**2. Conventional aquaculture studies with** *Solea senegalensis*

### **2.1 Early studies**

Studies of sole aquaculture began in Faro (S Portugal) and Cádiz (SW Spain) to produce good quality larvae and juveniles (Dinis et al., 1999). Broodstock spawning studies established optimal feeding regimes by combining squid (*Loligo vulgaris*) and polychaetes (*Hediste diversicolor*) at the final maturation stages. Spawning was studied in terms of temperature (stopped <16 ºC), duration (4-6 months), egg fertilisation rate (20-100%) and viable egg rate (72%). Larvae hatch at 2.4 mm and accept *Artemia nauplii* as the first prey two days after hatching (DAH). Metamorphosis spans from 11 to 19 DAH, at which point the fish are fed live *Artemia metanauplii*. They reach 16 mm at 40 DAH and 35 cm/450 g after 1 year, with 8% survival. Pasteurellosis can cause pigmentation abnormalities and malformations associated with eye migration and can progress to death (Dinis et al., 1999).

The potential of sole for aquaculture was reviewed some time later (Cañavate, 2005). Although important progress in reproduction techniques was reached, much basic knowledge remained lacking. Ongrowth was successfully carried out, but progress was limited by opportunistic diseases due to suboptimal rearing conditions resulting in an inability of the sole to achieve an adequate physiological status for resistance. Growth, survival and pigmentation were studied during sole growth in tanks with three bottom types (Rodiles et al., 2005). The final length and weight was similar in the sand, white and dark conditions, but different pigmentation patterns appeared on the sand (clear, dark) and white bottoms (clear, brown, dark). The homogeneous dark pattern, preferred by markets, is only obtained in tanks with a dark bottom. A lower survival rate was found on sand bottoms due to pathologies derived from the difficulties in maintaining the sand bed.

#### **2.2 Organ development and reproductive studies**

Digestive tract development was studied in larvae until 30 DAH, which involved the assessment of histology, digestive enzymes, lipids, proteins and carbohydrates in the buccopharyngeal cavity, oesophagus, early stomach, anterior and posterior intestine, pancreas and liver (Ribeiro et al.*,* 1999a). The digestive tract elongates in metamorphosis, increasing absorption. Phosphatases, lipase and aminopeptidase have been detected starting at 2 DAH and the levels increase during development. Proteins abound in the intestinal epithelium and exocrine pancreas, and neutral lipids are found at the yolk sac intestinal epithelium and liver. After 31 DAH larvae ingest, digest and absorb nutrients because they now have a complete digestive tract. A time course of pancreatic and intestinal enzymes was studied in larvae until 31 DAH (Ribeiro et al., 1999b). Digestive enzymes increase until 10 DAH then decrease until 18 DAH, a pattern typical of developing animals. Alkaline phosphatase abounds from 21-27 DAH, during the development of brush border membranes, with a parallel decrease in the cytosolic enzyme, Leu–Ala peptidase.

Thyroid development was studied in sole larvae by histo- and immunohistochemistry to synchronise larval development and improve fish production (Ortiz-Delgado et al.*,* 2006). The first follicle is visible by the first feeding; increases during metamorphosis and has adult characteristics by 30 DAH. Thyroid hormones decrease to undetectable levels at yolk-sac reabsorption. T3 and T4 are detected by 6 DAH and increase during metamorphosis.

Seasonal profiles of sex steroids –17β-estradiol (17β-E), testosterone (T), 11-ketotestosterone (11-KT), and 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P)– were studied in *S. senegalensis* in an attempt to achieve steroid-induced maturation (García-López et al.*,* 2006a). Females have six maturation stages, as follows: early, intermediate and final ovarian development, then partially, mid and spawned out. By summer´s end, a new gonadal cycle starts, as demonstrated by increased reproductive parameters. By mid-autumn some females reach advanced maturation stages, which coincide with a peak of running males. By the start of spring, ovarian development reaches its peak, and plasma steroid levels are maximal at the start of the spawning period, which occurs from March to June. In parallel with oocyte and sperm release, the proportion of spawned out fish and non-running males increases, and steroid levels decline. The high levels of 17,20β-P during spawning make it a candidate for a maturation-inducing steroid.

