The aim of this study was to determine the relative importance of the different processes/mechanisms by which the toxic haptophyte Prymnesium parvum, cultured under different nutrient conditions, affects non-toxic phytoplankton competitors and microzooplankton grazers. P. parvum was cultured under steady-state growth in different nutrient conditions: nitrogen depleted (-N), phosphorus depleted (-P) and balanced nitrogen and phosphorus (+NP). Cells from each nutrient condition and culture cell-free filtrates, alone and combined with non-toxic prey (Rhodomonas salina), were used as food for the rotifer Brachionus plicatilis. An additional experiment was carried out to test the effect of P. parvum cells and culture cell-free filtrate on R. salina. The highest haemolytic activity values were achieved by -P F parvum cultures, followed by -N. However, the negative effect of R parvum on R. salina and rotifers did not correlate with haemolytic activity but with the number of P. parvum cells. -N-cultured P. parvum were the most toxic for both R. salina and rotifers, followed by +NP. Therefore, haemolytic activity is not a good indicator of the total potential toxicity of R parvum. The growth rate of R. salina was negatively affected by cell-free filtrates but the effect of P, parvum predation was greater. Rotifers fed on both toxic and non-toxic algae, indicating that they did not select against the toxic alga. The P. parvum cell-free filtrate had an effect on B. plicatilis, although this was weak, B, plicatilis was also indirectly affected by P. parvum due to the negative effects of the toxic alga on their prey (R. salina). However, the greatest negative effect of P. parvum on the rotifers was due to ingestion of the toxic cells. Therefore, the phytoplankton competitor R. salina is more affected by P. parvum predation and the grazer B. plicatilis is more affected by ingestion of the toxic cells, the effects of excreted compounds being secondary.

Harmful algal bloom species can persist in the environment, impacting aquatic life and human health. One of the mechanisms by which some harmful algal bloom species are able to persist is by consumption of organic particles. Methods to demonstrate and measure consumption can yield insight into how populations thrive. Here, we combine flow cytometry and real-time PCR to demonstrate consumption of a cryptophyte species (Rhodomonas sp.) by a toxic mixotrophic haptophyte (Prymnesium parvum). Using flow cytometry, the feeding frequency of a population of P. parvum cells was calculated using the phycoerythrin (PE) fluorescence signal from Rhodomonas sp. and the fluorescence of an acidotropic probe labeling the food vacuoles. Feeding frequency increased in the beginning of the experiment and then began to decline, reaching a maximum of 47.5% of the whole P. parvum population after 212 min. The maximum number of consumed Rhodomonas sp. cells was 0.8 per P. parvum cell, and occurred after 114 min corresponding to an ingestion rate of 0.4 Rhodomonas sp. cells/P. parvum/h. Cells from the feeding P. parvum population were sorted, washed, and subjected to a real-time PCR assay targeting the cryptophyte 18S locus. There was a correlation between cycle threshold (Ct) values and number of consumed prey cells calculated by fluorescence. Overall, this study shows that flow cytometric analysis, of the acidotropic probe and prey pigments, is an efficient and rapid tool in enumerating food vacuoles and the number of prey cells consumed. Furthermore, we suggest that real-time PCR can be applied to cells sorted by flow cytometry, thus allowing for the detection and potential quantification of the targeted prey cells.

Mixotrophy in Prymnesium parvum was investigated using carbon (δ¹³C) and nitrogen (δ¹⁵N) stable isotopes. The experiment was performed in light and dark. In the dark treatment we expected that the mixotrophic P. parvum would rely solely on its prey and therefore reflect the prey isotopic signatures. In the light treatment P. parvum can perform photosynthesis as well as utilize its prey, thus we expect the isotopic signatures to be between the dark mixed cultures and the monocultures, depending on how much prey was utilized. In the light treatment, addition of the ciliate Myrionecta rubra resulted in higher P. parvum cell numbers compared to monocultures. During the experiment, cell numbers in the dark monocultures and the mixed dark cultures did not increase. P. parvum had 2.5-3 times higher cellular phosphorus and nitrogen content in the dark compared to the cultures in the light whereas no difference in carbon content between treatments could be observed. This suggests that P. parvum can utilize nitrogen and phosphorus but not carbon in the dark. It thus seems as if P. parvum relies on photosynthesis to meet the carbon and energy demand required for growth. The expected isotopic signatures “become what you eat…plus a few per mil” were not observed. In the dark treatment, the δ¹³C did not differ between monocultures and mixed cultures. In the light treatments P. parvum δ¹³C became less negative then the corresponding dark treatments indicating that P. parvum used CO₂ rather than carbon from the added prey. No difference in δ¹⁵N between monocultures and mixed cultures could be observed during the experiment. We argue that light is necessary for P. parvum growth and that the ability to utilize nutrients originating from their prey may be important in bloom formation.

