The ultrastructure of the otolithic membrane and otolith in the juvenile mummichog,Fundulus heteroclitus

The sagitta otolithic membrane of Fundulus heteroclitus consists of two different zones. A structured zone (gelatinous layer), which usually exhibits a reticulated or honeycomb-like architecture, is composed of tightly arranged fibrous material and covers only the sensory region of the macula. The gelatinous layer extends from the otolith surface to the tips of the sensory hairs, and probably functions primarily as a mechanoreceptor. The arrangement of this zone is closely associated with specific overlying structural features of the otolith surface and may also influence the pattern of mineral deposition to some degree. A nonstructured zone (subcupular meshwork) consists of fibers in very loose networks and covers both sensory and nonsensory regions of the macula. Over the sensory region, some of this fibrous material extends from the epithelial surface, through pores in the gelatinous layer, to the surface of the overlying otolith. In the nonsensory region, fibers of the subcupular meshwork are relatively more numerous and extend around the peripheral margin of the otolith. Evidence is presented which suggests that the fibrous material of the subcupular meshwork is incorporated into the otolith as an organic matrix constituent. New aspects on the ultrastructure of the otolith are presented and discussed.     

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.     

Fast-start escape performance across temperature and salinity gradients in mummichog Fundulus heteroclitus

Fast-start predator-escape performance of mummichogs Fundulus heteroclitus was tested across field-informed variation in temperature (24, 30 and 36°C) and salinity (2, 12 and 32 ppt). Performance was similar across temperatures and salinities when fish were allowed to acclimate to these conditions. However, when mummichogs experienced acute temperature changes, performance exhibited thermal dependence in two contrasting ways. Fast-start turning rates and linear speeds varied directly with the temperature at which the manoeuvre was executed, but these aspects of performance varied inversely with acclimation temperature, with cool-acclimated fish exhibiting faster starts across test temperatures. Temperature effects were consistent across salinities. These results suggest that while mummichogs increase performance with acute temperature increases, long-term rises in sea temperature may cause these fish to become more susceptible to predation during abrupt cooling events, such as when storm events flood shallow water estuaries with cool rainwater.     

Effects of heavy metals on development of the killifish, Fundulus heteroclitus

When F. heteroclitus embryos were exposed to inorganic mercury at concentrations of 0.03 or 0.1 mg/1 at the early blastula stage, the percentage of successful axis formation was reduced and a significant proportion of embryos developed cyclopia or intermediate conditions leading to cyclopia. Treatment at the late blastula stage reduced the severity of the defects. Embryos which developed in lead at concentrations of 1 and 10 mg/1 were normal in appearance until hatching, at which time they exhibited lordosis or were unable to uncurl from the position they had while still inside the chorion. No significant effects of cadmium at concentrations up to 10 mg/1 were noted.     

Food limitation of a Delaware salt marsh population of the mummichog,Fundulus heteroclitus (L.)

Summary The relationship of the mummichog, Fundulus heteroclitus, to its food supply was examined using 8 m x 20 m enclosures under field conditions. Density effects were examined by stocking these enclosures with fish at densities ranging from 1/8 to four times the natural population density. Mean growth rate in each pen was estimated from measurements on individually marked fish. Fish enclosed at normal density grew at the same rate as fish from the natural population, indicating that caging had minimal effects on growth rate. When enclosed below natural density, mummichogs had a growth rate that was 2–3 times higher than that of the natural population. Fish enclosed at four times normal density had a negative mean growth rate, a higher mortality rate than fish enclosed at natural density, and no egg production.In further experiments, food was added to the same type of enclosure at fish densities ranging from one-half to four times natural density. Food additions had a positive effect on growth rate at all densities. Food additions also caused egg production of fish enclosed at the highest density to increase to a rate equivalent with the natural population. We conclude that the total food supply may regulate the maximum size of the Canary Creek mummichog population by affecting the individual growth rate, mortality rate, and fecundity.     

Characterization of aquabirnaviruses from flounder Pseudopleuronectes americanus and mummichog Fundulus heteroclitus in the Chesapeake Bay, Virginia, USA

