Allelopathic effects of filamentous cyanobacteria on ... - Helda

FINNISH INSTITUTE OF MARINE RESEARCH – CONTRIBUTIONS

No. 15 Sanna Suikkanen

Allelopathic effects of filamentous cyanobacteria on phytoplankton in the Baltic Sea

Finnish Institute of Marine Research, Finland Helsinki 2008

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ISSN 1457-6805 ISBN 978-951-53-3022-2 (Paperback) ISBN 978-952-10-4457-1 (PDF)

Allelopathic effects of filamentous cyanobacteria on phytoplankton in the Baltic Sea Sanna Suikkanen

Academic dissertation in Hydrobiology to be presented, with the permission of the Faculty of Biosciences of the University of Helsinki, Department of Biological and Environmental Sciences, for public examination in Auditorium Aura, Dynamicum, Erik Palménin aukio 1, Helsinki, on February 8th, 2008, at 12 o’clock noon.

Supervisors: Doc. Jonna Engström-Öst Finnish Institute of Marine Research Helsinki, Finland Prof. Markku Viitasalo Finnish Institute of Marine Research Helsinki, Finland Reviewers: Doc. Pirjo Kuuppo Finnish Environment Institute Helsinki, Finland Dr. Norbert Wasmund Baltic Sea Research Institute Warnemünde, Germany Opponent: Assoc. Prof. Karin Rengefors Lund University Lund, Sweden

CONTENTS List of original articles..................................................................................................................................... 7 Contributions ................................................................................................................................................... 7 Abstract ............................................................................................................................................................ 9 1. Introduction................................................................................................................................................ 11 1.1 Allelopathy ............................................................................................................................................ 11 1.1.1 Phytoplankton allelopathy: evolutionary and ecological roles........................................................ 11 1.1.2 Allelochemicals and their modes of action ..................................................................................... 12 1.1.3 Cyanobacterial allelopathy.............................................................................................................. 17 1.2 Ecosystem changes in the Baltic Sea ..................................................................................................... 17 1.3 Bloom-forming cyanobacteria ............................................................................................................... 19 1.3.1 Anabaena spp.................................................................................................................................. 20 1.3.2 Aphanizomenon flos-aquae............................................................................................................. 20 1.3.3 Nodularia spumigena...................................................................................................................... 20 2. Objectives of the study .............................................................................................................................. 21 3. Methods ...................................................................................................................................................... 21 3.1 Laboratory studies (I–III)...................................................................................................................... 21 3.2 Long-term data analysis (IV)................................................................................................................. 23 4. Results and discussion ............................................................................................................................... 24 4.1 Allelopathy of Baltic cyanobacteria....................................................................................................... 24 4.1.1 Effects of cyanobacteria on monocultures ...................................................................................... 24 4.1.2 Role of nodularin in allelopathy...................................................................................................... 24 4.1.3 Mode of allelopathic action............................................................................................................. 26 4.1.4 Effects of cyanobacteria on a natural plankton community ............................................................ 26 4.2 Long-term trends of phytoplankton and environmental factors ............................................................. 28 5. Conclusions................................................................................................................................................. 29 Acknowledgements ........................................................................................................................................ 30 References....................................................................................................................................................... 31


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LIST OF ORIGINAL ARTICLES This thesis is based on the following papers, which are referred to in the text by their Roman numerals:

I

Suikkanen, S., Fistarol, G.O. & Granéli, E. 2004: Allelopathic effects of the Baltic cyanobacteria Nodularia spumigena, Aphanizomenon flos-aquae and Anabaena lemmermannii on algal monocultures. – Journal of Experimental Marine Biology and Ecology 308: 85–101.

II

Suikkanen, S., Engström-Öst, J., Jokela, J., Sivonen, K. & Viitasalo, M. 2006: Allelopathy of Baltic Sea cyanobacteria: no evidence for the role of nodularin. – Journal of Plankton Research 28: 543–550.

III

Suikkanen, S., Fistarol, G.O. & Granéli, E. 2005: Effects of cyanobacterial allelochemicals on a natural plankton community. – Marine Ecology Progress Series 287: 1–9.

IV

Suikkanen, S., Laamanen, M. & Huttunen, M. 2007: Long-term changes in summer phytoplankton communities of the open northern Baltic Sea. – Estuarine, Coastal and Shelf Science 71: 580–592.

The original communications were reproduced with the kind permission of Elsevier Science (I and IV), Oxford University Press (II) and Inter-Research Science Publisher (III).

CONTRIBUTIONS

I

II

III

IV

Original idea

S. Suikkanen G. Fistarol

S. Suikkanen J. Engström-Öst

G. Fistarol S. Suikkanen

M. Laamanen M. Viitasalo

Study design and methods


S. Suikkanen J. Engström-Öst M. Viitasalo

G. Fistarol S. Suikkanen

S. Suikkanen M. Laamanen

Data gathering


S. Suikkanen J. Jokela


M. Huttunen S. Suikkanen M. Laamanen

Responsible for manuscript preparation

S. Suikkanen

S. Suikkanen

S. Suikkanen

S. Suikkanen

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Suikkanen

Finnish Institute of Marine Research – Contributions No. 15


9

Allelopathic effects of filamentous cyanobacteria on phytoplankton in the Baltic Sea Sanna Suikkanen Finnish Institute of Marine Research, Erik Palménin aukio 1, P.O. Box 2, FI-00561 Helsinki, Finland Suikkanen, S. 2008: Allelopathic effects of filamentous cyanobacteria on phytoplankton in the Baltic Sea. Finnish Institute of Marine Research – Contributions No. 15, 2008.

