DOI: 10.7280/d1mt39
title: Multigenerational exposure to increased temperature reduces metabolic rate but increases boldness in Gambusia affinis

description: Acute exposure to warming temperatures increases minimum energetic
 requirements in ectotherms. However, over and within multiple generations,
 increased temperatures may cause plastic and evolved changes that modify
 the temperature sensitivity of energy demand and alter individual
 behaviours. Here, we aimed to test whether populations recently exposed to
 geothermally elevated temperatures express an altered temperature
 sensitivity of metabolism and behaviour. We expected that long-term
 exposure to warming would moderate metabolic rate, reducing the
 temperature sensitivity of metabolism, with concomitant reductions in
 boldness and activity. We compared the temperature sensitivity of
 metabolic rate (acclimation at 20 versus 30°C) and allometric slopes of
 routine, standard, and maximum metabolic rates, in addition to boldness
 and activity behaviours, across eight recently divergent populations of a
 widespread fish species (Gambusia affinis). Our data reveal that
 warm-source populations express a reduced temperature sensitivity of
 metabolism, with relatively high metabolic rates at cool acclimation
 temperatures and relatively low metabolic rates at warm acclimation
 temperatures compared to ambient-source populations. Allometric scaling of
 metabolism did not differ with thermal history. Across individuals from
 all populations combined, higher metabolic rates were associated with
 higher activity rates at 20°C and bolder behaviour at 30°C. However,
 warm-source populations displayed relatively bolder behaviour at both
 acclimation temperatures compared to ambient-source populations, despite
 their relatively low metabolic rates at warm acclimation temperatures.
 Overall, our data suggest that in response to warming, multigenerational
 exposure (e.g., plasticity, adaptation) may not result in trait change
 directed along a simple “pace-of-life syndrome” axis, instead causing
 relative decreases in metabolism and increases in boldness. Ultimately,
 our data suggest that multigenerational warming may produce a novel
 combination of physiological and behavioural traits, with consequences for
 animal performance in a warming world. 
description: Fish acclimation   Fish from eight populations
 were acclimated into 16 20L tanks (2 tanks per population) in the
 laboratory, with each tank containing fish from a single population. We
 randomly allocated ~12 fish from a population to a tank (n = 198 fish
 total; details in S1). In each tank, we separated males and females using
 dividers to minimise sexually antagonistic interactions that can affect
 survival; however, mosquitofish females store sperm, so most females were
 pregnant during the time of trait measurements, as they would be in
 nature. Each population was acclimated to two experimental temperatures
 (20 ± 0.5 and 30 ± 0.5°C) over four months. Tank temperatures were
 initially set to the collection temperature for a given population and
 then adjusted by increasing or decreasing the set temperature of aquarium
 heaters by a maximum of 1°C every two days until the target temperature
 was reached. We started with water from the appropriate field site in each
 aquarium combined with treated tap water to remove chlorine (API Stress
 Coat) and progressively replaced it with treated water over two weeks. We
 fed fish twice daily by hand to satiation with freeze-dried Daphnia and
 Nutrafin MAX small tropical fish micro-granules and maintained a light
 cycle of 12:12 throughout the experiment. Each aquarium had artificial
 macrophytes and stones to provide refuge. Water was continuously filtered
 using sponge air filters, which we cleaned every second day. Fish
 mortality was low in most populations (see Table S1). We fasted
 individuals for 24 hours before measuring behavioural and metabolic traits
 to control for food digestion. Metabolic Rate
 We measured metabolism as maximum metabolic rate (MMR), standard
 metabolic rate (SMR), and routine metabolic rate (RMR). MMR is the maximum
 metabolic rate of an individual and sets the upper limit on organismal
 metabolic performance (Fry, 1971). In contrast, SMR is the minimum
 metabolic rate, measured after rest, with no digestion cost, on
 non-stressed fish and sets the lower requirement of an animal to sustain
 life. RMR was measured under similar conditions as SMR but allowed for
 some activity and sits between SMR and MMR. As RMR incorporates variation
 in activity between individuals, it may closely relate to behavioural
 traits (Mathot & Dingemanse, 2015). We measured
 RMR and MMR using static respirometry and SMR using intermittent
 flow-through respirometry at each fish’s acclimation temperature
 (Steffensen, 1989; Clark, Sandblom, & Jutfelt, 2013). We used
 respirometers comprising 40 mL acrylic chambers with magnetic stir bars in
 the chamber base to ensure water mixing throughout our oxygen measures in
 all assays. We measured metabolic rate as oxygen consumption (MO2) using a
 FireSting four-channel oxygen logger with optical oxygen sensors
 (PyroScience, Germany). Respirometers were placed into 80 L aquaria,
 filled with treated tap water, fitted with a UV filtration system, an
 aerator, and a 100W aquarium heater.  Immediately
 following behavioural trials (see below), we measured RMR by placing
 individuals into chambers and measuring oxygen consumption over 15
 minutes. Chambers were then connected to a recirculating pump and slowly
 flushed with oxygenated water for five minutes before beginning SMR
 measurements. Oxygen consumption measurements for SMR were taken overnight
 over an approximately 18-hour period. A computer-controlled aquarium pump
 intermittently flushed chambers for five minutes to ensure a complete
 turnover of water inside the chambers, then an oxygen measurement period
 of 15 minutes began after a 30 second wait period. We controlled oxygen
 flow and data logging through a PC using the software ‘AquaResp’
 (Svendsen, 2017). Following SMR measurements, we measured MMR using an
 exhaustive chase protocol to induce maximum oxygen consumption (Clark et
 al., 2013; Norin & Clark, 2016). Fish were removed from chambers
 one by one and placed into a circular tank; in this tank, we used an
 aquarium net to chase the fish until exhaustion (defined as the lack of
 ability for burst swimming) (Norin & Clark, 2016). Fish were then
 immediately placed into a static respirometer, and oxygen consumption was
 measured for 5 minutes. We chose to measure MMR after SMR measurement to
 ensure our SMR measurement accuracy as metabolic rates may remain elevated
 for long periods after exhaustive exercise. We immediately euthanised the
 fish following the measurement of MMR using clove oil. Fish were then
 measured for mass, length, sex, and volume, then dried at 60°C for 48
 hours and re-weighed for dry mass. We controlled for
 microbial oxygen consumption in our metabolism assay water by subtracting
 the oxygen consumption in blanks (respirometers with water only), which
 were run before and after every trial. We assumed a linear increase in
 microbial oxygen consumption between measurements in blanks. 
 We calculated each SMR, MMR, and RMR as; 
 〖MO〗_2=(V_r-V_f )×〖ΔC〗_wO2/Δt                                   
                                                              
