The role of allelopathy in microbial food webs

Linnaeus University Dissertations

No 33/2011

THE ROLE OF ALLELOPATHY IN MICROBIAL FOOD WEBS

ASTRID WEISSBACH

LINNAEUS UNIVERSITY PRESS

THE ROLE OF ALLELOPATHY IN MICROBIAL FOOD WEBS Doctoral dissertation, School of Natural Sciences, Linnaeus University 2011. Series editor: Kerstin Brodén Cover picture by Camilla Fahlgren and Oliver Gast ISBN: 978-91-86491-62-8 Printed by: Intellecta Infolog, Gothenburg

To My Family

Shall I refuse my dinner because I do not fully understand the process of digestion?

Oliver Heaviside (1850-1925) English physicist.

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TABLE OF CONTENTS

LIST OF PAPERS .................................................................................................2 SUMMARY ............................................................................................................3 SAMMANFATTNING.................................................................................. 4 ZUSAMMENFASSUNG ............................................................................... 5 LIST OF PAPERS........................................................................................... 6 INTRODUCTION ......................................................................................... 7

Strain specific variability in allelopathy.......................................................... 8 Factors regulating allelopathy ........................................................................ 9 Interactions between phytoplankton and bacteria ....................................... 11 Impacts of allelopathy on the microbial food web ....................................... 12

AIMS OF THE THESIS.............................................................................. 13 METHODS ................................................................................................... 15

Study sites .................................................................................................... 15 Laboratory setup for papers 1 – 4 ................................................................ 16 Textbox: Allelochemicals, P. parvum and A. tamarense ................................ 17 Analytical Methods...................................................................................... 18

RESULTS AND DISCUSSION .................................................................. 20 Exudates of different strains of Alexandrium and Prymnesium vary in their allelopathic effects ............................................................................... 20 The effect of salinity on allelopathy ............................................................. 21 How do allelochemicals affect plankton communities? ............................... 23

CONCLUSIONS........................................................................................... 31 FUTURE PERSPECTIVES......................................................................... 32 ACKNOWLEDGEMENTS ........................................................................ 33 REFERENCES.............................................................................................. 34

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LIST OF PAPERS

I. Weissbach, A., Legrand, C. Hemolytic activity, allelopathy and growth rates of four strains of Prymnesium parvum in two different salinities. Manuscript

II. Weissbach, A., Tillmann, U., Legrand, C., (2010). Allelopathic

potential of the dinoflagellate Alexandrium tamarense on marine microbial communities. Harmful algae, 10, 9–18.

III. Weissbach, A., Rudström, M., Olofsson, M,. Béchemin, C., Iceley, J.,

Newton, A., Tillmann, U., Legrand, C. Allelochemical interactions change microbial food web dynamics. Limnology and Oceanography (accepted)

IV. Weissbach, A., Béchemin, C., Geneauzau, S., Rudström, M., Legrand,

C. Impact of Alexandrium tamarense allelochemicals on DOM dynamics in an estuarine microbial community. Submitted to Harmful Algae

Paper II is reprinted with the kind permission of Elsevier Science.

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SUMMARY

Phytoplankton produce and excrete chemical substances that are affecting other microorganisms in their direct environment. Those substances are referred to as allelochemicals.

In my thesis, I investigated strain specific variability in the expression of allelochemicals of Prymnesium parvum. Although P. parvum is euryhaline, blooms of the species are most frequently reported from brackish or low saline water-bodies. My studies showed large variation in allelopathy among strains, but further that all strains of P. parvum were more allelopathic in brackish water compared to marine water.

In a marine microbial community, allelochemicals can affect prey, competitors and grazers both directly and indirectly. For instance, in a food web where grazing controls prey abundance, the negative direct effect of allelochemicals on grazers will positively affect their prey. During my thesis, I also investigated how marine microbial communities responded to the addition of allelochemicals. I performed field experiments with microbial communities from seawater collected from different places in Europe, and tested how these communities respond to the addition of allelochemicals from the dinoflagellate Alexandrium tamarense. Before I incubated the microbial communities for several days with A. tamarense algal filtrate, I evaluated the allelopathic efficiency of the algal filtrates with an algal monoculture of Rhodomonas salina. This allowed me to compare the effect of A. tamarense filtrate between the different microbial communities.

In general, bacteria reached higher abundances when allelochemicals were present. As allelochemicals also inhibited nanoflagellates and ciliates, we concluded, that allelochemicals indirectly benefit bacteria by reducing grazing pressure. In microbial food webs with many heterotrophic grazers, allelochemicals further benefited other phytoplankton by inhibiting grazers. We also showed that bioavailable dissolved organic material (DOM) is released from a microbial community when allelochemicals are present. As most DOM was released from the seawater fraction > 60 μm, we concluded, that larger microorganisms are more affected by allelochemicals than smaller microorganisms. Larger organisms provide more contact surface for allelochemicals to attach to, and therefore probably are more vulnerable towards allelochemicals.

In conclusion, the effect of allelochemicals on a microbial community depends among others on the structure of the microbial food web, the amount of available DOM, the particle density in the seawater and the composition of the phytoplankton community.

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SAMMANFATTNING

Växtplankton producerar och utsöndrar kemiska ämnen som påverkar andra mikroorganismer i deras direkta omgivning. Dessa ämnen kallas allelokemikalier. I min avhandling har jag undersökt stamspecifik variation i uttrycket av allelokemikalier hos Prymnesium parvum. Även om P. parvum är euryhalin (art som kan anpassa sig till ett brett spektrum av salthalter) så har blomningar av den här arten rapporterats mer frekvent från vattenområden med bräckt vatten eller med låg salthalt. Mina studier visade stora variationer i allelopati (förmågan att avge ämnen som hindrar andra organismers utveckling) mellan olika stammar, men också att alla stammar av P. parvum var mer allelopatiska i bräckt vatten jämfört med saltvatten.

