The extent of aquatic hypoxia (low dissolved oxygen) has increased in recent times and now threatens aquatic habitats worldwide. Here, we review intraspecific variation in hypoxia tolerance in fish on various scales: geographic variation among populations; variation among genetic strains; variation due to laboratory acclimation; and variation among individuals coming from a given population and having similar exposure history. Metrics of hypoxia tolerance include the oxygen level below which the costs of maintenance can no longer be met by oxygen uptake from the environment; the time or oxygen level at which aquatic surface respiration is conducted; and the time or oxygen levels at which fish lose the ability for coordinated movement. There is great diversity in the range of intraspecific hypoxia tolerance and its association with potential underlying morphological, physiological, and genetic correlates. We advocate for greater standardization of experimental design, enhanced data reporting, and development and validation of new metrics of hypoxia tolerance. With greater understanding of intraspecific variation in hypoxia tolerance, it might be possible to predict which populations, strains, or individuals will fare better during hypoxic episodes; mitigate the effects of low oxygen on fishes; and design breeding protocols to enhance the hypoxia tolerance of fishes.

AbstractVariation in rates of oxygen uptake (Ṁo2) among individuals within a species is widespread and observed during both rest and activity. Such variation is expected to be important in animal physiology, ecology, and evolution, yet the mechanistic bases for this variation are incompletely understood. In the present study, we asked whether interindividual variation in Ṁo2 at rest (standard Ṁo2 [Ṁo2,standard]) and during an incremental swim test (peak swimming Ṁo2 [peak Ṁo2,swim]) in Gulf killifish (Fundulus grandis) is related to variation in mitochondrial Ṁo2 in five tissues: heart, oxidative skeletal muscle, glycolytic skeletal muscle, liver, and brain. After accounting for the effects of body mass, Ṁo2,standard was positively related to liver mass and its maximum capacity for oxygen flux by the electron transport system (ETS). Peak Ṁo2,swim was positively related to ETS respiration by heart ventricle and mitochondrial respiration required to offset the dissipation of the proton gradient in the absence of ATP synthesis (LEAK) by glycolytic skeletal muscle. The relationship between peak Ṁo2,swim and glycolytic muscle LEAK respiration prompted us to examine the relationship between the aerobic cost of transport and mitochondrial phosphorylation efficiency in glycolytic skeletal muscle. We found that individuals with a lower phosphorylation efficiency consumed more oxygen to travel a given distance (i.e., had a higher aerobic cost of transport). This result supports the idea that LEAK respiration represents an energetic cost during activity, which might be partially offset if higher LEAK results in less reactive oxygen species formation.

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 (Pcrit) of the corresponding species. Our results demonstrate that Pcrit 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 Pcrit. 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.

Low dissolved oxygen (hypoxia) poses significant challenges to aquatic ecosystems, affecting the behavior, reproduction, and survival of aquatic organisms. Some fishes respond to hypoxia by changes in gene expression, which may be regulated by the hypoxia inducible factor (HIF) family of transcription factors. HIF abundance and activity depends upon the post-translational modification of the alpha protein subunit, although several studies indicate that HIFA mRNA abundance increases in tissues of fishes exposed to hypoxia. This study reviewed reports of laboratory exposures of adult ray-finned fishes to hypoxia and used generalized linear mixed effects models to examine the influence of HIFA gene, tissue sampled, and exposure conditions in explaining the diversity of responses seen in HIFA mRNA abundance. The frequency of hypoxia-induced increases in HIFA mRNA was poorly explained by gene, tissue, or the severity of the hypoxic exposure. Rather, the frequency of reported increases was strongly related to the extent to which studies adhered to guidelines for documenting quantitative real-time PCR methods: the frequency of hypoxia-induced increases in HIFA mRNA decreased sharply in studies with more thorough description of experimental design. Future research should (a) adhere to stringent reporting of experimental design, (b) address the relative paucity of data on HIF2A and HIF3A, and (c) determine levels of HIF alpha protein subunits. By following these recommendations, it is hoped that a more complete understanding will be gained of the role of the HIF family of transcription factors in the response of fish to hypoxia.

ABSTRACT The maximum rate at which animals take up oxygen from their environment (ṀO2,max) is a crucial aspect of their physiology and ecology. In fishes, ṀO2,max is commonly quantified by measuring oxygen uptake either during incremental swimming tests or during recovery from an exhaustive chase. In this Commentary, we compile recent studies that apply both techniques to the same fish and show that the two methods typically yield different mean estimates of ṀO2,max for a group of individuals. Furthermore, within a group of fish, estimates of ṀO2,max determined during swimming are poorly correlated with estimates determined during recovery from chasing (i.e. an individual's ṀO2,max is not repeatable across methods). One explanation for the lack of agreement is that these methods measure different physiological states, each with their own behavioural, anatomical and biochemical determinants. We propose that these methods are not directly interchangeable but, rather, each is suited to address different questions in fish biology. We suggest that researchers select the method that reflects the biological contexts of their study, and we advocate for the use of accurate terminology that acknowledges the technique used to elevate ṀO2 (e.g. peak ṀO2,swim or peak ṀO2,recovery). If the study's objective is to estimate the ‘true’ ṀO2,max of an individual or species, we recommend that pilot studies compare methods, preferably using repeated-measures designs. We hope that these recommendations contribute new insights into the causes and consequences of variation in ṀO2,max within and among fish species.

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.

