The effects of copper on ionic regulation by the gills of the seawater-adapted flounder (Platichthys flesus L.)

Summary Potentials measured in isolated, perfused gills fromPlatichthys suggest that the electrogenic ion-pump(s) contribute significantly to the potential measured in seawater. Copper added to the perfusate causes a significant reduction in the potential measured in the isolated gill and it is suggested that this is due to a direct inhibition of the branchial ion pump(s). Vascular resistance was unaffected by the presence of copper. Ouabain-sensitive oxygen consumption was reduced in tissue exposed to copper whereas residual oxygen consumption was unaffected, which suggests that the in vitro action of copper is confined to the Na⁺, K⁺ ATPase dependent portion of branchial metabolism.In vitro application of copper to gill homogenates fromPlatichthys flesus adapted to seawater caused a marked reduction in Na⁺, K⁺ ATPase activity. In vivo a combination of ouabain binding (to determine the number of enzyme sites), together with measurements of Na⁺, K⁺ ATPase activity showed that exposure of the fish to ambient copper in seawater also inhibited enzyme activity. However this response was modulated by a reduction in the sensitivity of the enzyme to copper in copper-treated fish and also responses, possibly of an endocrine nature, modifying Na⁺, K⁺ ATPase activity and returning overall enzyme levels to values not significantly different from those in control fish.In addition, this study indicates the importance of obtaining information on both the number of enzyme sites as well as their overall activity in assessing the effects of agents modifying enzymes involved in ion transport.     

Oxygenation properties of haemoglobins from the flatfish plaice (Pleuronectes platessa) and flounder (Platichthys flesus)

Summary The oxygen binding properties and some haematological data of haemoglobins of plaice (Pleuronectes platessa) and flounder (Platichthys flesus) were measured and compared, with the view of discerning their adaptations to the benthic habitat.Compared to plaice, flounders posses higher haematocrit and haemoglobin levels, and their haemoglobin has a higher oxygen affinity, smaller Bohr and Root effects and appears to have a greater ATP sensitivity. At pH 7.6, the half-saturation oxygen tensions,P ₅₀, of stripped plaice and flounder haemoglobins amount to about 5.9 and 4.2 torr, and the Bohr shifts (logP ₅₀/ pH) to –0.51 and –0.34, respectively. The species differences in the oxygen binding patterns occur after stripping the haemoglobin in solution, from dissolved ions and thus appear to be inherent in the pigment molecules. The differences suggest that the haemoglobin of flounder is better adapted to hypoxic and hypercarbic environments than plaice haemoglobin, in accordance with the more inshore occurrence of the former species.The molar ratios of ATP to haemoglobin tetramers are low in plaice and flounder (about 1.6); in plaice this ratio corresponds to that where the cofactor effect is most pronounced. In contrast to previous evidence for an adaptive reduction in the temperature dependence of the haemoglobin of flounder but not of plaice from the Baltic Sea (Friedrich, 1935), the same temperature effects were found in representatives of the two species both from the Dutch North Sea and the Danish Kattegat.     

Hyper-osmotic cell volume regulation in vivo and in vitro in flounder (Platichthys flesus) heart ventricles

Summary Cell volume regulation in heart ventricles of the flounderPlatichthys flesus has been studied under hyper-osmotic conditions in vivo and in vitro.During reacclimation of flounders from fresh water to sea water the osmolality of blood plasma increased to the sea water level in 7 days. The water content of the heart ventricle cells remained constant after reacclimation while the intracellular concentration of K⁺ and taurine increased to values found in sea water flounders.Hearts taken from fresh water flounders were mounted in vitro and perfused with hyper-osmotic ringer solution. Upon perfusion the cellular water content of the ventricle decreased initially but then steadily increased. After 6 h the cellular water content was still below the control values reflecting that the ventricle cells have an incomplete volume regulation in a hyper-osmotic medium. The cellular amount of K⁺ and taurine increased during the volume regulation. The perfusion media did not contain taurine or amino acids and the gain in cellular taurine must be due to intracellular production.     

