Heidi Mathiessen: PhD project title: Understanding natural resistance in zebrafish against Ichthyophthirius multifiliis

PhD Student: Heidi Mathiessen
Email: heidi.mathiessen@sund.ku.dk

BACKGROUND: 
The parasite Ichthyophthirius multifiliis is responsible for white spot disease which is a major problem in global aquaculture production and for the ornamental fish industry. It causes high mortality and morbidity in almost all freshwater fish species, leading to significant economic losses and reduced animal welfare.

Due to the severity of this disease, a lot of research has focused on understanding how fish respond to the parasite and how the host and parasite interact. The reason for this focus has been to develop better treatments or vaccines, particularly for later stages of infection. However, despite extensive efforts, an effective vaccine has not been found, so chemical treatments remain the main option, raising concerns about their impact on the environment and human/animal health. This situation has led to interest in strengthening the fish’s own immune response as a way to prevent infection, which requires identifying genes linked to resistance against I. multifiliis.

THE PROJECT
The overall aim of this thesis was to uncover the mechanisms behind zebrafish's natural resistance to I. multifiliis and identify genes linked to this resistance. This includes elucidating the early response following infection, comparing the response between naturally resistant and susceptible fish and test susceptibility of fish with a knockdown of the suggested resistance gene. 

RESULT
It was examined how zebrafish larvae, relying only on their innate immune system, respond to infection by I. multifiliis using qPCR and in vivo imaging. Larvae at 12-day post-fertilization (dpf) were more resistant to the parasite than 5 dpf larvae, although all managed to clear the infection at an early stage. qPCR analysis revealed upregulation of immune genes such as cxcl8a and mpeg1.2, associated with neutrophils and macrophages, cells which were seen actively attacking and sometimes killing the parasite using in vivo imaging. This suggested that early immune responses, driven by these cells, play a key role in resistance.

Additionally a comparison of the response of resistant zebrafish and susceptible rainbow trout to I. multifiliis infection through RNA sequencing was conducted. At 24 hours post-infection (hpi), resistant zebrafish showed an immediate response, with 1231 differentially expressed genes (DEG), including key chemokines involved in immune cell activity. In contrast, susceptible rainbow trout exhibited a much weaker response with only 28 DEGs. This difference suggested that either I. multifiliis suppresses the trout's immune system or that the trout fails to recognize the parasite early enough to mount an effective defense. Further investigation identified cxcl8a as a key gene involved in zebrafish resistance, as its expression was consistently upregulated during infection and it is involved with the immune cells killing the parasite.

Using CRISPR/Cas, zebrafish with knockdown of cxcl8a was created and following infection, a significantly higher parasite burden was recovered, confirming its crucial role in the natural resistance against I. multifiliis.