**4.3 Real-time qRT-PCR**

Although the determination of absolute transcript abundance (the exact number of molecules of a transcript in all samples of the study) is the only adequate procedure to accurately assess the expression of a gene (Prieto-Álamo et al., 2003), these kinds of studies are infrequent due to the experimental difficulties that impair this type of analysis. A set of more than 20 sole transcripts was selected for the quantification of their levels by this rigorous approach (Table 6) (Prieto-Álamo et al., 2009; Osuna-Jiménez et al., 2009), based on the physiological relevance of their products and on the magnitude and specificity of their responses.

#### *Immune Response*

complement component C3, complement component C7, transferrin, haptoglobin, ferritin M, natural killer enhancing factor, tumor necrosis factor alpha-induced protein 9, hepcidin, nonspecific cytotoxic cell receptor protein-1, angiotensinogen, sequestosome 1, tumor necrosis factor receptor associated factor

*Response to stress* 

CCAAT/enhancer binding protein, cold inducible RNA binding protein, DNA-damageinducible transcript 4-like, NHP2 non-histone chromosome protein

*Energetic Metabolism* 

glyceraldehyde-3P-DHase, transketolase, NADH-DHase 1 alpha subcomplex 4

*Protein Synthesis, Folding and Degradation* 

asparaginil-tRNA synthetase, heat shock protein GP96, proteasome 26S non-ATPase subunit 3, cathepsin Z

*Transport* 

α-globin

Table 6. Biological functions of proteins encoded by the sole transcripts selected to be validated by real-time qRT-PCR (Prieto-Álamo et al., 2009; Osuna-Jiménez et al., 2009).

The absolute quantification of the selected transcripts was not limited to the samples used for global analysis, but rather extended to samples coming from different individuals, organs, treatments and exposure times. Consequently, this gene-to-gene analysis provided valuable additional information on transcriptional expression patterns of the selected genes. Individual quantification is also mandatory to prevent biased interpretations of specimens with abnormal expression levels. The absolute real-time qRT-PCR on individual samples demonstrated that inter-individual variations of most examined transcripts in treated animals was in the range of that in control fish, indicating similar susceptibility to LPS or CuSO4 challenge among individuals. The qRT-PCR quantifications confirm, in general, the results obtained with the subtractive libraries and the DNA microarrays. As expected, substantial differences in abundance were found depending on the transcript and tissue examined. In general, each transcript displayed a characteristic expression profile, distinguishing between constitutive or up-/down-regulated, early or late responsive, stressor-specific or not, etc., as a function of the organ analysed.

### **5. Proteomics studies of GBD**

376 Aquaculture

Consequently, the GENIPOL platform was used to assess the response to LPS in the headkidney. After 24 h of LPS treatment, a total of 224 genes was statistically differentially expressed in the head-kidney (117 up-regulated and 107 down-regulated). The functional analysis of the results revealed that the biological processes altered by LPS treatment in the head-kidney were very similar to those detected in the liver. Most notably amongst the up-regulated genes were the functional groups immune response, unfolded proteinbinding, intracellular transport/secretion and proteasome, and digestive enzymes in the list of down-regulated genes. In contrast, the glutathione transferases category, whose transcript levels were down-regulated by LPS in the liver, was not affected in the head-

Although the determination of absolute transcript abundance (the exact number of molecules of a transcript in all samples of the study) is the only adequate procedure to accurately assess the expression of a gene (Prieto-Álamo et al., 2003), these kinds of studies are infrequent due to the experimental difficulties that impair this type of analysis. A set of more than 20 sole transcripts was selected for the quantification of their levels by this rigorous approach (Table 6) (Prieto-Álamo et al., 2009; Osuna-Jiménez et al., 2009), based on the physiological relevance of their products and on the magnitude and specificity of their

complement component C3, complement component C7, transferrin, haptoglobin, ferritin M, natural killer enhancing factor, tumor necrosis factor alpha-induced protein 9, hepcidin, nonspecific cytotoxic cell receptor protein-1, angiotensinogen, sequestosome 1, tumor necrosis

CCAAT/enhancer binding protein, cold inducible RNA binding protein, DNA-damage-

asparaginil-tRNA synthetase, heat shock protein GP96, proteasome 26S non-ATPase subunit 3,

inducible transcript 4-like, NHP2 non-histone chromosome protein

glyceraldehyde-3P-DHase, transketolase, NADH-DHase 1 alpha subcomplex 4

Table 6. Biological functions of proteins encoded by the sole transcripts selected to be validated by real-time qRT-PCR (Prieto-Álamo et al., 2009; Osuna-Jiménez et al., 2009).

