Zebrafish larvae locomotion bioassay: application on nanofractionated <i>Naja nigricollis</i> venom

Snakebite envenoming remains a major global health issue, particularly in underserved regions. To better understand venom composition and toxic effects, crude snake venoms can be fractionated into distinct toxin groups using high-throughput nanofractionation analytics. These fractions can then be assessed using venomics to identify specific toxins, in conjunction with bioassays to evaluate their bioactivity. Traditional <i>in vivo</i> testing in rodents is limited by legal and ethical concerns, the high number of fractions, and the small quantities of toxins in each. In this study, zebrafish larvae were introduced as an efficient alternative model for <i>in vivo</i> analysis of toxins that impair locomotion. A behavioral bioassay was developed using a light-dark challenge to assess locomotion in 5-day post-fertilization (dpf) larvae. A Locomotion Index was created to compare movement during the dark phase between venom-exposed and control groups. Using venom from the spitting cobra <i>Naja nigricollis</i>, two fractions were found to reduce locomotor activity significantly. Microscopic screening also revealed three tissue-toxic fractions, one of which caused bradycardia. Dose-response testing was performed for each toxic fraction. Furthermore, oxygen consumption assays were conducted with venom fractions from <i>N. nigricollis, N.haje, N. siamensis,</i> and <i>N. subfulva</i>, identifying two fractions that elevated oxygen consumption. High-throughput venomics linked these toxic effects to three-finger toxins and phospholipases A₂. This integrative approach offers a scalable, efficient method for <i>in vivo</i> toxicity screening of venom components and shows promise for broader applications in the discovery and profiling of diverse bioactive compounds.