rodriguespm12_proteomics in aquaculture.pdf

J O U R N A L O F P R O T E O M I C S 7 5 ( 2 0 1 2 ) 4 3 2 5 – 4 3 4 5

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Review

PROTEOMICS in aquaculture: Applications and trends☆

Pedro M. Rodriguesa,⁎, Tomé S. Silvaa, Jorge Diasa, Flemming Jessenb

aCentro de Ciências do Mar do Algarve (CCMar), Universidade do Algarve, Campus de Gambelas, 8005-139 Faro, PortugalbNational Food Institute, Technical University of Denmark, 2800 Kgs. Lyngby, Denmark

A R T I C L E I N F O

☆ This article is part of a Special Issue entit⁎ Corresponding author. Tel.: +351 289800100

E-mail address: [email protected] (P.M. R

1874-3919/$ – see front matter © 2012 Elseviedoi:10.1016/j.jprot.2012.03.042

A B S T R A C T

Article history:Received 20 December 2011Accepted 24 March 2012Available online 4 April 2012

Over the last forty years global aquaculture presented a growth rate of 6.9% per annumwithan amazing production of 52.5 million tonnes in 2008, and a contribution of 43% of aquaticanimal food for human consumption. In order to meet the world's health requirements offish protein, a continuous growth in production is still expected for decades to come.Aquaculture is, though, a very competitivemarket, and a global awareness regarding the useofscientific knowledge and emerging technologies to obtain a better farmed organism through asustainable production has enhanced the importance of proteomics in seafood biologyresearch. Proteomics, as a powerful comparative tool, has therefore been increasingly usedover the last decade to address different questions in aquaculture, regardingwelfare, nutrition,health, quality, and safety. In this paper we will give an overview of these biological questionsand the role of proteomics in their investigation, outlining the advantages, disadvantages andfuture challenges. A brief description of the proteomics technical approaches will bepresented. Special focus will be on the latest trends related to the aquaculture production offish with defined nutritional, health or quality properties for functional foods and theintegration of proteomics techniques in addressing this challenging issue.This article is part of a Special Issue entitled: Farm animal proteomics.

© 2012 Elsevier B.V. All rights reserved.

Keywords:AquacultureFishProteomicsWelfareQualityNutrition

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43262. Aquaculture: key features, challenges and development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43273. Aquaculture species and fish model organisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4328

3.1. Aquaculture species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43293.1.1. Vertebrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43293.1.2. Invertebrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43293.1.3. Algae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4330

4. Proteomics technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43304.1. Gel based approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4330

4.1.1. Sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43304.1.2. Protein separation and quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4330

led: Farm animal proteomics.x7855; fax: +351 289818353.odrigues).

r B.V. All rights reserved.

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mailto:[email protected]

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4326 J O U R N A L O F P R O T E O M I C S 7 5 ( 2 0 1 2 ) 4 3 2 5 – 4 3 4 5

4.1.3. Protein identification and characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43314.2. Gel free approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43314.3. Identifying proteins from peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43314.4. Challenges and new technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4332

5. Fish welfare in aquaculture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43326. Nutrition and health in aquaculture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43347. Quality and safety of produced fish . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43368. Concluding remarks and outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4338References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4338

1. Introduction

During the last decade Omic technologies (i.e., genomics,metabolomics and proteomics) have been widely implemen-ted in the field of farm animal proteomics with a very positiveimpact in areas such as aquaculture. In Fig. 1 we give a briefoverview of the use of these Omics technologies in aquacul-ture, describing the target research topics and the mainaquaculture species. Proteomics in particular has emerged asa powerful tool towards a deep understanding of marineorganisms’ biology, helping aquaculture to reach its main goal;high productivity of a better quality product. Compared togenomics, proteomics provides not only information at amechanistic level but can also capture changes in proteinactivity measured as post-translational modifications (PTMs).In fact the transcriptome does not account for the post-transcriptional and post-translational regulation of proteinexpression. Most studies reveal a poor correlation betweenprotein expression and changes in transcript level [1]. Theproteome can then provide relevant information of an orga-nism's physiological state that is missed by the transcriptome.

Interpretation of proteomic data requires availability ofinformation on genomic DNA and expressed RNAs, so a majorlimiting factor in aquaculture proteomics is still the lack ofinformation at the genome level in most of the 310 cultured

Main Tools• Genetic linkage maps• Radiation hybrid maps• QTL -Quantitative Trait Loci• Marker Assisted Selection

•Salmonids: salmon, trout•Marine fish species: cod,

halibut, seabass•Warm water fish species:

tilapia, catfish•Shellfish: oyster, scallop

Broodstock• Breeding programmes• Traceability

Farming• Nutrition• Farming practic• Physiological li• Immunological• Disease resista

Main Tools• Transcriptomics (cDNA• Proteomics and Protei• Metabolomics

•Model organisms: zebrmedaka, pufferfish

•Salmonids: salmon, tro•Marine fish species: co

seabream•Warm water fish specie

tilapia•Shellfish: oyster, clams

Fig. 1 – Omics in aquaculture: targ

species reported by FAO in 2008 [2]. Full genomes are still onlyavailable for somemodel species (such as zebrafish, stickleback,medaka, coelacanth, fugu and Tetraodon), althoughmore focus isbeing given lately in studying the genomes of commercialspecies (like tilapia, cod and salmon) [3,4]. As a result, proteomicstudies on aquaculture species still face some challenges at thelevel of protein identification. Nevertheless, recent develop-ments in genome sequencing technologies have pushed the costof full genome sequencing from hundreds of thousands tothousands of dollars [5], suggesting that the availability ofgenomic and ESTdata onaquaculture organismswill not remainfor long a bottleneck in aquaculture proteomics.

Farmed seafood organisms are susceptible to a wide rangeof factors that can pose a major threat to a thrivingaquaculture industry with considerable economical repercus-sions. This industry has been going through major challengesin its effort to respond to the continuous higher consumerdemand, coupled with clear market global awareness of abetter quality product and also animal welfare. A goodbalance between these challenges may greatly benefit from abetter scientific understanding of the biological traits inseafood farming. This paper is the first review manuscriptspecifically dedicated to proteomics in farmed seafoodorganisms and aims to address a global perspective and notfocus on any specific aspect of this thematic. Throughout the

esfe stage statence

Quality & Safety• Selection of quality traits(fat deposition, pigmentation)

• Post-mortem degradation• Authentication• Spoilage indicators

microarrays)n arrays

afish,

utd, seabass,

s: carp,

Main Tools• Marker Assisted Selection• Proteomics

•Salmonids: salmon, trout•Marine fish species: cod,

seabass, seabream•Shellfish

et research topics and species.

4%

15%

31%40%

10%

Aquaculture

Welfare Nutrition Health Quality Safety

5%

14%

34%36%

11%

Aquaculture Proteomics

Welfare Nutrition Health Quality Safety

5353

5169

4784

4054

3583

3299

2609

2330

1571

1097

652

3181

2864

2817

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20112010200920082007200620052004200320022001

Nu

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er o

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Year

Proteomics Aquaculture Aquaculture Proteomics

Fig. 2 – Number of published manuscripts in the last decadewithin the keywords Proteomics, Aquaculture andAquaculture+Proteomics. Searches were processed onScience Direct (December 2011), and grouped according topublication year.

4327J O U R N A L O F P R O T E O M I C S 7 5 ( 2 0 1 2 ) 4 3 2 5 – 4 3 4 5

different sections of the manuscript we pinpoint the differentareas and the importance of proteomics in aquacultureresearch. We will address separately factors like welfare,nutrition, health, quality, and safety, introducing for each onesome of the major achievements of proteome analysis and itseffort in helping optimizing production in the aquacultureindustry. A special focus will be put on fish model organismsand their advantages and disadvantages. A brief overview onthe different proteomics technologies will also be addressedwith reference to the different methods, new approaches andfuture challenges.

Fig. 3 – Relative percentage of the number of publishedpapers in the last decade in each of the five different topics(Welfare, nutrition, health, quality and safety) both inAquaculture and Aquaculture+Proteomics studies Searcheswere processed on Science Direct (December 2011), andgrouped according to each topic.

