Daphnia s dilemma: adjustment of carbon budgets in the face of food and cholesterol limitation

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1 2014. Pulished y The Compny of Biologists Ltd The Journl of Experimentl Biology (2014) 217, doi: /je RESEARCH ARTICLE Dphni s dilemm: djustment of cron udgets in the fce of food nd cholesterol limittion Mrcus Luks* nd Alexnder Wcker ABSTRACT We studied the cron (C) metolism in Dphni when the mount of C (food quntity) nd/or the content of iochemicl nutrients (food qulity) ws limiting. Growth performnces nd C udgets of Dphni mgn (ssimiltion, feces egestion, excretion nd respirtion mesured y [ 14 C]-trcing) were nlysed when nimls were rised on different food quntities nd concentrtions of cholesterol, n essentil iochemicl food compound. Cholesterol is of specil interest ecuse it not only cts s limiting nutrient ut lso contriutes to the overll C pool of the nimls. As the tissue cholesterol concentrtion in Dphni is quite low, we hypothesized the selective exclusion of cholesterol from C udgeting nd tested this using rdiolelled cholesterol. Somtic growth rtes of D. mgn were highest t high quntity nd qulity nd were reduced to moderte vlue if either the food quntity or the cholesterol concentrtion ws low. Growth ws lowest t low food quntity nd qulity. The mesurements of C udgets reveled high regultive response to low food qulity t high food quntity only. Here, low dietry cholesterol cused ulk C ssimiltion (AE) to decrese nd ssimilted (excess) C to e incresingly respired. Additionlly, Dphni enhnced efficient djustment of C udgets when fcing cholesterol limittion y (1) incresing the AE of the cholesterol itself nd (2) not chnging cholesterol respirtion, which ws still not detectle. In contrst, t low food quntity, Dphni is unle to djust for low food qulity, emphsizing tht food limittion could overrule food qulity effects. KEY WORDS: Biochemicl limittion, Cron udgets, Zooplnkton, Cron pthwy, Food qulity, Food quntity INTRODUCTION Informtion on the flow of energy nd nutrients is necessry for the understnding of the individul performnce of consumers, trophic interctions nd regultion in food wes (Andersen, 1997; Gedke et l., 2002). In qutic ecosystems especilly there re two mjor issues controlling the interction etween primry producers nd primry consumers: first, the mount of energy [e.g. in terms of cron (C)] supplied y the phytoplnkton community (food quntity) nd, second, the content of nutrients (e.g. minerls or essentil iochemicls) in the lge (food qulity). Cldocerns, s predominnt filter-feeding zooplnkton, re limited y energy vilility ecuse of low C concentrtions, e.g. in the cler-wter phse in spring (Sommer et l., 1986; Jeppesen et l., 1999) or due to reduced ingestiility (e.g. Gliwicz nd Lmpert, 1990) nd/or digestiility (Vn Donk et l., 1997; DeMott et l., 2010). Besides energy vilility, Deprtment of Ecology nd Ecosystem Modelling, Institute of Biochemistry nd Biology, University of Potsdm, Am Neuen Plis 10, D Potsdm, Germny. *Author for corresponding (mrcus.luks@gmil.com) Received 18 July 2013; Accepted 18 Novemer 2013 zooplnkton might e ffected y the low nutritionl qulity of the food, ecuse nimls otin lrge set of essentil or nerly essentil requirements from their food (Sterner nd Schulz, 1998). Mny recent studies hve investigted the performnce of herivores t imlnced element to C rtios (Sterner nd Elser, 2002) or imlnced rtios of mcronutrients (Ruenheimer nd Simpson, 2004). Recent studies hve focused on herivore growth limittion y polyunsturted ftty cids, mino cids, vitmins nd sterols (Anderson et l., 2004; Wcker nd Mrtin-Creuzurg, 2012). Sterols re essentil food components for herivorous rthropods (Behmer nd Nes, 2003; von Elert et l., 2003), which cnnot synthesize cholesterol (the predominnt niml sterol) de novo ut metolize it from phytosterols in their diet (Svood nd Thompson, 1985). Cholesterol serves s precursor for moult-inducing ecdysteroids in rthropods (God, 1981). Moreover, cholesterol is n indispensle component of plsm memrnes nd, ecuse of its stilizing properties (Roertson nd Hzel, 1997), is necessry for memrne temperture dpttion (Crockett, 1998; Sperfeld nd Wcker, 2009; Sperfeld nd Wcker, 2011). Feeding on cynocteri cn cuse growth limittion of herivorous crustcens (von Elert et l., 2003) ecuse cynocteri usully lck sterols (Volkmn, 2003), n effect possily excerted during cynocteri looms (Wcker nd Mrtin- Creuzurg, 2007). However, herivore sterol limittion might not e restricted to cynocteri looms s eukryotic lge cn lso e poor in sterols. In prticulr, high light intensity nd low nutrient vilility (for instnce in summer) cn reduce sterol concentrtions in lge elow criticl levels for herivorous zooplnkton such s Dphni (Piepho et l., 2010; Piepho et l., 2012). Furthermore, not ll phytosterols of eukryotic lge re suitle precursors for cholesterol, nd they vry in their conversion to cholesterol (Mrtin-Creuzurg nd Von Elert, 2004). Thus, the growth of herivorous crustcens my depend not only on the mount of sterols in their diet ut lso on the phytosterol composition (Piepho et l., 2010), which is determined y the phytoplnkton community composition. Recent studies hve focused mostly on the effects of different food conditions on herivores growth, ut neglected mesurements of C prtitioning into different physiologicl frctions. However, such knowledge is very importnt to predict the contriution of herivores to the overll C cycling in freshwter systems (He nd Wng, 2006). Dphni is model orgnism of freshwter ecology for severl resons: for exmple, Dphni plys n importnt ecologicl role s keystone species in qutic ecosystems, there is welth of informtion out Dphni s iology, nd its complete genomic informtion is ville (Lmpert, 2011). Dphni hs severl ehviourl nd metolic dpttions for deling with food limittions. Low food quntity cuses Dphni to filter t mximl rte to increse the C ssimiltion, nd lso results in n increse in the retention time of food in the gut (Geller, 1975; DeMott et l., 2010). Moreover, losses of C re diminished y reducing respirtion (Lmpert, 1986; Ure nd Wtne, 1990; Schmoker nd The Journl of Experimentl Biology 1079

