Cytotoxicity of Prymnesium parvum extracts and prymnesin analogs on epithelial fish gill cells RTgill-W1 and the human colon cell line HCEC-1CT.

Harmful algal blooms kill fish populations worldwide, as exemplified by the haptophyte microalga Prymnesium parvum. The suspected causative agents are prymnesins, categorized as A-, B-, and C-types based on backbone carbon atoms. Impacts of P. parvum extracts and purified prymnesins were tested on the epithelial rainbow trout fish gill cell line RTgill-W1 and on the human colon epithelial cells HCEC-1CT. Cytotoxic potencies ranked A > C > B-type with concentrations spanning from low (A- and C-type) to middle (B-type) nM ranges. Although RTgill-W1 cells were about twofold more sensitive than HCEC-1CT, the cytotoxicity of prymnesins is not limited to fish gills. Both cell lines responded rapidly to prymnesins; with EC50 values for B-types in RTgill-W1 cells of 110 ± 11 nM and 41.5 ± 0.6 nM after incubations times of 3 and 24 h. Results of fluorescence imaging and measured lytic effects suggest plasma membrane interactions. Postulating an osmotic imbalance as mechanisms of toxicity, incubations with prymnesins in media lacking either Cl-, Na+, or Ca2+ were performed. Cl- removal reduced morphometric rearrangements observed in RTgill-W1 and cytotoxicity in HCEC-1CT cells. Ca2+-free medium in RTgill-W1 cells exacerbated effects on the cell nuclei. Prymnesin composition of different P. parvum strains showed that analog composition within one type scarcely influenced the cytotoxic potential, while analog type potentially dictate potency. Overall, A-type prymnesins were the most potent ones in both cell lines followed by the C-types, and lastly B-types. Disturbance of Ca2+ and Cl- ionoregulation may be integral to prymnesin toxicity.

Impacts of inorganic nutrients on the physiology of a mixoplanktonic ciliate and its cryptophyte prey.

Many marine planktonic ciliates retain functional chloroplasts from their photosynthetic prey and use them to incorporate inorganic carbon via photosynthesis. While this strategy provides the ciliates with carbon, little is known about their ability to incorporate major dissolved inorganic nutrients, such as nitrogen and phosphorus. Here, we studied how ciliates respond to different concentrations of dissolved inorganic nitrogen and phosphorus. Specifically, we tested the direct and indirect effects of nutrient availability on the ciliate Strombidium cf. basimorphum fed the cryptophyte prey Teleaulax amphioxeia. We assessed responses in the rates of growth, ingestion, photosynthesis, inorganic nutrient uptake, and excretion. Our results show that the prey changed its carbon content depending on the nutrient concentrations. Low inorganic nutrient concentrations increased S. cf. basimorphum growth and prey ingestion. The higher carbon content of the prey under these low nutrient conditions likely supported the growth of the ciliate, while the higher carbon:nutrient stoichiometry of the prey led to the higher ingestion rates. The low carbon content of the prey at high nutrient concentrations resulted in reduced growth of S. cf. basimorphum, which indicates that carbon acquired via photosynthesis in the ciliate cannot compensate for the ingestion of prey with low carbon content. In conclusion, our findings show S. cf. basimorphum is not able to utilize dissolved inorganic nitrogen and phosphorus for growth, and this species seems to be well adapted to exploit its prey when grown at low nutrient conditions.

Karmitoxin: An Amine-Containing Polyhydroxy-Polyene Toxin from the Marine Dinoflagellate Karlodinium armiger.

Marine algae from the genus Karlodinium are known to be involved in fish-killing events worldwide. Here we report for the first time the chemistry and bioactivity of a natural product from the newly described mixotrophic dinoflagellate Karlodinium armiger. Our work describes the isolation and structural characterization of a new polyhydroxy-polyene named karmitoxin. The structure elucidation work was facilitated by use of 13C enrichment and high-field 2D NMR spectroscopy, where 1H-13C long-range correlations turned out to be very informative. Karmitoxin is structurally related to amphidinols and karlotoxins; however it differs by containing the longest carbon-carbon backbone discovered for this class of compounds, as well as a primary amino group. Karmitoxin showed potent nanomolar cytotoxic activity in an RTgill-W1 cell assay as well as rapid immobilization and eventual mortality of the copepod Acartia tonsa, a natural grazer of K. armiger.