I ett marint mikrobiellt samhälle kan allelokemikalier påverka bytesdjur, konkurrenter och betare både direkt och indirekt. Till exempel påverkas bytesdjur positivt av den negativa direkta effekten som allelokemikalier har på betare i en näringskedja eftersom betare reglerar mängden av bytesdjur. Under min avhandling har jag undersökt hur marina mikrobiella samhällen svarar på tillskott av allelokemikalier. Jag gjorde fältförsök med mikrobiella samhällen från havsvatten som samlats in från olika platser i Europa, och testade hur dessa samhällen svarar på tillskott av allelokemikalier från dinoflagellaten Alexandrium tamarense. Innan jag inkuberade de mikrobiella samhällena i flera dagar med filtrat från algen A. tamarense så utvärderade jag den allelopatiska effektiviteten i algfiltraten med en monokultur av algen Rhodomonas salina. Därmed kunde jag jämföra effekten av A. tamarense filtrat mellan de olika mikrobiella samhällena.

Generellt uppmättes större mängder bakterier när allelokemikalier var närvarande. Eftersom allelokemikalier även hämmade små flagellater och ciliater så drog vi slutsatsen att allelokemikalier gynnar indirekt bakterier genom att minska betestrycket. I mikrobiella näringskedjor med många heterotrofa betare gynnade allelokemikalier dessutom andra växtplankton genom att hämma betarna. Vi visade också att biotillgängligt löst organiskt material (DOM) frisläpps från ett mikrobiellt samhälle när allelokemikalier är närvarande. Eftersom det mesta lösta organiska materialet frisläpptes från havsvattenfraktionen > 60 μm så drog vi slutsatsen att större mikroorganismer påverkas mer av allelokemikalier än mindre mikroorganismer. Större organismer tillhandahåller mer kontaktyta för allelokemikalier att fästa till och är därför troligen mer sårbara mot allelokemikalier.

Sammanfattningsvis beror effekten av allelokemikalier på mikrobiella samhällen bland annat på strukturen av den mikrobiella näringskedjan, mängden tillgängligt DOM, partikeltätheten i havsvattnet och sammansättningen av växtalgerna.

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ZUSAMMENFASSUNG

Einige Mikroalgen sondern chemische Substanzen ab, die andere Mikroorganismen in ihrer direkten Umgebung beeinflussen können.

Im Zuge meiner Arbeit habe ich unter anderem untersucht, in wie weit die Absonderung allelopathischer Substanzen im Flagellaten Prymnesium parvum klonpezifisch ist. Giftige Algenblüten der euryhalinen Goldalge P. parvum verursachen regelmässig fischereiwirtschaftliche Schäden. Die meisten dieser Algenblüten ereignen sich in Wasser mit geringer Salinität. Deshalb wurde von mir der Einfluss von Salinität auf die Absonderung von allelopathischen Substanzen von Prymnesium parvum untersucht. Die Klone wiesen eine hohe Variabilität in Allelopathy auf, aber im allgemeinen wurden in Brackwasser mehr allelopathische Substanzen abgesondert als in Meereswasser.

Ein weiterer Teilaspekt meiner Arbeit war die Untersuchung von direkten und indirekten Einflüssen allelopathischer Substanzen auf marine mikrobielle Gesellschaften. Wenn zum Beispiel in einer Nahrungskette einige Predatoren die Abundanz ihrer Beute kontrollieren, so wird die negative Einwirkung allelopathischer Substanzen auf die Jäger deren Beute positiv beeinflussen. Ich untersuchte wie marine mikrobielle Gesellschaften verschiedener Ökosysteme auf allelopathische Substanzen des Dinoflagellaten Alexandrium tamarense reagieren, indem ich diese für einige Tage allelopathischen Substanzen von A. tamarense aussetzte und Veränderungen in der mikrobiellen Gesellschaft beobachtete. Im allgemeinen profitierten Bakterien von allelopathischen Substanzen; da diese deren Frassfeinde, Nanoflagellaten und Ciliaten, im Wachstum behinderten. In Gesellschaften mit hohem Frassdruck auf Phytoplankton haben allelopathische Substanzen durch den gleichen Mechanismus auch indirekt das Wachstum von Phytoplankton gefördert. Desweiteren konnte ich während meiner Studien nachweisen, dass es durch das Vorhandensein allelopathischer Substanzen zur Freisetzung von bioverfügbarem gelöstem organischem Kohlenstoff kommt. Zusammenfassend konnte ich schlussfolgern, das allelopathische Substanzen mikrobielle Gemeinschaften verschiedener Ökosysteme unterschiedlich stark beieinflussen. Ihr Einfluss ist unter anderem abhängig von der Stuktur der Nahrungskette, dem Angebot an gelöstem organischem Kohlenstoff und der Artenzusammensetzung der mikrobiellen Gesellschaft.

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INTRODUCTION

The process of one plant producing chemical compounds, which affect other plants, is called allelopathy. The word origins from the greek “allel” and “pathos” and means “let the one suffer that looks like you”. The term itself was created by H. Molisch in 1937 and according to his definition, allelopathy is the summary of all biochemical interactions between all classes of plants. Rice (1984) expanded the definition of the term to “any indirect or direct harmful or beneficial effect by one plant on another plant by the production of chemical compounds that escape into the environment”. In aquatic systems, the definition of allelopathy is not limited to plant – plant interactions (Cembella, 2003; Gross, 2003; Legrand et al., 2003; Granéli, 2006; Ianora et al., 2006), but enhanced to all chemical interactions among unicellular organisms (Tillmann et al., 2007). Allelochemicals can cause lysis, blistering, growth inhibition or death of the target cells (Legrand et al., 2003). They may also positively affect the growth of other phytoplankton species and other microorganisms (Mohamed, 2002; Fistarol et al., 2004). The substances may even stimulate or inhibit the donor organism itself (Vardi et al., 2006; Olli and Trunov, 2007). Allelochemicals are understood to benefit the donor organism in several ways:

a) Allelochemicals eliminate competitors

In several laboratory studies, phytoplankton was either inhibited in growth or destructed after being exposed to exudates of allelopathic algae (Tillmann, 2003; Fistarol et al., 2004; Uronen et al., 2007). In nature, the absence of phytoplankton competitors may allow the allelopathic species to exploit available nutrients. The patchy distribution of cyanobacteria and dinoflagellates in the Sea of Galilee was interpreted as a consequence of allelopathy, as both species’ exudates inhibit each others growth, and nutrient conditions are equal among the observed patches (Vardi et al., 2002). By eliminating competitors, allelopathy may influence seasonal succession patterns (Keating, 1978).

b) Allelochemicals deter grazers

Some scientists argue that allelochemicals are primarily defense mechanisms against predators (Wolfe, 2000). The allelopathic substance is either deleterious to the predator when the algae are ingested (Ianora et al., 2004), or prevents ingestion by killing the predator (Tillmann, 2003). In that sense, effects we observe on other microorganisms than grazers (like the elimination of competitors mentioned in the first paragraph) might be irrelevant bi-effects for the donor species itself; as the actual target of the chemicals are algal predators, for instance copepods or rotifers.

