Antennapedia regulates metallic silver wing scale development and cell shape in Bicyclus anynana butterflies

Butterfly wing scale cells can develop very intricate cuticular nanostructures that interact with light to produce structural colors such as silver, but the genetic basis of such nanostructures is mostly unexplored. Here, we address the genetic basis of metallic silver scale development by leveraging existing crispants in the butterfly Bicyclus anynana, where knockouts of five genes – apterous A, Ultrabithorax, doublesex, Antennapedia and optix – either led to ectopic gains or losses of silver scales. Most wildtype silver scales had low amounts of pigmentation and exhibited a common ultrastructural modification for metallic broadband reflectance, i.e., an undulatory air layer enclosed by an upper and lower lamina. Crispant brown scales differed from wildtype silver scales via the loss of the continuous upper lamina, increased lower lamina thickness, and increased pigmentation. The reverse was seen when brown scales became silver. On the forewings, we identified Antennapedia as a high-level selector gene, acting through doublesex to induce silver scale development in males and having a novel, post-embryonic role in the determination of ridge and crossrib orientation and overall scale cell shape in both sexes. We propose that apterous A and Ultrabithorax repress Antennapedia on the dorsal forewings and ventral hindwings, respectively, thereby repressing silver scale development, whereas apterous A activates the same GRN on the dorsal hindwings, promoting silver scales.

Dissections of Larval, Pupal and Adult Butterfly Brains for Immunostaining and Molecular Analysis

Butterflies possess impressive cognitive abilities, and investigations into the neural mechanisms underlying these abilities are increasingly being conducted. Exploring butterfly neurobiology may require the isolation of larval, pupal, and/or adult brains for further molecular and histological experiments. This procedure has been largely described in the fruit fly, but a detailed description of butterfly brain dissections is still lacking. Here, we provide a detailed written and video protocol for the removal of Bicyclus anynana adult, pupal, and larval brains. This species is gradually becoming a popular model because it uses a large set of sensory modalities, displays plastic and hormonally controlled courtship behaviour, and learns visual mate preference and olfactory preferences that can be passed on to its offspring. The extracted brain can be used for downstream analyses, such as immunostaining, DNA or RNA extraction, and the procedure can be easily adapted to other lepidopteran species and life stages.

Stage- and sex-specific transcriptome analyses reveal distinctive sensory gene expression patterns in a butterfly

Background Animal behavior is largely driven by the information that animals are able to extract and process from their environment. However, the function and organization of sensory systems often change throughout ontogeny, particularly in animals that undergo indirect development. As an initial step toward investigating these ontogenetic changes at the molecular level, we characterized the sensory gene repertoire and examined the expression profiles of genes linked to vision and chemosensation in two life stages of an insect that goes through metamorphosis, the butterfly Bicyclus anynana. Results Using RNA-seq, we compared gene expression in the heads of late fifth instar larvae and newly eclosed adults that were reared under identical conditions. Over 50 % of all expressed genes were differentially expressed between the two developmental stages, with 4,036 genes upregulated in larval heads and 4,348 genes upregulated in adult heads. In larvae, upregulated vision-related genes were biased toward those involved with eye development, while phototransduction genes dominated the vision genes that were upregulated in adults. Moreover, the majority of the chemosensory genes we identified in the B. anynana genome were differentially expressed between larvae and adults, several of which share homology with genes linked to pheromone detection, host plant recognition, and foraging in other species of Lepidoptera. Conclusions These results revealed promising candidates for furthering our understanding of sensory processing and behavior in the disparate developmental stages of butterflies and other animals that undergo metamorphosis.

Developmental plasticity in male courtship in Bicyclus anynana butterflies is driven by hormone regulation of the yellow gene

The organizational role for hormones in the regulation of sexual behavior is currently poorly explored. Previous work showed that seasonal variation in levels of the steroid hormone 20-hydroxyecdysone (20E) during pupal development regulates plasticity in male courtship behavior in Bicyclus anynana butterflies. Wet season (WS) males, reared at high temperature, have high levels of 20-hydroxyecdysone (20E) during pupation and become active courters. Dry season (DS) males, reared at low temperatures, have lower levels of 20E and lower courtship rates. Rescue of WS courtship rates can be achieved via injection of 20E into DS male pupae, but it is still unknown whether 20E alters gene expression in the pupal brain, and if so, the identity of those targets. Using transcriptomics, qPCR, and behavioral assays with a transgenic knockout, we show that higher expression levels of the yellow gene in DS male pupal brains, relative to WS brains, represses courtship in DS males. Furthermore, injecting DS males with 20E downregulates yellow to WS levels 4 hours post-injection, revealing a hormone sensitive window that determines courtship behavior. These findings are in striking contrast to Drosophila, where yellow is required for active male courtship behavior. We conclude that 20E plays an organizational role during pupal brain development by regulating the expression of yellow, which is a repressor of the neural circuity for male courtship behavior in B. anynana. This work shows that similar to vertebrates, hormones can also play an organizational role in insect brains, leading to permanent changes in adult sexual behavior.Significance StatementBehavioral plasticity in adult insects is known to be regulated by hormones, which activate neural circuits in response to environmental cues. Here, we show that hormones can also regulate adult behavioral plasticity by altering gene expression during brain development, adjusting the insect’s behavior to predictable seasonal environmental variation. We show that seasonal changes in the hormone 20E alters expression of the yellow gene in the developing pupal brain of Bicyclus anynana butterflies, which leads to differences in male courtship behavior between the dry and wet seasonal forms. This work provides one of the first examples of the organizational role of hormones in altering gene expression and adult sexual behavior in the developing insect brain.

