Queen collection and maintenance

We collected hundreds of mated queens in July 2013 and 2014 from the IST Austria campus, Klosterneuburg, Austria. Queens were returned to the laboratory in plastic boxes containing damp tissue paper until they were used in experiments, which was within one day of collection. Mortality during this time period was minimal and appeared to result from damage caused during the mating flight or collection, e.g. because of missing limbs or damaged abdomens. Queens collected during mating flights can have natural infections (ca. 1% of queens have fungal infections; C.D. Pull, unpublished data), however, in none of our experiments did we observe any pathogenic growth other than Metarhizium. No food was provided to queens in any of the experiments as they survive solely on the breakdown of muscle and fat reserves during colony founding [34]. Queens were chosen for each experiment/treatment group haphazardly.

Fungal pathogen

We used the species M. brunneum (strain KVL-03-143), collected from and grown on sabaroud dextrose agar plates before each experiment for approximately two weeks, until plates were fully sporulating. Conidiospores were suspended in autoclaved 0.05% Triton-X (in water) and washed three times by centrifuging the conidiospores (3000 g for 5 min), pouring away the supernatant, and re-suspending them again 0.05% Triton-X. We confirmed the viability of the conidiospores by plating out 100 μl of the conidiospore suspension onto sabouraud dextrose agar plates and checking the number of conidiospores germinating after 18 h (always >90%).

Queen pathogen exposure

Queens were exposed to the fungal suspension or autoclaved Triton-X as a sham-exposure, by gently restraining them with soft forceps and pipetting 0.5 μl of the fungal suspension or Triton-X onto their thorax. Queens were then placed onto filter paper to remove excess liquid and allowed to dry alone for several minutes before being added to experiments. For all experiments, we applied a droplet of 0.5 μl (2 × 107 conidiospores ml−1), which causes high mortality in queens (30/35 queens) kept alone for 30 days (median survival time = 6 days).

Experiment 1: Effect of pathogen exposure on colony co-founding choice

All queens were individually colour-marked (paint “Uni Posca”) on one of their abdominal segments to differentiate the pathogen-exposed and sham-treated queens. We set up plastic boxes (10 × 3.5 cm; Bock GmbH & Co. KG) comprising three equally sized chambers and a transparent lid. The middle chamber was uncovered and had no substrate, whereas the two chambers on either side were covered in red transparent foil (to reduce light entering the chamber whilst allowing observations of the ants) and had a damp plaster substrate. A hole (5 mm Ø) in the walls of the middle chamber connected it to the two adjacent chambers, allowing the ant queens to move freely between them.

Into one of the plastered and darkened chambers, we placed either a sham-treated or pathogen-exposed queen, which we termed the “residing queen”. After allowing her time to settle (1 h), we then introduced a second queen to the middle chamber, which was either sham-treated or pathogen-exposed, and termed the “introduced queen”. We varied which of the two darkened chambers the first queen was placed into in case there was a directional bias towards one of the chambers. In the field, queens choose to co-found or not within the first few hours following a mating flight and about half of queens are underground within 40 min of landing [29, 58], so our set up of allowing the residing queens 1 h to settle approximates field conditions.

Thus, we had four experimental groups (i) a sham-treated queen introduced to a nest with a sham-treated residing queen (ii) a sham-treated queen introduced to a nest with a pathogen-exposed residing queen (iii) a pathogen-exposed queen introduced to a nest with a residing sham-treated queen and (iv) a pathogen-exposed queen introduced to a nest with a residing pathogen-exposed queen (n = 20 in all cases). Following the introduction of the second queen, we observed the locations of queens after 1, 12, 24, 48 and 72 h. We stopped observations at 72 h as ~75% of queens had produced eggs and queens started dying from the fungal exposure after this point. The experiment was run at 23 °C and 70% humidity, under continuous light, to encourage queens to choose one of the darkened chambers, as opposed to remaining in the middle chamber. All queens in this experiment were collected in 2013.

Experiment 2: Queen behaviour towards co-foundress corpses and disease transmission

We placed single, unpainted and untreated queens into petri dishes (Ø = 3.5 cm) filled with damp plaster that contained a rectangular cavity measuring 1 cm × 3.5 cm, to mimic the small chambers queen’s construct when founding a colony. Each chamber contained 1 g of loose plaster particles as a nest material. The lids of the dishes were covered with red transparent foil to keep the chamber darkened. We termed these dishes “closed nests”. Half of the dishes remained closed nests, whilst the other half were placed into a second, larger dish (Ø = 9 cm) with a plaster substrate. A small hole (Ø = 5 mm) in the side of the small dish allowed the queen access to this external arena, to create an “open nest”. We then added a second, paint-marked queen (allowing us to distinguish her from the untreated queen) to each dish in both the closed and opens nests, which was either sham-treated or pathogen-exposed.

