During the commercial slaughter of farmed turbot (Scophthalmus maximus), a total of 67 fish were, on six occasions, removed from their rearing conditions at 14°C and put, as is standard commercial practice, into chilled seawater (-1.5 to -0.8°C) to monitor behavioural, muscular, osmoregulatory and respiratory responses during chilling time (90 min). Results show that a thermal insult alters the iso-osmotic balance, leading not only to an Na+ influx and an intracellular release of Ca2+ and K+, but also to a disturbance of respiratory function, leading to acidosis as a result of H+ and CO2 accumulation, increased pCO2 and reduced HCO3− in the blood. Once the internal temperature dropped below 1°C, the muscles contracted (cold shortening) and, although the fish were still alive, they reverted to a state of rigor, leading to a complete breakdown in their ability to move or ventilate and resembling an unconscious condition or death. Remarkably, the fish were able to prevent themselves undergoing hypoxia as pO2 remained within acceptable limits. No changes in muscle pH were observed and, thus, no noted effects on textural properties. We conclude that live chilling from 14°C to approximately -1°C is a highly questionable practice. It causes physical and physiological changes that are generally associated with stress and, in the case of observed forced muscle contractions, could lead to severe pain. Furthermore, we conclude that cold shortening associated with chilling can be easily mistaken for rigor mortis and, as such, should be subject to further attention in future research on quality.

H+-pyrophosphatases (H+-PPases) are integral membrane proteins that couple pyrophosphate energy to an electrochemical gradient across biological membranes and promote the acidification of cellular compartments. Eukaryotic organisms, essentially plants and protozoan parasites, contain various types of H+-PPases associated with vacuoles, plasma membrane and acidic Ca+2 storage organelles called acidocalcisomes. We used Lysotracker Red DND-99 staining to identify two acidic cellular compartments in trophozoites of the marine scuticociliate parasite Philasterides dicentrarchi: the phagocytic vacuoles and the alveolar sacs. The membranes of these compartments also contain H+-PPase, which may promote acidification of these cell structures. We also demonstrated for the first time that the P. dicentrarchi H+-PPase has two isoforms: H+-PPase 1 and 2. Isoform 2, which is probably generated by splicing, is located in the membranes of the alveolar sacs and has an amino acid motif recognized by the H+-PPase-specific antibody PABHK. The amino acid sequences of different isolates of this ciliate are highly conserved. Gene and protein expression in this isoform are significantly regulated by variations in salinity, indicating a possible physiological role of this enzyme and the alveolar sacs in osmoregulation and salt tolerance in P. dicentrarchi.

In this study, we chose a carnivorous fish, turbot (Scophthalmus maximus L.), to examine its nutrient-sensing and metabolic responses after ingestion of diets with fishmeal (FM), or 45 % of FM replaced by soyabean meal (34·6 % dry diet) balanced with or without essential amino acids (EAA) to match the amino acid profile of FM diet for 30 d. After a 1-month feeding trial, fish growth, feed efficiency and nutrient retention were markedly reduced by soyabean meal-incorporated (SMI) diets. Compared with the FM diet, SMI led to a reduction of postprandial influx of free amino acids, hypoactivated target of rapamycin signalling and a hyperactivated amino acid response pathway after refeeding, a status associated with reduced protein synthesis, impaired postprandial glycolysis and lipogenesis. These differential effects were not ameliorated by matching an EAA profile of soyabean meal to that of the FM diet through dietary amino acid supplementation. Therefore, this study demonstrated that the FM diet and SMI diets led to distinct nutrient-sensing responses, which in turn modulated metabolism and determined the utilisation efficiency of diets. Our results provide a new molecular explanation for the role of nutrient sensing in the inferior performance of aquafeeds in which FM is replaced by soyabean meal.

