Cryptochrome 2 mediates directional magnetoreception in cockroaches

Abstract

The ability to perceive geomagnetic fields (GMFs) represents a fascinating biological phenomenon. Studies on transgenic flies have provided evidence that photosensitive Cryptochromes (Cry) are involved in the response to magnetic fields (MFs). However, none of the studies tackled the problem of whether the Cry-dependent magnetosensitivity is coupled to the sole MF presence or to the direction of MF vector. In this study, we used gene silencing and a directional MF to show that mammalian-like Cry2 is necessary for a genuine directional response to periodic rotations of the GMF vector in two insect species. Longer wavelengths of light required higher photon fluxes for a detectable behavioral response, and a sharp detection border was present in the cyan/green spectral region. Both observations are consistent with involvement of the FADox, FAD(•-) and FADH(-) redox forms of flavin. The response was lost upon covering the eyes, demonstrating that the signal is perceived in the eye region. Immunohistochemical staining detected Cry2 in the hemispherical layer of laminal glia cells underneath the retina. Together, these findings identified the eye-localized Cry2 as an indispensable component and a likely photoreceptor of the directional GMF response. Our study is thus a clear step forward in deciphering the in vivo effects of GMF and supports the interaction of underlying mechanism with the visual system.

Keywords: circadian genes; cryptochrome; light spectrum; locomotor activity; magnetoreception.

Fig. 1.

MIR. (A) Schematic illustration of the magnetoreception assay: When the geomagnetic horizontal vector is rotated back and forth 60° periodically, the cockroaches change their resting positions more frequently compared with during steady MF periods. Magnetoreception assay setup: Cockroaches were placed individually into Petri dishes with opaque walls (small circles) accommodated in arena (large circle). On the next day, MF was changing its direction every 5 min during 135-min intervals. The four black lines depict the position of the Merritt coil frames. (B) Body turn was scored if body rotation exceeded 15°. (C) MIR essay is selective to magnetic direction: The activity was scored as a number of body turns per 135-min interval during control (steady field, sf) and treatment (rotating field, rf) periods. Red line depicts average paired levels of all individual activities (±SEM) between control and treatment periods. In the Basic test setup, significant elevation of activity (Wilcoxon Match Pair Test) was found. Nonspecific effects of electric feeding of coils were eliminated using a double-wrapped design (DoWr) that allowed us to feed the coils without producing any external MF. The red lines indicate the mean values of all animals between control and treatment; intra- and intersample variations are irrespective of pair test significance. n, number of animals.

Fig. 2.

Phenotypes of P. americana. Magnetoreception scored as body turns under sf and rf (A–E, Left); red line depicts the average change of body turns (±SEM). Daily activity profiles at a 12 h light:12 h dark cycle are shown as a black line (±SD, thinner lines); horizontal blue bar under daily activity profiles indicates the time during which magnetoreception was assayed (B–E, Middle). Circadian activity in constant conditions is shown as double-plotted actograms (A–E, Right). (A) Constant light (LL) abolished circadian rhythmicity, leaving magnetic sensitivity intact, whereas DD abolished magnetic sensitivity with unaffected circadian locomotor activity. (B) The control RNAi and (C) buffer injected animals displayed normal magnetoreception, as well as circadian rhythmicity. (D) cry2 RNAi-treated animals lost both magnetoreception and circadian rhythmicity. (E) timeless RNAi cockroaches showed unaffected magnetoreception, but their circadian behavior was disrupted.

Fig. 3.

Phenotypes of B. germanica. Magnetoreception scored as body turns under sf and rf; red line depicts the average change of body turns (±SEM). Daily activity profiles (Middle) at LD 12:12 are shown as a black line (±SD, thinner lines); horizontal blue bar indicates the time during which magnetoreception was assayed. Circadian activity (Right) in DD is shown as double-plotted actograms. (A) The control RNAi- and (B) buffer-injected animals displayed normal magnetoreception as well as circadian rhythmicity. (C) cry2 RNAi-treated animals lost magnetoreception and have reduced rhythmicity in DD. (D) cry1 RNAi cockroaches showed unaffected magnetoreception, but their circadian behavior was disrupted. (E) cry1, cry2 double RNAi-treated animals lost magnetoreception and had reduced rhythmicity in DD.

Fig. 4.

Light sensitivity of Blattella magnetoreception is wavelength-dependent and restricted to the region from UV to cyan/green light. Green dots indicate functional MIR; red dots no MIR reaction. Blue line approximates low threshold of illumination necessary for MIR. y axis, light intensity; x axis, wavelength of light used in experiment. For details, see Discussion, Spectral Effects and Magnetic Signaling.

Fig. 5.

P. americana eye participates in magnetoreception and expresses Cry2. (A) Cockroaches with clear-painted eyes responded to MF rotation, whereas individuals with black-painted eyes did not. (B) Immunofluorescence and (C) Nomarski contrast of P. americana eye. Cry2 immunoreactivity (in red) is localized underneath the retina (RE) between the two basement membranes (BM1, BM2), according to Ribi (35). BM1, first basement membrane; BM2, second basement membrane; LA, lamina; RE, retina; blue, cell nuclei (DAPI stained).