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The morphology, 16S rRNA gene phylogeny and 16S–23S rRNA gene ITS secondary structures of three strains of marine Cyanobacteria, isolated from inter- and subtidal environments from north Portugal were studied, resulting in the description of Zarconia navalis gen. nov., sp. nov. (Oscillatoriales incertae sedis), Romeriopsis navalis gen. nov., sp. nov. (Leptolyngbyaceae) and Romeriopsis marina sp. nov., named under the International Code of Nomenclature for algae, fungi, and plants. No diacritical morphological characters were found for the new genera and species. The 16S rRNA gene maximum-likelihood and Bayesian phylogenies supported that the genus Zarconia is a member of the Oscillatoriales, morphologically similar to the genera Microcoleus and Phormidium, but distant from them. The genus Romeriopsis is positioned within the Leptolyngbyaceae (Synechococcales) and is closely related to Alkalinema . The secondary structures of the D1-D1′, Box B, V2 and V3 helices corroborate the phylogenetic results. Furthermore, our study supports previous observations of polyphyletic Oscillatoriales families and reinforces the need for their taxonomic revision.

Due to non-linear interactions, the effects of contaminant mixtures on aquatic ecosystems are difficult to assess, especially under temperature rise that will likely exacerbate the complexity of the responses. Yet, under the current climatic crisis, assessing the effects of water contaminants and temperature is paramount to understanding the biological impacts of mixtures of stressors on aquatic ecosystems. Here, we use an ecosystem model followed by global sensitivity analysis (GSA) to prioritize the effects of four single emerging contaminants (ECs) and their mixture, combined with two temperature rise scenarios, on the biomass production of a NE Atlantic estuary. Scenarios ran for 10 years with a time-step of 0.1 days. The results indicate that macroinvertebrate biomass was significantly explained by the effect of each single EC and by their mixture but not by temperature. Globally, the most adverse effects were induced by two ECs and by the mixture of the four ECs, although the sensitivity of macroinvertebrates to the tested scenarios differed. Overall, the present approach is useful to prioritize the effects of stressors and assess the sensitivity of the different trophic groups within food webs, which may be of relevance to support decision making linked to the sustainable management of estuaries and other aquatic systems.

Organisms from the Synechococcus genus constitute one of the major contributors to oceanic primary production, broadly distributed in waters with wide range of environmental conditions. This work investigated the influence of abiotic factors (temperature, irradiance, and salinity) on the strength of allelopathic interactions between different phenotypes of picoplanktonic cyanobacteria of the genus Synechococcus sp. (Type 1, Type 2, and Type 3a) employing mixed cultures and cell-free filtrate assays. The response variables studied were population growth and content of photosynthetic pigments: chlorophyll a (Chl a), carotenoids (Car), phycocyanin (PC), phycoerythrin (PE), and allophycocyanin (APC). Temperature was shown to be the most significant abiotic factor impacting the allelopathy of Synechococcus sp. phenotypes, with the Type 2 most significantly impacted. Irradiance also had a significant effect, having the largest effect on allelopathy of Type 3a phenotype. Changes in salinity had the greatest effect on allelopathy of Type 1. Our study has shown the significant influence of temperature, irradiance, and salinity on the strength of allelopathic compounds secreted by Synechococcus sp. phenotypes, with temperature the most significantly affecting allelopathic properties. Moreover, we discovered that the allelopathic response to changing environmental factors is highly phenotype-specific. This differential response of allelopathy could help different phenotypes of Synechococcus sp. to coexist in the water column.

