Profiling the Paralytic Effects and Lethality of Cone Snail Venom Toxins Using Nanofractionation Analytics with In Vivo Zebrafish Larvae Assays

This study presents nanofractionation analytics coupled with <i>in vivo</i> profiling of zebrafish embryo paralysis and lethality in response to toxins in cone snail venoms. The focus of this study is on the development of this approach using venoms of <i>Conus marmoreus</i>, <i>Conus ebraeus</i>, and <i>Conus bandanus</i>. In brief, cone snail venoms were separated using reversed-phase chromatography following high-resolution nanofractionation on microplates with parallel mass spectrometry, enabled via a post-column flow split. All collected fractions were dried overnight, followed by assays on zebrafish embryos. For the paralysis assessment, we monitored swimming behavior and swimming distance and found that exposure to cone snail toxins led to paralysis and decreased movement and swim distance. To correlate the masses of eluted toxins with their paralyzing effects and potency, we compared the fractionation retention time versus normalized swimming distance. This allowed identification of the masses of toxins with paralyzing bioactivity, which were predominantly conopeptides. To assess lethality, zebrafish embryos were exposed to fractionated toxins for 24 h, after which they were inspected. The lethal doses and correlated toxins were identified by comparing retention times of fractionation versus the lethal dose values calculated for each fraction. We found that the most lethal venom was from <i>C. bandanus</i>, displaying the largest number of lethal peptides, followed by <i>C. marmoreus</i> and <i>C. ebraeus</i>. On the other hand, the most paralytic venom was from <i>C. ebraeus</i>, presenting a higher number of peptides with non-lethal paralytic effects, followed by <i>C. bandanus</i> and <i>C. marmoreus</i>. This study provides a pipeline to rapidly identify paralytic and lethal cone snail venom toxins using the zebrafish embryo model.