(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.

Seawater carbonate chemistry and maximum growth rates of Skeletonema marinoi and Alexandrium ostenfeldii, toxin composition of Alexandrium ostenfeldii in a laboratory experiment

Phytoplankton populations can display high levels of genetic diversity that, when reflected by phenotypic variability, may stabilize a species response to environmental changes. We studied the effects of increased temperature and CO2 availability as predicted consequences of global change, on 16 genetically different isolates of the diatom Skeletonema marinoi from the Adriatic Sea and the Skagerrak (North Sea), and on eight strains of the PST (paralytic shellfish toxin)-producing dinoflagellate Alexandrium ostenfeldii from the Baltic Sea. Maximum growth rates were estimated in batch cultures of acclimated isolates grown for five to 10 generations in a factorial design at 20 and 24 °C, and present day and next century applied atmospheric pCO2, respectively. In both species, individual strains were affected in different ways by increased temperature and pCO2. The strongest response variability, buffering overall effects, was detected among Adriatic S. marinoi strains. Skagerrak strains showed a more uniform response, particularly to increased temperature, with an overall positive effect on growth. Increased temperature also caused a general growth stimulation in A. ostenfeldii, despite notable variability in strain-specific response patterns. Our data revealed a significant relationship between strain-specific growth rates and the impact of pCO2 on growth-slow growing cultures were generally positively affected, while fast growing cultures showed no or negative responses to increased pCO2. Toxin composition of A. ostenfeldii was consistently altered by elevated temperature and increased CO2 supply in the tested strains, resulting in overall promotion of saxitoxin production by both treatments. Our findings suggest that phenotypic variability within populations plays an important role in the adaptation of phytoplankton to changing environments, potentially attenuating short-term effects and forming the basis for selection. In particular, A. ostenfeldii blooms may expand and increase in toxicity under increased water temperature and atmospheric pCO2 conditions, with potentially severe consequences for the coastal ecosystem.

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.

Comparison of fecal indicators with pathogenic bacteria and rotavirus in rural Bangladesh groundwater

Groundwater is routinely analyzed for fecal indicators but direct comparisons of fecal indicators to the presence of bacterial and viral pathogens are rare. This study was conducted in rural Bangladesh where the human population density is high, sanitation is poor, and groundwater pumped from shallow tubewells is often contaminated with fecal bacteria. Five indicator microorganisms (E. coli, total coliform, F+RNA coliphage, Bacteroides and human-associated Bacteroides (HuBacteroides)) and various environmental parameters were compared to the direct detection of waterborne pathogens by quantitative PCR in groundwater pumped from 50 tubewells. Rotavirus was detected in groundwater filtrate from the largest proportion of tubewells (40%), followed by Shigella (10%), Vibrio (10%), and pathogenic E. coli (8%). Spearman rank correlations and sensitivity-specificity calculations indicate that some, but not all, combinations of indicators and environmental parameters can predict the presence of pathogens. Culture-dependent fecal indicator bacteria measured on a single date did not predict bacterial pathogens, but annually averaged monthly measurements of culturable E. coli did improve prediction for total bacterial pathogens. F+RNA coliphage were neither correlated nor sufficiently sensitive towards rotavirus, but were predictive of bacterial pathogens. A qPCR-based E. coli assay was the best indicator for the bacterial pathogens, rotavirus and all pathogens combined. Since groundwater cannot be excluded as a significant source of diarrheal disease in Bangladesh and neighboring countries with similar characteristics, the need to develop more effective methods for screening tubewells with respect to microbial contamination is necessary.

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.

Seawater carbonate chemistry and morphometric coordinates and morphology of the control 8-arm pluteus of brittlestar Ophiothrix fragilis, 2008

The world's oceans are slowly becoming more acidic. In the last 150 yr, the pH of the oceans has dropped by ~0.1 units, which is equivalent to a 25% increase in acidity. Modelling predicts the pH of the oceans to fall by 0.2 to 0.4 units by the year 2100. These changes will have significant effects on marine organisms, especially those with calcareous skeletons such as echinoderms. Little is known about the possible long-term impact of predicted pH changes on marine invertebrate larval development. Here we predict the consequences of increased CO2 (corresponding to pH drops of 0.2 and 0.4 units) on the larval development of the brittlestar Ophiothrix fragilis, which is a keystone species occurring in high densities and stable populations throughout the shelf seas of northwestern Europe (eastern Atlantic). Acidification by 0.2 units induced 100% larval mortality within 8 d while control larvae showed 70% survival over the same period. Exposure to low pH also resulted in a temporal decrease in larval size as well as abnormal development and skeletogenesis (abnormalities, asymmetry, altered skeletal proportions). If oceans continue to acidify as expected, ecosystems of the Atlantic dominated by this keystone species will be seriously threatened with major changes in many key benthic and pelagic ecosystems. Thus, it may be useful to monitor O. fragilis populations and initiate conservation if needed.