Evolution and Mechanism of Spectral Tuning of Blue-Absorbing Visual Pigments in Butterflies

Motohiro Wakakuwa,Yoshinori Shichida,Mitsumasa Koyanagi,Kentaro Arikawa,Akihisa Terakita,Doekele G Stavenga,Eric James Warrant

doi:10.1371/journal.pone.0015015

Motohiro Wakakuwa, Yoshinori Shichida + Show 5 more

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https://doi.org/10.1371/journal.pone.0015015

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Abstract

The eyes of flower-visiting butterflies are often spectrally highly complex with multiple opsin genes generated by gene duplication, providing an interesting system for a comparative study of color vision. The Small White butterfly, Pieris rapae, has duplicated blue opsins, PrB and PrV, which are expressed in the blue (λ max = 453 nm) and violet receptors (λ max = 425 nm), respectively. To reveal accurate absorption profiles and the molecular basis of the spectral tuning of these visual pigments, we successfully modified our honeybee opsin expression system based on HEK293s cells, and expressed PrB and PrV, the first lepidopteran opsins ever expressed in cultured cells. We reconstituted the expressed visual pigments in vitro, and analysed them spectroscopically. Both reconstituted visual pigments had two photointerconvertible states, rhodopsin and metarhodopsin, with absorption peak wavelengths 450 nm and 485 nm for PrB and 420 nm and 482 nm for PrV. We furthermore introduced site-directed mutations to the opsins and found that two amino acid substitutions, at positions 116 and 177, were crucial for the spectral tuning. This tuning mechanism appears to be specific for invertebrates and is partially shared by other pierid and lycaenid butterfly species.

Highlights

Visual pigment molecules consist of an opsin, an integral membrane protein with seven transmembrane helices, and a chromophore, most commonly 11-cis retinal, which is attached to a lysine in the seventh helix in all opsins so far identified
We looked for amino acids that are close to the chromophore having different polarity between PrB and PrV, and focused on two amino acid residues and tested their contributions by introducing site-directed mutations followed by spectroscopic analyses of the mutant molecules
Spectroscopy of expressed PrB and PrV We first confirmed that the HEK293s cell system properly functioned by immunoblot analyses using the anti-rhodopsin 1D4

Summary

Introduction

Visual pigment molecules consist of an opsin, an integral membrane protein with seven transmembrane helices, and a chromophore, most commonly 11-cis retinal, which is attached to a lysine in the seventh helix in all opsins so far identified. Upon absorption of a photon, the chromophore isomerizes into the alltrans form, which subsequently causes transformation of the whole visual pigment molecule into a metarhodopsin state. This triggers the intracellular transduction cascade, which eventually produces a receptor potential in the photoreceptor cell. Honeybees employ one opsin from each clade, expressed separately in the UV, B and green (G) receptors in the compound eye [1] This basic pattern is often modified in butterflies, presumably because their life style strongly depends on their color discrimination capacities. B opsins duplicated independently in the families Pieridae and Lycaenidae [2,3,4], duplication of L opsins was found in Papilionidae [5,6] and Riodinidae [7], and UV opsins duplicated in the genus Heliconius belonging to the Nymphalidae [8]

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