Maria Teresa Dinis

A review on the cultivation potential of Solea senegalensis in Spain and in Portugal

Abstract The sole, Solea senegalensis , is a common high-value flatfish in Southern Europe, commonly reared in extensive aquacultural production in Portugal and Spain. Research in recent years has focused on the production of larvae and juveniles of good quality. Natural spawning of broodstock in captivity has been accomplished, and is the only way viable eggs have been obtained to date. The broodstock feeding regime is based on squid ( Loligo vulgaris ), being supplemented with polychaetes ( Hediste diversicolor ) during final maturation. Temperature plays a very important role in the onset and duration of the spawning period, with egg emission stopping below 16°C. Observed duration of the spawning period has ranged from 4 to 6 months. The total daily egg collection during the spawning season ranged from 0 to 180 g for a broodstock of 15 fish. Egg fertilization rates varied between 20% and 100%, and the percentage of viable eggs had a mean of 72.1±26.5%. Variations in egg size between batches were detected, with egg size tending to decline during the spawning season. Larvae hatch with an average size of 2.4±0.1 mm total length, but this varies widely by batch. Larvae accept Artemia nauplii as first prey and reach a size of 8 mm by day 15. Metamorphosis starts on 11 days after hatching (DAH) and is completed by 19 DAH. Survival at 19 DAH range from 29% to 87%. From 19 DAH onwards fish are fed live Artemia metanauplii, and reach 16±0.8 mm on 40 DAH. Ponds stocked with unweaned juveniles produced after one year fish with 16.6±2.1 cm total length and 40.3±2.5 g wet weight, with a survival of 20%. Unweaned juveniles stocked together with Sparus aurata reached a size of 35.3±1.8 cm total length and 456.1±3.6 g wet weight after one year fish, with a survival of 8%. Epizootic mortalities due to pasteurellosis have been observed in juvenile S. senegalensis . As in other flatfish, pigmentation abnormalities as well as some malformations associated with the migration of the eye have also been observed. The optimization of the weaning and growout procedures, including the development of appropriate feeding regimes, and further studies into the causes of pigmentation abnormalities are necessary before this species can be reared intensively on a large scale.

A review of the culture potential of Solea solea and S. senegalensis

A number of scientific studies have investigated aspects of soles(Solea soleaandS. senegalensis) ecology, population genetics and biology in their natural environment, and the species have been extensively studied in captivity during the last decade. Studies on the genetic population structure of sole indicate that several distinct breeding populations exist within its distributional range in European waters. Recent studies suggest a phylogenetic relatedness ofS. soleaand S. senegalensis, being found as closest sister lineages in most reconstructions. However, studies on molecular genetics and morphological traits give diagnostic differences that consistently lead to their taxonomic separation at the specific rank. Studies show that sole spawn readily in captivity, and the buoyant, fertilized eggs are easily collected. Stocking density during maturation should be 1–1.5kg/m2, and temperature should be kept above 16°C (S. senegalensis) or between 8 and 12°C (S. solea). In nature, the onset of spawning is related to a rise in temperature occurring during spring (March–June). Salinity should be kept constant around 33–35‰ and the fish reared under simulated natural photoperiod (LDN). In other cultured flatfish species, a change in the photoperiod is the key environmental signal used to manipulate and control maturation, but at present time there are no published work that verifies or contradicts this for either S. senegalensisor S. solea. Studies indicate that a mixture of inert and live food may increase the weaning success of sole fry, and this can be further enhanced by using attractants in the dry feed. Future experiments are needed to determine the ideal time to commence weaning and determine the minimum duration of this period. Studies on alternative feeding strategies are also required. The effect of temperature and photoperiod on juvenile growth has not been studied systematically in neither of the two species and the relative importance of a direct photoperiod effect on growth in sole therefore remains to be defined.

