The Africanized honey bee is one of the most spectacular invasions in the Americas. African bees escaped from apiaries in Brazil in 1956, spread over Americas and by 1994 they were reported in Puerto Rico. In contrast to other places, the oceanic island conditions in Puerto Rico may mean a single introduction and different dynamics of the resident European and new-coming Africanized bees.To examine the genetic variation of honey bee feral populations and colonies from different locations in Puerto Rico, we used eight known polymorphic microsatellite loci.Fst = 0.0783), and is best described as one honey bee population, product of hybridization of AHB and EHB. The genetic variability in this Africanized population was similar to that reported in studies from Texas. We observed that European private allele frequencies are high in all but one locus. This contrasts with mainland Africanized populations, where European allele frequencies are diminished. Two loci with European private alleles, one on Linkage Group 7, known to carry two known defensiveness Quantitative Trait Loci (QTLs), and the other on Linkage Group 1, known to carry three functionally studied genes and 11 candidate genes associated with Varroa resistance mechanisms were respectively, significantly greater or lower in European allele frequency than the other loci with European private alleles.In Puerto Rico, gAHB population does not show any genetic structure (Genetic structure of Puerto Rico gAHB differs from mainland AHB populations, probably representing evolutionary processes on the island. Apis mellifera scutellata was introduced to America in 1956. Before the introduction of the African honey bees, a subset of European subspecies and other African genotypes were introduced into the New World, first by settlers and later by beekeepers [Varroa spp. [Humans and honey bees interacted over history with consequences for distribution of the honey bee, the provider of the first sweetener or honey for humans. Honey bees are naturally distributed throughout Africa, Europe, and the Middle East. In this broad range, they have evolved into 24 subspecies, which have been grouped into three , four 2 distinctekeepers . This waekeepers -16. On troa spp. -16. We droa spp. , and nowroa spp. -16, 112 roa spp. , and thiHybridization between AHB and EHB has been previously reported in Argentina, Brazil, Perú, and Mexico ,6,19,20.A. mellifera is unclear. Natural hybridization between EHB and AHB has been occurring over the last 17 years, and currently, based on mtDNA and morphology, Puerto Rico has a uniform geographic distribution of gAHB, both feral and domestic [A. mellifera . In the local selection scenario, alternate European or African traits that best respond to environmental pressure under different conditions would be selected in different regions across Puerto Rico. According to this hypothesis different levels of European genes could be maintained in populations of feral bees at geographically distinct parts of the island, due to the different environmental conditions. Puerto Rico has a topography that has a strong effect on the climate, with the wetter regions on the windward, northern side of the mountains, and drier climate in the leeward rain shadow [The “n shadow . Moreoven shadow ,29, coulHybridization hypothesis” based on previous studies, states that there is only one gAHB population with large introgression of European genes, as demonstrated in one nuclear locus [Alternately, the “ar locus -16. Accoar locus ,30 or RAar locus  is more 2 surface area, Puerto Rico is the smallest and eastern most of the Greater Antilles. Vieques and Culebra Islands lie a short distance, 10 km southeast and 27 km east of the main island, respectively. Vieques and Culebra are the westernmost of the Lesser Antilles, which extend in a southeasterly arc from Puerto Rico to the northern coast of South America [The main island of Puerto Rico lies between 17°45′ N and 18°30′ N, and its longitude ranges from about 65°45′ W to 67°15′ W . We include more samples from the Vieques island because it is separated from Puerto Rico by a mass of water and may provide an important test for the Structure hypothesis. In addition, in 1988 a program of producing European queen bees was established in Vieques. This discontinued program was established because since that date, Puerto Rico was quarantined against import of European bees from elsewhere (except Hawaii) to prevent introduction of diseases and parasites of the honey bee [A total of 72 colony samples were collected from several apiaries and feral colonies from different places in Puerto Rico: Main island (55), Vieques (17). This is a large sample for population genetic studies using microsatellite markers with high levels of polymorphism . We alsooney bee . Howeveroney bee -16 and tA.m. ligustica or Italian or EHB) bees, as described by [Briefly, we used the protocol from  to identribed by . Previouribed by . We amplBees were stored in 95% ethanol and kept at 4°C until processed. To prevent oversampling of maternal alleles, only one bee per colony was subjected to genetic analysis. Genomic DNA from an individual honey bee thorax was extracted using DNeasy extraction kit from QIAGEN® with the animal tissue protocol.We used eight  DNA microsatellite loci  that werWe optimized PCR conditions in a gradient thermo cycler (Biorad-MyCycler). Final 8 μl reactions were performed with following conditions: 10 ng/μL of genomic DNA, 2.5 units of QIAGEN Taq Master Mix, 0.30 μM of the unmodified primer, 0.02 μM of the primer with the M13 tail and 0.30 μM of the fluorescently labeled M13 primer . After a first 5-min denaturing step (94°C), our PCR protocol consisted of 30 cycles of: 30 s at 94°C, 30 s at the primer-specific annealing temperature (50 to 60°C) and 1 min at 72°C followed by a final extension step of 20 min at 72°C. Genotyping was run on an automated station ABI 3130xl Genetic Analyzer (Applied Biosystems®) using LIZ-500 size standard and allele sizes were scored using Gene mapper version 4.0 (Applied Biosystems®). On occasion a locus did not amplify in individual samples (see Table Hd), and allelic richness (Rs) per locus and population sample were computed using FSTAT version 2.9.3 package [The number of alleles, allele frequencies, and observed (Ho) and expected (He) heterozygosity of each microsatellite locus were estimated using GENEPOP 4.0 . GENEPOP package . In FSTA package ,42. This package ,42. In s package  to measu package . We anal package .t-test of honey bees from Puerto Rico and Texas showed bees did not differ in genetic diversity (Hd) with the population of first group or early Africanization period (TX 91-96) , and had a difference with the second group or late Africanization period (TX 97-01)  (Table Rs) shows the same pattern, with gAHB showing no significant differences from the first group  and significant differences from the second group .The number of alleles per locus varied between six (loci A88) and 19 (loci A79) Table . Our datFst = 0.0783)  Table , and is 3) Table , product3) Table . In fact Table , In Puerto Rico, we found that 18 percent of European alleles are shared with the Texas population, in eight microsatellite loci. Exact tests for linkage disequilibrium at 5% level for each pair of loci did not provide any significant P-value after Bonferroni corrections.For these eight markers, we identified an average of 12.75 alleles by loci with heterozygosities in concordance with the expectation based on the HWE. We identified 12 (11.8 %) European private alleles in six of the eight microsatellite loci sampled to be present in the gAHB population Table . The pre2 = 0.71, P = 0.03). We found that in Puerto Rico, four loci  show similar allelic frequencies as the Hawaii EHB population. In contrast, the locus A107 that represents 50 percent of private alleles, has a higher allelic frequency for these European alleles even than in the EHB reference population   , indicating a more northern source for AHB in Puerto Rico than Brazil, and consistent with speculated Texas origin .The population genetics analysis indicates the presence of a uniform genetic structure or a single gAHB population in Puerto Rico, consistent with the hybridization hypothesis. Interestingly, two marker loci show introgression levels for EHB alleles significantly different than for other loci tested. This is in agreement with a global selection scenario for AHB or EHB alleles for different traits.One issue with highly polymorphic marker loci has been the perceived need to obtain a large sample . This prThe continuous uniform population could be the result of the large range of movement of the bees in comparison to the size of the island. For instance, reproductive drone congregation areas, DCAs are found at about 1.5 miles radius around bee colonies, and less than 10 such DCAs can span the island from North to South . Two otht-test for eight loci used both in this study, and reported previously [Varroa mites both in the mainland and in Puerto Rico, could contribute to the similarity [The variability of the microsatellite loci in the honey bee population in Puerto Rico is similar to that found in the EHB populations for a detected number of alleles and the values of gene diversity , Table . Pinto aeviously  for hetemilarity . The PueIntrogression is a genetic invasion of a local genome by a foreign genome -52. AlthVarroa mite resistance related genes , showedon study -16. Varrsee also ).Varroa mite defenses. This hypothesis of fast evolution or selection was suggested previously [Varroa resistance on LG1, near the A79 marker. Interestingly, in the time this paper was under review, a new publication identified 11 more candidate genes on LG1 in a linkage study for Varroa resistance [more linked genes hypothesis (Figure The deviations in allele frequencies for specific loci may be tentative evidence for global selection for a mosaic of African-like and European-like alleles. This strong global selection may have followed soon after hybridization process since the markers and candidate genes are relatively distant considering the high rate of recombination observed in honey bees . If thiseviously . An altesistance , supports Figure . The twoThe long-term stability of distribution of EHB-AHB allele frequencies suggests a stable equilibrium with reduced effects of genetic drift . The higVarroa resistance mechanisms were respectively, significantly greater or lower in European allele frequency than the other loci with European private alleles. Taken together, this evidence supports retention of a mosaic of African and European alleles for certain genes, mirroring the mosaic of African and European traits observed in bees from this gAHB population.We observed that European private allele frequencies are high in all but one locus. This contrasts with mainland Africanized populations, where European allele frequencies are greatly diminished. Two loci with European private alleles, one linked to two known defensiveness QTLs, and the other to 11 known genes associated with We consider this work is important for population geneticists and evolutionary biologists, because Africanization of honey bee in Puerto Rico is relatively recent, and combination of alleles from European and African bees presented in this study has implications for evolutionary processes on islands. Sociogenomics researchers may be interested in this population with increasing use of functional genomics and next-gen sequencing to study mechanisms underlying described variation in behavior of honey bees.The authors declare that they have no competing interests.AGC generated the hypothesis and AGC and TG generated the study design. Sample collection was conducted by AGC and BRM. TG served as primary graduate mentor for AGC. Genotyping was completed by AGC and JPA. Statistical analysis was conducted by AGC in collaboration with TG. All authors were involved in scientific discussion of the project and wrote the paper. All authors read and approved the final manuscript.“Allele frequency data for eight microsatellite loci used to compare PR and VIE populations”.Click here for file


