Harmful Algae 8 (2008) 77–93

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Harmful Algae
journal homepage: www.elsevier.com/locate/hal

Mixotrophy, a major mode of nutrition for harmful algal species in

eutrophic waters
JoAnn M. Burkholder a,*, Patricia M. Glibert b, Hayley M. Skelton a,1
a
Center for Applied Aquatic Ecology, North Carolina State University, 620 Hutton Street – Suite 104, Raleigh, NC 27606, USA
b
University of Maryland Center for Environmental Science, Horn Point Laboratory, PO Box 775, Cambridge, MD 21613, USA

A R T I C L E I N F O A B S T R A C T

Article history: Historically most harmful algal species (HAS) have been thought to be strictly phototrophic. Mixotrophy,
Received 1 March 2008 the use of phototrophy and heterotrophy in combination, has been emphasized as operative mainly in
Received in revised form 30 July 2008 nutrient-poor habitats as a mechanism for augmenting nutrient supplies. Here we examine an alternate
Accepted 1 August 2008
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
Keywords:
stimulated by the elevated nutrients. From review and synthesis of the available data, mixotrophy occurs
Eutrophication
in all HAS examined thus far in the organic substrate- and prey-rich habitats of eutrophic estuarine and
Harmful algae
Mixotrophy
marine coastal waters. Where data are available comparing phototrophy versus mixotrophy, mixotrophy
Nutrients in eutrophic habitats generally is significant in nutrient acquisition and growth of HAS and, therefore,
Osmotrophy likely important in the development and maintenance of their blooms. In eutrophic habitats
Phagotrophy phagotrophic mixotrophs, in particular, have been shown to attain higher growth than when in
Phototroph 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.
ß 2008 Elsevier B.V. All rights reserved.

1. Introduction (Bockstahler and Coats, 1993a,b). Mixotrophy has long been

Mixotrophy, the combination of phototrophy (use of photo-
synthesis to obtain inorganic carbon and energy) and heterotrophy
– the latter referring to uptake of dissolved organic substrates, and/
or phagotrophy, feeding on particulate organic carbon – enables
some algal species to use organic nutrient pools, augment
photosynthetic energy, and function at multiple trophic levels
(Sanders et al., 1990; Cloern and Dufford, 2005). Thus, it can lend a
competitive advantage over strict phototrophs and heterotrophs
* Corresponding author. Tel.: +1 919 515 2726; fax: +1 919 513 3194.
E-mail address: joann_burkholder@ncsu.edu (J.M. Burkholder).
1
Present address: Department of Marine Science, University of Connecticut,
1080 Shennecossett Road, Groton, CT 06340, USA.
considered as an important mode of nutrition for phytoplankton in
oligotrophic habitats (reviewed in Jones, 1994, 2000). Yet,
increasingly it has been found in many microalgae from eutrophic
estuaries (e.g., Nygaard and Tobiesen, 1993; Jeong et al., 2004).
Here we consider the premise that many phototrophic harmful
algal species (HAS) in eutrophic estuaries are mixotrophs that
respond directly to inorganic nutrients and dissolved organic
substrates added by anthropogenic sources, and indirectly by
consuming more abundant bacterial and algal prey that respond
directly to the elevated nutrients. Harmful algae are defined
according to Smayda (1997) and GEOHAB (2006); they may be
high-biomass producers and/or toxin producers, and directly or
indirectly cause disease or death of humans or cause harm to
aquatic ecosystems. Eutrophic refers to waters that are nutrient-
enriched or nutrient over-enriched, generally from anthropogenic

