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Erythrocyte micronuclei in winter flounder (Pseudopleuronectes americanus): results of field surveys during 1980-1988 from Virginia to Nova Scotia and in Long Island Sound.

Erythrocyte micronuclei (MN) frequencies of 280 winter flounder (Pseudopleuronectes americanus) from Chesapeake Bay to the southern Scotian Shelf including Long Island Sound were measured. The MN data were combined with data previously reported for 224 flounder and the combined data sets were analyzed. Incidences of MN were elevated sixfold in flounder from the New York Bight Apex as compared to frequencies for fish from the inshore Gulf of Maine and Block Island Sound, and twice those found in Georges Bank and Long Island Sound flounder. Inshore New Jersey fish had higher MN frequencies than those from inshore Gulf Of Maine and Block Island Sound. The occurrence of MN in flounder from inshore Virginia was higher than in flounder from inshore Gulf of Maine and Block Island Sound. The large subset of stations from Long Island Sound indicated higher frequencies of MN in flounder from Hempstead and Shoreham as compared to most other sites in the Sound. There was no significant difference in the frequency of MN between males and females, but males had significantly more MN than sexually immature flounder. Flounder over the entire sampling area had increased levels of MN during the fall months of the year. This is probably influenced by the maternal cycle of gonadal maturation.

Animals

Southern flounder hepatic and intestinal metabolism and DNA binding of benzo[a]pyrene (BaP) metabolites following dietary administration of low doses of BaP, BaP-7,8-dihydrodiol or a BaP metabolite mixture.

Certain finfish species living in chemically polluted environments exhibit a high incidence of gastrointestinal tract tumors. Carnivorous fish in such environments are likely to consume invertebrates which contain chemical procarcinogens and the invertebrate biotransformation products of these compounds. The retention in tissues, extent of DNA adduct formation in liver and intestine, and metabolite composition of bile was investigated in southern flounder following gavage with pure [3H]- or [14C]benzo[a]pyrene (BaP), pure [14C]benzo[a]pyrene-7,8-dihydrodiol (BaP-7,8D), or hepatopancreas from spiny lobsters previously dosed with [3H]- or [14C]BaP (Metab.HP). Metab.HP contained mainly polar conjugates of BaP diols, triols and tetraols. BaP-7,8D was retained in fish tissues and bile at 24 h to a greater extent (33.6% of the dose), than either BaP (19.00%) or Metab.HP (6.6%). Hepatic and intestinal DNA isolated from all dosed fish contained covalently bound radioactivity, but exposure to BaP-7,8D or BaP resulted in significantly higher binding in both tissues than exposure to Metab.HP. Hepatic DNA from BaP and BaP-7,8D-dosed flounder contained 0.24 +/- 0.07 and 0.33 +/- 0.06 pmol BaP equivalents/mg DNA respectively (mean +/- S.E.), while hepatic DNA isolated from Metab.HP-dosed flounder contained 0.006 +/- 0.002 pmol BaP equivalents/mg DNA. Binding of radioactivity to intestinal DNA was significantly higher than to hepatic DNA for flounder dosed with Metab.HP (0.026 +/- 0.003) or with BaP (0.76 +/- 0.27) but not for flounder dosed with BaP-7,8D (0.44 +/- 0.09). These studies show that dietary BaP, and metabolites likely to be present in invertebrates, can be absorbed by the southern flounder and form DNA adducts in target organs.

Animals

Sex differences in hepatic monooxygenases in winter flounder (Pseudopleuronectes americanus) and scup (Stenotomus chrysops) and regulation of P450 forms by estradiol.

