Combined effects of cadmium exposure and temperature on the annual killifish (Nothobranchius furzeri).

Freshwater organisms are increasingly exposed to combinations of stressors. However, because it is time-consuming and costly, research on the interaction of stressors, such as compound toxicity and global warming on vertebrates, is scarce. Studies on multigenerational effects of these combined stressors are almost nonexistent. In the present study, we tested the combined effects of 4 °C warming and cadmium (Cd) exposure on life-history traits, biomarkers, bioaccumulation, and multigenerational tolerance in the turquoise killifish, Nothobranchius furzeri. The extremely short life cycle of this vertebrate model allows for assessment of sublethal and multigenerational effects within 4 mo. The applied Cd concentrations had only limited effects on the measured endpoints, which suggests that N. furzeri is more resistant to Cd than fathead minnow and rainbow trout. In contrast, the temperature increase of 4 °C was stressful: it delayed female maturation and lowered adult mass and fecundity. Finally, indications of synergistic effects were found on peak fecundity and embryonic survival. Overall, these results indicate the importance of studying chronic and multigenerational effects of combined stressors. Environ Toxicol Chem 2018;37:2361-2371. © 2018 SETAC.

Neurotrophin-4 in the brain of adult Nothobranchius furzeri.

Neurotrophin-4 (NT-4) is a member of the well-known family of neurotrophins that regulate the development of neuronal networks by participating in neuronal survival and differentiation, the growth of neuronal processes, synaptic development and plasticity, as well as myelination. NT-4 interacts with two distinct receptors: TrkB, high affinity receptor and p75 low-affinity neurotrophin receptor (p75(NTR)). In the present survey, we identified the gene encoding NT-4 in the teleost Nothobranchius furzeri, a model species for aging research. The identified gene shows a similarity of about 72% with medaka, the closest related species. The neuroanatomical localization of NT-4 mRNA is obtained by using an LNA probe. NT-4 mRNA expression is observed in neurons and glial cells of the forebrain and hindbrain, with very low signal found in the midbrain. This survey confirms that NT-4 is expressed in the brain of N. furzeri during adulthood, suggesting that it could also be implicated in the maintenance and regulation of neuronal functions.

Warm acclimation improves hypoxia tolerance in Fundulus heteroclitus.

Human activities are increasing both the frequency of hypoxic episodes and the mean temperature of aquatic ecosystems, but few studies have considered the possibility that acclimation to one of these stressors could improve the ability to cope with the other stressor. Here, we used Atlantic killifish, Fundulus heteroclitus, to test this hypothesis. Hypoxia tolerance was measured as time to loss of equilibrium in hypoxia (LOEhyp) at 0.4 kPa oxygen. Time to LOEhyp declined from 73.3 ± 6.9 min at 15 °C to 2.6 ± 3.8 min at 23 °C, and at 30 °C no fish could withstand this level of hypoxia. Prior acclimation to warm temperatures significantly increased time to LOEhyp. Hypoxia tolerance of the southern subspecies of killifish, F. heteroclitus heteroclitus, was greater than that of the northern subspecies, F. heteroclitus macrolepidotus, measured both as critical oxygen tension (Pcrit) and as time to LOEhyp. Warm acclimation offset the negative effects of temperature on time to LOEhyp to a similar extent in the two subspecies. Warm acclimation increased total lamellar surface area of the gill in both subspecies as a result of regression of an interlamellar cell mass (ILCM). However, differences in total lamellar surface area could not explain differences in time to LOEhyp between the subspecies, suggesting that the lower time to LOEhyp of northern fish is related to their higher routine metabolic rate. These data suggest that thermal plasticity in gill morphology can improve the capacity of this species to tolerate hypoxia, and shows how existing plasticity may help organisms to cope with the complex interacting stressors that they will encounter with increasing frequency as our climate changes.

Rivulus berovidesi, a new killifish species (Teleostei: Rivulidae) from western Cuba.

