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\begin{document}

\title{Habitat-specific variation in bacterial loads of wooden nest boxes in
the pre-breeding period}



\author[1]{Agnieszka Zabłotni}%
\author[1]{Adam Kaliński}%
\author[1]{Miroslawa Banbura}%
\author[1]{Michał Glądalski}%
\author[1]{Marcin Markowski}%
\author[1]{Joanna Skwarska}%
\author[1]{Jarosław Wawrzyniak}%
\author[1]{Jerzy Bańbura}%
\affil[1]{Uniwersytet Łódzki}%


\vspace{-1em}



  
  \date{May 24, 2024}


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\selectlanguage{english}
\begin{abstract}
Among environmental factors affecting life - history traits of birds
breeding in nest boxes, an influence of microbial communities is
relatively poorly understood. In this study, nest boxes used for
breeding by great tit (Parus major) and blue tit (Cyanistes caeruleus)
were sampled before the start of the breeding season to assess the
bacterial loads of the nest box. Samples from the entrance hole and the
interior of the nest box were taken at two different study sites: an
urban parkland and a natural forest. Nest boxes were sampled to check if
their bacterial loads differed between habitats. The second objective of
this study was to check whether the occupancy of the nest boxes during
the previous season would influence the bacterial load of the nest box.
To verify this prediction, two categories of nest boxes were sampled at
both study sites: nest boxes occupied by any of the two tit species in
the previous season for breeding and nest boxes that had remained empty
that year. The bacterial load of the nest box was significantly higher
in the forest study area in both the occupied and unoccupied nest boxes.
The nest boxes used for breeding in the previous season had
significantly higher bacterial loads, but only in the forest area. Our
results suggest that the bacterial load of the nest box can vary between
habitats and may be positively related to the presence of the nests in
the previous breeding season.%
\end{abstract}%



\sloppy


\textbf{Habitat-specific variation in bacterial loads of wooden nest
boxes in the pre-breeding period}

\selectlanguage{polish}\textbf{Agnieszka Zabłotni\textsuperscript{1}, Adam
Kaliński\textsuperscript{2*}, Mirosława Bańbura\textsuperscript{2},
Michał Glądalski\textsuperscript{2}, Marcin
Markowski\textsuperscript{2}, Joanna Skwarska\textsuperscript{2},
Jarosław Wawrzyniak\textsuperscript{2}, Jerzy
Bańbura\textsuperscript{2}}\selectlanguage{english}

\textsuperscript{1}Laboratory of General Microbiology, Department of
Biology of Bacteria, Faculty of Biology and Environmental Protection,
University of \selectlanguage{polish}Ł\selectlanguage{ngerman}ód\selectlanguage{polish}ź, \selectlanguage{english}Banacha 12/16, 90-237 \selectlanguage{polish}Ł\selectlanguage{ngerman}ód\selectlanguage{polish}ź, \selectlanguage{english}Poland

\textsuperscript{2} Department of Experimental Zoology and Evolutionary
Biology, Faculty of Biology and Environmental Protection, University of
\selectlanguage{polish}Ł\selectlanguage{ngerman}ód\selectlanguage{polish}ź, \selectlanguage{english}Banacha 12/16, 90-237 \selectlanguage{polish}Ł\selectlanguage{ngerman}ód\selectlanguage{polish}ź, \selectlanguage{english}Poland

* Corresponding author: adam.kalinski@biol.uni.lodz.pl

\textbf{ABSTRACT}

Among environmental factors affecting life - history traits of birds
breeding in nest boxes, an influence of microbial communities is
relatively poorly understood. In this study, nest boxes used for
breeding by great tit (\emph{Parus major} ) and blue tit
(\emph{Cyanistes caeruleus} ) were sampled before the start of the
breeding season to assess the bacterial loads of the nest box. Samples
from the entrance hole and the interior of the nest box were taken at
two different study sites: an urban parkland and a natural forest. Nest
boxes were sampled to check if their bacterial loads differed between
habitats. The second objective of this study was to check whether the
occupancy of the nest boxes during the previous season would influence
the bacterial load of the nest box. To verify this prediction, two
categories of nest boxes were sampled at both study sites: nest boxes
occupied by any of the two tit species in the previous season for
breeding and nest boxes that had remained empty that year. The bacterial
load of the nest box was significantly higher in the forest study area
in both the occupied and unoccupied nest boxes. The nest boxes used for
breeding in the previous season had significantly higher bacterial
loads, but only in the forest area. Our results suggest that the
bacterial load of the nest box can vary between habitats and may be
positively related to the presence of the nests in the previous breeding
season.

