Although sociability offers many advantages, a major drawback is the increased risk of exposure to contagious pathogens, like parasites, viruses, or bacteria []. Social species have evolved various behavioral strategies reducing the probability of pathogen exposure []. In rodents, sick conspecific avoidance can be induced by olfactory cues emitted by parasitized or infected conspecifics []. The neural circuits involved in this behavior remain largely unknown. We observed that olfactory cues present in bodily products of mice in an acute inflammatory state or infected with a viral pathogen are aversive to conspecifics. We found that these chemical signals trigger neural activity in the vomeronasal system, an olfactory subsystem controlling various innate behaviors []. Supporting the functional relevance of these observations, we show that preference toward healthy individuals is abolished in mice with impaired vomeronasal function. These findings reveal a novel function played by the vomeronasal system.

To put our observations to the test in a more natural setting, we evaluated the role of the vomeronasal system in the avoidance of mice infected with one of its natural pathogens, the mouse hepatitis virus (MHV). This single-stranded RNA (ssRNA) virus of the Coronaviridae family is extremely contagious and is present in both wild and laboratory mice []. Infection with an enterotropic strain of MHV induces an immune response but is usually asymptomatic in adults []. We first observed that control C57BL/6J mice displayed a preference for investigating uninfected mice over MHV-infected animals (loginvestigation time ratio C57BL/6J = 0.58 ± 0.05; Figure 4 A). Urinary cues from MHV-infected mice were sufficient to trigger avoidance behavior (loginvestigation time ratio C57BL/6J = 0.35 ± 0.18; Figure 4 B), paralleling what we previously observed with LPS-treated mice. The perception of these cues increased neural activity in the AOB relative to controls ( Figures S4 C–S4F), pointing again toward a role played by the vomeronasal system. This role was evaluated by exposing Trpc2, Trpc2, and Trpc2mice to uninfected versus MHV-infected animals. Unlike control animals, vomeronasal-deficient mice did not prefer healthy peers and even preferentially investigated MHV-infected mice (loginvestigation time ratio Trpc2= 0.11 ± 0.06; Trpc2= 0.52 ± 0.07; Trpc2= −0.63 ± 0.07; Figure 4 A). This was without altering the time spent investigating healthy conspecifics (mean investigation time of contact with healthy mouse by Trpc2and Trpc2mice = 88 ± 10 s, n = 19; Trpc2= 83 ± 13 s, n = 12; p = 0.815, Mann-Whitney U test), meaning that the inverted ratio resulted from an increased interest toward sick conspecifics. Together, these results show that the VNO plays a crucial role in the differential behavior mice adopt toward healthy or sick conspecifics.

(B) Urine preference assay comparing the time mice spent in compartments containing urine from uninfected and MHV-infected mice. The black bar indicates the mean value ± SEM, and dots indicate values of individuals. n = 10. ∗ p < 0.05, one-sample t test.

(A) Log 2 investigation time ratios of healthy/MHV-infected mice by C57BL/6J, Trpc2 +/+ , Trpc2 +/− , and Trpc2 −/− male mice. Gray bars indicate average values (±SEM), and each dot represents an individual. n = 8–19. p = 0.001, Kruskal-Wallis ANOVA; ∗ p < 0.017, ∗∗ p < 0.003, ∗∗∗ p < 0.0003, Mann-Whitney post hoc analysis with Bonferroni correction.

To evaluate the function played by the vomeronasal system in health status assessment, we took two different loss-of-function approaches. First, we evaluated the neural response and the behavior of mice with a genetic disruption of the Trpc2 ion channel [] (a mutation that results in an impairment of VNO function) when exposed to sick conspecifics or to inflammation-related compounds present in urine. We observed no increased neural activity in the AOB of Trpc2mutants upon exposure to LPS urine ( Figures 2 E, 2F, and 2H). When confronted with a healthy and an LPS-injected conspecific, control mice (Trpc2and Trpc2) spent more time investigating healthy animals (loginvestigation time ratio Trpc2= 0.53 ± 0.07; Trpc2= 0.57 ± 0.11), whereas Trpc2mutants displayed no preference (loginvestigation time ratio Trpc2= −0.16 ± 0.08; Figure 3 A). Second, in a complementary approach to the genetic alteration of VNO function (to address the fact that a small portion of sensory neurons from the main olfactory epithelium also express Trpc2 []), we tested the behavioral response of mice whose VNO was surgically removed (VNX mice; Figures S3 A and S3B) but whose general olfactory abilities were intact ( Figures S3 C and S3D). Sham-operated mice spent more time investigating healthy animals (loginvestigation time ratio Sham = 0.66 ± 0.25), and this preference was abolished in VNX mice (loginvestigation time ratio VNX = 0.03 ± 0.20; Figure 3 B). The lack of positive bias toward healthy individuals in Trpc2mice was also observed when mice were exposed to urinary cues (loginvestigation time ratio Trpc2= 0.23 ± 0.16; Trpc2= 0.20 ± 0.09; Trpc2= −0.70 ± 0.34; Figure 3 C).

