Document Type

Thesis

Date of Degree Completion

Summer 2021

Degree Name

Master of Science (MS)

Department

Biology

Committee Chair

Jim Johnson

Second Committee Member

Clay Arango

Third Committee Member

Paul James

Abstract

One of several factors contributing to amphibian decline are organisms called water molds (oomycetes), which parasitize and kill amphibian and fish eggs, larvae, and occasionally adults. Amphibian decline is one of the most difficult issues facing ecologists and conservationists to date; half of amphibian species are in severe decline and one third of amphibian species are facing extinction, threatening the stability of ecosystems globally. Multiple oomycete species are known to cause the disease saprolegniasis and contribute to amphibian decline; however, the full range of species involved is not presently known. This study evaluated the Oxford Nanopore minION DNA sequencer (R9.2 chemistry) for use in DNA barcoding multiplexed samples. This study aimed to develop new methods of environmental metabarcoding analysis to assess aquatic oomycete and fungal communities using the internal transcribed spacer (ITS) DNA barcode. A proof-of-concept survey was performed to assess community composition and diversity of aquatic oomycetes and Fungi in 4 freshwater habitats with saprolegniasis present. Samples were taken April 15, 2016 at 4 sites along Interstate-90 near Snoqualmie Pass (Wenatchee National forest, Washington) during Rana cascadae and Anaxyrus boreas breeding season near eggs with saprolegniasis.

The minION produced high but uneven throughput across all four sites. The sequence accuracy and error type prevented chimera checking and phylotype assignment. Sequences were matched using NanoOK to the UNITE database to assign operational taxonomic units (OTUs). FUNGuild was used to assign trophic modes to OTUs to evaluate functional diversity. The recovered oomycete OTUs were low (~1% of all reads) and represented plant pathogenic phyla. Fungal recovery was broad with aquatic taxa represented heavily. Diversity analysis showed little difference between sites, with evidence that uneven throughput affected beta-diversity indices from assessing compositional differences. Trophic mode analysis showed little difference between sites.

Overall, this study found that the minION sequence data presented multiple bioinformatic challenges for metabarcoding using the ITS region. With low recovery of oomycetes, further study is needed to determine the availability of oomycete zoospores for collection via the sampling methods used in this study. Additionally, oomycete specific primers are recommended for increased recovery.

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