It has been recognized for many years that variation in the rate at which primary production is converted to zooplankton biomass is quite large (Sterner and Hessen, 1994). Some aquatic food webs, such as found in many hypereutrophic lakes, have very high biomass of primary producers, but relatively low zooplankton and fish biomasses, as demonstrated by the Eltonian biomass pyramids (Fig. 1). Other systems, such as marine upwelling zones, are low in phytoplankton biomass, but have very high zooplankton and fish biomasses (Fig. 1). This variation in carbon transfer efficiency can be attributed to variation in food quality.
|Fig. 1 – Eltonian biomass pyramids for the hypereutrophic Clear Lake (Carney and Goldman, unpublished data) and Peruvian Upwelling Zone food web (Dortch and Packard, 1989). Modified from Brett and Müller-Navarra, 1997.|
The food quality of a planktonic prey organism is given by a combination of factors including morphological, physiological, behavioural, chemical, and biochemical features. Additionally, particular requirements of the consumer together with its ability to ingest and digest the prey organism contribute to the determination of the ultimate quality of a planktonic prey. The nutritional value of prey organisms is more specifically determined by the prey mineral (Kilham et al., 1997) and biochemical composition (Kleppel et al., 1998) as well as by the efficiency of a consumer in assimilating prey minerals and essential biochemical compounds (Mayzaud et al., 1992). It is well known, that some cyanobacteria species (blue-green algae), despite their reported edibility for zooplankton in many cases, are a poor food, primarily attributed to phosphorus limitation (Bernardi and Guissani, 1990). On the other hand, phytoplankton species may be neither phosphorus nor nitrogen limited, but lack biochemical compounds, which are essential for herbivorous predators (Müller-Navarra, 1995; Müller-Navarra et al., 2004). Most of the biochemical compounds, [page 11↓]which have been considered in studies of nutritional quality in aquatic food webs, are lipids (fatty acids and sterols) or proteins (especially essential amino acids).
The high-energy content of lipids relative to proteins or carbohydrates coupled with the small body size of most planktonic invertebrates makes lipids the energy storage biomolecules of choice for zooplankton (Arts, 1998). Zooplankton lipids often comprise 60–65% of their dry weight (Arts et al., 1993), and their cellular function depends on the molecular structure (Fig. 2). Triacylglycerols and phospholipids are biochemically related, as they have a glycerol backbone to which two or three fatty acids are esterified (Fig. 2). Triacylglycerols are very important energy storage molecules, whereas phospholipids are essential components of membranes. Sterols share with phospholipids a structural function in membranes, but in terms of polarity they are grouped with triacylglycerols in the neutral lipids. Phospholipids are grouped with polar lipids, including glycolipids. Glycolipids contain one or more molecules of a sugar and are found in bacteria, plants, and animals.
|Fig. 2 – The structure of some important lipids (modified from Parrish, 1999).|
Among lipid molecules, fatty acids have received considerable attention, because they are usually present in low amounts but serve very important physical and metabolic functions [page 12↓]in the cell. Especially highly unsaturated fatty acids (HUFA) of the ω3 and ω6 families, like eicosapentaenoic acid (20:5ω3, EPA) and docosahexaenoic acid (22:6ω3, DHA) as well as some polyunsaturated fatty acids (PUFA), like arachidonic acid (20:4ω6) and linoleic acid (18:2ω6) are considered essential for many zooplanktonic consumers, which are not able to synthesize fatty acids (see appendix 1 for EPA and DHA synthesis and fatty acid nomenclature). Enhanced growth and reproduction of cladocerans, especially Daphnia, have been associated with higher contents of HUFA and PUFA in cultured prey organisms (e.g. DeMott and Müller-Navarra, 1997; Park et al., 2002; Becker and Boersma, 2003) and in lake seston (e.g. Müller-Navarra et al., 2000; Park et al., 2003; Müller-Navarra et al., 2004).
Along with essential fatty acids, the sterol composition of prey organisms has been reported to be important in limiting zooplankton life history traits (Von Elert et al., 2003; Hasset, 2004). Besides controlling membrane fluidity and permeability, sterols also form sexual hormones, sterol alkaloids, and act as vitamins. Sterol limitation for growth and reproduction of zooplankton predators has been found to be mainly caused by dietary cholesterol shortage, which has been associated with decreased growth of copepods (Hasset, 2004) and cladocerans (Von Elert et al., 2003), and retarded larval development of crustaceans (Teshima, 1991).
