The Carcinus maenas (L.) crabs used in these trials were all males of 65-75 mm carapace width in the intermoult stage, and were obtained live from commercial suppliers (UMBSM Animal Supply Service Millport and Loch Fyne Sea Farms Ltd., UK). They were housed initially in communal tanks, within a closed seawater circulating system at 10°C, then retained individually in tanks for at least two weeks before experimentation. From this stock, 6 crabs were subjected to electrical stunning for electrophysiological recording, 4 crabs were used for measuring HR and another 6 were used as controls.

The European lobsters (Homarus gammarus (L.) were all males of carapace length 80-95 mm in the intermoult stage. They were captured by commercial fishermen using baited traps (creels) laid offshore from St Abbs on the east coast of Scotland. After banding the claws of the lobsters (a standard practice by fishermen) they were held initially in seawater tanks at the St Abbs Marine Station, then transferred in chilled containers to the University of Glasgow. As with the crabs, they were housed initially in communal tanks, within a closed seawater circulating system at 10°C, then retained individually in tanks for at least two weeks before experimentation. From this stock, 6 lobsters were subjected to electrical stunning for electrophysiological recording, 6 lobsters were used for measuring HR and another 6 used as controls.

Electrical stunning was applied to individuals of each species without prior anaesthesia using a commercially available device (Crustastun™; Studham Technologies Ltd, Cambridge, UK) following the manufacturer’s operating procedures. The chamber of the stunning device was filled with a salt solution (NaCl 3 g/L) and a stunning cycle of 110 V, 2–5 amp electrical charge for 5 s for the C. maenas crabs and 10 s for the larger H. gammarus lobsters was applied.

Crabs not to be subjected to electrical stunning were held on ice for 30 min prior to experimentation to reduce their metabolic activity and to induce torpor, while those subjected to electrical stunning were used directly after the stun.

In order to expose the central nervous system (CNS) of the crab for recording ( Figure 1 ), the carapace was removed and the preparation was submerged in a balanced salt solution corresponding in composition and osmolarity to crab haemolymph, at a temperature of 10°C. The internal organs were then displaced in order to expose the circumoesophageal connectives around the base of the stomach.

A similar procedure was employed for the lobster, but after the cephalothorax was separated from the abdomen. To expose the abdominal ventral nerve cord of the lobster for recording, the dorsal skeletal plates (terga) were detached and the bulk of the underlying deep flexor musculature was removed. The preparation was then submerged in a balanced salt solution corresponding in composition and osmolarity to lobster haemolymph, at a temperature of 10°C. Selective removal of muscle blocks then revealed the motor roots emerging from the ventral nerve cord.

Isolated legs of both crabs and lobsters were prepared for nerve recording and, in the case of crabs, also nerve stimulation, by inducing intact animals to shed a leg spontaneously (autotomy) by applying pressure to the basipodite segment (McVean, 1975; Smith and Hines, 1991). The same procedure was followed for the electrically-stunned animals, except that the legs were detached by amputation (since these animals did not express the autotomy reflex). In all cases the joint between the meropodite and carpopidite ( Figure 1A ) was then disarticulated, the muscle tendons spanning this joint were cut and the leg was separated at this point with the leg nerve still attached to the distal portion. The leg nerve was teased into a number of separate bundles, to facilitate selective recording or stimulation. This isolated leg preparation was then submerged in physiological saline at 10°C until required.

Cardiac activity was monitored in both C. maenas and H. gammarus using a standard impedance methodology (Wycoff et al., 2018; Harrington et al., 2020). About 3 h prior to experimentation the animal was held on ice for 30 min to reduce its metabolic activity and to induce torpor. Holes were drilled in the dorsal carapace using a syringe needle at positions on either side of the heart, and thin silver wires (0.2 mm diameter), insulated except for the tip, were inserted by 1-2 mm and secured in place, and then tethered along the external surface of the carapace, with cyanacrylate adhesive. The animal was then returned to a holding tank for 2 h. During this time the wires were connected to a purpose-built impedance converter and the output of this was fed to the input of the PowerLab interface. In some cases, a window discriminator function of the LabChart v7 software was used to display the heart rate as a running average of the beats per minute (BPM). In others, a calculation of the HR was made at specified time points by direct counts of the beats over a 15s interval, which were then transformed to BPM.

Immediately prior to electrical stunning the recording wires were isolated from the electronics and the animals were subjected to a standard electrical stun (5 s for crab and 10 s for lobster). The cardiac recording was resumed immediately after the stun. Additionally, in the case of some lobsters, immediately before stunning one leg was autotomised (routinely leg L2) and prepared for recording, and immediately after stunning another leg (routinely Leg R2) was also removed and prepared for recording, before cardiac monitoring was recommenced.

Ethical approval for procedures on decapod crustaceans is currently not required in the UK. Nevertheless, all the live crabs and lobsters used were treated with proper care in order to minimise their discomfort and distress. There was no practical alternative to the use of live animals. The total number used (16 crabs and 18 lobsters) was the minimum required to obtain a reasonable threshold of certainty, considering that the gain in knowledge and long-term benefit to the subject will be significant. Prior to experimentation they were housed communally to ensure behavioural enrichment. Experimental design considered the 3R’s (Replacement, Reduction and Refinement). Minimisation of suffering and distress was inherent to the experimental design in that the objective was to render the crabs and lobsters insensible by electrical stunning prior to, if necessary, inducing death. No anaesthesia was used. The exposure of subjects to experimental treatment did not exceed 5 s for crabs and 10 s for lobsters. To ensure welfare, behaviour of experimental subjects was monitored for indications of consciousness recovery every 15 min following stunning. None were apparent up to 4 h after stunning, when the animals were then euthanised by freezing.

In untreated lobsters, neuronal traffic was recorded in the CNS both anteriorly in the circumoesophageal connective and posteriorly in the abdominal nerve cord. In addition, evoked activity was stimulated in the leg sensory nerves by mechanical stimulation of cuticular receptors, and spontaneous activity was recorded in the segmental motor nerves that serve to maintain tone in the tail musculature. Consistent results were obtained from the 6 animals tested, and representative traces are shown Figures 5A–D , upper traces.

When first measured following electrical stunning (10 min) there was a total absence of activity at all levels within the lobster CNS (circumoesophageal connective and abdominal nerve cord) ( Figures 5A, B , lower traces), and also in both the sensory and motor nerves ( Figures 5C, D , lower traces). Again this was found to be the case in all 6 animals tested, indicating that all these animals were effectively stunned by this procedure. Moreover, no recovery of nerve activity (nor any limb movements) could be observed even 4 h later.

In both C. maenas and H. gammarus the same protocols that elicited successful electrical stunning were applied to intact animals prepared for recording cardiac activity. For the four crabs tested, after a 2 h period of acclimation following the insertion of electrodes their HR was in the range 56-66 bpm ( Figures 6 , 7A ). After an electrical stun of 5 s there was a silent period of 1-2 min, and then the heartbeat returned gradually, being initially arrhythmic but later more regular, and reached a rate that varied between individuals from 20% and 75% of their pre-stun values. Post-stun, the amplitude of the recorded waveform was also reduced ( Figure 6 ). This cardiac activity continued for as long as the recording was made (between 20 and 30 min) (8A).

Results for lobsters followed the same trends. For the four lobsters tested the acclimated prestun HR values ranged from 32-48 bpm ( Figures 7 , 8B ). After an electrostun of 10 s there was a silent period of 1-2 min, and then the HR returned, being irregular initially but subsequently more regular, and reached rates that varied between individuals from 40% and 70% of their pre-stun values ( Figures 7B , 8 ). Post-stun, the amplitude of the waveform was reduced, and also showed long-term variation ( Figure 8 ). This cardiac activity continued for as long as the recording was made (between 50 and 75 min) ( Figure 7B ).

In a separate trial using two lobsters, simultaneous recordings were made of neuronal activity (sensory responses in an autotomised leg), and cardiac activity in an individual, both before and after electrical stunning. The absence of leg sensory responses was taken as an indication of the lack of neuronal activity. The results for both lobsters were the same, and one is shown in Figure 9 . Before stunning, both the sensory responses to two different stimuli and the cardiac activity were normal ( Figure 9A ). After electrical stunning the neuronal responses ceased, but the heart continued to beat, albeit with a reduced frequency and amplitude, for as long as the recordings were made (30 min) ( Figure 9B ).

The results obtained here are consistent with the literature on the neurophysiology of crustacean nervous systems (Wiese, 2002) in showing that the central nervous systems of intact C. maenas and H. gammarus both display continuous nerve activity, and in turn receive inputs from sensory nerves in the periphery and produce outputs in the motor nerves to the body and limb muscles. It is also well established that this activity persists even when parts of the CNS are isolated from each other by severing the nerve cord at one or more levels (Marder and Bucher, 2007).

We found that following electrical stunning all normal neuronal functioning ceases, both centrally and peripherally. According to the manufacturer, the target stunning current of the Crustastun™ (1.3 amp) is achieved within 0.5 s and is maintained throughout the stun cycle. It is impossible to know with certainty if nerve conduction fails at this point, as all electrical signals are completely masked by the artefact generated by the stun current. However, it is a reasonable assumption that this occurs almost instantaneously, within the stun cycle (10 s). After electrical stunning we found no recovery of central nervous activity, sensory responsiveness or motor action in all the 6 crabs and all the 6 lobsters within the period examined (up to 4 h post-stun). This demonstrates that this method may be effective in rendering both C. maenas and H. gammarus rapidly and persistently insensible under the conditions used in this study.

A number of other studies have used neurological recordings to evaluate electrical stunning. Fregin and Bickmeyer (2016) used implanted electrodes in the abdominal nerve cord of the American lobster Homarus americanus and the crayfish Astacus leptodactylus, and analysed the signals using Fourier analysis (FFT). More recently, Kells et al. (2023) recorded global neural activity from superficially placed electrodes placed anteriorly and posteriorly on the spiny lobster Jasus edwardsii and the crayfish Paranephrops zealandicus. In our laboratory we have previously used the methods of selective extracellular recording described here to record from the central, sensory and motor systems of the crab Cancer pagurus (Neil et al., 2022) and the Norway lobster Nephrops norvegicus (Albalat et al., 2022). Taken together with the results of the present study on Carcinus maenas and Homarus gammarus, there is now available data for a number of crabs, lobsters and crayfish, which also encompass a range of sizes (body weights from around 50 g up to 1 kg) ( Table 1 ).

Despite some variation in their methodologies, all these studies demonstrate that a state of insensibility is achieved in this range of decapods within the applied electrostun cycle, i.e. 0.5 -10 s, and can last for up to several hours. This would allow time to take a blood or tissue sample, if required for biological analysis, or for an appropriate procedure for humane slaughter to be applied while the animal is in an insensible state (the concept of the ‘stun-to-kill’ time). In our previous study of brown crabs (Cancer pagurus) (Neil et al., 2022), lack of either nerve activity or behavioural responses at 4 h after stunning was taken to indicate that the crabs were also killed by this procedure, and this was corroborated by holding intact, electrically-stunned crabs for 24 h, and finding no signs of life. Other findings suggest that some caution needs to be applied to this interpretation. Firstly, Fregin and Bickmeyer (2016) found that neural activity begins to recover in electrically-stunned American lobsters (H. americanus) after 2-3 h (see their Figure 6 ). Secondly, in the present study on the related European lobster (H. gammarus) and also on the crab C. maenas we found that, despite there being no neuronal recovery within 4 h of electrical stunning, there is continued cardiac activity after stunning, which could sustain the metabolism of these animals and so facilitate a long-term neuronal recovery. Taken together, the most conservative interpretation of these findings is that electrical stunning provides a ‘stun-to-kill’ time window of several hours, which, in practical terms would be sufficient in most fishery or laboratory situations to enable a method of dispatch to be applied.

While EEG recording has become the norm for the evaluation of different states in vertebrates, the difficulty and invasiveness of EEG measures renders them impractical for use within commercial environments. Ideally, EEG measurements performed in a laboratory setting should be correlated with non-invasive animal-based measures (ABMs) of the state of sensibility, such as behavioural (i.e. changes in movement or loss of equilibrium), physical signs (e.g. changes in ventilation) and physiological reflexes or measures (e.g. cardiac activity), which can be applied by non-specialists outside laboratory settings. We adopted this approach in this study by monitoring heart rate before and after successful electrical stunning.

We have found that after electrical stunning of both C. maenas and H. gammarus, even though nerve activity ceased both centrally and peripherally, cardiac activity continued (though at a reduced rate) for a substantial time (>1h). This is a somewhat unexpected finding, since the heartbeat of decapod crustaceans is neurogenic in origin (i.e. it is generated by the actions of neurones in the cardiac ganglion, rather than from within the cardiac muscle itself), and as such might have been expected to be abolished by electrical stunning, as were the other neuronal actions of the nervous system. This is not an isolated observation, however, since Wycoff et al. (2018) found a similar effect in the responses of crayfish to anaesthesia by eugenol, namely that the heart continues to beat despite the sensory and motor responses of the animal being suppressed. Wycoff et al. (2018) speculate that the cardiac ganglion may in some way be protected or isolated from the circulating anaesthetic. Finding the same effect after electrical stunning, however, throws some doubt on this suggestion, since the electrical current would be expected to flow through all body tissues, including the cardiac ganglion.

