Nest Material Shapes Eggs Bacterial Environment

Abstract

Selective pressures imposed by pathogenic microorganisms to embryos have selected in hosts for a battery of antimicrobial lines of defenses that includes physical and chemical barriers. Due to the antimicrobial properties of volatile compounds of green plants and of chemicals of feather degrading bacteria, the use of aromatic plants and feathers for nest building has been suggested as one of these barriers. However, experimental evidence suggesting such effects is scarce in the literature. During two consecutive years, we explored experimentally the effects of these nest materials on loads of different groups of bacteria (mesophilic bacteria, Enterobacteriaceae, Staphylococcus and Enterococcus) of eggshells in nests of spotless starlings (Sturnus unicolor) at the beginning and at the end of the incubation period. This was also explored in artificial nests without incubation activity. We also experimentally increased bacterial density of eggs in natural and artificial nests and explored the effects of nest lining treatments on eggshell bacterial load. Support for the hypothetical antimicrobial function of nest materials was mainly detected for the year and location with larger average values of eggshell bacterial density. The beneficial effects of feathers and plants were more easily detected in artificial nests with no incubation activity, suggesting an active role of incubation against bacterial colonization of eggshells. Pigmented and unpigmented feathers reduced eggshell bacterial load in starling nests and artificial nest boxes. Results from artificial nests allowed us to discuss and discard alternative scenarios explaining the detected association, particularly those related to the possible sexual role of feathers and aromatic plants in starling nests. All these results considered together confirm the antimicrobial functionality mainly of feathers but also of plants used as nest materials, and highlight the importance of temporally and geographically environmental variation associated with risk of bacterial proliferation determining the strength of such effects. Because of costs associated to nest building, birds should adjust nest building effort to expected bacterial environments during incubation, a prediction that should be further explored.

Fig 1. Experimental design of artificial and natural nests.

Experimental protocols designed for exploring the effects of feathers and aromatic plants as nest materials on bacterial loads on spotless starling eggs (A) and on quail eggs in non-incubated nests (B). Numbers within the tables indicate sample sizes of different experimental treatment for 2012/2013 study years.

Fig 2. Nest material and experimental treatments effect on mesophilic bacterial load.

Statistically significant relationships between loads of mesophilic bacteria of starling eggshells at day 3 in relation to number of unpigmented feathers in 2012 (A) and date of sampling in 2013 (B). The relationships between eggshell mesophilic bacterial load at day 12 (± 95% CI) in relation to feather treatment in 2013 (C), unpigmented feathers at day 12 in 2012 (D), pigmented feathers at day 12 in 2012 (E), unpigmented feathers at day 3 in 2013 (F) and pigmented feathers at day 12 in 2013 (G) are also shown.

Fig 3. Results of mesophilic bacterial load along incubation (variation between day 3 and day 12).

Average values of eggshell mesophilic bacterial growth (± 95% CI) on starling eggshells in relation to feathers’ experimental treatments (unpigmented, pigmented or without feathers) in 2013 (A, D). The associations of eggshell mesophilic bacterial growth with number of pigmented (B) and unpigmented (C) feathers in starling nests in 2012 are also shown. Figure only shows the most relevant associations detected.

Fig 4. Nest material and experimental treatments effects on bacteria in specific media.

Average number (± 95% CI) of unpigmented feathers at day 3 in 2013 in relation to prevalence of Enterobacteriaceae (A), and effects of sampling date in relation to prevalence of Enterococcus (in 2012, B), Enterobacteriaceae (in 2013, C), and Enterococcus (in 2013, D). Prevalence of Enterococcus at day 12 in relation to feather treatment (in 2013, E), number of pigmented feathers at day 3 (in 2013, F) and number of unpigmented feathers at day 12 (in 2013, G) is also shown. Figures show the most relevant associations detected.

Fig 5. Results of mesophilic bacterial density of quail eggs.

Eggshell mesophilic bacterial density (± 95% CI) estimated for experimental quail eggs during the two sampling events in relation to feathers experimental treatment and area for samples collected in 2012 (A and B). The effects of feathers (C and D) and plants (E and F) experimental treatments for contaminated and non-contaminated quail eggs during the three sampling events in 2013 are shown.