The aim of this study was to evaluate the diagnostic performance of in-house FISH (fluorescence in situ hybridisation) procedures for the direct identification of invasive fungal infections in blood cultures and cerebrospinal fluid (CSF) samples and to compare these FISH results with those obtained using traditional microbiological techniques and PCR targeting of the ITS1 region of the rRNA gene. In total, 112 CSF samples and 30 positive blood cultures were investigated by microscopic examination, culture, PCR-RFLP and FISH. The sensitivity of FISH for fungal infections in CSF proved to be slightly better than that of conventional microscopy (India ink) under the experimental conditions, detecting 48 (instead of 46) infections in 112 samples. The discriminatory powers of traditional microbiology, PCR-RFLP and FISH for fungal bloodstream infections were equivalent, with the detection of 14 fungal infections in 30 samples. However, the mean times to diagnosis after the detection of microbial growth by automated blood culture systems were 5 hours, 20 hours and 6 days for FISH, PCR-RFLP and traditional microbiology, respectively. The results demonstrate that FISH is a valuable tool for the identification of invasive mycoses that can be implemented in the diagnostic routine of hospital laboratories. The number of invasive fungal infections has increased over the last few decades. This phenomenon is the result of the growing number of pathological or iatrogenic immunocompromising conditions, premature births, neoplasms, abdominal surgeries, medical device insertion procedures and antibiotic therapies ,2.In northern Brazil, specifically in the state of Amazonas, histoplasmosis and cryptococcosis are some of the most frequent causes of death in AIDS patients , and canMolecular techniques, particularly PCR-based approaches, have been developed to detect fungi in a short period of time; these approaches include nested PCR, multiplex PCR, real-time PCR and microarray techniques . AlthougFluorescence in situ hybridisation (FISH) has already been successfully implemented in clinical microbiology for the identification of various pathogens, including fungi -15. The The literature contains several studies that have evaluated FISH for the detection and identification of Candida spp. ,16,17 an2 production) in a blood culture system BACTEC 9120 ; however, these blood samples were negative for bacteria according to Gram stain analysis. Anonymised patient information  was obtained from the computerised system “iDoctor Hospital”, which is used at FMT-HVD.Samples were collected from patients who had been referred to the Mycology Laboratory of the Fundação de Medicina Tropical Dr. Heitor Vieira Dourado, Manaus, Brazil (FMT-HVD) between November 2013 and April 2014. In total, 142 biological samples were investigated, including 112 cerebrospinal fluid (CSF) samples from patients with a clinical diagnosis of cryptococcosis and 30 blood cultures that had presented positive results  and NL4 (5′-GGTCCGTGTTTCAAGACGG-3′) ) and fixed for 4 h with 4% w/v paraformaldehyde in PBS at 4°C. After fixation, the cells were washed twice with PBS, suspended in one volume of PBS and one volume of cold absolute ethanol and stored at −20°C until use.The CSF samples were centrifuged at 10000 × g for 5 min. Then, the supernatant was discarded, and the pellet containing cells was treated following the same procedures described for treating the pellet obtained with CSF samples.For the investigation of pre-incubated blood cultures, 0.5 ml acetic acid (100%) was added to 5 ml blood culture medium to lyse the erythrocytes. The suspension was centrifuged at 10000 × The FISH assay was performed as described by Amann . The whoEthical clearance for this study was obtained from the Ethical Committee at the Fundação de Medicina Tropical Heito Viera Dourado in accordance with Brazilian laws relating to research with human subjects.Conventional microscopy (India ink) and FISH were performed to confirm or exclude the presence of fungal agents in 112 CSF samples from patients with the clinical suspicion of cryptococcosis. Table + cell counts between 211 and 669 CD4+ lymphocytes/ml.Thirty blood cultures were investigated, and 14 showed positive results with traditional microbiological approaches and with PCR-RFLP and FISH analyses. The identified pathogens included the C. neoformans complex (n = 8), Histoplasma capsulatum (n = 2) and Candida albicans (n = 4). Traditional microbiological approaches and PCR-RFLP and FISH analyses showed identical results for the blood culture materials. However, the mean times to diagnosis after the detection of microbial growth in the automated blood culture system were 5 hours, 20 hours and 6 days for detection by FISH, PCR-RFLP and traditional microbiological approaches, respectively. Selected FISH images of the identified microorganisms are shown in Figure The present work demonstrated the application of FISH for the routine detection of the primary causative agents of invasive fungal infections in patients in a diagnostic laboratory in South America. Molecular methods, including PCR and FISH, have been developed for the rapid identification of fungal agents from primary sample materials. FISH is a comparatively inexpensive and easy-to-perform molecular method that has already been successfully implemented in clinical microbiology for the identification of pathogenic species of Candida and Cryptococcus ,16,25.The sensitivity of FISH for fungal infections in CSF proved to be slightly better than conventional microscopy (India ink) under the experimental conditions and allowed the detection of two cryptococcosis patients who were missed by conventional microscopy. Martins et al.  also demFourteen blood cultures from patients with suspected systemic fungal infections showed positive results. The traditional microbiological approaches and PCR-RFLP and FISH analyses gave the same results in terms of the detection (100% sensitivity) and identification of the fungal agents. However, traditional microbiological assays require the sub-culturing of blood culture samples; microscopic assays, including India ink, lactophenol cotton blue and Gram staining; and biochemical assays, including sugar assimilation, sugar fermentation and enzyme production tests. These assays are slow, requiring 3–10 days and trained microbiologists. PCR-RFLP requires DNA extraction, PCR and electrophoresis to assess the PCR products. In addition, the PCR products must be digested by restriction enzymes, and the RFLP profiles must be evaluated. These steps require at least 20 hours and trained staff with experience in molecular techniques. FISH is robust, easier to perform, and considerably faster, with a time-to-result of up to 5 hours.In addition to these results, FISH demonstrated the capability of identifying the fungi directly in the biological samples and did not require sub-cultures; these characteristics are essential for conventional diagnoses. This capability is a great advantage for histoplasmosis diagnosis because H. capsulatum cultures should be manipulated under high biosafety containment conditions . NotablyIn the present work, 16 blood cultures presented “microbial growth” in the blood culture system but did not present bacterial (Gram stain) or fungal growth . The false detection of microbial growth by blood culture systems has been previously described . The simRegarding the limitations of the present study, we did not use latex agglutination tests to investigate the presence of cryptococcal polysaccharide antigen in the CSF samples. However, Martins et al.  developePrevious studies have demonstrated that FISH is a useful tool for environmental microbiology studies . The impThis study is the first to assess the detection and identification of important fungal agents, i.e., Candida spp., Cryptococcus spp. and H. capsulatum, using the routine procedures of an infectious disease hospital in South America. These results suggest that FISH is a valuable, robust, fast and easy to perform tool that can be readily implemented in the diagnostic routine of hospital laboratories.


