FINNISH INSTITUTE OF MARINE RESEARCH – CONTRIBUTIONS

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

.

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


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

Allelopathic effects of filamentous cyanobacteria on phytoplankton in the Baltic Sea 7

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 monocul-tures. – 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 natu-ral 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 G. Fistarol

S. Suikkanen J. Engström-Öst M. Viitasalo

G. Fistarol S. Suikkanen

S. Suikkanen M. Laamanen

Data gathering S. Suikkanen G. Fistarol

S. Suikkanen J. Jokela

S. Suikkanen G. Fistarol

M. Huttunen S. Suikkanen M. Laamanen

Responsible for manuscript preparation

S. Suikkanen

S. Suikkanen

S. Suikkanen

S. Suikkanen

8 Suikkanen Finnish Institute of Marine Research – Contributions No. 15



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 re-flected 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 pro-duce chemicals that inhibit competing phytoplankton species. Allelopathy may lead to the for-mation 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 indica-tions of such interactions can be detected in the long-term development of the Baltic phyto-plankton 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 spe-cific modes of action or chemical structures of the allelochemicals remain to be studied, the re-sults 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 prolifera-tion 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 sa-linity has decreased, whereas the water temperature and concentration of dissolved inorganic ni-trogen have increased. In the phytoplankton community, the biomass of cyanobacteria, chryso-phytes and chlorophytes has significantly increased, and the late-summer phytoplankton com-munity has become increasingly cyanobacteria-dominated. In contrast, the biomass of crypto-phytes has decreased. The increased temperature and nutrient concentrations probably explain


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 nu-trient-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 commu-nity 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 de-scribe the biochemical interactions between all types of plants and microorganisms. Rice (1984) modified the definition to refer to any direct or indirect harm-ful or beneficial effect of one plant or microorgan-ism 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 in-clude 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 al-lelopathic, although the possibility of ‘positive al-lelopathy’ (stimulatory effects) is speculated (article III).

Competition for resources can occur as exploita-tion and/or interference. Exploitation means the di-rect 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 hierar-chies (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 ac-cumulation of cells of the target phytoplankton spe-cies. Here, ‘phytoplankton’ also includes cyanobac-teria, 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 com-plexity 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

freshwater macrophytes, but the interest in allelo-pathic 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 cyanobac-teria, dinoflagellates, haptophytes, diatoms, raphido-phytes 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 dis-tances 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 be-tween cells and dilution of compounds have been considered as major problems for allelopathy; thus, it was argued that allelopathy is not an evolutionar-ily stable strategy for phytoplankton (Lewis 1986). In the light of recent studies, however, the advan-tages versus costs from the production of allelo-pathic 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 con-centrations, 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 re-duce 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. Al-lelopathy 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 phyto-plankton communities, limited by only a few re-sources, contradicts the competitive exclusion prin-ciple that predicts the exclusion of all but the best adapted species for each limiting factor (Hardin 1960). The presence of allelopathic species was sug-gested to reduce the competition among other, non-toxic species, and thus prevent the competitive ex-clusion of species that would otherwise not coexist (Roy & Chattopadhyay 2007).


The importance of allelopathy is probably en-hanced in cases of abiotic stress (e.g. nutrient avail-ability, light), invasion by exotic organisms, synthe-sis of a new molecule by the producer and delayed adaptation of the target species, or continuous re-lease and limited (e.g microbial) degradation of al-lelochemicals, 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/detoxi-fication 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, al-lelopathy 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 nu-trient uptake (Rengefors & Legrand 2001). How-ever, the problem of distinguishing between allelo-pathy 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 macro-phytes 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 sub-strate 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 pro-ducer. In contrast, pelagic organisms need more hy-drophilic 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 nu-trient 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 acti-vate 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, Fis-tarol & al. 2004a). Allelochemicals tend to simulta-neously 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 physio-logical 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 photosyn-thesis interfere with the photosynthetic electron transport in photosystem II (PSII), located in the thylakoid membranes of the chloroplasts. This de-creases 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 cyano-bacterin from Scytonema hofmanni (Smith & Doan 1999). In addition, several yet unidentified allelo-chemicals apparently act similarly (Table 1 and ref-erences therein).

Both environmental factors and the physiological status of a phytoplankton cell can affect allelochem-istry (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 al-lelochemicals 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 re-dox conditions), as well as bacterial activity, may influence the stability of allelochemicals (Gross 2003).


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lar C

25 a

lkan

e w

ith a

ph

enol

gro

up a

nd a

n α,

β-

unsa

tura

ted

carb

onyl

resi

due

Cya

noba

cter

ia,

chlo

roph

ytes

G

row

th in

hibi

tion,

cel

l lys

is, i

nact

ivat

ion

of p

hoto

synt

hetic

PSI

I-m

edia

ted

reac

tions

and

O2 e

volu

tion,

dam

age

of th

ylak

oid

mem

bran

es, l

oss o

f chl

, pro

tein

s and

toxi

city

Bag

chi &

al.

1990

, 199

3, C

hauh

an

& a

l. 19

92, B

agch

i 199

5, M

arw

ah

& a

l. 19

95, R

ay &

Bag

chi 2

001

O. r

ubes

cens

FW

pla

nkto

n,

USA

C

ell-f

ree

filtra

te

Cya

noba

cter

ia,

diat

oms,

chlo

roph

ytes

G

row

th in

hibi

tion

Kea

ting

1977

, 197

8, 1

987

O. s

anct

a*

CC

C

ultu

re m

ediu

m

Cya

noba

cter

ia

Gro

wth

inhi

bitio

n V

olk

2005

O

scill

ator

ia sp

.*

FW p

lank

ton,

U

SA

Cel

l-fre

e fil

trate

C

yano

bact

eria

, di

atom

s, ch

loro

phyt

es

Gro

wth

inhi

bitio

n K

eatin

g 19

77, 1

987

R

iver

ine,

ep

ilith

ic, S

pain

M

etha

nol e

xtra

cts,

mic

rocy

stin

s C

yano

bact

eria

G

row

th in

hibi

tion,

mor

phol

ogic

al a

nd u

ltras

truct

ural

alte

ratio

ns

Val

dor &

Abo

al 2

007

Phor

mid

ium

fove

olar

um

CC

C

ultu

re m

ediu

m

Cya

noba

cter

ia

Gro

wth

inhi

bitio

n V

olk

2005

P.

tenu

e (L

epto

lyng

bya

tenu

is)*

FW

pla

nkto

n,

Japa

n

Poly

unsa

tura

ted

fatty

aci

ds

Itsel

f C

ell l

ysis

M

urak

ami &

al.

1990

, 199

1,

Yam

ada

& a

l. 19

93

Phor

mid

ium

sp.*

R

iver

ine,

ep

ipel

ic, S

pain

M

etha

nol e

xtra

cts,

mic

rocy

stin

s C

yano

bact

eria

G

row

th in

hibi

tion,

mor

phol

ogic

al a

nd u

ltras

truct

ural

alte

ratio

ns

Val

dor &

Abo

al 2

007

Pseu

dana

baen

a ga

leat

a FW

pla

nkto

n,

USA

C

ell-f

ree

filtra

te

Cya

noba

cter

ia,

diat

oms,

chlo

roph

ytes

G

row

th in

hibi

tion

Kea

ting

1978

, 198

7

Nos

toca

les

An

abae

na c

ylin

dric

a*

CC

C

ultu

re m

ediu

m

Cya

noba

cter

ia

Gro

wth

inhi

bitio

n V

olk

2005

A.

flos

-aqu

ae*

CC

Si

dero

phor

es p

rodu

ced

unde

r Fe-

limita

tion

C

hlor

ophy

tes

Gro

wth

inhi

bitio

n, b

y Fe

dep

rivat

ion

or d

irect

toxi

city

M

urph

y &

al.

1976

, Mat

z &

al.

2004

CC

C

ultu

re m

ediu

m, m

icro

cyst

in-L

R,

anat

oxin

-a (a

lkal

oid)

C

hlor

ophy

te

Gro

wth

inhi

bitio

n, p

aral

ysis

, inc

reas

ed se

ttlin

g ra

te

Kea

rns &

Hun

ter 2

000,

200

1

A. h

olsa

ticum

FW

pla

nkto

n,

USA

C

ell-f

ree

filtra

te

Cya

noba

cter

ia,

diat

oms

Gro

wth

inhi

bitio

n K

eatin

g 19

77, 1

978

A. in

aequ

alis

* C

C

Cul

ture

med

ium

C

yano

bact

eria

G

row

th in

hibi

tion

Vol

k 20

05

A. c

f. le

mm

erm

anni

i*

BW

pla

nkto

n,

Swed

en

Cel

l-fre

e fil

trate

C

rypt

ophy

te, d

iato

m

Gro

wth

inhi

bitio

n I,

III

A. sp

iroi

des*

FW

pla

nkto

n,

Thai

land

Sp

iroid

esin

(lin

ear l

ipop

eptid

e)

Cya

noba

cter

ium

G

row

th in

hibi

tion

Kay

a &

al.

2002

A. to

rulo

sa*

FW p

lank

ton,

A

ustri

a Li

ving

cel

ls

Cya

noba

cter

ia,

diat

om, c

hlor

ophy

tes

Gro

wth

inhi

bitio

n Sc

hage

rl &

al.

