DOI: 10.5061/dryad.zcrjdfnk0
title: Immune challenge changes social behaviour in the Guppy (Poecilia reticulata)

description: Increased exposure to pathogens is often considered to be one of the most
 significant costs of group living. As a result, animals typically avoid
 close association with individuals that manifest symptoms of disease. The
 question remains, however, whether avoidance behaviours are mediated by
 effects relating specifically to the disease itself, or through
 recognition of more general sickness behaviours that emerge from the
 activity of the immune system in response to infection. Here we examined
 the effects of an immune challenge, induced through exposure to bacterial
 lipopolysaccharides (LPS), on individual and social behaviour in female
 guppies (Poecilia reticulata). We found that in a binary choice
 presentation, healthy focal individuals showed a strong preference to
 associate with stimulus shoals of saline-injected fish rather than
 stimulus shoals of LPS-injected fish.  In a subsequent
 examination of group-level dynamics, shoals of LPS-injected females were
 more dispersed than similar shoals comprised of control fish, and showed
 less coherent collective behaviour despite the lack of any obvious
 difference in general activity between LPS and control fish. We discuss
 these findings in relation to the need for social animals to mitigate the
 risk of disease that is associated with living in proximity to
 conspecifics.
description: <em>Study Species</em> We used the
 guppy (<em>Poecilia reticulata</em>) as our study species
 because it is a widely used model species in behavioural ecology and
 physiology studies. For the present study we used only females because of
 their greater shoaling tendency and to avoid the confounding effects of
 courtship and mating behaviour. Fish used in the present study were adult
 females measuring 20-30 mm sourced from a feral population of guppies in
 Darwin, NT, Australia. Fish used in the experiments were housed in 90L
 aquaria where they were kept at 25°C on a 12:12 light:dark cycle. Fish
 were fed daily ad libitum with commercially available fish flakes
 (Nutrafin).  <em>Immune Challenge
 </em> Females were randomly assigned to either
 LPS or control groups (LPS-injected: N = 60, Control: N = 60). All fish
 were lightly anaesthetised using a clove oil solution (50mg/L) and then
 weighed at the time of injection. Fish assigned to the LPS treatment were
 injected intramuscularly in the dorsal region at a dosage of 10mg LPS
 (Sigma-Aldrich; Lipopolysaccharides from <em>Escherichia
 coli</em> serotype O111:B4) per gram of fish. LPS was dissolved in a
 solution of Milli-Q<sup>® </sup>water and salt to a standard
 saline concentration of 0.9%. Control fish underwent the same procedure
 but were injected with saline only. Following injection, fish were
 immediately transferred to individual tanks containing aged tap water
 where they recovered within 1-2 minutes. Following injection, fish were
 monitored for signs of stress for 48 hours prior to experiments. A 48 hour
 interval between injection and testing was chosen on the basis of
 precedent from previous published studies which show that the immune
 response peaks 48hrs post-injection in a closely-related species
 (<em>Gambusia holbrooki</em>). 
 <em>Experiment 1: Binary Choice </em>
 Trials were conducted in a white rectangular Perspex arena
 measuring 60x30x20cm, which was filled to a depth of 10cm. The arena was
 divided into three zones, comprising a central chamber which measured
 34x30cm as well as two chambers at opposite ends of the longest axis of
 the arena each measuring 13x30cm. Focal individuals were introduced into
 the central chamber, while stimulus shoals were placed in each of the end
 chambers. The barriers separating stimulus fish from the focal individual
 were constructed using transparent perforated plastic (PennPlax) to
 facilitate the transmission of visual and chemical cues. Each stimulus
 shoal, comprised of 6 fish, were injected intramuscularly with either
 saline or LPS 48 hours prior to testing. Focal fish were healthy
 individuals that had not exposed to either the LPS or control (saline)
 treatment prior to testing. Stimulus shoals were introduced to their
 designated chamber of the arena and allowed to acclimate for 1 hour. After
 this, a single focal fish was introduced to the central chamber of the
 arena and filmed for 10 minutes. The focal fish was then removed, and
 after 5 minutes a new focal fish was added and the procedure repeated. We
 conducted a total of 4 trials before replacing stimulus shoals. In total 8
 sets of stimulus shoals were used for 4 trials each for a total of N=32
 trials. There was one focal fish per trial (N=32), none of which were
 reused. Only the last 7 minutes were extracted for analysis in order to
 allow the focal fish an acclimation period of 3 minutes. The extracted
 video footage was then reviewed and the time each focal fish spent in
 association with each of the stimulus shoals was manually recorded.
 Association of the focal fish with a stimulus shoal was defined as the
 time (in seconds) spent within 5cm of the PennPlax barrier separating the
 central chamber from each of the end chambers containing the stimulus
 shoals. Focal fish which did not investigate both stimulus shoals during
 the trial were excluded. Only N=1 trial was excluded from analysis on this
 basis (the focal fish associated solely with the LPS-injected shoal). For
 completeness, a further analysis which included the outlying individual
 was conducted, however the statistical outcome and interpretation of the
 test was not affected. (For comparison, the statistical outcome without
 the outlier was χ<sup>2</sup>= 29.048, Df = 1, P &lt;
 0.001, while with the outlier included, the outcome
 was χ<sup>2</sup>= 18.375, Df = 1, P &lt;
 0.001.) <em>Data Analysis</em>
 Statistical analysis was performed using R (v4.1.2; R Core Team
 2022). Data were examined using Q-Q plots and Shapiro-Wilk tests, and
 satisfied the assumption of normality. We analysed the time in seconds
 spent by focal fish with each of the two stimulus shoals using a linear
 mixed effects regression model, using the package lme4 (Bates et al.,
 2015), specifying the treatment of the stimulus shoals as the fixed
 effect. Focal fish ID and the stimulus shoal batch (from 1-8) were
 included as random factors in the model.
