Normalized sensitivities of three types of photoreceptor and eyeshine in the adult compound eye of Danaus plexippus. (A) Solid lines represent rhodopsin absorbance spectra based on the Bernard rhodopsin template in Palacios et al. (Palacios et al., 1996). Dotted lines represent spectral sensitivities of the blue [B] and long wavelength [LW] photoreceptors measured intracellularly by Stalleicken et al. (Stalleicken et al., 2006). Vertical lines represent emission maxima for lights used in choice assays. (B) Monarch dorsal eye showing red- and orange-reflecting ommatidia. Courtesy of Dr Gary Bernard. (C) Monarch cryostat sectioned eyeshine showing ommatidia that contain an orange filtering pigment adjacent to ommatidia lacking this filtering pigment.
Colored papers, color space triangle and innate color preferences of the monarch butterfly. (A) Spectral reflectance profiles for each colored paper model used in training and testing, and black model used in innate trials, obtained using an Ocean Optics USB2000 spectrometer. (B) The loci of colored and gray papers, and monarch orange wing pigments, represented in the color triangle model. Color distances are calculated as the Euclidean distance between two loci, assuming a trichromatic visual system, with Styrofoam used in the behavioral experiments as the adapting background and the halogen light as the illuminating light source. The orange circles represent monarch orange wing pigments sampled from the dorsal and ventral surfaces of the forewing and hindwing. Dark gray circles represent the black, white, and 17 gray papers. Arrows indicate colored papers. Red circle indicates the Styrofoam background, The gray line indicates the loci of pure spectral lights at background intensity. Brightnesses of the colored papers are: Rw-HUE, 0.4; O-HUE, 0.52; Yw-HUE, 0.68; Gw-HUE, 0.63; B-HUE, 0.75; V-HUE, 0.57. Since the filtered lights experiments (see text) demonstrate that monarchs can discriminate between colors (589 nm and 620 nm) that fall on the same color locus, independent of intensity, the color space shown is missing one dimension, that of a fourth (LW-sensitive) photoreceptor. (C) The initial color probed by naïve monarch butterflies offered six unrewarding paper models showed significant deviations from random choice, with orange being the most strongly preferred and purple the least preferred color. (D) When offered three unrewarding colored paper models, naïve monarch butterflies showed an overwhelming preference for yellow.
Monarchs learned to associate colored model flowers with a sucrose reward. Following an assay for innate color preference, different cohorts of butterflies were trained to red (A; N=22), orange (B; N=18), yellow (C; N=13), green (D; N=20), blue (E; N=23) and purple (F; N=11) respectively, by feeding the monarchs on their assigned color for eight days. Asterisks (*) indicate the first day of training that differs significantly from innate preference for the trained color, by repeated measure ANOVA followed by Tukey post hoc comparisons. Boxes in A, D and F indicate non-significant differences (confusion) between the training color and another color at day 4. (G) Comparison of the time spent probing each training color after eight days of training did not differ across training color, indicating a comparable “success rate” following the training regime. Values are means ± 1 s.e.m.
Monarchs change preferences quickly when a new color becomes rewarding. Different cohorts of butterflies were trained to a single color for 8 days, at which time the color of the training model was changed. Monarchs were trained to blue followed by purple (A), purple followed by blue (B) or red followed by yellow (C). Asterisks (*) indicate values that differ significantly from innate preferences for each of the two training colors, by repeated measure ANOVA followed by Tukey post hoc comparisons. Values are means ± 1 s.e.m.; N=17, 17, and 16, respectively. (D–F) Comparing learning rates for colors trained singly (dashed line) with those for the same color trained as a second color (solid line), blue and yellow were learned at the same rate whether they were the first or second color trained; however, butterflies showed a slower rate of learning for purple when it was trained as the second color.
Monarchs discriminate colors based on wavelength and not intensity. After being fed on a colored model flower for 8 days, monarchs were offered a choice between their colored training model and 19 gray models of varying brightness in an array where none of the artificial flowers contained nectar. (A) Intensity of each model offered in the choice array. Cohorts of butterflies trained to different colors, purple (B), blue (C), yellow (D) or red (E) were able to accurately discriminate between the single colored model and all the gray models in the arena. Significance was assessed by single sample t-test. Values are means ± 1 s.e.m.
Choice frequencies of Danaus plexippus for three colors after training, as a function of the ratio between the intensities of the rewarded color and the unrewarded color. The symbols represent the individual performance and the line the average. (A) Four D. plexippus trained to 589 nm as the rewarded color and 450 nm as the unrewarded. All correct choices are significantly higher than chance (P<0.05) except for Monarch 18 at the intensity rewarded/unrewarded ratio 4. (B) Five D. plexippus trained to 620 nm as the rewarded color and 589 nm as the unrewarded. The correct choices of every animal are significantly higher than chance (P<0.05).