Mitogen activated protein kinase 14-1 regulates serum glucocorticoid kinase 1 during seawater acclimation in Atlantic killifish, Fundulus heteroclitus

The Atlantic killifish (Fundulus heteroclitus) is an environmental sentinel organism used extensively for studies of environmental toxicants and osmoregulation. Previous research in our laboratory has shown that acute acclimation to seawater is mediated by an increase in SGK1. SGK1 promotes the trafficking of CFTR chloride channels from intracellular vesicles to the plasma membrane of the gill within the first hour in seawater resulting in increased chloride secretion. Although we have shown that the increase in gill SGK1 does not require activation of the glucocorticoid receptor, the mechanisms that mediate the rise SGK1 during acute acclimation is unknown. To test the hypothesis that mitogen activated protein kinase (MAPK14) is responsible for the rise in SGK1 we identified the coding sequence of killifish MAPK14-1 and designed a translational blocking vivo-morpholino targeting MAPK14-1. Injection of the MAPK14-1 vivo-morpholino resulted in a 30% reduction of MAPK14-1 and a 45% reduction in phosphorylated-MAPK14-1 protein in the gill of killifish transitioned from freshwater to seawater. Knock down of phosphorlyated-MAPK14-1 completely blocked the rise in SGK1 mRNA and protein in the killifish gill, providing the first direct and in vivo evidence that MAPK14-1 is necessary for acute seawater acclimation.     

Expression of aquaporin 3 in gills of the Atlantic killifish (Fundulus heteroclitus): Effects of seawater acclimation

Estuarine fish, such as the Atlantic killifish (Fundulus heteroclitus), are constantly and rapidly exposed to changes in salinity. Although ion transport in killifish gills during acclimation to increased salinity has been studied extensively, no studies have examined the role of aquaglyceroporin 3 (AQP3), a water, glycerol, urea, and ammonia transporter, during acclimation to increased salinity in this sentinel environmental model organism. The goal of this study was to test the hypothesis that transfer from freshwater to seawater decreases AQP3 gene and protein expression in the gill of killifish. Transfer from freshwater to seawater decreased AQP3 mRNA in the gill after 1 day, but had no effect on total gill AQP3 protein abundance as determined by western blot. Quantitative confocal immunocytochemistry confirmed western blot studies that transfer from freshwater to seawater did not change total AQP3 abundance in the gill; however, immunocytochemistry revealed that the amount of AQP3 in pillar cells of secondary lamellae decreased in seawater fish, whereas the amount of AQP3 in mitochondrion rich cells (MRC) in primary filaments of the gill increased in seawater fish. This response of AQP3 expression is unique to killifish compared to other teleosts. Although the role of AQP3 in the gill of killifish has not been completely elucidated, these results suggest that AQP3 may play an important role in the ability of killifish to acclimate to increased salinity.     

Intraspecific variation in gene expression after seawater transfer in gills of the euryhaline killifish Fundulus heteroclitus

Previous research has suggested that northern populations of the euryhaline killifish (Fundulus heteroclitus) are better adapted to freshwater environments than their southern counterparts. In this study, we examined whether this adaptation has come at an ionoregulatory cost in seawater, by comparing published data for northern killifish to newly acquired data on the molecular responses of southern killifish to seawater transfer. After abrupt transfer from brackish water (10 per thousand) to seawater, Na,K-ATPase activity, Na,K-ATPase alpha(1a) mRNA expression, and NKCC1 mRNA expression increased 1 and 4 days after transfer in the gills of southern fish (by 2-3-fold), but increased at 1 day and not 4 days after transfer in northern fish. Small increases in mRNA expression were observed in both populations at 14 days. CFTR expression also increased in southern and northern fish at 1 and 4 days into seawater, and was also elevated at 14 days in northern fish. Because fish from both southern and northern populations maintained plasma Na(+) and Cl(-) balance after seawater transfer, the differences in activity and expression could not have been caused by differences in plasma ion levels. Instead, some other regulatory factor may account for the differences in expression between populations. This study shows that freshwater adaptation in northern populations of killifish has not necessarily come at a significant ionoregulatory cost in seawater, but has altered the molecular responses of their gills to seawater transfer compared to southern killifish.     

