RodriguesPM12 - Proteomics in Aquaculture PDF

J O U RN A L OF P ROT EO M I CS 7 5 ( 2 0 12 ) 43 2 5 4 34 5
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Review
PROTEOMICS in aquaculture: Applications and trends

Pedro M. Rodriguesa,, Tom S. Silvaa , Jorge Diasa , Flemming Jessenb
a b
Centro de Cincias do Mar do Algarve (CCMar), Universidade do Algarve, Campus de Gambelas, 8005-139 Faro, Portugal National Food Institute, Technical University of Denmark, 2800 Kgs. Lyngby, Denmark
AR TIC LE I N FO
Article history: Received 20 December 2011 Accepted 24 March 2012 Available online 4 April 2012 Keywords: Aquaculture Fish Proteomics Welfare Quality Nutrition
ABS TR ACT
Over the last forty years global aquaculture presented a growth rate of 6.9% per annum with an amazing production of 52.5 million tonnes in 2008, and a contribution of 43% of aquatic animal food for human consumption. In order to meet the world's health requirements of fish protein, a continuous growth in production is still expected for decades to come. Aquaculture is, though, a very competitive market, and a global awareness regarding the use of scientific knowledge and emerging technologies to obtain a better farmed organism through a sustainable production has enhanced the importance of proteomics in seafood biology research. Proteomics, as a powerful comparative tool, has therefore been increasingly used over the last decade to address different questions in aquaculture, regarding welfare, nutrition, health, quality, and safety. In this paper we will give an overview of these biological questions and the role of proteomics in their investigation, outlining the advantages, disadvantages and future challenges. A brief description of the proteomics technical approaches will be presented. Special focus will be on the latest trends related to the aquaculture production of fish with defined nutritional, health or quality properties for functional foods and the integration 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.
Contents
1. 2. 3. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4326 Aquaculture: key features, challenges and development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4327 Aquaculture species and fish model organisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4328 Aquaculture species 3.1.1. Vertebrates . 3.1.2. Invertebrates . 3.1.3. Algae . . . . . Proteomics technologies . 4.1. 3.1. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4329 4329 4329 4330 4330
4.
Gel based approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4330 4.1.1. Sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4330 4.1.2. Protein separation and quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4330
This article is part of a Special Issue entitled: Farm animal proteomics. Corresponding author. Tel.: +351 289800100x7855; fax: + 351 289818353. E-mail address: pmrodrig@ualg.pt (P.M. Rodrigues).
1874-3919/$ see front matter 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.jprot.2012.03.042
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4.1.3. Protein identification and characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4331 4.2. 4.3. Gel free approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4331 Identifying proteins from peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4331 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4332 4332 4334 4336 4338 4338
4.4. Challenges and new technologies 5. Fish welfare in aquaculture . . . . . . . 6. Nutrition and health in aquaculture . . . 7. Quality and safety of produced fish . . . 8. Concluding remarks and outlook . . . . References . . . . . . . . . . . . . . . . . . .
1.
Introduction
During the last decade Omic technologies (i.e., genomics, metabolomics and proteomics) have been widely implemented in the field of farm animal proteomics with a very positive impact in areas such as aquaculture. In Fig. 1 we give a brief overview of the use of these Omics technologies in aquaculture, describing the target research topics and the main aquaculture species. Proteomics in particular has emerged as a powerful tool towards a deep understanding of marine organisms biology, helping aquaculture to reach its main goal; high productivity of a better quality product. Compared to genomics, proteomics provides not only information at a mechanistic level but can also capture changes in protein activity measured as post-translational modifications (PTMs). In fact the transcriptome does not account for the posttranscriptional and post-translational regulation of protein expression. Most studies reveal a poor correlation between protein expression and changes in transcript level [1]. The proteome can then provide relevant information of an organism's physiological state that is missed by the transcriptome. Interpretation of proteomic data requires availability of information on genomic DNA and expressed RNAs, so a major limiting factor in aquaculture proteomics is still the lack of information at the genome level in most of the 310 cultured
species reported by FAO in 2008 [2]. Full genomes are still only available for some model species (such as zebrafish, stickleback, medaka, coelacanth, fugu and Tetraodon), although more focus is being given lately in studying the genomes of commercial species (like tilapia, cod and salmon) [3,4]. As a result, proteomic studies on aquaculture species still face some challenges at the level of protein identification. Nevertheless, recent developments in genome sequencing technologies have pushed the cost of full genome sequencing from hundreds of thousands to thousands of dollars [5], suggesting that the availability of genomic and EST data on aquaculture organisms will not remain for long a bottleneck in aquaculture proteomics. Farmed seafood organisms are susceptible to a wide range of factors that can pose a major threat to a thriving aquaculture industry with considerable economical repercussions. This industry has been going through major challenges in its effort to respond to the continuous higher consumer demand, coupled with clear market global awareness of a better quality product and also animal welfare. A good balance between these challenges may greatly benefit from a better scientific understanding of the biological traits in seafood farming. This paper is the first review manuscript specifically dedicated to proteomics in farmed seafood organisms and aims to address a global perspective and not focus on any specific aspect of this thematic. Throughout the
Main Tools
Genetic linkage maps Radiation hybrid maps QTL -Quantitative Trait Loci Marker Assisted Selection
Main Tools
Transcriptomics (cDNA microarrays) Proteomics and Protein arrays Metabolomics
Main Tools
Marker Assisted Selection Proteomics
Broodstock
Breeding programmes Traceability
Farming
Nutrition Farming practices Physiological life stage Immunological state Disease resistance
Quality & Safety

Selection of quality traits (fat deposition, pigmentation) Post-mortem degradation Authentication Spoilage indicators
Salmonids: salmon, trout Marine fish species: cod, halibut, seabass Warm water fish species: tilapia, catfish Shellfish: oyster, scallop
Model organisms: zebrafish, medaka, pufferfish Salmonids: salmon, trout Marine fish species: cod, seabass, seabream Warm water fish species: carp, tilapia Shellfish: oyster, clams
Salmonids: salmon, trout Marine fish species: cod, seabass, seabream Shellfish
Fig. 1 Omics in aquaculture: target research topics and species.
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Aquaculture
Proteomics
Aquaculture
Aquaculture Proteomics
5169 5353
6000
4784
Welfare
Nutrition
Health
Quality
Safety
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Number of publications
4054
3299
4000
2609
3583
10%
3181 2817 2864
4% 15%
2680
1697 1571
1791
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3
652
1265 1097
2000
1123
1638
2486
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53
0 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011
11
27
30
54
75
87
116
138
40%
31%
Year
Fig. 2 Number of published manuscripts in the last decade within the keywords Proteomics, Aquaculture and Aquaculture + Proteomics. Searches were processed on Science Direct (December 2011), and grouped according to publication year.
Aquaculture Proteomics
Welfare Nutrition Health Quality Safety
11%
5% 14%
different sections of the manuscript we pinpoint the different areas and the importance of proteomics in aquaculture research. We will address separately factors like welfare, nutrition, health, quality, and safety, introducing for each one some of the major achievements of proteome analysis and its effort in helping optimizing production in the aquaculture industry. A special focus will be put on fish model organisms and their advantages and disadvantages. A brief overview on the different proteomics technologies will also be addressed with reference to the different methods, new approaches and future challenges.
36%
34%
2. Aquaculture: key features, challenges and development

