Enhancement of immune proteins expression in skin mucus of Japanese flounder Paralicthys olivaceus upon feeding a diet supplemented with high concentration of ascorbic acid.

To search immune defense proteins in skin mucus of Japanese flounder fed with a diet containing high concentration of ascorbic acid, we carried out 2D-PAGE and compared the resolved pattern of proteins between control group that fed commercial diet and ascorbic acid supplemented group (AsA group) fed a diet supplemented with high concentration of ascorbic acid (2,000 mg/kg) for 7 days. The results revealed that there were many proteins exhibited distinct increase in AsA group. Among them, 6 regions that showed a dramatic elevation were chosen for protein identification using LC-MS/MS analysis and Mascot database search. Six proteins were identified, i.e. serotransferrin (Sero), transferrin (Trans), warm temperature acclimation-related 65 kDa protein (Wap65), complement component c3 (C3), hemoglobin beta-A chain (Hbß) and apolipoprotein A-1 (Apo). Quantitative RT-PCR analysis showed that the mRNA level of Hbß in epidermis of AsA group gave much higher increase (11.6 folds) than control group; the levels of Sero/Trans, Wap65, C3 and Apo showed no apparent difference between the two groups. The mRNA levels of wap65 and c3 in the liver and Apo in the kidney of AsA group exhibited significant increase in comparison to control group. In the case of secreted immunoglobulin M (IgM) and lysozyme (lyz), no difference of the mRNA levels of IgM in epidermis, gill, kidney, spleen and intestine, and lyz in epidermis, gill, spleen and intestine, was observed. The results of in situ hybridization confirmed the elevation of Hbß mRNA level in the epidermis tissue of AsA group. Our present study provided additional evidence showing the effectiveness of AsA in activating innate immune defense system in skin mucosal tissue of fish.

Species-specific metabolism of naphthalene and phenanthrene in 3 species of marine teleosts exposed to Deepwater Horizon crude oil.

The 2 most abundant polycyclic aromatic hydrocarbons (PAHs) measured in Deepwater Horizon crude oil, naphthalene and phenanthrene, and their associated homologs have both been shown to be acutely toxic in fish. Although fish have a relatively high metabolic capacity for PAHs, hydroxylated PAH (OH-PAH) derivatives formed during the initial metabolic response can negatively impact the health of fish. Species-specific metabolism of naphthalene and phenanthrene was evaluated in 3 marine teleosts, red drum (Scianops ocellatus), Florida pompano (Trachinotus carolinus), and southern flounder (Paralichthys lethostigma). Fish were exposed to Deepwater Horizon crude oil by intraperitoneal injections at time 0 and 48 h, with bile sampling events at 24 and 72 h post injection. The data suggested metabolic induction in Florida pompano and red drum, whereas southern flounder may have demonstrated metabolic fatigue. By 24 h post injection, overall profiles of red drum and southern flounder were dominated by hydroxylated phenanthrene metabolites; conversely, the Florida pompano profiles were dominated by monohydroxylated naphthalenes. In addition, Florida pompano had faster overall relative biotransformation rates, suggesting their potential decreased susceptibility to adverse effects. Red drum and southern flounder had much lower relative biotransformation rates, indicating their probable susceptibility to adverse outcomes after naphthalene and phenanthrene exposures. To our knowledge, the present study is the first to investigate monohydroxylated PAHs in fish exposed to Deepwater Horizon oil. Environ Toxicol Chem 2017;36:3168-3176. © 2017 © 2017 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

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.

RNA seq- and DEG-based comparison of developmental toxicity in fish embryos of two species exposed to Iranian heavy crude oil.

To determine and compare the toxic effects of Iranian heavy crude oil (IHCO) on the embryonic development of two fish species, we examined transcriptome profiles using RNA-seq. The assembled contigs were 66,070 unigenes in olive flounder embryos and 76,498 unigenes in spotted seabass embryos. In the differential gene expression (DEG) profiles, olive flounder embryos showed different up- and down-regulated patterns than spotted seabass embryos in response to fresh IHCO (FIHCO) and weathered IHCO (WIHCO). In this work, we categorized DEG profiles into six pathways: ribosome, oxidative phosphorylation, Parkinson's disease, Alzheimer's disease, Huntington's disease, and cardiac muscle contraction, validating the expression patterns of 13 DEGs using real-time quantitative RT-PCR. The expression of the CYP1A, CYP1B1, and CYP1C1 genes in spotted seabass embryos was higher than in olive flounder embryos, whereas genes related to cell processing, development, and the immune system showed the opposite trend. Orthologous gene cluster analysis showed that olive flounder embryos were sensitive (fold change of genes with cutoff P<0.05) to both FIHCO and WIHCO, but spotted seabass embryos exhibited higher sensitivity to WIHCO than FIHCO, indicating that species-specific differences are likely to be reflected in population levels after oil spills. Overall, our study provides new insight on the different embryonic susceptibilities of two marine fish species to FIHCO and WIHCO and a better understanding of the underlying molecular mechanisms via RNA-seq and DEGs.

