[page 93↓]

7  General Discussion

Although it has been generally accepted that protists are an important component linking the microbial to the classical food web, virtually nothing is known about their biochemical composition and nutritional quality. In this thesis, I examined (1) whether the biochemical composition of free-living protists depends on their dietary sources, (2) their trophic mode and (3) whether their biochemical composition determines their nutritional quality for zooplankton predators. In this context, four hypotheses were tested (see Outline of the Thesis). In the following, I refer to each hypothesis and give an overview of the most important findings and implications for the nutritional quality of protists. Finally, I summarize both aspects – biochemical composition and nutritional quality of protists – in an ecological context.

Hypothesis 1. The biochemical composition of heterotrophic protists depends on the biochemical composition of their diet.

To test this hypothesis I analysed the fatty acid and amino acid (chapter 2) as well as the sterol composition (chapter 3) of two protist species grown on an algal diet and two species grown on a bacterial diet. Given known differences in the biochemical composition between algae and bacteria (Harvey et al., 1997), I expected to find these differences reflected in the composition of algivorous and bacterivorous protists. By comparing two species grown on the same diet, I discussed species-specific differences in the biochemical metabolism of those heterotrophic protists.

The most important finding here was that the fatty acid, amino acid, and sterol composition of the studied protists generally reflected the composition of their diet, but absolute concentrations were often higher in the protists than in their diet (you are not what you eat!). Higher concentrations in the heterotrophic protists as compared to dietary concentrations suggest that accumulation of biochemical compounds takes place. Goulden and Place (1990) showed that adult daphniids accumulate lipids preferentially, relative to other biochemical components of their diet. To explain the accumulation of fatty acids, sterols, and amino acids in the here studied heterotrophic protists, as well as of lipids in the daphniids, one must assume that these organisms (1) preferentially assimilate lipids and amino acids, [page 94↓](2) preferentially metabolize assimilated carbohydrates while storing lipids and amino acids, or (3) synthesize lipids and amino acids.

Efficient assimilation may have been related to high ingestion rates. An estimate of the ingestion rates needed to reach the EPA concentrations we measured in the protists, versus known published ingestion rates for Balanion planctonicum and Cyclidium sp. (Müller and Schlegel, 1999; Šimek et al., 1994) makes this a reasonable assumption. However, similar carbon-specific concentrations in organisms of different size, like Cyclidium and Balanion, may not only rely on high ingestion rates. Preferential assimilation of fatty acids, sterols, and amino acids, or preferential metabolism of carbohydrates or other carbon compounds may have contributed to the accumulation of biochemical compounds in the studied heterotrophic protists. Carbohydrates are not as energetic as lipid or protein molecules (Stryer, 1995), but carbohydrate metabolism may have provided the protists with enough energy for basic processes such as growth and survival. In this case, the differential allocation of lipids and amino acids for cellular functions other than energy production would explain the accumulation of those biochemicals in the heterotrophic protists. These may possibly have stored biochemical compounds for building structural molecules, such as phospholipids, as suggested by the positive correlation between cell size and cellular fatty acid concentrations (chapter 2).

Synthesis of biochemical compounds such as fatty acids and sterols by protists has been already described (Koroly and Connor, 1976; Klein Breteler et al., 1999). First evidences for the synthesis of highly unsaturated fatty acids, like EPA and DHA, and also sterols by the heterotrophic dinoflagellate Oxyrrhis marina (Klein Breteler et al., 1999) suggest to reconsider the assumption that heterotrophic protists are unable to synthesize complex lipid molecules. For instance, because DHA was not observed in the bacterial diet of Chilomonas, the only explanation for the presence of DHA in this flagellate should be synthesis, probably through elongation of linolenic acid (see Appendix 1). However, synthesis of unsaturated fatty acids may be inefficient in phagotrophic protists. Cryptomonas and Chilomonas are both cryptomonads, and they both contained DHA. However, DHA concentrations were higher in Cryptomonas than in Chilomonas. Possibly, DHA synthesis in Chilomonas is not as efficient as in Cryptomonas, which could be due to species-specific differences in fatty acid metabolism or to the trophic mode of the organisms, since Cryptomonas grown as autotroph and Chilomonas as heterotroph.

