Phaeocryptopus gaeumannii, causing Swiss needle cast (SNC) occurs wherever Douglas‐fir is found but disease damage is believed to be limited in the U.S. Pacific Northwest (PNW) to the Coast Range of Oregon and Washington . However, knowledge remains limited on the history and spatial distribution of SNC impacts in the PNW. We reconstructed the history of SNC impacts on mature Douglas‐fir trees based on tree‐ring width chronologies from western Oregon. Our findings show that SNC impacts on growth occur wherever Douglas‐fir is found and is not limited to the coastal fog zone. The spatiotemporal patterns of growth impact from SNC disease were synchronous across the region, displayed periodicities of 12–40 years, and strongly correlated with winter and summer temperatures and summer precipitation. The primary climatic factor limiting pathogen dynamics varied spatially by location, topography, and elevation. SNC impacts were least severe in the first half of the 20th century when climatic conditions during the warm phase of the Pacific Decadal Oscillation (1924–1945) were less conducive to pathogen development. At low‐ to mid‐elevations, SNC impacts were most severe in 1984–1986 following several decades of warmer winters and cooler, wetter summers including a high summer precipitation anomaly in 1983. At high elevations on the west slope of the Cascade Range, SNC impacts peaked several years later and were the greatest in the 1990s, a period of warmer winter temperatures. Climate change is predicted to result in warmer winters and will likely continue to increase SNC severity at higher elevations, north along the coast from northern Oregon to British Columbia, and inland where low winter temperatures currently limit growth of the pathogen. Our findings indicate that SNC may become a significant forest health problem in areas of the PNW beyond the coastal fog zone.The fungal pathogen,  P. gaeumannii is indigenous in western North America and occurs wherever its host is found  is a foliar disease caused by the ascomycete fungus d Boyce, . In its d Boyce, . The affd Boyce, . In the P. gaeumannii on Douglas‐fir have been well studied in young plantations, knowledge remains limited on the history and spatial distribution of SNC impacts on mature trees in the U.S. Pacific Northwest (PNW) and elsewhere. Phaeocryptopus gaeumannii has long believed to have been pervasive but innocuous in Douglas‐fir forests prior to 1950  proliferate in winter from December to April; (2) sporulation occurs in synchrony with bud break and shoot elongation from May to July and only current‐year needles are infected  SNC is sensitive to winter and summer temperature, and summer precipitation, and so, spatial variability in SNC severity can be attributed to variations in site conditions, location, and elevation; (2) SNC impacts display primary periodicities of 20–30 years and secondary periodicities of 4–6 years as seen for the coast sites  was widespread in western hemlock (Tsuga heterophylla) at Horse Creek Trail Lower and Soapgrass Mountain but was absent at drier sites. Douglas‐fir dwarf mistletoe (Arceuthobium douglasii) was not observed at any site. As western hemlock is not a host of P. gaeumannii, SNC affects the growth of the two tree species differently. The sites will be referred to by their abbreviations as follows: CH = Cascade Head; HCTL & HCTU = Horse Creek Trail Lower & Upper; WC = Woods Creek; JP = Jackson Place; FC = Falls Creek; MM = Moose Mountain; SG = Soapgrass; TC = Toad Creek.Nine mature, closed‐canopy forest stands dominated by Douglas‐fir were located on state and federal lands in western Oregon, USA Figure . The ninThe coast sites  are situated within the coastal fog zone where humidity and precipitation levels are highest with total annual precipitation for CH averaging 2,041 mm/year, whereas the valley sites  are situated in the rainshadow of the Coast Range and represent the driest sites with annual precipitation totals for JP averaging 1,236 mm/year. As Pacific air masses move eastwards to the Cascade Range, humidity and precipitation levels increase slightly, with annual precipitation totals for FC and SG averaging 1,708 and 2,014 mm/year, respectively. This region experiences a Mediterranean climate regime characterized by an annual summer drought and moist‐cool winters with 89% of total annual precipitation falling between October and May. See Beedlow, Lee, Tingey, Waschmann, and Burdick  and Lee,2.2Tree core samples from 17 to 29 dominant and codominant Douglas‐fir trees and 4 to 16 western hemlock trees were taken at approximately breast height (1.4 m) using 5‐mm diameter increment borers, collecting one or two cores per tree at each site  for the period 1895–2012 was obtained from the National Oceanic and Atmospheric Administration's National Climate Data Center at http://www.ncdc.noaa.gov. Variables were obtained for the specific study site locations when spatially interpolated climate data were available and for the nearest location or division otherwise  has been described by Lee et al. (t) and disturbance (Dt), that is, E(Yt) = Ct + Dt. The climate component, Ct, represents the interactions of temperature, precipitation, soil moisture, and evapotranspiration demand on tree growth and is assumed to be the same for all trees within a stand  of the host species  where βi is the model parameter for the pulse disturbance event at time t = ti for i = 1, 2, … r. In general, the disturbance events t* =  are unknown and must be inferred. Some multiple testing adjustments such as Bonferroni or Tukey and/or specification of a critical value A are appropriate for detection of multiple outliers because every point is a potential outlier. Chang and Tiao . Cospectral analysis was used to determine whether the aclimatic growth anomalies of inland Douglas‐fir displayed a cyclical pattern having a primary periodicity of 20–30 years and a secondary periodicity of 4–6 years in synchrony with the SNC index for coastal Douglas‐fir.3p‐value < .05/r where r = number of outliers detected) from the climate‐based model prediction and were specific to Douglas fir. The site‐specific TSIA models with components for climate and disturbance explained between 60% and 85% of the total variation in the EW and LW width chronologies  reduced growth more in a disturbance year than in years when disturbance was absent Figure . This in3.3The outbreak reconstructions based on the SNC index indicated that reductions in Douglas‐fir stem growth attributable to SNC have occurred periodically in western Oregon for the period of study from 1895 to 2011 Figure . Synchro3.4Spectral analysis found both high‐ and low‐frequency variabilities in the climate‐adjusted SNC index for the common period 1895–2010 for six of the nine study sites Figure a. The siCospectral analysis indicated that the SNC indices for CH, HCTL, and FC were coherent and in phase at the key frequencies Figures  and A2. 3.5Cross‐correlation and canonical correlation analyses were used to determine the climatic factors associated with the SNC index for each site, focusing on EW growth for two coast (CH and HCTL) and two Cascade sites (FC and SG) where the magnitude and frequency of disease impacts and the statistical power of the test for correlation were greater. Disease impact related to winter temperature, summer precipitation, and temperature consistently across all study sites. The SNC index was generally positively correlated with current and past winter temperature and summer precipitation and negatively correlated with summer temperature. The impact of SNC on stem growth lagged the seasonal averages of winter and summer temperatures and summer precipitation by 0–24 years to varying degrees for all study sites. For three of the nine sites , the SNC index correlated best with summer precipitation lagged 1–3 years Figure , similarCanonical correlation analysis was used to generate a climate index for each seasonal climate factor and study site based on a weighting scheme of current and previous years to optimize the correlation with the SNC index of impact Table . Much stVariations in the intensity and duration of the episodic SNC outbreaks for individual sites were strongly associated with seasonal climatic factors lagged 0–24 years. The peak SNC outbreaks of 1959 and 1984–1986 were generally preceded by several decades of anomalously warm winters and cool, wet summers which allowed the pathogen abundance to build Figures  and 11. 4P. gaeumannii, is ubiquitous in western Oregon conifer forests and reduces the growth of Douglas‐fir by restricting gas and water exchange via stomatal occlusion and early needle abscission , flatheaded fir borer (Melanophila drummondi), western spruce budworm , and Douglas‐fir tussock moth (Orgyia pseudotsugata) have historically been asynchronous and sporadic across western Oregon and Washington  YC Dai) and Armillaria root disease. Both laminated root rot  trees in the Swiss National Park which were chronically infected with Armillaria .Prolonged periods of high VPD conditions during the summer drought reduce stomatal conductance and can lead to carbon starvation and ultimately reduced annual stem growth, more so during a period of increased SNC severity when carbon uptake is further reduced by stomatal occlusion and early needle abscission. For five study sites , isotopic measurements of δP. gaeumannii abundance as well as growth impacts in the PNW  vegetation zone of the Coast Range of Oregon and Washington where P. gaeumannii is most abundant and Douglas‐fir is a minor species compared to western hemlock, even though Douglas‐fir grow much more rapidly than hemlock where environmental conditions for P. gaeumannii are less favorable. The composition of the Sitka spruce vegetation zone is distinct and shaped not only by climate favorable to growth of Sitka spruce and western hemlock but also to P. gaeumannii and its negative effect on the competitive ability of Douglas‐fir.SNC reduces Douglas‐fir growth relative to other tree species and therefore can affect competitive outcomes between tree species. This is very well illustrated by the Sitka spruce , the Pacific‐North American (PNA) pattern, and the Pacific Decadal Oscillation (PDO) (Lee et al., P. gaeumannii to develop rapidly, resulting in a widespread SNC outbreak in 1984–1986. Following the SNC outbreak initiated in 1983, there was a delay of 1 year before the reduction in photosynthate production due to stomatal occlusion and early needle abscission was fully expressed in stem growth. The anomalously cool, wet summer in 1983 occurred during an extra strong El Niño event. However, broad‐scale forcing factors such as ENSO and PNA modulate seasonal temperature patterns but not precipitation patterns (Abatzoglou et al., P. gaeumannii in warm, dry environments with a changing climate.Swiss needle cast outbreaks tend to be more severe during periods of relatively warm winter and cool, wet summer conditions (Lee et al., Rapid warming in the PNW during December–February and March–May since ~1950 has been attributed to ENSO and PNA (Abatzoglou & Redmond, P. gaeumannii development and growth by reducing the ascospore germination rate at time of sporulation. Summer precipitation is strongly associated with the magnitude and frequency of SNC impacts on Douglas‐fir growth for all sites. Under future climate change scenarios, SNC impacts are likely to increase in regions where summer precipitation frequently exceeds the P. gauemannii‐limiting threshold of 110 mm (Hood, Swiss needle cast has been and will continue to be an important forest disease of Douglas‐fir in the PNW, New Zealand, Europe, Chile, Australia, and elsewhere in light of the broader spatial extent of SNC impact beyond the coastal fog zone. Douglas‐fir forests will respond differently to the interacting effects of SNC and climate, depending upon location and site condition, as temperatures rise and precipitation patterns shift toward wetter winters and springs and drier summers. Higher winter temperatures are expected to enhance pseudothecia production, more so in cool environments, but warmer, drier summers are expected to inhibit mm Hood,  and decrmm Hood, , SNC in None declared.EHL, PAB, and DTT conceived of and designed the study. RSW provided technical support for the network of long‐term monitoring field sites. EHL, PAB, RSW, SC, MB, and CC collected the field data and CC processed the tree core samples in the laboratory. EHL and CW analyzed the data. EHL wrote the first draft of the manuscript, and all authors contributed substantially to revisions.


