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5  Evidence For Biochemical Limitation On Reproduction Patterns Of The Rotifer Keratella quadrata Fed Freshwater Heterotrophic Protists

Iola G. Boëchat and Rita Adrian

(Submitted to Journal of Plankton Research)

Keywords: Biochemical composition; Nutritional quality; Freshwater heterotrophic protists; Rotifers


Recent studies have demonstrated that the trophic mode shapes the nutritional quality of heterotrophic protists for rotifers. However, the biochemical factors that determine protist nutritional quality for rotifers remain unclear. We evaluated population growth rates and egg numbers of the rotifer Keratella quadrata fed either algivorous or bacterivorous protist species. The cryptomonad Cryptomonas phaseolus, considered a good quality prey, was used as control. Population growth rates and egg numbers of K. quadrata were correlated with single biochemical compounds (fatty acids, amino acids, sterols) of the heterotrophic protists. Feeding on algivores and the alga Cryptomonas phaseolus resulted in positive population growth rates and high egg numbers of K. quadrata, whereas feeding on bacterivores resulted in moderate egg production but no population growth. K. quadrata egg numberswere significantly correlated with protist biochemical composition, including several polyunsaturated fatty acids, sterols, and the amino acid leucine. No significant relationship was observed between growth rates of the rotifers and protist biochemistry, suggesting that rotifer growth and reproduction probably have different nutritional requirements. Factors apart from the biochemical components analysed, like nutrient ratios, vitamins, trace-elements, as well as synergetic and antagonistic interactions between chemical substances may have additionally influenced protist nutritional quality for K. quadrata.

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5.1  Introduction

Rotifers represent an important fraction of the zooplankton biomass in lakes (Arndt, 1993), and their prey size spectrum (< 1 – 200 µm, Pourriot, 1977; Bogdan and Gilbert, 1987) covers a wide range of microbes, including bacteria and heterotrophic protists. In lakes during spring, the abundance of rotifers peaks shortly after the abundance peak of heterotrophic protists (Sommer et al., 1986); it is well known that predation by rotifers may potentially regulate protist population in situ (Bogdan et al., 1980; Carrick et al., 1991; Gilbert and Jack, 1993; Mohr and Adrian, 2002a). However, despite the ecological significance of heterotrophic protists – rotifer interaction, it is still not fully understood how heterotrophic protists contribute to growth and reproduction of rotifer populations.

The influence of protist-prey on the life history of rotifers has been recently reported (Gilbert and Jack, 1993; Mohr and Adrian, 2001; Weisse and Frahm, 2001; Mohr and Adrian, 2002b). Enhanced reproduction and growth rates of Brachionus calicyflorus Pallas 1766 were observed when offering the rotifers a mixture of algae and algivorous ciliates. However, no positive effect was reported when the rotifers were offered a mixture of algae and bacterivorous ciliates (Mohr and Adrian, 2002b). Moreover, enhanced growth of rotifer populations of the genus Keratella was observed when the rotifers were fed a mixture of autotrophic cryptophytes and algivorous ciliates (Weisse and Frahm, 2001). Taken together, these results suggest that the diet source of heterotrophic protists – algae or bacteria – shapes their nutritional quality for rotifers. Indeed, previous studies have shown that dietary biochemical composition influences protist fatty acid and amino acid (chapter 2) as well as the sterol composition (chapter 3). Nevertheless, even when fed the same prey the biochemical composition of predators can differ species-specifically. Such disparities in the biochemical composition between heterotrophic protists and their diet suggest species-specific differences in protist metabolism, which could additionally affect nutritional quality of heterotrophic protists for mesozooplankton predators.