Rivulus berovidesi, a new killifish species, is described from a small stream in Sierra de Cajalbana, northwestern Cuba. It is readily distinguished from Rivulus cylindraceus Poey by the combination of an exclusive color pattern and meristic characters such as a d-type frontal scalation pattern (versus e-type pattern in Rivulus cylindraceus). The current diagnosis of Rivulus berovidesi based on chromatic, morphological and meristic characters is consistent with a recent molecular analysis of this genus in Cuba.

Influence of sex and habitat on the size and shape of anal and dorsal fins of the blackstripe topminnow Fundulus notatus.

The size and shape of the anal and dorsal fin in the blackstripe topminnow Fundulus notatus from lake and stream habitats across multiple ages and sexes were examined. Differences in the size and shape of anal and dorsal fins were sex-specific and not related to habitat differences. Males have longer and more pointed anal fins and longer, larger and more pointed dorsal fins than females. These sex differences occur predominantly in the older age class. The angle (i.e. pointedness) of the dorsal and anal fins is tightly correlated suggesting that fins follow a similar growth trajectory as individuals become sexually mature.

Visual pigments and opsin expression in the juveniles of three species of fish (rainbow trout, zebrafish, and killifish) following prolonged exposure to thyroid hormone or retinoic acid.

Thyroid hormone (TH) and retinoic acid (RA) are powerful modulators of photoreceptor differentiation during vertebrate retinal development. In the embryos and young juveniles of salmonid fishes and rodents, TH induces switches in opsin expression within individual cones, a phenomenon that also occurs in adult rodents following prolonged (12 week) hypothyroidism. Whether changes in TH levels also modulate opsin expression in the differentiated retina of fish is unknown. Like TH, RA is essential for retinal development, but its role in inducing opsin switches, if any, has not been studied. Here we investigate the action of TH and RA on single-cone opsin expression in juvenile rainbow trout, zebrafish, and killifish and on the absorbance of visual pigments in rainbow trout and zebrafish. Prolonged TH exposure increased the wavelength of maximum absorbance (λmax ) of the rod and the medium (M, green) and long (L, red) wavelength visual pigments in all fish species examined. However, unlike the opsin switch that occurred following TH exposure in the single cones of small juvenile rainbow trout (alevin), opsin expression in large juvenile rainbow trout (smolt), zebrafish, or killifish remained unchanged. RA did not induce any opsin switches or change the visual pigment absorbance of photoreceptors. Neither ligand altered cone photoreceptor densities. We conclude that RA has no effect on opsin expression or visual pigment properties in the differentiated retina of these fishes. In contrast, TH affected both single-cone opsin expression and visual pigment absorbance in the rainbow trout alevin but only visual pigment absorbance in the smolt and in zebrafish. The latter results could be explained by a combination of opsin switches and chromophore shifts from vitamin A1 to vitamin A2.

A hydrodynamic sensory antenna used by killifish for nocturnal hunting.

The perception of sensory stimuli by an animal requires several steps, commencing with the capture of stimulus energy by an antenna that, as the interface between the physical world and the nervous system, modifies the stimulus in ways that enhance the animal's perception. The mammalian external ear, for example, collects sound and spectrally alters it to increase sensitivity and improve the detection of directionality. In view of the morphological diversity of the lateral-line system across species and its accessibility to observation and experimental intervention, we sought to investigate the role of antennal structures on the response characteristics of the lateral line. The surface-feeding killifish Aplocheilus lineatus is able to hunt in darkness by detecting surface capillary waves with the lateral-line system atop its head. This cephalic lateral line consists of a stereotyped array of 18 mechanosensitive neuromasts bordered by fleshy ridges. By recording microphonic potentials, we found that each neuromast has a unique receptive field defined by its sensitivity to stimulation of the water's surface. The ridges help determine these receptive fields by altering the flow of water over each neuromast. Modification of the hydrodynamic environment by the addition of a supplemental ridge changes the pattern of water movement, perturbs the receptive fields of adjacent neuromasts and impairs the fish's localization ability. On the basis of electrophysiological, hydrodynamic and behavioral evidence, we propose that the ridges constitute a hydrodynamic antenna for the cephalic lateral line.

