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Phytoplankton produce allelochemicals; excreted chemical substances that are affecting other microorganisms in their direct environment. In my thesis, I investigated strain specific variability in the expression of allelochemicals of the harmful flagellate Prymnesium parvum, that is euryhaline but mainly bloom forming in brackish water. I found a large variation among strains, but further showed 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 positive affect their prey. During my thesis, I investigated how marine microbial communities respond to the addition of allelochemicals. I performed field experiments with microbial communities from seawater collected from different places over Europe, and tested how this 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 spp. 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 benefitted other phytoplankton by inhibiting grazers. It was also shown that bioavailable 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. The results can be explained by the surface to volume ratio of microorganisms: Larger organisms provide more contact surface for allelochemicals, and therefore, are probably 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.

Nanoflagellates were found to be abundant in a coastal area of the northern Bothnian Sea. The maximum concentration of nanoflagellates, approximately 8000 cells ml-1, was observed in July, coinciding with a decrease in the abundance of cyanobacteria. Pigmented and non-pigmented nanoflagellates were approximately equally distributed throughout the year. Most of the identified genera are known as being phagotrophic, independent if autotrophic or not. A non-cyst-forming pigmented flagellate, Ochromonas sp., was isolated and nutritionally characterized. This chrysophycean flagellate was shown to be a mainly heterotrophic organism: Photosynthesis was too poor to support multiplication of the cells, whereas when feeding on bacteria, high growth rates were obtained. The biological function of the photosynthetic apparatus is suggested to be a survival mechanism during poor bacterial conditions. The flagellate grazed bacteria selectively, preferring cyanobacteria and large cells of heterotrophic bacteria, presumably depending on size-selective grazing. Despite higher growth rates of the bacteria in the sea during summer (July) than spring (May), heterotrophic bacteria in the sea was observed to be smaller in the summer. Nanoflagellates showed a maximum in July, and by selective grazing of large bacteria they might have caused the decrease in the average size of the bacteria and the decrease in the abundance of cyanobacteria. During the consumption of bacteria the flagellate was shown to remineralize nutrients at high rates and excrete dissolved free amino acids. Assuming the existence of a protozoan predator-prey chain of several trophic levels, it seems likely that a significant part of the nutrients fixed by primary producers is remineralized in the euphotic zone. Furthermore, data from this work indicate that flagellate activity may be a significant source of dissolved free amino acids, utilizable for the heterotrophic bacteria. / digitalisering@umu

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第17775号 / 理博第3898号 / 新制||理||1562(附属図書館) / 30582 / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 中野 伸一, 准教授 奥田 昇, 教授 疋田 努 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

All natural aquatic systems harbour a vast variety of microorganisms. In the aquatic microbial food web, the larger microorganisms (i.e. protozoa) feed on the smaller microorganisms (i.e. bacteria and phytoplankton). An increase in nutrient availability results in changes of the microbial food web structure, like altered community composition and blooms of toxic phytoplankton. In this thesis work I hypothesised that nutrient-rich aquatic environments, with strong protozoan predation, favour the occurrence of predation-resistant bacteria like F. tularensis, and that the microbial food web may provide a reservoir for the bacterium between outbreaks. By using a size-structured ecosystem food web model it was shown that the protozoan predation pressure on bacteria, defined as protozoan predation per bacterial biomass, increases with increasing nutrient availability in aquatic systems (estimated chlorophyll a 0.2 to 112 μg L-1). This dynamics was caused by increasing growth-rate of a relatively constant number of bacterial cells, maintaining the growth of an increasing number of protozoan cells. The results were supported by meta-analysis of field studies. Thus my results suggest that protozoa control the bacterial community by predation in nutrient-rich environments. In a field study in a natural productivity gradient (chlorophyll a 1.4 to 31 μg L-1) it was shown that intense selection pressure from protozoan predators, favours predation-resistant forms of bacteria. Thus, the abundance of predation-resistant bacteria increases with increasing nutrient availability in lakes. Furthermore, I could demonstrate that the bacterium Francisella tularensis, the causative agent of tularemia, was present in eutrophic aquatic systems in an emerging tularemia area. Isolated strains of the bacterium were found to be resistant to protozoan predation. In a microcosm study, using natural lake water, high nutrient availability in combination with high abundance of a small colourless flagellate predator favoured the occurrence of F. tularensis holarctica. In laboratory experiments F. tularensis strains were able to form biofilm at temperatures between 30-37°C, but not below 30°C. In conclusion, I have shown that the protozoan predation pressure on bacteria increases with increasing nutrient availability in aquatic systems. Predation-resistant forms of bacteria, such as F. tularensis are favoured in nutrient-rich environments. The complexity of the microbial food web and nutrient-richness of the water, influence the transmission of the pathogenic F. tularensis holarctica. However, over long periods of time, the bacterium survives in lake water but may lose its virulence. The temperature-regulated biofilm formation by F. tularensis may play a role in colonization of vectors or for colonization of hosts, rather than for survival in aquatic environments.

