Vol 5 21 August 2 doi:.3/nture713 LETTERS Cryptochrome medites light-dependent mgnetosensitivity in Drosophil Roert J. Geger 1, Amy Csselmn 1, Scott Wddell 1 & Steven M. Reppert 1 Although mny nimls use the Erth s mgnetic field for orienttion nd nvigtion 1,2, the precise iophysicl mechnisms underlying mgnetic sensing hve een elusive. One theoreticl model proposes tht geomgnetic fields re perceived y chemicl rections involving specilized photoreceptors 3. However, the specific photoreceptor involved in such mgnetoreception hs not een demonstrted conclusively in ny niml. Here we show tht the ultrviolet-a/lue-light photoreceptor cryptochrome (Cry) is necessry for light-dependent mgnetosensitive responses in Drosophil melnogster. In inry-choice ehviourl ssy for mgnetosensitivity, wild-type flies show significnt nive nd trined responses to mgnetic field under full-spectrum light ( 3 7 nm) ut do not respond to the field when wvelengths in the Cry-sensitive, ultrviolet-a/lue-light prt of the spectrum (,2 nm) re locked. Notly, Cry-deficient cry nd cry flies do not show either nive or trined responses to mgnetic field under full-spectrum light. Moreover, Cry-dependent mgnetosensitivity does not require functioning circdin clock. Our work provides, to our knowledge, the first genetic evidence for Crysed mgnetosensitive system in ny niml. The ility of n niml to detect geomgnetic fields hs sustntil iologicl relevnce s it is used y mny inverterte nd verterte species for orienttion nd nvigtion purposes, including homing, uilding ctivity nd long-distnce migrtion 2,. Three generl modes of mgnetoreception hve een proposed 5. One mode is electromgnetic induction y the Erth s mgnetic field, which my occur in electrosensitive mrine fish, lthough there is little evidence to support such sensing. The two other modes, for which experimentl evidence does exist, re mgnetite-sed process 6 nd chemicl-sed rections 9, tht re modulted y mgnetic fields. One chemicl model of mgnetoreception proposes tht mgnetic informtion is trnsmitted to the nervous system through the lightinduced product of mgneticlly sensitive rdicl-pir rections in specilized photoreceptors 3. Cry proteins re flvoproteins tht hve een postulted to generte mgnetosensitive rdicl pirs tht could provide photoinduced electron trnsfer rection for the detection of mgnetic fields 3. Cry proteins re est known for their roles in the regultion of circdin clocks, nd cn e ctegorized into two groups on the sis of current phylogenetic nd functionl reltionships 13,1. Drosophillike Cry proteins re sensitive to light in the ultrviolet-a/lue rnge 15 nd function primrily s photoreceptors tht synchronize (entrin) circdin clocks. Verterte-like Cry proteins, which hve lso een found in every non-drosophilid insect so fr exmined 1, do not seem to e directly light-sensitive. Insted, verterte-like Cry proteins re potent repressors of the Clock nd Bml1 (known s Cycle in insects) trnscription fctors which, s heterodimers, drive the intrcellulr trnscriptionl feedck loop of the circdin clock mechnism in ll nimls studied. Although there is good ehviourl evidence for the involvement of short-wvelength photoreceptors in the detection of geomgnetic field 5,16 1, n essentil link etween Cry nd mgnetoreception hs not een estlished in ny niml. Drosophil re idelly suited to investigte role for Cry s mgnetoreceptor, ecuse they only hve the light-sensitive Cry 1 in which the ction spectrum peks in the ultrviolet-a rnge (35 nm) with plteu in the ner lue rnge (3 5 nm) 19,2. Notly, flies tht lck Cry (cry ) 21 or hrour the chemiclly induced missense cry muttion 22,23 cn e used to evlute the role of Cry in mgnetosensitive responses. We initited our studies y developing ehviourl ssy for mgnetosensitivity in Drosophil (Fig. 1). In this illuminted pprtus, flies experience mgnetic