DOI: 10.5061/dryad.866t1g1w7
title: Immune challenge affects reproductive behaviour in the Guppy (Poecilia reticulata)

description: Immunocompetence and reproductive success are among the most important
 determinants of fitness in animals. However, energetic and metabolic
 constraints create conflict between these two priorities. The trade-off
 between immunity and reproductive fitness has been the subject of great
 scientific interest over many decades. While a number of studies exploring
 the relationship between immune activity and reproductive
 fitness/behaviour have been conducted using birds and mammals inoculated
 with bacterial endotoxin (LPS), relatively few have focussed on fish. This
 presents a conspicuous gap in our understanding of how immune activity
 affects reproduction in vertebrates more generally. Fish have been
 experimentally neglected in this area due, in part, to the claim that fish
 are largely resistant to the immune effects of endotoxins, and thus
 presumably suffer negligible behavioural effects. While the immune
 response to endotoxins may differ somewhat between fish and terrestrial
 vertebrates, the findings here suggest that they are nonetheless
 susceptible to significant behavioural effects with respect to
 reproduction. In this study, we show that although immune challenge does
 not suppress general activity in male guppies, it significantly reduces
 reproductive effort. As such, it is important to expand the examination of
 these processes to a greater diversity of vertebrate species, including
 fish.
description: <strong><em>Study
 Species</em></strong> The Trinidadian guppy (<em>Poecilia
 reticulata</em>) is a widely-used model organism in ecology,
 behaviour and physiology studies, and increasingly have been used in the context
 of disease ecology. They
 are ideal subjects for the study of reproductive behaviour due to their
 clear sexual dimorphism and stereotypical courtship displays. Fish used in
 the study were adults, females measuring 26.6 ± 1.4 mm and males, which expressed adult
 colouration and had fully formed gonopodia, measuring 17.3
 ± 1.2 mm. The body length of the fish were
 measured using stills taken from the videos collected during the trials.
 Fish were the progeny of wild stock collected from feral populations in
 the Northern Territory (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). 
 <strong><em>Immune
 Challenge </em></strong>
 Males were randomly assigned
 to either LPS or control treatments (LPS-exposed: N = 16, Control: N =
 16). Groups of 8 males were placed in a 500mL bath of either LPS solution
 or plain aged tap water and left for 60 minutes before being transferred
 to 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 on the basis of previous studies
 which induced immune reactions in fish using similar
 dosages. Following
 treatment, fish were monitored for signs of stress for 48 hours prior to
 experiments. A 48-hour interval between exposure and testing was chosen on
 the basis of precedent from previous published studies which show that the
 immune response peaks 48-168hrs post-injection in a closely related warm
 water species (<em>Gambusia
 holbrooki</em>). The
 bath method was chosen over an intraperitoneal or intramuscular injection
 due to the small size of the males and the success of this method in other
 trials using small individuals.
 <strong><em>Testing
 Protocols</em></strong>
 At 48hrs post-exposure fish
 were transferred from their individual holding tanks to a circular test
 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. Each fish was only used once, and the males and females
 were taken from separate holding aquaria and were unfamiliar with each
 other. We conducted two treatments, each involving test
 groups of 3 females and 1 male to a total of 4 fish per group. The
 treatments were LPS, in which the male guppy was LPS-exposed and control,
 in which the male was bathed in plain water. At the time of testing, one
 control male showed signs of ill health and was excluded on this basis.
 Hence, we performed (LPS-exposed: N=16, Control: N=15) trials to a total
 sample size of (N = 31). 
 <em><strong>Data
 Extraction and Analysis</strong></em>
 For each 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.
 XY coordinates for each fish over the 10 minute test period were
 extracted. From these, we calculated individual speed and the mean
 distance between the male and the three females within each trial.
 Furthermore, the number of displays made by each focal male and the number
 of mating attempts made by each focal male were counted. This process was
 performed blind by an observer who was not aware of the treatment. Both
 the displays of male guppies and their mating attempts are stereotypical
 and unmistakable to an observer. Displays involve the male approaching a
 female and then assuming a sigmoid body attitude, with fins fully spread,
 and quivering. Mating
 attempts involve the male approaching the female from the rear and
 accelerating toward her in an attempt to locate her genital pore with his
 gonopodium, which involves the two fish being in contact
 briefly. <strong><em>Analysis</em></strong> Statistical analysis was performed using R, using the packages MASS and effectsize, while figures were produced using ggplot2. Data were examined visually using QQ-plots and histograms, and with Shapiro-Wilk tests. We ascertained that the assumption of normality was met for mean male size, mean female size, male swimming speed,  the mean distance of males to females, and the mean distance between females. We analysed these using linear models, with treatment as the independent variable. The number of displays and of mating attempts were both count variables and were overdispersed. Consequently, we analysed these generalised linear models with a negative binomial error distribution, using the glm.nb from the MASS package. Finally, mean female swimming speed was non-normal, and right skewed. Using the model.sel feature from the package MuMIn we established that a generalised linear model using a gamma distribution was best suited to the analysis. To create encounter frequency heatplots, we examined the positions of neighbours relative to a focal individual by first transforming 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 5 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 4 mm. We smoothed the plots by overlapping the bins in a manner analogous to the moving window used to calculate a moving average. We counted the number of times that neighbours occupied each bin and then normalised these absolute counts by dividing by the total counts across all bins.