Assessing the effect of compounds from plantar foot sweat, nesting material, and urine on social behavior in male mice, Mus musculus.

Home cage aggression causes poor welfare in male laboratory mice and reduces data quality. One of the few proven strategies to reduce aggression involves preserving used nesting material at cage change. Volatile organic compounds from the nesting material and several body fluids not only correlate with less home cage aggression, but with more affiliative allo-grooming behavior. To date, these compounds have not been tested for a direct influence on male mouse social behavior. This study aimed to determine if 4 previously identified volatile compounds impact home cage interactions. A factorial design was used with cages equally split between C57BL/6N and SJL male mice (N = 40). Treatments were randomly assigned across cages and administered by spraying one compound solution on each cage's nesting material. Treatments were refreshed after day 3 and during cage change on day 7. Home cage social behavior was observed throughout the study week and immediately after cage change. Several hours after cage change, feces were collected from individual mice to measure corticosterone metabolites as an index of social stress. Wound severity was also assessed after euthanasia. Measures were analyzed with mixed models. Compound treatments did not impact most study measures. For behavior, SJL mice performed more aggression and submission, and C57BL/6N mice performed more allo-grooming. Wound severity was highest in the posterior region of both strains, and the middle back region of C57BL/6N mice. Posterior wounding also increased with more observed aggression. Corticosterone metabolites were higher in C57BL/6N mice and in mice treated with 3,4-dimethyl-1,2-cyclopentanedione with more wounding. These data confirm previous strain patterns in social behavior and further validates wound assessment as a measure of escalated aggression. The lack of observed treatment effects could be due to limitations in the compound administration procedure and/or the previous correlation study, which is further discussed.

Compounds from plantar foot sweat, nesting material, and urine show strain patterns associated with agonistic and affiliative behaviors in group housed male mice, Mus musculus.

Excessive home cage aggression often results in severe injury and subsequent premature euthanasia of male laboratory mice. Aggression can be reduced by transferring used nesting material during cage cleaning, which is thought to contain aggression appeasing odors from the plantar sweat glands. However, neither the composition of plantar sweat nor the deposits on used nesting material have been evaluated. The aims of this study were to (1) identify and quantify volatile compounds deposited in the nest site and (2) determine if nest and sweat compounds correlate with social behavior. Home cage aggression and affiliative behavior were evaluated in 3 strains: SJL, C57BL/6N, and A/J. Individual social rank was assessed via the tube test, because ranking may influence compound levels. Sweat and urine from the dominant and subordinate mouse in each cage, plus cage level nest samples were analyzed for volatile compound content using gas chromatography-mass spectrometry. Behavior data and odors from the nest, sweat, and urine were statistically analyzed with separate principal component analyses (PCA). Significant components, from each sample analysis, and strain were run in mixed models to test if odors were associated with behavior. Aggressive and affiliative behaviors were primarily impacted by strain. However, compound PCs were also impacted by strain, showing that strain accounts for any relationship between odors and behavior. C57BL/6N cages displayed the most allo-grooming behavior and had high scores on sweat PC1. SJL cages displayed the most aggression, with high scores on urine PC2 and low scores on nest PC1. These data show that certain compounds in nesting material, urine, and sweat display strain specific patterns which match strain specific behavior patterns. These results provide preliminary information about the connection between home cage compounds and behavior. Salient compounds will be candidates for future controlled studies to determine their direct effect on mouse social behavior.

Volatile fatty acid and aldehyde abundances evolve with behavior and habitat temperature in Sceloporus lizards.

