First isolation of Tenacibaculum maritimum from wedge sole, Dicologoglossa cuneata (Moreau)

The first isolation of Tenacibaculum maritimum from wedge sole, Dicologoglossa cuneata, is reported. The pathogen was recovered from ulcers of cultured fish, from three different outbreaks. The six isolates obtained were biochemically and serologically characterized and diagnosis was confirmed by polymerase chain reaction using specific primers and partial 16S rRNA gene sequencing. The isolates constituted a homogeneous phenotypic group; however, they belong to two of the different serotypes described within this species. A virulence evaluation of the isolates using Wedge sole fry was also performed.     

Physiological responses of juvenile wedge sole Dicologoglossa cuneata (Moreau) to high stocking density

Physiological responses to a high stocking density were tested in juvenile wedge sole Dicologoglossa cuneata (Moreau). Fish were kept at low (1 kg m⁻²), medium (3 kg m⁻²) and high (9 kg m⁻²) stocking densities for 22 days. No differences in the weight, length, survival and hepatosomatic index were observed among treatments. Basal plasma cortisol and osmolality were found to be directly and positively related to stocking density. A mild increase in plasma glucose was seen at medium density, and plasma protein was elevated at medium and high densities. The liver glucose and glycogen content was inversely related to stocking density. The liver triglyceride level was significantly elevated at the highest density, and the α-amino acid content decreased at the highest density. In muscle, glucose levels were significantly higher in fish kept at the lowest density; the α-amino acid content was elevated in fish kept at high density. In conclusion, plasma cortisol levels indicated an increasing stress level depending on the culture density, but significant changes in energy reserves did not occur in tissue (mainly liver and muscle glycogen and glucose reserves were significantly affected).     

Osmoregulatory changes in wedge sole (Dicologoglossa cuneata Moreau, 1881) after acclimation to different environmental salinities

The osmoregulatory responses of 20 days of acclimation to environmental salinities of 5‰, 15‰, 25‰, 35‰ and 55‰ were assessed in juveniles of wedge sole ( Dicologoglossa cuneata Moreau, 1881). This sole shows a good capacity to adapt to this range of environmental salinities. A direct linear relationship between environmental salinity and plasma osmolality was observed, with a calculated isosmotic point of 10.4‰ (284 mOsm kg⁻¹). Na⁺, K⁺-ATPase activity in the gills followed a 'U-shaped' relationship with environmental salinity, and a direct linear relationship in kidney tissue. Plasma cortisol levels were elevated in fish held in extreme salinities, and glucose levels were higher only in the group maintained at the highest environmental salinity. In the liver, a decrease in glycogen, lactate and amino acid contents was observed in specimens acclimated to extreme salinities (5‰ and 55‰), suggesting mobilization of liver metabolites. Metabolite levels in white muscle showed a pattern similar to the liver, with lower values in specimens acclimated to extreme salinities. We conclude that wedge sole is strongly euryhaline, but acclimation to extreme salinities comes with an energetic cost.     

Physiological stress responses to captivity in early developmental stages of the wedge sole Dicologoglossa cuneata (Moreau)

The stress responses in early growth stages of the wedge sole have been studied to determine whether the high cortisol levels described in juvenile fish are present from early developmental stages. Whole‐body cortisol, glucose and lactate contents, as well as biometric parameters in wedge sole larvae were measured at three different stocking densities. Stocking density affected growth‐related variables significantly, and larvae in lower stocking densities grew faster. Survival did not significantly differ among treatments. At hatching, the whole‐body cortisol concentration was 0.33 ± 0.01 ng g^?1 and varied significantly from 0 to 30 days after hatching (DAH) for each stocking density, though values remained stable for the remaining time in the low‐stocking density group. These hormone levels rose significantly (5.17 ± 2.43 to 22.10 ± 4.95 ng g^?1) at the end of the experiment, depending directly on the stocking density. Glucose and lactate‐body concentrations did not vary among treatments. We conclude that the stress responses of wedge sole larvae are detectable from 45 DAH and that stocking density already can be a stressor at that age. As described for juvenile stage, cortisol content values in wedge sole larvae under non‐stressful conditions are one of the highest among those reported in the literature. The captivity conditions could be responsible for this apparently stressful situation, though those values also could be normal in wild specimens.     