2. Aquaculture: key features, challenges anddevelopment

The rapid growth of aquaculture industry over the last halfcentury is expected to continue in the future, due to thehigher consumer demand for aquatic animal food, coupledwith capture fisheries restrictions mainly in developedcountries. In fact, the aquaculture industry is growing athigher rate than any other animal food-producing sector withAsia dominating this production [6]. On the other hand,development of aquaculture in Europe and North Americagrew quickly during the last two decades of the 20th centurybut has since stagnated. Nevertheless, markets for fish andseafood in these continents are still growing in demand. Also,globalization and the world's economical growth led to largescale intensive farming of species like salmon, shrimp orcatfish. To cope with this development that is affected amongother factors, by market demand, availability of environmen-tal resources, infrastructures and technical capability orinvestment, there has been, mainly in the last decade,awareness that research in this area is an imperative. Toprove this, we can monitor the number of studies in

aquaculture-related studies, namely in the genomics/proteo-mics area, with up to 50 research publications in the area anda significant increase in the number of manuscripts. In Fig. 2we have an overview on the number of publications recog-nized in Science Direct (December 2011) in the areas ofAquaculture, Proteomics and Aquaculture Proteomics in thelast decade. Interesting is the fact that over the last five yearsthe increase of publications in the Aquaculture and Proteo-mics areas is around 20% and 50% respectively whileAquaculture Proteomics registered an increase of 155%.Nevertheless these numbers are quite low compared to thesame type of studies that have been reported in other farmedanimals like cows or pigs and they do not reflect the researcheffort and the importance of proteomics in Aquaculture. Also,


a quite significant number of proteomics studies carried out inaquatic organisms are focus on wild species and not farmedanimals. Still, we anticipate that with progress in genomics,this number will soon be comparable. Nutrition, welfare andhealth management have proven to be major constrains to anefficient production in aquaculture systems. One of the mainobjectives of aquaculture is to produce fish with an optimalgrowth rate and health status. From a physiological point ofview, fish growth is a complex process that apart from geneticbackground depends on other mutually interdependentprocesses, such as development, nutrition, metabolism andphysiological stress. Since growth is a physiological processwith multiple tissue contributions (e.g. liver, intestine, pan-creas and muscle), a global and multidisciplinary approach isrequired in order to have a complete view of all the factorscontributing to growth. At the present time, the application oftranscriptomic, proteomic and metabolomic approaches riseas powerful integrative tools to reconstruct the pathways andfunctional networks that govern the process of growth in fish.In this process, an important aspect is to determine how geneexpression is correlated to protein translation. Several ap-proaches have been used to compare the response of thetranscriptome in relation to the proteome. Generally, a fairlygood similarity was achieved between genes/proteins,appearing there is some correlation between the steady statemRNA abundance and protein abundance. Nonetheless,several factors like the transcriptional/translational regula-tion, proteins PTMs, analytical methods used in the studies orthe temporal scale, can influence the relationship betweengenome and proteome.

Fig. 4 – Typical proteomics workflow used in aquaculture proteomcan then be followed by a gel-based or a gel-free proteomics appand techniques is presented in each one of the followed strategitop-down and gel-free proteomics bottom-up (shotgun proteomicharacterization of proteins of interest.

As production methods gain importance to many con-sumers, issues of ethical production, animal treatment andwelfare, and environment-friendly production systems aswell as sustainability, seem to gain more influence onseafood product choice [7]. This “ethical quality” concept offish is mainly associated to aquaculture products, but thesustainable exploitation conditions in the case of capturefisheries are also gaining importance in seafood markets.This is largely due to increasing public concern on sustain-ability issues in the food chain and anticipated regulatorychanges, but also because the welfare standards by which afish is reared and then slaughtered may produce an impactupon both production and flesh quality. These factors haveled to considerable interest both in indices of good welfareand in development of production systems or technologiesthat promote it. Balancing current productivity standardswith higher disease and stress resistance, feed efficiency tolower environmental impact, ability to use more sustain-able fish ingredients (e.g. vegetable proteins and oils) are allconcrete traits that may contribute to the sustainablegrowth of the fish farming industry.

3. Aquaculture species and fishmodel organisms

There were around 310 species cultured and recorded byFAO in 2008 [2]. However, the 20 most significant speciesproduced account for 74% of production by volume and 63%by value. Among these, fresh water fish production is

ics studies. The common feature is sample preparation thatroach. A workflow pointing the different possible pathwayses; gel-base proteomics top-down, gel-free proteomicscs). Final objective is in all cases the identification and


dominated by carp, while Atlantic salmon leads among theintensively cultured marine fish. In coastal aquaculture,production is dominated by whiteleg, tiger shrimp, oysters,scallop and mussels.

Regarding the main areas of research impact in aquacul-ture (welfare, nutrition, health, quality, or safety) proteomicsapproaches are, by its nature, easily applied to model or non-model species. A relative percentage of the number ofpublished papers in the last decade in these five differentareas, both in Aquaculture and Aquaculture Proteomicsstudies, is presented in Fig. 3. We can easily observe that themajority of studies have been focused on health and quality.Nevertheless, we must consider that health related publica-tions are not strictly on health issues in aquaculture species,but are mainly focused on human health related to aquacul-ture. This value is then well inflated in the health perspectivethat we will point out later in this paper, which is strictlyrelated to health in aquaculture species.

Fish models have been used successfully in many biolog-ical aspects of human diseases, in areas like cancer, neurol-ogy, toxicology, infectious diseases and drug development[8,9]. Undoubtedly, the freshwater teleost zebrafish (Daniorerio) has been selected as the model-fish organism. Small insize, good availability, low cost maintenance, genome se-quence coverage and an available large-scale proteome profile[10], makes zebrafish highly attractive in disciplines asgenetics, developmental biology and physiology with a vastnumber of publications [10–24]. In these studies we can verifyhow omics technologies have been applied to analyze thetranscriptome, proteome and metabolome and how they canbe strategically incorporated into chemical screening andperturbation studies. Proteomic data is proven to complementboth the transcriptomic studies by validating the relativeprotein abundance as reflected in the relative mRNA abun-dance and also the metabolomic studies by reflecting thefunctional state of the protein machineries responsible foraltering the metabolites in a biological system. As a concreteexample, De Witt's studies [15] clearly demonstrate thepotential of combining transcriptomic and proteomic data togenerate mechanistic toxicological information.

Nevertheless quite a few studies are available in non-modeled species with advantages and disadvantages. A clearbenefit from using a model species in a proteomics study isdue to the availability of the genome sequence coverage indatabases that reduces the uncertainty of protein identifica-tion associated with mismatching amino acid sequencingacross species. Still, there are some authors pointing out thatnot only a non-model species provides a better surrogate for abiotic response in a specific system, but also suggest the useof de novo sequencing approaches can be used to overcomethe limitations of sequence coverage [25].

3.1. Aquaculture species

3.1.1. VertebratesMost proteomic studies have been carried out in Atlanticsalmon (Salmo salar), rainbow trout (Oncorhynchus mykiss),catfish (Ictalurus punctatus) and cod (Gadus morhua), mainlydue to their commercial importance. Stress related to farmingconditions [26–28] has been one of the main themes reported

in studies with these species and also in other less studiedspecies like gilthead seabream (Sparus aurata) [29], togetherwith muscle quality [30–33]. Related to fish quality is the workon understanding the mechanisms involved in the develop-ment of skeletal malformations of the vertebral column ofnormal and deformed white seabream (Diplodus sargus) bycomparative proteomic analysis [34]. The effect of probioticsadministration in diets has also been reported in rainbowtrout and cod [35,36] with interesting results at the level ofimmune-stimulation. In vitro [37] and in vivo infectionsproteomic studies on cultured fish like gilthead seabream[38], rainbow trout [39] and Atlantic salmon [40,41] are alsocommonly addressed since infection and concomitant deathis one of the main causes of heavy economic losses in marinefish cultures. These works lead not only to a better under-standing of the molecular mechanisms of pathogenesis aswell as provide novel protein candidates for vaccines devel-opment. Toxicological studies related to evaluation of chem-ically induced protein expression response have also beenreported in Atlantic salmon [42], rainbow trout [43], cod [44,45]and rare minnow (Gobiocypris rarus) that has been recentlyselected as a model for aquatic toxicological studies [46–48].Mainly focused on environmental pollutants that affectgrowth, reproduction and health of marine farmed organismswith repercussions to human health, these reports try notonly to understand the mechanisms of toxicity but alsodiscover potential biomarkers for environmental monitoringand risk assessment. Also lately, some interesting work hasbeen done to distinguish specific characteristics within thesame species. Examples are the efforts to establish potentialbiomarkers in testis for the poor reproductive farmed femalesin Senegalese sole (Solea senegalesis) [49] or the use of 2Dprotein maps to differentiate between wild and farm cod [50].Also the correct identification of fish species with commercialinterest has been investigated with proteomics techniques[51,52] and more recently with the application of biomarkerpeptide monitoring in MS in hake and grenadier [53,54]. Infarmed sturgeon some research has also been done regardingproteins involved in the differentiation between sturgeonspecies [55] and sex differentiation [56].

