Sex-dependent synaptic behaviour in triploid turbot,Scophthalmus maximus (Pisces,Scophthalmidae)

A surface-spreading synaptonemal complex (SC) technique was used to analyse spermatocytes and oocytes of triploid turbot (Scophthalmus maximus) in order to visualise the process of chromosome synapsis. The most conspicuous characteristic of triploid oocytes is that, in the trivalents, the lateral elements of the SC were frequently associated in threes, either completely along the length of the trivalent, or partially, forming a variety of forked structures. In these nuclei, synapsis usually occurred among homologous chromosomes and the number of bivalents observed was significantly higher than that expected under the assumption of random chromosome association among all partners. However, the frequency of trivalents was very low in triploid spermatocytes, triple synapsis being also scarce. In these nuclei chromosomes that were excluded from homologous synapsis become engaged in random SC formation, and, therefore a considerable number of non-homologous associations are produced. The causes of the synaptic differences observed in triploid males and females of turbot and their possible relation to the sterility displayed by these animals are discussed.     

Synaptonemal complex analysis in spermatocytes of tilapia, Oreochromis niloticus (Pisces, Cichlidae).

Some adaptations of the synaptonemal complex (SC) whole-mounting technique first used in plants permitted its application to meiotic studies in tilapia, Oreochromis niloticus. Direct observation of the chromosome pairing process and bivalent structure during the meiotic prophase of this fish species by light and electron microscopy permitted the analysis of SCs in autosomes and the possible identification of sex chromosomes. The analysis of SCs in spermatocytes of O. niloticus revealed that all 22 bivalent chromosomes completely paired, except for the occurrence of a size heteromorphism in the terminal region of the largest bivalent associated with the presence of an incompletely paired segment during the synapsis process, which may be the cytological visualization of an XX/XY sex chromosome system in this species.     

Development and characterization of 248 novel microsatellite markers in turbot (Scophthalmus maximus).

The turbot is a flatfish species of great relevance to marine aquaculture in Europe. Only a limited number of microsatellites have been isolated to date in this species. To increase the number of potentially useful mapping markers, we screened simple sequence repeat (SSR)--enriched genomic libraries obtained from several di-, tri-, and tetranucleotide tandem repeat motifs. A total of 248 new polymorphic microsatellites were successfully optimized. The efficiency of the protocol applied (6.4%) was higher than that in other studies of fish that used the same method. Dinucleotide and perfect microsatellites were predominant in this species; the (AC)n motif was the most frequent class of repeat. Polymorphism and structural properties at these loci, together with 30 variable loci previously reported in turbot, were evaluated in 6 wild individuals. The number of alleles per locus ranged from 2 to 10, with an average of 4.046. The microsatellite markers characterized in this study will contribute to the development of the turbot genetic map, which can be used for quantitative trait locus (QTL) identification, marker-assisted selection programs, and other applications to improve its culture.     

Cryopreservation of turbot (Scophthalmus maximus) spermatozoa

The aim of this study was to develop a method for cryopreserving turbot semen and to compare sperm motility characteristics, metabolic status and fertilization capacity of frozenthawed and fresh semen. The best results were obtained when spermatozoa were diluted at a 1:2 ratio with a modified Mounib extender, supplemented with 10% BSA and 10% DMSO. For freezing sperm samples, straws were placed at 6.5 cm above the surface of liquid nitrogen (LN) and plunged in LN. The straws were thawed in water bath at 30 degrees C for 5 sec. Use of this simple method resulted in a 60 to 80% reactivation rate of the thawed spermatozoa. Although the percentage of motile spermatozoa in the frozen-thawed semen samples was significantly lower than in fresh semen, spermatozoa velocity and respiratory rate remained unchanged. The process of cryopreservation significantly decreased intracellular ATP content. The fertilization rate of frozen-thawed spermatozoa was significantly lower than that of fresh spermatozoa, but it increased with sperm concentration.     

