Inferring the ancestral dynamics of effective population size is a long-standing question in population genetics, which can now be tackled much more accurately thanks to the massive genomic data available in many species. Several promising methods that take advantage of whole-genome sequences have been recently developed in this context. However, they can only be applied to rather small samples, which limits their ability to estimate recent population size history. Besides, they can be very sensitive to sequencing or phasing errors. Here we introduce a new approximate Bayesian computation approach named PopSizeABC that allows estimating the evolution of the effective population size through time, using a large sample of complete genomes. This sample is summarized using the folded allele frequency spectrum and the average zygotic linkage disequilibrium at different bins of physical distance, two classes of statistics that are widely used in population genetics and can be easily computed from unphased and unpolarized SNP data. Our approach provides accurate estimations of past population sizes, from the very first generations before present back to the expected time to the most recent common ancestor of the sample, as shown by simulations under a wide range of demographic scenarios. When applied to samples of 15 or 25 complete genomes in four cattle breeds , PopSizeABC revealed a series of population declines, related to historical events such as domestication or modern breed creation. We further highlight that our approach is robust to sequencing errors, provided summary statistics are computed from SNPs with common alleles. Molecular data sampled from extant individuals contains considerable information about their demographic history. In particular, one classical question in population genetics is to reconstruct past population size changes from such data. Relating these changes to various climatic, geological or anthropogenic events allows characterizing the main factors driving genetic diversity and can have major outcomes for conservation. Until recently, mostly very simple histories, including one or two population size changes, could be estimated from genetic data. This has changed with the sequencing of entire genomes in many species, and several methods allow now inferring complex histories consisting of several tens of population size changes. However, analyzing entire genomes, while accounting for recombination, remains a statistical and numerical challenge. These methods, therefore, can only be applied to small samples with a few diploid genomes. We overcome this limitation by using an approximate estimation approach, where observed genomes are summarized using a small number of statistics related to allele frequencies and linkage disequilibrium. In contrast to previous approaches, we show that our method allows us to reconstruct also the most recent part (the last 100 generations) of the population size history. As an illustration, we apply it to large samples of whole-genome sequences in four cattle breeds. Reconstructing the ancestral dynamics of effective population size is important in several contexts. From a long term evolutionary perspective, the history of population size changes can be related to various climatic or geological events, and reconstructing this history allows studying the impact of such events on natural species . This deUntil recently, methods allowing to infer the history of population size changes from genetic data were designed for data sets consisting of a limited number of independent markers or non recombining DNA sequences –8. HowevIn this context, several promising approaches allowing to infer complex histories, including several tens of stepwise population size changes, have recently been proposed –13. SomeWhile the above methods take advantage of whole-genome data, they are so far restricted to the analysis of small sample sizes. In the case of SMC based methods, this implies a limited resolution for the estimation of recent population sizes. Indeed, the most recent time at which these methods can infer population size is determined by the time to the most recent coalescence event occurring in the sample, which is older for small samples. For instance in humans, PSMC cannot estimate population sizes more recently than 400 generations  before present (BP), and MSMC cannot estimate these sizes more recently than 40 generations  BP. The most recent time for which an inference is possible will differ between species. In populations with small recent population sizes, coalescence events will occur at a higher rate than in larger populations, so the inference of recent history will be more accurate. Inference approaches based on the distribution of IBS segment length may be less affected by the use of small samples. Using this approach, estimations of population size in the Holstein cattle breed were obtained from a single genome even for the first few generations BP , and werAnother concern of the above methods is their sensitivity to sequencing errors. False positive SNPs can lead to a strong overestimation of population sizes in the recent past, i.e. in the first few hundred generations BP, both with PSMC , 12 and Here we introduce a new statistical method named PopSizeABC, allowing estimating population size history from a sample of whole-genome sequences. One of the main motivations for developing this method is to take advantage of large sample sizes in order to reconstruct the recent history as well. Since statistical approaches based on the full likelihood of such samples seem currently out of reach, even with approximated models such as the SMC, we followed an Approximate Bayesian Computation (ABC)  approachABC is a popular approach in population genetics, which has already been applied to the analysis of large-scale population genetic data sets –25. HoweApplying our ABC approach to samples of 25 diploid genomes, simulated under a large number of random population size histories, we show that it provides, on average, accurate estimations of population sizes from the first few generations BP back to the expected time to the most recent common ancestor (TMRCA) of the sample. This result is confirmed by the study of several specific demographic scenarios, where our method is generally able to reconstruct the population size history from present time back to the expected TMRCA, while PSMC or MSMC reconstruct it only for a limited time window.We then apply this method to samples of 15 or 25 genomes in four different cattle breeds, which reveals interesting aspects of cattle history, from domestication to modern breed creation. Through this application to a real data set, we also illustrate how sequencing and phasing errors, if not taken into account, can have a dramatic influence on the estimated past population sizes. Our method is actually insensitive to phasing errors, because it uses unphased data. In addition, we show that a simple modification in the choice of summary statistics makes it robust to sequencing errors.Following several recent studies –12, we mn diploid genomes sampled from the same population. We summarized this data set using two classes of summary statistics: (i) the folded allele frequency spectrum (AFS) of the sample, which includes the overall proportion of polymorphic sites in the genome and the relative proportion of those polymorphic sites with i copies of the minor allele, for all values of i between 1 and n, and (ii) the average linkage disequilibrium (LD) for 18 bins of physical distance between SNPs, from approximately 500 bp to 1.5Mb. We generated a very large number of population size histories, by drawing the population size in each time window from a prior distribution. For each history, we simulated a sample of n diploid genomes and computed a distance between the summary statistics obtained from this simulated sample and those obtained from the observed sample. A given proportion  of the most likely histories was accepted based on this distance. Finally, the joint posterior distribution of population sizes was estimated from the population sizes of accepted histories. Different statistical approaches were compared for this last estimation step.Our estimation procedure was based on the observation of A detailed description of the model and of the ABC procedure described above is provided in the Methods.In order to optimize our ABC estimation procedure and to evaluate its average performance, we first applied it to a large number of genomic samples simulated under random population size histories. These pseudo-observed datasets (PODs) included 25 diploid genomes and 100 independent 2Mb-long regions. For each POD, population sizes were estimated by ABC, using 450,000 simulated datasets of the same size. These estimated values were compared with their true values for different tolerance rates and different ABC adjustment approaches to process the accepted histories. We found that the best procedure was to accept simulated histories with a tolerance rate of 0.005, to adjust their parameter values using a non linear neural network regression , and to ABC provided accurate estimations of population sizes for a large range of times in the past . The besn genotypes or as a correlation between two vectors of 2n alleles was equivalent in terms of PE  confidence intervals could be obtained using 200,000 simulated samples .We then considered the influence of the genome length of observed and simulated samples . As expen = 10 to n = 50, we observed that using large samples resulted in a more accurate estimation of population sizes in the first 100 generations BP. For instance, in the most recent time window, PE was equal to 0.153 for n = 50 versus 0.212 for n = 10, and the 90% credible interval was narrower (ratio between upper and lower bound of 36 versus 74). These improvements resulted from the fact that low frequency alleles, which are better captured from large samples, are very informative about recent population history. In contrast, population sizes at times more ancient than 10,000 generations BP were more accurately estimated from small samples, although the magnitude of this effect was lower than for recent population sizes (PE of 0.66 for n = 50 versus 0.63 for n = 10 in the most ancient window). This is likely due to statistical overfitting: increasing the sample size leads to increasing the number of AFS statistics, so if these additional statistics are not sufficiently informative they may introduce some noise and reduce the prediction ability of the model.We also studied the influence of sample size on population size estimations . CompariFinally, we found that computing AFS statistics only from SNPs exceeding a given minor allele frequency (MAF) threshold (from 5 to 20%) resulted in larger PEs and confidence intervals, except for the most ancient population sizes . Again, To illustrate the performance of our ABC approach, we then considered six specific demographic scenarios: a constant population size of 500, a constant population size of 50,000, a population size declining from 40,000 to 300 individuals between 3,600 and 100 generations BP, a population size increasing from 2,500 to 60,000 individuals between 1,500 and 250 generations BP, a population size experiencing one expansion from 6,000 to 60,000 individuals followed by a bottleneck of the same magnitude, between 34,000 and 900 generations BP, and a “zigzag” scenario similar to the previous one but including one additional bottleneck between 520 and 50 generations BP  often showed a slight bias towards intermediate values, as can be seen in the large constant size scenario, the decline scenario and the expansion scenario. This partly comes from the fact that we estimated population size by the median of the posterior distribution, which tends to shrink it away from our prior boundaries. When estimating population sizes from the mode of the posterior distribution, we were