Development of digestive enzymes in larvae of Solea senegalensis, Kaup 1858

Abstract The activities of some digestive enzymes were studied in sole larvae fed live prey from first feeding until the first month of life. Whole larvae body homogenates were used for enzymatic determination in larvae younger than 21 days after hatching (DAH). Older larvae were dissected in order to obtain the pancreatic and intestinal segment. Brush border membranes of enterocytes were purified from intestinal segment. From 2 DAH to 18 DAH, activities of pancreatic and intestinal enzymes exhibited a pattern characteristic of developing animals: an increase during the first 10 days post-hatching, followed by a decrease. From 21 DAH to 27 DAH, the strong increase in alkaline phosphatase activity reflected the development of the brush border membranes of enterocytes, which occurred concurrently with a decrease in a cytosolic enzyme, leucine–alanine peptidase. These opposite patterns indicate a maturation of enterocytes and the acquisition of an adult mode of digestion.

Effect of dietary non-protein energy levels on condition and oxidative status of Senegalese sole (Solea senegalensis) juveniles

The effects of dietary non-protein energy levels on growth, oxidative status and condition were studied in juveniles of Senegalese sole (Solea senegalensis). Four isonitrogenous diets with four energy levels were used: A-low (11% lipid, raw carbohydrate); B-intermediate (11% lipid, digestible carbohydrate); C-intermediate (21% lipid, raw carbohydrate); and D-high (21% lipid, digestible carbohydrate). Survival, relative growth rate (RGR) and feed conversion rate were not significantly affected by the dietary treatments. The fatty acid composition of the fish muscle varied little among the treatments, but trans 18:2n - 6, total n - 6 and EPA/DHA ratio, tended to be higher in fish fed diets with low lipid level. Cellular energy allocation (CEA) results (indicative of metabolic status and net energy budgets) showed significant differences in liver, but not in muscle samples. Livers of fish fed diet C contained the lowest carbohydrate, protein and CEA values, but the highest cellular energy consumption. Fish fed diet A had the highest CEA for growth, followed by fish fed diets B and D and then diet C. The liver and muscle peroxidation and antioxidant activity were measured by thiobarbituric acid reactive substances (TBARS) test and the enzymatic activities levels of catalase (CAT) and superoxide dismutase (SOD). TBARS values were higher for fish fed diets with high lipid content. However, no clear relation was found between HUFA level and TBARS value. The activity levels of the antioxidant enzymes CAT and SOD were higher in livers of fish fed diets with a high lipid level. Furthermore, CAT and SOD activity and TBARS values were influenced by the type of dietary starch in the diet. Higher oxidation rates were observed in fish fed diets containing raw carbohydrate. These data suggest that lipid and carbohydrate energy sources affect the oxidative status of Senegalese sole. Diets containing low levels of lipid and digestible starch reduce the susceptibility of the fish to oxidation and may enhance growth rate

Histological and histochemical development of the digestive system of Solea senegalensis (Kaup, 1858) larvae

Abstract The digestive system of Solea senegalensis was studied from hatching until 1 month of larval life. Histological and histochemical procedures were used to study the histomorphology, digestive enzymes, lipid, protein and carbohydrate distribution in the digestive larval tract. The major events in digestive system differentiation occurred during the early stages. At first feeding (2DAH—days after hatching) both the mouth and anus had opened and the digestive tract was differentiated in five portions: buccal–pharyngeal cavity, esophagus, incipient stomach, anterior and posterior intestine. The pancreas and liver were also differentiated at this stage. During metamorphosis (12 to 20DAH) an elongation of the digestive tract and increase in the absorption surface were observed. Gastric glands were observed around 27DAH. Alkaline and acid phosphatase, lipase and aminopeptidase activities were observed from the mouth opening onwards and increased with larval development. Proteins exhibited a similar pattern of increase being especially abundant in the intestinal epithelium and exocrine pancreas. Neutral lipids were abundant in the yolk sac (oil globule), intestinal epithelium and liver (hepatocytes). Esophageal mucous cells were rich in sulphated mucosubstances and intestinal mucous cells, intestinal epithelium and liver were rich in neutral mucosubstances. Glycogen deposition was observed after 7DAH in liver and intestinal epithelium. The analysis of data obtained in this study suggests that after 31DAH S. senegalensis larvae are capable of ingesting, digesting and absorbing nutrients, having a morphologically complete digestive tract, equipped with digestive enzymes.

