A successful method to restore seagrass habitats in coastal areas affected by consecutive natural events.

Background: Seagrass meadows, known for providing essential ecosystem services like supporting fishing, coastline protection from erosion, and acting as carbon sinks to mitigate climate change effects, are facing severe degradation. The current deteriorating state can be attributed to the combination of anthropogenic activities, biological factors (i.e., invasive species), and natural forces (i.e., hurricanes). Indeed, the global seagrass cover is diminishing at an alarming mean rate of 7% annually, jeopardizing the health of these vital ecosystems. However, in the Island Municipality of Culebra, Puerto Rico, losses are occurring at a faster pace. For instance, hurricanes have caused over 10% of cover seagrass losses, and the natural recovery of seagrasses across Culebra's coast has been slow due to the low growth rates of native seagrasses (Thalassia testudinum and Syringodium filiforme) and the invasion of the invasive species Halophila stipulacea. Restoration programs are, thus, necessary to revitalize the native seagrass communities and associated fauna while limiting the spread of the invasive species. Methods: Here, we present the results of a seagrass meadow restoration project carried out in Punta Melones (PTM), Culebra, Puerto Rico, in response to the impact of Hurricanes Irma and María during 2017. The restoration technique used was planting propagation units (PUs), each with an area of 900 cm2 of native seagrasses Thalassia testudinum and Syringodium filiforme, planted at a depth between 3.5 and 4.5 m. A total of 688 PUs were planted between August 2021 and August 2023, and a sub-sample of 88 PUs was monitored between August 2021 and April 2023. Results: PUs showed over 95% of the seagrass survived, with Hurricane Fiona causing most of the mortalities potentially due to PUs burial by sediment movement and uplifting by wave energy. The surface area of the planting units increased by approximately 200% (i.e., 2,459 cm2), while seagrass shoot density increased by 168% (i.e., 126 shoots by PU). Additionally, flowering and fruiting were observed in multiple planting units, indicating 1) that the action taken did not adversely affect the PUs units and 2) that the project was successful in revitalizing seagrass populations. The seagrass restoration project achieved remarkable success, primarily attributed to the substantial volume of each PUs. Likely this high volume played a crucial role in facilitating the connection among roots, shoots, and microfauna while providing a higher number of undamaged and active rhizome meristems and short shoots. These factors collectively contributed to the enhanced growth and survivorship of the PUs, ultimately leading to the favorable outcome observed in the seagrass restoration project.

Green turtles shape the seascape through grazing patch formation around habitat features: Experimental evidence.

Understanding how megaherbivores incorporate habitat features into their foraging behavior is key toward understanding how herbivores shape the surrounding landscape. While the role of habitat structure has been studied within the context of predator-prey dynamics and grazing behavior in terrestrial systems, there is a limited understanding of how structure influences megaherbivore grazing in marine ecosystems. To investigate the response of megaherbivores (green turtles) to habitat features, we experimentally introduced structure at two spatial scales in a shallow seagrass meadow in The Bahamas. Turtle density increased 50-fold (to 311 turtles ha-1 ) in response to the structures, and turtles were mainly grazing and resting (low vigilance behavior). This resulted in a grazing patch exceeding the size of the experimental setup (242 m2 ), with reduced seagrass shoot density and aboveground biomass. After structure removal, turtle density decreased and vigilance increased (more browsing and shorter surfacing times), while seagrass within the patch partly recovered. Even at a small scale (9 m2 ), artificial structures altered turtle grazing behavior, resulting in grazing patches in 60% of the plots. Our results demonstrate that marine megaherbivores select habitat features as foraging sites, likely to be a predator refuge, resulting in heterogeneity in seagrass bed structure at the landscape scale.

Climate change mitigation by coral reefs and seagrass beds at risk: How global change compromises coastal ecosystem services.

Seagrass meadows provide valuable ecosystem services of coastal protection and chemical habitat formation that could help mitigate the impact of sea level rise and ocean acidification. However, the intensification of hydrodynamic forces caused by sea level rise, in addition to habitat degradation threaten the provision of these ecosystem services. With quantitative field measurements of the coastal protection and chemical habitat formation services of seagrass meadows, we statistically model the relationships between hydrodynamic forces, vegetation density and the provision of these ecosystem services. Utilising a high-resolution hydrodynamic model that simulates end of the century hydrodynamic conditions and three scenarios of coral reef degradation (i.e., keep up, remain or loss) we quantify how the environmental conditions within a tropical bay will change given changes to the provision of ecosystem services. Our study shows that increasing hydrodynamic forces lead to a seafloor made up of a larger grain size that is increasingly unstable and more vulnerable to erosion. The loss of a fringing reef leads to larger hydrodynamic forces entering the bay, however, the 0.87 m increase in depth due to sea-level rise reduces the bed shear stress in shallower areas, which limits the change in the ecosystem services provided by the current benthic seagrass meadow. Loss of seagrass constitutes the greatest change in a bay ecosystem, resulting in the sediment surface where seagrass existed becoming unstable and the median sediment grain size increasing by 5-7 %. The loss of seagrass also leads to the disappearance of the unique fluctuating chemical habitat, which leaves the surrounding community vulnerable to ocean acidification. A reduction or complete loss of these ecosystem services would impact the entire community assemblage while also leaving the surrounding coastline vulnerable to erosion, thus exacerbating negative effects brought about by climate change.

Mechanistic support for increased primary production around artificial reefs.

Understanding factors controlling primary production is fundamental for the protection, management, and restoration of ecosystems. Tropical seagrass ecosystems are among the most productive ecosystems worldwide, yielding tremendous services for society. Yet they are also among the most impaired from anthropogenic stressors, prompting calls for ecosystem-based restoration approaches. Artificial reefs (ARs) are commonly applied in coastal marine ecosystems to rebuild failing fisheries and have recently gained attention for their potential to promote carbon sequestration. Nutrient hotspots formed via excretion from aggregating fishes have been empirically shown to enhance local primary production around ARs in seagrass systems. Yet, if and how increased local production affects primary production at ecosystem scale remains unclear, and empirical tests are challenging. We used a spatially explicit individual-based simulation model that combined a data-rich single-nutrient primary production model for seagrass and bioenergetics models for fish to test how aggregating fish on ARs affect seagrass primary production at patch and ecosystem scales. Specifically, we tested how the aggregation of fish alters (i) ecosystem seagrass primary production at varying fish densities and levels of ambient nutrient availability and (ii) the spatial distribution of seagrass primary production. Comparing model ecosystems with equivalent nutrient levels, we found that when fish aggregate around ARs, ecosystem-scale primary production is enhanced synergistically. This synergistic increase in production was caused by nonlinear dynamics associated with nutrient uptake and biomass allocation that enhances aboveground primary production more than belowground production. Seagrass production increased near the AR and decreased in areas away from the AR, despite marginal reductions in seagrass biomass at the ecosystem level. Our simulation's findings that ARs can increase ecosystem production provide novel support for ARs in seagrass ecosystems as an effective means to promote (i) fishery restoration (increased primary production can increase energy input to the food web) and (ii) carbon sequestration, via higher rates of primary production. Although our model represents a simplified, closed seagrass system without complex trophic interactions, it nonetheless provides an important first step in quantifying ecosystem-level implications of ARs as a tool for ecological restoration.

Seagrass structural and elemental indicators reveal high nutrient availability within a tropical lagoon in Panama.

Seagrass meadows are valued coastal habitats that provide ecological and economic benefits around the world. Despite their importance, many meadows are in decline, driven by a variety of anthropogenic impacts. While these declines have been well documented in some regions, other locations (particularly within the tropics) lack long-term monitoring programs needed to resolve seagrass trends over time. Effective and spatially-expansive monitoring within under-represented regions is critical to provide an accurate perspective on seagrass status and trends. We present a comprehensive dataset on seagrass coverage and composition across 24 sites in Bahía Almirante, a lagoon along the Caribbean coast of Panama. Using a single survey, we focus on capturing spatial variation in seagrass physical and elemental characteristics and provide data on key seagrass bio-indicators, such as leaf morphology (length and width), elemental content (% nitrogen and phosphorus) and stable isotopic signatures (δ 13C and δ 15N). We further explore relationships between these variables and water depth (proxy for light availability) and proximity to shore (proxy for terrestrial inputs). The seagrass assemblage was mostly monospecific (dominated by Thalassia testudinum) and restricted to shallow water (<3 m). Above-ground biomass varied widely, averaging 71.7 g dry mass m-2, yet ranging from 24.8 to 139.6 g dry mass m-2. Leaf nitrogen content averaged 2.2%, ranging from 1.76 to 2.57%, while phosphorus content averaged 0.19% and ranged from 0.15 to 0.23%. These values were high compared to other published reports for T. testudinum, indicating elevated nutrient availability within the lagoon. Seagrass stable isotopic characteristics varied slightly and were comparable with other published values. Leaf carbon signatures (δ 13C) ranged from -11.74 to -6.70‰ and were positively correlated to shoreline proximity, suggesting a contribution of terrestrial carbon to seagrass biomass. Leaf nitrogen signatures (δ 15N) ranged from -1.75 to 3.15‰ and showed no correlation with shoreline proximity, suggesting that N sources within the bay were not dominated by localized point-source discharge of treated sewage. Correlations between other seagrass bio-indicators and environmental metrics were mixed: seagrass cover declined with depth, while biomass was negatively correlated with N, indicating that light and nutrient availability may jointly regulate seagrass cover and biomass. Our work documents the response of seagrass in Bahía Almirante to light and nutrient availability and highlights the eutrophic status of this bay. Using the broad spatial coverage of our survey as a baseline, we suggest the future implementation of a continuous and spatially expansive seagrass monitoring program within this region to assess the health of these important systems subject to global and local stressors.

The takeover of Thalassia testudinum by Anadyomene sp. at Biscayne Bay, USA, cannot be simply explained by competition for nitrogen and phosphorous.

Apart from direct light effects, we tested whether the takeover of the seagrass Thalassia testudinum by the seaweed Anadyomene sp. in high nutrient areas of Biscayne Bay, Florida, USA, is related to a faster nutrient surge uptake capacity of the seaweed and/or a negative effect on the seagrass uptake rates. Anadyomene sp. and T. testudinum showed a similar ammonium surge uptake capacity, but the seagrass performed better than the seaweed in mixed incubations at high ammonium concentrations. T. testudinum was faster than Anadyomene sp. at taking up pulses of phosphate, but the uptake rates of the seagrass were significantly decreased in the presence of the seaweed. The takeover of T. testudinum by Anadyomene sp. at Biscayne Bay is likely dominated by light and cannot be simply explained by their single or mixed nutrient surge uptake rates, but the phosphate availability and the seagrass uptake inhibition by the seaweed may also play a key role in the process.

Composition of seagrass phyllosphere microbial communities suggests rapid environmental regulation of community structure.

