Biochemical nutrient requirements of the calyciflorus: co-limitation by sterols and amino acids

Alexander Wacker*,l and Dominik Martin-Creuzburg2

1 Institute of Biochemistry and Biology, University of Potsdam, Am Neuen Palais to, 14469, Potsdam, Germany; and 2Umnological lnstitute, University of Konstanz, Mainaustrasse 252, 78464, Konstanz, Germany

Summary

1. It has been proposed that growth and reproduction of is frequently limited by mul­ tiple nutrients simultaneously. To improve our understanding of the consequences of multiple nutrient limitations (i .e. co-limitation) for the performance of animals, we conducted standard­ ized popUlation growth experiments using an important aquatic consumer, the rotifer Brachi­ onus calyciflorus. 2. We compared nutrient profiles (sterols, fatty acids and amino acids) of and their diets to reveal consumer- diet imbalances and thus potentially limiting nutrients. In concomi­ tant growth experiments, we directly supplemented potentially limiting substances (sterols, fatty acids, amino acids) to a nutrient-deficient diet, the cyanobacterium Synechococcus elonga­ (us, and recorded population growth rates. 3. The results from the supplementation experiments corroborated the nutrient limitations pre­ dicted by assessing consumer-diet imbalances, but provided more detailed information on co-limiting nutrients. While the fatty acid deficiency of the cyanobacterium appeared to be of minor importance, the addition of both cholesterol and certain amino acids (leucine and iso­ leucine) improved population growth rates of rotifers, indicating a simultaneous limitation by sterols and amino acids. 4. Our results add to growing evidence that consumers frequently face multiple nutrient limita­ tions and suggest that the concept of co-limitation has to be considered in studies assessing nutrient-limited growth responses of consumers.

Key-words: consumer, consumer- diet imbalance, dietary mismatch, fatty acid, global change, lipid, nutrition, phytoplankton, tetrahymanol, zooplankton

yet, experiments providing clear evidence for a co-limita­ Introduction tion of an by two or more essential nutrients are The concept of co-limitation, that is, a simultaneous limi­ scarce, in particular in regard to biochemical nutrients. tation by multiple nutrients, has been frequently used to Recently, using the freshwater herbivore Daphnia as a describe nutrient-limited growth responses of primary pro­ model organism, we have shown that the growth of this ducers (Davidson & Howarth 2007; Elser el al. 2007; herbivore can be limited simultaneously by a low availabil­ Saito, Goepfert & Rill 2008; Harpole et al. 2011). As yet, ity of dietary sterols and polyunsaturated fatty acids however, it has been poorly applied to the nutrient-limited (PUFAs) (Martin-Creuzburg, Sperfeld & Wacker 2009; growth of animals (Martin-Creuzburg, Sperfeld & Wacker Sperfeld, Martin-Creuzburg & Wacker 2012). Both sterols 2009; Lukas, Sperfeld & Wacker 201 1; Sperfeld, Martin­ and PUFAs are indispensable structural components of Creuzburg & Wacker 2012), which besides elemental nutri­ cell membranes (Hulbert & Else 2005; Hulbert el al. 2005) ents also rely on a number of essential biochemicals (e.g. and both serve as precursors for a number of bioactive Beenakkers, Van der Horst & Van Marrewijk 1985). As molecules. Sterols, for instance, are precursors of steroid hormones, such as ecdysteroids, which in arthropods are ·Corresponding author. E-mail: Alexander.Wacker@uni-pots­ involved in the process of moulting (Grieneisen 1994). dam.de Long-chain PUFAs, such as arachidonic acid (ARA) and 1136

