Plastic and genomic change of a newly established lizard population following a founder event.

Understanding how phenotypic divergence arises among natural populations remains one of the major goals in evolutionary biology. As part of competitive exclusion experiment conducted in 1971, 10 individuals of Italian wall lizard (Podarcis siculus (Rafinesque-Schmaltz, 1810)) were transplanted from Pod Kopiste Island to the nearby island of Pod Mrcaru (Adriatic Sea). Merely 35 years after the introduction, the newly established population on Pod Mrcaru Island had shifted their diet from predominantly insectivorous towards omnivorous and changed significantly in a range of morphological, behavioural, physiological and ecological characteristics. Here, we combine genomic and quantitative genetic approaches to determine the relative roles of genetic adaptation and phenotypic plasticity in driving this rapid phenotypic shift. Our results show genome-wide genetic differentiation between ancestral and transplanted population, with weak genetic erosion on Pod Mrcaru Island. Adaptive processes following the founder event are indicated by highly differentiated genomic loci associating with ecologically relevant phenotypic traits, and/or having a putatively adaptive role across multiple lizard populations. Diverged traits related to head size and shape or bite force showed moderate heritability in a crossing experiment, but between-population differences in these traits did not persist in a common garden environment. Our results confirm the existence of sufficient additive genetic variance for traits to evolve under selection while also demonstrating that phenotypic plasticity and/or genotype by environment interactions are the main drivers of population differentiation at this early evolutionary stage.

Marine Pollutant Tributyltin Affects DNA Methylation and Fitness of Banded Murex (Hexaplex trunculus) Populations.

Banded murex, Hexaplex trunculus, is a marine gastropod whose reproductive fitness can be severely affected by very low concentrations of antifouling compound tributyltin (TBT). TBT has strong xenoandrogen impacts on snails, causing the development of imposex (e.g., the superimposition of male sexual characteristic in females), thereby affecting the fitness of entire populations. TBT is also known as a DNA-demethylating agent and an obesogenic factor. The aim of this study was to unravel the interactions between TBT bioaccumulation, phenotypic responses, and epigenetic and genetic endpoints in native populations of H. trunculus. Seven populations inhabiting environments along the pollution gradient were sampled in the coastal eastern Adriatic. These included sites of intense marine traffic and boat maintenance activity and sites with low anthropogenic impact. Populations inhabiting intermediately and highly polluted sites exhibited higher TBT burdens, higher incidences of imposex, and higher wet masses of snails than populations in lowly polluted sites. Other morphometric traits and cellular biomarker responses did not show clear differentiation among populations in relation to marine traffic/pollution intensity. An analysis of methylation sensitive amplification polymorphism (MSAP) revealed environmentally driven population differentiation and higher epigenetics than genetic within-population diversity. Moreover, decreases in genome-wide DNA methylation coincided with the imposex level and snail mass, suggesting an epigenetic background of the animal phenotypic response.

Relationships between dietary breadth and flexibility in jaw movement: A case study of two recently diverged insular populations of Podarcis lizards.

The kinematics of lizard feeding are the result of complex interactions between the craniocervical, the hyolingual, and the locomotor systems. The coordinated movement of these elements is driven by sensory feedback from the tongue and jaws during intraoral transport. The kinematics of jaw movements have been suggested to be correlated with the functional characteristics of the prey consumed, such as prey mobility and hardness. However, whether and how dietary breadth correlates with the flexibility in the behavioral response has rarely been tested, especially at the intraspecific level. Here we tested whether an increase in dietary breadth was associated with a greater behavioral flexibility by comparing two recently diverged populations of insular Podarcis lizards differing in dietary breadth. To do so, we used a stereoscopic high-speed camera set-up to analyze the jaw kinematics while offering them different prey types. Our results show that prey type impacts kinematics, especially maximum gape, and maximum opening and closing speed. Furthermore, the behavioral flexibility was greater in the population with the greater dietary breadth, suggesting that populations which naturally encounter and feed on more diverse prey items show a greater ability to modulate their movements to deal with variation in functionally relevant prey properties. Finally, the more generalist population showed more stereotyped movements suggesting a finer motor control.

