DOI: 10.5061/dryad.51c59zw8r
title: Gouldian finch metabolic data for Buttemer et al. 2021 jeb242577

description: Evidence from a number of species suggests behaviours associated with
 social rank are positively correlated with metabolic rate. These studies,
 however, are based on metabolic measurements of isolated individuals,
 thereby ignoring potential effects of social interactions on metabolic
 rates. Here, we characterised three pertinent metabolic indices in the two
 predominant genetic colour morphs of the Gouldian finch (Erythrura
 gouldiae): diurnal resting metabolic rate (RMR), nocturnal basal metabolic
 rate (BMR), and exercise-induced maximal metabolic rate (MMR). Research
 reveals red-headed morphs consistently dominate the less aggressive
 black-headed morphs and the two morphs to differ in other behavioural and
 physiological traits. We measured daytime RMR of intermorph naïve birds
 (first-year virgin males maintained in total isolation from opposite
 colour morphs) and their metabolic responses to viewing a socially
 unfamiliar bird of each colour. Subsequently, each bird was placed in a
 home cage with an opposite colour morph (intermorph exposed) and the
 series of measurements repeated. Daytime RMR was indistinguishable between
 the two morphs, whether intermorph naïve or intermorph exposed. However,
 both red- and black-headed birds showed a greater short-term increase in
 metabolic rate when viewing an unfamiliar red-headed bird than when seeing
 a black-headed bird, but only when intermorph naïve. Measurements of BMR
 and exercise-induced MMR did not differ between the two morphs, and
 consequently aerobic scope was indistinguishable between them. We propose
 that the suite of behavioural differences between these two sympatric
 morphs are functionally complementary and represent evolutionary stable
 strategies permitting establishment of dominance status in the absence of
 metabolic costs.
description: <b>Animals and experimental design</b>
 We used 24
 black-headed and 24 red-headed captive-reared wild-type male Gouldian
 finches. Birds had been raised by parents of the same colour and, upon
 fledging, were housed alone in complete social isolation (Pryke et al.
 2007). While in seclusion and prior to experiments, juveniles transitioned
 from the drab grey/olivaceous immature plumage common to both morphs to
 the brightly coloured adult plumage of their male parent. Two cohorts of
 first-year virgin males were studied (one in 2007 and one in 2009); both
 involved 12 intermorph naïve birds of each colour morph. Upon arrival at
 the University of Wollongong, visual isolation of the two colour morphs
 was maintained by partitions between cages. Birds of the same colour morph
 were placed 2 per cage (34 cm × 45 cm × 45 cm; Hoei Cage Co., Japan).
 Cages were fitted with multiple perches and one dispenser each for
 commercial finch seed (Golden Cob, Mars Birdcare Australia), mineralised
 grit, and water, all available <i>ad libitum</i>. Individuals
 of both cohorts remained with their initial cage partners for two weeks
 before metabolic measurements commenced.
 <b>Experiment 1: Metabolic response to viewing a socially
 unfamiliar bird of each colour morph</b>
 <i>Phase 1: All birds intermorph naive</i>
 This involved two sets of metabolic measurements that took place
 on consecutive days (Tests 1 and 2), with Test 1 involving a pair of birds
 of the same or opposite colour morphs; for Test 2 each bird was exposed to
 a morph of the opposite colour of that viewed in Test 1. Experimental
 pairs were randomly selected from all birds other than either bird’s cage
 partner. The sequence in which each bird viewed the same or opposite
 colour morph was varied among birds to balance morph colour combinations
 for Tests 1 and 2. <i>Phase 2:</i>
 <i>All birds intermorph exposed</i> Upon
 completion of Phase 1, birds were transferred to cages containing an
 individual of the opposite colour morph for five days. Subsequently, two
 sets of metabolic measurements (Tests 3 and 4) were made on consecutive
 days (we define these individuals as ‘intermorph exposed’). As in Phase 1,
 the sequence in which each bird viewed the same or opposite colour morph
 to itself on consecutive days was varied among all birds to balance morph
 colour combinations for Tests 3 and 4.
 <b>Experiment 2: Relation between colour morphology and
 aerobic scope in intermorph exposed birds. </b>
 Birds from the 2009 cohort were placed as mixed morph groups in
 outdoor flight cages (2.5 m × 3 m × 4 m, 12 birds per cage) following
 completion of Experiment 1. Cages had multiple feeding sites, extensive
 perching locations, and shelter from weather. After at least 4 days in
 aviaries, individuals were collected about mid-morning through midday to
 measure exercise-induced maximal metabolic rates (MMR). Upon completing
 MMR measurements, birds were placed in individual holding cages with free
 access to food and water. The following day, food was removed three hours
 before initiation of nocturnal measurements of basal metabolic rate
 (BMR).  <b>Metabolic Measurements
 </b> <i>Experiment 1</i>
 We measured resting metabolic rate
 (RMR) during the day using an open-circuit respirometry system that
 allowed us to continuously monitor two metabolic chambers simultaneously.
