The Sound of the Genes in the Silence of the Valleys

Abstract
Butterflies and moths are now at the center of a global genomic revolution. In the Alpine village of Malles, the 10kLepGenomes and Project Psyche expeditions begin, aiming to sequence all 11,000 European Lepidoptera. High-altitude huts become improvised laboratories where young researchers identify species without Internet access. Fieldwork blends intuition and experience as traps are placed across contrasting microenvironments. Collected specimens are then sent for advanced DNA and RNA analyses. A crucial role is played by the Spanish laboratories CNAG – National Center for Genomic Analysis, CRG – Centre for Genomic Regulation and the University of Barcelona, together with Andorra Research & Innovation. Here, long-read RNA sequencing has revealed thousands of previously hidden noncoding genes. These advances support the broader Earth BioGenome Project, which aims to map 1.8 million species. A major challenge remains the identification of micromoths, often indistinguishable to the naked eye. DNA barcoding enables increasingly precise discrimination between closely related species. The genomic data reveal how Lepidoptera evolve, migrate, and adapt to climate change. The information gathered may guide more effective agricultural and conservation strategies.
In the silence of the Alps, each moth becomes a vital fragment of the planet’s genetic library.

Researcher Joana Isabel Meier, group leader at the Wellcome Sanger Institute of Cambridge (UK), catches moths with a net in the woods of Malles Venosta, Bolzano, Italy. «By studying and comparing the genomes of different species, we can find out how they have evolved and how they’ve diversified, as there have been different climatic shifts in Europe. And the genomes can help to tell us why it is that some groups of Lepidoptera have evolved into a greater number of species than others».
The "Alte Pforzheimer Hütte", a stone house originally built in Malles Venosta in 1901, served as a base camp for lepidopterists who hunted rare moths in the Italian Alps.
Researchers check the types of moths they collected during a trapping session at the Alte Pforzheimer Hütte refuge in Malles Venosta, Bolzano, Italy.

Malles Venosta, the village where the mountains guard the moths’ secrets
The adventure of the 10kLepGenomes project, part of the international Project Psyche initiative, begins in Malles Venosta, a small village nestled among the mountains of the Upper Val Venosta in South Tyrol. The choice is no coincidence: for years, Malles has been a privileged study area for the Society for European Lepidopterology (SEL) thanks to its extraordinary biodiversity, shaped by its unique geographical position and its alpine–Mediterranean climate.
Thanks to the efforts of Gerhard Tarmann and his colleagues at the Tiroler Landesmuseen in Innsbruck, more than 3,000 butterfly species have been recorded here. But what impresses researchers even more is the open and collaborative attitude of the local population, which has long opposed the excessive use of pesticides in agriculture, thereby fostering an ideal environment for studying insects.

On an alpine mountain trail above Malles Venosta, Italy, Teo Valentino, a Ph.D. student in biology at the University of Neuchâtel, in Switzerland, captured a moth lured to a light trap. Samples taken from the moth were sent to Cambridgeshire, England, so that the creature’s genome could be sequenced. That sequence will eventually be added to a database maintained by the Earth BioGenome Project, which aims to sequence the genome of every species of plant, animal, fungus, and many other organisms.
Entomologist Benjamin Wiesmair [at right] uses his smartphone to consult a Lepidoptera database to identify the species of moths captured during a trapping session on an alpine trail above Malles Venosta, Italy. Clara Spilker and Alena Sucháčková [middle] consult a table to determine whether the species are needed for genome sequencing.
Some scientists consult a manual to identify the species of moths captured during a trapping session on an alpine trail above Malles Venosta, Italy.

Dawn has just broken on a crisp July morning. In a stone mountain hut at more than 2,000 meters of altitude, a small group of young European lepidopterists has gathered around a wooden table. On the table, dozens of plastic jars hold the catches from the previous night.
At one end of the table, Gioele Moro of the Czech Academy of Sciences gently frees the moths tangled in the nets. At the other, Laura Torrado-Blanco from the University of Oviedo flips through identification guides — there is no Internet at 2,300 meters. “It’s Erebia palarica,” she says, pointing to her tattoo. “Look at this one,” says Pep Lancho Silva from the Institute of Evolutionary Biology in Barcelona, holding up a large white moth. It could be Arctia flavia, the striking tiger moth that lives only at very high altitudes. And it is precisely for species like this that the group has climbed so high: to collect them, catalogue them, and analyze their DNA.