From micro to macroevolution: drivers of shape variation in an island radiation of Podarcis lizards.

Phenotypictraits have been shown to evolve in response to variation in the environment. However, the evolutionary processes underlying the emergence of phenotypic diversity can typically only be understood at the population level. Consequently, how subtle phenotypic differences at the intraspecific level can give rise to larger-scale changes in performance and ecology remains poorly understood. We here tested for the covariation between ecology, bite force, jaw muscle architecture, and the three-dimensional shape of the cranium and mandible in 16 insular populations of the lizards Podarcis melisellensis and P. sicula. We then compared the patterns observed at the among-population level with those observed at the interspecific level. We found that three-dimensional head shape as well as jaw musculature evolve similarly under similar ecological circumstances. Depending on the type of food consumed or on the level of sexual competition, different muscle groups were more developed and appeared to underlie changes in cranium and mandible shape. Our findings show that the local selective regimes are primary drivers of phenotypic variation resulting in predictable patterns of form and function. Moreover, intraspecific patterns of variation were generally consistent with those at the interspecific level, suggesting that microevolutionary variation may translate into macroevolutionary patterns of ecomorphological diversity.

Gold and silver nanoparticles effects to the earthworm Eisenia fetida - the importance of tissue over soil concentrations.

To address and to compare the respective impact of gold and silver nanoparticles (Au and Ag NPs) in soil invertebrate, the earthworm Eisenia fetida was exposed to soil containing 2, 10, and 50 mg/kg of Au and Ag in both nanoparticulate and ionic forms for 10 days. Both metal NPs were 2-15 times less bioavailable than their ionic forms, and displayed similar transfer coefficients from soil to earthworm tissues. Both metal NPs triggered the onset of an oxidative stress as illustrated by increased glutathione S-transferase levels, decreased catalase levels, and increased malondialdehyde concentrations. Protein carbonylation distinguished the nanoparticular from the ionic forms as its increase was observed only after exposure to the highest concentration of both metal NPs. Au and Ag NPs triggered DNA modifications even at the lowest concentration, and both repressed the expression of genes involved in the general defense and stress response at high concentrations as did their ionic counterparts. Despite the fact that both metal NPs were less bioavailable than their ionic forms, at equivalent concentrations accumulated within earthworms tissues they exerted equal or higher toxic potential than their ionic counterparts.Capsule: At equivalent concentrations accumulated within earthworm tissues Au and Ag NPs exert equal or higher toxic potential than their ionic forms.

Diet variability among insular populations of Podarcis lizards reveals diverse strategies to face resource-limited environments.

Access to resources is a dynamic and multicausal process that determines the success and survival of a population. It is therefore often challenging to disentangle the factors affecting ecological traits like diet. Insular habitats provide a good opportunity to study how variation in diet originates, in particular in populations of mesopredators such as lizards. Indeed, high levels of population density associated with low food abundance and low predation are selection pressures typically observed on islands. In the present study, the diet of eighteen insular populations of two closely related species of lacertid lizards (Podarcis sicula and Podarcis melisellensis) was assessed. Our results reveal that despite dietary variability among populations, diet taxonomic diversity is not impacted by island area. In contrast, however, diet disparity metrics, based on the variability in the physical (hardness) and behavioral (evasiveness) properties of ingested food items, are correlated with island size. These findings suggest that an increase in intraspecific competition for access to resources may induce shifts in functional components of the diet. Additionally, the two species differed in the relation between diet disparity and island area suggesting that different strategies exist to deal with low food abundance in these two species. Finally, sexual dimorphism in diet and head dimensions is not greater on smaller islands, in contrast to our predictions.

