## 2 Materials and Methods
Fish were placed in the chamber with the circulation of water from the reservoir to prevent hypoxia throughout the acclimation. The fish acclimated for 60 min since no change in FMR was observed after the acclimation of this duration. FMR was then measured for 10 min after the open-loop pump was turned off, with measurements being taken every second (modified from [PERSON] et al. [4]). The MMR was measured by holding fish out of water for two minutes and immediately placing fish into the respirometer where the initial decline in O\({}_{2}\) is the MMR. This was modified from the chase protocol due to darters not reacting to being chased [39]. The average temperature of each run was used to correct the measurements of FMR and MMR to 20 \({}^{\circ}\)C for comparison using:  
\[B/M_{20^{\circ}\mathrm{C}}\ =\ B/Mte^{\kappa(1/20-1/t)},\]  
where \(t\) is the body (water) temperature and \(\alpha\) (\(-5.02\)) is the slope of the line for fish for the equation comparing the effects of temperature to the mass normalized metabolic rate [40]. Trials started at 9:30 a.m. and lasted until approximately 3 p.m. each day, spreading out the use of each species throughout the day to minimize the effects of diurnal fluctuations. There was no significant effect of sex on the metabolic rate when data were separated. Additionally, there were low sample numbers; therefore, samples were pooled together for greater power (<4% of the variance in all species). Gill tissue was examined histologically for changes in morphology and evidence of pathologies at all sites, using US1 as the reference site. Archived gill samples from RBDs in October of 2013 and 2014 were used for comparison as a pre-upgrade reference. Gill tissue was fixed in Davidson's solution for 24 h, then moved to 70% ethanol. Samples were embedded in paraffin wax, sliced at 5 um until whole gill arches, filaments, and lamellae were seen, and then stained using an automated hematoxylin and eosin stain procedure. Images of the gills were captured using a Leica microscope (DM 1000 LED) and camera (MC170 HD) at 100\(\times\) using Leica Application Suite (LAS version 10) software (Nussloch, Stuttgart, Germany). A random image was taken of the gill section that had at least three primary filaments present with interior cartilage visible to standardize the location of analysis. Images were randomized and measured blindly. The morphological assessment used was described by [PERSON] et al. [41]. Each image had nine measurements of secondary lamellar length (SLL), secondary lamellar width/diameter (SLW), inter-lamellar diameter (ID), and basal epithelium thickness (BET) each, spread along the base, middle, and distal edge of the filament. Measurements were taken using the software Fiji (Fiji Is Just Image) version 1) [42]. Gills were scored for pathologies, using a scale from 1-4 on five gill pathologies to assess the degree of damage in the gill tissue (see Appendix A Table A1 and Figure A2). Hypertrophy is the increase in an individual cell's volume, hyperplasia is cell proliferation, epithelial lifting is the peeling of the epithelium from the sinus of the gill, fusion is the (complete or partial) connecting of secondary lamellae, and edema of the lamellae is the enlargement of the sub-epithelium spaces. The gill scoring was modified from Poleksic and Mitrovic-Tutundzic [43]. Scores of all pathologies were summed and the average total score for samples at that site and species was compared to the reference site for each species. Data were analyzed using the statistical software GraphPad Prism 8 (San Diego, CA, USA). Sex differences were calculated for body measurements but not for metabolic rate due to low \(n\) numbers. Figures present the data as means \(\pm\) standard error of the mean (SEM). Each species-site group was compared using a two-way analysis of variance (two-way ANOVA) separated by season. The data were tested for equal variance and normality with [PERSON]'s test and the Kolmogorov-Smirnov test, respectively, to determine whether they met the assumptions of an ANOVA.