(A) Log 2 investigation time ratios of healthy/LPS-injected mice by Trpc2 +/+ , Trpc2 +/− , and Trpc2 −/− mice. Gray bars indicate mean values (±SEM) for each genotype, and each dot represents an individual. n = 6–15. p = 0.001, Kruskal-Wallis ANOVA; ∗ p < 0.017, ∗∗ p < 0.003, Mann-Whitney post hoc analysis with Bonferroni correction.

In most mammals, the olfactory system is subdivided into two principal subsystems, the main olfactory and the vomeronasal systems, which can both mediate innate behaviors []. The sensory structure of the vomeronasal system, the vomeronasal organ (VNO), detects cues triggering various innate social behaviors []. To determine whether sickness-related compounds are detected by vomeronasal sensory neurons (VSNs), we evaluated the expression of the activity-driven cFos protein [] in the accessory olfactory bulb (AOB), the brain structure targeted by VSN axonal projections. We observed that exposure to LPS urine induced higher neural activity in the AOB relative to healthy urine exposure ( Figures 2 A–2D and 2G ). The most striking increase of cFos signal was observed in the granule cell layer, in both the anterior AOB (fold change: 2.3) and posterior AOB (fold change: 2.02; Figure 2 G). The number of activated AOB neurons following LPS urine exposure was, however, lower than the number observed in the posterior AOB after exposure to rat bedding (rats are mouse predators) ( Figure S2 ).

(G and H) Quantification of cFos-positive cells in the anterior AOB (aAOB) and posterior AOB (pAOB) of Trpc2 +/+ (G) and Trpc2 −/− (H) mice. Scale bars represent 50 μm. Bars indicate average values (mean ± SEM) from all individuals, and dots indicate average values from 3–4 sections pertaining to one individual. ∗∗ p < 0.01, ∗∗∗ p < 0.001, Mann-Whitney U test.

(A) cFos immunostaining (red) and DAPI staining (blue) on the AOB. a, anterior; p, posterior; gl, periglomerular cell layer; mi, mitral cell layer; gr, granular cell layer. The scale bar represents 100 μm. The white box indicates the region magnified in (B)–(F).

In mammals, information about sexual and social status is often conveyed by olfactory cues found in urine []. In mice, this is also true for sickness-related signals that induce avoidance behaviors, in particular those produced by parasitized peers []. To determine whether the urine of LPS-injected mice contains olfactory cues able to recapitulate the effect of whole animals, we performed an odor preference assay based exclusively on urine stimuli. We found that mice spent more time investigating urine from healthy individuals rather than from LPS-injected individuals (loginvestigation time ratio = 0.41 ± 0.15; Figure S1 A). In order to determine whether the urine of LPS-injected mice contained aversive olfactory cues, we established assays based on the animals’ propensity to manipulate nesting material (blotting paper) impregnated with urine samples. Initially, we observed that the strong natural inclination to manipulate and incorporate blotting papers into the nest could be counteracted by the addition of predator urine (a mixture that contains aversive cues) ( Figures S1 B–S1D). We then assessed the behavior of mice that had the choice to use blotting papers impregnated with urine from healthy mice (healthy urine) or LPS-injected mice (LPS urine) ( Figure 1 C). We observed a preferential interaction with healthy urine samples (fold change: 3.25; Figure 1 D), associated with an increased frequency of incorporation of the papers into the nest (fold change: 8; Figure 1 E). These results may be explained either by a decrease of attractive cues or by the presence of repulsive cues in the urine of mice in an inflammatory state. To test for the presence of these potential aversive olfactory signals, we evaluated whether addition of LPS urine to the nesting paper could reverse the natural propensity of mice to use the material. In this single-choice avoidance assay, we found that the addition of LPS urine reduced the interest toward the papers ( Figures 1 F–1H). Together, these results strongly suggest that urine from mice in an endotoxin-induced inflammatory state contains aversive cues.

