Dictyocaulus sp. cf. eckerti and D. viviparus (because of relative conservation). This contrasts the situation for COX3 (125 amino acids) and NAD5 (132 amino acids), for which maximum variation within D. viviparus was 1.6% and 2.3%, respectively  ,8,54. Cu2 region . A detaiviviparus,17 reveaDictyocaulus cf. eckerti) represents a distinct species from (or a population variant of) “D. eckerti”  might harbour a unique species [Dictyocaulus specimens from various species of wild and domestic bovid and cervid hosts from different continents, which could be achieved using a combined morphological and mt proteomic barcoding approach.Whether the parasite from red deer  remains Germany  is greatred deer , althougcited in ), which  species ,8,13,55.s  and thaDictyocaulus spp., given that there are few morphological features for the specific differentiation of some developmental stages  [Dictyocaulus . Within-species variation in cox1 was low (0.3–2.3%) [cox1 is revealed to be the gene with the lowest nucleotide diversity between the two Dictyocaulus species. In another study, Höglund et al. [D. viviparus populations from Sweden using four mt regions  covering 11% (1542 bp) of the mt genome of D. viviparus  used to date provided some resolution of genetic variation , the present sliding window analysis indicates other candidate genes for capturing greater diversity. That the cox1 region (of 393 bp) is the most conserved relative to other regions   and give larvae) ,57. In n larvae) , indicat larvae) ,56,58,59s Figure . Low varlatworms , for PCR.3–2.3%) -65 but d.3–2.3%) ,67. In td et al.  showed dDictyocaulus from different bovid and cervid hosts. Utilizing such primer sets, PCR-based single-strand conformation polymorphism (SSCP) analysis [D. viviparus in Sweden [Dictyocaulus specimens representing distinct operational taxonomic units (based on ITS-2) and different ungulate host species.PCR primers can be designed rationally in conserved regions flanking “variable sequence tracts” within the mt genome considered to be most informative for population genetic studies of analysis  could ben Sweden , and couDictyocaulus between/among cattle and cervid hosts, and their pathogenicity in different host species  of ies (cf. ). Furtheida (cf. ).Dictyocaulus sp. cf. eckerti (red deer) and D. viviparus indicate that these parasites are closely related species. Barcodes identified in the mt genomes and proteomes could serve as markers for future studies of the systematics, population genetics and/or epidemiology of a range of Dictyocaulus species as well as other lungworms.Comparative analyses of proteomic sequence datasets inferred from the mt genomes of The authors declare that they have no competing interests.RBG, ARJ & DTJL conceived the project and attracted the funding; AJ, ARJ, NM & JH carried out molecular laboratory work; NM, ARJ, DTJL, RSH & RBG carried out data analysis and interpretation; RBG, ARJ & DTJL wrote the draft manuscript with critical input from AJ and JH. All authors read and approved the final version of the manuscript.


