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\begin{document}

\title{Effect of urbanization and parasitism on the gut microbiota of Darwin's
finch nestlings}



\author[1]{Gabrielle Solomon}%
\author[1]{Ashley Love}%
\author[1]{Grace Vaziri}%
\author[1]{Johanna Harvey}%
\author[1]{Taylor Verrett}%
\author[1]{Kiley Chernicky}%
\author[1]{Shelby Simons}%
\author[1]{Lauren Albert}%
\author[2]{Jaime Chaves}%
\author[1]{Sarah Knutie}%
\affil[1]{University of Connecticut}%
\affil[2]{San Francisco State University}%


\vspace{-1em}



  
  \date{May 29, 2024}


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\selectlanguage{english}
\begin{abstract}
Host-associated microbiota can be affected by factors related to
environmental change, such as urbanization and invasive species. For
example, urban areas often affect food availability for animals, which
can change their gut microbiota. Invasive parasites can also influence
microbiota through either competition or indirectly through a change in
the host immune response. These interacting factors can have complex
effects on host fitness, but few studies have disentangled the
relationship between urbanization and parasitism on an organism's gut
microbial composition. To address this gap in knowledge, we investigated
the effects of urbanization and parasitism by the invasive avian vampire
fly (Philornis downsi) on the gut microbiota of nestling small ground
finches (Geospiza fuliginosa) on San Crist\selectlanguage{ngerman}óbal Island, Galápagos. We
conducted a factorial study in which we experimentally manipulated
parasite presence in an urban and non-urban area. Feces were then
collected when nestlings to characterize the gut microbiota (i.e., alpha
and beta diversity, community composition). Although we did not find an
interactive effect of urbanization and parasitism on the microbiota, we
did find main effects of each variable. Urban and parasitized nestlings
had lower bacterial diversity and differences in relative abundance of
bacterial phyla and genera compared to non-urban non-parasitized
nestlings, respectively. Overall, this study advances our understanding
of the complex effects of anthropogenic stressors on the gut microbiota
of birds.%
\end{abstract}\selectlanguage{ngerman}%



\sloppy


\textbf{Introduction}

The gut microbiota is a community of microorganisms inhabiting an
organism's digestive tract that can affect host physiology and health
(Gomaa, 2020; Grond et al., 2018). For example, specific gut bacterial
taxa, such as \emph{Ruminococcus} , can aid in host digestion by
breaking down indigestible food compounds, such as cellulose, and
increase digestive efficiency in humans (Arumugam et al., 2011; Thursby
\& Juge, 2017). Furthermore, variation in diet, such as a plant-based
diet versus a meat-based diet, can shape host gut microbiota as it
shifts in response to nutrient availability (Gomaa, 2020; Zhu et al.,
2015). Additionally, the host's immune system can interact with the gut
microbiota, specifically by shaping its community composition (Hooper et
al., 2012). For example, Kato et al., (2014) found that knockout mice
who are deficient in B-cell production have lower gut bacterial
diversity compared to non-knockout mice. The gut microbiota can also
initiate the production of immune cells to help maintain or develop an
effective immune response (Zhang et al., 2017). Studying the dynamic
relationships between physiological processes and gut microbiota will be
continually important, especially in light of increasing anthropogenic
change which can directly and indirectly affect these factors.

Parasites and pathogens, which also affect host health, can directly and
indirectly affect microbial communities in the gut (Stensvold \& Van Der
Giezen, 2018). Within the gut, parasites can directly affect the
microbiota by competing with or consuming commensal bacteria (Abt \&
Pamer, 2014). Parasites might also indirectly affect the gut microbiota
via the host's immune system. For example, gut parasites
(e.g.,\emph{Heligmosomoides polygyrus} ) can activate a non-specific
mucosal immune response (e.g., T and Th2 cells), which can select for or
against bacterial taxa, such as an increase in gram negative
Enterobacteriaceae and a decrease in \emph{Enterococcus faecium} (Rausch
et al., 2018). However, parasites located outside of the gut might also
elicit a systemic immune response that could affect the bacterial
community in the gut. For example, fish with ectoparasitic fluke
(\emph{Dactylogyrus lamellatus} ) infections have lower gut bacterial
diversity compared to uninfected fish, which was likely mediated by
elevated expression of immune genes related to the IgM antibody,
toll-like receptor 3, and Major histocompatibility factor II responses
(Wang et al., 2023). Knutie, (2020) found that parasitic nest fly
abundance is negatively correlated with gut bacterial diversity and
positively correlated with IgY antibody levels, in eastern bluebirds
(\emph{Sialia sialis} ). Overall, most of these non-model organism
studies are correlational and thus the causal effects of parasitism on
gut microbiota are not well understood.

