11 Communication by Chemical Signals: Behavior, Social Recognition, Hormones and the Role of the Vomeronasal and Olfactory Systems R E Johnston and J delBarco-Trillo, Cornell University, Ithaca, NY, USA ß 2009 Elsevier Inc. All rights reserved. Chapter Outline 11.1 11.1.1 11.2 11.2.1 11.2.1.1 11.2.1.2 11.2.1.3 11.2.1.4 11.2.2 11.2.3 11.2.4 11.2.5 11.2.5.1 11.2.5.2 11.2.5.3 11.2.5.4 11.2.6 11.2.6.1 11.2.6.2 11.2.6.3 11.2.7 11.2.7.1 11.2.7.2 11.2.7.3 11.2.8 11.3 11.3.1 11.4 11.4.1 11.4.2 11.4.3 11.4.4 11.4.4.1 11.4.4.2 11.4.4.3 11.4.4.4 References Introduction Chemical Signals, Terminology and Concepts Functions of Chemical Signals in Social Behavior Individual Recognition: Signals, Methods, and Functional Importance Odor signals for individual recognition Discrimination of odors of individuals The nature of memories for individuals MHC, MUPs and odors Kin Recognition Group Recognition Species Recognition Interactions between Males and Females Sexual discrimination, identification, and preferences Advertising sexual receptivity: Females attracting males for mating and responses of males to female odors Male odors that attract females Mate evaluation and mate choice by females Odors, Intrasexual Competition, and Status Role of odors in the formation and maintenance of status relationships Scent marking in male–male competition for status Odors and sperm competition Scent Marking and Scent Over-Marking: Aspects of Competition for Mates and Other Resources Functions and causation of scent marking Hormonal control of scent-marking behavior Costs of scent marking Odors and Aggression Roles of Odors in Modulating Hormones in Vertebrates Chemical Identification of Signals that Influence Hormones Olfactory and Vomeronasal Systems and Their Roles in Communication and Social Behavior Structure Receptor Cells and Genes for Receptor Proteins Neural Projections from the Olfactory Bulb to the CNS Conceptual Views of the Main Olfactory and VNO Systems Hormonal responses to odors Role of the MOS in nipple search and attachment in rabbit pups Role of VNO and MOS in sexual behavior and sexual motivation VNO and the discrimination and recognition of individuals 396 396 397 398 398 398 399 401 402 403 403 404 404 405 408 408 411 411 412 413 414 415 418 418 419 420 421 422 422 423 424 424 425 425 425 429 429 395 396 Communication by Chemical Signals 11.1 Introduction 11.1.1 Chemical Signals, Terminology and Concepts More than many other areas in biology, the field of chemical communication is troubled by a lack of agreement about the use and meaning of several basic concepts, most notably the word pheromone. Confusion often occurs because scientists in different disciplines often have different ideas in mind when they use this term. Some scientists use the word pheromone in an all-inclusive way to refer to any chemical that has a function in communication between members of the same species (Wyatt, 2003). In this chapter, the phrase odor signal is used to refer to odors that have one or more roles in communication. The original meaning of the word pheromone was quite limited and restricted, and many scientists use pheromone in the original sense. Pheromones were initially defined as a single chemical compound that elicited a specific behavioral or physiological response in a member of the same species (Karlson and Butenandt, 1959; Karlson and Lüscher, 1959, see Chapter 18, Hormonal Pheromones in Fish). The idea was that pheromones were similar to hormones, except that, unlike hormones, pheromones acted between individuals rather than between organ systems within the body. This original definition of pheromones is referred to as the classic pheromone concept in this chapter. This concept of a pheromone was also congruent with the ethological concept of signals that were termed releasers (see below). The discovery of a chemical (called bombycol) from the female silkworm moth Bombyx mori that attracted males for mating fit this ethological concept well (Karlson and Butenandt, 1959; Karlson and Lüscher, 1959). Although there are other cases among insects in which a single chemical compound constitutes the sexual attractant, chemical signals that function as sexual attractants in most insects are generally more complex than this definition of a pheromone suggests. The classical concept of a single chemical compound with a specific and automatic effect on behavior or physiology is relatively rare. The classic pheromone concept and the odor signal concept are quite different on several dimensions. First, the nature of the signal: in the original definition, the signal was a single chemical compound, whereas in the odor signal concept the chemical nature of the signal is indeterminate and can range from a single chemical compound to a mixture containing a very large number of chemical compounds. Second, the nature of the response: in the original definition, a classic pheromone elicited either a single behavioral response that occurred immediately after detection of the pheromone or a specific physiological response (e.g., a hormonal response) that might take some time to become apparent. In contrast, the odor signal concept includes information, such as individual identity or group membership, in the signal and there may not be any immediate response or the response might depend on contextual information. Third, the predictability of the response: in the original definition, a specific response was highly predictable and was apparently caused or released by the classic pheromone. In the odor signal concept, the signal might cause immediate reactions but it might also primarily provide information (e.g., individual identity or group membership) and thus may not result in any specific response, or the response might be extremely variable depending on which individual or group the stimulus came from and the context in which the signal was perceived. Fourth, the role of genetics and learning in the response: the original definition of pheromone posited that the signal and response were determined by genetically determined mechanisms, whereas the odor signal concept makes no explicit distinction between responses that are innate and ones that depend largely on learned significance of the signal. Fifth, the nature of the neural mechanisms mediating responses: in the original definition, genetically determined, hardwired mechanisms were proposed for the detection and interpretation of classic pheromones, for example, specific receptors for a specific molecule (the pheromone) and a dedicated neural pathway for the physiological or behavioral response. This notion is quite similar to the concept of an innate-releasing mechanism in the terminology of classical ethology (see below). In contrast, the inclusive definition of pheromone does not make any claims about the neural mechanisms involved or the importance of genetics and/or learning in the mechanisms mediating responses (e.g., a flight response to a dominant individual vs. attraction to a friend). There are two major intellectual contexts that were fundamental in shaping the original pheromone concept, namely endocrinology and the behavioral discipline of ethology. For example, it was originally thought that hormones tended to have one or at most only a few, very specific effects on a particular tissue. Likewise, the original definition of a pheromone Communication by Chemical Signals posited a single effect on the individual perceiving this signal. The field of ethology had a major influence on the study of animal behavior. Its major proponents developed concepts and methods of observation and categorization of behavior that greatly advanced this field, especially the effects of visual and auditory stimuli produced by one individual that influenced another individual (Hinde, 1966; Manning, 1967; Marler and Hamilton, 1966; Tinbergen, 1951, 1964). Two ethological concepts are essential for understanding the original concept of a pheromone: the releasing stimulus and the innate-releasing mechanism. Researchers in this field discovered that when two individuals were interacting, a sender would be producing many potential signals but it was often the case that only one, very limited stimulus actually caused a response. The concept of a releasing stimulus was developed to highlight these findings. On the receiver’s end of the communication, ethologists posited the existence of an innate-releasing mechanism, that is, a genetically inherited perceptual module that, when stimulated by the proper stimulus, would coordinate a specific response (Hinde, 1966; Manning, 1967; Marler and Hamilton, 1966; Tinbergen, 1951, 1964). These concepts were enormously popular and helped to found an entire field of study known as neuro-ethology. Although these concepts are still useful, most scientists investigating the neural mechanisms of behavior now find these concepts to be greatly oversimplified. The original pheromone concept is a classic example in this conceptual framework. From a chemical secretion in a tissue of an organism, generally consisting of a mixture of up to hundreds of compounds, a single chemical compound causes a particular response in a receiving individual. Although work with insects has identified individual chemical compounds that influence the behavior or physiology of a receiver, it turns out that the most effective signal is rarely a single compound, but rather, it is usually a mixture of compounds. Indeed, a common type of signal is a pheromone blend, defined as a signal that is composed of a small number of chemical compounds in relatively specific ratios. Pheromone blends are especially common among insects for attracting a mate (Linn and Roelofs, 1989). In some species a single constituent of the blend may have some attractive effect by itself but the blend is much more attractive. Thus, examples that fit the definition of a classic pheromone are not common, even in insects. 397 The usage of pheromone by some to mean chemical signal and by others to mean the classical pheromone causes confusion and muddled thinking about chemical signals and the neural mechanisms underlying responses mediated by chemical signals. At the very least, authors must carefully define what they mean by their use of the term pheromone. Clear thinking about chemical signals and communication by such signals would be greatly improved by either using this term only in the classical sense of the word or by eliminating the term pheromone entirely. One alternative proposal for terms to classify chemical signals suggests three classes of signals, based on the number of chemical compounds that constitute the signal: if a single chemical compound is effective in stimulating one or more responses, it would be called a classic pheromone. If the effective signal is a blend of several compounds in relatively specific proportions, it would be called a pheromone blend. If the signal contains a large number of chemical compounds and if many of these compounds are necessary or involved in influencing responses, it would be called a mosaic signal ( Johnston, 2000, 2001, 2003; Johnston and Bullock, 2001). An example of the latter case would be a secretion containing several to hundreds of compounds that create an individually distinctive odor, based on differences in the proportions of these compounds across individuals. This scheme has the advantage of classifying a small number of types of signal based on simple, objective criteria. It does not solve all of the problems of nomenclature and communication, but it does provide a scheme that identifies different types of signals that must involve different types of neural mechanisms underlying responses to different types of signals. It also acknowledges that there are different types of chemical signals, rather than classifying all chemical signals into a single category such as pheromone. 11.2 Functions of Chemical Signals in Social Behavior It is clear that animals use information contained in odors to determine many characteristics of other animals, such as species identity, sex, reproductive state, age, social status, individual identity, level of fear or stress, health status, and quality of the diet. One odor may contain information about several characteristics of the donor. For example, male mice respond with a surge in luteinizing hormone (LH) 398 Communication by Chemical Signals when exposed to female mouse odors, but not when exposed to male mouse odors or female hamster odors, indicating that male mice discriminate sex and species information from odors (Maruniak and Bronson, 1976). Also, different odors from the same animal may provide different types of information about that animal ( Johnston, 2003). For example, beavers produce two main types of scents, castoreum and anal gland secretions. Castoreum is involved in territorial demarcation, and may also mediate recognition of family members. The anal gland secretion contains individual, kin, and sex information (Sun and Müller-Schwarze, 1999). Alternatively, different odor sources may contain redundant information, for example, five different odors provide information about individual identity among golden hamsters, Mesocricetus auratus, and Djungarian hamsters, Phodopus campbelli ( Johnston et al., 1993; Lai and Johnston, 1994), whereas other sources of odors do not. The same odors that provide individual information may also provide information about sex, reproductive state, and social status (Ferkin and Johnston, 1993, 1995a,b; Ferkin et al., 1994; Johnston et al., 1993; Lai et al., 1996). Redundant information from several sources may enhance responses by another individual. For example, male hamsters show high levels of copulatory behavior when all of a female’s odors are present but they show declining levels of copulatory behavior as specific odor sources are removed ( Johnston, 1986). 11.2.1 Individual Recognition: Signals, Methods, and Functional Importance Individuals are the fundamental units of social interaction and social organization. Thus, discrimination and recognition of individuals, or classes of individuals, are fundamental to the understanding of social behavior. Chemical signals are important for such processes in many species, especially in social insects and terrestrial and flying mammals. All social behavior involves communication between individuals by means of signals detected by sensory systems. Thus, discrimination between cues from different individuals and recognition of familiar individuals by means of these signals are likely to be extremely important for survival and reproduction. In this section, we discuss discrimination and recognition of individuals and categories of individuals by chemical cues and the roles that such recognition have in regulating social behavior. This summary focuses on vertebrates, but selected examples from other taxonomic groups will also be mentioned. 11.2.1.1 Odor signals for individual recognition The signals for discrimination and recognition of individuals can come from many different specific sources, such as a variety of specialized scent glands (e.g., sebaceous glands, apocrine and eccriine sweat glands) as well as urine and feces. In golden hamsters, for example, there are five different sources of individually distinctive scents, namely flank gland, vaginal secretions, ear glands (inside the pinna), urine, and feces. Six other potential sources of odors were tested but were not individually distinctive, as measured by habituation–dishabituation tests (fur from the midline ventral surface, fur from the dorsal surface between the shoulders, saliva, feet, fur behind the ears, and the flank-gland area from flank-glandectomized males (Johnston et al., 1991)). Similar results were found with Djungarian hamsters (Lai and Johnston, 1994). The information that provides individually distinctive signatures as well as colony recognition, hive recognition, and kin recognition has been shown to be due to the differences in the proportions of individual chemical compounds in complex mixtures of many chemicals, as demonstrated in a variety of mammals and insects, including a species of mongoose, humans, house mice, bees, wasps, and termites (Arnold et al., 1996; Dani et al., 2001; Gorman, 1976; Haverty and Thorne, 1989; Howard, 1993; Smith and Breed, 1995; Smith et al., 2001; Sommerville et al., 1994). From ants to mongooses and primates, differences in the proportions of different chemicals produce a different odor gestalt or signature that is readily distinguished by other individuals of the same species. It has previously been proposed that such signals be called mosaic signals because, like a real mosaic made with colored tiles, the meaning (information) is not dependent on any one component, but rather depends on the relative abundance of different chemical compounds and the odor quality or percept that develops out of this mixture. Similar pattern-perception mechanisms are involved in the recognition of familiar odors from everyday objects and food items, such as fruits, wines, different blends of coffee, etc. Visual recognition of complex patterns, such as faces, also depends on pattern-recognition processes. Perception of such odor-mosaic signals, clearly, must involve higher-order neural and perceptual processes that integrate inputs from many different types of odor receptors. 11.2.1.2 Discrimination of odors of individuals The first studies to demonstrate discrimination of odors in mammalian species used training procedures with food reinforcement to demonstrate such abilities Communication by Chemical Signals (Bowers and Alexander, 1967; Rasa, 1973). Since then, most investigators have used techniques that are much more naturalistic and much easier, usually variations on some kind of habituation or habituation– dishabituation technique. In such methods, subjects are exposed to repeated samples of an odor from one individual; the time spent investigating this scent is recorded over three to five trials. With repeated presentations of new samples of the same odor stimulus, the behavioral response declines significantly, indicating that habituation has occurred. If a new stimulus of the same type but from a different individual is presented, the sniffing investigation increases (dishabituation has occurred). This indicates that the subjects have noticed the difference between the first and second stimuli – that is, they discriminated between the two odors. A variation on this method is to present two stimuli on the test trial, both the stimulus that the subjects have been habituated to and a novel stimulus. Simultaneous presentation of the familiar and novel stimuli is an easier task and may show differences in response to the novel stimulus that a single-stimulus test does not (Brown et al., 1987). With standard laboratory species, the duration of the interval between trials can vary considerably without much change in the magnitude of the investigation times observed. In one study, for example, the intertrial interval was varied from 1 s to 2 days and the results were quite similar ( Johnston, 1993). However, investigators should test different intervals to determine which intervals work best for each species. In experiments with dogs and captive wolves, it was found that intervals needed to be much longer to be effective. These animals would not even approach the stimulus on the second trial after an interval of 15–30 min, apparently because they could determine from a distance that the odor presented on the second trial was the same as that on the first trial and was not interesting. However, when the interval between trials was shifted to 24 h, both dogs and wolves showed the typical habituation–dishabituation pattern of results (Brown and Johnston, 1983). One disadvantage of habituation tasks is that a failure to show dishabituation to a novel stimulus may have two different interpretations: (1) the animal did not discriminate or (2) the animal did not investigate the stimulus because of other motivational/ emotional reasons, for example, if the novel stimulus came from an animal that had beaten the subject in a fight, then the subject might be reluctant to investigate an odor from the familiar winner. Thus, failure to discriminate in habituation–dishabituation tasks may be difficult to interpret. One example from the 399 literature is the surprising results obtained in the so-called juvenile recognition task that has been used with laboratory rats and mice. In rats, males exposed once to a juvenile male fail to show an increased response to a different juvenile rat after a delay of 1–2 h (Thor and Holloway, 1982). This result is surprising because an increase in investigation would be expected. Furthermore, after exposure to odors alone, hamsters remember them for at least 10 days ( Johnston, 1993). Perhaps the odors of juveniles are not yet highly distinctive; also, it has been shown that a novel context distracts rats from investigating juveniles (Burman and Mendl, 1999). On the other hand, habituation methods also have some compelling advantages over learned discriminations. First, they are simple, easy, fast, and do not require elaborate testing chambers, equipment for recording responses, or prolonged training procedures. Perhaps even more importantly, the responses obtained reflect the natural responses of the animals and do not involve training the animals to respond to a particular stimulus and not to respond to another stimulus. Trained discriminations are most appropriate when the question of importance is, what is the capacity or limits of the sensory system? and such methods do sometimes show that the animal does have the capacity to discriminate between stimuli that they did not demonstrate when using a habituation method (Schellinck and Brown, 1992; Schellinck et al., 1995; Yamazaki et al., 1990). On the other hand, if one is interested in the natural social behavior of animals, training methods may not reflect what animals will naturally do. It is important to distinguish between the ability to discriminate between the cues from different individuals and the ability to recognize individuals. Discrimination implies the ability to distinguish between two or more odors, either when they are both present simultaneously or when comparing the memory of an odor with the presentation of a new odor. However, discrimination and memory for a single odor of an individual do not necessarily indicate that the subject has a memory for that individual. 