Animal signals evolve by striking a balance between the need to convey information through particular habitats and the limitations of what types of signals can most easily be produced and perceived. Here, we present new results from field measures of undisturbed behavior and biochemical analyses of scent marks from 12 species of Sceloporus lizards to explore whether evolutionary changes in chemical composition are better predicted by measures of species behavior, particularly those associated with visual displays, chemoreception, and locomotion, or by measures of habitat climate (precipitation and temperature). We found that more active lizard species used fewer compounds in their volatile scent marks, perhaps conveying less specific information about individual and species identity. Scent marks from more active lizard species also had higher proportions of saturated fatty acids, and the evolution of these compounds has been tracking the phylogeny closely as we would expect for a metabolic byproduct. In contrast, the proportions of unsaturated fatty acids were better explained by evolutionary shifts in habitat temperature (and not precipitation), with species in warmer climates using almost no volatile unsaturated fatty acids. The proportion of aldehydes was explained by both behavior and environment, decreasing with behavioral activity and increasing with habitat temperature. Our results highlight the evolutionary flexibility of complex chemical signals, with different chemical compounds responding to different elements of the selective landscape over evolutionary time.

Structural Identification, Synthesis and Biological Activity of Two Volatile Cyclic Dipeptides in a Terrestrial Vertebrate.

Single substances within complex vertebrate chemical signals could be physiologically or behaviourally active. However, the vast diversity in chemical structure, physical properties and molecular size of semiochemicals makes identifying pheromonally active compounds no easy task. Here, we identified two volatile cyclic dipeptides, cyclo(L-Leu-L-Pro) and cyclo(L-Pro-L-Pro), from the complex mixture of a chemical signal in terrestrial vertebrates (lizard genus Sceloporus), synthesised one of them and investigated their biological activity in male intra-specific communication. In a series of behavioural trials, lizards performed more chemosensory behaviour (tongue flicks, lip smacks and substrate lickings) when presented with the synthesised cyclo(L-Pro-L-Pro) chemical blend, compared to the controls, the cyclo(L-Leu-L-Pro) blend, or a combined blend with both cyclic dipeptides. The results suggest a potential semiochemical role of cyclo(L-Pro-L-Pro) and a modulating effect of cyclo(L-Leu-L-Pro) that may depend on the relative concentration of both compounds in the chemical signal. In addition, our results stress how minor compounds in complex mixtures can produce a meaningful behavioural response, how small differences in structural design are crucial for biological activity, and highlight the need for more studies to determine the complete functional landscape of biologically relevant compounds.

Beta-caryophyllene enhances wound healing through multiple routes.

Beta-caryophyllene is an odoriferous bicyclic sesquiterpene found in various herbs and spices. Recently, it was found that beta-caryophyllene is a ligand of the cannabinoid receptor 2 (CB2). Activation of CB2 will decrease pain, a major signal for inflammatory responses. We hypothesized that beta-caryophyllene can affect wound healing by decreasing inflammation. Here we show that cutaneous wounds of mice treated with beta-caryophyllene had enhanced re-epithelialization. The treated tissue showed increased cell proliferation and cells treated with beta-caryophyllene showed enhanced cell migration, suggesting that the higher re-epithelialization is due to enhanced cell proliferation and cell migration. The treated tissues also had up-regulated gene expression for hair follicle bulge stem cells. Olfactory receptors were not involved in the enhanced wound healing. Transient Receptor Potential channel genes were up-regulated in the injured skin exposed to beta-caryophyllene. Interestingly, there were sex differences in the impact of beta- caryophyllene as only the injured skin of female mice had enhanced re-epithelialization after exposure to beta-caryophyllene. Our study suggests that chemical compounds included in essential oils have the capability to improve wound healing, an effect generated by synergetic impacts of multiple pathways.

Experimental evidence that symbiotic bacteria produce chemical cues in a songbird.

Symbiotic microbes that inhabit animal scent glands can produce volatile compounds used as chemical signals by the host animal. Though several studies have demonstrated correlations between scent gland bacterial community structure and host animal odour profiles, none have systematically demonstrated a causal relationship. In birds, volatile compounds in preen oil secreted by the uropygial gland serve as chemical cues and signals. Here, we tested whether manipulating the uropygial gland microbial community affects chemical profiles in the dark-eyed junco (Junco hyemalis). We found an effect of antibiotic treatment targeting the uropygial gland on both bacterial and volatile profiles. In a second experiment, we cultured bacteria from junco preen oil, and found that all of the cultivars produced at least one volatile compound common in junco preen oil, and that most cultivars produced multiple preen oil volatiles. In both experiments, we identified experimentally generated patterns in specific volatile compounds previously shown to predict junco reproductive success. Together, our data provide experimental support for the hypothesis that symbiotic bacteria produce behaviourally relevant volatile compounds within avian chemical cues and signals.

