Juvenile Flounder Paralichthys Olivaceus Research Articles

PCO2-driven seawater acidification affects aqueous-phase copper toxicity in juvenile flounder Paralichthys olivaceus: Metal accumulation, antioxidant defenses and detoxification in livers

Ocean acidification potentially influences the biotoxicity of metals and the antioxidant defense systems of marine organisms. This study investigated how pCO2-driven seawater acidification (SA) affected aqueous-phase copper (Cu) toxicity in the juvenile flounder Paralichthys olivaceus from the perspective of hepatic oxidative stress and damage to better understand the mechanisms underlying the biological effects produced by the two stressors. Fish were exposed to aqueous-phase Cu at relevant ambient and polluted concentrations (0, 5, 10, 50, 100 and 200 μg L−1) at different pH levels (no SA: pH 8.10; moderate SA: pH 7.70, pCO2 ∼1353.89 μatm; extreme SA: pH 7.30, pCO2 ∼3471.27 μatm) for 28 days. A battery of biomarkers in the livers was examined to investigate their roles in antioxidant defense and detoxification in response to coexposure. Hepatic Cu accumulation (30.22–184.90 mg kg−1) was positively correlated with Cu concentrations. The biomarkers responded adaptively to different redox states following SA and Cu exposure. In unacidified seawater, increases in Cu concentrations significantly induced hepatic lipid peroxidation (LPO, by up to 27.03 %), although compensatory responses in antioxidant defenses and detoxification were activated. Moderate SA helped maintain hepatic redox homeostasis and alleviated LPO through different defense strategies, depending on Cu concentrations. Under extreme SA, antioxidant-based defenses were activated to cope with oxidative stress at ambient-low Cu concentrations but failed to defend against Cu toxicity at polluted Cu levels, and LPO (by up to 63.90 %) was significantly induced. Additionally, thiols (GSH and MT) responded actively to cope with Cu toxicity under SA. SOD, CAT, EROD, and GST were also sensitively involved in defending against hepatic oxidative stress during coexposure. These findings highlight the notable interactive effects of SA and Cu and provide a basis for understanding antioxidant-based defenses in marine fish confronting environmental challenges.

ITRAQ-based proteomic analysis on the mitochondrial responses in gill tissues of juvenile olive flounder Paralichthys olivaceus exposed to cadmium

Cadmium (Cd) is an important heavy metal pollutant in the Bohai Sea. Mitochondria are recognized as the key target for Cd toxicity. However, mitochondrial responses to Cd have not been fully investigated in marine fishes. In this study, the mitochondrial responses were characterized in gills of juvenile flounder Paralichthys olivaceus treated with two environmentally relevant concentrations (5 and 50 μg/L) of Cd for 14 days by determination of mitochondrial membrane potential (MMP), observation of mitochondrial morphology and quantitative proteomic analysis. Both Cd treatments significantly decreased MMPs of mitochondria from flounder gills. Mitochondrial morphologies were altered in Cd-treated flounder samples, indicated by more and smaller mitochondria. iTRAQ-based proteomic analysis indicated that a total of 128 proteins were differentially expressed in both Cd treatments. These proteins were basically involved in various biological processes in gill mitochondria, including mitochondrial morphology and import, tricarboxylic acid (TCA) cycle, oxidative phosphorylation (OXPHOS), primary bile acid biosynthesis, stress resistance and apoptosis. These results indicated that dynamic regulations of energy homeostasis, cholesterol metabolism, stress resistance, apoptosis, and mitochondrial morphology in gill mitochondria might play significant roles in response to Cd toxicity. Overall, this study provided a global view on mitochondrial toxicity of Cd in flounder gills using iTRAQ-based proteomics.

Interactive effects of strontium and barium water concentration on otolith incorporation in juvenile flounder Paralichthys olivaceus.

Although the relationship between the incorporation of an element into otoliths and the concentration of the element in water has been extensively investigated in many fish species, the interactive effects of multiple elements in water on the otolith incorporation of an element are not adequately explored or well understood. In this study, 16 treatments in triplicate using strontium (Sr; 1, 2, 3 and 4 times the ambient baseline, 6.5 mg l-1) and barium (Ba; 1, 2, 4 and 6 times the ambient baseline, 40 μg l-1) as categorical variables in an orthogonal design were established to evaluate the relative or interactive effects of water elements on otolith elemental incorporation in juvenile flounder Paralichthys olivaceus (from 15 to 116 days post hatching). The results revealed that otolith incorporation (Me:CaOtolith) of Sr and Ba were positively dependent on the concentrations of the elements in water (Me:CaWater). Overall, Sr was incorporated into otoliths more efficiently than was Ba, and the partition efficiency (DMe) of both elements decreased with increasing water elemental concentrations. Increasing Sr concentrations in water appeared to negatively affect the uptake of Ba into otoliths rather than facilitate it, as previously reported in fish reared in freshwater and brackish water, or showed no effect on fish in seawater. Conversely, the Ba concentration in water did not influence the otolith uptake of Sr, which agrees with the findings for other fish species. When applying otolith microchemistry to fish ecology studies, it is essential to cautiously address the interactive effects of multiple elements in the environment on otolith elemental incorporation.

