For potential future human missions to the Moon or Mars and sustained presence in the International Space Station, a safe enclosed habitat environment for astronauts is required. Potential microbial contamination of closed habitats presents a risk for crewmembers due to reduced human immune response during long-term confinement. To make future habitat designs safer for crewmembers, lessons learned from characterizing analogous habitats is very critical. One of the key issues is that how human presence influences the accumulation of microorganisms in the closed habitat.Proteobacteria  and Firmicutes (esp. Bacillaceae) were most abundant before human occupation (Day 0), while other members of Firmicutes  and Actinobacteria (esp. Corynebacteriaceae) were abundant during the 30-day occupation. Treatment of samples with PMA  had a significant effect on the microbial diversity compared to non-PMA-treated samples.Molecular technologies, along with traditional microbiological methods, were utilized to catalog microbial succession during a 30-day human occupation of a simulated inflatable lunar/Mars habitat. Surface samples were collected at different time points to capture the complete spectrum of viable and potential opportunistic pathogenic bacterial population. Traditional cultivation, propidium monoazide (PMA)–quantitative polymerase chain reaction (qPCR), and adenosine triphosphate (ATP) assays were employed to estimate the cultivable, viable, and metabolically active microbial population, respectively. Next-generation sequencing was used to elucidate the microbial dynamics and community profiles at different locations of the habitat during varying time points. Statistical analyses confirm that occupation time has a strong influence on bacterial community profiles. The Day 0 samples (before human occupation) have a very different microbial diversity compared to the later three time points. Members of Actinobacteria (mainly Corynebacteriaceae) and Firmicutes  were shown to increase over the occupation time period. The results of this study revealed a strong relationship between human presence and succession of microbial diversity in a closed habitat. Consequently, it is necessary to develop methods and tools for effective maintenance of a closed system to enable safe human habitation in enclosed environments on Earth and beyond.Statistical analyses revealed a significant difference in community structure of samples over time, particularly of the bacteriomes existing before human occupation of the habitat (Day 0 sampling) and after occupation . The online version of this article (doi:10.1186/s40168-016-0167-0) contains supplementary material, which is available to authorized users. The predominant cultivable bacterial genera were related to humans (Staphylococcus sp. and Bacillus sp.), and environmental species, such as Sphingomonas paucimobilis, were also found in the air along with a few opportunistic pathogens.In the industrialized world, humans are spending the majority of their lives indoors—some up to 90 % of their time , 2. BuilAn air sampling study at Providence Milwaukie Hospital revealed that ventilation (air and airflow rates) influenced the microbial community composition. For this study, outdoor and indoor air samples were collected from mechanically ventilated and “naturally” ventilated rooms of the hospital. The relative abundance of bacteria closely related to human pathogens was higher indoors than outdoors and in rooms with lower airflow rates . This stNeisseria, Shigella, Streptococcus, and Staphylococcus) [Similarly, examination of surfaces in 30 different offices in three different cities  found significant microbial diversity based on geographical location. The main sources of contamination were human and several of the bacterial genera found were pathogens  . These rHumans have a reduced immune response when exposed to unfavorable environmental conditions like long-term confinement and spaceflight , 14. In In early 2009, a team led by the Department of Space Studies at the University of North Dakota (UND), USA, developed advanced inflatable habitat architecture concepts that could be adapted for use on surfaces of the Moon and Mars. An inflatable lunar/Mars analogous habitat (ILMAH) was built to occupy four student crews for mission durations up to 6 months. After completion of the habitat, three test subjects  occupied the ILMAH for two different periods of time (10 and 30 days) . Among oHere, we present the results of the microbial succession study in the ILMAH, which was conducted by monitoring the bacteriome of several surfaces before human occupation and during the 30-day occupation by the three student crew. To address the microbial divergence in closed indoor environments, surface samples from defined locations were sampled before and after the occupation, as well as three consecutive time periods during the habitation. In addition to the total bacteriome (iTag Illumina sequencing), cultivable and viable microbial populations of the ILMAH surfaces were elucidated. Viable but not-yet-cultivable microbial populations were determined by using well-established propidium monoazide (PMA) and adenosine triphosphate (ATP) assays , 24. Mic3 to 107 CFU/m2. After an initial decline of ~1 to 3 logs of colony counts from Day 0 (before occupation) to Day 13, the counts were exhibited an increase in order from Day 13, to Day 20, to Day 30 for locations in front of the sleeping area (bedroom) and bathroom, whereas the colony counts in the kitchen area decreased over these time points. However, the cultivable counts did not change during the 30-day occupation period in the lab locations. Changes in Day 0 cultivable bacterial population were significant  was at least twice more on the final day  than before occupation . The percent cultivable bacteria among viable bacterial burden were greater at the surfaces in front of the bathroom (~47 %) or in bedroom area (~55 %) when compared to other locations (~12 to 14 %). These differences were highly significant when considering qPCR results of PMA vs. non-PMA samples in a paired t test (p = 0.000106).The qPCR assay that measured 16S ribosomal RNA (rRNA) gene copies from both dead and live bacterial cells showed an increase in bacterial density over the 30-day occupation period. In general, samples treated with PMA  revealed that ~60 % of the bacterial population was dead at Day 0, whereas the reduction in bacterial population was ~90 % for the subsequent days of occupation. The viable bacterial burden as measured by the PMA-qPCR assay ranged from below detection limit to 105–106 relative luminescence unit per m2. In general, when samples from all sampling locations were pooled together, the total microbial burden did not show any changes in microbial accumulation over the time period. However, ~55 % of the microbes were viable at Day 1, and the viable microorganisms were reduced to ~28 % on Day 13 and ~35 % on the last day of occupation. The percentage of viable bacterial burden (PMA-qPCR assay) among viable microbial burden (intracellular ATP content) was highest in the lab area (~40 %) and lowest in the bathroom area (~10 %). A closer look at the microbial burden as measured by ATP content of the different locations revealed that the succession of bacterial burden was not linear throughout the 30-day occupation. The bacterial density initially decreased in all the locations (from Day 13 to Day 20) and then drastically increased (Day 30) in the bedroom area, the bathroom area, and the lab area, whereas samples from the kitchen area showed no fluctuation. From a statistical standpoint, the microbial density fluctuations in various locations were not significant  and viable microorganisms (intracellular ATP content) were in the range of 10Firmicutes (76 strains), 26 % to Proteobacteria (39 strains), and 20 % to Actinobacteria (30 strains). Bacillus species represented the highest number of isolates (43 strains), followed by Staphylococcus (24 strains) and Pseudomonas (17) species.Phylogenetic characterization of 150 strains isolated during this study and identified via 16S rRNA gene analysis revealed a total of 62 known bacterial species and eight lineages yet to be described , 28 % Firmicutes (8 strains), 8 % Actinobacteria (2 strains), and 3 % Bacteroidetes (1 strain). The majority of the proteobacterial isolates (13 out of 14 strains) belonged to Gammaproteobacteria and most Firmicute isolates (6 out of 8 strains) were Bacillus species.Throughout the occupation period, changes in the abundance of the cultivable bacterial species occurred. Before the student crew moved in (Day 0 sampling), almost two thirds of the isolates were Proteobacteria declined to 10 %, whereas Firmicutes and Actinobacteria increased to 60 and 29 %, respectively. None of the Gammaproteobacteria recovered at Day 0 were retrieved at Day 13 (or Day 20 or Day 30), instead Alphaproteobacteria was present. A few of the Bacillus species found at Day 0 recurred at Day 13  and additional Bacillus species were isolated . Actinobacteria that were recovered for the first time were represented by the genera Microbacterium, Curtobacterium, Micrococcus, and Rhodococcus.At Day 13, the relative abundance pattern of cultivable bacteria changed drastically. The frequency of Proteobacteria and almost no species of Actinobacteria (one exception: Curtobacterium faccumfaciens) from Day 13 were isolated again at Day 20. However, bacterial species from all of the same genera at Day 13 also recurred at Day 20. Some of the Firmicutes from Day 13 reappeared, whereas some Bacillus species emerged for the first time.Day 20 samples showed a similar relative abundance pattern of the phyla as the Day 13 samples. No species of Firmicutes (51 %) were retrieved in favor of Proteobacteria (21 %). The recovery rate of Actinobacteria did not change (24 %), and one Bacteroidetes was isolated (3 %). Consistently, almost no Proteobacteria and Actinobacteria from the earlier time points were detected again. However, other species of the previously recovered genera were found and some of the Firmicutes from Day 13 and/or Day 20 were isolated again and some undetected Firmicutes species emerged  were only detectable at species level, not at phyla level. Almost all representative species from Actinobacteria and Proteobacteria were isolated only once throughout the whole occupation period, whereas representatives of the Firmicutes were recovered multiple times.The analysis of the cultivable bacteria indicates a strong decline in the number of Sampling device control, environmental controls, DNA extraction reagent controls, and no-template PCR controls included in this study yielded no sequence reads. The colony counts, qPCR, and ATP-based analyses to estimate microbial burden also exhibited values below detection limit.t test based on number of operational taxonomic units (OTUs) revealed a p value <0.0001). Moreover, 208 genera were detected in the non-treated samples, 37 of which were not identified in the PMA-treated samples. The two sample types also differed significantly in community relationships  and their Shannon diversity index indicated a significant reduction (paired t test p < 0.001). Due to these differences in the total and viable bacteriome, it could be concluded that the total bacteriome (including dead cells) does not give a true picture of the causative bacterial agent(s) that trigger illness. Consequently, all following results are based on data generated from the viable bacteriome only.The bacterial richness between PMA and non-PMA samples declined significantly  and, in combination with the Bacteroidetes, these four phyla accounted for 99 % of the characterized OTUs. A closer examination of the OTUs on genus level indicated a predominance of nine taxa, which were primarily comprised of by Corynebacteria , Bacilli (15 %), Staphylococci (18 %), Anaerococci (11 %), and Fusobacterii (14 %). In sum, the sequences from the aforementioned five genera constituted 78 % of all OTUs found in the PMA-treated samples . Notably, the bacteriome of Day 0 samples was substantially different from the bacteriome of the later time points. For that reason, Day 0 samples were excluded in another analysis . The NMDS ordinations based on Bray-Curtis distances between all samples that were not treated with PMA : Adonis p value of 0.012, MRPP significance of delta = 0.004, A = 0.03886).To investigate differences in the bacteriome between samples, multivariate statistics were applied using ordination analyses and Monte Carlo-based permutation tests. Viable bacterial communities formed significantly different groups in NMDS plots based on the factor time, i.e., the different sampling days showed distinct microbiome profiles , followed by Proteobacteria (27 %), and Actinobacteria (9 %). By the end of the occupation (Day 30 sampling), the relative abundance of Firmicutes in the habitat had not changed significantly (61 %), whereas Proteobacteria decreased (16 %) and Actinobacteria increased (21 %) , the majority of the microbial community consisted of bacteria 7 %, and p value of 0.01. Most of the OTUs belonged to the phylum Firmicutes (23 OTUs), followed by Proteobacteria (20 OTUs) and Actinobacteria (19 OTUs); the rest of the taxa were classified as Bacteroidetes (9 OTUs) and Candidatus Sachcharibacteria (formerly known as TM7), Cyanobacteria, Deinococcus-Thermus, Fusobacteria and Acidobacteria . Actinobacteria and Firmicutes that increased throughout the occupation period were dominated by two families: Corynebacteriaceae (7 OTUs), Clostridiales Incertae Sedis XI (8 OTUs). Other OTUs that increased throughout the occupation period were mostly Proteobacteria (7 OTUs) belonging to different families. Members of Proteobacteria that decreased throughout time (13 OTUs) were dominated by Oxalobacteraceae (4 OTUs), Comamonadaceae (2 OTUs), and Pseudomonadaceae (2 OTUs). Among Firmicutes (9 OTUs), members of the family Bacillaceae (3 OTUs) increased and all but one Bacteroidetes (8 OTUs) decreased within the course of time.To identify viable taxa that significantly correlated with the various occupation time points, a Spearman rank correlation was applied individually to each OTU’s abundance pattern and sampling time. The results are displayed as a heat map ), followed by Firmicutes ), followed by Actinobacteria (3 OTUs), and Actinobacteria (2 OTUs). Of those 26 OTUs, three families were identical with the correlation analyses above, confirming that OTUs belonging to Clostridiales Incertae Sedis XI, Comamonadaceae, and Corynebacteriaceae truly increased.Since the Day 0 samples exhibited a fundamentally different bacteriome composition, two additional analyses were employed. First, Spearman rank correlation was applied on the PMA-treated dataset excluding Day 0 samples. At a p = 0.01). Thirty-four OTUs that were more abundant at Day 0 were identified as belonging to similar genera as OTUs identified in the correlation analysis. They belonged mainly to Oxalobacteraceae (8 genera) and different genera belonging to Proteobacteria, Actinobacteria, and Firmicutes. The predominant and differential bacterial taxa  of various time points of the ILMAH surfaces are depicted in Fig. Second, an ANOVA was applied on OTU abundances across all the PMA-treated samples comparing data at Day 0 with those at the later three time points (Actinobacteria (mainly Corynebacteriaceae) and Firmicutes  were shown to increase over the occupation time period.In sum, statistical analyses revealed a significant difference in community structure of samples over time, particularly of the bacteriomes existing before human occupation of the habitat (Day 0 sampling) and after occupation . Nitrososphaera represented the majority of all OTUs found in the non-PMA-treated samples (94 %). Along with Nitrososphaera, three other archaeal genera were detected: Methanocaldococcus, Methanosarcina, and Nitrosopumilus. However, in the PMA-treated sample set, Nitrososphaera was the only archaeal genera observed, indicating that the latter three genera might be dead highlighting the importance of using viability assays for archaea in indoor microbiomes.In general, archaea were of low abundance in the samples collected from the ILMAH surfaces. The archaeal microbial burden detected via specific qPCR was below detection level in most of the samples tested (28 out of 32 samples) and in positive samples, archaea were between one and two logs lower than the bacterial microbial burden. The overall low abundance of archaea in the community was confirmed by the iTag data, since 92 % of all the OTUs found with archaea-specific primers were identified as bacteria indicating low abundance of archaea resulting in unspecific priming. Due to the low number of sequences detected it was not possible to depict any change in the archaeal community structure throughout the occupation period  and had been reported before for a variety of microorganisms including viruses [Previous studies have shown that meaningful data can often only be retrieved if the dead fraction of the cells is excluded , 30. Oth viruses . This ap6 to 107 particles per day [3 to 104 droplets per (re)action; [Various studies report that human presence is the most common source of contamination in strictly controlled rooms , 32, 33. per day ; perspir)action; , 36. In )action; . The ILM)action; . The phy)action; .Flavobacteriaceae, Caulobacteraceae, and Oxalobacteraceae) were reported to be common in aquatic and soil habitats [Corynebacteriaceae, Clostridiales Incertae Sedis, and Staphylococcaceae [Actinobacteria  were more abundant on skin, with Firmicutes (Clostridium) and Bacteroidetes more abundant in the gastrointestinal tract. Sequences of notable pathogens, such as Brevibacteria  retrieved from samples at Day 0 , Actinomycosis; ), Propioa , Corynecomical; –52), Sta rashes; ), and Clotulism; ) were re6/m2) compared to the occupation period . The bacterial burden in cleanrooms was comparatively low (1- to 3-logs less) than the ILMAH and MARS 500 habitats [Several studies have already been conducted on the microbial population in confined habitats, including several office buildings , 55, 56,habitats  and may Proteobacteria, followed by Bacteroidetes and Firmicutes, decreased over the occupation period; this was also noticed in the ILMAH samples that were not treated with PMA (including both dead and viable bacteria). But such trend was not found for the viable community of the ILMAH. Furthermore, the MARS 500 microbial analysis did not contain control, i.e., preoccupation bacterial diversity measurements, to compare the microbial accumulation as measured in this study. We conclude that it is critical to compare the microbiome of habitat without human occupation to habitat with human occupation since humans appeared to be the primary source of contamination in such habitats. Such a comparison may enable development of cleaning and maintenance protocols. Nonetheless, the MARS 500 project [Of all the microbial characterizations studies in confined habitats, the MARS 500 project (June 2010 to November 2011) had the most similar study objectives to the ILMAH study. However, the MARS 500 study did not measure the viable portion of the community. Hence, the measured bioburden and diversity characterization of the MARS 500 report is an overestimation. This has been confirmed since the MARS 500 study revealed a pattern: members of the class  project  providedStaphylococcus and Aspergillus were the dominant cultivable species [Staphylococcus species are dominant but also reported retrieval of a large percent (>90 %) of molecular signatures of viable Corynebacterium/Propionibacterium [However, the microbial risk assessment in an Earth setting might underestimate the importance of humans living in stressed situations, such as microgravity and long-term travel in confined spaces . Under m species , 69. Howacterium .Thaumarchaeota and Euryarchaeota in one third of the collected surface samples (and none from the air samples). In this ILMAH study, only 4 out of the 80 samples (5 %) tested positive for archaea. The majority of the ILMAH archaeal community comprised OTUs of Thaumarchaeota (over 94 %) and the rest were Euryarchaeota (6 %), which supports the results from the aforementioned studies. Particularly, Thaumarchaeota have been recognized to inhabit human skin, another indication that the ILMAH microbiome was influenced by human activity [Studies on archaea have concentrated on natural biotopes where they have been found in overwhelming numbers and with versatile properties . Moreoveactivity .Firmicutes, Proteobacteria, and Actinobacteria phyla were dominant and such results also supported by the Illumina-based sequencing studies. Changes in the abundance of the cultivable bacteria and molecular signatures of viable bacterial species were noticed throughout the occupation period and are statistically significant. Actinobacteria (mainly Corynebacteriaceae) and Firmicutes  were shown to increase over the occupation time period. As seen in other studies where confined habitats were investigated, this study also concluded that humans are the primary source of contamination. A combination of cultivation-based analysis and viability assays is warranted to elucidate the significance of bioaccumulation that might be problematic to the inhabiting human health.Accumulation of cultivable and viable (as per molecular methods) bacteria was evident during the 30-day occupation period of the enclosed habitat. No observed change in the total microbial burden (including dead cells) stressed the importance of differentiating dead organic matter from live cells. Thorough maintenance procedures adapted to keep the ILMAH system clean might have eradicated the microorganisms but such processes would not have removed the biomolecules from the surfaces, hence implementation of genetic methods estimating total microbial community structures including dead cells would result in overestimation. However, when viability assays were used, only ~55 % of the microbes were viable at Day 0, and they were reduced to ~28 % on Day 13 and ~35 % on the last day and this might be due to the prolonged stay by the human and their microbial shedding. Among the cultivable bacterial diversity members of the The ILMAH is approximately 12 m long, 10 m wide, and 2.5 m high. The ILMAH interior consists of four sleeping compartments where the student crews are able to rest as well as stow their personal belongings, a small galley/dining room, a bathroom, and plenty of laboratory space , the strength recommended by the Center for Disease Control for inactivating viruses and other pathogens. Immediately after cleaning, the ILMAH habitat was closed and undisturbed until 10 min prior to inhabitation. The student crews cleaned all the rooms and surfaces of the habitat once a week after they had taken the microbial samples. Cleaning included wiping down the surfaces  with antibacterial wipes , dusting, sweeping, and wet mopping the floor. Finally, the student crews performed additional cleaning duties, as required to keep the ILMAH system tidy. Cleaning procedures utilizing bleach, as carried out prior to occupation (Day 0), were not allowed during human occupation.2 each), two sample locations were in the sleeping compartment area, two in the dining room area, one in front of the bathroom, and three in the lab area. Samples from the closed habitat were collected using premoistened biological sampling kits  from the eight prescribed locations at four time points . Sampling point Day 0 was taken prior to the occupation of the ILMAH system by the student crew, and subsequent samplings were conducted on Day 13, Day 20, and Day 30, the last day of the occupancy. When particulate materials were collected for microbiological examination from cleanroom surfaces, we have shown that at least 1 m2 surface area should be sampled so that sufficient biological matter was available to conduct several traditional microbiological and molecular microbial community analyses.The architectural scheme of the ILMAH system is depicted in Additional file One BiSKit was used to collect samples from one location. The selected area was wiped in three different directions  while rotating the sampling device. After the sampling, the dropper attachment handle of the BiSKit was turned to expel the sample, soaked in the macrofoam, into the attached collection bottle. The liquid from the collection bottle was transferred into a sterile 50-ml Falcon tube. To increase sample extraction from each macrofoam, the collection bottle was filled with 15 mL of sterile phosphate-buffered saline (PBS) and reattached to the BiSKit and the macrofoam soaked again in PBS. Then, the dropper attachment was turned one more time to release the sample in the collection tube. The liquid was then transferred into the respective sample collection tube. This step was repeated twice. The sampling liquids (45 mL for each BiSKit) were stored at 4 °C and transported to JPL via overnight courier for further processing. For each sampling time point, a field control  and BiSKit control (unused BiSKit prepared the same way as BiSKits used for sampling) were collected. The protocol for BiSKit sampling and processing was followed as published elsewhere . The timAt JPL, the 45-mL liquid samples from each BiSKit were concentrated to ~4-mL samples using the Innovaprep system . The concentrated samples were utilized for cultivation and molecular analyses to measure microbial burden and community structure analyses. Briefly, 200 μL of the concentrated