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Biochemical descriptors for Posidonia oceanica and other seagrasses

Marine phanerogams are one of the most productive marine systems and play a crucial role in the dynamics and maintenance of coastal ecosystems (water oxygenation, food and shelter for various interventions, reduction of hydrodynamism and therefore of coastal erosion). Angiosperms Monocotyledons producing fruits and seeds, they are descendants of terrestrial plants which colonized the marine environment about 100 million years ago. Present in all continents, but Antarctica, they include 72 species, some of them are endangered species, such as Posidonia oceanica, others are spreading colonizers, such as Halophila stipulacea, an invasive species.

They form large submarine meadows playing a pivotal role in the dynamics and maintenance of coastal marine ecosystems (water oxygenation, food and shelter for various environments, reduction of hydrodynamism and therefore of coastal erosion).

 Posidonia oceanica is a key species, listed in the Habitat Directive 92/43/EEC; it is a recognized bioindicator of coastal marine water quality, according to the Water Framework Directive (WFD 2000/60 / EU). Unfortunately, the increasing impact of human activities in the coastal areas is responsible for a widespread regression of this species; Posidonia meadows seem vulnerable to changes in hydrological regime and sediment transport, pollution/eutrophication (eg. intensive aquaculture) and mechanical damage (eg. mooring) but also to climate change.

 Halophila stipulacea is a small tropical seagrass species. It is the dominant seagrass species in the Gulf of Aqaba (GoA; northern Red Sea), where it grows in both shallow and deep environments. Native to the Indian Ocean, Persian Gulf, and Red Sea, this species is a Lessepsian migrant established the Mediterranean but has recently invaded the Caribbean Sea, displacing the native species. Due to its invasive nature, there is growing interest to understand this species’ capacity to adapt to new conditions, which might be attributed to its ability to thrive in a broad range of ecological niches. 

 The study of these (and other) phanerogams is therefore based on the development, choice and combination of sensitive and measurable descriptors, an essential approach for meadows monitoring. The most common popular indicators give indications at individual, population or community level; however, many of them have low sensitivity and it is often necessary to combine/integrate multiple indicators to get meaningful information.

 The identification of new indicators able to give ecologically relevant information in a short time is therefore a crucial problem. In 2004 a new descriptor was identified: the synthesis of phenolic compounds, an easy and cheap biochemical descriptor. Phenolic compounds are a class of secondary metabolites widely distributed in terrestrial and aquatic plants, and the concentration of total phenols was already considered a biochemical marker of environmental stress in terrestrial plants. High concentrations of phenolic compounds have been detected in Posidonia plants exposed to different environmental pressures: competition with the invasive algae Caulerpa taxifolia, response to metal contamination, proximity to intensive aquaculture facilities. The determination of total phenols has been also associated with two-dimensional electrophoretic analysis of proteins, to identify expression changes in plants exposed or not to environmental stress, a way to identify new putative markers of plant stress conditions.

 In addition to the role of indicator, total phenol concentrations in marine phanerogams can modify the herbivore pressure on the plants. The role of total phenols (and its variation according to environmental conditions) was studied on the grazing rate of the mollusc, Smaragdia souverbiana, which feeds on two species of phanerogams, Zostera muelleri and Halophila ovalis, which live synthopically in different sites and environmental conditions of the Australian east coast. The connection between phenol concentration and herbivore pressure showed how herbivores may regulate top-down the dynamic of the seagrass.

 Moreover recently, along with the quantification of phenolic compounds in rhizomes, other descriptors were proposed: photosynthetic pigments, markers of oxidative stress and measures of genetic diversity, were successfully used as biomarkers of environment quality. All these markers can be evaluated on a single shoot/leaf; this allows (i) a more accurate evaluation of the conservation status of the plant, (ii) the reduction of the impact of sampling and, last but not least, (iii) a reduction of the costs of the monitoring sampling campaigns. Two of these biochemical markers (phenolic compounds and photosynthetic pigments) have been successfully applied in other marine phanerogams such as Cymodocea nodosa, Zostera noltii and Halophila stipulacea collected from different sites and different environmental conditions (C. nodosa and Z. noltii, Ria formosa lagoon, Faro, Portugal; H. stipulacea, Gulf of Aqaba, Eilat, Israel, and Limassol, Cyprus).