Details concerning the endogenous regulation of hepatic cytochrome P450 monooxygenases in teleosts, and the features of this regulation common among fish species, are poorly known. Gonadally mature female winter flounder (Pseudopleuronectes americanus) have been reported to have severalfold lower levels of microsomal cytochromes P450 and b5 and NADPH-cytochrome c reductase than do males (Stegeman and Woodin ('84) Mar. Environ. Res., 14:422-425). These strong sex differences prompted more detailed study of P450 regulation in winter flounder liver, and a comparison with sex differences in another marine teleost, scup (Stenotomus chrysops). Ethoxyresorufin O-deethylase (EROD) activity/nmol P450 was less in gonadally mature females than in males of both species. Immunoblot analysis with MAb 1-12-3 to P450E (the EROD catalyst) showed that the content of P450E counterpart was also much less in females of both species. Aminopyrine N-demethylase (APND) and testosterone 6 beta-hydroxylase (6 beta-OHase) activities per nmol P450 were higher in gonadally mature female than in mature male flounder, differences not seen in scup. Polyclonal antibodies to scup P450A were shown to detect proteins in a number of teleosts. The levels of anti-P450A cross-reacting protein were greater in mature female than in male flounder, but as with 6 beta-OHase activity, the content of this protein was not sexually differentiated in scup. Estradiol treatment of winter flounder depressed the rates of EROD, APND, 6 beta-OHase, and estradiol 2-OHase activities per mg protein, but APND and 6 beta-OHase activities per nmol P450 were unchanged. Thus, E2 promotes general decreases in some hepatic P450-catalyzed activities, but in achieving sex differences there is also specific regulation of the P450E counterpart, and possibly of the 6 beta-OHase (P450A?). Other factors, temporal or hormonal, can modify the effect of E2 treatment, and may contribute to the specific regulation of P450 forms in naturally maturing fish, and to species differences in this regulation.

Aminopyrine N-Demethylase

Chromosome-level genome assembly of starry flounder (Platichthys stellatus).

Starry flounder (Platichthys stellatus) is widely distributed along the coastlines of the North Pacific. As an euryhaline flatfish, it can adapt to a wide range of environmental salinity ranging from freshwater to seawater, and is a promising aquaculture flatfish species in Korea and North China. However, no high-quality starry flounder reference genome has been reported to date, which greatly limits the studies of genetics and functional genomics. Here, we obtained a high-quality chromosome-level starry flounder genome assembly with a length of 643.56 Mb (scaffold N50: 26.19 Mb, contig N50: 10.00 Mb) combining short-reads sequencing, PacBio HiFi sequencing, and Hi-C sequencing. Approximately 94.02% of assembled sequences were anchored into 24 pseudochromosomes, and a total of 18 telomeres were detected. Totally 22,835 protein-coding genes and 227.87 Mb repetitive sequences were identified. In summary, the high-quality chromosome-level genome assembly not only provides valuable resources for genetic research in starry flounder, but also advances the development of molecular breeding technology of starry flounder.

Animals

The role of blood glucose in the restoration of muscle glycogen during recovery from exhaustive exercise in rainbow trout (Oncorhynchus mykiss) and winter flounder (Pseudopleuronectes americanus).

The role of blood-borne glucose in the restoration of white muscle glycogen following exhaustive exercise in the active, pelagic rainbow trout (Oncorhynchus mykiss) and the more sluggish, benthic winter flounder (Pseudopleuronectes americanus) were examined. During recovery from exhaustive exercise, the animals were injected with a bolus of universally labelled [14C]glucose via dorsal aortic (trout) or caudal artery (flounder) catheters. The bulk of the injected label (50-70%) remained as glucose in the extracellular fluid in both species. The major metabolic fates of the injected glucose were oxidation to CO2 (6-8%) and production of lactate (6-8%), the latter indicative of continued anaerobic metabolism post-exercise. Oxidation of labelled glucose could account for up to 40% and 15% of the post-exercise MO2 in trout and flounder, respectively. Exhaustive exercise resulted in a reduction of muscle glycogen stores and accumulation of muscle lactate. Glycogen restoration in trout began 2-4h after exercise, whereas in flounder, glycogen restoration began within 2h. Despite a significant labelling of the intramuscular glucose pool, less than 1% of the infused labelled glucose was incorporated into muscle glycogen. This suggests that blood-borne glucose does not contribute significantly to the restoration of muscle glycogen following exhaustive exercise in either trout or flounder and provides further evidence against a prominent role for the Cori cycle in these species.