2002


Gro

up/s

peci

es

Ori

gin

Act

ive

com

pone

nt

Tar

get

Act

ion

R

efer

ence

s

Anab

aena

sp.*

C

C

Side

roph

ores

pro

duce

d un

der F

e-lim

itatio

n C

hlor

ophy

te

Fe d

epriv

atio

n B

aile

y &

Tau

b 19

80

FW

pla

nkto

n,

USA

C

ell-f

ree

filtra

te

Cya

noba

cter

ia,

diat

oms,

chlo

roph

ytes

G

row

th in

hibi

tion

Kea

ting

1987

Anab

aeno

psis

siam

ensi

s C

C

Cul

ture

med

ium

C

yano

bact

eria

G

row

th in

hibi

tion

Vol

k 20

05

Apha

nizo

men

on

elen

kini

i FW

pla

nkto

n,

USA

C

ell-f

ree

filtra

te

Cya

noba

cter

ia,

diat

oms

Gro

wth

inhi

bitio

n K

eatin

g 19

77, 1

978

A. fl

exuo

sum

FW

pla

nkto

n,

Aus

tria

Livi

ng c

ells

C

hlor

ophy

te

Gro

wth

inhi

bitio

n Sc

hage

rl &

al.

2002

A. fl

os-a

quae

* FW

pla

nkto

n,

USA

C

ell-f

ree

filtra

te

Cya

noba

cter

ia,

diat

oms

Gro

wth

inhi

bitio

n K

eatin

g 19

78, 1

987

FW

pla

nkto

n,

USA

Lo

ng-c

hain

uns

atur

ated

fatty

ac

ids

Chl

orop

hyte

G

row

th in

hibi

tion

Ikaw

a &

al.

1994

B

W p

lank

ton,

Fi

nlan

d C

ell-f

ree

filtra

te

Cry

ptop

hyte

, dia

tom

G

row

th in

hibi

tion,

dec

reas

e of

cel

lula

r chl

a a

nd C

O2 u

ptak

e I,

II

Cal

othr

ix b

revi

ssim

a B

enth

ic, C

C

Ace

tone

or m

etha

nol /

chl

orof

orm

ex

tract

D

iato

m

Gro

wth

inhi

bitio

n A

barz

ua &

al.

1999

C. p

arie

tina

Edap

hic,

Egy

pt

Ant

ibio

tic, e

xtra

cted

with

eth

yl

acet

ate

Cya

noba

cter

ia,

chlo

roph

ytes

In

hibi

tion

of g

row

th a

nd O

2 evo

lutio

n Is

sa 1

999

Cal

othr

ix sp

. Se

ssile

, Aus

tralia

Li

ving

cel

ls

Cya

noba

cter

ia,

chlo

roph

ytes

C

ell l

ysis

Sc

hleg

el &

al.

1999

Cyl

indr

ospe

rmop

sis

raci

bors

kii

FW p

lank

ton,

B

razi

l C

ell-f

ree

filtra

te

Cya

noba

cter

ia,

chlo

roph

ytes

Ph

otos

ynth

esis

(PSI

I act

ivity

) inh

ibiti

on

Figu

ered

o &

al.

2007

Cyl

indr

ospe

rmum

sp.

FW p

lank

ton,

A

ustri

a Li

ving

cel

ls

Cya

noba

cter

ia,

chlo

roph

ytes

G

row

th in

hibi

tion

Scha

gerl

& a

l. 20

02

Fisc

here

lla a

mbi

gua

Ben

thic

, CC

Fi

sche

relli

n A

(a

min

oacy

lpol

yket

ide)

C

yano

bact

eria

, ch

loro

phyt

es

Inhi

bitio

n of

pho

tosy

nthe

tic e

lect

ron

trans

port

Gro

ss &

al.

1991

F. m

usci

cola

B

enth

ic, C

C

Fisc

here

llin

A, f

isch

erel

lin B

C

yano

bact

eria

, ch

loro

phyt

es

Cel

l lys

is, i

nhib

ition

of p

hoto

synt

hetic

ele

ctro

n tra

nspo

rt at

PSI

I Fl

ores

& W

olk

1986

, Gro

ss &

al.

1991

, 199

4, H

agm

ann

& Jü

ttner

19

96, P

apke

& a

l. 19

97, S

rivas

tava

&

al.

1998

F.

tiss

eran

tii

Ben

thic

, CC

Fi

sche

relli

n A

C

yano

bact

eria

, ch

loro

phyt

es

Inhi

bitio

n of

pho

tosy

nthe

tic e

lect

ron

trans

port,

G

ross

& a

l. 19

91

Fisc

here

lla sp

. Se

ssile

, A

ustra

lia,

Indo

nesi

a, N

epal

, V

ietn

am

Livi

ng c

ells

, 12

-epi

-hap

alin

dole

E is

onitr

ile

(alk

aloi

d)

Cya

noba

cter

ia,

chlo

roph

ytes

C

ell l

ysis

, inh

ibiti

on o

f pho

tosy

nthe

sis

Schl

egel

& a

l. 19

99,

Doa

n &

al.

2000

R

iver

ine,

be

nthi

c, B

razi

l Fi

sche

relli

n A

, 12

-epi

-hap

alin

dole

F

Cya

noba

cter

ia

Gro

wth

and

pho

tosy

nthe

sis i

nhib

ition

Et

cheg

aray

& a

l. 20

04

Hap

alos

ipho

n fo

ntin

alis

Ed

aphi

c,

Mar

shal

l Isl

ands

H

apal

indo

le A

, sm

alle

r am

ount

s of

seve

ral m

inor

indo

les

Cya

noba

cter

ia

Gro

wth

inhi

bitio

n M

oore

& a

l. 19

84, 1

987,

198

9

Nod

ular

ia h

arve

yana

* M

pla

nkto

n, It

aly

Lipo

phili

c su

bsta

nces

C

yano

bact

eria

, ch

loro

phyt

e G

row

th in

hibi

tion

Push

para

j & a

l. 19

99


Gro

up/s

peci

es

Ori

gin

Act

ive

com

pone

nt

Tar

get

Act

ion

R

efer

ence

s

C

C

Nor

harm

ane,

nor

harm

alan

e (in

dole

alk

aloi

ds)

Cya

noba

cter

ia

Gro

wth

inhi

bitio

n V

olk

2005

, 200

6, V

olk

& F

urke

rt 20

06

N. s

pum

igen

a*

BW

pla

nkto

n,

Swed

en

Cel

l-fre

e fil

trate

C

rypt

ophy

te, d

iato

m

Gro

wth

inhi

bitio

n, d

ecre

ase

of c

ellu

lar c

hl a

and

CO

2 upt

ake

I, II

, III

Nos

toc

carn

eum

C

C

Cul

ture

med

ium

C

yano

bact

eria

G

row

th in

hibi

tion

Vol

k 20

05

N. c

omm

une

CC

C

ultu

re m

ediu

m

Cya

noba

cter

ia

Gro

wth

inhi

bitio

n V

olk

2005

N

. ins

ular

e

CC

4,

4′-d

ihyd

roxy

biph

enyl

C

yano

bact

eria

G

row

th in

hibi

tion

Vol

k 20

05, 2

006,

Vol

k &

Fur

kert

2006

N

. lin

ckia

Ed

aphi

c, R

ussi

a C

yano

bact

erin

LU

-1 (p

heno

lic

com

poun

d)

Cya

noba

cter

ia,

chlo

roph

ytes

G

row

th a

nd p

hoto

synt

hesi

s inh

ibiti

on (O

2 evo

lutio

n, P

SII

elec

tron

trans

port)

G

rom

ov &

al.

1991

N. m

usco

rum

C

C

Cel

l-fre

e fil

trate

C

yano

bact

eria

, di

atom

s, ch

loro

phyt

es

Gro

wth

inhi

bitio

n K

eatin

g 19

78, 1

987

FW

pla

nkto

n,

Aus

tria

Livi

ng c

ells

C

yano

bact

eria

, ch

loro

phyt

es

Gro

wth

inhi

bitio

n Sc

hage

rl &

al.

2002

N. s

pong

iaef

orm

e

FW,

Thai

land

N

osto

cine

A (v

iole

t pig

men

t) C

yano

bact

eria

, ch

loro

phyt

es

Gro

wth

inhi

bitio

n, R

OS

gene

ratio

n H

irata

& a

l. 20

03

Nos

toc

sp.*

C

C

Livi

ng c

ells

C

yano

bact

eria

C

ell l

ysis

Fl

ores

& W

olk

1986

FW, b

enth

ic, C

C

Nos

tocy

clam

ide

(cyc

lic

hexa

pept

ide)

, nos

tocy

clam

ide

M

Cya

noba

cter

ia,

diat

om, c

hlor

ophy

tes

Inhi

bitio

n of

gro

wth

, chl

, car

oten

oid

and

prot

ein

synt

hesi

s, al

tere

d m

orph

olog

y To

doro

va &

al.

