The toxicity of mercuric chloride and methylmercuric chloride to Fundulus heteroclitus embryos in relation to exposure conditions

Synopsis Embryos in specific stage of the estuarine teleost, Fundulus heteroclitus, were exposed to mercuric chloride (MC) and methylmercuric chloride (MMC) under several distinct treatment conditions. Four-eight cell stage eggs (0-day old) were exposed for 4 days (continuous), 2 days and one day to each mercury compound. One-day old (mid-blastula), 2-day old (mid-neurula) and 5-day old (beating heart) embryos were exposed 4 days to MC and MMC. Mortality for the four days immediately following the initiation of exposure was the embryonic response measured. Under most exposure conditions to the 4–8 cell eggs, progressive and significant reductions in survival were observed at all concentrations above 40 and 30 gHg⁺⁺l^–1 as MC and MMC, respectively. Reducing the duration of exposure to 1 day most significantly increased the survival potential of the 4–8 cell eggs. For all exposure treatments to the 4–8 cell eggs, significant differences in survival, between eggs exposed to MC and MMC, were determined at 40, 60 and 80 gHg⁺⁺l^–1, indicating the presence of compound-dependent response differences. In all cases demonstrating response differences between MC and MMC exposed embryos, survival was significantly lower following exposure to MMC. Survival of embryos was progressively increased when the initiation of continuous exposure (4 days) was delayed 1, 2 and 5 days after fertilization. As a result, compound-dependent response differences were progressively shifted to higher He⁺⁺ concentrations. For both MC and MMC, survival of 1-day old embryos exposed for 4 days was greater than that of 0-day old eggs exposed for 1 day. Of the embryonic stages examined, it appears that the earlier cleavage stages are the most sensitive to mercury intoxication.     

Alkaline phosphatase activity in the human tonsils and its relation to tonsillar diseases.

Alkaline phosphatase activity was examined in the human tonsils in fetal life and after repeated attacks of acute tonsillitis and in quinsy. Gomori's metal precipitate technique was used to demonstrate the phosphatase activity using four different substrates: sodium beta-glycerophosphate and adenosine triphosphate at pH 9, riboflavin 5-phosphate at pH 9.2 and 5-monophosphoric acid at pH 8.3. (2) The phosphatase activity differs somewhat according to the phosphate ester used as a substrate illustrating an example of 'substrate specificity'. (3) Alkaline phosphatase activity was increased in the case of both acute and chronic inflammation. This increase has been discussed in relation to such phenomena as transformation of lymphocytes into macrophages and antibody formation.     

Heavy metals in relation to the biology of the mummichog, Fundulus heteroclitus

Heavy metal levels of cadmium, copper, mercury, manganese, and zinc were examined in the mummichog, Fundulus heteroditus from industrialized and non-industrialized environments. With one exception, the environment with the highest trace metal in its waters, had the fishes with the highest metal concentration. Except for mercury, the concentration factor varied inversely with the metal concentrations of the fish and water, suggesting a possible regulatory mechanism for metals in the tissues of mummichogs from environments with high metal concentrations. There was an inverse relationship between standard length and concentrations of zinc, manganese, copper and cadmium in whole male and female fishes. The viscera contained significantly greater concentrations of these metals than somatic muscle tissue. There were also significant differences between males and females with respect to whole-body zinc and copper concentrations, but no sex differences for manganese and cadmium.     

Rodlet cell distribution in the gall bladder epithelium of Fundulus heteroclitus

Rodlet cells within the epithelial mucosa of the gall bladder of the estuarine killifish Fundulus heteroclitus (L.) obtained from a highly contaminated creek adjacent to a landfill, were arranged within the constraints of the epithelium. Furthermore, the rodlet cells established a close intimate association with electron dense epithelioid cells. A comparison with fish from a non impacted estuary revealed a significantly greater number of rodlet cells in the 'contaminated' group. The abundance of rodlet cells within the gall bladder of the fish exposed to contaminants further strengthens the hypothesis that these cells participate in the fish's immune system.     

Diversity and distribution of gynogenetic hybrids between Fundulus diaphanus and Fundulus heteroclitus in Porter's Lake (Nova Scotia) in relation to salinity

Unisexual hybrids between Fundulus diaphanus and Fundulus heteroclitus were found in low proportions at intermediate salinity in Porter's Lake, Nova Scotia. One clone accounted for 72% of the hybrids, with most other hybrids being different at a single microsatellite allele. This clone thrives over a wide range of salinities, suggesting a general-purpose genotype.     