field generted y n electric coil system nd disply their mgnetosensitivity in inry-choice T-mze. The two-coil system is idel for ehviourl studies of mgnetosensitivity, ecuse it produces mgnetic field on one side of the T-mze, while producing no field on the opposite side. This design elimintes non-mgnetic differences such s het generted y the electric coils etween sides during test sessions 2. Flies were tested either for their response to the mgnetic field in the nive stte (nive group) or fter trining session piring the field with sucrose rewrd (trined group). Wild-type Cnton-S, white-eyed w;cnton-s, Oregon-R nd Berlin-K strins ll developed lerned preference for mgnetic field (Fig. 1). The trined groups in the two Cnton-S lines showed the gretest response to the field (P 5.2, one-wy nlysis of vrince (ANOVA)) nd were the only ones to show nive voidnce of the field (P,.1, one-smple t-test). Thus, Drosophil consistently show mgnetosensitivity tht vries in mgnitude in strin-dependent mnner. The similrity of ehviourl responses etween red-eyed, wild-type Cnton-S flies nd white-eyed w;cnton-s flies shows tht eye colour does not sustntilly lter ehviourl responses to the mgnetic field. Becuse wild-type Cnton-S flies showed the most roust trined nd nive responses of the strins tested, we used them to determine whether the mgnetic responses we oserved were light-dependent. We ssyed nive nd trined Cnton-S flies under different longwvelength pss filters tht trnsmitted wvelengths of light t.5 nm,.2 nm or. nm (Fig. 2). In contrst to flies ssyed under full-spectrum light (Fig. 1 nd Fig. 2), flies did not show either nive or trined responses to the field when wvelengths,2 nm were locked (Fig. 2). Becuse the filter tht locked light,2 nm lso cused 13% decrese in totl irrdince (Fig. 2c, red line), we exmined whether the filter-induced lck of ehviourl responses to the mgnetic field ws secondry to the decrement in irrdince. When Cnton-S flies were studied under full-spectrum light, with totl irrdince level lower thn tht imposed y the filter (Fig. 2c, lue line), the flies still showed significnt nive (P 5.5, one-smple t-test) nd trined responses to 1 Deprtment of Neuroiology, University of Msschusetts Medicl School, Worcester, Msschusetts 165, USA. 1 2 Mcmilln Pulishers Limited. All rights reserved
NATURE Vol 5 21 August 2 LETTERS the mgnetic field (Fig. 2d). Thus, the filter-induced loss of ehviourl responses to the mgnetic field is due to the loss of shortwvelength light. Behviourl responses to the mgnet were prtilly restored when 2 nm light ws included (Fig. 2), which is consistent with the ction spectrum of Drosophil Cry tiling into the ner lue 19, nd, s expected, the trined response ws weker thn tht under full-spectrum light (full spectrum versus. nm, P,.1, Student s t-test). This wvelength-dependent effect of the mgnetic field on ehviour suggests tht Drosophil hs photoreceptor-sed mgnetosensitive system. Moreover, ecuse the response to the mgnetic field requires ultrviolet-a/lue light (,2 nm; Fig. 2e), these Trin Blck ox Light source λ Filter Photons s 1 cm 2 nm 1 ( ).2 3 5 6 7 >5 3 5 6 7 >2 > 3 5 6 7 3 5 6 7 Wvelength (nm) Test.2.1.1 Elevtor 9 λ T-port Coil Trining tue.2 Cnton-S w;cnton-s Oregon-R Berlin-K Figure 1 Behviourl pprtus for mgnetosensitivity nd ehviourl responses in different Drosophil strins., Behviourl pprtus for mgnetosensitivity. The top digrm (Trin) shows the frontl view of the choice chmer pprtus positioned for trining. The chmer pprtus consisted of trining tue, n elevtor to trnsfer flies, nd duel-choice point (T-port). For trining, the pprtus, with trining tue only, ws plced upright in n illuminted lck ox contining two-coil system. A popultion of flies (dots) ws loded into the trining tue with or without sucrose reinforcement nd mgnetic field. The ottom digrm (Test) shows the frontl view of the choice chmer pprtus positioned for testing. For testing, the pprtus, with tues ttched to the T-port (Tmze), ws rotted to the horizontl nd flies were trnsferred from the elevtor section to the T-port. Wvelength dependence ws exmined using long-wvelength pss filters., Drosophil strins vry in their ehviourl response to mgnetic field under full-spectrum light (Fig. 2). Brs show the preference index of the nive (white) or trined (lck) groups. Numers represent the groups tested nd vlues re men 6 s.e.m., P,.5;, P,.1;, P,.1. Photons s 1 cm 2 nm 1 ( ) Photons s 1 cm 2 nm 1 ( ).1.1.2 >5 nm 3 5 6 7 Wvelength (nm) 2 15 5 3 3 3 2 Mcmilln Pulishers Limited. All rights reserved c e Wvelength (nm) 2 >2 nm d.2.1.1.2 9 > nm 9 (low intensity) Figure 2 Short-wvelength light is required for mgnetosensitivity in Cnton-S flies., Irrdince curves for different light conditions. Light mesurements were tken inside the trining nd test tue. Dshed lines denote cutoff points of the locking filters., Wvelength-dependence of the mgnetic response. Brs show the preference index of the nive (white) or trined (lck) groups. Full-spectrum dt re from Fig. 1. Numers represent the groups tested., P,.5;, P,.1. c, Irrdince curves depicting full-spectrum light (lck line), light.2 nm (red line) nd full-spectrum light with reduced totl irrdince (full spectrum, low intensity; lue line). d, Cnton-S flies still elicited significnt responses to the mgnetic field under full-spectrum, low intensity light., P,.1. e, Irrdince vlues from 3 2 nm. Dt re expnded scle from fullspectrum pttern in. The irrdince vlues in ultrviolet-a/lue light in our studies (3 2 nm) re in line with those reported for Drosophil Cry function using other iologicl responses 19,2 ; tht is, rnge of to photons s 21 cm 22 nm 21. Vlues from nd d re men 6 s.e.m. 15
LETTERS NATURE Vol 5 21 August 2 dt re consistent with the hypothesis tht Cry cn function s mgnetoreceptor in Drosophil. We next used Cry-deficient cry mutnt flies to exmine directly whether Cry is required for mgnetosensitive ehviour. We tested two of the newly generted cry fly lines, ecuse, in cry flies, the entire cry coding sequence hs een replced with mini-white 1 y homologous recomintion, ensuring tht, unlike in the more commonly used point-mutnt cry flies, there is no possiility of residul Cry ctivity 21. In ddition, the three cry fly lines (from cry 1 to cry 3 ) were ckcrossed independently into w 1 ckground 21. Thus, we were le to use the pproprite w 1 control flies to test the contriution of the cry gene in mgnetosensitive ehviour. Control w 1 flies showed cler nive preference for, rther thn voidnce of, the mgnetic field (Fig. 3). The difference in the direction of the nive response to the mgnetic field etween Cnton-S flies nd the w 1 line re-emphsizes the importnce of controlling for genetic ckground in studies of mgnetosensitivity in flies. Nonetheless, like Cnton-S flies, the nive response of w 1 flies to the mgnetic field ws light dependent; the nive preference for the mgnetic field ws olished in the sence of ultrviolet-a/lue light (,2 nm; Fig. 3). Homozygous cry 2 flies lcking Cry did not show nive response to the mgnet under full-spectrum light, in contrst to the significnt nive responses mnifested y oth w 1 nd heterozygous cry 2 /1 flies (Fig. 3). Trining control w 1 flies to prefer the mgnetic field under full-spectrum light significntly enhnced their nive preference for the field (Fig. 3c). In contrst, homozygous cry 1 flies did not show either nive preference for the field (like cry 2 flies) or n enhnced preference for the field fter trining (Fig. 3c). The loss of the response to the mgnetic field in the Cry-deficient flies resemled the ehviour when w 1 flies were deprived of ultrviolet-a/lue light (Fig. 3), which is consistent with Cry eing