The potential of dietary polyunsaturated fatty acids to modulate eicosanoid synthesis and reproduction in Daphnia magna: A gene expression approach

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1 The potental of detary polyunsaturated fatty acds to modulate ecosanod synthess and reproducton n Daphna magna: A gene expresson approach Nna Schlotz a.*, jesper Gvskov S"'lfensen b, Domnk Martn-Creuzburg a, Lmn%gca//nsttllte, Unversty of Konstanz, Manaustrasse 252, Konstanz, Germany b Department of Boscence, Aarhus Unversty, Vej/s0vej 25, 8600 Slkebolg, Denmark ABSTRACT Keywords: Arachdonc acd Daphna magna Ecosanods Ecosapentaenoc acd Food qualty Gene expresson Nutrton Vtellogenn Nutrtonal ecology of the aquatc model genus Daphna has receved much attenton n past years n partcular wth regard to detary polyunsaturated fatty acds (PUFAs) whch are crucal for growth and reproducton. Besdes ther sgnfcant role as membrane components, (20 PUFAs serve as precursors for ecosanods, hormone-lke medators of reproducton, mmunty and on transport physology. n the present study we nvestgate transcrptomc changes n Daphna magna n response to dfferent algal food organsms substantally dfferng n ther PUFA composton usng quanttatve real-tme PCR and relate them to concomtantly documented lfe hstory data. The selecton of target genes ncludes representatves that have prevously been shown to be responsve to the ecosanod bosynthess nhbtor buprofen. The benefcal effect of C20 PUFA-rch food on reproducton and populaton growth rates was accompaned by an ncreased vtellogenn (DmagVtgl) gene expresson n D. magna. Addtonally. genes nvolved n ecosanod sgnalng were partcularly nfluenced by detary C20 PUFA avalablty. For example. the cyclooxygenase gene (Cox), codng for a central enzyme n the ecosanod pathway, was hghly responsve to the food treatments. Our results suggest that detary PUFAs are fundamental n D. magna physology as substrate for ecosanod synthess and that these ecosanods are mportant for D. magna reproducton. 1. ntroducton Speces ofthe genus Daphna have become mportant model organsms n ecology. ecotoxcology and evolutonary bology (Ebelt. 2005; Lampert ). As keystone herbvores.n freshwater food webs they provde a crucal lnk between prmary and secondaly producton. Owng to the long hstory of ecologcal research. our knowledge of e.g. lfe hstory trats or phenotypc plastcty s vast compared to many other model organsms. Wth the fully sequenced genome of Daphna pulex another major advantage was gven to Daphna as a model (Colbourne et a., 2011; Ebert. 2011). However. our understandng of the physologcal mechansms nvolved n copng wth envronmental stress stuatons (e.g. temperature changes. predaton. pathogen challenge, nutrent lmtatons) s stll scarce. AbbrevatollS: AlJ, a-lnolenc acd: ARA, arachdonc acd: (LEG, C-type lectn lke: COX, cyclooxygenase: DGlJ, dhomo-'y-lnolenc acd: DHA, docosahexaenoc acd: DmagVTG, vtellogennl: EPA, ecosapentaenoc acd: FABP3. fatty acd bndng proten 3: GlJ. "-lnolenc acd: GPX, glutathone peroxdase: jhe, juvenle hormone esterase : LN. lnolec acd: LP, tracylglycerollpase: LOX, lpoxygenase: ltb4dh, leukotrene B4 12-hydroxydehydrogenase: MUFA, monounsaturated fatty acd: PUFA, polyunsaturated fatty acd : SAFA, saturated fatty acd: SDA, steardonc acd. Correspondng author. Tel.: : fax : E-mal addresses:nna.schlotz@un-konstanz.de(n.schlotz). jgs@d mu.dk (J.G. S0rensen), Domnk.Martn-Creuzburg@un-konstanz.de (D. Martn-Crellzbllrg), One of the factors greatly nfluencng Daphna's performance s nutrton. Besdes the obvous need to obtan enough food..e. the role offood quantty (Petrzak et al ). t also proved to be crucal to consume food of good qualty n order to meet the requrements for optmal growth and reproducton. Even food organsms that are nontoxc and readly assmlated can be of poor qualty (Lampert. 