Gill area and dimensions of gilthead sea bream Sparus aurata L.

Detailed measurements of gill area and constituent variables (total filament length, lamellar frequency and bilateral area) were performed on both hemibranchs of all eight arches in six specimens of gilthead sea bream Sparus aurata (mean ±s.e . 49·9 ± 0·2 g). Shrinkage was also quantified and results were corrected accordingly. Filament number decreased from the first to the fourth gill arch, and average bilateral area of secondary lamellae was higher in the second and third arches. Total and mean filament length, total number of secondary lamellae and total gill area (A_TG) were lower in posterior than in anterior hemibranchs of the second, third and fourth gill arches; while the opposite was observed for the first arch. Lamellar frequency was increased in posterior hemibranchs of all arches compared to that in anterior hemibranchs, especially at the fourth arch. Comparison of the actually measured A_TG and constituent variables with estimates revealed that the third gill arch is the most representative for appropriate measurements and that any of its components (even one hemibranch) approximates the best A_TG (within the range of 0·2–4·3%, P > 0·05) and related dimensions. Consequently, necessary measurements were restricted to the posterior hemibranch of the third gill arch, and A_TG and dimensions (y) were estimated in 21 specimens (23·5–217·6 g) and correlated to body mass (M) according to the allometric equation y = aM^b. As fish increased in size, A_TG (b= 0·664), total (b= 0·425) and mean (b= 0·323) filament length, total number of filaments (b= 0·103) and secondary lamellae (b= 0·377), as well as average lamellar bilateral area (b= 0·288), increased, while the opposite was observed for lamellar frequency (b=?0·049) and mass‐specific area (b=?0·336). Data obtained are discussed in relation to S. aurata activity and living ethology.     

Evaluation of DNA damage in rainbow trout (Oncorhynchus mykiss) and gilthead sea bream (Sparus aurata) cryopreserved sperm

Cryopreservation causes several types of damage to spermatozoa, such as loss of plasma membrane integrity and functionality, loss of motility, and ATP content, resulting in decrease of fertility rates. This spermatozoal damage has been widely investigated for several marine and freshwater fish species. However, not much attention has been paid to the nuclear DNA. The objective of this study was to determine the degree to which cryopreservation induces spermatozoal DNA damage in two commercially cultured species, rainbow trout (Oncorhynchus mykiss) and gilthead sea bream (Sparus aurata), both of which could benefit from the development of cryopreservation strategies on a large scale. We have used the single-cell gel electrophoresis, commonly known as Comet assay to detect strand breaks in DNA. This technique was performed on fresh and cryopreserved sperm from both species. In rainbow trout there was a significant increase in the averages of fragmented DNA and Olive tail moment after cryopreservation (11.19-30.29% tail DNA and 13.4-53.48% Olive tail moment in fresh and cryopreserved sperm, respectively), as well as in the proportion of cells with a high percentage of DNA fragmentation. For gilthead sea bream there were no significant differences in the percentage of tail DNA between the control samples and sperm diluted 1:6 and cryopreserved (28.23 and 31.3% DNA(t), respectively). However, an increase in the sperm dilution rate produced an increase in the percentage of DNA fragmentation (41.4%). Our study demonstrates that cryopreservation can induce DNA damage in these species, and that this fact should be taken into account in the evaluation of freezing/thawing protocols, especially when sperm cryopreservation will be used for gene bank purposes.     

Branchial release of free cortisol and melatonin by rainbow trout

Individual rainbow trout Oncorhynchus mykiss were held in a specially constructed tank that enabled water to be collected separately from the anterior and posterior ends of the fish. Measurement by radioimmunoassay showed that >95% of the cortisol and melatonin released into the water originated from the anterior end (dominated by the gills). High performance liquid chromatography confirmed the identity of both hormones.     

Effects of aluminium and cortisol on in vitro calcium deposition on otoliths in rainbow trout

Aluminium (1–100 μM) reduced in vitro calcium deposition on otoliths concentration-dependently in rainbow trout, but cortisol (1 and 10 μg ml⁻¹) had no effect.     

