Parasites of the flounder Platichthys flesus (L.) from the German Bight, North Sea, and their potential use in ecosystem monitoring

As part of integrated biological-effect monitoring, the parasite fauna of the flounder Platichthys flesus (L.) was investigated at five locations in the German Bight, with a view to using parasite species as bio-indicators. Over a period of 6 years, parasites from 30 different taxa were identified, but only 7 taxa of the parasite community occurred regularly at all locations and in sufficient abundance that they could be considered as potential indicator species. These species were the ciliophoran Trichodina spp., the copepods Acanthochondria cornuta, Lepeophtheirus pectoralis and Lernaeocera branchialis, the helminths Zoogonoides viviparus and Cucullanus heterochrous and metacercaria of an unidentified digenean species. Infection characteristics of these parasites are presented, with a comparison of the results from individual sampling periods and those of the long-term data set. Natural influences on the infection levels, such as temporal variations, habitat conditions and host-related factors, were evaluated. All of these parasite species showed significant differences in their infection levels between the Elbe estuary, as the most polluted site, and the less polluted coastal and marine locations: Helgoland, Outer Eider estuary and Spiekeroog, especially in the long-term data set. Gradual differences between the Elbe, the Outer Eider and Helgoland, which were not detected in individual sampling periods, also became evident in the pooled-data set. These were found in the prevalence of Trichodina spp., A. cornuta, Z. viviparus and C. heterochrous. Although salinity is considered as the most important natural factor, influencing the distribution pattern of the majority of the potential indicator species, infection levels of most of these species differed between locations with similar salinity conditions. Infection levels corresponded to a contamination gradient (Elbe > Inner Eider, Outer Eider > Helgoland) established across the locations. Seasonal variation in the infection parameters affected the spatial distribution of the copepod species Lepeophtheirus pectoralis and Lernaeocera branchialis. Annual variations are considered to occur in the range of natural variability, so no trend of increasing or decreasing infection levels of the parasites was found during the course of the study. This study underlined the idea that an analysis of fish-parasite fauna is very useful in ecosystem monitoring. Communicated by H. von Westerhagen, A. Diamant     

Parasites of flounder (Platichthys flesus L.) from the German Bight, North Sea, and their potential use in biological effects monitoring

In the frame of an integrated biological effect monitoring programme, the parasite community of flounder (Platichthys flesus) was investigated at different locations in the German Bight from 1995 to 2000. In order to assess the impact of environmental contamination caused by anthropogenic activities on the parasite community, selected parasitological parameters that displayed significant differences between the sampling sites were subjected to correlation analyses with site-specific contamination and individual pollution loads of their fish hosts. In addition, correlation analyses were conducted with the responses of selected genetic, biochemical, histopathological, physiological and immunological parameters of fish, used as potential biomarkers. In total, 802 flounder were analysed for these parameters. Information on the chemical background at the sampling sites was derived from sediment samples and from 120 samples of blue mussel (Mytilus edulis) tissue, collected at each of the sampling sites. Based on chemical data available from the sediment and blue mussel samples, a pollution gradient could be established between the sampling sites for individual contaminants. The relative abundance of Acanthochondria cornuta, Cucullanus heterochrous and Zoogonoides viviparus, and the community measures species richness and number of heteroxenous species decreased with increasing concentrations of individual heavy metals or hydrocarbons in sediment and blue mussel samples. Most of the parasitological parameters significantly reflected the established site-specific contamination gradient, when data were pooled over all sampling campaigns. Significant correlations were also found with the contamination level of individual flounder. The parasitological parameters included the parasite species Lepeophtheirus pectoralis and Lernaeocera branchialis, which were not correlated to site-specific contamination. Several biomarkers were significantly correlated to the abundance of parasitic copepods A. cornuta, Lernaeocera branchialis and Lepeophtheirus pectoralis and to parasite community parameters. The results showed that the abundance of several metazoan parasite species, species richness and parasite diversity were reduced in contaminated habitats, and that differences between sites were not only related to natural factors, such as salinity, but also to pollution-induced stress. Thus, it can be concluded that the parasite community of fish responds to the level of pollution at a specific site as well as to residues of xenobiotics in individual fish. These findings give indications that the parasite community of fish is a valuable parameter for the assessment of ecological consequences of chemical contamination in aquatic habitats.     

