The uropygial gland (preen gland) of birds plays an important role in maintaining feather integrity and hygiene. Although a few studies have demonstrated potential defensive roles of bacteria residing within these glands, the diversity and functions of the uropygial gland microbiota are largely unknown. Therefore, we investigated the microbiota of great tit (Parus major) uropygial glands through both isolation of bacteria (culture-dependent) and 16S rRNA amplicon sequencing (culture-independent). Co-culture experiments of selected bacterial isolates with four known feather-degrading bacteria (Bacillus licheniformis, Kocuria rhizophila, Pseudomonas monteilii, and Dermacoccus nishinomiyaensis), two non-feather degrading feather bacteria, one common soil bacterial pathogen and two common fungal pathogens enabled us to evaluate the potential antimicrobial properties of these isolates. Our results show major differences between bacterial communities characterized using culture-dependent and -independent approaches. In the former, we were only able to isolate 12 bacterial genera (dominated by members of the Firmicutes and Actinobacteria), while amplicon sequencing identified 110 bacterial genera (dominated by Firmicutes, Bacteroidetes, and Proteobacteria). Uropygial gland bacterial isolates belonging to the genera Bacillus and Kocuria were able to suppress the growth of four of the nine tested antagonists, attesting to potential defensive roles. However, these bacterial genera were infrequent in our MiSeq results suggesting that the isolated bacteria may not be obligate gland symbionts. Furthermore, bacterial functional predictions using 16S rRNA sequences also revealed the ability of uropygial gland bacteria to produce secondary metabolites with antimicrobial properties, such as terpenes. Our findings support that uropygial gland bacteria may play a role in feather health and that bacterial symbionts might act as defensive microbes. Future investigations of these bacterial communities, with targeted approaches (e.g., bacterial isolation and chemical analyses), are thus warranted to improve our understanding of the evolution and function of these host-microbe interactions.

Biology Centre of Czech Academy of Sciences Institute of Entomology České Budějovice Czechia

Faculty of Science University of South Bohemia České Budějovice Czechia

Natural History Museum of Denmark University of Copenhagen Copenhagen Denmark

Section for Ecology and Evolution Department of Biology University of Copenhagen Copenhagen Denmark

See more in PubMed

Bodawatta K. H., Puzejova K., Sam K., Poulsen M., Jønsson K. A. (2020). Cloacal swabs and alcohol bird specimens are good proxies for compositional analyses of gut microbial communities of Great tits (Parus major). BMC Anim. Microb. 2:13 10.1186/s42523-020-00026-8 PubMed DOI PMC

Bodawatta K. H., Sam K., Jønsson K. A., Poulsen M. (2018). Comparative analyses of the digestive tract microbiota of New Guinean passerine birds. Front. Microb. 9:1830. 10.3389/fmicb.2018.01830 PubMed DOI PMC

Bolyen E., Rideout J. R., Dillon M. R., Bokulich N. A., Abnet C. C., Al-Ghalith G. A., et al. (2019). Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. Nat. Biotechnol. 37 852–857. 10.1038/s41587-019-0209-9 PubMed DOI PMC

Braun M. S., Sporer F., Zimmermann S., Wink M. (2018a). Birds, feather-degrading bacteria and preen glands: the antimicrobial activity of preen gland secretions from turkeys (Meleagris gallopavo) is amplified by keratinase. FEMS Microbiol. Ecol. 94:fiy117. 10.1093/femsec/fiy117 PubMed DOI

Braun M. S., Wang E., Zimmermann S., Boutin S., Wink M. (2018b). Kocuria uropygioeca sp nov and Kocuria uropygialis sp nov., isolated from the preen glands of great spotted Woodpeckers (Dendrocopos major). Syst. Appl. Microbiol. 41 38–43. 10.1016/j.syapm.2017.09.005 PubMed DOI

Braun M. S., Wang E., Zimmermann S., Wink M. (2018c). Corynebacterium heidelbergense sp. nov., isolated from the preen glands of Egyptian geese (Alopochen aegyptiacus). Syst. Appl. Microbiol. 41 564–569. 10.1016/j.syapm.2018.06.002 PubMed DOI