HPLC-HRMS Quantification of the Ichthyotoxin Karmitoxin from Karlodinium armiger.

Being able to quantify ichthyotoxic metabolites from microalgae allows for the determination of ecologically-relevant concentrations that can be simulated in laboratory experiments, as well as to investigate bioaccumulation and degradation. Here, the ichthyotoxin karmitoxin, produced by Karlodinium armiger, was quantified in laboratory-grown cultures using high-performance liquid chromatography (HPLC) coupled to electrospray ionisation high-resolution time-of-flight mass spectrometry (HRMS). Prior to the quantification of karmitoxin, a standard of karmitoxin was purified from K. armiger cultures (80 L). The standard was quantified by fluorescent derivatisation using Waters AccQ-Fluor reagent and derivatised fumonisin B1 and fumonisin B2 as standards, as each contain a primary amine. Various sample preparation methods for whole culture samples were assessed, including six different solid phase extraction substrates. During analysis of culture samples, MS source conditions were monitored with chloramphenicol and valinomycin as external standards over prolonged injection sequences (>12 h) and karmitoxin concentrations were determined using the response factor of a closely eluting iturin A2 internal standard. Using this method the limit of quantification was 0.11 µg·mL-1, and the limit of detection was found to be 0.03 µg·mL-1. Matrix effects were determined with the use of K. armiger cultures grown with 13C-labelled bicarbonate as the primary carbon source.

Dynamic Cluster Analysis: An Unbiased Method for Identifying A + 2 Element Containing Compounds in Liquid Chromatographic High-Resolution Time-of-Flight Mass Spectrometric Data.

Dynamic cluster analysis (DCA) is an automated, unbiased technique which can identify Cl, Br, S, and other A + 2 element containing metabolites in liquid chromatographic high-resolution mass spectrometric data. DCA is based on three features, primarily the previously unutilized A + 1 to A + 2 isotope cluster spacing which is a strong classifier in itself but improved with the addition of the monoisotopic mass, and the well-known A:A+2 intensity ratio. Utilizing only the A + 1 to A + 2 isotope cluster spacing and the monoisotopic mass it was possible to filter a chromatogram for metabolites which contain Cl, Br, and S. Screening simulated isotope patterns of the Antibase Natural Products Database it was determined that the A + 1 to A + 2 isotope cluster spacing can be used to correctly classify 97.4% of molecular formulas containing these elements, only misclassifying a few metabolites which were either over 2800 u or metabolites which contained other A + 2 elements, such as Cu, Ni, Mg, and Zn. It was determined that with an interisotopic mass accuracy of 1 ppm, in a fully automated process, using all three parameters, it is possible to specifically filter a chromatogram for S containing metabolites with monoisotopic masses less than 825 u. Furthermore, it was possible to specifically filter a chromatogram for Cl and Br containing metabolites with monoisotopic masses less than 1613 u. Here DCA is applied on (i) simulated isotope patterns of the Antibase natural products databases, (ii) LC-QTOF data of reference standards, and (iii) LC-QTOF data of crude extracts of 10 strains of laboratory grown cultures of the microalga Prymnesium parvum where it identified known metabolites of the prymnesin series as well as over 20 previously undescribed prymnesin-like molecular features.

Chemodiversity of Ladder-Frame Prymnesin Polyethers in Prymnesium parvum.

