Unlocking coagulation mechanisms in caterpillar hemolymph for medical use

Blood is a remarkable material: it must remain fluid inside the blood vessels, but clot as quickly as possible outside them, to stop the bleeding. The chemical cascade that makes this possible is well understood for vertebrate blood. But hemolymph, the equivalent of blood in insects, has a very different composition, lacking mostly red blood cells, hemoglobin and platelets and having amoeba-like cells called hemocytes instead of white blood cells for immune defense.

Just like blood, hemolymph clots quickly outside the body. How it does it remains a mystery. Now, materials scientists have shown Limits to soft matter how do Carolina sphinx caterpillars manage this feat. This discovery has potential applications for human medicine, the authors said.

“Here we show that these caterpillars, called smoke horns, can seal wounds in a minute. They do this in two steps: first, within seconds, their thin, watery hemolymph becomes ‘viscoelastic,’ or slimy, and the dripping hemolymph is drawn back into the wound,” said senior author Dr. Konstantin Kornev, a professor in the Department of Science and Engineering Clemson University Materials.

“Hemocytes then aggregate, starting at the surface of the wound and moving upward to embrace the hemolymph membrane coating that eventually becomes a crust that seals the wound.”

Challenging to study

Fully grown smoke horns, ready to pupate, are between 7.5 cm and 10 cm long. They contain only a small amount of hemolymph, which usually coagulates within seconds, making it difficult to study with conventional methods.

For these reasons, Kornev and his colleagues had to develop new techniques for the present study and work quickly. Even so, the failure rate for the most difficult manipulations was huge (up to 95%), requiring many attempts.

They held individual hornworms in a plastic sleeve and made a light wound on one of each caterpillar’s pseudopods through a window in the sleeve. They then touched the dripping hemolymph with a metal ball, which was pulled away, creating a “bridge” of hemolymph (about two millimeters long and hundreds of micrometers wide) that then contracted and broke, producing satellite droplets. Kornev et al. filmed these events with a high frame rate camera and macro lens to study them in detail.

Instantaneous change in properties

These observations indicate that during the first approximately five seconds after the onset of flow, the hemolymph behaved similarly to water: in technical terms, like a low-viscosity Newtonian fluid. But within the next 10 seconds, the hemolymph underwent a remarkable change: now it did not break instantly, but formed a large bridge behind the falling drop. Typically, the bleeding stopped completely after 60 to 90 seconds, after a crust formed over the wound.

Kornev et al. further studied the flow properties of hemolymph by placing a 10-μm-long nickel nanorod in a drop of fresh hemolymph. When a rotating magnetic field caused the nanorod to rotate, its hysteresis with respect to the magnetism gave an estimate of the hemolymph’s ability to hold the rod back through viscosity.

They concluded that within seconds of leaving the body, the caterpillar’s hemolymph changes from a low density to a viscoelastic fluid.

A good example of a viscoelastic fluid is saliva. When you smudge a drop between your fingers, it behaves like water: materials scientists will say it’s purely viscous. But thanks to the very large molecules called mucins in it, the saliva forms a bridge when you move your fingers away. Therefore, it is properly called viscoelastic: thick when you cut it and elastic when you stretch it.”

Dr Konstantin Kornev, Professor in the Department of Materials Science and Engineering, Clemson University

The scientists further used optical phase contrast and polarized microscopy, X-ray imaging and materials science modeling to study the cellular processes by which blood cells aggregate to form a crust over a wound. They did this not only in Carolina sphinx moths and their caterpillars, but also in 18 other insect species.

Blood cells are the key

The results showed that the hemolymph of all species studied reacted similarly to shear. But its response to stretching differed drastically between the hemocyte-rich hemolymph of caterpillars and cockroaches on the one hand and the hemocyte-poor hemolymph of adult butterflies and moths on the other: droplets spread to form bridges for the former, but immediately broke for the last.

“Turning the hemolymph into a viscoelastic fluid seems to help the caterpillars and cockroaches stop any bleeding, drawing the dripping droplets back into the wound within seconds,” Kornev said. “We conclude that their hemolymph has an extraordinary ability to momentarily change the properties of its materials. “Unlike silk-producing insects and spiders, which have a specialized organ for producing fibers, these insects can generate hemolymph filaments at any point during injury.”

The scientists concluded that blood cells play a key role in all these processes. But why caterpillars and cockroaches need more blood cells than adult butterflies and moths is still unknown.

“Our discoveries open the door to the design of fast-acting human blood thickeners. We don’t necessarily need to copy the exact biochemistry, but we should focus on designing drugs that could turn blood into a viscoelastic material that stops bleeding. We hope our findings will help accomplish this goal in the near future,” said Kornev.