Endogenous clocks help organisms to adapt to the 24-h cycle of the earth. One of the main characteristics of endogenous clocks is that they continue to oscillate even in the absence of external time cues. Under such conditions, they “free-run” with their endogenous inherited periods that are slightly different from 24 h. Therefore, they are also called circadian clocks. Under natural conditions circadian clocks are synchronized to the 24-h cycle on Earth by different Zeitgebers. The most important Zeitgeber is the light–dark cycle, but temperature, humidity, and the social environment can also serve as Zeitgebers. Additionally, magnetic fields have been discussed as agents that synchronize circadian clocks since 1960, when Brown et al. [1] found that the circadian rhythms of fiddler crabs and other organisms are influenced by small changes in the intensity of Earth's magnetic field. In particular, the electromagnetic field with a frequency of 10 Hz is known to show a prominent 24-h oscillation [2]; thus, the field could serve as geophysical synchronizer of the circadian clock. Although electromagnetic fields are not consciously perceived by humans, Wever [3] demonstrated that the circadian activity rhythm of humans can be entrained by an artificial 10 Hz electromagnetic field, mimicking the natural oscillations on Earth. The activity of mice, house flies, and fruit flies could also be synchronized or phase-shifted by 10 Hz electric fields applied for 12 h daily [4,5]. Cyclically changing intensities of a static magnetic field can also work as weak synchronizers of the circadian clock, as shown by Bliss and Heppner [6] for house sparrows. The latter study was motivated by the idea that birds perform magnetic compass orientation and that this ability depends on a functional clock. Thus, the authors speculated about a link between the magnetosense and the circadian clock.

Despite the compelling effects of (electro)-magnetic fields on the circadian clock, the mechanisms by which the magnetic field is perceived and how it manipulates circadian clocks are unknown. Two main models of magnetic field sensing are currently under discussion: (1) the magnetite model proposing a primary process involving tiny crystals of permanently magnetic material [7], and (2) the radical-pair model suggesting a “chemical compass” based on singlet–triplet transitions in photopigments [8–10].

The radical-pair mechanism, in which the magnetosensor is a photopigment, was first suggested by Schulten and coworkers [8–10]. The first step of the reaction is the absorption of a photon of light energy by the pigment molecule, leading to the transient formation of radical pairs (unpaired electrons) with opposite electron spin states (singlet electrons). The model predicts that these electrons are close enough together that the unpaired electron spin states can undergo transition, at some frequency, from antiparallel to parallel spin states (singlet–triplet intersystem crossing). Such singlet–triplet intersystem crossing is a natural feature of many photochemical reactions that, in fact, may have nothing to do with magnetosensitivity and is the result of the system reaching the most energetically favorable state subsequent to absorption of photon energy. The critical feature of a magnetosensitive radical-pair reaction is that the radicals have a sufficiently long lifetime and are oriented in such a way that singlet–triplet transitions are modified by their alignment in the presence of an applied magnetic field. In such a case, product yield or rate of formation would be different depending on the relative amount of singlet or triplet formation, and these changes in output would provide a means for the organisms to detect the effects of even very weak applied fields. This presumed radical-pair mechanism has indeed been observed experimentally in reactions involving organic compounds [11] and in biological systems such as photosynthetic reaction centers [12,13].

Since the radical-pair mechanism of magnetosensitivity depends on the activation of the relevant photopigment by light, it is wavelength dependent. Magnetosensitivity is maximal at the absorbance maximum of the photopigment, and it depends on the strength of the magnetic field. Ritz et al. [10] calculated the yield of the triplet products in relation to the strength of the magnetic field and the angle of the field with respect to the alignment of the radical pair in space. They found that there is a small window of the magnetic field strength at which the radical-pair mechanism furnishes magnetosensory capacities. In agreement with these calculations, Wiltschko and Wiltschko found that the magnet compass orientation of birds works only at a certain range of magnetic intensities [14].