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Grazing deterrence is known from diatoms, where copepod recruitment is suppressed during blooms via aldehydes (Ianora et al., 2004) and several dinoflagellates reduce egg production and hatching success in copepods and rotifers (Xie et al., 2008).

c) Allelochemicals serve as a hunting device and enable mixotrophy

Additionally to gaining energy via photosynthesis, phytoplankton can gain energy by using organic carbon for nutrition (mixotrophy) (Legrand and Carlsson, 1998; Stoecker, 1999). Allelopathy might function as `pseudo – mixotrophy´ by enlarging the nutrient pool for allelopathic donor species (Roy, 2009). By lysis of prey cells, allelochemicals may supply the donor species with dissolved organic nutrients (Stoecker et al., 2006; Jonsson et al., 2009). Some motile mixotrophs ingest particulate organic material (phagotrophy) and for those, allelochemicals may be a hunting device. For instance the marine dinoflagellate Karlodinium veneficum uses its extracellular toxins as a means of immobilizing prey cells before ingestion (Sheng et al., 2010). Another example is Prymnesium parvum, whose feeding frequency was shown to be positively correlated with an allelopathic effect that caused lysis and immobility of the cells (Skovgaard et al. 2003). Whereas mixotrophy historically was seen as a strategy of phytoplankton to survive in oligotrophic environments, nowadays, many harmful algae frequently blooming in eutrophic areas are suspected to be mixotrophs (Burkholder et al., 2008).

Strain specific variability in allelopathy Geno- and phenotypic intraspecific variation is common among microalgae (Burkholder and Glibert, 2009). Allelopathy as well as toxicity has been found to differ significantly among algal strains of the same species (Larsen and Bryant, 1998; Alperman et al., 2010). The expression of allelopathy is a trade-off between the metabolic costs of production and the advantage allelochemicals provide to the donor species. The co-existence of strong allelopathic and weak allelopathic strains is possible if allelochemical interactions between the algae and its competitors or grazers are only important in certain stages of the development of the population. If additionally thereto other phenotypic traits are also only of selective importance for certain periods during population growth, the balancing selection on different traits at different developmental stages may lead to a broad phenotypic diversity over time (Alpermann et al., 2009). Strain variability is not only restricted to geographically distinct populations of the same species (Medlin et al., 2000) but also occurs within populations. For instance, clones from about 90 Alexandrium tamarense strains isolated “from the same hatch” during an A. tamarense bloom in the North Sea exhibited a high variation in allelopathy (Alpermann et al., 2010). Strain variability in

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allelopathy is an important aspect in understanding and predicting harmful algae bloom (HAB) dynamics. In my thesis, I investigated the clonal variability in allelopathy in four strains of the euryhaline harmful algae species Prymnesium parvum (Paper I).

Factors regulating allelopathy

Synthesis, exudation, stability and degradation rate of allelochemicals can be affected by both, abiotic and biotic environmental conditions. Generally, the production of allelochemicals is enhanced when the organism is stressed (Granéli and Hansen, 2006). For instance, nutrient deficiency as an abiotic stress factor has been proven to increase the production of allelochemicals in several algae (Legrand et al., 2003). Furthermore, for Raphidophytes it is known that salinity affects the production of allelochemicals - with lower salinities leading to higher levels of allelopathy (de Boer et al., 2004). As allelochemical compounds bind to surfaces, the allelopathic effect can also be influenced by the availability of surfaces (living and non living particulate organic material (POM)) in the environment (Tillmann 2003). For instance, the lytic activity of Prymnesium parvum was reduced by increasing the amount of target organisms (Tillmann, 2003). Furthermore, a weak target organism is more susceptible to allelochemicals than a healthy target organism (Fistarol et al., 2005). Thus, stress factors are not only increasing the production of allelochemicals in the donor organism but also the effect of allelochemicals on the target organism. In my thesis, I investigated how the environmental stress factor salinity influences allelopathy in Prymnesium parvum (Paper I).

How do allelochemicals affect plankton communities?

Most of the research on chemical interactions among microorganisms focuses on direct responses of specific target species towards allelochemicals, while information is scarce about responses of marine communities to algal bioactive substances (Fistarol et al., 2003; Fistarol et al., 2004; Strom, 2008; Weissbach et al., 2010). If the sensitivity of microorganisms towards allelochemicals differs among functional groups or species, the structure of the plankton community can change in presence of allelochemicals. Via trophic cascades, the removal of microbial grazers, such as ciliates, might affect the lowest trophic levels in the microbial food web, wich includes phytoplankton and bacteria. Further, due to the lysis of microorganisms, organic and inorganic nutrients are released with allelochemicals, changing the nutrient availability in the plankton community.

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In my thesis, I investigated the response of plankton communities towards the addition of allelochemicals of the harmful dinoflagellate Alexandrium tamarense (Paper II, III and IV).

Interactions between phytoplankton and bacteria

The release of dissolved organic material (DOM), either directly from phytoplankton or as a consequence of “sloppy feeding” by phytoplankton grazers, is an important source of labile organic carbon for bacteria. Bacteria consume about 40 – 50 % of the primary production across all aquatic systems, however, their uptake of nitrogen (N) and phosphorus (P) place bacteria and phytoplankton in competition for growth limiting nutrients. Few studies have addressed allelopathic interactions between bacteria and phytoplankton in the marine environment. In arctic sea ice, low primary production rates correlated well with bacterial abundance (probably due to the dependence of bacteria on DOM provided by phytoplankton); whereas bacterial abundance decreased significantly, when algal biomass exceeded a certain level (possibly allelopathy) (Monfort et al., 2000). Contrary thereto, many authors report an increased bacterial abundance in presence of allelochemicals (Fistarol et al., 2004; Suikkanen et al., 2005; Uronen et al., 2007).

