2.3.1 Fish origin and maintenance
The guppies used to experimentally test the effect of infection with Gyrodactylus turn-
bulli on escape response were second-generation laboratory-bred descendants of wild
guppies from the Caura River, Trinidad (UTM: 20 P 67952.77 m E, 118037.64 m N; elevation 112m). Wild guppies (n = ca. 600) were transported to Cardiff University in June 2012 (Cefas APB authorisation number CW054-D-187A), where they were prophylactically treated for infection using Binox® (Nitrofurazone; Jungle Laboratories Corporation®, Cibolo, Texas). Fish were housed in 70 L mixed sex stock tanks on a 12h light: 12h dark lighting regime (overhead fluorescent lighting) at 24±1°C and fed daily with Aquarian® tropical fish flakes supplemented with Artemia and bloodworm. Fry were removed from the breeding tanks soon after birth, reared in one of four juvenile tanks until sex determination was possible (ca. 6-8 weeks), and then separated by sex. All fish used in the experiment were therefore virgins, controlling for the fact that reproductive status influences the escape response of females (Ghalambor et al., 2004).
2.3.2 Experimental infections
We used 38 guppies (19 females, 19 males) as our ‘infected’ treatment group, and 30 guppies (19 females, 11 males) as the ‘uninfected’ treatment group. These sample sizes were dictated by the numbers of fish available due to an additional experiment that
Chapter 2. A further cost for the sicker sex 19 was running simultaneously. The weight and standard length of all individuals was recorded immediately prior to infection. To help discriminate between the effects of inherent differences between the sexes and those due purely to their different sizes, we sized-matched males and females (difference in mean standard length: 0.25 mm;
t(66) = -0.51; p = 0.61), as well as the infected and uninfected fish (difference in mean
standard length: 0.42 mm; t(66) = -0.88; p = 0.38). Fish (overall mean standard length, mm±SEM: 16.4±0.3; mean weight, mg±SEM: 73.4±3.9) were held individually in 1 L containers throughout the experiment. The Gt3 strain of G. turnbulli, originally isolated from guppies in a petshop in Nottingham, UK in 1997, was used to infect experimental fish. Fish to be infected were randomly assigned to batch 1 (12 females, 8 males) or batch 2 (7 females, 11 males) allowing all behavioural trials to be conducted at an equivalent stage of infection (Day 8: n=10; or 9: n=28). Fish were infected under anaesthetic (0.02% tricaine methanesulfonate; MS222; PHARMAQ Ltd., UK) on day 0 through the transfer of 2.3±0.1 (mean±SEM) parasites from a heavily infected, recently killed donor, as observed using a dissecting microscope and fibre optic illumination. We subsequently monitored infections under anaesthetic (0.02% MS222) on days 1, 3, 5, 7 and one day post-trial (Day 9: n=10; or 10: n=28). Golf%ball% Ball%release%% tube% Ball%release% pegs% 30cm% 60cm% 30cm% 20cm% 10cm% 10cm% 2cm%water%level% GoPro%camera%
Figure 2.1: The apparatus used to test for the effect of infection with Gyrodactylus turnbulli on guppy escape response. The left panel gives a side view, the right an
overhead view.
2.3.3 Experimental set-up and design
To assess their escape response, guppies were individually exposed to multiple startling stimuli within a trial arena. The experimental setup consisted of a 30 × 60 cm outer
tank with a 20 × 30 cm inner tank, both filled with dechlorinated water at 25-26°C to a depth of 2 cm (Fig. 2.1). An arena constructed from plastic mesh (10 × 10 × 16 cm; Darice® Inc. USA), was placed in the smaller tank on a waterproof white background. The side length was selected to approximate five body-lengths of the test fish (Ghalambor et al., 2004). A remotely operated ball release was situated above the gap between the inner and outer tanks, containing three golf balls at 35, 40 and 45 cm height, each with a separate release mechanism. No variation in the escape response was attributable to this difference in ball drop height (p > 0.05, included as a factor in the general linear mixed model described below), which is unsurprising given that escape responses are a threshold ‘all-or-nothing’ behaviour (Bergstrom, 2002). At the start of each trial, a fish was placed in the arena and left for a 10 min acclimatisation period. The first ball was released after acclimatisation, followed by the remaining two at 3 min intervals. A wirelessly operated camera (HERO3+ White Edition; GoPro Inc., USA), set to film at 60 frames per second, was suspended 20 cm above the arena and activated for approximately 15 s during the release of each ball. Between the trials of individual fish, the inner tank was emptied and cleaned with 70% ethanol in order to remove any chemical cues or dislodged parasites. The 68 fish were tested over the course of six days. We used EthoVision XT (Noldus) to record the distance the fish travelled (mm) in the 60 ms immediately following the initiation of the escape response for each video (3 videos per fish: n = 204). We also recorded whether the fish was in the half of the arena next to the ball, or the half away from the ball (position in the test arena), and its orientation (facing towards the ball, away from the ball, or neutral) when the ball dropped. If multiple escape responses occurred following a single ball drop only the first was analysed.
