Anna Navarro

Physiological responses to mercury in feral carp populations inhabiting the low Ebro River (NE Spain), a historically contaminated site

The low Ebro River course (Northeast Spain) is historically affected by mercury pollution due to a chlor-alkali plant operating at the town of Flix for more than a century. River sediments analysed during the last 10 years showed high mercury levels in the river section starting just downstream the factory and spanning some 90km, down to the river delta. The possible environmental impact was studied by a combination of field and laboratory studies. Mercury concentrations in liver, kidney and muscle of feral carp (Cyprinus carpio) sampled downstream Flix were one to two orders of magnitude higher than those from carps sampled upstream Flix. Elevated levels of mercury in these samples associated with significant increases on the concentration of reduced glutathione (GSH) in liver and on mRNA expression of two metallothionein genes, MT1 and MT2, in kidney and, partially, in scales, but not in liver. Conversely, no biochemical evidence for oxidative stress or DNA damage was found in these tissues. Non-contaminated carps subjected to intraperitoneal mercury injection resulted in a 20-fold increase of MT1 and MT2 mRNA levels in carp kidney, with minimal changes in liver levels. Our data suggests the coordinate increase of metallothionein mRNA in kidney and of GSH in liver constitutes an excellent marker of exposure to sub-toxic mercury levels in carps. This study also demonstrates that apparently healthy fish populations may exceed the mercury contamination acceptable for human consumption.

Transcriptional response of stress genes to metal exposure in zebra mussel larvae and adults.

Development of stress markers for the invader freshwater zebra mussel (Dreissena polymorpha) is of great interest for both conservation and biomonitoring purposes. Gene expression profiles of several putative or already established gene expression stress markers (Metallothionein, Superoxide dismutase, Catalase, Glutathione S transferase, Glutathione peroxidase, Cytochrome c oxidase, the multixenobiotic resistance P-gp1, and heat shock proteins HSP70 and HSP90) were analyzed by quantitative Real-Time PCR in adults and pediveliger larvae after exposure to metals (Hg, Cu, Cd). A defined pattern of coordinated responses to metal exposure and, presumably, to oxidative stress was observed in gills and digestive gland from adults. A similar, albeit partial response was observed in larvae, indicating an early development of stress-related gene responses in zebra mussel. The tools developed in this study may be useful both for future control strategies and for the use of zebra mussel as sentinel species in water courses with stable populations.

Abcb and Abcc transporter homologs are expressed and active in larvae and adults of zebra mussel and induced by chemical stress

Multixenobiotic resistance (MXR) of aquatic invertebrates has so far been associated with cellular efflux activity mediated by P-glycoprotein (ABCB1) and MRP (multidrug resistance protein; ABCC) type ABC (ATP binding cassette) transporters. Expression and activity of an abcb1/Abcb1 homolog has been shown in eggs and larvae of the zebra mussel Dreissena polymorpha. Here we report identification of a partial cDNA sequence of an abcc/Abcc homolog from zebra mussel that is transcribed and active as a cellular efflux transporter in embryos and gill tissue of adult mussels. Transcript expression levels were comparatively low in eggs and sharply increased after fertilization, then maintaining high expression levels in 1 and 2 dpf (days post fertilization) larvae. MK571, a known inhibitor of mammalian ABCC transporters, blocks efflux of calcein-am in larvae and gill tissue as indicated by elevated calcein fluorescence; this indicates the presence of active Abcc protein in cells of the larvae and gills. Dacthal and mercury used as chemical stressors both induced expression of abcb1 and abcc mRNAs in larvae; accordingly, assays with calcein-am and ABCB1 inhibitor reversin 205 and ABCC inhibitor MK571 indicated enhanced Abcb1 and Abcc efflux activities. Responses to chemicals were different in gills, where abcb1 transcript abundances were enhanced in dacthal and mercury treatments, whereas abcc mRNA was only increased with mercury. Abcb1 and Abcc activities did not in all cases show increases that were according to respective mRNA levels; thus, Abcc activity was significantly higher with dacthal, whereas Abcb1 activity was unchanged with mercury. Our data indicate that abcb1/Abcb1 and abcc/Abcc transporters are expressed and active in larvae and adult stages of zebra mussel. Expression of both genes is induced as cellular stress response, but regulation appears to differ in larvae and tissue of adult stages.

