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PNAS proof Embargoed Q:A Contribution of photoreceptor subtypes to spectral wavelength preference in Drosophila Q:1 Satoko Yamaguchi a,b,1 , Claude Desplan a,1 , and Martin Heisenberg b a Center for Developmental Genetics, Department of Biology, New York University, New York, NY 10003; and b Department of Genetics and Neurobiology, Biocenter, University of Würzburg, Würzburg 97074, Germany Edited* by Michael W. Young, The Rockefeller University, New York, NY, and approved February 4, 2010 (received for review September 20, 2008) The visual systems of most species contain photoreceptors with distinct spectral sensitivities that allow animals to distinguish lights by their spectral composition. In Drosophila, photoreceptors R1R6 have the same spectral sensitivity throughout the eye and are respon- sible for motion detection. In contrast, photoreceptors R7 and R8 exhibit heterogeneity and are important for color vision. We inves- tigated how photoreceptor types contribute to the attractiveness of light by blocking the function of Q:2 their subsets and by measuring dif- ferential phototaxis between spectrally different lights. In a UV vs. bluechoice, ies with only R1R6, as well as ies with only R7/R8 photoreceptors, preferred blue, suggesting a nonadditive interaction between the two major subsystems. Flies defective for UV-sensitive R7 function preferred blue, whereas ies defective for either type of R8 (blue- or green-sensitive) preferred UV. In a blue vs. greenchoice, ies defective for R8 (blue) preferred green, whereas those defective for R8 (green) preferred blue. Involvement of all photoreceptors [R1R6, R7, R8 (blue), R8 (green)] distinguishes phototaxis from motion detection that is mediated exclusively by R1R6. color vision | differential phototaxis | Q:3 photoreceptor | R7/R8 system D epending on its spectral composition and intensity, light can serve as an attractive or repulsive landmark for orientation. Accordingly, animals identify an object or a light source at a certain location in visual space and approach or retreat from it. Phototaxis in insects has been a useful paradigm to gain a better under- standing of this behavior. Here we take advantage of Drosophila genetics to investigate the function of the different subtypes of photoreceptors in this behavior. The Drosophila compound eye consists of about 750 ommatidia, each containing 8 photoreceptor cells (1). The six outer photo- receptors (R1R6) contain a blue-sensitive rhodopsin (Rh1) and show a second sensitivity peak in the UV due to a sensitizing pig- ment (15). They are specialized for vision at low light levels and low pattern contrast (1, 6). They are necessary and sufcient for motion vision, and y optomotor responses are abolished in their absence (7, 8). The two inner photoreceptors (R7 and R8) are heteroge- neous. Along the dorsal margin of the eye, both R7 and R8 express rhodopsin Rh3 that is sensitive to shorter wavelength UV (9). This part of the retina is specialized for the detection of the e-vector of polarized light (10, 11). The remaining part of the retina contains two types of ommatidia called pale (p) and yellow (y). In p-type ommatidia, R7 cells contain Rh3 (R7p) and R8 express blue- sensitive Rh5 (R8p). In y-type ommatidia, R7 cells express Rh4 sensitive to longer wavelength UV (R7y) and R8 cells contain green-sensitive Rh6 (R8y) (Fig. 1A) (5, 1215). R7y cells in the dorsal third of the eye coexpress Rh3 and Rh4 (16). Approximately 30% of the ommatidia are of the p- and 70% of the y-type, with the p- and y-subtypes distributed stochastically in the retina. R7/R8 cells in the main part of the retina appear to be involved in color vision (15), which requires the comparison of at least two photoreceptors with different spectral sensitivities. This comparison could be between R7 and R8 within one ommatidium or between different (p vs. y) ommatidia (17), or it could involve outer photoreceptors. Phototaxis can be tested in different ways (6, 18, 19). When ies in small tubes illuminated from one side (light/dark) are dis- turbed, they rush toward the light for an escape (fast phototaxis) (20). In a variant of this behavior, called differential phototaxis,ies choose between two light sources. Both paradigms have been used to determine the action spectra of phototaxis. Using a light/ dark assay (2123), two peaks of sensitivity were identied, one in the UV and one in the blue, representing mainly the sensitivity of the outer photoreceptors R1R6 (24). In differential phototaxis (2527), the UV peak is particularly pronounced. Mutants with defects in phototactic behavior (20) affect photo- transduction and/or cause photoreceptor degeneration [e.g., ninaE 17 (28) rdgB (29, 30)]. sevenless LY3 mutants (sev) (27) also display phototaxis defects. In these mutants, photoreceptor devel- opment is affected because R7 cells are missing, and photoreceptors R1R6 and R8 are largely intact, although the latter are deprived of their proper optics, the rhabdomere of R7. The sev mutation has been useful for investigating the contribution of R7 to phototaxis behavior. The sensitivity of sev ies to UV measured by ERG Q:7 (27) or light/dark phototaxis (23) is comparable to that of wild-type ies. In differential phototaxis, however, sev ies prefer visible light at intensities where wild-type ies have a strong preference for UV (7, 27, 31). The UV vs. visiblechoice paradigm is a robust func- tional assay that was used for genetic screens that identied other genes essential for R7 development (6, 19). Functional analyses of R8 cells have lagged behind. Due to their position behind R7 photoreceptors and their small size, their phys- iological properties are difcult to examine by electrophysiological recordings. The spectral properties of R8 opsins were measured by using the double-mutant rdgB sev, in which R8 were believed to be the only functional photoreceptors (27), and more recently by expressing R8 opsins in R1R6 of ninaE 17 mutant cells lacking Rh1 (5). It appears that R8 alone can mediate phototactic behavior at high intensities (32). However, the contribution of R8 in the presence of an intact R1R6 system has not been investigated because R8 specic mutants were previously not available. Moreover, the distribution of R8 subtypes is altered in sev ies (33, 34), possibly confounding results that had assessed the contribution of R8 to phototaxis. To study the contributions of photoreceptor types to phototaxis, we developed a variant of differential phototaxis involving choices between two different monochromatic lights: UV vs. blue or blue vs. green. We tested various mutants with altered or lost opsin expression, as well as ies in which specic types of photoreceptors were functionally switched off by specically blocking synaptic transmission. We showed that R1R6, R7, R8p, and R8y all affect phototaxis. The results are largely consistent with the prediction made by estimating the relative number of quanta captured by each photoreceptor subtype if each subtype is separately weighted. However, some photoreceptor combinations generated effects that Author contributions: S.Y. designed and performed research; S.Y., C.D., and M.H. ana- lyzed data; and S.Y., C.D., and M.H. wrote the paper Q:4; 5 . The authors declare no conict of interest. *This Direct Submission article had a prearranged editor Q:6 . 1 To whom correspondence may be addressed. E-mail: [email protected] or cd38@ nyu.edu. This article contains supporting information online at www.pnas.org/cgi/content/full/ 0809398107/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.0809398107 PNAS Early Edition | 1 of 7 NEUROSCIENCE