Trichuris trichiura, affects more than a billion people worldwide, causing significant morbidity and health problems especially in poverty-stricken developing countries. Despite extensive research, the role of the immune system in triggering parasite expulsion is incompletely understood which hinders the development of anti-parasite therapies. Trichuris muris infection in mice serves as a useful model of T. trichiura infection in humans and has proven to be an invaluable tool in increasing our understanding of the role of the immune system in promoting either susceptibility or resistance to infection. The old paradigm of a susceptibility-associated Th1 versus a resistance-associated Th2-type response has been supplemented in recent years with cell populations such as novel innate lymphoid cells, basophils, dendritic cells and regulatory T cells proposed to play an active role in responses to T. muris infection. Moreover, new immune-controlled mechanisms of expulsion, such as increased epithelial cell turnover and mucin secretion, have been described in recent years increasing the number of possible targets for anti-parasite therapies. In this review, we give a comprehensive overview of experimental work conducted on the T. muris infection model, focusing on important findings and the most recent reports on the role of the immune system in parasite expulsion.Infection with soil-transmitted gastrointestinal parasites, such as  Trichuris trichiura alone is believed to infect almost 800 million people worldwide, with the majority being children [Soil-transmitted helminth (STH) infections, mainly trichuriasis, ascariasis and hookworm, affect more than a billion people, especially in poverty-stricken areas in the developing world . Trichurchildren . Infectechildren . Moreovechildren . The burTrichuris muris, a mouse model of T. trichiura infection in humans, have greatly contributed to our knowledge on components of immune responses responsible for resistance and susceptibility to infection. Research conducted on T. muris has presented us with novel explanations on how the immune system induces parasite expulsion, which could have broader application for new treatment development against soil-transmitted parasite infections.In recent decades, studies on the gastrointestinal parasite T. muris occurs by the ingestion of infective eggs that accumulate in the caecum , L3 (17 days p.i.) and L4 stage (22 days p.i.). Moults may occur at slightly different time points depending on the strain of the host. During larval development, the parasite moves from solely within the epithelial layer to extend into the gut lumen. By day 32 p.i., adult worms are observed in the caecum and proximal colon of infected mice. Interestingly, the anterior part of the worm is buried in parasite-modified epithelial cells which form a structure resembling ‘syncitial tunnels’. Tilney et al. [e dwells ).T. muris infection [T. muris. Studies on two groups of congenic strains have demonstrated that certain genetic backgrounds are more resistant than others (e.g. mice on the BALB genetic background are more resistant than mice on B10 background) even if they share the same H-2 haplotype [q, H-2b) expel parasites faster than mice having a ‘susceptible’ H-2 phenotype . The influence of these alleles was further modulated by the differences in the D end of the H-2 complex between these strains [T. muris infection in mice. This is especially evident in BALB/k and AKR mice, both sharing the H-2k haplotype, with the former generating a resistant-associated Th2 response followed by parasite expulsion and the latter succumbing to chronic infection accompanied by the development of a Th1 response [Early studies on outbred and inbred strains of mice suggested that the genetic background of the host was an important element contributing to observed variation in susceptibility to nfection . Indeed,aplotype . Likewis strains . Thereforesponse T. muris infection, the gender of the host also affects the efficiency of worm clearance. Studies by Bancroft et al. have shown that IL-4-deficient BALB/c male and female mice respond differently to T. muris infection, with males developing a chronic infection whereas females expel the worms  [T. muris should be considered a contributing factor in the variation in resistance to infection with this parasite. Taken together, both host genetic background and gender can greatly influence the type of immune responses generated against T. muris and subsequent parasite expulsion.Beside the genetic background influence on resistance to ontrols) . Furtherontrols) . Interesontrols) . Similarontrols) . Moreoveontrols) . Furtherontrols) . Conversontrols) . These