Pmental Studies Hybridoma Bank (DSHB) developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242.Butterfly rearingB. anynana larvae were reared on young corn plants (Zea mays) in aluminum mesh cages in a 27uC environmental chamber with a 12:12 light: dark cycle with a gradual “sunrise” and “sunset,” each one hour in duration. J. coenia were reared on 18325633 narrowleaf plantain (Plantago lanceolata) leaves at room temperature (,25uC), under a natural photoperiod, and inside large plastic H the number of leukocytes in the NE and MC was containers. After injections, B. anynana were reared in mesh sleeve cages with no more than 15 larvae per corn plant. J. coenia were reared in the same plastic containers. Butterflies being raised to adults were transferred to hanging net cages after pupation, with no more than 15 pupae per cage, and adults were frozen upon emergence.Antibody injectionsHh activity was suppressed via injection of the 5E1 antibody into larvae. The 5E1 antibody prevents the Hh signaling ligand from binding to its receptor Patched (Ptc). When this happens, Ptc is able to inhibit Smoothened (Smo) resulting in interactions between Smo, Ci and other protein complexes, which results in the transformation of Ci into a Title Loaded From File repressor form of Ci (CiR). This repressor form acts as a transcription factor inhibiting the transcription of target genes such as en [22]. NS1 medium was used in control injections. Antibody injections were performed using a #701 Hamilton 10 mL syringe with 26?3 gauge needles. Fifth instar larvae were injected on the left side directly posterior to the third thoracic segment with 5 mL of either NS1 medium or 5E1 antibody solutions, at a concentration of 41 mg/mL (in B. anynana) and 100 mg/mL (in J. coenia). A higher concentration of 5E1 was used for J. coenia larvae, in proportion to their higher weight.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?: B. anynana NS1; 4?: B. anynana 5E1; 7?: J. coenia NS1; 9?0: 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. doi:10.1371/journal.pone.0051087.gHedgehog’s Role in Wing and Eyespot DevelopmentFigure 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 5E1injected 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 1655472 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. anyn.Pmental Studies Hybridoma Bank (DSHB) developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242.Butterfly rearingB. anynana larvae were reared on young corn plants (Zea mays) in aluminum mesh cages in a 27uC environmental chamber with a 12:12 light: dark cycle with a gradual “sunrise” and “sunset,” each one hour in duration. J. coenia were reared on 18325633 narrowleaf plantain (Plantago lanceolata) leaves at room temperature (,25uC), under a natural photoperiod, and inside large plastic containers. After injections, B. anynana were reared in mesh sleeve cages with no more than 15 larvae per corn plant. J. coenia were reared in the same plastic containers. Butterflies being raised to adults were transferred to hanging net cages after pupation, with no more than 15 pupae per cage, and adults were frozen upon emergence.Antibody injectionsHh activity was suppressed via injection of the 5E1 antibody into larvae. The 5E1 antibody prevents the Hh signaling ligand from binding to its receptor Patched (Ptc). When this happens, Ptc is able to inhibit Smoothened (Smo) resulting in interactions between Smo, Ci and other protein complexes, which results in the transformation of Ci into a repressor form of Ci (CiR). This repressor form acts as a transcription factor inhibiting the transcription of target genes such as en [22]. NS1 medium was used in control injections. Antibody injections were performed using a #701 Hamilton 10 mL syringe with 26?3 gauge needles. Fifth instar larvae were injected on the left side directly posterior to the third thoracic segment with 5 mL of either NS1 medium or 5E1 antibody solutions, at a concentration of 41 mg/mL (in B. anynana) and 100 mg/mL (in J. coenia). A higher concentration of 5E1 was used for J. coenia larvae, in proportion to their higher weight.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?: B. anynana NS1; 4?: B. anynana 5E1; 7?: J. coenia NS1; 9?0: 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. doi:10.1371/journal.pone.0051087.gHedgehog’s Role in Wing and Eyespot DevelopmentFigure 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 5E1injected 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 1655472 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. anyn.