Protist Diversity and Seasonal Dynamics in Skagerrak Plankton Communities as Revealed by Metabarcoding and Microscopy.

Protist community composition and seasonal dynamics are of major importance for the production of higher trophic levels, such as zooplankton and fish. Our aim was to reveal how the protist community in the Skagerrak changes through the seasons by combining high-throughput sequencing and microscopy of plankton collected monthly over two years. The V4 region of the 18S rRNA gene was amplified by eukaryote universal primers from the total RNA/cDNA. We found a strong seasonal variation in protist composition and proportional abundances, and a difference between two depths within the euphotic zone. Highest protist richness was found in late summer-early autumn, and lowest in winter. Temperature was the abiotic factor explaining most of the variation in diversity. Dinoflagellates was the most abundant and diverse group followed by ciliates and diatoms. We found about 70 new taxa recorded for the first time in the Skagerrak. The seasonal pattern in relative read abundance of major phytoplankton groups was well in accordance with microscopical biovolumes. This is the first metabarcoding study of the protist plankton community of all taxonomic groups and through seasons in the Skagerrak, which may serve as a baseline for future surveys to reveal effects of climate and environmental changes.

Cylindrospermopsin- and Deoxycylindrospermopsin-Producing Raphidiopsis raciborskii and Microcystin-Producing Microcystis spp. in Meiktila Lake, Myanmar.

Meiktila Lake is a shallow reservoir located close to Meiktila city in central Myanmar. Its water is used for irrigation, domestic purposes and drinking water. No detailed study of the presence of cyanobacteria and their potential toxin production has been conducted so far. To ascertain the cyanobacterial composition and presence of cyanobacterial toxins in Meiktila Lake, water samples were collected in March and November 2017 and investigated for physico-chemical and biological parameters. Phytoplankton composition and biomass determination revealed that most of the samples were dominated by the cyanobacterium Raphidiopsis raciborskii. In a polyphasic approach, seven isolated cyanobacterial strains were classified morphologically and phylogenetically as R. raciborskii, and Microcystis spp. and tested for microcystins (MCs), cylindrospermopsins (CYNs), saxitoxins and anatoxins by enzyme-linked immunosorbent assay (ELISA) and liquid chromatography-mass spectrometry (LC-MS). ELISA and LC-MS analyses confirmed CYNs in three of the five Raphidiopsis strains between 1.8 and 9.8 µg mg-1 fresh weight. Both Microcystis strains produced MCs, one strain 52 congeners and the other strain 20 congeners, including 22 previously unreported variants. Due to the presence of CYN- and MC-producing cyanobacteria, harmful effects on humans, domestic and wild animals cannot be excluded in Meiktila Lake.

Heteroxanthin as a pigment biomarker for Gonyostomum semen (Raphidophyceae).

The ability to identify drivers responsible for algal community shifts is an important aspect of environmental issues. The lack of long-term datasets, covering periods prior to these shifts, is often limiting our understanding of drivers responsible. The freshwater alga, Gonyostomum semen (Raphidophyceae), has significantly increased distribution and mass occurrences in Scandinavian lakes during the past few decades, often releasing a skin irritating slime that causes discomfort for swimmers. While the alga has been extensively studied, long-term data from individual lakes are often absent or greatly limited and drivers behind this species' success are still not clear. However, if specific and persistent taxa biomarkers for G. semen could be detected in dated sediment cores, long-term data would be improved and more useful. To test for biomarkers, we examined the pigment composition of several G. semen strains in culture. Further, dated sediment core samples from Lake Lundebyvann, Norway, were used to test the pigments' suitability as biomarkers in paleolimnological studies. Modifications to a common analysis allowed for the successful detection of the polar xanthophyll heteroxanthin and the non-polar chlorophyll a, as well as several other algal pigments by using high performance liquid chromatography-photometric diode arrays (HPLC-PDA). Heteroxanthin was confirmed by liquid chromatography-mass spectrometry (LC-MS) and detected by HPLC-PDA in all examined G. semen strains, along with chlorophyll a. Using HPLC-PDA, we also identified and confirmed the presence of the biomarker, xanthophyll heteroxanthin, in sediment core samples up to 60 years of age. The specificity of this xanthophyll was also tested by examining a wide range of algal strains from common Norwegian phytoplankton species. Heteroxanthin was not detected in any species commonly occurring in significant amounts in Norwegian lakes. We therefore conclude that heteroxanthin is a suitable pigment biomarker for G. semen and that this pigment can be successfully used for paleolimnological studies.

