Impact of elevated pCO2 on paralytic shellfish poisoning toxin content and composition in Alexandrium tamarense

Ocean acidification is considered a major threat to marine ecosystems and may particularly affect primary producers. Here we investigated the impact of elevated pCO2 on paralytic shellfish poisoning toxin (PST) content and composition in two strains of Alexandrium tamarense, Alex5 and Alex2. Experiments were carried out as dilute batch to keep carbonate chemistry unaltered over time. We observed only minor changes with respect to growth and elemental composition in response to elevated pCO2. For both strains, the cellular PST content, and in particular the associated cellular toxicity, was lower in the high CO2 treatments. In addition, Alex5 showed a shift in its PST composition from a nonsulfated analogue towards less toxic sulfated analogues with increasing pCO2. Transcriptomic analyses suggest that the ability of A. tamarense to maintain cellular homeostasis is predominantly regulated on the post-translational level rather than on the transcriptomic level. Furthermore, genes associated to secondary metabolite and amino acid metabolism in Alex5 were down-regulated in the high CO2 treatment, which may explain the lower PST content. Elevated pCO2 also induced up-regulation of a putative sulfotransferase sxtN homologue and a substantial down-regulation of several sulfatases. Such changes in sulfur metabolism may explain the shift in PST composition towards more sulfated analogues. All in all, our results indicate that elevated pCO2 will have minor consequences for growth and elemental composition, but may potentially reduce the cellular toxicity of A. tamarense.

Phytoplankton calcification as an effective mechanism to alleviate cellular calcium poisoning

Marine phytoplankton has developed the remarkable ability to tightly regulate the concentration of free calcium ions in the intracellular cytosol at a level of ~ 0.1 µmol /l in the presence of seawater Ca2+ concentrations of 10 mmol/1. The low cytosolic calcium ion concentration is of utmost importance for proper cell signalling function. While the regulatory mechanisms responsible for the tight control of intracellular Ca2+ concentration are not completely understood, phytoplankton taxonomic groups appear to have evolved different strategies, which may affect their ability to cope with changes in seawater Ca2+ concentrations in their environment on geological time scales. For example, the Cretaceous (145 to 66 Ma ago), an era known for the high abundance of coccolithophores and the production of enormous calcium carbonate deposits, exhibited seawater calcium concentrations up to four times present-day levels. We show that calcifying coccolithophore species (Emiliania huxleyi, Gephyrocapsa oceanica and Coccolithus braarudii) are able to maintain their relative fitness (in terms of growth rate and photosynthesis) at simulated Cretaceous seawater calcium concentrations, whereas these rates are severely reduced under these conditions in some non-calcareous phytoplankton species (Chaetoceros sp., Ceratoneis closterium and Heterosigma akashiwo). Most notably, this also applies to a non-calcifying strain of E. huxleyi which displays a calcium-sensitivity similar to the non-calcareous species. We hypothesize that the process of calcification in coccolithophores provides an efficient mechanism to alleviate cellular calcium poisoning and thereby offered a potential key evolutionary advantage, responsible for the proliferation of coccolithophores during times of high seawater calcium concentrations. The exact function of calcification and the reason behind the highly-ornate physical structures of coccoliths remain elusive.

(Table S2) Cell concentrations of individual and combined Alexandrium spp. and phylogenetic groups as obtained by qPCR assay and Utermöhl counts, as well as particulate PSP toxin concentrations from discrete water depths

