Recognition and avoidance of the odors of parasitized conspecifics and predators: Differential genomic correlates

Neuroscience and Biobehavioral Reviews 29 (2005) 1347–1359 www.elsevier.com/locate/neubiorev

Recognition and avoidance of the odors of parasitized conspecifics and predators: Differential genomic correlates Martin Kavaliersa,*, Elena Cholerisb, Donald W. Pfaff c a

Department of Psychology, University of Western Ontario, London, Ont., Canada N6A 5C2 b Department of Psychology, University of Guelph, Guelph, Ont., Canada N1G 2W1 c Laboratory of Neurobiology and Behavior, The Rockefeller University, New York, NY 10021, USA Received 1 December 2004; revised 30 March 2005; accepted 1 April 2005

Abstract In many species of animals chemical stimuli are an important source of information about the threats and dangers present in the social and non-social world. Olfactory cues play a fundamental role in modulating social recognition and interactions in a wide variety of mammals. Rodents, in particular, utilize chemical signals, to recognize and avoid conspecifics infected with parasites and other pathogens. Animals also respond to, and utilize, predator odor related information to assess and minimize their risk of predation. In this review, we briefly focus on the relations between odors, parasite recognition and avoidance and consider some of the associated hormonal, neural and genomic mechanisms. We describe how both male and female rodents distinguish between infected and uninfected males on the basis of odors, displaying aversive response to, and avoidance of, the urine odors of infected individuals. We further describe how the recognition and avoidance of the odors of infected individuals involves genes for the neuropeptide, oxytocin, (OT), and estrogenic mechanisms. We show that mice with deletions of the oxytocin gene (OT knockout mice (OTKO)) and mice whose genes for estrogen receptor (ER)-a or ER-b [ER knockout mice (ERKO), aERKO and bERKO] have been disrupted are specifically impaired in their recognition, avoidance, and memory of the odors of infected individuals. We contrast this with the recognition and display of aversive responses to predator (cat) odor that are insensitive to these genetic manipulations. These findings reveal some of the mechanisms associated with the olfactory mediated recognition of parasitized individuals and predators. q 2005 Elsevier Ltd. All rights reserved. Keywords: Mate choice; Parasite avoidance; Predator recognition; Pheromones; Odor cues; Individual recognition; Social recognition; Oxytocin; Estrogen receptors a,b

1. Introduction In the wild animals are exposed to a variety of socially and non-socially related threats and dangers including parasites, pathogens and predators. One of the major costs of social behavior is the increased risk of exposure to parasites and pathogens (Møller et al., 1993; Altizer et al., 2003). Social behaviors facilitate interactions between conspecifics and affect the likelihood of the transmission of parasites from infected to uninfected individuals. As such, during the course of host-parasite co-evolution hosts have evolved * Corresponding author. Tel.: C519 661 2111x86084; fax: C519 661 3961. E-mail address: [email protected] (M. Kavaliers).

0149-7634/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.neubiorev.2005.04.011

a variety of behavioral mechanisms and responses to minimize their exposure to parasitized individuals and to avoid contagion (Folstad and Carter, 1992; Able, 1996; Kavaliers et al., 2004). Indeed, it has been speculated that parasites are a major underlying determinant of social organization, including that of mating systems (Freeland, 1976; Møller et al., 1993; Altizer et al., 2003). Olfactory information has a predominant role in determining intraspecific social behaviors and interactions of non-primate mammals. Chemical signals provide information about sex, sexual and social status, individuality and condition (Brown, 1979; Halpin, 1986; Johnston, 2003; Wyatt, 2003). As such, olfactory information also plays a key role in the detection of, and defense against, parasites and pathogens. It has become increasingly evident that animals, and in particular small rodents, utilize chemical signals (e.g. urine odor) for the recognition and

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avoidance of infected conspecifics (Kavaliers and Colwell, 1995a,b; Penn and Potts, 1998; Kavaliers et al., 2003a–d, 2004). Animals also exhibit a variety of adaptations for detecting and defending themselves from predators. The roles of chemical signals in interspecific or heterospecific interactions, and in particular predator detection and subsequent avoidance, have been highlighted (e.g. Blanchard et al., 1990; Katz and Dill, 1998; Dielenberg and McGregor, 2001; Kavaliers and Choleris, 2001). In almost all systems examined prey are reported to use predator associated odors as cues to their risk of predation with animals displaying both innate and acquired active and passive defensive responses to predator odor (Katz and Dill, 1998). Here, we briefly discuss the relations between odors, parasite recognition and avoidance. We also consider aspects of the functional genomics of parasite recognition. In particular, we discuss the involvement of genes for the neuropeptide, oxytocin, and genes for estrogen receptors a and b, in mediating the recognition and avoidance of parasitized individuals. This is accompanied by a brief consideration of the similarities and differences in the expression and mediation of responses to predator odors. We begin with a short overview of terminology and some conceptual issues related to chemical signals.