Developmental toxicity in flounder embryos exposed to crude oils derived from different geographical regions.

Crude oils from distinct geographical regions have distinct chemical compositions, and, as a result, their toxicity may be different. However, developmental toxicity of crude oils derived from different geographical regions has not been extensively characterized. In this study, flounder embryos were separately exposed to effluents contaminated by three crude oils including: Basrah Light (BLO), Pyrenees (PCO), and Sakhalin Vityaz (SVO), in addition to a processed fuel oil (MFO-380), to measure developmental toxicity and for gene expressions. Each oil possessed a distinct chemical composition. Edema defect was highest in embryos exposed to PCO and MFO-380 that both have a greater fraction of three-ring PAHs (33% and 22%, respectively) compared to BLO and SVO. Observed caudal fin defects were higher in embryos exposed to SVO and MFO-380, which are both dominated by naphthalenes (81% and 52%, respectively). CYP1A gene expressions were also highest in embryos exposed to SVO and MFO-380. Higher incidence of cardiotoxicity and lower nkx 2.5 expression were detected in embryos exposed to PCO. Unique gene expression profiles were observed in embryos exposed to crude oils with distinct compositions. This study demonstrates that crude oils of different geographical origins with different compositional characteristics induce developmental toxicity to different degrees.

Transcriptomic response to water accommodated fraction of crude oil exposure in the gill of Japanese flounder, Paralichthys olivaceus.

Illumina-based RNA-seq was used to determine the short-term transcriptomic responses of Paralichthys olivaceus gill to an environmentally relevant level of water accommodated fraction (WAF) of crude oil. 213,979 transcripts and 128,482 unigenes were obtained. Differential expression analysis revealed that 1641 and 2142 genes were significantly up- and down-regulated. Enrichment analysis identified a set of GO terms and putative pathways involved in the response of P. olivaceus to WAF exposure. Analysis of the transcripts revealed the effective protective mechanisms of P. olivaceus to reduce the toxic effects of WAF. Moreover, WAF exposure induced the metabolism of energy substrates, and downstream pathway genes were modified to provide protection against toxic damage. Transcripts analysis demonstrated that the genes involved in circadian rhythm signaling were regulated in gills of P. olivaceus exposed to WAF. These results provide insights into the mechanisms of WAF-induced toxicity in fishes and into the WAF-sensitive biomarkers in P. olivaceus.

Differential regulation of taurine biosynthesis in rainbow trout and Japanese flounder.

Animals have varied taurine biosynthesis capability, which was determined by activities of key enzymes including cysteine dioxygenase (CDO) and cysteine sulfinate decarboxylase (CSD). However, whether CDO and CSD are differentially regulated across species remains unexplored. In the present study, we examined the regulations of CDO and CSD in rainbow trout and Japanese flounder, the two fish species with high and low taurine biosynthesis ability respectively. Our results showed that the expression of CDO was lower in rainbow trout but more responsive to cysteine stimulation compared to that in Japanese flounder. On the other hand, both the expression and catalytic efficiency (k(cat)) of CSD were higher in rainbow trout than those of Japanese flounder. A three-residue substrate recognition motif in rainbow trout CSD with sequence of F126/S146/Y148 was identified to be responsible for high k(cat), while that with sequence of F88/N108/F110 in Japanese flounder led to low k(cat), as suggested by site-directed mutagenesis studies. In summary, our results determined new aspects of taurine biosynthesis regulation across species.

Differential Toxicokinetics Determines the Sensitivity of Two Marine Embryonic Fish Exposed to Iranian Heavy Crude Oil.

