In vitro effects of the polyphenols resveratrol, mangiferin and (-)-epigallocatechin-3-gallate on the scuticociliate fish pathogen Philasterides dicentrarchi

This study investigated the in vitro effects of the polyphenols resveratrol, mangiferin and (-)-epigallocatechin-3-gallate (EGCG) on the histiophagous ciliate Philasterides dicentrarchi, which causes fatal scuticociliatosis in farmed turbot Scophthalmus maximus L. Of the 3 polyphenols, resveratrol showed strongest antiprotozoal activity, reducing ciliate density after 1 wk culture by, on average, 91% at 50 microM, and 96% at 500 microM. EGCG reduced ciliate density by, on average, 93% at 500 microM, with no significant effect at 50 microM. Mangiferin reduced ciliate density by, on average, 56% at 500 microM, again with no significant effect at 50 microM. In view of these findings, we discuss the potential utility of chemotherapy with polyphenols as a strategy for the control of scuticociliatosis in farmed turbot.     

Occurrence of scuticociliatosis in olive flounder Paralichthys olivaceus by Phiasterides dicentrarchi (Ciliophora: scuticociliatida)

In the course of identifying scuticociliates recently obtained from systemically infected olive flounder Paralichthys olivaceus in Korea, we found a scuticociliate species whose small subunit ribosomal RNA (SS rRNA) gene was not amplified by species-specific primers previously designed for Uronema marinum and Pseudocohnilembus persalinus. By studying morphological characteristics of wet-mounted and stained specimens, we identified the species as Philasterides dicentrarchi, which has been reported to cause systemic infection in the European sea bass Dicentrarchus labrax and turbot Scophthalmus maximus. In this study, we compared morphological characteristics of our specimens with previously reported Philasterides species, including P. dicentrarchi, and sequenced the SS rRNA gene in order to design P. dicentrarchi specific primers. This is the first report on scuticociliatosis caused by P. dicentrarchi from marine fish in Asia.     

A comparative study of blood oxygen transport in turbot and sea bass: effect of chronic hypoxia

A comparative study of blood oxygen binding and carrying capacities of turbot Scophthalmus maximus and sea bass Dicentrarchus labrax , two fish species differing in their demand for oxygen, was carried out under three levels of chronic hypoxia ( P o ₂ = 93, 65 and 40 mmHg) for 40 days. Blood O₂ affinity in normoxia was moderately high in both species ( P ₅₀ was c . 12–13 mmHg at pH 7·7). The Bohr factor was significantly lower in turbot (−0·52) than in sea bass (−0·85). In both species, blood O₂ affinity was not significantly affected by oxygen depletion whatever its level and duration. In turbot, however, P ₅₀ appeared to slightly decrease at the two more severe levels of hypoxia. In both species, blood O₂ carrying capacity was not affected by hypoxia and remained twice as high in sea bass than in turbot.     

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.     

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

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

Uncovering QTL for resistance and survival time to Philasterides dicentrarchi in turbot (Scophthalmus maximus)

Disease resistance‐related traits have received increasing importance in aquaculture breeding programs worldwide. Currently, genomic information offers new possibilities in breeding to address the improvement of this kind of traits. The turbot is one of the most promising European aquaculture species, and Philasterides dicentrarchi is a scuticociliate parasite causing fatal disease in farmed turbot. An appealing approach to fight against disease is to achieve a more robust broodstock, which could prevent or diminish the devastating effects of scuticociliatosis on farmed individuals. In the present study, a genome scan for quantitative trait loci (QTL) affecting resistance and survival time to P. dicentrarchi in four turbot families was carried out. The objectives were to identify QTL using different statistical approaches [linear regression (LR) and maximum likelihood (ML)] and to locate significantly associated markers for their application in genetic breeding strategies. Several genomic regions controlling resistance and survival time to P. dicentrarchi were detected. When analyzing each family separately, significant QTL for resistance were identified by the LR method in two linkage groups (LG1 and LG9) and for survival time in LG1, while the ML methodology identified QTL for resistance in LG9 and LG23 and for survival time in LG6 and LG23. The analysis of the total data set identified an additional significant QTL for resistance and survival time in LG3 with the LR method. Significant association between disease resistance‐related traits and genotypes was detected for several markers, a single one explaining up to 22% of the phenotypic variance. Obtained results will be essential to identify candidate genes for resistance and to apply them in marker‐assisted selection programs to improve turbot production.     

