Predominant heterotrophic diazotrophic bacteria are involved in Sargassum proliferation in the Great Atlantic Sargassum Belt.

Since 2011, the Caribbean coasts have been subject to episodic influxes of floating Sargassum seaweed of unprecedented magnitude originating from a new area "the Great Atlantic Sargassum Belt" (GASB), leading in episodic influxes and mass strandings of floating Sargassum. For the biofilm of both holopelagic and benthic Sargassum as well as in the surrounding waters, we characterized the main functional groups involved in the microbial nitrogen cycle. The abundance of genes representing nitrogen fixation (nifH), nitrification (amoA), and denitrification (nosZ) showed the predominance of diazotrophs, particularly within the GASB and the Sargasso Sea. In both location, the biofilm associated with holopelagic Sargassum harboured a more abundant proportion of diazotrophs than the surrounding water. The mean δ15N value of the GASB seaweed was very negative (-2.04‰), and lower than previously reported, reinforcing the hypothesis that the source of nitrogen comes from the nitrogen-fixing activity of diazotrophs within this new area of proliferation. Analysis of the diversity of diazotrophic communities revealed for the first time the predominance of heterotrophic diazotrophic bacteria belonging to the phylum Proteobacteria in holopelagic Sargassum biofilms. The nifH sequences belonging to Vibrio genus (Gammaproteobacteria) and Filomicrobium sp. (Alphaproteobacteria) were the most abundant and reached, respectively, up to 46.0% and 33.2% of the community. We highlighted the atmospheric origin of the nitrogen used during the growth of holopelagic Sargassum within the GASB and a contribution of heterotrophic nitrogen-fixing bacteria to a part of the Sargassum proliferation.

Impact of sea level rise on the Mediterranean Lithophyllum byssoides rims.

The calcified red macroalga Lithophyllum byssoides, a very common midlittoral species in the western Mediterranean Sea, is a significant ecosystem engineer capable, under exposed and dim light conditions, of building wide and solid endemic bioconstructions near the mean sea level: the L. byssoides rims or 'trottoirs à L. byssoides'. Although the growth of the species is relatively rapid for a calcified alga, the construction of a large rim requires several centuries of near stable or slowly rising sea level. As the time scale of their formation is measured in centuries, L. byssoides bioconstructions constitute valuable and sensitive sea level markers. The health status of L. byssoides rims has been studied at two sites located far apart from each other (Marseille and Corsica), both in areas heavily impacted by humans and in areas with little impact (MPAs and unprotected areas). A health index is proposed: Lithophylum byssoides Rims Health Index. The main and inevitable threat is the rise in the sea level. This ecosystem would be the first case worldwide of marine ecosystem collapse resulting, indirectly, from man-induced global change.

Trace metal content from holopelagic Sargassum spp. sampled in the tropical North Atlantic Ocean: Emphasis on spatial variation of arsenic and phosphorus.

We document for the first time, the spatial distribution at basin scale (North tropical Atlantic Ocean) of As, P and trace metal (TM) concentrations in the three morphotypes belonging to the two holopelagic species Sargassum natans and S. fluitans and three morphotypes: S. natans VIII, S. natans I and S. fluitans III. These samples collected in the North equatorial current (NEC) and in the subtropical Sargasso Sea (sSS) (∼25°N, 60°W) were also compared to coastal samples collected downwind Guadeloupe Island and on the strand of Martinique (mangrove and beach). Along the studied zonal oceanic transect, the highest values of As (range 120-240 µg g-1, dry weight, dw) were found in the sSS area where primary production is highly limited by phosphorus. At these stations, the P content of Sargassum spp. was minimal (range 500-1000 µg g-1, dw) as well as the content in Cd and Zn known for their nutrient-like oceanic behaviors and distributions very similar to P. This illustrates for the first time in the natural environment, the higher bioaccumulation of arsenic in Sargassum spp. in P-limiting conditions which is due to the competition in the phosphate transporter between arsenate and phosphate. As compared to samples collected at sea, the Sargassum spp. collected in the strand of Martinique had (1) lower As concentrations (typical range 30-45 µg g-1, dw) and (2) much higher Al, Fe, Mn, Cr and Co concentrations, showing a certain ability of Sargassum spp. to be depurated of its As content in the coastal zone following competitive exchange with terrigenous metals.

All shallow coastal habitats matter as nurseries for Mediterranean juvenile fish.

