Parasitized female mice display reduced aversive ... - Europe PMC

Parasitized female mice display reduced aversive responses to the odours of infected males Martin Kavaliers1,2*, Douglas D. Colwell3 and Elena Choleris4 1

Department of Psychology and 2Neuroscience Program, University of Western Ontario, London, Ontario, Canada N6A 5C2 ([email protected]) 3 Agriculture and Agri-Food Canada, Box 3000 Main, Lethbridge, Alberta, CanadaT1J 4B1 4 Lawson Research Institute, St Joseph's Health Centre and University of Western Ontario, London, Ontario, Canada N6A 4CL The present study showed that parasites in£uence both the responses of uninfected females to males and the responses of female hosts to infected males. In female laboratory mice one of the consequences of exposure to the olfactory cues associated with an infected male was a reduction of the reactivity to a thermal surface, i.e. pain inhibition or analgaesia. Uninfected oestrous and non-oestrous female mice displayed marked analgaesic responses after exposure to the odours of males infected with either the enteric single-host nematode parasite, Heligmosomoides polygyrus, or the protozoan parasite, Eimeria vermiformis. The uninfected oestrous females distinguished between infected and physically stressed males, displaying a greater analgaesic response to the odours of infected males. These analgaesic responses and their anxiety/ fearfulness-associated behavioural correlates could elicit either a reduced interest in, or avoidance of, parasitized males by females. Oestrous female mice infected with H. polygyrus displayed a reduced analgaesic response to the odours of the infected males and di¡erentially responded to the odours of males infected with either the same (H. polygyrus) or a di¡erent parasite (E. vermiformis). An exposure time of 1min elicited minimal responses to the odours of males infected with the same parasite, H. polygyrus, and an attenuated, though signi¢cant, non-opioid peptide-mediated analgaesic response to males infected with E. vermiformis. An exposure time of 30 min elicited similar markedly reduced endogenous opioid peptidemediated analgaesic responses to the odours of both of the categories of infected males. The responses to the odours of a stressed male were, however, una¡ected by the parasitic infection. The reduced analgaesic responses of the parasitized females to the odours of infected males may involve either enhanced odour familiarity and responses to group odour templates and/or neuromodulatory shifts resulting in reduced fearfulness and potentially greater interest in the infected males. Keywords: mate choice; sexual selection; parasites and behaviour; analgaesia; host behaviour

infected with either the murine protozoan parasite, Eimeria vermiformis, or the murine trichostrongylid nematode parasite, Heligmosomoides polygyrus (Kavaliers & Colwell 1995a,b). Resistant stages of both E. vermiformis and H. polygyrus are shed in the faeces of infected hosts and after a short development period they are infective to other mice that acquire them during feeding, grooming and other behavioural interactions (Blagburn & Todd 1984; Herandez & Sukhedo 1995). An additional consequence of exposure to the olfactory cues associated with an infected male was a reduction in the reactivity of females to a noxious thermal stimulus, i.e. pain inhibition or analgaesia. The nature of this pain inhibition varied as a function of the duration of exposure, with prolonged (15 min plus) exposures to the odours of infected males inducing a relatively longlasting, opioid peptide-mediated (i.e. endorphin, enkephalin) analgaesia and brief (1min) exposures eliciting a lower amplitude and short lasting non-opioidmediated analgaesia (Kavaliers & Colwell 1993, 1995b).

1. INTRODUCTION

Parasites have been reported to a¡ect mate choice and mating patterns (Hamilton & Zuk 1982; Zuk 1992, 1996), with various studies suggesting that females preferentially select parasite-free or resistant males on the basis of di¡erences in sexual ornamentation or coloration (e.g. Kennedy et al. 1987; Borgia & Collis 1989; Milinski & Bakker 1990; Wedekind 1992). In their original proposal Hamilton & Zuk (1982) speculated that during mate choice animals should examine urine and faecal cues in an attempt to detect mates that were disease- and parasite-free. In rodents, urine and other odour cues are of major importance in mate detection and choice (Lenington 1983; Brown 1995; Egid & Brown 1989; Hurst 1987, 1990a,b; Potts et al. 1994). We observed that female laboratory mice, Mus musculus domesticus, displayed a reduced interest in the urine and other odorous secretions of male mice *

Author and address for correspondence.

