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The maximum rate at which fish can take up oxygen from their environment to fuel aerobic metabolism is an important feature of their physiology and ecology. Methods to quantify maximum oxygen uptake rate (ṀO2), therefore, should reliably and reproducibly estimate the highest possible ṀO2 by an individual or species under a given set of conditions (peak ṀO2). This study determined peak ṀO2 and its repeatability in Gulf killifish, Fundulus grandis, subjected to three methods to elevate metabolism: swimming at increasing water speeds, during recovery after an exhaustive chase, and after ingestion of a large meal. Estimates of peak ṀO2 during swimming and after an exhaustive chase were repeatable across two trials, whereas peak ṀO2 after feeding was not. Peak ṀO2 determined by the three methods was significantly different from one another, being highest during swimming, lowest after an exhaustive chase, and intermediate after feeding. In addition, peak ṀO2 during recovery from an exhaustive chase depended on the length of time of recovery: in nearly 60% of the trials, values within the first hour of the chase were lower than those measured later. A novel and important finding was that an individual's peak ṀO2 was not repeatable when compared across methods. Therefore, the peak ṀO2 estimated for a group of fish, as well as the ranking of individual ṀO2 within that group, depends on the method used to elevate aerobic metabolism. [ABSTRACT FROM AUTHOR]

The capacity of fishes to tolerate low oxygen (hypoxia) through behavioral and physiological adjustments varies among species in a fashion that correlates with oxygen availability in their natural habitats. Less is known about variation in hypoxia tolerance within a species, but it is expressly this interindividual variation that will determine which individuals will survive during severe hypoxia. Here, we measured aquatic surface respiration (ASR) and loss of equilibrium (LOE), two common indexes of hypoxia tolerance of fishes, in gulf killifish, Fundulus grandis, subjected to multiple trials of a highly reproducible hypoxia protocol over a period of 6–8 wk. The time and PO2 at the first occurrence of ASR and the time and PO2 at LOE differed significantly among individuals in a repeatable fashion. This interindividual variation in ASR and LOE was significantly correlated with general body and gill morphology. The time to ASR was shorter and the PO2 at ASR was higher for fish with greater mass. After correcting for mass, fish with longer or more numerous gill filaments had longer times to ASR or LOE, respectively. Fish in better condition factor (heavier for their length) had lower PO2 at LOE. Repeatable interindividual variation in hypoxia tolerance, if genetically based, could influence the capacity of species to adapt as their habitats become increasingly threatened by aquatic hypoxia. [ABSTRACT FROM AUTHOR]

Abstract: Previous studies have suggested that the lactate dehydrogenase-B gene (Ldh-B) of the Atlantic killifish, Fundulus heteroclitus, is a hypoxia-responsive gene. Here, we demonstrate that the F. heteroclitus Ldh-B promoter confers hypoxia-dependence upon reporter gene expression in transiently transfected mammalian (Hep3B) and fish (RTG-2 and RTH-149) cells in culture. Mutation and deletion analyses identified a putative hypoxia-response element (HRE) between 109 and 90 nucleotides upstream of the major start site. This HRE is characterized by the sequence 5′-GATGTG-3′ spaced by 8 nucleotides from a perfect inverted repeat, and both sites are necessary for hypoxic induction of reporter gene expression in mammalian and fish cells. This HRE differs from the canonical sequence at one nucleotide position that is invariant among HREs from a wide range of hypoxia-sensitive genes. In fish cells, maximal induction of reporter gene expression driven by this HRE occurred at the lowest oxygen level tested (0.5%), took 48 h to 96 h, and was independent of glucose concentration (between 5.6 and 25 mM). Under all conditions tested, hypoxic induction of gene expression was lower in RTH-149 cells than in RTG-2, suggesting a potential defect in hypoxia signaling in RTH-149 cells. These results demonstrate that the F. heteroclitus Ldh-B promoter contains a novel HRE that is capable of driving reporter gene expression in a sequence-specific and oxygen-, time-, and cell line-dependent manner. [Copyright &y& Elsevier]

