DOI: 10.5061/dryad.0zpc866xw
title: Social Context Affects Camouflage in a Cryptic Fish Species

description: Crypsis, or the ability to avoid detection and/or recognition, is an
 important and widespread anti-predator strategy across the animal kingdom.
 Many animals are able to camouflage themselves by adapting their body
 colour to the local environment. In particular, rapid changes in body
 colour are often critical to the survival of cryptic prey which rely on
 evading detection by predators. This is especially pertinent for animals
 subject to spatiotemporal variability in their environment, as they must
 adapt to acute changes in their visual surroundings. However, which
 features of the local environment are most relevant is not well
 understood. In particular, little is known about how social context
 interacts with other environmental stimuli to influence crypsis. Here we
 use a common cryptic prey animal, the goby (Pseudogobius species 2) to
 examine how the presence and body colour of conspecifics influences the
 rate and extent to which gobies change colour. We find that solitary
 gobies change colour to match their background faster and to a greater
 extent than gobies in pairs. Further, we find that this relationship holds
 irrespective of the colour of nearby conspecifics. This study demonstrates
 the importance of social context in mediating colour change in cryptic
 animals.
description: <strong>Study Species and Holding
 Conditions</strong> The goby Pseudogobius species
 2 (part of a species complex undergoing taxonomic resolution) was used as
 the model species. Members of this genus are widely distributed throughout
 temperate, sub-tropical and tropical waters of the Indo- Pacific,
 occurring in shallow freshwater, marine and estuarine habitats47. The
 range of species 2 extends from southern Queensland to Victoria. It grows
 to a maximum body size of approximately 35mm. Due to the absence of any
 clear sexual dimorphism, adults of both sexes were used for
 experimentation. Like many fish species, this goby is capable of changing
 its body colour in response to variation in the physical environment. As
 is common among Gobiids, this species lacks a swim bladder and is adapted
 to a benthic lifestyle spent in close association with the substrate. This
 species is non-burrowing and demonstrates a characteristic saltatory
 pattern of locomotion, with relatively long periods of stillness
 punctuated by short hops forward. Fish were collected from a field site at
 Middle Creek, Narrabeen (33.718491°S, 151.270281°E) between August and
 October 2020 using large hand-held nets. Fish were captured in shallow
 waters approximately 5-20cm in depth. Permits for capture were obtained
 from the NSW Department of Primary Industries. Following capture, the fish
 were transported in oxygenated water to holding facilities at the
 University of Sydney. There, they were housed in tanks containing
 substrate composed of natural sand and variegated gravel, intended to
 represent a typical physical environment commonly encountered in the wild.
 They were fed daily with commercially available fish food (Nutrafin
 Tropical Flakes, Hagen Products, Germany) and their health was monitored
 prior to testing. <b>Experimental
 Design</b> Fish were exposed to one of two
 acclimation conditions (white or black background) and one of two test
 conditions (again, white or black background), for a total of four unique
 colour treatments. Additionally, fish were tested under one of two social
 treatments; a solitary treatment, in which the fish was tested alone, and
 a group treatment, in which the fish were tested in pairs.. In the group
 treatments fish were exposed to all four colour treatments as well as
 being either matched or mismatched to their partner, resulting in six
 unique social treatments for a total of ten treatments altogether (see
 Table 1). Each of the ten treatments were replicated six times to a total
 sample size of N = 60. Test arenas were constructed
 from acrylic plastic and comprised a central square chamber and two
 removable side chambers with sliding door mechanisms allowing entry to the
 central chamber. The central arena measured 20 x 20 x 20 (L x W x H),
 while the side chambers measured 6 x 6 x 15 (L x W x H). There were two
 arenas, one black and one white, and four side chambers, two black and two
 white. The removable side chambers allowed pairing of any combination of
 colours (e.g. two black, two white, or one of each) with each colour of
 test arena. When side chambers were not matched to the colour of the
 central test arena, black or white duct tape was used to cover the part of
 the side chamber that faced into the central arena. The whole arena was
 placed inside a large plastic tub, and a frame was built around the entire
 apparatus. This frame was covered in white Corflute® in order to minimise
 external disturbance. Within the screens, four LED light batons (Cool
 white, 3300K) were placed, two at each end of the arena along the short
 axes of the tub. Lights were positioned so as to prevent direct
 illumination of the test arena and instead provide diffuse
 light. <b>Experimental
 Protocol</b> For each replicate, fish were
 haphazardly selected from the holding aquaria and transferred to the test
 apparatus in beakers. The fish were initially added to the side chambers
 (maximum one fish per side chamber) and allowed to acclimate for 15
 minutes. This timing was based on pilot studies conducted prior to the
 experiment described here, which showed that most colour change occurs
 within 2-3 minutes, so this 15 minute acclimation was conservative. Once
 this period had elapsed, fish were released (simultaneously, in the case
 of pairs) into the central test arena by manually raising the sliding door
 of each side chamber. After the fish had exited the side chamber, the door
 was closed behind it. Fish were then allowed to move freely around the
 arena for a period of fifteen minutes. Each replicate was filmed using a
 Canon G1X camera positioned approximately 50cm above the central arena.