Coastal zones are ecosystems of high economic value but exposed to numerous disturbances, while they represent nurseries for many fish species, raising the issue of the preservation of their functions and services. In this context, the juvenile fish assemblages of all types of habitats present in shallow coastal zones were studied on the south-east coast of France using underwater visual censuses in warm (June-July 2014) and cold (April 2015) periods. A total of fourteen habitat types were characterized, which could be grouped into three broad categories, rocky substrates (natural and artificial), sedimentary bottoms with all levels of granulometry, and seagrass beds including Cymodocea nodosa and Posidonia oceanica meadows; the ecotones or interfaces between the three broad habitat categories were individualized as particular habitat types. The abiotic and biotic descriptors of the 14 habitat types individualized did not vary with time, except for a higher cover percentage and canopy height of macrophytes in the warm period, which increased the three-dimensional structure of some habitats. The taxonomic composition and density of juvenile fish assemblages were analyzed using both multivariate and univariate descriptors, after grouping the 57 fish species recorded into 41 well-individualized taxa. Juvenile fishes were recorded in all habitat types, with higher mean species richness and abundance during the warm than the cold period. The richest habitats in terms of both fish species richness and abundance were the natural rocky substrates and the interfaces between Posidonia beds and the other habitats. Although juvenile fish assemblage composition differed among habitat types and between periods, the most abundant fish species were Atherina sp., Sarpa salpa, Gobiidae, Symphodus spp., Pagellus spp. and several Diplodus species, which colonized 7 up to 14 different habitat types (depending on taxa) during their juvenile life. Most species settled in one or a few specific habitats but rapidly colonized adjacent habitats when growing. This study provided evidence of the role of all types of shallow coastal habitats as fish nurseries and their varying pattern of occupation in space and time by the different juvenile stages. It highlighted the importance of the mosaic of habitats and interfaces for the complete development of all juvenile life stages of fishes, and for the preservation of a high diversity of coastal fish assemblages and fisheries resources in the Mediterranean Sea.

Genomic signatures of clonality in the deep water kelp Laminaria rodriguezii.

The development of population genomic approaches in non-model species allows for renewed studies of the impact of reproductive systems and genetic drift on population diversity. Here, we investigate the genomic signatures of partial clonality in the deep water kelp Laminaria rodriguezii, known to reproduce by both sexual and asexual means. We compared these results with the species Laminaria digitata, a closely related species that differs by different traits, in particular its reproductive mode (no clonal reproduction). We analysed genome-wide variation with dd-RAD sequencing using 4,077 SNPs in L. rodriguezii and 7,364 SNPs in L. digitata. As predicted for partially clonal populations, we show that the distribution of FIS within populations of L. rodriguezii is shifted toward negative values, with a high number of loci showing heterozygote excess. This finding is the opposite of what we observed within sexual populations of L. digitata, characterized by a generalized deficit in heterozygotes. Furthermore, we observed distinct distributions of FIS among populations of L. rodriguezii, which is congruent with the predictions of theoretical models for different levels of clonality and genetic drift. These findings highlight that the empirical distribution of FIS is a promising feature for the genomic study of asexuality in natural populations. Our results also show that the populations of L. rodriguezii analysed here are genetically differentiated and probably isolated. Our study provides a conceptual framework to investigate partial clonality on the basis of RAD-sequencing SNPs. These results could be obtained without any reference genome, and are therefore of interest for various non-model species.

Analysis of organellar genomes in brown algae reveals an independent introduction of similar foreign sequences into the mitochondrial genome.

Kelp species (Laminariales, Phaeophyceae) are globally widespread along temperate to Polar rocky coastal lines. Here we analyse the mitochondrial and chloroplast genomes of Laminaria rodriguezii, in comparison to the organellar genomes of other kelp species. We also provide the complete mitochondrial genome sequence of another endemic kelp species from a Polar habitat, the Arctic Laminaria solidungula. We compare phylogenetic trees derived from twenty complete mitochondrial and seven complete chloroplast kelp genomes. Interestingly, we found a stretch of more than 700 bp in the mitochondrial genome of L.rodriguezii, which is not present in any other yet sequenced member of the Phaeophyceae. This stretch matches a protein coding region in the mitochondrial genome from Desmarestia viridis, another brown seaweed. Their high similarity suggests that these sequences originated through independent introduction into the two species. Their origin could have been by infection by yet unknown similar mitoviruses, currently only known from fungi and plants.

