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.