Proc. R. Soc. Lond. B (1998) 265, 1111^1118 Received 5 February 1998 Accepted 19 March 1998

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M. Kavaliers and others

Parasitized female mice responses to infected males

The non-opioid-mediated analgaesic responses and their neurochemical substrates were suggested to represent anxiety-related anticipatory defence reactions designed to prepare the female mouse for a potential encounter with a parasitized male, while the more protracted opioidmediated responses were proposed to be associated with stress and related defence responses. These analgaesic responses and their anxiety/fearfulness/stress-associated behavioural correlates could elicit either a reduced interest in, or avoidance of, parasitized male mice by the females and, thus, could potentially serve as a component of female mate preference. Under natural conditions it is likely that both male and female mice are harbouring parasites. Relatively little attention has, however, been given to the possible e¡ects of parasites on the responses of the host to sexual cues (Poulin & Vickery 1996). In two-host systems, male mice clinically infected with the nematode, Taenia crassiceps, and females infected with the nematode, Trichinella spiralis, were reported to be inhibited in their sexual behaviours (Edwards & Barnard 1987; Morales et al. 1996). Conversely, data from single-host systems suggest that parasitized individuals may be less choosy in terms of mate selection. Female upland bullies, Gobiomorphus breviceps, infected with the trematode, Telogaster opistschochis, were less particular in their mate preferences and more likely to choose lowerquality males than were non-parasitized individuals (Poulin 1994). In addition, we showed recently that subclinical infection with the protozoan, E. vermiformis, had, depending on the stage of the infection and associated neuromodulatory changes, either signi¢cant facilitatory or inhibitory e¡ects on male responses to females (Kavaliers et al. 1997a). These ¢ndings from single-host systems suggest that parasites may have signi¢cant e¡ects on the host's own mate responses or preferences. In the present study we addressed the possibility that parasitic infection may a¡ect the host's responses to other infected individuals and that a female's own parasites may in£uence her responses to males. H. polygyrus is a common natural nematode parasite of rodents that is used in elucidating host ^ parasite relationships (e.g. Cypess et al. 1977; Scott 1990). H. polygyrus has been shown to a¡ect the development of social status (Freeland 1981) and various behavioural attributes in mice (Kavaliers & Colwell 1995b; Kavaliers et al. 1997b). Here, we examined the thermal (nociceptive) responses and levels of analgaesia in H. polygyrus-infected and -uninfected female mice that were exposed for either 1 or 30 min to the urine and other odorous secretions of (i) uninfected male mice and (ii) male mice infected with either the same parasite, H. polygyrus, or a di¡erent parasite, E. vermiformis. As the results of previous studies had established that exposure to the odour cues of physically stressed mice also elicited signi¢cant analgaesic responses (e.g. Fanselow 1985; Kavaliers 1988; Colwell & Kavaliers 1990; Kavaliers & Colwell 1995b; Porro & Carli 1988), we further considered the responses of uninfected and infected female mice to the odour cues of uninfected restraint-stressed male mice. This addressed the possible non-speci¢c e¡ects of `stress odour' on the responses of females to infected males. Proc. R. Soc. Lond. B (1998)

2. METHODS

(a) Animals

Male and female mice [RML (CF-1 strain-derived)], 2^3 months old (25^30 g), were housed in separate rooms in clear polyethylene cages. Males and females were housed singly with a wood shavings bedding at 20 2 8C under a 12 h light : 12 h dark cycle. Food (Mouse Breeder Blox, Wayne Laboratory Animal Diets, Madison, WI) and water were available ad libitum.

(b) Parasite infection (i) H. polygyrus

Sexually naive female and male mice were infected by oral intubation with H. polygyrus larvae under light Metofane (Pitman-Moore, Canada) anaesthesia. Each mouse received approximately 400 infective (L3 stage) larvae of H. polygyrus suspended in 0.5 ml of distilled water. Sham-infected mice received 0.5 ml of distilled water delivered similarly. Parasite infection was determined by necropsy 30 d post-infection (p.i.). All of the mice given larvae were con¢rmed as infected by removing H. polygyrus from the small intestine: all of the shaminfected mice were uninfected. The infected mice displayed no evident signs of malaise (e.g. altered feeding, weight loss, poor grooming, disruption of oestrous cycles), or pathology at any stage of infection. Although the dose of L3 larvae used here is considered su¤cient to elicit immune depression in male mice (Wahid et al. 1989; Barnard et al. 1997), no behavioural responses associated with substantial changes in immune activity (e.g. Barnard et al. 1998; Maier & Watkins 1998) were evident in the female mice. Detailed descriptions of the life cycle of H. polygyrus are provided elsewhere (Cypess et al. 1977; Scott 1990).

