Experimental evolution in a tri-factorial system

Project Information

EXPEL

Grant agreement ID: 101108012

DOI

10.3030/101108012

Project closed

EC signature date 2 May 2023

Start date 1 March 2024

End date 28 February 2026

Funded under

• Marie Skłodowska-Curie Actions (MSCA)

Total cost

No data

EU contribution

€ 230 774,40

Coordinated by

AALBORG UNIVERSITET
Denmark

Periodic Reporting for period 1 - EXPEL (Experimental evolution in a tri-factorial system)

Reporting period: 2024-03-01 to 2026-02-28

Summary of the context and overall objectives of the project

Crop pests cause huge financial losses for farmers around the world. Traditionally, these pests are controlled with chemical pesticides, but this is becoming a concern because of harmful effects on the environment, possible risks to human health, and the fact that pests can become resistant to these chemicals over time. Because of this, there is a growing need for more sustainable ways to protect crops. Some alternatives include rotating crops instead of growing the same one repeatedly, planting different crops together, using pest-resistant plants (either naturally or through breeding or genetic modification), and using natural predators like entomopathogenic nematodes (EPNs). These EPNs are tiny worms that naturally infect and kill insect larvae and can help control pest populations.


EPNs are already available commercially and can be applied using equipment similar to pesticide sprayers. However, they are not yet widely used because many of them die shortly after being applied, which makes them less effective. So far, research has mainly focused on testing which combinations of crops, pests, and worm species work best, but results have been inconsistent. In this project (EXPEL), we created a controlled lab system using EPNs and a model host to better understand how these worms infect and interact with their hosts. This system can help improve how EPNs are used and potentially make pest control more effective and environmentally friendly.


Another challenge is identifying different EPN species. Although many species have been described, and new species are still being discovered around the world, they often look very similar. Their similar appearance makes it difficult to identify species using traditional microscopic methods. Modern genetic approaches are still not widely used but offer a promising way forward. While some EPN species have had their genomes studied, many remain uncharacterized. As part of EXPEL, we worked on improving DNA-based methods to identify and study EPN species. By understanding which species are naturally present in local agricultural soils and how they behave, we can better compare them to commercial strains and identify those best suited for pest control. This knowledge could help develop more effective, affordable, and sustainable ways to protect crops.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

This project successfully established a laboratory model using fruit flies and entomopathogenic worms (EPNs) to study infection processes. All organisms were set up and maintained, and a reliable infection method was developed through repeated testing and optimization. The results showed clear patterns: younger fruit fly larvae are more susceptible to infection, the health, quality and species of the EPNs strongly affect success, and temperature plays a major role in how infections develop. Standardizing the model was challenged by environmental conditions, including temperature. Conditions ideal for the host and the EPNs do not always overlap, making experiments harder to standardize. While these species-specific differences made it harder to establish the experimental model, they also highlight an opportunity. They allow us to test how the timing of treatment can be optimized under different environmental conditions to take advantage of factors that naturally weaken the target insect species.
Collecting flies and EPNs hatching after infection proved to be the main challenge of the project. As a result of the small fruit fly larval size, EPNs hatched in limited numbers and were hard to collect, even after various iterations of culturing containers. Because of this, the original plan to follow evolutionary changes over multiple generations could not be completed. However, alternative experiments using the established system still produced useful insights into how infection depends on host age, temperature, and other factors, including host co-infection with endosymbionts. New protocols for freezing and DNA analysis were successfully developed. The DNA analysis approach allows rapid identification of EPN species in soil samples, without the need for highly specialized and time-consuming morphological identification skills. With this approach the demography of known EPNs and discovery of new species is facilitated. Indeed, targeted small-scale field sampling identified several known worm species across different environments and uncovered two previously undescribed species using this approach. Overall, the project built a strong experimental foundation and generated valuable findings that can be exploited in further optimization studies for use of EPNs as biocontrol agents in large-scale agriculture.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

During this project (EXPEL) a new laboratory system was developed that uses fruit flies and beneficial insect-killing nematodes (microscopic worms used in biological pest control) to study how hosts and pathogens interact and evolve over time. Several important methods or long-term evolution experiments were developed, including protocols for infecting and tracking populations of flies, for freezing and long-term storage of nematodes, and a DNA-based approach to identify nematode species without the need for highly specialized knowledge of nematode morphology.

Research using the infection protocols showed that several factors strongly influence whether an infection is successful. Young fruit fly larvae were more vulnerable to infection than older larvae, and some nematode species were better at infecting hosts than others. The health and quality of the nematodes, as well as contamination by other microorganisms, also had a major impact on infection success. These findings improve our understanding of the factors that influence infections and provide a solid basis for future studies.

Beyond the laboratory model, the project significantly increased knowledge of beneficial nematodes living in Danish soils. Surveys across farmland, natural habitats, and agroforestry systems identified several populations of known species and revealed two potentially new species. These findings suggest that the diversity of these beneficial organisms in Denmark is greater than previously recognised and provide valuable material for future studies and biological control applications.

The project also developed genetic tools that make species identification faster and less dependent on specialist taxonomic expertise. Early results indicate that nematode presence may be detectable directly from soil samples using molecular methods, potentially providing a more efficient alternative to traditional sampling approaches.

Potential impact and future uptake:

The project provides new methods, datasets, and biological resources that can support future research on host–pathogen interactions, biodiversity, nematode ecology, and sustainable pest management.

The discovery of potentially new nematode species and the development of improved genetic identification tools will be valuable for researchers working on biological control and soil biodiversity. In the longer term, this knowledge may help improve the use of naturally occurring beneficial nematodes as environmentally friendly alternatives to chemical pesticides.

To maximise future impact, further work should focus on:
- Improving methods for recovering nematodes from infected fruit flies so that long-term evolution experiments can be carried out.
- Expanding genetic reference databases to improve species identification.
- Further developing soil-based detection methods for biodiversity monitoring.
- Studying differences among local nematode populations, including traits relevant for biological pest control.
- Exploring potential agricultural applications of locally adapted nematode populations through collaboration with biological control and crop protection stakeholders.

Overall, EXPEL established a versatile new research platform, generated valuable laboratory and genetic tools, and revealed previously undocumented diversity of beneficial insect-killing nematodes in Danish soils, creating a strong foundation for future research and potential agricultural applications.