Exploring priming strategies to improve stress resilience of Posidonia oceanica seedlings.

Seagrasses' ability to store information after exposure to stress (i.e. stress memory) and to better respond to further stress (i.e. priming) have recently been observed, although the temporal persistence of the memory and the mechanisms for priming induction remain to be defined. Here, we explored three priming strategies in Posidonia oceanica seedlings, each inducing a different level of stress, for temperature and salinity. We investigated changes in morphometry, growth rate and biomass between primed and non-primed seedlings. The results showed similar behaviour of seedlings when exposed to an acute stress event, regardless of whether they had been primed or not and of the priming strategy received. This opens the debate on the level of stress necessary for inducing a priming status and the persistence of the stress memory in P. oceanica seedlings. Although no priming-induced stress resistance was observed, seedlings showed unexpectedly high resilience to extreme levels of both abiotic stressors.

Carbon economy of Mediterranean seagrasses in response to thermal stress.

Increased plant mortality in temperate seagrass populations has been recently observed after summer heatwaves, although the underlying causes of plant death are yet unknown. The potential energetic constrains resulting from anomalous thermal events could be the reason that triggered seagrass mortality, as demonstrated for benthic invertebrates. To test this hypothesis, the carbon balance of Posidonia oceanica and Cymodocea nodosa plants from contrasting thermal environments was investigated during a simulated heatwave, by analyzing their photosynthetic performance, carbon balance (ratio photosynthesis:respiration), carbohydrates content, growth and mortality. Both species were able to overcome and recover from the thermal stress produced by the six-week exposure to temperatures 4 °C above mean summer levels, albeit plants from cold waters were more sensitive to warming than plants from warm waters as reflected by their inability to maintain their P:R ratio unaltered. The strategies through which plants tend to preserve their energetic status varied depending on the biology of the species and the thermal origin of plants. These included respiratory homeostasis (P. oceanica warm-plants), carbon diversion from growth to respiration (C. nodosa cold-plants) or storage (P. oceanica warm-plants) and changes in biomass allocation (C. nodosa warm-plants). Findings suggest an important geographic heterogeneity in the overall response of Mediterranean seagrasses to warming with potential negative impacts on the functions and services offered by seagrass meadows including among others their capacity for carbon sequestration and carbon export to adjacent ecosystems.

Experimental evidence of warming-induced flowering in the Mediterranean seagrass Posidonia oceanica.

Sexual reproduction in predominantly clonal marine plants increases recombination favoring adaptation and enhancing species resilience to environmental change. Recent studies of the seagrass Posidonia oceanica suggest that flowering intensity and frequency are correlated with warming events associated with global climate change, but these studies have been observational without direct experimental support. We used controlled experiments to test if warming can effectively trigger flowering in P. oceanica. A six-week heat wave was simulated under laboratory mesocosm conditions. Heating negatively impacted leaf growth rates, but by the end of the experiment most of the heated plants flowered, while controls plants did not. Heated and control plants were not genetically distinct and flowering intensity was significantly correlated with allelic richness and heterozygosity. This is an unprecedented finding, showing that the response of seagrasses to warming will be more plastic, more complex and potentially more resilient than previously imagined.

Antioxidant response to heat stress in seagrasses. A gene expression study.

