A man born in the 1990s who dropped out of school and quit his job did scientific research for his hobby, and his results were published in Science

He has loved observing insects since he was a child, especially butterflies. His whole family made nets for him to catch insects. When he grew up, he went to four countries to study and discovered the secret of regulating the color of butterfly wings. The relevant paper was published in Science on December 6.

Written by | Luffy

From undergraduate to doctoral studies, and then to postdoctoral work, Tian Shen has changed four countries, studying and conducting research in famous universities in Australia, Japan, Singapore and the United States. The only thing that remains unchanged is his fascination with butterflies.

Especially the color of butterfly wings, which can reflect colorful light in the sun, from deep blue to warm red, from mysterious purple to dazzling gold. These colors, like secret language hidden between wings, tell the mystery of life. These colors are not just for decoration, they are the result of the interaction between butterflies and the environment, a masterpiece of natural selection, and a manifestation of biological adaptability.

On December 6, Tian Shen, a postdoctoral fellow at the National University of Singapore, published a research paper in Science as the first author, revealing the mystery of regulating the color of butterfly wings.

At 8 a.m. Eastern Time, Fanpu connected with Tian Shen. When he turned on the camera, he smiled shyly and said, "It's okay, I'm not nervous."

Tian Shen | Source: Provided by myself

Five years of hard work

In August 2019, Tian Shen arrived at the National University of Singapore with his suitcase, and since then he has been working on his doctoral studies. In his words, "I never dared to take a break in the past five years. I didn't meet my parents until I graduated with my doctorate, and they came to see me."

Tian Shen lives in the cafeteria, dormitory and laboratory. His research project in Antonia Monteiro's laboratory at the National University of Singapore focuses on the genetic, developmental and evolutionary mechanisms of wing pattern polymorphism in Lepidoptera (butterflies and moths).

In biology, polymorphism refers to the different morphologies of organisms in the same natural population in a changing environment, such as the classic case of "industrial melanization" of the British birch moth.

This case is mentioned in high school biology textbooks: Before the Industrial Revolution in Britain, most birch moths were white so that they could blend in with the white lichens on tree trunks. After the Industrial Revolution, as pollution increased, soot killed the lichens and blackened the tree trunks, exposing the white birch moths to the black environment and making them more easily preyed on by birds. At this time, a black birch moth appeared in the population. Because the black type is more concealed and less likely to be discovered by predators in an environment polluted by soot, they quickly occupied the entire population in just a few decades.

Polymorphism is caused by changes in the DNA sequence, which results in multiple alleles on the same locus, thus producing different phenotypes. The reason why the blackened birch moth can be inherited from generation to generation is related to polymorphism.

In addition to the birch moth, there are many butterflies and moths that have evolved 100 million years apart and have similar wing color polymorphisms, such as the leaf-shaped mimicry of the dead leaf butterfly and the black and white color of the silkworm.

In a study of lepidopteran wing pattern polymorphism, a genomic region was identified as an evolutionary hotspot.

This genomic region revolves around a protein-coding gene called "cortex". Over the past decade, a large number of genome-wide association analyses have found that in the 100 million years of evolution of Lepidoptera insects, the formation of melanin-related wing pattern diversity is inseparable from this genomic region. Therefore, scientists have always believed that cortex is the key gene in this genomic region that controls whether melanin is produced or not.

However, genome-wide association analysis can only find genomic regions associated with different wing colors through statistical methods, but cannot accurately determine which gene controls the color.

A large amount of contradictory evidence makes people doubt whether cortex is really the key to controlling melanin formation? For example, after knocking out the cortex gene, only about 10% of individuals showed obvious differences. This inspired Tian Shen, and he began to test the functions of some genes other than cortex in this genomic region, including the previously ignored non-coding gene miRNA - this is Tian Shen's research "comfort zone". "When my doctoral student began to study the developmental evolution mechanism of Lepidoptera wing patterns, I subconsciously paid attention to the fact that in this hot genomic region, there is actually miRNA next to the cortex gene, but it has been ignored."

miRNAs are small non-coding RNAs that, although they do not encode proteins like most genes, play an important role in gene regulation by inhibiting the expression of target genes.