Results. Correlation between RSf and CHr, r = 0.85. ROC curve analysis for RSf in the diagnosis of iron restricted erythropoiesis definedas CHr < 28 pg: AUC 0.983; Cutoff 91.1%; Sensitivity 98.8%; Specificity 89.6% Correlation between LHD% and %Hypo, r = 0.869. ROC curve analysis for LHD% in the diagnosis of iron deficiency defined by%Hypo >5%: AUC 0.954; Cut off 6.0%; Sensitivity 96.6%; Specificity 83.2%Conclusions. RSf and LHD% could be reliable parameters for the study of ironmetabolism status.Reticulocyte hemoglobin content (CHr) and percentage of hypochromic cells(%Hypo) are restricted to the Siemens analysers. The aims of the study were to investigate the correlation of Red cells size factor(RSf) and low Hemoglobin density (LHD%), reported by Beckman-Coulteranalysers, with CHr and %Hypo in the assessment of iron status in the presence ofinflammation. 381 samples were run on both LH 750 (Beckman-Coulter) and Advia 2120 (Siemens)analysers. Correlation between parameters were calculated and the diagnosticperformance of the new parameters was assessed.  In iron deficiency anemia (IDA) iron supply depends on the quantity of iron storage in the body, while in functional iron deficiency (iron restricted erythropoiesis) supply depends on the rate of mobilization of iron from the stores. Functional iron deficiency is defined as an imbalance between the iron needs for erythropoiesis and the iron supply, with the latter not maintained at sufficient rate for adequate hemoglobinization of reticulocytes and mature erythrocytes .The diagnosis of iron deficiency or functional iron deficiency is particularly challenging in patients with acute or chronic inflammatory conditions because most of the biochemical markers for iron metabolism are affected by acute phase reaction. This is the case of the anemia of chronic disease (ACD) and the anemia associated to chronic renal failure (CKD).Serum ferritin, an indicator of iron storage but not of iron supply, is a positive acute phase reactant, while transferrin is a negative acute phase reactant, rendering the calculation of transferrin saturation unreliable in this case –4.For these reasons, an iron deficient erythropoiesis may occur despite that normal serum ferritin and transferrin saturation values and interest have been generated in the use of erythrocyte and reticulocyte parameters, available on the modern analysers based on flow cytometry technology.Direct consequence of an imbalance between the erythroid marrow iron requirements and the actual supply is a reduction of red cell hemoglobin content, which causes hypochromic mature red cells and reticulocytes. Reticulocyte hemoglobin content (CHr) and the percentage of hypochromic red blood cells (%Hypo) reflect iron availability and are reliable markers of functional iron deficiency , 6.The measurement of CHr is a direct assessment of the incorporation of iron into erythrocyte hemoglobin (Hb) and thus an estimate of the recent functional availability of iron into the erythron; due to the life span of the reticulocytes, CHr is a sensitive indicator of iron deficient erythropoiesis –9.The measurement of %Hypo (defined as the percentage of red blood cells with Hb concentration less than 280 g/L) is a sensitive method for quantifying the hemoglobinization of mature red cells. Because of the long circulating life span of mature erythrocytes %Hypo values are related to iron status in the last 2-3 months, and have been recognised as an indicator of iron deficiency , 11.CHr and %Hypo have been used as a diagnostic tool, together with biochemical markers, to distinguish IDA from ACD, and are incorporated to National Kidney Foundation (NKF-K/DOQI) guidelines for the monitoring of recombinant human erythropoietin rHuEPO therapy –14.To date, the measurement of CHr and %Hypo has been restricted to the analysers of a single manufacturer, Siemens . A second manufacturer has produced a comparable index, the so-called reticulocyte hemoglobin equivalent (Ret He) generated by the Sysmex XE 2100 analyser .Measurements of Ret He provides useful information in diagnosing anemia, iron restricted erythropoiesis, and functional iron deficiency and response to iron therapy during r-HuEPO. Twenty nine pg is the cutoff value that defines deficient erythropoiesis , 16. RetBeckman Coulter  has recently introduced on the LH series analysers two new parametersRed blood cell Size Factor (RSf) joins together the volume of mature red cells (MCV) and the volume of reticulocytes (MRV), both related to erythropoietic activity and hemoglobinizationl volume .Low hemoglobin density (LHD%) derives from the traditional mean cell hemoglobin concentration (MCHC), using the mathematical sigmoid transformationIn the same way, LHD% is related to iron availability and the hemoglobinization of the mature red cells.In this equation defining LHD%, in addition to the standard sigmoid function, a square root is applied to further enhance numerical resolution in the region corresponding to the lower end of %Hypo, to improve the differentiation between the normal and the abnormal among the blood samples having relatively low values of LHD% .The aims of this study wereto establish the reference range of RSf and LHD% and their values in different types of anemia; to evaluate these recently introduced reticulocyte and erythrocyte parameters provided by the LH series analysers in terms of correlation with CHr and %Hypo as well as their diagnostic efficiency assuming CHr < 28.0 pg or %Hypo > 5% to detect iron restricted erythropoiesis and iron deficiency. Samples from 120 healthy individuals, 72 iron deficiency anemia (IDA), 60 IDA with acute phase response (IDA APR), 71 chronic kidney disease (CKD), and 58 anemia of chronic disease (ACD) were randomly extracted from the routine workload and run sequentially on both LH 750  and Advia 2120 , analysers within 6 hours of collection.Healthy group: 54 male and 66 female adult subjects, with no clinical symptoms of disease and with results of the complete blood count and biochemical iron metabolism markers within reference ranges. μmol/L, transferrin saturation < 20%, ferritin < 50 μg/L, and Hb < 110 g/L, were included before iron treatment. A group of 132 IDA patients fulfilled traditional diagnostic criteria for iron deficiency anemia diagnosis, serum iron < 7.5 n = 72, CRP < 5 mg/L) and acute phase response group . Acute phase response included inflammation or infectious conditions, in addition to ferropenic status.This group was divided into a nonacute phase response group (CKD patients were managed according to the recommendations of the NKF-K/DOQI guidelines . All patACD group included patients with a variety of diseases: chronic infections (tuberculosis); neoplasic disorders ; noninfectious inflammatory diseases .ACD patients received treatment to maintain normal erythropoiesis and presented the traditional diagnostic criteria for “Functional iron-deficiency” diagnosis Transferrin saturation < 20%, Hb < 110 g/L, and serum ferritin values normal or over the reference range.Statistical software package SPSS  version 17.0 for Windows was applied for statistical analysis of the results.Reference ranges were calculated from the results obtained in the group of healthy subjects . Kolmogorov—Smirnoff test was applied to verify the Gaussian distribution of RSf and LHD% values.t-test was performed in order to detect statistical deviations between the groups of patients; P values less than  .05 were considered to be statistically significant.When the parameters under study presented a Gaussian distribution correlation, coefficients were calculated by Pearson method; independent samples U-test was performed; P values less than  .05 were considered to be statistically significant.When the parameters under study presented a non-Gaussian distribution correlation, coefficients were calculated by Spearman method; independent samples Mann-Whitney Receiver operating characteristic (ROC) curve analysis was utilized to illustrate the diagnostic performance of RSf and LHD% in the detection of iron deficiency status, defined by a CHr value 28 pg.Cutoff values were established based on the optimal combination of sensitivity and specificity.P =  .279)  . ReferenThe horizontal line in the center of the box shows the median value, the upper and lower limits of the box show the interquartile range, and the whiskers show the minimum and maximum values for each group.The horizontal line in the center of the box shows the median value, the upper and lower limits of the box show the interquartile range, and the whiskers show the minimum and maximum values for each group.P <  .001.Correlation between CHr and RSf values, Pearson regression coefficient 0.85, t-test was performed in order to detect statistical deviations between the groups of patients.Independent samples P <  .001) were detected when groups with iron restricted erythropoiesis  were compared with patients undergoing therapy  and the healthy subjects (mean 100.9 fL).Significant differences in RSf mean values (P =  .481).No statistic difference was found between IDA group and IDA patients with acute phase response  ACD and CKD patients receiving therapy maintained CHr levels higher than the cutoff value of 28 pg.ROC curve analysis for RSf in the diagnosis of restricted erythropoiesis, defined by CHr < 28 pg, results were AUC 0.983, Cutoff 91.1 fL, sensitivity 98.8%, specificity 89.6%. P = .034)  . ReferenThe horizontal line in the center of the box shows the median value, the upper and lower limits of the box show the interquartile range, and the whiskers show the minimum and maximum values for each group.r = 0.869 (Spearman method) (P <  .001). y = 1.338x + 4.40.Correlation between %Hypo and LHD% values, U-test was performed in order to detect statistical deviations between the groups of patients.Independent samples P <  .001) were detected when groups with iron deficiency  were compared with patients undergoing therapy  and the healthy subjects (median 2.1%).Significant differences in LHD% values (P =  .578).No statistic difference was found between IDA group and IDA patients with acute phase response (Receiver operating characteristic (ROC) curve analysis for LHD% in the diagnosis of iron deficiency, defined by %Hypo > 5% AUC 0.954, cutoff 6.0%, sensitivity 96.6%, specificity 83.3%. Discriminant efficiency of biochemical parameters and classical erythrocyte indices:mean cell hemoglobin (MCH), AUC 0.89; mean cell volume, (MCV), AUC 0.822; serum ferritin, AUC 0.722; serum iron, AUC 0.683 .Uncomplicated iron deficiency is not difficult to diagnose by means of the traditional laboratory tests. Biochemical markers are reliable parameters to diagnose iron deficiency in an uncomplicated clinical setting.Serum ferritin, as a potent positive acute phase reactant is often increased in ACD and CKD patients . TransfeEfforts have been made to evaluate some readily available and relatively inexpensive laboratory parameters as indirect markers of iron restricted erythropoiesis and iron availability in a clinical context influenced by inflammation and acute phase reaction. The best combination of hematological indices for iron deficiency is an increased percentage of hypochromic erythrocytes and a reduced hemoglobin content of reticulocytes . Measurements of reticulocyte hemoglobin content (CHr) have been shown to provide useful information in diagnosing functional iron deficiency during r-HuEPO therapy , 26 and Recent studies confirm the clinical reliability of mature erythrocyte parameters, such as hypochromic red cells (Hypo %), as markers of iron deficiency in hemodialysis patients , 30.Beckman Coulter  applies the Volume Conductivity Scatter technology to this field and new parameters are now available on the LH series analysers.The main purpose of the present study was to determine the diagnostic performance of RSf and LHD% and to assess whether the new parameters correlate with the existing diagnostic tests CHr and %Hypo for restricted erythropoiesis and functional iron deficiency diagnosis.This study shows a reasonable level of agreement between RSf and CHr. Despite the fact that CHr is a measurement of the reticulocyte hemoglobin content and RSf is a measurement of both mature red cells and reticulocyte size, a high concordance between these parameters has been observed and values of both parameters showed the same trend in the different groups.In patients with iron restricted erythropoiesis, (IDA and IDA APR) RSf values were lower than the reference range 91.1 fL.ACD and CKD patients were receiving therapy, they suffered a mild anemia but maintained bone marrow activity, as the CHr values state . RSf values in these patients were in the reference range of RSf, but the macrocytosis associated to CKD and hemodialysis treatment causes that 15% of the patients in this group had RSf values slightly over the reference range, 106.6 fL.Diagnostic sensitivity, specificity, and efficiency of RSf were good when compared to a 28 pg value of CHr. In particular, a cutoff value of 91.1 fL for RSf showed the best diagnostic efficiency, with a sensitivity of 98.8% and a specificity of 89.6%. This study shows a reasonable agreement between % Hypo and LHD% and their values showed the same trend in the different groups.%Hypo values in patients undergoing therapy were near the threshold of iron deficient erythropoiesis 5% .The LHD% values obtained in these groups of patients were statistically lower  than the iron deficient ones  but, as all of them had a mild anemia, LHD% values in these patients were above the reference range 4.4%.The optimal cutoff point for LHD% was 6.0%, which provided sensitivity 96.6%, specificity 83.2%, and area under curve 0.954, for iron deficiency detection, defined by %Hypo > 5%.In this study, it has been stated that RSf and LHD% could be reliable parameters for the study of iron status and the amount of the supply available for erythropoiesis.Iron metabolism is a dynamic process which cannot be defined by one test parameter only. The analysis of these new parameters can be performed simultaneously in the course of routine blood counts, with no incremental costs and no additional needs of more blood sampling. In conjunction with standard blood cell counts and iron, parameters could enable the diagnosis to be made rapidly and accurately. The new parameters derived from Beckman-Coulter technology seem to be an acceptable alternative to CHr and %Hypo in the routine practice, with the same clinical meaning, but more prospective and longitudinal studies are needed in order to verify the results obtained, to determine their reliability for clinical purposes or whether the additional information provided could be used in managing the iron requirements of patients.