Schistosoma mansoni is a parasite of profound medical importance. Current control focusses on mass praziquantel (PZQ) treatment of populations in endemic areas, termed Preventative Chemotherapy (PC). Large-scale PC programmes exert prolonged selection pressures on parasites with the potential for, direct and/or indirect, emergence of drug resistance. Molecular methods can help monitor genetic changes of schistosome populations over time and in response to drug treatment, as well as estimate adult worm burdens through parentage analysis. Furthermore, methods such as in vitro drug sensitivity assays help phenotype in vivo parasite genotypic drug efficacy.S. mansoni collected from children from two schools in 2010, five years after the introduction of a National Control Programme. Children at one school had received four annual PZQ treatments and the other school had received two mass treatments in total. We compared genetic differentiation, indices of genetic diversity, and estimated adult worm burden from parasites collected in 2010 with samples collected in 2005 (before the control programme began) and in 2006 (six months after the first PZQ treatment). Using 2010 larval samples, we also compared the genetic similarity of those with high and low in vitro sensitivity to PZQ.We conducted combined in vitro PZQ efficacy testing with population genetic analyses of We demonstrated that there were individual parasites with reduced PZQ susceptibility in the 2010 collections, as evidenced by our in vitro larval behavioural phenotypic assay. There was no evidence, however, that miracidia showing phenotypically reduced susceptibility clustered together genetically. Molecular analysis also demonstrated a significant reduction of adult worm load over time, despite little evidence of reduction in parasite infection intensity, as measured by egg output. Genetic diversity of infections did not reduce over time, despite changes in the genetic composition of the parasite populations.Genotypic and phenotypic monitoring did not indicate a selective sweep, as may be expected if PZQ treatment was selecting a small number of related “resistant” parasites, but there was evidence of genetic changes at the population level over time. Genetic data were used to estimate adult worm burdens, which unlike parasite infection intensity, showed reductions over time, suggesting the relaxation of negative density-dependent constraints on parasite fecundity with PZQ treatment. We thereby demonstrated that density-dependence in schistosome populations may complicate evaluation and monitoring of control programmes.The online version of this article (10.1186/s13071-017-2533-6) contains supplementary material, which is available to authorized users. Schistodecrease .per-capita rates of reproduction or survival, thereby potentially contributing to population persistence and resilience, and complicating the monitoring of chemotherapy success [Clinical efficacy of PZQ is measured as the egg reduction or cure rates and recent data around Lake Victoria have raised concerns that there may be a real possibility of reduced efficacy in communities with a more intensive history of PZQ treatment , 9. Howe success .A key problem in monitoring for PZQ resistance is that though such resistance can be selected for in the laboratory, its molecular basis has not yet been ascertained, and thus genotypic markers for PZQ resistance are currently not available. However, at the individual level, in vitro phenotypic monitoring of larval behaviour when exposed to PZQ has been developed to monitor the phenotypic status of individual parasites, with in vitro PZQ susceptibility correlating to in vivo treatment success . In addiSchistosoma mansoni parasite population genetics in two schools in the Lake Victoria region in Tanzania [refugia , we demonstrated a significant decrease of allelic richness and heterozygosity of the parasites after only one round of PC including in miracidia isolated from untreated children [S. mansoni ranging from no evidence of genetic change over time  to clea Senegal ).S. mansoni parasite population. Using the same experimental set-up as the baseline study, we compared genetic differentiation and indices of genetic diversity in 2010 samples with baseline samples collected in 2005 and 2006. We performed in vitro tests of PZQ sensitivity and, for the first time in a natural population, compared the genetic profiles of individual miracidia with low or high susceptibility to PZQ in vitro, to quantify genetic differentiation between them. Finally, we assessed evidence of the existence of density dependence in schistosome populations using genetic parentage analysis.The aim of this study was to return to Bukindo and Kisorya Primary schools to measure the effect of five years of the National Schistosomiasis Control Programme Tanzania on the phenotype and genotype of the We predicted that: (i) the genetic composition of parasite populations may change over time and in response to PZQ treatment; (ii) in vitro PZQ testing can identify individual parasites with reduced sensitivity to PZQ and that population genetics can ascertain if those with reduced sensitivity are closely related to each other; (iii) the genetic diversity of parasite populations will further reduce with additional PZQ treatment; and (iv) adult worm burdens are non-linearly related to egg output and that this relationship may be affected by PZQ treatment.S. mansoni miracidia were collected from children aged 7–11 years old at two schools in close proximity to Lake Victoria, Tanzania, in April 2005, six months prior to the first National Schistosomiasis Control Programme mass school based treatment with PZQ, and again one year later in April 2006. In 2005, miracidia were collected from 38 children at Bukindo Primary School  and 42 children from Kisorya Primary School , and in 2006 from 18 children at Bukindo Primary School and 29 children from Kisorya Primary School . Following data and sample collection, all children in both schools  were treated for schistosomiasis with PZQ (40 mg/kg) and soil-transmitted helminths with albendazole (400 mg).As reported elsewhere , S. mansIn order to determine the prevalence and intensity of schistosome infection, duplicate Kato-Katz thick smears were prepared from three individual stool samples, over three consecutive days, from each child. The infection intensity was expressed as eggs per gram stool (epg) and averaged over the two slides for each day, and then over the three days of collection. All infection data were collected pre-treatment at each time point and there was no direct measure of drug efficacy in terms of egg reduction rate or cure rate so that the selected populations had the same treatment history as the rest of the control programme.Schistosoma mansoni eggs were purified from individual stool samples of all Kato-Katz-positive children as previously described [escribed , 15 and S. mansoni miracidia when they are exposed to PZQ. In brief, susceptible miracidia first become tadpole and then dumbbell shaped upon PZQ exposure, with the degree of shape change and activity level correlated to drug susceptibility and in vivo drug efficacy [−6 M PZQ solution  were added, in order to expose every miracidium to a final PZQ concentration of 1 × 10−6 M in 20 μl total volume. After 5 min incubation at room temperature, resistance levels of individual miracidia were microscopically classified according to observed phenotypical changes: “less susceptible” - normal shape following PZQ exposure, “intermediate susceptibility/resistance” - tadpole shape, “fully susceptible” - dumbbell shape [Samples from five children, which yielded sufficient viable miracidia, were used to screen for susceptibility to PZQ using an in vitro phenotypic test based on the change in shape that occurs in susceptible efficacy . For eacll shape , and stoMolecular analysis was carried out on up to 20 miracidia per child from the 2010 collections depending on the infection intensity of the child. We replicated the identical methodologies of the original 2005 and 2006 surveys in order that the data were directly comparable , since tEstimates of prevalence and infection intensity were calculated using data from the 2010 survey of approximately 60 children at each school, and were compared with estimates of prevalence and infection intensity from 2005 and 2006. These were based on larger surveys of approximately 200 children per school undertaken as part of the National Schistosomiasis Control programme monitoring and evaluation (Schistosomiasis Control Initiative), unpublished data . To compχ2 = 1.04, df = 2, P = 0.41). All population genetic measures were calculated at an infrapopulation level , except where explicitly stated.Allele sizes were calculated in Genemapper (v4) and manually checked using the same bin sizes as for the 2005 and 2006 samples. Subsequent analysis was restricted to miracidia with at least 4 allele calls. There was an average of 7% missing data present per locus but no evidence that there were systematic differences between the years in the amount of missing data , incorporating an interaction term to investigate whether differences between years were similar at the two schools under study. Child age, sex, and miracidial sample size were used as covariates and survey commands were used to account for within-school clustering.Summary statistics for expected heterozygosity (He), observed heterozygosity (Ho) and allelic richness (Ar) across each population (each child at each time point) were created in FSTAT.2.9.3.2 . AllelicAs a measure of genetic distance between the parasite