3.1.2. InvertebratesWith the intensification of crustacean farming in the lastdecade, proteomics studies have also been reported,namely as a tool to better understand the immune responsemechanisms of these organisms. In Chinese shrimp(Fenneropenaeus chinensis) 33 different proteins have beenidentified as a result to stress exposure by hypoxia [57], whilein giant tiger shrimp (Penaeus monodon) a study revealed that theeffect of antibiotics on patterns of hemolymph protein expres-sion are overwhelmed by the effects of external farmingconditions [58]. Aquatic invertebrates, as bivalves, often serveas reservoirs for many environmental pollutants, due to theirenvironment and exposure to aquatic sediments. In earlyproteomics, focus was mainly on taxonomy [59–64] while latelyquite a lot of work has been published on environmentaltoxicological proteomics. In fact, these organisms are popularsentinel species used in estuarine and coastal monitoringprograms. Many authors have been trying to establish proteinpatterns of environmental contaminants exposure, as well as


comparing protein profiles of bivalves residing in polluted areasto those of reference sites [65–68]. Environmental contaminantsproteomics studies include exposure to cadmium [69] NP(nonylphenol) [70] and p,p-dichlorodiphenyldichloroethylene(DDE) in the clam Ruditapes decussates, benzo(α)pyrene in zebramussels (D. polymorpha) [71] PCBs, PAHs (polyaromatic hydro-carbons) and heavy metals in blue mussels (Mytilus edulis)[72,73], cylindrospermopsin (CYN) inMytilus galloprovincialis andCorbicula fluminea [74] and crude oil in blue mussels [75–77].

3.1.3. AlgaeThe cultivation of algae is a very important part of aquaculture(with about 15.8 million tonnes produced in 2008 worldwide),constituting about 94% of the global production of aquaticplants [2]. Macroalgae represent most of algae aquaculture bothby volume and value (about 99%), being mainly cultivated asa source of food or specific substances (like iodine, algin,carrageenan and agar) [2]. Not many proteomic studies exist inmacroalgae, but there are already a few [78–82], mostly focusingon optimizing viable proteomic workflows for algae, since theseorganisms tend to contain substances which make adequateprotein extraction more challenging.

Interestingly, despite these technical issues, there arealready a large number of proteomic studies on microalgae,on a wide variety of topics. A major focus concerns an issuethat is very relevant for aquaculture: the study of microalgaebiology and their response to environmental signals, in orderto understand the mechanisms that trigger the developmentof harmful algal blooms. This knowledge is important toensure that management practices are optimized to guaran-tee not only fish health, but food safety and public health (assome microalgae produce neurotoxins that can bioaccumu-late in marine animals, even in the absence of algal blooms).Most proteomics studies therefore focus on subjects such asthe response of microalgae to different stress sources orenvironmental signals [83–97] and issues related to taxonomyand identification of potentially harmful species [98,99].Understanding the metabolism of algae is also of the utmostimportance, given the numerous roles such organisms canhave in biotechnology, namely in production of bioactivecompounds and biofuel, as well as their use in bioremedia-tion/integrated aquaculture systems. Proteomics is increas-ingly seen as an important tool in this context [100–102].

4. Proteomics technologies

Data regarding teleost fish and invertebrate speciesobtained by proteome analyses from the last decade hasbeen published in recent reviews [103,104]. The commonworkflow (gel-based and gel-free) analysis includes: 1) sam-ple preparation; 2) protein separation and quantification;and, 3) Protein identification and characterization, and isrepresented in Fig. 4.

4.1. Gel based approach

4.1.1. Sample preparationThe first step is usually protein extraction, since mostanalytical techniques used in proteomics require prior

solubilization of proteins in an appropriate solvent (aque-ous buffers, organic solvents). This makes sample prepara-tion a critical step in the analytical workflow. The purposeof this step is to attempt thorough solubilization of all (or aspecific subset of) the proteins present in a given bodilyfluid, organ, tissue or cell extract. Commonly used aqueousextraction buffers often contain (besides buffering agents)detergents, chaotropes, reducing agents and protease in-hibitors, ensuring that enzymatic activity is halted duringextraction and that intra and inter-molecular interactionsbetween proteins are minimized, preventing aggregation.Although standard extraction buffer formulations workreasonably well for a broad range of samples (eukaryoticcell cultures, bodily fluids and soft tissues of animals), sometypes of organisms (algae, bacteria) or protein sub-populations (membrane proteins, proteins embedded in acalcified matrix) often require different types of extractionbuffers for optimal results.

To improve protein separation and identification, severalprocedures can be considered. Sample fractionation is often achoice for sample pre-processing prior to proteome analysis,simplifying protein extracts and/or improving the dynamicrange of a protein mixture. Fractionation methods are usuallybased on chromatography, electrophoresis, differential solu-bility and/or (ultra) centrifugation and the most popular areusually aimed at isolation of organelles and sub-cellularcompartments (nuclei, mitochondria), enrichment in particu-lar sub-populations of proteins (phosphorylated, glycosylated,secreted, and membranar) and depletion of highly abundantproteins (like albumin and immunoglobulins in blood plasma,or actin/myosin/tropomyosin in muscle).

4.1.2. Protein separation and quantificationThe two most important analytical techniques in proteomicsare two-dimensional gel electrophoresis (2-DE) and massspectrometry (MS). Due to the central role that these twomethods have in proteomic studies, most experiments arebroadly classified as either “gel-based proteomics” or “gel-freeproteomics”, depending on whether 2-DE is used for proteinseparation and quantification or not.

Currently, 2-DE is still, by far, the most common strategyfor protein separation and quantification, enabling theseparation, detection and quantification of hundreds (andsometimes thousands) of different proteins from a singleextract, taking advantage of the fact that the isoelectric pointof a protein is mostly uncorrelated to its molecular weight. Onthe other hand, issues like gel-to-gel variation, limited lineardynamic range, limited throughput, and protein co-migrationstill pose challenges to gel-based proteomics. Although mostsoftware geared toward the analysis of 2-DE gels, likeProgenesis SameSpots (Nonlinear Dynamics), PDQuest (Bio-Rad Laboratories) and DeCyder (GE Healthcare), tries toovercome some of these issues with a simplified, semi-automated analysis workflow, they still cannot achieve 100%unambiguous results without significant human interventionand visual inspection.

Nevertheless, there are also some advantages of gel-basedapproaches: first of all, they are generally less expensive thanpurely MS-based approaches. Besides that, information onsmall post-translational modifications (PMTs) and highly


homologous isoforms can often be obtained directly, sincethese tend to shift the isoelectric point of a protein withoutextensively changing its molecular weight.

Classically, detection and quantification methods for 2-DEare usually based on Coomassie Brilliant Blue (CBB) or silverstaining, which enable estimation of protein quantity byscanning 2-DE gels in the visible range. Recently, thedevelopment of multiplex 2-DE (dubbed “difference gelelectrophoresis” or DIGE), which instead involves tagging theprotein samples with different fluorophores prior to 2-DE, notonly allows several samples to be run on a single gel, but alsosignificantly improves gel-to-gel variability, by providing acommon reference channel across all gels of an experiment[105]. This recent improvement of the 2-DE method has beenincreasing in popularity in aquaculture species proteomestudies with some successful results in several species[24,49,58,106,107].