The hypoxia inducible factor 1 (HIF1) is a central regulator of the molecular responses to low oxygen in animals. It has been extensively studied in mammals, where its tissue levels are regulated by stabilization of the alpha subunit (HIF1α) when oxygen levels decrease. Consistent with this, the initial characterization of HIF1α in fish cells in culture indicated that protein abundance increased during hypoxia even when transcription was blocked. Subsequent studies, however, have reported an increase in HIF1α mRNA levels during hypoxia in certain tissues of selected species, raising the question whether hypoxic exposure brings about coordinated changes in HIF1α mRNA and protein in tissues when measured in the same fish. We have directly addressed this question by determining levels of HIF1α protein and mRNA in the tissues of Gulf killifish, Fundulus grandis , exposed to short-term hypoxia (24 h at 1 mg O 2 l −1 ). HIF1α protein was higher in brain, ovary, and skeletal muscle from fish exposed to hypoxia compared with normoxic controls by 6 h, and it remained elevated in brain and ovary at 24 h. In contrast, HIF1α mRNA levels were unaffected by hypoxia in any tissue. Moreover, levels of HIF1α protein and mRNA in the same tissues were not correlated with one another, during either normoxia or hypoxia. These results suggest that, during the initial response to low oxygen, HIF1α protein levels increase as the result of post-translational protein stabilization, rather than new transcription, as predicted from studies in mammalian and fish cells in culture. Summary Statement Parallel measurements of protein and mRNA of the hypoxia inducible factor support post-translational protein stabilization rather than new transcription in the initial response of fish to low oxygen

ABSTRACT The hypoxia inducible factor 1 (HIF1) is a central regulator of the molecular responses of animals to low oxygen. While the hypoxia-responsiveness of HIF1 is generally attributed to the stabilization of the alpha protein subunit (HIF1α) at low oxygen, several studies on fish report increased tissue levels of HIF1A mRNA during hypoxia, suggesting transcriptional regulation. In the current study, HIF1α protein and HIF1A mRNA were determined in parallel in tissues of Gulf killifish, Fundulus grandis, exposed to short-term hypoxia (24 h at 1 mg O2 l−1). HIF1α protein was higher in brain, ovary, and skeletal muscle from fish exposed to hypoxia compared with normoxic controls by 6 h, and it remained elevated in brain and ovary at 24 h. In contrast, HIF1A mRNA levels were unaffected by hypoxia in any tissue. Moreover, HIF1α protein and HIF1A mRNA levels in the same tissues were not correlated with one another during either normoxia or hypoxia. Hence, an increase in HIF1α protein does not depend upon an increase in HIF1A mRNA during acute exposure to low oxygen in this species. The results support the widely accepted mechanism of post-translational protein stabilization, rather than new transcription, during the initial response of fish to hypoxia.

This study asked whether interindividual variation in maximum and standard aerobic metabolic rates of the Gulf killifish, Fundulus grandis, correlates with gill morphology and cardiac mitochondrial bioenergetics, traits reflecting critical steps in the O2 transport cascade from the environment to the tissues. Maximum metabolic rate (MMR) was positively related to body mass, total gill filament length and myocardial oxygen consumption during maximum oxidative phosphorylation (multiple R2=0.836). Standard metabolic rate (SMR) was positively related to body mass, total gill filament length and myocardial oxygen consumption during maximum electron transport system activity (multiple R2=0.717). After controlling for body mass, individuals with longer gill filaments, summed over all gill arches, or greater cardiac respiratory capacity had higher whole-animal metabolic rates. The overall model fit and the explanatory power of individual predictor variables were better for MMR than for SMR, suggesting that gill morphology and myocardial bioenergetics are more important in determining active rather than resting metabolism. After accounting for body mass, heart ventricle mass was not related to variation in MMR or SMR, indicating that the quality of the heart (i.e. the capacity for mitochondrial metabolism) was more influential than heart size. Finally, the myocardial oxygen consumption required to offset the dissipation of the transmembrane proton gradient in the absence of ATP synthesis was not correlated with either MMR or SMR. The results support the idea that interindividual variation in aerobic metabolism, particularly MMR, is associated with variation in specific steps in the O2 transport cascade.

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.

This Honors Thesis-Restricted is protected by copyright and/or related rights. It has been brought to you by ScholarWorks@UNO with permission from the rights-holder(s). You are free to use this Honors Thesis-Restricted in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s) directly, unless additional rights are indicated by a Creative Commons license in the record and/or on the work itself.

Standard metabolic rate (SMR), maximum metabolic rate (MMR), absolute aerobic scope (AAS), and critical oxygen tension (Pcrit) were determined for the Gulf killifish, Fundulus grandis, an ecologically dominant estuarine fish, acclimated to lowered salinity, elevated temperature, and lowered oxygen concentration. Acclimation to low salinity resulted in a small, but significant, elevation of Pcrit (suggesting lower tolerance of hypoxia); acclimation to elevated temperature increased SMR, MMR, AAS, and Pcrit; acclimation to low oxygen led to a small increase in SMR, but substantial decreases in MMR, AAS, and Pcrit. Variation in these metabolic traits among individuals was consistent and repeatable when measured during multiple control exposures over seven months. Trait repeatability was unaffected by acclimation condition suggesting that repeatability of these traits is not context dependent. There were significant phenotypic correlations between specific metabolic traits: SMR was positively correlated with MMR and Pcrit; MMR was positively correlated with AAS; and AAS was negatively correlated with Pcrit. In general, within-individual variation contributed more than among-individual variation to these phenotypic correlations. The effects of acclimation on these traits demonstrate that aerobic metabolism is plastic and influenced by the conditions experienced by these fish in the dynamic habitats in which they occur; however, the repeatability of these traits and the correlations among them suggest that these traits change in ways that maintains the rank order of performance among individuals across a range of environmental variation.