The absolute quantification of the selected transcripts was not limited to the samples used for global analysis, but rather extended to samples coming from different individuals, organs, treatments and exposure times. Consequently, this gene-to-gene analysis provided valuable additional information on transcriptional expression patterns of the selected genes. Individual quantification is also mandatory to prevent biased interpretations of specimens with abnormal expression levels. The absolute real-time qRT-PCR on individual samples

kidney.

responses.

*Immune Response* 

*Response to stress* 

*Energetic Metabolism* 

cathepsin Z

α-globin

*Transport* 

factor receptor associated factor

*Protein Synthesis, Folding and Degradation* 

**4.3 Real-time qRT-PCR** 

Fish reared in earth ponds are eventually affected by Gas Bubble Disease (GBD) outbreaks if ponds are not correctly handled, particularly under high temperature and radiation conditions. GBD is a non-infectious pathology occurring when the partial pressures of atmospheric gases in solution exceed their respective partial pressures in the atmosphere. GBD was initially observed in farmed species, although outbreaks in wild fish, both freshwater and marine animals, have also been reported. GBD can have serious adverse economic repercussions in fish cultures by reducing productivity and the commercial value of the fish as well as the farm profitability. Thus, the development of biomarkers responsive to hyperoxia stress would be a valuable tool to apply in systems where oxygen supersaturation might be possible, and would also contribute to basic knowledge of oxidative stress. Oxygen supplementation is a common practice in intensive fish farming, in order to allow high density cultivation while reducing the amount of water demanded in aquaculture facilities. It is also required during fish transportation. In intensive aquaculture, the use of oxygen is regulated by sophisticated mechanisms to keep its concentration close to desired values. However, in open ponds, the likelihood of oxygen supersaturation conditions is higher, because primary producers are in a high nutrient environment, occasionally combined with high temperature and radiation. Photosynthetic oxygen overproduction is a factor of concern for pond aquaculture, particularly when species such as sole, which exhibit nocturnal and benthic habits, are considered. These features complicate water management compared to pond operation in pelagic fish farming.

Environmental and physicochemical conditions inducing hyperoxia, such as radiation, temperature and dissolved O2, were monitored in two independent land-based ponds of an aquaculture research centre (IFAPA Centro El Toruño, Puerto de Santa María, Cádiz, Spain) in which *S. senegalensis* were reared, after a GBD outbreak was detected in some of these animals (Salas-Leyton et al.*,* 2009). Fig. 2 (upper) shows the appearance of the earth pond used as control (100 m2, water renewed 4-fold/day) and of that in which GBD developed (900 m2, water unrenewed) in which algal blooms can be observed at its border. As shown in Fig. 2 (centre), the dissolved oxygen profile detected in the hyperoxic earth pond was typical of that in environments dominated by macroalgal biomass and high photosynthetic activity, where extreme oxygen levels are reached during a great part of the daily cycle (including night hours), always above saturation without a desaturation phase. As shown in Fig. 2 (lower), the following typical GBD symptoms were detected in fish from this pond: exophthalmia caused by retrobulbar bubbles (A), subcutaneous emphysemas, obstruction of gill lamellas (B), big bubbles located at caudal (C) and dorsal

Fig. 2. Earth ponds, dissolved oxygen monitoring and visible symptoms in GBD-affected soles. (I) The experiments were carried out in two independent land-based ponds, one a 900 m2 rectangular pond where oxygen supersaturation developed spontaneously, named the GBD pond (right) and the other a 100 m2 control pond (left) in which water was renewed four times per day. (II) Dissolved oxygen was simultaneously monitored throughout the daily cycle in both the control (green line) and GBD ponds (red line). (III) Visible symptoms observed in GBD-affected soles included the following: exophthalmia caused by retrobulbar bubbles (A), a bubble obstructing a gill lamella (B), and a large bubble located at caudal fin (C).

Fig. 2. Earth ponds, dissolved oxygen monitoring and visible symptoms in GBD-affected soles. (I) The experiments were carried out in two independent land-based ponds, one a 900 m2 rectangular pond where oxygen supersaturation developed spontaneously, named the GBD pond (right) and the other a 100 m2 control pond (left) in which water was renewed four times per day. (II) Dissolved oxygen was simultaneously monitored throughout the daily cycle in both the control (green line) and GBD ponds (red line). (III) Visible symptoms observed in GBD-affected soles included the following: exophthalmia caused by retrobulbar bubbles (A), a

bubble obstructing a gill lamella (B), and a large bubble located at caudal fin (C).

fins, haemorrhages, anomalous swimming accompanied by loss of orientation, nearlethargy status and individual isolation were the main effects of O2 supersaturation (Salas-Leyton et al., 2009).

