The circadian system has a major role in maintaining homeostasis and proper body functions including reproductive capacity. The aim of this study was to examine whether there is an association between genetic variability in the primary clock genes CLOCK and ARNTL and male infertility in humans.−5, q = 4·10−4, OR equaled 1.9 with 95% CI 1.4–2.7), rs6811520  and rs6850524 . Further analyses of haplotypes were consistent with genotyping results.We performed a case-control study, where we searched for an association between polymorphisms of CLOCK and ARNTL genes and male infertility in 961 Slovenian and Serbian Caucasian men. The study group consisted of 517 patients with idiopathic infertility and a control group of 444 fertile men. A statistically significant difference was found in genotype distribution between the two groups in the CLOCK gene: rs11932595 (p = 6·10We provide evidence that genetic variability in the CLOCK gene might be associated with male infertility warranting further confirmation and mechanistic investigations. Infertility affects about 9% couples There is a growing body of evidence that circadian rhythms have a major role in maintaining homeostasis and proper body function including reproductive capacity Molecular components of the circadian clock network represent positive and negative transcriptional-translational feedback loops of many genes. CLOCK and ARNTL genes represent the central node in the network, generating a positive loop, and heterodimerizeing and initiating the transcription of other clock genes. Resultant proteins forming the negative feedback loop inhibit the CLOCK and ARNTL transcriptional activity Therefore, we hypothesized that genetic variability of the CLOCK and ARNTL genes may be associated with male infertility in humans. To test the hypothesis we performed a retrospective case-control genetic association study of polymorphic sites in these two genes on a population of patients with male infertility in comparison with fertile male control population.The study was approved by committees in both countries participating in the study: Slovenian National medical ethics committee (reference number: 73/05/12) and by the Ethics committee at Medical faculty at University of Belgrade, Serbia (reference number: 29/II-3). All patients gave informed written consent to participate in the study.Male partners of infertile couples, attending the infertility outpatient clinic of the Department of Obstetrics and Gynecology, University Medical Centre Ljubljana, and the Institute of Human Genetics, and Institute of Urology and Nephrology, Faculty of Medicine, University of Belgrade, participated in the study. The diagnosis of infertility was based on clinical assessment (testicular volume was measured using Prader's orchidometer), semen analysis, follicle stimulating hormone (FSH) testing and histologic evaluation of testicular biopsy specimens. Exclusion criteria were: patients with a history of testicular carcinoma, obstructive azoospermia including congenital bilateral absence of vas deferens, cytogenetic abnormalities and Y chromosome microdeletions.Clinical, sperm and hormonal characteristics of the 517 infertile patients are summarized in The control group of proven fertility consisted of 444 healthy males, who were fathers of at least one child and who reported no history of infertility. The seminal parameters and biopsy were not assessed for subjects in the control group. All patients and control subjects were Caucasian of the Slavic (Slovene or Serbian) origin and were recruited in a consecutive manner.In the 517 infertile men, semen analysis was performed according to the World Health organization (WHO) criteria Serum FSH was measured by Microparticle Enzyme Immunoassay ; the reference interval for FSH was 1–8 mIU/ml.Testicular biopsy and histological evaluation was performed in 77 patients with diagnosis of non obstructive azoospermia. In all patients testicular biopsy was performed for diagnostic and therapeutic (sperm recovery followed by cryostorage) purposes.Following unilateral hemiscrototomy, a small incision was made and at least two samples of testicular tissue were taken from each testis. A systematic histological evaluation was performed under light microscopy; more than 100 seminiferous tubules were scored for each patient and results expressed as a relative number of tubules showing Sertoli cells, spermatogonia, spermatocytes, round and elongated spermatids, and spermatozoa. The diagnoses were as follows: Sertoli-cell only syndrome (SCOS) alone 46.8%, Maturation arrest (MA) alone 11.3%, Germinal hypoplasia (GH) alone 7.8%, and following combinations: GH+MA 14.1%, SCOS+MA 11.3%, GH+SCOS+MA 6.3% and GH+SCOS 2.1%.http://www.hapmap.org). The SNPs were regarded as proxies for neighbouring SNPs, when their pairwise r2 values exceeded 0.80. The set of most representative tagSNPs for ARNTL and CLOCK gene regions was obtained using the Tagger algorithm available through the Haploview software  Genetic variants in both key circadian rhythm-regulating genes, CLOCK and ARNTL, were genotyped in this study. Eight tagging single nucleotide polymorphisms (SNPs) were chosen from both genes. Of these, four SNPs, rs11932595, rs6811520, rs6850524, and rs13124436 were selected in the CLOCK gene, and four SNPs, rs3789327, rs1481892, rs4757144, and rs12363415 in the ARNTL gene. We based the selection of SNPs on the known genetic linkage in both genes, according to HapMap Phase 3  using KASPar SNP genotyping chemistry. The PCR reaction mix of 8 µL final volume consisted of 4 µL of DNA sample, 4 µL of Reaction Mix 2X, 0.11 µL Assay Mix, 0.064 µL 50 mM MgCl2, and 3.826 µL H2O. The protocol for PCR amplification was: initial denaturation step at 94°C for 15 minutes, then 20 cycles of denaturation at 94°C for 10 sec, followed by 57°C or 61°C for 5 sec, 72°C for 10 sec, 94°C for 10 sec, 57°C or 61°C for 20 sec, and final extension at 72°C for 40 sec.The allelic discrimination analysis was performed using SDS Software Version 1.2 .Genotype assignment was performed and interpreted independently by two investigators.Significances of associations between allelic and genotype frequencies and disease status were analyzed using the Chi-square test (χ2). Odds ratios (OR) and their respective 95% confidence intervals (CI) were also calculated to compare allelic and genotype distribution in patients and control subjects. Analyses were performed using R statistical language (R 2.15.0 for Windows). χ2 goodness-of-fit tests for deviation of genotype distribution from those predicted by Hardy-Weinberg equilibrium were also calculated, providing an additional quality control step of genotyping process. The investigated associations were regarded as significant when they reached p≤0.05.As multiple SNPs were investigated, appropriate corrections of significance values were also applied using the Benjamini-Hochberg correction method . We studied the extent of genetic linkage between SNPs using Haploview software version 4.2, which enables calculation of D’ and r2, representing the extent of pairwise linkage between two SNPs.http://mayoresearch.mayo.edu/mayo/research/schaid_lab/software.cfm). Functions in haplo.stats package permit the calculation of indirectly measured haplotypes, under the founding assumption that subjects in the study are not related and the linkage phase unknown haplo.score function was used to directly ascertain differences in haplotype distributions across the groups of infertile men and control individuals. A global test of association, and per-haplotype association test were performed for both investigated genes. To reduce the effects of multiple testing we excluded all haplotypes with a frequency below 5% from downstream tests.Haplotype frequencies and haplotype-disease associations were estimated using haplo.stats package for R .For power calculations, the Genotype frequencies of investigated polymorphisms were in accordance with those predicted by the Hardy-Weinberg equilibrium in the study and in the control group (p<0.05), with the exception of rs12363415, which was excluded from further analyses. Genotype and allelic distribution of the CLOCK and ARNTL polymorphisms of the 517 infertile man and 444 fertile controls are shown in −5, q = 4·10−4), rs6811520  and rs6850524 . However, we did not, find any significant association between rs13124436 polymorphism from CLOCK gene and male infertility. Under recessive genotype model, OR estimates ranged between 1.4 and 1.9 for CLOCK gene polymorphisms: rs11932595 , rs6811520  and rs6850524 .We found a statistically significant difference in the allelic distribution of rs11932595 , CCGG  and CGGG  between the infertile patients and fertile controls.A statistically significant difference in haplotype distributions was also confirmed at the CLOCK gene locus when comparing the frequencies of haplotypes TCAG  have shown that clock-mutant males produce significantly fewer progeny, and release smaller quantities of sperm The circadian clock is internal timing system which allows an organism to provide environmental changes and adapt to them. Therefore, circadian rhythms manage a large variety of physiological and metabolic functions; any disruption of these rhythms may affect human health. Only a few studies have investigated the role of the circadian system in male fertility. It has been shown that night shift workers have an increased risk of infertility In women, genetic variability in the circadian rhythm genes ARNTL and NPAS2 has been suggested to contribute to fertility and seasonality, whereas the genetic variability in the ARNTL gene has been related to a higher number of pregnancies and also to a higher number of miscarriages; polymorphisms in the Npas2 gene have been associated with a decreased number of miscarriages In addition to its direct effects, the indirect effect of the circadian system on many physiological processes might possibly influence the male reproductive function. For example, the genetic variability in the CLOCK gene has been associated with increased weight and obesity In conclusion, we provide evidence that genetic variability in the CLOCK gene might be associated with male infertility, and consequently imply the role of the circadian timing system in human reproduction. Further confirmation and mechanistic investigation of circadian system in male infertility is warranted.