In vitro efficacy of new antiprotozoals against Philasterides dicentrarchi (Ciliophora, Scuticociliatida)

Philasterides dicentrarchi is a histiophagous ciliate that causes severe losses in turbot and sea bass farming. This study investigated the in vitro efficacy against P. dicentrarchi of 85 newly synthesized compounds and 12 commercial compounds, of which 2 are fluoroquinolones (norfloxacine and lomefloxacine) with known antibacterial activity. Seventeen of the newly synthesized compounds (2 naphthyridines, 2 pyridothienodiazines and 13 pyridothienotriazines) and the fluoroquinolone norfloxacin showed good activity. The most promising compound was the pyridothienotriazine 12k, with activity similar to that of the salicylanilides niclosamide and oxiclozanide (MLC 0.8 mg l(-1) in PBS, 1.5 mg l(-1) in seawater; MLC = minimum 24 h lethal concentration).     

Fatty acids in liver and muscle of farmed and wild sea bass (Dicentrarchus labrax L.)

Growth rate and assimilation of essential fatty acids were analysed in sea bass (Dicentrarchus labrax L.) fed different diets. Gas Chromatographie analyses of total lipids in commercial diets indicated excessive presence of saturated, mono and dienoic fatty acids, while the content of to 3 HUFA was very low. Fatty acid patterns of liver and white muscle farmed sea bass reflected the contents of the dietary lipids. The concentration of polyunsaturated fatty acids, particularly ω3 HUFA was significantly lower when compared with samples from the wild population. The atherogenic and fish lipid quality indices confirmed the higher nutritional quality of wild sea bass when compared with farmed fishes.     

A microsatellite linkage map of the European sea bass Dicentrarchus labrax L

A genetic linkage map of the European sea bass (Dicentrarchus labrax) was constructed from 174 microsatellite markers, including 145 new markers reported in this study. The mapping panel was derived from farmed sea bass from the North Adriatic Sea and consisted of a single family including both parents and 50 full-sib progeny (biparental diploids). A total of 162 microsatellites were mapped in 25 linkage groups. Eleven loci represent type I (coding) markers; 2 loci are located within the peptide Y (linkage group 1) and cytochrome P450 aromatase (linkage group 6) genes. The sex-averaged map spans 814.5 cM of the sea bass genome. The female map covers 905.9 cM, whereas the male map covers only 567.4 cM. The constructed map represents the first linkage map of European sea bass, one of the most important aquaculture species in Europe.     

Immune response of turbot, Psetta maxima (L.) (Pisces: Teleostei), to formalin-killed scuticociliates (Ciliophora) and adjuvanted formulations

The increasing frequency of scuticociliatosis in turbot culture has stressed the need of knowledge on the immune responses to these parasites, for further developing of prevention and control strategies. The immune response of turbot to killed parasites, alone (Ag) or in combination with Montanide ISA 763A (MON), was studied in a laboratory-scale experiment. The variations of several innate immune factors and the antibody response were analysed in immunized vs. non-immunized fish at different times after immunization, and also after a challenge with live ciliates. Amongst innate immune factors, serum lysozyme increased progressively in all inoculated groups. Differences in innate immune factors in Ag and Ag-MON fish with respect to controls were mainly evidenced after challenge, especially for serum complement. Serum antibody levels increased in immunized fish after booster and particularly after challenge. In addition, certain protection was obtained for immunized groups compared to controls or to fish receiving MON alone, and the levels of specific antibodies were also the highest in immunized groups. The obtained information could be useful for further design of immunoprophylactic formulations against scuticociliatosis.     

By-products of farmed European sea bass (Dicentrarchus labrax L.) as a potential source of n-3 PUFA

Total by-products (TBP) obtained by filleting farmed and wild European sea bass (Dicentrarchus labrax) were analyzed to evaluate if, on the basis of the percentage yield, total lipid content and fatty acid composition, they can be considered a resource of n-3 polyunsaturated fatty acids (PUFA). Results show that TBP from intensively farmed fish (IFF) contain higher total lipid content and have a higher level of n-3 PUFA rich in eicosapentaenoic (EPA) and docosaexaenoic acid (DHA), compared to extensively farmed fish (EFF) and to wild fish (WF) (P < 0.05). This difference may suggest a way of promotion of TBP from IFF sea bass through the n-3 PUFA recovery by extraction.     