populations of individual children, a matrix of Cavalli-Sforza and Edwards’ chord distances  was estiP-values were calculated by 10,000 random permutations. A hierarchical analysis is the most robust analysis as it accounts for the potential relatedness of miracidia within individual children, which will tend to inflate estimates of classical Wrights’ FST statistics [Hierarchical Wrights’ FST statistics measuring evidence of genetic differentiation between years, and between children within years were calculated for each school separately using Hierfstat . P-valueatistics  but sepaatistics . In ordeS. mansoni in children aged 7 to 11 years is shown in Table t(65) = -4.04, P = 0.001). Combining these 2010 data with previously published baseline data (2005) and data six months after the first round of PC (2006) [Prevalence and infection intensity of C (2006) , we obse−6 M PZQ. As shown in Table Following screening in both schools, children with medium (100–399 epg) or high (>400 epg) infection intensities, and stool samples which yielded sufficient numbers of viable miracidia within 20 min, were selected for in vitro PZQ susceptibility testing. In 2010, five children complied with these criteria and 35–65 miracidia per child were tested being exposed to 1 × 10F = 1.66, P = 0.194). One hundred and thirty miracidia, which had been characterised as PZQ-susceptible or not by the in vitro phenotypic assay, were analysed separately.In 2010, miracidia were successfully stored from nine children at Bukindo Primary School and from 18 children at Kisorya Primary School. For the longitudinal analysis, we analysed up to 20 miracidia per child depending on availability and thus analysed 107 miracidia from Bukindo Primary School and 245 miracidia from Kisorya Primary School in 2010, with a school treatment history of four and two rounds of PC, respectively, and compared them with previously collected miracidia from the same schools in 2005 (baseline) and 2006 (6 months after first PC), to give a total dataset of 1936 miracidia. The mean number of miracidia per child  in 2010 was 13.0 ± 0.70 which was not significantly different from the mean number of miracidia in 2005 (12.2 ± 0.63) or 2006 (13.8 ± 0.56) . The number of miracidia was significantly associated with the estimated number of worms  but there was no significant association of child age  or sex  with the estimated number of adult worms in this dataset and the fitted model explained 92% of the variation in the estimated number of adult worms. The mean number of eggs per adult worm pair was higher in parasite infrapopulations in 2010 than 2006 and 2005 , and as shown in Fig. β = 41.4, t = 4.68, P < 0.001).Adult worm burdens were estimated from genetic data using parentage analysis. As shown in Fig. β = -0.067, t = 5.32, P < 0.001). This was not, however, maintained in 2010 and genetic diversity was slightly higher in the 2010 collections relative to the baseline collection . There was no difference in allelic richness between the schools . As shown in Fig. β = -0.07, t = -6.46, P < 0.001; 2010 compared to 2005: β = 0.05, t = 4.46, P < 0.001; Ho 2006 compared to 2005: β = -0.07, t = 6.62, P < 0.001; 2010 compared to 2005: β = 0.03, t = 2.62, P = 0.01), although there was a significant lower diversity at Bukindo school , which might reflect differences in infection intensities between the two schools, despite statistical attempts to correct for the miracidial sample size. Observed heterozygosity was lower than expected heterozygosity in all populations, although there was no evidence of change in this relationship over time. The significant differences between years were confirmed by non-parametric permutation tests .There was a significant difference in genetic diversity between parasite populations collected in 2006 and 2005, and between those collected in 2010 and 2005. As previously reported, and shown in Fig. The genetic similarity of parasite infrapopulations collected in the two schools at each time point are shown in Fig. One hundred and twenty one susceptible and eight less susceptible miracidia isolated from Kisorya Primary School were successfully genotyped. There was no evidence that less susceptible miracidia identified by in vitro PZQ testing were related and therefore might constitute a genetic cluster, as demonstrated using minimum spanning network analysis and a consensus NJ tree between individuals Fig. .Fig. 4GeS. mansoni strains that have lost susceptibility is a major concern. With the current lack of specific genetic markers for PZQ resistance [S. mansoni parasite populations. Our results provide evidence of the presence of parasites with reduced susceptibility to PZQ in the parasite population five years after the start of a National Control Programme using PC with PZQ , as evidenced by an in vitro phenotypic assay based on larval response to PZQ. However, there was no evidence that parasites showing phenotypically reduced susceptibility clustered together genetically, as would be expected following an ongoing selective sweep. Genetic data were also used to estimate adult worm burden, demonstrating a reduction of worm load over time, despite little evidence of long term reduction in parasite infection intensity. Genetic diversity of infections did not reduce further over time but there was evidence of change in the parasite populations in their genetic composition.In host populations that are subject to frequent MDA, the parasite population may be predicted to respond with genetic changes. In the case of PZQ, the only drug currently in distribution for schistosomiasis, the emergence of sistance , we relysistance , 12 and sistance . Here, fS. mansoni) or bladder (S. haematobium) and the only data that currently exists for human populations is from a single autopsy study [Prevalence and infection intensity of schistosomiasis remained high at both study sites, although infection intensity was significantly lower at Bukindo School on Ukewere Island, compared to the mainland school, Kisorya. Both schools received school-based PC in 2005 and 2007 under the National Control Programme, but Bukindo additionally received community-wide PC for all individuals over five years old in 2008 and 2009. Drug efficacy in terms of egg reduction or cure rate immediately after treatment was not measured, such that these schools had the same treatment history as the wider population undergoing schistosomiasis control. The temporal trends in the prevalence and intensity data suggest that the PC treatments were not having a major impact on the longer-term prevalence and intensity, possibly due to rapid re-infection in this high transmission area. It should be noted, however, that there is wide heterogeneity in individuals’ schistosomiasis infection levels, and this study was not powered to detect changes in prevalence and infection intensity following MDA treatment, for which much larger samples sizes are required (see e.g. ). Indeedsy study , thus posy study  is warraS. mansoni population as susceptible to PZQ, it was noted that all of the susceptible miracidia responded with a tadpole shape, indicative of intermediate susceptibility, rather than a dumbbell shape associated with full susceptibility, in contrast to samples from Uganda which consisted of equal proportions of tadpole and dumbbell shapes, i.e. a mixture of intermediately and fully susceptible [Clustering, hierarchical FST and pairwise FST analyses confirmed that parasite populations had changed over the five-year time period since the baseline collections. Re-analysis confirmed that a reduction in genetic diversity was seen between the first and second collections, but later collections showed no further reduction in diversity. One concern from the original study was that a reduction in parasite diversity may suggest the selection of parasite sub-populations able to survive MDA treatment. Thus a further aim of this study was to consider whether we were able to detect strains that are less susceptible to PZQ. We used an in vitro larval behaviour assay, which is positively correlated with in vivo treatment success . We democeptible . Moreoveceptible , identifceptible . Such stS. mansoni populations that had been investigated before treatment began, and six months after, the first school-based PC treatment [. Using the same experimental set-up as the original study, we identified no further decrease in the intensity or genetic diversity of S. mansoni infections within school children but observed density-dependent effects on the worm population following treatment, which would urge caution in interpreting infection intensity results as measured by egg output. Our longitudinal analysis showed further changes in the genetic make-up of schistosome populations which also included miracidia that were less susceptible to PZQ in vitro. However, we found no evidence of genetic similarity between parasites that were less susceptible to PZQ in vitro, as might be expected given a large scale selective sweep and the overall S. mansoni population at the study sites in Tanzania’s Lake Victoria region still be classified as susceptible in 2010. We suggest that monitoring of the parasite population and its responsiveness towards PZQ is strongly advisable and that studies on cost-of-resistance [This study revisited two schools in the Lake Victoria area of Tanzania five years after the introduction of a National Control Programme to conduct follow-up studies of the epidemiology and population genetics of reatment . Using tsistance  in natur