(ii) E. vermiformis

E. vermiformis were obtained from a stock culture maintained at the Animal Disease Research Institute (W), Lethbridge, Alberta, Canada. There, oocysts had been regularly passaged through Swiss Webster mice to retain infectivity. Oocysts were stored in 2.5% potassium dichromate at 4 8C. Prior to use, oocysts were washed twice in distilled water. Male mice were infected by oral intubation under light Metofane anaesthesia. Each mouse received, through serial dilution of a stock solution, approximately 4000 oocysts of E. vermiformis suspended in 0.5 ml of distilled water. Shaminfected mice received 0.5 ml of distilled water delivered similarly. Parasite infection was determined by examination of faecal samples collected 12 d p.i. All mice given oocysts were con¢rmed infective, whereas all sham-infected mice produced no oocysts. The pre-infective (pre-patent) period for E. vermiformis in RML mice is 7 d, with mice becoming infective (patent) and shedding oocysts on day 8 p.i. Maximum oocysts output is evident 10^11d p.i., declining to minimal levels by days 18^20 p.i. (non-infective, post-patent stage). The infected mice displayed no evident signs of malaise (e.g. altered feeding, weight loss, poor grooming), or pathology at any stage of infection. Detailed descriptions of the life cycle of E. vermiformis, whose time-course and infectivity were con¢rmed in RML mice, are provided elsewhere (Rose & Hesketh 1976; Blagburn & Todd 1984; Colwell & Kavaliers 1993).

(c) Nociceptive responses of females

During the mid-to-late light period, uninfected (sham) oestrous, uninfected non-oestrous or 20-d H. polygyrus-infected oestrous female mice (n 5, in each case), were individually placed in clean cages (25 cm 15 cm 20 cm) with vented

Parasitized female mice responses to infected males Plexiglas tubes (10 cm in length, 3 cm in diameter and sealed at each end with plastic mesh). The tubes were provided with ¢lter paper containing the urine and other odorous secretions of either (i) uninfected male mice; (ii) male mice infected for 20 d with H. polygyrus; (iii) male mice infected for 5 d with E. vermiformis; or (iv) di¡erent individual uninfected male mice that had been restrained for 15 min in a polypropylene centrifuge tube (Corning, New York) provided with air vents. Each female was presented with the odour of only one male, and the odour of a di¡erent male was used with each female. Isolated females were primed with substrate from cages of other uninfected males to induce oestrous and stimulate oestrous cycling (Marsden & Bronson 1965). Non-oestrous females received no exposure to male odours prior to experimentation. Wet mount vaginal smears were used to determine the oestrus state of the females (Allen 1922; Rugh 1968). H. polygyrus infection had no evident e¡ect on the oestrous cycle of the female mice. Urine and associated odours were obtained from single males that were placed for 1h in a clean cage (25 cm 15 cm 120 cm) lined with blank ¢lter paper (Whatman No. 4, England). Examination under an ultraviolet light con¢rmed that the ¢lter papers were scent-marked, with the males generally urinating within 5 min of arriving in the cage. Five-day E. vermiformis-infected male mice, which were not shedding any oocysts, were used to eliminate any possible odour cues that could arise from parasite components in the urinary products. After exposure to the male odours for either 1 or 30 min the nociceptive responses of individual female mice were determined using the `hot-plate' test (Woolfe & MacDonald 1944). Individual mice were placed on a metal surface (hot-plate, Accu-Scan (Omni-Tech), Columbus, OH), maintained at 50 0.5 8C, and the latency of a foot-lifting or jump response, whichever occurred ¢rst, was recorded. After this response was displayed, the mouse was quickly removed from the surface and returned to her home cage. Testing was terminated if the foot-lifting or jumping response was not observed within 60 s. Thermal responses of the uninfected oestrous and H. polygyrus-infected oestrous female mice were recorded at 0 min (10^15 s), 15, 30 and 60 min after exposure to the odours for 1 and 30 min, as well as at 30 min prior to exposure. Response latencies of the uninfected nonoestrous females were determined only prior to and immediately after exposure to the male odours. The measurement of the latency of response to a heated surface is a standard means of measuring pain or nociceptive sensitivity and has been shown to have no evident e¡ects on foot tissue even after repeated testing at higher temperatures (Hunskaar et al. 1986). Previous investigations showed that repeated handling procedures and exposure to clean, unmarked (blank) ¢lter paper had no signi¢cant e¡ects on the response latencies (Kavaliers & Colwell 1993). All of the odour exposures and nociceptive assessments were conducted in rooms separate from the holding room. Nociceptive responses were analysed with a `mixed-design repeated measures' analysis of variance (ANOVA), and leastsquares mean comparisons with a 0.05 signi¢cance level. 3. RESULTS

