Molecular cloning and characterization of chemokine C-C motif ligand 34 (CCL34) genes from olive flounder (Paralichthys olivaceus).

Chemokines are a superfamily of chemotactic cytokines that regulate the migration and immune responses of leukocytes. Depending on the arrangement of the first two cysteine residues, chemokines are divided into four groups: CXC (α), CC (ß), C (γ), and CX3C (δ). Chemokine C-C motif ligand 34 (CCL34) is a member of the CC chemokine family and is known as a fish-specific CC chemokine. In this experiment, we analyzed the molecular cloning and characterization of the PoCCL34 gene in olive flounder (Paralichthys olivaceus), including CCL34a.3 (PoCCL34a.3) and CCL34b.3 (PoCCL34b.3). The amino acid sequence of PoCCL34 has four highly conserved cysteine residues and it has a C-C motif. Phylogenetic analysis revealed that PoCCL34 was phylogenetically clustered in the fish CCL34 subcluster. Recombinant PoCCL34 induced chemotaxis of head kidney leukocytes in a dose-dependent manner. Head kidney leukocytes stimulated with PoCCL34 also exhibited significant respiratory burst activity and increased expression of pro-inflammatory cytokines (IL-1ß, IL-6, and CXCL8), but the overall expression of interferon-related genes (IFN-α/ß, IFN-γ, Mx, and ISG15) did not increase. Olive flounder injected with recombinant PoCCL34 demonstrated increased expression of pro-inflammatory cytokines (IL-1ß and IL-6) in the head kidney. However, there was no increase in the expression of interferon-related genes (IFN-α/ß, IFN-γ, Mx, and ISG15). Additionally, recombinant PoCCL34 induced high lysozyme activity in the serum of the flounder. These results indicate that although PoCCL34 is not involved in the antiviral response, it may play a significant role in the overall immune response of the flounder, particularly in mediating the inflammatory response.

A deep-sea pathogenic Bacillus subtilis isolate employs different strategies to escape the killing of teleost and murine complements.

Bacillus subtilis subsp. subtilis G7 was isolated from a deep-sea hydrothermal vent and is pathogenic to pathogenic to fish (Japanese flounder) and mice. G7 is able to survive in host sera and phagocytes. In this study, we investigated the underlying mechanism of G7 serum resistance. We found that (i) the remaining complement activity was very low in G7-incubated flounder serum but high in G7-incubated mouse serum; (ii) cleaved C3 and C5 components were detected on flounder serum-incubated G7 but not on mouse serum-incubated G7; (iii) abundant uncleaved C5 was localized in G7-incubated mouse, but not flounder, serum; (iv) G7-incubated flounder, but not mouse, serum exhibited strong chemotactic activity; (v) pre-treatment with low-dose lysozyme abolished the serum resistance of G7. Hence, G7 activates flounder complement but is protected from complement-mediated destruction by its cell wall structure, while G7 prevents the activation of mouse complement. These results indicate that G7 employs different mechanisms to avoid the complement killing of different hosts.

Salinity-mediated changes in hematological parameters, stress, antioxidant responses, and acetylcholinesterase of juvenile olive flounders (Paralichthys olivaceus).

The purpose of this study was to confirm the limit of salinity tolerance in juvenile olive flounders (Paralichthys olivaceus) by changes in blood parameters, AChE, antioxidant and stress responses. The P. olivaceus (mean weight 38.8 ± 4.2 g and mean length 16.4 ± 1.2 cm) were exposed to different concentrations of salinity (seawater, 16, 8, 4, 2, and 0 psu) for 2 weeks. Plasma osmotic pressure was significantly decreased in the P. olivaceus at 0 psu. Hematological parameters such as hematocrit and hemoglobin were significantly decreased in the P. olivaceus at low salinity. Plasma components also changed significantly in the low salinity environment. As a stress indicator, cortisol was significantly increased at low salinity. SOD and GST antioxidant responses, were significantly increased. GSH level in the liver was significantly increased, whereas a significant decrease was observed in the gill GSH level. AChE was significantly increased in P. olivaceus at low salinity. The results of this study indicate that exposure to salinities lower than 8 psu leads to changes in hematological parameters, neurotransmitter, antioxidant and stress responses of P. olivaceus.

