UC Berkeley

Pause for a moment, close your eyes, and picture a few of the most beautiful living organisms that come to your mind... What did you see? Perhaps the creatures were bright and shiny. Perhaps they were colorful and charismatic. Perhaps one of the beautiful creatures you pictured was a butterfly. Indeed, the diversity of colorful patterns in butterflies have captivated humans for centuries. Moreover, their wings have influenced studies in a variety of scientific fields, including evolutionary biology, ecology, and biophysics.Lepidopteran wings are covered with thousands of flat overlapping scales, each one of which derives from a single cell. The scales on an adult are cuticular projections that serve as the unit of color for the wing. Each scale can generate color through pigmentation, which results from molecules that selectively absorb certain wavelengths, or due to light interacting with physical nanoarchitecture on the scales, known as structural color. Thus far, researchers have made progress in understanding genetic pathways responsible for pigment production and the early transcription factors and signaling molecules that demarcate wing pattern positions. It remains less clear, however, what precise genes and pathways give rise to an individual wing scale cell, how such a novel cell type evolved, or what factors modulate cuticular micro- and nanostructures that generate specific optical properties. To better understand processes underlying scale and nanostructure development in Lepidoptera, my dissertation focuses on a unique optical strategy: wing transparency. The wings of butterflies and moths are typically covered with thousands of flat, overlapping scales that endow the wings with colorful patterns. Yet, numerous species of Lepidoptera have evolved highly transparent wings, which often possess scales of altered morphology and reduced size, and the presence of membrane surface nanostructures that dramatically reduce reflection. This trait has been interpreted as an adaptation in the context of camouflage, in which numerous lineages independently evolved transparent wings as a form of crypsis to reduce predation. In order to unravel the biological processes of wing transparency, I engaged in an interdisciplinary collaboration (including the labs of Nipam Patel, Marianne Elias, Doris Gomez and Serge Berthier) at the interface of physics, developmental biology and evolutionary ecology. Working in parallel with our collaborators, we aimed to investigate the structure, development and evolution of wing transparency in butterflies and moths by implementing experimental and phylogenetic comparative methods. We revealed a diversity of structural features that underlie transparent wings, notably modifications of scale morphology, size, and density, and the presence of finely-tuned nanostructures on the surface of the wing membrane that generate anti-reflective properties. We were able to characterize developmental processes of wing micro- and nanostructure formation of glasswing butterflies that were raised in the field and in the lab, and additionally utilized museum specimens and data to identify correlations between light transmission (a quantitative measure of transparency) and structural features. Together, our results provide insight into the development, ecology and evolutionary history of terrestrial transparency within Lepidoptera, highlighting multiple lineages that have independently evolved clearwing phenotypes, as well as potential trade-offs related to thermoregulation, water repellency and predation pressure. One of my main experimental systems became the so-called ‘glasswing butterfly’ Greta oto, which has thin, vertically oriented scales and nanopillars coating the wing membrane that enable omnidirectional anti-reflective properties. My collaborators and I employed a multitude of techniques, including confocal and electron microscopy, GC-MS, optical spectroscopy and analytical simulations to characterize wing development, comparing transparent and non-transparent wing regions. We found that during early wing development, scale precursor cell density was reduced in transparent regions, and cytoskeletal organization during scale growth differed between thin, bristle-like scale morphologies within transparent regions and flat, round scale morphologies within opaque regions. We also show that nanostructures on the wing membrane surface are composed of two layers: a lower layer of regularly arranged nipple-like nanostructures, and an upper layer of irregularly arranged wax-based nanopillars composed predominantly of long-chain n-alkanes. By chemically removing wax-based nanopillars, along with optical spectroscopy and analytical simulations, we demonstrate their role in generating anti-reflective properties. These findings provide insight into morphogenesis and composition of naturally organized microstructures and nanostructures, and may provide bioinspiration for new anti-reflective materials. Additionally, I undertook a comparative transcriptomic analysis to identify molecular pathways involved in scale cell development in the buckeye butterfly Junonia coenia and the giant silkmoth Antheraea polyphemus. I also investigated differential expression between two regions within the wing of A. polyphemus: a region we refer to as a transparent ‘window’ in which scale cells do not develop, and an adjacent region that undergoes canonical scale development. I then applied fluorescent in situ hybridization and CRISPR/Cas9 induced knockouts to characterize the spatiotemporal expression and function of genes involved in scale cell development. Comparative RNA-seq between J. coenia and A. polyphemus uncovered genes with similar expression levels during early pupal wing development and scale precursor differentiation, including proneural, cell cycle, and Notch signaling factors. At later pupal stages, when scale cell projections are forming and maturing, I identified genes with similar expression levels related to cytoskeletal organization, melanization, cuticle formation, and chitin-synthesis. Using stage-specific transcriptomic analysis followed by in situ hybridization, I uncover a suite of genes that likely play conserved roles in scale cell patterning and morphogenesis in butterflies and moths. I identified two achaete scute homologs (ASH1, ASH2) expressed at the scale cell precursor stage and loss of function of ASH2 resulted in the loss of scale cells. In contrast, loss of function of the Notch receptor led to overproduction and dense clusters of scale cells, likely due to improper lateral inhibition during scale precursor cell differentiation. I also identified that the ‘window’ scaleless region in A. polyphemus is associated with high expression levels of Wnt ligands, including wingless, and the bHLH transcription factor hairy, a negative regulator of sensory bristles, revealing how putative co-option of neurogenesis regulatory factors could contribute to scale cell patterning in Lepidoptera. Finally, I lay out a new and easy-to-follow protocol for portable, rapid, field-deployable amplicon sequencing through the use of new miniaturized lab equipment, which can be beneficial for biodiversity exploration and educational programs. Human-mediated environmental change is depleting biodiversity faster than it can be characterized, while invasive species cause agricultural damage, threaten human health, and disrupt native habitats. Consequently, the application of effective approaches for rapid surveillance and identification of biological samples is increasingly important to inform conservation efforts. Taxonomic assignments have been greatly advanced using sequence-based applications, such as DNA barcoding, a diagnostic technique that utilizes polymerase chain reaction (PCR) and DNA sequence analysis of standardized genetic regions. However, in many biodiversity hotspots, endeavors are often hindered by a lack of genomic infrastructure and funding for biodiversity research and restrictions on the transport of biological samples. A promising development is the advent of low-cost, miniaturized scientific equipment. Such tools can be assembled into functional laboratories to carry out genetic analyses in situ, at local institutions, field stations, or classrooms.