Many moths, attracted to the ultraviolet lights, were captured during a light-trapping excursion near Malles Venosta, Italy.
Shortly before sunset, researcher Gioele Moro, of the Czech Academy of Sciences, sets up a moth trap on a mountain slope above the stone hut (the Alte Pforzheimer Hütte) in the Italian Alps. «Catching moths relies heavily on intuition, which comes from experience, perception, and judgment. Placing traps in different "microenvironments" will likely yield a wider range of specimens. But there's no formula for this. In a sense, you have to think like a moth, imagining the path it might take, analyzing the light, vegetation, and wind that might influence its flight path».
One of the UV light traps used by lepidopterists to catch moths at night is located in the mountains of Malles Venosta, Bolzano, Italy. Moth hunting with UV light primarily exploits the natural behavior of moths and other nocturnal insects toward light, a phenomenon known as "positive phototaxis".

Between mountain slopes and genomes: a race against time
For centuries, lepidopterists have captured and classified winged insects, but today their work is part of an unprecedented scientific mission. Project Psyche aims to sequence the genome of all 11,000 European lepidopteran species, as part of the Darwin Tree of Life and the monumental Earth BioGenome Project. On the first afternoon of fieldwork, Gioele Moro places light traps on different mountain slopes to compare microhabitats. At dawn he returns with more than 150 specimens, including the striking yellow tiger moth. The selected species are photographed, frozen with dry ice, and shipped to the Wellcome Sanger Institute in England, where high-precision genetic analyses will take place.

A few of the scores of moths captured on a single night at a site in the Italian Alps are lined up on a bench in the stone hut. Researchers will identify the moths’ species and some of the insects will be sent on for tissue sampling and eventual genome sequencing.
A large specimen of “Hawk moth” is preserved in a jar prior to dissection at the "Alte Pforzheimer Hütte" in Malles Venosta, Bolzano, Italy.
A "Lomographa vestaliata" moth inside a jar ready to be dissected and sequenced at the Clusio forest, Bolzano, Italy.

It is here that the most urgent applications of these genomic data become clear. As Meier explains: “Every year, billions and billions of euros are lost because, in agriculture, some species cause enormous damage.” Sequencing helps reveal the genetic basis of these destructive capacities and helps develop strategies to mitigate them. Wright adds another crucial dimension: “Pests are moving into new regions where previously they weren’t present, causing huge losses because the crops there haven’t been developed to be protected against these new species.” Understanding why certain insects succeed in new environments — while others fail — is only possible by analysing many genomes. And, as Wright emphasizes: “It’s a dynamic situation, monitoring these pests’ movements”.
This challenge requires a small army of students, researchers, and citizen-scientists. One of the goals of the expedition is to refine best practices for collecting samples destined for genome sequencing and to train a new generation of lepidopterists who can navigate genetics, bioinformatics, and genome annotation. The success of these techniques underpins not only Project Psyche, but ultimately the entire Earth BioGenome Project.

At the crack of dawn in the stone hut, researchers [from left] Eric Toro Delgado, Laura Torrado-Blanco, Mónica Doblas-Bajo, and Gioele Moro (standing) unpack and examine the moths captured during the previous night in Alte Pforzheimer Hütte, Malles Venosta, Italy.
The researcher Gioele Moro handles a yellow tiger moth, Arctia flavia, is among the catch at the stone hut, at an altitude of 2,300 meters in the Alps of Malles Venosta, Italy.
Lepidopterist Charlotte Wright of the Wellcome Sanger Institute, a leader of Project Psyche, collects moths trapped in a UV light trap on mountain trails in Malles Venosta, Bolzano, Italy. «Pests are moving to new regions where previously they weren’t present and causing huge losses because the crops there haven’t been developed to be protected against these new species. The reasons why some species succeed in a new area as climate changes, and are able to adapt and thrive, are also understandable only by studying many genomes—of the creatures that succeed, as well as the ones that don’t».
At the Hotel Tyrol in Malles Venosta, lepidopterist Charlotte Wright, researcher at the Wellcome Sanger Institute of Cambridge (UK), dissects a specimen of "Tiger moth” in the temporal laboratory set up at the Hotel Tyrol in Malles Venosta, Bolzano, Italy. Packed with liquid nitrogen, the tissue samples will subsequently be sent to the institute in England for genome sequencing.
Charlotte Wright, researcher at the Wellcome Sanger Institute of Cambridge (UK), dissects a specimen of "Tiger moth” in the temporal laboratory set up at the Hotel Tyrol in Malles Venosta, Bolzano, Italy.
Researcher Joana Isabel Meier places the abdomen of a moth into a jar with an identification code at the temporal laboratory set up at the Hotel Tyrol in Malles Venosta, Bolzano, Italy. The barcode allows the location of the sample to be tracked, which is then entered into a database with all the associated information, such as collection location, species, and collector. The barcode allows multiple samples to be simultaneously selected from a freezer and used for large-scale DNA extraction.