Kinetic properties of liver pyruvate kinase from the flounder (Platichthys flesus L.)

Pyruvate kinase, purified from flounder liver, in two forms, i.e. PK I and PK II, is characterized by sigmoid kinetics with phosphoenolpyruvate as substrate at pH 6.3, 6.7 and 7.7. K0.5 for PEP increases with increasing pH. PK I and PK II show hyperbolic kinetics with ADP, but are inhibited by ADP concentrations above 1-2 mM. K0.5 for ADP decreases with increasing pH. PK I and PK II differ in their K0.5 values for PEP with a factor of at least 2, showing the highest figures for the latter. K0.5 for ADP is about the same for the two enzyme forms. Other nucleotide diphosphates can replace ADP as the substrate. When the nucleoside diphosphates are arranged in a rank order showing decreasing effectiveness as substrate, different rank orders are obtained for PK I and PK II.     

Ethoxyresorufin O-deethylase (EROD) activity in the liver of dab (Limanda limanda L.) and flounder (Platichthys flesus L.) from the German Bight. EROD expression and tissue contamination

Ethoxyresorufin O-deethylase (EROD) activity was measured in the liver of dab (Limanda limanda) and flounder (Platichthys flesus) from the German Bight (southern North Sea) and compared with muscle and liver polychlorinated biphenyl (PCB) concentrations in an attempt to relate EROD activity to PCB body burden. In none of the different datasets (species-, tissue- or matrix-dependent) was a significant (P<0.05) correlation between PCB tissue contamination and EROD activity found. Yet EROD activity was significantly correlated with polycyclic aromatic hydrocarbons (PAH) levels (phenanthrene, fluoranthene, pyrene) in muscle tissue, indicating a possible dependence of EROD expression on other ubiquitous organic contaminants, thus making it a suitable biomarker for general pollution. Received: 14 April 1999 / Received in revised form: 10 July 1999 / Accepted: 15 July 1999     

Tissue specific haemoglobin gene expression suggests adaptation to local marine conditions in North Sea flounder (Platichthys flesus L.)

Recent genetic analyses of candidate genes and gene expression in marine fishes have provided evidence of local adaptation in response to environmental differences, despite the lack of strong signals of population structure from conventional neutral genetic markers. In this study expression of the haemoglobin alpha and beta subunit genes was studied in reciprocally transplanted European flounder Platichthys flesus from the highly saline North Sea and the brackish Baltic Sea. Clear differences in expression patterns of haemoglobin alpha and beta subunit genes were found among different types of tissue in flounder. In gill tissue a plastic response to salinity treatments was observed with general up-regulation of these genes concomitant with higher salinity. For liver tissue a population specific expression differences was observed with lower expression at simulated non-native compared to native salinities. Finally, for kidney tissue a stress response was observed in one population, with gene up-regulation when North Sea flounders were transplanted to low salinity. This study underlines the importance of tissue specific gene expression and the significance of gene expression for evolution of local adaptation in high gene flow marine fishes.     

Annual variation in lymphocystis infection frequency in flounder, Platichthys flesus (L.)

The infection frequency of the virus induced lymphocystis disease has been registered in flounder from the Oslofjord, Norway, during a 4 year period. The disease has a clear regular seasonal variation, and also an annual variation. The percentage infection in the summer was 1–10%, and in the winter up to 57%. The possibility that the high pollution level of the inner Oslofjord could be the reason for the high frequency of lymphocystis disease, is discussed.     

Population dynamics of lymphocystis disease in estuarine flounder, Platichthys flesus (L.)

