Thermal tolerance and standard metabolic rate of juvenile gilthead seabream (Sparus aurata) acclimated to four temperatures

Temperature variation affects the growth, maturation and distribution of fish species due to increasing constraints on physiological functions therefore, the aim of the present study is to evaluate effect of temperature on thermal tolerance and standard metabolic rate (SMR) of gilthead seabream (Sparus aurata). For this purpose, tolerable temperature ranges of juvenile gilthead seabream acclimated at 15, 20, 25, and 30 °C for 30 days were estimated using dynamic and static thermal methodologies. The SMRs of the fish were also determined based on oxygen consumption rate (OCR). The dynamic and static thermal tolerance zones of gilthead seabream were calculated as 737 °C² and 500 °C², respectively, with a resistance zone area of 155.5 °C². The SMR of the fish at the above acclimation temperatures (AT) was determined as 138, 257, 510, and 797 mg O₂ h⁻¹ kg⁻¹, respectively and were significantly different (P < 0.01, n = 10). The temperature quotient (Q₁₀) in relation to the SMR of the fish was calculated as 3.45, 3.91, and 2.44 for acclimation temperature ranges of 15–20, 20–25, and 25–30 °C, respectively. The fact that the SMR increased with rising temperatures and then decreased gradually after 25 °C indicates that the temperature preference of juvenile gilthead seabream lies between 25 and 30 °C. This study shows that gilthead seabream tolerates a relatively narrow temperature range, and consequently, a low capacity for acclimatisation to survive in aquatic systems characterised by temperature variations.     

Testicular involution prior to sex change in gilthead seabream is characterized by a decrease in DMRT1 gene expression and by massive leukocyte infiltration

Background  

Leukocytes are found within the testis of most, if not all, mammals and are involved in immunological surveillance, physiological regulation and tissue remodelling. The testis of seasonal breeding fish undergoes a regression process. In the present study, the second reproductive cycle (RC) of the protandrous seasonal teleost fish, gilthead seabream, was investigated and the presence of leukocytes analysed. Special attention has been paid to the testicular degenerative process which is particularly active in the last stage of the second RC probably due to the immediacy of the sex change process.     

Mortality Caused by Bath Exposure of Zebrafish (Danio rerio) Larvae to Nervous Necrosis Virus Is Limited to the Fourth Day Postfertilization

Nervous necrosis virus (NNV) is a member of the Betanodavirus genus that causes fatal diseases in over 40 species of fish worldwide. Mortality among NNV-infected fish larvae is almost 100%. In order to elucidate the mechanisms responsible for the susceptibility of fish larvae to NNV, we exposed zebrafish larvae to NNV by bath immersion at 2, 4, 6, and 8 days postfertilization (dpf). Here, we demonstrate that developing zebrafish embryos are resistant to NNV at 2 dpf due to the protection afforded by the egg chorion and, to a lesser extent, by the perivitelline fluid. The zebrafish larvae succumbed to NNV infection during a narrow time window around the 4th dpf, while 6- and 8-day-old larvae were much less sensitive, with mortalities of 24% and 28%, respectively.     

Possible Transmission of Streptococcus iniae from Wild Fish to Cultured Marine Fish

Streptococcus iniae was isolated from diseased wild fish collected near a mariculture facility where gilthead sea bream and European sea bass exhibited a similar infection. Species-specific PCR and ribotyping confirmed that wild and cultured fish were infected by a single S. iniae clone. Wild fish are therefore potential amplifiers of pathogenic S. iniae strains.     

Effect of lipopolysaccharides from Vibrio alginolyticus on the Mx gene expression and virus recovery from gilthead sea bream (Sparus aurata L.) experimentally infected with Nodavirus

Infections with nodavirus affect a wild and farmed fish species throughout the world, mostly from the marine environment. The aim of this work was to determine the immune status of gilthead sea bream that comes as a result of a Nodavirus infection, induced by activation of the interferon response pathway by lipopolysaccharides from Vibrio alginolyticus and the expression of interferoninduced Mx protein in liver samples. The enhancement of Mx protein gene expression was detected in liver samples of experimentally nodavirus infected fish and, furthermore, the immunostimulant LPS of V. alginolyticus decreased almost three times the virus titration with respect to no-immunized or infected with nodavirus group of fish.     