Molecular methods provide promising tools for routine detection and quantification of toxic microalgae in plankton samples. To this end, novel TaqMan minor groove binding probes and primers targeting the small (SSU) or large (LSU) ribosomal subunit (rRNA) were developed for two species of the marine dinoflagellate genus Alexandrium (A. minutum, A. tamutum) and for three groups/ribotypes of the A. tamarense species complex: Group I/North American (NA), Group II/Mediterranean (ME) and Group III/Western European (WE). Primers and probes for real-time quantitative PCR (qPCR) were species-specific and highly efficient when tested in qPCR assays for cross-validation with pure DNA from cultured Alexandrium strains. Suitability of the qPCR assays as molecular tools for the detection and estimation of relative cell abundances of Alexandrium species and groups was evaluated from samples of natural plankton assemblages along the Scottish east coast. The results were compared with inverted microscope cell counts (Utermöhl technique) of Alexandrium spp. and associated paralytic shellfish poisoning (PSP) toxin concentrations. The qPCR assays indicated that A. tamarense (Group I) and A. tamutum were the most abundant Alexandrium taxa and both were highly positively correlated with PSP toxin content of plankton samples. Cells of A. tamarense (Group III) were present at nearly all stations but in low abundance. Alexandrium minutum and A. tamarense (Group II) cells were not detected in any of the samples, thereby arguing for their absence from the specific North Sea region, at least at the time of the survey. The sympatric occurrence of A. tamarense Group I and Group III gives further support to the hypothesis that the groups/ribotypes of the A. tamarense species complex are cryptic species rather than variants belonging to the same species.

Intraspecific facilitation by allelochemical mediated grazing protection within a toxigenic dinoflagellate population, link to supplementary material

Dinoflagellates are a major cause of harmful algal blooms, with consequences for coastal marine ecosystem functioning and services. Alexandrium tamarense is one of the most abundant and widespread toxigenic species in the temperate northern and southern hemisphere, and produces paralytic shellfish poisoning toxins as well as lytic allelochemical substances. These bioactive compounds may support the success of A. tamarense and its ability to form blooms. Here we investigate the impact of grazing on monoclonal and mixed set-ups of highly (Alex2) and moderately (Alex4) allelochemically active A. tamarense strains and on a non-allelochemically active conspecific (Alex5) by the heterotrophic dinoflagellate Polykrikos kofoidii. While Alex4 and particularly Alex5 were strongly grazed by P. kofoidii when offered alone, both strains grew well in the mixed assemblages (Alex4+Alex5 and Alex2+Alex5). Hence, the allelochemical active strains facilitated growth of the non-active strain by protecting the population as a whole against grazing. Based on our results, we argue that facilitation among clonal lineages within a species may partly explain the high genotypic and phenotypic diversity of Alexandrium populations. Populations of Alexandrium may comprise multiple cooperative traits that act in concert with intraspecific facilitation, and hence promote the success of this notorious harmful algal bloom species.

Seawater carbonate chemistry and toxicity of Pseudo-nitzschia fraudulenta in a laboratory experiment

Anthropogenic CO2 is progressively acidifying the ocean, but the responses of harmful algal bloom species that produce toxins that can bioaccumulate remain virtually unknown. The neurotoxin domoic acid is produced by the globally-distributed diatom genus Pseudo-nitzschia. This toxin is responsible for amnesic shellfish poisoning, which can result in illness or death in humans and regularly causes mass mortalities of marine mammals and birds. Domoic acid production by Pseudo-nitzschia cells is known to be regulated by nutrient availability, but potential interactions with increasing seawater CO2 concentrations are poorly understood. Here we present experiments measuring domoic acid production by acclimatized cultures of Pseudo-nitzschia fraudulenta that demonstrate a strong synergism between projected future CO2 levels (765 ppm) and silicate-limited growth, which greatly increases cellular toxicity relative to growth under modern atmospheric (360 ppm) or pre-industrial (200 ppm) CO2 conditions. Cellular Si:C ratios decrease with increasing CO2, in a trend opposite to that seen for domoic acid production. The coastal California upwelling system where this species was isolated currently exhibits rapidly increasing levels of anthropogenic acidification, as well as widespread episodic silicate limitation of diatom growth. Our results suggest that the current ecosystem and human health impacts of toxic Pseudo-nitzschia blooms could be greatly exacerbated by future ocean acidification and 'carbon fertilization' of the coastal ocean.

