Uropygial Gland-Secreted Alkanols Contribute to Olfactory Sex Signals in Budgerigars

Jian-Xu Zhang 0 Wei Wei 0 Jin-Hua Zhang 0 Wei-He Yang 0 0 The State Key Laboratory of Integrated Management and Research of Insect and Rodent Pests and the Center for Integrative Studies of Biology, the Institute of Zoology, the Chinese Academy of Sciences , Beichenxi Road 1-5, Beijing 100101 , China The possible role of uropygial gland-secreted compounds in olfactory discrimination of sex or sex attractants in the budgerigar, Melopsittacus undulatus, was investigated using behavioral 2-choice tests and gas chromatography-mass spectrometry analysis. Our data showed that female budgerigars were capable of distinguishing males from females in a Y maze via body odor, indicating its sexual dimorphism. When we conducted a chemical assay of the uropygial preen gland secretions, we found 4 times more volatile octadecanol, nonadecanol, and eicosanol in ratios in males than in females, making them putative male pheromone candidates. Female birds also showed overt preferences for the odor of male preen gland secretions or the 3-alkanol blend equivalent preened onto the plumage of a male over that of female counterparts. Removal of any one alkanol was associated with a loss of attractiveness to the female. In another test device (a test cage) with visible male bird stimulus, females chose the male with the 3-alkanol blend of males over the other male with female preen gland secretion, whereas did not differentiate their responses between the males with either this blend or male preen gland secretions. The behavioral data robustly suggested that the 3 alkanols synergistically created a female attractant odor or male pheromone in the budgerigar and that bird uropygial glands have broader implications in sexual behavior than previously known. This is the first investigation with bioassay of components of the gland in a bird species. Introduction Work on a number of bird species suggests that the capacity for chemical communication in birds is likely to be comparable with that of mammals with special reference to both olfactory reception such as food searching and predator assessment and odor production (Grubb 1972; Jones and Roper 1997; Weldon and Rappole 1997; Hagelin et al. 2003; Bonadonna and Nevitt 2004; Nevitt and Bonadonna 2005; Amo et al. 2008; Nevitt 2008; Steiger et al. 2008; Balthazart and Taziaux 2009). Sex recognition serves as the first step in breeding behavior of many animals and sex attractants or sex pheromones released by animals to attract opposite sex mates are therefore likely to exist in birds as in mammals and are worthy of exploration (Brennan and Zufall 2006; Hagelin and Jones 2007; Douglas et al. 2008). Some previous studies have shown that olfactory cues are involved in sexual behavior in birds. For example, sexual behavior was significantly inhibited in male domestic ducks (Anas platyrhynchos) with olfactory nerve section (Balthazart and Schoffeniels 1979); uropygial gland-removed females are less attractive to males in domestic chickens (Gallus gallus domesticus; Hirao et al. 2009). On the other hand, sexual differences in the chemical composition of the uropygial gland waxes and volatiles in some birds such as domestic ducks, dark-eyed Juncos (Junco hyemalis), and Bengalese finches (Lonchura striata) have been detected (Jacob et al. 1979; Soini et al. 2007; Zhang et al. 2009). However, it has not been directly demonstrated that birds can use body-emitted odor for sex discrimination (Bonadonna et al. 2009). The budgerigar (Melopsittacus undulatus) is a small parrot native to Australia that lives in flocks, is the most popular caged bird worldwide, and is known to use vocal behavior, plumage coloration, and fluorescent and ultraviolet colors for sexual attractiveness (Dooling et al. 1987; Arnold et al. 2002, Eda-Fujiwara et al. 2003; Zampiga et al. 2004). Because budgerigar males and females exhibit visible sex monomorphism except cere color and lack sexually dimorphic vocalization (Dooling et al. 1987), we hypothesized that sexual odor could contribute to sex discrimination in the budgerigar. Animal sebaceous glands, particularly in mammals, are important sources of secreted pheromones, in addition to other substances (e.g., oily wax esters) (Wyatt 2003; Brennan and Zufall 2006). In rodents, the sebum gland-secreted volatiles being either male specific or greater in relative concentrations in males than in females can be viewed as male pheromone candidates and are usually verified as pheromones by bioassay (Singer et al. 1997; Sun and M uller-Schwarze 1998; Zhang et al. 2007, 2008). The uropygial gland (also called preen, oil, or scent gland) is a large gland at the base of a birds tail and is found in the large majority of birds that secrete oil used in preening. A bird typically transfers this oil to its feathers by rubbing its head against the oil and then around the rest of the body. Like the preputial gland in rodents, the preen gland serves as the largest exocrine gland and is the most likely pheromone source in most birds. For 2 particular examples, a synthetic analog of uropygial secretion of mother hens increase growth and decrease stress of chicks (Madec et al. 2006); the role of uropygial glands in mediating sexual behavior relies on olfaction domestic chickens (Hirao et al. 2009). Previous research has mainly focused on the glandular nonvolatile wax composition and functional association with light reflectance and plumage waterproofing in birds (Arnold et al. 2002; Zampiga et al. 2004; Soini et al. 2007; Jacob and Ziswiler 1982; Zhang et al. 2009). Emerging evidence shows that the avian uropygial gland produces low molecular weight volatiles that quantitatively vary with reproductive status, structurally resemble insect and mammalian pheromones, and can be preened into body plumage for likely airborne chemosignal transmission (Wyatt 2003; Burger et al. 2004; Zampiga et al. 2004; Madec et al. 2006; Bonadonna et al. 2007; Soini et al. 2007; Zhang et al. 2007, 2008; Douglas 2008), implying that the secretions might contribute to chemical sexual signaling in budgerigars. We thus hypothesized that if the gland secretion of males could attract females, it must include some volatiles exhibiting male-specific properties and/or be quantitatively heightened in males and affect behavior. Therefore, we characterized the constituents of the gland secretions to ascertain the putative male pheromone components via qualitative and quantitative comparison using gas chromatographymass spectrometry (GC-MS) and then validated the activity of the pheromone candidates using behavioral 2-choice tests. Materials and methods Animals Twenty-four male and 24 female budgerigars (yellow and green-based coloration) at 614 months of age were obtained from 3 large colonies maintained by respective pet owners. Eight males and 8 females were assigned randomly to each of 3 sex-mixed groups. The color of the cere of the subjects showed an overt difference between the (...truncated)