An Odor That Does Not Lie

Our Japanese colleague Professor Endo puts aside the manuscript and asks: “Misha, why do you use the terms of everyday life in a research article? Honest signal, for example?” I take the textbook by I. Roitt: “Endo, look here, in the immunology, where you work, everyday language is quite common. For example, here one can find the terminology of mafia groups: “killer cells”, or military terms: “armed helper T cells” or even “presentation” of the marketing.

But let us return to the honest signals and immunology. Not so long ago it was found that infected males of different animal species display altered signals used for sexual advertising in the “fianc fair” (Hamilton, Zuk, 1982). Importantly, this effect is observed even when an infection is not obvious. Bright colours of fish and birds become paler, bird songs sound poorer and duller. Scent of infected rodent males becomes less attractive for females as compared to the one of their healthy rivals.

The decreased attractiveness of contagious sexual partners is of clear benefit for a population, since probability of spreading pathogens through sexual intercourse is consequently diminished. This serves as a general restriction of reproductive success for the individuals with immune mechanisms compromised by an infection. But is there any benefit for the infected males? It should be noted that there is an example of a strategy opposite to the described above: experimentally parasitized Drosophila males lived shorter lives, but they courted significantly more intensely, and despite the shorter life span produced more offspring (Polak, Starmer, 1998).

One can ask if it is possible to explain the “evasion” of infected males from reproductive competition entirely within the Darwinian paradigm of the intra-species struggle as the basis for evolution, and completely avoiding ethical and moral concepts. In other words, is it of any benefit to be honest?

Forced honesty?

To understand the nature of the phenomenon, let us consider the mechanisms of biological signal modification triggered by an infection. First of all, let us clarify what plays the major role in the decrease of male attractiveness at the initial, pre-clinical stages of infection. Is it due to the pathogene itself or to the mechanisms of immune defence that are mobilized by an organism when it recognizes the parasitic intervention (Moshkin et al., 2000; Moshkin et al., 2004)?

One of the approaches with a potential of resolving this dilemma is in taking into consideration the so-called non-replicating antigenes. By definition, an antigen is any foreign organic substance (including proteins or polysaccharides) that induces an immune response of an organism. Non-replicating, or in other words non-infectious antigens, are not hazardous for an animal, but they trigger a complex of defensive reactions similarly to the way it happens with the antigens that are a part of parasitic organisms (Abelev, 1996).

Erythrocytes of Ovis aries, or plainly speaking a common domestic ram, became one of the most popular and easily available immune response inducers. They have been used successfully in immunologic research for more than 50 years. But such an approach, evident for experimental biologists, allowing them to study single links and to separate them from a complex chain of processes, unexpectedly met objections from some zoologists. In fact, a reference to “an effect of ram blood on scent signals in mice” at first sounds odd: mice do not eat rams…

However, we have already published the first results obtained using this approach. We compared the time spent by a female of the common house mouse near a male-soiled bedding from the cage of a control male with the time she spent near the cage of a male injected with sheep red blood cells (SRBC) and came to the conclusion that in injected males, activation of immune response resulted in the decreased sexual attractiveness of scent marks (Moshkin et al., 2002).

Similar effect was observed in the experiments with males of the dwarf hamster: activation of the immune system made their scent less attractive for females and more offensive for other males. It was found that hamsters prefer to fill the cheek pouch with sunflower pips from a feeder box “contaminated” by the scent of a control male, but not by the sent of an antigen-stimulated one (Novikov et al., 2004; Kondratyuck et al., 2004).

Tight coordination of immune and neuroendocrine processes makes it impossible for the infected males to choose between providing either “honest” or “dishonest” information on their sad state. In the hierarchy of vital preferences – either to rapidly reproduce despite the threat of death, or to recover first and then breed – the desire for life usually prevails. Anyway, “better to be a dull-smelling male than a dead male” (Penn, Potts, 1998)

An effect of alteration of chemosignals upon activation of immune response was recently reproduced and studied in depth by the researchers from the Institute of Ethology, Vienna, Austria, and Utah University, USA. In their experiments a thermophilic bacterial strain C5TS of Salmonella enterica, which does not reproduce in the warm-blooded animals was used as a foreign antigen (Zala et al., 2004).

