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SPECIAL REVIEW

Algal and cyanobacterial secondary metabolitesin freshwaters: a comparison of allelopathic compounds

and toxins

J O S EP HI NE LEF LA I VE A ND LO I C TEN- HA GE

Laboratoire d’Ecologie des Hydrosystemes, Universite Paul Sabatier, Toulouse, France

SU M M A R Y

1. The photoautotrophic micro-organisms collectively termed ‘micro-algae’ (including

micro-eukaryotes and cyanobacteria) are known to produce a wide range of secondary

metabolites with various biological actions. A small subset of these compounds has been

identified. Some of them, termed allelopathic compounds, have been shown to play a role

in allelopathy, defined here as inhibitory effects of secondary metabolites against eithercompetitors or predators. Freshwater cyanobacteria also produce some secondary

metabolites, termed toxins, which are highly toxic for animals.

2. While allelopathic compounds play a role in the interactions between the emitter

organisms and their direct competitors or predators, toxins are categorised according to

their toxic effect on several organisms, including some that may not be present in their

immediate environment. However, these two definitions are not mutually exclusive. This

review considers the evolutionary, ecological and physiological aspects of the production

of allelopathic compounds by micro-algae in freshwaters, and compares the characteristics

of allelopathic compounds with those of toxins.

3. Allelopathic compounds include alkaloids, cyclic peptides, terpens and volatile organic

compounds. Toxins include alkaloids, cyclic peptides and lipopolysaccharides. No

allelopathic compound type is associated with a particular phylogenetic group of algae. Incontrast, freshwater toxins are only produced by cyanobacteria belonging to a restricted

number of genera. Allelopathic compounds have various modes of action, from inhibition

of photosynthesis to oxidative stress or cellular paralysis. Toxins are often enzyme

inhibitors, or interfere with cell membrane receptors.

4. The ecological roles of allelopathic compounds have been well identified in several

cases, but those of toxins are still debated. In the light of descriptions of negative effects of

toxins on both micro-invertebrates and photoautotrophic organisms, we suggest that at

least some toxins should actually be considered as allelopathic compounds. Further

research on toxic secondary metabolites in freshwaters is now needed, with emphasis

on the ecological effects of the compounds in the immediate environment of the emitter

algae.

Keywords: allelochemicals, allelopathy, micro-algae, phototrophic micro-organisms, toxins

Correspondence: Josephine Leflaive, Laboratoire d’Ecologie des Hydrosystemes, UMR CNRS 5177, Universite Paul Sabatier,

118 route de Narbonne, 31062 Toulouse Cedex 9, France. E-mail: [email protected]

Freshwater Biology (2007) 52, 199–214 doi:10.1111/j.1365-2427.2006.01689.x

Ó 2006 The Authors, Journal compilation Ó 2006 Blackwell Publishing Ltd 199

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Introduction

The term ‘allelopathy’, from the Greek word ‘allelon’

meaning mutual and ‘pathos’ meaning harm or affec-

tion, was introduced by Molisch (1937) to designate the

process by which one plant influences another by

chemical means. Rice (1984) has included micro-organisms (bacteria, fungi and micro-algae) in this

definition and considered both positive and negative

effects on the target organism. By analogy with plant–

insect interactions, predator defences are sometimes

included in the definition of allelopathy (Rizvi & Rizvi,

1992). Because of the complexity of the interactions in

natural ecosystems, definitive evidence for allelopathy

in the field is almost impossible to obtain. Nevertheless,

many field and laboratory studies have pointed to the

existence of allelopathic interactions, especially due to

secondary metabolites produced by micro-algae (see

reviews by Maestrini & Bonin, 1981; Lewis, 1986).

For convenience here we follow the traditional

practice of grouping both cyanobacteria and

photoautotrophic micro-eukaryotes under the term

‘micro-algae’ even though these two groups are

phylogenetically quite distinct. Indeed, we emphasise

ecological aspects and in that context no distinction is

needed between cyanobacteria and photoautotrophic

micro-eukaryotes. Freshwater algae, like marine algae,

are known to produce a wide range of secondary

metabolites (Table 1). These diverse compounds are

released into the environment during algal growth orat cell lysis, but for many no biological or physiological

activity has been ascribed to date. Those compounds

that affect in a positive or negative way other organ-

isms and hence affect the structure of ecosystems are

termed allelochemicals. Among allelochemicals, some

compounds that have been shown to have anti-algal,

antibiotic, antifungal and anti-predator activities are

allelopathic compounds (Smith & Doan, 1999; Gross,2003; Legrand et al., 2003). Allelopathic activity is a

widespread phenomenon amongst freshwater primary

producers (Inderjit & Dakshini, 1994). The production

of allelopathic compounds is highly species-, and even

strain-dependent. Few compounds have been chemi-

cally identified to date, despite the increasing number

of allelopathic interactions described between micro-

algae. In lakes, allelopathy is suspected to play an

important role in the establishment of algal successions

and in the formation and ending of blooms (Keating,

1977; Vardi et al., 2002; Takamo et al., 2003). In rivers

and streams, where algal exudates may be rapidly

carried away by the current, allelopathy may be less

important in planktonic communities, although it can

still be a factor in benthic communities. Some allelo-

chemicals which present acute toxicity against animals

(Jochimsen et al., 1998; Griffiths & Saker, 2003) are

grouped under the term ‘toxin’. In freshwaters these

are mainly produced by cyanobacteria (Carmichael,

1997). Because they represent a health hazard, increas-

ing with the eutrophication of inland waters which

facilitates the formation of dense blooms of toxic

cyanobacteria, these secondary metabolites have beenextensively studied during the last 20 years.

