Nann A. Fangue

Intraspecific variation in thermal tolerance and heat shock protein gene expression in common killifish, Fundulus heteroclitus

SUMMARY Populations of common killifish, Fundulus heteroclitus, are distributed along the Atlantic coast of North America through a steep latitudinal thermal gradient. We examined intraspecific variation in whole-animal thermal tolerance and its relationship to the heat shock response in killifish from the northern and southern extremes of the species range. Critical thermal maxima were significantly higher in southern than in northern fish by ∼1.5°C at a wide range of acclimation temperatures (from 2-34°C), and critical thermal minima differed by ∼1.5°C at acclimation temperatures above 22°C, converging on the freezing point of brackish water at lower acclimation temperatures. To determine whether these differences in whole-organism thermal tolerance were reflected in differences in either the sequence or regulation of the heat shock protein genes (hsps) we obtained complete cDNA sequences for hsc70, hsp70-1 and hsp70-2, and partial sequences of hsp90α and hsp90β. There were no fixed differences in amino acid sequence between populations in either hsp70-1 or hsp70-2, and only a single conservative substitution between populations in hsc70. By contrast, there were significant differences between populations in the expression of many, but not all, of these genes. Both northern and southern killifish significantly increased hsp70-2 levels above control values (Ton) at a heat shock temperature of 33°C, but the magnitude of this induction was greater in northern fish, suggesting that northern fish may be more susceptible to thermal damage than are southern fish. In contrast, hsp70-1 mRNA levels increased gradually and to the same extent in response to heat shock in both populations. Hsc70 mRNA levels were significantly elevated by heat shock in southern fish, but not in northern fish. Similarly, the more thermotolerant southern killifish had a Ton for hsp90α of 30°C, 2°C lower than that of northern fish. This observation combined with the ability of southern killifish to upregulate hsc70 in response to heat shock suggests a possible role for these hsps in whole-organism differences in thermal tolerance. These data highlight the importance of considering the complexity of the heat shock response across multiple isoforms when attempting to make linkages to whole-organism traits such as thermal tolerance.

Thermal Performance Curves, Phenotypic Plasticity, and the Time Scales of Temperature Exposure

Thermal performance curves (TPCs) describe the effects of temperature on biological rate processes. Here, we use examples from our work on common killifish (Fundulus heteroclitus) to illustrate some important conceptual issues relating to TPCs in the context of using these curves to predict the responses of organisms to climate change. Phenotypic plasticity has the capacity to alter the shape and position of the TPCs for acute exposures, but these changes can be obscured when rate processes are measured only following chronic exposures. For example, the acute TPC for mitochondrial respiration in killifish is exponential in shape, but this shape changes with acclimation. If respiration rate is measured only at the acclimation temperature, the TPC is linear, concealing the underlying mechanistic complexity at an acute time scale. These issues are particularly problematic when attempting to use TPCs to predict the responses of organisms to temperature change in natural environments. Many TPCs are generated using laboratory exposures to constant temperatures, but temperature fluctuates in the natural environment, and the mechanisms influencing performance at acute and chronic time scales, and the responses of the performance traits at these time scales may be quite different. Unfortunately, our current understanding of the mechanisms underlying the responses of organisms to temperature change is incomplete, particularly with respect to integrating from processes occurring at the level of single proteins up to whole-organism functions across different time scales, which is a challenge for the development of strongly grounded mechanistic models of responses to global climate change.

