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3  Sterol Composition Of Four Freshwater Heterotrophic Protists An Their Diet

Iola G. Boëchat, Angela Krüger and Rita Adrian

(Journal of Chemical Ecology, under revision)

Keywords:Heterotrophic protists; Sterol composition; Sterol metabolism; Triterpenoids; Consumer-diet interactions


Heterotrophic protists represent an important link in aquatic food webs as they transfer energy and biochemical matter from their bacterial and algal prey to mesozooplankton predators. Our aim was to understand how the sterol composition of the diet affects the sterol composition of four freshwater heterotrophic protists. We analyzed the sterol composition of two algivorous protists (Balanion planctonicum and Urotricha farcta) and two bacterivorous protists (Cyclidium sp. and Chilomonas paramecium) as well as of their diet – the cryptomonad Cryptomonas phaseolus for B. planctonicum and U. farcta, anda mixture of bacteria growing on two different rice types for Cyclidium sp. and C. paramecium. The sterol composition of the protists did not generally resemble that of their diet, since some discrepancies were observed. Ergosterol was the main sterol in C. phaseolus, whereas stigmasterol was dominant in both B. planctonicum and U. farcta. The diets consisting of “bacterial + rice” were both rich in cholesterol and sitosterol, whereas cholesterol and stigmasterol were the major sterols in Cyclidium sp. and C. paramecium respectively. The occasionally higher sterol concentrations in the protists than in their diet suggest high sterol accumulation efficiencies by the protists. Moreover, ergosterol synthesis is likely to occur in C. paramecium. We conclude that the dietary sterol composition influences the sterol composition of the four freshwater heterotrophic protists; however, species-specific differences in sterol metabolism ultimately determine the sterol composition of the protists.

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3.1  Introduction

Generally, the biochemical composition of a consumer is expected to resemble that of its diet. This was already shown for different mesozooplankton predators in food quality studies involving fatty acid (e.g. Harvey et al., 1997) and amino acid composition (e.g. Guisande et al., 2000). If this is also the case for heterotrophic protists, the biochemical composition of their diet will affect or even determine the biochemical composition of the protists. Consequently it may affect protist nutritional quality for zooplankton predators if we consider the potential limiting role of some biochemical compounds for zooplankton (DeMott and Müller-Navarra, 1997; Becker and Boersma, 2003; Ravet et al., 2003). If this is not the case, discrepancies in the biochemical composition between heterotrophic protists and their diet may result from differences in prey capture, ingestion, and digestion rates as well as in the capability of heterotrophic protists to synthesize biochemical compounds. Indeed, initial investigations of the biochemical composition of four freshwater heterotrophic protists showed that the fatty acid and amino acid composition of the protist species generally resembled that of their diet. Nevertheless, some polyunsaturated fatty acids were observed in the protists, which were not present in their diet (Boëchat and Adrian, submitted). With regard to nutritional quality, those heterotrophic protists can be considered as prey of upgraded quality for their zooplankton predators, since the polyunsaturated fatty acids referred to are known to be essential for many zooplankton predators (Brett and Müller-Navarra, 1997; Ravet et al., 2003).

Along with essential polyunsaturated fatty acids and amino acids, sterols have recently gained much attention as biochemical components which confer food quality of planktonic prey (Von Elert, 2002; Hassett, 2004; Martin-Creuzburg and Von Elert, 2004). Sterols share, along with phospholipids, a structural function in membranes, but in terms of polarity they are grouped with triacylglycerols in the neutral lipids (Parrish, 1999). In addition to controlling membrane fluidity and permeability, sterols also form sexual hormones, sterol alkaloids, and act as vitamins. In some higher plants, sterols have specific functions in cell proliferation, signal transduction, and also as modulators of the activity of membrane-bound enzymes (Volkman, 2003). Additionally, sterols have also been used as markers in marine and freshwater sediments, since some of them are typical for certain taxonomic groups (Harvey and McManus, 1991; Elhmmali et al., 2000; Hudson et al., 2001). While cholesterol is the predominant sterol in animal cells, plant membranes are rich in several types of ‘phytosterols’, which are similar in structure to cholesterol but include a methyl or ethyl group at [page 41↓]carbon-atom 24. Additionally, phytosterols are also thought to stabilize plant membranes (Moreau et al., 2002).