1568-9883/$ – see front matter ß 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.hal.2008.08.010
78 J.A.M. Burkholder et al. / Harmful Algae 8 (2008) 77–93
sources, and eutrophication is considered to be a process initiated
by the disturbance of exogenous nutrient enrichment (after
Smayda, 2008).
2. Historic views—mixotrophy in nutrient-poor habitats
For decades across the salinity gradient, phytoplankton were
regarded as strictly phototrophic organisms at the base of aquatic
food webs (Hutchinson, 1961; Raymont, 1980; Wetzel, 1983;
Thingstad et al., 1996). Consideration of heterotrophy in the
nutrition of phytoplankton was limited to tests with dissolved
organic substrates. Bacteria were regarded as superior competitors
for dissolved organic substrates because of their larger surface
area-to-volume ratio (Bratback and Thingstad, 1985). Short-term
experiments comparing uptake of dissolved organic substrates
such as glucose and acetate by bacteria versus pelagic phyto-
plankton species from light-replete oligotrophic waters led to the
inference that phytoplankton cannot compete with bacteria for
such organic substrates and thus, derive little benefit from
heterotrophic nutrition (e.g., Wright and Hobbie, 1966; Wetzel,
1983; Kirchman, 2000). As another important consideration,
dissolved organic substrates typically were tested at concentra-
tions far in excess of the concentrations in natural waters (see
Droop, 1974; Wright and Hobbie, 1966).
Laboratory experiments also emphasized culturing algae in
defined media, although many phytoplankton did not grow well in
such media (Provasoli et al., 1957). Soil extracts, added to promote
growth of these ‘‘difficult to culture’’ organisms, were believed to
provide dissolved or colloidal humic substances that act as
chelators to enhance trace metal availability (Provasoli et al.,
1957; Prakash et al., 1973; Anderson and Morel, 1982). The role of
humic substrates as growth promoters rather than sources of
nutrients recently was supported by al Gagnon et al. (2005), based
upon tests of non-axenic cultures of the dinoflagellate Alexandrium
tamarense to humic and fulvic acids isolated from river waters. On
the other hand, Granéli et al. (1985) showed that cell production of
the dinoflagellate Prorocentrum minimum increased when humic
acids and phosphate were added to the medium, and that P.
minimum cells grown with humic acid additions minus inorganic N
contained similar N concentrations as cells grown with inorganic N
minus humic acids. More recently, Heil (2005) reported complex
effects of humic acids on P. minimum, ranging from roles as
chelators to substrates interacting with dissolved inorganic
nutrients to support growth. Aside from the role of humic acids
as chelators and nutrients, historic emphasis on inorganic
nutrients and defined culture media in experiments to understand
algal nutrition inadvertently discouraged advances on the more
complex nutrition of mixotrophs.
Discovery of the ‘‘microbial loop’’ in the early 1980s revealed
that substantial dissolved organic carbon (DOC) excreted, secreted,
or leaked by photosynthetic algal cells is taken up by bacteria and
transferred up the food web through bacterivorous microfauna
(Azam et al., 1983; Azam, 1998). Phagotrophy also became
recognized as a major mode of ‘‘particulate’’ organic carbon and
nutrient (N, P) acquisition in well-lighted, nutrient-poor habitats
(e.g., Bird and Kalff, 1986; Sanders, 1991; Tittel et al., 2003).
Potentially limiting nutrients such as nitrogen (N) and phosphorus
(P) are much more concentrated in microbial prey than in the
water column (Vadstein, 2000). Thus many previous authors
suggested that mixotrophy is most operable and most advanta-
geous in nutrient-poor habitats as a mechanism to supplement
nutrient supplies (e.g., Granéli et al., 1999; Stibor and Sommer,
2003; Stoecker et al., 2006). Stibor and Sommer (2003) showed
that simultaneous P uptake by the harmful haptophyte, Chryso-
chromulina polylepis, from dissolved inorganic and particulate
(radiolabeled bacteria) sources followed basic predictions of
optimal foraging theory (Stephens and Krebs, 1986). The onset
of mixotrophy depended upon the dissolved inorganic P concen-
tration: At low concentrations of dissolved inorganic P (DIP), C.
polylepis took up P from both bacterial and dissolved sources,
whereas the major source was DIP under more water column-
enriched conditions. Based upon data for mixotrophs from
nutrient-poor systems, Raven (1997) expected mixotrophs to
have lower maximum growth rates than strict phototrophs or
heterotrophs because of higher energetic costs in maintaining
photosynthetic organelles, enzyme systems for assimilating
inorganic nutrients, and feeding apparatus. He hypothesized that
mixotrophs should be able to compete with strict phototrophs and
heterotrophs only under conditions of light, nutrient, and prey
limitation. A chemostat study of a freshwater plankter supported
that hypothesis (Rothhaupt, 1996).
Mixotrophic microorganisms were described in the early 1900s
(Pascher, 1917; Biecheler, 1936) and historically mixotrophy was
recognized as widespread among some flagellate groups (Granéli
and Carlsson, 1998). There was little interest in its ecological
importance, however, including its importance to harmful algae,
until the 1980s (Thingstad et al., 1996). At that time various
published reports began to describe mixotrophy for a wide array of
microalgae, including phagotrophy of other algae and microfauna
by some potentially harmful phototrophs (see reviews in Jacobson,
1999; Stoecker, 1999). While most reports described species that
were thought to thrive only in oligotrophic habitats, a few – such as
P. minimum and Karlodinium veneficum – are habitual bloom
formers in eutrophic waters (Burkholder, 1998; Anderson et al.,
2002; Adolf et al., 2008).
Since the early 1990s, there has been an evolving recognition of
the potential importance of mixotrophy in the nutritional ecology
of harmful estuarine and marine microalgae. Many more photo-
trophs from eutrophying estuaries and coastal waters are now
known to use dissolved or particulate organic substrates in their
nutrition, countering previous conceptions that are still reflected
in most present-day efforts to model the dynamics of phyto-
plankton assemblages as strict phototrophs (Thingstad et al., 1996;
Stoecker, 1998; Flynn, 2005).
3. Harmful algae in eutrophic habitats—evidence for an
important role of mixotrophy
Many ecosystems are increasingly characterized as nutrient
over-enriched and light-poor, due to suspended sediment loading
from watershed development and algal blooms that respond to
elevated nutrients (National Research Council, 2000; Glibert et al.,
2005a; Wassmann, 2005; GEOHAB, 2006). Beyond local estuaries
and coastal embayments where various HAS have been related to
cultural eutrophication or nutrification (Burkholder, 1998;
Smayda, 2008), the global distribution of nitrogen export (based
upon models of Seitzinger and Kroeze, 1998) coincides with the
documented occurrences of the high-biomass, potentially toxic
HAS P. minimum. This species is most common where anthro-
pogenic N export is high in estuaries of Asia, Europe, and North
America (Heil et al., 2005; Glibert and Burkholder, 2006; Glibert
et al., 2008a). Outbreaks of species that cause paralytic shellfish
poisoning, such as A. tamarense, A. minutum, Gymnodinium
catenatum and Pyrodinium bahamense var. compressum, have also
corresponded with regions of increased anthropogenic nutrient
loadings (Glibert and Burkholder, 2006; Glibert et al., 2005a).
Blooms of various species such as Heterosigma akashiwo and
Lingulodinium polyedrum have been related to elevated levels of
anthropogenic N substrates, as well (Kudela et al., 2008a, and
references therein). Increased occurrences of other HAS have been
 J.A.M. Burkholder et al. / Harmful Algae 8 (2008) 77–93
associated with shifts in nutrient supply ratios related to
anthropogenic sources, especially decreased N:P ratios that reflect
regional, disproportionate P loading relative to N (Glibert and
Burkholder, 2006). In Tolo Harbor, Hong Kong over a decadal
period, for example, increased harmful algal blooms (HABs)
dominated by Gonyaulax polygramma, Prorocentrum micans, P.
sigmoides, and P. triestinum coincided with a decrease in the
ambient N:P ratio from roughly 20:1 to <10:1 (atomic basis)
(Hodgkiss and Ho, 1997). All of the species in these examples are
mixotrophs (Tables 1 and 2).
Mixotrophy is promoted by low light and/or nutrient deficiency
(Legrand et al., 1998; Granéli et al., 1999; Stoecker, 1999; Stoecker
et al., 2006), conditions that commonly occur in eutrophic systems
(low light – after precipitation events, wind mixing or other
disturbance, especially in shallow waters; nutrient deficiency –
seasonally, during/following algal blooms, or during periods when
nutrient availability is out of stoichiometric balance) (Hecky and
Kilham, 1988; Cloern and Dufford, 2005). Eutrophic habitats offer a
wealth of dissolved organic substrates from high organic loads and
substances excreted, secreted or lysed by bacteria, algae, and other
aquatic organisms (e.g., Bjornsen, 1988; Bronk et al., 2006; Verity
et al., 2007), as well as abundant ‘‘particulate’’ bacterial, algal, and
other prey whose growth is stimulated by the elevated nutrients
(e.g., Stoecker et al., 1997; Lewitus et al., 1999a; Berg et al., 2003;
Adolf et al., 2008). Algal mixotrophs vary from species with poor
efficiency as phototrophs but high efficiency as phagotrophs, to
obligate phototrophs with minimal heterotrophy (Caron et al.,
1990, 1993; Stoecker, 1998). Some species only supplement their
nutrition with mixotrophy, while others are capable of using
mixotrophy to grow in complete darkness (Granéli et al., 1999).
Some initiate mixotrophy only if high prey densities are present
(Sanders et al., 1990); for others, light limitation triggers
mixotrophy (e.g., Caron et al., 1993; Jones et al., 1993, 1995).
Many harmful microalgal species thrive in eutrophying or
eutrophic estuarine and coastal marine habitats, including
cyanobacteria (Phylum Cyanobacteria), dinoflagellates (Phylum
Dinophyta), ochrophytes (Phylum Ochrophyta—diatoms, golden
flagellates, brown tide algae, and raphidophycean flagellates)
(Table 1). Some potentially toxic HAS have relatively low affinities
(high Ks values) for uptake of inorganic N and P substrates (DIN,
dissolved inorganic N; DIP, dissolved inorganic P), and commonly
express mixotrophy (Smayda, 1997). While most species are free-
living, mixotrophy also occurs in some parasitic dinoflagellates
with photosynthetic life stages, such as Blastodinium spp. (Chatton,
1920; Pasternak et al., 1984), Dissodinium psuedocalani (Drebes,
1969), and Crepidoodinium australe (Lom et al., 1993), from
estuarine and marine waters that include eutrophic habitats.
Mixotrophy is apparently operative, although not well studied,
among other photosynthetic parasitic dinoflagellates as well
(Cachon and Cachon, 1987; Gaines and Elbrächter, 1987; Coats,
1999). The importance of mixotrophy to overall nutrition is
unknown for most of these species, but could be significant—for
example, Pasternak et al. (1984) reported that the endoparasites
Blastodinium spp. acquire up to half of the energy needed for
growth through phagotrophy.
Three physiological types of protistan mixotrophs have been
proposed (Stoecker, 1998): Type I—‘‘ideal’’ mixotrophs that can use
phototrophy and phagotrophy equally well; Type II—predomi-
nantly phototrophic algae, including many of the harmful
estuarine and coastal marine microalgae that thrive in eutrophic
habitats; and Type III—predominantly heterotrophic algae (pro-
tozoans, e.g., Pfiesteria piscicida and Pfiesteria shumwayae (Noctiluca
scintillans) (Marshall et al., 2006). Various studies support the
premise that the many Type II species feed when dissolved
inorganic nutrients (DIN, DIP) become limiting; their photosyn-
79
thetic rates can be directly related to food concentration, and their
feeding rates can be directly related to available light (Stoecker
et al., 1997; Stoecker, 1998; Li et al., 2000a; Stickney et al., 2000)
and food concentration (Granéli and Carlsson, 1998). Type III
harmful algal mixotrophs either retain algal endosymbionts that
allow them to augment their nutrition with photosynthesis (N.
scintillans; Sweeney, 1971, 1976), or retain kleptochloroplasts from
algal prey (P. piscicida; Lewitus et al., 1999a,b) (Tables 1 and 2). For
these species, photosynthetic rates are related to feeding rates and
prey concentrations.
3.1. Dissolved organic substrates
The uptake of dissolved organic substrates is variously referred
to as osmotrophy, saprotrophy, or resorption. Osmotrophy has
been defined as uptake by osmosis, or as active uptake of dissolved
organic substances (Pringsheim, 1963) or, more recently, simply as
uptake of dissolved organic substrates (inferred, by any means, e.g.,
Glibert and Legrand, 2006). Saprotrophy refers to absorption of
dead or decaying organic matter, whereas resorption (literally, to
absorb again) is the process of breaking down or assimilating
organic matter; for example, some parasitic dinoflagellates digest
algae extracellularly and then take up the nutrients by resorbing
the molecules (Schnepf and Elbrächter, 1992; Schnepf, 2004).
Because all of these processes have been reported among harmful
algae, here we use the term ‘‘osmotrophy’’ following the broad
definition given by Glibert and Legrand (2006).
Although osmotrophy has been underemphasized (Stoecker,
1999; Stickney et al., 2000), uptake and growth of various HAS on
urea, dissolved amino acids and/or other substrates is well known
(note that while N is emphasized here, phosphoesterases that
release phosphate ions from some organic substrates are also
common; see Table 1). Dissolved organic nitrogen (DON) from
natural and anthropogenic sources represents 14–90% of the total
N in lower rivers (Seitzinger et al., 2002), and includes many labile
substrates of potential use by algae in eutrophic habitats (Benner,
2002; Berman and Bronk, 2003; Seitzinger et al., 2005; Lewitus,
2006; Bronk et al., 2006). DON substrates are acquired by direct
uptake, extracellular oxidation and hydrolysis, and pinocytosis
(less well known) (Glibert and Legrand, 2006; Lewitus, 2006).
Direct uptake of urea has been shown for many harmful microalgae
(Tables 1 and 2). Extracellular oxidation and hydrolysis of amino
acids and proteins apparently is also common (Palenik and Morel,
1990; Mulholland et al., 1998, 2002a,b; Stoecker and Gustafson,
2003; Dyhrman, 2005). For example, the haptophyte Prymnesium
parvum uses cell-surface L-amino acid oxidases to oxidize amino
acids and primary amines, and takes up the resulting NH4+ (Palenik
and Morel, 1990). Stoecker and Gustafson (2003) demonstrated
that leucine amino peptidase activity in a natural estuarine
phytoplankton assemblage was associated with a dinoflagellate
bloom, and in non-axenic cultures of bloom species Akashiwo
sanguinea, Gonyaulax grindleyi, Gyrodinium uncatenum, K. venefi-
cum, and P. minimum, the leucine amino peptidase activity was
associated with the dinoflagellates and not the bacteria. Mulhol-
land et al. (2002a,b) demonstrated that peptide hydrolysis and
amino acid oxidation were associated with the size fraction
containing brown tide cells (A. anophagefferens) in natural
plankton assemblages.
Quantification of the importance of mixotrophy to harmful
algae in estuarine and coastal marine environments has been
impeded because of the diverse array of dissolved organic
substrates, many of which are uncharacterized or poorly char-
acterized (Jones, 2000; Benner, 2002; Bronk, 2002; Karl and
Björkman, 2002; Sipler and Seitzinger, 2008). Among DON
substrates, urea can be an important nitrogen source for
80 J.A.M. Burkholder et al. / Harmful Algae 8 (2008) 77–93

Table 1
Examples of free-living mixotrophic harmful algal species that thrive in eutrophyinga or eutrophic estuarine or marine coastal watersb

Taxon Linkages to nutrient enrichment Mixotrophicc

Cyanobacteria (Phylum Cyanobacteria)

Microcystis aeruginosa, Synechococcus sp. Bloom in eutrophic and hypereutrophic brackish waters, or +Osmotrophy (amino acids—M. aeruginosa;
eutrophying watersa; high P optima (Marshall et al., 2005b; urea—Synechococcus sp.)
Heil et al., 2007)

Dinoflagellates (Phylum Dinophyta)