In my thesis, I investigated the response of bacterial abundance, production and composition in natural plankton communities towards the addition of allelochemicals of the harmful dinoflagellate Alexandrium tamarense (Paper II, III and IV).

Effects of allelopathy on the microbial food web

In aquatic ecosystems, energy is channeled among organisms in two ways (Azam et al., 1983): (1) via the classical food web, consisting of phytoplankton, zooplankton, and fish; and (2) via the microbial food web, a complex structured microhabitat, including several levels of heterotrophic grazers (nanoflagellates and ciliates) feeding on primary producers (phytoplankton) and bacteria (remineralizers of dissolved organic matter). The traditional view on the microbial food web in the ocean is shown in Figure 1A. Adding phytoplankton allelopathic interactions creates a more complex picture (Figure 1B) as bacteria, flagellates, phytoplankton and protozoans might be lysed by allelochemicals, but also release DOM, and therefore stimulate bacterial production in the microbial food web. In my thesis, I investigated how allelochemicals affect trophic interactions in the plankton food web (Paper II, III and IV).

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Figure 1. Sketch of the trophic interactions and organisms in the microbial food web: (A) traditional view and (B) including allelochemical interactions

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AIMS OF THE THESIS

The aims of my thesis are:

- to investigate interclonal variability in allelopathy and intracellular

toxicity in the harmful algae Prymnesium parvum - to investigate the effect of salinity on allelopathic activity of

Prymnesium parvum

- to investigate the impact of allelochemicals on a natural marine bacterial community

- to evaluate if labile DOM is released in a natural estuarine microbial

community due to the presence of allelochemicals

- to evaluate the relative importance of indirect versus direct effects of marine phytoplankton allelochemicals on natural microbial communities from different ecozones

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METHODS

In this chapter I will only present methods that are unusual in the field of phytoplankton ecology. All methods used are extensively described in the publications following the introduction.

Study sites

The physiology study on Prymnesium parvum (Paper I) was performed at the Linnaeus University, Kalmar, with four strains originating from marine (North Sea in England (Strain B) and Norway (Strain C)) and brackish water (river estuaries in Israel (Strain A) and England (Strain D) regions. For Papers II, III and IV, responses of the microbial community towards Alexandrium tamarense allelochemicals were tested. For the study in Paper II, seawater was collected in spring from the southern North Sea near Helgoland, Germany. The microbial community was dominated by Phaeocystis globosa, which is commonly forming blooms during spring whereas dinoflagellates dominate the phytoplankton community in the central North Sea during summer (Reid et al., 1990). For the study in Paper III, we collected a microbial community in the south of the Iberian Peninsula during a relaxing upwelling situation in autumn. The initial microbial community in the study in Paper III consisted of diatoms and dinoflagellates, but also contained a high proportion of heterotrophic and autotrophic nanoflagellates. In the study in Paper IV the microbial community originated from a eutrophic estuary of the Charente River (French Atlantic coast). The area is known for its high turbidity rates and high particle density in the water (Heral et al., 1984), and the microbial community collected for the study was dominated by diatoms (Figure 2).

Figure 2. Study sites for natural plankton community experiments

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Laboratory setup papers 1-4

Paper I aimed on identifying patterns in physiology, allelopathy and intracellular toxicity in the response of P. parvum towards salinity changes. To overcome strain variability four strains were incubated at two different salinities. Allelopathy was hereby understood as extracellular toxicity and was measured with the Rhodomonas bioassay, whereas the intracellular toxicity of P. parvum was measured with the hemolysis test. The stability of intraspecific strain characteristics was tested by repeating the experiment in slightly modified experimental conditions. The four strains of P. parvum were grown in triplicate batch cultures at Salinity 7 and 26. Cultures were investigated for hemolytic and allelopathic activity and physiological parameters (as intracellular C:N:P ratios and Chl a content) during exponential, stationary and senescent growth. In Paper II, the response of a plankton community to extracellular compounds produced by two strains of A. tamarense (lytic and non-lytic) was investigated. This comparative approach was chosen to distinguish between the impact of the added A. tamarense suspension per se and the impact of lytic compounds, produced by A. tamarense. Seawater was incubated with supernatant corresponding to 200 and 1000 cells ml-1 from a lytic and a non-lytic A. tamarense strain. All bottles were incubated for 96 h. During incubation, bacterial abundance and production was followed. After 96 h, the changes in the microbial community were monitored. The response of four experimental microbial food webs to the addition of A. tamarense allelochemicals was investigated in Paper III. In half of the treatments, the initial microbial community was filtered through a 1 μm net to remove grazers. Further, dissolved organic carbon (DOC) was added to half of the seawater. The resulting four manipulated food webs were incubated for 96 h either with or without lytic A. tamarense filtrate. The development of the microbial community was investigated. In Paper IV, the size class distribution in a natural plankton community that is most sensitive towards allelochemicals was investigated. Using nylon nets, seawater was separated into three fractions: < 150 μm; < 60 μm and < 20 μm and incubated with either lytic or non-lytic filtrate of A. tamarense. Aliquots (50 ml) of all replicates from all treatments were taken immediately (T0) and 6 h (T6) after A. tamarense filtrate addition. Bacterial seawater cultures were prepared and the growth of bacteria was followed for 48 h.

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Text box: Allelochemicals, P. parvum and A. tamarense

5 µm

5 µm

Allelochemicals

A. tamarense and P. parvum allelochemicals probablyattach to the target cells since both substances show amphipathic properties. Here, they punch holes intothe outer membrane and thereby increase membranepermeability, resulting in the lysis of the target cell (Prince et al., 2008; Ma et al., 2009; Manning and La Claire, 2010).

The cell lysis of a Rhodomonas cell provoked by allelochemicals is shown to the left. The structure of allelopathic compounds is unknown for A. tamarense, whereas the scientific community argues that substances isolated from P. parvum, calledPrymnesins, are allelopathic. Nowadays, P. parvum is suggested to produce at least six different compounds with allelopathic properties (Manning et al., 2010; Schug et al., 2010).