sample was mixed with 1.8 mL sterile PBS and thoroughly mixed before performing appropriate dilution(s) and subsequent plating onto suitable agar media and ATP assay. Remaining solutions were used for DNA-based analysis and ~800-μL sampling fluids were stored at 4 °C and used for any contingency.2. The identification and phylogenetic affiliations were carried out via Sanger sequencing targeting the 1–5-kb 16S rRNA gene sequencing. Initially, colony PCR was performed to generate appropriate PCR fragments. However, when the colony PCR step did not yield PCR amplicon, those isolates were subjected to a freeze (−80 °C)–thaw (+80 °C) cycle , automated DNA extraction system [For the analysis of cultivable bacterial population, 100 μL of appropriately diluted sample was spread onto two plates of R2A media (Difco). Plates were incubated at room temperature for 7 days. After the cultivation conditions, colony-forming units (CFUs) were counted and reported as CFU/mn system , and finn system  to breakn system . PCR conn system  and align system .Total ATP and intracellular ATP contents representing total and viable microbial population, respectively, were determined using the CheckLite HS kit  as previously described , 63. TheThe concentrated samples were divided into two aliquots and one of the aliquots was treated with 12.5 μL of PMA  to a final concentration of 25 μM , followeFor the analysis of the bacterial and archaeal burden in the samples, real-time quantitative polymerase chain reaction (qPCR) assay, which targets the 16S rRNA gene, was performed in triplicate with a qPCR instrument . Universal bacterial primers targeting the 16S gene, 1369F (5′-CGG TGA ATACGT TCY CGG-3′) and modified 1492R (5′-GGW TAC CTTGTT ACG ACT T-3′), and universal archaeal primers targeting the 16S gene, 344af (5′-ACG GGG YGC AGC AGG CGC GA-3′) and 517r (5′-GCC AGC AGC CGC GGT AA-3′), were used , 82 to mThe DNA samples were quantified using a Qubit 2.0 fluorometer . Bacterial primers 519wF and 1017R were used to amplify a ~500-bp fragment spanning the V4 hypervariable regions of the bacterial 16S rRNA gene . SequencThe bacterial and archaeal Illumina-generated MiSeq quality reads were processed and analyzed using the LotuS software . Sequencp value of 0.05). The change of diversity was measured via the Shannon-Wiener diversity index. OTUs that were unclassified at phylum level were removed. When warranted, the p value was decreased to only represent high-abundant OTUs and remove false positive results. Heat maps were presented at family level.An in-house R-script employing the libraries vegan, ape, gplots, mgcv, and GUniFrac was used to compare the bacterial and archaeal Illumina data , 86. EacThe data set supporting the results of this article is available in the NCBI SRA repository, under accession # SRP069729.


Universal screening for gestational diabetes mellitus (GDM) has not been implemented, and this has had substantial clinical implications. Biomarker-directed targeted screening might be feasible. We sought to determine the accuracy of circulating adiponectin for early prediction of GDM.A systematic review and meta-analysis of the literature to May 2015 identified studies in which circulating adiponectin was measured prior to a diagnosis of GDM. Data on diagnostic accuracy were synthesised by bivariate mixed effects and hierarchical summary receiver operating characteristic (HSROC) models.Thirteen studies met the eligibility criteria, 11 of which  had extractable data. Circulating adiponectin had a pooled diagnostic odds ratio (DOR) of 6.4 , a summary sensitivity of 64.7%  and a specificity of 77.8%  for predicting future GDM. The AUC of the HSROC was 0.78 . First trimester adiponectin had a pooled sensitivity of 60.3% , a specificity of 81.3%  and a DOR of 6.6 . The AUC was 0.79 . Pooled estimates were similar after adjustment for age, BMI or specific GDM diagnostic threshold.Pre-pregnancy and early pregnancy measurement of circulating adiponectin may improve the detection of women at high risk of developing GDM. Prospective evaluation of the combination of adiponectin and maternal characteristics for early identification of those who do and do not require OGTT is warranted.The online version of this article (doi:10.1007/s00125-015-3855-6) contains peer-reviewed but unedited supplementary material, which is available to authorised users. Gestational diabetes mellitus (GDM) can affect between 1% and 20% of pregnancies, depending on the diagnostic criteria used or the population studied, and is associated with a range of adverse maternal and neonatal outcomes . Randomi1c has not been shown to enhance GDM prediction, due to the substantial overlap of values between affected and non-affected individuals [Improvement of targeted screening by inclusion of first trimester biomarkers may be feasible, facilitating stratified care and early intervention. Yet assessment of fructosamine or HbAividuals , 9. A raividuals , as it sividuals . Contempividuals , but theividuals , 13, or ividuals . To provThe study was conducted according to the PRISMA guidelines , and folWe searched several electronic databases up to May 2015, namely, PubMed, EMBASE, MEDLINE, Web of Science and the Cochrane Library. The following search terms were combined using Boolean rules: gestation*, diabetes, pregnan*, adiponec*, with a filter for human studies without language restriction. The full search strategy is provided in the electronic supplementary material ESM . Two resWe included studies that met the following criteria: (1) the study population included women of reproductive age without existing diabetes; (2) serum adiponectin was measured prior (pre-pregnancy or index pregnancy) to the diagnosis of GDM; (3) the clinical outcome was GDM diagnosed by OGTT against reference criteria ; (4) any study design, apart from case reports, without a language restriction. In the meta-analysis, we included studies if 2 × 2 tables could be constructed from published or requested data.Two researchers (SI and JS) independently assessed the papers for final selection. If a study fulfilled the eligibility criteria, it was included in the systematic review. Any discrepancies were resolved with discussion. A third reviewer (SMN) was consulted if any unresolved issues persisted.http://plotdigitizer.sourceforge.net/), a computer software programme which can extract data from published plots, the articles and data therein were used in the meta-analysis [If a study had extractable data to construct a 2 × 2 table with the number of false positives, false negatives, true positives and true negatives, or the sensitivity, specificity or AUC of the receiver operating characteristic (ROC) curve of circulating adiponectin in the prognosis of GDM, then it was included in the meta-analysis. If a study was selected for the systematic review but did not provide data that could be included in the meta-analysis, the authors were contacted via e-mail. If the authors did not reply or did not provide the requested information, but the relevant information was extractable using a reverse engineering technique through Plot Digitizer , study sample characteristics , test characteristics  and reference standards for diagnosing GDM.p < 0.10) is suggestive of asymmetry and small study bias [We used the QUADAS-2 checklist to assess the quality of the selected studies and the risk of bias associated with the study protocol . We asseudy bias .I2 measure [We used STATA/SE  and SAS/STAT software  for statistical analysis. We used the random effects model for binary data to estimate a summary measure of diagnostic odds ratio (DOR) with 95% CIs. The DOR summarises the diagnostic accuracy of a test and can take values from 0 to infinity. A DOR of 1 represents an uninformative test; as the DOR increases it reflects a test with increasing discriminatory power. Herein, it expresses the odds of having low levels of adiponectin below a given threshold associated with GDM in each study (positive test results) among women with GDM relative to the odds of high adiponectin levels among women without GDM , 24. Het measure , and vis measure –28.Ethics approval was not required, as we pooled previously published studies.Figure The characteristics of the 13 studies included in the systematic review are listed in ESM Table ESM Fig. We present data from 11 studies with extractable data incorporating 2,865 women of whom 794 were diagnosed with GDM by OGTT following adiponectin testing. Figure Figure +0–17+6 weeks’ gestation. Sensitivity analysis of the remaining studies that assessed the value of adiponectin in the first trimester resulted in a sensitivity of 60.3% , a specificity of 81.3%  and a DOR of 6.6 . The AUC was 0.79 , which was not substantially different from the summary estimates that included all 11 studies, but with greater CI because of the smaller sample size.The majority of the pooled studies measured circulating adiponectin during the first trimester, with the exception of the study by Weerakiet et al , which mThree studies included patients at high risk  of developing GDM , 15, 32.I2 value was 70.8%. Univariate meta-regression analysis showed that different age of women among the studies explained 57.9% of the heterogeneity. Ethnicity distribution within the studies accounted for 59.4% of between-study heterogeneity. Multivariate meta-regression demonstrated that both covariates (ethnicity and age) accounted for 100% of between-study heterogeneity. The different methods of measuring adiponectin accounted for 5.4% of the between-study variation in the DOR, yet there was no suggestion of individual test superiority (p = 0.28). Between-study differences in the BMI, the threshold of adiponectin for discriminating women with GDM from women without GDM, the reference criteria of OGTT for diagnosing GDM, the timing of the adiponectin test or the study design had a negative r2, which suggests that each covariate explained less of the heterogeneity than would be expected by chance. The funnel plot shown in ESM Fig. p = 0.77).The estimated Our study suggests that early pregnancy measurement of circulating adiponectin in isolation has a moderate predictive accuracy for the development of GDM and could facilitate targeted OGTT screening . This wo1c, GDM diagnosis requires the much more cumbersome and costly OGTT. Thus, any simple test which can meaningfully lessen this burden has potential to improve clinical practice.This is the first study assessing the summary predictive value of low circulating adiponectin predating the diagnosis of GDM. Low adiponectin has been linked with type 2 diabetes: a 1−log μg/ml increase in serum adiponectin was associated with a 38% decrease in the risk of developing type 2 diabetes . CirculaAdiponectin is an adipocyte-derived hormone whose circulating levels are closely and inversely related to insulin concentration and insulin resistance, and is a good reflection of whole body insulin sensitivity . It is a1c has a sensitivity of 19% and a specificity of 95%; and fructosamine has a sensitivity of 12% and a specificity of 95% [Notably, combinations of multiple conventional risk factors in early pregnancy  often do not predict later GDM well. A Health Technology Assessment systematic review found that risk factors as a screening test produced sensitivities of 50–69% and specificities of 58–68% . MeasureI2 was 70.8%, suggesting a moderate degree of heterogeneity [n = 42 [n = 32 [This is the first study presenting pooled data in a large number of women to assess the predictive value of circulating adiponectin in early identification of women who subsequently develop GDM. The strengths of this review lie in its methodology. We used an extensive search strategy , did notogeneity ) and theogeneity . In addiogeneity . Our sysogeneity , 35, we  [n = 42  and n =  [n = 32  which wa [n = 32 . Our stu [n = 32 . However [n = 32  may enabFirst trimester biomarkers that can predict independently or additively late pregnancy complications have the potential to stratify women in different risk categories, initiate different surveillance patterns and trigger preventative measures. This approach would be complementary to universal screening for GDM at the end of the second trimester, with diagnosis and treatment of milder types of GDM improving perinatal outcomes. Furthermore, in many countries, including the UK, where universal screening is not available, accurate biomarkers can optimise targeted screening by reducing the numbers of false positives and false negatives, with a potential reduction in cost and the clinical implications of missing GDM cases. Current clinical guidelines suggest risk stratification solely based on maternal baseline characteristics and previous history, using a categorical approach rather than a risk score . HoweverESM Methods(PDF 68 kb)ESM Fig. 1(PDF 37 kb)ESM Fig. 2(PDF 120 kb)ESM Table 1(PDF 323 kb)ESM Table 2(PDF 82 kb)


