Diet quality impact on growth, reproduction and ... - Oxford Journals

JOURNAL OF PLANKTON RESEARCH

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Diet quality impact on growth, reproduction and digestive activity in Brachionus calyciflorus ´ 1,2*, JIR ˇ I´ NEDOMA2, JAROMI´R SED’A2,1 AND JAROSLAV VRBA1,2 MARTINA SˇTROJSOVA 1

´ FACULTY OF SCIENCE, UNIVERSITY OF SOUTH BOHEMIA, BRANISˇOVSKA

´ CH INSTITUTE OF HYDROBIOLOGY, NA SA´DKA

7,

31,

ˇ ESKE´ BUDE ˇ JOVICE, CZECH REPUBLIC AND 2BIOLOGY CENTRE AS CR, CZ-37005 C

ˇ ESKE´ BUDEˇJOVICE, CZECH REPUBLIC CZ-37005 C

*CORRESPONDING AUTHOR: [email protected] Received March 13, 2008; accepted in principle June 24, 2008; accepted for publication June 26, 2008; published online June 27, 2008 Corresponding editor: John Dolan

We examined population growth rate, reproduction and phosphatase activity in digestive tracts of individual Brachionus calyciflorus fed by P-replete (molar C:P ¼ 107) or P-depleted (C:P ¼ 920) algal food supplied at three carbon (C) concentrations (1, 2, and 4 mg C L21). Rotifer growth rate was significantly influenced by P content, whereas the effect of algal C concentration was not significant. Both algal P content and C concentration had a significant effect on the egg ratio. The individual amount of chlorophyll a (chl-a) was considered as a proxy for the ingested algal food occurring in the rotifer’s gut and phosphatase activity in the gut likely reflected variable individual assimilation of P under different food treatments. The amount of both chl-a and phosphatase activity in the rotifers fed by P-replete algae significantly increased with an increase in C concentration in the food suspension. In contrast, the amount of chl-a significantly decreased with an increase in food concentration, and phosphatase activity did not change significantly over different food quantity in the rotifers fed by P-depleted algae. Thus, the hypothesis that P-deficient rotifers use preabsorptive regulation by changing activity of the digestive enzymes is not supported by the data.

I N T RO D U C T I O N Rotifers often dominate the freshwater zooplankton during a short time period of the year and the size of their populations changes rapidly (Sommer, 1989). Since phosphorus (P) represents the main limiting element in freshwater systems (Sterner and Hessen, 1994; Sterner, 1997; Sterner and Elser, 2002), it likely becomes limiting for a specific period of individual development for fast-growing rotifers (Elser et al., 2000a). Results of recent studies have shown that not only food quantity but also food quality influence the rotifer growth rate, e.g. nutrient deficiency in algae grown under nutrient limitation (Rothhaupt, 1995; Conde-Porcuna, 2000; RamosRodrı´guez and Conde-Porcuna, 2003; Jensen and Verschoor, 2004; Lu¨rling, 2006; Hessen et al., 2007) or biochemical composition of protists (Boe¨chat et al., 2005; Boe¨chat and Adrian, 2006).

Elser et al. (Elser et al., 1996) developed the growth rate hypothesis (GRH), the key idea of ecological stoichiometry, that there are positive associations among organismal growth rate, the content of its RNA and P. Hence, P content of an organism varies due to differences in the amount of RNA. This suggests that fastgrowing organisms may have high P demands and a low carbon (C):P atomic ratio; therefore, their growth may be P-limited (Frost et al., 2006). Algae are a main rotifer food source (Arndt, 1993), whose C:P ratio increases considerably during P limitation (Hessen et al., 2004). On the other hand, zooplankton maintain their C:P ratio approximately constant and have generally lower C:nutrient ratios compared with the primary producers. Homeostatic consumers such as rotifers (Sterner, 1990) have to deal with such unbalances in their food. Jensen and Verschoor (Jensen and Verschoor, 2004) did

doi:10.1093/plankt/fbn069, available online at www.plankt.oxfordjournals.org # The Author 2008. Published by Oxford University Press. All rights reserved. For permissions, please email: [email protected]