1977). one reason beng the lack of essental elemental or bochemcal nutrents, whch mpars the ftness of the consumer. Among the elements. phosphorus (P) and ntrogen (N) are by far the best-studed representatves (Sterner and Elser. 2002). Among bochemcal food qualty constrants are. for example. essental lpds and amno acds. Amno acds have been reported recently to affect populaton growth and the reproductve mode of daphnds (Koch et al ). As yet. however. the majorty of studes on bochemcal food qualty constrants have focused on polyunsaturated fatty acds (PUFAs) and sterols (MUler-Navarra et al : von Elert. 2002; Martn-Creuzburg et al ). The man sterol n anmals s cholesterol. t s an ntegral part of membranes where t nfluences fludty and permeablty and thus plays a role n temperature acclmaton (Mourtsen and Zuckermann. 2004). Furthermore. n althropods. t serves as precursor for moultng hormones. the ecdysterods (Mykles. 2011). The lack of sterols has been proposed to be a major constrant of energy transfer at the Daphna-cyanobactera nterface (Martn-Creuzburg et al ) and has shown to be one reason for the poor food qualty of heterotrophc

2 450 bactera (Martn-Creuzburg et a., 2011). The role of PUFAs has been nvestgated usng dfferent approaches. Correlatve feld studes suggested lmtatons by dfferent PUFAs n the feld (Mller-Navarra, 1995; Mller-Navarra et a., 2000; Wacker and Von Elert, 2001) and results from laboratory growth experments corroborate the mportance of PUFAs for Daphna ftness by demonstratng for example a better performance under temperature stress and revealng allocaton patterns durng PUFA defcences (Wacker and Martn-Creuzburg, 2007; Masclaux et a., 2009; Martn-Creuzburg et a., 2012). However, there s a lack of knowledge concernng the mechansms underlyng these benefcal effects as most of the avalable research merely compares varous ftness parameters as a functon of the detary PUFA content. PUFAs not only are ndspensable components of membranes, where they alter membrane propertes and contrbute to, e.g., temperature acclmaton, three of them also are precursors of a famly of boactve molecules, the ecosanods (Stanley, 2000; Guschna and Harwood, 2006; van Meer et a., 2008). These three precursor PUFAs are dhomo-'y-lnolenc acd (DGLA, 20:3n-6), arachdonc acd (ARA, 20:4n-6), and ecosapentaenoc acd (EPA, 20:5n- 3). They can be metabolzed by a cascade of enzymes to prostaglandns, leukotrenes, and thromboxanes. n mammals, there are three dstnct pathways leadng to formaton of ecosanods (Stanley, 2000); bonformatcs data suggests that only two of these pathways are present n Daphna (Heckmann et a., 2008b). The genomc structure of the nvolved pathway ("arachdonc acd pathway", termed after the man substrate ARA) reveals sgnfcant expanson and contracton of relevant gene faml es n Pancrustacea and the lneage leadng to D. pulex (Colbourne et a., 2011). Ths gan or loss of genes ndcates evolutonary recent and extensve restructurng of an mportant metabolc pathway. Even though there are a consderable number of studes that argue for the mportance of PUFAs n nvertebrate physology, few are able to conclusvely attrbute the observed effects to actons exerted by these essental lpds or ther metaboltes. n ths frst attempt to elucdate mechansms underlyng the benefcal effects of detary lpds on Daphna growth and reproducton, we adopted and modfed the dea of Heckmann et al. (2008a) and related lfe hstoy parameters to gene expresson. Yet, nstead of applyng a substance nterferng wth the pathway of nterest we provded food organsms of dfferent lpd qualty. Heckmann et al. (2008a) used buprofen, one of the best-known non-sterodal ant-nflammatory drugs, to fnd out f the mode of acton of ths ecosanod bosynthess nhbtor n D. magna s akn to that n mammals. n mammals, buprofen works by nhbtng the