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Page 1: PNAS proof

PNAS proofEmbargoed

Q:A Contribution of photoreceptor subtypes to spectralwavelength preference in DrosophilaQ:1

Satoko Yamaguchia,b,1, Claude Desplana,1, and Martin Heisenbergb

aCenter for Developmental Genetics, Department of Biology, New York University, New York, NY 10003; and bDepartment of Genetics and Neurobiology,Biocenter, University of Würzburg, Würzburg 97074, Germany

Edited* by Michael W. Young, The Rockefeller University, New York, NY, and approved February 4, 2010 (received for review September 20, 2008)

The visual systems of most species contain photoreceptors withdistinct spectral sensitivities that allow animals to distinguish lightsby their spectral composition. In Drosophila, photoreceptors R1–R6have thesamespectral sensitivity throughout theeyeandare respon-sible for motion detection. In contrast, photoreceptors R7 and R8exhibit heterogeneity and are important for color vision. We inves-tigated how photoreceptor types contribute to the attractiveness oflight by blocking the function ofQ:2 their subsets and by measuring dif-ferential phototaxis between spectrally different lights. In a “UV vs.blue” choice, flies with only R1–R6, as well as flies with only R7/R8photoreceptors, preferredblue, suggesting a nonadditive interactionbetween the two major subsystems. Flies defective for UV-sensitiveR7 function preferred blue, whereas flies defective for either type ofR8 (blue-orgreen-sensitive)preferredUV. Ina “bluevs.green” choice,flies defective for R8 (blue) preferred green,whereas those defectivefor R8 (green) preferred blue. Involvement of all photoreceptors[R1–R6, R7, R8 (blue), R8 (green)] distinguishes phototaxis frommotion detection that is mediated exclusively by R1–R6.

color vision | differential phototaxis |Q:3 photoreceptor | R7/R8 system

Depending on its spectral composition and intensity, light canserve as an attractive or repulsive landmark for orientation.

Accordingly, animals identify anobject or a light source at a certainlocation in visual space and approach or retreat from it. Phototaxisin insects has been a useful paradigm to gain a better under-standing of this behavior. Here we take advantage of Drosophilagenetics to investigate the function of the different subtypes ofphotoreceptors in this behavior.The Drosophila compound eye consists of about 750 ommatidia,

each containing 8 photoreceptor cells (1). The six outer photo-receptors (R1–R6) contain a blue-sensitive rhodopsin (Rh1) andshow a second sensitivity peak in the UV due to a sensitizing pig-ment (1–5).Theyare specialized for visionat low light levels and lowpattern contrast (1, 6). They are necessary and sufficient formotionvision, and fly optomotor responses are abolished in their absence(7, 8). The two inner photoreceptors (R7 and R8) are heteroge-neous. Along the dorsal margin of the eye, both R7 and R8 expressrhodopsin Rh3 that is sensitive to shorter wavelength UV (9). Thispart of the retina is specialized for the detection of the e-vector ofpolarized light (10, 11). The remaining part of the retina containstwo types of ommatidia called pale (p) and yellow (y). In p-typeommatidia, R7 cells contain Rh3 (R7p) and R8 express blue-sensitive Rh5 (R8p). In y-type ommatidia, R7 cells express Rh4sensitive to longer wavelength UV (R7y) and R8 cells containgreen-sensitive Rh6 (R8y) (Fig. 1A) (5, 12–15). R7y cells in thedorsal third of the eye coexpress Rh3 and Rh4 (16). Approximately30% of the ommatidia are of the p- and 70% of the y-type, with thep- and y-subtypesdistributed stochastically in the retina.R7/R8 cellsin the main part of the retina appear to be involved in color vision(15), which requires the comparison of at least two photoreceptorswith different spectral sensitivities. This comparison could bebetween R7 and R8 within one ommatidium or between different(p vs. y) ommatidia (17), or it could involve outer photoreceptors.Phototaxis can be tested in different ways (6, 18, 19).When flies

in small tubes illuminated from one side (“light/dark”) are dis-

turbed, they rush toward the light for an escape (“fast phototaxis”)(20). In a variant of this behavior, called “differential phototaxis,”flies choose between two light sources. Both paradigms have beenused to determine the action spectra of phototaxis. Using a light/dark assay (21–23), two peaks of sensitivity were identified, one inthe UV and one in the blue, representing mainly the sensitivity ofthe outer photoreceptors R1–R6 (24). In differential phototaxis(25–27), the UV peak is particularly pronounced.Mutants with defects in phototactic behavior (20) affect photo-

transduction and/or cause photoreceptor degeneration [e.g.,ninaE17 (28) rdgB (29, 30)]. sevenlessLY3 mutants (sev) (27) alsodisplay phototaxis defects. In these mutants, photoreceptor devel-opment is affectedbecauseR7 cells aremissing, andphotoreceptorsR1–R6 and R8 are largely intact, although the latter are deprivedof their proper optics, the rhabdomere of R7. The sevmutation hasbeen useful for investigating the contribution of R7 to phototaxisbehavior. The sensitivity of sevflies toUVmeasuredbyERG Q:7(27) orlight/dark phototaxis (23) is comparable to that of wild-type flies. Indifferential phototaxis, however, sev flies prefer visible light atintensities where wild-type flies have a strong preference for UV(7, 27, 31). The “UV vs. visible” choice paradigm is a robust func-tional assay that was used for genetic screens that identified othergenes essential for R7 development (6, 19).Functional analyses of R8 cells have lagged behind. Due to their