Independently of rodent studies, research of visual and acoustic signals sent by male birds of different species to attract sexual partners was started. The most prominent result of these investigations was recently published in the “Science” with a meaningful title “Immune activation rapidly mirrored in a secondary sexual trait” (Faivre et al., 2003). It was found that in the European blackbird, the beak coloration was changing as a result of already mentioned immunization with SRBC. Beak coloration of these birds naturally varies from yellow to orange, and female birds prefer the “red-nosed” males. And activation of the immune response in males was leading to their “pale appearance” – both literally and figuratively speaking.

In another avian species – pied flycatchers – male sexual attractiveness is determined by a white patch on the forehead, and the higher it is, the better. Experimental activation of male immune defense by vaccination with novel antigens (diphtheric vaccine) led to the shrinking of the forehead patch (Kilpimaa et al., 2003). Moreover it was found that in another flycatcher species immunization of males with SRBC resulted in the reduction of song complexity and, consequently, in their attractiveness for females (Garamszegi et al., 2003).

It is better to be a dull-smelling male than a dead male

So, an activation of the mechanisms of immune defence, which is inevitably caused by a parasitic intervention, leads to the reduction in male sexual attractiveness. This is true not only for different animal species, but also for different types of sexual advertising — visual, acoustic, olfactory… Therefore, one should seek physiologic explanations for these phenomena at the linking points, where immune reactions are linked to the general neuro-endocrine processes responsible for the development of advertising traits — so-called secondary sexual traits.

The well known male sex hormone testosterone is a universal stimulus for the development of “masculinity”, which manifests itself either as red beaks of a blackbird, or as bushy antlers of a deer, or as an attractive scent of a male mouse. Its secretion is decreased upon infection or immunization with foreign antigenes. And it is quite reasonable since testosterone is known to suppress immune defence mechanisms. Besides, sparing the resources that could be spent for the testosterone-induced sexual advertising allows to channel them towards some more urgent needs such as body defences. For example, carotenoids responsible for the bright colour of “beaks”and “stomachs” in males of some fish and birds at the same time have certain immunostimulating and antioxidatnt effect (Lozano, 2001). Although their movement from the tissue to the blood stream leads to pale coloration of males, it increases the efficiency of their immune system.

Probably you would ask if the energy spent for the “production” of an olfactory signal is really that high. In the end “pooh!” is only “pooh!” and nothing serious. However you would be wrong to think of it as of “nothing serious”. For example for mice the “cost” is quite high, because male attractiveness is determined not only by the concentration of volatile odorants – sexual pheromones – in urinary marks, but also by their stability. For the pheromones not to vanish instantly into thin air, a considerable part of them is released in a protein-bound form. Pheromone-binding proteins from the lipocalin superfamily are predominant among the proteins of urine and for this reason are called major urinary proteins (MUPs).

Notably, about 4% of the total amount of proteins synthesized in the liver is lipocalins. An adult male excretes daily up to 10 mg of MUP in the urine, which is about 10% in the total nitrogen balance (Cavaggioni, Mucignat-Carretta, 2000). Comparing this percentage with the percentage of GDP Russia spends for research, culture and education together (i.e. everything that creates a positive image of a country), mice seem to “finance” their image much more generously.

Activation of the immune system results in almost twofold decrease in MUP concentration, which in turn results in decreasing olfactory attractiveness. However, when we experimentally stabilized the blood testosterone concentration, antigen stimulation affected neither the MUP concentration, nor the smell attractiveness of males (Litvinova et al., 2005).

In a healthy body…

It was found that activation of the immune system did suppress sexual display for the majority, but not for all males. In females sexual interest is decreased, first of all, in respect to the males with a weak immune response to foreign antigenes (Moshkin et al., 2001). At the same time, males actively producing specific antibodies (i.e. the ones with effectively working immune defence) practically do not lose their olfactory attractiveness and retain the opportunity to get the offspring.

The above examples illustrate that defensive function of the immune system is not restricted to the limits of a single individual but spread to a higher, “social”, level – the level of population. It happens because the reproductive success of males in a population also depends on their capability to avoid or to counteract effectively an infection.

