The molecular basis of scale development highlighted by a single-cell atlas of Bicyclus anynana butterfly pupal forewings.

Butterfly wings display a diversity of cell types, including large polyploid scale cells, yet the molecular basis of such diversity is poorly understood. To explore scale cell diversity at a transcriptomic level, we employ single-cell RNA sequencing of ∼5,200 large cells (>6 µm) from 22.5- to 25-h male pupal forewings of the butterfly Bicyclus anynana. Using unsupervised clustering, followed by in situ hybridization, immunofluorescence, and CRISPR-Cas9 editing of candidate genes, we annotate various cell types on the wing. We identify genes marking non-innervated scale cells, pheromone-producing glandular cells, and innervated sensory cell types. We show that senseless, a zinc-finger transcription factor, and HR38, a hormone receptor, determine the identity, size, and color of different scale cell types and are important regulators of scale cell differentiation. This dataset and the identification of various wing cell-type markers provide a foundation to compare and explore scale cell-type diversification across arthropod species.

Lepidopteran prolegs are novel traits, not leg homologs.

Lepidopteran larvae have both thoracic legs and abdominal prolegs, yet it is unclear whether these are serial homologs. A RNA-seq analysis with various appendages of Bicyclus anynana butterfly larvae indicated that the proleg transcriptome resembles the head-horn transcriptome, a novel trait in the lepidoptera, but not a thoracic leg. Under a partial segment abdominal-A (abd-A) knockout, both thoracic leg homologs (pleuropodia) and prolegs developed in the same segment, arguing that both traits are not serial homologs. Further, three of the four coxal marker genes, Sp5, Sp6-9, and araucan, were absent from prolegs, but two endite marker genes, gooseberry and Distal-less, were expressed in prolegs, suggesting that prolegs may be using a modular endite gene-regulatory network (GRN) for their development. We propose that larval prolegs are novel traits derived from the activation of a pre-existing modular endite GRN in the abdomen using abd-A, the same Hox gene that still represses legs in more lateral positions.

Antennapedia and optix regulate metallic silver wing scale development and cell shape in Bicyclus anynana butterflies.

Butterfly wing scales can develop intricate cuticular nanostructures that produce silver colors, but the underlying genetic and physical basis of such colors is mostly unexplored. Here, we characterize different types of wild-type silver scales in Bicyclus anynana butterflies and show that the varying thickness of the air layer between two cuticular laminas is most important for producing silvery broadband reflectance. We then address the function of five genes-apterous A, Ultrabithorax, doublesex, Antennapedia, and optix-in silver scale development by examining crispants with either ectopic gains or losses of silver scales. Simultaneous transformations of three parameters-loss of the upper lamina, increased lower lamina thickness, and increased pigmentation-occur when silver scales become brown and vice versa when brown scales become silver. Antennapedia and optix are high-level regulators of different silver scale types and determine cell shape in both sexes. Moreover, Antennapedia is involved in determining ridge and crossrib orientation.

Distal-less and spalt are distal organisers of pierid wing patterns.

Two genes, Distal-less (Dll) and spalt (sal), are known to be involved in establishing nymphalid butterfly wing patterns. They function in several ways: in the differentiation of the eyespot's central signalling cells, or foci; in the differentiation of the surrounding black disc; in overall scale melanisation (Dll); and in elaborating marginal patterns, such as parafocal elements. However, little is known about the functions of these genes in the development of wing patterns in other butterfly families. Here, we study the expression and function of Dll and sal in the development of spots and other melanic wing patterns of the Indian cabbage white, Pieris canidia, a pierid butterfly. In P. canidia, both Dll and Sal proteins are expressed in the scale-building cells at the wing tips, in chevron patterns along the pupal wing margins, and in areas of future scale melanisation. Additionally, Sal alone is expressed in the future black spots. CRISPR knockouts of Dll and sal showed that each gene is required for the development of melanic wing pattern elements, and repressing pteridine granule formation, in the areas where they are expressed. We conclude that both genes likely play ancestral roles in organising distal butterfly wing patterns, across pierid and nymphalid butterflies, but are unlikely to be differentiating signalling centres in pierids black spots. The genetic and developmental mechanisms that set up the location of spots and eyespots are likely distinct in each lineage.

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.

Doublesex Mediates the Development of Sex-Specific Pheromone Organs in Bicyclus Butterflies via Multiple Mechanisms.

