The Hows and Whys of Wing Transparency in Mimetic Lepidoptera

The hows and whys of wing transparency in mimetic Lepidoptera

Charline2018 © II PinnaJoint Congress, onAaron Evolutionary BiologyPomerantz 2018. All rights reserved, Serge - Any reproduction Berthier, even in part is prohibited.Christine Andraud, Doris Gomez, Nipam Patel and Marianne Elias

II Joint Congress on Evolutionary Biology 19th August 2018

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ODONATA

ORTHOPTERA

HEMIPTERA

DIPTERA 2018 © II Joint Congress on Evolutionary Biology 2018. All rights reserved - Any reproduction even in part is prohibited. 1 2018 © 48th European Contact Lens Society Of Ophthalmologists. All rights reserved - Any reproduction even in part is prohibited] .

ODONATA

ORTHOPTERA

HEMIPTERA

LEPIDOPTERA

ODONATA

ORTHOPTERA

HEMIPTERA

LEPIDOPTERA

PIERIDAE

NYMPHALIDAE

RIODINIDAE LEPIDOPTERA

SPHINGIDAE

SATURNIIDAE

EREBIDAE

NOCTUIDAE 2018 © II Joint Congress on Evolutionary Biology 2018. All rights reserved - Any reproduction even in part is prohibited. 2 2018 © 48th European Contact Lens Society Of Ophthalmologists. All rights reserved - Any reproduction even in part is prohibited] .

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= Yuck!

Pattern convergence in the same locality

Co-mimics of the mothone mimicry ring

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Pattern convergence in the same locality

Co-mimics of the mothone mimicry ring Co-mimics of the confusa mimicry ring

1) How is transparency achieved? Is there a link between structures and optical properties?

2018 © II Joint Congress on Evolutionary Biology 2018. All rights reserved - Any reproduction even in part is prohibited. 5 2018 © 48th European Contact Lens Society Of Ophthalmologists. All rights reserved - Any reproduction even in part is prohibited] .

1) How is transparency achieved? Is there a link between structures and optical properties?

2) How the diverse structures involved in transparency are generated during development ?

1) How is transparency achieved? Is there a link between structures and optical properties?

2) How the diverse structures involved in transparency are generated during development ?

3) Beyond general pattern perception, are transparent zones perceived similarly by predators? 2018 © II Joint Congress on Evolutionary Biology 2018. All rights reserved - Any reproduction even in part is prohibited.

confusa mimicry 2018 © II Joint Congress on Evolutionary Biologyring 2018. All rights reserved - Any reproduction even in part is prohibited. 5 2018 © 48th European Contact Lens Society Of Ophthalmologists. All rights reserved - Any reproduction even in part is prohibited] .

1) How is transparency achieved? Is there a link between structures and optical properties?

2) How the diverse structures involved in transparency are generated during development ?

Comparative study of : - 57 species, - belonging to 6 families - distributed among 10 mimicry rings confusa mimicry 2018 © II Joint Congress on Evolutionary Biologyring 2018. All rights reserved - Any reproduction even in part is prohibited. 5 2018 © 48th European Contact Lens Society Of Ophthalmologists. All rights reserved - Any reproduction even in part is prohibited] .

2018 © II Joint Congress on Evolutionary Biology 2018. All rights reserved - Any reproduction even in part is prohibited. 6 2018 © 48th European Contact Lens Society Of Ophthalmologists. All rights reserved - Any reproduction even in part is prohibited] .

Noctuidae 1 cm AGNOSIA BLUE Erebidae

1 cm Pieridae CONFUSA BANJANA-M

Riodinidae

EURIMEDIA AURELIANA

THEUDELINDA Nymphalidae PANTHYALE

HEWITSONI 1 cm LERIDA

Spectrogoniometre Computer

Xenon Lamp Transmitting fibre Collecting fibre 75W Spectro

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Spectrogoniometre Computer

Xenon Lamp Transmitting fibre Collecting fibre 75W Spectro

Spectra analyses 100

2018 © II Joint Congress on Evolutionary Biology 2018. All rights reserved - Any reproduction even in part is prohibited. Transmission (%) Transmission 0 300 400 700 UV Humain visible spectrum Wavelength (nm)2018 © II Joint Congress on Evolutionary Biology 2018. All rights reserved - Any reproduction even in part is prohibited. 7 2018 © 48th European Contact Lens Society Of Ophthalmologists. All rights reserved - Any reproduction even in part is prohibited] .