Studies on the nutritional quality of planktonic prey organisms have emphasized the importance of essential biochemical compounds in promoting growth and reproduction of zooplankton (e.g. Ahlgren et al., 1990; Brett and Müller-Navarra, 1997; Weers and Gulati, 1997; Becker and Boersma, 2003). Among the substances that have received considerable attention are 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 [page 72↓]fatty acids (PUFA), like arachidonic acid (20:4ω6) and linoleic acid (18:2ω6). 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 ω3 and ω6 fatty acids, amino acids (Kleppel et al., 1998; Guisande et al., 2000), and more recently the sterol composition of prey organisms (Von Elert et al., 2003; Hasset, 2004), have been reported to be nutritionally important for zooplankton predators. Other studies have suggested that prey mineral composition and stoichiometry, especially the P:C and N:C ratios, may play a more important role in zooplankton nutrition (e.g. Sterner et al., 1992; DeMott et al., 1998; Plath and Boersma, 2001).

Here, we investigated how algivorous and bacterivorous protists contribute to the nutrition of rotifers, and specifically, which biochemical components of the protists may be responsible for their quality as prey for rotifers. For these purposes, we performed population growth and reproduction experiments using the common species Keratella quadrata as predatorand two algivores and two bacterives as prey. Cryptomonas phaseolus, considered a good quality alga, as well as treatments without prey served as experimental controls. The biochemical composition of the heterotrophic protists (analysed in the chapters 2 and 3) was correlated with the results on population growth rates and egg numbers of K. quadrata. To our knowledge, this study is the first to consider a wide range of biochemical parameters and to explore nutritional quality of heterotrophic protists for rotifers. Our study highlights the importance of heterotrophic protists as prey for rotifers and suggests some biochemical factors influencing their nutritional quality for K. quadrata.

5.2 Methodology

5.2.1 Cultures

The algivorous ciliates Balanion planctonicum (3256 ± 1331 μm3, average biovolume ± SD) and Urotricha farcta (2778 ± 1707 μm3) were cultured in WC medium (Guillard and Lorenzen, 1972) in frequently diluted batch cultures [page 73↓]incubated at 17 ± 1°C under a 12:12 h light : dark regime. The ciliates were fed the cryptomonad Cryptomonas phaseolus (392 ± 125 μm3), obtained from the Algal Collection of the University of Göttingen, Germany, and cultured in WC medium at 17 ± 1°C under a 16:8 h light : dark regime.

The bacterivorous ciliate Cyclidium sp. (1315 ± 617 μm3) and the flagellate Chilomonas paramecium (403 ± 288 μm3) were cultivated in Volvic mineral water (a spring water, poor in minerals, sold worldwide by Société des Eaux de Volvic, Puy-de-Dôme, France) and were fed a bacterial assemblage grown on previously autoclaved rice corns. Bacteria generally comprised around 20 – 40 % of the total carbon content in the cultures of bacterivores. The cultures were kept at 18 ± 1°C under a 16:8 h light : dark regime.

The rotifer K. quadrata was originally isolated from the lake Müggelsee in Berlin, Germany, and cultivated in WC medium in frequently diluted batch cultures incubated at 18 ± 1°C under a 16:8 h light : dark regime. The rotifers were routinely grown on the cryptomonad C. phaseolus.Henceforth, prey and predator species are referred to by their genus names only.

5.2.2 Reproduction and Population Growth Experiments

We conducted a series of feeding experiments in which reproduction patterns and population growth of Keratella fed algivorous and bacterivorous protists were investigated. By choosing protist species of similar size, shape and mobility, we have minimized the influence of morphological and behavioural features of the prey on population growth and egg numbers produced by Keratella. Functional response experiments for Keratella fed each protist were conducted previously to the feeding experiments (data not shown) in order to determine the ILL (incipient limiting level) for Keratella fed each protist, which was the basis for determining prey concentrations used during the feeding experiments. Through the functional experiments we assured that the rotifers indeed ingest the heterotrophic protists. Four prey treatments were conducted, each one with five replicates: Treatment 1: Balanion + Cryptomonas (2 x 103Balanion cells mL-1 + 5 x 103Cryptomonas cells mL-1); Treatment 2: Urotricha + Cryptomonas (2 x 103Urotricha cells mL-1 + 5 x 103Cryptomonas cells mL-1); Treatment 3: Cyclidium + bacterial assemblage (3.5 x 103Cyclidium cells mL-1); and Treatment 4: Chilomonas + bacterial assemblage (5 x 103Chilomonas cells mL-1). Control treatments with Cryptomonas as sole prey (5 x 103 cells mL-1) as well as controls without prey were run in parallel.

