Posibles soluciones a las deficiencias detectadas en el diagnóstico

2.1.1 The model insect G. mellonella

Galleria mellonella (Lepidoptera, Pyralidae) is becoming increasingly popular as a laboratory model for investigating microbial infections in animals (Mylonakis et al., 2005; Bergin et al., 2006; Ramarao et al., 2012; Harding et al., 2013). In nature itis a pest of beehives where the larvae feed on pollen and wax (Hussein et al., 2012), which can lead to weak colonies being completely destroyed (Shimanuki, 1981). It can be reared between 20-30 ˚C and infection studies are performed over a wide range of environmentally relevant temperatures (15 ˚C-37 ˚C)(Ramarao et al., 2012). The large size of the larvae facilitates injection, which permits known doses of chemicals or microorganisms to be delivered into the insect (Glavis-Bloom et al., 2012). Recently a draft G. mellonella genome sequence was published (Lange et al., 2018), which will enable genetic manipulation and allow comparisons to other species. G. mellonella is particularly useful for studying the innate immune response against human pathogens (Mylonakis et al., 2005; Mukherjee et al., 2010; Harding et al., 2013), partly because of the conserved nature of the vertebrate and invertebrate innate immune systems (Vilmos and Kurucz, 1998) and also because studies can be performed at 37 ˚C. G. mellonella could also act as a useful tool for development of new control methods for Lepidopteran pests since the Pyralidae family contains a large number of species that are pests of a range of agricultural crops.

G. mellonella is very susceptible to EPF infection, possibly because beehives are a very clean environment due to the production of antimicrobial chemicals such as propolis by bees (Grange and Davey, 1990). As a result, there may not have been selection pressure for G. mellonella larvae to possess a robust immune system.

Because of this, they can also be used as ‘bait’ to acquire new isolates of entomopathogenic fungi and nematodes from soil (Chandler et al., 1997).

The immune response of G. mellonella after infection with EPF has been reasonably well studied. Plasmatocytes isolated from G. mellonella treated with M. anisopliae s.l.

were found to exhibit morphological and cytoskeletal changes upon infection (Vilcinskas et al., 1997b), in addition to impaired phagocytic activity (Vilcinskas et al., 1997c). Beauveria bassiana infection has also been studied in G. mellonella,

particularly the effect of this EPF on the systemic antifungal immune response (Vilcinskas and Matha, 1997; Wojda et al., 2009). This is discussed in more detail in later chapters.

2.1.2 Laboratory bioassays investigating the infection of insects by EPF

Due to the use of EPF as biological control agents their interaction with insects has been studied extensively using laboratory bioassays. In particular, B. bassiana and M. brunneum have been widely investigated (Copping and Menn, 2000), with isolates of both having been licenced for use as pest control agents (Section 1.4). In the U.K, two commercial products based on these species are B. bassiana in BotaniGard (Certis, USA) and M. brunneum in Met52 (Fargro, U.K).

In a bioassay EPF can be applied to insects through injection or surface application, with either conidia or blastospores. Conidia are the infectious stage of the fungal lifecycle commonly found in nature (Figure 1.3), whereas blastospores are vegetative cells that are produced during fungal growth in the insect haemolymph (Pendland and Boucias, 1997). Previous studies have treated G. mellonella by injection of B. bassiana

blastospores into the haemolymph to monitor its impact on the expression of immune- related genes using RT-qPCR or to monitor its impact on the cellular immune response (Vilcinskas and Matha, 1997; Wojda et al., 2009). Injection allows treatment of insects with an accurate dose of pathogen, but is not necessarily informative about EPF virulence as the ability to penetrate the cuticle is a major virulence determinant (Lacey, 2012).

Vertyporokh and Wojda, (2017) infected G. mellonella with B. bassiana by rolling the larvae onto the sporulating fungus in an agar plate. Alternatively, insects can be immersed in a suspension of conidia (Lacey, 2012). A disadvantage of these techniques is that the dose of EPF received by an insect cannot be measured precisely. EPF can also be applied topically using an apparatus such as a Potter tower air atomising sprayer (Bateman, 1999), where a rotary atomiser is used to apply uniform droplets to insects. The final technique to apply conidia to the insect surface is to place a droplet of spore suspension onto the insect (Lacey, 2012). This enables a precise dose to be administered, but is only possible for use with some larger insects.

2.1.3 Galleria mellonella as a tool for studying the effect of fungal secondary

metabolites on insects

In addition to its use to study EPF-insect interactions, G. mellonella has also been used as a model to study the impact of secondary metabolites on insects. Beauverolides have been found to have contrasting effects on the G. mellonella immune system following injection (Vilcinskas et al., 1999), having immunosuppressive and immunostimulatory effects depending on the quantification method. Injection of this metabolite stimulated lysozyme and antibacterial activity in vivo but inhibited phagocytic activity of plasmatocytes in vitro (Vilcinskas et al., 1999). Another study (Fiolka, 2008) found that cyclosporins (a secondary metabolite of Tolypocladium inflatum) have a strong immunosuppressive activity in G. mellonella.

Cordycepin, a secondary metabolite of the EPF C. militaris,is lethal when fed to P. xylostella larvae (Kim et al., 2002). It has also been reported to cause deformity when injected into G. mellonella pupae (Roberts, 1981). However, detailed studies on its mode of action have not been carried out and the reason for mortality and deformity have not been determined.

2.1.4 The insect gut as a barrier to infection

The cuticle of the external integument is a major barrier to EPF infection. One way in which pathogenic microorganisms may bypass this barrier is by entering the insect

through the gut following feeding. Lepidoptera have defensive mechanisms to prevent this route of infection, including a very low gut pH, digestive enzymes to prevent the growth of microorganisms, and the production of reactive oxygen species (ROS) (Vallet-Gely et al., 2008). If a potentially pathogenic microorganism is able to persist, AMP production may be triggered in the gut epithelial cells via the Toll (Ferreira et al., 2014) or IMD pathways (Tzou et al., 2000). AMP production has been found to be critical in resisting oral infection, for example D. melanogaster lacking an IMD pathway are more susceptible to oral infection by bacteria than wild-type flies (Liehl

et al., 2006).

Commensal bacteria also reside in the insect gut and are thought to have beneficial functions such as aiding in the digestion of some materials (Breznak and Switzer, 1986; Prem Anand et al., 2010), modulating development/ growth (Shin et al., 2011) and protecting against pathogenic microorganisms (Dillon and Charnley, 2002; Engel and Moran, 2013). The reason that these commensals are not targeted by the immune system in the gut is not clear (Vallet-Gely et al., 2008). In D. melanogaster it is thought that in gut epithelial cells the transcription factor Caudal acts as a master regulator for AMP expression, preventing overexpression of AMPs (Ryu et al., 2008) and allowing commensal organisms to survive. Bacteria have been isolated from the guts of healthy

G. mellonella, which suggests that there are either commensal bacteria or pathogens that are kept in check by the innate immune system (Bucher and Williams, 1967).

2.1.5 Aims

The first aim of this component of the Ph.D. was to quantify survival of G. mellonella

larvae in response to treatment with EPF and the secondary metabolite cordycepin. In order to do this, it was necessary to develop an assay that could be used to reliably infect G. mellonella and monitor their survival. The second aim was to elucidate the cause of larval death when treated with cordycepin.