Microsatellite primers for the Atlantic coastal killifish,Fundulus heteroclitus,with applicability to related Fundulus species

The mummichog (Fundulus heteroclitus), a common Atlantic coastal killifish, is a model vertebrate species for the study of molecular genetic variation in natural populations and of environmental toxicology. We report the development of a set of 20 microsatellite loci in this species. Average expected heterozygosity across all loci was 0.84 (range: 0.60–0.97), revealing a high level of variability at most loci. A survey of seven additional Fundulus species yielded one or two robust amplification products in over half (63%) of the species–primer combinations tested. Therefore, many of these loci will also prove useful in studies of other members of the genus Fundulus.     

Effects of environmental salinity on gill endothelin receptor expression in the killifish, Fundulus heteroclitus

We recently determined that rapid changes in environmental salinity alter endothelin-1 (EDN1) mRNA levels in the euryhaline killifish, Fundulus heteroclitus, so we hypothesized that EDN1 may be a local regulator of gill ion transport in teleost fishes. The purpose of the present study was to examine the effects of changes in environmental salinity on the gill endothelin receptors: EDNRA, EDNRB, and EDNRC. Using quantitative real-time PCR, we determined that after a fresh water (FW) to seawater (SW) transfer, there is a two to threefold increase in gill EDNRA and EDNRB mRNA levels. Likewise, we found a two to three fold increase in gill EDNRA and EDNRB protein concentration. In addition, killifish that have acclimated to FW for 30 days had significantly lower EDNRA mRNA and protein levels than SW killifish. ENDRA were immunolocalized to the mitochondrion-rich cells of the killifish gill, suggesting that EDN1 signaling cascades may affect MRC function. EDNRB were found throughout the gill vasculature and on lamellar pillar cells. We previously immunolocalized EDN1 to the pillar cell suggesting that EDN1 acts as an autocrine signaling molecule and potentially regulates pillar cell tone and lamellar perfusion. We conclude that EDN1 is physiologically active in the teleost gill, and regulated by environmental salinity. Future functional studies examining the physiological role of this system are necessary to completely understand EDN1 in the fish gill.     

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.     

Characterization of two Ca-ATPases in gill epithelium from the killifish (Fundulus heteroclitus)

1. Two Ca-ATPases in the gill microsomal fraction from the killifish (Fundulus heteroclitus) have been characterized. 2. A (Ca2+ + Mg2+)-ATPase which has a high affinity for Ca2+, requires Mg2+ for activity and may be stimulated by calmodulin. 3. A (Ca2+ + Na+)-ATPase which has a low affinity for Ca2+ requires Na+ for activity, does not require Mg2+ and is probably not stimulated by calmodulin. 4. These enzymes may play a physiological role in killifish calcium regulation.     

Hemoglobins of the killifish Fundulus heteroclitus. Separation, characterization, and a model for the subunit compositions.

Polyacrylamide and starch gel electrophoresis of the hemoglobin of the killifish Fundulus heteroclitus reveal the presence of four clearly distinguishable components. These isohemoglobins, each tetramers consisting of alpha and beta chains, can be preparatively separated by ion exchange chromatography on DEAE-cellulose and are homogeneous according to isoelectric focusing in polyacrylamide gels. Oxygen equilibria of the isolated hemoglobin components (Hb I, Hb II, Hb III, and Hb IV) show only minor differences in the magnitude of the Bohr effect and in the effect of ATP on the binding of oxygen. Four different globin chains, alphaa, alphab, betaa, and betab, can be separated by ion exchange on CM-cellulose. Hb I is a homotetramer of alphab and betab chains, Hb IV consists of alphaa and betaa subunits, and components II and III are heterotetramers consisting of all four chains. The alpha and beta chains differ significantly in amino acid composition. A model suggesting the existence of 10 different isohemoglobins, 6 of which have stable intersubunit contacts, has been proposed to account for the qualitative and quantitative aspects of the electrophoretic behavior of the components. Separations of the isohemoglobins on DEAE-cellulose under slightly modified conditions provide additional support for the model.     

