UNDERSTANDING BIOMARKERS IN FISH NUTRITION
UNDERSTANDINGBIOMARKERSIN FISH NUTRITIONTECHNICAL BOOKLET2
Technical content prepared byCSIC with contributions fromother ARRAINA project partnersAll photos and images supplied by CSIC, Wageningen University,INRA, NIFES, CCMAR and AquaTT.
UNDERSTANDINGBIOMARKERSIN FISH NUTRITIONTECHNICAL BOOKLETReliable use of biomarkers and methodologies forassessing nutritional status, and metabolic and healthconditions, in fish fed with diets based on plant ingredients.
Feed Ingredients in AquacultureINTRODUCTION 5About This Booklet 5About Aquaculture Feeds in Europe 5About the ARRAINA Project 5ARRAINA Biomarkers Database Tools to Predict Flesh Fatty Acid Profiles7 Biomarkers of Mineral and Vitamin Requirements Zinc and Bone Development in Freshwater and Seawater Salmon Markers of Vitamin B6 requirements in Freshwater and Seawater Salmon 9101011Markers of Methionine Metabolism in Rainbow Trout 12Markers of Lipid Metabolism and Growth Performance in European Sea Bass and Gilthead Sea Bream 14Molecular Markers of Lipid Metabolism in European Sea Bass and Gilthead Sea Bream 15Biomarkers of Lipid Metabolism in Gilthead Sea Bream 16Biomarkers of Growth Performance and Specific Nutritional Status 17Markers of Intestinal Function and Architecture in European Sea Bass and Gilthead Sea Bream Nutritionally Mediated Effects on the Intestinal Transcriptome 1920Determining the Potential Presence and Prevalence of Contaminants in Fish Fillets for Human Consumption 21Concluding Remarks 22ARRAINA References 23Acknowledgements 261
INTRODUCTIONAbout This BookletThis booklet is the final in a series of threetechnical booklets on the nutrition and feedingof fish farmed in Europe, and has been producedunder the framework of the European Union FP7funded ARRAINA project (Advanced ResearchInitiatives for Nutrition & Aquaculture). The aimis to make these booklets widely available bothto targeted stakeholders and society in general,in order to raise awareness of the science-basedknowledge supporting the development of highquality, safe, and environmentally sustainableaquaculture feeds.This booklet is specifically aimed at scientistsand feed producers, but it also targets otherindustrial segments (e.g. fish farmers, feedadditives companies, retailers) and individualsinterested in gaining further knowledge of thephysiological consequences of the raw materialsthat are currently used in the feeds of farmedfish. The species focussed on by the ARRAINAproject are: Atlantic salmon, rainbow trout,common carp, European sea bass andgilthead sea bream.The objective of this booklet is to providea reliable set of biomarkers and associatedmethodologies to assess the nutritionallymediated effects on growth performance,metabolic homeostasis, stress responsivenessand health condition of fish which are fednew diet formulations from early life stagesto completion of production cycle and sexualmaturation. Of particular value are predictiveand non-invasive biomarkers available at arelatively low cost, although the combinationof conventional and omics approaches isemerging as a user-friendly option.The first two ARRAINA technical bookletswere focused on feed ingredients and nutrientrequirements, and are available at www.arraina.eu.About Aquaculture Feeds in EuropeFish feeds are produced using a large varietyof feed ingredients that have different nutritionaland physical properties. They are formulatedon the basis of solid scientific knowledge,contributing to the development of anenvironmentally-friendly aquaculture sectorand to produce high quality nutritious foodfor humans1-3.The production of environmentally sustainableaquaculture feeds starts with the selection ofhigh-quality raw materials that may need to besupplemented with specific nutrients (aminoacids, phospholipids, vitamins, macro and microminerals) to support maximum growth, healthand well-being of fish. Given that the nutrientprofiles of marine feedstuffs and plant proteinsources can vary greatly, one of the aims ofthe ARRAINA project was to identify whichmicronutrients, and in which form, need to besupplemented to plant-based diets to supportmaximum growth and production of farmed fishfrom early life stages to completion of sexualmaturation.The ARRAINA project brings