Keywords: nest boxes, bacterial load, great tit, blue tit

\textbf{COVER LETTER}

On behalf of all coauthors, I am submitting a manuscript titled
`\textbf{Habitat-specific variation in bacterial loads of wooden nest
boxes in the pre-breeding period'} for possible publication in Ecology
and Evolution. The manuscript is new, approved by all coauthors, and is
not considered for publication elsewhere. This study is a research
article and is a continuation of our previous work (Zab\selectlanguage{polish}ł\selectlanguage{english}otni, A.,
Kali\selectlanguage{polish}ń\selectlanguage{english}ski, A., Ba\selectlanguage{polish}ń\selectlanguage{english}bura, M., Gl\selectlanguage{polish}ą\selectlanguage{english}dalski, M., Markowski, M., Skwarska, J.,
Wawrzyniak, J., \& Ba\selectlanguage{polish}ń\selectlanguage{english}bura, J. (2020). Experimental nest replacement
suggests that the bacterial load of nests may mediate nestling
physiological condition in cavity nesting Great Tits (\emph{Parus major}
). \emph{Journal of Ornithology} , 161, 819-828) on the influence of
bacterial loads on hole-nesting passerines. This study presents observed
differences in bacterial loads in nest boxes located in two distinct
habitats: urban parkland and natural forest. We showed that the
bacterial load was on average higher in the nest boxes at the forest
study site. We assume that this result is related to differences between
habitats, including the composition of plant species in parkland and
forest. We also showed that the presence of the nest in the nest box in
the previous season positively affected the bacterial load, but only in
the forest area. The results presented are relatively novel and
supplement our understanding of the factors that potentially can affect
the breeding performance of birds using artificial nest boxes with a new
dimension related to microorganism-host interactions. Therefore, we
consider Ecology and Evolution to be the most appropriate journal to
publish the findings.

\textbf{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; Zab\selectlanguage{polish}ł\selectlanguage{english}otni 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; Zab\selectlanguage{polish}ł\selectlanguage{english}otni 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 (Zab\selectlanguage{polish}ł\selectlanguage{english}otni 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 (Zab\selectlanguage{polish}ł\selectlanguage{english}otni 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 \emph{Parus major} and the
blue tit \emph{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.

\textbf{2. MATERIALS AND METHODS}

\textbf{2.1. Study sites}

This study was carried out in 2020 in two different habitats: an urban
parkland and a deciduous forest. The study sites are located
\textasciitilde{} 10 km apart and are separated by the city of \selectlanguage{polish}Ł\selectlanguage{ngerman}ód\selectlanguage{polish}ź. \selectlanguage{english}The
urban parkland study site (51\selectlanguage{ngerman}º45'N; 19º24'E) consists of \selectlanguage{polish}Ł\selectlanguage{ngerman}ód\selectlanguage{polish}ź \selectlanguage{english}Botanical
Garden, which covers a total area of approximately 67 ha. It is mainly
of anthropogenic origin and has fragmented tree and bush cover with very
few remnants of natural stands including birches (\emph{Betula pendula}
), beeches (\emph{Fagus sylvatica} ) and numerous alien species planted
intentionally by garden administrators (Gl\selectlanguage{polish}ą\selectlanguage{english}dalski et al., 2016). The
forest study site (51\selectlanguage{ngerman}°50' N;19°29' E) is an area of approximately 145 ha
located in the interior of a rich, mature mixed deciduous forest called
the \selectlanguage{polish}Ł\selectlanguage{english}agiewniki Forest (1250 ha in total). Oaks (\emph{Quercus robur} and
\emph{Q. petrea} ) are the dominant tree species in the forest. Both
study sites were supplied with standard wooden nest boxes, each made of
pinewood (200 in the parkland area and 300 in the forest area). After
each breeding season in mid-October, each nest box was cleaned with a
wire brush, so there were no visible nest remnants.