2. Chemical signals 2.1. Terminology Chemical signals that convey information between individuals of the same species are commonly known as pheromones (Wyatt, 2003). They represent a subclass of semiochemicals used for communicating within the species. Karlson and Luscher (1959) first defined pheromones as ‘substances secreted to the outside of an individual and received by a second individual of the same species in which they release a specific reaction, for example a definitive behavior (releaser pheromone) or developmental process (primer pheromone)’. Since then there have been put forth a variety of definitions/classification of pheromones based on factors such as; chemical composition, aspects relating to genetic control of response mechanisms, the efficacy of the signal in causing a response, and the type of responses elicited (Johnston, 2003; Wyatt, 2003). The use of the term pheromone has varied from that of a single chemical component to a broader mixture of components in the chemical signal (pheromone ‘blend’) through to ‘mosaic signal’ that contains a large number of compounds. This latter definition of pheromone has incorporated the broad array of chemical signals released by mice and other small rodents and used in intraspecific communication. Here, we will restrict our usage to the word ‘odors’ for describing the intraspecific chemical signals

(described below) produced by infected and uninfected individuals and used for the detection and/or recognition of parasitized individuals. 2.2. Volatile and non-volatile chemical signals in rodents Mammalian species release to their environment a variety of volatile and nonvolatile chemicals via skin glands and skin microorganisms, saliva, as well as via excretory products, such as urine and faeces (Brown, 1979; Wyatt, 2003). Chemical signals provide information about an individuals sex, sexual and social status, individuality and condition. Mouse urine contains fixed ‘genomic’ information about species, sex and individual identity as well as more flexible ‘metabolic’ information concerning the current owners social, reproductive, health and reproductive status (Singer et al., 1993; Hurst et al., 2001; Beauchamp and Yamazaki, 2003; Nevison et al., 2003). Information concerning species and sex of the scent owner is provided by a number of specific volatile compounds including, 2-sec-butyl-4-5-dihydrothiazole (‘thiazole’) and 2,3-dehydro exo-brevicomin (‘brevicomin’) which are present in male mouse urine and are under androgen control (Wyatt, 2003). In addition, two sesquiterpenes (‘farnesenes’) are produced by preputial gland and added to the urine on elimination. Recently, there has also been described an additional compound, (methylthio)methanethiol (MTMT) that is secreted in the urine of intact male mice (Lin et al., 2005) These male specific volatiles are attractive to females and can provide information regarding male sexual social status. Studies with rodents have revealed that two highly polymorphic and polygenic complexes contribute to genetically determined individual differences in urine odor. The major histocompatibility complex (MHC) encodes highly polymorphic glycoproteins involved in self- non-self recognition in the immune system (Beauchamp and Yamazaki, 2003). There are two classes of MHC molecules. MHC class I molecules are found on the surface of most cells and present proteins that are generated in the cytosol to T lymphocytes. MHC class II molecules are expressed only at the surface of activated antigen-presenting cells and they present peptides that have been degraded in cellular vesicles in T cells. MHC associated urinary odors are used by mice and other rodents in mate choice, discriminating kin from nonkin and recognizing familiar individuals (e.g. Yamazaki et al., 1976; Egid and Brown, 1989; Hurst, 1990; Manning et al., 1992; Potts et al., 1994; Roberts and Gosling, 2003). Yamazaki et al. (1976) first observed MHC-based disassortative mating patterns in mice. Since then MHC dependent mating preferences have been described in a variety of species, including that of humans (human MHC is termed Human Leukocyte Antigen (HLA)) (humans, e.g. Wedekind and Furi, 1997; Jacob et al., 2002). MHCdisassortative mating patterns produce MHC-heterozygous

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patterns, which may have enhanced immunocompetence and be more resistant to pathogens (Penn and Potts, 1999). MHC-disassortative mating preferences may also a help avoid inbreeding (Manning et al., 1992; Potts et al., 1994). It has also been suggested that rather than maximizing heterozygosity, there may be an optimum intermediate degree of MHC-heterozygosity that is used in mate selection (Milinski, 2003; Milinski et al., 2005) A number of hypotheses have been put forth to explain how an individuals MHC-genotype shapes its odor, with none proven to date (Penn and Potts, 1998). The exact molecular basis of MHC-associated odors is also not clearly established, but in mice it comprises a complex mixture of volatile metabolites bound and released by urinary proteins or peptides. This may include fragments of MHC molecules, MHC molecules themselves and/or specific peptides that bind MHC class 1 molecules (Singer et al., 1993; Beauchamp and Yamazaki, 2003; Leinders-Zufall et al., 2004). In addition, mice utilize odor cues provided by another highly polymorphic gene complex, that of the major urinary proteins (MUPs) for individual recognition (Hurst et al., 2001; Beynon and Hurst, 2003; Hurst and Beynon, 2004). The MUPs, and their counterparts in rats, termed a2U— globulins, are lipocalins whose tertiary structure consists of an eight-stranded anti-parallel b-barrel enclosing an internal binding site. MUP genes are largely expressed in the liver under stimulation by androgens and their products become concentrated in the urine by filtration from the blood serum. The MUPs bind and release small volatiles that can be used for individual recognition, with either the proteins themselves and, or protein-ligand complexes, providing long lasting nonvolatile odor cues found in scent marks (Brenan and Keverene, 2004). These proteins are also highly polymorphic and the pattern of polymorphic variation provides a stable signal that is considered to communicate gene-derived information on the individual identity of the scent owner. 2.3. Predator associated odors Animals also discriminate between chemosignals from their own species (conspecific) and those from other species (hetrospecific) (Wyatt, 2003; Meredith and Westberry, 2004). Chemical signals (semiochemicals) acting between individuals from different species are called allochemicals and are further divided depending on the costs and benefits to the signaler and receiver. Chemical signals that are received and utilized by unintended recipients are termed kairomones (Dickie and Grostal, 2001). This is considered as a semiochemical that is released by one species and has a favorable or adaptive effect on a different ‘receiving’ species but a non-favorable effect in the ‘transmitting’ species. This includes predator associated chemical stimuli that are detected by prey at the disadvantage of the predator. We will refer here to these chemical signals as ‘predator

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odors’ or ‘predator related odors’. These predator odors likely incorporate chemical signals that are used as intraspecific signals by the predators themselves.

