Composition of microbial communities
In this study, we have examined the bacterial communities on eggshells and nest materials. The composition of microbial community on the eggshell was significantly different that on the nest material at OTU level, although the major phylum composition was rather similar. The most abundant phylum found in this study was Proteobacteria, which is one of the dominant phyla found in soil [27], phyllosphere [28], potential nest material sources such as moss crusts [29], and also on avian eggshells [7, 8, 10, 30, 31].
Fimicutes, which was significantly more abundant in eggshell samples collected after incubation compared to the ones collected before incubation, is often found in animal’s gut microbiota including that of wild bird species [32]. Considering the lower abundance of Firmicutes in nest materials compared to eggshells, it is possible that the species belonging to Fimicutes, which might have originated from parent bird’s gastrointestinal tract, are competitively dominant over the other taxa on eggshells probably because the temperature on the eggshell is closer to that in gastrointestinal tract (due to incubation). Lee et al. (2014) [8] also found overall higher averaged relative abundance of Firmicutes in incubated eggs and found Bacillus (belonging to Firmicutes), which includes species producing antibiotics [33], being significantly more abundant after incubation. It is however also possible that there is a consistent load of Firmicutes from external sources other than nest materials during the incubation.
The most abundant genus in the eggshell microbiome was Psychrobacter and they were more abundant on the eggshell before incubation and their relative abundance decreased significantly with incubation. As its name suggests, they are mostly found in cold environments such as Arctic glacier or Antarctic [34-36]. However, they have also been found in other eggshell studies. Wang et al. (2011) [3] studied Western Bluebird, Tree Swallow, and Violet-green Swallow’s eggs in California and isolated Psychrobacter species from their samples. Grizard et al. (2014) [7] studied homing pigeons in Netherlands and found an OTU that has 99% sequence similarity with Psychrobacter glacinola. Although it has not been discussed in a detail in these studies, it seems Psychrobacter sp. can also be found in temperate environments. Further studies on the sources of Psychrobacter in the eggshells and their survival strategies in an ambient temperature would be interesting. Decrease in the relative abundance of Psychrobacter on eggshells during incubation might be due to increase in temperature both outside and inside of nests.
Massilia, which was found to be the most abundant genus in the nest material microbiome, has been isolated from a variety of environments, encompassing soil, atmosphere, freshwater, phyllosphere, rizosphere, and in blood samples [6, 37]. They have also been found in hen’s flea (Ceratophyllus gallinae) samples in Great tit’s nests {Aivelo, 2020 #55}. The presence of plants and rootlets in the nest may be related to the abundance of Massilla.
Goodenough and Stallwood (2010) {Goodenough, 2010 #71} accessed bacterial and fungal microbial composition of blue tit and great tit nest material using culture-based method and found species belonging to Bacillus, Pseudomonas, Enterobacter, and Staphylococcus dominating. Although some of those genera were also detected in our study, their relative abundance was much lower than it was found in Goodenough and Stallwood (2010) [38]. This could be due to the different methods used in the two studies. Animal pathogens have been studied very well and have been cultured for more than decades. In contrast, most of the bacterial taxa found in environmental samples are uncultured or recently culture lacking detailed information. The result from our study suggests that there are much more diverse bacterial groups present in bird’s nest and it is not necessarily dominated by pathogenic taxa.
Another finding in our study is that there is a lack of incubation effect on bacterial diversity even though there is an effect on bacterial composition. Grizard et al. (2014) [7] found reduction in bacterial diversity after incubation on the eggshells of homing pigeon Columba livia. Lee et al. (2014) [8] found the same pattern on the eggshells of Oriental magpie, Pica serica. The different pattern found in our study could be due to the ecological difference between studied species, for example, breeding season and the use of different types of nest materials. Below we discuss the remaining results in the framework of testing of the four predictions (see Introduction).
Testing the predictions
Prediction 1) Before the onset of incubation, the microbial communities formed on the nest material will be more diverse and will show stronger phylogenetic clustering than the microbial communities on the eggshells.
