Responses of alkaline phosphatase activity to phosphorus stress in Daphnia magna

The phosphorus (P) content of algal food can strongly affect the growth and reproduction of aquatic consumers (Sterner and Elser, 2002; Frost et al., 2005). While the effects of consumer P-limitation on growth and reproduction have received considerable attention (e.g. Sterner and Hessen, 1994; Gulati et al., 1997; Sterner and Schulz, 1998; Sterner and Elser, 2002), dietary P-stress also affects other aspects of animal metabolism (e.g. Balseiro et al., 2008). In particular, physiological processes involved in the disposal of excess carbon and the acquisition/retention of P can be especially sensitive to the P content of food (DeMott et al., 1998; Darchambeau et al., 2003; Jensen and Hessen, 2007). Given these physiological responses to P-deficient foods, we hypothesize that the activity of enzymes involved in P-metabolism is also affected by this consumer's dietary P intake. We further predict that this may especially be the case in P-rich invertebrate taxa such as the crustacean Daphnia, which is known to respond strongly to changes in the P content of its food (e.g. Urabe et al., 1997; Elser et al., 2001).

One biochemical mechanism involved in the acquisition of dietary P in crustacean zooplankton is the production of alkaline phosphatase (AP), an enzyme that hydrolyzes phosphate monoesters and which exhibits low substrate specificity (Boavida and Heath, 1984). Examples of this enzyme class are found in organisms ranging from bacteria to mammals. Animals express a variety of isotypes, including an intestinal isotype in mammals (Yeh et al., 1994) and a midgut isotype in insects (Azuma et al., 1991). Additionally, histological methods have located phosphatase activity in the midgut of the crustacean copepod Centropages typicus (Arnaud et al., 1984). Previous work has also shown Daphnia magna can produce soluble phosphatases that are distinct from, and in larger quantities than, those produced by their algal food sources (Boavida and Heath, 1984). In addition, AP activity has been found in the dissolved release from axenic Daphnia, which is further evidence that the soluble enzyme is not produced by associated gut flora (Rigler, 1961; Wynne and Gophen, 1981). Whether the activity of AP within the body or in their release from the body is altered by the P-content of the zooplankter's diet remains untested.

Most work examining AP in freshwater zooplankton has focused on the activity of the enzyme released by the crustacean Daphnia (e.g. Boavida and Heath, 1984). The measurement of significant AP activity in Daphnia release may reflect AP being flushed out of the gut along with waste products. Given its established role in digestion in other animals, the location of AP in the gut or its lining in Daphnia would not be surprising. Seven AP genes have been identified in the genome of Daphnia pulex; two of these genes appear to encode soluble AP and three other genes match types known to be membrane anchored (http://genome.jgi-psf.org/Dappu1/Dappu1.home.html). It is likely that one or more of these AP have roles in dietary P-acquisition and P-recycling within the Daphnia body and would be altered by dietary P-intake by this animal.

In this study, we examined whether AP activity in Daphnia bodies and their release would change as a function of algal food P content. We grew Daphnia on algal diets that varied in P content and measured the AP activity in their bodies at different ages. We considered the possibility that algal food and phosphate inhibition were potential regulators of AP activity in Daphnia body homogenates and designed additional experiments to examine these potential mechanisms. AP activity was also measured in the water containing the differentially P-nourished Daphnia. This was performed with animals raised entirely on a single P-diet and with animals that were shifted from P-rich to P-poor algae. We predicted that the AP activity would increase in the bodies of P-stressed Daphnia as P-acquisition would be relatively more important in P-limited animals. In addition, we expected that the AP activity in the materials released from Daphnia would increase with increasing food carbon:phosphorus (C:P) ratios. Our results demonstrate that AP activity is sensitive to P-limitation in the diets of this important aquatic consumer with effects that differ between animal body tissues and released materials.

