Effects of Environmental Stressors and Immunosuppressants on Immunity in Fundulus heteroclitus

Environmental stressors have been shown to alter immunocompetencein mammals. Similar effects have been reported in fish. Thepurpose of this paper is to review the literature concerningthe white blood cells and immunity in Fundulus heteroclitus.Evidence for immunosuppression following prolonged captivity,handling, hypophysectomy, radiation, changes in temperatureand salinity, and exposure to chemicals including environmentalpollutants is discussed. New evidence for immunosuppressionfollowing exposure to benzo-a-pyrene, pentachlorophenol, andhexachlorobenzene is presented. Possible mechanisms of immunealteration and the consequences to F. heteroclitus are discussed.     

The Role of Fundulus heteroclitus in Salt Marsh Trophic Dynamics

The common mummichog, Fundulus heteroclitus, functions as bothpredator and prey in the trophic structure of east coast tidalmarshes. Although mummichogs are generally considered importantto energy transformations within marshes, few studies have convincinglydemonstrated that predation by F. heteroclitus affects the abundanceof salt marsh benthic invertebrates. Thus far investigationsof this type have dealt only with the direct effects of adultmummichogs. The results of recent experiments have suggestedthat by controlling smaller predators, mummichogs may indirectlyhave a positive effect on the densities of some infaunal marshinvertebrates. Our current knowledge of larval and juvenilemummichogs in their natural habitat is minimal. Unlike the adults,which can utilize the intertidal zone only when it is flooded,the young remain on the marsh even at low tide, inhabiting shallowpuddles of residual tidal water that form between clumps ofvegetation and around fiddler crab (Uca sp.) burrows. The importanceof F. heteroclitus in salt marsh communities will remain incompletelyunderstood unless future studies consider the role of larvaland juvenile mummichogs. Although many species of fishes andwading birds feed on mummichogs, the blue crab (Callinectessapidus) is probably the major predator of adult F. heteroclitusin the intertidal salt marsh. Predation by adult mummichogsand xanthid crabs (e.g., Eurytium limosum) may contribute tothe high mortality of larval and juvenile Fundulus.     

Competition and Coexistence Among Four Estuarine Species of Fundulus

Four sympatric species of Fundulus (F. heteroclitus, F. majalis,F. diaphanus, and F. luciae) are distributed along tidal heightand salinity gradients such that F. heteroclitus co-occurs witheach species, while the remaining species rarely occur together.Feeding habitats of all the species are similar, and food hasbeen shown to limit population size of F. heteroclitus. Thispaper examines whether competition is an important structuringforce within this guild by addressing two questions: 1) is thespatial separation exhibited by three of the species due tophysiological barriers or due to competitive exlusion? and 2)when F. heteroclitus occurs with other Fundulus species doescompetition for resources take place? Laboratory studies indicate that all four species are tolerantof a wide range of environmental conditions; available evidencesuggests that physiological barriers are an unlikely explanationfor spatial segregation among some members of this guild. Competitiveexclusion seems a likely alternate explanation, but is supportedonly by studies of diet overlap. Similarly, only inference fromdiet overlap is available to answer the second question. A fieldexperiment is presented here in which F. heteroclitus and F.majalis were placed in enclosures separately and together. Competitionbetween these species appears to be important, and similar fieldexperiments are recommended to investigate competitive interactionsamong other species pairs within the genus     

FINE STRUCTURE OF CHLORIDE CELLS FROM THREE SPECIES OF FUNDULUS

A morphological basis for osmoregulation in the teleosts was studied by comparing the fine structure of chloride cells found in epithelia of the gills of three species of fish: Fundulus heteroclitus which can survive in a wide range of salinities, and F. similis and F. chrysotus which are usually restricted to salt water and fresh water environments, respectively. Gills were removed from F. heteroclitus which had been laboratory adapted to either sea water or pond water. For a comparison, gills were also removed from the marine F. similis and the fresh water F. chrysotus which had been adapted to their natural environments. Gill-filaments were fixed in Millonig's phosphate buffered (pH 7.4), 1 per cent osmium tetroxide and were embedded in Epon. Thin sections of filaments were stained with lead hydroxide. The cytoplasm of chloride cells of all three species of Fundulus is heavily populated with mitochondria and is filled with tubules of the agranular endoplasmic reticulum (ER). An orderly secretory cycle was indicated for chloride cells of salt water adapted F. heteroclitus and the marine F. similis. An amorphous material is observed in the agranular ER. Its density increases towards the apical end of the cell. In the apical cytoplasm, tubules of the agranular ER appear to converge and to discharge the amorphous material into an apical cavity. Except for the actual opening of the apical cavity, the distal end of salt water adapted chloride cells is characteristically shielded from the hypertonic environment by thin cytoplasmic flanges projecting from the neighboring epithelial cells. Chloride cells of the fresh water F. chrysotus resemble chloride cells of pond water adapted F. heteroclitus, in that these cells do not have apical cavities with the functional appearance of those in the sea water adapted forms. The distal end of fresh water adapted chloride cells is typically exposed to the free surface of the gill-filament. The possible function of the cell type is discussed.     

