DOI: 10.5061/dryad.xsj3tx9q8
title: Data from: Immune challenge affects risk sensitivity and locomotion in mosquitofish (Gambusia holbrooki)

description: The immune system is crucial in responding to disease-causing pathogens.
 However, immune responses also generally cause stereotypical changes in
 behaviour known as sickness behaviours, which often include reduced
 activity. Sickness behaviours are thought to have an important role in
 conserving energy required to support the immune response, however little
 is known about how they manifest over time or in relation to risk,
 particularly in fishes. Here, we induced an immune response in
 mosquitofish (Gambusia holbrooki) by inoculating them with E.coli
 lipopolysaccharide (LPS). We subsequently tested batches of fish at
 24-hour intervals and examined: locomotory behaviour, tendency to use a
 refuge and response to a threat stimulus. There was no difference in
 behaviour between control and LPS-treated fish 1, 3, or 4 days
 post-inoculation. However, 2 days post-inoculation, LPS fish swam more
 slowly and spent more time in the refuge than control fish, although no
 difference in peak acceleration was found. Our findings suggest that the
 expression of sickness behaviours peaks roughly 2 days following exposure
 to LPS and are relatively short-lived. Specifically, immune-challenged
 individuals exhibit reduced locomotion and exploratory behaviour. They
 also become more risk averse, but retain the ability to respond acutely to
 a threat stimulus.
description: <strong>Study Species</strong>
 Mosquitofish (<em>Gambusia holbrooki</em>) measuring
 20.4 ± 4.9 mm (mean ± standard deviation) were collected for use in the
 experiments in May 2023 from Manly Dam, Sydney (-33.806360 S, 151.235144
 E). The mosquitofish is a small species of fish that inhabits shallow
 freshwater habitats and is preyed upon by a wide variety of predators,
 including fish, birds and insects (Pyke 2005). Prior to experiments, the
 fish were held for 1 week in 180 L vats held at a temperature of
 23.1 ± 1°C and a 12:12 light:dark cycle in the animal holding rooms at the
 University of Sydney. Fish were fed daily ad libitum with commercially
 available fish flakes (Nutrafin). <strong>Immune
 Challenge</strong> Fish were randomly assigned to
 either LPS or control treatments (LPS-treated: N = 64, Control: N = 64).
 Groups of 8 individuals were placed in a 500mL bath of either LPS solution
 or plain aged tap water and left for 60 minutes before being transferred
 in their groups to separate 5L holding aquaria. Fish assigned to the LPS
 treatment were bathed in a solution of aged tap water and LPS at a
 concentration of 100mg/L (Sigma-Aldrich; Lipopolysaccharides
 from <em>Escherichia coli</em> serotype O111:B4). This
 concentration was selected based on previous studies which induced immune
 reactions in fish using similar dosages. LPS offers a valuable means of
 inducing an inflammatory response in vertebrates and has been widely used
 on subjects from various taxa for this purpose. The use of LPS allows the
 effects of innate immune responses to be decoupled from the variable and
 disease-specific effects of live pathogens, allowing broader inferences to
 be made. Following treatment, fish were monitored for signs of stress
 prior to experiments. Previous studies show that the immune response of
 mosquitofish peaks at approximately 48hrs post-exposure. The testing
 schedule (24, 48, 72, 96hrs) was chosen on this basis. The bath method was
 chosen over an intraperitoneal or intramuscular injection due to the small
 size of the fish and the success of this method in other trials using
 small teleosts. <strong>Experimental
 Protocol</strong> A single fish was netted from
 its holding tank and introduced to an adjacent circular test arena, which
 had a diameter of 64cm, and was filled to a depth of 8 cm with aged tap
 water at the same temperature as the holding tank (24°C). The arena was
 constructed from white Perspex. A single artificial aquarium plant was
 added to the arena. This had a circular base of diameter 3cm, spreading to
 a diameter of 11cm at the water’s surface. The plant was positioned 23 cm
 from the arena centre. The purpose of this was to provide a refuge for the
 fish. The arena was surrounded by white screens to minimise external
 disturbance and was lit by 2 x 20W cool white LED strips. Following its
 introduction, the fish was allowed to acclimate to the arena for 10
 minutes, after which we filmed for 5 minutes, using a Canon GX camera set
 into a recess in the ceiling of the screens, 1.2m above the arena, and
 filming at 24 fps at a resolution of 1920p. Halfway through the filming
 process (i.e. 12.5 minutes after introduction and 2.5 minutes after we
 began filming) we dropped a 3g cubic lead weight into the centre of the
 arena. The purpose of this was to simulate an attack by an aerial
 predator. At the completion of 5 minutes’ filming, the fish was removed
 and transferred to a new holding tank. We conducted tests on each of four
 days, respectively 24 hours, 48 hours, 72 hours, and 96 hours following
 control inoculation, or inoculation with LPS. On each day, we tested 32
 fish, comprising 16 LPS treated fish and 16 control fish. Each test was
 done with a naïve fish. Each fish was used only once. After testing, the
 sex and body length of the fish were recorded.
