Establishment of the thermotolerant Arabian killifish as a model for infection studies

Zebrafish have been widely used in recent years to study the interaction of the immune system with a range of microbes that cause human disease - yet none of these reflects the importance of temperature on the expression of virulence traits. We have therefore specifically chosen the Arabian killifish (Aphanius dispar) for this proposal as it is naturally acclimated to high temperature environments (37-40'C) - allowing us to investigate the pathobiology of microbial infections, and the behaviour of pathogens at temperatures consistent with our own.

The proposal builds upon work we have already undertaken in a doctoral project (Kudoh, Ramsdale & Hamied) to develop the model for fungal (Candida albicans) infections. So far our model has shown all of the benefits of the zebrafish model, including excellent live cell fluorescence and brightfield imaging, antifungal screens, and (as we have shown) the ability to create transgenic and CRISPR/Cas9 mutant lines. In the project we are proposing, we now have the following scientific aims:

(1) Generate transgenic fish lines that facilitate live cell imaging of key immune cell populations - replacing rodent models.

(2) Generate mutant fish lines with defects in genes linked to human disease that are predisposing factors for infection - replace rodent models for mechanistic infection studies.

(3) Establish a publicly available, searchable Arabian killifish genome database /portal based on our existing sequence data - to allow the community to design experiments in our system. Use RNAseq data from different stages of embryo development and infection at different temperatures (30'C and 37'C) to map out key markers of the immunome that can be made available as a track on the killifish genome viewer so that users of the model can assess stage specific pathobiology related events.

(4) Disseminate our tools e.g. (fish lines), data (immunome) and methods (imaging and infection) to the wider infection biology research community and provide opportunities to researchers in zebrafish, mice and other models to use the Arabian killifish as an alternative model. In relation to NC3Rs our model has the following advantages over rodents and zebrafish:

Replacement (Partial): Our Arabian killifish embryo model could replace many mice that are used for studies of interactions with immune cells and tissues, initial virulence testing and antifungal / antibacterial drug screens. TOTAL in local facilities, with 25% uptake within the MRC Centre for Medical Mycology at the University of Exeter over 5-years = 1,700 mice.

Reduction: In the Arabian killifish data can be collected from different stages of infection in a single live animal, greatly reducing the unnecessary culling of mice and associated high husbandry costs. Assuming 5% take-up over five years a reduction in use of 45,687 mice for infection studies could be achieved.

Refinement: Standard screens of human pathogens in zebrafish embryos are undertaken at 32-33'C , imposing unnecessary thermal stress. Scientifically the model has greater validity as human pathogens need to grow at 37'C in order to express their full suite of virulence traits. There is also a reduced need for tricaine anaesthesia due to less developmental twitching.

A TOTAL of 74,820 zebrafish are used p.a (based on literature analysis) so with a 5% take up over five years 18,750 zebrafish embryo studies could be refined.

As core members of the MRC Centre for Medical Mycology (CMM - Ramsdale and Farrer) and the Aquatic Resource Centre (ARC - Kudoh) at the University of Exeter we have state-of-the-art expertise in fish biology, fungal molecular and cell biology and cell biology, immunology and bioinformatics to support this work. Exeter therefore provides a vibrant, exciting and scientifically excellent research environment, and is the perfect place for these studies to be executed.