Marine microalgae attack and feed on metazoans

ABSTRACT Free-living microalgae from the dinoflagellate genus _Karlodinium_ are known to form massive blooms in eutrophic coastal waters worldwide and are often associated with fish kills.

Natural bloom populations, recently shown to consist of the two mixotrophic and toxic species _Karlodinium armiger_ and _Karlodinium veneficum_ have caused fast paralysis and mortality of

finfish and copepods in the laboratory, and have been associated with reduced metazooplankton biomass _in-situ_. Here we show that a strain of _K. armiger_ (K-0688) immobilises the common

marine copepod _Acartia tonsa_ in a density-dependent manner and collectively ingests the grazer to promote its own growth rate. In contrast, four strains of _K. veneficum_ did not attack or

affect the motility and survival of the copepods. Copepod immobilisation by the _K. armiger_ strain was fast (within 15 min) and caused by attacks of swarming cells, likely through the

transfer and action of a highly potent but uncharacterised neurotoxin. The copepods grazed and reproduced on a diet of _K. armiger_ at densities below 1000, cells ml−1, but above 3500 cells

ml−1 the mixotrophic dinoflagellates immobilised, fed on and killed the copepods. Switching the trophic role of the microalgae from prey to predator of copepods couples population growth to

reduced grazing pressure, promoting the persistence of blooms at high densities. _K. armiger_ also fed on three other metazoan organisms offered, suggesting that active predation by

mixotrophic dinoflagellates may be directly involved in causing mortalities at several trophic levels in the marine food web. SIMILAR CONTENT BEING VIEWED BY OTHERS EFFECTS OF PREY TROPHIC

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INTRODUCTION In terms of nutritional modes the free-living dinoflagellates represent one of the most diverse groups of unicellular eukaryotes in the marine food web. Half of the species are

primarily phototrophic and possess permanent chloroplasts (that is, they are microalgae), while the other half lacks pigments and are primarily heterotrophic grazers and predators. Many

species of both types have mixotrophic tendencies and can combine both phototrophy and heterotrophy in different ways (Stoecker et al., 2009; Hansen, 2011). The degree of mixotrophy varies

along a spectrum from purely phototrophic to purely heterotrophic species (Jones, 1994). While a few primarily heterotrophic species can acquire phototrophy through the retention of

chloroplasts from ingested prey for short periods of time (days) (Burkholder and Glasgow, 1997; Skovgaard, 1998; Lewitus et al., 1999; Jakobsen et al., 2000), several primarily phototrophic

species with permanent chloroplasts can supplement growth with heterotrophy by feeding on unicellular prey (Hansen, 2011). Mixotrophy in primarily phototrophic microalgae is especially

common in harmful and toxic bloom species from coastal eutrophic waters (Burkholder et al., 2008; Place et al., 2012). Typically, mixotrophic dinoflagellates catch motile prey using a

so-called capture filament that functions similarly to a harpoon. Depending on the feeding mechanisms, the prey is either engulfed directly by membrane engulfment, or parts are sucked out by

a microtubule supported feeding tube or peduncle (myzocytosis) (Hansen and Calado, 1999). In general, mixotrophic dinoflagellates differ from most grazers in the marine food web by having a

large optimal prey size, often corresponding to their own size (Hansen and Calado, 1999). Tube feeding enables dinoflagellates to feed on very large prey (Berge et al., 2008a) and a few

primarily heterotrophic dinoflagellates, generally not forming mono-specific high-density blooms, have been reported to ingest rotifers, nauplius and bivalve larvae, nematodes and even

finfish by the use of a feeding tube (Calado and Moestrup, 1997; Vogelbein et al., 2002; Shumway et al., 2006; Jeong et al., 2010). This includes ingestion of fish in the primarily

heterotrophic _Pfiesteria piscicida,_ which can retain functional plastids from cryptophyte prey and thereby become mixotrophic (Burkholder and Glasgow 1997; Lewitus et al., 1999). In

contrast, primarily phototrophic and potentially bloom forming dinoflagellates with permanent chloroplasts have not been reported to ingest metazoans (Hansen and Calado, 1999; Jeong et al.,

2010). However, we observed this in a field sample from The Sound (Denmark). Unidentified dinoflagellates possessing chloroplasts were tube feeding on a copepod nauplius larva (Supplementary