Particle and bacteria uptake by Japanese flounder (Paralichthys olivaceus) red blood cells: Size dependence and pathway specificity.

Red blood cells (RBCs) are traditionally considered non-professional phagocytes functioning predominately in oxygen transport. In the present study, we examined the ability of Japanese flounder (Paralichthys olivaceus), a teleost species with important economic values, RBCs to uptake inorganic particles and bacteria in different size/form, as well as the involving endocytic pathways. We found that flounder RBCs exhibited relatively high uptake/attachment capacities for 0.1 µm-1.0 µm (diameter) latex beads, but not for 2.0 µm beads. For the 0.1 µm beads, the uptake/attachment was executed through macropinocytosis and caveolae-mediated pathway, while for 0.5 µm and 1.0 µm beads, the uptake/attachment depended primarily on macropinocytosis and partially on the caveolin-mediated pathway. In addition to latex beads, flounder RBCs also exhibited an apparent capacity to engulf both live and inactivated bacteria. For live bacteria, the endocytosis was clathrin-mediated, while for inactivated bacteria, clathrin- as well as non-clathrin-mediated endocytosis were involved. Taken together, these results demonstrated that teleost RBCs possess particle uptake/attachment and bacteria phagocytosis capacities via different pathways that depend on the physical size and biological nature of the engulfed objects.

Distribution of vitellogenin in Japanese flounder (Paralichthys olivaceus) for biomarker analysis of marine environmental estrogens.

Estrogen pollution in marine environments has become a research hotspot due to its adverse effects on the reproduction of wild organisms. To early detection of estrogen pollution, this study developed two methods for detecting Japanese flounder vitellogenin (Vtg), a sensitive biomarker for environmental estrogens. Firstly, monoclonal antibodies (mAb) specific to Vtg were prepared using purified lipovitellin (Lv), a main Vtg-derived yolk protein. Anti-Lv mAb (C1F1) had the highest titer (1:256,000) and was labeled with fluorescein isothiocyanate to establish a direct immunofluorescence (DIF) method for histological detection of Vtg in tissues. Additionally, using the purified Lv and mAb, an enzyme-linked immunosorbent assay (ELISA) was developed and this assay had a detection limit of 0.75 ng/mL and a working range of 1.95-250 ng/mL. Furthermore, Vtg induction in the plasma of Japanese flounder exposed to 17ß-estradiol (E2), 17α-ethinylestradiol (EE2), and bisphenol A (BPA) were quantified by ELISA, and Vtg induction in the liver of EE2-exposed Japanese flounder were measured by DIF. Finally, the distribution of Vtg in Japanese flounder was detected using these two methods. The results revealed that Vtg mainly appeared in the terminal tail fin, liver, kidney, intestine, and spleen. Considering the high concentration of Vtg and easy sample collection, the terminal tail fin could be a new alternative to plasma for Vtg quantification, while kidney and liver are suitable for histological detection of Vtg.

Hematological parameters and antioxidant responses in olive flounder Paralichthys olivaceus in biofloc depend on water temperature.

The purpose of this study was to ascertain the optimum water temperature for breeding juvenile Paralichthys olivaceus in biofloc. Hemoglobin and hematocrit were significantly decreased when the temperature was higher than 28 °C. Plasma calcium, glucose, cholesterol, glutamic-oxaloacetic transaminase, and glutamic pyruvic transaminase were significantly elevated at high temperatures, whereas total protein was substantially lower. Superoxide dismutase and glutathione-S-transferase activities in the liver and gills were significantly elevated at high temperatures, whereas glutathione was significantly lower. This indicates that temperatures greater than 26 °C induced hematological changes and oxidative stress in the juvenile P. olivaceus in biofloc. We ascribe these changes to thermal stress.

