Discussion
Our study used a factorial experiment to determine the main and interactive effects of urbanization and parasitism on the gut microbiota of nestling small ground finches across two years. In contrast to a previous study (Michel et al., 2018), we did not find an effect of year on the gut microbiota of nestling small ground finches. Although we did not find an interactive effect of urbanization and parasitism on the microbiota, we did find main effects of each variable. Contrary to our prediction, urban nestlings had lower bacterial diversity (Shannon index) compared to non-urban nestlings. Parasitized (sham-fumigated) nestlings also had lower bacterial diversity (observed richness) compared to non-parasitized (fumigated) nestlings. However, parasitism did not affect Shannon index and urbanization did not affect observed richness, which suggests that parasitism affects the number of bacterial taxa but not evenness of those taxa, whereas urbanization influences gut microbiota evenness. This explanation is supported by our finding that urbanization, but not parasite treatment, affects the relative abundance of several bacterial phyla and genera. Although urbanization and parasitism did not have an overall effect on bacterial community membership and structure, changes in bacterial diversity or specific taxa can also affect physiological processes in hosts (Hooper et al., 2012; Round & Mazmanian, 2009), as discussed below.
We hypothesized that urban nestlings would have higher bacterial diversity because of their wide breadth of food items, including human-processed food, compared to a primarily insect-rich diet in non-urban nestlings. This hypothesis was based on several studies that found an increase in gut bacterial diversity in response to urbanization (Berlow et al., 2021; Knutie et al., 2019; Littleford‐Colquhoun et al., 2019; Phillips et al., 2018) and because diet can influence the gut microbiota (Bodawatta et al., 2022). However, we found that urban nestlings had lower bacterial diversity than non-urban nestlings, as found in Teyssier et al., (2018). One possible explanation is that the human-related food items select for particular bacterial taxa that dominate the microbiota, leading to fewer taxa in the gut. Teyssier et al., (2020) found that adult house sparrows (Passer domesticus ) that were experimentally fed an urban diet had lower gut bacterial diversity. Knutie, (2020) found that food supplementation with yellow mealworm beetle (Tenebrio molitor ) larvae increased the gut bacterial diversity of eastern bluebirds. Thus, an insect-rich diet in non-urban nestlings might maintain high bacterial diversity compared to diets of human-based foods in urban nestlings. Another possible explanation is that the food itself is introducing bacteria into the gut of the nestlings (Grond et al., 2018; Videvall et al., 2019) because human food items have different microbiota (Jarvis et al., 2018). To test these hypotheses, future studies may consider sequencing the microbiota of specific diet items and comparing these results with the gut microbiota.
Urban living also affected the relative abundance of bacterial genera and phyla in nestlings. For example, urban nestlings had higher abundances of the phylum Firmicutes and genus CandidatusArthromitus. These specific taxonomic changes in the gut microbiota can also facilitate functional changes to host physiology. For example, bacterial species from the phylum Firmicutes can aid in nutrient uptake and metabolism in chickens (Li et al., 2016; A. Zheng et al., 2016), which might be required for human-processed food.Candidatus Arthromitus is a well-studied, segmented filamentous bacterium that is non-pathogenic and attaches to the intestinal wall (Snel et al., 1995). Across host taxa, Candidatus Arthromitus influences the innate and adaptive immune responses in the gut (Macpherson & McCoy, 2015; Suzuki et al., 2004). Specifically, Liu et al., (2023) found that the relative abundance of CandidatusArthromitus is positively correlated with the innate immune response (e.g., T-lymphocytes) during avian development. In our system, urban finch nestlings upregulate the expression of genes related to the T-lymphocyte production (Knutie et al., 2023), which might explain why we observed higher relative abundance of Candidatus Arthromitus in urban nestlings.
Vertical transmission of microbiota from parent to nestling might also explain the observed differences in the microbiota of urban and non-urban nestlings. To feed their nestlings, finch parents regurgitate food from their crop into the mouth of the nestling (Koop et al., 2013; O’Connor et al., 2014; Price et al., 1983); thus, potentially transferring the crop and mouth microbiota from parent to offspring. Studies have found that crop microbiota can be transferred from parent to offspring through regurgitation in birds (Chen et al., 2020; Ding et al., 2020; Grond et al., 2018). However, the crop and fecal/cloacal microbiota of birds can differ in beta but not alpha diversity (Bodawatta et al., 2022; Wilkinson et al., 2017). To test whether our results are due, at least in part, to vertical transmission of microbiota, a future study could compare the crop and mouth microbiota to the gut microbiota of finch parents and nestlings.