Seawater warming associated to the ongoing climate change threatens functioning and survival of keystone coastal benthic species such as seagrasses. Under elevated temperatures, the production of reactive oxygen species (ROS) is increased and plants must activate their antioxidant defense mechanisms to protect themselves from oxidative damage. Here we explore from a molecular perspective the ability of Mediterranean seagrasses to activate heat stress response mechanisms, with particular focus on antioxidants. The level of expression of targeted genes was analyzed in shallow and deep plants of the species Posidonia oceanica and in shallow plants of Cymodocea nodosa along an acute heat exposure of several days and after recovery. The overall gene expression response of P. oceanica was more intense and complete than in C. nodosa and reflected a higher oxidative stress level during the experimental heat exposure. The strong activation of genes with chaperone activity (heat shock proteins and a luminal binding protein) just in P. oceanica plants, suggested the higher sensitivity of the species to increased temperatures. In spite of the interspecific differences, genes from the superoxide dismutase (SOD) family seem to play a pivotal role in the thermal stress response of Mediterranean seagrasses as previously reported for other marine plant species. Shallow and deep P. oceanica ecotypes showed a different timing of response to heat. Shallow plants early responded to heat and after a few days relaxed their response which suggests a successful early metabolic adjustment. The response of deep plants was delayed and their recovery incomplete evidencing a lower resilience to heat in respect to shallow ecotypes. Moreover, shallow ecotypes showed some degree of pre-adaptation to heat as most analyzed genes showed higher constitutive expression levels than in deep ecotypes. The recurrent exposure of shallow plants to elevated summer temperatures has likely endowed them with a higher basal level of antioxidant defense and a faster responsiveness to warming than deep plants. Our findings match with previous physiological studies and supported the idea that warming will differently impact Mediterranean seagrass meadows depending on the species as well as on the depth (i.e. thermal regimen) at which the meadow grows. The increase in the incidence of summer heat waves could therefore produce a significant change in the distribution and composition of Mediterranean seagrass meadows with considerable consequences for the functioning of the whole ecosystem and for the socio-economic services that these ecosystems offer to the riverine populations.

Transcriptome characterisation and simple sequence repeat marker discovery in the seagrass Posidonia oceanica.

Posidonia oceanica is an endemic seagrass in the Mediterranean Sea, where it provides important ecosystem services and sustains a rich and diverse ecosystem. P. oceanica meadows extend from the surface to 40 meters depth. With the aim of boosting research in this iconic species, we generated a comprehensive RNA-Seq data set for P. oceanica by sequencing specimens collected at two depths and two times during the day. With this approach we attempted to capture the transcriptional diversity associated with change in light and other depth-related environmental factors. Using this extensive data set we generated gene predictions and identified an extensive catalogue of potential Simple Sequence Repeats (SSR) markers. The data generated here will open new avenues for the analysis of population genetic features and functional variation in P. oceanica. In total, 79,235 contigs were obtained by the assembly of 70,453,120 paired end reads. 43,711 contigs were successfully annotated. A total of 17,436 SSR were identified within 13,912 contigs.

Response of the seagrass Posidonia oceanica to different light environments: Insights from a combined molecular and photo-physiological study.

Here we investigated mechanisms underlying the acclimation to light in the marine angiosperm Posidonia oceanica, along its bathymetric distribution (at -5 m and -25 m), combining molecular and photo-physiological approaches. Analyses were performed during two seasons, summer and autumn, in a meadow located in the Island of Ischia (Gulf of Naples, Italy), where a genetic distinction between plants growing above and below the summer thermocline was previously revealed. At molecular level, analyses carried out using cDNA-microarray and RT-qPCR, revealed the up-regulation of genes involved in photoacclimation (RuBisCO, ferredoxin, chlorophyll binding proteins), and photoprotection (antioxidant enzymes, xanthophyll-cycle related genes, tocopherol biosynthesis) in the upper stand of the meadow, indicating that shallow plants are under stressful light conditions. However, the lack of photo-damage, indicates the successful activation of defense mechanisms. This conclusion is also supported by several responses at physiological level as the lower antenna size, the higher number of reaction centers and the higher xanthophyll cycle pigment pool, which are common plant responses to high-light adaptation/acclimation. Deep plants, despite the lower available light, seem to be not light-limited, thanks to some shade-adaptation strategies (e.g. higher antenna size, lower Ek values). Furthermore, also at the molecular level there were no signs of stress response, indicating that, although the lower energy available, low-light environments are more favorable for P. oceanica growth. Globally, results of whole transcriptome analysis displayed two distinct gene expression signatures related to depth distribution, reflecting the different light-adaptation strategies adopted by P. oceanica along the depth gradient. This observation, also taking into account the genetic disjunction of clones along the bathymetry, might have important implications for micro-evolutionary processes happening at meadow scale. Further investigations in controlled conditions must be performed to respond to these questions.