In this study, Tian Shen discovered a miRNA - mir-193, which is adjacent to the cortex gene. So he used the gene editing tool CRISPR-Cas9 to knock out mir-193 in three deeply differentiated butterflies. The study found that after knocking out mir-193, the black and dark wing colors disappeared in the African butterfly (Bicyclus anynana), the Oriental cabbage butterfly (Pieris canidia) and the jade butterfly (Papilio polytes), leaving only white/light colors.

Comparison of butterfly colors before and after knocking out mir-193 | Source: Paper

Knocking out four other protein-coding genes in the same genomic region, including cortex, did not affect wing color. Controlled experiments showed that mir-193, not cortex or any other nearby protein-coding genes, is the key to controlling the polymorphism of melanin wing color patterns in lepidopterans.

Since mir-193 exists not only in Lepidoptera, but also in the entire animal kingdom, including humans, in order to expand the universality of the research results, the team worked with the University of Michigan to test the function of mir-193 in non-lepidoptera insects, fruit flies. Surprisingly, mir-193 can also control the generation of black in fruit flies, indicating that mir-193 can even control the color of organisms outside of Lepidoptera.

"There are many people involved in this study, including members of my laboratory and Antonia Monteiro's, as well as Cheng Kumiko's laboratory at the University of Tokyo (where Tian Shen is doing his master's degree), Fujiwara Haruhiko's laboratory, and Patricia Wittkopp's laboratory at the University of Michigan."

Protect butterflies, protect articles

Tian Shen at a lecture | Source: provided by myself

Why did Tian Shen not dare to rest for a moment in these five years?

Because he wants to protect the butterfly and the article.

In 2020, the butterflies in the laboratory were infected with fungi and died one after another, causing many studies to be shelved and half-written articles to stop there. Although the laboratory was disinfected several times, the butterflies still died in batches. Fortunately, Tian Shen's butterflies were safe, but this group of "elves" made him nervous all day long.

So he planned to find a new home for the butterfly, and an Indian professor happened to have an empty room on the same floor, so Tian asked him to borrow it. "Thanks to him, I expressed my gratitude to this professor at the end of the article."

Next, Tian Shen left the dormitory every day, and the first thing he did was to go to the borrowed room to conduct related experiments "before he was contaminated". After finishing the work, he would return to his own laboratory to complete other matters. When he returned to his residence at night, the first thing he did was to "clean himself" to avoid contaminating the butterflies the next day.

During the holidays, Tian Shen became the "stay-behind experimenter" in the laboratory, helping to take care of his fellow students' experimental butterflies. Fortunately, under his care, the butterflies survived.

While Tian Shen was conducting intensive experiments in Singapore, two American teams studying butterflies also made a major breakthrough in the same research field. They found that long non-coding RNA "ivory" in the same hot genomic region can also regulate melanin production.

"The atmosphere in the academic community is relatively inclusive and open. When we learned that the other party was also conducting research, the three laboratories agreed to publish a preprint simultaneously in February this year. Their two articles were published in PNAS (Proceedings of the National Academy of Sciences) in August and October respectively. I went to congratulate them." Tian Shen said confidently, "But I am sure that our work is more detailed and complete."

Later, Tian Shen found that ivory is actually the precursor of mir-193. There is an interesting episode here. At that time, Tian Shen wanted to add an additional experiment to fully prove that "ivory is the precursor of mir-193", but also wanted to publish the article as soon as possible. He was undecided, so he went to discuss with his supervisor and finally decided to submit the paper to the journal first, and then use the time of the first round of review to complete the additional experiment.

“I think the experiment I added is very important to support a certain conclusion, so even if the reviewer did not bring it up, I hope to add this result in the subsequent review process, because I like to be a reviewer of my own article, and I hope my work is watertight.”

Sure enough, after the first round of review, all three reviewers gave high praise to this achievement. One of the reviewers proposed to add an experiment to directly prove that the precursor of mir-193 is ivory, so that the whole chain of evidence will be more complete.

In fact, before this, Tian Shen had already completed the additional experiments, so after receiving the review comments, he quickly returned the second draft. "I was even worried that the reviewers would think we were cheating," Tian Shen said with a smile.