Table 1 Main algal groups and the chemical natures of associated toxic and allelopathic secondary metabolites produced in fresh-

water (bold) and marine environments

Taxonomic groups Phycotoxins Allelopathic compounds

Dinophytes Alkaloids, polyethers Fatty acids, polyethers, unidentified compounds

Euglenophytes – –

Green algae Alkaloids, esters Fatty acids, unidentified compounds

Haptophytes Polyethers Polyunsaturated aldehydes, polyethers

Ochrophytes

Bacillariophyceae (diatoms) Domoic acid Polyunsaturated fatty acids, polyunsaturated aldehydes

Phaeophyceae – Fatty acid derivatesRaphidophyceae Fatty acids? polyethers? Aldehydes

Rhodophytes Domoic acid Unidentified compounds

Cyanobacteria

Chroococcales Cyclic peptides, alkaloids, LPS Peptides

Nostocales Cyclic peptides, alkaloids, LPS Cyanobacterin, nostocyclamide, nostocyclamide M,

nostocine A

Oscillatoriales Cyclic peptides, alkaloids, LPS –

Stigonematales LPS Fischerellin A and B, alkaloids

LPS, lipopolysaccharides.

200 J. Leflaive and L. Ten-Hage

Ó 2006 The Authors, Journal compilation Ó 2006 Blackwell Publishing Ltd, Freshwater Biology, 52, 199–214

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This review considers the evolutionary, ecological

and physiological aspects of the production of allel-

opathic compounds by micro-algae (including cyano-

bacteria) in freshwater environments, and compares

the characteristics of allelopathic compounds with

those of toxins. In the light of recent studies on the

pattern of secreted cyanobacterial peptides (Welker,

Christiansen & van Dohren, 2004), toxins may appearas a part of a more important group of bioactive

compounds comprising both toxic and non toxic

compounds. We argue that some freshwater algal

toxins should be considered as allelochemicals active

against both competitors and predators, i.e. allelo-

pathic compounds (Fig. 1).

Conceptual and methodological aspects

of allelopathy

Evolutionary aspects

Allelopathy maybe either the result of a direct selection

of secondarymetabolism,or a secondaryprocess where

the biosynthesis of molecules was originally selected

for other purposes (Reigosa, Sanchez-Moreiras &

Gonzales, 1999). It may have developed when the

emitter organism first released some compounds in

order to avoid their autotoxicity or when mechanisms

of self-resistance evolved, which could then have led to

a secondary advantage. In the case of terrestrial plants,

allelopathy may have served primarily to protect the

plant against attack by fungi or micro-organisms. For

those organisms, the synthesis of defence metabolites is

constitutive or inducible (Tang et al., 1995). Most

authors adopt the view that allelopathy originated as

a byproduct of other ecological processes.A major issue in evaluating the impact of allelopathy

is that for long-term co-existence in the same habitat,

organisms are necessarily adapted to each other. This

implies that allelopathic interactions are transitory and

in most cases not apparent because of co-evolution

(Reigosa et al., 1999). Allelopathy should become

apparent in cases of abiotic stress, invasion by exotic

organisms, synthesis of a new molecule by the emitter

organism, delay in the target adaptation, or accumu-

lation of allelopathic compounds in the environment.

Stress can enhance both the production of allelopathic

compounds and the susceptibility of the target. In spite

of adaptations, Legrand et al. (2003) considered that

allelopathic interactions should be widespread in

aquatic environments. Natural selection should favour

allelopathic compound production, given that this

reduces competition and thus improves resource

availability. Some targets may become adapted to an

allelopathic compound, but in a complex community

Aquatic plants

Emitting algae

AlgaeZoobenthos

Zooplancton

Terrestrial vertebrates

Fishes

Toxins

Competitors Predators

Allelochemicals

Health hazard

Allelopathic compounds

Direct environment of the emitter

?

?

Fig. 1 Examples of interactions involvingallelochemicals (allelopathic compounds

and toxins) produced by freshwaters

micro-algae.

Allelopathic compounds and toxins in freshwaters 201


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with a mix of different species some will remain

sensitive to the compound. This can confer a weak but

sufficient advantage for the emitter. Costs associated

with the production of an allelopathic compound may

decrease this advantage. Their existence is suspected

but they are still unidentified (Legrand et al., 2003).

Several instances have been reported that illustrateco-evolution between aquatic freshwater micro-organ-

isms with respect to chemical interactions; for exam-

ple, physiological resistance to toxins in the freshwater

crustacean Daphnia magna compared with acute sen-

sitivity of zooplankton that do not coexist with toxin-

producing cyanobacteria (Kurmayer & Juttner, 1999).

In some cases co-evolution is marked by the existence

of reciprocal interactions, two organisms producing

some compounds acting each on the other organism

(Kearns & Hunter, 2000, 2001; Vardi et al., 2002).

In water, chemical information is transmitted by

diffusion and advective lamina flow (Wolfe, 2000).

Allelopathic compounds with a small molecular

weight are favoured because of their faster diffusion.