Interspecific difference in the developmental toxicity of crude oil to embryonic fish allows the prediction of injury extent to a number of resident fish species in oil spill sites. This study clarifies the comparative developmental effects of Iranian heavy crude oil (IHCO) on the differences of biouptake and toxic sensitivity between embryonic spotted sea bass (Lateolabrax maculates) and olive flounder (Paralichthys olivaceus). From 24 h after exposure to IHCO, several morphological defects were observed in both species of embryonic fish, including pericardial edema, dorsal curvature of the trunk, developmental delay, and reduced finfolds. The severity of defects was greater in flounder compared to that in sea bass. While flounder embryos accumulated higher embryo PAH concentrations than sea bass, the former showed significantly lower levels of CYP1A expression. Although bioconcentration ratios were similar between the two species for some PAHs, phenanthrenes and dibenzothiophenes showed selectively higher bioconcentration ratios in flounder, suggesting that this species has a reduced metabolic capacity for these compounds. While consistent with a conserved cardiotoxic mechanism for petrogenic PAHs across diverse marine and freshwater species, these findings indicate that species-specific differences in toxicokinetics can be an important factor underlying species' sensitivity to crude oil.

Ferritin M of Paralichthys olivaceus possesses antimicrobial and antioxidative properties.

Ferritin is an evolutionarily conserved protein that plays a vital role in maintaining iron homeostasis. In this study, we identified a ferritin M (PoFerM) from Japanese flounder (Paralichthys olivaceus) and analyzed its biological property. PoFerM is composed of 176 amino acid residues and contains the conserved ferroxidase diiron center and the ferrihydrite nucleation center typical of M ferritins. Expression of PoFerM occurred in multiple tissues and was most abundant in blood. Bacterial infection upregulated PoFerM expression in head kidney, spleen, and liver in a time-dependent manner. Recombinant PoFerM (rPoFerM) purified from Escherichia coli exhibited iron-chelating activity and inhibited bacterial growth, whereas rPoFerMM, the mutant protein that bears alanine substitution at two conserved residues of the ferroxidase center and the ferrihydrite nucleation center, failed to do so. Oxidative protection analysis showed that rPoFerM, but not rPoFerMM, was able to alleviate the deleterious effect of H2O2-induced free radicals on plasmid DNA and primary flounder cells. Together these results indicate that PoFerM is an iron chelator with antimicrobial and antioxidative properties, all which depend on the conserved ferroxidase center and the ferrihydrite nucleation site.

Molecular characterization and expression analysis of ATP-gated P2X7 receptor involved in Japanese flounder (Paralichthys olivaceus) innate immune response.

ATP-gated P2X7 receptor (P2RX7) channel is a key component for purinergic signaling and plays important roles in the innate immune response in mammals. However, the expression, molecular properties and immune significances of P2RX7 in lower vertebrates are still very limited. Here we identified and characterized a novel bony fish P2RX7 homologue cDNA, termed poP2RX7, in Japanese flounder (Paralichthys olivaceus). PoP2RX7 protein shares about 60-88% sequence similarity and 45-78% sequence identity with known vertebrate P2RX7 proteins. Phylogenetic analysis placed poP2RX7 and other P2RX7 proteins within their own cluster apart from other P2RX members. While the functional poP2RX7 channel shares structural features in common with known P2RX7 homologs, electrophysiological studies revealed that BzATP, the more potent agonist for known mammalian and fish P2RX7s, shows similar potency to ATP in poP2RX7 activation. poP2RX7 mRNA constitutively expressed in all examined tissues from unstimulated healthy Japanese flounder with dominant expression in hepatopancreas and the lowest expression in head kidney, trunk kidney, spleen and gill. poP2RX7 mRNA expression, however, was significantly induced in Japanese flounder head kidney primary cells by Poly(I:C) and bacterial endotoxin LPS stimulations. In vivo experiments further revealed that poP2RX7 gene expression was substantially up-regulated by immune challenge with infectious bacteria Edwardsiella tarda and Vibrio anguillarum. Moreover, activation of poP2RX7 results in an increased gene expression of multifunctional cytokines IL-1ß and IL-6 in the head kidney primary cells. Collectively, we identified and characterized a novel fish P2RX7 homolog which is engaged in Japanese flounder innate immune response probably through modulation of pro-inflammatory cytokines expression.

Biochemical characteristics of four marine fish skins in Korea.