enzyme cyclooxygenase (COX) whch comprses, together wth poxygenase (LOX), the central part of the ecosanod synthess pathway. The genes examned here are nvolved n lpd metabolsm and the mmune and/or endocrne system, all of whch mght be affected by the avalablty of detary PUFAs; furthermore, most of them were responsve to buprofen n the study by Heckmann et al. (2008a). By smultaneously nvestgatng lfe hstory trats and gene expresson responses of D. magna we hope to gan frst nsghts n how mportant pathways are nfluenced by PUFAmedated food qualty and to reveal new avenues that should be explored n future studes. 2. Materal and methods 2.1. Food organsms We chose three algae dfferng n ther lpd profles for the growth experments: the green alga Scenedesmus oblqtltls (SAG 276-3a), the eustgmatophyte Nannochloropss lmnetca (SAG 18.99), and the cryptophyte Cryptomonas sp. (SAG 26.80). Algae were obtaned from the culture collecton of the Unversty of Gottngen (SAG, Germany). The algae were each cultured sem-contnuously n modfed Woods Hole (WC) medum (Gullard, 1975) n aerated 5 L vessels (20 C; dluton rate: 0.2 d - 1 ; llumnaton: 100 mmol quanta m- 2 S- l). Food suspensons were produced by centrfugaton of the halvested algae and resuspenson n fresh medum. Carbon concentratons were estmated from photometrc lght extnctons (480 nm) and from prevously determned carbon-extncton equatons. The carbon- lght extncton regressons were confrmed by subsequent carbon analyss of the food suspensons Bochemcal analyses Fatty acds - for the analyss of fatty acds approxmately 1 mg partculate organc carbon (POC) was fltered separately onto precombusted GF/ F flters (Whatman, 25 mm). Total lpds were extracted three tmes from flters wth dchloromethane/ methanol (2:1, v/v). Pooled cell-free extracts were evaporated to dryness under a ntrogen stream. The lpd extracts were transesterfed wth 3 M methanolc Hel (60 (, 20 mn). Subsequently, fatty acd methyl esters (FAMEs) were extracted three tmes wth 2 ml of so-hexane. The lpd-contanng fracton was evaporated to dryness under ntrogen and resuspended n a volume of 20 J- so-hexane. Lpds were analyzed by gas chromatography on a HP 6890 GC equpped wth a flame onzaton detector (FlO) and a DB-225 (J&W Scentfc, 30 m x 0.25 mm 10 x 0.25 mm flm) capllaly column to analyze FAMEs. Detals of GC confguratons for the analyss of FAMEs are gven elsewhere (Martn-Creuzburg et a., 2010). FAMEs were quantfed by comparson wth the nternal standard (C23:0 ME) of known concentraton, consderng response factors determned prevously wth lpd standards (Sgma-Aldrch). FAMEs were dentfed by ther retenton tmes and ther mass spectra, whch were recorded wth a gas chromatograph-mass spectrometer (Aglent Technologes, 5975C) equpped wth a fused-slca capllary column (DB-225MS, J&W). Spectra were recorded between 50 and 600 Da n the electron mpact onzaton mode. The lmt for quanttaton of fatty acds was 20 ng. The absolute amount of each lpd was related to the POc. Elemental composton - alquots of food suspensons were fltered onto precombusted glass fber flters (Whatman GF/F, 25 mm dameter) and analyzed for POC and ntrogen usng an EuroEA3000 elemental analyzer (HEKAtech GmbH, Wegberg, Germany). For the determnaton of partculate phosphorus, alquots were collected on acd-rnsed polysulfone flters (HT-200; Pall, Ann Arbor, M, USA) and dgested wth a soluton of 10% potassum peroxodsulfate and 1.5% sodum hydroxde for 60 mn at 121 C. Soluble reactve phosphorus was determned usng the molybdate-ascorbc acd method (Greenberg et a., 1985) Expermental desgn For all experments the same clone of D. magna (HOz, orgnally solated n Hungary) was used. Stock cultures were cultvated n artfcal medum (Aachener Daphnen Medum (ADaM)), modfed after Klttgen et al. (1994) and fed wth saturatng amounts of S. oblqtltls. All experments were conducted wth thrd-clutch neonates born wthn 12 h at 20 C Lfe hstory experment Anmals were kept ndvdually n 80 ml ADaM. They were randomly assgned to one of the three food regmes: S. oblqtltls (Seen), N. lmnetca (Nanno), and Cryptomonas sp. (Crypto). Every other day, they were transferred to fresh medum and receved algal food suspensons correspondng to 3 mg carbon per ltre. Durng the experment mortalty and reproducton were recorded. Populaton growth rates were estmated from the Euler- Lotka equaton