Superior mesenteric artery (SMA) syndrome or Wilkie's syndrome is a rare but potentiallylife threatening gastrointestinal condition. This syndrome is a clinical phenomenonbelieved to be caused by compression of the third part of the duodenum between the SMAand the aorta, leading to obstruction. Patients may present symptoms of gastrointestinalobstruction, such as with recurrent episodes vomiting, upper abdominal distension andepigastric tenderness2, weight: 50 kg, length:160 cm), abdominal distension,epigastric tenderness. Laboratory investigations showed a total white cell count of 9500 mmcomputerized tomography scan revealed grosslydistended stomach and duodenum proximal to the third part of the duodenum at the levelof the origin of superior mesenteric artery with abrupt narrowing at this level,suggestive of Wilkie's syndrome. While, normally, the angle between the SMA and theaorta is 22° to 60°, in this case, the aortomesenteric angle was 13,5°(A 27 years old woman was referred to our hospital, with recurrent episodes of profusevomiting and upper abdominal pain associated with loss of appetite and dyspepsia sincetwo years. She had no other comorbidities. Had been treated at another hospital withproton pump inhibitors, analgesics and intravenous fluids. She had a history of chronicanorexia and progressive loss of weight along with recurrent episodes of vomiting andupper abdominal pain. Clinical examination revealed dehydration, asthenicity (body massindex 19,5 kg/mwas 13,5°. In this,,Wilkie's syndrome occurs when the third portion of the duodenum is compressed betweenthe SMA and the aorta. While, normally, the angle between the SMA and the aorta is 25°to 60°, it is narrowed in this syndromeThe diagnosis of Wilkie's Syndrome requires a high degree of clinical suspicionconfirmed by radiographic studies demonstrating compression of the third portion of theduodenum. CT of the abdomen typically shows gastric and duodenal dilation and narrowedaortomesenteric angle


Data pertaining to risk factors associated with stillbirths and very low Apgar scores is very sparse. This study was conducted to determine the prevalence of, and examine the socio-demographic and obstetric factors associated with stillbirths and very low Apgar scores among vaginal births in a tertiary health facility, Ghana.st January to 31st December, 2009. Background characteristics and obstetric history of the mother as well as the vital status of the baby at birth were extracted. Risk factors associated with stillbirths and very low Apgar scores were examined using binomial regression with a log-link function, and population attributable fractions calculated for significant risk factors.A retrospective cross-sectional review of vaginal deliveries conducted at a teaching hospital in Ghana from 1Of the 8,758 deliveries which met the inclusion criteria, 5.9% of the babies were stillbirths, and 6.5% and 1.9% of live births had very low Apgar scores in the first and fifth minutes respectively. Preterm delivery, hypertensive disorders in pregnancy, breech delivery and vacuum extraction were significant risk factors for stillbirths and very low Apgar scores in the fifth minute of life. Low birth weight was also a significant risk factor for very low Apgar scores.The prevalence of stillbirths and very low Apgar scores were high. Improving the quality of obstetric care during labour and delivery may help improve these adverse vaginal birth outcomes. Over 3 million stillbirths occur globally each year, nearly all of which are in low-income countries–4. StillThe vaginal route remains the commonest mode of delivery in all health care facilities. A few studies have reported the prevalence of adverse birth outcomes and the maternal and reproductive factors associated with these outcomes in sub-Saharan Africa, 10, 13.The main aim of this study was to determine the prevalence of stillbirths and very low Apgar scores among vaginal births in a tertiary health facility in Ghana, and examine the socio-demographic and obstetric factors associated with these adverse vaginal birth outcomes.st January to 31st December, 2009. The hospital receives referrals from at least five out of the 10 regions in the country. About 13,000 deliveries are conducted in the hospital annually and nearly three-quarters of these are vaginal deliveries. There are three labour wards in the hospital, one of which is located in the intensive care unit for hypertensive disorders in pregnancy. Women admitted to these labour wards are monitored at regular intervals for maternal and fetal conditions as well as the progress of labour using the modified WHO partograph[This is a retrospective cross-sectional study of vaginal deliveries conducted at Komfo Anokye Teaching Hospital (KATH), a tertiary referral facility in Kumasi, Ghana from 1rtograph. Intermirtograph, 16.The hospital keeps a delivery register of all deliveries conducted in its labour wards. All records in the register were reviewed. All women who delivered vaginally at KATH within the study period were eligible for inclusion. Births without gestational age and birth weights were excluded. For each birth record, we abstracted information on the date of delivery, socio-demographic characteristics of the mother, obstetric factors such as parity, presentation, number of foetuses (single or multiple pregnancy), hypertensive disorders in pregnancy  and the type of vaginal delivery . The sex, birth weight and Apgar scores of each baby at delivery were also extracted.The main outcomes examined in this study were stillbirth and very low Apgar scores. Stillbirth was defined as death of a fetus weighing at least 1000 g or after 28 completed weeks of gestation occurring before the complete expulsion or extraction from its mother in accordance with World Health Organisation-agreed definition of stillbirth for international comparison. A viabl2 test while continuous variables were compared using student t-tests. To quantify the risk of stillbirth or very low Apgar scores with the maternal and obstetric factors, crude and adjusted relative risks (RRs) with corresponding 95% confidence intervals (CIs) were calculated using univariable and multivariable binomial regression with a log-link function. Univariable analysis was performed to examine the association of each risk factor with stillbirth or very low Apgar scores and factors which reached statistical significance at P < 0.1 were entered into the multivariable model using the forward stepwise approach. Factors which remained significant (P < 0.1) were included the final model for each outcome. Excluded risk factors were retested in the final model one at a time to confirm lack of association. Statistical significance was assessed using the likelihood ratio and Wald tests. Tests for linear trend were performed for variables with more than two ordered categories.Data was double entered into Microsoft Excel database, cleaned and transferred to Stata 11.0  for statistical analysis. Categorical variables were compared using χThe study was approved by the Committee on Human Research, Publications and Ethics of the Kwame Nkrumah University of Science and Technology, Kumasi, Ghana. This research adhered to the STROBE guidelines for observational studies.A total of 12,190 deliveries were conducted in the period under study: 8,856 (72.6%) were vaginal deliveries, out of which 8,758 (98.9%) records met the inclusion criteria. Ninety-eight (1.1%) vaginal birth records were excluded for the following reasons; 67 (0.76%) babies weighed less than 1000 g at birth, 26 (0.29%) were born before 28 completed weeks of gestation and 5 (0.06%) lacked information on the birth weight and estimated gestational age at delivery.The demographic and reproductive characteristics of the women are shown in Table Overall, 517 (5.9%) women had stillbirths. About one quarter of preterm or breech deliveries and 11% of vacuum extractions were stillbirths. On univariable analysis, stillbirth was associated with older mothers (>35 years), being unemployed or in semiskilled employment, grand multiparity (parity ≥ 5), hypertensive disorders in pregnancy, breech delivery, vacuum extraction and preterm births. Table On multivariable analysis, stillbirths were significantly higher among grand multiparous women , breech delivery , vacuum extraction , hypertensive disorders in pregnancy  and preterm delivery . Adolescent mothers were less likely to have stillbirths compared to older mothers, although this was not statistically significant.Five hundred and twenty-four (6.5%) of 8,125 live births for whom Apgar scores were documented in the first minute of life had very low Apgar scores 0–3). The median Apgar score in the first minute was 6 (interquartile range 5–7). Nulliparity , preterm birth , breech delivery , vacuum extraction , low birth weight  and male sex  were predictive of very low Apgar scores in the first minute of life. The risk of having very low Apgar scores in the first minute increased with decreasing parity (P trend < 0.001), see Table . The medAt 5 minutes, 148 (1.9%) of 7,669 live births whose Apgar scores were recorded had very low Apgar scores. The median Apgar score at 5 minutes was 8 (interquartile range 7–8). A significant proportion of the neonates  initially with Apgar scores of 0–3 attained improved scores of 4–10 at 5 minutes. Breech delivery , vacuum extraction , preterm birth  and low birth weight  remained significantly associated with very low Apgar scores at 5 minutes. Maternal age (P trend = 0.009) and hypertensive disorders in pregnancy  which were not significantly associated with low Apgar scores in the first minute of life, became predictive of very low Apgar scores in the fifth minute, Table The prevalence of stillbirths and babies with very low Apgar scores are high among women with vaginal births in this study. Hypertensive disorders in pregnancy, preterm births, low birth weight, vacuum extraction and breech deliveries were not only important risk factors for stillbirth, but were also predictive of very low Apgar scores at 5 minutes. These maternal and obstetric risk factors associated with adverse vaginal birth outcomes require closer monitoring and timely interventions when necessary during delivery.The 5.9% stillbirths observed in this study compares well with reported stillbirth rates in other tertiary hospitals in sub-Saharan Africa, 18, 19.Although it was not possible to distinguish between intra-uterine fetal deaths and intrapartum stillbirths in this study, unpublished reports from the centre  indicate that about 40% of stillbirths occur during delivery, suggesting that about half of these stillbirths may not have occurred during labour. It has been suggested that up to 60% of intrapartum stillbirths can be avoided with the provision of better obstetric care including skilled attendance at birth, the use of partograph for earlier recognition of intrapartum complications (in low resource settings) and appropriate timely interventions to manage these complications, 21. DesAbout 6.5% and 2% of live births had very low Apgar scores at 1 and 5 minutes respectively which are consistent with scores from other studies in low-resource countries, 22. TheThe study has some limitations. As a retrospective analysis, the study was limited to the available data in the delivery register which excluded such factors as booking status, antepartum haemorrhage, prelabour rupture of membranes, arrival in late second stage, indication for vacuum delivery and obstetric practices such as induction and augmentation of labour. It would have been useful to categorise stillbirth into intra-uterine fetal death and intrapartum stillbirth, which is a better indicator of the quality of delivery care received. Our findings in this tertiary referral facility cannot be generalised to the rest of the population due to selection bias. Notwithstanding these limitations, the study identifies important factors associated with adverse vaginal birth outcomes in this low resource setting.The prevalence of stillbirths and very low Apgar scores associated with vaginal deliveries is high in the study setting. Although preterm birth, low birth weight and hypertensive disorders in pregnancy were significant risk factors for stillbirths and very low Apgar scores at 5 minutes, breech deliveries and vacuum extractions were the important obstetric practices associated with these adverse vaginal birth outcomes. Improving quality of obstetric care during labour (both within and outside the tertiary facility) and delivery can reduce these adverse birth outcomes. Strategies to reduce pre-labour and intrapartum stillbirths, and improve Apgar scores must be identified and strengthened.


