Energetics of osmoregulation: II. Water flux and osmoregulatory work in the euryhaline fish, Fundulus heteroclitus

Teleost fish experience passive osmotic water influx in fresh water (FW) and water outflux in salt water, which is normally compensated by water flow driven by active ion transport mechanisms. Euryhaline fish may also minimize osmotic energy demand by "behavioral osmoregulation", seeking a medium isotonic with their body fluids. Our goal was to evaluate the energy requirement for osmoregulation by the euryhaline fish Fundulus heteroclitus, to determine whether it is of sufficient magnitude to favor behavioral osmoregulation. We have developed a method of weighing small fish repetitively for long periods without apparent damage, which was used to assess changes in water content following changes in external salinity. We found that cold (4 degrees C) inhibits osmoregulatory active transport mechanisms in fish acclimated to warmer temperatures, leading to a net passive water flux which is reversed by rewarming the fish. A sudden change of salinity at room temperature triggers a transient change in water content and the initial slope can be used to measure the minimum passive flux at that temperature. With some reasonable assumptions as to the stoichiometry of the ion transport and ATP-generating processes, we can calculate the amount of respiration required for ion transport and compare it to the oxygen uptake measured previously under the same conditions. We conclude that osmoregulation in sea water requires from 6% to 10% of the total energy budget in sea water, with smaller percentages in FW, and that this fraction is probably sufficient to be a significant selective driving force favoring behavioral osmoregulation under some circumstances.     

Energetics of sodium transport in frog skin. I. Oxygen consumption in the short-circuited state

Sodium transport and oxygen consumption were studied simultaneously in the short-circuited frog skin. Sodium transport was evaluated from I _o /F, where I _o is the short-circuit current measured with standard Ringer''s solution bathing each surface and F is the Faraday constant. Oxygen tension was measured polarographically. Under a variety of circumstances the rate of oxygen consumption from the outer solution exceeded that from the inner solution, the ratio being constant (0.57 ± 0.09 SD). Both I _o and the associated rate of oxygen consumption J _ro declined nonlinearly with time, but the relationship between them was linear, suggesting that the basal oxygen consumption was constant. For each skin numerous experimental points were fitted by the best straight line. The intercept (J _ro)_Io=0 then gave the basal oxygen consumption, and the slope dNa/dO₂ gave an apparent stoichiometric ratio for a given skin. The basal oxygen consumption was about one-half the total oxygen consumption in a representative untreated short-circuited skin. Values of dNa/dO₂ in 10 skins varied significantly, ranging from 7.1 to 30.9 (as compared with Zerahn''s and Leaf and Renshaw''s values of about 18). KCN abolished both I _o and J _ro. 2,4-dinitrophenol (DNP) depressed I _o while increasing J _ro four- to fivefold. Anti-diuretic hormone stimulated and ouabain depressed both I _o and J _ro; in both cases apparent stoichiometric ratios were preserved.     

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     

Fundulus heteroclitus in the Laboratory: A History

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

Actin microfilaments in melanophores of Fundulus heteroclitus

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

Fundulus heteroclitus acutely transferred from seawater to high salinity require few adjustments to intestinal transport associated with osmoregulation

The common killifish, Fundulus heteroclitus, has historically been a favorite organism for the study of euryhalinity in teleost fish. Despite the species' large range of salinity tolerance, studies of osmoregulation in high salinity are rare, with most previous studies focused on fish transferred between freshwater and seawater. Similarly, while branchial transport properties have been studied extensively, there are relatively few studies investigating the role of the intestine in osmoregulation in killifish. This study sought to characterize the fluid and ion transport occurring in the intestinal tract of killifish adapted to seawater, and furthermore to investigate the adjustments that occur to these mechanisms following acute transfer to high salinity (70ppt). In vivo samples of blood plasma and intestinal fluids of seawater-acclimated killifish indicated absorption of Na(+), Cl(-), and water, the relative impermeability of the intestine to Mg(2+) and SO(4)(2-), and active secretion of HCO(3)(-) into the intestinal lumen. The details of these processes were investigated further using in vitro techniques of isolated intestinal sac preparations and an Ussing chamber pH-stat titration system. However, these methods were discovered to be of limited utility under physiologically relevant conditions due to tissue deterioration. Results that could be validly interpreted suggested that there are few changes to intestinal transport following transfer to high salinity, and that adjustments to epithelial permeability occur in the first 24h post-transfer.     

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.     

