Effects of the dioxin-like PCB 126 on larval summer flounder (Paralichthys dentatus).

Little is known about the sensitivity of teleost post-embryonic developmental stages (larval and metamorphic) to dioxin-like compounds. Larval and metamorphosing summer flounder (Paralichthys dentatus) were exposed to the dioxin-like polychlorinated biphenyl congener PCB 126, to compare their sensitivity to other fish species early life stages, and to document effects on metamorphic development, including degree of eye migration and gastric maturation. Median lethal doses (LD 50s) ranged between 30 and 220 ng/g wet mass, indicating that pre- and early-metamorphic stages of summer flounder are equally sensitive to the embryos of some of the most vulnerable fish species tested. Consistent with the presence of a functional aryl hydrocarbon receptor pathway, dose-dependent induction of cytochrome P-4501A (CYP1A) at four days post-exposure was observed in liver, stomach, intestine, and kidney of metamorphosing larvae. Stage-dependent differences in the epithelial distribution of CYP1A immunoreactivity were observed in the developing stomach of fish exposed to relatively high PCB 126 doses. A single sublethal dose (15 ng/g) delayed metamorphic progress (determined by the degree of eye migration), and resulted in abnormally high levels of cell proliferation and abnormal gastric gland morphology in late metamorphic stages. These results suggest that the post-embryonic larval and metamorphic stages of summer flounder, and potentially other fish species with complex life histories, are vulnerable to the effects of dioxin-like compounds, including lethality, developmental delay, and malformations.

Ghrelin in the summer flounder: immunolocalization to the gastric glands and action on plasma cortisol levels.

We searched for evidence of the hormone ghrelin in the stomach of a juvenile, marine teleost, the summer flounder. Using antiserum against the conserved core of the ghrelin peptide, immunoreactivity was observed in the simple, branching epithelium that comprises the gastric glands. The immunoreaction was especially strong in the glandular epithelium located deep in the tissue. Next, we assessed a possible connection between ghrelin and the hypothalamic-pituitary-interrenal axis through a series of three injection experiments in which acylated or non-acylated (des-acyl) ghrelin was injected into the peritoneum of summer flounder (Paralichthys dentatus). A significant increase in plasma cortisol relative to saline-injected controls was observed for the acylated form at a dose of 1000 ng g(-1) of body mass in one of the experiments. In another, there was a trend for des-acyl ghrelin at 1000 ng g(-1) of body mass to increase plasma cortisol. Taken together, this study provides evidence that gastric glands in the stomach of summer flounder are a site of ghrelin production and that peripherally administered ghrelin can stimulate the cortisol axis in a teleost.

Evidence for the onset of feedback regulation of cortisol in larval summer flounder.

We investigated the functional development of feedback regulation of cortisol levels during early development in a marine teleost, summer flounder, using a novel pharmaceutical approach. Larvae were immersed for 4h in the glucocorticoid agonist dexamethasone (20 microM) and/or the glucocorticoid-receptor antagonist RU486 (0.12 microM) at 1, 7, or 21 days after hatching. The hypothesis was that, if feedback regulation were operational, tissue cortisol concentrations would be suppressed by dexamethasone and stimulated by RU486. Whole-body cortisol content of 1-day-old larvae was significantly decreased from 0.32 ng/g body weight (mean) to 0.08-0.12 ng/g by immersion in dexamethasone, RU486, or both, perhaps due to displacement of cortisol from the yolk sac. There were no changes in cortisol content among treatment groups in 7-day-old larvae. The expectations of our hypothesis were met in 21-day-old larvae. Immersion in RU486 increased cortisol content from 0.29 ng/g (control) to 2.00 ng/g, whereas immersion in dexamethasone (with or without RU486) suppressed cortisol to 0.03-0.04 ng/g. The results indicate that a fully functional hypothalamic-pituitary-interrenal axis is established by 3 weeks after yolk-sac resorption, but before the onset of metamorphosis in summer flounder. This is the earliest detection of feedback regulation in a teleost fish.

The hypothalamic-pituitary-thyroid (HPT) axis in fish and its role in fish development and reproduction.

Bony fishes represent the largest vertebrate class and are a very diverse animal group. This chapter provides a thorough review of the available scientific literature on the thyroid system in these important vertebrate animals. The molecular components of the hypothalamic-pituitary-thyroid (HPT) axis in this group correspond closely to those of mammals. The thyroid tissue in the fishes is organized as diffuse follicles, with a few exceptions, rather than as an encapsulated gland as is found in most other vertebrate species. The features of this diffuse tissue in fishes are reviewed with an emphasis on feedback relationships within the HPT axis, the molecular biology of the thyroid system in fishes, and comparisons versus the thyroid systems of other vertebrate taxa. A review of the role of thyroid hormone in fish development and reproduction is included. Available information about the HPT axis in fishes is quite detailed for some species and rather limited or absent in others. This review focuses on species that have been intensively studied for their value as laboratory models in assays to investigate disruption in normal function of the thyroid system. In addition, in vitro and in vivo assay methods for screening chemicals for their potential to interfere with the thyroid system are reviewed. It is concluded that there are currently no in vitro or in vivo assays in fish species that are sufficiently developed to warrant recommendation for use to efficiently screen chemicals for thyroid disruption. Methods are available that can be used to measure thyroid hormones, although our ability to interpret the causes and implications of potential alterations in T4 or T3 levels in fishes is nonetheless limited without further research.

