| Summary: | Vargula tsujii is a bioluminescent cypridinid ostracod crustacean that is found off the Pacific Coast of North America. The ostracod secretes a bright blue luminescence that it uses as an anti???predator mechanism. The biochemical basis of the light reaction is oxidation of a cypridinid luciferin substrate by a cypridinid luciferase enzyme. Bioluminescence in ostracods might be affected by changing ocean conditions such as ocean acidification and increased temperature. To study the effects of the environment on bioluminescence, aquaculture and molecular methods needed to be developed. The goals of this project were to develop ostracod culture methods for laboratory aquaria in the absence of a flowing seawater source, and quantitative polymerase chain reaction (qPCR) assays for luciferase expression. This work designed and tested two effective primer sets for studying V. tsujii luciferase (VtL) gene expression by qPCR. Additionally, four candidate reference genes, actin, succinate dehydrogenase (SDH), clathrin, and glyceraldehyde???3???phosphate dehydrogenase (GAPDH), were tested to determine the most stable ones for normalization of RT???qPCR data using V. tsujii as model organism in future studies. From PCR and qPCR results, SDH was the most stable reference gene. Analysis of the relative gene expression of VtL indicated possible differences between developmental stages; however, there was no statistically significant difference between stages. Overall, this study made advances in the development of methods for luciferase gene expression studies and creates possibilities for future research in bioluminescence.
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