the relevnt light sensor. These dt using two cry null strins strongly suggest tht oth nive nd trined responses to the mgnetic field in Drosophil require Cry function. The Cry-defective cry mutnt flies re lso unle to respond to the mgnetic field; the cry muttion renders Cry B essentilly nonfunctionl 22,23. Becuse the genetic ckground of cry mutnt flies is not well defined, we compred ehviourl responses to the mgnetic field etween homozygous cry flies nd heterozygous cry /Cnton-S flies. Wheres homozygous cry flies did not show either nive or trined responses to the mgnetic field under full-spectrum light, heterozygous cry /Cnton-S flies showed significnt nive (P 5., one-smple t-test) nd trined responses (Fig. 3d); the trined response in the heterozygotes ws less thn tht of wild-type Cnton-S flies (Fig. 1) nd proly results from differences in genetic ckground. To rule out non-cry muttions s the reson for the lck of mgnetic responses in cry mutnts, we showed tht the cry muttion fils to complement the cry 1 null muttion. Trnsheterozygous cry / cry 1 flies did not show significnt nive or trined responses to the mgnet, wheres heterozygous cry 1 /Cnton-S nd cry /Cnton-S flies did (nive response, P 5.6, one-smple t-test; Fig. 3d). Tken together, these dt indicte tht the cry locus is necessry for light-dependent mgnetosensitivity in Drosophil. Furthermore, the lck of trined response in oth cry 1 nd cry mutnt flies is consistent with Cry eing n essentil component of the mgnetosensitive sensory input pthwy nd perhps the mgnetoreceptor itself. Becuse light-ctivted Cry intercts with the criticl circdin clock protein Timeless to reset the circdin clock mechnism 25,we exmined whether n intct circdin system is necessry for the Crydependent mgnetosensitive responses in wild-type Cnton-S flies. Circdin rrhythmicity ws induced y constnt light, which disrupts circdin clock function in Cry-contining cells y cusing the constnt degrdtion of not only Cry ut lso Timeless nd then Period 25. We susequently tested ehviourl responses to the 16 mgnetic field fter t lest 5 dys in constnt light when the flies were shown to express rrhythmic locomotor ehviour (Fig. ), to hve disrupted Period undnce rhythms (Fig. ), nd to express constntly low levels of Cry (Fig. c). Notly, these rrhythmic flies continued to show significnt nive (P 5., one-smple t-test).2.15..5.5..15 c.2.15..5.5.2.15..5.5.2.15..5.5 2 Mcmilln Pulishers Limited. All rights reserved d 15 w 1 w 1 w 1 1 w 1 >5 nm 1 w 1 >2 nm cry 2 cry 2 /+ 1 cry 1 1 cry cry /C-S cry /cry 1 cry 1 /C-S Figure 3 Drosophil Cry medites mgnetosensitivity., Mgnetosensitivity in w 1 flies depends on ultrviolet-a/lue light. Brs show preference index vlues for nive responses under full-spectrum light nd light.5 nm nd.2 nm. Numers represent the groups tested., P,.1., Nive response to mgnetic field is impired in Crydeficient cry 2 flies, ut not in cry 2 /1 flies. Brs show preference index vlues for nive responses., P,.1;, P,.1. c, Nive nd trined responses to mgnetic field re impired in Cry-deficient cry 1 flies. Brs re preference index vlues for nive (white) nd trined (lck) groups., P,.5. d, Nive nd trined responses to mgnetic field re impired in homozygous cry nd trnsheterozygous cry /cry 1 flies. Brs show preference index vlues for nive (white) or trined (lck) groups. C-S, Cnton-S., P,.5;, P,.1. Vlues from d re men 6 s.e.m.