Staphylococcus aureus isolates corresponding to a pan-European population snapshot, with epidemiological and resistance data. Geospatial visualization of the data is made possible by a generic software tool designed for public health purposes that is available at the project URL (http://www.microreact.org/project/EkUvg9uY?tt=rc). Our analysis demonstrates that high-risk clones can be identified on the basis of population level properties such as clonal relatedness, abundance, and spatial structuring and by inferring virulence and resistance properties on the basis of gene content. We also show that in silico predictions of antibiotic resistance profiles are at least as reliable as phenotypic testing. We argue that this work provides a comprehensive road map illustrating the three vital components for future molecular epidemiological surveillance: (i) large-scale structured surveys, (ii) WGS, and (iii) community-oriented database infrastructure and analysis tools.The implementation of routine whole-genome sequencing (WGS) promises to transform our ability to monitor the emergence and spread of bacterial pathogens. Here we combined WGS data from 308 invasive  The spread of antibiotic-resistant bacteria is a public health emergency of global concern, threatening medical intervention at every level of health care delivery. Several recent studies have demonstrated the promise of routine whole-genome sequencing (WGS) of bacterial pathogens for epidemiological surveillance, outbreak detection, and infection control. However, as this technology becomes more widely adopted, the key challenges of generating representative national and international data sets and the development of bioinformatic tools to manage and interpret the data become increasingly pertinent. This study provides a road map for the integration of WGS data into routine pathogen surveillance. We emphasize the importance of large-scale routine surveys to provide the population context for more targeted or localized investigation and the development of open-access bioinformatic tools to provide the means to combine and compare independently generated data with publicly available data sets. Most bacterial infections of humans are caused by organisms that have a large population size and short generation times, and lineages with novel properties emerge and expand within observable time scales . LineageStaphylococcus aureus represents an epidemiological paradigm because of the undisputed public health relevance of this species, which is characterized by multiple-antibiotic resistance and a potential for swift dissemination through health care, social, and farm animal production networks. Here we demonstrate the utility of WGS when applied to a continental-scale representative “population snapshot” by using a novel data visualization platform. We sequenced 308 S. aureus isolates responsible for invasive infections that were recovered from 186 hospitals in 21 countries across Europe in a 6-month period. We consider three analytical strands: (i) a representative phylogeographic analysis that defines HRCs on a population level, (ii) an analysis of the dynamics of virulence and resistance carried by mobile genetic elements (MGEs), and (iii) an in silico ascertainment of antibiotic resistance encompassing 19 antibiotic compounds of clinical relevance.http://www.microreact.org/project/EkUvg9uY?tt=rc). This tool can be used for any appropriate data set where a phylogenetic tree and associated metadata are available (see http://www.microreact.org).To underline the added value for public health decision making when WGS data are supplemented with epidemiological metadata collected through structured surveys, we developed a web application (Microreact) that allows easy access and visualization of our data by medical and public health audiences and is available at the project URL  of isolates collected as part of a European structured survey of S. aureus from invasive diseases (n = 185) of these isolates were methicillin-sensitive S. aureus (MSSA), and 40% (n = 123) were methicillin-resistant S. aureus (MRSA). A total of 235,226 SNP sites within the core genome were identified by mapping against a single reference genome, HO 5096 0412 (sequence type 22 [ST22]). We have divided the analysis and interpretation of the results into three parts: (i) a broad phylogenetic analysis and more fine-scaled consideration of exemplar lineages of high public health relevance, (ii) the distribution and dynamics of the accessory genes conferring virulence and resistance traits in the context of individual HRCs, and (iii) a comparison of antibiotic susceptibility profiles ascertained by in silico prediction from genome data with conventional susceptibility testing carried out in Staphylococcal Reference Laboratories (SRLs) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) Development Laboratory (EDL).We chose a random sample n = 08; 10.6%spa typing.Analysis of core SNPs resolved the population into 6 major  and 10 minor clonal complexes (CCs) and STs , as previously defined by multilocus sequence typing (MLST)/eBURST  and 2. TS. aureus mutation rate (–The distribution of MRSA and MSSA isolates is not random with respect to CCs . Ninety-ion rate  6–9). T–9. TS. aspa typing was that MRSA variants tend to show geographic clustering, whereas MSSA variants do not  by use of the Microreact tool; a detailed guide is provided in A key observation from our previous work on s do not . The WGSspa type t002, and this observation is consistent with our previous work showing that MSSA genotypes are widely distributed across Europe . The data thus point to the border between Germany and the Czech Republic as being a barrier to health care referral practices and thus MRSA spread.CC5 is the most abundant CC in this study, being represented by 70 isolates, 80% of which are MRSA (red branches) . Three Ms Europe . The 13 mec element. Nine of these isolates define a very tight subcluster corresponding to ST111 (n = 8) and ST1481 (n = 1). Eight of these nine isolates originate from Croatia, which is indicative of rapid epidemic radiation in that country, and three isolates from Split cluster together on the tree, reflecting geographic structuring on a national level. One ST111 isolate originates from Sweden, consistent with the probable importation of ST111 into Sweden from Croatia by travel. The MRSA ST125 cluster consists of eight MRSA isolates restricted to central and northern Spain. For further investigation, see the Microreact tool, where supplementary analysis and geographic detail can be explored.The MRSA cluster corresponding to ST228 and related STs consists of 15 isolates all harboring a type I SCCmec type IVh element and form a tightly clustered starlike phylogeny (CC22 contains epidemic MRSA-15 (EMRSA-15) , which ihylogeny . Fourteehylogeny . In the hylogeny . This clhylogeny . Similarhylogeny .spa types. Given the public health significance of this cluster, we tentatively suggest the designation EMSSA-ST30. All of the isolates in this cluster contain the SNPs in hla and agrC previously reported to attenuate virulence (CC30 contains successful HA-MRSA and community-associated MRSA (CA-MRSA) epidemic clones, including EMRSA-16 and the Panton-Valentine leukocidin (PVL) toxin-positive Southwest Pacific (SWP) clone. irulence  . We categorized these groups according to the types of mobile elements with which they are associated, specifically, phages, SCCmec elements, plasmids, S. aureus pathogenicity islands, and transposons (The accessory genome consists of genes that are variably present in a defined population and can be major drivers of adaptation . An undensposons . Fifty-smec is excluded (data not shown).mec elements were excluded , and Sa1int (27%). Sa4int was very rare, being observed in only four isolates (1.3%). The distribution of these phage types and associated cargo genes across the tree reveals important differences between the CCs and between different clusters belonging to the same CC  or sea/sep . Notably, chp and scn are common in CC15 even though the prophage itself is absent. The most likely explanation for this pattern is that the prophage was once integrated and then lost from the genome, leaving chp and scn behind.Phages are important from a public health perspective, as they are carriers of virulence genes and resistance determinants. We assayed the distribution of seven phage types on the basis of the presence/absence of their integrase genes as described previously . The ovesasX-positive ST239 isolate in our data is more likely to be a native Polish variant and most likely acquired sasX from the imported Asian strains cocirculating in Poland. The data thus point to the horizontal dissemination, presumably via lysogenic conversion, of the phage-borne sasX virulence factor within Europe. Other examples of the horizontal dissemination of virulence genes are evident in our data. We note only a single representative of USA300, an MRSA strain from Belgium that closely clusters with the reference USA300 genome and contains a type I arginine catabolic MGE (ACME)  see . HoweverE region . The dismec types  were present among the 120 MRSA isolates. Type IV elements are the most widely distributed among different lineages, being present in all five of the major CCs that contain MRSA strains. From the phylogenetic analyses, we estimate that a minimum of 20 independent acquisitions of this element have occurred in our sample. However, the rate of SCCmec acquisition clearly varies between CCs, whereas only a single acquisition of the SCCmec type IVh element is observed in CC22, in CC5, there have been a minimum of seven different acquisition events encompassing five different SCCmec types. For example, the Spanish ST125 cluster harbors both SCCmec type IVc and IVs elements, indicating multiple acquisitions. In contrast, no CC15 isolates have acquired SCCmec. The reversion of MRSA to MSSA because of the deletion of SCCmec appears to be a relatively rare event and was identified in only two CC8 isolates in our sample  of the individual tests , which contributed the isolates to our sample, with the EDL reference data. The SRLs submitted a total of 2,252 AST results. Of these, 2,203 (97.8%) were concordant with the EUCAST test results  results provided by the European  results ; Table 2S. aureus invasive disease isolates in Europe at the time of sampling. However, as with all sampling frameworks, important caveats remain. First, all of the isolates were recovered from invasive infections, primarily blood, meaning that we have underrepresented isolates causing skin and soft tissue infections. Second, laboratories submitted the first five MSSA isolates and the first five MRSA isolates from individual patients. A small number of laboratories did not receive five MRSA isolates during the 6-month survey because of a very low frequency of MRSA disease in their regions, for example in Scandinavia, and these laboratories