Evaluation of environmental impact on natural populations of the Mediterranean killifish Aphanius fasciatus by quantitative RNA biomarkers.

The Mediterranean killifish, Aphanius fasciatus (Valenciennes, 1821) represents an excellent sentinel species for analysis of environmental impact in coastal areas. Quantitative changes on liver mRNA levels of five stress-related genes--metallothionein (metal exposure), vitellogenin, (estrogenic effects), cytochrome P4501A (CYP1A, dioxin-like compounds), superoxide dismutase (oxidative stress) and HSP70 (general stress)--were tested as markers of exposure to pollutants both in natural populations and in experimental treatments with model effectors. Analysis of fish from a metal-contaminated site in the Tunisian coast (Sfax) indicated that fish presenting spinal deformities (eight times more frequent at this site than in reference sites) showed increased levels of hepatic CYP1A mRNA levels compared to non-deformed fish from the same site or from the reference Louza site, suggesting a combined impact by metals and organic pollutants. Due to the strategy used in their design, these quantitative RNA markers will likely be useful to analyze environmental impacts on other related fish species.

Integrated biological and chemical analysis of organochlorine compound pollution and of its biological effects in a riverine system downstream the discharge point.

Pollution in riverine systems, along with its biological effects, may propagate downstream even at considerable distances. We analyzed the organochlorine compound (OC) pollution in a section of the low Ebro River (Northeast Spain) downstream a long-operating chlor-alkali plant. Maximal levels of OCs and of their associated dioxin-like biological activity occurred in residue samples from the plant, and persisted in river sediments some 40km downstream (Xerta site). Biological analysis at multiple organization levels in local carp (Cyprinus carpio, EROD, Cyp1A mRNA expression in the liver, hepatosomatic index, condition factor, and micronuclei index in peripheral blood) showed a similar pattern, with a maximal impact in Ascó, few kilometers downstream the plant, and a clear reduction at Xerta. This combination of chemical, molecular, cellular and physiological data allowed the precise assessment of the negative impact of the chlor-alkali plant on the quality of river sediments and on fish, and suggests that sediments may be a reservoir for toxic substances even in dynamic environments like rivers.

A noninvasive test of exposition to toxicants: quantitative analysis of cytochrome P4501A expression in fish scales.

Elevated expression of cytochrome P4501A (CYP1A) is an established biomarker for exposition to a wide range of toxicants, particularly for dioxin and structurally similar compounds. Expression of CYP1A usually is analyzed in internal organs, which involves dissection of the specimen. To avoid unnecessary animal killing, we present here an alternative method based on the monitoring of CYP1A expression in fish scales. Using beta-naphthoflavone (BNF; 50 mg/kg body wt, intraperitoneal injection) as inducer in goldfish (Carassius auratus), we monitored levels of CYP1A mRNA both in scales and liver of treated and control specimens. Treatment with BNF resulted in a similar induction of CYP1A gene in both tissues, although scales responded faster (at 8 h after treatment) than liver (between 24 and 48 h). The scale-based test has the unique advantage of allowing sequential testing in the same specimen, which facilitates analysis of the time course of CYP1A induction and allows the study of individual variability. The method implies minimal suffering of the animals, because it only requires removal of a moderate (n = 1-3) number of scales for each time point. This nondestructive, fast, and relatively inexpensive test for toxic exposure therefore is suitable for environmental monitoring and food safety control programs in which specimen preservation is required.

Transcriptomic seasonal variations in a natural population of zebra mussel (Dreissena polymorpha).

The zebra mussel Dreissena polymorpha is a Caspian Sea bivalve that colonized freshwater bodies worldwide during the XX century. To analyze the impact of seasonal and environmental variations on the physiology and metabolism of this invasive species, we developed a custom microarray using 4057 publicly available DNA sequences from Dreissena and other related genera. Transcriptome profiles were analyzed using half-body samples from a relatively clean site (Riba-Roja, low Ebro River, N.E. Spain), at three different stages of the annual cycle: Pre-spawning (February), spawning (June), and gonad resorption (September). Transcripts from a total of 745 unique sequences showed significant changes among these three groups of samples. Functional characterization of these transcripts based on their closest known homologues showed that genes involved in stress defense (oxidative and infection) were overrepresented in September, whereas genes related to reproductive functions were overrepresented in the spawning and pre-spawning periods. This transcriptomic information can help to identify developmental stages at which the organism is more vulnerable for future control strategies. These data will also contribute to the implementation of gene expression-based assays for pollution monitoring in water bodies harboring stable zebra mussel populations.