NATURE Vol 5 21 August 2 LETTERS Dy Dy Reltive protein levels 1 5 1 5 2. 1.5 1..5 LD LL Per 1 5 9 13 17 21 Zeitgeer time/circdin time (h) Figure Constnt light disrupts circdin function ut not Cry-medited mgnetosensitivity in Cnton-S flies., Men ctivity records in h light: h drk lighting cycle (LD; top) or constnt light (LL; ottom) in doule-plotted formt (n 5 62 for ech group). The lighting conditions were identicl to those used for housing flies tested for responses to mgnet; light irrdince, 1.5 3 15 photons s 21 cm 22 nm 21. For the light-drk cycle, 9% expressed circdin rhythms when relesed in constnt drkness (period, 2.6 6.3 h). All the constnt-light flies were rrhythmic., Temporl profiles of Period (Per) in heds. Protein undnce ws rhythmic in the LD LL LD LL c d Reltive protein levels 2. 1.5 1..5.2.1.1.2 Cry LD LL LD LL cry 1 C-S light-drk cycle (P,.1, one-wy ANOVA), ut not in constnt light. Hed extrcts were nlysed y western lot 3 nd normlized ginst -tuulin. Vlues re men 6 s.e.m. from three sets of heds. c, Cry undnce is decresed in LL. Vlues re men 6 s.e.m. from three sets of heds collected over 2 h in the light-drk cycle or in constnt light. Right, western lot proed for Cry 3 showing presence (rrow) in the light-drk cycle or in constnt light in Cnton-S (C-S) heds, nd sence in cry 1 heds. d, Flies in constnt light elicit ehviourl responses to the mgnetic field. Vlues re men 6 s.e.m., P,.1. nd trined responses to the mgnetic field (Fig. d). Thus, the continuous ctivtion of Cry y light does not disrupt its ility to sense the mgnet, nd n intct circdin system is not required for the mgnetoreception mechnism to operte. There re two other pulished reports of mgnetosensitivity in dult Drosophil 26,27. One descries ehviourl evidence tht mle wild-type Oregon-R flies show light-dependent mgnetic compss response in rdil mze wheres femle flies did not respond to the mgnet 27. Additionlly, mle flies responded in opposite directions when tested under either 365 nm or 5 nm light. In our studies, oth mle nd femle flies showed mgnetic response. Regrdless of experimentl differences, oth the previous study 27 nd ours demonstrte tht fruitflies cn respond to mgnetic field in wvelength-dependent mnner. Our results extend sustntilly the presence of light-dependent mgnetic sense in Drosophil y showing the necessity of Cry. We cnnot distinguish unequivoclly whether fly Cry functions s the ctul mgnetoreceptor or whether it is n essentil component downstrem of the receptor. Cry is necessry for oth the nive nd trined responses to the mgnetic field, consistent with the notion tht Cry is in the input pthwy of mgnetic sensing. In ddition, the continued ehviourl responses to the mgnet in constnt light, in which the known Cry signlling components re eing constntly degrded nd the circdin clock is rendered non-functionl, is lso consistent with n input function. The most compelling evidence supporting mgnetoreceptor role for Cry is tht the Crydependent ehviourl responses to the mgnetic field require ultrviolet-a/lue light, which mtches the ction spectrum of Drosophil Cry 19,2. 2 Mcmilln Pulishers Limited. All rights reserved Our ehviourl ssy for mgnetosensitivity does not t present hve pure directionl component, nd therefore it is difficult to relte our findings directly to the use of geomgnetic fields for niml orienttion nd nvigtion. Nevertheless, it is prole tht the response we hve identified is the prototype for the involvement of Cry in chemicl-sed mgnetic sensing. Thus, our findings open new venues of investigtion into the cellulr nd moleculr sis of chemicl-sed mgnetic sensing in nimls. The powerful genetics of Drosophil will fcilitte n understnding of the precise mechnism of ction of Cry in mgnetosensitivity, such s the ctul involvement of mgnetosensitive rdicl pirs produced y photoinduced electron trnsfer rections 2. Our dt further show tht the iologicl functions of Drosophil Cry extend eyond those in circdin clocks. METHODS SUMMARY Fly stocks were rised on stndrd cornmel/gr medium t 25 uc nd 6% reltive humidity under h light: h drk lighting cycle. The w 1 ;;cry flies (from cry 1 to cry 3 ) were gift from J. C. Hll nd re descried in ref. 21. The w 1 stock used in our experiments ws the sme stock used to crete w 1 ;;cry flies 21. The cry