Amino acids are the basic structural unit of protein molecules. Some amino acids cannot be synthesized by animal cells at all or only at low rates (Kleppel et al., 1998; Guisande et al., 2000). These amino acids are considered essential and must therefore be supplied in the diet (see appendix 2 for the structure of the essential amino acids considered in this study).
There is an ongoing debate on the factors, which primarily determine the nutritional quality of a planktonic prey for its consumer – the mineral or the biochemical composition. Some authors have advocated the relevance of prey mineral composition and elemental stoichiometry, especially the P:C and N:C ratios, in determining prey nutritional quality, because mineral nutrients usually limit production and growth in nature (e.g. Sterner et al., 1992; DeMott et al., 1998; Plath and Boersma, 2001). Others have emphasized the importance of essential biochemical compounds in promoting enhanced growth and reproduction of zooplankton, based on correlative evidences derived from laboratory and in situ studies (e.g. Ahlgren et al., 1990; Brett and Müller-Navarra, 1997; Weers and Gulati, 1997; Becker and Boersma, 2003). Actually, elements may be present in a wide [page 13↓]range of biochemical compounds. Therefore, the biochemistry of the compound in which an element is present dictates how that element is processed by a consumer (Tang and Dam, 1999). Hence, the concept of elemental stoichiometry, although powerful and useful (see Sterner and Elser, 2002), may not be sufficient to the full understanding of the food-related limitation of zooplankton production.
Further, there is a controversy on the question, which biochemical compound is most important in limiting zooplankton life-history traits. Some authors showed evidence for the essentiality of the polyunsaturated fatty acid EPA for Daphnia (Müller-Navarra, 1995; Becker and Boersma, 2003), while others did not find any limitation by EPA on daphniids (Von Elert and Wollfrom, 2001; Weiler, 2001) and suggested that sterols may be more important lipids limiting Daphnia growth. Moreover, DHA and linoloenic acid have been reported to limit growth and reproduction of zooplankton (Wacker et al., 2002). It must be kept in mind that nutritional quality is not only a matter of prey composition, but also of predator requirements. The fact that EPA was found to limit Daphnia growth under a set of conditions does not necessarily imply limitation under different conditions. This does not mean that EPA is not limiting at all, but that its limitation may be modulated by other factors, which were not quantified. Living organisms are actually a complex “package” of nutrients interacting on molecular, biochemical, and physiological levels. Thus, prey organisms should be viewed as a dynamic pool of nutrients rather than as a static source of unaltered composition waiting to be eaten.
It is known that a large fraction of the primary production may not be consumed directly by herbivorous consumers but is channelled through detrital organic matter via bacterial production to phagotrophic microrganisms. This led to the concept of the “microbial loop” (Fig. 3) (Azam et al., 1983) and to the discovery that planktonic food chains include a higher number of trophic levels than hitherto believed (Fenchel, 1988).
|Fig. 3 – Trophic links between the microbial and the classical food webs. Modified from|
However, recent works have shown that the early view of the microbial loop was somehow too simplistic. Today we know that: (1) heterotrophic bacteria do not only rely on dissolved organic matter (DOM) released by autotrophs as a substrate, but also on DOM released by heterotrophs (Jumars et al., 1989); (2) heterotrophic protists comprise more than one trophic level, as predation of ciliates on flagellates is also known (Berninger et al., 1993); (3) grazing by protists may not simply affect bacterial numbers but also have an impact on the morphological structure and productivity of bacterial communities (Jürgens and Güde, 1994); (4) there are immense species-specific differences concerning the trophic roles of protists in a food web; some flagellates, for instance, show autotrophic, mixotrophic and heterotrophic growth under natural conditions (Fenchel, 1986); (5) the production of low trophic levels is not only channelled to higher trophic levels through consumption of bacterial biomass by ciliates and flagellates; but that herbivory by heterotrophic protists also contributes significantly to the matter transfer to higher trophic levels (Sherr and Sherr, 1994). Therefore interactions of heterotrophic protists in planktonic food webs are more various and complex than traditionally believed. The use of simplified “chains” or “loops” does not capture the complexity of microbial food webs (Arndt and Berninger, 1995).
Several laboratorial studies with cultured protists have shown that mesozooplankton predators are able to efficiently grow and reproduce on a diet consisting of heterotrophic protists (Sanders and Porter, 1990; Weisse and Frahm, 2001; Mohr and Adrian, 2001). [page 15↓]Moreover, heterotrophic protists are likely to be an important alternative food resource for zooplankton when phytoplankton abundances are low or when phytoplankton quality is reduced (e.g. during periods of nutrient limitation). High predation pressure by copepods and cladocerans has been observed as a mechanism regulating protist community structure and densities in situ (Carrick et al., 1991; Wiackowsky et al., 1994; Jürgens et al., 1999; Jürgens and Jeppesen, 2000; Adrian et al., 2001; Burns and Schallenberg, 2001). However, evidence for high predation rates by mesozooplankton on heterotrophic protists does not attribute them high nutritional quality. Unfortunately, little attention has been paid to the biochemical features of heterotrophic protists, which do confer their nutritional value as prey in aquatic food webs.
