Influence of egg size on embryos and larvae of Fundulus heteroclitus (L.)

Egg size for Fundulus heteroclitus (L.) populations is concordant with the distribution of the two F . heteroclitus subspecies, i.e. F. h. heteroclitus eggs are considerably larger than F. h. macrolepidotus eggs. The influence of egg size on survival of embryos during incubation and survival and growth of newly-hatched larvae was estimated for four populations representing both subspecies along the Atlantic coast of the United States and in Delaware Bay. Survival of embryos was determined for incubation periods of 14, 21 and 28 days. Greatest differences in survival were detected following the longest incubation period where less than 50 per cent of the smaller F. h. macrolepidotus eggs survived while little or no mortality was detected among the larger F. h. heteroclitus eggs . Influence of egg size on larval survival was also greatest among those larvae hatched after 28 days where F. h. macrolepidotus larvae survived without food, for an average of 6 days, while F. h. heteroclitus larvae survived 11–12 days. F. h. heteroclitus larvae were significantly larger at hatching than F. h. macrolepidotus larvae. Larval growth rates were the same (0.4 mm day⁻¹) in both subspecies. As a result, size differences at hatching were still maintained after 42 days of growth. The differences in egg size along with other morphological and reproductive characteristics of F. heteroclitus populations probably represent genetically based adaptations to environmental conditions, of which the length of the spawning season is one of the major components stimulating the coevolution of these traits.     

In vivo repair of rat liver DNA damaged by dimethylnitrosamine or diethylnitrosamine.

Effects of hepatocarcinogens dimethylnitrosamine (DMN) and diethylnitrosamine (DEN) on the sedimentation pattern of rat liver DNA in alkaline sucrose gradients were studied with regard to time and dose dependency. Both DMN (10 mg/kg body weight) and den (13.4 or 134 mg/kg) induced appreciably decreased DNA sedimentation rates at 24 h after injection. DMN at 10 mg/kg was as effective in decreasing the DNA sedimentation rate at 24 h after injection as was the higher dose of DEN (134 mg/kg). Sedimentation patterns at 1, 6 and 14 days after injection indicated that damage induced by DEN (134 mg/kg) was repaired at a substantially lower rate than DMN (10 mg/kg) induced damage. When effects of equimolar doses of DMN (10 mg/kg) and DEN (13.4 mg/kg) were compared at 1, 6 and 14 days after injection, it was observed that the more pronounced damage of rat liver DNA induced by DMN was repaired at a faster rate than was the DEN-induced damage. At the molecular level this difference in repair between damage induced by the two nitrosamines is probably related to different DNA alkylation patterns. The relatively persistent nitrosamine-induced DNA lesions (observed especially after DEN administration) are thought to represent phosphotriesters which give rise to single strand DNA breaks at strongly alkaline conditions of lysis on top of the gradient. The results are discussed in relation to the possible significance of alkylation and repair of DNA in the formation of (pre)cancerous lesions in rat liver.     

Karyotypic analysis of 2 sympatric species of fish, Fundulus heteroclitus L. and F. majalis

A chromosome study was performed on 2 sympatric species of marine fish, Fundulus heteroclitus L. and F. majalis . A modification of techniques which included the combination of the procedures of McPhail & Jones (1966) with those more recently developed by Ohnuki (1968), for demonstrating the spiral structure of cultured human leukocytes, resulted in clear and distinct chromosome spreads from gill epithelial cells of these 2 species. Optimum chromosome spreading and accumulation of metaphase figures resulted when specimens of F. heteroclitus received either a 0·28 ml or a 0·4 ml injection of a 0·1 % colcemid solution for 45 h and 48 h respectively. Best results in F. majalis occurred when specimens received a 0·1 ml dose of the 0·1 % colcemid solution for 22 1/2 h. Studies of the karyotypes of 18 specimens of F. heteroclitus and 9 specimens of F. majalis indicated that the 2 N chromosome number for each species was 48.     

Copper uptake by two sympatric species of Killifish Fundulus heteroclitus (L.) and F. majalis (Walbaum)

Differential copper uptake within brain, visceral, gill, somatic and residual tissue was examined in the two sympatric species of killifishes Fundulus heteroclitus and F. majalis . Various salinities and copper concentrations were tested on both juvenile and adult stages to examine their effects on copper uptake. Salinity played a significant role in copper uptake in both species of killifishes. However, the effect of salinity was specific for each species and life history stage. Copper content of various tissues was directly related to the uptake pattern exhibited by whole adult fishes. Fundulus majalis brain tissue and F. heteroclitus muscle tissue were exceptions. Severe body lesions and subcutaneous haemorrhaging were observed after 2–3 day exposure to both 2 and 8 ppm copper.     