1995

, Tod

orov

a &

Jü

ttner

199

6, Jü

ttner

& a

l. 20

01

Se

ssile

, A

ustra

lia,

Indo

nesi

a

Livi

ng c

ells

C

yano

bact

eria

, ch

loro

phyt

es

Cel

l lys

is, i

nhib

ition

of p

hoto

auto

troph

ic g

row

th

Schl

egel

& a

l. 19

99

FW

pla

nkto

n,

Aus

tria

Livi

ng c

ells

C

yano

bact

eria

, di

atom

, chl

orop

hyte

s G

row

th in

hibi

tion

Scha

gerl

& a

l. 20

02

C

C

Cul

ture

med

ium

C

yano

bact

eria

G

row

th in

hibi

tion

Vol

k 20

05

Rivu

lari

a ha

emat

ites

Riv

erin

e,

epili

thic

, Spa

in

Met

hano

l ext

ract

s, m

icro

cyst

ins

Cya

noba

cter

ia

Gro

wth

inhi

bitio

n, m

orph

olog

ical

and

ultr

astru

ctur

al a

ltera

tions

V

aldo

r & A

boal

200

7

Scyt

onem

a ho

fman

ni

FW, b

enth

ic, C

C

Cya

noba

cter

in

(chl

orin

e-co

ntai

ning

γ-la

cton

e)

Cya

noba

cter

ia,

diat

om, e

ugle

noph

yte,

ch

loro

phyt

es

Inhi

bitio

n of

pho

tosy

nthe

tic e

lect

ron

trans

port

in P

SII,

dete

riora

tion

of th

ylak

oid

mem

bran

es a

nd c

ell w

alls

, los

s of c

hl

Mas

on &

al.

1982

, Pig

nate

llo &

al.

1983

, Gle

ason

& P

auls

on 1

984,

G

leas

on &

Bax

a 19

86, G

leas

on

1990

, Lee

& G

leas

on 1

994,

A

barz

ua &

al.

1999

S.

myo

chro

us

Riv

erin

e,

epili

thic

, Spa

in

Met

hano

l ext

ract

s, m

icro

cyst

ins

Cya

noba

cter

ia

Gro

wth

inhi

bitio

n, m

orph

olog

ical

and

ultr

astru

ctur

al a

ltera

tions

V

aldo

r & A

boal

200

7

Toly

poth

rix

dist

orta

R

iver

ine,

ep

ilith

ic, S

pain

M

etha

nol e

xtra

cts,

mic

rocy

stin

s C

yano

bact

eria

, ch

loro

phyt

e G

row

th in

hibi

tion,

mor

phol

ogic

al a

nd u

ltras

truct

ural

alte

ratio

ns

Val

dor &

Abo

al 2

007

Tric

horm

us d

olio

lum

(s

yn. A

naba

ena

dolio

lum

)

CC

Li

ving

cel

ls

Cya

noba

cter

ia

Cel

l lys

is

Flor

es &

Wol

k 19

86

FW

pla

nkto

n, C

C

Livi

ng c

ells

, exu

date

C

yano

bact

eria

, ch

loro

phyt

es

Inhi

bitio

n of

gro

wth

, pho

tosy

nthe

tic e

lect

ron

trans

port

and

O2

prod

uctio

n, in

crea

sed

chl f

luor

esce

nce

von

Eler

t & Jü

ttner

199

6, 1

997


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, hapto-phytes and raphidophytes (Smayda 1997, Cembella 2003). Little is known of the allelopathic interac-tions 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 al-lelopathic against other cyanobacteria and a chloro-phyte (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 com-bining 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 cyano-bacteria on both their phytoplankton successors and predecessors. The filtrates of each cyanobacterial species generally inhibited its immediate predeces-sors 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 col-lected during cyanobacterial blooms, also inhibited the growth of diatoms isolated from the same lake, and diatom bloom populations in situ varied in-versely with the preceding cyanobacterial popula-tions over several years (Keating 1978).

In Lake Kinneret, Israel, the reciprocal allelo-pathic interactions of the cyanobacterium Microcys-tis sp. and the dinoflagellate Peridinium gatunense determine the species dominating the phytoplankton assemblage (Sukenik & al. 2002, Vardi & al. 2002). Cyanobacterial allelochemicals have also been sug-gested to contribute to a shift from macrophyte-dominated 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 pro-duction, together with increased turbidity and re-duced light availability for littoral and benthic pro-duction. Moreover, Figueredo & al. (2007) sug-gested that allelopathy contributed to the recent geo-graphical 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 com-pounds (cyanotoxins) that are extremely toxic to

vertebrates. However, the ecological role of cyanotoxin production is still largely unknown. One hypothesis concerns allelopathy, suggesting that the toxins are allelochemicals directed against compet-ing 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), in-cluding phytoplankton (Kearns & Hunter 2000, 2001, Sedmak & Eleršek 2005, Table 1), anatoxin-a on phytoplankton and aquatic plants (Kearns & Hunter 2001, Mitrovic & al. 2004), cylindrosper-mopsin 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 mecha-nism 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, Pflug-macher 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 mix-ing of the deep saline water with the less saline sur-face water layer. Consequently, O2 deficiency fre-quently occurs in the deep water. Only episodic in-tensive salt water inflows from the North Sea, largely governed by meteorological variability, oc-casionally renew the deep water (Schinke & Matthäus 1998). The flora and fauna of the Baltic Sea are mixtures of freshwater, brackish water and


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 industri-alized 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 under-gone considerable changes, which either directly or indirectly are associated with increased human ac-tivities (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 con-taminants [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 human-induced contaminants.

Global climate change will most likely be one of the most important factors shaping the future eco-system 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 Os-cillation, 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 increas-ing 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 pe-lagic 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 long-term 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 water-shed (HELCOM 2004). Significant increases in the surface water nitrogen and phosphorus concentra-tions have been recorded since the late 1960s (Larsson & al. 1985). Despite recent successful re-ductions 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 (chloro-phyll 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 (Fleming-Lehtinen & 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 cyano-bacterial 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 sedi-ment records (Poutanen & Nikkilä 2001). The in-crease in bloom intensity is most probably associ-ated with anthropogenic nutrient loading, and in some areas, such as the Gulf of Finland, especially with the recently enhanced internal P loading origi-nating from anoxic sediments (Pitkänen & al. 2003).

The bloom-forming cyanobacteria of the open Baltic Sea fix atmospheric N and are therefore inde-pendent of dissolved N, which is generally the lim-iting nutrient in the open sea area (Granéli & al. 1990). However, external N inputs enhance the in-ternal 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 ‘vi-cious circle’ regarding eutrophication and cyano-

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 phyto-plankton whose growth depends on dissolved N. A low N:P ratio may be a prerequisite for cyanobacte-rial 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 tem-peratures stabilize the water column and decrease the mixing depth, which increases the light irradi-ance 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 P-depleted 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, Ana-baena 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), nonhet-erocystous picocyanobacteria (e.g. Synechococcus spp.), may be much more important in terms of bio-mass (Stal & Walsby 2000). The filamentous cyano-bacteria in question possess gas vacuoles, which make them buoyant. During calm conditions, cyano-bacterial 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 <3 psu) include Microcystis aeruginosa (order Chroococcales) and Planktothrix agardhii (order Oscillatoriales) (Niemi 1988, Kauppila & al. 1995). However, most studies concerning Baltic cyanobacteria have focused on the filamentous species, and especially on the toxin-producing N. spumigena.


1.3.1 Anabaena spp.

The genus Anabaena Bory ex Bornet & Flahault is a cosmopolitan group of planktonic and benthic species that occur in fresh and brackish water habi-tats (Komárek & Hauer 2004). The taxonomy of the Baltic Anabaena spp. is still unclear, but the Ana-baena populations appear to be genetically more diverse than the Baltic Aphanizomenon or Nodularia populations, and there are probably several species present (Janson & Granéli 2002, Halinen & al. 2007). Based on recent phylogenetic analyses, the Baltic Anabaena strains are closely related to strains isolated from freshwater areas (Sivonen & al. 2007).

In lakes, Anabaena spp. often form dense mono-specific blooms, but in the Baltic Sea they coexist, mostly in small numbers, with A. flos-aquae and N. spumigena. The reason for the low abundance of Anabaena spp. in Baltic cyanobacterial blooms may be that many of the species occur as single fila-ments, in contrast to N. spumigena and A. flos-aquae, which form large colonies and aggregates. The high floating velocities of colonies, as opposed to filaments, allow them to spend more time in the euphotic zone (Stal & al. 2003). Anabaena spp. only occur in the Baltic plankton community during the warmest summer period (July–August; Laamanen & Kuosa 2005), and probably overwinter as akinetes (Olli & al. 2005).

Freshwater blooms of Anabaena are often toxic and the group belongs to the main genera responsi-ble for cyanobacterial intoxications worldwide (Carmichael 2001). Anabaena spp. produce both neurotoxins, such as anatoxin-a, anatoxin-a(s) and saxitoxins, and hepatotoxins, such as microcystins and cylindrospermopsin (Sivonen & Jones 1999, Schembri & al. 2001). The Baltic Anabaena species have commonly been considered nontoxic; however, microcystins have occasionally been recorded in water samples from the coastal southern Baltic Sea (Mazur & Pliński 2003, Luckas & al. 2005), as well as at the entrance to the Gulf of Finland, with Ana-baena sp. as the likely toxin producer (Karlsson & al. 2005). Recently, microcystin-producing Ana-baena strains were isolated from the Gulf of Finland (Halinen & al. 2007).