Effects of diazinon on mummichog (Fundulus heteroclitus) larvae produced from eggs differentially treated with PCB126.

During their formation, fish eggs receive a load of contaminants including polychlorinated biphenyls (PCBs) from their mother and then, after spawning, are exposed to pesticides present in water. This is the first study investigating the interaction between PCBs and organophosphorous pesticides in fish. The effect of diazinon was evaluated in mummichog (Fundulus heteroclitus) larvae produced from eggs differentially treated with 3,3',4,4',5 pentachlorobiphenyl (PCB126). A few hours after fertilization, eggs were treated topically with a solution of PCB126 (100 pg/microl) in dimethyl sulfoxide (DMSO) (Group P), DMSO (Group D), or not treated (Group N). Newly hatched larvae from Groups P and D were exposed to diazinon (125-12,900 ng/L) in saltwater and Group N larvae to saltwater alone. Diazinon caused a dose-responsive inhibition of cholinesterase (ChE) activity at environmentally realistic concentrations (> or =361 ng/L), with up to 85% inhibition at 12,900 ng/L. Body length was also inversely related to diazinon at concentrations > or =361 ng/L and was significantly reduced (by 4%) at 12,900 ng/L compared to controls. Mummichog larvae were highly sensitive to PCB126 with an eightfold induction of the activity of ethoxyresorufin-O-deethylase at a dose of 710 pg PCB126 or 3.6 pg TCDD-TEQ/g wet weight. Treatment with PCB126 also caused a slight reduction in body length but no effect on ChE activity. This study indicates that the effects of PCB126 and diazinon on body length are cumulative because no significant synergistic or antagonistic interactions were observed. Longer term studies with several doses of PCB126 are needed to fully assess the overall impact of joint exposure to diazinon and PCB126 on growth and survival of fish larvae.

Speciation in killifish and the role of salt tolerance.

Species pairs whose distributions are tied to environmental conditions provide intriguing candidates for the study of ecological speciation. Here, we examine the role that adaptation to salinity has played in the divergence between two closely related species, Lucania goodei and Lucania parva, whose distributions reflect salinity (L. goodei- fresh water, L. parva- euryhaline). We first tested whether these two species display local adaptation and, subsequently, tested for ecological, genic and behavioural isolation by performing crosses within and between L. goodei and L. parva and raising offspring under various salinities. We found strong evidence for differential adaptation to salinity and also for behavioural isolation where animals preferentially mated with conspecifics over heterospecifics. However, we found no evidence for F1 hybrid inviability. We discuss the general lack of evidence for genic isolation in teleost fish and whether this is a real phenomenon or simply a reflection of experimental design.

Gill morphology of the mangrove killifish (Kryptolebias marmoratus) is plastic and changes in response to terrestrial air exposure.

Amphibious mangrove killifish, Kryptolebias marmoratus (formerly Rivulus marmoratus), are frequently exposed to aerial conditions in their natural environment. We tested the hypothesis that gill structure is plastic and that metabolic rate is maintained in response to air exposure. During air exposure, when gills are no longer functional, we predicted that gill surface area would decrease. In the first experiment, K. marmoratus were exposed to either water (control) or air for 1 h, 1 day, 1 week, or 1 week followed by a return to water for 1 week (recovery). Scanning electron micrographs (SEM) and light micrographs of gill sections were taken, and morphometric analyses of lamellar width, lamellar length and interlamellar cell mass (ILCM) height were performed. Following 1 week of air exposure, SEM indicated that there was a decrease in lamellar surface area. Morphometric analysis of light micrographs revealed that there were significant changes in the height of the ILCM, but there were no significant differences in lamellae width and length between any of the treatments. Following 1 week of recovery in water, the ILCM regressed and gill lamellae were similar to control fish, indicating that the morphological changes were reversible. In the second experiment, V(CO(2)) was measured in fish continuously over a 5-day period in air and compared with previous measurements of oxygen uptake (V(O(2))) in water. V(CO(2)) varied between 6 and 10 micromol g(-1) h(-1) and was significantly higher on days 3, 4 and 5 relative to days 1 and 2. In contrast to V(O(2)) in water, V(CO(2)) in air showed no diurnal rhythm over a 24 h period. These findings indicate that K. marmoratus remodel their gill structures in response to air exposure and that these changes are completely reversible. Furthermore, over a similar time frame, changes in V(CO(2)) indicate that metabolic rate is maintained at a rate comparable to that of fish in water, underlying the remarkable ability of K. marmoratus to thrive in both aquatic and terrestrial habitats.

