Paralytic peptide reduces hemolymph viscoelasticity and promotes bulk-initiated coagulation in hornworm Manduca sexta

Hemolymph coagulation is a vital component of insect immunity, rapidly reducing fluid loss and limiting pathogen spread after injury. In Manduca sexta larvae, paralytic peptide (PP) is a fast-acting cytokine activated by an unknown serine protease, and its influence on hemolymph mechanics remains largely unexplored. Here, we examine how PP exposure alters the extensional and surface rheological properties of larval hemolymph, providing insight into the physical manifestation of the immune activation. Hemolymph collected from fifth-instar larvae exhibited weak viscoelastic behavior in 84% of extensional rheology experiments, with a mean characteristic relaxation time of 1.9±1.5ms. Treatment with 10 μM PP resulted in a pronounced shift toward a more Newtonian-like behavior: only 63% of samples remained viscoelastic, and the mean relaxation time (0.7±0.6ms) (p<0.001) was reduced by >2-fold. In samples subjected to repeated measurements, PP exposure caused a rapid loss of viscoelasticity on the timescale of seconds. Surface rheology was probed using magnetic rotational spectroscopy with magnetic microrods. In hemolymphs aged for > 10 min, the surface layer of untreated and PP-treated hemolymphs behaved as a Maxwellian viscoelastic fluid. The relaxation time of the viscoelastic reaction is measured in seconds, i.e., it is three orders of magnitude larger than that in the bulk. The surface drag coefficients of the microrods on PP-treated hemolymph are about twofold smaller than those of untreated ones. As the hemocyte aggregation ceased in ∼10 min after hemolymph extraction, these rheological signatures suggest that PP-stimulated coagulation initiates within the bulk hemolymph. STATEMENT OF SIGNIFICANCE: Hemolymph coagulation is essential for insect survival, preventing fluid loss, pathogen invasion, and loss of mechanical support following injury. While molecular components of insect immune responses have been extensively studied, physical transformations of hemolymph during early wound responses remain poorly understood. Using extensional rheology and magnetic rotational spectroscopy, we show that a cytokine named paralytic peptide (PP), which is proteolytically activated after wounding, rapidly alters the viscoelastic properties of the hornworm Manduca sexta hemolymph. PP reduces bulk viscoelasticity within seconds and modifies interfacial rheological behavior, revealing a direct link between immune signaling and hemolymph mechanics. These findings provide new insight into how proteolytic enzyme(s) convert hemolymph into a mechanically protective material during wound healing.