Response of Marine Food Webs to Climate-Induced Changes in Temperature and Inflow of Allochthonous Organic Matter

Home , Ecological stoichiometry, Microbial food web, Microbial loop

Response of marine food webs to climate-induced changes in temperature and inflow of allochthonous organic matter

Rickard Degerman

Department of Ecology and Environmental Science 901 87 Umeå Umeå 2015

Tillägnad

Maria, Emma och Isak

Table of Contents

Abstract 5

List of papers 6

Introduction 7 Aquatic food webs – different pathways Food web efficiency – a measure of ecosystem function Top predators cause cascade effects on lower trophic levels The Baltic Sea – a semi-enclosed sea exposed to multiple stressors Varying food web structures Climate-induced changes in the marine ecosystem Food web responses to increased temperature Responses to inputs of allochthonous organic matter

Objectives 14

Material and Methods 14 Paper I Paper II and III Paper IV

Results and Discussion 18 Effect of temperature and nutrient availability on heterotrophic bacteria Influence of food web length and labile DOC on pelagic productivity and FWE Consequences of changes in inputs of ADOM and temperature for pelagic productivity and FWE Control of pelagic productivity, FWE and ecosystem trophic balance by colored DOC

Conclusion and future perspectives 21

Author contributions 23

Acknowledgements 23

Thanks 24

References 25

Abstract

Global records of temperature show a warming trend both in the atmosphere and in the oceans. Current climate change scenarios indicate that global temperature will continue to increase in the future. The effects will however be very different in different geographic regions. In northern Europe precipitation is projected to increase along with temperature. Increased precipitation will lead to higher river discharge to the Baltic Sea, which will be accompanied by higher inflow of allochthonous organic matter (ADOM) from the terrestrial system. Both changes in temperature and ADOM may affect community composition, altering the ratio between heterotrophic and autotrophic organisms. Climate changes may thus have severe and complex effects in the Baltic Sea, which has low species diversity and is highly vulnerable to environmental change. The aim of my thesis was to acquire a conceptual understanding of aquatic food web responses to increased temperature and inputs of ADOM. These factors were chosen to reflect plausible climate change scenarios. I performed microcosm and mesocosm experiments as well as a theoretical modeling study. My studies had a holistic approach as they covered entire food webs, from bacteria and phytoplankton to planktivorous fish. The results indicate a strong positive effect of increased temperature and ADOM input on the bacterial community and the microbial food web. However, at the prevailing naturally low nutrient concentrations in the Baltic Sea, the effect of increased temperature may be hampered by nutrient deficiency. In general my results show that inputs of ADOM will cause an increase of the bacterial production. This in turn can negatively affect the production at higher trophic levels, due to establishment of an intermediate trophic level, consisting of protozoa. However, the described effects can be counteracted by a number of factors, as for example the relatively high temperature optimum of fish, which will lead to a more efficient exploitation of the system. Furthermore, the length of the food web was observed to be a strong regulating factor for food web responses and ecosystem functioning. Hence, the effect of environmental changes may differ quite drastically depending on the number of trophic levels and community composition of the system. The results of my thesis are of importance as they predict possible ecological consequences of climate change, and as they also demonstrate that variables cannot be examined separately.

List of papers

This thesis is a summary of the following papers, which henceforth will be referred to by roman numerals.

I. Degerman R., J. Dinasquet, L. Riemann, S. Sjöstedt de Luna and A. Andersson, 2013. Effect of resource availability on bacterial community responses to increased temperature. Aquatic Microbial Ecology, 68: 131-142.

II. Degerman R., R. Lefébure, P. Byström, U. Båmstedt, S. Larsson and A. Andersson Food web interactions determine transfer efficiency and top consumer responses to increased allochthonous carbon input. (Submitted Manuscript)

III. Lefébure R., R. Degerman, A. Andersson, S. Larsson, L.-O. Eriksson, U. Båmstedt and P. Byström, 2013. Impacts of elevated terrestrial nutrient loads and temperature on pelagic food-web efficiency and fish production. Global Change Biology, 19(5): 1358- 1372.

IV. Degerman R. and A. Andersson. Modelling effects of river inflow of allochthonous dissolved organic carbon on coastal production. (Manuscript)

Paper III have been reprinted with kind permission from the publisher.

