Turbot Larvae Research Articles

Aliphatic hydrocarbon levels in turbot and salmon farmed close to the site of the Aegean Sea oil spill.

After the Andros Patria oil spill, the most serious oil tanker accident to occur off the coast of Galicia (N.W. Spain) was the running aground and subsequent conflagration of the Aegean Sea supertanker outside the northern Spanish port of La Coruna (December 3rd 1992). Approximately 60,000 tonnes of Brent oil were spilled into the Atlantic Ocean in the cited coastal region. Subsequently, an impropitious combination of a high tide and a change in wind direction caused the resulting slick to rapidly spread into the port. Measures aimed at cleaning up affected areas and evacuating the ca. 11,215 tonnes of oil remaining in the supertanker were immediately implemented. However, within just a few days the resulting contamination had killed some 15000 turbot juveniles and larvae, which are cultivated in fish farms close to the accident site. The environmental impact of major oil spillages has been widely studied. Several scientists have suggested that, in terms of the negative effects on the seawater quality and productive capacity of the affected maritime regions, the magnitudes of the Aegean Sea and Amoco Cadiz accidents are comparable. This paper reports variations over time of aliphatic hydrocarbon levels in turbot and Atlantic salmon sampled from fish farmsmore » close to the site of the Aegean Sea oil spill. 6 refs., 2 figs., 1 tab.« less

Dietary ascorbic acid requirements during the hatchery production of turbot larvae

The effect of high ascorbic acid (AA) levels transferred through enriched live food was evaluated for turbot Scophthalmus maximus larvae in two consecutive feeding experiments. The same feeding strategy was applied to all treatments, except for the AA content of the live food which was manipulated through bioencapsulation with ascorbyl palmitate. This resulted finally in a low, medium and high‐AA treatment. The AA incorporation levels in the turbot larvae (up to 1400 μg AA g DW−1) were correlated with the AA content of the live food administered. However, feeding the high AA concentration resulted in the same values as for the medium treatment, indicating a saturation of the body AA reserves. Under standard culture conditions, no differences in growth nor overall survival could be detected among the different groups, illustrating that the dietary AA requirements of larval turbot are met by non‐enriched live food containing already 500 μg AA g DW−1. The larvae of the high‐AA treatment, however, showed a better pigmentation rate (47 and 32% for experiments 1 and 2, respectively) compared to the other groups (35 and 25%, respectively). Evaluation of the physiological condition applying a salinity stress test revealed an improvement by feeding extra AA, significantly in the medium‐AA treatment. Though not significantly different, cumulative mortalities after challenge with Vibrio anguillarum amounted to 50% for the control v. 40% for the fish fed medium and high‐AA diets, respectively. Moreover, the onset of mortalities in this study was slower (not significantly) for the fish fed the extra AA.

Dietary ascorbic acid requirements during the hatchery production of turbot larvae

The effect of high ascorbic acid (AA) levels transferred through enriched live food was evaluated for turbot Scophthalmus maximus larvae in two consecutive feeding experiments. The same feeding strategy was applied to all treatments, except for the AA content of the live food which was manipulated through bioencapsulation with ascorbyl palmitate. This resulted finally in a low, medium and high-AA treatment. The AA incorporation levels in the turbot larvae (up to 1400 μg AA g DW−1) were correlated with the AA content of the live food administered. However, feeding the high AA concentration resulted in the same values as for the medium treatment, indicating a saturation of the body AA reserves. Under standard culture conditions, no differences in growth nor overall survival could be detected among the different groups, illustrating that the dietary AA requirements of larval turbot are met by non-enriched live food containing already 500 μg AA g DW−1. The larvae of the high-AA treatment, however, showed a better pigmentation rate (47 and 32% for experiments 1 and 2, respectively) compared to the other groups (35 and 25%, respectively). Evaluation of the physiological condition applying a salinity stress test revealed an improvement by feeding extra AA, significantly in the medium-AA treatment. Though not significantly different, cumulative mortalities after challenge with Vibrio anguillarum amounted to 50% for the control v. 40% for the fish fed medium and high-AA diets, respectively. Moreover, the onset of mortalities in this study was slower (not significantly) for the fish fed the extra AA.