Pathogenic agents themselves are not necessary to drive sick conspecific avoidance []. We first evaluated the investigation pattern of mice in the presence of a healthy peer and in the presence of a conspecific in an acute inflammatory state. This state was induced by injecting mice with lipopolysaccharide (LPS), an endotoxin produced by Gram-negative bacteria that activates the immune system and mimics bacterial infection []. Stimulus animals were anesthetized to diminish non-olfactory cues such as motor behaviors or vocalizations. Mice displayed a strong investigation preference toward healthy conspecifics (loginvestigation time ratio = 0.48 ± 0.17; Figure 1 A). To assess whether this difference of investigation toward healthy and sick peers reflected a simple preference toward healthy animals and was a byproduct of a setting involving a choice between two animals, we measured the time spent by mice investigating animals presented individually. Again, mice spent more time inspecting healthy mice over LPS-injected mice (loginvestigation time ratio = 0.30 ± 0.13; Figure 1 B).

(C–E) Schematic of the odorized paper interaction assay with choice (C), where petri dishes containing blotting papers impregnated with healthy urine (light green) and LPS urine (light orange) are placed inside the mice’s home cage and the number of papers displaced (D) and brought inside the nest (E) after 5 min are counted. n = 8; mean ± SEM; ∗ p < 0.05, Wilcoxon matched-pairs test.

(B) Conspecific investigation assay without choice. Healthy and LPS-injected stimulus mice were presented individually on 2 consecutive days (randomized order). n = 15. The black trace shows an example of a recorded track in the presence of an LPS mouse.

(A) Left: schematic representation of the conspecific investigation assay with choice. Each mouse freely investigated two anesthetized conspecifics: one healthy mouse (investigation zone depicted in light green) and one LPS-injected mouse (light orange). The black trace shows an example of a recorded track from the investigating mouse. Right: log 2 investigation time ratios of healthy/LPS-injected mice, where the black line indicates mean value (±SEM), and each dot represents an individual. n = 10.

In the wild, animals are exposed to various pathogenic and transmissible agents, including viruses, bacteria, and parasites. Social animals, which interact closely with their conspecifics, are particularly prone to exposing themselves to a high risk of contagion []. From humans displaying disgust toward persons with symptoms of pathogenic infections [] to spiny lobsters shunning conspecifics carrying a lethal virus [], the behavior of social species has been shaped throughout evolution by host-pathogen interactions and has been selected to minimize the risk of exposure []. The initial step to avoid a pathogen is to identify the threat. Health status assessment is based on the perception and integration of various cues. For example, in some fish and birds, sexual ornamentation is altered in parasitized males; these visual cues play an important role for female mate choice []. In many mammals though, health assessment largely relies on olfactory signals. Rodents have an ability to discriminate between odorant cues from healthy and parasitized conspecifics []. This ability, which results from an altered balance between attractive and repulsive signals and translates into a limited investigation of unhealthy peers, is innate. We refer to such behavior as “sick conspecific avoidance.” Although extensive research has described sick conspecific avoidance behaviors, little is known about the neural circuits and mechanisms underlying this ability. Here, we show that in addition to mediating male sexual preference [], female sexual behavior [], predator avoidance [], and inhibition of sexual behavior toward juveniles [], the vomeronasal circuit mediates sick conspecific avoidance.

Discussion

14 Stowers L.

Holy T.E.

Meister M.

Dulac C.

Koentges G. Loss of sex discrimination and male-male aggression in mice deficient for TRP2. 15 Leypold B.G.

Yu C.R.

Leinders-Zufall T.

Kim M.M.

Zufall F.

Axel R. Altered sexual and social behaviors in trp2 mutant mice. 17 Papes F.

Logan D.W.

Stowers L. The vomeronasal organ mediates interspecies defensive behaviors through detection of protein pheromone homologs. 18 Ferrero D.M.