Introduction

Aquatic food webs - different pathways

Aquatic food webs are formed by organisms which can be grouped into trophic levels based on their size and role in the ecosystem (Legendre and Rassoulzadegan 1993). Microscopic bacteria and phytoplankton constitute the base of the food web. They are diffusion feeders and take up dissolved substances through their cell membranes. Phytoplankton are autotrophs, which use sunlight to get energy and assimilate inorganic carbon (carbon dioxide) from the water to create energy-rich molecules such as carbohydrates. This process is called primary production. Heterotrophic bacteria are on the other hand detrivores, which are vital in nutrient recycling. They use organic carbon as an energy source, either autochthonous organic carbon derived from phytoplankton or allochthonous organic carbon originating from outside the system. Bacteria are, due to their large surface-to-volume ratio, more efficient at absorbing nutrients at lower concentrations compared to phytoplankton and may therefore outcompete phytoplankton if an allochthonous carbon supply is available. The carbon produced at the base of the food web is transferred through a number of trophic levels before reaching the top consumers. Phytoplankton are grazed upon by zooplankton which are in turn eaten by zooplanktivorous fish. However, bacteria are too small to be readily eaten by many marine zooplankton. Instead, heterotrophic flagellates and ciliates prey heavily on bacteria, forming a link between bacteria and zooplankton (Azam et al. 1983). Hence, the energy produced by bacteria is transferred through more trophic levels before reaching top consumers than that of phytoplankton (Fig. 1). Phytoplankton-based food webs are generally named “classical” or the “herbrivorous food webs”, while bacteria-based food webs are called microbial food webs (Azam et al. 1983, Legendre and Rassoulzadegan 1993). The classical food web is known to dominate in nutrient rich waters, while microbial food webs dominate in nutrient poor waters or systems highly influenced by allochthonous organic matter. Thus, the food webs have quite different structures and pathways depending on nutrient levels and other drivers in the system.

Figure 1. Simplified view of two contrasting food webs. One is based on phytoplankton primary production and the other is based on bacterial production. Illustration: Mats Minnhagen.

Food web efficiency – a measure of ecosystem function

At every trophic level, a significant part of the consumed energy/carbon is lost due to e.g. respiration, excretion and sloppy feeding (e.g. Azam et al. 1983, Straile 1997). Consequently a smaller fraction of the basal production within the system would reach the highest trophic level in food webs dominated by heterotrophic bacterial production than in food webs based on phytoplankton production (Berglund et al. 2007, Eriksson-Wiklund et al. 2009). The ratio between production by the top trophic level and the basal trophic level, i.e. the food web efficiency (FWE) (Rand and Stewart 1998), can be calculated and used as a measure of the overall system efficiency. Accordingly, a bacteria dominated system will have lower FWE than a phytoplankton based system. The gross growth efficiency is defined as the growth of an organism divided by the food ingestion (Fenchel 1987). In ecosystem studies, this concept can be applied to trophic levels and is often called trophic transfer efficiency. The trophic transfer efficiency has been shown to be in the range of ~15-35% (e.g. Welch 1968, Straile 1997), depending on food availability. High food availability leads to lower assimilation efficiency and growth efficiency (Welch 1968), which in turn leads to decreased FWE. In aquatic food webs where edible phytoplankton constitute the base, zooplankton the intermediate level and planktivorous fish the highest trophic level, and the trophic transfer efficiency is 25%, the FWE will be ~6% (0.25^2). If instead bacteria constitute the base and an additional intermediate trophic level is established (protozoa), the FWE will end up being

1.6% (0.25^3). However, since bacterial production (BP) and primary production (PP) generally co-occur in natural systems, the FWE in systems with planktivorous fish as highest trophic level might be somewhere in-between 6 and 1%. Many other factors may affect the FWE, for example the edibility of the basal producers. In nutrient rich systems the phytoplankton community is often dominated by inedible or less edible forms, e.g. filamentous cyanobacteria or green algae, which result in a very low FWE (Andersson et al. 2013). On the contrary, the occurrence of omnivory, i.e. feeding by an organism on different trophic levels (e.g. Sprules and Bowerman 1988, Burns 1989, Thompson et al. 2007), would increase the FWE. Taken together, there are several factors which may govern the food web function. The regulation of the food web efficiency in different aquatic systems is still poorly known, which makes it difficult to understand, assess and predict how environmental change will affect the ecosystem function.

Top predators cause cascading effects on lower trophic levels Top predators are known to cause cascading effects on lower trophic levels (Carpenter et al. 1985). However, depending on what top predator is present in the system, varying effects on lower trophic levels can be expected. If mesozooplankton constitute the highest trophic level, their grazing on phytoplankton and ciliates is high, and the phytoplankton biomass is kept down. In such cases the nutrient availability per phytoplankton cell is high, which potentially make them healthy and of good food quality for zooplankton. In contrast, when planktivorous fish is present in the system, they graze down the zooplankton biomass leading to predation-release on the phytoplankton (Carpenter et al. 1985). This in turn leads to higher competition for nutrients as the phytoplankton biomass builds up, possibly yielding a community with less favorable CNP (Carbon, Nitrogen, and Phosphorous) stoichiometry as food source for zooplankton. Another effect of reduced zooplankton biomass may be decreased predation-pressure on bacteria, due to an increase in ciliate biomass resulting in higher grazing on their main prey flagellates. However, bacteria may be grazed upon by ciliates directly without the link through flagellates. Both protozoa and fish excretion plays a significant role in nutrient recycling and can benefit both primary- and secondary production (Andersson et al. 1985, Vanni and Layne 1997, Vanni et al. 2013). Hence, both bacterial and primary production can be larger when planktivorous fish is present in the system, resulting in a larger basal production or food quantity. Their quality as food source may however be poorer (Malzahn et al. 2007, Dickman et al. 2008, Malzahn and Boersma 2012). According to the ecological stoichiometry theory, the larger the difference in C:N and C:P ratios between predator and prey, the lower transfer efficiency between those trophic levels (Sterner and Elser 2002). In turn, these factors are likely to affect the overall function of the ecosystem, i.e. the FWE. The Baltic Sea – a semi-enclosed sea exposed to multiple stressors The Baltic Sea is a 415 000 km2 large semi-enclosed brackish sea in northern Europe, with a catchment area that covers 1.74 million km2 which is populated by about 85 million people (e.g. Kautsky and Kautsky 2000). Nine countries are situated along the Baltic Sea coast and five additional countries are partially located within the drainage area. Due to the geographical, climatological, and oceanographic characteristics, the Baltic Sea is highly sensitive to the environmental impacts of human activities. It consists of a series of sub-basins