The role of oxygen in regulating patterns of gene expression in mammalian development, physiology, and pathology has received increasing attention, especially after the discovery of the hypoxia-inducible factor (HIF), a transcription factor that has been likened to a ‘master switch’ in the transcriptional response of mammalian cells and tissues to low oxygen. At present, considerably less is known about the molecular responses of nonmammalian vertebrates and invertebrates to hypoxic exposure. Because many animals live in aquatic habitats that are variable in oxygen tension, it is relevant to study oxygen-dependent gene expression in these animals. The purpose of this review is to discuss hypoxia-induced gene expression in fishes from an evolutionary and ecological context. Recent studies have described homologs of HIF in fish and have begun to evaluate their function. A number of physiological processes are known to be altered by hypoxic exposure of fish, although the evidence linking them to HIF is less well developed. The diversity of fish presents many opportunities to evaluate if inter- and intraspecific variation in HIF structure and function correlate with hypoxia tolerance. Furthermore, as an aquatic group, fish offer the opportunity to examine the interactions between hypoxia and other stressors, including pollutants, common in aquatic environments. It is possible, if not likely, that results obtained by studying the molecular responses of fish to hypoxia will find parallels in the oxygen-dependent responses of mammals, including humans. Moreover, novel responses to hypoxia could be discovered through studies of this diverse and species-rich group. [ABSTRACT FROM AUTHOR]

Describes the presence of DNA elements in intron 2 of the lactate dehydrogenase-B gene from Fundulus (F.) heteroclitus that resemble hypoxia response elements described for mammalian genes. Identification of two consensus hypoxia-inducible transcription factor-1 binding sites; Targets of hypoxia-dependent gene regulation in the killifish, F. heteroclitus; Information on hypoxia response elements that have been characterized in mammalian genes.

Low dissolved oxygen (hypoxia) is recognized as a major threat to aquatic ecosystems worldwide. Because oxygen is paramount for the energy metabolism of animals, understanding the functional and genetic drivers of whole-animal hypoxia tolerance is critical to predicting the impacts of aquatic hypoxia. In this study, we investigate the molecular evolution of key genes involved in the detection of and response to hypoxia in ray-finned fishes: the prolyl hydroxylase domain (PHD)–hypoxia-inducible factor (HIF) oxygen-sensing system, also known as the EGLN (egg-laying nine)– HIF oxygen-sensing system. We searched fish genomes for HIFA and EGLN genes, discovered new paralogs from both gene families, and analyzed protein-coding sites under positive selection. The physicochemical properties of these positively selected amino acid sites were summarized using linear discriminants for each gene. We employed phylogenetic generalized least squares to assess the relationship between these linear discriminants for each HIFA and EGLN and hypoxia tolerance as reflected by the critical oxygen tension (P crit) of the corresponding species. Our results demonstrate that P crit in ray-finned fishes correlates with the physicochemical variation of positively selected sites in specific HIFA and EGLN genes. For HIF2A , two linear discriminants captured more than 90% of the physicochemical variation of these sites and explained between 20% and 39% of the variation in P crit. Thus, variation in HIF2A among fishes may contribute to their capacity to cope with aquatic hypoxia, similar to its proposed role in conferring tolerance to high-altitude hypoxia in certain lineages of terrestrial vertebrates. [ABSTRACT FROM AUTHOR]