position behind R7 photoreceptors and their small size, their phys-iological properties are difficult to examine by electrophysiologicalrecordings. The spectral properties of R8 opsins were measured byusing thedouble-mutant rdgB sev, inwhichR8werebelieved tobe theonly functional photoreceptors (27), andmore recently by expressingR8 opsins in R1–R6 of ninaE17 mutant cells lacking Rh1 (5). Itappears that R8 alone can mediate phototactic behavior at highintensities (32). However, the contribution of R8 in the presence ofan intactR1–R6systemhasnotbeen investigatedbecauseR8specificmutants were previously not available. Moreover, the distribution ofR8 subtypes is altered in sev flies (33, 34), possibly confoundingresults that had assessed the contribution of R8 to phototaxis.To study the contributions of photoreceptor types to phototaxis,

we developed a variant of differential phototaxis involving choicesbetween twodifferentmonochromatic lights:UVvs. blue or blue vs.green. We tested various mutants with altered or lost opsinexpression, as well as flies in which specific types of photoreceptorswere functionally switched off by specifically blocking synaptictransmission. We showed that R1–R6, R7, R8p, and R8y all affectphototaxis. The results are largely consistent with the predictionmade by estimating the relative number of quanta captured by eachphotoreceptor subtype if each subtype is separately weighted.However, some photoreceptor combinations generated effects that

Author contributions: S.Y. designed and performed research; S.Y., C.D., and M.H. ana-lyzed data; and S.Y., C.D., and M.H. wrote the paper Q:4; 5.

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor Q:6.1To whom correspondence may be addressed. E-mail: [email protected] or [email protected].

This article contains supporting information online at www.pnas.org/cgi/content/full/0809398107/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.0809398107 PNAS Early Edition | 1 of 7

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could not be explained by simple summation of the responses of allfunctional photoreceptors.Thesephototaxis experiments provideafirst glimpseof a spectral-

wavelength–dependent attractiveness function used in landmarkdiscrimination. A quantitative description of this function wouldrequire measuring all the intensity dependencies of spectral pref-erences. It will be of interest to compare this function to color visiondefined as intensity-invariant hue discrimination, which has beenmeasured by learning and generalization tasks (35, 36).

ResultsDifferential Phototaxis. To test the contribution of photoreceptortypes to phototaxis, we used a T-maze apparatus (37), adding twodifferentmonochromatic light sources (LEDs) at the end of each ofthe test tubes. Approximately 100 flies were placed at the choicepoint, and the number of flies that had moved toward each lightsource was counted 20 s later. A preference index (PI) was calcu-lated as PI = (NL − NS)/(NL + NS), where NL and NS were thenumber of flies on the side of the longer and shorter wavelengthlights, respectively. To test the ability of the flies to distinguish lightsof different spectral composition, we used two combinations ofmonochromatic lights:UVvs. blue (UV/B), to presumably assayR7vs. R8 function, and blue vs. green (B/G) for R8p vs. R8y (seebelow). The wavelength of the lights was chosen such that theystimulate the respective photoreceptor types effectively (Fig. 1).R7p and R7y contain UV-sensitive rhodopsins that are tuned fairlyclosely, and we did not attempt to test the ability of the fly to dis-tinguish in this range of wavelengths.For the UV/B tests, UV intensity was set to ∼1,000 times the

phototaxis threshold of wild-type flies to make sure that the lightscould be detected by the R7 and R8 cells. This was more than fivetimes the phototaxis threshold of ninaE17, a rh1 null mutant thatcompletely blocks R1–R6 function (3), as tested in separate “lightvs. dark” experiments. The intensity of the blue light was adjustedsuch that wild-type flies distributed equally between UV and blue(PI = 0). As UV is highly attractive for wild-type flies (7, 25–27), itsintensity was∼57 times lower than the blue (Fig. 1B,Upper). For B/G tests, green intensity was set to ∼500 times the phototaxisthreshold of wild-type flies, i.e., 20 times the phototaxis threshold ofninaE17 mutants in the “light vs. dark” experiment. The blueintensity was 7.8 times lower than the green, reflecting the innatepreference of flies for shorter wavelengths. In the UV/B test, blue

was about 85 times more intense than in the B/G test (see SIQ:8

Materials and Methods for Q:9further information).

Contributions of Photoreceptors in UV/B Choice Tests. InUV/B choicetests, theUV (peak wavelength 350 nm) was chosen tomaximizeR7responses. R7p contains Rh3 maximally absorbing around 345 nm,and R7y contains Rh4 with an absorption maximum around 375 nm(14). The UV light also optimally stimulated the R1–R6 photo-receptors in the spectral range of the sensitizing pigment (gray curvein Fig. 1A). The blue (peak wavelength 430 nm; Fig. 1B, Upper)effectively stimulated both subtypes of R8 cells as well as R1–R6(Rh1opsinpeakat 478nm).R8pexpressedblue-absorbingRh5 (437nm), andR8y a green-absorbingRh6 (508 nm) (5). Fig. 1A shows thenormalized spectral properties of each photoreceptor subtype(RogerHardie, personal communication Q:10and refs. 5 and 38). Fig. 1Bindicates the spectral profile of eachLEDforUV/BandB/Gchoices.The relative numbers of quanta captured by each subtype of the

photoreceptors were estimated for the light intensities used(Fig. 1C; see also SI Materials andMethods) by taking into accountthe spectral properties of each photoreceptor subtype (Fig. 1A)and the relative irradiance of each LED (Fig. 1B). Note that theseestimates refer to single photoreceptors and neither their absolutesensitivities nor their relative numbers (e.g., R8p:R8y = 3:7) aretaken into account.Both R8p and R8y photoreceptors respond to blue much more

strongly than to UV (Fig. 1C, Upper). Thus, in the UV/B choicetest, mutants impaired in R8p and/or R8y are expected to preferUV to blue, assuming a simple additive model of photoreceptorfunction. On the other hand, R7p receptors should respondmore to UV than to blue, whereas R7y should respond similarlyto UV and blue (Fig. 1C, Upper). Thus, switching off both R7pand R7y is expected to lead to a preference for blue. Theseassumptions imply, however, that the attractiveness function ofdifferential phototaxis is not dominated by the R1–R6 photo-receptors which, due to their size, optics, and number are about10–50 times more sensitive than R7/R8 cells.

