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Advanced Morphology Techniques Reveal Tobacco Hornworm’s Secrets

tobacco hornworm - Manduca sexta

The tobacco hornworm (Manduca sexta) is a model organism used in a range of biological study, but its lack of silk production has never been thoroughly researched. A new study in Insect Systematics and Diversity, however, models in new detail the caterpillar’s first instar silk-producing anatomy and subsequent loss of that capability as it molts to later instars (such as shown here). (Photo by Joseph Berger, Bugwood.org)

By Paige Embry

“Let the students discover,” says István Mikó, Ph.D., research scientist at the University of New Hampshire. The results of that philosophy can be found in a study published in November in Insect Systematics and Diversity that was born in Mikó’s classroom. Students bring in insects of their choice to study, and one discovered something surprising while investigating the mouthparts of the tobacco hornworm (Manduca sexta).

Paige Embry

Paige Embry

It wasn’t the first unexpected finding made in Mikó’s classroom; “We’ve discovered crazy, crazy things in my class,” he says. In his work, Mikó relies on old-school dissection coupled with cutting-edge visualization techniques and the newest technologies based on next-generation genetic sequencing.

This new discovery was surprising because the tobacco hornworm has been a heavily researched insect, both as a pest and a model organism. Mice, rats, and fruit flies are some of the best known model organisms, but scientists have been using M. sexta for decades to study everything from neurobiology and antimicrobial defenses to the mechanics of flight. A large part of M. sexta‘s allure as a model organism lies in the large size of its last instar, which may reach 80 millimeters (approximately 3 inches) long, though few people have investigated the tiny first instar. Mikó and colleagues looked at differences in the spinnerets and accessory labial glands (also known as the Lyonet’s glands) for all five instars, giving insight into how the tobacco hornworm lives at different stages of its larval life.

István Mikó, Ph.D.

István Mikó, Ph.D.

The later, more studied, instars of M. sexta lack mouthparts built for spinning silk, which makes sense because M. sexta, unlike many other Lepidopterans, doesn’t pupate inside a silken cocoon. (Instead, it digs down into the leaf litter or dirt to make a safe spot for the transition to adulthood.) The surprising finding of the study is that the first instar “has a typical, silk-producing tubelike spigot and well-developed Lyonet’s glands,” which are also believed to contribute to silk production. The little first instar uses its silk-making ability to create a pad on which to molt and has also been seen using silk strands as a means of descent—self-made rapelling ropes. In later instars, the spigot shrinks and is hidden by brush-like structures that may be used for spreading saliva.

Spinnerets and Lyonet’s glands are tiny, three-dimensional objects. Scientists can see and dissect them using a stereomicroscope, but, Mikó writes, “the real challenge is to communicate their observations with the audience, as conventional imaging techniques only provide two-dimensional images.” For this study the scientists used confocal laser scanning microscopes (CLSMs), which collect “virtual sections of the specimens,” similar to micro computed tomography (micro-CT). Mikó says that CLSMs allow scientists “to visualize and make discoveries accessible. … You can publish things using digital files and other people can see in 3-D what you see under the microscope.”

In addition to studying the morphology of the mouthparts that can produce silk, the authors looked for genes involved in silk production. Since a large amount of genomic information is available online for M. sexta, the authors were able to find appropriate datasets for each instar. Mikó says, “There are actually more than 40 transcritomes available for Manduca sexta, and we basically re-analyzed these data.” They found five silk-related genes in the genome. Three were highly expressed but only in the first instar. The other two had low expression in all instars.

The report from this research was published as part of a special collection in Insect Systematics and Diversity titled “Current Techniques in Morphology” that Mikó helped compile in 2019.

“I really want to emphasize,” Mikó says, “that 21st-century morphology is only possible with the combination of traditional and new technologies.” Part of the benefit of that 21st-century tech is the ability to share meaningful data, like 3-D images and genomic information, online for others to use. For this study, the researchers used that array of techniques to add to our understanding of a commonly used model organism—and it all began in a classroom.

Paige Embry is a freelance science writer based in Seattle and author of Our Native Bees: North America’s Endangered Pollinators and the Fight to Save Them. Website: www.paigeembry.com.

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