Growth,tissue metabolites and digestive functionality in Sparus aurata juveniles fed different levels of macroalgae,Gracilaria cornea and Ulva rigida

The aim of this study was to evaluate the inclusion of two macroalgae as dietary ingredients on growth, body composition, intermediate metabolism, intestinal enzymes and gut histology of Sparus aurata juveniles. Six isocaloric and isonitrogenous diets incorporating 5%, 15% or 25% of Gracilaria cornea or Ulva rigida meal were tested against a control diet. Duplicate groups of fifteen fish (14 g) were fed the experimental diets for 70 days. Growth performance showed an inverse linear relationship with the inclusion level of Gracilaria. Feed conversion ratio increased significantly in fish fed 25% Gracilaria. Ulva presented a quadratic relationship with the lowest SGR at 15% of inclusion. Fish fed 25% Ulva exhibited significantly lower lipid content than those fed control diet. Overall, metabolites did not show differences with specimens fed control diet, except for plasmatic glucose and muscle lactate. Intestinal proteolytic activity was reduced with the macroalgae inclusion level, but all fish showed the same enzyme pattern. The histological study revealed no negative effect on liver and intestine structure by the macroalgae inclusion. The present work demonstrated that these ingredients can be used in practical diets for sea bream, but a more comprehensive research is needed to determine the optimum dietary level in a longer feeding trial.     

Quantitative investigation of Enteromyxum leei (Myxozoa: Myxosporea) infection and relative condition factor in cultured olive flounder Paralichthys olivaceus (Temminck and Schlegel)

Enteromyxum leei has been reported to cause emaciation disease in various fish species. To determine the effect of parasite intensity on cultured olive flounder Paralichthys olivaceus, we investigated the relationship between the relative condition factor (rCF = CF/standard CF × 100) and parasite load with quantitative polymerase chain reaction (qPCR) and the challenge test. A total of 57 cultured olive flounders were obtained from 11 fish farms and divided into five groups based on their rCF. We investigated the parasite intensity in the posterior intestine of the fish. The parasite load was closely matched to severe loss of body weight. In addition, olive flounders were inoculated either orally or anally with intestinal scrapings of infected fish or phosphate‐buffered saline. The fish were reared at natural water temperature and transferred to different tanks, and the water temperature was adjusted to 20°C after 6 weeks of inoculation. When the water temperature was increased to 20°C, the rCF decreased in the experimentally infected group. The results demonstrated that qPCR can be utilized to determine the relative abundance of E. leei in olive flounders and water temperature is an important factor to track the progress of the emaciation disease.     

Early assessment of gilthead sea bream (Sparus aurata) spawning dynamics by mini‐broodstocks

The implementation of any sustainable breeding program requires a maximization of effective size (Ne) to maintain inbreeding rate at minimum levels. However, the control of Ne remains a major challenge for those species whose reproduction in captivity is based on a spawning strategy where there is no control over the established matings. The present study aims to assess the spawning dynamic of gilthead sea bream (Sparus aurata) at different dates from two mini‐broodstocks (4 ♀ and 4 ♂). Here we report the use of a simple protocol based on the genotyping of fertilized eggs from a reduced set of microsatellite loci which provides a quick evaluation of daily spawning in terms of family structuring (number of active breeders and number and family size). According to our results, the daily spawning dynamics is characterized by a fluctuation in the number of active breeders, reaching maximum values (until 100%) during peak production phase. However, the proportion of possible mating (families) in a single daily spawning never exceeded more than 81%. Unbalanced parental contributions were highly frequent, with extreme cases involving the assignment of 50%–90% of the offspring to a single family. Therefore, the use of mini‐broodstocks and the grouping of spawning harvested at different dates are proposed as strategies to minimize the inbreeding risk by providing a more optimal pattern of family structuring (increase in the number of families obtained and a more balanced parental contributions) that entails a more conservative Ne/N ratio.     

