Elsevier

Harmful Algae

Volume 93, March 2020, 101775
Harmful Algae

Original Article
A strain of the toxic dinoflagellate Karlodinium veneficum isolated from the East China Sea is an omnivorous phagotroph

https://doi.org/10.1016/j.hal.2020.101775Get rights and content

Highlights

  • Karlodinium veneficum is an omnivorous phagotroph with wide prey spectrum.

  • K. veneficum showed two phagotrophic modes, myzocytosis and direct engulfment.

  • K. veneficum exhibited cannibalism on its own species and micropredation on animals.

  • K. veneficum prefers to feed on non-motile, injured and newly died preys.

  • The phagotrophy is promoted by prey addition and nutrient limitation.

Abstract

Karlodinium veneficum is a cosmopolitan, toxic, and harmful algal bloom-forming dinoflagellate, of which the mixotrophy has been suggested to be a key factor in the formation and maintaining of HABs and thus deserves more intensive explorations. Here, we report an investigation on the phagotrophy of K. veneficum using a clonal culture isolated from the coastal water of East China Sea. We found K. veneficum is an omnivorous phagotroph feeding on both live and dead bodies/cells of a fish (Oryzias melastigma), brine shrimp (Artemia salina), rotifer (Brachionus plicatilis), co-cultivated microalgae Akashiwo sanguinea, Margalefidinium polykrikoides, Alexandrium leei, Rhodomonas salina, Isochrysis galbana, and its own species. Karlodinium veneficum extracted the cell contents of all species provided through either a peduncle (i.e. myzocytosis) or by engulfing the whole cell of small preys (i.e. phagotrophy sensu stricto). Karlodinium veneficum preferred to ingest non-motile or newly dead preys, no matter whether they were fish, zooplankton, or phytoplankton. Importantly, K. veneficum exhibited micropredation on animals with sizes much larger than itself (fish, rotifer, and brine shrimp), especially when they were injured or newly dead. The LysoSensor- and LysoTracker-stained lysosomes or/and phagolysosomes of K. veneficum increased when preys were added. Cannibalism in K. veneficum, i.e. a cell feeds on other unhealthy or dead cells of the same species, was observed as the first time in the study, which can help the growth and elongated maintaining of the population under nutrient deficiency (i.e. the culture maintained viable in culture plates without nutrient supplement up to a year). The growth rate of K. veneficum exhibited significant positive correlation with ingestion rate, which differed among prey species, and the highest growth rate was observed when feeding on R. salina. The ingest ability of K. veneficum was triggered by nutrient deficiency. In conclusion, the omnivorous mixotrophy is proposed to be a key autecological mechanism for K. veneficum to widen its ecological niche and succeed in forming a cosmopolitan distribution and frequent blooms.

Introduction

Karlodinium veneficum (Ballantine) Larsen, 2000 is a notorious harmful algal bloom-forming dinoflagellate, which has a cosmopolitan distribution and is responsible for numerous fish-killing events (Deeds et al., 2002; Place et al., 2012). The reported presence of K. veneficum covers coastal ecosystems of all continents except Antarctica (Fig. 1) (Braarud, 1957; Bjɵrnland and Tangen, 1979; Li et al., 2000a, b; Ajani et al., 2001; Mooney et al., 2009; Xu et al., 2012; Moreno-Pino et al., 2018; Yang et al., 2019), given that it might have even been overlooked in many cases of routine monitoring because of its small size (< 8‒12 μm).

Karlodinium veneficum utilizes many survival strategies to advance the growth, survive, and expand the distribution range, such as mixotrophy, wide tolerance to environmental stresses, special life history, toxicity to predators, and allelopathic effects on competitors (Li et al., 2000b; Adolf et al., 2006b, 2008; Hall et al., 2008; Place et al., 2012; Yang et al., 2019). Karlodinium veneficum produces karlotoxins which cause hemolytic, ichthyotoxic, and cytotoxic effects (Deeds et al., 2002; Deeds, 2003; Deeds and Place, 2006; Place et al., 2012) and the toxicity appeared to deacrease with the time of laboratorial monospecific culturing (Yang et al., 2019). Karlodinium veneficum also exhibited potent allelopathic effects on other co-occurring algae (Adolf et al., 2006b; Yang et al. 2019) and one of the derivatives of karlotoxins, KmTX2, has been demonstrated to be at least one of the allelochemicals (Adolf et al., 2006b).