Video S1). Copepods are millimeter-sized abundant planktonic crustaceans and typically represent the most important metazoan grazers for the transfer of primary production to higher trophic

levels in the sea (Ohman and Hirche, 2001). Generally in marine food webs, microalgae are prey and copepods grazers, and copepod grazing reduces microalgal biomass and controls the

formation of blooms (Ohman and Hirche, 2001; Armbrust, 2009). Based on inverted light microscopy (Supplementary Video S1) the microalgae feeding on the nauplius larva resembled members of

the relatively recently described genus of bloom-forming and toxic microalgae _Karlodinium_ (Daugbjerg et al., 2000). Species of _Karlodinium_ are less than 20 μm in length and 15 μm in

width and require electron microscopy and/or DNA sequence analyses for proper species identification (Bergholtz et al., 2005; de Salas et al., 2005), which was not available for this study.

Species of _Karlodinium_ possess permanent chloroplasts of haptophyte endosymbiotic origin (Bergholtz et al., 2005) and seem to be obligate phototrophs (that is, unable to survive without

light). However, the two species investigated in this study _Karlodinium armiger_ and _Karlodinium veneficum_ are mixotrophic and use feeding tubes to phagocytise microalgal prey to obtain

faster growth rates (Li et al., 1999; Berge et al., 2008a). The widespread species _K. veneficum_ represents one of the most problematic and well-studied toxic bloom-forming microalgae known

(Garcés et al., 2006; Place et al., 2008; Mooney et al., 2009; Calbet et al., 2011) and has been recognised for its ichtyotoxicity (toxic to fish) since the 1950s (Braarud, 1957). Several

aspects of its biology (Adolf et al., 2008), toxins (Deeds et al., 2002; Van Wagoner et al., 2010) and harmful effects on fish (Nielsen, 1993) have been described in detail and were recently

reviewed (Place _et al._, 2011). The haemolytic and neurotoxic Karlotoxins (Deeds et al., 2002; Van Wagoner et al., 2010) produced by _K. veneficum_ aid prey capture and are used directly

to stun cryptophytes before ingestion (Sheng et al., 2009). Karlotoxins also have anti-grazing properties towards important microzooplankton and copepod grazers (Adolf et al., 2007; Waggett

et al., 2008). Evidence suggests that fish kills because of _K. veneficum,_ are caused indirectly by cell-rupture and toxin release upon contact with the gills (Place et al., 2008; Mooney et

al., 2009), as opposed to the non-toxic heterotrophic dinoflagellate _Pseudopfiesteria shumwaye_ that may kill fish larvae by active tube-feeding (Vogelbein et al., 2002). _K. armiger_ is

much less studied than _K. veneficum_ (Daugbjerg et al., 2000; Bergholtz et al., 2005), and Karlotoxin production has not been reported in any strains of _K. armiger_. The species has been

reported to produce a different substance with neurotoxic activity (Garcés et al., 2006), but the specific toxin has not been isolated and chemically characterised. While _K. veneficum_

seems to prefer cryptophytes as food (Li et al., 1999; Adolf et al., 2008), _K. armiger_ ingests most types of unicellular prey offered. It displays pronounced swarming behaviour and forms

feeding aggregates when fed algal prey, allowing ingestion of prey several times larger than itself (Berge et al., 2008a). We studied if a strain of _K. armiger_ and four strains of _K.

veneficum_ attacked and ingested copepods. We also describe the interactions observed via light microscopy and report the effects of an ecologically relevant range of _K. armiger_ cell

densities on the trophic flow between the mixotrophic microalgae and copepods in more detail. MATERIALS AND METHODS MICROALGAL STRAINS AND COPEPOD CULTURE Monocultures of _K. armiger_

(strain K-0668) and _K. veneficum_ (strain K- 1640) were obtained from the Scandinavian Culture Collection of Algae and Protozoa (SCCAP). The three other _K. veneficum_ strains (K-1385,

K-1635, K-1386) were isolated from North–East Atlantic locations (http://www.sccap.dk) and established during this work. All _Karlodinium_ strains are available from SCCAP, except strain