 For a given metabolic series (Tests 1 through 4), birds were removed from
 holding cages, had body mass (M<sub>b</sub>) measured on a
 digital scale (±0.01g; Model OHAUS AV413C), and then placed individually
 in 1.5l chambers fashioned from sealable polycarbonate plastic food
 containers. Chambers were optically transparent, rectangular, and fitted
 with a perch at 1/3 height, as well as inlet and outlet tubes for airflow.
 The chamber containing the first bird was positioned in the
 constant-temperature cabinet and then screened with an opaque partition
 before positioning the second bird’s chamber within 2 cm of one another.
 The perches were oriented perpendicularly to the partition, resulting in
 mutual viewing irrespective of perching location. Overhead fluorescent
 lighting within the temperature-controlled cabinet illuminated the birds
 evenly and a webcam was used to monitor behaviour. Cabinet temperature was regulated at 30°C,
 which is within the thermoneutral zone for Gouldian finches (Burton and
 Weathers, 2003). Mass-flow controllers (Mykrolis, model FC-2902V-T)
 provided a constant air supply of 500 ml min<sup>–1 </sup>STP
 of dry air into each metabolic chamber. The outflows of each chamber and
 sequential sampling of inlet air were subsampled (approximately 100 ml
 min<sup>–1</sup>). Subsampled air was passed through Drierite
 and soda lime to remove water and CO<sub>2</sub>,
 respectively, en route to a two-channel O<sub>2</sub> analyser
 (Oxilla II; Sable Systems International; Henderson, NV, USA). We used
 LabHelper software (warthog.ucr.edu) to control the multiplexer outputs
 and read chamber O<sub>2</sub>-concentration at 1-sec
 intervals. We used LabAnalyst (warthog.ucr.edu) to correct oxygen readings
 for drift between consecutive baselines and to calculate
 O<sub>2</sub> consumption rates ( O<sub>2</sub>, ml min<sup>-1</sup>) according to Eqn. 2 of Hill (1972). Oxygen consumption was recorded continuously after closure of the cabinet door. Video monitoring showed that birds usually settled within a few minutes, and their metabolic rates declined steadily, reaching a plateau about 30 to 60 min after door closure. About 10 min after both birds exhibited a stable RMR, the partition was lifted so birds could see one another, and  O<sub>2</sub> was recorded for a further 60 minutes. Resting MR (RMR) was identified as the lowest 3-min running mean  O<sub>2</sub> recorded over the entire measurement period. This duration was selected as birds tended to be restive under full illumination and periods of sustained rest levels of metabolic rate rarely exceeded 5 min. Peak MR (MR<sub>peak</sub>) was designated as the highest 3-min running mean  O<sub>2</sub> recorded after the partition was lifted. These measurements were used to evaluate the maximum factorial increase in MR associated with birds viewing one another, which we calculated as MR<sub>peak</sub>/RMR. The average  O<sub>2</sub> over the entire 60-min period that birds viewed one another was designated as MR<sub>mean</sub>. <i>Experiment 2</i> Procedures used for measuring basal (BMR) and exercise-induced maximal metabolic rates (MMR) are described in detail elsewhere (Buttemer et al. 2019). In brief, for MMR tests, birds were collected from the flight cages and placed individually in a hop-flutter wheel which was rotated to elicit maximal oxygen consumption during exercise. Rotation speed was dynamically adjusted to each bird’s pattern of movement to achieve maximal activity until they exhibited exhaustion. Data were adjusted with ‘instantaneous’ conversion procedures to account for gas mixing characteristics of the wheel and accurately resolve short-term changes in  O<sub>2</sub> (Chappell et al. 1999; Buttemer et al. 2019). The peak 30-sec instantaneous rate of  O<sub>2</sub> during exercise was designated as MMR. Following MMR tests, birds were placed in holding cages (2 birds per cage) with free access to food and water. BMR tests were performed on these birds the following evening, with food removed from their holding cages three hours prior to measurements. Birds were placed in 2-l metal metabolic chambers for overnight measurements of  O<sub>2</sub> under thermoneutral conditions (30°C) in total darkness. BMR was calculated as the minimum 5-min running average  O<sub>2</sub> over the entire night; BMR obtained using this averaging interval was significantly repeatable in another study of similar-sized passerine birds (Careau et al. 2014a).
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