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The oxidative phosphorylation (OxPhos) pathway is responsible for most aerobic ATP production and is the only metabolic pathway with proteins encoded by both nuclear and mitochondrial genomes. In studies examining mitonuclear interactions among distant populations within a species or across species, the interactions between these two genomes can affect metabolism, growth, and fitness, depending on the environment. However, there is little data on whether these interactions impact natural populations within a single species. In an admixed Fundulus heteroclitus population with northern and southern mitochondrial haplotypes, there are significant differences in allele frequencies associated with mitochondrial haplotype. In this study, we investigate how mitochondrial haplotype and any associated nuclear differences affect six OxPhos parameters within a population. The data demonstrate significant OxPhos functional differences between the two mitochondrial genotypes. These differences are most apparent when individuals are acclimated to high temperatures with the southern mitochondrial genotype having a large acute response and the northern mitochondrial genotype having little, if any acute response. Furthermore, acute temperature effects and the relative contribution of Complex I and II depend on acclimation temperature: when individuals are acclimated to 12°C, the relative contribution of Complex I increases with higher acute temperatures, whereas at 28°C acclimation, the relative contribution of Complex I is unaffected by acute temperature change. These data demonstrate a complex gene by environmental interaction affecting the OxPhos pathway., (Copyright © 2016 the American Physiological Society.)

Regulation of internal ion homeostasis is essential for fishes inhabiting environments where salinities differ from their internal concentrations. It is hypothesized that selection will reduce energetic costs of osmoregulation in a population's native osmotic habitat, producing patterns of local adaptation. Killifish, Fundulus heteroclitus, occupy estuarine habitats where salinities range from fresh to seawater. Populations inhabiting an environmental salinity gradient differ in physiological traits associated with acclimation to acute salinity stress, consistent with local adaptation. Similarly, metabolic rates differ in populations adapted to different temperatures, but have not been studied in regard to salinity. We investigated evidence for local adaptation between populations of killifish native to fresh and brackish water habitats. Aerobic scope (the difference between minimum and maximum metabolic rates), excess post-exercise oxygen consumption, and swimming performance (time and distance to reach exhaustion) were used as proxies for fitness in fresh and brackish water treatments. Swimming performance results supported local adaptation; fish native to brackish water habitats performed significantly better than freshwater-native fish at high salinity while low salinity performance was similar between populations. However, results from metabolic measures did not support this conclusion; both populations showed an increase in resting metabolic rate and a decrease of aerobic scope in fresh water. Similarly, excess post-exercise oxygen consumption was higher for both populations in fresh than in brackish water. While swimming results suggest that environmentally dependent performance differences may be a result of selection in divergent osmotic environments, the differences between populations are not coupled with divergence in metabolic performance., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The Houston Ship Channel (HSC) in Houston, Texas is an aquatic environment with a long history of contamination, including polychlorinated dibenzodioxins (PCDD), polychlorinated dibenzofurans (PCDF), polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAHs), and heavy metals. Populations of Gulf killifish (Fundulus grandis) from the HSC have adapted to resist developmental cardiac deformities caused by dioxin-like compounds (DLCs). Contaminants in the HSC have acted as a strong selective pressure on resident Gulf killifish populations. Rapid adaptation can lead to fitness costs, some as a direct result of the mechanisms involved in the adaptive process, whereas other adaptations may be more general. To explore potential fitness costs, we evaluated two Gulf killifish populations with documented resistance to DLC-induced cardiac teratogenesis (Patrick Bayou and Vince Bayou), and one previously characterized reference population (Gangs Bayou). We also characterized a previously unstudied population from Galveston Bay as an additional reference population (Smith Point). We tested the sensitivity of F1 larvae from these four populations to two classes of pesticides (pyrethroid (permethrin) and carbamate (carbaryl)) and two model pro-oxidants (tert-butyl hydroquinone (tBHQ) and tert-butyl hydroperoxide (tBOOH)). In addition, we explored their responses to hypoxia and measured resting metabolic rates (M.O2). Both adapted populations were cross-resistant to the toxicity of carbaryl and both pro-oxidants tested. There were no population differences in sensitivity to permethrin. On the other hand, one reference population (Gangs Bayou) was less sensitive to hypoxia, and maintained a lower M.O2 . However, there were no differences in hypoxia tolerance or resting metabolic rate between the second reference and the two adapted populations. This investigation emphasizes the importance of including multiple reference populations to clearly link fitness costs or cross-resistance to pollution adaptation, rather than to unrelated environmental or ecological differences. When compared to previous literature on adapted populations of Fundulus heteroclitus, we see a mixture of similarities and differences, suggesting that F. grandis adapted phenotypes likely involve multiple mechanisms, which may not be completely consistent among adapted populations., (Copyright © 2016 Elsevier B.V. All rights reserved.)

17α-Ethinylestradiol (EE2) is a potent estrogen used in birth-control pills. Previous laboratory and field studies have shown negative impacts in a variety of fish species after exposure to low levels of EE2, most notably a nearly complete shutdown of egg production. The present study demonstrates that mummichog (Fundulus heteroclitus), a small-bodied estuarine species, is able to continue to produce eggs after exposure for 28 d to 100 ng of EE2/L. No effect of EE2 on egg production was observed, whereas a >35-fold increase in vitellogenin (vtg 1) gene expression in males was found. The lack of response in egg production in fish exposed to high levels of EE2 warrants further investigations on species-specific responses to estrogens and endocrine disruptors in general., (© 2015 SETAC.)

Many fish encounter hypoxia in their native environment, but the role of mitochondrial physiology in hypoxia acclimation and hypoxia tolerance is poorly understood. We investigated the effects of hypoxia acclimation on mitochondrial respiration, O2kinetics, emission of reactive oxygen species (ROS), and antioxidant capacity in the estuarine killifish ( ITALIC! Fundulus heteroclitus). Killifish were acclimated to normoxia, constant hypoxia (5 kPa O2) or intermittent diel cycles of nocturnal hypoxia (12 h:12 h normoxia:hypoxia) for 28-33 days and mitochondria were isolated from liver. Neither pattern of hypoxia acclimation affected the respiratory capacities for oxidative phosphorylation or electron transport, leak respiration, coupling control or phosphorylation efficiency. Hypoxia acclimation also had no effect on mitochondrial O2kinetics, but ITALIC! P50(the O2tension at which hypoxia inhibits respiration by 50%) was lower in the leak state than during maximal respiration, and killifish mitochondria endured anoxia-reoxygenation without any impact on mitochondrial respiration. However, both patterns of hypoxia acclimation reduced the rate of ROS emission from mitochondria when compared at a common O2tension. Hypoxia acclimation also increased the levels of protein carbonyls and the activities of superoxide dismutase and catalase in liver tissue (the latter only occurred in constant hypoxia). Our results suggest that hypoxia acclimation is associated with changes in mitochondrial physiology that decrease ROS production and may help improve hypoxia tolerance., (© 2016. Published by The Company of Biologists Ltd.)