Structural peculiarities of the tubercles in the skin of the turbot,Scophthalmus maximus (L., 1758) (Osteichthyes,Pleuronectiformes, Scophthalmidae)

The structure of the bony tubercles of the turbot, Scophthalmus maximus (L., 1758), was examined using ground sections, microradiography, SEM, and TEM. The tubercles are small, isolated, mineralized conical plates randomly distributed in the eyed side of the body. They are composed of three layers: the outer limiting layer, the external layer, and the basal plate, which make up the thin and flat elasmoid scales of Teleostei. The main difference between regular elasmoid scales and bony tubercles lies in the organization and the growth of the basal plate. Indeed, the conical shape of the tubercle is the result of a prominent thickening of the central part of the basal plate where the collagen matrix is organized in a complicated three-dimensional network. Densely packed thick collagen fibrils form superimposed plies organized in a plywood-like structure that resembles that of the elasmoid scales but it is criss-crossed by numerous vertical sheets of thin collagen fibrils. The tubercles originate from thin and flat plates located in the skin of larvae and juveniles, whose structure is that of regular-developing elasmoid scales. Thus, the tubercles of Scophthalmus maximus could be considered as modified elasmoid scales rather than bony structures. They might be the result of specific arrangements related to the general trend of reduction of the dermal skeleton in the teleostean lineage.     

Peracute vibriosis in juvenile turbot Scophthalmus maximus

An outbreak of an acute oedematous disease characterised by orbital and abdominal swelling, and high mortality, in young turbot, was studied. The clinical and pathological picture was consistent with a circulating toxaemia principally affecting the heart, orbit and kidney. Vibrio anguillarum , virtually identical with type strain NCMB6, was isolated in pure culture from all organs of all fish. Therapy was only marginally successful using antibiotics but in a small group subjected to temperature reduction below 10°C, losses were reduced from 20 % to 5 % compared with the controls.     

Mycobacterial infection in farmed turbot Scophthalmus maximus

Mycobacteriosis (piscine tuberculosis) has been reported to affect a wide range of freshwater and marine fish species; however, this is the first report describing mycobacterial infections in turbot Scophthalmus maximus. High numbers of granulomas were initially observed in the organs of moribund farmed turbot. Bacteriological analysis of organs with granulomas led to the isolation of Mycobacterium marinum. Further analysis, to determine the prevalence of the infection in the farm and to identify its source, showed the occurrence of a dual infection by M. marinum and M. chelonae. The presence of Nocardia sp. in some of the fish infected with mycobacteria was also detected. The presence of granulomas in internal organs of apparently healthy fish indicated a high prevalence of the disease, a conclusion that was supported by isolating mycobacteria from all fish with or without granulomas. The infection was probably responsible for the mortality observed (approximately 2% mo(-1)), as most of the recently dead fish presented high numbers of granulomas and isolation of mycobacteria was possible from all of the fish. The isolation of M. marinum from the inlet water suggested this as the most plausible source for the infection occurring in the farm.     

Cloning and analysis of a ferritin subunit from turbot (Scophthalmus maximus)

Ferritin is an evolutionarily conserved protein that plays an important role in iron storage and detoxification. In this study, the gene encoding a ferritin H subunit homologue (SmFer1) was cloned from turbot (Scophthalmus maximus) and analyzed at the expression and functional levels. The open reading frame of SmFer1 is 534 bp and preceded by a 5′-untranslated region that contains a putative Iron Regulatory Element (IRE). The deduced amino acid sequence of SmFer1 shares extensive sequence identities with the H ferritins of a number of fish species and contains the ferroxidase center that is preserved in ferritin H subunits. Examination of tissue specific expression indicated that SmFer1 expression was most abundant in muscle, liver, and blood. Experimental infection with bacterial pathogens induced significant induction of SmFer1; however, the magnitudes of induction effected by Gram-negative pathogens were much higher than that induced by Gram-positive pathogen. Consistently, lipopolysaccharide (LPS) challenge drastically augmented SmFer1 expression. In addition to bacterial pathogens and LPS, poly(I:C) also induced a strong but transient induction of SmFer1 which differs in profile from those induced by bacterial pathogens. In vitro iron-chelating analysis showed that recombinant SmFer1 purified from Escherichia coli was able to bind ferrous iron in a concentration-dependent manner. To examine whether SmFer1, with its iron-chelating capacity, could have any effect on the infection of bacterial pathogens, purified recombinant SmFer1 was subjected to bacteriostatic analysis and proved to be able to inhibit the growth of the fish pathogen Listonella anguillarum which enhanced SmFer1 expression upon infection. Taken together, these results suggest that SmFer1 is likely to play a role in both iron storage and immune defense against microbial infections.     