In addition to toxins, freshwater Anabaena spp. produce a number of bioactive compounds, mostly lipopeptides, that have antibiotic, antialgal, antican-cer, anti-inflammatory, cytotoxic and enzyme-inhib-iting effects (Burja & al. 2001, Fujii & al. 2002, Ta-ble 1). Benthic Anabaena spp. from the Baltic Sea are highly cytotoxic and induce programmed cell death (Herfindal & al. 2005, Surakka & al. 2005).

1.3.2 Aphanizomenon flos-aquae

Aphanizomenon flos-aquae (L.) Ralfs ex Bornet & Flahault is a euryhaline species, able to adapt to a wide range of salinities (Pankow & al. 1990) and

thus common in fresh and brackish water globally. In the Baltic Sea, A. flos-aquae is most abundant during July–September, although vegetative fila-ments occur in the water column throughout the year (Niemi 1973, Laamanen & Kuosa 2005). Therefore, akinetes probably do not play a role as a seed popu-lation for A. flos-aquae in this region, similar to the situation in some temperate lakes (Jones 1979, Head & al. 1999).

The growth of A. flos-aquae is promoted by short-term nutrient pulses from below thermocline, accompanied by upwelling (Kononen & al. 1996) and mixing events, whereas N. spumigena is fa-voured by a stable water column (Stal & Walsby 2000). Aphanizomenon flos-aquae prefers lower irradiances (25–45 µmol m-2 s-1), salinities (0–10 psu) and temperatures (16–22 °C) than N. spumi-gena (45–155 µmol m-2 s-1, 5–20 psu, 25–28 ºC, respectively; Lehtimäki & al. 1997). Consequently, A. flos-aquae often occurs in water layers below N. spumigena and is more common than N. spumigena in the northern parts of the Baltic Sea, such as the Gulf of Finland and the northern Baltic proper (Niemistö & al. 1989).

In lakes, A. flos-aquae produces neurotoxins, such as saxitoxins and anatoxin-a (Carmichael 1986, Sivonen & al. 1989a), and a hepatotoxin, cylidros-permopsin (Preuβel & al. 2006). The Baltic geno-type probably originates from lakes (Laamanen & al. 2002), but apparently produces no known hepato-toxins (Lehtimäki & al. 1997). Furthermore, the production of enzyme inhibitors (Cannell & al. 1988, Murakami & al. 2000), antibiotics (Falch & al. 1995, Østensvik & al. 1998), allelochemicals (Keating 1978, 1987, Ikawa & al. 1994), and com-pounds that inhibit feeding of Daphnia (Haney & al. 1995) and development of fish larvae (Papendorf & al. 1997) have also been documented in freshwater strains of A. flos-aquae. It is not known how the genotype affects allelopathy. Recently, Cox & al. (2005) reported widespread production of a neuro-toxic amino acid, β-N-methylamino-L-alanine (BMAA) within cyanobacterial taxa, including the Baltic A. flos-aquae and N. spumigena.

1.3.3 Nodularia spumigena

Nodularia spumigena Mertens ex Bornet & Flahault occurs in temperate and subtropical marine and brackish waters, shallow seas and bays, saline coastal and inland lakes and swamps, and in reser-voirs with high contents of mineral salts (Komárek & al. 1993). In the Baltic Sea, the N. spumigena biomass usually peaks during late summer (July–August), when the water temperature reaches its annual maximum. During the remaining year, N. spumigena is virtually absent from the water col-umn, although it can be encountered in the sea ice (Laamanen 1996). The overwintering strategy of


Baltic N. spumigena is currently unknown, since akinetes are only rarely found in natural samples (Kononen 2002).

Mass occurrences of N. spumigena have caused intoxications of domestic animals in the coastal ar-eas of the Baltic Sea (Persson & al. 1984, Edler & al. 1985, Nehring 1993), due to the production of a cyclic pentapeptide hepatotoxin, nodularin (Rinehart & al. 1988, Sivonen & al. 1989b,c). Nodularin in-hibits the eukaryotic serine/threonine-specific PPs (1 and 2A), in a manner similar to that of microcystin, causing liver damage, and potentially promoting tumours (Yoshizawa & al. 1990). As with most cyanobacterial toxins, nodularin mainly remains inside the cells and is only released when the cells die (Lehtimäki & al. 1997). Concentrations as high as 18 mg nodularin g-1 dry weight or 18 mg nodu-larin l-1 were measured in the Baltic Sea (Kononen & al. 1993, Mazur & Pliński 2003).

Although the effects of nodularin on zooplankton are variable (DeMott & al. 1991, Koski & al. 2002, Reinikainen & al. 2002), the toxin may accumulate at higher trophic levels via copepods (Engström-Öst & al. 2002b). Nodularin can be transferred to zoo-plankton (Karjalainen & al. 2006), littoral gam-marids (Korpinen & al. 2006), prawns (Van Buynder & al. 2001), fish (Sipiä & al. 2001), clams and mus-sels (Falconer & al. 1992, Sipiä & al. 2002), sea birds (Sipiä & al. 2006), as well as to sediments (Kankaanpää & al. 2001, Mazur-Marzec & al. 2007). Nodularin may induce severe liver damage in trout (Kankaanpää & al. 2002) and oxidative stress in mussels (Kankaanpää & al. 2007) and macroalgae (Pflugmacher & al. 2007). However, it has been suggested that compounds other than nodularin, pre-sent in N. spumigena extract, are responsible for its negative impact on fish larval growth (Karjalainen & al. 2005).

In addition to nodularin and its variants (Mazur-Marzec & al. 2006), other types of peptides, such as nodulapeptins A and B and spumigins A–C, have been isolated from N. spumigena (Fujii & al. 1997), but the potential bioactivity of these compounds is unknown. Baltic N. spumigena is also weakly antivi-ral (Mundt & al. 2001). In contrast, the related Mediterranean N. harveyana produces lipophilic substances with antibacterial, antifungal, allelopathic and toxic activities (Pushparaj & al. 1999). Cyto-toxicity was also observed in benthic Nodularia strains originating from the Baltic Sea (Surakka & al. 2005).

2. OBJECTIVES OF THE STUDY

The aim of my thesis was to increase the level of understanding of allelopathic interactions in phyto-plankton and, especially, to conduct the first known

studies on the allelopathy of brackish water bloom-forming cyanobacteria. This was done by studying the effects of Baltic filamentous cyanobacteria ex-perimentally in a natural phytoplankton community and in monocultures of other phytoplankton species, originating from the same habitat. Subsequently, the results of the experimental studies were evaluated against the long-term development of Baltic cyano-bacterial biomass in relation to other phytoplankton groups.

The more specific objectives of the individual studies were: 1. To study the potential allelopathic effects of Bal-

tic cyanobacteria on eukaryotic phytoplankton species, their duration and association with the growth phase and measured hepatotoxicity of cyanobacteria (I).

2. To compare the allelopathic effects of cyanobac-terial filtrates with those of purified nodularin, to determine whether nodularin acts as an allelo-chemical, and to study the potential mechanism of action of cyanobacterial allelochemicals (II).

3. To study the effects of cyanobacterial filtrates on the abundance and species composition of phytoplankton and other organisms in a natural microplankton community (III).

4. To reveal the long-term trends in summer bio-mass of cyanobacteria in relation to other phyto-plankton and environmental factors in the north-ern Baltic Sea and to determine whether evi-dence of allelopathic interactions can be detected on a time scale of several years (IV).

3. METHODS

3.1 Laboratory studies (I–III)

I conducted three experimental studies (I–III) to investigate the allelopathic effects of Baltic cyano-bacteria (Table 2). Two of these (I, III) were under-taken at Kalmar University, Sweden, and one (II) at Tvärminne Zoological Station, Finland (Fig. 1). In these experiments, I used the cyanobacteria Ana-baena sp. 1 (strain KAC 16, cited as A. lemmerman-nii in I), Aphanizomenon flos-aquae (Tr183) and Nodularia spumigena (KAC 13 and AV1) as allelo-pathic donor species, and the cryptophyte Rhodomo-nas sp. (KAC 30), the haptophyte Prymnesium par-vum (KAC 39) and the diatom Thalassiosira weiss-flogii (KAC 32) as target species, to have represen-tatives of different systematic groups and due to their availability in cultures. The strains KAC 13, KAC 16, KAC 30, KAC 32 and KAC 39 were ob-tained from the Kalmar Algal Collection (KAC), Kalmar University, and strains Tr183 and AV1 from


the Division of Microbiology, University of Hel-sinki. All species used in the experiments were nonaxenic monocultures originating from the Baltic Sea. The sample of a natural plankton community was collected from site NC in the central Baltic Sea (Fig. 1) and filtered through a 150-µm net to remove mesozooplankton (III).

Allelopathic effects can only be separated from those of exploitation competition in microcosm ex-periments, e.g. by cross-culturing. In this technique, the target species is cultured in medium enriched with cell-free filtrate from the donor species (Le-grand & al. 2003). I used cell-free filtrates of the

donor species to study the allelopathic influence of the selected cyanobacteria. The filtrates were pre-pared as gently as possible, i.e. by using as low pres-sure as possible and by keeping the filter wet. This was done to avoid cell lysis and the release of intra-cellular toxins, because the effects caused by chemi-cals excreted by the donor cells only are considered allelopathic (Rice 1984). Although cross-culturing, i.e. the use of filtrates, does not allow for direct identification of the allelochemicals, it is a starting point that has been widely employed for studying the allelopathic effects of a particular phytoplankton species (Legrand & al. 2003).