Ursus americanus) harvested in a large , geographically isolated population and used parentage analysis to identify mother-offspring dyads (n = 337). We quantified the effects of sex, age, habitat type and suitability, and local harvest density at the natal and settlement sites on the probability of natal dispersal, and on dispersal distances. Dispersal was male-biased  but a small proportion (21%) of females also dispersed, and female dispersal distances (mean ± SE  =  48.9±7.7 km) were comparable to male dispersal distances (59.0±3.2 km). Dispersal probabilities and dispersal distances were greatest for bears in areas with high habitat suitability and low harvest density. The inverse relationship between dispersal and harvest density in black bears suggests that 1) intensive harvest promotes restricted dispersal, or 2) high black bear population density decreases the propensity to disperse. Multigenerational genetic data collected over large landscape scales can be a powerful means of characterizing dispersal patterns and causal associations with demographic and landscape features in wild populations of elusive and wide-ranging species.Understanding the factors that affect dispersal is a fundamental question in ecology and conservation biology, particularly as populations are faced with increasing anthropogenic impacts. Here we collected georeferenced genetic samples  from three generations of black bears ( Dispersal is an important ecological process that allows individuals to exploit temporally and spatially variable resources, and has implications for population dynamics and population viability through the spatial redistribution of individuals Numerous hypotheses have been proposed to explain the evolution of, and variation in dispersal behavior, yet most explanations involve competition, inbreeding avoidance, and/or environmental stochasticity Dispersal probability may also be inversely related to maternal age. Whether or not a female offspring disperses or exhibits philopatry is dependent upon the costs of dispersal as well as the costs of kin competition. Theory predicts that as a mother ages, her investment in future reproductive events  declines The above hypotheses are not mutually exclusive, and thus patterns in data, particularly when based on small sample sizes, may not characterize underlying processes unambiguously. Traditional studies of dispersal require marking and tracking a large number of individuals over time, and complications can arise due to difficulty obtaining adequate sample sizes and tracking long distance dispersers. Further, the limited duration over which observations are collected, particularly for long-lived, iteroparous species, can affect the accuracy and power of inferences Ursus americanus) inhabiting a large geographical area in the Northern Lower Peninsula (NLP) of Michigan, USA . This area is considered an insular population, being bounded on three sides by the Great Lakes, and to the south by a landscape composed of intensive agricultural and expansive urban areas. The NLP landscape is a largely forested mix of northern and mixed hardwoods, pines, and forested and non-forested wetlands. The NLP is also highly fragmented by roads, forestry activities, and agriculture Our study area covered the northern two-thirds of the lower peninsula of Michigan . A pre-molar tooth is extracted for aging and DNA extraction. Bears are aged by the MDNR using the cementum annuli method During the annual NLP bear hunting season (September and October), hunters must register harvested bears at registration stations facilitated by the Michigan Department of Natural Resources (MDNR). At the check stations, hunters report the bear's sex and harvest location , 30s at the locus-specific annealing temperature , 1 min at 72°C, and a final 10 min extension step at 72°C. We used 6.5% denaturing acrylamide gels for electrophoresis visualized on a LI-COR 4200 Global IR2 System . For each gel, we included molecular weight standards and individual bears with known genotypes. All alleles were scored independently by two experienced lab personnel using Saga genotyping software . 10% of samples were randomly selected and genotyped twice to provide a genotyping error rate of <2%.We extracted DNA from bear teeth using Qiagen DNEasy Tissue Kits following manufacturer protocols . DNA was quantified using a Nanodrop spectrophotometer  and diluted to a 20 ng/μl working concentration. Using polymerase chain reaction (PCR), we amplified 12 microsatellite loci including Using MICRO-CHECKER SE47 and SE48 amelogenin primers To minimize potential errors in field sexing, we genetically sexed 1) all females that were identified as having had cubs in consecutive years  score Parentage analysis was conducted using the program FRANz 2 area). Euclidean distances between MO dyads were determined using ETGeowizards and Hawth's Tools Locations of mothers and offspring were georeferenced to the centroid of their reported harvest sections  into seven land cover classifications and ranked them according to bear habitat suitability (most (1) to least (5) suitable) based on an ecological model of bear habitat selection in Michigan , offspring age (offage), characteristics of the natal (mother's) site including habitat type (momhab), habitat suitability (momhs), and harvest density (momharv) and characteristics of the settled (offspring harvest) site including habitat type (offhab), habitat suitability (offhs), harvest density (offharv), and mother's age . Subsequently, using only those individuals classified as dispersers, we modeled dispersal distance as a function of offspring sex and age and characteristics of the settlement site  and the natal site . Lastly, again using only those individuals classified as dispersers, we investigated whether natal habitat-biased dispersal occurred. We modeled the habitat type at the settlement site as a function of offspring sex, offspring age, and the habitat type at the natal site. We tested 64 a priori hypothetical models of dispersal probability, 53 models of dispersal distance, and 7 models of habitat type (see iw) were calculated relative to the other models in each set. Models with ΔAIC values of ≤2 were considered to be supported.We used mixed models  for inclusion in the parentage analysis to identify MO dyads. Parentage analysis identified 337 MO dyads with posterior probabilities ≥0.9. Dyads were comprised of 159 female and 178 male offspring, from 214 mothers. The average age at time of death for offspring, and at the time of offspring birth for mothers was 3.0 years (range  = 1–12), 2.5 years (range  = 1–14), and 5.8 years (range  = 2–22) for female offspring, male offspring, and mothers, respectively.Euclidean distances averaged 14.2 km (median  = 7.7 km) between mother-female offspring dyads, and 41.1 km (median  = 34.9 km) for mother-male offspring dyads . The maxoffsexβ  = 2.24), offspring age (offageβ  = 0.18), and harvest density at the offspring settlement site (offharvβ  = −0.39) , harvest density  and habitat type at both the natal and the settlement sites  and non-forested wetland  and for individuals settling in more suitable habitat types like mixed forest , forested wetland , and evergreen forest .The best model of dispersal distance included an interaction between offspring age and sex  or settled in a site with lower harvest density than in their natal site (44%)  remained within 30 km of their natal ranges. The relatively high percentage of non-dispersing male bears is surprising considering black bears are thought to unequivocally exhibit strong male-biased dispersal, with 100% of males dispersing in some study systems Comparisons of studies using ecological and genetic methods often reveal disparities in their results, even when conducted in the same study area. In a long-term radio telemetry study of NLP bears, Etter et al. (2002) found that 30 of 31 male bears (95%) dispersed, compared to the 52 of 68 male bears (76%) ≥3 years old that dispersed in our study . The disFemale philopatry, like male dispersal, is not absolute. Approximately 21% of females ≥3 years old emigrated, often over large distances . A similAlthough maternal age was not a strong predictor of offspring dispersal probability in our models, this might be due more to the small proportion of females that did disperse  or to the intensive harvest that reduces the median age of mothers in the population. In Scandinavian brown bears, 41% of females disperse, and female dispersal probability is strongly negatively correlated with maternal age The putative alteration of natural dispersal patterns by intensive harvest is a situation deserving of management attention. Several studies comparing harvested and non-harvested bear populations have shown that vital rates, like reproductive success and survival, can be density-dependent and can be altered by intensive harvest File S1Methods and results of parentage analysis based on simulated black bear offspring.(DOCX)Click here for additional data file.File S2Full list of linear mixed effects models of black bear dispersal probability and dispersal distance.(XLSX)Click here for additional data file.


