And Nutrients JOTARO URABE* and ROBERT W

Proc. Natl. Acad. Sci. USA Vol. 93, pp. 8465-8469, August 1996 Ecology

Regulation of herbivore growth by the balance of light and nutrients JOTARO URABE* AND ROBERT W. STERNER Department of Ecology, Evolution, and Behavior, University of Minnesota, St. Paul, MN 55108 Communicated by Eville Gorham, University of Minnesota, St. Paul, MN, April 19, 1996 (received for review November 11, 1995)

ABSTRACT Experiments using planktonic organisms re- proportional to P supply/algal biomass. This ratio implies that vealed that the balance of radiant energy and available above a certain light intensity where P limitation becomes nutrients regulated herbivore growth rates through their increasingly severe, the algal P/C ratio would start to decline effects on abundance and chemical composition of primary (3, 4). As a result, the algal P/C ratio is expected to reach a low producers. Both algae and herbivores were energy limited at value at high light. low light/nutrient ratios, but both were nutrient limited at Herbivore responses were hypothesized based on ingestion high light/nutrient ratios. Herbivore growth increased with of C and P (Fig. IB). Because algal C content varies only increasing light intensity at low values of the light/nutrient slightly with growth conditions (18), C ingested by the herbi- ratio due to increases in algal biomass, but growth decreased vore per unit time (Ic) would be proportional to the rate of with increasing light at a high light/nutrient ratio due to ingestion of algal cells, which we took to be a rectilinear decreases in algal quality. Herbivore production therefore was functional response (19). P ingestion per unit time (Ip) is equal maximal at intermediate levels ofthe light/nutrient ratio. The results contribute to an understanding of mass transfer mech- to Ic multiplied by the algal P/C ratio. The small plateau ofIp anisms in ecosystems and illustrate the importance of integra- in Fig. 1B is due to our assumption that the light level tion of energy-based and material-based currencies in separating light limitation from combined limitation by light ecology. and P is less than the light level causing algal biomass to satiate the herbivore's functional response. Depending on the re- Both light and nutrients are essential in sustaining ecosystems, but very little is known about how relative changes in these sponse of algae to given light and nutrients regimes relative to abiotic factors extend into food chains (1, 2). Plants use solar the functional response of herbivores, alternate configurations radiation to fix carbon while they acquire nutrients at appro- without a plateau are possible. The critical feature here is that priate rates to maintain their biological integrity. However, Ip is expected to reach a maximum level at an intermediate photosynthesis and nutrient uptake are not perfectly coupled, light intensity due to the difference in the direction of response- and thus the contents of bioelements relative to carbon (C) in to light intensity between the algal biomass and P/C ratio. plant biomass vary within species (3-6). Because foraging and The net production of carbon by herbivores is given by the growth of many herbivore species respond to the chemical balance of assimilated carbon minus metabolic loss (mainly composition of their diet (7-10), the balance between photosyn- respiration). However, if Ip is too low compared with Ic, the thesis and nutrient uptake may in turn regulate herbivores carbon net production may be lower than otherwise expected. through the interplay of food quantity and quality. In this report, Under such a condition, the carbon net production would be we test the hypothesis that herbivore growth is dependent on the a product of net P intake divided by the P/C ratio of the body light/nutrient balance supplied to laboratory ecosystems. tissue. Because the P/C ratio of herbivore biomass is constant We focused on phosphorus (P) as a limiting nutrient because (20, 21) and because P excretion approaches zero when algal growth is frequently limited by P in freshwater systems herbivores ingest food with low P/C ratio (22), herbivore (11, 12), and because the algal P/C ratio has been most growth rate in carbon units (G) can be expressed as strongly implicated in regulating planktonic herbivores (13- 17). We first considered the likely responses of algae to light G = min[Ic x ac - ,3, Ip x ap/Zp/c] [1] intensity for a given P supply with a moderate but constant loss rate (Fig. 1A). Here, we expressed the response of algae by a where ac and ap are production efficiencies for C and P, Zp/c rectilinear form to show the essence