Detecting genome-wide gene transcription profiles associated with high pollution burden in the critically endangered European eel.

The European eel illustrates an example of a critically endangered fish species strongly affected by human stressors throughout its life cycle, in which pollution is considered to be one of the factors responsible for the decline of the stock. The objective of our study was to better understand the transcriptional response of European eels chronically exposed to pollutants in their natural environment. A total of 42 pre-migrating (silver) female eels from lowly, highly and extremely polluted environments in Belgium and, for comparative purposes, a lowly polluted habitat in Italy were measured for polychlorinated biphenyls (PCBs), organochlorine pesticides (OCPs) and brominated flame retardants (BFRs). Multipollutant level of bioaccumulation was linked to their genome-wide gene transcription using an eel-specific array of 14,913 annotated cDNAs. Shared responses to pollutant exposure were observed when comparing the highly polluted site in Belgium with the relatively clean sites in Belgium and Italy. First, an altered pattern of transcription of genes was associated with detoxification, with a novel European eel CYP3A gene and gluthatione S-transferase transcriptionally up-regulated. Second, an altered pattern of transcription of genes associated with the oxidative phosphorylation pathway, with the following genes involved in the generation of ATP being transcriptionally down-regulated in individuals from the highly polluted site: NADH dehydrogenase, succinate dehydrogenase, ubiquinol-cytochrome c reductase, cytochrome c oxidase and ATP synthase. Although we did not measure metabolism directly, seeing that the transcription level of many genes encoding enzymes involved in the mitochondrial respiratory chain and oxidative phosphorylation were down-regulated in the highly polluted site suggests that pollutants may have a significant effect on energy metabolism in these fish.

Genetic variability is unrelated to growth and parasite infestation in natural populations of the European eel (Anguilla anguilla).

Positive correlations between individual genetic heterozygosity and fitness-related traits (HFCs) have been observed in organisms as diverse as plants, marine bivalves, fish or mammals. HFCs are not universal and the strength and stability of HFCs seem to be variable across species, populations and ages. We analysed the relationship between individual genetic variability and two different estimators of fitness in natural samples of European eel, growth rate (using back-calculated length-at-age 1, 2 and 3) and parasite infestation by the swimbladder nematode Anguillicola crassus. Despite using a large data set of 22 expressed sequence tags-derived microsatellite loci and a large sample size of 346 individuals, no heterozygote advantage was observed in terms of growth rate or parasite load. The lack of association was evidenced by (i) nonsignificant global HFCs, (ii) a Multivariate General Linear Model showing no effect of heterozygosity on fitness components, (iii) single-locus analysis showing a lower number of significant tests than the expected false discovery rate, (iv) sign tests showing only a significant departure from expectations at one component, and, (v) a random distribution of significant single-locus HFCs that was not consistent across fitness components or sampling sites. This contrasts with the positive association observed in farmed eels in a previous study using allozymes, which can be explained by the nature of the markers used, with the allozyme study including many loci involved in metabolic energy pathways, while the expressed sequence tags-linked microsatellites might be located in genes or in the proximity of genes uncoupled with metabolism/growth.

Genetic composition of Atlantic and Mediterranean recruits of European eel Anguilla anguilla based on EST-linked microsatellite loci.

Anguilla anguilla glass eels arriving at two Mediterranean and two Atlantic sites were tested for differences in genetic composition between regions using a total of 23 microsatellite loci developed from an expressed sequence tag (EST) library. Hierarchical analysis of molecular variance indicated a non-significant difference between regions (Mediterranean v. Atlantic), which contrasted with the significant differences observed between samples within regions. The existence of a single spawning site for all A. anguilla individuals and extensive migration loop with great opportunity for mixing of individuals might explain the homogeneity in genetic composition found between regions. The observation of a (small-scale) pattern of genetic patchiness among intra-annual samples (arrival waves) within geographic regions does not conflict with the lack of (large-scale) geographic sub-structuring found between the Mediterranean and Atlantic regions, but most likely is a consequence of the strong dependence of A. anguilla on oceanic conditions in the Sargasso Sea that might result in a limited parental contribution to each spawning event. The comparison of Atlantic and Mediterranean A. anguilla glass eel recruits based on EST-linked microsatellite loci provides evidence supporting the hypothesis of panmixia A. anguilla across Europe.

Micronuclei induced in erythrocytes of Cyprinus carpio (teleostei, pisces) by X-rays and colchicine.

In the present work the induction of micronuclei in erythrocytes of Cyprinus carpio treated with X-rays and colchicine is studied for the evaluation of mutagenic effects of both clastogenic and mitoclastic (spindle poisoning) agents in this system. Three different experiments were performed treating groups of laboratory-reproduced animals with (1) single doses of X-rays (0.1, 0.5 and 2Gy); (2) a single i.p. injection of colchicine at the concentrations: 1.6x10(-2), 8x10(-2), 0.4 and 2mg/kg b.w. so as to mimic an acute exposure to the agent and (3) six repeated i.p. injections of the first three concentrations of colchicine, over a period of 18 days, so as to mimic a chronic exposure. Repeated blood samplings were performed by cardiac puncture over a period of about 2 months after the treatment and micronucleus frequencies were determined at multiple times on the same individuals after mutagen exposure. A dose-dependent increase in the micronucleus frequency was observed in irradiated fish and a peak value detected at 21 days. Slight increases of micronucleus frequencies were also observed in both colchicine experiments only for the highest concentrations at the earliest sampling time. Higher concentrations of colchicine clearly showed a lethal effect. According to the present data the micronucleus frequency induced by the highest colchicine dose is comparable to that observed after 0.1Gy of X-ray irradiation.

Micronucleus test in erythrocytes of Barbus plebejus (Teleostei, Pisces) from two natural environments: a bioassay for the in situ detection of mutagens in freshwater.

Erythrocyte micronucleus frequencies in wild fish from two riverine environments and in fish reproduced and reared under controlled conditions (control group) were compared, with the aim to evaluate the suitability of the MN test for the in situ detection of mutagens in freshwaters. Fish were caught in different months in two rivers of central Italy which have different pollution levels. As indicator species, the barbel (Barbus plebejus) was chosen because of its ecological significance. Blood samplings were performed on wild fish immediately after capture and repeated at different time intervals on the same individuals, which were maintained in controlled conditions after capture. A total of 10,000 erythrocytes per specimen were scored. No significant differences in micronucleus frequencies were observed between the control group and fish from the unpolluted river (Mignone). A significantly higher frequency of micronuclei was observed in fish caught in the polluted river (Tiber), in comparison to both the controls and the Mignone river fish. No significant seasonal differences were observed. Barbels examined 50 and 100 days after capture presented a remarkable decrease in micronucleus frequency in comparison with the frequency observed in barbels at capture. The micronucleus test in fish erythrocytes was shown to be a sensitive bioassay for detecting mutagenic pollution in fresh water environments.