Histopathology of experimental scuticociliatosis in turbot Scophthalmus maximus

A scuticociliate strain (B-2), originally isolated from an outbreak in a turbot Scophthalmus maximus (= Psetta maxima) farm in Galicia (northwestern Spain) and maintained in axenic culture, was injected intracoelomically (lethal dose 80 equivalent, LD80) in healthy turbot (50 g). Ciliate-injected fish were kept under controlled conditions in a recirculating seawater system and sampled on Days 1 through 8, 10, 12 and 14 postinfection (PI). Necropsies were conducted and included blood collection from the caudal vein and samples of liver, spleen, heart, digestive tract, kidney, gills, abdominal wall and neurocranium taken for routine histology. Mortality occurred from Day 6 until Day 12 PI and reached 66.7% by the end of the experiment. Presence of ciliates in the coelomic fluid was scarce until Day 4 PI. Parasitaemia was only observed from Day 5 until Day 10 PI and its incidence was always low. Presence of scuticociliates in tissue sections followed a progressive pattern of diffusion, with ciliates showing preference for loose connective tissue and also a clear haematophagous activity. The most severely affected organs were the pancreas and digestive tract. No special tropism for nervous tissues was observed in this study. The inflammatory reaction was variable depending on the tissue. After 3 wk, survivors had apparently managed to extinguish the infection.     

Synaptonemal complex analysis in Talpa occidentalis spermatocytes (Insectivora, Mammalia). II. Evolution of the X-chromosome self-pairing process

Zygotene and pachytene configurations of the X chromosome were studied in whole-mount, silver-stained preparations of spermatocytes from XY males from a population of Talpa occidentalis in which sex reversal has been described. The most striking finding in this study was a self-pairing conformation of the axial (differential) element of the X chromosome. This self-pairing was markedly constant in the site of initiation, which invariably involved the distal free end of the X and the region beyond the X-Y pairing segment, so that X-Y synapsis was never disturbed. In addition, self-pairing occurred later than autosomal synapsis and was accompanied by thickening of the axes, although this seemed to occur after the formation of an ordinary synaptonemal complex. The etiology of this phenomenon may be based on residual homology, possibly due to conservation of a primitive isochromosome throughout the karyotypic evolution of this species. However, the possible relationship between self-pairing and sex reversal remains obscure.     

Synaptonemal complex analysis in spermatocytes and oocytes of tertiary trisomic Ts(512)31H mice with male sterility

Whole-mount preparations of silver-stained spermatocytes and oocytes from Ts(512)31H mice were examined in the electron microscope. The 5(12) chromosome was associated with the XY bivalent in the large majority of spermatocytes, whereas in about one-half of the oocytes, the 5(12) was associated with either unpaired chromosomes or heterochromatic parts of chromosomes or showed self-synapsis. There was a tendency for 5(12) chromosomes to be more fully heterochromatic in oocytes than in spermatocytes. A large proportion of oocytes (50%) and a much smaller proportion of spermatocytes exhibited various errors of chromosome pairing, but these proportions were only marginally greater than in control gametocytes from mice with normal karyotypes. It is concluded that the observed errors of pairing bear no simple relation to the almost complete breakdown of spermatogenesis and the marked impairment of oogenesis that occur in tertiary trisomic Ts(5(12))31H mice.     