Recent studies have revealed that seagrass blade surfaces, also known as the phyllosphere, are rich habitats for microbes; however, the primary drivers of composition and structure in these microbial communities are largely unknown. This study utilized a reciprocal transplant approach between two sites with different environmental conditions combined with 16S rRNA gene sequencing (iTag) to examine the relative influence of environmental conditions and host plant on phyllosphere community composition of the seagrass Thalassia testudinum. After 30 days, identity of phyllosphere microbial community members was more similar within the transplant sites than between despite differences in the source of host plant. Additionally, the diversity and evenness of these communities was significantly different between the two sites. These results indicated that local environmental conditions can be a primary driver in structuring seagrass phyllosphere microbial communities over relatively short time scales. Composition of microbial community members in this study also deviated from those in previous seagrass phyllosphere studies with a higher representation of candidate bacterial phyla and archaea than previously observed. The capacity for seagrass phyllosphere microbial communities to shift dramatically with environmental conditions, including ecosystem perturbations, could significantly affect seagrass-microbe interactions in ways that may influence the health of the seagrass host.

Marine Seagrass Extract of Thalassia testudinum Suppresses Colorectal Tumor Growth, Motility and Angiogenesis by Autophagic Stress and Immunogenic Cell Death Pathways.

Marine plants have become an inexhaustible reservoir of new phytopharmaceuticals for cancer treatment. We demonstrate in vitro/in vivo antitumor efficacy of a standardized polyphenol extract from the marine angiosperm Thalassia testudinum (TTE) in colon tumor cell lines (RKO, SW480, and CT26) and a syngeneic allograft murine colorectal cancer model. MTT assays revealed a dose-dependent decrease of cell viability of RKO, CT26, and SW480 cells upon TTE treatment with IC50 values of, respectively, 175, 115, and 60 µg/mL. Furthermore, TTE significantly prevented basal and bFGF-induced angiogenesis in the chicken chorioallantoic membrane angiogenesis assay. In addition, TTE suppressed bFGF-induced migration of endothelial cells in a wound closure assay. Finally, TTE treatment abrogated CT26 colorectal cancer growth and increased overall organism survival in a syngeneic murine allograft model. Corresponding transcriptome profiling and pathway analysis allowed for the identification of the mechanism of action for the antitumor effects of TTE. In line with our in vitro/in vivo results, TTE treatment triggers ATF4-P53-NFκB specific gene expression and autophagy stress pathways. This results in suppression of colon cancer cell growth, cell motility, and angiogenesis pathways in vitro and in addition promotes antitumor immunogenic cell death in vivo.

Interaction of Thalassia testudinum Metabolites with Cytochrome P450 Enzymes and Its Effects on Benzo(a)pyrene-Induced Mutagenicity.

The aim of the present work was to evaluate the effects of Thalassia testudinum hydroethanolic extract, its polyphenolic fraction and thalassiolin B on the activity of phase I metabolizing enzymes as well as their antimutagenic effects. Spectrofluorometric techniques were used to evaluate the effect of tested products on rat and human CYP1A and CYP2B activity. The antimutagenic effect of tested products was evaluated in benzo[a]pyrene (BP)-induced mutagenicity assay by an Ames test. Finally, the antimutagenic effect of Thalassia testudinum (100 mg/kg) was assessed in BP-induced mutagenesis in mice. The tested products significantly (p < 0.05) inhibit rat CYP1A1 activity, acting as mixed-type inhibitors of rat CYP1A1 (Ki = 54.16 ± 9.09 µg/mL, 5.96 ± 1.55 µg/mL and 3.05 ± 0.89 µg/mL, respectively). Inhibition of human CYP1A1 was also observed (Ki = 197.1 ± 63.40 µg/mL and 203.10 ± 17.29 µg/mL for the polyphenolic fraction and for thalassiolin B, respectively). In addition, the evaluated products significantly inhibit (p < 0.05) BP-induced mutagenicity in vitro. Furthermore, oral doses of Thalassia testudinum (100 mg/kg) significantly reduced (p < 0.05) the BP-induced micronuclei and oxidative damage, together with an increase of reduced glutathione, in mice. In summary, Thalassia testudinum metabolites exhibit antigenotoxic activity mediated, at least, by the inhibition of CYP1A1-mediated BP biotransformation, arresting the oxidative and mutagenic damage. Thus, the metabolites of T. testudinum may represent a potential source of chemopreventive compounds for the adjuvant therapy of cancer.

Recovery of a large herbivore changes regulation of seagrass productivity in a naturally grazed Caribbean ecosystem.

What happens in meadows after populations of natural grazers rebound following centuries of low abundance? Many seagrass ecosystems are now experiencing this phenomenon with the recovery of green turtles (Chelonia mydas), large-bodied marine herbivores that feed on seagrasses. These seagrass ecosystems provide a rare opportunity to study ecosystem-wide shifts that result from a recovery of herbivores. We evaluate changes in regulation of seagrass productivity in a naturally grazed tropical ecosystem by (1) comparing Thalassia testudinum productivity in grazed and ungrazed areas and (2) evaluating potential regulating mechanisms of T. testudinum productivity. We established 129 green turtle exclusion cages in grazed and ungrazed areas to quantify T. testudinum growth (linear, area, mass, productivity : biomass [P:B]). In each exclosure, we recorded temperature, irradiance, water depth, nitrogen : phosphorus ratio (N:P) of blade tissue, grazing intensity before cage placement, and T. testudinum structural and nutrient characteristics. Thalassia testudinum exhibited compensatory growth in grazed areas via stimulated blade linear growth, blade area growth, and P:B across seasonal high and low growth periods and in shallow (3-4 m) and deep (9-10 m) seagrass meadows. Irradiance, depth, and N:P ratios had significant roles in regulating mass growth and P:B of T. testudinum in ungrazed areas. Depth was a significant regulating factor of mass growth and P:B in grazed areas; rates were higher and more variable in shallow meadows than in deep meadows. Grazing intensity was also a significant regulating factor for P:B, stimulating tissue turnover with increasing grazing pressure. This study provides important insights into how recovery of a large marine herbivore can result in dramatic, sustainable changes in the regulation of seagrass productivity. We also highlight the need for a historical perspective and use of appropriate indicators, including P:B and grazing intensity, when evaluating seagrass response to green turtle grazing as meadows are returned to a natural grazed state. In an age of green turtle recovery and global seagrass decline due to anthropogenic threats, a thorough understanding of green turtle-seagrass interactions at the ecosystem level is critical to ensure the restoration of seagrass ecosystems and continued recovery of green turtle populations.