line ws gift from P. Emery 22. Our choice pprtus ws sed on the olfctory conditioning pprtus s descried 29. Our two-coil system ws sed on the doule-wrpped coil system descried previously 2. We djusted the current flowing through the coils so tht the mgnetic field intensity ws no more thn 5 G in ny re long the tue. Coils were positioned 5u to the horizontl for experiments involving Cnton-S, w;cnton-s, Berlin-K nd Oregon-R flies. Coils were positioned prllel to the horizontl for ll other experiments, ecuse it produced more roust response nd eliminted polr grdient; tht is, there ws no horizontl mgnetic grdient, s the field ws perpendiculr to the T-port tues. To ssess the mgnetoresponse of flies, we used simple choice prdigm. Flies were plced in glss vil contining 17
LETTERS NATURE Vol 5 21 August 2 moistened Whtmn pper nd strved for 22 h efore trining. All experiments were performed etween : nd : EST. For ech popultion of flies tested ( 15 flies per group), we clculted preference index vlue on the sis of the eqution: (P M 2.5)/[(P M 1.5) 2 (2P M 3.5)], in which P M is the proportion of flies on the mgnetic field side of the T-port. To test whether flies responded to the experimentl mgnetic field, we used either Student s t-test to compre preference index vlues etween trined nd nive groups or onesmple t-test to compre preference index vlues to zero (tht is, preference index vlues expected with no response to the mgnetic field). Full Methods nd ny ssocited references re ville in the online version of the pper t www.nture.com/nture. Received 25 Ferury; ccepted 19 June 2. Pulished online 2 July 2. 1. Lohmnn, K. J., Lohmnn, C. M. F. & Putmn, N. F. Mgnetic mps in nimls: nture s GPS. J. Exp. Biol. 2, 3697 375 (27). 2. Wiltschko, W. & Wiltschko, R. Mgnetic orienttion nd mgnetoreception in irds nd other nimls. J. Comp. Physiol. A Neuroethol. Sens. Neurl Behv. Physiol. 191, 675 693 (25). 3. Ritz, T., Adem, S. & Schulten, K. A model for photoreceptor-sed mgnetoreception in irds. Biophys. J. 7, 77 71 (2).. Luschi, P. et l. Mrine turtles use geomgnetic cues during open-se homing. Curr. Biol. 17, 6 133 (27). 5. Johnsen, S. & Lohmnn, K. J. The physics nd neuroiology of mgnetoreception. Nture Rev. Neurosci. 6, 73 7 (25). 6. Kirschvink, J. L. & Gould, J. L. Biogenic mgnetite s sis for mgnetic-field detection in nimls. Biosystems 13, 11 21 (191). 7. Wlker, M. M. A model for encoding of mgnetic field intensity y mgnetitesed mgnetoreceptor cells. J. Theor. Biol. 25, 5 91 (2).. Kirschvink, J. L., Wlker, M. M. & Dieel, C. E. Mgnetite-sed mgnetoreception. Curr. Opin. Neuroiol., 62 67 (21). 9. Lesk, M. J. M. Physicochemicl mechnism for mgnetic-field detection y migrtory irds nd homing pigeons. Nture 267, 1 15 (1977).. Schulten, K., Swenerg, C. E. & Weller, A. Biomgnetic sensory mechnism sed on mgnetic-field modulted coherent electron-spin motion. Z. Phy. Chem. (Frnkfurt) 1, 1 5 (197).. Cshmore, A. R. Cryptochromes: Enling plnts nd nimls to determine circdin time. Cell, 537 53 (23).. Prtch, C. L. & Sncr, A. Photochemistry nd photoiology of cryptochrome lue-light photopigments: The serch for photocycle. Photochem. Photoiol. 1, 91 13 (25). 13. Zhu, H. et l. The two CRYs of the utterfly. Curr. Biol. 15, R953 R95 (25). 1. Yun, Q., Metterville, D., Briscoe, A. D. & Reppert, S. M. Insect cryptochromes: Gene dupliction nd loss define diverse wys to construct insect circdin clocks. Mol. Biol. Evol. 2, 9 955 (27). 15. Öztürk, N., Song, S. H., Sely, C. P. & Sncr, A. Animl type 1 cryptochromes nlysis of the redox stte of the flvin cofctor y site-directed mutgenesis. J. Biol. Chem. 23, 3256 3263 (2). 16. Phillips, J. B. & Borlnd, S. C. Behviorl evidence for use of light-dependent mgnetoreception mechnism y verterte. Nture 359, 12 1 (1992). 17. Ritz, T., Dommer, D. H. & Phillips, J. B. Shedding light on verterte mgnetoreception. Neuron 3, 53 56 (22). 