Characterisation of the G. mellonella cellular innate immune response and a comparison with the human neutrophil

The use of insects as alternative model organisms in areas of research such as pathogen virulence studies, the development of novel therapeutics and the screening of microorganism strains are increasing. Some of the reasons for this are the lower cost of insects, results are often acquired very quickly with insect experiments and there are relatively few ethical issues involved with the use of insects compared to the ethical issues that are involved when using the mammalians. Larvae of the Lepidopteran Greater Wax Moth, Galleria mellonella are used extensively in biological and chemical research. G. mellonella larvae possess an immune cell, the haemocyte, which is capable of participating in many of the processes that the human innate immune cell, the neutrophil participates in. The haemocyte and the neutrophil are compared to each other throughout this project in a bid to further elucidate similarities between the human innate immune system and the insect immune system. Experimental standardizations are studied and optimized in this work in order to characterize ideal conditions for G. mellonella larvae to be used in the laboratory and to study the effect that the nutritional status of the insect has on it’s ability to fight fungal infections. This was carried out by employing bioassays, proteomic studies and by employing the molecular biology technique, Real Time PCR. This project also studied a number of similarities between the two immune cells, such as phagocytosis, a crucial component of the immune system’s first line of defense. Proteomic similarities between the haemocyte and the neutrophil were studied extensively throughout this work, different techniques were utilised throughout, to identify proteins of interest in the haemocyte. These techniques included SDS-PAGE, mass spectrometry, Western blotting, confocal microscopy and phospho-proteomic staining techniques. The cytoskeletal properties of the haemocyte were also studied and compared to the neutrophil cytoskeleton due to it’s importance in killing processes such as reactive oxygen species production, phagocytosis and pseudopodia production. Work in this Thesis documents the separation of the G. mellonella larvae haemocyte population into sub-populations that differ to each other in size and granularity by employing Flow cytometry cell sorting techniques. It is possible that one of these sub-populations is more similar to the human neutrophil than the others. The presence of granules in the haemocyte was also studied, particularly the presence of MPO-like and lactoferrin-like material. This work outlines many parallels between the human innate immune system and the immune system of G. mellonella larvae and the results demonstrated throughout this Thesis further justify the use of insects as alternative model organisms