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.