Asexual and sexual hybrids between Fundulus diaphanus and F. heteroclitus in the Canadian Atlantic region.

Levels and origins of clonal diversity in asexual hybrid animals are critical to understanding how they can coexist with their sexual progenitor species. In this study, asexual gynogenetic hybrids between Fundulus diaphanus and Fundulus heteroclitus known from two sites in Nova Scotia (Canada) were characterized using discriminant morphological traits, eight microsatellite loci, and mitochondrial DNA. Fifteen clonal genotypes were uncovered, all bearing the same F. diaphanus maternal haplotye. Each site harboured a different dominant clone along with several rarer clones that all appear to be of recent origin. Unexpectedly, highly introgressed sexually reproducing hybrids (0.25 > q > 0.75) were also detected. Sexual hybrids with maternal ascendance in either species were also found at three other sites in the Atlantic region. Based on a single meristic trait (scale counts), it is shown that asexual clones can be significantly more variable than populations of sexual parental species. Also, species are morphologically more alike when living in sympatry, suggesting that introgression may occur via sexual hybrids. Altogether, these results confirm and refine the available knowledge on this hybrid system, and indicate that hybridization is probably a more widespread phenomenon than suspected, with implications for the phenotypic variability of a widely used model species, F. heteroclitus.

Estrogen receptor-related receptors in the killifish Fundulus heteroclitus: diversity, expression, and estrogen responsiveness.

The estrogen receptor-related receptors (ERRs) are a group of nuclear receptors that were originally identified on the basis of sequence similarity to the estrogen receptors. The three mammalian ERR genes have been implicated in diverse physiological processes ranging from placental development to maintenance of bone density, but the diversity, function, and regulation of ERRs in non-mammalian species are not well understood. In this study, we report the cloning of four ERR cDNAs from the Atlantic killifish, Fundulus heteroclitus, along with adult tissue expression and estrogen responsiveness. Phylogenetic analysis indicates that F. heteroclitus (Fh)ERRalpha is an ortholog of the single ERRalpha identified in mammals, pufferfish, and zebrafish. FhERRbetaa and FhERRbetab are co-orthologs of the mammalian ERRbeta. Phylogenetic placement of the fourth killifish ERR gene, tentatively identified as FhERRgammab, is less clear. The four ERRs showed distinct, partially overlapping mRNA expression patterns in adult tissues. FhERRalpha was broadly expressed. FhERRbetaa was expressed at apparently low levels in eye, brain, and ovary. FhERRbetab was expressed more broadly in liver, gonad, eye, brain, and kidney. FhERRgammab was expressed in multiple tissues including gill, heart, kidney, and eye. Distinct expression patterns of FhERRbetaa and FhERRbetab are consistent with subfunctionalization of the ERRbeta paralogs. Induction of ERRalpha mRNA by exogenous estrogen exposure has been reported in some mammalian tissues. In adult male killifish, ERR expression did not significantly change following estradiol injection, but showed a trend toward a slight induction (three- to five-fold) of ERRalpha expression in heart. In a second, more targeted experiment, expression of ERRalpha in adult female killifish was downregulated 2.5-fold in the heart following estradiol injection. In summary, our results indicate that killifish contain additional ERR genes relative to mammals, including ERRbeta paralogs. In addition, regulation of ERRalpha expression in killifish apparently differs from regulation in mammals. Together, these features may facilitate determination of both conserved and specialized ERR gene functions.

