A multiple endpoint analysis of the effects of chronic exposure to sediment contaminated with Deepwater Horizon oil on juvenile Southern flounder and their associated microbiomes.

Exposure to oiled sediments can negatively impact the health of fish species. Here, we examine the effects of chronic exposure of juvenile southern flounder, Paralichthys lethostigma, to a sediment-oil mixture. Oil:sediment mixtures are persistent over time and can become bioavailable following sediment perturbation or resuspension. Juvenile flounder were exposed for 32 days under controlled laboratory conditions to five concentrations of naturally weathered Macondo MC252 oil mixed into uncontaminated, field-collected sediments. The percent composition of individual polycyclic aromatic hydrocarbons (PAHs) of the weathered oil did not change after mixing with the sediment. Spiked exposure sediments contained 0.04-395mg/kg tPAH50 (sum of 50 individual PAH concentration measurements). Mortality increased with both exposure duration and concentration of sediment-associated PAHs, and flounder exposed to concentrations above 8mg/kg tPAH50 showed significantly reduced growth over the course of the experiment. Evident histopathologic changes were observed in liver and gill tissues of fish exposed to more than 8mg/kg tPAH50. All fish at these concentrations showed hepatic intravascular congestion, macrovesicular hepatic vacoulation, telangiectasia of secondary lamellae, and lamellar epithelial proliferation in gill tissues. Dose-dependent upregulation of Cyp1a expression in liver tissues was observed. Taxonomic analysis of gill and intestinal commensal bacterial assemblages showed that exposure to oiled sediments led to distinct shifts in commensal bacterial population structures. These data show that chronic exposure to environmentally-relevant concentrations of oiled sediments produces adverse effects in flounder at multiple biological levels.

Effects of resveratrol on growth and skeletal muscle physiology of juvenile southern flounder.

Resveratrol is a naturally occurring antioxidant that has been widely studied in mammals due to its potential to extend lifespan. However, antioxidants may also limit protein damage and therefore reduce rates of protein degradation, providing a potential avenue for enhancing growth in an aquaculture setting. The present study tested the hypotheses that in Southern flounder, Paralichthys lethostigma, resveratrol would decrease protein carbonylation and 4-HNE (indicators of protein and lipid oxidative damage, respectively), levels of ubiquitinylation and LC3 (indicators of non-lysosomal and lysosomal protein degradation, respectively), while having no effect on S6K activation (indicator of protein synthesis). These effects were predicted to increase growth rate. Mitochondrial volume density was also examined since resveratrol may lead to the proliferation of mitochondria, which are the principal source of reactive oxygen species (ROS) that cause oxidative damage. Juvenile fish (n=142) were fed a control diet or a diet supplemented with 600 µg resveratrol per g of food for 16 weeks. Fish treated with resveratrol had a 9% greater length and 33% greater body mass than control fish after 16 weeks. Additionally, there was lower protein carbonylation and lipid 4-HNE within the muscle tissues of treated fish, indicating decreased oxidative damage, and reduced protein ubiquitinylation in the resveratrol fed flounder, indicating less protein degradation. However, there was not a significant difference in LC3, S6K activation, or mitochondrial volume density. These results suggest that resveratrol has positive effects on growth due to its antioxidant properties that reduce non-lysosomal protein degradation.

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.

Food deprivation increases the expression of the prepro-orexin gene in the hypothalamus of the barfin flounder, Verasper moseri.

Orexins (orexin-A and -B) are involved in the regulation of food intake in mammals. In the barfin flounder, Verasper moseri, we previously reported that orexin-A-like-immunoreactive (ir) cell bodies are localized in the hypothalamus, which is a possible orexigenic center in fish. However, the physiological roles of orexin in the barfin flounder remain unclear. Here, we cloned prepro-orexin cDNA and examined the effects of feeding status on orexin gene expression in the barfin flounder to obtain a better insight into the roles of orexins in feeding regulation. A molecular cloning study showed that barfin flounder prepro-orexin cDNA encodes a 145 amino acid (aa) polypeptide containing orexin-A (43 aa) and orexin-B (28 aa). Prepro-orexin gene transcripts were detected in the hypothalamus, pituitary, and several peripheral organs such as the eyeball, gills, head kidney, body kidney, spleen, testis, and the skin on the eye-side of the flounder's body. Furthermore, the mean prepro-orexin mRNA expression level in the hypothalamus was significantly higher in fasted than in fed fish. These results show that fasting regulates orexin mRNA in the hypothalamus and suggest that orexin is involved in feeding regulation in barfin flounder.