The rapid growth of aquaculture industry over the last half century is expected to continue in the future, due to the higher consumer demand for aquatic animal food, coupled with capture fisheries restrictions mainly in developed countries. In fact, the aquaculture industry is growing at higher rate than any other animal food-producing sector with Asia dominating this production [6]. On the other hand, development of aquaculture in Europe and North America grew quickly during the last two decades of the 20th century but has since stagnated. Nevertheless, markets for fish and seafood in these continents are still growing in demand. Also, globalization and the world's economical growth led to large scale intensive farming of species like salmon, shrimp or catfish. To cope with this development that is affected among other factors, by market demand, availability of environmental resources, infrastructures and technical capability or investment, there has been, mainly in the last decade, awareness that research in this area is an imperative. To prove this, we can monitor the number of studies in
Fig. 3 Relative percentage of the number of published papers in the last decade in each of the five different topics (Welfare, nutrition, health, quality and safety) both in Aquaculture and Aquaculture + Proteomics studies Searches were processed on Science Direct (December 2011), and grouped according to each topic.
aquaculture-related studies, namely in the genomics/proteomics area, with up to 50 research publications in the area and a significant increase in the number of manuscripts. In Fig. 2 we have an overview on the number of publications recognized in Science Direct (December 2011) in the areas of Aquaculture, Proteomics and Aquaculture Proteomics in the last decade. Interesting is the fact that over the last five years the increase of publications in the Aquaculture and Proteomics areas is around 20% and 50% respectively while Aquaculture Proteomics registered an increase of 155%. Nevertheless these numbers are quite low compared to the same type of studies that have been reported in other farmed animals like cows or pigs and they do not reflect the research effort and the importance of proteomics in Aquaculture. Also,
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a quite significant number of proteomics studies carried out in aquatic organisms are focus on wild species and not farmed animals. Still, we anticipate that with progress in genomics, this number will soon be comparable. Nutrition, welfare and health management have proven to be major constrains to an efficient production in aquaculture systems. One of the main objectives of aquaculture is to produce fish with an optimal growth rate and health status. From a physiological point of view, fish growth is a complex process that apart from genetic background depends on other mutually interdependent processes, such as development, nutrition, metabolism and physiological stress. Since growth is a physiological process with multiple tissue contributions (e.g. liver, intestine, pancreas and muscle), a global and multidisciplinary approach is required in order to have a complete view of all the factors contributing to growth. At the present time, the application of transcriptomic, proteomic and metabolomic approaches rise as powerful integrative tools to reconstruct the pathways and functional networks that govern the process of growth in fish. In this process, an important aspect is to determine how gene expression is correlated to protein translation. Several approaches have been used to compare the response of the transcriptome in relation to the proteome. Generally, a fairly good similarity was achieved between genes/proteins, appearing there is some correlation between the steady state mRNA abundance and protein abundance. Nonetheless, several factors like the transcriptional/translational regulation, proteins PTMs, analytical methods used in the studies or the temporal scale, can influence the relationship between genome and proteome.
As production methods gain importance to many consumers, issues of ethical production, animal treatment and welfare, and environment-friendly production systems as well as sustainability, seem to gain more influence on seafood product choice [7]. This ethical quality concept of fish is mainly associated to aquaculture products, but the sustainable exploitation conditions in the case of capture fisheries are also gaining importance in seafood markets. This is largely due to increasing public concern on sustainability issues in the food chain and anticipated regulatory changes, but also because the welfare standards by which a fish is reared and then slaughtered may produce an impact upon both production and flesh quality. These factors have led to considerable interest both in indices of good welfare and in development of production systems or technologies that promote it. Balancing current productivity standards with higher disease and stress resistance, feed efficiency to lower environmental impact, ability to use more sustainable fish ingredients (e.g. vegetable proteins and oils) are all concrete traits that may contribute to the sustainable growth of the fish farming industry.
3. Aquaculture species and fish model organisms

