Extreme environmental temperatures pose significant metabolic challenges for ectothermic

organisms such as teleost fishes.

Pupfishes (genus Cyprinodon, Figure 1) in the Death Valley region of California and Nevada,

USA, occupy remote aquatic habitats that vary widely in temperature conditions making them an

ideal study organism to investigate metabolic acclimation and evolution in the face of varying

thermal environments.

In this study, we evaluate the aerobic and anaerobic metabolic responses of C. n. amargosae

pupfish from two geographically isolated habitats – the Amargosa River and Tecopa Bore – when

fish from these habitats are acclimated under laboratory conditions to high and low temperatures

(35°C and 23°C respectively).

•Both the Amargosa River and Tecopa Bore habitats support populations of C. n. amargosae

pupfish, but the habitats are the strikingly different in thermal regime (Table 1).

What is more, the Tecopa Bore habitat experienced an approximately 15°C increase in mean

temperature between 2008 and 2013 to the values shown in Table 1 due to a human-mediated

change in water flow.

Here, we quantified activity of Citrate Synthase (CS) and Lactate Dehydrogenase (LDH) as

measures of metabolic status. CS is a robust indicator of an animal's capacity for aerobic

metabolism (where CS activity is measured in μmol/ming) due to that fact that it is a

mitochondrial enzyme involved in the Krebs cycle. LDH is the final enzyme in the fermentation

pathway; thus LDH activity (μmol/ming) reflects an animal's capacity for anaerobic respiration.

Our results indicate that the two populations of pupfish differ in their acclimatory change in LDH

activity in response to varying temperatures:

1) Pupfish from the Tecopa Bore population showed decreased LDH activity in cold

temperatures, while pupfish from the Amargosa River population showed increased LDH

activity in cold temperatures.

2) Surprisingly, the Tecopa Bore and Amargosa River populations showed equivalent LDH

activity in high temperatures.

Our results showed no significant difference between low and high temperature acclimation in either

population’s CS activity.

There was, however, a significant linear regression observed in both populations when comparing body

size with CS activity. This body mass relationship is to be expected due to metabolic scaling (Clarke &

Johnston, 1999).

While predications were not entirely correct, the overall hypothesis was supported by the varying

anaerobic metabolic rate observed in both the Tecopa Bore and Amargosa River populations between

high and low temperatures. No significant conclusions can be drawn regarding changes in aerobic

metabolic rate due to variable temperature from the current data set.

Effects of temperature on metabolism in two populations of the desert pupfish

Cyprinodon nevadensis Emily J. Resner, Alex A. Westman, Sean C. Lema, Kristin M. Hardy

Biological Sciences, California Polytechnic State University, San Luis Obispo, CA 93407

Introduction

Acknowledgments

Population Temp. mean Temp. range

Amargosa River 19.4°C 16.1 to 22.5°C

Tecopa Bore 36.7°C 33.8 to 41.5°C

Table 1. Recorded temperature data from 48 hrs of observations during May 2014.

Conclusions

Results

Figure 1. Pupfish, Cyprinodon nevadensis amargosae, from the Death Valley region.

Table 2. Enzyme activity measurements (U/g tissue wet weight) in pupfish from the Amargosa River

and Tecopa Bore. There was no significant effect of population, temperature or their interaction on CS

activity (ANCOVA, p>0.05) Means with different letters within each enzyme are significantly different

from one another (ANCOVA; Tukey’s HSD; α=0.05)

References

We would like to acknowledge the Cal Poly Biological Sciences Department for funding assistance via

College Based Fee funds. Additional support was provided in the form of a New Investigator Award

from the CSU Program for Education and Research in Biotechnology (CSUPERB) to S.C.L. Pupfish

were collected with permission from the California Department of Wildlife (Permit # SC-4793).

Clarke, A and N.M. Johnston. 1999. Scaling of Metabolic Rate with Body Mass and Temperature in Teleost Fish. J Anim

Ecology 68.5:893-905.

Future Work

•Data collection will continue until all samples have been processed.

