Juvenile Flounder Research Articles

Pichia pastoris yeast as a vehicle for oral vaccination of larval and adult teleosts

Oral vaccination is of major interest because it can be used for mass vaccination of fish of various size and age. Given that their administration is relatively easy and stress-free, oral vaccines have both economic and animal welfare benefits. Yet, mostly due to their limited efficacy, only very few oral vaccines are available to aquaculture industry. Here we present a method for oral vaccine delivery based on the yeast Pichia pastoris. We could express a model antigen, green fluorescent protein (GFP), in this yeast and subsequently show delivery of the GFP protein to the intestine of juvenile flounder or adult carp and trout. We tested this approach in several commercially-relevant fish species, from juvenile to adult stage. To test the oral delivery of antigen to larval fish, the GFP-expressing Pichia pastoris was first fed to planktonic crustacean Daphnia or rotifers that served as ‘bioencapsulation vehicles’ and afterwards, fed to flounder larvae. Again, we could show delivery of intact GFP protein to the intestine. In rainbow trout, the orally-administered GFP-expressing yeast elicited a rapid local innate immune response in the intestine and a subsequent systemic response in the spleen. Our results show that Pichia pastoris is a good vehicle for oral antigen delivery and that it can be used in non-encapsulated form for older fish or in bioencapsulated form for larval fish. We discuss the immunomodulatory properties of the yeast itself, and its potential to enhance local immune responses and act as an adjuvant.

Effect of sediment, salinity, and velocity on the behavior of juvenile winter flounder (Pseudopleuronectes americanus)

Winter flounder (Pseudopleuronectes americanus) is a benthic flatfish that is economically important for recreational and commercial fishing in North America. In the last twenty years, the species has undergone a drastic decline, mainly due to anthropic influence. The goal of this study was to gain knowledge on habitat preferences and behavior of juvenile winter flounder to improve the management of natural stocks and optimize release sites of juveniles produced for stock enhancement. Three abiotic factors (sediment, current, and salinity) potentially influencing the distribution of flatfish species were tested in a recircurlating flume with juvenile winter flounder. Time budgets of observed behaviors including swimming, orientation, and burying capacity were analyzed. Sediment texture was the only factor that significantly influenced the burying behavior of winter flounder juveniles; shear velocity, salinity, and sediment had no effect on the orientation of juveniles.

Dynamic habitat use of an estuarine nursery seascape: Ontogenetic shifts in habitat suitability of the European flounder (Platichthys flesus)

Estuaries have long been recognized as nursery habitats for various marine fish species. As seascapes consisting of a mosaic of habitat patches, estuarine nurseries incorporate ecological processes such as ontogenetic shifts. To improve our understanding of the habitat requirement changes of nursery species within the estuarine seascape, habitat suitability models were built using boosted regression trees for three size classes of European flounder (Platichthys flesus) juveniles. The models combined several dynamic and static predictors, from physical and chemical water parameters to biological and physical habitat variables. The relationships between juveniles and predictor variables varied with size, potentially indicating the dynamics of habitat use for the juvenile flounder inside the estuarine nursery. Younger post-settled juveniles tended to be spatially constrained to a shallow upstream area and, with size, there were ontogenetic shifts to the middle estuary. This demonstrates the importance of considering different life stages when developing essential fish habitat maps, which reflect fine scale temporal and spatial distributions. Given the high level of anthropogenic pressures to which estuaries are subjected, the consideration of fish ontogenetic habitat requirements within the estuarine nursery is needed to identify high-value areas, and is valuable for developing effective spatial and temporal management plans. These are essential to fully protect the most sensitive life stages, such as early post-settlement stages occupying transient habitats. Additionally, and as the juvenile-habitat associations can rapidly change, fine-scale connectivity in the estuarine nursery becomes even more relevant in determining recruitment. Connectivity should therefore be central in conservation planning, in order to sustain estuarine and marine adult populations, and ultimately maintaining the fisheries.