Braun M. S., Wang E. J., Zimmermann S., Wagner H., Wink M. (2019). Kocuria tytonis sp. nov., isolated from the uropygial gland of an American barn owl (Tyto furcata). Int. J. Syst. Evol. Micr. 69 447–451. 10.1099/ijsem.0.003170 PubMed DOI

Braun M. S., Zimmermann S., Danner M., Rashid H. O., Wink M. (2016). Corynebacterium uropygiale sp nov., isolated from the preen gland of Turkeys (Meleagris gallopavo). Syst. Appl. Microbiol. 39 88–92. 10.1016/j.syapm.2015.12.001 PubMed DOI

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. 10.1023/B:JOEC.0000042071.65335.f3 PubMed DOI

Callahan B. J., McMurdie P. J., Rosen M. J., Han A. W., Johnson A. J., Holmes S. P. (2016). DADA2: high-resolution sample inference from Illumina amplicon data. Nat. Methods 13 581–583. 10.1038/nmeth.3869 PubMed DOI PMC

Caspi R., Foerster H., Fulcher C. A., Hopkinson R., Ingraham J., Kaipa P., et al. (2006). MetaCyc: a multiorganism database of metabolic pathways and enzymes. Nucleic Acids Res. 34 D511–D516. 10.1093/nar/gkj128 PubMed DOI PMC

Cotgreave P., Clayton D. H. (1994). Comparative-analysis of time spent grooming by birds in relation to parasite load. Behaviour 131 171–187. 10.1163/156853994x00424 DOI

Craig A. J. F. K. (1999). Anting in Afrotropical birds: a review. Ostrich 70 203–207. 10.1080/00306525.1999.9634237 DOI

Creevey C. J., Kelly W. J., Henderson G., Leahy S. C. (2014). Determining the culturability of the rumen bacterial microbiome. Microb. Biotechnol. 7 467–479. 10.1111/1751-7915.12141 PubMed DOI PMC

Darriba D., Taboada G. L., Doallo R., Posada D. (2012). jModelTest 2: more models, new heuristics and parallel computing. Nat. Methods 9 772–772. 10.1038/nmeth.2109 PubMed DOI PMC

Davidson S. K., Stahl D. A. (2008). Selective recruitment of bacteria during embryogenesis of an earthworm. ISME J. 2 510–518. 10.1038/ismej.2008.16 PubMed DOI

Delius J. D. (1988). Preening and associated comfort behavior in birds. Ann. N.Y. Acad. Sci. 525 40–55. 10.1111/j.1749-6632.1988.tb38594.x PubMed DOI

Douglas G. M., Maffei V. J., Zaneveld J. R., Yurgel S. N., Brown J. R., Taylor C. M., et al. (2019). PICRUSt2: an improved and extensible approach for metagenome inference. bioRxiv [Preprint]. 10.1101/672295 DOI

Drummond A. J., Suchard M. A., Xie D., Rambaut A. (2012). Bayesian phylogenetics with BEAUti and the BEAST 1.7. Mol. Biol. Evol. 29 1969–1973. 10.1093/molbev/mss075 PubMed DOI PMC

Fenske G. J., Ghimire S., Antony L., Christopher-Hennings J., Scaria J. (2020). Integration of culture-dependent and independent methods provides a more coherent picture of the pig gut microbiome. FEMS Microbiol. Ecol. 96:fiaa022. 10.1093/femsec/fiaa022 PubMed DOI

Fülöp A., Czirják G. Á, Pap P. L., Vágási I. (2016). Feather-degrading bacteria, uropygial gland size and feather quality in House sparrows Passer domesticus. Ibis 158 362–370. 10.1111/ibi.12342 DOI

Gond S. K., Bergen M. S., Torres M. S., White J. F., Jr. (2015). Endophytic Bacillus spp. produce antifungal lipopeptides and induce host defence gene expression in maize. Microbiol. Res. 172 79–87. 10.1016/j.micres.2014.11.004 PubMed DOI

Greay S. J., Hammer K. A. (2011). Recent developments in the bioactivity of mono- and diterpenes: anticancer and antimicrobial activity. Phytochem. Rev. 14 1–6. 10.1007/s11101-011-9212-6 DOI