 <em>Experiment 2: Collective Behaviour
 </em> At 48hrs post-injection fish were
 transferred from their individual holding tanks to a circular arena with a
 diameter of 70 cm and a depth of 5 cm. Arenas were lit using cool white
 LED strips (6500K) and surrounded with white screens to minimise external
 disturbance. All trials were filmed using a Canon G1X camera positioned
 1.1m above the arena at a frame rate of 24fps and a resolution of 1920p.
 Each trial was filmed for 12 minutes from the time of introduction.
 Following this, fish were removed and transferred to new holding tanks. No
 fish were reused.  We conducted two treatments, each
 involving test groups of 6 fish. In the wild, female guppies are social
 and tend to live in small shoals of between 2 and 10 individuals.  As
 such, a group size of 6 was selected on the basis of being ecologically
 realistic. The treatments were LPS, in which all fish were LPS-injected
 and Control, in which all fish were saline-injected. We performed (N=10)
 per treatment to a total of 20 trials. <em>Data
 Extraction and Analysis</em> For each group
 trial, we extracted the last 10 minutes of video footage for analysis.
 This allowed the fish an initial 2-minute acclimation period prior to data
 collection. Videos were tracked using TRex; x,y coordinates for each fish
 over the 10-minute test period were extracted and we used these values as
 the basis for subsequent analyses.  To examine the
 effect of LPS on individual activity and group cohesion, we calculated
 mean individual speed and mean nearest neighbour distance for each fish.
 Again, data were analysed using R (v4.1.2; R Core Team 2022). Data were
 examined using Q-Q plots and Shapiro-Wilk tests of normality. These
 dependent variables were analysed using linear mixed-effect models package
 lme4 with treatment as the independent variable and specifying group as
 the random factor. To examine the effects of LPS on the
 distribution of near neighbours relative to a focal fish, and to
 characterise the local interactions between that focal fish and its
 neighbours, we used the methods described in detail in Schaerf et al.,
 (2016) and which we previously employed in Encel et al., (2021)
 Specifically, we examined the local distribution of near neighbours, and
 the alignment, speed, change in speed and change in heading of a focal
 individual relative to the positions of near neighbours. To achieve this,
 we first transformed the position of each fish to a consistent coordinate
 system where a focal individual was located at the origin (0,0), and the
 direction of motion of the focal individual was parallel to the positive
 x-axis. A square domain centred on the focal individual was then
 sub-divided into smaller overlapping square bin regions. The larger square
 domain extended over the region where − 100 &lt; x ≤ 100, − 100
 &lt; y ≤ 100 millimetres, that is, out to distances of approximately 4
 body lengths to the front and back, and to the left and right, of the
 focal individual. The smaller square bin regions had side lengths of 15 mm
 (approximately half a body length for the guppies), with the leftmost
 and/or bottommost edges of adjacent bins separated by 3.75 mm (one-quarter
 of a bin width). Square bins and the rectangular (x, y) coordinate system
 were chosen so that each bin covered equal areas of the domain near the
 focal individual (as opposed to bins based on distances to, and angular
 ranges containing groupmates, where the area of bins would grow as the
 distance from the focal individual increased). The overlap of the bins is
 a means to smooth the resulting plot, analogous to the moving window used
 to calculate a moving average. The number of times that neighbours
 occupied each bin was counted, with data aggregated by treating all group
 members as the focal individual in turn. Absolute counts in each bin were
 then normalised by dividing by the total counts across all bins.
 We adapted the randomisation method based on the mean absolute
 difference between pairs of fitted functions (Schaerf et al., 2021) for
 across group comparisons to examine if there was a significant difference
 across treatments. We adjusted the thresholds for significance for the
 mean absolute difference randomisation tests according to the
 Holm-Bonferroni method (Holm 1979) to take into account multiple pairwise
 comparisons.
description: # Immune Challenge Changes Social Behaviour in the Guppy (Poecilia
 reticulata)
 [https://doi.org/10.5061/dryad.zcrjdfnk0](https://doi.org/10.5061/dryad.zcrjdfnk0) The dataset contains data from an experiment examining the effects of LPS exposure on 1) the social interactions and 2) collective behaviour in the female guppy (Poecilia reticulata). Experiment 1 examined whether unexposed fish preferred to associated with control or LPS exposed stimulus shoals, using a binary choice test. It was found that focal individuals significantly prefer to associated with control stimulus shoals. Experiment 2 examined the collective behaviour of exposed and unexposed shoals, and found that groups of LPS exposed fish showed reduced group cohesion. ## Description of the data and file structure There are two data files, one containing the data from (1) the binary choice experiment and (2) the collective behaviour experiment. The data are laid out as follows: 1\. Binary Choice Experiment \(file name LPSChoiceTests\.csv\) The data are arranged by treatment (LPS-exposed or control) and our measurements are: Time Spent Associating with each stimulus shoal (in seconds) Group cohesion, measured as nearest neighbour distance (in cm) Swimming speed (in cm/s) 2\. Collective Behaviour Experiment \(file name 'ArenaDataDryad\.csv'\) The data are arranged by treatment (column D; LPS-exposed or control) and our data refer to: ID - this is the individual identity of each fish (ranging from 1-6) in each replicate MeanDist - this is the mean distance (in cm) between a given fish and the other members of the group Speed - this is speed (measured in cm per second) Treat - this is the treatment - LPS or Control Trial - this is the ID for each trial. ## Sharing/Access information All data will be available from Dryad ## Code/Software All statistical analyses were conducted using R. The following packages were used: Lme4