Mutants Defective in R7 Function Prefer Blue in UV/B Choice. We firstexamined the contribution of R7 photoreceptors. sev mutant flies(27) preferred blue in the UV/B choice test (Fig. 2C), consistentwith earlier experiments using a UV vs. green choice test (7). Inaddition to the loss of R7 cells, sev flies also have defects in R8:

A

B C

Fig. 1. (A) Normalized spectral quantum sensitivity of thedifferent rhodopsins in the photoreceptor subtypes R7p, R7y,R8p, R8y, and R1–R6 (R. Hardie, personal communication).The height of the blue peak relative to the UV peak of R1–R6(gray curve) might be lower than shown depending upon theβ-carotene content of fly food (5, 38). (B) Spectral propertiesof the stimuli used in this study. The normalized spectralcurves of LEDs are shown in the Upper Inset. (C) The relativenumber of quanta captured by each photoreceptorQ:22 (PR)subtype was estimated for the light stimuli used in UV/B(Upper) and B/G experiments (Lower). Note that these nor-malized estimates refer to single photoreceptors. Neithertheir absolute sensitivities nor their relative numbers weretaken into account.

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BecauseR7 cells are required during development for R8p subtypespecification,∼93%ofR8 cells in sevmutant flies containRh6 (R8ysubtype) (33, 34). To assess the contribution of R7 alone withoutaffecting R8, we silenced R7 cells in the adult after they had cor-rectly instructed R8p cells. We used the Gal4/UAS system (39) toswitch off R7 cells using a late R7-specific driver (panR7-Gal4) (40)and, as effector, the temperature-sensitive semidominant allele ofshibire (UAS-shits) (41, 42) that switches off synaptic transmission inphotoreceptors (43). The result for panR7 > shits flies was not sig-nificantly different from that of sev flies: When R7 cells wereblocked in this way, flies showed a strong preference for blue overUV (Fig. 2A), indicating that it is the impairment of R7 functionalone that results in this phenotype.

Mutants Defective in R8 Function Prefer UV in UV/B Choice. Next, weinvestigated the contribution of R8 in the UV/B choice. As expec-ted, all mutants defective for R8 function showed preference forUV over blue (Fig. 2B). Mutant rh52 flies completely lack Rh5expression (8) and thus have nonfunctional R8p cells but havenormal R8y cells. In the UV/B choice, rh52 flies preferred UV.Mutant rh61 flies lacking functional R8y cells also showed increasedUV preference. The behavior of rh52 rh61 double-mutant flies(Fig. 2B) was not significantly different from that of rh61 flies.The melted (melt) gene affects the specification of the R8p

subset (40). In flies homozygous for a melt gain-of-function allele(meltGOF), R8p cells are dramatically expanded at the expense ofR8y cells, without affecting R7 or R1–R6 (40). Rh6 is therefore

nearly absent and almost all R8 express blue-sensitive Rh5, incontrast to rh61 mutants in which only R8p cells express Rh5 andthe R7p cells are Q:11empty. Unexpectedly, meltGOF

flies also hadenhanced preference for UV over blue (Fig. 2B). This result doesnot fit the assumption of a simple addition of the responses fromeach of the photoreceptor types. Because R8p photoreceptors areabout 2.5 times as sensitive to blue light as R8y cells (Fig. 1C),transforming the 70% R8y into R8p cells in meltGOF

flies shouldlead to Q:12an increased sensitivity to blue light compared to blue-lightsensitivity in wild type. Yet, the preference was shifted toward theUV (see Discussion for possible interpretation).

Mutants Defective in Both R7 and R8 Prefer Blue in UV/B Choice.Impairing R7 or R8 function resulted in opposite phenotypes.Therefore, impairing bothR7 andR8might cancel out their effects.To test this, we examined mutants defective for both R7 and R8function. Because sev mutant flies lack R7 and have very few R8pcells, sev rh61 doublemutants have only very fewR8 cells expressingRh5 Q:13; therefore, the sev rh52 rh61 triple mutant should have nofunctional opsins expressed in the inner photoreceptors and thecontributions of R7 andR8 to the attractiveness function should belost (8). These flies showed a preference for blue over UV. In boththe double and triple mutants, the preference for blue was smallerthan in sevflies (Fig. 2C), indicating a contribution ofR8y cells in sevmutants. Thepreference forblue inmutants lackingbothR7andR8functions indicates a low weight of the R1–R6 subsystem in theattractiveness function and a prevailing contribution of the R7/R8subsystem for UV in wild type, as had been reported earlier (7, 23).We wondered whether the replacement of R8y by R8p cells in

the absence of R7 (sev meltGOF) would also enhance UV attrac-tiveness in the UV/B choice. In this case, however, the preferencefor blue overUVwas increased compared to sev or to panR7> shits

flies (P < 0.05) (Fig. 2C). The distinct phenotypes ofmeltGOF in thepresence and absence of R7 support the notion of a nonadditiveinteraction between R7 and R8. Photoreceptor R7 might, forexample, enhance the input ofR8 to the attractiveness function (butsee Discussion formeltGOF phenotype).Taken together, these results show that flies impaired for R7

function prefer blue and that flies impaired for R8 function preferUV. All flies lacking R7 function preferred blue regardless of R8function (Fig. 2 A and C), confirming that the contribution of R7cells is essential for UV attractiveness. Surprisingly, the effects ofreplacing R8y with R8p depended on R7 function. The UV/Bchoice behavior revealed an interaction between R7 and R8.

Mutants Defective in Photoreceptors R1–R6. Flies lacking R1–R6cells walk more slowly and exhibit severe defects in orientationbehavior (44) not observedwith any of theR7/R8mutants, makingthe direct comparison of these mutants somewhat difficult. Yet,the outer photoreceptors R1–R6 are not essential for phototaxis(32). We confirmed this result by finding that Q:14ninaE17 and rh1 >shits flies preferred blue to UV in the UV/B choice (Fig. 2D). Wecould assess the attractiveness function of these mutants becausetheir phototaxis scores were normal. Remarkably, both majorsubsystems, R1–R6 and R7/R8, showed a blue preference bythemselves whereas their combination in wild type led to a balancebetween UV and blue preferences, indicating that these systemscontribute nonadditively to the attractiveness function.These results also show that the R1–R6 subsystem does con-

tribute to the attractiveness function and is not switched off by theR7/R8 subsystem, as suggested by Jacob et al. (22). This conclusionwas strengthened by replacing Rh1 in R1–R6 by the UV-sensitiveopsin Rh3{ninaE17 P[rh1 > 3] (14)}. These flies behaved similarlyto wild type in the UV/B choice (Fig. 2D), showing that the pres-ence ofRh3 inR1–R6does provide a preference shift toward shortwavelengths as compared to flies lacking a functional R1–R6subsystem (ninaE17or rh1> shits flies). As these flies did not show apreference shift toward theUVcompared towild type, as would be