The Atlantic killifish (Fundulus heteroclitus) is a resilient estuarine species that may be subjected to anthropogenic contamination of its natural habitat, by toxicants such as nickel (Ni). We investigated Ni accumulation and potential modes of Ni toxicity, in killifish, as a function of environmental salinity. Killifish were acclimated to 4 different salinities [0 freshwater (FW), 10, 30 and 100% seawater (SW)] and exposed to 5 mg/L of Ni for 96 h. Tissue Ni accumulation, whole body ions, critical swim speed and oxidative stress parameters were examined. SW was protective against Ni accumulation in the gills and kidney. Addition of Mg and Ca to FW protected against gill Ni accumulation, suggesting competition with Ni for uptake. Concentration-dependent Ni accumulation in the gill exhibited saturable relationships in both FW- and SW-acclimated fish. However SW fish displayed a lower Bmax (i.e. lower number of Ni binding sites) and a lower Km (i.e. higher affinity for Ni binding). No effect of Ni exposure was observed on critical swim speed (Ucrit) or maximum rate of oxygen consumption (MO2max). Markers of oxidative stress showed either no effect (e.g. protein carbonyl formation), or variable effects that appeared to depend more on salinity than on Ni exposure. These data indicate that the killifish is very tolerant to Ni toxicity, a characteristic that may facilitate the use of this species as a site-specific biomonitor of contaminated estuaries., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Polyamines are a family of low molecular weight organic cations produced in part by the coordinated actions of arginase II (Arg II) and ornithine decarboxylase (Odc). Although gill polyamine homeostasis is affected by acute transfer to fresh water, little is known of its function in fish osmoregulation. The current study investigated the role of polyamines in the compensatory response of hypoosmotic challenge in the euryhaline fish, Fundulus grandis. Adult F. grandis were acclimated to 5 ppt water, transferred abruptly to 5, 2, 1, 0.5 and 0.1 ppt water, and assessed for osmoregulatory function, gill morphology, and polyamine homeostasis. The plasma osmolality, Na(+) concentration, and Cl(-) concentration were only significantly reduced during exposure to salinities at or below 0.5 ppt, although these effects were transient except in the 0.1 ppt treatment. The phenotype of mitochondrion-rich cells (MRCs) shifted from a seawater-type to a freshwater-type only at salinities that also produced a plasma osmotic disturbance. Hypoosmotic exposure increased the concentrations of putrescine, spermidine, and spermine in the gill over the entire 7 day period. Exposure to 0.1 ppt water also transiently increased gill caspase-3 activity and gill mRNA levels of the immediate-early response genes, c-fos and c-myc, thus tightly associating polyamines with gill remodeling during freshwater acclimation. Furthermore, arginase II and ornithine decarboxylase mRNA levels were most highly expressed in MRCs, and these levels were further increased only in the 0.1 ppt treatment. Reduction of gill polyamine levels following administration of the Odc inhibitor, alpha-dl-difluoromethylornithine (DFMO), inhibited gill caspase-3 activity, but surprisingly reduced the magnitude of the plasma osmotic imbalance elicited by exposure to 0.1 ppt water. We used isolated opercular epithelia mounted on Ussing chambers to assess the influence of polyamines on the attenuating response of hypotonic shock on active Cl(-) secretion. Spermidine partially reduced the decrease of short-circuit current (Isc) and membrane conductance (Gt) produced by hypotonic exposure. These data suggest polyamines blunt the hypotonic inhibition of NaCl secretion and may lead to early apoptosis of seawater ionocytes and their replacement by FW-type ionocytes., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Human ageing is a fundamental biological process that leads to functional decay, increased risk for various diseases and, ultimately, death. Some of the basic biological mechanisms underlying human ageing are shared with other organisms; thus, animal models have been invaluable in providing key mechanistic and molecular insights into the common bases of biological ageing. In this Review, we briefly summarise the major applications of the most commonly used model organisms adopted in ageing research and highlight their relevance in understanding human ageing. We compare the strengths and limitations of different model organisms and discuss in detail an emerging ageing model, the short-lived African turquoise killifish. We review the recent progress made in using the turquoise killifish to study the biology of ageing and discuss potential future applications of this promising animal model., (© 2016. Published by The Company of Biologists Ltd.)

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., (© 2016. Published by The Company of Biologists Ltd.)

In this study, we explored the hypothesis that killifish acclimate to thermal extremes through regulation of genes involved in stress and metabolism. We examined the liver and gonadal transcription of heat shock proteins (hsp70, hsp90a, hsp90b), glucokinase (gck), and high mobility group b1 (hmgb1) protein in wild killifish species from hot springs and rivers using quantitative real-time PCR. Moreover, we exposed a river killifish species to a long-term thermal regime of hot spring (37-40°C) and examined the liver transcription of the heat shock genes. Our results showed that hot spring killifish showed a significant, strong upregulation of liver hsp90a. Moreover, the testicular transcript levels of hsp90a, hsp90b, and hsp70 were higher in hot spring killifish than the river ones. The results of the common garden experiments showed that the transcripts of hsp70, hsp90b, and hmgb1 were mildly induced (> twofold) at the time when temperature reached to 37-40°C, while the transcripts of hsp90a were strongly induced (17-fold increase). The level of hsp90a was dramatically more upregulated when fish were maintained in thermal extreme (42-fold change higher than in ambient temperature). Moreover, a significant downregulation of gck transcripts was observed at the time when temperature was raised to 37-40°C (80-fold decrease) and during exposure to long-term thermal extreme (56-fold decrease). It can be concluded that the regulation of heat shock genes particularly hsp90a might be a key factor of the acclimation of fish to high temperature environments like hot springs., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Mummichogs (Fundulus heteroclitus; Cyprinodontiformes) are intertidal killifish that can breathe air and locomote on land. Our goals were to characterize the terrestrial locomotion of mummichogs and determine their method of navigation towards water in a terrestrial environment. We used high-speed video to record behavior during stranding experiments and found that mummichogs use a tail-flip jump to move overland, similarly to other Cyprinodontiformes. However, mummichogs also prop themselves upright into a prone position between each jump, a previously undescribed behavior. After becoming prone, mummichogs rotate about their vertical axis, directing the caudal fin towards the water. Then, they roll back onto their lateral aspect and use a tail-flip behavior to leap into a caudally-directed, ballistic flight path. We conducted experiments to determine the sensory stimulus used to locate a body of water by placing mummichogs on a square platform with one side adjacent to a sea table. Under artificial light, mummichogs moved towards the sea table with a higher frequency than towards the other sides. Under dark conditions, mummichogs did not show a preference for moving towards the sea table. When the surface of the water was covered with reflective foil, mummichogs moved towards it as if it were a body of water. These results suggest that mummichogs primarily use visual cues, specifically reflected light, to orient towards the water. The uprighting behavior that mummichogs perform between terrestrial jumps may provide an opportunity for these fish to receive visual information that allows them to safely return to the water. J. Exp. Zool. 325A:57-64, 2016. © 2015 Wiley Periodicals, Inc., (© 2015 Wiley Periodicals, Inc.)