Physiological stress associated with urban environments could also be a mechanism through which urban environments affect nestling gut microbiota. A study comparing urban and rural great tits (Parus major ) found that urban birds had an upregulation of several genes linked to stress responses, innate and adaptive immunity, and detoxification and repair systems (Watson et al., 2017). Physiological changes in immunity associated with urban stress likely impact gut microbiota composition, as both innate and adaptive immune responses can regulate and respond to shifts in gut microbiota (Zheng et al., 2020). Additionally, hormones associated with the stress response, such as glucocorticoids, can alter gut microbiota. For example, higher glucocorticoid levels are associated with lower bacterial diversity in squirrels (Petrullo et al., 2022) and gulls (Noguera et al., 2018). However, the effect of urbanization on glucocorticoids is variable across avian studies (Brodin & Watson, 2023; Deviche et al., 2023), and the link between physiological stress and gut microbial taxa likely varies based on the metric being investigated and type of sample used (e.g., glucocorticoids measured from blood vs. feathers/hair; Stothart et al., 2019). Future work should explore whether physiological stress mediates the effects of urbanization on nestling gut microbiota diversity and the relative abundance of gut bacterial taxa in this system.
Given that past studies have not found an effect of parasitism on gut microbiota of non-urban finches (Addesso et al., 2020; Knutie, 2018), we did not expect to find different results in our non-urban nestlings. However, we found an overall effect of parasitism on the gut microbiota across both locations, with parasitized nestlings having lower bacterial diversity (via observed richness) compared to non-parasitized nestlings. One possible explanation for the contradictory results is that other species of Darwin’s finches (e.g., medium ground finch [Geospiza fortis ] and common cactus finch [Geospiza scandens ]), host different gut microbial communities due to their different diets (e.g., cactus flower pollen, different seed types; De León et al., 2014). The interactions between the immune system and gut microbiota can be determined by which microbes are recognized by immune molecules. Thus, a change in the gut microbiota in small ground finches, but not medium ground finches or common cactus finches, in response to parasitism could be because their specific members of the microbiota are recognized and removed by the immune system. We also hypothesized that only urban nestlings would be affected by parasitism since they are more resistant to parasites compared to non-urban nestlings (Knutie et al., 2023). This resistance is potentially related to expression of type 1 interferon (IFN) genes, which can activate natural killer cells and macrophages that can destroy bacteria (Perry et al., 2005). Since the bacterial diversity metrics of both urban and non-urban nestlings were affected by parasitism, this suggests that both populations are having a general response to the parasite that interacts with the gut microbes. To establish a causal relationship, further investigation is required to understand the interaction between gut microbiota and immune response.
Overall, our study suggests that both parasitism and urbanization affect the gut microbiota of small ground finches. Since these anthropogenic factors also affect the health of finches in the Galápagos Islands (Harvey et al., 2021; Knutie et al., 2023), the next question is whether the microbiota are mediating these effects or influencing other traits, such as the immune system, in developing finches. To causally test these interactions, an experimental manipulation of the gut microbiota is necessary, either with the introduction of relevant bacterial taxa or a disruption of the gut microbiota with antibiotics. Although birds of the Galápagos Islands have experienced many direct effects of human presence, such as the introduction of parasites and changes in diet (De León et al., 2019; Wikelski et al., 2004), many indirect effects that are more difficult to study, such as those on the gut microbiota, could have important implications for the fitness of many endemic birds.
Acknowledgements: We thank Corinne Arthur for field assistance, and Karla Vasco for her lab assistance and logistical support. We also thank the Galápagos Science Center and the Galápagos National Park for support. The work was supported by start-up funds and a Research Excellence Program Grant from the University of Connecticut, and a National Science Foundation Grant (DEB-1949858) to SAK. GS was supported by Research Experience for Post-Baccalaureate Students (REPS) Funds (DEB-1949858). All bird handling and work was conducted according to approved University of Connecticut IACUC (Institutional Animal Care and Use Committee) protocols (No. A17-044). Our work in 2018-2019 was done under GNP permits PC 03-18 and PC 28-19 and Genetic Access permit MAE-DNB-CM-2016-0041.
Data accessibility: Supporting information has been made available online. Data are available at FigShare (doi: available upon acceptance) and sequences have been uploaded to GenBank (BioProject accession number: available upon acceptance).
Authors’ contributions: Conceptualization: SAK; Experimental Methodology: SAK, GJV; Analyses: AL; Investigation: GS, AL, GJV, JH, TBV, KC, SS, LA, SAK; Visualization: AL; Funding acquisition: SAK; Project administration: JC, SAK; Supervision: SAK; Writing – original draft: GS, AL, SAK; Writing – review & editing: All authors.
Conflict of Interest: The authors declare that they have no conflict of interest.