 <strong>Analysis and Data
 Extraction</strong> Each video was tracked using
 TRex software, yielding a total of 7200 x,y coordinates for each fish (300
 seconds at 24 fps). From this, we were able to calculate our two key
 response variables, which were speed, and time in the refuge. We defined
 time in the refuge as being when a fish was within a radius of 5.5 cm of
 the centre of the artificial plant. We calculated speed based only on the
 time the fish spent outside the refuge. For the purpose of our analysis,
 we divided the trial into two halves: before the introduction of the lead
 weight, and after the introduction of the lead weight. The delineation
 between the two was determined as the point at which the lead weight first
 made contact with the water surface. Acceleration was measured by
 examining the trajectories of fish in the 10 seconds immediately following
 the threat stimulus. We took the 10 highest values for acceleration (i.e.
 peak acceleration) in this time and calculated the median of
 these. Statistical analysis was performed using R, and
 using packages lme4, car, MuMIn, and ggplot. We used the model.sel
 function in MuMIn to identify the most appropriate model for the error
 distributions in both cases, We subsequently used a generalised linear
 mixed model, specifying a gamma distribution, to analyse time in the
 refuge, accounting for the fact that this response variable was non-normal
 and right skewed. We used a general linear mixed model to analyse speed.
 Neither sex, nor size were statistically significant (p &gt; 0.6 in
 all cases), and so both were excluded from the final models. In each of
 our two models (‘Time in Refuge’ and ‘Mean Speed’), we specified three
 fixed effects: Treatment (Control or LPS), Day (1, 2, 3, or 4), and Threat
 (Pre-threat and Post-threat). We specified Fish ID as our random effect,
 to account for the repeated measures nature of the data in respect of
 quantifying behaviour before and after the threat stimulus. We used a
 general linear model to analyse acceleration, with Day and Treatment as
 our fixed factors.
description: # Immune challenge affects risk sensitivity and locomotion in mosquitofish
 (Gambusia holbrooki)
 [https://doi.org/10.5061/dryad.xsj3tx9q8](https://doi.org/10.5061/dryad.xsj3tx9q8) The dataset contains data from an experiment examining the effects of LPS exposure on locomotion, exploratory behaviour and risk sensitivity in the mosquitofish (*Gambusia holbrookii*) over a period of 4 days both before and after a threat stimulus. All fish responded strongly to the threat stimulus.  It was found that LPS-treated individuals had significantly lower swimming speeds and spent significantly more time in the refuge 2 days post-inoculation both before and after the threat stimulus, but not on any other day. No difference in acceleration after the threat stimulus was found.  ## Description of the data and file structure There are two data files, one (`LPS_Individual_RSOS.xlsx`) containing the data relating to: (1) mean time spent in refuge (before and after threat stimulus) (2) mean swimming speed (before and after threat stimulus) The second data file (`LPS_Individual_Accel_RSOS.xlsx`) contains the data relating to:  (3) median peak acceleration (in the 10s immediately after the threat stimulus) The data are laid out as follows: 1\. Mean time spent in refuge and mean swimming speed (file name `LPS_Individual_RSOS.xlsx`) The data are arranged by treatment (LPS-treated or control) and Day (1,2,3,4) and our measurements are: * Refuge Time Pre - this is the time in seconds spent in the refuge before the threat stimulus * Refuge Time Post - this is the time in seconds spent in the refuge after the threat stimulus * Speed Arena Pre - this is swimming speed (measured in cm per second) during the time the fish was outside the refuge before the threat stimulus * Speed Arena Post - this is swimming speed (measured in cm per second) during the time the fish was outside the refuge after the threat stimulus 2\. Median peak acceleration (file name '`LPS_Individual_Accel_RSOS.xlsx`') The data are arranged by treatment (LPS-treated or control) and Day (1,2,3,4) and our data refer to: * TrialID - this is the individual identity of each fish  * Accel - This is the median peak acceleration (measured in cm/s) of the fish in the 10 seconds immediately following the threat stimulus. ## Sharing/Access information All data will be available on Dryad. ## Code/Software All statistical analyses were conducted using R. The full script, including information about all the packages used, is available here on Zenodo (see link in Related Works) (file name: '`LPS_Individual_RSOS.R`')