Efficacy of anesthetics in an echinoderm based on multifaceted stress biomarkers

Abstract

Animal care committees remain ambiguous on the need for anesthetics during experimental procedures on invertebrate taxa due to long-standing questioning of their sentience and pain perception. When used, anesthetizing procedures for invertebrates have commonly been adapted from those developed for vertebrates, under the largely unverified assumption that they afford the same benefits. The present study formally tested the efficacy of four common anesthetics of aquatic invertebrates (ethanol, MgCl₂, clove oil, MS-222) using behavioural (reaction to physical contact and presence of a predator), physiological (respiration rate), cellular (coelomocytes), and hormonal (cortisol) biomarkers in the holothuroid Cucumaria frondosa (Echinodermata). While subjects recovered from exposures to all anesthetics tested, their responses differed markedly. Ethanol did not immobilize the individuals and concurrently increased their respiration rate, and cellular and hormonal stress markers. MgCl₂ and clove oil reduced the behavioural and physiological responses, and decreased the cellular markers, but increased the cortisol levels. Only MS-222 fully immobilized the treated individuals and decreased their respiration rate, both during exposure and throughout ulterior interactions with a predator, while keeping coelomocyte counts and cortisol concentrations at baseline levels. MS-222 thus appears to induce the loss of sensation, representing a promising anesthetic and sedative in soft-bodied aquatic invertebrates.

Introduction

Invertebrates are animals without backbones, composing most of the biodiversity on Earth, i.e. representing about 95% of species inhabiting marine, freshwater and terrestrial environments^1,2. In addition to their crucial ecosystem roles, they provide services to humans as sources of food and nutraceuticals, among others¹, they are consequently the object of countless scientific studies every year. However, the guidelines for ethical research with them are either poorly developed or non-existent, in stark contrast to the stringent animal care protocols developed for vertebrates, including amphibians, reptiles, fishes and mammals, largely owing to human-biased perceptions³.

An anesthetic is described as a chemical that induces the loss of consciousness or sensation, generally used to prevent an organism from feeling pain⁴. In humans, anesthetics are routinely used when providing lifesaving procedures that would otherwise be unbearably painful. In other vertebrates, such as fishes, anesthetics are used for humanely carrying out dissections, examinations, handling, and more, in a manner that limits pain and reduces stress. While the purpose of anesthesia is not questioned in humans and other members of the Chordata phylum (vertebrates), it remains ambiguous for nearly all other animals (mostly invertebrates). There is an ongoing debate in the scientific community about whether invertebrates are sentient and able to feel not only noxious stimuli, but emotions or pain, fuelling long-standing questions about the use of anesthetics with most taxa^5–8. Recently, Hamel et al.⁹ showed that holothuroid echinoderms (sea cucumbers) can appraise threatening cues (i.e. scent of a predator or alarm signals from injured conspecifics) and prepare themselves immunologically, presumably to cope more efficiently with potential future injuries. The responses share features with recently defined central emotional states¹⁰ and wane after prolonged stress in a manner akin to habituation, which are traits that have rarely been shown in non-vertebrates. Because echinoderms sit alongside chordates in the deuterostome clade, such findings offer unique insights into the adaptive value and evolution of stress responses in animals⁹.

Invertebrates are generally not subject to formal animal care protocols due to the ongoing ambiguity regarding their sentience¹¹, with the exception of cephalopod molluscs and decapod crustaceans¹². While not required, anesthetics are sometimes used for aquatic invertebrates being handled in research and public aquarium settings, including for various species of Mollusca (Cephalopoda, Bivalvia, Gastropoda), Arthropoda (Crustacea, Arachnida, Insecta), Echinodermata, and a few other phyla^1,13. However, while efficacy has sometimes been examined based on physical responses (e.g. Ref.¹⁴), stress levels have not been measured, with most studies tending to assume that if the animal seems relaxed (limp/unresponsive), it is anesthetized. Popular anesthetic procedures have been adapted from knowledge gathered with fishes, such as using tricaine methanesulfonate (MS-222, C₁₀H₁₅NO₅S) and eugenol (clove oil, C₁₀H₁₂O₂)^1,15, without confirming their mode of action in invertebrates. Other anesthetics have been used in aquatic invertebrates for some years, such as magnesium chloride (MgCl₂) and ethanol^1,16. In echinoderms, MgCl₂ and MS-222 emerge as preferred methods for anesthesia and euthanasia^1,13,17,18. Despite their occasional use, the true efficacy of these and other chemicals remains poorly understood, leading a recent review to underscore the limited availability of evidence-based options for invertebrate taxa¹³.

