Summary
Acute elevation of cortisol via activation of the hypothalamic-pituitary-interrenal (HPI) axis aids the fish in dealing with a stressor. However, chronic elevation of cortisol has detrimental effects and has been studied extensively in lab settings. However, data pertaining to wild teleosts are lacking. Here, we characterized the metabolic consequences of prolonged cortisol elevation (96 h) in wild-caught pumpkinseed (Lepomis gibbosus). Pumpkinseed were implanted with cocoa butter alone (sham) or containing cortisol (25 mg kg−1 body weight), and at 24, 48, 72, and 96 h, tissue samples were collected, whole-body ammonia excretion was determined, and whole-organism metabolism was assessed using intermittent flow respirometry. Cortisol-treated pumpkinseed exhibited the highest plasma cortisol concentration at 24 h post-implantation, with levels decreasing over the subsequent time points although remaining higher than in sham-treated fish. Cortisol-treated fish exhibited higher standard and maximal metabolic rates than sham-treated fish, but the effect of cortisol treatment on aerobic scope was negligible. Indices of energy synthesis/mobilization, including blood glucose concentrations, hepatosomatic index, hepatic glycogen concentrations, and ammonia excretion rates, were higher in cortisol-treated fish compared with controls. Our work suggests that although aerobic scope was not diminished by prolonged elevation of cortisol levels, higher metabolic expenditures may be of detriment to the animal’s performance in the longer term.
Methodology
Juvenile pumpkinseed (N = 285; mass = 24.3 ± 0.7 g; total length = 110.6 ± 0.4 mm) were captured in Lake Opinicon (44.5590° N, 76.3280° W) in the months of July, August, and September 2016. Animals were collected (OMNRF permit no. 1082340) in shallow weedy bays using a seine net and were transported to Queen’s University Biological Station (Chaffey’s Lock, ON, Canada). Fish were held in flow-through tanks (~ 435 L) supplied with natural lake water (T = 25.2 ± 0.1 °C; NH4+ < 0.25 mg L−1; pH = 7.5) under natural photoperiod. During holding, fish were not fed. Pumpkinseed were allowed to acclimate to these holding conditions for 48 h prior to experimentation. Experimental protocols were approved by the Carleton University Animal Care Committee (AUP no. 104262) in compliance with the guidelines of the Canadian Council for Animal Care.
Pumpkinseed were randomly selected and given an intraperitoneal implant of cocoa butter (5 mL kg−1 body weight (BW)) either alone as a control (sham) or containing cortisol (hydrocortisone 21-hemiscuccinate; 25 mg kg−1 BW). This method of elevating circulating cortisol has been validated for use in teleost fishes (Gamperl et al. 1994). Cocoa butter containing cortisol was prepared as described in Hoogenboom et al. (2011). Briefly, the hydrocortisone 21-hemiscuccinate salt was dissolved in ethanol to distribute the cortisol evenly throughout the cocoa butter (i.e., avoid clumping of the cortisol). Pre-warmed cocoa butter was then added to the mixture and heated to 75 °C to evaporate the ethanol. Handling procedures associated with implantation were not likely to have substantially affected the variables measured here as prior work with bluegill sunfish, a close relative of the pumpkinseed sunfish, demonstrated comparable stress-related parameters between no-treatment controls and sham-treated fish (see McConnachie et al. 2012).
The use of cortisol-containing cocoa butter implants for elevating plasma cortisol concentrations in teleosts is well-known to yield variable results in plasma cortisol titres (reviewed in Gamperl et al. 1994; Sopinka et al. 2015; Crossin et al. 2016). Such cortisol treatment can produce supraphysiological levels of cortisol in the blood that are not reflective of an endogenous cortisol response (e.g., see McConnachie et al. 2012 and Lawrence et al. 2017), or individuals may clear the hormone rapidly (i.e., in a few hours), failing to meet the criterion for chronic exposure and making interpretation of treatment effects difficult (see Foster and Moon 1986). Thus, to avoid confounding effects associated with fish in which the implant was ineffective (plasma cortisol concentration not elevated at the sampling time) or too effective (plasma cortisol concentration elevated beyond the desired maximum of 140 ng mL−1), individuals that exhibited plasma cortisol concentration outside of the desired range of 36 to 140 ng mL−1 within the first 24 h of implantation were excluded from analyses of blood or tissue variables. This approach was not possible for individuals used in respirometry or ammonia excretion trials because plasma samples could only be collected post-experiment, when circulating cortisol levels would have been vulnerable to stress associated with the experiment and handling, and the implants would have been past their effective period. Following implantation, all fish of a single treatment group (25 individuals) were transferred to a holding tank (~ 211 L) under holding conditions as described above. However, pumpkinseed used in 24 h respirometry and metabolite flux experiments (see below) were transferred immediately to respirometry chambers following implantation. All fish were maintained in a fasted state throughout the experiments.