How does co-infection influence parasite virulence?

Overview

In nature, animals often become multiply infected by different strains or species of pathogen at the same time. These pathogens then interact either directly or indirectly: they compete for resources inside the host’s body, they trigger independent but often overlapping host immune responses, furthermore some pathogens directly supress immune responses with consequences for their own replication and that of competitors.

Insect pathogenic fungi are obligate killers. Fungal spores germinate and penetrate the insect cuticle, the fungus grows inside the insect and must then kill their host to achieve transmission at the end of the infection cycle. In cases like this, where pathogen virulence is so intimately linked to pathogen fitness, when coinfection occurs the different pathogens engage in a race to hasten death to exploit transmission opportunities.

How coinfection influences animal health is widely researched and debated. In some cases coinfection exacerbates illness and hastens death, whilst in other cases interactions between pathogen can mean that combined infection virulence is reduced. This research arena is underpinned by a rich literature of empirical and modelling studies. This PhD project aims to determine effects of coinfection in insects exposed to multiple fungal parasites. The project will provide an important test case of fundamental evolutionary and ecological principles regarding how pathogens interact within hosts. However, the fungi we study are used as agricultural biological control agents: this project will also shed light on ways to enhance efficacy of biocontrol for environmentally sustainable crop protection.

Insect pests pose major agricultural problems by consuming crops and spreading plant diseases. To protect crops farmers typically resort to heavy chemical insecticide use, which can harm farmworker health, limit crop sales due to pesticide residues, and frequently causes environmental damage due to effects on non-target organisms. Biopesticides offer a sustainable alternative to traditional synthetic insecticides. These agents, made from insect pathogens such as fungi, can help deliver food security whilst also providing compelling health, environmental and economic benefits. A barrier to uptake of biopesticides is that a substantial fraction of the pest population often survives application, and those insects that do die tend to die relatively slowly over several days. By exploring the impact of coinfection by entomopathogenic fungi on overall infection virulence, this PhD will assist efforts to improve biopesticide efficacy for agriculture.

Methodology

Our lab group has a strong track record of working with a range of insect model organisms and agricultural pests including: Helicoverpa armigera (cotton bollworm); Trialeurodes vaporariorum (glasshouse whitefly); Adalia bipiunctata (2 spot ladybird); Bombus terrestris (buff-tailed bumblebee) and Drosophila melanogaster. The successful candidate could focus on any of these organisms (or a combination) in order to answer different questions during their PhD. Depending on the study species, virulence of infections can be studied by assessing: insect survival, feeding, growth rate, or fecundity. One benefit of studying entomopathogenic fungi is that it is possible to estimate pathogen fitness by studying onward transmission potential at the end of an infection cycle: the pathogen breaks out through the insect cuticle and sporulates on the dead insect, these spores can be counted to assess parasite fitness.

The key aims to be addressed during the PhD can be tailored to suit the interests and skills of the successful candidate. However, suitable project aims would include:

Aim 1. How does the virulence of single infections compare to mixed infections?

Whilst many studies have pitted two pathogens against each other in coinfection trails and compared virulence to single infections, these experiments rarely yield generalisable conclusions. In this PhD the successful candidate will study coinfection effects on hosts for a panel of ~10 different fungal pathogen strains, providing multiple pairwise estimates of coinfection virulence compared to single infections.  In nature most instances of coinfection probably result from sequential exposure to several different pathogens. This study will move on to investigate the impact of infection order on the outcome of coinfection by pairs of fungal pathogens.

Aim 2. A proof-of-concept development of novel fitness read-outs using high throughput mid-infrared spectroscopy and artificial intelligence to estimate ageing and infection burden. Working in close collaboration with Glasgow University the project will seek to develop new techniques to assess virulence and pathogenicity using infrared spectroscopy of insect bodies following different infection treatments. This will build on existing Glasgow expertise in determining insect age, species identity and infection status using infrared spectroscopy techniques. Once complete, this work will open up novel opportunities to use these teqchniques later in the PhD.

Aim 3. To what extent is the outcome of coinfection specific to individual host genotypes?

