Ontogenesis of opercular deformities in gilthead sea bream Sparus aurata: a histological description

The aim of this study was to characterize histological changes during opercular osteogenesis in farmed gilthead sea bream Sparus aurata larvae from 7 to 69 days post hatching (dph) and compare normal osteogenesis with that of deformed opercles. Mild opercular deformities were first detected in 19 dph larvae by folding of the opercle's distal edge into the gill chamber. Here, the variation in the phenotype and the irregular bone structure at the curled part of the opercles is described and compared with the histology of normal opercles. Results indicated that deformed opercles still undergo bone growth with the addition of new matrix by osteoblasts at the opercular surface, especially at its edges. No significant difference was found in bone thickness between deformed and normal opercles. In addition to differences in bone architecture, differences in collagen fibre thickness between normal and deformed opercles were also found.     

Morphometric characterization of sharpsnout sea bream (Diplodus puntazzo) and gilthead sea bream (Sparus aurata) spermatozoa using computer-assisted spermatozoa analysis (ASMA)

As part of a larger study on sperm quality and cryopreservation methods, the present study characterized the head morphometry of sharpsnout sea bream (Diplodus puntazzo) and gilthead sea bream (Sparus aurata) spermatozoa, using both scanning electron microscopy (SEM) and computer‐assisted morphology analysis (ASMA). The latter method has been used rarely in fish and this is its first application on sharpsnout sea bream and gilthead sea bream spermatozoa. Results obtained using SEM are expensive and time‐consuming, while ASMA provides a faster and automated evaluation of morphometric parameters of spermatozoa head. For sharpsnout sea bream spermatozoa, similar head measurement values were obtained using both ASMA and SEM, having a mean ± standard error length of 2.57 ± 0.01 μm vs 2.54 ± 0.02 μm, width of 2.22 ± 0.02 μm vs 2.26 ± 0.04 μm, surface area of 4.44 ± 0.02 μm² vs 4.50 ± 0.04 μm² and perimeter of 7.70 ± 0.02 μm vs 7.73 ± 0.04 μm using ASMA and SEM, respectively. Although gilthead sea bream spermatozoa were found to be smaller than those of sharpsnout sea bream, spermatozoal head morphometry parameters were also found to be similar regardless of evaluation method, having a mean head length of 1.97 ± 0.01 μm vs 1.94 ± 0.02 μm, head width of 1.80 ± 0.01 μm vs 1.78 ± 0.02 μm, surface area of 3.16 ± 0.03 μm² vs 3.18 ± 0.06 μm² and perimeter of 6.52 ± 0.04 μm vs 6.56 ± 0.08 μm using ASMA and SEM, respectively. The results demonstrate that ASMA can be considered as a reliable technique for spermatozoal morphology analysis, and can be a useful tool for studies on fish spermatozoa, providing quick and objective results.     

Visualization of skeletons and intervertebral disks in live fish larvae by fluorescent calcein staining and disk specific GFP expression

Zebrafish and medaka have become popular models for studying skeletal development because of high fecundity, shorter generation period, and transparency of fish embryo. The first step to study skeletal development is visualizing bone and cartilage. Live animal staining with fluorescent calcein have several advantages over the standard skeletal staining protocol by using alizarin red and alcian blue for bone and cartilage. However, there is no detailed study examining skeletal development of live marine fish larvae by calcein staining. Here we applied calcein staining to examine skeletal development in red sea bream larvae. In addition, green fluorescent protein (GFP) reporter zebrafish was employed to trace lineage analysis of intervertebral disk cells in live fish larvae. Calcein staining of red sea bream larvae successfully visualized development of craniofacial skeletons as well as urinary calculus. Histochemical detection of alkaline phosphatase (ALP) activity revealed that abnormal segmentation of notochord induced by RA during vertebral development in zebrafish. Immunohistochemistry clearly revealed that GFP‐positive cells in intervertebral space was nucleus polposus like cell in twhh‐GFP transgenic zebrafish. It was demonstrated usefulness of calcein and ALP staining and twhh‐GFP transgenic zebrafish for studying skeletal development in live fish larvae.     

