An analysis of the cellular and humoral immune responses of Galleria mellonella larvae

The invertebrate immune system is composed of the intertwined cellular and humoral components which have similar structural and functional attributes to the mammalian innate immune system. For this reason insects have served as useful screening tools in academic and industry research for the assessment of pathogenicity of microorganisms or the antimicrobial efficacy of drugs. Due to the low cost, fast turnover of results and lack of ethical constrictions the insect screening model has been used widely. The work presented here in this thesis sought to establish the effects of using Galleria mellonella larvae under different conditions to determine the impact these effects could have on larval survival following infection. A number of abiotic factors known to influence the immune response of insects were assessed including temperature and food abundance or factors such as injury and age. G.mellonella larvae exposed to thermal and physical stress demonstrated increased survival to a lethal dose of Aspergillus fumigatus or Staphylococcus aureus when compared to control larvae. The thermal and physical stress induced an immune priming effect on larvae which coincided with an increased abundance of immune proteins from haemolymph and haemocytes, increased circulating haemocytes, altered expression of genes and a change in the composition of haemocyte populations. These changes peaked 24h after the stress event but declined 72h after the stress event, demonstrating the short term priming of the larval immune response to thermal and physical stress. The effects of pre-incubation at 15oC on larval ability to survive an infection demonstrated a greater susceptibility to infection among larvae pre-incubated for > 3weeks. Larvae possessed reduced abundance of proteins associated with energy metabolism, immune function, decreased circulating haemocytes and AMP expression within larvae pre-incubated for periods of 3 weeks or more. The results highlight the use of larvae pre-incubated for different periods may impair inter-laboratory comparisons. Previous findings demonstrated that insufficient availability of nutrients for larvae also reduced survival to microbial infection when compared to larvae with adequate food. A reduced abundance of immune related proteins was detected in nutrient deprived larvae. The established use of G.mellonella in antimicrobial efficacy screening was demonstrated here through the assessment of a new silver based antimicrobial which was capable of increasing the survival of larvae to a microbial challenge when compared to larvae that received no compound. The assessment of similarities between the insect haemocytes NADPH oxidase activation profile to neutrophils demonstrated a number of proteins to be phosphorylated in response to fMLP and PMA stimulation which was previously demonstrated within mammalian neutrophils. The results preformed here have highlighted the similarities shared between the insect and mammalian immune systems and how inter-laboratory variations in larval use may have consequences on larvae in terms of immune competence and priming. The utility of the larvae as a screening model to study the efficacy of antimicrobial compounds has also been demonstrated in this work