4.1.3. Protein identification and characterizationAlthough immunostaining methods are a valid choice forprotein identification after 2-DE, the overwhelming majorityof gel-based proteomic studies rely on digestion of detectedproteins and analysis of the resulting peptides by MS for theiridentification and characterization, since it does not rely onhaving specific antibodies against every single protein to beidentified. Given the accuracy of current MS instruments andthe low number of proteins usually present on a single 2-DEspot, the results obtained can be quite reliable. Instrumentscurrently employed for this purpose include ESI-Ion Trap,MALDI-TOF/TOF and ESI-QTOFmass spectrometers to a lesserextent. Identification of proteins can be assessed eitherdirectly through its peptide mass fingerprint (PMF), for thecase of organisms with fully sequenced genome, or byanalysis of the fragmentation spectra of such peptides (PFF,peptide fragment fingerprinting or even de novo sequencing)obtained through tandem MS.

4.2. Gel free approach

Due to the above-mentioned issues associated with 2-DE andthe continuous improvement and cost-reduction of MS-basedmethods, gel-free strategies are becoming increasingly popular,since they generally allow higher analytical throughput andgenerally deeper proteome coverage than gel-based methods.In away, 2-DE can be seenas an elaborate pre-fractionation stepthat precedes MS analysis in a typical proteomic workflow and,as such, some researchers prefer to omit this step and applydifferent strategies which scale much better.

In contrast to the usual “top-down” approach of gel-basedproteomic studies, where proteins are separated and quantifiedbefore being digested and identified, most gel-free studiesemploy a “bottom-up” approach. Here, proteins are digestedfrom the start and analyses (separation, quantification, charac-terization) are done at the peptide level. Since digestion of acomplex protein mixture dramatically increases its complexity,gel-free methods are usually combined with fractionationmethods to reduce the number of different peptides enteringthe mass spectrometer at any given moment, maximizing thetotal number of distinct peptides detected over the course of asample run. By far, the most common pre-fractionation

methods used in gel-free proteomics are based on liquid-phasechromatography procedures, which can readily be coupled toESI-based mass spectrometers. Even multidimensional chro-matographic separations are commonly used, as in the case ofMudPIT, where peptides are separated by charge (SCX-HPLC)and hydrophobicity (RP-HPLC) prior to MS analysis. Despite thefact that label-free methods exist, most gel-free workflows relyon stable isotope labeling for peptide quantification, either bymetabolic incorporation of radioactive amino acids in proteins(SILAC) or by post-extraction chemicalmodification (ICAT, TMT,iTRAQ). With stable isotope labeling, several samples can beanalyzed in parallel on the sameMS run (with identical peptidesfrom distinct samples separated by a small mass shift) andrelative abundance can be estimated from the ratio of peakareas of identical peptides. At the endof a “bottom-up” analysis,information on protein abundance/identity is inferred from theobtained information on peptide abundance/identity, usingcomputational tools. This is, in itself, a challenging part of“bottom-up” approach and still a matter of research in the fieldof mass spectrometry.

Although a direct “top-down” approach is intrinsicallymore challenging, due to the difficulty of ionizing wholeproteins, the higher resolution of MS instruments using FT-ICR or Orbitrap technologies make “top-down” approachesincreasingly practical, beyond themore common “bottom-up”approaches.

4.3. Identifying proteins from peptides

Most proteomic studies (both gel-based and gel-free) attemptto identify proteins by looking at peptides, as large proteinsstill constitute a challenge for MS-based methods. Theclassical method used (dubbed “peptide mass fingerprinting”or PMF) is based on in silico digestion of genomic/ESTsequences following the pattern of a predictable endonucle-ase (usually trypsin) to obtain a list of peptide masses (or“mass fingerprint”) for each database entry. Identification isperformed by comparing experimentally obtained MS masslists with those generated in silico, and choosing significantlysimilar matches. Nonetheless, this is a reliable method whenworking with a model species (like zebrafish), for which thereis full genome data, and for simple digests (like 2DE spotdigests). On the other hand, by using tandemMS instruments,you obtain not only a peptide mass list, but also informationon the fragmentation mass spectra of those peptides, provid-ing a fingerprint for each peptide that directly reflects itssequence. The most common strategy here (“peptide frag-ment fingerprinting” or PFF) also involves starting with agenomic/EST database and performing in silico digestion witha standard endonuclease. Then, for each peptide, the massesof all likely fragments are deduced from prior models.Identification of peptides is then performed by comparisonof experimentally obtained MS/MS spectra against all possiblefragmentation spectra in the database. Since a fragmentationspectrum (unlike mass) is usually very specific for a certainpeptide sequence, identification of proteins can often beattained from a single high-quality peptide match. Neverthe-less, unless you work with species for which there is a greatwealth of genomic/EST data available, this strategy onlyworks for highly conserved peptides.


It is therefore important to underline that protein identi-fications depend not only on the quality of spectra, but also onthe quality of the sequence database used. Generally, a goodapproach is to start by searching genomic databases (sincethey tend to be smaller and therefore fast to query) and thenEST databases. The results obtained are often different, sincegenomic databases reflect genomic DNA sequences while ESTdatabases reflect mRNA sequences (which are closer to theactually translated amino acid sequences). Observations onthe frequency of alternative splicing events in teleostssuggests that this difference is particularly important forspecies with a compact genome (like Takifugu rubripes, forwhich ~43% of mapped genes seem to be affected byalternative splicing) and less so for species with a high levelof gene duplication (like zebrafish, for which only ~17% ofmapped genes seem to be affected by alternative splicing)[108]. Also, given current developments in DNA sequencingtechnology [5], it is expected that the coverage of genome/ESTinformation on commercially relevant species will be greatlyexpanded, certainly improving the quality of MS-basedpeptide/protein identification methods.

4.4. Challenges and new technologies

Looking at the results of proteomic experiments, we observethat there are several issues that prevent perfect consistencyof results across experiments on a single topic, despite thefact that there is usually significant overlap in terms ofaffected pathways. Some of these differences can be attribut-ed to technical limitations, like the influence of samplepreparation methods, the “user” factor, the difficulty ofionizing certain peptides, as well as other instrumental andanalytical limitations. Most of these can hopefully be over-come by increased automation, improvements in instrumentquality and a decrease in user intervention throughout theworkflow. On the other hand, the dynamic and complexnature of the proteome provides several intrinsic challengesduring interpretation of proteomic results. This can be seen,for example, in experiments coupling transcriptome andproteome observations, which often show a weak correlationbetween the two profiles (when sampled at the same time).This dynamism and the sensitivity of the proteome towardsenvironmental signals and subtle changes in physiologicalstate justifies the necessity of obtaining and using as muchcontextual information as possible (including both classicaland/or –omics observations) to support proteomic observa-tions and having into account the importance of time andlocality on the proteome.

The fact that most proteins display post-translationalmodifications (PTMs) further adds to the proteomes’ com-plexities and is increasingly seen as essential to study, giventhe biologically important roles some of these PTMs areknown to have. Although gel-based approaches can use eitherspecific dyes (like Pro-Q Diamond, for detection of phosphor-ylated proteins) or immunoblotting for detection of PTMs, thereal workhorse of PTM analysis is mass spectrometry. Specificdetection methods are often combined with protein/peptideenrichment steps, to target for some specifically modifiedproteins (like affinity chromatography with lectins, to enrichfor glycoproteins, or TiO2, to enrich for phosphopeptides).

As technology continuously develops, the future of prote-ome research is still uncertain, but there are already newapproaches which have good potential to become invaluabletools in aquaculture research (and marine biology in general).One of them is MS imaging. This technology involves thedirect digestion of histological samples fixed to a suitablesupport, followed by direct MS/MS analysis (for example, byapplying a matrix solution on the sample and doing MALDI-MS all over the surface, point by point). This is the equivalentof using immunocytochemistry methods, but in a non-specific way, providing information on a key variable:location. This is very important because, unless cell sortingor microdissection techniques are applied prior to proteomicanalysis of a given tissue, the profiles obtained are very likelyto represent an “average profile” of a heterogeneous popula-tion of cells, sincemost organs and tissues are heterogeneous.Most importantly, this heterogeneity is often location-dependent, which implies that MS imaging can provide atype of deep morpho-proteomic information that would notbe accessible otherwise. It's interesting to note that MSimaging doesn't necessarily require protein identification:using computational methods (dimensionality reductionmethods, classifiers, neural networks and similar machinelearning tools) it's possible to map all the MS and MS/MSinformation obtained for each point in space as a pixel, wherecolor information is defined so that it reflects the similarityrelations between proteomes. This means that MS imagingcan provide useful information on proteome distributionsover any tissue (regardless of source), distinguish betweensub-populations of cells with different proteome profiles andpinpoint exactly where proteome changes occur. The size ofmatrix crystals are a limiting factor preventing increasedresolution in MALDI imaging, so there is still a lot of researchon alternativematrices or ionizationmethods for MS imaging,like SIMS and DESI. New breakthroughs in this field willcertainly revolutionize proteomics and greatly increase ourunderstanding of biological processes which are intrinsicallymorphological and relevant for aquaculture research(like larval development or muscle growth).