The sequence of catabolic substrate oxidation and enthalpy balance of developing embryos and yolksac larvae of turbot (Scophthalmus maximus L.)

A model of catabolic metabolism during the endogenous developmental phase of turbot ( Scophthalmus maximus L.), a species whose pelagic eggs contain a single oil globule, is presented. Yolk-dependent routine rates of oxygen consumption and ammonia excretion and flux of ammonium ions were related stoichio-metrically to quantitative changes in the contents of free glucose, glycogen, lactate, free amino acids, proteins, and lipid classes with their associated fatty acids, in order to determine the rate and sequence of catabolic substrate oxidation that occurs with development. These data were further related to the changes in caloric contents of the eggs and larvae in order to derive an enthalpy balance equation for the period of yolk dependence. The stoichiometric relation of the oxygen consumption and ammonia production with the quantitative changes in substrates indicated that, following the first 18–19 hr of glycogen dependence, free amino acids (84%) together with a small amount of phosphatidyl choline (9%) and later wax esters (5%) comprised the metabolic fuels of embryonic development. Following hatch (day 4.4 post fertilisation), wax esters (33%) and triacylglycerols (25%) were initially catabolised with the remaining free amino acids (10%). Upon exhaustion of the free amino acids on day 6 post fertilisation, body proteins (32%) were recruited and catabolised together with wax esters and triacylglycerols. Thus, the catabolic metabolism of endogenously feeding turbot embryos and larvae were equally fuelled by amino acids (50%, with similar amounts being supplied from both the free and protein bound pools) and lipids (50%, mainly of neutral origin), while carbohydrates (predominantly glycogen) were only quantitatively important during the early cellular division stages. We argue that this pattern of catabolic substrate oxidation is also generally applicable to other marine fishes which spawn eggs containing oil globules. For an enthalpy balance equation of the form P = C + R + E, a turbot larva at 15°C, utilised 60% of yolk and oil globule enthalpy (C) for growth (P), 37% was dissipated due to metabolism (R) while only 3% was lost via excretion of nitrogenous end products (E).

Inducibility of Metallothionein mRNA Expression and Cadmium Tolerance in Larvae of a Marine Teleost, the Turbot (Scophthalmus maximus)

An acute (48-hr) lethal toxicity study of turbot at various stages from hatch to juveniles showed that prior to first exogenous feeding, larvae of turbot were an order of magnitude more sensitive to cadmium toxicity than later developmental stages (LC50 of 0.18–0.23 ppm Cd versus 2–5 ppm Cd). To investigate the possible role of metallothionein (MT) in Cd tolerance, the concentrations of MT mRNA were determined in control and Cd-exposed animals using a plaice MT cDNA probe. MT mRNA was expressed throughout embryonic and early larval development at levels approximately twice those in juvenile turbot liver apart from the period around hatching, when there was a decreased level of expression. During endogenous feeding, despite larval drinking, MT mRNA levels did not appear to be inducible by exposure to Cd in the water within 3 days of hatching but were induced three- to fivefold after 48 hr exposure to waterborne Cd at all later stages of development. The results suggest that Cd tolerance is associated with metal induction of MT gene transcription and that the enhanced sensitivity of very early stage larvae is attributable to a deficiency in MT gene transcription.

Benzo[a]pyrene metabolism and xenobiotic-stimulated reactive oxygen species generation by subcellular fraction of larvae of turbot ( Scophthalmus maximus L.)