Odour responses of uninfected and infected oestrous females were analysed with a mixed-design repeated measures ANOVA consisting of three between-group factors: female infection type (uninfected or infected); odour exposure time (1 or 30 min); and male odour exposure condition (uninfected, H. polygyrus-infected, Proc. R. Soc. Lond. B (1998)

M. Kavaliers and others 1113

E. vermiformis-infected, stressed and control (no male odour)); and one within-group factor: response latency (pre- and 0, 15, 30 and 60 min post-exposure). A secondary analysis compared the pre- and post-exposure responses of uninfected oestrous, uninfected non-oestrous and infected oestrous female mice. There was a signi¢cant main e¡ect of: female infection status (F1,80 28.2, p50.001); odour exposure (F4,80 44.0, p50.001); duration of exposure (F1,80 10.48, p 0.002), as well as an interaction between female infection type exposure time male exposure condition postexposure time (F16,320 2.149, p 0.007). (a) 1-min exposure

Uninfected oestrous female mice exposed for 1min to the odour of either a H. polygyrus- or E. vermiformis-infected male displayed a signi¢cant increase in response latency indicative of the induction of analgaesia (¢gures 1a, 3a). Maximum increases in response latency were evident at 0 (H. polygyrus-infected: t 6.95, p 0.0001; E. vermiformisinfected: t 5.39, p 0.0001) and 15 min (H. polygyrusinfected: t 3.19, p 0.002; E. vermiformis-infected: t 2.13, p 0.036) post-exposure, with a decline to basal (preexposure) levels by 30 min post-exposure. Exposure for 1min to the odour of a stressed male also elicited a signi¢cant analgaesic response at 0 (t 2.09, p 0.04), but not at 15 min (t 1.77, p 0.079) post-exposure. At 0 min post-exposure, the level of analgaesia induced by the odour of the stressed male was signi¢cantly (H. polygyrusinfected: t 4.90, p 0.0001; E. vermiformis-infected: t 4.07, p 0.0001) lower than that elicited by the infected males. Exposure to the odour of an uninfected, nonstressed male had no signi¢cant e¡ect on response latency immediately after exposure. Non-oestrous females also exhibited signi¢cant (E. vermiformis-infected: t 6.96, p 0.0001; H. polygyrusinfected: t 6.69, p 0.0001; at 0 min post-exposure) analgaesic responses to the odours of infected males (¢gure 3a). There were no signi¢cant di¡erences between the analgaesic responses of the uninfected oestrous and non-oestrous females to the odours of either the H. polygyrus- or E. vermiformis-infected males. Non-oestrous females also displayed a signi¢cant (0 min, t 5.79, p 0.0001) analgaesic response following exposure to the odour of a stressed male that was not signi¢cantly di¡erent from that elicited by the odours of the infected males (t 0.90, n.s., and t 1.176, n.s., for H. polygyrusand E. vermiformis-infected males, respectively). The analgaesia induced by the odour of a stressed male was signi¢cantly (t 2.89, p 0.004) greater in the nonoestrous than in the oestrous females. Non-oestrous females also exhibited a signi¢cant (t 2.08, p 0.039) analgaesic response to the odours of non-stressed uninfected males, with the magnitude of this response being signi¢cantly (t 3.891, p 0.0002) less than that elicited by the odours of stressed uninfected males. Non-oestrous females also displayed a signi¢cantly (t 2.69, p 0.008) lower basal response latency than oestrous females. Infected oestrous females exposed for 1min to the odours of either an E. vermiformis-infected or a stressed male displayed a signi¢cant analgaesic response at 0 min (E. vermiformis-infected: t 3.65, p 0.0005; stressed males t 3.44, p 0.0009), and 15 min (t 3.78, p 0.0003, for


both) post-exposure, with a decline to basal levels by 30 min post-exposure (¢gure 1b). The level of analgaesia at 0 min post-exposure was similar to that displayed by uninfected oestrous females exposed to the odour of a stressed male, and signi¢cantly lower (t 2.82, p 0.0061) than that displayed by the uninfected females exposed to the odour of an E. vermiformis-infected male. Exposure to the odour of an H. polygyrus-infected male had no signi¢cant e¡ect on the response latencies of the infected females immediately after exposure. A signi¢cant e¡ect (t 3.90, p 0.02) was, however, evident at 15 min postexposure, similar to that evident in the non-infected females. This was equivalent to the signi¢cant (t 2.95, p 0.01) increase in response latency elicited by exposure to the odour of an uninfected male at 15 min post-exposure. There were no signi¢cant di¡erences in the basal (pre-exposure) response latencies of the infected and uninfected oestrous females, and in their non-signi¢cant responses to the control odour. (b) 30-min exposure