Osteological development and abnormalities of the vertebral column and caudal skeleton in larval and juvenile stages of hatchery-reared Senegal sole (Solea senegalensis)

The Senegal sole is a species recently adapted to aquaculture for which little information on larval development is available. This study was designed to describe normal skeletal development and the occurrence of skeletal malformations in Senegal sole reared in captivity. Eggs were collected from natural spawning, incubated until hatching and larvae reared to the juvenile stage in a closed recirculating system. Samples were collected throughout development at regular intervals from hatching to fully formed juveniles. Specimens were stained with alcian blue and alizarin red and observed for skeletal development and detection of anomalies. A high number of malformations were detected, both in the caudal complex and the vertebral column. About 44% of the individuals observed showed at least one malformation and the highest occurrence of deformities was observed in the caudal region and in the vertebral column. Accordingly, 28% of the total deformities identified in this study were detected at those sites and in adjacent arches and spines. The causes were not identified in this study, but the high incidence of malformations may reflect culture problems due to rearing and/or feeding conditions that affect skeletal development.

Co-feeding microparticulate diets with algae: toward eliminating the need of zooplankton at first feeding in larval red drum (Sciaenops ocellatus)

The effect of adding algae to the culture water used to rear red drum larvae was evaluated in terms of growth, survival and digestive enzyme activity. Red drum larvae were subjected to one of the following dietary regimes from first feeding (day 3 post-hatch) to day 14: (1) zooplankton supplemented with algae (L-A), (2) zooplankton without algae (L-NA), (3) a microparticulate diet with algae (M-A) and (4) the microparticulate diet alone (M-NA). The presence of algae in the rearing tanks improved growth of red drum larvae for both types of feeds. Growth was significantly higher (P<0.05) in larvae reared in the presence of algae (L-A and M-A) than in larvae raised in the corresponding treatments without algae (L-NA and M-NA). Red drum larvae raised on the microparticulate diet and algae (M-A) grew as well as the zooplankton treatment with no algae (L-NA), and were not significantly different from the L-A treatment. The larvae fed the microparticulate diet in the absence of algae (M-NA) were significantly smaller than the other three treatments. These results were consistent for two separate feeding trials. Final survival was highly variable in all treatments; nevertheless, mean final survival values were 30% higher in treatment L-A compared to L-NA (14.1 and 10.1%, respectively) and 42% higher in M-A than for M-NA (6.2 and 4.0%, respectively). Significantly higher trypsin and aminopeptidase activity was observed in the presence of algae, which may have influenced the digestion of the diet. Our results demonstrate that red drum larvae may be raised on a microparticulate diet from first feeding without the use of zooplankton.

Glyconjugates in epidermal, branchial and digestive mucous cells and gastric glands of gilthead sea bream, Sparus aurata, Senegal sole, Solea senegalensis and Siberian sturgeon, Acipenser baeri development

Epidermal, branchial and digestive mucous cells, and the gastric glands of larvae/postlarvae (from hatching until 45 days posthatching) of three fish species (two teleostean and a chondrostean) were investigated using conventional histochemical methods (periodic acid schiff -PAS-, diastase-PAS; alcian blue pH 0.5, 1 and 2.5) in order to distinguish neutral and acidic (carboxylated and sulphated) glycoconjugates, as well as bromophenol blue reaction for identification of proteins. Additionally, the presence and distribution of sugar residues in the oligosaccharide side chains of glycoconjugates were investigated using horseradish peroxidase (HPR)-conjugated lectins (Con A, DBA, WGA and UEA-I). Most mucous cells (digestive, epidermal and branchial) of Siberian sturgeon, Acipenser baeri, sea bream, Sparus aurata and Senegal sole, Solea senegalensis larvae were PAS- and alcian blue- (pH 2.5 and 0.5) positive, with small variations between organs/tissues and species. Bromophenol blue reaction (general proteins) was positive in a minority of the mucous cells, usually in those cells which were PAS-negative. Proteins rich in sulphydryl (-SH) and/or disulphide (-S-S-) groups related with the glycoprotein nature of the glycoconjugates present in mucous cells were also observed. Epidermal, branchial and digestive mucous cells of all studied larvae did not contain glycogen or lipids. Con A lectin staining was negative in all mucous cells types of sea bream and sole, but oesophageal mucous cell of sturgeon were reactive to different lectin reactions, suggesting the presence of mannose -Man- and/or glucose -Glc-, L-fucose -Fuc- ; N-acetyl-D-galactosamine -GalNAc-, as well as N-acetyl-D-glucosamine- GlcNAc - and/or sialic acid -NANA- residues. Digestive mucous cells of all studied larvae were positive to WGA and DBA lectins. Epidermal and branchial mucous cells of sea bream and sole were Con A, DBA and UEA-I unreactive. However, mucous cells of sturgeon larvae were stained with UEA-I lectin. Gastric glands appear very early in sturgeon stomach larvae development (between 5-6 days posthatching) but rather late (around 40 days) during the ontogeny of sole and sea bream larvae. These glands contain neutral glycoproteins with Man and/or Glc, Fuc, GlcNAc- and/or sialic acid and rich in GalNAc- sugar residues, as well as proteins moderately rich in arginine, and others particularly rich in tyrosine and tryptophan.