Gene expression and hormone secretion profile of urotensin I associated with osmotic challenge in caudal neurosecretory system of the euryhaline flounder, Platichthys flesus.

The caudal neurosecretory system (CNSS) is a part of stress response system, a neuroendocrine structure unique to fish. To gain a better understanding of the physiological roles of CNSS in fluid homeostasis, we characterized the tissue distribution of urotensin I (UI) expression in European flounder (Platichthys flesus), analyzed the effect chronic exposure to seawater (SW) or freshwater (FW), transfer from SW to FW, and reverse transfer on mRNA levels of UI, L-type Ca2+ channels and Ca-activated K+ channels transcripts in CNSS. The tissue distribution demonstrated that the CNSS is dominant sites of UI expression, and UI mRNA level in fore brain appeared greater than other non-CNSS tissues. There were no consistent differences in CNSS UI expression or urophysis UI content between SW- and FW-adapted fish in July and September. After transfer from SW to FW, at 8 h CNSS UI expression was significantly increased, but urophysis UI content was no significantly changes. At 24 h transfer from SW to FW, expression of CNSS UI was no apparent change and urophysis UI content was reduced. At 8 h and 24 h after transfer from FW to SW UI expression and urophysis UI content was no significantly effect. The expression of bursting dependent L-type Ca2+ channels and Ca-activated K+ channels in SW-adapted fish significantly decreased compared to those in FW-adapted. However, there were no differences in transfer from SW to FW or from FW to SW at 8 h and 24 h. Thus, these results suggest CNSS UI acts as a modulator in response to osmotic stress and plays important roles in the body fluid homeostasis.

Pharmacokinetic profiles of florfenicol in spotted halibut, Verasper variegatus, at two water temperatures.

The pharmacokinetic profiles of florfenicol in the spotted halibut (Verasper variegatus) were investigated at 15 and 20°C water temperatures, respectively. Florfenicol content in plasma samples was analyzed using an HPLC method. Drug concentration versus time data were best fitted to a three-compartment model after a single intravenous administration (15 mg/kg BW), and fitted to a two-compartment model after an oral administration (30 mg/kg BW) at 15 and 20°C. The florfenicol concentration in the blood increased slowly during the 12 hr following an oral administration at 15°C, with a peak concentration (Cmax ) of 9.1 mg/L, and then declined gradually. The half-lives of absorption, distribution, and elimination phase were 2.18, 5.66 and 14.25 hr, respectively. The bioavailability (F) was calculated to be 24.14%. After an oral administration at 20°C, shorter half-lives of absorption (1.33 hr), distribution (2.51 hr) and elimination (9.71 hr), a higher Cmax (12.2 mg/L), and a similar F (23.98%) were found. Based on the pharmacokinetics and pharmacodynamics, an oral dose of 30 mg/kg BW was suggested to be efficacious for bacterial disease control in spotted halibut farming.

Characterization of UDP-Activated Purinergic Receptor P2Y6 Involved in Japanese Flounder Paralichthys olivaceus Innate Immunity.

Uridine 5'-diphosphate (UDP)-activated purinergic receptor P2Y6 is a member of a G-protein-coupled purinergic receptor family that plays an important role in mammalian innate immunity. However, the role of the P2Y6 receptor (P2Y6R) in fish immunity has not been investigated. In this report, we characterized a P2Y6R gene from Japanese flounder (Paralichthys olivaceus) and examined its role in fish innate immunity. Sequence analysis reveals that the Japanese flounder P2Y6R protein is conserved and possesses four potential glycosylation sites. Quantitative real-time RT-PCR analysis shows that P2Y6R is broadly distributed in all examined Japanese flounder tissues with dominant expression in the liver. In addition, P2Y6R gene expression was up-regulated in head kidney macrophages (HKMs) upon lipopolysaccharides (LPS) and poly(I:C) stimulations but down-regulated by LPS challenge in peripheral blood leukocytes (PBLs). Furthermore, pharmacological inhibition of the endogenous P2Y6 receptor activity by the potently selective P2Y6R antagonist, MRS 2578, greatly up-regulated pro-inflammatory cytokine interleukin (IL)-1ß, IL-6 and TNF-α gene expression in PBL cells treated with UDP. Moreover, LPS- and poly(I:C)-induced gene expression of IL-1ß and TNF-α in Japanese flounder PBL cells was attenuated significantly by inhibition of P2Y6R activity with antagonist MRS 2578. Collectively, we, for the first time, showed the involvement of functional purinergic P2Y6R in fish innate immunity.