Mixotrophy is another important strategy that benefits K. veneficum in many ways. Karlodinium veneficum has been proven to be mixotrophic by several means. For instance, cell-surface proteases may play a role in nutrition by providing amino acids for assimilation. Alternatively, released amino acids may be degraded by cell-surface amino acid oxidases to provide ammonium that can be taken up as a source of nitrogen (Stoecker and Gustafson, 2003). Karlodinium veneficum may be better adapted to utilize urea than other species based on high urease activity and large intracellular urea pools (Solomon and Glibert, 2008). Karlodinium veneficum was shown to increasingly ingest eubacteria when phosphate became limited (Nygaard and Tobiesen, 1993). Karlodinium veneficum was also shown to ingest other small-sized cryptophytes by phagocytosis, which, reportedly, enhanced the photosynthetic performance of K. veneficum (Li et al., 1999). However, with a more careful experimentation, Adolf et al. (2006a) demonstrated a much lowered photosynthesis and a dominance of heterotrophic metabolism when K. veneficum was under mixotrophic growth. Prey abundance, particularly the abundance of nano-planktonic cryptophytes, has been believed to be a key factor driving the formation of toxic K. veneficum blooms in eutrophic environments (Adolf et al., 2008; Place et al., 2012).

The feeding mechanisms for phagotrophic dinoflagellates can be categorized into three types: (1) direct engulfment: some species phagocytize an entire food particle, including the prey cell membrane (i.e. phagotrophy sensu stricto) (Biecheler, 1952; Schnepf and Elbrächter, 1992); (2) tube feeding: some species use an appendage to suck out some of the prey cell contents but leave the plasma membrane outside the predator (i.e. myzocytosis) (Hansen and Calado, 1999) or suck whatever comes (e.g. Peridiniopsis berolinensis) (Calado and Moestrup, 1997); and (3) pallium feeding: some dinoflagellates use a feeding veil (pallium) to surround the prey and digest it outside the cell body of the predator, then the cytoplasmic contents of prey are liquefied and taken up by the pallium until only an empty wall or frustule remains (e.g. Zygabikodinium lenticulatum, Oblea rotunda, and Protoperidinium conicum) (Jacobson and Anderson, 1986; Jeong and Latz, 1994; Hansen and Calado, 1999). Many other dinoflagellates have been observed to be phagotrophic (e.g. Peridiniopsis berolinensie, Katodinium fungiforme, Cystodinedria, and Stylodinium) (Frey and Stoermer, 1980; Spero, 1985; Calado and Moestrup, 1997), and possibly all dinoflagellates have this trophic mode under certain circumstances.

Benefiting from flexible trophic strategies, small dinoflagellates are capable of feeding on preys much larger than themselves (e.g. tube feeding). Pfiesteria shumwayae was demonstrated to kill fishes by myzocytosis (Vogelbein et al., 2002). This phenomenon, as a type of predation, has thus been named as "micropredation" (Vogelbein et al., 2002). This trophic strategy describes a situation in that a natural enemy attacks more than one victim during a life stage and does not necessarily eliminate the victim's fitness because the predators may just take a small meal before moving away (Lafferty and Kuris, 2002).

The dinoflagellate genus Karlodinium Larsen, 2000 includes at least nine species (Luo et al., 2018). Some of them were confirmed being mixotrophic, e.g. K. australe (de Salas et al., 2005), K. armiger (Berge et al., 2008a, b; Berge and Hansen, 2016) and K. veneficum (Li et al., 1999; Sheng et al., 2010). Other species were reported to at least have a peduncle-like structure, such as K. gentienii (Nezan et al., 2014) and K. zhouanum (Luo et al., 2018), suggesting them being mixotrophic also. Phagotrophy of K. australe was observed when the culture was offered live cells of Rhodomonas salina as prey and K. australe has been shown to have a thick, tubular peduncle-like structure located along the sulcus (de Salas et al., 2005). Karlodinium armiger has been observed to ingest major groups of marine protists (Berge et al., 2008b), via either the peduncle when preys are large or thecate, or engulfing whole cells when preys are small enough (Berge et al., 2008b). In K. veneficum, a peduncular microtubular strand was also reported (Taylor, 1992; Bergholtz et al., 2005), however, the feeding process of K. veneficum by peduncle has not been well examined. Although K. veneficum demonstrated feeding ability on different protists (Li et al., 1999), previous studies have been focusing on its feeding on small phytoplankton species such as cryptophytes.

