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• Use of flow cytometry to quantify ingestion rate of the toxic mixoplankton Prymnesium parvum. • Addition of algal prey significantly increased the growth rate of P. parvum both in inorganic phosphorus deficiency and sufficiency cultures. • Rates of ingestion and the lytic effects were stimulated by inorganic phosphorus deficiency in P. parvum. • P. parvum is able to use prey and/or debris derived from algae as phosphorus sources. Many harmful algae are mixoplanktonic, i.e. they combine phototrophy and phagotrophy, and this ability may explain their ecological success, especially when environmental conditions are not optimal for autotrophic growth. In this study, laboratory experiments were conducted with the mixotrophic and ichthyotoxic haptophyte Prymnesium parvum (strain CCAP 946/6) to test the effects of phosphorus (P) sufficiency and deficiency on its growth rate, phagotrophic and lytic activities. P-deficient P. parvum cultures were grown without or with addition of P in the form of inorganic phosphorus (nutrients) and/or living algal prey (i.e. the cryptophyte Teleaulax amphioxeia). The ingestion rate of P. parvum and prey mortality rate were calculated using flow cytometry measurements based on pigment-derived-fluorescence to distinguish between prey, predators and digesting predators. The first aim of the study was to develop a method taking into account the rate of digestion, allowing the calculation of ingestion rates over a two-week period. Growth rates of P. parvum were higher in the treatments with live prey, irrespective of the concentration of inorganic P, and maximum growth rates were found when both inorganic and organic P in form of prey were added (0.79 ± 0.07 d -1). In addition, the mortality rate of T. amphioxeia induced by lytic compounds was 0.2 ± 0.02 d -1 in the P-deficient treatment, while no mortality was observed under P-sufficiency in the present experiments. This study also revealed the mortality due to cell lysis exceeded that of prey ingestion. Therefore, additional experiments were conducted with lysed prey cells. When grown with debris from prey cells, the mean growth rate of P. parvum was similar to monocultures without additions of prey debris (0.30 ± 0.1 vs. 0.38 ± 0.03 d -1), suggesting that P. parvum is able to grow on prey debris, but not as fast as with live prey. These results provide the first quantitative evidence over two weeks of experiment that ingestion of organic P in the form of living prey and/or debris of prey plays an important role in P. parvum growth and may explain its ecological success in a nutrient-limited environments. [Display omitted] [ABSTRACT FROM AUTHOR]

Recently, a hitherto unknown feeding strategy, the toxic mucus trap, was discovered in the dinoflagellate Alexandrium pseudogonyaulax . In this study, over 40 strains of 8 different Alexandrium species ( A. ostenfeldii, A. tamarense, A. catenella, A. taylorii, A. margalefii, A. hiranoi, A. insuetum and A. pseudogonyaulax ) were screened for their ability to ingest prey and/or to form mucus traps. The mucus trap feeding strategy, where a mucus trap is towed by the longitudinal flagellum remains unique to A. pseudogonyaulax. In additional experiments, details of the trap were examined and quantified, such as speed and frequency of trap formation as well as what happens to the trap after the A. pseudogonyaulax cell detaches from it. The percentage of A. pseudogonyaulax cells producing a mucus trap and the number of prey cells caught increased with increasing prey concentration, whereas the physical size of the traps was independent of prey concentration. In one strain given an excess of prey, within 1 h over 90% of individual A. pseudogonyaulax cells had formed a trap, each containing an average of 45 prey cells. Individual A. pseudogonyaulax cells steadily produced traps and up to 5 traps were produced by a single A. pseudogonyaulax cell after only 24 h. The attachment of an A. pseudogonyaulax cell to the trap only ceased during, and just following, cell division. Prey cells were, to some extent, capable of escaping from the mucus trap, but the trap remained sticky and continued catching prey for up to 48 h after the trap had been abandoned by the A. pseudogonyaulax cell. These results reveal that the effects of the mucus trap extend far beyond the removal of prey through ingestion, and the potential impact of this strategy on surrounding cells is high. [ABSTRACT FROM AUTHOR]

