Virulence and molecular typing of Vibrio harveyi strains isolated from cultured dentex,gilthead sea bream and European sea bass

Vibrio harveyi was isolated from internal organs or ulcers of diseased and apparently healthy gilthead sea bream (Sparus aurata) and European sea bass (Dicentrarchus labrax) cultured in several fish farms located on the Spanish Mediterranean coast. The prevalence of the bacterium was significantly higher in European sea bass than in gilthead sea bream, and was closely related to the season in both fish species, occurring almost exclusively on warm months (June to November). After phenotypic characterization, a selection of forty five isolates from gilthead sea bream, sea bass, and several isolates previously obtained from common dentex (Dentex dentex) of the same area, were molecularly typed by automated ribotyping and random amplified polymorphic DNA (RAPD) analysis. Cluster analysis of data established 8 RAPD types and 13 ribotypes among wild isolates, and the combination of both techniques allowed to define fourteen different groups and a clear discrimination of all outbreaks and samplings. Several strains isolated from diseased gilthead sea bream and sea bass and also from asymptomatic sea bream, were tested for virulence in both fish species by intracoelomic injection. All the isolates (11) were pathogenic for sea bass, with nine out of the eleven LD50 values ranging from 1.5 x 10(5) to 1.6 x 10(6) cfu/fish. Gilthead sea bream was unaffected by the seven tested strains, even by those more virulent for sea bass, and only one strain caused a 10% mortality at 4.2 x 10(7) cfu/fish. This is the first report on virulence of V. harveyi for sea bass.     

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.     

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.     

Cymothoid parasite Ceratothoa parallela inflicts great losses on cultured gilthead sea bream sparus aurata in Greece

For the first time Ceratothoa parallela (Otto, 1828), a cymothoid isopod, is reported parasitizing cage-cultured gilthead sea bream Sparus aurata, in Greece. The specimens observed are larvae (Pullus secundus). They were found in the branchial and buccal cavity of young gilthead sea bream of 2 g mean body weight, upon introduction in the cages in an intensive cage farm facility. The species was previously known from several species of wild fish, particularly Sparidae (chiefly Boops boops) in the Mediterranean Sea, Adriatic Sea and Atlantic Ocean. However, this is the first documentation of this parasite in cage-cultured gilthead sea bream. Serious lesions were macroscopically visible and typical of a crustacean infection. The cumulative mortality over a 2 mo period was over 50%. The parasitic problem was not successfully dealt with due to high stocking densities and the non removal of the dead fishes, resulting in constant reinfection.     

In silico mining and characterization of simple sequence repeats from gilthead sea bream (Sparus aurata) expressed sequence tags (EST-SSRs); PCR amplification, polymorphism evaluation and multiplexing and cross-species assays

We screened for simple sequence repeats (SSRs) found in ESTs derived from an EST-database development project ('Marine Genomics Europe' Network of Excellence). Different motifs of di-, tri-, tetra-, penta- and hexanucleotide SSRs were evaluated for variation in length and position in the expressed sequences, relative abundance and distribution in gilthead sea bream (Sparus aurata). We found 899 ESTs that harbor 997 SSRs (4.94%). On average, one SSR was found per 2.95 kb of EST sequence and the dinucleotide SSRs are the most abundant accounting for 47.6% of the total number. EST-SSRs were used as template for primer design. 664 primer pairs could be successfully identified and a subset of 206 pairs of primers was synthesized, PCR-tested and visualized on ethidium bromide stained agarose gels. The main objective was to further assess the potential of EST-SSRs as informative markers and investigate their cross-species amplification in sixteen teleost fish species: seven sparid species and nine other species from different families. Approximately 78% of the primer pairs gave PCR products of expected size in gilthead sea bream, and as expected, the rate of successful amplification of sea bream EST-SSRs was higher in sparids, lower in other perciforms and even lower in species of the Clupeiform and Gadiform orders. We finally determined the polymorphism and the heterozygosity of 63 markers in a wild gilthead sea bream population; fifty-eight loci were found to be polymorphic with the expected heterozygosity and the number of alleles ranging from 0.089 to 0.946 and from 2 to 27, respectively. These tools and markers are expected to enhance the available genetic linkage map in gilthead sea bream, to assist comparative mapping and genome analyses for this species and further with other model fish species and finally to help advance genetic analysis for cultivated and wild populations and accelerate breeding programs.     