[page 95↓]

I found evidence for sterol synthesis by heterotrophic protists in my study, especially concerning the phytosterols ergosterol and brassicasterol. Both sterols were found in Chilomonas, although not present in their bacterial diet. De novo synthesis of sterols has been described for some amoebae species belonging to the genera Acanthamoeba and Naegleria (Raederstorff and Rohmer, 1987a,b). In those species, ergosterol was the predominant sterol, with cycloartenol serving as a precursor. Cycloartenol was also found in the here studied protist species, including Chilomonas. This finding also points to ergosterol synthesis in Chilomonas.

The presence of high phytosterol concentrations, especially stigmasterol, instead of cholesterol in all analyzed protists suggests that phytosterols may have analogous regulatory functions as cholesterol in cell membranes of these species. Cholesterol was only found in minor amounts in the protists, except for Cyclidium, which also contained the triterpenoids tetrahymanol, diplopterol, and diploptene, which are typically found in ciliates and some bacteria (Ourisson et al., 1987). In Tetrahymena, synthesis of tetrahymanol was shown to be hampered by exogenous supply of stigmasterol, suggesting that synthesis is regulated by energetic trade-off mechanisms or possibly through molecular concurrence between these triterpenoids for the same enzymatic bonding sites (Holler et al., 1993).

Conclusion: The answer to the question “Does the biochemical composition of free-living protists depend on their dietary sources?” is: “Yes, but not exclusively”. Dietary composition influences protist composition, but the studied species were able to accumulate dietary fatty acids, sterols, and amino acids. They could have been able to differentially allocate lipids and proteins for structural functions and to preferentially metabolise carbohydrates for generating energy. Moreover, synthesis of some compounds is likely, as important precursors of fatty acids and sterols were observed in the heterotrophic protists. The precursors may have been obtained from the diet, but the enzymatic accessories necessary for elongating fatty acids or cycling sterols must come from the protists themselves, as enzyme synthesis and activation is genetically determined. In other words: heterotrophic protists probably get the biochemical composition of their diet, but they are able to modify dietary compounds and to allocate them differentially. Moreover, these metabolic features seem to vary species-specifically, as suggested by the different biochemical composition in two species fed the same diet (e.g. Chilomonas had DHA but Cyclidium had not).

[page 96↓]

Future Perspectives: It would be very interesting to follow the fate of dietary compounds and differential allocation of biochemicals in heterotrophic protists. For these purposes experiments involving labelled target molecules which are followed throughout metabolic pathways (time series experiments) in the protist cells are necessary. Information provided by such experiments would contribute to the understanding of metabolic pathways in heterotrophic protists as well as the energetic trade-offs – allocation into structural molecules or energy reserves – which certainly influences the nutritional quality of heterotrophic protists, besides the biochemical composition itself.

Hypothesis 2.The biochemical composition of protists depends on the trophic mode.

In a second approach (chapter 4), I analysed the fatty acid and sterol composition of a single species – the flagellate Ochromonas sp. – cultured under three different trophic modes: autotrophic, mixotrophic, and heterotrophic. The aim here was to test the influence of the trophic mode on the biochemical profile of Ochromonas sp. and to get some insights on metabolic features related to synthesis and allocation of lipids in this flagellate.

