Cryptochrome 1a localisation in light- and dark-adapted retinae of several migratory and non-migratory bird species: no signs of light-dependent activation

Abstract

The magnetic compass of birds seems to be based on light-dependent radical-pair processes in the eyes. Cryptochromes are currently the only candidate proteins known in vertebrates that may serve as the primary radical-pair-based magnetoreceptor molecules. Previous immunohistochemical studies have suggested that cryptochrome 1a (Cry1a) is localised in the photoreceptor outer segments of the ultraviolet/violet (UV/V) cones, and it has been claimed that differences in Cry1a antibody staining intensities show that Cry1a is activated by light and that this should make Cry1a the most likely magnetoreceptive candidate molecule. Here, we present an independent study of Cry1a distribution within retinae of several bird species, ranging from non-migratory domestic chicken and rock pigeon to night-migratory passerines, using both the previously used antibody and two newly generated antibodies, one against the same epitope as the originally used antibody and one against a different epitope of Cry1a. We confirm the UV/V cone outer segment localisation of Cry1a in all the tested bird species. In some stainings, we found Cry1a immunoreactivity as a distinct punctate pattern throughout the whole length of the UV/V cone outer segments. These dots with a diameter of around 170 nm might suggest that many Cry1a molecules accumulate in distinct spots in the UV/V cone outer segments. However, we did not see any notable difference in Cry1a immunoreactivity between light- and dark-adapted retinae. We find no evidence whatsoever that a C-terminal antibody against Cry1a labels only a light-activated form of the Cry1a protein.

KEY WORDS:

• magnetic orientation
• magnetoreception
• magnetic sense
• cryptochrome
• bird navigation
• light adaptation
• Erithacus rubecula

ACKNOWLEDGEMENTS

We thank Christine Nießner and Prof. Roswitha Wiltschko for providing an aliquot of the original Frankfurt antibody used in Nießner et al. (2011). We thank Margrit Kanje, Irina Fomins, Jessica Schmidt and Bianca Brüggen for excellent technical support in the lab. The authors cordially acknowledge the Service Unit (Core Facility) Fluorescence Microscopy of the Carl-von-Ossietzky-University Oldenburg for the use of the imaging facilities.

DISCLOSURE STATEMENT

No potential conflict of interest was reported by the authors.

ETHICAL STANDARD

All animal procedures were performed in accordance with local, national, and EU guidelines for the use of animals in research and were approved by the Animal Care and Use Committees of the Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit (LAVES, Oldenburg, Germany), Az: 3314-42502-04-10/0121.

AUTHOR CONTRIBUTIONS

P. Bolte and A. Einwich contributed equally to this work.

P. Bolte, K. Dedek and H. Mouritsen designed and supervised the experiments, A. Einwich, I. Wojahn, P. Bolte, P.K. Seth, R. Chetverikova, D. Heyers and U. Janssen-Bienhold performed the experiments, P. Bolte, A. Einwich and H. Mouritsen analysed the data, R. Feederle generated monoclonal antibodies, P. Bolte, A. Einwich, K. Dedek, P.J. Hore and H. Mouritsen wrote the paper. All authors commented on the final manuscript.

SUPPLEMENTAL DATA

Supplemental data for this article can be accessed at https://doi.org/10.1080/03949370.2020.1870571

Additional information

Funding

This project was funded by a Lichtenberg Professorship from the Volkswagen Stiftung to H. Mouritsen, by a grant from the Niedersächsisches Ministerium für Wissenschaft und Kultur to H. Mouritsen, by the Deutsche Forschungsgemeinschaft (RTG 1885/1-2: Molecular Basis of Sensory Biology to U. Janssen-Bienhold, A. Einwich, P.K. Seth, K. Dedek and H. Mouritsen; HE6221/1-1 to D. Heyers; MO 1408/1-2 to H. Mouritsen; SFB 1372: Magnetoreception and Navigation in Vertebrates to K. Dedek, D. Heyers and H. Mouritsen), by an ERC Synergy Grant no. 810002 (“QuantumBirds”) to P.J. Hore and H. Mouritsen, by DARPA (QuBE N66001-10-1-4061) to H. Mouritsen and P.J. Hore, and by the Air Force Office of Scientific Research (Air Force Materiel Command USAF award FA9550-14-1-0095 to H. Mouritsen and P.J. Hore).