Isolation and characterization of polymorphic microsatellite markers in Pagellus bogaraveo,and cross‐species amplification in Sparus aurata and Dicentrarchus labrax

Several new fish species are currently being included in breeding programmes. However, as specific molecular markers have not yet been developed, this represents a commercial handicap with respect to traditional aquaculture species such as gilthead sea bream or Atlantic salmon. In the present study, 12 new microsatellite loci were developed for blackspot sea bream (Pagellus bogaraveo) that show high levels of polymorphism, especially useful in parentage assignment and individual identification. In addition, cross‐amplification was obtained for two important species for Spanish aquaculture, gilthead sea bream and sea bass.     

Respiratory responses and tolerance to hypoxia in two marine teleosts,Epinephelus akaara (Temminck & Schlegel) and Mylio macrocephalus (Basilewsky)

The respiratory responses and tolerance of hypoxia were studied in two marine teleosts, the red grouper (Epinephelus akaara, a sluggish species) and the black sea bream (Mylio macrocephalus, an active species). Neither species showed abnormal behaviour or mortality when exposed to 2 mg O₂ l^–1 for 7 h. The black sea bream was, however, comparatively more tolerant when exposed to 1 mg O₂ l^–1, but tolerance of both species became similar under extremely hypoxic conditions (i.e. 0.5 mg O₂ l^–1). In contrast to most other teleosts, both species showed a reduction in opercular beating rate during hypoxia, and oxygen conformity was found in the range of 0.5 to 7.0 mg O₂l ^–1. O₂ dissociation curves were constructed, and the P₅₀ value of the black sea breams (27 ± 5.6 mm Hg) was found to be much lower than that of the red groupers (50 ± 2.5 mm Hg). For both species, the general levels of venous PO₂ showed a direct relationship to ambient PO₂, and were markedly reduced after 1 h exposure to various levels of hypoxia. Compared with the red groupers, the black sea breams appeared to be more able to maintain its venous PO₂ levels during prolonged hypoxic exposure.     

Tissue Distribution, Effects of Salinity Acclimation, and Ontogeny of Aquaporin 3 in the Marine Teleost, Silver Sea Bream (Sparus sarba)

The purpose of the present study was to ascertain the tissue-specific expression of the water channel protein, aquaporin 3 (AQP3), during salinity acclimation and larval development of silver sea bream (Sparus sarba). A cDNA fragment encoding aquaporin 3 (aqp3) from silver sea bream gill was cloned and from the deduced amino acid sequence a polyclonal antibody was prepared. AQP3 was found to be present in gill, kidney, liver, brain, heart, and spleen but not in whole blood. The abundance of AQP3 was significantly highest in gills of hypoosmotic (6 ppt) and isoosmotic (12 ppt) acclimated sea bream when compared to seawater (33 ppt) and hypersaline (50 ppt)- acclimated sea bream. Spleen tissue also displayed significantly high levels of AQP3 protein in hypoosmotic and isoosmotic salinities whereas the AQP3 abundance in brain, liver, heart, and kidney remained unchanged across the range of salinities tested. The ontogenetic profile of AQP3 was also investigated from developing sea bream larvae and AQP3 was first detected at 14 days posthatch (dph) and increased steadily up to 28–46 dph. In conclusion, this study has demonstrated that AQP3 expression is modulated in gill and spleen tissue of salinity acclimated sea bream and that it can be detected relatively early during larval development.     

Effects of vegetable feed ingredients on bone health in Atlantic salmon

The aim of the present study was to examine if dietary inclusion of vegetable lipids (VL) and proteins (VP) influenced markers of bone health in Atlantic salmon. Triplicate groups were fed one of four different diets; 100% fish protein (FP) and fish lipids (FL) (FPFL), 80% VP and 35% VL (80VP35VL), 40% VP and 70% VL (40VP70VL), or 80% VP and 70% VL (80VP70VL) for 12 months on‐growth in sea water. Fish were analyzed for vertebral bone mineralization (mineral content, as % of bone dry weight), vertebral deformities (radiology), vertebral bone mRNA expression of factors involved in mineralization (bone gla protein, bgp) and growth regulation (igf‐I and growth hormone receptor), as well as plasma vitamin D metabolites. The fish grew from 0.35 to 4 kg during the experimental period. At the end of the experiment, significantly lower prevalence of fish with one or more deformed vertebrae was observed in the 80VP70VL group (11%) compared to the other groups (33–43%). There was a significant higher relative expression of igf‐I mRNA in vertebral bone of fish fed the 80VP70VL diet compared to control fish (FPFL), while the other genes studied were unaffected. Elevated plasma 25‐hydroxyvitamin D₃ recorded in the marine feed group is discussed as a predictor for later development of bone deformities. In conclusion, the present study shows that high inclusion levels of vegetable lipids and proteins may have a positive effect on bone health in Atlantic salmon postsmolts.     

