Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Molecular logic behind the three-way stochastic choices that expand butterfly colour vision

Abstract

Butterflies rely extensively on colour vision to adapt to the natural world. Most species express a broad range of colour-sensitive Rhodopsin proteins in three types of ommatidia (unit eyes), which are distributed stochastically across the retina1,2,3. The retinas of Drosophila melanogaster use just two main types, in which fate is controlled by the binary stochastic decision to express the transcription factor Spineless in R7 photoreceptors4. We investigated how butterflies instead generate three stochastically distributed ommatidial types, resulting in a more diverse retinal mosaic that provides the basis for additional colour comparisons and an expanded range of colour vision. We show that the Japanese yellow swallowtail (Papilio xuthus, Papilionidae) and the painted lady (Vanessa cardui, Nymphalidae) butterflies have a second R7-like photoreceptor in each ommatidium. Independent stochastic expression of Spineless in each R7-like cell results in expression of a blue-sensitive (SpinelessON) or an ultraviolet (UV)-sensitive (SpinelessOFF) Rhodopsin. In P. xuthus these choices of blue/blue, blue/UV or UV/UV sensitivity in the two R7 cells are coordinated with expression of additional Rhodopsin proteins in the remaining photoreceptors, and together define the three types of ommatidia. Knocking out spineless using CRISPR/Cas9 (refs 5, 6) leads to the loss of the blue-sensitive fate in R7-like cells and transforms retinas into homogeneous fields of UV/UV-type ommatidia, with corresponding changes in other coordinated features of ommatidial type. Hence, the three possible outcomes of Spineless expression define the three ommatidial types in butterflies. This developmental strategy allowed the deployment of an additional red-sensitive Rhodopsin in P. xuthus, allowing for the evolution of expanded colour vision with a greater variety of receptors7,8. This surprisingly simple mechanism that makes use of two binary stochastic decisions coupled with local coordination may prove to be a general means of generating an increased diversity of developmental outcomes.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Rhodopsin expression and ommatidial types in the P. xuthus retina.
Figure 2: Stochastic Spineless expression in flies and butterflies.
Figure 3: Targeted CRISPR/Cas9 knockout of yellow and spineless.
Figure 4: Outcome of spineless knockout and coordination of ommatidial fate.

Accession codes

Primary accessions

Sequence Read Archive

Data deposits

Data was deposited at SRA under accession code SRP071804.

References

  1. 1

    Arikawa, K. Spectral organization of the eye of a butterfly, Papilio. J. Comp. Physiol. A Neuroethol. Sens. Neural Behav. Physiol. 189, 791–800 (2003)

    CAS  Article  Google Scholar 

  2. 2

    Briscoe, A. D. Reconstructing the ancestral butterfly eye: focus on the opsins. J. Exp. Biol. 211, 1805–1813 (2008)

    CAS  Article  Google Scholar 

  3. 3

    Wernet, M. F., Perry, M. W. & Desplan, C. The evolutionary diversity of insect retinal mosaics: common design principles and emerging molecular logic. Trends Genet. 31, 316–328 (2015)

    CAS  Article  Google Scholar 

  4. 4

    Wernet, M. F. et al. Stochastic spineless expression creates the retinal mosaic for colour vision. Nature 440, 174–180 (2006)

    CAS  ADS  Article  Google Scholar 

  5. 5

    Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012)

    CAS  ADS  Article  Google Scholar 

  6. 6

    Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013)

    CAS  ADS  Article  Google Scholar 

  7. 7

    Briscoe, A. D. Six opsins from the butterfly Papilio glaucus: molecular phylogenetic evidence for paralogous origins of red-sensitive visual pigments in insects. J. Mol. Evol. 51, 110–121 (2000)

    CAS  ADS  Article  Google Scholar 

  8. 8

    Koshitaka, H., Kinoshita, M., Vorobyev, M. & Arikawa, K. Tetrachromacy in a butterfly that has eight varieties of spectral receptors. Proc. R. Soc. B 275, 947–954 (2008)

    Article  Google Scholar 

  9. 9

    Franceschini, N., Kirschfeld, K. & Minke, B. Fluorescence of photoreceptor cells observed in vivo. Science 213, 1264–1267 (1981)

    CAS  ADS  Article  Google Scholar 

  10. 10

    Rister, J. & Desplan, C. The retinal mosaics of opsin expression in invertebrates and vertebrates. Dev. Neurobiol. 71, 1212–1226 (2011)

    CAS  Article  Google Scholar 

  11. 11

    Chou, W. H. et al. Patterning of the R7 and R8 photoreceptor cells of Drosophila: evidence for induced and default cell-fate specification. Development 126, 607–616 (1999)

    CAS  PubMed  Google Scholar 

  12. 12

    Johnston, R. J. Jr et al. Interlocked feedforward loops control cell-type-specific Rhodopsin expression in the Drosophila eye. Cell 145, 956–968 (2011)

    CAS  Article  Google Scholar 

  13. 13

    Papatsenko, D., Sheng, G. & Desplan, C. A new rhodopsin in R8 photoreceptors of Drosophila: evidence for coordinate expression with Rh3 in R7 cells. Development 124, 1665–1673 (1997)