\textbf{2.2 Bacterial sampling}

In the first half of March 2020, 40 nest boxes (20 at each study site)
were randomly chosen for bacterial sampling. About half of the nest
boxes at each study site had been occupied by one of the two tit species
(great tit or blue tit) in the previous year for breeding, while the
other half had remained empty that year (5 occupied by blue tits, 6 by
great tits and 4 by blue tits, 6 by great tits in the parkland and in
the forest, respectively). Using disinfected latex gloves, an entrance
hole of each nest box was swabbed with circular movement for 30 s with a
sterile cotton swab previously moistened with sterile phosphate-buffered
saline (PBS, pH 7.2; Adlab, Poland). Subsequently, a restricted area of
ca. 2.5 cm\textsuperscript{2} of an internal rear wall of every nest box
in the middle of its height was swabbed in the same manner. A sample per
entrance hole and another sample per inner wall of each nest box were
taken (in a standardized manner with respect to time and area).

\textbf{2.3. Lab procedures}

Once in the laboratory, bacteria from the swabs were transferred to the
solution in the manner used in our previous study on the bacterial load
(see Zab\selectlanguage{polish}ł\selectlanguage{english}otni et al., 2020 for technical details). The bacteria were
cultivated and incubated on Tryptic Soy Agar medium (TSA) for 48 h at
37\selectlanguage{ngerman}±1°C, then for an additional 48 h at 25±1°C and the colony forming
units (CFU) were counted. The results were expressed as CFU/ml.

\textbf{2.4. Statistical analyzes}

The bacterial load of the nest box entrance hole and the nest box
interior variables were normalized by ln transformation prior to
analyses. A two-way ANOVA was used to test whether the study area and
the occupancy of the nest box affected the nest box entrance hole
bacterial load in the study year; the interaction term was also
calculated. This approach was repeated for nest box interior bacterial
load. Interactions, both significant and non-significant were included
in the models. Analyses were performed using Statistica ver. 12 software
(StatSoft Inc., 2014).

\textbf{3. RESULTS}

The mean, minimum and maximum ln transformed values of the bacterial
loads in the entrance hole and the interior bacterial loads of the nest
box in the parkland and forest, occupied for breeding and empty in the
previous year, are given in Tables 1 and 2.

The mean entrance hole bacterial load differed significantly between the
study areas and was higher in the forest study area (Table 1, Table 3,
Fig.1). Similarly, the interior bacterial load of the nest box differed
significantly between the nest boxes occupied and unoccupied in the
previous year (which was higher in the nest boxes occupied in the
previous season), but the difference was significant only in the forest
study area (Table 2, Table 3, Fig.2).

The correlation between the entrance bacterial load and the interior
bacterial load was non-significant in the parkland study area and in the
forest study area (r = 0.41, p = 0.075 and r = -0.43, p = 0.129,
respectively).

\textbf{FIGURE 1.} Mean (± standard errors) entrance hole bacterial load
in the nest boxes occupied and unoccupied in the previous season in two
study areas

\textbf{FIGURE 2.} Mean (± standard errors) nest box interior bacterial
load in the nest boxes occupied and unoccupied in the previous season in
two study areas

\textbf{4. DISCUSSION}

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 roosting
(Saag et al., 2011; Burtt and Ichida, 1999). Bacteria are found on avian
beaks and toes, however, the communities in the plumage are most
important in this context, since bacteria are both most numerous and
most diverse in feathers (Burtt and Ichida, 1999; Bison et al., 2007).
In our study system, the nest boxes were used mainly by the blue tit and
the great tit. Both species forage mainly on leaves and twigs. Plants,
as a source of bacteria, are suggested to play an important role in the
transmission of these organisms to plumage (Bison et al., 2007). This
impact of the plant host on the bacterial microbiota was shown between
predators and prey in the trophic networks of blue tit (Dion-Phenix et
al., 2021). Given that our study sites differ markedly floristically,
with deciduous forests being richer in plant species than urban
parkland, it seems very likely that the forest site may maintain richer
bacterial communities. Saag et al. (2011) showed such a pattern of
habitat-related differences in bacterial density and species richness.
They revealed that although the number of phylotypes per bird was higher
in coniferous habitat, bacterial densities were higher in deciduous
habitat. It is also supported by the results presented by Broughton and
Gross (2000) who suggested that microbial activity was positively
correlated with plant productivity. Similarly, Bisson et al. (2007)
found that microorganisms from particular groups were more abundant in
plumage sampled from American redstart \emph{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 \emph{Arion vulgaris} (see Ba\selectlanguage{polish}ń\selectlanguage{english}bura et al. 2019 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. In addition, other factors, such as the
presence of bird aggregations influence soil properties and microbial
community in the soil (Wang et al., 2020), which may be important in the
garden area where during the autumn-winter period, wintering birds
gather in relatively large quantities (own observations). For the above
reasons, the microbial profile of the soil itself may play an important
role in the acquisition of plumage bacterial (Bison et al., 2007; Lucas
et al., 2003; Llado et al., 2017). It may be important in the context of
this study, since the great tit relatively frequently forages on the
ground (Gosler, 1993; own observations), 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) (McComb and
Noble, 1982; Broughton et al., 2015; own observations). 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 (\emph{Plecotus auratus} ) and noctules (\emph{Nyctalus
noctule} ) 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 (\emph{Martes martes}
)\emph{,} 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
forest (Kali\selectlanguage{polish}ń\selectlanguage{english}ski et al., 2014), 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.