Effects of antinutritional factors on plasma lipoprotein levels in Japanese flounder Paralichthys olivaceus.

This study examined the effects of four types of antinutritional factor (phytic acid, stachyose, soy saponins and soy isoflavones) on lipoprotein levels in plasma of Japanese flounder Paralichthys olivaceus. A basal diet was prepared with fish meal as primary protein source, the other diets were supplemented with 0·2, 0·4 or 0·8% phytic acid, 0·4, 0·8 or 1·5% stachyose, 0·1, 0·35 or 0·7% soy saponins and 0·10, 0·35 or 0·70% soy isoflavones, by dry mass, in place of white flour in the basal diet. Total cholesterol (TC) and triglyceride (TG) levels in plasma of P. olivaceus were not affected by phytic acid or stachyose. In general, addition of 0·2-0·8% phytic acid or 0·4-1·5% stachyose decreased plasma high-density lipoprotein cholesterol (HDL-C) levels, increased plasma low-density lipoprotein cholesterol (LDL-C) levels, thereby increasing the LDL-C:HDL-C ratio. By contrast, supplementation with 0·35-0·7% soy saponins generally depressed plasma TC levels and the LDL-C:HDL-C ratio. Supplementation with 0·35-0·7% soy isoflavones, however, increased plasma TC and TG levels. These results indicate that soy saponins may be partly responsible for the cholesterol-lowering effects of soybean meal.

A time-course study of immune response in Japanese flounder Paralichthys olivaceus exposed to heavy oil.

PURPOSE: The immunotoxicities of oil and its components on fish immunities have been investigated, but there is little literature on the recovery of the fish from the immune suppression. Therefore, the recovery of Japanese flounder Paralichthys olivaceus from an immunosuppressive effect due to heavy oil (HO) exposure was investigated in this study. METHODS: Fish were exposed to HO at a concentration of 0.385 g/L for 2 days, while control fish received no exposure. Seven fish were sampled at 0, 3, 7, and 14 days post-exposure. The respiratory rate was measured everyday as an indicator of the acute effect of HO exposure. Fish serum was collected and used for antibacterial activity assay against Edwardsiella tarda. Expression changes of respiratory and immune-related genes were evaluated by real-time PCR. RESULTS AND DISCUSSION: The respiratory rate was significantly increased in the HO-exposed group until 4 days post-exposure. A respiratory-related gene, ß-hemoglobin, was also significantly downregulated in the spleen both at 0 and 7 days post-exposure and kidney at 3 days post-exposure in HO-exposed fish. Immunotoxicity, including suppression of antibacterial activities and downregulation of the IgM gene, was observed in HO-exposed fish until 3 days post-exposure, but not after that time. From these results, we conclude that the fish likely return to normal status around 1 week.

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.

Cloning, distribution and effects of season and nutritional status on the expression of neuropeptide Y (NPY), cocaine and amphetamine regulated transcript (CART) and cholecystokinin (CCK) in winter flounder (Pseudopleuronectes americanus).

cDNAs encoding for neuropeptide Y (NPY), cocaine and amphetamine regulated transcript (CART) and cholecystokinin (CCK) were cloned in winter flounder, a species that undergoes a period of natural fasting during the winter. Tissue distribution studies show that these peptides are present in several peripheral tissues, including gut and gonads, as well as within the brain. We assessed the effects of season and fasting on the expression of these peptides. Our results show that NPY and CCK, but not CART, show seasonal differences in expression with higher hypothalamic NPY and lower gut CCK expression levels in the winter. In the summer, fasting induced an increase in hypothalamic NPY expression levels and a decrease in gut CCK levels, but did not affect hypothalamic CART expression levels. None of the peptides examined was affected by fasting in the winter. Our results suggest that NPY and CCK, but maybe not CART, might have a major role in the regulation of feeding in winter flounder and might contribute to the seasonal fluctuations in appetite in this species.

Evidence for nitric oxide role in the caudal neurosecretory system of the European flounder, Platichthys flesus.