Do mitochondrial properties explain intraspecific variation in thermal tolerance

SUMMARY As global temperatures rise, there is a growing need to understand the physiological mechanisms that determine an organisms thermal niche. Here, we test the hypothesis that increases in mitochondrial capacity with cold acclimation and adaptation are associated with decreases in thermal tolerance using two subspecies of killifish (Fundulus heteroclitus) that differ in thermal niche. We assessed whole-organism metabolic rate, mitochondrial amount and mitochondrial function in killifish acclimated to several temperatures. Mitochondrial enzyme activities and mRNA levels were greater in fish from the northern subspecies, particularly in cold-acclimated fish, suggesting that the putatively cold-adapted northern subspecies has a greater capacity for increases in mitochondrial amount in response to cold acclimation. When tested at the fishs acclimation temperature, maximum ADP-stimulated (State III) rates of mitochondrial oxygen consumption in vitro were greater in cold-acclimated northern fish than in southern fish but did not differ between subspecies at higher acclimation temperatures. Whole-organism metabolic rate was greater in fish of the northern subspecies at all acclimation temperatures. Cold acclimation also changed the response of mitochondrial respiration to acute temperature challenge. Mitochondrial oxygen consumption was greater in cold-acclimated northern fish than in southern fish at low test temperatures, but the opposite was true at high test temperatures. These differences were reflected in whole-organism oxygen consumption. Our data indicate that the plasticity of mitochondrial function and amount differs between killifish subspecies, with the less high-temperature tolerant, and putatively cold adapted, northern subspecies having greater ability to increase mitochondrial capacity in the cold. However, there were few differences in mitochondrial properties between subspecies at warm acclimation temperatures, despite differences in both whole-organism oxygen consumption and thermal tolerance at these temperatures.

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.

Countergradient Variation in Temperature Preference in Populations of Killifish Fundulus heteroclitus

Behavioral thermoregulation can allow ectotherms to buffer the effects of changes in environmental temperature, and thus an organism’s preferred temperature is thought to be under strong selection. However, this contention has seldom been tested. We used common killifish Fundulus heteroclitus from high‐latitude (northern) and low‐latitude (southern) populations to investigate intraspecific variation in thermal preference and its relationship to habitat temperature. We quantified the preferred temperatures of northern and southern killifish populations acclimated to three temperatures (5°, 15°, and 25°C) to evaluate two alternative hypotheses for the evolution of differences in thermal preference among latitudinally separated populations: local thermal adaptation, which predicts that organisms from high latitudes should prefer lower temperatures than individuals from lower latitudes, versus countergradient variation, which predicts that high‐latitude organisms should prefer higher temperatures to compensate for shorter growing seasons. All killifish selected their final thermal preferendum within 4 h. Southern killifish and killifish acclimated to warmer temperatures had greater variability in selected temperature. This increase in variability was the result of an increase in interindividual variation in preferred temperature rather than a reduction in the precision of temperature selection in these groups. Northern killifish preferred significantly higher temperatures than southern fish (30.6° vs. 29.0°C, respectively, when calculated on the basis of the temperature selected consistently for at least 30 min; 28.4° vs. 26.5°C, respectively, when calculated on the basis of the mean temperature occupied), regardless of acclimation temperature. These data are not consistent with local adaptation in thermal preference but instead can be better explained by countergradient variation in thermal preference in killifish.

Ontogeny influences sensitivity to climate change stressors in an endangered fish

We assessed thermal and salinity limits in several ontogenetic stages and acclimation states of Delta Smelt to evaluate sensitivity to climate change stressors. Thermal tolerance decreased among successive stages, and juvenile tolerance limits were closest to current environmental conditions. Salinity impacted juvenile and adult survival in exposures over acute timescales.

The Onset Temperature of the Heat-Shock Response and Whole-Organism Thermal Tolerance Are Tightly Correlated in both Laboratory-Acclimated and Field-Acclimatized Tidepool Sculpins (Oligocottus maculosus)

We examined the relationship between thermal tolerance, measured as critical thermal maximum (CTmax), and aspects of the heat-shock response in tidepool sculpins (Oligocottus maculosus) acclimated to constant laboratory temperatures or acclimatized to field conditions. The CTmax of fish laboratory acclimated to 6°, 13°, and 20°C were , , and , respectively, increasing linearly by 0.2°C for each 1°C increase in acclimation temperature. The CTmax of field-acclimatized fish from the low intertidal () was significantly lower than that of fish from the mid- () and high () intertidal. CTmax and the onset temperature of hsp70 induction in gill (Ton) were highly correlated in both laboratory-acclimated and field-acclimatized sculpins, with Ton occurring at 2°C below CTmax in all cases. However, there was no consistent relationship between CTmax and the maximum levels of gill hsp70 mRNA. Predicted “acclimation” temperature () and mean habitat temperature () were similar for sculpins from low intertidal pools, but this relationship was not apparent in mid- and high intertidal fish. Mark-recapture experiments indicated that approximately 80% of fish from low intertidal pools were residents of that pool, but residency rates were less than 50% in mid- and high intertidal pools, which may explain the lack of correlation between CTmax and habitat variables in these groups. These data indicate that gill hsp70 Ton and CTmax are highly correlated indicators of the thermal performance of tidepool sculpins in both laboratory and field settings.

