The use of Galleria mellonella (Linnaeus) (Lepidoptera: Pyralidae), an economical insect model, for the study of enteropathogenic Escherichia coli (Migula) (EPEC), a diarrheagenic human pathogen, has been demonstrated previously but remains poorly understood. The present study characterizes the Galleria-EPEC system extensively for future studies using this system. We found that EPEC causes disease in G. mellonella larvae when injected intrahemocoelically but not orally. Disease manifests as increased mortality, decreased survival time, delayed pupation, decreased pupal mass, increased pupal duration, and hemocytopenia. Disease symptoms are dose-dependent and can be used as metrics for measuring EPEC virulence in future studies. The type III secretion system was only partially responsible for EPEC virulence in G. mellonella while the majority of the virulence remains unknown in origin. EPEC elicits insect anti-bacterial immune responses including melanization, hemolymph coagulation, nodulation, and phagocytosis. The immune responses were unable to control EPEC replication in the early stage of infection (≤3 h post-injection). EPEC clearance from the hemocoel does not guarantee insect survival. Overall, this study provided insights into EPEC virulence and pathogenesis in G. mellonella and identified areas of future research using this system.

Extracellular traps (ETs) released from vertebrate and invertebrate immune cells consist of chromatin and toxic granule contents that are capable of immobilizing and killing microbes. This recently described innate immune response is not well documented in insects. The present study found that ETs were released by hemocytes of Galleria mellonella (Linnaeus) (Lepidoptera: Pyralidae) in vivo and ex vivo after bacterial stimulation. ET release (ETosis), hemolymph coagulation, and melanization likely contributed to the immobilization and killing of the bacteria. The injection of G. mellonella hemocyte deoxyribonucleic acid (DNA) in the presence of bacteria increased bacterial clearance rate and prolonged insect survival. Taken together, these results indicate the presence of insect hemocyte extracellular traps (IHETs) that protect the insect against microbial infection in the hemocoel and represent the first documentation of ETs in insects in vivo.

No abstract