Fig. 2. UV/B choice tests. Pictograms below graphs represent the rhodopsincontentofeachphotoreceptor subtype (1–6 refer to rh1-rh6) for eachgenotype.R1–R6 are omitted in Figs. 2 and 3. (A) Contribution of R7. panR7-Gal4/UAS-shits

flies, inwhich R7 function is lost, preferred blue and control UAS-shits or panR7-Gal4 alone showed normal behavior. (B) Contribution of R8. All mutant fliesdefective for R8 (rh52, rh61, rh52 rh61, andmeltGOF) preferred UV. The behaviorof rh52 rh61 double-mutant flies was not significantly different from rh61 flies.(C) Mutant flies lacking R7 preferred blue. All mutant flies (sev, sev rh6, sev rh5rh6, and sev meltGOF) showed preference for blue. Distinct phenotypes ofmeltGOF were observed in the presence or absence of R7 (compare meltGOFin Band sev meltGOF in C). In B and C, results from each mutant were compared towild type inB. n.s, not significant; *P< 0.05, **P< 0.01, ***P< 0.001;n=5–6. (D)Contributionof the R1–R6 system indifferential phototaxis; both rh1-Gal4/UAS-shits and ninaE17flies preferred blue to UV, whereas ninaE17 P[rh1> 3] behavedsimilarly towild type. BothninaE17 and sev ninaE17flies preferred blue, withoutdetectable differences. BothninaE17 rh61andninaE17 rh52 rh61 showeda strongblue preference. n = 5–10. Error bars are SEMs.

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expected from the change in photopigment, these results again arenot compatible with a linear model.Interestingly, comparing ninaE17 to flies with only R8y photo-

receptors (sev ninaE17) did not reveal an effect of R7 cells. Bothstrains had the same blue preference in the UV/B choice (Fig. 2D),suggesting that without the R1–R6 subsystem the attractivenessfunction is dominated by theR8 photoreceptors: The contribution ofR7 to theattractiveness function seems to requirea functionalR1–R6system. If, however, in addition to R1–R6, R8y photoreceptors werealso inactivated (ninaE17, rh61), flies showed a very strong bluepreference, as did the flies of the triple mutant ninaE17, rh52, rh61. Inother words, with only R7 photoreceptors left, flies preferred blue intheUV/Bchoice, and thispreferencewasnotaffectedby thepresenceor the absence of input from the blue receptor R8p.

Contribution of Photoreceptors in B/G Choice Tests. In the B/Gchoice, the stimuli (blue peak wavelength 430 nm, green peakwavelength 565 nm; Fig. 1B,Lower) were chosen to characterize theattractiveness function in the spectral range of the two types of R8photoreceptors, R8p (blue) and R8y (green). The relative numbersof quanta captured by each subtype were estimated for the B/Gexperiments (Fig. 1C,Lower) in the sameway as forUV/B.WhereasR8p is expected to respond to bluemore than to green, the responseofR8y shouldbebiased towardgreen (Fig. 1C,Lower).This predictsthatmutants impaired inR8p should prefer green, whereasmutantsimpaired inR8y should prefer blue, again assuming that theR1–R6subsystem does not dominate the attractiveness function.Because R7 cells are mostly sensitive to UV, mutants defective

for R7 might show no preference shift in the B/G choice. To testthis, we used panR7 > shits flies tested at the restrictive tem-perature, which exhibited a weak preference for green over blue(Fig. 3A). Attractiveness of blue is likely mediated in part by R7ywhose spectrum of absorption overlaps with blue, but not withgreen (see also ninaE17 P[rh1 > 3] flies below).As shown above, in panR7 > shits flies, R8p and R8y almost

balanced each other in the B/G choice as they are sensitive toblue (Rh5) and to green (Rh6). We thus assayed their respectivecontribution. As predicted from Fig. 1C, rh52 mutant fliesshowed a preference for green (Fig. 3B). Consistent with the factthat sev flies lack all UV-sensitive R7 and most blue-sensitiveR8p, they showed a stronger preference for green over blue thanpanR7 > shits (P < 0.01) or rh52 alone (P < 0.001; Fig. 3 A and B).Both rh61 mutant and meltGOF

flies lack functional R8y but haveUV- and blue-sensitive photoreceptors, with meltGOF mutant fliesexhibiting an expansion of blue-sensitive Rh5 to all R8 cells (40).Both mutants showed a strong preference for blue (Fig. 3C). More-over, sev meltGOF

flies, which lacked R7 as well, still showed a strongpreference for blue, indicating that this preference is mostly due toR8p and that R7 cells contribute less in the B/G choice (Fig. 3C).Because loss of blue-sensitiveR8por green-sensitiveR8y results

in opposite phenotypes in B/G choice, we examined flies in whichboth R8 subtypes were nonfunctional (rh52, rh61 double mutant).These flies still preferred blue over green, similar to the rh61 singlemutant (Fig. 3D) and suggesting that R7 cells do contribute to thepreference for short wavelengths in these flies. To confirm this, weassayed the contribution of R7 in the absence of functional R8.Both sev rh61 double-mutant or sev rh52 rh61 triple-mutant flies, inwhich the function of both R7 and R8 is largely or completely lost,did not show a preference for either blue or green (Fig. 3D).Taken together, flies lacking R8p function preferred green,

whereas flies in which R8y function was impaired preferred blue. Itis also apparent that R8 cells play a major role in the B/G choicewhereas R7 cells play only a minor role. In the B/G choice, a sub-stantial shift to preference for blue lightQ:15 was observed for ninaE17,P[rh1> 3] (14) in comparison with ninaE17

flies (Fig. 3E; P< 0.05),demonstrating that Rh3 rhodopsin is sufficiently stimulated by bluelight in R1–R6 cells to contribute to the attractiveness function.