Chronic stress inhibits growth and induces proteolytic mechanisms through two different nonoverlapping pathways in the skeletal muscle of a teleost fish.

Chronic stress detrimentally affects animal health and homeostasis, with somatic growth, and thus skeletal muscle, being particularly affected. A detailed understanding of the underlying endocrine and molecular mechanisms of how chronic stress affects skeletal muscle growth remains lacking. To address this issue, the present study assessed primary (plasma cortisol), secondary (key components of the GH/IGF system, muscular proteolytic pathways, and apoptosis), and tertiary (growth performance) stress responses in fine flounder ( Paralichthys adspersus) exposed to crowding chronic stress. Levels of plasma cortisol, glucocorticoid receptor 2 ( gr2), and its target genes ( klf15 and redd1) mRNA increased significantly only at 4 wk of crowding ( P < 0.05). The components of the GH/IGF system, including ligands, receptors, and their signaling pathways, were significantly downregulated at 7 wk of crowding ( P < 0.05). Interestingly, chronic stress upregulated the ubiquitin-proteasome pathway and the intrinsic apoptosis pathways at 4wk ( P < 0.01), whereas autophagy was only significantly activated at 7 wk ( P < 0.05), and meanwhile the ubiquitin-proteasome and the apoptosis pathways returned to control levels. Overall growth was inhibited in fish in the 7-wk chronic stress trial ( P < 0.05). In conclusion, chronic stress directly affects muscle growth and downregulates the GH/IGF system, an action through which muscular catabolic mechanisms are promoted by two different and nonoverlapping proteolytic pathways. These findings provide new information on molecular mechanisms involved in the negative effects that chronic stress has on muscle anabolic/catabolic signaling balance.

Dynamic responses of prolactin, growth hormone and their receptors to hyposmotic acclimation in the olive flounder Paralichthys olivaceus.

Prolactin (PRL) and growth hormone (GH) play important roles in regulating salt and water balance through osmoregulatory organs in vertebrates. The aim of this study was to investigate the dynamic changes of GH/PRL hormone gene expressions in the pituitary gland and their receptors in gill and kidney, as well as the plasma osmolality when the olive flounder fish Paralichthys olivaceus were acclimated in freshwater (FW) conditions. After transfer from seawater (SW) to freshwater (FW), the osmolality of FW-adaption fish reached the lowest level at 1d which rose slightly afterwards. However, the hormone gene expression of PRL increased from 2d, reaching its peak at 5d, and then decreased at 14d. At this time, the value was still significantly higher than the control, showing a similar trend to the plasma hormone PRL. In contrast, the pituitary mRNA level of GH significantly decreased at 1d and then returned to normal levels. The mRNA levels of PRL receptor (PRLR) in both gill and kidney displayed a similar trend to the pituitary PRL. We also observed the synchronous expression trend of the renal PRLR with pituitary PRL (5d) and the asynchronous expression peaks between branchial (8d) and renal PRLR (5d). Significant responses of GH and its receptor (GHR) in both gill and kidney during the FW-acclimation were not observed. Nevertheless, the gene expression of GH receptor variant (GHR-V) in both gill and kidney declined at 2d, indicating unknown osmoregulatory functions of GHR-V. Collectively, our results provided more insights of the PRL, GH and their corresponding receptors in modulating osmoregulatory responses, representing an important aspect of FW-acclimation in flounder fish.