We monitored the survival of pathogen-exposed queens on a daily basis and noted when they died. So that we could compare the behaviours of naïve queens towards infected and non-infected queens when they died, we removed and froze sham-treated queens on the days that pathogen-exposed queens died to create non-infected dead queens (mean day of death ± standard deviation: pathogen-killed = 6.4 ± 1.7; freeze-killed = 6.5 ± 1.9). These queens were frozen for 5 min at −80 °C, before being added back to the dishes with the surviving queens. We did not freeze pathogen-exposed queens when they died in case this affected the outgrowth of the fungus. However, freezing as a method of killing has been used to study undertaking behaviour in other ant species, termites and bees, and elicits typical undertaking responses in nestmates [31, 50, 59, 60]; however, infected corpses may be more attractive or elicit more rapid undertaking behaviour [39, 61]. The pathogen-exposed queens died in the majority of nests (47 out of a total of 58 that we set up) and those where they did not were not included in the analysis. There was no difference between the survival of untreated queens in the control group when they were kept in closed or open nests (100% survival in both cases), meaning that nest type did not affect their mortality. Overall, once the queens were killed by the pathogen or freezing, we had four treatment groups: (i) untreated queens in closed nests with the corpse of a pathogen-exposed queen (n = 23); (ii) untreated queens in closed nests with the corpse of a sham-treated queen (n = 24); (iii) untreated queens in open nests and the corpse of a pathogen-exposed queen (n = 24); (iv) untreated queens in open nests and the corpse of a sham-treated queen (n = 21).

On a daily basis, each nest was inspected visually for several minutes to record the presence or absence of a behavioural response of untreated queens to corpses, as well as when sporulation occurred. Burial was recorded when queens had covered the corpses in plaster or had built a ‘wall’ that separated the untreated queen from the corpse, whilst biting was defined as the removal of limbs and/or body segments. In addition, we recorded the survival of the untreated queens and, when they died, if sporulation occurred on their corpse, which we always identified to be Metarhizium. In a few cases, the exact timing of the occurrence of the behaviour (2/36 for biting, 5/31 for burial, 1/35 for removal), or sporulation (1/45) was missed. Exact sample sizes per test are provided in the results section. The experiment was run at 23 °C and 70% humidity, under a 12 h light:dark schedule, though because dishes were covered in red foil, the closed nests and the smaller chamber in the open nests were always darkened, mimicking the dark chambers in which queens reside. The duration of the experiment, from pathogen exposure to the final inspection for fungal growth, was 30 days. All queens in this experiment were collected in 2014.

Data analysis

All statistical data analysis was carried out using R version 3.3.2 [62]. We analysed the colony co-founding choice of queens using a generalised linear mixed model (GLMM; ‘lme4’ R package [63]), including chamber choice as a logistic response and a predictor for when the introduced queen was pathogen-exposed, a predictor for when the residing queen was pathogen-exposed, a predictor for time (z-transformed) and a three-way interaction between all three predictors to assess if co-founding choice differs over time. To control for the repeated observation of the same replicate, a random intercept was included for each replicate, and their individual differences over time were explicitly modelled by including random slopes for each individual. General linear models (GLMs) with binomial error terms and logit-link functions were used to compare the behaviour of queens towards infected and non-infected corpses, including the presence/absence of the behaviour (biting, burial or removal) as the response and the type of corpse (infected or non-infected) as a predictor and day (log transformed to achieve normality) of treated queen death as a covariate (since queens died on different days). However, as the covariate was always non-significant (biting: LR χ2 = 0.28, df = 1, P = 0.6; burial: LR χ2 = 0.28, df = 1, P = 0.6; removal: LR χ2 = 0.98, df = 1, P = 0.32), we removed it from the models to gain better estimates for the remaining predictor. In these models, we analysed open and closed nests separately, given that there were clear differences in the types of behaviours performed between nest types. Mann-Whitney U tests were used to test for differences between the day of onset of undertaking behaviours between infected and non-infected corpses. Wilcoxon signed-rank tests were used to compare the days that the undertaking behaviours and fungal sporulation occurred, and to control for multiple testing, we corrected the resulting P values using the Benjamini-Hochberg procedure to protect against a false discovery rate of 0.05% [64]. Adjusted P values are reported. The survival of queens performing different behaviours was analysed using GLMs with binomial error terms and logit-link functions that included mortality of the untreated queen as the response and the presence/absence of the behaviour (biting, burial or removal) as the predictor. Again, we controlled for multiple testing by correcting the P values from these models using the Benjamini-Hochberg procedure [64]. We analysed whether the onset of the behaviour affected untreated queen survival by including the mortality of the untreated queen as the response and the day the behaviour was performed, relative to queen death, as the predictor. For these models, we log(x + 1) transformed day to achieve normality, and corrected the resulting P values using the Benjamini-Hochberg procedure. We also tested whether, overall, the duration that untreated queens (including both those from open and closed nests) were with the pathogen-exposed queens before they died affected survival in the same way, again, log(x + 1) transforming day. We ensured all data fit the assumptions of the models (i.e. normality of predictors, multicollinearity, Cook’s distance, dffits, dfbetas and leverage) and overall model significance, plus the effect of predictors, were tested by comparing full models to nested null and reduced models, respectively, where all predictors present occur in the full model (except those being tested), using likelihood ratio tests. All graphs were made using the ‘ggplot2’ R package [65].