1. Wiltschko, R. & Wiltschko, W. Mgnetoreception. Bioessys 2, 157 16 (26). 19. VnVickle-Chvez, S. J. & Vn Gelder, R. N. Action spectrum of Drosophil cryptochrome. J. Biol. Chem. 22, 561 566 (27). 2. Helfrich-Forster, C. et l. The extrretinl eyelet of Drosophil: Development, ultrstructure, nd puttive circdin function. J. Neurosci. 22, 9255 9266 (22). 21. Dolezelov, E., Dolezel, D. & Hll, J. C. Rhythm defects cused y newly engineered null muttions in Drosophil s cryptochrome gene. Genetics 177, 329 35 (27). 22. Emery, P. et l. CRY, Drosophil clock nd light-regulted cryptochrome, is mjor contriutor to circdin rhythm resetting nd photosensitivity. Cell 95, 669 679 (199). 23. Stnewsky, R. et l. The cry muttion identifies cryptochrome s circdin photoreceptor in Drosophil. Cell 95, 61 692 (199). 2. Kirschvink, J. L. Uniform mgnetic-fields nd doule-wrpped coil systems improved techniques for the design of ioelectromgnetic experiments. Bioelectromgnetics 13, 1 (1992). 25. Stnewsky, R. Genetic nlysis of the circdin system in Drosophil melnogster nd mmmls. J. Neuroiol. 5, 1 17 (23). 26. Wehner, R. & Lhrt, T. Perception of geomgnetic field in fly Drosophil melnogster. Experienti 26, 967 96 (197). 27. Phillips, J. B. & Syeed, O. Wvelength-dependent effects of light on mgnetic compss orienttion in Drosophil melnogster. J. Comp. Physiol. A Sens. Neurl Behv. Physiol. 172, 33 3 (1993). 2. Med, K. et l. Chemicl compss model of vin mgnetoreception. Nture 53, 37 39 (2). 29. Tully, T. & Quinn, W. G. Clssicl-conditioning nd retention in norml nd mutnt Drosophil melnogster. J. Comp. Physiol. A Sens. Neurl Behv. Physiol. 157, 263 277 (195). 3. Rush, B. L., Murd, A., Emery, P. & Gieultowicz, J. M. Ectopic CRYPTOCHROME renders TIM light sensitive in the Drosophil ovry. J. Biol. Rhythms 21, 272 27 (26). Supplementry Informtion is linked to the online version of the pper t www.nture.com/nture. Acknowledgements We thnk H. Zhu for the protein work in Fig., c; L. Foley for ssistnce; J. C. Hll for the cry flies; P. Emery for the Per nd Cry ntiodies; nd P. Emery, P. Perrt, B. Leung, S. DsGupt, M. Krshes nd H. Zhu for discussions. This work ws supported y grnts from the NIH. Author Contriutions S.M.R. nd R.J.G. conceived the ide of using Drosophil to study mgnetosensitivity; S.W. nd R.J.G. conceived the ide of using ppetitive conditioning to study mgnetoresponses; R.J.G. designed the experimentl pprtus; R.J.G., S.W., A.C. nd S.M.R. designed the experiments nd nlysed the dt; R.J.G. performed the experiments with help from A.C.; R.J.G., S.M.R., S.W. nd A.C wrote the pper. Author Informtion Reprints nd permissions informtion is ville t www.nture.com/reprints. Correspondence nd requests for mterils should e ddressed to S.M.R. (steven.reppert@umssmed.edu). 1 2 Mcmilln Pulishers Limited. 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doi:.3/nture713 METHODS Fly strins. Oregon-R, Berlin-K nd w 1 stocks were provided y the Bloomington Drosophil Stock Center (product numer 6326, 269 nd 522, respectively). Behviourl pprtus. The min chmer consisted of trining tue, centre elevtor section to trnsfer flies, nd two-tue choice point or T-port for testing the reltive response of flies to mgnetic field (Fig. 1). The trining nd T-port tues were round-ottom polystyrene tues nd could e removed from the min section of the pprtus. The mgnetic stimulus ws delivered to the trining nd T-port tues y plcing the choice chmer pprtus inside n opque housing ox tht contined the mgnetic coil system. The ox ws constructed such tht the min chmer could e plced etween the two coils in either n upright position (s shown in Fig. 1, top pnel) or horizontl position (s shown in Fig. 1, ottom pnel). The upright position of the chmer pprtus ws used for trining so tht the tue could e plced in the