Optimización del proceso de extracción de compuestos fenólicos de la angiosperma marina Thalassia testudinum / Optimization of the extraction process of phenolic compounds from the marine angiosperm Thalassia testudinum

RESUMEN Thalassia testudinum es la planta marina de mayor abundancia en el litoral de La Habana y del Caribe en general, conocida comúnmente como praderas submarinas o hierba de tortuga. Entre los compuestos de interés que se pueden encontrar en esta especie sobresalen los polifenoles, los cuales son componentes estructurales de su pared celular y poseen propiedades funcionales y bioactivas como antioxidante, anti-inflamatorio, neuroprotector y hepatoprotector. Investigaciones previas evaluaron diversos métodos de extracción de compuestos bioactivos para esta especie, por lo que este trabajo tuvo como objetivo optimizar las condiciones de extracción del contenido de polifenoles totales. Para ello se utilizó el método de Box y Hunter y se evaluó el efecto de tres factores influyentes en la extracción de compuestos fenólicos (velocidad de agitación, relación material vegetal/% alcohol y concentración de etanol). Como variable respuesta se empleó el contenido de polifenoles totales determinada por el método de Folin-Ciocalteu. Los resultados del diseño proporcionaron como condiciones óptimas en las variables estudiadas las siguientes: 1/11.5 p:v, 60% de EtOH y 800 r.p.m., alcanzando rendimiento de polifenoles totales, igual a 25.60 mg/g de extracto seco; superior a las restantes condiciones de extracción para un extracto bioactivo con potencialidades de uso en la industria farmacéutica o nutracéutica.

ABSTRACT Thalassia testudinum is the marine plant of greatest abundance along the coast of Havana and the Caribbean in general, commonly known as seagrass meadows or turtle grass. Among the compounds of interest that can be found in this species there are polyphenols, which are structural components of its cell wall and have functional and bioactive properties such as antioxidant, anti-inflammatory, neuroprotective and hepatoprotective. Previous research evaluated different methods of extracting bioactive compounds from this species, and this work aimed to optimize the extraction conditions of the total polyphenol content. For this, the Box and Hunter method was used and the effect of three influential factors in the extraction of phenolic compounds (agitation speed, vegetal material / solvent ratio and ethanol concentration) was evaluated. The total polyphenol content determined by the Folin-Ciocalteu method was used as a response variable. The design results provided as optimal conditions in the studied variables the following: 1/11.5 w: v, 60% EtOH and 800 r.p.m., reaching a total polyphenol yield of 25.60 mg/g of dry extract; which contributes to the obtaining of a better content of total phenols in a bioactive extract with potentialities of use in the pharmaceutical or nutraceutical industries.

Thalassia testudinum as a potential vector for incorporating microplastics into benthic marine food webs.

Seagrasses are among the most productive shallow water ecosystems, serving a diverse assemblage of fish and invertebrates. Tropical seagrass communities are dominated by the turtle grass Thalassia testudinum, whose wide, flattened blades host diverse epibiont communities. Amidst its epibionts, T. testudinum may also be accumulating microplastics, which are a ubiquitous marine pollutant even in remote locales. To assess the extent of microplastic accumulation, seagrass samples were collected from Turneffe Atoll, which lies offshore but parallel with a major urban center. Seventy-five percent of Thalassia blades had encrusted microplastics, with microfibers occurring more than microbeads and chips by a ratio of 59:14. Grazers consumed seagrasses with higher densities of epibionts. Potential mechanisms for microplastic accumulation include entrapment by epibionts, or attachment via biofilms. This study is the first to document microplastics on marine vascular plants, suggesting that macroherbivory is a viable pathway for microplastic pollution to enter marine food webs.

Intra- and inter-annual variation in a seagrass meadow on the Caribbean coast of Costa Rica: 2009-2015 / Variación intra e inter anual en una pradera de pastos marinos en la costa caribeña de Costa Rica: 2009-2015

Abstract Seagrass beds are an important ecosystem on the Caribbean coast of Costa Rica. At Cahuita National Park (CNP) a seagrass bed at Perezoso has been monitored continually since 1999 within the CARICOMP program. Thalassia testudinum is the dominant seagrass species, in some cases mixed with Syringodium filiforme. The results from the 2009 to 2015 monitoring period are presented here, and contrasted with data before 2009. Total (above and below ground tissue) mean biomass of T. testudinum was higher (1 255.4 ± 146.0 gm-2) than biomass before 2009, with an increasing tendency. However, productivity (1.5±0.59 gm-2d-1) and turnover rate (4.3 ± 1.22 %d-1) were lower than previous monitoring periods. In this period, mean leaf area diminished considerably (4.9 ± 2.30 m2), but leaf area index (LAI) increased (1.9 ± 0.80 m2leafm-2) in comparison to prior monitoring. Productivity, density, turnover rate, LAI and biomass showed intra-annual variations; while mean biomass of T. testudinum did not vary significantly among years. No correlations were found between water salinity, temperature and clarity with seagrass measurements. However, most seagrass parameters were strongly correlated with precipitation. These results highlight the effect of external environmental agents acting on the ecosystem. CNP presents a long-term stable seagrass meadow. However, there are indirect signals, such as high biomass and above-ground biomass proportion, along with low productivity and LAI, which point to a nutrient increment in Perezoso's seagrass bed. To continue protecting this seagrass bed, it is necessary to improve monitoring methods, and seagrass beds should be included in national conservation policies and monitoring programs. Rev. Biol. Trop. 66(3): 1149-1161. Epub 2018 September 01.

