Westen Archibald and Dr. Byron Adams, Biology

To better understand the diversity and distribution of entomophilic nematodes in Utah’s Mojave region, I have begun a project that’s purpose is to develop phylogenetic maps of 55 nematode species that I have already extracted from the soils of the Beaver Dam wash ecotone. This project involves both the reconstruction of evolutionary relationships to recover biodiversity, and generating a species distribution map for the Beaver Dam wash ecotone.

In early February I began working on this project. The project required extensive planning because it would be both a field study and lab research. I began by printing off a huge map of Utah’s ecoregions. I analyzed the different ecoregions by focusing on regions of ecological significance. I chose to focus on Utah’s Mojave region. The Mojave region of Utah is an area of particular ecological importance because it is a place of convergence for three separate ecoregions of Utah (Stevens, 2008). The Colorado plateau, Mojave Desert, and Great Basin all meet in this location that brings distinctive diversity to the Beaver Dam wash area. Many desert ecosystems are threatened due to changes in water and land use. Our project is focused on analyzing the abundant diversity of parasitic and necromenic nematodes in Utah’s Mojave region. Due to the abundance and ubiquity of nematodes they act as an excellent bio-indicator of ecosystem health. By focusing our study on parasitic and necomenic nematodes we will we begin to grasp the relationship between nematodes and other invertebrates in the ecosystem and look at how these creatures have evolved for their spectacular and harsh environment.

I decided to divide the project into three main objectives. The first objective involved sampling design. The fields plan required samples from various ecosystems around the wash. Accordingly, I sampled an East-West elevation transect across West Mountain, I started at about 2002-meters above see level and worked down to the beaver Dam wash. I took samples at every 200-meter drop in elevation, with care to sample the representative habitat diversity at each location. Soil samples were taken from 3 inches of topsoil beneath a variety of plants in the habitat as well as a variety of soil types in each habitat. The samples were poured into Whirlpac bags and stored in coolers in order to regulate temperature. A total 153 samples were gathered from all represented habitats. Objective two of my project required extracting entomophilic nematodes from the soil samples. To extract the nematodes from the soils I used a baiting method were I placed 31/2 Galleria mellonella larvae into the bottom of a clear plastic cup. Each cup was filled with ~100cc soil from each sample. Soil samples were observed daily for 7-14 days. Infected or decaying larvae were removed from the soil and placed on filter paper moats in petri dishes (White, 1927). I inspected the samples every day for 7 to 14 days to check for nematode emergence. I observed 55 positive samples for nematode emergence. Samples that were positive were moved to objective three of the project and the Koch’s postulates test.

For the Koch’s postulates test 4 Galleria mellonella larvae placed on filter paper in a 60mm petri dish then each dish was saturated in 1 ml of extracted nematode solution from each of the 55 samples. After the 7 days Galleria mellonella larvae were checked for infection or decay. Many samples were dead however none of the samples were infected. The first trial proved that 1mL was to much water and the worms died from drowning. The second trial took place weeks later and none of the nematodes appeared to infect the Galleria mellonella larvae. None of the nematode species were able to infect and then re-infect a new host thus according to Koch’s postulates none of the 55 samples were parasitic but rather necromenic and entomophillic. This data however was affected by a lot of variable and was very inaccurate so I needed to do molecular sequencing to identify the nematode species genetically.Objective three is the part of the project that I am still working on. It involves nematode identification and phylogenetic analysis. Nematodes from each sample were centrifuged into a pellet. Using the worm lysis buffer techniques (Adams, Peat, Dillman, 2007) the DNA was extracted and concentrated into a mass DNA pellet. Using universal primer sets for the rDNA LSU and ITS regions of the ribosomal RNA tandem array, these markers were PCR amplified and then sequenced by the BYU DNASC. Sequences will then be edited manually in Geneious R 6.1 (Biomatters. Available from http://www.geneious.com/ ). All sequences will be uploaded to Barcode of Life Database for data management purposes and preparation for submission to GenBank. The DNA sequences will be used to generate phylogenetic trees, which will allow for a clearer resolution of the taxonomic and phylogenetic diversity of the entomophilic nematodes of this region. From these data I will develop a distribution map using GPS data gathered from field sampling. This will allow me to explore patterns of distribution ecology within an evolutionary context.

A lot has been accomplished on this project, however Dr. Adams and I still have a long ways to go. I need to finish sequencing and begin really analyzing the data. My data will help us to answer many questions regarding nematode evolution in Utah’s Mojave region.

The reason that I changed projects for my ORCA grant was that Dr. Adams and I decided it was not very ethical of me to kill slugs at the rate that I was. It seemed ethically unnecessary and so we both decided to switch my focus to a new project.