Evaluation of genetically-improved (glandless) and genetically-modified low-gossypol cottonseed meal as alternative protein sources in the diet of juvenile southern flounder Paralichthys lethostigma reared in a recirculating aquaculture system

Cottonseed meal (CSM) proteins from genetically-improved (glandless) low-gossypol seed (GI-CSM, 52.1% crude protein, CP), genetically-modified low-gossypol seed (GMO-CSM, 56.0% CP) and from an untreated regular (glanded) seed (R-CSM 49.9% CP) were evaluated to replace fish meal (FM) protein (59.5% CP) in juvenile southern flounder Paralichthys lethostigma diets. Eight isonitrogenous (45% CP) and isolipidic (16% crude lipid, CL) diets were formulated. A control diet (0% CSM) contained 40% fishmeal (FM) and other practical protein sources. Six diets replaced 50, 75 and 100% FM protein with GI-CSM or GMO-CSM protein. One diet replaced 100% FM protein by R-CSM protein. Fifteen fish (mean = 1.81 g) were stocked in each of twenty-four 75-L tanks (N = 3 per treatment) and were fed the treatment diets for eight weeks. Final percent weight gain was not significantly (P > 0.05) different in fish fed the GI- and GMO- and R-CSM protein diets (898–1405%) compared with the control diet (0% CSM) (1242%), but percent weight gain was greater (P < 0.05) in fish fed the 50% GI-CSM diet (1405%) versus the 100% GI-CSM diet (898%). Feed conversion ratio was excellent for all treatments (FCR = 0.70–1.00), with no treatment differences (P < 0.05). Protein efficiency ratios (PER) were also not significantly different among the treatments (2.26–3.24), although the lowest value was for 100% R-CSM diets. After 8 weeks of feeding, survival of fish ranged from 80 to 91%, with no treatment differences. Apparent protein digestibility of diets was significantly higher for the fish fed 75% and 100% GI-CSM and 100% GMO-CSM protein diets (83.5, 83.5 and 86.5%, respectively) compared with the control diet (79.4%). After 8 weeks, no significant (P > 0.05) interactive effects between CSM sources and FM replacement levels on final weight, FCR and PER were observed. Arginine levels in the diets increased as CSM was increased, consistent with the high arginine concentrations found in CSM. Liver gossypol was only detectable in fish fed the 100% R-CSM diet (37 μg/g). Replacing up to 75% FM protein by GI- or GMO-CSM protein did not affect on whole body omega-3 PUFAs, or liver gossypol. The results suggest that up to 75% of fish meal protein may be replaced by GI- or GMO-CSM protein in the diet of juvenile flounder without adverse effects on growth performance and body composition. Statement of relevanceCottonseed meal (CSM) is a potentially cost-effective alternative plant protein source for use in aquafeeds. The results suggest that a 75% of fishmeal protein could be replaced by genetically-improved and genetically modified (GMO) low gossypol based cottonseed flour protein in the diet of southern flounder. These ultra-low gossypol cottonseed flour proteins could be inexpensive protein sources for the commercial culture of southern flounder and other finfish species.

Effect of sand grain size on substrate preference and burial behaviour in cultured Japanese flounder juvenile, Paralichthys olivaceus

To improve release techniques of Japanese flounder, Paralichthys olivaceus, for the purpose of stock enhancement, the substrate preference and burial behaviour over a range of sand grain sizes were investigated under laboratory conditions. Ten 12-hr trials (i.e. 10 experimental groups), which, respectively, contained two different levels of substrates, demonstrated that juvenile flounder (55–78 mm total length) selected substrate of grain size (GS) < 2.00 mm, and the degree (from high to low) of their preference could be listed as Level 3 (0.50–2.00 mm) > Level 4 (0.25–0.50 mm) > Level 5 (0.05–0.25 mm) > Level 2 (2.00–4.00 mm) > Level 1 (4.00–6.00 mm). The average burying of fish in substrates of GS < 2.00 mm was more than 93.1%. Additionally, along with food deprivation time increased from 12 to 24 hr, some flounder showed random off-bottom swimming behaviour, and the appearance of off-bottom swimming behaviour was <3.2%. These results suggest that releasing sites with appropriate substrate grain size (GS < 2.00 mm) are suitable for conducting mass releases of hatchery-reared flounder juveniles, and starvation prior to release should be minimized. The results of the present study are essential to improve stocking strategies of Japanese flounder and reduce the juvenile mortality after release.