eicosapentaenoic acid (EPA), a re precursors of eicosa­ (Weithoff & Wacker 2007; Wacker & Weithoff 2009). noids, which are thought to be relevant in invertebrate Thus, given their high ecological relevance for food web reproducti on, the immune system and o ther physiological processes rotifers a re, besides Daphnia, promising model processes (Stanley-Samuelson 1994; Heckmann et al. organi sms to investigate multiple resource limita tio ns a nd 2008). to deepen o ur knowledge o n the concept o f co-limitati on. In aquatic ecosystems, a low availa bility of essential lip­ Here, usi ng the rotifer Brachionus calyciflo/' us as a model ids a nd consequently a lipid limita tion of zooplankton has organi sm, we tested o ur hypothesis tha t also rotifers can been supposed to be associated wi th a high sha re of cyano­ be limited by mU ltiple nutrients simulta neously . In feeding bacteri a within the phyto plankto n (MUlier-N avarra el al. experiments, we compared nutrient profiles (sterols, fa tty 2000; Vo n Elert, M artin-Creuzburg & Le Coz 2003), acids a nd amino acids) of rotifers and their diets to detect because cyanobacteri a lack sterols a nd long-chain PUF As consumer- diet imbala nces a nd thus potentially limiting (> C 18) (Volkman 2003; Summons el al. 2006; Martin­ nutrients. In addition, we conducted population growth Creuzburg, Von Elert & Hoffma nn 2008). As cyanobacte­ experiments with rotifers feeding on a nutrient-defi cient rial bloom forma tion, with a ll its negative effects on wa ter diet, which was supplemented with cho lesterol, the most quality, is presumably favoured by global wa rming (J6hnk prominent sterol in a nimal tissues a nd/or EPA, e ither in et al. 2008; Paerl & Huisman 2008), a limitation of zoo­ the absence or in the presence of a n additional source of consumers by essential lipids may be intensified amino acids. T he significance of single dieta ry amino acid s in near future, in particul a r in eutrophic systems (Sperfeld for the performance of rotifers was assessed by experimen­ & Wacker 2009). tall y increasing the availability of several putatively In contrast to fa tty acids and sterols, the relevance of essentia l dieta ry amino acids. a mino acids as potentially limiting nutrients has been mostly neglected in food quality studies. However, it has been shown that the ava il ability of certain dietary amino Materials and methods acids correlates wi th the fecundity a nd hatching success of marine copepods (Guisande, Maneiro & Riveiro 1999; CULTIVATION OF FOOD ORGANISMS Guisa nde, Riveiro & Ma neiro 2000; Hell and el al. 2003) The coccoid cyanobacterium SYllecitococClis e/ongaltls (SAG 89·79; a nd a potential limitation of copepods by a low availabil­ culture collection of the University of Gottingen) and the fl agellate ity of the amino acid arginine has been suggested by Chiamy dolllOnas reillhardlii (SAG 11 - 32b) were cultured semicon­ Anderson, Boersma & Raubenheimer (2004) based on tinuously in modified Woods Hole Culture Medium (WC) with compari sons between amino acid profil es of copepods and vitamins (Guillard 1975) at 20 °C and an illumination of 30 and 120 m- 2s- 1 ( 16-h : 8-h light/dark cycle) in aerated I-L ves­ their food. Moreover, it has been suggested that specific ~lI no l sels, respecti vely. T he molar rati o of carbon to nitrogen to phos­ dietary amino acids, in pa rticular argin ine and histidine, phorus (CfN : P) of S. e/ongaLUs (130 : 16·5: I) was close to the are involved in triggering the switch between reproductive Redfield Ratio, indicating a high nitrogen and phosphorus content modes in Daphnia, that is, they have been found to avert of the food suspensions. Thus, a limitation by nitrogen or crowding-induced resting egg production a nd to enha nce phosphoru s was highly improbable. T he carbon concentrations of the food stock suspensio ns we re subita neous reproduction (Koch el al. 2011). As a mino estimated from photometric li ght exti nctions (a t 800 nm) using acid profiles of natural food sources vary seasona ll y, carbon-exti ncti on equations determ ined prior to the experiment. (Cowie & Hedges 1992; Petersson & Floderus 2001 ; Ali quots of algal or cyanobacteri al stock suspensions were added Kalachova e/ al. 2004), the perfo rma nce of freshwater her­ to sterile WC medium, and the resulting suspensions of bivores might be constrained not only by a low availability 2 mg C L - I were used as food/culture medium for rotifers. S. e/ollgatlls was used beca use it is a nontoxic, phosphorus-rich of essenti al dietary lipids, but also by the availa bility of cyanobacterium that has been shown to be of poor food qualit y certain amino acids, which potentiall y results in a simul­ for Dap/lllia due to the lack of sterols and long-chain PUFAs ta neous limita tion by mUltiple nutrient s. (Fig. I; Wacker & Martin-Creuzburg 2007; Martin-Creuzburg, Ro tifers a re importa nt components of freshwa ter food Sperfeld & Wacker 2009). webs (Walz 1995) tha t can domina te the zooplankton with densities often > 1000 individuals per litre (Modenutti 1994; LlPOSOM E PREPARATION Dagne el al. 2008). By feeding on bacteri a, algae, hetero­ trophic nano fl agell a tes and cilia tes (Po urriot 1977; Sanders The li posome stock solution was prepared from 3 mg I-palmitoyl- et al. 1989; A rndt 1993) a nd by serving as food, for exam­ 2-0Ieoyl-phosphatid ylglycerol (POPG) and 7 mg l-palmitoyl-2- oleoyl-phosphatidylcholin (POPC; Lipoid , Ludwigshafen, ple, for copepods a nd the larvae of insects and fi shes, they Germany) di ssolved in an aliquot of ethanol. Either 3·33 mg of a re significantly invo lved in transferring energy and nutri­ cholesterol or EPA (Sigma Aldrich, Deise nh ofen, Germany) was ents to higher trophic levels (Willia mson 1983; Walsh added from a cholesterol or EPA solution in etha nol. Solu tions 1995; Walz 1995). H owever, the efficiency with which roti­ were dried usi ng a rotary evaporator and dissolved in 10 mL of buffer ( 15 111 M [N-Tris(hydroxymethyl)met hyl-2-al11inoethanesulf­ fe rs use assimilated carbon for biomass productio n is onic acid, TES], 150 mM NaCI, pH 7·0). Afterwards, the liposome potentia lly constrained by a low avail ability of essential suspension was sonicated in an ultrasonic bath fo r 30 s and excess nutrients, tha t is, elements (Rothha upt 1995; Urabe, Cla­ free cholesterol or EPA was removed by washing the liposomes sen & Sterner 1997; Jensen & Verschoor 2004) or PU FAs in fresh buffe r using an ultra-speed centrifuge ( 15·000 g, 360 min, 1137