Subclinical herpesvirus infection in farmed turbot Scophthalmus maximus

Subclinical infections with a herpesvirus were detected by light and electron microscopic examination of juvenile turbot collected during a national surveillance programme. Virions detected in the epidermis of skin and in the epithelium of gills had a morphology consistent with those of Herpesvirus scophthalmi described from turbot in the United Kingdom and Denmark. This is the first report of herpesvirus infection in turbot in Norway.     

Immunomodulatory effects of nisin in turbot (Scophthalmus maximus L.)

In the present work, the effect of nisin on the non-specific immune response of turbot (Scophthalmus maximus L.) leukocytes has been studied both in vitro and in vivo. The head kidney macrophage chemiluminescent (CL) response was significantly increased with intermediate doses of nisin (2.5 and 0.025 micro g ml(-1)) whilst the higher dose (25 micro g ml(-1)) significantly decreased the response after 24h incubation. When the incubation time was extended to 72 h, significant differences between doses were observed and the lower nisin concentration (0.025 micro g ml(-1)) appeared to be the optimum dose for increasing the CL response. The phagocytic activity of HK macrophages was also affected by in vitro nisin treatments. Nisin at 0.25 micro g ml(-1) and 0.025 micro g ml(-1) significantly stimulated the response after 24 and 72 h incubation respectively. Nitric oxide (NO) production by HK macrophages was not influenced by any nisin concentration employed for 24 or 72 h incubationsIn vivo, one week post injection, a slightly but non-significant stimulation of the CL response was observed with the lowest nisin concentration (0.0025 micro g fish(-1)). NO in serum and serum antibacterial index were not significantly affected by nisin treatments. On the other hand, lysozyme concentration in serum was significantly augmented with the lowest nisin dose (0.0025 micro g fish(-1)).The antibacterial effect of nisin against the fish pathogenic bacteria Carnobacterium piscicola (CECT 4020) was also demonstrated in vitro.     

The hepato-renal syndrome in cultured turbot (Scophthalmus maximus L.)

The histopathology of biliary hyperplasia and renal calcinosis, associated with infections of two parasites, Myxidium incurvatum and Rhabdospora thelohani , is described in turbot, Scophthalmus maximus (L.), from Scottish marine fish farms and wild populations. Liver and kidney lesions were divided into four, and parasite infections into five grades of severity. A few of the fish examined were not parasitized, but every one showed some evidence of pathological change, even the apparently normal individuals from all populations. Although there is a positive association between the parasitic and pathological data, parasitism is not considered to be the principal cause of either the hepatic or renal lesion; the evidence suggests rather a secondary opportunist role for the parasites. The general severity of the condition in captive stocks suggests that the cause may lie in the fundamentals of present techniques of turbot husbandry, but no specific agent has been identified. A case is put forward for considering Rhabdospora to be a parasite rather than a specialized host cell.     

Cloning, characterization and expression analysis of a novel CXC chemokine from turbot (Scophthalmus maximus)

Chemokines represent a superfamily of chemotactic cytokines playing an important role in leucocyte chemotaxis. Here we report a novel turbot CXC chemokine screened from a turbot spleen cDNA library. The complete cDNA of the turbot CXC chemokine contains an 81bp 5' UTR, a 414bp open reading frame (ORF) encoding 137 amino acids and a 449bp 3' UTR. Four exons and three introns are identified in the turbot CXC chemokine gene. Phylogenetic analysis showed that the turbot CXC chemokine clustered apart from all other CXC chemokines. RT-PCR demonstrated that turbot CXC chemokine was expressed highly in spleen and head kidney. During the early stages of embryo development after fertilization, it appears that low expression level of turbot CXC chemokine was firstly observed at somites stage. Interestingly, the turbot chemokine was highly and rapidly (5h) induced in liver, spleen and head kidney of turbot after challenge with Vibrio anguillarum. Furthermore, the expression of CXC chemokine was also dramatically increased after challenge in turbot embryonic cells (TECs). These results indicated that the turbot CXC chemokine played an important role in turbot immune response.