Effect of β-glucooligosaccharides as a new prebiotic for dietary supplementation in olive flounder (Paralichthys olivaceus ) aquaculture

β-Glucooligosaccharides (BGO), produced from barley β-glucan, were used as a feed supplement (0.1%) for juvenile olive flounder (Paralichthys olivaceus) to identify and quantify its oral administration effects on innate immunomodulation and infectious disease protection. Juvenile flounders (14 ± 0.5 g) were divided into two groups fed either 0.1% BGO (treatment) or a standard diet (control) for 8 weeks. At the end of the experiment, investigation of the effects was carried out through systemic studies on growth performance, serum and mucus biochemical parameters, innate immunity, microvillus length, and relative pro-inflammatory cytokine gene expression. The results demonstrated that the BGO diet produced slightly higher levels of growth performance, serum protein, microvillus length and pro-inflammatory cytokine gene (tumour necrosis factor-α, interleukin [IL]-1β, and IL-6) expression without any significant differences (p > .05). All innate immunity parameters were up-regulated by BGO administration and, among these, respiratory burst, lysozyme and superoxide dismutase (SOD) were significantly different (p < .05). Fish in the both groups were challenged with Streptococcus iniae (1.35 × 108 CFU/ml), and BGO group was focused to confirm the promotion of innate immunity parameters. The results showed a significantly (p < .05) lower death rate compared with that of the control. Therefore, BGO could be used as a new prebiotic in future olive flounder aquaculture as well as to control streptococcosis.

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

ABSTRACTOtolith 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.

Survival, growth and physiological responses of juvenile Japanese flounder (Paralichthys olivaceus, Temminck &amp; Schlegel, 1846) exposed to different dissolved oxygen concentrations and stocking densities

The aim of this study was to determine to what extent juvenile Japanese flounder can adapt to different stocking densities in captivity and to examine whether growth and some physiological parameters critical for welfare might be affected by different dissolved oxygen levels. Japanese flounder (Paralichthys olivaceus) juveniles (initial weight 1.27 ± 0.04 g/fish) were reared at five stocking densities (500, 1,000, 1,500, 2,000, 2,500 ind/m3) and two levels of dissolved oxygen (DO) concentration (5.5 ± 0.5 mg/L or 14 ± 2 mg/L) with duplicate tanks for each treatment in water-recirculating systems for 40 days. Survival and activity of superoxide dismutase (SOD) were not affected either by stocking density or dissolved oxygen, whereas final body weight, specific growth rate (SGR) and feed conversion efficiency (FCE) of fish under low DO concentration decreased significantly with increasing stocking density. In contrast, growth of fish reared in high DO levels were unaffected by the stocking density. Furthermore, fish in this group had a higher feed intake and, consequently, grew faster (SGR) and achieved a higher final weight than fish reared at the low DO level. A significant reduction in hemoglobin (Hb) concentration and red blood cell (RBC) count of fish were recorded as DO concentration increased. Furthermore, the activity of protease decreased significantly with increasing stocking density and increased significantly with increasing DO concentration. The ventilation frequency results indicate that gill ventilation decreased significantly as DO levels increased. This study demonstrates that stocking density can affect the growth performance and physiological parameters critical for welfare of juvenile Japanese flounder. Also shown is that pure oxygen supplementation is an effective way to improve the growth of juvenile Japanese flounder when reared at a high stocking density.

Metabolite and gene expression responses in juvenile flounder Paralichthys olivaceus exposed to reduced salinities

Seawater salinity is one of the most important changeable environmental factors influencing the behavior, survival, growth and production of marine organisms. In this work, metabolite and gene expression profiles were used to elucidate the biological effects of reduced salinities in juvenile flounder Paralichthys olivaceus. Metabolic profiling indicated that both reduced salinities (23.3‰ and 15.6‰) enhanced proteolysis and disturbed osmotic regulation and energy metabolism in juvenile flounder P. olivaceus. Furthermore, the low salinity (15.6‰) enhanced anaerobic metabolism indicated by the elevated lactate in flounder tissue extracts. Gene expression profiles exhibited that reduced salinities could induce immune stress and oxidative stress and disturb energy metabolism in juvenile flounder P. olivaceus. In addition, reduced salinities might promote the growth and gonadal differentiation in juvenile flounder P. olivaceus.