Relative influences of historical and contemporary forces shaping the distribution of genetic variation in the Atlantic killifish, Fundulus heteroclitus

A major goal of population genetics research is to identify the relative influences of historical and contemporary processes that serve to structure genetic variation. Most population genetic models assume that populations exist in a state of migration-drift equilibrium. However, in the past this assumption has rarely been verified, and is likely rarely achieved in natural populations. We assessed the equilibrium status at both local and regional scales of the Atlantic killifish, Fundulus heteroclitus . This species is a model organism for the study of adaptive clinal variation, but has also experienced a complicated history of range expansion and secondary contact following allopatric divergence, potentially obscuring the influence of contemporary evolutionary processes. Presumptively neutral genetic markers (microsatellites) demonstrated zones of secondary intergradation among coastal populations centred around northern New Jersey and the Chesapeake Bay region. Analysis of genetic variation indicated isolation by distance among some populations and provided supporting evidence that the Delaware Bay, but not the Chesapeake Bay, has acted as a barrier to dispersal among coastal populations. Bayesian estimates indicated large effective population sizes and low migration rates, and were in good agreement with empirically derived estimates of population and neighbourhood size from mark–recapture studies. These data indicate that populations are not in migration-drift equilibrium at a regional scale, and suggest that contributing factors include large population size combined with relatively low migration rates. These conditions should be considered when interpreting the evolutionary significance of the distribution of genetic variation among F. heteroclitus populations.     

Structural diversity of occluding junctions in the low-resistance chloride-secreting opercular epithelium of seawater-adapted killifish (Fundulus heteroclitus)

The structural features of the chloride-secreting opercular epithelium of seawater-adapted killifish (Fundulus heteroclitus) were examined by thin-section and freeze-fracture electron microscopy, with particular emphasis on the morphological appearance of occluding junctions. This epithelium is a flat sheet consisting predominantly of groups of mitochondriarich chloride cells with their apices associated to form apical crypts. These multicellular groups are interspersed in an otherwise continuous pavement cell epithelial lining. The epithelium may be mounted in Ussing-type chambers, which allow ready access to mucosal and serosal solutions and measurement of electrocal properties. The mean short-circuit current, potential difference (mucosal-side negative), and DC resistance for 19 opercular epithelia were, respectively, 120.0 +/- 18.2 microA/cm2, 12.3 +/- 1.7 mV, and 132.5 +/- 26.4 omega cm2. Short-circuit current, a direct measure of Cl- transport, was inhibited by ouabain (5 micron) when introduced on the serosal side, but not when applied to the mucosal side alone. Autoradiographic analysis of [3H]-ouabain-binding sites demonstrated that Na+,K+-ATPase was localized exclusively to basolateral membranes of chloride cells; pavement cells were unlabeled. Occluding junctions between adjacent chloride cells were remarkably shallow (20-25 nm), consisting of two parallel and juxtaposed junctional strands. Junctional interactions between pavement cells or between pavement cells and chloride cells were considerably more elaborate, extending 0.3-0.5 micron in depth and consisting of five or more interlocking junctional strands. Chloride cells at the lateral margins of crypts make simple junctional contacts with neighboring chloride cells and extensive junctions with contiguous pavement cells. Accordingly, in this heterogeneous epithelium, only junctions between Na+,K+-ATPase- rich chloride cells are shallow. Apical crypts may serve, therefore, as focal areas of high cation conductivity across the junctional route. This view is consistent with the electrical data showing that transmural resistance across the opercular eptihelium is low, and with recent studies demonstrating that transepithelial Na+ fluxes are passive. The simplicity of these junctions parallels that described recently for secretory cells of avian salt gland (Riddle and Ernst, 1979, J. Membr. Biol., 45:21-35) and elasmobranch rectal gland (Ernst et al., 1979, J. Cell Biol., 83:(2, Pt. 2):83 a[Abstr.]) and lends morphological support to the concept that paracellular ion permeation plays a central role in ouabain-sensitive transepithelial NaCl secretion.     