As aquatic hypoxia worsens on a global scale, fishes will become increasingly challenged by low oxygen, and understanding the molecular basis of their response to hypoxia may help to better define the capacity of fishes to cope with this challenge. The hypoxia inducible factor (HIF) plays a critical role in the molecular response to hypoxia by activating the transcription of genes that serve to improve oxygen delivery to the tissues or enhance the capacity of tissues to function at low oxygen. The current study examines the molecular evolution of genes encoding the oxygen-dependent HIFα subunit (HIFA) in the ray-finned fishes (Actinopterygii). Genomic analyses demonstrate that several lineages retain four paralogs of HIFA predicted from two rounds of genome duplication at the base of vertebrate evolution, broaden the known distribution of teleost-specific HIFA paralogs, and provide evidence for salmonid-specific HIFA duplicates. Evolution of the HIFA gene family is characterized by widespread episodic positive selection at amino acid sites that potentially mediate protein stability, protein–protein interactions, and transcriptional regulation. HIFA transcript abundance depends upon paralog, tissue, and fish lineage. A phylogenetically-informed gene nomenclature is proposed along with avenues for future research on this critical family of transcription factors. [ABSTRACT FROM AUTHOR]

Abstract: In this study, patterns of protein expression in zebrafish (Danio rerio) white skeletal muscle after 48h exposure to hypoxia (PO2 =1.9 kPa) or normoxia (PO2 =18.6 kPa) were evaluated using two-dimensional fluorescence difference gel electrophoresis (2D-DIGE). Proteins were separated over two pH ranges in the first dimension (pH 4–7 and pH 7–11) prior to separation in the second dimension, resolving a total of 821 protein spots. Of these, 77 spots (9.4%) differed between hypoxia and normoxia (p ≤0.01), with approximately twice as many proteins being higher during hypoxia (56) compared to the number found to be higher in normoxic fish (26). Thirty-one protein spots were identified by MALDI-TOF/TOF mass spectrometry. The expression of several glycolytic enzymes was greater in hypoxia than in normoxia, whereas enzymes associated with mitochondrial ATP synthesis were lower during hypoxia. Among the more highly up-regulated proteins during hypoxia were two variants of hemoglobin α subunit. These patterns of protein expression are consistent with a hypoxic response that enhances anaerobic metabolism and O2 transport to tissues, with a concomitant suppression of mitochondrial metabolism. These proteomic changes may contribute to the acclimation of zebrafish to hypoxia, thereby increasing their tolerance of low oxygen concentrations. [Copyright &y& Elsevier]

We examined whether western fence lizards Sceloporus occidentalis occurring in thermally divergent environments display differential responses to high temperature in locomotor performance and heat-shock protein (Hsp) expression. We measured maximum sprint speed in S. occidentalis from four populations at paired latitudes and elevations before and after exposure to an experimental heat treatment and then quantified hind-limb muscle Hsp70 expression. Lizards collected from northern or high-elevation collection sites suffered a greater reduction in sprint speed after heat exposure than lizards collected from southern or low-elevation sites. In addition, lizards from northern collection sites also exhibited an increase in Hsp70 expression after heat exposure, whereas there was no effect of heat exposure on Hsp70 expression in lizards from southern collection sites. Across all groups, there was a negative relationship between Hsp70 expression and sprint speed after thermal stress. This result is significant because (a) it suggests that an increase in Hsp70 alone cannot compensate for the immediate negative effects of high-temperature exposure on sprint speed and (b) it demonstrates a novel correlation between an emergent property at the intersection of several physiological systems (locomotion) and a cellular response (Hsp70 expression). Ultimately, geographic variation in the effects of heat on sprint speed may translate into differential fitness and population viability during future increases in global air temperatures. [ABSTRACT FROM AUTHOR]