Oogenesis in Fundulus heteroclitus: I. Preliminary observations on oocyte maturation in vivo and in vitro

During the breeding season, the ovary of Fundulus heteroclitus contains follicles of all sizes up to around 1.8 mm in diameter and continuously ovulates eggs (1.7 mm) into the ovarian lumen. Vitellogenic follicles appear to extend up to 1.3 mm, while those larger than 1.4 mm are undergoing hydration concomitant with maturation. If feeding ceases, vitellogenesis appears to stop and maturational stages flush out of the ovary. Refeeding causes reappearance of maturational stages. A culture procedure was developed for dissected F. heteroclitus follicles and a size range (1.3–1.4 mm) was defined which responded to added hormones by undergoing the same maturational changes (germinal vesicle breakdown, fusion, and peripheral detachment of oil droplets, appearance of activability) as occurs in vivo, although the extent of hydration was not quite as extensive. Ovulation, however, did not consistently occur in vitro. Both 11-deoxycorticosterone and progesterone are potent inducers of maturation in vitro, with median effective doses being 10–20 ng/ml. Hydrocortisone is about one-tenth as active. Human chorionic gonadotropin promotes maturational changes in about 25% of the follicles over a broad concentration range (1–100 IU/ml). The environmental and hormonal mechanisms which influence oocyte maturation in teleosts are discussed in relation to our findings for F. heteroclitus.     

Programmed Endocytosis During Epiboly of Fundulus heteroclitus

A major question in the analysis of teleost epiboly is the fateof the yolk cytoplasmic layer. It diminishes during epibolyand eventually disappears at the completion of epiboly. Thispaper is concerned with the fate of the surface of the yolkcytoplasmic layer during epiboly. When gastrulae during epibolyare bathed in lucifer yellow (CH) and then observed with fluorescentmicroscopy or bathed in ferritin and then fixed and observedwith TEM, a thin circumferential ring of endocytic vesiclesis observed, confined to the external yolk syncytial layer justperipheral to the advancing margin of the blastoderm. Even thoughthe entire egg is immersed in the marker, endocytosis is confinedto this limited region. More precisely, this endocytosis occursonly within the region of the external yolk syncytial layer,where the surface is most folded. The endocytic vesicles thusformed move downward and settle on the surface of the membraneseparating the yolk from the cytoplasm in the yolk syncytiallayer. They do not join the surface of the internal yolk syncytiallayer; hence they do not contribute to its expansion. Priorto the onset of epiboly there is no such endocytosis at thesurface of the egg. Since this endocytosis occurs only duringepiboly and only at the surface of the external yolk syncytiallayer just peripheral to the advancing margin of the blastoderm,and in the absence of large molecules in the medium, we concludethat it is programmed. We, therefore, present this as a caseof programmed internalization of cell surface serving as themorphogenetic mechanism responsible for the disappearance ofthe surface of the yolk cytoplasmic layer during gastrulationof the teleost Fundulus heteroclitus     

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.     

The Molecular Ecology of Fundulus heteroclitus Hemoglobin-Oxygen Affinity

Animals faced with environmental perturbations must adapt orface extinction. The respiratory complex, specifically hemoglobins,is perhaps the best system to study such adaptation becauseit exists at the organism—environment interface. Fishare particularly useful models because they respond directlyto such environmental variables as temperature, oxygen, pH,carbon dioxide, and salinity. Our experiments have addressedthe molecular, cellular, and physiological mechanisms employedby fish to maintain respiratory homeostasis in the wake of changingtemperature and oxygen. Immediate, intermediate, and long-termadaptation can only be understood when the hemoglobin's ligandbinding properties and the cellular and hormonal regulationof various ligands are considered simultaneously. We describea detailed thermodynamic model for the binding of oxygen, protons,and organic phosphates to hemoglobin; discuss the role of multiplehemoglobins; and present evidence for physiological and geneticregulation of hemoglobin's major allosteric modifiers in responseto environmental stress in the mummichog, Fundulus heteroclitus     

Blood cells of Fundulus heteroclitus (L.)

Much of the parenchyma of the normal pronephros of the adult Fundulus heterodilus , a euryhaline teleost, is haematopoietic tissue which was examined in cytocentrifuge preparations and plastic embedded thick sections. As we have not characterized many of the blood cells functionally, the terminology used is based on their morphological resemblance to similarly named cells in higher vertebrates. Approximately 80% of the non-erythroid elements observed in the pronephros are mature eosinophilic granulocytes (48%), immature eosinophilic granulocytes (25%), or cells likely to be their precursors [e.g. small blast cells (6%) and large blast cells (2%)]. Although there are macrophages in the pronephros that are capable of endocytotic activity, the mature and immature granulocytes are not. The granulocytes are non-specific esterase positive, PAS positive, acid phosphatase negative, and are capable of being mobilized by a RES activating agent, Ecteinascidia turbinata .     