able to better reconstruct the very recent population size in these three scenarios . NeverthIn order to explore why ABC failed to fully reconstruct this zigzag history, we considered five variants of this scenario . For a zBecause one of our objectives was to estimate the population size history in taurine cattle, we studied more precisely the continuous decline scenario that is expected in this species , and evaFor each scenario of Finally, it is important to note that the simulated data that we used in these MSMC analyzes were assumed to be perfectly phased. However, real data consist generally in statistically inferred haplotypes, which can typically include from 1 to 10 switch errors per Mb and individual, even when using recent phasing algorithms and large sample sizes . In our We applied our ABC approach to estimate the population size history in four cattle breeds, using large samples of diploid genomes recently published by the 1,000 bull genomes project . An impoIn contrast, the average zygotic LD (i.e. the LD computed directly from genotype data) was identical for the NGS and the 800K data. We also observed a perfect match between the polymorphic site AFS obtained from the NGS data subsampled at 800K positions, and from 800K data. Finally, the overall proportion of SNPs was similar in the two types of data. More precisely, based on the 800K positions and the sample of 12 individuals, we found approximately 0.5% of false positive SNPs, i.e. positions that were found polymorphic when using NGS data but not when using 800K data , left, aTo overcome this limitation, we studied directly the influence of sequencing and phasing errors on ABC estimations, by analyzing one sample of 25 Holstein genomes with slightly different combinations of summary statistics . When LDBos taurus population which resulted from the domestication of the wild aurochs, Bos primigenius, approximately 10,000 years BP , where ti = exp(log(1 + aT)i/(I − 1)) − 1)/a generations BP for i from 0 to I − 1, with T = 130,000 and a = 0.06, and tI = +∞. These specific values of T and a were chosen to capture important periods of cattle history. Modifying T would allow population size changes to occur on a longer or shorter period in the past, and modifying a would allow to describe more precisely one specific part of the history, playing on the ratio between the length of recent versus old time windows. With our parametrization, the most recent time window ranged from present to 10 generations BP, the second most recent ranged from 10 to 25 generations BP, …the second oldest ranged from 83,000 to 130,000 generations BP and the oldest included all generations above 130,000 generations BP.Here the observed data bination . We assubination , 10, we Ni for i from 0 to I − 1, the per generation per site recombination rate r and the per generation per site mutation rate μ. Prior distributions for the population sizes were taken uniform in the log 10 scale, from 10 to 100,000. In order to avoid unrealistic trajectories, we also set that the ratio of population sizes between two consecutive time windows could not exceed 10. In practice, we thus sampled log10(N0) uniformly between 1 and 5, and iteratively computed log10(Ni) = max(min(log10(Ni − 1) + α, 5), 1), with α sampled uniformly between −1 and 1. For the recombination rate, we used an uniform prior between 1e-9 and 1e-8, consistent with recent estimations in cattle  for a given population size history was allocated to the rescaled interval . Applying the equations above to the specific situation of a piecewise constant population size process, ui was computed asN0 is the haploid population size here, while the population sizes mentioned anywhere else in this paper are always diploid population sizes. We used In Twenty scenarios with fixed population size history were considered for validation, see For 12 of the 129 Holstein bulls considered in this study, genotypes on the 800K Illumina bovine SNP chip were obtained from the Gembal project . Among thttps://forge-dga.jouy.inra.fr/projects/popsizeabc/. Simulated and observed summary statistics used in this study are also provided on this web page.Python and R scripts for the PopSizeABC method can be found at S1 FigEmpirical coverage (left) and width (right) of the 90% credible interval for the population size in each time window. The empirical coverage is the proportion of simulated histories for which the true population size was included in the 90% credible interval of the posterior distribution. If the posterior distribution was correctly estimated, this proportion should have been 90%, as shown by the black horizontal solid line. Parameter settings were the same as in (PDF)Click here for additional data file.S2 FigE[TMRCE], and the expected TMRCA, E[TMRCA]. Summary statistics considered in the ABC analysis were (i) the AFS and (ii) the average zygotic LD for several distance bins. These statistics were computed from n = 25 diploid individuals, using all SNPs for AFS statistics and SNPs with a MAF above 20% for LD statistics. The posterior distribution of each parameter was obtained by neural network regression [Prediction error for the estimated population size when time is measured in units of the expected time to the most recent common ancestor (TMRCA) of the sample. Prediction errors were evaluated from 2,000 random population size histories. Black vertical dotted lines indicate the expected time to the most recent coalescence event, gression , with a (PDF)Click here for additional data file.S3 FigEmpirical coverage (left) and width (right) of the 90% credible interval for the population size in each time window. Parameter settings were the same as in (PDF)Click here for additional data file.S4 Fign = 25 diploid individuals, using different MAF thresholds. Other parameter settings were the same as in Prediction error for the estimated population size in each time window, evaluated from 2,000 random population size histories. Summary statistics considered in the ABC analysis were the average gametic LD (triangles) or the average zygotic LD (circles) for several distance bins. These statistics were computed from (PDF)Click here for additional data file.S5 Fign = 25 diploid genomes was simulated, where each genome consisted in 100 independent 2Mb-long segments. Population size history was estimated from this POD by ABC, for various numbers of simulated datasets (see the legend) with the same sample size (n = 25) and genome length (100 independent 2MB segments). Summary statistics considered in the ABC analysis were (i) the AFS and (ii) the average zygotic LD for several distance bins. AFS statistics were computed using all SNPs and LD statistics were computed using SNPs with a MAF above 20%. The posterior distribution of each parameter was obtained by neural network regression, with the tolerance rate leading to the smallest prediction error. Population size point estimates were obtained from the median of the posterior distribution.Top: Prediction error for the estimated population size in each time window (left) and standard deviation of this error (right). Bottom: Empirical coverage (left) and width (right) of the 90% credible interval for the population size in each time window. These quantiles were evaluated from 2,000 random population size histories. For each of these histories, one POD of (PDF)Click here for additional data file.S6 Fign = 25 diploid genomes was simulated, where each genome consisted in 10, 50 or 100 independent 2Mb-long segments (see the legend). Population size history was estimated from this POD by ABC, using 450,000 simulated datasets with the same sample size (n = 25) and genome length. The posterior distribution of each parameter was obtained by neural network regression, with a tolerance rate of 0.005. All other settings are similar to Top: Prediction error for the estimated population size in each time window (left) and standard deviation of this error (right). Bottom: Empirical coverage (left) and width (right) of the 90% credible interval for the population size in each time window. These quantiles were evaluated from 2,000 random population size histories. For each of these histories, one POD of (PDF)Click here for additional data file.S7 Fign = 25 diploid genomes was simulated, where each genome consisted in 10 or 100 independent 2Mb-long segments (see the legend). Population size history was estimated from this POD by ABC, using 450,000 simulated datasets with the same sample size (n = 25) but a possibly different genome length (see the legend). The posterior distribution of each parameter was obtained by neural network regression, with a tolerance rate of 0.005. All other settings are similar to Top: Prediction error for the estimated population size in each time window (left) and standard deviation of this error (right). Bottom: Empirical coverage (left) and width (right) of the 90% credible interval for the population size in each time window. These quantiles were evaluated from 2,000 random population size histories. For each of these histories, one POD of (PDF)Click here for additional data file.S8 Fign diploid genomes was simulated, for different values of n between 10 and 50 (see the legend). Each genome consisted in 100 independent 2Mb-long segments. Population size history was estimated from this POD by ABC, using 450,000 simulated datasets with the same sample size and genome length. All other settings are similar to Top: Prediction error for the estimated population size in each time window (left) and standard deviation of this error (right). Bottom: Empirical coverage (left) and width (right) of the 90% credible interval for the population size in each time window. These quantiles were evaluated from 2,000 random population size histories. For each of these histories, one POD of (PDF)Click here for additional data file.S9 Fign = 25 diploid genomes was simulated, where each genome consisted in 100 independent 2Mb-long segments. Population size history was estimated from this POD by ABC, using 450,000 simulated datasets with the same sample size and genome length. Summary statistics considered in the ABC analysis were (i) the AFS and (ii) the average zygotic LD for several distance bins. AFS statistics were computed using different MAF thresholds, LD statistics were computed from SNPs with a MAF above 20%. The posterior distribution of each parameter was obtained by neural network regression, with a tolerance rate of 0.005. Population size point estimates were obtained from the median of the posterior distribution.Top: Prediction error for the estimated population size in each time window (left) and standard deviation of this error (right). Middle: Bias for the estimated population size in each time window. Bottom: Empirical coverage (left) and width (right) of the 90% credible interval for the population size in each time window. These quantiles were evaluated from 2,000 random population size histories. For each of these histories, one POD of (PDF)Click here for additional data file.S10 FigAll settings are similar to (PDF)Click here for additional data file.S11 Figa scenario where all population sizes are divided by ten compared to the original zigzag , a scenario where only the recent bottleneck of the original zigzag is simulated , a scenario corresponding to the history wrongly inferred by ABC based on data from the “bottleneck1 recent large” scenario (middle right), and two scenarios where only the recent (bottom left) or the old (bottom right) bottleneck of the “zigzag small” are simulated. All settings are similar to (PDF)Click here for additional data file.S12 FigFor one of the five PODs analyzed in this scenario, observed AFS (left) and LD (right) statistics are shown by green full circles. The average value of these statistics over the five best simulated data sets, i.e. the five simulated data sets leading to the smallest distance between observed and simulated statistics, are shown by blue crosses. The variation of these statistics over the five best simulated data sets is also indicated by blue dotted lines, which correspond to the average value plus (or minus) twice the standard deviation of each statistic.