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
boxes (Mainwaring, 2011; own observations) 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.

\textbf{5. CONCLUSIONS}

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.

\textbf{AUTHOR CONTRIBUTION}

\selectlanguage{polish}\textbf{Agnieszka Zabłotni:}\selectlanguage{english} laboratory procedures, writing -- review
and editing. \selectlanguage{polish}\textbf{Adam Kaliński:}\selectlanguage{english} investigation, conceptualization,
fieldwork, writing -- original draft, statistical
analyses.\selectlanguage{polish}\textbf{Mirosława Bańbura:}\selectlanguage{english} fieldwork, writing -- review and
editing.\selectlanguage{polish}\textbf{Michał Glądalski:}\selectlanguage{english} fieldwork, writing -- review and
editing.\textbf{Marcin Markowski:} writing -- review and editing.
\textbf{Joanna Skwarska:} writing -- review and editing.
\selectlanguage{polish}\textbf{Jarosław Wawrzyniak:}\selectlanguage{english}writing -- review and editing.
\selectlanguage{polish}\textbf{Jerzy Bańbura:}\selectlanguage{english}conceptualization, writing -- review and editing.

\textbf{ACKNOWLEDGEMENTS}

The authors thank E. Wr\selectlanguage{ngerman}óblewska, M. Kryzi\selectlanguage{polish}ń\selectlanguage{english}ska and D.Stobiecka for their
consent to conduct our research in the areas under their administration.
We are also greatly obliged to D. Ma\selectlanguage{polish}ń\selectlanguage{english}kowska, M. Janiszewska and J.
Bia\selectlanguage{polish}ł\selectlanguage{english}ek for their support.

\textbf{CONFLICT OF INTEREST}

The authors declare no conflict of interest.

\textbf{FUNDING}

This research did not receive any specific grant from funding agencies
in the public, commercial or not-for-profit sectors.

\textbf{ETHICAL STATEMENT}

All procedures were approved by the Local Ethical Committee in \selectlanguage{polish}Ł\selectlanguage{ngerman}ód\selectlanguage{polish}ź
\selectlanguage{english}(Lokalna Komisja Etyczna do Spraw Do\selectlanguage{polish}ś\selectlanguage{english}wiadcze\selectlanguage{polish}ń \selectlanguage{english}na Zwierz\selectlanguage{polish}ę\selectlanguage{english}tach w \selectlanguage{polish}Ł\selectlanguage{english}odzi,
Uniwersytet Medyczny w \selectlanguage{polish}Ł\selectlanguage{english}odzi -- Zak\selectlanguage{polish}ł\selectlanguage{english}ad Farmakodynamiki, ul. Muszy\selectlanguage{polish}ń\selectlanguage{english}skiego
1, 90-151 \selectlanguage{polish}Ł\selectlanguage{ngerman}ód\selectlanguage{polish}ź\selectlanguage{english}). All the methods were carried out under Polish law.

\textbf{DATA AVAILABILITY STATEMENT}

The dataset generated and analysed during the current study is available
in the figshare
repository,\emph{https://figshare.com/s/8cf266bd98ae89b5f394}.

\textbf{ORCID}

Agnieszka Zab\selectlanguage{polish}ł\selectlanguage{english}otni\emph{https://orcid.org/} 0000-0001-7982-9767

Adam Kali\selectlanguage{polish}ń\selectlanguage{english}ski\emph{https://orcid.org/}0000-0003-2743-6907

Miros\selectlanguage{polish}ł\selectlanguage{english}awa Ba\selectlanguage{polish}ń\selectlanguage{english}bura\emph{https://orcid.org/}0000-0003-1226-0398

Micha\selectlanguage{polish}ł \selectlanguage{english}Gl\selectlanguage{polish}ą\selectlanguage{english}dalski\emph{https://orcid.org/}0000-0002-6471-3080