Assessment and Potential Application of the Probiotic Strain, Bacillus amyloliquefaciens JFP2, Isolated from Fermented Seafood-Jeotgal in Flounder Paralichthys olivaceus Juveniles

The strain Bacillus amyloliquefaciens JFP2 was isolated from Jeotgal, traditional fermented seafood from Jeju Island, South Korea to assess its probiotic and antibacterial effect against fish pathogens. The isolate JFP2 was identified using scanning electron microscopic analysis and 16S rRNA gene sequence analysis. The isolated strain, B. amyloliquefaciens JFP2 showed a broad spectrum of antibacterial activity against both Gram-positive and Gram-negative fish pathogenic bacteria namely Aeromonas hydrophila, Edwardsiella tarda, Photobacterium damselase sup-sps, Photobacterium phosphoreum, Streptococcous parauberis, S. iniae and Vibrio anguillaram. The bacteriocin JFP2 with a molecular mass of 37 kDa that was purified by ammonium sulphate precipitation followed by carboxymethyl-Sephadex column chromatography, showed antibacterial activity at a range of temperatures, from 4º-45ºC and at a range of pH values from 2-12. However, loss of antibacterial activity was observed after treating the bacteriocin with proteolytic enzymes such as pepsin, proteinase K, and trypsin. The mode of antibacterial action shown by the bacteriocin JFP2 was bacteriolytic in nature, resulting in cell wall degradation of A. hydrophila. Dietary inclusion of the isolated JFP2 strain showed improved growth performance in juvenile flounder along with increased disease resistance against Streptococcus iniae. These results indicate that the bacteriocin JFP2 belongs to class IIIa bacteriocin that could be used as a potential alternative antibacterial agent to control pathogenic diseases in aquaculture and seafood industries.

Assessment and Potential Application of the Probiotic Strain, Bacillus amyloliquefaciens JFP2, Isolated from Fermented Seafood-Jeotgal in Flounder Paralichthys olivaceus Juveniles

The strain Bacillus amyloliquefaciens JFP2 was isolated from Jeotgal, traditional fermented seafood from Jeju Island, South Korea to assess its probiotic and antibacterial effect against fish pathogens. The isolate JFP2 was identified using scanning electron microscopic analysis and 16S rRNA gene sequence analysis. The isolated strain, B. amyloliquefaciens JFP2 showed a broad spectrum of antibacterial activity against both Gram-positive and Gram-negative fish pathogenic bacteria namely Aeromonas hydrophila, Edwardsiella tarda, Photobacterium damselase sup-sps, Photobacterium phosphoreum, Streptococcous parauberis, S. iniae and Vibrio anguillaram. The bacteriocin JFP2 with a molecular mass of 37 kDa that was purified by ammonium sulphate precipitation followed by carboxymethyl-Sephadex column chromatography, showed antibacterial activity at a range of temperatures, from 4º-45ºC and at a range of pH values from 2-12. However, loss of antibacterial activity was observed after treating the bacteriocin with proteolytic enzymes such as pepsin, proteinase K, and trypsin. The mode of antibacterial action shown by the bacteriocin JFP2 was bacteriolytic in nature, resulting in cell wall degradation of A. hydrophila. Dietary inclusion of the isolated JFP2 strain showed improved growth performance in juvenile flounder along with increased disease resistance against Streptococcus iniae. These results indicate that the bacteriocin JFP2 belongs to class IIIa bacteriocin that could be used as a potential alternative antibacterial agent to control pathogenic diseases in aquaculture and seafood industries.