11.2.1.3 The nature of memories for individuals Both methods outlined above (trained discrimination and the basic habituation–dishabituation tests) are primarily useful for determining whether animals discriminate between, and remember, individual signatures. If one wants to determine other types of information, such as the content of the memory or the emotional salience of a memory for an individual, other methods of testing are necessary. After 400 Communication by Chemical Signals individuals interact with one another, what do they remember about each other? Two types of information may be valuable to remember: (1) memories that incorporate several different characteristics of the individual, such as several separate odors or other cues (sound of voice, visual features, etc.) and (2) the emotional or functional significance of the individual to the subject. 11.2.1.3(i) Integrated, multi-odor representations of individuals Recognition involves the ability to discriminate between the cues/signals from one individual and another, but it also involves knowledge of the individuals involved (re-cognition – to know again). Recognition of an individual implies that the subject has knowledge of the stimulus animal. At the simplest level, this may just involve familiarity with one or more signals from another individual and the ability to categorize a stimulus into one of two categories (familiar or unfamiliar). In some cases, such as discriminating between own group and another group, this level of knowledge may be sufficient, for example, to accept an individual into the nest or not. True individual recognition, however, implies more thorough knowledge of the characteristics of other individuals. One type of knowledge about other individuals is to know several distinctive characteristics of others. Humans, for example, have been shown to recognize others on the basis of smell, faces, speaking-voice quality, singing-voice quality, feel of the skin, gait, and posture. These physical characteristics are integrated into memories of others that also include historical details about things that a particular individual has done, what their likes and dislikes are, the identity of their friends and family members, etc. All of this kind of information is integrated in the brain into multicomponent memories of other individuals. Observational studies of many species of animals suggest that individuals recognize many other individuals and that they know a lot about these individuals, including the type of social relationship they have with others: friends, allies, enemies, etc. (Beecher, 1991; Blaustein and Porter, 1990; Caldwell, 1985; Cheney and Seyfarth, 1990; De Waal, 1982; Halpin, 1986; McComb et al., 2000; Mennill et al., 2002; Payne, 2003; Rasmussen 1995; Tyack, 2003). Can experimental studies indicate the complexity of the memories that animals have of others? Specifically, do animals remember other familiar individuals using several different distinctive signals and integrate these separate memories into integrated representations of others? A series of experiments suggest that this does occur. In one series of experiments, male hamsters first had a series of brief interactions with females on four successive days. Then, using a unique variant of the habituation– dishabituation method, it was shown that, after habituation to one individually distinctive odor of a stimulus animal (e.g., that from vaginal secretions), males were also habituated to other odors from the same individual (e.g., sebaceous flank glands). That is, subjects showed an across-odor habituation ( Johnston and Jernigan, 1994). Since the odors themselves are composed of very different chemical compounds, it is not likely that this effect was due to chemical similarities in the two odors. Additional evidence for this conclusion comes from results of control experiments in which subjects were not familiar with the stimulus animals. If there was similarity in odor quality across different odor sources, subjects should show across-odor habituation without familiarity with the scent donor. However, if subjects had not previously interacted with the scent donors, no cross-odor habituation was observed ( Johnston and Bullock, 2001; Johnston and Jernigan, 1994). Subsequent experiments showed that the same effects were observed with other pairs of odors ( Johnston and Bullock, 2001). Thus, these experiments provide evidence for the existence of integrated, multi-odor memories of other individuals. Interestingly, it has recently been found that contact between the subjects and the stimulus animals is necessary for this acrossodor habituation effect to occur ( Johnston and Peng, 2008). It is not known as to what contact provides – one possibility is the exposure to individually distinctive proteins which could serve as a separate identity cue (Hurst et al., 2001). Another possibility is that the sense of touch provides a more salient emotional response to odors of others and enhances memory of odor characteristics ( Johnston and Peng, 2008). Memories that integrate numerous types of information about other individuals are the starting point for detailed knowledge of others, just the type of knowledge that is necessary for long-term social relationships and integrated group structure and stability. These results suggest that the memories of individuals provide integrated nodes of information about known individuals. Such knowledge is a prerequisite for development of complex societies, including nuanced relationships with different individuals. The functional significance of memories of individuals will be described in some of the following sections. Communication by Chemical Signals 11.2.1.3(ii) Emotional and functional significance of the individual to the subject In virtually all laboratory studies aimed at individual recognition, it has proven difficult to provide evidence for true individual recognition – that is, the unique significance of an individual, not just the significance of categories of individuals, such as familiar versus unfamiliar or dominant versus subordinate. In particular, it has been difficult to demonstrate the emotional significance of another individual without possible confounding interpretations, such as differences in familiarity or dominance status (Martin and Beauchamp, 1982). This is partly due to the fact that, in order to have some emotional significance, the subjects must have some interactions that have significance (e.g., a fight or mating experience). How does one determine if subsequent responses to a particular individual are due to this emotional significance or to familiarity or degrees of familiarity? Recent experiments with golden hamsters have provided some evidence for such true recognition by giving male hamsters exposures to two different males. One stimulus male beat the subject in a series of three brief fights whereas the subject became familiar with a second male by interacting across a wire-mesh screen. This latter type of exposure is sufficient for males to develop multicomponent memories of the stimulus animal ( Johnston and Peng, 2008). After both of these experiences, male subjects were then tested for their responses in a Y-maze with odors and other cues from the two stimulus animals. Subjects were attracted to the odors and other cues from a familiar neutral male but were hesitant to approach the odors of the familiar winner or stay near him (Lai et al., 2004, 2005). These results indicate specific types of response to two different individuals that have different significance to the subject but have equivalent (or at least very similar) levels of familiarity. These results are based on a relatively simple set of procedures and experiences compared to the number and potential complexity of experiences in the wild. Nonetheless, this study is the first that, to the authors’ knowledge, demonstrates true individual recognition and not just different degrees of familiarity. 11.2.1.4 MHC, MUPs and odors There are two primary polymorphic and multigenic complexes that are important in studies of olfactory communication that contribute to or determine individual differences in odors. These complexes are the major histocompatibility complex (MHC) and the 401 major urinary proteins (MUPs). MHC genes produce highly polymorphic glycoproteins involved in immune system function (Yamazaki et al., 1980). MUPs are mostly produced in the liver and become concentrated in the urine in mice. Even though MUPs have been described only in house mice, there are similar lipocalin proteins in scent-producing organs of other species, such as a-2u globulins in rats (Beynon and Hurst, 2004) and a similar lipocalin protein in hamster vaginal secretion (Macrides and Singer, 1991; Singer et al., 1986, 1989; Singer and Macrides, 1990). MUPs are nonvolatile molecules, but they bind smaller, volatile molecules and release them slowly (Hurst and Beynon, 2004), thus prolonging the effectiveness of volatile compounds contained in scent marks (Hurst et al., 1998). Without such binding, the volatile compounds might be lost from the scent mark relatively quickly (in minutes), which would render the scent mark uninformative (Hurst and Beynon, 2004). By being bound to MUPs, these signaling volatiles can emanate from the scent mark for up to 24 h (Humphries et al., 1999). MUPs also contain individually distinctive information on their own (Hurst et al., 2001). Several studies have shown that female mice show a preference for the odors of males with dissimilar MHC types compared to their own MHC type (Penn and Potts, 1999). There are two primary explanations for why the MHC type of males and females influence mate choices. First, by mating with males that have a different MHC type, females produce MHCheterozygous offspring and such offspring should be able to respond effectively to a wider range of pathogens than homozygous pups (Penn and Potts, 1998a). A second advantage is that a preference for a male with a different MHC type reduces inbreeding and produces offspring that are more heterozygous across all of the genome, not just in the MHC (Brown and Eklund, 1994; Potts and Wakeland, 1993). Most of the studies on the role of the MHC in mate choice have been conducted using inbred laboratory mice that are genetically identical except at the MHC locus (Yamazaki et al., 1980; Brown et al., 1990). In such studies, male mice show the ability to discriminate between the odor of two conspecific females that have a dissimilar MHC (Yamazaki et al., 1980). Studies with laboratory rats show similar results (Brown et al., 1990). Female mice in estrus also show a preference for the odors of males with a dissimilar MHC when tested in a Y-maze (Egid and Brown, 1989). Interestingly, females that were not in estrus did not show such a preference (Egid and Brown, 1989). 402 Communication by Chemical Signals When females had access to two tethered males, one with a similar MHC and another with a dissimilar MHC, females showed a preference for the male with a dissimilar MHC (Egid and Brown, 1989). Jumping to another taxonomic group, women in the fertile phase of their menstrual cycle find the odors of men who are the most dissimilar from them in their MHC to be the most attractive/pleasant (Wedekind et al., 1995). When women are taking oral contraceptives, such preferences are reversed; that is, women then prefer the odors of men with a similar MHC type. Contradicting the above literature on mice, a recent study using wild mice living in large enclosures has shown that MHC is not a relevant marker that animals use for inbreeding avoidance (Sherborne et al., 2007). MUPs, however, were sufficient to explain inbreeding avoidance in that study (Sherborne et al., 2007). The fact that mice and rats can discriminate between the odors of two conspecific individuals that differ genetically only in their MHC has been taken as an indication that the MHC is a major component of an individual’s chemical fingerprint (Brown et al., 1990; Yamaguchi et al., 1981). However, there is no clear evidence that the MHC of an animal offers an individual signature that is perceived by conspecifics. In fact, studies that specifically tested whether mice can use MHC-related odors to recognize the owner of a given scent found negative results (Cheetham et al., 2007; Hurst et al., 2005). In these studies, MUPs were involved in individual discrimination but the MHC was not (Cheetham et al., 2007). It is not surprising that this is the case, because MHC-related odors are affected by factors such as status and diet (Schellinck et al., 1997), and thus are not a good candidate to offer a stable individual chemical fingerprint (Hurst et al., 2005). MUPs appear to be a better candidate for individually distinctive information, at least in mice (Hurst and Beynon, 2004), because the pattern of MUPs expressed by an individual is consistent, and thus provides a constant individual signature unaffected by factors such as diet or social status (Nevison et al., 2003). For example, when two individuals share the same pattern of MUPs, odors of one mouse do not trigger competitive behaviors in the other (Hurst et al., 2001). Also, when a purified MUP is added to the urine of a male, he treats his own modified urine as if it belonged to an intruder male (Hurst et al., 2001). The components involved in individual discrimination are either the MUPs themselves or the MUP– ligand complexes, rather than the volatiles emanating from a scent mark (Nevison et al., 2003). 11.2.2 Kin Recognition Recognizing kin is important because it also allows an individual to avoid mating with closely related individuals and thus reduces inbreeding depression in offspring. Mutual recognition between a mother and her offspring may also be beneficial to both parties. Female Mongolian gerbils, Meriones unguiculatus, use their ventral gland to scent-mark their pups if they have been experimentally cleaned, and they will retrieve pups marked with their own secretion preferentially as compared to unmarked pups (Wallace et al., 1973). Female mice are more likely to retrieve pups with a similar MHC (Yamazaki et al., 2000). Pups also show a preference for the odors of the mother and siblings (thus sharing a similar MHC) over the odors of individuals with a dissimilar MHC (Yamazaki et al., 2000). Mutual recognition between mothers and their offspring is also well documented in ungulates (Grau, 1976). Kin recognition allows an individual to focus its cooperative behavior toward extended kin, and to focus aggressive behavior toward nonkin as a means of increasing its inclusive fitness. For example, odors of kin elicit lower levels of agonistic scent marking (flank marking) in hamsters than odors of nonkin (Heth et al., 1998). There are at least two mechanisms by which an individual can use odors to recognize kin. First, in recognition by association, animals learn the characteristics of others that they grow up with and, later in life, treat these individuals as kin. This type of learned recognition can only be useful for actual kin recognition in species in which litters of one female’s pups are physically separated from the litters of other females so that developing pups are only exposed to kin. Recognition by association can be demonstrated by using a cross-fostering design in which pups grow up with siblings and foster siblings from another litter. When these individuals are adult, results showing that individuals treat nonsiblings that shared their nest like kin indicate that the mechanism underlying such recognition is the association in the nest. A second mechanism is self-referent phenotype matching, in which an individual compares its own phenotype (e.g., odors) to that of other conspecifics. If there is a high correlation between the odors of self and another individual, that other individual may be treated as kin. This method of kin recognition may be especially important in species with multiple paternity or maternity, where full-siblings and halfsiblings may share a nest. It may also occur in species in which pups of unrelated females can mix early in Communication by Chemical Signals life. In this type of situation, recognition by association would not be a reliable method to recognize kinship. An example of self-referent phenotype matching has been shown in female golden hamsters. Using cross-fostering shortly after birth, it was found that estrous females were more attracted to unfamiliar nonkin than to unfamiliar kin (Mateo and Johnston, 2000). Odor similarities based on genetic similarities between close kin undoubtedly provide the basis for recognition of kinship (Heth and Todrank, 2000; Heth et al., 2001, 1998, 1999; Todrank and Heth, 2003; Todrank et al., 1998). Little is known about the specific mechanisms beyond the role of MUPs and MHC genes, discussed above. Nonetheless, it is clear that genetic similarity does translate into odor similarity. Many genes must have roles in producing odors, since many different metabolic processes can be reflected in the output of scent glands and other secretory and excretory products. 11.2.3 Group Recognition Group recognition is important in social species in which agonistic behaviors are directed preferentially at individuals from another group. Group discrimination may occur by individually recognizing each member of the group and/or by means of a group odor. A group odor can be produced either by mixing the scent of all individuals together on each individual, by scent marking one another (allomarking), or merely by being in contact with one another. For example, European badgers (Meles meles) produce a secretion in the subcaudal pouch which contains distinctive group membership information (Buesching et al., 2003). This secretion is partly produced by bacterial flora. Group members transfer such flora between one another by pressing the subcaudal pouch against the body of another group member. In some cases, two individuals press both pouches together, so that the bacterial flora and/or secretions are transferred between the subcaudal pouches of the two individuals. The authors observed 3021 instances of allomarking between 40 individuals in natural conditions, indicating the regularity and importance of this behavior (Buesching et al., 2003). Another example is the European rabbit, Oryctolagus cuniculus, which lives in close-knit social groups (Mykytowycz, 1968, 1970). Dominant males mark all members of the group with their chin glands. If a rabbit from one group is experimentally scented with scent from a dominant male of another group, and 403 then introduced back into its natal group, it is attacked (Mykytowycz, 1968). Dominant males even attack females of other groups and females of their own group that are scented with the odors of a male or female from another group (Mykytowycz, 1968). It is also possible that the scent of a group is conferred by a high degree of genetic similarity due to inbreeding, but we are not aware of any examples in vertebrates. 11.2.4 Species Recognition Discriminating between individuals of one’s own species and individuals of a closely related species is virtually universal across taxa since individuals who mate with other species usually do not produce young or may produce infertile young. Female preference for odors of conspecific males and male attraction only to odors of conspecific females may serve as a precopulatory isolating mechanism resulting in avoidance of interspecific mating. Indeed, there are many studies with rodent species showing female preferences for odors of conspecific males over odors of closely related heterospecific males ( Johnston, 1983). Some examples are deer mice, Peromyscus maniculatus (Doty, 1973; Moore, 1965); collared lemming, Dicrostonyx groenlandicus; brown lemming, Lemmus trimucronatus (Huck and Banks, 1980); and the fossorial mole rat, Spalax ehrenbergi (Nevo et al., 1976). Further support for the notion that female preferences for odors of conspecific males may have the ultimate function of avoiding interspecific matings comes from several studies showing that preference for conspecific odors occurs only when females are sexually receptive. This effect has been shown, for example, in fossorial mole rats (Nevo et al., 1976) and deer mice (Doty, 1972). Females of three populations of the striped mouse, Rhabdomys pumilio, showed a preference for the odors of males of the same population over those of other populations, but only when females were in estrus (Pillay, 2004). Species recognition can be innate or can be learned neonatally. In the brown and collared lemmings, males prefer odors of conspecific females. However, when pups of these two species are cross-fostered, males show a preference for the female odors of the cross-fostered species, that is, heterospecific odors (Huck and Banks, 1980). Cross-fostered females do not show a preference between conspecific and heterospecific male odors, whereas females that were not cross-fostered showed a strong preference for conspecific male odors (Huck and Banks, 1980). 