Plant protein and vegetable oil‐based diets modulate gilthead seabream (Sparus aurata) muscle biochemical status and proteolytic enzymes at an early postmortem stage

A 70 days feeding trial was carried out on juvenile gilthead seabream (Sparus aurata) (mean initial body weight: 362 ± 59 g) to evaluate whether the simultaneous replacement of fishmeal and fish oil by vegetable ingredients affected the muscle's proteolytic potential. Fish were fed either a fishmeal/fish oil diet, a fishmeal/vegetable oil diet, a plant protein/fish oil or a plant protein/vegetable oil diet. The use of dietary plant proteins resulted in lower sulphated glycosaminoglycans' content and lower glycogen phosphorylase activity in the muscle, while both plant proteins and vegetable oil increased muscle pH and reduced cathepsin B activity. Our results indicate that high replacement of fish meal and/or fish oil with plant ingredients has a significant impact on early postmortem metabolic processes and proteolytic potential of seabream muscle, especially when replacing both fish meal and fish oil.     

Effect of phytase supplementation of a plant‐based diet on phosphorus and nitrogen bioavailability in sea bream Sparus aurata

To assess to what extent addition of phytase to a plant‐based diet results in spatio‐temporal changes of phytate, available P, soluble protein, total amino acids and the activity of the main digestive proteases in gilthead sea bream, fish were fed two plant‐based diets with or without phytase. Stomach, proximal intestine and distal intestine contents were monitored for these parameters at 0, 1, 2, 4 and 6 h after feeding. A reduction (P < 0.0001) of the soluble P–IP6 in the stomach when phytase was added to the diet was observed. Within stomach, most of the total P–IP6 was precipitated (86%), possibly due to the low acidification capacity of the sea bream (pH > 4), but 57% of the dietary P–IP6 was dephosphorylated, suggesting that phytase could have the capacity to dephosphorylate insoluble IP6 at such pH. An increment (60%) (P < 0.01) in total gastric protease activity was observed by phytase addition, this being the first demonstration of the in vivo effect of IP6 on the pepsin activity in fish stomach. Gastric pH and residence time of the digesta inside the stomach are critical factors for an efficient phytase action and improve P and N bioavailability in plant‐based diets used in fish aquaculture.     

A behavioural approach of dominance establishment in tank‐held sea bream (Sparus aurata L.) under different feeding conditions

Dominance is a strong behaviour exhibited by farmed species that very often impinges on fish growth and welfare. This study presents a behavioural approach of dominance, where colour pattern differentiation was tested as a signal of dominance presence in small sea bream population. The phenotype of dominance was first described in detail, referring to vertical dark stripes and splayed fins. Fish were kept in triplicate tanks for 21 days, during which they were exposed to three feeding conditions (well fed, limited fed and no‐fed) and continuous video recordings. Each tank was stocked with 15 individuals (~30.34 ± 1.70 g), and they were confined to the half volume of the tank via a removable net pen. Percentage of dominants per population was found up to 40%, while duration was calculated to 53.52 ± 7.44 s. Dominance behaviour was further quantified via colour pattern differentiation on sea bream body (CDA, contrast of dominance appearance). The results demonstrated that the body colour of sea bream is directly linked to species social hierarchies and such variations are visual signal of dominance rank inside the population. The fish feeding conditions had an influence on the dominance presence, but not on the dominance rank (as measured by the CDA) between fish groups. The described study provides state‐of‐the‐art knowledge on sea bream dominance. In relevance to aquaculture, dominance quantification would be a reliable tool to evaluate non‐equal food distribution in tank‐held populations, thus, avoid non‐regulate growth performance of fish before transfer to sea‐cage installations.     

Genetic variation in farmed populations of the gilthead sea bream Sparus aurata in Greece using microsatellite DNA markers

Genetic variation in seven reared stocks of gilthead sea bream Sparus aurata, originating from Greek commercial farms, was assessed using five polymorphic microsatellite markers and was compared with that of two natural populations from the Ionian and the Adriatic Seas. The total number of alleles per marker ranged from 11 to 19 alleles, and hatchery samples showed the same levels of observed heterozygosity with samples from the wild but substantially smaller allelic diversity and expected heterozygosity. The global genetic differentiation for the cultivated samples was significant as indicated by F_st analysis, which might indicate random genetic drift and inbreeding events operating in the hatcheries. On the contrary, no significant difference was found between the two wild populations. Population pairwise tests between farmed and wild stocks were also significant, with the exception of one hatchery sample, the Central Greece 1, which was not significantly different from the two wild samples perhaps due to its recent use in aquaculture from wild‐caught animals. The UPGMA tree topology grouped the wild samples together with the Central Greece 1 stock, and showed a clear division between wild and farmed sample sets for the six remaining hatchery samples. Knowledge of the genetic variation in S. aurata cultured populations compared with that in the wild ones is essential for setting up appropriate guidelines for the proper monitoring and management of the stocks either under traditional practices or for the implementation of selective breeding programmes.     

