Radiological characterization of gilthead seabream (Sparus aurata) by X-ray computed tomography

In recent years, the increasing use of fish as new animal models in scientific research and the growth of fish farming (mainly for human consumption) have highlighted the need for advanced technology to deepen our knowledge of fish biology. Hence, the present study was carried out to radiologically analyse the whole body of gilthead seabream (Sparus aurata) specimens using X-ray computed tomography (CT). Images were acquired in an Albira SPECT/PET/CT tri-modal preclinical-scanner. Segmentation, measurements and three-dimensional reconstruction were made using the Carestream Molecular imaging Albira CT system in conjunction with Pmod, AMIDE and Amira software packages. The results showed that the density values of gilthead seabream are in the range −700 to +2500 HU for the whole body. We also determined the density ranges that topographically coincide with the swim bladder, soft tissues, fat, skin and skeleton. This work describes, validates and demonstrates the application of a fully automated image analysis technique to study and quantify fish body composition, whether segmented or as a whole. In addition, the basis for applying this image technique in other in vivo studies is established.     

Quantitative trait loci affecting morphology traits in gilthead seabream (Sparus aurata L.)

We report a quantitative trait loci (QTL) mapping study on 18 morphometric characters in gilthead seabream based on a total of 74 informative microsatellite markers genotyped in 409 offspring coming from 10 paternal half‐sib families. Statistical analysis was carried out using a linear regression approach, and various suggestive and significant morphology QTL were detected in three (9, 21 and 25) of nine linkage groups examined. Fitting body weight as a covariate reduced the significance of some QTL but revealed three new QTL in other linkage groups (LG6 and LG10). Current results combined with those obtained from previous studies underline highly significant loci affecting overall growth and morphology in S. aurata.     

Identification of a major locus determining a pigmentation defect in cultivated gilthead seabream (Sparus aurata)

The gilthead seabream (Sparus aurata) is an important cultivated species in the Mediterranean area. A major problem for the gilthead seabream aquaculture sector derives from the high frequency of phenotypic abnormalities, including discolorations. In this study, we applied a whole-genome resequencing approach to identify a genomic region affecting a pigmentation defect that occurred in a cultivated S. aurata population. Two equimolar DNA pools were constructed using DNA extracted from 30 normally coloured and 21 non-pigmented fish collected among the offspring of the same broodstock nucleus. Whole-genome resequencing reads from the two DNA pools were aligned to the S. aurata draft genome and variant calling was performed. A whole-genome heterozygosity scan from single pool sequencing data highlighted a peak of reduced heterozygosity of approximately 5 Mbp on chromosome 6 in the non-pigmented pool that was not present in the normally coloured pool. The comparison of the non-pigmented with the normally coloured fish using a whole-genome F_ST analysis detected three main regions within the coordinates previously detected with the heterozygosity analysis. The results support the presence of a major locus affecting this discoloration defect in this fish population. The results of this study have practical applications, including the possibility of eliminating this defect from the breeding stock, with direct economic advantages derived from the reduction of discarded fry. Other studies are needed to identify the candidate gene and the causative mutation, which could add information to understand the complex biology of fish pigmentation.     

Immune defence mechanisms presented in liver homogenates and bile of gilthead seabream (Sparus aurata)

Because the role of the liver of fishes in providing possible immunity remains largely unknown, the aim of this work was to identify and characterize different humoral defence mechanisms in the liver homogenates and bile of gilthead seabream (Sparus aurata) for the first time. Total protein levels and several immune parameters (complement activity, lysozyme and immunoglobulin M level) were studied. Furthermore, the activity of some lytic (proteases, antiproteases, esterase, alkaline phosphatase) and antioxidant (superoxide dismutase, catalase and peroxidase) enzymes was determined. Finally, bacteriostatic activity on three opportunist fish pathogens (Vibrio harveyi, Vibrio angillarum and Photobacterium damselae) was measured. Lysozyme and antiprotease activity were undetected in liver and bile, while natural haemolytic complement activity was only detected in bile, and immunoglobulin M was detected in both samples. The levels of proteases, esterase and antioxidant enzymes were greater in bile than in liver homogenates, while the level of alkaline phosphatase was very low in both samples. In addition, while no bacteriostatic activity was detected on liver homogenates, the bile revealed a very potent bacteriostatic activity against all the tested pathogenic bacteria. These results corroborate that fish liver – especially fish bile – contains many factors involved in innate immunity that could be useful for better understanding the role of the liver as an organ involved in fish immune functions as well as the possible contribution of bile to gut mucosal immunity.     