Uninfected oestrous females that were exposed for 30 min to the odours of infected males displayed marked analgaesic responses, with maximum analgaesia evident at 0 min (t 12.94, p 0.0001, for both) (¢gure 2a, 3b). Signi¢cant analgaesic responses were also evident at 15 min (H. polygyrus-infected: t 11.59, p 0.0001; E. vermiformis-infected: t 9.23, p 0.0001), and at 30 min postexposure (t 4.883, p 0.0001, for both), with a decline towards basal levels by 60 min post-exposure. The maximum level (0 min) of analgaesia induced by the 30min exposure to the infected males was signi¢cantly (E. vermiformis-infected: t 6.26, p 0.0001; H. polygyrusinfected: t 5.43, p 0.0001) greater than that induced by the 1-min exposure. Exposure to the odour of stressed males also elicited a signi¢cant analgaesic response immediately (t 4.49, p 0.0001), and 15 min (t 2.01, p 0.047) after exposure, with the peak level (0 min) of this analgaesia being signi¢cantly lower than that elicited by exposure to the odour of the infected males (0 min, t 8.45, p 0.0001, for both male infections). A 30-min exposure to the odour of non-stressed, uninfected males had no signi¢cant e¡ects on the response latency of the uninfected oestrous females. Non-oestrous uninfected females also exhibited signi¢cant (0 min; E. vermiformis-infected: t 12.57, p 0.0001; H. polygyrus-infected: t 11.22, p 0.0001) analgaesic responses following a 30-min exposure to the odours of the infected males (¢gure 3b). There were no signi¢cant di¡erences between the analgaesic responses of the oestrous and non-oestrous females to the odours of the infected males. Exposure of the non-oestrous females to the odour of stressed males also elicited a signi¢cant (t 5.70, p 0.0001) analgaesic response that was of significantly (0 min; E. vermiformis-infected: t 6.87, p 0.0001; H. polygyrus-infected: t 5.52, p 0.0001) lower magnitude than that elicited by the odours of the infected males. The analgaesic response elicited in the non-oestrous females by the 30-min exposure to the odours of the stressed males was signi¢cantly (t 1.90, p 0.05) greater than that elicited in the oestrous females by the odours of the stressed males. Exposure to the odour of a non-stressed uninfected male for 30 min also elicited a signi¢cant Proc. R. Soc. Lond. B (1998)

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Figure 1. Nociceptive responses of (a) uninfected and (b) H. polygyrus-infected oestrous female mice that were exposed for 1 min to the urine and other odorous secretions of male mice that were either uninfected (sham-infected), subclinically infected with E. vermiformis, subclinically infected with H. polygyrus, or subject to restraint stress (stressed) for 15 min. Responses to a blank odour (control, no odour) are also shown. Nociceptive sensitivity was measured as the latency of response to a 50 8C thermal surface. The response latencies of the female mice prior to (pre-) exposure are also shown. n 5, in all cases. Vertical lines denote a standard error of the mean.

(t 2.35, p 0.02) analgaesic response in the non-oestrous females. This analgaesic response was of signi¢cantly (t 3.34, p 0.001) lower magnitude than that elicited by the odours of the stressed males. The non-oestrous females, here, also displayed signi¢cantly (t 2.02, p 0.0453) lower pre-exposure basal response latencies than the oestrous females. Infected females that were exposed for 30 min to the odours of infected males also displayed signi¢cant (E. vermiformis-infected: t 4.90, p 0.0001; H. polygyrusinfected: t 5.638, p 0.0001) analgaesic responses immediately after exposure, with a decline to basal levels by 30 min post-exposure (¢gures 2b, 3b). The 30-min exposure elicited a signi¢cantly (E. vermiformis-infected: t 1.77, p 0.079; H. polygyrus-infected: t 4.48, p 0.0001) greater level of analgaesia in the infected females than the 1-min exposure. The peak level of analgaesia displayed by the parasitized females following exposure to the infected males was, however, still markedly lower (0 min; E. vermifomis-infected: t 7.30, p 0.0001; H. polygyrusinfected: t 6.57, p 0.0001) than that elicited by the 30min exposure in the uninfected oestrous females. Exposure of the infected females for 30 min to the odour of stressed males also elicited a signi¢cant (0 min, t 3.23, p 0.001) analgaesic response that was not signi¢cantly di¡erent from the analgaesia induced by exposure to the odours of the infected males. The level of analgaesia induced by exposure to the odour of the stressed males was not

