Martinez-Rubio et al. BMC Genomics 2014, 15:462

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1 Mrtinez-Rubio et l. BMC Genomics 2014, 15:462 RESEARCH ARTICLE Open Access Effects of functionl feeds on the lipid composition, trnscriptomic responses nd pthology in hert of Atlntic slmon (Slmo slr L.) before nd fter experimentl chllenge with Piscine Myocrditis Virus (PMCV) Lur Mrtinez-Rubio 1*, Øystein Evensen 2, Aleksei Krsnov 3, Sven Mrtin Jørgensen 3, Simon Wdsworth 4, Kri Ruohonen 4, Jose LG Vecino 4 nd Dougls R Tocher 1 Abstrct Bckground: Crdiomyopthy syndrome (CMS) is severe crdic disese of Atlntic slmon (Slmo slr) recently ssocited with double-strnded RNA virus, Piscine Myocrditis Virus (PMCV). The disese hs been dignosed in frms in Norwy ech yer over the lst decde resulting in nnul economic losses estimted t up to 9 million. Recently, we demonstrted tht functionl feeds led to milder inflmmtory response nd reduced severity of hert lesions in slmon experimentlly infected with Atlntic slmon reovirus, the cusl gent of hert nd skeletl muscle inflmmtion (HSMI). In the present study we employed similr strtegy to investigte the effects of functionl feeds, with reduced lipid content nd incresed eicospentenoic cid levels, in controlling CMS in slmon fter experimentl infection with PMCV. Results: Heptic stetosis ssocited with CMS ws significntly reduced over the time course of the infection in fish fed the functionl feeds. Significnt differences in immune nd inflmmtory responses nd pthology in hert tissue were found in fish fed the different dietry tretments over the course of the infection. Specificlly, fish fed the functionl feeds showed milder nd delyed inflmmtory response nd, consequently, less severity of hert lesions t erlier nd lter stges fter infection with PMCV. Decresing levels of phosphtidylinositol in cell membrnes combined with the incresed expression of genes relted with T-cell signlling pthwys reveled new interctions between dietry lipid composition nd the immune response in fish during virl infection. Dietry histidine supplementtion did not significntly ffect immune responses or levels of hert lesions. Conclusions: Combined with the previous findings on HSMI, the results of the present study highlight the potentil role of clinicl nutrition in controlling inflmmtory diseses in Atlntic slmon. In prticulr, dietry lipid content nd ftty cid composition my hve importnt immune-modultory effects in Atlntic slmon tht could be potentilly beneficil in fish blncing the immune nd tissue responses to virl infections. * Correspondence: lur.mrtinez@stir.c.uk 1 Institute of Aquculture, School of Nturl Sciences, University of Stirling, Stirling FK9 4LA, Scotlnd, UK Full list of uthor informtion is vilble t the end of the rticle 2014 Mrtinez-Rubio et l.; licensee BioMed Centrl Ltd. This is n Open Access rticle distributed under the terms of the Cretive Commons Attribution License ( which permits unrestricted use, distribution, nd reproduction in ny medium, provided the originl work is properly credited. The Cretive Commons Public Domin Dediction wiver ( pplies to the dt mde vilble in this rticle, unless otherwise stted.

2 Mrtinez-Rubio et l. BMC Genomics 2014, 15:462 Pge 2 of 20 Bckground Crdiomyopthy syndrome (CMS) is severe crdic disese of Atlntic slmon (Slmo slr) recently ssocited with double-strnded RNA virus termed piscine myocrditis virus (PMCV) [1]. The disese ws first described nd dignosed in Norwy in 1985 [2], nd hs since lso been dignosed in the Froe Islnds [3], Scotlnd [4] nd, possibly, Cnd [5]. In 2012, the Norwegin Veterinry Institute dignosed the presence of CMS t 89 slmon frming sites locted in the mid-cost of Norwy [6]. Erly stges of the disese hve been reported in dult Atlntic slmon close to hrvest, round 14 to 18 months fter trnsfer to sewter [7], nd so the economic impct cn be considerble [8]. Histopthologiclly, CMS is chrcterized by severe inflmmtion nd necrosis of the spongy myocrdium of the trium nd ventricle [9], but liver my lso be ffected due to the circultory disturbnce ssocited with the hert lesions. Mortlity is usully moderte lthough morbidity cn be very high with the ssocited chronic inflmmtory response lsting for severl months. The pthogenesis of experimentlly induced CMS ws previously ssessed by trnscriptomic profiling using oligonucleotide microrrys in ssocition with pthology [10,11]. In these studies, crdic pthology nd virl lod were correlted, with n up regultion of T cell response genes. It ppered tht the cellulr effector response medited by CD8+ T cells contributed to successful clernce of the virus infection, lthough it ws lso correlted with n up regultion of poptotic genes, which could contribute to tissue dmge. The lck of commercil vccines to CMS currently mkes the use of lterntive therpies crucil. Fctors tht modulte the inflmmtory process might be key to mitigte the clinicl symptoms nd improve the performnce of ffected fish. The concept of clinicl nutrition is well known in humns [12], nd is becoming of incresing interest in quculture. Functionl feeds re defined s high-qulity feeds tht, beyond their nutritionl composition, re formulted with helth promoting fetures tht could be beneficil in supporting disese resistnce nd mitigtion of clinicl disese symptoms. Thus clinicl nutrition pproch using functionl feeds potentilly enbles shift wy from chemotherpeutic nd ntibiotic tretments, lowering the costs of disese tretment nd mngement [13]. There re severl studies in fish linking nutrition nd immunology, recently reviewed by Kiron (14). The inclusion in quculture feeds of dditives such s prebiotics, probiotics, immunostimulnts, vitmins nd nucleotides, is reported to increse growth nd feed conversion efficiency, s well s hving positive effects on the immune system nd protection ginst bcteril infections [13]. Moreover, mcronutrients like proteins nd lipids re reported to ply key roles in the regultion of pthwys of the immune system nd hve been widely studied due to the necessity of the qufeed industry to replce dietry fishmel (FM) nd fish oil (FO) [14]. Polyunsturted ftty cids (PUFA) re involved in the regultion of both innte nd dptive immunity, nd the inflmmtory response through four potentil mechnisms including gene expression, eicosnoid metbolism, cellulr signlling nd membrne orgniztion [15]. Specific trnscription fctors re ctivted by number of PUFA, long-chin (LC)-PUFA nd eicosnoid lignds [16], nd subsequently regulte the expression of genes relted with inflmmtory, B-cell nd T-cell responses, which ply importnt roles in virl infections. The roles of PUFA in cell membrne orgniztion nd signlling pthwy mechnisms hve been widely studied in humns [15]. Inhibition of T-cell signlling pthwys by PUFA is minly linked to the suppression of the elevtion of cytoplsmic clcium concentrtion through the phosphtidylinositol (PI) signlling system, which is key event in T-cell ctivtion [17]. Eicosnoids re key meditors of inflmmtion nd the