There were around 310 species cultured and recorded by FAO in 2008 [2]. However, the 20 most significant species produced account for 74% of production by volume and 63% by value. Among these, fresh water fish production is
Fig. 4 Typical proteomics workflow used in aquaculture proteomics studies. The common feature is sample preparation that can then be followed by a gel-based or a gel-free proteomics approach. A workflow pointing the different possible pathways and techniques is presented in each one of the followed strategies; gel-base proteomics top-down, gel-free proteomics top-down and gel-free proteomics bottom-up (shotgun proteomics). Final objective is in all cases the identification and characterization of proteins of interest.
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dominated by carp, while Atlantic salmon leads among the intensively 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 aquaculture (welfare, nutrition, health, quality, or safety) proteomics approaches are, by its nature, easily applied to model or nonmodel species. A relative percentage of the number of published papers in the last decade in these five different areas, both in Aquaculture and Aquaculture Proteomics studies, is presented in Fig. 3. We can easily observe that the majority of studies have been focused on health and quality. Nevertheless, we must consider that health related publications are not strictly on health issues in aquaculture species, but are mainly focused on human health related to aquaculture. This value is then well inflated in the health perspective that we will point out later in this paper, which is strictly related to health in aquaculture species. Fish models have been used successfully in many biological aspects of human diseases, in areas like cancer, neurology, toxicology, infectious diseases and drug development [8,9]. Undoubtedly, the freshwater teleost zebrafish (Danio rerio) has been selected as the model-fish organism. Small in size, good availability, low cost maintenance, genome sequence coverage and an available large-scale proteome profile [10], makes zebrafish highly attractive in disciplines as genetics, developmental biology and physiology with a vast number of publications [1024]. In these studies we can verify how omics technologies have been applied to analyze the transcriptome, proteome and metabolome and how they can be strategically incorporated into chemical screening and perturbation studies. Proteomic data is proven to complement both the transcriptomic studies by validating the relative protein abundance as reflected in the relative mRNA abundance and also the metabolomic studies by reflecting the functional state of the protein machineries responsible for altering the metabolites in a biological system. As a concrete example, De Witt's studies [15] clearly demonstrate the potential of combining transcriptomic and proteomic data to generate mechanistic toxicological information. Nevertheless quite a few studies are available in nonmodeled species with advantages and disadvantages. A clear benefit from using a model species in a proteomics study is due to the availability of the genome sequence coverage in databases that reduces the uncertainty of protein identification associated with mismatching amino acid sequencing across species. Still, there are some authors pointing out that not only a non-model species provides a better surrogate for a biotic response in a specific system, but also suggest the use of de novo sequencing approaches can be used to overcome the limitations of sequence coverage [25].
in studies with these species and also in other less studied species like gilthead seabream (Sparus aurata) [29], together with muscle quality [3033]. Related to fish quality is the work on understanding the mechanisms involved in the development of skeletal malformations of the vertebral column of normal and deformed white seabream (Diplodus sargus) by comparative proteomic analysis [34]. The effect of probiotics administration in diets has also been reported in rainbow trout and cod [35,36] with interesting results at the level of immune-stimulation. In vitro [37] and in vivo infections proteomic studies on cultured fish like gilthead seabream [38], rainbow trout [39] and Atlantic salmon [40,41] are also commonly addressed since infection and concomitant death is one of the main causes of heavy economic losses in marine fish cultures. These works lead not only to a better understanding of the molecular mechanisms of pathogenesis as well as provide novel protein candidates for vaccines development. Toxicological studies related to evaluation of chemically induced protein expression response have also been reported in Atlantic salmon [42], rainbow trout [43], cod [44,45] and rare minnow (Gobiocypris rarus) that has been recently selected as a model for aquatic toxicological studies [4648]. Mainly focused on environmental pollutants that affect growth, reproduction and health of marine farmed organisms with repercussions to human health, these reports try not only to understand the mechanisms of toxicity but also discover potential biomarkers for environmental monitoring and risk assessment. Also lately, some interesting work has been done to distinguish specific characteristics within the same species. Examples are the efforts to establish potential biomarkers in testis for the poor reproductive farmed females in Senegalese sole (Solea senegalesis) [49] or the use of 2D protein maps to differentiate between wild and farm cod [50]. Also the correct identification of fish species with commercial interest has been investigated with proteomics techniques [51,52] and more recently with the application of biomarker peptide monitoring in MS in hake and grenadier [53,54]. In farmed sturgeon some research has also been done regarding proteins involved in the differentiation between sturgeon species [55] and sex differentiation [56].
3.1.2.
Invertebrates
3.1. 3.1.1.
Aquaculture species Vertebrates
Most proteomic studies have been carried out in Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss), catfish (Ictalurus punctatus) and cod (Gadus morhua), mainly due to their commercial importance. Stress related to farming conditions [2628] has been one of the main themes reported
With the intensification of crustacean farming in the last decade, proteomics studies have also been reported, namely as a tool to better understand the immune response mechanisms of these organisms. In Chinese shrimp (Fenneropenaeus chinensis) 33 different proteins have been identified as a result to stress exposure by hypoxia [57], while in giant tiger shrimp (Penaeus monodon) a study revealed that the effect of antibiotics on patterns of hemolymph protein expression are overwhelmed by the effects of external farming conditions [58]. Aquatic invertebrates, as bivalves, often serve as reservoirs for many environmental pollutants, due to their environment and exposure to aquatic sediments. In early proteomics, focus was mainly on taxonomy [5964] while lately quite a lot of work has been published on environmental toxicological proteomics. In fact, these organisms are popular sentinel species used in estuarine and coastal monitoring programs. Many authors have been trying to establish protein patterns of environmental contaminants exposure, as well as
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comparing protein profiles of bivalves residing in polluted areas to those of reference sites [6568]. Environmental contaminants proteomics studies include exposure to cadmium [69] NP (nonylphenol) [70] and p,p-dichlorodiphenyldichloroethylene (DDE) in the clam Ruditapes decussates, benzo()pyrene in zebra mussels (D. polymorpha) [71] PCBs, PAHs (polyaromatic hydrocarbons) and heavy metals in blue mussels (Mytilus edulis) [72,73], cylindrospermopsin (CYN) in Mytilus galloprovincialis and Corbicula fluminea [74] and crude oil in blue mussels [7577].
3.1.3.
Algae
The 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 aquatic plants [2]. Macroalgae represent most of algae aquaculture both by volume and value (about 99%), being mainly cultivated as a source of food or specific substances (like iodine, algin, carrageenan and agar) [2]. Not many proteomic studies exist in macroalgae, but there are already a few [7882], mostly focusing on optimizing viable proteomic workflows for algae, since these organisms tend to contain substances which make adequate protein extraction more challenging. Interestingly, despite these technical issues, there are already a large number of proteomic studies on microalgae, on a wide variety of topics. A major focus concerns an issue that is very relevant for aquaculture: the study of microalgae biology and their response to environmental signals, in order to understand the mechanisms that trigger the development of harmful algal blooms. This knowledge is important to ensure that management practices are optimized to guarantee not only fish health, but food safety and public health (as some microalgae produce neurotoxins that can bioaccumulate in marine animals, even in the absence of algal blooms). Most proteomics studies therefore focus on subjects such as the response of microalgae to different stress sources or environmental signals [8397] and issues related to taxonomy and identification of potentially harmful species [98,99]. Understanding the metabolism of algae is also of the utmost importance, given the numerous roles such organisms can have in biotechnology, namely in production of bioactive compounds and biofuel, as well as their use in bioremediation/integrated aquaculture systems. Proteomics is increasingly seen as an important tool in this context [100102].
solubilization of proteins in an appropriate solvent (aqueous buffers, organic solvents). This makes sample preparation a critical step in the analytical workflow. The purpose of this step is to attempt thorough solubilization of all (or a specific subset of) the proteins present in a given bodily fluid, organ, tissue or cell extract. Commonly used aqueous extraction buffers often contain (besides buffering agents) detergents, chaotropes, reducing agents and protease inhibitors, ensuring that enzymatic activity is halted during extraction and that intra and inter-molecular interactions between proteins are minimized, preventing aggregation. Although standard extraction buffer formulations work reasonably well for a broad range of samples (eukaryotic cell cultures, bodily fluids and soft tissues of animals), some types of organisms (algae, bacteria) or protein subpopulations (membrane proteins, proteins embedded in a calcified matrix) often require different types of extraction buffers for optimal results. To improve protein separation and identification, several procedures can be considered. Sample fractionation is often a choice for sample pre-processing prior to proteome analysis, simplifying protein extracts and/or improving the dynamic range of a protein mixture. Fractionation methods are usually based on chromatography, electrophoresis, differential solubility and/or (ultra) centrifugation and the most popular are usually aimed at isolation of organelles and sub-cellular compartments (nuclei, mitochondria), enrichment in particular sub-populations of proteins (phosphorylated, glycosylated, secreted, and membranar) and depletion of highly abundant proteins (like albumin and immunoglobulins in blood plasma, or actin/myosin/tropomyosin in muscle).