The population dynamics of viral lymphocystis disease in an age-structured population of flounder, Plarichthys flesus (Linnaeus 1758), is studied with a mathematical model. For a single cohort the model illustrates the influence of host density and acquired immunity on length-prevalence profiles. This case corresponds to a host population at demographic equilibrium. When the model is extended to several cohorts, seasonal recruitment of susceptible hosts is shown to drive seasonality in disease prevalence. In both cases, there is good qualitative agreement between model predictions and field data from the Elbe estuary, Germany.     

Purification and properties of pyruvate kinase from liver of the flounder (Platichthys flesus L.)

Two forms of pyruvate kinase, PK I and PK II, have been demonstrated in flounder liver. PK I, purified 991-fold to a specific activity of 105 units per mg of protein, has an unusually high molecular weight of about 2 X 10(6). PK II, purified 172-fold to a specific activity of 16.5 units per mg of protein, has a molecular weight of 210,000 when determined on a sucrose gradient but of 300,000 when derived from gel chromatography. PK I and PK II differ in sensitivity to the inhibitor L-phenylalanine, a fact which is used to evaluate the amount of each of them in a mixture. pH optimum for both forms is 6-6.6. PK I and PK II behave different in an Arrhenius plot--PK II showing a transition at 21 degrees C.     

Note: the effect of parasite infection on the innate immune response of European flounder (Platichthys flesus L.) in the southern North Sea

In a field study, infecting European flounder (Platichthys flesus L.) subclinically with different parasite species did not result in any alteration of the innate immune response. Due to the high variability in infection status and the immune parameters measured, no relationships of biological significance were found. The data indicate that copepods, as the most abundant parasites, most probably had no major influence on immune responses measured here. Thus it might be concluded that these parameters were not sensitive to parasite infections occurring under natural conditions. The immune parameters considered here are regarded as promising indicators of chemical contaminant-induced variation in piscine immune responses, which could be implemented in pollution monitoring programmes. Communicated by H. v. Westernhagen, A. Diamant     

Surfactant effects on adrenergic responses in the gills of the flounder (Platichthys flesus L.)

Summary The interactions between catecholamines and surfactants was investigated in perfused gills of the marine teleostPlatichthys flesus L. The activity of the branchial ion pumps was monitored via the electrogenic transepithelial potential (inside positive) measured in gills bathed and perfused with identical saline. Vascular resistance of the arterio-arterial and arterio-venous pathway was also recorded simultaneously by measuring respectively the afferent perfusion pressure and venous flow in gills perfused at constant flow and at constant efferent pressure. The specific effects of respective - and -adrenergic receptor stimulation was investigated by the administration of discrete doses of either adrenaline in the presence of 10 mol l^–1 propranolol or isoprenaline in the perfusate. In the absence of surfactants the -adrenergic effects were an inhibition of electrogenic ion transport, a decrease in venous flow and an increase in the vascular resistance of the arterioarterial vascular pathway. In contrast the -adrenergic effects consisted of a stimulation of electrogenic ion transport and a vasodilation of the arterio-arterial pathway. Both anionic (linear alkyl sulphonate; sodium lauryl sulphate) and non-ionic (nonyl phenol ethoxylate; synthetic alcohol ethoxylate) surfactants were administered in the perfusate at nominal concentrations of 1 mol l^–1 (0.3–0.5 mg l^–1). None of these compounds had any effect on the affinity or the efficacy of the -adrenergic responses. In contrast there was a significant reduction in the efficacy of isoprenaline in the presence of all of the surfactants used but only in the case of the synthetic alcohol ethoxylate was there an effect on the affinity of this agonist for the -adrenergic receptor. The results are discussed in the context of the mechanism of action of these environmental contaminants and the nature of adrenergic receptors in the gill.     

The effects of handling on flounder (Platichthys flesus L.) and Atlantic salmon (Salmo salar L.)