Kryptolebias marmoratus, a small killifish that lives in mangrove habitat from southern Florida to Brazil, is one of the planet's only known self-fertilizing hermaphroditic vertebrates. Generation after generation, hermaphroditic individuals simultaneously produce sperm and eggs and internally self-fertilize to produce what are, in effect, highly inbred clones of themselves. Although populations are composed primarily of hermaphrodites, they also contain some true males. The frequency of males in a population varies geographically, from <2% in Florida to as high as 25% in Belize. Males are known to mate occasionally with hermaphrodites, thereby releasing genetic variation that has profound consequences for population genetic structure. However, it is unknown whether hermaphrodites can or do sporadically mate with each other also. Here, we test whether hermaphroditic individuals of the killifish Kryptolebias marmoratus are capable of crossing with one another, in addition to their much more common habits of self-fertilization and occasional outcrossing with pure males. We employ an experimental design in which replicate hermaphrodite pairs were housed together and allowed to reproduce naturally. Among 173 embryos screened at diagnostic microsatellite loci, all were found to result from selfing (i.e., no embryos were the product of a hermaphrodite cross). We thus conclude that hermaphrodite pairs are unlikely to cross, or do so exceedingly rarely., (© The American Genetic Association. 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Persistent organic pollutants (POPs), including polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs), potentially target mitochondria and cause toxicity. We compared the effects of POPs on mitochondrial respiration by measuring oxidative phosphorylation (OxPhos) metabolism in hepatocytes isolated from lab-depurated Fundulus heteroclitus from a Superfund site contaminated with PAHs (Elizabeth River VA, USA) relative to OxPhos metabolism in individuals from a relatively clean, reference population (King's Creek VA, USA). In individuals from the polluted Elizabeth River population, OxPhos metabolism displayed lower LEAK and lower activities in complex III, complex IV, and E State, but higher activity in complex I compared to individuals from the reference King's Creek population. To test the supposition that these differences were due to or related to the chronic PAH contamination history of the Elizabeth River population, we compared the OxPhos functions of undosed individuals from the polluted and reference populations to individuals from these populations dosed with a PAH {benzo [α] pyrene (BaP)} or a PCB {PCB126 (3,3',4,4',5-pentachlorobiphenyl)}, respectively. Exposure to PAH or PCB affected OxPhos in the reference King's Creek population but had no detectable effects on the polluted Elizabeth River population. Thus, PAH exposure significantly increased LEAK, and exposure to PCB126 significantly decreased State 3, E state and complex I activity in the reference King's Creek population. These data strongly implicate an evolved tolerance in the Elizabeth River fish where dosed fish are not affected by PAH exposure and undosed fish show decreased LEAK and increased State 3 and E state., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Endocrine disrupting substances (EDSs) have the potential to disturb sensitive hormone pathways, particularly those involved in development and reproduction. Both fresh and estuarine water bodies receive inputs of EDSs from a variety of sources, including sewage effluent, industrial effluent and agricultural runoff. Based on current literature, freshwater species appear to respond to lower levels of EDSs than estuarine or marine species. Therefore, effects elicited by EDSs in freshwater teleosts may not be an accurate representation of how EDSs affect teleosts in estuarine and marine environments. To address this potential difference, a short-term reproductive bioassay was conducted under conditions of low and high salinity using mummichog (Fundulus heteroclitus), a euryhaline species that is native to the east coast of North America. The goals of this study were to determine the response of mummichog when exposed to an androgenic EDS and whether salinity affected the response. A model androgen, 5α-dihydrotestosterone (DHT), was selected for this experiment. Impacts on reproduction were evaluated at multiple biological levels, including physiological (sex steroid levels), organismal (gonad size and gonad morphology), and functional (egg production) endpoints. Under conditions of high salinity, egg production was significantly reduced at all exposure concentrations. Under conditions of low salinity, there were no significant differences based on DHT treatment; however, egg production in all treatment groups including the control were significantly reduced relative to the high salinity control group. Other reproductive endpoints, such as sex steroid production, showed stronger correlation to fecundity in females than males. This study demonstrates that mummichog fecundity is sensitive to androgenic endocrine disruption while also underscoring the importance of how changes in salinity, an environmental variable, can impact reproduction., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The osmorespiratory compromise, the trade-off between the requirements for respiratory and ionoregulatory homeostasis at the gills, becomes more intense during environmental hypoxia. One aspect that has been previously overlooked is possible change in transepithelial potential (TEP) caused by hypoxia, which will influence branchial ionic fluxes. Using the euryhaline killifish, we show that acute hypoxia reduces the TEP across the gills by approximately 10 mV in animals acclimated to both freshwater (FW) and seawater (SW), with a higher PO2  threshold in the former. TEP becomes negative in FW, and less positive in SW. The effects are immediate, stable for at least 3 h, and reverse immediately upon return to normoxia. Hypoxia also blocks the normal increase in TEP that occurs upon transfer from FW to SW, but does not reduce the fall in TEP that occurs with transfer in the opposite direction. These effects may be beneficial in FW but not in SW., (© 2015. Published by The Company of Biologists Ltd.)

Processes acting at the level of the mitochondria have been suggested to affect the thermal limits of organisms. To determine whether changes in mitochondrial properties could underlie shifts in thermal limits, we examined how mitochondrial properties are affected by thermal acclimation in the eurythermal killifish, Fundulus heteroclitus - a species with substantial plasticity in whole-organism thermal limits. We hypothesized that thermal acclimation would result in functional changes in the mitochondria that could result in trade-offs in function during acute thermal shifts. We measured the mitochondrial respiration rate (V̇O2 ) through multiple complexes of the electron transport system following thermal acclimation (to 5, 15, 33°C) and assessed maintenance of mitochondrial membrane potential (Δp) and rates of reactive oxygen species (ROS) production as an estimate of costs. Acclimation to 5°C resulted in a modest compensation of mitochondrial respiration at low temperatures, but these mitochondria were able to maintain Δp with acute exposure to high temperatures, and ROS production did not differ between acclimation groups, suggesting that these increases in mitochondrial capacity do not alter mitochondrial thermal sensitivity. Acclimation to 33°C suppressed mitochondrial respiration as a result of effects on NADH dehydrogenase (complex I). These high-temperature acclimated fish nonetheless maintained levels of Δp and ROS production similar to those of the other acclimation groups. This work demonstrates that killifish mitochondria can successfully acclimate to a wide range of temperatures without incurring major functional trade-offs during acute thermal shifts and that high-temperature acclimation results in a suppression of metabolism, consistent with patterns observed at the organismal level., (© 2015. Published by The Company of Biologists Ltd.)

Killifish (Fundulus heteroclitus) are euryhaline teleosts that are widely used in environmental and toxicological studies, and they are tolerant to arsenic, in part due to very low assimilation of arsenic from the environment. The mechanism of arsenic uptake by the intestine, a major route of arsenic uptake in humans is unknown. Thus, the goal of this study was to determine if aquaglyceroporins (AQPs), which transport water and other small molecules including arsenite across cell membranes, are expressed in the killifish intestine, and whether AQP expression is affected by osmotic stress. Through RT-PCR and sequence analysis of PCR amplicons, we demonstrated that the intestine expresses kfAQP3a and kfAQP3b, two previously identified variants, and also identified a novel variant of killifish AQP3 (kfAQP3c) in the intestine. The variants likely represent alternate splice forms. A BLAST search of the F. heteroclitus reference genome revealed that the AQP3 gene resides on a single locus, while an alignment of the AQP3 sequence among 384 individuals from eight population ranging from Rhode Island to North Carolina revealed that its coding sequence was remarkably conserved with no fixed polymorphism residing in the region that distinguishes these variants. We further demonstrate that the novel variant transports arsenite into HEK293T cells. Whereas kfAQP3a, which does not transport arsenite, was expressed in both freshwater (FW) and saltwater (SW) acclimated fish, kfAQP3b, an arsenic transporter, was expressed only in FW acclimated fish, and kfAQP3c was expressed only in SW acclimated fish. Thus, we have identified a novel, putative splice variant of kfAQP3, kfAQP3c, which transports arsenic and is expressed only in SW acclimated fish., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Many fish encounter hypoxia on a daily cycle, but the physiological effects of intermittent hypoxia are poorly understood. We investigated whether acclimation to constant (sustained) hypoxia or to intermittent diel cycles of nocturnal hypoxia (12 h normoxia:12 h hypoxia) had distinct effects on hypoxia tolerance or on several determinants of O2 transport and O2 utilization in estuarine killifish. Adult killifish were acclimated to normoxia, constant hypoxia, or intermittent hypoxia for 7 or 28 days in brackish water (4 ppt). Acclimation to both hypoxia patterns led to comparable reductions in critical O2 tension and resting O2 consumption rate, but only constant hypoxia reduced the O2 tension at loss of equilibrium. Constant (but not intermittent) hypoxia decreased filament length and the proportion of seawater-type mitochondrion-rich cells in the gills (which may reduce ion loss and the associated costs of active ion uptake), increased blood haemoglobin content, and reduced the abundance of oxidative fibres in the swimming muscle. In contrast, only intermittent hypoxia augmented the oxidative and gluconeogenic enzyme activities in the liver and increased the capillarity of glycolytic muscle, each of which should facilitate recovery between hypoxia bouts. Neither exposure pattern affected muscle myoglobin content or the activities of metabolic enzymes in the brain or heart, but intermittent hypoxia increased brain mass. We conclude that the pattern of hypoxia exposure has an important influence on the mechanisms of acclimation, and that the optimal strategies used to cope with intermittent hypoxia may be distinct from those for coping with constant hypoxia., (© 2015. Published by The Company of Biologists Ltd.)

The killifish Fundulus heteroclitus is an estuarine species with broad physiological plasticity, enabling acclimation to diverse stressors. Previous work suggests that freshwater populations expanded their physiology to accommodate low salinity environments; however, it is unknown whether this compromises their tolerance to high salinity. We used a comparative approach to investigate the mechanisms of a derived freshwater phenotype and the fate of an ancestral euryhaline phenotype after invasion of a freshwater environment. We compared physiological and transcriptomic responses to high- and low-salinity stress in fresh and brackish water populations and found an enhanced plasticity to low salinity in the freshwater population coupled with a reduced ability to acclimate to high salinity. Transcriptomic data identified genes with a conserved common response, a conserved salinity-dependent response and responses associated with population divergence. Conserved common acclimation responses revealed stress responses and alterations in cell-cycle regulation as important mechanisms in the general osmotic response. Salinity-specific responses included the regulation of genes involved in ion transport, intracellular calcium, energetic processes and cellular remodeling. Genes diverged between populations were primarily those showing salinity-specific expression and included those regulating polyamine homeostasis and the cell cycle. Additionally, when populations were matched with their native salinity, expression patterns were consistent with the concept of 'transcriptomic resilience', suggesting local adaptation. These findings provide insight into the fate of a plastic phenotype after a shift in environmental salinity and help to reveal mechanisms allowing for euryhalinity., (© 2015. Published by The Company of Biologists Ltd.)