 This research line has developed thanks to numerous collaborations, listed here in order of time, from the first to the most recent: Agata Giallongo, Maria Di Bernardo and Letizia Anello from CNR - IBIM (Institute of Biomedicine and Molecular Immunology) "Alberto Monroy" in Palermo; Andrea Peirano and Carla Micheli from ENEA (National Agency for new technologies, energy and economic development; Centro Ricerche Santa Teresa-La Spezia, e Centro Ricerche Casaccia-Roma, respectively); Isabel Barrote e João Silva dell'Università dell'Algarve/CCMAR di Faro (Portogallo); Gidon Winters del Dead Sea & Arava Science Center, Hazeva (Israele); Ian R. Tibbets and Renee A. Rossini of the School of Biological Sciences of the University of Queensland in Brisbane (Australia), where we worked thanks to an Erasmus Mundus mobility grant of the NESSIE Program; Marlene Vasquez from the Cyprus University of Technology in Limassol.

 

Beca-Carretero P., Rotini A., Mejia A.Y., Migliore L., Vizzini S., Winters G. (2019). Halophila stipulacea descriptors in the native area (Red Sea): a baseline for future comparisons with native and non-native populations. Marine Environmental Research, Available online, in press. DOI: 0.1016/j.marenvres.2019.104828

Halophila stipulacea is a small tropical seagrass species native to the Red Sea. Due to its invasive character, there is growing interest in understanding its ability to thrive in a broad range of ecological niches. We studied temporal (February 2014 and July 2014), depth (5, 9, 18 m) and spatial (NB and SB) related dynamics of H. stipulacea meadows in the northern Gulf of Aqaba. We evaluated changes in density, morphometry, biomass, and biochemical parameters alongside the reproductive effort. In both sites, maximal growth and vegetative performance occurred in the summer with a marked increase of 35% in shoot density and 18% in biomass; PAR reduction with season and depth induced a significant increase of 28% in leaf area. Sexual reproduction efforts were only observed in July, and the density of plants carrying male or female flowers decreased significantly with depth. The favorable growth responses of H. stipulacea plants observed in the N-enriched NB site suggests their capacity to acclimate to human-disturbed nearshore environments 

 

Rotini A., Tibbetts I.R., Migliore L., Rossini R.A. (2018) - THE TRADE-OFF BETWEEN DIGESTIBILITY AND PHENOL CONTENT INFLUENCES THE FOOD CHOICE OF THE OBLIGATE SEAGRASS-FEEDING NERITID SNAIL SMARAGDIA SOUVERBIANA. Journal of Molluscan Studies, 84(1): 12-18. DOI: 10.1093/mollus/eyx038

 Grazing influences the structure and function of seagrass meadows. Like terrestrial plants, seagrasses have evolved nutritional, structural and chemical mechanisms to either tolerate or resist grazing. Phenols are common secondary metabolites that have multiple roles, including grazing deterrence, and their content in seagrass tissues can vary both among species and within a single species in response to biotic and abiotic processes. Such variability influences the grazing behaviour of species that consume seagrass, but few studies have addressed mesograzers that consume or damage seagrass, such as the nerite Smaragdia souverbiana. We assessed the effect of environmentally-mediated differences in phenol concentration on these seagrass mesograzers by testing: (1) if phenol content is different between sites and seagrass species (Zostera muelleri vs Halophila ovalis) and (2) if S. souverbiana inflicts different amounts of damage on different seagrasses from different sites, according to their phenol content. Phenol content was consistently lower in Z. muelleri than H. ovalis, and concentrations were about 60% lower in both species at one of two sites in eastern Moreton Bay, Queensland. These differences corresponded to different foraging patterns of S. souverbiana, with the snails generally choosing the seagrass species with the lowest phenol content. These results suggest that S. souverbiana chooses among locally available seagrasses based on a trade-off between phenol content and digestibility (related to cell size). This work demonstrates how subtle local processes can create complex changes in seagrass–grazer interactions with potential ecological consequences at local and regional scales.

 
Rotini A., Belmonte A., Barrote I., Micheli C., Peirano A., Santos R.O., Silva J., Migliore L. (2013) EFFECTIVENESS AND CONSISTENCY OF A SUITE OF DESCRIPTORS FOR ASSESSING THE ECOLOGICAL STATUS OF SEAGRASS MEADOWS (POSIDONIA OCEANICA L. DELILE). Estuarine, Coastal and Shelf Science, 130: 252–259. DOI: 10.1016/j.ecss.2013.06.015
 
The increasing rate of human-induced environmental changes on coastal marine ecosystems has created a demand for effective descriptors, in particular for those suitable for monitoring the status of seagrass meadows. Growing evidence has supported the useful application of biochemical and genetic descriptors such as secondary metabolite synthesis, photosynthetic activity and genetic diversity. In the present study, we have investigated the effectiveness of different descriptors (traditional, biochemical and genetic) in monitoring seagrass meadow conservation status. The Posidonia oceanica meadow of Monterosso al Mare (Ligurian sea, NW Mediterranean) was subjected to the measurement of bed density, leaf biometry, total phenols, soluble protein and photosynthetic pigment content as well as to RAPD marker analysis. This suite of descriptors provided evidence of their effectiveness and convenient application as markers of the conservation status of P. oceanica and/or other seagrasses. Biochemical/genetic descriptors and those obtained by traditional methods depicted a well conserved meadow with seasonal variability and, particularly in summer, indicated a healthier condition in a portion of the bed (station C), which was in agreement with the physical and sedimentological features of the station. Our results support the usefulness of introducing biochemical and genetic approaches to seagrass monitoring programs since they are effective indicators of plant physiological stress and environmental disturbance.
 