Parasitized female mice responses to infected males signi¢cantly di¡erent between the infected and uninfected females. Exposure for 30 min to the odour of a nonstressed uninfected male had no signi¢cant e¡ect on the response latency of the infected females. There were no signi¢cant di¡erences between the pre-exposure basal response latencies and the non-signi¢cant responses to the control odours in the infected and uninfected females. 4. DISCUSSION

The present results demonstrate that parasites in£uence both the responses of uninfected females to males and the responses of female hosts to infected males. We show that (i) uninfected female mice can discriminate between the odours of parasitized and non-parasitized male mice, and display analgaesic responses to the odours of infected males; (ii) uninfected females can distinguish between the odours of infected and physically stressed mice, displaying greater analgaesic responses to the odours of infected males; (iii) parasitic infection has signi¢cant e¡ects on a female host's responses to infected males, reducing the analgaesic responses displayed by females to the odours of infected males; and (iv) parasitized females can distinguish between, and di¡erentially respond to, the odours of males infected with either the same or a di¡erent parasite. Consistent with prior ¢ndings (Kavaliers & Colwell 1993, 1995a,b; Kavaliers et al. 1997a), uninfected oestrous female mice distinguished between uninfected and infected males, displaying marked analgaesic responses to the odour cues of the infected males. Relatively prolonged exposures (30 min) to the odours of the infected males were associated with the induction of endogenous opioid-peptide-mediated analgaesia, whereas brief (1min) exposures elicited immediate and relatively shorter-lasting non-opioid-mediated analgaesic responses. These neurochemical mechanisms underly the expression of a variety of behavioural responses, of which pain inhibition is just one readily measurable component. The non-opioid-mediated responses and their neuromodulatory substrates have been associated with the mediation of anxiety(`anticipatory fear')-related defensive behavioural patterns, whereas the opioid-mediated responses were related to more prolonged stress-associated responses. These analgaesic responses are consistent with the attenuated preferences displayed by oestrous females for the odour cues of infected males in odour preference tests, with similar neurochemical mechanisms being implicated in the mediation of both responses (Kavaliers & Colwell 1995a,b). As such, these analgaesic responses and their neuromodulatory substrates are likely to be a part of the mechanism whereby females can detect parasitized individuals, reduce the transmission of parasites to themselves, and select parasite-free mates (Able 1996; Hamilton & Zuk 1982). Exposure to the odour of a physically stressed male also elicited signi¢cant analgaesic responses in oestrous females, consistent with prior results (Kavaliers & Colwell 1995b). The analgaesia induced by exposure to the odours of the stressed males was signi¢cantly lower than that elicited by the odours of the infected males, suggesting that oestrous females can distinguish the odours of stressed males from those of infected males. These ¢ndings also suggest that the odours of the infected Proc. R. Soc. Lond. B (1998)