Mummified human tissues are of great interest in forensics and biomolecular archaeology. The aim of this study was to analyse post mortem DNA alterations in soft tissues in order to improve our knowledge of the patterns of DNA degradation that occur during salt mummification. In this study, the lower limb of a female human donor was amputated within 24 h post mortem and mummified using a process designed to simulate the salt dehydration phase of natural or artificial mummification. Skin and skeletal muscle were sampled at multiple time points over a period of 322 days and subjected to genetic analysis. Patterns of genomic fragmentation, miscoding lesions, and overall DNA degradation in both nuclear and mitochondrial DNA was assessed by different methods: gel electrophoresis, multiplex comparative autosomal STR length amplification, cloning and sequence analysis, and PCR amplification of different fragment sizes using a damage sensitive recombinant polymerase. The study outcome reveals a very good level of DNA preservation in salt mummified tissues over the course of the experiment, with an overall slower rate of DNA fragmentation in skin compared to muscle. Mummification is the process by which soft tissue decomposition after death is halted or significantly slowed, resulting in long-term preservation. From a medical perspective, mummies enable the origins and evolution of human disease to be studied directly, thereby making them a unique reservoir of information about the human past Natural mummification can occur under a variety conditions. Examples of natural mummification by desiccation are typically found in hot and arid environments, such as Egypt Natural mummification can also occur under wet conditions such as with the Mawangdui female corpse of Changsha, China Examples of artificial mummification are known from both ancient Egypt and the Chinchorro culture of South America. During ancient Egyptian artificial mummification water was removed from the body using natron, a mixture of salts composed of sodium carbonate, sodium bicarbonate, sodium sulphate and sodium chloride. After desiccation, the body was then embalmed with resins and oils that chemically stabilized the tissues and further inhibited decay Although mummification can be described in broad terms, many aspects of the process remain poorly characterized and are only partially understood. Natural mummification is often a sporadic occurrence, and preservation can vary greatly both among and within individuals. During natural mummification by desiccation, for example, it is not uncommon for a head or a foot to preserve exceptionally well while the rest of the body is reduced to mere skeletal remains. By contrast among bog bodies, the skin and hair is often preserved in exquisite detail but the entire skeleton may be lacking if the pH of the bog is low. In addition to tissue-level differences, variation can also be observed in the preservation of biomolecules, such as DNA. Although ancient DNA can be extracted from Peruvian mummies or permafrost mummies like Ötzi, DNA recovery from ancient Egyptian mummies is more controversial Teasing apart the various factors that cause these differences is a difficult challenge, and relatively few studies have specifically investigated experimental mummification in chickens and pigeons In order to better characterize the mummification processes, we have isolated a crucial step common to several forms of artificial and natural mummification, salt desiccation, and replicated it under laboratory conditions. Natural salt mummies have been found from collapsed salt mines in Cherabad, Iran The study of post mortem DNA damage during the mummification process is critically important to ensure the generation of accurate data from ancient or forensic sources of DNA. The aim of this research is to characterize the effect of salt desiccation on the preservation of human tissues at the biomolecular level. This is the first study to apply molecular methodologies to the investigation of post mortem alterations in a human experimental mummification setting.intra vitam body donation declaration. Written informed consent from the donor was obtained by the Institute of Anatomy for use of this sample in research. Ethics committee approval of Canton Zurich was not required for this research project, since research projects on body parts of deceased donors, who donated their bodies during their lifetime to the Institute of Anatomy, University of Zurich for research projects do not require additional approval of the ethics committee (cf. §29 Abs.5 as well as §32 Patients law), and a letter of waiver was written to this effect.The following artificial mummification experiment was carried out in the Institute of Anatomy at the University of Zurich in accordance with medical ethics standards and permissions, including an A lower limb (LL) was amputated from an adult female donor 24 h post mortem (day 0) who had died of natural causes. The LL was desiccated in a container of natron salt, in simulation of the desiccation phase of an ancient Egyptian mummification. For our experiment 70 kg of artificial natron was prepared. Prior to use, the components of the artificial natron  were purSkin and muscle tissue samples were collected from the mid-calf sampling area at days 1, 3, 5, 7, 11, 14, 19, 25, 32, 38, 45, 52, 60, 73, 94, 125, 160, 208, 322 . Each tissue sample was separated into three parts, one to assess histological and the remaining two for assessing biomolecular post mortem changes. Following each tissue collection, the LL was transported to University Hospital Balgrist, Zurich for MRI and CT imaging to further characterize tissue changes. The LL was then replaced in the natron mixture. Tissue samples for genetic analysis were shock frozen in liquid nitrogen and stored at −80°C until analysis. For standard histological analysis, tissues were fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin wax and sectioned using microtome . Routine histological staining (Hematoxylin-Eosin) was performed as discussed in another publication R DNA mini extraction kit (Qiagen CA) following manufacturer protocols. Between 25 and 45 mg of each tissue was used in the extraction. Quantification of extracted DNA from each sample was performed using a NanoDrop ND-1000 spectrophotometer and a Qubit fluorometer (Invitrogen).Genomic DNA was extracted in the Institute of Anatomy, University Zurich, twice for each time point from both skin and muscle samples using a QIAampWhole genomic DNA was extracted from paired skin and muscle samples, in duplicate on separate occasions, and fragmentation was assessed using two methods. First, genomic DNA fragmentation in skin and muscle samples was visualized using gel electrophoresis by loading 5 µL DNA extract into a 0.8% agarose gel containing Gel-Red DNA stain and characterizing genomic DNA fragmentation across multiple time points.DNA fragmentation analysis was also performed using the Agilent DNA 12000 kit (PN 5067-1508) on the Agilent 2100 Bioanalyzer System (Agilent Technologies) following the manufacturer's protocols. The 12000 kit can measure DNA fragments up to 12,000 bp long and has the highest upper size limit among the Agilent kits.To characterize DNA fragmentation at a sub-genomic level on a scale most relevant for forensic and ancient DNA applications (50–400 bp) approximately 1 ng DNA was amplified with the SGM Plus multiplex kit (Applied Biosystems/Life Technologies) in a total reaction volume of 25 µL on a GeneAmp PCR System 9700 (Life Technologies) according to the manufacturer's protocol. PCR products were detected with a Genetic Analyzer 3130 xl (Life Technologies). One microliter of the amplified sample was then added to 12.5 µL Hi-Di Formamide and 0.5 ml of GeneScan-500 ROX (Life Technologies). The following electrophoresis conditions were used: 18 s injection time, 1.2 kV injection voltage, 15 kV run voltage, 60°C, 25 min. run time, Dye Set F . Raw data were analyzed with the Genemapper Software (Life Technologies). A peak detection threshold of 50 RFUs was used.2 (Life Technologies), 0.5 µM forward and 0.5 µM reverse primers (Microsynth), 0.2 mM each dNTP, 0.25 µL AmpliTaq Gold, and 10 ng template DNA. The PCR cycling conditions were as follows: 95°C for 10 min, 30 cycles of 95°C for 30 s, 58°C for 30 s, and 72°C for 30 s, and a final extension at 72°C for 10 min. Escherichia coli cells using standard protocols. Between 20 and 25 colonies from each sample were randomly picked and PCR amplified with Go Taq Flexi DNA Polymerase (Promega) in 25 µL reaction volume. Each reaction contained 1× Go Tag Flexi buffer (Promega), 0.5 µM T3 forward (ATTAACCCTCACTAAAGG) and 0.5 µM T7 (AATACGACTCACTATAGG) reverse primers (Microsynth), 0.2 mM each dNTP, 0.2 µL Go Taq Flexi DNA Polymerase. The PCR cycling conditions were as follows: 95°C for 2 min, 25 cycles of 95°C for 20 s, 50°C for 20 s, and 72°C for 30 s with final extension at 72°C for 10 min.To assess changes in DNA sequence quality  during salt mummification, 1000 bp long nuDNA and mtDNA targets spanning identity informative regions  were PCR-amplified from skin and muscle samples from day 0 and day 322. Each 25 µL reaction contained 1× AmpliTaq Gold buffer (Life Technologies), 1.5 mM MgClPositive PCR reactions were sequenced using a Genetic Analyzer 3100 (Life Technologies) and analyzed using CLC Main Workbench software. The resulting sequences were aligned against the mtDNA and nuDNA sequences obtained at day 0 to analyze for postmortem damage sites. All day 0 mtDNA sequences were aligned and compared to the Cambridge reference sequence Overall DNA damage (fragmentation and base modifications) in skin and muscle samples was assayed by PCR amplification using Phusion polymerase (Thermo Scientific), a highly accurate and thermostable recombinant DNA polymerase that stalls at most miscoding lesions, such as cytosine deaminations and abasic sites Chi-square statistics with Yates correction (χ2) was performed (GraphPad InStat3) to investigate whether the observed patterns of base transitions and transversions in mtDNA and nuDNA sequences were similarly distributed. All tests were performed at the 5% significance level.At day 322 of the mummification experiment the foot of the LL was substantially shrunken and dehydrated, whereas the thigh had not yet reached the complete dehydration and mummification. The weight of the LL decreased from 6.049 kg on day 0 to 3.232 kg on day 322 of the mummification experiment. During the experiment, the temperature fluctuated from 20–26°C and relative humidity ranged from 35–70%. The pH of the LL was observed to increase from pH 6 to 10 over the course of the experiment.The histological examination reveals generally good soft tissue preservation. The muscle fibres were shrunken, but otherwise morphologically unchanged. The epimysium, connective tissue that surrounds the muscle fibres, showed minimal alteration. The nuclei of the muscle cells stained clearly for the entire duration of the experiment. The skin was similarly well