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not find evidence that Brachionus calyciflorus is able to take up free mineral nutrients directly, hence cannot use mineral compensation. It is therefore likely that rotifers are dependent only on their food to obtain the needed amounts of essential elements. Nutrition is a multi-step process, which involves ingestion, gut transit time, digestion, assimilation, excretion and egestion. Stoichiometric regulations dealing with nutrient limitation of available food operate either pre- or postassimilation and involve changes in uptake, incorporation and release (Frost et al., 2005). There are two potential physiological solutions that operate before absorption. First, the ingestion rate could be decreased leading to the increase in the gut transit time, improving digestion and assimilation of the limiting elements (Sibly, 1981; Horn and Messer, 1992). Alternatively, nutrientdeficient consumers could increase their ingestion rate and decrease the gut transit time (Darchambeau, 2005; Mitra and Flynn, 2007) and also regulate the secretion of gut enzymes (Darchambeau, 2005). Likewise, theoretical studies suggest that maintaining a low gut transit time and maximizing the absorption gradient of digestion products are always advantageous to the consumer (Penry and Jumars, 1987; Jumars, 2000). On the other hand, the model by Anderson et al. (Anderson et al., 2005) suggests that consumers maintain their homeostasis mainly by using postabsorptive mechanisms rather than using regulation before absorption by the gut. Also, the experimental work by Darchambeau et al. (Darchambeau et al., 2003) suggests that Daphnia magna maintains its homeostasis mainly by transferring excess C via respiration and excretion of dissolved organic C. Phosphatases catalyze the release of orthophosphate from a variety of organic P compounds (Jansson et al., 1988). Gonza´les-Gil et al. (Gonza´les-Gil et al., 1998) were the first to use the fluorescently labeled enzyme activity (FLEA) technique (in earlier papers wrongly called the ELF technique) for direct detection of enzymatic activity in marine phytoplankton, and a number of studies on phosphatase activity in natural marine and freshwater environments and cultures have been published to date (Dyhrman and Palenik, 1999; Rengefors et al., 2001, 2003; Nedoma et al., 2003; Sˇtrojsova´ et al., 2003). Recently, the FLEA technique has been employed to study the individual variation and localization of enzyme activity in rotifers (Sˇtrojsova´ and Vrba, 2005, 2007). Artificial substrates can be consumed by rotifers; a fluorescent product of substrate hydrolysis tags the site of enzyme activity within the rotifer body. The FLEA technique, together with image analysis software, can be used for fluorescence quantification (Nedoma et al., 2003). The specific aim of this study was to determine the effects on population growth and reproduction of the

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rotifer B. calyciflorus when feeding on P-replete and P-depleted algal food. We also examined how the distinct food stoichiometry affected the activities of phosphatases in the gut of rotifers, using the FLEA technique. We tested the following hypotheses: (i) feeding by P-depleted algae will result in a lower population growth rate of B. calyciflorus. (ii) Phosphatase activity in the digestive tract of B. calyciflorus fed by P-depleted algae will increase to compensate for lowquality food.

METHODS Rotifer and algal cultures Brachionus calyciflorus Pallas, isolated from Lake Constance, was obtained from the Leibniz-Institute of Freshwater Ecology and Inland Fisheries (Berlin, Germany) and cultured in 6 mL chambers of 12-well microtiterplates in Z/4 medium (Zehnder and Gorham, 1960). Stock cultures were diluted weekly (1:2; culture/ medium) and incubated at 19 + 18C under a 12:12 h light/dark regime. Rotifers were fed with Chlorella kessleri Fott et Novakova (Culture Collection of Algal Laboratory, Institute of Botany AS CR, Trˇebonˇ, Czech Republic; food concentration 2 mg C L21) once a week. The algal food was cultured semi-continuously in a 2 L vessel at 20 + 28C under continuous light of 80 mmol quanta m22 s21 provided by cool-white tubes (Osram L 18W/20) at a dilution rate of 0.15 day21. The algae were cultured in two types of media: the P-replete medium with a phosphate concentration of 42 mmol L21 (unmodified Z/4) and the P-depleted medium with the phosphate concentration reduced to 2 mmol L21 (modified Z/4). The algae were in stable cultures for 3 months prior to the experiments. The non-axenic P-replete and P-depleted cultures differed in basic characteristics (Table I). Algae for the feeding experiment were harvested at 24 h intervals. Algal cells were counted by flow cytometry (FACSCalibur, Becton Dickinson, San Jose, CA, USA) before each preparation of fresh food suspensions, and the required C concentrations were calculated. The algal culture was diluted in bottled natural water (spring low-nutrient water) to avoid subsequent uptake of nutrients. Analytical procedures Characteristics of P-replete and P-depleted algal cultures (Table I) were determined before the addition of fresh medium and 24 h later (cultures were diluted