    CAS  PubMed  Google Scholar 

  14. 14

    Kitamoto, J., Sakamoto, K., Ozaki, K., Mishina, Y. & Arikawa, K. Two visual pigments in a single photoreceptor cell: identification and histological localization of three mRNAs encoding visual pigment opsins in the retina of the butterfly Papilio xuthus. J. Exp. Biol. 201, 1255–1261 (1998)

    CAS  PubMed  Google Scholar 

  15. 15

    Kinoshita, M. & Arikawa, K. Color and polarization vision in foraging Papilio. J. Comp. Physiol. A Neuroethol. Sens. Neural Behav. Physiol. 200, 513–526 (2014)

    Article  Google Scholar 

  16. 16

    Friedrich, M., Wood, E. J. & Wu, M. Developmental evolution of the insect retina: insights from standardized numbering of homologous photoreceptors. J. Exp. Zool. B Mol. Dev. Evol. 316B, 484–499 (2011)

    Article  Google Scholar 

  17. 17

    Mollereau, B. et al. Two-step process for photoreceptor formation in Drosophila. Nature 412, 911–913 (2001)

    CAS  ADS  Article  Google Scholar 

  18. 18

    Takemura, S.-Y., Kinoshita, M. & Arikawa, K. Photoreceptor projection reveals heterogeneity of lamina cartridges in the visual system of the Japanese yellow swallowtail butterfly, Papilio xuthus. J. Comp. Neurol. 483, 341–350 (2005)

    Article  Google Scholar 

  19. 19

    Xie, B., Charlton-Perkins, M., McDonald, E., Gebelein, B. & Cook, T. Senseless functions as a molecular switch for color photoreceptor differentiation in Drosophila. Development 134, 4243–4253 (2007)

    CAS  Article  Google Scholar 

  20. 20

    Cook, T., Pichaud, F., Sonneville, R., Papatsenko, D. & Desplan, C. Distinction between color photoreceptor cell fates is controlled by Prospero in Drosophila. Dev. Cell 4, 853–864 (2003)

    CAS  Article  Google Scholar 

  21. 21

    Arikawa, K., Mizuno, S., Kinoshita, M. & Stavenga, D. G. Coexpression of two visual pigments in a photoreceptor causes an abnormally broad spectral sensitivity in the eye of the butterfly Papilio xuthus. J. Neurosci. 23, 4527–4532 (2003)

    CAS  Article  Google Scholar 

  22. 22

    Thanawala, S. U. et al. Regional modulation of a stochastically expressed factor determines photoreceptor subtypes in the Drosophila retina. Dev. Cell 25, 93–105 (2013)

    CAS  Article  Google Scholar 

  23. 23

    Burgess, E. A. & Duncan, I. Direct control of antennal identity by the spineless-aristapedia gene of Drosophila. Mol. Gen. Genet. 221, 347–357 (1990)

    CAS  Article  Google Scholar 

  24. 24

    Kitamoto, J., Ozaki, K. & Arikawa, K. Ultraviolet and violet receptors express identical mRNA encoding an ultraviolet-absorbing opsin: identification and histological localization of two mRNAs encoding short-wavelength-absorbing opsins in the retina of the butterfly Papilio xuthus. J. Exp. Biol. 203, 2887–2894 (2000)

    CAS  PubMed  Google Scholar 

  25. 25

    Arikawa, K. & Stavenga, D. Random array of colour filters in the eyes of butterflies. J. Exp. Biol. 200, 2501–2506 (1997)

    CAS  PubMed  Google Scholar 

  26. 26

    Arikawa, K. et al. An ultraviolet absorbing pigment causes a narrow-band violet receptor and a single-peaked green receptor in the eye of the butterfly Papilio. Vision Res. 39, 1–8 (1999)

    CAS  Article  Google Scholar 

  27. 27

    Tomlinson, A., Bowtell, D. D. L., Hafen, E. & Rubin, G. M. Localization of the sevenless protein, a putative receptor for positional information, in the eye imaginal disc of Drosophila. Cell 51, 143–150 (1987)

    CAS  Article  Google Scholar 

  28. 28

    Ready, D. F. A multifaceted approach to neural development. Trends Neurosci. 12, 102–110 (1989)

    CAS  Article  Google Scholar 

  29. 29

    Wakakuwa, M., Kurasawa, M., Giurfa, M. & Arikawa, K. Spectral heterogeneity of honeybee ommatidia. Naturwissenschaften 92, 464–467 (2005)

    CAS  ADS  Article  Google Scholar 

  30. 30

    Rister, J., Desplan, C. & Vasiliauskas, D. Establishing and maintaining gene expression patterns: insights from sensory receptor patterning. Development 140, 493–503 (2013)

    CAS  Article  Google Scholar 

  31. 31

    Hsiao, H. Y. et al. Dissection and immunohistochemistry of larval, pupal and adult Drosophila retinas. J. Vis. Exp. http://dx.doi.org/10.3791/4347 (2012)

  32. 32

    Stoehr, A. M., Walker, J. F. & Monteiro, A. Spalt expression and the development of melanic color patterns in pierid butterflies. Evodevo 4, 6 (2013)