The cardiac ganglion is a classic central pattern generator (CPG), and has been researched extensively (see review by Cooke, 2002). It comprises only nine neurones which act as a pacemaker to drive the heartbeat, with both cycle period and amplitude being modulated by sensory feedback from cardiac muscle, and by the actions of numerous neuropeptides (Powell et al., 2023). Additionally, it has been found that homeostatic regulatory mechanisms exist that maintain a target level of excitability in the cardiac ganglion cells by altering their ionic conductances by up to four-fold (Ransdell et al., 2012). This restoration of intrinsic excitability, which can occur over time periods from a few milliseconds up to hours (Marom and Marder, 2023), represents a resilience that stabilises neuronal output of the cardiac CPG and maintains its overall function in the face of perturbations such as temperature or salinity changes, pathology, injury, trauma or experimental manipulations (Ransdell et al., 2013). It is therefore possible that these compensatory responses within the cardiac ganglion CPG also occur in the face of electrical stunning, resulting in the continuation of heart beating that we have observed post-stunning. This cannot be the full explanation, however, since to produce a heartbeat there must also be a functioning neuromuscular connections between the cardiac ganglion cells and the cardiac muscles, and these too must be resistant to the effect of electrical stunning.

More generally, we conclude that since cardiac activity does not cease after an electrostun that totally suppresses activity in the central and peripheral nervous systems, it cannot serve as a proxy indicator of the state of (in)sensibility. Thus, we recommend that future studies aiming to validate the effectiveness of stunning within decapod crustaceans should not be based alone on the assessment of cardiac activity as a physiological index.

Nevertheless, Wycoff et al. (2018) demonstrated that the heartbeat of crabs, crayfish and shrimps is subject to neuronal modulation in response to external stimuli, such as a rapid tap on the rostrum, and that this startle response no longer occurs after anaesthesia with eugenol. Since these changes in cardiac activity are mediated by pathways (sensory-CNS-cardiac) that would also be expected to be subject to suppression by electrical stunning, the disappearance of a cardiac response to startle stimuli could be a potential indicator of loss of central sensibility, even though, due to its continuation, cardiac activity, per se, cannot.

It is well accepted that the assessment of consciousness in vertebrate species is complex. In aquatic vertebrate species, such as fish, visual indicators of consciousness include equilibrium, changes in ventilation and reflexes such as the vestibulo-ocular reflex also known as ‘eye-roll’. However, the lack of movement and clinical reflexes does not necessarily mean that the animal is not sensible to pain or distress (Robb et al., 2000). In fact, recent studies have shown that following electrical stunning, the rainbow trout (Oncorhynchus mykiss) brain is able to respond to external stimuli before ventilation is resumed (Hjelmstedt et al., 2022). For this reason, it has been proposed that behavioural indicators of consciousness in fish need to be correlated to changes in EEG signal characteristics, which can vary in terms of both amplitude and frequency.

Similar considerations also apply to decapod crustacean species, but there has so far been little research in this area. In crabs, Roth and Øines (2010) used visual indicators of sensibility following electrical stunning, based on the level of response to mechanical stimulation of different parts of the body. However, no correlation with neuronal activity was conducted and it is therefore unclear if unresponsive animals were completely insensible or merely paralysed. On the other hand, Fregin and Bickmeyer (2016) analysed changes in neuronal activity in lobsters in response to mechanical stimulation to different body parts (antenna and eyes, telson, walking legs) following anaesthesia and various stunning methods, but made no simultaneous measures of behavioural indicators of sensibility. Other studies also divide into those that used behavioural indicators alone (Roth and Grimsbø, 2016; Weineck et al., 2018; Astanasoff et al., 2022) and those that used only neurological methods (Albalat et al., 2022; Neil et al., 2022; Kells et al., 2023) ( Table 1 ). Therefore, there is a need for a more comprehensive and integrated approach in which neuronal activity equivalent to the vertebrate EEG, which could include sensory evoked potentials to somatosensory and visual stimuli (Horridge and Sandeman, 1964; Gregory and Wotton, 1987; Hjelmstedt et al., 2022), is correlated with clearly observable body movements and other behavioural measures. It is envisaged that this will need to be done in a species-specific way, since decapod crustaceans vary greatly in their sensory capabilities and their behavioural responses. When validated in this way, these behavioural measures can then be employed as reliable indicators of sensibility following electrical stunning across the range of decapod crustaceans, providing tools for use in both research and commercial settings.

In conclusion, we have found that electrical stunning with the Crustastun™ can rapidly arrest spontaneous activity within the central nervous systems of the crab C. maenas and the lobster H. gammarus, with an accompanying loss of sensory responsiveness and a failure in neuromuscular activation. This stunning renders the animals rapidly insensible, with no recovery detectable for at least 4 h. For all these reasons this procedure may meet the criteria for being a humane method of slaughter for these decapod crustaceans. In contrast, in both species, cardiac activity continues after electrical stunning, making it inappropriate as a proxy indicator of insensibility. Behavioural indicators of effective electrical stunning may be more easily applied, and are indeed being sought, but these will require to be validated by reference to neurological criteria.