Much of the information concerning sterols in plankton organisms relates to the sterol composition of the phytoplankton (see Patterson, 1991 for a review). Algal sterol composition is generally similar to that observed in higher plants, but algal classes differ in their predominant sterols. By far the widest range of sterols has been found in Chlorophyceae (Patterson, 1991) including (22E)-ergosta-5,7,22-trien-3β-ol (ergosterol), cholest-5-en-3β-ol (cholesterol), (22E)-ergosta-5,22-dien-3β-ol (brassicasterol), [24(24’)E]-stigmasta-5,24(24’)-dien-3β-ol (fucosterol), and many others (Volkman et al., 1994). Although the occurrence of sterols in Cyanophyceae is controversial (Volkmann, 2003), cholesterol, 24α-ethylcholest-5-en-3β-ol (sitosterol), and brassicasterol were found in this group (Volkman, 1986), while (22E)-(24S)-24-methylcholesta-5,22-dien-3β-ol (epibrassicasterol) was the major sterol found in Cryptophyceae (Goad et al., 1983; Gladu et al., 1990). Nearly all dinoflagellates contain cholesterol and 4,23,24-trimethylcholest-22-trien-3β-ol (dinosterol) as the most common sterols (Mansour et al., 1999).

In sharp contrast to the vast literature on algal sterols, the sterol composition of bacteria and heterotrophic protists has been only marginally been considered. Except for some methanotrophic species (Schouten et al., 2000), bacteria are generally considered not to produce sterols, or only in non-significant amounts (but see Kohl et al., 1983; Sorkhoh et al., 1990). Despite studies on marine species (Harvey and McManus, 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 freshwater flagellates of the genus Chilomonas, which contained 24α-ethylcholesta-5,22(E)-dien-3β-ol (stigmasterol) as the predominant sterol (Patterson, 1991), and the freshwater 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). This alcohol as well as hopanoid terpenes has also been found in marine ciliates (Harvey and McManus, 1991; Harvey et al., 1997), and may exhibit equivalent regulatory functions as those attributed to sterols (Ferguson et al., 1975; Ourisson et al., 1987).

To determine whether the sterol composition of freshwater heterotrophic protists resembles the dietary sterol composition, we analyzed the sterols of four protist species (the ciliates Balanion planctonicum, Urotricha farcta, Cyclidium sp., and the flagellate Chilomonas paramecium) as well as the sterols of their diet (the [page 42↓]cryptomonad Cryptomonas phaseolus, which served as prey for B. planctonicum and U. farcta, andtwo different bacterial assemblages growing on different rice types – unpolished rice for Cyclidium sp. and polished rice for C. paramecium). We found discrepancies between protists and diet in both the composition and the relative and absolute concentrations of sterols. We discuss these discrepancies in light of possible metabolic mechanisms already described for other protists, including biosynthetic pathways and sterol conversion mechanisms. The present study suggests that heterotrophic protists are able to efficiently accumulate, convert, or even synthesize some sterols, especially phytosterols. Thus, our results support the hypothesis that freshwater heterotrophic protists are organisms of upgraded food quality, with respect to the essentiality of some sterols for zooplankton predators.

3.2 methodology

3.2.1 Cultures

The ciliates Balanion planctonicum (average biovolume 3256 ± 1331 μm3) and Urotricha farcta (2778 ± 1707 μm3) were cultured in WC medium (Guillard and Lorenzen, 1972) in weekly diluted batch cultures incubated at 17°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 feeding spectrum of both ciliates includes bacteria, small algae (Cryptomonas, Ochromonas), and organic debris (optimal particle size 8 – 12 μm; Müller, 1991; Foissner et al., 1999). Nevertheless, our ciliate cultures were only able to efficiently grow when fed on C. phaseolus, thus providing sufficient biomasses for biochemical analysis.

The flagellate Chilomonas paramecium (403 ± 288 μm3) and the bacterivorous ciliate Cyclidium sp. (1315 ± 617 μm3) were cultured in Volvic water (a spring water, poor in minerals, sold worldwide by Société des Eaux de Volvic, Puy- de-Dôme, France) at 18 ± 1°C under a 16:8 h light : dark regime. Chilomonas was fed on a mix of bacteria grown on polished rice (referred to as “bacteria + polished rice” diet, since the protists might have been able to ingest rice particles along with bacteria in the medium) and Cyclidium was fed a mix of bacteria grown on unpolished rice (referred to as “bacteria + unpolished rice”, for the same [page 43↓]reason). Bacteria and small rice particles could not be completely separated (see sample preparation and extraction). The reason why we used two different bacterial assemblages was because Cyclidium was only successfully cultured on bacteria growing on unpolished rice1, the same being the case for Chilomonas growing on polished rice. Attempts to cultivate both species on bacteria growing on the other respective type of rice as well as on wheat corns were not successful. The ciliate cultures were able to grow for a couple days before collapsing, but never yielded biomasses sufficiently high for biochemical analysis. Sterol analyses of dried rice samples of both types revealed significant differences (tested using the Mann-Whitney U-test, Statistica for Windows, version 5.01, Stat Soft). Thus, an ultimate comparison of the sterol composition between our Cyclidium and Chilomonas cultures is not possible. However, the fact that the two protist species were fed bacterial assemblages growing on rice of different biochemical composition provides additional information regarding species-specific differences on sterol metabolism and synthesis in these species.

