Temporal gene expression variation associated with eyespot size plasticity in Bicyclus anynana.

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 Bicyclus anynana. 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 earlier 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.

Oliver JC ，Ramos D ，Prudic KL ，Monteiro A ... -  《PLoS One》

Differential involvement of Hedgehog signaling in butterfly wing and eyespot development.

Butterfly eyespots may have evolved from the recruitment of pre-existent gene circuits or regulatory networks into novel locations on the wing. Gene expression data suggests one such circuit, the Hedgehog (Hh) signaling pathway and its target gene engrailed (en), was recruited from a role in patterning the anterior-posterior insect wing axis to a role patterning butterfly eyespots. However, while Junonia coenia expresses hh and en both in the posterior compartment of the wing and in eyespot centers, Bicyclus anynana lacks hh eyespot-specific expression. This suggests that Hh signaling may not be functioning in eyespot development in either species or that it functions in J. coenia but not in B. anynana. In order to test these hypotheses, we performed functional tests of Hh signaling in these species. We investigated the effects of Hh protein sequestration during the larval stage on en expression levels, and on wing size and eyespot size in adults. Hh sequestration led to significantly reduced en expression and to significantly smaller wings and eyespots in both species. But while eyespot size in B. anynana was reduced proportionately to wing size, in J. coenia, eyespots were reduced disproportionately, indicating an independent role of Hh signaling in eyespot development in J. coenia. We conclude that while Hh signaling retains a conserved role in promoting wing growth across nymphalid butterflies, it plays an additional role in eyespot development in some, but not all, lineages of nymphalid butterflies. We discuss our findings in the context of alternative evolutionary scenarios that led to the differential expression of hh and other Hh pathway signaling members across nymphalid species.

Tong X ，Lindemann A ，Monteiro A 《PLoS One》

Origin of the mechanism of phenotypic plasticity in satyrid butterfly eyespots.

Plasticity is often regarded as a derived adaptation to help organisms survive in variable but predictable environments, however, we currently lack a rigorous, mechanistic examination of how plasticity evolves in a large comparative framework. Here, we show that phenotypic plasticity in eyespot size in response to environmental temperature observed in atyrid butterflies is a complex derived adaptation of this lineage. By reconstructing the evolution of known physiological and molecular components of eyespot size plasticity in a comparative framework, we showed that 20E titer plasticity in response to temperature is a pre-adaptation shared by all butterfly species examined, whereas expression of EcR in eyespot centers, and eyespot sensitivity to 20E, are both derived traits found only in a subset of species with eyespots.

Bhardwaj S ，Jolander LS ，Wenk MR ，Oliver JC ，Nijhout HF ，Monteiro A ... -  《eLife》

Hox genes are essential for the development of eyespots in Bicyclus anynana butterflies.

The eyespot patterns found on the wings of nymphalid butterflies are novel traits that originated first in hindwings and subsequently in forewings, suggesting that eyespot development might be dependent on Hox genes. Hindwings differ from forewings in the expression of Ultrabithorax (Ubx), but the function of this Hox gene in eyespot development as well as that of another Hox gene Antennapedia (Antp), expressed specifically in eyespots centers on both wings, are still unclear. We used CRISPR-Cas9 to target both genes in Bicyclus anynana butterflies. We show that Antp is essential for eyespot development on the forewings and for the differentiation of white centers and larger eyespots on hindwings, whereas Ubx is essential not only for the development of at least some hindwing eyespots but also for repressing the size of other eyespots. Additionally, Antp is essential for the development of silver scales in male wings. In summary, Antp and Ubx, in addition to their conserved roles in modifying serially homologous segments along the anterior-posterior axis of insects, have acquired a novel role in promoting the development of a new set of serial homologs, the eyespot patterns, in both forewings (Antp) and hindwings (Antp and Ubx) of B. anynana butterflies. We propose that the peculiar pattern of eyespot origins on hindwings first, followed by forewings, could be due to an initial co-option of Ubx into eyespot development followed by a later, partially redundant, co-option of Antp into the same network.

Matsuoka Y ，Monteiro A 《-》

Over-expression of Ultrabithorax alters embryonic body plan and wing patterns in the butterfly Bicyclus anynana.

In insects, forewings and hindwings usually have different shapes, sizes, and color patterns. A variety of RNAi experiments across insect species have shown that the hox gene Ultrabithorax (Ubx) is necessary to promote hindwing identity. However, it remains unclear whether Ubx is sufficient to confer hindwing fate to forewings across insects. Here, we address this question by over-expressing Ubx in the butterfly Bicyclus anynana using a heat-shock promoter. Ubx whole-body over-expression during embryonic and larvae development led to body plan changes in larvae but to mere quantitative changes to adult morphology, respectively. Embryonic heat-shocks led to fused segments, loss of thoracic and abdominal limbs, and transformation of head limbs to larger appendages. Larval heat-shocks led to reduced eyespot size in the expected homeotic direction, but neither additional eyespots nor wing shape changes were observed in forewings as expected of a homeotic transformation. Interestingly, Ubx was found to be expressed in a novel, non-characteristic domain - in the hindwing eyespot centers. Furthermore, ectopic expression of Ubx on the pupal wing activated the eyespot-associated genes spalt and Distal-less, known to be directly repressed by Ubx in the fly׳s haltere and leg primordia, respectively, and led to the differentiation of black wing scales. These results suggest that Ubx has been co-opted into a novel eyespot gene regulatory network, and that it is capable of activating black pigmentation in butterflies.

Tong X ，Hrycaj S ，Podlaha O ，Popadic A ，Monteiro A ... -  《-》