Effects of sediment-bound zinc contamination on early life stages of the mummichog (Fundulus heteroclitus L.) in the Christina watershed, Delaware, U.S.A.

During the last century, the Christina River, the major estuarine river system in New Castle County (DE, USA), has received loadings of organic and inorganic chemicals, primarily from manufacturing facilities. Among the most abundant chemicals is zinc, which has accumulated in sediments at concentrations as high as 5,440 mg/kg. We studied the possible effects of zinc on early life stages of the mummichog (Fundulus heteroclitus), a resident species in the river and watershed. We conducted three different types of exposures. The first was a 96-h median lethal concentration (LC50) test with larvae exposed to waterborne zinc. The second was a larval exposure with zinc-spiked sediments (obtained from the relatively uncontaminated Magothy River in Anne Arundel County, MD, USA). The third was an embryo-larval exposure with Christina River sediments having a gradient of zinc concentrations. The average 96-h LC50 with newly hatched yolk sac larvae was 970 lig/L. In the larval tests, the average 7- and 21-d LC50s were 1154 and 1012 mg/kg, respectively. In the embryo-larval test, no significant difference was found in survival at concentrations between 38.8 and 1098 mg/kg. However, significant reductions were observed in condition factor at concentrations of 582, 799, and 1098 mg/kg. We calculated an average no-observed-effects concentration of 579 mg/kg and an average lowest-observed-effects concentration of 849 mg/kg for larval survival. Based on these results, we suggest that zinc in the Christina River may be affecting early life stages of the mummichog.

Effect of dispersant on the composition of the water-accommodated fraction of crude oil and its toxicity to larval marine fish.

Newly hatched mummichog (Fundulus heteroclitus) were exposed in a 96-h static renewal assay to water-accommodated fractions of dispersed crude oil (DWAF) or crude oil (WAF) to evaluate if the dispersant-induced changes in aqueous concentrations of polycyclic aromatic hydrocarbons (PAH) affected larval survival, body length, or ethoxyresorufin-O-deethylase (EROD) activity. Weathered Mesa light crude oil (0.05-1 g/L) and filtered seawater with or without the addition of Corexit 9500 were used to prepare DWAF and WAE At 0.2 g/L, the addition of dispersant caused a two- and fivefold increase in the concentrations of total PAH (sigmaPAH) and high-molecular-weight PAH (HMWPAH) with three or more benzene rings. Highest mortality rates (89%) were observed in larvae exposed to DWAF (0.5 g/L; sigmaPAH, 479 ng/ml). A reduction in body length was correlated with increased levels of sigmaPAH (r2 = 0.65, p = 0.02) and not with HMWPAH. The EROD activity increased linearly with HMWPAH (r2 = 0.99, p = 0.001) and not with sigmaPAH. Thus, chemical dispersion increased both the sigmaPAH concentrations and the proportion of HMWPAH in WAF. Dispersed HMWPAH were bioavailable, as indicated by a significantly increased EROD activity in exposed mummichog larvae, and this may represent a significant hazard for larval fish.

Vitellogenesis in male Fundulus heteroclitus (killifish) induced by selected estrogenic compounds.

The response of male Fundulus heteroclitus to estrogenic compounds was assessed in anticipation of using this species in endocrine disrupter field studies in the Chesapeake Bay. Measurements of plasma vitellogenin, gonadosomatic (GSI) and hepatosomatic (HSI) indices, and an assessment of changes in gonadal histology were made. Of the parameters assessed, vitellogenin was found to be the most sensitive biomarker. Plasma vitellogenin production occurred in a dose-dependent manner in males exposed to 4-nonylphenol, 4-(tert-octyl)phenol, bisphenol-A, and 17beta-estradiol. There was some indication that the effect on GSI may be influenced by the season in which the experiments are carried out. Two time course experiments revealed that vitellogenin is a fairly long-lived biomarker in male F. heteroclitus. There was also evidence that fish from two moderately contaminated areas injected with 4-nonylphenol or bisphenol-A produced less vitellogenin than those collected from a more pristine habitat. Production of vitellogenin in male F. heteroclitus appeared similar to two other species dosed with the same compounds.