completed their quota with MSSA isolates.A consistent and comprehensive sampling frame is a crucial component of effective pathogen surveillance in public health, though in reality, this may involve significant logistical and political challenges. By taking a random sample of a larger collection assembled as part of a pan-European structured survey, we have minimized the problems associated with phylogenetic discovery bias  and are Despite these caveats, the combined analysis of WGS with epidemiological metadata addresses the following three key elements of managing infectious disease threats in public health.S. aureus is readily partitioned into highly discrete subpopulations or CCs. These CCs vary in their potential to spawn HRCs such as EMRSA-15  than do the data generated by the SRLs  a consideration of clonal relatedness, abundance, and phylogeographic structure at the population level and (ii) mining of the accessory genome repertoire to ascertain likely virulence and resistance properties. These two perspectives should also be combined when considering appropriate containment measures. The recognition of an HRC through large-scale surveillance would enable the development of tailored rapid diagnostic tests based on distinguishing SNPs or accessory gene signatures. This will allow for selective screening and targeted containment by decontamination and isolation strategies that are easier to implement, more economical, and more likely to be effective than more generic procedures. The expedition of specifically tailored tests during outbreaks would allow health authorities or infection control practitioners to screen potential hosts for colonization or infection, thereby reducing the chance of onward transmission. WGS allows for rapid appraisal of outbreak sources and transmission pathways that will also help in weighing up infection control priorities. Moreover, information regarding the virulence gene repertoire associated with an emerging or outbreak HRC will inform clinicians and medical microbiologists of likely clinical manifestations.Ultimately, the key to managing HRCs lies in making the data available in an open and intuitive format for infection control and public health audiences. This democratization of the data increases the collective power of interpretation  while decreasing the necessity for local expertise in bioinformatics. The coupling of large-scale population sampling by WGS within open-access and freely available web resources empowers the community to identify clone-specific signatures , promoting the design of HRC-specific rapid and cost-effective molecular diagnostic tests .With this communication, we aimed to demonstrate how the integration of WGS into epidemiological surveillance programs provides the means for both the early warning of emerging HRCs and a robust assessment of associated public health threats. However, the advent of the underlying sequencing technology addresses only a small part of the challenge of managing the threat from infectious disease. In order to exploit this technical advance to its maximum potential, two things must happen. First, national reference laboratories need to agree to, and abide by, common standards for the contribution of isolates to structured surveys, ideally at 5-year or shorter intervals, on pancontinental and, ideally, global scales. Second, platforms for bioinformatics need to be developed to allow intuitive management and exploration of the data. These data sets should conform to internationally curated standards for sets of genes and mutations that are recognized as key virulence or resistance determinants, and we propose the initiation of internationally curated data sets to act as a gold standard resource for genomic antimicrobial resistance determinants.The establishment of cumulative databases will engender a far richer understanding of the detailed dynamics underpinning clonal emergence and replacement, national and international transmission, and the horizontal transfer of core genes and MGEs. The increasing public health threat from bacterial pathogens is, in large part, down to the ability of these organisms to rapidly adapt through the dissemination of genes and mobile elements. Our best chance of managing these threats in the future is to emulate, as far as possible, this resource and data sharing through the development of international surveillance networks and a common data exchange infrastructure.S. aureus infections, as described previously (n = 308) of these were randomly selected for sequencing. Isolate details are available for download at the project URL.From September 2006 to February 2007, 357 laboratories serving 450 hospitals in 26 countries collected nearly 3,000 MSSA and MRSA isolates from patients with invasive For each sample, index-tagged libraries were prepared and sequenced in Illumina Genome Analyzer GAII cells with 54-base paired-end reads or Illumina HiSeq with 75-bp paired-end reads. Downstream processing utilized the index tag sequence information to assign reads to individual samples.S. aureus reference genomes. The strain names and accession numbers are as follows: 04-02981, CP001844  against the chromosome of S. aureus strain HO 5096 0412  (https://www.broadinstitute.org/gatk/) (The paired-end reads for the survey isolates and the 26 publicly available genomes were mapped with SMALT (g/gatk/) . UnmappeIn silico reconstruction of STs conventionally generated by MLST for each isolate was carried out with the sequence data as described by Croucher et al.  correction of sequencing errors in reads with Quake v0.3 , (ii) dePdenovo2  by usingPdenovo2 . PredictPdenovo2 , and FASPdenovo2  search oPdenovo2   genes as described previously . Phenotypic resistance was defined by applying inhibition zone diameter epidemiological cutoff values (ECOFFs) and EUCAST clinical breakpoints . We pooled the intermediate and resistant categories into a single nonsusceptible category. For our sample, this partition was fully consistent with the classification into wild-type and non-wild-type isolates defined by the EUCAST ECOFFs. The antibiotics tested were penicillin, cefoxitin, ciprofloxacin, moxifloxacin, amikacin, gentamicin, tobramycin, erythromycin, clindamycin, tetracycline, tigecycline, fusidic acid, linezolid, mupirocin, rifampin, and trimethoprim. MRSA resistance to clinically relevant reserve antibiotics  not suitable for disk diffusion testing was determined by using the respective MIC ECOFFs  with E tests . Phenotypic test results were dichotomized and grouped into wild-type and non-wild-type categories. Results of genomic predictions were compared with the phenotypic classifications as resistant or susceptible as defined by the ECOFFS. Moreover, results that were made available by the SRLs were equivalently dichotomized and also compared to those generated by the EDL in a consistent manner.The antibiotic susceptibility of all http://www.microreact.org/project/EkUvg9uY?tt=rc.Microreact is a Node.js application written in JavaScript. Locational data are displayed by using the Google Maps Application Programming Interface, and phylogenetic data (in Newick tree format) are displayed by using a custom tree viewer developed for the HTML5 Canvas element (PhyloCanvas). Image files (.png) of annotated trees and subtrees can be saved. Instructions and examples are shown at the microreact.org website. The project URL for exploring the data described in this article is WGS data for all of the isolates tested in this study have been deposited in the Sequence Read Archive, and accession numbers are included in the metadata available at the project URL.Text S1 Appendix: Walkthrough of web application for genomic pathogen surveillance. Download Text S1, DOCX file, 1.2 MBFigure S1 mec IV subtypes are shown. Download Phylogenetic reconstruction of CC45. Branch color indicates MSSA (green) or MRSA (red). The symbols at branch tips indicate the geographic origins of these isolates. SCCFigure S1, PDF file, 0.2 MBFigure S2 mec IV clusters show their starlike radiation. SCCmec IV subtypes are shown. The third cluster represents USA300 isolates consisting of two reference isolates and one Belgian isolate. Download Phylogenetic reconstruction of CC8 (excluding ST239). Branch color indicates MSSA (green) or MRSA (red). The symbols at branch tips indicate the geographic origins of these isolates. Close-ups of the two SCCFigure S2, PDF file, 0.2 MBFigure S3 sasX gene are indicated by the letter X. Blue shading highlights isolates belonging to the Asian clade according to Harris et al. . (B) Phylogenetic tree of combined ST239 data obtained from isolates from the reports of Harris et al. and Castillo-Ramírez et al.  and isolates in panel A. Clusters are named in accordance with the reports of Harris et al. and Castillo-Ramírez et al. Download (A) Phylogenetic reconstruction of ST239 isolates in the sample. Red branches depict all of the MRSA isolates. The symbols at the tips of the branches indicate the geographic origins of the isolates. Isolates carrying the Figure S3, PDF file, 0.2 MBFigure S4 Phylogenetic reconstruction of CC15. Starlike phylogeny indicates the recent rapid expansion of this MSSA-only lineage. Symbols at the tips indicate the geographic origins of these isolates. The branch leading to an ST582 isolate—a single-locus variant of CC15—is shortened, and the number of SNPs defining that branch is shown. Download Figure S4, PDF file, 0.2 MBFigure S5 Number of ncHGs per major lineage. The bar chart shows the average number of ncHGs per isolate split into MGE type for each of the six major lineages. Each pie chart indicates the proportion of MGE types for each major lineage. Download Figure S5, PDF file, 0.2 MBFigure S6 mec, for each major lineage. Asterisks indicate lineages with a significant size difference of the accessory genome in MSSA and MRSA isolates. CC8, P = 0.039; CC5, P = 0.0325 ; CC22, P = 0.0539 . Download MRSA or MSSA genome size. The bar chart shows the average number of ncHGs per MSSA or MRSA isolate, respectively, excluding the HGs associated with SCCFigure S6, PDF file, 0.03 MBFigure S7 mec type and MSSA (green) or MRSA (red) (B), and seven prophage types classified on the basis of the presence or absence of their integrase genes (C). Colored boxes indicate presence. Black boxes indicate the presence of virulence genes associated with the prophage. (D) Colored boxes indicate the presence of two virulence genes (sasX and tstH) and acr—the gene commonly used to detect ACMEs. (E) Size and composition of the accessory genome based on the number of ncHGs with further categorization according to MGE type. (F) Close-up of phylogenetic trees of the six major lineages. Download Prophage distribution. Rooted neighbor-joining tree like that in Figure S7, PDF file, 0.8 MBTable S1 S. aureus used to predict genotypic resistance.Known genetic mechanisms of antibiotic resistance in Table S1, XLSX file, 0.04 MBTable S2 Affiliations of and contact information for members of the European SRL Working Group.Table S2, XLSX file, 0.02 MB


