Adaptive differences in gene expression in European flounder (Platichthys flesus)

Population structure was previously believed to be very limited or absent in classical marine fishes, but recently, evidence of weakly differentiated local populations has been accumulating using noncoding microsatellite markers. However, the evolutionary significance of such minute genetic differences remains unknown. Therefore, in order to elucidate the relationship between genetic markers and adaptive divergence among populations of marine fishes, we combined cDNA microarray and microsatellite analysis in European flounders (Platichthys flesus). We demonstrate that despite extremely low levels of neutral genetic divergence, a high number of genes were significantly differentially expressed between North Sea and Baltic Sea flounders maintained in a long-term reciprocal transplantation experiment mimicking natural salinities. Several of the differentially regulated genes could be directly linked to fitness traits. These findings demonstrate that flounders, despite little neutral genetic divergence between populations, are differently adapted to local environmental conditions and imply that adaptation in gene expression could be common in other marine organisms with similar low levels of population subdivision.     

Evidence of p53 mutation in an early stage of liver cancer in European flounder, Platichthys flesus (L.)

A number of flounders dwelling in highly contaminated coastal areas of Northern Europe develop liver tumours. In order to increase our understanding of the molecular pathogenesis of these sporadic tumours, we examined p53 mutations in eleven hyperplasia and six adenoma. p53 introns 4 to 8 were first sequenced to allow individual amplification of exons 5 to 8. DNA extracted from formalin-fixed livers was amplified and PCR products were directly sequenced. Two major results were obtained. (i) Flounders from different geographical areas displayed a high rate of sequence variation. Base substitutions were identified in both tumour and normal tissues and thus may be considered as polymorphic variations in individuals. (ii) One mutation was detected in two hyperplastic foci from the same flounder. This mutation was a T:A to A:T transversion at codon 147, resulting in the replacement of valine for glutamic acid. This residue took place in the L2 loop of the DNA binding surface. Its substitution by an hydrophilic and charged residue could thus impair p53 (protein) biological activity.     

DNA adduct analysis and histopathological biomarkers in European flounder (Platichthys flesus) sampled from UK estuaries

The presence of genotoxic and potentially carcinogenic chemical contaminants in the estuarine and coastal marine environment is well documented. In this study, European flounder (Platichthys flesus) sampled from eight UK estuaries were analysed for hepatic DNA adducts, using the 32P-postlabelling assay and liver histopathology as part of an on going survey to establish the health status of UK estuaries. Fish were collected from the estuaries Tyne, Mersey, Thames, Alde (reference site), Belfast, Forth, Clyde and Southampton. At the majority of contaminated sites (Southampton, Thames, Clyde, Tyne and Mersey) the predominant DNA adduct profile consisted of diagonal radioactive zones (DRZs). In contrast, flounder collected from the Forth, Alde and Belfast lacked DRZs with only background levels of DNA damage being observed. Statistically significant differences were observed between several of the sites sampled with the hepatic DNA adduct levels detected in flounder from Southampton, Thames and Clyde statistically elevated (P < 0.05) over those levels detected at the Tyne (Southampton and Thames only), Forth, Alde and Belfast. Histological analysis of these samples revealed a range of lesions including foci of cellular alteration, hepatocellular fibrillar inclusions, nuclear pleomorphisms along with non-toxicopathic changes/alterations, such as those associated with cell turnover (apoptosis, necrosis, regeneration) and immune-related functions (melanomacrophage aggregates, inflammation). Although it is difficult to associate higher prevalence of these lesion types with specific sites, generally, the lowest prevalence was seen in flounder captured from the Alde estuary, with higher prevalence (particularly of melanomacrophage aggregates, inflammation and necrotic foci) seen in fish from the contaminated sites.     