Turbidity and Salinity Affect Feeding Performance and Physiological Stress in the Endangered Delta Smelt

Coastal estuaries are among the most heavily impacted ecosystems worldwide with many keystone fauna critically endangered. The delta smelt (Hypomesus transpacificus) is an endangered pelagic fish species endemic to the Sacramento-San Joaquin Estuary in northern California, and is considered as an indicator species for ecosystem health. This ecosystem is characterized by tidal and seasonal gradients in water parameters (e.g., salinity, temperature, and turbidity), but is also subject to altered water-flow regimes due to water extraction. In this study, we evaluated the effects of turbidity and salinity on feeding performance and the stress response of delta smelt because both of these parameters are influenced by water flows through the San Francisco Bay Delta (SFBD) and are known to be of critical importance to the completion of the delta smelts life cycle. Juvenile delta smelt were exposed to a matrix of turbidities and salinities ranging from 5 to 250 nephelometric turbidity units (NTUs) and 0.2 to 15 parts per thousand (ppt), respectively, for 2 h. Best statistical models using Akaikes Information Criterion supported that increasing turbidities resulted in reduced feeding rates, especially at 250 NTU. In contrast, best explanatory models for gene transcription of sodium-potassium-ATPase (Na/K-ATPase)-an indicator of osmoregulatory stress, hypothalamic pro-opiomelanocortin-a precursor protein to adrenocorticotropic hormone (expressed in response to biological stress), and whole-body cortisol were affected by salinity alone. Only transcription of glutathione-S-transferase, a phase II detoxification enzyme that protects cells against reactive oxygen species, was affected by both salinity and turbidity. Taken together, these data suggest that turbidity is an important determinant of feeding, whereas salinity is an important abiotic factor influencing the cellular stress response in delta smelt. Our data support habitat association studies that have shown greater delta smelt abundances in the low-salinity zone (0.5-6.0 ppt) of San Francisco Bay, a zone that is also understood to have optimal turbidities. By determining the responses of juvenile delta smelt to key abiotic factors, we hope to aid resource managers in making informed decisions in support of delta smelt conservation.

Gene expression responses of threespine stickleback to salinity: implications for salt-sensitive hypertension

Despite recent success with genome-wide association studies (GWAS), identifying hypertension (HTN)-susceptibility loci in the general population remains difficult. Here, we present a novel strategy to address this challenge by studying salinity adaptation in the threespine stickleback, a fish species with diverse salt-handling ecotypes. We acclimated native freshwater (FW) and anadromous saltwater (SW) threespine sticklebacks to fresh, brackish, and sea water for 30 days, and applied RNA sequencing to determine the gene expression in fish kidneys. We identified 1844 salt-responsive genes that were differentially expressed between FW sticklebacks acclimated to different salinities and/or between SW and FW sticklebacks acclimated to full-strength sea water. Significant overlap between stickleback salt-responsive genes and human genes implicated in HTN was detected (P < 10−7, hypergeometric test), suggesting a possible similarity in genetic mechanisms of salt handling between threespine sticklebacks and humans. The overlapping genes included a newly discovered HTN gene—MAP3K15, whose expression in FW stickleback kidneys decreases with salinity. These also included genes located in the GWAS loci such as AGTRAP-PLOD1 and CYP1A1-ULK3, which contain multiple potentially causative genes contributing to HTN susceptibility that need to be prioritized for study. Taken together, we show that stickleback salt-responsive genes provide valuable information facilitating the identification of human HTN genes. Thus, threespine sticklebacks may be used as a model, complementary to existing animal models, in human HTN research.

Larval green and white sturgeon swimming performance in relation to water-diversion flows

Larval sturgeon swimming capacity has never been assessed. We measured critical swimming velocity of larval green and white sturgeon, and summarized published juvenile critical swimming velocity data for all sturgeon species. Recommendations for anthropogenic water diversion facility flow management were developed from the data, emphasizing Californian green and white sturgeon conservation.