In hemodialysis patients, elevated plasma troponin concentrations are a common finding that has even increased with the advent of newly developed sensitive assays. However, the interpretation and relevance of this is still under debate.In this cross-sectional study, we analyzed plasma concentrations of sensitive troponin I (TnI) and troponin T (TnT) in stable ambulatory hemodialysis patients (n = 239) and investigated their associations with clinical factors and mortality.In all of the enrolled patients, plasma TnI or TnT was detectable at a median concentration of 14 pg/ml (interquartile range: 7–29) using the Siemens TnI ultra assay and 49 pg/ml (31–74) using the Roche Elecsys high sensitive TnT assay. Markedly more patients exceeded the 99th percentile for TnT than for TnI . In a multivariate linear regression model, TnT was independently associated with age, gender, systolic dysfunction, time on dialysis, residual diuresis and systolic blood pressure, whereas TnI was independently associated with age, systolic dysfunction, pulse pressure, time on dialysis and duration of a HD session. During a follow-up period of nearly two years, TnT concentration above 38 pg/mL was associated with a 5-fold risk of death, whereas elevation of TnI had a gradual association to mortality.In hemodialysis patients, elevations of plasma troponin concentrations are explained by cardiac function and dialysis-related parameters, which contribute to cardiac strain. Both are highly predictive of increased risk of death. At this point, the analytical precision of conventional troponin measurements is reduced, and the coefficient of variation (CV) can reach 10–20% th percentile have been developed and have shown improved accuracy in the diagnosis of MI th percentile Typically, plasma troponin concentrations in ESRD patients are slightly elevated above the 99With the advent of sensitive assays, cardiac troponins are now detected to a larger extent in hemodialysis patients raising questions about interpretation and relevance to nephrologists and cardiologists alike. The increased sensitivity and resolution of the assays, however, also improve the chance to identify factors that are associated with elevated plasma troponin concentrations, such as loss of residual renal function This cross-sectional, prospective multicenter study was conducted in stable, ambulatory hemodialysis patients from four dialysis centers in southwest Germany between September 2009 and April 2010. Patients without evidence of an acute illness and cardiac event or procedure within two months were included in the study after providing written informed consent. Patients with cardiac diseases leading to increased plasma troponin concentrations independent of ESRD, such as amyloidosis, were excluded. The study was approved by the local ethics committee of the University hospital Tuebingen.th percentile at 40 pg/ml, and a CV of less than 10% at 30 pg/ml, as specified by the manufacturer. Plasma concentrations of troponin T were measured using an automated Roche assay on an Elecsys 2010 system with a detection limit of 2 pg/ml, a 99th percentile at 14 pg/ml, and a coefficient of variation of less than 10% at 13 pg/ml Plasma concentrations of troponin I and troponin T were measured in three independent samples collected within two weeks. Each sample was collected prior to the start of a dialysis session. Blood was collected in lithium-heparinized tubes , cooled at 4°C, and centrifuged within 4 hours. The sera were stored at −80°C until further analysis. Plasma concentrations of troponin I were measured using the Troponin I Ultra assay on a Siemens ADVIA Centaur system  with a detection limit of 6 pg/ml, a 99The following data were extracted from each patient: residual diuresis, as measured by 24 h urine collection: single pool Kt/V , interdialytic weight gain, predialytic systolic and diastolic blood pressure , dialysis access and membrane, length of time on dialysis, blood pump flow and shunt flow measured using a Transonic system . The left ventricular (LV) systolic function was classified from available echocardiography examinations in which class 1 indicated normal function, 2 was mildly reduced, and 3 was moderately or severely reduced. Determination of the LV systolic function was done at the discretion of the cardiologist and not standardized. Echocardiography was available in 84% of all patients within one year (plus-minus) relative to study enrollment.http://www.wessa.net/. Univariate and multivariate proportional hazards were calculated to analyze the risk ratios of each predictor and the independence of the predictors. Data analysis was performed using the statistical software package JMP 10.0.1 .Three samples were available in 87% of the patients and were averaged to calculate the arithmetic mean without excluding possible outliers. For further analysis, plasma troponin concentrations and continuous clinical data were log transformed to approximate normal distribution. The association of the plasma troponin concentrations with clinical or dialysis-related factors was analyzed by univariate parametric correlation. Multivariate linear regression analyses were performed to identify independent determinants of the plasma troponin concentrations. Selection of the variables entering the model were derived from forward, stepwise multiple linear regression, and all variables with a p-value <0.05 were included in the multivariate linear regression models. The residuals of each model were tested for normality. Averaged values of the deceased patients were compared to those from the surviving patients using t-Test or Wilcoxon's test. Kaplan-Meier curves were generated after stratification into tertiles of the variable according to its distribution. The follow-up period started on the first day of blood draw and was censored as of 31 December 2011. The diagnostic performance was analyzed using receiver-operator curves (ROC or c-index) and the best cut-off value was considered as the maximal difference of sensitivity and 1-specificity (Youden index). C-Index was calculated using Wessa, P. (2012), Free Statistics Software, Office for Research Development and Education, version 1.1.23-r7, Of the n = 250 available patients that were treated in the participating centers, n = 239 were included in the study. The reasons for exclusion of the n = 11 patients were the following: patients declined to participate (n = 6), deaths occurred following the initial plasma collection (n = 2), cardiac amyloidosis (n = 2) and a recent enrollment in dialysis (n = 1). The characteristics of the study cohort are provided in Cardiac comorbidities were present in a large proportion of the patients. Ischemic heart disease was the most frequent condition (31%). Systolic LV dysfunction was found in 26% of all of the patients .th percentile at 14 pg/ml, while only 14% of the patients had a plasma TnI concentration above the 99th percentile at 40 pg/ml. Expressed as multiples of the 99th percentile, the TnT concentration was elevated 3.5 fold (interquartile range: 2.2 to 5.2) compared to 0.4 fold for TnI , 44 patients died corresponding to an annual mortality rate of 9.2%. Compared to surviving patients, deceased patients were significantly older , were more likely to have LV dysfunction  and had lower diastolic blood pressure . They had higher plasma CRP and parathormone concentrations . Plasma troponin T and I concentrations were also significantly (p<0.0001) higher in deceased patients  compared to surviving patients  was above the 99th percentile.On average, patients in this cohort had a TnT value that was 3.5-fold greater than the 99The study is limited by the paucity of cardiac parameters that were collected, which might have contributed to an unexplained variability of roughly 70% and 50% in plasma troponin I and troponin T concentrations during multivariate modeling. Survival analyses might be limited by the low mortality during the follow-up period. This study focused on nephrological parameters that are commonly available and accessible during hemodialysis treatment, such residual diuresis or shunt flow. Systolic left ventricular function served as a surrogate for cardiac status and showed a high correlation to the plasma troponin concentration. Other cardiac morbidities, such as valvular disease or atrial fibrillation were also contributors of the plasma troponin concentration. Again it should be emphasized that association studies cannot prove causality between the studied parameters and elevated troponin concentrations and cannot distinguish between cause and consequence.In summary, elevations of plasma troponin concentrations in hemodialysis patients measured with sensitive assays are influenced by cardiac function and dialysis-related parameters. Both are strong predictors of mortality in the short-term and are useful markers to identify patients at risk, however, troponin T is more retained during ESRD compared to troponin I.