preserved, although structural changes were more pronounced compared to muscle. By day 208, the five layers of the epidermis were no longer easily distinguishable: the dermis was shrunken, and structures such as the vascular networks of the papillary dermis and the free sensory nerve endings, were not discernible  [38].Whole genomic DNA fragmentation in muscle and skin extracts from multiple time points was visualized using gel electrophoresis . High moIn order to evaluate viability of DNA amplification at a sub-genomic level on a scale most relevant for forensic and ancient DNA applications (50–400 bp), DNA was amplified using the AmpFlSTR SGM Plus PCR Amplification Kit, a multiplex STR assay commonly used in forensic analysis . The kitIn the multiplex STR assay, signal intensity is proportional to the quantity of DNA containing the targeted marker. In fresh tissue, the quantity of DNA should be equal for all markers, but in highly degraded tissues, DNA fragmentation results in an underrepresentation of longer markers. In order to compare the relative proportion of short to long targets, a ratio was calculated for the shorter locus D19S433 (amplicon range 102–135 bp) and the longer locus D18S51 (amplicon range 262–355 bp)  [42]. A 2χ values for comparison between days 0–322. No significant differences were observed in mitochondrial DNA between day 0 and 322 in any tissue. The same is true for nuclear DNA damage in skin. Slight, but non-significant, differences, however, were observed in muscle nuclear DNA  and two tissues (skin and muscle). All mtDNA and nuDNA sequences were aligned to the revised Cambridge Reference Sequence (rCRS) lear DNA . The mislear DNA  accordin−7). The error rate, determined by a modified lacI-based method In order to characterize overall DNA damage, we next amplified the DNA extracts for mitochondrial and nuclear DNA targets of four lengths  using Phusion, a synthetic DNA polymerase that is highly sensitive to many forms of DNA damage. Unlike Taq-based polymerases, as was used in the AmpFlSTR assay and to characterize miscoding lesions in amplicon clones, Phusion DNA Polymerases have extremely low error rates (4.4×10Long (8000 bp) mitochondrial and nuclear targets could be readily amplified from the day 0 extracts . Over ti2 test P = 0.0265 – <0.0001) across the date points and tissues , modified to extend the desiccation period from 40 days to 322 days In our study we simulated the natron salt desiccation step of ancient Egyptian mummification described by Herodotus  confirmed by studies using 454 sequencing and Illumina sequencing Although next generation sequencing (NGS) can be a powerful tool for evaluating DNA fragment length in highly degraded samples, NGS cannot be used to assess preservation in samples this well preserved. The observed fragment lengths in this study were so long that they would have required shearing prior to NGS analysis, a step that would have defeated the goals of this damage assessment. For this reason, NGS was not attempted.The PCR amplifiable level of each amplicon was assessed in comparison with the level at day 0. These data in turn enabled two complementary analyses to be performed, tracking of each target through the mummification time and the differential degradation between the four targets. These two measures therefore enable the assessment of the rate of degradation of single transcript and the relative rate of degradation of multiple transcripts, respectively. As expected in the beginning of the experiment all mitochondrial and nuclear targets could be successfully amplified from both tissues. But over time, the maximum amplifiable target length decreased, with earlier decline in muscle (day 11 post mortem) than in skin (day 38 post mortem). Nuclear DNA also failed to amplify earlier than mitochondrial, which is in agreement with experience on aDNA analysis Analysis showed that post mortem DNA degradation was present in both muscle and skin tissues. The types of damage identified include DNA fragmentation, nucleotide substitution and DNA loss. This resulted in band smearing on gel electrophoresis and increased ratio of shorter to longer fragments in STR analysis. In addition, the frequency of successful PCR of longer nuclear and mitochondrial PCR products declined earlier than for shorter products over time. These changes were first observed in muscles and then in skin, which can be due to the direct proximity of skin to the salt and thus the higher efficiency of dehydration of skin than muscles.We used a non-proofreading Taq (AmpliTaq Gold) to identify nucleotide miscoding lesions. The degree of nucleotide damage we observed was consistently low across both targets and both tissues. Although we observed more degradation in day 322 samples than the day 0, none of these increases are significant . This inMummification can occur under a wide range of desiccating or anoxic conditions, but only mummification by desiccation generally provides high quality DNA. The results of this pilot study in artificial salt mummification indicate that salt desiccation is an effective method for medium to long-term preservation of human tissues at the molecular level. Overall, DNA preservation in salt-desiccated skin and muscle was observed to be excellent, with damage-free, amplifiable DNA fragments in excess of 4000 bp nearly one year post mortem. Among tissues, preservation was greater in skin, most likely because this tissue was in direct contact with the natron salt and thus was more efficiently dehydrated than interior tissues, such as muscle. Although DNA fragmentation increased over the course of the experiment, with noticeably higher fragmentation in muscle than in skin, no significant increases in miscoding lesions were observed in either skin or muscle. These results suggest that under salt desiccation little nucleotide damage occurs, and DNA strand breaks are slowed in proportion to salt proximity. Natron mummification results in remarkable DNA preservation and likely contributes to the long-term survival of DNA in a range of natural and artificial mummified tissues.


Epos Lhc, which incorporates additional hydrodynamic evolution of the created system, is able to reproduce most of the data features, unlike Hijing and Ampt. The Spectra of identified charged hadrons are measured in pPb collisions with the CMS detector at the LHC at  The study of hadron production has a long history in high-energy particle and nuclear physics, as well as in cosmic-ray physics. The absolute yields and the transverse momentum  detector can be found in Ref. . The CMSThe reconstruction of charged particles in CMS is bounded by the acceptance of the tracker  is used. The Epos [Epos Lhc [Hijing and Ampt models for proton–nucleus collisions.In this paper, comparisons are made to predictions from three MC event generators. The Hijing  event geHijing , in addiThe Ampt  event geThe Epos  event geEpos Lhc  is an imThe data were taken in September 2012 during a 4-h-long pPb run with very low probability of multiple interactions (0.15 % “pileup”). A total of 2.0 million collisions were collected, corresponding to an integrated luminosity of approximately at the trigger level, the coincidence of signals from both BPTX devices, indicating the presence of both proton and lead bunches crossing the interaction point; in addition, at least one track with offline, the presence of at least one tower with energy above 3xel hits , are supThe efficiencies for event selection, tracking, and vertexing were evaluated using simulated event samples produced with the Hijing 2.1 MC event generator, where the CMS detector response simulation was based on Geant4 [The event selection consisted of the following requirements:n Geant4 . SimulatThe simulated ratio of the data selection efficiency to the DS selection efficiency is shown as a function of the reconstructed track multiplicity in the top panel of Fig. The extrapolation of particle spectra into the unmeasured gorithms , used ingorithms , 16, 19,The acceptance of the tracker (mentclass2pt{minimThe region where pPb collisions occur (beam spot) is measured by reconstructing vertices from many events. Since the bunches are very narrow in the transverse direction, the lgorithm  was usedThe vertex reconstruction resolution in the Hijing event generator, the corrections are reweighted by a The hadron spectra were corrected for particles of non-primary origin  The statistical uncertainties for the extracted yields are given by the fits. The observed local variations of parameters in the The measured yields in each rization  was usedThe corrected yields were obtained by applying corrections for acceptance , but change rapidly at very low The systematic uncertainties originating from the unfolding procedure were studied. Since the In previously published measurements of unidentified and identified particle spectra , 24, thedynamics , the parAs discussed earlier, pions and kaons cannot be unambiguously distinguished at higher momenta. Because of this, the pion-only, the kaon-only, and the joint pion and kaon η≈0, Eq.  becomes:Ampt 1.26/2.26 [Epos Lhc [Hijing 2.1 [The results discussed in the following are for laboratory rapidity .26/2.26 , Epos LhEpos Lhc , 15, andjing 2.1  MC eventjing 2.1 .Ampt, Epos Lhc, and Hijing predictions. Epos Lhc gives a good description, while other generators predict steeper The transverse momentum distributions of positively and negatively charged hadrons  are shown in Fig. entclass1pt{minimaAmpt simulation, only Epos Lhc is able to predict both Ratios of particle yields as a function of the transverse momentum are plotted in Fig. A study of the dependence on track multiplicity is motivated partly by the intriguing hadron correlations measured in pp and pPb collisions at high track multiplicities –31, suggollision . The strollision . In addiollision .Hijing simulation in The event multiplicity  hadrons .   is shown (0–5 %) . These A=0.2TeV, , 4, 6) t=0.2TeV,  suggestsFor low track multiplicity . While the data display a linear dependence on mass with a slope that increases with particle multiplicity, the models predict a flat or slowly rising behavior versus mass and only limited changes with track multiplicity. This is to be compared with pp results [pythia 6 [pythia 8 event generators show features very similar to those in pPb data. A similar trend is also observed in nucleus–nucleus collisions [The transverse momentum spectra could also be successfully fitted  predict steeper The results can be used to further constrain models of hadron production and contribute to the understanding of basic non-perturbative dynamics in hadron collisions. The Combined with similar results from pp collisions, the track multiplicity dependence of the average transverse momentum and particle ratios indicate that particle production at LHC energies is strongly correlated with event particle multiplicity in both pp and pPb interactions. For low track multiplicity, pPb collisions appear similar to pp collisions. At high multiplicities, the average