regultion of T nd B lymphocyte functions. Archidonic cid (20:4n-6; ARA) nd eicospentenoic cid (20:5n-3; EPA), relesed from membrne phospholipids through the ction of phospholipses, re converted to pro- or nti-inflmmtory eicosnoids, respectively nd, lthough the enzymes of eicosnoid metbolism hve preference for ARA, incresed levels of EPA in the membrnes of immune cells inhibit the production of proinflmmtory eicosnoids [18]. Studies hve demonstrted tht LC-PUFA hve similr roles in immune modultion in fish. Substitution of dietry FO by vegetble oil (VO), with consequent reduction in the rtio of n-3/n-6 LC- PUFA, promotes the synthesis of pro-inflmmtory eicosnoids [19,20], nd lters humorl immunity nd the expression of pro-inflmmtory cytokine genes [21]. Most studies hve been performed in vitro [22-24], nd so little is known bout the roles of LC-PUFA during virl infections in fish. In previous study, the use of functionl feeds led to milder inflmmtory response nd reduced severity of hert lesions in slmon experimentlly infected by Atlntic slmon reovirus (ASRV), the cusl gent of hert nd skeletl muscle inflmmtion (HSMI) [25]. As both HSMI nd CMS hve similr symptoms, we hve employed similr strtegy in the present study to investigte the effects of functionl feeds in controlling CMS. The functionl feeds contined incresed EPA levels, from 3.6% in reference feed to 14%, nd reduced dietry lipid, from 31% to 18% in the functionl feeds. Supplementtion of histidine ws lso ssessed in one of the functionl feeds s this mino cid nd relted compounds, such s N-αcetyl-histidine (NAH), hve importnt roles in muscle ph buffering [26,27] nd tissue ntioxidnt systems

3 Mrtinez-Rubio et l. BMC Genomics 2014, 15:462 Pge 3 of 20 [28,29]. In prticulr, histidine hs been ssocited with crdio-protective role in humn studies, being potentilly beneficil in the llevition of oxidtive stress ssocited with virl diseses [30]. Thus, we hypothesized tht dietry supplementtion of this mino cid could hve potentilly beneficil effect in fish suffering CMS. Fish were fed the experimentl feeds for 8 weeks prior to infection by PMCV nd throughout the study postinfection. The incorportion of LC-PUFA into membrne phospholipids of hert, the min orgn ffected by the disese, ws ssessed, nd both hert nd liver tissues were subjected to histologicl evlution. The inflmmtory nd immune sttus in hert tissue ws ssessed fter infection by determining gene expression through oligonucleotide microrry nlysis. Results Lipid nd ftty cid composition of hert tissue The totl lipid content of hert tissue ws unffected by dietry tretment nd did not show ny significnt chnges over the course of infection (Tble 1). Proportions of tricylglycerol (TAG) were lower nd those of totl PL nd the mjor PL clsses, phosphtidylcholine (PC) nd phosphtidylethnolmine (PE), significntly higher in hert tissue of fish fed the functionl feeds compred with fish fed the REF diet (2-wy ANOVA p- vlue diet, < 5) (Figure 1). These differences were prticulrly pprent t the end of the pre-feeding phse nd, fter PMCV infection, PL levels generlly decresed, significntly for phosphtidylinositol (PI) nd phosphtidylserine (PS), over the course of the infection. The ftty cid compositions of totl PL of hert tissue of fish fed the functionl feeds reflected the composition of the diets showing different proportions of the LC-PUFA tht could potentilly influence the immune response [31] (Tble 1). Thus, the proportions of EPA, ARA nd the EPA/ARA rtio were significntly higher in fish fed the functionl feeds compred to fish fed the REF diet (P vlue diet, <5). However, levels of docoshexenoic cid (22:6n-3; DHA) were similr between the different dietry groups, which ws consistent with our previous study using similr feeds in which tissue levels of DHA were more conserved despite differences in the composition of the diets [25]. Theproportionsoftheinflmmtion-reltedLC-PUFA in hert totl PL chnged during the time course of the infection but were similr in fish fed the three dietry tretments (Tble 1). The chnges in ARA postinfection were not significnt, but the proportions of EPA nd DHA were significntly higher t 6-wpc compred with pre-chllenge. After 6-wpc, levels of DHA progressively decresed during the time-course of the infection but proportions of EPA were significntly higher t 10-wpc. As consequence of the ltter, the EPA/ARA rtio in hert totl PL ws lso higher in ll dietry tretments t 10-wpc. There were no differences in the EPA/ARA rtio in hert PL between fish fed the different diets in the lter stges of the infection. Hert nd liver tissue histopthology Histologicl chnges were consistent with CMS nd first observed in the trium t 6-wpc (Figure 2), chrcterized by focl infiltrtion of inflmmtory cells dominted by lymphocytes. A degenertive process ssocited with the inflmmtory chnges ws lso observed in crdiomyocytes of the trium. The most mrked increse in histopthologicl scores ws found in the ventricle of fish from ll the dietry groups from 6- to 8-wpc. These chnges were typified by multifocl infiltrtion of inflmmtory cells, dominted by lymphocytes nd mcrophges, nd concomitnt degenertion nd necrosis of myocytes. Another typicl finding ws hyperplsi of endothelil cells in inflmed res. Inflmmtory chnges were gretest in the trium from 6- to 8-wpc nd significntly higher for ll groups t both time points. By 10- wpc, the inflmmtory chnges nd myocyte necrosis hd levelled off in ll groups, nd ws not different from 8-wpc. At 12- nd 14-wpc there ws moderte decline in inflmmtory scores (Figure 2). Differences between dietry groups were found minly in the erly stges of the disese, t 6- nd 8-wpc. Fish fed both functionl feeds showed significntly lower histoscores in the trium t both 6- nd 8-wpc compred to fish fed the REF diet (Figure 2). Lesions in the ventricle were lso lower in fish fed the functionl feeds, being significnt in both dietry groups t 8-wpc but only for the fish fed with the CMS2 diet t 6-wpc. There were no differences in inflmmtion in either prt of the hert between dietry groups t 10- nd 12-wpc. At the end of the tril, 14-wpc, lesions in the trium were not significntly different between groups, lthough fish fed with CMS2 hd lower histoscores compred with the other groups. In ventricle, fish fed both functionl feeds showed lower histoscores lthough they were not significntly different (Figure 2). The liver histology scores, bsed on the degree of stetosis were interpreted s vcuole-formtion in heptocyte cytoplsm chrcterized by both micro- nd mcrovesiculr lesions. There were cler differences between the fish fed the functionl feeds nd fish fed the REF diet t ll smplings points post-chllenge, with the ltter group presenting higher micro- nd mcro-vesiculr stetosis. Severity of the liver histopthology ws significntly higher t the beginning of the infection nd greter t the end of the chllenge (12- nd 14-wpc) in slmon fed the REF diet compred to fish fed the two functionl feeds (Figure 3).