Marcin Markowski\emph{https://orcid.org/}0000-0003-3113-9702

Joanna Skwarska\emph{https://orcid.org/}0000-0001-7806-3608

Jaros\selectlanguage{polish}ł\selectlanguage{english}aw Wawrzyniak\emph{https://orcid.org/}0000-0003-3450-9764

Jerzy Ba\selectlanguage{polish}ń\selectlanguage{english}bura\emph{https://orcid.org/}0000-0002-7479-3117

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\textbf{TABLE 1} . The mean, minimum, and maximum values of the entrance
hole bacterial load (CFU/ml) in the parkland and forest study areas in
both nest categories (occupied and unoccupied in the previous year).
Values are ln-transformed. SD values are given in parentheses.\selectlanguage{english}
\begin{longtable}[]{@{}lllllll@{}}
\toprule
& Parkland & Parkland & Parkland & Forest & Forest &
Forest\tabularnewline
\midrule
\endhead
& Mean & Min & Max & Mean & Min & Max\tabularnewline
Occupied & 7.54 (\selectlanguage{ngerman}±2.01) & 5.30 & 10.37 & 13.56 (\selectlanguage{ngerman}±1.91) & 10.40 &
15.40\tabularnewline
Unoccupied & 6.86 (\selectlanguage{ngerman}±0.65) & 5.99 & 7.78 & 14.98 (\selectlanguage{ngerman}±2.08) & 12.82 &
17.97\tabularnewline
\bottomrule
\end{longtable}

\textbf{TABLE 2} . The mean, minimum, and maximum values of the nest box
interior bacterial load (CFU/ml) in the parkland and forest study areas
in both nest categories (occupied and unoccupied in the previous year).
Values are ln-transformed. SD values are given in parentheses.\selectlanguage{english}
\begin{longtable}[]{@{}lllllll@{}}
\toprule
& Parkland & Parkland & Parkland & Forest & Forest &
Forest\tabularnewline
\midrule
\endhead
& Mean & Min & Max & Mean & Min & Max\tabularnewline
Occupied & 8.08 (\selectlanguage{ngerman}±2.01) & 5.30 & 12.47 & 14.35 (\selectlanguage{ngerman}±1.85) & 10.82 &
16.07\tabularnewline
Unoccupied & 8.58 (\selectlanguage{ngerman}±1.16) & 6.68 & 10.82 & 11.35 (\selectlanguage{ngerman}±1.98) & 8.99 &
15.40\tabularnewline
\bottomrule
\end{longtable}

\textbf{TABLE 3} . Summary of the bacterial load in the two-way ANOVA of
the entrance hole bacterial load (top) and the bacterial load in the
nest box (bottom). The effects of the study area, nest box occupancy in
the previous season, and the interaction between these factors are
given.\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
\begin{minipage}[b]{0.22\columnwidth}\raggedright\strut
Factor\strut
\end{minipage} & \begin{minipage}[b]{0.22\columnwidth}\raggedright\strut
Df\strut
\end{minipage} & \begin{minipage}[b]{0.22\columnwidth}\raggedright\strut
F\strut
\end{minipage} & \begin{minipage}[b]{0.22\columnwidth}\raggedright\strut
\emph{p}\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
\textbf{Entrance hole bacterial load} Intercept\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
1; 31\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
1550.001\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
\textbf{\textless{}0.001}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Study area\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
1; 31\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
168.089\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\textbf{\textless{}0.001}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Previous season occupancy\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
1; 31\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.465\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.500\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Study area* Previous season occupancy\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
1; 30\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
3.865\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.064\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
\textbf{Nest box interior bacterial load} Intercept\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
1; 31\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
1199.654\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
\textbf{\textless{}0.001}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Study area\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
1; 31\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
54.575\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\textbf{\textless{}0.001}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Previous season occupancy\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
1; 31\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
4.165\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\textbf{0.049}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Study area* Previous season occupancy\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
1; 30\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
8.207\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\textbf{0.007}\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

FIGURE CAPTIONS

\textbf{FIGURE 1.} Mean (\selectlanguage{ngerman}± standard errors) entrance hole bacterial load
in the nest boxes occupied and unoccupied in the previous season in two
study areas

\textbf{FIGURE 2.} Mean (± standard errors) nest box interior bacterial
load in the nest boxes occupied and unoccupied in the previous season in
two study areas

\selectlanguage{english}
\FloatBarrier
\end{document}