ABSTRACT Eutrophication and enhanced internal nutrient loading of the Baltic Sea are most clearly reflected by increased late-summer cyanobacterial blooms, which often are toxic. In addition to their toxicity to animals, phytoplankton species can be allelopathic, which means that they produce chemicals that inhibit competing phytoplankton species. Allelopathy may lead to the formation of harmful phytoplankton blooms and the spread of exotic species into new habitats. The aim of my thesis was to investigate whether the Baltic filamentous cyanobacteria Anabaena sp., Aphanizomenon flos-aquae and Nodularia spumigena have allelopathic properties, and if indications of such interactions can be detected in the long-term development of the Baltic phytoplankton community structure. My studies provide the first evidence for allelopathic effects in brackish water cyanobacteria. In laboratory experiments employing both monocultures of the target species and a natural phytoplankton community from the Baltic Sea, exudates of all three cyanobacteria inhibited cryptophytes. The allelopathic effects are rather transitory, and some co-occurring species show tolerance to them. The allelochemicals are excreted during active growth and they decrease cell numbers, chlorophyll a content and carbon uptake of the target species. Although the more specific modes of action or chemical structures of the allelochemicals remain to be studied, the results clearly indicate that the allelopathic effects are not caused by the hepatotoxin, nodularin, produced by N. spumigena. On the other hand, cyanobacteria stimulated the growth of bacteria, other cyanobacteria, chlorophytes and flagellates in a natural phytoplankton community. The stimulation is probably due to the ability of these taxa to utilize organic matter or bacteria, or nutrients provided by the bacteria or released from the damaged cryptophyte cells. Therefore, the allelochemicals may act via lysis of the target algal cells, making them release nutrients, which will lead to the proliferation of the allelopathic organism. In a long-term data analysis of phytoplankton abundances and hydrography of the northern Baltic Sea, a clear change was observed in the phytoplankton community structure, together with a transition in environmental factors, between the late 1970s and early 2000s. Surface water salinity has decreased, whereas the water temperature and concentration of dissolved inorganic nitrogen have increased. In the phytoplankton community, the biomass of cyanobacteria, chrysophytes and chlorophytes has significantly increased, and the late-summer phytoplankton community has become increasingly cyanobacteria-dominated. In contrast, the biomass of cryptophytes has decreased. The increased temperature and nutrient concentrations probably explain

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Suikkanen


most of the changes in the phytoplankton, but my results suggest that the possible effect of chemically mediated biological interactions should also be considered. Cyanobacterial allelochemicals can cause additional stress to other phytoplankton in the nutrient-depleted late-summer environment and thus contribute to the persistence of long-lasting cyanobacterial mass occurrences. On the other hand, cyanobacterial blooms may either directly or indirectly promote or retard the growth of some phytoplankton species. Therefore, a further increase in cyanobacteria will probably shape the late-summer pelagic phytoplankton community by stimulating some species, but inhibiting others.

Key words: allelopathy, cyanobacteria, Baltic Sea, eutrophication, long-term changes, Anabaena sp., Aphanizomenon flos-aquae, Nodularia spumigena, nodularin


1. INTRODUCTION 1.1 Allelopathy The term ‘allelopathy’, originating from the Greek words allelon (= of each other) and pathos (= to suffer), was introduced by Molisch (1937) to describe the biochemical interactions between all types of plants and microorganisms. Rice (1984) modified the definition to refer to any direct or indirect harmful or beneficial effect of one plant or microorganism on another through chemicals that are released into the environment. Most recently, allelopathy was formulated as ‘any process involving secondary metabolites produced by plants, algae, bacteria, and fungi that influence the growth and development of agricultural and biological systems’ (International Allelopathy Society 1996). Some authors also include grazer deterrence (e.g. Leflaive & Ten-Hage 2007), but in a strict sense and within this thesis, only the inhibitory effects among competing plants and autotrophic microorganisms are considered allelopathic, although the possibility of ‘positive allelopathy’ (stimulatory effects) is speculated (article III). Competition for resources can occur as exploitation and/or interference. Exploitation means the direct use of a resource, reducing its availability to a competing individual or species. In interference, access to a resource is denied to competitors by the dominant individual or species, due to the release of antibiotics, territorial behaviour and social hierarchies (Valiela 1995). Allelopathy is an example of interference competition with a passive character (Reigosa & al. 1999), compared with e.g. territorial behaviour. The allelopathic organism releases chemicals that inhibit the growth of a competing organism and thus indirectly prevents it from using common resources. In the present work, the term ‘growth inhibition’ is used widely to refer to the negative effects on either the growth rate or the accumulation of cells of the target phytoplankton species. Here, ‘phytoplankton’ also includes cyanobacteria, which are photoautotrophic prokaryotes that functionally belong to phytoplankton. Due to the economic importance of agricultural and forest ecosystems, terrestrial allelopathy has been widely studied (Rice 1984, Rizvi & al. 1999). In aquatic environments, studies are complicated e.g. by the high diffusive potential of compounds, as well as difficulties in collecting and culturing the organisms. Definitive evidence for allelopathy in the field is almost impossible to obtain due to the complexity of natural interactions. However, allelopathy is considered as an important process that occurs among all groups of marine and freshwater primary producers (Gross 2003, Legrand & al. 2003). Most of the studies on aquatic allelopathy have focused on

