Dynamic proteomic changes and ultrastructural insights into Pochonia chlamydosporia's parasitism of Parascaris equorum Eggs

Luyao Hao^1,2Fengmiao Zhao³Hongyou Liu^1,2Chengyu Ma^1,2Xiaoqing Jia^1,2Lili Jiang⁴Zhaobin Fan⁴Rui Wang^1,2*

• ¹College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot, Inner Mongolia Autonomous Region, China
• ²Key Laboratory of Clinical Diagnosis and Treatment of Animal Diseases, Ministry of Agriculture, National Animal Medicine Experimental Teaching Center, People’s Republic of China, Hohhot, China
• ³College of Grassland Science, Inner Mongolia Agricultural University, Hohhot, Inner Mongolia, People’s Republic of China, Hohhot, China
• ⁴College of phparmacy Heze University, University road 2269, Heze, 274015, People's Republic of China, Heze, China

Pochonia chlamydosporia (Goddard) Zare & Gams (Ascomycota, Sordariomycetes, Hypocreales, Pochoniaceae, Pochonia) is a nematophagous fungus with significant potential as a biocontrol agent against animal-parasitic nematode. However, the molecular and cellular mechanisms underlying its infection process remain poorly understood. This study aims to provide a comprehensive investigation of P. chlamydosporia infection dynamics in Parascaris equorum eggs using both microscopic and proteomic approaches. The infection was monitored at three distinct stages (early, middle, and late), with corresponding ultrastructural and molecular changes observed.Microscopic analysis using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and light microscopy (LM) revealed the progressive invasion of P. chlamydosporia into nematode eggs. These observations provided detailed insights into the morphological changes in both fungal structures and nematode eggs, highlighting key infection stages such as fungal attachment, germination, and egg degradation. Furthermore, the observations confirmed the stages of fungal colonization, emphasizing the dynamic host-pathogen interaction at the macroscopic level.To complement these observations, a 4D-DIA-based quantitative proteomics approach was employed to analyze the exoproteomic changes in P. chlamydosporia during infection. A total of 410 differentially expressed proteins (DEPs) were identified across the three infection stages, with 313 proteins upregulated and 403 proteins downregulated. Gene Ontology (GO) enrichment analysis revealed that these DEPs are involved in critical biological processes, including cellular stress response, proteolysis, metabalic process, and hydrolase activity. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis further identified key infection-associated pathways, such as signal transduction, cell wall biosynthesis, energy metabolism, and host-pathogen interactions. These findings suggest that P. chlamydosporia employs a highly coordinated molecular strategy to adapt to and exploit its host.Quantitative PCR (qPCR) validation of key genes involved in signal transduction and immune evasion mechanisms further supported the molecular basis of P. chlamydosporia's parasitic behavior. These findings contribute to our understanding of fungal-nematode interactions and lay a solid foundation for the development of P. chlamydosporia as a sustainable tool for integrated pest management.