(a)_ Diet (Chlamydomonas) 100 c::::::::J Rotifer

b 10 OJ E .9 "0 'u (II ; 0 c 'E , (II i ; ; r 0 0·1 , 1 I "0 ; ij I 'u (II (b)_ Diet (Synechococcus) ~ .J!! 100 c=::::J Rotifer e 2 If) '0 10 c 0 ~ C r QJ U C ; 0 ; 0 i

, , I J j r I I 0·1 I ! \ ij, n I ,

Fig, l. Concentrati ons of sterols, tetrahymanol, fatty acids, and essential amino acids in (a) Chiamydolll onas reillhardlii and Brachionlls calycijlol"lls Pa ll as fed C. reinflardlii, and (b) SYllechococcw' elollgalLis and B. calyci/lol"lls fed S. eiongallls (mean ± standard de viati on, 11 = 3). Concentrations a re given in log-scale.

4 °C). In addition, control liposomes without further in gredient s a lLIS were provided daily so that the food concentration in we re prepared. All liposome suspensions were sto red at -25°C cultures was c. 2 mg C L I . until preparation of food treatments. The cholesterol and EPA content of li posomes was determined from subsamples; I ~I L of the liposome suspensions contained either 0·52 ± 0·02 ~l g choles­ ROT I F ER GROWTH E X PERIM E NTS terol or 0·17 ± 0·01 ~l g EPA (mean ± SO), respecti vely . T he mea n diameter of li posomes was 2· 1- 2·2 I.lIn (determined by a particle To standardize ex perimental conditio ns, growth ex periments were coun ter; CASY, Schiirfesystem, Germany). cond ucted with newly hatched juveniles that hatched in S. elonga­ IllS wi thout supplementa ti ons. J uveniles were collected and trans­ felTed randomly to the different trea tments. Ex periments lasted CULTIVATION OF ROTIFERS 6 days and were conducted at 20 °C in microti tre wells fill ed with 10 mL food/culture suspension. The rotifer strain (B. calyc!florLis Pallas) that we used in the Prior to the co-limitation ex periments, we conducted prelimin­ reported ex periniel1l s was originally o btained from K.-O. Roth­ a ry growth experiments using (i) WC medium witho ut any algal haupt (University of Konstanz). Since then, this strain has been diet, (ii) as before but with control liposomes, (iii) WC medium used as a 'rotifer model' in numero us studies (e.g. Rothhaupt enriched with 25 mg L - I dissolved bovine serum albumin (BSA; 1988; Fussmann , Weithoff & Yoshida 2005). Prior to th e ex peri ­ Sigma-Aldrich) to provide an un specific source of amino acids, ment , the rotifers were raised in glass beakers fi lled with 200 mL (iv) the latter combined with liposomes and (v) c. reillhardlii as a of sterile WC medium with saturati ng concentrati ons of C. reill­ reference food with a carbon conce ntration of 2 mg C L - I. With­ hardlii (>3 mg C L - I), to standard ize experimental conditio ns o ut any particulate food sources such as C. reillhardl ii or S. elollg­ and to start a ll experiments with animals in comparable nutri­ alliS, a ll rotifers died within 4 days irrespecti ve of whether they tional state; no mi xis was o bserved neither during preculturing were kept on pure WC medium o r on pure WC medium supple­ nor during the experiments. Befo re diet changes, rotifers we re mented with BSA and/o r liposomes (Fig. S I, Supporting Informa­ repeatedly sieved usin g a 55- ~un mesh and rinsed with sterile cul­ ti o n). Previolls tests also revealed that different concentrations of lU re medium to remove food o rga ni sms. To obtain suffi cient bi o­ control Ii posomes, 0·33, 0·66, 1·3 ~I L mL - I food suspension con­ mass for chemi ca l analyses, rotifers were reared in taining 2 mg C L - I S. e/ollgallls, did not significantl y affect semicontinuous cultures in 700 mL of WC at a density of c. 10 growth rates of rotifers. B. calyci/lol"lls per millilitre and fed either with C. reillhardlii o r In the first co-limitati on experiment , rotifers were fed S. elollga­ S. elollgallls. Fresh food suspensio ns of C. reillhardlii o r S. elollg- IllS (2 mg C L - I) supplemented with cholesterol and /or 1138

