[Show abstract][Hide abstract] ABSTRACT: Ostreopsis ovata is a species-complex harbouring several cryptic species. The Mediterranean-Atlantic populations are homogeneous in terms of phylogenetic markers, but show differences in physiological and ecological properties among sub-regions of the area. The aim of our study was to set up and validate a genetic fingerprint method that can be applied to assess genetic diversity within and among populations of Ostreopsis ovata. To this end, we selected the AFLP (Amplified fragment length polymorphisms) technique. We successfully generated distinct AFLP fingerprints for a number of O. cf. ovata strains isolated from three different sites of the Italian coasts. Reproducibility was confirmed by very similar molecular profiles obtained from three replicates for each strain. A binary matrix was compiled and a cluster analysis of the clones was performed to build a dendrogram for the evaluation of the relationships among strains. Results of this first analysis show that strains from Ancona and Taormina (Adriatic and Ionian seas, respectively) are more closely related amongst themselves than with Neapolitan strains, and confirms AFLP as a powerful technique for the identification of Ostreopsis ovata populations at the regional scale.
15th Internation Conference on Harmful Algae; 03/2014
[Show abstract][Hide abstract] ABSTRACT: Studies on stress genes are fundamental to understand how marine organisms maintain or re-estabilish a normal metabolism in face of physical or chemical disturbances. Aquatic organisms are in fact constantly exposed to environmental stimuli and natural and/or dissolved anthropogenic variables/compounds, including both physical (e.g. cold, heat, salinity and pH) and chemical (e.g. heavy metals, hydrocarbons and other pollutants) stressors. Organisms may react to these stressors by activating a series of cellular defense systems, such as defense, antioxidant and/or condition-specific proteins.In this study Reverse Transcription-Quantitative Polymerase Chain Reaction (RT-qPCR) was used to characterize metabolic processes at the cellular level in response to different CO2 vents. We evaluated the differential expression of selected stress genes (e.g. Glutathione S-transferase, Glutathione reductase, synthase and peroxidase, Superoxide dismutase and HSPs) in the seagrass Posidonia oceanica occurring in the vicinity of natural CO2 vents in two different locations: Panarea and Ischia islands. Similar gene expression levels were observed (with some exceptions), suggesting that individuals may respond in the same way to the same environmental conditions, even at two such separate locations. These results help to understand how stress genes may be used as early indicators of stressful conditions at sea and during laboratory experiments.
Seagrasses in Europe: Threats, Responses and Management, Olhão, Portugal; 03/2014
[Show abstract][Hide abstract] ABSTRACT: Diatoms dominate productive regions in the oceans and have traditionally been regarded as sustaining the marine food chain to top consumers and fisheries. However, many of these unicellular algae produce cytotoxic oxylipins that impair reproductive and developmental processes in their main grazers, crustacean copepods. The molecular mode of action of diatoms and diatom oxylipins on copepods is still unclear. In the present study we generated two Expressed Sequence Tags (ESTs) libraries of the copepod Calanus helgolandicus feeding on the oxylipin-producing diatom Skeletonema marinoi and the cryptophyte Rhodomonas baltica as a control, using suppression subtractive hybridization (SSH). Our aim was to investigate differences in the transcriptome between females fed toxic and non-toxic food and identify differentially expressed genes and biological processes targeted by this diatom. We produced 947 high quality ESTs from both libraries, 475 of which were functionally annotated and deposited in GenBank. Clustering and assembling of ESTs resulted in 376 unique transcripts, 200 of which were functionally annotated. Functional enirchment analysis between the two SSH libraries showed that ESTs belonging to biological processes such as response to stimuli, signal transduction, and protein folding were significantly over-expressed in the S. marinoi-fed C. helgolandicus compared to R. baltica-fed C. helgolandicus library. These findings were confirmed by RT-qPCR analysis. In summary, 2 days of feeding on S. marinoi activated a generalized Cellular Stress Response (CSR) in C. helgolandicus, by over-expressing genes of molecular chaperones and signal transduction pathways that protect the copepod from the immediate effects of the diatom diet. Our results provide insights into the response of copepods to a harmful diatom diet at the transcriptome level, supporting the hypothesis that diatom oxylipins elicit a stress response in the receiving organism. They also increase the genomic resources for this copepod species, whose importance could become ever more relevant for pelagic ecosystem functioning in European waters due to global warming.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Seagrasses adaptation to light can imply changes at morphological, physiological and genetic levels. To investigate the molecular mechanisms of photo-acclimation in the seagrass Posidonia oceanica (Posidoniaceae), we selected nine genes of interest belonging to the photosynthetic apparatus as possible markers of the plant response to light availability and measured pigment composition as well as photosynthetic and photo-protective performances. Introduction -Posidonia oceanica (L.) Delile, meadows have declined extensively
43° Congresso della Società Italiana di Biologia Marina; 10/2013
[Show abstract][Hide abstract] ABSTRACT: 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 underwa-ter 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 distur-bances 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