Background: Few studies have been conducted to identify risk factors for balance and vestibular dysfunction in general populations, but previous studies have reported evidence of adverse effects of lead and cadmium on balance control in high-risk groups.Objective: We evaluated the relationship between blood lead and cadmium levels and balance and vestibular dysfunction in a general population study.Methods: We analyzed data from the 1999–2004 National Health and Nutrition Examination Survey (NHANES) of 5,574 adults ≥ 40 years of age. Balance dysfunction was evaluated by the Romberg Test of Standing Balance on Firm and Compliant Support Surfaces, which examines the ability to stand unassisted using four test conditions to evaluate vestibular system, vision, and proprioception inputs that contribute to balance. Blood levels of lead and cadmium were measured by atomic absorption spectrometry. Associations were estimated using logistic regression models adjusted for potential confounders. Associations with time to loss of balance were estimated using adjusted Cox proportional hazard models.Results: The adjusted odds ratio (OR) for balance dysfunction in association with the highest quintile (3.3–48 µg/dL) versus the lowest quintile (< 1.2 µg/dL) of lead was 1.42 . The corresponding OR for cadmium (0.9–7.4 µg/L vs. < 0.2 µg/L) was 1.27 . The adjusted hazard ratio for time to failure for the most physiologically challenging balance test among subjects with the highest vs. lowest quintiles of blood lead was 1.24 . Cadmium levels were not associated with time to failure.Conclusions: Our findings suggest that blood lead and cadmium levels may be associated with balance and vestibular dysfunction in a general sample of U.S. adults. Many studies have examined the effects of lead and cadmium exposures on neurobehavioral functions, such as declines in cognitive ability and visuomotor performance , but theAn individual’s ability to maintain balance is an essential component for nearly all daily life activities. The control of balance depends on sensory inputs from the vestibular and visual systems, neural processing centers throughout the central nervous system, and motor outputs (the proprioceptive system). Functional declines in any of these systems can lead to balance deficits . AlthougFew studies of risk factors for decreased balance andvestibular function have been conducted in the general population, despite the public health significance of these outcomes . FurtherStudy population. The NHANES, conducted by the Centers for Disease Control and Prevention (CDC), is a nationally representative survey of the noninstitutionalized civilian population in the United States. The 1999–2004 NHANES study protocols were approved by the National Center for Health Statistics Institutional Review Board. All participants gave oral and written informed consent.n = 851), and in all adult participants ≥ 40 years of age in the NHANES 2001–2002  and the 2003–2004  surveys. Of the 5,851 total participants, 5,574 (95.3% of those who were eligible) also had blood lead and cadmium measurements and data for all covariate variables.The present study used data on balance and blood levels of lead and cadmium that were measured concurrently in NHANES participants (1999–2004). The NHANES Balance Component was assessed in a 50% sample of U.S. adults 40–69 years of age in the 1999–2000 survey  is also recorded for test condition 4, with those who passed the test assigned the maximum value of 30 sec.Measurement of blood lead and cadmium levels. Whole blood specimens were stored frozen (–20°C) until they were shipped to the National Center for Environmental Health for testing. Blood lead and cadmium levels were measured using a multielement atomic absorption spectrometer with Zeeman background correction . The analytical laboratory  followed extensive quality control procedures.For external calibration of blood lead and cadmium levels , 2004, sOther variables of interests. We also evaluated other factors that might be associated with balance dysfunction and exposure to lead or cadmium, including age , sex , race/ethnicity , and education . Smoking and consumption of alcohol were modeled as pack-years and ounces per day, respectively . History of stroke was defined based on a self-reported physician’s diagnosis. History of diabetes mellitus was defined based on a self-reported physician’s diagnosis or an 8-hr fasting serum glucose level ≥ 126 mg/dL at the time of the examination. Dietary calcium intake was categorized as ≥ 1,000 mg or < 1,000 mg, and iron intake was categorized as ≥ 8 mg or < 8 mg.Statistical analysis. To account for the complex sampling design, weighted estimates of the population parameters were computed. Because of differences in the sampling frames for the balance examination, we combined the 1999–2000 and 2001–2002 balance data sets using special 4-year balance subsample weights; 6-year data analyses (1999–2004) were performed by combining the 4-year balance subsample weights with the 2003–2004 examination weights according to NHANES analytic guidelines  was conducted to compare the prevalence of balance dysfunction for each quintile of lead or cadmium distribution with the lowest quintile of exposure. We used the Hosmer–Lemeshow goodness-of-fit test , PearsonWe estimated associations between failure time for balance test condition 4 and blood lead and cadmium levels using Cox proportional hazards models , with ouStatistical models were adjusted for age, sex, race/ethnicity, education, pack-years of smoking, alcohol consumption (ounces per day), histories of stroke and diabetes, and intakes of calcium and iron.Of the 5,574 participants, 3,084 successfully completed all four balance test conditions and were designated as without balance dysfunction, whereas 2,490 failed to pass one of the test conditions, including 7 who failed to pass test condition 1 (case 1); 163 who passed condition 1 but failed to pass test condition 2 (case 2); 86 who passed conditions 1 and 2 but failed to pass test condition 3 (case 3); and 2,234 who passed conditions 1, 2, and 3 but failed to pass test condition 4 (case 4) . Comparep = 0.69 for lead and p = 0.47 for cadmium) indicated that models fit reasonably well (data not shown).Fully adjusted ORs for balance dysfunction, comparing blood lead quintiles 2, 3, 4, and 5 with the lowest quintile, were 1.14 , 0.96 , 1.18 , and 1.42 , respectively, . CorrespFully adjusted ORs for failure of each balance test comparing the highest with the lowest quintiles of blood lead concentrations were 1.38  for case 2, 2.17  for case 3, and 1.42  for case 4 . The corTime to failure for balance test 4 was significantly higher among participants with the highest versus lowest quintile of lead exposure , but cadmium levels were not significantly associated with time to failure .After adjusting for potential covariates, the prevalence of balance dysfunction was significantly higher among participants in the highest versus lowest quintiles of lead and cadmium . However, lower quintiles of lead were not clearly associated with the outcome, and associations with cadmium were comparable for quintiles 2, 4, and 5, but absent for quintile 3. The hazard ratio for time to failure for balance test 4 was significantly higher among participants in the highest versus lowest quintiles of blood lead  only, while cadmium levels were not clearly associated with time to failure for test condition 4.There is substantial evidence that implicates associations of impaired balance , 2002, hTo our knowledge, we are the first to demonstrate associations between impaired vestibular function and exposure to lead and cadmium in the general population. However, the clinical relevance of the outcomes we assessed is uncertain and it is not clear whether the balance test used reflects a defect in vestibular function specifically. Because postural balance is controlled by a complex interaction of multiple sensorimotor processes , balance, even with the limited input, can be maintained with the remaining afferents . For exaOur study has several limitations. First, because of its cross-sectional design, we can not confirm the temporal sequence of exposures and outcomes. However, it is unlikely that balance and vestibular dysfunction increases an individual’s exposure to lead and cadmium. Second, blood lead and cadmium levels alone are not an accurate reflection of lifetime exposures. Although blood measurements of both metals are commonly used in studies, and some studies have suggested that the effects of lead on postural stability or vestibular function are related more to recent increases in blood lead concentrations among exposed workers rather than to cumulative body burdens , studiesIn conclusion, we provide preliminary evidence of associations between blood levels of lead and cadmium and balance and vestibular dysfunction in a representative sample of U.S. adults ≥ 40 years of age. Although our findings require confirmation, they suggest that background exposures to lead and cadmium may be deleterious to the maintenance of balance and may be involved in the pathogenesis of vestibular dysfunction in the general population.