In aquatic environments the distances between cells

are quite important and a major problem in the pelagic

environment is dilution of the secreted products. Thus

Lewis (1986) assumed that allelopathy is not an

evolutionarily stable strategy for phytoplankton. Gi-

ven the distances between cells, the large number of

cells present and the importance of viscous forces at

that scale, algae of the same species but also those of

other species could benefit from the presence of anallelopathic compound (what Lewis, 1986, called

‘distributed benefits’). These algae avoid costs associ-

ated with the production of allelopathic compounds

but have all the benefits. Group selection of individuals

sharing the same genome is not a satisfactory explan-

ation because, unlike terrestrial plants and benthic

algae, phytoplankton lack any fixed spatial association

(Lewis, 1986). As a consequence, Lewis (1986) consid-

ered only the ‘allelochemical-signal hypothesis’ to be

realistic; for the emitter organism, the allelopathic

compound is a byproduct of the metabolism, but for

the target, it is an indicator of the position of the

environment which has an effect on its life cycle. In the

light of recent work on the diverse mode of action of

allelopathic compounds inside the target cell (see

Modes of action), Lewis’s theory looks quite unrealis-

tic. One of his postulates, the importance of viscous

forces, is now questioned by advances in flow

mechanics whereby the environment in the vicinity

of algal cells is now considered rather stable at the

relevant scale. The problem of ‘distributed benefits’ is

thus reduced and the advantages from production of

allelopathic compounds appear high enough for it to

be selected.

In benthic habitats, the physical constraints are

different. Epilithic biofilms in rivers are microbialaggregates formed by an association of both photo-

autotrophs and heterotrophs (both prokaryotes and

eukaryotes) surrounded by a polysaccharide matrix. In

such a habitat, cellular distances are shorter or even

zero (direct cell contacts). Molecules transferred by

direct contact or through the polysaccharide matrix can

be more lipophilic than in the water column, which

means reduced costs for the emitter cell. Inside the

biofilm, in addition to nutrient competition, the photo-

autotrophic micro-organisms are in competition for

space through the access to an anchor zone, to light or

to nutrient-enriched zones. Space competition adds a

supplementary selective pressure which can lead to the

formation of allelopathic interactions. Hence biofilms

appear quite favourable environments for the appear-

ance of allelopathy and for its study (Juttner, 1999).

Methods

Research on allelopathy in aquatic environments is

focused on: (1) demonstration of the production of

allelopathic compounds by an organism; (2) under-

standing factors influencing production of com-pounds; (3) identification and characterisation of the

compounds and their pathway of biosynthesis and (4)

estimation of the role and importance of allelopathic

interactions in the field. These objectives necessitate a

wide range of methods, from classical culturing to

modern chemical investigations. Use of molecular

methods will certainly increase in the near future. One

common difficulty, particularly for field studies, is to

separate competition from allelopathy. Yet first

reports of allelopathic interactions in aquatic environ-

ment often came from observations in the field

(Akehurst, 1931; Hutchinson, 1944; Keating, 1977).

One of the most widely used methods to identify

allelopathic interactions is cross-culturing: a target

alga is cultured in a medium enriched with filtrate

from the culture of another alga whose allelopathic

activity is being investigated. Whatever bioassay is

chosen, an important issue is the choice of the

indicator or target strain. Species that co-exist in the



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field should be well-adapted to each other and

consequently allelopathy is rarely apparent amongst

species belonging to the same community (Reigosa

et al., 1999). Allelopathic interactions that are not

detectable because of adaptation may become evident

under physico-chemical stress. In light-, nutrient-, or

space-limited conditions the production of an allelo-pathic compound may be enhanced while the target

may become more sensitive. Legrand et al. (2003)

suggested that nutrient depletion effects should be

avoided in studying allelopathy. Yet, environmentally

realistic, nutrient-depleted conditions for both donor

and target strains could yield useful information if the

effects of competition are well separated from those of

allelopathy. The isolation of allelopathic compounds

and the determination of their structure require

classical chemical methods such as nuclear magnetic

resonance, X-rays, UV and mass spectroscopy, high

performance liquid chromatography, gas chromatog-

raphy/mass spectrometry. These methods are coupled

with a bioassay to determine which fraction contains

the active compound. The bioassay needs to be

sensitive, easy to perform and environmentally rele-

vant. A major difficulty for the isolation of bioactive

compounds is that they are often produced in very low

amounts, as producing a low amount of a highly active

compound is a more cost-effective strategy.

Modes of action


The mode of action of the compound depends on the

nature of the interaction between donor and target

organisms, the activity of allelopathic compounds

being directed against either competitors or predators.

In the context of competition, which is mainly with

other photoautotrophic organisms, allelopathic com-

pounds may inhibit photosynthesis, kill the compet-

itor or exclude it from the donor vicinity (settling,

paralysis). As a predator defence, allelopathic com-

pounds would be efficient by poisoning grazers or by

inducing resistant forms in the other algae. In the

field, allelopathic compound modes of action are quite

various (Table 2). Some examples are given here to

illustrate this variability.

Inhibition of photosynthesis. Growth inhibition, and

eventually, death by inhibition of photosynthesis is a

quite widespread mode of action for cyanobacteria.

Cyanobacterial allelopathic compounds are generally

soluble in organic solvents, insoluble in water and

have a low molecular weight. These properties help

them to reach the thylakoid membranes where pho-

tosynthesis occurs (Smith & Doan, 1999). Allelopathic

compounds produced by the cyanobacteria Scytonemahofmanni (cyanobacterin) and Trichormus doliolum

(unidentified compound) both inhibit the photosys-

tem II-mediated photosynthetic electron transfer

(Gleason & Baxa, 1986; von Elert & Juttner, 1996,

1997). Fischerellin A, produced by Fischerella muscico-

la, is another compound acting against the PSII (Gross,

Wolk & Juttner, 1991) but here four different targeted

sites have been identified (Srivastava, Juttner &

Strasser, 1998). In addition to its action against

photosynthesis, fischerellin A is toxic for fungi at

higher concentrations, although the mode of that

action is still unknown (Hagmann & Juttner, 1996).