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freshwater macrophytes, but the interest in allelopathic interactions within the phytoplankton has recently been kindled (reviewed by Maestrini & Bonin 1981, Lewis 1986, Cembella 2003, Gross 2003, Legrand & al. 2003, Leflaive & Ten-Hage 2007, Macías & al. 2008). Among phytoplankton, allelopathic effects have been reported in cyanobacteria, dinoflagellates, haptophytes, diatoms, raphidophytes and chlorophytes, but not in cryptophytes, chrysophytes or euglenophytes. 1.1.1 Phytoplankton allelopathy: evolutionary and ecological roles Allelopathic interactions can occur in all aquatic habitats. In littoral or benthic ecosystems, the distances between organisms are smaller than in the pelagial and allelopathic interactions are probably a means of competing for space. The allelochemicals may be translocated by direct contact from the emitter species to targets in their vicinity (Gross 2003). In the pelagic zone, the larger distances between cells and dilution of compounds have been considered as major problems for allelopathy; thus, it was argued that allelopathy is not an evolutionarily stable strategy for phytoplankton (Lewis 1986). In the light of recent studies, however, the advantages versus costs from the production of allelopathic compounds appear high enough for it to also be adaptive in the pelagic environment (Leflaive & Ten-Hage 2007). Compared with terrestrial and benthic/littoral allelochemicals, pelagic metabolites are probably more efficient and work at lower concentrations, and/or their production and excretion rates are higher, thus ensuring their effects on the target species (Gross 2003). Coexisting organisms are probably adapted to each other’s presence, which was suggested to reduce the importance of allelopathic interactions in natural environments (Reigosa & al. 1999). On the other hand, Legrand & al. (2003) assumed that in a complex community with a mix of different species, some targets may become adapted to an allelopathic compound, and some will remain sensitive, which confers a sufficient advantage for the emitter. Allelopathy has even been proposed as one of the many mechanisms explaining ‘the paradox of the plankton’ (Hutchinson 1961), where the coexistence of a large number of competing species in phytoplankton communities, limited by only a few resources, contradicts the competitive exclusion principle that predicts the exclusion of all but the best adapted species for each limiting factor (Hardin 1960). The presence of allelopathic species was suggested to reduce the competition among other, nontoxic species, and thus prevent the competitive exclusion of species that would otherwise not coexist (Roy & Chattopadhyay 2007).

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Suikkanen

The importance of allelopathy is probably enhanced in cases of abiotic stress (e.g. nutrient availability, light), invasion by exotic organisms, synthesis of a new molecule by the producer and delayed adaptation of the target species, or continuous release and limited (e.g microbial) degradation of allelochemicals, which leads to their accumulation in the environment (Reigosa & al. 1999). For example, allelochemicals released by an invasive plant into a new environment may result in its predominance, because native organisms lack the defence/detoxification mechanisms developed through coevolution. Many terrestrial (Bais & al. 2003, Hierro & Callaway 2003) and also some aquatic plants are suggested to use allelopathy as a spreading mechanism (Macías & al. 2008). In addition to many environmental factors, allelopathy can be important in explaining community structure and its spatiotemporal changes, such as algal successions and the induction and termination of blooms (Vance 1965, Keating 1977, 1978, Schagerl & al. 2002, Sukenik & al. 2002, Vardi & al. 2002, Chiang & al. 2004, de Figueiredo & al. 2006). Some species, such as the dinoflagellate Peridinium aciculiferum, apparently use allelopathy to compensate for their low rates of growth and nutrient uptake (Rengefors & Legrand 2001). However, the problem of distinguishing between allelopathy and resource exploitation competition makes it difficult to evaluate the importance of allelopathy in natural environments. 1.1.2 Allelochemicals and their modes of action Allelochemicals produced by aquatic macrophytes resemble those produced by terrestrial plants (e.g. fatty acids, phenolic compounds, terpenoids, polysaccharides), whereas the allelochemicals of microalgae and cyanobacteria have apparently evolved in their own direction (Macías & al. 2008). Macrophytes usually live attached to a solid substrate and need a means to control epiphytes; thus, they produce lipophilic compounds that will remain attached to their surface or in the vicinity of the producer. In contrast, pelagic organisms need more hydrophilic compounds with a high degree of activity to overcome dilution effects (Gross 2003, Macías & al. 2008). Few microalgal allelochemicals have been chemically identified to date, and they include cyclic peptides, alkaloids, organic acids and long-chain polyunsaturated fatty acids (Legrand & al. 2003). The major difficulty for the isolation of bioactive compounds from phytoplankton is that they often are produced in very small amounts, because under nutrient limitation, the production of a highly active