9 which are separated by shallow sills. In the northern part the inflow of freshwater from rivers is large; while in the south, water from the North Sea occasionally flows in. This causes a distinct salinity gradient from north to south which, together with geographical differences in temperature and sun hours, affects all the organisms inhabiting the ecosystem. The Baltic Sea is exposed to multiple stressors. The low salinity causes very low species diversity, and the organisms inhabiting the system are exposed to osmotic stress (Kautsky and Kautsky 2000). Eutrophication is a large problem in the south, e.g. the Baltic proper, which cause huge problems with cyanobacterial blooms and anoxic bottoms. In the northernmost basin, the Bothnian Bay, the productivity is 10 times lower (Samuelsson et al. 2006), and in this basin organic pollutants is a more severe threat. Overfishing is a problem, especially in the southern parts of the Baltic. In the north huge amounts of freshwater containing colored terrestrial organic matter enters the system. This makes the seawater brown and the phosphorus concentrations are very low. It is a great challenge to try to understand how climate change will affect the Baltic Sea ecosystem. Varying food web structures

The nutrient availability and productivity are very different in different basins of the Baltic Sea. In the northernmost basin, the Bothnian Bay, strong phosphorus limitation is prevailing (Andersson et al. 1996), while further south nitrogen limitation is predominant. The annual primary production is 10 times higher in the south than in the north, while bacterial production is more similar in the north-south gradient (Samuelsson et al. 2006). This causes a quite different food web structure in the different basins. In the north phytoplankton and bacteria make up 50% of the basal production, respectively, while in the south phytoplankton contributes to 95% of the basal productivity (Samuelsson et al. 2006). The reason for the relatively high contribution of bacterial production in the north is likely the high availability of allochthonous organic matter (ADOM), which subsidizes bacterial growth (Sandberg et al. 2004). The low concentration of inorganic nutrients in the north, together with the external carbon source, allows heterotrophic bacteria to outcompete phytoplankton for nutrients and trace elements. This is probably also the reason to why the Bothnian Bay is net heterotrophic, while further south the system is more balanced (Algesten et al. 2006). By assuming full edibility of the basal producers, food web structures as presented in figure 1, and a trophic transfer efficiency of 25% (Welch 1968, Straile 1997), it is possible to calculate a hypothetical FWE from the basal producers to mesozooplankton in the different basins of the Baltic Sea (Table 1). The FWE would increase from 3.8 to 5.9% along the north south gradient. This is caused by 0.4 step longer food web in the north, which leads to higher losses. However, this is a theoretical calculation which may be contradicted by many factors like varying edibility and trophic transfer efficiency. Nevertheless, the calculation gives an indication that the food web function is rather different in different areas of the Baltic.

Table 1. Phytoplankton and bacterial production in different basins of the Baltic Sea, standard deviations within brackets. Hypothetical FWE and number of trophic levels from basal producers to zooplanktivorous fish.

Basin Bothnian Bay Bothnian Sea Baltic proper

Prim. prod.1 1.4 (0.3) 4.8 (2.4) 12.3 (2.2)

(molC/m2*year)

Bact. Prod.2 1.0 (0.1) 1.5 (0.1) 0.7

(molC/m2*year)

FWE3 (%) 3.8 4.9 5.9

Trophic levels3 3.4 3.2 3.0

1 Larsson et al. 2010 and Samuelsson et al. 2006. 2 Samuelsson et al. 2006. Only one data point for the Baltic proper. 3 Calculation is based on the phytoplankton and bacterial production, assuming full edibility of the basal producers, 25% trophic transfer efficiency and a food web channeling according to figure 1 with fish as top consumer.

Climate-induced changes in the marine ecosystem

Projections of future climate changes indicate a global atmospheric temperature increase of 1.4-5.8°C and changed precipitation during the coming century (Cubasch et al. 2001, HELCOM 2007). However, these changes are expected to vary both spatially and temporally. For the European northern hemisphere, a relatively large increase is anticipated for both temperature and precipitation (fig. 2), especially during the winter (Cubasch et al. 2001, HELCOM 2007). A direct consequence of increased precipitation is a higher riverine inflow to marine ecosystems, including an increased transport of dissolved organic matter containing massive amounts of carbon. Furthermore, the increased inflow of freshwater will affect salinity and pH. It may thus be speculated that the present environmental condition and ecosystem dynamics in the Bothnian Bay may in the future be transferred further south. If so, large productivity changes in the Baltic Sea can be expected.