Factors related to urbanization, such as changes in food availability,
can also influence the gut microbiota of wild hosts (Berlow et al.,
2021; Phillips et al., 2018; Teyssier et al., 2020). Studies have found
that urban hosts have a larger diet breadth, which can result in greater
diversity of the gut bacterial community (Littleford-Colquhoun et al.,
2017). For example, non-urban water dragons (\emph{Intellagama
lesueurii} ) feed primarily on invertebrates but urban water dragons
feed on invertebrates and plant material, which results in higher gut
bacterial diversity. These effects are either because plants introduce
additional bacterial taxa or a larger diet breadth selects for different
bacteria to aid in digestion. If the latter is supported, a more diverse
gut microbial community could help hosts be more equipped to deal with
environmental change (Littleford-Colquhoun et al., 2017). In contrast,
the gut of house sparrows (\emph{Passer domesticus} ) consuming a
non-urban diet, which is rich in protein, is correlated with higher
bacterial diversity compared to urban sparrows whose diet is poor in
protein (Teyssier et al., 2020). While increased gut bacterial diversity
is generally assumed to be beneficial to hosts, in some cases, such as
in organisms with highly specialized diets, lower bacterial diversity
can be associated with better health outcomes (Shade et al., 2017). For
example, urban coyotes consume carbohydrate-rich anthropogenic food
items and have higher bacterial diversity compared to their rural
counterparts, but are in poorer body condition and have increased
prevalence of the parasite \emph{Echinococcus multilocularis}(Sugden et
al., 2020). Ultimately, these effects of urbanization and other
stressors related to human activity (e.g., invasive parasites) are
complex and could result in positive or negative implications for host
health.

Given the complex effects of human activity on hosts, few studies have
examined the influence of multiple synergistic anthropogenic factors,
such as urbanization and invasive parasitism, on host gut microbiota.
The Gal\selectlanguage{ngerman}ápagos Islands of Ecuador provide an ideal study system to
investigate these complex effects. Since 1979, the number of residents
and tourists have increased, leading to changes in the natural habitat.
These changes include the introduction of non-native species, including
parasites (Kerr et al., 2004; Wikelski et al., 2004). For example, the
avian vampire fly (\emph{Philornis downsi} ; hereon, vampire fly) was
introduced to the Galápagos in the past several decades and is found on
nearly all islands, including human-inhabited islands such as San
Cristóbal. Adult flies are non-parasitic but lay their eggs in birds'
nests where the hematophagous larvae feed on nestling hosts and brooding
mothers (Fessl et al., 2001; Fessl \& Tebbich, 2002). Several studies
have found that the vampire fly can have detrimental effects on the
survival of nestling Darwin's finches (Fessl et al., 2010; Kleindorfer
\& Dudaniec, 2016; Knutie et al., 2016; Koop et al., 2011, 2013; McNew
\& Clayton, 2018; O'Connor et al., 2010). However, a recent study found
that urban finches on San Cristóbal Island are less affected by and more
resistant to the vampire fly than non-urban finches (Knutie et al.,
2023) who suffer up to 100\% mortality due to the fly (Koop et al.,
2013; O'Connor et al., 2014). For non-urban finches, the vampire fly
does not affect the gut microbiota (Addesso et al., 2020; Knutie, 2018;
Knutie et al., 2019). However, because urban finches are more resistant
to the fly, and immunological resistance can interact with gut
microbiota, parasitism may cause a greater change on the microbiota of
urban finches compared to non-urban finches. To date, no studies have
causally explored whether parasitism and urbanization interact to affect
the gut microbiota of hosts.