The effect of 9 min of net confinement on two marine teleost species, the flounder and the Atlantic salmon, was investigated in order to gauge how different species respond to the same stress stimulus. Net confinement in both species induced significant elevations in plasma cortisol, glucose, lactate, osmolality, and monovalent ion levels; the responses to net confinement in salmon were generally of a greater magnitude. In both species, handling induced significant alterations in PFFA levels though there were marked species differences in the qualitative and quantitative aspects of this response. Plasma protein levels were significantly elevated only in the flounders. In general, the two species responded similarly to the net confinement with differences only in the absolute levels and durations of the responses.     

The use of innate immune responses as biomarkers in a programme of integrated biological effects monitoring on flounder (Platichthys flesus) from the southern North Sea

Immunological biomarkers that reflect the effects of exposure to environmental contaminants in coastal marine habitats were sought in European flounder (Platichthys flesus) from five locations in the German Bight with different anthropogenic impacts. During a 2-year period of sampling, innate immune responses were monitored from a total of 331 individual flounder of a body length of 18 to 25 cm. From the fish, plasma lysozyme, phagocytosis and respiratory burst activity of head kidney leucocytes were analysed and implemented as part of an integrated biological effects monitoring programme. As the measurements of the parameters applied here varied within wide ranges at some locations, spatial differences could not always be established, but some general trends could be drawn: plasma lysozyme activity was decreased in flounder contaminated with DDT adducts and some PCBs, while cellular functions such as phagocytosis and respiratory burst were stimulated by some chlorinated hydrocarbons. Correlation analysis also revealed connections not only between the parameters applied here and some contaminants but also with some biochemical parameters used as biomarkers in pollution monitoring: in flounder with decreased integrity of hepatocyte lysosomal membranes, immune functions also were impaired, and plasma lysozyme as well as phagocytosis activity of head kidney cells were impaired when the activity of cytochrome P450 1A was induced. The data presented here indicate that innate immune responses may be useful parameters to monitor cellular functions in a battery of biomarkers of different levels of biological organisation. Communicated by H. v. Westernhagen, A. Diamant     

The diet and growth of O-group flounder,Platichthys flesus (L.), in the River Dee,North Wales

Many O-group flounder, Platichthys flesus, live in the lower reaches of rivers in North West Europe; while there is extensive information on the niche that they occupy in estuarine and marine environments, little is known of their life in freshwater. The diet of O-group flounder in the Lower Welsh River Dee was studied by stomach contents analysis of diel and seasonal samples. The dominant prey were chironomid larvae and pupae, tubificid oligochaetes and cyclopoid copepods. There was little quantitative variation in diet temporally, although a weak crepuscular pattern of stomach fullness was shown. There was wide variation in length and weight in all months, but the mean values at age I were 60 mm and 2.9 g respectively.     

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.     

Peptidergic and adrenergic regulation of electrogenic ion transport in isolated gills of the flounder (Platichthys flesus L.)

Summary Electrogenic potentials measured in isolated gills of seawater-adaptedPlatichthys flesus conform to the current model proposed for salt secretion by teleost chloride cells. Gills perfused and bathed with identical salines maintained a stable potential (blood-side positive) thought to represent the activity of a chloride pump. Furosemide added to the perfusate (1×10^–4 and 5×10^–4 mol l^–1) caused a large inhibition of the transepithelial potential. Cyclic 8-(4-chlorophenylthio) adenosine-3:5-monophosphate (5×10^–5 mol l^–1) stimulated the transepithelial potential and decreased the arterial vascular resistance. The adenylate cyclase activator forskolin mimicked the effects of the cAMP derivative on branchial vascular resistance and, at low concentrations, on electrogenic ion transport. At high concentration (>5×10^–7 mol l^–1) forskolin inhibited the transepithelial potential. These results implicate cAMP as an important intracellualr regulator of both ionoregulatory and haemodynamic functions in the teleost gill.The -adrenergic agonist isoprenaline administered as injected doses in the perfusate produced a stimulation of the transepithelial potential and a decrease in the arterial vascular resistance. A dose-response analysis showed that half-maximal haemodynamic effects occurred at significantly lower doses of agonist than those required for half-maximal stimulation of the potential. The pancreatic hormone glucagon also caused dose-dependent stimulation of the transepithelial potential but had no effect on arterial vascular resistance. It is suggested that regulation of the rate of branchial monovalent ion excretion may be under peptidergic as well as adrenergic control.     