Figure 2. Predicted climate-induced changes in temperature and precipitation in Europe during 100 years, using assembly analysis of 21 models (From Cubasch et al. 2001, HELCOM 2007). Top and mid panels show the simulated changes in temperature and precipitation from 1980-1999 to 2080-2099, respectively. Bottom panel shows the number of models out of 21 that project increases in precipitation. Left, mid and right-hand panels show annual, winter (DJF: December, January and February) and summer values (JJA: June, July and August), respectively.

Food web responses to increased temperature

Changes in temperature may lead to variations of the metabolic rates of the aquatic organisms, and in turn the community composition can be altered. The growth of autotrophic organisms has been suggested to be less favored by increased temperature than the heterotrophic organisms (e.g. Andersson et al. 1994, Hoppe et al. 2008). Thus, a temperature increase would influence the ratio between heterotrophic and autotrophic production at the base of the food web, as well as the trophic balance of the ecosystem. The ecosystem may turn in to net- heterotrophy. Increased temperature may also lead to changes in the size structure of the plankton community (Andersson et al. 1994, Suikkanen et al. 2013). Warming will cause increased nutrient limitation due to the higher metabolic costs, which in turn leads to cell size reduction in the plankton community (Kalista and Sommer 2013). At higher trophic levels, development times, consumption capacities and metabolic rates are affected, thereby altering the top down cascading-effect. Furthermore, the timing in the succession of organisms in aquatic systems can be expected to change, which may cause drastic alterations in the function of the ecosystem (Sommer et al. 2007). It is likely that there is an interaction between temperature and the food source availability for the growth of the aquatic organisms. For example, if nutrient concentrations are high, bacteria and phytoplankton can respond to increased temperature, while if nutrients are not available their growth will not be promoted. The response of aquatic food webs to increased temperature is therefore likely to be very complex.

Responses to inputs of allochthonous organic matter

In contrast to the autotrophs, heterotrophic bacteria are dependent on organic carbon sources, such as carbohydrates produced by the autotrophs. These compounds are often highly bioavailable autochthonous carbon sources for bacteria (Cole et al. 1982). An alternative, or supplementary, carbon source for bacteria originating from outside the aquatic system is allochthonous dissolved organic carbon (ADOC), from terrestrial sources, which can uncouple the bacteria from phytoplankton, and make them less dependent on autochthonous carbon (Sandberg et al. 2004). In systems with a high inflow of allochthonous carbon, heterotrophs tend to dominate at the basal production level, outcompeting phytoplankton for other essential nutrients and trace elements (Berglund et al. 2007 and Jansson et al. 2007). Even though only a minor part of the ADOC is bioavailable for bacterial growth (e.g. Lignell et al. 2008), the high concentrations entering estuarine systems can account for a significant part of the bacterial production (Sandberg et al. 2004). Under such conditions heterotrophic bacteria can outcompete phytoplankton for inorganic nutrients. The negative effect of ADOC on phytoplankton growth is further strengthened as the inflow of allochthonous matter can also induce significant browning of the water, depending on the source of the organic matter. With browner or more turbid water in lake ecosystems, the light condition for the phytoplankton becomes poorer, with decreased primary production as a result (Jansson et al. 2007). A previous study showed that similar effects may occur in estuarine systems. During a period with high precipitation the phytoplankton primary

13 production was shown to be reduced in the northern Baltic Sea, while bacterial production was unaffected (Wikner and Andersson 2012). The changes could be explained by increased inflows of ADOC to the Gulf of Bothnia. In my thesis I therefore hypothesized that climate- induced increased precipitation and inflows of ADOC would affect the trophic balance in estuarine marine systems.

Objectives

The aim of my thesis is to acquire a conceptual understanding of aquatic food web responses to increased temperature and availability of allochthonous dissolved organic carbon (ADOC). These factors were chosen to reflect a plausible climate change scenario. More specifically I wanted to:

I. Assess discrete and combined effects of increased temperature and nutrient availability on bacterial growth rates and community composition.

II. Conceptualize the roles of ADOC and food chain length for food web efficiency and structure in marine ecosystems, ultimately assessing the impact on fish production.

III. Find out what effects predicted climate-induced changes of increased inputs of ADOC and higher temperature will have on the pelagic food web structure and efficiency, focusing on the basal production of the system and its effect on the fish production.

IV. Elucidate how increasing inputs of ADOC, with varying bioavailability and light attenuation, affect the production and the balance between autotrophy and heterotrophy in marine systems of different nutrient states. These questions were addressed in four different studies.

Materials and methods

In my thesis work I have performed microcosm and mecocosm experiments as well as a theoretical study. I have used established and state of the art laboratory techniques to analyze plankton, fish and chemical-physical factors. I used a mechanistic ecosystem model to perform simulations of the effect of increased inflow of ADOC to estuarine environments with different nitrogen and phosphorus availability. Below follows a description of the used method in the four sub-studies: Paper I The seawater used for study I was collected at an offshore monitoring station in the northern Baltic Sea (62° 05' 99", 18° 32' 91"). Experiments using seawater from this location were performed during seven occasions over an annual cycle. The effects of a 4oC temperature increase above in situ and a range of nutrient additions on the growth rate and bacterial community composition were tested in microcosm experiments.