Blooms of the microalga Prymnesium parvum cause devastating fish kills worldwide, which are suspected to be caused by the supersized ladder-frame polyether toxins prymnesin-1 and -2. These toxins have, however, only been detected from P. parvum in rare cases since they were originally described two decades ago. Here, we report the isolation and characterization of a novel B-type prymnesin, based on extensive analysis of 2D- and 3D-NMR data of natural as well as 90% (13)C enriched material. B-type prymnesins lack a complete 1,6-dioxadecalin core unit, which is replaced by a short acyclic C2 linkage compared to the structure of the original prymnesins. Comparison of the bioactivity of prymnesin-2 with prymnesin-B1 in an RTgill-W1 cell line assay identified both compounds as toxic in the low nanomolar range. Chemical investigations by liquid chromatography high-resolution mass spectrometry (LC-HRMS) of 10 strains of P. parvum collected worldwide showed that only one strain produced the original prymnesin-1 and -2, whereas four strains produced the novel B-type prymnesin. In total 13 further prymnesin analogues differing in their core backbone and chlorination and glycosylation patterns could be tentatively detected by LC-MS/HRMS, including a likely C-type prymnesin in five strains. Altogether, our work indicates that evolution of prymnesins has yielded a diverse family of fish-killing toxins that occurs around the globe and has significant ecological and economic impact.

Chemical Diversity, Origin, and Analysis of Phycotoxins.

Microalgae, particularly those from the lineage Dinoflagellata, are very well-known for their ability to produce phycotoxins that may accumulate in the marine food chain and eventually cause poisoning in humans. This includes toxins accumulating in shellfish, such as saxitoxin, okadaic acid, yessotoxins, azaspiracids, brevetoxins, and pinnatoxins. Other toxins, such as ciguatoxins and maitotoxins, accumulate in fish, where, as is the case for the latter compounds, they can be metabolized to even more toxic metabolites. On the other hand, much less is known about the chemical nature of compounds that are toxic to fish, the so-called ichthyotoxins. Despite numerous reports of algal blooms causing massive fish kills worldwide, only a few types of compounds, such as the karlotoxins, have been proven to be true ichthyotoxins. This review will highlight marine microalgae as the source of some of the most complex natural compounds known to mankind, with chemical structures that show no resemblance to what has been characterized from plants, fungi, or bacteria. In addition, it will summarize algal species known to be related to fish-killing blooms, but from which ichthyotoxins are yet to be characterized.

Sublethal concentrations of ichthyotoxic alga Prymnesium parvum affect rainbow trout susceptibility to viral haemorrhagic septicaemia virus.

Ichthyotoxic algal blooms are normally considered a threat to maricultured fish only when blooms reach lethal cell concentrations. The degree to which sublethal algal concentrations challenge the health of the fish during blooms is practically unknown. In this study, we analysed whether sublethal concentrations of the ichthyotoxic alga Prymnesium parvum affect the susceptibility of rainbow trout Oncorhynchus mykiss to viral haemorrhagic septicaemia virus (VHSV). During exposure to sublethal algal concentrations, the fish increased production of mucus on their gills. When fish were exposed to the algae for 12 h prior to the addition of virus, a marginal decrease in the susceptibility to VHSV was observed compared to fish exposed to VHSV without algae. If virus and algae were added simultaneously, inclusion of the algae increased mortality by 50% compared to fish exposed to virus only, depending on the experimental setup. We concluded that depending on the local exposure conditions, sublethal concentrations of P. parvum could affect susceptibility of fish to infectious agents such as VHSV.

Ichthyotoxicity of the microalga Pseudochattonella farcimen under laboratory and field conditions in Danish waters.

Blooms of the marine dictyochophyte Pseudochattonella farcimen have been associated with fish kills, but attempts to verify ichthyotoxicity of this microalga under experimental conditions have not been successful. In the early spring of 2009 and 2011, P. farcimen bloomed in the inner Danish waters. The blooms occurred at a seawater temperature of ~2°C and correlated with extensive kills of farmed salmonid fish (2009) and wild populations (2011). Several strains of P. farcimen were isolated from the 2009 bloom. However, exposure of rainbow trout Oncorhynchus mykiss to laboratory-grown P. farcimen cultures did not reveal any toxic effects. During the 2011 bloom, fish were exposed to bloom water under both laboratory and field conditions. While no clinical effect was observed on fish incubated in bloom water in the laboratory trial, a remarkable difference was seen in the field trial between rainbow trout kept in tanks supplied with a continuous flow of filtered versus non-filtered bloom water. Histological examination of the gill tissue revealed karyorrhexis and epithelial loosening in the affected fish. Microscopy analysis of algal cell morphology suggested that mucocysts detected on the cell surface only in freshly sampled bloom water might be associated with ichtyotoxicity.