Point mutations in the coding region of the interleukin 28 gene (rs12979860) have recently been identified for predicting the outcome of treatment of hepatitis C virus infection. This polymorphism detection was based on whole blood DNA extraction. Alternatively, DNA for genetic diagnosis has been derived from buccal epithelial cells (BEC), dried blood spots (DBS), and genomic DNA from serum. The aim of the study was to investigate the reliability and accuracy of alternative routes of testing for single nucleotide polymorphism allele rs12979860CC.Blood, plasma, and sera samples from 200 patients were extracted (400 µL). Buccal smears were tested using an FTA card. To simulate postal delay, we tested the influence of storage at ambient temperature on the different sources of DNA at five time points There was 100% concordance between blood, plasma, sera, and BEC, validating the use of DNA extracted from BEC collected on cytology brushes for genetic testing. Genetic variations in HPTR1 gene were detected using smear technique in blood smear (3620 copies) as well as in buccal smears (5870 copies). These results are similar to those for whole blood diluted at 1/10. A minimum of 0.04 µL, 4 µL, and 40 µL was necessary to obtain exploitable results respectively for whole blood, sera, and plasma. No significant variation between each time point was observed for the different sources of DNA. IL28B SNPs analysis at these different time points showed the same results using the four sources of DNA.We demonstrated that genomic DNA extraction from buccal cells, small amounts of serum, and dried blood spots is an alternative to DNA extracted from peripheral blood cells and is helpful in retrospective and prospective studies for multiple genetic markers, specifically in hard-to-reach individuals. Recent studies have demonstrated that host genetics may be useful for predicting the spontaneous clearance of hepatitis C virus during acute hepatitis and the drug response to peginterferon and ribavirin in chronic hepatitis C However, all the IL28B-based studies used whole blood DNA extraction for SNP analysis. DNA circulates freely in blood plasma both in health and in disease, but the source of this DNA remains enigmatic. It is presumed that circulating DNA in healthy subjects is derived from lymphocytes or other nucleated cells The aim of this study was to investigate the reliability and accuracy of alternative routes of sampling for genetic testing, whether DNA from serum, buccal epithelial cells (BEC), or dried blood spots (DBS) for SNP allele (rs12979860 CC) and to blindly compare these results with those obtained with DNA extracted from whole blood as reference.Two hundred patients with chronic hepatitis C infection genotype 1 were included from three hepatology units in the south of France (two centres in Marseille and one in Saint Laurent du Var). Mean age was 56±11 years; there were 42 (21%) naïve patients and 40 (20%) non responders. In whole blood, 59 (30%) patients had rs12979860 CC genotype, 107 (53%) had rs12979860 CT genotype, and 34 (17%) had rs12979860 TT genotype.Written informed consent including for genetic testing was mandatory for inclusion. This study was approved by the French ethics committee “Comité de Protection des Personnes Sud-Méditerranée II” and had the reference number 210R22.The DBS sample consisted of three drops of 50 µL of whole blood absorbed onto a Protein Saver 903 Card  to completely fill 12-mm preprinted circular paper disks and then stored at room temperature. The FTA range of products has been developed to collect, store, and extract nucleic acid from many sample types, including blood, plasma/sera, and buccal smears. After application of the sample to the FTA card, microorganisms are inactivated, cell membranes lysed, and nucleic acids entrapped onto the FTA matrix.Blood, plasma, and sera samples were extracted with a MagNA Pure device (Roche) according to manufacturer recommendations; 400 µL of each sample type were used. Blood and buccal smears were tested on Whatman system . All sampling and tests were performed simultaneously and the amount of DNA was sufficient for molecular analysis. The study design is presented in Cells are captured on the foam applicator by swabbing the oral mucosa and then transferred to indicating FTA Cards To test the performance of the different systems, we used CELL Control r-gene  on a Light Cycler 480 II (Roche) according to manufacturer recommendations. The CELL Control r-gene™ is a ready-to-use premix designed to validate results obtained by real time amplification checking the presence of cells on extracted samples. CELL Control r-gene™ determines the number of cells in the sample by quantifying the HPRT1 gene (hypoxanthin phospho-ribosyl transferase) using standards included in the kit. The HPRT1 gene is a housekeeping gene for normalizing gene expression To simulate postal delay, we assessed the influence of storage at ambient temperature on the different sources of DNA . Ten patients were prospectively sampled and their corresponding sources of DNA were tested after storage at ambient temperature at five time points . The stability of HPRT1 gene quantification and the IL28B SNPs on BEC, DBS, and plasma was also investigated at these different time points. Whole blood served as a reference source of DNA.The rs12979860 SNPs were analysed in a Fret 5′ allelic discrimination assay using the Roche ‘LightCycler® FastStart DNA Master HybProbe’ with the LightCycler® 1.x/2.0/480 Instruments Statistical analyses were performed using SAS software . Wilcoxon paired test was used to compare results between whole blood, serum, DBS, and BEC. Significance level was set at P<0.05. and There was 100% concordance in detecting DNA between blood, plasma, sera, and BEC. Regarding the sensitivity of DNA detection of the different samplings, with reference the whole blood, sera and plasma appeared less efficient than DBS and BEC (Genetic variations in the HPTR1 gene were detected using smear technique in blood smear (3620 copies) as well as in buccal smears (5870 copies). These results are similar to those for whole blood diluted at 1/10.As 400 µL of each type of sample were extracted with MagnaPur, a minimum of 0.04 µL, 4 µL, and 40 µL was necessary to obtain exploitable results respectively for whole blood, sera, and plasma Table 1.IL28B SNPs analysis at these different time points showed the same results using the four source of DNA Table 2.Our study is the first to prospectively compare and validate different DNA sampling sources. Results show that plasma, serum, BEC, or DBS can be used as a source of DNA for IL28B genotyping, with full concordance (100% of sensitivity) with whole blood DNA extraction. The various extraction procedures gave identical SNP genotypes, which confirms that genomic DNA prepared from small amounts of serum or plasma can serve as a template to amplify DNA segments in order to detect genetic alterations, as described by Lin and Floros It is important to recall that the stability and integrity of genomic DNA depend on storage conditions such as temperature, freeze-thaw cycles, and buffer composition. We observed no influence of storage conditions at ambient temperature, incidently showing postal delay in DNA sample shipping may be avoided. There was no difference in DNA from baseline to 12 days for the different sources of DNA tested . FTA cards, plasma, and sera are appropriate tools for clinical study because of their long term DNA conservation.The combination of a micro-extraction procedure from serum and PCR genotyping provides a rapid and inexpensive tool for genetic analysis Cancer patients have a greater amount of circulating DNA than healthy subjects As an alternative to the usual venipuncture technique, several methods have been described that use DBS from finger-puncture or oral swabs collected on DNA-cards Another important issue is that, ideally, tissue sampling should be non-invasive and painless, especially for children and psychiatric groups. Preparation of DNA also needs to be rapid, reliable, and consistent. In any large-scale genetic association project, per-unit cost of DNA preparation becomes high. Also, DNA sample collection is needed in genotyping and in pharmacogenetic studies to understand the inter-individual variability of drug responses.We also analysed patients' acceptability of the various sampling conditions. Buccal smear had an excellent and acceptability rate, the highest compared with serum and dried blood spot (data not shown).In conclusion, we report that extracting genomic DNA from small amounts of serum, DBS, and BEC is an appropriate alternative to extracting DNA from peripheral blood cells and offers the possibility to analyse existing specimens. New patterns in genetic markers might be considered using serum as DNA source. DNA cards clearly facilitate genetic and pharmacogenetic testing for routine clinical practice. Moreover, collection and extraction of DNA from buccal smear are a noninvasive, convenient, and cost-effective alternative to blood sampling for participants and researchers, and can be mailed out for self-collection, overcoming geographical impediments. For patients with chronic hepatitis C infection, treatment response may be established through large screening for personalized medicine.


FMR1 mRNA, as those observed in premutation carriers can lead to Fragile X- associated tremor ataxia syndrome (FXTAS). Until recently, FXTAS had been observed only in carriers of an FMR1 premutation (55–200 CGG repeats); however the disorder has now been described in individuals carriers of an intermediate allele (45–54 CGG repeats) as well as in a subject with a full mutation with mosaicism.Lack of the fragile X mental retardation protein leads to Fragile X syndrome (FXS) while increased levels of FMR1 mosaic individual with full and premutation alleles. Molecular analysis of FMR1 and FMRP expression in this subject is consistent with a FXS phenotype. We observed reduced expression of FMRP in both peripheral blood and brain leading to the FXS diagnosis. In addition, a dramatic 90% depletion of both FMR1 mRNA and FMRP levels was observed in the blood, as normally observed in FXS cases, and an even greater depletion in the brain. A clinical report of this patient, at age 71, described neurodegenerative signs of parkinsonism that were likely, in retrospect, part of a FXTAS scenario as post-mortem examination shows the presence of intranuclear inclusions, the hallmark pathology of FXTAS.Here, we report on molecular and clinical data of a male FMR1 alleles. In addition, based on symptoms and pathological and molecular evidence, this report suggests the need to redefine the diagnostic criteria of FXTAS.The findings presented in this study indicate co-morbidity for both FXS and FXTAS in this individual carrying both full and premutation  FMR1 gene: Fragile X syndrome (FXS) and Fragile X -associated tremor ataxia syndrome (FXTAS).Two very different disorders arise from a CGG repeat expansion mutation at the promoter region of the X-linked FMR1 gene, which consequently becomes silenced, leading to the absence of the FMR1 protein (FMRP), the underlying cause of FXS.Full mutation (FM) individuals with greater than 200 CGG repeats invariably develop FXS, a neurodevelopmental disorder that is present from birth and produces cognitive impairment, behavioral, emotional and sleeping problems -3. AdditFMR1 gene, ranging from 55–200 CGG repeats, usually do not have developmental disabilities but are at high risk for developing FXTAS in late adulthood [Individuals with shorter ‘premutation’ (PM) expansions in the dulthood . FXTAS idulthood . The neudulthood ,9, in tedulthood  and in odulthood  and in tdulthood .FMR1 gene and toxic accumulation of CGG-repeat expanded mRNA that is thought to contribute to the formation of intranuclear inclusions and to the pathogenesis of PM-associated disorders, particularly FXTAS. The exact mechanism of mRNA-mediated neurotoxicity remains incompletely understood. One possibility is that CGG binding proteins are sequestered in the intranuclear inclusions, which also contain FMR1 mRNA [PM alleles are associated with increased transcription of the MR1 mRNA . More thMR1 mRNA -16. InclMR1 mRNA . HoweverMR1 mRNA -21, whicFMR1 mRNA an association, although less striking, between transcriptionally active FMR1 expanded alleles across the whole CGG repeat range and FXTAS could be made. Indeed, cases of individuals who meet diagnostic criteria of FXTAS but not falling within the “PM category” have been reported. These constitute a group of individuals in whom neurological manifestations seen in the PM related FXTAS spectrum exist. Another question concerns the presence of intranuclear inclusions in carriers of alleles outside the premutation range. In fact, rare and small intranuclear inclusions were observed in three males with FXS [Since the first FXTAS cases were described  it was twith FXS . Intranuwith FXS , but theFMR1 size mosaicism. FMR1 size mosaicism indicates that a person carries both FM and PM alleles. The subject developed FXS and showed signs of neurodegeneration during aging. A previous clinical study on this case reported that the patient developed parkinsonism that was not thought to be attributable to FXTAS [Here we report molecular, neuropathological and clinical characterization of a man with to FXTAS , althougOur molecular and neuropathological observations