First results on spawning, larval rearing and growth of the wedge sole (Dicologoglossa cuneata) in captivity, a candidate species for aquaculture

The wedge sole is a target species in the fisheries of the Gulf of Cadiz (Spain). Having reared them to commercial size, we have studied reproduction and breeding in captivity of the wedge sole in this work here. The breeders adapt easily to captivity, and they can spawn in less than 1 year in captivity. The relative fecundity is relatively high, 1.06–2.33 · 10⁶ eggs kg⁻¹ per spawning season (mean 1.6 ± 0.1). Larval SGR is high, 7.2 ± 0.2% day⁻¹ (range 5.8–9.1), similar to other cultured flatfish species. In 1 year, some individuals reach market size and can release eggs, registering SGRs of 1.39–1.66% day⁻¹ (mean 1.56 ± 0.01). This species presents some very different characteristics with respect to other farmed flatfishes (turbot, halibut and Senegal sole, mainly), so that it is necessary to develop new techniques to optimise its cultivation. In the present work, the first results are presented on the biology of the wedge sole in captivity.     

Changes in lipid classes,fatty acids,protein and amino acids during egg development and yolk‐sac larvae stage in brill (Scophthalmus rhombus L.)

Brill (Scophthalmus rhombus L.) is a flatfish considered of special interest for aquaculture diversification, but the high mortality observed during the early larval rearing is the main obstacle to commercial culture. The objective of this study was to contribute to the knowledge of nutrient utilization of early‐hatched larvae, characterizing the changes in lipid and protein contents during embryogenesis and the yolk‐sac larval stage of S. rhombus. Total lipid, lipid classes and fatty acid contents remained constant during embryogenesis and yolk‐sac larval development, except for phosphatidylethanolamine and phosphatidylinositol, which increased in quantity during the yolk‐sac larval stage. On the other hand, total protein (including non‐protein nitrogen) and amino acids decreased their contents in both periods, especially at hatching. The decrease only in the serine, glutamic acid, proline and lysine contents during embryogenesis suggests a selective use of amino acids during this phase. Unlike embryogenesis, amino acids loss during hatching appears to be non‐selective, and almost all amino acids (essential and non‐essential) decreased. Our results suggest that there is higher catabolism of protein vs. lipid during embryogenesis and the yolk‐sac larval stage of S. rhombus.     

Morphopathological features of a severe ulcerative disease outbreak associated with Tenacibaculum maritimum in cultivated sole, Solea senegalensis (L.)

This study describes morphopathologic changes in naturally infected farmed Senegalese sole affected by tenacibaculosis caused by Tenacibaculum maritimum. Macroscopic observation, in addition to light microscopy and scanning electron microscopy, was used to study the lesions. Main lesions were characterized by complete loss of epidermis and dermis, as well as extensive necrosis of muscle layers. Mild-to-moderate inflammatory response with the presence of macrophages was noted around hyaline degenerated muscle cells. Gram-negative filamentous bacteria could be detected only at the dermis. Under scanning electron microscopy, filamentous bacteria located over the scales without epithelium could be observed. These findings together with the isolation and PCR detection of the bacteria in kidney and skin tissues suggest that once the bacteria reach the dermis, probably through eroded epidermis, they are able to proliferate and produce enzymes that are responsible for the damage in the underlying tissues.     

Cellular and humoral immune responses of Senegalese sole,Solea senegalensis (Kaup), following challenge with two Photobacterium damselae subsp. piscicida strains from different geographical origins

The present study aimed to investigate leucocyte responses to inflammation as well as some innate immune parameters of Senegalese sole, Solea senegalensis, following challenge with two strains of Photobacterium damselae subsp. piscicida belonging to the European and Japanese clones described for this bacterium. Pathogenicity assays were performed to assess the virulence of each Photobacterium damselae subsp. piscicida strain for sole. Subsequently, fish were intraperitoneally injected with phosphate‐buffered saline (control) or two concentrations (2 × 10² and 2 × 10⁶ CFU mL^?1) of each bacterial strain and sampled after 6 and 24 h. Results showed that the European isolate induces a higher degree of response than the Japanese strain. While blood neutrophilia and monocytosis correlated well with the increase in neutrophil and macrophage numbers in the peritoneal cavity, fish infected with the European isolate presented higher peritoneal cell numbers than fish challenged with the Japanese strain. In addition, alternative complement pathway activity and respiratory burst of head kidney leucocytes increased significantly in fish infected with the European isolate. The enhanced innate immune response displayed by Senegalese sole challenged with the European isolate is probably due to the higher degree of virulence presented by this Photobacterium damselae subsp. piscicida strain.     

A comparison of fatty acid composition and quality aspects of eggs and larvae from cultured and wild broodstock of common sole (Solea solea L.)