Megalobrama amblycephala) is an important freshwater aquaculture fish throughout China. Because of widespread introductions of this species to many regions, the genetic diversity of wild and natural populations is now threatened. In the present study, SRAP (sequence-related amplified polymorphism) markers were used to assess genetic diversity of blunt snout bream. Three natural populations  and one genetic strain (‘Pujiang No. 1’) of blunt snout bream were screened with 88 SRAP primer combinations, of which 13 primer pairs produced stable and reproducible amplification patterns. In total, 172 bands were produced, of which 132 bands were polymorphic. Nei's gene diversity (h) and Shannon's information index (I) values provided evidence of differences in genetic diversity among the five populations (Poyang Lake>Liangzi Lake>Nanxian>‘Pujiang No. 1’>Yuni Lake). Based on cluster analysis conducted on genetic distance values, the five blunt snout bream populations were divided into three groups, Poyang Lake and Liangzi Lake , Nanxian and ‘Pujiang No. 1’ , and Yuni Lake . Significant genetic differentiation was found among the five populations using analysis of molecular variance (AMOVA), with more genetic divergence existing among populations (55.49%), than within populations (44.51%). This molecular marker technique is a simple and efficient method to quantify genetic diversity within and among fish populations, and is employed here to help manage and conserve germplasm variability of blunt snout bream and to support the ongoing selective breeding programme for this fish.The blunt snout bream ( It was originally distributed in the middle Yangtze River and a few accessory lakes, of which Yuni Lake, Liangzi Lake and Poyang Lake are the major sources The Yangtze River is the world's third longest river system, with more than 3000 tributaries and 4000 lakes Genetic diversity has been estimated in several natural and genetically selected populations of blunt snout bream using morphometrics and isozymes In this study, SRAP markers are used to investigate population genetic diversity and differentiation among three natural populations, a genetically selected strain and a cultured population of blunt snout bream in China. Our aim is to better understand the genetic diversity and integrity of natural populations based upon geography and the genetic affinities of selected strain and cultured populations based upon history. This work contributes to the critical need for germplasm conservation while at the same time supporting the ongoing selective breeding and improvement of blunt snout bream.No specific permits were required for the field studies described here. The sampling lakes and ponds are not privately-owned or protected in any way, and the field sampling activities did not involve endangered or protected species.2) was clipped from each individual without anesthesia. After sampling, the individuals of the selected and cultured populations were returned to their corresponding ponds, whereas the individuals of the three natural populations were transported to the Ezhou fish breeding base of Huazhong Agricultural University (HZAU) for further breeding programme. The clipped fin tissues were preserved in 95% ethanol. Total DNA was extracted from fin tissues using the phenol-chloroform method In total, 234 individuals from five blunt snout bream populations were included in this study. Samples were collected from three natural populations , from one selected strain (‘Pujiang No. 1’ from Shanghai), and from one cultured population (Nanxian County in Hunan Province). Details of the sampled populations are provided in A set of 19 SRAP primers , 0.5 µM each of primers, and 20 ng template DNA. All PCRs were conducted in a PTC-200 Thermocycler under the following conditions: an initial denaturing step was performed at 94°C for 5 min, followed by five cycles of 1 min at 94°C, 1 min at 35°C, 1 min at 72°C, then 35 cycles at 94°C for 1 min, 50°C for 1 min, 72°C for 1 min, followed by a final extension step at 72°C for 10 min. PCR products were separated on a 6% non-denaturing polyacrylamide gel via electrophoresis and silver staining was used to visualize the results.All SRAP PCR reactions were carried out in a final volume of 15 µL containing 1.5 µL 10×PCR buffer, 2.0 mM MgN), percent of polymorphic bands (P), Nei's gene diversity (h), Shannon's information index (I), Nei's unbiased genetic distance (D) and genetic similarity (S) estimates D, a NJ tree with bootstrap support values  was constructed using POPTREE2 FST) was used to evaluate the level of genetic differentiation among populations. Sequential Bonferroni corrections Using a 100 bp DNA marker ladder as a size standard, SRAP size-dependent fragments were scored as absent (0) or present (1) to obtain a binary matrix. Only clear, strong and reproducible bands in the size range of 60 to 1124 bp were scored. POPGENE 1.32 was used to calculate number of polymorphic bands  to determine the population assignment success for each individual fish based on its genetic diversity. The SRAP markers had 140 variable bands, a number too large for the DFA to work with, so we summarised the genetic variation using principal components analysis (PCA). The first 40 eigen vectors, which explained 82% of the variation in the SRAP data set, were used in the DFA. The DFA was based on original group size . The DFA and PCA were carried out using STATISTICA v7 software (StatSoft Ltd).Eighty-eight primer combinations were tested and of these, 13 primer pairs  showed clear, reproducible and polymorphic banding patterns. The number of amplified bands for each primer pair ranged from 8 to 21 (average 13). In total, 172 bands were detected, of which 132 bands showed polymorphism, giving a polymorphic value of 76.7%.N) and the percentage of polymorphic bands (P) were highest in the PYL population , and lowest in the YNL population . Nei's gene diversity (h) across the five populations ranged from 0.090 (YNL) to 0.185 (PYL), and Shannon's information index (I) ranged from 0.137 (YNL) to 0.280 (PYL) (For fish from the five populations (mean ± SD sample size of 46.8±2.17), the number of polymorphic bands (80 (PYL) .FST values ranged from 0.351 between the geographically adjacent natural populations of Liangzi Lake (LZL) and Poyang Lake (PYL), to 0.685 between the Yuni Lake (YNL) population and the genetically selected strain of ‘Pujiang No. 1’ (PJ-1). The differences in genotype frequencies between all pairs of populations were highly significant (P<0.001)  .FST = 0.555, P<0.001)  of hierarchical gene diversity indicated that 44.51% of the genetic variation was explained by within-population variation, whereas 55.49% of the variation was explained by among-population variation, with a significant fixation index (P<0.001) .D) among all populations ranged from 0.087 (LZL-PYL) to 0.270 (YNL-NX), and Nei's unbiased genetic similarities (S) ranged from 0.763 (YNL-NX) to 0.917 (LZL-PYL) (Nei's unbiased genetic distances (LZL-PYL) .The NJ dendrogram constructed using Nei's unbiased genetic distance  showed tFST, AMOVA and NJ analysis, all of which reveal a high level of genetic differentiation among fish from the five populations.Discriminant function analysis (DFA) of the first 40 eigen values from the PCA resulted in 100% correct assignment. That is, all fishes from all populations were correctly assigned to their populations of sampling. The DFA result is consistent with the et al. noted that the cluster of lakes within the middle Yangtze River region requires protection for the preservation of migratory fishes and that there is a need to identify areas of high fish biodiversity to mitigate loss of fish biodiversity in the Yangtze River basin China's freshwater fish diversity is high at 920 species in 302 genera N, percent of polymorphic bands P, Nei's gene diversity h, Shannon's information index I) showed the same order from largest to smallest for the five populations. Both h and I indicated that the highest genetic diversity level was observed in the PYL population, followed by the LZL population, and the lowest was in the YNL population, all three being natural populations of Megalobrama amblycephala. As suggested by Li et al.All four indices  and large yellow croaker (Pseudosciaena crocea) For the cultured (Nanxian) and selectively bred (‘Pujiang No. 1’) populations, the levels of genetic diversity were much lower than within the native PYL and LZL populations. These results are in agreement with investigations on largemouth bass . This surprising result may be due to the degradation of germplasm resources of YNL over a reasonably short period of time. Previously, Li FST is one of the most widely used measures for evaluating the genetic differentiation between/among populations, which can provide important insights into the evolutionary processes that cause the genetic differentiation among populations FST values is to some extent species- or even group-specific, as a general rule FST values of 0–0.05 represent little differentiation, values of 0.05–0.25 indicate moderate differentiation, and values higher than 0.25 indicate very great differentiation among populations FST between the five populations were >0.25, indicative of a high level of genetic differentiation among these five populations. The highest FST value detected (0.685) was between PJ-1 and YNL, suggesting that pronounced genetic differentiation now exists between the selectively bred strain (PJ-1) and the natural YNL population. These findings were supported by our discriminant function analysis (DFA) which was able to assign each and every fish to its population of origin, are comparable to the results reported by 7 strain). The five populations of blunt snout bream clustered into three groups, as revealed by NJ analysis. Two of the three wild populations, PYL and the geographically close population of LZL, clustered together, while the third wild population (YNL) was highly differentiated from the other two wild populations . In addition, we tried to construct a NJ tree based on pairwise FST between populations at the same time. The NJ tree based on pairwise FST was consistent with the results above (data not shown). These results confirm earlier observations that obvious genetic divergence occurred between fish from YNL and LZL or PYL, and no divergence occurred between LZL and PYL et al.In conclusion, the SRAP system has been successfully employed to assess population genetic diversity among domesticated and wild genotypes of blunt snout bream from China. Analysis revealed that cultivated, genetically selected and wild bream germplasm are highly differentiated and that wild lake populations have high levels of genetic diversity. Comparison of the genetic diversity of wild fish to that of cultured and selectively bred fish illustrates the need for greater care managing this important resource so that the aquaculture programme to support this species is not disadvantaged in the future and so that wild populations of fish are protected for both conservation and fisheries purposes.