Enzyme inhibition. Many aquatic organisms produce

extracellular enzymes that are essential for nutrition.

Juttner & Wu (2000) reported that 20% of the

cyanobacteria isolated from freshwater biofilms in

Taiwan could inhibit a-glucosidase activity. This may

be a means to inhibit the hydrolysis of the mucilage

produced by the cyanobacteria. The range of enzymes

targeted by this activity and the implied compounds

were not identified.

Cellular paralysis. The cyanobacterium Anabaena flos-

aquae can induce paralysis and thus faster settling of

the cells of the competing motile green alga Chlamydo-

monas reinhardtii (Kearns & Hunter, 2001). This may

create a competitor-free zone for the cyanobacterium.

Inhibition of nucleic acid synthesis. Two alkaloids

isolated from Fischerella sp. (12-epi-hapalindole E)

and Calothrix sp. (calothrixine A) exhibit an inhibitory

activity directed against the RNA polymerase of

bacteria, fungi and green algae (Doan et al., 2000).

This activity is strongly dependent on polymerase

concentration and leads to growth inhibition because

of protein synthesis inhibition. Calothrixine A also

inhibits DNA synthesis.

ROS generation. The violet pigment nostocine A,

produced by Nostoc spongiaeforme, is highly cytotoxic

for several micro-algae (Hirata et al., 2003). It has been



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found to accelerate the formation of reactive oxygen

species (ROS) in the green alga C. reinhardtii. Inside

the target cell, nostocine A is reduced specifically by

intracellular reductants such as NAD(P)H. When the

level of O2 is sufficiently higher than that of nostocine

A, the reduced product of nostocine A is oxidised by

O2 which generates the production of superoxideradical anion (OÀ

2 ). OÀ

2 and the ROS subsequently

derived from OÀ

2 may cause the cytotoxicity of the

nostocine A (Hirata et al., 2004). An unidentified

compound from cyanobacterium Microcystis sp. also

induces oxidative stress in the dinoflagellate Peridi-

nium gatunense. It inhibits carbonic anhydrase activity

which simulates CO2-limiting conditions (Sukenik

et al., 2002). In presence of light, this may lead to the

formation of ROS because photosynthetic electrons

could not be used to fix CO2. Depending on their

concentration, these ROS and especially H2O2 may

induce programmed cell death, a process close to

apoptosis of animal and plant cells (Vardi et al., 1999).

The alternative to death of the dinoflagellate cells is

cyst formation.

Toxins

Most toxins are classified as hepatotoxins, neurotoxins

or dermatotoxins after the symptoms they produce.

However, because those symptoms have been mainly

described in vertebrates, in the context of this review

it is more relevant to classify them according to theirchemical structures [cyclic peptides, alkaloids, lipo-

polysaccharides and polyunsaturated fatty acids (PU-

FAs) and their derivatives]. Some recent studies have

focused on the effects of toxins on plankton and on

macrophytes (Table 2). The effects of the toxin may be

direct or indirect, linked to the metabolism of the

molecule by the detoxification system.

Cyclic peptides. Two toxins are cyclic peptides: micro-

cystins and nodularins, microcystins being the most

widely distributed toxins. Microcystins are produced

by planktonic cyanobacteria belonging to the genera

Anabaena, Microcystis, Planktothrix and by some spe-

cies of the benthic Oscillatoria (Wiegand & Pflugma-

cher, 2005); nodularins are produced by Nodularia

spumigena (Briand et al., 2003). These peptides contain

unusual amino acids and show a strong structural

variability: more than 75 structural variants of micro-

cystin have been described to date. Microcystins and

nodularins have been shown to be inhibitors of the

serine/threonine protein phosphatases types 1 and 2A

(MacKintosh et al., 1990; Honkanan et al., 1994). This

activity has been demonstrated for mammals and

higher plant protein phosphatases. The toxin–enzyme

interactions are very strong, and binding is essentially

stoichiometric. The concentration required to inhibitprotein phosphatases in vitro is lower for nodularin

than for microcystins (Ohta et al., 1994). Inhibition of

protein phosphatases leads to hyperphosphorylation

of proteins associated with the cytoskeleton and

consequent redistribution of these proteins. In mam-

mals and birds, the toxic effects of microcystins are

almost restricted to the liver.