With 2 temperatures (in situ and in situ + 4oC, established from the start) and 4 different nutrient concentrations, 8 different treatments performed in 3 replicates were analyzed. In these experiments yeast extract was used as nutrient source, with the aim to mimic autochthonously produced organic substances. The microcosms were sampled regularly for bacterial abundance, biomass, growth rate and bacterial community composition. Standard laboratory techniques were used to analyze plankton and physico-chemical factors. State of the art molecular methods were used to analyze microbial communities, and the results were examined using varying types of analysis of variation.

Paper II and III

In study II and III an advanced indoor mesocosm facility, located at Umeå Marine Sciences Centre (UMSC), was used (Fig. 3). Mesocosm experiments are an excellent alternative when the aim is to study functions in natural ecosystems, and at the same time being able to control certain factors. Furthermore this method allows for replication. The facility at UMSC consists of 12 polyethylene mesocosms with a depth of 5 m and a diameter of 73 cm. Temperatures in these tubes are controlled by a heating-cooling system containing glycol, which was needed to obtain the 4°C temperature difference in study III. Light was provided by 150W halogen lamps (MasterColour CDM-T 150W/942 G12 1CT) positioned over each tank, individually adjusted to ensure the same light condition in each mesocosm and finally set to a 12 h light - 12 h dark regime. The mesocosms were filled simultaneously with unfiltered water from the northern Baltic Sea using a peristaltic pump system. This resulted in close to identical ecosystems containing natural assemblages of all planktonic organisms. In these mesocosm studies, I studied organisms from the base of the food web up to zooplanktivorous fish, juvenile stickleback, Gasterosteus aculeatus. In study II, we used either mesozooplankton or fish as top predator in the different treatments, while in study III fish constituted top trophic level in all treatments. At an early stage, before the experiments started, a large amount of juvenile G. aculeatus were caught with a beach seine from the coastal area outside of UMSC, in the Gulf of Bothnia. They were then kept in a 400 l tank at 10°C with a continuous water exchange and fed daily, but later starved before introduction into the tanks. For both experiments, biological variables at all levels of the food web were continuously measured, with emphasis on the bottom (primary- and bacterial production), and top (fish or zooplankton) of the food web. In study II, glucose, nitrogen and phosphorus were added to 6 of the mesocosm tanks in order to stimulate bacterial production, while nitrogen and phosphorus were added to the remaining tanks to stimulate phytoplankton production. This was expected to give quite different base of the food web in the two sets of treatments. Glucose differs from natural ADOC, as it is transparent and fully bioavailable for bacterial uptake. However, these were properties we wanted, as we needed a compound that increased bacterial growth but did not exclude light. One problem with using natural ADOC is that it is difficult to disentangle effects of light attenuation and nutrient subsidy on the interplay between bacteria and phytoplankton and other trophic interactions. In this study we wanted to elucidate bottom-up and top-down effects on food web efficiencies, where light attenuation was excluded as a variable factor.

With this nutrient manipulation we achieved a successful experimental design for the raised question. In study III, we attempted to simulate the present-day and future condition in an estuarine environment, according to a climate change scenario with higher temperature and increased inputs of terrestrial dissolved matter (TDM) (Meier 2006, HELCOM 2007, IPCC 2007), (Eriksson-Hägg 2010, Wikner and Andersson 2012). I therefore used natural ADOC extracted from a soil close to a river in the northern Baltic Sea. In accordance with Adams and Byrne (1989), the soil was mixed with MQ filtered water and an ion exchange resin (Amberlite IRC 748I) for 48 h and then filtered before introduction into the mesocosm tanks. This terrestrial dissolved matter, like natural DOM contained not only TDOC, but also N, P and colored substances reducing the light climate. To test the effect of increased inflow of DOM and higher temperature, according to climate change scenarios, four experimental treatments were created, where the control treatment reflected the current environmental condition in coastal areas of the northern Baltic Sea. The other three treatments had 4 ° C higher temperature, 30% higher inputs of TDM or a combination of both these factors. In both study II and III the effects of different treatments on the productivity of different organisms groups and FWE were analyzed using analysis of variation.

Figure 3. Indoor mesocosm facility at Umeå Marine Sciences Centre used in paper II and III. Photo K.Viklund.

Paper IV

Paper IV was a theoretical study where a mechanistic ecosystem model was used to study effects of increased inflows of colored ADOC to estuaries with different inorganic nitrogen and phosphorus availability. Also in this sub-study we used an outlined climate change scenario regarding increased inflows of ADOC into the northern Baltic Sea (Meier 2006, Eriksson-Hägg et al. 2010). For half of the simulations the light level was lowered with increasing ADOC, imitating light attenuation due to colored substances in natural DOM. The model we used was a size- structured ecosystem food web model, built on empirical parameters (Fig. 4). Organisms in the modeled food web, consisting of 5 trophic layers, were grouped by size and included bacteria, phytoplankton, protozoa and mesozooplankton. All organisms were defined by their radius, carbon density and C:N and C:P ratios. The processes driving the model were physical constraints set by osmotrophy, primary production, bacterial production, respiration, resting metabolism and predation. Since the model was designed as a population dynamic model in a homogeneous volume of water, without spatial resolution, no random movement of organisms was included. The model was set up as a flow-through system and we chose a water turn over time of 10 days, in order to mimic conditions in some bays. In total 96 simulations were run in a factorial design including six levels of DOC ranging from 0 to 16.7 μmol C L-1 d-1 and four different levels of inorganic nitrogen and phosphorus additions, corresponding to a daily addition of 10%, 50%, 100% and 200% of the summer concentrations in the off-shore northern Bothnian Sea (Andersson et al. 1996). Each simulation was run for 365 days in order to stabilize the food web processes, and the period 250-365 days was used for calculations. We analyzed the effects of colored DOC and nutrient availability on phytoplankton, bacterial and zooplankton production. Furthermore we used these results to estimate effects on the FWE and trophic balance of the ecosystem.

