Comparative analysis of gut microbiome among populations of Channel Island Fox, Urocyon littoralis

Materials and methodsWe 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

IntroductionThe 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 otheranimals.

Lauren Stoneburner, Nicole Adams, Suzanne EdmandsLife Sciences

Literature cited

Results Future Research

AcknowledgementsI 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.

____________________________________________________________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.

Hypotheses1. 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)