Movement patterns of Chilean flounder (Paralichthys adspersus) inside Tongoy Bay (central northern Chile): Observations using passive acoustic telemetry

The movement patterns of juvenile and adult Chilean flounder (P. adpersus) were investigated inside Tongoy Bay using ultrasound signal acoustic receivers from June 2012 to March, 2013. Flounder landings in Tongoy Bay and Puerto Aldea from December 2011 to March 2013 were examined. Multiple regression analysis indicated that the Catch per Unit of Effort of Chilean flounder was significantly and negatively related to temperature and depth. Analyses of site- and time-specific length-frequency distributions indicated movement of Chilean flounder on the time scale of weeks, which was likely due to emigration of fish >30 cm in total length. A mark-recapture study was performed. Visible elastomer paint was used to tag 7,510 Chilean flounder. A total of 12 Chilean flounder individuals of different lengths were tagged with an ultrasound transmission device to monitor their movement inside Tongoy Bay. Adults flounder showed increased activity inside Tongoy Bay during the study period, likely due of the differences in length among the released individuals. Although differences were detected in the area occupied by juvenile and adult flounders in Tongoy Bay, it was also noticed that the smaller sized individuals exhibited changes in behavior after implanting the transmitters that resulted in impaired capacity to move freely. Key words: Chilean flounder, acoustic telemetry, movement patters, landing, mark-recapture.

A simulation model for estimating optimum stocking density of cultured juvenile flounder Paralichthys olivaceus in relation to prey productivity

The determination of the optimum stocking density of hatchery-reared juveniles in relation to the carrying capacity of the nursery ground is important to the success of any stock enhancement programs. In order to estimate the surplus productivity of nursery grounds available to support hatchery-reared fish, a population growth model for juvenile Japanese flounder Paralichthys olivaceus was developed by extending an ecophysiology-based model on growth of juvenile fishes. Parameters of the model were adjusted using field-collected data from Ohno Bay in northeastern Japan. This model predicted the optimum stocking density, namely the maximum number of hatchery-reared juvenile Japanese flounder released into the bay without decreasing the growth of conspecific wild juveniles owing to food limitation. The optimum stocking density for juveniles of 89mm total length was estimated to be 2000 individuals per 250,000m2 in Ohno Bay in 1989. Under the model, the most important factors affecting the optimum stocking density of Japanese flounder were the abundance of mysids and wild juveniles at the time of release. The effect of abundance of a competing flatfish species on growth of wild Japanese flounder was less important.

Individuals Maintain Similar Rates of Protein Synthesis over Time on the Same Plane of Nutrition under Controlled Environmental Conditions.

Consistent individual differences in animal performance drive individual fitness under variable environmental conditions and provide the framework through which natural selection can operate. Underlying this concept is the assumption that individuals will display consistent levels of performance in fitness-related traits and interest has focused on individual variation and broad sense repeatability in a range of behavioural and physiological traits. Despite playing a central role in maintenance and growth, and with considerable inter-individual variation documented, broad sense repeatability in rates of protein synthesis has not been assessed. In this study we show for the first time that juvenile flounder Platichthys flesus reared under controlled environmental conditions on the same plane of nutrition for 46 days maintain consistent whole-animal absolute rates of protein synthesis (As). By feeding meals containing 15N-labelled protein and using a stochastic end-point model, two non-terminal measures of protein synthesis were made 32 days apart (d14 and d46). As values (mass-corrected to a standard mass of 12 g) showed 2- to 3-fold variation between individuals on d14 and d46 but individuals showed similar As values on both days with a broad sense repeatability estimate of 0.684 indicating significant consistency in physiological performance under controlled experimental conditions. The use of non-terminal methodologies in studies of animal ecophysiology to make repeat measures of physiological performance enables known individuals to be tracked across changing conditions. Adopting this approach, repeat measures of protein synthesis under controlled conditions will allow individual ontogenetic changes in protein metabolism to be assessed to better understand the ageing process and to determine individual physiological adaptive capacity, and associated energetic costs of adaptation, to global environmental change.