 The camera was set to take a sequence of still images, rather than to
 video, since the former allowed a higher resolution of 4352 x 3264 pixels
 per image. The frequency of images was approximately one image per half
 second in white tested treatments and approximately one image per 1.25
 seconds in black tested treatments. This was due to the longer exposure
 period required in the darker (black) arena. No flash was used. After
 testing, fish were removed and transferred to a separate holding tank. No
 fish were reused. <b>Analysis
 </b> From the sequence of images, we selected the
 first image following the entry of the fish to the arena. Subsequently, we
 selected images at thirty second intervals. Where this was not possible
 due to blurred images as a result of test subject movement, we selected
 the closest possible alternative in the series of images. Where a suitable
 image could not be selected within +/- 10 seconds of the scheduled
 sampling point, the data point was disregarded and the next image taken
 from the subsequent sampling point. This however was a rare occurrence,
 accounting for 5 data points out of 600 across all trials.
 Adobe Photoshop was used to measure the RGB values of each fish
 and the background against which it was tested over time. The RGB colour
 model is an additive 3D model for the mathematical and digital
 representation of colour, based on a trichromatic visual system in which
 the visual spectrum is perceived as an additive combination of red, blue
 and green light. In this model, colours can be represented within a 3D
 plane by a set of coordinates with values ranging from 0-255. A standard
 24-bit RGB colour display devotes 8 bits per pixel to each spectral
 channel (red, green, blue) in order to construct colour images. A binary
 encoded system (2<sup>8</sup> = 256) thus permits values from
 0-255 for each spectral channel, with 255 representing full saturation. As
 such, in the RGB colour model (0,0,0) denotes pure black and (255,255,255)
 denotes pure white. This system allows the expression of millions of
 unique shades as cartesian points within the 3D RGB colour plane
 (256<sup>3</sup> = 16,777,216). On this basis, the difference
 between any two colours can be quantified by calculating the Euclidean 3D
 distance between their RGB co-ordinates. In order to quantify the
 difference between a fish and its background, the magnetic lasso tool was
 used to trace the outline of the fish. After this, the fish could be cut
 and pasted onto a transparent background to allow the colour of the fish
 to be measured independent of its background. The eyedropper tool was then
 used to measure the average RGB values across the entire body of the fish.
 Similarly, the eyedropper tool was used to measure the RGB values of the
 background within one body length of the fish.
 Together, these measures were used to calculate the 3D Euclidean
 distance between the colour of the fish and the background colour over
 time. All analyses were conducted using R. Data were
 visually inspected using QQ plots and histograms. Outliers were identified
 using Cook’s distance. In total, three trials of the total sixty were
 removed due to anomalous colouration in the test subjects. Since there
 were differences in the ability of gobies to conform to black versus white
 backgrounds (see Supplementary Information), we conducted the analyses for
 these separately.  To test whether the presence (or absence) of
 conspecifics, and the background colour to which those conspecifics had
 been acclimated, affected the rate and extent of colour change of gobies,
 we used a linear mixed-effects model from the lme4 package for R. The
 response variable was the difference between gobies and their background.
 Fixed effects were treatments and time (as a continuous variable). For
 white acclimated gobies against a black background, we used treatments 3,
 7 and 9 (see Table 1). For black acclimated gobies tested against a white
 background, we used treatments 2, 6 and 8. In each case, therefore, we
 compared gobies tested on their own, gobies tested in a pair (where the
 gobies used in the analysis were acclimated to a different colour than
 their partner) and gobies tested in a pair (where both were acclimated to
 the same colour). Using model selection methods (model.sel from the MuMIn
 package), we determined that the colour changing response of fish as a
 function of time followed a quadratic function, rather than a linear
 function. Finally, we specified trial as the random effect to account for
 multiple observations being taken within each trial.
description: File names refer to acclimation and test backgrounds: 
 BWTest indicates black-habituated fish tested against a white
 background  BBTest indicates black-habituated fish
 tested against a black background  WBTest indicates
 white-habituated fish tested against a black background
 WWTest indicates white-habituated fish tested against a white
 background