2 The Journl of Experimentl Biology (2014) doi: /je Hernndez-Leon, 2003). This response of the respirtion rte to chnging food concentrtions refers to the increse in energy expenditure tht occurs during digestion, nd is defined s specific dynmic ction (SDA) (e.g. Kiøroe et l., 1985; Lmpert, 1986; Secor, 2009). In contrst, when food qulity is low, dphnids hve to either improve the ssimiltion of the potentilly limiting compound in their diet or get rid of the excess of other dietry ingredients, mostly C [see review y Hessen nd Anderson (Hessen nd Anderson, 2008) nd model pproch y Anderson et l. (Anderson et l., 2005)]. However, orgnisms my increse their fitness using this excess of C for other purposes, such s storge, structure nd defence (Hessen nd Anderson, 2008). Recent studies with phosphorus (P)-limited Dphni showed djustments in ingestion rte (Drchmeu nd Thys, 2005) nd ssimiltion of (excess) C nd P (DeMott et l., 1998). Furthermore, dphnids my compenste for poor food qulity y incresing C excretion (He nd Wng, 2008) nd respirtion (Drchmeu et l., 2003; Jensen nd Hessen, 2007), which depends on cclimtion time (Luks nd Wcker, 2014). Unfortuntely, food qulity spects sed on the iochemicl composition of the diet re missing in these studies. However, it is importnt to tke the iochemicl composition of food into ccount, ecuse the incorportion of C into proteins, lipids nd polyscchrides depends on it (Thor et l., 2002). Consequently, our purpose ws to improve our knowledge of the processes tht regulte C udgets for compounds such s sterols. Such informtion will e essentil to understnd the homeosttic regultion of sterols in dphnids (Sperfeld nd Wcker, 2009), prticulrly ecuse sterols (nd other essentil iochemicl food components) contin C nd re consequently prt of the overll C pool of the nimls. If Dphni were not le to spre cronic sterols from C losses y egestion of feces, excretion nd respirtion, the elimintion of excess C would e useless or even detrimentl. Therefore, we hypothesize tht Dphni re le to selectively exclude sterols from C losses. In this study, we cclimted Dphni mgn Strus 1820 to different regimes of food quntity nd iochemicl qulity (cholesterol) nd exmined their C udgets y mesuring egestion, excretion nd respirtion using the rdiolelled C method. Cholesterol udgeting ws tested using rdiolelled cholesterol. We predicted tht cholesterol would e spred from C losses (feces egestion, excretion nd respirtion) when the nimls fed on cholesterol-deficient diets. RESULTS Somtic growth The growth of D. mgn ws limited y food quntity s well s food qulity in terms of dietry cholesterol (two-wy ANOVA, cholesterol: F 1,8 =607.2, P<0.001, food quntity: F 1,8 =510.7, P<0.001; Fig. 1). When the cholesterol concentrtion nd food quntity ws high (HC/HQ), D. mgn reched the highest growth rte (P<0.001, Tukey s HSD). If either the quntity or the cholesterol content of food ws low, growth rtes of D. mgn were reduced to the sme moderte vlue (which pprently depends on the prticulr comintion of cholesterol deficiency nd food quntity limittion). At high food quntity (HQ), low cholesterol concentrtion (LC) cused strong decrese of D. mgn growth rte y 50% compred with growth under HC/HQ. In contrst, when food quntity ws low (LQ), low food qulity (LC) led to decrese in growth of only 37% compred with growth under HC/LQ (ANOVA, two-wy interction, cholesterol food quntity: F 1,8 =111.5, P<0.001). When oth food quntity nd cholesterol concentrtion were reduced simultneously (LC/LQ), the growth rte ws lowest. Somtic growth rte (dy 1 ) LC/LQ HC/LQ LC/HQ HC/HQ Fig. 1. Somtic growth rtes of Dphni mgn fed with food of different qulity nd quntity. LC, low cholesterol; HC, high cholesterol; LQ, low food quntity; HQ, high food quntity. Dt re mens ± 1 s.d.; n=3. Sttisticlly significnt differences re indicted s different letters (Tukey s post hoc test, P<0.001). Pulse-chse feeding experiment In generl, nerly ll of the mesured processes of D. mgn C udgets were ffected y the quntity of the food nd its cholesterol concentrtion (two-wy ANOVAs in Tle 1). ws the exception. Interestingly, neither food quntity nor food qulity significntly ffected C excretion in D. mgn, lthough there ws mrginl effect of cholesterol. The solute vlues of mesurements re displyed in supplementry mteril Tle S1. Assimiltion, feces nd gross growth Cron ssimiltion (AE C ) ws strongly ffected y oth food quntity nd dietry cholesterol concentrtion (Tle 1, Fig. 2). When the food quntity ws low (LQ), AE C ws highest nd did not differ etween high (HC) nd low cholesterol (LC) tretments (LC/LQ: 82.7±4.6%; HC/LQ: 88.6±3.5%, men ± 1 s.d., n=5). However, t high food quntity (HQ), AE C ws generlly lower nd, dditionlly, it vried etween high nd low dietry cholesterol concentrtions. Hence, we found the lowest AE C when cholesterol ws low nd food quntity ws high (LC/HQ: 50.7±3.1%; HC/HQ: 67.9±7.9%). This pttern of AE C led to concordnt results of feces mesurements nd gross growth (GGE C ) clcultions (Fig. 2). In greement with the high AE C t low food quntity, the egestion of feces ws low nd did not differ etween