Transition from a mixotrophic/heterotrophic protist community during the dark winter to a photoautotrophic spring community in surface waters of Disko Bay, Greenland.

Unicellular eukaryotic plankton communities (protists) are the major basis of the marine food web. The spring bloom is especially important, because of its high biomass. However, it is poorly described how the protist community composition in Arctic surface waters develops from winter to spring. We show that mixotrophic and parasitic organisms are prominent in the dark winter period. The transition period toward the spring bloom event was characterized by a high relative abundance of mixotrophic dinoflagellates, while centric diatoms and the haptophyte Phaeocystis pouchetii dominated the successive phototrophic spring bloom event during the study. The data shows a continuous community shift from winter to spring, and not just a dormant spring community waiting for the right environmental conditions. The spring bloom initiation commenced while sea ice was still scattering and absorbing the sunlight, inhibiting its penetration into the water column. The initial increase in fluorescence was detected relatively deep in the water column at ~55 m depth at the halocline, at which the photosynthetic cells accumulated, while a thick layer of snow and sea ice was still obstructing sunlight penetration of the surface water. This suggests that water column stratification and a complex interplay of abiotic factors eventually promote the spring bloom initiation.

Loss of phototaxis and degeneration of an eyespot in long-term algal cultures: evidence from ultrastructure and behaviour in the dinoflagellate Kryptoperidinium foliaceum.

Phototaxis provides phytoplankton with the means to orient themselves in a light gradient. This is accomplished using an eyespot and associated organelles. For the dinoflagellate Kryptoperidinium foliaceum, which has been described as having one of the most elaborate eyespot complexes known, positive phototaxis has hitherto not been reported. In this study, we show that a newly isolated strain of K. foliaceum is indeed capable of positive phototaxis with a mean vector (± 95% confidence interval) of 352°± 2.2, where 0/360° indicates the position of the light source. A study of three strains (UTEX 1688, CCMP 1326, and MBL07) of K. foliaceum showed that the eyespot in two of these strains has degenerated following decades in culture. Thus, previous studies have failed to report positive phototaxis due to loss of directionality caused by the degenerated eyespot. The results are discussed in a broader context and we conclude that studies on algal morphology and physiology may result in erroneous conclusions if based on algal cultures maintained under laboratory conditions for extended periods.

Effects of Glacial Flour on Marine Micro-plankton: Evidences from Natural Communities of Greenlandic Fjords and Experimental Studies.

Meltwater runoff from glaciers carries particles, so-called glacial flour that may affect planktonic organisms and the functioning of marine ecosystems. Protist microplankton is at the base of marine food webs and thus plays an important role in sustaining important ecosystem services. To assess the effect of glacial flour on photoautotrophic, heterotrophic and mixotrophic microplankton, the spatial distribution of these trophic groups was studied in four Greenlandic fjords during summer. The results suggest that the abundance of the autotrophic microplankton was affected by the glacier meltwater due to reduced light penetration and nutrient availability. The abundance of heterotrophic and mixotrophic microplankton were not apparently affected by the glacier meltwater. Incubation experiments were conducted on the natural population and in laboratory cultures of two mixoplanktonic ciliate species. The experiments on the natural population revealed that none of the trophic groups were affected by the suspended material at concentrations up to 50 mg L-1. The experiments on cultures gave no indication that glacial flour was ingested by the mixoplanktonic ciliates. Growth rates of cultured ciliates were not affected by the glacial flour addition. These results suggest that heterotrophic and mixotrophic microplankton are not affected by glacial flour as much as autotrophic microplankton.

Local adaptation through countergradient selection in northern populations of Skeletonema marinoi.