404 Communication by Chemical Signals 11.2.5 Interactions between Males and Females 11.2.5.1 Sexual discrimination, identification, and preferences Given the importance of finding potential mates of the opposite sex and competing with rivals of the same sex, identification of males and females is probably universal in all species that reproduce sexually. Chemical signals often provide crucial information about the sex of conspecifics; a few examples of species in which individuals are more attracted to odors of opposite-sex conspecifics than those of same-sex conspecifics include duikers, black-tailed deer, collared peccaries, brown bears, dogs, wood rats, golden hamsters, Djungarian hamsters, mice, rats, voles, and guinea pigs (Eisenberg and Kleiman, 1972; Ferkin and Johnston, 1995b; Johnston, 1983). In most species, there are several distinct sources of odor that provide information about sex and/or reproductive state, but at the same time not all odors contain such information. For example, in Djungarian hamsters, males showed a preference for the odors of receptive females over the odors of males when the odor source was urine, anogenital secretions, saliva, or midventral gland secretion, but not when the odor source was feces or secretions from the feet (Lai et al., 1996). Hormones, such as testosterone and estradiol, influence the attractiveness of odors of the opposite sex. Gonadectomy usually reduces the attractiveness of odors of the opposite sex and the attractive properties of such odors can be restored by replacement therapy. Ovariectomy often reduces male’s responses to female odors and replacement treatment with estradiol often restores the attractiveness of these odors – for example, in meadow voles, Microtus pennsylvanicus (Ferkin and Johnston, 1993). When several body odors are indicative of the sex of an individual, the degree to which each odor is affected by steroid hormones can vary. In meadow voles, odors from feces, mouth, and the posterolateral region were clearly affected by testosterone concentrations in the blood of males and by estradiol in females. Gonadectomy of scent donors eliminated preferences for these odors by opposite-sex subjects and these preferences were restored by replacement therapy with testosterone for male donors and estradiol for female donors. The attractiveness of urine and anogenital odors were reduced but not eliminated by gonadectomy (Ferkin and Johnston, 1993). In addition to showing a preference for odors of opposite-sex individuals, males usually show a preference for odors of females that are sexually receptive over those that are in other reproductive states (Brown, 1985b). For example, sexually experienced, female rats prefer odors of normal males over the odors of castrated males (Carr et al., 1965). Similarly, male rats prefer odors of receptive females over odors of nonreceptive females (Carr et al., 1965). These preferences, however, may depend on knowledge that males learn during interactions with females: for example, sexually experienced male rats prefer odors of receptive females over odors of nonreceptive females but sexually naive males do not show this preference (Carr et al., 1965). The preference for opposite-sex conspecifics is often also dependent on the reproductive state of the receiver. In domestic rats, males prefer the odors of estrous females to those of females in other reproductive states, but castrated males do not show this effect (Carr et al., 1965; Randall, 1986). Similarly, the preference that diestrous, estrous, and lactating female golden hamsters show for the odors of intact male hamsters over castrated males is not shown by pregnant females ( Johnston, 1979). One might expect that males of many species would generally be more attracted to the odors of females than those of males regardless of the reproductive state of the female (with the possible exception of lactation, when females are guarding their young and can be quite aggressive toward intruders). On the other hand, recent data suggest that the ability of males to discriminate between males and females and the preference for female odors by males are two distinct mechanisms. Experiments with house mice show that males with the vomeronasal organ (VNO) removed do not show a preference for the odors of estrous females over odors of males (Pankevich et al., 2004). However, these males were still able to discriminate between odors of males and estrous females when tested in a habituation–dishabituation method. These results make the important point that the ability to discriminate between odors of males and females does not necessarily lead to functionally relevant responses. Previous experience with particular individuals as mates can also influence responses to the odors of these individuals. For example, male rats show a preference for the odors of a novel female over the odors of a familiar, previous mate but female rats show a preference for the odors of a previous mate over the odors of a novel male (Carr et al., 1980). Communication by Chemical Signals Sex identification based on chemical signals must rely on sexual differences in the composition of specific scents but information about the chemical basis of such differences is surprisingly limited. There are, however, a few cases in which active compounds have been characterized. In mice, two farnesene compounds (E,E-a-farnesene and E-b-farnesene) are characteristic compounds found in male preputial glands, and sexually experienced female mice are attracted to farnesenes in bladder urine or water but sexually naive females do not show attraction to these odors (Jemiolo et al., 1991). Recently, a novel compound was identified in male urine that does not occur in female urine, (methylthio)methanethiol. This compound is attractive to females, and may be important in the attraction of estrous females to adult males, but it is not known whether this compound is testosterone dependent (Lin et al., 2005). It is essential that we learn more about the chemistry of all social signals in order to understand this type of communication. This field is in desperate need of more chemists so that we can better understand communication by odors. 11.2.5.2 Advertising sexual receptivity: Females attracting males for mating and responses of males to female odors In many mammals, it is essential for females approaching estrus or in estrus to attract males for mating; one common method of attracting males is for females to begin to release odors from the body into the air and/or to deposit scent marks. Several mechanisms may be used for this purpose: the quantity of odors produced and released into the air may be increased, the frequency of scent marking may increase, the composition of odors may change with the female’s reproductive state, or a combination of these mechanisms may be employed. Note that with the first two mechanisms there need not be a change in the quality of the odor, but often both the quality and the quantity do change. 11.2.5.2(i) Changes in odor associated with sexual receptivity in females Metabolic products of sexual hormones may be contained in the female’s odors, so that odors can be a direct reflection of the reproductive state of the female and the attractiveness of female odors may also vary directly with female reproductive state. However, male preferences for odors of estrous females over odors of nonestrous females vary across species, and sometimes even within species. 405 A preference for odors of receptive females has been shown to develop or to increase compared to other reproductive states in house mice (Hayashi and Kimura, 1974), brown and collared lemmings (Huck and Banks, 1984), meadow voles (Ferkin and Johnston, 1995a), Indian desert gerbils, Meriones hurrianae (Kumari and Prakash, 1984), Mongolian gerbils (Block et al., 1981), woodrats, Neotoma lepida (Fleming et al., 1981), Columbian ground squirrels, Spermophilus columbianus (Harris and Murie, 1984), dogs (Beach and Gilmore, 1949; Dunbar, 1977), rams (Lindsay, 1965), and pygmy marmosets, Cebuella pygmaea (Converse et al., 1995). A lack of preference for odors of estrous females over odors of nonestrous females has been reported in deer mice (Dewsbury et al., 1986) and guinea pigs (Nyby, 1983). In some species, sexual experience is necessary for males to show preferences for the odors of estrous females over those of females in other reproductive states, for example, domestic rats (Carr et al., 1965). Not all sources of odor vary with sexual receptivity. Male Djungarian hamsters show a preference for the urine, saliva, and vaginal secretion of estrous females over similar odors of diestrous females, whereas a preference is not shown when males investigate other types of body odor, such as feces and midventral gland secretions, even though these odors are important in other aspects of communication (Lai et al., 1996). Different odors from the same female may be most attractive at different times during the estrous cycle. In a unique study on Djungarian hamsters, it was found that the attractiveness of three different odors of females varied across the estrous cycle but that each odor was maximally attractive at a different stage of the cycle (Lai and Johnston, 1994). This type of pattern is especially interesting because such a pattern might allow males to accurately track and predict the timing of estrus. In nature, female rodents and other mammals are not likely to go through repeated estrous cycles. Rather, they usually breed during one season and are infertile at other times of the year. In the case of rodents with short periods of pregnancy and lactation, females will quickly become pregnant again, that is, during the breeding season there are usually cycles of pregnancy, lactation, and sexual receptivity rather than repeated estrous cycles. In meadow voles, for example, females mate, become pregnant, and give birth; this is followed, within 12–48 h, by a postpartum estrus, so that females are then simultaneously pregnant and lactating. During the gestational period, males do not show a preference for 406 Communication by Chemical Signals the odors of pregnant females compared to parous females, that is, mature and capable of mating within a few hours after being paired with a male (Ferkin and Johnston, 1995a). Males do show a strong preference for the odors of a parous female over the odors of a female on the day of birth of her litter. Males, however, prefer the odors of estrous females on the following 2 days after parturition (postpartum estrus) over the odors of parous females. In contrast, throughout all of these times, females preferred the odors of a male over those of a parous female (Ferkin and Johnston, 1995a). When meadow voles were tested in short-day conditions, females showed a preference for the odors of other short-photoperiod females over those of males, probably associated with the habit of often nesting with other females in winter to maximize warmth (Ferkin et al., 1995). Short-photoperiod males, in contrast, did not show a preference between odors of short-photoperiod males and females. Furthermore, neither short-photoperiod males nor short-photoperiod females showed sexspecific preferences for long-photoperiod males or females, and long-photoperiod voles (both sexes) similarly did not show sex-specific preferences for short-photoperiod males or females (Ferkin et al., 1995). These and other related findings in meadow voles suggest that attraction and lack of attraction to odors of males and females may have multiple functions throughout the year that are not yet fully understood. The endocrine basis of some of these seasonal changes has been partially analyzed. During long photoperiods, the attractiveness of the male scent to females depends on high titers of both testosterone and prolactin (Ferkin et al., 1997a). We are not aware of any studies that have carried out a complete analysis of seasonal changes in odor preferences. 11.2.5.2(ii) results Methodological issues that influence In species that have been studied by many investigators, such as mice, rats, and hamsters, some studies report male preferences for estrous females (Carr et al., 1965; Johnston, 1980; Lydell and Doty, 1972), whereas other studies report the lack of such a preference (Brown, 1977; Kwan and Johnston, 1980; Landauer et al., 1978; Taylor and Dewsbury, 1990). Such differing results are probably due to differences in methodology (Taylor and Dewsbury, 1990). First, the type of method used is very important; different methods often yield different results and the best method may differ for different species. It is unfortunately rare for investigators to first determine the best method for use with a particular species. One species in which different methods were compared is the golden hamster. In one set of experiments with females, six different methods were investigated using measures of investigation of a single stimulus and five similar methods were used to measure preference for one of two odor stimuli ( Johnston, 1981a). Results showed that tests in the subject’s home cage or in a moving air stream were relatively poor; that is, few preferences were demonstrated. Tests in which the stimuli were presented in the center of a relatively large arena or in a chamber outside the arena that allowed odors into the arena, however, often yielded data that demonstrated differences in attraction. A second variable that may influence the results is the source of the odor used, such as soiled bedding, awake stimulus animals, anesthetized animals, or odors collected directly from the animal (e.g., urine, feces, vaginal secretions, preputial secretions). Neither sexually experienced nor inexperienced golden hamster males, for example, showed a preference for odors emanating from awake females in estrus versus diestrus ( Johnston, 1980; Landauer et al., 1978), but sexually experienced males did prefer the odors from the bedding material of females in estrus over those from females in diestrus ( Johnston, 1980). Sexually naive males also showed differences in flank-marking frequency on different days of the female’s estrous cycle in response to odors in a female’s home cage. Specifically, males flank marked more on diestrous days 1 and 2 than when the females were in estrus ( Johnston, 1980). Another variable that may be important is that some odors are relatively easy to collect whereas others are more difficult to collect and, in these latter cases, it is often difficult or impossible to quantify the amount of secretion collected. Thus, variability in results may be due to unknown differences in the quantity of a scent source that is available. Another variable that can influence results is whether or not the subject has contact with the odor source; many experiments do not allow contact with an odor or a stimulus animal (Taylor and Dewsbury, 1990). If contact is not allowed, the subject only has access to volatile components of the scent source. It has been clearly demonstrated, especially in mice, that nonvolatile chemicals are essential for communication of information. For example, the MUPs in mouse urine provide information about individual identity (Hurst et al., 2001). In addition, contact with the MUPs of a male is essential for a female Communication by Chemical Signals mouse to later demonstrate a preference for volatile odors from this same male (Ramm et al., 2008). A fourth variable is whether the animals providing odors in different reproductive states are in those states due to natural changes in hormones or these states have been produced by the experimenter (e.g., ovariectomized and then either given hormone therapy or not). Males of some species react to hormonally treated, ovariectomized females differently than they do to intact, naturally cycling, estrous females (Taylor and Dewsbury, 1990). Similarly, the hormones of males may differ depending on their age (juvenile or adult males) or recent experience, such as interactions with females or their odors or aggressive interactions with males or females (Bronson and Macmillan, 1983; Huhman et al., 1992, 2003; Lloyd, 1971; Macrides et al., 1974; Maruniak and Bronson, 1976; Pfeiffer and Johnston, 1992). Previous sexual experience or lack of such experience can also have significant effects on responses to odors. In some studies, sexually experienced males showed a preference for the odors of receptive females but sexually naive males did not (Carr et al., 1965; Hayashi and Kimura, 1974; Huck and Banks, 1984; Johnston, 1980; Lydell and Doty, 1972). In other studies, however, both sexually naive and sexually experienced males show a similar preference for odors of estrous females over odors of nonestrous females, for example, in male mice (Rose and Drickamer, 1975). Most of the methods mentioned above used some type of a measure of interest, such as duration of investigation or time spent near a stimulus. In a few studies, more direct measures of sexual interest or motivation were employed. In rats, for example, males were exposed to the odors of estrous or diestrous females and the frequency of erections was measured. More erections were elicited by exposing males to the odors of receptive females than to the odors of unreceptive females (Sachs, 1999). Virtually all of the studies mentioned above have used either a mixture of all of the odors emanating or deposited by a stimulus animal or just one odor source. Relatively few experiments have investigated the interaction of multiple odor sources on male sexual arousal and behavior or to responses of animals in other contexts. In golden hamsters, many studies have shown that vaginal secretions are a source of attractive and sexually arousing odors ( Johnston, 1977b, 1983; Macrides et al., 1974; Meredith, 1986; Murphy, 1973; O’Connell et al., 1979, 1981; Singer et al., 1976, 1986, 1983). However, little was known about the effects of other odors on 407 male sexual arousal. This was investigated by measuring copulatory performance in male hamsters toward receptive females and anesthetized females that had one or more specific odor sources removed. Elimination of vaginal secretions significantly reduced male copulatory behavior and removal of flank, ear, and Harderian secretions caused additional decreases in this behavior. Copulatory behavior was, however, not eliminated. These experiments demonstrate that at least four different odor sources have some influence on male sexual arousal and performance ( Johnston, 1986). 