Biomarker response of Mediterranean mussels Mytilus galloprovincialis regarding environmental conditions, pollution impact and seasonal effects.

The complexity of seasonally and spatially variable environments, coupled with complex biological interactions, makes it difficult to pinpoint biological responses to specific environmental stressors, including chemical pollution. To disentangle causative factors and reveal biomarker responses, we applied biomarker-based multivariate approaches to 15 native populations of Mediterranean mussel Mytilus galloprovincialis in spring and autumn. In addition, we used a subset of these populations in transplant experiments between clean and polluted environments in nature and in lab mesocosms. The extent of biomarker responses in native populations is affected by season, and significantly lower variability across seasons was observed among mussels from clean than from polluted sites. Results of paired block designed transplant experiment demonstrated both regional and pollution effect, with mussels uniformly exhibiting higher responses on more impacted sites in each of the Adriatic regions. Biomarker status of mussels varied among Adriatic regions in dependence on the set of environmental variables, and between clean and polluted sites in dependence on measured concentrations of metals in mussels' tissue. Results of the mesocosm experiment revealed distinctive biomarker responses of two populations of different origin when exposed to common conditions. Multivariate description of biomarker activity and application of specific experiments allowed us to link environmental condition, exposure to pollution and seasonality to mussels' biomarker responses.

Electromagnetic fields at a mobile phone frequency (900 MHz) trigger the onset of general stress response along with DNA modifications in Eisenia fetida earthworms.

Eisenia fetida earthworms were exposed to electromagnetic field (EMF) at a mobile phone frequency (900 MHz) and at field levels ranging from 10 to 120 V m-1 for a period of two hours (corresponding to specific absorption rates ranging from 0.13 to 9.33 mW kg-1). Potential effects of longer exposure (four hours), field modulation, and a recovery period of 24 h after two hours of exposure were addressed at the field level of 23 V m-1. All exposure treatments induced significant DNA modifications as assessed by a quantitative random amplified polymorphic DNA-PCR. Even after 24 h of recovery following a two hour-exposure, the number of probe hybridisation sites displayed a significant two-fold decrease as compared to untreated control earthworms, implying a loss of hybridisation sites and a persistent genotoxic effect of EMF. Expression of genes involved in the response to general stress (HSP70 encoding the 70 kDa heat shock protein, and MEKK1 involved in signal transduction), oxidative stress (CAT, encoding catalase), and chemical and immune defence (LYS, encoding lysenin, and MYD, encoding a myeloid differentiation factor) were up-regulated after exposure to 10 and modulated 23 V m-1 field levels. Western blots showing an increased quantity of HSP70 and MTCO1 proteins confirmed this stress response. HSP70 and LYS genes were up-regulated after 24 h of recovery following a two hour-exposure, meaning that the effect of EMF exposure lasted for hours.

Prospects and challenges for the conservation of farm animal genomic resources, 2015-2025.

Livestock conservation practice is changing rapidly in light of policy developments, climate change and diversifying market demands. The last decade has seen a step change in technology and analytical approaches available to define, manage and conserve Farm Animal Genomic Resources (FAnGR). However, these rapid changes pose challenges for FAnGR conservation in terms of technological continuity, analytical capacity and integrative methodologies needed to fully exploit new, multidimensional data. The final conference of the ESF Genomic Resources program aimed to address these interdisciplinary problems in an attempt to contribute to the agenda for research and policy development directions during the coming decade. By 2020, according to the Convention on Biodiversity's Aichi Target 13, signatories should ensure that "…the genetic diversity of …farmed and domesticated animals and of wild relatives …is maintained, and strategies have been developed and implemented for minimizing genetic erosion and safeguarding their genetic diversity." However, the real extent of genetic erosion is very difficult to measure using current data. Therefore, this challenging target demands better coverage, understanding and utilization of genomic and environmental data, the development of optimized ways to integrate these data with social and other sciences and policy analysis to enable more flexible, evidence-based models to underpin FAnGR conservation. At the conference, we attempted to identify the most important problems for effective livestock genomic resource conservation during the next decade. Twenty priority questions were identified that could be broadly categorized into challenges related to methodology, analytical approaches, data management and conservation. It should be acknowledged here that while the focus of our meeting was predominantly around genetics, genomics and animal science, many of the practical challenges facing conservation of genomic resources are societal in origin and are predicated on the value (e.g., socio-economic and cultural) of these resources to farmers, rural communities and society as a whole. The overall conclusion is that despite the fact that the livestock sector has been relatively well-organized in the application of genetic methodologies to date, there is still a large gap between the current state-of-the-art in the use of tools to characterize genomic resources and its application to many non-commercial and local breeds, hampering the consistent utilization of genetic and genomic data as indicators of genetic erosion and diversity. The livestock genomic sector therefore needs to make a concerted effort in the coming decade to enable to the democratization of the powerful tools that are now at its disposal, and to ensure that they are applied in the context of breed conservation as well as development.