Centaurea stoebe and Potentilla recta invasions in the western USA to assess whether patterns were consistent with mycorrhizal-mediated invasions. We asked whether (1) AMF abundance and community composition differ between native and exotic forbs, (2) associations between native plants and AMF shift with invading exotic plants, and (3) AMF abundance and/or community composition differ in areas where exotic plants are highly invasive and in areas where they are not. We collected soil and roots from invaded and native forb communities along invasion gradients and in regions with different invasion densities. We used AMF root colonization as a measure of AMF abundance and characterized AMF communities in roots using 454-sequencing of the LSU-rDNA region. All plants were highly colonized (>60%), but exotic forbs tended to be more colonized than natives (P < 0.001). We identified 30 AMF operational taxonomic units (OTUs) across sites, and community composition was best predicted by abiotic factors . Two OTUs in the genera Glomus and Rhizophagus dominated in most communities, and their dominance increased with invasion density , while overall OTU richness decreased with invasion density . Samples along P. recta invasion gradients revealed small and reciprocal shifts in AMF communities with >45% fungal OTUs shared between neighboring native and P. recta plants. Overall, we observed significant, but modest, differences in AMF colonization and communities between co-occurring exotic and native forbs and among exotic forbs across regions that differ in invasion pressure. While experimental manipulations are required to assess functional consequences, the observed patterns are not consistent with those expected from strong mycorrhizal-mediated invasions.Controlled experiments show that arbuscular mycorrhizal fungi (AMF) can increase competitiveness of exotic plants, potentially increasing invasion success. We surveyed AMF abundance and community composition in  Non-native plant invasions can reduce plant diversity and alter ecosystem processes  in the phylum Glomeromycota that form a symbiosis, arbuscular mycorrhiza (AM), with the majority of land plants in which the fungi receive photosynthate in exchange for phosphorus, nitrogen, and other putative services (Smith and Read Euphorbia esula (spurge) and Centaurea jacea (knapweed) have a high AM dependency  and Bromus tectorum (cheatgrass) are nonmycorrhizal or have a low mycorrhizal dependency  the exotic forbs harbor a different AMF abundance and/or community composition than native forbs, (2) mycorrhizal associations in natives are altered in the presence of P. recta , and (3) AMF abundance and/or community composition differs between areas where the two forbs are highly invasive and areas where they are not. The observed patterns of AM were examined for consistency with patterns that might be expected in a mycorrhizal-mediated invasion. For example, increases in colonization or changes in composition of the AMF community in more densely invaded plots  and controlled experiments are therefore required to assess causation as well as any functional consequences of differences observed . Each site harbored P. recta and C. stoebe monocultures, mixed native grass and forb communities, and transition areas in which native and P. recta plants were growing side by side. Distance among sites ranged from 250 m to 1500 m, and within each site, all plant community types were within a 50 × 50 m area, which reduced spatial bias.To address questions 1 and 2, we identified three sites on MPG Ranch located south of Missoula, Montana  and one P. recta site . We also included one C. stoebe site located in Cheney, eastern Washington , approximately midway between MPG Ranch and Bellingham. These sites occur along a gradient of climate and invasion density with the highest rainfall, mildest temperatures, and lowest severity of invasion in western Washington.To address question 3, we located four additional sites near Bellingham in western Washington in which the exotic forbs were found in low abundance: three C. stoebe invasions, four P. recta invasions, three native forb communities, and three transition communities harboring co-occurring native forbs and P. recta) were surveyed in June of 2011 by establishing five, 4 m by 4 m plots in each community for a total of 85 plots. Plots within sites were separated by at least 3 m. Within each plot, percent cover of focal forb species was measured in sixteen 1 m by 1 m adjacent sections and averaged to determine mean percent cover . Native forbs included representative native species identified in the survey (Table S1 in Supporting Information), whereas samples from the P. recta and C. stoebe communities only included the focal species. Potentilla gracilis, a native Potentilla, was found at MPG3, and five plants were pooled into one sample to qualitatively assess whether the AMF community was more similar to the invasive P. recta than to the native forbs.Four plants were randomly selected and pooled from each of the five plots within each community, resulting in a total of 100 samples . Half of the roots were lyophilized and stored at −20°C prior to DNA extraction. We used AM colonization as an indirect measure of AMF abundance and AMF dependency, because intraradical root colonization has been shown to reflect differences in extraradical fungal abundance , sieved through a 2-mm sieve, and ultimately pooled within each plant community for a total of 17 soil samples (one from each plant community). Soil was air-dried prior to analysis for texture , pH, conductivity, macronutrients and micronutrients , and soil organic matter based on loss of ignition .P. recta samples were separated from native forbs in transition zones, for a total of 100 samples. DNA extractions were carried out on freeze-dried, ground roots (15 mg) using MoBio PowerPlant DNA isolation kits following the manufacturer's instructions with the double clean-up step . Optimized template concentrations (1:10–1:100) were amplified with the LSU rRNA primer set FLR3 and FLR4  were combined prior to sequencing based on DNA concentrations (nanodrop) to ensure similar representations from all sites prior to sequencing at the Genome Sequencing and Analysis Facility at University of Texas Austin.We extracted and amplified DNA from each of the five plots per community type separately, and C. stoebe community (MPG3) was excluded due to very low sequence numbers. OTU accumulation curves, generated using the “specaccum” function with the “rarefaction” method in the vegan package of R . One All statistical analyses were completed in R , and the nonmetric t tests (“t.test”), ANOVAs (“aov”), and post hoc tests (“TukeyHSD”) were completed in the “stats” package. Total AM colonization fractions were arcsine transformed prior to analyses to meet assumptions of normality. All other data sets met ANOVA assumptions of homogenous variances  in the “lawstat” package) and normal distributions (“qqnorm” and “qqline”) in the “stats” package.Community indices were completed with “diversity” function in the same vegan package. Network visualizations were completed using the “plotweb” function in the bipartite package . Total, arbuscular, and vesicular colonization data were analyzed with 3 × 3 fixed-factor ANOVAs (three plant communities × three sites and five replicates). There was a significant interaction between site and host plant , in which colonization was higher in C. stoebe and P. recta than native forbs at the MPG2 and MPG3 sites, but there was no difference in colonization between host species at MPG1. The density of arbuscules  varied only with host plant  and accounted for 50% of colonization in native forbs, which was 16% greater than in C. stoebe and 23% greater than in P. recta  or plant .We identified 25 OTUs across the native and invaded communities in Montana. All forbs were dominated by the same two taxa; OTU 1 and 16  in P. recta compared with native forbs. Colonization was higher overall in plants from the MPG2 site than MPG3 and MPG1 .Data on AMF colonization and OTU community metrics in the transition areas of the three Montana sites were used to address this question. All plants in the transition areas were highly colonized with an average AM colonization of 81% ± 14%. Total, arbuscular, and vesicular colonization data were analyzed with 2 × 3 fixed-factor ANOVAs (two plant communities × three sites and five replicates). There was a trend of higher colonization but a lower occurrence of arbuscules  and diversity . AM colonization varied by site ; overall we found higher AM colonization in Montana and Eastern Washington than Western Washington . Although our sample numbers are too low for a conclusive result , it appears that the AMF community is typically dominated by OTU 1 and 16, even across the climate and invasion gradient. P. recta maintained fewer but a more consistent mix of rare AMF taxa than C. stoebe. The singular Washington P. recta sample from WW4 stands out as different . In contrast, C. stoebe had a wide and consistent variation in rare taxa across sites , were covariates of texture and pH. The AMF communities from the sites in Montana were similar to each other, clustering near the center of the ordination, while the AMF communities from the sites in eastern and western Washington were divergent , with individual plant communities harboring anywhere from 6 to 14 OTUs. AMF communities were best predicted by soil texture (fine sand and silt) and pH  were not a significant predictor of OTU assemblage, invasion density  correlated positively with dominance by two most abundant OTUs (OTU 1 and OTU 16) and negatively with OTU richness. Dominance of OTU 1 and 16 increased from 12% to 78% with increasing cover of invasive species , while AMF richness decreased from 14 to 6 OTUs with increasing cover of invasive species . P. recta and C. stoebe differed in the strength of these relationships. P. recta were dominated by OTU 1 and OTU 16 across the invasion gradient, but lost species richness at higher densities, the exception to this trend was P. recta from WW4 in western Washington which had low richness even at low invasion density. C. stoebe maintained species richness, but had a large increase in OTU 1 and OTU 16 dominance with increasing cover. Additionally, the rare taxa in each C. stoebe sample varied significantly; of the 30 OTUs detected in C. stoebe roots, each site only hosted 8–11 different AMF taxa.While the categorical plant communities  whether native and exotic forbs at the same sites harbor different AM colonization and AMF communities, (2) whether the composition of AMF communities in native forbs is different in the presence of exotic forbs, and (3) whether exotic forbs harbor different AM colonization and AMF communities across regions (and simultaneously across a gradient of invasion density). The nature of the data sampled also allowed us to assess whether the AMF community composition correlated more strongly with the soil environment, plant communities, or spatial distance among sites.2-fixing, mycorrhizal forbs across multiple, independent invasions to isolate the effect of plant provenance from factors of mycorrhizal status and plant functional group.Previous comparisons between native and exotic invasive plants have focused on specific native species with selected invaders and found significant shifts in AMF communities associated with native plants when co-occurring with invaders , which accounted for 54% of all sequences recovered in this study. By considering the invasion gradient, we can infer how invading P. recta interacts with existing AM. In the pre-invasion (native community), OTU 1 and 16 made up 44% of the recovered sequences, but this increased to 62% of recovered sequences in the “post-invasion” (exotic monoculture community). These data suggest that this exotic forb forms symbioses with pre-existing dominant AMF rather than promoting rare AMF taxa. Whether or not these OTUs contribute to invasiveness or respond to an increased abundance of a preferred host is unknown and will require manipulating OTU abundances. It is interesting to note, however, that both Rhizophagus irregularis and Glomus microaggregatum are considered generalists in their host and environmental preferences, which is consistent with suggestions that exotic plants may associate with generalist, cosmopolitan mutualists to a greater extent than native plants  community and the “postinvasion” monoculture. Apparently, neighboring plants of the same functional group can influence each other's AMF communities. However, whether these shifts are significant enough to affect overall mycorrhizal function is debatable and will require more detailed in situ functional studies.Altering the AMF of native neighbors may be one mechanism by which exotic forbs increase competitiveness , not plant community type, adding to the growing body of evidence that some taxa are widespread generalists and environmental conditions are important filters. While controlled experiments are required to verify the functional consequences of the patterns observed here, we found only weak support for mycorrhizal-mediated invasions of C. stoebe and P. recta in the intermountain west.Utilizing survey data that can reveal the end result of long-term ecological processes, we observed greater mycorrhizal colonization in exotic than in native forbs, and in regions where exotics are highly invasive. This could imply that AMF directly benefit exotic plants and may contribute to invasions, although natives were only slightly less colonized and fungal abundance alone seems an unlikely explanation for invasive success. Samples collected along invasion gradients suggested that


