Optimization of anesthetic procedure in crustaceans: Evidence for sedative and analgesic-like effect of MS-222 using a semi-automated device for exposure to noxious stimulus.

Abstract

The implementation of anesthetic procedure in aquatic crustaceans remains mostly limited to studies dealing with sedation and survival from anesthesia, possibly owing to the debated question of pain in invertebrates. However, two important issues are generally overlooked: actual analgesic-like effect, and possible physiological post-anesthesial effects. Here we report on the anesthetic properties and possible after-effects of MS-222 (Tricaine Methanesulfonate or Ethyl 3-aminobenzoate methanesulfonate) and Eugenol in the freshwater amphipod Gammarus pulex. We first optimized the concentration of MS-222, and the induction and recovery time, based on preliminary tests and published studies. We then relied on the nociceptive modulation of sheltering behavior to assess the analgesic-like effect of the two drugs, using a new semi-automated electric shock device. In addition, we monitored the impact of anesthesia with MS-222 on locomotor activity and oxygen consumption and addressed potential adverse effects upon recovery using biomarkers related to metabolism and neurotoxicity. We provide evidence for the sedative and analgesic-like effects of MS-222 at 600mg.L-1 and, to a lesser extent, of Eugenol at 100 µL.L-1, with no decrease in survival rate at 6 days post anesthesia. Oxygen consumption was reduced -but not eliminated- under full anesthesia with 600mg.L-1 MS-222. No significant physiological effect of anesthesia was evidenced on the activity of the mitochondrial electron transfer system, or that of acetylcholine esterase, nor on total antioxidant capacity. We therefore conclude to the efficiency of MS-222 as an anesthetic drug in G. pulex. Eugenol should be tested at a higher concentration to reach the same efficiency, providing that increased concentration would not incur side-effects. Furthermore, the new and original semi-automated electric chock device used to induce nociception can be easily adapted to any species of aquatic invertebrates and small-sized fish and tadpoles, offering a standardized and flexible protocol to study nociceptive response and anesthesia in aquatic organisms.