Titanium dioxide (TiO2) and silver (Ag) NPs are among the most used engineered inorganic nanoparticles (NPs); however, their potential effects to marine demersal fish species, are not fully understood. Therefore, this study aimed to assess the proteomic alterations induced by sub-lethal concentrations citrate-coated 25 nm ("P25") TiO2 or polyvinylpyrrolidone (PVP) coated 15 nm Ag NPs to turbot, Scophthalmus maximus. Juvenile fish were exposed to the NPs through daily feeding for 14 days. The tested concentrations were 0, 0.75 or 1.5 mg of each NPs per kg of fish per day. The determination of NPs, Titanium and Ag levels (sp-ICP-MS/ICP-MS) and histological alterations (Transmission Electron Microscopy) supported proteomic analysis performed in the liver and kidney. Proteomic sample preparation procedure (SP3) was followed by LC-MS/MS. Label-free MS quantification methods were employed to assess differences in protein expression. Functional analysis was performed using STRING web-tool. KEGG Gene Ontology suggested terms were discussed and potential biomarkers of exposure were proposed. Overall, data shows that liver accumulated more elements than kidney, presented more histological alterations (lipid droplets counts and size) and proteomic alterations. The Differentially Expressed Proteins (DEPs) were higher in Ag NPs trial. The functional analysis revealed that both NPs caused enrichment of proteins related to generic processes (metabolic pathways). Ag NPs also affected protein synthesis and nucleic acid transcription, among other processes. Proteins related to thyroid hormone transport (Serpina7) and calcium ion binding (FAT2) were suggested as biomarkers of TiO2 NPs in liver. For Ag NPs, in kidney (and at a lower degree in liver) proteins related with metabolic activity, metabolism of exogenous substances and oxidative stress (e.g.: NADH dehydrogenase and Cytochrome P450) were suggested as potential biomarkers. Data suggests adverse effects in turbot after medium/long-term exposures and the need for additional studies to validate specific biological applications of these NPs. Funding: European Regional Development Fund (ERDF) through Interreg Atlantic Area Program, project NANO -CULTURE [EAPA 590/2018]; Portuguese Foundation for Science and Technology (Fundacao para a Ciencia e Tecnologia; FCT) [UIDB/04423/2020, UIDP/04423/2020]

Picocyanobacteria represent the main fraction of ocean primary production and, due to the effects of ocean acidification and an increase in seawater temperature, this group is expected to be favored in future scenarios. For this reason, we studied the ecophysiological response of picocyanobacteria to increases in water temperature by testing the allelopathic activity of a subpolar strain of Synechocystis sp., grown at a temperature range of 10–20°C, against coexisting microalgae. We showed that cell-free filtrates of Synechocystis sp. inhibited growth and the maximum quantum yields of PSII (Fv/Fm) of Porphyridium purpureum, Fistulifera sp., and Chlorella vulgaris, and the negative effect of the picocyanobacterium was stronger at the highest temperatures at which Synechocystis sp. was grown. Similarly, in P. purpureum, the effect of filtrates reduced chlorophyll a (Chl a) and carotenoids (Car) content, and the effect depended on the temperature at which the picocyanobacterium was grown. The lipid content was observed to increase in all species, and the allelopathic effect was the strongest at the highest temperature in which Synechocystis sp. was grown. The results of the present study predict a stronger allelopathic effect of picocyanobacteria against competitors with rising temperatures. They also suggest that the potential effect of climate change would benefit this group in future phytoplankton communities.

Abstract FigureThe morphology, 16S rRNA gene phylogeny and the 16S-23S rRNA gene ITS secondary structures of three strains of marine Cyanobacteria, isolated from inter- and subtidal environments from north Portugal were studied, resulting in the description of Zarkia subtidalensis gen. et. sp. nov. (Zarkiaceae fam. nov.) and Romeriopsis marina gen. et. sp. nov (Leptolyngbyaceae). No diacritical morphological characters were found either for the new family or for the new genera. The 16S rRNA gene Maximum Likelihood and Bayesian phylogenies supported that Zarkia and Zarkiaceae are members of the Oscillatoriales, positioned close to Microcoleaceae genera, but distant from Microcoleus. Romeriopsis is positioned within the Leptolyngbyaceae and is closely related to Alkalinema. The secondary structures of the D1-D1’, Box B, V2 and V3 helices corroborate with the phylogenetic results. Furthermore, our study supports previous observations of polyphyletic Oscillatoriales families and reinforces the need for their taxonomical revision.

