Testing Evolutionary Conservation of Sex Determination in Lepidoptera Using CRISPR/Cas9 Gene Editing
Date of Award
2023
Document Type
Honors Thesis (Open Access)
Department
Colby College. Biology Dept.
Advisor(s)
Dave R. Angelini
Second Advisor
Christina Cota
Third Advisor
Josh Martin
Abstract
The mechanism of sex determination in Lepidoptera is largely unexplored, and limited to knowledge of only a few genes. Adult Lepidoptera have obvious sexually dimorphic qualities, such as their genitalia. However, significant sex determination genes are not imperative for the adult developmental stage. Knockouts of some sex determination genes (doublesex isoforms) are shown to be lethal in the embryonic and larval stages of development in several animals. However, it is unclear whether this is due to their respective impact on sex determination and sexual dimorphism, or the existence of another function, such as pleiotropy, within a particular gene. Moreover, Little is known about the specific impacts of sex determination gene knockouts on Lepidopteran morphology and behavior. This honors thesis explores the effects of knockouts of key sex determination gene masculinizer (masc) in Lepidopteran species Vanessa cardui. masc is thought to be an important gene in some Lepidopteran species, specifically for the persistence of masculinization during embryonic development, and for dosage compensation of chromosomal activation on the Z chromosome. masc expression was knocked out in Vanessa cardui embryos using CRISPR/Cas9 gene editing which targeted the masc gene at two different positions within the V. cardui protein-coding sequence. Surviving adult butterflies were observed in order to determine phenotypic differences in sexually dimorphic anatomy and behavior. Specifically, vestigial forelegs were collected from each injected individual and analyzed for mosaic phenotypes. It was found through statistical and morphometric analysis that knockouts of the masc gene have the potential to decrease embryonic survival probability and adult fitness in Vanessa cardui, and produce mosaic phenotypes in surviving male animals. These findings support the hypothesis that masc is conserved across Lepidoptera, including Vanessa cardui.
Keywords
Lepidoptera, Sex Determination, Microinjection, masculinizer, CRISPR/Cas9
Recommended Citation
Schroeder, Sophia, "Testing Evolutionary Conservation of Sex Determination in Lepidoptera Using CRISPR/Cas9 Gene Editing" (2023). Honors Theses. Paper 1414.https://digitalcommons.colby.edu/honorstheses/1414