DOI: 10.5061/dryad.kwh70rz6t
title: Higher ultraviolet skin reflectance signals submissiveness in the anemonefish, Amphiprion akindynos

description: Ultraviolet (UV) vision is widespread among teleost fishes, of which many
 exhibit UV skin colours for communication. However, aside from its role in
 mate selection, few studies have examined the information UV-signalling
 conveys in other socio-behavioural contexts. Anemonefishes (subfamily,
 Amphiprioninae) live in a fascinating dominance hierarchy, in which a
 large female and male dominate over non-breeding subordinates, and body
 size is the primary cue for dominance. The iconic orange and white bars of
 anemonefishes are highly UV-reflective, and their colour vision is
 well-tuned to perceive the chromatic contrast of skin, which we show here
 decreases in the amount of UV-reflectance with increasing social rank. To
 test the function of their UV-skin signals, we compared the outcomes of
 staged contests over dominance between size-matched Barrier Reef
 anemonefish (Amphiprion akindynos) in aquarium chambers viewed under
 different UV-absorbing filters. Fish under UV-blocking filters were more
 likely to win contests, whereas fish under no-filter or neutral-density
 filter were more likely to submit. For contests between fish in no-filter
 and neutral density filter treatments, light treatment had no effect on
 contest outcome (win/lose). We also show that sub-adults were more
 aggressive towards smaller juveniles placed under a UV filter than a
 neutral density filter. Taken together, our results show that
 UV-reflectance or -contrast in anemonefish can modulate aggression and
 encode dominant and submissive cues when changes in overall intensity are
 controlled for.
description: <strong><em>Experiment 1: effect of UV on staged
 contest outcome</em></strong> To test the
 importance of UV-signalling in anemonefish interactions, fish were paired
 against size-matched opponents (n = 70 trials) within 1.0 cm SL
 (Δ<sub>SL</sub> range = 0.0 cm to 0.85 cm; mean
 Δ<sub>SL</sub> ± s.d. = 0.32 ± 0.24 cm). Fish were used in one
 to six contests, depending on the number of size-matched pairings (average
 number of contests per fish ± sd = 3.0 ± 1.5). No repeat pairings, nor
 pairs comprised of fish from the same anemone were made. Each pairing was
 pseudo-randomly assigned to one of three treatments: 1) no-filter versus
 UV-filter (n trials = 21), 2) no-filter versus ND (n trials = 15), and 3)
 ND versus UV-filter (n trials = 34). At the beginning of each contest,
 fish from each pair were transferred by hand-net into one section of the
 tank. They were given an adjustment period of 30-min with an opaque
 barrier placed in front of the acrylic window to prevent any pre-contest
 interactions. Immediately prior to contest commencement, the barrier was
 removed, and interactions were video recorded for 20-min using a Go-Pro
 (Hero 3) mounted on a tripod with a full view of the experimental tank. At
 20-min, the contest was terminated, and fish were returned to their
 aquaria. Analysis of the footage involved recording key
 agonistic and submissive behaviours when anemonefish interacted with each
 other at the acrylic window. The outcome (winner and loser) of contests
 was decided by the first observation of submissive posturing displayed by
 either fish, subsequently deemed the loser. Because of the dual role that
 head-shaking serves in agonism and submissiveness, and the occasional
 subtlety between dorsal leaning and ventral leaning, we avoided ambiguity
 by adhering to a strict criterion that postures were only submissive when
 immediately preceded by an attack (i.e., charge or bite) from the
 opponent.  Upon observing submission, the video time stamp (in seconds),
 and outcome (loser and winner fish IDs and corresponding treatments) was
 recorded and footage analysis was terminated. 
 <em><strong>Experiment 2: effect of UV on aggression
 directed at subordinates</strong></em> To
 further investigate the potential role of UV-signalling in anemonefish
 social interactions, we compared the effect of UV light and reduced UV
 light on the level of aggression directed by larger fish at smaller
 (less-dominant) fish. In this experiment, anemonefish pairings had a SL
 difference over 1.0 cm (Δ<sub>SL</sub> range = 1.1 cm – 3.5
 cm; mean Δ<sub>SL</sub> ± s.d. = 2.1 ± 0.72 cm), which
 exceeded the minimum threshold size difference between adjacent
 hierarchical ranks. This perceived difference in rank was further
 emphasised by almost exclusively pairing sub-adults with juveniles. Prior
 to trial commencement, individual fish were randomly assigned (via coin
 toss) to either the UV-filter or ND-filter treatment and similar to the
 previous experiment were given a 30-min adjustment period with an opaque
 barrier placed in front of the acrylic window to prevent any prior
 interaction. Trials commenced after removing the opaque barrier and were
 video recorded for 20-min, after which video recording stopped and fish
 were returned to their aquaria. Footage analysis
 involved counting the number of different agonistic behaviours directed by
 the larger fish towards the smaller fish at the acrylic window. Behaviours
 exhibited by smaller fish were limited to typical submissive postures in
 response to attacks. Trial footage was analysed for the full recorded
 20-min (n = 20). <em><strong>Skin colour
 measurement and analysis</strong></em>
 After the behavioural experiments were completed, the spectral
 reflectance of multiple skin patches (Figure 2) was measured for
 anemonefish (n = 47) used in the experiment. Measurements were done across
 a 300-700 nm range using an Ocean Optics Jaz spectrometer (Ocean Optics,
 Largo, FL, USA) with pulsed Xenon light source (Jaz-PX module), and a
 bifurcated 200 µm fibre optic cable (Thorlabs, NJ, USA). Reflectance
 measurements were taken at a 45° angle against skin patches, relative to a
 Spectralon 99% diffuse reflectance standard (Lab-sphere, North Sutton, NH,
 USA). All measurements were taken out-of-water and in a dark room by two
 personnel. One person maintained the fibre in position above skin patches,
 while the second person took measurements and gradually exposed skin
 patches caudal-rostrally by rolling back a seawater-soaked towel that
 wrapped fish to protect their skin from desiccation and eyes from the
 bright light. We ensured consistency among measurements by targeting the
 same locations across anemonefish and compensated for changes in body
 contour/relief by adjusting the held distance of the fibre accordingly.
 Three measurements were taken per skin patch (within 0.5
 cm<sup>2</sup> – 1 cm<sup>2</sup>) and the average
 was used in subsequent analyses. To minimise stress from air exposure, all
 measurements were taken within two min per fish and no stress-related
 colour changes were observed. Post-measurement, all fish promptly
 recovered in a bucket of seawater and were then returned to aquaria. A low
 number of measurements (39/225, ~ 17%) were discarded due to either being
 saturated or containing artefacts. Sea anemone
 (<em>Stichodactyla gigantea</em>) reflectance was based on
 averaged (n = 10) measurements recorded in-situ using a submersible
 spectrometer (USB2000 Ocean Optics) with a 100µm fibre and relative to a
 99% Spectralon reflectance standard positioned next to the anemone.
 Reflectance measurements of tentacles used natural daylight as a light
 source at midday during non-overcast conditions at ~3m depth.
 Downwelling spectral irradiance measurements were taken at ~5m
 depth on the reef (average of n = 3) using a submersible spectrometer
 (USB2000 Ocean Optics) with a 100µm fibre and cosine corrector.
description: Microsoft Excel Windows Media Player (or
 equivalent video player) File (de-)compression
 software.