Eggs from a F₁ cultured broodstock of sole were compared with eggs from wild‐caught breeders throughout one spawning season, to evaluate if egg quality may be affected by culture‐related conditions. Fourteen batches of eggs from cultured broodstock and 17 batches from wild‐caught sole were compared with respect to fatty acid (FA) composition, egg size, fertilization rate and hatching rate. Based on a multivariate analysis of the FA profiles, it was possible to discriminate between culture and wild inheritance. Eggs from cultured broodstock had high levels of C20:1(n‐9), C18:2(n‐6) and C18:3(n‐3), whereas eggs from wild fish had high levels of C16:1(n‐7), C20:4(n‐6) and C20:5(n‐3). Differences in FA profiles were most likely related to dietary differences. Fertilization and hatching rates were generally low and lowest in eggs from cultured broodstock, but not related to FA composition. Larval growth of one batch from each group was compared. Larval growth was not correlated to broodstock origin, FA composition or egg or larval size. However, larval survival was significantly lower for larvae from cultured broodstock.     

Vibrio species isolated from diseased farmed sole,Solea senegalensis (Kaup), and evaluation of the potential virulence role of their extracellular products

Bacteria isolated from an outbreak with moderate mortalities in farmed sole, Solea senegalensis (Kaup), in the south of Spain were identified as Vibrio harveyi and V. parahaemolyticus. Only bacterial strains showing swarming were virulent in sole and caused mortalities in experimentally inoculated fish. However, the signs of the disease were only reproduced with V. harveyi. The intramuscular inoculation of the extracellular products (ECPs) of both species produced mortalities in inoculated fish and the appearance of surface ulcers in the case of V. harveyi. However, the inoculation of sublethal doses of ECPs to fish showed a protective effect against V. harveyi.     

Morphology,seasonality and phylogeny of Zschokkella soleae sp. n. (Myxozoa,Myxosporea) parasite of Solea solea (L.) (Pleuronectiformes,Soleidae) from Ghar El Melh Lagoon,Tunisia

A new Myxosporea, Zschokkella soleae sp. n., was found in the gall bladder and the bile of common sole, Solea solea (L.), from Ghar El Melh Lagoon in north‐east Tunisia. This is the first record for the presence of Zschokkella species in Tunisian waters. The parasite's plasmodia are polysporic with variable size and shape. Some plasmodia appeared attached to the gall bladder epithelium while others were found free in bile. Mature spores are ellipsoidal in frontal view 13.8 ± 0.38 μm long and 10.86 ± 0.40 μm wide with two equal size spherical polar capsules 3.6 ± 0.43 μm in size. The prevalence of infection seems to correlate with host size and changes over the year with maximum percentage in summer. Based on the 18S rDNA sequence data, Z. soleae sp. n. is readily distinguishable from other myxozoan DNA sequences in GenBank. Phylogenetically, the new species is placed in the freshwater Myxidium clade including several Zschokkella spp. infecting the gall bladder. Morphology, histology as well as DNA sequence analysis indicate that the examined species differs from all previously described Zschokkella species.     

Growth,feed utilization and liver histology of juvenile common sole (Solea solea L.) fed isoenergetic diets with increasing protein levels

This study was undertaken to determine the influence of dietary protein levels on growth, feed utilization and liver histology in common sole. Four isoenergetic diets were formulated to contain four different crude protein levels: 39 (P39), 45 (P45), 51 (P51) and 57 (P57) % dry weight. Fifty animals per tank (initial weight 10.2 ± 0.4 g) were randomly distributed into twelve 500 litre square tanks (bottom surface: 5600 cm²) connected to a closed recirculation system. The diets were tested in triplicate for 84 days. At the end of the experiment, the final weight ranged from 19.6 (P39) to 25.4 g (P57). The specific growth rate showed statistical differences between groups, with the best results in the group fed diet P57 (1.07% day^?1). Significant differences between groups were also recorded for the feed conversion ratio, with values of 1.31, 1.28, 1.12 and 0.94 in P39, P45, P51 and P57 respectively. Gross lipid efficiency was also significantly affected by the dietary treatment, with the highest value (42.07%) found in P57. Ammonia excretion, expressed as g 100 g^?1 protein intake, was significantly lower for group P39 (2.46) than groups P51 (4.70) and P57 (4.75). Increased incidence of lipid droplets in hepatocytes was observed when the dietary protein levels increased and/or dietary lipid decreased.     

Recovery of Hafnia alvei from diseased brown trout,Salmo trutta L., and healthy noble crayfish,Astacus astacus (L.), in Bulgaria

Hafnia alvei was isolated in Bulgaria from healthy noble crayfish, Astacus astacus (L.), and then from farmed diseased brown trout, Salmo trutta L., with signs of haemorrhagic septicaemia. The isolates were identified initially with conventional phenotyping and commercial Merlin Micronaut and API 20E rapid identification systems, followed by sequencing of the 16S rRNA gene. Hafnia alvei Bt1, Bt2 and Aa4 were of low virulence to rainbow trout and brown trout, although cytotoxicity was demonstrated by Bt1 and Bt2, but not by Aa4.     