Sex-related variations of serum immunoglobulins during reproduction in gilthead sea bream and evidence for a transfer from the female to the eggs

A significant elevation of serum immunoglobulin (Ig) concentration occurred in female gilthead sea bream Sparus aurata during spawning. Furthermore, a progressive rise of serum Ig level was observed throughout the process of sexual inversion (from functional male to functional female), suggesting that the synthesis of Ig could be regulated by sex-related factors (probably sexual hormones) involved in the process of oogenesis. The immunoglobulins of eggs were purified by affinity chromatography on protein A-sepharose. SDS-PAGE and Western blot analysis showed reactivity of the antiserum Pab1 with the Ig heavy and light chains, and some degradation products. This purification process yielded detectable amounts of Ig. The sex-related increase of serum Ig during the reproductive period, and the detection of Ig in eggs suggest a transfer of Ig from the blood of the adult female.     

A non‐invasive stress assay based upon measurement of free cortisol released into the water by rainbow trout

A procedure previously used for sex steroids was adapted to extract free cortisol and cortisone from water samples taken from rainbow trout Oncorhynchus mykiss tanks. Both corticosteroids could be readily detected by radioimmunoassay (RIA), with cortisol being predominant. All stages of the sampling, extraction and RIA procedure were validated for cortisol. An intermittent problem with poor replication was traced to the use of diethyl ether during the extraction procedure, and was overcome by the use of ethyl acetate. Other modifications were also introduced to speed up the procedure. The concentration and time course of release of both corticosteroids were shown to be related to the degree of stress that the fish had been subjected to. It was confirmed that cortisol concentrations in water and estimated cortisol release rates increased in response to handling stress, and that both were correlated with plasma cortisol concentrations. The potential for using water cortisol concentration and release rates to assess the primary stress response of fishes as a non‐invasive alternative to blood sampling is discussed.     

Poststress levels of lysozyme and cortisol in adult rainbow trout: heritabilities and genetic correlations

Lysozyme response in stressed rainbow trout was compared with measurements of poststress cortisol activity. Estimates of heritability of lysozyme and cortisol were both moderate-to-high and both traits displayed positive genetic correlations in pair-wise comparisons of stress exposures. Genetic correlations between lysozyme and cortisol in stressed rainbow trout tended to be negative, although insignificant. Neither lysozyme, nor cortisol exhibited significant correlations with serum haemolytic activity. It is concluded that the data do not confirm earlier suggestions that lysozyme should be superior to cortisol in consistency of stress response in rainbow trout.     

Cortisol and immune characteristics in rainbow trout (Oncorhynchus mykiss) selected for high or low tolerance to stress

The influence of exposure to stressors on cortisol and the non-specific immune traits lysozyme and serum haemolytic activity were examined in second generation rainbow trout ( Oncorhynchus mykiss ) selected for either high or low serum cortisol level following a confinement stress. Lysozyme and serum haemolytic activity were also assessed, together with levels of specific antibodies against Aeromonas salmonicida A-layer, Vibrio salmonicida O-antigen and Vibrio anguillarum O-antigen, following injection of vaccines against these pathogens.
Significant differences in mean cortisol levels between the two selection lines were observed, but in only one of two stress experiments was the 'high-stress' line found to have the higher cortisol level; in the other experiment the 'high-stress' line had significantly lower cortisol levels than the 'low-stress' line. Lysozyme levels were in four of four assessments higher in the high-stress line than in the low-stress line, whereas components of serum haemolytic activity tended to be lower in the high-stress line than in the low-stress line. Levels of specific antibodies against all three bacterial pathogens were elevated following the injection of the vaccines. Only antibody production against A. salmonicida A-layer was significantly different between the two lines, the higher production of antibody being in the high-stress line.     