K-1386, which has been lost. The strain of _K. armiger_ was the same as the one that was used to describe the species (Bergholtz et al., 2005). It was isolated from Alfacs Bay (Spain) during

a harmful bloom event in 2000 and kindly submitted to SCCAP by Dr. Margarita Fernandéz-Tejedor. The microalgal stock cultures were grown in F/20 medium without silicate, based on sterile

filtered seawater with a salinity of 30. The same medium was used for the incubation experiments with the different _Karlodinium_ strains and copepods (see below). The copepod _Acartia

tonsa_ originated from Denmark and was grown in gently aerated, large 80-l tanks, fed the cryptophyte _Rhodomonas salina_ (strain K-0294, SCCAP) at saturating food conditions. The salinity

in the copepod stock-culture tanks was 32 and the temperature was 20 °C. As the detailed study of potential toxins involved was beyond the scope of this initial report, we did not measure

toxin production in our strains. However, we tested whether the _Karlodinium_ strains used were in a phagotrophic state under the provided experimental conditions (that is, mixotrophic and

presumably toxic (Sheng et al., 2009)). We offered all five _Karlodinium_ strains the cryptophyte _Rhodomonas salina_ and the dinoflagellate _Heterocapsa triquetra_ as food in predator:prey

cell density ratios of 1:5 and 2:5, respectively. For determination of mixotrophic tendency or feeding frequencies (% cells containing visible food vacuoles), the strains were mixed in

triplicate microwells (24-well plate, volume 2 ml) with the microalgal prey and fixed in glutaraldehyde (1% final concentration) after 14 h of incubation under the same experimental

conditions used throughout this study (see below). Feeding frequencies of the different _Karlodinium_ strains were determined as the number of cells containing visible food vacuoles in the

first 100 cells encountered, using an inverted microscope at 200–400 magnification. EXPERIMENTAL CONDITIONS The _Karlodinium_ and microalgal prey cultures were adapted to the experimental

light conditions (70 μmol photons m2 s−1 in a light/dark cycle of 14:10 h) for at least 5 days of exponential growth before the experiments. Adaptation to experimental salinity (30) and

temperature (20 °C) lasted at least 30 days, and F/20 medium was used (see above). One cohort of female _Acartia tonsa_ was used in all experiments. Immobilisation and survival of copepods

were observed via stereo and inverted microscopy. Immobilisation was recorded when the copepod was lying on the bottom of the microwell (containing from 2–15 ml total culture/medium volume;

see below) with erratic movements and without the ability to swim or jump, but with internal body movements still occurring, especially gut movements. Immobilisation caused the copepods to

sink to the bottom of the microwells, where they were observed lying still but alive for many hours. Death was recorded when no internal gut movement occurred. At termination of the

experiments (after 24–40 h), mixtures of microalgae and copepods were fixed in acid Lugol’s solution. Microalgal cell concentrations were counted manually in acid Lugol’s fixed samples using

a Sedgewick Rafter chamber (Graticules Ltd, Tonbridge, UK), and at least 300 cells were counted. To estimate copepod production during the incubation period we counted the number of eggs,

empty egg-shells and nauplius larvae at the end of the incubation period (24–40 h). To estimate copepod grazing, we counted the number of faecal pellets produced during the incubation

period. EFFECT ON COPEPOD MOTILITY AND MORTALITY OF STRAINS OF K. VENEFICUM AND K. ARMIGER To study potential immobilisation and mortality effects of four strains of _K. veneficum_ and one

strain of _K. armiger_ on copepods, we incubated two adult _Acartia tonsa_ females in five replicate 24-plate microwells containing 2 ml of dense _Karlodinium_ spp. cultures (>10 000 

cells ml−1) (Table 1). The densities used represent commonly occurring _Karlodinium_ bloom densities (blooms are often <10 000 cells ml−1 and sometimes 100 000 cells ml−1) (Garcés et al.,

2006; Place et al., 2012). As control copepod treatments, we included copepods fed 20 000 cells ml−1 of an unidentified phototrophic gymnodinoid dinoflagellate (North–East Atlantic origin;

strain ID unavailable) and the cryptophyte _Rhodomonas salina._ We also included a starved copepod control treatment without food in fresh medium. Additionally, to study potential

involvement of extracellular toxins that leaked into the water, we included a treatment of cell-free culture filtrate of the live _K. armiger_ culture (10 000 cells ml−1). The cell-free

culture filtrate was prepared by gently filtering the culture through 5 μm membrane filters (Poretics, Livermore, CA, USA) at a very low pressure<15 cm Hg vac and the filter did not dry

during this filtration, but was always covered by the culture medium. This was done to avoid potential physical disruption of cells and release of toxins. The copepods were starved for 2

days before setting up the experiment. For the initial 130 min, we observed copepod motility in all the treatments every 5–30 min, and recorded motility and mortality after 24 h. Copepod

grazing (that is, number of faecal pellets produced) was recorded in the control treatments, the culture filtrate treatment and the _K. armiger_ live cells treatment. ABILITY TO IMMOBILISE