Alexandrium tamarense

Members of the genus Alexandrium are dinoflagellates of the family Goniodomaceae. Alexandrium is common in upwelling regions worldwide. Blooms of Alexandrium have been reported worldwide, usually associated with shallow salt ponds, coastal bays and open coastal waters. Since it produces neurotoxins, causing Paralytic Shellfish Poisoning (PSP), it is classified as “harmful algae”.

Prymnesium parvum

Prymnesium parvum is a flagellatewithin the phylum Haptophyta. The species tolerates large variations in temperature and salinity, but bloomsare frequently occuring in brackishwater at Salinity 1 – 12. The blooms develop in the warm season at water temperatures above 10 °C (Edvardsen and Paasche, 1998). Although P. parvum toxins are not affecting humans, bloom events cause large economic losses due to fish kills, which classifies the species as “harmful algae”

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Allelochemicals

A. tamarense and P. parvum allelochemicals probablyattach to the target cells since both substances show amphipathic properties. Here, they punch holes intothe outer membrane and thereby increase membranepermeability, resulting in the lysis of the target cell (Prince et al., 2008; Ma et al., 2009; Manning and La Claire, 2010).

The cell lysis of a Rhodomonas cell provoked by allelochemicals is shown to the left. The structure of allelopathic compounds is unknown for A. tamarense, whereas the scientific community argues that substances isolated from P. parvum, calledPrymnesins, are allelopathic. Nowadays, P. parvum is suggested to produce at least six different compounds with allelopathic properties (Manning et al., 2010; Schug et al., 2010).

Alexandrium tamarense

Members of the genus Alexandrium are dinoflagellates of the family Goniodomaceae. Alexandrium is common in upwelling regions worldwide. Blooms of Alexandrium have been reported worldwide, usually associated with shallow salt ponds, coastal bays and open coastal waters. Since it produces neurotoxins, causing Paralytic Shellfish Poisoning (PSP), it is classified as “harmful algae”.

Prymnesium parvum

Prymnesium parvum is a flagellatewithin the phylum Haptophyta. The species tolerates large variations in temperature and salinity, but bloomsare frequently occuring in brackishwater at Salinity 1 – 12. The blooms develop in the warm season at water temperatures above 10 °C (Edvardsen and Paasche, 1998). Although P. parvum toxins are not affecting humans, bloom events cause large economic losses due to fish kills, which classifies the species as “harmful algae”

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Analytical methods

Allelopathy test

To quantify the allelopathic effect of an algal species, the Rhodomonas bioassay (Tillmann, 2004) was used. The survival of an even number of Rhodomonas cells is tested against a dilution series of the filtrate from allelopathic algae. The resulting sigmoidal curve gives information about the effective concentration EC50 of P. parvum filtrate (50 % Rhodomonas are dead).

Hemolytic activity test

Intracellular toxicity was measured as hemolytic activity of P. parvum cells on horse red blood cells based on Igarashi et al., 1998. Prior to the hemolysis test, the toxins are extracted from the algal cells with methanol. The lysis of an even number of blood cells is tested against a dilution series of the extracted toxin. The resulting sigmoidal curve gives information about the EC 50 value (50 % blood cells are lysed at this concentration of filtrate).

Bacterial production, seawater cultures and community composition

Bacterial production

The measurement of bacterial production provides information about the rate of synthesis of bacterial cells mass. Bacterial production is measured by using the [3H]Leucine incorporation centrifugation method (Smith and Azam, 1992). Leucine is an essential amino acid and incorporated into the bacterial protein. Bacterial biomass production can therefore be calculated from protein production rates, which are calculated from [3H]Leucine incorporation rates (Kemp et al., 1993).

Bacterial seawater culture

In a bacterial seawater culture, seawater can be tested for the amount of DOM available for bacterial growth. The seawater is sterile filtered prior to the incubation of a small amount of bacteria. In theory, bacteria will grow in the bacterial seawater culture until all bioavailable DOM is used. As grazers are excluded from the cultures, bacterial abundance can grow to the maximum capacity, the so called “bacterial yield”. The height of the bacterial yield will give information on the amount of bioavailable DOM in the seawater (Ammerman et al., 1984).

Bacterial community composition

Bacterial diversity was studied by using the 16s rDNA gene as a phylogenetic marker. This gene contains both, highly conservative regions (that are the same in all bacterial phyla) and regions that are variable in different bacterial

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phyla. With genetic fingerprinting methods, the 16s rDNA of different bacterial phyla can be separated and give information about the bacterial diversity in an environmental sample. The genetic fingerprinting methods used in my thesis were denaturing gradient gel electrophoresis (DGGE) and transverse restriction fragment length polymorphism (TRFLP). DNA fragments are hereby either separated based on GC content variations (DGGE) or based on different lengths after cutting sequences with restriction enzymes (TRFLP).

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RESULTS AND DISCUSSION

Exudates of different strains from Alexandrium and Prymnesium vary in their allelopathic effects

The results of Paper I showed that allelopathy and hemolytic activity differ between Prymnesium parvum strains (Figure 3). As the production of allelochemicals probably is a trade off between metabolic costs of production and benefits in competition or nutrient availability (Lewis, 1986), we hypothesized that high allelopathic P. parvum strains probably have a disadvantage in another physiological feature compared to low allelopathic P. parvum strains. However, we could not identify correlations between cell physiology and allelopathic efficiency between the strains. Most ecological studies on P. parvum are based on the results of single strain experiments and/ or logarythmic growth (Johansson and Granéli, 1999; Baker et al., 2007; Lindehoff et al., 2009). In accordance with Larssen and Bryant (1998), our study points out how important it is to consider strain specific behavior.

