Cryptochromes Define a Novel Circadian Clock Mechanism in Monarch Butterflies That May Underlie Sun Compass Navigation

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Abstract
The circadian clock plays a vital role in monarch butterfly (Danaus plexippus) migration by providing the timing component of time-compensated sun compass orientation, a process that is important for successful navigation. We therefore evaluated the monarch clockwork by focusing on the functions of a Drosophila-like cryptochrome (cry), designated cry1, and a vertebrate-like cry, designated cry2, that are both expressed in the butterfly and by placing these genes in the context of other relevant clock genes in vivo. We found that similar temporal patterns of clock gene expression and protein levels occur in the heads, as occur in DpN1 cells, of a monarch cell line that contains a light-driven clock. CRY1 mediates TIMELESS degradation by light in DpN1 cells, and a light-induced TIMELESS decrease occurs in putative clock cells in the pars lateralis (PL) in the brain. Moreover, monarch cry1 transgenes partially rescue both biochemical and behavioral light-input defects in cryb mutant Drosophila. CRY2 is the major transcriptional repressor of CLOCK:CYCLE-mediated transcription in DpN1 cells, and endogenous CRY2 potently inhibits transcription without involvement of PERIOD. CRY2 is co-localized with clock proteins in the PL, and there it translocates to the nucleus at the appropriate time for transcriptional repression. We also discovered CRY2-positive neural projections that oscillate in the central complex. The results define a novel, CRY-centric clock mechanism in the monarch in which CRY1 likely functions as a blue-light photoreceptor for entrainment, whereas CRY2 functions within the clockwork as the transcriptional repressor of a negative transcriptional feedback loop. Our data further suggest that CRY2 may have a dual role in the monarch butterfly's brain—as a core clock element and as an output that regulates circadian activity in the central complex, the likely site of the sun compass. During their spectacular fall migration, eastern North American monarch butterflies (Danaus plexippus) use a time-compensated sun compass to help them navigate to their overwintering sites in central Mexico. The circadian clock plays a critical role in monarch butterfly migration by providing the timing component to time-compensated sun compass orientation. Here we characterize a novel molecular clock mechanism in monarchs by focusing on the functions of two CRYPTOCHROME (CRY) proteins. In the monarch clock, CRY1, a Drosophila-like protein, functions as a blue-light photoreceptor for photic entrainment, whereas CRY2, a vertebrate-like protein, functions within the clockwork as the major transcriptional repressor of the self-sustaining feedback loop. An oscillating CRY2-positive neural pathway was also discovered in the monarch brain that may communicate circadian information directly from the circadian clock to the central complex, which is the likely site of the sun compass. The monarch clock may be the prototype of a clock mechanism shared by other invertebrates that express both CRY proteins, and its elucidation will help crack the code of sun compass orientation.