Another interesting technology is the development ofprotein array/protein chip approaches. These allow for veryhigh throughput at the expense of high specificity, whichcurrently makes the development of such tools relativelyexpensive and challenging, particularly for non-model or nonsequenced organisms. Nevertheless, as these types of tech-nologies develop, the potential for very large scale studiesregarding major issues in aquaculture suddenly becomesfeasible, opening the doors to new, more in-depth approachesfor comparative proteome studies.

5. Fish welfare in aquaculture

Animal welfare is a complex concept and it is by no meansstraightforward to define and measure it. Ashley has incomprehensive terms made a good definition of welfare asthe freedom from hunger and thirst, discomfort, pain, injury,disease, fear and distress, and the freedom to express normalbehaviour [109]. From this definition it is quite evident that no


easy and single measure of welfare is available. The mostgeneral accepted measure of welfare has until now beenphysical health where a variety of physiological and biochem-ical measures have been used. However, welfare also involveslack of mental suffering and in fish reliable measures of thisconstitute an important future challenge [109].

This complexity of the welfare concept underlines theimportance of a multidisciplinary and holistic approach to thestudy of fish welfare and results have shown that proteomicscan be an important part of the toolset required for suchstudies and to develop aquaculture practices that guaranteegood welfare and health and ensure that marine animals arereared in an environment that optimizes their capacity tocope with unavoidable challenges/stress. When it comesspecifically to fish in aquaculture the aspects of welfareissues can be divided in three groups: 1) aspects relating tofish health; 2) aspects relating to aquaculture managementprocedures; and, 3) aspects relating to stocking density,aggression and altered behaviour [109].

Given all the different number of factors involved inwelfare, it is clear that no single tissue encompasses allinformation required to make a complete assessment of anorganism's welfare status. Although most proteomic studieson welfare issues focus either on the liver (due to its centralrole in most key metabolic processes) or bodily fluids likeblood plasma (due to its value in providing non-lethalproteomics-based diagnostic tools), some of them also targetbrain, skeletal muscle, osmorregulatory and immune-relatedorgans and tissues.

A number of the existing proteomic studies within aquacul-ture and related research areas already concerns welfare. Heremost are on health aspects, with focus on viral diseases[41,110–118], bacterial diseases [38,110,119–128] and vaccinedevelopment [39,129–138] but also parasites [139–141], hepatictumours [142] and skeletal deformities in fish [34] have beenstudied. Besides these, immunoproteomic studies also encom-pass results on immunostimulants [35], antimicrobial peptides[143–145] and composition of mucosal secretions [146,147],along with other topics related to a better understanding ofmechanistic aspects of immune responses to certain stimuli[40,148–150]. The study of the impact of xenobiotics in aquacul-ture has also benefited from proteomics technologies, withseveral studies done on the impact of biotoxins [141,151–153],pharmaceuticals [58,154,155], hormones [15,46,156], metals[157,158] and other pollutants [43,45,67,159–163] on the pro-teomes of aquaculture aquatic organisms. Finally, there are alsosome studies correlating environmental sources of stress inaquaculture (hypoxia [57], anoxia [26,164–166], hyperoxygena-tion [167], osmotic [168–173] and temperature [174–177], as wellas stress induced by management practices (like high stockingdensities [29,178], handling [29,179] and pre-slaughter stress[180]), with proteome changes in several tissues.

Most sources of stress related to fish handling, crowding,harvesting and slaughter stress involve exposure to hypoxia/anoxia/normoxia cycles and acute cellular energy depletion,being characterized by increased formation of ROS andoxidation of proteins and lipids [181]. These events usuallyinduce cellular adaptations mediated (among others) byoxygen sensing systems, which include widely studied factorslike HIF-1α, and generally characterized by the induction of

cytoprotection systems (including increased expression ofchaperones, detoxification enzymes, protein turnover andantioxidant proteins) as well as adaptive changes in energyhomeostasis and cell cycle control [182,183]. Furthermore, theeffect of these physiologic stressors has slightly differenteffects on the organism's distinct organs, some of whichhormone-mediated by CNS signals, such as the release ofepinephrine and glucocorticoids (like cortisol) into the blood-stream [184].

The prior state of the cell, as well as the type, intensity andduration of exposure to these types of oxidative stressdetermine whether the described adaptations enable the cellto appropriately cope with the oxidative stress or if cell deathis inevitable, which implies the observed cellular response iscontext-dependent [184].

Comparative proteomic studies in fish reflect this:although most types of stress broadly induce changes interms of energy metabolism, cytoskeletal dynamics andexpression of cytoprotective proteins across most organs[26,29,57,164–167,180], there are specific particularities incellular responses depending on cellular/tissue type. Forexample, in energy-bound organs, like brain and muscles,changes in energy metabolism are usually quite evident,particularly at the level of glycolysis/carbohydrate metabo-lism and phosphotransfer networks [165,180]. Liver tissue alsodisplays energymetabolism shifts in response to stress, but ata much deeper level, reflecting its essential role in physiolog-ical detoxification and energy management: adaptationsusually cover amino acid and lipid metabolism, besidescarbohydrate metabolism. Other often affected pathways inliver include the urea cycle, the methionine/homocysteinecycle, the folate one-carbon pool and betaine metabolism,which are intimately connected to amino acid metabolismand detoxification processes [29,57,179]. Besides these mainpathways, cellular stress also induces changes in the expres-sion of proteins related to signaling, cell cycle regulation,transcription factors, ribosomal proteins and others. The factthat these last changes are not as consistent may simply be areflection of the negative bias towards low abundance pro-teins and does not necessarily imply that their role issecondary.

Exposure of fish to atypical environmental parameters (pH,temperature, salinity) can also induce general physiologicstress, which results in cellular responses overlapping withwhat is observed in proteomic studies with hypoxic-typestressors. Heat shock, for instance, is usually accompanied bychanges in the expression of chaperones and proteins of theubiquitin/proteasome pathway [174,175,177]. In the case ofsalinity changes, proteomic studies in fish often targetosmoregulatory organs (like gills and kidney) and generallyshow similar signs of cellular stress, particularly affectingcytoskeletal dynamics, betaine metabolism and aldehydedetoxification [169,171–173]. Regarding the effect of metals,biotoxins and other environmental toxicants on the proteomeof fish, most studies focus on the liver, generally displayingincreased signs of oxidative stress, changes in immune/inflammatory effectors, as well as induction of transportersand detoxification enzymes [43,154,157,158,160–163,185–187].But, again, there is a general overlap in terms of affectedpathways, when comparing to hypoxia-type stressors. When


looking at the proteomic impact of bacterial and viral in-fections on the liver, spleen and kidney of fish, there is again asignificant overlap with other sources of stress, in terms ofaffected pathways [38,41,111,112,115,121] underlining the ideathat these sources of stress have a cumulative effect at thecellular level. Also when looking at studies involving prote-ome analysis of plasma proteins, which usually focus ondetection and validation of potential welfare biomarkers,several proteins stand out as being affected by sources ofphysiologic stress (microglobulins, macroglobulins, apolipo-proteins, α1-antitrypsin, transferrin, plasminogen, comple-ment system proteins, among others) [160,188–190], althoughagain we see a wide overlap in the responses to very differentstressors (e.g. bacterial infection vs. high-density crowding).Combined, this suggests that proteomic studies on fishwelfare may benefit from holistic approaches, coveringseveral organs and using complementary measures of healthand stress status, which provide the necessary contextualinformation for correctly interpreting observed changes asadaptive or maladaptive.