Fine structure of spermatozoa in the gilthead sea bream (Sparus aurata Linnaeus, 1758) (Perciformes, Sparidae)

Scanning and transmission electron microscopy were used to investigate the fine structure of the sperm of the sparid fish Sparus aurata L. The mature spermatozoon of gilthead sea bream belongs, like that of the other sparid fish, to a "type I" as defined by Mattei (1970). It has a spherical head which lacks an acrosome, a short, irregularly-shaped midpiece and a long cylindrical tail. The nucleus reveals a deep invagination (nuclear fossa) in which the centriolar complex is located. The two centrioles are approximately perpendicular to each other and show a conventional "9+0" pattern. The proximal centriole is associated with a cross-striated cylindrical body lying inside a peculiar satellite nuclear notch which appears as a narrow invagination of the nuclear fossa. The distal centriole is attached to the nuclear envelope by means of a lateral plate and radial fibres made of an electron-dense material. The short midpiece houses one mitochondrion. The flagellum is inserted perpendicularly into the base of the nucleus and contains the conventional 9+2 axoneme.     

Selected plasma biochemistry parameters in gilthead seabream (Sparus aurata) juveniles

The aim of this study was to assess plasma biochemistry parameters with the potential of being used as indicators of the nutritional status for healthy gilthead seabream juveniles. Triplicate groups of 18 seabream (body weight of 58 g) were kept unfed for 24 h, 7 or 14 days. Nine fish per treatment were then sampled randomly for blood collection and the following parameters analyzed in the plasma using standard clinical methods: glucose; protein; triglycerides; cholesterol; calcium; magnesium; inorganic phosphorus; alkaline phosphatase (ALP); aspartate aminotransferase (AST); lactate dehydrogenase (LDH); gamma‐glutamyl transferase (GGT); creatine phosphokinase (CPK); and lipase. Biochemical parameters showed lower variability among individuals than did enzymatic parameters. Plasma glucose, protein, cholesterol, calcium and inorganic phosphorus levels were inversely related to the duration of starvation. On the contrary, plasma triglycerides decreased significantly during the first week of starvation and remained stable in the second week. Plasma ALP, AST and LDH decreased significantly after 1 week of starvation and then remained constant. In healthy seabream juveniles, plasma glucose, protein, cholesterol, calcium and inorganic phosphorus are responsive to starvation and may be useful indicators of the nutritional status of the animals. Indicative baseline reference values for gilthead seabream juveniles starved for 24 h and held at optimum temperature are: protein, 3.7–4.9 g dl^?1; cholesterol, 341–407 mg dl^?1; calcium, 13.1–8.0 mg dl^?1; and inorganic phosphorus, 10–14.2 mg dl^?1. Plasma triglycerides, along with plasma enzyme activities, may be useful as indicators of short term starvation. For these parameters baseline values after 1 week of starvation were: triglycerides: 138–230 mg dl^?1; ALP: 58–125 U L^?1; AST: 15–127 U L^?1; and LDH 61–677 U L^?1. Plasma glucose is only responsive to longer starvation periods, remaining relatively stable during the first week of starvation, and ranging from 59 to 196 mg dl^?1.     

Characterization of digestive carbohydrase activity in the gilthead seabream (Sparus aurata)

Six species of Phallodrilinae are reported from Hainan Island in southern China. The mesopsammic Aktedrilus yiboi sp. nov. is new to science. It is characterized by a combination of (1) small, straight, conical, strongly cuticularized penis sheaths, (2) posterior prostate glands being associated with the most ectal parts of the atria, and (3) small spermathecal ampulla. Bathydrilus ampliductus Erséus, 1997 is recorded for the first time since it was originally described from Australia's Northern Territory.     

