To explore whether preputial gland secretions and/or urine from the house mouse (Mus musculus) can be used for coding information about sex, individuality, and/or the genetic background of strain [ICR/albino, Kunming (KM), and C57BL/6], we compared the volatile compositions of mouse preputial glands and urine using a combination of dichloromethane extraction and gas chromatography coupled with mass spectrometry (GC–MS). Of the 40 identified compounds in preputial gland secretions, 31 were esters, 2 sesquiterpens, and 7 alcohols. We failed to find any compound unique to a specific sex, individual, or strain. However, many low molecular weight compounds between the sexes, most compounds among individuals, and several compounds among the 3 strains varied significantly in relative ratios. These quantitative differences in preputial gland volatiles (analog coding) are likely to convey information about sex, individual, and the genetic background of mouse strain. We identified 2 new main and male-elevated compounds, 1-hexadecanol (Z = 3.676, P = 0.000, N = 19 in ICR; Z = 3.576, P = 0.000, N = 18) and 1-hexadecanol acetate (Z = 3.429, P = 0.000, N = 19 in ICR; Z = 3.225, P = 0.001, N = 18), which were eluted in GC chromatogram after the 2 sesquiterpens. They might also be potential male pheromones, in addition to the well-known E-β-farnesene and E,E-α-farnesene. Additionally, a few compounds including 1-hexadecanol also varied with strains and might also code for genetic information. Of the 9 identified volatile compounds in male urine, (s)-2-sec-butyl-4,5-dihydrothiazole and R,R-3,4-dehydro-exo-brevicomin are known urine-originated male pheromones from previous studies. We also detected 6-hydroxy-6-methyl-3-heptanone, a male urinary pheromonal compound, which had not been directly detected by GC–MS previously. Chemical analysis shows that the genetically more closely related ICR and KM strains had a higher similarity in the volatile compositions of preputial glands and urine than that between ICR or KM and C57BL/6. R,R-3,4-dehydro-exo-brevicomin, in particular, was sensitive to genetic shifts and differed in relative abundance among the 3 strains, whereas (s)-2-sec-butyl-4,5-dihydrothiazole differed between ICR or Km and C57BL/6. Hence, these 2 compounds might code for information about their genetic background.

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.

This study was aimed at identifying sex pheromones of the rat (Rattus norvegicus). We characterized the volatiles and semivolatiles of rat preputial gland and voided urine by using gas chromatography–mass spectrometry (GC–MS) and quantified them by their GC areas (abundances) and percentage of GC areas (relative abundances). Although all the compounds other than 4-heptanone and phenol detected were shared by males and females, the quantities for some of these sex-common compounds exhibited sexual dimorphism and decreased with gonadectomy. Thus, these compounds might be sex pheromones. Among them, squalene from preputial glands and 2-heptanone and 4-ethyl phenol from urine were 3 major compounds. They were richer in males and could be suppressed by castration. Adding any of the 3 compounds (at a concentration higher than its physiological level in male urine) to castrated male urine (CMU) increased the attractiveness of CMU to sex-naive females. Adding the 3 together (at the levels in normal male urine) to CMU significantly increased the attractiveness of CMU to females. However, such combination did not fully restore females' preference for urine from intact males, suggesting that some other trace compounds such as 4-heptanone and phenol might also play some roles in sex attractiveness. Thus, squalene, 2-heptanone, and 4-ethyl phenol were indeed male pheromone molecules in rats. Our study also indicates that E,E-β-farnesene and E-α-farnesene, both richer in females than males, might be putative female pheromones.

Murine rodents are excellent models for study of adaptive radiations and speciation. Brown Norway rats (Rattus norvegicus) are successful global colonizers and the contributions of their domesticated laboratory strains to biomedical research are well established. To identify nucleotide-based speciation timing of the rat and genomic information contributing to its colonization capabilities, we analyzed 51 whole-genome sequences of wild-derived Brown Norway rats and their sibling species, R. nitidus, and identified over 20 million genetic variants in the wild Brown Norway rats that were absent in the laboratory strains, which substantially expand the reservoir of rat genetic diversity. We showed that divergence of the rat and its siblings coincided with drastic climatic changes that occurred during the Middle Pleistocene. Further, we revealed that there was a geographically widespread influx of genes between Brown Norway rats and the sibling species following the divergence, resulting in numerous introgressed regions in the genomes of admixed Brown Norway rats. Intriguing, genes related to chemical communications among these introgressed regions appeared to contribute to the population-specific adaptations of the admixed Brown Norway rats. Our data reveals evolutionary history of the Brown Norway rat, and offers new insights into the role of climatic changes in speciation of animals and the effect of interspecies introgression on animal adaptation.

Olfactory cues play a vital role in kin recognition and mate choice of the rat. Here, using 2 inbred strains of rats, Brown Norway (BN) and Lewis, as models to simulate kinship via genetic distance, we examined whether urine-derived volatiles are genetically determined, and, if so, how they code for olfactory information and the degree of genetic relatedness in mate choice. Binary choice tests showed that BN females preferred the urine odor of Lewis males over that of BN males, suggesting that they avoided males genetically similar to themselves and were able to assess this olfactorily. Gas chromatography–mass spectrometry analysis revealed that the composition of urine-derived volatiles was more similar within strains than between strains and suggests that odortypes may reflect genetic relatedness. Our data further show that BN males had lower ratios of 2-heptanone and 4-heptanone and higher ratios of dimethyl sulfone and 4-ethyl phenol than Lewis males. When we supplemented BN and Lewis male urine to make each similar, the preferences of BN females were reversed. We conclude that some urine-derived volatiles covary in relative abundance with degree of genetic relatedness, and this relationship may play a key role in chemical signaling and genetic identity in this species.