As in the UV/B choice, no differences were detected in the B/Gchoice between ninaE17 and sev ninaE17

flies. Both had a pro-nounced preference for green (Fig. 3E), suggesting that withoutR1–R6 the R7/R8 subsystem is dominated by R8 and in particularby R8y. No contribution of R7 to the attractiveness function wasdetectable under these conditions.Finally, flies that had R1–R6 as well as R8y (ninaE17, rh61) or

both types of R8 photoreceptors (ninaE17, rh52, rh61) inactivatedshowed a very strong blue preference as had been observed inthe UV/B choice for these genotypes. As the latter flies had onlyR7 photoreceptors left in the compound eyes, we assume that, inthe absence of functional R1–R6 and R8y photoreceptors, R7cells mediated this preference in phototaxis (Discussion).

DiscussionPhototaxis consists of at least three behavioral components: (i)detection of an object or a light source in visual space; (ii) theattractiveness or aversiveness of the light stimulus at this location;and (iii) a motor output, in our case goal-directed walking andturning. Our genetic dissection of the visual input to phototaxisrelies on the assumption that phototaxis stays intact even if some ofthese inputs are deleted, i.e., that the mutants can still detect thelocations of the two stimuli and move toward or away from them.Although inmutants affecting theR1–R6 subsystem,walking speed,turning, and orientation toward stationary objects are affected(44, 45), these behaviors are sufficiently intact to supportphototaxis.In our experiments, we chose light intensities such that all mutantshad at least one photoreceptor type that could detect the lightsources and guide walking and turning behavior. This allowed us tomeasure the second component, the differential attractiveness of

Fig. 3. B/G choice tests. (A) Contribution of R7; panR7-Gal4/UAS-shits fliesshowed preference for green. (B) Contribution of R8p. Flies in which R8pfunction is impaired (rh52and sev) preferredgreen. sevflies showeda strongerpreference for green over blue than panR7 > shits or rh52 alone. (C) Con-tribution of R8y. Flies in which R8y function is impaired (rh6,meltGOF, and sevmeltGOF) preferred blue. (D) Flies lacking functional R8; rh5 rh6 mutant fliesshowed a preference for blue, whereas sev rh6 and sev rh5 rh6flies showed noobvious preference. In B–D, the results from each mutant were compared tothe wild type in B. (E) Contribution of the R1–R6 system in differential pho-totaxis. BothninaE17 and sev ninaE17flies preferredgreen,without detectabledifferences. ninaE17 P[rh1 > 3] flies showed a preference for blue. As in UV/Bchoice, both ninaE17 rh61 and ninaE17 rh52 rh61 showed a strong blue pref-erence. n = 5–10. Error bars are SEMs.

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the two stimuli, which determined the choice between the twomonochromatic lights depending upon the photoreceptor typesavailable in the respective fly strains. Flies could judge the attrac-tiveness of a light source on the basis of its spectral composition and/or spectrally weighted brightness. In the following paragraphs wediscuss thecontributionsof the four typesofphotoreceptorsR1–R6,R7, R8p, and R8y to the attractiveness function.

All Four Photoreceptor Types Influence Attractiveness. Blocking orremoving any one of the photoreceptor types shifts the prefer-ence away from the point of neutrality in at least one of thechoice tests. Silencing the R1–R6 cells leaves the flies with amodest blue preference in the UV/B choice, and replacing theirbroadly sensitive opsin Rh1 by the UV-sensitive opsin Rh3 shiftsthe preference in the B/G choice to blue. Removing or silencingR7 in the presence of R1–R6 shifts the preference in the UV/Bchoice to blue and in the B/G choice to green. Inactivating thegreen-sensitive Rh6 opsin in R8y cells shifts the preference in theB/G choice to blue and in the UV/B choice to UV. Inactivatingblue-sensitive Rh5 in the R8p cells causes a green preference inthe B/G choice and a UV preference in the UV/B choice.

Independence of Subsystems. Each of the three photoreceptorsubsystems (R1–R6, R7, R8) alone can drive phototaxis. Flies withonly photoreceptors R1–R6 (sev rh52 rh61 triple mutants) show ablue preference in the UV/B choice, implying that this subsystemnot onlymediates optomotor responses andorientationbut also canmediate the attractiveness function inphototaxis, iQ:16 .e., that,withonlyR1–R6 photoreceptors, flies compare the quantum flux of two lightsources 180° apart.The R8 subsystem alone can also mediate the attractiveness

function in phototaxis (sev ninaE17), as had been shown before forthe sev rdgB double mutant (32). The sev ninaE17

flies have a pro-nounced green preference in the B/G choice test, consistent withthe absorption spectrumofRh6 expressed in all R8 photoreceptors.Flies that have only R7 photoreceptors operating (ninaE17, rh52,

rh61) show phototaxis. Surprisingly, they have a very strong pref-erence for blue in both choice tests. This could be explained ifQ:17 R7pinhibit R7y photoreceptors. Otherwise, the flies would show astrong UV preference in the UV/B choice. As an alternativeexplanation, however, it is possible that phototaxis is mediated bythe ocelli in flies with only R7 photoreceptors.

Nonlinear Interactions Between Subsystems. As long as only one ofthe central photoreceptor subsystems R7 or R8 is inactivated, theresults can be explained by a model in which the respective photo-receptors contribute roughly additively to the attractiveness func-tion. In the honeybee, all three subtypes of photoreceptors (UV,blue, and green) also feed into phototaxis (46, 47). Spectral mixingexperiments in phototaxis are consistent with simple summation ofquantal fluxes (47), and the action spectra of phototactic behavioralso suggest pooling of all three photoreceptor types. Color infor-mation, i.e., comparison between different photoreceptors, doesnot appear to be used in honeybee phototaxis (47).A simple summationmodel thus can no longer explain the results

of all of the genetic manipulations of photoreceptor typesin Drosophila presented here (7, 23). When all four photoreceptortypes are intact, UV light is more attractive than would be expectedfrom the sum of the UV attractiveness values of the two isolatedmajor subsystems,R1–R6andR7/R8. In theUV/Bchoice test that isbalanced for wild type, both flies with only the R1–R6 subsystem(sev rh52 rh61) and flies with only theR7/R8 subsystem (rh1> shits orninaE17) showabluepreference (Fig.2D).We thushave topostulatethat an interaction between the twomajor retinal subsystemsR1–R6and R7/R8 enhances UV attractiveness. The interaction cannot beexplained by an attenuation of the attractiveness of blue because noincreased blue preference of the twomutants is observed in the B/Gchoice (Fig. 3E). The interaction canunambiguously be traced to the