AND INGEST OTHER MARINE METAZOANS Because of the absence of attacks on copepods in four out of five investigated _Karlodinium_ strains, the rest of the incubations were only done for _K.

armiger_ (strain K-0688). To study if _K. armiger_ immobilise and kill other metazoan organisms, seawater samples (2 l) were brought to the laboratory and a subsample was gently filtered

through a 100-μm net to concentrate the organisms (the organisms were covered in water during filtration to avoid physical stress). An adult nematode, a trochophore and a later-stage

polychaete larva were isolated and transferred to multidish wells, containing _K. armiger_ cells at densities of 1000–2000 cells ml−1, and observed using an inverted microscope connected to

a video-camera. ACARTIA TONSA IMMOBILISATION AND SURVIVAL, AND TROPHIC ROLES OF MICROALGAE AND COPEPODS IN A RANGE OF K. ARMIGER CELL DENSITIES The relationship between _K. armiger_ cell

density and copepod immobilisation, mortality, grazing and reproduction was also investigated. We exposed adult female _Acartia tonsa_ to a range of cell densities in six-plate microwells.

Five copepods were added in each microwell containing 15 ml of culture or F/20 medium (control) in three replicate units. _K. armiger_ cell densities were, 0, 400, 1100, 3500, 5000 and 7300,

cells ml−1. The range was chose as these densities represent naturally occurring bloom densities of _K. armiger_ (Delgado and Alcaraz, 1999; Fernandez-Tejedor et al., 2004; Garcés et al.,

2006). Using a stereomicroscope and an inverted microscope, we recorded the number of immobile and dead copepods every 1–12 h for up to 2 days. We also measured copepod grazing (number of

faecal pellet produced) and copepod reproduction (number of eggs and nauplii produced) in the different _K. armiger_ cell density treatments. At a density of 3500 _K. armiger_ cells ml−1, we

compared the population growth rate of _K. armiger_ with the growth in cultures without copepods, by measuring the increase in cell density over the 2 days incubation. For comparison, _K.

armiger_ cells in mono-culture were grown under identical light conditions (see above) as in the copepod treatment. CALCULATIONS Mortality and immobilisation was calculated in the microwells

containing two or five copepods each, as the averaged percentage of dead and immobile copepods observed in the 3–5 replicate units (see above). Copepod production and grazing rates were

calculated as the change in numbers of eggs and nauplius larvae and faecal pellets, respectively, divided by the length of incubation period, or expressed as numbers produced during the

incubation period. Exponential growth rates of the _K. armiger_ were calculated according to, μ = (ln x t 2 -ln x t 1 )/( t 2 - t 1 ) , where X t 2 and X t 2 is the cell concentration at end

(time=t2) and start (time=t1) of the sampling interval, respectively. Cell densities were taken from three replicate units. Difference in growth rate between the cultures (3500 cells ml−1)

with and without copepods was tested using a _t_-test and evaluated at a significance level of 0.05. RESULTS AND DISCUSSION EFFECTS ON COPEPOD MOTILITY AND SURVIVAL OF STRAINS OF K.

VENEFICUM AND K. ARMIGER In the cultures with live _K. armiger_ cells (10 000 cells ml−1), half of the copepods were immobilised within only 1 h, and nearly all were unable to swim or

perform escape jumps after 135 min (Figure 1a). We observed no effects on the motility of _Acartia tonsa_ in high-density cultures of any of the four _K. veneficum_ strains, and the copepods

were healthy and swam normally after the 24 h incubation in these treatments (Figures 1a and b; densities from 25 000–50 000 cells ml−1; Table 1). Both starved and fed control copepods (fed

an unidentified gymnodinoid phototrophic dinoflagellate and the cryptophyte _Rhodomonas salina_) were all alive and healthy after 24 h, while all copepods in the treatment with _K. armiger_