3. Responses to the odors of parasitized males 3.1. Avoidance and analgesic responses of females The ability to recognize contagious animals is important for effective behavioral defence against infectious diseases. Animal species have developed a wide variety of characteristics that function to avoid infection by microparasites and macroparasites. Aspects of sexual selection, foraging strategies, social behavior and habitat selection often serve to avoid contact with infected individuals and infective stages of parasites. The likelihood of the acquisition of an infection during social interactions associated with mate selection and mating is especially high and costly. Parasites have been shown to affect mate choice and mating patterns with females preferentially selecting parasite-free or parasiteresistant males (Hamilton and Zuk, 1982; Zuk, 1995; Kavaliers et al., 2000, 2004). Choosy females could increase their fitness both directly by reducing their own risk of contagion and indirectly by enhancing the parasite and disease resistance in their offspring. Females may select males on the basis of secondary sexual traits (‘good genes’) which indicate resistance to disease (Zuk, 1995). Females may also choose unparasitized males, not necessarily to obtain ‘good genes’ for parasite resistance, but rather to directly avoid infection. In this case the degree of expression of variable male traits may indicate the parasite load of a male, and, indirectly the level of risk for a choosy female of acquiring these parasites (Able, 1996). Hamilton and Zuk (1982) first clearly suggested that, during mate choice, animals should examine urine and faecal cues in an attempt to detect mates that were disease and parasite free. It has now been shown that female rodents can readily discriminate between the odors of uninfected individuals and individuals subclinically infected with parasites such as the nematode, Heligmosomoides polygyrus (Kavaliers and Colwell, 1995a; Ehman and Scott, 2001; Ehman and Scott, 2002; Kavaliers et al., 2003a,c, 2005). Resistant stages of this gastrointestinal nematode are shed in the faeces of non-clinically infected hosts. After a short period they are infective to other mice that can acquire them directly during feeding, grooming and other social interactions (Herandez and Sukhedo, 1995). The parasitized animals here are not sick and do not display any behavioral or physiological responses (e.g. weight loss, poor grooming) characteristic of sickness. Animals that are acutely sick from systemic bacterial infections display what is termed an acute phase responses—a coordinated set of behavioral and physiological responses (including cytokine secretions, fever, anorexia, diminished activity) to facilitate

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recuperation (Hart, 1990). The acute phase response is an adaptation on the part of the host, and not that of the infective agent at the cost of the host. These ‘sickness’ related changes are not advantageous in the subclinical parasitic infections being considered here, where infected animals continue to engage in various social behaviors. Estrous and non-estrous female mice discriminated between the urine odors of H. polygyrus—parasitized and nonparasitized males in a variety of odor preference tests. The females displayed both an overall preference for, and initial choice of, the odors of the uninfected males (Kavaliers and Colwell, 1995a; Ehman and Scott, 2001; Kavaliers et al., 2003a, 2005). The initial choice results showed that the females found the odors of uninfected males, and by implication the males themselves, more attractive than those of infected males, whereas the total preference findings suggested that the females actively discriminated against, and avoided, the odors of infected males (Wagner, 1998; Kavaliers et al., 2003a). In a controlled laboratory setting female mice also mated preferentially with non-parasitized males and avoided the infected males as well as their odors (Ehman and Scott, 2002). As resistance to H. polygyrus is heritable (Wahid et al., 1989), this odor based mate selection may be construed as being consistent with the acquisition of ‘good genes’ by discriminating females. Control studies showed the avoidance responses displayed by the females to the odors of parasitized males were not directly associated with any shifts in testosterone levels or the production of stress associated odors by the infected males. As well, there were no parasite components in the urine. Female mice also discriminated the urine odors of males sub-clinically infected with influenza, a respiratory virus (which is not a natural infection of mice), from the odors of other uninfected mice (Penn et al., 1998). Female mice also distinguished between the odors of males infected with the protozoan Eimeria vermiformis from that of uninfected individuals and in an arena setting actively avoided the odors of infected males (Kavaliers and Colwell, 1993, 1995a,b; Kavaliers et al., 1997, 1998a). Although females displayed maximal avoidance of the odors of infective males they also avoided the odors of parasitized but preinfective males. This supports the direct recognition and avoidance of specific odors associated with a parasitized male. Results of a study by Yamazaki et al. (2002) revealed that mouse mammary tumor virus (MMTV) altered the urinary odor of male mice, regardless of the presence or absence of tumors and pathology, and lead to an active avoidance of the male odors by the females. In all cases, females recognized and avoided the odors of the virus infected males. It is likely that females here are responding to a combination of odor cues that relate to male condition and infection status. Female mice, rats and meadow voles also discriminated against males infected with the nematodes Hymenolepis diminuita and