The hypothesis was corroborated: the trophic mode determined the metabolic pathways of Ochromonas sp., resulting in different biochemical composition of the flagellates cultured under different trophic modes. Decreasing concentrations of polyunsaturated fatty acids were observed as the trophic mode changed from autotrophic via mixotrophic to heterotrophic growth. Saturated fatty acids were the most abundant fatty acid class in heterotrophs, while polyunsaturated fatty acids were most abundant in autotrophs. Interestingly, the highest concentration of total saturated fatty acids was observed in mixotrophs, suggesting ingestion of bacteria by mixotrophs. Nevertheless, mixotrophs showed similar total concentrations of monounsaturated fatty acids to those found in autotrophs. Moreover, total polyunsaturated fatty acid concentrations in mixotrophs were higher than in heterotrophs, but not as high as in autotrophs. Mixotrophs clearly synthesized some biochemical compounds, which were not present in their bacterial diet. Nevertheless a number of their biochemical compounds were equivalent to those found in the bacteria.

There is an ongoing debate on the meaning of mixotrophy; i.e. the question as to why flagellates, capable of photosynthesis, graze bacteria. Some authors argue [page 97↓]that mixotrophy is a mean of acquiring nutrients for photosynthesis during periods of limitation (Nygaard and Tobiesen, 1993). Other authors suggest that the prime purpose of mixotrophy is to obtain carbon to support growth (Bird and Kalff, 1986; Caron et al., 1990; Jones et al., 1993). If so, carbon obtained by phagotrophy may be primarily used for building structural biomolecules such as carbohydrates and phospholipids, whereas photosynthesis may be responsible for generating substrates for the synthesis of more complex energetic biomolecules, such as triglycerides.

The patterns observed for fatty acids suggest that fatty acid metabolism may be strongly influenced by the trophic mode and by the processes of energy supply – photosynthesis or phagotrophy. In superior plants, the enzyme acetyl CoA carboxylase catalyses the first step of fatty acid synthesis and its activity depends on both light and the rate of fatty acid synthesis in the chloroplasts (Post-Beitenmiller et al., 1992). De novo synthesis of lipids in plants also depends on NADPH generated in the light reactions of photosynthesis. Hence, it is expected that rates of lipid synthesis and the relative allocation of photosynthates into lipid synthesis both tend to increase with incubation irradiance (Wainman and Lean, 1992). My findings of an increasing tendency of fatty acid concentrations and degree of unsaturation from heterotrophs to autotrophs are consistent with this tendency of high lipid synthesis rates in the presence of light, if all other conditions remain unaltered.

The sterol pattern found for Ochromonas in my study corroborates findings of sterols in other Ochromonas species (Halevy et al., 1966; Avivi et al., 1967; Goodwin, 1974), where stigmasterol was also the most abundant sterol present. The sterol metabolism in Ochromonas seems to be less dependent on the trophic mode than fatty acids do, as autotrophs and heterotrophs contained similar concentrations of all sterols present. Mixotrophs, on the other hand, showed the lowest sterol concentrations among flagellates, although the relative amounts remained similar to those found in autotrophs and mixotrophs. The high metabolic costs of a mixotroph to run both an autotrophic and a phagotrophic metabolism (Rothhaupt, 1996a) may limit the synthesis rate of some sterols. In mixotrophic Ochromonas, sterol synthesis may have been mostly directed to stigmasterol synthesis, as stigmasterol concentrations in mixotrophs were similar to those fond in autotrophs and heterotrophs. It would be interesting to investigate the function of stigmasterol in Ochromonas, since stigmasterol synthesis seems to be maintained even at low synthesis rates of other sterols.

[page 98↓]

The fact that heterotrophs contained higher concentrations of sterols than mixotrophs may relate to the phagotrophic mode of heterotrophs. The metabolic processes discussed in the past chapters for fatty acids and amino acids, including high ingestion rates, preferential metabolism of carbohydrates, and self synthesis, could also be claimed to explain the higher sterol concentrations in heterotrophs than in their bacterial diet. The fact that these metabolic processes are energy-consuming might explain why mixotrophs did not contain sterol concentrations as high as heterotrophs did.

