Galleria mellonella: A Model for Studying Pathogen Virulence and Insect Immunity

• Author / Creator

  Chen, Robin Y

• The greater wax moth, Galleria mellonella L. (Lepidoptera: Pyralidae), has been used as a model organism to study numerous human pathogens due to low rearing costs, short generation time, and the ability to be reared at human body temperature. The use of G. mellonella for the study of enteropathogenic Escherichia coli (EPEC), an enterobacteria causing diarrhea in humans, has been proposed and demonstrated previously. However, very little is known regarding the virulence of EPEC in G. mellonella and G. mellonella immune responses against EPEC, both of which are essential in understanding the Galleria-EPEC model system.Chapter 1 of this thesis provides relevant background information on G. mellonella immunity, EPEC pathogenesis, and the Galleria-EPEC system.Chapter 2 examines: (1) the nature of EPEC virulence in the hemocoel of G. mellonella by monitoring insect mortality, survival time, time to pupation, pupal mass, pupal duration, fecundity, and egg hatch rate after injection; (2) the source of EPEC virulence in G. mellonella by comparing the intrahemocoelic median lethal dose (LD50) of EPEC to the LD50 of an EPEC mutant (ΔescN) with disabled type III secretion system (T3SS) and to the LD50 of a benign E. coli strain (DH5α); (3) the degree of EPEC virulence in G. mellonella by comparing the intrahemocoelic LD50 of EPEC to that of a known entomopathogen (Providencia rettgeri) and a benign soil bacterium (Bacillus clausii); (4) the oral pathogenicity of EPEC in G. mellonella by comparing the intrahemocoelic LD50 to the per os LD50 of EPEC; and (5) the virulence of EPEC in B. mori and its suitability an alternative insect model for the study of EPEC. I found that EPEC-induced disease in G. mellonella was dose-dependent and manifested as increased mortality, decreased survival time, delayed pupation, decreased pupal mass, and increased pupal duration. The T3SS contributes to EPEC virulence in G. mellonella but unknown factors are responsible for most of the virulence. EPEC displayed moderate virulence in G. mellonella relative to P. rettgeri and B. clausii but had low oral pathogenicity. Bombyx mori is inferior to G. mellonella for the study of EPEC virulence due to low EPEC susceptibility and low thermotolerance. Chapter 3 examines: (1) the immune responses of G. mellonella against EPEC in vivo by hemolymph examination and larval dissection following intrahemocoelic EPEC injection; and (2) the temporal dynamics of circulating hemocytes, melanized particles, nodules, and EPEC replication/clearance during EPEC infection in G. mellonella by quantification using hemocytometer and the plate-count method. In addition to the typical insect immune responses (i.e. melanization, hemolymph coagulation, phagocytosis, and nodulation), a novel insect immune response in the form of extracellular DNA release similar to neutrophil extracellular traps (NETs) was present. The extracellular DNA immobilized EPEC and appeared to be hemocytic in origin. Hemocytopenia was temporarily induced in G. mellonella by EPEC between 3h - 6h post-injection but the circulating hemocyte count recovered by 48h post-injection. The immune responses of G. mellonella were unable to control EPEC replication in the early stage of infection (i.e. within 3h post-injection) but was eventually able to clear EPEC from the hemolymph by 48h post-injection. The clearance of circulating EPEC corresponded to the appearance of melanized particles and nodules, implicating these insect immune responses in EPEC clearance.Chapter 4 examines: (1) the origin of the extracellular DNA by ex vivo stimulation of G. mellonella hemocytes using known inducers of NET release; and (2) the role of extracellular DNA in vivo in G. mellonella by monitoring disease severity after intrahemocoelic injection of EPEC in the presence and absence of DNase I, heat-inactivated DNase I, and G. mellonella hemocyte DNA. The results confirm the involvement of G. mellonella hemocytes (likely granulocytes and oenocytoids) in DNA release ex vivo and demonstrate that extracellular DNA confers protection (increased EPEC clearance rate and prolonged insect survival) to G. mellonella against EPEC infection of the hemocoel in vivo, providing support to the hypothesis that insect hemocytes release extracellular DNA that protects the insect against microbial infection in the hemocoel.Chapter 5 concludes this thesis and discusses future research utilizing the Galleria-EPEC model system.

• Subjects / Keywords

  • Pathogen virulence
  • Extracellular trap
  • Enteropathogenic Escherichia coli
  • Galleria-EPEC system
  • Insect immunity
  • Galleria mellonella

• Graduation date

  Fall 2020

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/r3-zc7e-c130

• License

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