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4  Trophic Mode Influences Fatty Acid And Sterol Composition Of The Chrysophyte Ochromonas sp.

Iola G. Boëchat, Guntram Weithoff, Angela Krüger, and Rita Adrian

(To be submitted to Journal of Phycology)

Keywords: Trophic mode; Ochromonas sp.; Fatty Acids; Sterols

Abstract

We investigated the influence of trophic mode – autotrophic, mixotrophic, and heterotrophic – on the fatty acid and sterol composition of the chrysophyte Ochromonas sp. Total fatty acid concentrations, especially of polyunsaturated fatty acids, decreased as the trophic mode changed from autotrophic via mixotrophic to heterotrophic. Saturated fatty acids were the most abundant fatty acid class found in heterotrophic Ochromonas, whereas polyunsaturated fatty acids were most abundant in autotrophic flagellates. Sterol concentrations were higher in autotrophs and heterotrophs than in mixotrophs, and stigmasterol was the most abundant sterol found in Ochromonas of all trophic modes. Discriminant analyses revealed that polyunsaturated fatty acids are the most efficient parameter discriminating among trophic modes. The analyses pointed out single fatty acids, which more efficiently discriminated among trophic modes. Besides disparities in the fatty acid patterns among Ochromonas of different trophic modes, differential lipid allocation for survival and cellular division in this protist may occur. Such differential investment in survival or reproduction may dictate the trophic mode, which will dominate within a species, under natural conditions. The dominance of one trophic mode over the other may, in turn, affect the nutritional quality available for predators in nature.


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4.1  Introduction

The fatty acid and sterol composition of non-autotrophic protists have been shown to partially rely on dietary composition (Ederington et al., 1995; chapters 2 and 3). Moreover, species-specific metabolism of fatty acids and sterols may differ among heterotrophic protist species fed a same diet (chapters 2 and 3). However, to which extent the fatty acid and sterol profile of a protist species may be dictated by the trophic mode is still unknown.

Living organisms require energy to drive the chemical reactions necessary for maintenance, growth, and reproduction. This energy is normally obtained by two routes, autotrophy and heterotrophy (Sanders, 1991). Light-energy is absorbed by photoautotrophic organisms, which use this energy to synthesize energy-rich organic molecules during photosynthesis. Heterotrophic organisms, otherwise, relay on the organic composition obtained from ingested particles (phagotrophy) or dissolved matter (osmotrophy). A special strategy of life – mixotrophy – combines both the ability of photosynthesis and the uptake of organic matter (Jones, 1994).

Flagellated protists display all basic trophic strategies known among autotrophy and heterotrophy, and also ensure the largest number of species presenting mixotrophy, especially within Chrysophyta (Sanders, 1991). While autotrophs are able to synthesize all biochemical compounds necessary for their metabolic activities, heterotrophs are unable to synthesize most biochemical molecules (Sleigh, 2000). They depend therefore on the uptake of many classes of organic molecules from their environment. Mixotrophs ought to invest in both a photosynthetic apparatus and mechanisms of prey uptake and digestion, which may imply high energetic costs (Tittel et al., 2003), but provide them with an ecological advantage when light or nutrients are limiting (Rothhaupt, 1996a,b). The shift of one growth pathway to another has profound metabolic implications, which drives adaptation (Sleigh, 2000) and may result in altered biochemical composition. Since the nutritional quality of planktonic prey organisms has been associated with the presence of some essential biochemical compounds, such as essential fatty acids and sterols (Brett and Müller-Navarra, 1997; Becker and Boersma, 2003; Von Elert, 2002; Hassett, 2004), the trophic mode may affect the nutritional quality of a prey organism, via changes in the biochemical composition.


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The chrysophyte Ochromonas sp. is a common flagellate in many aquatic systems with a broad range of habitats. It is also able to cope with extreme environments such as acidic water bodies, e.g. mining lakes (Wollmann et al., 2000). Ochromonas species have been commonly identified as mixotrophs in both marine and freshwater plankton (Sanders et al., 1990). Besides that, Ochromonas has been successfully cultured as autotrophs and heterotrophs (Tittel et al., 2003; this study).