malformations associated with eye migration and can progress to death (Dinis et al.,

The potential of sole for aquaculture was reviewed some time later (Cañavate, 2005). Although important progress in reproduction techniques was reached, much basic knowledge remained lacking. Ongrowth was successfully carried out, but progress was limited by opportunistic diseases due to suboptimal rearing conditions resulting in an inability of the sole to achieve an adequate physiological status for resistance. Growth, survival and pigmentation were studied during sole growth in tanks with three bottom types (Rodiles et al., 2005). The final length and weight was similar in the sand, white and dark conditions, but different pigmentation patterns appeared on the sand (clear, dark) and white bottoms (clear, brown, dark). The homogeneous dark pattern, preferred by markets, is only obtained in tanks with a dark bottom. A lower survival rate was found on sand bottoms due to pathologies derived from the difficulties in maintaining the sand bed.

Digestive tract development was studied in larvae until 30 DAH, which involved the assessment of histology, digestive enzymes, lipids, proteins and carbohydrates in the buccopharyngeal cavity, oesophagus, early stomach, anterior and posterior intestine, pancreas and liver (Ribeiro et al.*,* 1999a). The digestive tract elongates in metamorphosis, increasing absorption. Phosphatases, lipase and aminopeptidase have been detected starting at 2 DAH and the levels increase during development. Proteins abound in the intestinal epithelium and exocrine pancreas, and neutral lipids are found at the yolk sac intestinal epithelium and liver. After 31 DAH larvae ingest, digest and absorb nutrients because they now have a complete digestive tract. A time course of pancreatic and intestinal enzymes was studied in larvae until 31 DAH (Ribeiro et al., 1999b). Digestive enzymes increase until 10 DAH then decrease until 18 DAH, a pattern typical of developing animals. Alkaline phosphatase abounds from 21-27 DAH, during the development of brush border

membranes, with a parallel decrease in the cytosolic enzyme, Leu–Ala peptidase.

reabsorption. T3 and T4 are detected by 6 DAH and increase during metamorphosis.

Thyroid development was studied in sole larvae by histo- and immunohistochemistry to synchronise larval development and improve fish production (Ortiz-Delgado et al.*,* 2006). The first follicle is visible by the first feeding; increases during metamorphosis and has adult characteristics by 30 DAH. Thyroid hormones decrease to undetectable levels at yolk-sac

Seasonal profiles of sex steroids –17β-estradiol (17β-E), testosterone (T), 11-ketotestosterone (11-KT), and 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P)– were studied in *S. senegalensis* in an attempt to achieve steroid-induced maturation (García-López et al.*,* 2006a). Females have six maturation stages, as follows: early, intermediate and final ovarian development, then partially, mid and spawned out. By summer´s end, a new gonadal cycle starts, as demonstrated by increased reproductive parameters. By mid-autumn some females reach advanced maturation stages, which coincide with a peak of running males. By the start of spring, ovarian development reaches its peak, and plasma steroid levels are maximal at the start of the spawning period, which occurs from March to June. In parallel with oocyte and sperm release, the proportion of spawned out fish and non-running males increases, and steroid levels decline. The high levels of 17,20β-P during spawning make it a candidate for a

**2.2 Organ development and reproductive studies** 

maturation-inducing steroid.

1999).

Testicular development was also studied (García-López et al.*,* 2006b). The spermatogenetic cycle consists of the following five stages: early (I), mid (II), and late (III) spermatogenesis, maturation (IV), and recovery (V). In the summer, stage I and V testes are found with low values of sex steroids and IG (gonadosomatic index). Recrudescence begins in autumn, with an initial increase of IG 11-KT and T and the appearance of stage II and III testes. In the winter, 11-KT and T peak and soon decrease, and IG slightly declines. In the spring, 11-KT and T decline further, while IG slightly increases and running males peak with stage IV testes. Sperm production and quality was assessed in wild-captured and F1 broodstock fish (Cabrita et al.*,* 2006). Males produce motile sperm from February to November, with specific peaks of high spermiation and fluent males. Sperm volume and cell density is lower in F1 males than in wild-captured broodstock.