Table 2. Summary of the laboratory experiments (I–III) presented in this thesis.

Article Experimental unit (volume)

Donor species Extract Target species Chl a ratio donor:target

Analyses

I Scintillation vial (20 ml)

1. Anabaena sp. 1 (cf. lemmermannii)

2. Aphanizomenon flos-aquae

3. Nodularia spumigena

1. Filtrate, ex-ponential phase

2. Filtrate, sta-tionary phase (N. spumigena)

3. Filtrate, co-existing bacteria

1. Rhodomonas sp.

2. Prymnesium parvum

3. Thalassiosira weissflogii

1:1 Target cell counts

II Erlenmeyer flask (400 ml)

1. A. flos-aquae

2. N. spumigena

1. Filtrate

2. Purified nodularin

Rhodomonas sp.

2:1 1. Chl a concen-tration

2. 14CO2 uptake

3. Target cell counts

4. Transfer of nodularin

5. pH

III Erlenmeyer flask (400 ml)

1. Anabaena sp. 1

2. Aphanizomenon sp.

3. N. spumigena

Filtrate Plankton com-munity (<150 µm)

75:1 1. Chl a concen-tration

2. Bacteria, phyto-plankton and ciliate cell counts

Article Duration (days)

Sampling interval (days)

Addition of extract

Toxin analyses

Statistical methods # replicates

I 3 1 In the begin-ning vs. daily

HPLC, PPI Repeated measures ANOVA, Tukey’s HSD, t-test

3

II 6 1 (cells), 2 (chl a, 14CO2, pH), 6 (nodularin)

Every second day

ELISA Repeated measures ANOVA, Tukey’s HSD

3

III 4 1 (chl a, bacteria, ciliates), 4 (phytoplankton)

Daily None Repeated measures ANOVA, one-way ANOVA, Tukey’s HSD; hierarchical clustering, ANOSIM1, SIMPER2

3

1 Analysis of similarity 2 Similarity percentages


Identification of the exact allelochemicals would require a thorough chemical analysis of the filtrate and its active fraction(s), followed by isolation and testing of the effects of the individual extracts and compounds. One step in this direction was taken when the hepatotoxin nodularin was purified from Nodularia spumigena and its effect was tested on a target species and compared with that of the filtrate of the entire culture (II). Furthermore, I compared the effects of a filtrate of an exponentially growing N. spumigena culture with those of a filtrate of a culture in the stationary phase to determine how the growth phase affects the allelopathic potential of the species (I).

A modified version of the growth medium F/2 (‘F/10’, i.e. 20% of the nutrient concentrations in the original F/2 medium; Guillard 1975) was used as a control for the filtrate additions. The same medium was used for growing the cyanobacterial and target species to be tested. However, to ensure that the observed allelopathic effects were not due to nutrient deficiency in the filtrate treatments, compared with the nutrient-rich medium, I always measured the main inorganic nutrient concentrations, nitrate-nitro-gen (NO3-N) and phosphate-phosphorus (PO4-P), in the culture filtrates and adjusted them to the same level as in the control medium.

The experimental strains were nonaxenic. To en-sure that the observed effects were caused specifi-cally by the cyanobacteria, the potential effects of associated bacteria isolated from the cyanobacterial cultures were also tested on the target species (I). This was done by filtering out the cyanobacteria from the cultures with GF/C filters, incubating the filtrates (which contained only the associated bacte-ria) for 7 days, counting the bacteria and testing the effects of the incubated filtrates on the target algae, in a manner similar to the tests for cyanobacterial allelopathy.

I adjusted the biomass ratio of cyanobacte-ria:target species in the experiments using the chlo-rophyll a concentrations of the respective cultures. In III, the chlorophyll a ratio of cyanobacte-ria:natural community was 75:1, resembling a cyanobacterial bloom situation (Potter & al. 1983), whereas in I and II, the ratio was adjusted to 1:1 and 2:1, respectively, to study a more balanced situation between cyanobacteria and co-occurring species.

The duration of the experiments varied from 3 to 6 days. During a longer cross-culturing experiment, the effects of the cyanobacterial extracts may gradu-ally decrease due to degradation of the effective compounds (without continuous excretion from cells), e.g. by bacteria, if fresh filtrate is not added (Gleason & Paulson 1984). In a comparison between one or several additions of filtrate in an allelopathic

assay (I), stronger effects were detected when new filtrate was added continuously. Therefore, in the other experiments, I added fresh filtrate every sec-ond day, at least, when I took samples for the quanti-fication of allelopathic effects.

The allelopathic effects were quantified by cell counts of the target organisms in all three studies, using a flow cytometer (I), electronic particle ana-lyzer (II) and inverted microscope (III). In I and II, the target cultures were also checked qualitatively under a microscope, to see if the cyanobacterial ex-tracts had induced any changes in target cell mor-phology. However, no such changes were detected.

In addition to cell numbers, I measured the chlo-rophyll a concentration (Jespersen & Christoffersen 1987, II, III), 14CO2 uptake (Niemi & al. 1983, II) and pH (II). I hypothesized that the allelochemicals of the Baltic cyanobacteria act by inhibiting the photosynthesis of the target species (II). Thus I ex-pected that the effects are first expressed at the level of the photosynthetic pigments and primary produc-tion capacity, and finally, in cell numbers.

The toxin concentrations of the cyanobacterial cultures and their extracts were studied with high-performance liquid chromatography (HPLC; Lawton & al. 1994, Dahlmann & al. 2003, Fistarol & al. 2004b, I), PP1 inhibition assay (Fontal & al. 1999, I), and enzyme-linked immunosorbent assay (ELISA; Chu & al. 1990, II). Nodularin uptake of Rhodomonas cells or its adherence to their surface was monitored by ELISA (II).

I analysed the results of the laboratory studies (I–III) with repeated measures analysis of variance (ANOVA), followed by Tukey’s honestly significant difference (HSD) post hoc test. In III, species-de-pendent multivariate techniques of the PRIMER (v5) package were also applied (Clarke & Warwick 2001).

3.2 Long-term data analysis (IV)

I evaluated a 25-year (1979–2003) Helsinki Commission (HELCOM) monitoring dataset from five sampling sites in the northern Baltic Sea (Fig. 1), consisting of quantitative phytoplankton obser-vations, chlorophyll a, and environmental variables (temperature, salinity, inorganic P, N and Si), for the late-summer period (end of July – beginning of September). All data were analysed for the presence of monotonic increasing or decreasing trends by the nonparametric Mann-Kendall test (Gilbert 1987). In addition, I assessed the relationships between the phytoplankton composition and environmental fac-tors, using redundancy analysis (RDA), which is a linear method of direct ordination (ter Braak 1994).


4. RESULTS AND DISCUSSION

4.1 Allelopathy of Baltic cyanobacteria

Allelopathic effects were previously described for certain Baltic phytoplankton species, such as the haptophytes Chrysochromulina polylepis (Myklestad & al. 1995, Schmidt & Hansen 2001) and Prymne-sium parvum (Granéli & Johansson 2003, Fistarol & al. 2003), and the dinoflagellate Alexandrium tama-rense (Fistarol & al. 2004b). New research indicates that the Baltic spring-bloom diatoms Chaetoceros wighamii, Melosira arctica, Skeletonema costatum, Thalassiosira baltica and Diatoma tenuis may also be allelopathic towards co-occurring dinoflagellates (Spilling, in press). Although it had been suggested a few times that Baltic cyanobacteria have allelopathic properties (Sellner 1997, Balode & al. 1998, Engström-Öst & al. 2002a), it had not been ad-dressed in laboratory experiments prior to the studies presented in my thesis. Therefore, I is the first report on cyanobacterial allelopathy in the Baltic Sea and in brackish waters.

4.1.1 Effects of cyanobacteria on monocultures

Cross-culturing is commonly used to demon-strate the existence of allelopathic interactions be-tween two phytoplankton species. Although the con-ditions are far from natural, the advantage of the method is that detailed studies concerning the modes of action of allelochemicals can be conducted. In my thesis, I used the cross-culturing technique in two studies (I, II) to examine the inhibitory effects, caused by the cyanobacterial filtrates, on different phytoplankton species. The results showed that all studied cyanobacteria (Anabaena sp. 1, Apha-nizomenon flos-aquae and Nodularia spumigena) decreased the cell numbers of Rhodomonas sp., but none of them affected the cell numbers of Prymne-sium parvum. Thalassiosira weissflogii was also inhibited by all cyanobacteria, but only after re-peated filtrate additions (I, Table 3). With one fil-trate addition at the beginning of the experiment, T. weissflogii was only inhibited by N. spumigena in the end. Thus, the allelopathic effect of the other species only lasted for a couple of days, after which the chemicals were degraded, or the target species recovered.

Heterotrophic bacteria associated with the nonaxenic cyanobacteria were probably not involved

in the observed allelopathic effects, because the in-cubated filtrate with exudates of the heterotrophic bacteria did not inhibit Rhodomonas sp.