In the present study, a linear regression analysis between lysine intake and lysine retention was conducted to investigate the efficiency of lysine utilisation (kLys) at marginal lysine intake of either protein-bound or free lysine sources in juvenile turbot (Psetta maxima). For this purpose, nine isonitrogenous and isoenergetic diets were formulated to contain 2·25–4·12 g lysine/100 g crude protein (CP) to ensure that lysine was the first-limiting amino acid in all diets. The basal diet contained 2·25 g lysine/100 g CP. Graded levels of casein (Cas), fishmeal (FM) and l-lysine HCl (Lys) were added to the experimental diets to achieve stepwise lysine increments. A total of 240 fish (initial weight 50·1 g) were hand-fed all the experimental diets once daily until apparent satiation over a period of 56 d. Feed intake was significantly affected by dietary lysine concentration rather than by dietary lysine source. Specific growth rate increased significantly at higher lysine concentrations (P< 0·001). CP, crude lipid and crude ash contents in the whole body were affected by the dietary treatments. The linear regression slope between lysine retention and lysine intake (kLys) was similar between all the dietary lysine sources. The kLys values for the diets supplemented with Cas, Lys or FM were 0·833, 0·857 and 0·684, respectively. The bioavailability of lysine from the respective lysine sources was determined by a slope-ratio approach. The bioavailability of lysine (relative to the reference lysine source Cas) from FM and Lys was 82·1 and 103 %, respectively. Nutrient requirement for maintenance was in the range of 16·7–23·4 mg/kg0·8 per d, and did not differ between the treatments. There were no significant differences in lysine utilisation efficiency or bioavailability of protein-bound or crystalline lysine from the respective sources observed when lysine was confirmed to be the first-limiting nutrient.

Several species of opportunistic histophagous scuticociliates have been implicated in systemic infections of farmed fish. In turbot, scuticociliatosis is an emerging disease, and the identification of the parasite species involved is controversial. We have previously isolated Philasterides dicentrarchi from farmed turbot scuticociliatosis outbreaks in northwest Spain. In the present study, we report detailed ultrastructural studies of this parasite, and investigate phylogenetic relations with other members of the order Philasterida on the basis of sequence comparison of the small-subunit rRNA (SSUrRNA) gene. Ultrastructural study indicates the presence of dikinetids in the anterior two-thirds of the body; micronucleus closely associated with the macronucleus, though not physically connected; numerous mitochondria located below the cell cortex, parallel to the surface; numerous spherical and fusiform extrusomes located close to the plasma membrane. We consider that these characteristics are useful for diagnosis of infections by this parasite. A nested 350-bp nucleotide sequence of the SSUrRNA gene of the turbot P. dicentrachi isolate showed high identity with previously reported SSUrRNA gene sequences from 2 scuticociliates isolated from olive flounder Paralichthys olivaceus in Korea, namely P. dicentrarchi (98%) and Miamiensis avidus (99%); conversely, our P. dicentrarchi sequence showed low identity (86%) with that of Uronema marinum, a scuticociliate that has also been implicated in scuticociliatosis outbreaks in turbot in Europe and olive flounder in Asia. Phylogenetic tree construction on the basis of the SSUrRNA gene sequences, using the neighbour-joining method, confirm that the different P. dicentrarchi isolates and M. avidus are closely related and a possible synonymy between both ciliates species should be considered.

Turbot juveniles (45 g) were exposed for 41 d (17 °C, 34‰ salinity) to constant normoxic (100–100% air saturation, 100–100) or moderate hypoxic (75–75% air saturation, 75–75) conditions and to repeated hypoxic shocks (20% saturation for 1 h, 5 d per week) from normoxic (100–20% air saturation, 100–20) or moderate hypoxic (75–20% air saturation, 75–20) conditions. A normoxic group was feed restricted (100-FR). Mass increase of 100–100 and 75–75 groups fed to satiation was not significantly different. In comparison, it was significantly lower in the 100–20 and 75–20 groups (NS between the two hypoxic shocks groups). Intermediate results were obtained in the 100–100-FR group. The lowest mass increase under hypoxic shocks was explained by a significant decrease in both feed intake and food conversion efficiency (FCE). FCE was lower in the two hypoxic groups, but only the 75–20 group was significantly different from all the other groups. There was no sign of stress and no change in the physiological status of fish in any group. When challenged, pre-conditioning of turbot to regular hypoxic shocks extended survival time, slightly but significantly, for 50% of the population. It was 8 h longer in starved than in fed fish. When reared for 1 year in normoxic water, the growth rate of post-challenged survivors was dependent on pre-conditioning: day 0–375 specific growth rate was significantly higher in the two groups acclimated to repeated hypoxic shocks. In the second experiment, it was shown that exposure to 20% air saturation for 12 h led to major physiological changes within 4 h: a significant decrease in plasma total CO2 and increase in plasma lactate contributing in maintaining blood pH stable, and a significant increase in osmolarity and chloride concentration. When returned to normoxic water, the recovery capacity of the fish was high: plasma osmolarity and total CO2 returned to pre-exposure levels within 1 h. The results are discussed in terms of turbot capacity to cope with repeated hypoxic shocks and to acclimate.