In this study, we investigated the biochemical characteristics of the fish skins of four industrial species: olive flounder (Paralichthys olivaceus), black rockfish (Sebastes schlegeli), sea bass (Lateolabrax maculatus) and red sea bream (Pagrus major). There is high domestic demand in Korea for farming of these fish for human consumption. Crude protein contents in the skin of these fish ranged from 73% to 94% by dry weight; this was in part due to a high content of the structural protein, collagen. Among the four species, olive flounder had the thickest dermal and epidermal layers in the dorsal skin. This species was also associated with the highest extraction ratio of acid-soluble collagen. We also examined whether fish skin could be a cost-effective alternative to current fish meal sources. Our analysis indicates that, when supplemented with additional fish oils and essential amino acids, fish skin is a viable alternative for fish meal formulations.

Dietary Hizikia fusiformis glycoprotein-induced IGF-I and IGFBP-3 associated to somatic growth, polyunsaturated fatty acid metabolism, and immunity in juvenile olive flounder Paralichthys olivaceus.

This study was aimed to examine the effect of dietary glycoprotein extracted from the sea mustard Hizikia fusiformis (Phaeophyceae: Sargassaceae) as a dietary supplement on growth performance in association with somatotropin level, proximate compositions, and immunity in juvenile olive flounder Paralichthys olivaceus. Water-ethanol extracted glycoprotein from H. fusiformis was supplemented to three fishmeal-based diets at the concentration of 0, 5, and 10gkg(-1) diet (designated as H0, H5, and H10, respectively). After a 12week-long feeding trial, growth performance and biochemical responses were analyzed including proximate composition, and whole body amino acids and fatty acids. We also measured plasma insulin like growth factor (IGF), IGF-binding protein (IGFBP) and interleukin (IL). The fish fed H5 showed the greatest weight gain among the dietary treatments. In parallel with the growth, the fish fed the diets containing H. fusiformis glycoprotein showed an increased plasma IGF-I activity and increased expression of 43-kDa IGFBP-3 compared to that in the control, whereas an opposite trend was observed for 34-kDa IGFBP-1. Although no differences were found in the level of whole body linoleic acid (C18:2n-6) and linolenic acid (C18:3n-3) among treatments, increases in arachidonic acid (ARA, C20:4n-6), eicosapentaenoic acid (EPA, C20:5n-3) and docosahexaenoic acid (DHA, C22:6n-3) were observed in fish fed H5 compared to control. IL-2 and -6 levels increased significantly in fish fed H10 compared to those in the control indicating increased immunity. These results suggest that supplementation of H. fusiformis glycoprotein in fish diet may be beneficial for fish growth and immunity in juvenile olive flounder.

Microarray analysis of hepatic gene expression in juvenile Japanese flounder Paralichthys olivaceus fed diets supplemented with fish or vegetable oils.

Gene expression profiling was performed in Japanese flounder Paralichthys olivaceus fed diets supplemented with fish oil (FO), linseed oil (LO), or olive oil (OO) for 6 weeks. The LO and OO groups showed significantly retarded growth, lower feed intake, lower protein efficiency ratio, and lower hepatosomatic index (P < 0.05). Liver fatty acid composition reflected the dietary fatty acid composition. Microarray analysis revealed that dietary n - 3 highly unsaturated fatty acid (HUFA) deficiency affected 169 transcripts. In the LO group, 57 genes were up-regulated and 38 genes were down-regulated, whereas in the OO group nine genes were up-regulated and 87 genes were down-regulated. Analysis of the functional annotations suggested that dietary n - 3 HUFA affected genes involved in signal transduction (23.2 %), cellular processes (21.1 %), metabolism (including glucose, lipid, and nucleobase; 15.5 %), transport (11.3 %), regulation of transcription (10.5 %), and immune response (4.2 %). Several genes encoding serine/threonine kinases such as protein kinase C and calmodulin-dependent kinase and nuclear hormone receptors such as vitamin D receptor, retinoic acid receptor, and receptors for cytokines (bone morphogenic protein and transforming growth factor ß) were affected. Among 169 transcripts, 22 genes were affected in both LO and OO groups. The present study identified several genes involved in n - 3 HUFA deficiency-sensitive pathways, which will be useful for selective breeding of flounder strains able to adapt to n - 3 HUFA deficiency.

In vivo antimicrobial evaluation of an alanine-rich peptide derived from Pleuronectes americanus.