dT. muris. It has been demonstrated that decreasing the infective dose can alter the polarization of the immune response favouring the development of a susceptibility-associated Th1 immune response. Normally resistant BALB/k mice when infected with less than 40 T. muris eggs (low dose) instead of 400 eggs (high dose) develop chronic infection [Apart from host genetic background and gender, the size of infective dose and parasite genetics can also influence the variation in susceptibility to infection with nfection . Moreovenfection .T. muris can also elicit distinct reactions from the immune system of the host. There are three laboratory-used isolates of T. muris: E (Edinburgh), J (Japan) and S (Sobreda) isolates [T. muris has been associated with increased numbers of regulatory T cells (Tregs) in the gut of mice infected with S but not E isolate. It has been therefore suggested that Tregs can inhibit the development of protective immunity and promote chronic infection [In addition to differences in host immune responses to low and high antigen load, different isolates of isolates . It has isolates , 16. Thiisolates , 16. On isolates . Here sunfection .T. muris is critical in mediating either susceptibility or resistance to infection. The development of a Th2-type of response is associated with fast parasite expulsion whereas a Th1 response is linked to establishment of chronic infection and increased immunopathology. The use of different mouse strains and gene knockout animals has been crucial in determining important cellular and molecular pathways of importance during T. muris infection  mice, which lack the adaptive arm of the immune system and therefore are susceptible to infection, resulted in worm expulsion proving that CD4+ T cells, and not CD8+ T cells or B cells, are crucial for the development of protective immunity to T. muris [+ T cells are most effective against larval stages of the parasite and that they act locally at the site of infection, since inhibition of the gut homing receptors β7 and αE integrins and the gut homing ligand MAdCAM-1 completely abrogates the ability of transferred CD4+ T cells to expel infection in SCID mice [+ T cells in the MLN, did not prevent expulsion of infection [T cells were shown to be important in mediating nfection . In addinfection –22. AddiT. muris . It has CID mice , 25. IntT. muris-infected resistant BALB/c and susceptible AKR mice have shown that at the time of expulsion (around day 21 post infection) BALB/c mice have increased numbers of CD4+ IELs whereas IELs in AKR animals are predominantly CD8+ [+ T cells to respond to stimulation and to polarize into Th2 cells [T. muris depends almost completely on CD4+ T lymphocytes.Analysis of intestinal intraepithelial lymphocytes (IELs) in the large intestine of tly CD8+ . Interesh2 cells . Taken tT. muris infection is in agreement with recent reports on novel innate cell populations, driven mainly by IL-25 and IL-33 production, which can serve as a source of Th2 cytokines early during GI parasite infections [type2) has been suggested to be important in resistance to T. muris infection [type2 cells are linage− Sca-1+ c-kitint and can be found in all compartments of gut-associated lymphoid tissue (GALT) such as MLNs, Payer’s patches and caecal patches but not in the spleen or bone marrow of IL-25-treated mice. As their name suggests, they can give rise to many different cell populations, i.e. monocyte, macrophages, mast cells and basophils both in vitro and in vivo. They have also been shown to promote T cell proliferation and differentiation toward a Th2 phenotype in vivo. In terms of T. muris infection, mice deficient in IL-25 (IL-25 KO mice) are susceptible to infection, showing reduced Th2 cytokine production and impaired mucin responses, with the phenotype being reversed by MPPtype2 cell transfer [type2 cells had reduced worm numbers at day 20 post infection compared to the untreated controls. Consistently, treated mice showed increased production of resistance-associated IL-4, IL-5 and IL-13 in the MLNs and higher IgG1 levels in the serum [type2 cells have been shown to promote development of a Th2-type response, they can be responsible for triggering a resistance-associated T cell response essential for T. muris expulsion. Furthermore, recently described nuocytes [Nippostrongulus brasiliensis infection. Hence, it remains to be seen whether these novel innate cell types play a similar role to MPPtype2 cells during T. muris infection.The hypothesis that early immune events may be important in mounting protective responses during