Tolerance to Environmental Contaminants in the Mummichog,Fundulus heteroclitus

Mummichogs or killifish (Fundulus heteroclitus) are abundant estuarine fish that can tolerate widely varying environmental conditions. They are found in some highly contaminated sites, and their development of tolerance to toxicants has been studied. Populations have developed resistance to methylmercury, kepone, dioxins, polychlorinated biphenyls, and polyaromatic hydrocarbons. This article describes what is known about their tolerance, and discusses similarities and differences among these resistant populations. In some cases tolerance is seen only in early life stages, while in other cases tolerance is seen in adults also. In many of the populations, adults show signs of stress. The mechanism of embryo tolerance to meHg appears to be reduced chorionic permeability, and more rapid development through sensitive stages. One mechanism of resistance to dioxins, PCBs and PAHs is non-responsiveness (lack of inducibility) of CYP1A. In some populations it is already elevated, while in others it is not. Another mechanism in the PAH-resistant fish is elevation of the phase II metabolizing enzyme, GST. The tolerance appears to be genetic. In populations in which it has been studied, the age structure is skewed towards younger fish, and adults appear to put more energy into reproduction as a way of maintaining the population in the stressful environment.     

Hormones and Calcium Regulation in Fundulus heteroclitus

For the past 20 years, our laboratory has been involved in studyingthe endocrine control of calcium balance in the killifish, Fundulusheteroclitus. We have surveyed almost all endocrine systemsand discovered two main ones directly involved in plasma calciumregulation. They are the pituitary gland and the corpusclesof Stannius. When fish adapted to low-calcium seawater werehypophysectomized, hypocalcemia and tetany were observed. Whenfish adapted to high-calcium seawater were Stanniectomized,hypercalcemia was seen. In both cases, other electrolytes wereunaffected and replacement therapy corrected the plasma calciumchanges. We have tried to characterize the active principlesin both glands. We discovered that prolactin is hyperacalcemic.The pituitary gland also seems to contain a second hypercalcemicfactor which may be located in the PAS-positive pars intermediacells. For the Stannius corpuscle factors, we developed a bioassayand named the active substance(s) hypocalcin. In collaborationwith Dr. Hirofumi Sokabe at Jichi Medical School, Japan, weshowed that the Stannius corpuscles also contain a renin-likesubstance capable of generating a hypocalcemic angiotensin-likesubstance. The exact chemical nature of the hypocalcemic substanceis being investigated. Calcium balance in the whole fish wasalso studied with 47-calcium. These studies were carried outin collaboration with Dr. Nicole Mayer-Gostan at Villefranche-sur-Mer,France. We discovered that killifish depend on the environmentrather than bone as a calcium reservoir. Hormones may be involvedin the exchanges with the environment.     

Scaling of fast-start performance and its thermal dependence in mummichog Fundulus heteroclitus

Fast-start predator-escape performance and its sensitivity to temperature (24, 30, and 36°C) were evaluated in mummichog Fundulus heteroclitus across a range of body sizes spanning YOY to adult (35–68 mm standard length). Mummichogs exhibit isometry of body dimensions and areas of the dorsal and anal fins but negative allometry of the caudal fin area. These scaling relationships are consistent with observed decreases in fast-start angular velocities with increasing body size. Linear velocity, on the contrary, does not vary with size, and both large and small mummichogs are capable of traversing similar distances in a given amount of time. In addition, temperature influences fast-start performance in similar ways over the size range, though the magnitude of the effect varies with size for some performance measures. In general, fast-start performance increases with test temperature, but mummichogs acclimated to warmer temperatures exhibit lower performance at each test temperature. Altogether, our results suggest that mummichogs across the adult size range may suffer decreases in their predator-escape performance as increasing sea temperatures combine with short-term temperature fluctuations in the estuaries these fish occupy.     

