An On-Farm Workflow for Predictive Management of Paralytic Shellfish Toxin-Producing Harmful Algal Blooms for the Aquaculture Industry.

Paralytic shellfish toxins (PSTs) produced by marine dinoflagellates significantly impact shellfish industries worldwide. Early detection on-farm and with minimal training would allow additional time for management decisions to minimize economic losses. Here, we describe and test a standardized workflow based on the detection of sxtA4, an initial gene in the biosynthesis of PSTs. The workflow is simple and inexpensive and does not require a specialized laboratory. It consists of (1) water collection and filtration using a custom gravity sampler, (2) buffer selection for sample preservation and cell lysis for DNA, and (3) an assay based on a region of sxtA, DinoDtec lyophilized quantitative polymerase chain reaction (qPCR) assay. Water samples spiked with Alexandrium catenella showed a cell recovery of >90% when compared to light microscopy counts. The performance of the lysis method (90.3% efficient), Longmire's buffer, and the DinoDtec qPCR assay (tested across a range of Alexandrium species (90.7-106.9% efficiency; r2 > 0.99)) was found to be specific, sensitive, and efficient. We tested the application of this workflow weekly from May 2016 to 30th October 2017 to compare the relationship between sxtA4 copies L-1 in seawater and PSTs in mussel tissue (Mytilus galloprovincialis) on-farm and spatially (across multiple sites), effectively demonstrating an ∼2 week early warning of two A. catenella HABs (r = 0.95). Our tool provides an early, accurate, and efficient method for the identification of PST risk in shellfish aquaculture.

Emerging phylogeographic perspective on the toxigenic diatom genus Pseudo-nitzschia in coastal northern European waters and gateways to eastern Arctic seas: Causes, ecological consequences and socio-economic impacts.

The diatom Pseudo-nitzschia H. Peragallo is perhaps the most intensively researched genus of marine pennate diatoms, with respect to species diversity, life history strategies, toxigenicity, and biogeographical distribution. The global magnitude and consequences of harmful algal blooms (HABs) of Pseudo-nitzschia are particularly significant because of the high socioeconomic impacts and environmental and human health risks associated with the production of the neurotoxin domoic acid (DA) among populations of many (although not all) species. This has led to enhanced monitoring and mitigation strategies for toxigenic Pseudo-nitzschia blooms and their toxins in recent years. Nevertheless, human adaptive actions based on future scenarios of bloom dynamics and proposed shifts in biogeographical distribution under climate-change regimes have not been implemented on a regional scale. In the CoCliME (Co-development of climate services for adaptation to changing marine ecosystems) program these issues were addressed with respect to past, current and anticipated future status of key HAB genera such as Pseudo-nitzschia and expected benefits of enhanced monitoring. Data on the distribution and frequency of Pseudo-nitzschia blooms in relation to DA occurrence and associated amnesic shellfish toxin (AST) events were evaluated in a contemporary and historical context over the past several decades from key northern CoCliME Case Study areas. The regional studies comprised the greater North Sea and adjacent Kattegat-Skagerrak and Norwegian Sea, eastern North Atlantic marginal seas and Arctic gateways, and the Baltic Sea. The first evidence of possible biogeographical expansion of Pseudo-nitzschia taxa into frontier eastern Arctic gateways was provided from DNA barcoding signatures. Key climate change indicators, such as salinity, temperature, and water-column stratification were identified as drivers of upwelling and advection related to the distribution of regional Pseudo-nitzschia blooms. The possible influence of changing variables on bloom dynamics, magnitude, frequency and spatial and temporal distribution were interpreted in the context of regional ocean climate models. These climate change indicators may play key roles in selecting for the occurrence and diversity of Pseudo-nitzschia species within the broader microeukaryote communities. Shifts to higher temperature and lower salinity regimes predicted for the southern North Sea indicate the potential for high-magnitude Pseudo-nitzschia blooms, currently absent from this area. Ecological and socioeconomic impacts of Pseudo-nitzschia blooms are evaluated with reference to effects on fisheries and mariculture resources and coastal ecosystem function. Where feasible, effective adaptation strategies are proposed herein as emerging climate services for the northern CoCLiME region.