cholesterol concentrtions. In contrst, t high food quntity, low dietry cholesterol resulted in high egestion of feces (Fig. 2). Hence, when D. mgn were presented with high food quntity, GGE C decresed t low cholesterol. In contrst, GGE C did not differ etween food qulities nd ws generlly higher t low food quntity (Fig. 2). Net growth, excretion nd respirtion The C net growth (NGE C ), which contins informtion out the proportion of ssimilted C used for production, showed similr pttern s the GGE C when food quntity nd the cholesterol concentrtion in the food were chnged (Fig. 3). At low food quntity, NGE C ws high nd not different etween the two cholesterol concentrtions. At high food quntity, dphnids reched the sme high NGE C when cholesterol ws non-limiting. In contrst to the results t low quntity, lower cholesterol concentrtion led to lower NGE C when food quntity ws high. In the ltter scenrio, we found n incresed respirtion t low dietry cholesterol nd high food quntity (Fig. 3). At low food quntity, this effect of food qulity on respirtion ws not present. The excretion rtes of D. c The Journl of Experimentl Biology 1080

3 The Journl of Experimentl Biology (2014) doi: /je Tle 1. Results of two-wy ANOVA on the effect of food qulity (cholesterol) nd food quntity on severl cron pthwys in Dphni mgn Cholesterol Food quntity Interction Proportion (Efficiency) F 1,16 P F 1,16 P F 1,16 P Assimiltion/ingestion (AE) 24.4 < < Feces/ingestion < Production/ingestion (GGE) < Production/ssimiltion (NGE) /ssimiltion* Respirtion/ssimiltion < *d.f.=15. AE, ssimiltion ; GGE, gross growth ; NGE, net growth. Bold P-vlues indicte P<0.05. mgn were not significntly ffected either y the food quntity or y the cholesterol concentrtion in the food (Tle 1). Nevertheless, we found mrginl increse in excretion rte t low food qulity (two-wy ANOVA, P=0.073). Cholesterol in C udgets Compred with ssimiltion efficiencies (AE) of ulk C t high quntity (Fig. 2), the AE of cholesterol were high (c. 86%) nd did not differ etween the two cholesterol concentrtions (Fig. 4). Accordingly, the egestion of feces ws low t oth concentrtions, which resulted in high, non-vrying gross growth efficiencies (production per ingestion) for cholesterol. Furthermore, the proportion of ssimilted cholesterol used for production (net growth ) ws high, indicting respirtion losses tht were lower nd even negligile compred with ulk C losses. The only significnt effects of the dietry cholesterol concentrtion on the direct cholesterol metolism were those on the excretion, which ws higher t low dietry cholesterol compred with the non-limiting concentrtion (one-wy-anova, F 1,4 =3.2, P=0.022; Fig. 4). DISCUSSION The present study reveled strong effects of sterol vilility nd food quntity on C ssimiltion nd feces egestion s well s respirtion in D. mgn. Moreover, we found tht D. mgn selectively exclude cholesterol from C losses such s feces egestion nd respirtion. In the following we discuss the different C pthwys in ech of our four tretments nd use the high cholesterol (HC) high quntity (HQ) tretment s reference. High cholesterol high quntity In generl, we produced evidence tht effects of food qulity strongly depend on food quntity, ecuse D. mgn growth reduction due to cholesterol limittion ws diminished t low food quntity. The HC/HQ tretment hd the highest growth rtes of D. mgn, which is consistent with recent results (Sperfeld nd Wcker, 2009; Luks et l., 2011). Moreover, the results for lmost ll mesured C pthwys of the present study (Fig. 5A) were similr to those from previous experiments with Dphni grown under non-limiting food conditions (neither quntittively nor qulittively). Accordingly, we found comprle vlues for cron ssimiltion efficiencies (AE C ), cron gross growth efficiencies (GGE C, production per ingestion) nd cron net growth (production per ssimiltion, NGE C ) (DeMott et l., 1998; He nd Wng, 2008) s well s for respirtion (Fedorov nd Sorokin, 1967; He nd Wng, 2006) nd excretion of dissolved orgnic cron (DOC) (Drchmeu et l., 2003; He nd Wng, 2006). Only the results for the AE C were somewht contrsting, s we nd DeMott et l. (DeMott et l., 1998) showed high vlues, ut He nd Wng (He nd Wng, 2008) otined much lower AE C (indicting low nd high feces egestion, respectively). These differences in AE C might e due to differences in food concentrtions (DeMott et l., 2010), ut the food concentrtion in our study nd those from He nd Wng (He nd Wng, 2008) were more similr thn those of DeMott et l. (DeMott et l., 1998). Moreover, AE C might e ge dependent, ecuse nimls from the He nd Wng study (He nd Wng, 2008) were much older (11 dys) nd hd lredy trnsferred energy to their offspring with energy. In ny cse, we clerly show here tht feces egestion is non-negligile frction Percentge of ingestion c Assimiltion,c Feces c Gross growth LC/LQ HC/LQ LC/HQ HC/HQ Fig. 2. Cron ssimiltion (ssimiltion per ingestion), feces egestion (per ingestion) nd gross growth (production per ingestion) of D. mgn with food of different qulity nd quntity. Dt re mens ± 1 s.d.; n=5. Sttisticlly significnt differences re indicted s different letters (Tukey s post hoc test, P<0.05). c Percentge of ssimiltion Net growth LC/LQ HC/LQ LC/HQ HC/HQ Respirtion Fig. 3. Net growth (production per ssimiltion), excretion nd respirtion (oth s proportions of ssimiltion) of D. mgn with food of different qulity nd quntity. Dt re mens ± 1 s.d. with n=5, except for excretion t HC/LQ (n=4). Sttisticlly significnt differences re indicted s different letters (Tukey s post hoc test, P<0.05). The Journl of Experimentl Biology 1081