Observations on the relationship between “chloride-type” and “pseudobranch-type” cells in the gills of a fish,Oligocottus maculosus

Summary Chloride cells in gill epithelium of Oligocottus maintained in sea water have a much branched system of agranular cytoplasmic tubules, numerous mitochondria, and a prominent apical crypt. The mitochondria are randomly dispersed and do not show preferential orientation with respect to the tubules.After brief exposure of fish to sea water diluted 1/100 with glass distilled water, the mitochondria and tubules become rearranged into parallel arrays and the apical crypts disappear. The appearance of these cells is similar to that of pseudobranch cells in Fundulus heteroclitus (Copeland and Dalton, 1959).These data suggest that chloride cells and pseudobranch cells represent different adaptive forms of a single cell type and that transformation from the chloride cell configuration to that of pseudobranch cells can be induced by osmotic stress.This work was performed in 1963 when the author was enrolled in the Fine Structure Training program of the Department of Biological Structure, University of Washington. Financial aid was provided by the United States Public Health Service (Grant No. 5T1 GM-136).     

Environmental Influences on Thyroid Function in Teleost Fish

SYNOPSIS. A brief account of some of the influences that environmentalfactors, such as the lunar cycle, daylength and temperature,may have on thyroid activity in certain salmonids and the killifish,Fundulus heteroclitus, is presented. The rapid and immediatecharacterof temperature effects is discussed and a role for central neuroendocrinecontrolin mediating the effects of photoperiod and lunar phenomenainvolvement is suggested.     

Environmental and Endocrine Influences on Reproduction of Fundulus heteroclitus

Reproduction in Fundulus heteroclitus is adapted to the temperateclimate and the tidal environment of the coastal marshes whichare its primary habitat. Egg deposition and spawning are closelylinked to the tidal cycle. Concentration of spawning on springtides gives the fish access to sites in the high intertidalzone. Eggs are deposited near the high water mark, usually insand in New England populations and in Spartina alternifloraor empty Geukensia demissa shells in Middle Atlantic and southernpopulations. Aerial incubation of eggs appears to be essentialfor their survival in the salt marsh. Loss of eggs due to lackof fertilization, mortality during development and predationwas always less than 30 percent in our observations. Hatchingoccurs only when the eggs are inundated, usually on spring tides.An endogenous semilunar gonadal cycle appears to be involvedin control of spawning, but an effective environmental synchronizerhas not been found. Cyclic changes in estradiol-17ßaccompany the gonadal cycle, while spawning behavior is stimulatedby neurohypophyseal hormones. Both temperature and photoperiodhave been implicated in the control of seasonal reproductionin F. heteroclitus. Early workers focused on males and concludedthat increasing temperature was the stimulus that initiatedgonadal recrudescence in spring. These experiments were notrigorous tests of the effects of photoperiod. We have shownthat in females both warm temperatures and long photoperiodsare involved. In female F. heteroclitus, as in most mammalsand birds, it is the timing rather than the duration of thelight exposure which determines its effectiveness in stimulatinggonadal maturation. Maintenance of gonadal maturity in femaleF. heteroclitus depends on long photoperiods, but the ovarymay become refractory and regress even when stimulatory photoperiodsare sustained in the laboratory. Ovarian maturity can be stimulatedwith gonadotropin injection in refractory animals, indicatingthat the brain-pituitary axis is the site of refractoriness.The pathway by which light influences ovarian maturity may involvean encephalic photoreceptor, since neither the eyes nor thepineal gland is necessary.     