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Table I: Characteristics of P-replete and P-depleted algal cultures were determined daily for 10 days prior to the first feeding experiment Characteristics 5

21

Algal abundance (cells 10 mL ) Cell volume (mm3) Algal biomass (as DW, mg L21) Algal C content (mg C L21) Algal P content (mg P (mg DW)21) C:P atomic ratio Residual SRP concentration (mg P L21) Bacterial C content (mg C L21) Bacteria (% of the total organic C content)a

P-replete

P-depleted

66.1 32.3 373 180 12.0 107 11.4 0.05 0.03

7.77 42.5 296 145 1.4 920 2.1 0.25 0.18

(+3.4) (+15.7) (+20) (+9) (+1.9) (+18) (+4.1) (+0.001) (+0.001)

(+0.51) (+33.1) (+16) (+8) (+0.2) (+140) (+1.3) (+0.001) (+0.01)

Means (+SD) were calculated from n measurements; soluble reactive phosphorus (SRP) was measured in triplicate (n ¼ 30), other characteristics in duplicate (n ¼ 20). Algal cell volume and bacterial abundance were calculated from duplicate subsamples (n ¼ 20) containing 200 algal cells and .400 bacterial cells per subsample. a Bacteria were counted in an epiﬂuorescence microscope after DAPI staining (Porter and Feig, 1980); their C content was calculated using the conversion factor of 20 fg C cell21 (Lee and Fuhrman, 1987).

every second day) for 10 days prior to the first feeding experiment. Soluble reactive phosphorus (SRP) was measured in triplicate spectrophotometrically with the molybdate blue method (Murphy and Riley, 1962). Duplicate subsamples from the algal cultures were filtered through precombusted and preweighed glass-fiber filters (GF/C, Whatman) for the determination of dry weight (DW). Filters were dried at 1058C to a constant weight. The dried material was oxidized at 5008C for 2 h. The loss upon ignition is referred to as ash-free DW, which is converted to the C content of algal cells by a conversion factor of 0.5 (Ketchum and Redfield, 1949). Particulate P was further determined using the same ashed filters. Filters were extracted by diluted perchloric acid and after heat extraction at 1008C for 2 h, they were analyzed using the molybdate blue method (Murphy and Riley, 1962).

Feeding experiments Brachionus calyciflorus was fed for 5 days with the two types (P-replete and P-depleted) of C. kessleri at three C concentrations (1, 2 and 4 mg C L21); treatments without food served as controls. Food suspensions were mixed according to cell C and the current abundance of algal cells in the cultures. This experimental design resulted in seven food treatments as follows: P-replete C. kessleri (1 mg C L21), P-replete C. kessleri (2 mg C L21), P-replete C. kessleri (4 mg C L21), P-depleted C. kessleri (1 mg C L21), P-depleted C. kessleri (2 mg C L21), P-depleted C. kessleri (4 mg C L21) and a control with no food. All treatments were in duplicates, and the

experiments were repeated four times. All experiments were performed with rotifers from the same well-fed stock culture. B. calyciflorus was separated from its food by filtration through a 40 mm mesh and resuspended in bottled water without food 18 h prior to the experiment. The experiments were run in 6 mL chambers of 12-well micro-titerplates. The algal culture was diluted to the required C concentration in bottled natural water (spring low-nutrient water) to avoid subsequent uptake of nutrients. Ten rotifers with no attached eggs were transferred by micropipette into a single chamber with 5 mL of food suspension. Micro-titerplates were incubated at 19 + 18C under a 12:12 h light/dark regime. At 24 h intervals during the incubation period, the rotifers (no males were observed during the experimental period) and eggs in each chamber were counted under a stereoscope and transferred by micropipette into a new chamber with freshly diluted food suspension. Intrinsic population growth rates (r) of the rotifers for daily intervals were calculated as r ¼ ln(Nt)2ln(Nt21); where Nt and Nt21 are rotifer numbers on consecutive days. The growth rate for the experimental period was calculated from the average values of the last 3 days of the experiment. Egg ratio (eggs per rotifer) was calculated from the numbers of individuals and eggs on the last day of the experiment.