Spectrogoniometre Computer

Xenon Lamp Transmitting fibre Collecting fibre 75W Spectro

Spectra analyses 100

Brightness = area under the curve Transmission (%) Transmission 0 300 400 700 UV Humain visible spectrum Wavelength (nm)2018 © II Joint Congress on Evolutionary Biology 2018. All rights reserved - Any reproduction even in part is prohibited. 7 2018 © 48th European Contact Lens Society Of Ophthalmologists. All rights reserved - Any reproduction even in part is prohibited] .

Scale type Scale color

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Scale type Scale color Scale insertion Scale length Scale width Scale density

Membrane thickness

Godyris panthyale 60 (%) 40 Brightness

20 Methona curvifascia

0 Flat scale Erected Hair-like scale scale

Coverage decreases Vila azeca cacica

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Godyris panthyale 60 (%) 40 Brightness

20 Methona curvifascia

0 Flat scale Erected Hair-like scale scale

Coverage decreases Vila azeca cacica

➔ The less covered the membrane, the more transparent the wing 2018 © II Joint Congress on Evolutionary Biology 2018. All rights reserved - Any reproduction even in part is prohibited. 9 2018 © 48th European Contact Lens Society Of Ophthalmologists. All rights reserved - Any reproduction even in part is prohibited] .

1 µm Godyris panthyale 60 Greta ortygia (%) (%) 40 Brightness birgthness 20 1 µmMethona curvifascia Hyalurga grandis Mean Flat scale 2018 © II Joint Congress on Evolutionary Biology 2018. All rights reserved - Any reproduction even in part is prohibited. Mean Mean Erected scale Hair-like scale 0 Flat scale Erected Hair-like scale scale Nanostructures density (nb/µm) Coverage decreases Lycorea ilione 1 µmVila azeca cacica ➔ The less covered the membrane, the more transparent the wing 2018 © II Joint Congress on Evolutionary Biology 2018. All rights reserved - Any reproduction even in part is prohibited. 9 2018 © 48th European Contact Lens Society Of Ophthalmologists. All rights reserved - Any reproduction even in part is prohibited] .

transparency

1 µm Godyris panthyale 60 Greta ortygia (%)

40 (%) Brightness birgthness 20 1 µmMethona curvifascia Hyalurga grandis Mean Flat scale 2018 © II Joint Congress on Evolutionary Biology 2018. All rights reserved - Any reproduction even in part is prohibited. Mean Mean Erected scale Hair-like scale 0 Flat scale Erected Hair-like scale scale Nanostructures density (nb/µm) Coverage decreases Lycorea ilione 1 µmVila azeca cacica ➔ The less covered the membrane, the more transparent the wing ➔ The denser the nanostructures, the more transparent the wing2018 © II Joint Congress on Evolutionary Biology 2018. All rights reserved - Any reproduction even in part is prohibited. 9 2018 © 48th European Contact Lens Society Of Ophthalmologists. All rights reserved - Any reproduction even in part is prohibited] .

Cephonodes hylas 1 µm Without nanostructures Transmission -

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Cephonodes hylas 1 µm Without nanostructures Transmission -

➔ Nanostructures act as antireflective device, thereby increasing2018transparency © II Joint Congress on Evolutionary Biology 2018. All rights reserved - Any reproduction even in part is prohibited. 10 2018 © 48th European Contact Lens Society Of Ophthalmologists. All rights reserved - Any reproduction even in part is prohibited] .

Material & Methods N. Patel Injection on different dose of heparin, a protein enablingWnt diffusion in pupae near developpingwings A. Pomerantz

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2018 © II Joint Congress on Evolutionary Biology 2018. All rights reserved - Any reproduction even in part is prohibited] . Material Transparency Transparency Transparency Introduction Conclusion & Methods & Structures & Development & Mimicry How is the position of transparent zones determined during development ? WT [Heparin] increases Material & Methods N. Patel 1 2 Injection on different dose of heparin, a protein enablingWnt diffusion in pupae near developpingwings A. Pomerantz

➔ Wnt determines the position of coloured zones

➔ Wnt determines the position of coloured zones  Probably WntA, as shown by Martin et al. in Heliconius species

Injection of heparin in H. sara sara, adapted from Martin et al., PNAS (2012) 2018 © II Joint Congress on Evolutionary Biology 2018. All rights reserved - Any reproduction even in part is prohibited. 11 2018 © 48th European Contact Lens Society Of Ophthalmologists. All rights reserved - Any reproduction even in part is prohibited] .