(PDF)Click here for additional data file.S13 FigPopulation size varied between 60,000 and 6,000 individuals in the top panels, and between 6,000 and 600 individuals in the bottom panels. Population size changes occurred between 2,300 and 50 generations BP in the left panels, and between 34,000 and 900 generations BP in the right panels. All settings are similar to (PDF)Click here for additional data file.S14 Figa sudden (rather than continuous) decline from 40,000 to 300 individuals occurring 200 generations BP (top right), a sudden decline from 40,000 to 300 individuals occurring 1,000 generations BP (middle left), the same sudden decline followed by an expansion to 5,000 individuals occurring 580 generations BP (middle right) or an expansion to 1,000 individuals occurring 140 generations BP (bottom left), and a scenario similar to the continuous decline (top left) but including a sudden decline to 100 individuals between 230 and 140 generations BP, followed by an expansion to 1,000 individuals (bottom right). All settings are similar to (PDF)Click here for additional data file.S15 FigFor each scenario, the five PODs considered for MSMC estimation were the same as in (PDF)Click here for additional data file.S16 FigFor each scenario, the five PODs considered for MSMC estimation were the same as in (PDF)Click here for additional data file.S17 FigFor each scenario, the five PODs considered for MSMC estimation were the same as in (PDF)Click here for additional data file.S18 FigFor each scenario, the five PODs considered for MSMC estimation were the same as in (PDF)Click here for additional data file.S19 FigEstimation of past effective population size using MSMC with four haplotypes in the “small” scenario (top), the “decline” scenario (middle) and the “expansion” scenario (bottom). MSMC analyzes were run from perfectly phased data, phased data with 1 or 10 switch errors per Mb and diploid individual, or unphased data . All other settings are similar to (PDF)Click here for additional data file.S20 Figpolymorphic site AFS, i.e. without the overall proportion of SNPs (left), average gametic LD (middle) and average zygotic LD (right). These statistics were computed from 12 Holstein animals for which both NGS data and genotyping data were available, using only SNP positions from the 800K chip (even for the NGS data statistics). No MAF threshold was used.(PDF)Click here for additional data file.S21 FigError rates were computed from 12 Holstein animals for which both NGS data and genotyping data were available. False positive SNPs were positions that were found polymorphic in the NGS data but not in the 800K data. Their minor allele count in the NGS data was called the wrong minor allele count. False negative SNPs were positions that were found polymorphic in the 800K data but not in the NGS data. Their minor allele count in the 800K data was called the true minor allele count.(PDF)Click here for additional data file.S22 FigAll settings are similar to (PDF)Click here for additional data file.S23 FigHolstein (top left), Angus (top right), Fleckvieh (bottom left) and Jersey (bottom right). Parameter settings are the same as in (PDF)Click here for additional data file.S24 FigTen thousand genomic samples were simulated under population size histories that were sampled from the posterior distribution estimated in (PDF)Click here for additional data file.S25 FigObserved AFS (left) and LD (right) statistics are shown by green full circles. The average value of these statistics over the five best simulated data sets, i.e. the five simulated data sets leading to the smallest distance between observed and simulated statistics, are shown by blue crosses. The variation of these statistics over the five best simulated data sets is also indicated by blue dotted lines, which correspond to the average value plus (or minus) twice the standard deviation of each statistic.(PDF)Click here for additional data file.S26 Figr2 values, up to 1 if the topologies are exactly the same.LD statistics for several scenarios inplying a sudden expansion from 500 to 50,000 individuals (left) or a sudden decline from 50,000 to 500 individuals (right). Several expansion or decline times were considered, as well as two scenarios with a constant population size of 500 or 50,000 individuals (see the legend). For each scenario, LD statistics were averaged over 20 PODs including 25 diploid genomes and 100 2Mb-long regions. In contrast with expansion scenarios, some decline scenarios lead to even larger LD statistics than those obtained for a constant small population. Indeed, as these declines are very old compared to the expected TMRCA of a population of 500 individuals, their main effect is to increase, at some loci, the time during which the sample has only two ancestral lineages. Because this increase is very large , mutations occuring in this part of the coalescence tree eventually represent a large proportion of all oberved polymorphic sites. Besides, for two linked loci with similar topologies of the coalescence tree, mutations occuring in this part of the tree lead to very high (PDF)Click here for additional data file.S27 FigPrediction error for the estimated population size in each time window, focusing on scenarios with a population size below 1,000 (top left), above 10,000 (top right), below 1,000 in the last 200 generations and above 10,000 for times more ancient than 13,000 generations BP (bottom left) or above 10,000 in the last 200 generations and below 1,000 for times more ancient than 13,000 generations BP (bottom left). For the two latter scenarios, the time window where population size goes from above 10,000 to below 1,000 (or vice versa) is delimited by vertical dotted lines. For each scenario category, PE were evaluated from 2,000 random histories. Summary statistics considered in the ABC analysis were either the AFS statistics alone, the LD statistics alone or the AFS and LD statistics together (see the legend). All other settings are similar to (PDF)Click here for additional data file.S28 FigSummary statistics considered in the ABC analysis were either the AFS statistics alone (left column), the LD statistics alone (middle column), or the AFS and LD statistics together (right column). AFS statistics were computed using either all SNPs (top panels) or only those with a MAF above 20% (bottom panels). All other settings are similar to (PDF)Click here for additional data file.S29 Fign = 25 diploid individuals, using all SNPs for AFS and IBS statistics and SNPs with a MAF above 20% for LD statistics. Other parameter settings are the same as in Prediction error for the population size in each time window, evaluated from 2,000 random population size histories. Summary statistics considered in the ABC analysis included several combinations of (i) the AFS, (ii) the average zygotic LD for several distance bins and (iii) the distribution of IBS segment lengths within one diploid individual. These statistics were computed from (PDF)Click here for additional data file.S30 Fign = 25 animals. Summary statistics considered in the ABC analysis included different combinations of (i) the AFS and (ii) the average zygotic LD for several distance bins. These statistics were computed either from the SNPs that are included in the 800K SNP chip or from all SNPs found in the NGS data. A MAF threshold of 20% was used for all curves and statistics. Other parameter settings are the same as in Estimation of population size history in the Holstein cattle breed using ABC, based on whole genome NGS data from (PDF)Click here for additional data file.S31 Fign = 25 animals. Summary statistics considered in the ABC analysis were either both the AFS and the average zygotic LD for several distance bins, or the distribution of IBS segment lengths within one diploid individual. These statistics were computed using SNPs with a MAF above 20%. Other parameter settings are the same as in Estimation of population size history in the Holstein cattle breed using ABC, based on whole genome NGS data from (PDF)Click here for additional data file.


Mangroves are among the most diverse and productive coastal ecosystems in the tropical and subtropical regions. Environmental conditions particular to this biome make mangroves hotspots for microbial diversity, and the resident microbial communities play essential roles in maintenance of the ecosystem. Recently, there has been increasing interest to understand the composition and contribution of microorganisms in mangroves. In the present study, we have analyzed the diversity and distribution of archaea in the tropical mangrove sediments of Sundarbans using 16S rRNA gene amplicon sequencing. The extraction of DNA from sediment samples and the direct application of 16S rRNA gene amplicon sequencing resulted in approximately 142 Mb of data from three distinct mangrove areas . The taxonomic analysis revealed the dominance of phyla Euryarchaeota and Thaumarchaeota (Marine Group I) within our dataset. The distribution of different archaeal taxa and respective statistical analysis  revealed a clear community shift along the sampling stations. The sampling stations  with history of higher hydrocarbon/oil pollution showed different archaeal community pattern  compared to station (Dhulibhashani) with nearly pristine environment (dominated by methanogens). It is indicated that sediment archaeal community patterns were influenced by environmental conditions. Archaea, representing the third domain of life, were originally anticipated to thrive under extreme environments, such as hydrothermal vents, hot water springs, salt brines, and extremely acidic and anoxic environments, where they contribute significantly towards the maintenance of the biogeochemical cycles –7. HowevMangrove wetlands are typical example of mesophilic and moderately halophilic environmental niches. They are commonly situated at the intertidal zones along the tropical and subtropical coasts and play a very important role in shaping the coastal ecology . MangrovHeritiera fomes), goran (Ceriops decandra), geoa , keora , and so forth, and the world's famous endangered royal Bengal tiger (Panthera tigris tigris). Microbial communities play an important role in generation of detritus in mangrove areas and Sundarbans is one such ecotype where microbes have been shown to be involved in biogeochemical cycling of the nutrients [Sundarbans is the world's largest tidal halophytic mangrove ecosystem covering 20,400 square kilometers  of area and has been recognized as a UNESCO World Heritage site. Situated in the delta of Ganges, Meghna, and Brahmaputra rivers on the Bay of Bengal, Sundarbans is shared between India and Bangladesh. This mangrove ecosystem is the home for diverse flora and fauna, including mangrove plant species like sundari  authority of the state of West Bengal, India, has approved this experimental exercise.Samples were collected in triplicate from surface (2 cm) and subsurface (32 cm) sediments of the Sundarbans mangrove wetland, which is located on the northeastern coast of India . The samMicrobiological and biochemical analyses were performed with the field moist sediments. Physical and chemical analyses were carried out with air-dried sediment samples. The sediment pH was measured in 1 : 2.5 sediment-water suspensions and found to be alkaline. The total organic carbon (TOC) was measured by the methods described previously , 30. Bri in situ with Hach Portable Meters (HQ40d). Measured salinity was expressed in parts per thousand (ppt) or gm Kg−1 as described previously [Conductivity and salinity were measuredeviously . Nutrieneviously . BrieflyOrganic pollutants  were measured using a combined gas chromatography and mass spectrometry (GC/MS) method described