Laboratory-grown Daphnia magna Straus were fed algae of different C:P ratios (100–700) throughout these experiments, which is approximately the range of algal C:P ratios found in natural lakes (Guildford and Hecky, 2000). For all of the experiments, neonates (<1-day-old) were taken from second-generation monoclonal Daphnia fed P-rich food (algae C:P ratios of 60–100). Only neonates born after the first brood were used for experiments. In general, experimental Daphnia were raised in groups of 10 in replicate jars containing 200 ml (days 1–4), 300 ml (days 5–8) and 400 ml (days 9–15) of P-free freshwater culture medium (COMBO) (Kilham et al., 1998). Throughout, we provided Daphnia high concentrations of algal food (2–4 mg Cl⁻¹) by replenishing food every other day over the course of each experiment.

The algal food, Scenedesmus acutus (University of Toronto Culture Collection 10), was grown in semi-continuous cultures that were diluted daily with COMBO (Kilham et al., 1998). A range in algae C:P ratios was created by changing the media's concentration of P (KH₂PO₄) and the dilution rate (Table 1). The cultures were grown with a 16 h:8 h light:dark photoperiod at 20°C with two 400 W Lumalux bulbs (LU400/ECO, Sylvania Ltd, Mississauga, ON, Canada). Harvested algae were centrifuged for 20 min at 4000 g and resuspended in P-free COMBO. We estimated the C:P ratios of this concentrated algae, which allowed us to mix the algae to create targeted nominal food C:P ratios fed to the Daphnia. Algal %P was measured on dried and digested samples by the molybdate-blue ascorbic acid colormetric assay (APHA, 1992). The %C and %N of dried algae was determined on additional samples by a CN Elemental Analyzer (Vario EL III, Elementar Incorporated, Mt Laurel, NJ, USA) and was used to calculate more precisely nominal C:P ratios of algal food.

We measured the AP activity in the bodies of Daphnia fed algae at one of seven C:P ratios (100–700) and at three different ages (5-, 10- and 15-day-old Daphnia). For this experiment, Daphnia neonates were collected and randomly assigned to one of seven algal food C:P ratios (3–10 replicate jars per food level with 10 animals per jar). On the specified day (5, 10 or 15), AP activity was measured on 10 groups of Daphnia collected from replicate jars within each food level (Table 2). Because smaller Daphnia produce less AP, different numbers of Daphnia were grouped for AP activity measurements depending on the sampling day. Daphnia that were not used for body AP activity measurements were dried and weighed with an ultra-microbalance (MX5, Mettler-Toledo Inc., Columbus, OH, USA). With these dried Daphnia, %P was measured by the molybdate-blue ascorbic assay and their %C and %N was determined with a CN Elemental Analyzer as described above.

Daphnia were prepared for the body AP activity assay by washing them twice in P-free COMBO for 3–5 min to remove any AP in their holding water. Each group of Daphnia was placed in a 1.5 ml plastic ultracentrifuge tube and was homogenized in 0.75 ml of P-free COMBO. The volume was then brought up to 1.5 ml, and two 600 μl aliquots were removed for duplicate measurements during the enzyme assay. AP activity was measured in real-time by monitoring the hydrolysis of p-nitrophenyl phosphate (pNPP) into p-nitrophenol (pNP) and phosphate by detecting the absorbance of pNP formation at 400 nm. This colormetric assay was modified from the methods described by Boavida and Heath (Boavida and Heath, 1984). In 1 cm plastic cuvettes, the enzyme assay mixture contained: 600 μl of sample or P-free COMBO blank, 300 μl of 0.1 mol l⁻¹ Tris-HCl buffer (pH 8.0) and 100 μl of 0.5 mmol l⁻¹ pNPP (Bioshop Canada Inc., Burlington, ON, Canada). The assay was run within 2–5 h after the Daphnia had been homogenized and was performed at room temperature. The absorbance of pNP formation was measured by a UV-visible recording spectrophotometer (Shimadzu UV-160 U, Columbia, MD, USA). Readings were taken for 40 min at 10 min intervals, starting on the tenth minute. Phosphatase activity was measured as the moles of pNP produced per unit of Daphnia mass per unit of time (μmol pNP mg⁻¹Daphnia h⁻¹). We used a literature value for the extinction coefficient of pNP in our calculations (0.0173 μmol l⁻¹ cm⁻¹) (Bosron et al., 1975).