males may have some `stress odour' features, as well as components that are unique or speci¢c indicators of the parasitized individual. In this regard, the roles of the recently proposed hypothalamic ^ pituitary ^ exocrine axis merit consideration (Chen et al. 1997). The possible sources of the male odour cues (e.g. urine, faecal material, and scent gland products), and olfactory mediation mechanisms (e.g. olfactory systems, vomeronasal organ, and associated central neural systems), remain to be determined. Sexually unreceptive, non-oestrous females who had no prior exposure to males displayed analgaesic responses to control, uninfected as well as stressed, uninfected and infected males. Brief, 1-min exposures elicited similar analgaesic responses to the odours of the stressed and infected males, while the more prolonged exposure to the odours of the infected males elicited a markedly greater opioid-mediated response similar to those displayed by the oestrous females that were exposed to the odours of infected males. The neurochemical mechanisms underlying the mediation of non-opioid analgaesia in female mice have been indicated to be oestrogen-dependent and are suggested to di¡er between oestrous and non-oestrous females (Kavaliers & Galea 1995; Mogil et al. 1997). H. polygyrus-infected females displayed markedly reduced analgaesic responses to the odours of infected males with the extent of the analgaesic response being related to the nature of the male infection and duration of exposure. Following a 1-min exposure, infected females did not display an analgaesic response to the odours of males infected with the same parasite as themselves (H. polygyrus), and displayed an attenuated, though still signi¢cant, analgaesic response to males infected with a di¡erent parasite, E. vermiformis. The longer 30-min exposure to the odours of the E. vermiformis- and H. polygyrus-infected males elicited equivalent analgaesic responses that were, however, in both cases of a signi¢cantly lower amplitude than those displayed by the uninfected females. The parasitized females also displayed signi¢cant analgaesic responses to the odours of stressed males that were of comparable magnitude to the analgaesia induced by exposure to the infected males. The reduced analgaesia displayed by the H. polygyrusinfected females, following exposure to the odours of infected males, could be interpreted in terms of odour familiarity and responses to group odour templates (e.g. Barnard et al. 1991). Substantial evidence exists that mice and rats can distinguish urinary odours associated with di¡erences in the major histocompatibility complex (MHC), and use MHC-related odours for either kin recognition and/or mating preferences (Yamazaki et al. 1976; Egid & Brown 1989; Eklund et al. 1991; Potts et al. 1994; Brown 1995). The MHC activity is part of the immune system with infection activating the release of MHC antigens (Brown 1995; Hedrick 1994). H. polygyrus and E. vermiformis infections stimulate host responses with an accompanying in£ammation of mucosal tissues. Adult H. polygyrus also modulate host immunity, allowing persistence of infestations for more than 30 weeks in certain strains of mice (Robinson et al. 1989; Wahid et al. 1989). Thus, it is possible that parasite-induced alterations in endocrine, immune and neurochemical functions (e.g. Folstad & Karter 1992; Sheldon & Verhulst 1996; Zuk


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uninfected H. polygyrus E. vermiformis stressed infected infected

post

Figure 2. Nociceptive responses of (a) uninfected and (b) H. polygyrus-infected oestrous female mice that were exposed for 30 min to the urine and other odorous secretions of male mice that were either uninfected (sham-infected), subclinically infected with E. vermiformis, subclinically infected with H. polygyrus, or subject to restraint stress (stressed) for 15 min. Responses to a blank odour (control, no odour) are also shown. Nociceptive sensitivity was measured as the latency of response to a 50 8C thermal surface. The response latencies of females prior to (pre-)exposure are also shown. n 5, in all cases. Vertical lines denote a standard error of the mean.

Figure 3. Nociceptive responses of uninfected oestrous (oestrous), uninfected non-oestrous (non-oestrous), and H. polygyrus-infected oestrous female mice that were exposed for either (a) 1 min, or (b) 30 min, to the urine and other odorous secretions of male mice that were either uninfected (sham-infected), subclinically infected with E. vermiformis, subclinically infected with H. polygyrus, or subject to restraint stress (stressed) for 15 min. Nociceptive sensitivity, measured as the latency of response to a 50 8C thermal surface, was recorded before (pre-) and after (post-) exposure to the male odours. n 5, in all cases. Vertical lines denote a standard error of the mean.

1992, 1996), may a¡ect MHC activity and the expression of urinary or associated odour cues, and thus a¡ect subsequent analgaesic responses displayed by uninfected females. An uninfected oestrous and a non-oestrous female could, thus, be construed as displaying analgaesic responses to the odours of infected males, because the odours are di¡erent from the odour template (i.e. uninfected urine and her (uninfected) self ) which constitutes her group member pro¢le. The similarity in the aversive responses of the male-naive non-oestrous and oestrous females to the odours of infected males minimizes the likelihood of any confounding e¡ects arising from the male cues used for the induction of oestrous. Likewise, the infected females may display a greater similarity to the odour template of the infected male, such that the odour of the infected male is considered familiar (self ) and the analgaesic response is either reduced or eliminated. The similarity in MHC and urinary cues between H. polygyrus-infected males and females could lead to the absence of an analgaesic response after a brief exposure. Depending on the similarities of MHC-related shifts this could also temper the aversive responses of the infected females to E. vermiformis-infected males. Reduced odour familiarity and only partial group template matching is also consistent with the analgaesic responses exhibited following exposure to the odours of the