Songbird chemical signals reflect uropygial gland androgen sensitivity and predict aggression: implications for the role of the periphery in chemosignaling.

Chemical signals can provide useful information to potential mates and rivals. The production mechanisms of these signals are poorly understood in birds, despite emerging evidence that volatile compounds from preen oil may serve as chemosignals. Steroid hormones, including testosterone (T), may influence the production of these signals, yet variation in circulating T only partly accounts for this variation. We hypothesized that odor is a T-mediated signal of an individual's phenotype, regulated in part by androgen sensitivity in the uropygial gland. We quantified natural variation in chemosignals, T, uropygial gland androgen sensitivity, and aggressive behavior in dark-eyed juncos (Junco hyemalis). The interaction between circulating T and androgen receptor transcript abundance significantly correlated with volatile concentrations in male, but not female, preen oil. In both sexes, odorant variables correlated with aggressive response to an intruder. Our results suggest that preen oil volatiles could function as signals of aggressive intent, and, at least in males, may be regulated by local androgen receptor signaling in the uropygial gland. Because these behavioral and chemical traits have been linked with reproductive success, local regulation of androgen sensitivity in the periphery has the potential to be a target of selection in the evolution of avian olfactory signaling.

Evolutionary Interactions Between Visual and Chemical Signals: Chemosignals Compensate for the Loss of a Visual Signal in Male Sceloporus Lizards.

Animals rely on multimodal signals to obtain information from conspecifics through alternative sensory systems, and the evolutionary loss of a signal in one modality may lead to compensation through increased use of signals in an alternative modality. We investigated associations between chemical signaling and evolutionary loss of abdominal color patches in males of four species (two plain-bellied and two colorful-bellied) of Sceloporus lizards. We conducted field trials to compare behavioral responses of male lizards to swabs with femoral gland (FG) secretions from conspecific males and control swabs (clean paper). We also analyzed the volatile organic compound (VOC) composition of male FG secretions by stir bar extraction and gas chromatography-mass spectrometry (GC-MS) to test the hypothesis that loss of the visual signal is associated with elaboration of the chemical signal. Males of plain-bellied, but not colorful-bellied species exhibited different rates of visual displays when exposed to swabs of conspecific FG secretions relative to control swabs. The VOC composition of male Sceloporus FG secretions was similar across all four species, and no clear association between relative abundances of VOCs and evolutionary loss of abdominal color patches was observed. The emerging pattern is that behavioral responses to conspecific chemical signals are species- and context-specific in male Sceloporus, and compensatory changes in receivers, but not signalers may be involved in mediating increased responsiveness to chemical signals in males of plain-bellied species.

Urinary volatile compounds differ across reproductive phenotypes and following aggression in male Siberian hamsters.

Chemical communication plays an integral role in social behavior by facilitating social encounters, allowing for the evaluation of social partners, defining territories and advertising information such as species and sex. Odors provide information about the social environment for rodents and other mammals; however, studies identifying chemical compounds and their functions have thus far focused primarily on a few species. In addition, considerably less attention has been focused on how environmental factors and behavioral context alter these compounds during periods of reproductive quiescence. We examined the effects of photoperiod and social context on chemical communication in the seasonally breeding Siberian hamster which displays modest territorial aggression during long "summer-like" days, but increased aggression in short "winter-like" days. We collected urine samples from long- and short-day male hamsters to investigate how photoperiod and subsequent changes in reproductive phenotype alter urinary volatile compound profiles. Next, we identified changes in urinary compounds before and after an aggressive encounter. Male hamsters exhibited a diverse urinary profile across photoperiods; however, long-day reproductive males showed higher levels of individual compounds when compared to short-day non-reproductive males. In addition, individual compounds were altered following an aggressive encounter; some changed only in long days whereas others changed regardless of photoperiod. Further, aggression and circulating levels of testosterone were positively correlated with urinary compounds in long-, but not short-day males. These findings suggest both photoperiod- and aggression-specific physiological regulation of urinary compounds in this species and contribute to a greater understanding of chemical communication more broadly.