Predation favours Bicyclus anynana butterflies with fewer forewing eyespots

There are fewer eyespots on the forewings versus hindwings of nymphalids but the reasons for this uneven distribution remain unclear. One possibility is that, in many butterflies, the hindwing covers part of the ventral forewing at rest and there are fewer forewing sectors to display eyespots (covered eyespots are not continuously visible and are less likely to be under positive selection). A second explanation is that having fewer forewing eyespots confers a selective advantage against predators. We analysed wing overlap at rest in 275 nymphalid species with eyespots and found that many have exposed forewing sectors without eyespots: i.e. wing overlap does not constrain the forewing from having the same number or more eyespots than the hindwing. We performed two predation experiments with mantids to compare the relative fitness of and attack damage patterns on two forms of Bicyclus anynana butterflies, both with seven hindwing eyespots, but with two (in wild-type) or four (in Spotty) ventral forewing eyespots. Spotty experienced more intense predation on the forewings, were shorter-lived and laid fewer eggs. These results suggest that predation pressure limits forewing eyespot number in B. anynana . This may occur if attacks on forewing eyespots have more detrimental consequences for flight than attacks on hindwing eyespots.

optix is involved in eyespot development via a possible positional information mechanism

optix, a gene essential and sufficient for eye development in Drosophila melanogaster, also plays important roles in the development of both the structure and pigmentation of butterfly wing scales. In particular, optix regulates wing scale lower lamina thickness and ommochrome pigment synthesis. Here we explore the role of optix in wing pattern development of Bicyclus anynana butterflies by examining its expression using immunostainings and testing its function via CRISPR-Cas9. We found Optix to be expressed in multiple domains, most prominently in the orange ring of the eyespots and in other scattered orange scales, and to regulate the pigmentation and the development of the upper lamina of the orange scales. We further explored the interaction of Optix with Spalt, a protein involved in the development of black scales in the eyespots, and expressed adjacent to the Optix domain. CRISPR knockouts of optix or spalt, followed by immunostainings, showed that Spalt represses optix expression in cells of the central black region of the eyespot. This regulatory interaction mimics that found in the anterior compartment of the wing disc where both genes respond to Decapentaplegic (Dpp) signaling and play a role in venation patterning. Using in situ hybridizations we show that dpp is expressed in the center of the eyespots and propose that this same circuit might have been recruited for eyespot development where Decapentaplegic acts as a central morphogen, activating optix and spalt at different concentration thresholds, and where spalt cross-regulates optix resulting in the formation of a sharp boundary between the two eyespot color rings.

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.

Sexual selection drives maladaptive learning under climate warming

Current predictions for the effects of the climate crisis on biodiversity loss have so far ignored the effects of learning ability and sexual selection. Using the African butterfly Bicyclus anynana, which shows strong phenotypic plasticity in response to temperature, we show that learning produces a maladaptive mate preference under climate warming. We modelled climate warming and found that as temperature becomes an unreliable cue at the onset of the dry season, adult butterflies displayed the wet season rather than the dry season form. Female learning further suppressed their innate, adaptive sexual preference for dry season males. Instead, females learned to prefer a phenotype transiently present during the seasonal transition. Female fertility and longevity were also affected by learning, reducing female fitness following climate warming. Our results emphasize the importance of sexual selection, learning, and their fitness consequences for understanding (mal)adaptation of natural populations to climate warming.

Molecular mechanisms underlying simplification of venation patterns in holometabolous insects

ABSTRACTHow mechanisms of pattern formation evolve has remained a central research theme in the field of evolutionary and developmental biology. The mechanism of wing vein differentiation in Drosophila is a classic text-book example of pattern formation using a system of positional information, yet very little is known about how species with a different number of veins pattern their wings, and how insect venation patterns evolved. Here, we examine the expression pattern of genes previously implicated in vein differentiation in Drosophila in two butterfly species with more complex venation Bicyclus anynana and Pieris canidia. We also test the function of some of these genes in B. anynana. We identify both conserved as well as new domains of decapentaplegic, engrailed, invected, spalt, optix, wingless, armadillo, blistered and rhomboid gene expression in butterflies, and propose how the simplified venation in Drosophila might have evolved via loss of decapentaplegic, spalt and optix gene expression domains, via silencing of vein-inducing programs at Spalt-expression boundaries, and via changes in expression of vein maintenance genes.

Cell Dissociation from Butterfly Pupal Wing Tissues for Single-Cell RNA Sequencing

Butterflies are well known for their beautiful wings and have been great systems to understand the ecology, evolution, genetics, and development of patterning and coloration. These color patterns are mosaics on the wing created by the tiling of individual units called scales, which develop from single cells. Traditionally, bulk RNA sequencing (RNA-seq) has been used extensively to identify the loci involved in wing color development and pattern formation. RNA-seq provides an averaged gene expression landscape of the entire wing tissue or of small dissected wing regions under consideration. However, to understand the gene expression patterns of the units of color, which are the scales, and to identify different scale cell types within a wing that produce different colors and scale structures, it is necessary to study single cells. This has recently been facilitated by the advent of single-cell sequencing. Here, we provide a detailed protocol for the dissociation of cells from Bicyclus anynana pupal wings to obtain a viable single-cell suspension for downstream single-cell sequencing. We outline our experimental design and the use of fluorescence-activated cell sorting (FACS) to obtain putative scale-building and socket cells based on size. Finally, we discuss some of the current challenges of this technique in studying single-cell scale development and suggest future avenues to address these challenges.