Ovarian development was also studied (García-López et al.*,* 2007). In the autumn/winter, oocytes progress to vitellogenic stages in parallel with high levels of K (condition factor), IG, and plasma 17β-E and T. In the late winter/early spring, development is maximal, with females at intermediate and final maturation and K, IG, 17β-E and T peaking. Steroid levels are lower in cultured sole than in naturally spawning females, leading to atresia and lack of oocyte maturation, thus reducing ovary size with declining K, IG, and 17β-El and T levels and many perinucleolar oocytes. The amount of circulating 17,20β-P, the putative maturation-inducing steroid, remains near constant through the period, suggesting that oocytes are unresponsive to its stimulation.

Skeletal development and malformations are a bottleneck in sole aquaculture. Maturation and abnormalities of the vertebral column and caudal skeleton have been studied in sole (Gavaia et al.*,* 2002). Different defects are found in the caudal complex and the vertebral column, and 44% of fish show at least one defect. While the causes are unknown, their high incidence may reflect rearing and/or feeding problems. The tissue distribution and evolution of bone Gla (Bgp) and matrix Gla proteins (Mgp) and Ca2+ deposition were studied in zebrafish during larval development and in adult tissues as well as sole metamorphosis (Gavaia et al.*,* 2006). In zebrafish, Bpg and Mpg accumulate mainly in the matrix of skeletal structures already calcified or under calcification. In sole metamorphosis, Bpg and Mpg increase in parallel to the calcification of the axial skeleton. In both species, Mpg also accumulates in non-mineralised vessel walls.

#### **2.3 Nutrition studies**

Studies on the requirements, catabolism and assimilation of amino acids (AAs) were carried out in early larval, metamorphic and post-larval sole. Initial studies on indispensable (IAA) and dispensable (DAA) amino acids (Rønnestad et al.*,* 2001), showed that sole assimilated most (85%) of the dietary IAAs and catabolised most of the DAAs. Such results were confirmed after studying the bioavailability of several AAs in larvae (Conceição et al.*,* 2003). The demand and availability of AAs and proteins in relation to digestive capacity were reviewed, and AAs sources were described, highlighting the regulatory role of cholecystokinin and peristaltic activity (Rønnestad et al.*,* 2003). A balanced AA profile improved amino acid assimilation in post-larval sole (Aragão et al.*,* 2004a). Changes in AA requirements and dietary imbalances were studied in *Sparus aurata* and *S. senegalensis* (Aragão et al.*,* 2004b); the AA profiles of both changed during ontogeny, especially in sole due to its marked metamorphosis. AA imbalances were found during development. In both species, Phe/Tyr addition was studied to assess the effects on metamorphosis after their conversion into thyroid hormones (Pinto et al.*,* 2010). While Phe did not affect sole metamorphosis, dietary Tyr increased the production of thyroid hormones, which was beneficial for sole metamorphosis.

The nutritional physiology of sole development was studied to optimise diets and understand limiting factors in weaning (Conceiçao et al., 2007). Larvae have a high capacity to digest live prey, even at the early stages. Use of inert microdiets in co-feeding with *Artemia* resulted in the development of intestinal activity and enhanced survival, although it was also accompanied by low growth and high size dispersal. Fatty acid absorption increases with their degree of unsaturation, and larvae spare DHE from catabolism. Rotifers and *Artemia* are deficient in one or more AAs, such as His, Lys, Arg, Thr, or those containing sulphur and aromatic rings, depending on the larval stage; balancing the dietary AA profile with dipeptides increases retention and decreases catabolism in *Artemia*-fed larvae.

The effects of non-protein energy levels on growth and oxidative status were studied in sole fed diets with 4 energy levels (Rueda-Jasso et al.*,* 2004). Cellular energy allocation showed differences in liver, but not in muscle. TBARS were higher in fish fed a diet with high lipid content, in parallel to high CAT and SOD activity. Yet, the protein source or energy levels had no major impact on sole growth, nutrient utilisation or fatty acid composition (Valente et al.*,* 2011). Quantitative lipid imbalances and a low protein/neutral lipid ratio increased the accumulation of lipid droplets in the enterocytes and lowered fatty acid absorption in larvae (Morais et al.*,* 2005). The effects of a neutral lipid level and source were studied in marine fish larvae (Morais et al.*,* 2007). A growth-depressing effect of high neutral lipids, as assessed by lower digestive enzyme activity, absorption and/or food intake was reported. In larvae, lipid transport from enterocytes to the body is more critical than lipolytic activities. Phospholipid digestion is more efficient than that of neutral lipids, whose excess leads to the accumulation of large lipid droplets in the enterocytes, reducing fatty acid absorption and growth.