4 The Journl of Experimentl Biology (2014) doi: /je Percentge of ingestion or ssimiltion 120 Low cholesterol High cholesterol Assimiltion Feces Gross growth Net growth Respirtion Fig. 4. Assimiltion (ssimiltion per ingestion), egestion (feces per ingestion), gross growth (production per ingestion), net growth (production per ssimiltion) nd excretion nd respirtion (s proportions of ssimiltion) of cholesterol of D. mgn cclimted (48 h) to two different cholesterol concentrtions (low: 3.5 μg cholesterol mg 1 C, high: 14 μg cholesterol mg 1 C). Dt re mens ± 1 s.d.; n=3. Except for excretion (one-wy ANOVA, F 2,6 =13.2, P=0.022), ll remining one-wy ANOVAs reveled no differences etween low nd high dietry cholesterol (P>0.85). of C ingested even under good food conditions. In contrst with green lge, the cynocteri Synechococcus elongtus we used does not hve cell wlls nd is well digestile (Lmpert, 1977). If Dphni food supply consisted insted of green lge with strong cell wlls (e.g. Vn Donk et l., 1997) or geltinous coverings tht reduce digestiility (DeMott et l., 2010), feces egestion might hve een more pronounced. Unfortuntely, it hs not een well descried, until now, how limittions of food qulity (nd quntity) ffect Dphni s egestion of feces (neither independently nor simultneously). This knowledge gp shows how the defection processes in dphnids hve een neglected s n importnt prt in the regultion of C udgets. High cholesterol low quntity When we reduced the food quntity nd kept food qulity high (HC/LQ tretment), the growth of the nimls decresed significntly to moderte level. At low food quntity, the reduced ingestion ws prtly offset y incresed AE C (Fig. 5B); the higher AE C proly derives from longer gut pssge times t low food concentrtion (DeMott et l., 2010). Our results of very high AE C t low food quntity corroorte erlier studies, which found higher AE C t low compred with high food concentrtions (Ure nd Wtne, 1991; He nd Wng, 2006). The feces frction ws much smller under food limittion nd, therefore, it ppers s reltively minor route of C loss in energy-limited Dphni (He nd Wng, 2006). However, modelling pproch y Anderson et l. (Anderson et l., 2005) did not revel lower feces egestion t food limittion. Interestingly, the GGE C incresed mrkedly when food concentrtion decresed, ut the NGE C did not chnge. Consequently, we suggest tht in oth low quntity tretments the from ssimiltion to production is very high. Our low food concentrtion ws clerly limiting, ut still provided enough energy for moderte growth. Therefore, our interprettion of the high vlues of GGE C nd NGE C cnnot e generlized to situtions where food quntity pproches the threshold for zero growth. Then, the entire ssimilted C is consumed s metolic expenditure, nd gross (nd net) growth pproches zero (Lmpert, 1977). Aove such threshold concentrtions, nimls pper to respond differently to food limittion; we found still high NGE C, which might originte from very low excretion. As excretion of DOC did not chnge ecuse of ltered food quntity (see lso He nd Wng, A High cholesterol high quntity B High cholesterol low quntity Respirtion CO 2 Respirtion CO 2 Food Ingestion AE Assimiltion NGE Production Food Ingestion AE Assimiltion NGE Production Egestion Egestion Feces DOC Feces DOC C Food Ingestion Low cholesterol high quntity AE Egestion Feces Assimiltion Respirtion NGE CO 2 Production DOC D Food Ingestion Low cholesterol low quntity Egestion Feces Assimiltion Respirtion CO 2 Production Fig. 5. Schemtic of cron pthwys in D. mgn grown under different food conditions. The thickness of the rrows indictes the reltive vlue of the respective C pthwys. AE, ssimiltion ; DOC, dissolved orgnic cron; NGE, net growth. AE NGE DOC The Journl of Experimentl Biology 1082

5 The Journl of Experimentl Biology (2014) doi: /je ), we ssume low respirtion s nother potentil explntion for the high NGE C oserved in the HC/LQ tretment. In comprison with the HC/HQ tretment, HC/LQ respirtion vlues were not significntly different, ut, consistent with erlier studies, our results show generlly decresed respirtion t lower food vilility (food quntity effect in two-wy ANOVA; Tle 1) (Lmpert, 1986; Schmoker nd Hernndez-Leon, 2003; Anderson et l., 2005). This response of the respirtory rte to chnging food conditions (termed SDA) refers to the increse in energy expenditure tht occurs during mel digestion (e.g. Kiøroe et l., 1985; Secor, 2009). Low cholesterol high quntity When C ws ville in excess reltive to cholesterol (LC/HQ tretment), the growth rtes of D. mgn were reduced to the sme moderte vlue s in the high cholesterol (HC) low quntity (LQ) tretment. This indictes tht ecologiclly relevnt chnges in food quntity nd qulity (in terms of cholesterol) cn potentilly hve similr impct on Dphni, lthough this certinly depends on the prticulr comintion of cholesterol deficiency nd how much the food quntity is reduced elow the incipient limiting level. The comprle responses of growth rtes under food quntity or qulity limittion, however, were not sed on similr mechnisms. Insted they were the result of chnges in C pthwys. In generl, y using cholesterol s food qulity indictor, the present study differs from previous studies exmining the effect of P limittion on Dphni C pthwys. We conclude tht Dphni hs complex network of regultory mechnisms for different types of limittions, i.e. we suggest tht different strtegies re used to hndle limittions y cholesterol nd elementl P. Interestingly, two importnt studies on C udgets in Dphni, when dietry P supply ws chnged, differed in their results (DeMott et l., 1998; He nd Wng, 2008) nd mke comprisons difficult. Becuse we did not ddress P supply, in