Behav Ecol Sociobiol (2003) 54:423–430 DOI 10.1007/s00265-003-0631-2
ORIGINAL ARTICLE
Martin Kavaliers · Melissa A. Fudge · Douglas D. Colwell · Elena Choleris
Aversive and avoidance responses of female mice to the odors of males infected with an ectoparasite and the effects of prior familiarity Received: 23 December 2002 / Revised: 22 April 2003 / Accepted: 23 April 2003 / Published online: 24 May 2003 Springer-Verlag 2003
Abstract The detection and avoidance of parasitized males is a component of female mate choice. Here we show that female mice can distinguish between the odors of individual males infected with an ectoparasite, the murine louse, Polyplax serrata, and uninfected males. Female mice displayed aversive responses to, and avoided the odors of, parasitized males. A 15 min exposure to the urinary odors of infected males induced an endogenous opioid-peptide-mediated reduction in pain sensitivity or analgesia, while a brief 1 min exposure to the odors elicited a non-opioid-mediated analgesic response. These neuromodulatory mechanisms facilitate the expression of a variety of anxiety and stress associated responses of which pain inhibition is one component. Females further distinguished between novel and familiar infected males. Prior exposure to the odors of an infected males reduced the degree of analgesia expressed and the associated levels of anxiety and stress and their concomitant costs. In a Y-maze odor preference test females also displayed a marked overall preference for, and initial choice of, the odors of clean, uninfected males and an active discrimination against, and avoidance of, the odors of both familiar and novel infected males. These findings indicate that female mice can distinguish between males infected with an ectoparasite and clean uninfected males and display aversive and avoidance responses to infected Communicated by P. Heeb M. Kavaliers ()) · M. A. Fudge Department of Psychology and Neuroscience Program, University of Western Ontario, London, ON, N6A 5C2, Canada e-mail:
[email protected] Fax: +1-519-6613961 D. D. Colwell Agriculture and Agri-Food Canada, Lethbridge, AB, T1J4B1, Canada E. Choleris Department of Psychology, University of Guelph, Guelph, ON, N1G2W1, Canada
males. They also show that females can discriminate between individual infected males and modulate their aversive responses to the odors of infected males on the basis of prior familiarity. This is likely part of the mechanisms whereby females can both reduce the transmission of ectoparasites, such as lice, to themselves and select for parasite-free males. Keywords Analgesia · Mate choice · Parasites and behavior · Odor cues · Individual recognition
Introduction Parasites have been reported to affect mate choice and mating patterns with females preferentially selecting parasite-free or parasite-resistant males (Hamilton and Zuk 1982; Clayton 1991; Zuk 1992). Results from studies with birds, lizards and fishes have shown that ectoparasites (i.e. lice, mites and ticks) can have pronounced effects on host condition (e.g. sexual ornamentation, coloration and display) and fitness, and impact on female mate choice (e.g. Kennedy et al. 1987; Hart 1992; Clayton 1990, 1991; Clayton and Moore 1997; Kose et al. 1999). The advantages to a discriminating female include acquisition of parasite-resistant genes for her offspring (Hamilton and Zuk 1982), decreased risk of contagion (Borgia and Collis 1989; Able 1996), and increased parental care (Milinski and Bakker 1990). Clayton (1990) clearly showed that female rock doves selected against, and avoided, males that were infested with lice that could be transferred from one bird to another during contact in mating. He indicated that this selection by the females against the infected males was based on the reduced duration of behavioral displays shown by the lousy males. Mammals, and especially rodents, also harbor ectoparasites with a variety of behavioral, immunological and humoral effects (e.g. Murray 1990; Hart 1992; Lehmann 1992,1993; Price and Graham 1997). Since the transmission of ectoparasites can occur through body contact, the likelihood of the acquisition of an infection during mating
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is high. Consequently, one would expect mammals to select against ectoparasite infected mates. However, investigations of the effects of parasites on mate responses and choice in mammals have, to date, been restricted to endoparasites. In rodents, where urine and other odor cues are of major importance in mate detection and choice (Hurst 1987, 1990; Potts et al. 1994; Hurst et al. 2001), females can discriminate between infected and uninfected males on the basis of odor. Female laboratory mice, Mus musculus domesticus, displayed a reduced interest in, and avoidance of, the urine and other odorous secretions of males infected with either endoparasites such as the protozoan, Eimeria vermiformis, and the nematode, Heligmosomoides polygyrus, as well as influenza virus (Kavaliers and Colwell 1995a, 1995b; Kavaliers et al. 1997, 2000, 2003; Penn et al. 1998; Ehman and Scott 2001). An additional consequence of exposure to the olfactory cues associated with a subclinically infected male is a decrease in nociceptive or pain sensitivity and the induction of antinociception or analgesia. The nature of this pain inhibition varies as a function of the duration of exposure, with prolonged exposures to the odors of infected males inducing a relatively long-lasting endogenous opioid peptide (e.g. enkephalin, endorphin) mediated analgesia and brief exposures eliciting a shorter lasting non-opioid-mediated analgesia (Kavaliers and Colwell 1995b). In these non-pathological infections the