Variability in the sxt Gene Clusters of PSP Toxin Producing Aphanizomenon gracile Strains from Norway, Spain, Germany and North America.

Paralytic shellfish poisoning (PSP) toxin production has been detected worldwide in the cyanobacterial genera Anabaena, Lyngbya, Scytonema, Cuspidothrix and Aphanizomenon. In Europe Aphanizomenon gracile and Cuspidothrix issatschenkoi are the only known producers of PSP toxins and are found in Southwest and Central European freshwater bodies. In this study the PSP toxin producing Aphanizomenon sp. strain NIVA-CYA 851 was isolated from the Norwegian Lake Hillestadvannet. In a polyphasic approach NIVA-CYA 851 was morphologically and phylogenetically classified, and investigated for toxin production. The strain NIVA-CYA 851 was identified as A. gracile using 16S rRNA gene phylogeny and was confirmed to produce neosaxitoxin, saxitoxin and gonyautoxin 5 by LC-MS. The whole sxt gene clusters (circa 27.3 kb) of four A. gracile strains: NIVA-CYA 851 (Norway); NIVA-CYA 655 & NIVA-CYA 676 (Germany); and UAM 529 (Spain), all from latitudes between 40° and 59° North were sequenced and compared with the sxt gene cluster of reference strain A. gracile NH-5 from the USA. All five sxt gene clusters are highly conserved with similarities exceeding 99.4%, but they differ slightly in the number and presence of single nucleotide polymorphisms (SNPs) and insertions/deletions (In/Dels). Altogether 178 variable sites (44 SNPs and 4 In/Dels, comprising 134 nucleotides) were found in the sxt gene clusters of the Norwegian, German and Spanish strains compared to the reference strain. Thirty-nine SNPs were located in 16 of the 27 coding regions. The sxt gene clusters of NIVA-CYA 851, NIVA-CYA 655, NIVA-CYA 676 and UAM 529, were characterized by 15, 16, 19 and 23 SNPs respectively. Only the Norwegian strain NIVA-CYA 851 possessed an insertion of 126 base pairs (bp) in the noncoding area between the sxtA and sxtE genes and a deletion of 6 nucleotides in the sxtN gene. The sxtI gene showed the highest variability and is recommended as the best genetic marker for further phylogenetic studies of the sxt gene cluster of A. gracile. This study confirms for the first time the role of A. gracile as a PSP toxin producer in Norwegian waters, representing the northernmost occurrence of PSP toxin producing A. gracile in Europe known so far.

Seasonal dynamics of harmful algae in outer Oslofjorden monitored by microarray, qPCR, and microscopy.

Monitoring of marine microalgae is important to predict and manage harmful algal blooms. Microarray Detection of Toxic ALgae (MIDTAL) is an FP7-funded EU project aiming to establish a multi-species microarray as a tool to aid monitoring agencies. We tested the suitability of different prototype versions of the MIDTAL microarray for the monthly monitoring of a sampling station in outer Oslofjorden during a 1-year period. Microarray data from two different versions of the MIDTAL chip were compared to results from cell counts (several species) and quantitative real-time PCR (qPCR; only Pseudochattonella spp.). While results from generation 2.5 microarrays exhibited a high number of false positive signals, generation 3.3 microarray data generally correlated with microscopy and qPCR data, with three important limitations: (1) Pseudo-nitzschia cells were not reliably detected, possibly because cells were not sufficiently retained during filtration or lysed during the extraction, and because of low sensitivity of the probes; (2) in the case of samples with high concentrations of non-target species, the sensitivity of the arrays was decreased; (3) one occurrence of Alexandrium pseudogonyaulax was not detected due to a 1-bp mismatch with the genus probe represented on the microarray. In spite of these shortcomings our data demonstrate the overall progress made and the potential of the MIDTAL array. The case of Pseudochattonella - where two morphologically similar species impossible to separate by light microscopy were distinguished - in particular, underlines the added value of molecular methods such as microarrays in routine phytoplankton monitoring.