Phosphatase assay Phosphatase activity in the gut should reflect individual P needs of the rotifers under different food treatments or their variable individual P assimilation, or both. The amount of chlorophyll a (chl-a) was considered a proxy of ingested food in the rotifer gut after a 3 h incubation in a food suspension. Phosphatase activities of individual rotifers were examined using the FLEA technique, which allows for direct microscopic detection in rotifers and enables quantification of the resultant fluorescence by image cytometry. The water-soluble fluorogenic ELFw97 phosphate (ELFP, Molecular Probes, Eugene, OR, USA; 20 mmol L21 final concentration) is hydrolyzed by phosphatases into an insoluble, bright-green fluorescing ELF-alcohol (ELFA), which precipitates at the site of enzyme activity. ELFP was purchased as a 5 mM aqueous solution and stored at 2188C prior to use. It was filtered through a spin filter (Ultrafreew-MC, pore size 0.1 mm; Millipore, Bedford, MA, USA) before use. The FLEA protocol was modified from Nedoma et al. (Nedoma et al., 2003). Every rotifer from all food treatments, except for controls (rotifers were not alive), was assayed for phosphatases after the 5 day period of each feeding

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experiment. The rotifers were transferred into test tubes filled with 5 mL of the corresponding food suspension. Samples were incubated using ELFP at 208C in the light. After a 3 h incubation, the samples were filtered through a membrane filter ( pore size 12 mm) over a mild vacuum in a glass filter holder. The filters with retained rotifers were inspected for the presence of ELFA precipitates and chl-a autofluorescence in an epifluorescence microscope (Olympus AX – 70, Tokyo, Japan) supplied with a DVC-1300 digital camera (DVC, Austin, TX, USA). The green ELFA fluorescence and red autofluorescence of chl-a were quantified as described elsewhere (Nedoma et al., 2003). All individuals on the filter were photographed to quantify the ELFA fluorescence and chl-a autofluorescence in each rotifer’s stomach and intestine. The image of a rotifer was focused on its digestive tract. The Lucia G/F 4.8 image analysis software (Laboratory Imaging, Prague, Czech Republic) was used for image processing and fluorescence quantification. The quantification of chl-a autofluorescence in the single cells of C. kessleri was examined using the same technique described above.

Statistics Population growth rate and egg ratio of B. calyciflorus in the different food treatments were analyzed using two-way ANOVAs followed by the Bonferroni post hoc test. One-way ANOVA followed by the Tukey post hoc test was used for testing the influence of food quantity on egg ratio. Linear regression was used for testing whether the rotifer and egg numbers changed over time during the experiment and for testing whether the amount of chl-a and phosphatase activity changed over different food quantity in the rotifers fed with either P-replete or P-depleted algae. We checked the differences between the amount of chl-a per cell of P-replete and P-depleted C. kessleri as well as between the rotifer phosphatase activity of the P-replete and P-depleted treatments using an unpaired t-test. The analyses were performed and graphs were constructed using GraphPad Prism (version 4.00 for Windows; GraphPad Software, San Diego, CA, USA).

R E S U LT S Rotifer reproduction The rotifers had positive growth rates when fed with the P-replete algae at all three concentration, whereas the rotifers fed the P-depleted algae, as well as those in the control without food, had negative growth rates (Fig. 1).

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Fig. 1. Population growth rate of B. calyciflorus fed for 5 days with P-replete (solid symbols) and P-depleted (open symbols) algal food at three different food concentrations. Assays without food (asterisk symbol) served as controls. Population growth rates (mean + SD) were calculated from the average values of the last 3 days of the experiment.