Animals

Levels of environmental pollutants in male and female flounder (Platichthys flesus L.) and cod (Gadus morhua L.) caught during the year 1988 near or in the waterway of Glomma, the largest river of Norway. I. Polychlorinated biphenyls.

Levels of polychlorinated biphenyls, PCBs, in liver samples of 111 and 121 flounder (Platichthys flesus L.) and cod (Gadus morhua L.), respectively, were determined gas chromatographically by quantification of 9 individual PCB congeners. The congeners were 28, 52, 101, 118, 153, 138, 180, 170, and 209 (IUPAC numbering system, Ballschmiter and Zell 1980). The fish were caught during 1988 at 5 different stations near or in the Glomma estuary, in the Hvaler archipelago, Norway. The mean level of the sum of the congeners, 1,050 ng/g, found in cod was 3 times higher than the corresponding level found in flounder (361 ng/g). The influences on the variation in PCB levels of sex, season, and distance from the Glomma outlet and waterway were investigated using 1-, 2-, and 3-way ANOVA. The main effects of these factors as well as their possible interactions were considered. An expected decreasing PCB pollution gradient from the mouth of Glomma was found for cod, while PCB levels in flounder seemed to be less dependent on sampling site. However, the sexes in both fish species exhibited a different pattern in their PCB levels. The PCB levels in female cod were significantly affected by the season of sampling, with PCB levels in September/October significantly greater than the corresponding levels in June and November/December. No such effect was found for male cod. In flounder, a significant station effect on the PCB levels was found for females only, and a decreasing PCB pollution gradient was found to be significant. The interactions between the three factors station, season, and sex were different in the two species.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals

Steroidogenic role of the caudal neurosecretory system in the flounder, Platichthys flesus.

Transfer of flounders from seawater (SW) to fresh water (FW) resulted in a small reduction in circulating cortisol levels and urophysial protein storage. Transfer of flounders from FW to SW resulted in a larger increase in plasma cortisol and specific urophysial protein storage. Over the first 4 days after transfer from FW to SW there was a positive correlation between the observed changes in urophysial urotensin I (UI) content and plasma cortisol. This apparent steroidogenic effect of UI was supported by the increases in plasma cortisol observed following iv injection of crude flounder urophysial gland extract and synthetic Catostomus commersoni UI. The study supports a contribution of the caudal neurosecretory system to the control of interrenal steroidogenesis as part of the integrated osmoregulatory physiology of euryhaline species like the flounder.

Animals

Regulation of antifreeze protein production in winter flounder: a unique function for growth hormone.

Salmon pituitary extract and the protein fraction unabsorbed on concanavalin A-Sepharose, the carbohydrate-poor fraction, depressed plasma levels of antifreeze proteins (AFP) when the pituitary fractions were administered to flounder in late fall or winter. The active pituitary protein occurred in the fraction with a mean molecular weight of 25,000. The two major isohormones of growth hormone (GH) were the only biologically active proteins identified from the pituitary. Hypophysectomized flounder synthesize AFP in the spring and the two isohormones of GH suppress the synthesis. The fraction of flounder pituitaries containing putative GH depressed flounder plasma levels of AFP in late fall.

Acclimatization

8-BrcAMP does not affect Na-K-2Cl cotransport in winter flounder intestine.

The flounder intestinal epithelium possesses luminal Na-K-2Cl cotransport and K secretory mechanisms that account for the short-circuit current (Isc) across the tissue. This epithelium is also highly cation selective. Bumetanide or 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP) completely inhibit Isc, Na-K-2Cl cotransport, and K secretion, whereas 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) inhibits K secretion, partially inhibits Isc, and greatly increases Cl permeability. Although the direct effects of adenosine 3',5'-cyclic monophosphate (cAMP) on other Na-K-2Cl cotransport systems have been examined, the effects of 8-BrcAMP on flounder intestinal Na-K-2Cl cotransport have not been directly measured. In this study, the effects of 8-BrcAMP and bumetanide, either alone or in combination, on the influx (initial rates of uptake) of Cl, Rb, and Na across the luminal surface of the flounder intestine were examined. Bumetanide significantly inhibited Na (50%), Cl, and Rb influx (70% each). In contrast, 8-BrcAMP significantly increased Cl influx in the presence or absence of bumetanide and thereby did not effect the bumetanide-sensitive Cl influx. The nucleotide neither altered bumetanide-sensitive Rb influx nor net transpithelial Na absorption measured under short-circuit conditions but caused a 51-60% decrease in Isc. Measurements of bumetanide-sensitive Na influx exhibited large experimental variability but showed no statistically significant effects of 8-BrcAMP. Prostaglandin E1 (PGE1, 10 microM) and forskolin (10 microM) but not atrial natriuretic factor (1 microM) increased flounder intestinal [cAMP] 200 and 237%, respectively, and, like 8-BrcAMP, increased tissue conductance.(ABSTRACT TRUNCATED AT 250 WORDS)