Continuous laboratory production of fertile Fundulusheteroclitus (Walbaum) eggs lacking chorionic fibrils

A brood stock of Fundulus heteroclitus (Walbaum) minnows was established at theJohnson Space Center in Houston, Texas. By use of the aquarium maintenance systemdescribed in this report the fish were induced to produce fertile eggs throughout the year.Additional data is presented to confirm that F. heteroclitus found in the vicinity of Beaufort, North Carolina produce eggs which lack chorionic fibrils, making them especially suitablefor use in embryo development studies.     

First record of a population of the exotic mummichog Fundulus heteroclitus (L., 1766) in the Mediterranean Sea basin (Ebro River delta)

Until now, mummichog Fundulus heteroclitus have been recorded only from the estuaries of the southernmost river basins of the Iberian Peninsula (Guadiana and Guadalquivir Rivers). Data reported here indicate the first record of F. heteroclitus in the Ebro River delta (north-east Spain), and hence also the first citation for F. heteroclitus as an invasive species present in any estuary of the Mediterranean Sea basin.     

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.     

Acute toxicology to an estuarine teleost of mixtures of cadmium, copper and zinc salts

Effects of mixtures of chloride salts of cadmium, copper and zinc on survival, whole body residues, and histopathology of mummichog, Fundulus heteroclitus (L.), were investigated in synthetic sea water at 20‰ salinity and 20°C. Mixtures of Cu²⁺ and Zn²⁺ as indicated by 96 h bioassay studies produced more deaths than expected on the basis of toxicities of individual components. Concentrations of Cd²⁺ not ordinarily lethal exerted a negative effect on survival of fish intoxicated by salts of copper, zinc, or both.
Atomic absorption determinations of Cd, Cu, and Zn residues in mummichog which survived 96 h exposures to each of these toxicants provided useful indices of total body burdens for these metals. Residues from survivors held in mixtures, especially Cd²⁺ and Zn²⁺ mixtures, did not conform to patterns observed for single elements. Whole body aggregates of Cd, Cu, and Zn from dead mummichogs were of limited worth owing to possible accumulation of these metals from the medium after death.
Renal and lateral line canal lesions were noted in all fish subjected to copper concentrations of 1 mg/1 and higher. Renal lesions observed in fish immersed in mixtures of Cu²⁺ and Cd²⁺ assumed a damage pattern characteristic of Cd²⁺; with mixtures of Cu²⁺ and Zn²⁺, lesion were typical of Cu²⁺-induced damage. Lesions induced in lateral line epithelium by Cu²⁺ were not affected by either Cd²⁺ or Zn²⁺. Epithelia lining the oral cavity were necrotized by the caustic action of high levels of Zn²⁺ (60 mg/1) and of Cu²⁺ (8 mg/1).     

Development of rhythmic melatonin secretion from the pineal gland of embryonic mummichog (Fundulus heteroclitus)

The pineal gland of vertebrates produces and secretes the hormone melatonin in response to changes in the light-dark cycle, with high production at night and low production during the day. Melatonin is thought to play an important role in synchronizing daily and/or seasonal physiological, behavioral, and developmental rhythms in vertebrates. In this study, the functional development of the pineal melatonin-generating system was examined in the mummichog, Fundulus heteroclitus, an euryhaline teleost. In this species, the pineal gland contains an endogenous oscillator, ultimately responsible for timing the melatonin rhythm. Oocytes from gravid females were collected and fertilized in vitro from sperm collected from mature males. Skull caps containing attached pineal glands were obtained from F. heteroclitus embryos at different embryonic stages and placed in static or perfusion culture under various photoperiodic regimes. Rhythmic melatonin secretion from pineal glands of embryonic F. heteroclitus embryos exposed to a 12L:12D cycle in static culture was observed at five days post-fertilization. The ontogeny of circadian-controlled melatonin production from F. heteroclitus pineal glands exposed to constant darkness for five days was also seen at day five post-fertilization. These data show that early development of the pineal melatonin-generating system in this teleost occurs prior to hatching. Pre-hatching development of the melatonin-generating system may confer some selective advantage in this species in its interactions with the environment.     

Persistence and accumulation of (potential) single strand breaks in liver DNA of rats treated with diethylnitrosamine or dimethylnitrosamine: correlation with hepatocarcinogenicity.

