Inoculation of G mellonella Larvae

While many insect models are now available for use, the larvae of G. mellonella have many advantages. Due to their size it is possible to inoculate individual larvae with specific doses of the pathogen in question whereas it is difficult to quantify the dose per insect when using other models such as Drosophila. In addition G. mellonella larvae can be purchased commercially and yield results in 24-48 h.

Larvae of G. mellonella are easy to inoculate via injection into the haemocoel through the last left proleg (Cotter et al., 2000). The base of the proleg can be opened by applying gentle pressure to the sides of the leg and this aperture will reseal after removal of the syringe needle without leaving a scar. Inoculation of larvae with test micro-organisms must be accompanied by inoculation of larvae with the buffer used to re-suspend the test micro-organisms to ensure that this has no impact on larval viability. A number of workers also suggest the 'mock-inoculation' of a number of larvae per experiment to ensure that the handling and inoculation procedures are not deleterious to the health of the larvae (Dunphy & Webster, 1984; Cotter et al., 2000). Larvae can be stored at 15°C prior to use and, once inoculated, may be maintained at temperatures up to 37°C, as long as appropriate controls are implemented to quantify the effect of temperature on survival. Larvae should be handled with care, as rough handling affects survival and also leads to the expression of stress proteins.

While larval death is often used as the end point in an experiment, other parameters may also be employed particularly when dealing with a relatively 'weak' pathogen which may not actually kill the test larvae. Fluctuations in fungal load and haemocyte density have been used as accurate indicators of fungal virulence in larvae (Bergin et al., 2003) as have changes in the expression of antimicrobial peptides (AMPs) (Bergin et al., 2006). When analysing a variety of C. albicans strains in larvae of G. mellonella isolates of high virulence could be detected by a decrease in circulating haemocyte density and an increase in fungal load whereas isolates of low virulence were detectable by a high-haemocyte density and a low fungal load (Bergin et al., 2003). Changes in the expression of selected AMPs can be used as an indicator of the immune response to infection and assist in differentiating pathogenic from non-pathogenic infections. (Bergin et al., 2006). Irrespective of which endpoint is used (larval death, fungal load, haemocyte density, AMP expression) results can be obtained within 48 h (Cotter et al., 2000; Brennan et al., 2002; Bergin et al., 2003, 2006).

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