Many gaps in knowledge contribute to this uncertainty around the need for and suitability of invertebrate anesthesia. There have been few studies that have evaluated the efficacy of anesthetics used to immobilize and desensitize aquatic invertebrates, as mentioned by Lewbart et al.¹, and even fewer that tested the efficacy of anesthetics in conjunction with multi-stressors indicators. While the loss of consciousness (e.g. brain activity, ventilation, eye movements) can be measured in vertebrates such as fishes to determine if they are conscious and feeling any sort of pain¹⁹, the signs are not obvious in most invertebrate taxa, especially those devoid of a cephalic region, eyes, and central nervous system. For instance, the nervous system of echinoderms (sea stars, sea urchins, brittle stars, sea cucumbers and crinoids) typically consists of a nerve ring with several extending radial nerves²⁰, contrasting markedly with the brain and spinal cord of vertebrates. One role of an anesthetic is to render the subject immobile, however, it additionally needs to do so in a humane fashion and ultimately block all sensations, including pain and stress, or at least attenuate these sensations in the manner of a sedative. While some invertebrates may appear anesthetized (or sedated) at first glance (i.e. limp/unresponsive), they could still be exhibiting forms of distress that go unnoticed until a proper way is devised to measure such reactions at the physiological, cellular, or hormonal levels.

The present study sought to address some of the key questions surrounding the use of anesthetics in aquatic invertebrates, using behavioural, physiological, cellular, and hormonal biomarkers of stress. The model organism chosen is the sea cucumber Cucumaria frondosa, a member of the phylum Echinodermata, and the class Holothuroidea. Using the invertebrate phylum that is the most closely related to Chordata (vertebrates) seemed relevant for a preliminary assessment of the efficacy of common anesthetics. Another reason why C. frondosa is a worthy focal species is because it has recently been shown to exhibit measurable cellular and hormonal responses (spike in coelomocytes and cortisol) to perceived threats, which is akin to stress/anxiety^9,21. Practically speaking, their unique organ called the Polian vesicle acts as an easily accessible reserve of fluid and coelomocytes that can be used in standard stress assays²².

Four anesthetic agents and protocols described in the literature were tested to see if they worked to immobilize the subject, and if so, whether the procedure caused any measurable stress, to provide some of the first evidence in determining whether it would be more ethical to use them or not. Agents were selected based on their common use with marine/aquatic taxa, including ethanol (C₂H₅OH), magnesium chloride (MgCl₂), clove oil (Eugenol, C10H12O2) tricaine methanesulfonate (MS-222, C₁₀H₁₅NO₅S) and clove oil (Eugenol, C₁₀H₁₂O₂). It was hypothesized that if the anesthetic agent worked, the individuals would (i) become numb (e.g. unresponsive to poking) following anesthesia; (ii) be able to fully recover from the procedure; and (iii) display null or low stress markers during and immediately after exposure to the anesthetic. In addition, anesthetized subjects should not show signs of stress when presented with their predator, the sea star Solaster endeca. This study hoped to reveal whether certain anesthetic agents showed more promise than others, ultimately seeking to help researchers and animal care committees identify the most ethical anesthetics for echinoderms, and possibly other aquatic invertebrates.

Results

Behavioural and physiological responses

Response to a physical stimulus

Exposure to ethanol caused visible agitation along with an elevated response score to the physical stimulus by ~ 58% compared to that measured in the controls. MgCl₂ exposure decreased the score by ~ 23% from 1.7 ± 0.8 in controls to 1.3 ± 0.5 (Fig. 1). Clove oil also decreased the intensity of the physical response by ~ 66% from 1.8 ± 0.8 in the controls to 0.8 ± 0.8. Only individuals exposed to MS-222 showed complete unresponsiveness (score of 0 in all individuals) starting immediately after the beginning of the treatment (Fig. 1).

Fig. 1 [Images not available. See PDF.]

Behavioural scores (mean ± SD, n = 6) of sea cucumbers on a scale of 0–3 (from no reaction to the most intense response) for control and treatment groups exposed to the anesthetics (ethanol, MgCl₂, clove oil, and MS-222). The absence of a bar indicates a result of zero.

