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Final Report Summary - BIRDSCENTS (Role and function of olfaction in Seabirds)

The project titled ‘Birdscents’ assessed the role and function of olfaction in seabirds. Its specific objectives were to identify i) the nest odour and ii) the individual odour in blue petrels (Halobaena caerulea, Gmelin 1789), a seabird living in the Southern Ocean and breed on small oceanic islands around Antarctica.

After a foraging trip, petrels return to their nest at night (Mougeot & Bretagnolle 2000; Bonadonna et al. 2001). The characteristic scent of burrows, together with petrels’ olfactory capabilities, suggests that olfaction could be involved in the nocturnal locating of the burrow. In particular, experiments have shown that only species returning to their colony at night need olfaction to locate their nest; birds that return during the day find their nests regardless of whether or not their olfaction has been experimentally impaired (Bonadonna & Bretagnolle 2002). We cannot, however, exclude the possibility that in natural conditions birds rely on a combination of olfactory and visual cues to locate burrows. The fact that individuals return annually to the same nest in the same colony strongly suggests that nests carry a specific odour, which attracts and orientates birds towards their own nests and that this odour is sufficiently stable to last over time. Olfactory cues that lead blue petrels towards the burrow entrance might have a variety of sources such as owner’s feathers and glandular excretions and other material such as dead branches of Acaena magellanica, which the birds use to rub against (Bonadonna et al. 2004). The chemical composition of nest odour, the nature of components that facilitate recognition even after a year-long absence, as well as the extent and nature of variation between nests, years, seasons, etc. remain unknown. Indeed some components of individual scents might stay in the nest and aid in recognition of the burrow each year. The aim of the project is to identify and characterise the chemical components from nests and birds that constitute the odour of individual nests and facilitate homing.

Appropriateness of research methodology and approach.

Our investigation into chemical signalling in Petrels targeted nature and origin of the airborne chemical signals released directly from plumage of birds and nests. To date only a few studies on vertebrate chemical signalling have focused directly on the volatile chemicals emitted by an animal and no published study has focused on the actual airborne volatiles emitted by birds (Cardé & Millar 2004; Röck et al. 2006). The reasons for this gap lie, in part, in the relative recent interest into the role of chemical signalling in birds and, most likely, in a corresponding lack of an appropriate methodological framework and experimental difficulties associated with collecting such samples. This project proposed to develop a robust and reproducible methodology for capture, analysis and characterization of airborne volatiles.

Samples for the present study were collected during two austral summers (fields seasons 2014-2015 and 2015-2016), on “Ile Verte”, (49°51'S, 70°05'E), a small sub-Antarctic island of the Kerguelen archipelago, a French territory located in the southern Indian Ocean. In the field, birds were removed from their burrow and transported to the field laboratory established on the island, in an opaque cotton bag. Around 100 mg of feathers was cut from the bird’s ventral duvet, packed in nalophan® and aluminium foil placed in a sealed plastic bag. At the same time, samples of all nest materials (e.g. plant material, feathers and soil etc.) were collected in glass jars. Feathers and nests materials were stored at -8 °C in the field station and at -20 °C in the laboratory at Cardiff.

Initially, samples from birds and nests, collected as described above, were sampled using i) solid-phase microextraction (SPME) fibres, ii) direct thermal desorption (DTD). Then we explored a novel method to capture the nest airborne by headspace sampled in-situ on thermal desorption tubes following protocols that have been successfully tested and established (see below).

Specifying nest odours – Identifying a homing signal.

We studied nest materials (e.g. plant material and soil etc.) from blue petrels and Antarctic prions (closest sister clade (Rheindt & Austin 2005) with similar ecology and nesting behaviour) during the 2013-2014 and 2014-2015 field seasons. We collected material from nest i) with incubating bird, ii) with a cold egg (parents feeding in the sea), iii) with a bird alone, iv) with a chick and v) an empty nest. Also we collected soil and plant material from the ground of the colony as control. Preliminary analysis showed that a 2 cm DVB/CAR/PDMS composite fibre (50/30 µm, Supelco) catches a wider range of substances than any other fibre coating (C. Müller personal data). Subsequently, the fibre was exposed to the headspace of nesting material at 60 °C in water bath for 1 h before immediate desorption in the injection port of a GC-MS for analysis. GC-MS data were processed and integrated using the Automated Mass Detection and Identification System (AMDIS, NIST). Data were studied by using presence and absence analysis. Contaminants were removed for further statistical tests and evaluation.
We observed 118 volatiles compounds in all samples. We compared of chemical profiles of the same nests over two subsequent years. Results showed an effect of the year (Permanova test: F1,75=17.81 p=0.0001, Figure 1a). Also materials collected from nests and from the ground were significantly different (F1,75=5.19 p=0.0001, Figure 1b). When we considered the species factor, we had a significant variation between nest materials from burrow used by blue petrels and prions (F1,47=5.02 p=0.0005, Figure 1c). In addition, we observed a significant variation of chemical profiles between nest materials from blue petrel nest with an adult incubating, with alone egg, with a blue petrel chick, with a prion incubating and a prion chick (F4,42=2.32 p=0.0005, Figure 1d). Finally, results showed an effect of the nest origin on the chemical profiles (F32,64=1.46 p=0.04).