NADPH-dependent 3H-benzo[a]pyrene (BaP) metabolism and basal and xenobiotic-stimulated NAD(P)H-dependent reactive oxygen species (ROS) production were investigated in 11,600 g supernatants of 4-day-old (yolk sac) larvae of turbot ( Scophthalmus maximus L.). BaP metabolites were resolved by HPLC and detected radiometrically. ROS were quantified by the iron-EDTA mediated production of hydroxyl radical (·OH) that was detected by its oxidation of 2-keto-4-methiolbutyric acid (KMBA) to yield ethylene. BaP metabolism produced phenols, dihydrodiols and diones (quinones) (respectively, 54, 32, and 14% of free metabolites) and putative protein adducts. Metabolites identified by retention time included the 7,8-dihydrodiol, the 1,6-, 3,6- and 6,12-diones and the 3- and 9-phenols. Pre-exposure of turbot larvae to 5 ppb BaP for 24 hr caused an approximate 2-fold increase in both BaP metabolism and 7-ethoxyresorufin O-deethylase activity, indicative of the induction of cytochrome P4501A and its involvement in BaP metabolism. Basal KMBA oxidation rates were similar for NADH and NADPH. Inhibition studies indicate that ·OH was formed via the production of Superoxide anion radical and hydrogen peroxide. Basal ROS production was stimulated up to 3-fold by a range of redox cycling aromatic hydrocarbon quinones and indicated to be stimulated by other xenobiotics, including nitroaromatics. The results indicate biotransformation and ROS production as potential mechanisms of toxicity in larval fish.

Metallothionein protects against cadmium toxicity — proof from studies developing turbot larvae

Metallothionein protects against cadmium toxicity — proof from studies developing turbot larvae

Pre- and postmetamorphic organization of the vestibular nuclear complex in the turbot examined by retrograde tracer substances

During metamorphosis of flatfish larvae, eye migration leads to a 90 degrees misalignment of the visual and vestibular frames of reference. In order to maintain vestibular eye stabilization, the vestibulo-ocular (V-O) pathways have to be radically reorganized. Here, we have examined the vestibular projections in turbot larvae and juveniles by means of conventional neurohistological techniques using horseradish peroxidase and fluorescent dextranamines as tracers. We have found that the vestibular projections to the rostral eye motor nuclei consist of five densely clustered groups of neurons projecting to the rostral eye motor nuclei, some through the ipsilateral, others through the contralateral medial longitudinal fascicle (MLF). In addition, there are three groups of vestibulo-spinal neurons. The most prominent of these gives rise to the ipsilateral vestibulo-spinal tract. The other two project contralaterally, one descending in the MLF, the other more laterally in the anterior funiculus of the spinal cord. These subnuclei of the vestibular complex are easily identifiable in larvae before metamorphosis, as well as in juvenile turbots. The number of projection neurons in each of the subnuclei is approximately doubled over the period of metamorphosis. Applying different tracers to rostrally and caudally projecting pathways, we found no double-labeled neurons, indicating that the V-O and vestibulo-spinal groups are distinct entities. However, by applying the two tracers ipsi- and contralaterally in the terminal fields in the rostral eye motor nuclei after metamorphosis, we found many double-labeled neurons in all the V-O subgroups. In contrast, we found only a small fraction of double-labeled vestibular neurons when the same strategy was applied to larval preparations. We conclude that 1) the basic organization of the vestibular nuclei of the turbot is similar to that of other teleosts, in larvae as well as juveniles; 2) there is a substantial increase in projection neurons over the period of metamorphosis in all the subgroups of the vestibular nuclear complex; and 3) many more of the V-O neurons project bilaterally to the rostral eye motor nuclei in juvenile than in larval turbots.

Comparison of the Growth and Survival of Larval Turbot in the Absence of Culturable Bacteria with Those in the Presence of Vibrio anguillarum, Vibrio alginolyticus, or a Marine Aeromonas sp

Larval turbot (Scophthalmus maximus) were reared on rotifers (Brachionus plicatilis) in the absence of culturable bacteria for up to 14 days and exhibited growth and high rates of survival (>55% in five experiments). Low numbers of known bacteria were introduced into similar cultures by exposure of the rotifers to a suspension of bacteria prior to addition of rotifers to the larval cultures; Vibrio anguillarum 91079 caused a highly significant decrease (P <0.01) in the proportion of survivors in two separate trials. With an Aeromonas sp. previously isolated from a healthy batch of copepod-fed larvae, there was no significant difference in survival compared with control larvae, even though the density of bacteria in the water of larval cultures reached 10(sup7) ml(sup-1). Bacteria colonized the gut of larvae exposed to Aeromonas-treated rotifers to levels similar to those in conventionally reared fish (>4 x 10(sup4) CFU per larva). Rearing of larvae in the presence of known bacteria provides a means of investigating the interaction of specific bacteria with turbot larvae and could provide a method for the selection of bacteria which may restrict the growth of opportunistic pathogens which would be harmful to turbot larvae.