fections . A new, nfection . MMPtypetransfer . Thus, Ihe serum . Therefonuocytes , naturalnuocytes  and innanuocytes , have beBasophils can be found in draining lymph nodes upon activation of the immune system and have recently been shown to express MHC class II and produce Th2-inducing cytokines such as TSLP and IL-4 –35. IL-4T. muris infection. Depletion of FcεRI+ cells (which include basophils) results in reduced Th2 responses in the intestine and a trend for increased worm burden during T. muris infection [T. muris infection [Indeed, there is now data indicating that basophils can play an important role in induction of a Th2 response during parasitic infection, including nfection . Furthernfection . Interesnfection .T. muris infection. Additional treatment of mice with MHC class II restricted to DCs with IFN-γ neutralizing antibody led to the restoration of a resistant phenotype [T. muris infection if Th1 development is abrogated. However, in subsequent work using different parasite infections and alternative strategies to abrogate the function of basophils and DCs, DCs were found to be crucial in the generation of parasite-induced Th2 responses. Thus, several studies have shown that basophil depletion has no effect on the development of Th2 responses and parasite clearance during primary infection with N. brasiliensis [Schistosoma mansoni infection does not alter the development of Th2 responses [S. mansoni infection impairs the Th2 response [T. muris is due to the particular models used by Perrigoue et al. [Interestingly, restriction of MHCII expression to DCs is not sufficient to generate Th2 cytokine production and results in lack of parasite expulsion , suggesthenotype , suggestiliensis , althougiliensis . Similaresponses , 41. Insresponse . Thus, we et al.  or is a T. muris infection, evidence exists that susceptibility/resistance to infection is linked to differences in DC phenotype. Thus, mice that are resistant to infection have faster DC mobilization to the site of parasite infection than susceptible animals [T. muris infection DCs from resistant mice show higher expression of CD80/86, MHCII and CCR7 and lower endocytic activity than DCs from susceptible mice, indicating that DCs from resistant animals mature faster. Moreover, epithelial cells from resistant animals have higher expression of chemokines such as CCL2, CCL3, CCL5, CCL20 and TSLP early during infection which correlates with the rapid mobilization of DCs to the large intestine [Although there is some controversy about the importance of DCs in triggering Th2 responses during  animals . Th2-ind animals . Howeverntestine . Indeed,ntestine .+ DCs, which have been shown to have tolerogenic properties due to elevated production of retinoic acid [T. muris, expression of CD103 by these cells seems to be dispensable for the development of protective immunity against this parasite [Furthermore, increased frequency of CD103oic acid , 45 and oic acid , have beoic acid . Howeverparasite .T. muris infection [T. muris infection [B cells and B cell-produced antibodies have been suggested in the past to be involved in the generation of protective immunity against nfection , 48. IgGnfection , 50. Furnfection . These dT. muris infection and that their resistant phenotype can be restored by adoptive transfer of B cells or administration of IgG from resistant mice infected with the parasite [+ T cells alone into SCID mice results in successful expulsion of T. muris, indicating that expulsion can occur in the absence of B cells [T. muris [T. muris infection requires further investigation.In addition, it has been demonstrated that B-cell deficient mice (μMT mice) are susceptible to parasite . However B cells . Indeed,T. muris . Thus, tT. muris show an increase in mast cell numbers upon infection [T. muris results in increased mast cell numbers at the site of infection, elevated serum levels of mMCP-1 (mouse mast cell protease 1) and faster parasite expulsion [T. muris expulsion [T. muris is minor [T. muris.Mast cell infiltration and activation are one of the hallmarks of parasitic infections. Indeed, mice infected with nfection . Howevernfection . On the xpulsion . Moreovexpulsion . Howeveris minor . Furtheris minor . TherefoT. muris infection have elevated numbers of these cells in the colon [T. muris infection is under the control of IL-5 and the chemokine CCL11 which work in synergy to recruit eosinophils [T. muris infection has no effect on the development of the Th2 response and parasite expulsion [T. muris.Increased numbers of eosinophils are characteristic of parasitic helminth infections. Mice resistant to he colon  