Physiological response in the European flounder (Platichthys flesus) to variable salinity and oxygen conditions

Physiological mechanisms involved in acclimation to variable salinity and oxygen levels and their interaction were studied in European flounder. The fish were acclimated for 2 weeks to freshwater (1 per thousand salinity), brackish water (11 per thousand) or full strength seawater (35 per thousand) under normoxic conditions (water Po(2) = 158 mmHg) and then subjected to 48 h of continued normoxia or hypoxia at a level (Po(2) = 54 mmHg) close to but above the critical Po(2). Plasma osmolality, [Na(+)] and [Cl(-)] increased with increasing salinity, but the rises were limited, reflecting an effective extracellular osmoregulation. Muscle water content was the same at all three salinities, indicating complete cell volume regulation. Gill Na(+)/K(+)-ATPase activity did not change with salinity, but hypoxia caused a 25% decrease in branchial Na(+)/K(+)-ATPase activity at all three salinities. Furthermore, hypoxia induced a significant decrease in mRNA levels of the Na(+)/K(+)-ATPase alpha1-subunit, signifying a reduced expression of the transporter gene. The reduced ATPase activity did not influence extracellular ionic concentrations. Blood [Hb] was stable with salinity, and it was not increased by hypoxia. Instead, hypoxia decreased the erythrocytic nucleoside triphosphate content, a common mechanism for increasing blood O(2) affinity. It is concluded that moderate hypoxia induced an energy saving decrease in branchial Na(+)/K(+)-ATPase activity, which did not compromise extracellular osmoregulation.     

Characterization of microsatellite markers derived from sequence databases for the European flounder (Platichthys flesus)

The European flounder (Platichthys flesus; Pleuronectidae) is an important commercial fish species. Nevertheless, no microsatellites for this species have been published to date. The sequence of flounder genes present in the public database was searched for short tandem repeats. Out of 12 microsatellite pairs derived from Sequenced Databases, five amplified successfully with an expected heterozygosity ranging from 0.43 to 0.75. The average allele number of these microsatellites was 4.8 per locus with a range between two and nine.     

Adaptive divergence in a high gene flow environment: Hsc70 variation in the European flounder (Platichthys flesus L.)

Little is known about local adaptations in marine fishes since population genetic surveys in these species have typically not applied genetic markers subject to selection. In this study, we used a candidate gene approach to investigate adaptive population divergence in the European flounder (Platichthys flesus L.) throughout the northeastern Atlantic. We contrasted patterns of genetic variation in a presumably neutral microsatellite baseline to patterns from a heat-shock cognate protein gene, Hsc70. Using two different neutrality tests we found that the microsatellite data set most likely represented a neutral baseline. In contrast, Hsc70 strongly deviated from neutral expectations. Importantly, when estimating standardized levels of population divergence (F(ST)'), we also found a large discrepancy in the patterns of structuring in the two data sets. Thus, samples grouped according to geographical or historical proximity with regards to microsatellites, but according to environmental similarities with regards to Hsc70. The differences between the data sets were particularly pronounced in pairwise comparisons involving populations in the western and central Baltic Sea. For instance, the genetic differentiation between geographically close Baltic Sea and North Sea populations was found to be 0.02 and 0.45 for microsatellites and Hsc70 respectively. Our results strongly suggest adaptive population divergence and indicate local adaptations at the DNA level in a background of high levels of gene flow, typically found in many marine fish species. Furthermore, this study highlights the usefulness of the candidate gene approach for demonstrating local selection in non-model organisms such as most marine fishes.     

Interaction of benzo[a]pyrene, 2,3,3′,4,4′,5 hexachlorobiphenyl (PCB 156) and cadmium on biomarker responses in flounder (Platichthys flesus L.)

