Effect of amino acids on the swimming activity of newly hatched turbot larvae (Scophthalmus maximus)

The swimming behaviour of newly hatched turbot (Scophthalmus maximus L.) larvae was observed in artificial seawater (ASW) and in solutions of 21 l-amino acids at a concentration of 10⁻⁵  M. The behaviour of 20 larvae was analysed in each solution. Each larva was observed for 1 min. Individual movements were recorded on video and analysed using a computer-assisted program. The larvae swam in convoluted, randomised three-dimensional paths, rested and started swimming again. There were large variations in the swimming behaviour of turbot larvae during ontogeny. In ASW the mean frequency of trajectories longer than a body length of 4 mm larva⁻¹ min⁻¹ increased from 1.2 at Day 1, to 10 at Day 4. Analysing the data (Dunnett's method) revealed that the frequency of swimming trajectories increased in the presence of glycine, histidine and glutamine, and decreased in the presence of proline. The total distance swum increased for glycine but decreased for proline. The threshold concentration for glycine detected by turbot larvae was 10⁻⁵  M. The straightness index did not change in the presence of the amino acids. The possible role of these changes in behaviour is discussed. Received: 12 June 1997 / Accepted: 13 January 1998     

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.     

Temperature and development in larvae of the turbot Scophthalmus maximus

Turbot (Scophthalmus maximus L.) were reared at 12 and 16°C until 26 d after hatching. At both temperatures, starting at the neural plate stage, somites were initially formed every 75 min. Expressed as a percentage of development time (DT, fertilisation to 90% larvae hatching) somite formation occurred relatively earlier during embryogenesis at 12°C (45% DT) than at 16°C (55% DT). At 12°C, after the 32-somite stage the rate of somite formation decreased to one every 300 min. The larvae hatched after 6 d at 12°C and 3 d at 16°C at a relatively primitive stage of development, prior to the opening of the mouth and anus, with unpigmented eyes, and a straight gut. Temperature altered the relative timing of organogenesis in the larval stages. At 12°C, the following characters appeared (in this order): swimbladder>loop in the gut (at the time of yolk exhaustion)>caudal fin. In contrast, at 16°C, the caudal fin appeared at the same time as the loop in the gut. At 16°C, spines formed on the head in the region of the otic capsule at the time the swimbladder formed and the yolk was exhausted, but were absent in 12°C larvae. At both temperatures, in 1 d-old larvae the myotomes just behind the yolk-sac contained 200 inner muscle fibres (presumptive white muscle). The initial growth of inner muscle was largely due to hypertrophy, but by 26 d at 12°C and 11 d at 16°C hyperplastic growth became important, as evidenced by a significant increase in the number of small fibres (<10 m²). By 26 d the average number of inner muscle fibres had increased to 341 at 12°C and 988 at 16°C. New muscle fibres were added in distinct germinal zones at the dorsal and ventral apices of the myotomes. Metamorphosis was associated with a thickening of the superficial (presumptive red) muscle layer and the appearance of tonic muscle fibres.     

Free amino acid and protein contents of start-feeding larvae of turbot (Scophthalmus maximus) at three temperatures

The contents of free amino acids (FAA) and total protein, together with growth and gut-content, of turbot (Scophthalmus maximus L.) larvae reared at 14, 18 and 22 °C were studied from first-feeding to approximately 140 effective day-degrees post hatch (D_eff ^∘). Artemia franciscana nauplii and two species of rotifers were used as prey. Protein content accounted for about 42 and 26% of dry body mass in the A. franciscana nauplii and the rotifers, respectively. The FAA pool constituted 5.6 and 4.8% of the total amino acids in the same animals. The dry body mass of turbot larvae was exponentially related to D_eff ^∘. Protein and FAA contents were linearly related to dry body mass, and were independent of rearing temperature between 14 and 18 °C. At the end of the experiment, however, turbot larvae at 22 °C had lower gut content values, retarded growth rates, and decreased FAA contents and concentrations. Thus, at this high temperature, turbot larvae seem unable to catch and ingest sufficient prey, or to sustain an amino acid assimilation rate from the intestine sufficient to meet metabolic demands. Received: 2 January 1997 / Accepted: 25 September 1998     

Variations in carbohydrate metabolism during gonad maturation in female turbot (Scophthalmus maximus)

