Aeacus Formosanus

Diversity of the Photoreceptors and Spectral Opponency in the Compound Eye of the Golden Birdwing, Troides aeacus formosanus Pei-Ju Chen1, Kentaro Arikawa2, En-Cheng Yang1,3* 1 Department of Entomology, National Taiwan University, Taipei, Taiwan, 2 Laboratory of Neuroethology, Sokendai-Hayama (The Graduate University for Advanced Studies), Hayama, Japan, 3 Graduate Institute of Brain and Mind Sciences, National Taiwan University, Taipei, Taiwan Abstract The compound eye of the Golden Birdwing, Troides aeacus formosanus (Papilionidae, Lepidoptera), is furnished with three types of ommatidia, which are clearly different in pigmentation around the rhabdom. Each ommatidium contains nine photoreceptors, whose spectral sensitivities were analyzed electrophysiologically. We identified nine spectral types of photoreceptor with sensitivities peaking at 360 nm (UV), 390 nm (V), 440 nm (B), 510 nm (BG), 540 nm (sG), 550 nm (dG), 580 nm (O), 610 nm (R), and 630 nm (dR) respectively. The spectral sensitivities of the V, O, R and dR receptors did not match the predicted spectra of any visual pigments, but with the filtering effects of the pigments around the rhabdom, they can be reasonably explained. In some of the receptors, negative-going responses were observed when they were stimulated at certain wavelengths, indicating antagonistic interactions between photoreceptors. Citation: Chen P-J, Arikawa K, Yang E-C (2013) Diversity of the Photoreceptors and Spectral Opponency in the Compound Eye of the Golden Birdwing, Troides aeacus formosanus. PLoS ONE 8(4): e62240. doi:10.1371/journal.pone.0062240 Editor: Adrian G. Dyer, Monash University, Australia Received January 16, 2013; Accepted March 19, 2013; Published April 16, 2013 Copyright: ß 2013 Chen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported in part by the JSPS (Japanese Society for Promotion of Science) Grants-in-Aid for Scientific Research No. 21247009 and the MAFF (Ministry of Agriculture, Forestry and Fisheries of Japan) grant (Elucidation of biological mechanisms of photoresponse and development of advanced 1 technologies utilizing light) No. INSECT-1101 to KA. This work was supported by the Forestry Bureau, Council of Agriculture, Executive Yuan, Taiwan, ROC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] Introduction photoreceptors, causing an inhibitory effect on the photoreceptor in question [12]. Color vision is defined as the capability to discriminate visual As suggested by the number of literatures cited above, butterflies stimuli based solely on the difference in spectral distribution, have been an important group of insects in the study of color independent of the stimulus intensity [1]. In terms of physiology, vision. The first and the most extensively studied species is the the existence of at least two spectral types of photoreceptor in the Japanese yellow swallowtail, Papilio xuthus (tribe Papilionini, retina is a prerequisite for color vision [2]. To achieve better Papilionidae), whose eyes are furnished with at least six classes discrimination, several strategies have been developed to enhance of spectral receptors [8]. Accumulated evidence has suggested that the spectral resolution in the visual system, such as narrowing the the spectral organization of butterfly eyes is quite diverse [13–16]. spectral sensitivity, shifting the peak sensitivity, and diversifying the To address the question how such variability has evolved in spectral type of photoreceptor [3]. butterflies, we have conducted a comparative study in represen- The absorption spectrum of visual pigments is the primary tative species. factor in determining the spectral sensitivity of photoreceptors, but Troides is a genus of a peculiar group in Papilionidae, the it is largely modified by some other factors. The spectral sensitivity birdwing butterflies, which are large with the wing span of more can be widened by the self screening effect of the visual pigment in than 15 cm. The genus Troides contains 18 species. Although they the same photoreceptor [4], or by coexpressing multiple visual all look similar with black forewings and bright yellow hindwings pigments in a photoreceptor [5,6]. It can also be sharpened by the in both sexes, they still appear depending on vision when lateral filtering of neighboring photoreceptors [7], or by the discriminating conspecific mates. We therefore have initiated a filtering effect of fluorescing and colored pigments [8,9]. In work on their vision in the Golden Birdwing, Troides aeacus addition to the optical filtering by visual and non-visual pigments, formosanus. In