Trace element profiles of the sea anemone Anemonia viridis living nearby a natural CO2 vent

Ocean acidification (OA) is not an isolated threat, but acts in concert with other impacts on ecosystems and species. Coastal marine invertebrates will have to face the synergistic interactions of OA with other global and local stressors. One local factor, common in coastal environments, is trace element contamination. CO2 vent sites are extensively studied in the context of OA and are often considered analogous to the oceans in the next few decades. The CO2 vent found at Levante Bay (Vulcano, NE Sicily, Italy) also releases high concentrations of trace elements to its surrounding seawater, and is therefore a unique site to examine the effects of long-term exposure of nearby organisms to high pCO2 and trace element enrichment in situ. The sea anemone Anemonia viridis is prevalent next to the Vulcano vent and does not show signs of trace element poisoning/stress. The aim of our study was to compare A. viridis trace element profiles and compartmentalization between high pCO2 and control environments. Rather than examining whole anemone tissue, we analyzed two different body compartments-the pedal disc and the tentacles, and also examined the distribution of trace elements in the tentacles between the animal and the symbiotic algae. We found dramatic changes in trace element tissue concentrations between the high pCO2/high trace element and control sites, with strong accumulation of iron, lead, copper and cobalt, but decreased concentrations of cadmium, zinc and arsenic proximate to the vent. The pedal disc contained substantially more trace elements than the anemone's tentacles, suggesting the pedal disc may serve as a detoxification/storage site for excess trace elements. Within the tentacles, the various trace elements displayed different partitioning patterns between animal tissue and algal symbionts. At both sites iron was found primarily in the algae, whereas cadmium, zinc and arsenic were primarily found in the animal tissue. Our data suggests that A. viridis regulates its internal trace element concentrations by compartmentalization and excretion and that these features contribute to its resilience and potential success at the trace element-rich high pCO2 vent.

Molecular and isotopic properties of gases from ODP Holes of ODP Leg 204

We report and discuss molecular and isotopic properties of hydrate-bound gases from 55 samples and void gases from 494 samples collected during Ocean Drilling Program (ODP) Leg 204 at Hydrate Ridge offshore Oregon. Gas hydrates appear to crystallize in sediments from two end-member gas sources (deep allochthonous and in situ) as mixtures of different proportions. In an area of high gas flux at the Southern Summit of the ridge (Sites 1248-1250), shallow (0-40 m below the seafloor [mbsf]) gas hydrates are composed of mainly allochthonous mixed microbial and thermogenic methane and a small portion of thermogenic C2+ gases, which migrated vertically and laterally from as deep as 2- to 2.5-km depths. In contrast, deep (50-105 mbsf) gas hydrates at the Southern Summit (Sites 1248 and 1250) and on the flanks of the ridge (Sites 1244-1247) crystallize mainly from microbial methane and ethane generated dominantly in situ. A small contribution of allochthonous gas may also be present at sites where geologic and tectonic settings favor focused vertical gas migration from greater depth (e.g., Sites 1244 and 1245). Non-hydrocarbon gases such as CO2 and H2S are not abundant in sampled hydrates. The new gas geochemical data are inconsistent with earlier models suggesting that seafloor gas hydrates at Hydrate Ridge formed from gas derived from decomposition of deeper and older gas hydrates. Gas hydrate formation at the Southern Summit is explained by a model in which gas migrated from deep sediments, and perhaps was trapped by a gas hydrate seal at the base of the gas hydrate stability zone (GHSZ). Free gas migrated into the GHSZ when the overpressure in gas column exceeded sealing capacity of overlaying sediments, and precipitated as gas hydrate mainly within shallow sediments. The mushroom-like 3D shape of gas hydrate accumulation at the summit is possibly defined by the gas diffusion aureole surrounding the main migration conduit, the decrease of gas solubility in shallow sediment, and refocusing of gas by carbonate and gas hydrate seals near the seafloor to the crest of the local anticline structure.

