Faecal DNA-based genetic analysis is a suitable tool for assessing both population size and genetic diversity of threatened and elusive species. We applied microsatellite analysis and mtDNA sequencing for investigating the southernmost Italian (Sila Massif, Calabria Region) population of the Eurasian otter ( Lutra lutra ). This relict population, filed as extinct in the mid-1980s, is currently expanding but still quite isolated. On the two main rivers hosting otters permanently since 2014, we collected 47 spraints, out of which 24 (51.1%) were successfully genotyped (on average 2.0 alleles per locus ). Thirteen individuals were identified: seven females and three males (sex identification success of 76.9%). Population size was assessed as 16 individuals (13–22), corresponding to a density of 0.15 (0.13–0.21) ind/km. Successfully amplified mtDNA samples (N = 16) confirmed the occurrence of a haplotype—H10—which had been previously reported only for Southern Italy, bringing new evidence of the unicity of the Italian otter population. Although density values complied with those reported for the core area of otter Italian range, the small size and genetic isolation of this population require special attention. To assist the ongoing re-colonisation of the Sila Massif, habitat management should aim to enhance fish availability and connectivity with the core area.

Abstract On the River Ticino in northern Italy, a small number of captive Eurasian otters Lutra lutra, belonging to the European breeding programme for self-sustaining captive populations, were reintroduced in 1997, after the species had been declared locally extinct in the 1980s. We surveyed for otter signs in 2008, 2010, 2016–2017 and 2018, confirming the presence of what is probably a small population. To assess the abundance and viability of the population, we genotyped fresh spraints collected during the last two surveys, using 11 microsatellite markers, and modelled the population trend using Vortex. A minimum of six individuals were identified from 25 faecal samples. The analysis of mitochondrial DNA determined that the reintroduced otters share a transversion that is characteristic of the Asiatic subspecies Lutra lutra barang, confirming the contribution of the Asiatic subspecies to the genetic pool of the captive-bred founder population. Population size was consistent with the release of three pairs of otters and all models implied that the number of founders was too small to ensure the long-term survival of the population. Stochastic factors are therefore likely to threaten the success of this reintroduction.

On the River Ticino (Piedmont and Lombardy regions, N Italy), the Eurasian otter Lutra lutra became extinct in the 1980s and was reintroduced in 1997. Since then, the status of the reintroduced population has been assessed only occasionally, in 2008 and 2010. Between 2016 and 2018, we conducted an extensive survey for otter signs along the whole Italian stretch of the River Ticino, following the ‘Standard Method’ for otter surveys. In 2016–2017, we found 101 spraints (mean marking intensity: 0.40 spraints/100 m), spread over a 97-km long stretch of the river. In 2018 only five spraints were collected, the two furthermost marking sites being 32 km apart. Genotyping of nDNA extracted from 21 faecal samples enabled the identification of six different individuals. The surveys led to drawing a reliable picture of otter distribution and population size, with evidence of otter occurrence on a longer than previously recorded stretch of the river. The results of the 2018 survey suggest that stochastic factors may still threaten the survival of reintroduced otters and would suggest a reinforcement of the population is required to increase its genetic diversity.

Decreased inflammation and cardiovascular mortality are evident in patients with end-stage chronic kidney disease treated by online hemodiafiltration. Extracellular vesicles (EV) are mediators of cell-to-cell communication and contain different RNA types. This study investigated whether mixed online hemodiafiltration (mOL-HDF) beneficial effects associate with changes in the RNA content of plasma EV in chronic kidney disease patients. Thirty bicarbonate hemodialysis (BHD) patients were randomized 1:1 to continue BHD or switch to mOL-HDF. Concentration, size, and microRNA content of plasma EV were evaluated for 9 mo; we then studied EV effects on inflammation, angiogenesis, and apoptosis of endothelial cells (HUVEC) and on osteoblast mineralization of vascular smooth muscle cells (VSMC). mOL-HDF treatment reduced different inflammatory markers, including circulating CRP, IL-6, and NGAL. All hemodialysis patients showed higher plasma levels of endothelial-derived EV than healthy subjects, with no significant differences between BHD and mOL-HDF. However, BHD-derived EV had an increased expression of the proatherogenic miR-223 with respect to healthy subjects or mOL-HDF. Compared with EV from healthy subjects, those from hemodialysis patients reduced angiogenesis and increased HUVEC apoptosis and VSMC calcification; however, all these detrimental effects were reduced with mOL-HDF with respect to BHD. Cell transfection with miR-223 mimic or antagomiR proved the role of this microRNA in EV-induced HUVEC and VSMC dysfunction. The switch from BHD to mOL-HDF significantly reduced systemic inflammation and miR-223 expression in plasma EV, thus improving HUVEC angiogenesis and reducing VSMC calcification.