Photoperiod and aggression induce changes in ventral gland compounds exclusively in male Siberian hamsters.

Chemical communication is a critical component of social behavior as it facilitates social encounters, allows for evaluation of the social partner, defines territories and resources, and advertises information such as sex and physiological state of an animal. Odors provide a key source of information about the social environment to rodents; however, studies identifying chemical compounds have thus far focused primarily on few species, particularly the house mouse. Moreover, considerably less attention has been focused on how environmental factors, reproductive phenotype, and behavioral context alter these compounds outside of reproduction. We examined the effects of photoperiod, sex, and social context on chemical communication in the seasonally breeding Siberian hamster. We sampled ventral gland secretions in both male and female hamsters before and after an aggressive encounter and identified changes in a range of volatile compounds. Next, we investigated how photoperiod, reproductive phenotype, and aggression altered ventral gland volatile compound composition across the sexes. Males exhibited a more diverse chemical composition, more sex-specific volatiles, and showed higher levels of excretion compared to females. Individual volatiles were also differentially excreted across photoperiod and reproductive phenotype, as well as differentially altered in response to an aggressive encounter. Female volatile compound composition, in contrast, did not differ across photoperiods or in response to aggression. Collectively, these data contribute to a greater understanding of context-dependent changes in chemical communication in a seasonally breeding rodent.

Are single odorous components of a predator sufficient to elicit defensive behaviors in prey species?

When exposed to the odor of a sympatric predator, prey animals typically display escape or defensive responses. These phenomena have been well-documented, especially in rodents, when exposed to the odor of a cat, ferret, or fox. As a result of these experiments new discussions center on the following questions: (1) is a single volatile compound such as a major or a minor mixture constituent in urine or feces, emitted by the predator sufficient to cause defensive reactions in a potential prey species or (2) is a whole array of odors required to elicit a response and (3) will the relative size or escapability of the prey as compared to the predator influence responsiveness. Most predator-prey studies on this topic have been performed in the laboratory or under semi-natural conditions. Field studies could help to find answers to these questions. Australian mammals are completely naïve toward the introduced placental carnivores. That offers ideal opportunities to analyze in the field the responses of potential prey species to unknown predator odors. During the last decades researchers have accumulated an enormous amount of data exploring the effects of eutherian predator odors on native marsupial mammals. In this review, we will give a survey about the development of olfactory research, chemical signals and their influence on the behavior and-in some cases-physiology of prey species. In addition, we report on the effects of predator odor experiments performed under natural conditions in Australia. When studying all these literature we learned that data gained under controlled laboratory conditions elucidate the role of individual odors on brain structures and ultimately on a comparatively narrow range behaviors. In contrast to single odors odor arrays mimic much more the situation prey animals are confronted to in nature. Therefore, a broad range of methodology-from chemistry to ecology including anatomy, physiology, and behavior-is needed to understand all the different (relevant) stimuli that govern and guide the interactions between a predator and its potential prey.

Cross-generational impact of a male murine pheromone 2-sec-butyl-4,5- dihydrothiazole in female mice.

The current understanding of the activity of mammalian pheromones is that endocrine and behavioural effects are limited to the exposed individuals. Here, we demonstrate that the nasal exposure of female mice to a male murine pheromone stimulates expansion of mammary glands, leading to prolonged nursing of pups. Subsequent behavioural testing of the pups from pheromone-exposed dams exhibited enhanced learning. Sialic acid components in the milk are known to be involved in brain development. We hypothesized that the offspring might have received more of this key nutrient that promotes brain development. The mRNA for polysialyltransferase, which produces polysialylated neural cell adhesion molecules related to brain development,was increased in the brain of offspring of pheromone-exposed dams at post-natal day 10, while it was not different at embryonic stages, indicating possible differential brain development during early post-natal life.

Behavioral responses of predator-naïve dwarf hamsters (Phodopus campbelli) to odor cues of the European ferret fed with different prey species.