Actin microfilaments in melanophores of Fundulus heteroclitus

Summary In melanophores of Fundulus heteroclitus, hormone-stimulated melanosome aggregation is accompanied by cytoplasmic flow from the cellular processes to the perikaryon, and reversal of these events takes place upon hormone-induced melanosome dispersion. These cells contain parallel arrays of microtubules, the majority of which are located in the perikaryon and in cortical regions of the processes. Studies with heavy meromyosin binding demonstrated two types of actin filaments: 1) a decorated meshwork of filaments similar to those usually found in close association with plasma membranes, and 2) filaments decorated in a manner similar to that of stress fibers. There is an apparent increase in the amount of filaments during melanosome aggregation. These results are discussed in relation to intracellular movement.Supported, in part, by grants AM-5384 and AM-13724 from U.S.P.H.S., and grant 234046 from the Japanese Ministry of Education     

Fundulus heteroclitus in the Laboratory: A History

The mummichog, Fundulus heteroclitus, was first used in thelaboratory as a source of ripe eggs and sperm to provide developingbony-fish eggs. These in turn were used mostly to study embryologicalprocesses and responses to various chemicals. Other investigationsat the end of the nineteenth and beginning of the twentiethcenturies dealt with regeneration, developmental genetics andhybridization, osmoregulation, behavior, and pigmentation, especiallycolor change. Except for experiments on chromatophore control,endocrinological studies did not get underway until the 1930s.They have included the functioning of the thyroid, adrenal,and pineal glands, and the endocrine control of reproduction,growth, osmoregulation, and calcium metabolism. Among more recentlystudied subjects are survival at subzero temperatures, adaptationto stress, weightless orientation in space, circadian rhythms,and the bioassay of the exophthalmos-producing substance ofman as well as other physiologically active factors. The singleattribute of the mummichog that has been most responsible forits remarkable popularity as a laboratory animal is its hardinessin captivity. Despite the fact that it is not widely availablelike the goldfish, is not easily bred in aquaria like the livebearingguppy, and has no value as a food or game fish like the trout,the mummichog has made a most substantial contribution to experimentalbiology     

Photoperiodic regulation of thyroid responsiveness to TSH in Fundulus heteroclitus

Treatment of the killifish, Fundulus heteroclitus, with ovine thyrotropin (oTSH) produced elevations in serum thyroxin (T4) that varied seasonally with a strong inverse correlation to environmental photoperiod (P less than 0.001). At 20 degrees C, oTSH (0.2 IU) increased serum T4 to levels ranging from 1.5 micrograms/100 ml in midsummer to 7.5 micrograms/100 ml in midwinter despite relatively stable resting levels throughout the two years of study. Similar rates of clearance of T4 in summer and winter suggest that an alteration in thyroid response to oTSH accounts for this change. The serum T4 response to oTSH is both photoperiod- and temperature-dependent. In laboratory studies, fish exposed to a short artificial photoperiod (LD 8:16) for one month responded to oTSH with T4 levels about twice as high as those in fish exposed to long days (LD 14:10). On the other hand the T4 response to oTSH was sharply reduced at 5 degrees C from that seen at 20 degrees C. This suggests that, in nature, seasonal changes in photoperiod and temperature have opposite effects on thyroidal responsiveness to TSH. Overall, the annual variation in the T4 response to oTSH appears to be driven by photoperiod, inasmuch as the alterations preceded major temperature changes in the wild and can be shown to occur at constant temperature in captivity. Photoperiodic induction of changes in thyroid sensitivity may aid in the maintenance of basal T4 levels under changing thermal conditions.     

Use of Fundulus heteroclitus in Pollution Studies

The mummichog, Fundulus heteroclitus (L.), an estuarine cyprinodontiformteleost, is used extensively and increasingly as a bioassayorganism in toxicological investigations and as an indicatorof marine water quality owing, in part, to its wide geographicrange, abundance throughout most of that range, and adaptabilityto laboratory conditions. Available data are summarized foracute toxicities to mummichog adults of 118 contaminants at22±2% salinity, 19.5±0.5°C, pH 7.8±0.2,and dissolved oxygen >4.0 mg/liter. Organochlorine insecticideswere the most toxic chemicals tested under these conditions;approximate concentrations of 15 organochlorine pesticide compoundsfatal to 50% in 96 hr ranged between 0.0001 mg/liter and 0.1mg/liter. Comparable data for other groups of chemicals were:11 organophosphorus insecticides, 0.001 and 100.0 mg/ liter;30 trace metals and metalloids, 0.01 and 10,000.0 mg/liter;34 synthetic detergents, 1.0 and 1,000.0 mg/liter; 10 chemicaloil dispersants, 1.0 and 10,000.0 mg/liter; 13 oildispersantmixtures, 100.0 and 10,000.0 mg/liter; 3 household soaps, 100.0and 10,000.0 mg/liter; and 2 crude oils, 1,000.0 and>10,000.0mg/liter. Parameters currently used to evaluate sublethal effectsof pollutants in mummichogs include bioaccumulation, histopathology,growth and early development, teratology, liver enzymes, bloodchemistry, fin regeneration, organ weight, and behavior; theseare illustrated with appropriate examples. There is a need formore pertinent variables to assess toxicant-induced stress inmummichogs.     