Non‐invasive fast real‐time PCR assay for detection of the enteric parasite Enteromyxum scophthalmi in cultured turbot (Scophthalmus maximus L.)

Enteromyxum scophthalmi is a myxozoan parasite that causes severe parasitic diseases in cultured turbot affecting mainly the intestine of the host. It is characterized by producing acute enteritis, starvation and eventually death. Current diagnosis of E. scopthalmi use traditional techniques, based on the identification of the morphology of the parasite. These techniques take extended time to be carried out and do not favour the adoption of control measure at turbot farms and require the sacrifice of fish. This study develops a fast real‐time PCR molecular tool for the detection of E. scophthalmi in infected farmed turbot. This methodology is applicable for routine controls on the farm at every stage of the parasite infection. Results of the study demonstrate the robustness, specificity, efficiency and reliability of the technique. In addition, this study also provides a non‐invasive procedure of sampling through swaps. This allows control, prevention and diagnosis of the parasite infection at turbot farms while maintaining the welfare of the cultivated fish and avoiding sacrifice of the fish sampled.     

Study of the distribution of active caspase‐3‐positive cells in turbot,Scophthalmus maximus (L.), enteromyxosis

Enteromyxosis caused by Enteromyxum scophthalmi is one of the parasitizations with a higher economic impact on turbot, Scophthalmus maximus (L.), aquaculture. This myxosporean produces severe catarrhal enteritis with abundant inflammatory infiltrates in the lamina propria‐submucosa (LP), epithelial detachment and leucocyte depletion of the lymphohaematopoietic organs. Some advances made on the pathogenesis pointed to a role of apoptosis in the enteromyxosis. Therefore, the main aim of this work was to employ the TUNEL assay and the anti‐(active caspase‐3) immunohistochemical assay to detect apoptotic cells in both healthy and E. scophthalmi‐infected turbot in order to establish the presence and distribution of apoptotic cells during development of the disease. More apoptotic cells located within the gastrointestinal epithelium were observed in the initial stages of the infection in E. scophthalmi‐infected turbot compared with non‐infected turbot. As the infection progressed, a higher degree of apoptosis occurred in the epithelium of folds heavily parasitized. In the severely infected turbot, apoptosis was also found among the leucocytes of the intestinal inflammatory infiltrates. Moreover, the number of active caspase‐3‐positive cells in the lymphohaematopoietic organs tended to increase with disease severity. In view of the results, increased apoptosis in the epithelium may favour the scaling that occurs during enteromyxosis and cell death of leucocytes in the intestinal LP, contributing to leucocyte depletion in severe cases.     

Cottonseed oil as a complementary lipid source in diets for gilthead seabream Sparus aurata juveniles

The efficacy of using cottonseed oil (CSO) as a fish oil (FO) substitute in gilthead seabream (Sparus aurata) juveniles feed was evaluated. Fish (BW_i 4.0 ± 2.9 g) were fed one of four isoproteic (~48% CP) and isolipidic (~18% L) diets for 9 weeks. Added oil was either FO (control diet, CTRL) or CSO, replacing 50% (CSO50 diet), 60% (CSO60 diet) and 70% (CSO70 diet) of dietary FO. Results indicated that FO replacement by CSO up to 60% level had no detrimental effects on growth or nutritive utilization and composition in fish muscles. Higher CSO intake (CSO70 diet, 56 g kg^?1) led to a 16% reduction in weight gain, 14% in feed utilization (FCR) and 57% in muscle n‐3 long‐chain polyunsaturated fatty acids (lc PUFA) as compared with CTRL and to abundant accumulation of lipid within the hepatocytes. Use of CSO altered fatty acid (FA) profiles of muscle and liver. Data suggested utilization of linoleic acid (LOA) by fish and retain of docosahexaenoic acid (DHA) in muscles. Therefore, limits of CSO inclusion as the main source of supplementary dietary lipid, with no negative effects on fish performance or nutritive composition and utilization in muscles, are: 40–48 g kg^?1 feed for gilthead seabream juveniles.     

