Visualization of coral host--pathogen interactions using a stable GFP-labeled Vibrio coralliilyticus strain

The bacterium Vibrio coralliilyticus has been implicated as the causative agent of coral tissue loss diseases (collectively known as white syndromes) at sites across the Indo-Pacific and represents an emerging model pathogen for understanding the mechanisms linking bacterial infection and coral disease. In this study, we used a mini-Tn7 transposon delivery system to chromosomally label a strain of V. coralliilyticus isolated from a white syndrome disease lesion with a green fluorescent protein gene (GFP). We then tested the utility of this modified strain as a research tool for studies of coral host–pathogen interactions. A suite of biochemical assays and experimental infection trials in a range of model organisms confirmed that insertion of the GFP gene did not interfere with the labeled strain’s virulence. Using epifluorescence video microscopy, the GFP-labeled strain could be reliably distinguished from non-labeled bacteria present in the coral holobiont, and the pathogen’s interactions with the coral host could be visualized in real time. This study demonstrates that chromosomal GFP labeling is a useful technique for visualization and tracking of coral pathogens and provides a novel tool to investigate the role of V. coralliilyticus in coral disease pathogenesis.Human Frontier Science Program (Strasbourg, France) (No. RGY0089RS

Manual EcoDiver MAC: Gu\ueda al Monitoreo del Ambiente Costero Mediterr\ue1neo Mediterraneo. Ver. 5.2 Espa\uf1ol.

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Development of long-term primary cell aggregates from Mediterranean octocorals

In lower metazoans, the aggregative properties of dissociated cells leading to in vitro stable multicellular aggregates have furnished a remarkable experimental material to carry out investigations in various research fields. One of the main expectations is to find good models for the study in vitro of the first steps of biomineralization processes. In this study, we examined five common Mediterranean gorgonians (Paramuricea clavata, Corallium rubrum, Eunicella singularis, Eunicella cavolinii, and Eunicella verrucosa) using mechanical cell aggregate production techniques. In particular, we investigated the conditions of aggregate formation, their number and survival in experimental conditions, the DNA synthesizing activity using 5\u2032-bromo-2\u2032-deoxyuridine (BrdU) tests, and the response to calcein addition and observed the secretion of newly formed sclerites. The BrdU tests showed that cell proliferation depends on the size of aggregates and on the presence/absence of symbiotic zooxanthellae. With epifluorescent and confocal imaging from calcein addition assays, we observed the presence of calcium ions within cells, a possible clue for prediction of sclerite formation or calcium deposition. The species-specific efficiency in  production of cell aggregates is correlated to the size of polyps, showing that the higher density of polyps and their diameter correspond to higher  production of cell aggregates. Regarding the long-term maintenance, we obtained the best results from E. singularis, which formed multicellular aggregates of 0.245 mm \ub1 0.086 mm in size and maintained symbiotic association with zooxanthellae throughout the experimental run. Formation of sclerites within aggregates opens a wide field of investigation on biomineralization, since de novo sclerites were observed around 30 d after the beginning of the experiment

Testing methods to support management decisions in coralligenous and cave environments. A case study at Portofino MPA

12 páginas, 3 tablas, 6 figuras.Baseline data on the distribution, condition and extent of coralligenous and cave bioconcretions is one of the main requirements of the European Marine Strategy Framework Directive (MSFD) necessary to assess the achievement of a Good Environmental Status (GES) by 2020. In this study the potential of remote sensing and distribution modelling techniques to map, measure descriptors and choose indicators were tested, that could provide standard methods for the assessment of the health status and assist in monitoring activities. It is demonstrated how, by combining different methodologies, it is possible to map the distribution of the bioconcretions with acceptable accuracy and to discriminate the main habitat types and facies. In addition, zonal statistical analysis revealed that fishing activities primarily coincide with areas of high coverage of the bioconcretions. Results demonstrate that the presented methodology is a valuable simple tool to assess several MSFD descriptors and indicators, and could strengthen management efficiency when making informed, ecologically relevant decisions.This research was funded by the European project “Training Network for Monitoring Mediterranean Marine Protected Areas” (MMMPA: FP7-PEOPLE-2011-ITN, Grant Agreement no.: 290056) and 2010e11 PRIN project “Coastal bioconstructions: structures, functions, and management” prot. 2010Z8HJ5M_003.Peer reviewe

Testing methods to support management decisions in coralligenous and cave environments. A case study at Portofino MPA

Baseline data on the distribution, condition and extent of coralligenous and cave bioconcretions is one of the main requirements of the European Marine Strategy Framework Directive (MSFD) necessary to assess the achievement of a Good Environmental Status (GES) by 2020. In this study the potential of remote sensing and distribution modelling techniques to map, measure descriptors and choose Indicators were tested, that could provide standard methods for the assessment of the health status and assist in monitoring activities. It is demonstrated how, by combining different methodologies, it is possible to map the distribution of the bioconcretions with acceptable accuracy and to discriminate the main habitat types and facies. In addition, zonal statistical analysis revealed that fishing activities primarily coincide with areas of high coverage of the bioconcretions. Results demonstrate that the presented methodology is a valuable simple tool to assess several MSFD descriptors and indicators, and could strengthen management efficiency when making informed, ecologically relevant decisionsDepartamento Administrativo de Ciencia, Tecnología e Innovación [CO] Colciencias7277-569-33195Links between marine biotic evolution and carbonate platform and petroleum reservoir development in the Guajira Basing, Colombia Caribbeann

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

<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

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

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