Frontiers in Plant Science 01/2013; 4:38. · 3.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: For seagrasses, seasonal and daily variations in light and temperature represent the mains factors driving their distribution along the bathymetric cline. Changes in these environmental factors, due to climatic and anthropogenic effects, can compromise their survival. In a framework of conservation and restoration, it becomes crucial to improve our knowledge about the physiological plasticity of seagrass species along environmental gradients. Here, we aimed to identify differences in transcriptomic and proteomic profiles, involved in the acclimation along the depth gradient in the seagrass Posidonia oceanica, and to improve the available molecular resources in this species, which is an important requisite for the application of eco-genomic approaches. To do that, from plant growing in shallow (-5 m) and deep (-25 m) portions of a single meadow, (i) we generated two reciprocal Expressed Sequences Tags (EST) libraries using a Suppressive Subtractive Hybridization (SSH) approach, to obtain depth/specific transcriptional profiles, and (ii) we identified proteins differentially expressed, using the highly innovative USIS mass spectrometry methodology, coupled with 1D-SDS electrophoresis and labeling free approach. Mass spectra were searched in the open source Global Proteome Machine (GPM) engine against plant databases and with the X!Tandem algorithm against a local database. Transcriptional analysis showed both quantitative and qualitative differences between depths. EST libraries had only the 3% of transcripts in common. A total of 315 peptides belonging to 64 proteins were identified by mass spectrometry. ATP synthase subunits were among the most abundant proteins in both conditions. Both approaches identified genes and proteins in pathways related to energy metabolism, transport and genetic information processing, that appear to be the most involved in depth acclimation in P. oceanica. Their putative rules in acclimation to depth were discussed.
Frontiers in Plant Science 01/2013; 4:195. · 3.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Effects of the toxic Gulf of Mexico dinoflagellate Karenia brevis on its copepod grazers are equivocal, in that this dinoflagellate is ingested by various copepods, but may be toxic to, and/or nutritionally inadequate for grazers. Recent studies have shown that when the Mediterranean copepod Calanus helgolandicus fed on K. brevis, egg production rates and egg viability declined steadily over time, reaching almost zero levels after 5 days of feeding (Turner et al., 2012). The K. brevis strain used in that study was chemically analyzed and shown not to contain any brevetoxins, but may have contained other harmful metabolites. Here we present gene expression analyses by reverse transcription-quantitative PCR (RT-qPCR) in C. helgolandicus after different exposure times (3, 5 and 8 days) to the same non-brevetoxin-containing K. brevis strain examined previously. The analyzed genes have a primary role in generic stress responses, defense systems (e.g. aldehyde, free fatty acid and free radical detoxification) and apoptosis regulation in other organisms. After 3 days of feeding on K. brevis, gene expression levels in copepods did not show significant changes. However, after 5 days of exposure, expression of stress-response genes was significantly reduced. After prolonged exposure for 8 days, expression of genes for 5 of 6 aldehyde dehydrogenases was strongly reduced, but expression of other genes was not coherently reduced, or increased. Microscopic observations revealed that after only a single day of feeding on K. brevis, freshly-spawned eggs were morphologically similar to those produced by copepods feeding on control diets, but after 3 days of feeding on K. brevis, eggs exhibited altered membrane cell morphology and apoptotic features such as granulation and degeneration of the cytoplasm matrix, compared to the unaltered eggs produced by copepods feeding on control diets. The changes in gene expression levels in copepods feeding on K. brevis were similar to those noted previously for copepods feeding upon diatoms that contained oxylipins, which interfered with copepod reproductive success. Changes in gene expression in copepods feeding upon toxic algal diets may offer important early warning signals of copepod stress responses to these diets.
[Show abstract][Hide abstract] ABSTRACT: The diatom Pseudo-nitzschia multistriata exhibits a diplontic life cycle composed of an extensive phase of vegetative cell division and a brief phase of sexual reproduction. To explore genotypic stability, we genotyped seven polymorphic microsatellite loci in 26 monoclonal strains over 3-16 months in a culture maintenance regime. Moreover, to assess inheritance patterns of the microsatellite alleles, we genotyped 246 F1 strains resulting from four mating experiments between parental strains of know genotype. Results generally conformed expectations according to Mendelian inheritance patterns, but deviations were detected indicating mutations during sexual reproduction. A total of forty-two mutations were detected in the clonal cultures over time. Microsatellites with more core-repeats accumulated mutations faster. The mutation rate varied significantly across loci and strains. A binomial mass function and a computer simulation showed that the mutation rate was significantly higher during the first months of culture (μ≈3×10(-3) per locus per cell division) and decreased to μ≈1×10(-3) in the strains kept for 16 months. Our results suggest that genetic mutations acquired in both the vegetative phase and sexual reproduction add to the allelic diversity of microsatellites, and hence to the genotypic variation present in a natural population.