Concluding, although welfare biomarker discovery andvalidation is still a big challenge, even with all the –omicstools available, it is also clear that proteomics providesuntargeted information which is invaluable to meaningfullyunderstand fish welfare and work towards a sustainable andethical aquaculture. Nevertheless, there is still room forimprovements and adapting experimental designs to explic-itly address the influence of intrinsic confounding factors onfish welfare (e.g. through the use of regression discontinuitydesign or by using auxiliary welfare measures as blocking orstratification factors) would provide more refined insightsinto the problematic. The solution to these challenges alsolies, in part, in obtaining better data (i.e. deeper proteomecoverage) and better analytical tools (enabling data-miningand integration of classical and molecular data), whichsuggests that, as mass spectrometry technologies and bioin-formatics tools continuously improve, the use of proteomicsin aquaculture research is increasingly seen as, not onlyrelevant, but essential.

6. Nutrition and health in aquaculture

Although still at early stages, the proteomic approach is nowbeing successfully implemented in nutritional studies withfish. Nutrient deprivation, or starvation, is a commonly usedintervention when analyzing the metabolic and physiologiccapabilities of model organisms and tissues. Several studieshave evaluated the response of hepatic proteome undergoingfasting and refeeding. In response to a 14 days fasting period,24 differentially expressed proteins were detected in therainbow trout liver. Among the proteins with increasedabundance after fasting were enolase and cytochrome Coxidase, on one hand, and cathepsin D, on the other hand,most likely related to the higher energy requirements andprotein degradation, respectively, that takes place duringfasting [191]. More recently, when comparing the changes inhepatic mitochondrial proteome of zebrafish undergoingstarvation (15 days) and refeeding (7 days), the 18 identified

proteins indicated that starvation resulted in reduction inglycolysis and increase in gluconeogenesis, while refeedingcaused these activities to return to normal levels. Expressionpattern of several proteins related to fatty acid and aminoacid metabolism also suggested the utilization of non-carbohydrate resources for energy during starving conditions.Proteins with chaperoning and antioxidative roles such asglucose-regulated protein, paraxonase and heat-shock pro-tein were also upregulated in starved conditions [192]. Aproteomic approach with zebrafish was also used to assessthe metabolic effects of variable dietary energy intake levels.Twenty-nine protein spots differentially expressed betweentreatments were identified. The most significant proteinchanges associated with high-caloric intake were related to adecrease in oxygen-binding activity, namely heme bindingproteins [193].

A shift towards a lower usage of finite marine-harvestedresources is a major sustainability challenge facing theaquaculture industry. It is now consensual that vegetableprotein and oil sources are valid alternative ingredients in fishfeeds. Despite great advances achieved in this area in recentyears, high inclusion levels of vegetable ingredients, still elicita reduced growth performance and feed efficiency. The recentusage of genomic and post-transcriptomics tools are greatlycontributing to a better understanding of the metabolicpathways affected by dietary changes in terms of fishmealand fish oil replacement [194]. Proteome analysis identified anumber of metabolic pathways sensitive to plant proteinsubstitution in rainbow trout liver, for example, pathwaysinvolved in cellular protein degradation, fatty acid break-down, and NADPH metabolism [195,196]. Adaptation ofrainbow trout to a diet with a partial (30%) substitution offish meal with soybean meal for 12 weeks resulted inunaltered growth rates but increased protein catabolism andturnover, accompanied by changes in the abundance of 33protein spots. Identified spots referred to structural proteins(e.g. keratin and tubulin), lipid binding proteins (e.g. apolipo-protein A) and heat shock proteins, which are indicative of astress response [195]. In a subsequent trial, rainbow trout wasfed a fishmeal-free diet and showed a significant reduction ingrowth rate [196] and changes in 30 protein spots. Namely,several proteins involved in primary energy metabolism(production of NADPH, ATP, etc.) increased their abundancein liver, as well as two proteasome subunits, indicative of anincrease in protein degradation. The effects on the protea-some were particularly noteworthy. The proteasome is amulti subunit enzyme complex that catalyzes proteolysis viathe ATP-dependent ubiquitin-proteasome pathway, which inmammals, is thought to be responsible for a large fraction ofcellular proteolysis [197]. In rainbow trout, the ubiquitin-proteasome pathway has been shown to be down regulated inresponse to starvation [198] and to have a role in regulatingprotein deposition efficiency [199]. By correlating all findingsof the study, Vilhelmsson et al. (2006) [196] suggested that thedifferences found in fish regarding texture (sensory analysis)and post-mortem muscle free amino acid pool could beassociated to ante-mortem proteasome activity. Fish dietsare currently rich in fish oil that supplies both energy andhighly unsaturated n-3 fatty acids, but fish oil is becomingscarce. Hepatic proteome was analyzed to investigate global


metabolic changes induced by the absence of fish oil in troutdiets [200]. Fish fed the fish oil free diet exhibited lowergrowth rate and body fat deposition and 105 hepatic proteinswere found to be differentially expressed. The 13 identifiedproteins supported that fish oil suppression increased fattyacid transport, de novo lipid synthesis and fatty aciddesaturation, while decreasing fatty acid catabolism. Severalproteins involved in glycolysis, amino acid catabolism andenergy production were up-regulated, showing a shift innutrient utilization for energy production. Overall, changesin protein abundance in the livers of fish fed diets containingplant protein sources suggest that these fish have higherenergy demands than fish fed fish meal-based diets. It isnoteworthy to bear in mind that effects of a changed diet onfish revealed at the proteome level depend on the geneticbackground of the studied fish. Although, our knowledge ongenes and pathways involved in e.g. growth has increasedenormously by using genomic approaches, such approachescannot stand alone in studying environmental impacts on thegrowth related cellular functions as these are exerted by theproteins. Thus proteomics provide important complementaryunderstanding to the genomic based knowledge.

In this context of fish meal replacement, commonly usedplant ingredients in salmon feed such as soybeans and maizeare increasingly grown as genetically modified (GM) varieties.The question arises whether these are equally nutritious andsafe for the fish as conventional varieties. The proteinexpression in Atlantic salmon was screened by proteomicsto reveal potential effects onmetabolism or other processes inthe liver. One protein (calreticulin) was up-regulated in theGM group and two down-regulated (thymidine phosphorylaseprecursor/alpha-enolase and triosephosphate isomerase). Butgiven the low fold changes in the differentially expressedproteins, the overall proteomics data could not detect anybiologically meaningful differences between the livers ofsalmon fed non-GM or GM diets for 7 months [201].

One area in which proteomic studies show a high potentialis the interaction between specific nutrients or feed additivesand the immune system. An optimal dietary supply ofindispensable amino acid supply is of paramount importanceto fish growth. Lysine (Lys) is an indispensable amino acid(AA) and is generally the first limiting AA in most vegetableproteins used in fish feeds. Lys availability may thus limitprotein synthesis and accretion, and growth of fish. Metaboliceffects of various dietary Lys imbalance levels were examinedby 2D-proteomics using zebrafish as model [11]. Comparativeproteomic analysis of whole-body zebrafish showed 45 spotsdifferentially expressed. Twenty-nine of these proteins wereidentified revealing proteins involved in muscle growth,energy and lipid metabolism, eye lens differentiation, chap-erone activity and apoptosis. Lys deficiency was accompaniedby a down-regulation of muscle proteins and up-regulation ofproteins associated to fasting, energy deficit, growth arrestand apoptosis. Additionally, it was found that excess Lys wasaccompanied by an up-regulation of proteins related toglycolysis, steroidogenesis and sexual maturation [11]. Acomparative proteomic approach was used to assess theprotein expression profile in the liver of 34 days old pikeperchlarvae fed diets varying in their phospholipid (PL) contents[202]. From the 56 protein spots with a differential intensity