Several carbohydrate metabolism pathways were studied in four different tissues (liver, gonad, kidney and white muscle) of female turbot (Scophthalmus maximus) at three different stages of gonad maturation, i.e., previtellogenesis, early vitellogenesis and late vitellogenesis. During carbohydrate metabolism in the liver, the only major change noticed throughout vitellogenesis was increased glycolytic activity at early vitellogenesis. The major changes observed in the gonad during vitellogenesis were an increased use of exogenous glucose and increased gluconeogenesis, which were reflected in increased glucose levels throughout vitellogenesis accompanied by a small increase in glycogen levels. Changes observed in carbohydrate metabolism in the kidney pointed to increased gluconeogenesis during early vitellogenesis. Finally, during ovarian maturation, the muscle mobilized proteins whose constituent amino acids could have been used in other tissues either as substrates for gluconeogenesis or for protein synthesis. As an overall conclusion, changes in the carbohydrate metabolism of female turbot during maturation are quite marked in the gonad, kidney and white muscle, in contrast to other species, in which major changes have been previously reported to occur in the liver.     

Otolith ring deposition in relation to growth rate in herring (Clupea harengus) and turbot (Scophthalmus maximus) larvae

Larvae of Clyde spring-spawning Clupea harengus L. and hatchery-produced Scophthalmus maximus (L.) were reared from hatching through metamorphosis in 1980 and 1981 in laboratory tanks and in large enclosures under various light, temperature, and feeding regimes in order to study otolith ring deposition and growth under different conditions. Ring deposition and growth rates were significantly affected by rearing conditions in both species. The ring deposition rates observed under the conditions tested ranged from 0.34 to 0.92 rings d^-1 in herring larvae, and from 0.07 to 1.0 rings d^-1 in turbot larvae. Growth rates ranged from 0.11 to 0.42 mm d^-1 in herring and from 0.05 to 0.27 mm d^-1 in turbot. The number of otolith rings was dependent on the growth rate of the individual larva. At the population level, higher ring deposition rates were observed in faster growing populations. In herring larvae, the relationship between average growth rate and average ring deposition rate was logarthmic, reaching an asymptote at 1 ring d^-1 for growth rates approaching 0.40 mm d^-1. The relationship was linear for turbot larvae for the range of growth rates observed.     

Free amino acids and energy metabolism in eggs and larvae of seabass, Lates calcarifer

Contents of free amino acids (FAA), protein and ammonium ions together with rates of ammonia excretion and oxygen consumption were measured in order to study the role of FAA as an energy substrate in developing eggs and larvae of seabass (Lates calcarifer) maintained in seawater (30 ppt) at 28 °C without feeding. Initially eggs contained 25.3 nmol ind⁻¹ of FAA of which 21.5 nmol was rapidly utilised by the developing eggs and larvae during the period up to 40 h post spawning (PS) when nearly all the yolk had been resorbed. During the same period, a net increase in protein content of 1.7 μg ind⁻¹ was observed, indicating that the major part of the amino acids lost from the free pool had been polymerised into body proteins. Assuming that the balance of the FAA after protein synthesis was used entirely for energy metabolism, FAA appeared to be an important energy substrate during the embryonic stages (2 to 16 h PS); after hatching, the contribution of FAA to energy metabolism was less significant. From 50 h PS until the end of the study period at 100 h PS, amino acids derived from somatic protein were used for energy metabolism. For the overall period from just after spawning up to 100 h PS, the data indicate that ca. 14% of the total aerobic energy metabolism was derived from amino acid catabolism. Received: 26 September 1997 / Accepted: 1 April 1998     

Fatty-acid composition of ovulated eggs from wild and cultured turbot (Scophthalmus maximus) in relation to yolk and oil globule lipids

