Eva Garcia-Vazquez

Chromosomal mapping and nucleotide sequence of two tandem repeats of Atlantic salmon 5S rDNA

Atlantic salmon 5S ribosomal DNA (5S rDNA) was amplified by the polymerase chain reaction, using as primers conserved sequences from the coding region of rainbow trout 5S rRNA. Two amplified products of different molecular weights were obtained, cloned, and sequenced, revealing them to be tandemly arranged. The nucleotide sequences differed between the two clones in the length of the nontranscribed spacer (NTS) and in three nucleotides of the coding sequence. By means of fluorescence in situ hybridization the 5S rDNA was chromosomally located in the heterochromatic arm of the pair bearing the satellite, adjacent to the major ribosomal DNA locus (rDNA).

Induction of micronuclei and other nuclear abnormalities in European minnow Phoxinus phoxinus and mollie Poecilia latipinna: an assessment of the fish micronucleus test.

In this work, we have measured both micronuclei and other nuclear abnormalities in renal erythrocytes from European minnow Phoxinus phoxinus and mollie Poecilia latipinna, with the aim to contribute to the standardisation of the micronucleus test for fish species. Intraperitoneal injections of colchicine (10 mg/kg), cyclophosphamide (40 mg/kg), or mitomycin C (20 mg/kg) for 24 h induced diverse nuclear abnormalities in minnow erythrocytes, therefore nuclear abnormalities should be added to micronuclei as genotoxicity indicators in fish micronucleus tests. The adequacy of administration protocols based on intraperitoneal injections has been evaluated by injecting saline solution to both species: single or double injections have not induced neither micronuclei nor other nuclear abnormalities in any case. Finally, the differential sensitivity of both species to toxic heavy metals was evaluated by exposing individuals of both species to different doses (0.17, 1.7, 2x1.7, and 3.4 mg/kg) of cadmium and mercury for 24 h; we concluded that the mollie is sensitive to both metals whereas the minnow is not sensitive to mercury.

Sex chromosome linkage of 5S rDNA in rainbow trout (Oncorhynchus mykiss)

The karyotype of the rainbow trout is characterized by a primitive XX/XY sex-determining chromosomal system. (Thorgaard et al., 1977). In the present study using FISH we have physically linked the 5S rRNA genes to the partially undifferentiated X chromosome pair. PCR amplified 5S rDNA was used for FISH and hybridization signals indicated that the genes were duplicated, present in one acrocentric and one metacentric pair of chromosomes. After analyzing several individuals, the female metaphases showed four fluorescent signals whereas males presented only three signals. Two of the three signals obtained in males corresponded to the metacentric pair whereas the single signal was mapped to the heterochromatin that cytologically differentiates the X chromosome from the Y chromosome. Double FISH experiments demonstrated that the 5S rDNA which is not sex linked is located at the NOR bearing arm close to the major ribosomal RNA genes (5.8S, 18S and 28S), similar to the situation observed in Atlantic salmon (Pendas et al., 1994a).

Ribosomal RNA genes are interspersed throughout a heterochromatic chromosome arm in Atlantic salmon

The ribosomal RNA genes (rDNA) have been mapped by fluorescent in situ hybridization (FISH) using four rDNA probes (rDNA/FISH) to Atlantic salmon chromosomes. Signals appeared over the whole heterochromatic chromosome arm displaying the secondary constriction and satellites. The size polymorphism of this sort arm, revealed by C-banding, was confirmed by rDNA/FISH, supporting large interindividual differences in the number of rDNA copies. Conventional techniques for the detection of nucleolar organizer regions are discussed, and their results are compared with those of rDNA/FISH.

Applications of 5S rDNA in Atlantic salmon, brown trout, and in Atlantic salmon brown trout hybrid identification

The members of the salmonid family form perhaps the most economically important group of the world’s fish species. In Europe the most important species employed in fish farming are the introduced rainbow trout (Oncorhynchus mykiss) and the Atlantic salmon (Salmo salnr), whereas interest in the brown trout (Salmo trufta) is based on its ecological diversity and sport fishing. These two Salmo species are very difficult to distinguish using morphological characters, not only during their first months of life (eggs and alevins) but also at the returning adult stage. Isoenzyme genetic analysis, which has usually been used for their identification, has often demonstrated that anglers have confused adults of brown trout with Atlantic salmon, or with salmon x trout hybrids (Leaniz & Verspoor 1989). Caution must therefore be taken in enhancement programmes since the artificial spawning of adults caught in the river could lead to restocking with salmon x trout hybrids. Another area of interest is the identification of manufactured products of these species (i.e. smoked). Genetic analysis potentially provides the only reliable method to unambiguously determine their species identity. In addition, the genetic marker employed should be a nuclear marker if interspe cific hybrids among these species have to be recognized. From a practical point of view, the procedure chosen must be fast, extremely robust, and be able to utilize tiny quantities of any tissue. This last requirement excludes most isoenzyme techniques. At present, PCR-based methodologies constitute the most reliable techniques available for this purpose. Most of these approaches are, however, based on the use of conserved mitochondria1 DNA primers (for the cytochrome b gene) and sequencing of the amplified fragment (Paabo et al. 1989; Bartlett h Davidson 1992). Instead, we have focused on the 5s