A primary product of AP-catalyzed reactions is phosphate, which is known to inhibit AP enzyme activity. Product inhibition potentially reduces AP activity in the body homogenates of P-rich animals due to their greater P-content. To address the potential that phosphate inhibition affected AP activity in Daphnia tissue homogenates, we designed two experiments where we altered the phosphate found in homogenized Daphnia bodies and measured the change in the corresponding AP activity. In both experiments, Daphnia were either fed a P-rich diet (algae C:P ratio ∼150) or a P-poor diet (algae C:P ratio ∼525) for 5 days. For the first experiment, we made the soluble phosphate concentration equivalently high in tissue homogenates from animals consuming both food types. After measuring soluble phosphate on the homogenates of animals, we spiked homogenates from the Daphnia eating the P-poor diet with KH₂PO₄ to ensure that the P concentration in these samples was equivalent to that in the samples from the Daphnia eating the P-rich diet. AP activity in the homogenates was then measured as described above.

In the second experiment, we used gel filtration chromatography to reduce the soluble phosphate concentration of samples of 5-day-old Daphnia consuming P-rich and P-poor food to nearly undetectable levels. Groups of five animals were homogenized in 400 μl of P-free COMBO. 15 μl of protease inhibitor cocktail and 15 μl of Triton X-100 were added to each cell suspension and left to sit for 20 min. Cellular debris was removed by centrifuging samples at 12,000 g for 20 min. 300 μl of each supernatant was added to 10 ml spin columns containing 2.0 ml of desalting gel resin (P-6 DG, Bio-Gel, Hercules, CA, USA). Samples were centrifuged at 3000 g for 2 min, and 250 μl of flow through was assayed either for AP activity or soluble phosphate concentration.

We also designed two experiments to assess whether algal-associated AP contributes to Daphnia body AP activity. The first experiment involved feeding Daphnia heat-treated algal food. Heating algae can denature algae AP and reduce its contribution to the AP activity found in the animal's body. After heating algal food at 97°C for 10 min, we measured the AP activity and C:P ratios of saved subsamples of the food as previously described. After we found that algae AP activity was reduced by 95% using this method, we fed Daphnia a diet of heat-treated (C:P ratios 510 or 1480) or non-heat-treated algae (C:P ratios 180, 510 or 1640) for 5 days. Body AP activity was then measured on Daphnia homogenates as previously described. In the second experiment, we included a longer pre-measurement incubation period where Daphnia were held for 2 h without food to reduce the algal influence on AP activity. For this experiment, Daphnia were grown for 5 days under a P-sufficient (C:P ratio 170) or P-deficient (C:P ratio 550) algae food diet. Body AP was also measured on these animals as described above.

We also examined the AP activity on the materials released from Daphnia (age 15 days) fed algae of different C:P ratios (100–700). At the start of this experiment, we placed Daphnia neonates into one of the seven algae C:P food levels (100–700) with 10 replicate jars per food level and 10 animals per jar (Table 2). On the specified day, AP activity was measured in P-free COMBO water containing all of the Daphnia from each replicate jar within each food level. After a 60 min incubation, these animals were saved and analyzed for their dry mass, %C, %N and %P as described above.

In preparation for measuring AP activity of Daphnia release, animals were washed for 3–5 min in P-free COMBO to remove any AP in the water. After two rinses with fresh P-free COMBO, Daphnia from each jar were incubated together in 1.5 ml of P-free COMBO for 1 h. Subsequently, two 600 μl aliquots of media were removed for duplicate measurements in the enzyme assay. Phosphatase activity was monitored as the absorbance of pNP formation during 120 min with readings taken every 30 min. Because AP activity measurements on released dissolved enzymes required the crowding of animals to detect appreciable activity, we performed an initial trial to assess whether crowding affects Daphnia's release of AP. Daphnia were grown for 15 days on a low C:P algae diet (∼150) in this experiment. AP activity did not differ in the release from Daphnia that were incubated in groups of 1, 5 or 10 individuals in 1.5 ml of P-free COMBO for the duration of 60 min (data not shown).