levels of analgesia are relatively independent of the intensity of infection (Kavaliers et al. 2000), The nonopioid-mediated responses and their neurochemical substrates represent anxiety-related anticipatory defense reactions while the more protracted opioid-mediated responses are primarily associated with stress and related responses. These analgesic responses and their anxiety/ fearfulness/stress-associated behavioral correlates elicit either a reduced interest in, or avoidance of, parasitized male mice by the females, thus serving as an important neuromodulatory component of female mate choice (Kavaliers et al. 2000). In the present study we considered the responses of female mice to the odor cues of males infected with an ectoparasite, the directly transmitted murine anopluran louse, Polyplax serrata (Murray 1960, 1990; Price and Graham 1997). We examined the nociceptive responses of female mice that were exposed for either 1 or 15 min to the odors of P. serrata infested mice. Using an odor preference test (Coopersmith and Lenington 1992), the results of which are considered to be consistent with mating preferences and mate choice (Egid and Brown 1989; Krackow and Matusch 1991), we also examined the responses of females to the odors of infected and uninfected male mice. In view of the evidence for individual odor recognition in mice (e.g. Hurst 1990; Gheusi et al. 1994; Nevison et al. 2000; Hurst et al. 2001) and the findings that prior experience with a parasitized individual can influence subsequent responses (Kavaliers et al. 2000, 2003) we also examined the responses of
females that were re-exposed to the odors of either a novel or a familiar infected male.
Methods Animals Male and sexually naive female mice (CF-1, Rocky Mountain Laboratories, Hamilton, Mont.), 2–3 months old, were housed in clear polyethylene cages with a wood shavings bedding at 20€2C under a 12 h light:12 h dark cycle. Food (Mouse Breeder Blox, Wayne Laboratory Animal Diets, Madison, Wis.) and tap water were available ad libitum. Wet mount vaginal smears were used to determine the estrous state of the females at the test period. Louse infections P. serrata were obtained pathogen free from The Mouse Man, Medicine Hat, Alta. and colonies were established on individual CF-1 male mice. The life cycle of the louse consists of an egg, three nymphal stages and adult females and males (Murray 1960; Price and Graham 1997). This cycle can be completed in 13 days, as the eggs hatch in 6 days and nymphs develop into adults over 7 days. Eggs are attached to the hair close to the skin. Nymphs and adults move freely within the pelage from the skin surface to the hair tips. They feed at the skin surface obtaining blood from the venuoles. Scanning electron micrographs confirmed the lice as P. serrata based on the morphology of tergites, paratergites, legs, head, and dorsal principle seta. For this study individual mice were infected by placing approximately 250–300 nymphal and adult stages of P. serrata on the shoulders. The lice infected mice were held individually to prevent mutual grooming and enhance the level of infestation (Lodmell et al. 1970; Murray 1990). There were approximately 46– 100 lice per male at 4–6 weeks after infestation when the studies were conducted. This relatively low level of infestation, which is similar to that reported in natural populations, does not cause any symptoms of malaise in the mice (i.e. no weight loss or poor grooming, Murray 1960). The mice do display local skin immune responses and systemic humoral responses and may develop some immunity to infection after 50–60 days (Nelson et al. 1979, 1983; Ratzllaf and Wikel 1990). The infected males were held in a room that was isolated from clean, uninfected male mice. Uninfected sentinel males that were individually housed in the same room as the infected males remained clean throughout the course of the study. All of the infections and testing were carried out in accordance with the guidelines and approval of the Canadian Council of Animal Care. Urine collection During the early light period individual lousy (n=5) and clean (n=5) male mice were held by the scruff of the neck to stimulate urine production (0.2–0.3 ml). The urine from individual males was collected into tubes using a funnel and frozen immediately at 18oC until use. Prior to each day’s testing a fixed aliquot (0.5 ml) of the urine of an individual male was thawed, diluted 1:5 with deionized water and spotted on filter paper (1.0 cm diameter, Whatman No.5, England). Urine was used as the odors source as it contains no ectoparasite components or products. Experiment 1: aversive responses During the mid-light period on day 1, individual pro-estrous female mice, who display a heightened interest in males, were placed in clean cages (25 cm15 cm20 cm) with a vented Plexiglas tube (10 cm in length, 3 cm in diameter and sealed at each end with