In several organisms, the first barrier against microbial infections consists of antimicrobial peptides (AMPs) which are molecules that act as components of the innate immune system. Recent studies have demonstrated that AMPs can perform various functions in different tissues or physiological conditions. In this view, this study was carried out in order to evaluate the multifunctional activity in vivo of an alanine-rich peptide, known as Pa-MAP, derived from the polar fish Pleuronectes americanus. Pa-MAP was evaluated in intraperitoneally infected mice with a sub-lethal concentration of Escherichia coli at standard concentrations of 1 and 5 mg kg(-1). At both concentrations, Pa-MAPs exhibited an ability to prevent E. coli infection and increase mice survival, similar to the result observed in mice treated with ampicillin at 2 mg kg(-1). In addition, mice were monitored for weight loss. The results showed that mice treated with Pa-MAPs at 1 mg kg(-1) gained 0.8% of body weight during the 72 h of experiment. The same was observed with Pa-MAP at 5 mg kg(-1), which had a gain of 0.5% in body weight during the treatment. Mice treated with ampicillin at 2 mg kg(-1) show a significant weight loss of 5.6% of body weight. The untreated group exhibited a 5.5% loss of body weight. The immunomodulatory effects were also evaluated by the quantification of IL-10, IL-12, TNF-α, IFN-γ and nitric oxide cytokines in serum, but no immunomodulatory activity was observed. Data presented here suggest that Pa-MAP should be used as a novel antibiotic against infection control.

Neuropeptide Y gene expression around meal time in the Brazilian flounder Paralichthys orbignyanus.

Neuropeptide Y (NPY) is considered the major stimulant for food intake in mammals and fish. Previous results indicate that NPY is involved in the feeding behaviour of the Brazilian flounder, Paralichthys orbignyanus. In this study, we evaluated hypothalamic NPY expression before (-2 h), during (0 h) and after feeding (+2 h) in two independent experiments: (1) during a normal feeding schedule and (2) in fish fasted for 2 weeks. During normal feeding, changes in the levels of NPY mRNA were periprandial, with expression levels being significantly elevated at meal time (P less than 0.05) and significantly reduced 2 h later (P less than 0.05). Comparing the fasting and unfasted groups, NPY mRNA levels were significantly higher (P less than 0.05) at -2 h and +2 h in the fasting group, but there was no difference at 0 h. In addition, the higher NPY mRNA levels that were observed in the fasting group were maintained throughout the sampling period. In summary, our results show that NPY expression was associated with meal time (0 h) in food intake regulation.

Relationships between body burdens of trace metals (As, Cu, Fe, Hg, Mn, Se, and Zn) and the relative body size of small tooth flounder (Pseudorhombus jenynsii).

Several studies have described strong relationships between body size and the accumulation of trace metals in animal tissues. However, few of these studies have utilized aging techniques to control for age related effects. We utilized relative body size (gy(-1)) of a model flounder species, Pseudorhombus jenynsii, in order to control for age related effects on growth and size measurements. We investigated links between relative body size, concentrations of trace metals in flounder muscle tissue, physico-chemical variables (temperature, salinity, pH, and turbidity), and levels of trace metals in the sediment. Flounder were sampled using an otter trawl net in the inner areas of eight estuaries that were either heavily modified or relatively unmodified by urbanization and industrial activity. Our results indicate that this commonly eaten fish is accumulating significant levels of some trace metals in their muscle tissue, both in relatively unmodified and heavily modified estuaries. Concentrations of Cu, Zn and Fe in muscle tissue, as well as temperature, showed a negative relationship to the relative body size of flounder. In contrast, Se and Hg in muscle showed a positive relationship to relative body size. Observed growth patterns indicate that these effects are not driven by age related differences in metabolic activity. Instead, our results suggest that differences in food supply or toxicological effects may be responsible for the observed relationships between relative body size and concentrations of Cu, Zn, and Se in muscle tissues. The use of otolith aging and growth measurement techniques represents a novel method for assessing the relationships between trace metal accumulation and the relative body size of fish in a field environment.

A preliminary investigation of the role of melanin-concentrating hormone (MCH) and its receptors in appetite regulation of winter flounder (Pseudopleuronectes americanus).

In order to better understand the role of melanin-concentrating hormone (MCH) in the regulation of appetite in fish, the mRNAs of two forms of MCH, prepro-MCH and MCH2, and two forms of MCH receptors, MCH-R1 and MCH-R2, were isolated from winter flounder (Pseudopleuronectes americanus). In addition, the mRNA expressions of these peptides and their receptors were determined under fed and fasted conditions. Both MCHs are expressed in forebrain and midbrain, as well as peripheral tissues including gut and gonads. Both MCH-Rs are ubiquitously expressed in the brain and periphery. Fasting induced an increase in the expression levels of MCH and MCH-R1 mRNAs in optic tectum/thalamus and hypothalamus but had no effect on either MCH2 or MCH-R2 mRNA expressions. Our results suggest that MCH and MCH-R1, but not MCH2 and MCH-R2 might have a role in the regulation of appetite in flounder.