and re-assessment of clinical reports suggest that this individual was affected with both FXS and FXTAS.Written informed consent for publication of his case report and any accompanying images was obtained from the patient earlier in life or from a family member. Tissue harvest and brain autopsy was performed in accordance with University of California, Davis, Institutional Review Board-approved protocols.CGG repeat size was measured on genomic DNA extracted from peripheral blood and from brain regions using standard procedures  and determined by Southern Blot and PCR analysis as previously described ,29.FMR1 mRNA levels were as in [Total RNA from all brain regions was isolated using trizol . cDNA synthesis and QRT-PCR used to quantify re as in .Postmortem cerebellum, frontal pole, orbital frontal cortex, medial frontal cortex and dorsolateral pre-frontal cortex tissues from this case and from the cerebellum of 8 age matched controls (CGG ranging from 21 to 48 repeats) were pulverized in liquid nitrogen and resuspended in 1X RIPA buffer  that contained protease and phosphatase inhibitors. The lysates were sonicated and spun in a Sorval centrifuge at 18,000 rpm, 4°C. The supernatants were collected and protein concentrations measured using a BCA protein assay . Approximately 65 μg of protein were loaded in a 10% Criterion SDS/PAGE gel and ran in 1X MOPS at 25 mAmps for 30 minutes followed by 80 volts for 4 hours, and proteins were transferred into a PVDF membrane overnight at 4°C in 1X Tris/Glycine/SDS with 20% methanol. The membrane was blocked in LICOR blocking buffer and hybridized overnight at 4°C with 1:1,300 mouse anti-FMRP  and the next day for 2 hours at RT with 1:10,000 anti-mouse secondary antibody (LICOR wv 680 mm). Following detection the blot was re-hybridized with 1: 200,000 mouse anti-GAPDH  antibody and the same secondary antibody. Bands were detected at 169 μm resolution using the Odyssey infrared scanner.2O2 for twenty minutes at RT. Slides were rinsed in 0.1 M PBS and incubated for two hours at RT in blocking buffer containing (v/v) 10% fetal donkey serum (Millipore), 0.1% Triton X-100 (Acros), and (w/v) 0.2% gelatin (Acros). Slides were incubated in primary antibody incubation buffer containing mouse anti-FMRP clone 1C3 1:200 (Millipore), (v/v) 2% fetal donkey serum, 0.2% Triton X-100, and (w/v) 0.2% gelatin overnight at RT. Sections were rinsed in 0.1 M PBS and incubated in secondary antibody buffer containing biotin-conjugated donkey anti-mouse secondary antibodies 1:200 (Jackson), (v/v) 2% fetal donkey serum, 0.2% Triton X-100, and (w/v) 0.2% gelatin at RT for one hour. Sections were rinsed in 0.1 M PBS and the immunoenzymatic reaction was visualized with an ABC horseradish peroxidase kit using the chromagen DAB (Vector). Sections were washed in 0.1 M PBS and Milli-Q H2O. Slides were dehydrated in a series of fifteen minute incubations as follows: (v/v) 50% EtOH, 70% EtOH, 96% EtOH, 100% Chloroform, 96% EtOH, 100% EtOH and Safeclear (Fisher) and coverslipped in DPX mounting medium (EMS).Tissue was fixed in paraformaldehyde for at least 48 hours and paraffin embedded. Four μm paraffin-embedded sections of cerebellum and frontal cortex mounted on Thermo Scientific Permafrost Adhesion microscope slides were deparaffinized and hydrated in a series of fifteen minute incubations as follows: Safeclear Xylene substitute , (v/v) 100% ethyl alcohol (EtOH), 96% EtOH, 70% EtOH and 50% EtOH followed by a five minute incubation in Milli-Q H2O. Slides were washed in 0.1 M Phosphate Buffered Saline (PBS). Heat-mediated antigen retrieval was performed by submerging the slides in Citrate buffer  and (v/v) 0.05% Tween-20 (Acros) followed by boiling for 15 minutes in a microwave. Slides were allowed to cool to room temperature (RT) and rinsed in 0.1 M PBS. Endogenous peroxidase activity was quenched by incubating slides in 0.3% HNeuropathologic features from single H & E stained 4 μm paraffin sections were quantified from the frontal cortex, hippocampus, temporal cortex, and cerebellum. A priori selection criteria were applied to select anatomically constrained regions of interest and every cell within that region was counted at 1000X total magnification using a Nikon E600 ECLIPSE microscope in the same manner as previously described .FMR1 gene and his IQ at age 60 on the WAIS-R showed a verbal IQ of 69, a performance IQ of 67 and a full scale IQ of 67. His clinical examination demonstrated typical features of FXS including a mildly long face, large and mildly prominent ears, macroorchidism, in addition to perseverative speech, verbal outbursts, paranoid ideation and anxiety that interfered with sleep. He was placed on buspirone and aripiprazole for his behavioral outbursts. At age 71 he developed a history of balance difficulties with falling and slowness in his motor movements. He also described memory problems and his Mini-Mental Status Examination (MMSE) was 25/30. He had masked facies, hypotonic speech and increased tone in the upper extremities without rigidity or tremor. He demonstrated a stooped posture and shuffling gait. Because of his parkinsonian features the aripiprazole was discontinued. His MRI demonstrated subcortical white matter disease and dilated ventricles but he did not have the middle cerebellar peduncle (MCP) sign. Over the next few years he gradually developed more bradykinesia, rigidity and difficulties with walking. He also had depression, which had been an intermittent problem for him since age 60. He was treated with counseling and fluoxetine in addition to his buspirone. At age 77 he had significant cognitive deficits and his MMSE dropped to 13/30. Just before his death he developed an intermittent tremor and he had only limited communication abilities. He was reported in a medical journal [The patient was a high-functioning man with FXS who was initially diagnosed at age 60 because of behavioral outbursts, which required medical treatment. He was found to be a mosaic for the  journal  and was The family history was significant in that his brother had the full mutation and fragile X syndrome and was lower functioning cognitively than him. His mother, who was a carrier, died at age 75 and developed lung cancer but had a long history of depression and also had dementia before death.External examination of the cerebral hemisphere showed moderate cortical atrophy and mild atrophy of the superior and middle lobules of the cerebellar vermis. The lateral ventricle was moderately dilated as seen on serial coronal sections. Numerous representative sections from all regions of the brain were taken. H&E stains were performed on cerebellum, frontal cortex and hippocampus. A severe vascular hyalinopathy was present throughout the brain, and in the cerebrum involving even subcortical vessels. Congophilic angiopathy was not seen.FMR1 mosaicism was identified by Southern Blot and PCR analysis in both peripheral blood and brain tissues. DNA analysis demonstrated the presence of methylated FM alleles  and a PM allele in ~25% of cells (78 CGG repeats) in peripheral blood. Genotyping analysis of DNA isolated from post-mortem brain tissues showed that similar FMR1 mutation patterns  were conserved in the brain  and frontal cortex (0.05 ± 0.008). Brain sections from the same brain block that were used for determining CGG repeat size by Southern Blot and FMR1 mRNA levels by QRT-PCR were used for determining FMRP expression by Western Blot (WB) analysis and immunohistochemistry (IHC). WB analysis did not detect any FMRP  and 4.49% of astroglial cells (11 of 245 cells counted). The CA3 and the Hilus were, however, not well represented in these sections and were not counted. Every neuron and astroglial cell was counted in this section similar to the dentate gyrus granule cells.Similar to the quantifications in the hippocampus, the frontal cortex and temporal cortex were sampled. In this case, a single gyrus was chosen by marking the slide prior to placing under the microscope. The gyrus and sulci on either side were completely sampled and all neurons and astroglia were counted, as well as the number of intranuclear inclusions in each cell type  and 8.03% of astroglial cells (25 of 311 cells); Frontal cortex: 3.40% of neurons (7 of 206 cells) and 4.51% of astroglial cells (19 of 421 cells). The temporal cortex and frontal cortex were chosen for comparison with previous studies of inclusions in male and female subjects with FXTAS 9 cells a, as wellTo quantify the presence of intranuclear inclusions in the cerebellum, a protocol used to quantify neuropathological features in FXS  was modiFMR1 size mosaicism consisting of a FM and a PM allele who was diagnosed with FXS and whose phenotype was related to the striking reduction of FMRP expression in the brain. Later in life this individual also showed features congruent with the minor diagnostic criteria for FXTAS, but this diagnosis was not given due to lack of appreciable FMR1 mRNA levels and the presence of FXS symptomatology [FMR1 mRNA expression, most likely due to the presence of a PM allele in a small percent of cells, was detected in peripheral blood [FMR1 mRNA throughout the brain and in different brain regions  did not seem elevated, it was likely to be elevated in cells that were actually transcriptionally active  and therefore leading to RNA toxicity due to the presence of the CGG repeat expanded alleles as reported in other cases [This study examines the case of a male with matology . Howevermatology . FMR1 mRal blood  and in ter cases . Thus, tFMR1 premutation, as little as 5-6% in the cerebellum and in the frontal cortex, may contribute to a process of neural degeneration in this individual in addition to his parkinsonism. As cells in our brain are involved in many networks, alterations such as protein sequestration and mRNA toxicity in small pockets throughout the brain could potentially lead to significant pathway dysregulation  amounting to major signaling network breakdown. In addition, when the subject developed symptoms of FXTAS, he had already experienced a great deficit of FMRP and the consequent related phenotype. This may suggest that early in life the presence of the PM allele, producing small levels of FMRP, could have helped to ameliorating some of the FXS phenotype. In fact, the patient was not diagnosed with FXS until late in life at age 60.It has been shown that expansion of the CGG repeat leads to sequestration of a number of proteins, including DROSHA and DGCR8, which are key players in the microRNA biogenesis as described in  and indeThe present finding of symptoms and pathological and molecular evidence of FXTAS in a FXS mosaic male, raises a number of diagnostic issues for FXTAS. The finding also raises concerns since, during antemortem investigations this case received a diagnosis of parkinsonism with cognitive decline based on clinical features, rather than a diagnosis of FXTAS . What thFMR1 gene). In contrast, FXTAS is believed to result from a toxic RNA gain of function mechanism resulting in alterations of cellular function [The diagnosis provided by our group was based on the knowledge of the neurobiology of FXS and FXTAS at the time. Most working models of FXS pathogenesis posit that FXS is the result of dramatically reduced or absent expression of biologically active FMRP (i.e., no FMRP expression due to the expansion of the CGG repeats or deletion or mutation in the coding region of the function . In thisDuring antemortem investigations, the diagnosis of parkinsonism was made rather than FXTAS-though the potential for a patchy presentation of FXTAS-like symptoms was mentioned as a remote, albeit unlikely possibility at the time. After receiving these diagnoses and following the death of this individual, postmortem brain tissue was available for analysis. Similarly to the three cases previously reported , a sparsIn conclusion, it is likely that the neuropathological changes of FXTAS were present here and that this patient had FXS and FXTAS, and Parkinson Disease.Dr. Randi Hagerman has received funding from Roche, Novartis, Seaside Therapeutics, Forest, Curemark and the National Fragile X Foundation for clinical trials in fragile X syndrome and/or autism. She has also consulted with Novartis regarding treatment in fragile X syndrome. All other authors declare that they have no conflict of interests. This work is dedicated to the memory of Matteo.TF designed and coordinated the study, drafted the manuscript and participated in data analysis and interpretation of the results. DP participated in the design and coordination, ran experiments, drafted the manuscript and participated in data analysis and interpretation of the results. HMR ran experiments, drafted the manuscript and participated in data analysis and interpretation of the results. CLC ran experiments, drafted the manuscript and participated in data analysis and interpretation of the results. CMG performed pathological analysis and revision of the manuscript. RJH performed clinical evaluation, participated in drafting and revision of the manuscript and participated to the data analysis and clinical interpretation. SCN revised the manuscript and participated in data analysis and interpretation of the results. DAH performed clinical evaluation and participated in revision of the manuscript. PJH revised the manuscript and participated to the data analysis and interpretation of the results. All authors read and approved the final manuscript.