In the present study we investigate whether the fatty acid and sterol profile of Ochromonas sp. depends on their trophic mode. We discuss the differences in fatty acid and sterol composition among Ochromonas sp. cultured autotrophically, mixotrophically, and heterotrophically in light of metabolic features of the flagellates and consider the role of such differences in an ecological perspective.

4.2 Methodology

4.2.1 Cultures

Ochromonas was isolated from a mining lake (L 111) in Lausitz, Germany. L 111 is characterized by a low pH of ca. 2.7 and high concentrations of iron and sulphate (Wollmann et al., 2000). Ochromonas sp. is the dominant phytoflagellate in the lake and has its population maximum in summer in the epilimnion (Tittel et al., 2003; Kamjunke et al., in press). The prevailing trophic mode in the field is heterotrophy, potentially subsidised by autotrophy (Tittel et al., 2003). Ochromonas was cultured in a medium reflecting the ion composition of lake L 111 (Bissinger et al., 2000) at a temperature of 20 °C. Three different kinds of cultures were set up in duplicate. For autotrophic growth, the cultures were kept in the light at a light : dark regime of 16:8 h. Although the cultures were not axenic, bacteria growth and abundance were low as the medium was free of organic carbon. For mixotrophic growth, the cultures were under the same light conditions as for autotrophic growth and fed bacteria isolated from L 111. For heterotrophic growth the cultures were treated in the same way but kept in the dark. Bacteria cultures were cultured in the same medium as for Ochromonas sp., except for the addition of glucose to promote bacterial growth. Throughout the text mixotrophy is used for describing the strategy of combining phototrophy and heterotrophy.


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4.2.2  Sample Preparation

Flagellate and bacteria cultures were filtered in replicate on pre-combusted glass fibre filters. GF/C filters (Whatman) were used for collecting Ochromonas samples and GF/F filters (Whatman) were used for collecting bacterial samples. The density of flagellates and bacteria was determined in both the cultures and the filtrates, and the difference was calculated for determining the biomass captured on the filter. Samples were fixed with Lugol´s Iodine and flagellates were counted with an inverted microscope. Bacteria were stained with acridine orange and counted using a fluorescent microscope (Zeiss, Axioscope 2). The carbon content of the bacteria was calculated from size measurements according to Simon and Azam (1989). Ochromonas was measured with a computer-aided image analysis (TSO Thalheim). The volume of the cells was calculated assuming a rotational ellipsoid, and converted into carbon units assuming 0.23 pgC per µm³ cell volume (Kamjunke et al., in press).

4.2.3 Fatty Acid and Sterol Analysis

Filters containing autotrophic, mixotrophic, and heterotrophic Ochromonas as well as the bacterial diet were extracted in chloroform-methanol 2:1, v/v solution (Folch et al., 1957) and homogenized by sonication for 5 min at 5000 cycles per min (Ultrashall-Desintegrator USD 20, VDE Wiss. Gerätebau, Berlin). After sonication, an internal standard was added to the samples (tricosanoic acid, 0.2 mg mL-1 for fatty acids, and 5α-cholestane, 0.2 mg mL-1 for sterols). 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.

For fatty acid analysis, fatty acid methyl-esters (FAME) were formed by addition of 5 mL sulphuric acid (5% v/v) and heating the samples for 4 h at 80°C (Weiler, 2001). An aliquot of 0.2 μL of the samples was finally injected into a Varian Star 3600 CX series gas chromatograph, equipped with a fused silica capillary column (Omegawax 320, SUPELCO, 30 m x 0.32 mm). The following heating program was applied: initial temperature of 180°C (2 min), subsequent heating at 2°C min-1 to 200°C, which was held isothermally for 33 min. Injector and FID detector temperatures were 250°C and 260°C, respectively. Helium was used as a carrier gas. FAMEs identification were made by comparing the retention times with retention times of a calibration standard solution (Supelco FAME Mix 47885–4, PUFA Nr. 3–47085–4 and PUFA Nr. 1–47033) and quantified by comparing the peak areas with the peak area of the internal standard. For presentation, we selected some fatty acids from the total measured pool. It should be kept [page 60↓]in mind that the SAFA, MUFA, and PUFA sums refer to the whole pool of measured fatty acids, i.e. including those not presented.