I speculated that specific group characteristics, such as membrane permeability, contributes to the sensitivity of Rhodomonas sp. to cyanobacterial al-lelochemicals, compared with the other species studied (I). Thalassiosira spp. are vernal species (Edler 1979) that probably are not adapted to late-summer cyanobacterial metabolites, whereas Prymnesium parvum co-occurs with cyanobacteria (Lindholm & Virtanen 1992) and may therefore be resistant. Prymnesium parvum itself has deleterious effects on the natural phytoplankton community, but cyanobacteria are the least affected group (Fistarol & al. 2003). This further suggests that the two groups are reciprocally tolerant. Resistant species that co-occur with the allelopathic species may bene-fit from the production of allelochemicals via re-duced competition (Legrand & al. 2003). Prymne-sium parvum, being mixotrophic (Nygaard & Tobiesen 1993), may also escape the possible allelo-pathic effects on photosynthesis by switching to heterotrophic nutrition.

4.1.2 Role of nodularin in allelopathy

Nodularia spumigena was allelopathic only in the exponential growth phase, whereas the culture filtrate was more hepatotoxic in the stationary phase (135.2 compared with 28.7 ng nodularin (µg chl a)-1 in stationary and exponential phase culture filtrates, respectively; I). Nodularin mostly remains inside the intact cells during the exponential growth phase and is released from the decaying cells to the growth medium during the stationary phase (Sivonen & Jones 1999). In contrast, allelochemicals are ex-creted during active growth, thereby affecting the competitors of the producer (Vance 1965, von Elert & Jüttner 1996).

When tested with purified nodularin (10 µg l-1), no significant effect on Rhodomonas sp. could be observed, in contrast to the negative effects of the filtrate of N. spumigena (7 µg l-1 nodularin) (II). It has been observed that crude cyanobacterial extracts containing toxins cause stronger effects than purified toxins, suggesting that extracts contain a mix of compounds that act synergistically (Pietsch & al. 2001, Wiegand & al. 2002, Leflaive & Ten-Hage 2007). However, my results strongly suggest that nodularin is not the primary compound causing the allelopathic effects of N. spumigena.


Table 3. Allelopathic effects of cyanobacterial extracts, according to the studies included in this thesis. Statistically significant effects on the last experimental day [after 3 (I), 4 (III) or 6 (II) days] on cell numbers of different phytoplankton species (and cellular chlorophyll a concentration and 14CO2 uptake of Rhodomonas sp., II) are indicated as percentage decrease or increase of cells (or other parameters) in the filtrate treatment relative to the control (n = 3, mean ± SD). 0 = nonsignificant effect, n.a. = not analysed.

Anabaena sp. 1

Aphanizomenon sp.

Nodularia spumigena Extract # extract

additions Article

Cyanobacteria Snowella spp. +611 ± 68% +544 ± 37% +520 ± 112% Exponential

phase (exp.) filtrate

4 III

Other picocyanobacteria 0 0 0 Exp. Filtrate 4 III Pseudanabaena spp. +166 ± 21% +106 ± 28% +109 ± 35% Exp. Filtrate 4 III Anabaena spp. 0 0 +479 ± 129% Exp. Filtrate 4 III Aphanizomenon sp. 0 +5070 ± 458% 0 Exp. Filtrate 4 III Nodularia spumigena 0 0 +415 ± 209% Exp. Filtrate 4 III

Cryptophytes Rhodomonas sp. strain KAC 30

-51 ± 5% -55 ± 2% -46 ± 15% Exp. Filtrate 1 I

-67 ± 5% -57 ± 6% -64 ± 5% Exp. Filtrate 3 I n.a. n.a. 0 Stationary phase

(stat.) filtrate 1 I

n.a. -29 ± 5% -14 ± 4% Exp. Filtrate 3 II 0 Nodularin 3 II

-chl a cell-1 n.a. -34 ± 3% -12 ± 7% Exp. Filtrate 3 II 0 Nodularin 3 II -14CO2 uptake cell-1 n.a. -45 ± 5% -43 ± 11% Exp. Filtrate 3 II

0 Nodularin 3 II All cryptophytes -79 ± 9% 0 -72 ± 24% Exp. Filtrate 4 III

Dinoflagellates Dinophysis norvegica 0 0 0 Exp. Filtrate 4 III Amphidinium sp. +65 ± 17% +59 ± 19% 0 Exp. Filtrate 4 III Paulsenella sp. 0 0 0 Exp. Filtrate 4 III Other dinoflagellates 0 0 0 Exp. Filtrate 4 III Dinoflagellate cysts 0 0 0 Exp. Filtrate 4 III

Haptophytes Prymnesium parvum strain KAC 39

0 0 0 Exp. Filtrate 1 I

0 0 0 Exp. Filtrate 3 I Diatoms

Chaetoceros spp. 0 0 0 Exp. Filtrate 4 III Thalassiosira weissflogii strain KAC 32

+48 ± 8% 0 -41 ± 5% Exp. Filtrate 1 I

-69 ± 10% -32 ± 7% -27 ± 3% Exp. Filtrate 3 I n.a. n.a. 0 Stat. filtrate 1 I Other diatoms 0 0 0 Exp. Filtrate 4 III

Chlorophytes Oocystis sp. 0 0 +47 ± 21% Exp. Filtrate 4 III Planktonema lauterbornii 0 0 0 Exp. Filtrate 4 III

Nanoflagellates +110 ± 7% 0 0 Exp. Filtrate 4 III


This conclusion agrees with the review by Babica & al. (2006), who found no support for the hypothesis concerning an allelopathic function of microcystins at environmentally relevant toxin con-centrations (1–10 µg l-1). Since the biosynthesis of microcystins and nodularins developed way before the evolution of eukaryotic photoautotrophs (Rantala & al. 2004), it is unlikely that the primary ecological role of cyanobacterial hepatotoxins is to act as al-lelochemicals against these organisms (Welker & von Döhren 2006). Other suggestions for the natural role of cyanotoxins range from by-products of me-tabolism to cell-cell signalling, iron-scavenging, storage substrates, growth regulators and grazer de-terrence (Kaebernick & Neilan 2001, Babica & al. 2006 and references therein). Recently, Schatz & al. (2007) suggested that microcystins may act as in-fochemicals that are released following lysis of a fraction of the cyanobacterial population and sensed by the remaining cells, which increase their ability to produce microcystins. This would enhance their fitness due to the toxic effects on grazers and com-petitors. The specific ecological role of nodularin remains to be elucidated.

4.1.3 Mode of allelopathic action

The effects of cyanobacterial allelochemicals on the target cells may be manifold (Table 1), although they often lead to decreasing cell numbers of the target as an outcome. To more closely examine the mechanisms of allelopathic actions of Baltic cyano-bacteria, underlying the decrease in cell numbers, I monitored the cellular chlorophyll a concentration and 14CO2 uptake, pH, and nodularin incorporation of Rhodomonas sp. exposed to filtrates of Apha-nizomenon flos-aquae and Nodularia spumigena and to purified nodularin (II). As discussed above, nodularin showed no allelopathic effects, although it was incorporated into the Rhodomonas cells to some extent (73.5 x 10-6 pg nodularin equivalents cell-1 after the first nodularin addition at the beginning of the experiment). In contrast, filtrates of both A. flos-aquae and N. spumigena significantly decreased the cell numbers, cellular chlorophyll a content as well as 14CO2 uptake of the cryptophyte (Table 3).

Most of the cyanobacterial allelochemicals whose mode of action and target site have been clarified to date are directed against oxygenic photo-synthetic processes in other phytoplankton (Smith & Doan 1999). In II, the inhibition of cellular chloro-phyll a concentration and 14CO2 uptake, which may indicate inhibition of photosynthesis, only occurred some days after a decrease in cell numbers. There-fore, I concluded that the allelopathic effect of the cyanobacteria was not directly due to an inhibition of photosynthesis. Instead, the decrease in cell num-bers may have been caused e.g. by breakage of the cell membranes, which is also a common mode of

action of phytoplankton allelochemicals (Legrand & al. 2003). Based on the present data, however, the target function or site of the cyanobacterial al-lelochemicals cannot be definitely concluded.

The pH of the environment can affect the growth of phytoplankton species, depending on their pH tolerance (Hansen 2002). In some cases, the ‘allelo-pathic’ effects detected have actually been due to the elevation of pH by the donor species to a level that is intolerable to the target species (Kroes 1971, Gold-man & al. 1981, Schmidt & Hansen 2001). High pH tolerance (up to 10.6), instead of allelopathy, has also been argued as the explanation for the domi-nance of Nodularia spumigena over other phyto-plankton in mixed culture experiments (Møgelhøj & al. 2006). While it may be the case for some target species, there was no difference in pH of the Rhodomonas cultures treated with either N. spumi-gena or A. flos-aquae filtrates or the control cultures: the average pH was 8.3 ± 0.2 both in the control and the N. spumigena treatment and 8.1 ± 0.2 in the A. flos-aquae treatment (II). Therefore, the inhibitory effects were not caused by pH changes, but rather by allelopathy of the cyanobacteria.