and MLNshe colon  during iinophils . CCL11 Kinophils . Moreoveinophils . It has inophils . It is pxpulsion –56 indicT. muris infection [T. muris clearance [Both humans and wild-type animals are usually susceptible to infection with gastrointestinal parasites suggesting that worms are capable of modulating immune responses of their host to prevent expulsion. Indeed, it has been shown that development of an inappropriate Th1 response leads to chronic infection. Susceptible mouse strains produce high levels of IFN-γ, IL-12 and IL-18, cytokine characteristic of a Th1 response  and IFN-nfection . In addinfection . Intereslearance .T. muris may produce an IFN-γ-like molecule which could potentially contribute to regulation of the host immune response and promotion of parasite survival [T. muris infection lead to the development of host-detrimental immunopathology (e.g. colitis) and carry a risk of exacerbating bystander immune responses to different agents. For example, it has been shown that chronic T. muris infection resulted in systemic up-regulation of pro-inflammatory mediators, i.e. IFN-γ, TNF-α and IL-17, leading to augmented brain injury in a mouse model of ischemia-induced stroke [Additionally, mice which lack the IL-27 receptor WSX-1 (WSX-1 KO mice) do not develop chronic infection. IL-27 and WSX-1 are believed to interact in the early stages of infection to trigger Th1 responses in susceptible animals. WSX-1 KO animals have increased production of Th2-associated cytokines  and decreased levels of Th1-associated cytokines  . Interessurvival . Of noted stroke . Chronicd stroke .T. muris infection and lead to parasite expulsion by triggering expulsion mechanisms such as increased epithelial cell turnover, mucin production and muscle hypercontractility. Cytokines associated with protective immunity to T. muris infection are Th2-type cytokines. Two cytokines that play a major role in the resistance to infection are IL-4 and IL-13 [T. muris which is expelled by wild-type animals [T. muris infection can be stimulated in IL-4-deficient male mice by IL-13 administration [T. muris  become susceptible after IL-13 neutralization, indicating a dominant role for IL-13 in immunity against T. muris [+ T cells and DX5+ NK cells have been shown to be a source of IL-13 in IL-4 KO mice, although only depletion of the former results in complete abrogation of parasite clearance [Resistance-associated cytokines are usually produced against high-dose nd IL-13 . Mice la animals . Moreovestration . In addiT. muris . Both CDlearance .T. muris infection, a phenotype that can be restored by IL-13 administration [As mentioned before, male but not female TNF-α receptor deficient mice are susceptible to stration . Moreovestration . Howeverstration . TherefoT. muris, since adoptive transfer of IL-9-producting T cells into susceptible mice results in faster parasite expulsion, increased intestinal mast cell infiltration and mMCP-1 levels [T. muris infection [T. muris.It also has been reported that IL-9 is significant in early development of a protective immune response against 1 levels . Indeed,nfection . Furthernfection  further T. muris infection are IL-25 and IL-33 [type2 cells which have been shown to promote Th2 responses and T. muris expulsion [T. muris infection produce more IL-33 at day 3 post infection compared to susceptible animals. Furthermore, susceptible mice treated with recombinant IL-33 early during T. muris infection become resistant [Other cytokines important in the development of the protective immune response in early stages of nd IL-33 , 69. As xpulsion . In addixpulsion . Mice reesistant . Howeveresistant .T. muris [T. muris towards the resistance-associated Th2 response.In addition to these findings, increased production of IL-33 early during infection in resistant strains of mice has been correlated with increased production of the Th2-inducing cytokine TSLP . Indeed,T. muris , 71. ThiT. muris . In addition, phenotypic and translational similarities between chronic T. muris infection and mouse models of IBD and human IBD have been recently shown by transcriptional profiling studies [Trichuris suis eggs, a related parasite which normally causes trichuriasis in pigs, leads to remission of IBD symptoms in some patients [T. suis [Prolonged infection with gastrointestinal parasites, if not properly regulated, can result in severe damage to surrounding tissues. Immunopathology associated with susceptibility to disorder . Interes studies . It is spatients , 74. How[T. suis . Nonethe[T. suis . TherefoT. muris infection. A cytokine that plays an important role in resistance to T. muris infection