Monitoring ecdysteroid activities using genetically encoded reporter gene in Daphnia magna.

Ecdysteroid is an important hormone that regulates growth, reproduction, and embryogenesis in arthropods. However, little is known about its role and action mechanism in crustaceans, despite their pivotal role in aquatic ecosystem. Daphnia magna, a freshwater crustacean, is used as a classic model organism in ecology and ecotoxicology. Its ecdysteroid activity has been partially characterized previously. However, the spatio-temporal behavior of ecdysteroid, especially during early embryogenesis, when it is thought to have pivotal roles, is still unclear. Thus, we proposed a genetic modification approach by integrating a reporter gene exhibiting ecdysteroid activity in vivo. We used the clustered regularly interspaced palindromic repeats (CRISPR) genome editing technique, followed by non-homologous end-joining (NHEJ) pathway as the transgenesis method to generate the ecdysteroid reporter transgenic Daphnia. One transgenic Daphnia containing one copy of the ecdysone response element (EcRE)-controlled reporter gene mCherry was successfully obtained and was designated EcRE-mCh. The expression of mCherry was observed during early embryogenesis starting from 12 h after ovulation (hao). The time-lapse imaging during 12-24 hao showed the growing expression of mCherry signal originating from the posterior section of embryo and then migrating toward the anterior section. From 18 hao, the signal was detected around the developing thoracic appendages and localized between the first to third thoracic segments. The establishment of this EcRE-mCh line and its ability to exhibit ecdysteroid activity spatio-temporally might serve as convenient tool to elucidate the roles of ecdysteroid during the early stage of animal development. Moreover, the expression of mCherry in response to the presence of ecdysteroid in water suggests that EcRE-mCh could be used for monitoring ecdysteroid activities in environmental water.