A method for the early assessment of the quality of turbot larvae

The bacterial environment should be taken into account for assessing the quality of fish larvae, besides biochemical and developmental criteria. Detrimental effects of associated flora have been observed on the survival rates of larvae (e.g. Nicolas et al., 1989), and experimental infections were performed on fish larvae by introducing the pathogen either directly into the rearing medium or into live food organisms (e.g. Kusuda et ai, 1986). A challenge test was used for assessing the effect of a food additive on the ability of turbot (Scophthalmus maximus) larvae to resist infection, but that required rearing the larvae separately (Gatesoupe, 1993). Bergh et al. (1992) have proposed an infection test with transfer of eggs into polystyrene dishes, but it was not convenient for larvae with exogenous food source. The present paper was an attempt to develop a new test with transfer of larvae after feeding. Then this test was inserted into a simple survey of the quality of turbot larvae, with a view to drawing up relations between rearing success, associated bacteria and resistance against pathogens. For this purpose, seven batches of larvae were successively reared in 150 1 cylindrical tanks with conical bottoms from day 1 after hatching until day 10. Three tanks were used for batches 1, 3, 5 and 6, while five tanks were used for batches 4 and 7. The larvae of batch 2 were reared in one tank. The rearing temperature was gradually increased from 14 °C to 18 °C by day 5. The salinity was 35%. For feeding the larvae, rotifers (Brachionus plicatilis) were cultured in seawater according to the method already described (Gatesoupe, 1990). The rotifer diet was made up of baker's years, menhaden (Breuoortia tyrannus) oi l (Sigma F8020), soybean lecithin, DL-a-tocopherol (USB), and vitamin premix (Spyridakis et ai, 1988), at the daily rates of 60.8, 7.7, 0.7, 0.015, and 0.7 mg r1, respectively, on a dry matter basis. The larvae were fed with the rotifers from day 3 to day 9, at the daily rates of 20, 50, 75, 100, 150, 200, and 250 rotifers per initial larva, corresponding to the seven consecutive days of feeding. Each day, the rotifers were transferred from their culture medium into clean seawater tanks (200 rotifers m l 1 ) , where they were enriched with spray-dried fish autolysate, menhaden oi l , soybean lecithin, and DL-a-tocopherol, at the rates of 15, 7.7, 0.7, and 0.015 mg T 1 , respectively, on a dry matter basis. The rotifers were continuously distributed to turbot with a peristaltic pump, the flow rate of which was adjusted so that the whole volume was carried through in 24 h. At day 10, the survivors were counted and 30 fish were sampled in each tank,

Calorespirometry of developing embryos and yolk-sac larvae of turbot (Scophthalmus maximus)

The rates of oxygen consumption and heat dissipation were simultaneously measured and related to contents of glucose, glycogen and lactate in order to determine whether anaerobic processes contributed significantly to the energy metabolism of developing turbot embryos and larvae. The results suggest that metabolism is fully aerobic between Days 0 and 12 post fertilisation. The data further suggest that glycogen is the sole metabolic fuel during the first 18 to 19 h post fertilisation. After the commencement of epiboly, carbohydrates play an insignificant role in the energy metabolism of the developing embryo and yolk-sac larva.