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.     

Biodegradability of swine effluents I. Oxygen consumption

Positive and negative effects of dilution of raw effluents from swine breeding are discussed, as to the treatability of those waste waters. Its effects on oxygen consumption and on the variation of inhibitory actions possibly observed, are studied and quantified. Using BRADENDER and VANDEPUTTE techniques based on peptone degradation, and determining PHELPS'S equation parameters for different consumption curves observed, the advantages of dilution versus treatment of raw effluents is studied.     

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.     

Arsenic inhibits CFTR-mediated chloride secretion by killifish (Fundulus heteroclitus) opercular membrane.

Killifish are euryhaline teleosts that normally experience rapid changes in the salinity of the swim water. Acclimation to seawater is mediated by cortisol, which by activating glucocorticoid receptors, upregulates CFTR mediated Cl- secretion in the gill and operculum. Arsenic, a toxic metalloid that naturally occurs in the aquatic environment, has been shown to disrupt glucocorticoid hormone-mediated regulation of genes. Because little is known about the effects of environmentally relevant levels of arsenic on ion channels and salt homeostasis, studies were conducted to examine the effects of arsenic on the ability of killifish to acclimate to increased salinity. Arsenic in the swim water or administered by intraperitoneal injection prevented acclimation. To determine if arsenic blocked acclimation by inhibiting CFTR mediated Cl- secretion (Isc), opercular membranes were isolated and mounted in Ussing chambers and the effects of arsenic on Isc were measured. Arsenic (24 hr exposure) reduced Isc in opercular membranes isolated from salt water acclimated killifish. In addition, arsenic acutely (5-10 minutes) and reversibly inhibited Isc with an IC50 = 4.1 microM (305 ppb) when applied to the apical (seawater) side of the operculum, but not when added to the basolateral side of the operculum. Arsenic (4 microM for 60 minutes) also reduced mitochondrial respiration. Thus, environmentally relevant levels of arsenic block acclimation to seawater in killifish by reversibly inhibiting CFTR-mediated Cl- secretion by the opercular membrane, in part by inhibiting mitochondrial respiration.     

Leucophores and iridophores of Fundulus heteroclitus: Biophysical and ultrastructural properties

Classical light microscopic studies on pigmentation of Fundulus heteroclitus (killifish) indicated that there are three groups of light reflecting cells; one group on the surface of scales reflects white light, while two other deeper groups (the melaniridophores and the stratum argenteum) are iridescent. The results presented here show that: (1) The scale leucophores reflect white light by a Tyndall light-scattering mechanism, by virtue of the presence of randomly oriented organelles of “novel” morphology. (2) The iridophores of the melaniridophores contain stacks of irregularly-spaced, large reflecting platelets which function as an imperfect multiple thin layer interference system. (3) The stratum argenteum consists of a continuous layer(s) of iridophores with reflecting platelets which are so regularly packed as to approach an ideal multiple thin layer interference system. (4) In all three types of light reflecting cells, the dimensions and packing (orientation) of the reflecting organelles satisfactorily account for the chromogenic properties of the cells, including colors as viewed under transmitted, reflected, or polarized light. (5) The spacial relationships between these light reflecting cells and adjoining melanophores are different for each type of light reflecting cell. Furthermore, we propose to replace the term reflecting platelet with refractosome.     

Developmental genetics of the esterase isozymes of Fundulus heteroclitus

The ontogeny of the esterase isozymes of the teleost, Fundulus heteroclitus, has been investigated. One group of esterase isozymes is present at all stages of development, whereas other esterase isozymes only very gradually appear at later stages of development, or abruptly appear at such dramatic developmental events as hatching. The ontogeny of these isozyme patterns is interpreted as the expression of differential regulation of separate esterase genes. The general pattern of teleost esterase gene activation is similar to that reported for birds and mammals. Allelic variation was detected at two of the esterase loci. On the basis of electrophoretic mobility, substrate specificity, inhibitor specificity, genetic variation, and ontogeny of esterases, there appear to be at least 15 different esterase isozymes, which constitute 6–8 groups, each of which is probably encoded in one or more genetic loci.This study was supported by NSF Grant GB 544OX to Professor C. L. Markert and an NSF Graduate Fellowship to G. S. Whitt.     

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.