However, temporary “removal” of infected males from reproductive process is only the first stage in the system of penalties for low efficiency of defence against parasites. At the second stage complete “discarding” of the individuals that allowed massive attack of pathogens takes place. At this stage death of an individual is often caused by the parasite-induced defensive reaction of an organism rather than by parasites themselves.

Developing the concept of programmed death (from the death of individual cells, apoptosis, to the death of a whole organism, phenoptosis) V. P. Skulachev (2001) supposed that the death of infected individuals as a result of immune hyperactivation is the last line of defence in the population struggle against epidemics.

Coming back to the honest signals, we would like to emphasize that population protection from the parasites still starts with more “humane” restriction of sexual attractiveness of infected individuals and their temporary removal from the reproductive process. A well-known maxim of Democritus commonly cited in popular articles on the human health can be equally well used in the case of small mammals extensively exploiting chemical signals. In modern interpretation it sounds as, “in a healthy body, a healthy mind accommodates with much effort,” which is quite true for the advertising character of odor information.

A finishing spurt

Integration of any biologic system is based upon a number of certain positive and negative feedbacks. Therefore, a question arises: if activation of immune system affects chemical signals, do chemical signals affect mechanisms of immune response in turn?

An illustrious reader might say “Hey, you are trying to amaze Newcastle with coal”. However, take any immunological journal and read almost any article related to the experiments on mice (which are one of the major objects of immunological studies). In the “Materials and Methods” section one can find information about almost everything, like water and food, photoperiod and temperature… But you will not find a single word about animal abilities to exchange chemosignals with opposite-sex individuals from the neighbouring cages.

In fact, we started to study immunomodulation by sex pheromones not just from idle curiosity. In fact, during our long-term studies on the strength of immune response in male mice belonging to the same strain we have obtained significant variations in parameters in the control groups, which should not be the case. In experiments where only males were involved (and when they were kept separately from females), a specific immune response to foreign antigenes was always appreciable. If experiment involved animals of both sexes, immune response of males to immunization with SRBC was substantially lower as compared to the case with gender-based separation of quarters.

So we got an impression that the very presence of females affected immune reaction in males, and later this suggestion was confirmed. It was found that placement of a “scent-transparent” (mesh type) box with a bedding soiled by mature females over a cage with males was sufficient to produce a twofold decline of their immune response to foreign antigenes (Moshkin et al., 2001). Simultaneously and independently, similar results were obtained by B. P. Surinov et al (2001) from the Medical Radiology Research Centre, Russian Academy of Medical Sciences, Obninsk.

This relative immunosuppression in males who have “tested” the scent of females fits well into the concept of resource re-distribution with prioritising reproduction over the protection against infection (Folstad, Karter, 1992). This is a strategy of a finishing spurt: when one is close to the finish line, he should not spare his efforts but use them all to reach the goal.

Validity of the above concept was confirmed by M. Cavaliers et al. (2001), who showed that the presence of female sex pheromones made male mice not only less sensitive to pain, but also reduced their fear of predators. Responding to the odor of a cat, mice behaved themselves as if the cat were a guinea-pig! Notably, the strongest suppression of the fear reaction was caused by chemosignals of an unfamiliar female, which in a sense characterizes the “moral” make-up of the males.

Every cloud has a silver lining

Our studies have shown that female scent also causes some positive effects together with the suppression of humoral immunity. In particular, spontaneous synthesis of immunoglobulins by the spleen cells is also increased. But the most unexpected fact is that female scent-exposed males have lower mortality in aggressive bouts with their cage mates, despite having a higher number of wounds (Moshkin et al., 2004).

Mechanisms of such resistance to injury became clearer in our works conducted in collaboration with the Department of Medicine and Science in Sports and Exercise, Graduate School of Medicine, Tohoku University (Sendai, Japan). It was found that female scent causes an increase in the number of platelet cells in the blood of males. And the platelet cells are the corpuscles playing a major role in the processes of blood coagulation, or in other words, in the prevention of blood loss upon injury. Pheromones also appeared to change the distribution of immune cells in the body, causing them to move from the bloodstream to the periphery (air ways in particular).

Are the males so reckless when they rush to the finish? May be the male immunophysiologic reactions to the female scent have some elements of the adaptation to hazards accompanying reproduction-related behavioural fuss: a chance of being wounded in aggressive bouts or intervention of new pathogenes inhabiting potential mates?