Resumen Los lechos de pastos marinos son un ecosistema importante en la costa caribeña de Costa Rica. En el Parque Nacional Cahuita (PNC), un lecho de pastos marinos en Perezoso ha sido monitoreado continuamente desde 1999 dentro del programa CARICOMP. Thalassia testudinum es la especie dominante, en algunos casos mezclada con Syringodium filiforme. Se presentan los resultados del período de monitoreo del 2009 al 2015. La biomasa promedio de T. testudinum fue mayor (1 255.4 ± 146.0 gm-2) que la biomasa antes de 2009, mostrando una tendencia creciente. Sin embargo, la productividad (1.5 ± 0.59 gm-2d-1) y la tasa de recambio (4.3 ± 1.22 %d-1) fueron inferiores a los períodos de monitoreo previos. En este período, el área foliar media disminuyó considerablemente (4.9 ± 2.30 m2), pero el Índice de Área Foliar (LAI) aumentó (1.9 ± 0.80 m2 hoja m-2) en comparación con el monitoreo previo. El promedio de la productividad, la densidad, la tasa de recambio, el LAI y la biomasa mostraron variaciones intra-anuales, mientras que la biomasa promedio de T. testudinum no varió significativamente entre los años, lo que representa un período estable. Ningún parámetro del agua de mar (salinidad, temperatura o turbiedad) se correlacionó con los parámetros del pasto marino. Sin embargo, la mayoría de los parámetros del pasto marino estaban fuertemente correlacionados con la precipitación. Estos resultados ponen en evidencia el efecto de factores ambientales externos sobre el ecosistema. El PNC tiene condiciones estables a largo plazo en las que se establece el pasto marino sin que haya evidencia de pérdida de biomasa. Sin embargo, hay señales indirectas; como el aumento de la biomasa y de la proporción de biomasa sobre el sustrato, así como una baja productividad y LAI; que apuntan a un incremento de nutrientes en el lecho de pastos marinos de Perezoso. Para continuar protegiendo este lecho marino, es necesario mejorar la calidad del agua y los métodos de monitoreo, además de incluir a estos hábitats en las políticas nacionales de conservación.

Assessment of the cytotoxic potential of an aqueous-ethanolic extract from Thalassia testudinum angiosperm marine grown in the Caribbean Sea.

OBJECTIVES: Reported antioxidant, anti-inflammatory and neuroprotective properties for one aqueous-ethanolic extract from Thalassia testudinum which grows in the Caribbean Sea compelled us to explore about extract cytotoxic effects. METHODS: Cell viability was assayed on tumour (HepG2, PC12, Caco-2 and 4T1) and non-tumour (VERO, 3T3, CHO, MCDK and BHK2) cell lines. The extract effects upon primary cultures of rat and human hepatocytes and human lymphocytes were assayed. KEY FINDINGS: The extract exhibited cytotoxicity against cancer cells compared to normal cells, and the IC50 values were 102 µg/ml for HepG2, 135 µg/ml for PC12, 165 µg/ml for Caco-2 and 129 µg/ml for 4T1 cells after 48 h, whereas IC50 could not be calculated for normal cells. Additional data from a high-content screening multiparametric assay indicated that after 24-h exposure, the extract (up to 100 µg/ml) induced death in HepG2 cells through oxidative stress-associated mechanism, DNA damage and hypercalcaemia. Comet assay corroborated extract-induced DNA damage. CONCLUSIONS: Thalassia testudinum extract is more cytotoxic and produced more DNA damage on human hepatoma cells than to other non-tumour cells. A possible mechanism is suggested for extract-induced cytotoxicity based on oxidative stress, nuclear damage and hypercalcaemia in HepG2 cells. T. testudinum may be a source for antitumour agents.

Anthropogenic versus fish-derived nutrient effects on seagrass community structure and function.

Humans are altering nutrient dynamics through myriad pathways globally. Concurrent with the addition of nutrients via municipal, industrial, and agricultural sources, widespread consumer exploitation is changing consumer-mediated nutrient dynamics drastically. Thus, altered nutrient dynamics can occur through changes in the supply of multiple nutrients, as well as through changes in the sources of these nutrients. Seagrass ecosystems are heavily impacted by human activities, with highly altered nutrient dynamics from multiple causes. We simulate scenarios of altered nutrient supply and ratios, nitrogen:phosphorus (N:P), from two nutrient sources in seagrass ecosystems: anthropogenic fertilizer and fish excretion. In doing so we tested expectations rooted in ecological theory that suggest the importance of resource dynamics for predicting primary producer dynamics. Ecosystem functions were strongly altered by artificial fertilizer (e.g., seagrass growth increased by as much as 140%), whereas plant/algae community structure was most affected by fish-mediated nutrients or the interaction of both treatments (e.g., evenness increased by ~140% under conditions of low fish nutrients and high anthropogenic nutrients). Interactions between the nutrient sources were found for only two of six response variables, and the ratio of nutrient supply was the best predictor for only one response. These findings show that seagrass structure and function are well predicted by supply of a single nutrient (either N or P). Importantly, no single nutrient best explained the majority of responses-measures of community structure were best explained by the primary limiting nutrient to this system (P), whereas measures of growth and density of the dominant producer in the system were best explained by N. Thus, while our findings support aspects of theoretical expectations, the complexity of producer community responses belies broad generalities, underscoring the need to manage for multiple simultaneous nutrients in these imperiled coastal ecosystems.

