Drying eggs to inhibit bacteria: Incubation during laying in a cavity nesting passerine

Early incubation has been suggested as a defensive adaptation against potentially pathogenic bacteria colonizing avian eggshells in the wild. The inhibitory mechanisms underlying this adaptation are poorly understood and only recent experimental evidence demonstrates that keeping eggs dry is a proximate mechanism for the antimicrobial effects of avian incubation. We estimated partial incubation (the bouts of incubation that some birds perform during the egg-laying period, days of lay 3-5 in our population) intensity of female pied flycatchers breeding in nest-boxes using data loggers that allowed a precise measurement of temperature just between the eggs in the nest-cup. We also measured relative humidity within the nest-boxes and related it to incubation intensity, showing that more intense incubation during laying contributes to drying the air near the eggs. We analyzed separately the effects of incubation and of relative humidity on loads of three types of culturable bacteria known to be present on eggshells, heterotrophic bacteria, Gram-negative enterics and pseudomonads. Our results show an association of early incubation with an inhibition of bacterial proliferation through a drying effect on eggshells, as we found that incubation intensity was negatively and relative humidity positively associated with eggshell bacterial loads for heterotrophic bacteria, Gram-negative bacteria and pseudomonads, although the significance of these associations varied between bacterial groups. These results point to microclimatically driven effects of incubation on bacterial proliferation on eggshells during laying in a temperate cavity nesting passerine.