Barrett, The Honors College Thesis/Creative Project Collection

Oxymonas is a genus of Oxymonad protist found in the hindgut of drywood termites (family Kalotermitidae). Many genera of drywood termites are invasive pests globally. The hindgut microbiome of Cryptotermes brevis, the West Indian drywood termite, has not been described in detail, and only one published sequence exists of Oxymonas from C. brevis. This study aims to analyze Oxymonas sequences in C. brevis from whole gut genetic material, as well as to dissect its place in phylogenetic trees of Oxymonas and how it fits into specific and evolutionary patterns. To amplify the 18S rRNA gene Oxymonas from C. brevis, the MasterPure DNA extraction kit was used, followed by PCR amplification, followed by agarose gel electrophoresis, followed by purification of the resulting gel bands, followed by ligation/transformation on to an LB agar plate, followed by cloning the resulting bacterial colonies, and topped off by colony screening. The colony screening PCR products were then sequenced in the Genomics Core, assembled in Geneious, aligned and trimmed into a phylogenetic tree, along with several long-read amplicon sequences from Oxymonas in other drywood termites. All whole gut sequences and one amplicon from C. brevis formed a single clade, sharing an ancestor with a sister clade of Oxymonas sp. from C. cavifrons and Procryptotermes leewardensis, but the other long-read fell into its own clade in a different spot on the tree. It can be conjectured that the latter sequence was contaminated and that the C. brevis clones are a monophyletic group, a notion further corroborated by a distantly related clade featuring sequences from Cryptotermes dudleyi, which in turn has a sister taxon of Oxymonas clones from C. cavifrons and P. leewardensis, pointing toward a different kind of co-diversification of the hosts and symbionts rather than cospeciation.

The symbiosis between termites and their parabasalid hindgut protists centers around the wood digestion that is needed for both species to acquire the nutrients from wood. One of the important carbohydrate-active proteins required for the wood breakdown are glycoside hydrolase (GH) families. Previous studies have looked at the phylogeny of some of these protein families from a termite whole gut transcriptome or in a different context than lignocellulose digestion. In this study, we attempt to understand the function and evolution of these GH families in the context of protist evolution by using protist single cell transcriptomes. 14 families of interest were chosen to create phylogenetic trees: GH2, GH3, GH5, GH7, GH8, GH9, GH10, GH11, GH26, GH43, GH45, GH55, GH67, GH95 for their interesting expressions across different protists such as being present in all protists or being present in only termite-associated protists. The dbCAN2 (automated Carbohydrate-active enzyme ANnotation) program was used to find GH families in each of the protist single cell transcriptomes and additional characterized sequences registered on the National Center for Biotechnology Information to create phylogenetic trees for each of the GH families of interest. Results show that many of the GH families expressed in protists were acquired through horizontal gene transfer from fungi and bacteria. Additionally, comparison to the parabasalid phylogeny indicates most GH families evolved independently from the protists. Based on the pattern of expression of these GH families throughout different protist orders, conclusions can be made about whether the specific family was vertically or horizontally acquired in the termite symbionts.

Nonsense mediated decay is a pathway that selectively degrades mRNAs that contain premature termination codons (PTCs). The purpose of this study is to research the evolution of NMD in Parabasalia and infer whether they have a normal, functioning nonsense-mediated decay pathway. Parabasalia are single-celled, flagellated protists that have undergone evolutionary transitions as they become obligate symbionts of termites. The key proteins involved in nonsense-mediated decay, ATM, ATR, UPF1, SMG1, UPF2, UPF3A, UPF3B, were researched and used in order to build phylogenetic trees to analyze what other species of eukaryotes have these same genes and where they branch relative to the nonsense mediated decay proteins present in Parabasalia. The main question being asked in this research is if Parabasalia have enough of the main nonsense mediated decay proteins to have a functional nonsense-mediated decay process and if not, which proteins have been lost over evolutionary history. To carry out this research, phylogenic trees were built using transcriptomes from many different types of eukaryotes that contained the main proteins involved in the nonsense-mediated decay pathway. These transcriptomes were taken from the National Center for Biotechnology Information (NCBI) database using the BLAST algorithm, trimmed using TrimAl, aligned by utilizing AliView which utilizes Muscle. Sequoia was then used to remove redundant species from the trees, and IQ-TREE was used to form the phylogenic trees. This process was repeated four times to create well-rounded trees with various eukaryotic species present. The results of this research found that ATM, ATR, UPF1, SMG1, and UPF2 are present in Parabasalia as well as across many eukaryotic groups, whereas UPF3A and UPF3B were not found in many of the eukaryotes researched. This points to Parabasalia having a normal and functioning nonsense-mediated decay pathway as they have the majority of the essential proteins needed for a functional pathway.