The giant panda Ailuropoda melanoleuca belongs to the family of Ursidae; however, it is not carnivorous, feeding almost exclusively on bamboo. Being equipped with a typical carnivorous digestive apparatus, the giant panda cannot get enough energy for an active life and spends most of its time digesting food or sleeping. Feeding and mating are both regulated by odors and pheromones; therefore, a better knowledge of olfaction at the molecular level can help in designing strategies for the conservation of this species. In this context, we have identified the odorant-binding protein (OBP) repertoire of the giant panda and mapped the protein expression in nasal mucus and saliva through proteomics. Four OBPs have been identified in nasal mucus, while the other two were not detected in the samples examined. In particular, AimelOBP3 is similar to a subset of OBPs reported as pheromone carriers in the urine of rodents, saliva of the boar, and seminal fluid of the rabbit. We expressed this protein, mapped its binding specificity, and determined its crystal structure. Structural data guided the design and preparation of three protein mutants bearing single-amino acid replacements in the ligand-binding pocket, for which the corresponding binding affinity spectra were measured. We also expressed AimelOBP5, which is markedly different from AimelOBP3 and complementary in its binding spectrum. By comparing our binding data with the structures of bamboo volatiles and those of typical mammalian pheromones, we formulate hypotheses on which may be the most relevant semiochemicals for the giant panda.odorant-binding proteins | chemical communication | X-ray structure | proteomics | giant panda T he giant panda Ailuropoda melanoleuca is endemic of China and was formerly classified as an endangered species, now as a vulnerable species, but its population has remained rather stable, although very low, during the last centuries (1). Its phylogenetic classification has been a matter of debate for some time, but molecular genetic studies have recently shown that this species belongs to Ursidae, of which it represents an ancestral branch together with the spectacled bears, Tremarctos, and the sloth bear (1-3). The diets of these species are different from those of carnivorous bears: the giant panda is fully herbivorous, the spectacled bears are mainly herbivorous, and sloth bears feed on termites, fruits, and vegetables. The giant panda also shares with spectacled bears and sloth bears the absence of hibernation, an important characteristic that differentiates these species from other Ursidae (4).The obligate bamboo diet of the giant panda, which is not compatible with its carnivorous digestive system, is barely sufficient to provide the energy required for an active life, likely accounting for the slow movements and long periods of rest typical of this species (5). It has been also suggested that the reduced size of the brain, liver, and kidneys of the giant panda relative to other mammals could be a measure to further reduce the use of i...

Anogenital gland secretions play a major role in chemical communication by giant pandas, Ailuropoda melanoleuca (David, 1869). We analyzed 45 samples of anogenital gland secretions collected from 24 captive pandas (5 male adults, 6 female adults, 6 male subadults, and 7 female subadults) by gas chromatography and mass spectrometry. The secretions contained over 95 compounds. Based on 56 common compounds (relative abundances >0.1%) shared by more than three individuals, we identified steroids, long-chain fatty acids, fatty-acid esters, aldehydes, alkanes, alkenes, amines, terpenes, and furans. The chemical composition of each secretion was individual-specific. Although none of these individual compounds was age-or sex-specific, the relative abundances of several compounds differed between males and females and between adults and subadults. This result shows that information about sex and age could be coded in analog form. Information about age but not gender could also be digitally coded by the presence or absence of some of the 56 compounds, in addition to the analog coding.Résumé : Les sécrétions de la glande génito-anale jouent un rôle important dans la communication chimique chez les grands pandas, Ailuropoda melanoleuca (David, 1869). Nous avons analysé 45 prélèvements des sécrétions de la glande génito-anale provenant de 24 pandas en captivité (5 adultes mâles, 6 adultes femelles, 6 subadultes mâles, 7 subadultes femelles) par chromatographie gazeuse et spectrométrie de masse. Les sécrétions contiennent plus de 95 composés. Parmi les 56 des composés communs (d'abondance relative >0,1 %) présents chez plus de trois individus, nous avons identifié des stéroïdes, des acides gras à longue chaîne, des esters d'acides gras, des aldéhydes, des alcanes, des alcè-nes, des amines, des terpènes et des furanes. La composition chimique des sécrétions est propre à chaque individu. Bien qu'aucun de ces composés ne soit spécifique à un âge ou un sexe, l'abondance relative de plusieurs composés diffère entre les mâles et les femelles et entre les adultes et les subadultes. Ces résultats indiquent que l'information concernant le sexe et l'âge peut être codée de manière analogique. L'information concernant l'âge, mais non le sexe, peut aussi être codée de façon digitale d'après la présence oabsence de certains des 56 composés, en plus de la codification analogique.[Traduit par la Rédaction] Yuan et al. 1604

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With a combination of dichloromethane extraction and analysis by gas chromatography-mass spectrometry (GC-MS), we found 39 compounds (corresponding to 38 GC peaks) in the anogenital gland secretion (AGS) of captive adult giant pandas, Ailuropoda melanoleuca, during the non-mating season. In addition to indole, squalene, and some of the straight-chain fatty acids that had been characterized previously from the AGS of giant pandas, we identified several new compounds such as decenal, two isomers of decadienal, phenylacetic acid, 5-methylhydantoin, hydroquinone, phenylpropanoic acid, and erucic acid. Quantitative comparison of the relative abundances of the 20 main GC peaks revealed that 5-methylhydantoin, indole, and erucic acid are putative female pheromones, whereas squalene and hydroquinone are putative male pheromones. In addition to the presence of a few individual-specific compounds, the relative abundances of most of the 21 constituents varied more among individuals than within individuals. This suggests that individual identity might be coded in both digital and analog form. The chemical composition of different AGS samples from the same pandas consistently displayed a minimum cluster distance, much smaller than that between samples from different individuals in a hierarchical linkage cluster (average linkage) dendrogram. Our results indicate that the AGS might contain an "odor fingerprint." Although putative sex pheromones such as squalene and erucic acid should be assessed further by bioassay, our study suggests that synthetic chemosignals might be useful in modulating the behavior and physiology of giant pandas.