of trends and qualitative is the ratio of differences along the light gradient. Precise response to light P/C the herbivore, and C is the metabolic loss and nutrients would depend on the identity of the algal species rate of C (respiration). As an example, we show the response and other environmental factors. At low light, algal growth of G to light intensity by setting 0.8 for ac and ap and 5% of should be limited by irradiance such that algal biomass in- a maximum Ic for X3 (Fig. 1B). Eq. 1 suggests that herbivore creases with light intensity. At high light, algal growth should growth will decrease with increasing light if they cannot be limited by finite P and algal biomass should reach a plateau. compensate for decreased algal P/C by increasing P produc- At extremely high light, algal growth may decrease due to tion efficiency, which of course must be the case at 100% photoinhibition, but Fig. 1A assumes light is below the pho- production. Thus, herbivore growth may be maximal at inter- toinhibition point. The response of algal P/C ratio is also mediate light intensity at the point where algal composition shown in Fig. 1A. At low light, the algal P/C ratio is expected becomes deficient in P relative to herbivore demands. Fur- to be high, close to the Redfield ratio (0.0094 by atoms), thermore, the light intensity where herbivores show maximal because algal growth is limited by irradiance alone and thus P growth rate may decrease with decreasing P supply rate, supply is sufficient relative to algal biomass. Because the P because the algal P/C ratio at a given light intensity is expected supply rate is constant, per capita P availability depends on to be lower at lower P supply rate. algal biomass and the algal P/C ratio is expected to be Abbreviations: C, carbon; P, phosphorus; N, nitrogen. The publication costs of this article were defrayed in part by page charge *To whom reprint requests should be sent at the present address: payment. This article must therefore be hereby marked "advertisement" in Center for Ecological Research, Kyoto University, Shimosakamoto accordance with 18 U.S.C. §1734 solely to indicate this fact. 4-1-23, Otsu, 520-01, Japan. e-mail: [email protected]. 8465 Downloaded by guest on September 26, 2021 8466 Ecology: Urabe and Sterner Proc. Natl. Acad. Sci. USA 93 (1996) obtusa born within a 12-hr time span from the second clutch A of the maternal individuals were placed into each flask. Neonate dry mass was 1.80 ,ug (SD, 0.05). Because D. obtusa Light Nutrient initiates reproduction within 1 week under preferable food limitation limitation conditions and because it is difficult to quantify individual growth rate (somatic growth + reproduction rates) after release of offspring, we incubated Daphnia for 6 days and then measured their dry mass for estimation of growth rate. During the 6-day run, algae continued to be diluted every 2 days but animals were not diluted. Potential complications that could invalidate our results include the possibility for interference or blocking in food collection by very high food density (25) or a direct inhibitory effect of high light on animal performance. To overcome such difficulties, a second experiment was performed with an "adjusted" treatment and effects of food quality and quantity were separately assessed. In this experiment, semibatch algal cultures with six different nutrient concentrations (N/P ratio = 80:1) were established and maintained under the high (260 ,uE m-2.s-) and low (12 ,uE m-2.s-) irradiance as mentioned above. These treatments were used as controls. In parallel with these treatments, algal suspensions of adjusted treatments were made every 2 days by adding 100 ml of algal suspension from the high-light treatments to 900 ml COMBO medium without P and N, and placed at the low irradiance. Thus, animals in the adjusted treatments were offered food of similar composition to the high-light treatment, but algal biomass was reduced 10-fold. Twenty neonates born within 12 h were introduced to each treatment and body mass on day 6 was measured. C and P contents of algae were examined using 250-ml culture suspensions collected for replacement at 2-day inter- vals while D. obtusa was incubated. Known aliquots of the Light intensity suspension were filtered onto precombusted glass fiber filters and analyzed for algal P content by spectrophotometric means FIG. 1. Qualitative model showing responses of algal biomass and after oxidation P/C ratio (A) and herbivore ingestion and growth rate (B) to changes by persulfate (26), and analyzed for C content in light intensity. We assumed that all chemical elements besides P are using a Perkin-Elmer model 