The dinoflagellate Prorocentrum donghaiense is a dominant harmful algal bloom (HAB) species on the East China Sea (ECS) coast. The co-occurrence of Karlodinium veneficum with P. donghaiense is often observed and can later develop into dense blooms. However, the role of K. veneficum in P. donghaiense population dynamics is unknown. In the current study, three K. veneficum (GM1, GM2, and GM3) strains were isolated from the ECS with one (GM1) from a mixed, dense bloom of P. donghaiense and other HAB species. All three isolates had identical ITS sequences that were concordant with the species designation. Unique karlotoxin congeners were isolated from one strain (GM2). The sterol compositions of P. donghaiense and K. veneficum were consistent with sensitivity to karlotoxin in the former and insensitivity in the latter. Additional experimentation showed that: (1) in monocultures, higher growth rate of P. donghaiense than K. veneficum is observed in nutrient-enriched and nutrient-depleted media. In co-cultures, the growth of P. donghaiense is inhibited; (2) feeding on P. donghaiense by K. veneficum is clearly demonstrated by fluorescent dye tracking; and (3) the isolated karlotoxin is lethal to P. donghaiense in a concentration-dependent manner. From these studies we propose that K. veneficum may play a negative role in P. donghaiense bloom maintenance and that P. donghaiense may in turn be a bloom initiator as a prey item for K. veneficum . [ABSTRACT FROM AUTHOR]

Abstract: The genus Pfiesteria includes two toxigenic species, Pfiesteria piscicida and Pfiesteria shumwayae, that are thinly thecate dinoflagellates with apparently cosmopolitan distribution, especially in shallow, poorly flushed, eutrophic estuaries. They are heterotrophic prey generalists that typically feed via phagotrophy and prefer live fish or their fresh tissues as food. They can also engage in limited mixotrophy through temporary retention of kleptochloroplasts from algal prey. Toxicity is highly variable among strains, ranging from apparently nontoxic to highly toxic. Some strains produce a group of hydrophilic toxins with metal-mediated free radical production. Various metals can be involved in the toxin congeners, and the purified toxins are highly labile. These toxins can adversely affect mammalian cells as well as fish. Toxic strains are capable of killing fish by both toxins and physical attack from feeding upon epidermis and other tissues. Non-inducible strains do not produce sufficient toxin to kill fish, but some are capable of causing larval fish death by physical attack. From 1991 to 1998, Pfiesteria spp. were linked to major kills of juvenile Atlantic menhaden (Brevoortia tyrannus), mostly at densities of ≥4(3)×102 to 103 (rarely, 104)flagellatecellsmL−1. These kills mainly occurred in the second largest and largest estuaries on the U.S. mainland, especially two main tributaries of the Albemarle-Pamlico Estuarine System, following decades of hurricane-free conditions. Between kills, Pfiesteria abundance was low in surface waters (<10cellsmL−1), and the available evidence suggests that the populations were mostly in the lower water column and within surficial sediments. Apparently highly sensitive to scouring effects from major storms, Pfiesteria populations have been sparse in the affected estuaries since several hurricanes struck the Albemarle-Pamlico in the late 1990s. Recent research highlights include characterization of a novel group of Pfiesteria toxins, culture of a toxigenic strain on a sterile fish cell line, axenic culture on a semi-defined medium, the discovery of a new mode of heterotrophic feeding in dinoflagellates as manifested by Pfiesteria, and other advances in understanding the nutritional ecology and prey acquisition of these harmful dinoflagellates. [Copyright &y& Elsevier]

Abstract: Laboratory experiments were conducted to test the effects of nitrogen (N) and phosphorus (P) sufficiency and deficiency on mixotrophy in Prymnesium parvum (Haptophyta). P. parvum was grown with and without algal prey (Rhodomonas salina) and observed for 120h. 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 P. 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±0.3μM) incorporated by P. parvum in the N-deficient treatment, and 45±9% of the P (0.3±0μM) 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. [Copyright &y& Elsevier]