Griffiths R., Double M. C., Orr K., Dawson R. J. G. (1998). A DNA test to sex most birds. Mol. Ecol. 7 1071–1075. 10.1046/j.1365-294x.1998.00389.x PubMed DOI

Hagelin J. C., Jones I. L. (2007). Bird odors and other chemical substances: a defense mechanism or overlooked mode of intraspecific communication? Auk 124 741–761. 10.1093/auk/124.3.741 DOI

Haribal M., Dhondt A. A., Rodriguez E. (2009). Diversity in chemical compositions of preen gland secretions of tropical birds. Biochem. Syst. Ecol. 37 80–90. 10.1016/j.bse.2008.12.005 DOI

Haribal M., Dhondt A. A., Rosane D., Rodriguez E. (2005). Chemistry of preen gland secretions of passerines: different pathways to same goal? why? Chemoecology 15 251–260. 10.1007/s00049-005-0318-4 DOI

Hothorn T., Bretz F., Westfall P. (2008). Simultaneous inference in general parametric models. Biom. J. 50 346–363. 10.1002/bimj.200810425 PubMed DOI

Jacob J., Ziswiler V. (1982). “The uropygial gland,” in Avian Biology, eds Farner D. S., King J. R., Parkes K. C. (New York, NY: Academic Press; ), 199–324. 10.1016/b978-0-12-249406-2.50013-7 DOI

Jacob S., Immer A., Leclaire S., Parthuisot N., Ducamp C., Espinasse G., et al. (2014). Uropygial gland size and composition varies according to experimentally modified microbiome in Great tits. BMC Evol. Biol. 14:134. 10.1186/1471-2148-14-134 PubMed DOI PMC

Jacob S., Sallé L., Zinger L., Chaine A. S., Ducamp C., Boutault L., et al. (2018). Chemical regulation of body feather microbiota in a wild bird. Mol. Ecol. 27 1727–1738. 10.1111/mec.14551 PubMed DOI

Javurková V. G., Kreisinger J., Procházka P., Požgayová M., Ševèíková K., Brlík V., et al. (2019). Unveiled feather microcosm: feather microbiota of passerine birds is closely associated with host species identity and bacteriocin-producing bacteria. ISME J. 13 2363–2376. 10.1038/s41396-019-0438-4 PubMed DOI PMC

Kildgaard S., de Medeiros L. S., Phillips E., Gotfredsen C. H., Frisvad J. C., Nielsen K. F., et al. (2018). Cyclopiamines C and D: epoxide spiroindolinone alkaloids from Penicillium sp. CML 3020. J. Nat. Prod. 81 785–790. 10.1021/acs.jnatprod.7b00825 PubMed DOI

Kolattukudy P. E. (1981). Avian uropygial (preen) gland. Methods Enzymol. 72 714–720. 10.1016/s0076-6879(81)72061-5 PubMed DOI

Kovács ÁT. (2019). Bacillus subtilis. Trends Microbiol. 27 724–725. 10.1016/j.tim.2019.03.008 PubMed DOI

Lahti L., Shetty S. (2017). Tools for Microbiome Analysis in R. Version 2.1.24. Available online at: https://microbiome.github.io/tutorials/ (accecced June 17, 2020).

Law-Brown J., Meyers P. R. (2003). Enterococcus phoeniculicola sp nov., a novel member of the enterococci isolated from the uropygial gland of the Red-billed Woodhoopoe, Phoeniculus purpureus. Int. J. Syst. Evol. Micr. 53 683–685. 10.1099/ijs.0.02334-0 PubMed DOI

Leifert C., Li H., Chidburee S., Hampson S., Workman S., Sigee D., et al. (1995). Antibiotic production and biocontrol activity by Bacillus subtilis CL27 and Bacillus pumilus CL45. J. Appl. Bacteriol. 78 97–108. 10.1111/j.1365-2672.1995.tb02829.x PubMed DOI

Lezalova-Pialkova R. (2011). Molecular evidence for extra-pair paternity and intraspecific brood parasitism in the Black-headed Gull. J. Ornithol. 152 291–295. 10.1007/s10336-010-0581-1 DOI