NK-like and oxidative burst activities are the main early cellular innate immune responses activated after virus inoculation in reservoir fish

Viral diseases are a major problem in fish farming and a deeper knowledge of the immunological mechanisms playing a part in the antiviral defence is still important. Moreover, fish farming practices (high densities, new areas of culture and egg/larvae/adult transport) are significantly increasing the spread of viruses and the number of susceptible or reservoir fish species. In this last point, no studies have focused on the immunological mechanisms playing a part in the antiviral responses in reservoir and non-susceptible fish species. Thus, we have evaluated the very early innate immune responses of gilthead seabream (Sparus aurata) to the virus causing viral haemorrhagic septicaemia (VHSV) in salmonids since this virus has been found in seabream and neighbouring farmed marine fish species acting as a viral reservoir. The virus was detected in liver, head-kidney, spleen and peritoneal cavity suggesting that the virus reached these tissues but did not replicate as viral expression was almost absent by 72h post-inoculation. Interestingly, VHSV provoked an influx of leucocytes to the peritoneal cavity and a redistribution of peritoneal exudate (PELs) and head-kidney (HKLs) leucocytes and their innate immune responses (non-specific cytotoxic (NCC or NK-like) activity, phagocytosis, reactive oxygen intermediate (ROI) production and myeloperoxidase (MPO) activity) were generally increased demonstrating that the immune system is activated and involved in the clearance of the virus. Strikingly, NK-like, ROI and MPO were the most enhanced by the presence of VHSV in both PELs and HKLs suggesting that these early innate immune events are crucial during early viral infection stages in non-susceptible or reservoir species. Differences in the immunological mechanisms between susceptible and reservoir species and with other particulate antigens are discussed.     

Development and testing of a combined species SNP array for the European seabass (Dicentrarchus labrax) and gilthead seabream (Sparus aurata)

SNP arrays are powerful tools for high-resolution studies of the genetic basis of complex traits, facilitating both selective breeding and population genomic research. The European seabass (Dicentrarchus labrax) and the gilthead seabream (Sparus aurata) are the two most important fish species for Mediterranean aquaculture. While selective breeding programmes increasingly underpin stock supply for this industry, genomic selection is not yet widespread. Genomic selection has major potential to expedite genetic gain, particularly for traits practically impossible to measure on selection candidates, such as disease resistance and fillet characteristics. The aim of our study was to design a combined-species 60 K SNP array for European seabass and gilthead seabream, and to test its performance on farmed and wild populations from numerous locations throughout the species range. To achieve this, high coverage Illumina whole-genome sequencing of pooled samples was performed for 24 populations of European seabass and 27 populations of gilthead seabream. This resulted in a database of ~20 million SNPs per species, which were then filtered to identify high-quality variants and create the final set for the development of the ‘MedFish’ SNP array. The array was then tested by genotyping a subset of the discovery populations, highlighting a high conversion rate to functioning polymorphic assays on the array (92% in seabass; 89% in seabream) and repeatability (99.4–99.7%). The platform interrogates ~30 K markers in each species, includes features such as SNPs previously shown to be associated with performance traits, and is enriched for SNPs predicted to have high functional effects on proteins. The array was demonstrated to be effective at detecting population structure across a wide range of fish populations from diverse geographical origins, and to examine the extent of haplotype sharing among Mediterranean farmed fish populations. In conclusion, the new MedFish array enables efficient and accurate high-throughput genotyping for genome-wide distributed SNPs for each fish species, and will facilitate stock management, population genomics approaches, and acceleration of selective breeding through genomic selection.     

Effects of dietary vitamin D3 administration on innate immune parameters of seabream (Sparus aurata L.)

The present study assesses the in vivo effect of vitamin D₃ or cholecalciferol on some innate immune parameters of the gilthead seabream (Sparus aurata L.). Cholecalciferol was orally administered to seabream specimens in a commercial pellet food supplemented with 0 (control); 3750; 18,750 or 37,500 U kg^?1 and fish were sampled after 1, 2 and 4 weeks of treatment. Serum and head– kidney leucocytes were obtained and humoral (peroxidase and complement activity) and cellular (leucocyte peroxidase content, phagocytic, respiratory burst and natural cytotoxic activities) innate immune parameters were measured. Diet supplementation with 37,500 U kg^?1 cholecalciferol for 2 or 4 weeks resulted in a significant increase in phagocytic ability or serum peroxidase content, respectively, whereas the 3750 and 18,750 U kg^?1 supplemented diets led to significant increases in the phagocytic capacity of leucocytes at week 2 compared with the values found in control fish. Natural cytotoxic activity was increased in leucocytes from fish fed for 1 week with 3750 U kg^?1 cholecalciferol. No significant differences were observed in complement activity or in respiratory burst activity in the assayed conditions. These results suggested that dietary vitamin D₃ administration has an effect on the innate immune parameters of gilthead seabream. The immunostimulant effect was greater on the cellular innate immune parameters assayed, suggesting that similar receptors to those present in mammals are involved in the action of this vitamin in the fish immune system.     