Apparent biogeographical trends in Alexandrium blooms for northern Europe: identifying links to climate change and effective adaptive actions.

The marine dinoflagellate Alexandrium Halim represents perhaps the most significant and intensively studied genus with respect to species diversity, life history strategies, toxigenicity, biogeographical distribution, and global magnitude and consequences harmful algal blooms (HABs). The socioeconomic impacts, environmental and human health risks, and mitigation strategies for toxigenic Alexandrium blooms have also been explored in recent years. Human adaptive actions based on future scenarios of bloom dynamics and shifts in biogeographical distribution under climate-change parameters remain under development and not yet implemented on a regional scale. In the CoCliME (Co-development of climate services for adaptation to changing marine ecosystems) project these issues were addressed with respect to past, current and anticipated future status of key HAB genera and expected benefits of enhanced monitoring. Data on the distribution and frequency of Alexandrium blooms related to paralytic shellfish toxin (PST) events from key CoCliME Case Study areas, comprising the North Sea and adjacent Kattegat-Skagerrak, Norwegian Sea, and Baltic Sea, and eastern North Atlantic marginal seas, were evaluated in a contemporary and historical context over the past several decades. The first evidence of possible biogeographical expansion of Alexandrium taxa into eastern Arctic gateways was provided from DNA barcoding signatures. Various key climate change indicators, such as salinity, temperature, and water-column stratification, relevant to Alexandrium bloom initiation and development were identified. The possible influence of changing variables on bloom dynamics, magnitude, frequency and spatial and temporal distribution were interpreted in the context of regional ocean climate models. These climate change impact indicators may play key roles in selecting for the occurrence and diversity of Alexandrium species within the broader microeukaryote communities. For example, shifts to higher temperature and lower salinity regimes predicted for the southern North Sea indicate the potential for increased Alexandrium blooms, currently absent from this area. Ecological and socioeconomic impacts of Alexandrium blooms and effects on fisheries and aquaculture resources and coastal ecosystem function are evaluated, and, where feasible, effective adaptation strategies are proposed herein as emerging climate services.

Temporal and spatial distribution of epibenthic dinoflagellates in the Kattegat-Skagerrak, NE Atlantic-Focus on Prorocentrum lima and Coolia monotis.

Epibenthic dinoflagellates occur globally and include many toxin-producing species of concern to human health and benthic ecosystem function. Such benthic harmful algal blooms (BHABs) have been well described from tropical and sub-tropical coastal environments, but assessments from north temperate waters, e.g., northern Europe, and polar regions are scarce. The present study addressed the biodiversity and distribution of potentially toxic epibenthic dinoflagellate populations along the west coast of Sweden (Kattegat-Skagerrak) by morphological and molecular criteria. Morphological analysis conducted by light- and electron-microscopy was then linked by DNA barcoding of the V4 region of 18S rRNA gene sequences to interpret taxonomic and phylogenetic relationships. The presence of two potentially toxigenic epibenthic dinoflagellates, Prorocentrum lima (Ehrenberg) F.Stein and Coolia monotis Meunier was confirmed, along with a description of their spatial and temporal distribution. For P. lima, one third of the cell abundance values exceeded official alarm thresholds for potentially toxic BHAB events (>1000 cells gr-1 of macroalgae fresh weight). The same species were recorded consecutively for two summers, but without significant temporal variation in cell densities. SEM analyses confirmed the presence of other benthic Prorocentrum species: P. fukuyoi complex, P. cf. foraminosum and P. cf. hoffmannianum. Analyses of the V4 region of the 18S rRNA gene also indicated the presence P. compressum, P. hoffmannianum, P. foraminosum, P. fukuyoi, and P. nanum. These findings provide the first biogeographical evidence of toxigenic benthic dinoflagellates along the west coast of Sweden, in the absence of ongoing monitoring to include epibenthic dinoflagellates. Harmful events due to the presence of Coolia at shellfish aquaculture sites along the Kattegat-Skagerrak are likely to be rather marginal because C. monotis is not known to be toxigenic. In any case, as a preliminary assessment, the results highlight the risk of diarrhetic shellfish poisoning (DSP) events caused by P. lima, which may affect the development and sustainability of shellfish aquaculture in the region.