EPA-containing liposomes either in the absence or in the presence Wacker (2009), respectively, and normalized to the carbon content of BSA, that is, an additional source of amino acids. C. reinhardtii of the samples. was used as a reference food. Liposome supplemented diets were Prior to analysis of amino acids, the samples (store d under prepared by adding 0·167 !lL of liposome stock suspensions to' nitrogen at - 80°C until analysis) were hydrolysed with 6 M HCI I mL of food suspension resulting in 87 ng cholesterol mL - I andl at 110°C for 24 h, neutralized with 6 M NaOH and analysed or 28 ng EPA mL - I food suspension. This equals 43 ,lg choles­ according to Gzik (1996). The hydrolysed and lyophilized samples terol and 14 ,lg EPA mg- I cyanobacterial carbon, respectively. were dissolved in 80% methanol and aliquots were derivatized BSA supplemented diets were prepared by adding 25 !lg dissolved with o-phthaldialdehyde (800 mM borate butTer, pH 10·4, contain­ BSA to I mL of food suspension. ing 50 mg o-phthaldialdehyde, I mL methanol and 100 IJ,L 3-mer­ In the second co-limitation experiment, rotifers were fed either captopropionic acid) to yield stable, fluorescent derivatives. After unsupplemented S. elongatlls or S. elongaflls supplemented with a reaction time of 2 min, derivatives were separated by reversed­ one of 10 putatively essential amino acids (25 ,1M of food suspen­ phase high-performance liquid chromatography (Knauer GmbH, sion). A previous test showed that amino acid concentrations of Berlin, Germany) using an Aqua® column (5 !1m CI8 125 A LC, 25 !1M had no detrimental effect on rotifers but were in a sufficient 250 x 4·6 mm id; Phenomenex Inc. , Torrance, CA, USA), and a range to induce growth enhancing effects. Amino acids are abbre­ stepwise gradient of acetate buffer (pH 7·0) with increasing con­ viated as follows: histidine, His; arginine, Arg; threonine, Thr; centration of methanol. Eluents were detected using a fluorescence methionine, Met; valine, Val; phenylalanine, Phe; isoleucine, lie; detector (Model RF 551; Shimadzu, Kyoto, Japan). With this leucine, Leu; lysine, Lys and tryptophane, Try. C. reinhardtii was method, Try and Val as well as Gly and Thr, respectively, could again used as a reference food. not be separated. Amino acids were quantified using multipoint The initial density of rotifers in all growth experiments was one standard calibration curves previously determined for each amino rotifer per millilitre culture medium. At 24-h intervals, the food acid and normalized to the carbon content of the sample. suspensions were completely renewed and the rotifers were For the determination of POC and nitrogen aliquots containing counted under a stereo microscope. To avoid density dependent 1500 B. calycijlorus or 0·5 mg POC of C. reinhardtii and S. elong­ processes, 10 rotifers were randomly chosen and pipetted into new alus were filtered onto precombusted glass- fibre filters (25 mm food suspensions of 10 mL. In case of negative population growth diameter; Whatman GF/F) and after drying for at least 2 days at rates, all remaining individuals were transferred into new food sus­ 50°C analysed for carbon and nitrogen using an elemental analy­ pensions. Densities of rotifers in all treatments fluctuated below ser (EuroEA 3000; HEKAtech GmbH, Wegberg, Germany). For those reported to cause consumer-dependent effects by direct and the determination of particulate organic phosphorus concentra­ indirect consumer interference (Fussmann, Weithoff & Yoshida tions, aliquots of food suspensions were filtered onto polysulphone 2005). After 2 days of acclimatization, .the numbers of individuals filters (25 mm, 0-45 ,1m ; Pall Corporation, Ann Arbor, MI, USA) were used to calculate intrinsic growth rates for daily intervals and analysed using the molybdate-ascorbic acid method (M urphy using the equation: & Riley 1962) after dissolving tissues with sulphuric acid and oxidative hydrolysation by K 2S20 s at 120°C for 60 min. r = In(N,) - In(N, _I) eqn I