Akashiwo mikimotoi Nearshore blooms stimulated by nutrient enrichment (waters with low +Osmotrophy (urea)
N:P ratios; as Gymnodinium nagasakiense; Lam and Ho, 1989)
Akashiwo sanguinea Thrives in eutrophic waters; linked to N stimulation (Garcı́a-Hernández +Phagotrophy (cyanobacteria, algal and
et al., 2005; Rothenberger, 2007); blooms potentially linked to microfaunal prey); +osmotrophy (urea)
anthropogenic nutrients (Kudela et al., 2008a)
Alexandrium catenella, Alexandrium Cysts of the A. catenella/A. tamarense complex abundant in eutrophic +Phagotrophy (eubacteria—A. tamarense;
minutum, Alexandrium tamarense near-shore waters (Wang et al., 2004); blooms in nutrient-rich cyanobacteria—A. minutum, A. tamarense;
waters (A. minutum, A. amarense—Delgado et al., 1990; Giacobbe algal prey (A. tamarense) + osmotrophy
et al., 1996; Sorokin et al., 1996) (A. catenella—dextrans, DOM, urea; A. minutum,
A. tamarense—urea)
Alexandrium monilatum Thrives in eutrophic waters (Connell and Cross (1950), Juhl (2005) Data not available
Alexandrium taylori Blooms in eutrophic waters (Vila et al., 2001) Data not available
Ceratium furca Blooms in eutrophic waters (Smalley and Coats, 2002) +Phagotrophy (oligotrich ciliates)
Cochlodinium polykrikoides Blooms in eutrophic waters (Marshall et al., 2005a; Lee et al., +Phagotrophy (eubacteria, cyanobacteria;
2002; Lee, 2006) cryptophytes, other algae)
Dinophysis acuminata Blooms in eutrophic waters (e.g., Tango et al., 2004); linked indirectly +Phagotrophy (ciliate M. rubra)
to anthropogenic nutrient enrichment: thrives on Myrionecta rubra prey,
based upon culture experiments (Park et al., 2006), and M. rubra is
stimulated by increasing nutrient concentrations (Sagert et al., 2005)
Gambierdiscus toxicusa Linked indirectly to anthropogenic nutrient inputs via stimulation +Phagotrophy (unknown prey)d
of its macroalgal habitats (Lapointe and Thacker, 2002; Lapointe
et al., 2004; Briggs and Leff, 2007)
Gonyaulax polygramma Blooms in eutrophic waters with low N:P ratios (Hodgkiss and Ho, 1997) +Phagotrophy (cyanobacteria;
cryptophytes, other algae)
Gonyaulax spinifera Blooms in eutrophic waters (Penna et al., 2006); cyst records of +Phagotrophy (cyanobacteria)
G. spinifera complex linked to eutrophication (Pospelova et al., 2002)
Gymnodinium catenatum Thrives in eutrophic waters (Garcı́a-Hernández et al., 2005) +Phagotrophy (cyanobacteria, algae)
Gymnodinium impudicum Blooms in eutrophic waters (Vila et al., 2001, as Gyrodinium impudicum; +Phagotrophy (cyanobacteria,
Glibert et al., 2002) eukaryotic algae)
Heterocapsa triquetra Blooms in eutrophic estuaries; some blooms linked to anthropogenic +Phagotrophy (eubacteria, cyanobacteria;
N enrichment (Mallin, 1994; Rothenberger, 2007) cryptophytes, other eukaryotic algae;
+osmotrophy (urea, DOC)
Karenia brevis Nearshore blooms in some areas may be stimulated or supported by +Phagotrophy (cyanobacteria);
nutrient enrichment (Buskey et al., 1996; Denton and Contreras, +osmotrophy (urea)d
2004; Vargo et al., 2004, 2008)
Karlodinium armiger Blooms in eutrophic waters (Garcés et al., 1999—as Gyrodinium +Phagotrophy (algae)
corsicum; Garcés et al., 2006)
Karlodinium veneficum Blooms in eutrophic waters; link supported by laboratory +Phagotrophy (eubacteria; cryptophytes,
experiments (Li et al., 1999) other algae); +osmotrophy (amino acids,
urea)
Lingulodinium polyedrum Blooms linked to anthropogenic nutrients in surface waters +Phagotrophy (cyanobacteria, algae);
(Kudela et al., 2008a) +osmotrophy (urea)
Noctiluca scintillans (‘‘green’’ form) Blooms in eutrophic waters of Southeast Asia (Okaichi and +Phagotrophy (algae) and apparent
Nishio, 1976; Lam and Ho, 1989; Qi et al., 1993; Huang and kleptochloroplastidy
Qi, 1997; Wang et al., 2008)
Ostreopsis lenticularisa, Ostreopsis Linked indirectly to anthropogenic nutrient inputs via +Phagotrophy (unknown prey)e
ovataa, Ostreopsis siamensisa stimulation of macroalgal habitat (Lapointe and Thacker, 2002;
Lapointe et al., 2004; Briggs and Leff, 2007)

Pfiesteria piscicida
Prorocentrum donghaiense
Prorocentrum micans
Prorocentrum minimum
Prorocentrum sigmoides
Prorocentrum triestinum
Scrippsiella trochoidea
Toxic strains (Burkholder et al., 2005; Moeller et al., 2007) thrive +Phagotrophy (cryptophytes, other algae)
in eutrophic estuaries (Burkholder and Glasgow, 1997; Magnien while harboring cryptophyte
et al., 2000; Glasgow et al., 2001); linked both directly (via nutrient kleptochloroplasts; +osmotrophy (urea)
uptake—experiments by Lewitus et al., 1999b; Glibert et al., 2006b)
and indirectly to nutrient enrichment (experiments by Burkholder
and Glasgow, 1997; Lewitus et al., 1999a; Burkholder et al., 2001a,b)
Blooms in eutrophic estuaries (Lee et al., 2002; Lu et al., 2003; +Phagotrophy (cyanobacteria;
Zhou et al., 2003) cryptophytes, other algae);
Blooms in eutrophic waters with low N:P ratios (Hodgkiss +Phagotrophy (cyanobacteria, algae)
and Ho, 1997)
Blooms in eutrophic and eutrophyinga waters; supported by +Phagotrophy (cyanobacteria, algae);
laboratory experiments (Fan et al., 2003; Heil et al., 2005; +osmotrophy (urea, amino acids, DON)
Springer et al., 2005; Glibert et al., 2001, 2008a)
Blooms in eutrophic waters with low N:P ratios Data not available
(Hodgkiss and Ho, 1997)
Blooms in eutrophic waters with low N:P ratios +Phagotrophy (eubacteria, eukaryotic algae)
(Hodgkiss and Ho, 1997)
Blooms in eutrophic waters (Ismael, 2003); is strongly influenced by +Phagotrophy (cyanobacteria, algae)
nutrient enrichment (Wang et al., 2004, 2008)
 J.A.M. Burkholder et al. / Harmful Algae 8 (2008) 77–93 81

Table 1 (Continued )
Taxon Linkages to nutrient enrichment Mixotrophicc

Haptophytes (Phylum Haptophyta)

Chrysochromulina brevifilum
Chrysochromulina leadbeateri
Chrysochromulina polylepis
Phaeocystis spp.
Prymnesium parvum
Blooms in eutrophic waters; supported by laboratory experiments +Phagotrophy (algae)
(Edvardsen and Paasche, 1998)
Blooms in eutrophic waters; supported by laboratory experiments +Phagotrophy (eubacteria); +osmotrophy
(Edvardsen and Paasche, 1998) (linked to putracine)
Thrives in eutrophic waters; supported by laboratory experiments +Phagotrophy (eubacteria); +osmotrophy
(Edvardsen and Paasche, 1998) (urea)
Among many habitats, thrive in eutrophic waters affected by raw Suspected but not examined
sewage and animal wastes with low N:P ratios; supported by (Davidson and Marchant, 1992;
laboratory experiments (Phaeocystis pouchetti) (Riegman et al., Verity et al., 2007)
1993; Riegman, 1995; Burkholder et al., 1997; Schoemann et al.,
2005; Wang et al., 2008)
Blooms in eutrophic waters; supported by laboratory experiments +Phagotrophy (eubacteria; algae, fish,
(Edvardsen and Paasche, 1998; Johansson and Granéli, 1999) other prey)

Ochrophytes (Phylum Ochrophyta)

Brown tide algae (Class Pelagophyceae)
Aureococcus anophagefferens
Can be indirectly or directly stimulated by organic nutrient +Osmotrophy (urea, amino acids, acetamide,
enrichment (Berg et al., 1997; Mulholland et al., 2002a,b; peptides, DON)
Lomas et al., 2001, 2004; Glibert et al., 2007); supported by
laboratory experiments (Lomas et al., 1996; Pustizzi et al., 2004)

Diatoms (Class Bacillariophyceae)

Toxic Pseudo-nitzschia species complex
Long-term linkage to eutrophication, low N:Si ratios – +Osmotrophy (urea—P. australis)
Parsons et al., 2002; bloom linked to sewage – Smith et al., 1990;
blooms potentially linked to anthropogenic nutrients
(Kudela et al., 2008a); supported by laboratory experiments
(Bates et al., 1998; Cochlan et al., 2008)

Raphidophytes (Class Raphidophyceae)

Chattonella spp. Thrive in eutrophic waters; supported by laboratory experiments +Phagotrophy (C. ovata—eubacteria)
(Lam and Ho, 1999; Imai et al., 1998; Lewitus et al., 2003;
Garcı́a-Hernández et al., 2005; Zhang et al., 2006)
Heterosigma akashiwo Thrives in eutrophic waters; blooms potentially linked to +Phagotrophy (eubacteria) +osmotrophy
anthropogenic nutrients (Kudela and Cochlan, 2000; Livingston, (urea, glutamic acid)
2007; Wang et al., 2008); supported by laboratory experiments
(Smayda, 1998; Zhang et al., 2006)
a
The term ‘‘eutrophying’’ is used in reference to habitats such as coral reefs or some Gulf of Mexico waters that, while still relatively nutrient-poor, are sustaining
anthropogenic nutrient loadings. In some coral reefs, these nutrient loadings are stimulating macroalgal production that is increasing habitat for benthic ciguatera
dinoflagellates, representing an indirect link for stimulation of these HAS by eutrophication (Lapointe and Thacker, 2002; Lapointe et al., 2004).
b
Most phototrophic algae are auxotrophs, relying upon vitamins supplied by other organisms (e.g., Gaines and Elbrächter, 1987). Vitamins and other external organic
substances required in small amounts, likely as catalysts or other ‘‘growth factors’’, are not considered here. In addition, externally acting phosphatases, known to be
produced by most phototrophic algae (Jansson et al., 1988), are not considered here but have been found in many HAS of eutrophic estuarine and marine habitats (e.g., see
Glibert and Legrand, 2006). Most phytoplankton show increased alkaline phosphatase activity under P deficiency (Pettersson, 1980; Cembella et al., 1984; Jansson et al.,
1988). The term ‘‘alkaline phosphatases’’ mostly has been used in reference to phosphomonoesterases, a specific group of phosphatases that catalyze the hydrolysis of
monoesters of orthophosphoric acid (Cembella et al., 1984). The resulting soluble phosphate ion is then available for uptake. It should also be noted that there are many
examples in the literature of uptake of dissolved organic substrates by harmful algae and/or stimulation of growth that are not included here, because the studies used
unrealistically high concentrations that would not be expected to occur in natural habitats (e.g., glycerol stimulation of growth for Amphidinium carterae—Morrill and Loeblich
III, 1979; use of ethionine or methionine as the sole N source by Prymnesium parvum—Rahat and Hochberg, 1971).
c
See Tables 2–4 for further information and references.
d
K. brevis (as Gymnodinium breve) has also been shown to take up amino acids glycine and valine (Baden and Mende, 1979), glucose (Baden and Mende, 1978), and urea
(Shimizu et al., 1995), but at unrealistically high concentrations that would not be expected in its natural habitat (for natural concentration ranges see Gaines and Elbrächter,
1987; Van Leeuwenhoek, 1993; Glibert et al., 2005b, 2006a).
e
Phagotrophy was inferred based upon observations of food vacuoles; prey were not identified. Urea uptake and growth were reported for G. toxicus by Durand-Clement
(1987), but at unrealistically high concentrations that would not be expected to occur in its natural habitat (for natural concentration ranges see Glibert et al., 2005b, 2006a).