A comparison of fatty acid,cholesterol and vitamin composition in sea bass [Dicentrarchus labrax (Linnaeus, 1758)] and sea bream [Sparus aurata (Linnaeus, 1758)] from three cage farm areas: Antalya and Muğla (Turkey) and İskele (Northern Cyprus)

In the present study the fatty acids, cholesterol and vitamin composition in farmed sea bass (8 fish per species per farm; weight range: 389.6–395.8 g, total length range: 297–316 mm) and sea bream (8 fish per species per farm; weight range: 386.8–391.7 g, total length range: 263–268 mm) from three cage farms (?skele in northern Cyprus, Antalya and Mu?la in Turkey) were compared during the harvesting period in June–July 2011. The results showed that the muscles of D. labrax and S. aurata farmed fish were rich in n‐3 fatty acids, but with important differences. For example, the muscles of sea bass farmed in ?skele were rich in docosahexaenoic acid (DHA) and n‐3 polyunsaturated fatty acids (PUFA). Palmitic acid (C16:0) was the primary saturated fatty acid, and oleic acid (C18:1 n‐9) the primary monounsaturated fatty acid in the muscle and liver samples of the cage‐farmed sea bass and sea bream. There were no significant differences in the cholesterol content in the muscles of sea bream farmed in ?skele, Antalya or Mu?la. In conclusion, the n‐3/n‐6 ratio in the muscle of farmed S. aurata and D. labrax is within the recommended limits for a healthy human diet, being very suitable for human nutrition.     

Zooplankton preference of two species of freshwater ornamental fish larvae

The study aimed at evaluating the ascorbic acid (AA) concentrations in the liver and blood of the Mediterranean sea bream (Sparus aurata L.) after being fed Rovimix Stay C‐25 (RS C‐25) at three different levels (0, 200 and 800 mg kg^?1 of AA) over 45 days. RS C‐25 is a mixture of equal parts of AA monophosphate, diphosphate and polyphosphate, containing a 25% AA equivalent. Increasing RS C‐25 levels in the diet yielded an increasing ascorbic acid content in the plasma and liver, showing a good sea bream utilization of this source of vitamin C. Dietary vitamin C did not affect the growth rate or feed efficiency during the 45‐day experiment. After 75 days, fish fed a vitamin C‐free diet displayed a severe depletion of AA in the liver.     

Satellite data identify decadal trends in the quality of Pygoscelis penguin chick‐rearing habitat

Pygoscelis penguins are experiencing general population declines in their northernmost range whereas there are reported increases in their southernmost range. These changes are coincident with decadal‐scale trends in remote sensed observations of sea ice concentrations (SIC) and sea surface temperatures (SST) during the chick‐rearing season (austral summer). Using SIC, SST, and bathymetry, we identified separate chick‐rearing niche spaces for the three Pygoscelis penguin species and used a maximum entropy approach (MaxEnt) to spatially and temporally model suitable chick‐rearing habitats in the Southern Ocean. For all Pygoscelis penguin species, the MaxEnt models predict significant changes in the locations of suitable chick‐rearing habitats over the period of 1982–2010. In general, chick‐rearing habitat suitability at specific colony locations agreed with the corresponding increases or decreases in documented population trends over the same time period. These changes were the most pronounced along the West Antarctic Peninsula where there has been a rapid warming event during at least the last 50 years.     

