Schematic of the different temperature shift experiments performed in this study.

<p>Each temperature shift treatment has been assigned a different letter, and experiments are grouped by specific question and analysis (Exp 1–3). L, P, and A stand for larval, pupal, and adult development, respectively. Brown (the color of dead leaf litter) indicates the DS temperature of 17°C and green (the color of lush vegetation) represents the WS temperature of 27°C.</p

Courtship rate is influenced by the temperature the butterfly experiences during pupation and adulthood, but not during larval development.

<p>Vertical bars represent the mean courtship rate exhibited by males per observation period. Error bars represent 95% confidence intervals. The temperature treatment the butterfly experienced during development and adulthood is represented by horizontal bars along the x-axis. Symbols used below the x-axis are defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064061#pone-0064061-g001" target="_blank">Figure 1</a>. A: Bars represent the mean courtship rate of ten males in a 50-minute observation period. B, C: Bars represent the mean courtship rate of five males in a 30-minute observation period. C: Presents the mean courtship rate from pooled data from both types of larval temperature experienced during development (larval temperature was not a significant factor in the GLM analysis).</p

WS <i>B</i>. <i>anynana</i> males have higher titers of 20-hydroxyecdysone (20E) during early pupal development than DS males.

<p>T-test performed on hormone titer measurements show that there are significant differences in 20E titers in the hemolymph of <i>B</i>. <i>anynana</i> WS and DS male pupae at 14%, 30%, and 50% of pupal development but not at 65% pupal development. Error bars correspond to 95% confidence intervals for the means.</p

Steroid hormone signaling during development has a latent effect on adult male sexual behavior in the butterfly <i>Bicyclus anynana</i>

<div><p>It is well established that steroid hormones regulate sexual behavior in vertebrates via organizational and activational effects. However, whether the organizational/activational paradigm applies more broadly to the sexual behavior of other animals such as insects is not well established. Here we describe the hormonal regulation of a sexual behavior in the seasonally polyphenic butterfly <i>Bicyclus anynana</i> is consistent with the characteristics of an organizational effect. By measuring hormone titer levels, quantifying hormone receptor gene expression in the brain, and performing hormone manipulations, we demonstrate steroid hormone signaling early in pupal development has a latent effect on adult male sexual behavior in <i>B</i>. <i>anynana</i>. These findings suggest the organizational/activational paradigm may be more highly conserved across animal taxa than previously thought.</p></div

Raising 20E titers in DS males during pupal development significantly increases courtship rate.

<p>A) Butterflies reared at the WS typical temperature of 27°C have a significantly higher courtship rate than butterflies reared at the DS typical temperature of 17°C. B) Courtship rate of DS males injected with 6000 pg of 20E at 30% of pupal development (30%P) is significantly higher than the courtship rate of DS males injected with vehicle at the same time. There is no significant difference in the courtship rate of DS males injected with 20E at 50% of development (50%P) compared to DS males injected with vehicle at the same time (light brown). Error bars correspond to 95% confidence intervals for the means.</p

Graphical representation of B-values of offspring from three types of parental cross.

<p><b>a.</b> Average B-values (± SD) of offspring from three types of parental cross. Each cross is represented by five families arranged vertically by instar. <b>b.</b> Scatter plot to show overlap and spread of B-values for individuals from the families of the three types of parental cross.</p

Topical application of JHA to dl leads to a supernumerary instar.

<p><b>a</b>. normal 5^th instar larva that was treated with acetone. <b>b</b>. 6^th supernumerary instar larva that was treated with JHA on the 4^th day of the 4^th instar.</p

Method of measuring the cuticle brightness value (B-value) of caterpillars.

<p><b>a.</b> 5th instar dl. <b>b</b>. 5^th instar wt. Both original images have been separated from the background using the “lasso” tool, averaged using the “average” tool, and then converted to gray scale in Adobe Photoshop.</p

Results of topical Juvenile Hormone analogue (JHA) application on larval cuticle brightness (B-value) in the fifth instar and on the length of the fourth instar.

<p>F statistics represent results from Analysis of Variance to test for significant differences across experimental and control treatments in larval color and development time. Experiment; experiment number. DTI; developmental interval when JHA was applied. A ctrl; Acetone control. N; numbers treated. JHA; experimental groups treated with JHA. HCA; time of head cap apolysis.</p

Light microscope and TEM imaging of dl and wt cuticles.

<p><b>a,b.</b> Light micrograph of the dorsal cuticle of a fifth instar wt (a) and dl (b) larvae showing darkly pigmented bristles and star-like structures with concentrated pigmentation. <b>c,d</b>. TEM micrograph of dorsal cuticle of fifth instar wt (c) and dl (d) showing a star-like structure containing a homogeneous layer of diffuse pigment that is darker in dl (d).</p