previously , 34. Hean = 3) and aliquots of homogenized sediment of 0.5 g were subjected to DNA extraction using the MoBio PowerSoil DNA Isolation Kit . After the extraction, DNA from all three samples from each sampling station was pooled together (approximately 200 ng of DNA from each extraction), and the pooled DNA was concentrated in a speed vacuum centrifuge  to a final volume of 25 μL (approximately 24 ng μL−1). A NanoDrop  spectrophotometer was used to quantify the extracted pooled DNA and to measure other important parameters for DNA quality, such as the ratio of absorbance at 260/280 nm and 260/230 nm.For 454-pyrosequencing, each of the sediment samples from a station  contained 5 μL of 5-fold Phusion GC buffer , 200 μM of each of the four deoxynucleoside triphosphates, 1.5 mM MgCl2, 4 μM of each primer , 2.5% DMSO, 1 U of Phusion High Fidelity Hot Start DNA polymerase (Finnzymes), and 25 ng of pooled sediment DNA. The following thermal cycling scheme was used on a Veriti Thermal Cycler : initial denaturation at 98°C for 5 min, 25 cycles of denaturation at 98°C for 45 s, and annealing at 68°C for 45 s, followed by extension at 72°C for 30 s. The final extension was carried out at 72°C for 5 min. Negative controls were performed by using the reaction mixture without template. Primer sequences for amplification of the V3–V5 region [To analyze archaeal diversity, the V3–V5 region of archaeal 16S rRNA gene was amplified by PCR. The PCR reaction  and quantified by fluorometry using the Quant-iT PicoGreen dsDNA Assay Kit  according to the Roche 454 “Amplicon Library Preparation Method Manual” of the GS Junior Titanium Series . Pooled amplicons were diluted as recommended and amplified by emulsion PCR on a Thermal Cycler 9700 (Applied Biosystem) according to the Roche 454 “emPCR Amplification Method Manual Lib-L” .Pyrosequencing was performed for 200 cycles on a Roche 454 GS Junior sequencing instrument according to the manufacturer's protocol . All reads were filtered using the standard read rejecting filters of the GS Junior sequencer, namely, key pass filters, dot and mixed filters, signal intensity filters, and primer filters . Raw sequencing and processing data was carried out using a combination of mothur (software version 1.28.0) and Ribosomal Database Project (RDP-II). The raw data were subjected to initial quality trimming using the mothur software and all reads having an average quality value of <20 were discarded. Then we removed the number of chimeric sequences by using UCHIME algorithm. Then the chimeric free reads were further screened for the presence of ambiguous bases and any reads which have a length <200 were discarded and forward primer sequence was removed from the final dataset. The obtained processed reads were then demultiplexed to separate sample based on the 10 bp MID sequence and any reads which do not match the MID were discarded. The high quality reads were then aligned to archaeal 16S rRNA sequences and clustering was performed at 97% similarity; using the RDP pipeline, a representative sequence from each cluster was selected based on abundance using the sequence selection tool. The representative sequences obtained from each cluster were then classified using the naïve Bayesian Classifier  at a bootstrap confidence of 80.After quality trimming all the sequence reads were aligned against the SILVA, which is a curated ribosomal RNA sequence database using BLASTN , 37. TheAll six RMA files were normalized to the smallest data set size (without including reads classified as not having a taxonomic assignment) to allow intersample comparison of taxonomic abundances and to obtain comparative tree view for all samples. Comparative abundance of read counts at different levels of NCBI taxonomy  was exported from all sample comparison-file for later statistical analyses.Hierarchical cluster analyses have been performed using R 3.0.2 , with spComposition (presence/absence) data were also analyzed to compare the community structure. SIMPER analyses were used to determine which taxa/function contributed most to the observed differences. Further data were analyzed using common multivariate ordination techniques: Nonmetric Multidimensional Scaling (NMDS) using Bray Curtis distance. To understand the influence of physicochemical parameters in different sampling stations, Principal Component Analysis (PCA) was performed. Additionally, we have performed correspondence analyses on genomic data as well as on PAH (polyaromatic hydrocarbons) data.We have also plotted the genomic data using Voronoi diagram, which is a partitioning of a plane into regions based on distance to points in a specific subset of the plane. In this plot we have highlighted 6 major phyla.All 454-GS Junior sequence data from this study were submitted to the NCBI Sequence Read Archive (SRA) under accession numbers SRR1632262 , SRR1632263 , SRR1632258 (Bonnie camp_surface), SRR1632259 (Bonnie camp_subsurface), SRR1632260 (Dhulibhashani_surface), and SRR1632261 (Dhulibhashani_subsurface). in situ . Ammonia was found maximum in the sediment samples of Godkhali, while it was minimum in the sediment of Dhulibhashani (P < 0.05). Nitrite was found negligible in all the stations confirming very high rate of nitrification, resulting in nitrate formation, which was evenly detected in all three sampling stations (Table S1). Other parameters such as phosphate, sulphate, and silicate were found evenly distributed in all the sampling stations possibly due to regular inundation.Measurement of NOHeavy metals have been shown to play a pivotal anthropogenic role in the marine environment. Previously, it has been shown that heavy metals like Fe, Zn, Cu, Mn, Hg, Pb, Ni, Cr, and Cd are present in the Sundarbans estuaries –44. To a Halosarcina, Halorientalis, Halolamina, Halorhabdus, Halogranum, Haloferax, Halomarina, Halorussus, Haloplanus, and Halarchaeum, and of the class Methanomicrobia, for example, Methanosarcina, Methermicoccus, Methanocella, Methanococcoides, Methanosalsum, Methanolobus, and Methanogenium were detected  amplicons prepared from six sediment samples originated from three sampling stations at two different depths. After quality filtering, denoising, and removal of potential chimeric sequences of pyrosequencing dataset, this resulted in recovery of 1,41,816 sequences with an average length ranges between 500 and 510 bases (506 bp average) . Within detected . BesidesH′) index varied from 0.67 to 1.08 and the Simpson diversity (1 − λ′) index varied from 0.29 to 0.52 (J′) was between 0.23 and 0.37 and the Margalef (d) index was between 1.52 and 3.69 ; and simultaneously they also take the relative abundances of different species into account. We evaluated the archaeal diversity and evenness using the normalized dataset. Considering all the sampling sites, the Shannon-Weaver  were commonly shared between six samples  to compare the community composition using presence/absence data. NMDS employs an iterative algorithm that optimizes the position of samples into a low-dimensional space minimizing the difference between rank-order of multivariate ecological dissimilarity and distances in NMDS space . From thFurthermore, our PCA analysis revealed that the physicochemical parameters influence sampling stations differentially. The first PCA axis showed high positive correlations with heavy metals Zn, Pb, and Cu . SamplesFurther, we have performed correspondence analysis (CA) to compare the influence of abundant taxonomic groups and polyaromatic hydrocarbons (PAH) in these sampling sediments Figures . The corCorrespondence analysis, performed to understand the influence of PAHs in the sediments of the sampling stations, revealed that both the surface and subsurface sediments of Godkhali and Bonnie camp correlated with the detected PAH congeners . In contFinally, we have generated Voronoi plot , which d Methanosarcina, the relative abundance of which was higher in Bonnie camp. In general, SIMPER analysis targeting relative abundance of taxa in different stations revealed higher relative abundance of class Halobacteria in Godkhali and Bonnie camp, while class Methanomicrobia was found relatively more abundant in Dhulibhashani  analysis was performed  [ in situ growth chamber placed on a deep-sea hydrothermal vent on the Mid-Atlantic Ridge [ Halogranum genus. Besides, a significant number of sequences affiliated to Natronomonas, Haloferax, Halorhabdus, and Halolamina were identified. Methanomicrobia and Methanobacteria were the other two abundant euryarchaeal classes in the investigated samples. Methanomicrobia and Methanobacteria are known for their putative importance in sulfate reduction and methanogenesis in anoxic marine sediments, such as mangroves. The members of these classes are methanogens and are involved in carbon-cycle through methanogenesis.The present study focused on accessing the archaeal community structure and diversity in the world's largest tropical mangrove sediments of Sundarbans using 16S rRNA gene-based 454-amplicon sequencing. To our knowledge, this is the first study on understanding archaeal diversity in Sundarbans ecosystem. The majority of sequences obtained were affiliated to the Euryarchaeota. Previous studies by Sapp et al. on marine sediment and Wemheuer et al. on German Bight found high abundance of members of Euryarchaeota . We idenchaeota) . In our ic Ridge . BesidesBeside euryarchaeota, another archaeal group found in all samples was the Marine Group I (MG1) or Thaumarchaeota. Marine Group I (MGI) Thaumarchaeota are one of the most abundant and cosmopolitan chemoautotrophs and prokaryotic picoplankton within the global deep sea water. They were originally identified by sequencing of environmental 16S rRNA genes derived from sea water. All organisms of this lineage thus far identified are chemolithoautotrophic ammonia-oxidizers and may play important roles in biogeochemical cycles, such as the nitrogen cycle and the carbon cycle. Within our sequence dataset, the number of ammonia oxidizing Thaumarchaeota was identified.We observed an increased abundance of Halobacteriaceae in the investigated subsurface samples from Godkhali and Bonnie camp. In contrary, the number of Methanosarcinaceae and Methanobacteriaceae was higher in the samples derived from Dhulibhashani. This might be correlated with the high amounts of organic matter in resident algal blooms in Godkhali and Bonnie camp Figures . FurtherIn summary, the spatial variations of the sedimentary archaeal diversity have been studied in the Sundarbans mangrove ecosystem for the first time by means of 16S rRNA 454-pyrosequencing. We found highly diverse archaeal communities in the sediment of Sundarbans. Our analyses have confirmed the influence of environment in shaping the archaeal community diversity within the sediment. However, due to the lack of pure cultures and large comparative investigations, robust conclusions related to the involvement of identified archaeal communities in biogeochemical cycle cannot be drawn. Further research including analyses of expression of functional genes and determination of the active archaeal population within the sediment might unravel the role of archaea in Sundarbans mangrove ecosystem.Supplementary Figure 1: A phylogenetic tree was constructed with the assigned archaeal 16S rRNA sequences found in this study using MEGAN5 metagenome analyzer. The Euryarchaeota and Thaumarchaeota were the dominant representative phyla present within our dataset. In the Euryarchaeota phylum, Thermoplasmatales and Halobacteriaceae grouped the majority of the representative sequences of different samples.Supplementary Table 1: Primers used for amplification of the V3–V5 region of the archaeal 16S rRNA.Supplementary Table 2: Assessment of physico-chemical parameters, heavy metals and polyaromatic hydrocarbons (PAH) in Sundarbans sediments.Supplementary Table 3: Comparative percent detection of Euryarchaeota and Thaumarchaeota in Sundarbans.