Hindcasting the 2017 dispersal of Sargassum algae in the Tropical North Atlantic.

Since 2011, huge amounts of Sargassum algae are detected in the equatorial Atlantic, causing large strandings events on the coasts of the West Indies, Brazil and West Africa. The distribution of this stock shows strong annual and interannual variability, whose drivers are not settled yet. Here we use satellite Sargassum observations from MODIS and currents from an ocean reanalysis to simulate the passive transport of algae in 2017. Wind effect was necessary to fit the observed distribution. Simulations reasonably reproduce the satellite monthly distribution for up to seven months, confirming the prominent role of transport in the distribution cycle. Annual cycle appears as a zonal exchange between eastern (EAR) and western accumulation regions (WAR). EAR is well explained by advection alone, with sharp meridional distribution controlled by converging currents below the inter-tropical Convergence Zone. Instead, WAR is not explained by advection alone, suggesting local growth.

In situ observations and modelling revealed environmental factors favouring occurrence of Vibrio in microbiome of the pelagic Sargassum responsible for strandings.

Historically, pelagic Sargassum were only found in the Sargasso Sea. Since 2011, blooms were regularly observed in warmer water, further south. Their developments in Central Atlantic are associated with mass strandings on the coasts, causing important damages and potentially dispersion of new bacteria. Microbiomes associated with pelagic Sargassum were analysed at large scale in Central Atlantic and near Caribbean Islands with a focus on pathogenic bacteria. Vibrio appeared widely distributed among pelagic Sargassum microbiome of our samples with higher occurrence than previously found in Mexico Gulf. Six out the 16 Vibrio-OTUs (Operational Taxonomic Unit), representing 81.2 ± 13.1% of the sequences, felt in cluster containing pathogens. Among the four different microbial profiles of pelagic Sargassum microbiome, Vibrio attained about 2% in two profiles whereas it peaked, in the two others, at 6.5 and 26.8% respectively, largely above the concentrations found in seawater surrounding raft (0.5%). In addition to sampling and measurements, we performed backward Lagrangian modelling of trajectories of rafts, and rebuilt the sampled rafts environmental history allowing us to estimate Sargassum growth rates along raft displacements. We found that Vibrio was favoured by high Sargassum growth rate and in situ ammonium and nitrite, modelled phosphate and nitrate concentrations, whereas zooplankters, benthic copepods, and calm wind (proxy of raft buoyancy near the sea surface) were less favourable for them. Relations between Vibrio and other main bacterial groups identified a competition with Alteromonas. According to forward Lagrangian tracking, part of rafts containing Vibrio could strand on the Caribbean coasts, however the strong decreases of modelled Sargassum growth rates along this displacement suggest unfavourable environment for Vibrio. For the conditions and areas observed, the sanitary risk seemed in consequence minor, but in other areas or conditions where high Sargassum growth rate occurred near coasts, it could be more important.

From In Situ to satellite observations of pelagic Sargassum distribution and aggregation in the Tropical North Atlantic Ocean.

The present study reports on observations carried out in the Tropical North Atlantic in summer and autumn 2017, documenting Sargassum aggregations using both ship-deck observations and satellite sensor observations at three resolutions (MSI-10 m, OLCI-300 m, VIIRS-750 m and MODIS-1 km). Both datasets reported that in summer, Sargassum aggregations were mainly observed off Brazil and near the Caribbean Islands, while they accumulated near the African coast in autumn. Based on in situ observations, we propose a five-class typology allowing standardisation of the description of in situ Sargassum raft shapes and sizes. The most commonly observed Sargassum raft type was windrows, but large rafts composed of a quasi-circular patch hundreds of meters wide were also observed. Satellite imagery showed that these rafts formed larger Sargassum aggregations over a wide range of scales, with smaller aggregations (of tens of m2 area) nested within larger ones (of hundreds of km2). Match-ups between different satellite sensors and in situ observations were limited for this dataset, mainly because of high cloud cover during the periods of observation. Nevertheless, comparisons between the two datasets showed that satellite sensors successfully detected Sargassum abundance and aggregation patterns consistent with in situ observations. MODIS and VIIRS sensors were better suited to describing the Sargassum aggregation distribution and dynamics at Atlantic scale, while the new sensors, OLCI and MSI, proved their ability to detect Sargassum aggregations and to describe their (sub-) mesoscale nested structure. The high variability in raft shape, size, thickness, depth and biomass density observed in situ means that caution is called for when using satellite maps of Sargassum distribution and biomass estimation. Improvements would require additional in situ and airborne observations or very high-resolution satellite imagery.

