Figure 1.
Differential expression of Hh signaling pathway members in Junonia coenia and Bicyclus anynana larval hindwings.
Summary of mRNA (italics) and protein expression data from [8], [9]. hedgehog (hh) and its receptor patched (ptc) are expressed in primitive patterns throughout the posterior wing compartment and in a narrow anterior domain abutting that compartment, respectively. These two genes are also expressed in novel domains in J. coenia but not in B. anynana: flanking the developing eyespot centers, and in the centers, respectively. The other depicted genes share a similar expression pattern between J. coenia and B. anynana.
Figure 2.
Measurements taken of adult B. anynana and J. coenia wings.
Wing height and wing area (J. coenia only) and a series of eyespot trait diameters for the M1 and Cu1 eyespots were measured on both ventral (left) and dorsal surfaces (right). R5, R4, M1, M2, M3, Cu1, Cu1+Pc, and 1A+2A refer to the wing compartments that were individually measured in B. anynana wings only. Their combined height defined the B. anynana wing height. w: white center; b: black disc; g: gold ring; in: inner ring (from the distal border of the black patch to the proximal border of the orange patch).
Figure 3.
Western blots and similarity of Sonic-Hh and butterfly Hh sequences suggest that 5E1 antibody recognizes Hh in butterflies.
(A) Alignment of sequences corresponding to the Sonic-Hh peptide used to make the 5E1 monoclonal antibody [15]. Areas boxed in red correspond to the 5E1 epitope [26], [27]. (B) Western blot with B. anynana proteins extracted from wing discs showing three potential Hh fragments with the predicted sizes of 19 kD, 25 kD, and 37 kD (arrows) previously characterized from D. melanogaster Hh [21]. (C) No bands were detected with the control NS1 medium. The left lane of each photo is the protein standard.
Figure 4.
Injections of 5E1 antibody reduce the levels of en/inv transcripts one day later in both B. anynana and J. coenia.
(A) PCR amplification of en/inv (top) and the house-keeping gene EF1a (bottom) from the same samples after injection of either 5E1 antibody or NS1 vehicle. Samples 1–3: B. anynana NS1; 4–6: B. anynana 5E1; 7–8: J. coenia NS1; 9–10: J. coenia 5E1. (B) Quantification of brightness levels of en/inv PCR amplification relative to brightness levels of the EF1a housekeeping gene (averages from data in A). Asterisk (*) indicates a significant difference in en/inv relative levels between 5E1 and NS1 injections.
Figure 5.
Hh sequestration decreases wing size in both species and relative eyespot size in J. coenia.
Forewing height in B. anynana (A) and J. coenia (B) and forewing area in J. coenia (C) are smaller in 5E1-injected butterflies compared with NS1-injected controls. Relative eyespot size, e.g., the diameter of the black and gold rings of the Cu1 eyespot on both ventral (vent) and dorsal (dors) surfaces of J. coenia is also smaller in 5E1-injected individuals (D-F; mean trait values are displayed for a wing height of 16 mm). GLM analyses use sex as a grouping variable but here sexes are plotted together. (G) Regression of black disc diameter of Cu1 dorsal eyespot on wing height for B. anynana (left) and J. coenia (right). In J. coenia, 5E1-injected individuals (red dots) display significantly smaller Cu1 eyespots relative to NS1-injected individuals (green dots) of comparable size, while B. anynana eyespots are not significantly smaller for an individual of a given size.
Table 1.
F statistics and p-values for GLM analysis testing for differences in wing compartment size in B. anynana across treatments.
Table 2.
F statistics and p-values for GLM analysis testing for differences in eyespot trait sizes across treatments.
Table 3.
F statistics and p-values for GLM analysis testing for differences in eyespot trait sizes across treatments using wing size as a covariate.