Molecular cloning and characterization of gilthead sea bream (Sparus aurata) growth hormone receptor (GHR). Assessment of alternative splicing

The full-length growth hormone receptor (GHR) of gilthead sea bream (Sparus aurata) was cloned and sequenced by RT-PCR and rapid amplification of 5'and 3'ends. The open reading frame codes for a mature 609 amino acid protein with a hydrophobic transmembrane region and all the characteristic motifs of GHRs. Sequence analysis revealed a 96 and 76% of amino acid identity with black sea bream (Acanthopagrus schlegeli) and turbot (Scophthalmus maximus) GHRs, respectively, but this amino acid identity decreases up to 52% for goldfish (Carassius auratus) GHR. By means of real-time PCR assays, concurrent changes in the hepatic expression of GHRs and insulin-like growth factor-I (IGF-I) was evidenced. Moreover, their regulation occurred in conjunction with the summer spurt of growth rates and circulating levels of GH and IGF-I. Search of alternative splicing was carried out exhaustively for gilthead sea bream GHR, but Northern blot and 3' RACE failed to demonstrate the occurrence of short alternative messengers. Besides, RT-PCR screening did not reveal deletions or insertions that could lead to alternative reading frames. In agreement with this, cross-linking assays only evidenced two protein bands that match well with the size of glycosylated and non-glycosylated forms of the full-length GHR. If so, it appears that alternative splicing at the 3'end does not occur in gilthead sea bream, although different messengers for truncated or longer GHR variants already exist in turbot and black sea bream, respectively. The physiological relevance of this finding remains unclear, but perhaps it points out large inter-species differences in the heterogeneity of the GHR population.     

Insulin regulation of lipoprotein lipase (LPL) activity and expression in gilthead sea bream (Sparus aurata)

Lipoprotein lipase (LPL) is a key enzyme in lipoprotein metabolism by virtue of its capacity to hydrolyze triglycerides circulating in the form of lipoprotein particles. Here we analyzed the fasting effects of LPL in gilthead sea bream (Sparus aurata) and also present the first study in fish of the role of insulin as a potential modulator of both LPL activity and expression. Fasting for 2 weeks provoked a clear decrease in adipose tissue LPL activity, concomitant with lower levels of plasma insulin, while no effects were observed in red muscle. To elucidate the specific role of insulin, increases of plasma insulin were experimentally induced by arginine and insulin injections. However, arginine predominantly stimulated glucagon over insulin secretion in this fish species while LPL activity did not change significantly in adipose tissue. Instead, insulin administration induced an increase in adipose tissue LPL activity 3 h after the injection, whereas LPL activity in red muscle was not affected. Changes in LPL activity were accompanied by an increase in LPL mRNA levels in the adipose tissue of insulin-injected gilthead sea bream, although changes in LPL expression were delayed in time with respect to variations in LPL activity. Finally, LPL mRNA levels in red muscle were similar between control and insulin-injected gilthead sea bream, suggesting that insulin does not play a direct role in the regulation of LPL in this tissue. The current study shows that LPL activity is regulated by nutritional condition and underscores the importance of insulin as a modulator of LPL activity and expression in the adipose tissue of gilthead sea bream.     

Molecular mechanisms determining sperm motility initiation in two sparids (Sparus aurata and Lithognathus mormyrus)

