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 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 (see Bańbura 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 (Plecotus auratus ) and noctules (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 (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 forest (Kaliński 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.