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Research co-authored by ���������� Boulder PhD graduate Megan E. Zabinski and evolutionary biology Professor M. Deane Bowers reveals how museum butterfly specimens, some almost a century old, can still offer insight into chemical defense of insects and plants

You’re sitting in a field, a garden or another outdoor space, basking in a beautiful summer day. Clouds drift across the sky when something catches your eye. You turn to see a butterfly, its delicate wings and vibrant coloring shifting as it moves from flower to flower. For a moment it’s there, but soon, it moves too far away for you to see.

At first glance, butterflies appear to be just simple, dainty creatures that fly around feeding on plants. For University of Colorado Boulder PhD graduate�� and evolutionary biology �ʰ��Ǵڱ�����ǰ���M. Deane Bowers, however, butterflies are anything but simple. Beneath their wings lies a complex system that plays an integral role in their survival.

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In recently published research, ���������� Boulder PhD graduate Megan E. Zabinski (left) and evolutionary biology Professor M. Deane Bowers (right), emphasize the value that museum specimens have in current scientific research.

In a recently published study in the , Zabinski and Bowers researched how two Euphydrays butterfly species—E. phaeton and E. anicia—sequester certain chemical compounds, a process by which organisms capture and store substances from their host plants to defend themselves against their enemies. The researchers found that they were able to understand how these butterflies sequester substances using both historic specimens as well as fresh ones.

Their project points to the value museum specimens can have in scientific research. By comparing historic butterfly specimens from ���������� Boulder’s Museum of Natural History (����������MNH) with freshly collected and laboratory-reared butterflies, their research demonstrates the benefits, as well as the limitations, of using preserved insects to study chemical defenses decades after collection.

Hatching a plan

Although museum collections house billions of specimens, only a small fraction are used in research after they are acquired. Recognizing this gap inspired Zabinski to begin her research. While Zabinski was still a graduate student, an encounter with Bowers helped shape the trajectory of her academic career.

“Deane came up to me one day—I was in the EBIO club—and she told me she had a job for me. And I thought, ‘A job! You mean I can quit waiting tables at Applebee’s?’”

This opportunity allowed Zabinski to explore her interest in insects and plant-insect interactions within a laboratory setting.

“I absolutely loved being in the lab, doing the physical work with my hands, (whether it was) being able to be outside in the field or looking after the plants,” she says.

Working alongside Bowers—whose research also focuses on how insects interact with their environments—Zabinski began developing her own research questions. She specifically focused on how butterflies in different developmental stages consume and store defensive chemicals to use them later.

Zabinski became interested in whether museum butterfly specimens—which have rarely been investigated and examined for their chemical defenses—could still be helpful.

“We thought about how detecting sequestered defenses in museum specimens really has rarely been done,” she says. “The world of sequestration hadn’t really delved into museum collections. So, we were curious if there was utility there.”

The project was made possible in part by Bowers’ extensive research background and personal butterfly collection, which is housed at ����������MNH. The collection includes the species used in the study.��When combined with outside specimens, this collection, which includes the species used in the study, allowed Bowers and Zabinski to enrich their understanding of the butterflies.

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The Euphydryas anicia butterfly is able to sequester compounds that plants create in defense against herbivores. (Photo: Robert Webster/Wikimedia Commons)

“There has been work done on detecting chemical compounds in plants,” Bowers says. “But there had been less done on insects, and Megan’s thesis had centered on looking at how this particular group of compounds in my lab has worked on particular compounds. We thought it would be really interesting to see if we could find them in old specimens.”

For Zabinski, the combination of Bowers’ expertise and insects available for research made this experiment uniquely valuable.

“It’s kind of the perfect storm for a good experiment. You have a colony in the lab; you also know where there is a field lab where you can get fresh specimens. You know that the museum also has them, but one of the species we had sequestered a high amount, so we thought that … even if there was some degradation, we would still be able to detect them,” she says.

Crawling toward a new understanding

Zabinski and Bowers analyzed specimens from two checkerspot butterfly species in the genus Euphydryas: Euphydryas anicia��and Euphydryas phaeton.��The species were selected because they are known for their high sequestration ability, abundance in the ����������MNH entomology collection and the ease of obtaining live adult specimens. Their research aimed to better understand how the insects use and store these compounds after consuming them as larvae.