Acute and chronic toxicity effects of acrylonitrile to the juvenile marine flounder Paralichthys olivaceus.

Acrylonitrile (ACN) spills in marine environment have the potential to cause ecological hazards and consequences, but currently little is known about the disruptive effects of ACN on marine organisms. In the present study, we investigated the lethal and sublethal effects of ACN on juvenile flounder Paralichthys olivaceus. The results showed that the 96-h LC50 of ACN to P. olivaceus juveniles was 6.07mg/L. The fish were then exposed to different sublethal concentrations (0.1, 0.2, and 0.4mg/L) of ACN for 28days and then transferred to clean seawater and keep in clean seawater for 14days to simulate the conditions of a spill incident. Biomarkers (EROD, GST, SOD, AChE activity, and levels of LPO and DNA alkaline unwinding) were tested in liver and brain. The weight gain rates and specific growth rate of juvenile marine flounder exposed to ACN (≥ 0.1mg/L) for 28days decreased significantly, indicating that ACN had an inhibitory effect on juvenile growth. Deformity of fish tails was observed on individuals exposed to the highest concentration (0.4mg/L ACN) for 14days, and the malformation rate was 38% after 28-day exposure. The present study provides the first evidence that ACN causes inhibition of AChE activity in fish brain. Furthermore, the results showed that ACN can significantly inhibit SOD activity and cause lipid peroxidation and DNA damage in fish brain. The results indicated that brain is more sensitive to ACN toxicity compared to liver and provides a suitable tissue for biomonitoring. The biomarkers measured during the depuration period showed that the effects caused by ACN were reversible when the exposure concentration was lower than 0.4mg/L. These results highlight the adverse effects of ACN in brain of fish, which should be considered in environmental risk assessment. Biomarkers including AChE activity, LPO, and DNA damage of brain tissue should be included in fish bioassays for toxic effect assessment of ACN spills.

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.

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.

Compensatory Growth of Juvenile Flounder Paralichthys olivaceus L. and Changes in Biochemical Composition and Body Condition Indices during Starvation and after Refeeding in Winter Season

Compensatory growth and changes in biochemical composition, hematocrit and body condition indices of juvenile flounder Paralichthys olivaceus were assessed during starvation and after refeeding. Twenty juvenile fish were stocked into each 200-L flow-through tank to give five treatments with three replicates per treatment: control group fish (C) were hand fed to apparent satiation twice daily for 8 wk, whereas the Sl, S2, S3, and S4 fish were hand fed to apparent satiation twice daily for 7, 6, 5, and 4 wk after 1, 2, 3, and 4 wk of starvation, respectively. During starvation, weight decreased linearly with periods of feed deprivation up to 3 wk. Survival was not significantly different among treatments. At the end of the feeding trial, weight gain (g/fish) and specific growth rate (SGR) of flounder in S2 was significantly (P < 0.05) higher than those of fish in S3 or S4, but not significantly different from those of fish in C or Sl. Feed consumption of flounder (g/fish) was proportional to duration of feeding except for that of fish in S2. Feed efficiency ratio (FER) and protein efficiency ratio (PER) values for flounder in S2 were significantly (P < 0.05) higher than those for fish in C, but not significantly different from those for fish in Sl, S3, or S4. During starvation, hepatosomatic index (HSI) and lipid content of flounder without liver decreased with periods of feed deprivation. However, HSI and condition factor (CF) for flounder in S2 were significantly (P < 0.05) higher than those for fish in Sl, S3, S4 and C except for CF in Sl at the end of the feeding trial. Proximate composition of flounder without the liver was not significantly different among treatments at the end of the feeding trial. In considering above results, juvenile flounder achieved compensatory growth with up to 2-wk feed deprivation. Compensatory growth of flounder fed for 6 wk after 2-wk feed deprivation was well supported by improvement in SGR, FER, and PER. HSI could be a good index to monitor changes in body condition during starvation and after refeeding.

