Tiny RNA Molecule Controls Wing Coloration in Butterflies and Moths

Lepidopterans (butterflies and moths) exhibit a splendid diversity of wing color patterns, and many species display black and white, or dark and bright, wing color pattern variants associated with the presence and absence of melanin. Many of these wing color pattern variants are textbook examples of natural selection and evolution. Iconic examples include the rapid increase in frequency of the melanic form of the British peppered moth Biston betularia, driven by the sootier and darker environment caused by carbon burning and industrialisation in the late 1800s in the United Kingdom, and the mimetic radiation of Heliconius butterflies, among others.

Despite the often well-understood ecological drivers that favour the presence or absence of melanin in the wings of these lepidopterans, the genetic and developmental basis of changes in coloration has remained unclear.

How Do Butterflies and Moths Paint Their Wings Either Black or White?

Over the past two decades, scientists discovered that the majority of melanic wing color variants are controlled by a single genomic region surrounding the protein-coding gene 'cortex'. It was assumed, then, that cortex was the melanic color switch. A team of international researchers from Singapore, Japan, and the United States of America, led by Professor Antónia MONTEIRO and Dr. Shen Tian from the Department of Biological Sciences at the National University of Singapore (NUS), discovered that cortex does not affect melanic coloration. Instead, a previously ignored microRNA (miRNA), is the actual color switch.

The findings were published in the journal Science on 5 December 2024.

Dr. Tian, the lead author of this work said, "Piles of evidence from previous studies cast doubt on whether cortex was really the melanic color switch, which inspired me to test the function of some other genomic features within this genomic region – miRNAs." He conducted this research work as a PhD/postdoctoral researcher in Professor Monteiro's laboratory 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 do, yet they play essential roles in gene regulation by repressing the expression of target genes," added Dr Tian.

In this study, Dr. Tian and colleagues found a miRNA located next to cortex, mir-193. The team disrupted mir-193 using a gene editing tool CRISPR-Cas9 in three deeply diverged lineages of butterflies. The complete disruption of mir-193 eliminated 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, disrupting cortex and three other protein-coding genes from the same genomic region in B. anynana had no effect on wing colors. This indicated that mir-193, not cortex or any other nearby gene, is the key melanic color regulator across these Lepidoptera.

The team further confirmed that mir-193 is processed from a long non-protein-coding RNA, ivory, and it functions by directly repressing multiple pigmentation genes. Since the sequence of mir-193 is deeply conserved not only in Lepidoptera but across the animal kingdom, the team also tested the role of mir-193 in Drosophila flies. Surprisingly, mir-193 was also found to control melanic coloration in these flies, suggesting a deeply conserved role for mir-193 beyond Lepidoptera.

Prof. Monteiro said, "While previous studies exclusively focused on the role of cortex in generating melanic color variations, this work brings a twist to this long-standing hypothesis and demonstrates that a small, non-protein coding RNA is the switch that, by being expressed or not expressed, brings about the diverse melanic wing color variations in nature."

"This study shows that poorly annotated non-protein-coding RNAs, such as miRNAs, should never be ignored in genotype-phenotype association studies, which would otherwise lead to misleading conclusions," added Prof. Monteiro.

The role of non-coding RNAs in phenotypic diversification is largely understudied. This study prompts further investigations on how non-coding RNAs such as miRNAs can contribute to phenotypic diversifications in organisms."

Dr. Shen Tian, Department of Biological Sciences, National University of Singapore