Intraspecific variation in retinal cone distribution in the bluefin killifish, Lucania goodei.

Studies of visual ecology have typically focused on differences among species while paying less attention to variation among populations and/or individuals. Here, we show that the relative abundance of UV, violet, yellow, and red cones varies between two populations of bluefin killifish, Lucania goodei. Animals from a spring population (high-transmission UV/blue light) have a higher frequency of UV and violet cones and a lower frequency of yellow and red cones than animals from a swamp population (low-transmission UV/blue light). Visual sensitivity does not vary significantly between the populations, but spring animals tend to be more sensitive in the UV/blue wavelengths (360-440 nm) and less sensitive in longer wavelengths (560-600 nm) than swamp animals. The results have two important implications. First, the tight conservation of functional regions of opsin genes across taxa does not imply that visual systems are constrained in their evolution; differential sensitivity can arise through differential expression of cone classes within the retina. Second, intraspecific visual signals in this species may evolve to maximize contrast between the signaler and the background (as opposed to brightness); males with blue anal fins are most abundant in swamp habitats where animals express fewer UV and violet cones.

Vacuolar-type proton pump in the basolateral plasma membrane energizes ion uptake in branchial mitochondria-rich cells of killifish Fundulus heteroclitus, adapted to a low ion environment.

We examined the involvement of mitochondria-rich (MR) cells in ion uptake through gill epithelia in freshwater-adapted killifish Fundulus heteroclitus, by morphological observation of MR cells and molecular identification of the vacuolar-type proton pump (V-ATPase). MR cell morphology was compared in fish acclimated to defined freshwaters with different NaCl concentrations: low (0.1 mmol l(-1))-, mid (1 mmol l(-1))- and high (10 mmol l(-1))-NaCl environments. MR cells, mostly located on the afferent-vascular side of the gill filaments, were larger in low- and mid-NaCl environments than in the high-NaCl environment. Electron-microscopic observation revealed that the apical membrane of well-developed MR cells in low- and mid-NaCl environments was flat or slightly projecting, and equipped with microvilli to expand the surface area exposed to these environments. On the other hand, in the high-NaCl environment, the apical membrane was invaginated to form a pit, and MR cells often formed multicellular complexes with accessory cells, although the NaCl concentration was much lower than that in plasma. We cloned and sequenced a cDNA encoding the A-subunit of killifish V-ATPase. The deduced amino acid sequence showed high identity with V-ATPase A-subunits from other vertebrate species. Light-microscopic immunocytochemistry, using a homologous antibody, revealed V-ATPase-immunoreactivity in Na(+)/K(+)-ATPase-immunoreactive MR cells in low-NaCl freshwater, whereas the immunoreactivity was much weaker in higher NaCl environments. Furthermore, immuno-electron microscopy revealed V-ATPase to be located in the basolateral membrane of MR cells. These findings indicate that MR cells are the site responsible for active ion uptake in freshwater-adapted killifish, and that basolaterally located V-ATPase is involved in the Na(+) and/or Cl(-) absorbing mechanism of MR cells.

Lighting environment predicts the relative abundance of male colour morphs in bluefin killifish (Lucania goodei) populations.