Molecular mechanisms involved in sperm motility initiation in two sparids (Sparus aurata and Lithognathus mormyrus) have been studied. Our comparative study demonstrates that osmolality is the key signal in sperm motility activation in both species, whereas K(+) and Ca(2+) do not have any role. The straight-line velocity that resulted, however, was significantly different when measured in sperm activated with non-ionic and/or calcium-free solutions with respect to that measured in seawater-activated sperm. In both species, motility initiation depends on cAMP-dependent protein phosphorylation. The phosphorylation/dephosphorylation patterns that resulted in gilthead and striped sea bream were quite different. In gilthead sea bream, the phosphorylated proteins have molecular weights of 174, 147, 138, 70, and 9-15 kDa, whereas the dephosphorylated proteins have molecular weights of 76, 57, and 33 kDa. In striped sea bream, phosphorylation after sperm motility activation occurred on proteins of 174, 147, 103, 96, 61, 57, and 28 kDa, whereas only one protein of 70 kDa resulted from dephosphorylation. Matrix-assisted laser desorption ionization-time of flight analyses allowed identification of the following proteins: In gilthead sea bream, the 9-15 kDa proteins that were phosphorylated after motility activation include an A-kinase anchor protein (AKAP), an acetyl-coenzyme A synthetase, and a protein phosphatase inhibitor, and in striped sea bream, 103- and 61-kDa proteins that were phosphorylated after motility activation were identified as a phosphatase (myotubularin-related protein 1) and a kinase (DYRK3), respectively.     

Rearing temperature enhances hepatic glucokinase but not glucose-6-phosphatase activities in European sea bass (Dicentrarchus labrax) and gilthead sea bream (Sparus aurata) juveniles fed with the same level of glucose

The aim of this work was to elucidate if the previous results observed in hepatic glucokinase (GK) and glucose-6-phosphatase (G6Pase) activities in European sea bass and gilthead sea bream are due to temperature per se or to differences in feed intake at different water temperatures. For that purpose triplicate groups of fish (30 g initial body weight) were kept at 18 degrees C or 25 degrees C during two weeks and fed a fixed daily ration of a glucose-free or 20% glucose diet. At the end of the experimental period, plasma glucose levels in both species were not influenced by water temperature but were higher in fish fed the glucose diet. Higher hepatic GK activity was observed in the two fish species fed the glucose diet than the glucose-free diet. In the glucose fed groups, GK activity was higher at 25 degrees C than at 18 degrees C. Glucose-6-phosphatase activities in both species were not influenced by water temperature. In European sea bass and in contrast to gilthead sea bream it was observed an effect of dietary composition on G6Pase activities with surprising higher activities recorded in fish fed the glucose diet than in fish fed the glucose-free diet. Overall, our data strongly suggest that European sea bass and gilthead sea bream are apparently capable to strongly regulate glucose uptake by the liver but not glucose synthesis, which is even enhanced by dietary glucose in European sea bass. Within limits, increasing water temperature enhances liver GK but not G6Pase activities, suggesting that both species are more able to use dietary carbohydrates at higher rearing temperatures.     

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.     

Amino acid composition and endocrine control of vitelline envelope proteins in European sea bass (Dicentrarchus labrax) and gilthead sea bream (Sparus aurata)

The vitelline envelopes of European sea bass and gilthead sea bream are both composed of mainly four proteins with the molecular masses of 90, 52, 48, 45 kDa and 75, 50, 48, 44 kDa, respectively. Each protein has an amino acid composition that is characterized by a high content of proline and glutamic acid and a low content of cysteine, similar to the whole vitelline envelope of both species. The amino acid composition suggests that each protein is distinct but related to the other vitelline envelope proteins. The use of homologous antisera shows that both species have vitelline envelope proteins that are induced by estradiol-17β. As males of both species synthesize these proteins after treatment with estradiol-17β, the origin is not restricted to the ovaries. Vitellogenin of both Eurpoean sea bass and gilthead sea bream has the apparent molecular mass of 170 kDa. © 1995 Wiley-Liss, Inc.     

