DOI: 10.5526/erdr-00000243
title: Warming effects on trout energetic efficiency

description: Data and R code from metabolic and feeding rate experiments conducted across streams of different temperature in the Hengill geothermal valley in Iceland. Fish were collected from one cold and two warm streams in the system and acutely exposed to different experimental temperatures (using the natural temperature gradient of the streams) to measure the mass and temperature dependence of metabolic and feeding rates. Metabolism experiments lasted 2.5 hours and were conducted in situ in 7.2 litre plastic chambers containing a dissolved oxygen probe. Feeding rate experiments lasted approximately 24 hours and were conducted in situ in 250 mm diameter x 300 mm height cylindrical plastic arenas with 20 individuals of the snail Radix balthica or blackfly larvae from the Simuliidae family and a rock for shelter. The energetic efficiency of brown trout was calculated as the dimensionless ratio of feeding rate to metabolic rate. At the end of the experiments, a subset of fin clips were taken for population genetics, with 17 microsatellites genotyped.
description: The study was conducted from 20th May to 3rd June 2018 in the Hengill valley, Iceland, which consists of numerous spring-fed streams flowing into the Hengladalsá river. Mean annual stream temperatures range from 3-20 °C due to differential geothermal heating of groundwater through the bedrock. Importantly, the indirect nature of this heating means that physical and chemical characteristics are very similar across all streams, with no significant difference among streams in pH, sulphate, and key nutrients and minerals. A total of 86 brown trout (65–180 mm fork length) were collected from three streams in the system where they are particularly abundant: 44 from a cold stream (IS12 with a mean annual temperature of 7.8 ± 4.2 standard deviations °C); and 42 from two warm streams (21 from IS1 = 11.3 ± 4.0 °C and 21 from IS5 = 13.8 ± 1.6 °C).

Prior to transplantation to the experimental streams the trout were maintained in their home streams in cylindrical white plastic arenas (250 mm diameter, 300 mm height). Each arena contained two holes situated directly opposite each other (70 mm diameter, 10 mm from the base), which were covered by 500 μm mesh to allow in-flow of oxygenated stream water, whilst preventing in- and out-flow of potential macroinvertebrate prey. The arenas were partially submerged in the streams such that half of the interior was filled with water, leaving the trout access to the surface as in the natural system. Metal rebars were taped to the arenas and hammered into the stream bed to hold them in place, and a rock was placed inside the arena and on top of the lid to weigh it down. The fish were starved for 48 hours prior to experiments to standardise their hunger levels, whilst preventing metabolic down-regulation due to extended starvation.

Oxygen consumption rates were used as a proxy for metabolic rates and were measured in situ in five streams in the system: IS1, IS5, IS8, IS11, and IS16. Here, these experimental streams acted as a sort of natural laboratory, whereby we could conduct metabolic rate experiments at different temperatures (in the experimental streams to which fish from the three source streams were transplanted) without needing to bring fish back to the laboratory to do so in an artificial setting under temperature-controlled conditions. These experimental streams were chosen to best span the range of available temperatures during sampling (4.6 to 19.7 °C). Note that although fish from IS12 (cold source stream) were included as one of the three study populations, IS12 itself was not included as an experimental stream because it is much further from the others, so would have involved transporting fish over greater distances and was not needed to evenly span the temperature gradient. Static respirometry experiments were conducted in 7.2 L circular plastic chambers (LocknLock, South Korea) with a lid that could be opened and closed to create an airtight seal. Before placement in the streams, each chamber was fully submerged in a 50 L plastic container that had been filled with water from the experimental stream, filtered through a 250 µm sieve to remove small organisms or plant matter that may affect the level of background respiration. A single brown trout individual from a given source stream was placed inside each chamber along with a MiniDOT logger (PME, USA) to measure dissolved oxygen concentrations and water temperature every minute. The chamber was then sealed underwater to avoid any air bubbles that may interfere with dissolved oxygen readings and completely submerged in the experimental stream, with a rock on the lid to secure it to the stream bed. Up to ten chambers containing fish were placed in the experimental stream for each block of metabolic experiments, whilst an extra chamber without any fish was included as a control for measuring background respiration. This typically involved five fish from the cold source stream (IS12) and five fish from the warm source streams (e.g. two from IS1 and three from IS5, or vice versa). Occasionally, an experimental run had less than ten fish, but we ensured a balanced number of cold and warm origin fish were used in these cases. Note that fish were transported in buckets from their stream of origin and placed in the experimental stream for 30-60 minutes prior to being introduced into the metabolic chambers to help equalise temperature and avoid a stress response.
The experiments ran for at least 2.5 hours, after which time the fish was removed, its body length was measured, and it was subsequently used in a feeding rate experiment (see below). Oxygen depletion was noticeably linear and relatively stable, suggesting that the fish experienced minimal stress during the experiments. Nevertheless, the first half hour of data was excluded as an acclimation period for the fish to overcome any effects of handling and adjust to their new environment. To ensure a consistent duration across all experiments, we analysed the subsequent 2 hours of data using a linear regression of oxygen consumption over time to obtain the oxygen consumption rate [mg O2 L^-1 h^-1], which was corrected to a whole organismal rate [mg O2 h^-1] by multiplying by the volume of the chamber minus the volume of the fish (assuming a density of 1,000 kg m^-3). To account for background respiration, we subtracted the slope of the control data for the corresponding time period. We excluded any data where the final rate had r^2 &lt; 0.8 (5 of 91 experiments). Experiments typically took place in the morning to mid-afternoon when the fish were quite active in the chambers, and so the measurements can be considered an active field metabolic rate.

Following each metabolism experiment, the fish were immediately used in a feeding rate experiment conducted in the same experimental stream. Before each experiment, 200 freshwater snails (Radix balthica) and 200 blackfly larvae (Simulium vittatum) were hand-collected from an independent stream (IS7). Both taxa are naturally present in the streams and also well represented in the diet of trout in IS1, IS5, and IS12. Twenty individuals of a particular taxon were added to each of twenty cylindrical arenas (identical to those used to store fish before the metabolic experiments), with ten arenas containing snails and ten containing blackfly larvae. The arenas were secured in the experimental stream with metal rebars and rocks. An individual trout was then added to each arena, ensuring that five arenas for each prey taxon contained trout from the cold stream and five contained trout from the warm streams. The experiments ran for approximately 24 hours, after which time the remaining prey individuals were counted, and fish were released back into their home streams. Adipose fins were removed for genetic analysis prior to release and thus also ensuring that no fish would be reused in any further experiments. The feeding rate of fish [individuals h^-1] was calculated as the number of prey eaten by the fish in an arena divided by the duration of the experiment in hours.

To determine the potential impact of source-stream temperature on the energetic constraints of brown trout evaluated across different experimental temperatures, we calculated a dimensionless energetic efficiency as the ratio of feeding rate (multiplied by assimilation efficiency) to metabolic rate. This approach enabled us to calculate an energetic efficiency for both prey taxa in energetic equivalents [J h^-1]. Oxygen consumption rates were converted to energetic equivalents using the density of O2 (1.429 g L 1) and a standard energy conversion (1 ml O2 = 20.1 J). Feeding rates were converted to energetic equivalents using the average body mass of R. balthica and S. vittatum from IS7 and the caloric content of Gastropoda and Simulium spp. from an established database of energetic equivalents. At the end of all experiments, fin clips were taken for population genetics and preserved in 96% ethanol, with a total of 17 microsatellites genotyped.