Enhancement of antibody response by LAMP1 chimeric antigen in a DNA vaccine

Immunization by an antigen-encoding DNA (DNA vaccines) has shown promising results in rendering protection against some bacterial and viral diseases in fish. However, some studies showed that DNA vaccines induce variable levels of protection compared with the other types of vaccination. These results are driving the research interest against these infectious diseases to improve the efficacy of DNA vaccines. Recently, chimeric antigens in DNA vaccines have been reported to increase the protection against certain diseases by enhancing specific antibody responses. In this study, we assessed the use of a chimeric DNA vaccine by using lysosomeassociated membrane protein-1 (LAMP1) fused with the major capsule protein (MCP) from red seabream iridovirus. First, Japanese flounder (Paralichthys olivaceus) LAMP-1 (jfLAMP1) gene was cloned and characterized through tissue distribution and expression analysis. The ORF for jfLAMP1 gene has a length of 1248 bp that encodes for 415 aa (about 44 KDa) and shows highly conserved transmembrane and cytoplasmic domains as those of higher vertebrates. Then, jfLAMP1 was used as a carrier of MCP gene by replacing the intra-lysosomal domain, and subsequent inserting into an expression vector, pCI-neo. In the vaccination trial, juveniles of Japanese flounder were distributed into four groups: pCMCP (MCP in pCI-neo vector), pCLAMP1-MCP (chimeric DNA of jfLAMP1 and MCP in pCI-neo), rMCP (recombinant MCP) and pCI-neo (empty vector as a control). At 30 days post vaccination, blood samples were taken and the serum antibody titers were measured by ELISA. The 3 vaccinated groups showed higher antibody titers than pCI-neo group. The pCLAMP1-MCP vaccinated fish showed antibody levels significantly higher than pCMCP (p<0.05). These results will serve as a basis for the use of LAMP1-chimeric DNA vaccines to increase the immune response against certain infectious diseases, for which conventional DNA vaccines show marginal efficacy.

Can analysis of Platichthys flesus otoliths provide relevant data on historical metal pollution in estuaries? Experimental and in situ approaches

Despite recent efforts to manage them more efficiently, estuaries are natural sinks for a wide range of metal contaminants, many of which accumulate at potentially toxic concentrations for fish populations, posing a threat to recruitment and stocks. While analysis of metal concentrations in soft tissue and water samples calls for continuous and long-term sampling operations, the use of otoliths to study metal pollution may be one way of providing a historical record of pollutant exposure. In this study, we examine the potential use of otoliths as natural tracers of metal contamination. A “cocktail” of different metals (Cd, Pb and Ni) was used to test bio-accumulation in otoliths and tissue (liver, kidney, muscle and gills) extracted from juvenile flounder (Platichthys flesus). Assessment took place under controlled conditions over a three month period, with water exposure concentrations increasing every 3weeks. The concentrations used were natural (T1), X5 (T2), X10 (T3), and null (T4). Chemical analyses were carried out using an inductively coupled plasma optical emission spectrometer ICP-OES and atomic absorption spectrometer AAS for water and tissue, while otolith microchemistry analyses were performed using a femtosecond laser ablation-high resolution inductively coupled plasma mass spectrometer (fsLA-ICP-MS-HR). Significant differences between control and exposed individuals, as well as an increase in metal concentrations according to exposure level, were observed in all tissues except in muscle tissue. Significant increases in Pb were also detected in contaminated fish otoliths compared with control specimens, with the highest concentrations occurring in T3. Cartographies of Pb distribution in otoliths of both control and contaminated fish only showed high concentrations of Pb at the edge of contaminated fish otoliths, indicating an accumulation of metal during the experiment. Although the relationships between exposure level and Pb concentration in otoliths were complex, the concentrations were correlated with those in the water. Analysis of flounder specimens collected from 2007 to 2014 in the Gironde estuary (SW France) showed interannual variability in Pb concentrations, with higher values for fish otoliths from 2007 to 2010 than those from 2012 to 2014. This trend indicated a decrease in Pb in the Gironde estuary over the last decade, which is consistent with the results of other surveys on bivalves. Our study demonstrates that it is possible to use otolith microchemistry as a tool in assessing and retracing long-term metal pollution in estuarine systems.