The effect of fructose 1,6 diphosphate on pyruvate kinase from the liver of the flounder (Platichthys flesus L.)

1. Pyruvate kinase purified from flounder liver in two forms, i.e. PKI and PKII, is activated by fructose 1,6 diphosphate. 2. Two or more binding sites for FDP are demonstrated for PKII, the binding to which is influenced by the levels of substrates. 3. FDP reduces or abolishes the cooperative effect of PEP. 4. FDP increases the maximal activity. 5. The inhibition observed at higher levels of ADP is not abolished by FDP.     

Vascular pathways in the gill filaments of the flounder, Platichthys flesus L.

This study is concerned with functional organization of some of the blood pathways in the gill filament of the flounder, Platichthys flesus L. The existence of two independent vascular pathways has been confirmed. The blood from the efferent filament artery (EFA) enters the central venous sinus (CVS) through very small blood vessels which are characterized by the presence of sphincter-like structures. The existence of an independent chamber of the CVS mainly full of white blood cells provides evidence of an independent lymphatic system connected to the CVS. Gill rays support the afferent side of a gill filament whereas plasma and an extensive network of nutritive blood vessels in the CVS supports the efferent part.     

Evolutionary mechanisms shaping the genetic population structure of marine fishes; lessons from the European flounder (Platichthys flesus L.)

A number of evolutionary mechanisms have been suggested for generating low but significant genetic structuring among marine fish populations. We used nine microsatellite loci and recently developed methods in landscape genetics and coalescence-based estimation of historical gene flow and effective population sizes to assess temporal and spatial dynamics of the population structure in European flounder (Platichthys flesus L.). We collected 1062 flounders from 13 localities in the northeast Atlantic and Baltic Seas and found temporally stable and highly significant genetic differentiation among samples covering a large part of the species' range (global F(ST) = 0.024, P < 0.0001). In addition to historical processes, a number of contemporary acting evolutionary mechanisms were associated with genetic structuring. Physical forces, such as oceanographic and bathymetric barriers, were most likely related with the extreme isolation of the island population at the Faroe Islands. A sharp genetic break was associated with a change in life history from pelagic to benthic spawners in the Baltic Sea. Partial Mantel tests showed that geographical distance per se was not related with genetic structuring among Atlantic and western Baltic Sea samples. Alternative factors, such as dispersal potential and/or environmental gradients, could be important for generating genetic divergence in this region. The results show that the magnitude and scale of structuring generated by a specific mechanism depend critically on its interplay with other evolutionary mechanisms, highlighting the importance of investigating species with wide geographical and ecological distributions to increase our understanding of evolution in the marine environment.     

Benthic communities in the Southern Bight of the North Sea and their use in ecological monitoring

Macrobenthic and meiobenthic communities of an area off the Belgian coast of the North Sea were studied from 1970 until 1975 at 74 stations. On the basis of both macro- and meiobenthos, three zones can be distinguished in the area. The coastal zone is characterized by the macrobenthicAbra alba community, corresponding to the meiobenthicMicroarthridion littorale — Halectinosoma herdmani community, and the open sea zone by the macrobenthicVenus gallina community and the meiobenthicLeptastacus laticaudatus — Paramesochra helgolandica community. In between is a transient zone where elements of both other zones mix. The distribution of these zones is governed by the hydrodynamical regime of the region, especially by the residual and tidal current system of the Southern Bight. Within the coastal zone, the composition of the community is influenced by pollution which especially affects the epibenthic detritus-feeders of the meiobenthos. The spatial stability of parameters describing community structure can be used for monitoring changes. Temporal characteristics of these parameters could not be investigated properly, but diversity seems to be much stabler than biomass.