Marine microorganisms have the potential to disperse widely with few obvious barriers to gene flow. However, among microalgae, several studies have demonstrated that species can be highly genetically structured with limited gene flow among populations, despite hydrographic connectivity. Ecological differentiation and local adaptation have been suggested as drivers of such population structure. Here we tested whether multiple strains from two genetically distinct Baltic Sea populations of the diatom Skeletonema marinoi showed evidence of local adaptation to their local environments: the estuarine Bothnian Sea and the marine Kattegat Sea. We performed reciprocal transplants of multiple strains between culture media based on water from the respective environments, and we also allowed competition between strains of estuarine and marine origin in both salinities. When grown alone, both marine and estuarine strains performed best in the high-salinity environment, and estuarine strains always grew faster than marine strains. This result suggests local adaptation through countergradient selection, that is, genetic effects counteract environmental effects. However, the higher growth rate of the estuarine strains appears to have a cost in the marine environment and when strains were allowed to compete, marine strains performed better than estuarine strains in the marine environment. Thus, other traits are likely to also affect fitness. We provide evidence that tolerance to pH could be involved and that estuarine strains that are adapted to a more fluctuating pH continue growing at higher pH than marine strains.

The combined effect of pH and dissolved inorganic carbon concentrations on the physiology of plastidic ciliate Mesodinium rubrum and its cryptophyte prey.

Ocean acidification is caused by rising atmospheric partial pressure of CO2 (pCO2) and involves a lowering of pH combined with increased concentrations of CO2 and dissolved in organic carbon in ocean waters. Many studies investigated the consequences of these combined changes on marine phytoplankton, yet only few attempted to separate the effects of decreased pH and increased pCO2. Moreover, studies typically target photoautotrophic phytoplankton, while little is known of plastidic protists that depend on the ingestion of plastids from their prey. Therefore, we studied the separate and interactive effects of pH and DIC levels on the plastidic ciliate Mesodinium rubrum, which is known to form red tides in coastal waters worldwide. Also, we tested the effects on their prey, which typically are cryptophytes belonging to the Teleaulax/Plagioslemis/Geminigera species complex. These cryptophytes not only serve as food for the ciliate, but also as a supplier of chloroplasts and prey nuclei. We exposed M. rubrum and the two cryptophyte species, T. acuta, T. amphioxeia to different pH (6.8 - 8) and DIC levels (∼ 6.5 - 26 mg C L-1) and assessed their growth and photosynthetic rates, and cellular chlorophyll a and elemental contents. Our findings did not show consistent significant effects across the ranges in pH and/or DIC, except for M. rubrum, for which growth was negatively affected only by the lowest pH of 6.8 combined with lower DIC concentrations. It thus seems that M. rubrum is largely resilient to changes in pH and DIC, and its blooms may not be strongly impacted by the changes in ocean carbonate chemistry projected for the end of the 21st century.

Studies on the genus Mesodinium I: ultrastructure and description of Mesodinium chamaeleon n. sp., a benthic marine species with green or red chloroplasts.

We provide here the description of a new marine species that harbors green or red chloroplasts. In contrast to certain other species of the genus, Mesodinium chamaeleon n. sp. can be maintained in culture for short periods only. It captures and ingests flagellates including cryptomonads. The prey is ingested very rapidly into a food vacuole without the cryptomonad flagella being shed and the trichocysts being discharged. The individual food vacuoles subsequently serve as photosynthetic units, each containing the cryptomonad chloroplast, a nucleus, and some mitochondria. The ingested cells are eventually digested. This type of symbiosis differs from other plastid-bearing Mesodinium spp. in retaining ingested cryptomonad cells almost intact. The food strategy of the new species appears to be intermediate between heterotrophic species, such as Mesodinium pulex and Mesodinium pupula, and species with red cryptomonad endosymbionts, such as Mesodinium rubrum.

The strengths and weaknesses of Live Fluorescently Labelled Algae (LFLA) to estimate herbivory in protozooplankton and mixoplankton.