Magallanes S., Møller A. P., García-Longoria L., de Lope F., Marzal A. (2016). Volume and antimicrobial activity of secretions of the uropygial gland are correlated with malaria infection in house sparrows. Parasit. Vect. 9:232. 10.1186/s13071-016-1512-7 PubMed DOI PMC

Maraci Ö, Engel K., Caspers B. A. (2018). Olfactory communication via microbiota: what is known in birds? Genes 9:387. 10.3390/genes9080387 PubMed DOI PMC

Mardon J., Saunders S. M., Bonadonna F. (2011). From preen secretions to plumage: the chemical trajectory of blue petrels’ Halobaena caerulea social scent. J. Avian Biol. 42 29–38. 10.1111/j.1600-048x.2010.05113.x DOI

Martín-Vivaldi M., Peña A., Peralta-Sánchez J. M., Sánchez L., Ananou S., Ruiz-Rodríguez M., et al. (2010). Antimicrobial chemicals in hoopoe preen secretions are produced by symbiotic bacteria. Proc. R. Soc. B Biol. Sci. 277 123–130. 10.1098/rspb.2009.1377 PubMed DOI PMC

Martín-Vivaldi M., Soler J. J., MartÍnez-GarcÍa Á, Arco L., Juárez-García-Pelayo N., Ruiz-Rodríguez M., et al. (2018). Acquisition of uropygial gland microbiome by hoopoe nestlings. Microb. Ecol. 76 285–297. 10.1007/s00248-017-1125-5 PubMed DOI

Martínez-García Á, Martín-Vivaldi M., Rodríguez-Ruano S. M., Peralta-Sánchez J. M., Valdivia E., Soler J. J. (2016). Nest bacterial environment affects microbiome of hoopoe eggshells, but not that of the uropygial secretion. PLoS One 11:e0158158. 10.1371/journal.pone.0158158 PubMed DOI PMC

McMurdie P. J., Holmes S. (2013). phyloseq: an R package for reproducible interactive analysis and graphics of microbiome census data. PLoS One 8:e61217. 10.1371/journal.pone.0061217 PubMed DOI PMC

Miller C. S., Handley K. M., Wrighton K. C., Frischkorn K. R., Thomas B. C., Banfield J. F. (2013). Short-read assembly of full-length 16S amplicons reveals bacterial diversity in subsurface sediments. PLoS One 8:e56018. 10.1371/journal.pone.0056018 PubMed DOI PMC

Møller A. P., Czirjak G. Á, Heeb P. (2009). Feather micro-organisms and uropygial antimicrobial defences in a colonial passerine bird. Funct. Ecol. 23 1097–1102. 10.1111/j.1365-2435.2009.01594.x DOI

Møller A. P., Erritzøe J., Rózsa L. (2010). Ectoparasites, uropygial glands and hatching success in birds. Oecologia 163 303–311. 10.1007/s00442-009-1548-x PubMed DOI

Montalti D., Gutiérrez A. M., Reboredo G., Salibian A. (2005). The chemical composition of the uropygial gland secretion of rock dove Columba livia. Comp. Biochem. Phys. A 140 275–279. 10.1016/j.cbpb.2004.10.008 PubMed DOI

Moreno-Rueda G. (2010). House Sparrows Passer domesticus with larger uropygial glands show reduced feather wear. Ibis 153 195–198. 10.1111/j.1474-919x.2010.01082.x DOI

Moreno-Rueda G. (2016). Uropygial gland and bib colouration in the house sparrow. PeerJ 4:e2102. 10.7717/peerj.2102 PubMed DOI PMC

Moreno-Rueda G. (2017). Preen oil and bird fitness: a critical review of the evidence. Biol. Rev. 92 2131–2143. 10.1111/brv.12324 PubMed DOI

Oksanen J., Blanchet F. G., Friendly M., Kindt R., Legendre P., McGlinn D., et al. (2019). vegan: Community Ecology Package. R package version 2.5-4 [Online]. Available online at: https://CRAN.R-project.org/package=vegan (accessed September 1, 2019).