Identification of glycoproteins in goblet cells of epidermis and gill of plaice (Pleuronectes platessa L.), flounder (Platichthys flesus (L.)) and rainbow trout (Salmo gairdneri Richardson)

Synopsis A quantitative analysis has been made of the glycoproteins present in the goblet cells of the epidermis, gill filaments and gill lamellae of three species of teleost fish. The glycoproteins have been identified by a combination of techniques, including the use of the enzyme sialidase followed by Alcian Blue staining, at pH 2.6 or I. o, in combination with periodic acid-Schiff. The selected fish were representative of species living in marine, freshwater and estuarine environments.The range of glycoproteins identified in these fish was similar to that found in mammalian tissue in that both neutral and acid glycoproteins were present, the latter included both sialomucins sensitive and resistant to sialidase, and sulphomucin. A single goblet cell contained either neutral or acid glycoproteins alone or in combination. Only the epidermis of the plaice and rainbow trout contained uniform cell populations producing acid glycoproteins, the former sulphomucin and the latter mainly sialomucin. At each site in the flounder and in the gill epithelia of the plaice and rainbow trout, the goblet cell population was mixed, with cells producing each type of glycoprotein. The number of goblet cells producing each type of glycoprotein varied at each tissue site.     

Growth and condition of 0-group European flounder, Platichthys flesus as indicator of estuarine habitat quality

The purpose of this present study was to use the growth rates, RNA–DNA ratio and morphometric (Fulton’s K) condition indices measured on 0-group juveniles European flounder, as a means of comparing the quality of estuarine nursery habitats. The Seine estuary, heavily impacted by manmade modifications and one of the most contaminated in Europe, was compared to two smaller estuaries considered as clean (low domestic, agricultural and industrial effluents). Indicators of human disturbance (sediment chemical contaminants) as well as biotic data (macrobenthos abundances) were used to establish between-site differences in environmental quality. The abundance of macrobenthos (potential preys for juvenile) was high and not significantly different among the three estuaries confirming the important role played by estuaries as feeding grounds. Sediment metal concentrations (Cd, Cr, Cu, Pb, Zn) were significantly higher in the Seine than in the Canche and Authie estuaries. Otolith growth was closely related to somatic fish growth, and we used daily otolith increments from the previous 10 days before capture as an indicator of 0-group flounder recent growth (RG). RG did not vary significantly among flounders from the Canche and Authie estuaries and individuals from the Seine had slower RG. RNA–DNA ratio and K values were also significantly lower in individuals from the Seine compared to those of the Authie or the Canche. Pollution (e.g. metal contaminants) is the most likely cause of growth and condition differences among estuaries. The results of this study suggest that habitat-specific fish growth and condition are useful proxies for habitat quality since they integrate the effect of multiple environmental factors. Handling editor: P. Viaroli     

Detection of nodularin in European flounder (Platichthys flesus) in the west coast of Sweden: Evidence of nodularin mediated oxidative stress