Feeding by marine fish larvae: developmental and functional responses

Synopsis The relationship between prey consumption rate and prey concentration (functional response), and its change with growth (developmental response) were examined in the laboratory for three species of marine fish larvae: bay anchovy Anchoa mitchilli (Engraulidae), sea bream Archosargus rhomboidalis (Sparidae) and lined sole Achirus lineatus (Soleidae). The major objective was to determine relative predatory abilities of the larvae by fitting feeding rate data to developmental and functional response models. Feeding success, prey capture success, attack rates, handling times and search rates were estimated. Prey consumption rates and attack rates of bay anchovy usually were highest, but at the lowest prey level (50 per liter) first-feeding sea bream larvae had the highest consumption rate. Sea bream could consume prey at near-maximum rates at prey levels lower than those required by the other species. As larvae grew, time searching per attack decreased rapidly for all species, especially at low prey levels. Handling time also decreased, but most rapidly for bay anchovy. Search rates were highest for bay anchovy and lowest for lined sole. Bay anchovy had the best apparent predation ability, but when previous results on larval growth rates, survival rates and growth efficiencies were considered, sea bream larvae were the most efficient predators and the least likely of the three species to be limited by low prey levels.     

Effects of dietary protein and lipid levels on growth and feed utilization of wild‐caught striped sea bream,Lithognathus mormyrus

A feeding trial was carried out to determine the effects of dietary protein and lipid levels on the growth performance and feed utilization of wild‐caught striped sea bream (Lithognathus mormyrus). The experimental fish were collected from a local lagoon (Çardak Lagoon, Çanakkale, Turkey), transferred to the Marine Net Cage Unit and fed by hand to apparent satiation with a commercial sea bream feed (Biomar; 42% crude protein, 16% crude lipid). Approximately 4 weeks were needed to acclimate the fish to farming conditions. No pathological signs were observed and no fish losses occurred during the adaptation period. For the test trials four test diets with different levels of protein and lipid were formulated [low protein and low lipid (LP:LL), low protein and high lipid (LP:HL), high protein and low lipid (HP:LL), and high protein and high lipid (HP:HL)] and fed to L. mormyrus (mean weight 85.0 ± 4.6 g SEM) in the net cages (Ø 2 m, depth 2.5 m) for 60 days. During the experiment water temperature varied between 21.1 and 26.4°C; dissolved oxygen 8.4–9.6 mg L^?1; pH 7.2–8.6; and salinity 23.3–25.6‰. Growth performances of fish fed high protein diets were higher compared to fish fed low protein diets, irrespective of the dietary lipid level (P < 0.05). Feed conversion ratio (FCR) and protein efficiency ratio (PER) were not influenced by dietary protein or lipid levels (P > 0.05). Preliminary results indicate that striped sea bream can be easily adapted to farming conditions in net cages, and that a diet containing 50% crude protein and 15% crude lipid (HP:LL) levels with 23.0 g protein MJ^?1 gross energy of protein/energy ratio would be suitable for striped sea bream growth.     

Influence of genetic drift on patterns of genetic variation: The footprint of aquaculture practices in Sparus aurata (Teleostei: Sparidae)

Aquaculture finfish production based on floating cage technology has raised increasing concerns regarding the genetic integrity of natural populations. Accidental mass escapes can induce the loss of genetic diversity in wild populations by increasing genetic drift and inbreeding. Farm escapes probably represent an important issue in the gilthead sea bream (Sparus aurata), which accounted for 76.4% of total escapees recorded in Europe during a 3‐year survey. Here, we investigated patterns of genetic variation in farmed and wild populations of gilthead sea bream from the Western Mediterranean, a region of long gilthead sea bream farming. We focused on the role that genetic drift may play in shaping these patterns. Results based on microsatellite markers matched those observed in previous studies. Farmed populations showed lower levels of genetic diversity than wild populations and were genetically divergent from their wild counterparts. Overall, farmed populations showed the smallest effective population size and increased levels of relatedness compared to wild populations. The small broodstock size coupled with breeding practices that may favour the variance in individual reproductive success probably boosted genetic drift. This factor appeared to be a major driver of the genetic patterns observed in the gilthead sea bream populations analysed in the present study. These results further stress the importance of recommendations aimed at maintaining broodstock sizes as large as possible and equal sex‐ratios among breeders, as well as avoiding unequal contributions among parents.     