 Where: MO2 is oxygen consumption rate, Vr is respirometer volume,
 Vf is fish volume, ΔCwO2 is the change in oxygen concentration, Δt is the
 change in time. We calculated SMR using the mean of the
 lowest 10 % of all measurements, excluding any outliers (± two standard
 deviations [SD] from the mean), aerobic scope as MMR-SMR, and factorial
 aerobic scope as MMR/SMR (Clark et al., 2013; Chabot, Steffensen,
 & Farrell, 2016).  Behaviour
 Immediately before measuring metabolism, we conducted behavioural
 assays on individuals in a 60 L aquarium with a water depth of 20 cm and
 temperature set to the acclimation temperature. We fit the aquarium with
 an air pump and a UV filtration system to maintain high oxygen saturation
 and control microbial respiration. We measured individual’ boldness’ as
 latency to exit a refuge and individual ’‘activity' as time spent
 exploring a novel environment (Cote et al., 2010; Wilson, Godin, &
 Ward, 2010). For these behavioural measures, we placed individuals into a
 small enclosed and darkened area (‘refuge,’ 10cm × 30cm) at one end of the
 60 L aquarium. The aquarium was covered on all but one side to allow for
 observation. In the refuge, we provided artificial macrophytes and river
 stones. Fish were left in the refuge for 10 minutes before a 4 × 4 cm door
 was opened remotely, allowing fish to exit and explore the remainder of
 the tank (‘open area’). In the open area, we placed macrophytes opposite
 the refuge opening as a visual cue for exploration. We measured boldness
 using a stopwatch as the time it took the fish to leave the refuge. Fish
 that did not leave were assigned a maximum latency time of 600 seconds and
 were not measured for activity as forced tests may measure anxiety or fear
 traits (Brown, Burgess, & Braithwaite, 2007). Once the fish began
 exploring, we video-recorded their movement and later measured activity as
 time spent moving (versus remaining stationary) over five minutes
 following their emergence from the refuge
description: Males
 have no data in the pregnancy column 
 Individuals that
 did not leave the refuge were not measured for activity and have no data
 in the "Time_spent_exploring_s" column
 NA's in dataset indicate missing data
 Column descriptions/  units: 
 <strong>Site</strong> refers to the location where
 <em>Gambusia </em>were collected 
 <strong>Geothermal</strong> refers to the designation of sites
 as geothermal or ambient, geothermal sites received warm water inputs and
 ambient sites experience daily and seasonal changes in environmental
 temperature. 
 <strong>Source_Temp</strong> refers to the temperature of the
 site at the time of fish collection in degrees Celcius.
 <strong>Temp_lab</strong> refers to the temperature
 that fish were acclimated in the laboratory in degrees Celcius
 <strong>Run </strong>refers to the order in which
 metabolic rate was measured on fish 
 <strong>Fish_ID </strong>is a unique identifier for each fish
 in this study. <strong>Sex
 </strong>refers to the sex of the fish measurements were done on (M
 = male, F = female). <strong>Pregnancy
 </strong>describes if the fish was visibly pregnant at the time of
 metabolic rate measurement, where Y indicates yes or that the fish was
 pregnant and N indicates no or that the fish was not pregnant.
 <strong>length</strong>: fish length
 (mm) <strong>dry_mass_mg</strong>: fish
 dry mass (mg) 
 <strong>dry_mass_g</strong>: fish dry mass (g)
 <strong>Excretion_rate: </strong>Fish excretion
 rate (ammonium and NO<sub>X</sub>) measured immediately
 following RMR (µg N min<sup>-1</sup>
 fish<sup>-1</sup>) 
 <strong>Time_to_leave_s:</strong> Time the fish took to leave
 the refuge, maximum latency was 600 seconds (seconds)
 
 <strong>T</strong>i<strong>me_spent_exploring_s</strong>: Time the fish spent exploring after leaving the refuge (seconds) <strong>SMR_rate_fish</strong>: standard metabolic rate of each fish (µg min<sup>-1</sup> fish<sup>-1</sup>) <strong>RMR_rate_fish</strong>: routine metabolic rate of each fish (µg min<sup>-1</sup> fish<sup>-1</sup>) <strong>MMR_rate_fish</strong>: maximum metabolic rate of each fish (µg min<sup>-1</sup> fish<sup>-1</sup>)