Ichthyophonus infections in cultured marine fish from Spain

Ichthyophonus sp. is reported for the first time in Mugil capito (thinlip grey mullet) and Li a saliens (leaping grey mullet). The fungus was also found in L. aurata (golden grey mullet), Dicentrarchus labrax (sea bass), Sparus aurata (gilthead sea bream) and Scophthalmus maximus (turbot), whereas Mugil cephalus (grey mullet) was not parasitized. In fish sampled periodically, the highest prevalences were observed in sea bass and the lowest in turbot. Among the fish sampled occasionally, the fungus was found associated to an epizootic in thinlip grey mullet. Ichthyophonus was never found in fish weighing <0·5 g. An increase in the prevalence of infection with the age of turbot and gilthead sea bream was observed. Gilthead sea bream and sea bass showed higher prevalences in a closed system than in open and semi-intensive systems. Multinucleate spherical spores, hyphae and endospores of Ichthyophonus sp. parasitized different organs of thinlip and leaping grey mullets, though infection intensity was maximal in the spleen. In the remaining fish, the fungus was found mainly in the trunk kidney, where it appeared frequently in a necrotic form. Ichthyophonus sp. can be considered a potential threat for marine fish aquaculture, especially in culture conditions which may favour the introduction and transmission of the fungus.     

Evaluation of candidate probiotic strains for gilthead sea bream larvae (Sparus aurata) using an in vivo approach

AIMS: The aim of this study was to evaluate the effect of six bacterial strains on gilthead sea bream larvae (Sparus aurata). METHODS AND RESULTS: Six bacterial strains isolated from well-performing live food cultures were identified by sequencing fragments of their 16s rDNA genome to the genus level as Cytophaga sp., Roseobacter sp., Ruergeria sp., Paracoccus sp., Aeromonas sp. and Shewanella sp. Survival rates of gilthead sea bream larvae transferred to seawater added these bacterial strains at concentrations of 6 +/- 0.3 x 10(5) bacteria ml(-1) were similar to those of larvae transferred to sterilized seawater and showed an average of 86% at 9 days after hatching, whereas, survival rates of larvae transferred to filtered seawater were lower (P < 0.05), and showed an average of 39%, 9 days after hatching. CONCLUSION: Several bacterial strains isolated from well-performing live food cultures showed a positive effect for sea bream larvae when compared with filtered seawater. SIGNIFICANCE AND IMPACT OF THE STUDY: The approach used in this study could be applied as an in vivo evaluation method of candidate probiotic strains used in the rearing of marine fish larvae.     

Effect of water temperature and dietary starch on growth and metabolic utilization of diets in gilthead sea bream (Sparus aurata) juveniles

We evaluated the effect of dietary starch level on growth performance, feed utilization, whole-body composition and activity of selected key enzymes of intermediary metabolism in gilthead sea bream juveniles reared at 18 and 25 degrees C. A diet was formulated to contain 48% crude protein, 12% lipids and 30% gelatinized maize starch (diet 30GS). Two other diets were formulated to include the same level of ingredients as diet 30GS except for the gelatinized starch, which was included at 20% (diet 20GS) or 10% (diet 10GS). No adjustment to diet composition was otherwise made. Each diet was fed to triplicate groups of gilthead sea bream (30 g initial mass) for 8 weeks, on a pair-feeding scheme. The higher temperature improved growth performance but the opposite was true for feed efficiency and protein efficiency ratio. Independently of temperature, growth performance, feed efficiency and protein efficiency ratio were lower in fish fed diet 30GS. No effect of temperature or dietary starch level on whole-body composition was noticed. Hepatosomatic index and liver glycogen were higher at 18 degrees C and, within each temperature, in fish fed diet 30GS. Glycemia was not affected by temperature, but was lower in fish fed diet 10GS. Data on enzyme activities showed that increasing water temperature enhances liver glucokinase (GK) and pyruvate kinase (PK) activities, suggesting that gilthead sea bream is more apt to use dietary starch at higher temperatures. No effect of temperature was noticed on hexokinase (HK), fructose-1,6-bisphosphatase (FBPase), glucose-6-phosphate dehydrogenase (G6PD) and glutamate dehydrogenase (GDH) activities. Dietary starch enhanced PK and FBPase activities while depressed GDH activity, suggesting a lack of significant regulation of hepatic glucose utilization and production in this species. HK, GK and G6PD activities were unaffected by dietary composition. Irrespectively of water temperature, gelatinized starch may be included up to 20% in diets for gilthead sea bream juveniles; at higher dietary levels, growth and efficiency of feed utilization are depressed.     