In this study we have investigated whether the carbon isotopic signature differs between different groups and species of marine phytoplankton depending on growth phase, nutrient conditions and salinity. The 15 investigated algal species, representing the Bacillariophyceae, Chlorophyceae, Cryptophyceae, Cyanophyceae, Dinophyceae and Haptophyceae classes were grown in batch monocultures and analysed for delta(13)C in both exponential and stationary phase. For all the cultured species, delta(13)C signatures ranged from -23.5 parts per thousand (Imantonia sp.) to - 12.3 parts per thousand (Nodulania spumigena) in the exponential phase and from - 18.8 parts per thousand (Amphidinium carterae) to - 8.0 parts per thousand (Anabaena lemmermannii) in the stationary phase. Three species (Dunaliella tertiolecta, Rhodomonas sp.. Heterocapsa triquetra) were also grown under nutrient sufficient and nitrogen or phosphorus deficient conditions. Nitrogen limitation resulted in a more negative delta(13)C signature, whereas no effect could be observed during phosphorus limitation compared to nutrient sufficient conditions. Growth of Prymnesium parvum in two different salinities resulted in a more negative delta(13)C signature in the 26 parts per thousand-media compared to growth in 7 parts per thousand-media. Our results show that the carbon isotopic signature of phytoplankton may be affected by salinity, differ among different phytoplankton species, between exponential and stationary phase, as well as between nutrient treatments.

Bioassay experiments were performed two times (with 2 years in between) in order to investigate if nitrogen(N, ammonium), phosphorus (P, phosphate) and carbon (C, glucose) additions would stimulate the growth ofbacteria and phytoplankton differently in three different tropical aquatic environments. The water and theirindigenous microbial communities were taken from a freshwater coastal lake (Cabiunas), a coastal (Anjos),and an offshore marine station (Sonar) in the Atlantic outside Cabo Frio, Rio de Janeiro State, Brazil. Ammonium,phosphate and glucose were added alone or in combination to triplicate bottles. In the lake, P seemedto be the primary limiting factor during the first experiment, since both bacterial production and phytoplanktongrowth was stimulated by the P addition. Two years later, however, addition of P inhibited phytoplanktongrowth. During both years, C was closely co-limiting for bacteria since CP additions increased the responseconsiderably. For both the coastal and offshore seawater stations, phytoplankton growth was clearly stimulatedby N addition in both years and the bacteria responded either to the P, N or C additions (alone or incombination). To conclude, the results from these tropical aquatic systems show that it is possible that phytoplanktonand bacteria may compete for a common resource (P) in lakes, but can be limited by different inorganicnutrients in marine waters as well as lakes, suggesting that phytoplankton and bacteria do notnecessarily compete for the same growth limiting nutrient in these environments.

Several studies have proved that some Dinophysis species are capable of ingesting particulate organic matter besides of being photosynthetic, a form of nutrition termed mixotrophy. Phagotrophy may be an important aspect of the life history of the genus Dinophysis and the key to understand its ecology. We used modern techniques coupling flow cytometry and acidotropic probes to detect and score food vacuolated Dinophysis norvegica cells in natural samples. In addition, feeding experiments were conduced under controlled conditions to observe if D. norvegica would grow feeding on the cryptophyte Teleaulax amphioxeia. The results of the field observations showed a frequency of phagotrophy between 25 and 71% in a natural D. norvegica population from the Baltic Sea, which is higher than previous reports (1–20%). Although molecular methods have proved that the kleptoplastids of the D. norvegica from the Baltic Sea are from T. amphioxeia, the laboratory experiments showed that the presence of T. amphioxeia in the cultures did not enhance the survival rate of D. norvegica neither in phototrophic nor in heterotrophic conditions. We suggest that the D. norvegica Kleptoplats are obtained through a heterotrophic or mixotrophic protist, which have been feeding on cryptophytes, as it has recently been shown for Dinophysis acuminata. Our main conclusion is that D. norvegica, and probably all other species from the genus Dinophysis, is mainly phagotrophic and feeds on a larger prey than T. amphioxeia. Autotrophy through kleptoplastidy would be a secondary feature used as a complementary or short-term survival strategy. 