compound at low concentrations is a cost-effective strategy (Leflaive & Ten-Hage 2007). Allelochemicals may inhibit photosynthesis or protein activity of the target species, modify or activate its other physiological functions, damage cell membranes, kill the competitor or exclude it from the donor vicinity, e.g. by settling (Uchida & al. 1995, Smith & Doan 1999, Kearns & Hunter 2001, Schmidt & Hansen 2001, Legrand & al. 2003, Fistarol & al. 2004a). Allelochemicals tend to simultaneously affect many physiological processes, and one species can produce several allelochemicals that work synergistically in the environment (Reigosa & al. 1999). Inhibition of photosynthesis, the central physiological process of competing primary producers, is an especially widespread mode of allelopathic action among cyanobacteria (Smith & Doan 1999). The majority of the allelochemicals acting on photosynthesis interfere with the photosynthetic electron transport in photosystem II (PSII), located in the thylakoid membranes of the chloroplasts. This decreases oxygen evolution and carbon incorporation of the target cells, leading to a decreased growth rate and biomass accumulation. Examples of isolated and characterized cyanobacterial allelochemicals that are known to inhibit PSII include fischerellins from Fischerella spp., cyanobacterin LU-1 from Nostoc linckia, nostocyclamide from Nostoc sp. and cyanobacterin from Scytonema hofmanni (Smith & Doan 1999). In addition, several yet unidentified allelochemicals apparently act similarly (Table 1 and references therein). Both environmental factors and the physiological status of a phytoplankton cell can affect allelochemistry (Legrand & al. 2003). Abiotic stress, such as nutrient limitation (von Elert & Jüttner 1996, Ray & Bagchi 2001, Rengefors & Legrand 2001, Granéli & Johansson 2003, Fistarol & al. 2005), or extreme conditions of light (von Elert & Jüttner 1996, Hirata & al. 2003), temperature (Gromov & al. 1991, Issa 1999, Hirata & al. 2003) or pH (Ray & Bagchi 2001) can enhance both the production of the allelochemicals and the vulnerability of the target (Reigosa & al. 1999). The intensity of the interaction may also be dependent on biotic factors, such as growth phase or donor/target cell concentrations (Bagchi & al. 1990, Arzul & al. 1999, Kearns & Hunter 2000, Rengefors & Legrand 2001, Schmidt & Hansen 2001, Uronen & al. 2005, Volk 2007). After release, abiotic factors (light, oxygen and redox conditions), as well as bacterial activity, may influence the stability of allelochemicals (Gross 2003).

Synechocystis aquatilis Oscillatoriales Arthrospira laxissima A. maxima Geitlerinema splendidum

Synechococcus sp.*

Chroococcales Chroococcus minutus* Microcystis aeruginosa*

Group/species

Microcystin-LR, -RR

CC

CC CC Riverine, epipelic, Spain

Culture medium Culture medium Methanol extracts, microcystins

Culture medium

Cell-free filtrate

Microcystin-LR, -RR Microcystin-LR, -RR, -YR

FW, CC CC

FW plankton, USA CC

Microcystin-RR

Culture medium, microcystin-LR

Kasumigamide (linear tetrapeptide) Crude bloom extract, microcystin-LR, -RR Microcystin-LR

Microcystin-RR (cyclic heptapeptide)

Polyunsaturated fatty acids

Culture medium Extracellular metabolites

Active component

FW plankton, China

FW plankton, India FW plankton, Israel

FW plankton, Germany

FW, CC

CC FW plankton, USA, New Zealand FW plankton, USA FW plankton, Slovenia

Origin

Cyanobacteria Cyanobacteria Cyanobacteria, chlorophyte

Chrysophyte Cyanobacteria, chlorophyte Cyanobacterium, chlorophytes Cyanobacteria, diatoms, chlorophytes Cyanobacteria

Cyanobacterium

Cyanobacteria, chlorophytes Dinoflagellate

Chlorophyte

Cyanobacteria, cryptophyte, chlorophytes Chlorophyte

Cyanobacteria Cyanobacteria, cryptophytes, chlorophytes Chlorophyte

Target

Growth inhibition Growth inhibition Growth inhibition, morphological and ultrastructural alterations

Growth inhibition

Growth inhibition

Growth inhibition

Growth inhibition, cell lysis, loss of O2 evolution, reduction in 14 CO2 uptake, loss of nitrogenase activity Growth and photosynthesis inhibition, depression of internal carbonic anhydrase activity, activation of protein kinases, accumulation of reactive oxygen species (ROS), oxidative stress, cell death Growth inhibition, chlorosis, chlorophyll (chl) a and phycocyanin synthesis inhibition, PSII inhibition, changes in protein and carbohydrate concentrations and nitrate reductase activity, increases in ROS, malondialdehyde and detoxication enzymes, oxidative stress Growth stimulation, but oxidative stress Increased cell aggegation, volume and pigment production

Elevation of detoxication enzyme activity, inhibition of photosynthesis

Immobilization of flagella, settling

Growth inhibition or stimulation

Growth inhibition

Growth inhibition Growth inhibition

Action

Volk 2005 Volk 2005 Valdor & Aboal 2007

Volk 2005

Keating 1978, 1987

Babica & al. 2007

Ou & al. 2005 Sedmak & Eleršek 2005

Hu & al. 2004, 2005

Sukenik & al. 2002, Vardi & al. 2002

Singh & al. 2001

Pietsch & al. 2001

Ishida & Murakami 2000

Sedmak & Kosi 1998

Ikawa & al. 1996

Volk 2005 Vance 1965, Lam & Silvester 1979

References

Table 1. Allelopathic effects of cyanobacteria on microalgae. Species names are from the cited literature (with new names, when available, in brackets, according to Hällfors (2004)). * denotes species occurring in the Baltic Sea. CC = culture collection, BW = brackish water, FW = freshwater, M = marine. Names of isolated and characterized chemicals causing the effects are underlined.