4 Tble 1 Ftty cid compositions (percentge of totl ftty cids) of totl phospholipids of hert tissue from fish fed the reference (REF) nd functionl (CMS1 nd CMS2) feeds t different times before (PreCh) nd fter (6, 8, 10, 12 nd 14 weeks) infection with PMCV PreCh 6 wpc 8 wpc 10 wpc 12 wpc 14 wpc TWO WAY ANOVA P-vlue REF CMS1 CMS2 REF CMS1 CMS2 REF CMS1 CMS2 REF CMS1 CMS2 REF CMS1 CMS2 REF CMS1 CMS2 Diet Week Diet*Week Sturted 25.9 ± 18:1n ± Monounst 24.6 ± :2n ± 20:3n-6 ± 20:4n ± n-6 PUFA 7.3 ± 18:3n-3 ± 20:5n ± 22:6n-3 3 ± 1.4 n-3 PUFA 42.0 ± 1.4 PUFA 49.3 ± EPA/ARA 4.6 ± n-3/n ± Totl lipid 2.8 ± 31.2 ± ± 15.4 ± ± ± 4.7 ± ± 9.7 ± 33.8 ± ± ± ± 1 ± 3.5 ± 31.9 ± 6.8 ± 15.3 ± ± 1.6 ± 4.5 ± ± 9.6 ± 33.9 ± 48.0 ± ± 5.8 ± 1 ± 3.6 ± 27.6 ± 9.5 ± 18.0 ± ± ± ± 6.2 ± 1.1 ± 7.7 ± 35.8 ± ± ± ± 7.6 ± 3.32 ± 29.7 ± 6.8 ± 14.7 ± ± 4.5 ± ± 1 ± 35.3 ± ± 55.2 ± 6.3 ± 11.2 ± 3.34 ± 3 ± 7.2 ± 15.1 ± 1.1 ± 4.5 ± ± 1 ± 34.8 ± 5 ± ± ± 11.2 ± 3.49 ± ARA, rchidonic cid; DHA, docoshexenoic cid (DHA); EPA, eicospentenoic cid; PUFA, polyunsturted ftty cid. Diet*Week indictes the p vlue of the interction of those two fctors in the 2-wy ANOVA sttisticl nlysis ± 1 ± 19.5 ± 3.4 ± ± ± 6.2 ± 1.2 ± 8.0 ± 34.6 ± 46.8 ± 53.0 ± 5.5 ± 7.5 ± 3.8 ± 29.9 ± 7.2 ± 14.9 ± 1.8 ± ± 4.7 ± ± 1 ± 34.8 ± 5 ± 55.0 ± 6.1 ± 1 ± 3.34 ± 29.2 ± 8.0 ± 16.8 ± ± ± 1.6 ± 4.8 ± ± 1 ± 33.6 ± 48.8 ± 53.6 ± 6.5 ± 1 ± 3.63 ± 27.1 ± 1 ± 18.9 ± ± ± 1.4 ± 6.1 ± 1.3 ± 8.4 ± 34.5 ± 47.3 ± ± ± 7.7 ± 3.84 ± 29.5 ± 7.5 ± 15.6 ± ± ± 4.8 ± ± 11.3 ± 33.2 ± ± ± 6.6 ± 1 ± 3.55 ± 29.9 ± 7.5 ± 15.8 ± ± ± 4.7 ± ± 1 ± 33.6 ± 49.4 ± ± ± 1 ± 3.58 ± 27.6 ± 11.0 ± 2 ± ± ± 1.3 ± 6.0 ± 1.3 ± 8.0 ± 33.4 ± ± ± 7.7 ± ± 3 ± ± 15.5 ± ± 1.8 ± 4.6 ± ± 10.9 ± 32.9 ± ± ± ± 1 ± 3.56 ± 31.2 ± ± 15.8 ± ± 4.6 ± ± 1 ± 32.8 ± ± 52.6 ± 6.3 ± 1 ± 3.46 ± 27.9 ± 1 ± 2 ± ± ± 1.4 ± 6.4 ± 1.4 ± 7.7 ± ± ± 5.7 ± 6.9 ± 3.81 ± 31.3 ± 7.5 ± 15.7 ± 1.8 ± ± 1.8 ± 5.0 ± ± 1 ± 32.4 ± 47.5 ± 52.5 ± 5.7 ± 9.6 ± 3.44 ± 3 ± 7.3 ± 15.5 ± ± ± 1.8 ± 4.9 ± ± 11.0 ± 32.7 ± ± ± 6.0 ± 1 ± 3.46 ± ns ns 00 ns ns ns ns ns ns Mrtinez-Rubio et l. BMC Genomics 2014, 15:462 Pge 4 of 20

5 Mrtinez-Rubio et l. BMC Genomics 2014, 15:462 Pge 5 of 20 Figure 1 Proportions of lipid clsses in totl lipid of hert tissue from fish fed the reference (REF) nd functionl (CMS1 nd CMS2) feeds t different times before (PreCh) nd fter (6, 8, 10, 12 nd 14 weeks) infection with PMCV. Dt were nlysed by 2-wy ANOVA with time-course nd diet s the two fctors. Different letters bove time-points indicte significnt differences between time-points over the time-course of the infection. The fctor diet showed significnt differences in PC, PE, totl phospholipids nd TAG (not indicted). PC, phosphtidycholine; PE, phosphtidylethnolmine; PI, phosphtidylinositol; PS, phosphtidylserine; TAG, tricylglycerol. Virl lod Virl lod ws similr for the three dietry groups t 6- wpc, however by 8-wpc, lthough individul vrition in virl RNA ws observed, hert virl lod ws higher in fish fed the REF diet compred with fish fed the functionl feeds, significntly so in the cse of fish fed CMS2 (Figure 4). Crdic trnscriptomic responses Time-course of disese Comprison of gene expression chnges in fish fed the REF diet over the course of infection (6-, 8- nd 14-wpc) reltive to pre-chllenge levels showed highly coordinted up-regultion of genes involved in immune pthwys (Figure 5); dt for representtive genes included in Figure 5 re in Tble 2. The ntivirl nd IFN response (62 fetures), minly represented by previously nnotted virus-responsive genes [32], showed highest expression t 6- nd 8-wpc tht levelled off by 14-wpc. The lrgest group of genes ssocited with T cell responses (99 fetures) reched pek levels by 8-wpc coinciding with the pek of crdic histoscores. A similr profile ws observed for genes involved in ntigen presenttion vi mjor histocomptibility complexes (MHC clss I nd II), however their induced expression remined stble to 14-wpc. The B cell response, represented by vrious immunoglobulins (26 fetures), showed incresing expression from 6- nd 8-wpc until pek levels t 14-wpc. Recovery in crdic histopthology by 14-wpc ws consistent with significntly reduced expression of severl inflmmtory mrkers, such s grnzyme A, serum myloid A, TNF decoy receptor (tnfrsf6b) nd neutrophil cytosolic fctor 4. It is lso noteworthy tht negtive immune regultors such s suppressor of cytokine signling 3 nd lymphocyte ntigen 75/cd205 were induced by 14-wpc. Effects of functionl feeds pre- nd post-chllenge Effects on immune responses A lrge frction of differentilly expressed genes encoded proteins of the immune system nd genes from severl

6 Mrtinez-Rubio et l. BMC Genomics 2014, 15:462 Pge 6 of % 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% Atrium histoscores R 1 2 R 1 2 R 1 2 R 1 2 R wpc 8 wpc 10 wpc 12 wpc 14 wpc Atrium Sttisticl model wpc wpc wpc wpc wpc 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% Ventricle histoscores R 1 2 R 1 2 R 1 2 R 1 2 R wpc 8 wpc 10 wpc 12 wpc 14 wpc Ventricle Sttisticl model wpc wpc wpc wpc wpc Figure 2 Histopthology results in hert tissue. Figures on the left re representing incidence (percentge of fish smpled) nd severity of histopthology (bsed on the criteri described in methods) in both prts of the hert, trium nd ventriculum t 6-, 8-, 10-, 12-, 14-weeks post-chllenge in fish fed the Reference diet (R), nd the two functionl feeds CMS1 (1) nd CMS2 (2). Figures on the right re representing the sttisticl nlysis of the trium nd ventriculum histoscores. Estimted effects of CMS1 nd CMS2 diets in comprison to the REF diet by smpling weeks. Negtive estimtes men there re lower scores nd positive tht there re higher scores thn for the REF dietry group. Error brs denote pproximte 95% confidence limits. functionl groups showed similr expression profiles over the course of the infection (Figure 6; Additionl file 1: Tble S1); results for selected genes re given in Tble 3. Overll, the functionl feeds resulted in down-regulted levels of immune genes, suggestive of immune suppressive ctions (Figure 7). At pre-chllenge this ws most significnt for ntivirl nd IFN responses, which showed reduced expression in fish fed CMS1 nd CMS2 compred to fish fed the REF diet (Figure 7). This down-regultion ws lso significnt t 8-wpc, lthough with lower mgnitude, nd this coincided with the most significnt reduction in ventricle histoscore in fish fed the functionl feeds (Figure 2). Among ntivirl nd IFN genes, the lrgest expression differences were found in genes known for potent responses to viruses, such s very lrge inducible GTPse 1, IFN inducible MX protein nd receptor-trnsporting protein 3. Notble exceptions were two genes encoding IFN regultory fctors IRF4 nd IRF7 tht hd from 2.6 to 4.4-fold higher expression before chllenge in slmon fed with the functionl feeds compred to the REF diet. The expression profiles of immunoglobulin (Ig) genes most likely reflected the influx nd mount of B cells in the hert. Fish fed the functionl feeds hd slightly lower bundnce of 28 Ig trnscripts before chllenge tht were further reduced by 6-wpc, followed by normlized levels from 8- to 14-wpc. The most striking immunosuppressive effect of functionl feeds ws observed for T cell response genes, s suggested by significntly reduced expression of 64 trnscripts t pre-chllenge, 6- nd 8-wpc, nd modertely lower reduction t 14-wpc. Down-regultion of CD8 bet, grnzyme A/K nd IFN gmm could imply tht lrge frction of this popultion ws represented by cytotoxic T cells. Grnzyme A nd TNF decoy receptor tnfrsf6b, which were lso down-regulted by the functionl feeds, showed strongest correltion with hert histopthology level in previous CMS study [11].

