The chemical composition of the preen secretions of European hoopoes Upupa epops (Upupidae) has never been analysed. Despite these secretions are similar in colour and odour to those of the green woodhoopoe Phoeniculus purpureus, a species belonging to a close relative family (Phoeniculidae), whose composition is known (Burger et al 2004), it is important to describe the chemical profile of the secretion of the European hoopoes in order to study the role of symbiotic bacteria in its particular composition. Moreover, European hoopoes produce two very different secretions, namely dark and odorous secretions by nestlings and breeding females, and white and odourless secretions by males throughout the year and females out of the breeding season (Martín-Vivaldi et al 2009), which is a very different pattern of what happen in woodhoopoes in which the secretion is similar in all individuals throughout the life cycle.
Material and methods
For the descriptive analyses of the composition of brown and white hoopoe secretions we used 11 randomly selected nestlings of 11 different broods, and 7 adult birds (3 nonbreeding females and 4 males), respectively. For this descriptive analysis, the entire secretion available from each bird was extracted with 100 µl of dichloromethane in a glass chromatographic vial.
Chemical analyses
A Trace GC Ultra (Thermo-Finnigan) gas chromatograph was used, fitted with a CTC Analytics automatic injector and connected to a Trace DSQ (Thermo-Finnigan) mass spectrometer. A 1-mL volume of the extract was injected splitless into a fused silica DB-5 capillary column (J&W Scientific) (30 m, 0.25 mm i.d., 0.25 mm film thickness). The injector, transfer line and ion source temperatures were 250, 250, and 200 ºC, respectively. Helium was used as the carrier gas at a flow-rate of 0.7 mL min-1 and oven temperature was programmed starting at 40 ºC (1 min), ramp at 7 ºC min-1 to 250 ºC, where it was held for 5 min. A scan rate of 0.75 s/scan with an interval of 0.1 s between scans was employed, recording from m/z 1-650 in the electron impact mode, starting 3 min after injection.
Tentative identification of the volatile organic compounds was first carried out by comparison with those available in the NIST library. Then commercial standards were used and positive identification was confirmed by coincidence of spectra and retention times. Commercial compounds with purities ≥ 90% were used for comparison with constituents identified by the library. Calibration curves were prepared in dichloromethane, except for indole which was prepared in n-hexane.
Identification of all but one of the peaks obtained in the volatile fraction of chromatograms was confirmed by comparison with standards. One compound, however, has not yet been identified. This unknown compound was tentatively identified, by comparison with the library, as 2H-benzotriazole, 2-ethyl, with low matching probability, and the comparison with the standard showed that it was a misidentification. This compound was present in all the dark secretions analysed, but was absent from adult white secretions. In order to determine its properties, a secretion corresponding to a single nestling was also extracted with solvents of different polarity, i.e. methanol, ethyl acetate, isopropanol and hexane, and injected in the GC-MS. The unidentified compound did not appear in any of the extracts, except in that of dichloromethane. Given our failed identification, we have treated it as an unidentified chemical, although the predominant presence of a fragment at m/z = 91 in its mass spectrum is an indication that it includes a benzene ring in its structure.
Results and interespecific comparison
The chemical profile of a typical nestling dark secretion is shown in Figure 1. Compounds eluting between 21 and 25 min were tentatively identified as aldehydes by their mass spectra, while those eluting after 25 min were found to be waxes, and were not investigated further. Table 1 lists the volatile compounds identified and some quantitative data on the major constituents present in the secretion of 11 nestlings from 11 different broods. The list of volatiles included short-chain organic acids and benzolic and indolic derivatives. White secretions of adult birds lacked all volatile compounds.
The composition of dark secretions of hoopoes and woodhoopoes slightly differs in the volatile region of the chromatograms. While benzaldehyde, phenol, 4-methyl pentanoic acid, and indole were present in both species, butanoic, pentanoic, 2-methyl butanoic, and 3-phenyl propanoic acids, besides phenyl acetaldehyde, 4-chloro indole, and other unidentified compound of probable indolic or phenolic nature are exclusive to European hoopoes, and trimethylamine, propanoic acid, 3-methylbutanal, 3-methylbutanol, dimethyl di- and tri-sulfide, 2-phenylethanol and 2-phenylethyl acetate, are exclusive to woodhoopoes. Most of these differences were not due to methodological differences. We extracted secretions with the same solvent as Burger et al. (2004) immediately after sampling of birds transported to the laboratory, and the extract was injected only a few minutes after sampling (i.e. we did not loose any chemical due to chemical degradation, photodegradation or volatilisation). However, trimethylamine and propanoic acid were detected in woodhoopoe secretions exclusively when using the SPME technique (Burger et al. 2004), and, because we did not use this technique, it is possible that these compounds are also present in European hoopoes.
References
Burger, B. V., Reiter, B., Borzyk, O. & Du Plessis, M. A. 2004 Avian exocrine secretions. I. Chemical characterization of the volatile fraction of the uropygial secretion of the green woodhoopoe, Phoeniculus purpureus. J. Chem. Ecol. 30, 1603-1611.
Martín-Vivaldi, M., Ruiz-Rodriguez, M., Soler, J. J., Peralta-Sanchez, J. M., Méndez, M., Valdivia, E., Martín-Platero, A. M. & Martínez-Bueno, M. 2009 Seasonal, sexual and developmental differences in hoopoe preen gland morphology and secretions. Evidence for a role of bacteria. J. Avian Biol. 40: 191-205.
Table 1. Volatile compounds identified in the uropygial dark secretions of nestling hoopoes, Upupa epops. (n.d. denotes non-detected chemicals). Compounds are numbered in elution order in chromatogram of Figure 1.
(min) / Identification / Concentration (mg/100 ml)
Mean ± SD (range)
N = 11
1 / Butanoic acid / 6.9 / a, b / 0.5 ± 0.3 (0.2 - 0.9)
2 / 2-Methyl butanoic acid / 8.3 / a, b / 0.1 ± 0.1 (n.d. - 0.2)
3 / Pentanoic acid / 9.1 / a, b / 0.1 ± 0.2 (n.d. - 0.4)
4 / Benzaldehyde / 9.7 / a, b / 0.007 ± 0.012 (n.d. - 0.041)
5 / Phenol / 10.0 / a, b / 0.2 ± 0.2 (n.d. - 0.8)
6 / 4-Methyl pentanoic acid / 10.5 / a, b / 0.3 ± 0.2 (n.d. - 0.6)
7 / Phenyl acetaldehyde / 11.7 / a, b / 0.010 ± 0.005 (0.005 - 0.024)
8 / Unidentified* / 15.1 / --- / 0.002 ± 0.001 (0.001 - 0.003)
9 / Indole / 16.2 / a, b / 0.007 ± 0.004 (n.d. - 0.014)
10 / 3-Phenyl propanoic acid / 17.4 / a, b / 0.4 ± 0.2 (n.d. - 0.6)
11 / 4-Chloro indole / 20.1 / a, b / 0.02 ± 0.01 (n.d. - 0.04)
a. GC-MS analysis
b. retention time comparison with standard compounds
* Values for the unidentified compound are derived from the chromatogram areas assuming a linear response in the range studied
Figure 1. Chromatogram of the secretion of a nestling hoopoe. Peaks are numbered according to Table 1.