Mutagenicity of bithionol sulfoxide and its metabolites in the salmonella/mammalian microsome test

The mutagenic effects of bithionol sulfoxide and its two major metabolites, bithionol and bithionol sulfone, on 4 Salmonella typhimurium strains (TA97, TA98, TA100 and TA102) were investigated. Bithionol sulfoxide was found to be mutagenic to TA98 and TA100. However, mutagenicity was abolished in the presence of rat-liver S9 fractions.     

Studying sperm motility in marine fish: an overview on the state of the art

This contribution reviews existing literature and some new own findings on teleost sperm motility and factors controlling it, emphasizing selected marine species. In marine teleosts with external fertilization (halibut, turbot, sea bass, hake, cod and tuna serving as examples), mainly the osmolality controls sperm motility: movement is activated by transfer from the seminal fluid into sea water, representing a large upward step in osmolality. The exception are flatfishes (such as halibut or turbot) where CO₂ is responsible for flagellar immotility in seminal fluid. In all cases, the duration of motility is short and limited to minutes ranges due to partial exhaustion of the ATP energy and to increase of internal ionic concentration as suggested by studies with de‐membranated/ATP reactivated flagellae. In this overview, we compare motility characteristics (percentage of active spermatozoa, velocity, linearity), flagellar waves parameters (wave length and amplitude, number of waves) and energy content (respiration and ATP concentration) within species where these data have been established. All parameters show a rapid decrease after activation; therefore progressive forward movement needed by the sperm to effectively reach the egg surface, is limited to a short initial period following activation. In two species (turbot and sea bass) the rapid decrease of sperm motility is reflected by a corresponding decrease of the fertilizing ability. Exposure to external environments (sea water) at activation also leads to local defects of the sperm flagella posing additional limitations on motility duration. However, minor flagellar damages as well as energetic exhaustion are reversible: after a resting period in a non‐swimming solution at the end of the motility period, spermatozoa can be re‐activated for a second motility period. From these results and from additional data obtained from de‐membranated/ATP re‐activated spermatozoa, a paradigm has been developed which establishes a link between external osmolality (sea water), internal ionic concentration and control of axonemal activity.     

A vaccine based on biodegradable microspheres induces protective immunity against scuticociliatosis without producing side effects in turbot

The histiophagous scuticociliate parasite Philasterides dicentrarchi is an emergent pathogen in aquaculture and causes significant economic losses on turbot (Scophthalmus maximus) farms. In this study, the surface antigens (Ag) of the parasite were encapsulated and covalently linked to a polymeric microparticle formulation composed of two biodegradable polymers (chitosan and Gantrez). The antigenicity of the formulation and the protection provided were compared in mice and turbot. This formulation induced a higher antibody (Ab) response in mice at doses of 5mg of microspheres (MS) conjugated with approximately 230 μg of Ag (MS-Ag(c)). However, Ab levels were significantly lower than in mice vaccinated with the same concentration of Ag in complete Freund's adjuvant (FCA). In turbot, the MS-Ag(c) formulation induced a higher level of Abs than that induced by the same vaccine emulsified in FCA. The challenge experiments performed with P. dicentrarchi and vaccinated turbot also showed a clear correlation between Ab levels and survival levels. Growth was significantly affected in fish vaccinated with FCA, but not in fish vaccinated with MS. The high adjuvant capacity of MS, together with its biodegradability and low toxicity to fish, makes this new vaccine an economical, effective and safe alternative to oil-based adjuvants for the immunoprophylaxis of scuticociliatosis in turbot.     