The Live Fluorescently Labelled Algae (LFLA) technique has been used numerous times to estimate microzooplankton herbivory. Yet, it is unknown how mixoplankton (i.e., single-cell organisms that can combine phototrophy and phagotrophy) affect the outcome of this technique. Hence, we conducted a broad-spectrum assessment of the strengths and weaknesses of the LFLA technique, using several mixoplanktonic and protozooplanktonic grazers. Species from different taxonomic groups and different feeding mechanisms were tested in short-term experiments (ca. 5 h) in the laboratory, at different prey concentrations and during light and dark periods of the day. Overall, our findings suggest that the LFLA technique, due to its short-term nature, is an effective tracker of diel ingestion and digestion rates, and can detect new mixoplanktonic predators. We recommend that, irrespective of the prey concentration, incubations to measure grazing rates with this technique should generally be concluded within 1 h (adaptable to the environmental temperature). Nevertheless, our results also call for caution whenever using LFLA in the field: feeding mechanisms other than direct engulfment (like peduncle feeding) may provide severely biased ingestion rates. Furthermore, size and species selectivity are very hard to circumvent. To reduce the effects of selectivity, we propose the combined use of two distinctly coloured fluorochromes (i.e., distinct emission spectra). With this modification, one could either label different size ranges of prey or account for species-specific interactions in the food web.

The coupling between irradiance, growth, photosynthesis and prymnesin cell quota and production in two strains of the bloom-forming haptophyte, Prymnesium parvum.

Prymnesium parvum causes harmful algal blooms worldwide that are often associated with massive fish-kills and subsequent economic losses. Most of our knowledge of the toxicity of P. parvum derives from bioassays since methods for the identification and quantification of their toxins have been lacking. Recently, a quantitation method was developed for the causative lytic toxins, the prymnesins. Here, we for the first time present data on the influence of irradiance on cellular content and production of prymnesins under nutrient replete conditions in two P. parvum strains, which both produce B-type prymnesins. Large differences were observed between the two strains with regard to the influence of irradiance on prymnesin cell quota and production rates. At the highest irradiance level (550 µmol photons m-2 s-1), the cellular prymnesin quota was thirty times higher in strain K-0081 strain than in K-0374. The cellular prymnesin quota and production rates were closely linked to rates of growth and photosynthesis in strain K-0081, while this was not the case for K-0374. Yet, growth rate did explain the differences in prymnesin quota in the two strains. Consequently, the maximum prymnesin production rate (414 attomol cell-1 d-1) was only about three times higher in strain K-0081 than in K-0374, and revealed an optimum at the same irradiance of 200 µmol photons m-2 s-1 in both strains. At low irradiance levels, the difference in production rates between both strains became smaller, with 41 and 49 attomol cell-1 d-1 for K-0081 and K-0374, respectively. The carbon content of prymnesins made up for ∼3% and <1% of the total cellular carbon content in strains K-0081 and K-0374, respectively. The fraction of extracellular dissolved prymnesins was measured for strain K-0081, where it accounted for 14-30% of total prymnesin concentration in the cultures, irrespective of irradiance. The concentrations of prymnesins released to the water by the K-0081 strain were not significantly influenced by irradiance. Overall, we observed comparable responses in growth and photosynthesis of both tested strains toward changes in irradiance. However, the effects of irradiance on prymnesin quota and production rates were remarkably different between the two strains.

The role of photosynthesis and food uptake for the growth of marine mixotrophic dinoflagellates.

Mixotrophy (i.e. combined use of photosynthesis and food uptake for growth) is widespread among marine dinoflagellates. Species with permanent chloroplasts generally display a growth response towards irradiance like an ordinary autotrophic alga. However, some species cannot grow in the light on a standard inorganic nutrient medium, because they require the ingestion of prey for sustained growth. This includes species with various types of chloroplast origin. Only a few species have been shown to be able to grow in the dark if supplied prey. About half of the studied species were primarily phototrophic species, and food uptake marginally increased their growth rates at low irradiances. In the remaining species, food uptake increases to a large degree their growth rate when light is limiting and in some cases even when irradiance is not limiting growth. Some of these species grow relatively fast at high irradiances without food, while other species only grow slowly or cannot even maintain themselves at high irradiances without food. Dinoflagellates, which form symbioses with endo- and ectosymbionts are a very heterogeneous group, which have been studied only sporadically. Some species are clearly primarily phototrophs, while others rely heavily on food uptake for growth.