Figure 3. Differences in intracellular toxicity (A and B) and allelopathic activity (C and D) in 4 strains of P. parvum during exponential, stationary and senescent growth at Salinity 7 and 26 (Paper I)

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A main issue in studies investigating allelopathy is that the chemical compounds provoking the allelopathic effect are unknown and therefore, experiments with allelochemicals almost always use exudates of the allelopathic donor species (Legrand et al., 2003). The variability in allelopathy among algal strains enabled us in Paper II to establish a control based on exudates of a non-lytic A. tamarense strain. It could be argued that adding exudates of any algal species probably result in changes in the community when compared to adding medium. However, under the assumption that exudates of different A. tamarense strains are similar in chemical composition, we argue that the differences observed in a microbial community after the addition of allelopathic and/or non allelopathic A. tamarense exudates must be due to the allelopathic substances.

The effect of salinity on allelopathy

In Paper I, the effect of the environmental stress factor salinity on allelopathy and hemolytic activity was tested on four strains of Prymnesium parvum. The effect of salinity on hemolytic activity (intracellular toxicity) has been investigated before, however, results of different studies vary and no clear

Table 1. Salinity and cell density in P. parvum cultures prior allelopathy tests

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relation between hemolytic activity and salinity was found (Padilla, 1970; Larsen and Bryant, 1998). Recent studies suggest that salinities “at the edge of the niche of distribution” lead to higher hemolytic activities in the cell (Baker, 2009). In contrast, the effect of salinity on extracellular toxins has not been studied. Most studies investigating allelopathy in P. parvum are almost always performed at low salinities (Table 1). I found only 2 papers investigating allelopathy at higher salinities, but no comparative studies of several salinities. In Paper I, hemolytic activity in P. parvum was almost similar between Salinity 7 and 26, whereas allelopathy was strongly enhanced at low salinity (Salinity 7; Figure 4). Further, a large variation in allelopathy during culture growth was observed; confirming a recent publication by Skingel et al. (2010). Our study suggests, that P. parvum allelochemicals probably do not accumulate in the medium, but are degraded over time and need to be constantly exuded into the surrounding water as in Stolte et al., 2002; Granéli and Johansson, 2003(b); Fistarol et al., 2005. The mechanism of toxin exudation in P. parvum is unknown. P. parvum blooms frequently occur in brackish water but are not observed in marine seawater. As no physiological differences were observed between the incubations in the two salinities, and growth rate did not show a clear trend in both incubations, allelopathy might give a competitive advantage to P. parvum in brackish water compared to marine seawater.

Figure 4. Relation between allelopathic (extracellular) and hemolytic (intracellular) activity in Salinity 7 and 26 from all replicates measured during exponential,stationary and senescent growth in four strains of Prymnesium parvum (Paper I)

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How do allelochemicals affect plankton communities?

In Paper II, III and IV, the response of natural microbial communities towards the addition of allelochemicals was investigated. A special emphasis was laid on the effect of allelochemicals towards bacteria.

The effect of allelochemicals on natural bacterial communities

The bacterial community composition did not change significantly with the addition of allelochemicals (Paper II, Figure 5), and we did not find any indication for a significant inhibition of bacteria by allelochemicals. To the contrary, bacteria reached higher growth rates and bacterial yields in treatments with allelochemicals (Paper II and III). The release of DOM due to plankton lysis in allelopathic A. tamarense treatments may have increased the amount of bioavailable carbon and supported the growth of bacteria (Paper II and III). Similarly, mixed culture trials of Rhodomonas salina and P. parvum lead to a significant increase of DOC concentration after 30 min and an increase in bacterial biomass after 6 to 12 h (Uronen et al., 2007). In Paper IV, we investigated how much DOM is released from which size class of the microbial community if allelochemicals

Figure 5. A: Bacterial community composition data analysed by non-metric multidimensional scaling, showing the relative similarities between bacterialcommunities from natural seawater incubated with different doses of A. tamarenseor a medium control for 96 h (Paper II); B: Bacterial growth in seawater cultures (SC). Seawater was incubated with eitherallelopathic (ALEX) or non allelopathic algal filtrate (Control) prior to sterilefiltration. A natural bacterial community was incubated in the SC and bacterialgrowth was observed for 60 h (Paper IV)

A BA B

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are present. Although no differences were observed in DOC and dissolved organic nitrogen (DON) dynamics, bacterial seawater cultures showed a higher amount of bioavailable DOM in treatments incubated with allelochemicals (Figure 6). However, the bacterial yield only changed in the largest seawater fraction (60 – 150 μm). The study indicated that larger microorganisms are more affected by allelochemicals than smaller organisms: larger organisms provide more contact surface and might therefore be more vulnerable towards allelochemicals. Not only resources changed with the addition of A. tamarense filtrate, also the amount of bacterivores was affected. In Paper II and III, we observed a reduction in nanoflagellates and ciliates in allelopathic treatments. Other studies have shown that the presence of allelopathic substances could eliminate up to 80 % of nanoflagellates over 3–4 days, and therefore contribute to a high bacterial abundance in algal filtrate treatments (Fistarol et al., 2003, Fistarol et al., 2004). Our studies confirm these results. The impact of allelopathy on bacterivory was investigated in detail in Paper III. Herein, we manipulated resource availability (by addition of DOC) and grazing pressure (by 1 μm filtration). The addition of DOC lead to a higher bacterial production during

Figure 6. Bacterial abundance and production in natural seawater communitiesmanipulated by peptone and filtration during 96 h of incubation with andwithout allelochemicals; modified from Paper III

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the first 24 h of the experiment, but the resulting bacterial abundance was highest in allelopathic treatments. As bacterial production rates showed that the resource availability in both treatments was similar, the lower bacterial abundances in control treatments was most probably due to a lower grazing pressure on bacteria in allelopathic treatments. In treatments without grazers and allelochemicals, bacteria maintained higher abundances until the end of the experimental incubation. In control treatments, bacteria were grazed and decreased in abundance towards the end of the study. Probably, the recovery of bacterivores was reduced in allelopathic treatments compared to control treatments (Figure 6). Contrary thereto, bacteria did not increase in abundance with addition of allelochemicals during our study in Paper IV. However, the impact of allelochemicals on the whole natural community in this study was weak.