Appearance of cuboidal cells in relation to salinity in gills of Fundulus heteroclitus, a species exhibiting branchial Na+ but not Cl− uptake in freshwater

Fundulus heteroclitus (killifish) is a model organism for ionoregulatory studies, particularly because of its opercular epithelium, although the gills are the major sites of ion exchange. Whereas Na⁺ and Cl⁻ are excreted through the gills in seawater (SW), the killifish is unusual in taking up only Na⁺ and not Cl⁻ at the gills in freshwater (FW). We describe morphological changes in the branchial epithelium following transfer from an acclimation medium of 10% SW to 100% SW or FW. In 10% SW, mitochondria-rich cells resemble typical seawater chloride cells (SWCCs) with accessory cells. After transfer to 100% SW, no change occurs in pavement cell (PVC) morphology or mitotic rate (measured by bromo-deoxyuridine technique), although the density of SWCC apertures increases several fold because of the uncovering of buried SWCCs by PVCs, in accord with increased rates of Na⁺ and Cl⁻ efflux. After transfer to FW, PVC morphology remains unchanged, but SWCCs and accessory cells are quickly covered by PVCs, with many undergoing apoptosis or necrosis. The mitotic rate doubles by 10–14 h but typical freshwater chloride cells (FWCCs) do not appear. Instead, a wedge-shaped cell type that is moderately rich in apically oriented mitochondria, with a large ovoid nucleus, thin cytoplasmic layer, paucity of vesicular-tubular network, and variably villous surface rapidly (by 3 h) and progressively appears in the filament epithelium, by both uncovering and mitosis. This cell type is similar to that recently identified as the site of Na⁺ uptake in the FW trout gill. We propose the new term “cuboidal cell” for this cell, based on its morphology, to avoid confusion with traditional terminology (of PVC). We hypothesize that the cuboidal cells are the sites of active Na⁺ uptake in FW F. heteroclitus and suggest that the lack of Cl⁻ uptake is attributable to the absence of typical FWCCs previously described in teleosts.This work was supported by NSERC Discovery grants (to C.M.W.) and by an NSERC International Fellowship (to P.L.). C.M.W. is supported by the Canada Research Chair Program.     

Geographical Variation in Fundulus heteroclitus: Tests for Concordance Between Egg and Adult Morphologies

Recent studies have shown distinct patterns of variation inthe egg morphology (oil droplet number, diameter and densityof chorionic filaments) of Fundulus heteroclitus along the eastcoast (including Chesapeake and Delaware bays). We tested thehypothesis that variation in adult morphology and in electromorphsis concordant with that of egg morphology over the same rangeby subjecting data for these character sets to the Mantel test.This analysis was based on data for three egg characters, nineteenadult morphological (meristic and morphometric) characters andfive protein characters from locations along the Atlantic coast.If the geographical differences observed among locations reflectgenetic differences between two forms that have establishedsecondary contact after isolation, we would expect general concordancebetween the different character sets. We would expect littleconcordance between the sets if they are each affected by differentselectional and/or developmental responses to environmentalregimes. Our analyses indicate that patterns of variation amonglocalities for meristic, egg, and genetic character sets areall concordant, and thus the observed differences among locationsare likely the result of secondary intergradation between twopreviously isolated forms. These conclusions are supported bysimilar patterns of geographical variation in spawning sitepreference and body coloration. Thus, these results supportour earlier suggestion that these morphs should be treated asseparate taxa, with F. h. heteroclitus occurring along the eastcoast from New Jersey south to Florida including lower Chesapeakeand Delaware bays and F. h. macrolepidotus distributed fromConnecticut north to Newfoundland with disjunct populationsin upper Chesapeake and Delaware bays. Intergrade zones occurwhere the two morphs come together along the east coast in northernNew Jersey and on Long Island, as well as in Chesapeake andDelaware bays     

A Suite of Adaptations for Intertidal Spawning

Salt marshes and similar tide-dominated habitats present anunusual challenge for reproduction of resident aquatic organisms.Strong currents, siltation and hypoxia can potentially contributeto reproductive failure through mortality of the eggs or flushingof the eggs and larvae from the habitat. Fundulus heteroclitus,a small brackish water killifish, is a common resident of tidalmarshes along the east coast of North America from Newfoundlandto Florida. The reproductive strategy of this and related speciesis based on aerial incubation of eggs in the high intertidalzone. The eggs are resistant to desiccation and, when fullydeveloped, hatch on immersion. Copulatory behavior and anatomyof accessory reproductive structures are adapted to placementof eggs in protected incubation sites. The gonads of both malesand females mature rhythmically with an endogenous circasemilunarperiod, which is synchronized with the "spring" tides of newand full moons. Spawning occurs on high tides. Embryos developin 9–15 days, and usually hatch on the succeeding springtide series. Reproductive cyclicity in F. heteroclitus and relatedfishes ensures that spawning fish will have access to the highintertidal zone, thereby permitting aerial incubation of eggs.     

Aspects of the life history of a freshwater population of the mummichog,Fundulus heteroclitus (Pisces: Cyprinodontidae), in the Bronx River,New York,U.S.A.