4.1.4 Effects of cyanobacteria on a natural plankton community

Combining laboratory and field studies by em-ploying micro- or mesocosm experiments with natu-ral phytoplankton communities may provide more ecologically reliable data on allelopathy than if using isolated species combinations. On the other hand, the results of whole-community experiments may greatly differ from those of simple cross-culturing with a few species, due to the multitude of interac-tions between trophic levels and the various species in the community. Thus, unless the allelopathic ef-fects are very prominent, it may be difficult to detect them and confirm the causative organism.

To determine whether Anabaena sp. 1, Apha-nizomenon sp. and Nodularia spumigena are able to produce allelochemicals that affect natural plankton assemblages, I employed a microplankton commu-nity from the central Baltic Sea to study the effects of cyanobacterial filtrate additions on the phyto-plankton biomass (measured as chlorophyll a con-centration) of the entire community, and on the abundances of phytoplankton, bacteria and ciliates (III). The filtrates increased the cell numbers of other cyanobacteria (Snowella spp., Pseudanabaena spp., Anabaena spp., Aphanizomenon sp., N. spumi-gena), a chlorophyte (Oocystis sp.), a dinoflagellate (Amphidinium sp.) and nanoflagellates, but de-creased the cell numbers of cryptophytes (Fig. 2, Table 3). The numbers of bacteria were also in-creased at the beginning of the experiment, follow-ing the filtrate addition, whereas it had no effect on the chlorophyll a concentrations or ciliate abun-


dances. According to the cluster analysis (III), the various cyanobacterial filtrate treatments generated plankton communities that differed both from the original community and from each other.

In III, I used a high ratio of cyanobacteria:target chlorophyll a concentration to simulate a cyanobac-terial bloom event (Table 2). The high amount of added organic matter, originating from the filtrates, probably explains the observed high stimulation of bacteria and several phytoplankton species that ei-ther benefited from nutrients remineralized by the increased bacteria or directly from the dissolved organic matter. Alternatively, organically bound nutrients released from the degrading cryptophyte cells may have led to stimulation of the remaining phytoplankton species. The stimulation may also be considered as a case of ‘positive allelopathy’, be-cause allelopathy in a broad sense includes all ef-fects, both harmful and beneficial, caused by com-pounds that are released into the environment (Rice 1984). In the natural community, many coexisting species may have developed resistance to the in-hibitory metabolites of cyanobacteria and may even benefit from these or other compounds. The fact that reports on positive allelopathy are restricted to a few (Keating 1977, 1987, Rice 1986, Mohamed 2002), may simply reflect the shortage of allelopathic ex-periments done with natural communities.

I suggest that the negative effects of the cyano-bacterial filtrates on the cryptophytes were due to allelochemicals excreted by the cyanobacteria (III). This agrees with the consistent inhibition of Rhodo-monas sp. by the cyanobacterial filtrates, observed in

the laboratory studies (I, II). Otherwise it is unclear why the cryptophytes were negatively affected in the filtrate treatments, if there was something (e.g. nu-trients) in the filtrates that all the other phytoplank-ton were able to utilize. The cryptophytes may be less adapted to cyanobacterial exudates than other phytoplankton groups, because they were the least abundant group in the community used in the ex-periment. Nevertheless, cryptophytes are known to be sensitive to allelochemicals excreted by other phytoplankton, e.g. cyanobacteria (Vance 1965, In-fante & Abella 1985, Sedmak & Kosi 1998, I, II), dinoflagellates (Rengefors & Legrand 2001, Fistarol & al. 2004b, Tillmann & al. 2007), and haptophytes (Schmidt & Hansen 2001, Granéli & Johansson 2003, Skovgaard & al. 2003, Barreiro & al. 2005, Uronen & al. 2005, 2007), and because they are ubiquitous and easy to cultivate, they have recently become model organisms that are often used as tar-gets in allelopathy experiments.

In nature, ciliates may vigorously graze on pico- and nanoplankton (Setälä & Kivi 2003), e.g. crypto-phytes. As ciliates were present in the experimental community (III), it is possible that grazing contrib-uted to the loss of cryptophytes to some extent. However, the initial numbers of ciliates and their development were similar in all treatments and the control, although the amounts of cryptophytes de-creased only in the filtrate treatments. This strongly suggests that the filtrates, and not grazing, were mainly responsible for the decrease in the crypto-phytes.

Fig. 2. Effects of cyanobacterial (Anabaena sp.1, Aphanizomenon sp., Nodularia spumigena) filtrates on a natural Baltic phytoplank-ton community after a 4-day exposure, compared with those of the control medium (n = 3, mean + SD) (III, redrawn from Karjalainen & al. 2007).


In general, the cyanobacterial filtrates greatly in-creased the numbers of heterotrophic and small-celled species (bacteria, cyanobacteria, chlorophytes, dinoflagellates and nanoflagellates) in the plankton community, without affecting its total biomass (ex-pressed as chlorophyll a concentration). This is con-sistent with observations that cyanobacterial blooms support active microbial food webs due to the leak-age of dissolved organic compounds from decaying cells (Hoppe 1981, Engström-Öst & al. 2002a). Al-lelopathy is most probably the cause of inhibitory effects by the cyanobacteria on cryptophytes.

4.2 Long-term trends of phytoplankton and environmental factors

Both environmental conditions and the phyto-plankton community structure have changed consid-erably in the northern Baltic Sea during recent dec-ades. A transition has occurred from more saline summer conditions, with higher silicate concentra-tions in winter in the late 1970s and in the 1980s, towards higher summer temperatures and higher absolute and relative winter dissolved inorganic ni-trogen (DIN) concentrations in the 1990s and early 2000s (IV). Simultaneously, the summertime chlo-rophyll a concentration of the surface water has sig-nificantly increased, indicating that the eutrophica-tion process continues in the open sea. The decrease in salinity and silicate concentrations, as well as the

increase in temperature, DIN and summer chloro-phyll a in the northern Baltic Sea have also been reported by several other authors (Flinkman & al. 1998, HELCOM 2002, Rönkkönen & al. 2004, Raateoja & al. 2005, Fleming-Lehtinen & al. in press.).

In contrast to hydrography, nutrients and chloro-phyll a, changes in phytoplankton species composi-tion in the northern Baltic Sea have not been ana-lysed in detail since the late 1980s (Kononen 1988). The biomass of cyanobacteria in the surface water layer of the Gulf of Finland increased significantly between 1979 and 2003 (IV). At the same time, the biomasses of chrysophytes and chlorophytes have increased, whereas that of the cryptophytes has de-creased. In 1979–1981, cyanobacteria comprised on average 42% of the total late-summer phytoplankton biomass, whereas their proportion was 69% in 2001–2003 (Fig. 3). Meanwhile, the relative biomass of cryptophytes decreased from 26% to 2%.

These results agree with reports that late-summer blooms of cyanobacteria have increased both in fre-quency and intensity (Kahru & al. 1994, 2007, Finni & al. 2001), and with the occurrence of several ex-tensive cyanobacterial blooms in the late 1990s and early 2000s (Rantajärvi 2003). Other studies also report increases in flagellate and chlorophyte bio-masses in the Baltic Sea (Kononen 1988, Wrzołek 1996). However, the considerable decrease in cryp-tophyte biomass has not been described before.

Fig. 3. Late-summer phytoplankton community structure (% of total biomass) in the Gulf of Finland in 1979–1981 and 2001–2003 (IV). Cyano = Cyanobacteria, Crypto = Cryptophyceae, Dino = Dinophyceae, Chryso = Chrysophyceae, Diatom = Diatomophyceae, Eugleno = Euglenophyceae, Chloro = Chlorophyceae, Ebri = Ebriidae.


Temperature, salinity and nutrient concentrations are considered the most important environmental factors affecting the phytoplankton community structure throughout the Baltic Sea (Kononen 1988, Alheit & al. 2005, Gasiūnaitė & al. 2005). Multi-variate analysis (IV) showed that the elevated sum-mer temperature and winter DIN concentration were the most significant factors explaining the variance in phytoplankton data, and the biomass development of many phytoplankton groups, such as the increase in chrysophytes, could be attributed to changes in either of the two factors. In addition, the decreased salinity probably contributed to the increase in chlorophytes, many of which are of lacustrine origin (Niemi 1973). Therefore, I concluded that the changes in the phytoplankton community in the northern Baltic Sea reflect both changes in hydro-graphy and the ongoing eutrophication process.

Biological interactions, such as interspecific competition, may also play a role in the variability of phytoplankton community composition. For ex-ample, in a multivariate analysis of phytoplankton dynamics in a lake in Portugal, the variance ex-plained for the phytoplankton assemblage was in-creased when cyanobacterial blooms were also con-sidered as an explanatory variable, in addition to environmental parameters (de Figueiredo & al. 2006). The authors suggested that this correlation was due to the competitive advantage and/or allelo-pathy of the bloom-forming cyanobacteria towards microalgae.

In the present study (IV), the decrease in crypto-phytes, coinciding with the increase in cyanobacte-ria, suggests that allelopathic interactions are in-volved, because the sensitivity of the cryptophytes to cyanobacterial extracts was proven in laboratory experiments (I, II). The cryptophytes also consti-tuted the only taxon that was significantly inhibited by cyanobacterial cell-free filtrates added to a natu-ral phytoplankton community, whereas most of the other phytoplankton taxa were stimulated (III, Fig. 2). In a recent modelling study, it was concluded that allelopathy, together with mixotrophy, is crucial to the coexistence and seasonal dynamics of cyano-bacteria and cryptophytes in the southern Baltic Sea (Hammer & Pitchford 2006).