Cortisol receptor blockade and seawater adaptation in the euryhaline teleost Fundulus heteroclitus

To examine the role of cortisol in seawater osmoregulation in a euryhaline teleost, adult killifish were acclimated to brackish water (10 per thousand) and RU486 or vehicle was administered orally in peanut oil daily for five days at low (40 mg.kg(-1)) or high dose (200 mg.kg(-1)). Fish were transferred to 1.5 x seawater (45 per thousand) or to brackish water (control) and sampled at 24 h and 48 h after transfer, when Cl- secretion is upregulated. At 24 h, opercular membrane Cl- secretion rate, as Isc, was increased only in the high dose RU486 group. Stimulation of membranes by 3-isobutyl-1-methylxanthine and cAMP increased Isc in vehicle treated controls but those from RU486-treated animals were unchanged and membranes from brackish water animals showed a decrease in Isc. At 48 h, Isc increased and transepithelial resistance decreased in vehicle and RU486 groups, compared to brackish water controls. Plasma cortisol increased in all groups transferred to high salinity, compared to brackish water controls. RU486 treated animals had higher cortisol levels compared to vehicle controls. Vehicle treated controls had lower cortisol levels than untreated or RU486 treated animals, higher stimulation of Isc, and lower hematocrit at 24 h, beneficial effects attributed to increased caloric intake from the peanut oil vehicle. Chloride cell density was significantly increased in the high dose RU486 group at 48 hours, yet Isc was unchanged, suggesting a decrease in Cl- secretion per cell. Thus cortisol enhances NaCl secretion capacity in chloride cells, likely via glucocorticoid type receptors.     

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.     

Radiocadmium exchange with seawater by Fundulus heteroclitus (L.) (Pisces: Cyprinodontidae)

Mummichogs, Fundulus heteroclitus (L.) accumulated Cd 115m (NO₃)₂ from synthetic seawater solutions during a period of 25 days, but the rate of whole body accumulation decreased with increasing concentrations of stable Cd²⁺ in the medium. Viscera were the major repository of Cd 115m during uptake, especially gastrointestinal tract and to a lesser extent liver. Loss in whole body radioactivity over a post-treatment interval of 180 days in cadmium-free seawater was about 90%, with gallbladder and especially liver most influential in the excretion processes; this pattern was unchanged by the level of stable Cd²⁺ present in the medium during uptake. The significance of this and other observations in terms of field monitoring of cadmium utilizing teleosts as indicators is discussed.     

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     

Salinity affects growth but not thermal preference of adult mummichogs Fundulus heteroclitus

The effects of an ecologically relevant range of salinities (2, 12, 22, 32) on thermal preferences and growth of adult mummichogs Fundulus heteroclitus were determined for fish from a southern Chesapeake Bay population. Salinity did not affect the mean temperature selected by F. heteroclitus in a thermal gradient, which was identified as 26.6°C based on observations of 240 individuals. Salinity and temperature had significant and interacting effects on growth rates of F. heteroclitus measured over 12 weeks. Growth rates were highest overall and remained high over a broader range of temperatures at moderate salinities (12 and 22), while high growth rates were shifted toward lower temperatures for fish grown at a salinity of 2 and higher temperatures at a salinity of 32. Significant reductions in growth relative to the optimal conditions (28.6°C, salinity of 22) were observed at the coolest (19.6°C) and warmest (33.6°C) temperature tested at all salinities, as well as temperatures ≥ 26.6°C at a salinity of 2, ≥ 28.6°C at a salinity of 12 and ≤ 26.6°C at a salinity of 32. Growth rates provide a long-term, organismal measure of performance and results of this study indicate that performance may be reduced under conditions that the highly euryhaline F. heteroclitus can otherwise easily tolerate. The combination of reduced salinity and increased temperature that is predicted for temperate estuaries as a result of climate change may have negative effects on growth of this ecologically important species.     

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     

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.     