A new Myxozoa species causing enteritis and death in cultured turbot, Scophthalmus maximus, is described at light and electron microscope levels. In addition, small subunit ribosomal RNA gene sequences (SSU rDNA) from the new species and from similar myxozoans were obtained and used for phylogenetic inference, as complementary criteria to resolve its taxonomic classification. The new parasite is closely related to Myxidium leei, another enteric histozoic species from marine fish. However, the ascription of M. leei to the genus Myxidium was based on weak morphological evidence and is not supported by our rDNA data analysis. A close relationship with Zschokkella, the other morphologically related myxozoan genus is also not supported. The combined morphological and molecular study results in the establishment of the new genus Enteromyxum to accommodate the new species E. scophthalmi, and the former M. leei, which is transferred to the new genus as Enteromyxum leei (Diamant, Lom & Dyková 1994) n. comb. This genus of marine, histozoic and enteric myxozoans includes significant parasite species for marine finfish culture.

This study investigated the spatial distribution of Tetramicra brevifilum spores in the musculature of infected turbot Scophthalmus maximus, with the aim of identifying the most appropriate body locations for diagnostic assays. A PCR protocol optimized for the detection of T. brevifilum spores in turbot muscle is also described. In fish showing low- and moderate-intensity infection, the spatial distribution of spores was best fitted by a negative binomial distribution, indicating a clumped spatial pattern; the negative binomial coefficient k was lower for fish with low-intensity infection, indicating a more markedly clumped pattern in these fish. In fish with high-intensity infection, the spatial distribution of spores was best fitted by the Poisson distribution, indicating a random pattern. In both low- and moderate-intensity infection, spores were present at highest density in the musculature adjoining the dorsal fins. Samples for PCR were therefore obtained from this location. PCR amplification was of the small subunit ribosomal DNA (SSUrDNA), using a pair of species-specific primers that amplify the 1250 bp product. The PCR protocol developed showed better sensitivity than microscopical techniques (detection rate by microscopy 25%, versus 42% by PCR), suggesting that it may be useful for routine screening for Tetramicra brevifilum infection in cultured turbot.

The effect of different parameters on short-term storage capacity of turbot ova was assessed over a 45-h period after ova collection for fertilization rates and over a 9-h period after ova collection for hatching rates. Increasing the volume of ova sampling from 0.5 to 2.5 mL, as well as adding an antibiotic-antimicotic solution or oxygen did not significantly change the storage capacity of ova. Regarding the hatching rates, a higher storage ability was recorded at 8 and 13 °C, compared to 3 °C. The mean composition of the ovarian fluid was determined (n = 57 spawns). Use of a diluent mimicking the ovarian fluid significantly decreased the storage ability as assessed by the fertilization rates but did not modify the hatching rates. Diluting ova in an artificial ovarian fluid deprived of calcium significantly decreased the fertilization and hatching rates during the storage period. Furthermore, addition or not of soybean trypsin inhibitor (Sigma T 9003) to the artificial ovarian fluid deprived of calcium did not significantly change the results. Storage capacity of control batches of ova was low: at 13 °C, without any diluent and when ova were fertilized 3 h after stripping, the hatching rate was lowered to 62.4 ± 29.4 % (mean ± SD) of the initial value.

Techniques were developed for labelling prey, and for gut content analysis in fish larvae. Labelling consisted of a short feeding period of the prey (rotifer and Artemia metanauplii) on black-drawing ink before supplying them to the larvae. Prey labelling was rapid and remained stable for at least 3 h when transferred into seawater. In addition, a method was tested to facilitate the observation of larval gut contents. The method, based on Dawson's technique, involved fixation, removal of lipids and clearance of turbot (Psetta maxima) larvae, which become transparent and ready for gut content analysis. This treatment did not interfere with the black ink label of the ingested prey.

The survival of turbot eggs and the rearing capacities of larvae stemmed from artificial fertilization practices using frozen-thawed spermatozoa were evaluated. Furthermore, the viability of sperm samples stored during a 9 month period in liquid nitrogen was assessed. No significant difference in the fertilization rate, hatching rate, survival and wet weight of 10-day old larvae were observed using fresh or frozen-thawed spermatozoa. The motility recorded at 10 s and 60 s post-activation and the fertilization capacity of frozen-thawed spermatozoa were not significantly decreased during a 9 month storage period in liquid nitrogen. These results confirm the high quality of the turbot spermatozoa stemmed from the cryopreservation process, allowing their use for routine aquaculture practices.

