Animal cryptochromes mediate magnetoreception by an unconventional photochemical mechanism.

Understanding the biophysical basis of animal magnetoreception has been one of the greatest challenges in sensory biology. Recently it was discovered that the light-dependent magnetic sense of Drosophila melanogaster is mediated by the ultraviolet (UV)-A/blue light photoreceptor cryptochrome (Cry). Here we show, using a transgenic approach, that the photoreceptive, Drosophila-like type 1 Cry and the transcriptionally repressive, vertebrate-like type 2 Cry of the monarch butterfly (Danaus plexippus) can both function in the magnetoreception system of Drosophila and require UV-A/blue light (wavelength below 420 nm) to do so. The lack of magnetic responses for both Cry types at wavelengths above 420 nm does not fit the widely held view that tryptophan triad-generated radical pairs mediate the ability of Cry to sense a magnetic field. We bolster this assessment by using a mutant form of Drosophila and monarch type 1 Cry and confirm that the tryptophan triad pathway is not crucial in magnetic transduction. Together, these results suggest that animal Crys mediate light-dependent magnetoreception through an unconventional photochemical mechanism. This work emphasizes the utility of Drosophila transgenesis for elucidating the precise mechanisms of Cry-mediated magnetosensitivity in insects and also in vertebrates such as migrating birds.

Cryptochrome mediates light-dependent magnetosensitivity in Drosophila.

Although many animals use the Earth's magnetic field for orientation and navigation, the precise biophysical mechanisms underlying magnetic sensing have been elusive. One theoretical model proposes that geomagnetic fields are perceived by chemical reactions involving specialized photoreceptors. However, the specific photoreceptor involved in such magnetoreception has not been demonstrated conclusively in any animal. Here we show that the ultraviolet-A/blue-light photoreceptor cryptochrome (Cry) is necessary for light-dependent magnetosensitive responses in Drosophila melanogaster. In a binary-choice behavioural assay for magnetosensitivity, wild-type flies show significant naive and trained responses to a magnetic field under full-spectrum light ( approximately 300-700 nm) but do not respond to the field when wavelengths in the Cry-sensitive, ultraviolet-A/blue-light part of the spectrum (<420 nm) are blocked. Notably, Cry-deficient cry(0) and cry(b) flies do not show either naive or trained responses to a magnetic field under full-spectrum light. Moreover, Cry-dependent magnetosensitivity does not require a functioning circadian clock. Our work provides, to our knowledge, the first genetic evidence for a Cry-based magnetosensitive system in any animal.

Cryptochromes define a novel circadian clock mechanism in monarch butterflies that may underlie sun compass navigation.

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 cry(b) 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.

Defining behavioral and molecular differences between summer and migratory monarch butterflies.

BACKGROUND: In the fall, Eastern North American monarch butterflies (Danaus plexippus) undergo a magnificent long-range migration. In contrast to spring and summer butterflies, fall migrants are juvenile hormone deficient, which leads to reproductive arrest and increased longevity. Migrants also use a time-compensated sun compass to help them navigate in the south/southwesterly direction en route for Mexico. Central issues in this area are defining the relationship between juvenile hormone status and oriented flight, critical features that differentiate summer monarchs from fall migrants, and identifying molecular correlates of behavioral state. RESULTS: Here we show that increasing juvenile hormone activity to induce summer-like reproductive development in fall migrants does not alter directional flight behavior or its time-compensated orientation, as monitored in a flight simulator. Reproductive summer butterflies, in contrast, uniformly fail to exhibit directional, oriented flight. To define molecular correlates of behavioral state, we used microarray analysis of 9417 unique cDNA sequences. Gene expression profiles reveal a suite of 40 genes whose differential expression in brain correlates with oriented flight behavior in individual migrants, independent of juvenile hormone activity, thereby molecularly separating fall migrants from summer butterflies. Intriguing genes that are differentially regulated include the clock gene vrille and the locomotion-relevant tyramine beta hydroxylase gene. In addition, several differentially regulated genes (37.5% of total) are not annotated. We also identified 23 juvenile hormone-dependent genes in brain, which separate reproductive from non-reproductive monarchs; genes involved in longevity, fatty acid metabolism, and innate immunity are upregulated in non-reproductive (juvenile-hormone deficient) migrants. CONCLUSION: The results link key behavioral traits with gene expression profiles in brain that differentiate migratory from summer butterflies and thus show that seasonal changes in genomic function help define the migratory state.