Sterols are less appropriate to differentiate Ochromonas sp. by its different trophic modes, as no significant differences were observed between autotrophs and heterotrophs. Fatty acid composition, by contrary, seems to be a very efficient marker of the trophic mode, probably because fatty acid metabolism may be influenced more strongly by the mechanisms of energy uptake: photosynthesis or phagotrophy. Additionally, fatty acids and sterols are expected to respond differently to environmental factors such as light and nutrients, because these lipid classes exhibit differences in their functions and biosynthetic pathways (Parish and Wangersky, 1987; Smith and D’Souza, 1993).

Conclusion: The question “Does the biochemical composition of protists depend on their trophic mode?” could clearly be answered with “Yes, it does” for Ochromonas sp. This became especially evident for fatty acids, which differed significantly among Ochromonas sp. grown as autotroph, mixotroph, and heterotroph. For sterols, the differences observed between trophic modes seemed to be caused by energetic trade-offs as mixotrophs, living more energy-consuming, had lower sterol concentrations than specialized autotrophs or heterotrophs.

Future Perspectives: It would be interesting to investigate under which set of conditions lipids, especially fatty acids, are allocated into structural (phospholipids in the membranes) or energy reserve molecules (triglycerides) in Ochromonas of different trophic modes. Analyses of fatty acid contents in triglycerides and phospholipids would be an adequate method to test for differential lipid allocation. Differential allocation of lipids may affect cellular division rates, and consequently, population growth. If there is a defined allocation pattern, which is dictated by the trophic mode, this would help to understand the dominance of a trophic mode over another within the same species, under natural conditions. The dominance of one trophic mode may, in turn, profoundly affect trophic interactions between Ochromonas and its predators in nature.

[page 99↓]

Hypothesis 3. The biochemical composition of heterotrophic protists determines their nutritional quality for the rotifer Keratella quadrata.

With this hypothesis I examined the second aspect of this thesis: whether the biochemical composition of heterotrophic protists determines their nutritional quality for zooplankton predators. Here I carried out population growth experiments, whereby the rotifer Keratella quadrata was offered four heterotrophic protist species as prey (Balanion planctonicum, Urotricha farcta, Cyclidium sp., and Chilomonas paramecium). The cryptomonad Cryptomonas phaseolus was used as a control prey of good nutritional quality. Population growth rates and egg numbers of the rotifer were evaluated for 5 days periods, and were subsequently correlated to the fatty acid, amino acid, and sterol composition of the protists (presented in chapters 2 and 3).

The biochemical composition of the protists was significantly correlated to the cumulative egg numbers of Keratella but not to population growth rates. This suggests that biochemical requirements for egg production differ from those for population growth, in the case of Keratella fed heterotrophic protists. These findings corroborate the view that growth, reproduction, and survivorship have different metabolic demands (Sterner and Hessen, 1994).

Among the biochemical compounds, which were significantly correlated to the egg production of Keratella, polyunsaturated fatty acids were by far the most important, evidenced by the higher number of significant correlations (a total of eight, including γ-linolenic acid, arachidonic acid, EPA, DHA, and DPA), as compared to those found for sterols (ergosterol, desmosterol, and stigmastanol) and amino acids (leucine only). This result suggests that fatty acid limitation surpass sterol or amino acid limitation for Keratella, since polyunsaturated fatty acids were the biochemical class that exhibited the largest differences among protists species (chapter 2). Supplementation tests can help to elucidate the limiting effects of specific PUFAs for Keratella (see hypothesis 4).

As no population growth of Keratella was observed when feeding the rotifers with bacterivorous protists, there must be at least one additional factor constraining growth of the rotifers, when these were fed bacterivorous protists. This suggests that single biochemical factors become limiting only under non-limitation by another factor. This is along the lines of the Liebig’s Law of the Minimum (Von Liebig, 1855) that states that an organism will become limited by whatever resource that is in lowest supply compared to the organism’s needs. Negative [page 100↓]effects of bacterivorous protists on the survival of Keratella individuals could have led to the negative growth rates of Keratella’s populations.