Quantitative trait loci for resistance to fish pasteurellosis in gilthead sea bream (Sparus aurata)

Fish pasteurellosis is a bacterial disease causing important losses in farmed fish, including gilthead sea bream, a teleost fish of great relevance in marine aquaculture. We report in this study a QTL analysis for resistance to fish pasteurellosis in this species. An experimental population of 500 offspring originating from eight sires and six dams in a single mass‐spawning event was subjected to a disease challenge with Photobacterium damselae subsp. piscicida (Phdp), the causative agent of fish pasteurellosis. A total of 151 microsatellite loci were genotyped in the experimental population, and half‐sib regression QTL analysis was carried out on two continuous traits, body length at time of death and survival, and for two binary traits, survival at day 7 and survival at day 15, when the highest peaks of mortality were observed. Two significant QTLs were detected for disease resistance. The first one was located on linkage group LG3 affecting late survival (survival at day 15). The second one, for overall survival, was located on LG21, which allowed us to highlight a potential marker (Id13) linked to disease resistance. A significant QTL was also found for body length at death on LG6 explaining 5–8% of the phenotypic variation.     

Differences in interrenal tissue, biosynthetic capacity and ACTH sensitivity in progeny of sea bream from parents selected for high or low cortisol response

Progeny of sea bream Sparus aurata from parents selected for high or low cortisol response to stress also showed similar divergent cortisol response after 48 h of handling and confinement stress. The high response progeny, however, had a lower basal unstimulated cortisol release and ACTH-stimulated cortisol output in vitro .     

Contrasting seasonal modulation of the stress response in male and female rainbow trout

Between June and September the magnitude of the plasma cortisol response of maturing male and female rainbow trout to confinement was indistinguishable. A progressive increase in confinement-induced cortisol levels in both sexes occurred during May to September, associated with the seasonal rise in water temperature. Between September and January a reduction of >50% in the magnitude of the cortisol response to confinement in male fish (but no decline in females) coincided with declining water temperature and significant increases in plasma 11-ketotestosterone and elevated plasma testosterone levels. Plasma oestradiol-17β levels were significantly greater in females than males throughout the study period and this difference was maximal between September and January. However, plasma testosterone was also elevated in females during this period and levels overall were higher than those in male fish. Previous studies have shown oestradiol-17β and testosterone to have diametrically opposed effects on stress responsiveness in trout, with the former enhancing, and the latter suppressing, the cortisol response to a stressor. The relative roles of androgens, estrogen and water temperature in modulating the stress responsiveness of rainbow trout are discussed.     

Food deprivation alters osmoregulatory and metabolic responses to salinity acclimation in gilthead sea bream Sparus auratus

The influence of acclimation to different environmental salinities (low salinity water, LSW; seawater, SW; and hyper saline water, HSW) and feeding conditions (fed and food deprived) for 14 days was assessed on osmoregulation and energy metabolism of several tissues of gilthead sea bream Sparus auratus. Fish were randomly assigned to one of six treatments: fed fish in LSW, SW, and HSW, and food-deprived fish in LSW, SW, and HSW. After 14 days, plasma, liver, gills, kidney and brain were taken for the assessment of plasma osmolality, plasma cortisol, metabolites and the activity of several enzymes involved in energy metabolism. Food deprivation abolished or attenuated the increase in gill Na⁺,K⁺-ATPase activity observed in LSW- and HSW-acclimated fish, respectively. In addition, a linear relationship between renal Na⁺,K⁺-ATPase activity and environmental salinity was observed after food deprivation, but values decreased with respect to fed fish. Food-deprived fish acclimated to extreme salinities increased production of glucose through hepatic gluconeogenesis, and the glucose produced was apparently exported to other tissues and served to sustain plasma glucose levels. Salinity acclimation to extreme salinities enhanced activity of osmoregulatory organs, which is probably sustained by higher glucose use in fed fish but by increased use of other fuels, such as lactate and amino acids in food-deprived fish.     