Rotifers died more frequently in the chambers with the highest food concentration (4 mg C L21, both P-replete and P-depleted). The two-way ANOVA revealed a significant effect of algal P content (P , 0.0001); the effect of algal C concentration was not significant (P . 0.05). The Bonferroni post hoc test revealed significant differences in the growth rate between the P-replete and P-depleted treatments at a C concentration of 2 mg C L21 (P , 0.001), but insignificant differences between the P-replete and P-depleted treatments at C concentrations of 1 and 4 mg C L21 (P . 0.05). There was a significant loss of individuals fed by P-depleted food at C concentrations of 1, 2 and 4 mg C L21 and unfed rotifers over time of the experiment (linear regression, Table II). The number of rotifers fed by P-replete food at a C concentration of 2 mg C L21 significantly increased (Table II). The number of rotifers fed by P-replete food at C concentrations of 1 and 4 mg C L21 did not change over time (Table II). While the rotifers fed by P-replete algae produced more eggs at the end than at the beginning of the feeding experiment, the rotifers fed by P-depleted algae produced more eggs in the first and second days compared with the rest of the experiment. Rotifers from the control without food produced eggs only in the first 2 days and then stopped egg production (data not shown). Similar to rotifer numbers, egg numbers significantly decreased in the P-depleted treatment and control and increased in the P-replete treatment of a 2 mg C L21 food concentration. The egg number for P-replete treatment at C concentrations of 1 and 4 mg C L21 did not change over time (Table II). The egg ratio was significantly higher in the rotifers fed P-replete algae than in those fed P-depleted food

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Table II: Results of linear regression testing whether the rotifer and egg numbers of rotifers fed by different food change over time of the experiment Rotifer number

Egg number

Food treatment

P

Slope

y-intercept

R2

P

Slope

y-intercept

R2

P-replete 1 mg C L21 P-replete 2 mg C L21 P-replete 4 mg C L21 P-depleted 1 mg C L21 P-depleted 2 mg C L21 P-depleted 4 mg C L21 Control

0.09 0.02 0.08 0.002 0.001 0.01 0.002

0.42 1.40 0.47 2 0.57 2 0.80 2 0.54 2 2.27

9.11 8.20 9.20 10.36 10.30 9.80 11.19

0.56 0.80 0.58 0.92 0.94 0.84 0.92

0.62 0.02 0.23 0.05 0.28 0.46 < 0.0001

0.09 0.60 0.32 20.30 20.13 20.10 2 0.23

1.97 1.80 2.98 1.98 1.55 1.45 0.98

0.01 0.13 0.04 0.09 0.03 0.01 0.35

Statistically signiﬁcant differences are indicated in bold.

(Fig. 2) and zero in the control without food. The egg ratio significantly differed between the smallest and highest C concentration of P-replete food (one-way ANOVA, Tukey post hoc test; P , 0.001), whereas it was not affected by the C concentrations of P-depleted food (P . 0.05). Algal P content and C concentration had significant effects on the egg ratio (two-way ANOVA; P , 0.0001 and P , 0.01, respectively). The egg ratio differed significantly between the P-replete and P-depleted treatments at C concentrations of 2 and 4 mg C L21 (P , 0.05 and P , 0.001, respectively), but was not significant at a C concentration of 1 mg C L21 (P . 0.05).

over different food quantity in the rotifers fed with P-depleted algae (linear regression; P . 0.05, slope ¼ 20.71; Fig. 3B). Algal chl-a autofluorescence in fluorescence units (FUR in red channel) differed significantly between the P-replete and P-depleted treatments (t-test; P , 0.05), but only by a factor of 1.2, whereas C content differed by a factor of 6.9 (27.2 and 187 pg C cell21, respectively; Fig. 4).

DISCUSSION The population growth rate of B. calyciflorus was reduced on the diet composed of P-depleted algae (molar C:P

Physiology of rotifer digestion There was no significant difference in phosphatase activity between the P-replete and P-depleted treatments (t-test; P . 0.05). The amount of both chl-a and phosphatase activity in the gut of rotifers fed with P-replete algae significantly increased with increase in concentration of the food suspension (linear regression; P , 0.0001, slope ¼ 479 and P , 0.0001, slope ¼ 66.2, respectively; Fig. 3). In contrast, the amount of chl-a significantly decreased with increase in food concentration (linear regression; P , 0.001, slope ¼ 2170; Fig. 3A), and phosphatase activity did not change significantly

Fig. 2. Egg ratio of B. calyciflorus fed P-replete (solid bars) and P-depleted (open bars) algal food at different food concentrations on the last day of the experimental period (mean + SD).