Micromoths, DNA barcoding, and the frontier of genomic discovery
Among the veterans of the expedition is Niklas Wahlberg from Lund University, Sweden, the European sampling coordinator. With a wry smile, he remarks: “Butterflies are just day-flying moths.” Indeed, butterflies represent only about 10% of all known lepidopterans: out of roughly 200,000 species, over 180,000 are moths. The true challenge of Project Psyche, Wahlberg explains, lies in the micromoths — tiny moths, some no larger than a pinhead, often indistinguishable to the naked eye.
To identify them, researchers rely on DNA barcoding: a method that analyzes a mitochondrial gene called CO1, which varies from species to species. Across Europe, databases already contain barcodes for 99% of lepidopterans, and only 5% of micromoths share the same CO1 sequence.
It is a quiet revolution: a fragment of DNA just 600 base pairs long can reveal the true identity of a species.
While the team in Malles refines collection techniques, another group of researchers in Spain and Andorra has made a breakthrough that could reshape genomics: sequencing the genome of the violet copper butterfly (Lycaena helle) using long-read RNA sequencing.

A specimen of the "Mottled Beauty" moth is ready for museum preservation after the preparation in the Hotel Tyrol in Malles Venosta, Italy. The most beautiful specimens are saved for museum archives or collectors.
Genetics researcher Noé Dogbo of the Institute of Research on Insect Biology in Tours, France, chases a butterfly during a hunting session in the Roja mountains near Curon Venosta, Bolzano, Italy.
Un esemplare di Falena della pimpinella alpina (Zygaena transalpina) si nutre su un fiore di ambretta (Knautia arvensis) tra le montagne di Curon Venosta, Bolzano, Italy.
Researcher Marcin Wiorek of the Institute of Systematics and Evolution of Animals of the Polish Academy of Sciences puts his head through a net to check a moth he caught near Malles Venosta, Bolzano, Italy.

This approach allows scientists to map not only protein-coding genes but also non-coding genes, often overlooked but essential for cellular function. The genome of Lycaena helle, which ranges from the Pyrenees to Siberia, contains 25 pairs of chromosomes and over 547 million base pairs. Long-read sequencing revealed more than 20,000 coding genes and over 4,000 non-coding genes, providing an unprecedented view of lepidopteran genetic organization.
As Roderic Guigó Serra of the Centre for Genomic Regulation in Barcelona explains:
“With this technique, we can see genes that had never been identified before. It’s like turning on a light where there was only shadow.” Researchers are counting on these advances to power the largest scientific endeavor in history: the sequencing and annotation of all living organisms. With nearly 3,000 European lepidopteran species already sampled and more than 1,000 sequenced, Project Psyche has made butterflies and moths the most widely sequenced insects in the world — yet hundreds of thousands of species remain to be discovered, sampled, and understood. It is a colossal task, but also an exhilarating challenge. As the writer and lepidopterist Vladimir Nabokov wrote:
“My pleasures are the most intense known to man: writing and butterfly hunting.”
Today, those pleasures merge with modern science — where every beat of a wing can tell a millennia-old story of evolution, adaptation, and wonder.

Some lepidopterists identify a specimen of Platypteryx falcataria through a manual in the paths of Curon Venosta, Bolzano, Italy.
Biologist Clara Spilker, 26, of the Senckenberg German Entomological Institute of Muencheberg identifies the sex of a moth specimen at a temporary laboratory set up at the Hotel Tyrol in Malles Venosta, Bolzano, Italy.
A specimen of the "Diamondback moth" (Plutella xylostella) seen under a microscope at the temporary laboratory set up at the Hotel Tyrol in Malles Venosta, Bolzano, Italy.
Scientists at work in the Long Read Sequencing Unit of the Centro Nacional de Análisis Genómico in Barcelona, ​​Spain. The CNAG's Long Read Sequencing Unit uses advanced technologies to read long sections of DNA, essential for mapping complex genomic structures and identifying genetic variations associated with diseases. The research center processes many of the biological samples from Project Psyche.
Specialist technician Cyntia Fernández Cuesta is loading a library into a sequencer at the Long Read Sequencing Unit of the Centro Nacional de Análisis Genómico in Barcelona, ​​Spain. Absolutely. Long Read Sequencing allows for the assembly of complex genomes with greater accuracy, resolving repetitive regions and effectively identifying large structural variations in DNA.
A technician at the Centro Nacional de Análisis Genómico, in Barcelona, introduces a sample of fragmented DNA for sequencing in a PromethION machine from Oxford Nanopore Technologies.
Specialized Technician Alexandre Bote Tronchoni monitors the progress of cell sorting at the “Center of Genomic Regulation” (CGR) in Barcelona, Spain.
Sensitive biological samples containing DNA, RNA or cells are kept at -80 degrees Celsius to preserve their integrity at the “Guigò Lab” of the “Center of Genomic Regulation” (CGR) in Barcelona, Spain.
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