To investigate if the biochemical composition of heterotrophic protists resembles that of their diet, one could either (1) compare different species to their respective diets or (2) compare several cultures of one protist species raised on different diets. We applied the first approach because our species had very specific diet requirements and did not grow on alternative diets. Further, our study aimed at acquiring basic information about the biochemistry of a broader number of stable protist cultures, to facilitate future research on the nutritional quality of these protists for zooplankton predators.

Although our results suggest a series of possible mechanisms involved in the biochemical metabolism of the different protist species, a firm conclusion about the extent in which dietary composition determines biochemical composition of heterotrophic protists is hardly possible. For doing that, the next step should be to investigate the biochemical composition of single protist species raised on different diets or grown under different trophic modes.

3.2.2 Sample Preparation and Extraction

Sterol analyses were performed for heterotrophic protist species and their diet, and are given as percentage on the total sterol concentration (referred to as relative concentrations) and as carbon-specific concentrations (referred to as absolute concentrations). As organisms may show similar relative sterol concentrations, [page 44↓]despite different absolute concentrations, both concentration forms are considered when comparing protists and their diet. Additionally, the sterol composition of both polished and unpolished dried rice corns was analyzed and the results are given as µg per milligram dry weight.

For biomass estimates, samples of ciliates, flagellates, and the algal diet were fixed with Lugol’s solution and counted under a stereomicroscope at x20 magnification. Bacteria samples (1 mL) were taken from all protist cultures as well as from the algal and “bacteria + rice” diets. Samples were then fixed in 2% formaldehyde, filtered onto 0.2 μm pore size black polycarbonate membranes, stained with 4,6-diamidino-2-phenylindol (DAPI), and counted with an epifluorescent microscope (excitation wavelength 450 – 490nm combined with an FT 510 beam splitter and a LP 520 suppression filter) (Porter and Feig, 1980). Carbon concentrations in heterotrophic protists and their diet were derived from cell volume estimates using carbon : biovolume conversion factors of 0.10 pgC μm-3 for the alga Cryptomonas (Montagnes, 1994), 0.19 pgC μm-3 for heterotrophic protists (Putt and Stoecker, 1989), and 0.125 pgC μm-3 for the bacteria (Pelegri et al., 1999).

Due to their similar cell dimensions, Balanion and Urotricha could not be completely separated from their algal food via filtration and/or centrifugation. Cryptomonas biomass accounted for 4–16% of the carbon biomass in the protist samples for analyses. To a lesser extent, this was also the case for the protist cultures growing on bacteria and rice corns, although they were previously filtered successively over a 10 μm and a 5 μm mesh. Due to this previous filtration, bacterial biomass was reduced to less than 20% of the total carbon content in the cultures. Nevertheless, rice particles and bacterial cell smaller than 5 μm were still present in the samples for sterol analyses.

Since a complete separation of heterotrophic protists and their diet was not possible, we adopted a subtraction method, in order to accurately estimate the sterol composition and concentration of the protists. By knowing the mass-specific sterol contents of the diet, obtained from repeated analysis of either the algal or bacteria + rice cultures, we determined the sterol concentration of the protists indirectly through subtraction. By reducing dietary biomass to less than 20% of the total biomass in the protist samples for analysis, dietary sterol concentrations in these samples corresponded to less than 3% of the total concentration measured for the sample. Hence, subtracting dietary biomass concentrations from the overall concentration measured in the incompletely separated “protist + diet” samples minimized small biases due to incomplete [page 45↓]separation. Further, the accuracy of the correction was guaranteed by analyzing a large number of samples (at least 2–3 samples per culture, for a minimum of four cultures per protist species).

For sterol analyses, 300 to 700 mL of each culture of heterotrophic protists and the algal diet were filtered in 2–3 replicates on pre-incinerated (550°C, 4 h) GF/C Whatman glass fiber filters. Samples of bacterial cultures were collected on GF/F Whatman glass fiber filters. The analyses were performed for at least four different batches of each protist-diet combination and the individual dietary cultures. We collected protist cells at the end of the exponential growth phase because algal and bacterial densities were minimal in the cultures by this time (see sample preparation). Nevertheless, protist cells were still within the exponential growth phase. During this growth phase the food quality of green algae (Scenedesmus) had been found to be the highest for Daphnia (Sterner, 1993). For the sterol analyses of both rice types in dried form, 10 mg of homogenized rice sample were analyzed in triplicate. Rice samples and filters containing the organisms were immediately extracted with chloroform-methanol 2:1, v/v (Folch et al., 1957) and homogenized by sonication for 10 min at 5000 cycles per min. (Ultrashall-Desintegrator USD 20, VDE Wiss. Gerätebau, Berlin). After sonication, an internal standard was added to the samples (5α-cholestane, 0.2 mg mL-1). The samples were then allowed to extract for 3 h at 20°C.After extraction, samples were dried under nitrogen flux and promptly stored at –20°C until analysis.