Pearce D. S., Hoover B. A., Jennings S., Nevitt G. A., Docherty K. M. (2017). Morphological and genetic factors shape the microbiome of a seabird species (Oceanodroma leucorhoa) more than environmental and social factors. BMC Microbiome 5:146. 10.1186/s40168-017-0365-4 PubMed DOI PMC

Quast C., Pruesse E., Yilmaz P., Gerken J., Schweer T., Yarza P., et al. (2013). The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res. 41 D590–D596. 10.1093/nar/gks1219 PubMed DOI PMC

Rambaut A., Suchard M. A., Xie D., Drummond A. J. (2014). Tracer v1.6 [Online]. Available online at: http://beast.bio.ed.ac.uk/Tracer (accessed March, 2020).

Reneerkens J., Versteegh M. A., Schneider A. M. (2008). Seasonally changing preen-wax composition: red knots’ (Calidris canutus) flexible defense against feather-degrading bacteria? Auk 125 285–290. 10.1525/auk.2008.06217 DOI

Rodríguez-Ruano S. M., Martín-Vivaldi M., Martín-Platero A. M., López-López J. P., Peralta-Sánchez J. M., Ruiz-Rodríguez M., et al. (2015). The Hoopoe’s uropygial gland hosts a bacterial community influenced by the living conditions of the bird. PLoS One 10:e0139734. 10.1371/journal.pone.0139734 PubMed DOI PMC

Rodríguez-Ruano S. M., Martín-Vivaldi M., Peralta-Sánchez J. M., García-Martin A. B., Martínez-García A., Soler J. J., et al. (2018). Seasonal and sexual differences in the microbiota of the hoopoe uropygial secretion. Genes 9:407. 10.3390/genes9080407 PubMed DOI PMC

Ruiz-Rodríguez M., Tomás G., Martín-Gálvez D., Ruiz-Castellano C., Soler J. J. (2015). Bacteria and the evolution of honest signals. The case of ornamental throat feathers in spotless starlings. Funct. Ecol. 29 701–709. 10.1111/1365-2435.12376 DOI

Ruiz-Rodríguez M., Valdivia E., Martín-Vivaldi M., Martín-Platero A. M., Martínez-Bueno M., Méndez M., et al. (2012). Antimicrobial activity and genetic profile of enteroccoci isolated from hoopoes uropygial gland. PLoS One 7:e41843. 10.1371/journal.pone.0041843 PubMed DOI PMC

Ruiz-Rodríguez M., Valdivia E., Soler J. J., Martín-Vivaldi M., Martín-Platero A. M., Martínez-Bueno M. (2009). Symbiotic bacteria living in the hoopoe’s uropygial gland prevent feather degradation. J. Exp. Biol. 212 3621–3626. 10.1242/jeb.031336 PubMed DOI

Sanders J. G., Lukasik P., Frederickson M. E., Russell J. A., Koga R., Knight R., et al. (2017). Dramatic differences in gut bacterial densities correlate with diet and habitat in rainforest ants. Integr. Comp. Biol. 57 705–722. 10.1093/icb/icx088 PubMed DOI

Schneider C. A., Rasband W. S., Eliceiri K. W. (2012). NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9 671–675. 10.1038/nmeth.2089 PubMed DOI PMC

Shawkey M. D., Pillai S. R., Hill G. E. (2003). Chemical warfare? Effects of uropygial oil on feather-degrading bacteria. J. Avian Biol. 34 345–349. 10.1111/j.0908-8857.2003.03193.x DOI

Sivakumar G., Rangeshwaran R., Yandigeri M. S., Mohan M., Venkatesan T., Ballal C. R., et al. (2017). Characterization and role of gut bacterium Bacillus pumilus on nutrition and defense of leafhopper (Amrasca biguttula biguttula) of cotton. Indian J. Agr. Sci. 87 534–539.