The effects of passive integrated transponder (PIT) tagging on cortisol release, standard metabolic rate (SMR) and daily specific growth rate (GS) were evaluated in the Gulf killifish, Fundulus grandis, a small estuarine fish native to the Gulf of Mexico. Cortisol release by individual fish was measured non‐invasively prior to PIT tagging, immediately after tagging and once per week for 1 month following tagging. Within the first 2 h of tagging, cortisol release rates were significantly elevated compared with values measured prior to tagging and significantly higher than that of fish handled identically except not implanted with PIT tags. By 1 week after PIT tagging, cortisol release rates returned to control levels. SMR, determined by intermittent‐flow respirometry and GS, defined as per cent change in body mass per day, were measured prior to PIT tagging and weekly for 1 month after tagging. Neither SMR nor GS was significantly different in tagged v. untagged fish for the duration of the study. One month after tagging, haematocrit, plasma cortisol, blood glucose and blood lactate did not differ between tagged and untagged individuals. Therefore, after a transient stress response that subsides within 1 week, PIT tagging had no significant effects on these physiological variables in F. grandis, validating its use as a method of marking this and other small fishes. [ABSTRACT FROM AUTHOR]

The hypoxia-inducible factor (HIF) family of transcription factors plays central roles in the development, physiology, pathology, and environmental adaptation of animals. Because many aquatic habitats are characterized by episodes of low dissolved oxygen, fish represent ideal models to study the roles of HIF in the response to aquatic hypoxia. The estuarine fish Fundulus heteroclitus is found in habitats prone to hypoxia. It responds to low oxygen via behavioral, physiological, and molecular changes, and one member of the HIF family, HIF2α, has been previously described. Herein, cDNA sequencing, phylogenetic analyses, and genomic approaches were used to determine other members of the HIFα family from F. heteroclitus and their relationships to HIFα subunits from other vertebrates. In vitro and cellular approaches demonstrated that full-length forms of HIF1α, HIF2α, and HIF3α independently formed complexes with the β-subunit, aryl hydrocarbon receptor nuclear translocator, to bind to hypoxia response elements and activate reporter gene expression. Quantitative PCR showed that HIFα mRNA abundance varied among organs of normoxic fish in an isoform-specific fashion. Analysis of the F. heteroclitus genome revealed a locus encoding a second HIF2α-HIF2αb-a predicted protein lacking oxygen sensing and transactivation domains. Finally, sequence analyses demonstrated polymorphism in the coding sequence of each F. heteroclitus HIFα subunit, suggesting that genetic variation in these transcription factors may play a role in the variation in hypoxia responses among individuals or populations. [ABSTRACT FROM AUTHOR]

The Gulf killifish, Fundulus grandis, is a small teleost fish that inhabits marshes of the Gulf of Mexico and demonstrates high tolerance of environmental variation, making it an excellent subject for the study of physiological and molecular adaptations to environmental stress. In the present study, two-dimensional (2D) gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry were used to resolve and identify proteins from five tissues: skeletal muscle, liver, brain, heart, and gill. Of 864 protein features excised from 2D gels, 424 proteins were identified, corresponding to a 49% identification rate. For any given tissue, several protein features were identified as the same protein, resulting in a total of 254 nonredundant proteins. These nonredundant proteins were categorized into a total of 11 molecular functions, including catalytic activity, structural molecule, binding, and transport. In all tissues, catalytic activity and binding were the most highly represented molecular functions. Comparing across the tissues, proteome coverage was lowest in skeletal muscle, due to a combination of a low number of gel spots excised for analysis and a high redundancy of identifications among these spots. Nevertheless, the identification of a substantial number of proteins with high statistical confidence from other tissues suggests that F. grandis may serve as a model fish for future studies of environmental proteomics and ultimately help to elucidate proteomic responses of fish and other vertebrates to environmental stress. [ABSTRACT FROM AUTHOR]

When Hurricane Katrina struck the U.S. Gulf Coast, levees surrounding New Orleans, Louisiana, USA, were breached, leading to widespread flooding of the city and potential contamination from industrial spills, residential sources, and redistribution of pre-existing pollutants. We chemically characterized sediment samples from five New Orleans locations and used early development and mutagenesis in Japanese medaka (Oryzias latipes) as metrics of the toxic effects of these sediments. Sediment samples were analyzed for organohalogen pesticides, polycyclic aromatic hydrocarbons, polychlorinated biphenyls, and metals. One month after Hurricane Katrina, four of the five sites had unsafe concentrations of arsenic and one or more pesticides, pesticide metabolites, or polycyclic aromatic hydrocarbons. Medaka embryonic mortality and time to hatching both increased during exposure to aqueous extracts of sediments, with the greatest toxicity observed for the most heavily contaminated sediment. Exposure to sediment extracts did not, however, result in significantly elevated rates of mutagenesis. When the most contaminated site was resampled 4.5 months later, the sediment had lower contaminant concentrations and fewer deleterious effects on medaka development. Using the medaka bioassay, therefore, we demonstrate toxic effects of post-Hurricane Katrina sediments immediately following the storm, with some amelioration over time of contaminant concentrations and their negative biological effects. [ABSTRACT FROM AUTHOR]

Reports of anticancer and immunosuppressive properties have spurred recent interest in the bacterially produced prodiginines. We use electrospray tandem mass spectrometry (ES-MS/MS) to investigate prodigiosin, undecylprodiginine, and streptorubin B (butyl-meta-cycloheptylprodiginine) and to explore their fragmentation pathways to explain the unusual methyl radical loss and consecutive fragment ions that dominate low-energy collision-induced dissociation (CID) mass spectra. The competition between the formation of even-electron ions and radical ions is examined in detail. Theoretical calculations are used to optimize the structures and calculate the energies of both reactants and products using the Gaussian 03 program. Results indicate that protonation occurs on the nitrogen atom that initially held no hydrogen, thus allowing formation of a pseudo-seven-membered ring that constitutes the most stable ground state [M + H]+ structure. From this precursor, experimental data show that methyl radical loss has the lowest apparent threshold but, alternatively, even-electron fragment ions can be formed by loss of a methanol molecule. Computational modeling indicates that methyl radical loss is the more endothermic process in this competition, but the lower apparent threshold associated with methyl radical loss points to a lower kinetic barrier. Additionally, this characteristic and unusual loss of methyl radical (in combination with weaker methanol loss) from each prodiginine is useful for performing constant neutral loss scans to quickly and efficiently identify all prodiginines in a complex biological mixture without any clean-up or purification. The feasibility of this approach has been proven through the identification of a new, low-abundance prodigiosin analog arising from Hahella chejuensis. [Copyright &y& Elsevier]

The goal of the current study was to generate a comprehensive, multi-tissue perspective of the effects of chronic hypoxic exposure on carbohydrate metabolism in the Gulf killifish Fundulus grandis. Fish were held at approximately 1.3 mg l-1 dissolved oxygen (∼3.6 kPa) for 4 weeks, after which maximal activities were measured for all glycolytic enzymes in four tissues (white skeletal muscle, liver, heart and brain), as well as for enzymes of glycogen metabolism (in muscle and liver) and gluconeogenesis (in liver). The specific activities of enzymes of glycolysis and glycogen metabolism were strongly suppressed by hypoxia in white skeletal muscle, which may reflect decreased energy demand in this tissue during chronic hypoxia. In contrast, several enzyme specific activities were higher in liver tissue after hypoxic exposure, suggesting increased capacity for carbohydrate metabolism. Hypoxic exposure affected fewer enzymes in heart and brain than in skeletal muscle and liver, and the changes were smaller in magnitude, perhaps due to preferential perfusion of heart and brain during hypoxia. The specific activities of some gluconeogenic enzymes increased in liver during long-term hypoxic exposure, which may be coupled to increased protein catabolism in skeletal muscle. These results demonstrate that when intact fish are subjected to prolonged hypoxia, enzyme activities respond in a tissue-specific fashion reflecting the balance of energetic demands, metabolic role and oxygen supply of particular tissues. Furthermore, within glycolysis, the effects of hypoxia varied among enzymes, rather than being uniformly distributed among pathway enzymes. [ABSTRACT FROM AUTHOR]