Size (µm):

L i gh t

175 Pred4 (Mes o zoo pl a nk to n )

Pred3 (C ili a t es) 30 PP3 (M i cr o ph yto pl a nk to n) 20 Pred2 (M i cr ofl age ll a t es) 15 PP2 (Na n op hy to pl a n kto n ) 8 Pred1 (Na no fl agell a t es) 4

PP1 ( Pi c op hy to pl a n kto n) 1 Bact er i a 0,8

DOC ADOC DIN DIP

Figure 4. Schematic view of the food web used in the simulations in paper IV. Primary producers (PP) used light and inorganic nutrients (dissolved inorganic nitrogen [DIN], dissolved inorganic phosphate [DIP]) for growth. Bacteria consumed dissolved organic carbon (DOC), excreted by PPs or predators (Pred) and assimilated inorganic nutrients. Bacteria and primary producers were grazed by heterotrophic flagellates, ciliates or mesozooplankton. Predators also grazed other predators 1 size class below them. Organisms are arranged according to cell size, which is indicated along the arrow to the right. Uptake, grazing or consumption (solid lines); excretion (dot-dash lines).

Results and discussion

Effect of temperature and nutrient availability on heterotrophic bacteria Both increased temperature and higher nutrient levels had a positive effect on the bacterial growth rate, but at the lowest nutrient concentration (non-supplemented seawater) the bacterial growth remained low at all tested temperatures. This suggests that resource availability has a stronger effect on bacterial growth than temperature in natural low- productive seawater. The lag-phase, i.e. the bacterial response time, was markedly shortened in response to increased temperature as well as nutrient additions. These results support the view that elevated temperatures, e.g. due to climate change, will stimulate bacterial growth. However, the temperature-response will be highest in nutrient-rich areas or in areas where nutrient levels will increase concomitantly with increased temperature. In coastal areas in northern Europe an elevation of both may be expected. In oligotrophic regions, which are not significantly influenced by freshwater inflow, the impact will probably be less marked. Furthermore, our results indicate that community composition is affected by increased temperature and nutrient availability, although relatively large changes in nutrient concentrations and/or temperature are needed to induce a bacterial community shift.

It is highly likely that due to the combined effect of elevated temperatures and higher nutrient inflow, heterotrophic processes mediated by bacterial processing of organic matter will increase while autotrophy may decrease or remain at the same level (Andersson et al. 1994, Hoppe et al 2008). Since food webs dominated by bacterial production generally contain more trophic levels (e.g. Azam et al. 1983, Berglund et al. 2007), the food web efficiency and production at the higher trophic layers may consequently decrease.

Influence of food web length and labile DOC on pelagic productivity and FWE All mesocosms receiving labile DOC (glucose) had significantly higher bacterial production and lower food web efficiency regardless if the top consumer was constituted by mesozooplankton or planktivorous fish. The primary production was not affected to the same extent and therefore the basal production increased as well in the DOC treatment. Production of the top-consumer, on the other hand, differed in response to DOC addition. When zooplankton was the top-consumer, the production was higher in mesocosms with glucose, whereas when fish was the top-consumer, the production was higher in the NP treatment (addition of inorganic nitrogen and phosphorus, but no carbon enrichment). In the system where zooplankton constituted the highest trophic level, zooplankton abundance was high resulting in a high grazing on phytoplankton. Consequently the resource was suppressed by predation and clearly not nutrient limited. Thus, the increased bacterial production following the DOC addition may have acted as a subsidy, which resulted in a higher zooplankton production in this treatment than in the NP treatment. However, these results differ from a previous study (Berglund et al. 2007), where addition of labile DOC resulted in lower zooplankton production. The higher energy loss, when fish was the highest trophic level and labile DOC was added to the system, might be due to a to top-down effect, which indirectly through predator release of the lowest trophic level, increased the bacterial production. The predation on zooplankton was high in all fish treatments, which in turn lead to nutrient limitation for the thriving basal production. In the fish treatment with DOC, the basal production was totally dominated by bacterial production, which means that the energy had to pass more trophic levels before reaching the fish. Our results indicate that inputs of DOC to systems where planktivorous fish constitute the highest trophic level will induce cascading effects on the food web, affecting the magnitude and ratio of bacterial and primary production. This will in turn lead to feedbacks in the food web and ultimately lead to a negative effect on the fishes own production. We suggest that effects of increased DOC inflows on pelagic systems are dependent on the existing food web structure of the system.