Glossostemon bruguieri) as an alternative reversal therapy for JO. Phytochemical screening analysis showed that moghat contains 5.8% alkaloids, 1.5% flavonoids and 13.2% total phenols. Juvenile osteopenia was induced in 15 days old Sprague- Dawley female rats by feeding them free Ca and vitamin D synthetic diet for 21 days. Osteopenic rats were either treated with moghat , or with a reference nutritional supplements of calcium chloride (14 mg Ca/Kg) and vitamin D3 (7 IU/Kg), for extra 21 days. Both untreated and treated groups were compared to a control group that fed a regular pelleted food. Our results showed that osteopenic rats lost normal bone tissue architecture, 30 % of body mass, 54 % of bone mass and finally 93% of bone calcium mass. Furthermore, these rats showed a markedly increase in serum phosphate, PTH, alkaline phosphatase, aspartate transaminase activities and creatinine level as compared to the control group. Moghat administration was successfully reversed osteopenia by normalizing body and bone masses to the reference ranges, increased the bone calcium mass by 17 fold without any detectable side effects on liver and kidney physiological performance. Therefore, moghat could be considered as potent safe –JO- reversal extract. This study aimed to explore and validate a new juvenile osteopenic (JO) rat model then examine the efficacy of moghat ( Osteopenia is a skeletal disorder characterized by low bone mass and micro-architectural deterioration of bone tissue with a consequent increase in bone fragility and susceptibility to fracture risk. Osteopenia in children is rare and mostly is a secondary concomitant to conditions such as prolonged immobilization, malabsorption syndromes, corticosteroid excess, osteogenesis imperfecta, Celiac disease, Turner’s syndrome, malignancy and homocystinuria. After the exclusion of decreased bone mass causes, one form of osteopenia remains over which is idiopathic juvenile osteopenia (IJO) that was firstly described by Schippers in 1938.nd and 13th year of the life, however very young patients who are 9 months, 1.5 years and 2.0 years of age with IJO have also been reported  initial manifestation. Furthermore, such disease progression could be spontaneously arrested during or even after puberty. Only few adult patients with severe conditions of marked skeletal deformities may remain disabled  and density (BMD) where the physical activity level, the amount of meal calcium and other food ingredients are accounting as important factors that influence the development of peak bone mass and the rate at which bone is lost later in life . Diet den adults . Sodium n adults .Despite protein is very important ingredient for human diet, high protein intake, especially those rich in animal protein, is reported as a determinant factor for urinary calcium excretion and thus it is inadvisable for long-term bone health .The challenge that faced the scientists who worked in JO area is how to produce a valid animal model to track this disease, which in turn, give a chance to examine and develop new preventive and curative strategies. So the first motivation in this study was to establish a new JO animal model by using a simulator human unhealthy –imbalanced- poverty food ingredient. This diet of imbalanced calcium- phosphorus ratio contributed high sodium level and calcium deficient protein, egg albumin instead of casein. These ingredients when collected in one diet accounted as insurance to osteopenia incidence.etc. But these therapeutic managements possess several down-sides and available evidence suggests that long-term use of estrogen replacement therapy as example may have serious side effects such as breast or uterus cancer . The plant grows wild in Iraq and Iran from where it was imported. It was introduced to Egypt in 1932 and continued to be cultivated in very small area . In 2003, Meselhy  isolatedIsoflavone is naturally occurring substance such as genistein (isoflavone isolated from soybeans) that has estrogenic-like activity, which binds to osteoblastic estrogen receptors and plays an unique role in bone mass maintenance in ovariectomized rats . TherefoAs moghat contains phytoestrogen; such as isoflavone (biflavone), so it could be used as an osteopenia preventer. This study was aimed to explore and validate a new juvenile osteopenic (JO) rat model then examine the efficacy of Moghat as alternative therapy to reverse JO in this model in comparison to reference nutritional calcium and vitamin D supplement, the first therapeutic regimen used in JO treatment. Indeed the use of this reference drug gave the chance to deduce if the treatment with calcium and vitamin D combination reversed the detriment effect of synthetic imbalanced diet on bone tissue. MaterialsGlossostemon bruguieri) was purchased from Egyptian local market and authenticated by Prof. Dr. Eldareir, S. Prof. of plant ecology, Faculty of Science, University of Alexandria, Egypt. Calcium chloride and Vitamin D3 were purchased from Sigma Chemical Company . All other vitamins were bought from RP Scherer, Cairo, Egypt. Commercial kits for AST, ALT, ALP, creatinine and urea were bought from Randox Company (France). All other chemicals and reagents of highest quality were available commercially. Moghat . The animals were examined at every 30 min up to a period of 3 h and then occasionally for additional 4 h period, finally 24 h mortality was recorded. For sub-chronic toxicity or extract safety determination, four mice group (n=10) was orally administrated with 0.2 mL of aqueous suspension of moghat at concentration of  for one month. Liver and kidney function were determined in mice sera and comparing with control group that received 0.2 mL water.The acute and chronic toxicity study was performed as per the method described by Litchfield and Wilcoxon (1949), and LDThe antiosteoporotic activity was performed on female Sprague–Dawley rats at dose of 800 mg/Kg of body weight.Level of blood estrogen as a function of ageoC for estrogen analysis. Fourteen groups (6 rats/ group) of normal female rats aged from 7 to 98 days old (the age difference between groups was 7 days old) were fasted overnight, decapitated, then their sera were collected and stored at -20 Free calcium and vitamin D – synthetic – diet was prepared according to Ghareeb et al., , which were fed a regular pelleted diet and allowed for free access to tap water. JO was induced by feeding forty rats (15 days old) the free calcium and vitamin D synthetic diet for 21 days (100 g/Kg). After this induction period, ten rats were scarified (induced-untreated 21) and the other 30 rats were divided into three subgroups, all subgroups were kept on the same synthetic diet, as follow; 1) 10 rats were orally administrated 0.5 mL distilled water (induced- untreated 42), 2) another 10 rats were orally treated with a 0.5 mL of water suspension of powdered moghat at dose of 0.8 g/Kg, 3) and the last 10 rats were orally administrated with 0.5 mL reference nutritional supplement which containing 0.75 mM CaCloC for further biochemical analyses.At the end of experiment period, rats in all groups were fasted overnight and then decapitated. Blood was collected and sera were separated off to estimate estrogen level, parathyroid hormone (PTH), calcium and phosphate levels. Furthermore, the two ilia bones from each rat were isolated then one of them was fixed in10 % formalin for histological studies, while the other one and the collected sera were kept at -20 Serum calcium concentrationDiluted serum sample was introduced to an atomic absorption spectroscopy  flame where the calcium in its energized form absorbed radiation at 423 m .Bone calcium amountoC for 2 h. The dried powdered bones were mixed with acid solution , covered and stored at 37 ᵒC for one week. The samples were diluted with deionized water and centrifuged at 3000 rpm for 15 min. The precipitate was ashen for 2 h at 500 ᵒC and the amount of bone calcium was then calculated as follows / weight of the same iliac bone)*100Serum inorganic phosphate concentration2SO4, 0.36 M and NaCl, 154 mM) were added to 40 µL serum. Then 400 µL of phosphorus reagent  were added and incubated for five minutes at 37 ºC. The absorbances of standard and test were measured at 340 nm against blank  was carried out according to manufacturer's instruction . Serum estrogen and PTH concentrationsThe estrogen and PTH levels was estimated by the enzyme- linked- immunosorbent assay (ELISA) according to manufacturer's guidelines   and 23)23). s.Serum ALT , AST 2424, Urea Histological techniquesStaining of bone cells2SO4. Bones were dehydrated in 95% ethanol then embedded in paraffin. Three 5-mm-thick paraffin-embedded horizontal bone sections were cut from the proximal end of the diaphysis, stained with haematoxylin–eosin and examined by light microscopy  using Primer of Biostatistics (Version 5) software program. Significance of means ± SD was detected groups by the multiple comparisons Student-Newman-keuls test at p < 0.05. Glossostemon bruguieri contained 5.8 % alkaloids, 1.5% flavonoids, 13.5% phenols and 4.5 pg/g steroid compounds. Furthermore, each one gram of moghat contained 6.2 mg calcium ions.The phytochemical characters of moghat are summarized in 50 which was higher than 10 g/Kg. In the other side, sub-chronic toxicity is the gate to assess the extract safety, our data considered the moghat safety mentioned in In order to determine the safe medicinal dose of aqueous moghat suspension, acute and sub-chronic toxicity were preformed. Acute toxicity study gives indication about LDWithin the row, means with different letters (a or b) were significantly different at p < 0.05. Mean with letter (a) was significantly the lowest value while mean with the letter (b) was significantly the highest value. If two or three groups have the same letters that means there is no significant difference detected at p < 0.05.Serum estrogen level was measured as function of age to determine the puberty time for female rats. Free Ca and vitamin D synthetic diet was successfully induced alteration in bone mineralization and body parameters where the body, bone and bone calcium weights were decreased by 59, 55 and 94%, respectively after 21 days induction period and by 35, 37 and 83.6%, respectively after 42 days induction period than that of control groups at p < 0.05. Furthermore, the percentage of bone calcium/bone weight was decreased by 84 and 76% after 21 and 42 days of synthetic diet intake than the normal control ratio. Moreover, diet intake for 21 days decreased serum calcium level by 35 % that was combined with a moderate elevation in serum phosphate level by 21 % and extensive elevation in serum PTH by 266 % than those of control group. On the other hand, 42 days diet intake reduced the serum calcium by 31% and elevated both serum phosphate and PTH levels by 32 and 339 %, respectively than those of the control one, at p < 0.05 .The histological studies  shows thDiet intake lead to sever alteration on liver and kidney functions parameters as shown in Treatment with moghat suspension at a dose of 0.8 g/k/day successfully alleviated the body mass, ilia bone and bone calcium weights to normal control value, at p < 0.05 . FurtherThese results were confirmed by the histological studies  which shThe same biochemical  and histThe estrogen levels of JO-induced, moghat treated and reference nutritional supplement treated groups were similar to control estrogen level, at p < 0.05 .Urea level was significantly increased in rat received moghat treatment by 40%, while creatinine level was decreased by 16% in rat received reference nutritional supplement than those of the induced untreated 42 levels at p < 0.05 . UnfortuBoth treatment regiments successfully normalized liver function parameters where they decreased the AST, ALT and ALP levels by 81, 41 and 81 % in moghat regiment and 91, 47 and 50 % in reference nutritional supplement regiment, respectively than those showed in the induced untreated 42 group, at p < 0.05, Within the row, means with different letters  were significantly different at p<0.05. Mean with letter (a) was significantly the lowest value while mean with the letter (e) was significantly the highest value. Letters b within the row is significantly different from a and c and so on. If two or three groups have the same letters that means there is no significant difference detected at p < 0.05.The World Health Organization (WHO) estimates that herbal medicine care is about 75-80% of the world's population, mainly in the developing countries, because of better cultural acceptability, better compatibility with the human body and fewer side effects .In Egypt, moghat root powder is used as a tonic, nutritive and gout curative agent due to its highly mucilage content, furthermore it is prescribed as a demulcent agent . SeveralPlants derived phenolic compounds manifested beneficial effects and potential inhibition of several stages carcinogenesis and several serious diseases . FurtherBased on our phytochemical results, we speculated that moghat could be used as vital nutritional and medicinal agents due to the presence of estrogenic like compounds, alkaloid and flavoind. Furthermore, as it contains isoflavonid and sesemin, it could be a good nutritional reversal therapy for osteopenia. Osteopenia or bone mass loss is due to increased bone resorption and decreased bone formation . The two obstacles in this study were the lack of an animal model to study JO and the natural reversal therapeutic effect of ovarian estrogen during the onset of puberty. Unfortunately, the successful reported procedures to induce osteoporosis are ovariectomization and corticosteroids administration  that pro3 and calcium deficiency shall lead to rickets and bone mass loss, respectively , body mass loss, bone and bone calcium masses reduction as shown in Body mass loss could be attributed to the loss in the appetite which resulted from low calcium intake . Low calThe treatment with reference nutritional supplement or moghat was successfully returning the body and bone masses back to the control values, furthermore, no side effects on liver was detected. So, the treatment seemed sufficient to treat bone loss.3 normalized the ALP activity, serum levels of calcium, phosphate and PTH and increased calcium mass by 1.5 fold, but did not return it back to the control value. It was reported that treatment with calcium and vitamin D was able to ameliorate bone loss but unable to normalize the bone density  our work.