Trichinella spiralis (Klein et al., 1999; Willis and Poulin, 2002). In contrast to E. vermiformis and H. polygyrus, these parasites have a complex life cycle involving intermediate hosts and are not directly transmissible and infective to females. This again suggests that the females are directly responding to male condition. Ectoparasites (i.e. lice, mites, ticks) also have pronounced effects on host condition and impact on female mate choice (Lehmann, 1993). Since the transmission of ectoparasites occurs through body contact, the likelihood of the acquisition of an infection during mating and other social interactions is particularly high. Female mice discriminated between the urine odors of either familiar or unfamilar males infested with a low level of the mouse louse, Polyplax serrata, and uninfested males and displayed aversive responses to the odors of louse infected males (Kavaliers et al., 2003b,c). This allows females to both reduce the transmission of lice to themselves by avoiding contact with infected males, and to select for parasite resistant males. Odor preference tests do not test mating preferences directly, rather they examine an expression of social interest. However, preferences in Y-maze and related tests are consistent with the appetitive components of mating preferences and are suggested to give reliable indications of mate choice in mice (e.g. Egid and Brown, 1989; Krackow and Matuschak, 1991; Wagner, 1998). Moreover, the positive reproductive consequences of the social preferences displayed by female mice have been shown in mating tests (Drickamer et al., 2000). It is becoming evident that mating preferences and mate choice, including that of odor responses, are not always fixed. Rather, they are context and condition sensitive (Jennions and Petrie, 1997). The mating preferences of females and their levels of choosiness can be affected by their prior experience with infected and uninfected males in what is known as a ‘previous male effect’. Female mice that were briefly (1 min) exposed to the odors of a H. polygyrus infected male displayed a subsequent reduced discrimination between the odors of infected and uninfected males (Kavaliers et al., 2003a). Brief exposure to the odor cues associated with a parasitized male, may modify the condition of a female such that she finds all males and their odors aversive, or at least unattractive, reducing her likelihood of mating and acquiring an infection. In these choice situations ‘uninfected’ does not necessarily imply a parasite resistant and, or better quality male. High quality males may have both greater health and more parasites than low quality males (Getty, 2002). For example, dominant males are more susceptible to infection by H. polygyrus with reduced parasite clearance being associated with higher testosterone levels in males (Barnard et al., 1998). Recently, it was shown that the attraction of sexually inexperienced estrous female mice and their responses to, the urine odors of a H. polygyrus male can be modulated by the presence

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of the odor cues of another female in conjunction with that of the male (Kavaliers et al., 2003a–d). This permits some plasticity in female choice and allows the incorporation of public information (Danchin et al., 2004) regarding the possible mate choices of other females. Use of this information may be particularly adaptive for young inexperienced females. This ‘nonindependent’ mate choice may also explain why in seminatural and field setting females have been reported to have mated with both uninfected and infected males (Zuk, 1995). An additional response to exposure to the odors of an infected male is a decrease in nociceptive or pain sensitivity and the induction of antinociception or analgesia. Analgesia is an important component of the suite of defensive responses to stimuli associated with real or potential danger and is advantageous in situations, where a direct response to noxious stimuli might otherwise disrupt other adaptive behavioral responses. The activation of these analgesic mechanisms represents an important part of animal defensive systems that can be modulated by fear and anxiety. Manipulations that reduce anxiety (e.g. administration of benzodiazepines) can attenuate the analgesia elicited by threatening situations, while manipulations that induce anxiety associated with threatening environmental cues provoke analgesia (Kavaliers et al., 2000). It was found that relatively prolonged (15 min) exposure to the volatile and non-volatile odors of novel males infected with either H. polygyrus, E. vermiformis, or P. serrata induced an endogenous opioid mediated analgesia, while brief exposure (30 s—1 m) elicited a shorter lasting non-opioid mediated analgesia (Kavaliers and Colwell, 1993, 1995a; Kavaliers et al., 2003a,b, 2005). The initial non opioid-mediated responses and their neurochemical substrates represent anxiety-related anticipatory defense reactions, while the more protracted opioidmediated responses are primarily associated with stress and related responses. These analgesic responses and their fearfulness/stress-associated behavioral correlates elicit either a reduced interest in and avoidance of parasitized males by females. The magnitude of the analgesia displayed was also affected by prior familiarity. Females that were exposed to the odors of familiar H. polygyrus or P. serrata infected males displayed attenuated analgesia responses, while the odors of novel males elicited heightened analgesia (Kavaliers et al., 2003a,b, 2005). However, in all cases the females still avoided and selected against, the odors of the infected males in a choice test. This recognition of individual infected males has potentially important implications for the understanding of the dynamics of interactions between infected and uninfected individuals and parasite transmission in the wild. Exposure to the odors of either gonadally intact males with low levels of testosterone or castrated males did not elicit any significant analgesic responses, while exposure to

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the odors of an acutely (30 min) physically stressed males elicited levels of analgesia that were significantly lower than those elicited by exposure to parasitized males (Kavaliers et al., 2003c). This indicates that the females were responding to specific odor cues associated with infection and not simply to either any changes in male testosterone levels or the presence of the odors of a stressed male per. 3.2. Avoidance and analgesic responses of males Although studies of parasite recognition and avoidance have primarily focused on females, particularly in relation to mate preferences, males also face the threat of parasitic infection during social interactions. Consequently, the ability to use odor cues for the discrimination and avoidance of infected individuals is equally important for both sexes. Male mice can distinguish the identity and status of other males on the basis of odor (e.g. Hurst et al., 2001; Nevison et al., 2003). They can distinguish dominants from subordinates, as well as kin and familiar from non-kin and unfamiliar individuals. Male mice are also able to recognize infected conspecifics on the basis of odors. Male mice discriminated and displayed aversive and avoidance responses to the urine odors of other males infected with the louse, P. serrata (Kavaliers et al., 2004).This recognition is highly adaptive as social interactions with other lice infested males could readily lead to infestation and reduce male fitness and sexual attractiveness. Likewise, males distinguished and displayed aversive responses to the odors of other males infected with H. polygyrus (Kavaliers et al., 2004). Avoidance of, and aversive responses to, odor cues associated with infected males would also likely reduce the likelihood of social interactions and as such reduce the risk of contagion. There is accumulating evidence suggesting that males also gain from mate choice (Kokko et al., 2003). Uninfected healthy male mice were shown to avoid and refuse to copulate with, females infected with the indirectly transmitted parasite, Trichinella spiralis (Edwards and Barnard, 1987). Males were also able to detect, select and avoid females infected with the directly transmitted nematode, Taenia crassiceps (Gourbal and Gabrion, 2004). Male mice also discriminated against, and displayed aversive response to, the odors of H. polygyrus infected females (Kavaliers et al., 1998b). These responses were also dependent on prior sexual experience with significantly greater aversive responses being displayed by sexually experienced than sexually inexperienced males. The odors of estrous females can elicit in males a number of behavioral, hormonal and physiological responses that are affected by, or dependent on, a male’s prior sexual experience (e.g. Lydell and Doty, 1972; Kavaliers et al., 2001). These shifts may modulate the nature and magnitude of the responses displayed by males to the odors of infected and uninfected females.