Parents should differentially allocate resources to the production of offspring of either sex depending on their expected fitness return. In sexually promiscuous females, offspring sex ratio should be affected by the sexual attractiveness of biological fathers because sons, but not daughters, will benefit from inheriting genes for sexual attractiveness. Females may acquire benefits for the offspring if the extra-pair male is of superior genetic quality as compared to the social mate, if it carries compatible genes or genes that enhance offspring genetic diversity. If sexually selected ornaments reflect male quality, extra-pair offspring should be more frequently males and of higher quality compared to their half-siblings. Furthermore, the probability of extra-pair offspring to be male should increase with an increasing difference in sexual ornamentation between the extra-pair and the social mate. In this study, we tested if barn swallow offspring sex ratio depends on paternity and on sexual ornamentation of the extra-pair father, and whether paternity predicts offspring phenotypic quality. The results partially fulfilled our prediction. We did not provide support for the hypothesis of a differential sex allocation to the production of male offspring by promiscuous females, both at the individual and at the within-brood level. Moreover, the difference in the ornamentation of the extra-pair and the social male did not affect the sex ratio of individual offspring. However, in accordance with the good genes and the genetic compatibility hypotheses, extra-pair offspring were of superior quality as compared to their half-sibling, in terms of body size and feather growth.

Natural and sexual selection arise when individual fitness varies according to focal traits. Extra-pair paternities (EPPs) can affect the intensity of selection by influencing variance in fitness among individuals. Studies of selection require that individual fitness is estimated using proxies of lifetime reproductive success (LRS). However, estimating LRS is difficult in large, open populations where EPPs cause reallocation of biological paternity. Here, we used extensive field sampling to estimate LRS in a population of barn swallows (Hirundo rustica) to estimate selection on lifespan and ornamental traits of males. We found selection on lifespan mediated both by within-and extra-pair fertilization success and selection on tail length mediated by within-but not extra-pair fertilization success. In addition, we found selection on tail white spots via extra-pair fertilization success after controlling for selection on other traits. These results were not confounded by factors that hamper studies of LRS, including nonexhaustive sampling of offspring and biased sampling of males. Hence, natural and sexual selection mediated by LRS operates on lifespan, tail length, and size of the tail white spots in barn swallows.

Reproductive promiscuity, whereby females are fertilized by extrapair mates, is common. The frequency of extrapair fertilizations (EPFs) depends on at least three sources of variation. First, females may differ in their proneness to being fertilized by extrapair males. Second, males may differ in traits that affect realized promiscuity of females. Third, EPF decisions depend on the combined effects of the identity of social mates. Here, we relied on extensive genetic parentage analysis of the offspring of a socially monogamous bird, the barn swallow, Hirundo rustica, to assess which of the above sources of variation predict the occurrence of EPFs. When we controlled for pair composition and social mate attractiveness, EPFs covaried with morphological and coloration traits of feathers in females. As expected, females mated with highly ornamented, long-tailed males had fewer EPFs. The composition of the breeding pair also accounted for variation in EPFs, implying that the ability of individual males to secure genetic parentage varies between female mates. These results show that females differ in promiscuity, and phenotypic traits of females that are visible to males are associated with promiscuity, potentially serving as cues to prospecting males. Hence, contrary to common interpretations of the negative relationship between male sexual attractiveness and female promiscuity, it can be speculated that larger genetic parentage by highly ornamented males results from their ability to secure the less promiscuous mates rather than from females being less promiscuous when mated to them. Moreover, our study shows that EPFs also depend on the composition of the social pair, as expected if a component of female promiscuity decisions depends on genetic or behavioural compatibility with the social male mate. Our study emphasizes that female promiscuity and its phenotypic correlates, and composition of the social pair, deserve closer attention in studies of sexual selection mediated by EPFs. (C) 2017 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.

Individuals often considerably differ in the timing of their life-cycle events, with major consequences for individual fitness, and, ultimately, for population dynamics. Phenological variation can arise from genetic effects but also from epigenetic modifications in DNA expression and translation. Here, we tested if CpG methylation at the poly-Q and 5′-UTR loci of the photoperiodic Clock gene predicted migration and breeding phenology of long-distance migratory barn swallows ( Hirundo rustica ) that were tracked year-round using light-level geolocators. Increasing methylation at Clock poly-Q was associated with earlier spring departure from the African wintering area, arrival date at the European breeding site, and breeding date. Higher methylation levels also predicted increased breeding success. Thus, we showed for the first time in any species that CpG methylation at a candidate gene may affect phenology and breeding performance. Methylation at Clock may be a candidate mechanism mediating phenological responses of migratory birds to ongoing climate change.

