M. Carmen Alvarez

A continuous cell line from the cultured marine fish gilt-head seabream (Sparus aurata L.)

Abstract Conspecific cell lines provide an efficient experimental system with many applications for improving the productivity of commercial fish species. A continuous cell line SAF-1 has been developed from fin tissues of an adult gilt-head seabream ( Sparus aurata ) without immortalising treatments. The cells grow in DME-F12 basal medium supplemented with 5% foetal bovine serum at the optimal temperature of 25 °C and have been sub-cultured approximately 70 times over a 1.5 year period. They show a fibroblast-like morphology, high efficiency of plating, doubling time of 2 days and high survival after being frozen. Karyotype and DNA content of the cell line reveal a mostly euploid and rather stable cell population, which is of great interest for cytogenetic studies. This cell line has been shown to be susceptible to several fish viruses and to bacterial extracellular products.

An ES-like cell line from the marine fish Sparus aurata: characterization and chimaera production.

Embryonic stem (ES) cells provide a unique tool for cell-mediated gene transfer and targeted gene mutations due to the possibility of in vitro selection of desired genotypes. When selected cells contribute to the germ line in chimaeric embryos, transgenic animals may be generated with modified genetic traits. Though the ES cell approach has up to now been limited to mice, there is an increasing interest to develop this technology in both model and commercial fish species, with so far promising results in the medaka and zebrafish. In this study, we present evidence regarding a long-term stable cell line (SaBE-1c), derived from embryonic cells of the aquaculture marine fish Sparus aurata which has been characterized for (i) cell proliferation, (ii) chromosome complement, (iii) molecular markers, and (iv) in vitro tests of pluripotency by alkaline phosphatase (AP) staining, telomerase activity, and induced cell differentiation. These cells have proved their pluripotent capacities by in vitro tests. Furthermore, we have demonstrated their ability to produce chimaeras and to contribute to the formation of tissues from all three embryonic germ layers. These features suggest that SaBE-1c cells have the potential for multiple applications for the ES technology in fish, with the added value of originating from an economically important species.

Interspecific genetic differentiation in Western Mediterranean sparid fish

Genetic differentiation and phylogenetic relationships among Diplodus bellottii, D. cervinus, Sparus aurata, Pagrus pagrus, D. sargus and D. vulgaris were investigated by examining the electrophoretic patterns of 22 enzymes, from which 35 loci were detected. The six species were fixed for different alleles at the ADH locus which has been suggested to be the most reliable species-specific marker. Average genetic distances (D) among the six species increased in proportion to the level of taxonomic status (D = 0.54 between congeneric species and D = 1.04 between different genera). Distances among the six species along with the dendrogram obtained, generally agree with the conventional systematics. They fit better with the view including S. aurata and P. pagrus in different genera, in spite of their high morphological similarity. This study reveals useful information for progressive aquaculture.

Multiple Chromosome Polymorphism in Gobius paganellus (Teleostei, Perciformes)

Chromosome preparations from 46 specimens of G. paganellus were studied, revealing seven different karyomorphs: D2 (2n=46, NF=46), D1 (2n=47, NF=47), B (2n=48, NF=48), F1 (2n=47, NF=48), F2 (2n=46, NF=48), D1F1 (2n=46, NF=47), I1 (2n=48, NF=49). It is proposed that the mechanisms involved are deletions, fusions and inversions. The meiotic results suggest that the same chromosome pair was engaged in both the fusion and the deletion rearrangements. Intra-individual polymorphism was found in only one specimen. This and the range of karyomorphs suggest that this variation is regularly inherited.

Fish ES Cells and Applications to Biotechnology

ES cells provide a promising tool for the generation of transgenic animals with site-directed mutations. When ES cells colonize germ cells in chimeras, transgenic animals with modified phenotypes are generated and used either for functional genomics studies or for improving productivity in commercial settings. Although the ES cell approach has been limited to mice, there is strong interest for developing the technology in fish. We describe the step-by-step procedure for developing ES cells in fish. Key aspects include avoiding cell differentiation, specific in vitro traits of pluripotency, and, most importantly, testing for production of chimeric animals as the main evidence of pluripotency. The entire process focuses on two model species, zebrafish and medaka, in which most work has been done. The achievements attained in these species, as well as their applicability to other commercial fish, are discussed. Because of the difficulties relating to germ line competence, mostly of long-term fish ES cells, alternative cell-based approaches such as primordial germ cells and nuclear transfer need to be considered. Although progress to date has been slow, there are promising achievements in homologous recombination and alternative avenues yet to be explored that can bring ES technology in fish to fruition.