Both species sequester iridoid glycosides (), which Zabinski explains are “compounds created by the plants in defense against the herbivores. They’re trying not to get eaten, but there are certain insects— including these butterflies—that capitalize off this process.” Bowers adds, “I’ve tasted (iridoid glycosides), and they’re really bitter. So they are a really good defense against predators and diseases.”

“They’ve been able to find a way to store these compounds in their own bodies and then they can confer some defense against predators,” Zabinski says.

In an initial pilot experiment, the researchers chemically extracted from only one set of wings—a forewing and a hindwing—from historic specimens to determine whether IGs could be detected from the wings alone. Previous experiments have determined that, because in butterfly wings there’s hemolymph (a circulatory fluid similar to blood), it’s possible to detect IGs there. Unfortunately, the results showed extremely low concentrations. To obtain detectable amounts, they found it necessary to analyze both the body and a pair of wings together. For documentation and future research, the set of right wings from each specimen was removed and preserved.

With their methodology established, they chose six E. phaeton��specimens from the ����������MNH that had been collected from 1936–1977. For comparison, E. phaeton larvae were collected from Burlington County, Vermont, brought back to Boulder and raised in the laboratory with their host plant, white turtlehead, Chelone glabra. Once the butterflies reached adulthood, they were freeze-killed and analyzed for their IG content.

Zabinski and Bowers also examined nine historic E. anicia specimens collected between 1933–1998. Fresh adult E. anicia��were collected from Crescent Meadows in Eldorado Springs, Colorado, freeze-killed and immediately underwent extraction for chemical analysis. Although it’s almost impossible to tell what plant the freshly caught butterflies consumed as larvae, the field they were collected from is known to have four catalpol-containing host plants. Catalpol, an IG that is found in these plants, allowed the researchers to determine whether the butterflies were sequestering these compounds, even if they weren’t sure what specific plant was the butterflies’ food source.

“Raising butterflies is not easy,” Zabinski says. “Plants can’t just be alive and available—they have to be high quality, because it’s been shown in studies with these plants that if the plant is not happy, it will not allocate energy to create those compounds. Then your caterpillars are not going to want to eat it.”

Shifting predetermined perceptions

Despite being preserved for decades, the historic specimens still contained detectable traces of sequestered chemical defenses. While IG concentrations were significantly lower in museum specimens than in freshly collected butterflies, Zabinski’s results demonstrate that even after nearly a century, chemical traces of larval diets can still be detected in preserved specimens.

By focusing on the detectability of chemical compounds in older specimens, Zabinski’s work contributes to a broader discussion about preservation methods. She notes that museums often have little control over how donated specimens were originally collected or preserved. She says that despite this, “If you’re a collections manager and you have a researcher that conducted a research experiment and would like to donate them to your collection, if you have the capacity to access them, you’re probably not going to say ‘no.’”

Zabinski explains that previous research demonstrating how preservation methods affect scientists’ ability to detect DNA in museum specimens really shifted how people preserve certain organisms.

“Most insects are preserved as dried specimens, although some are preserved in alcohol,” she says. “In other groups of organisms, like vertebrates and other invertebrates besides insects, they’re often preserved in alcohol or formaldehyde. We now know that using formaldehyde destroys DNA, and so I think the protocol for specimen preservation has changed, trying to preserve the DNA. That’s been one change that museums have been trying.”

Zabinski’s project and others like it are creating an incentive. “As more research comes out about the extended museum specimen and the utility of specimens—particularly with standardization—museums will find a draw to create some uniformity,” she says.

Soaring to new heights

On that summer day, someone who was watching the butterflies move was Bowers.

“I started collecting insects when I was a little kid,” she says. “In undergrad, I did some independent research on butterflies, [and later,] in graduate school, I had a really supportive advisor who told me to spend my first summer going out and looking at butterflies and seeing if I could find some interesting questions. That’s been the focus of my research since.”

Recognizing Zabinski’s curiosity and potential, Bowers recalls, “I brought Megan into the fold.”

“We hear a lot about climate change and we don’t really hear about these smaller interactions that are quite literally under our feet every day,” Zabinski reflects. She says this paper offers one example of how museum specimens are not just remnants of the past, but tools that can be used to better understand specimens today. As technology advances and more research is conducted into chemical defenses, Zabinski says museum specimens can prove to be even more valuable in understanding how organisms interact with their environments long after they’ve been collected.

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