phytoplankton growth (Tables 1 and 2). Elevated urea is also
considered as an indicator of coastal runoff and anthropogenic
nutrient enrichment (Kudela et al., 2008a) because it is a likely
contaminant in heavily urbanized regions (Antia et al., 1991) and
watersheds with intensive animal feed operations or high cropland
fertilizer application (Glibert et al., 2001, 2005b, 2006a). Recently
urea has become of special interest because of its accelerated
global use (Glibert et al., 2006a), and its positive correlation with
increased blooms of some harmful estuarine and marine micro-
algae (Glibert et al., 2005b, 2006a and references therein).
In a turbid, shallow eutrophic estuary, for example, urea was
estimated to contribute about 45% of the total N taken up by
phytoplankton assemblages (Twomey et al., 2005). A bloom of
Cochlodinium sp. acquired an estimated 55–62% of its N supply
from urea (Kudela et al., 2008b), and 38% of the N demand for a
bloom of L. polyedrum was met by urea (ambient concentration
0.5 mM) along with NH4+ (ambient concentrations 0.5 and
1 mM, respectively) (Howard et al., 2007; Kudela et al., 2008a). In
supporting laboratory experiments, maximal uptake rates of urea-
N were 2-fold higher than maximal rates for uptake of NH4+ or
NO3 (Howard et al., 2007). Urea has also been related to increased
toxicity in some harmful algae. For example, in a Pseudo-nitzschia
australis-dominated bloom, urea comprised 17% of the ambient N
substrates (Armstrong-Howard et al., 2007). Data from supporting
experiments indicated that the natural phytoplankton assemblage
could potentially double toxin production when growing on urea-
N in comparison to growth on ammonium or nitrate (Howard et al.,
2007). Although little is known about the role of other dissolved
organic substrates in supporting estuarine and coastal marine
HABs, the data for this substrate suggest the potential importance
of osmotrophy in bloom dynamics under eutrophic conditions
(Cochlan et al., 2008).
82 J.A.M. Burkholder et al. / Harmful Algae 8 (2008) 77–93

Table 2
Examples of mixotrophy or mixotrophic potential in harmful estuarine and marine algal flagellates from eutrophic habitats, considering osmotrophy (at realistic substrate
concentrations—Van Leeuwenhoek, 1993; Glibert et al., 2005b, 2006a) and phagotrophy of eukaryotic prey

Cyanobacteria
Microcystis aeruginosa  Uptake of amino acids and related growth (Kamjunke and Jähnichen, 2000)
Synechococcus sp.  Uptake of urea (Glibert et al., 2004; Heil et al., 2007), amino acids (Flynn, 1990) and related growth

Dinoflagellates
Akashiwo sanguinea  Leucine amino acid peptidase activity was associated with dinoflagellate (not bacteria) cells in culture (Stoecker and
Gustafson, 2003)
 Urea uptake and growth (Levasseur et al., 1993)
 Food vacuoles, ingestion of small oligotrich ciliates (Bockstahler and Coats, 1993a,b)
 Ingestion of Pyrenomonas salina (Hansen, 1998)
 Ingestion of Alexandrium tamarense, Amphidinium carterae, cryptophytes, Isochrysis galbana, Heterocapsa triquetra,
Heterosigma akashiwo, Prorocentrum minimum, Prorocentrum triestinum, Prorocentrum donghaiense, Scrippsiella
trochoidea (Jeong et al., 2005b)

Alexandrium catenella  Uptake of high-molecular-weight dextrans (Legrand and Carlsson, 1998)

 Uptake of humic substances (Doblin et al., 2001), N bound to humic substances (Carlsson et al., 1999)
 Urea uptake and growth (axenic culture, +nickel; Dyhrman and Anderson, 2005)
 Urea supported bloom (Collos et al., 2004)

Alexandrium minutum  Urea uptake and growth (Chang and McClean, 1997)
 Ingestion of cyanobacteria (Synechococcus sp.; Jeong et al., 2005a)

Alexandrium tamarense  Urea uptake and growth (Leong et al., 2004)

 Food vacuoles (Jacobson and Anderson, 1996)
 Ingestion of eubacteria (Nygaard and Tobiesen, 1993), cyanobacteria (Synechococcus sp.; Jeong et al., 2005a)
 Ingestion of Amphidinium carterae, unidentified cryptophytes, Isochrysis galbana, Heterosigma akashiwo,
Rhodomonas salina, Prorocentrum minimum (Jeong et al., 2005b)

Ceratium furca  Remains of oligotrich ciliates in food vacuoles (Bockstahler and Coats, 1993a,b)
 Ingestion of oligotrich ciliates (Smalley and Coats, 2002; Smalley et al., 2003)

Cochlodinium polykrikoides  Photosynthetic, ingestion of cryptophytes, Isochrysis galbana, Heterosigma akashiwo, Amphidinium carterae
(Jeong et al., 2004)
Dinophysis acuminata  Food vacuoles (Jacobson and Andersen, 1994; Gisselson et al., 2002; documented feeding on ciliate
Myrionecta rubra and its cryptophyte chloroplasts (Park et al., 2006)
Gambierdiscus toxicus  Food vacuoles (Faust, 1998)
Gonyaulax polygramma  Ingestion of various algae (cryptophyte species, Amphidinium carterae, Heterosigma akashiwo, Heterocapsa
triquetra, Isochrysis galbana, Prorocentrum minimum, Scrippsiella sp.), and related growth where tested
(with cryptophyte prey) (Jeong et al., 2005c)
Gonyaulax spinifera  Ingestion of cyanobacteria (Synechococcus sp.; Jeong et al., 2005a)

Gymnodinium catenatum  Ingestion of cyanobacteria (Synechococcus sp.; Jeong et al., 2005a)

 Food vacuoles containing Amphidinium carterae, unidentified cryptophytes, Heterosigma akashiwo,
Isochrysis galbana, Rhodomonas salina, Prorocentrum minimum (Jeong et al., 2005b)

Gymnodinium impudicum  Ingestion of cyanobacteria (Synechococcus sp.; Jeong et al., 2005c)

 Ingestion of Amphidinium carterae, unidentified cryptophyte species, Heterosigma akashiwo,
Prorocentrum minimum, Rhodomonas salina (Jeong et al., 2005b)

Heterocapsa triquetra
 Urea uptake and growth (Solomon and Glibert, 2008)
 Ingestion of eubacteria (Seong et al., 2006)
 Ingestion of cyanobacteria (Synechococcus sp.; Jeong et al., 2005a)
 Food vacuoles containing small unidentified flagellates, Thalassiosira pseudonana (Legrand et al., 1998)
 Ingestion of Amphidinium carterae, Heterosigma akashiwo, Isochrysis galbana, unidentified cryptophytes,
Prorocentrum minimum, Rhodomonas salina (Jeong et al., 2005b)

Karenia brevis  Uptake of 15N-DON from Trichodesmium blooms (Mulholland et al., 2002b; 2006)
 Urea and glutamate uptake (Bronk et al., 2004); urea uptake and growth (Glibert et al., in press; Sinclair, 2008)
 Ingestion of cyanobacteria (Synechococcus sp.; Jeong et al., 2005a; Glibert et al., in press)

Karlodinium armiger  Ingestion of cryptophytes (Rhodomonas marina) in the light (170 mmol photons m 2 s 1) significantly
increased growth (0.6 d 1) relative to growth without prey (0.01–0.10 d 1) (Berge et al., 2008)
Karlodinium veneficum  Leucine amino acid peptidase activity was associated with dinoflagellate (not bacteria) cells in culture
(Stoecker and Gustafson, 2003); urea uptake and growth (Solomon and Glibert, 2008)
 Ingestion of eubacteria (Nygaard and Tobiesen, 1993)
 Ingestion of various cryptophytes (e.g., Chroomonas salina, Cryptomonas appendiculata, C. calceiformis, C. maculata,
Hemiselmis brunnescens, Hemiselmis rufescens, Hemiselmis sp., Rhinomonas reticulata, Rhodomonas salina, Rhodomonas sp.,
Storeatula major, Isochrysis galbana). In mixotrophic mode when fed cryptophytes, growth rates were 2- to 3-fold higher
than the maximum growth rate without prey (Li et al., 1996, 1999, 2000a,b; Adolf et al., 2003, 2006a, 2008)

Lingulodinium polyedrum
 Urea uptake and growth; urea supported growth during a bloom (Kudela and Cochlan, 2000)
 Ingestion of cyanobacteria (Synechococcus sp.; Jeong et al., 2005a)
 Ingestion of Alexandrium tamarense, Amphidinium carterae, cryptophytes, Heterocapsa triquetra, Heterosigma
akashiwo, Isochrysis galbana, Prorocentrum minimum, P. triestinum, P. donghaiense, Scrippsiella trochoidea
(Jeong et al., 2005b)