For sterol analysis 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 (Gordon and Collins, 1982). 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 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 generated mass spectra were compared with mass spectra of a self-generated spectra library (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 along the text and at 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).

4.2.4 Statistical Analyses

Differences in the fatty acid and sterol composition among Ochromonas samples of different trophic modes were tested with one-way ANOVA followed by a pairwise post-hoc Tukey – HSD test (Statistica for Windows, version 5.01, Stat Soft). Percentage data were arcsin root square transformed prior to ANOVA. To single out the fatty acids and sterols that mostly differed among Ochromonas of different trophic modes we performed discriminant analyses considering the [page 61↓]absolute concentrations of the biochemical compounds. Discriminant analysis is an ordination method used to determine which single variables within a pool of variables better discriminate between two or more defined groups. In our case, the analyses were performed to determine which fatty acids and sterols (variables) better separated Ochromonas of different trophic modes (groups). In the case of a multiple group stepwise discriminant analysis (3 different groups – autotrophs, mixotrophs, and heterotrophs), the ultimate calculation correspond to a canonical correlation analysis, which provides the successive functions and canonical roots containing the variables mostly responsible for separating the groups. For the fatty acids we performed the discriminant analyses separately for saturated, monounsaturated, and polyunsaturated fatty acids. Although we do not present the entire pool of fatty acids measured, all fatty acids were considered for the statistical analyses. All statistical procedures were run in Statistica for Windows (version 5.01, Stat Soft).

4.3 Results

4.3.1 Fatty Acids

Fatty acid relative composition varied among Ochromonas of different trophic modes (Fig. 1). Autotrophic Ochromonas exhibited higher percentages of polyunsaturated fatty acids (44%) than mixotrophs (15%) and heterotrophs (16%) (one-way ANOVA F=38.77, P<0.01). Percentages of monounsaturated acids were similar in autotrophic (21%), mixotrophic (28%), and heterotrophic flagellates (31%) (one-way ANOVA F=6.52, P=0.13). Lower percentages of saturated fatty acids were found in autotrophic (28%) when compared to mixotrophic (64%) and heterotrophic (53%) flagellates (one-way ANOVA F=29.79, P<0.01).

Mixotrophic and heterotrophic Ochromonas showed both higher percentages of saturated and lower percentages of monounsaturated fatty acids than those observed in the bacterial diet (one-way ANOVA FSAFA=13.07, P<0.01; FMUFA=27.38, P<0.01). The total percentage of polyunsaturated fatty acids in the flagellates reflected the total percentage found in the bacterial diet (one-way ANOVA F=0.17, P=0.84; Fig.1).


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Fig. 1 – Percentages of saturated (SAFA), monounsaturated (MUFA), and polyunsaturated (PUFA) fatty acids in the total fatty acid content of autotrophic, mixotrophic, and heterotrophic Ochromonas sp. as well as in the bacterial diet of heterotrophic and mixotrophic Ochromonas sp. Significant differences at 95% confidence are represented by an astherisk.