Densidad poblacional y estructura de talla del pepino de mar Holothuria (Halodeima) grisea (Aspidochirotida: Holothuriidae) en aguas someras del sur del golfo de Morrosquillo, Caribe Colombiano / Density population and size structure of the sea cucumber Holothuria (Halodeima) grisea (Aspidochirotida: Holothuriidae) in shallow waters south of the gulf of Morrosquillo, Colombian Caribbean

Resumen Holothuria grisea, a pesar de no ser considerado un recurso comercial, ha sido objeto de extracciones ilegales en Colombia, hecho que se vuelve más grave debido al vació de información biológica de estos organismos. Por esta razón, se estimó la densidad poblacional y estructura de talla de H. grisea en tres sectores del sur del golfo de Morrosquillo de abril a noviembre 2015. Para ello, se ubicaron mensualmente tres transectos circulares en cada sector de muestreo abarcando un área de 300 m2, donde se contabilizaron y midieron in situ los individuos encontrados. La densidad media de H. grisea en el área estudiada fue 0.4 ind m-2, presentándose los mayores valores en el sector La Ahumadera (1.09 ± 0.11 ind m-2) y los menores valores en el Banco de Arena (0.02 ± 0.004 ind m-2). El análisis de varianza señalo una diferencia significativa en los valores de densidad y talla de H. grisea en los sectores estudiados. La especie presentó una distribución de tallas unimodal, registrándose una talla promedio de 13.5 ± 0.91 cm con una talla mínima de 2.5 y una máxima de 30 cm, en general las tallas intermedias presentaron una mayor representatividad a lo largo de este estudio (77.2 %). Los aspectos biológicos presentados en este documento son de gran importancia para la conservación de esta especie y corresponde a un avance en el conocimiento de la clase Holothuroidea en el Caribe Colombiano.

Abstract Holothuria grisea, despite not being considered a commercial resource has been the subject of illegal extractions in Colombia, a fact that becomes more serious due to the empty of biological information of these organisms. For this reason the density population and size structure of H. grisea were evaluated in three sectors south of the gulf of Morrosquillo from April to November 2015. To this end, three circular transects were located each month in each sampling site covering an area of 300 m2, where individuals were counted and measured in situ. The average density of H. grisea in the study area was 0.4 ind m-2, with the highest values in the La Ahumadera (1.09 ± 0.11 ind m-2) and the lowest values in Banco de Arena (0.02 ± 0.004 ind m-2). The analysis of variance showed a significance in the values of density and size of H. grisea in the studied sectors. The species presented a unimodal distribution of sizes with an average size of 13.5 ± 0.9 cm with a minimum size of 2.5 and a maximum of 30 cm, intermediate sizes generally showed a higher representativeness throughout this study (77.2 %). The biological aspects presented in this paper are of great importance for the conservation of this species and corresponds to an advance in the knowledge of the Holothuroidea class in the Colombian Caribbean. Rev. Biol. Trop. 66(2): 776-787. Epub 2018 June 01.

Leaf Spectral Reflectance Shows Thalassia testudinum Seedlings More Sensitive to Hypersalinity than Hyposalinity.

Thalassia testudinum (turtle grass) is the dominant and climax-successional seagrass species in the subtropical/tropical Atlantic and Caribbean region. Two die-offs of T. testudinum in Florida Bay, United States have raised concerns regarding the resilience of this species to environmental disturbances. Seedlings are important in recovery of T. testudinum, following disturbance events. Leaf spectral reflectance [R(λ)] was measured in T. testudinum seedlings exposed for 2 weeks to three salinities (20, 35, and 50) and two light levels (full sun and 50-70% light reduction) in experimental mesocosms. Multivariate analyses indicated that hypersalinity had a greater effect on spectral reflectance than hyposalinity or light reduction. There was an increase in variability and flattening of reflectance spectra at the highest salinity. All three salinity treatments had distinct reflectance spectra across green wavelengths (530-580 nm), with additional discrimination between 20 versus 50 and 35 versus 50 treatments across red wavelengths (630-690 nm). Red:Green reflectance ratios were highest and photochemical reflective index values were lowest for the salinity 50 treatment, but were not significantly different between the salinity 20 and 35 treatments. The changes in the R(λ) spectra for the salinity 50 seedlings were consistent with previously observed reductions in leaf pigments and maximum photochemical efficiency of photosystem II. These observations indicate that leaf spectral reflectance is a sensitive indicator of plant stress in T. testudinum seedlings and that seedlings are more sensitive to short-term exposures to hypersalinity than hyposalinity.