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.     

Effect of dietary lipids on plasma fatty acid profiles and prostaglandin and leptin production in gilthead seabream (Sparus aurata)

The aim of this study was to investigate the effects of different levels of substitution of fish oil by vegetable oils rich in oleic, linoleic and linolenic acids on gilthead seabream plasma and leukocyte fatty acid compositions and prostaglandin (PG) and leptin production. Juvenile seabream of 24 g initial body mass were fed four iso-energetic and iso-proteic experimental diets for 281 days. Fatty acid composition of plasma lipids was markedly affected by the inclusion of vegetable oils (VO). ARA (arachidonate), EPA (eicosapentaenoate) and DHA (docosahexaenoate) were preferentially incorporated into polar lipids of plasma, and DHGLA (di-homogammalinoleate) accumulated with increased vegetable oil inclusion. Dietary treatments resulted in alterations of DHGLA/ARA ratios, but not ARA/EPA. ARA-derived PGE₂ production in plasma was not affected by vegetable oils, in agreement with similar eicosanoid precursor ratio (ARA/EPA) in leukocytes total lipids and plasma phospholipids among fish fed with the different dietary treatments. Feeding vegetable oils leads to a decrease in plasma EPA which in turn reduced plasma PGE₃ concentration. Moreover, PGE₃ was the major prostaglandin produced in plasma of fish fed fish oil based diet. Such findings point out the importance of EPA as a precursor of prostaglandins in marine fish, at least for the correct function of the blood cells, and correlates well with the predominant role of this fatty acid in immune regulation in this species. A negative correlation was found between plasma PGE₂ and leptin plasma concentration, suggesting that circulating levels of leptin may act as a metabolic signal modulating PGE₂ release. The present study has shown that increased inclusion of vegetable oils in diet for gilthead seabream may profoundly affect the fatty acid composition of plasma and leukocytes, specially HUFA (highly unsaturated fatty acids), and consequently the production of PGE₃, which can be a major PG in plasma. Alteration in the amount and type of PG produced can be at least partially responsible for the changes in the immune system and health parameters of fish fed diets with high inclusion of VO.     

Modulatory effects of deltamethrin-exposure on the immune status,metabolism and oxidative stress in gilthead seabream (Sparus aurata L.)

Deltamethrin, a sintetic pyrethroid, is the insecticide that has been replacing recently to others like organochlorines, organophosphates and carbamates which are less toxic for birds and mammals, although, unfortunately, all of them are highly toxic to various non-targeted aquatic organisms including fish. In the present study, the consequences of the exposition of gilthead seabream (Sparus aurata L.) specimens to sublethal bath dose of deltamethrin (0.1 ppb) on organo-somatic indexes, immunity, seric metabolic parameters, oxidative stress and liver histology were determined after 1, 3, 7 and 14 days of exposure. Deltamethrin alters gilthead seabream immune status, the hepato-somatic index and various seric metabolic parameters since the first exposure day while important progressive deleterious morphological changes in liver were also observed. However, no statistically significant deviation was detected in the expression of oxidative stress-related genes whilst the expression of cytochrome P450 gene was up-regulated in head-kidney and liver of exposed fish. Overall, the present results indicate severe immunotoxicological and metabolic effects of deltamethrin in gilthead seabream, the species with the highest rate of production in Mediterranean aquaculture. In general, the values obtained for the tested parameters during the trial seem to indicate that specimens try to adapt to this adverse situation although the continuous presence of the toxic impede the hypothetic recovery of homoeostasis. The use of deltamethrin in the proximities of seabream farms should be carefully considered.     