After pregnant mummichog were implanted with 1-nitronaphthalene or 1-nitropyrene via cholesterol pellet, we investigated the effects of the chemicals on embryo normality, hatchability and days to hatch of normal embryos, and growth and survival of hatched larvae from normal embryos of the implanted mummichog. Hatchability was the parameter most sensitive to the effects of both 1-nitronaphthalene and 1-nitropyrene. The 4-week lowest-observed-effect concentration (LOEC) of 1-nitronaphthalene, based on the actual concentrations in the eggs in the test, was 447 ng g(-1) wet wt.; and the LOEC and no-observed-effect concentration (NOEC) of 1-nitropyrene were 958 and 344 ng g(-1) wet wt., respectively. The 4-week LOEC of 1-nitronaphthalene, based on the concentration in the water, was estimated at 4.8 µg L(-1) by using the reported bioconcentration factor; and the LOEC and NOEC of 1-nitropyrene, based on the concentration in the water, were estimated at 3.1 and 8.6 µg L(-1), respectively. The reported environmental concentrations of 1-nitropyrene and 1-nitronaphthalene are over three magnitudes lower than the toxicity values we obtained. Therefore, the effects of environmental levels of 1-nitropyrene and 1-nitronaphthalene on fish reproduction, not including genomic effects on embryos, appear to be almost negligible. However, DNA damage has been detected in marine organisms exposed to 1-nitropyrene. Further studies of the genotoxicity of nitrated polycyclic aromatic hydrocarbons at environmental levels are therefore needed to evaluate their ecotoxicological risks.

Reinforcement occurs when behavioral isolation is strengthened between species due to selection against hybridization in sympatry. Mate preferences and their target traits may change in sympatry as a consequence of reinforcement. This can potentially generate further behavioral isolation within species if sympatric populations evolve extreme preferences or traits that cause them to reject individuals from foreign populations as mates or be rejected as mates. This process is known as cascade reinforcement. We measured behavioral isolation between sympatric and allopatric populations of Lucania killifish to determine whether isolation evolves due to reinforcement between species and whether reinforcement affects preferences within species, consistent with the cascade reinforcement hypothesis. We measured mate preferences in both sexes between species (Lucania parva vs. Lucania goodei) and within species (among populations of L. parva). Between species, both male and female preferences for conspecifics were highest in sympatric populations. Within species, L. parva females from sympatric populations preferred their own native males over foreign males. Allopatric L. parva females and all L. parva males showed no preferences within species. Within species, behavioral isolation showed no association with ecological variables, such as salinity. Thus, reinforcement is a primary factor generating behavioral isolation in Lucania killifish, creating strong preferences in both sexes among species and leading to cascade reinforcement of female mate preference within species.

In freshwater (FW), many of the main mechanisms of copper (Cu) toxicity have been characterized; however, toxicity mechanisms in seawater (SW) are less well understood. We investigated the effects of salinity on Cu-induced oxidative stress and metabolic responses in adult killifish, Fundulus heteroclitus. We exposed FW and SW-acclimated killifish to either low Cu (LC, 50 μg/L) or high Cu (HC, 200 μg/L) for 96 h and compared them to controls (CTRL) under the same salinities without added Cu. Cu exerted minimal influence on tissue ion levels in either FW or SW. Salinity generally protected against Cu bioaccumulation in the gills and liver, but not in the carcass. Hematocrit (Hct) and hemoglobin (Hb) levels were increased by LC and HC in both FW and SW, and blood lactate was reduced in FW-killifish exposed to LC and HC. Rates of oxygen consumption were similar across treatments. Salinity reduced Cu load in gill, liver and intestine at LC but only in the gills at HC. In general, Cu increased gill, liver, and intestine catalase (CAT) activity, while superoxide dismutase (SOD) either decreased or remained unchanged depending on tissue-type. These changes did not directly correlate with levels of protein carbonyls, used as an index of oxidative stress. Cu-induced changes in carbohydrate metabolic enzymes were low across tissues and the effect of salinity was variable. Thus, while salinity clearly protects against Cu bioaccumulation in some tissues, it is unclear whether salinity protects against Cu-induced oxidative stress and metabolic responses., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Atlantic killifish (Fundulus heteroclitus) inhabiting the Atlantic Wood Superfund site on the Elizabeth River (Portsmouth, VA, USA) are exposed to a complex mixture of polycyclic aromatic hydrocarbons (PAHs) from former creosote operations, but are resistant to the acute toxicity and cardiac teratogenesis caused by PAHs. The resistance is associated with a dramatic recalcitrance to induction of cytochrome P450 (CYP1) metabolism enzymes following exposure to aryl hydrocarbon receptor (AHR) agonists, along with an elevated antioxidant response and increased expression of several other xenobiotic metabolism and excretion enzymes. However, the heritability of the resistance in the absence of chemical stressors has been inconsistently demonstrated. Understanding the heritability of this resistance will help clarify the nature of population-level responses to chronic exposure to PAH mixtures and aid in identifying the important mechanistic components of resistance to aryl hydrocarbons. We compared the response of Atlantic Wood F1 and F2 embryos to benzo[k]fluoranthene (BkF), benzo[a]pyrene (BaP), 3,3',4,4',5-pentachlorobiphenyl (PCB-126), and a mixture of BkF and fluoranthene (Fl) to that of F1 embryos of reference site killifish. Resistance to cardiac teratogenesis and induction of CYP mRNA expression and CYP activity was determined. We found that both Atlantic Wood F1 and F2 embryos were highly resistance to cardiac teratogenesis. However, the resistance by Atlantic Wood F2 embryos to induction of CYP mRNA expression and enzyme activity was intermediate between that of Atlantic Wood F1 embryos and reference embryos. These results suggest that resistance to cardiac teratogenesis in Atlantic Wood fish is conferred by multiple factors, not all of which appear to be fully genetically heritable.

The experimental integration of bioinspired robots in groups of social animals has become a valuable tool to understand the basis of social behavior and uncover the fundamental determinants of animal communication. In this study, we measured the preference of fertile female bluefin killifish (Lucania goodei) for robotic replicas whose aspect ratio, body size, motion pattern, and color morph were inspired by adult male killifish. The motion of the fish replica was controlled via a robotic platform, which simulated the typical courtship behavior observed in killifish males. The positional preferences of females were measured for three different color morphs (red, yellow, and blue). While variation in preference was high among females, females tend to spend more time in the vicinity of the yellow painted robot replicas. This preference may have emerged because the yellow robot replicas were very bright, particularly in the longer wavelengths (550–700 nm) compared to the red and blue replicas. These findings are in agreement with previous observations in mosquitofish and zebrafish on fish preference for artificially enhanced yellow pigmentation.

Freshwater and marine fishes exhibit a dichotomy in biosynthesis of long-chain polyunsaturated fatty acids (LC-PUFAs) from shorter-chain precursors. Marine species generally lack this ability due to deficiencies in requisite desaturase or elongase enzymes. Gulf killifish Fundulus grandis is a euryhaline cyprinodont whose fatty acid (FA) dynamics have not been previously examined. We utilized experimental variations in exogenous FAs available to spawning F. grandis in order to construct a multitissue evaluation of FA allocation and quantify effects on reproductive output and offspring fitness. No significant decrease in fecundity occurred in animals consuming low levels of LC-PUFAs, although embryo viability rates were affected. Maternal dietary FA variation did produce differences in starvation tolerance, hypoosmoregulatory ability, and acute thermal stress tolerance for larvae. These variations occurred at elevated salinities and temperatures seldom if ever encountered by F. grandis in culture or natural environments and are unlikely to affect larval survival in these settings. FA composition of eggs and tissues from spawning females suggested potential biosynthesis of LC-PUFAs from shorter-chain precursors in F. grandis. Results of this study suggest that F. grandis possesses physiological mechanisms allowing maintenance of reproductive function when subjected to dietary deficiencies in FAs generally considered essential for marine fishes.