Differences in the absolute concentration of individual fatty acids also reflected the trophic mode (Table 7). Autotrophic and mixotrophic flagellates had much higher concentrations of the saturated acids 16:0, 17:0, and 18:0 than heterotrophic flagellates (Table 7). The acid 20:0 was not found in heterotrophic flagellates, which in turn had minor amounts of 19:0, not detected in their bacterial diet. The concentrations of individual monounsaturated fatty acids such as 15:1, 16:1ω5, and 17:1 were higher in both autotrophic and mixotrophic flagellates than in heterotrophic ones (Table 7). Heterotrophic flagellates were rich in the monounsaturated fatty acid 18:1ω9 also found in high concentrations in the bacterial diet. The acid 22:1ω9 was only found in autotrophic and mixotrophic flagellates. The highest discrepancies were observed for individual polyunsaturated fatty acids. Heterotrophic flagellates had no polyunsaturated acid with a carbon-chain longer than 18 carbons, and the absolute concentrations of all polyunsaturated acids present reflected the concentrations found in the bacterial diet (Table 7). Autotrophic and mixotrophic flagellates in turn contained high concentrations of long-chain PUFA (more than 20 carbon-atoms chain length), especially of 20:3ω6, 21:5ω3 (DPA), and 20:5ω3 (EPA) in case of mixotrophs, [page 63↓]and 22:6ω3 (DHA) in the case of autotrophs (Table 7). Total polyunsaturated fatty acid concentrations in autotrophic and in mixotrophic flagellates were around 18 and 7 times higher than those found in heterotrophic flagellates. Ratios of ω6 to ω3 fatty acids were similar among mixotrophic and heterotrophic flagellates, but lower in autotrophic flagellates (one-way ANOVA F=7.51, P<0.01; Table 7).

The discriminant analyses run separately for saturated, monounsaturated, and polyunsaturated fatty acids provided the fatty acid combinations which discriminated most efficiently among Ochromonas’ trophic mode (Fig. 2).

Fig. 2 – Canonical roots provided by discriminant analyses run for saturated (SAFA), monounsaturated (MUFA), and polyunsaturated fatty acids (PUFA) of autotrophic (●), mixotrophic (▲), and heterotrophic (□) Ochromonas sp.See text and Table 8 for the fatty acids discriminating within each root.

For saturated fatty acids only the first root was significant (Wilk’s Lambda = 0.023; F = 6.16, P<0.001). The three trophic modes were separated at this root, but higher differences were observed between autotrophs and heterotrophs, whereas mixotrophs occupied an intermediate position (Fig. 2). The separation was mostly due to differences in 16:0, 18:0, 20:0, 21:0, and 24:0 (Table 8). The discriminant analysis on monounsaturated fatty acids provided two significant roots (Wilk’s Lambda=0.008; F=6.01, P<0.001). The first root primarily discriminated mixotrophs from the other groups, due to differences in the fatty [page 64↓]acids 15:1 and 20:1ω9 (Table 8). The second root better separated autotrophs from heterotrophs (Fig. 2), due to differences in the monounsaturated 17:1, 20:1ω11, and 22:1ω9 (Table 8). Polyunsaturated fatty acid were the most efficient variables in separating the three trophic modes of Ochromonas, as revealed by the two significant roots extracted by the discriminant analysis (Wilk’s Lambda=0.0004; F=31.09, P<0.001). The first root discriminated mixotrophs from the other trophic groups (Fig. 2). Differences in the polyunsaturated 20:3ω3, 16:3ω4, 18:3ω3, 18:3ω4, and EPA were responsible for separating mixotrophs at this level (Table 8). The second root better separated heterotrophs from autotrophs due to differences in the polyunsaturated acids 16:2ω4, DHA, 18:3ω6, 18:4ω3, 20:3ω6, 22:5ω3, DPA, and 20:4ω3 (Table 8).

4.3.2 Sterols

Total sterol concentration did not differ significantly among Ochromonas of different trophic mode, although significant differences were found for single sterols (Table 9). Overall, mixotrophic Ochromonas had the lowest absolute sterol concentrations among the flagellates (ANOVA, all P<0.05; Table 9). The only exception was stigmasterol, which was similar in mixotrophic and autotrophic flagellates (ANOVA F=4.84, P<0.05). Squalene, the sterol precursor in all sterol biosynthetic pathways, was found in all Ochromonas in significant different concentrations (ANOVA F=48.97, P<0.001). Interestingly, although absolute concentrations in mixotrophic Ochromonas were generally lower than in autotrophs and heterotrophs, the relative amounts did not differ with trophic mode (Fig. 3).