Nutritional and hormonal control of lipolysis in isolated gilthead seabream (Sparus aurata) adipocytes

We examined the effects of diet composition and fasting on lipolysis of freshly isolated adipocytes from gilthead seabream (Sparus aurata). We also analyzed the effects of insulin, glucagon, and growth hormone (GH) in adipocytes isolated from fish fed with different diets. Basal lipolysis, measured as glycerol release, increased proportionally with cell concentration and time of incubation, which validates the suitability of these cell preparations for the study of hormonal regulation of this metabolic process. Gilthead seabream were fed two different diets, FM (100% of fish meal) and PP (100% of plant protein supplied by plant sources) for 6 wk. After this period, each diet group was divided into two groups: fed and fasted (for 11 days). Lipolysis was significantly higher in adipocytes from PP-fed fish than in adipocytes from FM-fed fish. Fasting provoked a significant increase in the lipolytic rate, about threefold in isolated adipocytes regardless of nutritional history. Hormone effects were similar in the different groups: glucagon increased the lipolytic rate, whereas insulin had almost no effect. GH was clearly lipolytic, although the relative increase in glycerol over control was lower in isolated adipocytes from fasted fish compared with fed fish. Together, we demonstrate for the first time that lipolysis, measured in isolated seabream adipocytes, is affected by the nutritional state of the fish. Furthermore, our data suggest that glucagon and especially GH play a major role in the control of adipocyte lipolysis.     

Effect of dietary supplementation with yeast Saccharomyces cerevisiae on skin,serum and liver of gilthead seabream (Sparus aurata L)

The effect of dietary supplementation with Saccharomyces cerevisiae on gilthead seabream (Sparus aurata L.) was studied. Four replicates of fish (n = 6) were fed with a commercial diet containing 0 (control, no yeast added) or 10 mg per kilogram of heat-killed (30 min, 60°C) S. cerevisiae. After 4 weeks, half of the fish (two replicates) were injured and continued with the same diet. At 3 and 7 days post-wounding, samples of blood, skin mucus, skin and liver were obtained from each fish. The results showed that calcium concentrations were significantly higher (with respect to control fish) in the serum from fish sampled at 3 days post-wounding, whereas antioxidant enzymes in the skin mucus were altered after wounding (at both 3 and 7 days). Histological analyses revealed oedema, signs of inflammation and white cell recruitment together with a reduction in the epidermis layer in the wounded regions of fish fed control diet. Yeast supplementation did not change growth performance and helped maintain the normal serum calcium concentrations in wounded fish. Furthermore, a reduction in inflammation around wounds in the animals fed yeast with respect to that fed control diet was evident in the histological study. Furthermore, increased levels of stress-related gene expression in liver and skin from wounded fish were obtained. Overall, yeast supplementation seemed to be a functional and appropriate dietary additive to improve skin recovery reducing the stress resulting from wounds.     

Chromosome set manipulations and hybridization experiments in gilthead seabream (Sparus aurata). II. Assessment of diploid and triploid hybrids between gilthead seabream and red seabream (Pagrus major)

The experiments were designed to evaluate the suitability for mariculture of the diploid and triploid hybrids of gilthead seabream Sparus aurata and red seabream Pagrus major , both of which are members of the Sparidae family. Performance testing of the hybrids was carried out in comparison with the parental species under the same controlled environment. The reciprocal diploid hybrids as well as the triploid hybrid did not exhibit any significant growth advantage over diploid parental species either before or after sexual maturity. The adults, both reciprocal diploid hybrids and triploid hybrid (female S. aurata × male P. major ), were clearly immature and had only vestigial gonads; neither ovaries nor released milts were observed. Histological examination of the gonads, carried out during associated periods of the first and second maturation cycles of the parental species, showed that all the hybrids were completely sterile. Despite their sterility, the growth of the hybrids did not display any positive effect, and for this reason their commercial culture appears to be questionable. On the other hand, the use of sterilization through hybridization and chromosome set manipulation may be important when there is a need to restrict the ecological impact on a wild population.     

Development of the first standardised panel of two new microsatellite multiplex PCRs for gilthead seabream (Sparus aurata L.)