An oral challenge for turbot larvae with Vibrio anguillarum

An oral challenge for turbot larvae with Vibrio anguillarum

Comparison of the gut bacterial flora of start‐feeding larval turbot reared under different conditions

P.D. MUNRO, A. BARBOUR AND T.H. BIRKBECK. 1994. Bacterial colonization of the gut of turbot larvae coincided with the start of feeding and the gut microflora was then dominated by Vibrio and Aeromonas species. The detection of similar bacterial isolates over several days indicated the presence of a stable microflora during the rotifer feeding stage, probably reflecting a stable flora in the rotifer culture. There was no correlation between the number of bacteria in the gut and larval survival rates; incidences of high mortalities were not associated with high numbers of recognized fish pathogens, although an Aeromonas species was implicated as an opportunistic pathogen. Potential pathogens were isolated, albeit in low numbers, from batches of apparently healthy larvae with high survival rates. It is probable that the bacterial flora plays an important role in determining the survival of larval fish and that the establishment of a beneficial flora is possible, although the criteria for such a flora have still to be elucidated.

Lactic acid bacteria increase the resistance of turbot larvae,Scophthalmus maximus,against pathogenic vibrio

Characteristics of Lactic Acid Bacteria (LAB) isolated from rotifers, Brachionus plicutilis, resembled those of Lactobacillus plantarum or Carnobacterium sp. It was investigated whether the artificial maintenance of a high concentration of LAB in rotifers might increase their dietary value for turbot larvae, particularly when the fish were infected with pathogenic Vibrio sp. One LAB strain was cultivated, then introduced daily into the enrichment medium of rotifers. In this way, LAB were retrieved in large amounts in turbot, and a significant limitation of larval mortality rate was observed when turbot were challenged with pathogenic vibrio at day 9. The inoculum concentration of LAB had a decisive effect on survival rate, and the optimum was between 107 and 2 × 107 Colony Forming Units (CFU) daily added per ml of the enrichment medium (53% survival rate after 72 h of challenge, versus 8% for the infected control group without LAB).

Feeding at different illumination levels in larvae of three marine teleost species: cod, Gadus morhua L., plaice, Pleuronectes platessa L., and turbot, Scophthalmus maximus (L.)

Abstract. Optimal illumination levels for feeding vary in different fish larvae according to feeding strategy, prey type and habitat. Determining optimal illumination conditions is important in aquaculture, as illumination seems to be the dominating factor affecting the distribution of many pelagic fish larvae. Cod, Gadus morhua L., plaice, Pleuronectes platessa L., and turbot, Scophthalmus maximus (L.), have all been considered for aquaculture, and have different vertical positioning strategies. Three parallel groups of larvae of each of these species were exposed to planktonic feed organisms at seven illumination levels ranging from darkness to 10 800 lux. The number of ingested organisms was counted. Cod had maximal feed uptake at 1·0 lux, and higher uptake at 0·1 lux than at 87 lux. Plaice reached a threshold level at 87 lux, and turbot at 860 lux. Cod larvae, which are slowly searching dusk/ dawn predators, preferred very low illumination levels. Plaice larvae, being constant swimmers with a vertically orientated behaviour, fed equally well over a wide illumination range. The near-surface-feeding turbot larvae preferred high illumination levels.

The development of functional digestive and metabolic organs in turbot, Scophthalmus maximus

The functional status of organ systems involved into the processing of exogenous food is critical for the survival and growth of fish early life stages. The present study on laboratory-reared larval turbot, Scophthalmus maximus, provides an overview on the ontogeny of structure and functions involved in digestion, absorption and metabolism of nutrients. At start of exogenous feeding the intestine of larval turbot is anatomically differentiated, with enterocytes displaying an adult-type ultrastructure and being able to process lipids. At the microvillous border of the enterocytes, enzymes of contact digestion such as aminopeptidase are found. The ultrastructure of the exocrine pancreatic cells is fully differentiated from hatching onwards. Likewise, substantial activities of trypsin-type proteases are present. A stomach anlage exists in first-feeding larvae; however, the stomach becomes functional (appearance of gastric glands and pepsin secretion) only during metamorphosis. Liver parenchymal cells already display a functional ultrastructure during the endotrophic phase; with onset of exogenous feeding they develop pronounced diet-related changes of their energy stores. Larval respiration is not executed by the gills since respiratory surface of these structures develops only towards metamorphosis. The energy generation of larval muscle tissue depends on aerobic metabolism, whereas glycolytic activities start to increase at metamorphosis. In conclusion, two important patterns can be recognized in the development of turbot larvae: (1) The structure/function is differentiated at hatching or at the onset of exogenous feeding (afterwards it experiences mainly quantitative but not qualitative growth, i.e., intestine, exocrine pancreas, liver); or (2) the structure/function is absent in larvae and develops only during metamorphosis (i.e., gills, glycolytic muscle metabolism, stomach).