Noctiluca scintillans  Ingestion of Pyrodinium bahamense var. compressum and subsequent photosynthetic capability (Hansen et al., 2004)
Ostreopsis lenticularis, Ostreopsis  Observation of food vacuoles (Faust, 1998)
ovata, Ostreopsis siamensis

Pfiesteria piscicida  Urea uptake (Lewitus et al., 1999b; Glibert et al., 2006b)
 J.A.M. Burkholder et al. / Harmful Algae 8 (2008) 77–93 83

Table 2 (Continued )
 Ingestion of cryptophytes (e.g., Cryptomonas sp., Rhodomonas sp.), Dunaliella tertiolecta, Isochrysis galbana,
Thalassiosira pseudonana (Burkholder and Glasgow, 1995, 1997; Burkholder et al., 2001a,b)

Prorocentrum donghaiense  Ingestion of cyanobacteria (Synechococcus sp.; Jeong et al., 2005a)

 Ingestion of Amphidinium carterae, unidentified cryptophytes, Heterosigma akashiwo, Isochrysis galbana,
Prorocentrum minimum, Rhodomonas salina (Jeong et al., 2005b)

Prorocentrum micans  Observation of food vacuoles (Jacobson and Anderson, 1996)

 Ingestion of cyanobacteria (Synechococcus sp.; Jeong et al., 2005a)
 Ingestion of Amphidinium carterae, unidentified cryptophytes, Heterocapsa triquetra, Heterosigma akashiwo,
Isochrysis galbana, Prorocentrum donghaiense, Prorocentrum minimum, Prorocentrum triestinum, Rhodomonas
salina (Jeong et al., 2005b)

Prorocentrum minimum  Used 35% of the DON from humic acids added to cultures (Carlsson et al., 1999); cells grown with additions
of humic substances contained similar concentrations of N as cells grown with inorganic N sources (Granéli et al., 1985)
 Urease activity was high enough to meet the cellular N demand for growth (Fan et al., 2003); urea
uptake and growth (Solomon and Glibert, 2008)
 Leucine amino acid peptidase activity was associated with dinoflagellate (not bacteria) cells in culture
(Stoecker and Gustafson, 2003)
 Ingestion of cyanobacteria (Synechococcus sp.; Jeong et al., 2005a)
 Ingestion of unidentified cryptophytes (Li et al., 1996; Stoecker et al., 1997)
 Ingestion of Amphidinium carterae, unidentified cryptophytes, Heterosigma akashiwo, Isochrysis galbana,
Rhodomonas salina (Jeong et al., 2005b)

Prorocentrum triestinum  Ingestion of eubacteria (Seong et al., 2006)

 Ingestion of Amphidinium carterae, unidentified cryptophytes, Heterosigma akashiwo, Isochrysis galbana,
Prorocentrum minimum, Rhodomonas salina (Jeong et al., 2005b)

Scrippsiella trochoidea  Ingestion of cyanobacteria (Synechococcus sp.; Jeong et al., 2005a)

 Ingestion of Amphidinium carterae, unidentified cryptophytes, Heterosigma akashiwo, Isochrysis galbana,
Prorocentrum minimum, Rhodomonas salina (Jeong et al., 2005b)

Haptophytes
Chrysochromulina brevifilum  Ingestion of Marsupiomonas pelliculata (Jones et al., 1995)

Chrysochromulina leadbeateri  Enhanced growth with high concentrations of putracine simulating conditions in fish kills (Johnsen et al., 1999)
 Observation of uptake of chloroplast fragments (see Johnsen et al., 1999)

Chrysochromulina polylepis  Ingestion of eubacteria (Nygaard and Tobiesen, 1993)

 Suspected to have consumed coccolithophorids (Manton and Parke, 1962)

Prymnesium parvum (including  Consumes many eukaryotic prey (e.g., algae such as Dunaliella sp., Gyrodinum sp., Heterocapsa rotundata, Oxyrrhis marina,
P. parvum f. patelliferum) Rhodomonas baltica, diatoms Minidiscus sp., Thalassiosira pseudonana, Thalassiosira sp.; unknown amoeba; fish substrates;
mammalian red corpuscles) (Tillmann, 1998, 2003; Martin-Cereceda et al., 2003; Skovgaard and Hansen, 2003; Skovgaard
et al., 2003; E. Granéli, Kalmar University, unpublished data)

Ochrophytes
Aureococcus anophagefferens  Uptake of urea, glutamate (Lomas et al., 1996; Fan et al., 2003)
 Peptide hydrolysis, amino acid oxidation (Mulholland et al., 2002a,b)
 Uptake of urea, amino acids especially under low light promotes blooms (Lomas et al., 1996; Pustizzi et al., 2004);
growth on amino acids in axenic cultures (Mulholland et al., 2002a,b)
 High uptake rates of DON during blooms (Berg et al., 1997)
 Urease activity was high enough to meet the cellular N demand for growth (Fan et al., 2003)

Pseudo-nitzschia australis  Uptake and growth on urea (Armstrong-Howard et al., 2007; Cochlan et al., 2008; Kudela et al., 2008a)

Raphidophytes
Chattonella ovata  Ingestion of eubacteria (Seong et al., 2006)
Heterosigma akashiwo  Uptake and growth on urea, glutamic acid (Zhang et al., 2006; Herndon and Cochlan, 2007)

Humic acids are included here as sources of nutrients.

3.2. Particulate organic substrates—phagotrophy
Phagotrophy, consumption of particulate food or prey, refers to
ingestion of discrete particles wherein digestion occurs in
specialized phagocytic (food) vacuoles (Gaines and Elbrächter,
1987). In eutrophic estuaries and marine coasts, phagotrophy is
most well known for dinoflagellates, haptophytes, and raphido-
phytes (Tables 1 and 2), and can significantly augment C, N, and P
supplies in nutrient-enriched waters (Nygaard and Tobiesen,
1993; Jones et al., 1995; Li et al., 2000a; Smalley and Coats, 2002;
Adolf et al., 2006a). The particle size ranges from high-molecular-
weight organic colloidal material in humic-rich estuaries (Legrand
and Carlsson, 1998; Lewitus, 2006) to prey that are larger than the
mixotroph predator (Stoecker et al., 2006 and references therein).
Most of these mixotrophic species can grow using only
phototrophy, but the few available studies that have compared
growth with and without phagotrophy generally have shown that
phagotrophy significantly increases growth (e.g., Li et al., 1999;
Hansen et al., 2004; Jeong et al., 2004, 2005a,b,c; Adolf et al., 2006a)
(Table 3), sometimes even under nutrient- and light-replete
conditions (e.g., P. parvum—Martin-Cereceda et al., 2003). In
contrast, P. parvum (as Prymnesium patelliferum) and Chrysochro-
mulina spp. that ingested prey under light- and nutrient-sufficient
conditions maintained similar growth rates with versus without
phagotrophy (Pintner and Provasoli, 1968; Larsen et al., 1993;
Granéli and Carlsson, 1998).
While exhausted nutrient supplies, shifts in nutrient supply
ratios (N:P, C:P, etc.) or low light stimulate phagotrophy in some
HAS, others (e.g., dinoflagellates Ceratium furca, K. veneficum)
apparently only feed or increase feeding under P or N limitation but
not under C or light limitation (Li et al., 1999, 2000a; Smalley et al.,
2003—described in detail below). The toxicity of the algal strain
also can influence phagotrophic activity as well as protection from
grazing. For example, Tillmann (2003) examined interactions
84 J.A.M. Burkholder et al. / Harmful Algae 8 (2008) 77–93

Table 3
Comparison of mixotrophy vs. phototrophy in toxic and otherwise harmful algae (one strain of each species) at saturated eukaryotic algal prey densities under nutrient-
replete conditions (f/2 medium) (from Jeong et al., 2004, 2005b,c)

Taxon Light regime Preya (ESD) Maximum Sp.Gr.Rateb Maximum Sp.Gr.Ratec Estimates for field populations
(mmol photons as mixotroph as phototroph
m 2 s 1) Grazing Potential effectd
coefficient (prey population)
1
(maximum h )
1 1 1
Cochlodinium polykrikoides 50 (14:10 h Various algae 0.324 day 0.166 day 0.745 53% removal h
(ESD 23.1  3.2 mm) L:D cycle) (5–<11 mm) (9 cryptophytes (on cryptophytes)
grazer 1 day 1)
Gonyaulax polygramma 50 (14:10 h Cryptophytes, other 0.278 day 1 0.186 day 1 0.479 38% removal h 1

(ESD 32.5  5.4 mm) L:D cycle) algae (5–<17 mm) (10.6 cryptophytes (on cryptophytes)
grazer 1 day 1)
Heterocapsa triquetra 20 (14:10 h Cryptophytes, other 0.283 day 1 0.184 day 1 0.091 9.1% removal h 1

(ESD 15.0  4.3 mm) L:D cycle) algae (12.1 mm) (2.2 cryptophytes (on cryptophytes)
grazer 1 day 1)

Lingulodinium polyedrum 50 (14:10 h Scrippsiella trochoidea, 0.303 day 1 0.182 day 1 0.011 1.1% removal h 1

(ESD 38.2  3.6 mm) L:D cycle) other algae (28.1 mm) (0.5 S.troc (on S. trochoidea)
grazer 1 day 1)
0.254 day 1 0.157 day 1 0.026 2.6% removed h 1

(1.5 P.min + P.triest. (on Proro. spp.)

grazer 1 day 1)

Prorocentrum donghaiense 20 (14:10 h Cryptophytes, other 0.510 day 1 0.375 day 1 2.67 93% removal h 1

(ESD 13.3  2.0 mm) L:D cycle) algae (12.1 mm) (1.5 cryptophytes (on cryptophytes)
grazer 1 day 1)
Prorocentrum micans 20 (14:10 h Cryptophytes, other 0.197 day 1 0.106 day 1 0.041 4.2% removal h 1

(ESD 26.6  2.8 mm) L:D cycle) algae (15 mm) (2.4 cryptophytes (on cryptophytes)
grazer 1 day 1)
a
Maximum prey dimension or equivalent spherical diameter (ESD) in parentheses.
b
Sp.Gr.Rate  specific growth rate.
c
Maximum initial prey concentrations were: cryptophytes, 104–105 cells ml 1; Scrippsiella trochoidea, 103 cells ml 1
; Prorocentrum minimum, 104 cells ml 1
.
d
Maximum percentage (%) of the prey population removed h 1.