where r is the intrinsic growth rate (per day), and Nt and Nt _ 1 DATA ANALYSES are the animal numbers on consecutive days. The population growth rate of each replicate and treatment was estimated by cal­ Differences in population growth rates were analysed by two-way culating the average value of the intrinsic growth rates of rotifers ANOVA (co-limitation experiment I) or one-way ANOVA (co-limita­ on consecutive days. tion experiment 2) with subsequent post hoc comparisons (Tukey's HSD), when a factor or an interaction was significant (P < 0·05). Main factors were either BSA treatment and type of liposome ANALYTICAL PROCEDURES treatment (co-limitation experiment I) or amino acid treatment (co-limitation experiment 2). Normality was tested using the Shap­ For analytical purposes, rotifers fed with C. reinhardlii were iro- Wilk's test and homogeneity of variances using Fligner­ sieved using a 55-,nTI mesh and repeatedly resuspended and rinsed Killeen's test. Additionally, statistical models were tested for with sterile culture medium to remove food organisms. Then roti­ linearity and constant variances. According to Crawley (2002), fers were transferred to the target food suspensions C. reinhardtii data of co-limitation experiment 2 were boxcox transformed with and S. elongaflls (2 mg C L - I). Each rotifer culture was grown lambda = 3, that is, by using «I+y)' - 1)/3 to meet assumptions. for 3 days with food suspensions renewed on the second day. The All calculations .and statistical tests were conducted with the soft­ cultures were sampled I day after the last supply of food to mini­ ware package R, which is under general public licence (R Project mize potential contamination of the rotifer sterol, fatty acid and for Statistical Computing, www.R-project.org). amino acid profiles by undigested food particles from the digestive tract. Again the rotifers were sieved, repeatedly re-suspended and rinsed with sterile culture medium. Subsamples of rotifers were Results counted under a stereo microscope to determine rotifer densities, and aliquots containing 7500 individuals were filtered onto a 25- mm glass- fibre filter (What man, GF!C; Whaiman International CONSUMER - DIET IMBALANCE IN NUTRIENTS Ltd, Maidstone, UK). Likewise, aliquots of the C. reinhardtii and S. elongatus food suspensions, corresponding to 0·5 or 1·0 mg Considerable consumer-diet imbalances were detected for particulate organic carbon (POC). were filtered onto Whatman sterols, some n-3 PUFAs and in particular certain amino GFIF filters (25 mm). Prior to extraction of sterols and fatty acids, acids (Fig. I), suggesting a dietary deficiency and thus a 10 !lg of 51X-cholestan or 20 ,lg of tricosanoic acid methyl ester, potential limitation of rotifer growth by these individual die­ respectively (Sigma-Aldrich), were added as internal standards. tary compounds. The sterol composition of rotifers fed Filters were ex tracted with 7 mL dichloromethane/methanol (2: I; v: v) and stored under nitrogen at - 25°C until apalysis. Fatty C. reinhardtii was characterized by ergosterol (IUPAC name; acids and sterols of diets and rotifers were analysed according to (22E)-ergosta-5, 7,22- trien-3p-ol), 5-dihydroergosterol [(22E)- Wacker & Weithoff (2009) and Martin-Creuzburg, Sperfeld & 5cx-ergosta-7,22-dien-3p-ol], fungisterol (5cx-ergost-7-en-3p-ol), 1139

chondrill asterol [( 22E)-5cx-por iferasta-7,22-dien-3~-0 11, 22- ance in C I8 PUFAs, in particular in et- linolenic acid dihydrochondrillasterol (5cx-poriferast-7-en-3 ~-ol), and 7-de­ (ALA) and ' linoleic acid (UN) (Fig. I). Except for small hydroporiferasterol [(22E)-poriferasta-5, 7 ,22-trien-3 ~-oll. differences in the concentrations of Arg and lie, the amin o Ergosterol and 7-dehydroporiferasterol were the principal acid compositions and concentrations did not differ sterols detected in C. reinhard/ii and thus were presumably of between C. reinhard/a and rotifers fed C. reinhardlii. In dietary origin; the other sterols detected in the rotifers were contrast, a comparison of amino acid concentrations of absent from the diet. A ll these sterols, except 7-dehydroporif­ S. e/onga/us and rotifers fed S. e/ongatus revealed a high erasterol, were also detected in rotifers fed the sterol-free consumer- diet imbalance in lie, fo llowed by a somewhat S. e/ongatus (Fig. I), which suggest that these sterols were smaller imbalance in Leu, Met, Phe and Arg, suggesting a ei ther synthesized de novo or origin ated from preexperimen­ potential growth limitation by these dietary amino acids. tal feedi ng and were retained in the animals, (Fig. 2). This consumer-diet imbalance was evident for all sterols detected CO-LIMITATION EXPERIMENT 1 in the rotifers. In addition to sterols, the penta cyclic triterpe­ noid alcohol tetrahymanol was detected in the rotifers. Tetra­ Population growth rates of rotifers significantly increased hymanol was detected in somewhat higher concentrations in upon BSA supplementation by c. 0·3 day- I (differences animals fed the sterol-free S. e/ongatus than in animals fed between means; Fig. 2; Table I), irrespective of simulta­ the sterol-containing C. reinhardlii. neous supplementation with control liposomes or EPA­ The rotifers contained several long-chain PUFAs that containing liposomes. Similarly, population growth rates were absent from the diets (Fig. I). For instance, rotifers significantly increased upon supplementation with choles­ fed C. reinhard/ii contained significant amounts of C20 terol-containing Iiposomes by c. 0·25 day- I, irrespective of PUFA that could not be detected in C. reinhardlii. After simultaneous supplementation with BSA (Fig. 2; Table I, the diet switch to S. eionga/us, a comparison of PUFA Tukey's HSD, P < 0·001 following ANOVA). Population compositions revealed an additi onal consumer- diet imbal- growth rates significantly increased also by the s imulta­ neous supplementation with cholesterol- and EPA-contain ­ ing liposomes, but the obtained growth rates did not differ NoBSA significantly from those obtained by cholesterol supple­ 0·6 UIEJ With BSA mentation alone. The supplementation with control lipo­ somes or EPA-containing Iiposomes did not significantly B B increase population growth rates (Tukey's HSD, 0·4 - ':b P > 0·19). Q) fi A A A .c 0·2 CO-LIMITATION EXPERIMENT 2 ~ E Population growth rates of rotifers fed S. eionga/us OJ c increased significantly upon isoleucine and leucine sup­ 0 .~ 0·0 plementation (Fig. 3, Table I, Tukey's HSD, P < 0·0 I 'S a. and P < 0·05, respectively). All other amino acids did a...0 not significantly improve population growth rates com­ -0·2 pared to animals fed unsupplemented S. e/ongatus (Fig. 3, Tukey's HSD, P > 0·9). Growth rates increased upon isoleucine and leucine supplementation by 0·29 and -0-4 ,.,. 0·25 day- I compared with unsupplemented S. e/ongat/./s ~ 8> 9.'" 9.'" ~ ~ .~ (j"f ~ (Fig. 3, Table I), that is, in a simil ar range as observed ~Cf; 'f ,.,.'f ~ ~ ?' ~ ~ .~ upon BSA supplementation in co-limitation experiment I iii V:;~ V:;~ 0 "f ~ V:;. (j (Fig. 2). ~ CJ' V:;~