3.2.3 Sterol Analyses

Extracts were subjected to alkaline hydrolysis (saponification) by the addition of 5 mL 1 N potassium hydroxide (KOH) solution in 80% methanol (MeOH), followed by heating at 80°C for 30 min. After cooling, 10 mL distilled water and 5 mL n-hexane were added to the samples (Gordon and Collins, 1982). The n-hexane phase containing the free neutral lipids were separated and subsequently silylated by adding 25 μL Bis (trimethylsilyl)trifluoroacetamide + 1% trimethylchlorosilane (BSTFA), 75 μL pyridine, and heating the sample at 60°C for 30 min (Breteler et al., 1999). Sterol silylether derivatives were analyzed using a gas chromatograph (Agilent 6890) equipped with a mass selective detector (Agilent 5973-N) and a fused silica capillary column (HP-5MS; 60 m x 0.32 mm x 0.25 μm). The carrier gas (helium) was held constant at 1.3 mL min-1. The temperature of the PTV (programmed temperature vaporization) inlet – operating in splitless mode – was 300°C (initial temperature 100°C, 720°C min-1). The temperature of the detector interface was 280°C. The following temperature [page 46↓]program was employed: 150°C initial temperature for 3 min, than heating at 4°C min-1 up to 300°C, and maintained for 40 min. Sterol silylether derivatives were identified by their retention times and their mass spectra in full scan mode (SCAN) previously calibrated with individual sterol standards (Sigma-Aldrich). The mass spectra of the detected sterols were compared with the mass spectra of our self-generated spectra library stored in a dedicated data system (Agilent Chemstation). Sterol silylether derivatives were quantified by selective ion monitoring (SIM) at the two most intensive ions at the molecular ion cluster. Calibration curves ranged between 0.04 and 0.4 μg sterol per mL injected sample. Sterols are provided as percentages of the total sterol pool (relative amounts) and as absolute concentration per carbon biomass, whenever a commercial standard was available. We refer to the sterols by their trivial names throughout the text and in the tables, in order to facilitate reading and comparisons. However, we indicated the classic nomenclature when first referencing to the sterol common name (there are currently two main nomenclatures following the IUPAC-IUB recommendations; see Moreau et al. [2002] for a list of synonyms currently used). Although not quantified in terms of absolute concentrations, some additional neutral lipids were identified by their mass spectra and are provided as percentages of the neutral lipid fraction.

3.2.4 Statistical Analyses

Considering the non-normality of data distribution, differences in sterol absolute concentrations and relative amounts (percentages) between the heterotrophic protists and their diet were tested with the Mann-Whitney U-test (Statistica for Windows, version 5.01, Stat Soft). Differences in the sterol composition between polished and unpolished rice samples were tested with the Mann-Whitney U-test as well.

3.3 Results

In contrary to our hypothesis, the sterol composition of the heterotrophic protists did not generally resemble that of their diet (Tables 5 and 6). Discrepancies in sterol occurrence and concentration between heterotrophic protists and their diet were observed for Balanion and Urotricha, and especially for Chilomonas and Cyclidium. Considering relative concentrations, ergosterol was the predominant sterol in the algal diet Cryptomonas (42.8%), followed by brassicasterol (15.9%), [page 47↓]cholesta-5,24-dien-3β-ol (desmosterol, 12.4%), stigmasterol (9.8%), cholesterol (6.5%), and 24α-ethylcholestan-3β-ol (stigmastanol, 5.1%) (Table 5). Stigmasterol was the predominant sterol in Balanion (49.3%), along with brassicasterol (20.6%), desmosterol (18.4%), (24R)-24-methylcholest-5-en-3β-ol (campesterol, 5.2%), and cholesterol (1.9%). Interestingly, ergosterol was not detected in Balanion as well as 3β-hidroxy-5α-cholestane (dihydrocholesterol). Balanion contained higher relative amounts of stigmasterol, campesterol, desmosterol, 24-stigmasta-5,7,24(28)-trien-3β-ol, and of another non-identified sterol than its algal diet Cryptomonas (Mann Whitney U-test, P<0.05; Table 5). Stigmasterol was also the predominant sterol in Urotricha (26.7%), followed by ergosterol (20.9%), brassicasterol (13.9%), desmosterol (12.2%), sitosterol (9.0%), and cholesterol (7.5%) (Table 5). Urotricha presented higher relative amounts of stigmasterol, sitosterol, 24-stigmasta-5,7,24(28)-trien-3β-ol, and of the non-identified sterol than its algal diet Cryptomonas (P<0.05; Table 5).