3 451 where x s the age-specfc survvorshp: mx s the age-specfc fecundty (number of neonates per ndvdual): and x s the age at reproducton (n days) Gene expresson experment Smultaneously to the lfe hstory experment we rased anmals for the gene expresson study: treatments conssted of the same three algae S. oblquus, N. lmnetca, and Oyptomonas sp. Anmals were rased n 1.5 L beakers each contanng 20 ndvduals. Cultvaton, frequency of transfer and amounts of food were as descrbed for the lfe hstory experment. After releasng ther second clutch offsprng daphnds were sampled usng a "cylndrcal seve system" (Heckmann et a., 2007) and stored at - 80 C n 400 fll- RNAlateT (Ambon) for subsequent RNA extracton Gene expresson analyss RNA extracton and DNA synthess Total RNA was extracted usng the RNeasyMn kt wth on-column DNase treatment (Qagen) accordng to the manufacturer's nstructons. RNA concentratons were determned usng a NanoDropTM spectrophotometer (NanoDrop Technologes). Agarose (1.5%) gel electrophoress was used to verfy the qualty of RNA. cdna was syntheszed from 4 J.Jg total RNA usng the Frst Strand cdna Synthess Kt (Fermentas) followng the manufacturer's nstructons. Subsequently, cdna was dluted 50-fold to a concentraton equvalent to 4 ng total RNA fll-- 1 and stored at - 20 C Relatve expresson of mrna Analyss comprsed eght genes: Cox (cyclooxygenase), Gpx (glutathone peroxdase), Clect (C-type lectn lke), Ltb4dh (eukotrene B4 12-hydroxydehydrogenase), DmagVtgl (vtellogenn 1), Jhe (juvenle hormone esterase), Lp (tracylglycerollpase), Fabp3 (fatty acd bndng proten 3). Prmer sequences were taken from Heckmann et al. (2008a) (addtonal data fles 3, 8): prmers were syntheszed by bomers.net (Ulm, Germany). Real-tme quanttatve polymerase chan reactons (qpcr) were conducted on a Stratagene MX3005P (AH Dagnostcs) usng Stratagene Brllant SYBR Green qpcr Mastermx (AH Dagnostcs). Each reacton was run n duplcate and contaned 5 pl of cdna template (equvalent to 20 ng total RNA) along wth 900 nm prmers n a fnal volume of 15 tl. The amplfcaton was performed under the followng condtons: 95 C for 10 mn to actvate the DNA polymerase, then 40 cycles of 95 C for los and 60 C for 60 s. Meltng curves were vsually nspected to verfy a sngle amplfcaton product wth no prmer- dmers. No nconsstences were detected n the present data set Data analyss and statstcs Raw qpcr data were analyzed usng Data Analyss fo r Real-Tme PCR (DART-PCR) (Person et a., 2003). The calculated reacton effcences verfed the expected amplfcaton of around 2-fold for all genes. The few outlers detected were removed. The assocated meltng culves were nspected to verfy the presence of a sngle and specfc amplcon. The resultng data set was normalzed by NORMA-Gene (Heckmann et a., 2011). Dfferences n relatve normalzed expresson of the target genes among treatments were assessed usng one-way analyss of varance (ANOVA) f assumptons of normalty and homogenety ofvarances were met. f assumptons were volated, Kruskal- Walls one-way ANOVA on ranks was performed. For llustraton, gene expressons of anmals fed N. lmnetca or Oyptomonas sp. were calbrated to those of anmals fed S. oblquus,.e. the relatve expresson for the S. oblquus treatment was fxed to 1. All statstcs were carred out usng Sgma plot (v 12.0, Systat Software) or Statstca (v 6.0, StatSoft). 