How Butterflies Stick to Surfaces During Metamorphosis (And Other Silk Science)

Many of us will remember that the leap from caterpillar to butterfly requires the creature to reassemble itself within a chrysalis, but have you ever given much thought to the structure of the little pod that cloaks this remarkable biological transformation?

Unlike silk moths, whose larvae can craft a full cocoon from silk alone, butterflies are unable to produce enough silk to completely cover themselves and need to get a bit more creative: instead of producing a full covering, butterfly caterpillars in their last larval instar generate a thick layer of silk around their hanging area, known as the silk pad or button.¹ To add to the challenge, butterfly silk is thinner and weaker than that of the traditional silkworm (the larvae of the Bombyx mori moth), and scientists have been interested in how this thinner material can successfully keep the chrysalis securely attached to branches and other surfaces during metamorphosis. Now, new research published in ACS Biomaterials Science & Engineering shows that part of the strength in the butterfly chrysalis comes from their expertly knitted support structures and a perfectly shaped, built-in attachment tool.

Caterpillars have a handy appendage called the cremaster on their hind end, which is covered in little anchor-shaped hooks that latch onto the silk pad like Velcro (the pupa adheraena). Some also use a silk girdle around their midsection (the pupa contigua). The authors set out to observe and compare these two different attachment systems, and to find out more generally about the structure and properties of butterfly silk in comparison to silkworms.²

The researchers used Fourier-transform infrared (FTIR) spectroscopy for conformational structural analysis, with mechanical properties evaluated by tensile strength. FTIR spectroscopy has previously been used to examine silk from wild silkworms and spiders, with data from 2011 showing it can provide both qualitative and quantitative information about protein conformations.³ Prior to this, silkworm and spider silk had also been evaluated with mechanical deformation and Raman spectroscopy.⁴

One of the key findings in this new study was that the relative weakness in butterfly silk is likely because the primary structure has fewer beta sheet structures compared to that of the silkworm. However, butterflies have unique knitting strategies to make the most of their limited, more delicate silk. In addition to the hook-and-loop fastening that helps them hold on, caterpillars can also spin about 20 strands together into a rope-like structure, which provides an 8-fold increase in strength. The overall mechanism ends up being quite structurally sound after dozens of the cremaster hooks are connected to the silk pad, creating a secure interlock that firmly connects the two surfaces.

Members of this research team have also spent time exploring the structural and mechanical properties of silkworm silk during different points in the lifecycle. In contrast to that of butterflies, silkworm silk fibers have excellent mechanical properties and biocompatibility. Silkworms spin silk at the beginning and end of each of their five larval instar stages, and each of these silks has unique properties that serve different purposes for the caterpillar. For example, instar beginning silk is used to hold the larvae to the substrate to prevent them from falling during feeding and moving, scaffold silk is used to attach the cocoon to the substrate, and cocoon silk acts as a protective covering for the pupa. The group later expanded upon their research, using comparative proteomics to reveal 500 different proteins across seven types of silkworm silk fibers as well as abundant protease inhibitors, enzymes, and other proteins of unknown function.

Together, these studies are more than just biological interest. For example, understanding the structure and function of the cremaster could be useful for advancing nano- and millirobot design, which has previously mimicked the movement of caterpillars to move and grab. The authors note that a better understanding of structure and property relationships in silk could also be valuable for developing superior fibrous materials—either from synthetic or natural silks—with promising applications in areas such as biomedicine and cosmetics.

References

1. Santra, T. and Mandal, S. Studies on the life history of Yamfly butterfly (Loxura atymnus Stoll, 1780) (Lepidoptera: Lycaenidae). Bangabasi Acad. J. 2018, 17, 34–42.
2. Lu, M. et al. How Do Butterflies Use Silk to Attach their Pupae to Trees? ACS Biomater. Sci. Eng. 2024, 10, 8, 4855–4864.
3. Ling, S. et al. Synchrotron FTIR Microspectroscopy of Single Natural Silk Fibers. Biomacromolecules 2011, 12, 9, 3344–3349.
4. Sirichaisit, J. et al. Analysis of Structure/Property Relationships in Silkworm (Bombyx mori) and Spider Dragline (Nephila edulis) Silks Using Raman Spectroscopy. Biomacromolecules 2003, 4, 2, 387–394.
5. Peng, Z. et al. Structural and Mechanical Properties of Silk from Different Instars of Bombyx mori. Biomacromolecules 2019, 20, 3, 1203–1216.
6. Dong, Z. et al. Comparative Proteomics Reveal Diverse Functions and Dynamic Changes of Bombyx mori Silk Proteins Spun from Different Development Stages. J. Proteome Res. 2013, 12, 11, 5213–5222.