The article was quickly accepted, and the reviewers gave it a very high evaluation. "This will have a significant impact in the field of butterfly wing patterning, and more broadly in evolutionary development, laying the foundation for a series of new studies on this topic." "This study warns that we cannot only focus on protein-coding genes in genome-wide association analysis studies."

My grandfather made me a fishing net since I was a child.

Tian Shen was born in Qinhuangdao, Hebei Province in 1993. He has been extremely interested in insects since childhood. When other children were still crying over toys, he could spend half a day alone in the small flower bed staring at butterflies and moths with great interest. His parents not only did not blame him for getting covered in mud, but also bought him many illustrated books and butterfly specimens.

Because his mother lived in Japan and his father was busy with his bank work, he lived with his grandparents since he was a child. His grandfather made him many nets for catching butterflies. At first, he used bamboo poles, but he didn't know how many of them were broken. Later, he simply replaced them with steel pipes, "which were impossible to lift."

Biology was still his favorite subject until high school. "I always put biology homework last, just like delicious food that you always want to save for last." In June 2012, Tian Shen was admitted to Nankai University. Biology was his first choice, but the result was contrary to his expectations. He was admitted to his second choice - stomatology.

Tian Shen was very frustrated that he didn't get to study biology, which he wanted. So two months after enrollment, he decided to drop out and focus on preparing for the IELTS and applying to foreign schools. In February of the following year, Tian Shen went to Australia to study biology and eventually obtained a bachelor's degree in science from the Australian National University. Due to the long separation from his mother, when considering a master's school, he chose the University of Tokyo in Japan to conduct miRNA research in Cheng Kumiko's laboratory.

In Japan, the master's program is called the "pre-doctoral program" and usually lasts for 2 years. After completing the master's program, students can continue with the "post-doctoral program", which usually lasts for 3 years and is equivalent to the doctoral degree program in China.

When he graduated with a master's degree, Tian Shen was not sure whether to continue his doctoral studies or work, so he applied for a doctorate, aiming at the world-famous "Butterfly Lab"; he also applied for positions in the industry. As a result, he received admission offers from the University of Tokyo, Cambridge University, and the National University of Singapore, and a strategic consulting company also extended an olive branch to him.

Considering that he would be admitted to the doctoral program later, Tian Shen decided to join a consulting company first and become a well-paid white-collar worker. Floating in a cubicle high in the sky all day long, holding a cup of coffee and working on a computer, if you look out into the distance, you can even see the imperial palace. Isn't this a wonderful life? But Tian Shen was at a loss for such a life. "Only after trial and error did I realize that what I really love is scientific research."

After working for three months, Tian Shen made up his mind to pursue a doctorate, but he was very conflicted about choosing between the University of Tokyo, the National University of Singapore, and the University of Cambridge.

In July 2019, Tian Shen received an email from Antonia Monteiro, his supervisor at the National University of Singapore, which asked, "School is about to start in August, when will you arrive?" So on a sunny Saturday, Tian Shen called his parents to discuss the matter and finally decided to quit his consulting job, which he had been doing for four months, and go to Singapore to study for a doctorate.

Antonia Monteiro is a Portuguese with rich artistic talent and is also obsessed with butterflies. On one wall of her house, she designed a butterfly wing pattern made of mosaic tiles, which fascinated Tian Shen. In fact, he has received art training since he was a child. When he was in junior high school, he took training classes with art exam candidates. His paintings were often the best among art exam candidates. "The teacher even suggested that I take the art exam."

"Antonia Monteiro and I got along very well, and she gave me a broad space to develop my talents. I worked here for four years as a doctoral student and one year as a postdoctoral fellow. Antonia supported me both mentally and financially. She provided a lot of support in terms of research funding, and we were able to resonate with each other when discussing issues," Tian Shen could not help but express his gratitude to his mentor, "and I remained in close contact with her until graduation."

In October this year, Tian Shen received the Hargitt fellowship from Duke University in the United States, and is now continuing his postdoctoral research on butterfly research in Anyi Mazo-Vargas' laboratory. Tian Shen said, "I hope to establish my own laboratory (in the future) and continue to do the research I like."

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