Our objectives were to determine the most effective water stratum for eDNA detection, estimate true and false positive eDNA detection rates, and assess the number of water samples necessary to minimize the risk of false negatives. There were 28 eDNA detections in 324, 1-L, water samples collected from four experimental ponds. The best-approximating model indicated that the per-L-sample probability of eDNA detection was 4.86 times more likely for every 2.53 fish/m3 (1 SD) increase in fish density and 1.67 times less likely for every 1.02 C (1 SD) increase in water temperature. The best section of the water column to detect eDNA was the surface and to a lesser extent the bottom. Although no false positives were detected, the estimated likely number of false positives in samples from ponds that contained fish averaged 3.62. At high densities of African jewelfish, 3–5 L of water provided a >95% probability for the presence/absence of its eDNA. Conversely, at moderate and low densities, the number of water samples necessary to achieve a >95% probability of eDNA detection approximated 42–73 and >100 L, respectively. Potential biases associated with incomplete detection of eDNA could be alleviated via formal estimation of eDNA detection probabilities under an occupancy modeling framework; alternatively, the filtration of hundreds of liters of water may be required to achieve a high  level of certainty that African jewelfish eDNA will be detected at low densities .Little consideration has been given to environmental DNA (eDNA) sampling strategies for rare species. The certainty of species detection relies on understanding false positive and false negative error rates. We used artificial ponds together with logistic regression models to assess the detection of African jewelfish eDNA at varying fish densities (0, 0.32, 1.75, and 5.25 fish/m Assessing the distribution, abundance, and dynamics of populations or species frequently requires the collection and identification of individuals from sample locations. As such, species detection is fundamental to scientific disciplines such as phylogenetics, conservation biology, and ecology. The idea of a species being either present or absent from a collection of sites has a long history in ecology, as it provides the foundation for assessing the status and dynamics of species at local and landscape scales. Reliable species detection during sampling, however, can be difficult to achieve, especially for species that are present in low abundances such as threatened and endangered taxa and, in some cases, newly invaded species Recent advances in molecular and forensic methods have provided innovative tools for detecting marine and aquatic organisms that may circumvent the aforementioned limitations Recently, there has been increased attention and scrutiny regarding eDNA detection methodologies Occupancy modeling approaches are widely used in ecological research and management because they can effectively and efficiently account for potential biases associated with imperfect detection Hemichromis letourneuxi) is an aquatic invasive species that has spread throughout southern Florida since its introduction in 1965 3. Difficulties associated with the detection of African jewelfish eDNA when densities are less than 13 fish/m3 is concerning given that the spread or introduction of this species could transpire at densities much lower than the observed value The African jewelfish  determine the most effective water stratum for the detection of eDNA, 2) estimate true and false positive eDNA detection rates at varying fish densities, and 3) assess the number of water samples necessary to minimize the risk of false negative errors when developing eDNA sampling protocols.We used four artificial ponds as mesocosms to estimate and compare detection probabilities of the African jewelfish at differing densities. The four earthen ponds were located at the University of Florida's Tropical Aquaculture Laboratory, in Ruskin, Florida. The dimensions of each pond were approximately 18 m×7.5 m, with an average depth of 1.4 m. On June 10, 2013, 30 days prior to the introduction of fish, ponds were drained, pond bottoms and banks pressure washed, and remaining excess debris removed. Hydrated lime (CaOH) was then applied at a rate of 22.6 kg/pond to ensure that there were no remaining live fish in each pond. Over the course of 72 hrs, ponds were allowed to fill naturally from ground water. Once ponds were full, pH, dissolved oxygen, and temperature were monitored weekly. Three, 1-L eDNA water samples were taken from each pond 15 days before the introduction of the fish to check for any potential contamination. Samples were processed as outlined below, and African jewelfish DNA was absent in each pond of the study system before the fish introduction.3 or 1.7 g/m3), pond III contained 330 fish (moderate density: 1.75 fish/m3 or 9.35 g/m3), and pond IV contained 990 fish : pond I contained 0 fish (control), pond II contained 60 fish (low density: 0.32 fish/mEach water sample was treated with 1 mL of 3M sodium acetate (pH 5.2) and 33 mL 95% non-denature ethanol for DNA/tissue preservation and refrigerated on site until filtration. Each water sample was filtered on site and filter paper frozen until extraction date. DNA was extracted following the protocol of Díaz-Ferguson et al. Taq reaction buffer , 2.5 uL MgCl2 (25 mM), 0.5 uL of each dNTP (1 mM), 1 uL of each primer (10 uM each), 0.20 uL probe Pr028373859 (10 uM), 0.5 uL AmpErase (Uracil-N-glycosylase), and 0.20 uL Amplitaq Gold Taq polymerase .Taqman qPCR assays were employed to detect the presence of African jewelfish eDNA in each water sample collected from the four experimental ponds using primers AJFq3 and AJFRq2 and probe Pr028373859 designed for the target species . Taqman assays were optimized for 20 uL reactions using DNA normalized to a concentration of 25 ng/uL and Taqman core reagents as follows: 2.0 uL of 5× H. letourneuxi, filtered, DNA extracted, and DNA used as a positive confirmation that our qPCR reactions were working correctly in the presence of potential inhibitors. In addition, we sequenced 25% of the positive Taqman assays  for confirmation that the qPCR product was truly that of African jewelfish. All sequences were imported into GENEIOUS v4.8.5 alignment editor , ends trimmed, aligned by eye, and compared for base pair composition and similarity with other African jewelfish sequences previously deposited in the GenBank.All assays were conducted using the following thermal profile: 60 C (1 min), initial denaturation at 95°C for 10 min., followed by 35 cycles of 95 C (15 s) and 60 C (1 min.) Detection of DNA from each sample was performed using a 7500 Fast Real Time PCR machine . Taqman assay quality controls consisted of repetition of all qPCR results and inclusion of two negative qPCR controls (substitution of distilled water for DNA) and a positive qPCR inhibition control for each qPCR plate. The positive control consisted of a water sample taken from each pond, spiked with 5–10 mg/uL lyophilized tissue from Our primary interest was to assess per-sample eDNA detection rates ; however, we also evaluated the prevalence of false positive errors in the sample data. False-negative errors represented instances where, for a given 1 L water sample, qPCR DNA amplification failed to detect African jewelfish when it was known to be present in a pond. In contrast, false-positive errors represented instances where the species was detected when it was known to be absent from a pond . To estimate eDNA detection probabilities, we fitted logistic regression models h) or sample-level (k) factors  i) and transect (j)-level random effects, respectively, that were assumed to be normally distributed with mean of zero and random effect-specific variance 0iu and 1ju represented unique effects associated with sections and transects, respectively, that were unexplained by pond- and sample-level covariates. Because there were no DNA detections in the control pond  among repeated samples, and we suspected that repeated water samples taken from particular sections and transects were dependent pond see , it was We used an information-theoretic approach Prior to fitting candidate models, we evaluated the relative-fit of four different variance structures using the global  model by fitting models that contained several combinations of random effects for sections and transects. The four variance structures included (1) no random effects, (2) a random intercept associated with individual sections, (3) a random intercept associated with individual transects, and (4) a random intercept associated with individual sections and transects. The best approximating variance structure was identified using the Deviance Information Criterion (DIC). The DIC is a Bayesian measure of model fit or adequacy, with smaller DIC indicating a better approximating model We assessed the precision of parameter estimates for each model by calculating 95% Bayesian credible intervals Using parameter estimates from the best-approximating model, we also calculated cumulative detection probabilities to evaluate the number of 1-L water samples required to achieve a specified level of certainty that low, moderate, and high density African jewelfish populations would be detected at least once. We also calculated per-sample detection probabilities as a function of fish density.Hemichromis .A total of 28 detections of African jewelfish eDNA were made across 324 individual 1-L water samples collected from the four experimental ponds . SequencExcluding 81 water samples collected from the control pond , there were 28 detections across the 243 collections taken from ponds that contained varying densities of fish, which corresponded to an overall eDNA detection rate of ∼12%. The detection of eDNA was more prevalent on the surface and bottom (each with 39% of the detections) when compared to the middle of the water column. Notably, there were no false positive detections among the 81 control samples.The best-approximating error structure for the logistic regression models relating eDNA detection/non-detection data to pond- and sample-level covariates included no random effects associated model intercepts and slopes, indicating no substantial dependence among pond transects or quadrants. The assessment of model adequacy using the discrepancy measure method indicated that the global model provided an adequate description of the data, with a Bayesian p-value of 0.58. The confidence model set consisted of four models that contained various combinations fish density, water temperature, and sample position in the water column . The bes3) increase in fish density, whereas the species was 1.67 times less likely to be detected for every 1 SD (1.02 C) increase in pond temperature . The parameter estimate from the logistic regression model fit to the