A freshwater population of the mummichog, Fundulus heteroclitus, was sampled during summer and autumn 1979, from the Bronx River, New York. Other occurrences in freshwater of this common estuarine cyprinodontid fish have been noted in Pennsylvania, New Hampshire and Canada. In the Bronx River, this species is a significant part of the fauna and is one of the few species found throughout the river system. Specimens were examined to determine the growth and diet. It appears that this freshwater population does not differ from its brackish water conspecifics with respect to age structure, growth rate and feeding behavior.     

The effects of salinity on swimming performance of two estuarine fishes,Fundulus heteroclitus and Fundulus majalis

Prolonged and high‐speed swimming performance measurements were used to explore the swimming abilities of two species of estuarine fishes, the mummichog Fundulus heteroclitus and the striped killifish Fundulus majalis, under different salinities. Critical swimming performance was significantly higher for F. majalis in high salinity than in low salinity, but no difference was observed in brief constant acceleration swimming trials in this species; however, the swimming performance of F. heteroclitus was not significantly affected by salinity changes, indicating that this species is well adapted to regular estuarine salinity oscillations. Fundulus majalis displayed higher swimming speeds than F. heteroclitus in both high and low salinities, and while this cannot be explained by their respective salinity preferences, the specific habitat preferences of F. majalis for sandy subtidal habitats and F. heteroclitus for vegetated marshes could explain the better swimming performance of F. majalis.     

Shoaling preferences of two common killifish (Fundulus heteroclitus and F. diaphanus) in the laboratory and in the field: A new analysis of heterospecific shoaling

Heterospecific grouping behavior of mummichogs (Fundulus heteroclitus) and banded killifish (Fundulus diaphanus) was analyzed in the laboratory and in a freshwater tidal marsh in Cremona, Maryland. Several parameters of wild, intact shoals were measured, including species composition, body length, parasite load, gender, and any physical abnormalities. Fish collected were used for laboratory analysis of shoaling preferences. When size was equal, banded killifish and mummichogs preferred conspecific shoals to heterospecific shoals, consisting of mummichogs, banded killifish, and sheepshead minnows (Cyprinodon variegates). Shoal collection from the field resulted in mixed species shoals with individuals predominantly unaffected by parasites or other physical abnormalities. Size appeared to be a sorting mechanism. A temporal shift in lengths was evident. Initial shoals caught contained significantly smaller fish compared to the final shoals caught. Results are compared with previous studies on heterospecific shoaling in killifish and new characteristics of heterospecific shoals inhabiting freshwater tidal marshes are discussed.     

A divergent CFTR homologue: highly regulated salt transport in the euryhaline teleost F.heteroclitus

The killifish,Fundulus heteroclitus, is a euryhalineteleost fish capable of adapting rapidly to transfer from freshwater (FW) to four times seawater (SW). To investigate osmoregulation at amolecular level, a 5.7-kilobase cDNA homologous to human cysticfibrosis transmembrane conductance regulator (hCFTR) was isolated froma gill cDNA library from SW-adapted killifish. This cDNA encodes aprotein product (kfCFTR) that is 59% identical to hCFTR,the most divergent form of CFTR characterized to date. Expression ofkfCFTR in Xenopus oocytes generatedadenosine 3',5'-cyclic monophosphate-activated,Cl-selective currentssimilar to those generated by hCFTR. In SW-adapted killifish,kfCFTR was expressed at high levels in the gill, opercular epithelium, and intestine. After abrupt exposure of FW-adapted killifish to SW, kfCFTR expression in the gill increasedseveralfold, suggesting a role for kfCFTR in salinity adaptation. Undersimilar conditions, plasma Na⁺levels rose significantly after 8 h and then fell, although it is notknown whether these changes are directly responsible for the changes inkfCFTR expression. The killifish provides a unique opportunity to understand teleost osmoregulation and the role of CFTR.

     

Developmental uncoupling between blastoderm and yolk cell in the embryo of the teleost Fundulus

During cleavage and blastula stages of embryos of the teleost Fundulus heteroclitus all of the cells are both electotonically coupled and dye coupled to one another, as determined by microelectrode impalements and spread of Lucifer Yellow. At about the time that gastrulation begins we observed a specific loss of junctional coupling between the yolk cell and cells of the blastoderm. Passage of Lucifer Yellow between the yolk cell and blastoderm was reduced at stage 12 (late blastula), and not detected at stage 13 and thereafter, although cells of the blastoderm remain dye coupled to one another through gastrula stages. Also, junctional electrical coupling between the yolk cell and blastoderm became substantially reduced at stage 13 and thereafter. The loss of coupling at this specific cell apposition and time and the large size of the yolk cell may prove useful in analyzing the underlying cellular mechanisms.     