Primary and secondary indices of stress in the progeny of rainbow trout (Oncorhynchus mykiss) selected for high and low responsiveness to stress

A series of pooled gamete matings was carried out employing eggs and milt from mature male and female rainbow trout selected for a consistently high- or low-responsiveness to stress, as indicated by post-stress plasma cortisol elevation. Development of the progeny was closely monitored and the responsiveness to stress of the progeny of high-responding parents and the progeny of low-responding parents was assessed by two methods. For a period of 14 months, at approximately monthly intervals, the plasma cortisol elevation evoked by a standardized confinement stress was determined in fish from each group, and secondly, on one occasion, the time-course of the plasma cortisol response to a 24-h period of confinement was monitored. Progeny of high-responding parents snowed a significantly greater cortisol response to stress than the progeny of low-responding parents during both testing procedures. However, when the effect of a 14-day confinement stress was examined, high-responding progeny showed a more rapid recovery of plasma cortisol levels, while levels in the low-responding progeny, although initially lower, showed a more sustained elevation. To assess the possible functional implications of these observations, circulating lymphocyte numbers, an immunologically important cortisol-sensitive component of the blood cell complement, were determined. The duration of the lymphocytopenia observed following the onset of confinement was found to be related to the initial, not the sustained, cortisol response. These data suggest that manipulation of the sensitivity to stress of fish is feasible by selective breeding, but that careful.choice of the indices employed to identify traits considered desirable is necessary.     

Homologous growth hormone (GH) binding in gilthead sea bream (Sparus aurata). Effect of fasting and refeeding on hepatic GH-binding and plasma somatomedin-like immunoreactivity

Specific binding of gilthead sea bream growth hormone (sbGH) to liver membrane preparations was a time and temperature dependent process, and was saturable by increasing amounts of membrane proteins. Scatchard analysis evidenced a single class of high-affinity and lowcapacity binding sites. Ovine prolactin, recombinant tilapia prolactin, carp gonadotropin and chinook salmon gonadotropin did not compete for the¹²⁵I-sbGH binding sites, while recombinant trout GH, bovine GH and human GH displaced iodinated sbGH in a dose dependent-manner. IGF-I-like immunoreactivity was detected after acidification of plasma and removal of IGF-I binding activity. A parallel displacement to the rhIGF-1 standard was observed with extracted plasma samples. Free and total hepatic GH-binding decreased during long-term starvation (3–9 weeks), returning to control values during the refeeding period. Plasma IGF-I-like immunoreactivity showed a similar trend. To our knowledge, this is the first report that indicates a coordinated regulation of GH-binding and plasma somatomedin-like activity in a typical marine fish.     

The influence of social interaction on the acclimation of rainbow trout, Oncorhynchus mykiss (Walbaum) to chronic stress

To establish the time required for fish to acclimate to experimental conditions, comprising holding within a spatially restrictive environment, rainbow trout were transferred from rearing tanks (1500 1) to smaller volume holding tanks (50 1). The fish were maintained singly, in pairs, in groups of five fish, and in groups of 10 fish, and selected indices of physiological stress were monitored at intervals following the onset of confinement. In all cases transfer and confinement provoked a stress response in the fish, indicated by significantly elevated plasma cortisol levels, and significantly reduced numbers of circulating lymphocytes. Recovery of these parameters to levels similar to unstressed control fish, was most rapid among the fish confined singly, requiring 2 to 4 weeks, although weight gain, indicative of feeding, was apparent within 1 to 2 weeks. Acclimation of the fish confined as pairs was severely confounded by the development of hierarchical relationships between the fish. This resulted in some individuals displaying rapid acclimation and growth, in contrast to other fish within the groups which failed to acclimate, exhibited signs of chronic stress, and ultimately succumbed to bacterial infections. Similar, though less severe, evidence of social stress was observed among the fish confined as groups of five. A decline in the coefficient of variance for weight within the fish confined as groups of 10 suggested an absence of growth inhibition due to social interaction among these fish. The implications of these results for experimental design are discussed.     

The genetic population structure and temporal genetic stability of gilthead sea bream Sparus aurata populations in the Aegean and Ionian Seas,using microsatellite DNA markers

We examined 662 gilthead sea bream Sparus aurata from wild samples of the species in the Aegean and Ionian Seas, using 20 EST-linked microsatellite markers, in three multiplex panels, as well as seven anonymous loci. Most of the markers were revealed to be highly polymorphic. We found low genetic differentiation between the sampling stations/areas with total F_ST 0.002 (P < 0.05). Based on comparison of five temporal samples, our results indicate genetic data consistency over time for all tested samples, pointing to stable populations, despite reported repeated escape events. Our results confirm the genetic population structure previously observed in these specific areas, using by far more markers than in previous studies in both coding and non-coding DNA loci. The limited genetic structure and the temporal genetic stability indicate neither major genetic differentiation of local populations by geographic isolation nor influence from anthropogenic factors. These results provide a baseline for future reference in any management programme of both wild and farmed population of S. aurata as well as of other aquaculture species with a potential introgression among farmed and wild populations.     