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Bacterivorous protists were separated from their bacterial prey by filtering the cultures through a 10 μm net. Due to their similar size dimensions, we could not separate algivorous ciliates from their algal food, which explains why treatments 1 and 2 contained mixed diets of ciliates and Cryptomonas. To test whether differences in rotifer growth rates and egg production in these treatments were due to the additional presence of the algivorous ciliates, algal density in the treatments was kept as in the control treatment with Cryptomonas as sole prey in densities above the ILL (5 x 103 cells mL-1). As both the ciliates and the algae were offered in concentrations above the incipient limiting level (ILL, 0.8 – 1.5 mgC L-1) this excluded a major limitation by food quantity instead of food quality. It is important to note, that the ILL for Keratella fed algivorous ciliates was adequately estimated using cilate food suspensions containing the same algal densities as used for the treatments with Cryptomonas as sole prey. Under such conditions, Keratella efficiently ingested the algivorous ciliates (in a rate of 8 ciliates rotifer-1 h-1), even though the algal density present was sufficient to sustain rotifer’s populations.

Feeding experiments were run in macrotiter plates (final volume per chamber 12 mL), incubated at 18 ± 1°C under a 16:8 h light : dark regime. Twenty well-fed K. quadrata individuals without eggs were initially transferred into each chamber. The rotifers were separated from their algal diet by filtration through a 70 μm mesh and resuspended in WC medium 12 h prior to the experiments. The addition of cetyl-alcohol pellets reduced the superficial tension in the experimental chambers, thus preventing rotifer mortality through adherence to the surface film. The rotifers received fresh prey suspensions daily over a 5 d experimental period. As a common practice in such feeding experiments, the predators are daily transferred into a new chamber with fresh prey suspension. However, due to the high fragility of Keratella to manual handling, we decided not to remove them from the experimental chambers. Instead, we added fresh prey suspensions to the chambers, starting the experiments with an initial volume of 4 mL and adjusting the prey densities daily to a final volume of 12 mL at day 5. Rotifers and eggs were enumerated at x20 magnification (stereoscope). Population growth rates (r) of the rotifers were calculated assuming exponential growth according to:

Where Nt is the number of rotifers at the end of the experimental time interval (Δt) and Nt-1is the number of rotifers at the beginning of the experimental time interval[page 75↓] (Δt). Keratella numbers were log-transformed to assure the normality of data distribution. The overall growth rate for the whole experimental period (five days) was calculated as a mean of daily growth rates. Egg numbers are presented as cumulative curves, which represent the daily increase in egg numbers, fitted through non-linear regression models using the program Table Curve 2D, version 5.0.

5.2.3 Statistical Analyses

Differences in total population growth rates of Keratella were tested for significance using one-way ANOVA followed by the post-hoc Dunnett’s t-test, which pairwise compares experimental treatments against control treatments. Increases in rotifers’ egg numbers in experimental treatments were tested against control treatments using the non-parametric Mann-Whitney U-test. Rotifers’ growth rates and cumulative egg numbers were correlated with prey absolute and relative amounts of fatty acids, amino acids, and sterols using the non-parametric Spearman rank correlation method in cases where positive growth and egg production of the rotifers were observed. All statistical tests were perfomed using Statistica for Windows, version 5.01 (Stat Soft). A detailed description of the biochemical analyses as well as the biochemical data used in the present study for investigating nutritional quality of heterotrophic protists are provided elsewhere (chapters 2 and 3). For this part we additionally calculated the relative amounts of fatty acids and amino acids, based on the concentration data presented in chapter 2.