Rotini A., Anello L., Di Bernardo M., Giallongo A., Valiante L., Migliore L. (2013) COMPARATIVE ANALYSIS OF BED DENSITY, TOTAL PHENOL CONTENT AND PROTEIN EXPRESSION PATTERN IN POSIDONIA OCEANICA (L.) Delile. Open Journal of Ecology, 3(6): 438-444. DOI: 10.4236/oje.2013.36050
 
Posidonia oceanica meadows are experiencing a progressive decline, and monitoring their status is crucial for the maintenance of these ecosystems. We performed a comparative analysis of bed density, total phenol content and protein expression pattern to assess the conservation status of Posidonia plants from the S. Marinella (Rome, Italy) meadow. The total phenol content was inversely related to maximum bed density, confirming the relationship between high phenol content and stressful conditions. In addition, protein expression pattern profiles showed that the number of differentially expressed proteins was dramatically reduced in the latest years compared to previous analyses. Our results support the usefulness of integrating solid descriptors, such as phenol content, with novel biochemical/molecular approaches in the monitoring of meadows.
 
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Rotini A., Micheli C., Valiante L., Migliore L. (2011) ASSESSMENT OF POSIDONIA OCEANICA (L.) DELILE CONSERVATION STATUS BY STANDARD AND PUTATIVE APPROACHES: THE CASE STUDY OF SANTA MARINELLA MEADOW (ITALY, W MEDITERRANEAN). Open Journal of Ecology, 1(2): 48-56 doi:10.4236/oje.2011.12006
 
The conservation status of the Posidonia oceanica meadow at Santa Marinella (Rome) was evaluated through both standard (bed density, leaf biometry, “A” coefficient, Leaf Area Index, rhizome production) and biochemical/genetic approaches (total phenol content and Random Amplified Polymorphic DNA marker). The biochemical/genetic results are in agreement with those obtained by standard approaches. The bed under study was ranked as a disturbed one, due to its low density, and high heterogeneity in leaf biometry, LAI values, “A” coefficient and primary production. This low quality ranking is confirmed by both mean phenol content in plants, quite high and scattered, and by the low genetic variability in the meadow, with a very high similarity of specimen at a local scale. Hence, these two putative approaches clearly identify the endangered conservation status of the meadow. They link plant biodiversity and ecophysiology to ecosystem ‘health’. Further- more, they are repeatable and standardizable and could be usefully introduced in meadows monitoring to check environmental quality.
 
Migliore L., Rotini A., Randazzo D., Albanese N.N., Giallongo A. (2007) PHENOLS CONTENT AND 2-D ELECTROPHORESIS PROTEIN PATTERN: A PROMISING TOOL TO MONITOR POSIDONIA MEADOWS HEALTH STATE. BMC Ecology, 7:6 doi:10.1186/1472-6785-7-6
 
The endemic seagrass Posidonia oceanica (L.) Delile colonizes soft bottoms producing highly productive meadows that play a crucial role in coastal ecosystems dynamics. Human activities and natural events are responsible for a widespread meadows regression; to date the identification of "diagnostic" tools to monitor conservation status is a critical issue. In this study the feasibility of a novel tool to evaluate ecological impacts on Posidonia meadows has been tested. Quantification of a putative stress indicator, i.e. phenols content, has been coupled to 2-D electrophoretic protein analysis of rhizome samples. The overall expression pattern from Posidonia rhizome was determined using a preliminary proteomic approach, 437 protein spots were characterized by pI and molecular weight. We found that protein expression differs in samples belonging to sites with high or low phenols: 22 unique protein spots are peculiar of "low phenols" and 27 other spots characterize "high phenols" samples. Posidonia showed phenols variations within the meadow, that probably reflect the heterogeneity of environmental pressures. In addition, comparison of the 2-D electrophoresis patterns allowed to highlight qualitative protein expression differences in response to these pressures. These differences may account for changes in metabolic/physiological pathways as adaptation to stress. A combined approach, based on phenols content determination and 2-D electrophoresis protein pattern, seems a promising tool to monitor Posidonia meadows health state.
 
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