The goal of this study is to compare the effects of avian vampire flies
and urbanization on the gut microbiota of nestling small ground finches
(\emph{Geospiza fulignosa} ) in 2018 and 2019. Specifically, we
experimentally manipulated parasite abundance in urban and non-urban
finch nests and then characterized the gut microbiota (i.e., alpha and
beta diversity, community composition, relative abundance of taxa).
Because diet can influence the gut microbiota (Davidson et al., 2020)
and urban finches have a more diverse diet compared to non-urban finches
(De León et al., 2019), we hypothesize that urban nestlings will have a
different gut microbiota than non-urban nestlings (Loo et al., 2019).
Because immunological resistance can be linked to the gut microbiota
(Hooper et al., 2012), we predict parasitism will affect and ultimately
change the gut microbiota of urban nestlings. Past studies have found
that parasitism by vampire flies does not affect the gut microbiota of
finch nestlings in non-urban areas (Addesso et al., 2020; Knutie, 2018;
Knutie et al., 2019), but their diet is not supplemented. Therefore, we
predict that parasitism will not affect the gut microbiota of non-urban
nestlings. Further investigation will allow for a more thorough
understanding of environmental change on the gut microbiota of small
ground finch nestlings.

\textbf{Methods}

\emph{Study system}

The study was conducted between February -- May 2018 and 2019 (during
the breeding season) in the arid lowlands of San Cristóbal (557
km\textsuperscript{2}) in the Galápagos Islands. The urban area was in
the capital city of Puerto Baquerizo Moreno (0°54'9''S, 89°36'33''W)
(hereon, urban area), which is the only large town on San Cristóbal and
the second largest city in the Galápagos archipelago with a human
population of 7,199 (INEC, 2016). The urban area primarily consists of
impermeable concrete or stone surfaces and human built structures in
altered landscapes. Our urban study area measured 0.79
km\textsuperscript{2} (\textasciitilde{}1.2 km by 0.62 km) and included
tourist and residential zones. The non-urban area was in Jardín de
Opuntias (0°56'18.92''S, 89°32'54.93''W) (hereon, non-urban area), which
is a Galápagos National Park site located 4.5 km southeast of the urban
area. This site consisted of vegetated natural habitats with no
unnatural impermeable surfaces present. Our non-urban study area
measured 0.21 km\textsuperscript{2} and covered 1.4 km of the main trail
and 0.15 km to each side.

Small ground finches are abundant at both field sites (Harvey et al.,
2021). Small ground finches build domed-shaped nests in native and
non-native trees and shrubs as well as human-built structures, depending
on the location. On San Cristóbal Island, they lay 1-4 eggs per clutch
(mean = 2.84 eggs) and have an average of 2.83 nestlings per brood
across urban and non-urban areas. However, in some years, nests in urban
areas contained more eggs than non-urban areas, yielding higher nestling
survival (Harvey et al., 2021).

\emph{Experimental manipulation of parasites}

We searched field sites daily for evidence of nest building by small
ground finches. Once eggs were laid, we checked nests every other day
until the nestlings hatched. Prior to egg hatching, we experimentally
manipulated fly abundance via adding approximately 10 mL of 1\%
permethrin solution (Permacap; hereon, fumigated) or water (hereon,
sham-fumigated) to the nest. Within two days of nestling hatching, we
treated nests again to ensure the removal of all parasites. Briefly, we
removed the contents of the nests (including the nestlings, unhatched
eggs, and the nest liner), and treated them with either a permethrin
solution or water by spraying approximately 5 mL into the nest where the
larvae live. After treatment, we returned the dry nest liner, and placed
nestlings back into the nest. We used permethrin because it is highly
effective at removing vampire flies from the nests (Kleindorfer \&
Dudaniec, 2016). However, studies have found that permethrin can have
sublethal effects on nestling birds (Bulgarella et al., 2020);
therefore, we ensured that nestlings did not come into contact with the
insecticide. Additionally, adults returned to their nests with no cases
of abandonment from the treatment (Koop et al., 2013; Knutie et al.,
2016).

\emph{Sample collection}

We banded nestlings at 6-8 days old with a unique color band combination
and a numbered metal band (National Band and Tag, Kentucky, USA). We
collected fecal samples from nestlings opportunistically at this time.
To collect fecal samples, we removed nestlings from the nest and held
them over a sterile weigh boat until they defecated. We then moved the
fecal sample from the tray to a sterile tube, placed it on ice in the
field for up to 6 hours, and then stored it in a -20 °C freezer until
the bacterial DNA was extracted. We transported the samples to the
University of Connecticut and stored them in a -80 °C freezer for
downstream 16S sequencing. Although studies show that the bacterial
community in avian feces does not always represent the entire digesta of
the host (e.g., in the cecum; Wilkinson et al., 2017), fecal samples are
generally representative of the bacterial community in the large
intestines (Videvall et al., 2018; Wilkinson et al., 2017) and are used
when hosts cannot be euthanized. When nests were empty, they were
collected and examined for \emph{P. downsi} ; the full data set was
reported in Knutie et al., (2023).