Besides these well-studied effects of microcystins

against vertebrates, several cases of negative effects of

these hepatotoxins against micro-algae or aquatic

plants have been reported. The harmful effect

observed in these cases may not be linked to the

inhibition of protein phosphatases as for mammals

but to the elevated formation of ROS which seems to

occur in each case. Indeed, in the picocyanobacterium

Synechococcus elongatus, the toxicity of the microcystin-

RR seems to be linked to the induction of oxidative

stress manifested by elevated ROS levels and mal-

ondialdehyde content (Hu et al., 2005). The oxidative

stress induced in the dinoflagellate P. gatunense by

microcystin-LR is linked to the activation of mitogen-

activated protein kinases, enzymes known to play a

role in cellular responses to biotic and abiotic signalsin mammals and higher plants cells (Vardi et al., 1999;

Vardi et al., 2002). Concerning aquatic macrophytes,

several studies on the effects of microcystin-LR on

Ceratophyllum dermesum demonstrated the existence of

uptake of the toxin by the plant accompanied by a

subsequent increase in ROS concentration and fol-

lowed by an increase in the gluthatione S-transferase

and several antioxidant enzyme activities, a growth

inhibition and changes in pigments pattern (Pflugma-

cher, Codd & Steinberg, 1999; Pflugmacher, 2002,

2004). Interestingly, an increase in the amount of ROS

was observed in rat liver after administration of

cyanobacterial crude extract (Ding et al., 2000). The

harmful effects of microcystins on mammals could be

linked to both the inhibition of protein phophatases

and the formation of ROS.

Alkaloids. The alkaloid toxins include anatoxin-a (and

homoanatoxin-a), anatoxin-a(s), cylindrospermopsins



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and saxitoxins. Anatoxins are mainly produced by

Anabaena species, but also by Microcystis and Oscilla-

toria (Park et al., 1993; Sivonen & Jones, 1999).

Cylindrospermopsins are produced by Aphanizomenon

ovalisporum, Cylindrospermopsis raciborskii, Raphidiopsis

curvata and Umezakia natans (Briand et al., 2003).

Saxitoxins were first described in marine dinoflagel-lates but they have been recently identified in five

freshwater cyanobacterial species: Aphanizomenon flos-

aquae, Anabaena circinalis, C. raciborskii, Lyngbia wollei

and Planktothrix sp. (Briand et al., 2003). Anatoxin-a

and anatoxin-a(s), two unrelated compounds, both

inhibit transmission at the neuromuscular junction.

Anatoxin-a is a cholinergic agonist that binds to

nicotinic acetylcholine receptor while anatoxin-a(s) is

an acetylcholinesterase inhibitor with a mechanism

similar to that of organo-phosphorus insecticides

(Carmichael, 1994). Toxic effects observed on the

aquatic plant Lemna minor may be linked to the

metabolism of the toxin by the plant that may produce

either reactive species of oxygen or a new compound

toxic for the plant (Mitrovic et al., 2004). The toxicity

of the hepatotoxin cylindrospermopsin (CYN) seems

to be exerted through interference with protein/

enzyme synthesis (Griffiths & Saker, 2003). The

nucleotidic structure of CYN suggests that this toxin

may have effects on DNA and RNA, and a some

covalent interactions between CYN and DNA have

been reported in treated mice, with significant DNA

strand breakage (Shen et al., 2002). Saxitoxins bind tosite 1 of the sodium channels in cell membranes,

which blocks nervous transmission (Carmichael,

1994). To date there is no known effect of the

saxitoxins on aquatic plants or on micro-algae.

Polyunsaturated fatty acids and their derivatives.

Compounds containing the a)b)c)d-unsaturated

aldehyde structure (2,4-heptadienal, 2,4-octadienal

from diatoms) (Wendel & Juttner, 1996) act against

herbivores. Cell division is blocked by the aldehydes,

certainly because of microtubule de-polymerisation

whereby tubulin cannot organise into filaments

(Buttino et al., 1999). Moreover, no DNA replication

can occur in presence of the aldehyde (Hansen,

Even & Geneviere, 2004). In copepods, the poly-

unsaturated aldehyde induces a caspase-independent

programmed cell death as revealed by cytochemical

and biochemical approaches (Romano et al., 2003).

In sea urchin, the aldehyde (2E,4E-decadienal)

induces apoptosis and activates a caspase 3-like

protease.

Regulation and influence of environmental and

physiological factors


It has been shown for terrestrial plants that stress

conditions decrease the importance of competition in

favour of allelopathy in community structuring (In-

derjit & Del Moral, 1997). An environmental stress

(nutrients, light, temperature) may increase either

allelopathic compound production or target sensitiv-

ity (Reigosa et al., 1999). The same phenomenon may

exist for aquatic photoautotrophs: the importance of

allelopathy is enhanced when the environmental

conditions are suboptimal. In some cases this may

be directly linked to the mode of action of the

compound. The allelopathic compound produced by

the cyanobacterium T. doliolum, which inhibits photo-

synthesis, is more toxic to Anabaena variabilis under

light limitation (von Elert & Juttner, 1996). This result

points to the fact that experiments conducted under

light saturation may underestimate the impact of

allelopathic interactions given that many allelopathic

compounds act against photosynthesis.

Little is known about the mechanisms regulating

production or excretion. It has been shown for

T. doliolum that regulation of the release of allelopathiccompounds was decoupled from the release of

dissolved organic carbon (DOC) (von Elert & Juttner,

1997). Indeed, an increase in irradiance under

P-limited condition led to elevated of DOC with no

increase in the release of the allelopathic compound.

This suggests the existence of two different regulation

pathways, with allelopathic compounds having their

own regulation pathways.

The influence of several parameters on allelopathic

interactions has been studied in different cases. It

appears that allelopathic interactions are affected in

various ways by a great number of environmental

factors. Nutrient concentration has an important

influence on the interactions. Nutrient stress can

enhance the production of allelopathic compounds

by various algae (Ray & Bagchi, 2001; Rengefors &

Legrand, 2001) and subsequently modify the

equilibrium between taxa. For instance, under phos-

phorus-limited conditions, the release of allelopathic



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compounds by T. doliolum increases 30-fold (von Elert

& Juttner, 1996). Conversely, the allelopathic inter-

action between the dinoflagellate P. gatunense and the

cyanobacterium Microcystis sp. seems to be indepen-

dent of nutrient availability (Vardi et al., 1999).