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3.3. Odors, MHC and MUPs Parasite-induced alterations in host immune responses, and possibly other associated endocrine factors reflecting host status, influence the production of MHC and MUP related volatile and non-volatile odor cues (Penn and Potts, 1997, 1998). Mice are able to distinguish between individuals based on differences in MHC-associated volatiles and MUPs and infection may alter these cues. This is supported by the evidence indicating that acute antigenic challenge and immune activation can affect the relative attractiveness of males and their odor cues to females (e.g. Klein and Nelson, 1999; Yamazki et al., 2002; Zala et al., 2004; Litvinova et al., 2005). In the case of H. polygyrus, sub-clinical infection stimulates host immune response with an accompanying inflammation of mucosal tissues. Adult H. polygyrus modulate host immunity, allowing persistence of infection for more than 30 weeks in certain strains of mice (Wahid et al., 1989; Barnard et al., 1998). Similarly, MMTV has profound effects on the T-cell repertoire of infected individuals and likely affects MHC activity (Yamazaki et al., 2002). Louse infections also affect host immune response through local skin responses leading to systemic humoral responses along with changes associated with a gradual development of immunity. There is also intriguing evidence from transgenic mice suggesting that disease, and likely infection, can alter MUPs in male mice (Zabel et al., 2002) As such, the MHC and possibly MUP associated odor make up of the infected individuals is likely to differ from that of the uninfected individuals. Females, and likely males, may use all of these odor cues both to distinguish between infected and uninfected individuals and to actively avoid infected individuals.

4. Responses to predator odors Animals generally respond to the threat of predation and predator risk with a number of defensive behaviors, including immobilization or freezing and risk assessment (i.e. decision making as to when and how to feed, etc. in the presence of a predator), increased wariness and the suppression of nondefensive behaviors (Blanchard et al., 1990, 2001; Kavaliers and Choleris, 2001). The ability to assess predation risk requires the presence of temporally and spatially reliable information. Both chemical and visual cues are capable of providing such information. These cues represent, however, very different levels of risk and information. Although visual cues are reliable they are also very risky as the prey and predator must be within close proximity. Chemical information, while spatially and temporally less reliable, has a lower associated risk. The prey and predator need not be in close proximity for the prey to acquire information about the possible presence of a predator.

Increasingly, predator-related odor stimuli have been used to examine defensive responses of rodents. The seminal work of the Blanchards using a visible burrow system provided the first detailed description of the responses of rats to cat odor (Blanchard et al., 1990). Results of a variety of laboratory and seminatural studies have now shown that rodents display aversive and avoidance responses to the odors of predators, and in particular that of the domestic cat (e.g. Dielenberg and McGregor, 2001; Dielenberg et al., 2001; Perrot-Sinal et al., 2004). Results of field studies have also indicated that rodents, including mice, display avoidance responses to predator (scat, faecal) odors, though the extent of the response is habitat dependent (see reviews in Powell and Banks, 2004; Arthur et al., 2005). Here, we will very briefly consider the results of a few studies with predator odor that parallel the investigations carried out with the odors of parasitized animals. Male and female laboratory mice displayed an aversion to, and avoidance of cat odor (soiled cat bedding (urinary and faecal odors), cat collar odor), in a Y-maze choice situation (Kavaliers et al., 1994, 2001, 2003c). These responses were in many regards similar to those obtained with mice that were presented with the odors of parasitized male mice. Likewise, mice and other rodents displayed both opioid and non-opioid mediated analgesic responses following exposure to either cat odor or an actual cat (Lester and Fanselow, 1985; Lichtman and Fanselow, 1990; Kavaliers and Colwell, 1991; Kavaliers et al., 2001, 2003a). These analgesic responses were of a similar pattern as those elicited in male and female mice by exposure to the odors of infected conspecifics. Exposure to synthetic fox odor (2,5dihydro-2,4,5-trimethylethiazoline (TMT)) has also been shown elicit behavioral changes and aversive responses in rodents (e.g. Perrot-Sinal et al.,1996; Hotsenpiller and Williams, 1997; Walf and Frye, 2003). However, as discussed by McGregor and colleagues (2001) those findings may also reflect responses to aversive putrid odors and not predator odors per se. This is reinforced by the findings that the central nervous responses to TMT are substantially different from that reported to cat odor (Day et al., 2004). The responses to predator odor can also be affected by the social and sexual context. Brief, but not prolonged, preexposure to the urine odors of a novel estrous female decreased the analgesic and avoidance responses of male mice to cat odor. (Kavaliers et al., 2001). Likewise, the presence of female odors blocked the increases in corticosterone and decrease in testosterone levels elicited by exposure to cat odor. In all cases, sexually experienced males displayed a greater sensitivity to female odors than did sexually naı¨ve males. The male mice were considered to be ‘emboldened’ following brief exposure to a novel estrous female, displaying markedly reduced anxiety, stress responses, and avoidance responses to a predator odor and the implied risk of predation. This may result in a greater

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sexual motivation and interest in any novel sexually receptive females that may be in the immediate vicinity of the male.