While we could not demonstrate a significant contribution of FZD3 gene, we identified five rare FZD6 variants that were absent in all controls and predicted to have a functional effect by computational analysis: one de novo frameshift mutation (c.1843_1844insA), three missense changes , and one substitution (c.*20C>T) affecting the 3′-untranslated region (UTR) of the gene. The overall rate of predicted deleterious variants of FZD6 was 5.1-fold higher in cases compared to controls, resulting in a significantly increased NTDs mutation burden. This study demonstrates that rare nonsynonymous variants in FZD6 may contribute to NTDs in humans and enlarges the spectrum of mutations that link PCP pathway to NTDs. Hum Mutat 33:384–390, 2012. © 2011 Wiley Periodicals, Inc.Neural tube defects (NTDs) are severe malformations of the central nervous system, affecting 1 of 1,000 live births. Mouse models were instrumental in defining the signaling pathways defective in NTDs, including the planar cell polarity (PCP), also called noncanonical Frizzled/Disheveled pathway. Based on the highly penetrant occurrence of NTDs in double  Neural tube defects  are congenital malformations due to the incomplete closure of the neural tube during the early development. NTDs are known to occur in 1 of every 1,000 pregnancies, with varying rates reported among the world populations . The mosNTDs have a complex etiology involving both environmental and genetic factors. Although folic acid, supplemented in the mother periconceptionally, appears to have dramatically reduced the frequency of NTDs , the mecStrabismus/Van Gogh (Stbm/Vang), Frizzled (Fz), Dishevelled (Dsh), Flamingo (Fmi), Prickle (Pk), and Diego (Dgo) ), three are missense changes , and last one is a single nucleotide substitution (c.*20C>T), affecting the 3′ UTR of the FZD6 gene. (P = 0.036).We identified five rare D6 gene. ; Table 1FZD6 p.Cys615X (c.1843_1844insA) was a de novo mutation (absent in the parents), deriving from the insertion of an A nucleotide at position 1,843 of the coding region that introduces a premature stop codon at position 615  was found in two unrelated patients both affected with myelomeningocele (The 05Gln c.114G>A wasingocele . This vaingocele . AlthougFZD6 p.Arg511His (c.1532G>A) was found in an Italian patient with Caudal Regression syndrome, including vertebral and costal anomalies, meningocele, hydromyelia, tethered cord, left kidney agenesis  was identified in an Italian patient with myelomeningocele (MMC) (The le (MMC) ; Table 2le (MMC) . The ideFZD6 c.*20C>T localizes at the 3′ UTR of the gene, outside of protein coding sequence. It was identified in an Italian patient with MMC  and p.Ala388Asp (c.1163C>A) mutations, even if they were detected in one and two of 639 controls, respectively, could affect protein function, demonstrating that these variants could represent predisposing risk factors  and FZD6 p.Ser620Thr   .FZD3 and FZD6 coding regions and splice sites for small alterations in patients with NTDs. Resequencing analysis in 473 NTD patients and 639 controls identified a statistically significant overrepresentation of rare presumed pathological variants among the patients group. Prediction and understanding of the downstream effects of the nonsynonymous and noncoding variants was done using computational methods. Five mutations of FZD6, one de novo nonsense mutation (FZD6 p.Cys615X), three missense variants  affecting highly conserved residues, and a 3′UTR substitution (FZD6 c.*20C>T) were absent in all controls analyzed and have detrimental effects on the basis of bioinformatic tools. Thus, our findings support a significant involvement of FZD6 in the pathogenesis of a minority of NTDs patients and underscore the value of candidate gene resequencing to understand the genetic contribution in complex diseases such as NTDs. NTDs are the second human disease to be linked to mutations of FZD genes after familial exudative vitreoretinopathy  that arise from loss of function mutations of FZD4, a FZD gene that has not far been implicated in neural tube closure [This is the first study that systematically screened all  closure .FZD6 mutations account only for 1% of the NTDs cases. This mutation frequency is comparable to that detected in our previous studies in other human PCP genes [FZD6 gene represent low penetrance variants that must interact with other genes and/or environmental factors to modulate the incidence and the severity of NTDs. All the unique mutations were heterozygous and private except for FZD6 p.Arg405Gln found in two unrelated patients: one Italian familial case and one sporadic Canadian patient. The majority of the mutations were located in the intracellular domains of the FZD proteins. Mutagenesis studies have demonstrated that several residues in the first and third intracellular loops as well as in C-terminal domain of FZD are crucial for signaling [CP genes , 2010. Tignaling .FZD6 p.Cys615X arises from an A insertion in the coding region, which introduces a premature stop codon resulting in a truncated protein lacking the last 51 residues in its C-terminal tail. Genotype–phenotype correlation shows that this de novo and severe FZD6 mutation is associated with a complex dysraphic state in a patient presenting with thoracic meningocele, spondylo-costal malformations, hydromyelia, and lipoma. This is the first frameshift truncating mutation reported in PCP genes, since all previous reported mutations were missense [FZD6 p.Cys615X is a new germinal event, strongly implicating that this mutation might contribute to the NTD occurrence in this patient.Importantly, the missense , 2011. MFZD6 gene in a patient with myelomeningocele adds to the already complex picture of NTDs pathogenesis.Mutations in the 3′ UTR of mRNAs can lead to the removal or to the de novo generation of a target recognition site for a specific miRNA . miRNAs FZD6 mutations were detected both in open and closed spinal NTDs. This suggested that FZD6 gene could contribute in the pathogenesis of both open and closed forms of NTDs, as reported for other PCP genes, VANGL1 and VANGL2 [Unique nonsynonymous d VANGL2 , 2010, fFZD3 gene in the pathogenesis of NTDs. In fact, only one mutation was identified having an evident pathogenetic effect on protein function. Further studies would be required to determine whether FZD3 may play an independent role.By contrast, we could not demonstrate a significant contribution of Fzd3−/−/Fzd6−/− mice mutants provided a successful entry point for identifying human orthologs involved in NTDs. However, comparison of mouse and human data provide some limited insights, in that differently from double mutant mice that develop a severe form of NTDs, mutations of both FZD are unlikely to be found in the same affected individuals, probably due to their lethality. In addition, as reported in mice [FZD3 and FZD6 genes in neural tube closure, since that mutations at one single gene are not sufficient to develop the defect, being the majority of them inherited from an healthy parent, probably because of a compensatory effect of the wild-type gene. Given that single-locus allelism is insufficient to explain the variable penetrance and expressivity of such malformations, genetic variation across multiple sites of the PCP proteome may occur to influence clinical outcome.Mouse models represent powerful tools for gene discovery in human disease.  in mice , we confFZD6 gene in NTD represents a further step forward to our understanding of the intricate genetic puzzle underlying these complex malformations. Functional studies of the FZD6 variants will be necessary to confirm their implication in NTDs risk. Finally, our promising results induce us to pursue the investigation of other core PCP genes in large human NTDs cohorts.In conclusion, our findings are consistent with the emerging model that the cumulative contributions of multiple rare alleles with large genetic effects are found among individuals with complex trait. Identification of the role of


Anopheles (Kerteszia) cruzii (Diptera: Culicidae) is a primary vector of human and simian malaria parasites in southern and southeastern Brazil. Earlier studies using chromosome inversions, isoenzymes and a number of molecular markers have suggested that An. cruzii is a species complex.An. cruzii populations from Florianópolis–Santa Catarina (southern Brazil) and Itatiaia–Rio de Janeiro States (southeastern Brazil). The analyses were performed first comparing Florianópolis and Itatiaia, and then comparing the two putative sympatric incipient species from Itatiaia (Itatiaia A and Itatiaia B). The analysis revealed high FST values between Florianópolis and Itatiaia (considering Itatiaia A and B together) and also between the sympatric Itatiaia A and Itatiaia B, irrespective of their function. Also, using the IM program, no strong indication of migration was found between Florianópolis and Itatiaia (considering Itatiaia A and B together) using all loci together, but between Itatiaia A and Itatiaia B, the results show evidence of migration only in the direction of Itatiaia B.In this study, a multilocus approach using six loci, three circadian clock genes and three encoding ribosomal proteins, was carried out to investigate in more detail the genetic differentiation between the An. cruzii complex that diverged around 0.6 Mya, and also that the Itatiaia sample is composed of two sympatric incipient species A and B, which diverged around 0.2 Mya. Asymmetric introgression was found between the latter two species despite strong divergence in some loci.The results of the multilocus analysis indicate that Florianópolis and Itatiaia represent different species of the  Anopheles mosquitoes are insects belonging to order Diptera, family Culicidae and subfamily Anophelinae [Anopheles comprises almost 500 formally named species, although many of them are complexes of sibling species [Kerteszia[Anopheles (Kerteszia) cruzii is a Neotropical mosquito highly specialized in using bromeliad plants as breeding reservoirs during the development of immature stages. This bromeliad-breeding mosquito is mainly found in Atlantic forest areas along the Brazilian coast, a habitat rich in plants from the Bromeliaceae family [The phelinae . The gen species ,3. This Kerteszia,4. Anophe family . This moe family ,8, essene family -11.Anopheles species complexes, which are virtually identical in adult morphology, but exhibit differences in their malaria transmission competence, resting habitats, host preference and insecticide resistance [Anopheles species  in an effort to resolve their taxonomic status [An. cruzii and most of them suggest that An. cruzii is a species complex. Differences in X chromosome inversions frequencies from populations of southeastern and southern Brazil, suggested the existence of three An. cruzii sibling species [timeless gene, a locus involved in the control of circadian rhythms, was used as a marker to assess the genetic differentiation among a number of An. cruzii populations. The results indicated that a population of An. cruzii from northeast Brazil constitutes a highly differentiated group when compared with other five populations from the south and southeast regions of the country [FST values and fixed differences between these two cryptic species in all six loci, irrespective of their function. This analysis also indicated that they probably have not exchanged migrants since their separation, which was roughly estimated to have occurred around 2.4 million of years ago [Different studies have been made for identification of sistance ,12-14. Ic status ,15-21. D species ,23. On t species . These s country . In addiears ago .cpr gene, a locus involved in metabolic insecticide resistance and odorant clearance in insects, was used to analyze the divergence between An. cruzii populations from south and southeast Brazil . The cpr gene revealed very high FST values and fixed differences between Itatiaia and the other four populations studied. The data also provided preliminary evidence for the possible occurrence of two sympatric incipient sibling species in Itatiaia, called by the authors Itatiaia A and Itatiaia B [Besides, a fragment of the atiaia B .In the current study, a multilocus analysis using six different nuclear gene fragments was performed comparing Florianópolis and Itatiaia populations. Additional multilocus analyses were also carried out considering Itatiaia as two different incipient species (Itatiaia A and Itatiaia B). The aim of the study was to determine if there is gene flow between the putative sibling species and to estimate their divergence time.RM) and the length of the whole fragment and for the NR block of each gene . The alignments of the whole sequences of each gene are presented in Additional file cyc gene fragment, which was composed entirely of an exon. Besides, Additional file An. cruzii sibling species and two measures of nucleotide diversity: π, based on the average number of pairwise differences and θ, based on the total number of mutations . Pairwise estimates of population differentiation between the An. cruzii sibling species are shown in Table Dxy), the number of net nucleotide substitutions per site between species (Da) and the distribution of the four mutually exclusive categories of segregating sites observed in each comparison: the number of exclusive polymorphisms for each species (S1 and S2), the number of shared polymorphisms (Ss) and the number of fixed differences (Sf).One of the