5α-Dihydrotestosterone (DHT) is a potent androgen in mammals with multiple roles; however the physiological actions of DHT in male fishes are not well known. To address this knowledge gap, male mummichog (Fundulus heteroclitus) were continuously exposed to 0, 5, and 50 μg/L DHT for 21 days. Following exposure, testes were separated for histology, ex vivo incubation to measure steroidogenic capacity, and gene expression analyses (real-time PCR and microarray). DHT significantly decreased ex vivo 11-ketotestosterone (11KT) production in males exposed to 50 μg/L DHT but not 5 μg/L DHT, and DHT exposure did not affect ex vivo testosterone production. Histological examination revealed that the amount of interlobular and connective tissue present in the testes was increased in the 50 μg/L DHT treatment. Despite reductions in the production of 11KT, DHT did not affect the expression of targeted genes in the steroidogenic pathway such as steroidogenic acute regulatory protein (star), P450 side chain cleavage (cyp11a1) and 11β-hydroxysteroid dehydrogenase (hsd11b3). Microarray analysis in the testes of individuals from control and 50 μg/L DHT revealed that males exposed to 50 μg/L DHT showed regulated transcriptional sub-networks that were related to immunity, regulation of blood flow, lipids and xenobiotic clearance, suggesting that DHT may be involved in the physiological regulation of these processes in the fish testes. A second objective of this study was to determine the feasibility of measuring mRNA levels in tissues used for ex vivo steroid production by comparing RNA integrity and transcript levels in testes of both immediately flash frozen tissue and incubated tissue. There was no significant difference in RNA quality between the two time points, indicating RNA integrity can remain intact for at least 18 h in ex vivo assays, thereby providing a viable option for researchers assessing multi-level biological reproductive endpoints when limited tissue is available. While the gene expression levels of actb, efla, rps12, rps18, star, and hsd11b3 remained unchanged, esr2a (esrba), esr2b (esrbb) and cyp11a1 were significantly lower in incubated tissue compared to flash frozen tissue. Therefore caution must be used as the steady-state levels of select genes may change over time. This study improves our understanding of DHT action in the teleostean testis and generates new hypotheses regarding cell processes that are regulated by this underexplored and potent androgen., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The Houston Ship Channel (HSC), connecting Houston, Texas to Galveston Bay and ultimately the Gulf of Mexico, is heavily industrialized and includes several areas that have historically been identified as containing significant levels of mercury, dioxins, furans, polychlorinated biphenyls (PCBs), and polycyclic aromatic hydrocarbons (PAHs). Gulf killifish, Fundulus grandis, inhabit this entire estuarine system, including the most contaminated areas. F. grandis is the sister species of the well-established estuarine model organism Fundulus heteroclitus, for which heritable resistance to both PCB and PAH toxicity has been documented in several populations. F. grandis collected from two Superfund sites on the HSC and from a reference population were used to establish breeding colonies. F1 embryos from HSC populations were approximately 1000-fold more resistant to PCB126- and 2-5-fold more resistant to coal tar-induced cardiovascular teratogenesis, relative to embryos from the reference population. Reciprocal crosses between reference and contaminated populations exhibit an intermediate level of resistance, confirming that observed protection is genetic and biparentally inherited. Ethoxyresorufin-O-deethylase (EROD) data confirm a reduction in basal and induced cytochrome P4501A (CYP1A) activity in resistant populations of F. grandis. This result is consistent with responses previously described for resistant populations of F. heteroclitus, specifically a recalcitrant aryl hydrocarbon receptor (AHR) pathway. The decreased levels of cardiovascular teratogenesis, and decrease in CYP1A inducibility in response to PCB126 and a PAH mixture, suggest that HSC F. grandis populations have adapted to chronic contaminants exposures via a mechanism similar to that previously described for F. heteroclitus. To the best of our knowledge, this is the first documentation of evolved pollution resistance in F. grandis. Additionally, the mechanistic similarities between the population adaptation observed in this study and previous work in F. heteroclitus suggest that genetic variation predating the evolutionary divergence of these two species may best explain the apparent rapid parallel evolution of pollution resistance in genetically and geographically distinct species and populations., (Copyright © 2014 Elsevier B.V. All rights reserved.)

In ephemeral habitats, the same genotypes cope with unpredictable environmental conditions, favouring the evolution of developmental plasticity and alternative life-history strategies (ALHS). We tested the existence of intrapopulation ALHS in an annual killifish, Nothobranchius furzeri, inhabiting temporary pools. The pools are either primary (persisting throughout the whole rainy season) or secondary (refilled after desiccation of the initial pool), representing alternative niches. The unpredictable conditions led to the evolution of reproductive bet-hedging with asynchronous embryonic development. We used a common garden experiment to test whether the duration of embryonic period is associated with post-embryonic life-history traits. Fish with rapid embryonic development (secondary pool strategy, high risk of desiccation) produced phenotypes with more rapid life-history traits than fish with slow embryonic development (primary pool strategy). The fast fish were smaller at hatching but had larger yolk sac reserves. Their post-hatching growth was more rapid, and they matured earlier. Further, fast fish grew to a smaller body size and died earlier than slow fish. No differences in fecundity, propensity to mate or physiological ageing were found, demonstrating a combination of plastic responses and constraints. Such developmentally related within-population plasticity in life history is exceptional among vertebrates., (© 2014 The Authors. Journal of Evolutionary Biology © 2014 European Society For Evolutionary Biology.)

The 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxic equivalent quantity (TCDD-TEQ) approach was used successfully to predict lethal embryotoxicity in salmonids, but its applicability to sublethal effects of mixtures of organohalogenated compounds in other fish species is poorly known. The sublethal toxicity of two dioxin-like compounds (DLCs), 3,3',4,4'-tetrachlorobiphenyl (PCB77) and 2,3,4,7,8-pentachlorodibenzofuran (2,3,4,7,8-PnCDF), two non-dioxin-like (NDL) polychlorinated biphenyls (PCBs), 2,2',5,5'-tetrachlorobiphenyl (PCB52) and 2,3,3',4',6-pentachlorobiphenyl (PCB110), and of Aroclor 1254, a complex commercial mixture of PCBs, was assessed in Fundulus heteroclitus embryos exposed by intravitelline injection. At 16 days post-fertilization, the two DLCs and Aroclor 1254 altered prey capture ability in addition to inducing classical aryl hydrocarbon receptor-mediated responses: ethoxyresorufin-O-deethylase (EROD) induction, craniofacial deformities and reduction in body length. None of these responses was induced by the two NDL PCBs, at doses up to 5400 ng g(-1)wet weight. Dose-response curves for prey capture ability for the 2 DLCs tested were not parallel to that of TCDD, violating a fundamental assumption for relative potency (ReP) estimation. Dose-response curves for EROD induction were parallel for 2,3,4,7,8-PnCDF and TCDD, but the ReP of 2,3,4,7,8-PnCDF for F. heteroclitus was 5-fold higher than the World Health Organization (WHO) fish toxic equivalent factor (TEF) based on embryolethality in salmonids. The chemically derived TCDD-TEQs of Aroclor 1254, calculated using 3,3',4,4',5-pentachlorobiphenyl (PCB126) concentrations and it ReP for F. heteroclitus, overestimated its potency to induce EROD activity possibly due to antagonistic interactions among PCBs. This study highlights the limitations of using TEFs based on salmonid toxicity data alone for risk assessment to other fish species. There is a need to assess the variability of RePs of DLCs in different species for a variety of endpoints and to better understand interactions between DLCs and other toxic chemicals., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Continued development, use, and disposal of quantum dots (QDs) ensure their entrance into aquatic environments where they could pose a risk to biological organisms as whole nanoparticles or as degraded metal constituents. Reproductive Fundulus heteroclitus were fed a control diet with lecithin, diets containing 1 or 10 μg of lecithin-encapsulated CdSe/ZnS QD/day, or a diet containing 5.9 μg CdCl2/day for 85 days. Cadmium concentrations in liver, intestine, and eggs were quantified with inductively coupled plasma mass spectrometry. In fish fed 10 μg QD/day, QDs or their degradation products traversed the intestinal epithelia and accumulated in the liver. Less than 0.01% of the QD's cadmium was retained in the liver or intestinal tissues. This compares to 0.9% and 0.5% of the cadmium in the intestine and liver, respectively of fish fed a CdCl2 diet. Cadmium was also detected in the eggs from parents fed 10 μg QD/day. No significant changes in hepatic total glutathione, lipid peroxidation, or expression of genes involved in metal metabolism or oxidative stress were observed. While QDs in the diet are minimally bioavailable, unusual levels of vitellogenin transcription in male fish as well as declining fecundity require further investigation to determine if endocrine disruption is of environmental concern., (Copyright © 2013 Elsevier B.V. All rights reserved.)