The high number of multiplex PCRs developed for gilthead seabream (Sparus aurata L.) from many different microsatellite markers does not allow comparison among populations. This highlights the need for developing a reproducible panel of markers, which can be used with safety and reliability by all users. In this study, the first standardised panel of two new microsatellite multiplex PCRs was developed for this species. Primers of 138 specific microsatellites from the genetic linkage map were redesigned and evaluated according to their genetic variability, allele size range and genotyping reliability. A protocol to identify and classify genotyping errors or potential errors was proposed to assess the reliability of each marker. Two new multiplex PCRs from the best assessed markers were designed with 11 markers in each, named SMsa1 and SMsa2 (SuperMultiplex Sparus aurata). Three broodstocks (59, 47 and 98 breeders) from different Spanish companies, and a sample of 80 offspring from each one, were analysed to validate the usefulness of these multiplexes in the parental assignation. It was possible to assign each offspring to a single parent pair (100% success) using the exclusion method with SMsa1 and/or SMsa2. In each genotyped a reference sample (Ref‐sa) was used, and its DNA is available on request similar to the kits of bin set to genotype by genemapper (v.3.7) software (kit‐SMsa1 and kit‐SMsa2). This will be a robust and effective tool for pedigree analysis or characterisation of populations and will be proposed as an international panel for this species.     

Genetic structure of gilthead seabream, Sparus aurata, in the Central Mediterranean Sea

The gilthead seabream, Sparus aurata, represents an important economic resource for Mediterranean aquaculture. In spite of its wide geographic distribution and economic importance, only recently studies have been carried out on the genetic composition of natural populations, which have revealed a picture of a heterogeneous degree of genetic differentiation among S. aurata populations. In this study an allozyme analysis of samples from six different collecting sites along the Italian and Croatian coasts was carried out, covering an area in the Central Mediterranean sea that has yet to be investigated through gene-enzyme systems. Data on 26 gene loci, 10 of which are polymorphic, indicate a slight but significant genetic structure (F_ST = 0.0167) of the species. A hierarchical analysis of population subdivision made it possible to identify three different assemblages found in the Adriatic Sea, Tyrrhenian Sea and Sardinian Channel, though an isolation by distance model can be rejected. The results are discussed in the light of previous literature and taking conservation into consideration.     

Low vitamin E in diet reduces stress resistance of gilthead seabream (Sparus aurata) juveniles

This study investigates the effect of dietary vitamin E on juveniles of gilthead seabream under stressful situations, focusing on the effects on growth, haematology, some immune parameters and plasma cortisol as indicators of stress. Two sardine meal-based experimental diets, one of them supplemented with 150 mg of alpha tocopherol kg(-1) of diet (control) and another one without vitamin E supplementation (diet NE), were assayed under two different stress conditions: overcrowding as a chronic stressor (during 15 weeks) and repetitive chasing as an acute repetitive stressor. Low levels of vitamin E in the diet depleted alternative complement pathway activity [from 167.23 U ml(-1) (control fish) down to 100.99 U ml(-1)] and also nonspecific haemagglutination. Also, fish fed a non-supplemented diet showed an elevation of plasma cortisol basal levels without a stressor influence [from 3.91 ng cortisol ml(-1) plasma (control fish) up to 21.70 ng cortisol ml(-1) plasma]. Low levels of vitamin E in the diet also produced an increase of erythrocyte fragility. Under chronic stress, fish fed the vitamin E-deficient diet showed a reduction in growth and survival, and alterations in haematological parameters, such as an additional haemoconcentration in response to overcrowding when compared with control fish. Under repetitive stress, fish fed the vitamin E deficient diet showed faster elevation of plasma cortisol levels in response to stress and a lower survival rate than control fish. Production of oxygen radicals by blood neutrophils was reduced under repetitive stress in fish fed the non-supplemented diet. These results suggest that fish fed the vitamin E-deficient diet had lower stress resistance.     

Maximum length and age report of Sparus aurata (Linnaeus, 1758) in the Black Sea

The presence of gilthead sea bream (Sparus aurata) in the Black Sea, albeit sparse, has been known for many years. In recent years, it has been encountered in fishing nets, most commonly as by‐catch. In this study conducted in the southern Black Sea (Ordu) region, three female individuals were caught in trammel nets on October 9, 2017, and the largest individual was found to have a total length of 61.4 cm, a weight of 3080.6 g, and a gonad weight of 115.59 g. The metric and meristic characteristics of the largest individual sampled were also determined.     