There is increasing interest in the proximate factors that underpin individual variation in suites of correlated behaviours. In this paper, we propose that dietary macronutrient composition, an underexplored environmental factor, might play a key role. Variation in macronutrient composition can lead to among-individual differentiation in single behaviours  as well as among-individual covariation between behaviours . Here, we argue that the nutritional balance during any life stage might affect the development of syndrome structure and the expression of genes with pleiotropic effects that influence development of multiple behaviours, hence genetic syndrome structure. We further suggest that males and females should typically differ in diet-dependent genetic syndrome structure despite a shared genetic basis. We detail how such diet-dependent multivariate gene-environment interactions can have major repercussions for the evolution of behavioural syndromes. Typike target, 4 . HowIn ecology and evolution, there has been considerable interest in the effect of nutritional condition on life-history traits, morphology and longevity , 17–21. Teleogryllus commodus) is, for example, increased by a low-protein high-carbohydrate diet [Drosophila males, furthermore, increase their mating frequency and courtship when fed on a protein-high diet [Anabrus simplex) decreases when individuals can access high-protein food sources [Despite the well-known existence of nutritional effects on behavioural development, only a few studies have used experiments to investigate effects of macronutrient composition  on behaviour and its development , with most research to date focusing on invertebrates , for example, because diet can induce long-term effects in morphology or physiology, hence behaviour. For example, deficiencies or excesses of certain nutrients might facilitate or attenuate the expression of behaviour of most individuals, thereby increasing or decreasing the among-individual variance in behaviour. Although one study showed that the extent of among-individual differentiation in behaviours was not a function of nutritional environment , the relNutritional balance can also affect the amount of among-individual covariance between functionally distinct behaviours. Behavioural correlations exist not just due to pleiotropic effect of genes (or linkage disequilibrium) but also due to pleiotropic environmental effects (such as macronutrient composition) . The covFurthermore, because of their shared genetic basis, males and females within a species are typically not thought to differ in the expression of genetic (co)variation in behaviours. However, given sexual differences in the optimal diet for fitness maximisation , we expeIn this opinion paper, we discuss the effects of extreme biological scenarios where the composition of macronutrients in the nutritional environment is so biased that individuals are unable to consume the optimal amount of nutrients for the intake target (e.g. a protein-deficient environment). In the natural environment, this situation may occur when animals consistently stay within one habitat, which might occur when animals face restrictions in dispersion , 34, 35.Over the last decade, a large number of behavioural studies have shown that individuals of the same population differ consistently in their behaviour . IndividBehavioural correlations are the product of the joint influences of ‘among-individual’ and ‘within-individual’ correlations , 44, 47,. The amoAmong-individual correlations are themselves shaped by two main contributors: genetic (G) and environmental effects (E)  Figure . First, There is already considerable evidence for a major role of genetic correlations in shaping behavioural syndromes , 57, 58.Given that nutrition is known to affect behaviour and its development , 13–16, ad libitum balanced food intake, among-individual variation in behavioural traits might be much higher than in a nutritionally impoverished environment  because the expression of genetic variation is typically increased under favourable conditions  which has an important role in constraining evolution of phenotypes in response to selection or genetic drift [G-matrix remains incompletely understood [G-matrices are highly conserved among populations of certain species [G can change [G-matrices for behavioural traits vary across environments. Given that behavioural traits are likely to be subject to permanent environmental effects, we predict that G-matrices for behavioural traits can change across different environments such as those differing in availability of different types of nutrients .Quantitative genetic analyses can be utilized to simultaneously quantify the relative importance of environmental effects (e.g. due to diet) versus diet-specific genetic correlations underlying among-individual behavioural correlations. Quantitative genetic analyses and animal models enable estimation of the ic drift . Despitederstood –82. Alth species , empiricn change –89. It iG-matrices measured for different macronutrient composition environments can be used to study cross-environment  genetic correlations as well as diet-specific genetic correlations, and reveal the genetic architecture and stability of the G-matrix; this is a powerful approach that has rarely been applied in the context of behaviour (but see [(but see ). The ge. Li of each species, the magnitude of sex differences in diet-dependent genetic correlations should be species-specific.Animals need multiple nutrients to maximize their fitness. A variety of nutrients must be ingested in an optimal blend required for best performance. If the animal fails to attain the optimal balance of nutrients, nutritional imbalance will likely exert an effect on the expression of behavioural correlations, morphology (e.g. body composition), and physiology at the among-individual level. Though behavioural traits are heritable –108, thePublication costs for this article were funded by the German Research Foundation (FOR 1232) and the Open Access Publication Fund of Bielefeld University and Muenster University.


Melilotus is an important genus of legume plants and an herbage with excellent nitrogen fixation; it can tolerate extreme environmental conditions and possesses important medicinal value. However, there is limited genetic information about the genus; thus, we analysed four chloroplast loci  and one nuclear region (ITS) to determine the genetic diversity of 199 accessions from 18 Melilotus species. The rbcL and matK sequences were highly conserved, whereas the trnL-F and ITS sequences contained variable and parsimony-informative sites. In our analyses of the single and combined regions, we calculated the pairwise distance, haplotype and nucleotide diversity and gaps and then constructed phylogenetic trees to assess the genetic diversity, and our results revealed significant variations among the different accessions. The genetic distance values were between zero and nine, and based on the combined regions, the highest frequency value was approximately four. Melilotus showed high haplotype and nucleotide diversity, particularly in the ITS sequences, with values of 0.86 and 0.0087, respectively. The single ITS sequence, psbA-trnH, and the combined matK+rbcL+trnL-F (MRT) and matK+rbcL+psbA-trnH+trnL-F+ITS (MRPTI) regions showed interspecific variation in the gap analysis. Phylogenetic trees calculated using ITS, psbA-trnH and MRPTI sequences indicated distinct genetic relationship in 18 species, and these species could be divided into two groups. By determining the genetic diversity of plants, we can evaluate the genetic relationships among species and accessions, providing a basis for preserving and utilizing the genetic resources of Melilotus. Melilotus (sweet clover) consists of 19 annual and biennial species and belongs to the tribe Trifolieae of the legume family. Almost all species are native to North Africa or Eurasia, and manys can be found in North of China and Central Asia [Melilotus is an important forage crop, and certain species, such as M. albus, M. officinalis and M. indicus have been cultivated in many regions [Melilotus can tolerate extreme environmental conditions, such as drought, cold, and high salinity [Melilotus is valuable because of its coumarin content[Melilotus is worthy of further investigation [The genus ral Asia . Melilot regions . Comparesalinity , and itssalinity . Addition content, and thutigation .http://www.cigar.org/center/index.html).) The purpose of this organization is to maintain genetic diversity and to provide tools for population monitoring and assessment that can be used for conservation planning [Brassica napus [Genetic diversity within a particular species helps plants adapt to various environmental conditions, such as fluctuating climate and soil conditions; thus, assessing the diversity of available plant genetic resources is necessary to identify the genes associated with useful biological functions that can then be rationally integrated to design new varieties . Plant gplanning . Forage planning ,11. Geneplanning , restricplanning , amplifiplanning , simple planning , single planning , and chlca napus ,18.Melilotus present extensive variation [Melilotus species have been evaluated [Melilotus because previous research has concentrated on morphology, cultivation techniques and chemical composition, but SSR marker analyses have shown that Melilotus is highly diverse, which is indicative of high allelic richness in the accessions [Brassica napus, Brassica rapa and Brassica oleracea and their genetic relationships have been finely determined at the cpDNA level [Ferula assa-foetida L.) populations [Melilotus, we used five sequences, including rbcL, matK, psbA-trnH