between a toxic strain of P. parvum and its potential predator, the
heterotrophic dinoflagellate Oxyrrhis marina, in P-limited semi-
continuous cultures and nutrient-replete batch culture experi-
ments. When toxicity was low, P. parvum was consumed by
Oxyrrhis marina, but at high toxicity levels, the prey became the
predator as P. parvum killed and ingested O. marina.
3.2.1. Prokaryote prey
Harmful estuarine and marine algae have been reported to
phagocytize eubacteria and cyanobacteria as well as a wide range of
eukaryotic prey (Tables 1–4). Experiments on phagotrophy of
fluorescently labeled bacteria (FLB) by Nygaard and Tobiesen (1993)
suggested that bacterivory can be an important source of P for the
haptophyte C. polylepis during blooms in coastal Norwegian waters.
Ingestion of P-rich bacteria by C. polylepis (6 cells h 1) in the
natural phytoplankton assemblage from surface waters was
estimated to have comprised about 60% of the total bacterial
grazing. Supporting laboratory experiments on several HAS fed FLB
or radiolabeled bacteria (RLB, 14C-amino acids) indicated that
bacterivory increased under P limitation, and provided significantly
more P than was needed to maintain equilibrium population growth
rates (k = 0.3 day 1) (Table 4). P-limited continuous cultures of H.
akashiwo and K. veneficum (as Gyrodinium galatheanum) consumed
up to 113 and 48 bacteria cell 1 h 1, respectively, and A. tamarense
consumed up to 706 bacteria cell 1 h 1 (Table 4). It should be noted,
however, that despite the short incubation time (up to 20 min), it is
possible that the HAS may have acquired radiolabel via excreted
bacterial carbon, which could have over-estimated bacterial
consumption.
Grazing on eubacteria by natural assemblages of HAS under
eutrophic conditions was examined more recently by Seong et al.
(2006), supported by culture experiments (Table 4). Ingestion rates
(1.2–20.6 cells alga 1 h 1; bacterial density 105–106 cells ml 1)
were comparable to those of co-occurring heterotrophic nano-
flagellates (HNFs), but lower than those of co-occurring ciliates (15–
713 cells ciliate 1 h 1). Combined grazing coefficients for these
algae considered collectively (0.04–1.71 day 1; potential removal of
4–82% [mean 38%] of the bacterial flora day 1) usually were higher
than those for HNFs or ciliates. Maximum ingestion and clearance
rates (except for low rates by Cochlodinium polykrikoides in coastal
waters) were comparable to those reported for HNFs on bacteria.
Ingestion rates of these dominant bloom species were positively
correlated with bacterial concentrations, but were not affected by
NO3 + NO2 or PO43 concentrations. Unlike Nygaard and Tobiesen
(1993), Seong et al. (2006) reported that H. akashiwo did ingest
bacteria at high PO43 concentrations, and suggested that the
contrasting results may have reflected intraspecific variability in the
different strains that were used (see Wood and Leatham, 1992;
Burkholder and Glibert, 2006), or differences in methodology (use of
FLBs versus RLBs). Seong et al. (2006) also noted that despite the
short incubation time (10–30 min), it was possible that the HAA may
have acquired the bacteria secondarily by preying upon HNFs that
had ingested them, so that there may have been indirect bacterial
consumption mediated through consumption of other prey. Thus,
the data suggest that directly or indirectly, HAS can be important
protistan bacterivores.
Substantial phagotrophy on the cyanobacterium Synechococcus
sp. was reported by Jeong et al. (2005a) for an array of HAS based
upon culture experiments, and (for several Prorocentrum spp.),
based upon application of the grazing coefficients to field
abundances of the algal grazers and prey to estimate potential
effects on the prey populations (Table 3). P. micans was estimated
to remove up to 17% of the Synechococcus prey population in 1 h,
whereas a mixture of Prorocentrum donghaiense and P. minimum
was estimated to remove potentially up to 98% of the Synecho-
coccus population within 1 h. Algal ingestion rates increased with
increasing prey concentrations up to prey saturation at 1.1–
1.4  106 cells ml 1. Their ingestion rates were comparable to
literature reports for ingestion rates of HNFs and ciliates on
Synechococcus spp., supporting Seong et al.’s (2006) inferences
 J.A.M. Burkholder et al. / Harmful Algae 8 (2008) 77–93 85

Table 4
Reported rates of ingestion of prokaryotes (eubacteria or cyanobacteria at saturating densities, 106 ml 1) by harmful algae at bloom densities (103–104 cells ml 1)a. Carbon
acquired refers to the percent (%) of carbon obtained from prey by the algal bacteriovores on a per-algal-cell basis; FLB – fluorescently labeled bacteria; n.a. – data not available

Taxon Ingestion rate Potential growth Grazing Carbon Conditions (reference)

(cells ind. 1 h 1) on bacteria coefficient acquired
(divisions day 1) (day 1) (% cell 1)

Dinoflagellates
Akashiwo sanguinea 62.9  5.4 n.a. n.a. n.a. Culture (bottle incubation method—cyanobacteria at low
light; Jeong et al., 2005a)b
Alexandrium catenella 29.5  6.7 n.a. n.a. n.a. Culture (bottle incubation method—cyanobacteria at low
light; Jeong et al., 2005a)
Alexandrium minutum 3.2  2.2 n.a. n.a. n.a. Culture (prey inclusion method—cyanobacteria at low
light; Jeong et al., 2005a)

Alexandrium tamarense 103.0–706.0 0.27–0.55 n.a. n.a. Culture (FLB or RLB; bacterivory at lower external phosphate
level; Nygaard and Tobiesen, 1993)
13.7  0.9 n.a. n.a. n.a. Culture (bottle incubation method—cyanobacteria at low
light; Jeong et al., 2005a)

Cochlodinium polykrikoides 6.7  1.1 n.a. 0.040–0.067b 1.6 Coastal waters—eubacteria (FLB; Seong et al., 2006)
(m 17.4) Culture (nutrient replete; FLB; Seong et al., 2006)
38.7  1.1 n.a. n.a. n.a. Culture (bottle incubation method—cyanobacteria at low
light; Jeong et al., 2005a)

Gonyaulax polygramma 42.4  2.8 n.a. n.a. n.a. Culture (bottle incubation method—cyanobacteria at low
light; Jeong et al., 2005a)
Gonyaulax spinifera 24.3  3.5 n.a. n.a. n.a. Culture (bottle incubation method—cyanobacteria at low
light; Jeong et al., 2005a)
Gymnodinium catenatum 30.2  2.8 n.a. n.a. n.a. Culture (bottle incubation method—cyanobacteria at low
light; Jeong et al., 2005a)
Gymnodinium impudicum 14.5  1.5 n.a. n.a. n.a. Culture (bottle incubation method—cyanobacteria at low
light; Jeong et al., 2005a)

Heterocapsa triquetra 3.1  0.4 n.a. 0.019–0.151b 3.6 Eutrophic estuaries—eubacteria (FLB; Seong et al., 2006)
(m 6.0) Culture (nutrient replete; FLB; Seong et al., 2006)
4.4  0.3 n.a. n.a. n.a. Culture (nutrient-replete; prey inclusion method—cyanobacteria
at low light – Jeong et al., 2005a; but note that Legrand et al., 1998
did not discern ingestion of Synechococcus sp.)

Karenia brevis-enriched 5.0  0.1 n.a. n.a. n.a. Culture (bottle incubation method—cyanobacteria at low light;
Jeong et al., 2005a)
N-depleted (14:10 L:D):
300 mmol photons m 2 s 1 0c m 0.35c 0 0 Culture (prey inclusion and 15N bottle incubation methods) –
43 mmol photons m 2 s 1 1.0–84.4 m 0.58 n.a. n.a Cyanobacteria at low to moderate light (Glibert et al., in press)

Karlodinium veneficum 0–48.0 0.00–1.89 n.a. n.a. Culture (RLB or FLB); bacterivory at lower external phosphate
level (Nygaard and Tobiesen, 1993)
Lingulodinium polyedrum 64.2  2.2 n.a. n.a. n.a. Culture (bottle incubation method—cyanobacteria at low
light; Jeong et al., 2005a)

Prorocentrum donghaiense 8.2  0.4 n.a. n.a. n.a. Culture (bottle incubation method—cyanobacteria at low
light; Jeong et al., 2005a)
7.4 n.a. n.a. n.a. Culture (prey inclusion method—cyanobacteria at low
light; Jeong et al., 2005a)

Prorocentrum micans 35.4  2.1 n.a. n.a. n.a. Culture (prey inclusion method—cyanobacteria at low
light; Jeong et al., 2005a)

Prorocentrum minimum 5.6  1.3d n.a. 0.113–0.850b 27.1 Eutrophic estuaries—eubacteria (FLB; Seong et al., 2006)
(m 21.9) Culture (nutrient replete; FLB; Seong et al., 2006)
5.9  1.2 n.a. n.a. n.a. Culture (cyanobacteria at low light; Jeong et al., 2005a)

Prorocentrum triestinum 5.6  1.3d n.a. 0.498b n.a. Eutrophic estuaries—eubacteria (FLB; Seong et al., 2006)
Scrippsiella trochoidea 7.1  1.1 n.a. n.a. n.a. Culture (bottle incubation method—cyanobacteria at low
light; Jeong et al., 2005a)

Haptophytese
Chrysochromulina polylepis 6.8–57 1.13–3.90 n.a. n.a. Culture (RLB or FLB); lower bacterivory at higher external
phosphate level (Nygaard and Tobiesen, 1993)

Chrysochromulina ericina
0–18.0 n.a. n.a. n.a. Eutrophic estuary (FLB); high bacterivory coincided with
lack of water-column phosphate at depths 3 m (Nygaard
and Tobiesen, 1993)
Culture (RLB or FLB); lower bacterivory at higher external
phosphate levels (Nygaard and Tobiesen, 1993)