Many mammalian predators are able to identify their prey by odors and, vice versa, numerous prey species recognize predator odors as well. The present paper reports on the behavioral responses of predator-naïve dwarf hamsters (Phodopus campbelli) towards the urine odors of carnivorous ferrets, which were raised on either a chicken, mouse or hamster diet. Chemical composition from ferret urines of the different diet groups was analyzed, while quantitative differences in urinary volatile constituents were observed through capillary gas chromatography­mass spectrometry. In a Y-maze arrangement, hamsters were offered several two-choice odor discrimination tasks and their behavior was quantified. Hamsters were easily able to discriminate the urine odor of ferrets fed with mice against ferrets fed with hamsters. This is probably the first report indicating that a prey species can distinguish urine odors of even an unknown predator species that has been fed different prey species. The analytical data complemented behavioral assays.

Variation in preen oil composition pertaining to season, sex, and genotype in the polymorphic white-throated sparrow.

Evidence for the the ability of birds to detect olfactory signals is now well documented, yet it remains unclear whether birds secrete chemicals that can be used as social cues. A potential source of chemical cues in birds is the secretion from the uropygial gland, or preen gland, which is thought to waterproof, maintain, and protect feathers from ectoparasites. However, it is possible that preen oil also may be used for individual recognition, mate choice, and signalling social/sexual status. If preen oil secretions can be used as socio-olfactory signals, we should be able to identify the volatile components that could make the secretions more detectable, determine the seasonality of these secretions, and determine whether olfactory signals differ among relevant social groups. We examined the seasonal differences in volatile compounds of the preen oil of captive white-throated sparrows, Zonotrichia albicollis. This species is polymorphic and has genetically determined morphs that occur in both sexes. Mating is almost exclusively disassortative with respect to morph, suggesting strong mate choice. By sampling the preen oil from captive birds in breeding and non-breeding conditions, we identified candidate chemical signals that varied according to season, sex, morph, and species. Linear alcohols with a 10-18 carbon chains, as well as methyl ketones and carboxylic acids, were the most abundant volatile compounds. Both the variety and abundances of some of these compounds were different between the sexes and morphs, with one morph secreting more volatile compounds in the non-breeding season than the other. In addition, 12 compounds were seasonally elevated in amount, and were secreted in high amounts in males. Finally, we found that preen oil signatures tended to be species-specific, with white-throated sparrows differing from the closely related Junco in the abundances and/or prevalence of at least three compounds. Our data suggest roles for preen oil secretions and avian olfaction in both non-social as well as social interactions.

Pheromone-induced cell proliferation in the murine subventricular zone.

Enhancement of adult neurogenesis in female mice was previously demonstrated through exposure to soiled bedding from males, although the identity of relevant chemosignals has remained unknown. The farnesenes and SBT (2-sec-butyl-4,5-dihydrothiazole) are male murine pheromones that dominant males secrete at higher levels. Previous studies have shown that they induce oestrus in female mice. We have recently shown that these pheromones strongly increase cell proliferation in the SVZ (subventricular zone) of adult female mice. In addition, we found that a female murine pheromone, 2,5-dimethylpyrazine, facilitates similar changes in males. 2,5-dimethylpyrazine is a female pheromone that is secreted when females are housed in large groups and it was originally found to suppress oestrus in females. We found that it does not have suppressive effect on the cell proliferation in the SVZ of females. Similarly, male murine pheromones, SBT and the farnesenes, do not show a suppressive effect on the cell proliferation in the SVZ of males. Our results demonstrated that pheromonal communication between males and females has strong stimulatory effect on both the reproductive physiology and brain cell proliferation, but intrasex pheromonal exchanges do not reduce progenitor proliferation in these brain regions.

Modulation of social behavior by the agouti pigmentation gene.