Establishing research strategies, methodologies and technologies to link genomics and proteomics to seagrass productivity, community metabolism, and ecosystem carbon fluxes.

A complete understanding of the mechanistic basis of marine ecosystem functioning is only possible through integrative and interdisciplinary research. This enables the prediction of change and possibly the mitigation of the consequences of anthropogenic impacts. One major aim of the European Cooperation in Science and Technology (COST) Action ES0609 "Seagrasses productivity. From genes to ecosystem management," is the calibration and synthesis of various methods and the development of innovative techniques and protocols for studying seagrass ecosystems. During 10 days, 20 researchers representing a range of disciplines (molecular biology, physiology, botany, ecology, oceanography, and underwater acoustics) gathered at The Station de Recherches Sous-marines et Océanographiques (STARESO, Corsica) to study together the nearby Posidonia oceanica meadow. STARESO is located in an oligotrophic area classified as "pristine site" where environmental disturbances caused by anthropogenic pressure are exceptionally low. The healthy P. oceanica meadow, which grows in front of the research station, colonizes the sea bottom from the surface to 37 m depth. During the study, genomic and proteomic approaches were integrated with ecophysiological and physical approaches with the aim of understanding changes in seagrass productivity and metabolism at different depths and along daily cycles. In this paper we report details on the approaches utilized and we forecast the potential of the data that will come from this synergistic approach not only for P. oceanica but for seagrasses in general.

Genetic structure in the Mediterranean seagrass Posidonia oceanica: disentangling past vicariance events from contemporary patterns of gene flow.

The Mediterranean Sea is a two-basin system, with the boundary zone restricted to the Strait of Sicily and the narrow Strait of Messina. Two main population groups are recognized in the Mediterranean endemic seagrass Posidonia oceanica, corresponding to the Western and the Eastern basins. To address the nature of the East-West cleavage in P. oceanica, the main aims of this study were: (i) to define the genetic structure within the potential contact zone (i.e. the Strait of Sicily) and clarify the extent of gene flow between the two population groups, and (ii) to investigate the role of present water circulation patterns vs. past evolutionary events on the observed genetic pattern. To achieve these goals, we utilized SSR markers and we simulated, with respect to current regime, the possible present-day dispersal pattern of Posidonia floating fruits using 28-day numerical Lagrangian trajectories. The results obtained confirm the presence of the two main population groups, without any indices of reproductive isolation, with the break zone located at the level of the Southern tip of Calabria. The populations in the Strait of Sicily showed higher affinity with Western than with Eastern populations. This pattern of genetic structure probably reflects historical avenues of recolonization from relict glacial areas and past vicariance events, but seems to persist as a result of the low connectivity among populations via marine currents, as suggested by our dispersal simulation analysis.

Dr. Zompo: an online data repository for Zostera marina and Posidonia oceanica ESTs.

As ecosystem engineers, seagrasses are angiosperms of paramount ecological importance in shallow shoreline habitats around the globe. Furthermore, the ancestors of independent seagrass lineages have secondarily returned into the sea in separate, independent evolutionary events. Thus, understanding the molecular adaptation of this clade not only makes significant contributions to the field of ecology, but also to principles of parallel evolution as well. With the use of Dr. Zompo, the first interactive seagrass sequence database presented here, new insights into the molecular adaptation of marine environments can be inferred. The database is based on a total of 14 597 ESTs obtained from two seagrass species, Zostera marina and Posidonia oceanica, which have been processed, assembled and comprehensively annotated. Dr. Zompo provides experimentalists with a broad foundation to build experiments and consider challenges associated with the investigation of this class of non-domesticated monocotyledon systems. Our database, based on the Ruby on Rails framework, is rich in features including the retrieval of experimentally determined heat-responsive transcripts, mining for molecular markers (SSRs and SNPs), and weighted key word searches that allow access to annotation gathered on several levels including Pfam domains, GeneOntology and KEGG pathways. Well established plant genome sites such as The Arabidopsis Information Resource (TAIR) and the Rice Genome Annotation Project are interfaced by Dr. Zompo. With this project, we have initialized a valuable resource for plant biologists in general and the seagrass community in particular. The database is expected to grow together with more data to come in the near future, particularly with the recent initiation of the Zostera genome sequencing project.The Dr. Zompo database is available at http://drzompo.uni-muenster.de/