That magnetoperception is wavelength dependent has been shown not only in birds but also in newts and fruit flies. As photopigments, cryptochromes (CRYs) have been proposed [9,15,16]. CRYs are UV- and blue-absorbing photoreceptors, found in plants and animals, that contain flavin adenine dinucleotide (FAD) as chromophore [17] and have been shown to form radical pairs subsequent to light activation [18]. CRYs are expressed in the eyes of mammals [19] and migratory birds [20,21], where crucial magnetoreceptors have been localized [22,23]. However, CRYs are also expressed in the circadian clock neurons of mice and flies [24–27]. This raises the possibility that magnetic fields can interact directly with the circadian clock.

The fruit fly Drosophila melanogaster is best suited to reveal a putative role of CRYs in the magnetosensitivity of the circadian clock, because there is only one CRY, and because mutants are available that either knock out the binding domain of the FAD (cry^b mutants, [28]) or the entire CRY molecule (cry⁰ mutants, [29]; cry^OUT mutants, [25]; cry^Δ mutants, [30]). The molecular mechanisms of the circadian clock are largely unraveled. The main players of the clock are the clock genes period, timeless, Clock, and cycle, which participate in complex molecular feedback loops to generate the circadian oscillation [31,32]. CRY is known to play an important role in the photoreception pathway to Drosophila's endogenous clock [27]. Light promotes the binding of CRY to TIMELESS (TIM), leading to the degradation of TIM [33,34]. Under light–dark cycles the action of CRY leads to a “reset” of the clock every morning. However, under constant light (LL), TIM-mediated degradation by CRY occurs continuously. Therefore, the amount of TIM is reduced and the clock slows down (i.e., the free-running period of the locomotor rhythm is lengthened). When the constant illumination exceeds a critical intensity (> 10 lx), TIM disappears completely and the clock stops running (the flies become arrhythmic [35]).

On the basis of the background described above we speculated that a magnetic field might influence the circadian clock via CRY under blue light but not red light conditions. To investigate this possibility, we measured the free-running periods of the flies' locomotor rhythms before and during exposure to a magnetic field under constant weak blue light or red light illumination. The blue and red lights were adjusted to such low intensities that in the absence of the magnetic field the flies showed only a slightly longer period than when they were under constant dark (DD) conditions. We found that 40% of the flies further lengthened the free-running periods during the exposure to a magnetic field of 300 μT under blue light illumination. This effect was completely absent under red light conditions. We also demonstrate that the response of fruit flies to the magnetic field is not only wavelength dependent, but also dependent on the strength of the magnetic field, as is implied by the radical-pair model. Furthermore, the magnetic field could not induce any period lengthening in cry^b and cry^OUT mutant flies; but period lengthening was enhanced after overexpressing CRY in the clock neurons. Our results indicate that the magnetic field influences the circadian clock via CRY.

We show here that Drosophila's clock is magnetosensitive, and that this sensitivity depends on CRY. When the first studies were performed that showed magnetosensitivity of the circadian clock, CRY had not been detected yet and the mechanism of how the clock is influenced by magnetic fields was completely elusive. Now, we know that CRY is expressed in the clock neurons of Drosophila [25,30], that CRY absorbs UV and blue-green light [39], and that CRY consecutively binds to TIM and induces its degradation [34]. Under constant conditions this process leads to period lengthening or arrhythmicity [35]. The magnetic field effect that we observed in our study led to further lengthening of the period, in the same way as an increased intensity of blue light would lead to enhanced CRY function. The effect of a magnetic field was particularly pronounced in CRY-overexpressing flies, in which small differences in CRY function should be significantly amplified. Additionally, we could show that this reaction depends on the strength of the magnetic field and on the wavelength of the light. Red light that is outside of the absorption and action spectra of CRY [39–42] did not provoke significant responses. Consistent with the involvement of cryptochrome, cry^b and cry^OUT mutants respond neither to blue-green light nor to the magnetic field.