stressed males. In this regard, the relative contributions of stress odour to the odour cues of the infected mice and similarities/di¡erences between the odour constituents of the H. polygyrus- and E. vermiformis-infected male mice need to be examined. It should be noted that we have recently observed that both sexually naive and sexually experienced uninfected male mice display signi¢cant analgaesic responses to the odours of infected females, and that sexually naive H. polygyrus-infected male mice display reduced analgaesic responses to the odours of parasitized females (Kavaliers et al. 1998). This further mitigates against the possible confounding e¡ects on odour response matching that may arise from prior exposure to an uninfected male during the induction of oestrous. Odour matching is not, however, directly consistent with the similar levels of analgaesia evident in the infected females after 30 min of exposure to the odours of either stressed, E. vermiformis- or H. polygrus-infected male mice. The attenuation in analgaesia displayed by the parasitized females may also be part of a general reduction in fearfulness and anxiety evident in H. polygyrus-infected individuals (Kavaliers et al. 1997b), decreasing avoidance of conspeci¢cs and increasing interactions between the infected and other individuals, thereby facilitating parasite transmission. It should be emphasized that the H. polygyrus infection does not just simply compromise the responses of

Proc. R. Soc. Lond. B (1998)

Parasitized female mice responses to infected males the infected individual to stress. Uninfected and infected individuals display similar, relatively high-level analgaesic responses to physical stressors such as restraint (Colwell & Kavaliers 1990; Kavaliers & Colwell 1995b). Results of studies with laboratory rats have, however, implicated di¡erent brain regions and possibly neurochemical components in the mediation of responses to physical and `psychological' stressors (Sullivan & Gratton 1998). The altered responses of the H. polygyrus-infected females are not due to any pathology. There were no evident signs of malaise in the infected females that displayed normal oestrous cycles. Moreover, non-oestrous females displayed either equivalent or slightly greater analgaesic responses to the odours of infected males than oestrous females. In addition, the infected females did not display any alterations in their basal response latencies. Also, the responses of the infected females to the odours of stressed males remained unchanged by infection, which again does not support a pathology interpretation and further indicates that the parasitic infection does not signi¢cantly a¡ect the olfactory sensitivity of the infected females. Our ¢ndings from a single-host system show that a nonpathological H. polygyrus infection can signi¢cantly a¡ect the female host's mate's responses. These observations are consistent with, and extend, prior observations that E. vermiformis and H. polygyrus infections can a¡ect the responses of male hosts to females (Kavaliers et al. 1997a,b, 1998). Thus, the present ¢ndings with parasitized and non-parasitized females have important implications for the proposed role of parasites in mate choice and preferences. They suggest that parasitic infection may a¡ect the host's responses to other infected individuals and that a female's own parasites may in£uence mate preference and selection. We thank Greg Ti¤n for supplying E. vermiformis and Dr M. Scott for providing H. polygyrus. We thank Terry Seely, Joan Ingoldsby, Dawn Gray and Christine Himsl-Rayner for their assistance. We also thank two anonymous reviewers for their valuable comments. This study was supported by Agriculture and Agri-Food Canada and the Natural Sciences and Engineering Research Council of Canada. REFERENCES Able, D. J. 1996 The contagion indicator hypothesis for parasitemediated sexual selection. Proc. Natn. Acad. Sci. USA 93, 2229^2233. Allen, E. 1922 The oestrus cycle in the mouse. Am. J. Anat. 30, 297^371. Barnard, C. J., Hurst, J. L. & Aldhous, P. 1991 Of mice and kin: the functional signi¢cance of kin bias in social behaviour. Biol. Rev. 66, 379^430. Barnard, C. J., Behnke, J. M., Gage, A. R., Brown, H. & Smithurst, P. R. 1997 Immunity costs and behavioural modulation in male laboratory mice (Mus musculus) exposed to the odours of females. Physiol. Behav. 62, 857^866. Barnard, C. J., Behnke, J. M., Gage, A. R., Brown, H. & Smithurst, P. R. 1998 The role of parasite-induced immunodepression, rank and social environment in the modulation of behaviour and hormone concentration in male laboratory mice (Mus musculus). Proc. R. Soc. Lond. B 265, 693^701. Behnke, J. M., Barnard, C. J. & Wakelin, D. 1992 Understanding chronic nematode infections: evolutionary considerations, current hypotheses and the way forward. Int. J. Parasitol. 22, 861^907. Proc. R. Soc. Lond. B (1998)


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