the following we focus on the iochemicl scope of our study. We found the lowest AE C t low food qulity nd high quntity (Fig. 5C), which confirmed the results of DeMott nd Müller- Nvrr (DeMott nd Müller-Nvrr, 1997), who found reduced AE C for Dphni feeding on the cynocterium S. elongtus lone, ut higher AE C when S. elongtus ws provided in comintion with green lge of high food qulity. Low AE C in the present study led to high egestion of feces, stressing the importnce of feces egestion in Dphni C udgets, i.e. Dphni uses feces s highly effective mens to get rid of excess C when food qulity, in terms of cholesterol, is limiting. In correspondence with low AE C nd high feces egestion, we found the lowest GGE C t high food quntity ut low cholesterol vilility. Low GGE C nd high feces egestion t low food qulity were supported y DeMott et l. (DeMott et l., 1998), ut not y He nd Wng (He nd Wng, 2008). The ltter showed longer gut pssge times for P-limited nimls, fct tht suggests lower feces egestion. We lso found the lowest NGE C t limiting cholesterol concentrtions compred with our other tretments. Low NGE C could e the result of high DOC excretion, which cn e the predominnt component of C relese under P-limited conditions (Drchmeu et l., 2003; Anderson et l., 2005; He nd Wng, 2008). However, in contrst to results for P-deficient diets, we did not find significnt effects of cholesterol on the excretion of DOC. This clerly indictes tht Dphni responds differently to iochemicl limittion such s cholesterol, compred with P limittion. At ny rte, our dt suggest mrginl increse in excretion rte s mechnism to get rid of excess C t low food qulity in terms of iochemicls, e.g. sterols (two-wy ANOVA, effect of cholesterol on DOC excretion: P=0.073). Nevertheless, cre should e tken when interpreting excretion dt, s one hs to distinguish etween direct excretion of DOC nd the relese of DOC from fecl mteril. He nd Wng (He nd Wng, 2006) suggested tht feces lekge ws only smll frction of totl DOC relese. Our results re consistent with this, ecuse we did not find correltion etween the proportions of feces nd excretion (liner regression, R 2 =0.14, P=0.12). Hence we ssume lekge of feces into the DOC pool ws negligile. A further reson for Dphni s low NGE C in our study is certinly the strong increse in respirtion when cholesterol ws low, s previously shown for Dphni fed with P-limited diets (Drchmeu et l., 2003; Jensen nd Hessen, 2007). Nevertheless, the results of Jensen nd Hessen (Jensen nd Hessen, 2007) re not directly comprle with ours nd those of Drchmeu et l. (Drchmeu et l., 2003). The differences my stem from different methods of determintion of respirtion rtes, i.e. consumption of O 2 (Jensen nd Hessen, 2007) versus relese of 14 CO 2 (Drchmeu et l., 2003; present study). The respirtory quotient (defined s the volume of CO 2 produced per volume of O 2 consumed) will e ffected y the iochemicl mkeup of the lgl food (Jensen nd Hessen, 2007). Low cholesterol low quntity When Dphni ws grown in the worst food tretment (low quntity nd qulity, LC/LQ) the ptterns in C pthwys were not different from those of the HC/LQ tretment. Accordingly, Dphni C pthwys re not ffected y food qulity (in terms of cholesterol) s long s food quntity is lso limiting. As C nd cholesterol ssimiltion efficiencies were high in oth low quntity tretments (LC/LQ nd HC/LQ), the nimls grew etter t higher cholesterol vilility (HC/LQ) thn t lower cholesterol vilility (LC/LQ). The C losses of Dphni re reduced y decresing ll C-costly processes such s excretion, respirtion nd feces egestion (Fig. 5D). Less egestion of feces is proly the result of low ingestion rte nd slow gut pssge t low food concentrtion. To our knowledge, the present study is the first to show concurrent effects of low food quntity nd qulity on Dphni nd how nimls djust C udgets nd regulte growth in response to it. We illustrte the dilemm of nimls of djusting C udgets for low qulity t low quntity. When food quntity ws high, Dphni ws le to djust C pthwys for differences in dietry cholesterol (e.g. y higher feces egestion or respirtion), ut the constrints imposed y low food quntity superseded other possile djustments due to low food qulity. Pthwys of essentil orgnic compounds Until now, discussions on the regultion of zooplnkton C pthwys hve focused on ulk C, ut here we strt investigting the pthwys of essentil iochemicl compounds. The pthwys of ulk C differ significntly from those of cholesterol. We found AE C to e lowest t low cholesterol concentrtions, ut the ssimiltion efficiencies of cholesterol itself were much higher. Thus, our results show strong retention of this essentil food compound which increses our knowledge out the rection of Dphni to low iochemicl food qulity. Comprle to our findings, recent results descrie lower AE C ut higher ssimiltion efficiencies for P in P-limited Dphni (e.g. DeMott et l., 1998). Only when we set cholesterol concentrtion to very high vlues (ove 55 μg cholesterol mg 1 C), proly not found in nture, did the AE of cholesterol decrese nd the excretion of cholesterol increse (60% nd 30%, respectively). This response explins the ccumultion nd loss of cholesterol t low versus high cholesterol levels, respectively, nd enles Dphni to mintin suitle cholesterol concentrtion in the tissues (within ecologiclly relevnt scles of c μg cholesterol mg 1 C) (Sperfeld nd Wcker, 2009). The Journl of Experimentl Biology 1083