The Amazon River: A Major Source of Organic Plastic Additives to the Tropical North Atlantic?

The release of emerging organic contaminants is identified among the most critical hazards to the marine environment, and plastic additives have received growing attention due to their worldwide distribution and potential deleterious effects. Here, we report dissolved surface water concentrations of two important families of plastic additives (organophosphate esters (OPEs) and bisphenols) and other related organic compounds (perfluorinated chemicals) measured in the North Atlantic from Cape Verde to the West Indies. We found that OPEs were the most abundant contaminants, reaching remarkably high concentrations in open ocean waters (1200 km offshore of the American Coast, at the location of the Amazon river plume during the sampling period), with up to 1.3 µg L-1 (Σ9OPEs). A Lagrangian analysis confirmed that these high concentrations of contaminants originated from the Amazon River plume and were transported more than 3000 km by the North Brazil Current and its retroflection. We thus consider the Amazon River as a major source of organic contaminants of emerging concern to the tropical North Atlantic Ocean and suggest that medium-/long-range contaminant transport occurs, most certainly facilitated by the highly stratified conditions offered by the river plume.

The ups and downs of a canopy-forming seaweed over a span of more than one century.

Canopy-forming seaweeds constitute marine forests that deliver ecosystem services. The worldwide range shift, sharp decline or loss of many of these forests, caused by the cumulative impact of increasing human pressure and climate change, have been widely documented. Contrasting examples, reflecting higher than expected resilience, have been more rarely reported. Here, we took the opportunity of having at our disposal a two-century suite of documents (herbarium vouchers, articles) and a ~120-year observation period, dealing with a long-lived brown seaweed, Cystoseira mediterranea, along a well-explored Mediterranean coastline in the Gulf of Lions, to depict the fate of its populations. In addition, we provided baselines for future surveys, with a high degree of accuracy. The northernmost population, scattered on rare suitable substrates, gradually declined and has been extinct since the 1980s. The length of shore occupied by the southern population showed a long-term decline trend, with two sharp minima followed by partial recovery. The causes of the decline differ between sites and periods: coastal development, pollution, competition with mussels, heatwaves and exceptional storms. Overall, the Gulf of Lions populations reflects long-lasting resilience, higher than expected, and a health status that is better than that reported for many other canopy-forming seaweeds.

An ecosystem-based approach to assess the status of Mediterranean algae-dominated shallow rocky reefs.

A conceptual model was constructed for the functioning the algae-dominated rocky reef ecosystem of the Mediterranean Sea. The Ecosystem-Based Quality Index (reef-EBQI) is based upon this model. This index meets the objectives of the EU Marine Strategy Framework Directive. It is based upon (i) the weighting of each compartment, according to its importance in the functioning of the ecosystem; (ii) biological parameters assessing the state of each compartment; (iii) the aggregation of these parameters, assessing the quality of the ecosystem functioning, for each site; (iv) and a Confidence Index measuring the reliability of the index, for each site. The reef-EBQI was used at 40 sites in the northwestern Mediterranean. It constitutes an efficient tool, because it is based upon a wide set of functional compartments, rather than upon just a few species; it is easy and inexpensive to implement, robust and not redundant with regard to already existing indices.

Response of rocky shore communities to anthropogenic pressures in Albania (Mediterranean Sea): Ecological status assessment through the CARLIT method.

The lower mid-littoral and shallow subtidal communities were studied in the district of Vlora (Albania), three years after the establishment of a Marine Protected Area, with particular attention to the long-lived species. The bioconstructions built in the mid-littoral zone by the calcified rhodobiont Lithophyllum byssoides were in poor condition and sometimes even dead. In contrast, the brown alga Cystoseira amentacea constituted lush stands. For assessing the ecological status of the studied area, the CARLIT method, based upon macroalgal communities, was applied. The observed range of ecological status was wide ('high' through 'bad') and was overall among the lowest assessed to date in the Mediterranean Sea. The occurrence of extensive sea-urchin barren-grounds, though not taken into consideration by the CARLIT index, confirmed the poor condition of large sectors of the study area. Overall, the CARLIT index is well correlated with anthropogenic pressures, as assessed by the LUSI index.