Most of the information on the nutritional composition of heterotrophic protists dates back to the 60’s and 70’s, mostly related to studies of the ciliate Tetrahymena. The works of Holz, Connor, and Koroly on Tetrahymena were the first to consider the composition and metabolism of lipids and nucleic acids in this ciliate (see Elliott, 1973).
It is well known that heterotrophic protists often have lower C:N ratios than algae and mixotrophic protists (Stoecker and Capuzzo, 1990). The lower C:N ratios suggest that heterotrophic protists may be a source of nitrogen-rich compounds such as amino acids and proteins. Nevertheless, the amino acid composition of free-living heterotrophic protists is virtually unknown, except for studies on Tetrahymena (Holz, 1973).
It is has been observed that the fatty acid composition of heterotrophic protists is strongly influenced by the food resources (Ederington et al., 1995; Harvey et al., 1997). However, there are too few studies on the fatty acid composition of heterotrophic protists to permit such a generalization. Indeed, synthesis of long-chain polyunsaturated fatty acids has been reported for marine flagellates (Klein Breteler et al., 1999), suggesting that this metabolic ability should not be excluded for other species.
Despite studies on marine species (Harvey andMcManus, 1991; Ederington et al., 1995; Harvey et al., 1997; Breteler et al., 1999; Sul et al., 2000), the sterol composition of freshwater heterotrophic protists is still a ‘black box’. The only exceptions are studies on [page 16↓]freshwater flagellates of the genus Chilomonas, for which 24α-ethylcholesta-5,22(E)-dien-3β-ol (stigmasterol) was described as the predominant sterol (Patterson, 1991), and the bacterivorous ciliate Tetrahymena pyiriformis, for which a triterpenoid alcohol – tetrahymanol (gammaceran-3β-ol) – was first isolated and described as its major neutral lipid (Mallory et al., 1963).
It has been observed that the biochemical composition of heterotrophic protists resembles dietary composition (Ederington et al., 1995). For bacterivorous ciliates a fatty acid profile typical for bacteria has been reported, with high concentrations of odd chain-length and branched fatty acids (Ederington et al., 1995). Algivorous protists, in turn, have been reported to show a broader spectrum of polyunsaturated fatty acids, which are not typically found in bacteria (Desvilettes et al., 1997). However, some authors reported the presence of compounds in heterotrophic species, which were not identified in their diet (Ederington et al., 1995; Klein Breteler et al., 1999). The question remains, whether or not heterotrophic protists “are really what they eat”, or if they are able to metabolize some compounds obtained from the diet, thus modifying their biochemical composition (heterotrophic protists “are not what they eat”).
Some protist species have a fascinatingly complex nutritional ecology. Some flagellated species are able to adapt to a broad range of external conditions, because they exhibit a wide range of trophic modes, such as autotrophy, heterotrophy, and mixotrophy (Sleigh, 2000). Assuming that the trophic mode will ultimately determine the nutritional composition of such species, a large variability in the biochemical composition is expected to be found in these protists. As autotrophs, they are able to synthesize a broad palette of compounds, which are necessary for growth and maintenance. As heterotrophs, they ingest particulate material (phagotrophy) or absorb (osmotrophy) a wide diversity of molecules from their environment, thus extracting organic and inorganic building blocks for their own biosynthesis reactions as well as energy for growth and maintenance (Sterner and Elser, 2002). As mixotrophs, phagotrophy or osmotrophy supplements photosynthesis to generate energy and provide carbohydrate building blocks (Sanders, 1991).
The nutritional complexity of protists raises interesting questions about the nature of the mechanisms, which influence their biochemical composition, and subsequent nutritional quality as prey. Does the biochemical composition of protists depend only on their trophic mode? And within the same trophic mode, e.g. heterotroph/phagotroph, are there [page 17↓]differences between algivores and bacterivores, i.e. does the dietary composition determine protist biochemical composition? Between two algivores fed the same algae, are there species-specific differences in the biochemical composition? Those questions should be answered before one decides to evaluate the nutritional quality of protists for zooplankton predators. Only by knowing the biochemical composition of protists and to which extent it can be dictated by dietary composition or by the trophic mode, it is possible to evaluate what really determines the nutritional quality of protists as prey for planktonic predators.