7 Mrtinez-Rubio et l. BMC Genomics 2014, 15:462 Pge 7 of % 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% Liver histoscores R 1 2 R 1 2 R 1 2 R 1 2 R wpc 8 wpc 10 wpc 12 wpc 14 wpc Liver Sttisticl model wpc wpc wpc wpc wpc Figure 3 Histopthology results in liver tissue. Figure on the left is representing incidence (percentge of fish smpled) nd severity of histopthology (bsed on the criteri described in methods) t 6-, 8-, 10-, 12-, 14-weeks post-chllenge in fish fed the Reference diet (R), nd the two functionl feeds CMS1 (1) nd CMS2 (2). Figure on the right is representing the sttisticl nlysis of liver histoscores. Estimted effects of CMS1 nd CMS2 diets in comprison to the REF diet by smpling weeks. Negtive estimtes men there re lower scores nd positive tht there re higher scores thn for the REF dietry group. Error brs denote pproximte 95% confidence limits. Effects on lipid metbolism Differences in expression of genes involved in lipid metbolism tht could potentilly modulte the immune response were lso observed over the course of the infection (Additionl file 1: Tble S1). Among the significntly relevnt genes were those relted to the PI signlling system (Figure 8) nd the biosynthesis of LC-PUFA. In generl, the expression of the genes relted to the PI signlling pthwy ws not correlted with tissue pthology or the expression of immune-relted genes, s Figure 4 Virl lod in hert tissue of the different dietry groups t 6- nd 8-weeks post-chllenge. Virl lod ws determined by quntittive rel-time PCR nlysis of Piscine Myocrditis Virus. Results re presented s CT vlues (normlized) s bsis for showing the reltive level of virus expression (n = 6) Levels of significnt differences (t-test) were clculted using the reltive expression softwre tool (REST 2009). there ws higher ctivtion of those genes t ll smpling points fter PMCV infection in fish fed the functionl feeds compred with fish fed the REF diet. Of the lipid metbolism genes (Additionl file 1: Tble S1), the most significnt effect ws detected with probes for the delt- 6 ftty cyl desturse (Fdsd6) gene (Figure 9A). Differences between the three dietry groups were most prominent t 6-wpc, when expression of this gene ws higher in the fish fed with the functionl feeds compred with fish fed the REF diet. However, these differences ppered to correlte more with hert tissue pthology rther thn diet s the expression of this gene significntly decresed over the time course of the infection in ll dietry groups (Figure 9B). Discussion The present study demonstrted the potentil of dietry immunomodultion for reducing the pthologicl outcome of virus-ssocited hert diseses in slmon. Specificlly, incresed dietry levels of EPA nd reduced lipid content were ssocited with ltered expression of genes relted with the immune response fter n infection with PMCV, significntly reduced pthology in hert nd liver tissue, nd reduced virl lods t 8 wpc when there ws pek in hert pthology. In contrst, the ddition of histidine did not pper to improve the performnce of the fish s the level of hert lesions nd the expression of genes relted with the immune response were not further reduced in fish fed the histidine supplemented diet. The specific mechnism explining the potentil role (s) of reduced dietry lipid content in the effects of the

8 Mrtinez-Rubio et l. BMC Genomics 2014, 15:462 Pge 8 of Antivirl nd IFN response (61 fetures) b 5 4 Antigen presenttion (25 fetures) b b Log 2 expression rtio B cell response (26 fetures) b T cell response (99 fetures) b c Weeks post PMCV infection Figure 5 Time-course of four immune pthwys to PMCV infection exmined with microrry in slmon fed with REF diet. Boxes show medin log 2 gene expression rtio (totl number of fetures for ech pthwy indicted in pnel heder) between pre-chllenge nd time points post infection, with the 25 th (drk grey) nd 75 th (light gry) percentile, nd whiskers indicte minimum nd mximum vlues. Letters indicte significnt differences between time points (p < 5, Student t-test). functionl feeds is not clerly estblished [25,33]. Previous studies on HSMI showed tht there ws reduced lipid deposition (stetosis) in livers of fish fed the functionl feeds with lower lipid content t initil stges of the disese nd the stetosis ws usully more frequent when the severity of the hert lesions ws high [33]. Thus, there ws some ssocition between liver lipid metbolism nd on-going virl infection, lthough erlier studies hd found no correltion between the severity of hert nd liver lesions during different stges of HSMI disese, ssociting the liver lesions to the circultory disturbnces s consequence of hert pthology [34]. Therefore, lthough the precise mechnism of the reduced liver stetosis index t initil stges in fish fed the functionl feeds ws not identified in detil, lower dietry lipid ws likely contributing fctor [33]. The functionl feeds used in the present study were beneficil for the prevention of liver pthology ssocited with CMS. Fish fed functionl feeds showed lower liver histoscores over the whole course of the infection, significntly so t 6-, 12- nd 14-wpc, possibly due to the lower dietry lipid content tht could reduce liver lipid metbolism when circultory disturbnces re potentilly ffecting this orgn. Levels of EPA nd ARA, nd the EPA/ARA rtio, in hert tissue PL were significntly ltered by the functionl feeds, nd hence one of the min strtegies of the experimentl design ws chieved. Specificlly, the potentil biovilbility of EPA nd its proportion reltive to ARA ws lwys higher over the time-course of the infection in fish fed the functionl feeds compred with fish fed the REF diet. Interestingly, the use of Southern hemisphere FO in the functionl feeds in the present tril lso incresed the level of ARA in hert tissue PL nd thus the EPA/ARA rtio ws ctully lower thn in hert tissue of fish in the previous tril on HSMI [25]. The reltive proportions of ARA nd EPA remined generlly constnt during the time-course of the PMCV infection lthough, s described bove for DHA, there were incresed levels of both ARA nd EPA when, ccording to gene expression nlysis, there ws ctivtion of the immune response. However, this ws only significnt for EPA, possibly reflecting the higher bsolute levels of EPA generted by the functionl feeds in the present tril. As previously described in humns [35] nd fish [21,36,37], the ftty cid compositions of membrne PL plycriticlroleintheregultionoftheinntend dptive immune response. Ftty cids such s ARA, EPA nd DHA, relesed from membrne PL through the ction of phospholipses, modulte the immune response not only through eicosnoids, but lso by