Fig. 3. Chlorophyll a autofluorescence (A) and ELFA fluorescence (B) in fluorescence units (FUR in red channel or FUG in green channel, respectively) in the digestive tract of B. calyciflorus fed P-replete (solid bars) and P-depleted (open bars) algae at different food concentrations (mean + SD).

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Fig. 4. Chlorophyll a autofluorescence (left, in fluorescence units as in Fig. 3A) in P-replete and P-depleted algal cell (mean + SD; n ¼ 84 and 104, respectively) and C content (right), which was calculated from algal cell volume, algal abundance and C content in the culture (Table I).

ratio of 920) when compared with P-replete algae (C:P ¼ 107). This result is in agreement with some other studies on B. calyciflorus (Jensen and Verschoor, 2004; Lu¨rling, 2006; Hessen et al., 2007), Brachionus rubens (Rothhaupt, 1995) and Anureopsis fissa (Conde-Porcuna, 2000). For Keratella cochlearis, however, Ramos-Rodrı´guez and Conde-Porcuna (Ramos-Rodrı´guez and CondePorcuna, 2003) observed the opposite pattern: the lowest growth rates for rotifers fed P-replete algae, but enhanced growth rates in P-depleted algae. Whereas Jensen and Verschoor (Jensen and Verschoor, 2004) observed a positive population growth of B. calyciflorus fed P-depleted algae (C:P ¼ 626), we observed a negative population growth of B. calyciflorus fed more P-depleted algae (C:P ¼ 920). Also, Rothhaupt (Rothhaupt, 1995) found negative population growth in B. rubens fed with two kinds of P-depleted algae (C:P ¼ 684 and 874). The C:P threshold element ratio of food for B. calyciflorus is considered to be 119 (Frost et al., 2006), which is close to the stoichiometry of the P-replete food (C:P ¼ 107) in this study. Thus, it is likely that the higher C:P ratio of the P-depleted food caused the pronounced decrease in population growth of B. calyciflorus. Since all of the C food concentrations used in our experiment were above the threshold for zero population growth for B. calyciflorus (0.38 mg C L21; Stemberger and Gilbert, 1985), the observed effects of the algal food on the rotifers’ reproduction-related parameters should be caused by its low P content (high C:P). Nitrogen, polyunsaturated fatty acids, amino acids and vitamins have also been suggested as limiting factors of rotifer growth rates (Jensen and Verschoor, 2004). It is possible that the P-depleted algae used in our experiments could lack such substances due to P limitation; thus, another kind of limitation could come in effect in addition to P limitation.

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Population growth rate was rather low in the rotifers fed with the highest food concentration, likely due to neglected mortality in this treatment (either P-replete or P-depleted). Thus, the apparent growth rate was lower than expected. We have no explanation for this mortality; one can only speculate that it could be due to intense respiration by abundant algae present in small experimental chambers and consequent oxygen depletion during dark periods. Rotifers of the genus Brachionus have very definite sizedependent feeding effectiveness; B. calyciflorus feeds most efficiently on particles about 10-mm equivalent spherical diameter (Rothhaupt, 1990). Although the algae used in our experiment were below this optimal size, their sizes were still in the range that B. calyciflorus ingests. In our experiments, P-depleted algae were slightly larger (4.33 mm) than P-replete algae (3.95 mm), i.e. closer to the optimal size for B. calyciflorus. While algal cultures were not axenic, some bacteria (,0.5% of total C) were also available as a food for rotifers. Nevertheless, since B. calyciflorus has very low feeding efficiencies for small food items such as bacteria (Rothhaupt, 1990), bacteria did not compose a significant part of the rotifer food in this study. The rotifers fed P-replete food had significantly higher egg ratios than the rotifers fed P-depleted food (Fig. 2). While the production of eggs by rotifers in the P-replete variant increased after the second day of the feeding experiment, rotifer egg production in the P-depleted variant reached its maximum in the first and second day and then decreased. Rotifers from the control without food produced eggs only in the first 2 days and then stopped completely. Although the rotifers were left for 18 h without food prior to the feeding experiment, they possibly might have a certain amount of stored P from the previous feeding with P-replete algae. It is likely that rotifers utilize these P reserves for egg formation. Since the duration of embryonic development of B. calyciflorus is about 18 h at 208C (Galkovskaja, 1987), a small part of the initial egg production in the experiment could be dependent on the cultivation condition of the stock culture. Although the rotifers fed P-depleted food showed strongly reduced egg production, mortality was not as high as for the control rotifers without food (Table II). Apparently, the C concentrations were high enough to support the maintenance metabolism that demanded mainly C-rich substances (Sterner and Elser, 2002). Jensen and Verschoor (Jensen and Verschoor, 2004) did not find a change in lifespan due to algal nutrient limitation and they suggested that the reduction in reproduction, but not individual survival, could be a pattern in B. calyciflorus under a nutrient limitation. This strategy