6 The Journl of Experimentl Biology (2014) doi: /je The egestion of ulk feces incresed when the cholesterol concentrtion in the food ws low, ut the egestion of cholesterol vi feces ws very low. Accordingly, Dphni chieved strong regultion (fter ingestion) of this essentil iochemicl y improving low cholesterol:c rtios: (1) Dphni incresed egestion of excess C nd (2) simultneously retined cholesterol from egestion. Becuse of low egestion nd selective retention of cholesterol, ~80% of the ingested cholesterol ws used for production. A similr vlue ws found for the GGE of P when Dphni ws fed P-limited lge (89%, DeMott et l., 1998). Interestingly, our NGEs for cholesterol were consistently high, while DeMott et l. (DeMott et l., 1998) found decresing phosphorus NGEs for P-limited Dphni. They explined this y low, ut consistent, P excretion even in strongly P-limited Dphni. Similrly, we found mesurle cholesterol excretion even though cholesterol ws limiting. Hence, the mechnism of higher DOC excretion to get rid of excess C t low cholesterol concentrtions (s indicted in our study) seems to e inopertive. Consequently, excretion of C derived from essentil cronic compounds is not independent from ulk C excretion, which is clerly in contrst to non-cronic compounds such s minerls (Frost et l., 2004; He nd Wng, 2008). As n explntion, we suggest tht Dphni does not distinguish etween essentil nd non-essentil C compounds in the excretion pthwy: higher excretion of excess C t low food qulity simultneously cuses higher excretion of cronic cholesterol. In contrst, lthough the isotope method we used provides very sensitive mesure, we did not oserve detectle respirtion of cholesterol; therefore, cholesterol respirtion is very low nd not significntly different from zero. Hence, in ddition to the feces regultion, we found two further mechnisms y which Dphni cn improve low cholesterol:c rtios they increse respirtion of excess C, while spring cholesterol from respirtion t the sme time. With this conclusion, we emphsize tht mny iochemicl food components contin C nd re consequently prt of the overll C pool of the nimls. Especilly when these components re only smll prt of the overll C content, Dphni should hndle such cronic essentil molecules efficiently. This fct needs to e considered for nlyses of C udgets, when essentil food compounds tht contin C re investigted. Additionlly, co-limiting scenrios (e.g. co-limittion y cholesterol nd P or y cholesterol nd polyunsturted ftty cids) led to interctions etween colimiting nutrients (Luks et l., 2011; Sperfeld et l., 2012) nd consequently lso C udgeting of co-limiting cronic nutrients my interct. Such interctive effects on C udgeting of nimls in situ might e identified y nutritionl indictors (Wgner et l., 2013). In conclusion, our results clerly indicte tht Dphni vries its regultion of C losses in response to different food conditions. In prticulr, the effects of food qulity in terms of cholesterol re importnt in severl C pthwys, given tht food quntity ws nonlimiting. This provides further evidence of stronger effects of food qulity on zooplnkton t non-limiting food quntities, which were previously descried for growth only (see Sterner nd Schulz, 1998). Moreover, incresed dischrge of ulk C nd simultneous high retention of cholesterol imply tht Dphni is le to djust C Tle 2. Conditions used during the growth nd pulse-chse experiment udgets nd chieve moderte growth rtes t low cholesterol vilility. MATERIALS AND METHODS Orgnisms The stock culture of D. mgn ws grown in filtered wter (0.2 μm poresized memrne filter) from Lke Stechlin (northest Germny) with 2 mg C l 1 of the green lge Scenedesmus oliquus (SAG 276-3, culture collection Goettingen, Germny) s food. For the growth experiment, the esily ingestile, non-toxic nd P-sturted cynocterium Synechococcus elongtus ws used s food for D. mgn. Synechococcus elongtus (SYN; SAG 89.79), lcking sterols nd polyunsturted ftty cids (von Elert et l., 2003), ws cultured in erted 2-l flsks contining Wright s cryptophyte (WC) medium with vitmins (Guillrd, 1975) nd diluted dily (dilution rte 0.2 dy 1 ) in order to ensure nutrient repletion. The culture ws mintined t n illumintion of 40 μmol photons m 2 s 1 using 16 h:8 h light:drk cycle. All orgnisms were rised t 20 C. Experimentl design nd procedure In order to exmine the simultneous dependency of D. mgn s growth nd C udgets on food qulity nd quntity, we supplied Dphni with two different dietry concentrtions of cholesterol nd two food concentrtions in full-fctoril design (Tle 2). The high qulity tretments provided enough cholesterol so tht it ws not limiting (Sperfeld nd Wcker, 2009), ut ws not sustntil excess. We used liposomes loded with cholesterol to control food qulity (see Liposome preprtion, elow). Third-clutch juveniles used for the experiment were collected within 12 h from mothers tht were trnsferred to jrs with cholesterol-deficient food (SYN, 2 mg C l 1 ) in the eginning of the 12 h. By doing so, we voided temporlly different cholesterol supply to newly htched juveniles, nd thus potentilly confounding vrition in cholesterol storge s previously shown for P (Luks et l., 2013). A suset of these juveniles ws dried nd weighed for the determintion of the initil dry mss. The tretments with food quntity nd/or qulity limittion ech strted with 320 neontes tht were rndomly distriuted into eight replicte jrs. For the tretment without ny limittion, 160 neontes were used. In order to consider the different sizes of the dphnids nd to void depletion of food, the volumes of food suspension were djusted s follows: nimls with high food concentrtion were rised in jrs contining etween 30 ml per individul t the eginning nd 60 ml per individul t the end of the experiment. Animls with low food concentrtion were rised in jrs contining etween 80 ml per individul