Animal communication occurs when an animal emits a signal, the signal is transmitted through the environment, and then detected by the receiver. The environment in which signalling occurs should govern the efficacy of this process. In this study, I examine the relationship of lighting environment (light transmission and tree cover), location and the relative abundances of male colour morphs across seven drainages and 30 populations in the bluefin killifish, Lucania goodei. I found that males with blue anal fins were more common in populations with low transmission of ultraviolet (UV) and blue wavelengths. By contrast, males with red anal fins (and to a lesser extent, males with yellow anal fins) were more common in populations with high transmission of UV and blue wavelengths. High UV-blue light transmission should create a blue visual background and may make blue males less conspicuous and red males more conspicuous to conspecifics. Colour contrast with the visual background may be more important than total brightness of the colour pattern. These results indicate that natural selection for effective intraspecific communication drives the relative abundance of male colour morphs in different lighting habitats.

Morphological studies on microfilaments and their organizing center in killifish (Fundulus heteroclitus L.) melanophores.

Fish chromatophores serve as excellent study models for cytoskeleton-dependent organelle translocations because the distribution of pigmentary organelles can be observed against a time frame by microscopy. In this study the distribution of microfilaments along with microtubules in cultured melanophores of the killifish (Fundulus heteroclitus Linneaus) are examined using whole-cell transmission electron microscopy (WCTEM), fluorescence, and laser scanning confocal microscopy. Dispersing, dispersed, aggregating and aggregated states of pigment are induced by adding either caffeine (for dispersion) or epinephrine (for aggregation) to the cells in a standard culture medium. The cells that exhibited a random melanosome distribution in the standard culture media without these two reagents, served as the control. The results indicate that: (i) a structure considered to be the actin-filament organizing center (AFOC) is in close proximity to the microtubule-organizing center (MTOC); (ii) the radial layout of microfilaments remains similar over four physiological states of pigmentary response with the exception of epinephrine-aggregated pigment, in which the aggregate blocks the viewing of the AFOC and central microfilament rays, yet radial microfilaments, whether central and/or peripheral, are apparent in all physiological states of distribution; and (iii) microfilaments serve, together with microtubules, as scaffolding for melanosomes which migrate in bi-directional rows on cross-bridges, thus shedding light on the mechanisms for orderly melanosome translocations in a structural continuum.

Redistribution of immunofluorescence of CFTR anion channel and NKCC cotransporter in chloride cells during adaptation of the killifish Fundulus heteroclitus to sea water.

Cellular distribution of cystic fibrosis transmembrane conductance regulator (CFTR) immunofluorescence was detected by monoclonal antibody directed to the C terminus of killifish CFTR (kfCFTR) in chloride cells of fresh water (FW) adapted fish and animals transferred to sea water (SW) for 24h, 48h and 14+ days. Confocal microscopy allowed localization within mitochondria-rich (MR) cells to be determined as superficial (i.e. in the apical membrane) or deeper within the cytoplasm of the cells. In FW, 90 % of MR cells had diffuse kfCFTR immunofluorescence in the central part of the cytosol, with only 8.1 % having apical kfCFTR, which was 6.6+/-0.54 microm below the microridges of surrounding pavement cells. Curiously, FW but not SW pavement cells also had positive immunofluorescence to kfCFTR. After 24h in SW, a time when kfCFTR expression is elevated, a condensed punctate immunofluorescence appeared among 18.8 % of MR cells, 13.4+/-0.66 microm (mean +/- S.E.M.) below the surface of the cells. By 48h, a majority (76.3 %) of MR cells had punctate kfCFTR distribution and the distance from the surface was less (7.8+/-0.2 microm), a distribution approaching the SW-acclimated condition (i.e. all MR cells showing kfCFTR immunofluorescence, 6.1+/-0.04 microm below the surface). In contrast, NKCC immunofluorescence was condensed and localized in lateral parts of MR cell complexes in FW animals and then redistributed to the whole basal cytoplasm after acclimation to SW. CFTR, the anion channel responsible for Cl(-) secretion in marine teleosts, redistributes in MR cells during SW acclimation by condensation of a diffuse distribution below the apical crypt, followed by translocation and insertion in the apical membrane. NKCC, the cotransporter that translocates Cl(-) across the basolateral membrane, moves from an eccentric cytosolic location in FW to a diffuse basolateral localization in SW chloride cells.