Consequences of changes in inputs of ADOM and temperature for pelagic productivity and FWE In accordance with the previous study where labile DOC (glucose) was added to the seawater, addition of natural terrestrial/allochthonous dissolved matter ADOM was also found to stimulate the bacterial production and decrease the primary production. Less pronounced was the effect of increased temperature, although this factor also had a positive effect on bacterial

19 production and a negative effect on primary production. Although both these factors separately promoted bacterial growth and had a negative effect on the phytoplankton growth, the highest trophic level was not negatively affected. The simulated climate change scenario, i.e. the combined effect of temperature and ADOM addition, yielded significantly higher fish production and food web efficiency. These results were un-expected, but might be explained by promotion of the omnivorous ciliates (Bell et al. 1993, Berglund et al. 2007, Faithfull et al. 2012), which directly eat bacteria. Therefore the number of trophic levels may not be increased and consequently FWE and fish production is kept unchanged. Another contributing factor may be the difference in ADOC. As mentioned before, glucose was used as ADOC in the first mesocosm experiment. This highly bioavailable carbon source was quickly utilized by the bacteria, clearly visible in bacterial production peaks after each addition. The more natural, terrestrial derived, ADOC used in the second mesocosm experiment was a less bioavailable source of carbon, again noticeable in the bacterial production. Although additions of this form of ADOC also led to higher bacterial production, the production remained stable at a higher level than in the treatment with low ADOC addition without distinct peaks. These contrasting bioavailabilities lead to a difference in nutrient uptake of the bacteria; the highly available glucose is cause to a fast depletion of N and P, while the more slowly working natural ADOC is less likely to induce N and P limitation of the same magnitude. Additionally, the natural ADOC was not added as excessively as the glucose was. After all, the purpose of the glucose addition was to establish a food web dominated by bacteria, with no thought about natural ADOC levels. Furthermore, the optimal temperature for growth of three-spine sticklebacks is ~18oC, i.e. as in our high temperature treatment. Our results therefore suggests that climate change predictions of increased temperature and ADOC influx through rivers might actually have a positive effect on food web efficiency and fish production depending on actual species occurring in the system.

Control of pelagic productivity, FWE and ecosystem trophic balance by ADOC The theoretical model simulations showed that NP inputs in general had a positive effect on the production. ADOC inputs had a clear positive effect on the BP, while it affected the production at the top trophic level negatively and in some cases it also caused a decrease of the PP. No clear effects of light attenuation could be seen on the production. In general food web efficiency was negatively affected by DOC additions, probably due to the establishment of 1-2 extra trophic levels in the food web. Increased light attenuation induced by colored substances in the ADOM had minor effect, only marginally increasing the heterotrophy. Our results indicate that coastal systems receiving allochthonous carbon can end up being dominated by heterotrophic production depending on the nutritional status of the system and the magnitude of carbon reaching the food web. A switch to heterotrophy would likely lead to reduced productivity at higher trophic levels ultimately affecting fish production negatively. This scenario can be expected to occur in northern Europe where climate change simulations predict increased precipitation with higher river discharge as result. This increased river inflow containing high concentrations of colored terrestrial DOC may thus cause reduced production in coastal systems.

Conclusions and future perspectives

Most of the work presented in this thesis applies to the effect of allochthonous carbon availability and temperature on food webs in a climate change context. In the first study both temperature and nutrients significantly boosted bacterial growth rates, but evidently nutrients play a larger role as no difference was detected at any temperature at the lowest nutrient condition. This condition would prevail in naturally low-productive seawater. Furthermore, the results in this study indicate that a large change in either factor may lead to changes in bacterial community composition. Depending on if the predictions of climate in the future comes true, bacterial production and the microbial loop may well dominate future food webs receiving ADOM through river run-off. This would according to theories (Azam et al. 1983, Ducklow et al. 1986, Landry and Calbet 2004) have impact on the production at higher trophic levels as well as food web efficiency. However, many questions remain unanswered. How bioavailable is the ADOM that reaches the sea? Microorganisms in the river might already have consumed the bioavailable part of the matter which is transported a long way, leaving mostly a recalcitrant proportion. Will the increase in heterotrophic production affect food web efficiency and production at higher trophic levels negatively or could the higher production actually serve as a subsidy promoting production higher up in the food web? Our second study expanded on the ADOC topic by looking at the carbon effects on food webs with different top consumer. The glucose used as carbon source boosted the bacterial production and shifted the systems towards heterotrophy. However, depending on what top consumer was present in the food web, effects on food web efficiency and production at the highest trophic level were quite different. Results from paper II suggests that food webs with zooplankton as top consumer function equally well independent of the PP:BP ratio and might even yield higher zooplankton production following ADOC additions. In this food web the energy transferred through the microbial loop seems to have acted as a subsidy. In the food webs where fish was top consumer we saw a much larger effect of the ADOC treatment. These systems turned almost completely heterotrophic (89-98%) and both food web efficiency and fish production decreased significantly. This indicates that planktivorous fish impose cascading effects on the food web, ultimately altering the system, affecting their own production negatively. Translated to in situ conditions, our study suggests that if climate change will cause increased inputs of ADOC into aquatic systems, planktivorous fish production may be severely reduced. Yet again the importance of knowing the short term as well as the long term bioavailability of the ADOC is of outermost importance. Furthermore it is clear that in order to fully understand effects of ADOC, as well as DON and DOP, on pelagic systems, more studies on interaction effects between different top consumers and basal production are needed. The further development of the study of climate change related abiotic factors in paper III included temperature along with a natural source of ADOC. Although both factors independently affected the food web through higher bacterial production, promoting heterotrophy, the combination of these treatments had the largest effect, yielding a significantly higher fish production and food web efficiency. We hypothesize that the increase in temperature leads to a higher production potential for zooplankton and fish, which only can be met by increasing the nutrient availability. Therefore the increased basal production due to ADOC additions promoted fish production at the higher temperature. Evidently, in order to fully understand the effects of the abiotic factors influenced by climate change, these variables must be studied together. The obvious next step would be to include changes in salinity and pH. In addition to the previously mentioned question of ADOC bioavailability,