Host populations are usually very genetically variable with respect to infection defence. Resistance genes are often specific in their effects, such that a given genotype may effectively defend against one pathogen strain/species but be susceptible to a second. Host genetic variability has been largely ignored thus far in studies of coinfection biology. This PhD will study different families of insect host (representing different genotypes) and investigate the consistency of coinfection effects across a panel of host genotypes.

Aim 4. How does coinfection alter pathogen virulence evolution?

Coinfection can influence the virulence that an individual host experiences. However, it can also influence the course of pathogen evolution. Within-host competition between pathogen strains/species for transmission opportunities can alter selection pressures on pathogens, potentially selecting for both increased and decreased virulence depending on the infection scenario. This PhD could culminate with an experimental evolution study where pathogen strains are repeatedly passaged through hosts either under coinfection or single infection treatments, before changes in pathogen virulence are assessed.

The PhD will use a combination of laboratory techniques including: insect rearing and infection studies, pathogen culture and preparation, microscopic quantification of pathogen spores, molecular quantification of pathogen loads under coinfection (PCR/qPCR/microsatellites). Furthermore, subject to the interest of the candidate, host immune response activity could be assayed both by phenotypic means (eg phenoloxidase, haemocyte numbers & encapsulation assays) and by assessing immune gene expression profiles by qPCR.

Project Timeline

Year 1

This 3.5 year studentship will begin with 6 months for project planning, literature review and training in key lab skills for working with insects and fungal pathogens. The literature review could be developed into a published review article/meta-analysis. Aim 1 will be addressed in the second half of year 1.

Year 2

During year 2, the PhD will move on to address aim 2 (how infection order influences coinfection dynamics) and will begin working towards aim 4 (experimental study of virulence evolution under coinfection).

Year 3

During year 3, the PhD will work towards aim 3 (how host genotype influences coinfection dynamics) and compete work on aim 4.

Year 3.5

Thesis write up and submission. Paper publication.

Training
& Skills

This PhD will principally be based in the vibrant multidisciplinary research environment at the University of Stirling. The PhD student will become a member of the ‘Evolving Organisms’ research group and will be able to attend regular lab group meetings, along with weekly seminar series giving informal and formal opportunities for research presentation.

The candidate will receive training in subject specific and generic skills. These will include: insect husbandry, pathogen handling and infection experimentation; advanced statistics (Stirling R course and other NERC training); pathogen identification and quantification using morphological and molecular techniques (eg qPCR).

Training will be delivered at Glasgow in infrared spectroscopy and associated machine learning techniques for analysis.

The candidate will be expected to participate in training opportunities in a range of research and transferrable skills offered at Stirling University and through the IAPETUS DTP.

Training will be given in scientific communication skills, both writing and conference presentation, so that the candidate can successfully publish research papers and attend conferences during the PhD.

References & further reading

For an exciting review article on the consequences of coinfection for virulence evolution see:
Alizon, S, de Roode JC, Michalakis Y (2013) Multiple infections and the evolution of virulence. Ecology Letters. 16: 556–567. https://doi.org/10.1111/ele.12076

For an interesting account of the biology of one group of fungal entomopathogens see:
St. Leger RJ, Wang JB. (2020) Metarhizium: jack of all trades, master of many. Open Biol. 10: 200307. https://doi.org/10.1098/rsob.200307

For a couple of recent articles with conflicting results, see the following:

Sheehan G, Tully L, Kavanagh KA. (2020) Candida albicans increases the pathogenicity of Staphylococcus aureus during polymicrobial infection of Galleria mellonella larvae. Microbiology. 166: 375-385. https://doi.org/10.1099/mic.0.000892

Shiqin Li S, Yi W, Chen s, Wang C. (2021) Empirical support for the pattern of competitive exclusion between insect parasitic fungi. J. Fungi. 7, 385. https://doi.org/10.3390/jof7050385

For recent work on the applications of IR spectra see:

Siria DJ, et al. (2020) Rapid ageing and species identification of natural mosquitoes for malaria surveillance. bioRxiv 2020.06.11.144253. doi: https://doi.org/10.1101/2020.06.11.144253

Further Information

For further information please contact Matt Tinsley on matthew.tinsley@stir.ac.uk

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