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

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.

Quantum Mechanical/Molecular Mechanical Structure, Enantioselectivity, and Spectroscopy of Hydroxyretinals and Insights into the Evolution of Color Vision in Small White Butterflies

“…Wakakuwa et al showed that amino acid substitutions at sites 116 and 177 are crucial for spectral tuning in the Pr B and Pr V pigments. 24 As already mentioned before, within 5.0 Å environment of the retinal, seven sites in Pr B opsin and three sites in Pr V opsin differ in their identity compared to the squid opsin. However, due to difference in their polarity, only sites 116 and 177 were mutated in the experimental study.…”

“…In contrast, mutation of serine with alanine at site 116 (S116A) induces a red shift of 10 nm in agreement with experimental findings. 24 Apparently, only half of the blue shift (20 nm) induced by S116 in Pr B is recovered with A116 in Pr V. The calculated spectral shifts are non-additive as introduction of all three mutants decreases the λ max by only 7 nm.…”

“…We aligned the amino acid residues of Pr B and Pr V pigments with that of the Japanese common squid rhodopsin ( Todarodes pacificus ), 30 and this approach is adopted from the experimental study by Wakakuwa et al 24 Comparison of the amino acid sequence within 5.0 Å radius of any atom of the retinal (see Table 1) reveals that, seven amino acids at sites 112 (G112A), 113 (F113T), 116 (G116S), 120 (F120I), 123 (I123A), 177 (Y177F), and 185 (N185T) are different in their identity between squid and Pr B pigments and three amino acids at sites 113 (T113V), 116 (S116A) and 177 (F177Y) are different in their identity between Pr B and Pr V pigments.…”

“…21 (ii) Small white butterfly ( Pieris rapae ) opsins contain A 3 -retinal and their eyes have six classes of photoreceptors peaking at 360 nm (UV), 420 nm (violet), 450 nm (blue), 563 nm (green), 620 nm (red) and deep-red (640 nm); respectively. 22–24 …”

Optogenetic Potentials of Diverse Animal Opsins

“…In the visual cells of invertebrates, Gq-coupled opsin-based pigments light dependently activate a Gq-type G protein (Terakita et al 1993 ;Koyanagi and Terakita 2008 ), which results in the depolarization of these cells (Yau and Hardie 2009 We successfully obtained several Gq-coupled opsin-based pigments, which had different wavelength sensitivities, by expressing these opsins in cultured cells. These included UV-and blue-sensitive honeybee opsin-based pigments , blue-and violet-sensitive butterfl y opsin-based pigments (Wakakuwa et al 2010 ), green-sensitive spider opsin-based pigment (Nagata et al 2012 ), and blue-sensitive amphioxus melanopsin (Koyanagi et al 2005 ).…”