Spatial and biological oceanographic insights into the massive fish-killing bloom of the haptophyte Chrysochromulina leadbeateri in northern Norway.

A bloom of the fish-killing haptophyte Chrysochromulina leadbeateri in northern Norway during May and June 2019 was the most harmful algal event ever recorded in the region, causing massive mortalities of farmed salmon. Accordingly, oceanographic and biodiversity aspects of the bloom were studied in unprecedented detail, based on metabarcoding and physico-chemical and biotic factors related with the dynamics and distribution of the bloom. Light- and electron-microscopical observations of nanoplankton samples from diverse locations confirmed that C. leadbeateri was dominant in the bloom and the primary cause of associated fish mortalities. Cell counts by light microscopy and flow cytometry were obtained throughout the regional bloom within and adjacent to five fjord systems. Metabarcoding sequences of the V4 region of the 18S rRNA gene from field material collected during the bloom and a cultured isolate from offshore of Tromsøy island confirmed the species identification. Sequences from three genetic markers (18S, 28S rRNA gene and ITS region) verified the close if not identical genetic similarity to C. leadbeateri from a previous massive fish-killing bloom in 1991 in northern Norway. The distribution and cell abundance of C. leadbeateri and related Chrysochromulina species in the recent incident were tracked by integrating observations from metabarcoding sequences of the V4 region of the 18S rRNA gene. Metabarcoding revealed at least 14 distinct Chrysochromulina variants, including putative cryptic species. C. leadbeateri was by far the most abundant of these species, but with high intraspecific genetic variability. Highest cell abundance of up to 2.7 × 107 cells L - 1 of C. leadbeateri was found in Balsfjorden; the high cell densities were associated with stratification near the pycnocline (at ca. 12 m depth) within the fjord. The cell abundance of C. leadbeateri showed positive correlations with temperature, negative correlation with salinity, and a slightly positive correlation with ambient phosphate and nitrate concentrations. The spatio-temporal succession of the C. leadbeateri bloom suggests independent initiation from existing pre-bloom populations in local zones, perhaps sustained and supplemented over time by northeastward advection of the bloom from the fjords.

Retention of Prey Genetic Material by the Kleptoplastidic Ciliate Strombidium cf. basimorphum.

Many marine ciliate species retain functional chloroplasts from their photosynthetic prey. In some species, the functionality of the acquired plastids is connected to the simultaneous retention of prey nuclei. To date, this has never been documented in plastidic Strombidium species. The functionality of the sequestered chloroplasts in Strombidium species is thought to be independent from any nuclear control and only maintained via frequent replacement of chloroplasts from newly ingested prey. Chloroplasts sequestered from the cryptophyte prey Teleaulax amphioxeia have been shown to keep their functionality for several days in the ciliate Strombidium cf. basimorphum. To investigate the potential retention of prey genetic material in this ciliate, we applied a molecular marker specific for this cryptophyte prey. Here, we demonstrate that the genetic material from prey nuclei, nucleomorphs, and ribosomes is detectable inside the ciliate for at least 5 days after prey ingestion. Moreover, single-cell transcriptomics revealed the presence of transcripts of prey nuclear origin in the ciliate after 4 days of prey starvation. These new findings might lead to the reconsideration of the mechanisms regulating chloroplasts retention in Strombidium ciliates. The development and application of molecular tools appear promising to improve our understanding on chloroplasts retention in planktonic protists.

Harmful algal blooms and their effects in coastal seas of Northern Europe.