t the eginning nd 200 ml per individul t the end of the experiment. In order to do so, we used up to five 2000 ml jrs for one replicte. Throughout the experiment, dphnids were trnsferred dily into jrs with renewed food suspensions. The growth experiment ws terminted fter 5 or 6 dys for high food quntity (HQ) nd low food quntity (LQ), respectively, in order to llow nimls with LQ to rech size comprle to nimls with HQ. The dphnids of ech tretment were split into two groups. One group (five replictes) ws used for the pulse-chse feeding experiment (see elow). The remining dphnids (three replictes) were rinsed with ultrpure wter nd trnsferred into pre-weighed luminium ots. After drying for 48 h t 50 C, dphnids were weighed on n electronic lnce (±1 μg; CP2P, Srtorius, Goettingen, Germny). The somtic growth rtes (g) were clculted s the chnge in dry mss per individul from the eginning (M dry,0 ) to the end of the experiment (M dry,t ) using the eqution: g = [ln(m dry,t ) ln(m dry,0 )] t 1, (1) where t is the durtion of the experiment in dys. Tretment Cholesterol (µg mg 1 C) Food quntity (mg C l 1 ) Low cholesterol low quntity (LC/LQ) Low cholesterol high quntity (LC/HQ) High cholesterol low quntity (HC/LQ) High cholesterol high quntity (HC/HQ) We used cross-wy scheme of concentrtions of dietry cholesterol nd food quntity. The Journl of Experimentl Biology 1084

7 The Journl of Experimentl Biology (2014) doi: /je Liposome preprtion Cholesterol contining liposomes nd empty liposomes without further ingredients were prepred ccording to Wcker nd Mrtin-Creuzurg (Wcker nd Mrtin-Creuzurg, 2012). Cholesterol liposomes were used s food supplements in growth experiments nd during the pulse-chse feeding experiment. The overll C content of the liposome solution (liposomes plus cholesterol in uffer) ws 2 mg C ml 1. Accordingly to the supplemented volumes of liposomes (low cholesterol: 10 μl liposomes mg 1 C, high cholesterol: 50 μl liposomes mg 1 C), the C concentrtions of the low qulity tretments incresed y 2%, nd those of the high qulity tretments y 10% (i.e. the C increse from low to high qulity ccounted for 8% in ech food quntity). This dditionl C could e considered negligile when compred with the increse y 900% when food quntity ws chnged from low to high concentrtions. The mount of cholesterol in susmples of liposomes ws determined using gs chromtogrphy ccording to Mrtin-Creuzurg et l. (Mrtin-Creuzurg et l., 2009). For the clcultion of C-sed cholesterol concentrtions, the mount of cholesterol dded y liposomes ws relted to the POC concentrtions of S. elongtus in food suspensions. To otin rdiolelled cholesterol liposomes we loded empty liposomes with rdiolelled ( 14 C) cholesterol (Americn Rdioleled Chemicls Inc., St Louis, MO, USA); empty liposomes were sonicted for 15 min, followed y 1 h incution with 14 C-cholesterol (50 mci mmol 1 ). To this we dded 14 C- cholesterol in the sme concentrtion s tht used for the non-rdiolelled cholesterol liposomes (333 μg ml 1 ). To verify the efficient incorportion of 14 C-cholesterol into the liposomes, we filtered the ressemled liposomes on Nucleopore filters (0.2 μm, 25 mm, Whtmn Interntionl Ltd, Midstone, UK). Less thn 5% of the initil 14 C-cholesterol concentrtion ws detected in the filtrte nd more thn 95% in the ressemled liposomes on the filter. Pulse-chse feeding experiment In order to investigte the C udgets of D. mgn, we used the rdiotrcer technique (rdioctively lelled C; 14 C) to follow the lloction of C into different comprtments including respirtion of dissolved inorgnic cron (DIC), excretion of dissolved orgnic cron (DOC) nd egestion of prticulte orgnic cron (POC) s feces. Exponentilly growing SYN ws lelled with 14 C from NH 14 CO 3 (1 mci l 1 ) until cells were uniformly rdiolelled fter 4 dys (specific rdioctivity of dpm mg C 1 ). Before using them s diet, the food suspension ws supplemented with liposomes contining cholesterol ccording to the experimentl protocol (Tle 2). For the pulse-chse feeding experiment, D. mgn ws previously cclimted on non-rdiolelled experimentl diets (see Experimentl design nd procedure, ove) nd then exposed to the rdiolelled diets (five times replicted) for 5 min (pulse). This is expected to e much shorter thn the gut pssge time of pproximtely min nd voids the defection of rdiolelled feces (He nd Wng, 2008). After such pulse feeding, the nimls were rinsed with rdioctive-free medium nd trnsferred into nonrdiolelled experimentl food suspensions (chse) under dim light using completely filled 5 ml snp cp vils. During pulse nd chse phses, food suspensions hd the sme quntity nd qulity chrcteristics s during the growth experiment. To void recycling of 14 C (e.g. re-uptke y dphnids), dphnids were rinsed with rdioctive-free medium to void crry-over of 14 C nd were trnsferred into vils with new food suspension in regulr time intervls (fter 0.5, 1, 2, 4 nd 6 h). Using preliminry test (see supplementry mteril Fig. S1) we concluded tht Dphni stopped the incorportion of 14 C into the somtic tissue fter 6 h, for which reson we confined our mesurements to this time. To mesure the mount of egested, excreted nd respired 14 C during ech time intervl, susmples of the food suspension including feces (totl frction) were tken immeditely fter ech time intervl nd the ctivity ws instntly determined vi liquid scintilltion counting (0.5 ml smple ml Hionic Fluor scintilltion fluid in liquid scintilltion counter Tri-Cr 2810Tr, oth PerkinElmer, Rodgu, Germny). The externl stndrd rtio method ws used for quenching nd conversion from counts per minute (cpm) to disintegrtions per minute (dpm) ws corrected. By