Fig. 3 – Sterol percentages in autotrophic, mixotrophic, and heterotrophic Ochromonas sp. as well as in the bacterial diet of heterotrophic and mixotrophic Ochromonas sp. Significant differences at 95% confidence are represented by an astherisk.


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Hence, stigmasterol was the predominant sterol in all Ochromonas, covering up to 98% of the total sterol pool. In contrast to the relative sterol composition of heterotrophic and mixotrophic flagellates, the bacterial diet presented sitosterol and cholesterol as predominant sterols (Fig. 3). Non-sterol tertepenoids were not observed in Ochromonas or in the bacterial diet.

Two significant roots were extracted from the discriminant analysis run for the sterol absolute concentrations in Ochromonas of different trophic modes (Wilk’s Lambda=0.151; F=4.73, P<0.001) (Fig. 4). The first root separated autotrophs from the other trophic modes (Fig. 4), due to differences in cholesterol and sitosterol concentrations (Table 10). The second root discriminated between heterotrophs and mixotrophs (Fig. 4), mainly due to differences in campesterol and lathosterol (Table 10).

Fig. 4 – Canonical roots provided by discriminant analyses run for the sterol composition of autotrophic (●), mixotrophic (▲), and heterotrophic (□) Ochromonas sp.See text and Table 10 for the sterols discriminating within each root.

4.4 Discussion

The fatty acid profiles in Ochromonas sp. reflected the trophic mode of the flagellates, with concentrations of polyunsaturated fatty acids decreasing when the growth pathway changed from autotrophic via mixotrophic to heterotrophic. A similar pattern was found for several other protists (Vazhappilly and Chen, 1998; Heifetz et al., 2000; Poerschmann et al., 2004), which probably reflect metabolic adaptations imposed to organisms growing on fluctuating environmental conditions of light and nutrients (Sanders et al., 2001; [page 66↓]Hochachka and Somero, 2002). In a study carried out with the chlorophyceae Chlamydomonas sp., also isolated from the mining lake L111, a principal component analysis (PCA) proved to be a good tool for separating groups of fatty acids distinguishing among autotrophic, mixotrophic, and heterotrophic modes (Poerschmann et al., 2004). By using the discriminant analysis we did not only show that polyunsaturated fatty acids are a better indicator of the trophic mode than saturated and monounsaturated fatty acids, as suggested by the PCA in the aforementioned study, but we were also able to identify single polyunsaturated fatty acid accounting for the best separation of Ochromonas grown under different trophic conditions. For instance, the first canonical root of the DA for polyunsaturated fatty acids showed that fatty acids of 18 and 20 carbon-atoms belonging to the ω3 family (e.g. 18:3ω3, 20:3ω3) were the most appropriate to separate mixotrophs from autotrophs. On the other hand, long-chain acids with 20 or more carbon-atoms mainly separated heterotrophs at the second root of the discriminant analysis. Heterotrophic flagellates, in contrast to autotrophs or mixotrophs, are possibly unable to elongate the hydrocarbon chain, given the absence of polyunsaturated fatty acids with more than 18 carbons in heterotrophic Ochromonas.

According to the discriminant analysis, long-chain polyunsaturated fatty acids provided the best discrimination between autotrophs and mixotrophs, suggesting that polyunsaturated fatty acids may be a better indicator of the trophic mode, than saturated or monounsaturated acids. This is not surprising, since energetic costs to build polyunsaturated fatty acids are much higher than those for monounsaturated or saturated ones. Also the presence of a broader number of accessory enzymatic systems is necessary to insert double bounds into the hydrocarbon chain (Ratledge and Wilkinson, 1988; Stryer, 1995). It is reasonable to think that mixotrophs will preferentially use carbon compounds such as glucose obtained from the medium or through bacterial consumption to build structural biomolecules, such as carbohydrates and phospholipids, and to generate energy, instead of synthesizing complex long-chain polyunsaturated fatty acids. On the other hand, the ability to do photosynthesis enables mixotrophs to synthesize some compounds, which can be stored as energy reserve molecules to be used at times of light limitation (e.g. EPA, DHA, and DPA in this study). Some authors suggest that the prime purpose of mixotrophy is to obtain carbon for growth (Bird and Kalff, 1986; Caron et al., 1990; Jones et al., 1993). If so, carbon obtained by phagotrophy may be primarily used for building structural biomolecules such as carbohydrates and phospholipids, whereas photosynthesis may be responsible for generating [page 67↓]substrates for the synthesis of more complex energetic biomolecules, such as triglycerydes.

