The stoichiometric knife-edge refers to the reduced performance of consumers encountering food with excess phosphorus (P) relative to carbon (C) or nitrogen (N). Studies that provide evidence for such knife-edge in aquatic systems often apply phosphate supplementation to create P-rich food treatments. However, this method may suffer from artefacts, because after uptake algae may store P in a form different from the P-rich biomolecules typically consumed by zooplankton. Our aim was to test if P supplementation results in potential biases. We experimentally exposed populations of the herbivore rotifer species, Brachionus calyciflorus (Pallas), to four different food quality treatments: algae grown under P-saturating  (HP_chem, molar C:P ratio=59.7±2.7) and P-sufficient (MP_chem, molar C:P=116.3±5.2) conditions in chemostats, and algae grown under P-limiting conditions, but with molar C:P ratios equal to  HP_chem and MP_chem treatments, respectively (HP_LP+P, molar C:P =59.8±0.14; MP_LP+P, molar C:P=121.0±4.3). The latter two treatments were achieved through P-supplementation of P-limited algae. Results show that for rotifers fed algae with either excess or intermediate P content, population growth rates were consistently higher on algae grown in chemostats than algae treated with the P supplementation method. Importantly, growth rates were also consistently lower in HP than in MP treatments and the magnitude of this negative impact was independent on algal growth history. The latter result confirms the existence of a stoichiometric knife-edge, and indicates that P supplementation is a reliable method to study the relative effect of excess P on zooplankton performance in a standardized way.

We cultured C. reinhardtii in nine continuous 2L-chemostats at 23±1℃ using modified WC (Woods Hole Chu-10) medium (Guillard and Lorenzen, 1972) at a dilution rate of 0.33 day-1. More specifically, we created algae with contrasting C:P ratios by culturing them under different combinations of nutrient and light conditions: (1) high P supply (120 µmol P L-1, using K2HPO4 ) at 40 µmol quanta m-2 s-1 continuous light, resulting in phytoplankton cultures with a molar C:P  ratio of 60, further referred to as 'HPchem'; (2) intermediate P supply (65 µmol P L-1) at 40 µmol quanta m-2 s-1 of continuous light, yielding algae with a molar C:P of 115 (‘MPchem’); (3) low P supply (15 µmol L-1) at 120 µmol quanta m-2 s-1 continuous light, resulting in algae with a C:P of 600 (‘LPchem’).

During the experiment, we created two food quality treatments through P-supplementation. One treatment was created by enriching the LPchem algae with an amount of inorganic phosphate so that it achieved a molar C:P ratio similar to that of the MPchem algae (further referred to as the ‘MPLP+P’ treatment). The other treatment was created by enriching the LPchem algae so that they achieved a molar C:P ratio similar to that of the HPchem algae (‘HPLP+P’). Together with the MPchem and HPchem algae, the MPLP+P and HPLP+P algae provided four different food quality treatments with the MPLP+P and HPLP+P treatments combining a history of growth under P-limited conditions with an artificially elevated cell P content through P supplementation. The algae for HPchem and MPchem treatments were harvested daily from the chemostats and diluted with nutrient free WC medium. To create the MPLP+P and HPLP+P treatments, we first harvested algae from LP chemostats and then added inorganic phosphate (K2HP04, 0.05 mol L-1) to LP algae 90 minutes before feeding them to experimental rotifer cultures. Estimates of the amount of P needed for the algae to obtain the target molar ratios were based on the algal C content estimated from cell counts (Multisizer 3TM Coulter Counter, Beckman Coulter) and a C – biovolume regression relationship. For all the four food quality treatments, algae were kept in the dark for 90 minutes before being fed to the rotifers.

Experimental rotifer populations were fed with the four food quality treatments and their daily population growth rates calculated. Elemental composition of the food quality treatments were also assessed: algae were collected on GF/F filters and dried in an oven at 60℃. Algal C and N contents were determined using a FLASH 2000 organic element analyzer (Interscience B.V., Breda, The Netherlands), while P content was determined by a QuAAtro segmented flow autoanalyzer (Beun de Ronde, Abcoude, The Netherlands). Food C:P, N:P and C:N ratios were calculated as molar ratios.