associated to dietary changes, 11 proteins were un-ambiguously identified using nano LC-MS/MS tandem massspectrometry. Results showed that under high PL intake, theglycolytic pathway was down-regulated due to the under-expression of the fructose biphosphate aldolase B and thephosphoglucomutase 1, while propionyl coenzyme A carbox-ylase (a gluconeogenic enzyme) was also under-expressed. Ahigh dietary PL level increased the expression of sarcosinedehydrogenase, an enzyme involved in methionine metabo-lism, along with vinculin, a structural protein. In the larvaefed with the lowest dietary PL content, over-expression ofboth glutathione S-transferase M, glucose regulated protein 75might indicate a cellular stress, while the under-expression ofperoxiredoxin-1 might indicate a lower defence againstoxidative stress [202]. Dietary nucleotides supplementationcaused differential expression of muscle metabolic proteinsincluding glyceraldehyde-3-phosphate dehydrogenase, crea-tine kinase, adenylate kinase, nucleoside diphosphate kinase,and triosephosphate isomerase. In addition to metabolicenzymes, troponin-T-1 as a structural protein was found toincrease in abundance in fish fed the nucleotide supplemen-ted feeds [203]. Similarly, the intake of maslinic acid, atriterpene used as a feed additive to stimulate growth andprotein turnover rates, was found to regulate giltheadseabream hepatic proteins involved in the metabolism ofglucose, lipids, amino acids, and purines, as well as indetoxification and xenobiotics, oxidative stress, immunesystem, protein synthesis, protein folding, signaling, andcytoskeleton formation [204]. Moreover, the cellular-signaling pathways associated to the dietary use of alpha-ketoglutarate (AKG), an intermediate metabolite in the Krebscycle and a feed additive, were assessed on the pituitaryproteome of gilthead sea bream [205]. Metabolic proteins up-regulated with AKG supplementation included fructose-bis-phosphate aldolase, glyceraldehyde-phosphate dehydroge-nase and malate dehydrogenase. Protein folding-related pro-teins up-regulated with AKG supplementation, included twoisoforms of heat shock proteins as well as cyclophylin andchaperonin. Proteins found to be associated with regenerationof neural function namely cofilin and Vat-protein were up-regulated by AKG supplementation. One hormone modifiedby AKG treatment was somatolactin [205].

Proteomics holds great promise for discoveries in nutritionresearch. Integrated with other advanced technologies (geno-mics, transcriptomics, metabolomics, and bioinformatics) andsystems biology, proteomics will greatly facilitate the discov-ery of key proteins that function to regulate metabolicpathways and whose synthesis, degradation, and modifica-tions are affected by specific nutrients or other dietary factors.This will aid in rapidly enhancing our knowledge of thecomplex mechanisms responsible for nutrient utilization,identifying new biomarkers for nutritional status and diseaseprogression, and designing a contemporary paradigm fordietary prevention and intervention of disease.

Virus, parasites and bacteria have caused over the yearstremendous economic losses in the aquaculture industry invarious parts of the world. Farmed aquatic species aresusceptible to a wide range of pathogens (bacteria, virus,parasites and fungus) responsible for disease and losses withsignificant impact on the quality and volume of production


throughout the world [206]. In this section we will mainlyfocus on pathogen infections and diseases. Pollutants andcontaminants are also detrimental to a healthy aquacultureindustry. An effective health management program in aqua-culture industry should cover several steps, from up-dateinformation on health status, reducing exposure and spreadcontrol of pathogens and management of drugs or chemicals[206]. A rapid and accurate diagnosis of disease is thenessential for an effective outbreak control. Pathogen detectiontechniques have been widely used in this industry and can beachieved using a variety of methods – traditional (bacteriol-ogy, virology, parasitology and mycology culture and bio-chemical identification), immunological (FAT (fluorescentantibody technique), IFAT (indirect fluorescent antibodytechnique), IHC (immunohistochemistry), ELISA (enzyme-linked immunosorbent assay), dot blot/western blot, molecu-lar methods (PCR (polymerase chain reaction), serology,lateral-flow immunoassays, LAMP (Loop-mediated isothermalamplification) and multiplex technologies like LuminexxMAP, Multiplex-PCR assays and Micro-arrays) [134]. Vaccina-tion has also been a major research area for the impact it mayhave on disease prevention in aquaculture. Presently, vac-cines are already available against bacterial and some viraldiseases [207–209].

In this context, proteomics technologies can play a majorrole in aquaculture species health, since it can assist both inthe development of new vaccines and diagnosis of disease. Afew research papers have been published recently withpromising results. A recent study on three of the major fishspecies of mariculture in southeast Asia describes theisolation of a pathogenic iridovirus and proteomics analysisof its envelope proteins [37]. This virus is responsible from30% (adult fish) to 100% (fry) mortality in cultured grouper andhas the ability of inducing an advanced cytopathic effect in agrouper cell line (GP). Several envelop proteins were identifiedwith protein VP088 further characterized with the aim tobetter understand the molecular mechanisms of iridoviralpathogenesis and disease prevention in marine aquacultureindustry. Dumetz in his 2008 paper [39] reveals a number ofnovel candidate proteins for developing vaccines againstflavobacteriosis infection in aquaculture, using 2-DE and LC-MS/MS techniques. In bivalves, the majority of studies on itsinteraction molecular mechanisms with bacteria are mainlyon mRNA and recombinant protein levels. In 2011 a pioneerstudy by Huan focused on the anti-Vibrio immune response ofZhikong scallop (Chlamys farreri) through proteomics tech-niques [210]. Identified proteins include some immuno-related proteins, metabolism enzymes and new moleculesnot pinpointed in previous immunity studies in C. farreri. Theresults also indicated that molecular chaperons and theantioxidant system are key elements in the anti-Vibrioimmune response of hepatopancreas of C. farreri. Also inbivalves, a proteomics approach based in the two-dimensional gel electrophoresis and mass spectrometry wasused to study the effects of the exposure of two bivalvespecies, Mytilus galloprovincialis and Corbicula fluminea, to CYNproducing (CYN+) and non-producing (CYN−) Cylindrospermop-sis raciborskii cells [74]. Results obtained suggest the inductionof physiological stress and tissue injury in bivalves by C.raciborskii, also supported by the changes observed in GPx

(Glutathione peroxidase) and GST (Glutathione S-transferase)activities indicating alterations in the oxidative stress defensemechanisms.

Regarding contaminants, proteomics has also been shownto be an effective tool to evaluate its toxic effects inaquaculture species, as already mentioned in this paper. Allthese works aimed not only to further understand themechanisms of action, but also the identification of potentialprotein biomarkers for contaminant exposure. A summary oflaboratory controlled and field/caged studies that haveutilized proteomics techniques in toxicological studies inTeleost fish and aquatic invertebrate species is available inthe review paper by Sanchez et al. [104].

7. Quality and safety of produced fish

The early years of this century saw the first publications usingproteomics as an application to assess food quality. Thefuture potential for proteomics within food quality and safetyareas such as technological quality, allergy prevention, bio-actives, bioavailability, and nutrition prediction of end-product quality, was pinpointed in a review by Cabonaro in2004 [211].

In a very recent review by D'Alessandro and Zolla [212],the safety concept has been considered to encompass notonly food safety per se, but also traceability (food origin) andfood quality, the last of which these authors more or lessregard as identical with food composition and nutritionalvalue. However, in the present paper, quality is consideredas “eating quality” and “technological quality” (suitabilityfor certain products, production processes and types ofstorage conditions). A central objective in fish qualityresearch is, therefore, to obtain an understanding of theinvolved mechanisms when quality changes. This can bedue to either ante mortem biological aspect or to post mortemconditions, individually or in combination. The influenceon quality by pre-harvest parameters as feed composition[213–216], temperature [217], stress [218], slaughter method[219] and genetics [220], as well as post mortem parametersas processing [221], rigor mortis [222], and storage temper-ature and time [223], is well established. However, the useof proteomics to gather knowledge at a molecular level ofthe involved mechanisms responsible for a certain qualityin aquaculture species is only in the making, with only afew published studies.

Although some studies have investigated the influence offish feed composition on the proteome of fish muscle or liveronly a single proteomics study focusing directly on fish dietand quality have been performed. In a combined proteomicsand transcriptomics approach on rainbow trout (Oncorhynchusmykiss) liver, it was shown that changes in liver metabolismare likely to manage fat allocation in the muscle of fish fed ahigh energy/high fat diet [220]. Proteins involved in intra-cellular lipid transport, the respiratory chain, glycolysis/gluconeogenesis and amino-acid metabolism were expressedin lower levels in fish fed the high energy diet compared to fishfed a low energy diet. The found proteins could be used asquality markers to prevent excess muscle fat accumulation[220].


The detrimental impact of pre-harvest stress on quality offish muscle is well documented and the interest in revealingthe underlying mechanisms of the involved physiologicalresponses to different stressors has therefore constitutedbasis for a number of proteomics studies. A common stressorin aquaculture is crowding at different fish densities fordifferent periods of time (e.g. when fish are gathered in orderto be transported and/or slaughtered).

