1. Comparative analysis of gut microbiome among populations of Channel Island Fox, Urocyon littoralis
Materials and methods
We obtained scat samples from all six inhabited Channel Islands.
We extracted total genomic DNA from U. littoralis scat using
E.Z.N.A Stool DNA Kit. Genomic DNA was quantified using a
QuBit 2.0 Fluorometer. We verified scat contained
Introduction
The Island Fox (Urocyon littoralis), an endemic species to
California’s Channel Islands, underwent severe population
declines in the 1990s and four of the six subspecies were
listed on the Federal endangered species list in 2004.
Recent population bottlenecks may have implications for
those populations’ genetic variability and pathogen
prevalence. In particular, we are interested in quantifying
the diversity of the island foxes’ gut microbiome, since
recent studies have begun
to use microbiota diversity
as a useful proxy for
evaluating health in other
animals.
Lauren Stoneburner, Nicole Adams, Suzanne Edmands
Life Sciences
Literature cited
Results Future Research
Acknowledgements
I thank Dr. Suzanne Edmands and Nicole Adams for mentorship, and USC
Undergraduate Research Associates Program for funding. We thank the following
institutions for sample contributions: Institute for Wildlife Studies, Santa Catalina
Island Conservancy, U.S. National Park Service, and U.S. Navy
island fox DNA using canid-specific
cytochrome b (cytb) primers and
sequencing a subset of cytb-amplified
fragments. We are currently amplifying the
16S rRNA V4 hypervariable region of
available genomes for library preparation.
Figure 2. Raw U. littoralis scat sample.
Figure 1. Channel Island
Fox (Urocyon littoralis)
Figure 3. Map of Channel
Islands. Urocyon littoralis
scat samples collected from
locations indicated by red
landmarks.
Table 2. DNA concentrations from EZNA Stool Kit
Pathogen Extraction Protocol.
Figure 4. Gel electrophoresis of PCR
amplified sequences of total genomic DNA
(column 2), mitochondrial cytochrome b
(columns 3 - 4), and 16S rRNA V4 region of
all available DNA (columns 6 - 9).
Table 1. Scat samples collected and
standardized body condition data for
each island.
(Adams, 2015)
(Stoneburner, 2015)
Hofman CA, Rick TC, Hawkins MTR, Funk
WC, Ralls K, et al. (2015) Mitochondrial
Genomes Suggest Rapid Evolution of Dwarf
California Channel Islands Foxes (Urocyon
littoralis). PLoS ONE 10(2): e0118240.
Caporaso, JG; CL Lauber; WA Walters; D
Berg-Lyons; CA Lozupone; PJ Turnbaugh;
N Fierer; R Knight. 2011. Global patterns of
16S rRNA diversity at a depth of millions of
sequences per sample. PNAS 108 (1): 4516–
4522.
Aguilar, A; G Roemer; S Debenham; M
Binns; D Garcelon; RK Wayne. 2004. High
MHC diversity maintained by balancing
selection in an otherwise genetically
monomorphic mammal. PNAS 101(10):3490–
3494.
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We extracted sufficient DNA to sequence the 16S rRNA V4 region.
Fragment sizes of approximately 400 bp are being amplified by PRC using
cytb and 16S primers. This confirms that the samples collected contain canid
DNA and suggests microorganisms with the 16S rRNA V4 region which we
will sequence to quantify gut microbial diversity.
Our next step is to sequence the amplified 16S
rRNA V4 region of available genomes using
Illumina MiSeq 300PE and quantify the
microbial diversity in each individual.
Methods for quantifying and comparing microbial
diversity among populations follow the Earth
Microbiome Project (EMP) established pipeline. We
will then cross-reference the PATRIC database to filter
out possible pathogenic sequences.
Hypotheses
1. More phylogenetically similar populations,
according to mtDNA phylogeny in Hofman et
al. (2015), will have more similar gut
microbiomes (share more OTUs).
2. Foxes with lower body conditions will have
less proportional microbiota (lower
taxonomic evenness), whereas higher body
condition will correlate with greater evenness.
The results will likely have valuable implications
that guide the efforts of restoring the island fox
populations by broadening our understanding of
the relationship between U. littoralis gut
microbiota, individuals’ health, and strong
species-wide evolutionary forces.
Figure 5. Extracting
genomic DNA from
U. littoralis scat.
(Adams, 2015)