Zebrafish genome instability after exposure to model genotoxicants.

Sublethal exposure to environmental genotoxicants may impact genome integrity in affected organisms. It is therefore necessary to develop tools to measure the extent and longevity of genotoxicant-induced DNA damage, and choose appropriate model organisms for biomonitoring. To this end, markers of DNA damage were measured in zebrafish larvae and adults following exposure to model genotoxicants (benzo[a]pyrene and ethyl methanesulfonate). Specifically, we assessed primary DNA damage and the existence of potentially persistent genomic alterations through application of the comet assay, quantitative random amplified polymorphic DNA (qRAPD) and amplified fragment length polymorphism (AFLP) assays. Furthermore, expression of genes involved in DNA repair, oxidative stress response and xenobiotic metabolism was evaluated as well. Additionally, the AFLP method was applied to adult specimens 1 year after larval exposure to the genotoxicants to evaluate the longevity of the observed DNA alterations. Large numbers of DNA alterations were detected in larval DNA using the comet assay, qRAPD and AFLP, demonstrating that zebrafish larvae are a sensitive model for revealing genotoxic effects. Furthermore, some of these genomic alterations persisted into adulthood, indicating the formation of stable genomic modifications. qRAPD and AFLP methods proved to be highly sensitive to genotoxic effects, even in cases when the comet assay indicated a lack of significant damage. These results thus support the use of zebrafish larvae as a sensitive model for monitoring the impact of genotoxic insult and give evidence of the longevity of genomic modifications induced by genotoxic agents.

Genomic and gene expression responses to genotoxic stress in PAC2 zebrafish embryonic cell line.

PAC2 cell line is, along most of the developed zebrafish cell lines, poorly characterized concerning its response to genotoxicants. To define the PAC2 cell line response to different forms of genotoxic stress, we exposed the cells to model genotoxic agents (benzo[a]pyrene, B[a]P, and ethyl methanesulfonate) and subsequently monitored DNA damage and alterations by using the battery of tests, including the Comet assay, quantitative random-amplified polymorphic DNA and amplified fragment length polymorphism. The expression of several DNA repair (xpc, xpd, hr23b, rad51, msh2) and oxidative stress response (sod (Cu/Zn)) genes was monitored as well. To obtain an indication of the PAC2 cell line metabolizing capacity, the expression of genes belonging to cyp1, cyp2 and cyp3 families was assessed upon exposure to B[a]P. Genotoxic responses were observed in all the used methods, and quantitative random-amplified polymorphic DNA and amplified fragment length polymorphism proved to be more sensitive by revealing DNA alterations even when the Comet assay indicated lack of significant damage. The PAC2 cell line demonstrated basal and B[a]P-induced expression of several cyp genes, suggesting its ability to metabolize indirect acting xenobiotics to a certain point. Based on these results, PAC2 cells seem to be sensitive zebrafish in vitro model in the genotoxicity assessment of the direct acting genotoxicant; however, they are less sensitive toward the indirect acting genotoxicant due to their limited metabolizing properties.