As expected, nest material microbial communities had higher diversity compared to eggshell microbial communities. Also, the co-occurrence of closely related taxa (which have high possibility to share genes with similar functions) was stronger in the nest material microbiome (lower SES.MNTD). These results support the idea that the nest materials contain already established microbiome whereas the eggshell microbiome did not have sufficient time to be established. However, greater diversity in nest material microbial communities than that on eggshells remained similar even after incubation. This suggests that, even with direct contact, eggshell microbiome experiences independent change from nest materials (see below).
Prediction 2) The initial communities of microbes on the eggshells and on nest materials will become more similar to each other during the incubation period.
We found that the eggshell microbial community does not necessarily become more similar to nest material microbial community after incubation. In fact, the changes in nest material microbial communities during incubation were minimal compared to those of the eggshell microbial communities. The nest materials might have had less chance to be affected by incubation, because they are placed under the eggs and do not interact directly with the female’s brood patch. Another possibility is that the nest materials, as they are not in direct contact with the female’s body, they are maintained at lower temperature than eggshells throughout the incubation period, which may differentially limit the proliferation of certain bacteria. The huge changes in the eggshell microbiome during incubation may imply rapid successional processes occurring during incubation. It could be useful to collect another sets of samples in the middle of incubation to have a glance at the spectrum of the changes.
Prediction 3) There will be higher relative abundance of beneficial bacteria and lower relative abundance of pathogenic bacteria after incubation both in the eggshell and nest materials.
Overall, we found high variability between the samples in their taxonomic composition. However, we were still able to find Psychrobacter and Paenibacillus showing significant differences in their relative abundance after incubation.Psychrobacter are rare opportunistic human pathogens [39] and their pathogenicity against birds has not been reported. Their relationship with birds has been described only in a limited number of studies, so it is difficult to conclude if they are beneficial or harmful to birds in general.
The relative abundance of Paenibacillus was higher in the eggshell samples collected after incubation than the ones collected before incubation. Many of Paenibacillus species are known to produce antibiotics [40, 41]. Their ability to produce antibiotics might be related to prevention of pathogenic bacterial growth and it may contribute to overall change in the composition of bacterial communities of eggshells and nest materials. Some of the species belonging to Paenibacillus are known to have keratinolytic activity, potentially contributing to bird feather degradation [42]. The increase in Paenibacillus after incubation might be originated from the feather of female birds through continuous contact with their eggs and could possibly affect chick’s feather development.
In conclusion, there is no evidence that potentially pathogenic taxa decreased their relative abundance. However, as we did not measure the absolute abundance, we cannot exclude the possibility that indeed the abundance of potentially pathogenic bacteria decreased assuming that that general abundance of bacteria might have decreased like in some previous studies [1, 4, 9]. We have evidence suggesting that the proportion of antibiotic-producing, hence potentially beneficial for birds, bacteria increased in the eggshell communities during incubation.
Prediction 4) Eggshell microbial community will experience greater phylogenetic turnover compared to nest material microbial community during incubation.
As expected, the phylogenetic turnover in nest material microbial communities were minimal. Similarly, with what we mentioned in Prediction 1, this result might be due to their relative “age”, as they have been already developed and established long before the nest materials are transported to the nest during nest building stage. On the other hand, eggshell microbial community showed similar level of phylogenetic turnover from what can be expected by chance, implying relatively new microbial communities were developing during incubation. This implies that the phylogenetic turnover occurring in eggshell microbial community is not related with (or influenced by) the dynamics in nest material microbial community. More likely is that the microbes originating from parents shape the dynamics in eggshell microbial community during incubation [10, 11, 43, 44]. For instance, we found noticeable increase in the proportion of Firmicutes on the eggshell and Firmicutes include species of microbes present in uropygial gland [45] gastrointestinal tract [38, 46, 47], or feathers [48]. It is plausible that the microbes originating from parents, either through contact with cloaca (when laying) or with uropygial gland and feathers (during incubation), are transferred once or continuously to the eggshell and shape the dynamics and phylogenetic turnover in eggshell microbial community.