Our first thought on discovering these effects was: what a pity that mice do not kiss their mates! Because in such a case immune cells from the lungs would provide animals with additional defence against respiratory infection. However, the second, more balanced idea restored our optimism: yes, mice do not kiss like people, but they behave in a much more “extreme” way (at least from the point of view of infectious hazards). For example, a courtship ritual of mice, as well as of many other animals, includes a thorough olfactory inspection of faeces, urinary marks and genital area of potential mating partner – a complete “lack of sanitation” from the human point of view! At the same time, such a preventive defence against respiratory infection apart from an evident benefit for an individual, generates other hazards. In particular, male mice exposed to the female scent are more susceptible to the allergy as compared to their companions isolated from chemosignals of the “fair gender”.

So on the one hand, internal resources necessary to achieve the main goal for a male (that is, reproduction) are mobilised as a result of the female chemosignal exposure. On the other hand, a male body becomes better prepared to possible risks of sexual contacts. These changes can be referred to as mobilization and adaptation effects of sexual signals. Their manifestation is different for different species and depends on a particular reproductive strategy.

For animals with a short life span and preferentially “uniseasonal” reproduction, maximal mobilization of resources for reproduction is most justified evolutionarily, even when it happens at the expense of protection against parasites. A striking example of such strategy can be found in the behaviour of Australian marsupial mice. Their males are completely exhausted and die out by the end of a two-week rut. Prevalence of adaptation effects of sexual signals over the mobilization ones could be expected for the species, which persist and reproduce for several seasons, since in this case males would be able to compensate for their reproductive failure in the future – if still alive. In the nearest future we plan to check this hypothesis studying the animals inhabiting West Siberia.

How does a mediocrity smell

A traditional question asked by the students after a lecture about chemosignals is: how does it work with humans? Another question we constantly ask ourselves is: does our research have any practical value, besides a contribution to the basic science of evolutionary ecology?

One can answer the first question with confidence: though both dogs and rodents distinguish scents much better than humans, we release and perceive sex pheromones. And this is substantiated by a number of studies (Stern, McClintock, 1998; Wysocki, Preti, 2004; Grammer et al., 2005). Karl Grammer (Grammer et al., 2002) considers pheromones as a major component of the so-called non-verbal communication, playing an important role in the relations between men and women. Olfactory clues evidently complement visual and voice pitch characteristics, playing a significant role in the choice of a sexual partner.

For example, in an experimental study students were evaluating sets of photos of opposite-sex individuals varying in “masculinity” and “femininity”. Students were also asked to determine their attitude to the scent of male and female pheromones. It was found that young women with preference to a more masculine image evaluated attractiveness of male pheromone higher than those apt to less masculine face types. Studies also have revealed strong correlation between the preference for visual pattern and scent in young men (Cornwell et al., 2004).

Olfactory attractiveness depends not only on the gender but also on the physiologic state of the pheromone donor. For example, men perceived the smell of women at ovulatory (receptive) phase of the menstrual cycle as most attractive, (Kuukasjarvi et al., 2004). This was only true for the case when women did not use contraceptives. If they did, the situation was exactly the opposite. Watching a horror movie also affects an “odor” of the audience, it becomes less attractive (Arckel et al., 2002).

Olfactory sexual signals carry information not only about reproductive state of a potential mate, but also about its current health and ability to counteract infection. The same signals trigger a wide spectrum of behavioral and immunophysiologic processes. This resembles a track towards a mountain-pass, which takes in smaller tracks on the way up, and which branches again on the way down to a valley. Crucial moment of adopting either a romantic or a pragmatic strategy in courtship in a blessed affair of production of the next generation is like such a pivotal mountain pass- point on the lane of life

Our studies carried out in collaboration with the colleagues from Kemerovo University have demonstrated that exam stress also influenced chemosignals of male students. The scent of young men before an exam session received higher ratings among female students at a “receptive” phase of the menstrual cycle.