Analysis of symptoms of rural workers exposed to pesticides

This cross-sectional study was conducted in southern Minas Gerais, in two counties: São Gonçalo do Sapucaí and Silvianópolis. Presented as objective to verify the important variables associated with the occurrence of symptoms of subacute intoxication related to pesticides exposure. A questionnaire was dedicated to a sample of 412 workers. An analysis of non-conditional logistic regression was applied gradually. The likelihood ratio method was used to define the significant variables in the final model. Of the analysed population, 59.2% reported symptoms typical of subacute intoxication. Of the respondents, 91.5% reported knowing the deleterious effects associated with exposure to pesticides. The adjusted model was found with the significant variables: being male that presented Prevalence Odds Ratio (POR) adjusted . PORof 0.54 (95% CI 0.36 to 0.81), already hospitalized for poisoning with pesticides, POR of 3.26 (95% CI 1.08 to 9.82), living in the rural area of residence., POR of 2.17 (95% CI 1.20 to 3.93) and type of employment relationship or temporary employment, POR of 2.32 (95% CI 1.08 to 4.95).

Experiment on wound activated production of Prostaglandin E2 and related toxic compounds in populations of Gracilaria vermiculophylla

The capacity of the East Asian seaweed Gracilaria vermiculophylla ("Ogonori") for production of prostaglandin E2 from arachidonic acid occasionally causes food poisoning after ingestion. During the last two decades the alga has been introduced to Europe and North America. Non-native populations have been shown to be generally less palatable to marine herbivores than native populations. We hypothesized that the difference in palatability among populations could be due to differences in the algal content of prostaglandins. We therefore compared the capacity for wound-activated production of prostaglandins and other eicosatetraenoid oxylipins among five native populations in East Asia and seven non-native populations in Europe and NW Mexico, using a targeted metabolomics approach. In two independent experiments non-native populations exhibited a significant tendency to produce more eicosatetraenoids than native populations after acclimation to identical conditions and subsequent artificial wounding. Fourteen out of 15 eicosatetraenoids that were detected in experiment I and all 19 eicosatetraenoids that were detected in experiment II reached higher mean concentrations in non-native than in native specimens. The datasets generated in both experiments are contained in http://doi.pangaea.de/10.1594/PANGAEA.855008. Wounding of non-native specimens resulted on average in 390 % more 15-keto-PGE2, in 90 % more PGE2, in 37 % more PGA2 and in 96 % more 7,8-di-hydroxy eicosatetraenoic acid than wounding of native specimens. The dataset underlying this statement is contained in http://doi.pangaea.de/10.1594/PANGAEA.854847. Not only PGE2, but also PGA2 and dihydroxylated eicosatetraenoic acid are known to deter various biological enemies of G. vermiculophylla that cause tissue or cell wounding, and in the present study the latter two compounds also repelled the mesograzer Littorina brevicula. The dataset underlying this statement is contained in http://doi.pangaea.de/10.1594/PANGAEA.854922. Non-native populations of G. vermiculophylla are thus more defended against herbivory than native populations. This increased capacity for activated chemical defense may have contributed to their invasion success and at the same time it poses an elevated risk for human food safety.

Data compilation on the biological response to ocean acidification: environmental and experimental context of data sets and related literature

The exponential growth of studies on the biological response to ocean acidification over the last few decades has generated a large amount of data. To facilitate data comparison, a data compilation hosted at the data publisher PANGAEA was initiated in 2008 and is updated on a regular basis (doi:10.1594/PANGAEA.149999). By January 2015, a total of 581 data sets (over 4 000 000 data points) from 539 papers had been archived. Here we present the developments of this data compilation five years since its first description by Nisumaa et al. (2010). Most of study sites from which data archived are still in the Northern Hemisphere and the number of archived data from studies from the Southern Hemisphere and polar oceans are still relatively low. Data from 60 studies that investigated the response of a mix of organisms or natural communities were all added after 2010, indicating a welcomed shift from the study of individual organisms to communities and ecosystems. The initial imbalance of considerably more data archived on calcification and primary production than on other processes has improved. There is also a clear tendency towards more data archived from multifactorial studies after 2010. For easier and more effective access to ocean acidification data, the ocean acidification community is strongly encouraged to contribute to the data archiving effort, and help develop standard vocabularies describing the variables and define best practices for archiving ocean acidification data.