Regarding the digestion of the phagotrophically-ingested particles, phagolysosome, an acidic and hydrolytic organelle, has been believed to ultimately degrade the food particles (Cosson and Soldati, 2008). Phagosomes, a membrane-bound compartment containing particles that have been internalized from the cell surface by the process of phagocytosis, have membrane-bound proteins to recruit and fuse with lysosomes, a membrane-bound acidic organelle that contain hydrolytic enzymes and degrade many kinds of macromolecules, to form phagolysosomes (Luzio et al., 2007). Lysosomes were reported to present in dinoflagellates which were once termed as PAS/accumulation bodies (Taylor, 1968; Zhou and Fritz, 1994). These organelles (Phagosome, lysosome, and phagolysosome) play vital roles in phagotrophy. Whether or not there is an association between the activity in lysosomes/phagosomes and the phagotrophy in dinoflagellates, however, has not been investigated yet.

In this study, we examined the phagotrophy of a strain of K. veneficum isolated from the coastal water of East China Sea using multiple species (including microalgal species of different sizes, zooplankton, and fish) at different physiological statuses (live or dead) as "prey", and observed the lysosomes/phagolysosomes via staining cells with LysoSensor and LysoTracker. Further, we investigated the ingestion ability and growth performance of K. veneficum fed with different preys and/or under different nutrient levels. We found the strain of K. veneficum is a truly omnivorous phagotroph capable of feeding on everything provided, including the cells of its own species, which is believed to provide further insights into the population ecology and bloom dynamics of this species.

Section snippets

Cultures and culturing conditions

Karlodinium veneficum was isolated using single-cell pipetting from the coastal waters of Ningde, Fujian Province (26.88 ˚N, 120.19˚E), East China Sea, and confirmed for the identity as K. veneficum using their partial sequences of the large subunit (LSU) rRNA gene (D1‒D6 domains) (Yang et al., 2019). The existence of two types of cells with different sizes and pigmentations (see the descriptions in Results section) also led us to re-identify these two types of cells via single-cell isolation,

Karlodinium veneficum fed on all "preys" provided

We observed that K. veneficum could feed on the microalgae Akashiwo sanguinea (Fig. 3A‒F, Supplementary Video S1), Margalefidinium polykrikoides (Fig. 3G‒L; Supplementary Video S2), Isochrysis galbana (Fig. 4A‒F; Supplementary Video S3, S4), and Rhodomonas salina (Fig. 4G‒L; Supplementary Video S5, S6), the marine zooplankton Artemia salina (Fig. 5A, C, D) and Brachionus plicatilis (Figs. 5B, 6; Supplementary Video S7), and the fingerlings of finfish Oryzias melastigma (Fig. 5E, F). More

Karlodinium veneficum is a feeding generalist

The ability of dinoflagellates to feed on prey relies on a number of factors, including prey size, chemoattraction, prey recognition, and the swimming behavior of prey (Hansen and Calado, 1999). Previous studies confirmed that prey preference exists in some mixotrophic dinoflagellates, e.g. Heterocapsa triquetra (Legrand et al., 1998), Ceratium furca (Bockstahler and Coats, 1993), Prorocentrum minimum (Li et al., 1996), and Fragilidium subglobosum (Skovgaard, 1996). However, size selection

Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was financially supported by the National Science Foundation of China (Nos. 61533011, 41776125,41976134); the National Key R&D Program of China (No. 2017YFC1404300). Thank Mr. Yuyang Liu for the molecular identification of K. veneficum, and Dr. Yuanyuan Sun, Ms. Xinyu Zhai and Xiaoying Song for the help in SEM sample preparation.

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