3. Results 3.1. Elemental stochometry and lpd composton of food organsm The elemental composton of the food organsms was characterzed by hgh contents of ntrogen and phosphorus a nd low C:N and C:P ratos (means ± s.d.: S. oblquus: C:N 17.2 ± 0.6, C:P ± 9.7: N. lmnetca: C:N 10.5 ± 3.6, C:P ± 5.1: Oyptomonas sp.: C:N 5.4 ± 0.l, C:P ± 8.7). A lmtaton of D. magna by N or P could therefore be excluded. The three food organsms dffered notably n ther PUFA composton (Table 1). S. ob/quus contaned lnolec acd (LN, 18:2n-6), "V-lnolenc acd (GLA, 18:3n-6), a -lnolenc acd (ALA, 18:3n-3), and steardonc acd (SDA, 18:4n-3), but no PUFAs wth more than 18 carbon atoms were detected. N. lmnetca contaned LN and the C20 PUFAs ARA and EPA, the latter n exceptonally hgh amounts. n Cryptomonas sp., we found LN, ALA, SDA, EPA and docosahexaenoc acd (DHA, 22:6n- 3). The amounts of total saturated fatty acds (SAFAs) and total monounsaturated fatty acds (MUFAs) were hghest n N. lmnetca: S. oblquus and Cryptomonas sp. were comparable concernng the total amount of SAFAs, but Cryptomonas sp. contaned less MUFAs. The total PUFA content was generally hgher than the SAFA or MUFA content n all three algae. Cryptomonas sp. contaned the hghest amounts of total PUFA followed by N. lmnetca: both algae contaned more PUFAs than S. oblquus. Only N. lmnetca and Cryptomonas sp. contaned C20 PUFAs, the amount n N. lmnetca beng almost four tmes that n Cryptomonas sp Reproducton and populaton growth rates Cumulatve numbers of vable offsprng produced wthn the frst three reproducton cycles and the estmated populaton growth rates ncreased sgnfcantly from anmals fed S. oblquus to anmals fed N. lmnetca and agan to those fed Cryptomonas sp, (Fg. la and b). The dfferences n cumulatve offsprng numbers caused by dfferent algal dets were already apparent n the frst clutch, where both N. lmnetca and Cryptomonas sp. as det for D. magna led to sgnfcantly larger clutch szes compared to a S. oblquus det, but they were most pronounced after three reproducton cycles (Fg. 1a) Expresson of target genes The expresson of all examned genes was nfluenced by the food treatments (Fg. 2), only two were not sgnfcantly affected (DmagVtgl, p = 0.156: Ltb4dh, p = 0,228). The most promnent changes n gene expressons were observed for Cox, Fabp3, and Clect. Cox was up-regulated - 3-fold n both the N. lmnetca and Cryptomonas sp. treatment. Fabp3, n contrast, was down-regulated -4-fold when N. lmnetca or Cryptomonas sp. was used Table t Fatty acd composton of S. oblqlllls, N. lmnerca and Ctypromonas sp. Data are means of three replcates ±s.d. expressed n fg mg C- 1 (n.d. = not detectable). S. oblqlllls N. lmnerca Ctyptolllonas sp. 18:2n-6 (UN ) ± ± ± :3n-6 (GLA) 3.0 ± 0.1 n.d. 'td. 18:3n-3 (ALA) 60.1 ± 0.9 n.d ± :4n-3 (STA) 8.2± 0.1 n.d ± :4n-6 (ARA) n.d. 24.1±2.5 n.d. 20: 5n- 3 (E PA) n.d ± ± :6n-3 (DHA) n.d. n.d. 4.4 ± 0.0 Total SAFAs 46.6 ± ± ± 0.3 Total MUFAs 82.9 ± ± ±0.1 Total PUFAs ± ± ±2.8 Total (20 PUFAs n.d ± ± 0.7 Total FA ± ± ±2.4

4 r a 1.;; 60 Seen B V Ntmrn) 1 50 C ""'. ' ; c A 0.l J 1st " " g 2nd clutch r"b ,- 0.4 lrd Seen Nanne Crypto food treatment 0.3 ' Fg. 1. Cumulatve numbers of vable neonates produced per ndvdual (a) and estmated populaton growth rates (b) ofd. magna rased on S. ob/quus (Seen). N. /mnecca (Nanno) or CrypComonas sp. (Crypto). Data are means of 16 replcates ± s.e. Symbols or bars labelled wth the same letters are not sgnfcantly dfferent (Tukey's HSD test. p<o.05 fo llowng ANOVA). Comparsons were carred out wthn each clutch. as food. Smlarly. Clect was down-regulated -3- and 4-fold wth N. lmnetca and Oyptomonas sp. as food. respectvely. Changes n the expresson of Lp. Gpx. and }he were less dstnct. Whle Lp was clearly up-regulated n the N. lmnetca treatment (- 3.5-fold). ths was less pronounced n the Cryptomonas sp. treatment (- 2-fold). The expresson of Gpx dd not dffer n anmals fed S. oblqtltls and N. lmnetca. but was slghtly reduced n anmals fed c 0 '0; '" a. Q) r.;-"' ' :7'7 :: L m..w.. _"'-''''-'... '--''... 