Effects of diethylnitrosamine (DEN) and dimethylnitrosamine (DMN) on the sedimentation pattern of [3H]thymidine-labelled Sprague-Dawley female rat liver DNA in alkaline sucrose gradients were studied with regard to time and dose dependency. In experiments at 1--56 days after a single injection it was observed that (potential) single strand breaks induced by DEN were repaired at a low rate. At 56 days the sedimentation pattern was still grossly abnormal. Half-life values of 27 and 46 days were observed after 134 mg/kg DEN (approx. 45% of the LD50) and 13.4 mg/kg DEN, respectively. Identical experiments after DMN (10 mg/kg, corresponding to about 35% of the LD50) showed return to (almost) completely control sedimentation patterns within 56 days after injection (t 1/2 = 8 days). Experiments at 6 or 56 days after the last of a series of 5 or 10 weekly injections of DEN (13.4 mg/kg) showed that a major part of DEN-induced damage (measured as single strand breaks) is of a persistent and accumulating character. No accumulation of DMN-induced rat liver lesions was observed. It is concluded that DNA fragmentation and lack of DNA repair is not a consequence of hepatotoxicity. Since at equimolar doses DEN gives appreciably less DNA alkylation (including O6-alkylguanine) but is much more effective both as an inducer of preneoplastic liver lesions and as a hepatocarcinogen when compared with DMN, we believe that the formation of persistent (and accumulating) DNA damage after DEN administration might be relevant in the process of liver tumour formation.     

Temporal dynamics in growth and white skeletal muscle composition of the mummichog Fundulus heteroclitus during chronic hypoxia and hyperoxia

Specific growth rate (G(S) ) and white skeletal muscle composition were measured in the mummichog Fundulus heteroclitus over a period of 28 days at four levels of dissolved oxygen (DO): severe hypoxia (c. 1.2 mg O(2) l(-1) ), moderate hypoxia (3.0 mg O(2) l(-1) ), normoxia (7.1 mg O(2) l(-1) ) and hyperoxia (10.6 mg O(2) l(-1) ). The G(S) was calculated over 0-8, 0-14, 0-28 and 14-28 days, and muscle protein, lactate dehydrogenase (LDH), DNA, RNA and water were measured at 0, 8, 14 and 28 days. Exposure of fish to severe hypoxia was associated with significantly reduced G(S) , lower muscle protein content and lower RNA:DNA compared with other DO treatments. When calculated over the first and second half of the 28 day exposure, however, G(S) of fish in severe hypoxia increased significantly during the second two-week interval, to the same rate as that of normoxic fish. Muscle LDH activity and water content were not significantly affected by DO level. Neither moderate hypoxia nor hyperoxia significantly affected G(S) or any biochemical variable. The results demonstrate that F. heteroclitus can tolerate wide variation in ambient oxygen concentration and, during prolonged exposure to severe hypoxia, shows significant compensation for the initial negative effects on growth. The capacity of F. heteroclitus to grow over a wide range of DO probably contributes to its ability to exploit habitats characterized by marked variation in oxygen availability.     

Increased prevalence of larval Eustronglylides (Nematoda) in the mummichog, Fundulus heteroclitus (L.), from the discharge canal of a power plant in the Chesapeake Bay

The prevalence of larval Eustrongylides sp. in the mummichog, Fundulus heteroclitus , was compared for two populations, one in the vicinity of the intake and one in the discharge canal of a power plant at Chalk Point on the Patuxent River, Maryland. The parasite was four times as prevalent in female F. heteroclitus collected from the discharge canal as in those collected from the intake area. Almost all infected fish were 70 mm or greater in total length. The larvae occurred in capsules and free in the mesentery, or associated with the gut, liver, or gonad of the host; multiple infections were common. Infection by Eustrongylides resulted in pronounced negative effects on F. heteroclitus . The dry weight of somatic tissue was reduced 5% and ovary dry weight was reduced 50% in infected individuals, compared to uninfected individuals of the same length. The proportion of infected females carrying ripe eggs during the breeding season was less than half the value for uninfected females. It is hypothesized that the greater prevalence of the parasite in the discharge canal results from increased abundances of oligochaetes (suspected first intermediate hosts of the parasite), presumably as a consequence of elevated temperatures and organic enrichment from the power plant.     

The long amplicon quantitative PCR for DNA damage assay as a sensitive method of assessing DNA damage in the environmental model, Atlantic killifish (Fundulus heteroclitus)

DNA damage is an important mechanism of toxicity for a variety of pollutants, and therefore, is often used as an indicator of pollutant effects in ecotoxicological studies. Here, we adapted a PCR-based assay for nuclear and mitochondrial DNA damage for use in an important environmental model, the Atlantic killifish (Fundulus heteroclitus). We refer to this assay as the long amplicon quantitative PCR (LA-QPCR) assay. To validate this method in killifish, DNA damage was measured in liver, brain, and muscle of fish dosed with 10 mg/kg benzo[a]pyrene. This exposure caused 0.4-0.8 lesions/10 kb. We also measured DNA damage in liver and muscle tissues from killifish inhabiting a Superfund site, confirming the utility of this method for biomonitoring. In both cases, damage levels were comparable in nuclear DNA (nDNA) and mitochondrial DNA (mtDNA). Since extensive nDNA sequence data are not readily available for many environmentally relevant species, but mitochondrial genomes are frequently fully sequenced, this assay can be adapted to examine mtDNA damage in virtually any species with little development. Therefore, we argue that this assay will be a valuable tool in assessing DNA damage in ecotoxicological studies.     