The diet consisting of “bacteria + polished rice” displayed a smaller spectrum of sterols than the diet consisting of “bacteria + unpolished rice” (Table 6). Cholesterol was the predominant sterol (65.8%) identified in “bacteria + polished rice” samples (Chilomonas’ diet), followed by low relative concentrations of dihydrocholesterol (13.5%), stigmastanol (10.9%), and stigmasterol (6.1%). Sitosterol was the predominant sterol (49.9%) found in “bacteria + unpolished rice” samples (Cyclidium’s diet), followed by cholesterol (17.3%), campesterol (12.7%), stigmasterol (12.2%), and stigmastanol (6.4%). Other sterols found in low relative concentrations were 5α-cholest-7-en-3β-ol (lathosterol, 0.5%), desmosterol (0.5%), dihydrocholesterol (0.3%), and ergosterol (0.3%). Unlike the sterol composition found in “bacteria + unpolished rice”, campesterol, sitosterol, and ergosterol were not detected in the samples of “bacteria + polished rice” (Table 6). Differences in the sterol composition between both “bacteria + rice” samples basically stemmed from the differences found for the two types of rice (Fig. 1). This should be considered when comparing our results with natural bacterial assemblages. The significantly higher concentrations of campesterol, stigmasterol, sitosterol, and stigmastanol in unpolished rice (P<0.05; Fig. 1) were probably due to sterols attached to the shell of this rice type. All other sterols were detected in both rice types in similar concentrations, while ergosterol and brassicasterol were not detected in any rice type. Due to the discrepancies in the sterol composition of the two “bacteria +rice” assemblages, a direct comparison of the two protist species fed “bacteria + rice” was not possible (i.e. Cyclidium and Chilomonas).

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Fig. 1 – Absolute sterol concentrations (mean ± S.D.) in polished versus unpolished rice used as food sources for the bacterial diet in the cultures of Cyclidium sp. and Chilomonas paramecium. Asterisks represent significant differences at 95% confidence (P<0.05, Mann-Whitney U-test).

In contrast to the sterol composition observed in its “bacteria + polished rice” diet, Chilomonas contained stigmasterol (39.7%) and sitosterol (32.1%) as the main sterols, followed by campesterol, ergosterol, cholesterol, desmosterol, and lathosterol (Table 6). Interestingly, campesterol, sitosterol, and ergosterol were not found in the diet of Chilomonas. On the other hand, although dominant in its diet, cholesterol, and stigmastanol were of minor importance in Chilomonas (P<0.05; Table 6). Chilomonas did not contain dihydrocholesterol, although this sterol was observed in the “bacteria + polished rice” diet. Relative amounts of stigmasterol were higher in Chilomonas than in its diet consisting of “bacteria + polished rice” (P<0.05; Table 6). Moreover, Chilomonas contained brassicasterol and the 24-stigmasta-5,7,24(28)-trien-3β-ol, which were not observed in the “bacteria + polished rice” diet. Cyclidium fed “bacteria + unpolished rice” produced a different sterol composition than that observed for Chilomonas, which was fed “bacteria + polished rice” (Table 6). Cholesterol was the major sterol found in Cyclidium (70.6%) followed by sitosterol, stigmasterol, and dihydrocholesterol. Nevertheless, all sterols detected in Cyclidium were also observed in its diet, although not necessarily in the same proportions (Table 6). For example, cholesterol was the second dominant sterol in the [page 49↓]diet (17.3%), but represented the major fraction of the sterol pool in Cyclidium (70.6%). Interestingly, dihydrocholesterol was found in the algal and in both “bacteria + rice” diets, but it was not detected in the heterotrophic protists, except for Cyclidium.

When considering the absolute sterol concentration – per carbon units – in the heterotrophic protists and their diet, some discrepancies were observed (Tables 5 and 6, values in parentheses). The concentrations of campesterol and stigmasterol were much higher in Balanion than in the algal diet, while stigmasterol and sitosterol reached higher concentrationsin Urotricha than in Cryptomonas (P<0.05).Cryptomonas had higher concentrations of desmosterol and ergosterol than Urotricha (P < 0.05). However, overall sterol concentrations in Cryptomonas (26.8±21.2 μg mgC-1), Balanion (27.6 ± 2.2 μg mgC-1), and Urotricha (20.6 ± 12.1 μg mgC-1) did not differ significantly (P>0.05).