and trnL-F and ITS, to assess the genetic diversity within 18 species.A previous study showed that the leaves, flower colour and structure, and pod and seed characteristics of ariation , and thevaluated . Howevercessions . Understcessions . Additiocessions . Cytoplacessions , and it cessions ,25. The NA level , and theulations . Accordiulations ,28. To fMelilotus species  and 48 accessions from the National Plant Germplasm System  for a total of 199 accessions representing 18  species . The NGBrbcL [matK [psbA-trnH [trnL-F [Total genomic DNA was extracted from whole seedlings using the SDS (sodium dodecyl sulphate) method . For eacrbcL , matK [3cL [matK , psbA-trsbA-trnH  and trnL [trnL-F  and one  [trnL-F ) were amMelilotus was analysed using DnaSP software, and we estimated the genetic diversity of the species based on the five sequences by calculating the pairwise distances for each locus using MEGA 6.0 according to the number of differences model [http://www.ebi.ac.uk/Tools/psa/emboss_needle/nucleotide.html) was employed to analyse the dissimilarities in nucleotide deletions via a pairwise sequence alignment [Vicia sativa was adopted as outgroup [We used the Contig Express module of Vector NTI Suite 6.0  to assemble and edit the contigs . Sequences model . In addilignment . We usedoutgroup .Melilotus. The results of the amplification were preliminarily validated by agarose gel electrophoresis . The average GC content of trnL-F was 33.28%. Melilotus indicus and M. albus had the highest and lowest GC contents, which were 34.60% and 31.56%, respectively.The sequence length, GC content, variable sites and genetic distances based on the five sequences in 18 species were analysed and summarized Tables . The lenMelilotus was summarized, and the genetic diversity in each region of the 18 species was calculated , matK+rbcL+trnL-F (MRT), matK+rbcL+psbA-trnH+trnL-F (MRPT), matK+rbcL+ITS+trnL-F (MRIT), and matK+rbcL+psbA-trnH+trnL-F+ITS (MRPTI). Our results revealed that the highest frequency was approximately four in all combinations. The genetic distance value ranged from zero to nine according to the different accessions. For the five single regions, more than half of the species exhibited zero intraspecific distances within the ITS and matK sequences. Several species, including M. albus, M. italicus, M. officinalis, M. polonicus, M. speciosus and M. tauricus, showed small intraspecific distances. M. indicus, M. infestus, M. italicus, M. siculus, M. speciosus and M. sulcatus had similar interspecific distances because of the large distances between these species calculated using four loci .To determine the average distances among the 18 species, we combined several sequences to calculate the pairwise distances . These cmatK and rbcL, zero gaps, and approximately 100% similarity. A diversity of gaps was observed in the other three single sequences and combined regions. The intraspecific gap value of M. infestus and M. segetalis was large in the ITS sequence and close to zero in the other sequences. Approximately half of the species showed differences in interspecific gaps. M. speciosus had the highest gap value of 0.069. According to the alignment of psbA-trnH, almost all species showed variations in interspecific gaps. The four species, M. dentatus, M. segetalis, M. spicatus and M. wolgicus, showed high intraspecific diversity in gap values. For the trnL-F sequence, the intraspecific gap value was higher in M. altissimus, M. indicus, M. infestus and M. spicatus, and all species showed high interspecific variation. Only a small difference in the interspecific gap value was observed among the 18 species based on the five sequences except in M. dentatus. After removing the trnL-F sequence, the variation was apparent. A higher diversity was found after removing the ITS and psbA-trnH sequences. To a certain extent, the gap value could reveal the diversity among the sequences in 18 species.Furthermore, we calculated the similarity, gaps and scores through a pairwise sequence alignment to analyse the intraspecific and interspecific divergence Figs  and 3. WpsbA-trnH showed high discriminating in Melilotus. And phylogenetic trees were constructed using the Bayesian method based on the ITS, psbA-trnH respectively, and a combined region of the five sequences  didn’t get together with other accessions from NGBFG (China). And accessions from the same area tend to be together, such as Mo_CF… and Mo_PI… in M. officinalis.Based on genetic distance and similarity analysis, the ITS and ces Figs , S2 Fig.Melilotus is an important forage and green manure crop with high protein content and the ability to fix nitrogen [nitrogen , and it nitrogen ,41. HowetrnL-F and ITS sequences provided more variation sites and informative characteristics than the other three sequences. Many studies have reported that ITS sequences have more variable and informative sites than cpDNA [trnL-F region contained more variation sites and greater genetic distances than the other regions in this study. The rbcL and matK sequences were highly conserved in Melilotus, and the similarity among accessions was greater than 99.20% and 98.60%, respectively. The analysis of the five loci showed that the ITS and psbA-trnH sequences had more genetic diversity in Melilotus, whereas rbcL and matK could be used in combination with other sequences. In addition, the intraspecific and interspecific distances calculated using the rbcL and matK sequences were smaller than those of the other sequences. Our results were similar to the findings obtained by Chen, Yao et al [psbA-trnH, matK and rbcL. Previous researches showed that the rbcL and matK sequences were suitable for analyzing relationship at higher taxonomic levels [psbA-trnH sequences worked better and the combination of several sequences vielded the best result. Moreover, the sequence characteristics in Melilotus could be used in further genetic diversity studies.As shown in Tables an cpDNA ,42: Whitan cpDNA . Chloropan cpDNA  and presan cpDNA ,45. Due ao et al , who fouc levels ,48, and Melilotus species accessions [Melilotus showed higher diversity, particularly within the ITS sequences, than Euphrasia and Rhododendron [psbA-trnH and combined MRT and MRPI sequences. Additionally, the distance in Melilotus was significantly higher than that in species of Rhodiola [rbcL, matK and psbA-trnH regions. Compared with Medicago sativa, Melilotus has not been cultivated commercially and shows higher diversity [Melilotus into two groups according to flower colour. The white group contains four species, M. albus, M. tauricus, M. wolgicus and M. speciosus, and the other species composed the yellow group [M. albus, M. altissimus, M. elegans, M. officinalis, M. polonicus, M. suaveolens, M. wolgicus were included in the first group and M. italicus, M. speciosus, M. siculus, M. indicus, M. sulcatus, M. infestus, M. spicatus were included in the second group. These Melilotus species clustering within the same group may have closer genetic relationships.Our previous study showed that the SSR marker analysis revealed highly significant differences in genetic differentiation among accessions within cessions . Accordiodendron ,50, whicRhodiola  calculativersity ,51. In aiversity ,34. Tradiversity , but plaiversity . This reow group . Howeverow group ,34, M. aMelilotus species, and several, such as M. infestus and M. segetails, showed higher diversity than the others. To reduce the loss of Melilotus genetic resources, it is necessary to strengthen the collection and protection of wild germplasm resources. Current situation of the conservation of genetic resources in Melilotus is too many individuals of M. albus and M. officinalis were collected but the germplasm collections of other species were insufficient. Imbalance of these species is the main problem we are facing in germplasm collection and conservation. Surveys should occur worldwide and more individuals of the species with high diversity revealed by Tables Melilotus genetic resources for breeding purpose. The loss of genetic diversity has been recognized as the result of a genetic bottleneck imposed on crop plants during domestication and by modern plant-breeding practices [The significant differences were revealed among ractices ; thus, tMelilotus and indicated that analyses of these regions represent a valuable method for assessing genetic diversity. The analysis of the five loci provided important genetic information that will assist in germplasm collection and conservation of Melilotus.Our results identified the characteristics of five sequences in S1 FigM. albus, M. altissimus, M. dentatus, M. elegans, M. hirsutus, M. indicus, M. infestus, M. italicus, M. officinalis, M. polonicus, M. segetalis, M. siculus, M. speciosus, M. spicatus, M. suaveolens, M. sulcatus, M. tauricus, and M. wolgicus.From one to eighteen: (TIF)Click here for additional data file.S2 FigM. albus, Mm-M. altissimus, Md-M. dentatus, Me-M. elegans, Mh-M. hirsutus, Mi-M. indicus, Mf-M. infestus, Mr-M. italicus, Mo-M. officinalis, Mp-M. polonicus, Mg-M. segetalis, Mc-M. siculus, Ms-M. speciosus, Mu-M. spicatus, Mn-M. suaveolens, Ml-M. sulcatus, Mt-M. tauricus, Mw-M. wolgicus. See The abbreviations represent 18 species: Ma-(TIF)Click here for additional data file.S1 Table(XLS)Click here for additional data file.S2 Table(XLS)Click here for additional data file.S3 TableThe four colours represent the four canonical bases: green for adenine (A), blue for cytosine (C), purple for guanine (G) and orange-red for thymine (T). The symbol “▲” represents the sequence deletions from 1 to 188.(XLS)Click here for additional data file.


