Study system

The mice used in this experiment were F2 descendants of wild-trapped house mice (Mus musculus musculus) from Vienna (48° 12’ 38” N; 16°16’54” E). The parental generation were trapped at 14 different locations within a 500 m radius and then bred between locations. F1 mice were tested in a social partner preference and either a female’s preferred (P) or the non-preferred (NP) male was assessed and paired accordingly (see below) [following 9]. The resulting offspring (F2 generation) were used for experimental infection (see below). All F2 mice were weaned at the age of 21 ± 1 days and were kept individually in standard mouse cages (26.5 × 20.7 × 14 cm) with wooden bedding (Abedd: aspen wood chips), enrichment material consisting of nesting material (Abedd: aspen wood shavings), and nest boxes. Food (Altromin rodent diet 1324) and water were provided ad libitum. A standard 12:12 h light cycle was maintained and temperature ranged from 22 to 25°C. After experimental infection animals were housed individually in ventilated cages (36.5 × 20.7 × 14 cm, IVC). All other housing conditions remained identical to conditions prior to infection.

Social preference test

To test partner preference in wild-derived house mice, F1 mice were assigned to 30 triplets each containing one female (N = 30) and two unrelated males (N = 60). Prior to the social preference test, all females successfully mated and raised one litter to avoid any confounds from females’ sexual experience. Males were sexually mature (16.52 ± 0.35, mean ± SE), however they had no mating experience. On the day of the experiment, all mice were weighed. Mice were brought to the testing room at 12:00 to allow the animals to acclimate for six hours (five triplets were tested per day). During acclimation, male mice were stimulated with 5 μl of pooled female’s urine on filter paper (collected and mixed from seven females over five consecutive days). The experiments started in the dark phase (under red light conditions) and were conducted between 18:00 and 22:00. All trials were recorded with a D-link camera (DCS-3710 Day & Night WDR network camera) sensitive to red light. To avoid any effects from the presence of an observer on mouse behavior all experiments were simultaneously observed outside the experimental room on a monitor connected to the video camera system. Later, recorded videos were analyzed using the program Noldus Observer XD 9.0 to verify initial partner preference of females based on direct monitor observations.

The experimental apparatus consisted of three cages (Figure 3): one central female cage (26.5 × 20.5 × 14 cm) was connected with plastic tubes to two male cages on the left and right side, each measuring 36.5 × 20.5 × 14 cm (Figure 3). Male cages were separated into two compartments (i, ii) using an acrylic glass divider with several holes. This setup allowed exchange of acoustic, tactile and odour signals, however prevented mating. First, at the beginning of the experiment males were placed individually into their compartments (i), and bedding material from the male’s original cage was scattered in the other compartment (ii) of the male cages to provide direct odour cues for the female (Figure 3). Afterwards females were placed in the central cage. The connecting tubes were closed with regular inflated balloons to prevent the female from 1) entering male cages until the habituation period for all animals ended and 2) obtaining odour cues from male’s bedding material before the experiment started. After 10 minutes of acclimatization, video recording started and the balloons were simultaneously deflated to make the tubes (and cages) accessible for the female. The female was considered to have entered or left a cage when its nose was visible in the cage. The social preference recording began once the female had visited both male cages. This procedure ensured that the female was aware of both males (when she left the second male’s cage). Each trial lasted for 10 minutes, which has been shown to be a good predictor for partner preference [9]. Time the female had spent in each male cage was recorded by using a stopwatch. A male was classified as ‘preferred’ if the female spend more than 60% of the time in its compartment [following 9]. Subsequently, half of the females were paired with their preferred (P), the other half with their non-preferred (NP) male in a new cage and returned to their housing room. Females that were paired with P or NP males did not differ in weight (Mann-Whitney U test: U = 43, P = 0.631, r = 0.11). All pairs were separated after five days. The litter sizes, individual F2-pup weight and sex ratio were determined at weaning.