Viruses were isolated in cell culture from tissue homogenates of flounder Pseudopleuronectes americanus and mummichog Fundulus heteroclitus in the Chesapeake Bay, Virginia, USA. Neutralization and immunofluorescence tests with aquabirnavirus (West Buxton strain)-specific polyclonal antisera indicated that both viruses were aquabirnaviruses belonging to Serogroup A, the most common aquabirnavirus serogroup in the United States. This was confirmed by RT-PCR, with primers targeting the VP3 and VP2 gene of aquabirnaviruses. The VP2-specific RT-PCR cDNA amplification product was sequenced and deduced amino-acid sequences were compared with known sequences of the type strains of the 9 serotypes of aquabirnavirus Serogroup A. This demonstrated that the viruses from both flounder and mummichog belong to aquabirnavirus Genogroup 1. The flounder isolate exhibited deduced amino acid sequence similarities of 98.1% with the Jasper strain of serotype A9, and 97.7% with the West Buxton strain of serotype A1. The isolate from mummichog exhibited deduced amino acid sequence similarities of 99.1% with the West Buxton strain of Serotype A1 and 94.8% with the Jasper isolate of Serotype A9. Similarities of deduced amino acid sequences ranged from 79.9 to 86.9%, with representatives of the other 7 serotypes. This is the first report of an aquabirnavirus from mummichog F. heteroclitus and only the fifth report of an aquabirnavirus from a flounder species.     

Turnover rates of nitrogen stable isotopes in the salt marsh mummichog, Fundulus heteroclitus, following a laboratory diet switch

Nitrogen stable isotopes are frequently used in ecological studies to estimate trophic position and determine movement patterns. Knowledge of tissue-specific turnover and nitrogen discrimination for the study organisms is important for accurate interpretation of isotopic data. We measured δ¹⁵ N turnover in liver and muscle tissue in juvenile mummichogs, Fundulus heteroclitus, following a laboratory diet switch. Liver tissue turned over significantly faster than muscle tissue suggesting the potential for a multiple tissue stable isotope approach to study movement and trophic position over different time scales; metabolism contributed significantly to isotopic turnover for both liver and muscle. Nitrogen diet-tissue discrimination was estimated at between 0.0 and 1.2‰ for liver and –1.0 and 0.2‰ for muscle. This is the first experiment to demonstrate a significant variation in δ¹⁵ N turnover between liver and muscle tissues in a fish species.     

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.     

A new species of Swingleus (Monogenea: Gyrodactylidae) from the mummichog Fundulus heteroclitus, in the Delaware Bay

Swingleus ancistrus n. sp. (Monogenea: Gyrodactylidae) is described from the skin and fins of Fundulus heteroclitus from Canary Creek Marsh, Lewes, Delaware. Subsequent records from New Jersey, Maryland, Virginia, and North Carolina are reported. Swingleus ancistrus is differentiated from S. polyclithroides, the only other species of the genus, primarily by its size, haptoral morphology, host, and locality. Swingleus ancistrus is larger in almost every dimension and exhibits a distinct notch on the anterior border of the opisthaptor, and several features of the haptoral anatomy and peduncular bar are unique. The prevalence of infection in hosts collected from the type locality in August was 100%. The maximum number of S. ancistrus recovered from a single host was 134. The average intensity of infection was 24 S. ancistrus per host from the type locality.     

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.     

Gametogenesis in Fundulus heteroclitus

Our present understanding of the structure of the gonads andof gametogenesis in Fundulus helerodilus is briefly reviewed. The testes contain spermatogenic tubules which distally arecomposed of linearly arranged germinal cysts with clones ofsynchronously developing germ cells, and proximally of efferentducts with mature sperm. Within the tubules, those cysts containingspermatogonia are found close to the testicular periphery, whereascysts with mature sperm are contiguous with the efferent ducts.Cytological details of the three principal stages of spermatogenesis,namely spermatocytogenesis, meiosis, and spermiogenesis, arepresented along with examples of the various somatic cells ofthe testis. The ovary consists of numerous ovigerous lamellae which containrandomly arranged follicles in various stages of development.Since follicular growth is asynchronous, follicles of all sizesare present in the ovary during the breeding season and eggsare continuously ovulated into the ovarian lumen. Oocyte growthis divided into five distinct stages: Primary growth (substagesA and B), cortical alveolus formation, vitellogenesis, oocytematuration (substages A and B), and the ovulated egg. This seriesof stages is based both on cytological observations and physiologicalevents.     

Latitudinal variability in lunar spawning rhythms: absence of a lunar pattern in the northern mummichog Fundulus heteroclitus macrolepidotus

In this study, the natural spawning season of the northern mummichog Fundulus heteroclitus macrolepidotus was examined from May to July in the lower St John River, New Brunswick, Canada (45° N), through measurements of gonado-somatic index ( I _G), liver-somatic index ( I _L) and condition factor ( K ). I _G increased during the prespawning phase (mean ± s.e. 5·49 ± 0·20%), peaked during the spawning phase (14·68 ± 0·51%) and dropped during the regression phase (3·23 ± 0·29%). A single peak of spawning was observed in F. h. macrolepidotus , contrasting with multiple peaks observed in studies south of 41° N in North America. Within the prespawning phase, there were significant differences between sampling dates in female body mass, gonad mass, liver mass and K . On the basis of results from this study, water temperature is believed to be the predominant factor controlling initiation of spawning in F. h. macrolepidotus .     