Cortisol is necessary for seawater tolerance in larvae of a marine teleost the summer flounder.

Larval-stage summer flounder (Paralichthys dentatus) were immersed in the corticosteroid-receptor blocker RU486 to test the effects of cortisol deficiency on salinity tolerance. Premetamorphic larvae held at 10 (near isosmotic) or 30 (hyperosmotic) parts per thousand ( per thousand) seawater survived well over 5d in 0, 0.012, or 0.12 microM RU486. However, at concentrations of 1.2 or 3.6 microM RU486, mortality was significantly greater for larvae in 30 per thousand compared to larvae in 10 per thousand. In a separate experiment, the ability of RU486 to inhibit tolerance to hyperosmotic medium (30 per thousand) was confirmed; immersion at 1.2 microM RU486 induced mortality of larvae in the metamorphic climax stage held at 30 per thousand, but not 0 or 10 per thousand. Mortality due to RU486 in pre- or prometamorphic stage larvae was prevented by concurrent immersion in cortisol at concentrations approximately 10-200 times greater than RU486, indicating that the action of RU486 was specific to antagonism of cortisol. The efficacy of 1.2 microM RU486 in reducing survival in 30 per thousand was found to be stage-dependent and exhibited the following hierarchy for fastest time to 50% mortality: prometamorphosis>metamorphic climax>premetamorphosis. In a 5-d pretreatment of pre- or prometamorphic larvae by immersion in 20 microM cortisol and/or 0.12 microM RU486 at 30 per thousand, only RU486 had a limited effect on decreasing survival when larvae were challenged with abrupt exposure to 50 per thousand. In total, the results evidence for the first time a necessary role for cortisol in seawater tolerance of a larval marine teleost.

Adaptation for water balance in the partial gastrointestinal tract of summer flounder.

Marine teleosts continually drink and absorb water across the intestine to prevent dehydration. Surprisingly, summer flounder that are missing most of their intestine, due to necrotizing enteritis, maintain osmotic homeostasis. Here, we tested the hypothesis that this remnant gastrointestinal tract undergoes compensatory adaptation for fluid uptake. Flounder (Paralicthys dentatus) with a partial gastrointestinal tract had an emaciated liver. Moisture content of muscle however was similar to healthy cohorts with an intact gastrointestinal tract, indicative of an undisturbed osmoregulatory status. Mass-specific rates of fluid uptake across all segments of the partial gastrointestinal tract were less than or similar to rates in corresponding segments from intact flounder. In contrast, weights (percent of body mass) were doubled in stomach and partial intestine of the remnant gastrointestinal tract. Consequently, total capacity for fluid uptake (microL h(-1) g body mass(-1)) was similar for both groups. The functional capacity of the remnant gastrointestinal tract was therefore of a magnitude sufficient to maintain osmoregulatory ability, further evidencing a critical role of the intestine in salt and water balance of marine teleosts.

Osmoregulatory physiology of pyloric ceca: regulated and adaptive changes in chinook salmon.

Functions of the anatomically obvious, yet peculiar, pyloric ceca of the fish gut have been a source of conjecture for over two millennia since Aristotle hypothesized on digestive utilities. Here, we demonstrate regulated and adaptive changes in osmoregulatory physiology of ceca from chinook salmon (Onchorhynchus tshawytscha). Transfer of salmon from freshwater to seawater (both short- and long-term) significantly stimulated both fluid uptake from 5.1 to 8.3-9.3 microl/cm2/hr and also Na+/K+ -ATPase from 6.5 to 8.3-9.6 micromol/ADP/mg protein/hr. Similar changes were induced with implants of cortisol, which resulted in high physiological cortisol levels in plasma. Ceca, which can number about 200 in chinook salmon, were estimated to account for the majority of fluid uptake capacity of the intestine and, after long-term seawater adaptation, the proportion of uptake capacity was sixfold higher. Transport physiology of ceca is thus under environmental and endocrine control indicative of an important role in salt and water homeostasis.

Age-dependent changes in the response of the stomach to thyroidal signaling in developmentally arrested larval summer flounder.

Thyroid-dependent stomach development occurs between approximately 35 and 50 days post-hatch (dph) in laboratory-reared summer flounder larvae. The process can be blocked by thiourea (TU), and TU effects are reversed by exogenous thyroxine (T4). To establish whether a window of sensitivity exists for T4-dependent gastric development, we arrested development of larvae with 0.39 mM TU given from 26 to 61dph, and measured weekly the developmental response of the stomach to 13 nM T4 for 0, 2, or 7 days. We examined cell proliferation in surface epithelium, gastric glands, and connective tissue, pepsinogen immunoreactivity in gastric glands, and Ulex Europaeus I (UEA I) lectin staining of gastric mucous neck cells, indicative of mucous content. In 26-47dph larvae, cell proliferation was increased 5- to 10-fold in all cell types after 2 days in T4, and returned to pretreatment values by 7 days of treatment. In 54dph fish, however, the proliferative response of gastric gland and surface epithelial cells decreased significantly from that of younger fish, while that of connective tissue was unchanged. For the differentiation markers, T4-induced mucous content increased at 54dph, but not in older fish, while pepsinogen induction was not different at any of the ages tested. The age interval between 47 and 54dph corresponds with the completion of gastric development in spontaneously metamorphosing larvae. The findings suggest that a critical window exists for the mitogenic actions of T4 in epithelial cells, but not for connective tissue cells, whereas no critical period was found for markers of differentiation.