Proteomics has been recently introduced in aquaculture research, and more methodological studies are needed to improve the quality of proteomics studies. Therefore, this work aims to compare three sample preparation methods for shotgun LC–MS/MS proteomics using tissues of two aquaculture species: liver of turbot Scophthalmus maximus and hepatopancreas of Mediterranean mussel Mytilus galloprovincialis. We compared the three most common sample preparation workflows for shotgun analysis: filter-aided sample preparation (FASP), suspension-trapping (S-Trap), and solid-phase-enhanced sample preparations (SP3). FASP showed the highest number of protein identifications for turbot samples, and S-Trap outperformed other methods for mussel samples. Subsequent functional analysis revealed a large number of Gene Ontology (GO) terms in turbot liver proteins (nearly 300 GO terms), while fewer GOs were found in mussel proteins (nearly 150 GO terms for FASP and S-Trap and 107 for SP3). This result may reflect the poor annotation of the genomic information in this specific group of animals. FASP was confirmed as the most consistent method for shotgun proteomic studies, however, the use of the other two methods might be important in specific experimental conditions (e.g., when samples have a very low amount of protein).

Allelopathy is widespread in marine, brackish, and freshwater habitats. Literature data indicate that allelopathy could offer a competitive advantage for some phytoplankton species by reducing the growth of competitors. It is also believed that allelopathy may affect species succession. Thus, allelopathy may play a role in the development of blooms. Over the past few decades, the world's coastal waters have experienced increases in the numbers of cyanobacterial and microalgal blooming events. Understanding how allelopathy is implicated with other biological and environmental factors as a bloom-development mechanism is an important topic for future research. This review focuses on a taxonomic overview of allelopathic cyanobacteria and microalgae, the biological and environmental factors that affect allelochemical production, their role in ecological dynamics, and their physiological modes of action, as well as potential industrial applications of allelopathic compounds.

The authors would like to thank Fundaçao para a Ciencia e a Tecnologia (FCT) for funding the research via project grants PTDC/CTAAMB/30056/2017 (OPTOX), PTDC/AAG-MAA/1302/2014 (PROTEOME), UID/MAR/04292/2019 and UID/MULTI/04046/2013. B. Duarte and V. F. Fonseca were supported by investigation contracts (CEECIND/00511/2017 and DL57/2016/CP1479/CT0024). P. Reis-Santos was supported by FCT through a postdoctoral grant (SFRH/BPD/95784/ 2013). M.T.Cabrita is also supported by a DL57 investigation contract. Work was also funded by the Integrated Programme of SR&TD SmartBioR (reference Centro-01-0145-FEDER-000018), co-funded by Centro 2020 program, Portugal 2020, European Union, through the European Regional Development Fund. Emerging pollutants such as pharmaceuticals are continuously released to aquatic environments posing a rising threat to marine ecosystems. Yet, monitoring routines and ecotoxicity data on biota worldwide for these substances are lacking. Non-steroidal anti-inflammatory drugs are among the most prescribed and found pharmaceuticals in aquatic environments. The toxicity effects of environmentally relevant concentrations of ibuprofen on primary productivity, oxidative stress and lipid metabolism of the diatom Phaeodactylum tricornutum were assessed. Diatom cultures were exposed to 0, 0.8, 3, 40, 100 and 300 μg L-1 ibuprofen concentrations, usually found in the vicinity of wastewater treatment plants and coastal environments. Higher concentrations (100 and 300 μg L-1) had a negative impact in P. triconutum growth, inhibiting the chloroplastic energy transduction in the electron transport chain resulting in lower energy reaching the PS I (r2 = - 0.55, p < 0.05). In contrast, the mitochondrial electron transport and available energy increased (r2 = 0.68 and r2 = 0.85, p < 0.05 respectively), mostly due to enhancements in lipid and protein contents as opposed to reduction of carbohydrates. A general up-regulation of the antioxidant enzymes could contributed to alleviate oxidative stress resulting in the decrease of lipid peroxidation products (r2 = 0.77, p < 0.05). Canonical analysis of principal components was performed and successfully discriminated exposure groups, with optical data excelling in classifying samples to different ibuprofen concentrations, being potentially used as environmental indicators. Finally, the identified mild to severe effects of ibuprofen on diatoms are likely to be exacerbated by the sustained use of this drug worldwide, underpinning the urgency of evaluating the impacts of this pharmaceutical on coastal and marine trophic webs. info:eu-repo/semantics/publishedVersion