Surveillance of Viruses in Wild Fish Populations in Areas around the Gulf of Cadiz (South Atlantic Iberian Peninsula)

This report describes a viral epidemiological study of wild fish around the Gulf of Cadiz (southwestern Iberian Peninsula) and is focused on infectious pancreatic necrosis virus (IPNV), viral hemorrhagic septicemia virus (VHSV), and viral nervous necrosis virus (VNNV). One fish species (Chelon labrosus) was sampled inside the gulf, at the mouth of the San Pedro River. Another 29 were sampled, in three oceanographic campaigns, at sites around the Bay of Cadiz. The fish were processed individually and subjected to isolation in cell culture and molecular diagnosis. VHSV was not isolated from any species. Thirteen IPNV-type isolates were obtained from barracuda (Sphyraena sphyraena), axillary seabream (Pagellus acarne), common two-banded seabream (Diplodus vulgaris), common pandora (P. erythrinus), Senegal seabream (D. bellottii), and surmullet (Mullus surmuletus). Six VNNV isolates were obtained from axillary seabream, common pandora, black seabream (Spondyliosoma cantharus), red mullet (Mullet barbatus), Lusitanian toadfish (Halobatrachus didactylus), and tub gurnard (Chelidonichtys lucerna). In the river mouth, viruses were detected only after reamplification, obtaining prevalence percentages of IPNV and VNNV (44.4 and 63.0%, respectively) much higher than those observed in the oceanographic campaigns (25.7 and 19.6%, respectively). The opposite results were obtained in the case of VHSV after reamplification: 11.1% in the river mouth and 43.6% in the oceanic locations. Analyzing the results with respect to the proximity of the sampling sites to the coast, an anthropogenic influence on wild fish is suggested and discussed. The type of viruses and the presence of natural reassortants are also discussed.     

How does fish metamorphosis affect aromatic amino acid metabolism?

Aromatic amino acids (AAs, phenylalanine and tyrosine) may be specifically required during fish metamorphosis, since they are the precursors of thyroid hormones which regulate this process. This project attempted to evaluate aromatic AA metabolism during the ontogenesis of fish species with a marked (Senegalese sole; Solea senegalensis) and a less accentuated metamorphosis (gilthead seabream; Sparus aurata). Fish were tube-fed with three l-[U-¹⁴C] AA solutions at pre-metamorphic, metamorphic and post-metamorphic stages of development: controlled AA mixture (Mix), phenylalanine (Phe) and tyrosine (Tyr). Results showed a preferential aromatic AA retention during the metamorphosis of Senegalese sole, rather than in gilthead seabream. Senegalese sole’s highly accentuated metamorphosis seems to increase aromatic AA physiological requirements, possibly for thyroid hormone production. Thus, Senegalese sole seems to be especially susceptible to dietary aromatic AA deficiencies during the metamorphosis period, and these findings may be important for physiologists, fish nutritionists and the flatfish aquaculture industry.     

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.     

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.     

Antimicrobial peptides (AMP) with antiviral activity against fish nodavirus

Nervous necrosis virus (NNV) is classified as betanodavirus of Nodaviridae, and has caused mass mortality of numerous marine fish species at larval stage. Antimicrobial peptides (AMPs) play an important role of innate immunity either against bacterial pathogens or viruses. Up to date, little is known if any AMP could effectively inhibit fish nodaviruses and its mechanism. In this study, the antiviral activities of three antimicrobial peptides (AMPs) against grouper NNV (GNNV) were screened in the fish cell line. Two of the three AMPs, tilapia hepcidin 1-5 (TH 1-5) and cyclic shrimp anti-lipopolysaccharide factor (cSALF), were able to agglutinate purified NNV particles into clump, and the clumps were further confirmed to be viral proteins by TEM and Western blot. The NNV solution, separately pre-mixed with AMP (TH 1-5 or cSALF) or deionized-distilled water for 1 h, was used to infect GF-1 cells, and the levels of capsid protein in the GNNV-AMP-infected cells at 1 h post infection were much lower than that in the GNNV-H₂O-infected cells, indicating that only a small portion of viral particles in the GNNV-AMP mixture could successfully infected the cells. Treatment of cBB cells with TH 1-5 and cSALF did not induce Mx gene expression; however, grouper epinecidin-1 (CP643-1) could induce the expression of Mx in the pre-treated cBB cells. This study revealed three AMPs with anti-NNV activity through two different mechanisms, and shed light on the future application in aquaculture.