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

Temporal Gene Expression Variation Associated with Eyespot Size Plasticity in <i>Bicyclus anynana</i>

<div><p>Seasonal polyphenism demonstrates an organism's ability to respond to predictable environmental variation with alternative phenotypes, each presumably better suited to its respective environment. However, the molecular mechanisms linking environmental variation to alternative phenotypes via shifts in development remain relatively unknown. Here we investigate temporal gene expression variation in the seasonally polyphenic butterfly <i>Bicyclus anynana</i>. This species shows drastic changes in eyespot size depending on the temperature experienced during larval development. The wet season form (larvae reared over 24°C) has large ventral wing eyespots while the dry season form (larvae reared under 19°C) has much smaller eyespots. We compared the expression of three proteins, Notch, Engrailed, and Distal-less, in the future eyespot centers of the two forms to determine if eyespot size variation is associated with heterochronic shifts in the onset of their expression. For two of these proteins, Notch and Engrailed, expression in eyespot centers occurred <i>earlier</i> in dry season than in wet season larvae, while Distal-less showed no temporal difference between the two forms. These results suggest that differences between dry and wet season adult wings could be due to a delay in the onset of expression of these eyespot-associated genes. Early in eyespot development, Notch and Engrailed may be functioning as repressors rather than activators of the eyespot gene network. Alternatively, temporal variation in the onset of early expressed genes between forms may have no functional consequences to eyespot size regulation and may indicate the presence of an 'hourglass' model of development in butterfly eyespots.</p></div

PrudicEtAl_PRSB_Data

All data (3 experiments) reported in the pape

Temporal variation in expression of protein products of three eyespot-associated genes.

<p>Comparisons of Notch (A), Engrailed (B), and Distal-less (C) expression in two forms of <i>B. anynana</i>. Graphs show logistic curves fit to observed expression in each of the two forms. Images show expression in dry season and wet season wing discs of the same developmental stage; N_D and N_W reflect total compartments examined in dry and wet season wing discs, respectively. In (A) and (B), white arrows in dry season images indicate compartments with focal expression of respective proteins; black arrows indicate corresponding compartments in wet season wing discs, which lack focal expression at this stage.</p

Temporal dynamics of three eyespot-associated genes in different forms of <i>B. anynana</i>.

<p>In both dry season (A) and wet season (B) larvae, genes are expressed in future eyespot centers in the same order as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065830#pone.0065830-Oliver1" target="_blank">[25]</a>.</p

Differential Expression of Ecdysone Receptor Leads to Variation in Phenotypic Plasticity across Serial Homologs

<div><p>Bodies are often made of repeated units, or serial homologs, that develop using the same core gene regulatory network. Local inputs and modifications to this network allow serial homologs to evolve different morphologies, but currently we do not understand which modifications allow these repeated traits to evolve different levels of phenotypic plasticity. Here we describe variation in phenotypic plasticity across serial homologous eyespots of the butterfly <i>Bicyclus anynana</i>, hypothesized to be under selection for similar or different functions in the wet and dry seasonal forms. Specifically, we document the presence of eyespot size and scale brightness plasticity in hindwing eyespots hypothesized to vary in function across seasons, and reduced size plasticity and absence of brightness plasticity in forewing eyespots hypothesized to have the same function across seasons. By exploring the molecular and physiological causes of this variation in plasticity across fore and hindwing serial homologs we discover that: 1) temperature experienced during the wandering stages of larval development alters titers of an ecdysteroid hormone, 20-hydroxyecdysone (20E), in the hemolymph of wet and dry seasonal forms at that stage; 2) the 20E receptor (EcR) is differentially expressed in the forewing and hindwing eyespot centers of both seasonal forms during this critical developmental stage; and 3) manipulations of EcR signaling disproportionately affected hindwing eyespots relative to forewing eyespots. We propose that differential EcR expression across forewing and hindwing eyespots at a critical stage of development explains the variation in levels of phenotypic plasticity across these serial homologues. This finding provides a novel signaling pathway, 20E, and a novel molecular candidate, EcR, for the regulation of levels of phenotypic plasticity across body parts or serial homologs.</p></div

Plasticity in eyespot center brightness is due to variation in pigment deposition in the white central scales.

<p>Epi-illumination microscopy and reflection measurements for Cu1 eyespot centers in forewings (FW) and hindwings (HW) of both WS and DS forms. A) WS FW; B) DS FW; C) WS HW; D) DS HW. E-H) Reflection measurements of corresponding eyespot centers before (colored lines) and after (black lines) application of silicone oil.</p

20E hormone titers are higher in WS forms at the wanderer stage of development and the 20E receptor (EcR) is present in hindwing but absent from forewing eyespot centers at that stage.

<p>A) Titers of 20-hydroecdysone (20E) at several stages of larval, pre-pupal and pupal development. DS forms = brown line; WS forms = green line; Stages described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005529#pgen.1005529.t002" target="_blank">Table 2</a>. Error bars correspond to 95% CI for means. B) During the wanderer (W) stage of development EcR (green) is expressed in hindwing eyespot centers of both seasonal forms (WS, DS) but not in forewing eyespots, whereas a second eyespot-associated protein, spalt (sal; red), is expressed in all eyespot centers. C) Earlier during the 5^th instar, however, EcR is expressed in both forewing (left) and hindwing (right) eyespot centers on the ventral surface. Arrows point to the Cu1 eyespot.</p