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.     

Altered expression of the xenobiotic transporter P-glycoprotein in liver and liver tumours of mummichog Fundulus heteroclitus from a creosote-contaminated environment

P-glycoproteins (Pgps) are involved in efflux of xenobiotics from drug-resistant cell lines and tumours, and in excretion of toxicants from normal tissues. Recently, investigators have proposed that Pgp activity contributes to resistance or tolerance of certain aquatic species to pollutants. In the present study using immunoblot and immunohistochemical techniques, we found elevation of Pgp in liver and liver tumours of creosote-resistant mummichog from a contaminated site in the Elizabeth River, Virginia. Immunoblots of mummichog liver extracts showed an immunoreactive band at 170 kDa and indicated two- to three-fold elevation of Pgp in livers of resistant fish relative to those from a reference site. Laboratory exposures of reference site fish to a model PAH (3-methylcholanthrene), however, produced no increase in liver Pgp levels as measured by immunoblot. Normal mummichog liver sections showed specific immunohistochemical staining for Pgp on the canalicular surface of hepatocytes. In the majority of hepatic neoplasms we observed a high level of over-expression and altered patterns of Pgp expression. However we did not observe Pgp over-expression in early proliferative lesions. Elevation of Pgp in livers and liver tumoursof these resistant mummichog may contribute to their survival in a heavily contaminated environment.     

Methylmercury teratogenesis in the killifish, Fundulus heteroclitus.

Exposure of developing eggs of the killifish, Fundulus heteroclitus, to 0.03 or 0.04 mg/l of methylmercuric chloride resulted in a variety of abnormalities. Percentage of axis formation was reduced somewhat, and many embryos developed cyclopia or intermediate conditions leading to cyclopia, reflecting interference with induction of the forebrain. Defects in the cardiovascular system also appeared in the form of failure of the heart to differentiate properly into chambers. The heart was a thin, feebly beating tube, incapable of causing the blood to circulate. Other tissues, however, continued developing fairly normally, and embryos showed spontaneous movement comparable to controls. Embryos with severe cardiovascular or optic defects did not hatch. Upon hatching, some embryos which had previously appeared normal were found to have skeletal malformations in the form of vertebral bends or the inability to uncurl from the position which they had while still inside the chorion. Exposure to the toxicant for shorter periods of time (6, 12, or 24 hours) reduced the incidence of abnormalities. The second day of development was found to be the most sensitive period.     

Altered expression of the xenobiotic transporter P-glycoprotein in liver and liver tumours of mummichog Fundulus heteroclitus from a creosote-contaminated environment

P-glycoproteins (Pgps) are involved in efflux of xenobiotics from drug-resistant cell lines and tumours, and in excretion of toxicants from normal tissues. Recently, investigators have proposed that Pgp activity contributes to resistance or tolerance of certain aquatic species to pollutants. In the present study using immunoblot and immunohistochemical techniques, we found elevation of Pgp in liver and liver tumours of creosote-resistant mummichog from a contaminated site in the Elizabeth River, Virginia. Immunoblots of mummichog liver extracts showed an immunoreactive band at 170 kDa and indicated two- to three-fold elevation of Pgp in livers of resistant fish relative to those from a reference site. Laboratory exposures of reference site fish to a model PAH (3-methylcholanthrene), however, produced no increase in liver Pgp levels as measured by immunoblot. Normal mummichog liver sections showed specific immunohistochemical staining for Pgp on the canalicular surface of hepatocytes. In the majority of hepatic neoplasms we observed a high level of over-expression and altered patterns of Pgp expression. However we did not observe Pgp over-expression in early proliferative lesions. Elevation of Pgp in livers and liver tumoursof these resistant mummichog may contribute to their survival in a heavily contaminated environment.     

Microsatellite primers for the Atlantic coastal killifish,Fundulus heteroclitus,with applicability to related Fundulus species

The mummichog (Fundulus heteroclitus), a common Atlantic coastal killifish, is a model vertebrate species for the study of molecular genetic variation in natural populations and of environmental toxicology. We report the development of a set of 20 microsatellite loci in this species. Average expected heterozygosity across all loci was 0.84 (range: 0.60–0.97), revealing a high level of variability at most loci. A survey of seven additional Fundulus species yielded one or two robust amplification products in over half (63%) of the species–primer combinations tested. Therefore, many of these loci will also prove useful in studies of other members of the genus Fundulus.