[Show abstract][Hide abstract] ABSTRACT: Seagrass ecosystems rank with rainforests and mangroves in their many ecosystem services, yet are drastically declining worldwide as a consequence of both anthropogenic and natural pressures. In spite of this, the level of awareness of seagrass-loss is low – seagrass research is fragmented and there is little integration between researchers and coastal zone managers. The Cost Action network ES0906 'Seagrass productivity, from genes to ecosystem management' was therefore created to assess two main questions: 1) How is the productivity of seagrass meadows along European coastlines affected by anthropogenic disturbances that alter the light, temperature and pH environment and, consequently, how does this affect their capacity to adapt while still providing ecological services and function? 2) How can we implement a scientifi cally-based management system across Europe that will provide both baseline and predictive information to help to prevent seagrass decline? What makes this project so unique? European research about the goods and services arising from seagrass productivity is fragmented and there is little integration between researchers and coastal zone managers. This Action will be unique for Europe, consolidating existing knowledge and coordinating research efforts in physiological ecology, ecological genomics and conservation-resource management, fostering a multilevel approach to the problems from molecules to the ecosystem. Can you discuss some of the in situ measurement devices that you have employed to provide continuous analysis of seagrass productivity? A recent training school took place at Stareso, Corsica where several methods to measure seagrass production at levels varying from molecular, plant and community to ecosystem production were tested. Some of these methods provide continuous measurements of seagrass production, such as the use of multi-channel PAM fl uorometry (which measures electron transport rates at leaf scale), the use of oxygen optodes (which monitor the concentration of oxygen in the water column), and the use of an acoustic method (to detect water density changes related to the concentration of oxygen). The data obtained by these continuous methods will be compared with data obtained by discrete methods, such as traditional production versus irradiance (P/I) curves at the plant level, PAM fl uorometry versus O 2 measurements at leaf scale, incubation methods for measuring seagrass community production (and respiration) and measurements of air/water CO 2 fl uxes to measure production (and respiration) at ecosystem level. Could you describe how you have evaluated the effi cacy of existing and new seagrass health ecological indicators?
[Show abstract][Hide abstract] ABSTRACT: Diatoms are key phytoplankton organisms and one of the main primary producers in aquatic ecosystems. However, many diatom species produce a series of secondary metabolites, collectively termed oxylipins, that disrupt development in the offspring of grazers, such as copepods, that feed on these unicellular algae. We hypothesized that different populations of copepods may deal differently with the same oxylipin-producing diatom diet. Here we provide comparative studies of expression level analyses of selected genes of interest for three Calanus helgolandicus populations (North Sea, Atlantic Ocean and Mediterranean Sea) exposed to the same strain of the oxylipin-producing diatom Skeletonema marinoi using as control algae the flagellate Rhodomonas baltica. Expression levels of detoxification enzymes and stress proteins (e.g. glutathione S-transferase, glutathione synthase, superoxide dismutase, catalase, aldehyde dehydrogenases and heat shock proteins) and proteins involved in apoptosis regulation and cell cycle progression were analyzed in copepods after both 24 and 48 hours of feeding on the diatom or on a control diet. Strong differences occurred among copepod populations, with the Mediterranean population of C. helgolandicus being more susceptible to the toxic diet compared to the others. This study opens new perspectives for understanding copepod population-specific responses to diatom toxins and may help in underpinning the cellular mechanisms underlying copepod toxicity during diatom blooms.
PLoS ONE 01/2012; 7(10):e47262. · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 2010, attracting some 120 participants from 18 Euro-pean countries plus Israel and Australia. The goal of this 4-year research network is to integrate fundamental research strategies in order to devise pro-active tools for monitoring and management of seagrass ecosystems. At the fundamental research level, the aim is to integrate the fields of seagrass ecophysiology and ecogenomics in order to address questions related to anthropogenic disturbances that alter light, temperature and pH, thereby affecting pri-mary production as well as permanent stress (selection) regimes that may exceed the capacity of seagrass to respond (and eventually adapt) while still providing eco-logical services. In March 2011, the Ecophysiology and Genetics Working Groups of the Action organised an exploratory workshop entitled 'Linking Ecophysiology and Ecogenomics in Seagrass Systems' at the Stazione Zoolo-gica Anton Dohrn, in Naples (Italy), that discussed how to integrate comparative gene expression studies with physiological processes such as photosynthetic perfor-mance, carbon and nitrogen utilisation and environmental adaptation. Although seagrasses are angiosperms, their physiological mechanisms and pathways are not identical to those found in their terrestrial counterparts. Adaptation to a completely submerged life in the marine environment has occurred on at least three occasions, involving three different lineages (Waycott et al. 2006). Some of the major morphological adaptations include: gas-filled lacunal