19 The injection of laboratory animals with pathogenic microorganisms poses a significant safety 20 risk because of the potential for injury by accidental needlestick. This is especially true for 21 researchers using invertebrate models of disease due to the small size of the animals and the 22 required precision and accuracy of the injection. The immobilization of the greater wax moth 23 larvae (Galleria mellonella) is often achieved by grasping a larva firmly between finger and 24 thumb. Needle resistant gloves or forceps can be used to reduce the risk of a needlestick but can 25 result in animal injury, a loss of throughput, and inconsistencies in experimental data.26 Immobilization devices are commonly used for the manipulation of small mammals, and in this 27 manuscript, we describe the construction of injection chambers that can be used to entrap and 28 restrain G. mellonella larvae prior to injection with pathogenic microbes. These devices 29 significantly reduce the manual handling of larvae and provide an engineering control to 30 protection against accidental needlestick injury, while maintaining a high rate of injection. 31 32 33 34 35 36 37 38 39 40 3 41 Introduction 42 The larvae of the greater wax moth Galleria mellonella is an important animal model for 43 studying host-pathogen interactions and for the discovery of novel antimicrobial therapeutics. 44 The popularity of this model organism is driven by the low cost of purchase and the reduced 45 ethical concerns for the experimental manipulation of insects. This allows the challenge of a 46 large number of larvae in a single experiment, which can improve the statistical power of an 47 assay. Starting in the 1940s, a diversity of viral, bacterial, fungal, and nematode pathogens, have 48 been studied for their ability to cause disease in G. mellonella larvae [1-15]. Importantly, G.49 mellonella can be maintained at mammalian body temperature and the outcomes of infection can 50 reproduce that of mammalian animal models [16][17][18]. This is likely due to similarities in the 51 innate immune response to pathogens mediated by elements of cellular and humoral immunity 52 between insects and mammals [19][20][21]. There are several methods to initiate an infection of a 53 larva with a pathogen, including topical application, feeding, oral gavage, and direct injection 54 into the hemocoel. The latter method is favored because of the ability to control the timing of 55 infection and the dosage of pathogen.
5657 Despite the benefits of G. mellonella larvae, there are challenges with using this animal model, 58 most notably standardizing the health and developmental stage of the larvae. This is especially 59 challenging when they are purchased from commercial sources that are primarily focused on 60 providing feed for the pet and angling communities [22,23], although there are efforts to develop 61 commercial pipelines for scientific-grade larvae [24]. Another difficulty is the manipulation and 62 restraint of small larvae during experimental injection of pat...