\emph{Bacterial DNA extraction and sequencing}

We extracted total DNA from feces using a ZymoBIONICS DNA kit and sent
DNA extractions to the University of Connecticut Microbial Analysis,
Resources and Services for sequencing with an Illumina MiSeq platform
and v2 2x250 base pair kit (Illumina, Inc.). We also sequenced a
laboratory extraction blank to control for kit contamination. We
conducted bacterial inventories via amplification of the V4 region of
the 16S rRNA gene using primers 515F and 806R and with Illumina adapters
and dual indices (Kozich et al., 2013). We used the DADA2 (v. 1.22.0)
pipeline (Callahan et al., 2016) in R (v. 4.2.0) to process sequence
data. After quality assessment, we trimmed sequences to remove low
quality read areas and chimeric reads. We classified amplicon sequence
variant (ASV) taxonomies using RDP's Naive Bayesian Classifier (Q. Wang
et al., 2007) with the Silva reference database (v. 138.1) (Quast et
al., 2012). After classification, we removed sequences identified as
chloroplast and mitochondria from the dataset. We identified and removed
likely bacterial contaminants with the package \emph{decontam} (Davis et
al., 2018) in R using the kit extraction blank that was processed in
parallel with the other samples as a control. Sequences were aligned
using the \emph{DECIPHER} package (v. 2.22.0) in R (Wright, 2015), and a
generalized time-reversible maximum likelihood tree of the remaining
ASVs was constructed with the \emph{phangorn} package version 2.9.0
(Schliep, 2011). The ASV table, taxonomic information, phylogeny, and
sample metadata were joined for bacterial community analyses using the
package \emph{phyloseq} (McMurdie \& Holmes, 2013). We filtered the
feature table to retain samples with at least 1,500 total reads which
reduced the dataset to 58 samples containing 2,526 unique ASVs. The
resulting data set had an average of 51,056 ± 6,178 reads per sample
(min: 1,732; max: 294,720). For alpha and beta diversity analyses, we
rarefied samples to the sample with the lowest read count (1,732). The
filtered dataset contained 1,448 ASVs after random subsampling.

\emph{Statistical analyses}

\emph{Alpha Diversity}

We used the R package \emph{vegan} (Oksanen et al., 2022) to compute
alpha diversity (observed ASV richness, Shannon diversity index) on the
filtered dataset. Observed richness describes the number of observed
species and evenness describes the distribution of abundance across the
species. The Shannon diversity index is an estimator of species richness
and species evenness. We ran generalized linear mixed effects models
with a negative binomial error structure for observed ASV richness and a
Gaussian distribution for Shannon diversity using the glmmTMB package
(Brooks et al., 2017). Location (urban, non-urban), parasite treatment
(fumigated, sham-fumigated) and the interaction between location and
parasite treatment were considered in all models. Because the microbiota
of finches can vary across years (Michel et al., 2018), we also included
year (2018, 2019) as a covariate in all models. Nest identification was
included as a random effect in all models. We used the Anova function in
the \emph{car} package (Fox \& Weisberg, 2018) to determine
significance.

\emph{Beta Diversity}

To deal with unequal sequence coverage and compositional variation
within the data, we used Cumulative Sum Scaling (CSS) normalization on
the filtered dataset using the R package \emph{metagenomeseq} (Paulson
et al., 2013) following the methods in Maraci et al., (2021). We then
log (x + 0.0001) transformed the data and later corrected the
transformed values by subtracting the log of the pseudo count (Thorsen
et al., 2016). Dissimilarity matrices were computed based on
Bray--Curtis (Bray \& Curtis, 1957), unweighted UniFrac (C. Lozupone \&
Knight, 2005), and weighted UniFrac (Lozupone et al., 2007). To
visualize the dissimilarities between nestlings based on parasite
treatment and urbanization, we used a principal coordinate analysis
(PCoA) using the ordinate function in the \emph{phyloseq} package
(McMurdie \& Holmes, 2013). To determine the effect of parasitism and
urbanization on beta diversity metrics, we used PERMANOVAs with parasite
treatment, location, year (2018, 2019), and the interaction between
parasite treatment and location as fixed effects and beta diversity
metrics as response variables. Nest identification was included as a
random effect in all models. We used the adonis2 function with
the\emph{vegan} package for these analyses (Oksanen et al., 2022).