4,..-:------::---:-:--, A..._ _ H.H. _... H Fal>p3 B 0.0? "" Gpx A Seen Nanno CrWlO 4 fc;,; , ::t;;; ,... Lp ['COC j 0.0 L... Seeo food treatment B B NaollO Crypto Fg. 2. Relatve expresson of eght selected genes of D. magna ra sed on S. oblqulls ( Seen). N. lmnecca ( Nanno) or Cryptomonas sp. (Crypto). For graphc presentaton. expresson tevels are related to gene expresson observed on a S. oblqlllls det (= calbrator). Shown are mean relatve expressons (N = 3 ±s.e.). Bars labelled wth the same letters are not Sgnfcantly dfferent (Tukey's HSD test. p<o.05 followng ANOVA or Kruskal Walls). N.5. = not sgnfcant...1 Cryptomonas Sp. (- 2-fold).}he-expresson dffered n anmals feedng on N. lmnetca and Cryptomonas sp., but both treatments dd not dffer sgnfcantly from the expresson level obtaned wt h S. oblqtltls as food. Although not sgnfcant. the expresson levels of Ltb4dh as well as DmagVtgl were clearly affected by the food sources. Ltb4dh expresson was almost halved n anmals feedng on N. lmnetca or Cryptomonas sp. whereas DmagVtgl dsplayed the opposte trend..e. ts expresson was up-regulated 2- and 3-fold n anmals fed N. lmnetca and Cryptomonas sp.. respectvely. compared to anmals fed S. oblqtltls. 4. Dscusson As a frst approach to lnk bochemcal nutrent avalabltes to gene expresson. we rased Daphna magna on food sources dfferng n ther PUFA composton and assessed assocated changes n the expresson of selected canddate genes. We found all of these canddate genes to be responsve to the dfferent food sources and propose that the observed effects on gene expresson can be manly attrbuted to dfferences n the avalablty of detary fatty acds. Dfferences n fatty acd profles among the three algae used as food for D. magna are substantal and are expected to be responsble for the superor food qualty of N. lmnetca and Cryptomonas sp. as was observed n the concomtantly performed lfe hstory experment where both algae rch n PUFAs (> 18 C) yelded hgher offsprng numbers and populaton growth rates than S. oblqtltls. Although the elemental nutrent ratos (C:P and C:N) found n the three algae dffered slghtly. both Nand P are present n a supply far from what s thought to be lmtng for Daphna (Sterner and Elser. 2002). n contrast to S. oblqtltls. N. lmnetca and Oyptomonas sp. contan C20 PUFAs whch n Daphna can serve as precursors for ecosanod bosynthess. Only N. lmnetca contans ARA. the man substrate for ths bosynthess. n addton. the amounts of EPA found n ths alga are exceptonally hgh. Genes examned here ncluded representatves of dfferent pathways possbly nfluenced by changng detary PUFA avalabltes. as ndcated by ther response to the ecosanod bosynthess nhbtor buprofen (Heckmann et al a). Hayash et al. (2008) reported that reproducton of daphnds s mpared upon exposure to buprofen. whch s n lne wth the fndng that detaly C20 PUFAs..e. ecosanod precursors. ncrease the reproductve success of daphnds (Martn-Creuzburg et al ). Vtellogenn. a lpoproten and precursor of the major egg yolk proten vtelln. s ndspensable for oogeness and thus reproducton (Bownes. 