It infection begins with penetration of the

It is often easy to overlook the roles of microbes in terms of wider ecosystems, with such keen focus on their human medicinal aspects. Not only do microbes have pivotal roles in maintenance of stability in ecosystems on a large scale, but the applications of their intricate molecular functions can often induce outstanding biotechnological progress. With a particular focus on the insect pathogenic fungi and plant symbiont Metarhizium acridum, this essay will aim to investigate the role of the microbe in the ecosystem both as a pathogen and as a non-pathogenic symbiont, while applying its mechanisms and evolution to its potential for advances in biotechnology. (105 words)I will firstly explore how the dual life cycles of this fungi as a pathogen and an endophyte- a symbiont of a plant- are coupled by discussing their mechanisms of action and evolutionary history. The mechanism of action is complex both in M. acridum’s role as an insect pathogen and endophyte. As a pathogen, the mode of infection begins with penetration of the insect cuticle, by cuticular degradation using employment of enzymes such as proteases and lipases (Barreili et al., 2015, Pedrini et al., 2013). This technique is non-specific to M. acridum, but opens the opportunity of the general family of insect infecting fungi to infect a wider range of insects. The pathogens enter the insect’s body by transgression of the cuticle rather than ingestion, meaning that insects with sucking mouth parts such as Aphids can also be affected (Chandler et al., 1997). The major proteases produced by the Metarhizium genus and used in this way are Pr1A, a cuticle degrading subtilisin-like protease, and Pr2, a trypsin like serine proteinase. (St. Leger et al., 1998). Not only do these proteases play a key role in penetration, but they also contribute to necessary evasion of host defences. St. Leger et al. (1999), explained how they aided evasion of the host defence by degrading key antifungal proteins in the insect, Following this, hydrophobic conidial spores adhere to the cuticle (Small and Bidochka, 2005). These then germinate, forming outgrowing germ tubes and appressoria (Holder et al., 2007, Deising et al., 2000), which are then capable of further penetration. It is the structure of these appressoria that links the roles of insect pathogenic fungi with plant symbiont. Deising et al. (2000) identified that the appressoria present in the insect fungi showed significant similarity to those found in plant infecting fungi. From this, we can speculate that there may be a morphological link between the two, coupling their roles. The genus Metarhizium utilises the proteins MAD1 and ssgA to facilitate adherence of the conidia onto the insect cuticle surfaces (Wang and St. Leger., 2007a; St. Leger et al., 1992a). A similar protein, MAD2 has been found to be responsible for adherence to plant surfaces (Wang and St. Leger, 2007a). Once again, the presence of each of these genes suggests a dual role of the organism in the different hosts. Once Metarhizium has finally entered the main body cavity- the haemocoel, expression of a collagen-like protein MCLI evades the immune system of the insect (Wang and St. Leger, 2006), allowing it to produce toxins and absorb nutrients, depleting them to fatal levels and eventually resulting in retreat of the fungal hyphae and mummification of the host (Small and Bidochka, 2005; Schrank and Vainstein, 2010). Once inside the haemocoel, in order to survive the osmotic pressure of the haemolymph in the host Metarhizium must adapt and it does this through expression of the osmosensor-like protein Mos1 (Wang et al., 2008).The mechanism of colonisation of M. acridum in plants as an endophyte is not dissimilar to that of insect infection. Once again, the successful association depends on adherence of the fungi to the plant surface, this time facilitated by the protein MAD2 (Nicholson and Epstein, 1991; Wang and St. Leger, 2007a). Sequencing of the Pr1 substilisin-like protease by Reddy et al. (1996) indicated that this gene in the Metarhizium was in fact homologous to the protease At1 from grass endophyte Acremonium typhinum. This alternative fungal protease At1 functions as a facilitator of plant colonisation by cell wall degradation. The similarity of these two proteases indicates a similar functional role of Metarhizium, enabling it to successfully colonise as an endophyte. So why do the plant hosts’ defence pathways not result in expulsion or death of these invasions? It has been found that fungal endophytes such as M. acridum are capable of communicating with the plant, indicating that they are in fact not pathogens. The molecule responsible for this, mycorrhixal factor (Myc) induces transcriptional and morphological changes in the plant roots, such as activation of the symbiotic signalling pathway and increased root hair growth to raise likelihood of contact between the fungal hyphae and the plant roots (Maillet et al., 2011). By doing this prior to and during root colonisation, the fungi is able to trick the desired host into withdrawing its defences, and is then able to successfully carry out symbiosis.