\emph{Relative Abundance of Bacterial Taxa}

We calculated the relative abundances of phyla and genera from the
unrarefied dataset. For analyses, phyla and genera with mean abundances
\textless{}1\% were lumped into the ``Other'' category. This data
stringency limited analyses to the top three most abundant phyla
(Proteobacteria, Firmicutes, Actinobacteriota) and the top twelve most
abundant genera (Table 1). We ran non-parametric Kruskal-Wallis tests
using the kruskal.test function in R to compare abundances of bacterial
taxa between locations (urban, non-urban), parasite treatments
(sham-fumigated, fumigated), and locations within the parasitized
(sham-fumigated) treatment group. \emph{P} -values were adjusted for
false discovery rate with a Benjamini--Hochberg correction where
significance was determined as \emph{P} \textsubscript{adj} \textless{}
0.05.

\textbf{Results}

\emph{Bacterial diversity}

Nestlings from fumigated nests had higher bacterial richness than
nestlings from sham-fumigated nests (Figure 1A, \selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2} =
4.04, \emph{P} = 0.04). Shannon diversity index was not significantly
affected by parasite treatment (Figure 1B, \selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2} =
1.10,\emph{P} = 0.29). Urbanization did not significantly affect
observed richness (Figure 1C, \selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2} = 2.30, \emph{P} =
0.13), but did influence the Shannon index (Figure 1D,
\selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2} = 7.50,\emph{P} = 0.006). Non-urban nestlings had
higher Shannon diversity index values compared to urban nestlings.
Observed richness and Shannon index were not affected by the interaction
between parasite treatment and urbanization (Figure S1, Observed
richness: \selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2} = 0.28, \emph{P} = 0.60, Shannon Index:
\selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2} = 0.21,\emph{P} = 0.65). The year that samples were
collected (2018, 2019) did not significantly affect the observed
richness (Table S1, \selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2} = 2.55, \emph{P} = 0.11) or
Shannon index (\selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2} = 0.38, \emph{P} = 0.54).

\emph{Bacterial community structure and membership}

Using the Bray-Curtis Dissimilarity distances, parasitise treatment,
urbanization, and the interaction between the two variables did not
significantly affect bacterial community structure (Figure S2A, parasite
treatment: \emph{F} = 1.52, \emph{P} = 1.00, urbanization: \emph{F} =
2.05, \emph{P} = 1.00, Interaction: \emph{F} = 1.24, \emph{P} = 0.48).
Using the unweighted UniFrac distances, parasitism, urbanization, and
the interaction between the two variables did not significantly affect
bacterial community membership (Figure S2B, parasite treatment:
\emph{F}= 1.45, \emph{P} = 1.00, urbanization: \emph{F} = 2.26, \emph{P}
= 0.52, Interaction: \emph{F} = 1.77, \emph{P} = 0.52). Using the
weighted UniFrac distances, parasite treatment, urbanization, and the
interaction between the two variables did not significantly affect
bacterial community structure (Figure S2C, parasite treatment\emph{: F}
= 2.09,\emph{P} = 1.00, urbanization: \emph{F} = 2.73, \emph{P} = 1.00,
interaction: \emph{F} = 3.14, \emph{P} = 0.50). Year did not affect
Bray-Curtis Dissimilarity, weighted UniFrac, or unweighted UniFrac
(Table S2).

\emph{Microbiota taxonomic composition}

There were 2,526 unique ASVs present in the samples. Twenty-four phyla
were identified and samples were dominated by Firmicutes (52.6\%),
Proteobactiera (35.1\%), and Actinobacteriota (8.5\%; Fig. 2). All other
phyla had mean relative abundances of \textless{}1\%. Across the 58
samples, there were 551 genera identified and twelve genera had relative
abundances \textgreater{}1\%. The most commonly observed genera
were\emph{Candidatus} Arthromitus (22.3\%),
\emph{Escherichia-Shigella}(13.8\%), \emph{Enterococcus} (13.5\%),
\emph{Ligilactobacillus}(9.1\%), \emph{Klebsiella} (7.8\%),
\emph{Cronobacter} (4.8\%),\emph{Erysipelatoclostridium} (2.3\%),
\emph{Rothia} (2.2\%),\emph{Clostridium sensu stricto 1} (1.9\%),
\emph{Campylobacter}(1.6\%), \emph{Corynebacterium} (1.4\%), and
\emph{Kocuria} (1.1\%).