2018 © II Joint Congress on Evolutionary Biology 2018. All rights reserved - Any reproduction even in part is prohibited] . Material Transparency Transparency Transparency Introduction Conclusion & Methods & Structures & Development & Mimicry How are the transformed scales in the transparent zones generated during development ? Material & Methods N. Patel Dissection and stainingof developingwings in pupae and confocal imaging

A. Pomerantz

2018 © II Joint Congress on Evolutionary Biology 2018. All rights reserved - Any reproduction even in part is prohibited. 12 2018 © 48th European Contact Lens Society Of Ophthalmologists. All rights reserved - Any reproduction even in part is prohibited] .

2018 © II Joint Congress on Evolutionary Biology 2018. All rights reserved - Any reproduction even in part is prohibited] . Material Transparency Transparency Transparency Introduction Conclusion & Methods & Structures & Development & Mimicry How are the transformed scales in the transparent zones generated during development ? Material & Methods N. Patel Dissection and stainingof developingwings in pupae and confocal imaging Nuclei (DAPI) Actin (Phalloidin) Chitin (WGA) Adult A. Pomerantz

➔ Reduction in density of scale cells in transparent regions ➔ Differences in scale shape formation due to cytoskeletal organization2018 © II Joint Congressduring on Evolutionaryscale Biologygrowth 2018. All rights reserved - Any reproduction even in part is prohibited. 12 2018 © 48th European Contact Lens Society Of Ophthalmologists. All rights reserved - Any reproduction even in part is prohibited] .

2018 © II Joint Congress on Evolutionary Biology 2018. All rights reserved - Any reproduction even in part is prohibited] . Material Transparency Transparency Transparency Introduction Conclusion & Methods & Structures & Development & Mimicry How are the transformed scales in the transparent zones generated during development ? Chitin (WGA) Actin (Phalloidin) Merge N. Patel

A. Pomerantz

Scale growth depends on the cytoskeleton organization, mainly actin which guides chitin polymerisation 2018 © II Joint CongressDinwiddie on Evolutionary Biologyet 2018. al. All, rightsDevelopmental reserved - Any reproductionbiology even in part is(2014) prohibited. 13 2018 © 48th European Contact Lens Society Of Ophthalmologists. All rights reserved - Any reproduction even in part is prohibited] .

AURELIANA MIMICRY RING

CONFUSA MIMICRY RING 2018 © II Joint Congress on Evolutionary Biology 2018. All rights reserved - Any reproduction even in part is prohibited. 14 2018 © 48th European Contact Lens Society Of Ophthalmologists. All rights reserved - Any reproduction even in part is prohibited] .

1 cm

AGNOSIA BLUE

1 cm CONFUSA BANJANA-M

AURELIANA MIMICRY RING

EURIMEDIA AURELIANA

THEUDELINDA PANTHYALE

HEWITSONI 1 cm LERIDA CONFUSA MIMICRY RING 2018 © II Joint Congress on Evolutionary Biology 2018. All rights reserved - Any reproduction even in part is prohibited. 14 2018 © 48th European Contact Lens Society Of Ophthalmologists. All rights reserved - Any reproduction even in part is prohibited] .

➔ chromatic and achromaticconstrasts in JNDs (just noticeable2018differences © II Joint Congress )on Evolutionary Biology 2018. All rights reserved - Any reproduction even in part is prohibited. 15 2018 © 48th European Contact Lens Society Of Ophthalmologists. All rights reserved - Any reproduction even in part is prohibited] .

2018 © II Joint Congress on Evolutionary Biology 2018. All rights reserved - Any reproduction even in part is prohibited. 16 2018 © 48th European Contact Lens Society Of Ophthalmologists. All rights reserved - Any reproduction even in part is prohibited] .

2018 © II Joint Congress on Evolutionary Biology 2018. All rights reserved - Any reproduction even in part is prohibited] . Material Transparency Transparency Transparency Introduction Conclusion & Methods & Structures & Development & Mimicry Are transparent zones perceived similarly by predators? MANOVA Without phylogeneticcorrection: With phylogeneticcorrection: W = 0.487 (p = 0.011) W = 0.487 (p = 0.107)

➔Predators perceive butterflies differently, but it is not convergence2018 © II Joint Congress on Evolutionary Biology 2018. All rights reserved - Any reproduction even in part is prohibited. 16 2018 © 48th European Contact Lens Society Of Ophthalmologists. All rights reserved - Any reproduction even in part is prohibited] .