Soler J. J., Martín-Vivaldi M., Peralta-Sánchez J. M., Ruiz-Rodríguez M. (2010). Antibiotic-producing bacteria as a possible defence of birds against pathogenic microorganisms. Open Ornithol. 3 93–100. 10.2174/1874453201003010093 DOI

Soler J. J., Martín-Vivaldi M., Ruiz-Rodríguez M., Valdivia E., Martín-Platero A. M., Martínez-Bueno M., et al. (2008). Symbiotic association between hoopoes and antibiotic-producing bacteria that live in their uropygial gland. Funct. Ecol. 22 864–871. 10.1111/j.1365-2435.2008.01448.x DOI

Stein T. (2005). Bacillus subtilis antibiotics: structures, syntheses and specific functions. Mol. Microbiol. 56 845–857. 10.1111/j.1365-2958.2005.04587.x PubMed DOI

Stettenheim P. (1972). “The integument of birds,” in Avian Biology, eds Farner D. S., King J. R., Parkes K. C. (New York, NY: Academic Press; ).

R Core Team (2019). R: A Language and Environment for Statistical Computing. [Online]. Vienna: R Foundation for Statistical Computing.

Um S., Fraimout A., Sapountzis P., Oh D. C., Poulsen M. (2013). The fungus-growing termite Macrotermes natalensis harbors bacillaene-producing Bacillus sp that inhibit potentially antagonistic fungi. Sci. Rep. 3:3250. 10.1038/srep03250 PubMed DOI PMC

van Liere D. W., Bokma S. (1987). Short-term feather maintenance as a function of dust-bathing in laying hens. Appl. Anim. Behav. Sci. 18 197–204. 10.1016/0168-1591(87)90193-6 DOI

van Veelen H. P. J., Falcao Salles J., Tieleman B. I. (2017). Multi-level comparisons of cloacal, skin, feather and nest-associated microbiota suggest considerable influence of horizontal acquisition on the microbiota assembly of sympatric woodlarks and skylarks. BMC Microbiome 5:156. 10.1186/s40168-017-0371-6 PubMed DOI PMC

Verea C., Vitelli-Flores J., Ituriz T., Rodríguez-Lemoine V., Bosque C. (2017). The effect of uropygial gland secretions of Spectacled Thrushes (Turdus nudigenis) on feather degradation and bacterial growth in vitro. J. Ornithol. 158 1035–1043. 10.1007/s10336-017-1461-8 DOI

Vincze O., Vágási C. I., Kovács I., Galván I., Pap P. L. (2013). Sources of variation in uropygial gland size in European birds. Biol. J. Linn. Soc. 110 543–563. 10.1111/bij.12139 DOI

Whittaker D. J., Gerlach N. M., Slowinski S. P., Corcoran K. P., Winters A. D., Soini H. A., et al. (2016). Social environment has a primary influence on the microbial and odor profiles of a chemically signaling songbird. Front. Ecol. Evol. 4:90 10.3389/fevo.2016.00090 DOI

Whittaker D. J., Theis K. R. (2016). “Bacterial communities associated with Junco preen glands: preliminary ramifications for chemical signaling,” in Chemical Signals in Vertebrates, eds Schulte B., Goodwin T., Ferkin M. (Cham: Springer; ).

Wickham H., François R., Henry L., Müller K. (2019). dplyr: A Grammar of Data Manipulation [Online]. Available online at: https://CRAN.R-project.org/package=dplyr (accessed May 29, 2020).

Yamada Y., Kuzuyama T., Komatsu M., Shin-Ya K., Omura S., Cane D. E., et al. (2015). Terpene synthases are widely distributed in bacteria. Proc. Natl. Acad. Sci. U.S.A. 112 857–862. 10.1073/pnas.1422108112 PubMed DOI PMC

Zhang J., Wei W., Zhang J., Yang W. (2010). Uropygial gland-secreted alkanols contribute to olfactory sex signals in budgerigars. Chem. Senses 35 375–382. 10.1093/chemse/bjq025 PubMed DOI

Zhukova M., Sapountzis P., Schiøtt M., Boomsma J. J. (2017). Diversity and transmission of gut bacteria in Atta and Acromyrmex leaf-cutting ants during development. Front. Microbiol. 8:1942. 10.3389/fmicb.2017.01942 PubMed DOI PMC

Host traits rather than migration and molting strategies explain feather bacterial load in Palearctic passerines

Effects of genotype, sex, and feed restriction on the biochemical composition of chicken preen gland secretions and their implications for commercial poultry production