is the regulatory molecule IL-10, with mice deficient in IL-10 becoming susceptible to T. muris infection and developing severe and fatal intestinal pathology [T. muris. Recent studies have shown that CD4-dnTGF-βRII mice, whose CD4+ T cell have reduced ability to respond to TGF-β due to expression of truncated version of TGF-β receptor II (dnTGF-βII), have increased worm burden and down-regulated levels of IL-4 and IL-9 but normal IL-13 production [T. muris infection. A specific isolate of T. muris, isolate S, causes chronic infection in normally resistant C57BL/6 mouse strain that is associated with increased numbers of Foxp3+ Tregs in the gut of these mice [T. muris infection [T. muris stimulates a more regulatory environment to aid its survival. Moreover, a relatively new subset of regulatory cells called iTR35 (CD4+Foxp3−Ebi3+p35+IL-10−TGF-β−) has been proposed to be important in regulating immune responses during T. muris infection [T. muris infection [T. muris infection. However, more work is required to further assess regulatory mechanisms operating during T. muris infection.However, it is still unclear how the immune system is regulated during athology . In addioduction . It has ese mice . Moreoveese mice . Interesese mice . Since Iese mice  and IL-1nfection , it is pnfection . These cnfection . Taken tT. muris has been associated with crypt hyperplasia accompanied by both increased epithelial cell proliferation and apoptosis [T. muris infection, seem to be responsible for epithelial cell turnover down-regulation [T. muris clearance [T. muris infection, in controlling epithelial cell turnover ratio [T. muris infection [T. muris expulsion.Chronic infection with poptosis . Both ofpoptosis , 83. On poptosis . These fpoptosis . Interesgulation . Both AKlearance . In addier ratio . Normallnfection . Taken tT. muris infection in both resistance and susceptible animals [T. muris expulsion [T. muris worms with Muc5ac had detrimental effect on worm viability, as measured by parasite ATP levels [Goblet cells are major producers of mucins (the major protein component of mucus) and form an important element of the innate defence in the gastrointestinal tract. Goblet cell hyperplasia is observed during  animals . However animals –89. Up-r animals . Interesxpulsion , 88. MicT. muris. Interestingly, higher expression of Muc17 has also been observed in chronically infected animals [T. muris.The physical properties of the mucus barrier also changes during infection and correlates with responses to infection. Thus, the mucus barrier in resistant animals is less permeable, thicker and more highly charged than in susceptible mice , 89. TheT. muris infection with increased levels of this protein found in resistant animals [T. muris infection normally, RELMβ seems to be dispensable for the generation of Th2 response in resistant animals [Another goblet cell-derived molecule, resistin-like molecule β (RELMβ), has also been associated with immunity to  animals . Inducti animals . It has  animals . However animals .T. muris infection development [Conversely, studies have shown that RELMβ can actually promote chronic elopment . Thus, Relopment . RELMβ Kelopment .T. muris and seem to be indispensable for effective parasite clearance from the intestine.Taken together, goblet cells and their products are important elements of the host immune responses against Trichinella spiralis have shown that parasite expulsion is associated with muscle hypercontractility, via a STAT-6 and CD40-CD40L signalling-dependent mechanism controlled by the immune system [T. muris infection [T. muris. Indeed, neutralization of IL-9 by antibody treatment or by immunization with the OVA-IL-9 complex resulted in decreased smooth muscle contractility and the lack of parasite expulsion [T. muris have been reported to have decreased muscle contractility which could be partially restored by treatment with the immunosuppressive drug dexamethason [T. muris expulsion.It has been demonstrated that increased contractility of smooth muscle cells lining the wall of the intestine can be an important mechanism of gastrointestinal parasite expulsion. Studies on the small intestine-dwelling parasite e system , 93. Intnfection . This prxpulsion . Moreovemethason . Thus, sT. muris expulsion (Fig. Taken together, faster epithelial cell turnover, increased mucus production and muscle contractility are three known and characterised as immune-mediated mechanisms of ion Fig. .Fig. 3MeT. muris, as a mouse model of T. trichiura in humans, has contributed greatly to an increase in our understanding of the relation between gastrointestinal parasite and