Another factor that must be taken into account is

temperature. The antibiotic production by two cyanobacteria, Oscillatoria angustissima and Calothrix parie-

tina is not proportional to the biomass but depends

essentially on the temperature of the culture (Issa,

1999). A last factor in this non-exhaustive list is

growth medium pH: the algicidal activity of Oscilla-

toria laetevirens was negatively correlated with pH

(Ray & Bagchi, 2001).

The integration of the influence of environmental

factors on the production of allelopathic compounds

is essential for understanding the ecological role of

allelopathy. It is necessary to know what compound

production is in situ. Few studies focus on the

variation in target sensitivity, even though it is the

second part of the allelopathic interaction. Besides

these abiotic factors, the intensity of the interaction

may depend on biotic factors such as the concentra-

tion of the target or the composition of the bacterial

community. Effects of the target on production of

allelopathic compounds have rarely been studied,

presumably because production of allelopathic com-

pounds is often observed in the absence of the target.

However, the production of antifungal molecules by

the cyanobacterium Scytonema ocellatum was shown to be induced by fungal cell-wall polysaccharides (Pat-

terson & Bolis, 1997). As allelopathic compounds may

be metabolised by micro-organisms, their actual con-

centration may depend on the microbial activity.

Finally, the cellular phase is also an important

physiological factor that influences the interaction.

The toxicity of Peridinium aciculiferum against compet-

itors is maximal during stationary phase (Rengefors &

Legrand, 2001). In contrast, several studies have

indicated that the donor alga is more (or only)

allelopathic when the culture is in exponential growth

phase (Suikkanen, Fistarol & Graneli, 2004).

Toxins

Environmental parameters may influence both the

production of the toxin (intracellular amount) and its

release into the environment (extracellular amount

compared with cellular concentration). Generally,

toxins are released into the environment by cell lysis.

Nevertheless, Rapala et al. (1997) showed in micro-

cosm experiments that even if time is the most

important factor that controls the release of microcys-

tins into a growth medium, the concentration of

dissolved toxins was increased by light flux above

25 lmol m)2

s)1

and by addition of nitrogen.The effects of environmental conditions on toxin

production have been reviewed by Sivonen & Jones

(1999). In many cases, the production of freshwater

toxin is negatively correlated with nitrogen concen-

tration and positively with phosphorus concentration

(Rapala et al., 1997; Kaebernick & Neilan, 2001).

Highest production of toxin is generally found in

optimal conditions for cell growth (Kaebernick &

Neilan, 2001). However, the production of cylindros-

permopsin by C. raciborskii is negatively correlated

with growth rate (Griffiths & Saker, 2003), as for

several allelopathic compounds. The identification of

the genes of the microcystin synthetase, implied in

microcystins synthesis, provided a new powerful tool

for the investigation of the regulation of toxin

synthesis. A recent study demonstrated that the

transcription of two of these genes was controlled by

light quality and initiated at certain threshold in-

tensities (Kaebernick et al., 2000) which confirms the

results of Rapala et al. (1997). These authors found no

correlation between the transcription of one of these

genes and cellular toxin content. This made them

hypothesise that microcystins may be released fromthe cell and play a putative role under high light

conditions.

Biotic factors, such as the presence of a competitor,

may also influence the production of toxin (Vardi

et al., 2002). The regulation pathways may depend on

the toxin involved. For example, the extracellular

production of anatoxin-a by Anabaena flos-aquae is

increased while the production of microcystin is

totally inhibited by high concentration of C. reinhardtii

extracellular products (Kearns & Hunter, 2000).

Ecological roles


How can some species dominate the whole algal

community? What factors control the dynamic of the

benthic and planktonic algal communities and the

formation and disappearance of blooms? The



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persistence of a species depends on its competitive

capacity and species succession has often been

explained as a consequence of competition. A species

that produces allelopathic compounds will have an

advantage over its competitors (Wolfe, 2000). Thus,

allelopathy, as competition, should partly explain

species succession. However, the problem of distin-guishing between competition and allelopathy makes

it difficult to evaluate the real importance of allelo-

pathy in natural environments. Allelopathic effects

can only be separated from those of competition in

microcosm experiments.

Several cases have been well described where algal

succession and the formation of blooms are related to

the production of allelopathic compounds (Kearns &

Hunter, 2001; Rengefors & Legrand, 2001; Vardi et al.,

2002). A few examples are given here. Keating (1977,

Keating 1978) combined field observations and labor-

atory studies to show that allelopathic interactions

may be implicated in the establishment of bloom

sequences in a eutrophic lake. Cyanobacteria that

were dominant could inhibit both their predecessors

and their successors and there was a negative corre-

lation between diatom blooms and cyanobacterial

blooms. More recently, similar results were found for

diatom-cyanobacteria succession in a eutrophic lake

in Japan (Takamo et al., 2003). By comparing the

growth of the cyanobacterium Phormidium tenue in the

presence of diatoms with and without germanium, a

specific growth inhibitor of diatoms, the authorsdemonstrated that cyanobacterial development was

restrained by the production of inhibitory com-

pounds. In the lake, the decrease in diatoms due to

consumption of the available phosphorus allowed the

development of the cyanobacteria previously inhib-

ited. A freshwater bloom-forming green alga, Botryo-

coccus braunii, excretes free fatty acids which have

adverse effects on various phytoplankton and

zooplankton species (Chiang, Huang & Wu, 2004).

