Structure and Function of the Chloride Cell of Fundulus heteroclitus and Other Teleosts

Teleosts, the bony fishes, inhabit both freshwater and seawaterenvironments. Some euryhaline fish, such as Fundulus heteroclitus,alternate between the two milieux several times daily. Regardlessof adaptation, the gills of these animals possess a highly specializedcell type called the chloride cell. This cell contains numerousmitochondria and exhibits a greatly amplified basolateral cellsurface richly endowed with Na,K-ATPase. Recent studies on isolatedopercular epithelia containing chloride cells have demonstratedactive chloride secretion and passive transepithelial sodiummovements, and have established the chloride secretory roleof this cell type in seawater-adapted teleosts. Current modelssuggest that chloride transport occurs via a transcellular route.Seawater chloride cells exist in multicellular units and sharesimple, shallow tight junctions which are thought to be theroute for passive sodium movement. Freshwater chloride cells,whose function remains to be elucidated, are generally describedas existing in a unicellular configuration. However, recentobservations in Fundulus heteroclitus adapted to salinitiesas low as 1% sea water reveal that chloride cells persist inmulticellular complexes with apical crypts. Strikingly, tightjunctions between chloride cells in this freshwater environmentare deep     

Genetic Variation in Fundulus heteroclitus: Geographic Distribution

Studies of 16 polymorphic loci in the fish Fundulus heteroclitushave uncovered significant directional changes in gene frequencieswith latitude (i.e., clines). These spatial patterns could havearisen by primary and/or secondary intergradation. While wecannot presently distinguish between these two models, mitochondrialDNA analyses indicate that if previous isolation occurred asrequired for secondary intergradation, it must have been relativelyrecent and of short duration. Herein we discuss the roles ofgenetic drift, random migration, nonrandom migration, selectionand others as potential driving forces for both modes of clineformation. In addition, we address the potential role of thelast glacial period in (1) creating isolating barriers, (2)the opening of unoccupied habitats for range extension, and(3) as a mechanism for thermal selection. While some evidenceexists that at least one of the driving forces is selection,the extent to which it and other deterministic forces participateas opposed to various stochastic processes must await furtheranalysis     

Chloride transport activation by plasma osmolarity during rapid adaptation to high salinity of Fundulus heteroclitus

Transition from low salt water to sea water of the euryhaline fish, Fundulus heteroclitus, involves a rapid signal that induces salt secretion by the gill chloride cells. An increase of 65 mOsm in plasma osmolarity was found during the transition. The isolated, chloridecell-rich opercular epithelium of sea-water-adapted Fundulus exposed to 50 mOsm mannitol on the basolateral side showed a 100% increase in chloride secretion, which was inhibited by bumetanide 10^–4 m and 10^–4 m DPC (N-Phenylanthranilic acid). No effect of these drugs was found on apical side exposure. A Na⁺/H⁺ exchanger, demonstrated by NH₄Cl exposure, was inhibited by amiloride and its analogues and stimulated by IBMX, phorbol esters, and epithelial growth factor (EGF). Inhibition of the Na⁺/H⁺ exchanger blocks the chloride secretion increase due to basolateral hypertonicity. A Cl^–/HCO ₃ ^– exchanger was also found in the chloride cells, inhibited by 10^–4 m DIDS but not involved in the hyperosmotic response. Ca²⁺ concentration in the medium was critical for the stimulation of Cl^– secretion to occur. Chloride cell volume shrinks in response to hypertonicity of the basolateral side in sea-water-adapted operculi; no effect was found on the apical side. Freshwater-adapted fish chloride cells show increased water permeability of the apical side. It is concluded that the rapid signal for adaptation to higher salinities is an increased tonicity of the plasma that induces chloride cell shrinkage, increased chloride secretion with activation of the Na⁺K⁺2Cl^– cotransporter, the Na⁺/H⁺ exchanger and opening of Cl^– channels.The work was supported by the National Institutes of Health, Research Grant EYO1340 to J.A.Z. Part of this research was performed while Dr. Zadunaisky was a Scholar In Residence at the Fogarty International Center of The National Institutes of Health in Bethesda, Maryland. Ms. Dawn Roberts was a fellow of the Grass Foundation and Pew Foundation during this work. Grants from the National Science Foundation and the National Institutes of Health to the Mount Desert Island Biological Laboratory also provided assistance for this research.     