Philasterides dicentrarchi (Ciliophora, Scuticociliatida) as the causative agent of scuticociliatosis in farmed turbot Scophthalmus maximus in Galicia (NW Spain)

Two outbreaks of scuticociliatosis affecting farmed turbot Scophthalmus maximus in Galicia are described. Moribund fish showed cutaneous ulcers, darkened skin, swimming behaviour alterations, exophthalmos, and/or abdominal distension as a result of accumulation of ascitic fluid in the body cavity. Ciliates were detected in fresh mounts of practically all organs and tissues, including the blood and ascitic fluid. Histopathological studies revealed severe encephalitis and meningitis (associated with different degrees of softening or liquefaction of the brain), necrosis of the hepatic parenchyme, severe oedema of the intestinal wall, degeneration of muscle fibres, hyperplasia of the branchial epithelium, and/or vascular and perivascular inflammation. In some cases, parasites are surrounded by abundant monocytic and lymphocytic infiltrate. We report the morphological and biometric characteristics of this ciliate, which allow identification as Philasterides dicentrarchi. We discuss possible routes of entry into the host, and environmental factors possibly facilitating infection.     

Are biochemical biomarker responses related to physiological performance of juvenile sea bass (Dicentrarchus labrax) and turbot (Scophthalmus maximus) caged in a polluted harbour?

Biomarker responses to toxic exposure have been used for decades to indicate stress in aquatic organisms, or the magnitude of environmental pollution. However, little has been done to compare the simultaneous responses of both biochemical and physiological biomarkers. The purpose of this study was twofold. Firstly to analyse the responses of several biochemical biomarkers measured on juvenile sea bass and turbot caged in a northern France harbour at a reference and contaminated stations. Several biotransformation parameters (Ethoxyresorufin-O-deethylase - EROD - and Glutathione S-transferase -GST) and an antioxidant enzyme (Catalase -CAT) were analysed. Secondly, to compare their responses to several growth and condition indices, measured on the same fish. In the contaminated station, EROD and GST activities were found to be significantly higher, and a decrease of CAT activity was observed for both species. For individual sea bass, biochemical biomarkers showed numerous significant correlations with growth and condition indices, such as the Fulton's K condition index, the RNA:DNA ratio and the lipid storage index. On the contrary, there were only a few significant correlations for turbot, suggesting a species-specific response. Our study indicates that the analysis of the simultaneous responses of both biochemical and physiological biomarkers can be useful for monitoring complex exposure and to assess habitat quality.     

Sexual differentiation and juvenile intersexuality in the European sea bass (Dicentrarchus labrax)

Sexual differentiation was studied at the histological level using a mixture of 30 families of sea bass Dicentrarchus labrax . Most of the fish (93%) differentiated into males as usually observed in farmed populations. All testes were differentiated when the males reached 12 cm and no more undifferentiated fish were found from 419 days post-fertilization (p.f.). In 28% of the males, among the biggest, sexual differentiation had already begun at 168 days p.f. (8.3–9.5 cm) and these fish started spermatogenesis in their first year of life. The other males differentiated later and remained immature at the end of their first year of life. Ovaries could be identified at the histological level from the age of 168 days p.f. (7.9–9.0 cm) and the females became significantly longer than the males from the age of 191 days p.f., i.e. during the process of ovarian differentiation. In the studied group, 62% of the males developed intratesticular oocytes. Such intersexuality had no consequence on growth rate. Intratesticular oocytes were also recorded in testes of wild males originating from Atlantic (Britain and Gulf of Gascogne) and West Mediterranean showing that juvenile intersexuality is not restricted to farmed populations but is a widespread phenomenon in sea bass.     

Comparative effects of long-term hypoxia on growth, feeding and oxygen consumption in juvenile turbot and European sea bass

When juvenile turbot Scophthalmus maximus and sea bass Dicentrarchus labrax were fed to satiation, growth and food intake were depressed under hypoxia (3·2±0·3 and 4·5±0·2 mg O₂ l^-1). However, no significant difference in growth was observed between fishes maintained in hypoxia and fed to satiation and fishes reared in normoxia (7·4±0·3 mg O₂ l^-1) and fed restricted rations (same food intake of fishes at 3·2 mg O₂ l^-1). Routine oxygen consumption of fishes fed to satiation was higher in normoxia than in hypoxia due to the decrease in food intake in the latter. Of the physiological parameters measured, no significant changes were observed in the two species maintained in hypoxia. This study confirms the significant interaction between environmental oxygen concentrations, feeding and growth in fishes. Decrease in food intake could be an indirect mechanism by which prolonged hypoxia reduces growth in turbot and sea bass, and may be a way to reduce energy and thus oxygen demand.