Microsatellites markers to depict the reproductive and genetic patterns of farmed gilthead seabream (Sparus aurata): illustration by a case study on mass spawning

In the absence of breeding strategy, natural spawning constitutes the breeding ground for fish farmers to empirically manage their commercial broodstock. In this context, we used six microsatellite markers to characterize the genetic pattern of six commercial seabream broodfish tanks having a common history spanning four generations. The progeny of one tank single‐day mass‐spawning event, reared in two different environments, was used to estimate the genetic parameters for body weight. Limited genetic differentiation was observed among broodfish groups. A panel of nine loci allowed us to unambiguously assign 95.4% of the offspring (1692) and identify 37 parents (65% of the total broodfish). The limited effective population size (N_e = 15.3) was due to the elevated variance of parental contributions and to broodfish failing to contribute to the progeny. The fluctuation of the allele frequency highlighted the risks of genetic drift and reduction in the heterozygosity in the next generations. Heritability for body weight was moderate at commercial size (0.40 ± 0.10) and the high genetic correlation at later stages laid the groundwork for precocious selection criteria for growth. The discussion opens on the opportunity to use mass spawning for selective breeding.     

Comparative genome analysis reveals a distinct influence of nucleotide composition on virus–host species‐specific interaction of prawn‐infecting nodavirus

Discovery of species‐specific interaction between the host and virus has drawn the interest of many researchers to study the evolution of the newly emerged virus. Comparative genome analysis provides insights of the virus functional genome evolution and the underlying mechanisms of virus–host interactions. The analysis of nucleotide composition signified the evolution of nodavirus towards host specialization in a host‐specific mutation manner. GC‐rich genome of betanodavirus was significantly deficient in UpA and UpU dinucleotides composition, whilst the AU‐rich genome of gammanodavirus was deficient in CpG dinucleotide. The capsid of MrNV and PvNV of gammanodavirus retains the highest abundance of adenine and uracil at the second codon position, respectively, which were found to be very distinctive from the other genera. ENC‐GC3 plot inferred the influence of natural selection and mutational pressure in shaping the evolution of MrNV RdRp and capsid, respectively. Furthermore, CAI/eCAI analysis predicts a comparable adaptability of MrNV in squid, Sepia officinalis than its natural host, Macrobrachium rosenbergii. Thus, further study is warranted to investigate the capacity of MrNV replication in S. officinalis owing to its high codon adaptation index.     

Influence of a massive aquaculture escape event on artisanal fisheries

Spatiotemporal patterns of distribution of sea bass, Dicentrarchus labrax, and sea bream, Sparus aurata, and their influence on artisanal fisheries are explored before and after an escape event that released 1.5 million fish into the wild off La Palma (Canary Islands). Data were collected by in situ visual census and first sale data as a proxy of artisanal fisheries landings. Permutational anova of escapee abundances in shallow coastal habitats revealed consistent spatial patterns that linked densities of these fish to distance from escape point, whereas temporal patterns were related to a higher biomass released during winter. A nearby marine protected area did not show different densities of escaped fish. Local artisanal fleet catches accurately reflected the massive escape event and offer the main contingency force to mitigate the potential negative effects of massive escape events over shallow coastal habitats.     

Quality of reared gilthead sea bream (Sparus aurata) during ice storage,as affected by dietary fish oil substitution; an instrumental and sensory designation approach

The fillet fatty acids (FAs) and volatile compounds (VCs) of gilthead sea bream, fed either fish oil‐based diet (FO) or plant oils (PO), were studied in dorsal and ventral fillet parts over a 12‐day ice storage. Fillet FA reflected the FA composition of the respective diets. Monounsaturated FAs were reduced with storage, while no FA differentiations occurred between fillet parts. VCs varied between diet treatments, with food chain‐transported compounds (α‐pinene and dimethylsulphide) showing higher abundance in the FO group. VCs proposed as fish spoilage indicators (trimethylamine, pentanal, propanal, 3‐methylbutanal and 1‐penten‐3‐ol) increased with storage, while concentration of carbon disulphide associated with fresh fish aroma decreased during the same period. VCs varied between fillet parts, with ventral part exhibiting higher concentrations in fat‐soluble (terpenes, aromatic hydrocarbons) and spoilage‐associated VCs. Sensory analysis revealed no significant differences between diet groups with the exception of fattiness, which was found significantly higher in the plant oil group.     

