Eyespot Centers Research Articles

Expression of Multiple engrailed Family Genes in Eyespots of Bicyclus anynana Butterflies Does Not Implicate the Duplication Events in the Evolution of This Morphological Novelty

Gene duplication events often create genetic redundancy that can either lead to the appearance of pseudogenes or, instead create opportunities for the evolution of novel proteins that can take on new functions. One of the genes which has been widely studied with respect to gene duplication is engrailed (en). En-family proteins are expressed in a morphological novelty, eyespots (in the center and in the outer gold ring), in the African squinting bush brown butterfly Bicyclus anynana, as well as in a more conserved pattern, the posterior compartment of a wing. In the present study, we used whole-genome sequencing and transcriptome data to show the presence of three en-family genes and their differential expression on the pupal wings of B. anynana using in-situ hybridization. The results suggest two duplication events of en-family genes, the first evidence of a two-fold duplication in the Lepidoptera. We propose that all copies initially had posterior wing compartment expression and all copies subsequently gained a novel expression domain associated with eyespot centers. Two copies secondarily lost the posterior compartment expression, and one copy alone gained the outer ring expression domain. By dating the origin of both duplication events, however, we conclude that they predate the origin of eyespots by at least 60 mya, and hence our data does not support the retention of the multiple en gene duplicates in the genome via their involvement with the novel eyespot evolutionary innovation.

Interacting Effects of Eyespot Number and Ultraviolet Reflectivity on Predation Risk in Bicyclus anynana (Lepidoptera: Nymphalidae).

Small marginal eyespots on lepidopteran wings are conspicuous elements that attract a predator’s attention to deflect attacks away from the body, but the role of ultraviolet (UV) reflectivity at the center of these patterns and variation in eyespot number in altering the function of eyespots remains unclear. Here, we performed a field-based predation experiment with artificial prey items based on the appearance of squinting bush brown butterflies Bicyclus anynana (Butler, 1879). We tested how two visual properties of the wing pattern affect predation risk: i) the number of eyespots on the ventral forewing surface—two or four; and ii) the UV reflectivity of eyespot centers—normal (where the UV reflectivity of the centers contrasts strongly with that of the darker surrounding ring) or blocked (where this contrast is reduced). In total, 807 prey items were deployed at two sites. We found a significant interaction between the number of ventral forewing eyespots and UV reflectivity in the eyespot centers: in items with fewer eyespots, blocking UV resulted in increased predation risk whereas in items with more eyespots, blocking UV resulted in decreased predation risk. If higher predation of paper models can be equated with higher levels of wing margin/eyespot conspicuity, these results demonstrate that UV reflectivity is an important factor in making eyespots more conspicuous to predators and suggest that the fitness of particular butterfly eyespot number variants may depend on the presence or absence of UV in their centers and on the ability of local predator guilds to detect UV.

Male Bicyclus anynana Butterflies Choose Females on the Basis of Their Ventral UV-Reflective Eyespot Centers.

Butterflies often use their dorsal and ventral wing color patterns for distinct signaling functions. Color patterns on hidden dorsal wing surfaces are often used in sexual signaling, while exposed ventral patterns are often used to ward off predator attacks. At rest, however, part of the ventral forewings are often hidden by the hindwings, allowing individuals to also use the patterns on this wing surface for sexual signaling. Here, we test this hypothesis in Bicyclus anynana (Butler, Lepidoptera, Nymphalidae) butterflies by first determining the degree of sexual dimorphism in ventral forewing patterns, focusing on the eyespots, from both wet and dry season forms, and then testing the role of the larger ventral forewing eyespots of dry season females in male mate choice. We also test male investment in reproduction. We show that ventral forewing UV-reflective eyespot centers, in addition to dorsal forewing eyespot centers previously examined in this species, play a role in sexual signaling as males preferentially mated with females with their ventral eyespot centers intact instead of blocked with black paint. This male preference, however, did not translate into a detectable higher reproductive investment via a single mating toward ornamented females. This study provides an example of how ventral forewing patterns, often hidden by hindwings, are used in sexual communication, in this case by females to attract males.

Wingless is a positive regulator of eyespot color patterns in Bicyclus anynana butterflies

Eyespot patterns of nymphalid butterflies are an example of a novel trait yet, the developmental origin of eyespots is still not well understood. Several genes have been associated with eyespot development but few have been tested for function. One of these genes is the signaling ligand, wingless, which is expressed in the eyespot centers during early pupation and may function in eyespot signaling and color ring differentiation. Here we tested the function of wingless in wing and eyespot development by down-regulating it in transgenic Bicyclus anynana butterflies via RNAi driven by an inducible heat-shock promoter. Heat-shocks applied during larval and early pupal development led to significant decreases in wingless mRNA levels and to decreases in eyespot size and wing size in adult butterflies. We conclude that wingless is a positive regulator of eyespot and wing development in B. anynana butterflies.

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

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 Bicyclus anynana, 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.

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.

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.

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.

The combined effect of two mutations that alter serially homologous color pattern elements on the fore and hindwings of a butterfly

BackgroundThe ability for serially homologous structures to acquire a separate identity has been primarily investigated for structures dependent on Hox gene input but is still incompletely understood in other systems. The fore and hindwings of butterflies are serially homologous structures as are the serially homologous eyespots that can decorate each of these wings. Eyespots can vary in number between fore and hindwings of the same individual and mutations of large effect can control the total number of eyespots that each of the wings displays. Here we investigate the genetics of a new spontaneous color pattern mutation, Missing, that alters eyespot number in the nymphalid butterfly, Bicyclus anynana. We further test the interaction of Missing with a previously described mutation, Spotty, describe the developmental stage affected by Missing, and test whether Missing is a mutant variant of the gene Distal-less via a linkage association study.ResultsMissing removes or greatly reduces the size of two of the hindwing eyespots from the row of seven eyespots, with no detectable effect on the rest of the wing pattern. Offspring carrying a single Missing allele display intermediate sized eyespots at these positions. Spotty has the opposite effect of Missing, i.e., it introduces two extra eyespots in homologous wing positions to those affected by Missing, but on the forewing. When Missing is combined with Spotty the size of the two forewing eyespots decreases but the size of the hindwing spots stays the same, suggesting that these two mutations have a combined effect on the forewing such that Missing reduces eyespot size when in the presence of a Spotty mutant allele, but that Spotty has no effect on the hindwing. Missing prevents the complete differentiation of two of the eyespot foci on the hindwing. We found no evidence for any linkage between the Distal-less and Missing genes.ConclusionThe spontaneous mutation Missing controls the differentiation of the signaling centers of a subset of the serial homologous eyespots present on both the fore and the hindwing in a dose-dependent fashion. The effect of Missing on the forewing, however, is only observed when the mutation Spotty introduces additional eyespots on this wing. Spotty, on the other hand, controls the differentiation of eyespot centers only on the forewing. Spotty, unlike Missing, may be under Ubx gene regulation, since it affects a subset of eyespots on only one of the serially homologous wings.