togetherdifferent competences for developing toolsand methodologies based on targeted andnon-targeted biomarker approaches in the fivespecies of interest for the assessment of nutrientrequirements or status of fish, going furtherthan just identifying differences in growthparameters.Additionally, the project considers thepotential presence and prevalence ofcontaminants associated with plant-basedfeedstuffs on fish fillets for human consumption,developing targeted and broad screeningmethods for fast and sensitive analyses.About the ARRAINA ProjectIt is well recognised that the sustainability andcompetitiveness of aquaculture depend stronglyon the replacement of capture fishery-derivedfish meals and oils with alternative feedstuffs ofplant origin. ARRAINA has been responding tothis need by measuring the long-term effectsthat these changes in diet composition will haveon the full life cycle of fish for which presentlylittle is known.5
INTRODUCTION CONTD.One major aim of ARRAINA is to developapplicable tools and solutions of technologicalinterest to the European fish feed industry. Incollaboration with SMEs, ARRAINA strengthensthe links between the scientific community andthe EU feed industry and its contribution couldincrease the productivity and performance ofthe aquaculture sector leading to competitiveadvantage at a global level.For general information on the project, youmay download the project factsheet from theARRAINA website www.arraina.eu. For moreinformation, please contact the ARRAINA ProjectCoordinator, Sadasivam Kaushik (sadasivam.kaushik@inra.fr) or the Project Manager, VincentTroillard (vincent.troillard@inra.fr).1 Benedito-Palos L.; Navarro J.C.; Sitjà-Bobadilla A, Bell J.G.;Kaushik S.; Pérez-Sánchez J. (2008). High levels of vegetableoils in plant protein-rich diets fed to gilthead sea bream (Sparusaurata L.): growth performance, muscle fatty acid profiles andhistological alterations of target tissues. British Journalof Nutrition 454, 8-18.2 Nasopoulou C.; Zabetakis L. (2012). Benefits of fish oilreplacement by plant originated oils in compounded fishfeeds. A review. IWT Food Sci. Technol. 47, 217-224.3 Tacon S.G.J.; Metian M. (2008). Global overview on the useof fish meal and fish oil in industrially compounded aquafeeds:trends and future prospects. Aquaculture 285, 146-1586
ARRAINA Biomarkers DatabaseBased on the literature and ARRAINA projectresults, several sets of biomarkers have beenidentified to assess nutrient requirements andto metabolically phenotype the nutritionallymediated effects of feeds on health andperformance status in the five fish species of theproject (Atlantic salmon, rainbow trout, commoncarp, European sea bass and gilthead sea bream).Examples of use include histopathological,biochemical and molecular markers of tissues andbody fluids to be indicative of a wide range ofbiological/metabolic processes linked with protein,carbohydrate and lipid metabolism.Relevant information from ARRAINA isperiodically uploaded to the online ARRAINAbiomarkers database (www.nutrigroup-iats.org/arraina-biomarkers) designed for singleor combined searches of biomarkers providinginformation on nutrient requirements and specificbiological processes or metabolic pathways,including those related to health and welfare,growth and reproductive performance, qualityat harvest, safety, and stress responses.Ideal biomarkers:Fish Species in focus: Non-invasive Atlantic salmon (Salmo salar) Easy to measure Common carp (Cyprinus carpio) Cost effective European sea bass (Dicentrarchuslabrax) H ighly sensitive and specific whenused alone or in combination withother biomarkers C onsistent across developmentalstage, season, reproductive andnutritional condition, geneticbackground, etc. G ilthead sea bream (Sparus aurata) R ainbow trout (Oncorhynchusmykiss)At this stage, the ARRAINA biomarkers databasecontains more than 700 independent entriessubmitted by all the partners of the project. Thedatabase is periodically updated with new partnerinputs, and especially valuable are the reportingdata to establish the normal range of variationand robustness of selected markers in nutritionallychallenged fish.7
User-friendly Database of Biomarkers in Fish sExample of use1238