The higher percentages of saturated fatty acid in mixotrophs and heterotrophs than in autotrophs (Fig. 1) probably reflected the phagotrophic mode of nutrition, with bacterial fatty acids, mostly saturated ones, covering a large fraction of the fatty acid pool of mixotrophs and heterotrophs. A number of factors are expected to affect the degree of phagotrophy including light, dissolved organic carbon concentration, and food concentration (Sanders et al., 1990; Jones et al., 1993). On the other hand, loss of chlorophyll occurs with increased dependency on phagotrophy (Caron et al., 1990; Sanders et al., 1990). The overall lower content of fatty acids found in heterotrophic species compared to the corresponding mixotrophs and autotrophs may indicate changes in extra-chloroplastic membranes as well as loss of chloroplast lipids (Lösel, 1988).

There is a debate as to why flagellates graze bacteria rather than just carring out photosynthesis. Some workers argue that it is a mean of acquiring nutrients for photosynthesis during periods of limitation (Nygaard and Tobiesen, 1993). However, under optimal culture conditions, nutrients are provided in sufficient quantities to support cellular growth and reproduction. Nevertheless, mixotrophic flagellates in our cultures adopted both strategies, as reflected by their fatty acid composition. Mixotrophs contained high concentrations of saturated fatty acids, a typical pattern for organisms feeding on bacteria, but they also yield polyunsaturated fatty acids, which were not found in heterotrophs. Hence, light should be the most important factor driving metabolic patterns of fatty acid synthesis and allocation in autotrophs and mixotrophs. The enzyme acetyl CoA carboxylase catalyses the first step of fatty acid synthesis and its activity depends on both light and the rate of fatty acid synthesis in the chloroplast of higher plants (Post-Beitenmiller et al., 1992). De novo synthesis of lipids in plants also depends on NADPH generated in the light reactions of photosynthesis. Hence it is expected that lipid synthesis rate and the relative allocation of photosynthates into lipids both tend to increase with incubation irradiance (Wainman and Lean, 1992). Our findings on fatty acid composition support this transition from heterotrophy to autotrophy. It is important to note that fatty acid composition is strongly influenced by external temperatures, due to the function of these lipids in regulating membrane fluidity as an adaptation mechanism to temperature shifts (Davidson, 1991). At low temperatures, the fatty acid pattern may be primarily dictated via membrane fluidity regulation rather than by the trophic mode (Poerschmann et al., 2004).


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The sterol pattern identified for Ochromonas sp., with stigmasterol as the predominant sterol, was already described for Ochromonas danica (Halevy et al., 1966), Ochromonas malhamensis (Avivi et al., 1967), and Ochromonas sociabilis (Goodwin, 1974). Gershengorn et al. (1968) found porifesterol (the 24β-epimer of stigmasterol) to be the predominant sterol in O. malhamensis (98% of the total sterol pool). The presence of sitosterol (or its 24β-epimer – clionasterol), cholesterol, brassicasterol, and ergosterol was also shown in previous studies (Gershengorn et al., 1968; Tsai et al., 1975). Interestingly, except for stigmasterol, sterol composition in mixotrophic flagellates did resemble that of bacteria, whereas autotrophs and heterotrophs showed rather similar composition. Possibly, mixotrophy leads to a shortage in sterol synthesis and storage. The high efforts of a mixotroph to run both an autotrophic and a phagotrophic metabolism may cause higher basic metabolic costs and limit reproductive rates as compared to more specialized organisms, such as heterotrophs and autotrophs (Rothhaupt, 1996a). One of those metabolic costs may be decreased synthesis rates of some sterols, which may have analogous function to other molecules, such as fatty acids. In mixotrophic Ochromonas, sterol synthesis may have been mostly directed to stigmasterol synthesis, as stigmasterol concentrations in mixotrophs were similar to those found in autotrophs and heterotrophs. Stigmasterol has been found to be a major sterol in phagotrophic and phototrophic protists (chapter 3, Tables 5 and 6), suggesting its importance for protists. Apart from stigmasterol synthesis, synthesis of other sterols may have been reduced to the minimum necessary to guarantee cellular functioning in mixotrophs.