9 Mrtinez-Rubio et l. BMC Genomics 2014, 15:462 Pge 9 of 20 Tble 2 Exmples of immune genes induced in CMS chllenged slmon. Dt re chllenge to pre-chllenged expression rtios (fold chnges) on the fish fed with the REF diet Genbnk Gene, symbol 6 wpc 8 wpc 14 wpc Antigen presenttion nd VRG U20945 MHCII bet chin DY CD AF MHC clss I ntigen DW IFN-induced protein 44, ifit Brrier-to-utointegrtion fctor, binf EG GTPse IMAP fmily member 7, gimp T cells AJ Interferon gmm, ifng DY Grnzyme A, gr NM_ CD8 lph CD EG Cytotoxic T-lymphocyte protein 4, ctl Inflmmtory mrkers, regultors CX TRAF intercting protein, trip Chemokine CCL-C AJ TNFR ssocited fctor 3, trf CU Serum myloid A5, s EG TNF decoy receptor, tnfrsf6b EG IL1b Neutrophil cytosolic fctor 4, ncl TNF lph-induced protein 2, tnfip S Lymphocyte ntigen 75, Ly75/CD Helicse lymphoid-specific, hells DW Suppressor of cytokine signling 3b, socs 3b A B wpi 6 wpi 8 wpi 14 wpi 0 wpc, REF 0 wpc, CMS1 0 wpc, CMS2 6 wpc, REF 6 wpc, CMS1 6 wpc, CMS2 8 wpc, REF 8 wpc, CMS1 8 wpc, CMS2 14 wpc, REF 14 wpc, CMS1 14 wpc, CMS Figure 6 Effect of functionl feeds on gene expression in hert tissue of Atlntic slmon exmined with microrry. A: Number of differentil expressed genes (y-xis) per time point post infection (x-xis). B: Hierrchicl clustering by 2419 fetures (Person r, Wrd s method)

10 Mrtinez-Rubio et l. BMC Genomics 2014, 15:462 Pge 10 of 20 Tble 3 Exmples of immune genes tht responded to the functionl feeds Genbnk Or Probenme Gene, Symbol 0 wpc 6 wpc 8 wpc 14 wpc CMS1 CMS2 CMS1 CMS2 CMS1 CMS2 CMS1 CMS2 Antivirl responses BQ Very lrge inducible GTPse 1-3, vlig ? IFN inducible mx protein DY STAT CB Pyrin, bty BX IRF IRF DW Receptor-trnsporting protein T cells Ss#TC MHC clss I ntigen ? IFNg AY CD8 bet CA Kidney injury molecule 1, hvcr DY Grnzyme A-1, gr ? Grnzyme A-2, gr CR NF ctivted T-cells clcineurin-dependent 1, nftc Inflmmtory mrkers, regultors TNF lph-induced protein 2, tnfip EG c3 nphyltoxin chemotctic receptor, c3r DW Cytochrome P450 fmily 2 subfmily V, cyp2u FcRgmm-like protein, fcer1g EG TNF decoy receptor, tnfrsf6b Helicse lymphoid-specific, hells CK fetuin B S Ly75/CD Dt re expression rtios (fold chnges) of fish fed the functionl feeds (CMS1 nd CMS2) reltive to the REF diet during the course of the infection. modifying signlling pthwys nd stimulting immunerelted nucler trnscription fctors (PPARs nd NFkβ) nd the production of cytokines [15]. The n-6 LC-PUFA, ARA, is ssocited with pro-inflmmtory responses s the precursor of pro-inflmmtory eicosnoids, nd EPA, s well s being the precursor of nti-inflmmtory eicosnoids, cn lso decrese the production of ARA-derived eicosnoids through competition for the eicosnoidsynthesising enzymes. DHA hs lso n nti-inflmmtory role in humns, prticulrly relevnt t the recovery phse of n inflmmtory process, s it is precursor of immune-resolving resolvins nd protectins [31]. Although mcrophges, primrily produced in the hed kidney of fish, re mjor source of eicosnoids, production of eicosnoids t the site of the infection (e.g. hert in CMS) is lso highly relevnt due to the short life of these LC-PUFA derivtives [38]. As previously reported in unchllenged [36] nd chllenged fish [25], DHA levels in hert tissue PL did not reflect dietry levels, being generlly similr in fish fed the three diets in the present study. This my reflect the fundmentl role of DHA in the mintennce of cellulr membrne structure nd fluidity [39]. In our previous study investigting the effects of similr diets on HSMI, levels of DHA were not significntly ffected during the time-course of the infection fter chllenge with ASRV [25]. In contrst, the levels of DHA in hert tissue PL chnged significntly over the time-course of the infection fter PMCV chllenge. Levels of DHA were higher when there ws enhnced immune response nd the hert lesions were more prominent. Whether DHA levels were relted with the ctivtion of immune pthwys involved in the control of the inflmmtory process requires further investigtion in fish. However, the present results suggest tht chnges in the levels of DHA my be ssocited with PMCV infection nd therefore this ftty cid could hve role in the immune response in fish s hs been described in humns [31]. The ssocitions between levels of ARA, EPA nd DHA in hert PL, the extent of hert histologicl lesions, nd crdic expression of genes relted to

11 Mrtinez-Rubio et l. BMC Genomics 2014, 15:462 Pge 11 of 20 0,50 Antivirl nd IFN response (34 fetures) 1,00 B cell response (28 fetures) 0,00-0,50-1, b c 0,50 0,00-0,50-1, b c, b Log 2 expression rtio -1,50-2,00 1,00 0,50 T cell response (62 fetures) b b -1,50-2,00 0,00-0, ,00-1,50-2,00 Weeks post PMCV infection Figure 7 Effect of the two functionl feeds on immune gene expression in hert tissue of Atlntic slmon pre (week 0) nd post (weeks 6, 8, 14) PMCV infection, s exmined with microrry. Dt re log 2 expression rtios between functionl feeds (CMS1 nd 2, noted respectively s -1 nd -2 fter ech time points) nd reference feed (REF). Boxes show medin vlues (totl number of fetures for ech pthwy indicted in pnel heder) with the 25 th (drk grey) nd 75 th (light gry) percentile, nd whiskers indicting minimum nd mximum vlues. Letters indicte significnt differences between time points (p < 5, Student t-test). the immune response, indicted tht dietry effects on incorportion of LC-PUFA into cell membrnes of ffected tissues could hve importnt immunomodultory roles in virl infections. The relevnce of dietry supplementtion with EPA ws supported when evluting the expression of key enzyme of LC-PUFA biosynthesis, delt-6 ftty cyl desturse over the course of the infection. Higher expression of this enzyme nd potentilly higher biosynthesis of n-3 LC-PUFA ws reported previously in liver of fish fed similr functionl feeds compred with fish fed reference diet [25], lter linked to dietry lipid/energy levels [40]. However, the present study is the first to report generlly decresed expression of this enzyme in ll dietry groups over the course of PMCV infection fter 6-wpc, possibly indicting negtive effect of the virl infection on LC-PUFA biosynthesis. This highlights the importnce of dietry supplementtion of ntiinflmmtory n-3 LC-PUFA when fish re suffering virl hert disese lthough further studies re required. Figure 8 Expression between functionl feeds (CMS1 nd CMS2) nd reference diet (REF) of genes relted with phosphtidyl inositol signlling pthwy. At cut off log2-er = (1.75-fold). Red/ornge colour intensity indictes higher expression nd green/blue colour intensity indictes lower expression.

