We found that both the bacterial load of the entrance hole and the bacterial load of the nest box were significantly higher in the forest than the bacterial loads in the parkland, both in the nest boxes occupied and unoccupied in the previous season. We also found that the interior bacterial load of the nest box was higher in the nest boxes used for breeding in the previous season, but the difference was significant only at the forest site.
We revealed a clear difference in the interior bacterial load of the nest box between deciduous forest and urban parkland. This result suggests that the bacterial loads in nest boxes are strongly habitat dependent. The interior of the nest box is probably readily colonized by a variety of microorganisms shortly after placing the nest box in a particular environment. Birds play the main role of transferring microorganisms to nest boxes when visiting them, as well as to other various cavities as places potentially suitable for breeding or roosting19,20. Bacteria are found alike on avian beaks and toes, however, these communities in the plumage are most important in this context, since bacteria are both most numerous and most diverse in feathers20,21. In our study system, nest boxes were used mainly by the Blue Tit and the Great Tit. Both species forage mainly on trees leaves and twigs. Plants, as a source of bacteria, are suggested to play an important role in the transmission of these organisms to plumage21. This impact of the plant host on the bacterial microbiota was shown between predators and prey in the trophic networks of blue tit22. Given that our study sites differ markedly floristically with deciduous forest being richer in plant species than urban parkland, it seems very likely that the forest site may maintain richer bacterial communities. Saag et al.19 showed such a pattern of habitat-related differences in bacterial density and species richness. They revealed that while the number of phylotypes per bird was higher in coniferous habitat, the bacterial densities were higher in deciduous habitat. It is also supported by the results presented by Broughton and Gross23 who suggested that microbial activity was positively correlated with plant productivity. Similarly, Bisson et al.21 found that microorganisms from particular groups were more abundant in plumage sampled from American redstart (Setophaga ruticilla) in wet versus dry habitats. It is in line with our results, since rich, mature forests retain more moisture than urban parkland with its many open areas with no or little tree cover and, therefore, open to direct sunlight. However, the richness of plant species is not the only distinction between the two sites. The Botanical Garden is a place where various agrotechnological works are conducted throughout the year (own observations). These procedures include the application of different chemicals, such as insecticides, which probably affect different organisms, including bacteria that are present not only in plants but also in soil. For example, in 2013 and 2014, a large-scale molluscicide treatment was applied in the Garden to eradicate the invasive Spanish slug (Arion vulgaris) (see24 for details). Presumably, regular use of chemicals in parkland, contrary to the forest area, can disturb microbial communities, including the sheer number of bacteria in different ways. For the above reasons, the microbial profile of the soil itself may play an important role in plumage bacterial acquisition21,25,26. It may be important in the context of this study, since the great tit relatively frequently forages on the ground27, own obs., which means that the birds acquire bacteria directly from the soil and then carry them into nest boxes.
In addition to basic habitat characteristics including plant species composition and soil characteristics, there are probably other factors that can contribute to bacterial loads in nest boxes. It is known that different taxa of animals visit nest boxes for a variety of purposes. Many invertebrates (snails, spiders, or insects) are opportunistic species that use nest boxes year-round as shelters, roosting places, or to build the nests and raise their offspring (i.e. wasps)28,29, own.obs.. Invertebrate species colonizing nest boxes carry their bacteria acquired from the environment. Since the forest area is a more diverse habitat than the parkland area in terms of plant species composition, it is likely that the invertebrate assemblies are also richer in the forest. The next potential factor is the presence of particular mammal species, which visit nest boxes for two main reasons. In both our study areas, brown long-eared bats (Plecotus auritus) and noctules (Nyctalus noctula) occasionally use empty nest boxes for breeding and roosting during the summer / autumn period, and they are more frequent in the forest (own observations). The pine marten (Martes martes), in turn, is a predatory species that tries to reach the nest in the nest box and grab an adult bird or nestlings with its paws as a prey. In some breeding seasons, the predation rate was very high in the forest30, but not in the parkland where marten predation occurred only exceptionally (own observations). Both bats and martens carry their bacterial flora which is transferred to nest boxes; however, in the case of martens, this transfer is restricted mainly to the entrance hole size.
We also tested whether the occupancy of the nest box in the previous year had affected the bacterial load of the nest box. Our results are ambiguous on that point since this effect was significant in the case of the nest box interior but not in the case of the nest box entrance and only in the forest study site. It is not clear why this effect was found only at one study site. A possible explanation may be related to the intensity of using nest boxes as roosting sites during an autumn-winter period. It is well known that wintering tits spend the nights in nest boxes18, own obs. and thus probably transfer bacteria there. In the parkland, contrary to the forest area, both tit species are more abundant during winter (own observations) and probably use nest boxes as roosting sites more intensely. If this assumption is true, it should at least partially eliminate the expected difference in bacterial load between nest boxes occupied and unoccupied for breeding. However, it seems that this effect is not strong enough to nullify the striking difference between the study sites. Yet, we do not have any quantitative data on the intensity of using nest boxes as roosting sites out of the breeding season and, therefore, we cannot make any plausible conclusion on that issue.
In conclusion, our study demonstrates that the observed difference in the bacterial load of the nest box is probably related to habitat-related differences between the two study sites. The most important factors are probably associated with the richness of plant species and the physical properties of the soil, with additional factors resulting from the specificity of the two study sites. However, taking into account the complex and yet relatively weakly understood relations between microorganisms, their hosts, and the environment, probably other factors play their role and we are still far from understanding this issue. Furthermore, we should treat our results with care since we do not have data on the richness of bacterial species at both study sites. However, our study suggests that the bacterial load may be a potentially important factor for the life-history traits of secondary cavity nesting birds, since it may vary considerably between habitats. Therefore, since studies on cavity nesting birds with the use of artificial wooden nest boxes are widely conducted across a geographical range and a variety of habitats, and given that bacterial load may affect host fitness components, we consider our study perspective as a promising field for future research.