Alkaline phosphatase activity of the mouse

Plasma alkaline phosphatase (PAP) activity of young adult CD mice is low (e.g. 1/10th of that of rat) and is lower in males than in females. The variability of PAP was assessed in groups of mice kept under different husbandry conditions (e.g. caging, diet, SPF). PAP activities of fed and fasted mice are similar. After comparing their acrylamide electrophoresis and the action of several inhibitors upon the PAP and the alkaline phosphatase of various organ extracts, it is suggested that the two isoenzymes of PAP (indicated by heat inhibition) probably arise from bone and liver.     

Anomalous tolerance to low pH in the estuarine killifish Fundulus heteroclitus

1. The euryhaline fish Fundulus heteroclitus has an incipient lethal pH between 3.75 and 4.0 in fresh water.2. Fish exposed to pH 3.5 in sea water or fresh water died in about 3 hr, and had greatly elevated or depressed body sodium concentrations, respectively. The direction and degree of change in body sodium level depended on the sodium diffusion gradient between the environment and the fish. This is the first time that the death of fish in sea water at low pH has been shown to be associated with hypernatremia.3. Yet, sodium fluxes during the first hour of exposure to pH 3.5 in water of 3.5 or 35 ppt salinity were not different from controls, and body and plasma sodium concentration did not change during 2hr exposure to pH 3.5. This initial insensitivity of gill sodium regulation to blockage by low pH is quite different from the response of previously studied freshwater fish.4. The degree of acid tolerance displayed by F. heteroclitus is surprising considering its estuarine habits. This paradoxical tolerance appears to be a secondary consequence of its ability to adjust sodium balance in relation to rapid changes in salinity.     

Development of Eimeria funduli in Fundulus heteroclitus1

Laboratory-reared Fundulus grandis and F. heteroclitus were experimentally infected with Eimeria funduli by being fed Palaemonetes pugio (grass shrimp) collected from endemic areas. Histological sections were made of heart, liver, hepatopancreas, spleen, gall bladder, kidney, intestine, peri-intestinal fat, reproductive organs, and brain from F. grandis sacrificed at 1, 2, 6, 12, 18, and 24 h and from F. heteroclitus at 5, 6, 9, 14, 19, 24, 29, 34, 39, and 44 days after consuming naturally infected shrimp. We first found merogonous stages at day 9 postinfection (p.i.). No developmental stages of the parasite could be positively identified in the tissues of experimentally infected fish prior to day 9 p.i. Mature meronts were found 14 days p.i. The majority contained 8–16 (mean, 13) merozoites, but a few meronts had 18–26 (22) merozoites. Gamonts first appeared on day 14, were mature by day 19, and fertilization was completed by day 24 p.i. After sporoblast formation, sporopodia appeared during sporocyst wall formation, between days 24 and 29 p.i. Sporozoite formation was completed by day 44 p.i. in most sporocysts. Most endogenous stages occurred in hepatocytes; however, pancreatic and spleen cells were sometimes infected with gamonts.     

Cathepsin B differential expression and enzyme processing and activity during Fundulus heteroclitus embryogenesis

The present study aimed to test the effects of melatonin on oxidative stress in the yellowtail clownfish, Amphiprion clarkii, as produced by light emitting diodes (LEDs): red, green, and blue. We investigated the effects of the different LEDs on oxidative stress by measuring the mRNA expression of arylalkylamine N-acetyltransferase (AANAT2), the expression and activities of antioxidant enzymes (superoxide dismutase, SOD (EC 1.15.1.1); and catalase, CAT (EC 1.11.1.6)), and plasma H2O2 and plasma melatonin levels. In red light, the expression of AANAT2, SOD, and CAT mRNA was significantly higher than those under the other light spectra. SOD and CAT activities and plasma H2O2 and melatonin levels were also significantly higher for the red spectra than those for the other light spectra. These results indicate that red light induces oxidative stress. To investigate the effects of melatonin on oxidative stress, we injected melatonin into live fish (in vivo) or treated cultured pineal organ (in vitro) with melatonin. We found that AANAT2, SOD, and CAT mRNA expression levels, SOD and CAT activities, and plasma H2O2, lipid peroxidation (LPO) and melatonin levels were significantly lower than those for the controls. Therefore, our results indicate that red light induces oxidative stress and melatonin plays the role of a strong antioxidant in yellowtail clownfish.