(722 words)Having looked in depth at the mechanisms of the coupled functions of M. acridum, it is obvious that the fungus provides a multitude of beneficial services to its ecosystem. The primary benefits to the plant hosts are simply that they acquire insect derived nitrogen in areas where soil nitrogen may be limited (Behie et al., 2012)- they are therefore able to regain nitrogen lost to insects through herbivory. In return from the plant, M. acridum is receiving access to simple plant carbohydrates, those which are usually very difficult to access in the soil due to being bound into complex carbohydrates such as cellulose and lignin. This discovery was outlined in the work by Barelli et al. (2015), where they introduced 13CO2 to plants colonised with Metarhizium, tracking the 13C. Through doing this, they found that Metarhizium mutants lacking raffinose transporter gene mrt showed a reduced competency in the rhizosphere. This not only leads us to the suggestion that carbon acquisition by fungi from plant hosts is critical to their symbiotic relationship, but also that the mrt is a possible uptake route of these plant derived carbohydrates. The acquisition of nitrogen, however, is not the only benefit of M. acridum symbiosis for the plant. Increased foliage biomass in corn seeds, greater plant height and root length, and higher dry weights of shoots and roots are a few other proven benefits. (Liao et al., 2014; Elena et al., 2011). These would obviously contribute strongly to crop yield in agriculture, but also in terms of the natural ecosystem, herbivores would benefit from increased availability of food sources, leaving a cascading abundance increase on their higher trophic levels.Alongside the beneficial ecosystem services to the plant symbionts, we cannot ignore the negative impacts that exclusion of vital nutrients from the insects will ensue. As previously mentioned, eventual death and mummification of the insects is the primary consequence of their pathogenesis, and on the large scale this could impact food webs by removal of M. acridum specific hosts such as locusts, and the broader host ranges of other species in the genus such as M. robertsii (Barelli et al., 2015).  Reduction in herbivory predation by depleted insect populations at the same time as proliferation of plant abundance and yield with M. acridum and other similar symbioses may mean that imbalance to food webs could lead to increases in abundance of primary consumers, and therefore declines in their other prey which are not impacted by M. acridum. (411 words)The key biotechnological applications of M. acridum lie in the fields of agriculture and pharmaceuticals. The potential for individuals from the genera Metarhizium and Beauveria to be used in the control of insect pests in agricultural ecosystems has been known for over 100 years, leading to the approval of many different formulations of these species to be used in protection of crops (Madelin et al., 1963; Faria and Wraight, 2007). Leading to the increased use and approval of these methods in pest control is a 2007 study by Kabaluk and Ericsson, in which they compared yields of corn from samples treated with only conventional insecticide with those treated with Metarhizium in addition to the conventional technique. Their findings showed that the highest yield came undoubtedly from those which had been treated with both treatments, not only showing progress from the previously used methods but also providing significant evidence to that aforementioned that the Metarhizium symbiosis and its supply of nitrogen is the key factor implementing this increased health of the crop, rather than the removal of the insect pests, which was effectively carried out by the insecticide originally. A conversion from these traditional insecticidal treatments to those involving a larger amount of natural control through Metarhizium would have positive climatic implications, reducing run-off of toxic chemicals from the insecticides which have potential to accumulate in organisms and also cause damaging eutrophication in aquatic systems.Pharmaceutical advances are another area where biotechnology of our example pathogen are crucial. Genomic data of Metarhizium species compared to other fungi show enrichment of secondary metabolite gene clusters, with 52 core genes involved in biosynthesis of secondary metabolites in M.acridium. Clusters of these important genes have been shown to have potential for exploitation in biotransformation and biocatalysis, as well as in novel drug discovery. Garyali and Reddy (2014) outlined the capability of endophytes such as M. acridum synthesising important metabolites with pharmaceutical activity when associated with their plant hosts. An example of this they gave is Taxol, an anti-cancer drug that has been found to be present in the association of endophytic fungi with yew trees. The implication of this is that further areas of research are outlined, and with such high diversity of microbes, not only endophytic fungi, present in the soil microbiome there seems limitless exploitations in the case of secondary metabolites in novel drug synthesis. 393 words