The fatty-acid composition of lipids from ovulated eggs of wild and cultured turbot was investigated in order to estimate the nutritional requirements during embryonic and early larval development. Lipid comprised 13.8±0.5% (n=5) and 13.2±0.7% (n=7) of the egg dry weight in wild and cultured turbot, respectively. Polyunsaturated fatty acids (PUFA) of the (n-3) series accounted for 39% of total fatty acids in total lipid of both wild and cultured fish. The predominant (n-3) FUFA was docosahexaenoic acid (22:6 n-3), which also was the most abundant fatty acid in turbot eggs and comprised 24 and 23% of the total egg fatty acids in wild and cultured fish, respectively. Phospholipids, triacylglycerols and cholesterol-wax esters of turbot eggs all exhibited a specific fatty-acid profile distinctly different from that of total lipid. The general pattern of the fatty-acid distribution in lipids of eggs from wild and cultured turbot was similar, but the relative amount of 18:2(n-6) was considerably higher and 20:1(n-9) slightly higher in cultured fish. These differences were extended to all lipid classes and probably reflect the dietary intake of certain vegetable and marine fish oils. Calculations based on light microscopical studies showed that 55 to 60% of the total lipids in cultured turbot eggs are confined to the oil globule. The size of the oil globule remained constant during embryogenesis, and a reduction in size occurred first after hatching and mainly after yolk depletion. This implies that the total amount of lipids utilised during the embryonic development is considerably less than the total lipids present in ovulated turbot eggs. Comparison of the fatty-acid composition of total lipids from eggs and vitellogenin of wild turbot reveals that egg lipids contained a lower level of saturated and a higher level of monounsaturated fatty acids. Eggs also contained wax esters, which were not detected in vitellogenin, suggesting that vitellogenin is not the only source of lipids for turbot eggs.     

Feeding behaviour of North Sea turbot (Scophthalmus maximus) and Dover sole (Solea solea) larvae elicited by chemical stimuli

The effect of chemical stimuli on the feeding behaviour of turbot (Scophthalmus maximus L.) and sole (Solea solea L.) larvae was investigated under controlled laboratory conditions. Laboratory-reared flatfish larvae exposed to chemical stimuli showed significant differences in the frequency of various behaviour patterns related to feeding (such as swimming, snapping and darting) compared to larvae exposed to blanks of filtrated seawater. The chemical substances Il-asparagine, glycine, inosine 5-monophosphate and betaine evoked the strongest behavioural responses in turbot larvae. In sole larvae the most potent substances were Il-phenylalanine, Il-lysine, Il-asparagine, inosine 5-monophosphate and betaine. These results show that feeding of turbot and sole larvae is influenced by chemosensory processes at an early larval stage, and indicate that chemoreception may be an integrated part of turbot and sole larvae feeding strategy.     

Amino acid metabolism and protein turnover in larval turbot (Scophthalmus maximus) fed natural zooplankton or Artemia

The present paper studied the influence of different food regimes on the free amino acid (FAA) pool, the rate of protein turnover, the flux of amino acids, and their relation to growth of larval turbot (Scophthalmus maximus L.) from first feeding until metamorphosis. The amino acid profile of protein was stable during the larval period although some small, but significant, differences were found. Turbot larvae had proteins which were rich in leucine and aspartate, and poor in glutamate, suggesting a high leucine requirement. The profile of the FAA pool was highly variable and quite different from the amino acid profile in protein. The proportion of essential FAA decreased with development. High contents of free tyrosine and phenylalanine were found on Day 3, while free taurine was present at high levels throughout the experimental period. Larval growth rates were positively correlated with taurine levels, suggesting a dietary dependency for taurine and/or sulphur amino acids. Reduced growth rates in Artemia-fed larvae were associated with lower levels of free methionine, indicating that this diet is deficient in methionine for turbot larvae. Leucine might also be limiting turbot growth as the different diet organisms had lower levels of this amino acid in the free pool than was found in the larval protein. A previously presented model was used to describe the flux of amino acids in growing turbot larvae. The FAA pool was found to be small and variable. It was estimated that the daily dietary amino acid intake might be up to ten times the larval FAA pool. In addition, protein synthesis and protein degradation might daily remove and return, respectively, the equivalent of up to 20 and 10 times the size of the FAA pool. In an early phase (Day 11) high growth rates were associated with a relatively low protein turnover, while at a later stage (Day 17), a much higher turnover was observed. Received: 19 March 1997 / Accepted: 14 April 1997     

Weaning experiments with turbot (Scophthalmus maximus): Electron microscopic study of liver

