Practical Euthanasia Method for Common Sea Stars (Asterias rubens) That Allows for High-Quality RNA Sampling

SIMPLE SUMMARY: Sea stars are iconic marine invertebrates and are important for maintaining the biodiversity in their ecosystems. As humans, we interact with sea stars when they are used as research animals or displayed at public or private aquaria. Molecular research requires fresh tissues that have thus far been considered to be of the best quality if collected without euthanasia. This is the first paper describing a method to euthanize sea stars that still allows for sampling of high-quality tissue that can be used for advanced research. Since it can be difficult to tell if an invertebrate has died, it is important to use a two-step method where the first step makes it non-responsive and the next step ensures it has died. Sea stars were placed in a solution of magnesium chloride until they no longer reacted to having their underside or oral surface tapped. Sea stars did not seemingly find the magnesium chloride solution unpleasant. After they were no longer reactive, the sea stars were immediately sampled. Tissue from their digestive glands was extracted and interpreted to be of adequate quality for molecular research techniques. This study promotes animal welfare while maintaining high-quality tissue sampling for molecular research. ABSTRACT: Sea stars in research are often lethally sampled without available methodology to render them insensible prior to sampling due to concerns over sufficient sample quality for applied molecular techniques. The objectives of this study were to describe an inexpensive and effective two-step euthanasia method for adult common sea stars (Asterias rubens) and to demonstrate that high-quality RNA samples for further use in downstream molecular analyses can be obtained from pyloric ceca of MgCl(2)-immersed sea stars. Adult common sea stars (n = 15) were immersed in a 75 g/L magnesium chloride solution until they were no longer reactive to having their oral surface tapped with forceps (mean: 4 min, range 2–7 min), left immersed for an additional minute, and then sampled with sharp scissors. RNA from pyloric ceca (n = 10) was isolated using a liquid–liquid method, then samples were treated with DNase and analyzed for evaluation of RNA integrity number (RIN) for assessment of the quantity and purity of intact RNA. Aversive reactions to magnesium chloride solution were not observed and no sea stars regained spontaneous movement or reacted to sampling. The calculated RIN ranged from 7.3–9.8, demonstrating that the combination of animal welfare via the use of anesthesia and sampling for advanced molecular techniques is possible using this low-cost technique.