The effects of the toxic dinoflagellate Alexandrium on feeding, reproduction and mortality of the copepod Acartia: A systematic review employing weighted linear models.

The study of interactions between copepods of the genus Acartia and toxic dinoflagellates of the genus Alexandrium has been an important topic during the last four decades. Feeding behavior and physiological responses of copepods have been studied in laboratory and field experiments, sometimes with contradictory results. More recently, an evolutionary adaptive mechanism leading to enhanced tolerance of Alexandrium toxins in a population of Acartia experiencing chronic exposure to these dinoflagellates has been reported. In the present work, we collected data from the existing studies on the effects of Alexandrium on feeding, reproduction and mortality of Acartia. With these data, we performed a systematic review consisting of a secondary analysis employing general or generalized linear models, weighting data from different studies by the reciprocal of their standard deviation. Our first aim was to overcome shortcomings of individual studies: limited ranges of the variables and overlooked variables (experiment length, population adaptation). These shortcomings could have led to inconsistent conclusions by missing heterogeneous patterns in copepod responses and in the interactions between variables. Our second aim was to test the enhanced physiological performance of chronically exposed relative to naïve copepod populations over a wide geographic range. We found that the feeding rate is enhanced by increased food biomass, irrespective of the food type. Toxins do not have a clear effect on egg production and have a bi-phasic effect on egg hatching success, which was negative above a specific threshold. Toxins also increased mortality. Experiment length had a positive effect on egg production and negative on egg hatching. Naïve copepod populations showed consistently lower ingestion of Alexandrium and egg hatching rates, thereby supporting the spread of the aforementioned mechanism across populations over a wide geographic range.

Bioprospecting for industrially relevant exopolysaccharide-producing cyanobacteria under Portuguese simulated climate.

Cyanobacterial exopolysaccharides (EPS) are potential candidates for the production of sustainable biopolymers. Although the bioactive and physicochemical properties of cyanobacterial-based EPS are attractive, their commercial exploitation is limited by the high production costs. Bioprospecting and characterizing novel EPS-producing strains for industrially relevant conditions is key to facilitate their implementation in various biotechnological applications and fields. In the present work, we selected twenty-five Portuguese cyanobacterial strains from a diverse taxonomic range (including some genera studied for the first time) to be grown in diel light and temperature, simulating the Portuguese climate conditions, and evaluated their growth performance and proximal composition of macronutrients. Synechocystis and Cyanobium genera, from marine and freshwater origin, were highlighted as fast-growing (0.1-0.2 g L-1 day-1) with distinct biomass composition. Synechocystis sp. LEGE 07367 and Chroococcales cyanobacterium LEGE 19970, showed a production of 0.3 and 0.4 g L-1 of released polysaccharides (RPS). These were found to be glucan-based polymers with high molecular weight and a low number of monosaccharides than usually reported for cyanobacterial EPS. In addition, the absence of known cyanotoxins in these two RPS producers was also confirmed. This work provides the initial steps for the development of cyanobacterial EPS bioprocesses under the Portuguese climate.

Proteomics reveals multiple effects of titanium dioxide and silver nanoparticles in the metabolism of turbot, Scophthalmus maximus.

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.

Effects of light intensity, temperature, and salinity in allelopathic interactions between coexisting Synechococcus sp. phenotypes.

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.

Comparison of Sample Preparation Methods for Shotgun Proteomic Studies in Aquaculture Species.

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).

Structure-Antifouling Activity Relationship and Molecular Targets of Bio-Inspired(thio)xanthones.

The development of alternative ecological and effective antifouling technologies is still challenging. Synthesis of nature-inspired compounds has been exploited, given the potential to assure commercial supplies of potential ecofriendly antifouling agents. In this direction, the antifouling activity of a series of nineteen synthetic small molecules, with chemical similarities with natural products, were exploited in this work. Six (4, 5, 7, 10, 15 and 17) of the tested xanthones showed in vivo activity toward the settlement of Mytilus galloprovincialis larvae (EC50: 3.53-28.60 µM) and low toxicity to this macrofouling species (LC50 > 500 µM and LC50/EC50: 17.42-141.64), and two of them (7 and 10) showed no general marine ecotoxicity (<10% of Artemia salina mortality) after 48 h of exposure. Regarding the mechanism of action in mussel larvae, the best performance compounds 4 and 5 might be acting by the inhibition of acetylcholinesterase activity (in vitro and in silico studies), while 7 and 10 showed specific targets (proteomic studies) directly related with the mussel adhesive structure (byssal threads), given by the alterations in the expression of Mytilus collagen proteins (PreCols) and proximal thread proteins (TMPs). A quantitative structure-activity relationship (QSAR) model was built with predictive capacity to enable speeding the design of new potential active compounds.

Assessment of the Allelochemical Activity and Biochemical Profile of Different Phenotypes of Picocyanobacteria from the Genus Synechococcus.

Organisms belonging to Synechococcus sp. genera are observed in all freshwater, brackish, and marine waters of the world. They play a relevant role in these ecosystems, since they are one of the main primary producers, especially in open ocean. Eventually, they form mass blooms in coastal areas, which are potentially dangerous for the functioning of marine ecosystems. Allelopathy could be an important factor promoting the proliferation of these organisms. According to the authors' best knowledge, there is no information on the allelopathic activity and allelopathic compounds exhibited by different Synechococcus sp. phenotypes. Therefore, the research conducted here aimed to study the bioactivity of compounds produced by three phenotypes of Synechococcus sp. by studying their influence on the growth, chlorophyll fluorescence, and photosynthetic pigments of eighteen cyanobacteria and microalgae species. We demonstrated that three different Synechococcus sp. phenotypes, including a phycocyanin (PC)-rich strain (Type 1; green strain) and phycoerythrin (PE)-rich strains containing phycoerythrobilin (PEB) and phycocyanobilin (PCB) (Type 2; red strain and Type 3a; brown strain), had a significant allelopathic effect on the selected species of cyanobacteria, diatoms, and green algae. For all green algae, a decrease in cell abundance under the influence of phenotypes of donor cyanobacteria was shown, whereas, among some target cyanobacteria and diatom species, the cell-free filtrate was observed to have a stimulatory effect. Our estimates of the stress on photosystem II (Fv/Fm) showed a similar pattern, although for some diatoms, there was an effect of stress on photosynthesis, while a stimulatory effect on growth was also displayed. The pigment content was affected by allelopathy in most cases, particularly for chlorophyll a, whilst it was a bit less significant for carotenoids. Our results showed that Synechococcus sp. Type 3a had the strongest effect on target species, while Synechococcus sp. Type 1 had the weakest allelopathic effect. Furthermore, GC-MS analysis produced different biochemical profiles for the Synechococcus strains. For every phenotype, the most abundant compound was different, with oxime-, methoxy-phenyl- being the most abundant substance for Synechococcus Type 1, eicosane for Synechococcus Type 2, and silanediol for Synechococcus Type 3a.

Physiological and Metabolic Responses of Marine Mussels Exposed to Toxic Cyanobacteria Microcystis aeruginosa and Chrysosporum ovalisporum.

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 × 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.

Physiological Effects on Coexisting Microalgae of the Allelochemicals Produced by the Bloom-Forming Cyanobacteria Synechococcus sp. and Nodularia Spumigena.

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.