Electrophysiological Investigation of Different Methods of Anesthesia in Lobster and Crayfish

Abstract

Objectives: In search for methods of anesthesia of crustaceans, an implanted electrode into lobster and crayfish CNS enabled us to monitor signal propagation in the nerve system of animals undergoing different protocols.

Results: Cooling (tap water 0°C, sea water -1,8°C) and anesthesia with MgCl2 (10%) were both discarded as anesthetic procedures because responses to external stimuli were still detectable under treatment. Contrarily, bubbling the aquarium water with CO2 can be considered a "partially successful" anesthesia, because signal propagation is inhibited but before that the animals show discomfort. The procedure of "electro-stunning" induces epileptic-form seizures in the crustacean CNS (lobster, crayfish), which overlay but do not mitigate the response to external stimuli. After several minutes the activity declines before the nervous system starts to recover. A feasible way to sacrifice lobsters is to slowly raise the water temperature (1°C min-1), as all electrical activities in the CNS cease at temperatures above ~30°C, whereas below this temperature the animals do not show signs of stress or escape behavior (e.g. tail flips) in the warming water.

Conclusion: CO2 is efficient to anaesthetize lobster and crayfish but due to low pH in water is stressful to the animals previous to anesthesia. Electrical stunning induces epileptiform seizures but paralyses the animals and leads to a reversible decline of nerve system activity after seizure. Electric stunning or slowly warming just before preparation may meet ethical expectations regarding anaesthesia and to sacrifice crustaceans.

Fig 1

A: Juvenile lobster with implanted hook electrode between the third and fourth abdominal segment. The hook electrode is fixed around the connective between the ganglia. B: Recording trace during control conditions and mechanical stimulation of the head, the telson and the walking legs (blue marker). C-F: Power spectra of the FFTs performed during 5 s of the traces in B during the indicated conditions control (C), head- (D), telson- (E), and leg-stimulation (F). G: The integral of FFTs (black: head; red: telson; green: legs) were divided by the integral of control (without stimulation) to reveal the ratio of integral FFT Power Spectra (IFFTPS). If the ratio is one (green line) the animals were regarded as anaesthetized.

Fig 2. Relative response, the integral of FFT power spectra IFFTPS ratios (stimulated/not stimulated), during control conditions and treatment with 10% MgCl₂ after 5 min, 30 min and 60 min and corresponding recovery (washout).

Responses to mechanical stimulation of head (black), telson (red) and legs (green) are shown.

Fig 3

Recording traces of adult lobster (A) at 7°C (control) and (B) after 1h in frozen seawater slurry (-1,8°C). Marking lines reflect mechanical stimulation at the head, telson and legs, respectively. Red arrow marks electrical stimulation. IFFTPS ratios of (C) juvenile and (D) adult lobster during 15 min, 30 min and 60 min in frozen seawater.

Fig 4

A: Recording traces at time 0 min, 5 min, 10 min and 15 min (A, B, C, D) in seawater bubbled with CO₂ (pH ~5). E: IFFTPS ratios after 0, 15, 30, 45, and 60 min of CO₂ exposure.

Fig 5. Recording traces previous to (A) shortly after (B) and after electrical stunning (1 min, 3 min) (C, D) and with mechanical stimulation and recovery after up to 18 h (E-H).

Fig 6. IFFTPS ratios during different time points before and after electrical stunning.

A: Adult lobster stunned with Crustastun during 5 s. B: Adult lobster stunned with Crustastun during 10 s. C: Adult crayfish stunned with Crustastun (5 s).

Fig 7

A: Adult lobster CNS activity without stimulation during slowly heating (1°C/min) of water. IFFTPS ratios of recording traces baselines. B: IFFTPS ratios of adult lobster during slow heating (1°C / min) starting from 7°C up to 45°C during stimulation at head, telson and legs. C: Adult crayfish CNS activity without stimulation during slowly heating (1°C/min) of water. IFFTPS ratios of recording traces baselines. D: IFFTPS ratios during slow heating (1°C / min) starting from 7°C up to 45°C during stimulation at head, telson and legs.

Fig 8

A: Lobster CNS recordings before and during exposure to boiling hot water. Blue lines mark mechanical stimulations. Red line marks the hot water treatment. Green arrows indicate tail flips. B: Following electrical stunning: Lobster CNS recording trace before and during application of hot water. Red line marks treatment. Red arrows indicate electrical stimulation before and during treatment (only first one indicated; regular stimulation artefacts in trace app. all 15 seconds). C: Following CO₂ treatment: Lobster CNS recording trace before and during application of hot water. Red line marks treatment, red arrow begin of electrical stimulation.

Fig 9

A: Crayfish CNS recording traces before and during application of hot water. Red line marks treatment (100°C). B: Following electrical stunning: Crayfish CNS recording traces before and during application of hot water. Red line marks treatment. C: Averaged FFTPS of adult lobsters in hot water without prior treatment (control), and after treatment electrical stunning with Crustastun, slow warming, and (visually) dead animals (did not recover after surgery).

Fig 10

A: Control recording trace during stimulation of a dead lobster. B: Control recording of a dead animal in hot water. Touching of animal during transfer to hot water cause some distortions.