The presence of these species during B. braunii blooms

was negatively correlated with their sensitivity to the

fatty acids. The end of the bloom coincided with a

decrease in the production of free fatty acids. These

results suggest a relationship between the formation

and disappearance of the bloom and the production of

allelopathic compounds that eliminate both compet-

itors and predators.

Allelopathy may also be a way to compensate

competitive disadvantage (low growth rate, low

nutrient uptake). This is the case for the freshwater

dinoflagellate P. aciculiferum which produces allelo-

chemicals that would compensate for the disadvan-

tage of its large size in terms of nutrient uptake and

help it to dominate in winter the phytoplankton

biomass (Rengefors & Legrand, 2001). Moreover, the

lysed target cells release nutrients that can support thedinoflagellate growth.

Toxins

The ecological role of toxins is still debated. Several

hypotheses have been proposed, in particular for the

microcystins, but to date no consensus has emerged.

The term ‘toxin’ groups some molecules of quite

different chemical nature and with various biosyn-

thetic pathways, and thus they may have diverse

functions. From an evolutionary viewpoint, and given

the high costs of their production supported by the

cell, toxins must be presumed to have an ecological

role. Most research in this field has focused on

microcystins and several hypotheses have been pro-

posed to explain their production. Initially it had been

proposed that the toxins may have originally had a

critical function that is now lost (Carmichael, 1994).

This is supported by the fact that the activity of

microcystins is directed against the protein phospha-

tases that regulate eukaryote proliferation, and by the

absence of participation of the toxins in cell function

and cell division. Nevertheless, given the importantcost of production linked to the enzymatic complexes

involved in their synthesis, it seems probable that

toxins have an actual function in cyanobacterial

physiology or ecology. A second early hypotheses

was that toxins may be predator defences, as toxic or

deterrent compounds (DeMott, Zhang & Carmichael,

1991). Indeed some studies demonstrated that micro-

cystins can be toxic for zooplankton (copepods,

cladocerans) or may induce avoidance behaviour

(Kurmayer & Juttner, 1999; Ghadouani et al., 2004).

This is also true for the toxin cylindrospermopsin

(Nogueira et al., 2004). A third hypothesis is a role for

the toxin in the regulation of light harvesting and

chromatic adaptation. This is supported by the genetic

study of Kaebernick et al. (2000) who demonstrated

regulation by light quality and intensity of the genes

involved in microcystin synthesis. A fourth hypothe-

sis is that microcystins may be iron-scavenging

molecules and thus may be associated with iron



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transport (Utkilen & Gjolme, 1995). A Fifth hypothesis

is that toxins represent some storage substances

(Carmichael, 1997). Toxins have also been proposed

to act as allelopathic compounds (Christoffersen, 1996;

Pflugmacher, 2002). A final hypothesis was proposed

by Sedmak & Kosi (1998) who suggested that micro-

cystins could act as growth regulators helping cyanobacteria to multiply and giving them a better

opportunity for successful adaptation. Their hypothe-

sis is based on experiments that showed stimulating

effects of microcystins on the growth of various green

algae and cyanobacteria.

Among the hypotheses proposed to explain the

production of microcystins, some may be equally

applied to the others toxins: protection against pred-

ators and allelopathic function.

In addition to the issue of understanding the role of

toxins, a big question is to explain the co-existence of

toxic and non-toxic strains. Non-toxic strains may

either take advantage of the production of toxins by

co-occurring strains, without supporting the costs of

synthesis, or they may produce compounds with an

activity similar to that of toxins but that are not toxic

to the animals that are the focus of most toxicity

testing. Recently, peptide production by 18 clonal

strains of Planktothrix sp. from a single water sample

has been investigated by MALDI-TOF mass spectr-

ometry and HPLC (Welker et al., 2004). Each strain

appeared to produce between three and eight major

compounds comprising microcystins and otherknown peptides. The putative biological activity of

most of these peptides remains unknown but could be

the same for both toxic and non-toxic compounds.

Thus, microcin SF608, a peptide produced by Micro-

cystis sp., induced typical stress reaction in aquatic

plant and zooplankton, as microcystin-LR (Wiegand

et al., 2002).

The production of toxins may be a protection mean

against predators. Some PUFAs and their derivatives

have been shown to have anti-predator properties (see

review by Watson, 2003). When the freshwater

anostracan Thamnocephalus platyurus fed on diatom-

dominated biofilms it showed high mortality linked to

the production of various PUFAs with 5,8,11,14,17-

eicosapentaenoic acid being responsible of most of the

toxicity (Juttner, 2001). Such fatty acids are only

produced by the hydrolysis of lipids in the environ-

ment or within the digestive tract of the grazer when

cell walls are disrupted. This makes the use of PUFAs

a very efficient strategy for grazer defence: basic

cellular components (e.g. membrane lipids) are

rapidly transformed into highly toxic molecules when

the alga is grazed. This means no additional cost for

the synthesis of a specialised defence molecule or no

problem of autotoxicity and of transfer to the target.

Within the PUFAs derivatives produced by diatoms,a)b)c)d-unsaturated aldehydes (2E,4Z-decadienal,

2,4-heptadienal) have adverse effects on grazers,

mainly on the next generation (Miralto et al., 1999;

Pohnert, 2000).

Toxins or allelopathic compounds?