Seasonal behavior of Thalassia testudinum (Hydrocharitaceae) metabolites / Comportamiento estacional de los metabolitos de Thalassia testudinum (Hydrocharital: Hydrocharitaceae)

Abstract:The marine angiosperm Thalassia testudinum, commonly known as turtle grass, is a dominant sea- grass that grows in the Caribbean Sea shelf associated to Syringodium filiforme. The hydroalcoholic extract of T. testudinum is rich in polyphenols; the most abundant metabolite in this extract is thalassiolin B, a glycosilated flavonoid with skin damage repairing properties, and antioxidant capacity among others. The present study aimed at generating information about the seasonal behavior of secondary metabolites, as well as to study the antioxidant capacity of the T. testudinum leaves extract, collected monthly during 2012 from the Northeast coastline of Havana, Cuba. For this study, spectrophotometric methods were used to determine the concentrations of polyphenols, flavonoids, anthocyanins, soluble carbohydrates and proteins, chlorophylls a and b, and antioxidant activity of the extracts. In general, results demonstrated seasonal variations of the analyzed parameters. Extracts prepared from the vegetal material collected in October and November showed the highest values of polyphenols (58.81 ± 1.53 and 52.39 ± 0.63 mg/g bs, respectivally) and flavonoids (44.12 ± 1.30 and 51.30 ± 0.67 mg/g dw, respectively). On the contrary, the lowest values of polyphenols were found in extracts of leaves collected in July and August (15.51 ± 0.84 and 13.86 ± 0.48 mg/g, respectively). In accordance with these results, the lower value of Inhibitory Concentration (IC50) was obtained to get a 50 % of maximal effect on free radical scavenging activity with the extracts prepared from leaves collected in October and November, and less significant IC50 was obtained from the extract prepared from leaves collected in August (5.63 mg/mL). A negative correlation (r= -0.694) was observed in this study between the content of polyphenols and the IC50 necessary to get the half of its antioxidant maximal effect. The high correspondence between the maximum values of polyphenols, flavonoids, carbohydrates and proteins in October and November, revealed a close relationship between these metabolites found in the extract of T. testudinum. Our hypothesis about the annual variation in the concentration of these metabolites was validated; and these results will support the correct harvesting of T. testudinum leaves for biotechnology and industrial purposes. Rev. Biol. Trop. 64 (4): 1527-1535. Epub 2016 December 01.

Resumen:La angiosperma marina Thalassia testudinum, comúnmente conocida como "hierba tortuga", es un pasto marino dominante que crece en el Mar Caribe asociada a Syringodium filiforme. El extracto hidroalcohólico de T. testudinum es rico en polifenoles; el metabolito más abundante en este extracto es thalassiolina B, un flavonoide glicosilado con propiedades para la reparación de daños en la piel y la capacidad antioxidante, entre otros. El objetivo del presente estudio fue conocer el comportamiento estacional de los metabolitos secundarios, así como la capacidad antioxidante del extracto de hojas de T. testudinum recolectadas mensualmente durante el 2012, en la costa noreste de La Habana, Cuba. Para este estudio fueron empleados métodos espectrofotométricos para la determinación de la concentración de polifenoles, flavonoides, carbohidratos y proteínas solubles, clorofilas a y b y la actividad antioxidante del extracto. En sentido general, los resultados demostraron las variaciones estacionales de los parámetros analizados; los extractos preparados a partir de material vegetal recolectado en octubre y noviembre mostraron los mayores valores de polifenoles y flavonoides (44.12 ± 1.30 y 51.30 ± 0.67 mg/g bs respectivamente) y se encontraron los valores más bajos en los extractos de hojas recolectadas en julio y agosto. (15.51 ± 0.84 y 13.86 ± 0.48 mg/g respectivamente). De acuerdo con los resultados, se obtuvo el valor más bajo de la concentración inhibitoria (CI50 ) necesaria para obtener un 50 % de efecto máximo en la actividad de captación de radicales libres con los extractos preparados a partir de las hojas recolectadas en octubre y noviembre y la CI50 menos significativa se obtuvo a partir del extracto preparado a partir de las hojas recolectadas en agosto (5.63 mg/ml). Se observó una correlación negativa (r= -0,694) entre el contenido de polifenoles y la CI50 necesaria para obtener la mitad del efecto antioxidante máximo. La alta correspondencia que existe entre los valores máximos de polifenoles, flavonoides, carbohidratos y proteínas en octubre y noviembre reveló una estrecha relación entre estos metabolitos, encontrados en el extracto de T. testudinum. La hipótesis de la variación anual de la concentración de estos metabolitos fue validada. Estos resultados se tendrán en cuenta con el fin de seleccionar el momento de recolecta de las hojas de T. testudinum para su uso con fines biotecnológicos e industriales.

Distribución espacial y estructura morfométrica de las praderas de Thalassia testudinum (Hydrocharitaceae) en dos arrecifes del Parque Nacional Sistema Arrecifal Veracruzano, México / Spatial distribution and ecological structure of Thalassia testudinum (Hydrocharitaceae) in two reefs of Veracruz Reef System National Park, Mexico

ResumenLos pastos marinos son ambientes costeros que se han visto amenazados por el incremento de las actividades humanas. Alterando de forma negativa los procesos y servicios ambientales que presentan, así como la disminución de praderas. El objetivo es generar conocimiento de la distribución, estado de la estructura y nivel de fragmentación en dos arrecifes del Parque Nacional Sistema Arrecifal Veracruzano (PNSAV). Se eligieron dos praderas en arrecifes con características distintas: Sacrificios en el Norte que se encuentra cerca de la costa y Cabezo en el Sur que está alejado de la costa. Se determinaron las características específicas de haz y área de las praderas de macrófitas sumergidas presentes e identificaron cuatro grupos de cobertura morfofuncionales. Se comprobaron diferencias significativas entre coberturas (ANOVA no paramétrico, prueba de Kruskal-Wallis). Se realizó una clasificación supervisada de una imagen espacial de alta resolución verificada con datos de campo (55 Sacrificios y 290 Cabezo). El nivel de fragmentación se calculó usando métricas de paisaje a nivel de clase y se realizaron mapas temáticos en función de las cuatro coberturas. Las praderas se encuentran dominadas por Thalassia testudinum; se tuvieron densidades máximas de 208 haces/m2 para Cabezo y 176 haces/m2 en Sacrificios. Cabezo presentó pastos de hojas cortas (9 cm) y delgadas (0.55 cm) en promedio; Sacrificios tuvo hojas más largas (23.5 cm) y gruesas (1 cm). Sacrificios mostró menor grado de fragmentación que Cabezo; en ambos casos la fragmentación de la cobertura vegetal corresponde a menos del 50 %. Aunque el arrecife Cabezo presenta una mayor fragmentación, que crea un gran número de microambientes, siendo reconocido por su importancia como zona de reclutamiento. Este trabajo sirve como una línea de base para la creación de un plan de manejo adecuado (formación de una zona núcleo de Cabezo). Es necesario complementar este trabajo con nuevos esfuerzos del reconocimiento de las praderas de pastos marinos en todos los arrecifes del PNSAV, así como de monitoreos periódicos y reconocimiento de sus servicios ecosistémicos.