12 Mrtinez-Rubio et l. BMC Genomics 2014, 15:462 Pge 12 of 20 A P-vlue PreCh 6 wpc 8 wpc 14 wpc time CMS1-REF CMS2-REF CMS1-REF CMS2-REF CMS1-REF CMS2-REF CMS1-REF CMS2-REF Slmo slr delt-6 ftty cyl desturse (Fdsd6) 8.41E Slmo slr delt-6 ftty cyl desturse (Fdsd6) gene 4.44E Slmo slr delt-6 ftty cyl desturse (Fdsd6) gene 4.57E Delt-6 ftty cyl desturse D6fd_ 2.00E Slmo slr delt-6 ftty cyl desturse (Fdsd6) gene 2.18E B REF CMS1 CMS2 Normlized expression PreCh 6 wpc 8 wpc 14 wpc Normlized expression PreCh 6 wpc 8 wpc 14 wpc Normlized expression PreCh 6 wpc 8 wpc 14 wpc Figure 9 Expression of ftty cyl desturse (Fdsd6) gene. A) Normlized expression of different probes of the oligorry from delt-6 ftty cyl desturse (Fdsd6) gene over the time course of the PMCV infection on fish fed with the REF diet nd the functionl feeds (CMS1 nd CMS2). B) Expression rtios (fold chnges) of different probes of the oligorry from delt-6 ftty cyl desturse (Fdsd6) gene. Dt re fish fed the functionl feeds (CMS1 nd CMS2) reltive to the REF diet during the course of the infection. Red/ornge colour intensity indictes higher expression nd green/blue colour intensity indictes lower expression. In ddition to ftty cid composition, the PL clss composition ws lso ffected during virl infection, with decresed proportions of PI nd PS s the infection progressed. Phosphorylted derivtives of PI such s phosphtidylinositol 4,5-bisphosphte (PIP 2 ) re involved in the production of the intrcellulr second messengers dicylglycerol (DAG) nd inositol 1,4,5-triphosphte (IP 3 ) through the ction of the phospholipse C [17]. DAG nd IP 3 ctivte clcium chnnels, incresing C +2 concentrtion in the cytosol, with this being n essentil step for the survivl of some humn viruses [41]. PS hs lso been relted with this signlling pthwy s it is n importnt ctivtor of protein kinse C [42,43]. The difference in expression of genes relted with PI signlling between the dietry groups long with decresed proportions of PI nd PS, suggested relevnce of this pthwy during the infection, nd could reflect utiliztion of these PL clsses ssocited with n immunomodultory role. This is novel finding nd the composition/content of specific PL clsses could be n interesting re for future studies on clinicl nutrition in fish. Diet lone hd mrked effects on the crdic expression of immune genes prior to chllenge with functionl feeds suppressing genes relted with innte ntivirl responses nd even those specific for lymphocytes. Brodly similr effects were recently reported in tril investigting substitution of dietry FO with VO in slmon [44]. In tht study, expression levels of immune-relted genes in liver of fish fed FO diet, with ftty cid composition similr to the functionl feeds, were generlly lower thn in fish fed VO diet, with composition similr to the present REF diet. However, in the erlier study, genes relted with the T-cell response were expressed t lower level in fish fed the VO diet [44]. This my be explined by the higher levels of EPA present in the functionl feeds used in the present study compred with the FO diet used in the erlier study, s the influence of EPA in controlling T-cell signlling pthwys hs been documented previously [15,18]. Expression of genes relted with non-specific immune responses were lso higher in grouper (Epinephelus mlbricus) fed VO/FO blend compred with fish fed FO lone [37]. So it ppers tht, even without ny pprent infection, inclusion of FO could dmpen both innte nd dptive immune responses in fish. Thus, the generlly higher immune sttus of the fish fed the REF diet could be fctor in the erlier development of inflmmtion nd hert lesions ssocited with CMS in these fish compred with the fish fed the functionl feeds. Put nother wy, the functionl feeds generlly lowered the immune/inflmmtory sttus prior to virl chllenge nd, in doing so, delyed nd moderted the response to the infection. This ws supported by the recent finding tht showed low-responder fish to CMS, which developed high infection levels in the

13 Mrtinez-Rubio et l. BMC Genomics 2014, 15:462 Pge 13 of 20 bsence of crdic pthology, hd blted expression of dptive immunity genes nd genes involved in T cell responses [11]. However, it must be emphsised tht immune nd inflmmtory responses re essentil components of the host defence ginst infections [45]. Therefore, strtegies modulting the inflmmtory response must be blnce between promoting resolution nd mitigting negtive effects of chronic inflmmtion tht cn led to dmging fibrosis without preventing the initil onset of the response [31]. Interestingly, IFN regultory fctors 4 nd 7 were mong the few innte ntivirl genes tht were induced by the functionl feeds prior to infection. While IRF7 presumbly is n importnt regultor of the ntivirl IFN response in fish, s in mmmls [46], the suggested role of IRF4 s negtive regultor of TLR signlling nd pro-inflmmtory cytokines in mice peritonel mcrophges [47] is interesting in view of the down-regulted ntivirl stte pre-chllenge of fish fed the functionl feeds nd hence the possible implictions for the positive effects post-infection. Whether IRF4 hs similr function in fish hs yet to be confirmed. The time-courses for the development nd resolution of histopthology nd immune gene responses were similr to those observed previously [10,11]. In ddition, there were correltions between the expression of genes relted with immune responses, virl lod nd hert lesions, in greement with Timmerhus et l. [10]. Importntly, however, the temporl differences observed between the different dietry tretments in the present study strengthened these correltions. Thus, the chnges in the progression of CMS hert pthology, gene expression nd virl lod, in fish fed the REF diet were temporlly similr to the development of the infection described previously [10], wheres there ws cler dely in the ppernce of those chnges in fish fed the functionl feeds. Although virl lod ws not monitored t lter stges of the PMCV infection, the forementioned correltion between histopthology nd gene expression in the REF group llow us to hypothesize tht virl lod in fish fed the functionl feeds would be higher t the lter stges. Therefore, the lower virl lod in hert of fish fed the functionl feeds t erly stges of PMCV infection ws probbly direct effect of diet on initil virl repliction s reported previously fter ASRV infection [25]. Furthermore, in ddition to dely in the ppernce of hert pthology in fish fed the functionl feeds, slightly improved performnce of fish fed the CMS2 diet ws observed, which could be ssocited with reduced expression of genes ssocited with the innte immune response. At 6-wpc, gene expression nd hert tissue histopthology of fish fed the REF diet were in greement with those reported previously [10]. Thus, trium ws ffected by the infection t 6-wpc with higher histoscores in fish fed the REF diet compred to fish fed the functionl feeds, wheres ventricle ws only slightly ffected. A pek in the expression of genes relted with the complement response, B nd T-cell responses, nd poptosis ws expected t this time post-infection [10], nd mny of the genes relted to these pthwys showed lower expression in fish fed the functionl feeds compred to fish fed the REF diet. In ddition, genes relted with ntivirl nd interferon responses often showed higher expression in fish fed the functionl feeds compred with fish fed the REF diet. The pek in expression of the ltter genes ws reported to be t 2-4 wpc in the erlier study [10], supporting the conclusion tht there ws delyed response in fish fed the functionl feeds. A similr delyed response to virl infection of round 2 weeks ws observed previously fter ASRV infection in slmon fed functionl feeds [25]. As the CMS disese progressed the extent of lesions in the hert incresed in both trium nd ventricle with the differences between the fish fed the functionl feeds nd fish fed the REF diet being most pronounced t 8-wpc. At this time-point, fish fed the functionl feeds presented lower histoscores in both trium nd ventricle compred with fish fed the REF diet, which could be due to the upregultion of genes relted with host immune response observed t 6-wpc in fish fed the REF diet. At 8-wpc, following the hypothesis of delyed development of CMS in fish fed the functionl feeds, higher expression of genes involved in ntivirl nd innte immune pthwys could hve been expected in these fish compred with fish fed the REF diet but, in contrst, these groups showed lower ctivtion of these pthwys. Dietry modultion of the inflmmtory response hs, of course, been described previously in fish. Higher incorportion of n-3 LC-PUFA in biologicl membrnes of immune cells led to lower expression of pro-inflmmtory cytokines in gilthed sebrem (Sprus urt) [21], decresed ntibody production nd mcrophge killing ctivity in rinbow trout [48], nd lower production of pro-inflmmtory eicosnoids in Atlntic slmon [19,20]. Furthermore, dietry EPA suppressed production nd relese of TNF, interleukins nd IFN in humns [49]. Dietry EPA lso suppressed key components of the ntivirl response in gilthed se brem, nd dietry VO inclusion modulted the expression of Mx proteins, interferon-induced meditors of innte resistnce to RNA virus [50]. According to these previous studies, the incresed proportions of dietry EPA in the functionl feeds used in the present study could be fctor in preventing n uncontrolled immune response tht could be more hrmful to the fish. As expected, mny genes relted with the T-cell response showed lower expression in fish fed the functionl feeds compred with fish fed the REF diet [10].