Differences in plasma cortisol and cortisone dynamics during stress in two strains of rainbow trout (Oncorhynchus mykiss)

Plasma levels of cortisone, a steroid hormone of potential physiological significance in fish, have rarely been measured. This study examines the interrelationship between circulating levels of cortisone and the major teleost corticosteroid, cortisol, in the blood of two strains of rainbow trout subject to confinement stress, a condition know to stimulate corticosteroidogenic activity. In unstressed fish from both strains, mean plasma cortisol levels were within the range 0.4–7.5 ng ml⁻¹. Mean plasma cortisone levels were within the range 7.1–15.9 ng ml⁻¹. Plasma cortisol levels were elevated within 5 min of the onset of strees and reached peak values within 45 min, although there was a marked difference betweed the maxima observed in the two strains (strain 1:70 ng ml⁻¹; strain 2:150 ng ml⁻¹). The rate of increase of plasma cortisone levels during strees was more rapid than that of cortisol, maximum values (strain 1:100ng ml⁻¹; strain 2:160 ng ml⁻¹) being reached within 10 to 20 min of the onset of stress. This rapid stress-induced elevation of plasme cortisone has not previously been reported in fish. We suggest that rapid conversion of cortisol to cortisone during the initial response to stress accounts for the appearance of large amounts of cortisone in the blood, indicating that circulating for the appearance of large amounts of cortisone in the blood, indicating that circulating levels of cortisol alone do not fully reflect the secretory activity of the interregnal during the initial of cortisol alone do not fully the secretory activity of the interregnal during the initial phase of the stress response. The results also indicate that the rate of clearance of cortisone from the circulation may be a major factor in determining stress-stimulated levels of plasma cortisol.     

Patterns of gastric evacuation, digesta characteristics and pH changes along the gastrointestinal tract of gilthead sea bream (Sparus aurata L.) and European sea bass (Dicentrarchus labrax L.)

A comparative study of gastric evacuation rates (GERs) and digesta content, moisture and pH values along the gastrointestinal tract was performed between gilthead sea bream and European sea bass. In order to distinguish species-specific differences from diet-elicited effects, all parameters were determined under either a fishmeal diet or a carob seed germ meal diet that contained high levels of total and soluble non-starch polysaccharides. GERs were significantly different between species and they were not affected by diet. Similarly, species-specific patterns were revealed in the distribution of digesta and water content along the gastrointestinal tract. In sea bream, stomach digesta and water content decreased with time, whereas in sea bass stomach retained the highest digesta and water content throughout the sampling period. The anterior and distal intestine exhibited the lowest accommodating capacities of digesta and water in either species. Overall, sea bream performed stomach digestion at lower hydration levels and higher pH compared with sea bass. Diet affected stomach moisture in both species and pH of stomach digesta in sea bass and of all intestinal sections in sea bream. The results obtained indicated that water and inorganic ion exchanges through the gut may differentiate between the species and warrant further investigation.     

Development of a 37 k high-density oligonucleotide microarray: a new tool for functional genome research in rainbow trout

A rainbow trout high-density oligonucleotide microarray was constructed using all tentative consensus (TC) sequences that are publicly available from all international rainbow trout Oncorhynchus mykiss genomic research projects through the Rainbow Trout Gene Index database. The new array contains 60-mer oligonucleotide probes representing 37 394 unique TC sequences and 1417 control spots. The array (4 × 44 format) was manufactured according to the design by Agilent Technologies using the inkjet-based SurePrint technology (design number 016320). The performance of the new microarray platform was evaluated by analysing gene expression associated with rainbow trout, vitellogenesis-induced muscle atrophy. This microarray will open new avenues of research that will aid in the development of novel strategies for genetic improvement for economically important traits benefiting the salmonid aquaculture industries.