Deeper understanding of the mechanisms by which bacterial pathogens evade the innate immune response to establish infection will underpin the discovery of new ways to prevent and treat disease.One method that can be applied to uncover gene function in bacteria is transposon mutagenesis,where the random insertion of a single transposon into the target genome disrupts gene transcription and prevents expression.The gene disrupted may cause a detectable phenotype thus indicating possible function,and the transposon insertion site can be identified by arbitrary PCR.This technique can be used to screen transposon insertion mutants for reduced virulence,therefore permitting the discovery of novel bacterial virulence factors.We prepared a library of transposon mutants for an aquaculture pathogen Vibrio anguillarum and screened strains for reduced virulence in a Galleria mellonella (an insect) model of infection.This alternative model is ethically more acceptable than performing studies in vertebrate hosts and it also allows for the rapid screening of large mutant libraries.Bacteria were introduced by injection and the screening protocol led to the successful identification of virulence factors known or thought to play a role in fish infections,in addition to one surface protein of previously undetermined function.The screening oftransposon mutants in G.mellonella may be applied to other bacterial pathogens (e.g.,human foodborne pathogens) to identify strains with reduced virulence in vivo,and such knowledge may be applied to improve the prevention and treatment of bacterial diseases.