Harmful algal blooms (HAB) are recurrent phenomena in northern Europe along the coasts of the Baltic Sea, Kattegat-Skagerrak, eastern North Sea, Norwegian Sea and the Barents Sea. These HABs have caused occasional massive losses for the aquaculture industry and have chronically affected socioeconomic interests in several ways. This status review gives an overview of historical HAB events and summarises reports to the Harmful Algae Event Database from 1986 to the end of year 2019 and observations made in long term monitoring programmes of potentially harmful phytoplankton and of phycotoxins in bivalve shellfish. Major HAB taxa causing fish mortalities in the region include blooms of the prymnesiophyte Chrysochromulina leadbeateri in northern Norway in 1991 and 2019, resulting in huge economic losses for fish farmers. A bloom of the prymesiophyte Prymnesium polylepis (syn. Chrysochromulina polylepis) in the Kattegat-Skagerrak in 1988 was ecosystem disruptive. Blooms of the prymnesiophyte Phaeocystis spp. have caused accumulations of foam on beaches in the southwestern North Sea and Wadden Sea coasts and shellfish mortality has been linked to their occurrence. Mortality of shellfish linked to HAB events has been observed in estuarine waters associated with influx of water from the southern North Sea. The first bloom of the dictyochophyte genus Pseudochattonella was observed in 1998, and since then such blooms have been observed in high cell densities in spring causing fish mortalities some years. Dinoflagellates, primarily Dinophysis spp., intermittently yield concentrations of Diarrhetic Shellfish Toxins (DST) in blue mussels, Mytilus edulis, above regulatory limits along the coasts of Norway, Denmark and the Swedish west coast. On average, DST levels in shellfish have decreased along the Swedish and Norwegian Skagerrak coasts since approximately 2006, coinciding with a decrease in the cell abundance of D. acuta. Among dinoflagellates, Alexandrium species are the major source of Paralytic Shellfish Toxins (PST) in the region. PST concentrations above regulatory levels were rare in the Skagerrak-Kattegat during the three decadal review period, but frequent and often abundant findings of Alexandrium resting cysts in surface sediments indicate a high potential risk for blooms. PST levels often above regulatory limits along the west coast of Norway are associated with A. catenella (ribotype Group 1) as the main toxin producer. Other Alexandrium species, such as A. ostenfeldii and A. minutum, are capable of producing PST among some populations but are usually not associated with PSP events in the region. The cell abundance of A. pseudogonyaulax, a producer of the ichthyotoxin goniodomin (GD), has increased in the Skagerrak-Kattegat since 2010, and may constitute an emerging threat. The dinoflagellate Azadinium spp. have been unequivocally linked to the presence of azaspiracid toxins (AZT) responsible for Azaspiracid Shellfish Poisoning (AZP) in northern Europe. These toxins were detected in bivalve shellfish at concentrations above regulatory limits for the first time in Norway in blue mussels in 2005 and in Sweden in blue mussels and oysters (Ostrea edulis and Crassostrea gigas) in 2018. Certain members of the diatom genus Pseudo-nitzschia produce the neurotoxin domoic acid and analogs known as Amnesic Shellfish Toxins (AST). Blooms of Pseudo-nitzschia were common in the North Sea and the Skagerrak-Kattegat, but levels of AST in bivalve shellfish were rarely above regulatory limits during the review period. Summer cyanobacteria blooms in the Baltic Sea are a concern mainly for tourism by causing massive fouling of bathing water and beaches. Some of the cyanobacteria produce toxins, e.g. Nodularia spumigena, producer of nodularin, which may be a human health problem and cause occasional dog mortalities. Coastal and shelf sea regions in northern Europe provide a key supply of seafood, socioeconomic well-being and ecosystem services. Increasing anthropogenic influence and climate change create environmental stressors causing shifts in the biogeography and intensity of HABs. Continued monitoring of HAB and phycotoxins and the operation of historical databases such as HAEDAT provide not only an ongoing status report but also provide a way to interpret causes and mechanisms of HABs.

A dataset on trophic modes of aquatic protists.