Our results strongly support the radical-pair model suggesting light-activated flavin-based photoreceptors as sensors for magnetic fields. In several animals magnetoreception has been shown to depend preferentially on blue light [43–47]. Ritz et al. [10] stated first that CRYs are suitable molecules for such photoreceptor-based magnetoreception. CRYs are blue-green photoreceptors in plants and flies [17,28,48,49] and have been shown to form radical pairs upon photoexcitation [18] as is true for the closely related DNA photolyases [50]. Other classes of photoreceptors, such as phototropins [51] and chlorophylls [52] found in plants, also undergo radical-pair reactions. However, rhodopsins, which are the major photosensory pigments in flies and other animals, are not able to form radical pairs because photoexcitation leads to cis–trans isomerization of retinal rather than an electron transfer [53]. Thus, CRYs are the only currently known class of molecules found in animals that are likely to fulfill the physical and chemical characteristics that are required for functioning as the primary magnetic sensor.

The first evidence that CRYs may work as magnetosensors comes from the plant Arabidopsis thaliana, where it was shown that CRYs mediate magnetic field-dependent hypocotyl growth inhibition under blue light [37]. In these experiments, the effect of the magnetic field was to increase the plant's response to perceived blue light by enhancing cryptochrome activity, similarly to the presently observed magnetic field effect in flies. The second evidence comes from the fly D. melanogaster (that paper was published during the preparation of the present manuscript [54]). Gegear et al. [54] showed clearly that fruit flies need functional CRY to perceive a magnetic field that was paired with a sugar reward during a classical learning experiment. Although this study did not analyze the effect of magnetic field strength on CRY activity, these data are highly consistent with our present observations that the magnetic field intensifies the effects of blue light on the clock. The radical-pair mechanism predicts that the rate or yield of product formation from such a reaction will be altered by an applied magnetic field. Therefore, the pairing of blue light and the magnetic field may lead to the perception of more intense (brighter) light as compared to blue light of the same intensity presented alone. Consequently, hypocotyl growth was inhibited to a higher degree in Arabidopsis [37], and flies learned to correlate a more intense light signal under magnetic field presence with the sucrose award [54]. In our experiments the circadian clock lengthened its period or became arrhythmic as if it experienced blue light of a higher intensity. All three phenomena clearly do not depend on directional effects of the magnetic field.

That flies can also perform magnetic compass orientation was shown first by Wehner and Labhart [55] and further elaborated by Phillips and Sayeed [43] and Dommer et al. [47]. The latter two studies showed that Drosophila adult males and larvae are capable of learning the alignment of important environmental gradients (i.e., light, food, and humidity) with respect to the geomagnetic field [43,47]. These responses were maximal under UV light, but the involvement of CRY was not tested.

The ability of animals to detect geomagnetic fields has substantial biological importance, because it is used by invertebrate and vertebrate species for orientation and navigation [56]. Almost certainly CRY also plays a crucial role in this process (see [10,16,20,21,57] for discussion). Fruit flies may be well suited to further unraveling the involvement of CRY in the mechanisms of magnetic compass orientation. Since CRY is also present outside the clock neurons in tissues such as the compound eye and the ellipsoid body [25], in which it might show a directional alignment, it will be most rewarding to test the roles of these structures in magnetic compass orientation.

In conclusion, we provide the first evidence of a mechanistic link between CRY and magnetic sensitivity in the circadian clock of Drosophila. Our data provide powerful support for the radical-pair hypothesis, because they show the predicted relationship between strength of the applied field and the reaction (here period of the clock). Furthermore, the fact that the magnetic field leads to enhanced photosensitivity of CRY in both plants and flies argues for a similar mechanism of magnetoperception in all organisms that contain CRYs, including humans. Finally, given the recent observations that sun compass orientation in the monarch butterfly is dependent on the circadian clock and on CRY [57], this effect of magnetic field on the circadian clock may in fact play a role in fly orientation.