Connecting the navigational clock to sun compass input in monarch butterfly brain.

Migratory monarch butterflies (Danaus plexippus) use a time-compensated sun compass to navigate to their overwintering grounds in Mexico. Although polarized light is one of the celestial cues used for orientation, the spectral content (color) of that light has not been fully explored. We cloned the cDNAs of three visual pigment-encoding opsins (ultraviolet [UV], blue, and long wavelength) and found that all three are expressed uniformly in main retina. The photoreceptors of the polarization-specialized dorsal rim area, on the other hand, are monochromatic for the UV opsin. Behavioral studies support the importance of polarized UV light for flight orientation. Next, we used clock protein expression patterns to identify the location of a circadian clock in the dorsolateral protocerebrum of butterfly brain. To provide a link between the clock and the sun compass, we identified a CRYPTOCHROME-staining neural pathway that likely connects the circadian clock to polarized light input entering brain.

Chasing migration genes: a brain expressed sequence tag resource for summer and migratory monarch butterflies (Danaus plexippus).

North American monarch butterflies (Danaus plexippus) undergo a spectacular fall migration. In contrast to summer butterflies, migrants are juvenile hormone (JH) deficient, which leads to reproductive diapause and increased longevity. Migrants also utilize time-compensated sun compass orientation to help them navigate to their overwintering grounds. Here, we describe a brain expressed sequence tag (EST) resource to identify genes involved in migratory behaviors. A brain EST library was constructed from summer and migrating butterflies. Of 9,484 unique sequences, 6068 had positive hits with the non-redundant protein database; the EST database likely represents approximately 52% of the gene-encoding potential of the monarch genome. The brain transcriptome was cataloged using Gene Ontology and compared to Drosophila. Monarch genes were well represented, including those implicated in behavior. Three genes involved in increased JH activity (allatotropin, juvenile hormone acid methyltransfersase, and takeout) were upregulated in summer butterflies, compared to migrants. The locomotion-relevant turtle gene was marginally upregulated in migrants, while the foraging and single-minded genes were not differentially regulated. Many of the genes important for the monarch circadian clock mechanism (involved in sun compass orientation) were in the EST resource, including the newly identified cryptochrome 2. The EST database also revealed a novel Na+/K+ ATPase allele predicted to be more resistant to the toxic effects of milkweed than that reported previously. Potential genetic markers were identified from 3,486 EST contigs and included 1599 double-hit single nucleotide polymorphisms (SNPs) and 98 microsatellite polymorphisms. These data provide a template of the brain transcriptome for the monarch butterfly. Our "snap-shot" analysis of the differential regulation of candidate genes between summer and migratory butterflies suggests that unbiased, comprehensive transcriptional profiling will inform the molecular basis of migration. The identified SNPs and microsatellite polymorphisms can be used as genetic markers to address questions of population and subspecies structure.

Illuminating the circadian clock in monarch butterfly migration.

Migratory monarch butterflies use a time-compensated Sun compass to navigate to their overwintering grounds in Mexico. Here, we report that constant light, which disrupts circadian clock function at both the behavioral and molecular levels in monarchs, also disrupts the time-compensated component of flight navigation. We further show that ultraviolet light is important for flight navigation but is not required for photic entrainment of circadian rhythms. Tracing these distinct light-input pathways into the brain should aid our understanding of the clock-compass mechanisms necessary for successful migration.