The brackish, bloom-forming cyanobacterium Nodularia spumigena produces a peptide called nodularin, which may induce liver damage in fish. In the summer of 2007, nodularin was detected in liver tissue of European flounder caught in Swedish waters of Öresund, within the upper salinity limit for N. spumigena. Nodularin concentrations ranging between 22 and 557 μg kg⁻¹ liver (d.w.) were detected in fish liver. Nodularin was not detected in blue mussels (Mytilus edulis). Although N. spumigena blooms can occur in the area, the cyanobacteria were only present in very small amounts in 2007. Results suggested that nodularin accumulated in flounder livers during the summer of 2006, when vast N. spumigena blooms were observed in Öresund, and persisted over several months. Nodularin has previously been shown to induce oxidative stress in mice, crustaceans and mollusks but work on the potential negative effects of nodularin on fish is still scarce. To examine the dynamics of nodularin induced oxidative stress in liver tissue of flounder, the differential responses of the antioxidant enzymes glutathione-S-transferase catalase (CAT) and the formation of malondialdehyde (MDA) were monitored during 14 days in flounder exposed to an intraperitoneal injection of nodularin (0, 2, 10 and 50 μg nodularin kg⁻¹ body weight). The activities of GST and CAT in the liver decreased significantly in the 50 μg nodularin kg⁻¹ exposure after 7 days, but were restored to control levels after an additional 10 days of recovery. The results suggested that nodularin induced oxidative stress in terms of decreased GST and CAT activity, which can result in increased vulnerability of the cell to reactive oxygen species (ROS). No significant changes could be found in MDA levels between the treatments. Thus, the antioxidant defense system presumably managed to prevent oxygen mediated toxicity as seen by the unchanged levels of MDA. Alteration of the enzymatic defense system may increase energetic costs, thus reducing fish growth and survival. The present study also suggests that oxidative stress biomarkers can be used in fish to detect early responses to nodularin.     

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.     

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.     

Effects of different types of acidosis and Ca2+ on cardiac contractility in the flounder (Pleuronectes flesus)

Summary In contrast to most other fish the isolated myocardium of flounder (Pleuronectes flesus) was able to maintain contractility well under acidosis. The response to acidosis was biphasic, the initial force decline being followed by spontaneous recovery under ongoing acidification. This reaction took place only under hypercapnia and did not appear when extracellular pH was decreased to the same extent by lowering bicarbonate. Biphasic response to hypercapnia was more pronounced with low than with high extracellular Ca²⁺. A depressed sensitivity towards extracellular Ca²⁺ was seen in hypercapnia in contrast to acidification by lowering bicarbonate. This indicates that hypercapnia in inducing severe intracellular acidification, triggers an efficient compensatory reaction which is modified by Ca²⁺-availability.     

Reducing the allelopathic effect of Partheniumhysterophorus L. on wheat (Triticumaestivum L.) by Pseudomonasputida

The present study evaluates the role of Pseudomonas putida NBRIC19 in alleviating biotic stress of Parthenium hysterophorus (Parthenium) in Triticum aestivum. Due to presence of Parthenium there was 43.76, 53.08 and 78.65% inhibition in root length, shoot length and dry weight of wheat respectively. This inhibition was recovered when P. putida NBRIC19 treatment resulted in 52.29, 28.73 and 76.31% increase in root length, shoot length and dry weight respectively as compared to control. P. putida NBRIC19 was able to form more biofilm under toxic environment of allelochemicals and enhanced expression of stress responsive genes in wheat. Inoculated wheat plants showed lower activity of catalase and ascorbate peroxidase under biotic stress of Parthenium indicating that inoculated plants felt less stress as compared to uninoculated plants. Microbial community structure in bacterized and nonbacterized wheat rhizosphere in presence and absence of Parthenium, was investigated using Biolog. There was significant increase in microbial diversity in P. putida NBRIC19 bacterized wheat rhizosphere. Functional microbial diversity revealed that P. putida NBRIC19 had shifted the microflora in such a manner that utilization of phytotoxic allelochemicals increased to lessen its toxic effect and finally it resulted in better growth of wheat in presence of Parthenium. Principal component analysis showed that microbial community function in nonbacterized wheat rhizosphere in presence (WPC) and absence (WC) of Parthenium is totally different from each other but due to P. putida NBRIC19 treatment there was close clustering of WPT and WT indicating a total shift in microbial community structure.     

The effect of temperature on chyme proteinase activities in perch Perca fluviatilis L. and roach Rutilus rutilus (L.)