In the last decades, due to monitoring programs and strict legislation poisoning incidents occurrence provoked by ingestion of naturally contaminated marine organisms has decreased. However, climate change and anthropogenic interference contributed to the expansion and establishment of toxic alien species to more temperate ecosystems. In this work, the coasts of Madeira, São Miguel islands and the northwestern Moroccan coast were surveyed for four groups of lipophilic toxins (yessotoxins, azaspiracids, pectenotoxins, and spirolides), searching for new vectors and geographical tendencies. Twenty-four species benthic organisms were screened using UHPLC-MS/MS technique. We report 19 new vectors for these toxins, six of them with commercial interest (P. aspera, P. ordinaria, C. lampas, P. pollicipes, H. tuberculata and P. lividus). Regarding toxin uptake a south-north gradient was detected. This study contributes to the update of monitoring routines and legislation policies, comprising a wider range of vectors, to better serve consumers and ecosystems preservation. This research was partially funded by the Portuguese Foundation of Science and Technology (FCT) project UID/Multi/04423/2013 and by the projects ALERTOXNET (EAPA_317/2016), funded by the Interreg Atlantic program. The research leading to these results has received funding from the following FEDER co-funded-grants. From Consellería de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia, 2017 GRC GI1682 (ED431C 2017/01). From CDTI and Technological Funds, supported by Ministerio de Economía, Industria y Competitividad AGL2014-58210- R, AGL2016-78728-R (AEI/FEDER, UE), ISCIII/PI16/01830 and RTC2016-5507-2, ITC-20161072. From European Union POCTEP 0161- Nanoeaters-1-E-1, Interreg Agritox EAPA-998-2018. Additional funding was provided by National Funds through FCT—Fundação para a Ciência e a Tecnologia, under the projects UID/BIA/00329/2013, 2015–2018 and UID/BIA/00329/2019. This is a contribution to project MIMAR MAC/ 4.6d/066 funded by the EU program INTERREG MAC 2014-2020. MS acknowledges FCT (SFRH/BD/73269/2010). info:eu-repo/semantics/publishedVersion

Toxic cyanobacterial blooms are a major contaminant in inland aquatic ecosystems. Furthermore, toxic blooms are carried downstream by rivers and waterways to estuarine and coastal ecosystems. Concerning marine and estuarine animal species, very little is known about how these species are affected by the exposure to freshwater cyanobacteria and cyanotoxins. So far, most of the knowledge has been gathered from freshwater bivalve molluscs. This work aimed to infer the sensitivity of the marine mussel Mytilus galloprovincialis to single as well as mixed toxic cyanobacterial cultures and the underlying molecular responses mediated by toxic cyanobacteria. For this purpose, a mussel exposure experiment was outlined with two toxic cyanobacteria species, Microcystis aeruginosa and Chrysosporum ovalisporum at 1 &times, 105 cells/mL, resembling a natural cyanobacteria bloom. The estimated amount of toxins produced by M. aeruginosa and C. ovalisporum were respectively 0.023 pg/cell of microcystin-LR (MC-LR) and 7.854 pg/cell of cylindrospermopsin (CYN). After 15 days of exposure to single and mixed cyanobacteria, a depuration phase followed, during which mussels were fed only non-toxic microalga Parachlorella kessleri. The results showed that the marine mussel is able to filter toxic cyanobacteria at a rate equal or higher than the non-toxic microalga P. kessleri. Filtration rates observed after 15 days of feeding toxic microalgae were 1773.04 mL/ind.h (for M. aeruginosa), 2151.83 mL/ind.h (for C. ovalisporum), 1673.29 mL/ind.h (for the mixture of the 2 cyanobacteria) and 2539.25 mL/ind.h (for the non-toxic P. kessleri). Filtering toxic microalgae in combination resulted in the accumulation of 14.17 ng/g dw MC-LR and 92.08 ng/g dw CYN. Other physiological and biochemical endpoints (dry weight, byssus production, total protein and glycogen) measured in this work did not change significantly in the groups exposed to toxic cyanobacteria with regard to control group, suggesting that mussels were not affected with the toxic microalgae. Nevertheless, proteomics revealed changes in metabolism of mussels related to diet, specially evident in those fed on combined cyanobacteria. Changes in metabolic pathways related with protein folding and stabilization, cytoskeleton structure, and gene transcription/translation were observed after exposure and feeding toxic cyanobacteria. These changes occur in vital metabolic processes and may contribute to protect mussels from toxic effects of the toxins MC-LR and CYN.