Halide localization in the teleost chloride cell and its identification by selected area electron diffraction

Further evidence defining an active role for the chloride cell in teleost osmoregulation is presented in this study. Gill filaments from salt water adaptedFundulus heteroclitus were fixed in a solution of silver acetate-osmium tetroxide in an attempt to localize chloride at the level of light and electron microscopes. Characteristically, a reaction product in the form of dense granules appeared concentrated and localized near the margin of the chloride cell apical cavity. Selected area electron diffraction patterns obtained from the localized accumulations of reaction product match diffraction patterns from control (known) silver chloride preparations. It should be emphasized that since the reaction product is not concentrated in other regions of the branchial epithelium, these observations strongly support an electrolyte regulating function for the chloride cell.Supported by a Training Grant (2 G-707) to K. R. Porter from the United States Public Health Service.     

Effects of spironolactone and RU486 on gene expression and cell proliferation after freshwater transfer in the euryhaline killifish

We have explored the possible mechanisms by which mineralocorticoid (MR) and glucocorticoid (GR) receptors regulate the response to freshwater transfer in the gills of the euryhaline killifish Fundulus heteroclitus. Killifish were implanted with RU486 (GR antagonist) or spironolactone (MR antagonist) at doses of 0.1–1.0 mg g⁻¹, and subsequently transferred from 10‰ brackish water to freshwater. Compared to brackish water sham fish, mRNA expression of CFTR and NKCC1 decreased in the gills of sham fish transferred to freshwater, whereas Na⁺,K⁺–ATPase α_1a mRNA expression and α protein abundance, as well as cell proliferation (detected using BrdU) increased. Spironolactone inhibited the normal increase in cell proliferation and Na⁺,K⁺-ATPase expression after freshwater transfer. RU486 increased plasma cortisol levels and may have slightly inhibited Na⁺,K⁺–ATPase activity, but did not change α _1a expression. RU486 had no effect on cell proliferation in the non-lamellar region of the gills, but increased proliferation in the lamellar region. Neither antagonist inhibited the suppression of CFTR or NKCC1 expression after freshwater transfer. Glucocorticoid receptor expression was reduced in all sham and antagonist treatments compared to untreated controls, but no other consistent differences were observed. The effects of spironolactone suggest that MR is important for regulating ion transport in killifish gills after freshwater transfer.     

Responses to environmental stressors in an estuarine fish: Interacting stressors and the impacts of local adaptation

Common killifish, Fundulus heteroclitus, are found in marshes and estuaries along the Atlantic coast of North America from Newfoundland to Florida. Although these habitats are highly productive, they are also characterized by variation in a number of abiotic stressors, including temperature, salinity, oxygen, and anthropogenic toxicants, which vary substantially in both space and time. In order to survive in these habitats, killifish must be able to cope with these stressors, both individually and in combination. There is substantial evidence to suggest that populations of F. heteroclitus have undergone local adaptation to multiple abiotic stressors, including temperature, salinity, and toxicants, but most studies have examined the effects of single stressors in isolation. Here I review some of the studies on local adaptation in F. heteroclitus, focusing on the molecular basis of local adaptation to abiotic stressors, and the acute responses to these stressors both singly and in combination. This work demonstrates that there are substantial interactions between the responses to both natural and anthropogenic stressors at the cellular level.     

Occupation of submerged aquatic vegetation by fishes: testing the roles of food and refuge

Summary We conducted a series of field experiments to examine the roles of refuge and food availability in explaining the distribution and abundance of fish in tidal freshwater marsh creeks. Two hypotheses were tested: (1) relative predation pressure is less in SAV than in unvegetated areas and (2) fish food availability is greater in SAV than in nearby unvegetated areas. Tethering experiments using mummichogs (Fundulus heteroclitus) in vegetated and unvegetated areas revealed that relative predation pressure was significantly less in areas with SAV. Banded killifish (Fundulus diaphanus) maintained in vegetated enclosures consumed prey associated with SAV, whereas those held in unvegetated pens had empty stomachs. No differences were found in the number of prey eaten by bluespotted sunfish (Enneacanthus gloriosus) or mummichogs when confined in vegetated or unvegetated enclosures. However, larger prey were consumed by bluespotted sunfish and mummichogs maintained in vegetated enclosures. These data suggest that foraging profitability is significantly enhanced by feeding in the SAV. Submerged plant beds in tidal freshwater marsh creeks not only afford protection from predators, but also provide a rich foraging habitat. By foraging in SAV, fish consume larger prey and may have higher growth rates, lower mortality, and higher fecundity.