Environmental enrichment is usually applied immediately after weaning or in adulthood, with strong effects on CNS anatomy and behavior. To examine the hypothesis that a pre-reproductive environmental enrichment of females could affect the motor development of their offspring, female rats were reared in an enriched environment from weaning to sexual maturity, while other female rats used as controls were reared under standard conditions. Following mating with standard-reared males, all females were housed individually. To evaluate the eventual transgenerational influence of positive pre-reproductive maternal experiences, postural and motor development of male pups was analyzed from birth to weaning. Moreover, expression of Brain Derived Neurotrophic Factor and Nerve Growth Factor in different brain regions was evaluated at birth and weaning. Pre-reproductive environmental enrichment of females affected the offspring motor development, as indicated by the earlier acquisition of complex motor abilities displayed by the pups of enriched females. The earlier acquisition of motor abilities was associated with enhanced neurotrophin levels in striatum and cerebellum. In conclusion, maternal positive experiences were transgenerationally transmitted, and influenced offspring phenotype at both behavioral and biochemical levels. The development of mammalian brain is subjected to complex genetic and environmental influences. Specific environmental effects result from motor, cognitive, emotional, and social stimuli the individuals encounter. Enhancement of environmental stimulations, physical exercise and social interactions as occurs through the exposure to an enriched environment (EE) causes dramatic brain changes. Functional , macro-structural , micro-structural , and molecular  modifications have been described following EE exposure  and the Nerve Growth Factor (NGF), influence brain development, functional plasticity , as well as plastic mechanisms involved in learning and memory, and in responses to stress or injury Dreyfus, . The expWith regard to behavioral modification, parental manipulations either negative, as food restriction, benzodioxin exposure or maternal stress, or positive, as environmental enrichment, alter the maturation of reflexes and motor coordination as well as the cognitive and emotional offspring development  and on the pup . To this aim, female rats were reared in an EE from weaning to sexual maturity, then housed in standard condition from mating with standard-reared males onwards. In offspring, postural, locomotor and behavioral maturation was analyzed from the first postnatal day (pnd 1) to the weaning (pnd 25). Examining the maturation of motor behaviors has allowed a proper assessment of the influence of pre-reproductive maternal enrichment on the developmental motor sequences of offspring  were reared in groups of ten in a large cage (100 × 50 × 80 cm) with an extra level constructed of galvanized wire mesh and connected by ramps to create two interconnected levels, following the enrichment conditions previously described  were pair-housed in a standard cage containing wood shavings and a red plastic tube. Feeding boxes and water bottles were kept in the same position. SF received the usual care by the animal house staff without prolonged manipulations. This procedure avoided an impoverished rearing and also SF were accustomed to the human contact.ad libitum. All efforts were made to minimize animal suffering and reduce the number of animals that were used, per the European Directive (2010/63/EU). All procedures were approved by the Italian Ministry of Health.A 12/12 h dark/light cycle (light on between 07:00 and 19:00 h) was applied to both enriched and standard conditions. Food and water were provided Before mating all females were weighted. For mating, from pnd 80 to pnd 85 (six days), each EF and SF in oestrus stage was caged with a standard-reared male rat (≈300 g). Afterwards, the males were removed, and the females were maintained in the standard home cages (40 × 26 × 18 cm) throughout pregnancy, delivery and until offspring's weaning, at pnd 25.The day of delivery (pnd 0), litter size and sex ratio were noted. Then, culling was quickly performed by reducing the litters to six males and two females. The litters with less than six males were not used.Depending on the maternal rearing conditions, two groups of pups were obtained: “enriched” group (EG), encompassing male pups of enriched females; standard group (SG), encompassing male pups of standard females. Note that the difference in rearing conditions concerned the mothers in their pre-reproductive phase and not the pups that were all reared in standard conditions.For neurotrophin determination at pnd 0, 5 culled male pups from each group (2–3 pups/litter from two litters) were sacrificed.For behavioral analyses, 24 male pups of each group  were used. At the end of behavioral evaluation, 5 male pups from each group were sacrificed for neurotrophin determination.From pnd 1 to pnd 25, EG and SG pups were separated daily from the dams one by one for a maximum of 20 min. They were tested for their postural, locomotor, and complex behaviors in a warmed environment (30–32°C) between 9 AM and 3 PM ; reflex appearance ; development of quadrupedal stance (head and shoulder elevation) and locomotion ; development of complex motor behaviors . Swimming performance (direction and limb use) were also evaluated.To supplement direct behavioral observations, videos were recorded throughout the entire testing cycle. To avoid any eventual order effect, the testing sequence was in random order for each pup on each measure. Researchers unaware of the individual pup group assignment attributed the dominant behavior in each observation period to a specific category. Categorization was considered reliable only when judgments were consistent (inter-rate reliability > 0.9).Body weight was measured daily until pnd 25. Eye opening, fur appearance, and incisor eruption were evaluated by visual inspection.Physical development: Reflex appearance. Cliff avoidance: the pup was placed on an edge with forepaws and nose just over the edge. The time to retract itself by means of backward and/or sideward movement was recorded. The max allotted time was 60 s.—Negative geotaxis: the rat was placed on an inclined plane (25°) with its head pointing downwards. The time to turn to face up the slope was recorded. The max allotted time was 60 s.—Vestibular drop: the animal was suspended by its tail and it raised its head to tail level by arching its body sideways. The time to arch its body left and right to tail level was recorded. For all these reflexes, the pnd of appearance was noted.Development of quadrupedal stance and locomotion. Each animal was placed on a board and video-recorded for 180 s to analyze quadrupedal stance (head and shoulder elevation times) and locomotion categories  by using Ethovision XT  from pnd 3 to pnd 14. Pivoting consisted of turning movements by broad swipes with the forepaws, body weight supported by only one hindlimb used as a pivot, and pelvis anchored to the ground. Crawling consisted of dragging themselves forward or pushing backward by undulating movements of the trunk, hindlimbs were often dragged in extended position with soles of the feet facing upward. Quadrupedal locomotion consisted of fluent and swift forward movements, with all limbs supporting the whole body and the pelvis elevated. The appearance (or disappearance) of locomotion categories was scored as the prevalent behavior according to the rating scale described in Table Development of complex motor behavior. Ascending a ladder: the rat was placed on a steel ladder  at an angle of 25° with its top in contact with a platform, containing a female littermate of the tested animal. The max allotted time was 180 s. The day of the successful arrival on the platform was recorded.—Crossing a narrow bridge: the pup was placed on the start platform connected by a plywood bridge (60 × 1 × 3 cm) to a goal platform containing a female littermate of the tested animal. The ability to traverse the bridge within the allotted 180 s was evaluated and the day of the first successful crossing was recorded.—Suspension on a wire: the rat was suspended by the forepaws on a wire (3 mm thin and 100 cm long) extended horizontally between two poles (50 cm high). The max allotted time was 180 s. Suspension time was recorded.—Swimming performance: the rat was immersed in a glass tank (100 × 50 × 20 cm) filled with 35°C water. Water level was chosen to avoid contact with the tank bottom during swimming. The animal was gently released by hand in the water and allowed to swim freely about the tank. Two swimming parameters (swimming direction and limb use) were evaluated and scored according to the rating scale described in Table The animals were decapitated and the brains were quickly removed and dissected on ice using a binocular dissection microscope. The following brain regions were collected according to Glowinski and Iversen's method (1966): cortex and hippocampus (pnd 0); frontal cortex, hippocampus, striatum and cerebellum (pnd 25). Striatal and cerebellar neurotrophin expression was not analyzed at pnd 0, because of the very weak BDNF and NGF expression in these extremely immature areas in the very early postnatal days, as previously described . In brief, 96-well immunoplates (NUNC) were coated with 50 μ l/well with the corresponding capture antibody which binds the neurotrophin of interest, and stored overnight at 4°C. The next day serial dilutions of known amounts of BDNF and NGF ranging from 0 to 500 pg/ml were performed in duplicate to generate a standard curve. Then the plates were washed three times with wash buffer and the standard curves and supernatants of brain tissue homogenates were incubated in the coated wells (100 μ l each) for 2 h at room temperature (RT) with shaking. After additional washes, the antigen was incubated with second specific antibody for 2 h at RT (BDNF) or overnight at 4°C (NGF), as specified in the protocol. The plates were washed again with wash buffer and then incubated with an anti-IgY HRP for 1 h at RT. After another wash, the plates were incubated with a TMB/Peroxidase substrate solution for 15 min and phosphoric acid 1M (100 μ l/well) was added to the wells. The colorimetric reaction product was measured at 450 nm using a microplate reader . Neurotrophin concentrations were determined from the regression line for the neurotrophin standard (ranging