Marine species can demonstrate strong genetic differentiation and population structure despite the hypothesis of open seas and high connectivity. Some suggested drivers causing the genetic breaks are oceanographic barriers and the species’ biology. We assessed the relevance of seven major oceanographic fronts on species connectivity while considering their dispersal capacity and life strategy.We systematically reviewed the scientific articles reporting population genetic differentiation along the Mediterranean Sea and across the Atlantic-Mediterranean transition. We retained those considering at least one sampling locality at each side of an oceanographic front, and at least two localities with no-front between them to correctly assess the effect of the front. To estimate the impact of life history characteristics affecting connectivity we considered the planktonic larval duration (PLD) and adult life strategy.Oceanographic barriers in the Mediterranean Sea seem to reduce gene flow globally; however, this effect is not homogeneous considering the life history traits of the species. The effect of the oceanographic fronts reduces gene flow in highly mobile species with PLD larger than 2–4 weeks. Benthic sessile species and/or with short PLD (< 2 weeks) have more significant genetic breaks between localities than species with higher motility; however, genetic differentiation occurs independently of the presence of a front.Genetic connectivity is important for populations to recover from anthropogenic or natural impacts. We show that species with low mobility, mostly habitat-formers, have high genetic differentiation but low gene flow reduction mediated by the front, therefore, considering the importance of these species, we emphasize the vulnerability of the Mediterranean ecosystems and the necessity of protection strategies based on the whole ecosystem. The Mediterranean Sea displays one of the world’s richest diversity . This baModelling studies have indicated the great importance of spatial configurations of MPAs and stock identification units to promote population and ecosystem persistence ,5. AlthoThe majority of marine animals show life histories characterized by a long planktonic larval phase potentially allowing long-distance dispersal by marine currents. Therefore, we would expect to see low genetic structuring between localities of many marine species ,12. ThisThe length of the pelagic phase or planktonic larval duration (PLD) could be considered as proxy of the species’ dispersal potential, being one of the most important homogenising factors in the population structure . In the In the Mediterranean Sea, oceanographic processes, such as current patterns and oceanographic discontinuities are crucial factors influencing population genetic connectivity ,21,22. HRecently, some studies have provided interesting models defining hydrodynamic provinces, by coupling Lagrangian simulations of particles with oceanographic currents to modelate the transport of larvae in order to characterize marine connectivity ,33. The In the present review we aim to assess the effect of life history characteristics on population differentiation considering the impact of oceanographic discontinuities. In order to correctly evaluate whether a front is the cause for genetic differences between localities we screened all population genetic studies based in the Mediterranean Sea considering at least one sampling locality at each side of the oceanographic front, and at least two localities with no-front between them. We focus our review on the seven most important Mediterranean oceanographic discontinuities: Gibraltar Strait (GS), Almeria-Oran Front (AOF), Ibiza Channel (IC), Balearic Front (BF), Sicily Channel (SC), Otranto Channel (ADR) and the southern margin of the Aegean Sea (AEG) ,35,36. OWe searched the ISI Web of Knowledge database for scientific articles dated until 2016 which evaluated population genetic differentiation along the Mediterranean Sea and across the Atlantic-Mediterranean transition. Keywords were selected to identify these studies: “Mediterranean Sea”, “gene flow”, “genetic/population structure”, “genetic differentiation”, “connectivity”, which resulted in a total of 718 studies . We asseST values between localities against other less frequently used indices.Different molecular markers have been suggested to be more adequate at identifying historical (e.g. mtDNA) or contemporary (e.g. microsatellites) processes . We ther-1 [Between the Atlantic Ocean and Mediterranean Sea there are 7 major fronts frequently analysed in population genetic and biogeographic studies see : Gibralt-1 .To evaluate the impact of life history characteristics affecting connectivity we considered the planktonic larval duration (PLD), as a proxy of larval dispersal, and adult life behaviour. Each species was assigned to one of the following three groups according to their PLD: 1–15 days (S), 16–30 days (M), ≥31 days (L). Another categorical variable was constructed according to adult life strategy (LIFE): species were considered to be benthic sessile or with limited motility (BS), benthic vagile (BM), or pelagic (PEL). Finally, we also categorized the species according to major taxonomic groups: Angiosperm, Porifera, Cnidaria, Echinodermata, Mollusca, Crustacea, Tunicata and Pisces and grouped into a phylogenetic tree . BiologiST), comparing localities separated by fronts and one control pairwise comparison representative of no-fronts, were extracted from the selected scientific articles. Geographic distances between locations were approximated following the coastline with Google Earth.Pairwise genetic population distances  we tested differences in frequency of significant genetic distances between localities, separated by fronts or control sites, by chi-square tests .ST) were also standardized by geographic distance in Km as in Galarza et al.[ST between localities separated by front was larger than between control sites. We compared the two standardized pairwise FST (with front and no-front) with a Wilcoxon matched pairs test for each variable  using the programme STATISTICA V 8 [In order to avoid differences in significance due to isolation by distance, for each selected article all pairwise genetic distances  using the statistical package PRIMER-E v6 [To analyse the interaction between the front effect and the other variables, we created a continuous variable ranging between 0 and 1 called NDIF. This variable was calculated as (x-xmin) / (xmax-xmin) were x represents the difference between front and no-front standardized FMER-E v6 . We usedIn total 72 scientific papers passed our requirements to assess the effect of the fronts along the Mediterranean Sea and across the Atlantic-Mediterranean transition. These papers measured genetic differentiation between localities influenced by a front as well as a control comparison between localities without no-front . The meaWe observed that 66% of comparisons involving species with short PLD (S) had significant differences between localities, while only 41% and 28% were significant for species with medium (M) and long (L) PLD, respectively . This diST distances between localities separated by the front (F) and those without front (NF) and considered gene flow reduction when the former distance was larger. The frequency of comparisons with reduction increased with PLD  on the connectivity reduction mediated by fronts using Wilcoxon matched pairs tests . InteresLife history traits such as the length of PLD or the adult habitat show similar patterns across different taxa that seem to relate with genetic differentiation independent of phylogenetic relationships . For insST distances between localities separated by the front (F) and those without front (NF). No significant differences were observed for each front analysed separately or all together, despite having more species with higher standardized differentiation between populations when separated by a front  present a genetic structure generated by the presence of fronts [Tripterygion delaisi the presence of continuous rocky habitat between localities prevents genetic differentiation, while large discontinuities of sand or deep-water channels seem to reduce gene flow [For most species with longer PLDs (> two weeks) larvae move along the continental shelf and slope . The disf fronts ,21,25,28f fronts ,76,77. Hene flow . The larene flow . These dSerranus cabrilla [Epinephelus marginatus [The oceanographic discontinuities in the Mediterranean Sea restrict gene flow in numerous species. However, the dynamic behaviour of these fronts shows significant intra- and inter-annual variability ,81. Whencabrilla , and notrginatus .The identification of population units and the definition of population boundaries are one of the highest priorities in the management of marine ecosystems ,86. StudHowever, the establishment of MPA networks must contemplate the whole ecosystem, with special caution to those species with low dispersal capabilities. Oceanographic discontinuities did not generally affect the connectivity patterns in species with low dispersal larval capabilities (PLD < 2 weeks). These species include numerous sponges, gorgonians, angiosperms that are habitat formers ,89 and eAn optimal MPA network should protect areas of adequate size, including different home ranges, to help engineer species survive and flourish, thus providing habitat and food for larger species such as commercial fish species. Our results confirm the study of Shanks et al. that sugGenetic connectivity is important for recovery from anthropogenic of natural impacts. We show that both larval and adult life characteristics impact connectivity among localities and across oceanographic discontinuities. We observed that species with lower mobility potential have higher frequency of pairwise localities showing significant genetic differentiation than species with higher motility capacity, but independent of the existence of fronts and more subject to genetic drift. Moreover, oceanographic discontinuities reduce gene flow in species with medium to high dispersal abilities . We encoS1 Checklist(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.