The effect of food type (formulated diet vs. natural) and fish size on feed utilization in common sole,Solea solea (L.)

This study compares the effect of food type (formulated diet vs. natural food) and fish size on protein and energy utilization efficiencies for growth in common sole, Solea solea (L.). Replicate groups of common sole (mean initial body weight ± SD was 45.7 g ± 2.1 and 111.2 g ± 4.2) received the diets at five (natural feed) or four (formulated diet) feeding levels. The protein utilization efficiency for growth (k_gCP) was higher (P > 0.001) in common sole fed ragworms than in common sole fed the formulated diet (respectively, 0.40 and 0.31). Likewise, the energy utilization efficiency for growth (k_gGE) was higher (P = 0.001) in common sole fed ragworms than in common sole fed the formulated diet (respectively, 0.57 and 0.33). The protein maintenance requirement was not different between food types (P = 0.64) or fish size (P = 0.41) being on average 0.82 g kg^?0.8 day^?1. The energy maintenance requirement was not different between food type (P = 0.390) but differed between fish size (P = 0.036). The gross energy maintenance requirement of small common sole was 35 kJ g^?0.8 day^?1. The gross energy maintenance requirement of large common sole was 25 kJ g^?0.8 day^?1. In conclusion, the low growth of common sole fed formulated diets was related to reduced feed utilization.     

Putative virulence factors of atypical Aeromonas salmonicida isolated from Arctic charr,Salvelinus alpinus (L.), and European grayling,Thymallus thymallus (L.)

Atypical Aeromonas salmonicida (AAS) causes generalized lethal infections in farmed Arctic charr, Salvelinus alpinus (L.), and European grayling, Thymallus thymallus (L.), and is thus a serious threat for culture of these fish species. Virulence factors were studied among isolates of AAS from Arctic charr (n = 20), European grayling (n = 19) and other fish species (n = 20), of which 48 were of Finnish and 11 of Swedish origin. All isolates produced an A-layer. Extracellular products (ECP) of the AAS isolates did not produce detectable gelatinase and caseinase activity in test assays. Analysis of the same ECP preparations with substrate sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed weak proteolytic activity, indicating the different sensitivity of the detection methods used. The ECP from AAS isolates showed low cytotoxic activity against cultured cells. However, the ECP did not induce mortality in challenged Arctic charr. The results suggest that toxic components, like ECP, secreted by the bacterium may not be the major virulence factor in AAS-infection in Arctic charr and European grayling, and hence the pathogenesis also differs from the pathogenesis of AAS-infection in Atlantic salmon, Salmo salar L.     

First report of isolation and complete genome of Vibrio rotiferianus strain SSVR1601 from cage‐cultured black rockfish (Sebastes schlegelii) associated with skin ulcer

Vibrio rotiferianus is an important marine pathogen of various aquatic organisms and can be found widely distributed in the marine environment. To further characterize this pathogen, the pathogenic properties and genome of V. rotiferianus SSVR1601 isolated from Sebastes schlegelii with skin ulcer were analysed. SSVR1601 was shown to be short rod‐shaped cell with a single polar flagellum. Different degrees of pathological changes in fish kidney, intestine, gills and liver were observed after SSVR1601 challenge. The SSVR1601 genome consists of two chromosomes and two plasmids with a total of 5,717,113 bp, 42.04%–44.93% GC content, 5,269 predicted CDSs, 134 tRNAs and 40 rRNAs. The common virulence factors including OMPs, haemolysin, flagellin, DNase, entF, algU, tcpI, acfB and rfaD were found in strain SSVR1601. Furthermore, factors responsible for iron uptake (fur, fepC and ccmC) and types II, IV and VI secretion systems were detected, which are likely responsible for the pathogenicity of SSVR1601. The antimicrobial resistance genes, bacA, tet34 and norM, were detected based on Antibiotic Resistance Genes Database. The phylogenetic analysis revealed SSVR1601 to be most closely related to V. rotiferianus strains CAIM577 and B64D1.     

Characterization of β-haemolytic Group B Streptococcus agalactiae in cultured seabream, Sparus auratus L., and wild mullet, Liza klunzingeri (Day), in Kuwait

Streptococcus agalactiae was isolated from cultured gilthead seabream, Sparus auratus L., and diseased wild Klunzinger's mullet, Liza klunzingeri (Day), in Kuwait Bay, Arabian Gulf. Isolates were catalase negative, β‐haemolytic, Gram‐positive cocci and serogroup B. Experimental infectivity trials with mullet and seabream brain isolates in Nile tilapia, Oreochromis niloticus L., caused 100 and 90% mortality, respectively, within 7 days post‐inoculation indicating virulent S. agalactiae as the bacterial pathogen responsible for the epizootic in Kuwait Bay.