(10 000 cells ml−1) were dead (Figure 1b) and covered with dinoflagellates tube feeding on the carcasses. The copepods grazed and produced faecal pellets in the fed control treatments

(Figure 1c). No pellets were produced in the starved control or in the cell-free _K. armiger_ culture filtrate, but starvation did not cause mortality or affected the motility of the

copepods (Figures 1a and b). The lack of motility and mortality effects after 24 h of the cell-free _K. armiger_ culture filtrate of the same culture that caused 100% mortality (10 000 cells

 ml−1), suggests that direct cell-contact by live _K. armiger_ cells is required for copepod immobilisation and mortality to occur (Figures 1a and b). Previous studies involving harmful

dinoflagellates and metazoan mortality have also reported that effects require direct contact with live cells; for example, _Heterocapsa circularisquama_ caused mortality of bivalve larvae

and rotifers (Nagai et al., 1996) and _Cochlodiinium polykrikoides_ caused rapid mortality of diverse species of bivalve larvae, only by direct cell-contact (Tang and Gobler, 2009).

Similarly, natural bloom populations containing both _K. armiger_ and _K. veneficum_ (Garcés et al., 2006) studied in the laboratory by Delgado and Alcaraz (1999) also required live cells to

cause immobilisation and mortality of the copepod _Acartia granii_. These authors suggested a paralytic toxin as the cause and showed scanning electron microscopy of the affected copepods

with attached microalgal cells. The possibility of microalgal feeding on the copepods as the cause of mortality was not discussed, probably because the causative organisms were putatively

identified as _Gyrodinium corsicum_ and thought to be entirely phototrophic. However, all the cells in their micrographs were facing the copepod with the ventral parts where the feeding tube

emerges (Delgado and Alcaraz, 1999). The fact that none of the _K. veneficum_ strains tested here attacked _Acartia tonsa_ agrees well with previous experimental studies on other toxic

strains of this species, reporting no short-term (<48h) motility and mortality effects on different species of copepods in high-density cultures (Waggett et al., 2008; da Costa, Fernandez

2002; da Costa et al., 2005; Vaque et al., 2006). However, strain variation in cell quotas and types of Karlotoxins is extensive in _K. veneficum_ (Bachvaroff et al., 2009). Some strains

are non-toxic and these seem to be unable to catch and feed on microalgal prey and are therefore primarily phototrophic (Sheng et al., 2009; Place _et al._, 2011). Although, we did not

investigate toxin production in our strains, three of the four _K. veneficum_ strains were able to feed on both types of microalgal prey offered under the experimental conditions provided.

This indicates that our three _K. veneficum_ strains were probably toxic. Further studies of the presence, types and cell quotas of Karlotoxins are needed to confirm this assumption. The

lack of copepod attacks by _K. veneficum_ strains may also be linked to the relatively low mixotrophic tendencies observed under the experimental conditions provided (Table 1). Toxicity of

_K. veneficum_ strains has been found to vary according to salinity, temperature and nutrient limitation (Adolf et al., 2009; Place _et al._, 2011). Also the balance between heterotrophy and

phototrophy varies significantly as functions of light intensity, prey concentration and nutrient concentrations (Li et al., 1999; Adolf et al., 2006; Place _et al.,_ 2011). The mixotrophic

tendency, in terms of % cells containing food vacuoles, was much higher in the _K. armiger_ strain than in any of the _K. veneficum_ strains when fed microalgal prey (Table 1). Therefore we

cannot reject the possibility that some strains or natural populations of _K. veneficum_ may be triggered to attack copepods under other environmental conditions. In fact, _K. veneficum_

was recently reported to attempt feeding on _Acartia tonsa_ from field observations (USA), although conclusive evidence was not provided (Place _et al._, 2011). Our field observation of tube

feeding on a nauplius larva (Figure 2a; Supplementary Video S1), also support that feeding on copepods takes place in other species. INTERACTIONS BETWEEN K. ARMIGER, COPEPODS AND OTHER