4.1.2.
Protein separation and quantification
4.
Proteomics technologies
Data regarding teleost fish and invertebrate species obtained by proteome analyses from the last decade has been published in recent reviews [103,104]. The common workflow (gel-based and gel-free) analysis includes: 1) sample preparation; 2) protein separation and quantification; and, 3) Protein identification and characterization, and is represented in Fig. 4.
4.1. 4.1.1.
Gel based approach Sample preparation
The first step is usually protein extraction, since most analytical techniques used in proteomics require prior
The two most important analytical techniques in proteomics are two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS). Due to the central role that these two methods have in proteomic studies, most experiments are broadly classified as either gel-based proteomics or gel-free proteomics, depending on whether 2-DE is used for protein separation and quantification or not. Currently, 2-DE is still, by far, the most common strategy for protein separation and quantification, enabling the separation, detection and quantification of hundreds (and sometimes thousands) of different proteins from a single extract, taking advantage of the fact that the isoelectric point of a protein is mostly uncorrelated to its molecular weight. On the other hand, issues like gel-to-gel variation, limited linear dynamic range, limited throughput, and protein co-migration still pose challenges to gel-based proteomics. Although most software geared toward the analysis of 2-DE gels, like Progenesis SameSpots (Nonlinear Dynamics), PDQuest (BioRad Laboratories) and DeCyder (GE Healthcare), tries to overcome some of these issues with a simplified, semiautomated analysis workflow, they still cannot achieve 100% unambiguous results without significant human intervention and visual inspection. Nevertheless, there are also some advantages of gel-based approaches: first of all, they are generally less expensive than purely MS-based approaches. Besides that, information on small post-translational modifications (PMTs) and highly
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homologous isoforms can often be obtained directly, since these tend to shift the isoelectric point of a protein without extensively changing its molecular weight. Classically, detection and quantification methods for 2-DE are usually based on Coomassie Brilliant Blue (CBB) or silver staining, which enable estimation of protein quantity by scanning 2-DE gels in the visible range. Recently, the development of multiplex 2-DE (dubbed difference gel electrophoresis or DIGE), which instead involves tagging the protein samples with different fluorophores prior to 2-DE, not only allows several samples to be run on a single gel, but also significantly improves gel-to-gel variability, by providing a common reference channel across all gels of an experiment [105]. This recent improvement of the 2-DE method has been increasing in popularity in aquaculture species proteome studies with some successful results in several species [24,49,58,106,107].
4.1.3.
Protein identification and characterization
Although immunostaining methods are a valid choice for protein identification after 2-DE, the overwhelming majority of gel-based proteomic studies rely on digestion of detected proteins and analysis of the resulting peptides by MS for their identification and characterization, since it does not rely on having specific antibodies against every single protein to be identified. Given the accuracy of current MS instruments and the low number of proteins usually present on a single 2-DE spot, the results obtained can be quite reliable. Instruments currently employed for this purpose include ESI-Ion Trap, MALDI-TOF/TOF and ESI-QTOF mass spectrometers to a lesser extent. Identification of proteins can be assessed either directly through its peptide mass fingerprint (PMF), for the case of organisms with fully sequenced genome, or by analysis of the fragmentation spectra of such peptides (PFF, peptide fragment fingerprinting or even de novo sequencing) obtained through tandem MS.
methods used in gel-free proteomics are based on liquid-phase chromatography procedures, which can readily be coupled to ESI-based mass spectrometers. Even multidimensional chromatographic separations are commonly used, as in the case of MudPIT, where peptides are separated by charge (SCX-HPLC) and hydrophobicity (RP-HPLC) prior to MS analysis. Despite the fact that label-free methods exist, most gel-free workflows rely on stable isotope labeling for peptide quantification, either by metabolic incorporation of radioactive amino acids in proteins (SILAC) or by post-extraction chemical modification (ICAT, TMT, iTRAQ). With stable isotope labeling, several samples can be analyzed in parallel on the same MS run (with identical peptides from distinct samples separated by a small mass shift) and relative abundance can be estimated from the ratio of peak areas of identical peptides. At the end of a bottom-up analysis, information on protein abundance/identity is inferred from the obtained information on peptide abundance/identity, using computational tools. This is, in itself, a challenging part of bottom-up approach and still a matter of research in the field of mass spectrometry. Although a direct top-down approach is intrinsically more challenging, due to the difficulty of ionizing whole proteins, the higher resolution of MS instruments using FTICR or Orbitrap technologies make top-down approaches increasingly practical, beyond the more common bottom-up approaches.
4.3.
Identifying proteins from peptides
4.2.
Gel free approach
Due to the above-mentioned issues associated with 2-DE and the continuous improvement and cost-reduction of MS-based methods, gel-free strategies are becoming increasingly popular, since they generally allow higher analytical throughput and generally deeper proteome coverage than gel-based methods. In a way, 2-DE can be seen as an elaborate pre-fractionation step that precedes MS analysis in a typical proteomic workflow and, as such, some researchers prefer to omit this step and apply different strategies which scale much better. In contrast to the usual top-down approach of gel-based proteomic studies, where proteins are separated and quantified before being digested and identified, most gel-free studies employ a bottom-up approach. Here, proteins are digested from the start and analyses (separation, quantification, characterization) are done at the peptide level. Since digestion of a complex protein mixture dramatically increases its complexity, gel-free methods are usually combined with fractionation methods to reduce the number of different peptides entering the mass spectrometer at any given moment, maximizing the total number of distinct peptides detected over the course of a sample run. By far, the most common pre-fractionation
Most proteomic studies (both gel-based and gel-free) attempt to identify proteins by looking at peptides, as large proteins still constitute a challenge for MS-based methods. The classical method used (dubbed peptide mass fingerprinting or PMF) is based on in silico digestion of genomic/EST sequences following the pattern of a predictable endonuclease (usually trypsin) to obtain a list of peptide masses (or mass fingerprint) for each database entry. Identification is performed by comparing experimentally obtained MS mass lists with those generated in silico, and choosing significantly similar matches. Nonetheless, this is a reliable method when working with a model species (like zebrafish), for which there is full genome data, and for simple digests (like 2DE spot digests). On the other hand, by using tandem MS instruments, you obtain not only a peptide mass list, but also information on the fragmentation mass spectra of those peptides, providing a fingerprint for each peptide that directly reflects its sequence. The most common strategy here (peptide fragment fingerprinting or PFF) also involves starting with a genomic/EST database and performing in silico digestion with a standard endonuclease. Then, for each peptide, the masses of all likely fragments are deduced from prior models. Identification of peptides is then performed by comparison of experimentally obtained MS/MS spectra against all possible fragmentation spectra in the database. Since a fragmentation spectrum (unlike mass) is usually very specific for a certain peptide sequence, identification of proteins can often be attained from a single high-quality peptide match. Nevertheless, unless you work with species for which there is a great wealth of genomic/EST data available, this strategy only works for highly conserved peptides.
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It is therefore important to underline that protein identifications depend not only on the quality of spectra, but also on the quality of the sequence database used. Generally, a good approach is to start by searching genomic databases (since they tend to be smaller and therefore fast to query) and then EST databases. The results obtained are often different, since genomic databases reflect genomic DNA sequences while EST databases reflect mRNA sequences (which are closer to the actually translated amino acid sequences). Observations on the frequency of alternative splicing events in teleosts suggests that this difference is particularly important for species with a compact genome (like Takifugu rubripes, for which ~ 43% of mapped genes seem to be affected by alternative splicing) and less so for species with a high level of gene duplication (like zebrafish, for which only ~17% of mapped genes seem to be affected by alternative splicing) [108]. Also, given current developments in DNA sequencing technology [5], it is expected that the coverage of genome/EST information on commercially relevant species will be greatly expanded, certainly improving the quality of MS-based peptide/protein identification methods.
4.4.
Challenges and new technologies