1986). Therefore. one would expect that a hgher egg number s accompaned by an ncrease n the expresson of DmagVtgl. Although the ncrease n transcrpton of DmagVtgl from S. oblqtltls to N. lmnetca and further to Cryptomonas sp. was not sgnfcant due to the comparatvely large wthn-treatment varance. ths ncrease ncely reflects the ncreasng reproductve output of anmals feedng on these dets. Besdes DmagVtgl. }he expresson should be consdered when lookng at oogeness as the encoded esterase cleaves the actve juvenle hormone thereby renderng t nactve and as a consequence releases vtellogenn from the suppressve nfluence of juvenle hormone (Tokshta et al ). f an ncrease n DmagVtgl expresson would requre a synchronous ncrease n JHE. we would expect to fnd a smlar expresson pattern for both genes. However. we were examnng mature. reproducng anmals and as ncreased levels of juvenle hormone may be dsruptve of reproducton we would not expect to fnd any dfferences n }he expresson among the three algae. Methyl farnesoate. the crustacean juvenle hormone. has been shown to nduce male producton n D. magna (Olmstead and Leblanc. 2002) and hence should be suppressed under favourabl e standard expermental condtons. n ths study. all expermental anmals reproduced parthenogenetcally..e. no males were obselved. and the expresson of }he n anmals rased on N. lmnetca or Cryptomonas sp. was not sgnfcantly dfferent from that n anmals rased on S. oblquus. Hence. one mght

5 453 argue aganst a connecton between vtellogeness and juvenle hormone synthess and, therefore, aganst a role for PUFAs n juvenle hormone sgnallng. Lp codes for a tracylglycerollpase and hence s drectly nvolved n lpd metabolsm, partcularly n the glycerolpd metabolsm (Prentk and Madraju, 2008). Lp was up-regulated n anmals feedng on N. lmnetca, and also to a smaller extend n anmals feedng on Cryptomonas sp. The strong ncrease n Lp expresson n anmals feedng on N. lmnetca s most lkely a general response to the hgh SAFA and/or total fatty acd content of N. lmnetca rather than a specfc, PUFA-related response. LP presumably s a secreted proten as suggested by the presence of a predcted sgnal sequence; hence, the observed ncrease n Lp expresson mght ndcate an ncreased lpd assmlaton process, n whch avalable fatty acds n the det are released from tracylglycerdes n the gut through the actvty of pases, absorbed through the epthelum, and subsequently processed and stored n lpd droplets (Mu and Hoy, 2004; Lass et a., 2011). Lectns act as recognton molecules wthn the mmune system and are, through ther ablty to bnd glucoprotens and glycolpds and to medate e.g. endocytoss, mportant modules of pathogen defence (Klpatrck, 2002). C1ect expresson was down-regulated n both N. lmnetca and Cryptomonas sp. consumng anmals. Ths pattern may represent a trade-off stuaton between mmune response and reproductve output,.e. an ncreased allocaton of resources toward reproducton n anmals fed N. lmnetca or Cryptomonas sp. mght lead to a reduced nvestment n components of the mmune system. ndcatons for a trade-off of mmunty for other ftness parameters have been descrbed n many organsms (Zuk and Stoehr, 2002; Allen and Lttle, 2011). Rono et al. (2010) observed nterference of vtellogenn wth the mmune response to an nvadng pathogen n Anopheles gambae. Another group of genes can be assocated drectly wth ecosanod metabolsm as they are codng for the requred enzymes: COX, one of the central enzymes n the bosynthess of ecosanods and LTB4DH, an enzyme responsble for renderng some ecosanods nactve (Stanley, 2000); GPX provdes protecton aganst toxc oxygen dervatves and s able to reduce lpd hydroperoxdes formed durng ecosanod synthess (Schoene, 1985; Arthur, 2000). As key enzyme wthn the ecosanod bosynthess pathway, we expected the Cox gene to be hghly responsve to changes n substrate avalablty,.e. the avalablty of the three relevant C20 PUFAs. n both the N. lmnetca and Cryptomonas sp. treatment, where these C20 precursors are present, gene expresson levels were clearly elevated compared to S. oblqtltls. Ths mght reflect an nducton of the Cox gene by the presence of the substrate (ARA or EPA) of the encoded enzyme and would thus ndcate a hgher rate of ecosano'd synthess. Supportng ths possblty s the concomtantly slghtly lowered Ltb4dh expresson n anmals fed N. lmnetca or Oyptomonas sp., as the enzyme encoded by ths gene s, at least