Milica Mandic

Swimming Performance and Energetics as a Function of Temperature in Killifish Fundulus heteroclitus

Populations of the common killifish Fundulus heteroclitus are found along a latitudinal temperature gradient in habitats with high thermal variability. The objectives of this study were to assess the effects of temperature and population of origin on killifish swimming performance (assessed as critical swimming speed, Ucrit). Acclimated fish from northern and southern killifish populations demonstrated a wide zone (from 7° to 33°C) over which Ucrit showed little change with temperature, with performance declining significantly only at lower temperatures. Although we observed significant differences in swimming performance between a northern and a southern population of killifish in one experiment, with northern fish having an ∼1.5‐fold‐greater Ucrit than southern fish across all acclimation temperatures, we were unable to replicate this finding in other populations or collection years, and performance was consistently high across all populations and at both low (7°C) and high (23°C) acclimation temperatures. The poor swimming performance of southern killifish from a single collection year was correlated with low muscle [glycogen] rather than with other indicators of fuel stores or body condition. Killifish acclimated to 18°C and acutely challenged at temperatures of 5°, 18°, 25°, or 34°C showed modest thermal sensitivity of Ucrit between 18° and 34°C, with performance declining substantially at 5°C. Thus, much of the zone of relative thermal insensitivity of swimming performance is intrinsic in this species rather than acquired as a result of acclimation. These data suggest that killifish are broadly tolerant of changing temperatures, whether acute or chronic, and demonstrate little evidence of local adaptation in endurance swimming performance in populations from different thermal habitats.

MIGRATION, MITOCHONDRIA, AND THE YELLOW-RUMPED WARBLER

Discordance between mitochondrial and nuclear DNA has been noted in many systems. Asymmetric introgression of mitochondria is a common cause of such discordances, although in most cases the drivers of introgression are unknown. In the yellow‐rumped warbler, evidence suggests that mtDNA from the eastern, myrtle warbler, has introgressed across much of the range of the western form, the Audubons warbler. Within the southwestern United States myrtle mtDNA comes into contact with another clade that occurs in the Mexican black‐fronted warbler. Both northern forms exhibit seasonal migration, whereas black‐fronted warblers are nonmigratory. We investigated the link between mitochondrial introgression, mitochondrial function, and migration using novel genetic, isotopic, biochemical, and phenotypic data obtained from populations in the transition zone. Isotopes suggest the zone is coincident with a shift in migration, with individuals in the south being resident and populations further north becoming increasingly more migratory. Mitochondrial respiration in flight muscles demonstrates that myrtle‐type individuals have a significantly greater acceptor control ratio of mitochondria, suggesting it may be more metabolically efficient. To our knowledge this is the first time this type of intraspecific variation in mitochondrial respiration has been measured in wild birds and we discuss how such mitochondrial adaptations may have facilitated introgression.

The Osmorespiratory Compromise in Sculpins: Impaired Gas Exchange Is Associated with Freshwater Tolerance

We acclimated two species of sculpin, the freshwater prickly sculpin (Cottus asper) and the closely related marine Pacific staghorn sculpin (Leptocottus armatus) to freshwater (∼0 g/L), brackish water (15 g/L), and seawater (30 g/L) for at least 4 wk and examined the relationships between respiration, ion regulation, gill morphology, and freshwater tolerance. The prickly sculpin successfully acclimated to all three salinities and did not experience appreciable changes in plasma osmolality, [Cl−], or mortality. Gill Na+/K+‐ATPase activity was lowest in prickly sculpins acclimated to freshwater, their native salinity, and increased during acclimation to seawater. Furthermore, prickly sculpins acclimated to freshwater had a 30% higher Pcrit than fish acclimated to brackish water or seawater; Pcrit is the environmental Po2 below which an animal can no longer maintain a routine Ṁo2, and an increase in Pcrit represents a compromise of respiratory gas exchange. The higher Pcrit observed in prickly sculpins acclimated to freshwater is likely a consequence of their having small, relatively thick gills that increase in thickness (by ∼1 μm) during freshwater exposure. In contrast, the marine Pacific staghorn sculpin successfully acclimated to brackish water and seawater, but high mortality (25%) was observed after 3 wk of exposure to freshwater. Pacific staghorn sculpins exposed to freshwater suffered significant, 15%–20%, reductions in plasma osmolality and [Cl−], and these losses in plasma ions resulted in a 1.4‐fold increase in gill Na+/K+‐ATPase activity. Pacific staghorn sculpins have large, thin gills that are not modified in response to salinity acclimation, and as a result, these animals show no respiratory compromise during freshwater acclimation, as evidenced by the lack of change in Pcrit, but show significant ion regulatory disturbance. Overall, this study suggests that gill thickening and the resulting respiratory compromise are necessary for freshwater tolerance in sculpins.