Allelopathic effects of filamentous cyanobacteria on phytoplankton in the Baltic Sea 13

O. elegans

A. torulosa*

A. spiroides*

A. inaequalis* A. cf. lemmermannii*

A. holsaticum

Nostocales Anabaena cylindrica* A. flos-aquae*

FW plankton, USA CC BW plankton, Sweden FW plankton, Thailand FW plankton, Austria

CC

CC CC

Cyanobacteria, chlorophytes

Extracellular C25 alkane with a phenol group and an α, βunsaturated carbonyl residue Cell-free filtrate

Living cells

Spiroidesin (linear lipopeptide)

Culture medium Cell-free filtrate

Culture medium Siderophores produced under Felimitation Culture medium, microcystin-LR, anatoxin-a (alkaloid) Cell-free filtrate

Cyanobacteria, diatom, chlorophytes

Cyanobacterium

Cyanobacteria, diatoms Cyanobacteria Cryptophyte, diatom

Chlorophyte

Cyanobacteria Chlorophytes

Cyanobacteria, diatoms, chlorophytes

Growth inhibition

Growth inhibition


Growth inhibition

Growth inhibition, paralysis, increased settling rate

Growth inhibition Growth inhibition, by Fe deprivation or direct toxicity

Growth inhibition

Growth inhibition, morphological and ultrastructural alterations

Cyanobacteria

Methanol extracts, microcystins

Cell-free filtrate

Growth inhibition Cell lysis

Schagerl & al. 2002

Kaya & al. 2002

Volk 2005 I, III

Keating 1977, 1978

Volk 2005 Murphy & al. 1976, Matz & al. 2004 Kearns & Hunter 2000, 2001

Keating 1978, 1987

Volk 2005 Murakami & al. 1990, 1991, Yamada & al. 1993 Valdor & Aboal 2007

Valdor & Aboal 2007


Cyanobacteria Itself

Volk 2005 Keating 1977, 1987

Bagchi & al. 1990, 1993, Chauhan & al. 1992, Bagchi 1995, Marwah & al. 1995, Ray & Bagchi 2001 Keating 1977, 1978, 1987

Keating 1978

Infante & Abella 1985 Issa 1999

Keating 1978, 1987

Berry & al. 2004

References


Growth inhibition, cell lysis, inactivation of photosynthetic PSIImediated reactions and O2 evolution, damage of thylakoid membranes, loss of chl, proteins and toxicity Growth inhibition

Growth inhibition

Growth inhibition Inhibition of growth and O2 evolution

Growth inhibition

Inhibition of growth and hormogonia development

Action

Culture medium Polyunsaturated fatty acids



Cyanobacteria, diatoms, chlorophytes Cyanobacteria Cyanobacteria, diatoms, chlorophytes Cyanobacteria

Cryptophyte Cyanobacteria, chlorophytes Diatoms

Living cells Antibiotic, extracted with ethyl acetate Cell-free filtrate

Cell-free filtrate

Cyanobacterium, chlorophytes Diatoms

Target

Pahayokolide A (cyclic peptide)

Active component

Suikkanen

Pseudanabaena galeata

Phormidium foveolarum P. tenue (Leptolyngbya tenuis)* Phormidium sp.*

O. sancta* Oscillatoria sp.*

O. rubescens

FW plankton, USA CC FW plankton, USA Riverine, epilithic, Spain CC FW plankton, Japan Riverine, epipelic, Spain FW plankton, USA

FW plankton, USA FW plankton, India

O. angustissima

O. laetevirens

Edaphic, Egypt

Oscillatoria agardhii (Planktothrix agardhii)*

Origin

FW periphyton, USA FW plankton, USA

Lyngbya sp.*

Group/species

14 Finnish Institute of Marine Research – Contributions No. 15

Sessile, Australia

FW plankton, Brazil FW plankton, Austria Benthic, CC

Benthic, CC

Benthic, CC

Sessile, Australia, Indonesia, Nepal, Vietnam Riverine, benthic, Brazil

Edaphic, Marshall Islands M plankton, Italy

Calothrix sp.

Cylindrospermopsis raciborskii Cylindrospermum sp.

F. muscicola

F. tisserantii

Fischerella sp.

Hapalosiphon fontinalis

Nodularia harveyana*

Fischerella ambigua

Edaphic, Egypt

FW plankton, USA CC FW plankton, USA FW plankton, Austria FW plankton, USA FW plankton, USA BW plankton, Finland Benthic, CC

CC

Origin

C. parietina

Calothrix brevissima

A. flos-aquae*

Anabaenopsis siamensis Aphanizomenon elenkinii A. flexuosum

Anabaena sp.*

Group/species

Fischerellin A, 12-epi-hapalindole F Hapalindole A, smaller amounts of several minor indoles Lipophilic substances

Living cells, 12-epi-hapalindole E isonitrile (alkaloid)

Fischerellin A

Fischerellin A (aminoacylpolyketide) Fischerellin A, fischerellin B

Living cells

Cell-free filtrate

Acetone or methanol / chloroform extract Antibiotic, extracted with ethyl acetate Living cells

Long-chain unsaturated fatty acids Cell-free filtrate

Cell-free filtrate

Living cells


Siderophores produced under Felimitation Cell-free filtrate

Active component

Cyanobacteria, chlorophyte

Cyanobacteria

Cyanobacteria

Cyanobacteria, chlorophytes Cyanobacteria, chlorophytes

Cyanobacteria, chlorophytes Cyanobacteria, chlorophytes Cyanobacteria, chlorophytes Cyanobacteria, chlorophytes Cyanobacteria, chlorophytes Cyanobacteria, chlorophytes

Flores & Wolk 1986, Gross & al. 1991, 1994, Hagmann & Jüttner 1996, Papke & al. 1997, Srivastava & al. 1998 Gross & al. 1991 Schlegel & al. 1999, Doan & al. 2000