5.3 Results

5.3.1 Growth and Reproduction Experiments

Population growth rates of Keratella fed algivores (Treatments 1 and 2) and the alga Cryptomonas as a sole prey (Treatment 3) were significantly higher than population growth rates of Keratella fed bacterivorous protists (Treatments 4 and 5; one-way ANOVA, F=24.01, Dunnetts t-test P<0.001; Fig. 1). No significant differences were observed when comparing population growth rates of Keratella fed bacterivorous protists versus growth rates in the control treatment without prey (one-way ANOVA, F=24.01, Dunnetts t-test, all P>0.05; Fig. 1).

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Fig. 1 – Population growth curves of Keratella quadrata feeding on the alga Cryptomonas phaseolus, the heterotrophic protists,and in the control treatments without prey. Log numbers of K. quadrata individuals (average ± upper S.D.) represent a total of at least 5 replicates per treatment. Same letters indicate similar population growth rates (r) of the rotifers when compared to the growth rates obtained in the control treatments with C. phaseolus and without prey (one-way ANOVA followed by the Dunnetts t-test).

Curves of cumulative egg numbers of Keratella were similar for rotifers fed Balanion and Cryptomonas (Treatments 1 and 3; Mann-Whitney U-test, P>0.05; Fig. 2). However, the shape of the curves differed slightly, suggesting different daily accumulation rates (see Table 11 for equations).

Fig. 2 – Cumulative curves of egg numbers of Keratella quadrata feeding on the alga Cryptomonas phaseolus, the heterotrophic protists, and in the control treatments without prey. Curves were fitted according to non-linear regression models (see Table 11 for equations). K. quadrata cumulative egg numbers (average ± upper S.D.) represent a total of at least 5 replicates per treatment.

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A diet of Urotricha (Treatment 2) resulted in lower cumulative egg numbers than those observed on a diet of Cryptomonas and Balanion (Mann-Whitney U-test, P<0.05), but higher than those observed in the control treatment without prey (Mann-Whitney U–test, P<0.05). Cumulative egg numbers of Keratella in the Cyclidium and Chilomonas treatments (Treatments 4 and 5) were lower than those observed on a diet of Balanion or Cryptomonas as sole prey (Treatments 1 and 3; Mann-Whitney U-test, P<0.05), but they were higher than the cumulative egg numbers observed in the control treatment without prey (Mann-Whitney U-test, P<0.05).

5.3.2 Relationship between Prey Biochemical Composition and Keratella’s Growth and Reproduction

Significant positive correlations were found between cumulative egg numbers and both the absolute concentration and the relative amounts of total polyunsaturated fatty acids (PUFA), γ-linolenic acid (18:3ω6), 20:3ω6, arachidonic acid (20:4ω6), and docosapentaenoic acid (22:5ω3) (Table 12). Significant positive relationships were additionally found for the absolute EPA (20:5ω3) concentrations and the relative amounts of the PUFA 22:2ω6, 22:5ω6, and DHA. Significant negative correlations were observed between Keratella’s egg numbers and the absolute concentration and relative amount of linoleic acid (18:2ω6), the absolute concentration of stearidonic acid (18:4ω3), and the relative amount of 22:3ω6 (Table 12).

Desmosterol and ergosterol absolute concentrations and relative amounts as well as stigmastanol relative amounts were positively correlated with the cumulative egg numbers of Keratella (Table 13). A significant positive relationship was observed between the relative amounts of the amino acid leucine and the cumulative egg numbers of Keratella (Table 13).

Because algivorous ciliates and the alga Cryptomonas were the only prey capable of sustaining positive population growth rates of the rotifers throughout the experiments, we performed non-parametric Spearman rank correlation between population growth rates and biochemical composition considering only feeding experiments on algivorous ciliates and Cryptomonas. Nevertheless, we did not detect any significant correlation between Keratella’s population growth rates and the fatty acid, amino acid, and sterol composition of the algivorous ciliates nor the alga Cryptomonas (Spearman Rank correlation, all P>0.05).