control data indicated that false positive errors were very unlikely, with an estimated per-sample probability of 0.014 . Across Using parameter estimates from the best-approximating model, the number of 1-L water samples required to achieve a specified level of certainty varied considerably depending on the density of African jewelfish aggregates . Per-samNote that our best-approximating model included temperature and not time; yet, we expected a positive relationship between time and eDNA detection. Unfortunately it was difficult to differentiate the effects of temperature and time because temperature declined over the 10-day period in the entire system . To addrThe ability to detect individuals at low densities in aquatic habitats is critical for successful control and management of invasive species While our study and numerous others have illustrated a positive and often significant relationship between organismal density and eDNA detection The eDNA from living macrofauna most likely originates from urine and feces, epidermal tissues, or other secretions such as reproductive fluids and reproductive cells. Most of this material is introduced into the water column as large particles (>1000 um) that remain at the surface for a limited amount of time before sinking or breaking apart Elevated temperature can accelerate the rate of eDNA degradation. Degradation can occur directly by denaturing the DNA or indirectly by increasing enzymatic activity and microbial metabolism 3), the filtration of 3–5 L of water  should provide a high degree of confidence (95–100% probability) to confirm the presence or absence of its eDNA. However, if only a 1-L water sample was collected from our pond containing 990 fish, then our ability to detect eDNA would be approximately 55%. For our ponds that contained 330 and 60 fish, we had a 7% and a 3% chance of detecting African jewelfish eDNA if a 1-L water sample was taken. Conversely, at moderate and low densities, the number of water samples necessary to achieve a 95–100% probability of eDNA detection would approximate 42–73 L and >100 L, respectively. Our findings highlight a well-known and important concept with hypothesis testing – statistical power At high densities of African jewelfish  will come at the cost of filtering more water (either in the form of more volume or more samples). The filtration of hundreds of liters of water through a small micron filter tends to be problematic (filters clog) and often expensive ; alternatively, devices that maximize water volume such as inline filters or plankton nets, may prove valuable for eDNA monitoring of rare species While our study was not specifically designed to address the issue of taking numerous smaller samples vs. one large sample , we recommend the former, especially for rare species in lentic systems. In a lentic environment, the taxon in question may have specific (patchy) habitat requirements; thus, stratifying by habitat and conducting numerous smaller-volume samples would be preferred over taking one large-volume sample. Furthermore, more habitat and sample-specific covariates  are available for potential use in an occupancy model when collecting numerous smaller-volume samples vs. obtaining one large-volume sample.3 (or 70.85 g/m3) in our pond experiment and was a value greater than our highest density pond. Thus, if the species of concern is rare, then the estimation of detection probabilities should be conducted in a larger controlled system that can simulate the rarity of the organism in its natural setting. For an organism inhabiting a lotic system, the use of artificial streams 3Finally, our study emphasizes the difficulties of inferring detection probabilities for an organism inhabiting a natural system simply from aquarium trials. First, even when one fish is placed in an aquarium, it is difficult to simulate lower densities necessary for inferring accurate detection probabilities  with a desired level of confidence.Our results and those of Díaz-Ferguson et al.


Lithium is a drug for treatment of bipolar disorder by correcting mania and reducing depressive mood swings.In this study, effects of Lithium on volumetric parameters of cerebellum were investigated using stereological methods.In this experimental study, 20 sexually mature wistar male rats were selected and divided in two groups randomly (n = 10). Administration and control groups received continuously 0.1 percent Lithium carbonate solution and distilled water respectively with drinking water during a period of 12 weeks. Rat’s cerebellum excised and fixed in modified Lillie’s solution. Then tissues were dehydrated, cleared, and embedded in paraplast in random orientation, and exhaustively sectioned. Ten to twelve sections of ~ 5μm were sampled and stained from each cerebellum by systematic uniform random sampling. The whole section image projected on the table, and using Cavalier’s principle point-counting was employed to estimate the volumetric parameters. Data analyzed by nonparametric statistical test of Mann-Whitney, and differences between groups less than 0.05 considered significant.There were no significant difference in terms of total volume but gray matter volume of cerebellum increased and white matter decreased in administration group significantly (P < 0.05).Administration of 0.1% Lithium carbonate for a period of 12 weeks can affect cerebellar gray and white matters in rat. Lithium is a mood-stabilizer drug that is used to treat manic depressive and mixed episodes of bipolar mood disorders -3. TheraIn this study, effects of Lithium on volumetric parameters of cerebellum were investigated using stereological methods.In this experimental study, 20 male Wistar rats weighing 250-300 g were selected from animal house of Research center of Zahedan University of Medical Sciences (Iran). There was a constant cycle of 12 h light and 12 h darkness; the temperature was maintained at 22 ± 2 ºC, and rats were permitted to access freely to standard laboratory diet and water ad libitum. After 2 weeks of acclimatization, they were divided randomly into control and experimental groups (n = 10); former group received distilled water and the latter received 0.1% (w/v) Lithium carbonate solution through drinking water for a period of 12 weeks. Then the rats were anaesthetized and their cerebellum were excised and fixed in modified Lillie’s solution for 72 hours at room temperature. The cerebellums were dehydrated, cleared, and embedded in paraplast in random orientation, and then exhaustively were sectioned. Ten to twelve sections of ~ 5 μm were sampled from each cerebellum by systematic uniform random sampling. Each of these sections was stained with hematoxylin and eosin and mounted. In order to project whole section image on the screen, a modified slide projector was used . Point counting using Cavalier’s principle was employed to estimate the volume of cerebellum using Where ΣP stands for the number of points hitting the object transects, a/p for the area associated with each point in grid, t¯ for mean distance between sections and, M for linear magnification of image. Coefficient error of point counting was calculated as described previously . All steTotal cerebellar, gray matter, and white matter volumes, and gray/white matter ratio in rats of Lithium-treated and control groups are shown in The present study showed that administration of Lithium carbonate 0.1% (w/v) by drinking water in rat for a period of 12 weeks could cause an increase in gray matter volume and a decrease in white matter volume. There were no statistically significant differences in total volume of cerebellum between two groups. It has been shown that the cerebellum is particularly vulnerable to intoxication and poisoning, particularly at cerebellar cortex and Purkinje neurons. In humans, although the most common cause of a toxic lesion of cerebellar circuitry is associated to alcohol ,the cerebellum is also prone to drug exposure , drug abuse and addiction, and environmental toxins . In line


Petromyzon marinus) in Lake Champlain has been heavily debated over the past decade. Given the lack of historical documentation, two competing hypotheses have emerged in the literature. First, it has been argued that the relatively recent population size increase and concomitant rise in wounding rates on prey populations are indicative of an invasive population that entered the lake through the Champlain Canal. Second, recent genetic evidence suggests a post-glacial colonization at the end of the Pleistocene, approximately 11,000 years ago. One limitation to resolving the origin of sea lamprey in Lake Champlain is a lack of historical and current measures of population size. In this study, the issue of population size was explicitly addressed using nuclear (nDNA) and mitochondrial DNA (mtDNA) markers to estimate historical demography with genetic models. Haplotype network analysis, mismatch analysis, and summary statistics based on mtDNA noncoding sequences for NCI (479 bp) and NCII (173 bp) all indicate a recent population expansion. Coalescent models based on mtDNA and nDNA identified two potential demographic events: a population decline followed by a very recent population expansion. The decline in effective population size may correlate with land-use and fishing pressure changes post-European settlement, while the recent expansion may be associated with the implementation of the salmonid stocking program in the 1970s. These results are most consistent with the hypothesis that sea lamprey are native to Lake Champlain; however, the credibility intervals around parameter estimates demonstrate that there is uncertainty regarding the magnitude and timing of past demographic events.The origin of sea lamprey ( Petromyzon marinus) in Lake Champlain has been the subject of an ongoing debate in recent years , Atlantic salmon , and lake whitefish (Coregonus clupeaformis), as well as lake sturgeon (Acipenser fulvescens). Recent research has suggested that native coastal populations of sea lamprey may have positive environmental impacts on freshwater streams. For example, post-spawning sea lamprey carcasses may be important sources of marine-derived nutrients and materials in oligotrophic streams  is generally proportional to the effective population size (eN). Effective population size is the size of an idealized population , that is subject to the same level of genetic drift and inbreeding as the census population  is 491-bp long and non-coding region two (NCII) is 199-bp long . Second, two coalescent MCMC models were used to explicitly estimate changes in effective population size over time. Mitochondrial sequence data (NCII) were used to generate a Bayesian Skyline Plot (BSP) in the program BEAST to model effective population size history while taking into account coalescent and phylogenetic uncertainty. Previously-published allele frequency data for eight nuclear microsatellite loci , and employed both moment-based methods as