In particular we explored a novel method to sample airborne volatiles in situ from nest of blue petrels. Volatile Organic Compounds (VOCs) were directly collected from the burrow onto a sequence of two thermo-desorption tubes the first packed with Tenax-TA and the second with Sulficarb over 3 h active sampling at a flow rate of 50ml/min. We collected samples during two field seasons (2014-2015 and 2015-2016) from: i) nests with birds and egg, ii) empty nests without bird and egg and iii) nests with only the egg. VOCs from controls of tubes and colony were also collected. All tubes were capped after collection and stored at ambient temperature until laboratory analysis. TD-GC-MS-TOF analyses clearly showed that the method was able to catch the scent of nests. GC-MS data were processed and integrated using the Automated Mass Detection and Identification System (AMDIS, NIST). Integrated areas were standardized by adding an internal standard before the analysis of tubes. Contaminants were removed for further statistical tests and evaluation.
Results showed the nests yielded approx. 98 compounds. The comparison of relative proportion of volatile organic compounds displayed an effect of the year (Permanova test: F1,75=14.61 p=0.0001). Also chemical profiles were significantly different between nest with a bird incubating, with a cold egg, with a chick and empty nest (F3,68=1.55 p=0.03). Finally the chemical profiles from nest airborne were affected by the nest origin (F22,68=1.56 p=0.003).

What is the real odour of blue petrel? Identifying individual scents.

Similarly to the nest study, we explored different laboratory methods for extraction of VOCs from feathers. Firstly we tested solid-phase microextraction (SPME). Exposure to the feathers in a small glass vial at 60 °C in water bath for 1h, 2h and 4h yielded no discernibly different profile compared to empty vials as control.
Secondly, we tested direct thermal desorption of feathers in TD-GC-MS-TOF. Feathers were directly placed in a ‘Loose Fit’ Teflon® insert (Markes International) and transferred into empty TD tube for analysis. A low desorption temperature was applied to the tube and VOCs collected in a secondary trap for transfer into the GC-MS. First trials showed an abundant significant chemical profile. GC-MS data were processed and integrated using the Automated Mass Detection and Identification System (AMDIS, NIST). Integrated areas were standardized by adding an internal standard before the analysis of tubes. Contaminants were removed for further statistical tests and evaluation. We compared the chemical profiles from feathers of two consequently years for same individual (n=10). We found 120 compounds in all profiles. We observed an effect of the year (F1,19=2.19 p=0.03) and also an effect of individual (F9,19=1.53 p=0.02).

Final considerations.

Both methods for the volatile compounds emanating from nests showed that nest odours might change or evaluate each year. Also nest odour seems to differ with the species and, more interestingly, with the occupancy of nest (incubating bird, egg, bird alone or empty nest). Importantly, our data with these two methods displayed a nest label, which might be related with the homing behaviour and characteristics of these birds. VOCs emanating from feathers displayed an individual label which might play a role in the social communication and in the homing recognition for this species. We are currently identifying all volatile compounds found in these methods from nest and feathers. The next step will be to compare these VOCs from nest to the VOCs from feathers of individuals breeding in each nest sampled will allow for the presence of a homing label. For example samples from nest airborne and feathers share 26 compounds. Some compounds from the individual label might be present in the nest odour and facilitate the homing behaviour.

References: Mougeot, F., & Bretagnolle, V. 2000. J.Avian Biol. 31:376-387. Bonadonna, F., Spaggiari, J., & Weimerskirch, H. 2001. J.Exp.Biol. 204:1485-89. Bonadonna, F., & Bretagnolle, V. 2002. J. Exp. Biol. 205:2519-2523. Bonadonna, F., Villafane, M., Bajzak, C., & Jouventin, P. 2004. Anim. Behav. 67:893-898. Cardé, R.T., & Millar, J.G. 2004. Advances in Insect Chemical Ecology. CUP, Cambridge. 34. Röck, F., Mueller, S., Weimar, U., Rammensee, H.G., & Overath, P. 2006. J.Chem.Ecol. 32:1333-46.

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