Because of their potential health hazard, most toxic

compounds have been described and studied as

toxins. Nevertheless, as a function that could be

fulfilled by both toxins and other related compounds,

allelopathy has received much attention in the recent

years. Defence against grazers and inhibition of

competitors can confer strong competitive advantages

to the producer which may have been sufficient for

the selection of toxin-producing strains. It should be

noted that nostocyclamide, a cyclic peptide having a

biosynthesis pathway close to that of microcystins and

nodularins (Kaebernick & Neilan, 2001), has been

identified and studied on the base of its allelopathic

properties (Todorova & Juttner, 1995).

Several cases where toxins act as allelopathic

compounds have been reported. The negative effectof Anabaena flos-aquae on the green alga C. reinhardtii

is mediated by both microcystin-LR and anatoxin-a

(Kearns & Hunter, 2000). Singh et al. (2001) reported

that purified microcystin-LR from Microcystis aerugi-

nosa had a negative effect on the growth of several

green algae and cyanobacteria. Growth of S. elongatus

was inhibited by microcystin-RR (100 lg L)1) with a

decrease in photosystem II efficiency (Hu, Liu & Li,

2004). The effects of microcystin-LR against the

aquatic plant Ceratophyllum demersum have been fully

described by Pflugmacher (2002, 2004). He demon-

strated that the toxin could inhibit growth and had

adverse effects on photosynthesis and pigment

pattern at environmentally relevant concentrations

(5 lg L)1). The plant exhibited an oxidative stress

while detoxication and antioxidative enzymes were

induced (Pflugmacher, 2004). Pflugmacher pointed to

the fact that gluthatione S-transferase can recognise

microcystins as natural substrates and thus initiate



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the detoxication/elimination process. This empha-

sises the fact that an allelopathic role of toxins could

be considered in some cases. Microcystin-LR was

also found to influence growth and morphology of

the aquatic plant Spirodela oligorrhiza (Romanowska-

Duda & Tarczynska, 2002). The inhibitory effects

were observed with cellular extracts containing0.334 mg L)1 of microcystin-LR and with

0.1 g mL)1 of commercial-grade microcystins-LR. In

some of these studies, the concentrations of micro-

cystin were rather high compared to those measured

in the field (below 10 lg L)1) (Sivonen & Jones,

1999). Nevertheless the possibility that elevated

concentrations occur in the micro-environment sur-

rounding algal cells must be considered. Moreover,

cellular extracts containing toxins are often more

active than purified toxin, which suggests that

cellular extracts contain a mix of active the toxin

that may act synergistically.

The effects of anatoxins on aquatic plants have been

less studied. Negative effects on an aquatic plant have

been reported (Mitrovic et al., 2004). A 4-day exposure

of the free-floating plant L. minor to anatoxin-a led to

an increase in both peroxidase activity and gluthati-

one S-transferase activity (two detoxification enzyme

activities) while photosynthetic oxygen production

was reduced. In that study the mode of action of the

toxin is unknown. Anatoxin-a concentrations required

for the observation of a significant effect were quite

high (25 lg mL)1

) compared to natural concentration(rarely above 3 lg mL)1), which limits the relevance

of the described interaction.

In this review we have defined predator defences as

allelopathy. Under that definition, there are many

examples that support the allelopathic role of certain

toxins. Cylindrospermopsis raciborskii appeared to re-

duce the fitness and the growth of juvenile D. magna,

which was partly due to the production of cylindro-

spermopsin; a non toxic C. raciborskii strain affected

the same crustacean zooplankton species to a lesser

extent (Nogueira et al., 2004). In vitro experiments

demonstrated that microcystin-LR can induce oxida-

tive stress enzymes and cause death to D. magna

(Wiegand et al., 2002). As for activity against photo-

autotrophic organisms, toxins may also act synergis-

tically with other compounds. Indeed, the negative

effect of PUFAs on D. magna was enhanced by the

addition of microcystin-LR at a concentration at which

it was not active alone (Reinikainen et al., 2001). The

toxicity of microcystin-LR may also be more pro-

nounced when it is delivered via food rather than via

water (Reinikainen, Ketola & Walls, 1994).

Effects of toxins on predators and competitors very

much depend on the target tested; results cannot be

generalised. In many studies a high concentration of

the toxin is needed to observe an effect, one reasonwhy some authors exclude the hypothesis of an

allelopathic role of toxin. However, as stated before,

toxins may act synergistically with other compounds,

at much lower concentrations. Some studies also

implicate toxins in algae–algae interactions (Sivonen

& Jones, 1999; Kearns & Hunter, 2001; Vardi et al.,

2002). Moreover, in some cases, toxin production is

affected by the presence of various photoautotrophic

organisms which is consistent with a role for these

compounds in biotic interactions (Kearns & Hunter,

2000; LeBlanc, Pock & Aranda-Rodriguez, 2005).

Finally, many studies have demonstrated that toxins

can have adverse effects on grazers (see review by

Wiegand & Pflugmacher, 2005). Among the other

secondary metabolites produced by cyanobacteria,

some share the same biosynthesis pathway as toxins.

Those compounds are not toxic for vertebrates and

thus they have not been studied as toxins. Neverthe-

less they may have a similar function in predator

defence or competitor inhibition. All these results

support the view that toxins may be allelopathic

compounds. Although further research is needed to

clarify the allelopathic effects of toxins, the allelo-pathic hypothesis remains relevant.

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(Manuscript accepted 31 October 2006)



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