Looking at the results of proteomic experiments, we observe that there are several issues that prevent perfect consistency of results across experiments on a single topic, despite the fact that there is usually significant overlap in terms of affected pathways. Some of these differences can be attributed to technical limitations, like the influence of sample preparation methods, the user factor, the difficulty of ionizing certain peptides, as well as other instrumental and analytical limitations. Most of these can hopefully be overcome by increased automation, improvements in instrument quality and a decrease in user intervention throughout the workflow. On the other hand, the dynamic and complex nature of the proteome provides several intrinsic challenges during interpretation of proteomic results. This can be seen, for example, in experiments coupling transcriptome and proteome observations, which often show a weak correlation between the two profiles (when sampled at the same time). This dynamism and the sensitivity of the proteome towards environmental signals and subtle changes in physiological state justifies the necessity of obtaining and using as much contextual information as possible (including both classical and/or omics observations) to support proteomic observations and having into account the importance of time and locality on the proteome. The fact that most proteins display post-translational modifications (PTMs) further adds to the proteomes complexities and is increasingly seen as essential to study, given the biologically important roles some of these PTMs are known to have. Although gel-based approaches can use either specific dyes (like Pro-Q Diamond, for detection of phosphorylated proteins) or immunoblotting for detection of PTMs, the real workhorse of PTM analysis is mass spectrometry. Specific detection methods are often combined with protein/peptide enrichment steps, to target for some specifically modified proteins (like affinity chromatography with lectins, to enrich for glycoproteins, or TiO2, to enrich for phosphopeptides).
As technology continuously develops, the future of proteome research is still uncertain, but there are already new approaches which have good potential to become invaluable tools in aquaculture research (and marine biology in general). One of them is MS imaging. This technology involves the direct digestion of histological samples fixed to a suitable support, followed by direct MS/MS analysis (for example, by applying a matrix solution on the sample and doing MALDIMS all over the surface, point by point). This is the equivalent of using immunocytochemistry methods, but in a nonspecific way, providing information on a key variable: location. This is very important because, unless cell sorting or microdissection techniques are applied prior to proteomic analysis of a given tissue, the profiles obtained are very likely to represent an average profile of a heterogeneous population of cells, since most organs and tissues are heterogeneous. Most importantly, this heterogeneity is often locationdependent, which implies that MS imaging can provide a type of deep morpho-proteomic information that would not be accessible otherwise. It's interesting to note that MS imaging doesn't necessarily require protein identification: using computational methods (dimensionality reduction methods, classifiers, neural networks and similar machine learning tools) it's possible to map all the MS and MS/MS information obtained for each point in space as a pixel, where color information is defined so that it reflects the similarity relations between proteomes. This means that MS imaging can provide useful information on proteome distributions over any tissue (regardless of source), distinguish between sub-populations of cells with different proteome profiles and pinpoint exactly where proteome changes occur. The size of matrix crystals are a limiting factor preventing increased resolution in MALDI imaging, so there is still a lot of research on alternative matrices or ionization methods for MS imaging, like SIMS and DESI. New breakthroughs in this field will certainly revolutionize proteomics and greatly increase our understanding of biological processes which are intrinsically morphological and relevant for aquaculture research (like larval development or muscle growth). Another interesting technology is the development of protein array/protein chip approaches. These allow for very high throughput at the expense of high specificity, which currently makes the development of such tools relatively expensive and challenging, particularly for non-model or non sequenced organisms. Nevertheless, as these types of technologies develop, the potential for very large scale studies regarding major issues in aquaculture suddenly becomes feasible, opening the doors to new, more in-depth approaches for comparative proteome studies.
5.
Fish welfare in aquaculture
Animal welfare is a complex concept and it is by no means straightforward to define and measure it. Ashley has in comprehensive terms made a good definition of welfare as the freedom from hunger and thirst, discomfort, pain, injury, disease, fear and distress, and the freedom to express normal behaviour [109]. From this definition it is quite evident that no
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easy and single measure of welfare is available. The most general accepted measure of welfare has until now been physical health where a variety of physiological and biochemical measures have been used. However, welfare also involves lack of mental suffering and in fish reliable measures of this constitute an important future challenge [109]. This complexity of the welfare concept underlines the importance of a multidisciplinary and holistic approach to the study of fish welfare and results have shown that proteomics can be an important part of the toolset required for such studies and to develop aquaculture practices that guarantee good welfare and health and ensure that marine animals are reared in an environment that optimizes their capacity to cope with unavoidable challenges/stress. When it comes specifically to fish in aquaculture the aspects of welfare issues can be divided in three groups: 1) aspects relating to fish health; 2) aspects relating to aquaculture management procedures; and, 3) aspects relating to stocking density, aggression and altered behaviour [109]. Given all the different number of factors involved in welfare, it is clear that no single tissue encompasses all information required to make a complete assessment of an organism's welfare status. Although most proteomic studies on welfare issues focus either on the liver (due to its central role in most key metabolic processes) or bodily fluids like blood plasma (due to its value in providing non-lethal proteomics-based diagnostic tools), some of them also target brain, skeletal muscle, osmorregulatory and immune-related organs and tissues. A number of the existing proteomic studies within aquaculture and related research areas already concerns welfare. Here most are on health aspects, with focus on viral diseases [41,110118], bacterial diseases [38,110,119128] and vaccine development [39,129138] but also parasites [139141], hepatic tumours [142] and skeletal deformities in fish [34] have been studied. Besides these, immunoproteomic studies also encompass results on immunostimulants [35], antimicrobial peptides [143145] and composition of mucosal secretions [146,147], along with other topics related to a better understanding of mechanistic aspects of immune responses to certain stimuli [40,148150]. The study of the impact of xenobiotics in aquaculture has also benefited from proteomics technologies, with several studies done on the impact of biotoxins [141,151153], pharmaceuticals [58,154,155], hormones [15,46,156], metals [157,158] and other pollutants [43,45,67,159163] on the proteomes of aquaculture aquatic organisms. Finally, there are also some studies correlating environmental sources of stress in aquaculture (hypoxia [57], anoxia [26,164166], hyperoxygenation [167], osmotic [168173] and temperature [174177], as well as stress induced by management practices (like high stocking densities [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 and oxidation of proteins and lipids [181]. These events usually induce cellular adaptations mediated (among others) by oxygen sensing systems, which include widely studied factors like HIF-1, and generally characterized by the induction of
cytoprotection systems (including increased expression of chaperones, detoxification enzymes, protein turnover and antioxidant proteins) as well as adaptive changes in energy homeostasis and cell cycle control [182,183]. Furthermore, the effect of these physiologic stressors has slightly different effects on the organism's distinct organs, some of which hormone-mediated by CNS signals, such as the release of epinephrine and glucocorticoids (like cortisol) into the bloodstream [184]. The prior state of the cell, as well as the type, intensity and duration of exposure to these types of oxidative stress determine whether the described adaptations enable the cell to appropriately cope with the oxidative stress or if cell death is inevitable, which implies the observed cellular response is context-dependent [184]. Comparative proteomic studies in fish reflect this: although most types of stress broadly induce changes in terms of energy metabolism, cytoskeletal dynamics and expression of cytoprotective proteins across most organs [26,29,57,164167,180], there are specific particularities in cellular responses depending on cellular/tissue type. For example, in energy-bound organs, like brain and muscles, changes in energy metabolism are usually quite evident, particularly at the level of glycolysis/carbohydrate metabolism and phosphotransfer networks [165,180]. Liver tissue also displays energy metabolism shifts in response to stress, but at a much deeper level, reflecting its essential role in physiological detoxification and energy management: adaptations usually cover amino acid and lipid metabolism, besides carbohydrate metabolism. Other often affected pathways in liver include the urea cycle, the methionine/homocysteine cycle, the folate one-carbon pool and betaine metabolism, which are intimately connected to amino acid metabolism and detoxification processes [29,57,179]. Besides these main pathways, cellular stress also induces changes in the expression of proteins related to signaling, cell cycle regulation, transcription factors, ribosomal proteins and others. The fact that these last changes are not as consistent may simply be a reflection of the negative bias towards low abundance proteins and does not necessarily imply that their role is secondary. Exposure of fish to atypical environmental parameters (pH, temperature, salinity) can also induce general physiologic stress, which results in cellular responses overlapping with what is observed in proteomic studies with hypoxic-type stressors. Heat shock, for instance, is usually accompanied by changes in the expression of chaperones and proteins of the ubiquitin/proteasome pathway [174,175,177]. In the case of salinity changes, proteomic studies in fish often target osmoregulatory organs (like gills and kidney) and generally show similar signs of cellular stress, particularly affecting cytoskeletal dynamics, betaine metabolism and aldehyde detoxification [169,171173]. Regarding the effect of metals, biotoxins and other environmental toxicants on the proteome of fish, most studies focus on the liver, generally displaying increased signs of oxidative stress, changes in immune/ inflammatory effectors, as well as induction of transporters and detoxification enzymes [43,154,157,158,160163,185187]. But, again, there is a general overlap in terms of affected pathways, when comparing to hypoxia-type stressors. When