8-Bromo Cyclic Adenosine Monophosphate

Volume regulation by flounder red blood cells in anisotonic media.

The nucleated high K, low Na red blood cells of the winter flounder demonstrated a volume regulatory response subsequent to osmotic swelling or shrinkage. During volume regulation the net water flow was secondary to net inorganic cation flux. Volume regulation the net water flow was secondary to net inorganic cation flux. Volume regulation after osmotic swelling is referred to as regulatory volume decrease (RVD) and was characterized by net K and water loss. Since the electrochemical gradient for K is directed out of the cell there is no need to invoke active processes to explain RVD. When osmotically shrunken, the flounder erythrocyte demonstrated a regulatory volume increase (RVI) back toward control cell volume. The water movements characteristic of RVI were a consequence of net cellular NaCl and KCl uptake with Na accounting for 75 percent of the increase in intracellular cation content. Since the Na electrochemical gradient is directed into the cell, net Na uptake was the result of Na flux via dissipative pathways. The addition of 10(-4)M ouabain to suspensions of flounder erythrocytes was without effect upon net water movements during volume regulation. The presence of ouabain did however lead to a decreased ration of intracellular K:Na. Analysis of net Na and K fluxes in the presence and absence of ouabain led to the conclusion that Na and K fluxes via both conservative and dissipative pathways are increased in response to osmotic swelling or shrinkage. In addition, the Na and K flux rate through both pump and leak pathways decreased in a parallel fashion as cell volume was regulated. Taken as a whole, the Na and K movements through the flounder erythrocyte membrane demonstrated a functional dependence during volume regulation.

Animals

Thyroid hormone regulates developmental changes in muscle during flounder metamorphosis.

Morphological and biochemical changes in the muscular tissue of metamorphosing flounder were studied in relation to the regulatory role of thyroid hormone. Premetamorphic larvae were reared in seawater alone or seawater containing either thyroxine (T4) or an antithyroid drug (thiourea, TU). Histological changes in the muscle were examined and biochemical changes in the muscle proteins were evaluated by SDS-PAGE and immunoblotting for troponin T (TNT). The muscle tissue of premetamorphic larvae was characterized by abundant vacuoles and basophilic sarcoplasm. In control fish, the larval muscle transformed into the adult type during metamorphic climax; the fibers were filled with abundant myofibrils and the vacuoles disappeared. Analysis by SDS-PAGE showed that the bands at 41.5, 35.5, 34.0, 33.5, 25.5, 23.0, 20.0, and 19.0 kDa clearly increased in density from the climax stage. Premetamorphic larvae possessed two immunoreactive TNT isoforms of 41.5 and 34.0 kDa, the former being predominant. At the climax stage an additional isoform appeared at 33.5 kDa, and the 34.0- and 33.5-kDa TNT became predominant. The administration of T4 precociously induced these histological and biochemical changes in the muscle tissue of flounder larvae. In contrast, TU treatment inhibited these developmental changes in the larval muscle. Our results suggest that the developmental changes in the muscular tissue of metamorphosing flounder are regulated by thyroid hormone.

Animals

Interaction of carbon tetrachloride with beta-naphthoflavone-mediated cytochrome P450 induction in winter flounder (Pseudopleuronectes americanus).