Fig. 2. Population growth rates (IJleans ± standard deviations, Discussion /I = 4) of Brachianus ca/ycijla/'lls Pallas raised on different food Our study shows that population growth of the rotifer regimes in a two-factorial design. One factorial treatment was the dietary presence of bovine serum albumin (BSA) in the food sus­ B. ca/ycijforus can be limited by more than just one nutri­ pensions and the second factor was the treatment with different ent simultaneously, that is, by sterols and certain amino kinds of lipids supplemented via liposomes. Treatment with BSA acids, which adds to growing evidence that multiple has a significant effect on growth (ANOVA, P < 0·001), regardless of resource limitation of consumers is a common phenome­ any interaction with the li posome supplementations. Since there non in aquatic ecosystems. We used a controll ed labora­ was no interaction between both factors, bars with different letters (A or B) display significant dilTerent levels of factor two, Ihal is. tory system with unialgal diets to control, for example, differences between lipid supplementations (P < 0·00 I ; Tukey prey selecti on and other confounding factors potentiall y HSD following two-way ANOVA). associated with the diversity of natural alga l diets. 1140

Table I. Statistical analysis of the obtained population growth rates of Brachiolllis calyciflorus Pallas raised on different diets

Population growth rate (per day)

Experiment Factor d .f. F-value P-value

Co-limitation experiment I Protein treatment (BSA) 1,30 312·3 <0·0001 Liposome treatments (Lipo) 4, 30 26·59 <0·0001 BSA x Lipo 4, 30 1·63 0·19 Co-limitation experiment 2 Amino acid treatment (AA) 10,33 6·73 <0·0001

In .co-limitation experiment I, differences in population growth rates were analysed by two-way ANOVA. The factor 'Iiposome tl-eatment' includes different factor levels consisting of different kinds of lipids supplemented to the diet via liposomes. The factor 'bovine serum albu­ min (BSA) treatment' represents the presence or absence of BSA in the food suspensions. In co-limitation experiment 2, the factor 'amino acid treatment' represents the presence or absence of single amino acids in the food suspensions (one-way design).

not C28- and C29- sterols, from radio-labelled acetate, but 1.21 Iil at the same time argued that cholesterol and the C28- and 0·4 C29- sterols detected were mostly of dietary origin_ In our study, we did not find any cholesterol in B. calyciflorus but several C28- and C29-sterols, suggesting that ch"Olesterol is 0·2 B not synthesized by B. calyciflorus even under sterol limita­ 1- Be tion. One would expect that the putative sterol synthesising ~ pathway is highly conserved among rotifers and that, .<: A A A therefore, the two species B. plicatilis and B. calyciflorus ~ 0·0 e should have similar capabilities. However, although we Cl c o cannot definitely infer from our data whether B. calyciflo­ ~ rus is capable of synthesising sterols de novo, the growth :; -0·2 c. enhancing effect of sterol supplementation suggests that ~ the animals are either incapable of synthesising sterols de novo or that the rates of synthesis are too low to meet -0·4 physiological demands. The finding that B. calyciflorus maintained relatively high body sterol levels even in the ,5' :p ,p~ ~ .:;,.'& !!' ::g; i' ~ ~