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5.4  Discussion

Interestingly, protist prey species grown on different dietary sources, but showing similar concentration ranges of several biochemical compounds, led to significantly different population growth rates and egg production of Keratella. For instance, relative amounts of polyunsaturated fatty acids and absolute sterol concentrations in the bacterivores Cyclidium and Chilomonas were partially within the same range, or even higher (e.g. cholesterol, sitosterol, and campesterol) than the concentrations found in at least one algivorous species (chapters 2 and 3). However, bacterivores led to negative population growth rates, which did not significantly differ from the growth rates in control treatments without prey (Fig. 1). This finding is in contrast to previous reports on good nutritional quality of bacterivorous protists for daphnids (Sanders and Porter, 1990; Lair and Picard, 2000). On the other hand, inadequate nutritional quality of bacterivorous protists has already been demonstrated for the rotifer B.calyciflorus fed the flagellate C.paramecium or the ciliate Tetrahymena pyriformis Ehrenberg 1830 (Mohr and Adrian, 2002b), and for cladocerans fed the ciliates Cyclidium glaucoma Müller, 1773 (Bec et al., 2003) and Cyclidium sp. (De Biase et al.,1990). The opposite patterns observed for Keratella’s growth rates between feeding experiments with algivores and bacterivores, despite the similar biochemical profiles partially exhibited by algivores and bacterivores, led us to conclude that prey biochemical composition was not constraining Keratella’s population growth. When feeding on bacterivores, the biochemical parameters we investigated had obviously no primary influence on rotifer population growth rates, since these were as negative as in the control treatments without prey. Even in cases of positive population growth rates of Keratella (feeding on Balanion, Urotricha, and Cryptomonas) we did not observe significant relationships with prey biochemical composition, supporting a possible non-limitation of population growth by biochemical compounds. The alga Cryptomonas may have compensated for deficiencies in the algivorous ciliates, since no significant differences were observed among population growth rates of Keratella fed either the algivores or Cryptomonas only. However, the slight tendency of higher population growth rates of Keratella fed Balanion compared to Keratella fed Cryptomonas and Urotricha, along with significant higher cumulative egg numbers supported by a diet of Balanion than those provided by Urotricha, suggests a supplementary effect of Balanion to a sole Cryptomonas diet. It remains unclear if the supplementation effect of Balanion was due to [page 79↓]its biochemical features or to other factors, such as mineral composition, feeding behaviour of the rotifers, or interactions among different food quality aspects.

On the other hand, the significant positive relationship between Keratella’s egg numbers and prey biochemical composition suggests a primary influence of prey biochemistry on Keratella’s nutrition. For instance, the positive relationship with both the absolute concentrations and the relative amounts of total polyunsaturated fatty acids suggests the importance of those compounds for Keratella’s reproduction. Particularly the relationships with essential ω3 polyunsaturated fatty acids, such as EPA (20:5ω3), DPA (21:5ω3), and DHA (22:6ω3) find credence in the literature, as those fatty acids have been found to limit growth and reproduction of a number of planktonic predators, such as daphniids (DeMott and Müller-Navarra, 1997; Von Elert, 2002; Bec et al., 2003) and copepods (Jónasdóttir, 1994). Feeding experimentsof Keratella on protist cells supplemented with single fatty acid will be necessary to support this finding.