Effects of freshwater pollution on the genetics of zebra mussels (Dreissena polymorpha) at the molecular and population level.

Revealing long-term effects of contaminants on the genetic structure of organisms inhabiting polluted environments should encompass analyses at the population, molecular, and cellular level. Following this concept, we studied the genetic constitution of zebra mussel populations from a polluted (Dp) and reference sites (Cl) at the river Drava, Croatia, and applied microsatellite and DNA damage analyses (Comet assay, micronucleus test (MNT)). Additionally, mussels from both populations were exposed to polluted wastewater in the laboratory for three days, and DNA damage was analyzed to evaluate acclimatization and genetic adaptation of the investigated populations to the polluted environment. The two populations differed in their genetic constitution. Microsatellite analysis suggested that Dp had undergone a genetic bottleneck. Comet assay did not indicate any difference in DNA damage between the two populations, but MNT revealed that Dp had an increased percentage of micronuclei in hemocytes in comparison to Cl. The laboratory experiment revealed that Dp had a lower percentage of tail DNA and a higher percentage of micronuclei than Cl. These differences between populations were possibly caused by an overall decreased fitness of Dp due to genetic drift and by an enhanced DNA repair mechanism due to acclimatization to pollution in the source habitat.

Gene flow vs. pollution pressure: genetic diversity of Mytilus galloprovincialis in eastern Adriatic.

Environmental pollution may modify all the evolutionary processes involved in shaping the genetic patterns of exposed populations. In order to evaluate the pollution impact on the genetic diversity of Mediterranean mussel Mytilus galloprovincialis ten populations inhabiting differently polluted sites along the eastern Adriatic coast, from pristine bays to heavily trafficked harbours, were studied. Pollution pressure was assessed through an integrated study of biological effects and responses across different levels of biological organization. Eight microsatellite markers were analysed to assess genetic diversity of investigated populations. Both the principal component analysis (PCA) of the biomarker data set as well as the biomarker response index (BRI) confirmed substantial pollution pressure at the highly polluted sites, and very low pollution exposure at the three reference sites. Very shallow genetic differentiation was found in respect to maritime distances or pollution status, and this was attributed to a high gene flow among the populations. However, populations inhabiting polluted sites exhibited higher levels of genetic diversity and evolutionary mechanisms underlying this phenomenon are discussed.

What is Comet assay not telling us: AFLP reveals wider aspects of genotoxicity.

DNA damage detected by genotoxicity biomarkers such as the Comet assay is not always a reliable indicator of the consequences that genotoxic agents can have on the genome integrity of the exposed organisms. Therefore, to reveal the existence of more permanent alterations of DNA structure after genotoxic stress, the RTG-2 rainbow trout cell line was exposed for 3 days to benzo[a]pyrene (B[a]P, 0.1-10 µM) and ethyl methanesulfonate (EMS, 0.1-1mM) followed by 3 days of recovery period. Primary DNA damage was evaluated by the Comet assay and DNA alterations were assessed using AFLP (amplified fragment length polymorphism). Qualitative and quantitative modifications in AFLP profiles were analyzed in order to detect genetic alterations arising from mutation events and/or DNA damage. Significant induction in DNA damage measured by the Comet assay was noticed after B[a]P treatment at all concentrations but values returned to the control level after recovery. Exposure to EMS induced significant DNA damage only at the highest concentration and damage persisted after the recovery period. AFLP profiles detected DNA alterations even when Comet assay indicated complete DNA repair, revealing more persistent damage. Since such DNA damage can impair its structure and function, Comet assay results should preferably be supplemented with other methods in order to predict the consequences of genotoxic insult more accurately.

Oxidative and genotoxic effects of 900 MHz electromagnetic fields in the earthworm Eisenia fetida.