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100 µm 100 µm 600 µm

1 µm 1 µm 1 µm

100 µm 100 µm 600 µm

1 µm 1 µm 1 µm

• Diversity of ways of achieving transparency (more or less effective) 2018 © II Joint Congress on Evolutionary Biology100 2018. All rights reserved - Any reproduction even in part is prohibited. Transmission 0 UV 300 400 Human2018 ©visible II Joint Congressspectrum on Evolutionary Biology700 2018. All rights reserved - Any reproduction even in part is prohibited. Wavelength (nm) 17 2018 © 48th European Contact Lens Society Of Ophthalmologists. All rights reserved - Any reproduction even in part is prohibited] .

2018 © II Joint Congress on Evolutionary Biology 2018. All rights reserved - Any reproduction even in part is prohibited] . Material Transparency Transparency Transparency Introduction Conclusion & Methods & Structures & Development & Mimicry Conclusion ➢ Optical properties depend on the structures in transparent areas ➢ Transparent zones are genetically determined during development and the typical scales of transparent zones result from modification of cytoskeleton organization

Nuclei (DAPI) Actin (Phalloidin) Chitin (WGA) Adult

2018 © II Joint Congress on Evolutionary Biology 2018. All rights reserved - Any reproduction even in part is prohibited] . Material Transparency Transparency Transparency Introduction Conclusion & Methods & Structures & Development & Mimicry Conclusion ➢ Optical properties depend on the structures in transparent areas ➢ Transparent zones are genetically determined during development and the typical scales of transparent zones result from modification of cytoskeleton organization ➢ Predators perceive differently mimicry rings but transparency is not convergent among co-mimics

➢ Development of structures involved in transparency N. Patel

• Comparative transcriptomics between transparent and opaque areas to find genes involved in the devlopment of structures typical of transparent areas A. Pomerantz

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➢ Development of structures involved in transparency N. Patel

• Comparative transcriptomics between transparent and opaque areas to find genes involved in the devlopment of structures typical of transparent areas A. Pomerantz

• Candidate gene approach using Crispr/Cas9 system to knock out genes assumed to be involved in developement of structures typical of transparent areas 2018 © II Joint Congress on Evolutionary Biology 2018. All rights reserved - Any reproduction even in part is prohibited.

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• Increases constrasts in aposematic signal

1 cm

• Increases constrasts in aposematic signal

1 cm

• Mixed strategy between camouflage and aposematism

• Increases constrasts in aposematic signal

1 cm

• Mixed strategy between camouflage and aposematism  Mónica Arias’ poster P-1260 (S-77) 21st of August 17:30, Level 3 2018 © II Joint Congress on Evolutionary Biology 2018. All rights reserved - Any reproduction even in part is prohibited.

• Increases constrasts in aposematic signal

1 cm

• Forms intraspecific signals without impairing aposematic signal

2018 © II Joint Congress on Evolutionary Biology 2018. All rights reserved - Any reproduction even in part is prohibited] . Fundings Agence Nationale Human Frontier de la Recherche Science Program Acknowledgments

Collaborators UC Berkeley

Doris Gomez Willy Serge Berthier Nipam Patel Aaron Pomerantz Daney de Marcillac

ISYEB

Christine Andraud Maëlle Vilbert Florence Prunier Marianne Elias Stephan Borenstjan

THANK YOU FOR YOUR2018 © II JointATTENTION Congress on Evolutionary Biology 2018. All rights reserved - Any reproduction even in part is prohibited. 2018 © 48th European Contact Lens Society Of Ophthalmologists. All rights reserved - Any reproduction even in part is prohibited] .

Linear model taking into account phylogeny (PGLS) package caper

Brightness = αtype.insertion.scale + β1 * scale.density+ β2 * nanostructure.density + γ type.insertion.scale * nanostructure.density

1 µm Hair-like scale

1 µm Flat scale

500 nm 500 nm 500 nm

Nanostructures at the surface of the wing membrane Greta oto

Modelisation of wing reflection

Modelisation of wing refractive index ➔ Nanostructures = continuousgradient of refractive index limitingrelflexion → Increasing transparency 2018 © II Joint Congress on Evolutionary Biology 2018. AllSiddique rights reserved - Anyet reproduction al., Nature even in part Com is prohibited. (2015) 2018 © 48th European Contact Lens Society Of Ophthalmologists. All rights reserved - Any reproduction even in part is prohibited] .

Female drosophila prefer some iridescent patterns → Able to detect + sexual selection ➔ Intraspecific communication Katayama et al., PNAS (2014)

Wing Interference Patterns of different Drosophila species

Shevtsova et al., PNAS (2011) Iridescent pattern on the wings of Greta oto butterfly