Phalacrocorax atriceps). Video footage was obtained from 12 animals and 49 dives comprising a total of 8.1 h of foraging data. Video information revealed that Imperial cormorants are almost exclusively benthic feeders. While foraging along the seafloor, animals did not necessarily keep their body horizontal but inclined it downwards. The head of the instrumented animal was always visible in the videos and in the majority of the dives it was moved constantly forward and backward by extending and contracting the neck while travelling on the seafloor. Animals detected prey at very short distances, performed quick capture attempts and spent the majority of their time on the seafloor searching for prey. Cormorants foraged at three different sea bottom habitats and the way in which they searched for food differed between habitats. Dives were frequently performed under low luminosity levels suggesting that cormorants would locate prey with other sensory systems in addition to sight. Our video data support the idea that Imperial cormorants’ efficient hunting involves the use of specialized foraging techniques to compensate for their poor underwater vision.During the last few years, the development of animal-borne still cameras and video recorders has enabled researchers to observe what a wild animal sees in the field. In the present study, we deployed miniaturized video recorders to investigate the underwater foraging behavior of Imperial cormorants ( The foraging behavior of seabirds has been difficult to study, principally because they spend most of their time at sea and obtain their food underwater. Fortunately, over the last 30 years, technological advances have resulted in the development of recording devices that can be attached to marine animals, providing information about their movements and behaviors at sea e.g. , 2, 3). , 3. 2, 3During the last few years, development of animal-borne still cameras and video recorders has enabled researchers to observe what a wild animal sees in the field e.g. , 8, 9). , 9. 8, 9Phalacrocorax atriceps) from Punta León  forage benthically [Raneya fluminensis), benthic fish (Riberoclinus eigenmanni), and toadfish  throughout the breeding season [Imperial cormorants , and ifg season  or to wag season . The geng season , 27 but g season ). Secondg season , so it iFieldwork was conducted at Punta León provincial protected area  in December 2011 and 2012 under the project “"Ecología pelágica de aves marinas buceadoras: determinación de movimientos y comportamiento en el mar mediante la utilización de registradores electrónicos de alta resolución" directed by Flavio Quintana and authorized by La Secretaria de Turismo y Áreas Protegidas (STyAP) and La Dirección de Fauna y Flora Silvestre (DFyFS) of the Province of Chubut. Thirty-three male breeding Imperial cormorants were instrumented with 12 POV MAC10 Mini waterproof action video cameras designed to be water resistant at depth < 10 m . Because Imperial cormorants dive deeper than 10 m, the cameras were protected in underwater custom housings (8 cm in length and 2.5 cm in diameter) . The total weight of the camera plus the housing was 49.3 g, less than 2.2% of the average adult male body weight (2.3 kg) . The camhttp://www.kinovea.org/). Footage included cormorant behavior both at the colony, immediately prior to foraging trips, and during the first hour of foraging. However, for the purposes of this study, we focused only on video segments that were recorded after the animals left the colony to forage.Video footage was viewed using Kinovea free video player /0.7. This equation was constructed with the significant parameters obtained from a mixed effect model that incorporated 992 dives performed by 20 Imperial cormorants instrumented with daily diaries (DD) units [For each U-shaped dive, we calculated the amount of time the bird spent descending in the water column , the amount of time on the sea floor , the duration of the ascent phase , and the post-dive interval . The descent duration was used to calculate the maximum dive depth reached during a particular dive, following the equation: D) units  with preD) units .Only dives deeper than 2 m were included in the diving behavior analysis, since shallower dives are known to be associated with activities other than foraging  . Foragindownward when the 100% of the image showed the sea floor, slightly downward (approximately a 20° angle) when the image showed between 1/4 and 3/8 of water, horizontal when the image was 50% water and 50% seabed, and upward if only water was visible.The estimation of cormorant body angle during the bottom phase of dives was determined following the methodology presented by Watanuki et al. . The oripredominantly gravel , rocky,t bottom ) and grajpeg in R v. 3.0.2 [To determine the luminosity level of the microhabitats visited by cormorants, firstly we extracted an image of the bottom phase of each dive. The red, green, and blue (RGB) values for each pixel were obtained using the function readJPEG from the package v. 3.0.2 . RGB valZone 1), 2) a zone with a limited amount of texture and detail that represented very dark and light objects with some texture  and 3) a zone with full texture and detail . Each bottom image was classified into one of the previously defined zones by looking at the predominant values of the luminosity histogram. The head of the bird was present in all these images. Special care was taken so that the proportion of pixels occupied by the head was similar between images.Once all pixels had been converted to gray scale, we created a luminosity histogram for each image. In this histogram the vertical axis represented the number of pixels (frequency) while the horizontal axis represented the brightness value. Brightness values ranged between 0 (pure black) and 1 (pure white). Following , the horlme function from the package lnme of the open source statistical package R version 3.0.2 [glmer function from the package lme4. In all the mixed effect models bird identity was set as a random factor. Significant effects were detected applying a backward selection procedure and using the anova function from the package stats.For each diving parameter the presented grand mean was calculated as the mean of means of each individual. Differences in several diving parameters  between cormorants instrumented with cameras and cormorants instrumented with DD during 2005 and 2006 we tested by means of linear mixed effects models (LMM) with Gaussian distribution. These models were run using the on 3.0.2 . A generP<0.05. Results are shown as the grand mean ± standard error. The median and the range of each calculated variable are also presented.Statistical analysis were performed with a level of significance of Of a total of 33 instrumented cormorants, only 14 left the colony while their cameras were on. Two of these cormorants remained in the coastal area in front of the colony, collecting algae in shallow waters. Consequently, we obtained video footage from 12 foraging animals . We recoDuring the recorded period, the equipped animals performed a total of 38 U-shaped bottom dives . The maxDuring the U-shaped benthic dives, the average amount of time spent foraging along the sea floor was 111.1 ± 9.4 s . No diffSpheniscus magellanicus) was recorded in the ascent phase of a dive (In only one dive (2.9%), another cormorant was registered in the field of view of the camera, while in two occasions (4.1%) a Magellanic penguin . Male Imperial cormorants spent on average 92.6% of their time at the bottom searching for food and 6.7% capturing prey. The rest of the bottom time (less than 1%) involved manipulating and swallowing prey.We did not see any evidence that cormorants probed into the substrate with their bills. Rather birds seemed to detect prey at short distances and employ a prey-flushing strategy. In other words, cormorants exploited the escape response that they triggered on prey.-1 .During capture attempts, cormorants made quick turns and maintained their body in a vertical position facing downwards . Capture-1 . MaximumIn those capture attempts where the capture, manipulation, and swallow events took place underwater and could be clearly distinguished from one another (n = 6), the manipulation time ranged between 1 and 17 s, while the amount of time spent swallowing prey ranged between 1 and 2 s. In three other occasions, cormorants were observed to capture prey in the last few seconds of the bottom phase, subsequently carrying it in the bill to the surface. Once on the surface, the amount of time animals spent manipulating prey ranged between 3 and 22 s. We should note here that because Imperial cormorants mostly consume small and cryptic prey (see next section), manipulation and swallowing times could only be determined when large, visible prey were captured. The success rate where prey were observed being capture was of 100%.Pleoticus muelleri) during the ascent phase. Although we were able to clearly observe the cormorants feeding, it was difficult to detect and identify specific prey items by using the video cameras. We were able to identify prey species on only four occasions. Surprisingly, benthic fish (Riberoclinus eigenmanni) and toadfish  were not found in any of the videos despite their prevalence in the diet of Imperial cormorants at Punta León [Video images revealed that cormorants are almost exclusively benthic feeders; we only observed one occasion in which an animal captured and swallowed a Patagonian red shrimp (nta León . We attrAcanthistius brasilianus) in two cases (Raneya brasiliensis) in one case (Cottoperca gobio) in one case . This habitat supported patches of anemones, gorgonians, snails, and algae (rocky habitats (grasslike habitat (Instrumented cormorants foraged at three different sea bottom habitats. Most of the dives (77.8%) reached depths between 20 and 60 m on a seafloor composed of a matrix of predominantly gravel that also contained broken mollusk shells, and pebbles , navigating between rocks and foraging by inserting the neck and head between reef ledges  [Even though Imperial cormorants from Punta León frequently flew with conspecifics to their foraging grounds, we did not detect any evidence that they fed in group. Solitary feeding might be favored by birds feeding on sparse and/or cryptic benthic prey in rocky and gravel habitats, where the chances of encountering and capturing other nearby prey items are small. In such cases, Imperial cormorants might rely on underwater topographic cues to find prey, similar to other species in this genus  . These tnogenis) , 42.Our video data on cormorant underwater body orientation clearly support previous findings obtained by the use of accelerometers , 43. To The frequency of capture attempts and the speed reached on pursuits is critical for air breathing predators , 45. SwiEven though the videos only allow us to detect and identify large prey species , they undoubtedly represent the most comprehensive data so far collected on microhabitat use by Imperial cormorants while foraging. Our study shows that cormorants use at least three different microhabitats of varying biodiversity and physical features. The most commonly used microhabitat was a gravel bottom characterized by low levels of marine life, followed by an area with numerous reefs and rocky outcrops. In one instance, we observed cormorants diving to a bottom covered by polychaete tubes and algae. Because the cormorants overwhelmingly used one microhabitat over the other two, we were unable to make statistical comparisons of their diving behavior  across microhabitats. However, in a general sense, cormorant body inclination and foraging technique were quite different between the two most frequently visited habitats suggesting different foraging strategies among habitats.The amphibious lifestyle of cormorants imposes important optical challenges because aquatic optical requirements differ from those in air . CornealWhile foraging along the seafloor, cormorants were frequently observed to move their head constantly forward and backward. This rhythmic movement of the head called “head-bobbing” is observed in many avian species , 53 and Cormorants´poor visual acuity is further reduced under low light conditions . SeveralUse of animal-borne video cameras corroborated several aspects of Imperial cormorants’ foraging behavior previously determined by other electronic devices. At the same time, the use of cameras offered new insights into Imperial cormorants foraging behavior and seems to be a powerful tool for investigating how these predators capture prey. In addition, cameras provide information about the biological and physical characteristics of the underwater habitats where cormorants foraged. In agreement with Martin et al.  and Whit