Tools to Predict Flesh Fatty Acid ProfilesFillet fatty acid composition of fatty fish and,to a lesser extent, lean fish, highly reflects thecomposition of diet. The replacement of fish oilwith vegetable oils can compromise the highnutritive value of farmed fish as an importantsource of omega-3 long-chain poly-unsaturatedfatty acids (PUFA) in the human diet. This is moreevident in marine fish due to their limited capacityto elongate and desaturate C18 fatty acids into longchain C20 and C22 PUFA.To ensure the compatibility of fish feeds withhealthy fish for human consumers, an interactiveand publicly accessible web interface (www.nutrigroup-iats.org/aquafat) has been developedwithin the framework of ARRAINA and nationalprojects (AQUAFAT) to predict the fatty acidcomposition of farmed fish flesh.The tool has been initially developed and validatedfor European sea bass, gilthead sea bream,turbot, and sole. The model is based on dummyregression equations, and it is able to predict theproportion of saturated, mono-unsaturated andpoly-unsaturated fatty acids in a fish fillet whenkey data on diet composition and body fat contentare provided.9
Biomarkers of Mineral and Vitamin RequirementSZinc and Bone Development inFreshwater and Seawater Salmonfreshwater fish. In fresh water, dietary levels of 62 mg Zn/kg were sufficient for a vertebral andwhole fish Zn concentration comparable to normallevels in Atlantic salmon. This is in line with dataon Zn requirement for Atlantic salmon reared infresh water (Maage et al. 1993). However, in salmonreared in seawater, 160 mg Zn/kg diet resultedin vertebral and whole fish Zn concentrationscomparable to normal levels found in seawaterreared Atlantic salmon.Zinc (Zn) is a mineral that functions as a cofactorin several enzyme systems and is crucial for normalbone development. However, high levels of dietaryZn increase the cost of feed, increasing also therelease of minerals to the aquatic environment.Therefore, optimal additions of Zn to the feed areneeded to secure normal fish development withminimal environmental impact.Regarding Zn-dependent enzymes involvedin bone metabolism, the ALP/TRAP1 ratio infreshwater fish did not change when extra ZnARRAINA studies indicate that total and vertebralZn content of seawater fish is lower than inwas added to their diets. This supports therecommendations made by Maage et al. (1993)(37-67 mg Zn/kg diet) for freshwater Atlanticsalmon.In seawater grown salmon, the ALP/TRAP ratio wassignificantly reduced when more Zn was added tothe diet. Along with the increases in whole fish andvertebral Zn content, this provides strong evidencethat the dietary Zn addition to seawater salmonshould be around 160-190 mg/kg to supporthealthy bone development.Zinc in vertebraeALP/TRAP ratio Seawater4000.4Zn (mg kg -1 dw)p –0.00200100200Dietary Zn (mg kg -1 ww)101A lkaline3000.002550100 150200 400NP addition (% of requirement)phosphatase (ALP) – marker of bone forming cells, osteoblasts -.Tartrate resistant acid phospahatase (TRAP) – marker of bone-resorbing cells, osteoclasts -.Maage A.; Julshamn K. (1993). Assessment of zinc status in juvenile Atlantic salmon (Salmo salar) by measurement of whole body and tissue levels of zinc. Aquaculture 117, 179-191.
Markers of Vitamin B6 Requirementsin Freshwater and Seawater SalmonThe use of alternative ingredients as fish mealreplacers in feeds for farmed fish requires increasedfocus on water soluble vitamin supplies. Fishmeal is rich in most B vitamins, whereas otheringredients may contain variable amounts bothof these and other chemical forms that differ inbioavailability.ARRAINA biomarkers to determine vitamin B6(pyridoxine) requirements include classic muscletissue measures of B6 vitamin status and thecorresponding activity of ASAT1 in muscle, ametabolic enzyme that uses vitamin B6 as cofactor.Alternatively, vitamin B6 takes part in the 1-Cmetabolism through the transsulfuration pathway,and the resulting metabolic outcome (organlipid accumulation, metabolite profiles and geneexpression patterns) should be used as a marker ofvitamin B6 deficiency, although it is unspecific.The results showed that the currentrecommendations for vitamin B6 in practicalplant-based feeds for Atlantic salmon are too low,both in freshwater and seawater.Vitamin B6 in muscleASAT in muscle activated or not by B6880006Activity (U/kg)mg/kg wet wt100% NP4200HO6000O4000Not ated2000p 0.0001NP addition (% of requirement)1A spartate100% NPNVitamin B6500NP addition (% of requirement)aminotransferases (ASAT) uses vitamin B6 as cofactor and its activation is a good marker of vitamin B6 status.11