Accumulating evidence suggests that exposure to radiofrequency electromagnetic field (RF-EMF) can have various biological effects. In this study the oxidative and genotoxic effects were investigated in earthworms Eisenia fetida exposed in vivo to RF-EMF at the mobile phone frequency (900 MHz). Earthworms were exposed to the homogeneous RF-EMF at field levels of 10, 23, 41 and 120 V m(-1) for a period of 2h using a Gigahertz Transversal Electromagnetic (GTEM) cell. At the field level of 23 V m(-1) the effect of longer exposure (4h) and field modulation (80% AM 1 kHz sinusoidal) was investigated as well. All exposure treatments induced significant genotoxic effect in earthworms coelomocytes detected by the Comet assay, demonstrating DNA damaging capacity of 900 MHz electromagnetic radiation. Field modulation additionally increased the genotoxic effect. Moreover, our results indicated the induction of antioxidant stress response in terms of enhanced catalase and glutathione reductase activity as a result of the RF-EMF exposure, and demonstrated the generation of lipid and protein oxidative damage. Antioxidant responses and the potential of RF-EMF to induce damage to lipids, proteins and DNA differed depending on the field level applied, modulation of the field and duration of E. fetida exposure to 900 MHz electromagnetic radiation. Nature of detected DNA lesions and oxidative stress as the mechanism of action for the induction of DNA damage are discussed.

Genotoxicity monitoring of freshwater environments using caged crayfish (Astacus leptodactylus).

Genotoxicity of freshwater pollution was assessed by measuring DNA damage in haemocytes of caged freshwater crayfish Astacus leptodactylus by the means of Comet assay and micronucleus test, integrated with the measurements of physiological (total protein concentration) and immunological (total haemocyte count) haemolymph parameters as biomarkers of undergone stress. Crayfish were collected at the reference site (River Mreznica) and exposed in cages for 1 week at three polluted sites along the Sava River (Zagreb, Sisak, Krapje). The long term pollution status of these locations was confirmed by chemical analyses of sediments. Statistically significant increase in DNA damage measured by the Comet assay was observed at all three polluted sites comparing to the crayfish from reference site. In addition, native crayfish from the mildly polluted site (Krapje) cage-exposed on another polluted site (Zagreb) showed lower DNA damage than crayfish from the reference site exposed at the same location indicating adaptation and acclimatisation of crayfish to lower levels of pollution. Micronuclei induction showed similar gradient of DNA damage as Comet assay, but did not reach the statistical significance. Observed increase in total haemocyte count and total protein content in crayfish from polluted environments in the Sava River also confirmed stress caused by exposure to pollution. The results of this study have proved the applicability of caging exposure of freshwater crayfish A. leptodactylus in environmental genotoxicity monitoring using Comet assay and micronucleus test.

Distribution of mono-, di- and trisialo gangliosides in the brain of Actinopterygian fishes.

BACKGROUND: Mono-, di- and trisialo gangliosides are major glycosphingolipids in the brain of higher vertebrates involved in lipid raft assembly. In contrast, the fish brain is abundant in polisialo-gangliosides, whose function is implicated in the modulation of repulsive and attractive intercellular interactions during embryonic development and a temperature adaptation process. The histological distribution of gangliosides is usually studied in rodent and mammalian brains, but to date it has not been described in the case of fish brain. METHODS: Gangliosides were extracted from adult brains of trout, carp and zebrafish and separated by TLC. High-affinity anti-ganglioside (GM1, GD1a, GD1b, GT1b) IgG antibodies were used for immunohistochemistry. RESULTS: In trout and carp brains GM1 and GT1b are expressed in the same neuronal cell bodies from the telencephalon to the spinal cord. In zebrafish brain GM1 was not detected, whereas GT1b is a general neuropil staining. GD1a is specific for unmyelinated parallel fibers in carp and zebrafish brains as well as parallel fibers in the molecular layer of all cerebellar divisions. In trout brain GD1b is found in parallel fibers of the cerebellum, but not in the tectum mesencephali. GD1b is expressed in zebrafish neuronal cell bodies. CONCLUSIONS: Each studied species has a different expression of complex gangliosides. GT1b is widely present, whereas GD1a and GD1b appear in a specific group of unmyelinated fibers and could be used as their specific marker. GENERAL SIGNIFICANCE: This is the first report on mono-, di- and trisialo ganglioside (GM1, GD1a, GD1b and GT1b) distribution in the brain of adult Actinopterygian fishes. This article is part of a Special Issue entitled Glycoproteomics.