    Article  Google Scholar 

  33. 33

    de Celis, J. F., Barrio, R. & Kafatos, F. C. Regulation of the Spalt/Spalt-related gene complex and its function during sensory organ development in the Drosophila thorax. Development 126, 2653–2662 (1999)

    CAS  PubMed  Google Scholar 

  34. 34

    Chen, B. et al. Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system. Cell 155, 1479–1491 (2013)

    Article  Google Scholar 

  35. 35

    Arikawa, K., Scholten, D. G. W., Kinoshita, M. & Stavenga, D. G. Tuning of photoreceptor spectral sensitivities by red and yellow pigments in the butterfly Papilio xuthus. Zoolog. Sci. 16, 17–24 (1999)

    Article  Google Scholar 

  36. 36

    Kinoshita, M., Shimohigasshi, M., Tominaga, Y., Arikawa, K. & Homberg, U. Topographically distinct visual and olfactory inputs to the mushroom body in the Swallowtail butterfly, Papilio xuthus. J. Comp. Neurol. 523, 162–182 (2015)

    Article  Google Scholar 

Download references

Acknowledgements

We thank members of the Desplan and Arikawa laboratories for discussion, and especially M. Wernet and J. Rister for suggestions. We thank M. Friedrich for clarifying insect eye homologies, A. Stolfi for discussing CRISPR/Cas9 protocols, C. Merlin for discussing butterfly injection technique, and J. Bothma for discussion. We thank A. Monteiro for providing anti-Sal and K. Shi at Genscript for help with antibody design. This work was supported by NIH grant EY13010 and the Center for Genomics and Systems Biology of NYU Abu Dhabi to C.D., and the JSPS Kakenhi grant numbers 26251036 and 20167232 to K.A. M.P. was supported by an NIH Ruth L. Kirschstein NRSA, a JSPS Short Term Fellowship award, and the Revson Biomedical Research Foundation Postdoctoral Fellowship.

Author information

Affiliations

Authors

Contributions

M.P. and C.D. jointly conceived the project with input from K.A. M.P., C.D., and K.A. designed experiments. M.P., M.K., and L.H. performed experiments. G.S. was responsible for genomic data analysis and de novo assembly. C.D. and K.A. supervised the project. M.P., K.A., and C.D. wrote the paper. All authors discussed the results and manuscript.

Corresponding author

Correspondence to Claude Desplan.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Additional expression data and CRISPR/Cas9 spineless knock out results for V. cardui.

a, Antibodies to P. xuthus Pros and Dve (PxPros and PxDve) cross-react in V. cardui. As in P. xuthus, Pros labels two R7-like photoreceptors per ommatidium (green) while Dve labels bR3–8, the SVF photoreceptors equivalent to the outer photoreceptors of D. melanogaster (red), along with a stochastic subset of Pros-expressing R7-like PRs (yellow co-expression). b, Co-expression of Dve in Pros-positive R7-like photoreceptors is lost in spineless CRISPR/Cas9 knockout tissue; compare to yellow co-expression in wild-type in a. c, Sal antibodies label three photoreceptors per ommatidium in V. cardui (red). d, As in P. xuthus, Spineless antibodies (magenta) label a stochastic subset of R7-like Pros-expressing photoreceptors (green).

Extended Data Figure 2 Pleiotropic effects of the spineless mutation and sequencing of CRISPR/Cas9 generated mutations.

a, b, The spineless mutation affects antennal development in P. xuthus (a) and V. cardui (b), as shown previously for D. melanogaster. This effect is first visible at pupal stages, where missing/shortened antennae are absent in their normal channels (red arrow) and shortened structures protrude in their place (blue arrow). Strongly affected individuals have little to no remaining antennae in adult stages (at right). c, The spineless mutation affects bristle development in V. cardui, as shown previously for D. melanogaster, where some bristles are missing or reduced. in the left image, the regular comb of bristles on the tibia is interrupted in mosaic spineless mutant adults, or almost completely missing (right image). d, Mutation of spineless produced an unexpected effect on wing colour pattern. The wing of an animal injected with guide RNAs targeting both spineless and yellow during the late blastoderm stage (7–9 h after egg lay) is shown on the left. A brown-coloured yellow mutant patch of tissue is visible (yellow arrowhead), as shown in the yellow mutation in Fig. 3, but lighter patches of wing scales lacking both melanin (black) and ommachromes (oranges) are also visible, example marked with a white arrow. Similar clones were observed when only spineless sgRNAs were injected (middle and right). This effect was observed for two independent sgRNAs targeting Spineless. e, Cloning and sequencing of target regions from mutant tissues reveals a mixture of CRISPR/Cas9 generated mutations. Unmodified cDNA sequences are shown on the top line for comparison.

Extended Data Table 1 Specific probabilities of Spineless expression yield specific ratios of ommatidial type

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Perry, M., Kinoshita, M., Saldi, G. et al. Molecular logic behind the three-way stochastic choices that expand butterfly colour vision. Nature 535, 280–284 (2016). https://doi.org/10.1038/nature18616

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing