Comparative analysis of the bacterial communities associated with <i>P.</i><i>clavata</i>.

<p>16S rDNA pyrosequencing-based comparative analysis of dominant bacterial groups associated with <i>P. clavata</i> samples collected in different geographic areas, at different depth and showing different health status condition. (a) a comparative map is shown, where the numbers of normalised reads taken by each taxon (the tree is collapsed to the ‘genus’ level) in each year are represented as colour bar. The cumulative number of normalised reads across the different coral samples is also shown for each taxon <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067745#pone.0067745-Speck1" target="_blank">[29]</a>. Genus shared across all samples are in bold (b) agglomerative hierarchical clustering (CLUSTER analysis) and (c) non-Metric multi-Dimensional Scaling (nMDS) of the different sample datasets.</p

<i>P. clavata</i> samples collected in this study.

<p>Collected samples from different coral populations living in different geographic areas, at different depth and showing different health status condition. The level of direct anthropogenic Impact on coral populations at each site was quantified by measuring.</p>*<p>the number of coral colonies affected by fishing lines and nets wrapped around: absent = 0 entangled colonies/dive (P-pristine); occasional = 1–5 entangled colonies/dive and frequent = >5 entangled colonies/dive (I-impacted).</p>**<p>the number of dives per year is also reported: low = <1000 dives/year; high >5000 dives/year.</p>***<p>Diseased corals (D) are defined as those showing early symptoms of disease such as colonies displaying necrotic coenenchyme (pale pink to grayish colour) and patchy tissue loss exposing bare areas of the skeletal axis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067745#pone.0067745-Vezzulli1" target="_blank">[7]</a>. Healthy corals (H) are defined as those with no apparent symptoms of disease displaying a purple coloration of the coral tissue <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067745#pone.0067745-Vezzulli1" target="_blank">[7]</a>.</p

Analysis of <i>Vibrio</i> populations.

<p><i>Vibrio</i> relative abundance index (VAI) calculated on <i>P. clavata</i> samples collected in different geographic areas, at different depth and showing different health status condition. Z values are obtained by subtracting the population mean and dividing the difference by the s.d. * ANOVA p<0.05;+presence of the species <i>V. coralliilyticus</i>.</p

Bacterial diversity associated with <i>P.</i><i>clavata</i>.

<p>Alpha diversity metrics derived from 16S rDNA pyrosequencing of bacteria associated with <i>P. clavata</i> samples: (a) Rarefaction curves; (b) number of OTUs predicted (Chao1, Ace, jackknife) and observed (sobs); (c) Shannon-Weiner diversity of bacteria from each coral sample. OTU’s were grouped at >97% similarity based on mothur classified results.</p

<i>P.</i><i>clavata</i> population impacted by human activity.

<p>Human impacted <i>P. clavata</i> population sampled at “Cala Levante” (Pantelleria island) site in October 2011 at a depth of 63 m: (a) Coralligenous assemblage with a rope and a fishing line (arrows) entangling sea fans. The abrasive effect on sea-fan coenechime expose the bare skeleton to fouling organisms. (b) Sampling phase of a <i>P. clavata</i> colony with evident sign of necrotic tissue (arrows).</p

<i>V</i>. <i>cholerae</i> detection in CPR samples from endemic Cholera regions (South West Africa).

<p>Relative abundance of <i>Vibrio</i> spp. and <i>Vibrio cholerae</i> in CPR samples collected in cholera endemic areas of the Benguela Current Large Marine Ecosystem region (BCLME, South West Africa) as a proof of concept for the possible use of the CPR technology and the developed qPCR assay in cholera studies (see main text for details).</p

Performance of the qPCR assay for detection of <i>V</i>. <i>cholerae</i> in artificially degraded DNA samples.

<p>(A) Electropherogram plot obtained by Agilent Bioanalyzer analysis of artificially fragmented genomic DNA of <i>V</i>. <i>cholerae</i> ATCC 39315. (B) Plot of mean Cq-values from three replicates tested against the <i>V</i>. <i>cholerae</i> artificially fragmented DNA inputs. Error bars indicate the standard deviations of the means.</p

qPCR detection of <i>V</i>. <i>cholerae</i> ATCC 39315 cells in mixed cultures containing <i>V</i>. <i>mimicus</i> UM 6812.

<p>qPCR detection of <i>V</i>. <i>cholerae</i> ATCC 39315 cells in mixed cultures containing 0.5 ng/rx (corresponding to 8x10⁴ GU/rx) of <i>V</i>. <i>mimicus</i> UM 6812. Quantification cycle (Cq) is expressed as mean ± standard deviation calculated from two spiking experiments each quantified in triplicate on the same run (n = 6)</p><p>qPCR detection of <i>V</i>. <i>cholerae</i> ATCC 39315 cells in mixed cultures containing <i>V</i>. <i>mimicus</i> UM 6812.</p

Sensitivity of the qPCR assay for detection of <i>V</i>. <i>cholerae</i> O1 El Tor N16961.

<p>DNA was amplified with the gbpA TaqMan primers in the presence of the gbpA fluorogenic probe. (A) Amplification plot of <i>V</i>. <i>cholerae</i> ATCC 39315 sample dilutions containing 10⁶, 10⁵, 10⁴, 10³, 10², 10¹ genome copies per reaction (GU/rx). (B) Plot of mean Cq-values from three replicates tested against the <i>V</i>. <i>cholerae</i> DNA inputs. Error bars indicate the standard deviations of the means.</p

Amplification parameters for the qPCR assay applied to environmental and stool samples.

<p>The values of Efficiency, Slope and Intercept are expressed as mean ± standard deviation calculated from two spiking experiments with <i>V</i>. <i>cholerae</i> ATCC 39315 cells, each quantified in triplicate on the same run (n = 6)</p><p>Amplification parameters for the qPCR assay applied to environmental and stool samples.</p