Respiration rate

Exposure to ethanol elicited mean respiration rates that were higher than controls both at the onset of the trial (2.4 ± 0.57 vs 1.9 ± 0.75 respirations min^-1) and at the end (2.9 ± 0.93 vs 2.4 ± 1.1 respirations min^-1; Fig. 2), though without statistical support (Table 1). Individuals exposed to MgCl₂ initially maintained mean values (2.1 ± 0.64 respirations min^-1) close to the controls (2.1 ± 0.33 respirations min^-1), and this was still the case at the end of the trial (2.5 ± 1.3 vs. 1.9 ± 0.5 respirations min^-1; Table 1, Fig. 2). In contrast, respiration rates decreased immediately and significantly (Table 1) in the presence of MS-222 (0.10 ± 0.17 respirations min^-1) when compared to controls (1.9 ± 0.79 respirations min^-1); by then end of the trial, exposed individuals had almost ceased respiring (0.08 ± 0.16 vs 2.7 ± 0.55 respirations min^-1; Fig. 2). Finally, clove oil results showed a significant interaction between treatment and time, with respiration rates having decreased significantly relative to controls only at the end of the exposure (1.1 ± 0.95 vs 2.6 ± 0.59 respirations min^-1; Table 1).

Fig. 2 [Images not available. See PDF.]

Respiration rates (openings min^–1; mean ± SD, n = 6) of control and treatment groups at the beginning (0–5 min) and end (15–20 min) of the exposure period to the different anesthetics (ethanol, MgCl₂, clove oil, MS-222). For each pair, the black bar on the left is the control group and the coloured bar on the right is the treatment group.

Table 1. Statistical results of two-way analysis of variance (ANOVA) comparing respiration rates in the control and treatment groups (exposure) at the beginning and end (time) of each trial.

Cases	F statistic	df	p value
Ethanol
Exposure	1.307	1	0.200
Time	1.307	1	0.200
Interaction (exposure and time)	0.000	1	1.000
MgCl2
Exposure	0.703	1	0.412
Time	0.099	1	0.757
Interaction (exposure and time)	0.889	1	0.357
Clove oil
Exposure	9.346	1	0.006
Time	0.321	1	0.578
Interaction (exposure and time)	4.628	1	0.044
Exposure at start (0–5 min)	0.519	1	0.488
Exposure at end (15–20 min)	11.208	1	0.007
MS-222
Exposure	120.537	1	< 0.001
Time	3.174	1	0.090
Interaction (exposure and time)	3.476	1	0.077

Cellular response

Coelomocytes (all types combined)

While trends emerged in the cellular response, variability was high, and none of the departures from control conditions were statistically clear (Table 2). Sea cucumbers exposed to ethanol showed higher mean coelomocyte densities (16.3 ± 9.3 × 10⁵ cells ml^-1) compared to controls (10.1 ± 4.1 × 10⁵ cells ml^-1; Fig. 3). Individuals exposed to MgCl₂ and clove oil both showed coelomocyte densities similar to their controls, with 12.5 ± 8.5 vs 13.6 ± 4.9 × 10⁵ cells ml^-1 and 9.4 ± 5.5 vs 9.8 ± 4.4 × 10⁵ cells ml^-1, respectively (Fig. 3). Inversely, individuals submitted to MS-222 had lower mean coelomocytes densities than their controls (9.6 ± 4.3 vs 12.7 ± 3.4 × 10⁵ cells ml^-1; Fig. 3).

Table 2. Statistical t-test results for comparisons of coelomocyte and phagocyte density between control and treatment groups for each chemical agent.

Chemical agent	t statistic	df	p value
Free coelomocytes
Ethanol	− 1.502	10	0.164
Clove oil	0.167	10	0.871
MS-222	− 0.235	10	0.767
MgCl2	0.286	10	0.781
Phagocytes
Ethanol	− 1.496	10	0.166
Clove oil	− 1.328	10	0.214
MS-222	− 0.167	10	0.871
MgCl2	0.139	10	0.892

Fig. 3 [Images not available. See PDF.]

Coelomocyte densities (cells × 10⁵ ml^-1, mean ± SD, n = 6) of control and treatment groups following 20-min exposure to each anesthetic (ethanol, MgCl₂, clove oil, MS-222).