The decrease in cryptophytes may also have af-fected the dinoflagellates, which showed a trend toward decrease (IV, Fig. 3). Some dinoflagellate species, such as Dinophysis spp., need temporary kleptoplastids from cryptophytes (Hackett & al. 2003, Minnhagen & Janson 2006), and therefore, the abundance of Dinophysis may be dependent on the abundance of cryptophytes (Nishitani & al. 2005). It is even possible that Baltic cyanobacteria compete with late-summer dinoflagellates by inhibiting cryptophytes, their essential endosymbionts.

Furthermore, top-down effects, such as grazing by microzooplankton, probably affect the late-sum-

mer phytoplankton community structure. For exam-ple, certain species of ciliates (e.g. Euplotes sp.), potentially important as grazers of cryptophyte-sized phytoplankton, thrive in the cyanobacterial aggre-gates (Engström-Öst & al. 2002a). An increase in cyanobacteria may have favoured these organisms, which have in turn contributed to the decline in cryptophytes. Unfortunately, long-term data con-cerning the microzooplankton abundances are scarce, and no definite conclusions on their role in the phytoplankton community changes can be drawn.

Several authors have described cases in which field observations of proliferation or decline in cer-tain phytoplankton groups or species have been sup-ported by laboratory experiments revealing allelo-pathic interactions between the species or groups in question (Keating 1977, 1978, Sukenik & al. 2002, Vardi & al. 2002). Most of these field observations have concerned seasonal phytoplankton succession, and it is probable that the outcome of allelopathy is more easily detected on a seasonal than on a long-term scale. Nevertheless, in Lake Neusiedlersee (Austria), for example, allelopathy was suggested to contribute to the massive development of cyano-bacteria during the late 1980s, coinciding with a strong decline in chlorophytes (Schagerl & al. 2002). The field observations agreed with laboratory ex-periments, where the dominant cyanobacteria inhib-ited the growth of chlorophytes isolated from the same lake.

5. CONCLUSIONS

This study shows, on one hand, that the late-summer phytoplankton community in the northern Baltic Sea has changed towards domination by cyanobacteria during recent decades and, on the other hand, that the most common filamentous cyanobacteria have species- or group-specific effects on co-occurring phytoplankton, probably mediated via extracellular metabolites. The most important new finding is the evidence for Baltic cyanobacterial allelopathy towards cryptophytes, obtained from both laboratory experiments and field studies.

The negative allelopathic effects of the cyano-bacteria studied are rather transitory, exerted during active growth, and not caused by the hepatotoxin, nodularin, in Nodularia spumigena. The specific allelochemicals, as well as their mode of action re-main uncertain, but they apparently act by reducing cell numbers, cellular chlorophyll a content and car-bon uptake of the target species. Exudates of cyano-bacteria may also stimulate bacteria, other cyano-bacteria, chlorophytes and flagellates, which is due to the ability of these taxa to utilize organic matter or bacteria, or nutrients remineralized by the bacte-


ria, their adaptation to cyanobacterial metabolites, organically bound nutrients released from damaged cryptophyte cells, unknown stimulatory chemicals or a combination of several factors.

It is unlikely that allelopathy is the key factor in the initial phases of phytoplankton bloom formation, due to the low density of the producer cells (Solé & al. 2005). However, cyanobacteria may use allelo-pathy in the maintenance of the bloom to suppress competing phytoplankton, after the development and concentration of an adequate amount of cells in the surface layer during favourable hydrographic condi-tions. In the nutrient-depleted late-summer environ-ment, allelopathy may be an additional stressor for many phytoplankton species, leading to long-lasting blooms of cyanobacteria.

Cyanobacterial allelopathy may not only be a means to reduce nutrient competition by preventing other species from attaining high biomasses, but also to gain more nutrients through stimulation of bacte-ria or by breaking cryptophyte cells, as suggested for the dinoflagellate Peridinium aciculiferum (Renge-fors & Legrand 2001). Furthermore, the inhibitory effects of cyanobacteria on cryptophytes may be associated with the interactions between cyanobacte-ria and dinoflagellates, which need cryptophytes for their plastids, or indirectly with grazing of crypto-phytes by microzooplankton, which benefit from the presence of cyanobacteria.

The long-term development of phytoplankton communities reflects a combination of ongoing eu-trophication, expressed as increasing dissolved inor-ganic nutrient concentrations of the surface water, climate-driven changes in hydrography, i.e. de-creasing salinity and increasing temperature, and possibly, biological interactions among species of phytoplankton and between phyto- and zooplankton. The resulting significant increase in phytoplankton biomass is due to a considerable increase in cyano-bacteria, various flagellates and chlorophytes. Nev-ertheless, the biomass of some phytoplankton groups, e.g. cryptophytes, has declined.

The allelopathic effects detected in the laboratory studies of this thesis are generally in line with the observed long-term increase in cyanobacteria, chlorophytes and chrysophytes, and the decrease in cryptophytes. Thus, while the proliferation of cyanobacteria in the northern Baltic Sea is known to be enhanced by internal P loading and favourable hydrographic factors, my results show that chemi-cally mediated biological interactions should also be considered as factors affecting the structure of Baltic phytoplankton communities.

Acknowledgements

This work was funded by the Maj and Tor Nessling Foundation, the Walter and Andrée de

Nottbeck Foundation, University of Helsinki, So-cietas Pro Fauna and Flora Fennica, the Nordic Academy for Advanced Study and the Finnish In-stitute of Marine Research (FIMR).

I am grateful to my both supervisors, Jonna Eng-ström-Öst and Markku Viitasalo for their never-ending optimism, confidence and guidance through all the years, despite my own, somewhat unrealistic, picture of the amount of side-projects one is able to handle while concentrating on a PhD thesis. Special thanks to Make for initially accepting me as a PhD student in his experimental zooplankton ecology (EZECO) group, although I had no intentions what-soever to study experimental zooplankton ecology, and, consequently, for all the scientific freedom. Jonna with all her deadlines was crucial in finally putting an end to this work.

I thank the pre-examiners of this thesis, Pirjo Kuuppo and Norbert Wasmund, for an efficient re-view considering my tight schedule and especially Purjo for all the constructive criticism. Harri Kuosa and Sari Repka kindly commented on an early ver-sion of the manuscript. Leena Parkkonen and Leena Roine are thanked for editing the text, and Jorma Kuparinen for taking care of the administrative is-sues.

I wish to express my gratitude to Edna Granéli for inviting me to visit her lab at Kalmar University, to conduct some experiments on allelopathy. It was an opportunity that really helped me to get started with my scientific activities. I am indebted to Gio-vana and Paulo Salomon for making my visit so en-joyable and for arranging all practical things con-cerning work and freetime. Thank you Giovana for our joint experiments and for being an advisor, a co-author as well as a friend. I’m also thankful to Catherine Legrand in Kalmar for advice concerning allelopathic experimental set-ups.

Thanks to all the members of the late EZECO (R.I.P.): Eve, J.-P., Jonna, Maiju, Marja, Make, Mii-na, Roope, Samuli, Sandra, SannaR, Satu, Tarja and Tomi, for various hilarious activities both at work and in the freetime, and especially to SannaR, my room mate during so many years and in three differ-ent places. Futhermore, I wish to thank two exem-plary women of science, Maria Laamanen and Anke Kremp for all their support, advice and co-operation. Anke skillfully supervised my MSc thesis, concern-ing vernal dinoflagellates, and is thus to a large part responsible for my ending up as a phytoplankton researcher.

Division of Hydrobiology at the University of Helsinki provided me with a place to work during my first steps as a PhD student. Later, I was warmly welcomed by the Department of Biological Ocean-ography at FIMR, and I want to thank all the people there for creating an extraordinarily dynamic and inspiring working environment. A lot of the experi-mental work was done during long summer and au-


tumn days and nights at the Tvärminne Zoological Station. I am indebted to the staff there for all their help, especially to Anne-Marie Åström and Ulla Sjölund in the lab, and to Laila Keynäs, Raija Myllymäki and Jouko Pokki concerning all imagin-able practical things.

Kaarina Sivonen is acknowledged for kindly let-ting me use her cyanobacteria cultures, nodularin and lab facilities, and Matti Wahlsten and Jouni Jokela in her lab for helping with all practicalities. Sari Repka provided the inspiration for my first al-lelopathic experiments and patiently advised me in my early attempts to culture cyanobacteria. Miina Karjalainen is acknowledged for all help and discus-sions concerning nodularin analysis, as well as for sharing so many conference trips. Riitta Autio helped me to get started with primary production analysis, and Maija Huttunen assisted with all mat-ters concerning phytoplankton microscopy.

All my co-authors are acknowledged for their help in the various phases of experimental design, data gathering and article preparation.

During the freetime, the biologist gang Marjorie has been essential in escaping the world of (hydro-biological) science. Thanks to my nonbiologist friends from the good old Kannelmäki-Haaga region as well!

My parents have always believed in me and en-couraged me to make my own choices, for which I’m grateful. Thank you, and Aili as well, for always taking care of our little Eino when needed. Finally, my warmest thanks to Jarno for all the help and in-terest concerning science, statistics and computing, and above all, for your love and patience all the way.

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