Interactive effects of dietary leucine and isoleucine on growth, blood parameters, and amino acid profile of Japanese flounder Paralichthys olivaceus.

A 60-day feeding trial was conducted to assess the interactions of dietary leucine (Leu) and isoleucine (Ile) on Japanese flounder. Fish of 2.69 ± 0.04 g were fed experimental diets containing two levels of Leu (2.58 and 5.08% of diet) combined with three levels of Ile (1.44, 2.21, and 4.44% of diet), respectively. After the feeding trial, growth, proximate composition, muscle total amino acid profile, blood parameters, mucus lysozyme activity, and stress tolerance to freshwater were measured. Statistically significant (P < 0.05) interactive effects of Leu and Ile were found on growth parameters (final body weight, body weight gain, and special growth rate) of Japanese flounder. Antagonism was discovered in high dietary Leu groups, while stimulatory effects were obtained for increased dietary Ile in low Leu groups. Interactive effects of these two branched-chain amino acids were also found on hepatosomatic index of test fish. In addition, crude lipid content of fish whole body was significantly altered by various diets, with antagonism observed in low dietary Leu groups. Interactive effects also existed in muscle amino acid profiles for low fish meal diets, but no interactive impacts were observed on blood parameters. Furthermore, lysozyme activities and freshwater stress were significantly affected by different diets. And antagonism was found on lysozyme activities in low Leu groups. Moreover, high Leu and high Ile levels of diet significantly altered freshwater stress tolerance of Japanese flounder. These findings suggested that dietary Leu and Ile can effect interactively, and fish fed with diets containing 2.58% Leu with 4.44% Ile and 5.08% Leu with 1.44% Ile showed better growth performance.

Molecular characterisation and expression of parathyroid hormone-related protein in the caudal neurosecretory system of the euryhaline flounder, Platichthys flesus.

Parathyroid hormone-related protein (PTHrP) is a hypercalcemic factor in fish, but the source of circulating PTHrP remains unclear. In this study investigation of the caudal neurosecretory system (CNSS), considered one of major sources of PTHrP in fish, provided valuable insights into this regulatory system. We report pthrpa and pthrpb gene cloning, characterization, expression, and responses to low salinity and hypocalcemia challenge in flounder. The pthrpa and pthrpb precursors, isolated from a European flounder CNSS library, consist of 166 and 192 amino acid residues, respectively, with an overall homology of approximately 59.2%. Both precursors contain a signal peptide and a mature peptide with cleavage and amidation sites. The flounder PTHrPA and PTHrPB peptides share only 41% sequence identity with human PTHrPA. Quantitative PCR analysis demonstrated that the bone and bladder, are respectively major sites of pthrpa and pthrpb expression in flounder. Urophysectomy confirmed the CNSS as a likely contributor to circulating PTHrP peptides. There were no significant differences in CNSS pthrpa and pthrpb mRNA expression or plasma PTHrP levels between seawater (SW) and freshwater (FW)-adapted fish, though plasma total calcium concentrations were higher in FW animals. The intraperitonial administration of EGTA rapidly induced hypocalcemia and concomitant elevation in plasma PTHrP accompanied by increases in both pthrpa and pthrpb expression in the CNSS. Together, these findings support an evolutionary conserved role for PTHrP in the endocrine regulation of calcium.

Evidences for involvement of growth hormone and insulin-like growth factor in ovarian development of starry flounder (Platichthys stellatus).