Discrepancies between protist and dietary absolute sterol concentrations were especially evident for Cyclidium and Chilomonas. Chilomonas had higher concentrations of all sterols than its diet (“bacteria + polished rice”) except for cholesterol, desmosterol, and stigmastanol (P<0.05; Table 6). The opposite was found for Cyclidium, which had lower concentrations of all sterols compared with those in its diet (“bacteria + unpolished rice”), with the exception of the 27-carbon sterols cholesterol, dihydrocholesterol, desmosterol, and lathosterol (P<0.05; Table 6). The overall sterol concentration in Chilomonas (46.1 ± 19.1 μg mgC-1) was higher than in its “bacteria + polished rice” diet (16.5 ± 8.3 μg mgC-1), while the overall sterol concentration in Cyclidium (37.7 ± 20.7 μg mgC-1) was much lower than in its “bacteria + unpolished rice” diet (90.4 ± 5.6 μg mgC-1).

Two sterol precursors, the alcohol terpenoid tetrahymanol, and two tertepenoids of the hopane class (diplopterol and diploptene) were identified by their mass spectrometric patterns. Squalene, the sterol precursor in all biosynthetic pathways (Fig. 2), was detected in similar concentrations in all heterotrophic protists and in their diets (Tables 5 and 6). Cycloartenol was identified in Cryptomonas, Urotricha, Chilomonas, and Cyclidium. Cycloartenol is an intermediate precursor in the biosynthesis of phytosterols (Moreau et al., 2002). The alcohol tertepenoid Tetrahymanol, the hopanoids hopan-22-ol (diplopterol), and hop-22(29)-ene (diploptene) were detected in Cyclidium (11.5 %, 30.2 %, and 2.5 % of the total neutral lipids fraction). Trace amounts of hopan-22-ol were found in Cryptomonas and Urotricha (1.1 % of the total neutral lipids).

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3.4  Discussion

Along with certain essential fatty acids, amino acids, and other biochemical compounds, sterols can be viewed as nutritional quality indicators, since some of them are not synthesized de novo in animal tissues, and thus must be obtained from the diet. Consequently, the diet of a consumer is likely to determine its sterol composition, unless the consumer is able to modify dietary sterols and/or is able to synthesize sterols, given the presence of intermediate precursors. Here, we show that the dietary sterol composition influences the sterol composition of heterotrophic protists, since most sterols found in the diet were also found in the protists. For instance, high relative and absolute desmosterol concentrations were found in both Balanion and Urotricha as well as in their algal diet Cryptomonas. Stigmasterol was present in high relative and absolute concentrations in both Cyclidium and Chilomonas as well as in their diet consisting of “bacteria + rice”. However, the predominant sterols in the protists – based on relative concentrations – were not necessarily the same as those observed in their diet. For example, stigmasterol comprised about 39 and 61% of the overall sterol composition in Urotricha and Balanion, respectively, but only 9.8% of the total sterols found in the algal diet Cryptomonas. Moreover, except for Cyclidium absolute sterol concentrations were generally higher in the protists than in their respective diets (Tables 5 and 6). Finally, the occurrence of some sterols in the heterotrophic protists (e.g. campesterol, ergosterol, sitosterol, brassicasterol, and 24-stigmasta-5,7,24(28)-trien-3β-ol in Chilomonas) along with the absence of others (e.g. ergosterol in Balanion) characterizes disparities from the expected resemblance between consumer and dietary composition. These discrepancies in both sterol composition and concentration between heterotrophic protists and their diet may stem from species-specific differences in mechanisms which are involved in sterol metabolism, like sterol accumulation, assimilation, and synthesis. Evidence for such differences concerning fatty acid and amino acid metabolism have already been found for the same protist species (chapter 1).

In the following we singled out the sterols whose occurrence and concentration between heterotrophic protists and their diet were highly different. We then discuss these discrepancies in light of possible metabolic and functional mechanisms based on evidence provided by our results along with mechanisms already described for other protist species.

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The elevated absolute and relative stigmasterol concentrations especially in Balanion, Urotricha, and Chilomonas may have reflected the age of our cultures. In plants, the molar ratio of stigmasterol to other phytosterols was shown to increase during senescence (Stalleart and Geuns, 1994). By analyzing protist cultures originating from the end of the exponential growth phase, we may have included cells entering a “senescence” stage. On the other hand, the high relative and absolute stigmasterol concentration in the heterotrophic protists, despite low stigmasterol occurrence in their diets, makes us to ponder whether our protists are efficient in ingesting, assimilating, and/or synthesizing stigmasterol. High stigmasterol amounts in the protists suggest the importance of this phytosterol for growth within heterotrophic protists, as already demonstrated for the ciliate Trimyema compressum (Holler et al., 1993). Previous studies suggested that stigmasterol undertakes cholesterol analogous function in controlling permeability of cellular membranes (Piironen et al., 2000). Further efforts will be necessary to verify the role of stigmasterol in protist cells.