AbstractSeagrasses in coastal environments have been threatened by increased human activities; these have negatively altered processes and environmental services, and have decreased grassland areas. The aim of this study was to generate knowledge of Thalassia testudinum distribution, state of the structure and fragmentation level in two reefs of the Veracruz Reef System National Park (PNSAV). Two different reefs were selected: Sacrificios in the North and near the coast, and Cabezo in the South and away from the coast. Shoot-specific and area-specific characteristics of submerged macrophytes meadows present were determined, and four morpho-functional groups were identified. Significant differences between plant coverage were tested through nonparametric ANOVA, Kruskal-Wallis test. A supervised classification of spatial high-resolution image verified with field data was performed (55 Sacrificios and 290 Cabezo). The fragmentation level was calculated using landscape metrics, class level and thematic maps were made based on four covers. The meadows were dominated by Thalassia testudinum; maximum densities were 208 shoot/m2 in Cabezo, and 176 shoot/m2 in Sacrificios. Cabezo presented grasses with short (9 cm) and thin leaves (0.55 cm) on average; while Sacrificios showed longer (23.5 cm) and thicker (1 cm) leaves. Sacrificios showed lower fragmentation degree than Cabezo; in both cases, the vegetation cover fragmentation corresponded to less than 50 %. Although Cabezo reef presents further fragmentation, which creates a large number of microenvironments, being recognized for its importance as recruitment area. This work serves as a baseline for the creation of an adequate management plan (formation of a core area of Cabezo). It is necessary to complement this work with new efforts for the recognition of seagrass prairies in all PNSAV reefs, as well as periodic monitoring and recognition of ecosystem services. Rev. Biol. Trop. 64 (2): 427-448. Epub 2016 June 01.

Equinodermos enffondos someros del sector la ahmadera, bahía de cispatá, córdooba, caribe Colombiano / Echinoderms in Shallow-Bottom from Ahumadera Sector, Cispatá Bay, Cordoba, Colombian Caribbean

Este estudio representa un reporte de la fauna de equinodermos que se encuentran en fondos someros del sector La Ahumadera, bahía de Cispatá, Caribe colombiano. El área está formada por un sustrato de tipo fango-arenoso colonizado por esponjas en punta Terraplén, y arenoso con pasto marino (Thalassia testudinum), macroalgas y esponjas en punta Robalo. Se identificaron diez especies de equinodermos que pertenecen a cuatro clases (Asteroidea, Ophiuroidea, Echinoidea y Holothuroidea), distribuidas en ocho familias y nueve géneros. El número de taxones identificados corresponde al 4,1% de las especies registradas para el Caribe colombiano, y muestra la representatividad de especies de equinodermos en este sector. Las especies más abundantes en punta Robalo fueron Encope michelini (24,3%) y Ophiothrix angulata, mientras que en punta Terraplén fueron los ofiuros O. angulata (36,7%) y Ophiactis savignyi (16,5%). Se determinó mayor riqueza (S= 9), diversidad (H'= 2,64) y uniformidad (U= 0,92) de especies en punta Robalo al ser comparado con los valores obtenidos en Terraplén (S= 6, H'= 2,39 y U= 0,83). Ambas estaciones mostraron un índice de similitud (Is) de 0,66. Entre las especies identificadas, las siguientes son nuevos registros para el departamento de Córdoba: Holothuria floridana, Encope michelini y Mellita quinquiesperforata.

This study represents a report of the echinoderm fauna found in shallow waters of the sector La Ahumadera, Cispatá Bay, Colombian Caribbean. The area is formed by a plain substrate type soft sand-mud colonized by sponges in punta Terraplén and sandy with seagrass (Thalassia testudinum), macroalgae and sponges in punta Robalo. Were identified ten species of echinoderms belonging to four classes (Asteroidea, Ophiuroidea, Echinoidea and Holothuroidea), distributed in eight families and nine genera. The number of taxa identified corresponds to 4.1% of the species recorded for the Colombian Caribbean, and shows the representation of species of echinoderms in this sector. The most abundant species in punta Robalo were Encope michelini (24.3%) and Ophiothrix angulata, while in punta Terraplén were O. angulata (36.7%) and Ophiactis savignyi (16.5%). Higher richness (S= 9), diversity (H'= 2.64) and evenness (U= 0.92) were found in punta Robalo compared to values from Terraplén (S= 6, H'= 2.39 and U= 0.83). Both stations showed a similarity index (Is) of 0.66.Among the species identified, the following are new records for the department of Córdoba: Holothuria floridana, Encope michelini and Mellita quinquiesperforata.