Distribution and abundance of Ostreopsis spp. and associated species (Dinophyceae) in the northwestern Mediterranean: the region and the macroalgal substrate matter.

The range of Ostreopsis (Dinophyceae), a taxon harmful to both human health and ecosystems, has spread from a tropical and subtropical range of distribution to temperate areas, such as the Mediterranean Sea. This study has evidenced widespread summer occurrence in the northwestern Mediterranean, from French Catalonia to the French Riviera and Corsica. Ostreopsis spp. are usually associated with two other dinophycean taxa, Prorocentrum lima and Coolia spp. No obvious correlation (indicative of competition and/or facilitation) between the abundance of the three taxa was evidenced. In addition to local variability, we observed regional variability, with low abundance and local absence in French Catalonia and Languedoc, which contrasted with overall abundance and blooms in Provence, the French Riviera and Corsica, especially in late summer. Possible causes for this regional variability are discussed. Furthermore, the three taxa can grow on a variety of macroalgal substrates: 34 taxa belonging to the fleshy, bushy, flat and erect morpho-functional groups. Some macroalgal species were correlated with either high or low abundance of the studied dinophycean taxa and could therefore enhance or hinder their blooming.

Unexpected abundance and long-term relative stability of the brown alga Cystoseira amentacea, hitherto regarded as a threatened species, in the north-western Mediterranean Sea.

Cystoseira amentacea is a Mediterranean endemic alga thriving on very shallow rocky substrates. It has been considered as a threatened species, having experienced a steady decline and is therefore protected by international conventions. The historical distribution of the species has been assessed along the French Mediterranean coast, on the basis of 467 articles and herbarium vouchers. We have produced an accurate map of its current distribution and abundance along 1832 km of coastline, through in situ surveys. C. amentacea was observed along 1125 km of shoreline, including 33% of almost continuous or continuous belt. In most of its range, there is no evidence of loss, except in 4 areas of Provence, French Riviera and Corsica. A significant relation was found between the absence or low abundance of C. amentacea and the vicinity of ports and large sewage outfalls. The status of conservation of the species should therefore be reassessed.

An ecosystem-based approach to assess the status of a Mediterranean ecosystem, the Posidonia oceanica seagrass meadow.

Biotic indices, which reflect the quality of the environment, are widely used in the marine realm. Sometimes, key species or ecosystem engineers are selected for this purpose. This is the case of the Mediterranean seagrass Posidonia oceanica, widely used as a biological quality element in the context of the European Union Water Framework Directive (WFD). The good quality of a water body and the apparent health of a species, whether or not an ecosystem engineer such as P. oceanica, is not always indicative of the good structure and functioning of the whole ecosystem. A key point of the recent Marine Strategy Framework Directive (MSFD) is the ecosystem-based approach. Here, on the basis of a simplified conceptual model of the P. oceanica ecosystem, we have proposed an ecosystem-based index of the quality of its functioning, compliant with the MSFD requirements. This index (EBQI) is based upon a set of representative functional compartments, the weighting of these compartments and the assessment of the quality of each compartment by comparison of a supposed baseline. The index well discriminated 17 sites in the north-western Mediterranean (French Riviera, Provence, Corsica, Catalonia and Balearic Islands) covering a wide range of human pressure levels. The strong points of the EBQI are that it is easy to implement, non-destructive, relatively robust, according to the selection of the compartments and to their weighting, and associated with confidence indices that indicate possible weakness and biases and therefore the need for further field data acquisition.

Occurrence of the toxic dinoflagellate Ostreopsis cf. ovata in relation with environmental factors in Monaco (NW Mediterranean).

To study environment characteristics favoring the toxic benthic dinoflagellate Ostreopsis cf. ovata, a survey was conducted in Monaco (NW Mediterranean Sea), in summers 2007 and 2008. Epiphytic and planktonic blooms occurred almost simultaneously and a high variation of abundances at low spatial scales was observed. An early and very marked bloom occurred in 2007, compared to a later and less abundant development in 2008. These distinct patterns in bloom timing corresponded with very different hydroclimatic scenarios in 2007 (hot spring and relatively cold summer) and 2008 (standard year compared to the median year profile estimated with data from 1995 to 2008). No clear impacts of summer seawater temperature, rainfall or nutrient concentrations were evident. Strong wind may favor the dispersal of benthic and planktonic cells. Our study suggests that further investigations are needed to examine the potential role of Ostreopsis nutritional mode (i.e. autotrophy vs. mixotrophy).