Cryptosporidium molnari n. sp. (Apicomplexa: Cryptosporidiidae) infecting two marine fish species,Sparus aurata L. and Dicentrarchus labrax L

Cryptosporidium molnari n. sp. is described from two teleost fish, the gilthead sea bream (Sparus aurata L.) and the European sea bass (Dicentrarchus labrax L.). The parasite was found mainly in the stomach epithelium and seldom in the intestine. Oocysts were almost spherical, with four naked sporozoites and a prominent residuum, and measured 3.23-5.45 x 3.02-5.04 (mean 4.72 x 4.47) microm in the type host, gilthead sea bream (shape index 1-1.17, mean 1.05). Sporulation was endogenous, as fully sporulated oocysts were found within the fish, both in the stomach epithelium and lumen, and in faeces. Oocysts and other stages of C. molnari fit most of the diagnostic features of the genus Cryptosporidium, but differ from hitherto described species, including piscine ones. All stages were located within a host contributed parasitophorous vacuole lined by a double host microvillar membrane. Merogonial and gamogonial stages appeared in the typical extracytoplasmic position, whereas oogonial and sporogonial stages were located deeply within the epithelium. Ultrastructural features, including the characteristic contact zone of the parasite with the host epithelial surface, were mostly coincident with those of other Cryptosporidium spp. Mitochondria were found in dividing meronts, merozoites, microgamonts and sporozoites. Pathological effects were more evident in gilthead sea bream, which also exhibited a clearly higher prevalence (24.4 versus 4.64% in sea bass). External clinical signs, consisting of whitish faeces, abdominal swelling and ascites, were rarely observed, in contrast with important histopathological damage. The wide zones of epithelium invaded by oogonial and sporogonial stages appeared necrotic, with abundant cell debris, and sloughing of epithelial cells, which detached to the lumen. No inflammation reaction was observed and the cellular reaction was limited to the cells involved in the engulfing of intraepithelial stages and debris, probably macrophages.     

Feeding Responses of Hatchery-Reared Gilthead Sea Bream (Sparus aurata L.) to a Commercial Diet and Natural Prey Items

Many fish species have evolved feeding mechanisms and behaviours enabling them to feed on specific prey. However, such mechanisms may not be optimal for feeding on commercial-pelleted diets in aquaculture. Gilthead sea bream chew and occasionally eject pellets or parts of pellets from the mouth when feeding on commercial diets. This may result in an increase in nutritional waste from the intensive culture of this species. In this study we examined the prevalence of this food processing behaviour in two sizes of sea bream, feeding on three types of natural prey items in comparison to a commercial pellet, to give an insight into the circumstances in which excess chewing and ejection of food items from the mouth occurred. These included two hard-textured food items (commercial pellet and hard-shelled prey) and two soft-textured food items (larvae and small crustacean). Both sizes of sea bream frequently consumed the soft-textured food types, however large sea bream also frequently consumed hard-textured pellets. Hard-textured food required longer handling times and elicited more chewing and the ejection of food items from the mouth. These results suggest that future investigations on the food processing behaviour and consequent waste when fed commercial diets differing in texture could give an insight into improving diets and feeding efficiency for intensively cultivated gilthead sea bream.     

Comparison of two techniques for the morphometry study on gilthead seabream (Sparus aurata) spermatozoa and evaluation of changes induced by cryopreservation

The development of powerful software has made possible spermatozoa morphology studies. However, some problems have emerged in relation to protocol standardization to compare results from different laboratories. This study was carried out to compare two techniques commonly used (staining vs phase contrast technique) for the morphometry study of gilthead sea bream spermatozoa using an integrated sperm analysis system (ISAS). Spermatozoa morphometry values were significantly affected by the technique used, and phase contrast technique was found to be the more accurate method, showing lower coefficients of variation on spermatozoa morphometry parameters measurements. Moreover, it has been shown that cryopreservation process produces damage in gilthead sea bream spermatozoa, causing negative effects in sperm parameters as spermatozoa morphometry (a decrease in cell volume), motility (from 95 to 68% motile cells) and viability (from 95 to 87% of live cells), being the addition of freezing medium containing cryoprotectant (DMSO) an important factor that caused the morphometry changes.