Migratory behaviour is controlled by endogenous circannual rhythms that are synchronized by external cues, such as photoperiod. Investigations on the genetic basis of circannual rhythmicity in vertebrates have highlighted that variation at candidate 'circadian clock' genes may play a major role in regulating photoperiodic responses and timing of life cycle events, such as reproduction and migration. In this comparative study of 23 trans-Saharan migratory bird species, we investigated the relationships between species-level genetic variation at two candidate genes, Clock and Adcyap1, and species' traits related to migration and geographic distribution, including timing of spring migration across the Mediterranean Sea, migration distance and breeding latitude. Consistently with previous evidence showing latitudinal clines in 'circadian clock' genotype frequencies, Clock allele size increased with breeding latitude across species. However, early-and late-migrating species had similar Clock allele size. Species migrating over longer distances, showing delayed spring migration and smaller phenotypic variance in spring migration timing, had significantly reduced Clock (but not Adcyap1) gene diversity. Phylogenetic confirmatory path analysis suggested that migration date and distance were the most important variables directly affecting Clock gene diversity. Hence, our study supports the hypothesis that Clock allele size increases poleward as a consequence of adaptation to the photoperiodic regime of the breeding areas. Moreover, we show that long-distance migration is associated with lower Clock diversity, coherently with strong stabilizing selection acting on timing of life cycle events in long-distance migratory species, likely resulting from the time constraints imposed by late spring migration.

The timing of major life-history events, such as migration and moult, is set by endogenous circadian and circannual clocks, that have been well characterized at the molecular level. Conversely, the genetic sources of variation in phenology and in other behavioral traits have been sparsely addressed. It has been proposed that inter-individual variability in the timing of seasonal events may arise from allelic polymorphism at phenological candidate genes involved in the signaling cascade of the endogenous clocks. In this study of a long-distance migratory passerine bird, the willow warbler Phylloscopus trochilus, we investigated whether allelic variation at 5 polymorphic loci of 4 candidate genes (Adcyap1, Clock, Creb1, and Npas2), predicted 2 major components of the annual schedule, namely timing of spring migration across the central Mediterranean sea and moult speed, the latter gauged from ptilochronological analyses of tail feathers moulted in the African winter quarters. We identified a novel Clock gene locus (Clock region 3) showing polyQ polymorphism, which was however not significantly associated with any phenotypic trait. Npas2 allele size predicted male (but not female) spring migration date, with males bearing longer alleles migrating significantly earlier than those bearing shorter alleles. Creb1 allele size significantly predicted male (but not female) moult speed, longer alleles being associated with faster moult. All other genotype-phenotype associations were statistically non-significant. These findings provide new evidence for a role of candidate genes in modulating the phenology of different circannual activities in long-distance migratory birds, and for the occurrence of sex-specific candidate gene effects.

Anemia management, based on erythropoiesis stimulating agents (ESA) and iron supplementation, has become an increasingly challenging problem in hemodialysis patients. Maintaining hemodialysis patients within narrow hemoglobin targets, preventing cycling outside target, and reducing ESA dosing to prevent adverse outcomes requires considerable attention from caregivers. Anticipation of the long-term response (i.e. at 3 months) to the ESA/iron therapy would be of fundamental importance for planning a successful treatment strategy. To this end, we developed a predictive model designed to support decision-making regarding anemia management in hemodialysis (HD) patients treated in center. An Artificial Neural Network (ANN) algorithm for predicting hemoglobin concentrations three months into the future was developed and evaluated in a retrospective study on a sample population of 1558 HD patients treated with intravenous (IV) darbepoetin alfa, and IV iron (sucrose or gluconate). Model inputs were the last 90 days of patients’ medical history and the subsequent 90 days of darbepoetin/iron prescription. Our model was able to predict individual variation of hemoglobin concentration 3 months in the future with a Mean Absolute Error (MAE) of 0.75 g/dL. Error analysis showed a narrow Gaussian distribution centered in 0 g/dL; a root cause analysis identified intercurrent and/or unpredictable events associated with hospitalization, blood transfusion, and laboratory error or misreported hemoglobin values as the main reasons for large discrepancy between predicted versus observed hemoglobin values. Our ANN predictive model offers a simple and reliable tool applicable in daily clinical practice for predicting the long-term response to ESA/iron therapy of HD patients.