assumptions of the Isolation with Migration model used in this study is the absence of recombination within the studied loci. In order to fulfill this requirement, the optimal recombination-filtered block was extracted from each gene alignment. Additional file θ1, θ2, θA, t, m1, m2) from the An. cruzii sibling species through an "Isolation with Migration" model using multiple loci [t and θA in the simulations between Itatiaia A and Itatiaia B (see below).The IM program was used to simultaneously estimate six demographic parameters  and NR blocks (not shown) were estimated using the Neighbor-Joining method (NJ) with very similar results. The most suitable model selected using jModelTest 0.1.1 ,33 was Ktim and cyc and this was due to the higher number of recombination events identified in these two genes (RM = 13 and 8 respectively). Except for the tim gene, all base substitutions were synonymous or occurred within introns   [Mya .The divergence time parameter was estimated for all combined loci in four different IM runs Table . This pactively) . Using tDrosophila synonymous substitution rate mentioned above and the average Da values from the six loci .Itatiaia A was the least polymorphic, showing the lowest values of θ and π, as well as the smaller number of polymorphic sites (S) in most cases . The tim locus also shown a high FST value (FST = 0.34) between these two putative species and fixed differences as well, but shared polymorphisms were also observed in this gene. Among the other loci, there were shared polymorphisms and no fixed differences . In the RpS2 tree, there is a haplotype (Ita05a) from Itatiaia B among the Itatiaia A sequences. Also, the individuals Ita12  and Ita13 (that was not typed with cpr) appeared among Itatiaia A sequences. On the other hand, in the Rp49 tree, these two mosquitoes showed a hybrid aspect, in agreement with cpr gene results, where Ita12 was the only potential "hybrid" [Clk, cyc and RpS29, did not show an evident separation in NJ trees between Itatiaia A and Itatiaia B.The NJ trees Figures  and 3 grvs Itatiaia (considering Itatiaia A and B together) described above, analyses were also carried out comparing Florianópolis with Itatiaia A and Itatiaia B, separately.In addition to the analyses performed between Florianópolis FST values using the Clk and RpS29 genes, in agreement with the values found between Florianópolis and Itatiaia (considering Itatiaia A and B together). The further four genes shown FST values ranging from ~ 0.18 to 0.42. In all cases the FST values were significant. Interestingly, the clock genes  shown the higher FST values between Florianópolis and Itatiaia A, whereas the Rp49 and RpS2 loci shown the higher FST values between Florianópolis x Itatiaia B. The RpS29 showed very high and similar values in both comparisons (FST = 0.73 and 0.76).The pairwise estimates of population differentiation between the two groups Table  shown veClk, RpS29 and tim gene fragments. Between Florianópolis and Itatiaia B, the loci Clk, RpS29 and Rp49 shown fixed differences and few or inexistent shared polymorphisms .Additional file n Figure . Migrativs Itatiaia A, Florianópolis vs Itatiaia B and Florianópolis vs Itatiaia (considering A and B together), which suggest that the Itatiaia lineage separated from Florianópolis before splitting into A and B  and the FST > 0.35 to species diagnosis. Among the six FST values calculated for the pairwise Florianópolis and Itatiaia (including A and B), only two of them are higher than 0.35 (FST = 0.67 and 0.71). Nevertheless, the mean FST among the six loci (0.38) and both measures of evolutionary independence (gene flow and divergence time) are above the threshold criteria. The comparison made between Itatiaia A and Itatiaia B showed similar results, but the migration parameter in the direction of Itatiaia B shown values higher than the suggested threshold, consistent with the idea of incipient speciation.The multilocus results presented here revealed a high level of differentiation between Florianópolis, Itatiaia A and Itatiaia B indicating that these populations represent indeed different species in the  species . Hey & P species  discuss Clk and RpS29 showed the highest FST values between Florianópolis and Itatiaia (considering Itatiaia A and B together), while Rp49 and RpS2 showed the highest FST values between Itatiaia A and B. Interestingly, these four genes are located on the chromosome 2 of Anopheles gambiae, albeit the three ribosomal protein genes are on the right arm while Clk is on the left arm. Many authors have postulated that chromosomal inversions could promote speciation since their recombination suppressing effects facilitate the maintenance and accumulation of differences between interbreeding populations [Anopheles cruzii from south and southeast regions of Brazil, revealed the existence of three putative species, which differed mainly in the banding patterns of the X chromosome. Nevertheless, different autosomal inversions seemed to be associated with each form of X chromosome and it is tempting to speculate that some of the markers we used and showed high levels of differentiation between the sibling species are located within or nearby the inversions studied by Ramirez & Dessem [in situ hybridization experiments using these markers as probes might confirm if that is the case.Previously, we observed no evidence of association between function  in the divergence levels between the sibling species of Florianópolis and Itaparica . In the ulations -39. Ramiulations ,23, stud& Dessem ,23. FutuAnopheles gambiae complex [An. cruzii siblings have two main explanations: i) the maintenance of ancestral polymorphisms as these cryptic species have separated recently and ii) differential introgression in different genomic regions between the species reflecting locations where gene flow occurs freely or is restricted by selection.The observation of a putative hybrid  and the  complex -42 that The two conflicting but no mutually exclusive hypotheses of retention of ancestral polymorphism and introgression between closely related species are often difficult to distinguished. Introgression can sometimes be excluded based on the geographic separation of two species and in this case, the shared polymorphisms can be classified as ancestral . This iset al. [An. arabiensis and An. gambiae, found unidirectional introgression, from the former into the latter species, only in sympatric populations. Gomes et al. [Culex pipiens complex, in spite of the high levels of genetic differentiation between its forms.Asymmetric introgression has been documented in a variety of taxa -48, inclet al. , comparis et al.  found diet al. [An. gambiae complex, between M and S molecular forms, and they argue, as one of the possible explanations, that the asymmetric introgression may be a consequence of the differences in relative abundance of the two taxa , which could result in higher levels of backcrossing between the hybrids and the more abundant species. Our results are also consistent with this explanation as the estimates of population sizes and migration parameters from the IM program suggest that Itatiaia A (the genetic donor) has a smaller estimated population size than Itatiaia B (the genetic recipient).Marsden et al.  found siet al. [Marsden et al.  also proDa values) between Itatiaia A and Itatiaia B is ~ 0.2 Mya and between Florianópolis and Itatiaia (including A and B) is ~ 0.6 Mya. This indicates an earlier speciation process splitting the populations of Florianópolis and Itatiaia, followed by a more recent separation between the two sympatric species in the latter locality.The estimated divergence time (based on the Mya) [An. cruzii species complex. Since temperature and water are essential for the development of Anopheles spp. immature stages [Anopheles diversification seems plausible. Furthermore, in the case of An. cruzii (Kerteszia subgenus), its larval development is associated with water trapped in Bromeliads plants, which are restricted to the rainforest [An. cruzii, have revealed patterns of genetic divergence associated with the Pleistocene environmental changes [The time of divergence of these three sibling species as well as that between the Bahia and the south and southeast populations ~ 2.4 Mya) , points Mya)  as facto Mya , poinforest . So, a n changes ,51-55.et al. [The process of successive rainforest contractions and expansions was a very important consequence of these environmental modifications possibly favoring the differentiation and speciation of forest obligate species -58. Accoet al. , studyinet al. [Schizolobium parahyba (Fabaceae), a widespread tree in the Brazilian Atlantic forest, found high levels of genetic diversity in populations from the southeast region and low levels in southern populations. They argue that the low genetic diversity in the south may be the result of a founder effect followed by a range expansion after glacial periods. Lorenz-Lemke et al. [et al. [An. cruzii, Florianópolis (south sibling species) showed a higher level of genetic diversity than the Itatiaia (southeast) sympatric sibling species. Therefore, it is likely that there was a persistence of An. cruzii populations in the south during the contraction of the forest, as was proposed for toads [Turchetto-Zolet et al.  studyinge et al.  and Palm [et al.  also repor toads , insteadAn. cruzii complex suggest so far three main events of cladogenesis originating the different sibling species. The first one originated the Bahia (Itaparica) species and occurred toward the end of the Pliocene (~2.4 Mya) [Mya probably separated the Itatiaia lineage from other south and southeastern populations [Mya) originated the new sympatric incipient species found in this locality. Analysis of a number of other populations of An. cruzii from Rio de Janeiro State, where Itatiaia is located, might allow us to determine the distribution of these two new sibling species and to investigated whether they might have possibly originated by a process of sympatric or parapatric speciation.In summary, the results obtained with the studies of the 2.4 Mya) ,26, the ulations ,27, and Drosophila melanogaster genes involved in the control of circadian rhythms: timeless (tim), Clock (Clk) and cycle (cyc); and three code for ribosomal proteins: Rp49 , RpS2  and RpS29 .The mosquitoes used in this study were females captured in Florianópolis, Santa Catarina State (SC) (27°31′S / 48°30′W) and Itatiaia, Rio de Janeiro State (RJ) (22°27′S / 44°36′W). They were identified on the basis of their morphology according to Consoli and Lourenço-de-Oliveira [tim gene from Itatiaia were those previously published by our group [The sequences of the six genes from Florianópolis and the sequences of the ur group ,26 (AcceAn. cruzii genomic DNA extracted according to Jowett [Pfu DNA polymerase (Biotools).The primers used in this study were those previously published by our group ,26. The o Jowett  was usedPCR products were purified and cloned using either Zero Blunt TOPO PCR cloning kit (Invitrogen) or pMOS Blue vector blunt-ended cloning kit . Sequencing of positive clones was carried out in an ABI Prism 3730 DNA sequencer at the Oswaldo Cruz Institute using the ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems). The identity of the cloned fragments was determined by BlastX analysis using GenBank database .At least eight clones were sequenced for each mosquito. Sequences were edited and in most cases consensus sequences representing the two alleles were generated. In a number of individuals only one haplotype was observed among the eight sequences and in these cases mosquitoes were classified as homozygotes. The probability of incorrectly classifying a heterozygote as a homozygote individual with this procedure is less than 1%. The sequences from homozygote mosquitoes were duplicated prior to analysis. However, when carried out without duplicating homozygote sequences, the analysis rendered similar results. Sequences were submitted to GenBank (Accession numbers: JX129234–JX129351).ROSEQ v 2.91 [The sequences were aligned using ClustalX software  and popuQ v 2.91  softwareThe jModelTest version 0.1.1 ,33 was uθA, θ1, and θ2, respectively), divergence time (t) and migration parameters in both directions (m1 and m2). Initial IM runs were performed in order to establish appropriate upper limits for the priors of each demographic parameter. These preliminary simulations generated marginal distributions that facilitated the choice of parameter values used in the final IM analyses. The convergence was assessed through multiple long runs  and by monitoring the ESS values, the update acceptance rates and the trend lines.The IM program is an implementation of the Isolation with Migration model and is based on the MCMC (Markov Chain Monte Carlo) simulations of genealogies ,74. It sThe Infinite Sites model  was chosGC program, which also removes haplotypes that represent likely recombinant sequences [The optimal recombination-filtered block was extracted from each gene alignment using the IMequences ,77.The authors declare that they have no competing interests.LDPR participated in data generation and analysis, and drafted the manuscript. She also helped capture mosquitoes in Florianópolis. CJCP carried out the capture and morphological identification of mosquitoes. AAP is the principal investigator, participated in its design and coordination, and helped to write the manuscript. All authors read and approved the final manuscript.GC program and based on alignments presented in Additional file NR blocks and sequences excluded from the IM analysis. Edition of sequences prior to IM analysis using the IMClick here for fileAn. cruzii sibling species. RM, the minimum number of recombination events; n, number of DNA sequences of each sibling species; S, number of polymorphic (segregating) sites; θ, nucleotide diversity based on the total number of mutations (Eta); π, nucleotide diversity based on the average number of pair-wise differences; DT, Tajima’s D [DFL, Fu & Li’s D [FFL, Fu & Li’s F [Polymorphism summaries of jima’s D ; DFL, Fu& Li’s D  and FFL,& Li’s F , based oClick here for filetimeless, Clock, cycle, Rp49, RpS2 and RpS29 gene fragments from Florianópolis and Itatiaia. The translated amino acid sequences are shown above the alignments and the introns are highlighted in grey. Dots represent identity and dashed represent gaps. Flo: individuals from Florianópolis; Ita: individuals from Itatiaia.Alignments of the DNA sequences of the Click here for filevs Itatiaia.Summarized features of the marginal histograms for each parameter for the pairwise comparison Florianópolis Click here for filevs Itatiaia B.Summarized features of the marginal histograms for each parameter for the pairwise comparison Itatiaia A Click here for filevs Itatiaia A (A) and Florianópolis vs Itatiaia B (B).Summarized features of the marginal histograms for each parameter to the pairwise comparison Florianópolis Click here for file