2.3.4 Ethical note
This work was conducted under the UK Home Office license (PPL 30/2876) with approval by the Cardiff University Animal Ethics Committee. As described above, during the course of this experiment guppies were subjected to social isolation, and some were additionally experimentally infected with parasites. Although social isolation is likely to have temporarily elevated their stress levels, previous work using this method indicates that guppies show no sign of having suffered lasting harm. To further mitigate the effects of this isolation, fish were held in close proximity in transparent tanks; they were therefore in visual contact with conspecifics throughout. Among the infected fish, none were infected for longer than 10 days, which is the point at which mortality increases (Dargent et al., 2013). We monitored infected fish carefully and none exhibited severe pathology at any point during the experiment (fused fin rays, difficulty swimming, loss
Chapter 2. A further cost for the sicker sex 21 Response variable Error family Link function
Main effects Two-way interactions Random
effect Distance
covered in 60 ms (mm)
Gaussian Identity Infection status (c)*
Infection load (n) Sex (c) Position (c) Orientation (c) Drop (o) No. responses (n) Weight (n)*
Infection status × Sex Infection status × Weight*
Fish identity
Table 2.1: The starting model used to test the hypothesis that infection with
Gyrodactylus turnbulli affects guppy escape response. This starting model was simplified using backwards stepwise deletion of non-significant fixed effects to minimise the Akaike’s Information Criterion (AIC); those that remained in the final model are denoted with an asterisk. See the main text for an explanation of the fixed effects,
which were included as categorical (c), numeric (n), or ordinal (o) variables.
of appetite Cable, 2011). Throughout the experiment, mortality was low (<5%), and infected fish recovered fully with treatment.
2.3.5 Data analysis
We used a general linear mixed model (Gaussian error family with an identity link function) in the lme4 package in R (3.0.2; R Core Team, 2013; Bates et al., 2014) to test whether infection with G. turnbulli affected the escape response of guppies (Table 2.1). We used the distance (mm) the fish moved in the 60 ms immediately following the initiation of the escape response as the response variable, and included fish identity as a random effect because we recorded three escape responses per fish. Fixed effects included: infection status (infected or uninfected); infection load (how many G. turnbulli the fish was carrying when tested); fish weight (as a measure of size); fish sex; fish position in the test arena; fish orientation when the ball was dropped; drop number (1, 2 or 3); total number of responses to each ball; two way interactions between infection status and sex, and infection status and fish weight. We simplified the model by sequentially removing non-significant fixed effects from the full model to minimise Akaike’s Information Criterion (AIC). The analysis was conducted using weight as a continuous variable, but for Figure 2.2 we divided the fish into ‘small’ and ‘large’ categories (divided at the median value of 72 mg) for clarity.
15 16 17 18 19 20 21 Uninfected Infected Distance co v ered in 60ms (mm) Large Small
Figure 2.2: Infection with Gyrodactylus turnbulli reduced the distance that small fish (up to 72 mg; white squares) covered during the escape response by an average of 20.1%. There was no effect of infection on the escape response of large fish (over 72 mg; black squares). The error bars are the standard error of the mean. The analysis was conducted using weight as a continuous variable, but for the figure we divided the fish into ‘small’ and ‘large’ categories (divided at the median value of 72 mg) for clarity.