Laboratory experiments were conducted to test the effects of nitrogen (N) and phosphorus (P) sufficiency and deficiency on mixotrophy in Prymnesium parvum (Haptophyta). A parvum was grown with and without algal prey (Rhodomonas salina) and observed for 120 h. Detection and enumeration of cells containing food vacuoles with prey (i.e. phagotrophy) was based on flow cytometric detection of fluorescence of an acidotropic probe. Overall, the presence of R. salina increased phagotrophy in P. parvum suggesting that, at least in this strain of P. parvum, the presence of suitable prey can stimulate phagotrophic behavior in P. parvum. Feeding frequency (the percentage of A parvum cells containing food vacuoles in a given time) was significantly higher under N and P deficiency than in the nutrient-sufficient treatments. A nutrient budget constructed from the data indicated that ingestion of organic matter (OM) supplied with 78 +/- 7% of the N (3.9 +/- 0.3 mu M) incorporated by P. parvum in the N-deficient treatment, and 45 +/- 9% of the P (0.3 +/- 0 mu M) acquired in the P-deficient cultures. Even under nutrient sufficiency, ingestion of OM was estimated to have supplied 43 +/- 16% of the N and 48 +/- 16% of the P incorporated into P. parvum cells. Phagotrophy was observed even in the NP-sufficient cultures (non-axenic mixed and monocultures), although P. parvum cells did not lose their photosynthetic capability, suggesting that phagotrophy is probably a permanent nutritional adaptation to this species. The ingestion of organic nutrients played an important role in P. parvum growth, being a reliable source of nutrition for P. parvum inorganic nutrient limitation, and could explain its capabilities to form persistent blooms. (C) 2009 Elsevier B.V. All rights reserved.

The allelopathic effect of Prymnesium parvum (Prymnesiophyta), which produces toxins with haemolytic, ichthyotoxic and cytotoxic properties, was investigated on a natural plankton community. Under controlled conditions, 3 laboratory bioassays were performed by adding cell-free filtrate from a P. parvum culture into different size fractions (<150, <100 and 20 to 150 mum) of a natural Baltic Sea plankton community. The effect of P. parvum cell-free filtrate was determined by measuring chlorophyll a, cell numbers (phytoplankton, ciliates, bacteria), carbon (C-14) uptake by phytoplankton and the incorporation of H-3-leucine by bacteria. P. parvum cell-free filtrate affected the whole phytoplankton community, resulting in a decrease in both chlorophyll a and carbon uptake. Furthermore, the plankton groups present in the community exhibited different sensitivity to the cell-free filtrate. While growth of cyanobacteria and dinoflagellates was inhibited, that of diatoms and ciliates was not only completely suppressed, but no cells were present at the end of the experiment in the bottles with P. parvum filtrate. In all experiments, therefore, cyanobacteria and dinoflagellates were the most resistant groups, which led to their dominance in the treatments with filtrate compared to controls. Bacterial production was also negatively affected by P. parvum filtrate. The results show that compounds released by P. parvum induce changes in the plankton community structure, killing other members of the marine food-web, especially other phytoplankton (allelopathy), and suggest that secreted compounds of P. parvum are inhibitory to potential grazers (ciliates). It is proposed that allelopathy is an important process in the ecology of P. parvum.

Competition among phytoplankton for limiting resources may involve direct or indirect interactions. A direct interaction of competitors is the release of chemicals that inhibit other species, a process known as allelopathy. Here, we investigated the allelopathic effect of three toxic microalgae species (Alexandrium tamarense, Karenia mikimotoi and Chrysochromulina polylepis) on a natural population of the dinoflagellate Scrippsiella trochoidea. Our major findings were that in addition to causing death of S. trochoidea cells, the allelopathic species also induced the formation of temporary cysts in S. trochoidea. Because cysts were not lysed, encystment may act as a defence mechanism for S. trochoidea to resist allelochemicals, especially when the allelopathic effect is moderate. By forming temporary cysts, S. trochoidea may be able to overcome the effect of allelochemicals, and thereby have an adaptive advantage over other organisms unable to do so.

We studied allelopathy in the dinoflagellate genus Alexandrium by testing the effect of A. tamarense on a natural plankton community from Hopavagen Bay, Trondheimsfjord, Norway, and the effect of toxic and non-toxic strains of A. tamarense and a toxic strain of A. minutum on algal monocultures. Also, a possible relation between the allelopathic effect and the production of paralytic shellfish poison (PSP) toxin was investigated. A. tamarense affected the whole phytoplankton community by decreasing the growth rate and changing the community structure (relative abundance of each species, dominant species). A negative effect of A. tamarense was also observed on ciliates, but not on bacteria numbers, In the bioassay with algal monocultures, the diatom Thalassiosira weissflogii and the cryptophyte Rhodomonas sp. were exposed to the filtrate of Alexandrium spp. All tested Alexandrium strains negatively affected T weissflogii and Rhodomonas sp. cultures, independent of whether PSP toxins were produced. The compounds released by Alexandrium caused lysis of natural and cultured algal cells, suggesting that the allelopathic effect may be connected with previously described ichthyotoxic and haemolytic properties of Alexandrium. Furthermore, the observation that several components of the plankton community were affected by compounds released by A. tamarense emphasizes the importance of allelopathy for the ecology of this species.