Interactions between grazing, DOC and allelochemicals

In Paper III, the effect of allelochemicals on the microorganism groups flagellates, ciliates, bacteria and phytoplankton was compared between food webs manipulated in grazing pressure and the amount of DOC. Allelochemicals had a negative effect on all microorganism groups except bacteria in food webs with reduced grazing pressure. In food webs without manipulation in grazing pressure or DOC, allelochemicals did not negatively affect small nanoflagellates compared to the control. We concluded that the grazing pressure on large nanoflagellates and ciliates in these food webs was reduced by allelochemicals, and therefore, the negative direct effect of the compounds on small nanoflagellates was counteracted by this positive indirect effect. With the addition of DOC, heterotrophic microorganisms increased in abundance, enhancing the grazing pressure on autotrophic microorganisms. When allelochemicals were introduced to this system, the release from heterotrophic grazers benefited autotrophs compared to the control. The study showed that allelochemicals can change microbial dynamics and the population structure by interfering with trophic interactions. However, net effects of allelochemical interactions might be strongly affected by the availability of labile DOC and the density of grazers (Figure 7).

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Figure 7. Schematic sketch of the main effects of allelochemicals on food webs; either unchanged or manipulated in DOC and grazing pressure. The outcome of both, direct and indirect effects of allelochemicals on the different components of the microbial food web are either positive (+) or negative (-) after 96 h exposure. The size of the components is relative to the unchanged control after 96 h in manipulated controls and allelochemical treatments; modified from Paper III

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The effect of allelochemicals on marine microbial communities from different ecozones

I will hereby compare how plankton communities originating from different environmental types, with a gradient from flagellate-dominated pelagic communities to diatom-dominated estuarine communities, responded to the addition of A. tamarense allelochemicals. The flagellate-group in this sense consists of any species that do not use Silicate (Si) for frustule-building. The marine pelagic community investigated in Paper II showed the strongest inhibition in response to allelochemicals; whereas the plankton communities collected from the Trondheimsfjord (Fistarol et al., 2004) and from an Upwelling region south the Iberian Peninsula (Paper III) showed a moderate inhibition. Contrary thereto, the plankton community from the Charente estuary at the French Atlantic coast was not affected (Paper IV). There are different factors that affect the outcome of the previous studies. The strong impact of allelochemicals on the plankton community in Paper II is probably related to the high dose of allelochemicals used. However, the experimental outcomes of Paper III and Paper IV diverged, even if communities from both studies were incubated with comparable amounts of allelochemicals. Further, the estuarine plankton community from our study in Paper IV originated from an area known for its high turbidity rates and high particle density in the water (Heral et al., 1984). Since allelochemicals target the surface of organisms, their effectiveness is inversely proportional to the amount of absorbing particles in the water (Tillmann, 2003; Tillmann et al., 2007) and therefore, may have been lower in the study in Paper IV compared to the other studies. Organisms in the microbial community also differ in their susceptibility towards A. tamarense allelochemicals. The plankton community of Paper II was dominated by the flagellate Phaeocystis globosa. Plankton communities in Paper III and Fistarol et al., (2004), consisted of a mixed community of small flagellates and diatoms, whereas the plankton community of Paper IV was clearly diatom dominated. When all studies are compared, ciliates and nanoflagellates (heterotrophic and autotrophic) were almost always negatively affected by allelochemicals. Larger diatoms also were inhibited by allelochemicals, whereas smaller diatoms benefited from the addition. The differences in susceptibility might be due to differences in cell wall structures; for instance, diatoms possess silicon frustules that are probably well protected towards allelochemicals (Fistarol et al., 2004). Diatoms may furthermore be able to alter allelopathy in planktonic systems; as in the case of the allelopathic algae Karenia brevis. Growth inhibiting effects of bloom exudates were reduced in presence of the diatom Skeletonema costatum, and when both species co-occurred in the field, allelopathy of Karenia brevis was low (Prince et al.,

25

2008). My studies indicate that the species composition of the plankton community can impact the outcome of allelopathic interactions. In the oceans, blooms of allelopathic dinoflagellates alternate with diatom blooms (Cullen and McIntyre, 1998). Dinoflagellates have a low affinity for inorganic nutrients and low growth rates (Smayda, 1997), while diatoms in turn possess high nutrient affinities and high growth rates, which allows them to quickly dominate phytoplankton communities if conditions are favorable (Furnas, 1990). However, diatoms need turbulent water bodies, as the cells are heavy and sink to the ground in stratified water conditions. They further depend on the availability of silica, which they incorporate in their frustules (Valiela, 1995). If these conditions are fulfilled, diatoms are more competitive than dinoflagellates. It further seems that dinoflagellate allelochemicals do not impact diatom dominated plankton communities. The plankton communities studied in Paper III and Fistarol et al., 2004, are comparable to plankton communities dominating the seawater in times of water stratification and silica depletion (Smayda and Reynolds; 2001). Those consist of smaller motile hetero-, auto-, and mixotrophic plankters, capable to grow at lower nutrient levels. Together with ciliates and mesozooplankton, these flagellates create a highly efficient microbial food web that can reach a “steady state”, where nutrient loss due to biomass production and metabolism is balanced by inputs of nutrients due to grazing or cell lysis (Cermeno et al., 2006). Based on the results of my studies, I hypothesize that allelochemicals are more deleterious towards plankton communities dominated by flagellates (Figure 8, Table 2). Through allelochemicals, the donor organism might be able to disrupt the trophic interactions of the microbial food web and reduce the transfer of energy and nutrients to higher trophic levels. Instead, the access to released nutrients could allow the allelopathic species to establish dominance in the microbial food web (Sunda et al., 2006)

Figure 8: Schematic sketch of the allelopathic effect of A. tamarense on plankton communities studied during my thesis

Pelagic communityFlagellate dominatedPoor in particles

Estuarine communityDiatom dominatedRich in particles

Pelagic communityFlagellate dominatedPoor in particles

Estuarine communityDiatom dominatedRich in particles

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Eutrophication is expected to decrease the diatom:flagellate ratio (by increasing the N:Si ratio, which limits the growth of diatoms in the element Si) (Smayda and Trainer, 2010). These communities are closer to the described “non bloom community” in nutrient limited conditions, but, if nutrients are available, autotrophic flagellates will probably gain dominance. Bloom frequency of Phaeocystis globosa in the North Sea increased with lower N: Si ratios (Cloern, 2001). In Paper II, a P. globosa bloom established in the control treatments of the experiment, whereas bloom formation was inhibited in treatments with allelochemicals. In the North Sea, dinoflagellate blooms occur after the P. globosa bloom and are certainly dependent on abiotic factors as the presence of organic and inorganic nutrients, winds and temperature. However, my studies show that additionally, dinoflagellate allelochemicals inhibit flagellate dominated microbial communities. Jonsson et al. (2009) argue that allelochemicals do not explain the formation of harmful algae blooms in the field, because allelopathic effects are only observed at high cell densities. Cell concentrations provoking allelopathic effects were also high in our studies. However, in the field, dinoflagellates accumulate in horizontal layers, along thermoclines or the water surface which may possess high concentrations of secondary metabolites. (Macintyre et al., 1997; Mouritsen and Richardson, 2003; Ryan et al., 2008). The allelopathic potential in these layers may be underestimated.