different frctiontion we gined informtion out the mount of feces egested (POC, PO 14 C), respired 14 CO 2 nd excreted DOC (DO 14 C). Therefore, the rdioctivity in the totl frction (DI 14 C+DO 14 C+PO 14 C) ws mesured directly, nd fter ddition of hydrochloric cid (100 μl of 1 mol l 1 HCl in 4 ml of the smple) plus uling with ir. By dding HCl, DIC (DI 14 C) ws ll dehydrted to 14 CO 2, which out-gssed; DOC (DO 14 C) nd POC (PO 14 C) remined in the solution (DO 14 C+PO 14 C-frction). Rdioctivity of this frction ws mesured. Susmples of the DO 14 C+PO 14 C frction were memrne filtered (2 ml), nd the retined prticles on memrne filter were trnsferred into scintilltion vils. After the filter ws dissolved in 0.5 ml Soluene 350 (PerkinElmer), the rdioctivity of POC (PO 14 C) ws determined. This prticulte frction ws used s mesure for the feces, ecuse lgl C in chse suspensions ws not lelled with 14 C. We ensured tht uling the totl frction with ir hd no influence on the mount of mesured feces, s we found no differences etween the erted und unerted PO 14 C frctions (two-smple t-test, t=1.03, d.f.=198, P=0.31). By using the difference etween the totl frction (DI 14 C+DO 14 C+PO 14 C) nd the DO 14 C+PO 14 C frction, the DI 14 C (=respirtion) ws clculted. The DO 14 C (=excretion) ws clculted s the difference etween the DO 14 C+PO 14 C frction nd the PO 14 C frction. Immeditely fter the pulse-feeding (5 min) nd fter the lst time intervl (6 h), susmples of the dphnids were tken: 10 nimls ech for the tretment without ny limittion nd 20 nimls for ech tretment with food quntity nd/or qulity limittion. Animls were then instntly digested in soluilizer (0.5 ml Soluene 350) nd their rdioctivity ws determined vi liquid scintilltion counting. The mesurement of nimls fter the pulsefeeding ws used s vlue for ingested 14 C. After 6 h, we ssumed tht the mesured 14 C in the nimls ws used for iomss production. We clculted the mount of C in ech frction y dividing the mount of mesured 14 C y the rtio etween 14 C nd ulk C tht ws found in the rdiolelled diet. Resulting vlues were relted to the C content of the nimls, which ws clculted using the determined dry mss of n unlelled susmple of the dphnids nd previously determined conversion fctor of 0.41 μg C μg 1 dry mss. Cholesterol in C udgets To follow the fte of the iochemicl in the mesured C pthwys nd test the hypothesis of cholesterol exclusion from C udgeting, we rn seprte experiment nd used rdiolelled cholesterol (in liposomes, see Liposome preprtion, ove) nd the sterol-free cynocteri SYN. Using eukryotic diets insted (e.g. green lge) would e prolemtic s these contin phytosterols tht re rdiolelled y incorporting ulk 14 C derived from NH 14 CO 3 incution. Consequently, the 14 C signl of phytosterols nd of other C compounds in the different C frctions of Dphni would not hve een seprted from ech other. Juvenile dphnids (three replictes, 32 nimls ech) were cclimted on two different cholesterol concentrtions (low: 3.5 μg cholesterol mg 1 C, high: 14 μg cholesterol mg 1 C) for 48 h nd then used for pulse-chse feeding experiment s ove, except tht rdiolelled cholesterol (in liposomes) ws used during the pulse prt insted of ulk 14 C. We ssumed homogeneous distriution of the rdiolelled liposomes in the prepred food suspensions nd, ccordingly, ssumed Dphni s food to e uniformly lelled. The 14 C mesured fterwrds in ech frction ws directly derived from 14 C-cholesterol. Furthermore, we ssumed tht the cclimtion periods to sterol-limited conditions (48 h for the experiment with rdiolelled cholesterol s well s 5 6 dys for the experiment with rdiolelled ulk C) re pproprite time scles for detecting differences in C pthwys due to cholesterol limittion, though different times of cclimtion to the limittion my hve influenced the experimentl outcome (Luks nd Wcker, 2014). Sttisticl nlysis The dependency of Dphni s growth rte on high/low food quntity s well s high/low cholesterol ws nlysed using full-fctoril two-wy ANOVA. We lso nlysed the influence of the different food conditions on the C udgets of the nimls. Therefore, we clculted the proportions of ssimiltion (=ingestion feces), feces nd production of ingestion nd the proportions of production, excretion nd respirtion of ssimiltion. We defined cron ssimiltion (AE C )=ssimiltion/ingestion, cron gross growth (GGE C )=production/ingestion nd cron net growth (NGE C )=production/ssimiltion. For the ANOVA, proportions were trnsformed y rcsine-squre root nd the significnce of differences mong mens ws tested using multiple comprisons (post hoc The Journl of Experimentl Biology 1085

8 The Journl of Experimentl Biology (2014) doi: /je Tukey s test). All sttisticl nlyses were crried out using the sttisticl softwre pckge R version (R Development Core Tem, 2007). Acknowledgements We thnk Y. Jessen nd especilly S. Heim for technicl ssistnce, nd B. Lischke for helpful discussions. We lso thnk two nonymous reviewers for vlule comments on this mnuscript nd pprecite linguistic improvements y A. Montn nd F. de Cstro. Competing interests The uthors declre no competing finncil interests. Author contriutions Both uthors contriuted significntly to the conception, design nd execution of the study, interprettion of the findings s well s drfting the rticle. Funding The study ws supported y the Germn Reserch Foundtion (DFG, WA 2445/ 5-1 to A.W.). M.L. received postgrdute scholrship from the University Potsdm. Supplementry mteril Supplementry mteril ville online t References Andersen, T. (1997). Pelgic Nutrient Cycles. Ecologicl Studies. Berlin: Springer Verlg. Anderson, T. 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