Weaning, i.e. the change from live to dry food, is a critical stage in the rearing of turbot (Scophthalmus maximus). In order to obtain a more detailed understanding of the associated functional processes, an electron microscopical study of liver morphological alterations before, during and after the weaning process was conducted. Three days before the start of weaning, slight malnutrition of fish was suggested by histological features such as nuclear glycogen deposition, mitochondrial swelling, together with a low level of glycogen and lipid stores. Lipid material was repeatedly located within cisternae of endoplasmic reticulum. Intra-cisternal lipid deposition (steatosis) in the liver was dramatically increased 3 d after the start of weaning. At the end of the weaning period, steatosis disappeared while cytoplasmic lipid deposition was enhanced. Although the weaning process dramatically increased hepatocellular steatosis, this phenomenon is obviously not caused by weaning — since it was already present during the pre-weaning period. As a speculation, steatosis in the liver of larval turbot may be the result of ontogenetic changes in intermediary metabolism, with the magnitude of this process being controlled by nutritional factors.     

Individual growth and size-selective mortality of larval turbot (Scophthalmus maximus) reared in enclosures

Larval turbot (Scophthalmus maximus) were reared in a large marine enclosure and in plastic bags in southern Norway. Samples of larvae in the enclosure were taken during the first 12 d of life to estimate individual body growth based on back calculations from daily growth rings on their otoliths. Size selective mortality was documented for these larvae in the predator-free enclosure. Starvation in the laboratory occurred on the seventh day. In the enclosure, a mortality rate of 18.1% d^-1 prevailed. Our data indicates that the survivors beyond the starvation period are larger by 0.18 mm on average. This result is important with regard to the question of whether starvation is an important mechanism for larval mortality in the sea. A possible means of estimating the relative effects of starvation versus predation based on these results and the backcalculation technique is suggested.     

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).     

Karotypic characterization of turbot (Scophthalmus maximus) with conventional,fluorochrome and restriction endonuclease-banding techniques

A karyotypic analysis was carried out in turbot (Scophthalmus maximus) using conventional banding techniques, fluorochromes and restriction endonucleasebanding. The standard karyotype of turbot is 2n=44 chromosomes, with 48 chromosome arms. Nucleolar organizer regions, which were localized in the short arm of Chromosome Pair No. 3, showed C-and chromomycin A₃-positive staining. C-bands were mainly located at the centromeres, but were also present in some interstitial and telomeric locations. No differences were revealed among constitutive heterochromatin bands after treatment of fixed chromosomes with fluorochrome staining and restriction endonucleases. Digestion with DdeI restriction enzyme produced a pattern of interstitial banding which suggested some degree of differentiation along the chromosome arms in turbot. Neither variation in chromosome number of arm number nor polymorphism in nucleolar organizer regions was revealed in the turbot analyzed.     

Free amino acids as energy substrate in developing eggs and larvae of the cod Gadus morhua

The content of free amino acids (FAA) in the cod (Gadus morhua L.) egg is about 200 nmol at spawning, decreasing by about 100 nmol/egg during the egg stage and about 75 nmol/larva during the yolksac larval stage. Together, alanine, leucine, serine, isoleucine, lysine, and valine account for about 75% of the decrease. Ammonium accumulates gradually during the egg stage and is quickly excreted after hatching. The body protein content is maintained during the egg and yolksac larval stages. The measured oxygen uptake of the cod embryo during the egg and yolksac larval stages accounts for about 85% of the oxygen necessary to catabolize the FAA disappearing during this period. Ammonia excretion of the cod embryo, as taken from literature data, is similar to the expected ammonia production from catabolism of the FAA. Our data suggest that FAA are a major substrate for aerobic energy production in cod eggs and yolksac larvae. The implication of this finding for the production of a favourable first-feed for cod and other cultivated marine fish larvae, and for the selection of high quality eggs of marine fishes, is stressed.     

The renal and serum concentrations of calcium,magnesium and phosphorus in captive and wild turbot (Scophthalmus maximus)

Turbot (Scophthalmus maximus L.) reared in captivity suffer a hepato-renal syndrome, one of the characteristics of which is, on the basis of histological evidence, calcification of the renal tubules. The concentrations of calcium, magnesium and phosphorus were therefore compared in the kidney, the serum, and ultrafiltrates of the serum of wild turbot and of turbot reared in captivity at two separate sites. No differences in renal calcium, magnesium and phosphorus levels were found. Renal calcium levels were normal, being comparable to those found in other marine and freshwater fish. Serum from wild turbot contained significantly higher concentrations of both total and ultrafilterable magnesium than did serum from turbot reared in captivity. Less of the serum calcium of wild turbot was ultrafilterable than was the serum calcium of captive turbot. No other differences in serum levels of these elements were found between wild and captive turbot. The analyses do not suggest any relationship, either causal or indirect, between the hepato-renal syndrome and a disturbance of calcium/magnesium metabolism.     

