Eyespots deflect predator attack increasing fitness and promoting the evolution of phenotypic plasticity

Some eyespots are thought to deflect attack away from the vulnerable body, yet there is limited empirical evidence for this function and its adaptive advantage. Here, we demonstrate the conspicuous ventral hindwing eyespots found on Bicyclus anynana butterflies protect against invertebrate predators, specifically praying mantids. Wet season (WS) butterflies with larger, brighter eyespots were easier for mantids to detect, but more difficult to capture compared to dry season (DS) butterflies with small, dull eyespots. Mantids attacked the wing eyespots of WS butterflies more frequently resulting in greater butterfly survival and reproductive success. With a reciprocal eyespot transplant, we demonstrated the fitness benefits of eyespots were independent of butterfly behaviour. Regardless of whether the butterfly was WS or DS, large marginal eyespots pasted on the hindwings increased butterfly survival and successful oviposition during predation encounters. In previous studies, DS B. anynana experienced delayed detection by vertebrate predators, but both forms suffered low survival once detected. Our results suggest predator abundance, identity and phenology may all be important selective forces for B. anynana. Thus, reciprocal selection between invertebrate and vertebrate predators across seasons may contribute to the evolution of the B. anynana polyphenism.This is the publisher’s final pdf. The published article is copyrighted by the author(s) and published by the Royal Society. The published article can be found at: http://rspb.royalsocietypublishing.org/.Keywords: adaptive coloration, wing patterns, visual signallin

A high-coverage draft genome of the mycalesine butterfly <i>Bicyclus anynana</i>

The mycalesine butterfly Bicyclus anynana, the "Squinting bush brown," is a model organism in the study of lepidopteran ecology, development, and evolution. Here, we present a draft genome sequence for B. anynana to serve as a genomics resource for current and future studies of this important model species. Seven libraries with insert sizes ranging from 350 bp to 20 kb were constructed using DNA from an inbred female and sequenced using both Illumina and PacBio technology; 128 Gb of raw Illumina data was filtered to 124 Gb and assembled to a final size of 475 Mb (∼×260 assembly coverage). Contigs were scaffolded using mate-pair, transcriptome, and PacBio data into 10 800 sequences with an N50 of 638 kb (longest scaffold 5 Mb). The genome is comprised of 26% repetitive elements and encodes a total of 22 642 predicted protein-coding genes. Recovery of a BUSCO set of core metazoan genes was almost complete (98%). Overall, these metrics compare well with other recently published lepidopteran genomes. We report a high-quality draft genome sequence for Bicyclus anynana. The genome assembly and annotated gene models are available at LepBase (http://ensembl.lepbase.org/index.html)

decapentaplegic (dpp) regulates the growth of a morphological novelty, beetle horns. Development Genes and Evolution

Abstract Studies focusing on the development of morphological novelties suggest that patterning genes underlying traditional appendage development (i.e. mouthparts, legs, and wings) also play important roles in patterning novel morphological structures. In this study, we examine whether the expression and function of a member of the TGF-β signaling pathway, decapentaplegic (dpp), promotes development of a morphologically novel structure: beetle horns. Beetle horns are complex secondary sexual structures that develop in the head and/or prothorax, lack obvious homology to other insect outgrowths, and vary remarkably between species and sexes. We studied dpp expression through in situ hybridization, performed functional analyses with RNA interference, and gathered allometric measurements to determine the role of dpp during both pronotal and head horn development in both sexes of two morphologically dissimilar species in the Onthophagus genus, Onthophagus binodis and Onthophagus sagittarius. Our findings show that in addition to affecting growth and patterning of traditional appendages, dpp regulates beetle horn growth and remodeling

PrudicEtAl_PRSB_Data

All data (3 experiments) reported in the pape

Sex Differences in 20-Hydroxyecdysone Hormone Levels Control Sexual Dimorphism in Bicyclus anynana Wing Patterns

10.1093/molbev/msx301MOLECULAR BIOLOGY AND EVOLUTION352465-47

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

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