5. Functional genomics and the recognition of parasitized individuals The ability of animals to identify and recognize other individuals, is a crucial prerequisite for a wide range of social behavior. Social recognition, where animals identify and recognize other individual conspecifics, provides a foundation for mammalian social relationships. It permits the establishment of social bonds, hierarchies and facilitates interactions, allowing for group living (Choleris et al., 2004). Social recognition is also crucial for identifying, avoiding and coping with parasitized individuals and defending against infection. Unique modifications in the way an animal behaves toward another animal based on past experiences with either that specific individual or cues associated with that individual are considered evidence of true individual recognition (Johnston, 2003). True individual recognition has been shown in a number of species of rodents, including mice and rats who display not only longer term social memory but also form transient short-term memories of encountered individuals. 5.1. Vasopressin The neurobiology of social behavior and the functional genomics of social recognition and processing of social information are the subjects of increasing interest (e.g. Ferguson et al., 2002; Choleris et al., 2004; Keverne and Curley, 2004; Winslow and Insel, 2004). The neuropeptides, oxytocin (OT) and vasopressin (AVP), have been associated with the expression of social behaviors and the mediation of social recognition in several species of rodents (Winslow and Insel, 2004). Vasopressin is more abundant in the male brain than the female brain, and has in males been associated with social recognition and the expression of other aspects of social behavior. Intra-cerebral administrations of AVP and AVP antagonists have been shown to affect male territorial marking, aggressive behavior, social recognition and anxiety (review in Keverne and Curley, 2004). Of the genes for the three receptors for AVP (V1a, V1b and V2) the V1a receptor gene is, to date, suggested to play the primary role in male social behavior. Mutant mice null for the V1a receptor gene exhibited reduced anxietylike behavior and impaired social recognition (Bielsky et al., 2004). As well, the V1a receptor gene has been shown to be involved in the mediation of pair bonding and male parental care in the monogamous prairie vole, Microtus ochrogaster (Young et al., 1999). Vasopressin 1a receptor gene transfer has been reported to enhance male social affiliation in the non-monogamous meadow vole, Microtus pennsylvanicus (Lim et al., 2004). These facilitatory effects of vasopressin

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receptor activation on social bonding effects were associated with enhanced dopamine activity (D2 receptors) at the level of the ventral striatum. Central administration of a V1a receptor antagonist has, however, minimal effects on the recognition and avoidance of parasitized individuals by male mice (in preparation). Recognition of and display of aversive responses to the urine odors of infected males were unaffected by the AVP antagonist. As well, analgesic and avoidance responses to predator odor (cat collar) were unaffected by AVP manipulations. Further investigations are, however, required before any definite conclusions can be drawn regarding the involvement (or lack of direct involvement) of AVP in mediating the recognition of and responses to threat associated odors in mice and other species. 5.2. Oxytocin Oxytocin is equally expressed in female and male brains and is involved in the mediation of several social behaviors including that of mate and maternal bonds (e.g. Carter, 1998; Dulzen et al., 1998, 2000; Engelamnn et al., 1998; Ferguson et al., 2000, 2001; Pedersen and Boccia, 2002; Choleris et al., 2003a–c). Oxytocin is also involved in the mediation of olfactory based social recognition. Both male and female mice with deletions of the oxytocin gene (OT knockout (OTKO) mice) were impaired in tests of social recognition despite normal olfactory and learning abilities (Ferguson et al., 2000; Choleris et al., 2003a–c). Infusion of OT into the medial amygdala prior to social interaction restored social recognition in OTKO male mice, while infusion of an OT antagonist inhibited social recognition in wildtype (WT) male mice (Ferguson et al., 2001). Wildtype female mice that bilaterally received antisense DNA targeted against the oxytocin receptor (OTR) into the medial amygdala were as impaired in social recognition as their littermate OTKO mice in an habituation/dishabituation procedure (Choleris et al., 2003b). This habituation/dishabituation protocol is based on the natural propensity of a mouse to investigate another mouse (e.g. anogenital sniffing) that is introduced into its home cage (Gheusi et al., 1994; Choleris et al., 2004). The social responses decline to a low level (habituation) with repeated presentations of the same conspecific. However, when a novel individual is presented, the initial level of social investigation is re-instated (dishabituation). In this test the OTKO females, unlike their wild-type littermates, did not show a habituation response to a repeatedly presented individual, nor did they show a dishabituation response when presented a new individual. Recently, evidence was presented showing that the oxytocin gene is also associated with olfactory mediated recognition, memory and display of avoidance responses to the odors of parasitized male mice. OTKO female mice were impaired in their discrimination of, and display of aversive responses to, the odors of either H. polygyrus or

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P. serrata infected males (Kavaliers et al., 2003c, 2004, 2005). OT wild-type (OTWT) and OT heterozygous (OTHZ), but not OT knock-out females, readily distinguished the urine odors of uninfected male mice from those of infected males. Exposure to the odors of infected males also induced aversive analgesic responses in OTWT and OTHZ females, with the levels of analgesia in the OTKO females being markedly lower than those of the OTWT and OTHT females. The magnitude of the analgesic responses displayed by the OTWT and OTHZ females was further affected by prior familiarity, consistent with the involvement of OT in the recognition of specific individuals. The odors of a familiar infected male elicited attenuated, though significant, analgesic responses while the odors of a novel infected male elicited heightened analgesic responses in the OTWT and OTHT females. In both cases the OTWT and OTHZ females still avoided the odors of the infected males in a choice test. The OTKO females, in contrast, displayed equivalent analgesic responses to the odors of novel and familiar infected males and failed to discriminate against infected males in a choice test. These impairments in the recognition and avoidance of odors associated with infected individuals were neither associated with any impairments in olfactory abilities nor in the expression of analgesic and stress responses in the OTKO females. The OTKO females could, at various phases of their estrous cycle, distinguish between the odors of intact and castrated males. Moreover, OTKO females show normal (i.e. like the WT), and indeed, in certain cases heightened, analgesic and humoral stress responses to physical stressors (e.g. Neumann et al., 2000a, b; Kavaliers et al., 2003c; Mantella et al., 2004). These impairments in recognition and the displays of aversive responses were specific to the odors of infected individuals. Oxytocin manipulation had no evident effects both on the recognition of, and display of aversive responses to cat odor. OTKO, OTWT and OTHZ male and female mice displayed equivalent analgesic and avoidance and defensive avoidance responses to cat odor. The OTKO and OTWT females also displayed equivalent associative olfactory conditioning to predator odor (Kavaliers et al., 2003c). Mice that were exposed to cat odor in conjunction with a novel odor (almond), displayed 24 h later equivalent aversive responses to the cat and almond odors. This shows that the impairments in the ability of the OTKO mice to distinguish between novel and familiar mice were not due to either any non specific olfactory deficits or the ability to form olfactory based memories. These findings suggest that at least one copy of the oxytocin gene is required for the central mechanism(s), whereby female mice can specifically detect and assess parasitized individuals on the basis of odor. This is likely part of the socially relevant means, whereby females can both reduce the transmission of parasites to themselves, and select for parasite-free male. These findings also underscore

