Snake venom toxicity in Artemia salina

Introduction

In Argentina, accidents caused by venomous animals are a public health problem. Snake venom toxicity tests are usually performed in mice and rats, which causes pain and suffering. These tests are not only expensive but also highly regulated. To overcome these limitations, we evaluated the potential use of alternative invertebrate models for snake venom testing. Artemia salina is a small crustacean that lives in high-salinity environments and is commonly used by aquarists as fish food.

Methodology

A. salina cysts were incubated in a thermostatic bath at 28 °C in a 3.2% w/v NaCl solution until hatching. Ten nauplii larvae per well were pipetted into a 96-well microplate. Venoms from four snake species—Naja kaouthia, Micrurus pyrrhocryptus, Bothrops neuwiedi, and Bothrops diporus—were added to each well. Larval motility was quantified after 24 hours of incubation at 28 °C using the Wmicrotracker device in the 3.2% w/v NaCl solution. Each well was also video recorded to monitor potential morphological and/or motility changes caused by the venoms.

Results

The observed EC₅₀s values on motility inhibition suggest that the highest toxicity was observed in N. kaouthia, followed by B. neuwiedi, M. pyrrhocryptus, and B. diporus. Nauplii incubated with N. kaouthia and M. pyrrhocryptus venoms began to show signs of damage at 1.0 µg/mL. B. diporus and B. neuwiedi induced damage at higher concentrations. Structural damage was observed, including appendage destruction and cuticle detachment.

Conclusions

Venoms from four different species were tested for their effects on A. salina motility and morphology. All venoms caused structural damage and complete cessation of larval motility at the highest concentrations tested. These results suggest that A. salina is sensitive to snake venoms in a dose-dependent manner. Further studies will be necessary to test their usefulness in correlating the action of venom and its neutralization by antivenoms in the murine model.