R7 cells. In fact, the strength of the contribution of R7 to theattractiveness function in the UV/B choice depends upon which ofthe other photoreceptor types are functional. We cannot detect anyeffect of R7 on spectral preference without a functional R1–R6subsystem (comparing ninaE17 and sev ninaE17 in both choice tests;Fig. 2D). We find a moderate effect in the presence of R1–R6 andR8 (comparing wild type and sev; Fig. 2A andC) whereas the effectof R7 is large in the absence of the R8 subsystem (comparing rh52

rh61 and sev rh52 rh61 in UV/B choice; Fig. 2 B and C).The most parsimonious explanation of our data are to assume

that, in thepresenceofa functionalR8subsystem,neither theR1–R6nor the R7 subsystems on their own have a direct input to theattractiveness function (Fig. 4 and Fig. S1) Q:18. In the absence of R7(mutant sev), the R1–R6 subsystem seems not to matter for attrac-tiveness although the R8 subsystem is lacking spectral sensitivity inthe UV. Likewise, in the absence of a functional R1–R6 subsystem,the R7 subsystem seems not to contribute. Interestingly, as soon asR8y photoreceptors (or R8p andR8y) are inactivated, the other twosubsystems exert their influence on the attractiveness functionindependently of each other. A full model of these interactionswould require a more complete investigation.R1–R6 rhabdomeres degenerate in ninaE17 mutants (48), and

this degeneration may have secondary effects on the R7/R8 cells.However, as shown in Fig. 2D, there is no significant differencebetween ninaE17 mutants and flies expressing rh1>shits (P > 0.05),inwhichR1–R6 function is disruptedwithout affecting rhabdomerestructures. Moreover, it was recently reported that R8y are func-tional in ninaE17 mutants because circadian entrainment to redlight, which is still observed in ninaE17, is abolished in ninaE17 rh61

double mutants (49).Our data reveal a further deviation from a simple summation of

photoreceptor inputs to the attractiveness function. In themeltGOF

mutant, green-sensitive R8y cells are transformed into blue-sensi-tiveR8p cells. This should shift their preference in theUV/B choiceto blue.Yet, aUVshift is observed. In contrast, in the absence ofR7cells, the transformation of R8y to R8p photoreceptors (compar-ison between sev and sev meltGOF) increases the blue preference inthe UV/B choice as would be expected from spectral sensitivities(Fig. 1C). This might indicate that R8p cells enhance the input oftheUVchannel (R1–R6×R7) to the attractiveness function. Thesefindings, however, should be treated with caution as unknowndevelopmental defects in the sev and meltGOF mutants mightaccount for the phenotypes (50).Using a different phototaxis paradigm, Jacob et al. (22) pro-

posed a model to explain the nonadditive effects observed intheir phototaxis experiments. They postulated an inhibition ofR1–R6 by R7/R8 and suggested that R7 cells function only whenthe R1–R6 system is intact. Our results ask for revision of thismodel. We found no general inhibition of the R1–R6 receptorsubsystem by R7/R8. Our data suggest that neither the R1–R6nor the R7 subsystems have access to the attractiveness functionon their own, except in the absence of functional R8y photo-receptors (Fig. 3D). Moreover, R8p cells in our tests had aneffect only in the presence of functional R8y cells.Rh3-expressing R8 cells in the dorsal rim area account for about

10%ofallRh3expressing cells.TheseR8are still present in sevflies,but are switched off in panR7> shits flies. The comparison of thesetwo lines does not reveal an effect of theRh3-expressingR8 cells inthe UV/B choice. Taking these cells into consideration thereforedoes not change any of the above conclusions.

Concluding Remarks. We have shown that all four photoreceptortypes [R1–R6,R7(p, y),R8p, andR8y] are involved inphototaxis, incontrast to motion detection, which relies exclusively on R1–R6photoreceptors (7, 8). The wild-type attractiveness function cannotbe described as the sum of the attractiveness functions of flieslacking one or more functional photoreceptor types. We observe amultiplicative interaction between photoreceptors R1–R6 and R7.

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In the absence of functional R1–R6 or R7, the attractivenessfunction is governed byR8.Only in the absence ofR8 andoneof theother two subsystems does the remaining subsystem govern theattractiveness function. A recent study on the neuronal substrate ofspectral preference identified postsynaptic interneurons in themedulla (51) that are good candidates for mediating some of theseinteractions. Their involvement in differential phototaxis can nowbe tested.The attractiveness function of differential phototaxis is easy to

record, robust, and sensitive (for example, Rh5-expressing blue-sensitive cells account for only ∼4% of all photoreceptors, yetyield a significant phenotype when they are not functional). Theattractiveness function may lend itself to mutant screens affect-ingQ:19 other chromaticity computations in the brain, including colorvision, at the circuit level.

Materials and MethodsFly Stocks. Fly stocks were raised as described previously (8). See SI Materialsand Methods for more details. Mutant lines used are as follows: rh52 (8), rh61

(52), sevLY3, UAS-lacZ[melt] (meltGOF) (40), ninaE17 (Berlin background), andninaE17 P[rh1 > 3] (14). Gal4 driver and effector lines used were panR7-Gal4driver (40), rh1-Gal4 driver (43), and UAS-shibirets1 (41, 42)

Behavioral Assays. Flies that were 2–7 days old were used. Two fresh plastictubes (Falcon 352017) were attached to the T-maze, which is similar to the oneused for theolfactory paradigm (37) but is black toavoid light transmission. Flies(50–150) were put in the hole of the lift, the light stimuli were turned on, andthen the lift was opened for 20 s. The number of flies found in each tube was

counted. The PI was calculated as described in Results. See SI Materials andMethods for details about “light vs. dark” experiments and shibire experiments.

LEDs and Spectroradiometry. The LEDs used in this study are the following: UV(RT350-05–15; Roithner Laser Technik), blue (LED435-12–30; Roithner LaserTechnik), and green (E1503SGC, eLED). The relative quantal emission andspectral curves of the LEDs were measured with the spectroradiometer, asdescribed previously (8). The relative quantum capture of each subclass ofphotoreceptors is shown in Fig. 1C for UV/B (Upper) or for B/G (Lower). See SIMaterials and Methods for more details.