Figure 3 Social preference test. The central cage (A), where the female was located, was connected with the two male cages (B1, B2) by plastic tubes. The tubes were closed at the beginning of the experiment, after acclimatization the barriers were removed (C) to enable females (A) to access the two males’ cages. Males cages were separated into two compartments using perforated plastic dividers (d1, d2), which allowed olfactory, acoustic and visual inspection between sexes, however preventing a mating event. Each male was restrained to only one compartment of their cage (i, ii). Full size image

Salmonellainfection

To assess pathogen clearance of F2 offspring from P versus NP sires, a total of 72 offspring resulting from 23 families were experimentally infected on two consecutive days. The number of individuals was balanced for partner preference (P/NP) and sex. All mice were adults (15-21 weeks old) and received an intraperitoneal (IP) infection of 200 μl Salmonella enterica serovar Typhimurium (103 cfu mL-1, strain LT2). The bacteria (stored as slants at 4°C and originated from frozen stocks at -80°C) were cultured in 7.5 ml of heart-brain infusion at 37°C for 13 h (overnight) while shaking at 170 rpm. The overnight solution was diluted with sterile phosphate buffered saline (PBS) until the desired dilution of 103. To verify the concentration, serial plating of the dilution (50 μl per plate and three plates per solution) was performed.

All animals (N = 72) were divided into three groups to obtain a sample size of 24 individuals per week during the experimental period of three weeks. Groups were balanced for partner preference and sex. Individuals were euthanized and dissected over three weeks: group 1 after seven days (N = 24), group 2 after 14 days (N = 24), and group 3 21 days post infection (N = 24). The health condition of mice was examined daily until the end of the experiment. All animals were weighed weekly and euthanized humanely using an overdose of CO 2 . Additionally, we also monitored the survival of additional P (N = 64) and NP (N = 44) offspring in the colony (same age class), which were not experimentally infected. To determine pathogen loads of experimentally infected animals, spleens of euthanized animals were immediately removed and homogenized (Dispergierstation, T 8.10, IKA®-Werke) in 1 ml PBS under sterile conditions. Afterwards, 50 μl of each homogenate was plated on selective agar plates (Önöz Salmonella agar Merck, Darmstadt, Germany), and incubated overnight for 18 hours at 36°C. Pathogen loads per spleen were determined by calculating bacterial concentration (cfu/spleen) of spleen homogenates [25, 37] using the mean of three replicate plates. The experimental procedure was in accordance with ethical standards and guidelines in the care and use of experimental animals of the Ethical and Animal Welfare Commission of the University of Veterinary Medicine Vienna (Permit No. BMWF-68.205/0261-II/3b/2011).

Statistical analyses

To assess differences in female reproductive success when paired with P versus NP we compared the number of litters produced in the different treatments using a χ2 test. Differences in litter sizes were analysed using a general linear model (GLM) with pairing (P or NP male) as a fixed factor and female body mass as a covariate. Mean pup weight (offspring quality) was calculated as the total weight of litters divides by litter size at weaning. We compared mean pup weight of litters sired from P versus NP males in using a GLM with pairing (P or NP male) as a fixed factor and female body mass as a covariate. Sex ratio was analysed using a generalized linear model (GZLM) with a binomial distribution and a logit link function. The number of males within a litter was included as the dependant variable and litter size as binomial denominator. Pairing (P or NP male) was integrated as a fixed factor and female body mass as a covariate. These statistical analyses were performed using IBM SPSS® version 19 (SPSS Inc., Chicago, Illinois) software.

For subsequent pathogen and survival models we tested whether initial body mass was randomly distributed across experimental groups by using a linear mixed-effects model (LMM) with offspring sex and partner preference (P, NP) as fixed effects and week (time to euthanasia, see above) as a covariate. We also entered the day of experimental infection as a random effect to control for statistical non-independence of trials started on one or the other date. Additionally, to control for sex difference in body mass, we calculated residuals of body mass on sex and used these residuals as sex-independent measure of body mass. We transformed individual body mass and count data (pathogen load) using log-transformation to enhance normality and homogeneity of variances.

To assess individual pathogen load, we applied a LMM, entering partner preference (P/NP), and sex as fixed effects, week as a covariate and starting date of the experiments and family (N = 23) as random effects. We analysed survival using a generalized linear mixed-effects model (GZLMM) with binomial error distribution, entering partner preference treatment, and sex as fixed effects, week and body mass as covariates and starting date of the experiments and family ID as random effects. Statistical analyses were performed using ‘R’ (version 2.14.1). We implemented LMMs using the ‘lme’ function of the ‘nlme’ package, and GZLMM using the ‘glmmPQL’ function of the ‘MASS’ package.

We included all two-way interactions into initial models and applied a backward stepwise removal procedure [63] to avoid problems because of inclusion of non-significant terms (P < 0.05) [64]. Removed variables were re-entered one by one to the final model to obtain relevant statistics.