The Bicyclus lineage of satyrid butterflies exhibits male-specific traits, the scent organs, used for chemical communication during courtship. These organs consist of tightly packed brush-like scales (hair-pencils) that rub against scent patches to disperse pheromones, but the evolution and molecular basis of these organ's male-limited development remains unknown. Here, we examine the evolution of the number and location of the scent patches and hair-pencils within 53 species of Bicyclus butterflies, and the involvement of the sex determinant gene doublesex (dsx) in scent organ development in Bicyclus anynana using CRISPR/Cas9. We show that scent patches and hair-pencils arose via multiple, independent gains, in a correlated manner. Further, an initially nonsex-specific Dsx protein expression pattern in developing wing discs becomes male-specific and spatially refined to areas that develop the scent patches. Functional perturbations of dsx show that this gene activates patch development in males whereas hair-pencils develop in both sexes without Dsx input. Dsx in females is, instead, required to repress hair-pencils whereas Dsx in males regulates minor aspects of its development. These findings suggest that the patches and hair-pencils evolve as correlated composite organs presumably due to their functional integration. Divergence in the function of dsx isoforms occurred across the sexes, where the male isoform promotes patch development in males and the female isoform represses hair-pencil development in females, both leading to the development of male-limited traits. Furthermore, evolution in number of patches in males is due to the evolution of spatial regulation of dsx.

Ptychographic X-ray computed tomography at a high-brilliance X-ray source.

Ptychographic X-ray computed tomography is a phase-contrast imaging technique capable of retrieving three-dimensional maps of the index of refraction of the imaged volumes with nanometric resolution. Despite its unmatched reach, its application remains prerogative of a limited number of laboratories at synchrotron sources. We present a detailed description of an experimental procedure and a data analysis pipeline which can be both exploited for ptychographic X-ray computed tomography experiments at any high-brilliance X-ray source. These have been validated at the I13-1 Coherence Branchline within the first experiment of its kind to be successfully carried out on a biological sample at Diamond Light Source.

apterous A specifies dorsal wing patterns and sexual traits in butterflies.

Butterflies have evolved different colour patterns on their dorsal and ventral wing surfaces to serve different signalling functions, yet the developmental mechanisms controlling surface-specific patterning are still unknown. Here, we mutate both copies of the transcription factor apterous in Bicyclus anynana butterflies using CRISPR/Cas9 and show that apterous A, expressed dorsally, functions both as a repressor and modifier of ventral wing colour patterns, as well as a promoter of dorsal sexual ornaments in males. We propose that the surface-specific diversification of wing patterns in butterflies proceeded via the co-option of apterous A or its downstream effectors into various gene regulatory networks involved in the differentiation of discrete wing traits. Further, interactions between apterous and sex-specific factors such as doublesex may have contributed to the origin of sexually dimorphic surface-specific patterns. Finally, we discuss the evolution of eyespot number diversity in the family Nymphalidae within the context of developmental constraints due to apterous regulation.

Evolutionary Assembly of Communities in Butterfly Mimicry Rings.

Species co-occurrence in ecological communities is thought to be influenced by multiple ecological and evolutionary processes, especially colonization and competition. However, effects of other interspecific interactions and evolutionary relationships are less explored. We examined evolutionary histories of community members and roles of mutualistic and parasitic interactions (Müllerian and Batesian mimicry, respectively) in the assembly of mimetic butterfly communities called mimicry rings in tropical forests of the Western Ghats, India. We found that Müllerian mimics were phylogenetically clustered, sharing aposematic signals due to common ancestry. On the other hand, Batesian mimics joined mimicry rings through convergent evolution and random phylogenetic assembly. Since the Western Ghats are a habitat island, we compared species diversity and composition in its mimicry rings with those of habitat mainland to test effects of biogeographic connectivity. The Western Ghats consisted of fewer mimicry rings and an overall smaller number of aposematic species and mimics compared to habitat mainland. The depauperate mimicry rings in the Western Ghats could have resulted from stochastic processes, reflecting their long temporal and spatial isolation and trickling colonization by the mimetic butterfly communities. These results highlight how evolutionary history, biogeographic isolation, and stochastic colonization influence the evolutionary assembly and diversity of ecological communities.

Molecular mechanisms of secondary sexual trait development in insects.

Secondary sexual traits are those traits other than the primary gametes that distinguish the sexes of a species. The development of secondary sexual traits occurs when sexually dimorphic factors, that is, molecules differentially produced by primary sex determination systems in males and females, are integrated into the gene regulatory networks responsible for sexual trait development. In insects, these molecular asymmetric factors were always considered to originate inside the trait-building cells, but recent work points to external factors, such as hormones, as potential candidates mediating secondary sexual trait development. Here, we review examples of the different molecular mechanisms producing sexually dimorphic traits in insects, and suggest a need to revise our understanding of secondary sexual trait development within the insect lineage.