1. INTRODUCTION
Microorganisms are ubiquitous in a variety of habitats, and they inhabit all available spaces there. One of these habitats, avian nests, constitutes a unique environment for a variety of them (Gonzalez-Braojos et al., 2012; 2015; Goodenough and Stalwood, 2010; Zabłotni et al. 2020). Bacteria colonizing avian nests and spaces, which birds can potentially choose to breed, affect their avian hosts in both positive and negative ways (Goodenough and Stalwood, 2010; Singleton and Harper, 2010; Benskin et al., 2009). Some of the bacteria that colonize avian nests are commensals that feed on nest components consisting mainly of organic matter, but many bacterial species, including pathogenic strains, can exert a negative impact on birds (see Benskin et al., 2009 for the review). Recent research has shown that the nest microbiota plays an important role in mediating the life histories of birds. In particular, microorganisms shape the microbiome of their hosts and play a key role in the digestion of food (see Grond et al., 2009 for the review), pathogen defence (Soler et al., 2008; 2010), as well as influence plumage quality (Gunderson, 2008; Al Rubaiee et al., 2021). There is also a growing body of evidence showing the importance of microbial species for traits of avian condition and, consequently, for their reproductive success (Gonzales-Braojos et al., 2015; Goodenough and Stalwood, 2010; Zabłotni et al. 2020). However, relationships between nest bacterial load and life - history traits of birds are relatively poorly understood, mainly because the majority of studies on animal-associated microbiota are conducted on captive animals (Carina-Audisio et al., 2000). Little is still known about the complex relationships between different species of birds and their microbiota under particular environmental conditions in the wild. Given the ubiquity of bacterial communities and the complexity of host-microbiota interactions, it is important to document both the differences in abundance and diversity of microorganisms in different habitats (Horner-Devine et al., 2004; Levin et al., 2021).
Among a wide variety of locations where different species of birds breed, nests located in cavities are unique, as cavities constitute a particularly suitable environment for different types of microorganism (Gonzalez-Braojos et al., 2012; Goodenough and Stalwood, 2010; Berger et al., 2003; Goodenough and Stalwood, 2012). The special characteristics of nest boxes (and other cavities, including natural ones) result from maintaining relatively stable physical conditions, such as humidity and temperature inside them, which make them suitable for microbial colonization and growth (Gonzalez-Braojos et al., 2015; Devaynes et al., 2018). Additionally, nest boxes are frequently used for breeding in several consecutive seasons, ensuring steady delivery of organic matter (feathers, peeled fragments of the epidermis, or excreted feces) essential for microbial growth. It should also be mentioned that outside the breeding season, several species of birds use nest boxes as roosting places during an autumn-winter period (Mainwaring, 2011). Furthermore, available data suggest that the prevalent practice of cleaning nest boxes after the breeding season does not remove all litter, leaving enough residues for bacterial growth (Zabłotni et al., 2020). Furthermore, it is highly probable that the bacterial load of the nest box itself depends on a variety of environmental characteristics of a particular habitat. The physical properties of habitats include several traits such as humidity, thermal conditions, chemical properties of soil, and probably many other characteristics that influence the assemblages of bacteria within different spatial scales (see Horner-Devine et al., 2004 for a review). Furthermore, the richness of plant species in a particular habitat can also play an important role (Saag et al., 2011). Since the available studies on natural microbial diversity in nest boxes placed in different habitats are rather scant (Burtt and Ichida, 1999), there is a need to focus on microorganisms colonizing nest boxes which are used not only for breeding but also as roosting sites of many avian species.
For the above reasons and given that in our previous study we had demonstrated a negative influence of bacteria on the physiological condition of wild birds (Zabłotni et al., 2020), we conducted the study in two sets of wooden nest boxes that are used by the two secondary cavity nesting bird species, the great tit Parus major and the blue tit Cyanistes caeruleus in two different habitats: urban parkland and a deciduous forest. Both these tit species interchangeably use nest boxes for breeding in consecutive breeding seasons; however, in a particular year some of the nest boxes may remain unoccupied throughout the breeding season. This may potentially lead to a variation in the abundance of bacteria in nest boxes. The second potential source of variation in bacterial loads in nest boxes may be due to habitat differences between the two study sites. Probably, the different physical conditions that prevail at either site (temperature, humidity, soil characteristics, and similar characteristics) can shape the microbial communities in nest boxes. Since the nest boxes itself offer diverse conditions for bacterial communities, we decided to sample both the nest box interior and the nest box entrance hole. Therefore, we estimated the interior bacterial loads of the nest box and the entrance hole of the nest box to (i) check if there was a significant difference between the two habitats and (ii) test the following prediction: the nest boxes used for breeding during the preceding year had higher bacterial loads than the nest boxes that had remained empty that year.