Phytoplankton community composition in relation to environmental variability in the Urdaibai estuary (SE Bay of Biscay): Microscopy and eDNA metabarcoding.

Phytoplankton monitoring is essential for the global understanding of aquatic ecosystems. The present research studies the phytoplankton community of the Urdaibai estuary, combining microscopy and eDNA metabarcoding for the first time in the area. The main aims were to describe the phytoplankton community composition in relation to the environmental conditions of the estuary, and to compare the two methods used. Diatoms Minutocellus polymorphus and Chaetoceros tenuissimus dominated the outer estuary, being replaced by Teleaulax acuta (cryptophyte), Kryptoperidinium foliaceum (dinoflagellate) and Cyclotella spp. (diatom) towards the inner area. This change was mainly prompted by salinity and nutrients. Metabarcoding revealed the presence of 223 species that were not observed by microscopy in previous studies in the estuary. However, several characteristic species (e.g., K. foliaceum) were only detected with microscopy. Additionally, microscopy covered the limitations of eDNA metabarcoding concerning quantification. Thus, to give a full insight, a combination of techniques is recommended.

Bioactives Overproduction through Operational Strategies in the Ichthyotoxic Microalga Heterosigma akashiwo Culture.

The red tide-forming microalga Heterosigma akashiwo has been associated with massive events of fish deaths, both wild and cultured. Culture conditions are responsible for the synthesis or accumulation of some metabolites with different interesting bioactivities. H. akashiwo LC269919 strain was grown in a 10 L bubble column photobioreactor artificially illuminated with multi-coloured LED lights. Growth and production of exopolysaccharides, polyunsaturated fatty acids (PUFAs), and carotenoids were evaluated under different culture modes (batch, fed-batch, semicontinuous, and continuous) at two irradiance levels (300 and 700 µE·s-1·m-2). Continuous mode at the dilution rate of 0.2·day-1 and 700 µE·s-1·m-2 provided the highest production of biomass, PUFAs (132.6 and 2.3 mg·L-1·day-1), and maximum fucoxanthin productivity (0.16 mg·L-1·day-1). The fed-batch mode accumulated exopolysaccharides in a concentration (1.02 g·L-1) 10-fold over the batch mode. An extraction process based on a sequential gradient partition with water and four water-immiscible organic solvents allowed the isolation of bioactive fucoxanthin from methanolic extracts of H. akashiwo. Metabolites present in H. akashiwo, fucoxanthin and polar lipids (i.e., eicosapentaenoic acid (EPA)), or probably such as phytosterol (ß-Sitosterol) from other microalgae, were responsible for the antitumor activity obtained.

Evaluation of algaecide effectiveness of five different oxidants applied on harmful phytoplankton.

Harmful algal blooms (HABs) in coastal areas similarly impact both ecosystems and human health. The translocation of phytoplankton species via maritime transport can potentially promote the growth of HABs in coastal systems. Accordingly, ballast water must be disinfected. The main goal of this study is to assess the effectiveness of different emerging biocides, including H2O2, peracetic acid (PAA), peroxymonosulfate (PMS), and peroxydisulfate (PDS). The effectiveness of these biocides is compared with that of conventional chlorination methods. Their effects on two ichthyotoxic microalgae with worldwide distribution, i.e., Prymnesium parvum and Heterosigma akashiwo, are examined. To ensure the prolonged effectiveness of the different reagents, their concentration-response curves for 14 days are constructed and examined. The results suggest a strong but shorter effect by PMS (EC50 = 0.40-1.99 mg·L-1) and PAA (EC50 = 0.32-2.70 mg·L-1), a maintained effect by H2O2 (EC50 = 6.67-7.08 mg·L-1), and a negligible effect by PDS. H. akashiwo indicates higher resistance than P. parvum, except when H2O2 is used. Based on the growth inhibition performance and consumption of the reagents as well as a review of important aspects regarding their application, using H2O2, PAA, or PMS can be a feasible alternative to chlorine-based reagents for inhibiting the growth of harmful phytoplankton.

Olisthodiscus represents a new class of Ochrophyta.

The phylogenetic diversity of Ochrophyta, a diverse and ecologically important radiation of algae, is still incompletely understood even at the level of the principal lineages. One taxon that has eluded simple classification is the marine flagellate genus Olisthodiscus. We investigated Olisthodiscus luteus K-0444 and documented its morphological and genetic differences from the NIES-15 strain, which we described as Olisthodiscus tomasii sp. nov. Phylogenetic analyses of combined 18S and 28S rRNA sequences confirmed that Olisthodiscus constitutes a separate, deep, ochrophyte lineage, but its position could not be resolved. To overcome this problem, we sequenced the plastid genome of O. luteus K-0444 and used the new data in multigene phylogenetic analyses, which suggested that Olisthodiscus is a sister lineage of the class Pinguiophyceae within a broader clade additionally including Chrysophyceae, Synchromophyceae, and Eustigmatophyceae. Surprisingly, the Olisthodiscus plastid genome contained three genes, ycf80, cysT, and cysW, inherited from the rhodophyte ancestor of the ochrophyte plastid yet lost from all other ochrophyte groups studied so far. Combined with nuclear genes for CysA and Sbp proteins, Olisthodiscus is the only known ochrophyte possessing a plastidial sulfate transporter SulT. In addition, the finding of a cemA gene in the Olisthodiscus plastid genome and an updated phylogenetic analysis ruled out the previously proposed hypothesis invoking horizontal cemA transfer from a green algal plastid into Synurales. Altogether, Olisthodiscus clearly represents a novel phylogenetically distinct ochrophyte lineage, which we have proposed as a new class, Olisthodiscophyceae.