Angiostrongylus vasorum infection diagnosed in canine patients during clinical practice, to clinically and paraclinically monitor infected dogs after treatment, and to monitor the presence of L1 larvae in faecal samples by Baermann's test. Of the total 210 enrolled animals A. vasorum infection was diagnosed in 7 dogs. These dogs were clinically and paraclinically investigated and monitored after specific treatment. Further 3 symptomatic dogs were retrospectively included in the monitoring. Results suggest that the computed scoring system can help to increase the clinical suspicion of infection particularly in asymptomatic dogs before the onset of potentially lethal lesions. Data of faecal monitoring suggested that treatment may control parasite burden but be unable to eradicate infection. Thus, a continued faecal monitoring after treatment is advisable for identification of still infected or reinfected dogs.Canine angiostrongylosis is an increasingly reported disease in Europe which can be fatal if left untreated. The wide range of clinical presentation along with the absence of pathognomonic alterations can make the diagnosis challenging; thus any additional information that may provide clues to an early diagnosis may be of value, in order to ensure adequate anthelmintic treatment. Aim of the study was to assess a clinicopathological scoring system associated with natural  Canine angiostrongylosis is an increasingly reported disease, possibly due to climatic factors , to the Angiostrongylus vasorum defined as the “great imitator” [The parasite mitator”  is respomitator” . The sevmitator” , 11–13. mitator” –16, radimitator” , 18, echmitator” –21, and mitator” , 23 are The gold standard to achieve the diagnosis still remains the first-stage larvae (L1) detection using the Baermann technique on faecal samples, preferably collected over three consecutive days because of the intermittent larval detection in faeces . MoleculA. vasorum are of great importance in veterinary medicine, being potentially fatal if left untreated [Treatment of canine angiostrongylosis is performed through oral weekly application of milbemycin oxime for four weeks  or a sinntreated , 11, 33.ntreated , 32, anyA. vasorum infection diagnosed in canine patients; to collect the clinical, laboratory, and imagining data associated with natural A. vasorum infection and follow up animals after treatment; and to monitor the presence of L1 in faecal samples evaluated by Baermann's test in a long term followup after treatment.Therefore, the aims of this work were to assess a clinicopathological score associated with natural From November 2010 to November 2011 a faecal sample was routinely collected directly from the rectal ampulla of owned dogs presented for different problems or for a routine control visit at the Clinical Unit of the Veterinary Faculty of Bari, Italy. In case of empty ampulla, a fresh faecal sample was collected by the owner.Dogs of all ages, breeds, and living conditions were randomly enrolled in the study, except for animals living exclusively in apartments, which were excluded. Anamnestic data including dog history, living conditions, location, and date and duration of travelling into other regions or abroad as well as the medical history were collected and registered on individual clinical forms, along with data from clinical examination performed at the enrolment. A direct faecal smear was prepared and microscopically examined and the Baermann technique was performed. For patients tested negative but which during the clinical examination showed a clinicopathological score equal to or higher than 3 (see below) the direct faecal smear examination and the Baermann test were also performed on a 3-day faecal pool. Positive dogs were enrolled in the clinical study and treated . The infμL), mild eosinophilia (eosinophils 1.2–1.8 × 10³/μL), hyperglobulinaemia (>4.4 g/dL), or specific increase of β globulin (β1 + β2 > 1.7 g/dL) and any abnormalities in hemostatic parameters: 1 point each; severe eosinophilia (eosinophils > 1.8 × 10³/μL), lung interstitial/alveolar pattern on thoracic radiographs, pulmonary hypertension at echocardio-Doppler examination: 3 points each. Dogs living in the same environment of diagnosed positive dogs were also enrolled in the study, to which a score of 3 was assigned.For each dog clinicopathological changes suggestive for angiostrongylosis were evaluated and expressed with a score obtained by adding points as follows: presence of signs of coagulation deficits, respiratory disease, neurological signs: 3 points each; laboratory alterations such as anemia (hematocrit < 36%), leukocytosis , and a coagulation profile were performed in these dogs before anthelmintic treatment as already described .Further 3 symptomatic dogs (dogs 8-9-10) with a previous diagnosis of canine angiostrongylosis on faecal examination were included in the clinical study; their medical records and radiographs were retrospectively reviewed and clinical and paraclinical findings collected.0, T+15, T+30 . Symptomatic dogs were treated with fenbendazole 25 mg/kg BW/12 hourly/per os for 21 days . In symptomatic animals clinical examination was performed once a week until clinical recovery, afterwards twice a month, while asymptomatic animals were reexamined only after the third spot-on treatment. Laboratory, radiographic, and echocardiographic controls were performed after treatment according to owner disposability.Asymptomatic dogs were treated with an imidacloprid/moxidectin spot-on formulation (Im/Mox) in a dosage of 25 mg moxidectin/kg BW at TAfter treatment, dogs were weekly monitored using the Baermann test on a 3-day faecal pool, both in symptomatic and asymptomatic animals, for one month, afterwards once a month until possible, firstly depending on owner compliance. When infection persisted, a second-line treatment was considered .The study monitoring was stopped when normalization of any clinicopathological alterations and/or 2 consecutive parasitological negative results were reached. The animals were kept under their usual housing conditions and handled and sampled with the owners' consent and with the approval of the Ethical Committee of the Faculty of Veterinary Medicine of the University of Bari .A total of 210 owned dogs were enrolled in the study. The study population consisted of 97 females and 113 males; 84 were crossbred and 126 purebred animals. The age ranged between 4 months and 16 years (mean 6.2 years) and the weight ranged from 1 to 55 Kg (mean 22.9 Kg). The clinical and pathological abnormalities and scores for dogs included in the study are reported in A. vasorum larvae at least at one test . The highest score (11) was reached by a hunting dog (dog 6) presenting severe dyspnoea, while a score of 9 was reached by a dog (dog 7) presented for epistaxis and hyphema. The remaining five positive dogs were normal at clinical examination; nevertheless, they reached a score ≥ 3.A direct faecal smear was evaluated in all enrolled dogs (210 samples) and Baermann's test was performed on 162 single samples; in the other 48 dogs the sample volume was not enough to perform the test. A 3-day faecal pool was investigated by means of both direct fecal smear and Baermann's test, in 67 out of 74 dogs showing a clinical score ≥ 3, depending on owner compliance. On the whole, 7 dogs resulted positive for test see . A. vasoA. vasorum infection (n = 10)   were ageescribed .Fenbendazole was administered in monotherapy in five cases; Im/Mox was administered in monotherapy in the other five cases . In geneResults of faecal monitoring are reported in A. vasorum infection in the literature [Given the geographical difference with the previously reported case series  and prevterature , 20, 21.Combining the scoring system with faecal tests introduced as routine tests in clinical practice permitted to identify asymptomatic dogs. Diagnosis of angiostrongylosis was performed in all dogs by L1 detection in faecal samples and identification based on morphometric characters and measurements . The occA. vasorum in dogs, confirming that angiostrongylosis should be considered as a possible etiological cause of several from mild to severe clinical conditions [β globulin fraction at serum protein electrophoresis and the serpiginous/circular areas of radiopacity on thoracic radiographs were documented as useful findings that may help to reveal asymptomatic infections [The reported cases show different clinical presentations associated with the presence of nditions . As prevfections .Dirofilaria immitis infections, possibly leading to a reduced pathogenicity of A. vasorum infections. In experimental studies [A. vasorum presenting continuous or irregular pattern in larval output.Given its proven efficacy , 38, fen studies  comparin studies . However studies  reportedA. vasorum infections, possibly associated with the host and/or to the parasite biology. Moreover, to monitor the response to treatment, serial tests are needed since the limits of Baermann's tests are known and a single negative result does not mean absence of larval excretion.The highly variable response to treatment registered in this study suggests that under natural conditions several factors could affect the response to treatment and the diagnosis of Assessing a clinicopathological score can help to increase the clinical suspicion of infection particularly in asymptomatic dogs before the onset of potentially lethal lesions. Early detection of infected dogs is important because pathological changes may be already present in asymptomatic dogs . Further


In the adult males of helminthomorph millipedes, one or two pairs of legs in the anterior part of the trunk are strongly modified into sexual appendages (gonopods) used for sperm transfer during the copula. Gonopods differentiate in an advanced phase of post-embryonic development, in most cases as replacement for the walking legs of the seventh trunk ring, as these first regress to tiny primordia, to eventually develop into gonopods at a subsequent stadium. These extremely localized but dramatic changes have been described as a non-systemic metamorphosis. In the present study we describe morphological and anatomical changes of trunk ring VII associated with non-systemic metamorphosis in four helminthomorph species.As documented here for the first time by means of traditional histology methods and new techniques based on confocal laser scanning microscopy, the external modifications caused by non-systemic metamorphosis are associated to a huge rearrangement of internal anatomy, mostly due to the development of gonopod apodemes and extrinsic muscles.Nopoiulus kochii and Blaniulus guttulatus, with likely major functional consequences.Internal changes in the seventh trunk ring, locally leading to the dorsal displacement of the ventral nerve cord and the digestive tract, are modulated in a taxon-specific manner, and are very conspicuous in the blaniulids  Because of the independent segmentation of dorsal and ventral structures during millipede embryogenesis )al setup ,57, care®X 100 at least six times, each time for 20 min, and treated overnight with chitinase from Streptomyces griseus (0.7 u/ml at 37°C in HEPES buffer) to soften the exoskeleton; samples were then washed in PBS+0.1% Triton®X 100 and decalcified overnight in EDTA 0.5 M pH 8 at 4°C. After washing in PBS+0.1% Triton®X 100, the specimens were dehydrated through a graded ethanol series, then immersed in xylene and finally embedded in paraffin. Sagittal, frontal and transversal sections were made at a thickness from 7 to 12 μm  and stained with Mallory's triple stain [Soft parts anatomy was studied through serial paraffin sections. Fixed specimens were washed in PBS+0.1% Tritonle stain . Slides Digital images were processed with the software CorelPHOTO-PAINT, CorelDRAW (ver. 14) and Adobe Photoshop (ver. 12).The authors declare that they have no competing interests.LD carried out the morphological investigations on non-systemic metamorphosis in the four millipede species, assisted by EG. LD, GF and AM implemented data analysis and interpretation, and drafted the article. All authors participated in the design of the study, and read and approved the final manuscript.


