Toxoplasma gondii infection enhances the kairomonal valence of rat urine

Introduction

The protozoan parasite Toxoplasma gondii manipulates the behavior of its rat host in two important ways. First, it not only abolishes the rats’ innate fear of cat odors, it can also induce attraction to cat odors^1–4. This plausibly increases parasite transmission to cats that serve as its definitive hosts. Second, Toxoplasma gondii infection enhances the attractiveness of infected males to females⁵. Interestingly, enhanced attractiveness could benefit both the parasite and its host. This plausibly increases the parasites’ transmission through sexual and vertical routes^5–8 and benefits the hosts by increasing their reproductive opportunities.

Host-parasite interaction is typically characterized by significant trade-offs. Behavioral manipulation itself can impose substantial direct and opportunity costs on the parasite^9–11. For example, manipulation of innate fear in infected rats is optimized and not maximized¹²; suggesting that a dynamic balance exists for the parasite between the costs and benefits of the manipulation. From the perspective of the rat host, one of the most important trade-offs is the reproductive benefit obtained from increased attractiveness and the reproductive cost incurred by greater predation. An experimental study of this trade-off would require an ethically tenuous comparison of predation rates between control and infected animals. Another important trade-off for the host arises from the fact that pheromones produced to communicate male attractiveness are openly broadcasted, liable to be used by both the intended female audience and unintended prey or predator species. We examine this trade-off in the current study.

House mice are predated by rats. Studies have shown that around 70% of wild rats^13,14 kill mice. Mouse-killing or muricide has been shown to be influenced by rearing¹⁵, availability of food¹⁴ social¹⁶ and environmental¹⁷ conditions. In addition, mice express an innate fear of rats^18,19. This is characterized by the display of defensive behavior, secretion of stress hormones and activation of brain pathways dedicated to defensive behaviors. In fact, exposure to rat urine or even a recombinant rat urinary protein is sufficient to produce aversion in mice^20,21, with the presence of an actual rat not being necessary. Interestingly, in the case of rats, exposure to soiled bedding is sufficient for females to infer greater attractiveness of Toxoplasma gondii-infected males⁵. This suggests enhanced pheromonal production in infected males, which could result in greater kairomonal aversion in mice considering the openly broadcasted nature of urinary signals. Enhanced aversion in prey species could constitute an opportunity cost for the infected rats that need to be ‘traded-off’ with any incremental benefit of enhanced pheromone production. In light of this, we investigated whether Toxoplasma gondii infection increased the kairomonal valence of rat urine to its prey, mice.

Materials and methods

Results

Avoidance of mice in response to control or infected rat scent marks was determined using a avoidance-avoidance conflict task (n = 12 uninfected control mice). Kairomonal response to fresh urine and aged urine marks was studied separately and in sequence (aged followed by fresh marks).

ANOVA was conducted with time spent in each bisect (control or infected) and age of urine (fresh or aged) as two sources of within subject factors. ANOVA revealed a significant main effect for the infection status of scent donors (F_(1,11) = 8.049, p = 0.016). The difference between fresh and aged scent marks did not reach statistical significance (F_(1,11) = 0.005, p > 0.9). The interaction between the infection status of the donor and the age of the scent marks also did not reach statistical significance (F_(1,22) = 0.314, p > 0.5).

Over a twenty-minute trial, mice spent more time in the bisect containing fresh urine from control rats (planned comparison; paired t-test: t₂₂ = 2.13, p < 0.05; 674 ± 55 s in control bisect versus 510 ± 49 s in infected bisect; Figure 1A). A preference score of mice for control rat urine was computed for each trial by dividing the time spent in the control bisect by the time spent in the infected bisect. In 75% of trials, mice spent more time in the control bisect (Figure 1B; mean preference score = 1.36 ± 0.22; one outlier removed). Similar results were obtained when using aged urine marks. Mice spent more time in the bisect containing aged urine marks from control rats (planned comparison; t₂₂ = 3.36, p < 0.01; 715 ± 52 s in the control bisect versus 469 ± 51 s in the infected rat bisect; Figure 2A). Just like the fresh urine experiment, 9 out of 12 mice spent more time near the urine marks obtained from control animals (Figure 2B; mean preference score = 1.94 ± 0.36).