We also measured the AP activity in the released enzymes of adult Daphnia that experienced a dietary shift from high quality (C:P ratio 100) to low quality (C:P 700) algal food. We collected Daphnia neonates and fed them low C:P algae (100) for 10 days. Starting on day 11, one-half of the individuals was switched to higher algal C:P ratios (300, 500 or 700) with 13 replicate jars for each food level. These Daphnia were fed P-deficient algae for four days. Starting on day 13, another portion of the remaining Daphnia were switched to higher algal C:P ratios (300, 500 or 700) with 13 jars per food C:P level. These Daphnia were fed P-deficient algae for two days. The remaining Daphnia were fed C:P 100 algae for the entire duration of the experiment and served as a control group (N=13 jars). On day 15, AP activity was measured on the water containing Daphnia incubated together for 60 min in 1.5 ml of P-free COMBO. Daphnia dry mass, %C, %N and %P were measured as described above.

We found that P-nutrition of Daphnia can significantly affect the AP activity in their bodies. For all three ages of Daphnia in this study, AP activity increased in the bodies of animals consuming higher food C:P ratios (Fig. 1). Proximally, the increase in mass-specific body AP activity reflected both an increase in AP activity per animal and the smaller mass of P-limited Daphnia bodies (data not shown). The greater AP activity in Daphnia bodies may be attributed to an increased expression of AP, increased activity of existing AP pools or a combination of the two. Our results also indicate that Daphnia under P-stress maintain or increase AP activity despite having less total protein in their bodies (as evidenced by a concurrent reduction in body %N content, data not shown).

To assess the extent to which phosphate inhibition of AP accounts for the lower AP activity found in P-rich Daphnia homogenates, we measured the AP activity in P-spiked and P-free extracts of homogenized bodies of Daphnia. We found that increasing the [PO₄^3–] in homogenates of P-poor Daphnia slightly reduced the AP activity of these samples but this reduction could not account for the differences seen with P-rich animals (Fig. 2A). In addition, we found the removal of endogenous phosphate in Daphnia homogenates by gel filtration chromatography (Fig. 2B) did not eliminate the differences in AP activity produced by P-limitation in Daphnia. Rather, this treatment enhanced the observed differences in AP activity between samples derived from animals fed different P-quality food (Fig. 2C). Together, these results provide strong evidence that P-limitation elevates the quantity and activity of AP in the bodies of D. magna.

Another potential explanation for the differences in AP activity seen in the bodies of differentially P-nourished Daphnia is that consumed algae are a significant source of AP activity in animal body homogenates. Initial trials demonstrated that relatively elevated temperatures (>90°C) were required to significantly reduce the AP activity of algal food. This heating treatment increased the C:P ratios of both of our food types (P-rich from ∼150 to ∼500 and P-deficient from ∼500 to ∼1500), perhaps due to cell lysis and loss of internal pools of P. Nonetheless, animals fed on these relatively AP-free (but P-deficient) foods retained elevated body AP activity relative to animals consuming P-rich food (Fig. 3A). We also measured the AP activity of differentially nourished Daphnia after a short (5 min) and a long (2 h) period of starvation. While gut residence time of algal material can be as long as 12–24 h in starving animals (e.g. Gillis et al., 2005), even short periods of starvation should nonetheless reduce the quantity of algal material in Daphnia due to both egestion and digestion. We found no difference in AP activity of either P-rich or P-poor Daphnia bodies when these animals were starved for 5 min or 2 h (Fig. 3B). Moreover, by directly measuring AP activity on algae samples, we estimated that algae could only contribute up to 8% of Daphnia body AP activity, if we conservatively assume that 10% of a daphnid's dry mass is derived from live algal cells in the gut. These results, coupled with those from the heat-killed algal experiment, suggest that algal associated AP is not the primary source of AP activity within Daphnia bodies and does not likely account for the increased AP activity that we observe in animals fed P-poor food.

The AP activity in the bodies of Daphnia also varied with their age. Body AP activity was highest in the 5-day-old Daphnia and lowest for the other two ages tested, particularly for animals consuming P-rich food. While this increased AP activity may reflect the higher P demands of the faster-growing, juvenile Daphnia (Main et al., 1997), animals of different ages having the same body C:P ratios varied considerably in their mass-specific body AP activity (Fig. 1). One possible explanation for this ontogenetic disconnection between body C:P ratios and AP activity in Daphnia bodies is that juveniles allocate more proteins into digestive activities, including AP activity. By contrast, adult Daphnia may reallocate protein resources from digestion to reproduction and/or maintenance. In any case, age-related changes to AP activity in Daphnia potentially confounds simple interpretation of variable AP activity in these animals as it relates to food C:P ratios in natural environments.