Trends in Ostreopsis proliferation along the Northern Mediterranean coasts.

Harmful benthic microalgae blooms represent an emergent phenomenon in temperate zones, causing health, ecological and economic concern. The main goal of this work was to compile records of Ostreopsis at large temporal and spatial scales, in order to study the relationship between cell abundances, the periodicity and intensity of the blooms and the role of sea water temperature in 14 Spanish, French, Monegasque and Italian sites located along the northern limits of the Mediterranean Sea. General trends were observed in the two considered basins: the north-western Mediterranean Sea, in which higher cell abundances were mostly recorded in mid-summer (end of July), and the northern Adriatic Sea where they occur in early fall (end of September). The sea-water temperature does not seem to be a primary driver, and the maximal abundance periods were site and year specific. Such results represent an important step in the understanding of harmful benthic microalgae blooms in temperate areas, and provide a good base for policy makers and managers in the attempt to monitor and forecast benthic harmful microalgae blooms.

Long-term decline of the populations of Fucales (Cystoseira spp. and Sargassum spp.) in the Albères coast (France, North-western Mediterranean).

Only five of fourteen species of Fucales reported at the end of the XIXth century are currently present in the Albères Coast (France, NW Mediterranean). According to historical data there has been a steady decrease of all the populations since the 1940s. Seven taxa now extinct (Cystoseira crinita, Cystoseira barbata, Cystoseira foeniculacea f. tenuiramosa, Cystoseira spinosa, Cystoseira spinosa var. compressa, Sargassum hornschuchii and Sargassum vulgare) were considered frequent and some of them were the dominant and engineering species in several phytobenthic assemblages. Moreover, only one of the five species left, shows no signs of regression (Cystoseira compressa), two are considered as rare (Cystoseira caespitosa, Cystoseira zosteroides), and one is very rare (Cystoseira elegans). Cystoseira mediterranea, a species that was reported to make a continuous belt along the shores of the Albères coast, has almost disappeared from some areas. Overgrazing by sea urchins, outcompetition by mussels, habitat destruction, scientific research sampling and, probably, human trampling and chemical pollution are to be blamed for the decline of populations thriving in shallow waters. Deep-water species have been affected by an increase in water turbidity and, probably, chemical pollution and direct plant destruction attributed to net fishing. If degradation of the environmental conditions continues, the remaining Cystoseira species will face a most unwelcome prospect. Even after the removal of the causes that led to its die-off, natural restoration of extinct species seems not to be possible because the decline has also affected populations from nearby areas and zygotes are unable to disperse over long distances. Urgent management actions have to be designed in order to improve the current situation of the populations of Fucales in the Albères coast.

Comparison of the wound-activated transformation of caulerpenyne by invasive and noninvasive Caulerpa species of the Mediterranean.

The invasive green alga, Caulerpa taxifolia, that has spread rapidly after its introduction into the Mediterranean and the North American Pacific, reacts to wounding by transforming its major metabolite caulerpenyne. This wound-activated reaction involves the transformation of the bis-enol acetate moiety of 1, releasing reactive 1,4-dialdehydes. The ability to perform this transformation is found also in both the noninvasive Mediterranean C. prolifera and the invasive C. racemosa. Trapping experiments, as well as transformation of the model substrate geranyl acetate, suggest that all three investigated Caulerpa spp. rely on esterases that act upon wounding of the algae by subsequently removing the three acetate residues of caulerpenyne. The resulting reactive 1,4-dialdehyde oxytoxin 2 can be identified by liquid chromatography-mass spectrometry and is unstable in the wounded tissue. Caulerpenyne transformation occurs rapidly, and severe tissue damage caused degradation of more than 50% of the stored caulerpenyne within 1 min in all three algae. Prevention of the enzymatic reaction before extraction, by shock freezing the tissue with liquid nitrogen, was used for the determination of the caulerpenyne content in intact algae. It gives about twofold higher values compared to an established methanol extraction protocol. The speed and mechanism of the wound-activated transformation, as well as the caulerpenyne content in intact tissue of invasive and noninvasive Caulerpa spp., are comparable. Thus, this enzymatic transformation, despite being fast and efficient, is likely not the key for the success of the investigated invasive species.