Aporrectodea caliginosa, a suitable earthworm species for field based genotoxicity assessment?

There is a growing interest for the application of biomakers to field-collected earthworms. Therefore we have evaluated the usability of native populations of endogeic, widely distributed earthworm Aporrectodea caliginosa in the assessment of soil genotoxicity using the Comet assay. Validation of the Comet assay on earthworm coelomocytes has been established using commercially available Eisenia fetida exposed to copper, cadmium, and pentachlorophenol, along with A. caliginosa exposed to copper in a filter paper contact test. Neutral red retention time (NRRT) assay was conducted on copper exposed and field-collected earthworms. Significant DNA and lysosomal damage was measured using Comet and NRRT assays in native populations of A. caliginosa sampled from the polluted soils in the urban area in comparison to the earthworms from the reference site. The results of this study confirm the employment of A. caliginosa as a suitable species for the in situ soil toxicity and genotoxicity field surveys.

Genotoxicity of marine sediments in the fish hepatoma cell line PLHC-1 as assessed by the Comet assay.

The main goal of this study was to test the usefulness of the Comet assay in the PLHC-1 hepatoma fish cell line as a tool for detecting the presence of genotoxic compounds in contaminated marine sediments. The system has been tested using both model chemicals (benzo[a]pyrene (B[a]P) and ethyl methanesulfonate (EMS)) and extracts of sediment samples obtained with solvent dichloromethane/methanol. For all of the analysed sediment extracts as well as for the model chemicals a concentration dependent genotoxic effect was observed. The sediment with the highest observed genotoxic potential was additionally extracted using various solvents in order to test which class of compounds, according to their polarity, is most responsible for the observed genotoxic effect. Non-polar solvents (cyclohexane and dichloromethane) yielded stronger genotoxic effect but the highest level of DNA damage was determined after exposure to sediment extract obtained with the solvent mixture dichloromethane/methanol which extracts a wide range of contaminants. Our results indicate that the PLHC-1 cell line is a suitable in vitro model in sediment genotoxicity assessment and encourage the use of fish cell lines as versatile tools in ecogenotoxicology.

DNA integrity of chub erythrocytes (Squalius cephalus L.) as an indicator of pollution-related genotoxicity in the River Sava.

An alkaline comet assay and a micronucleus test were carried out on erythrocytes of the European chub, Squalius cephalus L., collected in spring and autumn in 2005 and 2006 at three sampling sites in River Sava, near Zagreb, Croatia. The results of comet assay showed the lowest genotoxic influence at the least polluted site, while higher DNA damage was observed at the polluted sites. Although the basal levels of DNA damage were elevated, a clear gradation of DNA damage was found due to pollution intensity in all sampling periods. The lowest cytogenetic damage as revealed by the micronucleus test (MNT) was observed as well at the least polluted site. High variations in MN frequency were observed between sampling periods, although the number of micronucleated erythrocytes was consistently the highest one at the polluted site. The comet assay as a biomarker of genotoxic effect exhibited higher sensitivity in discriminating the genotoxic capacity of studied polluted sites while the MNT was less sensitive. However, both tests should be used together in biomonitoring studies because they can reveal different aspects of DNA damage; comet assay, the early event of genotoxic exposure, and MNT, its final result as a mutagenic potential.