from 7.8 to 500 pg/ml-purified mouse BDNF or NGF) incubated under similar conditions in each assay. Cross-reactivity with other related neurotrophic factors, for example, NT-3 and NT-4 was less than 3%. Neurotrophin concentration was expressed as pg/g wet weight and all assays were performed in triplicate.Statistical analyses were performed by using STATISTICA 8 (StatSoft). The data expressed as mean ± s.e.m. were first tested for normality (Wilk-Shapiro's test) and homoscedasticity (Levene's test), and then analyzed by one-way or two-way ANOVAs for independent (group) and repeated (pnd) measures followed by Tukey's HSD test.U test.For locomotion and swimming performance, the comparisons between groups were performed by means of the Mann-Whitney To control for the alpha inflation the proportion of type I errors among all rejected null hypotheses, the False Discovery Rate (FDR) was set to 0.05. The FDR was estimated through the procedure described by Storey and Tibshirani . The booF = 1.77, p = 0.19. EF: x 51.94 ± 2.10 g; SF: x 57.16 ± 3.31 g], while at end of the EE exposure (pnd 72), EF weighted significantly less than SF .Enriched Females and SF showed similar body weights at weaning  or the male/female ratio .Pre-reproductive maternal enrichment did not affect either the litter size , without a significant group effect  and interaction   performed on body weight of EG and SG pups, all pups increased their weight with days  Figure .F = 2.09; p = 0.04], incisors at pnd 7 , and opened their eyes at pnd 14 , with no differences between groups  = 0.002; p = 0.96], negative geotaxis at pnd 6 , and cliff avoidance at pnd 7   = 0.77, p = 0.39; pnd effect F = 705.59, p < 0.00001; interaction F = 1.09, p = 0.16. Shoulder elevation time: group effect F = 2.85, p = 0.10; pnd effect F = 1508.42, p < 0.00001; interaction F = 1.94, p = 0.03]   and crossed the narrow bridge from pnd 15 , while SG pups acquired these behaviors at pnd 13 and pnd 17, respectively  = 9.49, p < 0.001]. Moreover, with days EG and SG pups increased the suspension time . The interaction was not significant  . At pnd 3 most EG pups already swam in circles, while most SG pups still floated . At pnd 13, most EG pups swam in a straight line, while most SG pups still swam in circles  . At pnd 21 most EG pups already swam moving only the hindlimbs, holding the forelimbs stationary (adult pattern of swimming), while most SG still exhibited swimming movements with all four limbs   = 1.87; p = 0.21; NGF: F = 1.82; p = 0.21] and hippocampal  regions  = 16.14; p < 0.01] and cerebellum  significantly higher than SG pups. EG pups also showed higher cerebellar NGF levels  than SG pups = 0.89; p = 0.37] and hippocampus . Similarly, no significant differences between groups were found in NGF levels in the frontal cortex , hippocampus , and striatum  . These abilities require proper sequencing and coordination of motor output, and exteroceptive and motivational cues. They depend on hierarchically related cortico-subcortical systems where striatum and cerebellum play crucial roles. Interestingly, the maturation of these structures lasts the first two post-natal weeks, at the end of which complex motor acts begin to be correctly executed (Gramsbergen, Interestingly, the accelerated acquisition of complex behavioral performance exhibited by EG pups was associated with increased expression of BDNF and NGF in cerebellum and striatum. However, it is hard to determine whether the biochemical differences at cerebellar and striatal level caused the differences in motor behavior or whether the different motor behavior caused the biochemical differences.The first hypothesis is based on the possibility that the increased neurotrophin expression in cerebellum and striatum allowed developing motor skills, by promoting neuronal growth and formation of synapses and connections. Accordingly, transgenic mice over-expressing BDNF display an accelerated maturation of granule cells (Bao et al., On the other hand, it is not possible to neglect the opposite hypothesis on the inverse relationship between complex motor performances and neurotrophin expression in cerebellum and striatum. In fact, the early acquired complex motor abilities may have caused an increase in neurotrophin levels as a consequence of increased neuronal activity just in the structures mostly involved in the complex motor behaviors. In accordance with this hypothesis, it has been previously demonstrated that animals reared in an EE that allows the acquisition of improved complex motor behaviors show activity-dependent enhancement of BDNF and NGF levels in cerebellar and striatal areas (Angelucci et al., Unexpectedly, the pre-reproductive maternal rearing conditions did not influence neurotrophin levels in neocortical and hippocampal regions, despite the high sensitivity of these regions to parental experiences (Arai et al., Knowing the mechanisms by which environment-gene interplay is achieved allows clarifying the dynamic nature of gene regulation and biological link between experiences of an organism and individual differences in neurodevelopment and behavior (Franklin and Mansuy, Epigenetic marks on chromatin can be inherited through maternal and paternal germ cells. Even if DNA methylation sites are generally erased during gonadal sex determination in embryogenesis, some of them survive and are integrated in the gametes persisting until the embryos become adult. In the present experimental model in which environmental manipulation was applied to mothers and not to fathers, epigenetic transgenerational effects occurred only through maternal germ-line, excluding any paternal influence.in utero and after the birth by lactation.Heritable epigenetic changes occur also through factors passed from mother to offspring via the placenta or milk, such as hormones, antibodies, antioxidants (Ho and Burggren, Transgenerational epigenetic changes occur also through altered maternal nurturing. In fact, mother-infant interactions during the early life have a profound impact on offspring phenotype, as indicated by studies demonstrating that natural variations in the quality or quantity of maternal care leave long-lasting epigenetic marks on offspring molecular factors crucial for plasticity, such as BDNF and NMDA receptors (Weaver et al., Overall, the present results demonstrating that pre-reproductive maternal environmental enrichment shapes the offspring phenotype indicate that females that experience a particular environment transgenerationally prepare their progeny to cope with that environment. Intriguingly, the transmission of maternal experience could have broad implications for progeny, improving their adaptive competencies and sculpting their behaviors (Petrosini et al., All authors designed research; Paola Caporali, Debora Cutuli, Daniela Laricchiuta, Francesca Foti and Laura Mancini performed behavioral evaluation; Francesca Gelfo, Francesco Angelucci, and Paola De Bartolo performed biochemical analyses; all authors analyzed data and discussed data; Laura Petrosini, Paola Caporali, Francesca Gelfo wrote the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


West Nile virus (WNV) has a wide geographical distribution and has been associated to cause neurological disease in humans and horses. Mosquitoes are the traditional vectors for WNV; however, the virus has also been isolated from tick species in North Africa and Europe which could be a means of introduction and spread of the virus over long distances through migratory birds. Although WNV has been isolated in mosquitoes in Kenya, paucity of genetic and pathogenicity data exists. We previously reported the isolation of WNV from ticks collected from livestock and wildlife in Ijara District of Kenya, a hotspot for arbovirus activity. Here we report the full genome sequence and phylogenetic investigation of their origin and relation to strains from other regions.A total of 10,488 ticks were sampled from animal hosts, classified to species and processed in pools of up to eight ticks per pool. Virus screening was performed by cell culture, RT-PCR and sequencing. Phylogenetic analysis was carried out to determine the evolutionary relationships of our isolate.Rhipicephalus pulchellus sampled from cattle, sequenced and submitted to GenBank (Accession number: KC243146). Comparative analysis with 27 different strains revealed that our isolate belongs to lineage 1 and clustered relatively closely to isolates from North Africa and Europe, Russia and the United States. Overall, Bayesian analysis based on nucleotide sequences showed that lineage 1 strains including the Kenyan strain had diverged 200 years ago from lineage 2 strains of southern Africa. Ijara strain collected from a tick sampled on livestock was closest to another Kenyan strain and had diverged 20 years ago from strains detected in Morocco and Europe and 30 years ago from strains identified in the USA.Among other viruses, WNV was isolated from a pool of R. pulchellus. The epidemiological role of this tick in WNV transmission and dissemination remains equivocal but presents tick verses mosquito virus transmission has been neglected. Genetic data of this strain suggest that lineage 1 strains from Africa could be dispersed through tick vectors by wild migratory birds to Europe and beyond.To our knowledge, this is the first characterized WNV strain isolated from The online version of this article (doi:10.1186/s13071-014-0542-2) contains supplementary material, which is available to authorized users. Flaviviridae, genus Flavivirus which is closely related to viruses such as Japanese encephalitis, Saint Louis encephalitis, Usutu, Kunjin, Kokobera, Stratford, Alfuy and Murray Valley encephalitis that belong to the Japanese encephalitis serocomplex [West Nile virus (WNV) is classified in the family ocomplex . WNV hasocomplex -4. Recenocomplex -10.Culex  [Argas persicus, A. hermanni [Ornithodoros moubata [WNV is known to be transmitted primarily by mosquitoes of the genus pipiens) -14 althopipiens) ,16. Vecthermanni  and Orni moubata  indicate moubata . The abiWNV causes mainly mild febrile illness but can result in meningo-encephalitis, acute paralysis and death in severe cases in humans and horses ,18-22. IWNV genome is composed of an 11 kb positive RNA fragment that is translated to form a polyprotein. This polyprotein consists of three structural  and seven non-structural proteins  . PhylogeRh. pulchellus ticks sampled from cattle and warthog has been documented in Kenya [Previous studies indicate that WNV