the role of the OT gene in olfactory mediated mate selection and ultimately the successful reproduction of females in an ecologically and evolutionary relevant context. 5.3. Integration of olfactory inputs Olfactory mediated social recognition begins with individual specific volatile and non-volatile odor cues (i.e. MHC and MUP associated odors, sex specific signals) being received by the main and accessory olfactory (Vomeronasal (VMO)) systems. In the main olfactory system, olfactory receptor neurons in the nasal epithelium are activated by small volatile odorants either passively by respiration or actively through sniffing (Dulac and Torrelo 2003; Mombaerts, 2004). There are a large number of odorant receptors (OR) genes, approximately 1000 in mice. There are a number of suggestions as to how the transcription of this OR genes superfamily is organized across olfactory sensory neurons (OSNs). A one gene—one neuron hypothesis has been put forth but is not clearly supported (Mombaerts, 2004). Recently, Lin et al. (2005) have shown that the male specific urine compound, MTMT, activates a large fraction of cells (mitral cells) in a relatively restricted region of the main olfactory bulb. There is also evidence that olfactory receptor neurons project to bilaterally symmetrical, spatially conserved glomeruli in the main olfactory bulb (MOB) creating a topographic map of olfactory activation in the MOB (Dulac and Torello, 2003). There is an abundance of oxytocin receptors in the olfactory bulb that may modulate various aspects of input sensitivity (Dulzen et al., 2000). There is also a distinct small island of chemosensory neuroepithelium, called the septal organ, located bilaterally at the ventral base of the nasal septum at the entrance of the nasopharynx (Ma et al., 2003). Although the septal organ resembles the main olfactory epithelium (MOE) in terms of response properties, it is physically separated from the MOE by the surrounding respiratory epithelium. By virtue of its location, it has been speculated that the septal organ may serve an alerting function by sensing odors in the environment during quiet respiration, when the airstream does not reach the main olfactory epithelium. In the accessory olfactory system, sensory neurons in the VNO, located along the base of the nasal septum, actively access nonvolatile stimuli (Dulac and Torello, 2003; Brennan and Keverne, 2004). The rodent VNO might also respond to some of the same volatile odorants recognized by the main olfactory system (Sam et al., 2001) suggesting some functional overlap between the two systems. In the VNO there are two major gene families of receptors, V1R and V2R. The sensory neurons of the apical compartment of the VNO express members of the V1R gene family, while neurons of the basal compartment express members of the V2R family. V1R s appear to bind liphophilic ligands with high affinity and specificity (Boschat et al., 2002; Brennan and Keverne, 2004). V2R

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receptors are co-expressed with non-classical MCH molecules suggesting a possible link an individuals own MHC type and their responsiveness to MHC-associated odors cues. Recently, small peptides that serve as ligands for MHC I molecules were shown to function as direct sensory stimuli for a subset of V2R receptors (Leinders-Zufall et al., 2004). Signals from the main and the accessory olfactory bulbs, converge at the medial and cortical nuclei of the amygdala, where it is proposed that OT receptors mediate OT gene effects on social recognition. This is supported by the findings that OT infusions into the medial amygdala restore social recognition in OTKO mice (Ferguson et al., 2001) and administration of OT antisense into the medial amygdala impairs social recognition (Choleris et al., 2003b). The amygdala has reciprocal connections with the nucleus accumbens (NAcc), with both areas showing enhanced dopamine activity after exposure to socially relevant odors. In female prairie voles partner preference is blocked by administration of a dopamine D2 receptor antagonist administered into the NAcc (Liu and Wang, 2003). This has led to suggestions that both dopamine and oxytocin, and likely AVP, are involved in the modulation of rewarding social interactions and recognition. How this may relate to the recognition and display of responses to socially relevant, but aversive, odors of parasitized individuals remains to be determined. In view of the plasticity in responses to the odors of infected individuals (non-independent mate choice) and the effects of prior sexual (odor) experience it is of interest to determine how this activation/processing is affected by prior experience and learning. This is of particular relevance in view of the evidence suggesting that the relative role of the VNO in mediating the expression of sexual behavior and responses can be affected by prior experience (Fewell and Meredith, 2002, Meredith and Westberry, 2002). Predator odor associated stimuli are also conveyed by the main and accessory olfactory systems to the medial amygdala. Exposure to cat odor (cat collar, cloth, bedding) and TMT elicits specific gene expression in the medial amygdala of mice and rats (Dielenberg et al., 2001; Fendt et al., 2003; Hebb et al., 2003, 2004; Koks et al., 2003; McGregor et al., 2004) while manipulations of either the entire amygdala or only of the medial amygdala reduce defensive responses (i.e. freezing) to cats and cat odor (Blanchard and Blanchard, 1972; Li et al., 2004).Results of recent studies have suggested that there is a categorization or delineation of chemical stimuli at the level of the medial amygdala. In hamsters and mice chemical signals (urine odors) from conspecifics activate the anterior medial amygdala and the posterior amygdala. However, chemical signals from heterospecifics (including cat urine odor) apparently activate only the anterior medial amygdala (Meredith and Westberry, 2004; Samuelson et al., 2004). The posterior medial amygdala is not activated by nonsocially relevant input (i.e. hetrospecific, predator odors) and may be suppressed by GABAergic inhibition from