Stereotypic behavior is exhibited by a wide range of captive animals. Its association with hormones, especially elevated cortisol level and lack of naturalistic stimuli in the environment, has been little studied. This study hypothesizes that stereotypic behavior is caused by stress due to lack of appropriate, naturalistic stimuli in the environment. Using four adult pandas (Ailuropoda melanoleuca) in the Beijing Zoo from March-July in 2003, we tested the following predictions: 1) stereotypic behavior and fecal cortisol level will not change associated with the progress of reproductive state; 2) there is a positive correlation between the occurrence of stereotypic behavior and fecal cortisol level; and 3) environmental enrichment by adding a naturalistic stimulus will reduce both stereotypic behavior and fecal cortisol level. We did not find any significant differences in the occurrence of stereotypic behavior and fecal cortisol level but did find a significant difference in the total time engaged in displaying the stereotypic behavior among the three estrous periods. The level of stereotypic behavior was correlated with elevated fecal cortisol level. Enrichment simply by adding a naturalistic stimulus did not show significant effects on stereotypic behavior, or on fecal cortisol level. Our results supported the second prediction, but did not completely support the first and the third ones perhaps because of the small sample size. Additionally, our results showed that stereotypic behavior might be a response to a heightened level of cortisol.

Seasonally reproducing animals show many behavioral and physiological changes during the mating period, including increased signaling for intrasexual competition and mate attraction. We collected 102 anogenital gland secretions (AGS) from marking trees in Foping Nature Reserve, and used gas chromatography mass spectrometry to analyze these chemical composition. Of these marks, all but one were from males, confirmed with DNA analysis. We found that several chemical constituents, especially volatile compounds, were present only during the mating season and that the relative abundance of many compounds changed as a function of breeding season, whereas nonvolatile compounds were lower in the mating season. This seasonal variation in chemical composition of AGS most likely plays an important role in governing giant panda reproduction, including mate location, attraction, and male-male competition. The chemical properties of many of these putative chemosignals-such as volatility and longevity-are suggestive of these roles, and undoubtedly contribute to successful reproduction for this species with a characteristically sophisticated chemical communication system. We also found a number of important differences between the chemical constituents of AGS from wild pandas and those found in previous studies with captive pandas, suggesting that inappropriate chemosignal composition may contribute to poor reproductive success in captive breeding programs.

The behavior of giant pandas (Ailuropoda melanoleuca) was studied under captive conditions. Both male and female pandas spent similar amounts of time engaged in eating and locomotion. Males performed anogenital-marking more but rested less than females, which suggests a sexually dimorphic pattern of behavior. Furthermore, females housed in the seminatural environment spent significantly less time engaged in stereotyped behavior than did females housed in the traditional enclosure, indicating that an enclosure environment affects the behavior of giant pandas. These data illustrate the importance of careful management and facility design for captive giant pandas.

Chemical communication plays an important role in kin selection and mate choice in mammals. The covariance of odor-genes of rodents has been documented and kinship odor has been proposed andtermed, yet little is known of the relationship between genetic relatedness and chemical composition of kinship odors. Giant pandas (Ailuropoda melanoleuca) rely substantially on chemical communication to mediate their social interactions. To examine the relationship between genetic relatedness and compounds in the urine/anogenital gland secretions, we compared the similarities between genetic relatedness and the chemical profiles of anogenital gland secretions and urine via lineage construction and GC-MS (gas chromatography and mass spectrometry). We found that information about kinship odors was present only in the urine of male adults in the mating season but absent in the non-mating season. Adult females and all sub-adults did not have such kinship odors in either mating or non-mating season. Therefore, kinship odor in the panda was contingent on age, sex, and season. This is the first report about the condition-dependent expression of kinship odor, which may have a significant implication in the practice of panda conservation in relation to chemical communication and sexual selection.kinship odor, giant panda, Ailuropoda melanoleuca, anogenital gland secretions, urine Most mammals use chemical signals to communicate a wide range of information in their social interactions. Chemical communication is particularly important in rodents, carnivores, and ungulates, which have highly sensitive olfactory systems [1] . The coevolutionary process between signalers and receivers drives chemical signals to become diverse and specific. Many mammals have evolved several glandular and non-glandular sources to communicate different types of information [2,3] . Carnivores, in particular, use chemical communication substantially and anal (or anogenital) gland is present in most carnivores [4] , although it is still unclear whether it is due to convergence or divergence evolution. The gland secretion itself and its contaminating feces are rich sources of chemical signals. In addition, urine also widely contains chemical information in carnivores as well as other mammals [5,6] .Odors that serve to distinguish one individual from another member of the species are determined by polymorphic genes [7] . Genetically related individuals show similar odors, and kinship odor was first demonstrated to be present in golden hamsters (Mesocricetus auratus) [8] and wild beavers (Castor canadensis) [9] . Similar results