While WHO guidelines recommend iron supplements to only iron-deficient children in high infection pressure areas, these are rarely implemented. One of the reasons for this is the commonly held view that iron supplementation increases the susceptibility to some infectious diseases including malaria. Secondly, currently used markers to diagnose iron deficiency are also modified by infections. With the objective of improving iron deficiency diagnosis and thus, its management, we evaluated the performance of iron markers in children exposed to high infection pressure.ROC) , and were the most sensitive markers in detecting iron deficiency , but with moderate specificities .Iron markers were compared to bone marrow findings in 180 anaemic children attending a rural hospital in southern Mozambique. Eighty percent (144/180) of the children had iron deficiency by bone marrow examination, 88% (155/176) had an inflammatory process, 66% (119/180) had moderate anaemia, 25% (45/180) severe anaemia and 9% (16/180) very severe anaemia. Mean cell haemoglobin concentration had a sensitivity of 51% and specificity of 71% for detecting iron deficiency. Soluble transferrin receptor (sTfR) and soluble transferrin receptor/log ferritin (TfR-F) index (adjusted by C reactive protein) showed the highest areas under the ROC curve (AUCIron deficiency by bone marrow examination was extremely frequent in these children exposed to high prevalence of infections. However, even the best markers of bone marrow iron deficiency did not identify around a quarter of iron-deficient children. Tough not directly extrapolated to the community, these findings urge for more reliable, affordable and easy to measure iron indicators to reduce the burden of iron deficiency anaemia in resource-poor settings where it is most prevalent. Iron deficiency (ID) is the most common and widespread nutrient deficiency, affecting approximately two billion people worldwide and resulting in over 500 million cases of anaemia By six months of age there is a physiological depletion of the iron stores that were accumulated by the foetus in the last months of pregnancy. If the infant’s diet does not provide enough iron, there is a significant risk to develop IDA. This physiological iron deficiency is often exacerbated by the early introduction of weaning foods The controversy around the risk-benefit ratio of giving iron supplements to individuals exposed to malaria is still unresolved The diagnosis of IDA may be suggested by some signs and symptoms, but specially by blood tests indicating low haemoglobin, ferritin, and plasma iron levels. However, it has long been recognized that in developing countries interpretation of these and other biochemical tests is limited by the confounding effects of infection, inflammation and malnutrition A reliable, non-invasive tool for the assessment of ID in these populations remains elusive. The use of the ratio of soluble transferrin receptor to log ferritin concentrations (sTfR/log ferritin index) has been advocated to assess iron status Until now, the microscopic examination of Perl’s Prussian blue stained bone marrow aspirate remains the “gold standard” for the assessment of iron stores A previous study among severely anaemic Malawian children comparing various iron markers against bone marrow iron content found that TfR-F index was the best predictor of bone marrow iron stores deficiency (sensitivity 74% and specificity 73%) The study protocol was approved by the National Mozambican Ethics Committee and the Hospital Clínic of Barcelona Ethics Review Committee. Parents-guardians were informed of the goals, procedures, benefits and risks of taking a bone marrow sample from their child, and it was never offered to them any financial or material inducement to agree on it. They were also given the choice of consenting to the participation of their child in the anaemia case-control study but refusing to bone marrow sample donation for the iron biomarkers study here presented. All the explanations were given in Portuguese  and when required in Changana . The parents-guardians of all children included in the study provided written informed consent.Plasmodium falciparumThe study was carried out at the Centro de Investigação em Saúde de Manhiça (CISM) in Manhiça District, southern Mozambique. The characteristics of the area have been described in detail elsewhere +, Änghelom, Sweden). A complete clinical examination was performed and the information was entered onto standardized questionnaires together with demographic data. Four ml of venous blood were collected by venipuncture for malaria parasitaemia examination, bacterial culture, full blood count and biochemical and molecular determinations. Participating children were offered voluntary HIV counselling and testing. A bone marrow aspiration was performed from the anterior-superior iliac crest or the tibia, under conscious sedation with parenteral ketamine, atropine and diazepam bronchial hyper-secretion and bone marrow aspirate was then not performed. Two other children vomited during the aspirate, also due to the administration of sedatives. Resuscitation equipment was always available during the procedure. All children received treatment according to their clinical condition and following national guidelines.The study was undertaken as part of a case-control study on the aetiology and risk factors of anaemia in children less than 5 years of age. Children aged 1 to 59 months, attending the MDH emergency department between October 2008 to August 2010 with anaemia (haemoglobin (Hb) <11 g/dl), and with no history of blood transfusion in the preceding 4 weeks, were recruited as cases if their parents-guardians gave written informed consent. Haemoglobin concentration was measured at the time of recruitment by the HemoCue® system  was performed on microscopically negative samples A complete blood count was performed on an automated haematology analyzer Sysmex XT-2000i . Plasma was stored at −80°C until iron biochemical markers were determined. Plasma iron, transferrin and C reactive protein (CRP) were measured in an ADVIA 2400 analyser . Ferritin was measured in an ADVIA Centaur analyser . sTfR was measured in a BN-II nephelometer . Transferrin saturation and TIBC were calculated from the transferrin and iron data according to a standard formula Bone marrow smears were air-dried, fixed with formaldehyde vapour and stained by the Perls' Prussian blue method using clorhidric solution of potassium ferrocyanide and Harris haematoxylin. Bone marrow iron content was semi-quantitatively estimated classifying the amount of blue stained haemosiderin perls in bone marrow fragments (aggregates of bone marrow cells) according to 4 categories: 0 (absent), 1 (diminished), 2  and 3 (abundant) P. falciparum infection was defined as presence of asexual parasites in blood detected either by microscopy or real time qPCR. Clinical malaria was defined as the above plus fever (axillary temperature ≥37°C) or history of fever in the preceding 24 hours. Inflammation was defined as CRP≥1 mg/dl Moderate anaemia was defined as an Hb concentration <11 and ≥7 g/dl, severe anaemia as Hb <7 and ≥5 g/dl, and very severe anaemia as Hb <5 g/dl. Internationally accepted cut-off values for the iron status markers used in the analysis were as follows: i) ferritin <12 ng/ml if CRP<1 mg/dl, and <30 ng/ml if CRP≥1 mg/dl ROC) were calculated ROC is close to 0.5. Therefore an AUCROC not statistically different to 0.5 indicates an ineffective test ROC≥0.75, ROC curves were used to identify new cut-off values with maximal sensitivity to detect ID maintaining the specificity as high as possible over 50%. All comparisons were made for a two-tailed significance level of 0.05. The analysis was performed using the statistical software STATA .The prevalence of iron stores deficiency diagnosed by each marker was estimated as the percentage of children with a value of that marker outside the internationally accepted normal range. The classification of ID by each marker was compared with the classification obtained using the “gold standard” (iron content in the bone marrow) to determine sensitivity, specificity and accuracy of each of them. To visualize the efficacy of each marker to detect ID, Receiver Operating Characteristics (ROC) curves were constructed and the areas under the resulting ROC curves  underwent a bone marrow aspiration. Reasons for not performing the bone marrow aspiration were: age below 3 months in 32 (7%) cases, potential risks of sedation in 65 (15%) cases , adverse effects of sedation in 1 (0.2%) case, technical problems that did not allow the bone marrow aspiration in 47 (11%) cases, and parental withdraw of consent in 6 (1%) cases. Of those children with a bone marrow sample, bone marrow iron content could not be assessed in 112 (38%) cases because of absence of marrow fragments in the bone marrow smears. Thus, the analysis is restricted to the 180 (62%) cases with bone marrow smears assessable for iron content.Children included in the analysis had an average age, gender distribution and mean Hb concentration similar to that of children who were not included. The mean ages of children included and not included in the study were (mean±SD) 22.06±13.67 and 19.97±13.56 months, respectively (p = 0.1229); the percentage of males was 57% and 59%, respectively (p = 0.6421); and the mean Hb concentrations were (mean±SD) 7.73±1.97 g/dl and 7.85±2.03 g/dl, respectively (p = 0.5584).P. falciparum infection and 43% (73/170) had clinical malaria. An EBV infection was detected in 31% (56/180) of the children, PV-B19 was found in 8% (15/180), 24% (40/164) were HIV positive and 8% (13/173) had bacteraemia. Seventy eight per cent (32/41) of the children were α-thalassaemia carriers.Demographic and clinical characteristics of the study participants are shown in Prevalence of iron stores deficiency by the different markers according to their internationally accepted normal levels is shown in ROC for each marker are shown in ROC than 0.5. Among them, sTfR and TfR-F index showed AUCROC≥0.75 (0.75 and 0.76 respectively) (The AUCctively) , thus ROThis is the first study on the evaluation of iron markers to identify ID in a high infection pressure setting among anaemic children with any degree of anaemia. The study compares iron markers to bone marrow iron content as the “gold standard”, and shows that detection of ID still remains unresolved in settings with high infection pressure, where ID is most prevalent and its diagnosis and management most needed.In agreement with a previous report, in this study ferritin, transferrin and TIBC had the lowest sensitivities to diagnose ID The TfR-F index has been suggested as a useful parameter for the identification of iron depletion even in settings with high infection pressure ROC (≥0.75). The sTfR ROC curve indicated that there was no alternative cut-off with higher sensitivity than that of the current one (1.76 mg/l) without lowering the specificity below 50%. For the TfR-F index, the ROC curve showed that the sensitivity of this marker could be improved from 42% to 78% by changing the current cut-off from 1.5 