The feed transit, protein and energy digestibility of practical feed were assessed in sole (Dias et al.*,* 2010). Protein digestibility is high for fishmeal and corn gluten, intermediate for soybean meal, and moderate for wheat meal. Energy digestibility varies from 88 to 93% for soybean meal, corn gluten and anchovy fishmeal and is 73% in wheat meal. Thus, flatfish, despite its high dietary protein requirement, digest vegetable ingredients quite well, suggesting that the development of practical feeds with high levels of plant-protein sources would be beneficial.

#### **2.4 Conventional biomarker studies in** *S. senegalensis* **aquaculture**

Nearly 15 years after the studies of the UCO group, the use of biomarkers to follow fish physiology and pollution effects has become popular and is now applied by most groups. Antioxidant enzymes, stress proteins, lipid peroxides and histology were studied in sole larvae (Fernández-Díaz et al.*,* 2006) fed on the following 3 diets: live *Artemia nauplii*, microcapsules, and vitamin A-supplemented microcapsules. Live-fed larvae grow larger and undergo faster metamorphosis than microcapsule-fed larvae, although all groups have near 80% survival. Vitamin A improves the growth and development compared to an inert diet. *Artemia*-fed larvae have organs with normal development, but histological alterations

species, Phe/Tyr addition was studied to assess the effects on metamorphosis after their conversion into thyroid hormones (Pinto et al.*,* 2010). While Phe did not affect sole metamorphosis, dietary Tyr increased the production of thyroid hormones, which was

The nutritional physiology of sole development was studied to optimise diets and understand limiting factors in weaning (Conceiçao et al., 2007). Larvae have a high capacity to digest live prey, even at the early stages. Use of inert microdiets in co-feeding with *Artemia* resulted in the development of intestinal activity and enhanced survival, although it was also accompanied by low growth and high size dispersal. Fatty acid absorption increases with their degree of unsaturation, and larvae spare DHE from catabolism. Rotifers and *Artemia* are deficient in one or more AAs, such as His, Lys, Arg, Thr, or those containing sulphur and aromatic rings, depending on the larval stage; balancing the dietary AA profile

The effects of non-protein energy levels on growth and oxidative status were studied in sole fed diets with 4 energy levels (Rueda-Jasso et al.*,* 2004). Cellular energy allocation showed differences in liver, but not in muscle. TBARS were higher in fish fed a diet with high lipid content, in parallel to high CAT and SOD activity. Yet, the protein source or energy levels had no major impact on sole growth, nutrient utilisation or fatty acid composition (Valente et al.*,* 2011). Quantitative lipid imbalances and a low protein/neutral lipid ratio increased the accumulation of lipid droplets in the enterocytes and lowered fatty acid absorption in larvae (Morais et al.*,* 2005). The effects of a neutral lipid level and source were studied in marine fish larvae (Morais et al.*,* 2007). A growth-depressing effect of high neutral lipids, as assessed by lower digestive enzyme activity, absorption and/or food intake was reported. In larvae, lipid transport from enterocytes to the body is more critical than lipolytic activities. Phospholipid digestion is more efficient than that of neutral lipids, whose excess leads to the accumulation of large lipid droplets in the enterocytes, reducing fatty acid

The feed transit, protein and energy digestibility of practical feed were assessed in sole (Dias et al.*,* 2010). Protein digestibility is high for fishmeal and corn gluten, intermediate for soybean meal, and moderate for wheat meal. Energy digestibility varies from 88 to 93% for soybean meal, corn gluten and anchovy fishmeal and is 73% in wheat meal. Thus, flatfish, despite its high dietary protein requirement, digest vegetable ingredients quite well, suggesting that the development of practical feeds with high levels of plant-protein sources