Moeller L.M.

Osakada T.

Horio N.

Li Q.

Roy D.S.

Cichy A.

Spehr M.

Touhara K.

Liberles S.D. A juvenile mouse pheromone inhibits sexual behaviour through the vomeronasal system. Social species, which are particularly exposed to transmissible pathogens, display a wide range of behavioral strategies to avoid infected conspecifics. The signals that act as stimuli triggering sick conspecific avoidance behaviors can include visual, auditory, and olfactory cues. We observed that mice avoid contact with conspecifics infected by a virus or conspecifics in an artificially induced inflammatory state and found that olfactory cues perceived by the VNO are driving this behavior. This adds to the few innate behaviors that have been shown to rely on this olfactory subsystem [].

6 Arakawa H.

Arakawa K.

Deak T. Sickness-related odor communication signals as determinants of social behavior in rat: a role for inflammatory processes. 13 Kavaliers M.

Choleris E.

Pfaff D.W. Recognition and avoidance of the odors of parasitized conspecifics and predators: differential genomic correlates. 30 Avitsur R.

Cohen E.

Yirmiya R. Effects of interleukin-1 on sexual attractivity in a model of sickness behavior. 31 Penn D.

Potts W.K. Chemical signals and parasite-mediated sexual selection. 32 Zala S.M.

Potts W.K.

Penn D.J. Scent-marking displays provide honest signals of health and infection. 33 Litvinova E.A.

Kudaeva O.T.

Mershieva L.V.

Moshkin M.P. High level of circulating testosterone abolishes decline in scent attractiveness in antigen-treated male mice. We observed that the urine of immune-challenged mice contains cues that trigger decreased interest relative to urine from healthy individuals. The identity of these cues is unknown. Our results with LPS-treated stimulus animals are consistent with previous studies showing that inflammatory processes can result in the production of olfactory signals inducing avoidance behaviors []. While pathogens, like viruses or bacteria, are very diverse, the immune response they generate is in most cases stereotyped and conserved. From an evolutionary perspective, the emergence of molecular sensory tools recognizing the constantly evolving cues deriving directly from numerous and different pathogens is far more complex than the emergence of sensory tools allowing the recognition of a few conserved molecules related to the immune response triggered by the pathogens. Chemical compounds involved in health assessment could be directly involved in or could result from inflammatory processes. For example, modifications in the concentration of hormones and urinary proteins would represent interesting candidates since they have been linked to infection and inflammation []. Of particular interest is the fact that the aversive cues could be related to stress induced by sickness and could thus signal danger in a broader sense. However, we made two observations that make this latter possibility unlikely: (1) the hypothalamic-pituitary-adrenal stress axis is not activated in LPS and MHV stimulus animals ( Figure S1 E), and (2) urine of stressed mice (by exposure to a predator odor) is not aversive to conspecifics ( Figures S1 F and S1G).

How the benefit of this health status assessment translates into wild rodent populations is unknown. Sick conspecific discrimination could represent an advantage at several levels, but the capability to avoid sick peers is likely to be crucial for mate choice. Indeed, it is decisive both to ensure the fitness of the parents that take care of the progeny and to enhance the genetic predisposition for pathogen resistance in offspring. The avoidance behavior toward sick conspecifics that we described is not as absolute as the avoidance that is observed, for example, toward predators. It is conceivable that sick conspecific avoidance is not categorical but that the magnitude of the aversion toward sick peers depends on several contextual variables, such as the availability of partners, the degree of infection, or the pathogenic load of the social group.

−/− mice not only lack avoidance behavior but also are rather attracted to sick conspecifics. This possibly reflects the natural interest exhibited by mice toward novelty [ 34 Barnett S.A.

Cowan P.E. Activity, exploration, curiosity and fear: an ethological study. 35 Isles A.R.

Baum M.J.

Ma D.

Keverne E.B.

Allen N.D. Urinary odour preferences in mice. Interestingly, we observed that Trpc2mice not only lack avoidance behavior but also are rather attracted to sick conspecifics. This possibly reflects the natural interest exhibited by mice toward novelty [], a curiosity that in this specific case is highly undesirable and that is revealed here by the absence of vomeronasal brake. If this explanation is correct, vomeronasal function not only makes sick individuals less desirable but also prevents them from being of particular interest.