11.2.5.2(iii) receptivity Scent marking to advertise sexual Scent marking advertises sexual receptivity by an increased frequency of marking and/or by changes in the quality of the odor with reproductive state. By distributing an odor signal more widely, females increase the probability that males will encounter these marks. Females may also leave a trail of marks in the direction of their home or burrow, as described in captivity for golden hamsters in a semi-natural enclosure (Huck et al., 1985a; Lisk et al., 1983). Female plains garter snakes, Thamnophis radix, deposit a pheromone on vertical structures as they move, and males can use these marks to determine a female’s trail and eventually to locate her (Ford and Low, 1984). In some rodent species, females scent-mark more around estrus than in other reproductive states, suggesting that females advertise their reproductive state to surrounding males ( Johnston, 1983). Female golden hamsters mark at the highest levels during their active period 12–24 h before sexual receptivity begins. This strategy seems appropriate for a solitary species in which individuals are relatively widely dispersed. Females vaginal mark more in the presence of male odors than in the presence of female odors or clean areas ( Johnston, 1977a, 1979), and produce trails of vaginal marks toward their burrows (Lisk et al., 1983). Females also vaginal scent-mark more in the presence of odors of conspecific males than those of heterospecific males ( Johnston and Brenner, 1982). Vaginal scent marks of females have also been shown to be attractive to males from a distance (Kwan and Johnston, 1980) and to stimulate ultrasonic courtship calls by males ( Johnston and Kwan, 1984). These behaviors indicate that one function of vaginal marking is attracting males for mating. In large enclosures holding a female and three males, the female created a clear trail of vaginal secretion between the alpha-male’s burrow and her burrow and more diffuse trails with fewer scent marks between 408 Communication by Chemical Signals her burrow and the other two males’ burrows (Huck et al., 1986). In other species, the peak rate of marking occurs when females are in estrus. For example, female Indian desert gerbils scent-mark most frequently during estrus (Kumari and Prakash, 1981); since gerbils live in social groups, it may not be necessary to use scent marking to advertise well in advance of receptivity. In many large mammals (e.g., ungulates), information about estrus appears to be in the urine and/or in other secretions in the genital region, and females usually urinate more often when in estrus (Ewer, 1968). Males investigate genital odors and places where females urinate and, in many species, males engage in flehmen to promote access of the odors to the VNO (Estes, 1967; Ewer, 1968). In elephants, males also test urine deposits by placing the trunk over the urine and delivering a sample to the VNO by placing the tip of the trunk over the duct to the VNO and forcing chemicals into the organ. One chemical component of elephant urine that stimulates this sampling behavior and may also increase males’ sexual arousal is Z-7-dodencen-1-yl acetate (Rasmussen et al., 1993, 1996, 1997). It would be valuable to know more about advertisement of sexual receptivity within specific taxonomic groups in relation to social organization, mating system, and patterns of spatial distribution. Given the large variation in social organization, spacing patterns, and mating systems across mammals, it would be extremely interesting to discover if there were systematic differences between species that were related to social organization. 11.2.5.3 Male odors that attract females There are also species in which males produce odors that attract females. For example, female frogs of the genus Hymenochirus are attracted to secretions produced by male breeding glands (Pearl et al., 2000). Females are attracted to water that contained males, but not to water that contained females or males without breeding glands. Males are not attracted to any of these odors, indicating that the attractant is specific to females and it is not a general signal that promotes aggregation (Pearl et al., 2000). Sexually mature, terrestrial female toadlets, Pseudophryne bibronii, tested in a two-choice Y-maze, were attracted to the odors deposited by males compared to the clean arm of the Y-maze (Byrne and Keogh, 2007). Similarly, male red-bellied newts, Cynops pyrrhogaster, produce a peptide pheromone in a cloacal gland that has a female-attracting function (Kikuyama et al., 1995). In contrast, female palmate newts, Triturus helveticus, are equally attracted to male and female chemical cues (Secondi et al., 2005). In this species, males and females aggregate on breeding sites at high densities. Attraction to odors of conspecifics, irrespective of sex, may serve to locate a breeding site; short-range chemical or visual signals may be used to locate mates (Secondi et al., 2005). When male and female terrestrial salamanders, Plethodon jordani, have located one another, courtship starts. Males use a proteinaceous secretion from the mental gland to increase female receptivity and to ease insemination (Feldhoff et al., 1999). Such so-called courtship pheromones occur in many arthropod species, but among vertebrates they have only been found in salamanders (Feldhoff et al., 1999). 11.2.5.4 Mate evaluation and mate choice by females Sexual selection and parental investment theories predict that the sex with the biggest investment in gametes and young (females) will be the sex that is most demanding of high quality when choosing a mate and that males will be less choosy than females. There is abundant evidence that females carefully evaluate the relative quality of potential mates on the basis of male odors. Because odors may contain information about the physiological condition of an animal, they can be an honest source of information about the relative quality of different males. For example, male odors can contain information about differences in steroid levels, quality of the diet, and other features related to male phenotypic quality. Thus, a female may increase her reproductive success by choosing mates with odors indicative of male quality. For example, female brown lemmings show a preference for the odors of males that have high levels of circulating testosterone and/or males that are dominant and have larger testes and possibly larger sperm stores (Huck et al., 1981). Female pigs in estrus show a preference for intact males over castrated males but anestrous sows do not show such preferences (Signoret, 1976). Female mice (Brown, 1977) and golden hamsters ( Johnston, 1981a) also prefer the odors of intact males over those from castrated males. In nature, females would usually be confronted with adult males that varied more subtly in androgen levels than the difference between castrated and intact males or between juvenile and adult males. At least one study has addressed how females respond to males with graded levels of circulating testosterone. The responses of female meadow voles to the odors Communication by Chemical Signals of males show a dose-dependent response pattern: using five different dosages of testosterone in groups of castrated males, females showed stronger preferences for odors of males injected with higher doses of testosterone (Ferkin et al., 1994). Female meadow voles also prefer the odors of older males (Ferkin, 1999a) that tend to be heavier and have larger testes (delBarco-Trillo, personal observation). High testosterone levels suppress immune system function, so, from the perspective of sexual selection theory, when males have high testosterone and are also in good health, this advertises their genetic and phenotypic quality. In species that live in permanent social groups, there are often well-established dominance relationships between males in a local area and dominance rank is often related to the ability of one male to monopolize or have preferential access to resources. Females should prefer dominant males as mates because at least some of the qualities of dominant males may be based on genetic characteristics that may be passed on to offspring. If odor parameters (e.g., testosterone metabolites) are related to male dominance, females may be able to distinguish between dominant and subordinate males just by investigating their odors. Female brown lemmings show a preference for the odors of dominant males over the odors of subordinate males, even when the females are not familiar with the two male stimulus animals before testing (Huck and Banks, 1982; Huck et al., 1981). Female brown lemmings also preferred odors of socially naive males that later became dominant over the odors of socially naive males that later became subordinate, suggesting that the differences between males were at least partially determined by genetic differences (Huck et al., 1981). Female European rabbits also show a preference for the odors of dominant males (Engel, 1990). These results all indicate that there are some constituents of male odors that correlate with social status, possibly related to levels of testosterone, cortisol, or other stress-related hormones (Engel, 1990; Huck et al., 1981). High-quality males are also likely to defend a territory that contains better nesting habitat and higher-quality food resources and thus will consume a higher-quality diet than subordinate males. Females of several species have been shown to prefer males or the odors of males that have been feeding on a highquality diet compared to those on a lower-quality diet. Both male and female meadow voles are more attracted to odors of opposite-sex conspecifics that were fed a high-protein diet compared to those fed a diet containing the standard level of protein in 409 commercial rodent food or a low level of protein in the food (Ferkin et al., 1997b). Female meadow voles are also more attracted to odors of males consuming a normal lab diet or males that were food deprived for no more than 18 h compared to odors of males food deprived for 24 h (Pierce et al., 2005). Similarly, gravid female salamanders, Plethodon cinereus, preferred to associate with males that fed on a high-quality diet (termites) compared to a low-quality diet (ants) and were attracted to the feces of males on the high-quality diet ( Jaeger and Wise, 1991; Walls et al., 1989). Female swordtail fish, Xiphophorus birchmanni, also show a preference for odors of males that are well fed over the odors of males that were food deprived for 5 days (Fisher and Rosenthal, 2006). To the extent that a male can defend or obtain highquality food resources and to the extent that this ability is heritable, females who choose such males should have higher-quality offspring and greater reproductive success than females that do not discriminate between males on the basis of the diet they consume. In the quest for choosing the highest-quality males as mates, females may also use odors to assess the health status of males. In several species, females have been shown to be more attracted to the odors of unparasitized males over the odors of parasitized males. For example, female mice avoid the urine of males infected with lice, compared to the urine of males that do not have lice (Kavaliers et al., 2003). Female mice and rats also show a preference for the urine of males without endoparasites over the urine of males infected with several species of such parasites (Kavaliers et al., 1998; Willis and Poulin, 2000). In addition, female mice are as attracted to water as to the urine of males infected with the influenza virus, whereas the same females are more attracted to urine of uninfected males than to the urine of infected males, indicating directly that viral infection can reduce the attractiveness of male odors (Penn et al., 1998). Familiarity of a female with a male and/or his odors can also be a factor in mate choice. A female repeatedly exposed to odors of a male may show a preference for that male over a male that is unfamiliar, possibly because familiarity indicates the ability to defend a territory or living area for some period of time. Female pygmy loris, Nycticebus pygmaeus, in captivity were first exposed to urine marks from one male over 14–20 weeks. When the female was close to ovulation, males confined in small cages were placed in the much larger living areas of females. Using three different behavioral measures, females showed strong preferences for the familiar male (Fisher et al., 2003b). 410 Communication by Chemical Signals In another example, female collared lemmings were first housed with a male for 30 days and were then separated for 1, 12, or 24 days. Females showed a strong preference for their partners’ odors over the odors of an unfamiliar male 12 days after separation, but they did not show such a preference after 24 days of separation (Huck and Banks, 1979). Female rats also show a preference for the odors of a previous, familiar mate over the odors of a novel male. In contrast, male rats show a preference for the odors of a novel female over the odors of a previous mate (Carr et al., 1980). 11.2.5.4(i) MHC and mate choice As potential parents, all species should have evolved mechanisms to maximize their own reproductive success and the success of their young. In addition to choosing mates that are healthy, have good genes as measured by secondary sexual characteristics, symmetry, social status, etc., individuals should choose mates that will provide their young with greater resistance to parasites and disease organisms. A mechanism that allows such choice was discovered by chance in 1976. Informal observations of mating between mice of different MHC types suggested that mice prefer to mate with other mice that have a different MHC type than their own. Experiments showed that when a male was introduced into a cage with two females differing in their MHC type, males showed clear preferences for females with an MHC type different from their own by means of observing which females had vaginal plugs (Yamazaki et al., 1991, 1976). Subsequently, it was shown that mice can discriminate between the odors of other mice of different MHC types. Mice were successfully trained to discriminate between the odors of mice with different MHC types (Yamaguchi et al., 1981; Yamazaki et al., 1983, 1990, 1986). Indeed, highly trained mice could distinguish the body odors of two individuals that were genetically identical except for one allele that resulted in a difference in three amino acids in one MHC-dependent protein (Yamaguchi et al., 1981; Yamazaki et al., 1980). Such learned discriminations do not show, however, whether mice spontaneously notice the differences in odors of mice with different MHC types. Using standard habituation–dishabituation methods, it was shown that mice do spontaneously notice differences in the odors of mice with different MHC types (Penn and Potts, 1998b). Norway rats also discriminate body odors based on slight genetic differences in the MHC (Brown et al., 1987, 1989, 1990). These results demonstrated that genes in the MHC complex do influence body odors and that mice and rats can discriminate between the odors of individuals with different MHC types. Why should the MHC influence the individual body odors of mice? Since the MHC is the part of the genome that is involved in producing antigens that detect foreign tissue and pathogenic organisms in the body, the theory is that if the MHC influences body odor depending on the specific alleles in the genes of the MHC, an individual could maximize the diversity of the MHC genes in its offspring by choosing mates that have an odor that is different from their own. The greater the diversity of the MHC, the greater the ability that an individual has to combat a variety of pathogens. Other experiments suggest that the preferences of mice for mates with a different MHC type than their own is based on the differences in odors. In one ingenious series of experiments it was determined that infant mice have characteristic differences in odor based on their MHC type (Yamazaki et al., 1992) and that these fetal odor types are evident in the urine of pregnant females (Beauchamp et al., 1994). Males showed preferences for females that depended on the genotype of the developing fetus – they preferred pregnant females carrying developing young that were most genetically different from themselves (Beauchamp et al., 2000). All of the preceding results were carried out in carefully controlled laboratory experiments. Are mate choices influenced by MHC-determined odors in nature or in more naturalistic experiments? Although the evidence is limited, some studies suggest that MHC type does influence mate choice. In a study of mice in relatively large, naturalistic enclosures, it was shown that males established territories that included several females. Based on genetic analysis, there were 41% fewer MHC-homozygous offspring than expected if females mated only with their territorial male, indicating that females left their home territories to mate with other males with a different MHC type than their own (Potts et al., 1991). Results from natural populations yield mixed results. In one study of a free-living population of sheep, no evidence was found for selective mating with individuals with differing MHC types (e.g., nonrandom patterns of mating), but there was evidence for a role for MHC type in juvenile survival (Paterson and Pemberton, 1997; Paterson et al., 1998). Communication by Chemical Signals In stickleback fish, Gasterosteus aculeatus, the diversity of the MHC complex is related to parasite resistance and immunity (Kurtz et al., 2004). There is also suggestive evidence that the MHC could be involved in mate-choice decisions in humans. Several studies have shown that the odors of men were rated as more pleasant by women when the male scent donors had an MHC type different from themselves compared to men with MHC types similar to the woman’s MHC type (Eggert et al., 1999; Wedekind and Furi, 1997; Wedekind et al., 1995). One study also suggests that MHC type may influence actual choices of a marriage partner, based on historical and contemporary records in a Hutterite community (Ober et al., 1997). In addition, odors of men who show a high degree of symmetry (technically, low fluctuating asymmetry, i.e., symmetry in basic body structure) are more attractive to women than odors of men who are less symmetrical in basic body structure (Rikowski and Grammer, 1999; Thornhill and Gangestad, 1999). Theoretically, symmetry is an indication of developmental stability in the face of various challenges during development (disease, poor diet, stress, etc.), and thus symmetrical individuals are likely to have better genetic quality than individuals that are less symmetrical (Møller, 1997; Møller and Swaddle, 1997). In one study, symmetry in basic structural features (e.g., ankle width, elbow width, length and width of ear lobes) was first assessed in male volunteers. The attractiveness of photographs of the men’s faces and the odors of these men were assessed. To obtain odors, men wore T-shirts for three consecutive nights (Rikowski and Grammer, 1999). Separate groups of women rated photographs of the faces and the odors from the T-shirts. The authors found a positive correlation between both measures of attractiveness and symmetry, but only when women were in the fertile phase of the menstrual cycle (Rikowski and Grammer, 1999). Unlike the results for women, men did not find the scent of symmetrical women more attractive than those from less symmetrical women (Thornhill and Gangestad, 1999). This study suggests that women may obtain information about the phenotypic quality of men by investigating their odors and faces. This result is not surprising to those who have studied symmetry in animals, since in nonhumans it is usually males that show fluctuating asymmetry. However, the asymmetries that are usually seen in animals that are related to mate choice are mostly observed in sexually dimorphic traits and not in basic structural traits. 411 11.2.6 Odors, Intrasexual Competition, and Status 11.2.6.1 Role of odors in the formation and maintenance of status relationships The establishment of a dominant–subordinate relationship between two individuals is common in many species. Here we consider how the odors of individuals may function in the establishment and maintenance of such relationships. Normally, larger males are dominant to smaller males because size gives them an advantage during competitive displays and fights. However, other factors may also influence which male becomes dominant. Smaller male mice, for example, may initially become dominant over larger males by investing more in preputial glands and scent marking than larger males (Gosling et al., 2000). However, this strategy is energetically very costly – a high investment in scent marking leads to slower growth and lower adult body weight, so these small males are vulnerable to reversal of dominance status later in life (Gosling et al., 2000). Another factor that influences which individual becomes dominant in laboratory experiments is where the encounter takes place. If the encounter takes place in the territory or home cage of male A or in a neutral arena that contains odors of male A, that male is more likely to win the initial encounter and become dominant. The results of the initial encounter have a strong effect on latter encounters between the same two individuals, even if a later encounter is in a neutral arena (Lai et al., 2004; Lai and Johnston, 2002). The odors that a male normally deposits in his territory, home range, or in the vicinity of a burrow can serve as a confidence-building context in aggressive interactions. For example, male golden hamsters entered an enclosure with a longer latency when another male’s odors were present, compared to a clean area or an area scented by a flank-glandectomized male; subjects also spent less time in an arena scented by an intact male compared to a flank-glandectomized male (Alderson and Johnston, 1975). The effect of an own-odor or home-odor context should be maximal when the odor in one location is consistent with other information about location (familiar landmarks, etc.). In experiments in which agonistic interactions take place in an unfamiliar, neutral area, however, odors may take on an especially important role and give an advantage to the male whose odors are present. 