Compartmental distribution of free amino acids and protein in developing yolk-sac larvae of Atlantic halibut (Hippoglossus hippoglossus)

Free amino acids (FAA) and protein have been measured in whole laboratory-readed halibut larvae and on dissected individuals separated into yolk and body compartments. At hatching both FAA and protein are mainly located in the yolk compartment. During the first 12 d of the yolk-sac stage more than 70% of the FAA pool disappeared from the yolk without any significant changes in the yolk protein pool. This suggests different uptake mechanisms for FAA and protein from the yolk, and a sequential utilisation of the endogeneous reservoirs of free and protein amino acids in Atlantic halibut larvae. The data suggest that in the early yolk-sac stage FAA enter the embryo from the yolk and are utilised both for energy and protein synthesis. Later on when the free pool cannot fulfil the nutritional requirements, additional amino acids are recruited from yolk protein. Of the total amino acids (free + protein amino acids) present at hatching ca. 60% will be used as precursors for body protein synthesis while the remaining 40% are used as fuel in the larval energy metabolism.     

Sequential utilization of free amino acids, yolk proteins and lipids in developing eggs and yolk-sac larvae of barfin flounder Verasper moseri

Changes in the contents of free amino acids (FAA), M_r 170,000 lipovitellin (oLv B), that is the major yolk protein in ovulated eggs, and lipids were measured in developing eggs and yolk-sac larvae of barfin flounder (Verasper moseri) to elucidate the sequential utilization of these nutrient stocks before the first feed. Hatching takes place on the 10th day after fertilization at a water temperature of 8°C, and the hatched larvae absorb almost all of their yolk masses within 21 days after fertilization. The total FAA content showed no change during the first 4 days, then decreased to about 13% of the initial level by the 13th day after fertilization. During the consumption of FAA, non-essential amino acids tend to decrease earlier than essential amino acids. The native molecular weight and immunoreactivity of M_r 170,000 oLv B did not show any change during the first 16 days after fertilization, though the sodium dodecyl sulphate-polyacrylamide gel electrophoresis patterns of oLv B showed a serial change. The oLv B contents, measured by quantitative immunodiffusion using antiserum against oLv B of ovulated eggs, were approximately stable during the 13 days after fertilization, then decreased rapidly until the end of yolk-sac absorption. Phospholipids (PL), which seem mostly to bind with lipovitellin-proteins, decreased gradually after hatching coincident with the decrease in oLv B and M_r 330,000 lipoprotein. From these results, we conclude that there are three periods for sequential nutrient utilization in barfin flounder embryos and larvae: (1) pre-FAA utilization period, 0-4th day; (2) FAA utilization period, 4th-13th day; (3) oLv B and PL utilization period, 16th-21st day post-fertilization.     

Growth,under laboratory conditions,of turbot,Scophthalmus maximus,from the Ría de Vigo (north-west Spain)

Egg production rates of Calanus glacialis (Jaschnov) were measured in the laboratory (in 1985) and in the field (in 1983–1984). In the laboratory, daily egg production was studied over one month at alternating feeding and fasting conditions. Spawning ceased after 3 d of starvation and started as soon as females were reintroduced to food. Egg production increased stepwise at 3-d intervals. Females survived more than nine months in captivity. In the field, egg production was measured during PREMIZEX 1983 and MIZEX 1984 at 5 and 16 stations, respectively. High egg production was found in polynyas on the East Greenland Shelf, where melt water induced stratification which supported a spring bloom. Highest egg production was converted into 6.1% body carbon female^-1 d^-1. Under thick pack-ice no eggs were spawned. Spawning was induced in females from a station with low food abundance by feeding them on board ship. These results from both experiments and field studies show that egg production in C. glacialis is closely related to food availability. Thus, C. glacialis exhibits a reproductive behavior similar to that of C. finmarchicus, but not C. hyperboreus, the other two dominant species in this region.