Abstract: An understanding of eye development is necessary to comprehend proper functioning of the eye, capacity for regeneration, and the developmental basis of disease. Many model systems, both vertebrate and invertebrate, have historically provided much important data about the developmental and genetic basis of eye formation. However, hurdles exist studying eye development in model systems such as inherent biases in mutant screens and the minimal amount of naturally occurring variation in eye size. One novel approach allowing for a greater understanding of the developmental and genetic bases of eye formation is to examine naturally occurring eye size variation in non-model organisms, such as that occurs in cave animals. We initially chose the crustacean, Asellus aquaticus, as a model for studying eye variation because of the extreme difference in eye size between cave and surface dwelling populations. Our work on the species has shown multiple independent mechanisms of eye reduction/ loss within a single cave population and a large genetic component influencing eye size. Our current grant utilized comparative transcriptome sequencing of cave and surface embryonic samples as an unbiased method to identify genes and pathways responsible for eye degeneration in two different cave populations. Additionally, we sequenced transcriptomes of hybrid embryos to identify genes with allele-specific expression containing putative cis-regulatory changes. One major next step is to obtain positional information for all three potentially different genetic mechanisms responsible for eye loss present in two different cave populations and in a surface intercross. Additionally, the genes identified from the previous grant showing allele-specific expression support the idea that genetic variants responsible for eye loss could be responsible for additional phenotypes. Therefore, an understanding of the genetic basis of eye loss could be furthered with an understanding of the other phenotypes that might be linked. To address these two major questions we will first utilize restriction site-associated DNA sequencing (RADseq) to map regions responsible for eye loss in the two cave populations and the surface intercross. Second, we will identify phenotypes that are linked to eye loss in Asellus aquaticus through the same genetic variants or different but linked genetic variants. Finally, we will investigate two genes from our previous allele-specific expression work, efr3 and mut-7, examining them functionally in flies for eye phenotype and additional behaviors. Our work provides a novel perspective on the developmental and genetic basis of eye size differences and furthers the development of A. aquaticus as an important model for eye degeneration and disease.