Although gonadotrophins are major regulators of ovarian function in teleosts and other vertebrates, accumulating evidence indicates that the growth hormone (GH)-insulin-like growth factor (IGF) axis also plays an important role in fish reproduction. As a first step to understand the physiological role of the GH-IGF system in the ovarian development of starry flounder (Platichthys stellatus), the expression profiles of GH and IGF messenger RNAs (mRNAs) and plasma GH, IGF-I, estradiol-17ß (E2), and testosterone (T) levels during the ovarian development were investigated. The developmental stages of ovaries were divided into five stages (II, III, IV, V, and VI) by histological analysis. The hepatosomatic index (HSI) and gonadosomatic index (GSI) values increased and peaked at stage IV and stage V, respectively, and then declined at stage VI. Pituitary GH mRNA levels decreased sharply at stage III and raised to top level at stage VI. The hepatic IGF-I mRNA levels ascended to maximum value at stage V and then declined significantly at stage VI. However, the hepatic IGF-II mRNA levels remained stable and increased significantly at stage VI. In contrast, the ovarian IGF-I mRNA levels increased gradually and peaked at stage VI. The ovarian IGF-II mRNA levels were initially stable and increased significantly at stage V until the top level at stage VI. Consistent with the pituitary GH mRNA levels, plasma GH levels reduced sharply at stage III and remained depressed until stage V and then raised remarkably at stage VI. Plasma IGF-I level peaked at stage V and then declined to initial level. Plasma E2 level peaked at stage IV and then dramatically descended to the basal level. Plasma T level peaked at stage V and then declined significantly back to the basal level. Based on statistical analysis, significant positive correlations between hepatic IGF-I mRNA and GSI, ovarian IGF-II mRNA and hepatic IGF-II mRNA, ovarian IGF-I mRNA and ovarian IGF-II mRNA, and plasma IGF-I and plasma T were observed, respectively. These results suggest that the GH-IGF system may be involved in the ovarian development of starry flounder; GH and IGFs appear to play distinct roles in the regulation of the ovarian development in paracrine/autocrine manners. These findings extend our knowledge of the roles of the GH-IGF axis on reproduction regulation in fish.

Comparison of the effects of dietary single and multi-probiotics on growth, non-specific immune responses and disease resistance in starry flounder, Platichthys stellatus.

An 8-week feeding trial was conducted to evaluate the effects of dietary probiotics on growth performance and non-specific immune responses in starry flounder, Platichthys stellatus. Fish averaging 46.5 ± 0.65 g (mean ± SD) were fed one of the six experimental diets; one control (Cont), and five other diets were prepared by supplementing single-probiotics 1 (Bacillus subtilis; SP1, 2 × 109 CFU kg-1 diet), single-probiotics 2 (Bacillus licheniformis; SP2, 2 × 109 CFU kg-1 diet), multi-probiotics 1 (Bacillus subtilis + Bacillus licheniformis; MP1, 2 × 109 CFU kg-1 diet), multi-probiotics 2 (commercial probiotics; Bacillus subtills + Bacillus licheniformis + Paenibacillus polymyxa + Aspergillus oryzae + Saccharomyces cerevisiae; MP2, 2 × 109 CFU kg-1 diet) and oxytetracycline (OTC) at 5 g OTC kg-1 diet. At the end of 8 weeks feeding trial, weight gain (WG) and specific growth rate (SGR) of fish fed SP1, MP1 and MP2 diets were significantly higher than those of fish fed control diet (P < 0.05). Superoxide dismutase (SOD) activity of fish fed MP2 diet was significantly higher than those of fish fed OTC diet (P < 0.05). Nitro blue tetrazolium (NBT) activity and lysozyme activity of fish fed SP1, MP1 and MP2 diets were significantly higher than those of fish fed OTC diet (P < 0.05). However, there was no significant difference among fish fed SP1, SP2, MP1 and MP2 diets. During the Edwardsiella tarda challenge test, the first mortality occurred on day 2. After the 14 days challenge test, cumulative survival rate of fish fed MP1 and MP2 diets were significantly higher than those of fish fed control diet (P < 0.05). However, there was no significant difference among fish fed SP1, SP2, MP1, MP2 and OTC diets in survival rate at the termination of the challenge test. Although there was little advantage in immunological parameters with fish fed MP diets, single and multi-probiotics were equally effective statistically. These results demonstrated that single or multi-probiotics had equal beneficial effect as an antibiotic replacer in terms of growth performance, non-specific immune responses and disease resistance in starry flounder.