412 Communication by Chemical Signals This effect has been shown in a variety of laboratory experiments. For example, when two European rabbits are placed in a neutral arena containing feces from one of the two contestants, the owner of the feces is more aggressive, initiates more interactions, and is more likely to win the contest (Mykytowycz, 1973). Thus, the factor determining dominance was not any intrinsic feature of the winner, but the fact of being in an environment containing the odors of one of the males. In house mice, an intruder is more submissive when there is a match between the odors on the substrate and the other individual (e.g., a resident), than when there is no match between the substrate odors and the other male (Gosling and McKay, 1990). When two female golden hamsters that are unfamiliar with each other are placed in a neutral arena scented with the vaginal secretion of one of the females, the female whose odor is in the arena is more likely to become dominant; subsequently the winner does all of the vaginal marking and most of the flank marking (Fischer and McQuiston, 1991). When two females with an established dominant–subordinate relationship are placed in a neutral arena scented with vaginal secretion from the subordinate female, a reversal in the dominance relationship was observed in 8 of 12 pairs (Fischer and McQuiston, 1991). After the establishment of a status relationship, there are usually differences between the dominant and subordinate individuals in how they respond to the odors of the other individual. For example, subordinate males may avoid odors of dominant males whereas dominant males usually do not avoid odors of subordinate males. Male cavies, Cavia aperea, that were dominant to one male and subordinate to another male spent more time in the side of an arena containing odors of the subordinate male than in the side containing odors of the dominant male (Martin and Beauchamp, 1982). A male golden hamster avoids odors of the male that beat him in a fight, but this subject does not avoid the odors of either a familiar male with whom he did not fight or an unfamiliar dominant male (Lai et al., 2004, 2005; Lai and Johnston, 2002; Petrulis et al., 2004). These latter results indicate that the identity of the individual and the memories associated with a specific individual are crucial factors that determine the type of response observed toward an individual’s odor. They also provide, perhaps for the first time in rodents, data indicating true individual recognition in an agonistic context that was not confounded by other possible explanations (Lai et al., 2005). On the other hand, reactions to odors of dominant or subordinate males may also be determined by information in the odor that identifies an animal as having a dominant status. Before the establishment of a polarized status relationship between two male snow voles, Chionomys nivalis, both males investigated areas scent-marked by the other male similarly. However, after the establishment of a dominant– subordinate relationship, the subordinate male investigated the area scented by the dominant male much less than the dominant male investigated the area scented by the subordinate male (Luque-Larena et al., 2002). These results can be explained either by individual recognition or by a reaction to information in the odors about status. Other results show that cues to status do influence the behavior of animals. For example, group-housed male mice avoid areas containing the urine of a dominant male, whereas urine of a subordinate male or water in the area results in much more time spent in the test chamber ( Jones and Nowell, 1973). Also, the urine of a dominant male spread on a castrated male triggers aggressive behavior in other conspecifics, whereas subordinate urine spread on a castrated male does not promote aggression in other conspecifics ( Jones and Nowell, 1973). Another aspect to consider is how odors are used to advertise a dominance relationship after this relationship is established. In many species, dominant individuals scent-mark more than subordinate individuals (Ralls, 1971). Some specific examples are house mice (Desjardins et al., 1973; Hurst, 1990b), golden hamsters (Ferris et al., 1987; Johnston, 1975a,b), Mongolian gerbils (Thiessen et al., 1971), stoats, Mustela erminea (Erlinge et al., 1982), European rabbits (Mykytowycz, 1965), sugar gliders, Petaurus breviceps (SchultzeWestrum, 1965, 1969), marmosets, Callithrix jacchus (Ralls, 1971), and ringtailed lemurs, Lemur catta (Kappeler, 1990). A larger number of marks deposited by one male may signal dominant status within the area that was marked (Gosling, 1990; Hurst, 1987; Hurst and Rich, 1999). 11.2.6.2 Scent marking in male–male competition for status Males in most species compete for sexual access to females and/or to prevent other males from gaining access to their mates. Scent marks are often used in such competitions. An advantage of assessing rivals via their odors is that dangerous physical interactions may be reduced or avoided. Males may recognize each other’s scent, and either withdraw or continue competing depending on the information obtained. Communication by Chemical Signals For example, after male snow voles investigated an arena containing the odors of another male, they behaved less aggressively toward that male (i.e., the resident male) than toward an unfamiliar male (Luque-Larena et al., 2001). Some lemur species, such as ringtailed lemur, spread odors on their tails and direct the odors toward competing males by waving their tails in the direction of their opponent, a behavioral interaction called a stink fight ( Jolly, 1966). Many ungulates mark or urinate as part of territorial and/or aggressive contests for dominance. Some species, such as the hartebeest, Alcelaphus buselaphus, perform scentmarking fights at territorial boundaries, in which two males scent-mark alternately and also attempt to remove the scent marks of their opponents (Gosling, 1982). In addition to scent marking in his territory, a male may rub his scent-gland secretions, urine, or other scents onto his own body; other males can then match the scent marks deposited in the territory with the male that deposited them. For example, territorial male hartebeest rub their antorbital gland onto their side, and also rub their body in their own feces (Gosling and Roberts, 2001). Blacktailed deer urinate on their hocks and then rub their hocks against one another (Müller-Schwarze, 1971). Many ungulate species produce wallows in which they urinate and then roll around in the wallow (Grau, 1976), for example, the American bison, Bison bison (Coppedge and Shaw, 2000). Capuchin monkeys (Cebus) urine wash their hands and then rub their hands up their flanks (Zeller, 1987). 11.2.6.3 Odors and sperm competition Sperm competition occurs when two or more males copulate with the same female and their sperm compete within the female’s reproductive tract for the fertilization of her ova (Parker, 1970). Males may change their sexual behavior (Stockley and Preston, 2004) or the number of sperm ejaculated (delBarcoTrillo and Ferkin, 2006) depending on the risk of sperm competition (the probability that a female will mate with other males) or the intensity of sperm competition (number of males mating with a female). Some studies have shown that males assess the risk and intensity of sperm competition by paying attention to the odors of other males on females or in the immediate environment of a female. For example, a ram will reduce his inter-ejaculatory interval when semen of another male is spread on the female’s vulva (Lezama et al., 2001). The ram’s own semen does not trigger the same response, suggesting that a male is 413 able to discriminate the odor of his own semen from the semen of other males or discriminate other odors that are transferred to females during mating. Reducing the inter-ejaculatory interval after detecting signs of a recent mating by another male may increase the ram’s own reproductive success, since, as the time between two mating events increases, the proportion of offspring fathered by the first male also increases (Huck et al., 1985b). A high risk of sperm competition can also be signaled by the presence of the odors of a conspecific male in the testing arena. A series of experiments investigated this possibility in meadow voles (delBarco-Trillo and Ferkin, 2004, 2006). Male voles mated either in a context with high risk of sperm competition (conveyed solely by the presence of odors of another male) or in a context with low risk of sperm competition (no odors of conspecifics present during the trial). When another male’s odors were present, male voles increased the number of sperm that they delivered to their mate (delBarco-Trillo and Ferkin, 2004). This increase was not due to any change in copulatory behavior (delBarco-Trillo and Ferkin, 2004, 2007b), which suggests that the odors of another male trigger a physiological response that ultimately produces an increase in the contractility of the cauda epididymidis and vas deferens. Even though it is not yet known whether this physiological response is caused by hormonal and/or neural mechanisms (delBarco-Trillo and Ferkin, 2005), there is evidence that some mechanism results in an increase in the number of sperm mobilized from the sperm reserves (epididymis) to the vas deferens prior to mating, which results in a larger number of sperm being ejaculated (delBarco-Trillo and Ferkin, 2007a). In addition, when a male vole mates while being exposed to the odors of many other males, he decreases the number of sperm ejaculated in comparison to when he is exposed to the odors of only one competing male (delBarco-Trillo and Ferkin, 2006). This result is in agreement with current spermcompetition theory – that is, as the number of competing males increases, the advantage of increasing the number of sperm decreases. Thus the best strategy is to decrease sperm investment in proportion to the number of other males or male odors detected in the immediate environment. The response of a male may depend on the possibility that surrounding males will compete. That is, if the odors of surrounding males come primarily from subordinate males that are not likely to mate, a subject male may respond differently than if those odors 414 Communication by Chemical Signals belong to a highly competitive male. For example, when a male meadow vole mates with a female in the presence of odors from an ad libitum-fed male, he increases the number of sperm ejaculated, but he will not do this in the presence of odors of a food-deprived male, indicating that subject males assign a lower competitive threat to odors of food-deprived males than to odors of ad libitum-fed males (Vaughn et al., 2008). Given that the risk of sperm competition is lower in unmated females than in recently mated females, males are generally expected to show a preference for unmated females. Indeed, in collared lemmings, sexually experienced males prefer the odors of an estrous unmated female over the odors of a female recently mated with another male, whereas sexually inexperienced males do not (Huck et al., 1984). Male golden hamsters, however, do not show a preference when exposed to unmated and mated estrous females ( Johnston and Rasmussen, 1984). In promiscuous species, males that mate with several females may become sperm-depleted, and females mating with such males may incur a reproductive cost (Wedell et al., 2002). If females are able to discriminate between recently mated and unmated males, they may reduce the cost of mating with a sperm-depleted male by avoiding mating with recently mated males. Female rats, for example, prefer the odors of males that have not copulated over those of males that have recently mated with another female (Krames and Mastromatteo, 1973). 11.2.7 Scent Marking and Scent Over-Marking: Aspects of Competition for Mates and Other Resources Scent glands and associated scent-marking behaviors are nearly ubiquitous among terrestrial mammals. Although mammals leave distinctive odors in the environment when they urinate and defecate, most mammals also have specialized scent-marking behaviors that have evolved to distribute scent-gland secretions, urine, and feces. Scent marks have both advantages and disadvantages as a means of signaling. The most obvious disadvantage is that regular marking in a territory, home range, or near a burrow or nest can alert predators to the existence of prey in a particular area. If predators are sensitive to the freshness of scent marks, marking may be particularly dangerous. Nonetheless, there has been little research on this topic and the only examples we are aware of in which mammalian scent marks are known to be used by predators are the urine marks of voles that reflect ultraviolet light. This visual information is detected by kestrels and used when searching for prey (Viitala et al., 1995). The lack of knowledge about use of odors by predators is no doubt due to the difficulty of studying predation, but it might not be difficult to carry out experiments with confined animals. There are several advantages of scent marking as a means of communication compared to communication by other types of signals. In many species, marking appears to be a low-cost activity because individuals mark in the course of normal daily activities and they may not carry out separate scent-marking patrols. Another advantage of scent marking is that in some environments (especially dry environments) the signals may last for a long time and may not need frequent replenishing ( Johnston and Lee, 1976; Roberts, 1998). This is especially true if proteins or other large molecules are present that can bind smaller molecules and release them slowly into the environment (Hurst et al., 1998). On the other hand, scent marking can be relatively costly in some species and situations. For example, in species with relatively large territories and a high degree of competition for territories, individuals may make regular patrols for the purposes of marking borders (Gorman, 1990; Gorman and Mills, 1984). Some patterns of scent marking may also be inherently costly. Male house mice that live in dense populations urine mark virtually all surfaces within a territory (Hurst, 1987, 1990a). Such patterns of marking are likely to be expensive, possibly in the energy expenditure to physically distribute marks in such a dense pattern and perhaps in water loss. In addition, mouse urine marks contain relatively high concentrations of major urinary proteins (MUPs), and production of these proteins may be metabolically costly (Nevison et al., 2003). Another situation in which marking may incur high costs are when males are engaged in overmarking competitions for status and/or advertising their vigor to females (see below). One type of scent marking that is particularly interesting is scent over-marking, in which one animal marks on top of the scent previously deposited by other members of the same species. This usually appears to be a means of indirect competition by which individuals (particularly males) advertise their own vigor and persistence without having to engage in actual aggressive interactions. At least some species appear to have evolved special perceptual mechanisms for analyzing and interpreting such over-marks ( Johnston, 1999, 2003, 2005; Johnston and Bhorade, 1998). Communication by Chemical Signals 11.2.7.1 Functions and causation of scent marking As in any other type of communication, scent marking has a variety of functions. One type of scent marking may have several functions that differ depending on the context (Brown and Macdonald, 1985; Ralls, 1971). These functions include (1) delimitation of territorial boundaries, core areas, or home ranges; (2) advertisement of presence in an area and/or dominant status; (3) use as beacons or landmarks for orientation; (4) defense of resources other than territory, such as food resources, home burrow and/or nest, and food hoard; and (5) to attract mates or advertise to potential mates. An individual may use different types of scent marking for different purposes. For example, female golden hamsters show agonistic motivation and competitiveness toward other females and males by flank marking, but vaginal-mark to attract mates ( Johnston, 1977a, 1979; Johnston and Kwan, 1984). Despite a variety of functions, scent marking is ultimately related to reproductive success and thus is shaped by selection for survival and sexual selection for reproductive success. Scent glands and scent marking are usually sexually dimorphic, and scent marking is usually dependent on, or influenced by, sex steroids ( Johnston, 1981b). In addition, scent marking is normally exhibited to its greatest extent during the reproductive season. Scent marks are usually not passively deposited (although there are exceptions, such as interdigital glands). Instead, animals actively deposit odors, often in prominent locations and/or by visually distinctive behaviors. Scent marking is usually stimulated by the odors of conspecifics or the presence of these individuals. Female rats urine-mark more in an area containing the odors of a sexually experienced male than in an area containing the odors of a sexually inexperienced or castrated male (Taylor et al., 1983). Male golden hamsters flank-mark much more frequently in response to conspecific males than to males of a closely related species, and similarly, females vaginalmark to attract mates more frequently in response to conspecific, than to heterospecific, males ( Johnston and Brenner, 1982). In most species, dominant individuals scent-mark more than subordinate individuals (Ralls, 1971). Male gerbils that scent-mark more than average are likely to become socially dominant (Ralls, 1971), indicating that there is a tight link between scent-marking rate and potential dominance status. Dominant stoats mark more than subordinates, especially in the presence of subordinates, whereas subordinate stoats mark less in the 415 presence of dominant individuals than when they are by themselves (Erlinge et al., 1982). In species that live in social groups, the alpha male normally scent-marks more than other males in the group. Some examples are the red-fronted lemur, Eulemur fulvus rufus (Ostner and Kappeler, 1998), ringtailed lemur (Kappeler, 1990), golden lion tamarins, Leontopithecus rosalia (Miller et al., 2003), wolves (Ryon and Brown, 1990), capybaras, Hydrochaeris hydrochaeris (Herrera and Macdonald, 1994), European rabbit (Mykytowycz, 1965), Mongolian gerbils (Shimozuru et al., 2006), house mice (Desjardins et al., 1973), African dwarf mongoose, Helogale undulata rufula (Rasa, 1973), and hyenas (Gorman and Mills, 1984). Scent marking may also occur during agonistic encounters. For example, flank marking in golden hamsters is associated with agonistic motivation. Males flank-mark in response to other males and diestrous females (both of which will behave aggressively), whereas males do not flank-mark in response to estrous females (which do not behave aggressively). Dominant hamsters of both sexes flank-mark more than subordinate hamsters and diestrous females flank-mark more than estrous females. In European rabbits, some scent-marking behaviors, such as paw scraping and latrine depositions, are frequently preceded by agonistic interactions (Bell, 1980). Blacktailed deer urinate on and rub their rear legs together during aggressive encounters (Müller-Schwarze, 1971). Similarly, bison urinate in wet, muddy areas and roll around (wallow) in this area, becoming covered in smelly mud (Coppedge and Shaw, 2000). The most common types of scent marking are urine marking and rubbing a scent gland against a substrate. However, there are other types of scent marking. One unusual type of scent marking is ball marking in sand rats (Psammomys obesus); sand rats urinate on sand and make a ball, which is then investigated by conspecifics. Male sand rats are particularly interested in investigating scented balls made by estrous females (Daly and Daly, 1975). Another unusual type of scent marking is carried out by dominant male hippos, who use their small tail as a fan to spray their semi-liquid feces on the vegetation and river banks to advertise their status and territories. There are a variety of patterns of spatial distribution of scent marks, and these patterns depend on a number of variables, including the size of the territory or home range, the pattern of use of this space, the type of social organization, the number of animals in the group, and many other factors. Animals may 416 Communication by Chemical Signals mark primarily near the boundaries of a territory, in the core area of use, along trails, at the entrance or along tunnels in burrow systems, etc. One species that marks most in the center of their territory is Thomson’s gazelle, Gazella thomsonii (Walther, 1964). Scent marking by wolves and coyotes has been carefully studied in nature (Allen et al., 1999; Peters and Mech, 1975). Both species have relatively exclusive territories with little overlap or intrusion of outsiders into territories, and they have similar patterns of marking. A predominance of scent marks (raised-leg urination) occurs at or near boundaries between territories and lower levels of marking occur within territories, especially along often-used trails. Another species that shows a similar pattern in nature is a marmoset, Callithrix humeralifer. These animals scent-mark in the periphery of the territory more than in the central areas of the territory (Rylands, 1990). Meadow voles, in contrast, preferentially make use of trails through dense vegetation and they scentmark in prominent locations along these trails. Yet another pattern is displayed by house mice, in which males saturate their territories with urine marks at least in cases of high-density populations with relatively small territories (Desjardins et al., 1973; Hurst, 1987, 1990a). Brown hyenas use an anal gland for one form of scent marking, called pasting, and concentrate these marks in the core area where they spend most of their time. In contrast, other depositions of urine and feces, called latrines, are placed near, but not at, territorial boundaries (Gorman, 1990; Gorman and Mills, 1984). Golden lion tamarins primarily mark fruit trees that they use as food sources, which may help them relocate these food sources (Miller et al. 2003). Alternatively, it could be that they mark these trees to indicate ownership of these resources. Scent marking may also provide honest signals of health and lack of infection. Male house mice that have been infected by a nonreplicating form of Salmonella enterica scent-mark at significantly lower levels than control males, and the scent that is deposited in these scent marks is less attractive to females compared to scent marks of control males that had a sham infection (Zala et al., 2004). 