Comparative skin mucus and serum humoral defence mechanisms in the teleost gilthead seabream (Sparus aurata)

Mucosal surfaces of fish, including skin, gill and gut, contain numerous immune substances poorly studied that act as the first line of defence against a broad spectrum of pathogens. This study aimed to identify and characterize for the first time different constitutive humoral defence mechanisms of the skin mucus of gilthead seabream (Sparus aurata). To do this, the levels of total immunoglobulin M, several enzymes and proteins (peroxidase, lysozyme, alkaline phosphatase, esterases, proteases and antiproteases), as well as the bactericidal activity against opportunist fish pathogens (Vibrio harveyi, Vibrio angillarum, Photobacterium damselae) and non-pathogenic bacteria (Escherichia coli, Bacillus subtilis) were measured in the skin mucus and compared with those found in the serum. This study demonstrates that gilthead seabream skin mucus contains lower levels of IgM, similar levels of lysozyme, alkaline phosphatase and proteases, and higher esterase, peroxidase and antiprotease activities than serum. In addition, skin mucus revealed stronger bactericidal activity against tested fish pathogen bacteria compared to the serum activity, while human bacteria can even grow more in the presence of mucus. The results could be useful for better understanding the role of the skin mucus as a key component of the innate immune system with potential application for the aquaculture.     

Immunocytochemical distribution of cytochrome P4501A (CYP1A) in developing gilthead seabream, Sparus aurata

CYP1A is a major inducible enzyme in the metabolism of xenobiotic substrates. In this paper we investigate by means of immunohistochemistry, the tissue distribution of constitutive cytochrome P4501A (CYP1A) during the period of endogenous nutrition (from hatching until day 4) in developing gilthead seabream, Sparus aurata larvae. For this purpose, a polyclonal antiserum (BN-1, Biosense Laboratories) directed against conserved piscine CYP1A sequences was used on paraffin-embedded sections from seabream larvae. From hatching onward, CYP1A immunoreactivity was observed in the following tissues and cells: syncytial, oil-globule envelopes and matrix of the yolk-sac, kidney (epithelia of renal tubules), cardiac muscle cells, skin epidermal cells, troncal musculature, enterocytes of different intestinal regions, goblet cells of the bucco-pharyngeal region, gill epithelial cells and the endothelia of the vascular system of various tissues (especially from liver and brain). Moreover, eye (retina), olfactory epithelium and some positive nerve fibers located in the proximity of the olfactory bulbs and running ventrally toward the posterior brain were strongly CYP1A immunoreactive. In general, the intensity of immunostaining increased with larval development.     

Analysis of vitelline envelope synthesis and composition during early oocyte development in gilthead seabream (Sparus aurata)

The oocyte vitelline envelope (VE) of gilthead seabream is composed of four known zona pellucida (ZP) proteins, ZPBa, ZPBb, ZPC, and ZPX. We have previously shown that the gilthead seabream ZP proteins are differentially transcribed in liver and ovary, with the expression in liver being under estrogenic control. However, although mRNA was found in both liver and ovary, only low ZPBa protein levels were detected in liver and plasma. Using isoform-specific ZP antibodies we show that ZPBa and ZPX translation products are present in the cytosol of stage I and II oocytes. In addition, the zpBa and zpX mRNAs were detected in early developing oocytes. During oocyte growth (vitellogenesis), the VE increased in thickness (>10 microm), and we show that the four ZP isoforms are present in different regions of the VE. ZPX was detected closest to the oocyte plasma membrane while the intermediate region was composed of ZPBa, ZPBb, and ZPC. At the outer layer, only ZPC was detected. When oocytes reach the fully grown stage they resume meiosis and hydration. As the oocyte expands, thinning to 4 microm, the VE acquire a striped and compact appearance at the electron microscopy level. This study provides further evidence for the oocyte origin of some ZP proteins in the gilthead seabream and suggests that the ZP proteins are differentially distributed within the VE.