Molecular cloning and characterization of two putative appetite regulators in winter flounder (Pleuronectes americanus): preprothyrotropin-releasing hormone (TRH) and preproorexin (OX).

cDNAs encoding for preproTRH and preproorexin were cloned in winter flounder, a species that undergoes a period of natural fasting during the winter. For both peptides, the deduced amino acid structure of the hormone precursor shows 30-70% similarities with their homologs in other fish species. RT-PCR studies show that these peptides are present not only in the brain, but also in several peripheral tissues, including gastrointestinal tract and testes. Fasting induced increases in both preproorexin and preproTRH expressions in the hypothalamus, but did not affect their expression levels in the telencephalon/preoptic area. In addition, the mRNA expressions of both preproorexin and preproTRH were higher in the winter than in the summer in both hypothalamus and telencephalon/preoptic area. Our results suggest that orexin and thyrotropin-releasing hormone (TRH) might have a role in the seasonal regulation of food intake in winter flounder.

Responses of metabolic pathways to polycyclic aromatic compounds in flounder following oil spill in the Baltic Sea near the Estonian coast.

In January 2006 an oil spill that involved approximately 40tons of heavy fuel oil affected more than 30km of the north-west coast of Estonia. The aquatic pollution of the coastal area of the Baltic Sea was monitored by measuring the content of selected polycyclic aromatic hydrocarbons (PAHs and PAH metabolites) in flounder (Platichthys flesus trachurus Duncker). One hundred and thirty-one fish were collected: muscle and liver tissues were analyzed by high-performance liquid chromatography (HPLC); bile and urine samples were analyzed using fixed wavelengths fluorescence. Fifteen different types of PAHs were analyzed in liver and muscle, and four types of PAH metabolites were analyzed in bile and urine (2-, 3-, 4- and 5-ringed PAH metabolites represented by naphthalene, phenanthrene, pyrene and benzo(a)pyrene). Fluorescence analyses were carried out using excitation/emission wavelength pairs: 290/380, 256/380, 341/383 and 380/430nm, respectively. There was a time-dependent decrease of PAH concentrations in liver (83%), bile (82%) and urine (113%). HPLC analysis of muscle tissues demonstrated low concentrations of single PAHs, but a decrease of concentrations during the study period was not observed. During the analyses concentrations of PAH metabolites in bile and urine were compared. Liver metabolic transformation activity is believed to exceed that of the kidney but the analyses demonstrated high metabolite concentration in fish urine, particularly of 4- and 5-ring PAH metabolites. The results indicate remarkable buffer capacity of hydrodynamically active sea as well as considerable importance of kidney-urine metabolic pathways in flounder physiology.

Lipid content and fatty acid composition of the monogenean Neobenedenia girellae and comparison between the parasite and host fish species.

Neobenedenia girellae, a capsalid monogenean, is a destructive fish parasite. We studied the lipid content and fatty acid composition of N. girellae and the skin and cutaneous mucus of a host fish, the amberjack Seriola dumerili (Carangidae). The lipid content of adult N. girellae was less than one-fourth that of both the skin and cutaneous mucus of its host. Adult N. girellae, S. dumerili skin and mucus had a relatively high weight-percentage of C16:0, C18:1(n-9), C18:0 and C22:6(n-3) fatty acids. When S. dumerili were fed a diet supplemented with [13C] fatty acids, [13C] fatty acids were detected in S. dumerili skin and adult N. girellae on S. dumerili, but no [13C] fatty acids were detected in the S. dumerili cutaneous mucus. In addition, the epidermis of S. dumerili, attached with N. girellae, was markedly thin. These results suggest that N. girellae feeds primarily on host epithelial cells. We then infected 2 host fishes, S. dumerili and the spotted halibut Verasper variegatus (Pleuronectidae; a host less susceptible to N. girellae infection), and compared the fatty acid composition of N. girellae with that of the skin and cutaneous mucus of the hosts. The fatty acid profiles from all samples were qualitatively and quantitatively similar. Thus, the fatty acid composition of the host may not contribute to the difference in susceptibility between S. dumerili and V. variegatus. These results may serve to develop new strategies for the control of N. girellae infections.