2400 CHN analyzer. Animals in not limiting and that algae suffer from a moderate but constant loss each treatment were pooled into samples of 3 to 5 individuals, rate. The model does not incorporate feedbacks from the herbivore to placed in preweighed aluminum boats, and dried at 60°C algal biomass or physiology. The scale of they axis differed among the overnight. Dry mass was measured with a Mettler model parameters and was not specified. Herbivore growth was found using UMT2 microbalance. Eq. 1 with ac = ap = 0.8, and 5% of a maximum Ic for 13- MATERIALS AND METHODS RESULTS AND DISCUSSION To test this set of predictions, we performed semibatch culture Both algae and herbivores showed responses consistent with experiments using the alga Scenedesmus acutus (Chlorophyta) our predictions (Fig. 2). In all nutrient concentrations, algal as a resource and Daphnia obtusa (Crustacea, Cladocera) as an abundance increased with light intensity, and at high light, herbivore. S. acutus and D. obtusa were obtained from stock algal abundance also increased with increasing phosphorus cultures maintained for >4 yr under constant lab conditions concentration. The algal P/C ratio was high at low light, being (23). We used several P concentrations and light intensities similar to or somewhat higher than the Redfield ratio at high typically found in freshwater lakes (24). The semibatch cultures P supply, and decreased with increasing irradiance. These were initiated by inoculating the algae into flasks containing quantitative and qualitative changes in the algae show that with 1 liter of medium. We used increasing light intensity, factors limiting algal growth shifted growth COMBO mediumt, which from light, which controls C uptake through photosynthesis, to supports long-term growth of both phyto- and zooplankton. P availability, which limits P uptake. At all P concentrations, Nutrient concentrations were adjusted by adding the desired body mass of 6-day-old D. obtusa was greatest at intermediate concentration of P as K2HPO4 and nitrogen (N) as NaNO3. light/phosphorus ratios (Fig. 2). In our experiments, body The N/P ratio was held at 80:1 (molar), so that N was sufficient mass is a good indicator of production rate because in no cases relative to P. Light was provided by cool-white fluorescent had animals released any offspring. Both maximum body mass bulbs, and light intensity was adjusted by a black window and the ligh-t intensity at which this maximum was reached screen placed over the bulbs. Light intensity was measured were greater in higher nutrient treatments. Low growth rates using a Li-Cor (Lincoln, NB) quantameter (LI-1000 with 2ir of herbivores at low light can be explained by low algal biomass. collector) placed just outside of the experimental flasks. Flasks However, we cannot explain lowered herbivore growth at high were shaken once per day by hand to homogenize the culture light intensity in the same way, because algal biomass was suspension. Every 2 days, 25% of the culture suspension was similar to, or even higher than that at, intermediate light. replaced by fresh growth medium. Algal density reached near Previous studies estimated a threshold food P/C ratio of maximum level within 6 to 8 d, after which 20 neonates of D. =0.0032 below which net production of Daphnia would be P limited even if they assimilate 100% of the P in the food (17). *Kilham, S., Annual Meeting of the American Society of Limnology Daphnia in the experiments showed a decreased growth rate and Oceanography, June 11-15, 1995, Nevada. when the algal P/C ratio was less than this threshold. Thus, it Downloaded by guest on September 26, 2021 Ecology: Urabe and Sterner Proc. Natl. Acad. Sci. USA 93 (1996) 8467 A 0.1 P 3 OpiM 6 6 0 10 Y (X - _~~~~~ ~ E _ 2 4 4 0 0 0. D 0 0 0 E 5 x 1 ~~ t / ~~~P:Cratio 2 2 ~~~~0 , CD - ;; 0 0 0 0

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FIG. 2. Responses of algae and herbivores to varying light intensity at different concentrations of phosphorus. Algal biomass (0) and P/C atomic ratio (a) are mean values during the incubation of the herbivore consumer. Herbivore growth is given as body mass at age 6 days (O). Error bars indicate standard deviation. The dashed horizontal line is the food P/C ratio below which the growth rate of the herbivore is expected to be limited by P rather than by C (17). Algal biomass, algal P/C ratio, and herbivore body mass differed significantly among the light intensities in all nutrient concentrations (P < 0.001, data log transformed). Maximum body mass at each nutrient concentrations was also significantly different among the nutrient concentrations (F3,12 = 37.6, P < 0.001).