circulates in Kenya, having been isolated from diverse mosquito species -33. Evidin Kenya , althougThis study aimed to investigate circulation, transmission and diversity of WNV in Ijara District and test the hypothesis of its spread through migratory birds and tick vectors across the continent. This study will also aid in assessing the risk of disease, monitoring emerging tick-borne viral diseases and providing a better understanding of the patterns and processes of evolution.This was a field-based descriptive cross-sectional and laboratory-based study conducted between 2010 and 2012.icipe), the hosting institution for the Arbovirus Incidence and Diversity (AVID) Project Consortium.This study was approved by the Kenya Medical Research Institute Ethical Review Committee protocol number SSC 2050. The Committee of the Department of Veterinary and Capture Services (DVCS) of the Kenya Wildlife Service (KWS) approved the study including animal immobilization capturing protocols. KWS guidelines on Wildlife Veterinary Practice-2006 were followed. All KWS veterinarians were guided by the Veterinary Surgeons Act Cap 366 Laws of Kenya that regulates veterinary practices in Kenya. For purposes of livestock use, permission was obtained from the owners involved. We worked in collaboration with the department of veterinary services and veterinarians mandated by the government to do livestock sampling and research. The above terms were stipulated well in an agreement between the farmers and the International Centre of Insect Physiology and Ecology  arid and semi-arid region where 90% of the people practice nomadic pastoralism, keeping indigenous cattle, goats, sheep, donkeys and camels. Approximately one-quarter of the district is covered by the Boni forest bordering the Indian ocean, which is an indigenous open canopy forest that forms part of the Northern Zanzibar-Inhamdare coastal forest mosaic. A section of the forest, the Boni National Reserve is under the management of the Kenya Wildlife Service as a protected conservation area and is home to a range of wildlife species, including hirola antelope , reticulated giraffe, elephant, buffalo, lion, leopard, cheetah, African wild dog, lesser kudu, desert warthog and bushbuck.Ticks sampling from both domestic animals and wildlife was undertaken at various sites of the Ijara District, including the Boni National Game Reserve as described previously in . QualifiμL/mL amphotericin B). Tick identification was performed using appropriate identification keys. The ticks were transferred to sterile vials and stored at -80°C until processed for virus isolation. Ticks were later thawed in ice (4°C), identified and pooled into groups of one to eight (depending on size) by species, sex, and animal host. Each pool was homogenized using 90-mesh alundum in a prechilled, sterile mortar and pestle with 1.6–2 mL ice-cold MEM containing 15% fetal bovine serum (FBS), 2% glutamine, 100U/mL penicillin, 100 lg/mL streptomycin, and 1 μL/mL amphotericin B. The homogenates were clarified by low-speed centrifugation at 1500 rpm for 15 min at 4°C, and supernatants aliquoted and stored at -80°C. In the case of Hyalomma spp., the primary vectors of Crimean–Congo hemorrhagic fever virus (CCHFV), each pool was prescreened for CCHF by reverse transcription-polymerase chain reaction (RT-PCR) to exclude this virus prior to cell culture screening.Tick identification and processing was performed as described previously in . The tic2 cell culture flasks to 80% confluency in MEM containing 10% FBS, 2% glutamine, 100U/mL penicillin, 100 lg/mL streptomycin, and 1 μ/mL amphotericin B. The cells were then rinsed with sterile phosphate-buffered saline (PBS) and 0.2 mL of clarified tick homogenate was added followed by incubation at 37°C for 45 min to allow virus adsorption. After incubation, MEM supplemented with 2% FBS, 2% glutamine, 100 U/mL penicillin, 100 lg/mL streptomycin, and 1 μ/mL amphotericin B was added into the flasks and the cells allowed to incubate at 37°C for 14 days while observing cytopathic effect on a daily basis. The supernatants of virus-infected Vero cell cultures exhibiting cytopathic effect of approximately 70% were harvested from the flasks for virus identification. The pooled infection rate program  was used to compare virus infection rates in the tick species collected and processed in this study.Virus isolation was performed as described in . BrieflyTick homogenates with positive cytopathic effect were filtered through a 0.22 μm filter, followed by RNA extraction using TRIzol reagent (Invitrogen). RNA was amplified using the modified random priming mediated sequence independent single primer amplification (RP-SISPA) methodology .http://www.repeatmasker.org) and sequences with more than 50% repeats were excluded. The sequences in each pool were assembled using the Newbler assembler version 2.5.3 with default settings . Contiguous sequences and reads which did not assemble into contigs were categorized using BLASTN and BLASTX homology searches against the non-redundant nucleotide and amino acid databases from NCBI (version June 2011). Taxonomic classification of each contig/read was investigated using MEGAN 4.0.Each amplified sample was further processed as described for shotgun library preparation in GS FLX 454 technology. The sequencing reads were trimmed to remove SISPA primers and barcodes and only reads with a length greater than 50 bp were retained. Low complexity repeats were masked using Repeatmasker (Repeat-Masker Open-3.0.1996-2010 R. pulchellus ticks in this survey in relation to strains isolated from other vectors (available in GenBank). The assembled sequence data isolated from ticks collected in Ijara District, Kenya were combined with available global full genome WNV reference sequences retrieved from the NCBI GenBank database. The final dataset comprised of 28 full genome sequences approximately 11,000 bp long. These were aligned using MUSCLE multiple sequence alignment program and manually edited and visualized using MacClade v4.08a [Comparative phylogenetic analyses were performed to describe and infer the lineage and occurrence of any selective pressures (amino acid substitutions) in the WNV strain isolated from e v4.08a . The gene v4.08a .Phylogenies were obtained using RAxML v7.4.4  implemenhttp://beast.bio.ed.ac.uk) under the GTR + gamma and proportion of invariant sites. BEAST employs Bayesian Markov Chain Monte Carlo MCMC tree estimation strategies. Two independent chain runs were conducted for 100 million generations, sampling trees and parameters at intervals of 10,000 generations under the uncorrelated lognormal relaxed clock model. To gauge the convergence of the runs, the log files were visualized in Tracer software included in the BEAST software package.To infer the evolutionary relationships among the selected WNV strains isolated from different vectors, dated ancestral states were reconstructed using BEAST available at  for prediction of site specific amino acid changes and genome distribution of sites under positive and negative selection pressure, respectively [Comprehensive site-specific amino acid substitution analysis was done using SLAC and FUBAR methods available in Hyphy suite of selection pressure analysis tools accessible through data-monkey web server as  model. The clustering observed under this method was similar to that observed under maximum likelihood providing further support that the WNV isolate KC243146.1) from ticks collected in Ijara district in Northern Kenya clustered closely to Lineage 1 strains from Kenya as well as Europe, North Africa and the USA. The time to the most recent common ancestor of Lineage 1 viruses was estimated to be approximately 200 years  pointing to cross-species transmission of this virus in Kenya.Further phylogenetic evolutionary analysis shows that the WNV strain (KC243146.1) isolated from Kenya is evolutionarily related as it clusters together in Bayesian phylogenetic reconstruction. Further observations made from this analysis point to the Japanese encephalitis virus isolate as the most common recent ancestor of the WNV Lineage 1 viruses with the divergence dating back approximately 200 years. Full genome analysis also suggested that the Lineage 1 strains used in this study from Kenya, North Africa and Europe diverged approximately 20 years ago which does not suggest that the Kenyan strain (KC243146.1) was involved in recent introduction. The closest ancestor to the European strains was from Morocco which diverged nine years ago from strains of France and Italy. This does emphasize a possible role of inter- and cross-continental spread for WNV strains. Transfer of ticks through migratory birds remains a potential mechanism to achieve this. Lineage 2 strains diverged more than 200 years ago from Lineage 1. The South African strains diverged 23 years ago from those in Europe. However the Greek strain diverged only nine years ago from those in Hungary; this may suggest that the Central European strains were the origin of this outbreak rather than recent introduction from Africa.R. pulchellus infesting birds is lacking. The phylogenetic analysis suggests that Morocco rather than Kenya was the source of recent introduction to Europe. Both highly and less neuroinvasive strains have been shown to exist in both Lineage 1 and 2 [WNV Lineage 1 strains have a widespread distribution in Africa, Europe, the Middle East, America, Australia and India. It is possible that WNV Lineage 1 could have been exported from Africa to Europe in particular, by migratory birds that might have had ticks attached to them though evidence of  1 and 2 . ExperimNeurovirulence is associated with specific genotypes and not related to lineage, source of isolate, geographic distribution, passage level, or year of isolation -49. SeleR. pulchellus species sampled from cattle in Ijara District might imply that either this tick species may have acquired the virus after ingesting a viraemic blood meal from the infected animal or the virus was present in the tick which may imply its potential as vector, reservoir and disseminator of WNV. In general, birds are known as reservoirs of WNV while most mammals have relatively low viremia and act as dead-end hosts [The isolation of WNV from nd hosts . The rolnd hosts .The identification of WNV Lineage 1 in Ijara District further elucidates the geographic range of Lineage 1 and 2 strains in Africa. Lineage 2 strains have been described in Tanzania and are almost exclusively found in South Africa [