Interactive effects of a mixed pollutant exposure on biomarker responses were studied in European flounder (Platichthys flesus L.). The model chemicals, benzo[a]pyrene (BaP, 2.5 mg kg^-1), 2,3,3′,4,4′5 hexachlorobiphenyl (PCB-156, 2.5 mg kg^-1), and cadmium (cadmium, 1 mg kg^-1), were administered to fish by subcutaneous injections. Biomarker responses were quantified both following administration of single chemicals and sequential combinations of the chemicals in pairs. Significant induction of CYP1A protein levels and corresponding ethoxyresorufin-O-deethylase (EROD) activities was observed in BaP and PCB treated flounder after 2 and 8 days, respectively. The strongest induction (44 fold) was caused by BaP. No further induction was observed after additional treatment with PCB 156. CYP1A induction caused by BaP was inhibited (40% compared with BaP treatment alone) in flounder pre treated with cadmium, whereas induction by PCB 156 appeared to be unaffected by pre treatment with cadmium. Flounder treated with cadmium only had significantly elevated hepatic levels of metallothionein (MT) after 15 days. Pre treatment with BaP and PCB prior to cadmium inhibited the MT induction (30-50%) compared with cadmium alone. Furthermore, significantly higher glutathione S transferase activities were observed in flounder administered cadmium alone, and in flounder treated with BaP or PCB 156 prior to cadmium. GST selenium independent peroxidase activities appeared to be unaffected by any of the treatments in the present study. The results indicate that chemical mixtures may affect biomarker responses differently from compounds administered alone, and that the sensitivity of both CYP1A and MT are influenced by pollutants other than their primary inducers.     

Excretion of benzo[a]pyrene-Gua adduct in the urine of benzo[a]pyrene-treated rats

A benzo[a]pyrene(BP)-Gua adduct was extracted in the urine of rats treated with BP. Some (0.15%) of the administered dose of BP was excreted as BP-Gua within 48 h. A double labelling experiment demonstrated that the excreted product contained both a BP and a Gua moiety. Partially hepatectomized rats treated with [14C]Gua during the regenerative phase were injected with [3H]BP and the urine collected and processed by chromatographic procedures. The adduct had similar chromatographic properties to the adduct released from human PLC/5 cells treated with 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) and co-chromatographed with 7-BPDE-Gua released from BPDE-adducted DNA under aqueous conditions. Detection and quantitation of BP-Gua offers an alternative, non-invasive method of monitoring individuals exposed to carcinogenic polycyclic aromatic hydrocarbons (PAHs).     

Binding and movement of silver in the intestinal epithelium of a marine teleost fish,the European flounder (Platichthys flesus)

The intestine has been indicated as a site of waterborne silver toxicity in marine fish and chronic effects at the intestine have been observed at concentrations far below acutely toxic level. Thus, models of silver toxicity to marine fish need to consider the intestine as a biotic ligand. The present study characterises binding of silver to the intestine of the European flounder (Platichthys flesus). Everted intestinal sacks were prepared and submersed in a solution mimicking the intestinal fluid of the fish at the acclimation salinity (21 per thousand). Silver was added as (110m)AgNO(3) or (110m)AgNO(3)/AgNO(3) mixtures at concentrations ranging from 1.6 to 950 nM total silver. Appearance of (110m)Ag was analysed in mucosal scrapings, muscle layers, and in the plasma saline on the serosal side of the intestine. The latter represented uptake into blood and other extra-intestinal compartments. Mucosal scrapings consisted of both epithelial cells and mucus and, thus contained adsorbed as well as absorbed silver. Most of the silver in mucosal scrapings was bound to mucus. There was no difference in silver binding between the anterior, mid, and posterior regions of the intestine. Concentration-dependency of silver binding was sigmoidal and saturated above 100 nM total silver. The saturable appearance of (110m)Ag in the plasma saline suggest that silver passage across the intestine is transcellular and carrier mediated. Mucus likely influences uptake of silver by altering its speciation from that in the lumen and by serving as physical barrier for silver binding to the brushborder membrane. A biotic ligand model for marine fish to silver may have to consider these interactions.     