Interestingly, significant positive relationships were detected with ω6 fatty acids, suggesting the importance of those fatty acids for Keratella’s reproduction. A limited ability for elongation and desaturation of 18-ω6 precursors such as γ-linolenic acid (18:3ω6) into arachidonic acid (20:4ω6) has been suggested for the rotifer Brachionus plicatilis (Lubtzens et al., 1985). It would be interesting to follow the fate of dietary ω6 fatty acids in Keratella’s metabolism in order to test the ability for PUFA synthesis in this species and to understand of the role of ω6 PUFA for the rotifer’s nutrition. On the other hand, a possible limitation by ω3 fatty acids is suggested indirectly because of significant negative relationships between Keratella’s cumulative egg numbers and the absolute and relative amounts of linoleic acid (18:2ω6) and the relative amounts of 22:3ω6. Due to their molecular resemblance, ω3 and ω6 fatty acids may compete for the same bonding sites in membranes (Singer, 1994). Hence, an increase of some ω6 fatty acids and the resultant decrease in the relative amount of their concurrent ω3 fatty acids may lead to a shortfall of active bonded ω3 fatty acids, which then become limiting even though they are available in high absolute concentrations. Since we used living prey organisms instead of artificial diets, complex co-limitation mechanisms involving synergistic and/or antagonistic interactions among biochemical substances may have affected the correlations with single fatty acids.

Sterol limitation of growth and reproduction of zooplankton predators has been found to be mainly caused by dietary cholesterol shortage (Von Elert et al., 2003; Hasset, 2004). Cholesterol plays an important role in membrane stabilization and acts as a precursor in hormone synthesis (Moreau et al., 2002). Cholesterol [page 80↓]deficiency has been associated with decreased growth of copepods (Hasset, 2004) and crustaceans (Von Elert et al., 2003), and with retarded development of crustacean larvae (Teshima, 1991). Using our experimental setup, no direct influence of cholesterol on population growth and reproduction patterns of Keratella was detected. However, we did observe a strong positive relationship between Keratella’s cumulative egg numbers and the sterol desmosterol. Except for acting as a precursor of cholesterol, no particular biological function has been attributed to desmosterol in invertebrates (Teshima, 1991). The positive relationship found between desmosterol and Keratella egg numbers may suggest an important function of desmosterol in this rotifer, perhaps as a precursor of cholesterol. Assuming that cholesterol concentrations were limiting rotifers reproduction, Keratella may have converted prey desmosterol to obtain cholesterol. Conversion of desmosterol into cholesterol has been described for a wide range of organisms, including crustaceans (Teshima et al., 1982), molluscs (Knauer et al., 1998), and insects (Ikekawa, 1985).

The significant positive relationship with ergosterol, a sterol with 29 carbon atoms (C-29) only detected in Cryptomonas, Urotricha, and Chilomonas (chapter 3, Tables 5 and 6), suggests its influence on Keratella’s reproduction. Ergosterol is a typical sterol of fungi, and its functions have been mainly elucidated in studies on yeast. High dietary ergosterol levels have been associated with enhanced growth and reproduction of crustacean larvae (Kanazawa et al., 1971; Teshima and Kanasawa, 1986), suggesting a possible dealkylation at the carbon C-24 of ergosterol into cholesterol. Teshima (1982) proposed a pathway of dealkylation of C-28 e C-29 sterols via desmosterol to cholesterol in crustaceans. Whether or not ergosterol is converted to cholesterol in Keratella, thus supplying prey cholesterol deficiencies is still an open question.

The positive relationship observed between Keratella egg numbers and the relative amounts of stigmastanol – the only saturated sterol we found in the studied prey organisms, is rather difficult to explain. It may indicate a limitation originating from increased sterol : stanol ratios. The effect of another phytostanol – sitostanol – on the solubilization of cholesterol was examined under in vitro conditions (Mel'nikov et al., 2004). Free sitostanol was shown to reduce the concentration of cholesterol in artificial diets (micelles) via a dynamic competition mechanism (Mel'nikov et al., 2004). However, since stigmastanol was the only saturated sterol observed in our samples, such a competition mechanism is unlikely in our prey organisms.