Cell lysis, inhibition of photosynthetic electron transport at PSII

Cell lysis, inhibition of photosynthesis

Growth inhibition

Growth inhibition

Growth and photosynthesis inhibition

Pushparaj & al. 1999

Moore & al. 1984, 1987, 1989

Etchegaray & al. 2004

Gross & al. 1991

Inhibition of photosynthetic electron transport

Inhibition of photosynthetic electron transport,

Schagerl & al. 2002

Figueredo & al. 2007

Growth inhibition

Photosynthesis (PSII activity) inhibition

Schlegel & al. 1999

Issa 1999

Inhibition of growth and O2 evolution Cell lysis

Abarzua & al. 1999

Growth inhibition

I, II

Growth inhibition, decrease of cellular chl a and CO2 uptake

Cryptophyte, diatom Diatom

Ikawa & al. 1994

Keating 1978, 1987

Schagerl & al. 2002

Volk 2005 Keating 1977, 1978

Keating 1987

Bailey & Taub 1980

References

Growth inhibition

Growth inhibition

Growth inhibition


Growth inhibition

Fe deprivation

Action

Cyanobacteria, diatoms Chlorophyte

Cyanobacteria, diatoms, chlorophytes Cyanobacteria Cyanobacteria, diatoms Chlorophyte

Chlorophyte

Target

Allelopathic effects of filamentous cyanobacteria on phytoplankton in the Baltic Sea 15

N. spongiaeforme

Trichormus doliolum (syn. Anabaena doliolum)

Tolypothrix distorta

S. myochrous

Scytonema hofmanni

Rivularia haematites

Living cells

Living cells, exudate

FW plankton, CC



Cyanobacterin (chlorine-containing γ-lactone)

Culture medium Methanol extracts, microcystins

Living cells

Riverine, epilithic, Spain Riverine, epilithic, Spain CC

Sessile, Australia, Indonesia FW plankton, Austria CC Riverine, epilithic, Spain FW, benthic, CC

Living cells Nostocyclamide (cyclic hexapeptide), nostocyclamide M Living cells


Cyanobacteria, chlorophyte Cyanobacteria

Cyanobacteria, chlorophytes


Cyanobacteria

Inhibition of growth, photosynthetic electron transport and O2 production, increased chl fluorescence

Cell lysis

Inhibition of photosynthetic electron transport in PSII, deterioration of thylakoid membranes and cell walls, loss of chl

Cyanobacteria, diatom, euglenophyte, chlorophytes

Growth inhibition Growth inhibition, morphological and ultrastructural alterations

Growth inhibition

Cell lysis Inhibition of growth, chl, carotenoid and protein synthesis, altered morphology Cell lysis, inhibition of photoautotrophic growth

Growth inhibition, ROS generation

Growth inhibition

Growth and photosynthesis inhibition (O2 evolution, PSII electron transport) Growth inhibition

Growth inhibition Growth inhibition Growth inhibition

Growth inhibition, decrease of cellular chl a and CO2 uptake

Growth inhibition

Action

von Elert & Jüttner 1996, 1997

Flores & Wolk 1986

Valdor & Aboal 2007

Mason & al. 1982, Pignatello & al. 1983, Gleason & Paulson 1984, Gleason & Baxa 1986, Gleason 1990, Lee & Gleason 1994, Abarzua & al. 1999 Valdor & Aboal 2007

Volk 2005 Valdor & Aboal 2007

Schagerl & al. 2002

Flores & Wolk 1986 Todorova & al. 1995, Todorova & Jüttner 1996, Jüttner & al. 2001 Schlegel & al. 1999

Hirata & al. 2003

Schagerl & al. 2002

Keating 1978, 1987

Volk 2005 Volk 2005 Volk 2005, 2006, Volk & Furkert 2006 Gromov & al. 1991

Volk 2005, 2006, Volk & Furkert 2006 I, II, III

References

Suikkanen

Cyanobacteria, diatom, chlorophytes Cyanobacteria Cyanobacteria

FW plankton, Austria FW, Thailand

CC FW, benthic, CC

Living cells

CC

N. muscorum

Nostoc sp.*

Cyanobacteria, chlorophytes Cyanobacteria, diatoms, chlorophytes Cyanobacteria, chlorophytes Cyanobacteria, chlorophytes Cyanobacteria Cyanobacteria, diatom, chlorophytes Cyanobacteria, chlorophytes

Cyanobacterin LU-1 (phenolic compound) Cell-free filtrate

Edaphic, Russia

Nostocine A (violet pigment)