Assessing genetic diversity in clonal organisms: low diversity or low resolution? Combining power and cost efficiency in selecting markers.

The increasing use of molecular tools to study populations of clonal organisms leads us to question whether the low polymorphism found in many studies reflects limited genetic diversity in populations or the limitations of the markers used. Here we used microsatellite datasets for two sea grass species to provide a combinatory statistic, combined with a likelihood approach to estimate the probability of identical multilocus genotypes (MLGs) to be shared by distinct individuals, in order to ascertain the efficiency of the markers used and to optimize cost-efficiently the choice of markers to use for deriving unbiased estimates of genetic diversity. These results strongly indicate that conclusions from studies on clonal organisms derived using markers showing low polymorphism, including microsatellites, should be reassessed using appropriate polymorphic markers.

Local genetic structure in a clonal dioecious angiosperm.

We used seven microsatellite loci to characterize genetic structure and clonal architecture at three different spatial scales (from meters to centimetres) of a Cymodocea nodosa population. C. nodosa exhibits both sexual reproduction and vegetative propagation by rhizome elongation. Seeds remain buried in the sediment nearby the mother plant in a dormant stage until germination. Seed dispersal potential is therefore expected to be extremely restricted. High clonal diversity (up to 67% of distinct genotypes) and a highly intermingled configuration of genets at different spatial scales were found. No significant differences in genetic structure were found among the three spatial scales, indicating that genetic diversity is evenly distributed along the meadow. Autocorrelation analyses of kinship estimates confirmed the absence of spatial clumping of genets at small spatial scale and the expectations of a very restricted seed dispersal (observed dispersal range 1-21 m) in this species.

Spatial patterns of genetic diversity in Posidonia oceanica, an endemic Mediterranean seagrass.

Posidonia oceanica is an endemic seagrass species in the Mediterranean Sea. In order to assess levels of genetic structure in this species, the microsatellite polymorphism was analysed from meadows collected in several localities, along the coasts of the Tyrrhenian Sea (Mediterranean Sea). The existence of single population units and the recruitment of seedlings collected in some localities were investigated. Moreover, genetic structure at different spatial scales and biogeographic relationships among populations were also assessed. Our analysis showed the existence of clear patterns of genetic structure in P. oceanica in the area considered in the analysis. P. oceanica, in fact, is present in separate meadows that represent discrete populations, characterized by low genetic diversity. Comparable levels of genetic variability between mature meadows and seedlings were found. Patterns of genetic relatedness among populations seem to be in accord with direction of dominant current flux in the whole area, separating South Tyrrhenian from North Tyrrhenian populations. Moderate levels of gene flow between populations and genetic substructure within populations, together with the finding of the limited role of sexual reproduction in increasing genetic variability, should be a cause for concern for the persistence of this essential resource in the Mediterranean basin.

Assessment of genetic diversity of seagrass populations using DNA fingerprinting: implications for population stability and management.

