PrudicEtAl_PRSB_Data

All data (3 experiments) reported in the pape

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

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

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

Developmental staging used in current study.

<p>Total larval development from 5^th instar ecdysis to pupation for female <i>B</i>. <i>anynana</i> takes approximately 8 days at 27°C (WS) and 20 days at 17°C (DS). Total pupal development until adult emergence takes an average of 6.4 days for WS and 16 days for DS females. The bright green wanderer stage occurs, on average, on day 6 for WS forms, and on day 17 for DS forms. Temperature shifts, hemolymph collection, and injections were all performed at 2pm of noted day. Note the difference in stage nomenclature for temperature shifts during the pupal stage and hormone titer quantification (equivalent % development time).</p

Manipulations of ecdysone signaling during the wanderer stages of development alters hindwing eyespots more extensively than forewing eyespots.

<p>Injections of 20-hydroxyecdysone (20E) and vehicle (V) were performed in DS forms (brown). Injections of Cucurbitacin B (CucB) and vehicle were performed in WS forms (green). Measurements of eyespot size (A, B), eyespot center size (C, D) and eyespot center darkness (K value) were performed in forewing eyespots (A, C, E; left graphs) and hindwing eyespots (B, D, F; right graphs). Error bars = 95% CI of means. Significant differences between treatments are represented by asterisks: *, p < 0.05; **, p < 0.01, ***, p < 0.001, ns = non significant. Values for trait size in A-D are evaluated at a wing area of 227 mm².</p

Forewing eyespots display lower levels of plasticity relative to hindwing eyespots.

<p>A) Traits measured in this study were eyespot size (mm²), center size (mm²), and center brightness (reflectance) for Cu1 homologous eyespots on fore and hindwings of DS and WS forms. B-D) Graphs on the left depict the allometric relationship between log trait size (or log brightness) and log wing size. Green and brown symbols represent WS and DS forms respectively. Lines of best fit are blue for forewings, fws, and orange for hindwings, hws. Right-hand graphs depict the estimated marginal means from each eyespot type, evaluated at a wing size of 245 mm². Notice how hindwing eyespots display steeper slopes, e.g., stronger plasticity, relative to forewing eyespots. A significant wing by seasonal form interaction (i.e., significantly different slopes for forewings and hindwings) is present in each case (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005529#pgen.1005529.t001" target="_blank">Table 1</a> for test statistics). No plasticity is present in the brightness of forewing eyespot centers (D). Error bars represent 95% CI for the means.</p