Conclusion: The answer to the question “Does the biochemical composition of heterotrophic protists determine their nutritional value as prey for the rotifer Keratella quadrata?”is “Yes, but not necessarily”. Although we found a significant effect of biochemical compounds, especially fatty acids, on the egg production of the rotifers, there were no relationships between the protists’ biochemistry and the rotifers’ growth rates. Additionally the rotifer was not able to grow on a diet of bacterivores. This means that additional factors may be primarily limiting Keratella fed bacterivorous protists, according to the Liebig’s Law of the minimum. The results suggest that the biochemistry is only one factor in a pool of factors driving the nutrition of Keratella fed heterotrophic protists.

Future Perspectives: The significant correlations observed between prey biochemical compounds and cumulative egg numbers of Keratella clearly indicate the necessity for testing limitation by single biochemical compounds for Keratella’s growth and reproduction. This is possible through supplementation of a poor quality diet, such as Chilomonas or Cyclidium with the target biochemical compound (see hypothesis 4). There is a need for working with axenically cultured prey organisms, such as osmotrophs. It would minimize possible harmful effects due to high bacterial densities on the survival of the rotifers, which may have led to the negative growth rates of Keratella’s population grown on bacterivorous protists.

Hypothesis 4. Chilomonas paramecium can be successfully supplemented with essential fatty acids (EPA and DHA). Chilomonas paramecium supplemented with EPA and DHA will enhance growth and reproduction of Keratella quadrata.

Given the correlative evidence between Keratella’s egg numbers and several polyunsaturated fatty acids, the need to test limiting effects of single fatty acids on Keratella’s growth and reproduction became clear. An efficient method to test limiting effects of single biochemical compounds is to supplement a poor quality diet with the target compound. However, such a method has not been tested for heterotrophic protists before.

I chose the polyunsaturated fatty acids EPA and DHA, based on current information concerning limiting effects of both fatty acids for zooplankton species (Von Elert, 2000; Müller-Navarra et al., 2004). I chose Chilomonas paramecium[page 101↓]to be supplemented with EPA and DHA because Chilomonas showed the lowest EPA concentrations among all heterotrophic protists I have analysed (chapter 2). Secondly, Chilomonas is of similar size and shape as the control alga Cryptomonas. Rotifers may exhibit different ingestion rates for protists of different size and shape (Bogdan and Gilber, 1982; Rothhaupt, 1990); thus, I minimized artefacts due to differential ingestion by choosing a target organism and a control of similar size and shape.

The supplementation technique proposed by Von Elert (2002) proved to be adequate for supplementing Chilomonas with defined concentrations of the polyunsaturated fatty acids EPA and DHA. Hence, the method allowed the evaluation of direct limiting effects by specific fatty acids on the performanceof Keratella fed Chilomonas, qualitatively and quantitatively.

Feeding on Chilomonas supplemented with DHA slightly improved rotifers’ population growth rates, but strongly enhanced egg production as compared to non-supplemented Chilomonas. EPA enriched Chilomonas did not significantly improve population growth rates and egg production of the rotifer. A slightly enhanced effect on population growth was observed when feeding the rotifers with Chilomonas previously incubated with BSA, but without fatty acids. This suggests limiting effects of certain amino acids for Keratella fed Chilomonas.

DHA may be, more than EPA, the limiting factor in the interface bacterivorous protists – zooplankton. Dietary DHA has been found in high amounts in copepod’s eggs (Sargent and Falk-Petersen, 1988). In some crustaceans DHA has been found in 2 – 5 fold higher amounts in eggs and ovaries than in other female tissues (Hayashi, 1976), indicating the importance of DHA for embryonic survival and growth. DHA is also believed to play an important role during larval development and metamorphosis of marine molluscs (Delaunay et al., 1993) and to promote enhanced larval growth of the zebra mussel Dreissena polymorpha (Wacker et al., 2002).

