ISSUE 19

Testing for “loss of equilibrium” in rainbow darters:

Assessing the impacts of multiple stressors

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Paul Craig
Department of Biology

Introduction

Fish routinely experience environmental and physiological disruptions that interfere with their biological stability or homeostasis. Direct comparisons of metabolic recovery from different stressors such as warming, hypoxia, and exhaustive exercise are rare, though such comparisons could be valuable in uncovering the distinct or shared mechanisms for coping with physiological challenges and predicting their ecological implications. Assessing the relative threat imposed by different stressors is difficult, as stressors vary in their mechanisms, effects, and timescales.

This study applies the fact that acute exposure to different stressors can result in the same whole-animal outcome of loss of equilibrium (LOE) to (i) compare the metabolic recovery profiles of rainbow darters in response to high temperature, hypoxia, and exhaustive exercise and (ii) test the concept that LOE could be used as a physiological calibration for the intensity of different stressors.

Methodology

The study focused on rainbow darters (Etheostoma caeruleum), a widespread benthic species that routinely copes with the stressors of interest as a routine part of its life history. Adult fish were collected from the wild, housed in 10-L acrylic tanks in a 385-L rack system (Aquatic Habitats Z-HAB System) and used for experiments. Using stop-flow (intermittent) respirometry, oxygen consumption rates in an undisturbed, control group and in fish before and after exposure to warming-, hypoxia-, or exercise-based physiological challenges sufficient to induce LOE were tracked.

The study distinguished between resting and routine MO₂ – the latter being representative of the “operational” MO₂ of the fish. Routine MO₂ was used as the baseline to calculate aerobic scope, total O₂ debt and recovery time. Both resting MO₂ and routine MO₂ data were collected prior to the stressor trials. The recovery profile of fish exposed to warming, hypoxia or exercise were quantified through two metrics: total O₂ debt and recovery time. Total O₂ debt was determined by calculating the area under the curve of measured MO₂ above routine MO₂ over time. Recovery time was defined as the time required for MO₂ to return to routine MO₂ after reintroduction of fish to the respirometry chamber.

Datasets that passed a D'Agostino-Pearson normality test were analyzed with a one-way ANOVA with Tukey's multiple comparisons test. Datasets that did not pass the normality test were analyzed with a non-parametric ANOVA on ranks (Kruskal-Wallis test) with a Dunn's multiple comparisons test or a mixed effects model followed by a Sidak multiple comparisons test.

darter lab Fish testing

Outcomes

The fish showed a considerable degree of natural variation in oxygen consumption rate on a daily as well as spontaneous basis (Figure 1). Routine MO₂was significantly higher than resting MO₂with oxygen consumption rates recorded when the animals were active during the day, typically ~30% higher than resting rates recorded late at night. Recovery parameters quantified with resting MO₂ instead of routine MO₂ had slightly different absolute values, but there were no differences in the relative statistical relationships between groups (Figure 2).

Whole-animal oxygen consumption rate (MO2).

Figure 1: Whole-animal oxygen consumption rate (MO₂). The dark phase of the photoperiod is indicated by gray shading. The thermal, hypoxia or exercise challenges occurred ~0900 to ~1100 local time, as indicated by the break in the of MO₂ trace. The black horizontal line indicates the mean resting MO₂ of each group, with the dot-shaded region indicating the standard error.

Diurnal variation in resting and routine oxygen consumption rate (MO2).

Figure 2: Diurnal variation in resting and routine oxygen consumption rate (MO₂). (a) Routine MO₂ immediately prior to the challenge window was consistently higher than resting MO₂ during the overnight period, and this is reflected in factorial (b) and absolute (c) aerobic activity scope. Dissimilar letters indicate groups that are statistically different for each other; the lowercase “a” above all groups indicates that they are not different from each other. * indicates routine MO2 is statistically different from resting MO2 within that group.

All fish survived and successfully recovered from exposure to warming-, hypoxia- or exercise-based physiological challenges sufficient to induce LOE. Regardless of the stressor used, the fish recovered rapidly, returning to routine MO₂ within about 3 hours. Fish recovering from hypoxia and warming had similar maximum MO₂, aerobic scopes, recovery times and total excess oxygen consumption post challenge, while fish exposed to the exhaustive chase challenge had a 52% increase in maximum MO₂, a 75% and 151% increase in factorial and absolute aerobic scopes respectively (Figure 3).

Maximum MO2 (a), factorial aerobic scope (b), and absolute aerobic scope (c) of fish recovering from a thermal, hypoxia, or exercise challenge that induced loss of equilibrium.

Figure 3: Maximum MO₂ (a), factorial aerobic scope (b), and absolute aerobic scope (c) of fish recovering from a thermal, hypoxia, or exercise challenge that induced loss of equilibrium.

Fish recovering from a hypoxia or exhaustive chase challenge maintained elevated MO₂ for longer periods than control animals (Figure 4). Although previous work has demonstrated that MO₂ increases with temperature, this study may be the first to successfully track MO₂ during recovery from a temperature trial and thus confirms the existence of an analogous excess post-warming oxygen consumption.

Recovery time and total O2 debt of fish recovering from a thermal, hypoxia, or exercise challenge that induced loss of equilibrium.

Figure 4: Recovery time and total O₂ debt of fish recovering from a thermal, hypoxia, or exercise challenge that induced loss of equilibrium.

Conclusions

Characterizing the dynamics of metabolic rate during recovery has important implications for understanding the ecophysiology of fish. Extended recovery from natural- or human-induced challenges such as angling, wastewater exposure, or surmounting barriers can impact fitness by constraining the aerobic scope available for digestion, locomotion and reproduction. Quantifying and comparing the recovery processes is especially important in the context of multiple stressors. Prolonged recovery times effectively extend the effects of a stressor and put animals at an increased risk of interactive effects such as increased vulnerability to a subsequent stressor or the development of cross-tolerance.

The study found that rainbow darter showed considerable daily variation in MO₂ and are capable of rapid recovery from severe warming, hypoxia, and exercise, which may reflect the physiological demands of its life history. Though exhaustive exercise induced a greater maximum MO₂ than warming or hypoxia, all three stressors led to the same level of disruption and impairment. Thus, using LOE as an unambiguous and reliable indicator may be a viable conceptual approach for investigators interested in questions related to multiple stressors, cross tolerance and how animals cope with challenges to homeostasis. Future comparisons between excess post-exercise, hypoxia or warming oxygen consumption may explain the shared or distinct molecular biology, biochemistry or physiology involved in coping with environmentally relevant stressors.