This thesis is based on the four following questions treated in form of 5 chapters:
To answer the first question I investigated the fatty acid, amino acid, and sterol composition of four phagotrophic protist species and their diet: the algivorous ciliates Balanion planctonicum and Urotricha farcta were fed the cryptomonad Cryptomonas phaseolus; the bacterivorous ciliate Cyclidium sp. and the flagellate Chilomonas paramecium were fed bacteria grown on rice corns (Fig. 4).
The second question was addressed by analysing the fatty acid and sterol composition of the flagellate Ochromonas sp. cultured under different trophic modes – autotrophic, mixotrophic, and heterotrophic (Fig. 4).
To evaluate the influence of the biochemical composition of heterotrophic protists on their nutritional quality as prey I performed population growth and reproduction experiments using the rotifer Keratella quadrata as a predator (Fig. 4). I chose K. quadrata as a model predator, because (1) of the ecological significance of this species in many lakes as a potential predator of heterotrophic protists and because of (2) the lack of knowledge concerning the nutritional value of heterotrophic protists for rotifers. The rotifer was [page 18↓]offered the four protist species analysed in chapters 2 and 3, and the biochemical composition of the protists was correlated to population growth rates and to the cumulative number of eggs produced by K. quadrata during the experiments.
The last question was formulated because I found evidence for the potential of some biochemicals of the protists to limit egg production of the rotifers. To test the effect of single biochemical compounds directly I artificially supplementated one protist species – Chilomonas paramecium – with two selected polyunsaturated fatty acids (EPA and DHA) and evaluated the effect of supplemented versus non-supplemented flagellates on population growth and egg production of K. quadrata (Fig. 4).
|Fig. 4 – Outline of the thesis. Upper box: Experiments related to the influence of|
dietary composition (chapters 2 and 3) and trophic mode (chapter 4) on the
biochemical composition of protists. Lower box: Experiments related to the nutritional value of heterotrophic protists for Keratella quadrata (chapters 5 and 6).
The following hypotheses were tested:
Hypothesis 1. Dietary biochemical composition determines the biochemical composition of heterotrophic protists.
To address this hypothesis, I selected four species, which were fed different diets: the algivorous ciliates Balanion planctonicum and Urotricha farcta were fed the cryptomonad Cryptomonas phaseolus. The ciliate Cyclidium sp. and the flagellate Chilomonas paramecium were fed bacteria grown on rice corns. The fatty acid and amino acid composition (chapter 2) as well as the sterol composition (chapter 3) were analysed in both the protists and their diet. Discrepancies between protist and dietary biochemical composition are discussed in light of metabolic features of the protists. Differences between two species fed the same diet are discussed in terms of species-specific metabolic features of the protists.
Hypothesis 2.The trophic mode determines the biochemical composition of the flagellate Ochromonas sp.
To test this hypothesis, the fatty acid and sterol composition of the flagellate Ochromonas sp. grown as autotroph, mixotroph, and heterotroph was analysed (chapter 4). The role of photosynthesis and phagotrophy in determining metabolic patterns of fatty acid and sterol synthesis and accumulation are discussed.
Hypothesis 3. Heterotrophic protists fed different diets have different nutritional quality for a rotifer predator due to differences in their biochemical compounds.
To test this hypothesis, the rotifer K. quadrata was separately offered two algivorous and two bacterivorous protists as prey (chapter 5). The cryptomonad C. phaseolus was used as a good quality control food. The biochemical composition of the protists, which was analysed in the previous chapters, was correlated with population growth rates and the egg numbers of the rotifer.
Hypothesis 4.Chilomonas paramecium can be sucesssfully supplemented with essential fatty acids (EPA and DHA), in order to test the influence of these biochemicals on rotifers’ life-history traits.
Correlation analyses between prey biochemical composition and the cumulative number of eggs produced by the rotifer K. quadrata (see chapter 5), provided evidence for limiting effects of EPA and DHA, among other compounds. I tested a new supplementation technique (Von Elert, 2002) for supplementing the heterotrophic flagellate Chilomonas [page 20↓]paramecium with EPA and DHA (chapter 6). Up to now, this supplementation technique has been only used for supplementing algal cells. The efficiency of the method was tested by supplementing C. paramecium with different EPA and DHA incubation concentrations. To test whether the nutritional quality of C. paramecium was enhanced through EPA and DHA supplementation, the effects of supplemented versus non-supplemented Chilomonas on population growth rates and egg numbers of the rotifer K. quadrata were evaluated.
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