Both temperature and terrestrial dissolved organic carbon (TDOC) have strong impacts on aquatic food web dynamics and production. Temperature affects vital rates of all organisms and terrestrial carbon has been shown to alter the dynamics of phytoplankton and bacterial production and affect the trophic structure of planktonic food webs. As climate change predictions for the Baltic Sea suggests future increases in both terrestrial carbon run-off and increases in temperature, the aim of thesis was to adopt a system-ecological approach and study effects of these abiotic variables, not only on interactions within planktonic food webs, but also on the growth and consumption rates of one of the most common zooplanktivorous fish in the Baltic Sea, the three-spined stickleback Gasterosteus aculeatus. Results showed that three-spined sticklebacks display a high degree of resilience against increasing temperatures, as both growth rates as well as consumption rates on zooplankton were high at temperatures well over 20 °C. Furthermore, it was shown that the minimal resource densities required to sustain individual and population growth, actually decreased with increasing temperatures, implying that sticklebacks around their optimum temperature for growth at 21 °C will actually have an increased scope for growth. As stickleback population densities have increased over the last decade in the Baltic Sea and are now suggested to out-compete other coastal fish species for shared zooplankton resources, the results presented in this thesis suggest that increased water temperatures would only serve to increase sticklebacks competitive advantage. As the structuring role of this small zooplanktivore on pelagic communities might be considerable, further studies investigating competitive interactions as well as patterns of population abundances are definitely warranted. TDOC was overall shown to stimulate bacterial production and the microbial food web. Because of the longer trophic pathways required to transport carbon from bacterial production to higher trophic levels, the addition of TDOC always reduced food web transfer efficiency. However, it became apparent that the full effect of TDOC additions on pelagic food webs was complex and depended heavily not only on the existing trophic structure to which the carbon was introduced, but also on ambient temperature levels. When three-spined sticklebacks were part of food webs with significant TDOC inputs, the presence of fish, indirectly, through predator release of lower trophic levels, amplified the magnitude of the effects of carbon addition on bacterial production, turning the base of the system significantly more heterotrophic, which ultimately, impacted negatively on their own production. However, when a pelagic food web containing sticklebacks was simultaneously subjected to realistic increases in temperature and TDOC concentrations, food web efficiency and fish production increased compared to present day conditions. These results were explained by a temperature dependent increased production potential of zooplankton, sustained by an increased production of heterotropic microzooplankton via TDOC additions, which lead to higher fish production. Although the increased number of trophic linkages in heterotrophic food webs should have reduced energy transfer efficiency, these negative effects seem here to have been overridden by the positive increases in zooplankton production as a result of increased temperature. These results show that heterotrophic carbon transfer can be a viable pathway to top-consumers, but also indicates that in order to understand the full effects of climate change on trophic dynamics and fish production, abiotic variables cannot be studied in isolation.

<p>Temporal and spatial variations of pelagic microorganisms in the northern Baltic Sea were studied, as well as factors influencing their abundance and growth rates. Three main questions were asked 1) How does increased productivity influence the structure of the microbial food web? 2) Does predation limitation vary between trophic levels? 3) What is the relative importance of resource and predation limitation at different trophic levels?</p><p>A field study in the northern Baltic Sea showed that dominating protozoa, flagellates and ciliates, increased with increasing primary productivity from north to south. Furthermore, relatively small protozoan cells dominated in the low productive north, while larger cells became more dominant in the south. The relationship between plankton size structure and productivity was further studied in an experimental system. In agreement with present theories regarding nutrient status of pelagic food webs, increased productivity caused a lengthening of the food chain as well as a change in plankton size structure. While microplankton dominated in nutrient rich treatments pico- and nanoplankton dominated during nutrient poor treament. The flagellate community was dominated by a potentially mixotroph, <i>Chrysochromulina</i> sp., at low nutrient concentrations. To our knowledge this is the first experimental study showing that <i>Chrysochromulina</i> sp. in resemblance with other mixotrophs is favoured by nutrient poor conditions compared to strict autotrophs and heterotrophs.</p><p>During a stratified summer period autotrophic microorganisms in the northern Baltic Sea did not respond to removal of potential predators, indicating that they were primarily limited by inorganic nutrients. An exception was small eucaryotic picoplankton that showed a large response to predator removal. Among the heterotrophic microorganisms direct effect of predation seemed to increase from ciliates, heterotrophic bacteria, small heterotrophic flagellates, medium flagellates to large flagellates. No quick indirect effect was observed, but after four days trophic cascades were detected.</p><p>The relative importance of resource and predation limitation was studied among heterotrophic bacteria, flagellates and ciliates in the northern Baltic Sea. For all these groups, resource limitation seemed to prevail during the summer period. The results also indicated that the relative importance of predation increased with the productivity of the system. To our knowledge there are no earlier measurements on the relative importance of resource and predation limitation for micoorganisms in the pelagic environment.</p>