Current distribution and potential expansion of the harmful benthic dinoflagellate Ostreopsis cf. siamensis towards the warming waters of the Bay of Biscay, North-East Atlantic.

In a future scenario of increasing temperatures in North-Atlantic waters, the risk associated with the expansion of the harmful, benthic dinoflagellate Ostreopsis cf. siamensis has to be evaluated and monitored. Microscopy observations and spatio-temporal surveys of environmental DNA (eDNA) were associated with Lagrangian particle dispersal simulations to: (i) establish the current colonization of the species in the Bay of Biscay, (ii) assess the spatial connectivity among sampling zones that explain this distribution, and (iii) identify the sentinel zones to monitor future expansion. Throughout a sampling campaign carried out in August to September 2018, microscope analysis showed that the species develops in the south-east of the bay where optimal temperatures foster blooms. Quantitative PCR analyses revealed its presence across almost the whole bay to the western English Channel. An eDNA time-series collected on plastic samplers showed that the species occurs in the bay from April to September. Due to the water circulation, colonization of the whole bay from the southern blooming zones is explained by inter-site connectivity. Key areas in the middle of the bay permit continuous dispersal connectivity towards the north. These key areas are proposed as sentinel zones to monitor O. cf. siamensis invasions towards the presumably warming water of the North-East Atlantic.

Altererythrobacter muriae sp. nov., isolated from hypersaline Añana Salt Valley spring water, a continental thalassohaline-type solar saltern.

A novel salt-tolerant alpha-proteobacterium, designated SALINAS58T, was isolated from Santa Engracia hypersaline spring water in the Añana Salt Valley, Álava, Spain. The isolate was Gram-negative, aerobic, non-motile, catalase-positive, oxidase-negative, rod-shaped and formed orange colonies on marine agar. Optimal growth was observed at pH 6.0-6.5, at 30 °C and in the presence of 1% (w/v) NaCl. The main cellular fatty acids (>20%) were summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c) and summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c). The major respiratory quinone was ubiquinone Q-10 and the major polar lipids detected were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidilglycerol, four unidentified glycolipids and one unidentified phospholipid. Strain SALINAS58T had the highest 16S rRNA gene sequence similarity to Altererythrobacter marensis MSW-14T (96.6%), Altererythrobacter aquaemixtae JSSK-8T (96.5%) and Pontixanthobacter luteolus SW-109T (96.5%) followed by Altererythrobacter atlanticus 26DY36T (96.4%). Results of the phylogenetic analysis, based on 16S rRNA gene sequences, and phylogenetic approaches based on whole genome nucleotide differences, showed that strain SALINAS58T could be distinguished from recognized species of the genus Altererythrobacter. The genomic DNA G+C content was 61.4 mol%. Digital DNA-DNA hybridization, average nucleotide identity and average aminoacid identity values between the genome of strain SALINAS58T and A. marensis MSW-14T were 18.4, 73.1 and 68.1%, respectively. Based on data from this polyphasic characterization, strain SALINAS58T (=CECT 30029T=LMG 31726T) is considered to be classified as representing a novel species in the genus Altererythrobacter, for which the name Altererythrobacter muriae sp. nov. is proposed.

Spectral Characterization of Eight Marine Phytoplankton Phyla and Assessing a Pigment-Based Taxonomic Discriminant Analysis for the in Situ Classification of Phytoplankton Blooms.

Early stage identification of harmful algal blooms (HABs) has gained significance for marine monitoring systems over the years. Various approaches for in situ classification have been developed. Among them, pigment-based taxonomic classification is one promising technique for in situ characterization of bloom compositions, although it is yet underutilized in marine monitoring programs. To demonstrate the applicability and importance of this powerful approach for monitoring programs, we combined an ultra low-cost and miniaturized multichannel fluorometer with Fisher's linear discriminant analysis (LDA). This enables the real-time characterization of algal blooms at order level based on their spectral properties. The classification capability of the algorithm was examined with a leave-one-out cross validation of 53 different unialgal cultures conducted in terms of standard statistical measures and independent figures of merit. The separation capability of the linear discriminant analysis was further successfully examined in mixed algal suspensions. Besides this, the impact of the growing status on the classification capability was assessed. Further, we provide a comprehensive study of spectral features of eight different phytoplankton phyla including an extensive study of fluorescence excitation spectra and marker pigments analyzed via HPLC. The analyzed phytoplankton species belong to the phyla of Cyanobacteria, Dinophyta (Dinoflagellates), Bacillariophyta (Diatoms), Haptophyta, Chlorophyta, Ochrophyta, Cryptophyta, and Euglenophyta.