PNA クランピングを用いたヒラメ稚魚と稚アオリイカにおける食性解析の試み

PNA クランピングを用いたヒラメ稚魚と稚アオリイカにおける食性解析の試み

The use of biomarkers as integrative tools for transitional water bodies monitoring in the Water Framework Directive context — A holistic approach in Minho river transitional waters

The Water Framework Directive (WFD) provides an important legislative opportunity to promote and implement an integrated approach for the protection of inland surface waters, transitional waters, coastal waters and groundwaters. The transitional waters constitute a central piece as they are usually under high environmental pressure and by their inherent characteristics present monitoring challenges. Integrating water quality monitoring with biological monitoring can increase the cost-effectiveness of monitoring efforts. One way of doing this is with biomarkers, which effectively integrate physical–chemical status and biological quality elements, dealing holistically with adverse consequences on the health of water bodies. The new Marine Strategy Framework Directive (MSFD) already incorporates the biomarker approach. Given the recent activities of OSPAR and HELCOM to harmonize existing monitoring guidelines between MSFD and WFD the use of similar methodologies should be fostered. To illustrate the potential of the biomarker approach, juveniles of flounder (Platichthys flesus) were used to evaluate the quality of the Minho river-estuary water bodies. The use of juveniles instead of adults eliminates several confounding factors such changes on the biological responses associated with reproduction. Here, a panel of well-established biomarkers, EROD, AChE, SOD, CAT, GST, LPO, ENA and FACs (1-Hydroxyrene) were selected and measured along with a gradient of different physical conditions, and integrated with trace elements characterization on both biota and sediments. In general, a clear profile along the water bodies was found, with low seasonal and spatial variation, consistent with a low impacted area. Overall, the results support the use of both the battery of biomarkers and the use of juvenile flounders in the monitoring of the water quality status within the WFD.

Turnover rates of carbon and nitrogen stable isotopes in juvenile marbled flounder Pleuronectes yokohamae estimated by diet switch

Many fish species generally migrate among habitats because of ontogenetic changes in their resource requirements; therefore, clarifying linkages among habitats throughout their life cycle is important for the management and conservation of their populations. Although carbon (δ13C) and nitrogen (δ15N) stable isotope ratios are widely used for elucidating fish migrations, information is still scarce how each fish species, including marbled flounder (Pleuronectes yokohamae), change the isotopic composition in their body tissues after they switch to a different diet during migrations. This study clarified isotopic turnover rates of marbled flounder during the juvenile stage using a diet-switch experiment in an aquarium. Furthermore, we statistically compared bulk samples (i.e., whole body without head and viscera) to partial muscle tissue samples to estimate the difference in the turnover rate as an index for diet switches. An exponential model was used to describe the temporal changes in δ13C and δ15N isotopic rates of juvenile marbled flounder. No significant difference was noted between the exponential models for changes in both δ13C and δ15N between muscle tissue and bulk samples, and the half-life values were similar for muscle and bulk samples for both δ13C and δ15N (14.0–18.5 days). These results suggest that bulk samples are a useful substitute for muscle tissues in isotopic analysis of juvenile fish and that stable isotope analysis and the estimated isotopic turnover rates in tissues of marbled flounder can be used to clarify their migration route in coastal areas.

Oxidative stress parameters in juvenile Brazilian flounder Paralichthys orbignyanus (Valenciennes, 1839) (Pleuronectiformes: Paralichthyidae) exposed to cold and heat shocks

The aim of this study was to determine oxidative stress parameters in the liver and gill of Brazilian flounder juveniles (307.0 ± 16.0 g and 30.0 ± 4.0 cm) submitted to different water temperature (17.1, 23.0 and 28.8ºC) for 72 h and maintained at salinity 25‰. After the acclimation of 7 days, in 23ºC, fish were transferred to 200 L tanks containing seawater (salinity 25‰) at 28.8ºC (heat shock), 17.1ºC (cold shock) or 23.0ºC (control), five replicates (five fish tank-1). The sampled collection occurred in 0 (pre-challenge), 3, 24, 48 and 72 h after temperature shock. Flounder exposed to 17.1ºC and 28.8ºC showed significantly higher TBARS levels and GST activity in the liver post-exposition (PE) in relation to the control (23ºC). CAT activity in liver present a significantly increase at 17.1ºC, in first 48 h, and subsequently decrease in 72 h PE in relation to 28.8ºC. The gills of flounder showed significantly higher TBARS levels, GST and CAT activity when submitted at 17.1 and 28.8ºC in relation to 23.0ºC. There were observed changes in lipid peroxidation levels (LPO), CAT and GST activities in the liver and gill of Brazilian flounder in response to reactive oxygen species (ROS) produced by thermal shocks.