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may increase the chance of individuals overcoming periods of limiting food quality (Jensen and Verschoor, 2004). The depressed growth rate of B. calyciflorus fed P-depleted food (this study) is in agreement with the GRH, which states that an increase in growth rate is connected with an increase in P demands because of increased production of P-rich ribosomal RNA required for the protein synthesis needs under rapid growth rates (Elser et al., 1996, 2000b, 2003; Sterner and Elser, 2002). A similar relationship was also observed for the egg production rate (Vrede et al., 2004). Also, Frost et al. (Frost et al., 2006) suggested that there is a general relationship between P content and maximum growth rates in organisms. Fast-growing organisms, such as rotifers, might therefore be sensitive to the P-content of their food, because of the need to maintain production of P-rich ribosomal RNA (Sterner and Elser, 2002). It is also known that rotifers contain a lot of P in their bodies (low C:P atomic ratio of 92; Jensen and Verschoor, 2004). It is likely that the rotifers fed P-depleted food did not have enough P necessary for protein synthesis in this study; consequently, they had a negative growth rate and low egg ratio. Autofluorescence of chl-a was located in the rotifer gut cavity and inside the cells of the stomach and intestine. The amount of chl-a in the gut can be a proxy of rotifer ingestion. The P-depleted algae had slightly higher chl-a autofluorescence than P-replete algae. However, they had a much higher amount of cellular C compared with P-replete algae (Fig. 4). This means that, in fact, rotifers fed by P-deplete algae might ingest almost six times higher amount of food C than indicated the amount of chl-a as shown in Fig. 3A (cf. Figure 4). In reality, rotifers fed by P-replete algae likely ingested less food than rotifers fed by P-depleted algae. Hence, the rotifers showed a compensatory feeding when food was P-depleted. While the amount of chl-a in the gut of rotifers fed P-replete algae increased with increasing algal biomass in the food suspension, the amount of chl-a in the gut of rotifers fed P-depleted algae exhibited the opposite trend. In our opinion, the rotifers might ingest fewer P-depleted algae due to a stress caused by higher concentrations of algal and bacterial secondary metabolites in the rotifer food suspension (cf. Stemberger and Gilbert, 1985). Activation of toxicity has been confirmed in several algal species under P limitation (Shilo, 1967; Dafni et al., 1972; Edvardsen et al., 1990; Johansson and Grane´li, 1999). Herbivorous zooplankton have low C:nutrients ratio, thus they often face an excess of C in their food. Regulation of this C excess may operate via