11.2.7.1(i) Counter-marking Counter-marking is the general term to indicate scent-marking in which an individual scent-marks on top of (over-marks) or close to (adjacent-marks) the scent marks of a conspecific that were previously deposited. Counter-marking occurs in a wide range of species (Brown and Macdonald, 1985; Ewer, 1968; Ferkin, 1999b; Fisher et al., 2003a). The major hypothesis for the function of over-marking is that it is a form of indirect competition (i.e., competition without fighting) between individuals of the same sex (Johnston, 1999). The immediate goal is for an individual to advertise its presence, status, or quality by keeping his/her scent marks on top most of the time. This strategy could be a straightforward measure of an individual’s vigor and persistence, especially since other same-sex individuals are usually trying to do the same thing. Success at over-marking may thus be an honest indicator of an individual’s vigor and quality. This hypothesis depends, however, on the ability of other individuals to determine which individual has the top scent marks and which individuals have underlying marks. One of the most surprising findings having to do with scent marking is that many experiments have shown that at least several species, including hamsters, voles, and the pigmy loris, can determine which individuals have their scent on top in an over-mark. After investigating scent over-marks, both hamsters and voles have a preferential memory of the top scent and behave as if they do not remember the bottom scent and/or value it less than the top scent (Ferkin, 1999b, 1999; Fisher et al., 2003a; Johnston and Bhorade, 1998; Johnston et al., 1994, 1995, 1997a,b). Preferential memory for the top scent in an overmark occurs when the scent marks are deposited in a wide range of configurations of the top and bottom marks (e.g., an L-shaped pattern with overlap in the corner, a pattern in the shape of a cross, a small spot on top of a large area of scent, etc. ( Johnston, 1999; Johnston et al., 1997a,b). Results indicate that when an individual over-marks the scent mark of another individual, the over-mark causes the bottom scent to have less importance to an individual that investigates the over-mark ( Johnston, 1999, 2003, 2005). The perceptual mechanisms involved in determining which scent is on top are not known. Since animals can determine which individual’s scent is on top when the odors are in a wide array of patterns, this suggests that there may be several perceptual mechanisms involved. Some obvious, possible mechanisms have been ruled out. For example, the relative freshness or age of marks from two individuals does not, by itself, result in preferential memory of one individual or preferences for one individual (Cohen et al., 2001; Ferkin et al., 1999; Johnston and Bhorade, 1998). The proposed function for over-marking as a means of advertising male vigor and quality is Communication by Chemical Signals supported by experiments in meadow voles, pigmy loris, and golden hamster. After females of these species investigate an area containing over-marks, they prefer the odor of the top-scent male (Ferkin et al., 2001, 2003a; Johnston et al., 1997a,b). These results indicate the existence of amazing and previously unsuspected perceptual mechanisms for the analysis of complex patterns of scent marks. Regardless of the perceptual and neural processes that are taking place, it is clear that after meadow voles have investigated a wide range of types of over-marks, they preferentially remember the top scent and also they prefer this odor over that of the bottom-scent donor. In addition, if the subjects are female meadow voles and the scent donors are male, the female subjects also display a preference for odors of the top-scent male ( Johnston, 1995; Johnston et al., 1997a,b). Similar preferences have been demonstrated by female pigmy loris (Fisher et al., 2003a) and female hamsters (Johnston, Song, and Gattermann, in preparation). Female meadow voles also demonstrate a preference for the male that has a greater number of over-marks compared to a male with fewer over-marks (Ferkin et al., 2005). This ability was observed with a range of differences in the number of over-marks (from 7:0 to 4:3), suggesting that voles can determine small differences in numerosity and can make fine distinctions in the relative number of over-marks produced by two males (Ferkin et al., 2005). These results again support the general hypothesis that animals evaluate the quality of other individuals based on the performance of courtship displays and competitions, only in this case, direct interaction between males is not necessary. Rather, some species have evolved amazing and highly sensitive mechanisms for evaluating the quality of potential mates based on their scent-marking performance. Additional support for the hypothesis that overmarking is a competitive behavior is the result that dominant individuals over-mark scents by subordinates much more frequently than subordinates overmark scents of dominant individuals. In addition, individuals over-mark odors of unfamiliar, nonrelated individuals more than odors of siblings (Ferkin, 1999b). These results also suggest the evolution of specialized, higher-order mechanisms in the analysis of scent marks. It is not at all clear how hamsters and voles can determine which of two individuals’ scent is on top and do so when the marks are in a variety of spatial configurations (Ferkin et al., 1999, 2005; Johnston, 1999, 2005; Johnston and Bhorade, 1998; Johnston 417 et al., 1997a,b). Three examples illustrate the problems involved in discrimination of the relative position (top or bottom) of two individuals. A particular problem is that the identity of an individual is coded in the relative proportions of chemical compounds in each individual’s scent, yet when scent marks overlap, the scents of the two individuals may become mixed together. In experiments on this topic, experimenters often collect the scent and make scent marks in specific configurations. One example is that of two individuals’ scents that are deposited such that the two scents form an L. In this case one could hypothesize that the odor that is perceived from the region of overlap of the two scents is more similar to the adjacent odor of the top scent than the adjacent odor of the bottom scent because the top scent probably masks some of the bottom scent. On the other hand, in the case of a small spot of scent placed on top of a larger area of scent, the perceiver cannot compare the over-marked region to the two pure scents, and thus it is not clear how voles can determine top scent. One might expect that females investigating a small spot of scent from one male on a field of scent from another male might prefer the scent of the male that was in the greatest quantity, but in fact they prefer the male that deposited the small, top scent ( Johnston, 1999, 2005; Johnston et al., 1997a,b). 11.2.7.1(ii) Allomarking Allomarking refers to one animal marking other individuals of the same species. This kind of marking may have a variety of functions, although it has not been thoroughly evaluated in any species. It may have a sexual function. In some rabbits and hares, males spray urine on the female, often while leaping in the air, sometimes leaping over the female; this type of behavior was originally termed harnspritzen or enurination (Ewer, 1968). Examples occur in lagomorphs, hystricomorphs, and South American rodents, for example, mara, Dolichotis patagonum, and green acouchi, Myoprocta pratti (Kleiman, 1971). Enurination may be part of courtship or may be a form of mate guarding. Allomarking of individuals within a group may help to produce a group odor and thus help individuals to recognize others in the group, promote group cohesion, or reduce intragroup competition. Group cohesion seems to be the main function of allomarking in European badgers, where all members within a group are involved in mutual allomarking (Buesching et al., 2003). Allomarking may also be used in courtship or to maintain a bond between a male and a female. For example, pairs of 418 Communication by Chemical Signals Maxwell’s duikers often rub their anteorbital glands together; this behavior is not observed between the male and other females (Leuthold, 1977; Ralls, 1971). European rabbits live in warrens containing a number of individuals and the dominant male often marks other individuals by rubbing them with the chin gland and/or spraying them with urine. If an individual is removed from the group and marked with urine from another group, this animal is attacked when it is returned to its native group; a mother will even attack her offspring if it has been marked with urine from another group (Mykytowycz, 1968, 1970). Thus, allomarking in these species serves to identify group members. 11.2.7.2 Hormonal control of scent-marking behavior Male scent-marking behavior is generally stimulated by androgens and by odors of other males and, in some species, by odors of females as well (Yahr, 1983). In adult male hamsters, castration reduces scent marking toward cues from other males and replacement therapy restores marking (Albers et al., 2002; Johnston, 1981b; Vandenbergh, 1973). Similarly, urine marking by male house mice and midventral gland marking by Mongolian gerbils is controlled by androgen levels (Thiessen et al., 1968, 1970) and these effects are due to the action of testosterone in the preoptic area (POA) of the hypothalamus and especially the sexually dimorphic area in the posterior part of the POA (Yahr and Stephens, 1987). Androgen influences on male scent marking in many large mammals is also suggested by the correlation of increased scent marking with the breeding season (Grau, 1976). In golden hamsters, further neural mechanisms underlying the control of flank marking have been thoroughly investigated and involve the action of arginine vasopressin (AVP) acting on areas in the hypothalamus. Microinjection of AVP into the medial and lateral aspects of the posterior hypothalamus is effective in stimulating an immediate bout of intense flank marking (Albers et al., 2002). The mechanisms underlying scent marking by female mammals is not as easily characterized. In female golden hamsters, the rate of flank marking is controlled both by hormone levels and the odors in the environment. Flank marking varies regularly with the estrous cycle in an arena that does not contain hamster odors and in an arena that contains male odors; in both cases flank-marking rates are high on nonestrous days and low when females are in estrus, a reflection of low levels of agonistic motivation when they are in estrus and higher levels of such motivation on other days of the cycle. In an area containing another female’s odor, however, the rate of flank marking is high across all days of the estrous cycle, reflecting high levels of agonistic motivation stimulated by another female’s odors ( Johnston, 1972, 1977a, 1979). Vaginal scent marking by female hamsters is related to advertising reproductive receptivity. When females are experiencing regular estrous cycles, vaginal marking is highest during the active (dark) period starting 24 h before receptivity. The lowest rate of vaginal marking is on the day after receptivity; marking occurs at intermediate levels on the other 2 days of the cycle. In addition, vaginal scent marking is very low during pregnancy and early lactation but begins to increase to high levels in late lactation, near weaning, and the reemergence of estrous cycles ( Johnston, 1979). Patterns of flank and vaginal marking similar to those described were also obtained in semi-natural enclosures that housed one male and female hamster (Takahashi and Lisk, 1983). The hormonal basis of vaginal scent marking by female hamsters has also been investigated. Estrogen implants into the ventromedial hypothalamus facilitated vaginal marking and a decrease in agonistic behavior. Similar implants into the anterior hypothalamus had no effect (Takahashi and Lisk, 1985). Female Mongolian gerbils, like males, scent-mark with the midventral gland, and this marking behavior is stimulated by ovarian steroids. In ovariectomized gerbils, sequential injections of estradiol benzoate and progesterone stimulated marking. There was, however, considerable variation in the responses of females to hormone treatments (Yahr and Thiessen, 1975). This may be related to different functions of marking in males and females. In males, this marking appears to be related to agonistic motivation and defense of a home area, whereas females use the midventral gland to mark their young and may use this scent to facilitate retrieval of pups (Wallace et al., 1973; Yahr and Thiessen, 1975). 11.2.7.3 Costs of scent marking A main cost of scent marking is eavesdropping by predators and parasites, which can use scents left by the prey/host to find such prey/host. Predators, such as raptors, may visualize scent marks during flight and search for prey around scented areas (Viitala et al., 1995). Mammalian predators can track scent marks using their olfactory system (Koivula and Korpimaki, 2001). Because scent-marking animals Communication by Chemical Signals carry their scents with them and the animal itself can be much smellier than the scent marks, predators and parasites may follow a scent gradient, where the smelliest point is the prey itself. Also, because scents are species specific, predators can avoid nonprey animals and focus on their chosen prey. Because there is information in odors about the health of animals (Zala et al., 2004), predators may use such information to pinpoint and find easier prey. However, it is not clear how discriminative predators are toward scent marks of different categories of a prey species. Weasels, Mustela nivalis, have been shown to prefer odors of estrous deer-mice females over odors of diestrous females (Cushing, 1985). However, least weasels, Mustela nivalis nivalis, offer contradictory results (Ylonen et al., 2003). Even though least weasels show a preference for bank or field vole odors over control odors in a Y-maze, they do not show a preference for dominant or subordinate males, nor between postpartum estrous or pregnant/lactating females (Ylonen et al., 2003). If predators track scent marks of prey, it is also possible that prey adjust their scent marking depending on the presence of predators. Indeed, outbred laboratory mice reduced their interest in over-marking conspecific scent marks in the presence of ferret urine, but not in the absence of ferret urine or in the presence of the scent of a nonpredator species (naked mole rat, Heterocephalus glaber; Roberts et al., 2001). Interestingly, only mice that usually mark at a high rate reduced their over-marking in the presence of ferret urine, whereas mice that usually mark at a low rate actually increased their over-marking, suggesting that subordinate males may take advantage of riskier situations (Roberts et al., 2001). In another experiment in which large, unfenced plots were treated with either vole scent or water, it was found that predation was higher in the scented plots, supporting the idea that scent marking carries a predation cost (Koivula and Korpimaki, 2001). However, neither the density nor the mobility of voles decreased in the scented plots (Koivula and Korpimaki, 2001). There may also be energetic costs associated with the production of some components of scent marks, such as the MUPs. Also, investing in large glands may result in slower growth rates. For example, in house mice there is a negative correlation between scentmarking rate and growth in young mice, suggesting a tradeoff between the level of scent marking and growth, and/or scent-gland investment and growth (Gosling et al., 2000). However, a later study did not find evidence of a tradeoff between higher scent 419 marking or MUP excretion and rate of body growth (Malone et al., 2005). 11.2.8 Odors and Aggression Agonistic interactions are normally preceded by sniffing the other animal, suggesting that information gained through olfactory investigation may determine or influence the behavior that follows. Dominant individuals often have larger scent glands and/or may produce more glandular secretion (Mykytowycz, 1968, 1970). In addition, odors of intact males and/or dominant males may have different chemical constituents that are characteristic of dominant status. Other conspecifics may use this information to avoid encounters with aggressive individuals or to determine the likelihood of winning an aggressive encounter with such individuals and adjust their behavior accordingly. Scent glands are sometimes the actual target for aggressive attacks, indicating that the odors from the glands may be a stimulus that elicits aggression. Male montane voles, Microtus montanus, for example, direct their attacks predominantly against the flank glands of other individuals. When male stimulus animals had their scent glands unilaterally removed, resident males directed more attacks to the side with the intact scent gland than to the side with the scent gland removed, indicating that the odors produced by the scent gland trigger aggression in other males ( Jannett, 1981). Similarly, when the urine of intact male mice was spread on individuals that do not usually elicit aggression (juveniles, castrated males, and females), increased aggressive attacks were elicited, indicating that the urine of intact males triggers aggression in other males (Chamero et al., 2007; Connor, 1972; Mugford and Nowell, 1970). In contrast, some odors may actually reduce aggressive responses by stimulating other behaviors or motivational systems. For example, odors of female mice reduce male–male aggression (Brown, 1985b; Mugford and Nowell, 1970). Intruder males swabbed with female urine were not attacked by other males, whereas intruder males swabbed with saline were attacked by other males (Connor, 1972). Male hamsters scented with vaginal secretion also elicit less aggression from other males (Murphy, 1973). When male hamsters are tested daily for flank marking in a female’s cage, the lowest rates of marking occur on the female’s estrous day, when females are most likely to extrude vaginal secretions, suggesting that vaginal secretions may inhibit male flank marking ( Johnston, 1975a,c, 1980, 1986). 420 Communication by Chemical Signals Scent marking may also occur during agonistic encounters. For example, male hamsters flank-mark in response to sniffing other males and diestrous females (both of which will behave aggressively), whereas males do not flank-mark in response to estrous females (which do not behave aggressively). Dominant hamsters of both sexes flank-mark more than subordinates and diestrous females flank-mark more than estrous females (Johnston, 1972, 1975a,b, 1977a). In European rabbits, some scent-marking behaviors, such as paw scraping and latrine depositions, are frequently preceded by agonistic interactions (Bell, 1980). Black-tailed deer urinate on and rub their rear legs together during aggressive encounters (Müller-Schwarze, 1971). 