is most likely that the lower herbivore growth at high light was that animals at low light, given food with low P/C ratio, still due to low P content in the food. showed low production rates. Again, herbivore growth was In the second experiment, in addition to low- and high-light high in foods with a P/C ratio greater than the calculated treatments, we measured Daphnia growth in an "adjusted" threshold for P limitation. In contrast, herbivore growth was treatment where algae from the high-light treatments were low in foods with a P/C ratio below this threshold. Thus, diluted and offered to the herbivores at the low-light level. quality was far more important than quantity in determining Regardless of nutrient concentrations, algal abundance in the animal production at high food biomass. adjusted treatment was close to the low-light treatment (Fig. However, when the P/C ratio was above the threshold, 3A). On the other hand, the algal P/C ratio in the adjusted chemical substances other than P may be important in deter- treatment was much lower than that in the low-light treatment mining food quality because animal mass in the two highest but was almost the same as the high-light treatment even after nutrient concentrations in the low light treatment (Fig. 3C, 2 days incubation (Fig. 3B). We found little or no difference in solid circles) was less than at the same nutrient concentrations animal mass at any nutrient concentrations between the high- in the high light treatment (Fig. 3C, open circles). This may be light and the adjusted treatments (Fig. 3C), indicating that the due to differences in C quality in the algal cell, such as the interference effect of high algal biomass was inconsequential. relative composition of cellulose and essential fatty acids. Furthermore, these results reject the possibility of direct Our experiments demonstrate for the first time that there is effects of light intensity on animal performance due to the fact an optimum light intensity relative to nutrient supply to Downloaded by guest on September 26, 2021 8468 Ecology: Urabe and Sterner Proc. Natl. Acad. Sci. USA 93 (1996) 8 Light/nutrient regimes can now be seen to affect ecological transfer efficiency from plant to herbivore. Existing theories ...... {.. .+ have pointed out that ecological transfer efficiency is a key 0- 6 parameter in regulating trophic dynamics and exploitation (27-29), but few studies have attempted to elucidate the factors 0E -, determining such transfer efficiencies. In Lake Biwa, the 0 .... 4 - &------largest lake in Japan, ecological transfer efficiency from 0C primary producers to zooplankton was higher for P than for C 2 because of elemental imbalances (30). Under such situations, changes in abiotic factors increasing C fixation rates alone could lead to further reductions in C transfer efficiency 0 because herbivores must eliminate a greater amount of C to maintain their own rather strict homeostasis. We induced these changes in chemical composition of algae 10 through direct manipulations in the light regime. However, we 0 r can speculate that increases in C fixation by plants due to globally increased availability of CO2 might have similar (a 0 effects, such that ecological transfer efficiencies may either O.)x increase or decrease, depending upon the stoichiometric bal- 6>--' 5 ance between producers and consumers for C, nutrients, and energy. For example, in a system where primary production is limited by nutrient supply rate, increase in the availability of CO2 is likely to decrease mineral/C ratio in producers and may 0 result in lowered quality of food for consumers. In addition, increased light transmission owing to the reduction in deoxy- cholate in lakes and streams affected by acid rain might be expected to show similar responses. 20 0) Early writers concerned with applying thermodynamic prin- .....0. High light .. . ciples to ecological systems wrote optimistically of how laws of * Low igtit energy and mass transfer would lay bare the workings of co .0....