This study simulated terrestrial incubation and measured rates of embryogenesis, nitrogen elimination, heart rate, lactate production, maximum length of time a hatch could be delayed and developmental responses of terrestrially incubated Gulf killifish Fundulus grandis embryos at temperatures ranging from 20 to 30° C. Temperature had a positive relationship with rate of embryogenesis, but a negative relationship with extent of extended incubation. The 30° C treatment reached embryonic maturity 6 days before the 20° C treatment. Embryos hatched between intervals of 240 and 336, 144 and 288, 96 and 240 and 96 and 192 h after reaching developmental maturity for the 20, 23, 26 and 30° C treatments. Significantly higher concentrations of total nitrogen, in the form of ammonia and urea, were recorded in the 20 and 30° C treatments. While temperature significantly influenced lactate and ATP concentrations, no significant influence of time of incubation was detected. Terrestrial embryos displayed an ability to develop quickly during embryogenesis and prolong incubation for an extended period of time after reaching embryonic maturity. This adaptation may be a life-history trait used to minimize asynchronous hatching, cannibalism and cohort size heterogeneity., (© 2014 The Fisheries Society of the British Isles.)

Active chloride secretion, measured as short-circuit current (Isc) in ionocytes of opercular epithelia (OE) in the eurythermic, euryoxic, and euryhaline killifish or mummichog (Fundulus heteroclitus) was studied in cold (5°C) and warm (20°C) acclimated fish to determine if homeoviscous adaptation aided chloride secretion in the cold. Isolated opercular epithelia were cooled from 30°C to 0.2°C for warm and cold acclimated fish; from 30 to 8°C, Isc decreased with Q10=1.68 for warm and Q10=1.56 for cold acclimated tissues. By Arrhenius plots, there is a critical temperature, 8°C, below which aerobic Isc decreased sharply (Q10=6.90 for warm and 4.23 for cold acclimated tissues), suggesting a shift in mitochondrial efficiency of oxidative phosphorylation. In anaerobic conditions (0.5mM NaCN; N2 saturation), chloride transport continued at a lower rate, and Isc decrease with cooling below 8°C was less pronounced (Q10=2.95 for warm and 3.08 for cold), suggesting a shift in transporter function in plasma membrane. Under anaerobic conditions, NaCl secretion at 20°C was reversibly inhibited by hypotonic shock, indicating normal regulation of transport. Chloride secretion in warm-acclimated fish was supported mostly (75% at 20°C) by aerobic metabolism, whereas that for cold-acclimated fish was lower (55% at 20°C), suggesting a greater reliance on anaerobic metabolism in the cold. Once acclimated to cold, ionocytes may be temporarily incapable of increasing their aerobic ATP supply, even when warmed to 30°C. In cold acclimated fish there was increased polyunsaturated fatty acid composition of gill epithelium (consistent with homeoviscous adaptation) and gill remodeling, wherein epithelial cells filled the interlamellar space (interlamellar cell mass, ILCM) by as much as 70%, thus increasing diffusion distance against passive ion gain. Most ionocytes in these thickened epithelial masses became taller, still connecting basal lamina with the environment, consistent with the continuing transport rates at low temperatures. Whereas the low aerobic scope of cold-acclimated fish and thickened gill epithelium is appropriate to winter inactivity, metabolic depression and anaerobiosis, the large aerobic scope of warm-acclimated fish favors active foraging at high temperatures., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: The non-migratory killifish Fundulus heteroclitus inhabits clean and polluted environments interspersed throughout its range along the Atlantic coast of North America. Several populations of this species have successfully adapted to environments contaminated with toxic aromatic hydrocarbon pollutants such as polychlorinated biphenyls (PCBs). Previous studies suggest that the mechanism of resistance to these and other "dioxin-like compounds" (DLCs) may involve reduced signaling through the aryl hydrocarbon receptor (AHR) pathway. Here we investigated gene diversity and evidence for positive selection at three AHR-related loci (AHR1, AHR2, AHRR) in F. heteroclitus by comparing alleles from seven locations ranging over 600 km along the northeastern US, including extremely polluted and reference estuaries, with a focus on New Bedford Harbor (MA, USA), a PCB Superfund site, and nearby reference sites., Results: We identified 98 single nucleotide polymorphisms within three AHR-related loci among all populations, including synonymous and nonsynonymous substitutions. Haplotype distributions were spatially segregated and F-statistics suggested strong population genetic structure at these loci, consistent with previous studies showing strong population genetic structure at other F. heteroclitus loci. Genetic diversity at these three loci was not significantly different in contaminated sites as compared to reference sites. However, for AHR2 the New Bedford Harbor population had significant FST values in comparison to the nearest reference populations. Tests for positive selection revealed ten nonsynonymous polymorphisms in AHR1 and four in AHR2. Four nonsynonymous SNPs in AHR1 and three in AHR2 showed large differences in base frequency between New Bedford Harbor and its reference site. Tests for isolation-by-distance revealed evidence for non-neutral change at the AHR2 locus., Conclusion: Together, these data suggest that F. heteroclitus populations in reference and polluted sites have similar genetic diversity, providing no evidence for strong genetic bottlenecks for populations in polluted locations. However, the data provide evidence for genetic differentiation among sites, selection at specific nucleotides in AHR1 and AHR2, and specific AHR2 SNPs and haplotypes that are associated with the PCB-resistant phenotype in the New Bedford Harbor population. The results suggest that AHRs, and especially AHR2, may be important, recurring targets for selection in local adaptation to dioxin-like aromatic hydrocarbon contaminants.

Adaptation to salinity affects species distributions, promotes speciation, and guides many evolutionary patterns in fishes. To uncover the basis of a complex trait like osmoregulation, genome-level analyses are sensible. We combine population genomic scans with genome expression profiling to discover candidate genes and pathways associated with divergence between osmotic environments. We compared transcriptome sequence divergence between multiple freshwater and saltwater populations of the rainwater killifish, Lucania parva. We also compared sequence divergence between L. parva and its sister species, Lucania goodei, a freshwater specialist. We found highly differentiated single nucleotide polymorphisms (SNPs) between freshwater and saltwater L. parva populations in cell junction and ion transport genes, including V-type H(+) ATPase. Between species, we found divergence in reproduction and osmotic stress genes. Genes that were differentially expressed between species during osmotic acclimation included genes involved in ion transport and cell volume regulation. Gene sets that were divergent in coding sequence and divergent in expression did not overlap, although they did converge in function. Like many studies using genomic scans, our approach may miss some loci that contribute to adaptation but have complicated patterns of allelic variation. Our study suggests that gene expression and coding sequence may evolve independently as populations adapt to a complex physiological challenge., (© 2013 The Author(s). Evolution © 2013 The Society for the Study of Evolution.)

Although the term environmental stress is used across multiple fields in biology, the inherent ambiguity associated with its definition has caused confusion when attempting to understand organismal responses to environmental change. Here I provide a brief summary of existing definitions of the term stress, and the related concepts of homeostasis and allostasis, and attempt to unify them to develop a general framework for understanding how organisms respond to environmental stressors. I suggest that viewing stressors as environmental changes that cause reductions in performance or fitness provides the broadest and most useful conception of the phenomenon of stress. I examine this framework in the context of animals that have evolved in highly variable environments, using the Atlantic killifish, Fundulus heteroclitus, as a case study. Consistent with the extreme environmental variation that they experience in their salt marsh habitats, killifish have substantial capacity for both short-term resistance and long-term plasticity in the face of changing temperature, salinity and oxygenation. There is inter-population variation in the sensitivity of killifish to environmental stressors, and in their ability to acclimate, suggesting that local adaptation can shape the stress response even in organisms that are broadly tolerant and highly plastic. Whole-organism differences between populations in stressor sensitivity and phenotypic plasticity are reflected at the biochemical and molecular levels in killifish, emphasizing the integrative nature of the response to environmental stressors. Examination of this empirical example highlights the utility of using an evolutionary perspective on stressors, stress and stress responses.