Dirofilaria immitis infection in dogs from Figueira da Foz, located in the central region of Portugal. In the period between November 2009 and January 2011, 304 blood samples were obtained from dogs over 1 year of age, with no previous history of heartworm prevention or diagnosis. Every blood sample was analyzed using varied laboratory techniques . In the samples in which microfilaremia was detected, a histochemical technique using acid phosphatase staining was applied to identify the species of microfilariae. A total prevalence of 27.3% (83 out of 304) was found. We also found that 73.5% of all positive cases (61 out of 83) were microfilaremic, and 26.5% were occult infections (22 out of 83). By means of a histochemical technique Dirofilaria immitis was identified in 96.7% of microfilaremic samples. A multivariate model allowed us to identify the following risk factors for the presence of heartworm disease: age between 4 and 9 years, dogs living in a rural environment, large breed dogs, and living outdoors. This study shows for the first time the high prevalence of heartworm disease in a central area of Portugal and emphasizes the importance of systematic screening for this disease, as well as the need to prevent it in dogs in this area.The aim of the present study was to determine the prevalence and risk factors concerning  Dirofilaria immitis   iy, 1856) , 30Culex, Aedes, and Anopheles, among others, have been implicated in the transmission of this parasite, allowing for its intermediate stage to complete its life cycle [Different species of culicid mosquitoes belonging to the genera fe cycle , 30. Weafe cycle .Dirofilaria immitis in the pulmonary arteries and right ventricle of the heart [Wolbachia (Rickettsiaceae) stimulates an inflammatory response from the host’s immune system, amplifying disease severity [Heartworm infection is a severe and life-threatening disease. The pathophysiological response to heartworm infection is mainly due to the presence of adult worms of he heart , 17. Thehe heart , 28. Furseverity .Clinical manifestations include cough, dyspnea, weight loss, poor exercise tolerance, weakness, hemoptysis, cyanosis, and congestive heart failure , 17.Dirofilaria immitis third-stage larvae (L3) and L4, which have developed within the previous 30 days, and thus prevent disease caused by adult worms [Canine heartworm infection is preventable and chemoprophylaxis is a priority in heartworm endemic areas. One of the goals of prevention is to reduce the reservoir population and decrease the prevalence of infection among unprotected dogs. The macrocyclic lactones  are the most commonly used chemoprophylaxis agents: they are safe, effective and are administered either in oral, topical, or parenteral formulations at monthly or six-month intervals , 21, 22.lt worms . Preventlt worms . Before lt worms , 22.The treatment of heartworm infection is neither simple nor safe in itself. The American Heartworm Society recommends the melarsomine dihydrochloride three-injection protocol (2.5 mg/kg followed 1 month later by two similar treatments 24 h apart) instead of the two-injection protocol of 2.5 mg/kg 24 h apart, because the former is safer in terms of thromboembolism and shock, and has a higher efficacy , 46, 47.Dirofilaria immitis in dogs supposes a risk for the human population. Epidemiological, molecular, and immunological studies as well as clinical practice have discovered an increasing number of countries in which clinical cases of human dirofilariasis are reported, while seroepidemiological studies suggest that humans frequently become infected with Dirofilaria spp. at an early age. This information has changed the picture of human dirofilariasis from a sporadic to an emerging disease [D. immitis infection in humans were reported in Portugal as a consequence of surgical removal and histological examination of larval lung nodules [The presence of  disease , 41. In  disease , and twoDirofilaria immitis is the causative agent of pulmonary dirofilariasis, and in many cases produces benign pulmonary nodules usually identified incidentally by chest radiography in asymptomatic patients, which can initially be misidentified as malignant tumors [Dirofilaria immitis worms were also found in cranial, hepatic, intraocular and mesenteric adipose tissue, testicular arteries, and conjunctival regions [In the human host, t tumors –32, 44.  regions , 41. The regions , 41.The epidemiological status of dirofilariasis is currently undergoing rapid evolution. In spite of efforts made to prevent and control the infection in dogs, disease prevalence is rising in endemic areas as well as spreading into new areas reported as dirofilariasis-free until recently . FactorsDirofilaria immitis in dogs living in the central area of Portugal and explore some of the epidemiological conditions that may increase the infection.In the present study we will analyze the current prevalence and the seroprevalence of 2 area) located near the Atlantic coast and crossed by the Mondego river. The climate is temperate. Winter temperatures range between 7 °C and 14 °C, rarely reaching below 0 °C, while in summer the temperatures range between 15 °C and 25 °C, sometimes exceeding 35 °C during heat waves. The average annual temperature is around 15 °C, while the mean annual precipitation is about 600 mm. The climate and socioeconomic life in the region with farming activity (rice culture) provide suitable conditions for development of culicid mosquitoes, vectors of Dirofilaria.The study was conducted in the county of Figueira da Foz, a central region of Portugal, in 18 contiguous parishes  were sampled: 247 animals were privately owned dogs which had been taken to the veterinary clinic for a routine health examination and 57 animals came from an animal rescue shelter , throughout the whole year .Figure 1Animals were selected to fulfill the following criteria: (1) native animals that had never been moved outside the study area according to the owners or the shelter’s representative, (2) animals more than 12 months old, (3) animals not submitted to chemoprophylaxis against heartworm, (4) animals with no past history or diagnosis of heartworm infection, and (5) owners who were willing to participate in this survey and authorized it.The clinical signs and a detailed history  were obtained together with a complete physical examination looking for the presence of cardiopulmonary signs. This check-up included a complete cardiac and pulmonary auscultation, pulse evaluation, capillary refill time, jugular distension, and abdominal palpation.A minimum of 5 mL of blood was collected from the jugular vein of all 304 dogs. Of this blood volume, at least 3 mL were collected in tubes with anticoagulant and at least 2 mL in tubes without anticoagulant and stored under refrigeration until analysis.This study was approved by the Ethics Committee of the Clinicão – Veterinary Hospital and all of the dogs’ owners signed an informed consent document.Dirofilaria immitis circulating antigen using a canine antigen test kit  with sensitivity of 84% (78–89), specificity of 97% (84–100), and accuracy of 86% (81–90) [All samples of canine blood were tested for detection of  (81–90) . The proBlood samples were processed on the day of collection. First, fresh blood from each sample was examined under a microscope to determine the presence of microfilariae by the thick drop technique. A modified Knott test was also performed on all samples. Blood was hemolyzed with 9 mL of 2% formalin and centrifuged at 1500 rpm for 5 min. The supernatant was then decanted, and the sediment was stained with a drop of methylene blue 0.1% [©, Merck, Darmstadt, Germany) according to Peribánez et al. [D. immitis show two acid phosphatase activity spots localized around the anal and excretory pores, whereas D. repens shows only one acid phosphatase activity spot localized around the anal pore and microfilariae with enzyme activity throughout the body, shown to be Dipetalonema reconditum [For species determination, microfilaremic-positive samples were analyzed using histochemical staining bases with different acid phosphatase activity using a commercial kit test , Alentejo (16.5%), and Algarve (12%). The island of Madeira had the highest prevalence, with 30% of the dogs tested being positive for Dirofilaria immitis microfilaremia [The information on the distribution and prevalence of atejo 16.%, Alenteatejo 16.%, Alenteatejo 16.%, Alente95%: 22.4%–32.7%). This value is higher than the previously estimated prevalence for Coimbra district and Portugal. Only the island of Madeira showed a similarly high prevalence.In the current study we demonstrate for the first time the existence of canine heartworm infection in Figueira da Foz  with 83 out of 304 dog samples analyzed testing positive in at least one diagnostic test, thus resulting in a prevalence of 27.3% (CID. immitis in one or more diagnostic tests was variable according to the techniques considered (Dirofilaria immitis were 19.4% (59/304) by the direct smear test, 20.1% by the modified Knott technique (61/304), and 25.7% (78/304) by the Snapp® IDEXX test. The resulting sensitivities were therefore 71.7% for the direct smear test, 73.5% for the modified Knott technique and 94% for the Snapp® IDEXX test. Specificity was 100% for the three techniques employed.The number of dogs that tested positive for nsidered . The preThe different sensitivities among the techniques could be explained by the fact that microfilariae-detecting techniques are not able to detect occult infections (amicrofilaremic infections). These infections could arise due to several causes: low parasite burdens, prepatent infection by young adults, infections by aging adult females with impaired fertility, infections with male-only parasites, or immune response from the host against microfilariae. The resulting percentage of occult infections was 26.5%. This high percentage of occult infections is not uncommon and was previously reported by several studies , 25, 39.© IDEXX test but tested positive on the modified Knott test and were identified as belonging to D. immitis species by histochemical staining (acid phosphatase activity). Many studies have reported that commercial serological kits have low sensitivity when parasite burdens are low , when the worms show low fertility, when the presence of microfilariae persists for 1 to 3 years after the death of adult females, prepatent infection or only male infection [Five of the positive (6%) samples tested negative when using the Snappnfection . This confection –37.D. immitis . The acid phosphatase activity offers an easily observable and reliable method for differentiation of microfilariae [The samples of the 61 microfilaremic dogs underwent histochemical staining and 59 (96.7%) of them revealed acid phosphatase distribution matching with microfilariae of filariae , 49.p = 1) and a high level of agreement with the dogs’ status of Dirofilaria immitis infection . The Snapp IDEXX® test demonstrated a much higher diagnostic capability, with a substantial level of agreement (Kappa = 0.958).In regard to the diagnostic method, the McNemar statistical test allowed us to compare the different diagnostic techniques. The direct smear test and the modified Knott technique showed the same ability to perform the diagnosis, with no statistical differences (p < .05), whereas no significant differences were found related to ownership, pure versus mixed breed, sex or hair length.The following risk factors were therefore determined: dogs aged between 4 and 9 years old (37.8%), medium breed dogs (34.7%), dogs living in a rural environment (38%), dogs living outdoors (37.7%), watchdogs (52.2%), and hunting dogs (36.2%) , dogs living in a rural environment , large breed dogs , and living outdoors .A multivariate model identified the following risk factors for the presence of heartworm disease: age between 4 and 9 years  caspius, and they are broadly distributed among the 18 districts of Portugal [Culex pipiens (57%), Culex theileri (33.7%), and Ochlerotatus caspius (6.2%) [Culex theileri and Ochlerotatus caspius species fed on both hosts: Canis familiaris and Homo sapiens [Culex pipiens is a natural and efficient vector of Dirofilaria immitis with a host-feeding pattern that includes humans, and is therefore of particular concern for animal and public health [Aedes caspius was also described as a vector of Dirofilaria immitis [Dirofilaria immitis in mosquitoes of the species Culex theileri was found in Funchal [In Portugal there are 41 identified species of mosquitoes. The most abundant (92%) are Portugal . Osório theileri .7%, and  sapiens . Culex ptheileri 3.7%, and pipiens 7%, Culexc health . Aedes c caspius .2% [Cule Funchal  and in t Funchal , 37.In this study, living in a rural environment and living outdoors are important risk factors and the activity of the dog is also a relevant health risk. Hunting dogs and watch dogs show higher prevalence. This influence is presumably due to vector exposure rates as they have a greater chance of being bitten by mosquitoes , 49, 50.Dirofilaria immitis-positive dogs were asymptomatic, with the risk of being silent carriers, which reinforces the importance of screening all dogs that live in Figueira da Foz that have not started any prevention strategy for Dirofilaria immitis yet.No significant difference was found in the results throughout the year. Although the period of transmission is restricted to a period of the year because of the relationship of the life of the vector with the weather, the long parasite development in dogs (7–9 months) and the fact that many of the infected dogs are asymptomatic for months or years makes it difficult to establish a curve during the year for prevalence  Figure . We founp < 0.05). These physical examination findings are compatible with the pathophysiology of dirofilariasis disease [The most common physical examination findings in this study were cough (38%), low body condition index (38.2%), increased capillary repletion time (66.7%), cardiac (48.8%), and pulmonary (66.6%) auscultation abnormalities, cardiac murmur (48.7%), and abdominal distention (48.1%). All these findings showed a significantly higher prevalence ( disease , 39, 47.Dirofilaria immitis. Given the impact on animal and human health, it is advisable that this infection should be included in the differential diagnosis of canine cardiopulmonary disease in the central area of Portugal. The epidemiological knowledge of human dirofilariasis in Portugal is still scarce. In Portugal, a case of ocular dirofilariasis in a human was reported [D. immitis infection in humans as a consequence of surgical removal and histological examination of larval lung nodules [The present study provides evidence that dogs in Figueira da Foz, Portugal, are at risk of becoming infected with reported , as well nodules . TherefoContinuing veterinary education and developing awareness will possibly avoid the spread of this parasitic disease throughout the country; for instance, by improving diagnostic and preventive measures against the vectors. This is particularly true given the possible lack of symptoms in microfilaremic animals and long incubation periods during which the animals are able to infect mosquitoes. The finding of positive microfilaremic but asymptomatic animals highlights the potential risk that exists. The presence of asymptomatic and microfilaremic animals in the present work demonstrates this potential risk.Dirofilaria immitis in the central area of Portugal, a situation that can be extended to other regions of the country where the information is also missing, thus contributing to the establishment of future control programs.Finally, this study is expected to give veterinary and public health authorities an increased awareness about the data concerning We declare that we have no conflict of interest.