For aquatic systems, studies on allelopathic interactions among phytoplankton have increased over recent years, with the main focus on the role of the donor organism. In this study, we report on the response of a target organism to allelochemicals and whether this response was affected by stress conditions (nutrient limitation). We exposed the diatom Thalassiosira weissflogii, grown under different nitrogen (N) and phosphorus (P) conditions (NP, -N, or -P), to single or daily additions of a cell-free filtrate of Prymnesium parvum (grown with no nutrient limitation). When we exposed T weissflogii to a single addition of filtrate, all 3 treatments were inhibited by P. parvum. However, T weissflogii NP was the most resistant, while T weissflogii -N showed the highest sensitivity to P. parvum filtrate, followed by T weissflogii -P. When T weissflogii was exposed. to daily additions of P. parvum, the degree of inhibition of all T weissflogii treatments was higher than when only 1 initial addition was made. In this case, even the treatment that had the highest resistance (T weissflogii NP) was not only inhibited by the filtrate, but also showed a decrease in cell numbers. Nevertheless, T weissflogii -N was still more sensitive than the other treatments. Therefore, nutrient-limiting conditions may increase allelopathic effects, by making the target more susceptive to allelopathic compounds. Under these conditions, allelopathy may play a strong role in phytoplankton competition, especially in natural environments where the allelochemicals are continuously released and, thus, the target species do not have time to recover.

The classic portrayal of plankton is dominated by phytoplanktonic primary producersand zooplanktonic secondary producers. In reality, many if not most planktontraditionally labelled as phytoplankton or microzooplankton should be identifiedas mixotrophs, contributing to both primary and secondary production. Mixotrophicprotists (i.e. single-celled eukaryotes that perform photosynthesis and grazeon particles) do not represent a minor component of the plankton, as some formof inferior representatives of the past evolution of protists; they represent a majorcomponent of the extant protist plankton, and one which could become moredominant with climate change. The implications for this mistaken identification, ofthe incorrect labelling of mixotrophs as “phytoplankton” or “microzooplankton”,are great. It extends from the (mis)use of photopigments as indicators of primaryproduction performed by strict photoautotrophs rather than also (co)locating mixotrophicactivity, through to the inadequacy of plankton functional type descriptionsin models (noting that mixotrophic production in the individual organism is not asimple sum of phototrophy and heterotrophy). We propose that mixotrophy shouldbe recognized as a major contributor to plankton dynamics, with due effortexpended in field and laboratory studies, and should no longer be side-lined inconceptual food webs or in mathematical models.

Faecal pellet production and content along with egg production of the dominant copepod species Acartia clausi were studied in the Thermaikos Gulf (NW Aegean Sea) during a pre-bloom and a bloom of the toxic dinoflagellate Dinophysis acuminata. Both faecal pellet production (6.8-8.6 ind(-1) d(-1)) and egg production (15.8-47.6 ind(-1) d(-1)) appeared unrelated to the D. acuminata bloom. Less than 11% of the copepod faecal pellets contained one or two D. acuminata cells, almost intact, whereas the other material in the pellets was broken into small pieces or amorphous shapes. The toxin outflux seemed to be insignificant when compared to the mean toxin concentration from the whole D. acuminata population. Finally, the potential grazing impact of A. clausi on D. acuminata during the study period was low.

The proposed plan for enrichment of the Sulu Sea, Philippines, a region of rich marine biodiversity, with thousands of tonnes of urea in order to stimulate algal blooms and sequester carbon is flawed for multiple reasons. Urea is preferentially used as a nitrogen source by some cyanobacteria and dinoflagellates, many of which are neutrally or positively buoyant. Biological pumps to the deep sea are classically leaky, and the inefficient burial of new biomass makes the estimation of a net loss of carbon from the atmosphere questionable at best. The potential for growth of toxic dinoflagellates is also high, as many grow well on urea and some even increase their toxicity when grown on urea. Many toxic dinoflagellates form cysts which can settle to the sediment and germinate in subsequent years, forming new blooms even without further fertilization. If large-scale blooms do occur, it is likely that they will contribute to hypoxia in the bottom waters upon decomposition. Lastly, urea production requires fossil fuel usage, further limiting the potential for net carbon sequestration. The environmental and economic impacts are potentially great and need to be rigorously assessed.