Kinetic characterization of outer-ring deiodinase activity (ORD) in the liver, gill and retina of the killifish Fundulus heteroclitus

Conversion of T4 to T3 is the first step in TH action and deiodinases are the major determinants of TH tissue availability and disposal. We here report the kinetic characterization of the outer-ring deiodinating (ORD) enzymes in the liver, gill and retina of sea water-adapted killifish, by using both rT3 and T4 as substrates. In liver, by using rT3, we detected a high Km (84 nM) and a low Km (1.3 nM) component with kinetic characteristics similar to mammalian deiodinases DI and DII. In contrast, T4-ORD only generated a low Km (0.5 nM) component. As judged by its Vmax (920 fmol 125I/mg per h) this DII enzyme is very abundant, approximately five and 20 times higher than that found in trout liver and hypothyroid rat, respectively. Kinetic analysis in killifish gill showed only one enzymatic component, with a high rT3 Km (430 nM) and a relatively low Vmax (4.3 pmol 125I/mg per h). Our results in killifish retina show the expression of a T4-low Km (0.6 nM) deiodinase with high cofactor requirements akin to the mammalian DII. The Vmax value for this enzyme is 182 fmol 125I/mg per h, five times lower than the one found in killifish liver, but comparable to that in hypothyroid rat pituitary. The biochemical similarities between fish and mammalian deiodinases could reflect their high conservation during vertebrate evolution and thus their importance in the regulation of thyroid hormone action.     

Real-time quantitative PCR assay on bacterial DNA: In a model soil system and environmental samples

Real-time quantitative PCR (RTQ-PCR) was used to quantify the bacterial target DNA extracted by three commonly used DNA extraction protocols (bead mill homogenization, grinding in presence of liquid nitrogen and hot detergent SDS based enzymatic lysis). For the purpose of our study, pure culture of Bacillus cereus (model organism), sterilized soil seeded with a known amount of B. cereus (model soil system) and samples from woodland and grassland (environmental samples) were chosen to extract DNA by three different protocols. The extracted DNA was then quantified by RTQ-PCR using 16S rDNA specific universal bacterial primers. The standard curve used for the quantification by RTQ-PCR was linear and revealed a strong linear relationship (r(2)=0.9968) with a higher amplification efficiency, e5=1.02. High resolution gel electrophoresis was also carried out to observe the effect of these extraction methods on diversity analysis. For the model soil system, the liquid nitrogen method showed the highest target DNA copy number (1.3 x 10(9) copies/microl). However, for both the environmental samples, the bead beating method was found to be suitable on the basis of the high target DNA copy numbers (5.38 x 10(9) and 4.01 x 10(8) copies/ml for woodland and grassland respectively), high yield (6.4 microg/g and 1.76 microg/g of soil for woodland and grassland respectively) and different band patterns on high resolution gel electrophoresis suggesting an overall high extraction efficiency. This difference in the extraction efficiency between the model soil system and environmental samples may be attributed to different affinity of seeded and native DNA to soil particles.     

Development and characterization of polyclonal antibodies against the aryl hydrocarbon receptor protein family (AHR1, AHR2, and AHR repressor) of Atlantic killifish Fundulus heteroclitus

The aryl hydrocarbon receptor (AHR) and AHR repressor (AHRR) proteins regulate gene expression in response to some halogenated aromatic hydrocarbons and polycyclic aromatic hydrocarbons. The Atlantic killifish is a valuable model of the AHR signaling pathway, but antibodies are not available to fully characterize AHR and AHRR proteins. Using bacterially expressed AHRs, we developed specific and sensitive polyclonal antisera against the killifish AHR1, AHR2, and AHRR. In immunoblots, these antibodies recognized full-length killifish AHR and AHRR proteins synthesized in rabbit reticulocyte lysate, proteins expressed in mammalian cells transfected with killifish AHR and AHRR constructs, and AHR proteins in cytosol preparations from killifish tissues. Killifish AHR1 and AHR2 proteins were detected in brain, gill, kidney, heart, liver, and spleen. Antisera specifically precipitated their respective target proteins in immunoprecipitation experiments with in vitro-expressed proteins. Killifish ARNT2 co-precipitated with AHR1 and AHR2. These sensitive, specific, and versatile antibodies will be valuable to researchers investigating AHR signaling and other physiological processes involving AHR and AHRR proteins.     