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looking at the proteomic impact of bacterial and viral infections on the liver, spleen and kidney of fish, there is again a significant overlap with other sources of stress, in terms of affected pathways [38,41,111,112,115,121] underlining the idea that these sources of stress have a cumulative effect at the cellular level. Also when looking at studies involving proteome analysis of plasma proteins, which usually focus on detection and validation of potential welfare biomarkers, several proteins stand out as being affected by sources of physiologic stress (microglobulins, macroglobulins, apolipoproteins, 1-antitrypsin, transferrin, plasminogen, complement system proteins, among others) [160,188190], although again we see a wide overlap in the responses to very different stressors (e.g. bacterial infection vs. high-density crowding). Combined, this suggests that proteomic studies on fish welfare may benefit from holistic approaches, covering several organs and using complementary measures of health and stress status, which provide the necessary contextual information for correctly interpreting observed changes as adaptive or maladaptive. Concluding, although welfare biomarker discovery and validation is still a big challenge, even with all the omics tools available, it is also clear that proteomics provides untargeted information which is invaluable to meaningfully understand fish welfare and work towards a sustainable and ethical aquaculture. Nevertheless, there is still room for improvements and adapting experimental designs to explicitly address the influence of intrinsic confounding factors on fish welfare (e.g. through the use of regression discontinuity design or by using auxiliary welfare measures as blocking or stratification factors) would provide more refined insights into the problematic. The solution to these challenges also lies, in part, in obtaining better data (i.e. deeper proteome coverage) and better analytical tools (enabling data-mining and integration of classical and molecular data), which suggests that, as mass spectrometry technologies and bioinformatics tools continuously improve, the use of proteomics in aquaculture research is increasingly seen as, not only relevant, but essential.
6.
Nutrition and health in aquaculture
Although still at early stages, the proteomic approach is now being successfully implemented in nutritional studies with fish. Nutrient deprivation, or starvation, is a commonly used intervention when analyzing the metabolic and physiologic capabilities of model organisms and tissues. Several studies have evaluated the response of hepatic proteome undergoing fasting and refeeding. In response to a 14 days fasting period, 24 differentially expressed proteins were detected in the rainbow trout liver. Among the proteins with increased abundance after fasting were enolase and cytochrome C oxidase, on one hand, and cathepsin D, on the other hand, most likely related to the higher energy requirements and protein degradation, respectively, that takes place during fasting [191]. More recently, when comparing the changes in hepatic mitochondrial proteome of zebrafish undergoing starvation (15 days) and refeeding (7 days), the 18 identified
proteins indicated that starvation resulted in reduction in glycolysis and increase in gluconeogenesis, while refeeding caused these activities to return to normal levels. Expression pattern of several proteins related to fatty acid and amino acid metabolism also suggested the utilization of noncarbohydrate resources for energy during starving conditions. Proteins with chaperoning and antioxidative roles such as glucose-regulated protein, paraxonase and heat-shock protein were also upregulated in starved conditions [192]. A proteomic approach with zebrafish was also used to assess the metabolic effects of variable dietary energy intake levels. Twenty-nine protein spots differentially expressed between treatments were identified. The most significant protein changes associated with high-caloric intake were related to a decrease in oxygen-binding activity, namely heme binding proteins [193]. A shift towards a lower usage of finite marine-harvested resources is a major sustainability challenge facing the aquaculture industry. It is now consensual that vegetable protein and oil sources are valid alternative ingredients in fish feeds. Despite great advances achieved in this area in recent years, high inclusion levels of vegetable ingredients, still elicit a reduced growth performance and feed efficiency. The recent usage of genomic and post-transcriptomics tools are greatly contributing to a better understanding of the metabolic pathways affected by dietary changes in terms of fishmeal and fish oil replacement [194]. Proteome analysis identified a number of metabolic pathways sensitive to plant protein substitution in rainbow trout liver, for example, pathways involved in cellular protein degradation, fatty acid breakdown, and NADPH metabolism [195,196]. Adaptation of rainbow trout to a diet with a partial (30%) substitution of fish meal with soybean meal for 12 weeks resulted in unaltered growth rates but increased protein catabolism and turnover, accompanied by changes in the abundance of 33 protein spots. Identified spots referred to structural proteins (e.g. keratin and tubulin), lipid binding proteins (e.g. apolipoprotein A) and heat shock proteins, which are indicative of a stress response [195]. In a subsequent trial, rainbow trout was fed a fishmeal-free diet and showed a significant reduction in growth rate [196] and changes in 30 protein spots. Namely, several proteins involved in primary energy metabolism (production of NADPH, ATP, etc.) increased their abundance in liver, as well as two proteasome subunits, indicative of an increase in protein degradation. The effects on the proteasome were particularly noteworthy. The proteasome is a multi subunit enzyme complex that catalyzes proteolysis via the ATP-dependent ubiquitin-proteasome pathway, which in mammals, is thought to be responsible for a large fraction of cellular proteolysis [197]. In rainbow trout, the ubiquitinproteasome pathway has been shown to be down regulated in response to starvation [198] and to have a role in regulating protein deposition efficiency [199]. By correlating all findings of the study, Vilhelmsson et al. (2006) [196] suggested that the differences found in fish regarding texture (sensory analysis) and post-mortem muscle free amino acid pool could be associated to ante-mortem proteasome activity. Fish diets are currently rich in fish oil that supplies both energy and highly unsaturated n-3 fatty acids, but fish oil is becoming scarce. Hepatic proteome was analyzed to investigate global
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metabolic changes induced by the absence of fish oil in trout diets [200]. Fish fed the fish oil free diet exhibited lower growth rate and body fat deposition and 105 hepatic proteins were found to be differentially expressed. The 13 identified proteins supported that fish oil suppression increased fatty acid transport, de novo lipid synthesis and fatty acid desaturation, while decreasing fatty acid catabolism. Several proteins involved in glycolysis, amino acid catabolism and energy production were up-regulated, showing a shift in nutrient utilization for energy production. Overall, changes in protein abundance in the livers of fish fed diets containing plant protein sources suggest that these fish have higher energy demands than fish fed fish meal-based diets. It is noteworthy to bear in mind that effects of a changed diet on fish revealed at the proteome level depend on the genetic background of the studied fish. Although, our knowledge on genes and pathways involved in e.g. growth has increased enormously by using genomic approaches, such approaches cannot stand alone in studying environmental impacts on the growth related cellular functions as these are exerted by the proteins. Thus proteomics provide important complementary understanding to the genomic based knowledge. In this context of fish meal replacement, commonly used plant ingredients in salmon feed such as soybeans and maize are increasingly grown as genetically modified (GM) varieties. The question arises whether these are equally nutritious and safe for the fish as conventional varieties. The protein expression in Atlantic salmon was screened by proteomics to reveal potential effects on metabolism or other processes in the liver. One protein (calreticulin) was up-regulated in the GM group and two down-regulated (thymidine phosphorylase precursor/alpha-enolase and triosephosphate isomerase). But given the low fold changes in the differentially expressed proteins, the overall proteomics data could not detect any biologically meaningful differences between the livers of salmon fed non-GM or GM diets for 7 months [201]. One area in which proteomic studies show a high potential is the interaction between specific nutrients or feed additives and the immune system. An optimal dietary supply of indispensable amino acid supply is of paramount importance to fish growth. Lysine (Lys) is an indispensable amino acid (AA) and is generally the first limiting AA in most vegetable proteins used in fish feeds. Lys availability may thus limit protein synthesis and accretion, and growth of fish. Metabolic effects of various dietary Lys imbalance levels were examined by 2D-proteomics using zebrafish as model [11]. Comparative proteomic analysis of whole-body zebrafish showed 45 spots differentially expressed. Twenty-nine of these proteins were identified revealing proteins involved in muscle growth, energy and lipid metabolism, eye lens differentiation, chaperone activity and apoptosis. Lys deficiency was accompanied by a down-regulation of muscle proteins and up-regulation of proteins associated to fasting, energy deficit, growth arrest and apoptosis. Additionally, it was found that excess Lys was accompanied