Using 2-DE Morzel et al. [180] characterised the modifica-tion of protein expression in rainbow trout (Oncorhynchusmykiss) white muscle as induced by a pre-slaughter crowding(>50 kg/m3 for 15 min). In samples taken 45 min post-mortemthey found 29 protein spots differentially expressed in groupsof crowded and un-crowded fish. The identified spots weremainly proteins from energy-producing pathways indicatinga rapid increase in energy demand or structural proteins [180].These findings matches what could be expected, because amain quality-related effect found due to crowding is an earlieronset of rigor that is closely related to both energymetabolismand structural proteins.

That proteins involved in energy metabolism changed inexpression as an effect of crowding was also found in Atlanticsalmon by Veiset-Kent et al. [224]. They investigated the effectof a more extensive crowding (>200 kg/m3 for 40 min) onmuscle and blood plasma proteomes of Atlantic salmon inorder to identify possible links to fillet quality attributes.Specifically proteins as enolase 3 and phosphoglyceratekinase 1 involved in glycolysis were found to be up-regulatedin muscle from the crowded salmon. As such this is inaccordance with the findings by Alves et al. [29] who founddown-regulation of gylcolytic enzymes in gilthead seabream(Sparus aurata) liver due to crowding (~50 kg/m3) and ascribedthis to an effect of stress activated liver glycogenolysis andgluconeogenesis supplying glucose to other organs with anincreased demand.

To establish a methodological approach to monitor thequality of the edible parts of fish Monti et al. [225] investigatedprotein expression changes induced by aquaculture. UsingSDS-PAGE, in situ protein hydrolysis, de novo sequencing ofpeptides by MALDI and ESI-MS/MS, protein identification andrelative quantitation of protein by denaturing capillaryelectrophoresis, they compared water-soluble muscle pro-teins from edible parts of wild and farmed sea bass (Dicen-trarchus labrax). They found that in aquaculture fish theexpression of enzymes involved in carbohydrate metabolismwere over-expressed, whereas among others the expressionof the major fish allergen, parvalbumin, was reduced by 22%.The obtained differences were by the authors ascribed todifferences in growth conditions, feeding strategies or to both[225]. However, in contrast to this, others found that farmingof sea bream (Sparus aurata) in offshore floating cages(mariculture) enabled production of fish comparable to wildfish, with no differences in muscle protein expressiondetected between the two groups [176].

Muscle texture is one of the very important quality aspectsof fish that are tightly connected to proteins. Texture changesduring chilled as well as frozen storage, and the biochemicalprocesses involved in this post-mortem tenderization havebeen extensively studied during many years. It is generallyaccepted knowledge that different proteolytic systems are

involved in this process, and also several of the structuralproteins being affected as substrates for these enzymes areknown. However, many important details remain to bediscovered, and a better and deeper understanding of thequality related post-mortem processes during storage might berevealed using proteomics.

Although the poly unsaturated fatty acids of fish are themain target for deteriorative oxidation during storage, pro-teins are oxidised as well, potentially causing protein aggre-gation resulting in decreased protein solubility withimplications on the textural quality [226,227]. All reactiveoxygen species (ROS) that react with proteins forms carbonylsand this can be used analytically. In a 2-DE approach withimmunoblotting of 2,4-dinitrophenyl labelled protein carbon-yl groups it was shown that not proteins in general butspecific proteins, especially myofibrillar proteins, were oxi-dised in rainbow trout during tainting [228]. Others have usedfluorescein-S-thiosemicarbazide to tag carbonyl groups insimilar approaches [229]. The complexity of specific proteinoxidation during frozen storage of rainbow trout was charac-terized by Kjaersgard et al. [230]. They found that although theoverall protein carbonylation level was increased at highfrozen storage temperatures the carbonylation pattern wasmore or less the same irrespective of storage temperature.Many abundant proteins were found to be carbonylated butsome less abundant proteins such as adenylate kinase wererather heavily oxidized indicating differentially susceptibilityof the proteins to carbonylation [230].

Amore extensive insight into changes in protein profiles insea bass due to protein degradation during chilled storage wassought by Terova et al. [231] in a study where effects of storagetemperature were investigated. As expected, they found thegreatest alteration in muscle protein pattern (composition) atthe highest storage temperature with clear degradation ofparticularly myosin heavy chain and glyceraldehydes-3-phosphate dehydrogenase. However, also at a low chillingtemperature (1 °C) several proteins changed in abundanceduring storage, although to a lesser extent. Additionally theyfound that nucleoside diphosphate kinase B decreased inabundance during storage but in a temperature independentmanner. This actually supported an earlier finding from a 2-DE study by Verrez-Bagnis et al. [232] of a gradual disappear-ance during storage of a 16 kDa (pI 6.67) protein suitable as afreshness marker.

A proteomics driven understanding of quality could lead tothe inclusion of quality as a breeding parameter together withthe parameters health, growth, and feed conversion which areprimarily used today. The perspective in this respect is to findcorrelations between protein expression in a tissue and theeating or technological quality of this tissue. This would openup for a biomarker guided selective breeding directed towardsa better and less variable quality. Moreover, when the qualityrelated biomarkers are combined with biomarkers relating toproduction parameters such as diet, feeding regime, temper-ature and handling procedures (stressors) it constitutes astrong tool for design of fish having certain product specificproperties. No investigations have been published yet inwhich proteomics and eating quality measured in detailedassessments by sensory taste panels are compared directly.However, the presence of a multivariate correlation between


certain protein expression profiles from 2-DE of shrimpmuscle and an eating quality attribute has been shown [233]indicating that such a scenario is a realistic future possibility.

An essential aspect of seafood safety is a rapid andaccurate identification of bacterial species and Bohme et al.[234] demonstrated that matrix-assisted laser desorptionionization-time of flight mass spectrometry is a feasible andcost-effective technique for this. By comparison of unknownspectra to reference spectra from food-borne pathogens andspoilage bacteria (fingerprinting), 26 bacterial strains isolatedfrom fresh fish and seafood products that have beenprocessed (vacuum packed and mild heat treatment) wereidentified.

From a health point of view food allergy is an importantissue and knowing that fish and crustacean (includingaquaculture species) are among the eight foods that havebeen identified as causing the majority of food allergenicreactions, pinpoints the need for suitable analytical tech-niques for the detection and measurement of the involvedallergens. LC/MS has by now shown its high potential withinthis area in general [235].

Another safety aspect is food authentication where prote-omic based approaches have already shown their strengthswithin the seafood area (see reviews [51,236,237]). Recentlyefforts have been done to increase speed and capability of theanalyses to differentiate between closely related species. Todistinguish between seven closely related shrimp species,Ortea et al. [238] used a high intensity focused ultrasoundtechnique to get a very fast trypsin digestion of sarcoplasmaproteins during sample preparation. This was followed by ashotgun proteomic approach combining LC separation andpeptide identification by selected MS/MS ion monitoring.Using this sensitive procedure, they were able to completean unambiguous identification of a shrimp product within90 min [238].

8. Concluding remarks and outlook

This review illustrates the growing importance of proteomicstechnologies in aquaculture. Even with limited genomicinformation to date, the studies carried out so far clearlydemonstrate the potential of proteomics to identify importantproteins related to topics as welfare, nutrition, health, qualityor safety, and unravel potential biomarkers and mechanismsrelated to aquaculture species biology.

Proteomics has undoubtedly shown to provide a holisticmeasure of an organism response, in this particular case anaquaculture species, to an external stimulus. Nevertheless,proteomics has their own limitations and an integration of thistechnology, together with others like transcriptomics ormetabolomics, will definitely be the direction of future researchin the area, since awider vision aswell as a validation of resultscan be obtained in thismanner.We can foresee in this way thatareas like proteogenomics might become a powerful approachin identifying protein biomarkers and to study genomevariation. Proteogenomics refers to the correlation of theproteomic data with the genomic and transcriptomic datawith the goal of enhancing the understanding of the genome

[239]. One of the important outcomes is the identification ofnovel genes that are missed by gene prediction programs. Thiscan be a powerful approachmore routinely employed in a nearfuture. Also the use of new technologies like MALDI imaging orprotein array/protein chip approaches will greatly contribute toa better understanding of the biological processes in aquacul-ture species.

As a final remark, we propose that a major challenge stillexists, which is to annotate the proteomes of species com-monly used in aquaculture. We consider this a major gap inproteomics studies, not only in aquaculture related research,but also in all the other areas using proteomics. However, wealso have to keep inmind that in the mid-short term the use ofde novo sequencing approaches might overcome this issue.

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