Ergosterol has been found to be the major sterol of several flagellates (Nes and McKean, 1977), amoebas (Raederstorff and Rohmer, 1987a,b), and some parasitic protozoa (Dixon et al., 1972; Furlong, 1989). Ergosterol, followed by brassicasterol, was the predominant sterol in our Cryptomonas cultures. Similar findings of brassicasterol and ergosterol as major sterols in other cryptomonads have already been reported (Goad et al., 1983). Ergosterol was the second major sterol found in our Urotricha. Cryptomonas can probably synthesize ergosterol, whereas Urotricha more probably obtained its ergosterol from Cryptomonas. However, ergosterol synthesis in Urotricha should not be excluded, since we detected the sterol precursor “cycloartenol” in Urotricha. 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, the Δ5,7 sterol ergosterol was the predominant sterol, with cycloartenol serving as a precursor (the numbers after the symbol Δ represent the position of the double bonds in the ring A of the sterol molecule).

In all sterol biosynthetic pathways, squalene is the 30-carbon precursor (Fig. 2), being a derivate from either mevalonate (the classic Bloch-Lynen mevalonate or MVA pathway) or pyruvate and glyceraldehyde (recently elucidated methylerythritol-phosphateor MEP pathway; Rohmer et al., 1996). Hence, we expected to find squalene in all organisms we analyzed, as actually observed. In animals, fungi, and dinoflagellates, squalene is converted to lanosterol, which is the precursor of most 27-carbon sterols, such as desmosterol and cholesterol (Volkman, 2003) (Fig. 2).

[page 52↓]

Fig. 2 – Sterol biosynthetic pathways. The Mevalonate pathway (MVA-pathway) and the recently elucidated Methylerythritol-Phosphate pathway (MEP-pathway) culminate in the synthesis of squalene. In animals, fungi, and dinoflagellates, squalene is converted to lanosterol, which is the precursor of desmosterol and cholesterol. In higher plants, most microalgae, and many heterotrophic protists squalene is converted to cycloartenol, which in turn is converted to phytosterols, like campesterol and ergosterol (see text for references).

In higher plants, most microalgae, and in many heterotrophic protists squalene is converted to the intermediate dimethylsterol cycloartenol through cyclization steps (e.g. Giner et al., 1991) (Fig. 2). Cycloartenol is then converted to cycloartanol, which is later converted to 28- and 29-carbon desmethyl phytosterols, such as campesterol and ergosterol. The presence of cycloartenol in our Cryptomonas and Urotricha samples may suggest ergosterol synthesis through conversion of cycloartenol in these species. Interestingly, ergosterol was not detected in Balanion, which probably metabolizes this sterol into another Δ5 sterol, according to the conventional sterol biosynthetic sequence Δ7 →Δ5,7→Δ5 (Goad, 1981).

Campesterol, sitosterol, ergosterol, and brassicasterol were detected in Chilomonas, despite their absence in its “bacteria + polished rice” diet. Direct [page 53↓]assimilation of campesterol and sitosterol through ingestion of rice particles from the medium could explain the presence of these sterols in Chilomonas, since they were detected in the analyses of dried polished rice. The reason why those sterols were detected in the samples of dried polished rice but not in the “bacteria + polished rice” samples (medium samples) is unclear. Maybe the concentration of those sterols in the “bacteria + polished rice” samples were below the detection limits of our method. Although we could not quantify it, the biomass of rice particles present in the “bacteria + polished rice” samples might have been very low, as the cultures were successively filtered over 10 and 5 μm prior to the analyses. However, the dried polished rice and the “bacteria + polished rice” samples did not contain ergosterol and brassicasterol. The conversion of a Δ5 sterol, such as cholesterol into ergosterol in Chilomonas could explain this discrepancy. The conversion of Δ5 to Δ5,7 sterols is already known to take place in some heterotrophic protists (Goad, 1981) and de novo synthesis of ergosterol has recently been demonstrated to occur via incorporation of leucine as a major precursor in some trypanosomatids (Ginger et al., 2000). Alternatively, cycloartenol present in Chilomonas may have served as an intermediate precursor in the synthesis of ergosterol and brassicasterol in this flagellate (see the discussion above for Cryptomonas and Urotricha). The presence of cycloartenol in Urotricha and Chilomonas is an interesting finding and suggests that some protists may be able to synthesize phytosterols. Studies involving a broader number of protists and labeled sterols are still necessary to test this hypothesis.