11.3 Roles of Odors in Modulating Hormones in Vertebrates In many species, odors cause hormonal changes in conspecific receivers. The term primer pheromones was originally coined to indicate substances that influence physiological processes, but it is more appropriate to say that these are primer effects because a particular odor that influences the hormonal status of other animals may also have a variety of other effects (e.g., influences on behavior). Such primer effects can also influence behavior due to a change in hormone level or to a sequence of changes in hormone levels. Among mammals, primer effects were first discovered in house mice (Bronson, 1968, 1971; Vandenbergh, 1967, 1969; Whitten, 1956, 1959, 1966). These effects included acceleration and inhibition of reproductive maturation, stimulation of hormone release, disruption of pregnancy, and acceleration or retardation of the estrous cycle of females. In general, odors of one sex positively affect opposite-sex individuals, whereas odors of same-sex individuals usually have a negative or suppressive effect on hormone levels (Brown, 1985a; Koyama, 2004). For example, female odors cause pseudopregnancy or longer estrous cycles in other females (Lee-Boot effect). Female odors increase sperm density in male mice, but disrupt spermatogenesis in bank voles, Clethrionomys glareolus (Koyama, 2004). Male odors may induce female estrus and ovulation (Whitten effect), and may accelerate female puberty (Vandenbergh effect). Odors of dominant males are more efficient at accelerating puberty than odors of subordinate males (Lombardi and Vandenbergh, 1977). An amount as small as 0.0001 ml of male urine has been shown to accelerate puberty in female mice (Vandenbergh and Coppola, 1986). Odors of an unfamiliar male also block pregnancy (Bruce effect). For this early literature, see reviews by Bronson and Macmillan (1983), Vandenbergh (1983b), and Whitten (1966). Male odors can also have negative effects on other males. For example, adult male odors disrupt spermatogenesis in juvenile house mice (Koyama, 2004) and can also retard growth of the testes and seminal vesicles in male deer mice (Lawton and Whitsett, 1979). Some of these primer effects can have important consequences for social behavior and reproductive success. In house mice, puberty can be delayed in offspring by the odors of the parents (Lepri and Vandenbergh, 1986). Such reproductive suppression may reduce the likelihood of reproduction between the parents and their offspring and thus avoid the costs of inbreeding. Reproductive suppression is more likely to occur in species in which populations are dense and availability of territories is limited, and thus offspring are likely to remain close to their parents for a longer time. In contrast, in those species with high levels of dispersal and/or low population density, reproductive suppression may not occur (Lepri and Vandenbergh, 1986). These early discoveries with mice were exciting and stimulated additional research in mice (Koyama 2004) and in other species (Albone, 1984; Brown and Macdonald, 1985; Doty, 1976; Vandenbergh, 1983b). In some ways it is odd that mice have continued to be an important model species in this field, since the magnitude of the effects in house mice tend to be much smaller in magnitude than the effects in other species. For example, the effects on acceleration or deceleration of the estrous cycle in mice tend to be on the order of a day and acceleration or deceleration of puberty is on the order of about a week. In contrast, the effects in microtine rodents are much more robust. Female prairie voles, Microtus ochrogaster, for example, do not show spontaneous estrous cycles; under light cycles typical of the breeding season, adult female voles housed alone or in female groups remain in an anestrous state with an imperforate vagina. Exposure adjacent to males, odors of males, or actual contact with males is sufficient to induce estrus in females. Other changes in their living conditions have much less of an effect (Richmond and Stehn, 1976). Female prairie voles lacking olfactory bulbs maintain anestrous vaginal smears and imperforate vaginas when they are housed adjacent to males. Furthermore, a male living with a female and Communication by Chemical Signals her litter, whether he is the father of the litter or not, does not have an estrus-inducing effect on the daughters of the litter (Richmond and Stehn, 1976). In house mice, females that mate generally become pregnant, although exposure to a strange male or his odors can induce failure of implantation into the uterus and thus a pregnancy block occurs. In prairie voles, the father must stay with the female for at least 4 days in order for her to maintain her pregnancy (Richmond and Stehn, 1976). Among different species of hamsters a wide range of social effects on puberty are found. In golden hamsters, there was no effect of housing weanling female pups alone or with littermates on several measures of sexual maturation (Levin and Johnston, 1986). In contrast, in Djungarian hamsters, a somewhat more social species in that males and females cooperate in raising young, there were dramatic effects. Housing females at weaning with an adult male accelerated uterine and ovarian development, whereas castration of the male eliminated this effect (Levin and Johnston, 1986; Reasner and Johnston, 1988). Young Djungarian females housed with an adult male at weaning began estrous cycles 8.5 days after weaning, those housed alone or with the mother and siblings began cycling 26 days later, and those housed with a female sibling matured 38 days after weaning (Levin and Johnston, 1986). In another study with this species, weanling females (18 days of age) were housed either alone, with an adult male, or with a littermate sister. Similar results were obtained – using 50% of a group of females ovulating as a criterion of sexual maturity, this criterion was reached at 8 days postweaning when females were housed with a male, 24 days postweaning when housed alone, and 38 days postweaning when housed with a sister (Gudermuth, 1989; Gudermuth et al., 1992). Hormone levels and vaginal cytology were also affected. Furthermore, there were effects of living conditions on the survival of pups after these females had given birth and were living with their own litter (Gudermuth et al., 1992). The golden hamster is strictly solitary and juveniles disperse from the natal burrow relatively early in life (about 20–25 days of age; Johnston, observations of golden hamsters in southern Turkey 2005–07). The Djungarian hamster, however, is more gregarious. In an experiment in which this species was housed in semi-natural enclosures in the lab, and in which young females could move freely between areas but adults could not, female pups preferred to live at home with their parents for several months rather than leave and join an adult male (Gudermuth, 1989). 421 One of the most dramatic examples of suppression of reproductive function by odors, however, occurs in a primate. In social groups of a species of marmoset, Callithrix jacchus, there is one dominant female and several subordinate females. Odor signals appear to be important in maintaining the status quo. If the dominant female is removed from the group, one of the other females will eventually become the new breeding female (Abbott, 1984). However, if the odors of the original dominant female are regularly introduced into the living area of the group, the subordinate females remain in a suppressed state of gonadal functioning (Barrett et al., 1990, 1993). Most cases in which reproductive function is accelerated are caused by stimuli from the opposite sex, whereas inhibitory effects are primarily from individuals of the same sex. There are, however, exceptions to this rule. In the California vole, Microtus californicus, males raised in cages containing the bedding from their own families were delayed in attainment of adult androgen levels and seminal vesicle weights compared to males raised in clean bedding (Rissman et al., 1984). When males were raised in cages containing bedding from their fathers, their mothers, or unrelated males, the surprising result was that both androgen levels and seminal vesicle weights were lowest in males exposed to bedding from their mothers. Males raised with bedding from their own families were also not able to stimulate reproductive development in female voles (Rissman and Johnston, 1985). The evolution of this mechanism may be related to the adaptive value of delaying reproduction under some conditions, such as when population density is high or when there is a lack of green vegetation and/or water. In a subsequent study it was found that supplementing the diet of developing voles with lettuce eliminated the suppressive effects of odors from their mothers (Rissman and Johnston, 1986). In species in which population size can grow dramatically, odors produced by individuals in crowded populations may affect other conspecifics. For example, male mice in crowded populations have heavier adrenal glands, and, when soiled bedding of a crowded population is regularly introduced in the cage of an isolated male, his adrenal glands grow to reach the same weight as in crowded males (Wuensch, 1979). 11.3.1 Chemical Identification of Signals that Influence Hormones The laboratory of Milos Novotny is responsible for most of what is known about specific chemicals that 422 Communication by Chemical Signals influence the endocrine system (Novotny et al., 1999b). This section reviews the major findings from this laboratory. Vandenbergh (1967, 1969) discovered that puberty could be accelerated in female mice if they were exposed to an adult male or to bedding from the cage of an adult male compared to conditions in which no male stimuli were available . Identification of a molecule that had an effect by itself was extremely difficult because the active compound or compounds were bound to other molecules and purified fractions often retained the odor of mouse urine. The active compound was eventually identified as 5,5-dimethyl-2-ethyltetrahydrofuran2-ol and/or its open-chain tautomer, 6-hydroxy6-methyl-3-heptanone. The synthetic heptanone had a strong effect on puberty in young mice, whereas other similar molecules did not have such activity (Novotny et al., 1999a). Puberty can also be delayed in female mouse pups by rearing them with other female pups compared to raising them alone; once again chemical signals were shown to be the cause. Pups exposed to bedding material or urine collected from pups living in a group inhibits puberty, whereas bedding or urine from a female pup housed alone does not have this effect (Drickamer, 1982). The chemical compound 2,5-dimethylpyrazine, found in male urine, has been found to delay sexual maturation in juvenile males and also delay maturation in juvenile females. This later result is quite curious and it is not entirely clear how this relates to the natural situation and why this substance should have suppressive effects on young of both sexes. The development of testes and accessory glands can also be delayed in juvenile male mice and California voles by chemical cues (Rissman et al., 1984). Another phenomenon in mice is that when females are housed together, they develop longer and more irregular estrous cycles. When grouphoused females are exposed to a male, however, a majority of females come into estrus 3 days later, indicating a major stimulation of the ovaries. Although there are differences of opinion in the literature, substances in male urine, contributed from the preputial gland, appear to be involved. Two farnesene compounds are produced in the preputial gland, and synthetic E,E-a-farnesene and E-b-farnesene have effects similar to extracts of the preputial gland on inducing estrous cycling (Ma et al., 1999). A more detailed review of these effects in mice is in a recent paper by Koyama (2004). 11.4 Olfactory and Vomeronasal Systems and Their Roles in Communication and Social Behavior 11.4.1 Structure In most species of terrestrial vertebrates there are two anatomically separate ‘olfactory’ systems, the main olfactory system (MOS) and the accessory olfactory or vomeronasal (VNO) system (Negus, 1958). Birds, however, lack the VNO. These two systems have different receptor cell types, different families of genes for receptor proteins, and separate neural projections from the sensory epithelia to the olfactory bulb. The olfactory epithelium projects to the main olfactory bulb (MOB) and the VNO projects to the accessory olfactory bulb (AOB) ( Johnston, 1998, 2000, 2001; Keverne, 1998, 1999, 2004; McCotter, 1912; Meredith and Fernandez-Fewell, 1994; Scalia and Winans, 1975; Wysocki, 1989; Wysocki and Meredith, 1987). In addition, the neural projections from the MOB and AOB to the central nervous system (CNS) are also nonoverlapping. These anatomical differences suggest that the two systems have some unique functions. However, these separate neural inputs are integrated by the next set of projections into the amygdala and hypothalamus, and both the MOS and VNO systems often have influences on a particular behavioral or physiological response. The MOS in all terrestrial vertebrates is situated in the nasal cavity. Stimuli usually reach the sensory mucosa via the nares (orthonasal access) but in some taxonomic groups stimuli may reach the olfactory receptors from the mouth via the pharynx (retronasal access). The olfactory mucosa, containing olfactory receptor cells, generally lines the posterior and dorsal septum on the midline of the nasal cavity and it also lines some parts of the complex, cartilaginous turbinates in the nasal cavity. Turbinates that have olfactory receptors are generally situated in the most dorsal and posterior parts of the nasal cavity (Negus, 1958). In amphibians, there is no separate VNO; rather, olfactory and accessory olfactory receptor types are located in the nasal cavity, often in separate but connected lobes (Bertmar, 1981; Eisthen, 1992, 1997). In reptiles, access to the VNO is usually via the mouth and nasopalatine canal; evidence suggests that chemical stimuli are picked up on the tongue and then transferred to the mouth and nasopalatine canal (Halpern, 1987; Halpern and Kubie, 1980). Although fish do not have two anatomically distinct sensory organs, they do have the two characteristic Communication by Chemical Signals receptor cell types, suggesting that the origins of two separate types of olfactory chemoreception are evolutionarily quite ancient (Eisthen, 1992, 1997). This two-part structure of the chemical senses in vertebrates has an interesting parallel among insects, in which there are also two parallel olfactory systems with different types of receptors, different projections to CNS, and different processing within the brain (Ache and Young, 2005; Hildebrand and Shepherd, 1997). In insects there is extensive evidence that chemicals involved in social communication, mostly communication between the sexes for mating, is mediated by one system and nonsocial communication is mediated by the other system. The division of both insect and vertebrate chemosensory functions into two parallel systems that have functional similarities is extraordinary, especially since they are widely separated taxonomically. This suggests that there is an adaptive advantage in separating the processing of functionally distinct types of chemical information and/or different classes of chemical compounds used as signals. The accessory olfactory receptors in mammals and reptiles are located in the VNO. This organ is a tube that usually has a small opening at the front but is closed at the rear. In many species the VNO is located on the midline in the nasal cavity at the base of the septum. The location of this organ in the nasal cavity varies considerably along the anterior–posterior axis. The opening into the VNO also varies in relation to the position of the nasopalatine canal that runs between the mouth and nasal cavity (Bertmar, 1981). The VNO may be located just posterior to the opening of this canal in the nasal cavity, presumably permitting ready access of chemicals taken into the mouth. In many species, however, the VNO is anterior to the nasopalatine canal (e.g., mouse and hamster), suggesting either that chemicals rarely enter the VNO from the mouth or that there are as yet undiscovered mechanisms for transport of chemicals from the mouth through the nasopalatine canal and then forward to the opening to the VNO. In many larger mammals (e.g., ungulates), the VNO is situated between the mouth and nasal cavities and access to it is directly off the nasopalatine canal or there may be an additional tube connecting from the nasopalatine canal to the VNO, for example, in pigs (Dorries et al., 1997), and goats (Melese-d’Hospital and Hart, 1985). Little comparative work has been done to carefully assess the functional consequences, if any, of the differences in location of the VNO in relation to the nasopalatine canal. 423 The mechanisms by which chemicals gain access to the VNO have been studied in detail in several species. The most detailed studies have been in the golden hamster. In this species the VNO lies within a cartilaginous capsule and a vascular pump draws chemicals into the VNO by changes in blood pressure within this capsule. When blood pressure is high, the VNO is compressed; a sudden drop in blood pressure surrounding the VNO causes the VNO to expand and produce suction, which draws mucus and chemicals dissolved in the mucus into the lumen of the VNO (Meredith, 1980, 1991; Meredith et al., 1980). In elephants, individuals pick up chemical signals by placing the end of the trunk on a scent and drawing in air and scent; they then place the end of the trunk into the mouth and over the nasopalatine canal, and apparently force air and chemicals into this canal (Rasmussen et al., 1993; Rasmussen and Schulte 1999). Many species of mammals, including hyenas, bats, some viverrids, ungulates, and most species in the cat family, often engage in a behavioral pattern called flehmen after investigating conspecific odors (Ewer, 1968); this behavior is characterized by raising of the head, retraction of the lips, and remaining still in this posture for a short period of time (a few to many seconds). In some species this behavior is associated with rapid licking (Estes, 1972). This author hypothesized that flehmen was associated with muscular action that opened the nasopalatine canal and thereby increased access of chemicals to the VNO (Estes, 1972). Experimental evidence for this role of flehmen was provided by studies in goats (Ladewig and Hart, 1980; Melese-d’Hospital and Hart, 1985). Flehmen is most commonly observed in male mammals when investigating odors from females, but in some species, such as domestic cats, both sexes engage in flehmen when investigating female odors (Hart and Leedy, 1987). The tendency of females to exhibit this behavior is greatly increased by treating them with testosterone proprionate (op. cit.). 11.4.2 Receptor Cells and Genes for Receptor Proteins The two olfactory systems differ in the morphology of the receptor cells and the families of genes that code for receptor proteins. The receptor cells of the main olfactory epithelium (MOE) have long cilia that form a dense, intertwined layer bathed in nasal mucus and glandular secretions. In contrast, the receptor cells in the VNO have microvilli rather 424 Communication by Chemical Signals than cilia; these microvilli are also bathed in mucus and glandular secretions (Bertmar, 1981; Kratzing, 1971). The receptor cells in the MOE project to the MOB; each receptor cell expresses just one olfactory receptor gene which in turn results in one type of receptor protein. All of the receptor cells that express the same gene project to two, usually symmetrical glomeruli on either side of the MOB (Buck, 1996; Firestein, 2001; Rodriguez et al., 1999). Since the receptor cells that express the same gene are distributed widely in the olfactory mucosa, the convergence onto just two glomeruli represents an amazing feat of targeting and a dramatic conversion of inputs onto the appropriate glomeruli. In contrast, the terminals of receptor neurons in the VNO project more broadly to glomeruli in the accessory olfactory bulb. Receptor cells expressing V1r genes project to 10–30 glomeruli in the rostral AOB (Belluscio et al., 1999; Rodriguez et al., 1999), whereas receptor cells expressing V2r genes project to 6–10 glomeruli in the caudal part of the AOB (Del Punta et al., 2002). These distinct patterns of projection onto the MOB and AOB indicate very different principles of organization and processing in the two divisions of the olfactory bulbs. 11.4.3 Neural Projections from the Olfactory Bulb to the CNS The MOB and AOB project to completely nonoverlapping target areas in the CNS ( Johnston, 2000; Scalia and Winans, 1975, 1976; Wysocki and Meredith, 1987). The AOB projects to a small group of anatomically related targets, including the bed nucleus of the stria terminalis, the nucleus of the accessory olfactory tract, the medial and posteromedial nuclei of the amygdala, and three areas in the hypothalamus, the medial POA, the ventromedial hypothalamus, and the premammilary nucleus ( Johnston, 2000; Wysocki and Meredith, 1987). Projections from the MOB are distributed widely to the base of the brain, including the anterior olfactory nucleus, olfactory tubercle, piriform cortex, entorhinal cortex, nucleus of the olfactory tract, and the anterior central and posterolateral nuclei of the amygdala ( Johnston, 2000; Wysocki and Meredith, 1987). These two separate pathways do eventually converge and integrate their information in the medial POA–anterior hypothalamus, ventromedial hypothalamus, and the premammillary nucleus (Kevetter and Winans, 1981a,b; Lehman and Winans, 1982; Scalia and Winans, 1975, 1976; Wood and Newman, 1995b). 