Agouti is a secreted neuropeptide that acts as an endogenous antagonist of melanocortin receptors. Mice and rats lacking agouti (called non-agouti) have dark fur due to a disinhibition of melanocortin signaling and pigment deposition in the hair follicle. Non-agouti animals have also been reported to exhibit altered behavior, despite no evidence for the expression of agouti outside the skin. Here we confirm that non-agouti mice show altered social behavior and uncover expression of agouti in the preputial gland, a sebaceous organ in the urinary tract that secretes molecules involved in social behavior. Non-agouti mice had enlarged preputial glands and altered levels of putative preputial pheromones and surgical removal of the gland reversed the behavioral phenotype. These findings demonstrate the existence of an autologous, out-of-skin pathway for the modulation of social behavior.

Chemosignaling diversity in songbirds: chromatographic profiling of preen oil volatiles in different species.

Large foraging seabirds are known to navigate to food sources using their excellent sense of smell, but much less is known about the use of olfaction by the songbirds (passerine birds). Some evidence of individual recognition based on the bird preen oil volatile organic compound (VOC) compositions, which is the main odor source in birds, have been reported for dark-eyed junco and house finch. In this study we have investigated preen oil VOCs in 16 different songbird species and two other small bird species in order to determine whether the VOC compositions follow phylogenetic and evolutionary relatedness. We have used the stir bar sorptive extraction (SBSE) methodology followed by gas chromatography-mass spectrometry (GC-MS) to determine preen oil VOCs during the long light summer conditions for mostly wild caught birds. Large diversity among the VOC compositions was observed, while some compound classes were found in almost all species. The divergent VOC profiles did not follow the phylogenetic family lines among the bird species. This suggests that songbirds may use VOC odors as a mate recognition cue.

Analysis of volatile mouse pheromones by gas chromatography mass spectrometry.

High-precision quantitative profiling of volatile organic constituents in rodent physiological fluids and glandular secretions is needed to relate olfactory signals to physiology and behavior. Whereas capillary gas chromatography-mass spectrometry (GC-MS) analysis has become the most widely applied in such investigations, the extraction and preconcentration of volatile organics is arguably the most critical step in the overall analytical task. In this chapter, we describe technical details of two main sample extraction procedures used in our laboratory: dynamic headspace trapping, and stir bar sorptive extraction (SBSE). They have been demonstrated here for the chromatographic analysis of mouse urine, serum, saliva, and preputial gland specimens.

Stimulation of cell proliferation in the subventricular zone by synthetic murine pheromones.

Adult neurogenesis in female mice is known to be enhanced by exposure to soiled bedding from males, although the identity of the relevant chemosignals has remained unknown. Here we show that the previously recognized male murine pheromones, the farnesenes and 2-sec-butyl-4,5-dihydrothiazole (SBT), strongly increase cell proliferation in the subventricular zone (SVZ) of adult female mice, but not younger female mice. In addition, we found that a unique female murine pheromone, 2,5-dimethylpyrazine, facilitates similar changes in males. SBT stimulated cell proliferation in the SVZ of only adult females and not in young adult or pre- and post-puberty females. Our study suggests that pheromonal communication between males and females is enhancing reproductive success by controlling the estrous cycle and by promoting cell proliferation in a reciprocal manner.

Investigation of scents on cheeks and foreheads of large felines in connection to the facial marking behavior.

We investigated head- and cheek-rubbing behavior in four species of large felines, lions (Leo panther), leopards (Panthera pardus), tigers (Panthera tigris), and cougars (Puma concolor), in captivity. Preliminary behavioral observations found that lions and tigers, but not leopards and cougars, showed behavioral responses to cardboard rubbing samples from head and cheek areas from conspecific felines, compared to the blank cardboard controls. In this context, surface samples on the facial areas of each species were collected to analyze volatile organic compounds that could be involved in the facial marking of felines. Previously developed stir bar surface sampling methodology was used. From all cheek and forehead samples, 100 volatile organic compounds were identified or tentatively identified. Among these, 41 have been previously reported to be present in feline urine and marking secretions. Several new compounds were identified on facial surfaces. Some of the compounds showed substantial quantitative differences among the species. One compound, that has not been reported previously in mammals, 3-acetamidofuran, was found in all investigated species. It was synthesized and tested for behavioral responses. No responses were elicited in a preliminary test. Future research will test other potential signaling compounds and their mixtures for ability to elicit behavioral responses.