Chloroplast Genome Sequences of Seven Strains of the Bloom-Forming Raphidophyte Heterosigma akashiwo.

We report here the complete chloroplast genome sequences of seven strains of the bloom-forming raphidophyte Heterosigma akashiwo These ~160-kb sequences contain 124 protein-, 6 rRNA-, and 34 tRNA-coding sequences. Notable sequence variations were observed among these seven sequenced and two previously characterized strains.

A hypervariable mitochondrial protein coding sequence associated with geographical origin in a cosmopolitan bloom-forming alga, Heterosigma akashiwo.

Geographical distributions of phytoplankton species can be defined by events on both evolutionary time and shorter scales, e.g. recent climate changes. Additionally, modern industrial activity, including the transport of live fish and spat for aquaculture and aquatic microorganisms in ship ballast water, may aid the spread of phytoplankton. Obtaining a reliable marker is key to gaining insight into the phylogeographic history of a species. Here, we report a hypervariable mitochondrial gene in the cosmopolitan bloom-forming alga, Heterosigma akashiwo We compared the entire mitochondrial genome sequences of seven H. akashiwo strains from Japanese and North American coastal waters and identified a hypervariable segment. The region codes for a hypothetical protein with no defined function, and its variations between Japanese and North American isolates were prominent, while the sequences were more conserved among Japanese strains and North American isolates. Comparison of the sequence in isolates obtained from different geographical points in the Northern Hemisphere revealed that the sequence variations largely correlated with latitude and longitude (i.e. Pacific/Atlantic oceans). Our results demonstrate the usefulness of the sequence in determining the phylogeographic history of H. akashiwo.

The genus Pseudo-nitzschia (Bacillariophyceae) in a temperate estuary with description of two new species: Pseudo-nitzschia plurisecta sp. nov. and Pseudo-nitzschia abrensis sp. nov.

The genus Pseudo-nitzschia contains potentially toxic species of problematic taxonomy, making it one of the most intensively studied diatom genera. The study of 35 clonal strains isolated from the Bilbao estuary, an area that experiences recurrent blooms of Pseudo-nitzschia, revealed the presence of two new species, P. abrensis and P. plurisecta, differing from their congeners in both morphology and gene sequence. The morphological features were analyzed by LM and EM, whereas molecular analyses were based on the internal transcribed spacer (ITS) and large subunit (LSU) regions of the rDNA. P. plurisecta appears closely related to P. cuspidata/P. pseudodelicatissima in the phylogenetic tree, whereas P. abrensis forms a moderately supported clade with P. heimii/P. subpacifica and P. caciantha/P. circumpora. Comparison of the secondary structure of ITS2 regions reveals marked differences in the most highly conserved regions among related taxa. Morphologically, the new species differ from their closest congeners in the arrangement of the poroid sectors and the density of valve striae and fibulae. The two species share similar pigment composition, and belong to the group of Pseudo-nitzschia species containing only chlorophyll c2 and c3 .

Bloom forming and toxic phytoplankton in transitional and coastal waters of Cantabria region coast (Southeastern Bay of Biscay, Spain).

Phytoplankton monitoring has extended to practically all the regions of the European coast due to the implementation of the European Water Framework Directive. In this way, the study of phytoplankton taxonomic composition and dynamic is being performed in many areas poorly studied or not studied before. During the last years, a monitoring programme has been carried out at the coast of Cantabria region (SE Bay of Biscay); the presence of some potentially toxic and bloom forming species (>7.5 × 105 cells per litre) has been observed. Diatoms and cryptophytes are the main blooming taxa in this region in the majority of the estuaries and in some of the coastal sites. All estuaries and coastal stations showed at least one potentially toxic species, being the dinoflagellates the group with the highest number of taxa observed. The potentially toxic species found in highest concentrations were the genera Pseudo-nitzschia and Chrysochromulina.

Phytoplankton pigments and epifluorescence microscopy as tools for ecological status assessment in coastal and estuarine waters, within the Water Framework Directive.

Inverted microscopy is widespread employed for the analysis of phytoplankton composition within water quality monitoring networks. However, the analysis at the lowest taxonomical level is not always required for ecological status assessment. In addition, inverted microscopy can underestimate the small phytoplankton, and not always distinguish photoautotrophic from heterotrophic cells. In this study, as alternative tools, epifluorescence microscopy and High Performance Liquid Chromatography (HPLC) were employed to characterize phytoplankton communities within waters of different trophic condition. Epifluorescence microscopy confirmed its effectiveness to count the small phytoplankton. Furthermore, significant correlations between nutrients of anthropogenic origin and nanoplankton abundances were found. However, this technique resulted very time-consuming. HPLC together with the CHEMTAX program was more appropriate than inverted microscopy, in terms of cost-effectiveness. Also, the main variability patterns observed in the phytoplankton community structure by HPLC coincided with previous findings in the study area. Nevertheless, a rapid screening at the inverted microscope is recommended.