Avian Magnetorecption: Behavioral Tests in view of the Cryptochrome Cycle Wolfgang Wiltschko, Roswitha Wiltschko J.W. Goethe-Universität, Frankfurt a.M., Germany

Bird Migration: Directional tendencies during the migration season European Robin Erithacus rubecula Distribution

Funnel paper on a light desk: Analysis of the Avian Magnetic Compass in Behavioral Experiments with captive Birds: Funnel cage lined with coated paper Funnel paper on a light desk: Funnel cage by Emlen & Emlen (1966)

magnetic North turned 120° to ESE Demonstrating magnetic compass orientation in robins with the help of migratory orientation: local geomagnetic field Control magnetic North turned 120° to ESE N = mN SE = mN

This range is flexible, however. Characteristics of the avian magnetic compass: (1) Functional window : It works only in an intensity range around the intensity where the birds live. This range is flexible, however. intensity in the housing room Robins Chickens

(2) Inclination compass: Characteristics of the avian magnetic compass: (2) Inclination compass: Birds do not distinguish between magnetic North (»mN «) and South (»mS «), but between "poleward" (»p«) and "equatorward" (»e«)

Ritz, Adem & Schulten (2000) proposed : These characteristics of the avian magnetic compass - the functional window, not using polarity - imply an unusual reception mechanism Ritz, Adem & Schulten (2000) proposed : Spin-chemical processes in photopigments: Radical Pair Model

'Radical Pair' model of magnetoreception Ritz et al. (2000) leads to response patterns on the retina, centrally symmetric to the magnetic vector angular difference 0° angular difference 40°

Effect of a Broad Band Field: geomagnetic field: 46 µT 0.1 – 10 MHz, 0.085 µT

Identifying a radical pair mechanism: Radio frequency-fields applied in different orientation with respect to the static geomagnetic field: up down

7.0 MHz-field in three different alignments: up S N down no unspecific effect!

Coherence time of the radical pair: 2-10 µs Orientation behavior at different frequencies: added vertically, intensity 480 nT Effect of RF-field: Coherence time of the radical pair: 2-10 µs

Resonance at the Larmor frequency Effect of the RF-field added verticaly to the geomagnetic field:

Ritz et al. (2000) suggested: Question: Which photopigment forms the magnetosensitive radical pairs? Ritz et al. (2000) suggested: Cryptochrome chromatophore Flavin (from Solov‘yov et al. 2007) Four Cryptochromes have been found in the eyes of birds: Cry1a, Cry1b, Cry2 and Cry4

Immunochemical studies of the retinae of chickens and robins: Cryptochrome 1a and UV Opsin labelled with a specific antiserum: Chicken, Gallus gallus Robin, Erithacus rubecula Cryptochrome 1a found in the outer segment of the UV/V cones UV/V cones identified as magnetoreceptors

Immunochemical studies in the electron-microscop:

Whole Mounts of the retina: Cry1a UV/V-opsin Merge Chicken, Gallus gallus Robin, Erithacus rubecula

Cryptochrome chromatophore: Flavin Absorbance of Flavin: Flavin cycle: (after Müller and Ahmad 2011, modified) Wavelengths to be tested (courtesy of M. Ahmad)

(quantal flux about 0.8 [UV] and 8·1015 quanta / s) Orientation of Robins tested under different wavelengths (quantal flux about 0.8 [UV] and 8·1015 quanta / s) 424 nm 510 nm 565 nm 590 nm 635 nm 373 nm Bird orientation requires short-wavelength light

The antiserum marks only light-activated Cry1a What form of Cry1a is marked by the antiserum? The antiserum marks only light-activated Cry1a

Activated Cry1a at the various wavelengths:

Which radical pair in the Cry-cycle mediates magnetic directional information? We observe oriented behavior and activated Cry1a under 565 nm green light

Effect of pre-exposure (1) 1 h pre-exposure under the same light as in test: Blue, Turquoise: 1st h 2nd h Green:

Orientation after 1 h pre-exposure under the same light as in test: 1st h 2nd h

Activated Cry1a at the various wavelengths: After 30 min exposure After 60 min exposure

Effect of pre-exposure (2) Pre-exposure in total darkness for 1 h: Cry-cycle during pre-exposure: 1 h pre-exposure, 1 h test

Orientation after pre-exposure in total darkness 1 h pre-exp. 1 h test Orientation after pre-exposure in total darkness

Activated Cry1a at the various wavelengths: after 30 min exposure to darkness B T G Followed by 30 min exposure to the various colors

Form labelled by the antiserum Pre-exposure experiments: Robins are oriented under green light, but no longer, if they have been exposed to darkness or green light before Form labelled by the antiserum Crucial radical pair?

Light and magnetic field alternatively Effect of pulsed stimuli: Control light magnetic field Pulsed magnetic field Light and magnetic field alternatively Flickering light

Flickering light: Pulsed magnetic field Pulsed magnetic field and flickering light allow orientation

Effect of alternatively flickering light and pulsed magnetic field: The crucial radical pair occurs in the dark!

Findings: Birds can still sense magnetic directions (1) under 565 nm green light, but only if they have been in daylight before, not when they have been in green light or in the dark. (2) in flickering light if the magnetic field is present only during the dark phase

Conclusion: Not the radical pair FADH°/ Trp° is the crucial one, but probably the FADH°/O2°- generated during reoxidation

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