Variation in gene expression in response to stress in two populations of Fundulus heteroclitus

We used differential display PCR to identify hepatic genes responsive to handling stress and genes that differ in expression between populations of a fish, Fundulus heteroclitus, from different thermal environments. Despite substantial inter-individual variation, we cloned 20 putatively stress-regulated bands from Northern fish, 10 of which had high similarity to genes of known function. We selected five of these genes for further analysis based on their known roles in the stress response. Three of these genes (glucokinase, serine-threonine kinase 10 and cRAF) were confirmed as stress-responsive using real-time PCR. These genes increased in expression in response to a 7-day chronic stress protocol in fish from the Southern population of F. heteroclitus, but did not change significantly in fish from the Northern population. These three genes also differed in expression between populations in control fish, suggesting a link between the response to chronic stress and inter-population differences in gene expression in unstressed laboratory-acclimated fish. Two genes that did not respond to stress (glycogen synthase kinase and warm acclimation-related protein (WAP)) also differed between populations. Expression of WAP was eight-fold higher in Southern than in Northern fish, consistent with a previously suggested role for this gene in thermal acclimation or adaptation in fish.     

The effect of acute warming and thermal acclimation on maximum heart rate of the common killifish Fundulus heteroclitus

Common killifish Fundulus heteroclitus were acclimated to ecologically relevant temperatures (5, 15 and 33°C) and their maximum heart rate (f_Hmax) was measured at each acclimation temperature during an acute warming protocol. Acclimation to 33°C increased peak f_Hmax by up to 32% and allowed the heart to beat rhythmically at a temperature 10°C higher when compared with acclimation to 5°C. Independent of acclimation temperature, peak f_Hmax occurred about 3°C cooler than the temperature that first produced cardiac arrhythmias. Thus, when compared with previously published values for the critical thermal maximum of F. heteroclitus, the temperature for peak f_Hmax was cooler and the temperature that first produced cardiac arrhythmias was similar to these critical thermal maxima. The considerable thermal plasticity of f_Hmax demonstrated in the present study is entirely consistent with eurythermal ecology of killifish, as shown previously for another eurythermal fish Gillichthys mirabilis.     

Phenotypic plasticity in gene expression contributes to divergence of locally adapted populations of Fundulus heteroclitus

We examine the interaction between phenotypic plasticity and evolutionary adaptation using muscle gene expression levels among populations of the fish Fundulus heteroclitus acclimated to three temperatures. Our analysis reveals shared patterns of phenotypic plasticity due to thermal acclimation as well as non‐neutral patterns of variation among populations adapted to different thermal environments. For the majority of significant differences in gene expression levels, phenotypic plasticity and adaptation operate on different suites of genes. The subset of genes that demonstrate both adaptive differences and phenotypic plasticity, however, exhibit countergradient variation of expression. Thus, expression differences among populations counteract environmental effects, reducing the phenotypic differentiation between populations. Finally, gene‐by‐environment interactions among genes with non‐neutral patterns of expression suggest that the penetrance of adaptive variation depends on the environmental conditions experienced by the individual.     

Photoperiodic regulation of thyroid responsiveness to TSH in Fundulus heteroclitus

Treatment of the killifish, Fundulus heteroclitus, with ovine thyrotropin (oTSH) produced elevations in serum thyroxin (T4) that varied seasonally with a strong inverse correlation to environmental photoperiod (P less than 0.001). At 20 degrees C, oTSH (0.2 IU) increased serum T4 to levels ranging from 1.5 micrograms/100 ml in midsummer to 7.5 micrograms/100 ml in midwinter despite relatively stable resting levels throughout the two years of study. Similar rates of clearance of T4 in summer and winter suggest that an alteration in thyroid response to oTSH accounts for this change. The serum T4 response to oTSH is both photoperiod- and temperature-dependent. In laboratory studies, fish exposed to a short artificial photoperiod (LD 8:16) for one month responded to oTSH with T4 levels about twice as high as those in fish exposed to long days (LD 14:10). On the other hand the T4 response to oTSH was sharply reduced at 5 degrees C from that seen at 20 degrees C. This suggests that, in nature, seasonal changes in photoperiod and temperature have opposite effects on thyroidal responsiveness to TSH. Overall, the annual variation in the T4 response to oTSH appears to be driven by photoperiod, inasmuch as the alterations preceded major temperature changes in the wild and can be shown to occur at constant temperature in captivity. Photoperiodic induction of changes in thyroid sensitivity may aid in the maintenance of basal T4 levels under changing thermal conditions.