Genetic identification of sparid species by isozyme markers: application to interspecific hybrids

Abstract The successful performance of Sparus aurata in Mediterranean aquaculture has prompted the domestication of other sparid species and the production of their interspecific hybrids. Allozyme markers have proved to be very powerful to study genetic variation at interspecific level and have been applied to differentiate the sparid species: Sparus aurata , Pagrus pagrus , Pagellus bogaraveo , Diplodus sargus , Diplodus puntazzo , Lithognatus mormyrus , Dentex dentex and Spondyliosoma cantharus . Seventeen enzymes, revealing 24 loci were suitable for comparison purposes. Among them, only the AK * locus does not allow differentiation of species, while the remaining revealed qualitative allelic differences in a variable number of species. The most resolutive loci were: ENDOP *, EST *, G3PDH *, and LDH-1 *, which discriminated 24 out of 28 species pairs in a dual species comparison matrix. A practical application of this study, was to demonstrate the hybrid nature of the offspring resulting from crossing female S. aurata and male P. pagrus . The ADH * locus, revealed from liver, proved to be very resolutive in terms of band separation. In addition the EST * showed to be very convenient, as it can be revealed from fin and mucus, avoiding sacrificing the animals.

Viral nervous necrosis virus persistently replicates in the central nervous system of asymptomatic gilthead seabream and promotes a transient inflammatory response followed by the infiltration of IgM+ B lymphocytes

The viral nervous necrosis virus (VNNV) is the causal agent of viral encephalopathy and retinopathy (VER), a worldwide fish disease that is responsible for high mortality in both marine and freshwater species. Infected fish suffer from encephalitis, which leads to abnormal swimming behavior and extensive cellular vacuolation and neuronal degeneration in the central nervous system (CNS) and retina. The marine fish gilthead seabream (Sparus aurata) does not develop VER but it is an asymptomatic carrier of VNNV. In this study, we report that VNNV was able to replicate and persist for up to 3 months in the CNS of the gilthead seabream without causing any neural damage. In addition, we found an early inflammatory response in the CNS that was characterized by the induction of genes encoding pro-inflammatory cytokines, a delayed but persistent induction of anti-inflammatory cytokines, and the infiltration of IgM(+) B lymphocytes, suggesting that local adaptive immunity played a major role in the control of VNNV in the CNS of this species.

Antiviral Specificity of the Solea senegalensis Mx Protein Constitutively Expressed in CHSE-214 Cells

Interferons play a key role in fish resistance to viral infections by inducing the expression of antiviral proteins, such as Mx. The aim of the present study was to test the antiviral activity of the Senegalese sole Mx protein (SsMx) against RNA and DNA viruses pathogenic to fish, i.e. the infectious pancreatic necrosis virus (IPNV, dsRNA), the viral haemorrhagic septicaemia virus (VHSV, ssRNA), and the European sheatfish virus (ESV, dsDNA), using a CHSE-214 cell clone expressing this antiviral protein. A strong inhibition of IPNV and VHSV replication was recorded in SsMx-expressing cells, as has been shown by the virus yield reduction and the decrease in the synthesis of the viral RNA encoding the polyprotein (for IPNV) and the nucleoprotein (for VHSV). The titres of these viruses replicating on SsMx-expressing cells were 100 times lower than those recorded on non-transfected cells. In contrast, SsMx did not inhibit ESV replication since no significant differences were observed regarding the virus yield or the major capsid protein gene transcription in transfected and non-transfected cells.

A new Robertsonian fusion in the multiple chromosome polymorphism of a mediterranean population of Gobius paganellus (Gobiidae, Perciformes)

Karyotypic analysis of 67 individuals of a Mediterranean population of G. paganellus has revealed three new karyomorphs: D2F′1, (2n = 45, NF = 46), F2F′1, (2n = 45, NF = 48), D1F1F′1, (2n = 45, NF = 47), in addition to the seven previously reported by Thode et al. (1985). The C-banding pattern and the meiotic configurations clearly show that the mechanism involved is a new centromeric fusion. The high level of intra-individual variability for this rearrangement suggests a possible origin within this population. The involvements of this fusion in the polymorphism of G. paganellus are discussed mainly in comparison with another centromeric fusion.

The piscine SAF-1 cell line: genetic stability and labeling.

Fish cell lines are increasingly important research tools. The SAF-1 cell line, fibroblast-like culture derived from the marine fish gilthead seabream (Sparus aurata), has proved useful in many applications, especially in viral research. For cell lines intended as in vitro models, characterization of their properties and authentication are essential for deeper understanding of their performance and thus more precise experimental design and applicability. In this study we characterized the SAF-1 cell line in terms of genetic stability through time and genetic labeling. Methods for determining stability include telomerase activity, karyotyping, mapping of ribosomal RNA regions, and DNA content. For genetic labeling 12 microsatellite loci were used. The results indicate that telomerase has been activated in the course of SAF-1 development, and the highest levels of telomerase activity correlate with an increase in cell proliferation, thus supporting a permanent cell line. This stability is in agreement with the normal situation presented by the cytogenetic traits and DNA content values, and the genotypic profile allows SAF-1 authentication at the single individual level. This study increases the value of SAF-1 as an in vitro system, which is now one of the few well-characterized cell lines from a marine fish.