Cyanobacteria Cyanobacteria Cyanobacteria

Cryptophyte, diatom

Cyanobacteria

Target

Culture medium Culture medium 4,4′-dihydroxybiphenyl

N. linckia

Norharmane, norharmalane (indole alkaloids) Cell-free filtrate

CC

Nostoc carneum N. commune N. insulare

Active component

Origin

BW plankton, Sweden CC CC CC

N. spumigena*

Group/species

16 Finnish Institute of Marine Research – Contributions No. 15


1.1.3 Cyanobacterial allelopathy Most observations on phytoplankton allelopathy originate from freshwater habitats and most of them concern cyanobacteria (Gross 2003). In contrast, allelopathy in marine ecosystems was described mainly for bloom-forming dinoflagellates, haptophytes and raphidophytes (Smayda 1997, Cembella 2003). Little is known of the allelopathic interactions of marine or brackish water cyanobacteria, which were the group of interest in the present study, due to their annual mass occurrences in the Baltic Sea (described below). An exception is the Mediterranean Nodularia harveyana, which is allelopathic against other cyanobacteria and a chlorophyte (Pushparaj & al. 1999). Observations on the allelopathic effects among cyanobacteria, including their isolated allelochemicals and modes of action, are listed in Table 1. It has been argued that allelopathy is one of the main factors contributing to the formation and/or persistence of cyanobacterial blooms in eutrophic lakes (Keating 1978, Bagchi & al. 1990). By combining laboratory experiments and field studies, Keating (1977, 1978) showed that the phytoplankton bloom sequence in a eutrophic lake correlated with the effects of cell-free filtrates of dominant cyanobacteria on both their phytoplankton successors and predecessors. The filtrates of each cyanobacterial species generally inhibited its immediate predecessors in the natural phytoplankton bloom sequence, whereas filtrates of the same species generally stimulated their immediate successors. Filtrates of cultured cyanobacteria, as well as lake waters collected during cyanobacterial blooms, also inhibited the growth of diatoms isolated from the same lake, and diatom bloom populations in situ varied inversely with the preceding cyanobacterial populations over several years (Keating 1978). In Lake Kinneret, Israel, the reciprocal allelopathic interactions of the cyanobacterium Microcystis sp. and the dinoflagellate Peridinium gatunense determine the species dominating the phytoplankton assemblage (Sukenik & al. 2002, Vardi & al. 2002). Cyanobacterial allelochemicals have also been suggested to contribute to a shift from macrophytedominated to more phytoplankton-dominated lakes (van Vierssen & Prins 1985, Pflugmacher 2002), although the principal cause of such a change in eutrophic lakes is probably increased pelagic production, together with increased turbidity and reduced light availability for littoral and benthic production. Moreover, Figueredo & al. (2007) suggested that allelopathy contributed to the recent geographical expansion of the toxic, bloom-forming cyanobacterium Cylindrospermopsis raciborskii from tropical and subtropical regions to temperate lakes and rivers. Cyanobacteria produce a wide array of compounds (cyanotoxins) that are extremely toxic to

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vertebrates. However, the ecological role of cyanotoxin production is still largely unknown. One hypothesis concerns allelopathy, suggesting that the toxins are allelochemicals directed against competing photoautotrophic organisms (Sedmak & Kosi 1998, Pflugmacher 2002). Indeed, several cases were reported in which cyanobacterial toxins exerted inhibitory effects on photoautotrophs. These include negative effects of microcystins on terrestrial (Abe & al. 1996, Gehringer & al. 2003) and aquatic plants (Pflugmacher 2002, 2004, Yin & al. 2005b), including phytoplankton (Kearns & Hunter 2000, 2001, Sedmak & Eleršek 2005, Table 1), anatoxin-a on phytoplankton and aquatic plants (Kearns & Hunter 2001, Mitrovic & al. 2004), cylindrospermopsin on a terrestrial plant (white mustard, Sinapis alba; Vasas & al. 2002) and nodularin on a brown macroalga (bladder wrack, Fucus vesiculosus; Pflugmacher & al. 2007). The observed harmful effects of cyanotoxins may not occur as a consequence of the same mechanism as for mammals [e.g. protein phosphatase 1 (PP1) inhibition of microcystins and nodularin], but to the enhanced production of reactive O2 species and oxidative stress (Mitrovic & al. 2004, Pflugmacher 2004, Hu & al. 2005, Yin & al. 2005a, Pflugmacher & al. 2007). However, the evidence concerning the role of toxins as allelochemicals is inconclusive, since the concentrations of toxins used in the experiments have often been higher than those occurring in natural waters (reviewed by Babica & al. 2006).

1.2 Ecosystem changes in the Baltic Sea The Baltic Sea is a brackish water sea (about 422 000 km2, mean depth 55 m), with a restricted connection to the North Sea and the Atlantic Ocean. The residence time of the water is long, more than 30 years (Dybern & Fonselius 1981). The Baltic Sea area is characterized by strong seasonal temperature variation and there is a steep north-south surface water salinity gradient, from 1–2 psu in the northern and eastern areas to ca. 20 psu in the Kattegat. The northern and eastern parts (most of the Gulfs of Bothnia and Finland, Fig. 1) are usually ice-covered between January and March. The water is stratified, with a deep, permanent halocline at a depth of ca. 60 m (except in the Gulf of Bothnia) and a thermocline at ca. 20 m in summer. The halocline prevents mixing of the deep saline water with the less saline surface water layer. Consequently, O2 deficiency frequently occurs in the deep water. Only episodic intensive salt water inflows from the North Sea, largely governed by meteorological variability, occasionally renew the deep water (Schinke & Matthäus 1998). The flora and fauna of the Baltic Sea are mixtures of freshwater, brackish water and

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marine species, although the species diversity is low and food webs are simple compared with in the oceans (e.g. Furman & al. 1998). Approximately 85 million people in 14 industrialized countries inhabit the Baltic Sea catchment area (1 720 000 km2), and thus the sea is heavily influenced by anthropogenic pressures. In recent decades, the ecosystem of the Baltic Sea has undergone considerable changes, which either directly or indirectly are associated with increased human activities (HELCOM 2002). For example, increased shipping has contributed to introductions of alien species via ballast water and increased the risk of oil spills and other accidents. Overfishing has not only affected the target fish stocks (e.g. salmon, cod), but probably also the remaining marine food web by removing key species from the top of the food chain (HELCOM 2002). Heavy metals and organic contaminants [e.g. dichloro-diphenyl-trichloroethanes (DDTs), polychlorinated biphenyls (PCBs) and hexachlorocyclohexanes (HCHs)], have accumu-


lated in and affected especially long-lived organisms such as the white-tailed sea eagle and grey seal. The cold climate and slow water exchange of the Baltic Sea further slow down the decay of these humaninduced contaminants. Global climate change will most likely be one of the most important factors shaping the future ecosystem of the Baltic Sea (HELCOM 2007a). There is a close association between the functioning of the pelagic ecosystem and hydrographic features (such as ice, stratification and landbased runoff), as well as large-scale weather patterns (Northern Atlantic Oscillation, NAO) over the runoff area of the Baltic Sea (Alheit & al. 2005). The change in the NAO index during the late 1980s from a negative to a more positive phase was attributed to both increasing freshwater runoffs and a decreasing salinity of the Baltic Sea (Hänninen & al. 2000), as well as a regime shift involving all trophic levels in the pelagic areas of the central Baltic Sea and the North Sea (Alheit & al. 2005).