27

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28

CONCLUSIONS

The main conclusions of this thesis are: - Allelopathy and intracellular toxicity vary between different strains of

the algae Prymnesium parvum - Low salinity increases allelopathy in P. parvum

With the addition of allelochemicals, in a natural microbial community

- bacteria reach higher abundances - grazing pressure on lower trophic levels (as bacteria and phytoplankton)

is reduced - labile DOM is released

The initial conditions in the microbial food web influence the impact of allelochemicals. If the microbial community is strongly or weakly affected, depends, among others, on

- the structure of the microbial food web - the amount of available DOM - the particle density in the seawater - the composition of the phytoplankton community

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FUTURE PERSPECTIVES

The identification of allelopathic substances is of considerable interest, as those may provide natural toxins to control harmful algal blooms. In comparison to synthetic herbicides, allelochemicals often target specific organisms (Tillmann and John, 2002; Kubanek et al., 2005, Weissbach et al., 2010) and further, often possess multi side action, for instance the destruction of the cell structure, inhibition of photosynthesis, respiration and protein production (Hong et al., 2009; Paper III). However, the ecological effects of allelochemicals on microbial communities are often not sufficiently described and need to be investigated before allelochemicals can be used as natural toxins. Our studies contribute to the expansion of this knowledge.

Furthermore, the benefit of the production of allelochemicals for the donor organism is still discussed. Our studies support the hypothesis, that allelochemicals provoke the release of DOM, which can be taken up by the donor, or directly act as a hunting device for phagotrophic species (Tillmann et al., 1998; Stoecker et al., 2006; Roy, 2009). Mixotrophy and allelopathy are well investigated for many algal taxa, and both are considered as important capabilities of harmful bloom forming species (Smayda, 1997; Burkholder, 2008). However, the link between both mechanisms has only recently gained attention for a few species (Prymnesium parvum, Karlodinium veneficum). Further experiments should investigate correlations between DOM uptake rates and the allelopathic potential of different algal strains. For Prymnesium parvum, our studies show strong enhancements of allelopathy at low salinity. As the species is mixotrophic and has been shown to feed preferably on immobilized prey (Skovgaard et al., 2003), a model study on the relationship between allelopathy and mixotrophy in Prymnesium parvum in different salinities could further undermine the hypothesis that allelochemicals are part of a feeding strategy.

Until today, most of the research regarding allelopathic interactions is still limited to laboratory work. However, culture conditions may change characters of algal strains, and laboratory conditions fail to simulate natural environments (Lakeman et al., 2009). For Karenia brevis, investigations on allelopathy during bloom conditions are available (Prince et al., 2008), but missing for other dinoflagellates. Further, for P. parvum, no investigations on phenotypic variation of allelopathy and hemolysis during blooms have been made, and it is not known, if blooms consist primarily of clones of the same individual or several individuals. Comparable studies of the phenotypic and genotypic diversity of P. parvum cells originating from distinct geographic populations may also help to understand what triggers bloom formation in this species.

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ACKNOWLEDGEMENTS

My time as a PhD student was a valuable experience for that I am grateful. Without the help of many people, it wouldn’t have been the same and I want to thank you all! First of all, I want to thank my supervisor, Catherine Legrand, for the constant support, guidance and all the help during my work. I couldn’t have accomplished a PhD with another supervisor! I feel that you have done everything in your power to help me through this time here and I am sincerely grateful to you. I also want to thank Urban Tillmann for helping me a lot with the manuscripts and the work in Bremerhaven. It was a pleasure to meet you and see your enthusiasm when working with the little creatures you can only see in the microscope. Thanks to Edna Graneli, for welcoming me in her group and for always being friendly and supportive. I also want to thank Jarone Pinhassi for showing me how to measure bacterial production and for discussing bacteria from time to time. THANKS TO THE MARINE GROUP! I want to thank all of you because you made it fun to go to work every day! For my practical work, Maria and Martin, thank you for all the help with the experiments, thank you for being great people, for all your patience and all your support! Sabina and Christina, thanks for all the help in the Kalmar lab. For my fights with the computer, thanks to my little Mc Gyver Camilla and Joakim, thank you so much for always helping me with everything! All of you and Laura, Julie, Oli, Veronika, Claudia, Neelam, Markus, Nayani, Johanna, Dusco and all the others, I want to thank you for your friendship, I am grateful for all the times we shared together. Thanks also to the rest of the people from the lab in the harbour, Florence, Elsa, Fred, Sarfraz, Quiao, Terney, Fatima, Anders, the Secretaries and all others, it was nice to get to know you, drink coffee with you or fight with you in front of the coffee machine every morning ☺!

Thanks also to the ecological chemistry group from the Alfred Wegener Institute in Bremerhaven, the diversity group from the Max Planck Institute for marine microbiology in Bremen and the people that helped for the experiments in Trondheim, La Rochelle and Sagres; the good memories from all these trips are due to you! I also want to thank the fundings providing money for my studies: the Faculty of Sciences and Technology from the University of Kalmar and Linnaeus University, the European Commission through the project Grants from ALGBACT and HYDRALAB and the LPPC resources that made my work at the Ifremer institute in La Rochelle possible. Last but not least, my biggest thanks to friends and FAMILY, for your love, encouragement and support, that means everything.

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