Under the described aquaculture conditions, a parallel proteomic study was carried out in search of protein alteration patterns that might be used as potential new and unbiased biomarkers of hyperoxic stress (Fig. 3) (Salas-Leyton et al., 2009). The following three health statuses were studied in sole individuals: (1) healthy control fish, (2) GBD-affected but asymptomatic fish, and (3) GBD-affected fish with visible symptoms. Protein expression profiles were studied by 2-DE in cytosolic fractions of gills and livers. A total of 1,525 and 1,632 spots were detected in the four gill and liver gels, respectively, that were run in each of the three situations studied. Fig. 3 (upper) shows the master gels of cytosolic gill (left) and liver (right) proteins. A total of 205 protein spots were differentially expressed in the gills and 498 in the liver in each health status. Fig. 3 (middle) shows the number of spots which are present only, absent, increased or diminished in each of the studied conditions, and the total number of changes found. A significantly higher number of differentially expressed spots were found in GBD-affected soles, mainly in fish with visible symptoms. Of these, 25 spots in the gills and 23 in the liver were selected for identification using tandem mass spectrometry (nESI-IT MS/MS), *de novo* sequencing and a bioinformatics search. Fig. 3 (lower) shows the percentage of the relative intensity of each spot in each health status. Sequence tags were obtained from 9 (gills) and 5 (liver) of the selected spots, resulting in a total of 14 identified spots in the GBD status, which are indicated in Fig. 3 (lower).

Due to the central role of gills in oxygen exchange, the proteins identified in the gills of GBD-affected fish were, unsurprisingly, related to oxidative alteration of the cytoskeleton structure or function (β-tubulin and β-actin), motility (light myosin chain and αtropomyosin), regulatory pathways (calmodulin, Raf kinase inhibitor protein [RKIP]) and carbohydrate metabolism (glyceraldehyde 3-P dehydrogenase). The hyperoxia-linked effects of these proteins and higher-level responses, related to inflammatory response, apoptosis or cell death, or derived from alterations in the regulatory proteins, have been discussed in depth (Salas-Leyton et al., 2009). In gills, only RKIP was most intense in GBD-affected animals without symptoms, while the other seven identified proteins were most intense in GBD-affected fish with symptoms. Proteins identified in the liver were related to protein oxidative damages (β-globin and fatty acid-binding protein [FABP]), protection from oxidative stress (dicarbonyl/L-xylulose reductase and glycine Nmethyltransferase) and inflammatory response (complement component C3), in agreement with the predominant metabolic role of this organ. In the liver, only FABP was most intense in healthy fish, while the other four identified proteins were most intense in GBD-affected fish with visible symptoms. Approximately 50% of the identified proteins corresponded to "unusual" proteins not often found in proteomics screens, while the rest were preferential targets of oxidative stress. Most of these latter proteins were found as truncated forms of oxidised proteins that might trigger cellular defences against hyperoxygenation preceding GBD outbreaks. Some of the identified proteins might be considered to be good hyperoxia stress biomarkers and could be used in the earlywarning detection of GBD outbreaks. Massive proteomic approaches have been applied for the first time to the study of this non-infectious pathological condition, gas bubble disease, in fish.

Fig. 3. Proteomic study of GBD-affected juvenile soles after 2-DE of cytosolic gill (left) and liver (right) proteins. (I) Master gels. (II) Number of proteins showing distinct protein expression patterns in healthy, GBD-asymptomatic and GBD-symptomatic fish. Bars represent the number of protein spots present only (■), absent (■), increased (■), diminished (■) and the total number of changes (■) found in each of the conditions. (III) Spots selected for possible identification by mass spectrometry analysis. Bars represent the percentage of the relative intensity of each spot in every status: healthy (■), GBD-asymptomatic (■) and GBD-symptomatic (■) animals. Successfully identified proteins are highlighted.

#### **6. Conclusions**

380 Aquaculture

Fig. 3. Proteomic study of GBD-affected juvenile soles after 2-DE of cytosolic gill (left) and liver (right) proteins. (I) Master gels. (II) Number of proteins showing distinct protein expression patterns in healthy, GBD-asymptomatic and GBD-symptomatic fish. Bars represent the number of protein spots present only (■), absent (■), increased (■), diminished (■) and the total number of changes (■) found in each of the conditions. (III) Spots selected for possible identification by mass spectrometry analysis. Bars represent the percentage of the relative intensity of each spot in every status: healthy (■), GBD-asymptomatic (■) and

GBD-symptomatic (■) animals. Successfully identified proteins are highlighted.

Given the economic importance of *S. senegalensis*, an aquaculture species that remains largely unexplored at the genomic level, the application of postgenomic methodologies provides results that may be highly relevant at a genetic, immunological and toxicological level, contributing to the improvement of management and welfare of this organism in aquaculture. The utility of high-throughput proteomic methods for unveiling the molecular basis of a cumbersome disease in aquaculture has been also demonstrated.