Toxic effects and depuration after the dietary lead(II) exposure on the bioaccumulation and hematological parameters in starry flounder (Platichthys stellatus).

Platichthys stellatus (mean length 20±2cm, mean weight 160.15±15g) were exposed to the different levels of dietary lead(II) at the concentrations of 0, 30, 60, 120, 240mg/kg for 4 weeks. Depuration was conducted for 2 weeks after exposure. The lead exposure over 60mg Pb/kg induced the significant bioaccumulation in tissues of P. stellatus (5-30µg/g tissue), except for brain and muscle where the exposure to 240mg Pb/kg caused the bioaccumulation (2-4µg/g tissue). The hematological parameters such as red blood cell (RBC) counts, hematocrit (Ht) value and hemoglobin (Hb) concentration were substantially decreased over 60mg Pb/kg, and lasted even after the depuration period. For plasma components, calcium and magnesium levels in plasma were generally decreased over 60mg Pb/kg, and glucose level was also mainly increased over 60mg Pb/kg. Total protein was significantly decreased over 120mg Pb/kg after 4 weeks exposure. Glucose and total protein showed the restoration after the depuration period in groups of fish exposed previously to over 60 and 120mg Pb/kg, respectively. However, other parameters that changed during the exposure over 60mg Pb/kg did not recovered. For enzymatic components in plasma, glutamic oxalate transminase (GOT), glutamic pyruvate transminase (GPT) and alkaline phosphatase (ALP) were significantly increased over 120mg Pb/kg, and there was only restoration observed after the depuration for ALP over 120mg Pb/kg.

Stress responses of starry flounder, Platichthys stellatus (Pallas) following water temperature rise.

Stress responses of starry flounder, Platichthys stellatus (Pallas) following water temperature rise were investigated to establish the influence of ambient temperature on this species. The physiological indicators of stress were plasma cortisol, glucose, aspartate aminotransferase, alanine aminotransferase, sodium, chloride, osmolality and triiodothyronine (T3). No significant difference in plasma parameters were observed among the experimental groups of 15 degrees C, 18 degrees C and 21 degrees C. Level of plasma cortisol (49.0-95.0 ng ml(-1)) and glucose (56.1-58.1 mg dl(-1)) of starry flounders kept at 24 degrees C-27 degrees C were significantly higher than those (cortisol: 20.4-23.6 ng ml(-1), glucose: 40.6-47.1 mg dl(-1)) observed in the 15 degrees C-21 degrees C groups. Changes in aspartate aminotransferase and alanine aminotransferase following water temperature rise showed a similar pattern to plasma cortisol and glucose. Starry flounders exposed to 27 degrees C exhibited higher plasma sodium (164.7 mmol l(-1)), chloride (147.6 mmol l(-1)), and osmolality (450.7 mOsm kg(-1)) than those (sodium: 154.0-158.7 mmol l(-1), chloride: 139.1-140.4 mmol l(-1), osmolality: 375.1-383.8 mOsm kg(-1)) fish exposed to 15-21 degrees C. Though plasma T3 (29.4 ng ml(-1)) of starry flounder increased at 24 degrees C, this hormone was significantly lower (19.3 ng ml(-1)) in fish kept at 27 degrees C than those (24.6 ng ml(-1)) the fish at 15 degrees C. This phenomenon seems to be directly associated with long-term fasting. Accordingly, the results suggested that starry flounders got stressed with osmoregulatory disturbances above 24 degrees C.

Hexestrol- and nonylphenol-induced differential vitellogenin synthesis in female and male barfin plaice Liopsetta pinnifasciata.