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Since we found only minor differences in the concentrations of amino acids among prey organisms (i.e. those in tryptophan and leucine in Cryptomonas and Balanion, respectively), we did not expect to find significant associations between prey amino acid and Keratella life history parameters.Top predators, such as the rotifer Brachionus plicatilis Müller 1786, showed rather constant amino acid composition when fed different algal species or the yeast Saccharomyces cerevisaeMeyen and Hansen, 1883 (Frolov et al., 1991). Guisande et al. (1999) concluded that the selective retention of amino acids in Euterpina acutifronsDana, 1848 was based on a chemical homeostasis of essential amino acids in this copepod. In our study we found a positive correlation between the cumulative egg numbers of Keratella and leucine relative amounts. Leucine has been found to be an essential amino acid involved in egg production and larval development in many species including fish (Fyhn, 1989; Dayal et al., 2003), crustacean (Reddy, 2000), copepods (Laabir et al., 1999), and insects (Chang, 2004).

The fact that biochemical compounds were not correlated with population growth rates but with cumulative egg numbers suggests that biochemical requirements for population growth differ from those for egg production, in the case of Keratella fed heterotrophic protists. This is in accordance with the stoichiometric theory that states that growth, reproduction, and survivorship have different metabolic demands (Sterner and Hessen, 1994). In our study, the observed effects of biochemical compounds were related to Keratella reproduction patterns, measured as the cumulative numbers of produced eggs. Effects of fatty acid and sterol may be related to egg production and maintenance until hatching. Increased egg production of the copepod Acartia tonsa Dana, 1848 was found to be positively influenced by the ω3 fatty acid composition of its algal diet (Jónasdóttir, 1994). Teshima et al. (1982) showed that cholesterol is indispensable for the metamorphosis from nauplii to post-larvae and the survival of the larval prawn Panulirus japonica Von Siebold, 1824.

Although algivorous and bacterivorous protists ensured partially similar biochemical composition (chapters 2 and 3), only feeding on algivores resulted in positive population growth rates of Keratella. In this case, the differences in nutritional quality of algivores versus bacterivores could not be explained by the investigated biochemical compounds. Our results suggest 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: an organism will become limited by whatever resource that is in lowest supplies compared to the organism’s needs. In a recent study, Becker and Boersma (2003) showed that limitation effects of an essential fatty acid on Daphnia life history [page 82↓]could only be detected below a specific prey nutrient threshold. Above this threshold, the daphniids were limited by nutrient ratios. It seems that, depending on the development stage of the daphniids, mineral limitation may occupy a higher position than biochemical compounds do in limiting daphniid nutrition. However, such limitation effects are more apparent in studies in which limitation of combined mineral and biochemical components is artificially induced, generally by supplementing prey organisms with one component in detriment to another. Unfortunately, despite the undoubted importance of those experimental studies, such extreme limitation conditions may not necessarily correspond to natural conditions. In nature, predator nutrient deficiencies resulting from ingesting a low quality prey may be compensated for by other strategies, like mixed ingestion of different prey. Here, we found support for biohchemical limitation in a trophic relationship between a rotifer species and different heterotrophic protist prey, whose original biochemical or mineral composition were not previously modified or artificially supplemented. Our data suggest a primary role of protist biochemistry for Keratella reproduction but only a secondary role for Keratella population growth, although we could not identify the primary limitation factors in this later case.

Interestingly, biochemical limitation effects were only evident for Keratella’s reproduction but not for population growth. In nutritional quality studies, whereby prey composition is kept unaltered, the necessity of considering synergetic and/or antagonistic aspects of simultaneous mineral and biochemical limitation for predators becomes clear, and may have been responsible for the absence of significant correlations between Keratella’s population growth and prey biochemistry in our study. Further, the influence of non-quantified chemicals, like vitamins and trace-elements, and especially of synergetic and/or antagonistic interactions among biochemical compounds cannot be excluded.

In a broader ecological sense, our study emphasizes heterotrophic protists as an alternative source of essential substances for mesozooplankton predators and an important link between microbial and classic food-webs. However, requirements of the predator may be not completely supplied by feeding on bacterivorous protists, as evidenced by the negative population growth rates of Keratella fed Chilomonas or Cyclidium.

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