This paper describes the development of a prototype of a low-cost digital fluorescent microscope built from commercial off-the-shelf (COTS) components. The prototype was tested to detect malignant tumor cells taken from a living organism in a preclinical setting. This experiment was accomplished by using Alexa Fluor 488 conjugate dye attached to the cancer cells. Our prototype utilizes a torch along with an excitation filter as a light source for fluorophore excitation, a dichroic mirror to reflect the excitation and pass the emitted green light from the sample under test and a barrier filter to permit only appropriate wavelength. The system is designed out of a microscope using its optical zooming property and an assembly of exciter filter, dichroic mirror and transmitter filter. The microscope is connected to a computer or laptop through universal serial bus (USB) that allows real-time transmission of captured florescence images; this also offers real-time control of the microscope. The designed system has comparable features of high-end commercial fluorescent microscopes while reducing cost, power, weight and size. Fluorescence microscopy is a powerful and popular technique used by modern biologists, especially neurobiologists as it allows them to distinguish between the object of interest to those of not interest. Because of its inherent selectivity, it has become a very important part of microscopy in the field of biology. Due to the large spectral range of fluorophores, it is often possible to image different cellular, subcellular and molecular structures simultaneously . Also, tHowever, for a person who is inexperienced in fluorescent microscopy, matching the best approach to the biological experiment can be difficult. Optimal and efficient use of fluorescence microscopy demands a basic knowledge of strengths and weaknesses of the different approaches as well as an understanding of the elementary trade-offs of the variables related to it. Familiarity with the principle of fluorescence is a prerequisite for taking advantage of many of these technological advancements towards fluorescence microscopy. One way forward is to start exposing the students (or learners) at schools and colleges to different fluorescent approaches. However, the size, cost and complexity of current fluorescence microscope requires sophisticated laboratories that are well funded and have trained personnel to maintain and operate it , 9. HencAs a result, even if the students have sound theoretical knowledge, they lack in practical knowledge. Therefore, the objective is to design a low-cost fluorescence microscope to be used in schools and academic institutions. In this research, we have designed a prototype of a fluorescence microscope using inexpensive commercial off-the-shelf components that is digital in nature and has real-time diagnostic capability.The proposed device consists of a dichroic mirror, excitation filter, barrier filter, microscope and flashlight as its major component. These components are inexpensive and can be easily found or at parts store. In this work, fluorophore is used as a stimulant to differentiate between cancer and normal cells. Samples with both malignant and healthy cells were prepared by using amphotropic HEK293-derived from phoenix packaging cells to package pBabe-puro retroviral system in order to generate stable cells. The packaging cells were cultured on 100 mm cell culture plates in 10 ml of Dulbecco’s Modified Eagle’s Medium (DMEM) which was supplemented with the absorption percentage of bovine calf serum to produce retrovirus. For transfection, 10 ug of retroviral DNA was gently mixed. 430 ul of 0.15M NaCl and 60 ul of 1% Polyethylenimine (PEI) were added to the mixture followed by a gentle vortex. The solution was led to stand by for 10 minute at room temperature and was then added to the 10 mm culture plates. After 24 hour and 48 hour respectively, virus-containing supernatant was collected and filtered through 0.45 um syringe filter and was then supplemented with polybrene, and overlaid on the target cells MDA-MB-231 (a triple negative breast cancer cell lines). After 24 hour, the viral supernatant was replaced by fresh culture medium and stable expressing GFP-BRK-YF fusions were used as negative controls.Different concentrations of fluorescein samples were made and stored in a dark place to avoid bleaching. Images from each sample were captured through the microscope on to a computer via a USB. The absorption or excitation spectrum peak and emission peak of the used fluorophore are 495 nm and 519 nm. So, we can exploit the common spectral wavelength for absorption and emission measurements. This means we need a blue light for excitation of the fluorophore and we must measure green light at the image sensor.The resulting device is a small, light-weight, and portable microscope which provides an alternative to conventional fluorescence microscope with no requirement of electric power to operate (as shown in Moreover, the proposed device enables us to take color images directly on computer while the commercial microscope  is limitThe efficient use of the device requires some practice as focusing into the sample and placing the flashlight at appropriate position can be tricky during the initial phase. In addition to that, the software consists of a user-friendly interface which helps in configuring image parameters. The system also provides the user with an option to select the output image format, frame rate, management of the collected images, ability to focus and select a section of an image for further enhancement. The user selected configurations are saved for future use. The effect on performance of different configuration of LEDs, filters and lenses were evaluated and overall performance was also compared with the conventional fluorescent microscope. The application of this portable microscope is not only limited to detect cancer cells but can also be used in various other purposes as mentioned before, such as detecting water and food quality, molecular imaging and optical biopsy in gastrointestinal endoscopy.Fluorescence microscope designed in this research is a simple, yet essential, imaging technology. Commercial fluorescence microscopes are bulky, costly and requires sophisticated laboratory and trained manpower which restricts their accessibility in the regions where there are limited resources. With the aim of overcoming these limitations, we have successfully designed and tested a digital fluorescence microscope. Our design is capable of imaging micrometer-scale resolution in real-time along with controlled mechanical actuation of both the lens and sample. Combination of mechanical actuation and software into a compact and inexpensive microscope system is an important initial step towards the objective of making fluorescence microscopy universally accessible, portable, and easy to use.


Lynx canadensis), American marten (Martes americana), fisher (Pekania pennanti), and southern flying squirrel (Glaucomys volans) to evaluate multi-species genetic connectivity across Ontario, Canada.Habitat fragmentation reduces genetic connectivity for multiple species, yet conservation efforts tend to rely heavily on single-species connectivity estimates to inform land-use planning. Such conservation activities may benefit from multi-species connectivity estimates, which provide a simple and practical means to mitigate the effects of habitat fragmentation for a larger number of species. To test the validity of a multi-species connectivity model, we used neutral microsatellite genetic datasets of Canada lynx (We used linear models to compare node-based estimates of genetic connectivity for each species to point-based estimates of landscape connectivity (current density) derived from circuit theory. To our knowledge, we are the first to evaluate current density as a measure of genetic connectivity. Our results depended on landscape context: habitat amount was more important than current density in explaining multi-species genetic connectivity in the northern part of our study area, where habitat was abundant and fragmentation was low. In the south however, where fragmentation was prevalent, genetic connectivity was correlated with current density. Contrary to our expectations however, locations with a high probability of movement as reflected by high current density were negatively associated with gene flow. Subsequent analyses of circuit theory outputs showed that high current density was also associated with high effective resistance, underscoring that the presence of pinch points is not necessarily indicative of gene flow.Overall, our study appears to provide support for the hypothesis that landscape pattern is important when habitat amount is low. We also conclude that while current density is proportional to the probability of movement per unit area, this does not imply increased gene flow, since high current density tends to be a result of neighbouring pixels with high cost of movement . In other words, pinch points with high current density appear to constrict gene flow.The online version of this article (10.1186/s40462-017-0112-2) contains supplementary material, which is available to authorized users. Much recent thinking about maintaining biodiversity in the face of environmental change suggests that ensuring adequate landscape connectivity is important. Indeed, managing connectivity is considered by some to be one of the key strategies for creating resilient landscapes and adapting to climate change . LandscaLynx lynx), evaluate the effectiveness of vaccination barrier strategies to control the transmission of rabies virus, and identify the appropriate placement of road mitigation measures to facilitate wildlife crossings in jaguars (Panthera onca) [Given the species-specific nature of the landscape connectivity concept, it is not surprising that many studies evaluate single-species landscape connectivity. For example, single-species connectivity estimates have been used as a means to assess suitable habitat for Eurasian lynx (ra onca) –7. Singlra onca) , 9. Therra onca) . This apra onca) . Such muMyodes gapperi) and deer mice (Peromyscus maniculatus) [Functional connectivity implies that animals are successfully moving through landscape elements and functioning as effective members of the local population via successful reproduction and gene flow . Thus, mculatus) , and morculatus) . In contculatus) –25.ST). Similarly, measures of landscape connectivity may also be pairwise. For example, the most popular methods in recent years for measuring connectivity of landscapes are least cost paths [One challenge in evaluating how landscape structure affects gene flow arises due to the pairwise nature of gene flow estimates  involves estimating the optimal, lowest cost route between source and destination nodes . This meThere are three key features of circuit theory important to highlight for our purposes. First, one output of circuit theory is called current density, which is spatially referenced, and is proportional to the probability of movement by a random walker , 40. CurPekania pennanti) telemetry and herpetofaunal roadkill data to validate the results for a study area in eastern Ontario. The methods were subsequently applied by Bowman and Cordes [Martes americana), southern flying squirrel (Glaucomys volans), and Canada lynx (Lynx canadensis) sampled in Ontario to test the hypothesis. We also compared the current density map and genetic connectivity estimates to a more traditional measure of landscape structure based on buffered estimates of habitat amount.Our