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.     

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.     

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.     

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.     

The effect of adrenaline on potentials in the isolated gills of the flounder (Platichthys flesus L.)

Summary A technique is described for simultaneously measuring perfusion pressure and transepithelial potentials in isolated perfused gills.In this preparation the potentials measured in external media of Ringer or sea water were both reduced by 4 mV to 5 mV in the presence of 10^–6 M adrenaline. This effect was reversed by 10^–5 M phentolamine in both situations; 10^–5 M propanolol was without effect (Figs. 2, 3). Both potentials were reduced by oxygen lack (Figs. 1, 6).The preparations showed inconsistent vasomotor responses to adrenaline but 10^–5 M acetylcholine consistently produced an increase in branchial vascular resistance (Figs. 7, 8).It is suggested that adrenaline directly inhibits the branchial ion extrusion pump via a mechanism involving -adrenergic receptors. It is further suggested that the biogenic potential produced by the activity of the branchial ion pump makes a significant contribution to the total potential in sea water. The significance of these findings in relation to other work is discussed.     

The accumulation of cadmium by the plaice, Pleuronectes platessa L. and the thornback ray, Raja clavata L.

The accumulation of cadmium (^115mCd) from both food and sea water by the plaice, Pleuronectes platessa L., and thornback ray, Raja clavata L., has been studied in relation to their measured cadmium concentrations. Plaice accumulated cadmium from sea water at a faster rate than rays, although the concentrations factors attained by both species as a result of such direct accumulation were very low. Both species retained cadmium from food and accumulated it in the liver, the rays relatively more than the plaice. From analyses of North Sea fish, some evidence was found for a positive linear relation between the concentrations of cadmium in plaice liver and age, but not weight, of the fish.     

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.     

Copper metabolism in the plaice, Pleuronectes platessa (L.)

Copper metabolism in a teleost, the plaice, Pleuronectes platessa (L.) from a natural environment has been studied. Distribution in the various tissues of the metal and of five key copper-dependent enzymes: ceruloplasmin EC 1.16.3.1; Superoxide dismutase EC 1.15.11; tryptophan oxygenase EC 1.13.1.12; cytochrome oxidase EC 1.9.3.1 and monoamine oxidase EC 1.4.3.4 have been determined. The copper distribution was found to be similar to that in mammals with the greatest concentrations in the brain and heart. Distribution of the copper enzymes is also similar to that found in mammals. A preliminary characterization of the copper enzymes showed that plaice cytochrome oxidase has a pH maximum 2 pH units more alkaline than the mammalian enzyme and that plaice tryptophan oxygenase is more sensitive to heat denaturation than the mammalian enzyme. The present data form a base-line against which studies on factors affecting the copper metabolism in a teleost can be assessed.     

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.     

Sexual maturation in triploid male plaice (Pleuronectes platessa) and plaice x flounder (Platichthys flesus) hybrids

Triploid male plaice, Pleuronectes platessa , and plaice x flounder, Platichthys flesus , hybrids produced from cold-shocked eggs were reared to maturity at three years of age. The testes of triploid hybrids were similar in macroscopic appearance and size to those from control fish but in histological section spermatocyte and spermatid cell formation was grossly abnormal. In two fish the majority of cells appeared to be aborted spermatids and spermatocytes although there were some spermatozoa with enlarged heads and thick bent flagellae.
The testes of mature triploid plaice were similar to controls both in shape and size and in histological section. Spermatocyte and spermatid cell formation was normal, apart from an increase in cell and nuclear size, and fully motile spermatozoa were produced from several fish. Plaice eggs fertilized with semen from triploids showed a slight reduction in fertility compared with controls but developed normally to gastrulation with little mortality. Beyond gastrulation there was increasing mortality associated with abnormalities in the appearance of embryos. Few hatched and none of these survived longer than 48 h. The value of triploidy in fish farming is briefly discussed.     

The accumulation of inorganic mercury from sea water by the plaice, Pleuronectes platessa L

The accumulation of inorganic mercury from sea water by plaice eggs, larvae, and adult fish has been studied using ²⁰³HgCl₂ as a tracer. The isotope was rapidly accumulated and the levels of accumulation have been related to stable element concentrations. High concentration factors were attained by many organs, but the distribution of the ²⁰³Hg was markedly different from that of the stable element. Whereas the largest fraction of the body burden of mercury is contained by muscle, only a slow rate of accumulation into this tissue was observed. In addition, the mercury in fish muscle is of the methyl form: no evidence for the methylation of the tracer was obtained.     

The accumulation of organic mercury from sea water by the plaice, Pleuronectes platessa L

The accumulation of organic mercury from sea water by plaice eggs, larvae and adult fish has been studied using CH₃²⁰³HgCl as a tracer. The isotope was rapidly accumulated, the largest fraction being taken up by muscle tissue. High concentration factors were attained by many internal organs, particularly blood, spleen, and kidney. Longer biological half-times than previous estimates with ²⁰³HgCl₂ were obtained: the possible consequences of inorganic mercury accumulated from sea water being excreted at the rate for methylmercury have been calculated.     