METAZOANS OBSERVED IN THE MICROSCOPE We directly observed copepod immobilisation by _K. armiger_ (Figure 2b) using an inverted microscope. These observations confirmed the importance of

direct cell-contact to cause immobilisation and mortality. Shortly after adding the copepods into the _K. armiger_ culture, the cells were attracted to the copepods (Figure 2c), and

frequently attached to them facing the metazoan with their ventral side (Supplementary Video S2). Initially, several cell-copepod contacts seemed to be directed towards the innervated first

antennae (Figure 2d) and the urosome or telson (Supplementary Video S2), which are involved in copepod motility and sensing. Copepods sense hydrodynamical disturbances caused by predators

and prey in the surrounding fluid with hairs and setae situated at their pair of first antennae and the telson (Strickler and Bal, 1973; Kiørboe and Visser, 1999). Depending on the swimming

speed and size of an encountering organism, copepods can respond to predators by performing powerful escape jumps (Kiørboe and Visser, 1999). The microscopic size of _K. armiger_ may

disguise it as a prey and enable the predator to attack the copepod unnoticed. The attacks on _Acartia tonsa_ resulted in erratic cramp-like movements and inability to swim (Supplementary

Video S2). This suggests that a fast-acting paralytic toxin is transferred directly into the nervous system. Potential toxin transfer could be brought about by the numerous extrusosomes

covering the cell surface of _K. armiger_ (Bergholtz et al., 2005) or through the feeding tube itself. The effect of _K. armiger_ attacks on the copepods was very fast, and the first

immobilised individual was observed within minutes (Figure 1a, Supplementary Video S2). After some time the microalgae swarmed more aggressively, accumulated and collectively fed on the

immobilised but still live copepods (Figures 2e and f) (gut movements still occurring). Finally, this collective feeding resulted in the death of the copepod (Supplementary Video S3). The

ligaments between copepod carapace segments seemed to be preferential zones for initial food uptake by the microalgae (Figure 2f). This may be related to the capacity of the feeding tube to

penetrate the exoskeleton. Feeding on other microalgae, _K. armiger_ has difficulties ingesting heavily armoured dinoflagellate prey, such as species of _Alexandrium_, and is hardly able to

ingest diatoms with tough silica cell coverings (Berge et al., 2008a). A single tube feeding event on the copepod lasted from seconds to several minutes and a _K. armiger_ cell was able to

feed several times. After 2 days of incubation with _Acartia tonsa_, the microalgal population had ingested almost all of the copepod body tissue (Supplementary Video S3). Food uptake

resulted in the formation of different sized food vacuoles and substantial cell swelling (a large food vacuole is shown in Figure 2g). When _K. armiger_ feeds on microalgal prey, it can

increase its cell volume to more than five times its original cell volume (Berge et al., 2008a). We inoculated an adult nematode, a polychaete trochophore and a later stage polychaete-larva

in cultures of _K. armiger_ (1000–2000 cells ml−1), to investigate if the carnivorous behaviour only affected the copepod _Acartia tonsa._ All metazoans offered were attacked and immobilised

within hours (Figures 2g–l, Supplementary Video S4).The entire body content of the trochophore larvae was nearly removed by tube feeding within 24 h (Figure 2i, Supplementary Video S4).

Feeding on the nematode was not directly observed, but the _K. armiger_ cells were clearly attached to it and attempted to penetrate the cuticle with the feeding tube (Figure 2l). The

swarming behaviour of _K. armiger_ toward metazoans was most likely by chemotaxis (Supplementary Video S3–S4), and similar swarming behaviour was reported when _K. armiger_ fed on microalgae

(Berge et al., 2008a). Chemotaxis has also been reported in primarily heterotrophic dinoflagellates (Spero, 1985; Schnepf and Drebes, 1986); for example, the tube feeding freshwater species

_Peridiniopsis berolinensis_ ingesting tissue of an injured nematode (Calado and Moestrup, 1997) and the marine _Pseudopfiesteria shumwayae_ tube feeding on fish larvae (Vogelbein et al.,

2002). EFFECT OF K. ARMIGER DENSITIES ON THE TROPHIC INTERACTIONS BETWEEN MICROALGAE AND COPEPODS Copepod immobilisation, mortality, grazing and reproduction depended on _K. armiger_ cell

densities in a dose–response manner (Figures 3a–d). At cell densities over 3500, cells ml−1, 50% of the copepods were immobilised within 2 h of exposure (Figure 3a), and after ca. 8 h, 50%

of the copepods were dead (Figure 3b). At densities below 1100, cells ml−1, the copepods grazed and reproduced on a diet of _K. armiger_, but above 3500, cells ml−1, copepod grazing and

egg-production were suppressed (Figures 3c and d). The number of nauplius larvae, eggs and faecal pellets produced in the lower _K. armiger_ densities, were lower after 40 h than after 24 h

