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Evolution and mechanism of spectral tuning of blue-absorbing visual pigments in butterflies.

Wakakuwa M, Terakita A, Koyanagi M, Stavenga DG, Shichida Y, Arikawa K - PLoS ONE (2010)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Neuroethology, Sokendai-Hayama, Hayama, Japan.

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.

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Expression of Pieris visual pigments in HEK293s cells.(A) Immunoblot analysis of 1D4 epitope-tagged proteins. The bands of about 38 kD (arrowhead) correspond to the full-length PrUV, PrB and PrV proteins expressed with three vectors, PcDNA, SRα and pEF. (B) Bistability of the crude extracts of PrB. Absorbance spectra were subsequently recorded in the dark, after irradiation with 460 nm and with 550 nm. Curve 1 is the difference before and after 460 nm irradiation, and curve 2 is the difference before and after 550 nm irradiation. The inset shows the average of the two difference spectra (black line), the predicted absorbance spectra of an R450 visual pigment (R) taken from Fig. 2B, and its metarhodopsin peaking at 485 nm derived from the Govardovskii template (M). (C) Bistability of the crude extracts of PrV. Samples were irradiated subsequently with 420 nm and 500 nm. Curves 3 and 4 are the differences of the absorbance spectra before and after 420 nm irradiation and before and after 500 nm irradiation, respectively. The inset shows the average of the two difference spectra (black line), the predicted absorbance spectra of an R420 rhodopsin (R), taken from Fig. 2C, and its metarhodopsin peaking at 482 nm derived from the Govardovskii template (M).
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pone-0015015-g001: Expression of Pieris visual pigments in HEK293s cells.(A) Immunoblot analysis of 1D4 epitope-tagged proteins. The bands of about 38 kD (arrowhead) correspond to the full-length PrUV, PrB and PrV proteins expressed with three vectors, PcDNA, SRα and pEF. (B) Bistability of the crude extracts of PrB. Absorbance spectra were subsequently recorded in the dark, after irradiation with 460 nm and with 550 nm. Curve 1 is the difference before and after 460 nm irradiation, and curve 2 is the difference before and after 550 nm irradiation. The inset shows the average of the two difference spectra (black line), the predicted absorbance spectra of an R450 visual pigment (R) taken from Fig. 2B, and its metarhodopsin peaking at 485 nm derived from the Govardovskii template (M). (C) Bistability of the crude extracts of PrV. Samples were irradiated subsequently with 420 nm and 500 nm. Curves 3 and 4 are the differences of the absorbance spectra before and after 420 nm irradiation and before and after 500 nm irradiation, respectively. The inset shows the average of the two difference spectra (black line), the predicted absorbance spectra of an R420 rhodopsin (R), taken from Fig. 2C, and its metarhodopsin peaking at 482 nm derived from the Govardovskii template (M).

Mentions: We first confirmed that the HEK293s cell system properly functioned by immunoblot analyses using the anti-rhodopsin 1D4. The 1D4 antibody predominantly labeled bands around 40k (arrowhead in Fig. 1A), which are most likely PrUV, PrB and PrV opsins. We could not detect any bands around 40k for PrL (data not shown) (Fig. 1A). The bands corresponding to smaller and larger molecular weights are probably degraded and aggregated products, respectively.


Evolution and mechanism of spectral tuning of blue-absorbing visual pigments in butterflies.

Wakakuwa M, Terakita A, Koyanagi M, Stavenga DG, Shichida Y, Arikawa K - PLoS ONE (2010)

Expression of Pieris visual pigments in HEK293s cells.(A) Immunoblot analysis of 1D4 epitope-tagged proteins. The bands of about 38 kD (arrowhead) correspond to the full-length PrUV, PrB and PrV proteins expressed with three vectors, PcDNA, SRα and pEF. (B) Bistability of the crude extracts of PrB. Absorbance spectra were subsequently recorded in the dark, after irradiation with 460 nm and with 550 nm. Curve 1 is the difference before and after 460 nm irradiation, and curve 2 is the difference before and after 550 nm irradiation. The inset shows the average of the two difference spectra (black line), the predicted absorbance spectra of an R450 visual pigment (R) taken from Fig. 2B, and its metarhodopsin peaking at 485 nm derived from the Govardovskii template (M). (C) Bistability of the crude extracts of PrV. Samples were irradiated subsequently with 420 nm and 500 nm. Curves 3 and 4 are the differences of the absorbance spectra before and after 420 nm irradiation and before and after 500 nm irradiation, respectively. The inset shows the average of the two difference spectra (black line), the predicted absorbance spectra of an R420 rhodopsin (R), taken from Fig. 2C, and its metarhodopsin peaking at 482 nm derived from the Govardovskii template (M).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2991335&req=5

pone-0015015-g001: Expression of Pieris visual pigments in HEK293s cells.(A) Immunoblot analysis of 1D4 epitope-tagged proteins. The bands of about 38 kD (arrowhead) correspond to the full-length PrUV, PrB and PrV proteins expressed with three vectors, PcDNA, SRα and pEF. (B) Bistability of the crude extracts of PrB. Absorbance spectra were subsequently recorded in the dark, after irradiation with 460 nm and with 550 nm. Curve 1 is the difference before and after 460 nm irradiation, and curve 2 is the difference before and after 550 nm irradiation. The inset shows the average of the two difference spectra (black line), the predicted absorbance spectra of an R450 visual pigment (R) taken from Fig. 2B, and its metarhodopsin peaking at 485 nm derived from the Govardovskii template (M). (C) Bistability of the crude extracts of PrV. Samples were irradiated subsequently with 420 nm and 500 nm. Curves 3 and 4 are the differences of the absorbance spectra before and after 420 nm irradiation and before and after 500 nm irradiation, respectively. The inset shows the average of the two difference spectra (black line), the predicted absorbance spectra of an R420 rhodopsin (R), taken from Fig. 2C, and its metarhodopsin peaking at 482 nm derived from the Govardovskii template (M).
Mentions: We first confirmed that the HEK293s cell system properly functioned by immunoblot analyses using the anti-rhodopsin 1D4. The 1D4 antibody predominantly labeled bands around 40k (arrowhead in Fig. 1A), which are most likely PrUV, PrB and PrV opsins. We could not detect any bands around 40k for PrL (data not shown) (Fig. 1A). The bands corresponding to smaller and larger molecular weights are probably degraded and aggregated products, respectively.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Neuroethology, Sokendai-Hayama, Hayama, Japan.

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.

Show MeSH