well as two classes of coalescent models to explore historical demography. Taken together, these data can provide insight into signatures of demographic events within two separate genomes.The purpose of this study was to use multiple analytical approaches and two sets of genetic markers to investigate historical population fluctuations in Lake Champlain sea lamprey. First, moment-based methods—including a mismatch distribution and Fu’s ite loci  were alsn = 33), Malletts Creek (n = 33), and Beaver Brook (n = 28), representing the northern, central, and southern regions of Lake Champlain, respectively. A broad geographic sampling regime was used in order to test for population structure. All tissue samples were stored in 95% non-denatured ethanol and genomic DNA was extracted using DNeasy Blood and Tissue Kits .To estimate historical population fluctuations within Lake Champlain, fin tissue samples from spawning-phase sea lamprey were obtained from the US Fish and Wildlife Service (USFWS) in May–June 2009. All tissue collection was conducted as part of routine USFWS sampling; the authors of this study received tissue, but did not handle any vertebrate specimens. First, to estimate fluctuations based on mtDNA sequence data, samples were collected at three Lake Champlain tributaries: Great Chazy River (taq polymerase (5 units/µl), 2.5 µl 10X PCR buffer (with 1.5 mM MgCl2), 10 µM each primer, 10 µM dNTP, and 50–100 ng template DNA. Thermal cycler settings were set at an initial denaturation at 95 °C for 5 min followed by 40 cycles of 95 °C for 45 s, 56.5 °C for 45 s, and 72 °C for 1 min; and a final extension at 72 °C for 10 min. PCR products were cleaned using the Wizard SV Gel and PCR Clean-Up Kit  and eluted in 25 µl H2O.The two non-coding regions of the mtDNA genome, NCI and NCII, were amplified in all Lake Champlain samples using PCR with the lamprey-specific primers CR1  and LampTo sequence the Lake Champlain samples at both non-coding regions, forward extension reactions were run for each individual using the forward primers CR1 (for NCI) and CASSFN (for NCII) (5′- GACCCCTAAGTTCATTGC-3′). All primers designed specifically for this study, including LampR, CR1, and CASSFN, were designed using Primer3 . Extensin = 54) were concatenated into a single sequence denoted by NCtotal. Concatenated sequences were then trimmed to a 652-bp region to exclude repetitive portions of reads prone to slippage . Regions prone to slippage were identified by superimposed sequences on the chromatograms. All trimmed sequences were aligned with the MUSCLE algorithm  and Lewis Creek (n = 40) at 8 microsatellite loci. There was no evidence for linkage disequilibrium or significant deviations from Hardy–Weinberg equilibrium  was used to test whether the observed distribution was significantly different from the expected theoretical distribution under a model of expansion  of haplotypes, given the observed haplotype diversity, thereby indicating a population expansion (or contraction).Historical population fluctuations were inferred using three moment-based methods. First, the mismatch distribution of pairwise differences between NCin v.3.1 . Second,xpansion . Third, utations . Fu’s FsP. marinus in Lake Champlain over time. First, the program BEAST v.1.6.2 , based on previous estimates of divergence in mtDNA noncoding regions in fishes (m) was set to 15. The parameter m allows adjacent coalescent intervals to be grouped so that they can have the same eN—it serves to smooth the resulting BSP. The maximum effective population size was set at 10,000, a high estimate based on preliminary coalescent model runs. MCMC chains were run for 506 iterations in triplicate, sampling the posterior distribution every 1,000 iterations. The sampling distribution of the model was evaluated in TRACER v.1.5, with the first 10% discarded as burn-in . To determine how sensitive model results were to the priors, runs with altered priors were compared.A sensitivity analysis for the BEAST modeling was carried out on two parameters—clock rate and maximum effective population size—to assess whether priors biased parameter estimates. These parameters were selected because the priors were based on our preliminary estimates. For each parameter change, three additional MCMC chains were run keeping all other baseline input values constant. We used two alternative maximum population sizes  and one alternate clock rate (2.0 × 10N0), historical effective population size (N1), mutation rate (μ), and time since the demographic change began (t)  credibility intervals. A Bayes factor was also calculated to test whether there was more support for a population expansion or contraction using the method described by Historical demographic changes were also inferred from coalescent modeling of nuclear microsatellite data. Allele frequencies at eight microsatellite loci, previously published by egan (t) . Broad pegan (t) . Five inegan (t)  under a egan (t) , and a No of 0.2 . Each chθ0 = 4N0μ and θ1 = 4N1μ ; ft = t/(2N0) .Sensitivity analyses were not conducted for Msvar because the model is computationally demanding; however, simulation modeling has revealed that Msvar has the power to detect true population contractions even with broad priors . These sTotal mitochondrial sequences . Thus, the Lake Champlain population is considered to be panmictic, consistent with results from previous studies . This indicates that there was no significant deviation from the theoretical model of expansion. Additionally, Fu’s sF statistic was significantly negative , indicating an excess of rare haplotypes, which would be predicted under a scenario of a recent population expansion. Overall, these moment-based methods of studying historical demography with mtDNA data strongly support a population expansion, without explicit estimates of the timing and magnitude of the event.The mismatch distribution of NCxpansion . The diseN estimate; when doubled, the eN estimates also doubled. However, changes in the maximum population size and clock rate priors did not affect the overall pattern of the BSP  + I, where I means there is a significant proportion of invariable sites. The BSP derived from the NCII data shows a decrease in effective population size starting around 400 years ago that continued until 50 to 100 years ago, at which point effective population size slowly began to increase . The sen the BSP .∗ln(BF) = 10.3)  down to approximately 50 currently     , with th = 10.3) . The pos = 10.3) –4D. Ther6, 1440) . The den, 71558) . Finally × 10−3) .θ estimates corresponded with a decline from approximately 2,335 individuals historically  to 60 individuals currently . Despite increased precision, there was still some overlap in the 95% credibility intervals for θ   .P. marinus in Lake Champlain. Synthesizing the results to draw conclusions about the most likely historic scenario of demographic change requires a critical analysis of the results generated by each method, an exploration of sea lamprey ecology, and consideration of the history of land-use and management practices in the region.Genetic-based methods can be useful tools for studying demographic changes in the absence of historical population records, though their precision is linked to the timing and magnitude of the events themselves . In thisMismatch distribution analysis is based on the assumption that demographic changes leave corresponding genetic signatures in neutral sequence data and gene trees . The miseN could be due to the limited number of segregating sites within the NCII sequence data. The sensitivity analysis also showed that the maximum population size prior affected the BSP results, with the estimated eN being proportional to the prior. However, the overall pattern of demographic change was consistent regardless of the priors  remain uncertain. The BSP generated using BEAST provides credibility intervals at every time point that reflect both coalescent and phylogenetic uncertainty. This proved to be important, as the BSP shows that the credibility intervals for effective population size ranged from 51 to 9,500. These wide credibility intervals around iors see , indicatIn contrast to BEAST, Msvar estimates four parameters simultaneously and can only detect the single most likely demographic trend over time based on the strongest genetic signal. Bayesian models, such as Msvar, require prior distributions for each parameter . GeneratCapitalizing on multiple analytical approaches and two unique genetic data sets used in this study, it is possible to infer multiple demographic fluctuations. While the mismatch distribution detected a population expansion and coalescent modeling using Msvar detected a decline, the results may not be in conflict. Instead, they may be detecting the two different demographic changes that are both evident in the BSP generated by BEAST. By considering known events in the ecological history of Lake Champlain and the approximate time scale estimated by each method, we present the most parsimonious demographic history of a native sea lamprey population in Lake Champlain.The decline in effective population size detected by both coalescent models may correlate with land-use changes and fishing pressures that began after the arrival of European settlers in the mid-18th century. The BSP from BEAST indicated that the decline began approximately 400 years ago. Given that there is uncertainty around this time estimate, one hypothesis is that a gradual decline in population size began around 1753 when a large number of European settlers arrived in the area. At this time, human impact on the landscape increased with the onset of large-scale agricultural and clear-cutting practices and, later, the construction of mills and dams that would have limited upstream lamprey spawning migrations . MoreoveThe mismatch distribution analysis may be detecting a more recent population expansion associated with salmonid stocking in Lake Champlain. This recent expansion is supported by the upward trend in effective population detected in the BSP in the last 50 years. A stocking program began for both Atlantic salmon and lake trout in 1972 and now focuses on stocking the main part of the lake with yearlings . The rei−4 mutations/site/generation. Nevertheless, Msvar, a model that uses microsatellite markers exclusively, has been shown to have a bias towards detecting ancient declines in effective population size . With the exception of mutation rate, broad priors were used for the parameters to test whether the model could detect population changes from the data.Click here for additional data file.10.7717/peerj.1369/supp-2Table S2Three replicates of each altered prior were run in BEAST and analyzed using Tracer. Manually altered priors are shaded. Replicates yielded similarly patterned BSPs. Effective population size in 2009 and the range of time when the credibility interval was within 95% HPD were used to compare each run.Click here for additional data file.