preabsorptive regulation, such as C assimilation efficiency, or postabsorptive regulation via increased respiration and/or excretion of dissolved organic C. Jensen et al. (Jensen et al., 2006) observed that the excess C was not released by respiration in B. calyciflorus and that nutrient balance must therefore be maintained by other means such as excretion of dissolved organic C or egestion in fecal material. On the other hand, Jensen and Hessen (Jensen and Hessen, 2007) observed that daphnids released the excess C by respiration, but they supposed another way of stoichiometric regulation as well. He and Wang (He and Wang, 2006) observed a negative relationship between the C assimilation efficiency of daphnids and food concentration of the same quality. DeMott et al. (DeMott et al., 1998) observed a decrease in the C assimilation efficiency as well as in the C:P ratio of excreted particulates of the daphnids when their food was P-depleted. However, rotifers may use different mechanisms than daphnids for regulating their C:nutrients ratio. The measurement of ELFA fluorescence that shows phosphatase activity in the digestive systems of rotifers could indicate the pattern of rotifer P utilization. Phosphatase activity increased with increase in food quantity in the rotifers fed with P-replete algae. Similarly, food concentration affected the activity of digestive enzymes of copepods (Mayzaud and Poulet, 1978). On the other hand, phosphatase activity did not change over different food quantity in the rotifers fed with P-depleted algae (this study). While the model by Darchambeau (Darchambeau, 2005) predicts a positive relationship between the food C:P ratio and optimal secretion of digestive enzymes, and assumes that the consumer can regulate the secretion of gut enzymes according to food concentration and nutrient content, we did not observe such regulation in our study. Our results agree with the model by Anderson et al. (Anderson et al., 2005), which suggests that consumers use postabsorptive mechanisms for regulation of nutritional imbalance rather than using regulation prior to absorption by the gut. They indeed proposed that using postabsorptive mechanisms needs not be universal, but will occur in nonselective grazers when food is changing rapidly, whereas using preabsorptive regulation by digestive activity might occur in responding to long-term changes in food quality. In conclusion, we observed a significant effect of the P content of algal food on population growth and reproduction of B. calyciflorus, whereas the effect of food quantity was significant only for the egg ratio of rotifers fed by P-replete food. Our results showed that the population growth rate and reproduction of B. calyciflorus was reduced on the diet composed of P-depleted algae when compared with P-replete algae. There was no

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significant difference in digestion activity between the P-replete and P-depleted treatments. Our results indicate that B. calyciflorus do not regulate nutrient balance via preabsorptive mechanisms such as an optimization of digestive activity, and thus postabsorptive mechanisms offer an alternative.

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Dyhrman, S. T. and Palenik, B. (1999) Phosphate stress in cultures and field populations of the Dinoflagellate Prorocentrum minimum detected by a single-cell alkaline phosphatase assay. Appl. Environ. Microbiol., 65, 3205– 3212. Edvardsen, B., Moy, F. and Paasche, E. (1990) Haemolytic activity in extracts of Chrysochromulina polylepis grown at different levels of selenite and phosphate. In Grane´li, E., Sundstro¨m, B., Edler, L., Anderson, D. M. et al. (eds), Toxic Marine Phytoplankton. Elsevier, New York. Elser, J. J., Dobberfuhl, D. R., MacKay, N. A. et al. (1996) Organism size, life history, and N:P stoichiometry. Bioscience, 46, 674–684.

AC K N OW L E D G E M E N T S We thank M. Kupkova´ and J. Zemanova´ for helping with cultivations and V. Hejzlarova´ and J. Kroupova´ for assistance with the chemical analyses. We acknowledge U. Newen for providing the B. calyciflorus stock culture. We really appreciate the helpful critical comments of two anonymous reviewers.

Elser, J. J., Dowling, T., Dobberfuhl, D. A. et al. (2000a) The evolution of ecosystem processes: ecological stoichiometry of a key herbivore in temperate and arctic habitats. J. Evol. Biol., 13, 845–853. Elser, J. J., Sterner, R. W., Gorokhova, E. et al. (2000b) Biological stoichiometry from genes to ecosystems. Ecol. Lett., 3, 540–550. Elser, J. J., Acharya, K., Kyle, M. et al. (2003) Growth ratestoichiometry couplings in diverse biota. Ecol. Lett., 6, 936–943. Frost, P. C., Evans-White, M. A., Finkel, Z. V. et al. (2005) Are you what you eat? Physiological constraints on organismal stoichiometry in an elementally imbalanced world. Oikos, 109, 18–28.

FUNDING This study was partly supported from the following projects: A600170602 (J.N.), Z60170517 and MSM 6007665801.

Frost, P. C., Benstead, J. P., Cross, W. F. et al. (2006) Threshold elemental ratios of carbon and phosphorus in aquatic consumers. Ecol. Lett., 9, 774 –779. Galkovskaja, G. A. (1987) Planktonic rotifers and temperature. Hydrobiologia, 147, 307– 317.

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