Supplementation with EPA did not affect population where no larvae survived (Chang 2004). The beneficial growth rates of B. calyelfiorus (Fig. 2), suggesting that a effect of leucine on population growth of B. ealyeiflorus is dietary source of EPA is not required. However, this is in in accordance with a previous study showing that egg pro­ contrast to previous studies reporting that egg production duction of the rotifer Keratella quadrata was correlated, of the rotifer Keralella quadrata fed ciliates was correlated among other biochemical nutrients (fatty acids and ster­ with the availability of dietary PUFAs, in particular EPA ols), with the leucine content of its protist food source (Boechat & Adrian 2006). Recently it has been shown that (Boechat & Adrian 2006). environmentally induced changes in dietary PUFA avail­ The availability of amino acids might correlate well with abilities (Spijkerman & Wacker 2011) strongly affect the that of nitrogen, and nitrogen was suggested as surrogate growth of rotifer from acidic mining lakes (Weit­ measurement for ami·no acids (Jensen et al. 2006; Hessen hoff & Wacker 2007) and their competitive abilities (Hart­ et al. 2007). Therefore, one might speculate that the wich, Wacker & Weithoff 20 I0). These contradictory observed positive effects of BSA or amino acid supplemen­ findings suggest that different dietary constraints arise tation are in fact due to the increased availability of nitro­ from feeding on cyanobacteria, as in the present study, gen rather than to the addition of essential amino acids. and eukaryotic food sources, as in the cited studies. However, in our direct supplementation approach we did The ecological relevance of a dietary deficiency in essen­ not find evidence for a potential limitation of the rotifers tial amino acids is potentially high, as suggested by large by particularly nitrogen-rich amino acids, such as arginine consumer-diet imbalances in certain amino acids found in or histidine. Only the addition of leucine and isoleucine several aquatic and terrestrial consumers (Anderson, (with low nitrogen contents) improved population growth Boersma & Raubenheimer 2004). In general, the amino rates of B. ealyelfiorus (Fig. 3), clearly indicating that the acid content of invertebrate (0ie & Olsen 1997; Tidwell rotifers were limited by the low dietary availability of these et al. 1998; Helland et al. 2003) and vertebrate (Alam essential biochemical molecules and not by the availability et al. 2002) consumers tends to be regulated homoeostati­ of dietary nitrogen. In general, the dietary supply of ele­ cally, that is, despite considerable changes in dietary amino mental nutrients was high in the present study and thus a acid availabilities, changes in the amino acid content of limitation of B. calyeijforus by phosphorus or nitrogen consumers are rather low. The tendency of consumers to appears unlikely. Moreover, the supplementation of maintain certain amino acid concentrations within their S. elongatus with control liposomes, which provide an body may increase the .risk of a limitation by single amino extra source of carbon and phosphorus (Martin-Creuz­ acids, in particular by those amino acids which are tempo­ burg, Von Elert & Hoffmann 2008), did not improve pop­ rarily underrepresented in the diet (Kalachova et al. 2004). ulation growth rates, showing that phosphorus supply and This assumption is supported by the observation that the food concentrations in general were sufficient. Hence, stud­ hatching success of copepods is improved when differences ies investigating nutritional requirements of rotifers should in the amino acid composition between females and their also consider essential biochemicals as potentially limiting diet are less pronounced (Guisande, Maneiro & Riveiro nutrients and should not merely focus on insufficient die­ 1999; Guisande, Riveiro & Maneiro 2000; Helland el al. tary supplies of minerals such as phosphorus and nitrogen 2003) . In the theoretical approach of Anderson, Boersma as has been done in the past (Rothhaupt 1995; Strojsova & Raubenheimer (2004), a comparison of amino acid pro­ et al. 20 I 0). files of copepods and their phytoplankton prey revealed a We show here, for the first time, that the rotifer B. ealy­ significant consumer - diet imbalance for arginine and elf/orus relies on a dietary source of sterols and that a die­ threonine, suggesting that the performance of copepods is tary sterol-deficiency results in reduced population growth potentially constrained by the dietary availability of these rates, as indicated by a growth enhancing effect of sterol amino acids. Following the same approach, our data supplementation. Similarly, we show that population revealed considerable differences in the concentrations of" growth rates of B. ealyeijforus, on the same nutrient-defi­ certain amino acids, in particular leucine and isoleucine, cient diet, can be improved by amino acid (leucine, isoleu­ between S. elongatus and rotifers fed S. elongatus, suggest­ cine) supplementation, indicating a simultaneous ing a potential growth limitation of rotifers by leucine and limitation by sterols and amino acids. The substantial isoleucine (Fig. I). This was corroborated by the amino increase in growth rates as a response to sterol and amino acid supplementation experiment, clearly showing that the acid supplementation demonstrates that a dietary defi­ population growth of B. ealye!florus on S. elongatus was ciency and not the production of toxins or growth inhibi­ in fact constrained by the availability of leucine and iso­ tors are responsible for the low food quality of leucine. S. elongatus. Rotifers significantly feed on cyanobacteria, The hypothesis that isoleucine is of general importance even if these are toxic (Snell 1980; Walz 1995; Soares, for invertebrate growth and reproduction is supported by Lurling & Huszar 2010). Hence, our data suggest that the the finding that a lack of isoleucine in the diet of the Medi­ growth of rotifers is potentially constrained simultaneously terranean fruit fly , Ceratitis eapitata, resulted in lowest lar­ by a deficiency in dietary sterols and certain amino acids. val growth compared to the absence of other potentially This might be particularly relevant when the phytoplank­ essential amino acids, except for threonine and tryptophan ton is dominated by cyanobacteria. Although the addition 1142