Fig. 1. Map of the Baltic Sea and the study locations. The laboratory experiments were performed at Kalmar University (KU), Sweden (I, III), and Tvärminne Zoological Station (TZS), Finland (II). Sampling sites for the natural community experiment (NC; III) and longterm data analysis (F1, F3, H1, H2 and H3; IV) are indicated.


Eutrophication, caused by anthropogenic nutrient inputs, is currently the most serious problem in the Baltic Sea ecosystem. The Baltic Sea is especially susceptible to eutrophication due to its shallowness, stratification and slow water exchange, combined with heavy external nutrient loading from the watershed (HELCOM 2004). Significant increases in the surface water nitrogen and phosphorus concentrations have been recorded since the late 1960s (Larsson & al. 1985). Despite recent successful reductions in point-source nutrient (especially P) loading and a slight decrease in dissolved nutrient concentrations in some areas (Fleming-Lehtinen & al. in press), the eutrophication process continues, due to external inputs of N and P and internal P loading from anoxic sediments (Pitkänen & al. 2001, Vahtera & al. 2007a, HELCOM 2007b). The most common signs of eutrophication, i.e. reduced water transparency, as well as increased primary production, phytoplankton biomass (chlorophyll a concentrations), algal blooms, deposition of organic matter and O2 deficiency of bottom waters, and dead bottom fauna, are clearly detected in the Baltic Sea (Bonsdorff & al. 2002). Perhaps the most conspicuous sign of eutrophication is the increased late-summer phytoplankton biomass (FlemingLehtinen & al. in press), expressed to a large degree as annual mass occurrences of filamentous N-fixing cyanobacteria.

1.3 Bloom-forming cyanobacteria A late-summer phytoplankton biomass peak, dominated by filamentous cyanobacteria, is a typical feature of the Baltic Sea (Niemi 1973, Bianchi & al. 2000). However, the extent and frequency of cyanobacterial mass occurrences have increased during the last half of the 20th century, which has been verified by satellite observations (Kahru & al. 1994, 2007), plankton monitoring (Finni & al. 2001) and sediment records (Poutanen & Nikkilä 2001). The increase in bloom intensity is most probably associated with anthropogenic nutrient loading, and in some areas, such as the Gulf of Finland, especially with the recently enhanced internal P loading originating from anoxic sediments (Pitkänen & al. 2003). The bloom-forming cyanobacteria of the open Baltic Sea fix atmospheric N and are therefore independent of dissolved N, which is generally the limiting nutrient in the open sea area (Granéli & al. 1990). However, external N inputs enhance the internal P loading by increasing the magnitude of the spring bloom and thereby the amount of sedimenting organic material. The decomposition of organic matter depletes the oxygen in the bottom waters, facilitating phosphate (PO4) release. The Baltic Sea is considered to be in a state of a self-sustaining ‘vicious circle’ regarding eutrophication and cyano-

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bacterial blooms, due to the feedback between the cycles of O2, P and N (Tamminen & Andersen 2007, Vahtera & al. 2007a). Diazotrophic cyanobacterial blooms develop in areas where the N:P ratio is below the Redfield ratio of 16 (Niemi 1979, Stal & al. 2003), because in these conditions, the N-fixing cyanobacteria are better competitors than other species of phytoplankton whose growth depends on dissolved N. A low N:P ratio may be a prerequisite for cyanobacterial blooms, while the temperature (>16 °C) was claimed to be the main factor determining the onset and intensity of toxic Nodularia spumigena blooms (Wasmund 1997, Kanoshina & al. 2003). High temperatures stabilize the water column and decrease the mixing depth, which increases the light irradiance available for the cyanobacterial community (Stal & al. 2003). In addition to N-fixation, the bloom-forming cyanobacteria have several competitive advantages compared with other phytoplankton species. They are able to store significant quantities of P early in the growing season to sustain later growth in a Pdepleted mixed layer (Larsson & al. 2001, Vahtera & al. 2007b). The cyanobacteria have gas vacuoles in their cells, which allow them to regulate their buoyancy and vertical position in the water column (Walsby & al. 1997). Furthermore, their large size and excretion of bioactive substances such as toxins, antibiotics and allelochemicals may deter grazers and competing microorganisms (Sellner 1997). In the Baltic Sea, the most conspicuous blooms are formed by the large diazotrophic, heterocystous, akinete-forming filamentous cyanobacteria of the order Nostocales: Aphanizomenon flos-aquae and Nodularia spumigena, and to a minor part, Anabaena spp. (contributing to ca. 10% of the total Nostocalean late-summer biomass, based on the HELCOM data presented in IV), although another functional group, the small-sized (ca. 2 µm), nonheterocystous picocyanobacteria (e.g. Synechococcus spp.), may be much more important in terms of biomass (Stal & Walsby 2000). The filamentous cyanobacteria in question possess gas vacuoles, which make them buoyant. During calm conditions, cyanobacterial filaments and aggregates concentrate in the uppermost (ca. 0–5 m) water layers, forming visible surface scums that may also drift ashore. Other cyanobacterial species, mostly of freshwater origin, that may also form visible mass occurrences in the coastal zone (salinity