At the phylum level, location significantly affected the relative
abundance of Firmicutes and Proteobacteria (Table 1). Specifically,
nestlings from urban sites had higher relative abundances of Firmicutes
(\selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2} = 13.74, \emph{P\textsubscript{adj}} = 0.008) and
lower abundances of Proteobacteria (\selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2} =
8.35,\emph{P\textsubscript{adj}} = 0.01) compared to nestlings from
non-urban field sites (Figure 2). At the genus level, location
significantly affected the abundance of \emph{Candidatus}
Arthromitus,\emph{Klebsiella} , \emph{Erysipelatoclostridium} , and
\emph{Rothia}(Table 1, Figure S3). \emph{Candidatus} Arthromitus was
highly abundant in urban nestlings, but rarely observed in fecal samples
collected from non-urban nestlings (Figure 3, \selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2} =
10.94,\emph{P\textsubscript{adj}} = 0.01). Conversely, \emph{Klebsiella}
( \selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2}=7.13, \emph{P\textsubscript{adj}} = 0.03)
and\emph{Erysipelatoclostridium} (\selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2} =
6.62,\emph{P\textsubscript{ad j}} = 0.03) were observed at higher
abundances in fecal samples from non-urban nestlings (Figure 3). The
genus\emph{Rothia} was more common in nestlings from urban nests and was
rarely observed in non-urban nestlings (\selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2} =
7.29,\emph{P\textsubscript{ad j}} = 0.03). This trend was difficult to
visualize due to two individuals that had very high relative abundance
values (Figure S4: one non-urban nestling: 41.89\% \emph{Rothia} , and
one urban nestling: 63.43\% \emph{Rothia} ). Additional outliers were
detected and removed using the boxplot.stats function in R (non-urban:
1.25\%, urban nestlings: 8.36\%, 2.77\%, 2.15\%, 0.73\%). Location
(urban, non-urban) still significantly affected the relative abundance
of \emph{Rothia} with the two greatest outliers removed (Figure S4:
\selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2} = 10.11, \emph{P\textsubscript{ad j}} = 0.01) and
with the five additional outliers identified by the boxplot.stats
function removed (shown in Fig 3, \selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2} =
12.57,\emph{P\textsubscript{ad j}} = 0.005).

Parasite treatment did not significantly influence the relative
abundance of the three most abundant phyla or the abundance of any of
the twelve most abundant genera (Table 1). When analyzing only nestling
samples from sham-fumigated nests, there was still a significant effect
of location on the abundance of Firmicutes and Proteobacteria (Figure
S5). However, there was no effect of urbanization on the abundance of
the top twelve genera within the sham-fumigated nests (Table 1).

\textbf{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 (\emph{Passer domesticus} )
that were experimentally fed an urban diet had lower gut bacterial
diversity. Knutie, (2020) found that food supplementation with yellow
mealworm beetle (\emph{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
\emph{Candidatus}Arthromitus\emph{.} 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.\emph{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, \emph{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
\emph{Candidatus}Arthromitus 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 \emph{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 (\emph{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 {[}\emph{Geospiza
fortis} {]} and common cactus finch {[}\emph{Geospiza scandens} {]}),
host different gut microbial communities due to their different diets
(e.g., cactus flower pollen, different seed types; De Le\selectlanguage{ngerman}ón 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.

\textbf{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.

\textbf{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).

\textbf{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.

\textbf{Conflict of Interest:} The authors declare that they have no
conflict of interest.

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\textbf{Figure Legends}

\textbf{Figure 1.} Effect of parasite treatment (sham-fumigated,
fumigated) on (a) observed ASV richness and (b) Shannon diversity of the
microbiota in nestling small ground finches. Individuals from fumigated
nests are represented by red circles and individuals from sham-fumigated
nests are represented by blue circles. Effect of location (urban,
non-urban) on (c) the observed ASV richness and (d) Shannon diversity of
the microbiota in nestling small ground finches. Individuals from
non-urban nests are represented by green circles and individuals from
urban nests are represented by gray circles. Black circles denote the
mean values (±SE) of birds from each treatment.