The effects of hypophysectomy on the retinomotor responses of the killifish, Fundulus heteroclitus

This paper presents the results of an investigation concerned with the effects of long-term hypophysectomy on the retinomotor responses of the euryhaline killifish, Fundulus heteroclitus: 1. The eye of hypophysectomised Fundulus heteroclitus responds to light and dark in the same manner as that of intact controls: the retina is not in a state of permanent light-adaptation as claimed by Vilter (1942) for the hypophysectomised eel. 2. There is no evidence of a persistent circadian rhythm during continous darkness. 3. Unilateral illumination of the eye of intact fish results in dispersion of retinal pigment in both illuminated and unilluminated eyes, as in the goldfish (Ali, 1964), but no such contralateral response was evident after hypophysectomy. The cones are unresponsive.     

Cell and species differences in metabolic activation of chemical carcinogens

Metabolic activation and DNA binding of aflatoxin B1 (AFB1), N-nitrosodimethylamine (DMN) and benzo[a]pyrene (B[a]P) were compared in human, rat and mouse hepatocytes and human pulmonary alveolar macrophages (PAM). The degree of carcinogen activation by hepatocytes and PAM was measured by cell-mediated mutagenesis assays in which co-cultivated Chinese hamster V79 cells were used to monitor mutagenic metabolites. Hepatocytes from human, mouse and rat metabolized DMN and released the active metabolites to induce either ouabain- or 6-thioguanine-resistant mutation. The mutation frequencies mediated by hepatocytes of the 3 animal species were approximately 3-9 mutants/10(5) survivors at a concentration of 0.2 mM DMN. The variations of radioactivity bound to liver cell DNA were relatively small in cultured mouse, rat, and human hepatocytes exposed to 14C label DMN (0.5 mM) and the binding values were in a range of 6-12 X 10(3) pmoles/mg DNA. However, rat hepatocytes were at least 10-fold more effective than either human or mouse hepatocytes in generating mutagenic metabolites of AFB1 and also had a much higher AFB1 metabolite DNA-binding value. The AFB1 DNA-binding levels were 4.1, 12-27 (range), 120 pmoles/mg DNA respectively in mouse, human, and rat liver cells following AFB1 (3.3 microM) exposure for 20 h. Hepatocytes from the 3 animal species were unable to mediate mutation in the presence of 4 microM B[a]P; PAM activated B[a]P and effectively mediated mutation in the co-cultivated V79 cells. In contrast to results with hepatocytes, PAM failed to generate enough mutagenic metabolites of AFB1 (3.3 microM) and the mediation of mutations was seen only at very high concentration of DMN (80 mM). The genotoxic effects of the 3 carcinogens on hepatocytes from different species in vitro were in agreement with the in vivo animal experiments in that mice are relatively resistant to AFB1 carcinogenesis whereas rats are sensitive; B[a]P is not effective as a complete liver carcinogen in adult rat and mouse whereas DMN induces liver cancer.     

Effect of 1,10-phenanthroline on acute liver injury induced by dimethylnitrosamine in the rat

Acute liver damage was induced in rats by intragastric doses of dimethylnitrosamine (DMN, 3 mg/100 g body weight) and measured 24 hours later by morphological and biochemical methods. 1, 10-Phenanthroline (1, 10-P, 2 mg/100 g) administered simultaneously with DMN prevented the development of the characteristic morphological picture of liver injury. At the same time, the amount and synthesis of total liver proteins, the activity and distribution of liver β-glucuronidase, and the level of seromucoid and isocitric dehydrogenase (ICDH) activity in the serum, significantly changed by DMN, was within the range of control values when 1, 10-P was simultaneously administered. The protective effect of 1, 10-P against acute DMN hepatotoxicity paralleled the inhibition of some liver microsomal drug-metabolizing enzymes (aniline hydroxylase, morphine demethylase, content of cytochrome P-450). At higher doses of DMN (6 mg and 10 mg/100 g), the administration of 1, 10-P was no longer protective, although the content of cytochrome P-450 was only 20% of the value for normal liver microsomes. Therefore, in acute administration within a certain range of concentration of DMN, 1, 10-P might inhibit the microsomal drug-oxidizing enzymes, thus inhibiting the metabolism of the drug to a more toxic product.