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the adjacent intercalated nucleus. Interestingly, maximum oxytocin receptor gene expression was reported to occur in the posterior part of the medial amygdala (Gould and Zingg, 2003). This differential activation/processing of odors at the level of the amygdala may, in part, contribute to the differential involvement of OT in the modulation of responses to the odors of parasitized conspecifics and predators. 5.4. Interplay of oxytocin and estrogens in the recognition and avoidance of parasitized individuals Estrogenic regulation of social behavior has been examined in male and female mice whose genes for estrogen receptor (ER)-a or ER-b were disrupted [ER knockout mice (ERKO), aERKO and bERKO] (e.g. Ogawa et al., 1997; Nomura et al., 2002a,b; Imwalle et al., 2002). These receptors were shown to be involved in the mediation of social recognition. Both aERKO and bERKO female mice were found to be deficient in social recognition in a manner that paralleled that seen in OTKO mice (Choleris et al., 2003a,b). This led to the proposal of a four gene ‘micronet’ (two estrogen receptors, OT and its receptor) model for the mediation of olfactory based social recognition in the brain (Fig. 1). According to this model ER-b gene expression is responsible for estrogens effects on OT gene transcription in the paraventricular (PVN) nucleus of the hypothalamus (Patisaul et al., 2003), with only ER-b expression being found in oxytocinergic cells (Shughrue et al., 1997). OT through axonal projections of the PVN neurons, reaches the amygdala, where ER-a gene products regulate the expression of OT receptors (De Kloet et al., 1986; Young et al., 1998; 1999). In this manner ER- a and ER-b and OT gene activation and products are associated with the expression of social recognition. OT as well as a-ER and b-ER genes are similarly involved in the recognition and mediation of aversive responses to the odors of parasitized males by uninfected male mice. It was found that OTKO males were specifically impaired in the recognition and display of aversive responses to the odors of P. serrata and H. polygyrus infected male mice (Kavaliers et al., 2003c, 2004, 2005). Moreover, central administration of a selective oxytocin, but not vasopressin, antagonist also impaired the recognition of, and display of aversive response to, P. serrata infested males (in preparation). However, both the discrimination of and display of, aversive and defensive responses to, cat odor were unimpaired in the OTKO males. This further reinforces the differential involvement of OT in the mediation of aversive responses to predator and parasitic infection associated odors. aERKO and bERKO males also displayed impaired recognition of, and aversive responses to, the odors of H. polygyrus infected males (Kavaliers et al., 2004). This was not associated with any impairments in olfactory

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Individual-specific olfactory cues (e.g. MHC,MUPs)

Main Vomeronasal Olfactory Organ Bulb/System OT Accessory Olfactory Bulb/System

OT

Hypothalamus PVN OT ERβ

Estrogens

OTR

the simple recognition of familiar individuals to strong familial and mate bonds. Understanding of the neurobiology of social behavior in relation to its evolutionary functions could aid in the eventual resolution of the genetic and environmental factors involved in human social disorders (e.g. autism schizophrenia, social phobias, depression). Parasites and parasite avoidance are considered key determinants of social behavior, organization and interactions. An understanding of the interplay between sociality, pathogen detection and avoidance could provide further insights into the evolution of contemporary social cognition, including that of anti-social behavior and discrimination (e.g. Faulkner et al., 2004). As well, this could further the understanding of how social interactions and socio-sexual behaviors influence the spread of parasites and novel pathogens.

Amygdala

Acknowledgements Social Recognition (e.g. kin, familiar, infected, non infected)

Fig. 1. Schematic of the 4-gene micronet regulating olfactory mediated parasite recognition and avoidance. Estrogens regulate oxytocin (OT) secretion in the paraventricular nucleus (PVN) through their binding of the estrogen receptor b (ER-b). Oxytocin, through axonal projections of the PVN neurons, reaches the amygdala, where estrogens regulate the expression of oxytocin receptors (OTR) through their binding of estrogen receptor a (ER-a). Estrogen-mediated oxytocin-OTR activation in the amygdala ultimately facilitates social recognition and the recognition and avoidance of parasitized individuals (adapted from Choleris et al., 2003a).

abilities. The a- and b-ERKO males distinguished between the odors of intact and castrated males and displayed aversive responses to cat odor. This suggests that OT and ER-a and ER-b genes and their products are part of the central mechanism(s), whereby mice can distinguish and avoid parasitized males on the basis of volatile and nonvolatile odor cues (Fig. 1). These findings are relevant to the understanding of the evolution, expression and regulation of social behavior and parasite and pathogen avoidance by oxytocin and ER-a and ER-b and their gene products.

6. Conclusions The defensive behaviors of rodents exposed to predators and their odors have been proposed as models for the analysis and understanding of defense related human psychopathology, including anxiety and panic disorders (Blanchard et al., 2001). Likewise, responses to infected individuals can serve as a model for aspects of human social behavior. Humans, as social animals, exhibit a wide range of behaviors that entail social recognition. This ranges from

Supported by The Natural Sciences and Engineering Research Council of Canada (MK, EC) and NIH MH38273 (DWP).

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