The interaction between beta-naphthoflavone induction (BNF: 100 mg/kg) and carbon tetrachloride (CCl4; 1 ml/kg) hepatotoxicity was examined in the flounder. Treatment groups composed of control, BNF, CCl4, and BNF/CCl4 were compared in terms of cytochrome P450 isozyme content (LM4b; LM2), catalytic activity, isozyme distribution. SGOT-SGPT levels, and pathology. CCl4 administration resulted in significant reductions in both the constitutive P450 (LM2) and the BNF-inducible isozyme (LM4b) as well as elevations in SGPT and SGOT levels. The decline in LM4b isozyme content was reflected by stoichiometric decreases in ethoxyresorufin-O-deethylase activities. BNF/CCl4 coadministration was protective in part against CCl4 hepatotoxicity. Immunohistochemistry indicated that LM4b was diffusely distributed throughout the liver. These interactions have demonstrated a multiple P450 isozyme involvement, the protective nature of BNF against CCl4 hepatotoxicity in the flounder, the ability to maintain an inductive response in face of CCl4 coadministration, and the diffuse distributional pattern of LM4b in the flounder liver.

Alanine Transaminase

P-glycoprotein genes in the winter flounder, Pleuronectes americanus: isolation of two types of genomic clones carrying 3' terminal exons.

In mammals, P-glycoprotein (P-gp) is encoded by two or more highly conserved genes that differ in their abilities to transport drugs. One isoform class (class I) is consistently associated with the multidrug resistance phenotype, while the other (class III) is not. This study was designed to enumerate the P-gp genes in fish and determine how they are related to the two functional classes already defined in mammals. Southern blot analysis using a conserved single exon from the 3' terminal region of hamster P-gp cDNA (pEX1-172) as a probe indicated that there were two P-gp genes in right-eye flounders. Subsequently, two sets of clones were isolated from a winter flounder genomic library that correspond to the 3' ends of the two flounder P-gp genes. Sequence analysis was done on two key areas: the 3' ATP binding site and the 3' terminal exon, both of which were found to be homologous with their mammalian counterparts. Despite high levels of sequence identity in the predicted coding regions of the gene fragments it has not been possible to use these sequences to relate the homologs to particular mammalian classes of P-gp genes, perhaps because of gene conversion between mammalian P-gp genes. These cloned sequences are the first set of P-gp genes reported in lower vertebrates and will be useful for delineating the expression of P-gp genes in fish and understanding the role of P-gp in fish physiology.

ATP Binding Cassette Transporter, Subfamily B, Mem

Electrical and mechanical activity in heart tissue of flounder and rainbow trout during acidosis.

1. Twitch force and voltage across the sarcolemma were measured in heart tissue of flounder and rainbow trout. 2. For the trout heart, hypercapnia was followed by a loss of force and an action potential prolongation. 3. This was also observed for the flounder heart, but only initially. 4. About 5 min after the onset of hypercapnia, an increase in force and a shortening of the action potential occurred in the flounder heart. 5. After about 30 min of hypercapnia a decrease in force and a prolongation of the action potential slowly appeared. 6. These results can be interpreted in terms of a species-dependent effect of acidosis on the cellular Ca2+ handling and the influence of intracellular Ca2+ on the action potential.

Acidosis

Hypoxia-induced changes in electrophysiological responses and associated calcium movements of flounder (Platichthys flesus) heart and gut.

1. Flounder hearts subjected to hypoxic conditions showed a reduction in contractile force. After 85 min in hypoxia-inducing salines, hearts were almost totally quiescent. 2. Membrane potentials were seen to change in hearts during hypoxic conditions. The action potential gradually declined in height but increased in duration and the resting membrane potential was also seen to decrease. 3. Sections of flounder gut, when subjected to identical hypoxic conditions as the hearts, showed no significant changes in either mechanical activity or membrane potential. 4. Heart and gut tissues were placed in hypoxia-inducing salines containing 45Ca either immediately or after a previous 3 hr exposure to hypoxia-inducing salines. Uptake of 45Ca increased beyond control values in both heart and gut subjected immediately to hypoxia. After 3 hr hypoxia exposure, uptake of 45Ca was reduced in heart but was not significantly different from controls in gut. 5. The changes seen in 45Ca uptake were far greater in heart tissue than in gut tissue. 6. In all physiological parameters examined, the gut tissue of the flounder is far more tolerant to hypoxic conditions than heart tissue.