to 0.86. It can be noticed that the performance of TfR-F index with the cut-off of 0.86 is similar to the performance of TfR-F index corrected by the CRP level . However, this similarity is not coincidental, since 88% of the study participants had a CRP≥1 mg/dl. This observation is in contrast with that of a previous study, whereby in spite of a similar prevalence of inflammation (89%) it was found that the TfR-F index unadjusted by the CRP level was a good marker of ID We found that sTfR, TfR-F index (adjusted by the level of CRP), and transferrin saturation showed the highest sensitivities. Moreover, sTfR and TfR-F index showed the highest AUCROC of only 0.59 (p = 0.3382). This finding is also in contrast with the performance of this marker observed in the Malawian study where the AUCROC of MCHC was 0.68 (p = 0.001) In this study, the MCHC, which could be a potentially feasible iron marker for resource poor settings, had an AUCDifferences in the participant’s selection criteria between the Malawian study and the present one may explain the discrepancies observed in the performance of the different iron markers studied. In the aforementioned study only severely anaemic children were included (Hb<5 g/dl), which may preclude its general applicability to the majority of anaemic children who do not have severe anaemia. In the present study all children with anaemia of any degree were recruited (Hb<11 g/dl). They were children with clinical conditions that required hospital admission and for whom investigation of anaemia is recommended in other less resource-limited settings. The physiopathology of anaemia may vary by its severity The findings of this study show that the majority (80%) of the anaemic children were iron deficient by direct assessment of iron stores, and that sTfR and TfR-F index adjusted by CRP are the most sensitive markers with specificities of at least 50% to identify ID in this study population. However, even with these markers, 17% and 25% of children, respectively, will not be diagnosed of ID and therefore adequately treated. The fact that the children included in the study were those attending the hospital may limit the extrapolation of the findings to children in the community. However, obvious ethical reasons would not have allowed to perform bone marrow aspirations in healthy (though may be iron-deficient) children; on the other hand, children attending the hospital with anaemia are likely to be those with the greatest need to be diagnosed and adequately treated.In summary, even the best indirect indicators of ID not only failed to detect an important proportion of iron-deficient cases, but also their assessment is not feasible in most developing settings where the majority of ID occurs. Thus, more reliable, affordable, and easy to measure iron markers are urgently needed to reduce the burden of ID anaemia in resource-poor settings where it is more frequent and severe.


Porphyra umbilicalis Kützing from Schoodic Point, ME, 2) determine whether there are seasonal trends in species diversity but a core group of bacteria that are always present, and 3) to determine how the microbial community associated with a laboratory strain (P.um.1) established in the presence of antibiotics has changed. P. umbilicalis blades  were analyzed by pyrosequencing over two variable regions of the 16 S rDNA . The bacterial taxa present were classified at an 80% confidence threshold into eight phyla . The Bacteroidetes comprised the majority of bacterial sequences on both field and lab blades, but the Proteobacteria  were also abundant. Sphingobacteria (Bacteroidetes) and Flavobacteria (Bacteroidetes) had inverse abundances on natural versus P.um.1 blades. Bacterial communities were richer and more diverse on blades sampled in fall compared to winter. Significant differences were observed between microbial communities among all three groups of blades examined. Only two OTUs were found on all 12 blades, and only one of these, belonging to the Saprospiraceae (Bacteroidetes), was abundant. Lewinella (as 66 OTUs) was found on all field blades and was the most abundant genus. Bacteria from the Bacteroidetes, Proteobacteria and Planctomycetes that are known to digest the galactan sulfates of red algal cell walls were well-represented. Some of these taxa likely provide essential morphogenetic and beneficial nutritive factors to P. umbilicalis and may have had unexpected effects upon evolution of macroalgal form as well as function.Macroalgae harbor microbial communities whose bacterial biodiversity remains largely uncharacterized. The goals of this study were 1) to examine the composition of the bacterial community associated with  Ulva spp. and related green algae) was lost in axenic cultures. Normal algal morphology could sometimes be restored by reinfection with mixed bacterial cultures or by the application of plant hormones Cytophaga sp. (Bacteroidetes), crystallized, and demonstrated to be effective in restoring normal morphology in axenic cultures of ulvalean algae Porphyra umbilicalis, because the blade phase of its close relative Pyropia yezoensis, does not develop in axenic culture Algal-bacterial associations are important to the biology of both groups of organisms, but the biodiversity of these associations remains largely unexplored and the ways in which bacterial-algal interactions impact metabolic and developmental features of the partner organisms are not well understood. A range of symbiotic, pathological and opportunistic interactions between macroalgae and bacteria are being discovered Ulva spp. Fucus vesiculosus, Saccharina latissimaDelisea pulchra, Gracilaria vermiculophyllaFew studies have attempted to characterize the microbial communities of macroalgae with techniques that surmount the well-known under-sampling of communities by culture techniques. Studies at appropriate geographic scales with 16 S rDNA DGGE, 16 S rDNA clone libraries, and more recent metagenomic approaches have expanded our understanding of microbial communities associated with P. umbilicalis by high-throughput pyrosequencing of the 16 S rDNA. This alga is commercially important as human food (“laver”), and it is widely distributed on rocky intertidal shores of the north Atlantic Porphyra sensu lato  are limited to culture and cloning studies or intensive analysis of a few pathogenic bacteria belonging to Flavobacteria and Gammaproteobacteria .Porphyra umbilicalis was collected from the intertidal zone at Schoodic Point, Acadia National Park, Maine  on October 28, 2010  and January 3, 2011 (winter). Five P. umbilicalis blades were collected individually in plastic bags using random numbers' sampling along a 30 m transect placed through the middle of the intertidal zone in each season. Positions for fall blades along the transect were 0 m (F1), 3 m (F2), 5 m (F3), 7 m (F4), 13 m (F5); winter blades were collected at 3 m (W1), 10 m (W2), 16 m (W3), 26 m (W4) and 28 m (W5). Blades were transported back to the laboratory on ice, rinsed briefly in 15°C autoclaved seawater within 2 h of collection to remove any superficially-attached microbes, frozen in liquid nitrogen, and stored at −80°C. The P. umbilicalis blade samples that were collected in the winter had fewer epiphytes based on observation with bright-field microscopy before blades were frozen.Porphyra umbilicalis laboratory blades were derived originally from a single blade collected at Schoodic Point on April 3, 2008; this is the same site where the field samples for this study were collected. Laboratory cultures in WES-PES seawater −1 penicillin G and 25 mg L−1 dihydrostreptomycin  using aquarium seaweed clips. The cultures were incubated in Percival 136LL VL incubators  at 10°C, a 14:10 L:D photoperiod and irradiance of 60 µmol photon m−2 s−1. Two clonal blades (hereafter “Lab”) were frozen and stored for DNA extraction. These blades experienced identical laboratory growth conditions and reproduction in the laboratory was exclusively by the generation of neutral spores P. umbilicalisEach sample consisted of an entire blade, allowing recovery of bacteria living on or inside any part of the blade. Each blade was ground in liquid nitrogen using a sterile mortar and pestle. About a 200 µl volume of the powdered tissue was transferred into an 1.5 ml microcentrifuge tube containing lysis buffer and RNase A solution. Tubes were placed on a rotator for 12 min at room temperature, and then genomic DNA was extracted using the DNeasy® Plant Mini Kit .P. umbilicalis, we obtained sequences of the V5–V7 and V8–V9 regions of the 16 S rRNA gene. Whole blade material was used for DNA extraction to minimize the potential loss of bacteria living inside blades, but this also leads to the extraction of chloroplast 16 S rDNA, which would be amplified by most primers that amplify bacterial 16 S rDNA sequences. To overcome this problem, we used a modified primer set 799f [5′-AMCVGGATTAGATACCCBG] To explore the diversity of bacteria associated with −1. Two pyrotag sequence libraries were generated by RTL from each of the 12 Porphyra blades using the primers given above, with barcoded sequences in each library recovered by sequencing from V5 through V7 or from V8 through V9. Raw sff files were provided by RTL for analysis, and these are available in the Sequence Read Archive  under accession SRA065667/SRP018227. We used the mothur (v.1.27.0) software package P. umbilicalis pyrosequencing data as an internal reference to detect chimeras with the chimera.uchime command Bacterial tag-encoded FLX 16 S rDNA amplicon pyrosequencing (bTEFAP) E. coli position 800 E. coli position 1190 and ending near position 1407. This means that these sequences include most of the conserved region before V8 and end just prior to the V9 region; this dataset is referred to as V8 in the rest of this study.Curation of the V5V7 sequences to reduce pyrosequencing errors resulted in a dataset in which sequences had a mean length of 266 bases (range: 249–281). Sequences began near Sequences were classified using the Ribosomal Database Project's rdp9 reference set in the mothur pipeline at an 80% confidence level, which is regarded as 89% accurate in classification at the genus level p-value ≤0.05) associated with the axes of the non-metric dimensional scaling (nmds) ordination used to plot relationships of the 12 samples were plotted using the statistical computing package R , winter (n = 5) and lab (n = 2). V8 samples were normalized to the number of sequences in the smallest sample  from October 1–31, 2010  and December 1–31, 2010 (winter). Photoperiod was estimated from the Eldridge Tide and Pilot Book 2012We obtained relevant environmental data for the Maine coast close to our study site from a coastal GoMOOS environmental station  and removed  from trimmed and chimera-checked sequences before tabulation of curated sequences  and 41,514 (V8) sequences  were obtequences . Most ofequences  probablyequences . Some V5equences .Seven phyla  were identified in the V8 library . The claLewinella (Saprospiraceae) was particularly abundant on P. umbilicalis blades, representing a total of 3.35% of all sequences recovered in the V8 library , which clustered into 66 OTUs, by far the most OTUs within a described genus  taxa . Most ofP. umbilicalis at the fall sampling date; this sample size was more suitable for the winter time point, although additional sampling would clearly find additional OTUs (0.03 distance). The group rarefaction analysis showed that analysis of 2 blades from the clonal laboratory cultures was satisfactory , 0.05 (“genus”) and 0.10 (“family”) distance was compared to estimates of the minimum true predicted richness with the Chao1 estimator . The numThe Bacteroidetes (866 OTUs) and Proteobacteria  were OTU-rich over all samples . Unclassp<0.006; F-W, p<0.001; L-W, p<0.001). Microbial communities of lab blades segregated from other samples, and the position of microbial communities on fall and winter blades in the dendrogram did not relate closely to blade positions along the linear transect in the intertidal zone. A dendrogram based upon distances from the Jaccard calculator . Lab communities and fall communities formed two discrete clusters, and there was more trend in the dendrogram toward relevance of the blade's transect position. The microbial communities from the three groups  were significantly different , the normalized community remained representative of the parent sample cf.  and was lculator  also shoWe identified shared OTUs (0.03 distance) in each library  without sample normalization in order to use the greatest sequencing depth across samples to identify the number of OTUs shared among groups . OTUs (0.03 distance) formed from V5V6 sequences for this analysis represented 4,599 OTUs, and 2,309 of these were found in Bacteria that classified to phyla and lower taxonomic levels.P. umbilicalis based upon the V8 analysis found 13 OTUs in common, and 11 OTUs had an abundance of at least 1% of all seqeuences on at least one of the 12 blades, including Granulosicoccus (Gammaproteobacteria). Eight of the core taxa were members of the Sphingobacteria; four of these belong to the Saprospiraceae, including one found across all 12 blades at high abundance, being represented by more than 1% of all sequences from each blade ; Aureispira, Haliscomenobacter, Lewinella, and unclassified Sphingobacteriales including Saprospiraceae and Chitinophagaceae (Sphingobacteria); Tenacibaculum (Flavobacteria); Rhodopirellula (Planctomycetacia); Hellea, Loktanella, and a large proportion of unclassified Rhodobacteraceae (Alphaproteobacteria); and Granulosicoccus (Gammaproteobacteria).Fall and winter microbial communities on blades shared 269 OTUs . The shaLewinella (Sphingopyxis in Alphaproteobacteria (p<0.05) associated with one or more of the nmds axes demonstrated that: 1) different OTUs belonging to the Alphaproteobacteria  were at least 3× more abundant in fall compared to winter , but not the same OTU. Several of the most abundant OTUs in Lewinella were sequence-rich, and these OTUs affect distances between samples within groups in nmds plots (Lewinella were represented by single sequences.Lacinutrix), Chloroflexi, Planctomycetes (both classses including Phycisphaera in Phycisphaerae), and an OTU in the Alphaproteobacteria  and water (11–13°C) temperatures were higher, and the photoperiod was longer (12 L:12 D) in contrast to December when air (−5–10°C) and water (7–9.5°C) temperatures were lower and the photoperiod was shorter (9 L:15 D)Porphyra umbilicalis identified 8 phyla containing 76 genera, including 48 genera that have been isolated and formally described. Bacteria belonging to the Bacteroidetes  were most abundant and also comprised the most species-level OTUs; however, the Proteobacteria, especially Alphaproteobacteria, were also abundant and OTU-rich. Clonal laboratory blades of “P.um.1”, the P. umbilicalis isolate that is part of a whole genome sequencing project, had much reduced microbial richness and diversity compared to field blades, and most bacteria remaining on blades were Bacteroidetes belonging to the Flavobacteria. The two days when we sampled blades from the Maine shore were in mid-fall and winter, and several statistical comparisons found that the microbial communities were different from each other on these two days and also that each was different from the microbial community on lab blades. Fall blades had the highest observed and predicted (Chao1) richness and the highest diversity (inverse Simpson Index). Only two OTUs were found on all blades in this study, and only an OTU belonging to the Saprospiraceae (Bacteroidetes) was found abundantly on all blades. Although the genus Lewinella was found on all field blades, none of its 66 OTUs was universally present on these blades. This pyrosequencing study increases our understanding of the microbial communities of the red algae and makes it possible to compare the bacteria associated with marine red and green algae, which have different cell wall compositions but are two of the three known groups of algae formed in primary endosymbiosis.Pyrosequencing of microbial communities associated with Delisea pulchraGracilaria vermiculophyllaUlva australis (Chlorophyta). These offer among the best comparisons available to our results on P. umbilicalis. The microbial community of G. vermiculophylla is of special interest because it has agar-rich cell walls, sufated galactans that are structurally similar to porphyran in the Porphyra cell wall Gracilaria was richest in Bacteroidetes followed by Proteobacteria, as found for P. umbilicalis in the present study. They found temporally consistent differences in the composition of the microbial community at the level of phylum between Gracilaria, the green alga Ulva and the brown alga Fucus vesiculosus from individuals collected within 20 m of each other. Longford et al. 's similar study Delisea pulchara (Rhodophyta); Bacteroidetes and Proteobacteria were also prominent on the red alga Delisea in that study. More recently, Burke et al. U. australis with over 5,000 nearly full-length 16 S rDNA sequences, finding that 74% of all sequences classified to Alphaproteobacteria (Proteobacteria) and 13% to Bacteroidetes. Given that only 6 OTUs  were found in common across the 6 samples of Ulva blades in their study, they proposed Ulva's obligately symbiotic microbes must operate as a lottery that in functionally-equivalent, but OTU-distinctive, subsets can supply the host with its microbial requirements . This concept was supported by their recent metagenomic study Porphyra blades, because even using the higher sequencing depth of pyrosequencing, only 1 OTU from the Saprospiraceae was found in common over all 12 blades in our study. However, the natural microbial community of the temperate Northern Hemisphere P. umbilicalis has some striking similarity to that reported from the temperate Southern Hemisphere U. australisLewinella and Haliscomenobacter, unclassified Flavobacteriaceae, unclassified Planctomycetaceae , and the Proteobacteria Krokinobacter, Loktanella, Maribacter, Roseovarius, unclassified Roseobacteriaceae, and Winogradskyella. The widespread level of horizontal gene transfer among taxa within and among microbial taxa The microbial communities of two red macrophytes from temperate latitudes, There was little variation in the taxa found to be present between the two libraries, especially when taking into account the nearly 2× higher coverage of the V5V6 library. However, Chloroflexi were abundant in the V5V6 library compared to being a small component of the microbial community in the V8 library. Both Deinococcus-Thermus and TM7 were recognized at trace levels in sequences within the V8 and V5V6 libraries, respectively; additional sampling depth in either might reveal both phyla. It is well-known that the 16 S rDNA evolves at different rates along its length Granulosicoccus). Nearly all of the Sphingobacteria were lost due to antibiotic treatment of blades but the core OTU in the Saprospiraceae (OTU 2521) remains. Because the laboratory blades continue to grow and reproduce normally, although grown in medium with vitamins  and Planctomycetes  blades experience many stresses ; neutral spores that are made are not viable and blades frequently have higher epiphyte loads before dying back to smaller blades Our analysis showed that the microbial communities on Littorina littorea snails. Further, migrating ducks and geese pass through this area in the fall, and they often float above the intertidal zone at high tide. Blade position on the transect showed some trend to be important in community similarity between blades based upon OTU presence or absence  but little such trend in the analysis (thetayc) that considered abundance as well as membership of OTUs in calculating dissimilarity of blade communities. A larger number of blades is required to examine the spatial relationships of microbial communities, especially in fall, based upon the rarefaction analysis done over blades within each group.The observed variability in the number of OTUs and species diversity within blades collected within the same season may be due in part to biotic factors that differentially affect blades. For example, there is differential grazing pressure from amphipods and isopods (Crustacea) as well as small Macroalgae live in a very dynamic environment that is affected by diverse biotic and abiotic factors; all of these will contribute to shaping the bacterial community associated with the blade and producing diversity in the microbial communities of different blades within a species. Despite these diversifying factors, some patterns in the assemblages of bacteria found on macroalgae that are attributable to the different cell wall materials found in host walls are evident, and also some conservation of the most abundant phyla of microbes on the host .Ulva (Chlorophyta) and Porphyra (Rhodophyta). Without particular bacteria, macroalgae literally fall apart, into simple filaments and single cell suspensions Zobellia and CytophagaGiven the greater understanding of the microbiome of macroalgae that is emerging from this study and others e.g., , there iPorphyra belongs to are an ancient group in a phylum in which classes closer to the ancestral rhodophyte are composed nearly entirely of taxa with simple unicellular and filamentous morphologies . It is important to point out that there appear to be “cheaters” among the bacteria in terms of our model; not all strains of Cytophaga (Bacteroidetes) supported normal morphogenetic development in Ulva linzaThe Bangiophyceae that Figure S1Richness of V8 OTUs (0.03 distance) observed and those predicted (Chao1) for normalized samples.(TIF)Click here for additional data file.Figure S2Dendrogram of the microbial community on each blade in the V8 library as clustered (Jaccard coefficient) to examine dissimilarity by group or by position along the intertidal transect for fall and winter blades.(TIF)Click here for additional data file.Figure S3Biplot of all 67 OTUs significantly associated with an nmds axis in a plot of V8 blade samples. See Table S9 for additional information on each OTU.(TIF)Click here for additional data file.File S1Combined file for the supporting information tables.(DOCX)Click here for additional data file.