the course of studying the spectral sensitivity of the electrical interaction between photoreceptors can also modify photoreceptors in the compound eye retina, we found nine the spectral sensitivity [10]. Electrical interactions between spectrally distinct photoreceptors, which is the most for butterflies photoreceptors have been reported in the retina of the Australian studied in this respect. We also frequently encountered photore- orchard butterfly, Papilio aegeus [11,12], where some photorecep- ceptors that exhibit spectral opponency. In the present study we tors depolarize at certain wavelengths and hyperpolarize at describe the various photoreceptors in Troides aeacus formosanus, different wavelengths. The hyperpolarization, or negative-going with a particular focus on the optical filtering effects of various responses, may originate from other simultaneously stimulated pigments as well as the physiological background of the negative- going responses in the spectrally-opponent photoreceptors. PLOS ONE | www.plosone.org 1 April 2013 | Volume 8 | Issue 4 | e62240 Spectral Sensitivity in Troides Materials and Methods If the recorded photoreceptors showed a negative-going (hyperpolarizing) response in any wavelength range, the normal- Animals ized angular responses and temporal impulse responses were Both sexes of the Golden Birdwing, Troides aeacus formosanus, recorded at the positive-peak and negative-peak wavelengths. For were obtained from laboratory culture stock derived from eggs laid recording the angular responses, the Cardan arm was moved at by females caught in the field around Taitung County, Taiwan. 0.6 deg intervals. The response amplitude induced by a 50 ms The hatched larvae were fed fresh Aristolochia kankaoensis leaves, and stimulus at each angle of the field is referred to as the angular raised under a constant photo period (L: D = 8 h: 16 h) and response. For recording the temporal impulse response, the shutter temperature (25uC) in the laboratory. The butterflies were fed a was set to deliver 20 ms light pulses at 5 s intervals. In order to sucrose solution daily and were used for experiments within two compare the temporal properties of the positive and the negative weeks after emergence. responses, the ND wedge was adjusted to give a similar amplitude response around 2–3 mV of positive and negative responses using Electrophysiology two wavelengths each eliciting either a positive or a negative Photoreceptor spectral sensitivities were determined by intra- response. The responses to 10 pulses were averaged for each cell. cellular electrophysiology [17].The wings and legs of the butterfly Finally, the time from the stimulus onset to the maximal response were amputated, and the thorax and the head were mounted on a amplitude, tp, of the positive and negative responses was metal pedestal with a beeswax and resin (3:1) mixture. A silver compared. The recorded V-log I data were fitted to the Naka-Rushton wire was inserted into the stump of one of the antennae to serve as n n n the reference electrode. To insert a glass micropipette into the eye, function [18]: V/Vmax = I /(I – K ), where I is the stimulus a small triangular hole covering about 10–20 facets was made in intensity, V is the amplitude of responses, Vmax is the peak the dorsal region of eye and sealed with a drop of Vaseline in order amplitude at the maximum positive response, K is the stimulus to prevent coagulation of haemolymph. The eye was positioned at intensity eliciting 50% Vmax, and n is the exponential slope. the center of a Cardan arm perimeter device set in a Faraday cage. Amplitudes of the spectral responses were extrapolated to the V-log I function of a given unit to transform the amplitudes into The electrophysiological recording was performed in the dark so photon numbers required for the responses. The normalized as to keep the butterfly nearly dark-adapted. reciprocal of the relative photon numbers then yielded the spectral A glass micropipette filled with 2M potassium acetate, with sensitivity. resistance about 100–120 MV, was inserted vertically into the retina using a micromanipulator. After impaling a single photoreceptor, the ommatidium was stimulated with a series of Anatomy monochromatic lights between 300–700 nm, with 10 nm steps, We first examined the intact eyes under the epi-fluorescence provided by a 1000 W xenon arc lamp through a monochromator microscope (BX-60, Olympus) under UV excitation (dichroic cube (SP-150-M with 150-030-300 grating, Acton Research Co.). The U-MWU, excitation

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