Statistical Analyses. For comparison between genotypes, Kruskal–Wallis testwas performed to detect overall significance. For pair-wise comparisons, theMann–Whitney U test was performed. For all multiple comparisons, Bonfer-roni correction was applied. In Figs. 1–3, one Q:20, two, and three asterisks indicatean α-level of 0.05, 0.01, and 0.001, respectively. The significance before Bon-ferroni correction is indicated in parentheses. Error bars are SEMs.

ACKNOWLEDGMENTS. We thank Konrad Öchsner and Daniel Vasiliauskasfor building the experimental apparatus; Terry Blackman for help with theflies; Reinhard Wolf for helpful advice and constant support; Roger Hardiefor providing data for the normalized spectral properties of each photo-receptor subtype; and people in the Desplan and Heisenberg laboratory,in particular Nina Vogt for establishing the rh52 rh61 ninaE17 mutant andJens Rister for helpful discussion. We thank Tetsuya Tabata for his kindsupport and the people in his laboratory, in particular Yuko Maeyama-Kamoshida for her generous assistance and Satoshi Murakami for his help.We thank Shuji Hanai for providing us with the rh61 ninaE17 double mutant.Funding for this work was provided by the Uehara memorial foundationpostdoctoral fellowship and a Human Frontier Science Program Q:21postdoctoralfellowship (to S.Y.), National Institutes of Health Grant EY017916 (to C.D.),and Sonder-Forschungs-Bereich Grant 554 (to M.H.).

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Supporting InformationYamaguchi et al. 10.1073/pnas.0809398107SI Materials and MethodsFly Stocks. Fly stocks were raised on standard medium under aconstant light and dark cycle (14 h/10 h). Flies were raised at 25 °C,60% relative humidity, unless described otherwise. Wild-typeCanton-S (CS) and Berlin were used, and no significant differencewas observed between the twowild types in the assays.Mutants arein a CS background unless stated otherwise. All flies are wild typefor eye color.

Behavioral Assays.Preferenceindex(PI)wascalculatedasdescribedin Results. The PI ranged from −1 to 1; when all flies preferredlonger wavelength (blue > UV or green > blue), PI was 1. Whenthere was no preference (i.e., flies distribute 50:50), PI was 0, andwhen all flies preferred the shorter wavelength, PI was −1.In the “light vs. dark” experiments, flies were placed in a T-maze

with two tubes, with a light on one side and no light on the other.After the flies were placed at the choice point of the T-maze, theymoved toward the illuminated tube when the light intensity wasabove the phototaxis threshold.shibire experiments were performed as follows: UAS-shits flies

(41,42) were raised at 18 °C until adulthood. Flies were in-cubated at 34–36 °C for 30 min before the experiments. Ex-periments were performed at 34–36 °C.

LEDs and Spectroradiometry. An OceanOptics USB2000 spectror-adiometer was used. The relative irradiance and spectral curveswere measured for UV, blue, and green LEDs. The spectroradi-ometer was calibrated using a standard calibration lamp (LS-1-CAL; OceanOptics) for measuring absolute intensities of visible(blue and green) stimuli. Because the calibration underestimatesthe irradiance of UV light, a photomultiplier (international light,PM270/IL700) was additionally used for the correction of UVirradiance. The irradiance ratio between UV and blue LEDs was

measuredby thephotomultiplier, andthespectral curvesmeasuredby the spectroradiometer were corrected accordingly.The relativesensitivity of each subclass of photoreceptors is shown in Fig. 1C,which indicates the relative number of quanta captured by eachphotoreceptor subtype for UV/B (Upper) or for B/G (Lower). Itwas calculated as follows: the spectral curves of LEDsmeasured byspectroradiometer were corrected by the photomultiplier to esti-mate the precise irradiance ratio between UV/B LEDs (Fig. 1B,Upper) and B/G LEDs (Fig. 1B, Lower). The relative sensitivity ofeach photoreceptor subtype in Fig. 1Awas taken from refs. 1 and 2and R. Hardie (personal communication). For each PR subtype,the PR sensitivity was multiplied by the relative irradiance at eachwavelength for each LED, and the value was integrated over Q:1allwavelengths.Intensities of the LEDs were adjusted electronically. The

intensities used are as follows: UV—2.1 × 1012 quanta Q:2·cm−2·s−1;blue—1.2 × 1014 quanta·cm−2·s−1 for UV/B experiments; blue—1.4×1012 quanta·cm−2·s−1; and green—1.1× 1013 quanta·cm−2·s−1

for B/G experiments.

Calculating an Additive Model. For each genotype, the expected rel-ative sensitivities for UV, blue, and green can be calculated byassuming additive contributions and by choosing appropriate weightfactors for the photoreceptor types. Taking into account the 30:70 p:yratiosinR7andR8aswellastheratioofinner-to-outerphotoreceptors(1:6), one would arrive at a sensitivity of SWT = [a*R7p*0.3 +b*R7y*0.7+ c*R8p*0.3+d*R8y*0.7+e*(R1–R6)*6] for wild typeand Smelt = [a*R7p*0.3 + b*R7y*0.7 + c*R8p*1 + e*(R1–R6)*6]for meltGOF

flies. The R values (e.g., R7p) would represent the sen-sitivities of the photoreceptor types as shown in Fig. 1C, the factors(a–e) would give their relative weights, and the numbers at the end ofeach term would give their relative abundance in the eye.

1. Salcedo E, et al. (1999) Blue- and green-absorbing visual pigments of Drosophila:Ectopic expression and physiological characterization of the R8 photoreceptor cell-specific Rh5 and Rh6 rhodopsins. J Neurosci 19:10716–10726.

2. Govardovskii VI, Fyhrquist N, Reuter T, Kuzmin DG, Donner K (2000) In search of thevisual pigment template. Vis Neurosci 17:509–528.

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R1-

6, R

8p, R

8y

R1-

6

R1-

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C

Fig. S1. Effects of adding photoreceptor subsystems on spectral preference in differential phototaxis were calculated by subtracting the score of one mutantfrom that of another. The other photoreceptor types present in the respective flies are indicated beneath the columns. (A) Effects of adding R7 photoreceptors.(B) Adding R1–R6. (C) Adding R8.

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Q: 1_"integrated over all wavelengths" ok as edited?

Q: 2_Use of multidots as meant?

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