Arsenic is a contaminant of drinking water and has been correlated with adverse developmental outcomes such as low birth weight, reduced weight gain, and altered locomotor activity. Previous research has shown that killifish (Fundulus heteroclitus) exposed to high arsenic levels during embryogenesis had smaller muscle fiber diameters. The current study was designed to determine whether changes in muscle fibers persisted, were exacerbated, or resolved over time. Killifish embryos were exposed to 0-5 ppm arsenite and, upon hatching, were transferred into either clean water or continued receiving the same exposure to arsenic for up to 16 weeks. Arsenic significantly decreased the weight of both embryonic-only exposed juveniles and continuously exposed juveniles between 4 and 16 weeks of development at concentrations as low as 0.8 ppm. Although arsenite exposure increased the percentage of small diameter fibers during the early weeks, fiber diameters returned to control levels in the embryonic-only exposed fish. However, muscle fiber density was still reduced to 85.7%, 80.3%, and 73.8% of control for the 0.8, 2, and 5 ppm embryonic-only exposure groups, respectively, even after 16 weeks of development. These results indicate that while continuous exposure to arsenic may alter the size of muscle fibers, embryonic-only exposure to arsenic has lasting effects on the number of muscle fibers formed., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Atlantic killifish (Fundulus heteroclitus) inhabiting the Atlantic Wood Industries Superfund Site (Elizabeth River, Portsmouth, VA, USA) are resistant to the acute toxicity and cardiac teratogenesis caused by high levels of polycyclic aromatic hydrocarbons (PAHs) from creosote. The resistance is linked to down regulation of the aryl hydrocarbon receptor (AHR) pathway. We investigated the association between CYP1 activity, as a marker of potential AHR pathway suppression, and contaminant resistance in killifish subpopulations from sites throughout the estuary that varied significantly in PAH contamination level. Adult killifish and sediments were collected from seven sites across approximately 13.7 km in river length within the estuary and from a nearby reference site. Sediment PAH levels were determined using gas chromatography mass spectrometry. Embryos obtained via manual spawning were exposed to individual AHR agonists and PAH mixtures 24 h post fertilization (hpf); CYP1 activity was determined by in ovo ethoxyresorufin-o-deethylase (EROD) at 96 hpf, and cardiac deformity severity was scored at 144 hpf. The total PAH levels measured among the sites varied from approximately 200 to 125,000 ng/g dry sediment. Overall, the resistance to teratogenesis was strongest in the subpopulations from sites in or closest to the major PAH contamination sites, but even embryos from less-contaminated sites within the Elizabeth River demonstrated at least partial resistance to many challenges. Surprisingly, all of the subpopulations tested were highly resistant to PCB-126 (3,3',4,4',5-pentachlorobiphenyl). However, the degree of CYP1 activity response varied significantly among subpopulations and did not always correlate strongly with resistance to teratogenesis; some subpopulations resisted the cardiac teratogenesis caused by the challenges at doses that still elicited strong EROD induction. Our results suggest that there is variation in the adaptive phenotype exhibited by laboratory-spawned embryos from killifish subpopulations throughout the estuary. Furthermore, the results show that contaminants have affected killifish subpopulations throughout the estuary, even in sites with lower levels of PAHs.

Many animals show lateral bias in various behaviors. In fish, behavioral laterality has been studied from the perspective of its relation with brain lateralization. On the other hands, in some fishes, head dimorphism (righty or lefty) corresponds to behavioral laterality, such as foraging behavior. To examine the correlation between morphological asymmetry and behavioral laterality associated with brain lateralization, we conducted two behavioral tests (the detour test and fast-start test) using a poeciliid fish, Girardinus metallicus. In both behavioral tests, some individuals tended to move rightward, while others tended to move leftward, in a manner associated with head incline. In the detour-test, righty individuals primarily detoured leftward, whereas lefty individuals primarily detoured rightward. In the fast-start tests, the reverse tendency was seen; righty individuals tended to escape rightward, whereas lefty individuals tended to escape leftward. Such results indicate that brain lateralization may be also associated with morphological asymmetry.

Environmental salinity presents a key barrier to dispersal for most aquatic organisms, and adaptation to alternate osmotic environments likely enables species diversification. Little is known of the functional basis for derived tolerance to environmental salinity. We integrate comparative physiology and functional genomics to explore the mechanistic underpinnings of evolved variation in osmotic plasticity within and among two species of killifish; Fundulus majalis harbours the ancestral mainly salt-tolerant phenotype, whereas Fundulus heteroclitus harbours a derived physiology that retains extreme salt tolerance but with expanded osmotic plasticity towards the freshwater end of the osmotic continuum. Common-garden comparative hypo-osmotic challenge experiments show that F. heteroclitus is capable of remodelling gill epithelia more quickly and at more extreme osmotic challenge than F. majalis. We detect an unusual pattern of baseline transcriptome divergence, where neutral evolutionary processes appear to govern expression divergence within species, but patterns of divergence for these genes between species do not follow neutral expectations. During acclimation, genome expression profiling identifies mechanisms of acclimation-associated response that are conserved within the genus including regulation of paracellular permeability. In contrast, several responses vary among species including those putatively associated with cell volume regulation, and these same mechanisms are targets for adaptive physiological divergence along osmotic gradients within F. heteroclitus. As such, the genomic and physiological mechanisms that are associated with adaptive fine-tuning within species also contribute to macro-evolutionary divergence as species diversify across osmotic niches., (© 2013 John Wiley & Sons Ltd.)

Estuarine eutrophication as a result of agricultural land use, including the use of chemical fertilizers, is increasing worldwide. Prince Edward Island (PEI), Canada has very high agricultural intensity by international standards with approximately 44% of the land area under production, and some watersheds in excess of 75% agricultural land-use. The type of agriculture is also intensive with primarily row crops that have high chemical fertilizer and pesticide usage. In light of these stressors, the hypothesis of this study was that mummichog (Fundulus heteroclitus) population parameters would change with point and nonpoint source pollution, and that multivariate statistics could be used to draw associations with specific stressors. Fish were sampled on a monthly basis from May through August at 7 estuaries spanning a range of land use, nutrient, and contaminant loadings. A suite of environmental variables were simplified into 3 principal components: PC1 representing agricultural land use, N loading, and plant habitat, PC2 being dominated by sediment sand and silt distribution, and PC3 largely reflecting P loading and sediment organic matter. There were significant differences in abundance of both adult and young-of-the-year mummichog, and these changes associated most strongly with PC1, the largely N-driven agricultural influences. In contrast, somatic variables such as liver and gonad size did not show strong association with the environmental quality principal component scores. The sand and silt PC2 appeared to have the opposite association with the biological data, with siltier environments correlating to older, larger, less dense populations of mummichog. Although pesticide residues were detected in estuarine sediment, there was no clear relationship between these and watershed agricultural intensity or biochemical indicators. There was, however, a strong relationship between agricultural environmental variables (PC1) and in vitro steroid production that is suggestive of a potential chemical effect. Eutrophication appeared to be a primary stressor affecting mummichog populations, as nutrient enrichment was associated with changes in habitat variables and these in turn were associated with high mummichog density. Thus, mummichog population demographics appear to have use as an indicator of adverse or worsening conditions in estuaries. We concluded that, based on the subset of environmental factors evaluated, the nonpoint-source inputs of sediments and nutrients exerted the greatest influence on mummichog populations in PEI estuaries., (Copyright © 2013 SETAC.)