Variation in resource use among species determines their potential for competition and co-existence, as well as their impact on ecosystem processes. Planktonic crustaceans consume a range of micro-organisms that vary among habitats and species, but these differences in resource consumption are difficult to characterize due to the small size of the organisms. Consumers acquire amino acids from their diet, and the composition of tissues reflects both the use of different resources and their assimilation in proteins. We examined the amino acid composition of common crustacean zooplankton from 14 tropical lakes in Colombia in three regions (the Amazon floodplain, the eastern range of the Andes, and the Caribbean coast). Amino acid composition varied significantly among taxonomic groups and the three regions. Functional richness in amino acid space was greatest in the Amazon, the most productive region, and tended to be positively related to lake trophic status, suggesting the niche breadth of the community could increase with ecosystem productivity. Functional evenness increased with lake trophic status, indicating that species were more regularly distributed within community-wide niche space in more productive lakes. These results show that zooplankton resource use in tropical lakes varies with both habitat and taxonomy, and that lake productivity may affect community functional diversity and the distribution of species within niche space. © 2018, Springer-Verlag GmbH Germany, part of Springer Nature. Acknowledgements NA was supported by a graduate fellowship from Colciencias 1892–2006. JS was supported by a Fulbright Colombia Fellowship, JS and NTJ were supported by NSF DEB 1457737.

Only a few studies have documented the physiological effects of allelopathy from cyanobacteria against coexisting microalgae. We investigated the allelopathic ability of the bloom-forming cyanobacteria Synechococcus sp. and Nodularia spumigena filtrates on several aspects related to the physiology of the target species: population growth, cell morphology, and several indexes of photosynthesis rate and respiration. The target species were the following: two species of green algae (Oocystis submarina, Chlorella vulgaris) and two species of diatoms (Bacillaria paxillifer, Skeletonema marinoi). These four species coexist in the natural environment with the employed strains of Synechococcus sp. and N. spumigena employed. The tests were performed with single and repeated addition of cyanobacterial cell-free filtrate. We also tested the importance of the growth phase in the strength of the allelopathic effect. The negative effects of both cyanobacteria were the strongest with repeated exudates addition, and generally, Synechococcus sp. and N. spumigena were allelopathic only in the exponential growth phase. O. submarina was not negatively affected by Synechococcus filtrates in any of the parameters studied, while C. vulgaris, B. paxillifer, and S. marinoi were affected in several ways. N. spumigena was characterized by a stronger allelopathic activity than Synechococcus sp., showing a negative effect on all target species. The highest decline in growth, as well as the most apparent cell physical damage, was observed for the diatom S. marinoi. Our findings suggest that cyanobacterial allelochemicals are associated with the cell physical damage, as well as a reduced performance in respiration and photosynthesis system in the studied microalgae which cause the inhibition of the population growth. Moreover, our study has shown that some biotic factors that increase the intensity of allelopathic effects may also alter the ratio between bloom-forming cyanobacteria and some phytoplankton species that occur in the same aquatic ecosystem. This research were funded by BMN grant number 538-G245-B568-17 and FCT Project UID/Multi/04423/2019. The APC was funded by DS 530-G245-D717-18.

Cyanobacteria and cyanotoxins constitute a serious environmental and human health problem. Moreover, concerns are raised with the use of contaminated water in agriculture and vegetable production as this can lead to food contamination and human exposure to toxins as well as impairment in crop development and productivity. The objective of this work was to assess the susceptibility of two green vegetables, spinach and lettuce, to the cyanotoxins microcystin (MC) and cylindrospermopsin (CYN), individually and in mixture. The study consisted of growing both vegetables in hydroponics, under controlled conditions, for 21 days in nutrient medium doped with MC or CYN at 10 &mu, g/L and 50 &mu, g/L, or CYN/MC mixture at 5 + 5 &mu, g/L and 25 + 25 &mu, g/L. Extracts from M. aeruginosa and C. ovalisporum were used as sources of toxins. The study revealed growth inhibition of the aerial part (Leaves) in both species when treated with 50&micro, g/L of MC, CYN and CYN/MC mixture. MC showed to be more harmful to plant growth than CYN. Moreover spinach leaves growth was inhibited by both 5 + 5 and 25 + 25 &micro, g/L CYN/MC mixtures, whereas lettuce leaves growth was inhibited only by 25 + 25 &micro, g/L CYN/MC mixture. Overall, growth data evidence increased sensitivity of spinach to cyanotoxins in comparison to lettuce. On the other hand, plants exposed to CYN/MC mixture showed differential accumulation of CYN and MC. In addition, CYN, but not MC, was translocated from the roots to the leaves. CYN and MC affected the levels of minerals particularly in plant roots. The elements most affected were Ca, K and Mg. However, in leaves K was the mineral that was affected by exposure to cyanotoxins.