Genetic integrity of black sea bream (Acanthopagrus schlegeli) sperm following cryopreservation

Although semen cryopreservation has been applied successfully in many fish species, extensive variation in post‐thaw semen quality exists between species and individuals. AFLP (amplified restriction fragment length polymorphism) is a powerful method for detecting DNA polymorphisms at the individual, population, and species levels. The method has been successfully applied to boars (Sus domestica, Suidae, Artiodactyla, Mammalia) to detect and evaluate differences in DNA sequences that correspond with semen integretiy after employing various freezing techniques. Freezing and thawing of semen has also an effect of selecting for freezing‐resistant (or intact) and eliminating non‐viable or defective sperm. Only the fully intact and functional sperm, despite potential compromise by adverse freezing and osmotic stresses, retain fertility after thawing. The objective of this study was to use AFLP to assess any genetic changes associated with the effect of employed cryo‐methodology on the genetic integrity of sperm of the black sea bream (Acanthopagrus schlegeli) under different cryopreservation treatments. The cryopreservation protocols had no significant effect on sperm motility or survival rate of fertilized ova regardless of using fresh (% motile sperm 89.6 ± 3.0; % embryonic survival rate 54.4 ± 2.9) and frozen‐thawed semen (% motile sperm 80.2 ± 2.0; % embryonic survival rate 51.8 ± 2.0). The post‐thaw sperm motility and survival rates were not significantly different among the sperm samples of the five black sea bream males examined. In the present study, the remaining black sea bream sperm that survive the cryopreservation limit the power of AFLP to trace the genetic markers which correlate with the differences in the sensitivity of sperm to cryo‐injury. It is also possible that point mutations outside the AFLP priming sites may not have been detected. More thorough investigations are needed to determine whether such DNA fingerprints would be found in fish species.     

Development of a microtitre plate indirect ELISA for measuring cortisol in teleosts, and evaluation of stress responses in rainbow trout and gilthead sea bream

A microtitre plate indirect enzyme‐linked immunoassay (ELISA) was developed for measuring plasma cortisol levels in rainbow trout Oncorhynchus mykiss, gilthead sea bream Sparus auratus sea bass Dicentrarchus labrax and Senegalese sole Solea senegalensis. Covalink microplates pretreated with disuccinimidyl suberate were coated with bovine serum albumin (BSA) conjugated to cortisol‐3‐carboxymethyl oxime. After blocking with BSA, competition was started by addition of plasma samples and anti‐cortisol antibody raised in rabbit. Goat anti‐rabbit IgG conjugated‐peroxidase was added as second antibody and then incubated with orthophenylenediamine as substrate. Reaction was stopped with 0·1 M HCl and absorbance was read at 450 nm in an automatic plate reader. The standard curve was linear from the lower limit of sensitivity of the assay (c. 0·3 ng ml^?1) to c. 3000 ng ml^?1. Dose‐response inhibition curves using serially diluted plasma samples of four species consistently showed parallelism with the standard curve using cortisol. The ELISA satisfied the strictest criteria of specificity (cross‐reactivity of anti‐cortisol antibody with testosterone, progesterone and 17ß‐oestradiol was negligible, cross‐reactivity with cortisone, corticosterone and 11‐deoxycortisol, was 1·5, 1 and 0·1%, respectively), reproducibility (interassay CV <6%), precision (intra‐assay CV <4%), and accuracy (average recovery >98%). Plasma cortisol concentration in rested fishes was in the range of 5–30 ng ml^?1. To physiologically validate the technique, changes in plasma cortisol concentrations were also measured in plasma of rainbow trout and gilthead sea bream following an acute 15 min chasing or 3 min air‐exposure stress, respectively. In both species plasma concentrations of cortisol, glucose and lactate rose significantly with respect to controls, showing concentrations similar to those reported previously for these species under similar stress conditions. Furthermore, gilthead sea bream chronically stressed by maintaining for 14 days under increased stocking density conditions also showed increased concentrations of plasma cortisol and glucose. These results validate the indirect ELISA technique developed for use in the evaluation of plasma cortisol concentration of at least four fish species.     

The population density, growth rate and production of bream, Abramis brama, in Tjeukemeer, the Netherlands

A method of estimating the population density of bream in Tjeukemeer (21.3 km²) using 16 690 introduced fish (fin-clipped and opercular tagged) is described. Gill nets of the winter fishery proved to be a more effective method of sampling the population for marked fish than fyke nets. The population density of bream (⋝25 cm) was estimated to be 180 000. There was no significant difference between the estimates derived from fin-clipped and opercular tagged fish. The growth rate of bream in Tjeukemeer (L_∞=41 cm) is poor compared with that of bream in other waters, due to its high density and the scarcity of zoobenthos available to it.
The production of bream (I–XV) is estimated to be 34 kg ha⁻¹ of which 25% is contributed by two (1959, 1963) strong year classes. Of a total biomass of 37.5 kg ha⁻¹ available to the fishery in 1969 only 2.7 kg ha^−l was removed as yield. The average P/B ratio for the population was low (0.39). It is concluded that a major flow of energy to bream is through zooplankton.     