High incidence of otolith abnormality in juvenile European flounder Platichthys flesus from a tidal freshwater area

Otolith abnormality has been reported for a wide range of freshwater and marine fish species. In this study, the sagittal otolith morphology and mineralogy of juvenile European flounder, Platichthys flesus, were examined to describe the incidence and types of structural deformities in this species. Juvenile flounder were collected over the years 2013–2015 in the tidal freshwater section of an estuarine nursery (Minho estuary, NW Portugal). Otolith abnormality was found in 43% of the individuals and, in most cases, occurred in both otoliths. Despite an abnormal mineralogy confirmed by scanning electron microscopy, the morphometry and the mineral polymorph composition of abnormal otoliths did not differ from normal ones (i.e. aragonite). This contrasts with other studies where vaterite and/or calcite were found to replace aragonite in abnormal otoliths. Further studies are needed to elucidate whether abrupt salinity changes associated with habitat transitions may have played a role in the abnormal otolith biomineralization in this species.     

The detection of biomarkers of genotoxin exposure in the European flounder (Platichthys flesus) collected from the River Tyne Estuary

The Tyne Estuary (North East England) is known to contain elevated levels of polycyclic aromatic hydrocarbons (PAH), compared with other less industrialised English waterways. Previous studies suggest that such contamination is responsible for the toxicity detected in invertebrate bioassays conducted on water and sediment samples collected from the Tyne. Here we present data from a biomonitoring study using hepatic DNA adducts (32P-postlabelling assay) and bile metabolites (synchronous fluorescence spectrometry) to investigate genotoxic exposure in flounder (Platichthys flesus) collected from three sites (Scotswood, Newcastle and Redheugh) along the Tyne Estuary. Flounder were also collected from a clean reference site, the Alde Estuary. Levels of bile metabolites (microgram kg-1 wet weight 1-OH pyrene equivalents) were elevated in flounder caught from the Tyne (Scotswood = 22,247 +/- 3408; Newcastle = 14,572 +/- 1888; Redheugh = 21,872 +/- 2935) compared with those collected from the Alde (632 +/- 56). The levels of DNA adducts (adducted nucleotides per 10(8) normal nucleotides) were also elevated in Tyne flounder (Scotswood = 24.6 +/- 3.2; Newcastle = 34.4 +/- 3.7; Redheugh = 27.6 +/- 6.3) compared with fish collected from the Alde (10.1 +/- 4.8), suggesting that a proportion of the bioavailable PAH was being converted into genotoxic metabolites. All DNA adduct profiles in flounder collected from the Tyne consisted of diagonal radioactive zones of radiolabelled adducts, which were not present in fish sampled from the Alde. The in vivo dosing of flounder with benzo[a]pyrene (BaP) to produced DNA adducts in similar chromatographic positions to the diagonal radioactive zones in the Tyne caught flounder are also described.     

Interaction of benzo[a]pyrene, 2,3,3',4,4',5 hexachlorobiphenyl (PCB 156) and cadmium on biomarker responses in flounder (Platichthys flesus L.)

Interactive effects of a mixed pollutant exposure on biomarker responses were studied in European flounder (Platichthys flesus L.). The model chemicals, benzo[a]pyrene (BaP, 2.5 mg kg^-1), 2,3,3',4,4'5 hexachlorobiphenyl (PCB-156, 2.5 mg kg^-1), and cadmium (cadmium, 1 mg kg^-1), were administered to fish by subcutaneous injections. Biomarker responses were quantified both following administration of single chemicals and sequential combinations of the chemicals in pairs. Significant induction of CYP1A protein levels and corresponding ethoxyresorufin-O-deethylase (EROD) activities was observed in BaP and PCB treated flounder after 2 and 8 days, respectively. The strongest induction (44 fold) was caused by BaP. No further induction was observed after additional treatment with PCB 156. CYP1A induction caused by BaP was inhibited (40% compared with BaP treatment alone) in flounder pre treated with cadmium, whereas induction by PCB 156 appeared to be unaffected by pre treatment with cadmium. Flounder treated with cadmium only had significantly elevated hepatic levels of metallothionein (MT) after 15 days. Pre treatment with BaP and PCB prior to cadmium inhibited the MT induction (30-50%) compared with cadmium alone. Furthermore, significantly higher glutathione S transferase activities were observed in flounder administered cadmium alone, and in flounder treated with BaP or PCB 156 prior to cadmium. GST selenium independent peroxidase activities appeared to be unaffected by any of the treatments in the present study. The results indicate that chemical mixtures may affect biomarker responses differently from compounds administered alone, and that the sensitivity of both CYP1A and MT are influenced by pollutants other than their primary inducers.     