by an up-regulation of proteins related to glycolysis, steroidogenesis and sexual maturation [11]. A comparative proteomic approach was used to assess the protein expression profile in the liver of 34 days old pikeperch larvae 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 unambiguously identified using nano LC-MS/MS tandem mass spectrometry. Results showed that under high PL intake, the glycolytic pathway was down-regulated due to the underexpression of the fructose biphosphate aldolase B and the phosphoglucomutase 1, while propionyl coenzyme A carboxylase (a gluconeogenic enzyme) was also under-expressed. A high dietary PL level increased the expression of sarcosine dehydrogenase, an enzyme involved in methionine metabolism, along with vinculin, a structural protein. In the larvae fed with the lowest dietary PL content, over-expression of both glutathione S-transferase M, glucose regulated protein 75 might indicate a cellular stress, while the under-expression of peroxiredoxin-1 might indicate a lower defence against oxidative stress [202]. Dietary nucleotides supplementation caused differential expression of muscle metabolic proteins including glyceraldehyde-3-phosphate dehydrogenase, creatine kinase, adenylate kinase, nucleoside diphosphate kinase, and triosephosphate isomerase. In addition to metabolic enzymes, troponin-T-1 as a structural protein was found to increase in abundance in fish fed the nucleotide supplemented feeds [203]. Similarly, the intake of maslinic acid, a triterpene used as a feed additive to stimulate growth and protein turnover rates, was found to regulate gilthead seabream hepatic proteins involved in the metabolism of glucose, lipids, amino acids, and purines, as well as in detoxification and xenobiotics, oxidative stress, immune system, protein synthesis, protein folding, signaling, and cytoskeleton formation [204]. Moreover, the cellularsignaling pathways associated to the dietary use of alphaketoglutarate (AKG), an intermediate metabolite in the Krebs cycle and a feed additive, were assessed on the pituitary proteome of gilthead sea bream [205]. Metabolic proteins upregulated with AKG supplementation included fructose-bisphosphate aldolase, glyceraldehyde-phosphate dehydrogenase and malate dehydrogenase. Protein folding-related proteins up-regulated with AKG supplementation, included two isoforms of heat shock proteins as well as cyclophylin and chaperonin. Proteins found to be associated with regeneration of neural function namely cofilin and Vat-protein were upregulated by AKG supplementation. One hormone modified by AKG treatment was somatolactin [205]. Proteomics holds great promise for discoveries in nutrition research. Integrated with other advanced technologies (genomics, transcriptomics, metabolomics, and bioinformatics) and systems biology, proteomics will greatly facilitate the discovery of key proteins that function to regulate metabolic pathways and whose synthesis, degradation, and modifications are affected by specific nutrients or other dietary factors. This will aid in rapidly enhancing our knowledge of the complex mechanisms responsible for nutrient utilization, identifying new biomarkers for nutritional status and disease progression, and designing a contemporary paradigm for dietary prevention and intervention of disease. Virus, parasites and bacteria have caused over the years tremendous economic losses in the aquaculture industry in various parts of the world. Farmed aquatic species are susceptible to a wide range of pathogens (bacteria, virus, parasites and fungus) responsible for disease and losses with significant impact on the quality and volume of production
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throughout the world [206]. In this section we will mainly focus on pathogen infections and diseases. Pollutants and contaminants are also detrimental to a healthy aquaculture industry. An effective health management program in aquaculture industry should cover several steps, from up-date information on health status, reducing exposure and spread control of pathogens and management of drugs or chemicals [206]. A rapid and accurate diagnosis of disease is then essential for an effective outbreak control. Pathogen detection techniques have been widely used in this industry and can be achieved using a variety of methods traditional (bacteriology, virology, parasitology and mycology culture and biochemical identification), immunological (FAT (fluorescent antibody technique), IFAT (indirect fluorescent antibody technique), IHC (immunohistochemistry), ELISA (enzymelinked immunosorbent assay), dot blot/western blot, molecular methods (PCR (polymerase chain reaction), serology, lateral-flow immunoassays, LAMP (Loop-mediated isothermal amplification) and multiplex technologies like Luminex xMAP, Multiplex-PCR assays and Micro-arrays) [134]. Vaccination has also been a major research area for the impact it may have on disease prevention in aquaculture. Presently, vaccines are already available against bacterial and some viral diseases [207209]. In this context, proteomics technologies can play a major role in aquaculture species health, since it can assist both in the development of new vaccines and diagnosis of disease. A few research papers have been published recently with promising results. A recent study on three of the major fish species of mariculture in southeast Asia describes the isolation of a pathogenic iridovirus and proteomics analysis of its envelope proteins [37]. This virus is responsible from 30% (adult fish) to 100% (fry) mortality in cultured grouper and has the ability of inducing an advanced cytopathic effect in a grouper cell line (GP). Several envelop proteins were identified with protein VP088 further characterized with the aim to better understand the molecular mechanisms of iridoviral pathogenesis and disease prevention in marine aquaculture industry. Dumetz in his 2008 paper [39] reveals a number of novel candidate proteins for developing vaccines against flavobacteriosis infection in aquaculture, using 2-DE and LCMS/MS techniques. In bivalves, the majority of studies on its interaction molecular mechanisms with bacteria are mainly on mRNA and recombinant protein levels. In 2011 a pioneer study by Huan focused on the anti-Vibrio immune response of Zhikong scallop (Chlamys farreri) through proteomics techniques [210]. Identified proteins include some immunorelated proteins, metabolism enzymes and new molecules not pinpointed in previous immunity studies in C. farreri. The results also indicated that molecular chaperons and the antioxidant system are key elements in the anti-Vibrio immune response of hepatopancreas of C. farreri. Also in bivalves, a proteomics approach based in the twodimensional gel electrophoresis and mass spectrometry was used to study the effects of the exposure of two bivalve species, Mytilus galloprovincialis and Corbicula fluminea, to CYN producing (CYN+) and non-producing (CYN) Cylindrospermopsis raciborskii cells [74]. Results obtained suggest the induction of 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 defense mechanisms. Regarding contaminants, proteomics has also been shown to be an effective tool to evaluate its toxic effects in aquaculture species, as already mentioned in this paper. All these works aimed not only to further understand the mechanisms of action, but also the identification of potential protein biomarkers for contaminant exposure. A summary of laboratory controlled and field/caged studies that have utilized proteomics techniques in toxicological studies in Teleost fish and aquatic invertebrate species is available in the review paper by Sanchez et al. [104].
7.
Quality and safety of produced fish
The early years of this century saw the first publications using proteomics as an application to assess food quality. The future potential for proteomics within food quality and safety areas such as technological quality, allergy prevention, bioactives, bioavailability, and nutrition prediction of endproduct quality, was pinpointed in a review by Cabonaro in 2004 [211]. In a very recent review by D'Alessandro and Zolla [212], the safety concept has been considered to encompass not only food safety per se, but also traceability (food origin) and food quality, the last of which these authors more or less regard as identical with food composition and nutritional value. However, in the present paper, quality is considered as eating quality and technological quality (suitability for certain products, production processes and types of storage conditions). A central objective in fish quality research is, therefore, to obtain an understanding of the involved mechanisms when quality changes. This can be due to either ante mortem biological aspect or to post mortem conditions, individually or in combination. The influence on quality by pre-harvest parameters as feed composition [213216], temperature [217], stress [218], slaughter method [219] and genetics [220], as well as post mortem parameters as processing [221], rigor mortis [222], and storage temperature and time [223], is well established. However, the use of proteomics to gather knowledge at a molecular level of the involved mechanisms responsible for a certain quality in aquaculture species is only in the making, with only a few published studies. Although some studies have investigated the influence of fish feed composition on the proteome of fish muscle or liver only a single proteomics study focusing directly on fish diet and quality have been performed. In a combined proteomics and transcriptomics approach on rainbow trout (Oncorhynchus mykiss) liver, it was shown that changes in liver metabolism are likely to manage fat allocation in the muscle of fish fed a high energy/high fat diet [220]. Proteins involved in intracellular lipid transport, the respiratory chain, glycolysis/ gluconeogenesis and amino-acid metabolism were expressed in lower levels in fish fed the high energy diet compared to fish fed a low energy diet. The found proteins could be used as quality markers to prevent excess muscle fat accumulation [220].