Abstract: Historically most harmful algal species (HAS) have been thought to be strictly phototrophic. Mixotrophy, the use of phototrophy and heterotrophy in combination, has been emphasized as operative mainly in nutrient-poor habitats as a mechanism for augmenting nutrient supplies. Here we examine an alternate premise, that many harmful algae which thrive in eutrophic habitats are mixotrophs that respond both directly to nutrient inputs, and indirectly through high abundance of bacterial and algal prey that are stimulated by the elevated nutrients. From review and synthesis of the available data, mixotrophy occurs in all HAS examined thus far in the organic substrate- and prey-rich habitats of eutrophic estuarine and marine coastal waters. Where data are available comparing phototrophy versus mixotrophy, mixotrophy in eutrophic habitats generally is significant in nutrient acquisition and growth of HAS and, therefore, likely important in the development and maintenance of their blooms. In eutrophic habitats phagotrophic mixotrophs, in particular, have been shown to attain higher growth than when in phototrophic mode. Yet for many HAS, quantitative data about the role of mixotrophy in nutrition, growth, and blooms are lacking, especially relating laboratory information to natural field assemblages, so that the relative importance of photosynthesis, dissolved organic nutrients, and ingestion of prey largely remain unknown. Research is needed to assess simultaneously the roles of phototrophy, osmotrophy and phagotrophy in the nutritional ecology of HAS in eutrophic habitats, spanning bloom initiation, development and senescence. From these data, models that include the role of mixotrophy can be developed to gain more realistic insights about the nutritional factors that control harmful algae in eutrophic waters, and to strengthen predictive capability in predicting their blooms. An overall forecast that can be tested, as well, is that harmful mixotrophic algae will become more abundant as their food supplies increase in many estuaries and coastal waters that are sustaining chronic, increasing cultural eutrophication. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

• Karlodinium australe feeds on a diverse range of preys by direct engulfment and tube feeding, including microalgae and zooplankton. • Micropredation (or phagotrophy) played a more important role than toxicity did in the lethal effects of Karlodinium australe on marine animals. • Karlodinium australe has a much higher potency than Karlodinium veneficum in causing mortality of marine animals. Multiple dinoflagellate species from the genus Karlodinium have been well known to form massive and toxic blooms that consequently cause fish kills in many coastal waters around the world. Karlodinium australe is a mixotrophic and potentially ichthyotoxic species associated with fish kills. Here, we investigated phagotrophy of K. australe (isolate KaJb05) established from a bloom event in the West Johor Strait, Malaysia, using several prey species (phytoplankton, zooplankton, and larval fish). The results showed that K. australe ingested relatively small prey cells of co-occurring microalgae by direct engulfment, while it fed on larger prey cells of microalgae by tube feeding. The results of animal exposure bioassays using rotifer (Brachionus plicatilis), brine shrimp (Artemia salina), and larval fish (Oryzias melastigma) demonstrated that phagotrophy (in terms of the trophic mode of the dinoflagellate), or micropredation (in terms of the mechanism of lethal effects on prey), played a more important role than the toxicity did in causing the lethal effects of K. australe on these aquatic animals under low cell densities of K. australe , while the mortalities of animals observed in the exposure to cell lysates of K. australe were solely caused by the toxicity. A comparison of the lethal effects between K. australe and K. veneficum revealed that the lethal effect of K. australe on rotifers was much stronger than that of K. veneficum at all cell densities applied in the experiments and the more "aggressive" micropredation of K. australe is suggested to explain the difference in lethal effect between K. austale and K. veneficum. Our results may explain why K. australe exhibited fish killings during moderate blooms at cell densities < 2.34 × 106 cells L−1, whereas K. veneficum was observed to cause massive fish kills only if the cell density was above 107 cells L−1. We believe these findings provide new insights into the ecological consequences of phagotrophy exhibited in some mixotrophic and harmful algae such as species of Karlodinium and of HAB events in general. [ABSTRACT FROM AUTHOR]