Survey of the metazoan ectoparasites of the European flounder Platichthys flesus (Linnaeus, 1758) along the north-central Portuguese coast

A survey was undertaken to identify metazoan ectoparasite species on the European flounder, Platichthys flesus (Linnaeus, 1758), in 4 different locations off the north-central Portuguese coast. Parasites of 7 different taxa were found: Caligus diaphanus, Caligus sp., and Lepeophtheirus pectoralis (Copepoda: Caligidae); Acanthochondria cornuta (Copepoda: Chondracanthidae); Holobomolochus confusus (Copepoda: Bomolochidae); Nerocila orbignyi (Isopoda: Cymotholdae); and praniza larvae (Isopoda: Gnathiidae). Lernaeocera branchialis, a common European flounder parasite in the North and Baltic Seas, was not observed among the surveyed fish. Caligus diaphanus, Caligus sp., and Nerocila orbignyi are new host records. The high prevalence and intensity values recorded for L. pectoralis and A. cornuta suggest that both parasite species are common to the European flounder along the north-central Portuguese coast. In contrast, infection levels with respect to the other parasite taxa were, in most cases, comparatively lower, thereby indicating that they only occur occasionally among flounders in the surveyed area.     

Isolation and characterization of 28 new microsatellite markers for European flounder (Platichthys flesus L.)

The European flounder (Platichthys flesus L.) is used in ecotoxicological studies to provide detailed information on the effects of pollution on individual fish. Data on population and evolutionary level effects are, however, limited. Here, the isolation and characterization of 28 novel species specific microsatellite loci are presented. The number of alleles ranged from 8 to 38, and observed heterozygosity from 0.542 to 1.     

Cell volume regulation in isolated,perfused heart ventricle of the flounder (Platichthys flesus)

• 1.1. The ventricle cells of the flounder (Platichthys flesus) maintained the cell volume regulation mechanism in vitro, when isolated hearts were submitted to a hyposmotic solution.
• 2.2. The initial osmotic swelling of the cells was followed by a secondary shrinking. A new steady state volume (water content) was established in the course of 6 hr. The water content was 3% above control.
• 3.3. The cellular amount of K⁺ and taurine decreased concomitantly with the decline in cellular water.
• 4.4. The decrement in cellular taurine was explained by leakage of taurine, per se, into the perfusion media.

     

Influence of temperature on clove oil anaesthesia in flounder (Platichthys flesus Linnaeus, 1758)

The efficacy of clove oil as an anaesthetic in juvenile flounder was evaluated at two temperatures (10–12 and 16–18 °C) and the effective concentration determined. Flounder juveniles with a body length of 9.0–10.2 cm and a weight of 6–14 g were individually exposed to clove oil at concentrations ranging from 200 to 1000 mg L^?1 in a 5‐L bath with three replicates per treatment with 10 fish. The induction and recovery times were recorded in seconds for all doses and temperatures. Temperature had no effect on induction, but higher temperature shortened the recovery time. The effective concentration (EC₅₀) was estimated as 753 (692–820) mg L^?1 for ≤3 min with 95% confidence limits. The study demonstrated that clove oil can be used as an effective anaesthetic in juvenile flounder and that temperature has an effect on recovery time from anaesthesia with clove oil.     

Effect of angiotensin II on transepithelial potential in the isolated perfused flounder (Platichthys flesus) gill

Angiotensin II (Asp¹, Val⁵) perfused through isolated flounder gills inhibited the transepithelial potential by up to 25 per cent at a concentration of 10⁻⁹M. There was no effect on gill haemodynamics and the subsequent response to 10⁻⁵ M adrenaline was normal.