Animals

Steroids involved with final oocyte maturation in the winter flounder.

A number of androgens and progestogens including 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17,20-P) were examined in female winter flounder as possible maturation inducing steroids (MIS). During final oocyte maturation serum levels of testosterone (T) and 17 beta-hydroxy-5 beta-androsten-3-one (5 beta-T) peaking at over 200 ng/ml and pregnenolone (PE) at 40 ng/ml were the predominant steroids found from each major group. High levels of T and 5 beta-T were correlated with oocyte stages characterized by germinal vesicle migration. Of the PEs measured, maximum serum levels of PE, 3 beta,17 alpha-hydroxy-5-pregnen-20-one (17-PE) and 3 beta,17 alpha, 20 beta-dihydroxy-5-pregnene (17,20-PE) were found during later oocytes stages associated with germinal vesicle breakdown. Levels of 17,20-P, an established MIS in most fish, were almost non-detectable (less than 0.1 ng/ml serum) in females throughout all stages of final oocyte maturation. Incubations of ovarian follicles in vitro with physiological concentrations of T and 5 beta-T indicated that these steroids could induce all stages of final oocyte maturation. Similar in vitro incubations showed that 17-PE and 17,20-PE were only effective on germinal vesicle breakdown. The principal conclusions are that T, 5 beta-T and the PEs can be considered as MISs in winter flounder and the PE pathway predominates during the final stages of oocyte maturation in winter flounder in contrast to progesterones which predominate in other fish species, mostly salmonids, studies to date.

Androgens

Structural characterization of peptides derived from prosomatostatins I and II isolated from the pancreatic islets of two species of teleostean fish: the daddy sculpin and the flounder.

The primary structures of three peptides from extracts from the pancreatic islets of the daddy sculpin (Cottus scorpius) and three analogous peptides from the islets of the flounder (Platichthys flesus), two species of teleostean fish, have been determined by automated Edman degradation. The structures of the flounder peptides were confirmed by fast-atom bombardment mass spectrometry. The peptides show strong homology to residues (49-60), (63-96) and (98-125) of the predicted sequence of preprosomatostatin II from the anglerfish (Lophius americanus). The amino acid sequences of the peptides suggest that, in the sculpin, prosomatostatin II is cleaved at a dibasic amino acid residue processing site (corresponding to Lys61-Arg62 in anglerfish preprosomatostatin II). The resulting fragments are further cleaved at monobasic residue processing sites (corresponding to Arg48 and Arg97 in anglerfish preprosomatostatin II). In the flounder the same dibasic residue processing site is utilised but cleavage at different monobasic sites takes place (corresponding to Arg50 and Arg97 in anglerfish preprosomatostatin II). A peptide identical to mammalian somatostatin-14 was also isolated from the islets of both species and is presumed to represent a cleavage product of prosomatostatin I.

Amino Acid Sequence

Do fish sniff? A new mechanism of olfactory sampling in pleuronectid flounders.

Although olfaction is known to be a highly developed sense in a variety of fishes, little information is available about behavioral mechanisms by which fishes sample the olfactory environment. This study was undertaken to assess the role of spontaneous jaw protrusion ('coughing') as a potential mechanism for olfactory sampling in pleuronectid flounders. Investigations were carried out using a combination of physiological, morphological and behavioral techniques. Physiological results show (1) that typical respirations are coupled to measurable olfactory sac pressure fluctuations and (2) that during a coughing event, water is rapidly sucked into the olfactory sac. Morphological results indicate (3) a direct linkage system between the protrusion apparatus and the olfactory or associated accessory sacs, and (4) that coughing is associated with a rapid expansion or stretching of these sacs. Lastly, behavioral studies demonstrate (5) that coughing rates increase significantly over background activity when flounders are presented with attractive food odorants. From these results, I propose that coughing in pleuronectid flounders represents a behavior truly analogous to sniffing in certain air-breathing organisms.

Animals