(Figures 3c and d). This suggests that _K. armiger_ also ingested copepod eggs and faecal pellets (Supplementary Video S5). Ingestion of copepod faecal pellets by _K. armiger_ and other

phagotrophic dinoflagellates has recently been reported (Poulsen et al., 2011). Our data show that the trophic roles of _K. armiger_ as prey and copepods as consumers are switched, when

densities of the mixotrophic microalgae reach levels above ca. 1000, cells ml−1. _K. armiger_ is known to occur at higher cell densities in eutrophic estuarine waters (Fernandez-Tejedor et

al., 2004; Garcés et al., 2006). Field studies have reported maximum densities in the range 10 000–100 000 cells ml−1 (Delgado and Alcaraz, 1999; Deeds et al., 2002) for _K. armiger_ and _K.

veneficum_. This indicates that microalgal feeding on copepods and other metazoans can occur in coastal areas. The carnivorous strategy of _K. armiger_ on copepods is likely to be an

important mechanism when high cell densities have developed, coupling population growth to reduced grazing and promoting the persistence of blooms. It may be less important during the

initiation of blooms at lower cell densities, but _K. armiger_ may also benefit from food uptake of other metazooplankton organisms at lower cell densities without being able of

immobilisation. This is because, although _K. armiger_ harbours permanent chloroplast and is unable to survive without light, its growth rate is very low when growing entirely phototrophic

(<0.1 day−1). Fast growth rates (0.5–0.7 day−1) are only obtained when ingesting prey under mixotrophic growth. As feeding seems to stimulate the photosynthetic machinery of this

mixotroph (possibly by acquiring essential growth factors), only small volumes of food may be required to significantly increase the growth rate (Berge et al., 2008a, 2008b). In the

treatment with 3500 cells ml−1 of the density experiment, the presence of copepods resulted in 85% higher population growth rate of _K. armiger,_ compared with the phototrophic growth rate

in monocultures without copepods (Figure 4). This shows that ingestion of metazoan tissue may also supply the growth factors necessary for faster growth in _K. armiger_. Thus, by feeding on

live individuals with no immobilisation and mortality effects on the metazoan population as a whole, _K. armiger_ may obtain increased growth rates and therefore also benefit during bloom

initiation at lower cell densities. TOXICITY, PREY SELECTION AND SPECIFICITY Although we did not measure toxin production in any of our tested strains, our direct observations of the

interactions between _K. armiger_ and different metazoans via microscopy, combined with the data on timescales of copepod immobilisation (within minutes) and the density-dependent

immobilisation and mortality (dose–response relation) strongly support the involvement of a potent neurotoxin in this species. Thus, _K. armiger_ seems to use toxins to stun and feed on prey

in a similar manner to _K. veneficum_ preying on cryptophytes (Sheng et al., 2009), but that the range of prey types extends to diverse metazoan organisms. This difference in prey

specificity may indicate that the activity of the toxins produced by _K. armiger_ differ from the well-known Karlotoxins produced by _K. veneficum_ (Deeds et al., 2002; Mooney et al., 2009;

Sheng et al., 2009). Karlotoxin specificity has been shown to be related to the sterol composition of cell membranes (Deeds et al., 2002). While most studies involving strains of _K.

veneficum_ have involved cryptophytes as food, recent reports suggest that the species is able to switch to other types of prey such as diatoms (Place _et al.,_ 2011). In our study,

mixotrophic _K. veneficum_ strains ingested both cryptophytes and thecate dinoflagellates (Table 1). These observations suggests that prey specificity may be more general in _K. veneficum_

than previously recognised. The trophic roles of the omnivorous tube feeding _K. armiger_ are multiple; it may simultaneously act as a primary producers, grazer of microalgal food, a

predator of unicellular (Berge et al., 2008a) and metazoan zooplankton and as a detritivore on faecal pellets (Poulsen et al., 2011). This feeding flexibility indicates that _K. armiger_