Effect of various levels of lipid exchanged with dextrin at different protein level in diet on growth and body composition of juvenile flounder Paralichthys olivaceus

A 3 × 3 factorial experiment was conducted to determine proper levels of dietary protein, lipid and dextrin for juvenile flounder. Nine experimental diets were formulated to contain three protein levels (410, 460 and 510 g kg−1) and three lipid levels (60, 130 and 190 g kg−1) with corresponding dextrin levels (250, 150 and 50 g kg−1). Triplicate groups of fish (8.9 ± 0.4 g) were hand-fed the diets to apparent satiation for 7 weeks in flow-through system. Specific growth rate was the highest in fish fed the 510 g kg−1 protein diet with 60 g kg−1 lipid, and was not significantly different from that of fish fed 460 g kg−1 protein diet with 60 g kg−1 lipid. Feed efficiency ratio tended to increase as dietary protein level increased. The feed efficiency ratio of fish fed the 510 g kg−1 protein diets with 60–190 g kg−1 lipid levels was not significantly different from that of fish fed 460 g kg−1 protein diet with 60 g kg−1 lipid. Daily feed intake tended to decrease with increasing dietary lipid level at each protein level. Daily protein intake increased with increasing dietary protein level at 60 g kg−1 lipid level. Hepatosomatic index and visceralsomatic index increased with increasing dietary lipid level at each protein level. The lipid contents of liver, viscera and whole body, and concentrations of plasma total cholesterol and triglyceride increased with increasing dietary lipid levels; however, no significant difference was observed in the contents of dorsal muscle lipid. The results of this study suggest that the diet containing 460–510 g kg−1 protein with low lipid level (60 g kg−1) is optimal for growth and efficient feed utilization of juvenile flounder.

Size dominance affecting the social interactions and growth of juvenile Japanese flounder, Paralichthys olivaceus

Juvenile flounder Paralichthys olivaceus were size‐graded into three groups (small, large and mixing of small and large) and social interactions (feeding, aggressive attacking and activity) and growth of the fish were investigated. Growth of the subordinates (small flounder) was markedly suppressed by the presence of the dominants (large flounder). Dominants did not significantly suppress the overall food intake of the subordinates, but exhibited high aggressive attacking on the subordinates and consequently inhibited their activity. Size dominant interactions had little influence on the aggressive behaviour, feeding, activity and growth of the dominants. Results substantiated the hypothesis that the retardation in the growth of the subordinate flounder was attributed to the physiological costs of ‘stress’ by the aggression of the dominants. Individual differences in aggressive behaviour, feeding and activity were at least partly responsible for the growth depensation of the juvenile flounder. The dominants could not effectively defend the food in excess and disproportional food acquisition of the subordinates and dominants was not the primary mechanism responsible for the size hierarchy effect. In culture, size grading of the early juvenile flounder could markedly improve the growth and survival and thus possibly increase the overall biomass production of the flounder.

Effects of feeding frequency and dietary energy level on growth and body composition of juvenile flounder, Paralichthys olivaceus (Temminck &amp; Schlegel)

A two (dietary energy levels; low energy, LE and high energy, HE) × 4 (feeding frequency; one feeding in 2 days, one feeding daily, two times daily and three times daily) factorial experiment was performed to determine the effects of dietary energy level and/or feeding frequency on the growth and body composition of juvenile flounder Paralichthys olivaceus. The survival rate was not significantly different among treatments. Weight gain of fish fed the LE and HE diets significantly increased as feeding frequency increased. The weight gain of fish fed the LE diet was higher at each level of increasing feeding frequency. The weight gain of fish fed the HE diet was higher than that of fish fed the LE diet once in 2 days and once daily, but weight gain of fish fed the HE diet was lower than the LE diet three times daily. Daily feed intake was significantly influenced by feeding frequency, but not by dietary energy level. Feed efficiency of fish fed the HE diet once daily was significantly higher than that of fish fed the LE diet once in 2 days, but no significant difference in feed efficiency was observed among other groups of fish. The protein efficiency ratio of fish fed the LE diet once in 2 days was the lowest. Feeding frequency and dietary energy level had a significant effect on the body lipid content. A feeding frequency of two times or three times daily would be effective, depending on dietary energy level for maximum growth of juvenile flounder grown from 3.5 to 15 g.

Effects of Dietary Protein and Energy Levels on Growth and Body Composition of Juvenile Flounder Paralichthys olivaceus

