Sex and stress modulate pupal defense response in tobacco hornworm

In insects, larval and adult defenses against predators have been well studied. However, pupal (also known as resting stage) defenses have been overlooked and not examined thoroughly. Although some pupa possess antipredator strategies such as hairs, spines, cryptic coloration, and exudation of chemicals, few studies have tested these responses and the factors affecting them. Here, we investigated the behavioral responses in tobacco hornworm Manduca sexta that pupates in soil by introducing an external stimulus using vibrations from an electric toothbrush to mimic predation. We observed that M. sexta made violent wriggling (twitching), followed by pulsating movements in response to the vibrational stimulus. Detailed examination showed that these twitches and pulsating events occurred more frequently and for longer periods of time in male pupa and were dependent on the magnitude of the stress (high and low frequency). However, when we estimated the angular force exerted by pupa using radian and angular momentum of twitches, it was found to be independent of pupal sex. A follow-up experiment on possible cascading effects of stress exposure on eclosion success revealed that low- and high-frequency stress exposure didn’t cause any of the common defects in eclosed adults. Our study clearly demonstrates that the so-called defenseless pupal stage uses a wide range of measurable defense behaviors that can actively defend against predators and should be examined further-linking observed behavior with underlying mechanisms.