Cholesterol, the main sterol in most crustaceans (Pakrashi et al., 1989), molluscs (Gordon, 1982), and mammals seems to play a secondary role in the organisms we studied, as suggested by its low relative concentrations in the heterotrophic protists and their diets analyzed here (but see “bacteria + polished rice” and Cyclidium, Table 6). The function of cholesterol in regulating membrane fluidity is well established (Bloch, 1992). Nevertheless, it has been generally accepted that phytosterols, detected in large amounts in our protists, also stabilize cell membranes (Moreau et al., 2002). The presence of high phytosterol concentrations in all heterotrophic protist species we analyzed, especially stigmasterol, may suggest analogous regulatory function to cholesterol functions in the cell membranes of heterotrophic protists.

In addition to sterols, the triterpenoids tetrahymanol, diplopterol, and diploptene represented an important fraction of the neutral lipid fraction found in Cyclidium. Ciliates as well as bacteria and other microorganisms have been found to be rich in tetrahymanol, a neutral lipid structurally similar to cholesterol, and also in the hopanoids diplopterol and diploptene (see Ourisson et al., 1987 for a review). [page 54↓]These microbial lipids are derived from the tertepene metabolism and are thought to play the same role in prokaryotic membranes that cholesterol does in eukaryotic membranes (Ourisson et al., 1987). Recent studies have demonstrated that some ciliates fed bacteria are able to de novo synthesize tetrahymanol (Harvey and McManus, 1991). In the ciliate Tetrahymena pyriformis, the presence of cholesterol inhibited tetrahymanol synthesis (Conner et al., 1968), and the authors suggested that tetrahymanol may carry out a function similar to cholesterol in the plasma membrane of T. pyriformis. In our study, cholesterol and tetrahymanol were both found in Cyclidium. Tetrahymanol synthesis was shown to be hampered by exogenous supply of stigmasterol in the ciliate Trimyema compressum (Holler et al., 1993). Stigmasterol was found in low concentrations in Cyclidium, but in high concentrations in all other species we investigated (Balanion, Urotricha, Chilomonas), which in turn did not contain tetrahymanol. It could be possible that stigmasterol may hamper tetrahymanol synthesis in Balanion, Urotricha, and Chilomonas but not in Cyclidium. Further research is needed in order to determine whether the presence of particular phytosterols interfere with the synthesis of other neutral lipids in protist cells.

Recent studies have revealed the importance of free-living freshwater heterotrophic protists as both consumers of bacteria and algae, and as prey for metazoan grazers (Stoecker et al., 1986, De Biase et al., 1990; Stoecker and Capuzzo, 1990; Wickham et al., 1993; Sanders et al., 1996; Adrian and Schneider-Olt, 1999; Adrian et al., 2001; Burns and Schallenberg, 2001; Mohr and Adrian, 2002a,b).Therefore, heterotrophic protists constitute a trophic link between primary producers and higher trophic level consumers. Depending on their ability to modify the biochemical composition obtained from their diet, heterotrophic protists can affect the transfer of matter and energy to higher trophic levels at an early stage in aquatic food webs. Here we analyzed several different consumer – diet combinations as a first step to understand the biochemical composition of different heterotrophic protists fed a known diet. The next step should include a single protists growing on different food resources to elucidate how the biochemical composition of heterotrophic protists responds to changes in dietary composition. Given the difficulty in cultivating our protist species on different food resources we may probably have to select other protist species, which are easier to culture on different diets.

Overall, our study contributes to the understanding of sterol composition in freshwater heterotrophic protists and suggests that species-specific variation occurs. We have discussed some possible mechanisms which may underlie the mismatch between protist and dietary sterol composition, including direct [page 55↓]assimilation of medium sterols for Cyclidium and Chilomonas, high ingestion and incorporation efficiencies of dietary sterols as well as sterol synthesis in Balanion, Urotricha, and in Chilomonas. Our results may stimulate further studies involving single labeled sterols in heterotrophic protists and their diet. Such studies are necessary to elucidate the mechanisms of sterol synthesis and metabolism in freshwater heterotrophic protists, thus contributing to our comprehension on the role of heterotrophic protists in the transfer of biochemical matter in aquatic food webs.

Footnotes and Endnotes

1 The Cyclidium culture analysed in the present study is not the same considered in the chapter 3. This new culture, obtained from Prof. K.O. Rothhaupt, was not able to grow efficiently on polished rice corns.

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