Markers of Methionine Metabolismin Rainbow TroutThe sustainability of the growing aquacultureindustry depends on the progressive reductionof wild-fishery derived feedstuffs in fish feeds.Plant proteins have therefore been proposed assustainable alternatives to fish meal. However, theessential amino acid profile in plant proteins differsfrom that of fish meal. As a result, plant-based dietsdo not generally meet the essential amino acidrequirements for some fish species, rainbow troutfor example, and often necessitate the additionof one or several crystalline amino acids in orderto meet the essential amino acid requirements.Methionine is considered one of the most limitingessential amino acids in many plant-proteinsources.Studies performed in the framework of theARRAINA project indicate that methioninedeficiency negatively affects growth performancesbut also impacts cellular mechanisms regulatingmetabolism and growth. In juvenile trout,methionine deficiency increased proteolyticpathways as indicated by the higher white musclelevels of autophagy-related markers at bothprotein (LC3-II and beclin 1) and gene expressionlevels (ATG4b, ATG12 l, UVEAG, SQSTM1, Mul1and Bnip3). Several genes related to proteasomeproteolytic system (Fbx32, MuRF2, MuRF3,ZNF216 and Trim32) were also significantlyupregulated by methionine limitation.Regarding hepatic metabolism, the ARRAINAstudies highlighted cholesterol metabolism as atarget of methionine deficiency with a reducedexpression of genes involved in the synthesisof cholesterol* in the liver of female broodstockand an enhanced expression of genes related tocholesterol elimination** (CYP7A1, ABCG8) inyoung fry.Together, these results extend our understandingof mechanisms regulating the reduction of musclegrowth induced by dietary methionine deficiency,and provide valuable information on biomarkers ofthe effects of plant-based diets.Markers of cholesterol metabolism affected by dietary methionine imbalance:*Cholesterol synthesis: HMGCS, HMGCR, DHCR7 and CYP51**Cholesterol elimination: CYP7A1 and UGT1A3 (bile acid synthesis) and ABCA1, ABCG8 and LXR (cholesterol efflux and excretion)12
aa,b1.0b0.50.0Beclin 1X-TubulinDEFADQEXC9483.576543*210ABCG8/geomean of HSK genesBeclin 1:X-tubulin1.5Hepatic gene expression level of CYP7A1and ABCG8 in trout fed deficient (D) oradequate (A) methionine dietsCYP7A1/geomean of HSK genesBeclin protein level in white muscle of troutfed diet with deficient (DEF), adequate(ADQ) or excess (EXC) levels of methionine32.52*1.510.50DADA13
Markers of Lipid Metabolism and Growth Performancein European Sea Bass and Gilthead Sea BreamExperimental evidence indicates that tissuehistopathological scoring, and measurementof circulating levels of electrolytes, metabolitesand hormones accurately reflect impaired growthperformance, stress condition, and diseaseoutcomes in European sea bass and giltheadsea bream. However, the use of these analysesas diagnostic or predictive tools is not yet wellestablished. This is mostly due to the lack ofreliable information on reference values, whichis especially evident for the new biomarkersemerging from “omic” approaches.200017501750 Fasting and re-feeding of fish which have beenfed commercial diet formulations. Identification of the normal range ofvariation for selected markers in fish whichhave been fed both standard feed andsustainable feed formulations based onplant ingredients.1000100010010000JanMay SepJan MayMay SepSep JanSep JanJan MayMay SepSep JanMay20162015201320142013201420152016BW 300g300 gBWSGR 0.70SGR 0.70 0.81FEFE 0.31FE 0.72FE 0.72606040402020 1000 gBWBW 1000g 0.51SGRSGR 0.51Body weight (g)Body weight (g)808050050025025014CTRL ARRAINA DIETCTRL SEMI-PURIFIED DIETMetn-3 LC-PUFAPLPMin Mix (1/2)Vit Mix (1/2)750750FE 0.990.99FE Long-term trials with ARRAINA diets formulatedby BIOMAR (industrial partner within the project)with different proportions of marine feedstuffsand plant ingredients. Definition of the most informative markersand tissues for the functional assessment ofa