Early estrogen exposure induces abnormal development of Fundulus heteroclitus

Many chemicals released into the environment exhibit estrogenic activity, having the potential to disrupt development and the functioning of the endocrine system. In order to establish a model system to study the effects of such environmental chemicals on aquatic animals, we examined the effects of a natural estrogen, 17 beta-estradiol (E(2)), on early development of Fundulus heteroclitus. Embryos of F. heteroclitus were reared in seawater containing 10(-10), 10(-8), and 10(-6) M E(2) throughout the experiment. Hatching and survival rates decreased in a dose-dependent manner, and fry treated with 10(-6) M E(2) and 10(-8) M E(2) were dead by two weeks and 12 weeks after hatching, respectively. More than 85% of fry treated with 10(-8) M E(2) showed malformations: i.e., eye extrusion, crooked vertebral column, faded lateral-stripe pattern eight weeks after hatching. Body weight and head and body lengths were significantly reduced in E(2)-treated fry when compared to controls. Ossification was not completed in vertebrae, cranial bones, and other bones in fry treated with 10(-8) M E(2) even 12 weeks after hatching. Sex ratio of control fry was 57% male and 43% female, whereas fry treated with 10(-8) M E(2) were 100% female eight weeks after hatching. The present results demonstrate that exogenous estrogen induced death of embryos and fry, malformations, sex reversal, and incomplete ossification of vertebrae and cranial bones, which would result in shorter body and head lengths and in malformed vertebrae leading to a hunchback condition.     

Regulation of pyruvate dehydrogenase in the common killifish, Fundulus heteroclitus, during hypoxia exposure

We examined the metabolic responses of the hypoxia-tolerant killifish (Fundulus heteroclitus) to 15 h of severe hypoxia and recovery with emphasis on muscle substrate usage and the regulation of the mitochondrial protein pyruvate dehydrogenase (PDH), which controls carbohydrate oxidation. Hypoxia survival involved a transient activation of substrate-level phosphorylation in muscle (decreases in [creatine phospate] and increases in [lactate]) during which time mechanisms to reduce overall ATP consumption were initiated. This metabolic transition did not affect total cellular [ATP], but had an impact on cellular energy status as indicated by large decreases in [ATP]/[ADP(free)] and [ATP]/[AMP(free)] and a significant loss of phosphorylation potential and Gibbs free energy of ATP hydrolysis (DeltafG'). The activity of PDH was rapidly (within 3 h) decreased by approximately 50% upon hypoxia exposure and remained depressed relative to normoxic samples throughout. Inactivation of PDH was primarily mediated via posttranslational modification following the accumulation of acetyl-CoA and subsequent activation of pyruvate dehydrogenase kinase (PDK). Estimated changes in cytoplasmic and mitochondrial [NAD(+)]/[NADH] did not parallel one another, suggesting the mitochondrial NADH shuttles do not function during hypoxia exposure. Large increases in the expression of PDK (PDK isoform 2) were consistent with decreased PDH activity; however, these changes in mRNA were not associated with changes in total PDK-2 protein content assessed using mammalian antibodies. No other changes in the expression of other known hypoxia-responsive genes (e.g., lactate dehydrogenase-A or -B) were observed in either muscle or liver.     

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     

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.     

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.     

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.     

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.     

Crcadian and Seasonal Variations of Lipogenesis in the Gulf Killifish, Fundulus Grandis

Lipogenesis was measured in male Gulf killifish at three seasons under different environmental conditions. Cold temperature is stimulatory for lipogenesis in the fall and spring, but warm temperature is more stimulatory during the summer. The highest rate of lipogenesis occurs during the spring. However, total body fat content is relatively low at that time, which suggests that lipids are being actively mobilized as well. A high rate of lipogenesis also occurs at a cold temperature (20°C) during the fall, and more lipids are stored at that time of year, as evidenced by higher liver and body fat stores. Both lipogenesis and total body fat content are at a minimum during the summer. These results indicate that high levels of lipid synthesis do not necessarily result in higher body fat content, as the latter is the product of both lipid synthesis and lipid mobilization. This study is also in agreement with previous studies which had indicated a changing responsiveness to temperature during the annual cycle of the Gulf killfish.     

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.     

Crcadian and Seasonal Variations of Lipogenesis in the Gulf Killifish,Fundulus Grandis

Lipogenesis was measured in male Gulf killifish at three seasons under different environmental conditions. Cold temperature is stimulatory for lipogenesis in the fall and spring, but warm temperature is more stimulatory during the summer. The highest rate of lipogenesis occurs during the spring. However, total body fat content is relatively low at that time, which suggests that lipids are being actively mobilized as well. A high rate of lipogenesis also occurs at a cold temperature (20°C) during the fall, and more lipids are stored at that time of year, as evidenced by higher liver and body fat stores. Both lipogenesis and total body fat content are at a minimum during the summer. These results indicate that high levels of lipid synthesis do not necessarily result in higher body fat content, as the latter is the product of both lipid synthesis and lipid mobilization. This study is also in agreement with previous studies which had indicated a changing responsiveness to temperature during the annual cycle of the Gulf killfish.     

