Biological Sciences, School of


First Advisor

Kenneth Nickerson

Second Advisor

Wayne Riekhof

Date of this Version



Dillard, B. (2020) "Bioinformatic Analysis of the Gut Microbiota Derived from the Oil Fly Helaeomyia petrolei from the La Brea Tar Pits"


A THESIS Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Master of Science, Major: Biological Sciences, Under the Supervision of Professor Kenneth W. Nickerson. Lincoln, Nebraska: July, 2020

Copyright 2020 Brian A. Dillard


In the early 1930s, Thorpe a prominent entomologist, called for more research into Helaeomyia petrolei larva. These larvae live in the Californian La Brea tar pits where they are exposed to large amounts of polyaromatic hydrocarbons. Molecules like anthracene, phenanthrene, and toluene which should be highly toxic to both the oil fly larvae and its enteric bacteria. This extremophilic gut microbiome has yet to be studied using current day next gen sequencing and bioinformatic techniques. In fact, since Thorpe’s work in the 1930s, there have been only two publications characterizing the oil fly larvae. Both in the early 2000s by Kadavy, characterizing the abundance of enteric bacteria in the oil fly, and another describing the surprising antibiotic resistance these gut isolates possess. Almost every isolate described was resistant to over half of the 22 antibiotics tested. We hypothesize that characterizing this larval microbiota on a more intimate level could identify important extremophilic enzymes that would be useful in an industrial capacity, provide insights into the rapid natural development of antibiotic resistance, and identify an organism or collection of organisms that can metabolize the aromatic hydrocarbons that make up tar.

Using a combination of 16S and whole genomes sequencing, we have solidified taxonomic classifications for a majority of the original isolates tested, even establishing a new genus Candidatus Petroalcaligenes. We have also identified a large array of putative drug efflux pumps which might confer both tolerance to solvent stress and antibiotic resistance in our OF2 isolate. When looking for evidence of OF2’s 16S sequence in metagenomic data sets we see that OF2 is only found in specific subsets of sample types and are found at extremely low percentages. Using Pangenomics, where you can compare genetic content amongst a large variety of genomes, we have determined that OF2 has enriched functions in the categories of transport, osmoregulation, metabolism, and antibiotic resistance.

We have also worked to show that the stress imposed by the tar pit rather than the oil gut are to blame for the increased antibiotic resistance and solvent tolerance. Using our isolates OF5, OF6, and OF10 from Providencia, we made a pangenome of 56 genomes comparing every known species of the Providencia. With particular interest on P. rettgeri and P. vermicola, gene enrichment analysis was done to multiple different groupings of genomes. These groups had an emphasis on bacterial host and host environment. While there were some enriched genes across the groups, the results were hard to parse because of the large number of species misclassifications within Providencia rettgeri. Through phylogenetic analysis, we highlighted some of the genomes sampled as candidates for a reclassification as P. vermicola.

Advisor: Kenneth W. Nickerson

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