•In the face of evidence that wild fish from these populations vary in thyroid hormone physiology, we

will also examine the role of temperature in modulating the potential regulation of metabolism by

thyroid hormones by treating fish from both populations under high and low temperatures to

triiodothyronine (T3) for 16 hours.

•We hypothesize that T3 exposure may influence the way that temperature affects metabolism in each

of these populations, and will test for such an influence as we complete data collection.

Figure 4. Effect of temperature (high=23oC; low=35oC) on lactate dehydrogenase (LDH) activity

(U/g of tissue wet wt) in C. nevadensis amargosae pupfish from the Amargosa River and Tecopa

Bore habitats. The effect of temperature on LDH activity differed between the two populations

(population*temperature interaction; ANCOVA with body mass as a covariate; F1,43=5.8656,

p=0.0197). Analysis performed on log transformed data; n = 12-18 per treatment group. Columns

with different letters are significantly different from one another (Tukey’s HSD, α=0.05) Error bars

represent S.E.M.

AB AB

A

B

•For LDH analysis, a 1:10 diluted sample supernatant was combined with Tris-HCl buffer (pH 7.6)

with NADH. The reaction was started with the addition of pyruvate. Analysis was run at 340 nm for 5

min in an Implen NanoPhotometer P-Class P300. Enzyme activity (μmol/min*g) was calculated using

the slope of the absorbance change following addition of pyruvate. All samples were run in duplicate.

•For CS analysis, sample supernatant was combined with Tris-HCl buffer (pH 8.1) with DTNB and

Acetyl CoA. The reaction was started with the addition of oxaloacetate. Analysis was run at 412 nm for

5 min in a NanoPhotometer P300. Enzyme activity (μmol/min*g) calculated using the slope of the

absorbance change immediately following addition of oxaloacetate. All samples were run in singles.

•Statistical comparisons between temperatures and populations were conducted using two-way

ANCOVA models using ‘population’, ‘temperature’, and their interaction as factors and body mass

(g) as a covariate (JMP v. 11 software).

Methods

Figure 2. Amargosa River on left, Tecopa Bore on right.

Figure 5. Effect of body mass on citrate synthase (CS) activity (U/g tissue wet wt) in C. nevadensis

amargosae fish from Amargosa River (n = 27) and Tecopa Bore (n = 21) habitats. Linear regression

analysis revealed a significant scaling effect of body mass on CS activity in both Amargosa (y=-

0.47x+4.32, r2=0.128, F1,25=3.68, p<0.001) and Tecopa Bore (y=-0.886x+5.05; r2=0.37, F1,19=11.265,

p=0.0033) populations. There is a decrease in CS activity with an increase in body mass, which is expected

based on metabolic scaling relationships (Clark & Johnson, 1999).

Hypothesis:

Acclimation to various temperatures will effect metabolic enzyme activity

similarly in pupfish from the Tecopa Bore and Amargosa River

populations.

Prediction 1:

Both populations of pupfish should have higher CS and LDH activity levels

at the higher temperature.

Prediction 2:

The cooler adapted Amargosa River pupfish population will have higher

CS and LDH activity at either temperature relative to the warm adapted

Tecopa Bore pupfish.

Habitat Temperature n CS activity (U/g) LDH activity (U/g)

Amargosa River High 18 3.3±0.26a 200.17±12.9a,b

Low 11 3.43±0.23a

261.43±37.4b

Tecopa Bore High 11 3.93±0.29a 211.40±13.35a,b

Low 12 3.25±0.35a 148.52±16.38a

Objectives

•Adult pupfish were collected from the Amargosa River

and Tecopa Bore using minnow traps. Fish from each

population were maintained in captivity under either low

(23°C) or high (35°C) temperature conditions for 4

months. Select fish from each temperature treatment were

given exogenous thyroid hormone (triiodothyronine, T3)

for 16 hrs. Liver tissue was collected from each fish for

CS and LDH activity measures.

population (AR or TB)

control T3-treated

low temp (23°C) high temp (35°C)

control T3-treated

Figure 3. Illustration of the three factor (population

origin, temperature regime, T3 hormone treatment)

design of the experiment.