# Hypoxia-induced oxidative stress and mitochondrial damage initiate ferroptosis in Cryptocaryon irritans, a protozoan parasite of marine fish

## TL;DR

This study shows that hypoxia causes metabolic changes and ferroptosis in the marine fish parasite Cryptocaryon irritans, offering new ways to control its spread.

## Contribution

The study reveals that hypoxia-induced ferroptosis is a novel mechanism of vulnerability in Cryptocaryon irritans tomonts.

## Key findings

- Hypoxia increases glycolytic activity and alters metabolite profiles in C. irritans tomonts.
- Ferroptosis is induced under hypoxia, marked by ROS accumulation, lipid peroxidation, and mitochondrial damage.
- Ferroptosis-related genes are upregulated, while gpx4 and mitochondrial components are downregulated under hypoxia.

## Abstract

Cryptocaryon irritans is an obligate parasitic ciliate that significantly endangers marine fish. Hypoxia suppresses the development and hatchability of C. irritans during the tomont stage, which often develops on the seafloor under hypoxic conditions. Despite this knowledge, the underlying adaptation mechanisms of tomonts remain poorly understood. We aimed to determine how hypoxia reprograms tomont metabolism and whether ferroptosis contributes to hypoxia-induced vulnerability. Herein, metabolomic profiling revealed 2,964 differential metabolites under hypoxia. Notably, there were significantly elevated glucose levels, suggesting enhanced glycolytic activity. Enzymatic and qRT-PCR analyses further confirmed hypoxia-induced metabolic reprogramming, including increased hexokinase and pyruvate kinase activities and upregulation of glycolysis-related genes. Hypoxia also induced surface depressions, disrupted cell walls, mitochondrial deformation, reduced mitochondrial membrane potential, disrupted energy homeostasis, and increased NAD⁺/NADH ratio fluctuations and lactate accumulation. To probe ferroptotic susceptibility under hypoxia, hypoxic tomonts were exposed to the ferroptosis inducer erastin, resulting in a hatchability of 13% and promoting reactive oxygen species (ROS) accumulation, lipid peroxidation, and mitochondrial damage. Fluorescence staining revealed strong PI and ROS signals in hypoxic tomonts exposed to the ferroptosis inducer erastin. Notably, mitochondrial dysfunction was accompanied by Ca2⁺ and Fe2⁺ accumulation. Ferroptosis-related genes were upregulated at 24 h post-hypoxia induction. In contrast, gpx4 and mitochondrial electron transport chain components were downregulated at 48 h post-hypoxia induction. These findings demonstrate that hypoxia triggers glycolytic reprogramming and mitochondrial dysfunction in C. irritans, whereas erastin induces ferroptosis under hypoxic stress. This study provides new insights into protozoan hypoxia adaptation and highlights ferroptosis as a potential therapeutic target for controlling parasitic infections in marine aquaculture.

The online version contains supplementary material available at 10.1007/s44154-025-00275-0.