Online hemodiafiltration (OL-HDF), the most efficient renal replacement therapy, enables enhanced removal of small and large uremic toxins by combining diffusive and convective solute transport. Randomized controlled trials on prevalent chronic kidney disease (CKD) patients showed improved patient survival with high-volume OL-HDF, underlining the effect of convection volume (CV). This retrospective international study was conducted in a large cohort of incident CKD patients to determine the CV threshold and range associated with survival advantage. Data were extracted from a cohort of adult CKD patients treated by post-dilution OL-HDF over a 101-month period. In total, 2293 patients with a minimum of 2 years of follow-up were analyzed using advanced statistical tools, including cubic spline analyses for determination of the CV range over which a survival increase was observed. The relative survival rate of OL-HDF patients, adjusted for age, gender, comorbidities, vascular access, albumin, C-reactive protein, and dialysis dose, was found to increase at about 55 l/week of CV and to stay increased up to about 75 l/week. Similar analysis of pre-dialysis β2-microglobin (marker of middle-molecule uremic toxins) concentrations found a nearly linear decrease in marker concentration as CV increased from 40 to 75 l/week. Analysis of log C-reactive protein levels showed a decrease over the same CV range. Thus, a convection dose target based on convection volume should be considered and needs to be confirmed by prospective trials as a new determinant of dialysis adequacy.

BACKGROUND:Haemodiafiltration (HDF) is the preferred dialysis modality in many countries. The aim of the study was to compare the survival of incident patients on high-volume HDF (HV-HDF) with high-flux haemodialysis (HD) in a large-scale European dialysis population.METHODS:The study population was extracted from 47,979 patients in 369 NephroCare centres throughout 12 countries. Baseline was six months after dialysis initiation; maximum follow-up was 5 years. Patients were either on HV-HDF (defined as with ≥21 litres substitution fluid volume per session) or on HD if on that treatment for ≥75% of the 3 months before baseline. The main predictor was treatment modality. Other parameters included country, age, gender, BMI, haemoglobin, albumin and Charlson comorbidity index. Propensity score matching and Inverse Probability of Censoring Weighting (IPCW) were applied to reduce bias by indication and consider modality crossover, respectively.RESULTS:After propensity score matching, 1,590 incident patients remained. Kaplan-Meier and proportional Cox regression analyses revealed no significant survival advantage of HV-HDF. Results were biased by modality crossover: during the 5-year study period, 7% of HV-HDF patients switched to HD, and 55% of HD patients switched to HV-HDF. IPCW uncovered a statistically significant survival advantage of HV-HDF (OR 0.501; CI 0.366-0.684; p < 0.001). A higher benefit of HV-HDF for some subgroups was revealed, for example, non-diabetics, patients 65-74 years, patients with obesity or high blood pressure.CONCLUSIONS:This large-scale study supports the generalizability of previous RCT findings regarding the survival benefit of HV-HDF. Sub-group analysis showed that some sub-cohorts appear to benefit more from HV-HDF than others.

Dissecting phenotypic variance in life history traits into its genetic and environmental components is at the focus of evolutionary studies and of pivotal importance to identify the mechanisms and predict the consequences of human-driven environmental change. The timing of recurrent life history events (phenology) is under strong selection, but the study of the genes that control potential environmental canalization in phenological traits is at its infancy. Candidate genes for circadian behaviour entrained by photoperiod have been screened as potential controllers of phenological variation of breeding and moult in birds, with inconsistent results. Despite photoperiodic control of migration is well established, no study has reported on migration phenology in relation to polymorphism at candidate genes in birds. We analysed variation in spring migration dates within four trans-Saharan migratory species (Luscinia megarhynchos; Ficedula hypoleuca; Anthus trivialis; Saxicola rubetra) at a Mediterranean island in relation to Clock and Adcyap1 polymorphism. Individuals with larger number of glutamine residues in the poly-Q region of Clock gene migrated significantly later in one or, respectively, two species depending on sex and whether the within-individual mean length or the length of the longer Clock allele was considered. The results hinted at dominance of the longer Clock allele. No significant evidence for migration date to covary with Adcyap1 polymorphism emerged. This is the first evidence that migration phenology is associated with Clock in birds. This finding is important for evolutionary studies of migration and sheds light on the mechanisms that drive bird phenological changes and population trends in response to climate change.

