Abstract
Galleria mellonella larvae are increasingly used to study the mechanisms of virulence of microbial pathogens and to assess the efficacy of antimicrobials. The G. mellonella model can faithfully reproduce many aspects of microbial disease which are seen in mammals, and therefore allows a reduction in the use of mammals. The model is now being widely used by researchers in universities, research institutes and industry. An attraction of the model is the interaction between pathogen and host. Hemocytes are specialised phagocytic cells which resemble neutrophils in mammals and play a major role in the response of the larvae to infection. However, the detailed interactions of hemocytes with pathogens is poorly understood, and is complicated by the presence of different sub-populations of cells. We report here a method for the isolation of hemocytes from Galleria mellonella. A needle-stick injury of larvae, before harvesting, markedly increased the recovery of hemocytes in the hemolymph. The majority of the hemocytes recovered were granulocyte-like cells. The hemocytes survived for at least 7 days in culture at either 28°C or 37°C. Pre-treatment of larvae with antibiotics did not enhance the survival of the cultured hemocytes. Our studies highlight the importance of including sham injected, rather than un-injected, controls when the G. mellonella model is used to test antimicrobial compounds. Our method will now allow investigations of the interactions of microbial pathogens with insect hemocytes enhancing the value of G. mellonella as an alternative model to replace the use of mammals, and for studies on hemocyte biology.
Highlights
Improving our understanding of infectious disease, developing new pre-treatments and therapies for diseases and testing the safety of biological and chemical materials often requires the use of regulated vertebrate animals
We initially investigated whether dosing larvae with an antibiotic affected the recovery of hemocytes compared to sham injected, phosphate-buffered saline (PBS) dosed or untreated control larvae
Pre-treating larvae with doxycycline, ciprofloxacin or PBS increased by 2–3-fold the number of hemocytes we recovered in hemolymph, compared to untreated controls (Figure 3)
Summary
Improving our understanding of infectious disease, developing new pre-treatments and therapies for diseases and testing the safety of biological and chemical materials often requires the use of regulated vertebrate animals. There is a need to replace, refine and reduce the use of regulated animal species in the UK and one approach is to develop alternative test systems and models for infectious disease. This work reported here is part of a project to develop the G. mellonella model to reduce the number of mammals used in research associated with insect-vectored viral pathogens. To assess the use of mammals for virus research in the UK we carried out a PubMed search using the key words “virus+UK+mice” This returned 118 relevant publications in 2018. In addition eight rabbits and six marmosets had been used in these studies Assuming that these numbers are representative for all publications in 2018, we calculate that in the UK at least 5546 mice were used for studies in viral diseases in 2018. Studies carried out in G. mellonella larvae will never completely replace studies in mammals, they will allow any subsequent work using regulated mammals to be better designed, providing refinement of experiments
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