Improvement of nutritional quality of cultured sobaity sea bream,Sparidentex hasta (Valenciennes) muscle by preharvest feeding of finisher feeds

A 6‐month long study was conducted to improve the nutritional quality of the cultured sobaity bream, Sparidentex hasta by feeding them finisher feeds containing high docosahexaenoic acid (DHA) at the last two months of the grow‐out stage so that the muscle DHA level be increased at par to the wild. A grow‐out feed used from the beginning until the end of the trial was considered as the control (Diet 1). Experimental diets 2 and 3 were formulated to contain 9.0% DHA (e.g. 1.68 g DHA/100 g feed) and 10.5% DHA (2.20 g DHA/100 g feed), by incorporating high DHA tuna oil into a sea bream grow‐out diet. For comparison, a commercial finisher feed (Diet 4) from Skretting, Italy was also used. The results of this study demonstrated that fish fed DHA enriched finisher diets resulted in significantly (p < .05) better growth, feed utilization and higher muscle eicosapentaenoic acid (EPA) and DHA content compared to those fed grow‐out diet. The muscle DHA and EPA of fish fed finisher diets were also higher than those of the whole year average DHA and EPA content of wild sobaity. An organoleptic evaluation showed no significant (p > .05) differences between sensory attributes of muscle from cultured and wild sea bream. The results of the study demonstrated that feeding finisher feed enriched with DHA at the later part of the grow‐out operation, the n‐3 PUFA levels of cultured sobaity can cost‐effectively be increased at par to the wild.     

Consistent melanophore spot patterns allow long‐term individual recognition of Atlantic salmon Salmo salar

The present study shows that permanent melanophore spot patterns in Atlantic salmon Salmo salar make it possible to use images of the operculum to keep track of individual fish over extended periods of their life history. Post‐smolt S. salar (n = 246) were initially photographed at an average mass of 98 g and again 10 months later after rearing in a sea cage, at an average mass of 3088 g. Spots that were present initially remained and were the most overt (largest) 10 months later, while new and less overt spots had developed. Visual recognition of spot size and position showed that fish with at least four initial spots were relatively easy to identify, while identifying fish with less than four spots could be challenging. An automatic image analysis method was developed and shows potential for fast match processing of large numbers of fish. The current findings promote visual recognition of opercular spots as a welfare‐friendly alternative to tagging in experiments involving salmonid fishes.     

Comparison of UV-B tolerance between wild and hatchery-reared juveniles of red sea bream (Pagrus major) and black sea bream (Acanthopagrus schlegeli)

The amount of ultraviolet (UV)-B radiation reaching the sea surface has increased due to ozone depletion. Several laboratory studies have highlighted the negative impacts of UV radiation on fish using hatchery-reared specimens. However, potential differences in UV tolerance between wild and hatchery-reared fish have been given little consideration. Wild and reared juveniles of red sea bream and black sea bream were exposed to one of four different UV-B radiation levels (1.8; 1.1; 0.4; 0?W/m²) for 4?h. Survival rate was measured every 2?h for a period of 24?h (red sea bream) or 48?h (black sea bream) following exposure. Wild and reared juvenile red sea bream were characterized by similar survival rate, with survival declining to almost 0?% 24?h after exposure at the 1.1 and 1.8?W/m² levels. In black sea bream, wild individuals showed significantly higher survival than reared fish in levels 1.1 and 1.8?W/m². Melanophore density was also measured since melanin absorbs UV radiation. Wild black sea bream showed higher melanophore density compared to reared individuals, while no such difference was observed in red sea bream. We conclude that wild black sea bream juveniles acquire higher UV tolerance partly by increasing melanophore density through exposure to UV radiation. Our results indicate that the predicted impacts of UV radiation on fish populations solely based on experimentation with hatchery-reared specimens may be overestimated for some species.