Graduation Year
2022
Document Type
Master's Thesis
Degree
Master of Science
Program
Biological Science
Program Director
Meredith Protas, PhD
First Reader
Meredith Protas, PhD
Second Reader
Obed Hernandez-Gomez, PhD
Abstract
The use of model organisms in genetic research is a well-established and effective method to further understand genetic disease and phenotypic variation in humans. One example of a group of model organisms that can help to understand human disease is cave animals. Cave animals are organisms that have evolved in a subterranean environment and have predictable variation in their phenotypes when compared to their corresponding surface populations. The phenotypic variations can include, but are not limited to: elongated limbs, pigment loss, and loss or reduction of eyes. One particular cave organism with the potential to be an excellent model for human disease is Asellus aquaticus. A. aquaticus is an aquatic crustacean found in Europe, that has cave and surface populations which can interbreed. There are a variety of phenotypic differences between the cave and surface populations, including elongated second antennae, reduction or loss of eyes, and reduced or absent pigment. For this study, our goal was to determine the genetic and transcriptomic basis of eye loss in A. aquaticus. We compared transcriptomes generated from RNA sequencing of Slovenian cave and surface populations and Romanian cave and surface populations to look for differences in gene expression between cave and surface samples. Gene ontology enrichment analysis showed an enrichment of differentially expressed genes between cave and surface samples belonging to the TCA cycle, amino sugar and nucleotide sugar metabolism, and the biosynthesis of amino acids pathways, among others. We also analyzed whether a subset of genes known as Light Interacting Genes showed differential expression between cave and surface individuals. Genes within the categories of crystallins, diurnal clock, heme synthesis, melanin synthesis, ommochrome synthesis, photoreceptor specification, phototransduction, pterin synthesis, 4 retinal determination network, and retinoid pathway showed differential expression between the cave and surface populations. Coding sequences of the light interacting genes that were differentially expressed between cave and surface samples were compared and amino acid substitutions were found though no drastic changes to the size of the coding region were seen. Candidate genes of particular interest were those that were differentially expressed between cave and surface samples and involved in phototransduction including retinal rod rhodopsin- sensitive cGMP 3’,5’-cyclic, rhodopsin, and arrestin 2. Next, we tracked embryonic development through mounting and morphometric measuring of hatchlings from both the Romanian cave and surface populations. We investigated hatchlings from wild caught individuals and found that the cave hatchlings were significantly larger and had longer relative antennae II length (longest antennae) compared to their surface counterparts. In addition, we found that the Romanian cave hatchlings were unpigmented and did not appear to have ommatidia. Finally, we worked on establishing the use of CRISPR technology for studying A. aquaticus, with the goal of functionally investigating candidate genes responsible for the eye loss phenotype. Though we have not successfully established a pipeline to silence genes using CRISPR in A. aquaticus, we have been able to harvest, inject, and electroporate embryos with some embryo survival. We have also designed targets for two genes, scarlet and dll, that when mutated have predictable phenotypes, and developed PCR assays that could be used to detect CRISPR- induced excisions. Establishing a functional technique, such as CRISPR, will potentially allow us to identify the genes or genetic pathways responsible for eye variation between the cave and the surface form.