its host in terms of generated immune responses and expulsion mechanisms. Moreover, knowledge acquired while studying T. muris model may be applied to other soil-transmitted gastrointestinal parasite infections. This is especially important since despite their evident public-health importance, soil-transmitted gastrointestinal infections remain largely neglected by medical and international societies. Part of the problem is that most affected individuals are also the world’s most impoverished and since helminth infections cause chronic illnesses most people cannot afford the prolonged and repeated treatment. Moreover, people in endemic areas are usually infected with more than one parasite causing more obscure and hard to diagnose clinical presentation [entation . Treatmeentation . Moreoveentation , insuffi


Leishmania tropica in samples from jackals and foxes. Infection was most prevalent in ear and spleen samples. Jackals and foxes may play a role in the spread of zoonotic L. tropica.During a survey of wild canids, internal transcribed spacer 1 real-time PCR and high-resolution melt analysis identified  Leishmania tropica is a major cause of cutaneous leishmaniasis in the Old World. Although cutaneous leishmaniasis associated with L. tropica usually is considered an anthroponotic infection (Procavia capensis)  in southern Italy (Canis lupus) in southwestern Europe  infected with L. infantum were reported in Iraq  real-time PCR and high-resolution melt analysis (ITS1-HRM) PCR , L. tropica (MHOM/IL/2005/LRC-L1239), and L. major (MHOM/TM/1973/5ASKH). Negative controls included samples from jackals born and reared at a zoo in central Israel, as well as from foxes and wolves from areas in which leishmaniasis is not endemic, that were tested by PCR and found negative. All positive PCR products were purified by using ExoSAP-IT  and sequenced at the Center for Genomic Technologies, Hebrew University of Jerusalem. Sequences obtained were compared for similarity to sequences in GenBank by using the BLAST program (www.ncbi.nlm.nih.gov/BLAST). Positive samples also were verified by kDNA PCR as described . A phylogenetic tree was constructed by using the neighbor-joining method in agreement with maximum-parsimony and maximum-evolution algorithms and by using the Kimura 2-parameter model with uniform rates for transitions and transversions. Bootstrap replicates were performed to estimate the node reliability, and values were obtained from 1,000 randomly selected samples of the aligned sequence data. Sequences were compared with the following Leishmania sequences deposited in GenBank: L. tropica FJ595949 and FJ595950 from central Israel and IARA/IL/02/LRC-L910 and ISER/IL/02/LRC-L909 from northern Israel; L. infantum (MHOM/TN/1980/IPT1) and L. major (MHOM/TM/1973/5ASKH) were used as outgroups.Sequences were analyzed by using MEGA version 3.0  and for foxes 8% (2/25). All wolves were negative. Fourteen tissue samples  tested positive. Five (63%) of the 8 animals positive for Leishmania spp. had >2 infected tissues  to L. tropica sequences deposited in GenBank , and the sample amplified from the jackal blood clustered with L. infantum (MHOM/TN/1980/IPT1).Thirteen sequences from positive DNA products obtained by ITS1-HRM PCR were identified as belonging to  GenBank . Only 1 infantum . DNA seqinfantum . By usinL. tropica infections in jackals and foxes from Israel. Sequence analysis  of ITS1 fragments showed perfect correlation with L. tropica isolates from hyraxes in central Israel. The finding of L. tropica positivity in >1 tissue sample from infected asymptomatic animals implies that wild canid species could be natural hosts for this parasite. Furthermore, the relatively high percentage of infected spleens indicates that this parasite can visceralize in foxes and jackals. Unlike hyraxes, which generally stay close to their burrows in caves or boulders, jackals and foxes travel long distances, potentially transmitting L. tropica from 1 area to another, provided that competent sandfly vectors are found. The home range of golden jackals in Israel is adapted to the food resources available. For golden jackals, it was 6.6 km2 near settlements and 21.2 km2 in sparsely inhabited settings (L. tropica from an area with an infected population of hyraxes to a remote naive hyrax population or be responsible for infecting humans because they tend to live peridomestically and frequently rely on human waste. The involvement of wild canids in the sylvatic life cycle of L. tropica could be crucial to understanding disease emergence in Israel, Jordan, and the Palestinian Authority (L. tropica in humans (We report