Steep,coincident, and concordant clines in mitochondrial and nuclear‐encoded genes in a hybrid zone between subspecies of Atlantic killifish,Fundulus heteroclitus

Steep genetic clines resulting from recent secondary contact between previously isolated taxa can either gradually erode over time or be stabilized by factors such as ecological selection or selection against hybrids. We used patterns of variation in 30 nuclear and two mitochondrial SNPs to examine the factors that could be involved in stabilizing clines across a hybrid zone between two subspecies of the Atlantic killifish, Fundulus heteroclitus. Increased heterozygote deficit and cytonuclear disequilibrium in populations near the center of the mtDNA cline suggest that some form of reproductive isolation such as assortative mating or selection against hybrids may be acting in this hybrid zone. However, only a small number of loci exhibited these signatures, suggesting locus‐specific, rather than genomewide, factors. Fourteen of the 32 loci surveyed had cline widths inconsistent with neutral expectations, with two SNPs in the mitochondrial genome exhibiting the steepest clines. Seven of the 12 putatively non‐neutral nuclear clines were for SNPs in genes related to oxidative metabolism. Among these putatively non‐neutral nuclear clines, SNPs in two nuclear‐encoded mitochondrial genes (SLC25A3 and HDDC2), as well as SNPs in the myoglobin, 40S ribosomal protein S17, and actin‐binding LIM protein genes, had clines that were coincident and concordant with the mitochondrial clines. When hybrid index was calculated using this subset of loci, the frequency distribution of hybrid indices for a population located at the mtDNA cline center was non‐unimodal, suggesting selection against advanced‐generation hybrids, possibly due to effects on processes involved in oxidative metabolism.     

Thermal acclimation is not necessary to maintain a wide thermal breadth of aerobic scope in the common killifish (Fundulus heteroclitus)

Loss of aerobic scope at high and low temperatures is a physiological mechanism proposed to limit the thermal performance and tolerance of organisms, a theory known as oxygen- and capacity-limited thermal tolerance (OCLTT). Eurythermal organisms maintain aerobic scope over wide ranges of temperatures, but it is unknown whether acclimation is necessary to maintain this breadth. The objective of this study was to examine changes in aerobic scope in Fundulus heteroclitus, a eurythermal fish, after acclimation and acute exposure to temperatures from 5° to 33°C. The range of temperatures over which aerobic scope was nonzero was similar in acclimated and acutely exposed fish, suggesting that acclimation has modest effects on the thermal breadth of aerobic scope. However, in acclimated fish, there was a clear optimum temperature range for aerobic scope between 25° and 30°C, whereas aerobic scope was relatively constant across the entire temperature range with acute temperature exposure. Therefore, the primary effect of acclimation was to increase aerobic scope between 25° and 30°C, which paradoxically resulted in a narrower temperature range of optimal performance in acclimated fish compared to acutely exposed fish. There was only weak evidence for correlations between the thermal optimum of aerobic scope and the thermal optimum of measures of performance (specific growth rate and gonadosomatic index), and indicators of anaerobic metabolism (lactate accumulation and lactate dehydrogenase activity) only increased at high temperatures. Together these data fit many, but not all, of the predictions made by OCLTT.     

