The interactions between hosts and their parasites and pathogens are omnipresent in the natural world. These symbioses are not only key players in ecosystem functioning, but also drive genetic diversity through co-evolutionary adaptations. Within the speciose invertebrates, a plethora of interactions with obligate fungal and microsporidian pathogens exist, however the known interactions is likely only a fraction of the true diversity. Obligate invertebrate fungal and microsporidian pathogen require a host to continue their life cycle, some of which have specialised in certain host species and require host death to transmit to new hosts. Due to their requirement to kill a host to spread to a new one, obligate fungal and microsporidian pathogens regulate invertebrate host populations. Pathogen specialisation to a single or very few hosts has led to some fungi evolving the ability to manipulate their host’s behaviour to maximise transmission. The entomopathogenic fungus, Entomophthora muscae, infects houseflies (Musca domestica) over a week-long proliferation cycle, resulting in flies climbing to elevated positions, gluing their mouthparts to the substrate surface, and raising their wings to allow for a clear exit from fungal conidia through the host abdomen. These sequential behaviours are all timed to occur within a few hours of sunset. The E. muscae mechanisms used in controlling the mind of the fly remain relatively unknown, and whether other fitness costs ensue from an infection are understudied.

In Chapter 2 of this Ph.D thesis, we provide a formal introduction to Entomophthora muscae’s biology and state of affairs of scientific knowledge. We highlight aspects of evolutionary and ecological significance, which still remain unknown about E. muscae.

In Chapter 3 of this thesis, we address a knowledge gap surrounding the reproductive fitness costs faced by male hosts infected with E. muscae during the week-long infection period. Using the terminal investment hypothesis to deduce potential reproductive outcomes, we found that infected male flies have a reduced desire to mate, that uninfected female refuse to mate with them, and that sperm viability in testis decreases. Surprisingly, sperm viability was recovered to normal levels in an apparent terminal investment on days four and five post infection, but with reduced mating, this investment seems to be to no avail. Overall, we found that with increasing fungal proliferation, male hosts experience increasing fitness costs in terms of activity and mating potential, the infection ultimately leading to certain death.

For Chapter 4, we investigated the mechanisms behind stereotypical end of life behaviours observed around sunset after six days of an E. muscae infection. Using detailed observations of the manipulated flies, we sampled flies during specific behaviours to identify putative candidate genes using transcriptomics. Through pairwise comparisons, we identified a handful of genes likely involved in the summiting phenotype. Of particular interest is a homologue of ecdysteroid UDP-glucosyltransferase (egt), a gene known to be involved in summiting in baculoviruses. This suggests possible convergent evolution of both the summiting phenotype and mechanism behind it. Moreover, we identify that an Iflavirus abundantly present in host heads during transcriptomic analyses. This Iflavirus associated with E. muscae has been reported before, but we find that it is phylogenetically divergent to known insect-infecting Iflaviruses and systemically infects hosts. This leaves us brooding over whether the fungus, the virus or both may be responsible for the observed moribund displays.

In Chapter 5, we assessed the potential of non-destructively monitoring methods can detect insect health using the E. muscae-housefly system. Specifically we developed a method using machine learning in combination with pre-existing LED sensors. We were able to determine infection with increasing accuracy as the fungal infection progressed. These results support the idea that non-invasive monitoring systems could be developed to inform efforts on insects of One Health concern, pest management and preserve beneficial insects.

E. muscae is a species complex, where genetically distinct fungal genotypes are found in different hosts. For Chapter 6, we isolated E. muscae from different host species in the wild, and used them in a cross-infection experiment to assess the interactions between host and fungal genotypes. We found that all E. muscae genotypes could infect and induce summiting disease in all host species and genotypes. This demonstrated that virulence in host-E. muscae systems is governed by genotype-by-genotype interactions. Furthermore, we found that each fungal genotype carried at least one symbiotic Iflavirus as identified in Chapter 4. Interestingly, phylogenies of E. muscae and their Iflaviruses show no apparent co-evolutionary relationship, but expected relationships between E. muscae and insect host species were present.

For Chapter 7, we mined publically available assembled genomics and transcriptomics data for microsporidian sequences to address whether the claim that “there may be a microsporidian in every living invertebrate” is true. Microsporidia are obligate intracellular parasites, classified as a sister group of Fungi, and are of zoonotic concern for domesticated animals and humans. In this chapter, we focused on the assembled project of the Panarthropoda specifically, and we identified almost 500 different host species that are potentially infected from across the panarthropod tree from a truly global scale. Within these identified potential hosts, multitudes of them are new host species. Furthermore, we also uncover novel host spectrums of known microsporidia clades, and we find potentially novel microsporidian clades associated with Araneae (spiders) and Lepidoptera host species.

For Chapter 8, we designed a protocol outlining how to isolate, and maintain in vivo and in vitro cultures of E. muscae. Furthermore, we describe best practices that were adopted during my PhD relating to make infections for experiments or rescuing a collapsed system. We hope that this protocol will promote a growing interest in using E. muscae to continue addressing questions left unanswered in this system, which have implications for helping develop our understanding of the evolutionary ecology of host/fungal pathogen interactions as a whole.

This thesis steps into many unknowns of the evolutionary biology behind invertebrate hosts and their fungal and microsporidian pathogens and contributes to our understanding of them. However, as is typical to science, we find that our efforts to leave no stone unturned in one aspect of the biology of E. muscae resulted in yet more left to be turned. This is particularly true of the mechanisms involved in behavioural manipulation, where our findings end in us needing to investigate the purpose of a virus apparently ubiquitously associated with E. muscae. Furthermore, the finding of microsporidia being present in numerous hosts, leaves us needing to confirm whether the pathogens are being infectious or simply happen to be in a host species through ecological reasons such as predation. The continuation of studies of these understudied groups of pathogens will only keep delivering new answers and more questions about hostpathogen evolutionary ecology.