competes for resources with organisms at several different trophic levels in the marine food web. Prey selection and prey preference by _K. armiger_ in natural communities may affect

metazoan food web structure and function. The efficiency of ingestion by _K. armiger_ feeding on phytoplankton species has been shown to be highly dependent on the food quality of the prey

and ingestion rates are lower when fed armoured compared with unarmoured microalgal prey (Berge et al., 2008b). Copepods have rigid exoskeletons, which may increase prey-handling time (that

is, time taken for immobilisation and piercing the prey). Thus, harmful effects of blooms of _K. armiger_ on more easily handled organisms (for example, unarmoured planktonic larvae) may

occur at even lower cell densities than reported here for _Acartia tonsa_. Support for this comes from an apparent higher sensitivity towards natural bloom populations containing _K.

armiger_ of two species of finfish compared with the copepod _Acartia grani_ (Delgado and Alcaraz, 1999; Fernandez-Tejedor et al., 2004). In these studies, paralysis of juvenile finfish (2–5

 cm in length) occurred within 2 min, and the fishes displayed erratic movements and loss of equilibrium and lethargy (Fernandez-Tejedor et al., 2004). CONCLUSIONS Four strains of _K.

veneficum_ (three of these were mixotrophic) tested did not attack and feed on the copepod _Acartia tonsa_. However, _K. armiger_ (strain K-0688) was able to paralyse metazoan prey and feed

on them when the dinoflagellate occurred at bloom densities (>1000 cells ml−1). _K. armiger_ thus represents the first example of mixotrophic microalgae that attack and eat live metazoans

(Hansen and Calado, 1999; Jeong et al., 2010), and obtain increased growth rate from it. The strategy seems to be accomplished by means of collective swarming behaviour (chemotaxis) and the

involvement of a potent but unknown neurotoxin. Following paralysis, feeding aggregates of _K. armiger_ cells accumulated on the metazoan body. _K. armiger_ may turn the food web upside

down and release top–down control by immobilising and feeding on important copepod grazers. Most importantly, _K. armiger_ ingests different types of metazooplankton organisms, suggesting

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supported by PhD grants from the Faculty of Science, University of Copenhagen, Denmark and LKP was supported by The Carlsberg Foundation, grant no. 2007-01-0495. We thank two anonymous

referees whose comments and suggestions significantly improved this manuscript. TB thanks Bente Aaberg Voigt for financial support. ND thanks the Carlsberg and Villum Kann Rasmussen

foundations for equipment grants. PJH was supported by the Danish Agency for Science and Technology, grant no. 2101-07-0086. We thank Julie Rindung for assistance with graphical presentation

and Tom Fenchel for critically reading and commenting on an earlier version of the manuscript. We also thank Gert Hansen at the Scandinavian Culture Collection for Algae and Protozoa

(SCCAP) for providing and receiving culture strains used in the present study. AUTHOR INFORMATION Author notes * Terje Berge Present address: Marine Biological Section, Department of

Biology, University of Copenhagen, Strandpromenaden 5, 3000 Helsingør, Denmark, AUTHORS AND AFFILIATIONS * Department of Biology, Marine Biological Section, University of Copenhagen,

Copenhagen K, Denmark Terje Berge & Niels Daugbjerg * Department of Biology, Marine Biological Section, University of Copenhagen, Helsingør, Denmark Terje Berge, Morten Moldrup & Per

Juel Hansen * DTU Aqua, Technical University of Denmark, Charlottenlund Castle, Charlottenlund, Denmark Louise K Poulsen Authors * Terje Berge View author publications You can also search

for this author inPubMed Google Scholar * Louise K Poulsen View author publications You can also search for this author inPubMed Google Scholar * Morten Moldrup View author publications You

can also search for this author inPubMed Google Scholar * Niels Daugbjerg View author publications You can also search for this author inPubMed Google Scholar * Per Juel Hansen View author

publications You can also search for this author inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to Terje Berge. ADDITIONAL INFORMATION Supplementary Information accompanies the

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_et al._ Marine microalgae attack and feed on metazoans. _ISME J_ 6, 1926–1936 (2012). https://doi.org/10.1038/ismej.2012.29 Download citation * Received: 21 November 2011 * Revised: 23

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Nature SharedIt content-sharing initiative KEYWORDS * algal blooms * copepods * marine food web * mixotrophy * phytoplankton * prey size