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The detrimental impact of pre-harvest stress on quality of fish muscle is well documented and the interest in revealing the underlying mechanisms of the involved physiological responses to different stressors has therefore constituted basis for a number of proteomics studies. A common stressor in aquaculture is crowding at different fish densities for different periods of time (e.g. when fish are gathered in order to be transported and/or slaughtered). Using 2-DE Morzel et al. [180] characterised the modification of protein expression in rainbow trout (Oncorhynchus mykiss) white muscle as induced by a pre-slaughter crowding (>50 kg/m3 for 15 min). In samples taken 45 min post-mortem they found 29 protein spots differentially expressed in groups of crowded and un-crowded fish. The identified spots were mainly proteins from energy-producing pathways indicating a rapid increase in energy demand or structural proteins [180]. These findings matches what could be expected, because a main quality-related effect found due to crowding is an earlier onset of rigor that is closely related to both energy metabolism and structural proteins. That proteins involved in energy metabolism changed in expression as an effect of crowding was also found in Atlantic salmon by Veiset-Kent et al. [224]. They investigated the effect of a more extensive crowding (>200 kg/m3 for 40 min) on muscle and blood plasma proteomes of Atlantic salmon in order to identify possible links to fillet quality attributes. Specifically proteins as enolase 3 and phosphoglycerate kinase 1 involved in glycolysis were found to be up-regulated in muscle from the crowded salmon. As such this is in accordance with the findings by Alves et al. [29] who found down-regulation of gylcolytic enzymes in gilthead seabream (Sparus aurata) liver due to crowding (~50 kg/m3) and ascribed this to an effect of stress activated liver glycogenolysis and gluconeogenesis supplying glucose to other organs with an increased demand. To establish a methodological approach to monitor the quality of the edible parts of fish Monti et al. [225] investigated protein expression changes induced by aquaculture. Using SDS-PAGE, in situ protein hydrolysis, de novo sequencing of peptides by MALDI and ESI-MS/MS, protein identification and relative quantitation of protein by denaturing capillary electrophoresis, they compared water-soluble muscle proteins from edible parts of wild and farmed sea bass (Dicentrarchus labrax). They found that in aquaculture fish the expression of enzymes involved in carbohydrate metabolism were over-expressed, whereas among others the expression of the major fish allergen, parvalbumin, was reduced by 22%. The obtained differences were by the authors ascribed to differences in growth conditions, feeding strategies or to both [225]. However, in contrast to this, others found that farming of sea bream (Sparus aurata) in offshore floating cages (mariculture) enabled production of fish comparable to wild fish, with no differences in muscle protein expression detected between the two groups [176]. Muscle texture is one of the very important quality aspects of fish that are tightly connected to proteins. Texture changes during chilled as well as frozen storage, and the biochemical processes involved in this post-mortem tenderization have been extensively studied during many years. It is generally accepted knowledge that different proteolytic systems are
involved in this process, and also several of the structural proteins being affected as substrates for these enzymes are known. However, many important details remain to be discovered, and a better and deeper understanding of the quality related post-mortem processes during storage might be revealed using proteomics. Although the poly unsaturated fatty acids of fish are the main target for deteriorative oxidation during storage, proteins are oxidised as well, potentially causing protein aggregation resulting in decreased protein solubility with implications on the textural quality [226,227]. All reactive oxygen species (ROS) that react with proteins forms carbonyls and this can be used analytically. In a 2-DE approach with immunoblotting of 2,4-dinitrophenyl labelled protein carbonyl groups it was shown that not proteins in general but specific proteins, especially myofibrillar proteins, were oxidised in rainbow trout during tainting [228]. Others have used fluorescein-S-thiosemicarbazide to tag carbonyl groups in similar approaches [229]. The complexity of specific protein oxidation during frozen storage of rainbow trout was characterized by Kjaersgard et al. [230]. They found that although the overall protein carbonylation level was increased at high frozen storage temperatures the carbonylation pattern was more or less the same irrespective of storage temperature. Many abundant proteins were found to be carbonylated but some less abundant proteins such as adenylate kinase were rather heavily oxidized indicating differentially susceptibility of the proteins to carbonylation [230]. A more extensive insight into changes in protein profiles in sea bass due to protein degradation during chilled storage was sought by Terova et al. [231] in a study where effects of storage temperature were investigated. As expected, they found the greatest alteration in muscle protein pattern (composition) at the highest storage temperature with clear degradation of particularly myosin heavy chain and glyceraldehydes-3phosphate dehydrogenase. However, also at a low chilling temperature (1 C) several proteins changed in abundance during storage, although to a lesser extent. Additionally they found that nucleoside diphosphate kinase B decreased in abundance during storage but in a temperature independent manner. This actually supported an earlier finding from a 2DE study by Verrez-Bagnis et al. [232] of a gradual disappearance during storage of a 16 kDa (pI 6.67) protein suitable as a freshness marker. A proteomics driven understanding of quality could lead to the inclusion of quality as a breeding parameter together with the parameters health, growth, and feed conversion which are primarily used today. The perspective in this respect is to find correlations between protein expression in a tissue and the eating or technological quality of this tissue. This would open up for a biomarker guided selective breeding directed towards a better and less variable quality. Moreover, when the quality related biomarkers are combined with biomarkers relating to production parameters such as diet, feeding regime, temperature and handling procedures (stressors) it constitutes a strong tool for design of fish having certain product specific properties. No investigations have been published yet in which proteomics and eating quality measured in detailed assessments by sensory taste panels are compared directly. However, the presence of a multivariate correlation between
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certain protein expression profiles from 2-DE of shrimp muscle 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 and accurate identification of bacterial species and Bohme et al. [234] demonstrated that matrix-assisted laser desorption ionization-time of flight mass spectrometry is a feasible and cost-effective technique for this. By comparison of unknown spectra to reference spectra from food-borne pathogens and spoilage bacteria (fingerprinting), 26 bacterial strains isolated from fresh fish and seafood products that have been processed (vacuum packed and mild heat treatment) were identified. From a health point of view food allergy is an important issue and knowing that fish and crustacean (including aquaculture species) are among the eight foods that have been identified as causing the majority of food allergenic reactions, pinpoints the need for suitable analytical techniques for the detection and measurement of the involved allergens. LC/MS has by now shown its high potential within this area in general [235]. Another safety aspect is food authentication where proteomic based approaches have already shown their strengths within the seafood area (see reviews [51,236,237]). Recently efforts have been done to increase speed and capability of the analyses to differentiate between closely related species. To distinguish between seven closely related shrimp species, Ortea et al. [238] used a high intensity focused ultrasound technique to get a very fast trypsin digestion of sarcoplasma proteins during sample preparation. This was followed by a shotgun proteomic approach combining LC separation and peptide identification by selected MS/MS ion monitoring. Using this sensitive procedure, they were able to complete an unambiguous identification of a shrimp product within 90 min [238].
[239]. One of the important outcomes is the identification of novel genes that are missed by gene prediction programs. This can be a powerful approach more routinely employed in a near future. Also the use of new technologies like MALDI imaging or protein array/protein chip approaches will greatly contribute to a better understanding of the biological processes in aquaculture species. As a final remark, we propose that a major challenge still exists, which is to annotate the proteomes of species commonly used in aquaculture. We consider this a major gap in proteomics studies, not only in aquaculture related research, but also in all the other areas using proteomics. However, we also have to keep in mind that in the mid-short term the use of de novo sequencing approaches might overcome this issue.
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8.
Concluding remarks and outlook
This review illustrates the growing importance of proteomics technologies in aquaculture. Even with limited genomic information to date, the studies carried out so far clearly demonstrate the potential of proteomics to identify important proteins related to topics as welfare, nutrition, health, quality or safety, and unravel potential biomarkers and mechanisms related to aquaculture species biology. Proteomics has undoubtedly shown to provide a holistic measure of an organism response, in this particular case an aquaculture species, to an external stimulus. Nevertheless, proteomics has their own limitations and an integration of this technology, together with others like transcriptomics or metabolomics, will definitely be the direction of future research in the area, since a wider vision as well as a validation of results can be obtained in this manner. We can foresee in this way that areas like proteogenomics might become a powerful approach in identifying protein biomarkers and to study genome variation. Proteogenomics refers to the correlation of the proteomic data with the genomic and transcriptomic data with the goal of enhancing the understanding of the genome
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PROTEOMICS in aquaculture: Applications and trends

Quality & Safety

Fig. 1 Omics in aquaculture: target research topics and species.

2. Aquaculture: key features, challenges and development

3. Aquaculture species and fish model organisms

Aquaculture species Vertebrates

Protein separation and quantification

Gel based approach Sample preparation

Protein identification and characterization

Identifying proteins from peptides

Gel free approach

Challenges and new technologies

Fish welfare in aquaculture

Nutrition and health in aquaculture

Quality and safety of produced fish

Concluding remarks and outlook

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