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

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 MgCl₂-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.

Keywords: animal welfare; invertebrate; killing; magnesium chloride; sea star; starfish.

Figure 1

Gel-like image and electropherogram of intact total RNA (RNA integrity number = 9.8) from Asterias rubens pyloric ceca analyzed by the Agilent 2100 Bioanalyzer RNA 6000 Nanochip.