of these biochemical- compounds considerably improved Bee nakk ers. A.M.Th .. Van der Horst. OJ. & Van Marrewij k. W.1 .A. the food quality of S. elongatlls for B. calyciflol'lIs, popula­ ( 1985) Insect lipids and lipopro.tein s. and their physiological processes. Progress ill Lipid Resellrch. 24. 19- 67. tion growth rates obtained on the supplemented di ets were Boechat. I.G . & Adrian. R. (2006) Ev idence for biochemica l li mitati on of significantly lower than those obtained on a sole diet of popu lation growth and reproduction of the rotifer Kel'o/ella quai/rata fed Chlamydomonas (Figs 2 a nd 3), suggesting that other food with fres hwater protists. } olll'llal '!f'PlllllkIOIl Research, 28. 1027- 1038. Chang. e.L. (2004) EfTect of amino acids on larvae and adults of Cerlililis quality constraints become important once the sterol and Cllpilalll (Diptera: Tephritidae). Allllllls of'lhe Ell w mologiclil Sociely 0/ amino acid requirements are met. Americli. 97. 529- 535 . Although our approach is straightforward in identifying Cowie. G .L. & Hedges. J .1. (1992) Sources and reactivities of amino acids in a coastal marine environment. Limnology & Oceanography, 37. 703- 724. limiting dietary compounds it does not consider the poss ibil­ Crawley. M.1 . (2002) S lali."iclil Complilillg: All 1II II'OdllClioll 10 Data Allllly­ ity of using complementary food sources to obtain nutrient sis Usillg S-PlIIS. John Wil ey & Sons. Chichester. balanced diets or compensatory feeding, that is, the Dagne. A .. Herzig. A .. Jersabek. C. D. & Tadesse. Z. (2008) Abundance. species composition and spatial di stribution of planktonic rotifcrs and increased uptake of certain food sources to satisfy the cru stacea ns in Lake Ziway (Rirt Valley, Ethi opia). International RelJieltl requirements for single di etary nutrients (Raubenheimer & o/ llydrobiology, 93. 2 10- 226. Jones 2006). However, during extensive cyanobacteria Davidso n. E.A. & Howa rth . R.W. (2007) Environmental science: Nutrients in synergy. NIIIIII'e. 449. 1000- 1001. blooms complementary and compensatory feeding might be Elser. J.1 .. Bracken. M.E.S .. Cleland . E.E .. Gruner. D.S .• Harpole. W.S .. of minor importance because rotifers potentially do not have Hillebrand . H .. Ngai. J.T.. Seabloom. E.W .. Shurin. J.B. & Smith. J. E. sufficient alternative food sources and may depend on poten­ (2007) Global analysis of nitrogen and phosphorus limi ta ti on o f primary producers in freshwater. marine Hnd terrestria l ecosyste ms. Ecology Lel­ tial improvement of the sterol and amino acid avail ability leI'S. 10. 1135- 1142. through seasonal changes of the phytoplankton community. Fussnlann, G.F., Weithon'. G. & Yoshida. T. (2005) A direct. experimcntal We show here, for the first time, that an aquatic inverte­ test of resource vs. consumer dependence. Ecology. 86. 2924-2930. Grieneisen. M.L. 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Riveiro. I. & Maneiro. I. (2000) Compari sons a mong the is more widespread as expected, and that (ii) biochemical amino acid compositi on of females. eggs and food to determine the re la­ nutrient requirements differ among phylogenetic groups. A tive importance of food quantity and food qua li ty to copepod reproduc­ tion. Mal'ille Ecology,Pmgl'es., Serie.\'. 202. 135- 142. limitation of rotifers by these essential biochemical nutri­ Gzik . A. (1996) Accumulation or proline and pattern of a lph a-amin o acids ents may also affect the functioning of freshwater food in sligar beet plants in response to osmo tic, wa ter and salt st ress. EIIII;­ webs, as rotifers are significantly involved in the carbon roltmellial and Experill1eJllal BOlrmy, 36. 29- 38. Ha giw::lfct . A., Hamada. K .. Nishi , A .. Im aizumi . K, & Hirayam a. K. transfer to higher trophic levels. 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