This research was supported by the Portuguese project SEEINGSHORE (NORTE-01-0145-FEDER-031893), co-financed by NORTE 2020, Portugal 2020, and the European Union through the ERDF, and by FCT through national funds and received additional funding from the project MarInfo - Integrated Platform for Marine Data Acquisition and Analysis (reference NORTE-01-0145-FEDER-000031), supported by North Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). João N. Franco was supported by a FCT PhD Grant (SFRH/BD/84933/2012). Herbivory plays a major role in shaping community dynamics across freshwater, marine and terrestrial habitats, by controlling patterns of abundance and distribution of primary producers, including seaweeds. In the context of biological invasions, the proliferation of non-native seaweeds has been often attributed to limited grazing by native herbivores on introduced species (“Enemy Release Hypothesis”, ERH). In our study, we aimed to explore the potential of an abundant generalist herbivore (the sea urchin Paracentrotus lividus) to graze on non-native in comparison to native macroalgae species. For this purpose, we used manipulative experiments to assess sea urchin preference on native and non-native seaweed species present in the northwest coast of Portugal. Specifically, we determined the preferences of P. lividus on brown seaweeds i.e. Laminaria ochroleuca, Saccorhiza polyschides and the non-native Undaria pinnatifida, and red seaweeds i.e. Chondrus crispus, Mastocarpus stellatus and the non-native Grateloupia turuturu. For each group of seaweed species, sea urchin preference and biomass consumption were examined. The nutritional (organic carbon and nitrogen) and chemical (phenolic content) features were also analysed, in order to assess their effect on sea urchin feeding. According to the results, P. lividus did not show a specific preference for any of the different seaweeds of each phylum. These results suggest that P. lividus is a generalist herbivore, not exerting a differential grazing pressure on non-native seaweeds when compared to native ones. info:eu-repo/semantics/publishedVersion

Sea anemones are among the most ubiquitous organisms inhabiting the rocky shores of the Atlantic Ocean. We assessed the occurrence of intertidal sea anemones along several beaches on the Portuguese coast, identifying three families: (i) Actiniidae, including Actinia equina, Actinia fragacea, Anemonia viridis, Aulactinia verrucosa, and Anthopleura krebsi species, (ii) Hormathiidae, including Calliactis parasitica, and (iii) Sagartiidae , including Cereus pedunculatus. Analyses of concatenated sequences from one nuclear (18S) and four mitochondrial (16S, COI, COIII and ND6) genes revealed minimal intraspecific phenotypic or genotypic heterogeneity among the sampled locations or relative with species records in NCBI. A comparison among preservation methods across several different tissues demonstrated that tissues preserved with ethanol, and in particular column tissue, yielded higher amounts of DNA compared with samples from other body parts, including tentacles, gastrodermis and ectodermis preserved at −80 °C.

Paralytic shellfish toxins (PSTs) are potent alkaloids of microalgal and cyanobacterial origin, with worldwide distribution. Over the last 20 years, the number of poisoning incidents has declined as a result of the implementation of legislation and monitoring programs based on bivalves. In the summer of 2012 and 2013, we collected a total of 98 samples from 23 different species belonging to benthic and subtidal organisms, such as echinoderms, crustaceans, bivalves, and gastropods. The sampling locations were Madeira, S&atilde, o Miguel Island (Azores archipelago), and the northwestern coast of Morocco. The samples were analyzed using post-column oxidation liquid chromatography with a fluorescence detection method. Our main goal was to detect new vectors for these biotoxins. After reporting a total of 59 positive results for PSTs with 14 new vectors identified, we verified that some of the amounts exceeded the limit value established in the EU. These results suggest that routine monitoring of saxitoxin and its analogs should be extended to more potential vectors other than bivalves, including other edible organisms, for a better protection of public health.