When testing different protists as prey for Keratella (chapter 5) egg production did significantly correlate with biochemical composition of the protists, whereas population growth did not. This trend was also observed here, when testing single fatty acids. The fact that DHA supplementation lead to increased egg numbers but only slightly enhanced Keratella population growth rates confirmed my previous results, that reproduction and population growth have different metabolic demands, in accordance with the stoichiometric theory (Sterner and Hessen, 1994).

[page 102↓]

The most important result here was that Chilomonas supplemented with fatty acid concentrations similar to those found in the algae Cryptomonas did not support population growth and egg production of Keratella as Cryptomonas did. Although supplementation has enhanced the nutritional quality of Chilomonas for Keratella, this heterotrophic flagellate was still of poor quality when compared to the algae. Hence, additional factors, such as other biochemical compounds, but perhaps also minerals as well as synergetic and antagonistic interactions among biomolecules may limit the performance of Keratella grown on a Chilomonas diet.

Conclusion: The question “Does the supplementation of Chilomonas paramecium with essential fatty acids enhance their nutritional value for Keratella quadrata?” can be answered with “YES, it does” but only with respect to DHA. DHA may primarily limit the nutritional quality at the interface bacterivorous protists – rotifers. Nevertheless, DHA supplementation of Chilomonas did not result in a nutritional quality comparable with that of the algae Cryptomonas.

Future Perspectives: An important approach should be to combine different biochemical compounds as well as mineral and biochemical supplementation to test for limitation of one compound under different concentrations of another. This would help to clarify, under which set of conditions a biochemical compound will limit the nutritionof Keratella fed heterotrophic protists.

Biochemical composition and nutritional quality of protists

To search for the biochemical or mineral compound, which primarily limits nutrition of a predator, is like looking for a needle in a haystack. Current methodological assays enable us to test the nutritional quality of different prey organisms and the results obtained often provide correlative evidence for certain compounds, which are likely candidates of causing limitation for a predator. As a next step, single compounds, for which correlative evidence was found, are directly tested through supplementation techniques. This methodological approach is very efficient for providing insights on the nutritional quality of a prey in a defined “prey – predator” model, and under a defined set of conditions. However, by using this method we will probably never exactly and undoubtfully identify that compound, which is primarily limiting a predator, because limitation may change with altering environmental conditions and among different prey-predator systems. This assumption is sustained by findings of contrasting limiting factors for a same predator species. For Daphnia galeata different biochemical and [page 103↓]mineral components have been claimed to be the “most important limiting factors” (Müller-Navarra, 1995; Weers and Gulati, 1997; Von Elert and Wollfrom, 2001; Von Elert, 2002). Such contrasting findings probably relate to different experimental conditions, as different prey organisms were used, cultures were submitted to different physical conditions, or the daphniids were isolated from different environments. Maybe we have been formulating the wrong question. Instead of searching for that one factor, which primary limits growth and reproduction of zooplankton organisms, efforts should be directed to understand under which set of conditions one single biochemical compound may be limiting. That some fatty acids or amino acids are essential for aquatic invertebrates is well known. A more realistic question could be: under which set of conditions a certain fatty acid or amino acid may play a more important limiting effect?

Müller-Navarra et al. (2004), for instance, elegantly showed that the degree of EPA limitation for Daphnia growth in nature depends on the phosphorus levels in the system. In phosphorus-poor lakes, sestonic EPA was able to efficiently limit growth rates of Daphnia populations. In phosphorus-rich systems, EPA limitation could not be discriminated from other limitation sources. Becker and Boersma (2003) showed that limitation effects of EPA on Daphnia life history could only be detected below a specific prey nutrient threshold. Above this threshold, the daphniids were limited by nutrient ratios. Both results make clear that limitation of a single factor depends on a constellation of factors, even for the same predator species.