centre of one coil, nd the horizontl position of the pprtus ws used to suspend the two tues of the T-mze in the sme re of the coil during test sessions. In this wy, flies were sujected to the sme light conditions nd intensity of mgnetic field during trining nd test sessions. Ech of the two coils ws wrpped with two wires; the wires were wrpped in one direction on one coil nd in the opposite direction on the other. Current flow through the coils produced mgnetic field in one coil (prllel current flow) ut not in the other (opposite current flow). A doule-pole, doule-throw switch reversed the current flow in one wire loop ut not the other, which llowed us to move the mgnetic field esily from the right to the left side of the T-mze. A DC power supply with current nd voltge controls ws used so tht we could chnge the intensity of the mgnetic field produced y the coils. Ech coil ws mounted on plstic trck so tht it could e positioned directly under the tues (field perpendiculr to the tues), t the end of the tues t 5u ngle (s shown in Fig. 1), or t the end of the tues (field prllel to the tues). We used mgnetic field intensity of 5 G for our experiments, ecuse it gve the most consistent nive response; decresing to 1 G incresed vriility to the point tht responses were no longer significnt. The housing ox for the test or choice chmer ws open on the top so tht the chmer, regrdless of position, could e illuminted y one ZooMed Reptisun. UVB fluorescent tue (F2T) nd one Agrorite full spectrum fluorescent grow tue. Wvelengths entering the ox were restricted y covering the top of the ox with long-wvelength filter tht trnsmitted wvelengths of light.5 nm (Edmund Optics) or.2 nm or. nm (E nd E2 from Gentex). Irrdince mesurements (from n Ocen Optics USB 2 fire optic spectrometer) were tken from inside one rm of the T-mze portion of the choice chmer pprtus; thus, lighting conditions represent those experienced y flies while eing either trined or ssyed for sensitivity to mgnetic field. Experimentl procedure. For the trining group, popultion of 15 flies ws loded into the elevtor section of the choice pprtus with n empty trining tue fcing one of the coils (Fig. 1, top pnel). Flies were trnsferred to the trining tue for 2 min nd then trnsferred ck to the elevtor nd held for 1-min rest period. The empty trining tue ws next replced with tue contining sucrose reinforcement nd flies were llowed to feed for 2 min in the presence of mgnetic field. Flies were then trnsferred ck to the elevtor nd held for 1 min while the coil system ws turned off. During this time, the trining tue ws lso removed, nd two empty tues were dded to form the two rms of the T-port. The choice chmer ws then positioned horizontlly in the ox (Fig. 1, ottom pnel). The coil system ws turned on, nd flies were trnsferred to the T-port, in which they were llowed to choose etween the sides with or without mgnetic field. After 2 min, the two rms of the T-port were locked nd flies from ech side were collected into seprte empty vils nd counted. For the nive group, second popultion of 15 flies ws immeditely loded into the elevtor section of the horizontlly plced choice chmer nd the coil system ws turned on. After 1 min, flies were trnsferred directly to the T-port for 2 min. Trined nd nive groups were tested consecutively nd with the mgnetic field on the sme side. This ws done to control for the possiility tht the choice ehviour of flies reflected preference for one rm of the T-port nd not response to the mgnetic field. As n extr control for side preferences independent of mgnetic stimuli, we lternted the side of the T-port contining the field fter ech consecutive set of trined nd nive flies (tht is, trined nd nive with mgnet on the left side nd then trined nd nive with mgnet on the right). 2 Mcmilln Pulishers Limited. All rights reserved