given nutrient deficiency and metabolicstatus. Short-term trials with semi-purified dietsformulated for specific nutrient deficiencies in12501250SGR 1.41.4SGRmethionine, essential fatty acids, phospholipids,phosphorous or minerals.EXPECTED RESULTSD1 (FM25/FO15)D2 (FM5/FO6)D3 (FM5/FO2.5)D4 (FM5/FO2.5/BUT)15001500Body weight (g)Body weight (g)The ARRAINA project aims to fill some of theseknowledge gaps, focusing on the definition ofnormal range values for the most robust andinformative biomarkers with emphasis on the liver,skeletal muscle, adipose tissue and the intestine.This is a meta-analysis approach with complexdata mining of results from different feeding/growth trials: 1600 gBW BW 1600g000020204040DaysDays60608080100100
Customised LIPID-chip PCR arrays with morethan 40 selected markers have been used in theARRAINA project for the transcriptomic profilingof lipid metabolic disorders in both European seabass and gilthead sea breamIn the liver, up to 24 differentially expressed(DE) genes (including desaturases, elongases,phospholipases and enzymes of lipoproteinand cholesterol metabolism) have been initiallyrecognised as robust markers of lipid metabolicdisorders in juveniles of gilthead sea bream withclear signs of deficiencies in essential fatty acidsor phospholipids. Among them, five genes areunspecific markers of lipid metabolic disorders,whereas the rest are emerging as robust andspecific markers of fatty acid (15) or phospholipid(4) deficiencies.The same trend was found in the skeletal muscleand adipose tissue, although the number of DEgenes decreased from 24 to 9.LIPID-CHIP15 43 genes5(24)4 Elongases, 4 Desaturates, 33 P L metabolism, 10 L ipase-related genes, 9 β-oxidation, 4 C holesterol & lipoproteinmetabolism, 10 T ranscription factors, 3Benedito Palas et al., 2013; 2014Rimoldi et al., 2016DE GenesMolecular Markers of LipidMetabolism in European Sea Bassand Gilthead Sea Bream30252015105051(9)6LiverSKM3(9)ATPL DeficiencyFA DeficiencyThis different tissue responsiveness is even moreevident in the brain, with a gene expression patternthat remains mostly unaltered during fasting andrefeeding in both European sea bass and giltheadsea bream.15
Biomarkers of Lipid Metabolismin Gilthead Sea BreamThe number of DE genes was practically reducedby half when the LIPID-chip PCR-array was usedto assess the differential gene expression pattern inthe liver of gilthead sea bream fed ARRAINA dietssupporting maximum growth from early life stages.Overall, the magnitude of changes was alsoreduced consistently.Together, all these obs
Coordinator, Sadasivam Kaushik (sadasivam. kaushik@inra.fr) or the Project Manager, Vincent Troillard (vincent.troillard@inra.fr). 1 Benedito-Palos L.; Navarro J.C.; Sitjà-Bobadilla A, Bell J.G.; Kaushik S.; Pérez-Sánchez J. (2008). High levels of vegetable oils in p
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r. Seuss's One fish, two fish, red fish, blue fish is a clas-sic children's story, a simple rhyming book for beginning readers. We need a similar rhyme to help people grasp the problems afflicting Alberta's native fish species. It might read like this: Two fish, one fish, dead fish, no fish, No grayling or goldeye, something's amiss .
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biomarkers that may predict sensitivity to immunotherapy is an area of active research. It is envisaged that a deeper . and emerging technologies showing promise for deeper profiling and insights. Biomarkers and immunotherapy modalities Peripheral immune-based biomarkers . † Uses millions of short reads (sequence strings), so all RNA in a .
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Page 1 of 37 2012 HOME ECONOMICS SEC MARKING SCHEME PAPER I 1a. Two other types of fish (2 x 0.5 marks) oily fish, shell fish. b.i. Type of fish which has tough fibres (0.5 mark) oily fish b.ii. Type of fish which is easily digested (0.5 mark) white fish c. Nutritive value of fish
Fish Dichotomous Key Step 1 If fish shape is long and skinny then go to step 2 If fish shape is not long and skinny, then go to step 3 Step 5 If fish has spots, then go to step 6 If fish does not have spots, then go to step 7 Step 2 If fish has pointed fins, it is a trumpet fish If fish has smooth fins, it is a spotted moray eel Step 6