425 plastic mesh) with filter paper containing the urinary odors of either a lousy or clean male. After exposure to the male odors for either 1 or 15 min the nociceptive responses of each female mouse (n=10, in each case) were determined. Individual mice were placed on a metal surface (analgesiometer, Accu-Scan, Columbus, Ohio) maintained at 50€0.5C and the latency of a foot-lifting or a jumping response, whichever occurred first, was recorded. After this response was displayed the mouse was quickly removed and returned to her home cage. Testing was terminated if a response was not observed within 60 s. Thermal responses were recorded at 0 min, 10–15 s, 15, 30, and 60 min after exposure to the odors for 1 and 15 min, as well as immediately prior to exposure. Twenty-four hours later (day 2) females were again exposed to male odors for 1 and 15 min after which their nociceptive responses were determined. Half of the females (n=5, for each time group) were exposed to the odors of the same (“familiar”) male (lousy or clean) and the other half (n=5, for each time) were exposed to the odors of a different (“novel”) male (lousy or clean). Previous investigations showed that repeated handling procedures and exposure to clean, unmarked filter paper had no significant effects on response latencies (Kavaliers and Colwell 1995b). All of the odor exposures and nociceptive assessments were conducted in rooms separate from the holding room. The measurement of the latency of response to a heated surface is a standard means of measuring pain or nociceptive sensitivity. This procedure has been shown to have no evident effects on foot tissue even after repeated testing for durations of up to 2 min at temperatures to 60C (Hunskarr et al. 1986). Experiment 2: neuromodulatory substrates of aversive responses Prior to their exposure to the odors of lousy or clean males, other female mice (n=5, per group) received intra-peritoneal (i.p.) injections of either: (1) the prototypic opiate antagonist, naloxone hydrochloride (1.0 mg/kg, Sigma, St. Louis, Mo.); (2) the Nmethyl-d-aspartate (NMDA) receptor antagonist, MK-801 (0.10 mg/kg, Sigma) or (3) the saline vehicle (10 ml/kg). Drugs were administered either 30 or 15 min before the 1 and 15 min exposures, respectively. Nociceptive responses were determined as previously described both prior to drug injection and immediately after the odor exposures. Doses of the drugs were based on the results of prior studies where naloxone was shown to reduce opioidpeptide-mediated analgesia and MK-801 to reduce non-opioidmediated responses (Kavaliers and Colwell 1993; Kavaliers and Galea 1995). Nociceptive responses in experiments 1 and 2 were analyzed with a repeated measures analysis of variance (ANOVA) and Tukey’s post hoc test with a 0.05 level of significance. Experiment 3: odor choices and preferences Initial odor choices and preferences of individual female mice were tested in a translucent Plexiglas Y-maze apparatus (5 cm diameter) with 30 cm arms (after Coopersmith and Lenington 1992). The stimulus compartments in the two arms of the Y in which odor cues were placed and the start box, in which a female mouse was placed, were each 14 cm long. A solid Plexiglas barrier restricted the mouse to the start box, while perforated Plexiglas barriers at the ends of the two stimulus arms prevented physical contact with the odor sources. Removable solid Plexiglas barriers were also present at “seams” 8 cm into each of the stimulus arms. The barriers prevented exposure of the mouse to the odors until the designated test times. To minimize novelty responses, individual females were placed in the apparatus and allowed to explore the various arms (after being held for 5 min in the start box) for 30 min on 3 consecutive days prior to the test day. For all of the choice/preference tests an individual female mouse was placed in the start box of the apparatus for 1 min after which the solid barrier was removed allowing the mouse access to the two arms of the Y-maze. Approximately 1 min later after the
test mouse had entered the stimulus arms and re-entered the neutral arm, the Plexiglas barrier in each of the arms was removed, exposing the mouse to the test odors. During the subsequent 5 min period the first odor chosen, the time taken to choose the first odor, and the duration of time a mouse spent within each stimulus arm, 8 cm or closer to the odor (as determined by the position of the mouse relative to a seam in each arm of the apparatus), was recorded. This distance was chosen on the basis of the results of prior studies (e.g. Coopersmith and Lenington 1992) that took into account the volatility of the odor constituents. On day 1 there were two different experimental groups of female mice (n=16, in each group). The stimulus odor sources for these mice were either: (1) the urinary odors of a lousy and clean male; or (2) the odors of two different clean males. Twenty-four hours later (day 2) the odor responses of the females were again determined with each of the experimental groups being subdivided into two experimental groups (n=8, in each of the four groups). The stimulus odors for these groups were from: (1) the clean familiar male and either the original familiar or a novel lousy male; and (2) a familiar clean male and either a familiar or a novel clean male. The first odor chosen was defined as the “initial odor choice.” “Odor preference” was defined as the duration of time the test mouse spent in the one stimulus arm of interest (within 8 cm of the odor source) divided by the total time spent in the two stimulus arms. Odor and control arms were randomized between trials and subjects. Trials were discontinued and an alternate female was used if she spent less than a total of 30 s of the 5 min test period in the distal ends of the arms. The Y-maze was washed thoroughly with hot water and unscented soap between trials. The results of previous studies had established that the results obtained in a 5 min test were comparable to those obtained with longer tests (Kavaliers and Colwell 1995b). First choice data were analyzed by a chi-square goodness-of-fit tests and time taken to make the first choice by a repeated measures ANOVA. Preference ratios were transformed to natural log (ln) values prior to analysis by ANOVA. A 0.05 level of significance was used throughout.
Results Experiment 1: aversive responses On day 1 female mice that were exposed to the odors of the infected lousy males for either 1 or 15 min displayed significant increases (1 min, F1,9=57.3, P