Genetic and phenoptypic differentiation of zebra mussel populations colonizing Spanish river basins

Zebra mussel populations in Ebro and Mijares Rivers (northern Spain) were analyzed to study the mechanisms by which this aquatic species deals with pollution. Variability analyses of mitochondrial cytochrome oxidase I gene and of one nuclear microsatellite were performed for ten populations from the Ebro River and one from the Mijares River. Comparison of these results with those from five additional European populations indicated that the Spanish populations constitute a homogeneous gene pool. Transcriptome analyses of gill samples from a subset of the Spanish populations showed changes on expression levels that correlated with variations in general fitness and loads of heavy metals. The less polluted upstream Ebro populations showed overexpression of mitochondrial and cell proliferation-related genes compared to the more polluted, downstream Ebro populations. Our data indicate that heavy metals were the main factors explaining these transcriptomic patterns, and that zebra mussel is resilient to pollutants (like mercury and organochlorine compounds) proved to be extremely toxic to vertebrates. We propose that zebra mussel populations sharing a common gene pool may acclimate to different levels and forms of pollution through modulations in their transcriptomic profile, although direct selection on genes showing differential expression patterns cannot be ruled out.

qRT-PCR evaluation of the transcriptional response of zebra mussel to heavy metals

BackgroundThe transcriptional response of adult zebra mussels (Dreissena polymorpha) to heavy metals (mercury, copper, and cadmium) was analyzed by quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) to study the coordinated regulation of different metal-, oxidative stress- and xenobiotic defence-related genes in gills and digestive gland. Regulatory network analyses allowed the comparison of this response between different species and taxa.ResultsChemometric analyses allowed identifying the effects of these metals clearly separating control and treated samples of both tissues. Interactions between the different genes, either in the same or between both tissues, were analysed to identify correlations and to propose stress-related genes’ regulatory networks. These networks were finally compared with existing data from human, mouse, zebrafish, Drosophila and the roundworm to evaluate their mechanistically-known response to metals (and to stressors in general) with the correlations observed in the still poorly-known, invasive zebra mussel.ConclusionsOur analyses found a general conservation of regulation genes and of their interactions among the different considered species, and may serve as a guide to extrapolate regulatory data from model species to lesser-known environmentally (or medically) relevant species.

Structural and molecular analysis of pollution-linked deformities in a natural Aphanius fasciatus (Valenciennes, 1821) population from the Tunisian coast

The Eastern coast of Tunisia hosts a mosaic of populations of the Mediterranean killifish A. fasciatus, an excellent monitoring species for the analysis of environmental impact in coastal Mediterranean areas. Among these populations, fish from the Sfax area, in the Gulf of Gabès, are known for presenting a high prevalence of different physiological alterations, including spinal deformation. Compared to a reference clean area, Luza, Sfax water and sediments are polluted with heavy metals (20 to 40 times more Cd, for example) and other compounds like PAHs (10 times higher levels). Histological analyses of deformed fish from this area showed an abnormal abundance of collagen fibers infiltrated in the spinal area, either as a physiological mechanism to alleviate the consequences of the deformation of the bones or as a secondary effect of the same pathological/toxicity mechanism inducing bone deformation. Deformed fish from Sfax showed elevated (4-5 fold) levels of transcripts from the collagen COL1A2 gene in bone tissue, compared to either non-deformed animals from the same location or normal animals from the reference Luza site. These results are consistent with an overexpression of collagen in the bone tissue, in agreement with the histological results. We propose that the deformation observed in the Sfax Aphanius population is a degenerative process occurring in adult animals and probably terminal, as it results in severe limitations on the fish capacity to swim, to catch food, and ultimately to survive.