Background. Despite a lack of clear evidence, multiple neurostimulants are commonly provided after severe brain injury (BI). The purpose of this study is to determine if the number of neurostimulants received during rehabilitation was associated with recovery of full consciousness or improved neurobehavioral function after severe BI. Method. Data from 115 participants were extracted from a neurobehavioral observational study database for this exploratory, retrospective analysis. Univariate optimal data analysis was conducted to determine if the number of neurostimulants influenced classification of four outcomes: recovery of full consciousness during rehabilitation, recovery of full consciousness within one year of injury, and meaningful neurobehavioral improvement during rehabilitation defined as either at least a 4.7 unit  or 2.58 unit  gain on the Disorders of Consciousness Scale-25 (DOCS-25). Results. Number of neurostimulants was not significantly (P > 0.05) associated with recovery of full consciousness during rehabilitation, within one year of injury, or meaningful neurobehavioral improvement using the DOCS-25. Conclusions. Receiving multiple neurostimulants during rehabilitation may not influence recovery of full consciousness or meaningful neurobehavioral improvement. Given costs associated with additional medication, future research is needed to guide physicians about the merits of prescribing multiple neurostimulants during rehabilitation after severe BI. Severe brain injury (BI) results in loss of consciousness for a period of time greater than 24 hours. The transition from unconsciousness to recovery of full consciousness is described clinically according to three states of disordered consciousness: comatose, vegetative state (VS), and the minimally conscious state (MCS). Each state is defined by varying levels of arousal and awareness, ranging from the absence of arousal and sleep wake cycles in the comatose state to inconsistent but definite behavioral indicators of self- or environmental awareness in MCS –6.Common practice in medical rehabilitation of people in states of disordered consciousness is to provide neuropharmacological interventions while the patient is transitioning through the states of disordered consciousness and provision is usually continued after emergence from MCS to a state of full consciousness . NeurostHowever, neurostimulants are commonly provided to this patient population, and, until recently, there was limited evidence to support the use of neurostimulants to improve neurobehavioral function and facilitate recovery of consciousness among people in states of disordered consciousness. A randomized placebo-controlled trial published in 2012 demonstrated that a widely used neurostimulant, amantadine, is efficacious in accelerating neurobehavioral function among people in VS or MCS between 4 and 16 weeks after traumatic brain injury (TBI) . Evidenc multiple neurostimulants to people in states of disordered consciousness after BI for the purpose of enhancing arousal, awareness, and neurobehavioral function, we conducted exploratory analyses to provide insights about the relationship between the provision of multiple neurostimulants during rehabilitation hospitalization and recovery. The purpose of this study is to assess whether receiving multiple neurostimulants is associated with recovery of full consciousness or improved neurobehavioral function after severe BI.Since, there is no evidence to date supporting prescription ofThe study sample for this paper included 115 participants selected from a larger study database of 191 people with severe brain injury (BI) who (a) were admitted to a rehabilitation hospital within 180 days of injury, (b) were ≥18 years of age at time of injury, (c) experienced unconsciousness for ≥28 consecutive days, and (d) had a severe BI that was not due to cancer, tumors, and inflammatory, infectious, and/or toxic metabolic encephalopathies. For our study sample, we included only those individuals from the larger study who had medication data and were provided at least one neurostimulant  during rehabilitation. Participants for the larger study were recruited from two freestanding inpatient rehabilitation facilities, one long-term acute care hospital providing rehabilitation, two Department of Veterans Affairs hospitals providing acute rehabilitation, and one subacute nursing facility. The study was approved by each facility's human subjects institutional review board. Each research participant was followed from time of rehabilitation admission through the first year of recovery to monitor time to full consciousness.Data collection procedures for the larger study included medical record reviews and bedside neurobehavioral assessments during rehabilitation hospitalization and monthly telephone follow-up to assess the recovery of full consciousness up to one year after injury. The data for the study sample were collected from 1997 to 2010.At time of study enrollment, each participant's emergency room, intensive care, and acute care records were reviewed for sociodemographics, medical history, cause of injury, etiology, and injury-related medical conditions. After review of each subject's records, a family/surrogate interview was conducted to collect any information not obtainable from the records and/or to confirm information regarding cause of injury.At time of rehabilitation discharge, all medications received each day of hospitalization as part of the participant's routine medical care were abstracted from the electronic medical records including start date, stop date, start time, and stop time for each dose.κ = .95)  × 100; and Sp = [true negatives/] × 100.Univariate optimal data analysis (UniODA) was conducted using UniODA software . UnivariESS values, reflecting stability after LOO procedures, were compared for each UniODA model. An explanatory variable is considered LOO stable if ESS does not vary between the total sample and LOO analyses on the resampled total sample.n = 115) was comprised predominantly of participants who are young (average age in years = 37 ± 16), Caucasian (75%), and males (66%). The majority of participants (57%) incurred a closed head injury. The average number of days between injury and rehabilitation was 65 ± 75. The average baseline total DOCS-25 measure was 48.46 ± 13.80 for the total sample, 50.39 ± 16.57 for the one neurostimulant group, and 47.77 ± 12.71 for the multiple neurostimulants groups. The average total DOCS-25 change measure was 2.35 ± 13.82 for the total sample, 4.12 ± 12.69 for the one stimulant group, and 1.79 ± 14.20 for the multiple neurostimulants groups. Recovery of full consciousness during rehabilitation occurred in 43% of the total sample, 43% of the one neurostimulant group, and 42% of the multiple neurostimulants group. Recovery of full consciousness within one year of injury occurred in 62% of the total sample, 57% of the one neurostimulant group, and 64% of the multiple neurostimulants group.P > 0.05) associated with recovery of full consciousness during rehabilitation, recovery of full consciousness within one year, or meaningful neurobehavioral improvement defined by the DOCS-25 MDC90 and MCID  as the explanatory variable in each UniODA model. Number of neurostimulants received was not significantly  and neurophysiological outcomes , for example, would allow elucidation of the relationship between receiving multiple neurostimulants and recovery. Among these neurobehavioral outcomes, it may be of value to also include additional measures of neurobehavioral function in future studies such as those examined in a systematic review in 2010  includinFuture analyses should also consider the cost of these neurostimulants, as it may be more cost-effective to use only one neurostimulant rather than more than one if outcomes are not significantly different . FurtherStudy findings suggest that receiving multiple neurostimulants during rehabilitation hospitalization does not influence recovery of full consciousness or meaningful neurobehavioral improvement. Future research is needed to determine whether it is advisable for clinicians to prescribe multiple neurostimulants during rehabilitation. Given positive findings that amantadine facilitates neurobehavioral function , it may