Prymnesium parvum 0–5.8 n.a. n.a. n.a. Eutrophic estuary (FLB); high bacterivory coincided with
lack of water-column phosphate at depths 3 m (Nygaard
and Tobiesen, 1993)
0.7–4.6 0.20–0.33 n.a. n.a. Culture (RLB or FLB); lower bacterivory at higher external
phosphate level (Nygaard and Tobiesen, 1993)
0.01–4.6 n.a. n.a. n.a. Culture (FLB); higher bacterivory under P stress
(Legrand et al., 2001)
86 J.A.M. Burkholder et al. / Harmful Algae 8 (2008) 77–93

Table 4 (Continued )
Taxon Ingestion rate Potential growth Grazing Carbon Conditions (reference)
(cells ind. 1 h 1) on bacteria coefficient acquired
(divisions day 1) (day 1) (% cell 1)

Raphidophytes
Chattonella ovata 11.2  1.6 n.a. n.a. 1.4 Eutrophic estuaries—eubacteria (FLB; Seong et al., 2006)
(m 24.5) Culture (nutrient replete; FLB; Seong et al., 2006)

Heterosigma akashiwo 6.9  0.9 0.00–1.84 0.046–0.857 12.5 Eutrophic estuaries—eubacteria (FLB; Seong et al., 2006)
(m 11.7) Culture (nutrient replete; FLB; Seong et al., 2006)
0–113.0 n.a. n.a. n.a. Culture (RLB or FLB); bacterivory at lower external phosphate
level (Nygaard and Tobiesen, 1993)
a
Response to eubacteria was tested by Nygaard and Tobiesen (1993) in a natural assemblage from depths 0–3 m (low-P environment, P < 0.05 mM PO43 P) and from 4 m
(higher P environment, 0.35 mM PO43 P), along with supporting data from selected harmful algal species maintained in culture (130–300 mmol photons m 2 s 1; low-P or
high-P conditions ranging from 0.3 to 1.6 mM PO43 P). Seong et al. (2006) also tested harmful algal response to eubacteria in natural assemblages and cultures (nutrient-
enriched conditions except for field populations of Cochlodinium polykrikoides; culture light at 30 mmol photons m 2 s 1). Legrand et al. (2001) examined bacterivory by
Prymnesium parvum in low-P and P-replete cultures. Harmful algal response to Synechococcus prey was tested under nutrient-replete conditions by Jeong et al. (2005a) (f/2
medium, low light [30 mmol photons m 2 s 1, saturating prey densities at 1.1 to 2.3  106 cells ml 1), and under N-depleted conditions by Glibert et al. (in press) (43–
300 mmol photons m 2 s 1, initial prey densities 105 to 108 cells ml 1). Ingestion rates (cells individual 1 h 1) are given as means  1 SE, or as maxima (m) depending upon
available data.
b
Means given from various eutrophic areas.
c
Growth of K. brevis at 300 mmol photons m 2 s 1 was phototrophic, with negligible bacterial consumption.
d
For field populations of P. minimum and/or P. triestinum.
e
Chrysochromulina leadbeateri has been observed to take up fluorescently labeled bacteria (Eikrem and Nygaard, unpublished data—see Johnsen et al., 1999).