BACKGROUND: An important functional trait of organisms is their trophic mode. It determines their position within food webs, as well as their function within an ecosystem. For the better part of the 20th century, aquatic protist communities were thought to consist mainly of producers (phytoplankton) and consumers (protozooplankton). Phytoplankton cover their energy requirements through photosynthesis (phototrophy), while protozooplankton graze on prey and organic particles (phagotrophy). However, over the past decades, it was shown that another trophic group (mixoplankton) comprise a notable part of aquatic protist communities. Mixoplankton employ a third trophic mode by combining phototrophy and phagotrophy (mixotrophy). Due to the historical dichotomy, it is not straightforward to gain adequate and correct information on the trophic mode of aquatic protists. Long hours of literature research or expert knowledge are needed to correctly assign trophic modes. Additionally, aquatic protists also have a long history of undergoing taxonomic changes which make it difficult to compare past and present literature. While WoRMS, the World Register of Marine Species, keeps track of the taxonomic changes and assigns each species a unique AphiaID that can be linked to its various historic and present taxonomic hierarchy, there is currently no machine-readable database to query aquatic protists for their trophic modes. NEW INFORMATION: This paper describes a dataset that was submitted to WoRMS and links aquatic protist taxa, with a focus on marine taxa, to their AphiaID and their trophic mode. The bulk of the data used for this dataset stems from (routine) monitoring stations in the North Sea and the Baltic Sea. The data were augmented and checked against state-of-the-art knowledge on mixoplankton taxa by consulting literature and experts. Thus, this dataset provides a first attempt to make the trophic mode of aquatic protists easily accessible in both a human- and machine-readable format.

Adaptive divergence across Southern Ocean gradients in the pelagic diatom Fragilariopsis kerguelensis.

The Southern Ocean is characterized by longitudinal water circulations crossed by strong latitudinal gradients. How this oceanographic background shapes planktonic populations is largely unknown, despite the significance of this region for global biogeochemical cycles. Here, we show, based on genomic, morphometric, ecophysiological and mating compatibility data, an example of ecotypic differentiation and speciation within an endemic pelagic inhabitant, the diatom Fragilariopsis kerguelensis. We discovered three genotypic variants, one present throughout the latitudinal transect sampled, the others restricted to the north and south, respectively. The latter two showed reciprocal monophyly across all three genomes and significant ecophysiological differences consistent with local adaptation, but produced viable offspring in laboratory crosses. The third group was also reproductively isolated from the latter two. We hypothesize that this pattern originated by an adaptive expansion accompanied by ecotypic divergence, followed by sympatric speciation.

Ocean acidification increases domoic acid contents during a spring to summer succession of coastal phytoplankton.

Enrichment of the oceans with CO2 may be beneficial for some marine phytoplankton, including harmful algae. Numerous laboratory experiments provided valuable insights into the effects of elevated pCO2 on the growth and physiology of harmful algal species, including the production of phycotoxins. Experiments close to natural conditions are the next step to improve predictions, as they consider the complex interplay between biotic and abiotic factors that can confound the direct effects of ocean acidification. We therefore investigated the effect of ocean acidification on the occurrence and abundance of phycotoxins in bulk plankton samples during a long-term mesocosm experiment in the Gullmar Fjord, Sweden, an area frequently experiencing harmful algal blooms. During the experimental period, a total of seven phycotoxin-producing harmful algal genera were identified in the fjord, and in accordance, six toxin classes were detected. However, within the mesocosms, only domoic acid and the corresponding producer Pseudo-nitzschia spp. was observed. Despite high variation within treatments, significantly higher particulate domoic acid contents were measured in the mesocosms with elevated pCO2. Higher particulate domoic acid contents were additionally associated with macronutrient limitation. The risks associated with potentially higher phycotoxin levels in the future ocean warrants attention and should be considered in prospective monitoring strategies for coastal marine waters.

Occurrence and distribution of Amphidomataceae (Dinophyceae) in Danish coastal waters of the North Sea, the Limfjord and the Kattegat/Belt area.