A neuromodulatory role for nitric oxide has been reported for magnocellular neuroendocrine cells in mammalian hypothalamus. We examined its potential as a local intercellular messenger in the neuroendocrine Dahlgren cell population of the caudal neurosecretory system (CNSS) of the euryhaline flounder. Immunocytochemistry using an antibody raised against human neuronal nitric oxide synthase (NOS) indicated the presence of NOS in the Dahlgren cells. Quantitative RT-PCR, using a flounder-specific probe, revealed NOS mRNA expression in the CNSS. In July, though not in September, NOS mRNA expression was significantly higher in fish fully adapted to seawater, compared to freshwater-adapted fish. Following acute transfer of fish from freshwater to seawater, NOS mRNA expression was elevated at 8h and then recovered by 24h. In pharmacological experiments in vitro, application of NO donors (SNAP, SNP) caused an increase in electrical activity (firing frequency) of Dahlgren cells, recruitment of previously silent cells, together with a greater proportion of cells showing phasic (irregular) activity. The NOS substrate, l-arginine, led to increased firing frequency, cell recruitment and enhanced bursting activity. However, this effect was not blocked by the NOS inhibitor L-NAME. These findings suggest that NO acts as a modulator within the CNSS, potentially enhancing electrical activity and hence secretory output. A role in supporting adaptation to hyperosmotic conditions is also indicated.

Evaluation of the relationships between biochemical endpoints of PAH exposure and physiological endpoints of reproduction in male California Halibut (Paralichthys californicus) exposed to sediments from a natural oil seep.

Coal Oil Point (COP) is a natural oil seep off the coast of Santa Barbara, California. Although most studies examining the fate and effects of petroleum have focused upon urbanized or anthropogenic sources of inputs, few have examined the effects of polyaromatic hydrocarbons (PAHs) derived from natural seeps. In order to evaluate the effects of PAHs derived from COP on marine fish populations, hatchery-reared California Halibut (Platichthys californicus) were exposed for 30 days to seven dilutions of sediments collected from COP. Hepatic cytochrome P450 1A (CYP1A), biliary fluorescent aromatic compounds (FACs), gonadal somatic indices, and plasma steroid concentrations. Sixteen USEPA priority PAHs were targeted for analysis in each sediment dilution. In general, biochemical responses were somewhat recalcitrant to dose-response relationships and were less sensitive than the literature values established for the same indicators following exposure to urbanized PAHs. Trends toward reductions in plasma 17beta-estradiol concentrations were observed, but reductions in gonadal somatic indices were not observed. FAC values for naphthalene, benzo(a)pyrene, phenanthrene-related compounds reached maximums at 33-100% COP sediment. The resulting insensitivity may be unique for exposure to "natural" petroleum due to a higher concentration of lower molecular weight PAHs or uncharacterized inhibitors.

Possible involvement of melanin-concentrating hormone in food intake in a teleost fish, barfin flounder.

We investigated the involvement of MCH in food intake in barfin flounder. The structure of barfin flounder MCH was determined by cDNA cloning and mass spectrometry. In fasted fish, the MCH gene expression and the number of MCH neurons in the brain were greater than controls. In white-reared fish, the MCH gene expression and the number of MCH neurons in the brain were greater than black-reared fish. Furthermore, white-reared fish grew faster than black-reared fish. These results indicate that a white background stimulated production of MCH and MCH, in turn, enhanced body growth, probably by stimulating food intake.

Hepatic CYP1A levels and EROD activity in English sole: biomonitoring of marine contaminants in Vancouver Harbour.

To assess chemical contaminant stress in the marine environment, ethoxyresorufin-O-deethylase (EROD) activity and cytochrome P450 1A (CYP1A) expression were measured in 88 English Sole (Pleuronectes vetulus) collected during May and June 1999 from four sites in Vancouver Harbour and at an expected reference site outside the harbour. Hepatic microsomes were prepared from the fish and analyzed for total CYP content, EROD activity, and CYP1A protein levels. Hepatic EROD activity and CYP1A protein levels were elevated in fish from two sites in the inner harbour. A comparison with sediment chemistry data showed that fish with increased EROD activity and CYP1A levels came from sites containing relatively high levels of polycyclic aromatic hydrocarbons and polychlorinated biphenyls. Unexpectedly high levels of EROD activity and CYP1A protein were also found in fish from a reference site near Gibsons, in Howe Sound. The elevated EROD activity and CYP1A expression in fish from this site cannot be explained by the chemical analysis data collected.