Pseudocapillaria tomentosa infections in laboratory larval and Adult Zebrafish (Danio rerio): Development and advances in an in vivo anthelmintic drug discovery model.

Abstract

Zebrafish (Danio rerio) are a widely used biomedical model and offers powerful high-throughput screening capabilities for assessing chemical bioactivity. We have previously employed adult zebrafish infected with the intestinal nematode Pseudocapillaria tomentosa to investigate nematode-microbiome interactions, nematode-promoted intestinal neoplasia, and anthelmintic drug discovery. Here we transition this model to a larval zebrafish infection infection to enable larger-scale experimentation and ultimately accelerate anthelmintic discovery. Infection conditions were optimized across 5-30 days post fertilization (dpf). The 30 dpf larvae exhibited the most robust and reproducible infections in multi-well formats, as well as the highest survival relative to younger stages. We described worm development from hatching through larval progression and maturation, addressing a major gap in foundational data with fish capillarids. Using in vitro-hatched larvae and infected larval and adult zebrafish, we documented developmental trajectories from 1 to 37 days post-exposure. Change-point analysis identified putative ecdysis transitions at the following worm lengths (mm): L1/L2 = 0.220, L2/L3 = 0.571, L3/L4 = 1.174, and L4/L5 = 1.584. Finally, we demonstrated proof-of-concept for anthelmintic screening by exposing fish to larvated eggs in the presence of emamectin benzoate (macrocyclic lactone) or fenbendazole (benzimidazole). Both compounds reduced worm burdens after 3 days, with the strongest effects at higher concentrations (0.7 µM emamectin benzoate; 0.3 µM fenbendazole). Together, these findings establishes a proof of concept for larval zebrafish infection platform which bridges the gap between in vitro and mammalian in vivo assays, enabling scalable, efficient, and biologically relevant screening for anthelmintic drug discovery.