\textbf{Figure 2.} (a) Proportional abundance of bacterial phyla across
location (urban, non-urban) and parasite treatment (fumigated,
sham-fumigated). Each bar represents a sample from an individual bird.
Samples are divided into treatment groups as follows: non-urban,
fumigated: \emph{n} = 7 nestlings; non-urban, sham-fumigated: \emph{n} =
7; urban, fumigated: \emph{n} = 23; urban, sham-fumigated: \emph{n} =
21. Phyla with \textless{}1\% relative abundance are collapsed into the
category ``\textless{}1\% abundance.'' (b) Relative abundance of the
three most common phyla separated by location (urban, non-urban).
Individual points represent the relative abundance of each phylum from
an individual nestling. Black circles denote the mean (±SE) relative
abundances across treatments.

\textbf{Figure 3.} Relative abundance of the four genera
(\emph{Candidatus} Arthromitus, \emph{Klebsiella}
,\emph{Erysipelatoclostridium} , \emph{Rothia} ) that significantly
varied by location (urban, non-urban). Individual points represent the
relative abundance of each genus from an individual nestling. Black
circles denote the mean (±SE) across treatments.

\textbf{Tables}

\textbf{Table 1.} Kruskal-Wallis chi-square (\selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2}) test
statistics and p-values for comparisons of bacterial taxa across
location (urban, non-urban), parasite treatment (sham-fumigated,
fumigated), and location (urban, non-urban) across parasitized
(sham-fumigated only) nests. \emph{P} -values were adjusted for false
discovery rate with a Benjamini--Hochberg correction where significance
was determined as \emph{P} \textsubscript{adj} \textless{} 0.05.
Significant differences between groups are represented in bold.\selectlanguage{english}
\begin{longtable}[]{@{}lllllll@{}}
\toprule
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{Taxonomic Group}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{Location}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{Location}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{Parasite} \textbf{Treatment}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{Parasite} \textbf{Treatment}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{Location} \textbf{(Sham-fumigated only)}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{Location} \textbf{(Sham-fumigated only)}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\(\chi^{2}\)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\emph{P}\textsubscript{adj}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\(\chi^{2}\)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\emph{P}\textsubscript{adj}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\(\chi^{2}\)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\emph{P}\textsubscript{adj}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Phylum\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Actinobacteriota\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
4.14\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.06\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
4.03\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.18\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.68\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.55\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Firmicutes\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{13.74}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{0.0008}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.65\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.42\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{9.63}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{0.004}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Proteobacteria\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{8.35}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{0.01}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.93\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.42\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{9.63}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{0.004}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Other (\textless{} 1\%)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
1.31\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.25\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
1.35\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.42\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.00\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.48\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Genus\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\emph{Campylobacter}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.65\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.55\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
2.18\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.56\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.69\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.53\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\emph{Candidatus} Arthromitus\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{10.94}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{0.01}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
1.94\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.56\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
5.71\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.11\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\emph{Clostridium} sensu stricto 1\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.46\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.59\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.36\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.71\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
2.62\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.28\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\emph{Corynebacterium}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
1.17\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.44\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
1.07\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.56\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.04\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.85\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\emph{Cronobacter}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.14\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.71\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
4.59\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.42\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
1.49\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.36\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\emph{Enterococcus}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.27\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.66\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
1.11\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.56\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.21\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.76\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\emph{Erysipelatoclostridium}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{6.62}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{0.03}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.98\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.56\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
3.32\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.22\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\emph{Escherichia-Shigella}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
1.06\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.44\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.90\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.56\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.09\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.82\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\emph{Klebsiella}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{7.13}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{0.03}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.74\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.56\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
6.76\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.11\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\emph{Kocuria}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
1.57\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.44\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.05\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.82\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
3.30\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.22\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\emph{Ligilactobacillus}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
1.30\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.44\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.07\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.82\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.72\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.53\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\emph{Rothia}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{7.29}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{0.03}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.17\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.80\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
2.10\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.32\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Other (\textless{} 1\%)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
4.07\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.11\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
1.79\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.56\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
1.83\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.33\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}



\textbf{Fig. 1}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Fig. 2}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Fig. 3}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

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

