During the Industrial Revolution, the light, speckled British peppered moth stood out against the soot-darkened trees and city walls. This stark contrast led to the evolution of darker coloration as lighter moths were more likely to get picked off by predators, a classic example of natural selection. While the ecological factors behind this shift are well-studied, the genetic mechanisms remain unclear.
Researchers have long thought that black and darker wing color variants in Lepidoptera (butterflies and moths) were regulated by the protein-coding gene cortex.1,2 However, in 2019, Shen Tian, then a graduate student in Antónia Monteiro’s group at the National University of Singapore, made a surprising discovery. When he knocked out cortex, the expected change from black to white butterfly wings didn’t happen at a high frequency. Puzzled, he wondered whether another genetic component near the gene was also affected. This motivated him to explore an overlooked player in gene regulation—microRNAs (miRNAs), key noncoding regulators of gene expression—as a potential driver of wing color patterns.
Shen Tian, now a postdoctoral researcher at Duke University, chased his love of butterflies and combined his background in microRNAs to study the evolutionary development of butterfly wing color patterns.
Joye Zhou
Now, in a paper published in Science, Tian and his colleagues identified mir-193, a miRNA encoded near cortex in the genome, as the key regulator of wing pigmentation.3 Their findings show that disrupting mir-193 expression eliminates black and dark wing colors in three butterfly species. These results reveal that a miRNA, not cortex, controls wing coloration and underscores the role of noncoding RNAs in this phenotypic diversity.
Tian’s childhood fascination with butterflies, inspired by their beauty as he watched them rest on flowers in his backyard, ignited an early passion for biology. This curiosity eventually led him to the study of miRNAs. During graduate school, he saw an opportunity to merge his expertise in miRNA with his lifelong interest in butterflies, leading him to explore the largely understudied role of miRNAs in shaping butterfly and moth wing coloration.
Detecting miRNA in standard mRNA transcriptome libraries is challenging due to minimal annotation. To overcome this, Tian sequenced and annotated RNA and miRNA from the model butterfly Bicyclus anynana to build his own miRNA libraries. As he investigated the cortex locus, the proposed genetic regulator of wing patterns, he noticed two neighboring miRNAs: mir-193 and mir-2788.
“I thought these miRNAs would be minor regulators of gene expression—maybe they would knock the gene down a little bit, but they’re not going to have a major effect,” said Monteiro. Nevertheless, Tian was convinced this was a promising lead, so Monteiro encouraged him to explore the two miRNAs.
Homing in to elucidate the function of these two miRNAs, Tian used CRISPR-Cas9 gene editing to knockout mir-193 or mir-2788 in three different lineages of butterflies: the African squinting bush brown butterfly (B. anynana), the Indian cabbage white butterfly (Pieris canidia), and the common Mormon butterfly (Papilio polytes). This resulted in mosaic butterflies that displayed varying levels of color. However, when Tian disrupted cortex and three other protein-encoding genes in the same genomic region, there was little color change in the dark wings of mosaic butterflies. To investigate further, he crossed the mosaic mutants to generate pure homozygous miRNA knockout lines. Between mir-193 and mir-2788, Tian found that homozygous mir-193 butterflies exhibited lighter wing colors, indicating that mir-193 is the key dark color regulator across these Lepidoptera.
“I still remember when I walked into the insectary and I saw B. anynana butterflies emerging from their pupa, and I saw the [pure homozygous mutant] phenotype—the whole light brown butterfly—which was really exciting.”
Although Tian identified the miRNA, the initial gene transcript that processed mir-193 was unknown. “[Mir-193] should first be transcribed as a long sequence and there’s machinery to cut a small hairpin structure to produce these miRNAs,” said Tian. The team used RNA sequencing and identified a region where mir-193 and mir-2788 overlap in the intron of a long noncoding RNA (lncRNA) called “ivory,” which was recently discovered by two other groups.4,5
Mosaic knockout experiments demonstrated variations in wing color phenotypes in the common Mormon butterfly.
Shen Tian
Tian compared mir-193 and ivory mutants and found similar phenotypic patterns in the butterflies, suggesting these genes play similar roles and that mir-193 is derived from ivory. This observation led Tian to investigate whether mir-193 directly targeted multiple pigmentation genes by analyzing transcriptome differences between wild type and mutant butterfly wings.
“We figured out that there was this ebony gene that was misregulated in the mutants,” explained Monteiro. “Ebony has a very well-understood link to the melanin pathway and that’s how we could close the loop.”
“These lncRNA and miRNA were [previously] invisible to us due to experimental biases. They did not code for protein and were not annotated, as we didn’t have the tools to initially see their importance,” remarked Arnaud Martin, a developmental biologist at The George Washington University who was not involved in the study. Martin’s group, along with evolutionary biologist Robert Reed and his team at Cornell University, previously demonstrated ivory’s role in regulating color patterns in a different set of butterflies.4,5 Martin noted that Tian’s findings support and add to this previous research, adding that lncRNA and miRNA are two sides of the same coin where ivory produces mir-193 which targets the ebony gene.
Building on this, Tian explored whether the role of mir-193 observed in Lepidoptera extended to an outgroup like Drosophila melanogaster, despite differences in its genomic location and the absence of the ivory gene. Tian found similar effects: overexpressing mir-193 increased pigmentation, while repressing it resulted in lighter coloration, underscoring the conserved role of mir-193.
“It definitely raises new questions in whether this feature is seen in other patterned insects like fly abdomens or bumblebees,” said Martin.
- Van't Hof AE, et al. The industrial melanism mutation in British peppered moths is a transposable element. Nature. 2016;534(7605):102-105.
- Nadeau NJ, et al. The gene cortex controls mimicry and crypsis in butterflies and moths. Nature. 2016;534(7605):106-110.
- Tian S, et al. A microRNA is the effector gene of a classic evolutionary hotspot locus. Science. 2024;386(6726):1135-1141.
- Livraghi L, et al. A long noncoding RNA at the cortex locus controls adaptive coloration in butterflies. Proc Natl Acad Sci USA. 2024;121(36):e2403326121.
- Fandino RA, et al. The ivory lncRNA regulates seasonal color patterns in buckeye butterflies. Proc Natl Acad Sci USA. 2024;121(41):e2403426121.