Nickel (Ni) is a common pollutant found in aquatic environments and may be harmful at elevated concentrations. Increasing salinity has been shown to decrease the bioavailability and toxicity of other metals to aquatic organisms. In the present study, acute Ni toxicity experiments (96-h) were conducted at various salinities (0-36 ppt) to determine the effects of salinity on Ni toxicity to 2 euryhaline fish species, Kryptolebias marmoratus and Fundulus heteroclitus. Nickel concentrations causing lethality to 50% of the fish ranged from 2 mg/L in moderately hard freshwater to 66.6 mg/L in 36 ppt saltwater. Nickel toxicity to F. heteroclitus decreased linearly with increasing salinity; however, Ni toxicity to K. marmoratus was only lowered by salinities above 6 ppt, demonstrating potential physiological differences between the 2 species when they are functioning as freshwater fish. Furthermore, the authors investigated the influence of Mg(2+) , Ca(2+) , Na(+) , and Cl(-) on Ni toxicity to F. heteroclitus. Freshwater with up to 120 mg/L Ca(2+) as CaSO4 , 250 mg/L Mg(2+) as MgSO4 , or 250 mg/L Na(+) as NaHCO3 did not provide protection against Ni toxicity. Alternatively, 250 mg/L Na(+) , as NaCl, was protective against Ni toxicity; and the extent of protection was similar to that demonstrated from salt water with the same Cl(-) concentration. These results suggest that Cl(-) is the predominant ion responsible for reducing Ni toxicity to K. marmoratus and F. heteroclitus in higher salinity waters., (Copyright © 2013 SETAC.)

Density-dependent selection is one of earliest topics of joint interest to both ecologists and evolutionary biologists and thus occupies an important position in the histories of these disciplines. This joint interest is driven by the fact that density-dependent selection is the simplest form of feedback between an ecological effect of an organism's own making (crowding due to sustained population growth) and the selective response to the resulting conditions. This makes density-dependent selection perhaps the simplest process through which we see the full reciprocity between ecology and evolution. In this article, we begin by tracing the history of studying the reciprocity between ecology and evolution, which we see as combining the questions of evolutionary ecology with the assumptions and approaches of ecological genetics. In particular, density-dependent fitness and density-dependent selection were critical concepts underlying ideas about adaptation to biotic selection pressures and the coadaptation of interacting species. However, theory points to a critical distinction between density-dependent fitness and density-dependent selection in their influences on complex evolutionary and ecological interactions among coexisting species. Although density-dependent fitness is manifestly evident in empirical studies, evidence of density-dependent selection is much less common. This leads to the larger question of how prevalent and important density-dependent selection might really be. Life-history variation in the least killifish Heterandria formosa appears to reflect the action of density-dependent selection, and yet compelling evidence is elusive, even in this well-studied system, which suggests some important challenges for understanding density-driven feedbacks between ecology and evolution.

Fundulus heteroclitus preference for association with familiar conspecifics of similar body length was impacted by benzyl butyl phthalate (BBP); this was found to be a statically significant result with a p < 0.0001. When presented with equally sized shoals consisting of either large or small fish, the majority of unexposed (84%) and acetone exposed control (82%) fish selected the shoal of large fish. A small number of control fish chose either the shoal of small fish (6% and 10%) or the neutral zone (10% and 8%) where they were clear morphological outliers. Fish exposed to 0.1 mg/L BBP exposure daily for four weeks selected the shoal of small fish more often than unexposed or acetone controls (7.5- and 4.5-fold respectively). They also remained in the neutral zone and displayed agitation at levels more than twice that of control. Agitation and shoal choice disruption are quantifiable behavioral responses that support the use of F. heteroclitus as a model for detecting sub-lethal BBP exposure., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Dioxin-like compounds (DLC) induce toxic responses in early life stages of fish through activation of the aryl hydrocarbon receptor (AhR) which is frequently assessed by ethoxyresorufin-O-deethylase (EROD) activity. A novel spectrofluorimetric method was developed to quantitatively assess EROD activity in individual living embryos and prolarvae of a marine model fish species, the mummichog Fundulus heteroclitus. This in vivo method is based on the measurement of the production of resorufin by single live embryos or prolarvae after 5 h incubation with ethoxyresorufin. Freshly fertilized eggs were treated topically from 2.5 to 50 pg egg⁻¹, with 3,3',4,4',5-pentachlorobiphenyl (PCB126), a prototypical DLC. EROD activity was assessed in embryos (7 days post-fertilization) and prolarvae (16 days post-fertilization). Resorufin was measured both in the culture medium (25‰ seawater) and in whole fish homogenates, to assess the percentage retained in the body. Approximately 95% and 17% of the resorufin produced in vivo was retained in embryos and prolarvae respectively. EROD activity in homogenates of embryos and in the culture medium of prolarvae increased linearly with dose. EROD activity measured by the in vivo method was highly correlated to that measured by a traditional in vitro technique using S9 fractions for both embryos and prolarvae. Both in vivo and in vitro EROD activity were higher in prolarvae than in embryos pretreated with PCB126. EROD induction measured in prolarvae by the in vivo and in vitro methods were similar whereas higher induction was measured in vivo than in vitro in embryos. The in vivo method was more sensitive and as reliable as the in vitro technique, and required a lower number of fish (4 compared to 3 pools of 5). This in vivo method is useful to link EROD induction in individual embryos or prolarvae to other organism-level responses. Further studies with other categories of xenobiotics should be performed to assess potential toxic effects on resorufin absorption/excretion processes which could affect in vivo measurements., (Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.)

The biological consequences of the Deepwater Horizon oil spill are unknown, especially for resident organisms. Here, we report results from a field study tracking the effects of contaminating oil across space and time in resident killifish during the first 4 mo of the spill event. Remote sensing and analytical chemistry identified exposures, which were linked to effects in fish characterized by genome expression and associated gill immunohistochemistry, despite very low concentrations of hydrocarbons remaining in water and tissues. Divergence in genome expression coincides with contaminating oil and is consistent with genome responses that are predictive of exposure to hydrocarbon-like chemicals and indicative of physiological and reproductive impairment. Oil-contaminated waters are also associated with aberrant protein expression in gill tissues of larval and adult fish. These data suggest that heavily weathered crude oil from the spill imparts significant biological impacts in sensitive Louisiana marshes, some of which remain for over 2 mo following initial exposures.

Embryos of the annual killifish Austrofundulus limnaeus can survive for months in the complete absence of oxygen. Survival of anoxia is associated with entry into a state of metabolic dormancy known as diapause. However, extreme tolerance of anoxia is retained for several days of post-diapause development. Rates of heat dissipation in diapause II and 4 days post-diapause II embryos were measured under aerobic conditions and during the transition into anoxia. Phosphorylated adenylate compounds were quantified in embryos during entry into anoxia and after 12 hr of aerobic recovery. Rates of heat dissipation were not affected by exposure to anoxia in diapause II embryos, while post-diapause II embryos experienced a profound decrease in heat dissipation. ATP decreased substantially in both developmental stages upon exposure to anoxia, and all indicators of cellular energetic status indicated energetic stress, at least based on the mammalian paradigm. The rate of decline in ATP is the most acute reported for any vertebrate. The mechanisms responsible for cellular survival despite a clear dysregulation between energy production and energy consumption remain to be identified. Necrotic and apoptotic cell death in response to hypoxia contribute to poor survival during many diseases and pathological conditions in mammals. Understanding the mechanisms that are in place to prevent maladaptive cell death in embryos of A. limnaeus may greatly improve treatment strategies in diseases that involve hypoxia and reperfusion injuries., (2012 Wiley Periodicals, Inc)

What is the effect of a variable environment on phenotypic variation? Does the physiological response to a new environment increase or decrease the differences among individuals? We provide a speculative hypothesis suggesting that the induction of a physiological response to environmental change minimizes phenotypic differences among individuals in outbred genetically variable populations. Although this suggestion runs counter to the general idea that environmental variation induces phenotypic variation, we provide evidence that this is not always the case. One explanation for this counterintuitive hypothesis is that in a variable environment, the physiological mechanism that maintains homeostasis changes the concentrations of active transcription factors (TFs). This change in TFs reduces the effectiveness of nucleotide polymorphisms in TF binding sites and thus reduces the variation among individuals in mRNA expression and in the phenotypes affected by these mRNA transcripts. Thus, there are fewer differences among individuals in a variable environment compared with the variation observed in a constant environment. Our conjecture is that the physiological mechanisms that maintain homeostasis in response to environmental variation canalize phenotypic variation. If our hypothesis is correct, then the physiological canalization of gene expression in a variable environment hides genetic variation and thereby reduces the evolutionary costs of polymorphism. This hypothesis provides a new perspective on the mechanisms by which high levels of genetic variation can persist in real-world populations.