Cryptochrome-1 is important for photosensitivity and post-translation resetting of the circadian clock in Aedes aegypti

鄭鴻源Hong-Guan Tee1,2, Jian-Chiuan Li1, Kun-Lin Liu1, YuMin M Loh3, Matthew P Su3,4, Chih-Fei Kao2，Chun-Hong Chen1

1 National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Taiwan

2 Department of Biological Science and Technology, National Yang Ming Chiao Tung University, Taiwan

3 Graduate School of Science, Nagoya University, Japan

4 Institute for Advanced Research, Nagoya University, Japan 

Light acts as a major zeitgeber for the circadian clock of Aedes aegypti (Ae. aegypti). The timing and duration of important Ae. aegypti behaviors, such as host-seeking, are thus strongly influenced by the environmental photoperiod. Members of the cryptochrome (CRY) protein family, which act as blue-lighted photoreceptors with high photosensitivity, are important for light-dependent regulation of the clock. Unusually amongst insects however, two CRY genes (CRY1 and CRY2) have been identified in Ae. aegypti, and their relative roles and importance remain unclear. Here, we investigated potential differences between Ae. aegypti CRY1 and CRY2 using molecular and behavioral assays. First, we generated CRY1 (AAEL004146) and CRY2 (AAEL011967) knock-out lines via CRISPR/Cas9. Next, we tested for changes in expression of core clock genes at both RNA and protein levels using newly developed antibodies. Finally we used locomotor monitoring assay to assay for changes in activity in different light:dark (LD) regimes. Interestingly, we found that only the CRY1 KO line showed significant differences in the cycling of circadian clock-relevant protein post-translation levels as compared to controls. CRY1 KO mutants also demonstrated distinct locomotor profiles and activity indexes as compared to controls. We are now generating CRY1 and CRY2 antibodies to further investigate the mechanisms by which CRY1 influences the circadian clock.