<p>Heterotrophic microorganisms are important for the flow of carbon and nutrients in the sea. Bacteria, nanoflagellates and ciliates are relevant components of the pelagic food web. In order to be able to predict the outcome of e.g. eutrophication or climate change we need to know how the different components of the pelagic food web are regulated. With the focus on the northern Baltic Sea food web, this thesis deals with limitation and control of heterotrophic protists, the effect of resource heterogeneity on food web efficiency and diversity of nanoflagellates.</p><p>In-situ microcosm experiments showed that the net growth of heterotrophic flagellates were resource limited throughout the year. Field data confirmed that the abundance of flagellates was bottom-up controlled. Furthermore, field data also showed that the annual average biomass of protists, flagellates and ciliates increased with primary productivity. On a smaller seasonal scale temperature and bacterial biomass were able to explain most of the variation in flagellate biovolume. The temporal variation in ciliate biovolume could not be explained by any bottom-up factors like bacterial biomass, flagellate biomass or chlorophyll a. This and an in-situ microcosm experiment implied that the seasonal dynamics of ciliates were more regulated by predators like mesozooplankton.</p><p>The food web efficiency i.e. how much of production at the resource level is converted to production at the top trophic level, may be affected by specific size or type of resource. Indoor mesocosms revealed that the food web efficiency was 11 times lower when heterotrophic bacteria dominated basal production instead of nano- and micro-sized phytoplankton. This was due to a lengthening of the food web when pico-sized bacteria constituted the main resource.</p><p>The PCR-DGGE molecular biological method was used to study the diversity of heterotrophic or mixotrophic chrysomonads. The focus was set on chrysomonads due to their relatively large contribution to the nanoflagellate community. Group-specific PCR primers were optimized for the target group. A field survey in the northern Baltic Sea showed that a handful of chrysomonad sequences were present throughout the year. Significantly more chrysomonads were recorded in the basin with higher primary productive and salinity. In total 15-16 different chrysomonad sequences were recorded. Most of them matched uncultured chrysomonad clones.</p>

Temporal and spatial variations of pelagic microorganisms in the northern Baltic Sea were studied, as well as factors influencing their abundance and growth rates. Three main questions were asked 1) How does increased productivity influence the structure of the microbial food web? 2) Does predation limitation vary between trophic levels? 3) What is the relative importance of resource and predation limitation at different trophic levels? A field study in the northern Baltic Sea showed that dominating protozoa, flagellates and ciliates, increased with increasing primary productivity from north to south. Furthermore, relatively small protozoan cells dominated in the low productive north, while larger cells became more dominant in the south. The relationship between plankton size structure and productivity was further studied in an experimental system. In agreement with present theories regarding nutrient status of pelagic food webs, increased productivity caused a lengthening of the food chain as well as a change in plankton size structure. While microplankton dominated in nutrient rich treatments pico- and nanoplankton dominated during nutrient poor treament. The flagellate community was dominated by a potentially mixotroph, Chrysochromulina sp., at low nutrient concentrations. To our knowledge this is the first experimental study showing that Chrysochromulina sp. in resemblance with other mixotrophs is favoured by nutrient poor conditions compared to strict autotrophs and heterotrophs. During a stratified summer period autotrophic microorganisms in the northern Baltic Sea did not respond to removal of potential predators, indicating that they were primarily limited by inorganic nutrients. An exception was small eucaryotic picoplankton that showed a large response to predator removal. Among the heterotrophic microorganisms direct effect of predation seemed to increase from ciliates, heterotrophic bacteria, small heterotrophic flagellates, medium flagellates to large flagellates. No quick indirect effect was observed, but after four days trophic cascades were detected. The relative importance of resource and predation limitation was studied among heterotrophic bacteria, flagellates and ciliates in the northern Baltic Sea. For all these groups, resource limitation seemed to prevail during the summer period. The results also indicated that the relative importance of predation increased with the productivity of the system. To our knowledge there are no earlier measurements on the relative importance of resource and predation limitation for micoorganisms in the pelagic environment.