BACKGROUND AND OBJECTIVES:High body mass index appears protective in hemodialysis patients, but uncertainty prevails regarding which components of body composition, fat or lean body mass, are primarily associated with survival.DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS:Data between April 2006 and December 2012 were extracted from the Fresenius Medical Care Europe subset of the international MONitoring Dialysis Outcomes initiative. Fresenius Medical Care Europe archives a unique repository of predialysis body composition measurements determined by multifrequency bioimpedance (BCM Body Composition Monitor). The BCM Body Composition Monitor reports lean tissue indices (LTIs) and fat tissue indices (FTIs), which are the respective tissue masses normalized to height squared, relative to an age- and sex-matched healthy population. The relationship between LTI and FTI and all-cause mortality was studied by Kaplan-Meier analysis, multivariate Cox regression, and smoothing spline ANOVA logistic regression.RESULTS:In 37,345 hemodialysis patients, median (25th-75th percentile) LTI and FTI were 12.2 (10.3-14.5) and 9.8 (6.6-12.4) kg/m(2), respectively. Median (25th-75th percentile) follow-up time was 266 (132-379) days; 3458 (9.2%) patients died during follow-up. Mortality was lowest with both LTI and FTI in the 10th-90th percentile (reference group) and significantly higher at the lower LTI and FTI extreme (hazard ratio [HR], 3.37; 95% confidence interval [95% CI], 2.94 to 3.87; P<0.001). Survival was best with LTI between 15 and 20 kg/m(2) and FTI between 4 and 15 kg/m(2) (probability of death during follow-up: <5%). When taking the relation between both compartments into account, the interaction was significant (P=0.01). Higher FTI appeared protective in patients with low LTI (HR, 3.37; 95% CI, 2.94 to 3.87; P<0.001 at low LTI-low FTI, decreasing to HR, 1.79; 95% CI, 1.47 to 2.17; P<0.001 at low LTI-high FTI).CONCLUSIONS:This large international study indicates best survival in patients with both LTI and FTI in the 10th-90th percentiles of a healthy population. In analyses of body composition, both lean tissue and fat tissue compartments and also their relationship should be considered.

Chronic Kidney Disease (CKD) anemia is one of the main common comorbidities in patients undergoing End Stage Renal Disease (ESRD). Iron supplement and especially Erythropoiesis Stimulating Agents (ESA) have become the treatment of choice for that anemia. However, it is very complicated to find an adequate treatment for every patient in each particular situation since dosage guidelines are based on average behaviors, and thus, they do not take into account the particular response to those drugs by different patients, although that response may vary enormously from one patient to another and even for the same patient in different stages of the anemia. This work proposes an advance with respect to previous works that have faced this problem using different methodologies (Machine Learning (ML), among others), since the diversity of the CKD population has been explicitly taken into account in order to produce a general and reliable model for the prediction of ESA/Iron therapy response. Furthermore, the ML model makes use of both human physiology and drug pharmacology to produce a model that outperforms previous approaches, yielding Mean Absolute Errors (MAE) of the Hemoglobin (Hb) prediction around or lower than 0.6 g/dl in the three countries analyzed in the study, namely, Spain, Italy and Portugal.

Circannual rhythms often rely on endogenous seasonal photoperiodic timers involving ‘clock’ genes and Clock gene polymorphism has been associated to variation in phenology in some bird species. In the long-distance migratory barn swallow Hirundo rustica , individuals bearing the rare Clock allele with the largest number of C-terminal polyglutamine repeats found in this species (Q 8 ) show a delayed reproduction and moult later. We explored the association between Clock polymorphism and migration scheduling, as gauged by light-level geolocators, in two barn swallow populations (Switzerland; Po Plain, Italy). Genetic polymorphism was low: 91% of the 64 individuals tracked year-round were Q 7 /Q 7 homozygotes. We compared the phenology of the rare genotypes with the phenotypic distribution of Q 7 /Q 7 homozygotes within each population. In Switzerland, compared to Q 7 /Q 7 , two Q 6 /Q 7 males departed earlier from the wintering grounds and arrived earlier to their colony in spring, while a single Q 7 /Q 8 female was delayed for both phenophases. On the other hand, in the Po Plain, three Q 6 /Q 7 individuals had a similar phenology compared to Q 7 /Q 7 . The Swiss data are suggestive for a role of genetic polymorphism at a candidate phenological gene in shaping migration traits and support the idea that Clock polymorphism underlies phenological variation in birds.