Nearly 15 years after the studies of the UCO group, the use of biomarkers to follow fish physiology and pollution effects has become popular and is now applied by most groups. Antioxidant enzymes, stress proteins, lipid peroxides and histology were studied in sole larvae (Fernández-Díaz et al.*,* 2006) fed on the following 3 diets: live *Artemia nauplii*, microcapsules, and vitamin A-supplemented microcapsules. Live-fed larvae grow larger and undergo faster metamorphosis than microcapsule-fed larvae, although all groups have near 80% survival. Vitamin A improves the growth and development compared to an inert diet. *Artemia*-fed larvae have organs with normal development, but histological alterations

**2.4 Conventional biomarker studies in** *S. senegalensis* **aquaculture** 

with dipeptides increases retention and decreases catabolism in *Artemia*-fed larvae.

beneficial for sole metamorphosis.

absorption and growth.

would be beneficial.

are seen in larvae fed an inert diet. Catalase (CAT), superoxide dismutase (SOD), total GSHperoxidase (t-GPX), lipid peroxides (MDA) and stress proteins (HSP70, not HSP60) are dietand age-dependent. Inert diet-fed larvae have similar biomarker responses, but different (p<0.05) from *Artemia*-fed larvae. Higher antioxidant defences are attributed to the start of metamorphosis and the use of inert food.

A similar approach was used by Cañavate et al. (2007) to assess the effect of light on the development of sole larvae with or without adding -carotene-rich *Dunaliella salina*. SOD, CAT, t-GPX and MDA were used as biomarkers. Growth and survival after metamorphosis were unaffected by light or *D. salina*. Light affects CAT and t-GPX throughout development but does not affect MDA, and SOD is only affected in metamorphosis. *D. salina* does not affect SOD, CAT or t-GPX and no interaction with light intensity was found. MDA lowers significantly only when *D. salina* is added, and its effect was found only in metamorphosing larvae, whose MDA levels are much higher than in earlier stages. These results confirm the antiperoxidative effect of -carotene from live algae in the larval rearing process.

#### **2.5 The Pleurogene project**

After the initial studies on the growth and reproduction of the Senegalese sole, "Genoma España" and "Genome Canada" promoted the "Pleurogene" project, in order to develop new technology to assess gene and protein expression during the reproduction and breeding of two flatfish, Senegalese sole and Atlantic halibut. The project [http://www.genes.org] aimed to improve basic knowledge of reproduction, larval development and survival, and had de following objectives: 1) Establishment of an EST database and shotgun proteome analysis; 2) Construction of a microarray for high-throughput analysis of gene expression; 3) Construction of genetic linkage map; 4) Development of methods for gene expression profiling through laser capture microdissection RNA; 5) Identification of changes in gene expression during gamete development and maturation; 6) Genomic analysis of sex determination and differentiation; 7) Determination of the pattern of gene expression during larval metamorphosis, the ontogeny of the gastrointestinal tract and the effects of dietary treatments; and 8) Development of E-mold, an integrative bioinformatics platform for genomic, proteomic and morphological information from flatfish.

The Pleurogene project developed genomic and proteomic tools to help achieve these goals (Douglas et al., 2007; Cerdà et al., 2008), and also had the following major research results: 1) Development of genomics tools for the Senegalese sole, including 10 different cDNA libraries from adult and larvae tissues and 1 normalised multi-tissue library, 10,300 new sole EST sequences and nearly 500 peptides, and a sole oligonucleotide microarray with probes to detect 4550 different RNAs; 2) Development of a hormone treatment to increase sperm motility in sole; 3) Generation of a sole genetic linkage map; 4) Development of a progeny test or paternity kit; 5) Development of indirect approaches for sex control; 6) Production of recombinant gonadotropin hormone; 7) Generation of gene and protein expression maps associated to larval development, metamorphosis, and nutrition; 8) Generation of gene and protein expression maps of testes producing high quality sperm; 9) Generation of gene expression maps for sexual differentiation and maturation; and 10) Development of the "Solea-mold" that allows for new data to be included, *in silico* experiments to be performed, and comparative studies to be undertaken.