The effect of temperature on the chyme proteinase activities of the perch Perca fluviatilis L. and the roach Rutilus rutilus (L.) was investigated. The two fish species showed different characters of temperature dependence and different values of activation energy for caseinolytic and hemoglobinolytic chyme proteinases. The perch was characterized by a higher level of relative proteinase activities and lower activation energy for protein hydrolysis at a temperature of 0–10°C than the roach.     

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.     

Patterns in abundance and distribution of juvenile flounder, Platichthys flesus, in Minho estuary (NW Iberian Peninsula)

For 2 years, the flatfish community of Minho estuary (Northwest of Iberian Peninsula) was surveyed monthly with a beam trawl. Flounder, Platichthys flesus, was the most representative species contributing the most (74%) to the total flatfish abundance and was the only species occurring throughout the sampling period and along the entire salinity gradient. The majority of flounder sampled consisted of 0- and 1-year group individuals. In both years, a peak in abundance was recorded in late spring–summer months (from May to August) which coincided with seasonal settlement events. Higher densities of newly-settled flounder were obtained in the most upstream site, located in the tidal freshwater wetland, indicating that this area is an important nursery area. Of the environmental variables tested, temperature and salinity were the best predictors for 0-year group abundance explaining around 30% of the variability, whereas sediment was also important when considering all age groups together. This study supports the generally prevalent idea that estuaries act as nurseries for the early life stages of flounder, with particular relevance to the tidal freshwater estuarine wetlands.     

Schneeammern (Passerina nivalis L.) und Zwergtrappe (Otis tetrax L.) in Krain 1914/1915

Ohne Zusammenfassung     

The structure of the gills of the elasmobranch,Scyliorhinus canicula (L.)

Summary The anatomy of the blood supply to the gills of the dogfish, Scyliorhinus canicula, is described. The anatomical basis for a counter-current exchange system at the respiratory surfaces is reported. Within the interbranchial septum there is a capillary network joining all the afferent branchial arterioles of the gill. The structure of the walls of the corpus cavernosum is found to be of smooth muscle cells supported by a basal lamina and connective tissue and lined by endothelial cells containing phagocytic vesicles. Both the capillary network and corpus cavernosum are suggested to function in smoothing the pressure pulses of the blood flow. Pre- and post-lamellar vessels and pre- and post-lamellar sphincters are described. The sphincters are thought to control the number of secondary lamellae physiologically in the respiratory circuit, and by retaining blood within nonperfused lamellae to act in conjunction with pillar cells (contracting in antagonism to the hydrostatic skeleton of the blood) to maintain the rigidity of secondary lamellae in the water current.Whorls of cells of unknown function are found within the interbranchial septum. In the epithelium lining the water channel large cells having a complexly branching plasma membrane and a very large central vacuole occurs. The cytoplasm lining the lumen contains numerous vacuoles each surrounded by a double membrane.This work formed part of a thesis submitted for the degree of Master of Science at the University of Bristol. I should like to thank Professor G.M. Hughes for the use of facilities in the Department of Zoology, University of Bristol.     

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.     

The effects of dissolved zinc on the gills of the stickleback Gasterosteus aculeatus (L.)

Concentrations of 0.5–1.0 mg Zn²⁺/dm³ distilled water killed sticklebacks after 1–3 days, producing detachment and sloughing of epithelial cells, and coalescing of adjacent secondary lamellar epithelia. Cytoplasmic abnormalities included extensive vacuolation, followed by swelling of nuclei and mitochondria leading to cellular disintegration. Many acutely poisoned fish recovered in zinc-free hard water, regeneration of epithelia being accompanied by a temporary appearance of chloride cells on the secondary lamellae. Concentrations of 2.0–6.0 mg Zn²⁺/dm³ hard water were not toxic over periods of up to 700 h. Extensive cytoplasmic abnormalities appeared, however, including the formation of membrane-bounded vesicles and dense accumulations of metabolites. The most pronounced effect was the appearance of active chloride cells on the secondary lamellae. The possible involvement of chroride cells in the excretion of ions other than chloride is briefly discussed.