main objective was to compare two node-based estimates of connectivity. We compared a measure of multi-species genetic connectivity to another measure derived from omnidirectional current density maps. We used a previously developed terrestrial multi-species connectivity map developed from circuit theory for natural heritage planning in Ontario, Canada , 44. KoeWe used existing population genetic datasets of four terrestrial mammal species to model multi-species genetic connectivity across Ontario, Canada Fig. . These dSamples were collected across the Ontario study area between about 42.3°N and 51.3°N. The southern portion of the study area was dominated by agriculture and urban development. Forests in this region represented a transition between southern temperate forests and boreal forests. The northern end of the study area was boreal forest, with a much lower human population density, lower agricultural intensity and little urban development. Fisher and flying squirrel samples were concentrated in the southern portion of the study area, whereas marten and lynx samples were concentrated in northern Ontario . In eacpopgraph . The shopopgraph .www.circuitscape.org) which models a rasterized landscape as a circuit board where each pixel represents resistance to wildlife movement. Expert opinion was used to assign land cover to one of three resistance values as outlined by Koen et al. [We used a previously published current density map for Ontario that was derived from a circuit theory approach to model omnidirectional, multispecies connectivity based on landscape resistance , 44. Then et al.  and at an et al. . The plan et al. , and then et al. . The expn et al. . TherefoWe calculated our measure of node-based genetic connectivity using the average inverse edge weight of cGD for each sampled node, which has been shown to be positively correlated to genetic connectivity . We stanOur population graphs indicated that there was relatively high connectivity within each of the mammal species considered, given the generally well connected graph topologies Fig. . Sample We found differing trends with respect to the relationship between buffer size and mean cost estimated for each species, which resulted from both the distribution of each species and the strategy taken for collecting DNA samples. Specifically, for the more northern distributed species (lynx and marten), mean cost increased with increasing buffer size. Alternatively, for the southern distributed species (fisher and southern flying squirrel), mean cost generally decreased as buffer size increased. Specifically, for southern flying squirrel, there was a consistent decline in mean cost as the buffer size increased from 6 km to 20 km to 120 km . PC1 had a negative loading of the mean cost and its standard deviation at all three spatial extents  , the only predictor of genetic connectivity (estimated as average inverse edge weight) whose confidence intervals did not overlap 0 was sample size , whereas the southern model, including fisher and southern flying squirrel, explained less variation . In the northern model, sample size was once again an important predictor of genetic connectivity, as was PC3, which showed a negative effect of cost on genetic connectivity. In contrast, PC2 (related to current density) was an important predictor of genetic connectivity in the southern model (including fisher and southern flying squirrel), as was sample size. The direction of the relationship in the southern model suggested that current density was negatively related to gene flow . We assume that this effect was a consequence of cGD being sensitive to under sampled sites [Perhaps not surprisingly, sample effort was the most important variable associated with genetic connectivity in all of our models. More samples at a site were associated with higher estimates of genetic connectivity


Biophysical and enzymatic analyses show that UDP- and ADP-glucose are more stable than claimed in a controversial study that questions the generally accepted pathway of starch synthesis in plants. 2+ and that this instability can lead to unreliable results from in vitro assays of enzyme activities. If substantiated, this claim would have far-reaching implications for many published studies that report on the activities of NDP-sugar metabolizing enzymes. To resolve this controversy, we investigated the stability of UDP- and ADP-glucose using biophysical, namely nuclear magnetic resonance (NMR), and highly specific enzymatic methods. Results obtained with both techniques indicate that NDP-sugars are not as unstable as previously suggested. Moreover, their calculated in vitro half-lives are significantly higher than estimates of their in planta turnover times. This indicates that the physico-chemical stability of NDP-sugars has little impact on their concentrations in vivo and that NDP-sugar levels are determined primarily by the relative rates of enzymatic synthesis and consumption. Our results refute one of the main arguments for the controversial pathway of starch synthesis from imported ADP-glucose produced by sucrose synthase in the cytosol.Nucleoside diphosphate sugars (NDP-sugars) are the substrates for biosynthesis of oligo- and polysaccharides, such as starch and cellulose, and are also required for biosynthesis of nucleotides, ascorbic acid, several cofactors, glycoproteins and many secondary metabolites. A controversial study that questions the generally accepted pathway of ADP-glucose and starch synthesis in plants is based, in part, on claims that NDP-sugars are unstable at alkaline pH in the presence of Mg Nucleoside diphosphate sugars (NDP-sugars) were discovered by Luis F Leloir and colleagues around the middle of the twentieth century . This seStarch is the most important carbon reserve in plants and the major source of calories in our staple crops. Transitory starch reserves are accumulated and degraded on a daily basis in the leaves of many plants to provide carbon and energy during the hours of darkness. Starch is also stored in many seeds, fruits, and tubers, as well as in the stems of woody perennials, providing longer term reserves for germination and regrowth after winter. In leaves, starch is synthesized in the chloroplasts from fructose 6-phosphate that is withdrawn from the Calvin-Benson cycle and converted to ADP-Glc via plastidial phosphoglucose isomerase (pPGI), plastidial phosphoglucomutase (pPGM), and ADP-Glc pyrophosphorylase (ADP-Glc PPase), which is restricted to the chloroplasts in leaves .2  mutant that lacks SUS expression in mesophyll cells was found to accumulate wild-type levels of starch , and enzymatic methods to investigate the stability of UDP-Glc and ADP-Glc over a range of pH and Mg+, Glc 1,6-bisphosphate, phosphoglucomutase from rabbit muscle, Glc-6-phosphate dehydrogenase from Baker’s yeast, BSA and MgCl2 were obtained from Sigma-Aldrich . Deuterium oxide was from Cambridge Isotope Laboratories Inc. . All other chemicals were of the highest quality available. Recombinant ADP-Glc and UDP-Glc pyrophosphorylases from Escherichia coli were expressed in E. coli and purified to near homogeneity as previously described , and cyclic Glc 1,2-phosphate concentrations were performed using a Varian Inova 500 MHz NMR spectrometer. A dilute phosphoric acid sample served as the external standard, denoting 0 ppm. At least 6000 scans were performed on all samples using a 33° tip angle and a 3.2 s acquisition time, while at least 12 000 scans were performed on samples with more than 80% apparent NDP-Glc degradation. The relative integration values of the observed species were used to calculate the percentage of NDP-Glc degradation that occurred during the given time period. As NMPs are known to be stable in alkaline solutions containing Mg2+ (ANMP)/[(2×ANMP)+ANDP-Glc]×100, where ANMP is the integrated area for the NMP and ANDP-Glc is the area of NDP-Glc. Half-lives for UDP-Glc in each of the pH conditions were calculated using the formula: P=(1−2−t/z)×100, where P is the percentage of UDP-Glc degradation, t is time in minutes, and z is the calculated half-life.Samples 1 ml in volume containing 5 mM UDP-Glc, 10 mM MgCling Mg2+ , but the2, and 10% deuterium oxide, to which 250 μl of a solution containing 1 M HEPES-NaOH at pH 7.0 and 150 mM EDTA were added. Peaks were found to be near 3 ppm for the NMP and near -11 and -13 ppm for the two phosphates of the NDP-Glc. Consistent with previously published results , experiments of T1 inversion recovery were performed as previously described  was 3.3 s. As described elsewhere  measuring the absorbance at 340 nm every 15 s. The absorbance was measured for at least 4 min after there was no further change to ensure that the reaction had reached completion, resulting in measurements spanning approximately 8 min in total. The absorbance value of the blank samples without NDP-Glc was subtracted from all readings.Four 1 ml solutions, each containing 0, 1.25, 2.5, or 5 mM NDP-Glc, 10 mM MgClescribed , with th2, and measuring the amount remaining after 90 min by enzymatic analysis with UDP-Glc pyrophosphorylase and ADP-Glc PPase, respectively. These conditions were chosen as a physiologically extreme scenario, combining high pH and high Mg2+ concentration with high temperature. Under these extreme conditions, 40–48% of the UDP-Glc was degraded within 90 min , suggesting that the reaction depends on the concentration of hydroxyl ions (2+, a similar value to that observed for UDP-Glc (We used [hosphate , the chahosphate . The cle mM Mg2+ . No dete mM Mg2+ . Calculaxyl ions , inset.  UDP-Glc .2+ concentration (2 and 37°C, over 50% of the initial UDP-Glc and ADP-Glc survived incubation for 90 min (2+ of 0.2–0.4 mM (in vivo will therefore be determined by the relative rates of UDP-Glc synthesis and consumption by UDP-Glc metabolizing enzymes, with its chemical instability at alkaline pH having little or no significant impact on in vivo levels.These results confirm the previous reports from ntration  and 4, ar 90 min  and 4. Ir 90 min  and a co2+ concentration to around 6 mM (in planta (in planta (in vivo.During photosynthesis, ion movements across the thylakoid membranes result in alkalinization of the stroma to around pH 8.3 and a rise in Mgund 6 mM . Althougn planta  and 4; t2+ and 37°C, losses of UDP-Glc and ADP-Glc were less than 50% after 90 min incubation. The disputed SUS activity data reported by in vitro assays with reaction mixtures containing 2.2 mM MgCl2, which were incubated at 20°C for only 20 min. Based on the data (2+ concentration alone would reduce the loss of UDP-Glc by a factor of three compared with the most extreme conditions tested, and that the shorter incubation time of 20 min versus 90 min would reduce losses by a further factor of 4.5. Thus, the maximal loss of UDP-Glc to be expected under the assay conditions used by sus mutants (in vitro assay conditions, is not supported by our results. It is also worth reiterating that even if residual SUS activity in the quadruple sus1234 mutant was underestimated by Our results also demonstrate that even under the most extreme conditions tested, namely pH 9.0, 10 mM Mgthe data  and 4, w2+ is unlikely to have any significant impact on in vivo concentrations of these metabolites in plants. Arguments in favor of the alternative pathway of starch synthesis based on NDP-sugar instability therefore have little merit.In conclusion, the chemical instability of UDP-Glc and ADP-Glc at alkaline pH in the presence of MgAAI and MAB conceived the project; BLH, CMF and MDAD performed experiments; all authors analyzed data and wrote the manuscript.