n vertebrates, responsble for the nactvaton of ecosanods. Moreover, leukotrene B4 (LTB4) has been shown to playa role n yolk formaton durng oogeness n nsects (Mederos et a., 2004). As Ltb4dh expresson s downregulated n anmals rased on a N. lmnetca or Cryptomonas sp. det, there should be more LTB4 present, whch n turn would (accordng to Mederos et al.) support yolk formaton. Together wth the ncreased expresson of the vtellogenn gene DmagVtgl n anmals fed the C20 PUFA-rch dets n our experment, ths argues for a smlar role of LTB4 n nsect and Daphna reproducton. Glutathone peroxdases reduce the hydroperoxdes formed by ARA metabolsm durng ecosanod synthess. Although ecosanods exert crucal bologcal actvtes ther peroxde nature can also cause severe damage to membranes. Hence, one would expect a more pronounced expresson of Gpx n anmals that are able to extensvely synthesze ecosanods,.e. n our case, n anmals feedng on the C20 PUFA-rch algae N. lmnetca or Cryptomonas sp. Yet, we dd not fnd the expected up-regulaton of Gpx expresson. Whle Gpx expresson levels dd not dffer between anmals fed S. oblqtltls and N. lmnetca, they were slghtly lower n the Cryptomonas sp. treatment Whether or not anmals feedng on Cryptomonas sp. are n fact exposed to lower hydroperoxde-medated stress than anmals feedng on S. oblqtltls and N. lmnetca remans unclear and has to be re- assessed n future studes. Another gene closely related to the ecosanod pathway s Fabp3. Fatty acd bndng protens (FABPs) re nvolved n sgnallng processes of vertebrates and nvertebrates as ntracellular carrer protens for fatty acds, ecosanods and other lpophlc substances (Zmmerman and Veerkamp, 2002; Esteves and Ehrlch, 2006). Possble destnatons for both free fatty acds and ecosanods bound to FABPs can be peroxsome prolferator-actvated receptors (PPARs). These nuclear receptors dmerze upon lgand bndng (.e. actvaton) wth the retnod X receptor (RXR) and the resultng complex consequently bnds to the promoter regon of target genes (Berger and Moller, 2002). n our study, the expresson pattern of Fabp3 n anmals fed the dfferent food sources was smlar to the expresson pattern of Ltb4dh, but reversed to that of Cox, whch mples a connecton to ecosanod sgnallng. However, the reason for the sgnfcant down-regulaton of Fabp3 n anmals feedng on the C20 PUFA-contanng algae N. lmnetca and Cryptol11onas sp. requres further nvestgaton, n whch receptors nvolved n the above descrbed sgnalng cascade, e.g. PPAR or RXR, are consdered. Also, as for all genes examned, t remans to be tested f the observed changes n gene expresson are reflected n changes n the functonal proten. Ecosanods play an mportant role n reproducton and mmunty of many nvertebrates (Stanley, 2000). So far there are only few hnts as to ther sgnfcance n Daphna. Hayash et al. (2008) used the ecosanod synthess nhbtor buprofen to assess ts toxcty for D. magna and found strong concentraton dependent effects on reproducton. Here, we demonstrated effects of food qualty on reproducton n a lfe hstory experment and hypotheszed that PUFAs, as one of the major factors dstngushng the employed algae, exert ther nfluence on offsprng producton n part through ecosanod actons. As the relevance of PUFAs for Daphna reproducton s well-establshed (e.g. Martn-Creuzburg et a., 2010) and the necessary enzymatc machnely for ecosanod synthess exsts n Daphna (Heckmann et a., 2008b; Colbourne et a., 2011), we are drectng future studes along ths lne of thought. To elucdate the role of PUFAs n ecosanod producton further studes are requred n whch the detaly PUFA supply s expermentally modfed by specfc PUFA supplementaton. t also has to be consdered that the actvty of COX s potentally nfluenced by other detary substances, such as carotenods (Reddy et a., 2005). 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