At the same time, scent samples collected during the exams appeared to be perceived as less pleasant. In this case the decrease in attractiveness of chemosignals produced by students did not depend on their hormonal reaction to examination-induced stress (which was measured via the concentration of stress hormone, cortisol, in saliva), but it depended on their psycho-emotional status. The smell of below-the-average students commonly indifferent to their examination results as well as the smell of the fully confident excellent students remained practically unchanged, while for the all-average students it became much less pleasant.

Basing on these results we recommend the students to stop temporarily dating a nice girl until the end of the exam session. This is one of the first practical recommendations coming from our studies. Seriously speaking, the study of mechanisms of interrelation between odor signals and immunity opens a broad perspective for the development of several lines of application.

When odor would learn to lie

One of the important areas of applications (although not evident for wide audience) is standardization of the studies carried out with laboratory mice. Laboratory mice are actually the main objects in the modern medical and immunologic studies. This problem has become more acute in recent years because of new equipment installed in many animal facilities. This equipment already allows maintaining effective isolation of each cage from the scents released by its neighbors.

The second promising area of application is also related to medicine. More specifically it relates to the non-invasive diagnostics. Such an approach is plausible, and this is already supported by the studies into the capability of animals not only to detect scents, but even to evaluate immune and physiologic status of a scent donor. For example, dogs can be trained to perform scent-based diagnostics of a bladder cancer (Willis et al., 2004). At the same time, the methods of physico- chemical analysis of complex odor compositions and their individual components are being constantly improved. In particular, modern gas analyzers in combination with methods of multivariate statistics — one of the numerous versions of the “artificial nose” – are capable of distinguishing scents of mice with different genetic constitution of the Major Histocompatibility Complex (Montag et al., 2001). And the artificial system is almost as efficient as the mice themselves (known experiments by Yamazaki et al., 1976; 1979).

The last but not the least application area directly follows from Russia’s priority in the studies of the effect of sex pheromone upon immunity. Here one can speak about the potential use of sex pheromones for improving the health of domestic animals and, possibly, humans. This should not be considered as the task for the remote future. Moreover, it is critical that the research into the discussed phenomena should not be suspended, since human sex pheromones are already produced and distributed as perfumes – without sufficient prior examination of the consequences of their long-term application.

We live in the time of rapid dissemination of information and technological gadgets. Evidently the time is not that far away when among the common accessories in the lady’s handbag or the man’s pocket one will find a scent “potion” — in addition to the mobile phone, a handkerchief and an aspirin. And from this moment the scent will “lie” — for the benefit of mankind.

Literature

Moshkin M.P., Gerlinskaya L.A., Evsikov V.I. [Immune system and behavioural strategies of reproduction under parasitic pressure] Zh Obshch Biol 2003, 64: 23-44. Russian.

Grammer K., Fink B., Neave N. Human pheromones and sexual attraction. Eur J Obstet Gynec Reprod Biol 2005, 118: 135-142.

Litvinova E.A., Kudaeva O.T., Mershieva L.V., Moshkin M.P. High level of circulating testosterone abolishes decline of scent attractiveness in antigen-treated male mice. Animal Behaviour 2005, 69: 511-517.

Moshkin M.P., Gerlinskaya L.A., Morozova O.V., Bakhvalova V.N., Evsikov V.I. Behaviour, chemosignals, and endocrine functions in male mice infected with tick-borne encephalitis virus. Psychoneuroendocrinology 2002, 27: 603-608.

Penn D.J., Potts W.K. Chemical signals and parasite-mediated sexual selection. Trends Ecol Evol 1998, 13: 391-396.

Wysocki C.J., Preti G. Facts, fallacies, fears and frustrations with human pheromones. The Anatomical Record. Part A 2004, 281A: 1201-1211.

This work was supported by Russian Foundation of Fundamental Research (Grants No 99-04-49927; 02-04-49253; Scientific School-1038.2003.4), Integration Project of the Presidium of Siberian Branch of Russian Academy of Sciences (RAS) No 56, “Biosphere Origin and Evolution”, and Heiwa Nakajima Foundation Grant (Japan).

Our special thanks are to Dr. Andrey V. Koptioug for his generous assistance in preparation of English version of our paper. It was not just a translation from Russian to English but a narration of our story in terms readable to both English-speaking people, due to his long-time teaching career in Great Britain, and to non-biologists, due to his educational background as a physicist. Anyway, after his double translation we still understand what we wrote.