AbstractA feeding trial of three protein levels (30, 40 and 50%) and two energy levels (300 and 400 kcal/100‐g diet) factorial design with three replications was carried out to investigate the proper dietary protein and energy levels for the growth of juvenile flounder Paralichthys olivaceus. Weight gain of fish tended to improve with increasing dietary protein level. Weight gain of fish fed either the 40% or 50% protein diet with 300 kcal/100‐g diet was significantly higher (P &lt; 0.05) than with 400 kcal/100‐g diet. The best weight gain was obtained from fish fed the 50% protein diet with 300 kcal/100‐g diet. Feed efficiency tended to improve with increasing dietary protein level. However, dietary energy level had no significant effect on feed efficiency of fish fed the 30% or 50% protein diet, but that of fish fed the 40% protein diet with 300 kcal/100‐g diet was significantly higher than with 400 kcal/100‐g diet. Protein retention tended to increase as dietary protein level increased and energy level decreased. Lipid content of fish fed the diet containing 400 kcal/100‐g diet was significantly higher than that of fish fed the diet containing 300 kcal/100‐g diet at all protein levels. Fatty acid compositions such as linoleic acid, EPA (20:5n‐3) and DHA (22:6n‐3) offish were directly affected by dietary lipid (squid liver oil and/or soybean oil) used for energy source. Based on the above results, it can be concluded that the proper dietary protein and energy levels for the growth of juvenile flounder are 50% and 300 kcal/100‐g diet, respectively.

ヒラメの白化個体出現に及ぼす変態期の餌料の影響

The dietary-related albinism in juvenile flounder Paralichthys olivaceus was investigated by offering several foods to the larvae during metamorphosis. Newly hatched larvae were reared on rotifer Brachionus plicatilis alone until they reached 6.8mm in total length. Thereafter, the larvae were divided into 8 groups with duplicates and reared on rotifer, vitamin-enriched rotifer, Artemia salina nauplii, vitamin-enriched Artemia nauplii, fertilized eggs of red sea bream Pagrus major and micro formulated diet, singly or combined, until the juveniles reached develop-mental stage G (nearly 10mm in TL), and then were reared on rotifer and Artemia nauplii until they had completely metamorphosed. The appearance of albinism was monitored among the juveniles accepting various foods. The occurrence of albinism was the highest in the juveniles fed on rotifer and/or Artemia (88-98%), the intermediate in the juveniles fed on the vitamin-enriched rotifer (74-76%) and on the micro formulated diet (54-60%), and the lowest in the juveniles fed on the eggs of the red sea bream (4-20%). The red sea bream egg was the best food for reducing the albinism which occurs in hatchery-reared flounder.

ヒラメ稚魚が摂餌したアミの大きさと摂餌日周期性

Juvenile flounder Paralichthys olivaceus in the Sea of Japan mainly fed on mysids. Five mysid species were identified in the 197 stomachs of the flounder examined, and they contained 1673 specimens of mysids. Acanthomysis robusta was most numerous (93.5%). Other species were Neomysis japonica (5.3%), A. nakazatoi (0.9%), Nipponomysis perminuta (0.2%). Ni. toriumii (0.1%). Total length of broken mysid in stomach was estimated using the regression equations between total length and the six part lengths i.e. carapace length, carapace width, length of 6th abdominal segment, exopod length of uropod, endopod length of uropod and telson length. Maxi-mum size of A. robusta in stomach increased with size of juvenile flounder. Mysid eaten by the flounder less than 20mm in body length was 5.4mm, and ones consumed by the20-25mm and 25-30mm flounder became rapidly larger to 7.4mm and 9.7mm, respectively. Juveniles less than 35mm mainly fed on small mysids less than 5mm, whereas ones greater than 35mm actively fed on large mysids (>30% of mysid number in stomachs). The percentage of mysid weight in stomachs to body weight of flounder less than 30mm was peaked (4.9%) in the evening, whereas the percentage dropped to 0% after midnight. A similar diurnal feeding pattern was found for juveniles greater than 30mm.

Estimation of larval stage determining the appearance of albinism in hatchery-reared juvenile flounder Paralichthys olivaceus.

The larval stage of the flounder P. olivaceus, when their body color (normal coloration or albinism of juveniles) was determined, was estimated by two ways of feeding. One was to feed Brazilian Artemia nauplii and rotifers which induce albinism easily in a high percentage. The other was to feed wild zooplankton and rotifers which usually induce normal coloration in a high percentage. Larvae of 10 days old were divided into 8 lots of 1, 000 larvae each and were fed for 29-30 days on different feeding schedules at different growth stages by a combination of the 2 feeding methods. Based upon the percentage of normal coloration and albinism at the end of experiment, 8 experimental lots were divided into 3 groups. In the first group (Tanks 1-3) in which Artemia nauplii were changed to wild zooplankton before the larvae grew up to D stage, the highest percentage of normal coloration was observed (60.2-75.4%). In the third group (Tanks 5-8) fish began to accept wild zooplankton from E stage, the highest percentage of albinism was ob-served (90.9-100.0%). In the second group (Tank 4 only), wild zooplankton were given to larvae in place of Artemia nauplii when the larval developmental stages consisted of 25% D and 75% E, and this composition agreed well with the percentage occurrences of normal coloration (27%) and albinism (73%). From these results, D stage was estimated to be the critical stage deter-mining the juvenile's coloration in terms of kinds of live foods.