The effects of hexestrol (HXS) and nonylphenol (NP) on plasma vitellogenin (Vtg) concentration in barfin plaice Liopsetta pinnifasciata was studied during spring and autumn experiment. In L. pinnifasciata two "complete" forms of Vtgs, namely VtgAa and VtgAb, were previously described which may be separated due to molecular mass of their largest polypeptide in SDS-PAGE. In spring, the injection of HXS led to an increase in Vtg concentrations in both females and males. SDS-PAGE analysis of plasma from HXS-exposed fish produced only one prominent band at a molecular mass of 180 kDa that corresponds to an increase in VtgAb levels. NP injected in fish in spring induced statistically significant increasing of Vtg concentration in males, and only one type of Vtg, as in case of HXS, accumulated in plasma. In autumn, the injection of HXS results to the increase of Vtg concentration in the plasma of females and males, electrophoretic analysis of plasma proteins showed that only a 98 kDa polypeptide, corresponding to the VtgAa-type showed a significant increase. The blood plasma ratios of VtgAa and VtgAb in experimental fish are discussed in relation to the season and stage of reproductive cycle.

Pharmacokinetics of amoxicillin trihydrate in cultured olive flounder (Paralichthys olivaceus).

The study was aimed at investigating the pharmacokinetics of amoxicillin trihydrate (AMOX) in olive flounder (Paralichthys olivaceus) following oral, intramuscular, and intravenous administration, using high-performance liquid chromatography following. The maximum plasma concentration (Cmax ), following oral administration of 40 and 80 mg/kg body weight (b.w.), AMOX was 1.14 (Tmax , 1.7 h) and 0.76 µg/mL (Tmax , 1.6 h), respectively. Intramuscular administration of 30 and 60 mg/kg of AMOX resulted in Cmax values of 4 and 4.3 µg/mL, respectively, with the corresponding Tmax values of 29 and 38 h. Intravenous administration of 6 mg/kg AMOX resulted in a Cmax of 9 µg/mL 2 h after administration. Following oral administration of 40 and 80 mg/kg AMOX, area under the curve (AUC) values were 52.257 and 41.219 µg/mL·h, respectively. Intramuscular 30 and 60 mg/kg doses resulted in AUC values of 370.274 and 453.655 µg/mL·h, respectively, while the AUC following intravenous administration was 86.274 µg/mL·h. AMOX bioavailability was calculated to be 9% and 3.6% following oral administration of 40 and 80 mg/kg, respectively, and the corresponding values following intramuscular administration were 86% and 53%. In conclusion, this study demonstrated high bioavailability of AMOX following oral administration in olive flounder.

Pharmacokinetics of difloxacin in olive flounder Paralichthys olivaceus at two water temperatures.

In this study, the pharmacokinetics profiles of difloxacin in the olive flounder (Paralichthys olivaceus) were investigated following intravenous and oral administration (10 mg/kg BW) at 14 and 22 °C water temperatures. Plasma and tissue samples (muscle, liver, and kidney) were analyzed using an HPLC method. The results showed that the plasma concentration-time data for difloxacin were described commendably by two-compartment open model at the two water temperatures. The absorption half-life (t(1/2ka)) of difloxacin after oral administration were 2.08 and 1.10 h at 14 and 22 °C, respectively; whereas the elimination half-life (t(1/2ß)) was 4.41 and 2.38 h, respectively. The muscle concentration of 1.35 ± 0.19 µg/g was observed at 9 h at 14 °C, and 2.11 ± 0.33 µg/g at 6 h at 22 °C, respectively. For liver, the peak concentration of difloxacin 2.43 ± 0.30 µg/g occurred at 6 h at 14 °C, which was lower than the 3.34 ± 0.24 µg/g peak that occurred at 4 h at 22 °C. The calculated bioavailability of difloxacin was 68.07% at 22 °C, which was higher than the 53.43% calculated for 14 °C. After intravenous administration, the t(1/2ß) were 4.79 and 2.81 h at 14 and 22 °C, respectively. The results indicate that the peak concentrations in muscle and liver at 14 °C are approximately half of those achieved at 22 °C. However, the C(max) in kidney at 14 and 22 °C were similar. The Vd values were 1.20 and 1.75 L/kg at 14 and 22 °C, respectively. These data indicated that both temperature and drug administration had significant effects on the elimination of difloxacin, and lower temperature or oral administration resulted in lower elimination.