Specific coelomocyte types

Coelomocytes can be further sorted into different cell types: phagocyte, morula and fusiform. Like for pooled coelomocytes, no statistical differences were detected for any of the individual coelomocyte types (Table 2), and trends thus remained unclear. Mean phagocyte densities in individuals exposed to ethanol were perhaps higher (14.8 ± 8.9 × 10⁵ cells ml^-1) than in the controls (8.9 ± 3.7 × 10⁵ cells ml^-1; Fig. 4). Sea cucumbers exposed to MgCl₂ showed similar values as their controls (12.1 ± 8.2 vs 12.6 × 10⁵ cells ml^-1 ± 4.1; Fig. 4). While masked by variability, mean phagocyte density in the clove oil treatment group (12.0 ± 2.4 × 10⁵ cells ml^-1) seemed higher than in the control (9.2 ± 4.5 × 10⁵ cells ml^-1). The MS-222 treatment did not elicit any phagocyte increase relative to control (12.1 ± 4.7 vs 12.4 ± 2.8 × 10⁵ cells ml^-1, Fig. 4).

Fig. 4 [Images not available. See PDF.]

Phagocyte densities (cells × 10⁵ ml^-1, mean ± SD, n = 6) in control and treatment groups following 20-min exposure to each anesthetic (ethanol, MgCl₂, clove oil, MS-222).

Some trends emerged in other cell types, though they were not statistically significant. Fusiform cells showed decreases in ethanol and MgCl₂ exposures, and stable values in clove oil and MS-222 individuals, compared with their respective controls (Supporting Information Fig. S1). For morula cells, there was an increase under ethanol, a decrease under clove oil, and stable values under MS-222, and complete disappearance under MgCl₂. The fusiform cells showed a decrease in mean abundance during exposures to ethanol and MgCl₂ compared to controls, whereas individuals exposed to clove oil and MS-222 showed stable densities relative to controls. Differently, the morula cells appeared to rise in individuals exposed to ethanol, decrease under clove oil, show no change under MS-222 and disappear under MgCl₂ (Supporting Information Fig. S1).

Hormonal response

There were no statistically significant differences between treatment groups (F_3,9 = 0.892, p = 0.482). The controls for all trials yielded a cortisol level of 11 ± 4 pg ml^-1 (n = 4, Fig. 5A). Individuals sampled immediately after exposure to MgCl₂ displayed the highest mean cortisol level (20 ± 11 pg ml^-1, n = 3) and those having been exposed to clove oil the lowest levels (7 ± 0.5 pg ml^-1, n = 3). Cortisol levels in individuals exposed to ethanol were the same as the controls (11 ± 4 pg ml^-1, n = 3), whereas individuals exposed to MS-222 had slightly higher cortisol levels (13 ± 3 pg ml^-1, n = 2; Fig. 5A).

Fig. 5 [Images not available. See PDF.]

(A) Cortisol response (pg ml⁻¹, mean ± SD) after 20-min exposure to anesthetics (ethanol, MgCl₂, clove oil, MS-222). Control (n = 5) is shown in black, and treatments are in various colours. (B) Cortisol response after 20-min exposure to anesthetic followed by exposure to a predator for 1 h. Control (n = 15) is shown in black, and groups exposed to the various chemicals (n = 6) are shown in colour. Note the different scales in the two panels.

Recovery from anesthesia

There were no mortalities recorded during the trials or during the recovery period. All individuals in all trials were able to fully recover from exposure to anesthetics. They were extending their podia and feeding as sea cucumbers do under normal conditions, and they had returned to baseline behavioural scores and respiration rates within 24 h post exposure (Supporting Information Table S1).

Anesthesia efficacy during predator exposure

Behavioural and physiological responses

Control sea cucumbers exhibited escape behaviours when exposed to the predator, showing moderate levels of ABA and respiration rates (Table 3). The ABA scores for individuals treated with clove oil were elevated above those of control individuals both at time 0 and 55 min, but the respiration rates remained unchanged relative to controls (Tables 3, 4). Both the ABA scores and respiration rates of individuals treated with MgCl₂ remained similar to those of controls. Individuals treated with MS-222 had ABA scores that were lower than those of all other groups with scores as low as 0. Their respiration rate was also the lowest as 0.6 ± 0.7 at the beginning, recovering slightly to show 1.2 ± 0.3 at the end of the trial (Tables 3, 4).

Table 3. Behavioural response of sea cucumbers that had either been exposed to seawater (control) or to an anesthetic agent (MgCl₂, clove oil, MS-222) before being exposed to a predator, as well as ABA score and respiration rates, initially and at the end of the 1-h trial.

