Over the last twenty years, researchers have found that most melanic wing color variations in butterflies and moths are governed by a specific genomic area around the ‘cortex’ protein-coding gene. Initially, it was believed that this gene acted as the melanic color switch. However, a team of global scientists has now revealed that the cortex gene does not influence melanic coloration. Instead, they discovered that a previously overlooked microRNA (miRNA) serves as the true color regulator.
Lepidopterans (butterflies and moths) showcase a remarkable array of wing color patterns. Many species feature variations of black and white or dark and bright colors that relate to the presence or absence of melanin. These color variations are often prime examples of natural selection and evolutionary processes. Notable cases include the swift rise of the melanic version of the British peppered moth Biston betularia, a change driven by the darker environments created by carbon emissions and industrialization in the late 1800s in the UK, and the mimetic evolution of Heliconius butterflies among others.
Even though the ecological factors that contribute to melanin’s presence or absence in the wings of these lepidopterans are generally understood, the genetic and developmental processes involved in color changes have been somewhat obscure.
How do butterflies and moths create black or white wings?
After extensive research over the past twenty years, scientists determined that most melanic wing color variations are linked to a specific genomic area around the protein-coding gene ‘cortex‘. The assumption was that cortex functioned as the melanic color switch. A research team from Singapore, Japan, and the USA, led by Professor Antónia MONTEIRO and Dr. Shen TIAN from the Department of Biological Sciences at the National University of Singapore (NUS), found that cortex does not play a role in melanic coloration. Instead, they identified a previously neglected microRNA (miRNA) as the real color switch.
The results were published in the journal Science on December 5, 2024.
Dr. Tian, the lead author of the study, commented, “Various pieces of evidence from earlier research cast doubt on whether cortex was indeed the melanic color switch, motivating me to explore the function of other genomic features in this area—specifically miRNAs.” He performed this research while pursuing his PhD/postdoctoral studies in Professor Monteiro’s lab at NUS, and is now a postdoctoral researcher at Duke University, USA.
“MiRNAs are small RNA molecules that do not encode proteins like most genes; however, they play vital roles in regulating genes by inhibiting the expression of target genes,” Dr. Tian explained.
In this research, Dr. Tian and his colleagues identified a miRNA adjacent to cortex, named mir-193. Using the CRISPR-Cas9 gene editing tool, they interrupted mir-193 in three distantly related butterfly lineages. This complete disruption led to the loss of black and dark wing colors in the African squinting bush brown butterfly, Bicyclus anynana, the Indian cabbage white butterfly, Pieris canidia, and the common mornon butterfly, Papilio polytes. In contrast, interrupting cortex and three other protein-coding genes from the same genomic area in B. anynana had no effect on wing colors, indicating that mir-193—not cortex or any nearby gene—is the primary regulator of melanic color in these Lepidoptera.
Further, the researchers confirmed that mir-193 comes from a long non-protein-coding RNA called ivory, and its function lies in directly suppressing several pigmentation genes. Because the sequence of mir-193 is highly conserved not just in Lepidoptera but throughout the animal kingdom, the team also examined its role in Drosophila flies. They found that mir-193 also regulates melanic coloration in these flies, suggesting that its function is preserved well beyond Lepidoptera.
Professor Monteiro remarked, “While previous studies have focused solely on the role of cortex in creating melanic color variations, this research adds a new perspective to this longstanding hypothesis and shows that a tiny, non-protein coding RNA is the actual switch that causes the rich melanic wing color diversity we observe in nature.”
“This study highlights the importance of not disregarding poorly annotated non-protein-coding RNAs, like miRNAs, in genotype-phenotype association studies; overlooking them could lead to erroneous conclusions,” Professor Monteiro further added.
Dr. Tian concluded, “The influence of non-coding RNAs on phenotypic changes remains largely unexplored. This research encourages more in-depth study into how non-coding RNAs, particularly miRNAs, may contribute to the diversification of traits in various organisms.”