Differences in cholesterol concentrations separated autotrophs from heterotrophs, whereas the phytosterol sitosterol separated autotrophs from mixotrophs. Cholesterol synthesis requires molecular oxygen and NADPH (Stryer, 1995) and is thus supposed to be strongly regulated by photosynthesis and respiration rates (Stryer, 1995). On the other hand, synthesis of phytosterols is regulated by the presence of some precursors, which differ from those needed for cholesterol synthesis (Moreau et al., 2002). The presence and abundance of such phytosterol precursors may have accounted for the observed differences in sitosterol concentrations in autotrophs versus mixotrophs. Discriminant analysis on sterols was by far less efficient to differenciate trophic mode of Ochromonas under laboratorial conditions when compared to the analysis on polyunsaturated fatty acids. This means that sterol concentration may be an inadequate parameter to distinguish trophic mode at non-limiting nutrient conditions. This hypothesis is supported by the finding that, despite differences in sterol absolute concentrations, relative amounts remained the same, independently of the trophic mode (Fig. 3). It [page 69↓]remains the question, how efficient are these lipids in differentiating trophic mode under conditions of nutrient limitation.

Interestingly, discriminant analyses on monounsaturated and polyunsaturated fatty acids separated mixotrophic flagellates from the other trophic modes, while sterols separated autotrophs from heterotrophs and mixotrophs at the first root. This may be an effect of differentiated patterns of lipid synthesis and allocation in Ochromonas growing under different trophic modes. Given the differences in function and biosynthetic pathways between fatty acids and sterols, these lipid classes may be expected to respond differently to environmental factors such as light and nutrients (Parish and Wangersky, 1987; Smith and D’Souza, 1993).

Lipid allocation may reflect differential ecological strategies in protists. Higher percentages of the polyunsaturated fatty acids 20:4ω6 and 18:3ω3 have been found to be allocated into triglycerides instead of phospholipids in autotrophic protists of the genus Cryptomonas (Boëchat, unpublished data). In heterotrophic protists (e.g. Cyclidium sp) higher percentages of these PUFA were found in the phospholipid fraction (Boëchat, unpublished data). Triglycerides are very important energy storage molecules, whereas phospholipids are essential components of membranes. A differential allocation of PUFA into triglycerides or phospholipids may suggest a differential investment into reproduction or growth in protists. Under light limitation lipid allocation in membranes may be stimulated to enable higher division rates. In fact, heterotrophic growth of Ochromonas did not deviate from mixotrophic growth (0.32 ± 0.06 day-1 and 0.27 ± 0.02 day-1, respectively; mean ± SE) whereas autototrophic growth was close to zero (0.07 ± 0.07 day-1) under sufficient nutrient conditions (Tittel et al., 2003). Autotrophy would than imply lower division rates and high energy allocation if nutrients are not limiting. The ecological theory predicts that specialization should be the most successful strategy for survival under non-limiting conditions (Rothhaupt, 1996a). The question remains to be answered, whether survival may represent a more important investment than high division rates for autotrophic protists under non-limiting conditions in nature.

The fact that differences in the fatty acid and sterol composition found for Ochromonas sp. were probably caused by the trophic mode may strongly affect the nutritional quality of this protist as prey. The fact that one trophic mode may predominate over the others in extreme ecosytems, such as mixotrophy in antartic polar lakes (Bell and Laybourn-Parry, 2003), may have profound nutritional consequences for predators living in such ecosystems.


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