-0.... Adjusted ecosystems (31-33). Since Lindeman's (34) classic work, ef- co E 10 forts to understand ecosystems based solely or primarily on energy flows have been made, but their success and impact have arguably been limited (35-37). Our study demonstrates 0 that a greater integration of energetics and nutrient-based analyses may provide more predictive and explanatory power 0 to mass transfer and trophic structure. 0.1 1 10 We thank N. George for technical assistance, and J. Elser, P supplied [ pM ] A. Galford, T. Hara, S. Kilham, E. Litchman, S. Naeem, and D. Tilman for comments and suggestions. This work was FIG. 3. Biomass (A) and P/C ratio (B) of S. acutus and body mass supported by grants from the National Science Foundation. of D. obtusa at age 6 days at various nutrient concentrations in high-light treatment (260 ,uE m-2.s-1; 0), low-light treatment (12 ,uE 1. Hill, W. R., Ryon, M. G. & Schilling, E. M. (1995) Ecology 76, m-2.s-l; *), and adjusted treatment (OI) where biomass of high-light 1297-1309. algae was diluted to 10% and incubation was made at the low light 2. Wootton, J. T. & Power, M. E. (1993) Proc. Natl. Acad. Sci. USA (mean ± SD). The dashed horizontal line in B is the threshold food 90, 1384-1387. P/C ratio (17). In all nutrient concentrations, algal biomass in the 3. Goldman, J. C., McCarthy, J. J. & Peavey, D. G. (1979) Nature high-light treatment was significantly higher than the other two (London) 279, 210-215. treatments (P < 0.001), whereas algal P/C ratio in the low-light 4. Sommer, U. (1989) in Plankton Ecology: Succession in Plankton treatment was significantly higher than the other two treatments (P < Communities, ed. Sommer, U. (Springer, Berlin), pp. 57-106. 0.001). The body mass in the low-light treatment was significantly 5. Harrison, P. J., Thompson, P. A. & Calderwcrod, G. S. (1990) higher (P < 0.01) at the lower three nutrient concentrations but lower J. Appl. Phycol. 2, 45-56. (P < 0.001) at the higher two nutrient concentrations than the 6. Rhee, G.-Y. & Gotham, I. J. (1981) Limnol. Oceanogr. 26, high-light and adjusted treatments. 635-648. 7. Checkley, D. M., Jr. (1980) Limnol. Oceanogr. 25, 430-446. maximize production of animal biomass. At low light relative 8. Dale, D. (1988) in Plant Stress-Insect Interactions, ed. Heinrichs, to nutrients, herbivore production is governed by production of E. A. (Wiley, New York), pp. 35-110. autotrophs. Such systems are clearly best thought of as energy 9. Sterner, R. W. (1993) Ecology 74, 2351-2360. limited, both in primary and secondary production (1, 2). In 10. McNaugliton, S. J. (1988) Nature (London) 334, 343-345. contrast, beyond a certain point of light/nutrient balance, 11. Schindler, D. W. (1977) Science 195, 260-262. increases in light energy input for given nutrient supply actually 12. Elser, J. J., Marzolf, E. R. & Goldman, C. R. (1990) Can. J. reduce animal growth rates. These systems are nutrient lim- Fisheries Aquatic Sci. 47, 1468-1477. ited, and surprisingly, increasing influx of energy to the system 13. Hessen, D. 0. (1992) Am. Nat. 140, 799-814. is detrimental to herbivore the 14. Urabe, J. (1993) Arch. Hydrobiol. 126, 417-428. production. Thus, decoupling 15. Elser, J. J. & Hassett, R. P. (1994)Nature (London) 370,211-213. of nutrient uptake and photosynthesis by plants modulates a 16. Sterner, R. W. & Hessen, D. 0. (1994)Annu. Rev. Ecol. Systemat. balance between energy and nutrients that influences food 25, 1-29. chain dynamics in ways that at first seem paradoxical from the 17. Urabe, J. & Watanabe, Y. (1992) Limnol. Oceanogr. 37,244-251. view of energy input, but are reasonable from a standpoint of 18. Goldman, J. C. & McCarthy, J. J. (1978) Limnol. Oceanogr. 23, energy/nutrient balance. 695-703. Downloaded by guest on September 26, 2021 Ecology: Urabe and Sterner Proc. Natl. Acad. Sci. USA 93 (1996) 8469

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