AbstractGerbillus for which i) the taxonomy is still highly debated, ii) collection of fresh specimens may prove difficult. Here we address precise taxonomic questions using a small portion of the cytochrome b gene obtained from 45 dry skin/skull museum samples (from 1913 to 1974) originating from two African and three Asian countries. The specimens were labelled Gerbillusgerbillus, Gerbillusandersoni, Gerbillusnanus, Gerbillusamoenus, Gerbillusperpallidus and Gerbilluspyramidum, and molecular results mostly confirmed these assignations. The close relationship between Gerbillusnanus (Asian origin) and Gerbillusamoenus (African origin) confirmed that they represent vicariant sibling species which differentiated in allopatry on either side of the Red Sea. In the closely related Gerbillusperpallidus and Gerbilluspyramidum, specimens considered as belonging to one Gerbilluspyramidum subspecies (Gerbilluspyramidumfloweri) appeared closer to Gerbillusperpallidus suggesting that they  may represent a unique species, distributed on both sides of the Nile River, for which the correct name should be Gerbillusfloweri. Furthermore, the three other Gerbilluspyramidum subspecies grouped together with no apparent genetic structure suggesting that they may not yet represent genetically differentiated lineages. This study confirms the importance of using these methods on museum samples, which can open new perspectives in this particular group as well as in other groups of interest.Methodological improvements now allow routine analyses of highly degraded DNA samples as found in museum specimens. Using these methods could be useful in studying such groups as rodents of the genus PageBreak The latter was represented in our sample by the subspecies Gerbilluspyramidumelbaensis (N = 5), Gerbilluspyramidumfloweri (N = 4), Gerbilluspyramidumgedeedus (N = 5) and Gerbilluspyramidumpyramidum (N = 5). These specimens were collected from 1913 to 1974 in Egypt (N = 38), Sudan (N = 1), Afghanistan, India and Pakistan  and GERBCYTB-R3 (5’-CAT TCT ACR ATT GTT GGG CCA - 3’). These primers are respectively located at positions 250 and 488 of the cytb gene, delimiting a 239 base pair (bp) fragment. The 25μl reaction solution was prepared by mixing 14.5μl of DNase-RNase free water (Qiagen), 2.5μl of buffer (1X concentration), 2μl MgCl2 (2mM), 2.5μl dNTP , 0.5μl of each primer (0.5μM), 0.5μl PageBreakof AmpliTaq Gold . 1μl and 2μl of DNA aliquots of the extracted samples were amplified separately, and used for further comparisons. The cytb amplification was done at the CBGP molecular biology platform  using PCR programs on a Master Cycle rep Gradient (eppendorf), including an activation step of 95 °C for 10 min followed by 55 cycles comprising a first denaturation at 94 °C for 30 s, hybridization at 50 °C for 30 s and elongation at 72 °C for 45s. The last step was a final extension at 72 °C for 7min. Three negative controls were used to check for contamination during DNA extraction, preparation of the mix and DNA distribution. In the first control (extraction control), no tissue was added to the tube; the second control (PCR mix control) was a closed tube, with only the PCR mix; the last control (DNA distribution control) was a tube with only the PCR mix, which was open during the entire process of DNA distribution, in order to check for the presence of DNA in the air. We verified the size and quality of each amplified DNA sequence fragment by performing an electrophoretic migration on a 2% agarose gel. The PCR products obtained at both DNA concentrations were sent to Eurofins MWG (Germany) for sequencing, and the results were compared among individuals to ensure that we obtained the same amplified sequence fragment.DNA was extracted in the Labex CeMEB degraded DNA platform  using the QiaAmp DNA micro kit (Qiagen). Due to the degradation of DNA in museum samples, we amplified a short fragment of cytwww.ncbi.nlm.nih.gov/genbank) of various well-characterized Gerbillus species as a reference, and Sekeetamyscalurus was used as an outgroup  and Bayesian inference (BI) using SeaView v.4.2.12  and posterior probabilities (PP) used as node support in respective analyses. The best fit models for Neighbor-Joining and Bayesian reconstructions were K2P and GTR+I+G, respectively. We tested it using jModeltest v2.1.4 (Markov chain Monte Carlo (MCMC) runs were carried out for one million generations each. Trees were sampled every 100 generations and convergence was reached when the average standard deviation of split frequencies remained under 0.01, thus reflecting the fact that the two tree samples become increasingly similar. Finally we applied a 25% burn-in. Pairwise Kimura 2-Parameter genetic distances were obtained for our cytb dataset under MEGA v6 . Unambiguous sequences (239 bp) of 37 individuals were obtained, to which we added the sequences of 40 “reference” individuals of different, well-characterized, Gerbillus species taken from GenBank and a representative of the outgroup Sekeetamyscalurus, leading to a final cytb dataset comprising 77 individuals.No contamination was recorded during this series of experiments, as testified by examination of the content of the control tubes. DNA could not be amplified from only NJ  presented here shows a similar topology to the BI tree with our museum specimens distributed in four main clades. The first two are unambiguously identified as Gerbillusandersoni (98/0.97) and Gerbillusgerbillus (99/1). The other two correspond to a Gerbillusnanus / Gerbillusamoenus (82/-), and a Gerbillusperpallidus / Gerbilluspyramidum (80/0.96) clade, respectively, each of which appears structured in two sub-clades. In the former, specimens referable to Gerbillusnanus from Asia (Pakistan and Afghanistan) cluster together (97/-) as a sister group to specimens referable to PageBreakGerbillusamoenus from Africa . The latter is divided into two sub-clades  made it possible to obtain generally robust reconstructions of the phylogenetic relationships between the study specimens, thus confirming the findings of ad hoc primers designed for this particular experiment are well-adapted for a molecular barcoding approach based on potentially degraded DNA in the study group.Overall, the labelling of the museum samples was remarkably in line with current taxonomy, and all corresponding specimens appear coherently placed in the phylogenetic tree produced (but see below for further details). This validates Gerbillusgerbillus and Gerbillusandersoni from Egypt clustered unambiguously with “fresh” specimens of the same species from other origins. As for the distinction between Asian Gerbillusnanus and African Gerbillusamoenus, even if supported here by non-optimal BP values, it confirms the findings of Gerbillusamoenus following Dipodillusamoenus) PageBreakas a distinct species from Gerbillusnanus (= Dipodillusnanus). This taxonomy was not followed by many subsequent studies (see details in Gerbillusamoenus) and the presence among the Gerbillusnanus sample of one reference specimen from Israel reinforce this conclusion, and confirmed that the Red Sea probably represents the geographical limit separating these two vicariant species.Museum specimens of Gerbillusperpallidus and Gerbilluspyramidum ssp. were distributed into two moderately well supported genetic clades: the first one includes all Gerbilluspyramidumfloweri and Gerbillusperpallidus samples, together with a reference specimen of Gerbillusperpallidus  as a subspecies of Gerbilluspyramidum, but it is generally listed as a valid species following the review of Gerbilluspyramidumfloweri and Gerbillusperpallidus are strikingly similar in color, bulla shape and, in some individuals, posterior margin of nasals”. Our results confirm these observations and suggest that these two taxa may represent a single species, the name of which should be Gerbillusfloweri, according to his first description. This species would be characterized by a karyotype with a diploid number of chromosomes of 2n = 40, and an autosomal fundamental number NFa = 76, as described in Gerbillusperpallidus. The distribution of this species would then encompass both sides of the Nile delta in Northern Egypt, and extend through the whole of Northern Sinai . It was larger in two other studies based on complete cytb sequences of samples of different individuals of Gerbilluspyramidum and Gerbillusperpallidus only , but they were intermixed in this clade, suggesting that these subspecies do not represent genetic lineages with independent evolutionary histories, at least based on partial sequences of cytb. Their current allopatPageBreakric distributions may be of relatively recent origin, following the last episode of drying of the Sahara, between 6,000 and 4,000 years ago (b fragments sequenced here may not reflect this differentiation yet.In addition to ears ago . The geoGerbillus, up to more than 100 years after their collection, may give useful information, and address testable hypotheses, about the systematics of the genus. This could aid in the completion of the taxonomic understanding of this complex and speciose genus, which is well represented in museum collections worldwide. The new primers specifically designed here, may prove useful for this purpose.In conclusion, we show here that molecular analysis of historic museum samples of the genus