I was first surprised to find no correlative evidence between the biochemical composition of the heterotrophic protists and population growth of Keratella quadrata. To my knowledge, the present study includes the widest range of biochemical compounds to test the nutritional quality of heterotrophic protists. Nevertheless, I did not find any evidence about the factors, which may have limited population growth of Keratella. It seems likely that the lower concentrations and relative amounts of several biochemical compounds (e.g. amino acids and sterols) found in the good quality algae Cryptomonas as compared to Balanion and Urotricha were already adequate for supporting Keratella’s population growth. If so, why did Chilomonas not support population growth of Keratella? Chilomonas had concentrations and relative amounts of several biochemical compounds similar to those found in Cryptomonas. And even when this was not the case (e.g. DHA), artificial supplementation did not enhance the nutritional quality of Chilomonas equivalent to that found for Cryptomonas. It is likely that a non-chemical harmful effect on Keratella’s survival, caused by high bacterial densities in the treatments (personal observation), has resulted in [page 104↓]the observed negative growth rates of the rotifers. Given that, I could not include bacterivores into the statistical analysis, which were performed for the algivores only. This reduced statistical variability may have contributed to the absence of significant relationships between Keratella’s growth rate and prey biochemistry. Working with axenic cultures (e.g. osmotrophs) would minimize such undesirable experimental artifacts.

Despite the absence of correlations between prey biochemical compounds and population growth rates of Keratella, the strong positive correlations found with egg production of Keratella suggests that the biochemistry of the protists, especially fatty acids, is indeed an important factor limiting Keratella’s performance.

The variation in terms of absolute concentrations found for polyunsaturated fatty acids in protists grown on different diets (algivores/bacterivores) and within a protist species grown under different trophic modes (autotrophic, mixotrophic, heterotrophic) suggests that polyunsaturated fatty acids most likely limit life history-traits of a predator fed protists. Fatty acid limitation for zooplankton has been observed for many species (Ahlgren et al., 1990; Brett and Müller-Navarra, 1997; Weers and Gulati, 1997; Becker and Boersma, 2003), mostly feeding on algae. I found positive relationships between several polyunsaturated fatty acids, such as γ-linolenic acid, EPA, DHA as well as arachidonic acid, and the egg production of Keratella quadrata (chapter 5). The number of polyunsaturated fatty acids which were significantly correlated with Keratella’s egg production was much higher than the number of sterols and amino acids. This probably means that polyunsaturated fatty acids are most likely limiting factors for Keratella feeding on heterotrophic protists. However, not only a single fatty acid is likely to be responsible for the entire limitation of Keratella fed heterotrophic protists, as suggested by the findings on Chilomonas supplemented with DHA (chapter 6). Probably a combination of biochemical compounds was responsible for the higher nutritional quality of algivores as compared to bacterivores. This could be tested by supplementing Chilomonas and Cyclidium with other single biochemical compounds, as well as with combined supplements of several fatty acids, sterols, and amino acids.

In this study, I showed that the biochemical composition of protists may be influenced by the dietary sources, but the biochemical concentrations are more dependent of metabolic features of the protists, such as efficient accumulation and synthesis. Moreover, the trophic mode exhibited by a protist species has an important effect on its lipid composition, especially that of fatty acids. It was [page 105↓]shown that Keratella was limited by the biochemical composition of heterotrophic protists. A direct test using supplementation of DHA confirmed the correlative evidence found for the limiting effects of DHA on Keratella’s reproduction, when the rotifers fed on bacterivorous protists.

My results on the biochemical composition of protists highlight the necessity to incorporate heterotrophic protists in food quality studies, because of their ability to modify the biochemical composition at an early stage in aquatic food webs, i.e. at the interface between algae/bacteria and the mesozooplankton. Considering the high energy density and essentiality of some biochemical compounds (such as lipids), small biochemical modifications at this early stage may have profound consequences for matter and energy transfer through the entire food web.

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