The response of the tidepool sculpin, Oligocottus maculosus, to hypoxia in laboratory, mesocosm and field environments

Animals living in the intertidal zone experience regular, predictable fluctuations in physical parameters including temperature, oxygen and salinity and rely on behavioural, physiological and biochemical mechanisms to cope with environmental variation. In the present study, behavioural strategies induced by aquatic hypoxia (e.g. emergence) were performed at similar oxygen tensions across laboratory, mesocosm and field environments; the number of individuals performing these behaviours at any one time was similar in mesocosms and the field. The use of aquatic surface respiration (ASR) was more plastic than emergence behaviour, occurring at a lower oxygen tension in juveniles than adults and being influenced by the addition of alarm substance. Oxygen uptake was lower in air than in water in adults but, in contrast, oxygen uptake was not influenced by the respiratory medium in juveniles. In the laboratory, 72 h of forced emergence did not affect whole body concentrations of lactate but when ASR and emergence were prevented within mesocosm environments there was a significant elevation of lactate. The present study highlights the benefits of transcending traditional laboratory/field boundaries allowing the responses of laboratory-held animals to environmental fluctuation to be integrated with how these animals perform in their natural environment.

Hypoxia Tolerance in Sculpins Is Associated with High Anaerobic Enzyme Activity in Brain but Not in Liver or Muscle

We assessed hypoxia tolerance in 11 species of fish from the superfamily Cottoidea (commonly called sculpins) that are known to differ in their critical O2 tensions (Pcrit) and examined whether hypoxia tolerance correlated with larger substrate stores and higher maximal activity of enzymes associated with anaerobic adenosine triphosphate production (especially glycolysis). Among the sculpins studied, there was large variation in time to loss of equilibrium (LOE50) at torr, with values ranging between 25 and 538 min, and the variation in LOE50 was correlated with Pcrit. Our measures of time to LOE50 and Pcrit were regressed against maximal enzyme activities of lactate dehydrogenase (LDH), pyruvate kinase (PK), creatine phosphokinase (CPK), and citrate synthase (CS) as well as the concentrations of glycogen, glucose, and creatine phosphate in the brain, liver, and white muscle. In the brain, there was a phylogenetically independent relationship between Pcrit and tissue LDH, PK, CPK, and CS activities expressed relative to tissue mass. Hypoxia-tolerant sculpins (those with low Pcrit values) had higher levels of brain LDH, PK, CPK, and CS than did hypoxia-sensitive sculpins. Similarly, LOE50 regressed against brain LDH, PK, and CPK activities expressed relative to tissue mass, with the more hypoxia-tolerant species (i.e., those with higher LOE50) having higher enzyme activities. However, when the phylogenetic relationship among our sculpins was taken into account, only the relationship between hypoxia tolerance and LDH activity remained significant. When enzyme activities were expressed relative to total soluble protein in the tissue, the only relationships that remained were between brain LDH activity and Pcrit and LOE50. In liver and white muscle, there were no relationships between the measures of hypoxia tolerance and enzyme activity or metabolite content. Overall, our analysis suggests that hypoxia-tolerant sculpins maintain higher maximal activities of some of the enzymes involved in anaerobic metabolism in the brain, and this may be an adaptation to hypoxia.

Escaping to the Surface: A Phylogenetically Independent Analysis of Hypoxia‐Induced Respiratory Behaviors in Sculpins

Behavioral responses to progressive hypoxia exposure were assessed in several species of fish from the family Cottidae (sculpins), which are distributed along the near‐shore marine environment and differ in their hypoxia tolerance. The use of aquatic surface respiration (ASR) and aerial emergence as a response to progressive decreases in environmental O2 differed between intertidal and subtidal sculpins. Intertidal sculpins consistently displayed ASR followed by emergence behaviors, while the subtidal species performed these behaviors at low frequency or not at all. There was a significant negative correlation between the O2 thresholds for the onset of ASR and critical O2 tensions (Pcrit, a measure of hypoxia tolerance), such that the hypoxia‐tolerant species performed ASR at higher O2 tensions than the more hypoxia‐sensitive species. There was no relationship between the O2 thresholds for emergence and Pcrit. When restricted from accessing the water surface during progressive decreases in O2, all species of sculpin displayed the same sequence of behaviors including agitation, attempts to escape, quiescence, and unresponsiveness. The only behavior from this suite that correlated with Pcrit was the first sign of agitation, which occurred at a lower O2 tension in the most hypoxia‐tolerant species. With the application of phylogenetically independent contrasts, all the relationships between behavior and Pcrit were nonsignificant, which limits our capacity to separate selection‐driven changes in behavior from the phylogenetic signal.