Populations of the temperate seagrass, Zostera marina L. (eelgrass), often exist as discontinuous beds in estuaries, harbors, and bays where they can reproduce sexually or vegetatively through clonal propagation. We examined the genetic structure of three geographically and morphologically distinct populations from central California (Elkhorn Slough, Tomales Bay, and Del Monte Beach), using multilocus restriction fragment length polymorphisms (DNA fingerprints). Within-population genetic similarity (Sw) values for the three eelgrass populations ranged from 0.44 to 0.68. The Tomales Bay population located in an undisturbed, littoral site possessed a within-population genetic similarity (Sw = 0.44) that was significantly lower than those of the other two populations. Cluster analysis identified genetic substructure in only the undisturbed subtidal population (Del Monte Beach). Between-population similarity values (Sb) for all pairwise comparisons ranged from 0.47 to 0.51. The three eelgrass populations show significantly less between locale genetic similarity than found within populations, indicating that gene flow is restricted between locales even though two of the populations are separated by only 30 km. The study demonstrates that (i) natural populations of Z. marina from both disturbed and undisturbed habitats possess high genetic diversity and are not primarily clonal, (ii) gene flow is restricted even between populations in close proximity, (iii) an intertidal population from a highly disturbed habital shows much lower genetic diversity than an intertidal population from an undisturbed site, and (iv) DNA fingerprinting techniques can be exploited to understand gene flow and population genetic structure in Z. marina, a widespread and ecologically important species, and as such are relevant to the management of this coastal resource.

[The use of the Daylight System in mammography]. / Impiego del Daylight in mammografia.

It is widely known that Daylight film handling has yielded several benefits to diagnostic radiology, that is, reduction in exam duration, thus improving both efficiency and productivity; space saving in departments, by minimizing darkroom spaces; improved working conditions for the staff. This paper reports on the study of the application of a Daylight system to mammography. For this purpose, the authors used an X-ray unit (Senographe 500 T-CGR) and a Dupont Daylight unit, whose diagnostic yield was compared to that of "vacuum" and cassettes units. The results of our experience confirmed the well-known advantages DDS yields to diagnostic radiology. In particular, the use of Daylight rather than vacuum system allowed a considerable reduction in exam duration (10 to 2 minutes). Moreover, the new Dupont screen-film system allowed a reduction in average whole breast dose by about 28% if compared to the conventional recording system employed in our department. Mammograms with high contrast resolution, sensitivity, and good spatial resolution were thus obtained, as shown in the analysis of quality image. The Daylight system allowed a marked improvement to be made in efficiency, productivity, and organization, as well as a reduction in whole breast dose and high-quality mammographic images.

[The factor of magnification in mammography]. / Considerazioni sul fattore di ingrandimento in mammografia.

The last generation of mammographic equipment allows mammography to be performed with direct magnification techniques, thanks to such technical features as microfocus, high focus-film distance, high-power generators. The authors compared the diagnostic yield of two different equipment sets, with 1.4x and 2x magnification respectively, and verified the utility of magnification radiography. A significant reduction was obtained in questionable diagnoses (42 to 18) in a group of 63 patients, with no evidence of substantial differences in the diagnostic yield of the images obtained with different magnification ratios. An objective analysis of the system resolution power privileges 2x magnification ratio, which however implies an increase in the average dose to the breast. The use of faster recording systems reduces the dose by 50%, though maintaining good image quality.

[Role of photon absorption densitometry of the bone in uremic osteodystrophy]. / Ruolo della densitometria ossea ad assorbimento fotonico nella osteodistrofia uremica.

The aim of this investigation is to evaluate the role of bone photonic densitometry in uremic osteodystrophy. Bone mineral content (BMC) and bone density (BD) have been measured in 80 hemodialyzed patients by double photonic emission densitometry. Photonic densitometry shows an higher sensibility to quantitative changes in bone mineral content than metacarpal index (IM). Photonic densitometry is unable to differentiate osteoporosis from osteomalacia; this differential diagnosis can be obtained by radiological analysis: low BD and low IM means osteoporosis, low BD and resorptive changes in cortical bone means osteomalacia and/or hyperparathyroidism. Photonic densitometry is particularly suitable for uremic osteodystrophy follow-up because of its easy repetitiveness and innocuousness and for its close correlation with iPTH variations.