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.

Study Species

The Trinidadian guppy (Poecilia reticulata) 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). 

Immune Challenge

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 Escherichia coli 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 (Gambusia holbrooki). 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.

Testing Protocols

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). 

Data Extraction and Analysis

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.

Analysis

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 < x ≤ 100, − 100 < 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.