11.4.4 Conceptual Views of the Main Olfactory and VNO Systems The differing properties of the two chemosensory systems suggest that the two systems may be specialized for detecting different types of molecules and/or that they have different functions. Indeed, many publications have provided evidence for different functions of these two systems. However, there is a strong tendency to oversimplify the functions of these two sensory systems and to create rigid dichotomies in proposed functions. Examples of such oversimplified categorizations include: (1) the VNO only responds to large, nonvolatile chemicals whereas the MOS detects small, volatile chemicals; (2) the VNO is responsible for detecting and responding to pheromones in the classical sense of this word (i.e., a chemical that automatically releases a specific behavioral or physiological response), whereas the MOS responds to other odors that are not pheromones – some authors actually define pheromones as substances detected by the VNO, but this is clearly not correct; and (3) the VNO is specialized for mediating hormonal responses whereas the MOS mediates behavioral responses. Some of these hypotheses are simply incorrect and others are misleading because they are stated as if these categories are sufficient to describe the roles of these systems in all species and in all situations. Furthermore, such categorizations do not reflect the degree to which the two systems are complementary and often work together in mediating responses to chemical signals. Despite many excellent reviews that have pointed out the broad range of functions for these two systems ( Johnston, 1998, 2001, 2003; Keverne, 2004; Restrepo et al., 2004; Wysocki, 1989; Wysocki and Meredith, 1987), many authors seem to ignore this wealth of information and the diversity of functions that both systems have. In this section, we review the functions of the MOS and the VNO and stress that, although there are some general trends in the functions of the two systems, there are also significant (1) species differences in the functions of the two systems; (2) sex differences in the functions of the two systems; (3) differences within a species depending on which particular odor source is being studied; (4) differences between species or between particular functions within a species on the importance of previous experience (e.g., sexually experienced Communication by Chemical Signals or sexually naive animals); and (5) differences in the degree to which the two systems have independent effects, no effects, or both are involved in a particular function. 425 block the increase in LH secretion (Gelez et al., 2004c). It is also interesting that sexual experience with rams is important for the endocrine and behavioral responses of ewes to the odors of rams (Gelez et al., 2004a,b). 11.4.4.1 Hormonal responses to odors Lesions of the VNO are especially likely to influence hormonal responses caused by exposure of subjects to opposite-sex conspecifics or odors from such animals (see Table 1). In female mammals a few examples of these effects include: induction of estrus by males or male odors in gray short-tailed opossum, Monodelphis domestica (Jackson and Harder, 1996) and acceleration of puberty onset by odors of the opposite sex in mice (Kaneko et al., 1980; Koyama, 2004; Lomas and Keverne, 1982; Vandenbergh, 1983a), Djungarian hamsters (Gudermuth et al., 1992), and several species of voles (Richmond and Stehn, 1976). Additional effects include delay of reproductive development by odors of conspecifics (Rissman et al., 1984; Vandenbergh, 1983a), and modulation of estrous cycles in female rats by exposure to male and female odors (Beltramino and Taleisnik, 1983; McClintock, 1983). Table 1 provides an extensive list of specific effects. In male mammals the VNO is also important in modulation of hormones; for example, lesions of the VNO or the AOB eliminate the following effects: (1) an increase in LH and testosterone in male mice after exposure to female mice or their odors (Coquelin and Bronson, 1980; Coquelin et al., 1984) and (2) increases in testosterone in male hamsters in response to female hamster vaginal secretions (Macrides et al., 1974; Pfeiffer and Johnston, 1992, 1994). It is clear from the summary of results shown in Table 1 that there are some general differences in the function of the main and accessory olfactory systems but, also, that there is often an overlap in functions. One trend that emerges from this summary is that the VNO and accessory olfactory system appear to be more often involved in hormonal responses to odors than the MOS. Nonetheless, the VNO is not always responsible for hormonal responses to odors. One notable exception to this generalization is the ram effect in ewes, in which exposure of ewes to odors from rams hastens the onset of fertility and sexual receptivity by increases in secretion of LH into the circulation (Cohen-Tannoudji et al., 1994, 1989). Lesions of the VNO have no effect on LH secretion in ewes in response to ram odors, whereas inactivation of areas involved in main olfactory inputs do 11.4.4.2 Role of the MOS in nipple search and attachment in rabbit pups One striking and extremely important example from Table 1 is the work on the mechanisms underlying the ability of rabbit pups to find and attach to the mother’s nipples. Rabbit pups are stimulated to search for and locate the nipple via the MOS; when this system is disrupted the pups do not succeed in finding the nipple or attaching to it. If the VNO is removed, pups have no difficulty finding and attaching to the nipple (Schaal et al., 2003). The signal involved is a classic pheromone: one chemical substance, 2-methylbut-2-enal, elicits searching and attachment to the nipple, and the degree of response is concentration dependent (Coureaud et al., 2004; Schaal et al., 2003). Milk from other species of mammals does not elicit these responses in rabbit pups. Furthermore, this signal is species specific and does not elicit nipple search or attachment in other, closely related species. Finally, pups delivered by cesarean section responded selectively to 2-methylbut-2-enal, demonstrating that pups naive to this odor nonetheless responded appropriately without experience or reinforcement, indicating genetic determination of the response. None of the other compounds found in rabbit milk had significant effects on these behaviors. This is the most completely characterized mammalian pheromone in terms of chemistry, functions, species specificity, and sensory mechanisms, and this classic pheromone is perceived through the MOS (Schaal et al., 2003). It is the only case in which a single substance has been identified that has the full effect of the raw secretion, the species specificity has been determined, a role for learning has been ruled out, and the sensory system involved in the response to this classic pheromone is the MOS. 11.4.4.3 Role of VNO and MOS in sexual behavior and sexual motivation Removal of the VNO has effects on a number of aspects of sexual behavior and motivation in both male and female mammals. These include effects on copulatory behavior, attraction to a sexual partner, discrimination and recognition of male versus female, 426 Communication by Chemical Signals Table 1 Effect of lesions of the vomeronasal system (VNO) or main olfactory system (MOS) on hormonal and behavioral responses to odors of other individuals Expected effect of VNO/MOS lesion Hormonal effects Blocks LH surge in response to female odors Blocks androgen surge in response to female odors Lesion Result Species Sex/experience References VNO þ Mouse Male Coquelin et al. (1984) VNO þ Hamster Pfeiffer and Johnston (1994) Hamster Male, both sexually experienced and naı̈ve Male Mouse Rat Male Female Sheep Female Mouse Female Mouse Female þ Mouse Female Female þ Marmoset monkey Mouse MOS Blocks LH surge in response to male odors VNO VNO/MOS þ þ VNO Developmental effects Eliminates pregnancy block by unfamiliar male VNO þ MOS Eliminates the acceleration of puberty by male odors VNO Eliminates estrous suppression by dominant female Eliminates the induction of estrus in suppressed females Eliminates the induction of estrus VNO and/ or MOS VNO Behavioral effects Increases latency to investigate vaginal secretion Reduces attraction to female odors Eliminates preference for novel female Eliminates preference for estrous female urine over male urine Blocks discrimination of estrous vs. nonestrous urine Blocks discrimination of intact vs. castrated male urine (both volatile and nonvolatile) (only nonvolatile) VNO VNO þ VNO þ VNO MOS Female Pfeiffer and Johnson (1994) Wysocki et al. (1983) Beltramino and Taleisnik (1983) Cohen-Tannoudji et al. (1989) Bellringer et al. (1980), Lloyd-Thomas and Keverne (1982) Lloyd-Thomas and Keverne (1982) Kaneko et al. (1980), Lomas and Keverne (1982) Barrett et al. (1993) Reynolds and Keverne (1979) Wysocki et al. (1991) Meek et al. (1994) Prairie vole Meadow vole Gray shorttailed opossum Female Female Female Jackson and Harder (1996) þ Hamster þ Hamster Pfeiffer and Johnston (1994) Pfeiffer and Johnston (1994) Hamster Male, sexually experienced Male, both sexually experienced and naı̈ve Male VNO MOS þ Hamster Male VNO VNO þ Mouse Hamster MOS þ Hamster VNO þ Mouse Male Sexually satiated male Sexually satiated male Male VNO VNO MOS þ Sheep Hamster Mouse Male Female Female O’Connell and Meredith (1984) O’Connell and Meredith (1984) Pankevich et al. (2006) Johnston and Rasmussen (1984) Johnston and Rasmussen (1984) Keller et al. (2006b), Pankevich et al. (2006) Blissitt et al. (1990) Petrulis et al. (1999) Keller et al. (2006a) VNO þ Mouse Female Keller et al. (2006b) Continued Communication by Chemical Signals Table 1 427 Continued Expected effect of VNO/MOS lesion Lesion (only volatile) Blocks discrimination of conspecific vs. heterospecific odors Blocks discrimination of individual odors VNO VNO MOS VNO Blocks the learned discrimination of MHC differences in congenic mice Reduces sexual behavior Result Species Sex/experience References þ þ Mouse Hamster Hamster Hamster Female Male Male Male VNO Hamster Female VNO Mouse Female Keller et al. (2006b), Murphy (1980) Murphy (1980) Johnston and Peng (2000) Johnston and Peng (2000), Petrulis et al. (1999) Wysocki et al. (2004) Hamster Male Hamster Male Mouse Prairie vole Rat Mouse Pig Meadow vole Hamster Male Male Female Female Female Female Mouse Hamster Mouse Lesser mouse lemur Mouse Female Male Male Male Female Female Female Female VNO þ MOS VNO VNO VNO MOS VNO VNO þ þ þ þ VNO þ VNO VNO/MOS VNO VNO þ þ þ þ VNO þ VNOþMOS VNO/MOS VNO þ þ Hamster Rat Rat Blocks pup recognition VNO þ Sheep Female Reduces nest building VNO Mouse Female Reduces pup retrieval VNOþMOS VNO Hamster Mouse Female Female Rat Rabbit Female Pups Reduces percentage of females mating Reduces male–male aggression Reduces maternal aggression toward males Blocks maternal behaviors Reduces nipple search and attachment Affects scent marking þ VNO/MOS VNO Female MOS þ Rabbit Pups MOS þ Hamster Male Hamster Male Hamster Hamster Hamster Mouse Lesser mouse lemur Female Female Female Male Male VNO VNO MOS VNO VNO VNO þ þ O’Connell and Meredith (1984), Powers and Winans (1975) O’Connell and Meredith (1984) Clancy et al. (1984) Wekesa and Lepri (1994) Saito and Moltz (1986) Keller et al. (2006a) Dorries et al. (1997) Meek et al. (1994) Mackay-Sim and Rose (1986) Keller et al. (2006b), Murphy (1976) Clancy et al. (1984), Aujard (1997) Bean and Wysocki (1989) Marques (1979) Jirik-Babb et al. (1984) Brouette-Lahlou et al. (1999) Booth and Katz (2000) Bean and Wysocki (1989) Marques (1979) Bean and Wysocki (1989) Jirik-Babb et al. (1984) Hudson and Distel (1986) Hudson and Distel (1986) Johnston and Mueller (1990) Johnston and Mueller (1990) Johnston (1992) Johnston (1992) Petrulis et al. (1999) Clancy et al. (1984) Aujard (1997) 428 Communication by Chemical Signals and differential responses to animals in different reproductive states (see Table 1). The following brief treatment of the role of the olfactory system and VNO in sexual behavior shows that chemical signals are extremely important for recognizing males and females, recognizing the reproductive states of males and females, attracting mates, and engaging in copulatory behavior. Both the VNO and the MOS are involved in these processes. Although there are similarities across some behaviors in different species, no simple rules apply to all mammals or even to the small number of rodent species that have been tested. Claims for such allinclusive rules should be viewed with considerable skepticism. 11.4.4.3(i) Male behavior The first demonstration of the necessity of odor information for the sexual behavior of male mammals was the finding that ablation of the olfactory bulbs completely eliminated mating behavior in male hamsters (Murphy and Schneider, 1970). The importance of the VNO in sexual behavior of male hamsters was demonstrated by showing that cutting the neural projections from the VNO to the AOB resulted in severe deficits in copulatory behavior in some males and that damage to the MOE in addition to the VNO eliminated copulatory behavior completely in male hamsters (Powers and Winans, 1975; Winans and Powers, 1977). In subsequent studies it was shown that previous sexual experience influenced the effects of lesions to the VNO, with sexually experienced males showing less severe deficits after removal of the VNO. Males with no previous sexual experience showed much greater deficits (Meredith, 1986, 1991; Meredith et al., 1980). In other studies, it was shown that lesions of both the VNO and the MOE eliminated male sexual behavior in sexually experienced and sexually naive males and that lesions of only the VNO or the MOE had a lesser influence on mating with an estrous female (Pfeiffer and Johnston, 1994). Lesions to either system also decreased behavioral responses to vaginal secretions in both sexually naive and sexually experienced males (Pfeiffer and Johnston, 1994; Powers et al., 1979). Further work has shown that the integration of information from the MOS, the VNO, and hormones occurs in the medial amygdaloid nucleus, bed nucleus of the stria terminalis, medial POA, and other associated areas (Kevetter and Winans, 1981a,b; Lehman and Winans, 1982; Lehman et al., 1980; Wood and Newman, 1995a,b). Female golden hamsters advertise their approaching state of estrus by greatly increasing the frequency of vaginal scent-marking behavior, beginning 24 h prior to estrus ( Johnston, 1977a, 1979). Females selectively direct such marking at dominant males (Huck et al., 1985a) and males that are not close kin (Heth et al., 1998; Mateo and Johnston, 2000), suggesting that females are using this behavior to attract certain classes of males. Odor-stimulated vaginal marking is mediated via the MOS – lesions of this system result in dramatic decreases in vaginal marking in response to male odors but removal of the VNO has no effect. Similarly, the MOS is essential for ultrasonic calling that hamsters use to attract and locate one another for mating. Calling was significantly decreased by lesions of the MOS but was not influenced by removal of the VNO ( Johnston, 1992). The VNO and the MOS play important roles in sexual behavior of other male mammals as well ( Johnston, 1998, 2000, 2001, 2003; Kelliher, 2007; Keverne, 2004; Restrepo et al., 2004; Wysocki, 1989). Male mice with their VNO removed are able to discriminate between volatile urinary odors from estrous females versus intact males in habituation– dishabituation tests. Such males also discriminate between volatile urinary odors from estrous females and ovariectomized females, thus indicating that these discriminations were carried out by the MOS. When males could contact these odor sources, however, males with vomeronasal lesions (VNX) did not show a preference for odors of estrous females over those from males whereas males with an intact VNO did show a preference for female odors. These results indicate that preferences for females were mediated via the VNO (Pankevich et al., 2006; Keller et al., 2006). Both VNO intact and VNX males did mate with estrous females, demonstrating that, although vomeronasal input does influence behavior toward male and female conspecifics, it is not necessary for mating (Pankevich et al., 2004). Male mice prefer female urine over a number of other odors. For example, they run more quickly toward female urine than male urine. The VNO is involved in these preferences, since VNX males did not show this difference in behavior (Pankevich et al., 2006). 11.4.4.3(ii) Female sexual behavior Female mammals also depend on the VNO for some aspects of sexual interactions. In female mice tested in a Y-maze, VNX females distinguished between the volatile odors from an intact, anesthetized male and an anesthetized, gonadectomized male. Females also Communication by Chemical Signals discriminated between volatile odors from urine of an intact male versus those from a gonadectomized male, indicating that the MOE mediated this discrimination (Keller et al., 2006b). When tested with nonvolatile odors from urine (MUPS), however, females with an intact VNO discriminated between these proteins from an intact male and a gonadectomized male but VNX females did not. VNX females also failed to discriminate between the MUPS from males versus females, showing that the VNO was necessary for discrimination of these proteins (Keller et al., 2006b). Most strikingly, VNX females showed very little lordosis behavior compared to sham-operated control females (Keller et al., 2006b). Injections of GnRH into VNX females did restore some sexual receptivity and the frequency of lordosis posture, but this behavior was still significantly less than in control females. Female hamsters and rats also showed deficits in lordosis and mating behavior after removal of the VNO; hormone therapy with GnRH (hamsters) or with GnRH plus estrogen (rats) restored female receptivity to levels of behavior shown by control females (Mackay-Sim and Rose, 1986; Saito and Moltz, 1986). 11.4.4.4 VNO and the discrimination and recognition of individuals Although few have touted individual discrimination and recognition as a function of the VNO, it is involved in such functions in some species. In the house mouse, MUPs are essential for individual recognition (Cheetham et al., 2007). Contact with these proteins is necessary for such recognition, which is consistent with processing by the VNO (Cheetham et al., 2007; Luo et al., 2003). Although lesions of the VNO in golden hamster females did not influence discrimination between individual odors using either flank-gland secretions or vaginal secretions, lesions of the VNO did decrease the ability of male hamsters to discriminate between flank-gland odors of other males. These lesions did not affect the ability of males to discriminate between female vaginal secretions ( Johnston and Peng, 2000). These results indicate that both the MOS and the VNO are involved in discrimination between the odors of different individuals. In many studies investigating individual discrimination, chemical stimuli from all of the body are used as the stimuli, and thus previous experiments may have missed a role for the VNO in discrimination of one or more specific odors that provide individually distinctive information. 429 References Abbott DH (1984) Behavioral and physiological suppression of fertility in subordinate marmoset monkeys. American Journal of Primatology 6: 169–186. 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Chicago, IL: The University of Chicago Press. Biographical Sketch As a beginning graduate student at Brown University and Rockefeller University, Robert Johnston was interested in neurophysiology and the cellular and neural mechanisms underlying behavior. However, he became fascinated by the possible existence of mammalian pheromones and switched to behavioral studies of communication by odors and scent marking for his graduate thesis. He accepted a job at Cornell University and has continued to be fascinated by animal communication and behavior. Collaboration with undergraduates, graduate students, and postdocs have been especially exciting and rewarding, and have led to some of his most important discoveries (e.g., odor influences on hormones, hormone influences on behavior and development, new concepts and information about scent marking and over-marking, individual recognition and kin recognition, the nature of representations of individuals and the functional neuroanatomy underlying individual recognition, and sexual and aggressive behavior). With colleague Bob Kraut he also enjoyed taking an ethological approach to the causation and functions of facial expression, particularly smiling. Most fundamentally, he loves the diversity of life and the amazing adaptations of animals, the beauty and pure sensory delight of nature, and the sometimes subtle but surprising and marvelous events that can occur at any time. Javier delBarco-Trillo was born in Barcelona, Spain, in 1973. He did his undergraduate studies at the Universidad de Barcelona. He did his PhD at the University of Memphis on how mammals use chemical signals of conspecific males to assess and respond to different levels of sperm competition. In 2004 he started his postdoctoral work at Cornell University, investigating the neural underlying of individual recognition and conspecific/heterospecific discrimination, the effects that captivity may have in female reproductive behavior, the role of familiarity in decreasing aggression, and the finding that juveniles before a threshold age do not elicit aggression on adult males.