Insect larvae as feed ingredient selectively increase arachidonic acid content in farmed gilthead sea bream (Sparus aurata L.)

This work is aimed at obtaining farmed fish designed to contain targeted PUFAs. To this end, an experiment was conducted with farmed specimens of gilthead sea bream (Sparus aurata L.), which were fed meal containing different percentages of common green bottle fly (Lucilia sericata Meigen) larvae, cultured on appropriate substrates, and then the fatty acid profiles in fish muscles were determined. Results indicate that arachidonic acid content in fish muscle is significantly increased when replacing different proportions of fishmeal by larvae from L. sericata, which contains arachidonic acid up to 10.6% of total fatty acids. Thus, larvae seem to be a suitable vector for introducing target PUFA in fish muscle and it could also contribute to reduce the use of wild fish stocks for fishmeal production.     

Total nitrogen,carbon and phosphorus digestibility in gilthead seabream (Sparus aurata) and European seabass (Dicentrarchus labrax) fed with conventional and organic commercial feeds: implications for particulate waste production

Organic fish farming is postulated as a more environmentally friendly activity than conventional activities. However, environmental sustainability as regards waste output has not been addressed. In this respect, the most important environmental problems associated with fish farming are those related with the discharge of particulate wastes. In this study, total nitrogen, carbon and phosphorus digestibility was estimated in a wide size range of gilthead seabream (Sparus aurata) and European seabass (Dicentrarchus labrax) fed a commercially available conventional diet and an organic feed, as a previous step to assessing particulate waste output. Neither fish size nor feed nutrient content was seen to influence nutrient digestibility in either species. The organic diet provided higher carbon but lower nitrogen and phosphorus digestibility than conventional feed in both species. Simulations under real ongrowing conditions resulted in lower values for nitrogen and higher values for carbon particulate wastes when conventional feed was supplied to both species. Particulate phosphorus wastes were 2.6‐ to 3.4‐fold higher for fish fed the organic diet. Restrictions concerning NO the raw materials used for organic diet formulation and differences in nutrient bioavailability between the diets seemed to be the underlying reason for the differences found.     

Morphological and histological characterization of an ectopically mineralized structure in a gilthead sea bream Sparus aurata with opercular deformation

In this study, we describe an abnormal ectopically mineralized structure (EMS) that was found inside the skull of a juvenile Sparus aurata that also showed a bilateral opercular deformation. The overall phenotype and tissue composition were studied using micro‐CT scanning and histological analyses. The ectopic structure occupies a large volume of the brain cavity, partially extruding into the gill cavity. It shows a dense mineralization and an extracellular matrix‐rich phenotype, with variation in both the morphology and size of the cell lacunae, combined with an irregular fibre organization inside the matrix. This study is the first to report such an EMS in a juvenile teleost fish, where the tissue does not resemble any other connective tissue type described in bony fish so far. The tissue phenotype seems to rule out that the EMS corresponds to a tumorous cartilage. Yet, it is rather reminiscent of a highly mineralized structure found in cartilaginous fish, where it is suggested to be associated with damage repair.     

Study of the dose of thyme essential oil in feed to prolong the shelf life of gilthead seabream (Sparus aurata)

The effect of thyme essential oil (Thymus zygis, subspecies gracilis) on the quality and shelf life of gilthead seabream was studied, when added to the diet: a control diet and four experimental diets (T500, T1000, T1500, T2000) with 500, 1000, 1500 and 2000 mg kg^?1 of thyme essential oil, respectively. After 12 weeks of experimentation, the fish were stored on ice at 4 °C for 0, 7, 14 and 21 days. Physical–chemical, microbiological and sensory analyses were carried out at each sampling point to determine the degree of deterioration. A dose‐dependent effect was observed on the colour, TBARS and total volatile basic nitrogen during the storage. Microbiological counts were lower for Enterobacteriaceae and coliforms at high doses of the essential oil. The sensory analysis showed an effect on the quality index that was not dependent on the dose, which extended the shelf life of gilthead seabream from 17 to 18 days for all doses.