Origins and functional diversification of salinity-responsive Na+, K+ ATPase α1 paralogs in salmonids

The Salmoniform whole‐genome duplication is hypothesized to have facilitated the evolution of anadromy, but little is known about the contribution of paralogs from this event to the physiological performance traits required for anadromy, such as salinity tolerance. Here, we determined when two candidate, salinity‐responsive paralogs of the Na+, K+ ATPase α subunit (α1a and α1b) evolved and studied their evolutionary trajectories and tissue‐specific expression patterns. We found that these paralogs arose during a small‐scale duplication event prior to the Salmoniform, but after the teleost, whole‐genome duplication. The ‘freshwater paralog’ (α1a) is primarily expressed in the gills of Salmoniformes and an unduplicated freshwater sister species (Esox lucius) and experienced positive selection in the freshwater ancestor of Salmoniformes and Esociformes. Contrary to our predictions, the ‘saltwater paralog’ (α1b), which is more widely expressed than α1a, did not experience positive selection during the evolution of anadromy in the Coregoninae and Salmonine. To determine whether parallel mutations in Na+, K+ ATPase α1 may contribute to salinity tolerance in other fishes, we studied independently evolved salinity‐responsive Na+, K+ ATPase α1 paralogs in Anabas testudineus and Oreochromis mossambicus. We found that a quarter of the mutations occurring between salmonid α1a and α1b in functionally important sites also evolved in parallel in at least one of these species. Together, these data argue that paralogs contributing to salinity tolerance evolved prior to the Salmoniform whole‐genome duplication and that strong selection and/or functional constraints have led to parallel evolution in salinity‐responsive Na+, K+ ATPase α1 paralogs in fishes.

Divergent transcriptional patterns are related to differences in hypoxia tolerance between the intertidal and the subtidal sculpins

Transcriptionally mediated phenotypic plasticity as a mechanism of modifying traits in response to an environmental challenge remains an important area of study. We compared the transcriptional responses to low oxygen (hypoxia) of the hypoxia‐tolerant intertidal fish, the tidepool sculpin (Oligocottus maculosus) with the closely related hypoxia‐intolerant subtidal fish, the silverspotted sculpin (Blepsias cirrhosus) to determine whether these species use different mechanisms to cope with hypoxia. Individuals from each species were exposed to environmental O2 tensions chosen to yield a similar level of tissue hypoxia, and gene transcription was assessed in the liver over time. There was an effect of time in hypoxia, where the greatest transcriptional change in the silverspotted sculpin occurred between 3 and 24 h in contrast to the tidepool sculpin where the largest transcriptional change occurred between 24 and 72 h of hypoxia. A number of genes showed similar hypoxia‐induced transcription patterns in both species (e.g. genes associated with glycolysis and apoptosis) suggesting they are involved in a conserved hypoxia response. A large set of genes showed divergent transcriptional patterns in the two species, including fatty acid oxidation and oxidative phosphorylation, suggesting that these biological processes may contribute to explaining variation in hypoxia tolerance in these species. When both species were exposed to a single environmental O2 tension, large transcriptional responses were seen in the hypoxia‐intolerant silverspotted sculpin while almost no response was observed in the hypoxia‐tolerant tidepool sculpin. Overall, divergent transcription patterns in response to both magnitude and duration of hypoxia provide insights into the processes that may determine an animals capacity to tolerate frequent bouts of hypoxia in the wild.

Evolution of Cytochrome c Oxidase in Hypoxia Tolerant Sculpins (Cottidae, Actinopterygii)

Vertebrate hypoxia tolerance can emerge from modifications to the oxygen (O2) transport cascade, but whether there is adaptive variation to O2 binding at the terminus of this cascade, mitochondrial cytochrome c oxidase (COX), is not known. In order to address the hypothesis that hypoxia tolerance is associated with enhanced O2 binding by mitochondria we undertook a comparative analysis of COX O2 kinetics across species of intertidal sculpins (Cottidae, Actinopterygii) that vary in hypoxia tolerance. Our analysis revealed a significant relationship between hypoxia tolerance (critical O2 tension of O2 consumption rate; Pcrit), mitochondrial O2 binding affinity (O2 tension at which mitochondrial respiration was half maximal; P50), and COX O2-binding affinity (apparent Michaelis-Menten constant for O2 binding to COX; Km,app O2). The more hypoxia tolerant species had both a lower mitochondrial P50 and lower COX Km,app O2, facilitating the maintenance of mitochondrial function to a lower O2 tension than in hypoxia intolerant species. Additionally, hypoxia tolerant species had a lower overall COX Vmax but higher mitochondrial COX respiration rate when expressed relative to maximal electron transport system respiration rate. In silico analyses of the COX3 subunit postulated as the entry point for O2 into the COX protein catalytic core, points to variation in COX3 protein stability (estimated as free energy of unfolding) contributing to the variation in COX Km,app O2. We propose that interactions between COX3 and cardiolipin at four amino acid positions along the same alpha-helix forming the COX3 v-cleft represent likely determinants of interspecific differences in COX Km,app O2.