Some species of the dinophytes Azadinium and Amphidoma (Amphidomataceae) produce azaspiracids (AZA), lipophilic polyether compounds responsible for Azaspiracid Shellfish Poisoning (AZP) in humans after consumption of contaminated seafood. Toxigenic Amphidomataceae are known to occur in the North Atlantic and the North Sea area, but little is known about their importance in Danish coastal waters. In 2016, 44 Stations were sampled on a survey along the Danish coastline, covering the German Bight, Limfjord, the Kattegat area, Great Belt and Kiel Bight. Samples were analysed by live microscopy, liquid chromatography-tandem mass spectrometry (LC-MS/MS) and quantitative polymerase-chain-reaction (qPCR) on the presence of Amphidomataceae and AZA. Amphidomataceae were widely distributed in the area, but were below detection limit on most of the inner Limfjord stations. Cell abundances of the three toxigenic species, determined with species-specific qPCR assays on Azadinium spinosum, Az. poporum and Amphidoma languida, were generally low and restricted to the North Sea and the northern Kattegat, which was in agreement with the distribution of the generally low AZA abundances in plankton samples. Among the toxigenic species, Amphidoma languida was dominant with highest cell densities up to 3 × 103 cells L-1 on North Sea stations and at the western entrance of the Limfjord. Azaspiracids detected in plankton samples include low levels of AZA-1 at one station of the North Sea, and higher levels of AZA-38 and -39 (up to 1.5 ng L-1) in the North Sea and the Limfjord entrance. Furthermore, one new AZA (named AZA-63) was discovered in plankton of two North Sea stations. Morphological, molecular, and toxinological characterisation of 26 newly established strains from the area confirmed the presence of four amphidomatacean species (Az. obesum, Az. dalianense, Az. poporum and Am. languida). The single new strain of Az. poporum turned out as a member of Ribotype A2, which was previously only known from the Mediterranean. Consistent with some of these Mediterranean A2 strains, but different to the previously established AZA-37 producing Az. poporum Ribotype A1 strains from Denmark, the new strain did not contain any AZA. Azaspiracids were also absent in all Az. obesum and Az. dalianense strains, but AZA-38 and -39 were found in all Am. languida strains with total AZA cell quotas ranging from 0.08 up to 94 fg cell-1. In conclusion, AZA-producing microalgae and their respective toxins were low in abundance but widely present in the area, and thus might be considered in local monitoring programs to preserve seafood safety in Danish coastal waters.

Detection of mercury in the 411-year-old beard hairs of the astronomer Tycho Brahe by elemental analysis in electron microscopy.

Hairs more than 400 years old of the famous astronomer Tycho Brahe were studied by electron microscopy to evaluate the hypothesis that Johannes Kepler murdered his teacher Brahe by mercury intoxication. The beard hairs showed a well-preserved ultrastructure with typical hair scales and melanosomes. The authors detected an accumulation of electron-dense granules of about 10 nm inside the outer hair scales, but not in the hair shaft and roots. At the places of these heavy-metal-containing granules they detected mercury besides other elements by energy dispersive X-ray analysis (EDX, Oxford, UK) in a field cathode scanning electron microscope (SEM, Gemini, Zeiss). The mercury-containing granules were found over the whole length of hairs, but only in the outer hair scales. Nevertheless, surface coatings of hairs were free of mercury. This distribution of mercury does not support the murder hypothesis, but could be related to precipitation of mercury dust from the air during long-term alchemistic activities.

(R)-(+)-palasonin, a cantharidin-related plant toxin, also occurs in insect hemolymph and tissues.

Gas chromatographic and mass spectroscopic analyses of extracts of cantharidin-containing meloid, clerid, and staphylinid beetles revealed the presence of minor to significant amounts of palasonin, previously only known from seeds and fruits of the Indian shrub Buteafrondosa (Leguminaceae). Unlike (S)-(-)-palasonin (> 99% ee) from B. frondosa, the insects produce palasonin of low ee with the (R)-(+)-enantiomer (0-50% ee) prevailing. The ee of palasonin from individual specimens of predatory insects (Trichodes apiarius), which acquire their chemical protection from cantharidin-producing insects, may vary considerably. The absolute configuration of (S)-(-)-palasonin, previously deduced from indirect chemical and spectroscopic methods, was confirmed by X-ray crystal structure analysis of a cyclic imide derived from (S)-(-)-palasonin and (S)-(-)- 1 -(4-nitrophenyl)-ethylamine.