The capability of turbots to use self-feeders was studied using 3 groups of 100 juveniles. Feeding activity reached a plateau after 20 days. Almost all feeding activity was observed during the photophase, and the acrophase was associated with dawn. The proportion of nocturnal demands was the highest in the replicate with the lowest feed efficiency, suggesting that feed waste was mainly occurring during nocturnal feeding activity. During the last 30 days of the experiment, demands were only rewarded during two periods of 2.5 h.day−1. Feed efficiency was improved and was homogeneous between the three tanks, but the voluntary feed intake was reduced. It is concluded that turbots are able to use self-feeders but they do not adapt quickly, and they are not able to compensate a reduction of feed access by increasing demand activity during the access period.

Gastrointestinal evacuation of inert X-ray dense marker by turbot, Psetta maxima, was followed by use of radiography. Groups of individually tagged turbot aged 1 + years (136.5 g), were held at 16 °C and acclimatized to a feeding period of 4 hours per day. Fish were fed on pelleted feed in excess. The experimental design allowed a test of the effects of handling stress and post-prandial feeding on gastrointestinal evacuation. Repeated handling affected feed intake, but there were no significant treatment effects on gastrointestinal evacuation rate. Fish that ingested large meals displayed higher evacuation rates of markers than fish which ingested smaller meals, but total evacuation time was longer for the fish that consumed the largestmeals. Marker particles started to be voided from the gastrointestinal tract approximately 10 hours following initiation of a meal. and evacuation of marker was completed within 52 hours under the holding conditions employed in the present study.

Short-term storage over several days as well as cryopreservation of turbot (Scophthalmus maximus) sperm were studied. Two extenders, Ringer 200 and artificial seminal liquid (ASL2), are suggested for semen collection in order to avoid the deleterious effect of urine contamination, and for the purpose of short-term storage between 0 and 15 °C. Oxygen atmosphere is not suitable for turbot sperm storage. Turbot spermatozoa undergo cryopreservation with a high rate of success especially in a sucrose solution with 10% dimethyl sulfoxide (DMSO) and 10% egg yolk.

The proportion of vibrio E, dominant in healthy turbot larvae, was artificially increased in the rotifer enrichment medium. The main effect of this vibrio enrichment was to improve the resistance of larval turbot challenged with a pathogenic strain of Vibrio splendidus, vibrio P. The improvement of the survival rates was significant with 1.5 × 106 CFU of vibrio E added per ml of enrichment medium, and it was still maximum at the highest dose tested (5 × 107 CFU.ml−1; 80 ± 2% of survivors after 48 h of challenge, versus 50 ± 2% for the control group).

Further in vitro characterisation showed that vibrio E was able to grow in the presence of the chelating agents EDDHA and EDTA, while producing siderophore, as revealed by chrome azurol S dyeing. The pathogenic vibrio P did not grow on iron-depleted medium, and it did not seem to produce siderophore. Consequently, a second experiment attempted to appraise the role of iron availability in the interaction between the pathogenic and probiotic vibrios. To this end, turbot larvae were fed with rotifers treated by the bacterial siderophore deferoxamine. This treatment significantly increased the resistance of larvae chal-lenged with vibrio P in comparison with the control group, but to a lesser extent than the group enriched with vibrio E. There was also an increase of the growth rate due to either the siderophore treatment or thevibrio enrichment, as well. No further improvement was observed when the rotifers were simultaneously both treated and enriched. It was concluded that the probiotic effect of vibrio E may be at least partly due to competition for iron with the pathogen.

This paper presents some features of turbot sperm and compares them with data reported in the literature on other freshwater and marine fish. In captivity, turbot spermiation period lasts 6 months. When compared to other fish species, turbot males are characterized by low values of maximum gonadosomatic index (0.6-0.8%) and are poor sperm producers in terms of volume (0.2-2.2 ml), concentration (0.7-11.0 × 109 spermatozoa/ml) and total sperm number released per stripping (0.2-12.0 × 109 spematozoa). Compared to most freshwater species, the duration of cell movement is long (1-17 min). The spermatozoon of turbot can be considered to be of a primitive type. A very high protein content (8.8 mg/ml) is found in the seminal fluid of turbot. Increasing collection frequency of turbot males, from monthly to fortnightly and weekly stripping, results in the release of successive samples presenting decreasing semen volume and spermatozoa concentration. Similar spermatozoa concentration and total volume of semen produced per turbot, during a 2 month experimental period, are released from males submitted to an annual light and temperature cycle, compared to those receiving a 6 month contracted light and temperature schedule.

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).