Seasonal Differences in Hypoxia Tolerance in Gulf Killifish, Fundulus Grandis (Fundulidae)

Many estuarine habitats are characterized by episodes of hypoxia, the frequency and severity of which may vary seasonally. Accordingly, resident fish species may show seasonal differences in their capacity to tolerate hypoxia. We have tested this hypothesis in the gulf killifish, Fundulus grandis, sampled from the Lake Pontchartrain estuary (Louisiana) at different times of the year. We measured 2 indicators of hypoxia tolerance, the frequency of aquatic surface respiration (ASR) during gradual reduction in dissolved oxygen (D.O.) and survival time during severe hypoxic stress, and found both to be significantly affected by season. Fish collected during the summer did not engage in ASR until the D.O. concentration dropped to values lower than that required to elicit ASR by fish collected during other seasons. Laboratory acclimation of fish to low oxygen did not change the relationship between ASR behavior and D.O., suggesting that the observed seasonal effect on ASR was not simply due to previous exposure of summer fish to environmental hypoxia. Furthermore, fish collected during the summer and winter had significantly longer survival times during exposure to severe hypoxia than fish collected during the fall. Survival analysis indicated that the condition of fish was positively associated with survival time, and seasonal variation in condition accounted for about half of the observed difference between survival times of fish collected during the summer and fall. Seasonal variation in ASR and survival, when taken together, demonstrate that hypoxia tolerance in F. grandis may be subject to acclimatization. An increase in hypoxia tolerance during the summer could increase survivorship of fish when exposed to elevated temperatures and low oxygen concentrations which prevail during the summer months.     

A fine structural study of cytodifferentiation during cleavage, blastula, and gastrula stages of Fundulus heteroclitus

The fine structure of cleavage, blastula, and gastrula stages of Fundulus heteroclitus was investigated. Cleavage blastomeres are relatively unspecialized, containing few or poorly developed organelles. Beginning in blastula stages, signs of differentiation were noted, including development of the endoplasmic reticulum and Golgi apparatus and appearance of a primary nucleolus and polyribosomes. More extensive structural specializations occur in gastrula stages, including further development of the endoplasmic reticulum and appearance of a granular component in the nucleolus. These changes are associated with cell differentiation and an increased capacity for protein synthesis, and may be preparatory to subsequent histogenesis. The periblast is a continuous syncytial cytoplasmic layer located between the blastodisc and yolk and is formed during late cleavage by incomplete division of the cytoplasm of the blastodisc. Cytoplasmic projections extend from the periblast (and from the basal region of cleavage blastomeres prior to formation of the periblast) into the yolk and function in uptake of yolk material in the absence of pinocytosis. Yolk material appears to be digested by the periblast and transferred into the segmentation cavity where it is available to the blastomeres. Protein granules, lipid droplets, glycogen, crystalline arrays, and multivesicular bodies are related to food storage and utilization by blastomeres. The yolk gel layer enclosing the yolk sphere was found to be a thin layer of cytoplasm continuous with the margin of the periblast and is renamed the yolk cytoplasmic layer.     

The vitelline envelope,chorion, and micropyle of Fundulus heteroclitus eggs

The architecture and transformation of the vitelline envelope of the developing oocyte into the chorion of the mature egg of Fundulus heteroclitus have been examined by scanning and transmission electron microscopy. The mature vitelline envelope is structurally complex and consists of about nine strata. The envelope is penetrated by pore canals that contain microvilli arising from the oocyte and macrovilli from follicle cells. During the envelope's transformation into the chorion, the pore canals are lost and the envelope becomes more fibrous and compact and its stratified nature less apparent. The micropyle, of pore, through which the sperm gains access to the enclosed egg is located at the bottom of a small funnel-shaped depression in the envelope. Internally, the micropyle opens on the apex of a cone-like elevation of the chorion. During the development of the envelope, structured chorionic fibrils, the components of which are presumed to be synthesized by the follicle cells, become attached to its surface. These chorionic fibrils are though to aid in the attachment of the egg to the substratum and perhaps to help prevent water loss during low tides when the egg may be exposed.     

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.