Spontaneous occurrence of a synchronous hermaphrodite in the banded killifish, Fundulus diaphanus (Lesueur)

An intersex specimen of the banded killifish, Fundulus diaphanus , was encountered during the analysis of the annual gametogenic cycle of this species. The simultaneous presence of mature spermatozoa and ova suggests that the potential for self-fertilization existed in this fish. The spontaneous occurrence of this intersex individual, although a rare event, indicates the potential for the development of the synchronous hermaphroditic condition under natural environmental conditions in this normally gonochoristic species.     

Detection of DNA adducts using a quantitative long PCR technique and the fluorogenic 5′ nuclease assay (TaqMan)

The detection of DNA adducts is an important component in assessing the mutagenic potential of exogenous and endogenous compounds. Here, we report an in vitro quantitative long PCR (XL-PCR) assay to measure DNA adducts in human genomic DNA based on their ability to block and inhibit PCR amplification. Human genomic DNA was exposed to test compounds and then a target sequence was amplified by XL-PCR. The amplified sequence was then quantified using fluorogenic 5′ nuclease PCR (TaqMan^®) and normalized to a solvent-treated control. The extent of DNA adduction was determined based on the reduction in amplification of the target sequence in the treated sample. A 17.7 kb β-globin fragment was chosen as the target sequence for these studies, since preliminary experiments revealed a two-fold increased sensitivity of this target compared to a 10.4 kb HPRT fragment for detecting hydrogen peroxide-induced DNA damage. Validation of the XL-PCR assay with various compounds demonstrated the versatility of the assay for detecting a wide range of adducts formed by direct acting or S9-activated mutagens. The same DNA samples were also analyzed using ³²P-postlabeling techniques (thin-layer chromatography or high-performance liquid chromatography) to confirm the presence of DNA adducts and estimate their levels. Whereas ³²P-postlabeling with nuclease P₁ enrichment was more sensitive for detecting bulky adducts induced by the compounds benzo[a]pyrene, dimethylbenzanthracene, 3-methylindole, indole 3-carbinol, or 2-acetylaminofluorene, the XL-PCR procedure was more sensitive for detecting smaller or labile DNA adducts formed by the compounds methyl methanesulfonate, diethyl nitrosamine, ethylnitrosourea, diepoxybutane, ICR-191, styrene oxide, or aflatoxin B₁. Compounds not expected to form adducts in DNA, such as clofibrate, phenobarbital, chloroform or acetone, did not produce a positive response in the XL-PCR assay. Thus, quantitative XL-PCR provides a rapid, high-throughput assay for detecting DNA damage that complements the existing ³²P-postlabeling assay with nuclease P₁ enrichment.     

The effect of environmental hypercapnia and salinity on the expression of NHE-like isoforms in the gills of a euryhaline fish (Fundulus heteroclitus)

The current models for branchial acid excretion in fishes include Na(+)/H(+) exchange and the electrogenic excretion of H+ via H+-ATPase. The predominant route of acid excretion in some freshwater fishes is thought to be via the H+-ATPase/Na+ channel system. The euryhaline Fundulus heteroclitus may not fit this profile even when adapted to freshwater (FW). We hypothesize that the Na+/H+ exchanger (NHE) in this species may play a predominant role in acid-base regulation for both marine and FW adapted animals. Acidosis induced by ambient hypercapnia (1% CO2 in air), resulted in an increase in net H+ excretion to the water in F. heteroclitus pre-adapted to FW, brackish (isoosmotic; BW) and seawater (SW). Both FW and SW adapted mummichogs were tested for NHE protein expression using mammalian NHE antibodies, and we identified NHE-like immunoreactive proteins in gill membrane preparations from both groups. Hypercapnia induced a approximately three-fold elevation in gill NHE2-like protein in FW animals but SW adapted fish showed inconsistent NHE3-like protein expression. There was no change in NHE-1 levels in FW fish. In contrast, SW animals demonstrated a significant increase in both NHE1 and NHE3-like proteins following hypercapnia but limited expression of the NHE2 protein. We hypothesize that different isoforms of NHE may be preferentially expressed depending on the salinity to which the animals are adapted. Net H+ transfers during acidosis may be driven, at least in part by the action of these transporters.