Influence of dietary bulk agents (silica, cellulose and a natural zeolite) on protein digestibility, growth, feed intake and feed transit time in European seabass (Dicentrarchus labrax) juveniles

The incorporation of various bulk agents by substitution of an equivalent amount of the basal mixture was studied in terms of protein digestibility, growth performance and body composition in European seabass (Dicentrarchus labrax) juveniles. During the growth trial, triplicate groups of 40 seabass (mean initial weight: 7.0 g) were grown in seawater (salinity: 35; temperature: 18 degrees C) over 60 days. Fish were hand-fed, three times a day, one of seven experimental diets. Bulk agents tested at 10 or 20 % level of incorporation were silica, cellulose and a natural zeolite (chabamin). Feeding rates were adjusted in proportion to the percent dilution of the control diet without bulk incorporation. The incorporation of the bulk agents, at a 10 and 20 % level, did not affect protein digestibility or growth performance. Dietary bulk incorporation reduced feed efficiency values, particularly at the 20 % incorporation level. However, this reduction was mostly caused by the dietary nutrient dilution of the bulk-incorporated diets, rather than by a negative effect of the bulk agents as dietary ingredients. In comparison to the control treatment, bulk incorporation at 10 and 20 % level did not affect protein retention values. When compared with the control diet, 20 % bulk agent incorporation changed the evacuation profile of faeces and increased faecal egestion time.

Multivariate cluster analysis to study motility activation of Solea senegalensis spermatozoa: a model for marine teleosts.

Computer-assisted sperm analysis (CASA) and clustering analysis have enabled to study sperm subpopulations in mammals, but their use in fish sperm has been limited. We have used spermatozoa from Senegalese sole (Solea senegalensis) as a model for subpopulation analysis in teleostei using two different activating solutions. Semen from six males was activated using 1100 mOsm/kg solutions: artificial seawater (ASW) or sucrose solution (SUC). Motility was acquired at 15, 30, 45, and 60 s post-activation. CASA parameters were combined into two principal components, which were used in a non-hierarchical clustering analysis, obtaining four subpopulations (CL): CL1 (slow/non-linear), CL2 (slow/linear), CL3 (fast/non-linear), and CL4 (fast/linear). We detected spermatozoa lysis, especially in ASW. Sperm motility was higher for SUC and decreased with time. The subpopulation proportions varied with time and activating treatment, showing both an increase in CL1 and CL2 and a decrease in CL3 and CL4 with time. Both CL3 and CL4 were higher in samples activated with SUC, at least in early post-activation. Proportions of CL3 and CL4 at 15 s were associated with higher quality at 60 s and with lower lysis. A second clustering analysis was conducted, classifying the males accordingly to their motility subpopulations. This analysis showed a high heterogeneity between samples. Subpopulation analysis of CASA data can be applied to Solea spermatozoa, allowing identification of potentially interesting sperm subpopulations. Future studies might benefit from these techniques to establish the relationship of these subpopulations with fish sperm quality and fertility, helping to characterize males according to their reproductive potential.