21 the choice of fish (three-spined stickleback) in study II and III affected the presented results. The stickleback has been shown to thrive in warm coastal areas and therefore effects seen for these zooplanktivorous fish might not translate directly to other fish species. A more thorough examination of the effect of ADOC was possible in the ecosystem model used in paper IV. Results from this study indicated that carbon increasingly affected the system with rising bioavailability through promoted bacterial production, but the effect of increased light attenuation was minor. Furthermore, it supported our previous observation that the nutrients nitrogen and phosphorus played an important role in determining the effect of DOC additions. Consequently, in a climate change scenario, aquatic systems receiving water with a high C to P and/or C to N ratio, may turn heterotrophic which would reduce productivity at higher trophic levels. This situation is likely to occur in northern Europe, where climate change is expected to lead to more precipitation. The food web model used in this study only included mesozooplankton as the highest trophic level and should naturally be continued by including fish. Additionally both light and nutrient data should as a next step follow the annual cycle; this would probably increase the importance of increased light attenuation as it would shorten the growth season. Taken together, my thesis indicates that climate change may alter food web structure and function in coastal areas in northern Europe due to increased inflow of colored ADOC and higher temperature. Bacterial growth may be favored, while phytoplankton growth will be disfavored. This in turn can lead to lower fish production. However, if enough nitrogen and phosphorus are available these effects may be less dramatic. In un-productive systems like the northern Baltic Sea, where the inflowing river water have high C to P ratios, climate change may lead to an overall oligotrophication.

Author contributions

I. RD and AA planned and designed the study. RD performed the microcosm experiments and analyzed bacterial responses. JD performed molecular analyses of bacterial species composition. RD, JD and SSL performed the statistical analyses. RD, AA, JD, LR and SSL wrote the paper.

II. RD, RL, AA, UB and SL planned and designed the study. RD and RL performed the mesocosm experiment and analyzed plankton and fish responses. RD performed statistical analyses. RD, AA, RL, PB and UB wrote the paper.

III. LF, RD, AA, UB, SL and LOE planned and designed the study. RD and RL performed the mesocosm experiment and analyzed plankton and fish responses. RL performed the statistical analyses. RD, AA, RL, PB and UB wrote the paper.

IV. RD and AA planned and designed the study. RD performed the model simulations and the statistical analyses. RD and AA wrote the paper.

Authors: AA = Agneta Andersson, PB = Pär Byström, UB = Ulf Båmstedt, RD = Rickard Degerman, JD = Julie Dinsaquet, LOE = Lars-Ove Eriksson, SL = Stefan Larsson, LR = Lasse Riemann, RL = Robert Lefebure, SSL = Sara Sjöstedt de Luna

Acknowledgements

This thesis was supported by grants from the Swedish Research Council FORMAS to AA and SL (217-2006-674), the Centre for Environmental Research in Umeå (CMF) to UB, AA and SL, and by the Swedish strategic research program ECOCHANGE to Umeå University. I would like to thank Agneta Andersson, Kristin Dahlgren and Pia Haecky for valuable comments on earlier versions of this thesis.

Thanks! My first and biggest gratitude goes to my supervisor Agneta who gave me the wonderful opportunity to join her research group and later did not give up on me. Thank you for your support and determination to see me through, without you I wouldn’t have made it this far. I also owe a big thanks to the wonderful staff at Umeå Marine Sciences Centre, especially Calle, Siv, Gun and Anna P for all their help. Of course, a huge thanks also goes to my co- worker, roommate and thesis collaborator Robert Lefébure. I had great fun most of the time working with you and I really miss our loony discussions and ideas. To my co-authors, thank you for your excellent cooperation, help and most of all your valuable opinions and comments. Thanks also to the other PhDs working at UMSC during my years there: Peder, Kristin, Anna, Rose and Ignacio. I would further like to thank all the people I at some time or the other played “innebandy” with, I had a lot of fun with you competitive lot. Last but not least I would like to thank my family, foremost my wife Maria for her help and support and our lovely kids Emma and Isak just for being you.

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