P-nutrition also controls the amount of AP activity that is released into the water with lower levels of AP released from P-starved animals (Fig. 4). Daphnia may produce a greater quantity of membrane-anchored AP under P-starvation and reduce their production of secreted AP that moves into, through and out of the gut. Alternatively, release of membrane-anchored AP into the gut may be restricted under P-starved conditions. Although intestinal AP in animals is attached to the cell membrane by a glycosylphosphatidylinositol anchor at the carboxy-terminus of the protein chain, membrane-anchored AP can be released in mammals by the action of phospholipases or by solubilization with endogenous surfactants in response to an increase in dietary fat (Deng et al., 1992). The responses of Daphnia that we observe are broadly consistent with this mechanism, in that AP released to the water is higher in well-fed Daphnia and is lower in P-starved animals. From a materials conservation standpoint, this reduced AP loss would be advantageous for P-limited Daphnia, given the strong constraints on their protein production imposed by P-constraints on ribosomal RNA content (Elser et al., 2003).

We also examined AP activity in the materials released from P-sufficient Daphnia in response to short-term changes in food C:P ratios. Body P content showed a consistent decrease with increasing duration of exposure to P-poor food in Daphnia consuming foods of all three ratios but this decrease was relatively greater for animals consuming food C:P 700 (Fig. 5A). Longer exposure to P-limited food also reduced the AP activity in Daphnia release, but in contrast to their body P-content, these changes in released AP activity did not differ among animals consuming different food C:P ratios (Fig. 5B). The reduced AP activity in released materials with increasing duration of P-poor exposure matches the result of lower AP activity in the release from P-stressed Daphnia observed in the previous experiment (Fig. 4). These results are further evidence of the P-sensitivity of Daphnia to even short periods of nutritional deprivation, which can be detected with analysis of AP activity in their tissues and/or released materials.

The responses of AP activity in the bodies of Daphnia to variable P-nutrition could lead to the development and refinement of biochemical indicators of zooplankton nutrition. Such bioreporters are being developed for phytoplankton (e.g. McKay et al., 2005) and could prove useful for the study of zooplankton nutrition. Elemental bioreporters would allow for immediate and relatively rapid assessment of the in situ nutritional state of an animal. However, there are a range of potential complications to the development and use of metabolic indicators of nutrition including: (1) many digestive enzymes are used for the acquisition of multiple elements from diverse natural substrates and thus lack element-specificity, (2) enzyme activities may vary within a taxa due to ontogeny or other environmental variables [as we found in this study; also see Balseiro et al. (Balseiro et al., 2008)], and (3) detailed knowledge of many target enzymes and their molecular regulation remains lacking. Consequently, there is a need to further study the regulation of metabolic enzymes, such as exploring the gene expression of nutritionally-relevant proteins in animals consuming foods of contrasting elemental quality under controlled laboratory and more realistic field conditions. Ultimately, elemental bioreporters, developed and refined, would provide an important and needed tool to assess and understand the nutrition of animals in nature.

 
• AP

  alkaline phosphatase

 
• C

  carbon

 
• COMBO

  freshwater culture medium

 
• C:P

  elemental carbon to phosphorus molar ratio

 
• N

  nitrogen

 
• pNP

  para-nitrophenol

 
• pNPP

  para-nitrophenyl phosphate

 
• P

  phosphorus

 
• PO₄^3–

  phosphate

 
• UV

  ultraviolet

We thank Nicole Wagner, Ashley Sanders, Alexandra Zalewski, Melanie Kingsbury and Michelle Marcus for their assistance during the experiments and for assisting with sample analysis. This work was supported by the Natural Sciences and Engineering Research Council of Canada through its Discovery grant program (P.C.F. and S.P.R.) and its USRA program (S.D.S.M.).