Multi-chromosomal location of ribosomal RNA genes and heterochromatin association in brown trout.

The ribosomal rRNA genes have been mapped by fluorescentin situ hybridization (FISH) to brown trout chromosomes. One major NOR chromosome pair and 8 novel minor NOR chromosome pairs have been found. Both major and minor NORs were closely related to polymorphic heterochromatin, as revealed by FISH and C-banding. These results are discussed with respect to NOR expression, the relationship between rDNA and heterochromatin, and evolutionary aspects.

Multiple paternity increases effective size of southern Atlantic salmon populations

Genetic analyses were performed on the progeny of Atlantic salmon (Salmo salar L.) sampled in natural redds of three rivers flowing into the Bay of Biscay, the Nivelle, the Mandeo and the Sella. These rivers are at the southern limit of the European distribution of the species and their populations are small and endangered by human activities. Nine variable number of tandem repeat (VNTR) loci (five minisatellites and four microsatellites) were used for parentage analysis. Multiple male participation was recognized in the fertilization of eggs. A large proportion was fertilized by precociously mature parr. We demonstrate that multiple paternity derived from mature parr is crucial for the conservation of genetic variability in small populations of Atlantic salmon.

MICRONUCLEUS TEST IN FRESHWATER FISH SPECIES: AN EVALUATION OF ITS SENSITIVITY FOR APPLICATION IN FIELD SURVEYS

Brown trout, Salmo trutta, European eel, Anguilla anguilla, and European minnow, Phoxinus phoxinus, three fish species inhabiting European freshwater ecosystems, were evaluated for their use as in situ pollution biomarkers using the micronucleus test in renal erythrocytes. Experimental exposure (by immersion) to different concentrations of cyclophosphamide, colchicine, and cadmium showed that brown trout are more sensitive to the three compounds than minnows and eels. In situ surveys of wild freshwater ecosystems with different levels of pollution showed that minnows and eels living in polluted sites do not present higher micronuclei averages than those caught in clean rivers systems, whereas micronuclei are induced in brown trout inhabiting polluted sites. Our results demonstrated the suitability of brown trout for in situ biomonitoring of freshwater ecosystems as well as for laboratory tests using the micronucleus test.

Chromosomal localization of the major and 5S rRNA genes in the European eel (Anguilla anguilla)

The major (18S, 5.8S, and 28S) and 5S rRNA genes have been mapped by double fluorescent in situ hybridization to European eel metaphase chromosomes. The major rRNA genes were localized to a submetacentric pair of chromosomes that showed a consistent size polymorphism among the individuals studied. The 5S rRNA genes were clustered in a single locus that mapped to the centromeric region of an acrocentric pair. In contrast to the major rRNA genes, no detectable polymorphism, in either size or intensity of the fluorescent signal, was observed. The chromosomal organization of both families of rRNA genes are discussed in terms of genomic organization and chromosomal evolution.

Micronuclei and fluctuating asymmetry in brown trout (Salmo trutta): complementary methods to biomonitor freshwater ecosystems

In this work we measured both micronuclei number in kidney erythrocytes and fluctuating asymmetry in wild brown trout (Salmo trutta), caught in different fluvial ecosystems of Asturias (northern Spain) characterized by different levels of anthropic influence. Brown trout samples from rivers with high anthropic influence possessed significantly higher averages of both micronuclei and fluctuating asymmetry than brown trout samples from less anthropic-influenced rivers. These findings demonstrated the sensitivity of the micronucleus test in kidney erythrocytes to biomonitor freshwater ecosystems. The positive association found between micronuclei average and fluctuating asymmetry at the populational level suggests that fluctuating asymmetry tests could be potential indicators of environmental threat. Variation of fish asymmetry with ageing indicates that fluctuating asymmetry surveys of wild populations should be carried out in trouts of the same age class.