14 Mrtinez-Rubio et l. BMC Genomics 2014, 15:462 Pge 14 of 20 Although gene expression ws not ssessed t 10- nd 12-wpc, incresed trnscription of genes involved in virus clernce my be predicted bsed on histopthology of hert lesions t these time-points. By 14-wpc, there ws cler remission in the lesions in both prts of the hert in ll dietry groups nd therefore no significnt differences in the pthology were observed between them. Even though the list of genes differentilly expressed t 14-wpc in fish fed the different diets ws shorter thn the ones from previous smpling points, some pthwys represented in the list of genes differentilly expressed t 14- wpc in fish fed the different diets could still indicte more effective remedition of the inflmmtory response by the group of fish fed with the functionl feeds. Thus, importnt mrkers of the T-cell response such s T cell receptor lph, CD8 bet nd lph nd CD82 ntigen showed lower expression in fish fed the functionl feeds, but expression of genes relted with PI signlling ws higher in those fish compred with fish fed the REF diet. As mentioned bove, the PI signlling pthwy is intimtely involved in the regultion of the T-cell response [17], nd dependent on the levels of membrne PL clsses nd their ftty cid compositions. As the lesions in hert tissue in CMS were ssocited with the ction of CD8+ cytotoxic T-cells [10], fctors controlling this response, such s nti-inflmmtory n-3 LC-PUFA, could be key to the generl better performnce observed in the fish fed the functionl feeds. Furthermore, results from the previous study evluting the use of functionl feeds in slmon infected with HSMI were consistent with this hypothesis. Thus, milder expression of genes relted with inflmmtory response nd virus clernce, including those relted with T-cell response, were reported in slmon fed the functionl feeds leding to better performnce of those fish over the course of the HSMI infection [25]. Conclusions The present study is the first to describe the effects of functionl feeds on the expression of genes relted with the immune response fter infection of Atlntic slmon with PMCV. Significnt differences in immune nd inflmmtory responses nd pthology in hert tissue were found in fish fed the different dietry tretments over the course of the infection, highlighting the potentil immune modultory role of dietry ftty cid composition nd lipid content in virl infections in slmon. The strtegy ws effective in significntly reducing hert pthology nd lso decresing the time-course of the virl infection. Furthermore, liver pthology ws significntly reduced during the time course of the infection in fish fed the functionl feeds. Moleculr signtures showed lower expression of immune-inflmmtory genes, especilly those reported to be correlted with hert lesions in the clinicl phse (i.e. T cells). Moreover, trnscriptome nlysis reveled novel pthwy of the fish immune response, the PI signlling pthwy, tht functionl feeds could modulte, s well s potentil suppression of LC-PUFA biosynthesis s the infection progresses, stressing the importnce of dietry supplementtion with n-3 LC-PUFA. Methods Experimentl feeds nd fish Three fishmel-bsed diets, bsed on previous study investigting HSMI [25], were formulted nd mnufctured by EWOS Innovtion (Dirdl, Norwy) (Tble 4). The reference diet (REF) ws essentilly stndrd, commercil formultion with 31% lipid with the dded oil being blend of Northern hemisphere FO nd rpeseed oil. The two functionl feeds (CMS1 nd CMS2) both contined lower level of lipid (18%) tht ws blnced by incresed protein, provided by fishmel nd krill mel. Therefore, s mjor fctor differing between the REF nd functionl feeds ws dietry lipid level, the feeds were not isolipidic or isoproteic. The dded oil in the functionl feeds ws provided by Southern hemisphere FO. As result the CMS1 nd CMS2 feeds hd similr ftty cid profile contining higher proportions of EPA (lmost 14%) nd n-3/n-6 PUFA rtio round 4, in comprison to the REF feed (<4% EPA nd n n-3/n-6 rtio of 1.4) (Tble 5). The only mjor difference between the functionl feeds ws tht CMS1 ws supplemented with dditionl histidine for resons described bove [30]. Tble 4 Formultion (g/kg) nd proximte composition (percentge) of the reference (REF) nd functionl (CMS1 nd CMS2) feeds Component (g/100 g) 1 REF CMS1 CMS2 Fish mel nd hydrolystes Plnt protein concentrtes Northern fish oil 13.0 Southern fish oil 1 1 Rpeseed oil 1 Crbohydrte-bsed binders Micro premixes Krill mel Histidine (synthetic) Totl Proximte composition Moisture Ft Protein ) All ingredients sourced from EWOS stocks unless otherwise stted. 2) Includes soy protein concentrte, pe protein concentrte, whet gluten nd sunflower mel. 3) Includes whet grin. 4) Includes vitmins, minerls, crystlline mino cids, mmonium phosphte. 5) Aker Biomrine AS.