Lipid composition in turbot larvae fed live feed cultured by emulsions of different lipid classes

Fatty acid and lipid class compositions were determined in eggs and larvae of turbot ( Scophthalmus maximus L.). The larvae were fed on rotifers and Anemia fed on various lipid emulsions. Starving larvae were also studied. The lipid class compositions of the emulsions affect the fatty acid composition of the rotifers. Ethyl ester-based emulsion exhibited the highest assimilation by the rotifers and Artemia and were possibly incorporated in their triacylglycerol fraction. During larval starvation docosahexaenoic acid and arachidonic acid levels tended to be conserved whereas eicosapentaenoic acid was highly reduced. Moreover, the relative proportion of phosphatidylethanolamine increased whereas that of phosphatidylcholine decreased in starved larvae. Increasing the n-3 highly unsaturated fatty acid levels in rotifers and Artemia did not influence the survival and growth of the turbot larvae. A positive correlation was found between pigmentation success and the ratios of docosahexaenoic/ eicosapentaenoic acids in the total and polar lipid fractions of the turbot larvae. Furthermore, the amounts of these polyunsaturated fatty acids in the phosphatidylethanolamine fraction of the larvae are suggested to be of particular importance in turbot pigmentation.

Pathways of bacterial contamination during egg incubation and larval rearing of turbot, Scophthalmus maximus

Summary Overall microbial levels in the water system of a turbot farm were similar to those found in regular sea-water. At the end of an incubation period, however, the numbers of colony-forming units (CFU) in Artemiu cultures and turbot eg incubation jars were up to four orders of magnitude higher than sea-water level. Rinsing of the foot organisms (rotifers) prior to feeding them to turbot larvae, however, significantly reduced bacterial numbers, thus reducing the number of cross-contaminations. Several species of Aeromonas, Pseudomonas and Vibrio among others, were identified in the eggs and larvae, or in the water in which these were incubated. Scanning electron microscop shows that the surface of unfertilized turbot eggs is a breeding ground for bacteria; the removal of these eggs from incubation jars is therefore recommended. The epidermis of turbot larvae was virtually free ofmicroorganisms.

Influence of lipid composition of live feed on growth, survival and pigmentation of turbot larvae

The effect of different lipid compositions of live feed on the survival, growth rate and pigmentation success of turbot larvae, Scophthalmus maximus (L.), was investigated. Rotifers, Brachionus plicatilis, together with the algae Tetraselmis sp., were administered until day 12, and Artemia was fed until day 27. The experimentally treated live feeds were enriched with four formulated emulsions, resulting in a gradient in the relative contents of Ω3 HUFA (highly unsaturated fatty acids) and in DHA (docosahexaenoic acid, 22:6 Ω3)/EPA (eicosapentaenoic acid, 20:5 Ω3) ratios in both the rotifers and Artemia.

Histological changes in starved turbot larvae (Scophthalmus maximus) quantified by digital image analysis

In turbot larvae, Scophthalmus maximus, deprived of food for 24 h there was a significant increase in the specific surface of the epithelium and the corresponding microvillous border of the foregut accompanied by slight cellular degeneration. Following 48 h starvation Sarvae showed severe tissue degeneration in the foregut mucosa, progressing to extensive mucosal desquamation and cellular sloughing. Intracellular vacuolation of the epithelium and loss of microvilli was also extensive and the ability of the gut to absorb food must be severely impaired, with further starvation probably resulting in larval death. There was no evidence of parasitic infection in any of the larvae sampled and all observed alterations are attributable to food deprivation.