Figure 1. Uninfected mice avoid scent marks obtained from infected rats.

Preference was quantified by comparing time spent by a mouse in two opposing bisects of an arena, with each bisect containing fresh urine from either control rats or rats infected six weeks earlier (panel A; trial duration = 1200 s, n = 12 mice). Ordinate and abscissa depict time spent in infected and control bisect in seconds, respectively (p < 0.05, paired t-test). Mean and SEM of data used in scatter-plot are depicted by dot and whiskers. A preference score was computed for each mouse by dividing the time spent in the control bisect with time spent in the infected bisect (panel B; chance = 1). Each dot represents preference data from one mouse (1 outlier removed). Box plots depict median, 25^th percentile and 75^th percentile. Mean and SEM of data used in scatter-plot are depicted in dot and whiskers.

Figure 2. Uninfected mice avoid aged scent marks from infected rats.

Preference of mice for control urine marks was retained when aged (3 days old), rather than fresh, rat urine was used. Panel A depicts time spent in the control and infected bisects; Panel B depicts preference ratio (see Figure 1 for further details on what the graphs denote).

Thus, fresh and aged urine marks obtained from infected rats evoke a greater kairomonal response than urine obtained from uninfected rats in mice.

Discussion

As a result of intense predation pressure^13,14, mice have developed an innate sensitivity to rat kairomones. Rat odors evoke immediate and intense defensive behaviors in laboratory mice, coupled with activation of brain pathways typical of defensive behavior and secretion of stress hormones^18–20. Here we report that urine obtained from rats infected with the parasite Toxoplasma gondii generate greater avoidance in mice compared to control rats. Furthermore, we demonstrate that the active ingredient involved in parasite-induced kairomonal changes is most likely non-volatile, leading us to speculate that major urinary proteins are involved. This is in agreement with a prior report that purified recombinant rat major urinary proteins can induce kairomonal aversion in mice, precluding an essential role of urinary volatiles²⁰.

Pheromones are chemical substances produced by an individual animal, which affect the behavior of conspecifics (chemical communication). Pheromones are widely used by insects to communicate danger, the presence of food or sexual receptiveness. In the mammalian world, pheromones are often used to signal dominance and to influence female mate choice^22–24. Moreover, pheromones can act as kairomones when these chemical substances are received by individuals of another species²⁵. As such, kairomones are used to the detriment of the emitter and for the benefit of the receiver²⁶. This is because, once produced, pheromones are an openly broadcasted information system. Apart from the intended receivers of the same species, they can also be perceived by unintended receivers of a different species. These unintended receivers typically fall into two categories: predators and prey. Predators routinely use pheromones produced by their prey to locate their food^27–29. Prey can also use this information to reduce their danger of predation^20,30, although this possibility is relatively less well studied. Thus, pheromones of a species can be used as kairomones by both the prey and predator of that species.

Parasitic infections can drastically affect pheromonal production. For example, female mice typically avoid the odor of male mice infected with an array of bacteria, viruses, protozoa and nematodes³¹. Atypically, male rats infected with the protozoan parasite Toxoplasma gondii produce urine that is more attractive to receptive females, suggesting enhanced pheromonal production⁵. We have also observed that Toxoplasma gondii can be transmitted during sexual intercourse in rats⁵ (see^6,32 for sexual transmission in other species). It is likely that increased male attractiveness is a parasitic manipulation, aiming to increase the frequency of parasite transmission between males and females (but also see³³). This atypical host manipulation by Toxoplasma gondii opens a rather interesting trade-off for the infected host. An increase in pheromonal communication might lead to enhanced reproductive benefits for the infected male. At the same time, intra-species pheromonal communication can be co-opted by prey to initiate defensive behaviors, placing a probabilistic cost on the predator. Consistent with this trade-off, the data presented here suggests that male rats infected with Toxoplasma gondii suffer opportunity costs in terms of greater aversion by mice, a prey species.

Data availability

figshare: Proportion of time spent by mice near urine from rats infected with Toxoplasma gondii vs urine from uninfected rats, doi: 10.6084/m9.figshare.993871³⁴