about the potential importance of harmful algae as bacteriovores
in eutrophic environments. From laboratory experiments, Jeong
et al. (2005a) estimated that 5 Synechococcus cells harmful algal
cell 1 h 1 could be grazed by the mixotroph Karenia brevis, while
Glibert et al. (in press) estimated that this HAS could potentially
remove ca. 1–84 Synechococcus cells h 1 (0.5–40% of the cellular N
requirements for K. brevis), depending upon the grazer:prey ratio.
3.2.2. Eukaryote prey
Harmful algae in eutrophic estuaries and coastal marine waters
consume diverse eukaryotic prey (Table 2). Although some of the
grazer/prey relationships were observed decades ago many
mixotrophs that were thought to be strictly phototrophs have
been uncovered only recently (e.g., Jeong et al., 2004, 2005a,b,c).
Phagotrophy is important in the nutritional ecology of these
harmful algae, and the combination of phototrophy + phagotrophy
supports higher growth than phototrophy alone, as illustrated by
the following examples.
In situ grazing rates were estimated by Bockstahler and Coats
(1993a,b) in digestion experiments with Protargol stain for the
large dinoflagellate A. sanguinea (as Gymnodinium sanguineum;
cells 60 mm  45 mm, biovolume 79,000 mm3) grazing on
oligotrich ciliates (length < 20 mm) in eutrophic Chesapeake
Bay. From integrated station and transect averages, A. sanguinea
removed 34% and 24%, respectively (medians 17% and 22%,
respectively) of the oligotrich ciliate population daily. Daily
consumption of ciliates supplied an average of 2.5% (maximum
11.6%) of the grazer carbon cell 1. Mixotrophy also was estimated
to supply 4% (maximum, 18.5%) of the grazer cellular N daily, and
15% of its N requirements for asexual reproduction. Thus,
mixotrophy may help balance the N requirements for A. sanguinea,
and may confer an advantage over strictly phototrophic algae
under N stress (Bockstahler and Coats, 1993a,b).
Grazing by another large dinoflagellate species, C. furca (cells
250 mm  25 mm, biovolume 3400 mm3) was estimated in the
same ecosystem using fluorescently labeled ciliate prey (Smalley
and Coats, 2002). Feeding rates ranged from 0 to 0.11 prey
dinoflagellate 1 h 1, and grazing by C. furca was estimated to
remove an average of 67% of the ciliates Strombilidium spp. per day.
Ingestion rates were positively correlated with prey abundance
and DON, but negatively correlated with depth, DIP, and the
DIP:DIN ratio. Prey consumption contributed an average of 4.6% of
cell C (maximum 36%), 6.5% of cell N (maximum 51%), and 4.0% of
cell P (maximum 32%). The authors inferred that phagotrophy can
be important to this species when inorganic nutrients are limiting,
providing a competitive advantage over strictly phototrophic
species.
Many phagotrophic dinoflagellates consume cryptophyte prey
(Hansen, 1991; Burkholder and Glasgow, 1995; Li et al., 1996,
2000a; Lewitus et al., 1999a; Jeong et al., 2004, 2005b,c; Park et al.,
2006; Adolf et al., 2008), which are often abundant in eutrophic
estuaries (Mallin, 1994; Li et al., 1996, 2000b; Bergmann, 2004;
Jeong et al., 2005b,c; Rothenberger, 2007). Some dinoflagellates
even have chloroplasts or pigments that clearly were derived from
cryptophytes (e.g., Hu et al., 1980; Wilcox and Wedemayer, 1984;
Schnepf et al., 1989; Skovgaard, 1998; Hackett et al., 2003). Various
studies of feeding on cryptophytes by HAS have been conducted in
natural phytoplankton assemblages in Chesapeake Bay, focusing
on the small dinoflagellates P. minimum (cells 22 mm  15 mm,
biovolume 2600 mm3) and K. veneficum (cells 15 mm  10 mm,
biovolume 340 mm3). P. minimum was examined for ingestion of
cryptophyte material as orange-fluorescent inclusions (OFIs)
(Stoecker et al., 1997). Mixotrophy was higher in spring than in
summer (frequency of OFIs 510% and 50%, respectively). Thus, in
natural assemblages, ingested cryptophyte material was observed
in up to 50% of the P. minimum cells. The frequency of OFIs was
positively correlated with cryptophyte densities, but OFIs were not
abundant in all populations of P. minimum when cryptophyte
densities were high. Ingestion was highest in the afternoon and
evening and lowest in the morning, and addition of NO3 + PO43
inhibited feeding. In P. minimum, then, feeding apparently is a
mechanism for obtaining organic nutrients rather than for
supplementing C during light limitation, and ingestion of
competitors for light and nutrients may facilitate bloom formation
in fluctuating nutrient regimes (Stoecker et al., 1997).
Phagotrophy in K. veneficum (reported as G. galatheanum or
Karlodinium micrum) has been the subject of intensive research
focus in Chesapeake Bay. Li et al. (2000a) found that K. veneficum
became phagotrophic at sub-optimal light and/or nutrient condi-
tions. Maximal rates of ingested cryptophytes per dinoflagellate
cell in field assemblages ranged from 0.04 to 0.47, and the mean
ingested prey was positively correlated with cryptophyte abun-
 J.A.M. Burkholder et al. / Harmful Algae 8 (2008) 77–93
dance. From field and laboratory data, incidence of K. veneficum
feeding was positively correlated with prey density and NO3
concentration, and negatively correlated with depth, salinity, and
PO43 concentration. Feeding activity of K. veneficum also
increased hyperbolically with increasing light intensity to a
maximum at 60 mmol photons m 2 s 1, with no apparent photo-
inhibition (800 mmol photons m 2 s 1). Addition of NO3 or
NO3 + PO43 inhibited feeding by K. veneficum in a natural
assemblage, suggesting that N limitation may induce feeding.
Incidence of feeding was negatively related to the DIP:DIN ratio,
suggesting that P limitation may also induce feeding. Ingestion of
cryptophyte prey by cultured K. veneficum was higher in low-NO3
or low PO43 or low N + P media. Nevertheless, feeding, although at
reduced rates, still occurred in nutrient-replete cultures. Overall, N
and/or P deficiency, or N:P ratios that were substantially higher or
lower than the optimum 10:1 ratio for this dinoflagellate led to an
increase in cellular C content and increased feeding activity. Thus,
feeding in K. veneficum supplements major nutrients (N, P) needed
for photosynthetic C assimilation, and may also supplement C
metabolism or assist in acquiring trace organic growth factors (Li
et al., 2000a).
Adolf et al. (2008) determined that K. veneficum blooms in
Chesapeake Bay were positively correlated with cryptophyte
abundance. Ingestion rates among multiple strains of cultured K.
veneficum ranged from 0 to 4 prey dinoflagellate 1 day 1, and
cultured toxic strains were capable of consuming an array of
cryptophyte species (31–421 mm3 cell 1). The authors hypothe-
sized from these data and supporting studies (e.g., Adolf et al.,
2003, 2006a) that cryptophyte prey abundance is a key factor that
support blooms of K. veneficum in eutrophic habitats, and perhaps
blooms of other dinoflagellates as well. This work built from a
previous intensive effort with cultures to examine the balance
between phototrophy and heterotrophy during mixotrophic
growth of K. veneficum on radiolabeled cryptophyte prey (Adolf
et al., 2006a). The authors used this dinoflagellate to experimen-
tally assess the physiological role of feeding by Type II mixotrophs,
the category, as mentioned, that includes many harmful algal
mixotrophs (Stoecker, 1998). Growth rates of mixotrophic K.
veneficum (0.52–0.75 divisions day 1) were comparable to or
exceeded its maximum growth rate in phototrophic mode
(0.55 divisions day 1). During mixotrophic growth, cellular photo-
synthetic performance (pg C cell 1 day 1) was 24–52% lower than
during phototrophic growth; thus, reduced photosynthetic effi-
ciency accompanied grazing. Thus, heterotrophic metabolism can
dominate mixotrophic growth in this species (Adolf et al., 2006a).
In a series of laboratory experiments, Jeong et al. (2004,
2005b,c) estimated ingestion rates and grazing coefficients for one
strain each of six cultured harmful algae (C. polykrikoides, G.
polygramma, Heterocapsa triquetra, P. donghaiense, P. micans, L.
polyedrum), from eutrophic coastal waters of Korea, given
cryptophytes or small dinoflagellates (Scrippsiella trochoidea,
Prorocentrum spp.) as prey (Table 4). The data were considered
in combination with field abundances of the algal grazers and prey
to estimate potential effects on prey populations in natural
assemblages. Estimated prey removal was lowest for L. polyedrum
(1.1% of the S. trochoidea population and 2.6% of the Prorocentrum
spp. removed per hour) and H. triquetra (9.1% of the cryptophytes
removed per hour), intermediate for G. polygramma and C.
polykrikoides (38–53% of the cryptophytes removed per hour),
and highest for P. donghaiense (93% of the cryptophytes removed
per hour). The data suggest that grazing activity by these harmful
algae can substantially affect algal prey populations (Jeong et al.,
2004, 2005b,c).
A final example focuses upon N. scintillans (‘‘green form’’ from
southeast Asia) as described by Hansen et al. (2004). The growth
87
rate of freshly collected phototrophic cells without prey was 0.058
and 0.14 day 1 at 45 and 150 mmol photons m 2 s 1, respectively
(12 h:12 h L:D). In mixotrophic mode when given potentially toxic
P. bahamense var. compressum as prey, the growth rate of N.
scintillans increased to 0.9 and 0.24 day 1, respectively, at these
light intensities. Ingestion rates measured at the higher light
intensity increased linearly with prey concentration. Phagotrophy
was estimated to contribute significantly (30%) to growth of N.
scintillans only at high prey concentrations. Thus, blooms of N.
scintillans (1–10 cells ml 1) may significantly affect population
dynamics of the HAS P. bahamense var. compressum (Hansen et al.,
2004).
4. Overall findings, future research targets, and forecast
The available data indicate that mixotrophy occurs in most
harmful algae examined thus far from eutrophic estuarine and
marine coastal waters, through both direct and indirect responses
to increased food supplies. The points that feeding can be sporadic,
chloroplasts can obscure food vacuoles, and many harmful algae
are still treated routinely as strict phototrophs in culture, support
Stoecker et al.’s (2006) prediction that more HAS probably will be
found to be mixotrophs. Based upon studies such as the above
examples, mixotrophy is important in the nutrient acquisition and
growth of HAS and, therefore, likely is important in the
development and maintenance of their blooms in eutrophic
habitats. In addition, the data support the premise that mixo-
trophic harmful algae are significant grazers and predators in
eutrophic habitats. Yet, for most of these species quantitative data
are lacking, especially relating laboratory information to natural
field assemblages, and the relative importance of photosynthesis,
dissolved organic nutrients, and feeding remain unknown. Studies
are needed that simultaneously, quantitatively assess the roles of
phototrophy, osmotrophy and phagotrophy in the nutritional
ecology of HAS in organic substrate-rich and prey-rich eutrophic
habitats. These data should span bloom initiation, development,
and senescence.
Mixotrophy may increase trophic efficiency (Sanders, 1991),
and is an important pathway for nutrient cycling in some systems
(Bird and Kalff, 1987; Maranger et al., 1998; Stibor and Sommer,
2003). Beyond HAS, the phytoplankton assemblages of turbid,
eutrophic coastal habitats may fundamentally function within a
multi-tiered system of mixotrophy. Cloern and Dufford (2005), for
example, reported that in San Francisco Bay, mixotrophic algae are
major components of the phytoplankton assemblage including
two abundant non-diatom groups, dinoflagellates (notably,
mixotrophic species of Dinophysis, Prorocentrum, Alexandrium)
and cryptophytes. They suggested that in such turbid, light-limited
estuaries, the ubiquity and persistence of cryptophytes may reflect
their ability to consume bacteria to supplement their nutrition.
Models that include the role of mixotrophy are needed to gain
insights about the nutritional influences on harmful algae in
eutrophic habitats in efforts to forecast HABs (Hood et al., 2006).
Thingstad et al. (1996, p. 2108) described the historic and ongoing
approach: ‘‘. . .conceptual understanding of the organic matter
dynamics in aquatic microbial communities comprises two major
pathways: one passing through the classical grazer food chain, and
the other through the microbial loop. . .This scenario is still used as
a simple way of compartmentalizing grazing experiments and
carbon budgets, and in dynamic ecological models.. . .It is usually
deemed an important [modeling] prerequisite that photoauto-
trophic and heterotrophic microorganisms are distinguished.
Because mixotrophs play a minor role in the conceptual frame-
work, they are most often not considered’’.
88 J.A.M. Burkholder et al. / Harmful Algae 8 (2008) 77–93
In attempts to gain predictive capability about HABs in
eutrophic estuarine and marine waters, a few conceptual models
have been developed that invoke the importance of cryptophytes
and mixotrophy. For example, Lewitus et al. (1999a) stressed the
importance of predatory grazing on cryptophytes by toxic strains
of P. piscicida in eutrophic habitats where this harmful alga thrives
(Burkholder and Glasgow, 1997; Burkholder et al., 2001a; Table 1).
The conceptual model was expanded by Glasgow et al. (2001) to
consider the seasonal importance of cryptophytes and other algal
prey in supporting Pfiesteria populations in the absence of other
preferred prey such as schools of juvenile Atlantic menhaden
(Brevortia tyrannus). More recently, Adolf et al. (2008) suggested
that cryptophytes may drive the overall population toxicity of
blooms of K. veneficum. The authors described a conceptual model
of key factors that promote blooms of K. veneficum including
eutrophic estuarine environments; co-occurrence of cryptophytes
and K. veneficum; rapid response of cryptophytes to nutrient inputs
and other favorable environmental factors, and increased cell
production; and mixotrophic predation of K. veneficum on
cryptophytes, assisted by an allelopathic effect of its toxin on
the prey. Schools of juvenile menhaden and cryptophyte blooms
occur in eutrophic estuaries (Mallin, 1994; Burkholder et al.,
2001a). Whereas toxic strains of Pfiesteria spp. prefer fish over
cryptophytes and other prey relative to nontoxic strains (Bur-
kholder et al., 2001a,b), toxic strains of K. veneficum have shown
higher feeding capacity on cryptophytes relative to nontoxic
strains (Adolf et al., 2008). For these and other HAS, mixotrophic
capability, together with allelopathic effects on predators and
phytoplankton competitors from the toxins produced by these
organisms, could help to promote blooms of toxic strains (e.g.,
Carlsson et al., 1990; Lewitus et al., 1999a, 2006; Burkholder et al.,
2001b; Granéli and Johansson, 2003; Stoecker et al., 2002;
Skovgaard and Hansen, 2003; Adolf et al., 2006b, 2007; Granéli
et al., 2008). Positive feedbacks from reduced grazing rates could
further promote blooms (Sunda et al., 2006).
Beyond conceptual models, few attempts have been made to
model the population dynamics of mixotrophic algal protists
(Thingstad et al., 1996) or, more specifically, mixotrophic harmful
algae. As examples of the latter, Stickney et al. (2000) developed
mathematical formulations to assess impacts of the three
previously mentioned types of mixotrophs (with harmful estuar-
ine and marine algae mostly in Type II) on microbial food webs.
Their approach considered idealized steady-state open-ocean and
estuarine/coastal habitats. The models indicated that mixotrophy
represents a unique resource niche during the summer season;
that mixotrophs tend to decrease primary production because of
uptake of N from the DON pool; and that this decrease may be
compensated for by the DON-supported primary production. Hood
et al. (2006) developed a semi-idealized marine ecosystem model
to examine the population dynamics of strains of Pfiesteria that
were in actively toxic versus nontoxic mode (the latter, with
kleptochloroplasts). The model indicated that nontoxic blooms
would occur in more turbulent, inorganic nutrient-rich conditions,
whereas toxic blooms would be more likely in calm, organic
nutrient-rich conditions under low grazing pressure.
Despite the fact that mixotrophy is widespread across nutrient
gradients from oligotrophic to eutrophic and salinity gradients
from freshwater to marine, the importance of mixotrophy to bloom
formation and toxicity of most HAS has received little attention
(Stoecker et al., 2006). Through insights gained in recent years
about the nutritional ecology of some HAS in eutrophic habitats,
and through the strength of long-term data bases that are available
for some systems, it is now possible to explore, from a comparative
perspective across major estuarine and marine coastal ecosystems,
how nutrient loading is contributing to the increasing abundance
of HAS. Detailed time series analyses can also be used to examine
whether prey abundance triggers blooms of mixotrophic HAB
species (Adolf et al., 2008). Specific hypotheses can be tested about
influences of land use changes, water quality changes and
interactive factors, through application of GIS models, multivariate
statistics, and high-frequency measurements (e.g., Rothenberger,
2007; Glibert et al., 2008b). An overall forecast that can be tested,
as well, is that harmful mixotrophic algae will increase in
abundance as their food supplies increase in many estuaries and
coastal waters of the world that are sustaining chronic, increasing
anthropogenic nutrient enrichment.
Acknowledgments
This is a contribution of the Global Ecology and Oceanography
of Harmful Algal Blooms (GEOHAB) core research project on HABs
and eutrophication. Funding support was provided by the Park
Foundation, the Center for Environmental Science Foundation of
the University of Maryland, and the U.S. EPA. This work is
University of Maryland Center for Environmental Science (UMCES)
contribution number 4205.[SS]
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