Gill ventilation and O2 extraction during graded hypoxia in two ecologically distinct species of flatfish,the flounder (Platichthys flesus) and the plaice (Pleuronectes platessa)

Synopsis Gill ventilation, breathing frequency, breath volume, oxygen extraction from the ventilatory water current and oxygen uptake through the gills were measured in flounder, Platichthys flesus, and plaice, Pleuronectes platessa, at water O₂ tensions ranging from 35 to 155 mm Hg at 10° C. Ventilation volumes were similar in the two species at high water O₂ tension. Exposure to hypoxic water elicited a larger increase in ventilation in the flounder. The per cent extraction of O₂ from water decreased slightly in both species as water O₂ tension was lowered. At comparable levels of ventilation O₂ extraction was higher in flounder. At the higher levels of water O₂ tension, O₂ uptake across the gills of flounder was stable, the critical O₂ tension being between 60 and 100 mm Hg. The plaice behaved as an oxygen conformer over the entire range of O₂ tensions investigated. The superior ability of the flounder in maintaining OZ uptake across the gills during a reduction in water O₂ tension may in part explain why the species, unlike plaice, inhabits very shallow waters with large fluctuations in dissolved oxygen.     

A quantitative description of the behaviour of wild juvenile plaice (Pleuronectes platessa L.)

The behaviour of young plaice (Pleuronectes platessa L.) as they migrate up and down sandy beaches with the tide is described. Their behaviour during this migration consists mainly of swimming and feeding interspersed with rarer behavioural acts. Over short periods swimming behaviour can be described by a random model in which the probability of a swim occurring remains constant. This probability varies markedly, however, from hour to hour. Two types of swimming movement are recognized: one of very short duration represents searching for food and the other, longer, category serves to transport the fish up and down the shore. Variations in the feeding rate and in the frequency, duration, and direction of swimming movements over the tidal cycle are described and related to the changing physical and biological conditions that the fish experience during their intertidal movements.     

Physiological impact of salinity increase at organism and red blood cell levels in the European flounder (Platichthys flesus)

Blood respiratory, acid-base, and ionic changes in response to hyperosmotic shock were studied in vivo and in vitro in the European flounder. One primary aim was to evaluate regulatory changes in red blood cell (RBC) volume and its interrelationship with blood O₂ transporting properties. An acute increase in the ambient salinity from 10 to 30 ppt caused small but significant increases in extracellular osmolality (<20 mosM kg⁻¹), [Na⁺], and [Cl⁻], which were corrected within 48 h. RBC volume was not significantly changed 3 h after the in vivo exposure to elevated salinity. A small metabolic acidosis was fully developed within 3 h, and this acidosis seemed responsible for a modest decrease in blood O₂ affinity (i.e., increased P₅₀-O₂ tension at 50% O₂ saturation). RBC organic phosphates were unchanged. In vitro elevation of whole blood extracellular osmolality by 60 mosM kg⁻¹ caused immediate RBC shrinkage. The subsequent regulatory volume increase (RVI) showed a graded dependency on blood O₂ saturation (S_O2). At S_O2 values of 0% and 20%, there were full RBC volume recoveries within 120 min, RVI was partial at S_O2 values of 45% and 55%, and RVI was absent at a S_O2 of 100%. S_O2 and P₅₀ did not change significantly during RBC shrinkage and RVI. Thus, the up-concentration of cellular haemoglobin and organic phosphates in hyperosmotically shrunken RBCs had minimal influence on blood O₂ transporting properties. The degree of cell shrinkage and time needed for RVI were positively correlated with the magnitude of the rise in extracellular osmolality. The RVI proceeded via elevation of cellular [Na⁺], [Cl⁻], and to some extent also [K⁺]. Cell volume regulatory mechanisms are only needed to correct minor volume disturbances in vivo, because changes in extracellular osmolality were limited by an efficient osmotic regulation at the epithelial interface between extracellular compartment and environment.     

The development of the retinomotor reactions in larval plaice (Pleuronectes platessa, L.) and turbot (Scophthalmus maximus, L.)

The development of the retinomotor reaction was examined histologically in the larvae of plaice (Pleuronectes platessa, L.) and turbot (Scophthalmus maximus, L.). In plaice retinal pigment migration is present from hatching, but, in turbot, does not occur until just before metamorphosis. In both species rods first develop at metamorphosis. Plaice show cone migration, but this does not occur in turbot. During dark-adaptation retinal pigment contraction started at ≈ 100 lux in all stages of plaice. Before metamorphosis pigment was fully contractedimmediately below 1000 lux, but after metamorphosis full contraction did not occur until the light intensity had fallen to < 1.0 lux. The thresholds for cone migration in plaice showed similar changes, although the actual light intensities were about an order of magnitude lower. In turbot there was always an abrupt transition between light- and dark-adaptation; the thresholds for this fell from ≈ 10 lux at the start of metamorphosis to between 1.0 and 10 lux at the end. The lower threshold light intensities in turbot compared to plaice may indicate that vision is of greater importance in the feeding of the former species.