Treatment conditions	ABA score (± SD)		Respiration rate (± SD)
	0–5 min	55–60 min	0–5 min	55–60 min
Control	0.6 ± 0.8	1.2 ± 0.9	2.2 ± 0.3	2.5 ± 0.5
MgCl2	0.2 ± 0.4	1.3 ± 1	2.9 ± 0.6	2.7 ± 0.2
Clove oil	1.3 ± 1	1.8 ± 0.8	2.1 ± 0.6	1.7 ± 1
MS-222	0 ± 0	0.34 ± 0.5	0.65 ± 0.7	1.2 ± 0.3

Table 4. Statistical results of two-way analysis of variance (ANOVA) comparing respiration rates in the predator exposed control and treatment groups (exposure) at the beginning (0–5 min) and end (55–60 min) (time) of each trial.

Cases	F statistic	df	p value
Clove oil
Exposure	0.755	1	0.398
Time	0.849	1	0.371
Interaction (exposure and time)	0.017	1	0.897
MgCl2
Exposure	0.356	1	0.560
Time	2.344	1	0.148
Interaction (exposure and time)	3.589	1	0.079
MS-222
Exposure	26.834	1	< 0.001
Time	5.301	1	0.040
Interaction (exposure and time)	0.037	1	0.851

Coelomocytes

Control individuals exposed to the predator showed coelomocyte densities of 9.3 ± 6.5 × 10⁵ cells ml^-1 (Fig. 6). Overall, there was a significant effect of treatment on the individuals (F_3,28 = 5.116, p = 0.006). Upon using a post-hoc test, only clove oil treated individuals showed significantly higher coelomocyte densities during exposure to the predator, with 20.8 ± 12 × 10⁵ cells ml^-1 (t = 3.210, p = 0.020). Coelomocyte densities for individuals exposed to MS-222 and MgCl₂ did not depart significantly from those of controls (MS-222: 7.4 ± 2.2 × 10⁵ cells ml^-1; t = 0.599, p = 1.00; MgCl₂: 6.5 ± 5.1 × 10⁵ cells ml^-1; t = 0.823, p = 1.00).

Fig. 6 [Images not available. See PDF.]

Coelomocyte densities (cells × 10⁵ ml^-1, mean ± SD) after 20-min exposure to anesthetics (MgCl₂, clove oil, MS-222) followed by exposure to a predator for 1 h. Control (n = 14) is shown in black, and groups exposed to the various anesthetic agents (n = 6) are shown in colour.

Hormonal response

Control individuals displayed the highest mean cortisol level recorded when exposed to predators (789 ± 1394 pg ml^-1; Fig. 5B). However, due to high variability, there were no statistically significant differences between the controls and any of the treatment groups (F_5,33 = 0.863, p = 0.516). Clove oil treated individuals showed cortisol levels of 277 ± 528 pg ml^-1, the MS-222 treated individuals showed the overall lowest mean values with 12 ± 3 pg ml^-1, and the MgCl₂ treated group had values closest to those of controls (686 ± 936 pg ml^-1; Fig. 5B).

Discussion

The anesthetic agents tested in the present study were chosen based on their documented use. In humans, ethanol functions as a known depressant, dissolving through the blood stream and affecting the brain²³, while in invertebrate science, its use has been reported in various molluscs such as cephalopods (cuttlefish and octopus) and gastropods (abalones and snails)¹. MgCl₂ is naturally found at low levels in seawater²⁴ and has been used widely as an anesthetic and muscle relaxant in marine invertebrates such as gastropods, cephalopods, bivalves, flatworms, and echinoderms¹. MgCl₂ and ethanol have both achieved anesthesia in cephalopods by blocking neural signals to the pallial nerve¹⁶. Clove oil is a new and popular anesthetic in fish because it is effective at very low doses²⁵, whereas its use in invertebrates has so far mainly been documented in crustaceans¹, where it is thought to block sodium and calcium channels and relax muscles^26,27. MS-222 is one of the two anesthetics recommended for use in echinoderms by Lewbart et al.¹, the other being MgCl₂. In other invertebrates (crabs, crayfish, flies), MS-222 has been thought to temporarily inhibit sensory and motor neurons by blocking sodium ion channels²⁸.

In the present study, immobilization was the first criterion used to measure the efficacy of the anesthetic agents. This metric operates under the assumption that the unresponsiveness or limpness of an individual is consistent with its full relaxation. However, there remains a possibility that an individual could be paralyzed by the anesthetic but still experience sensation or alertness, which would mean it is not relaxed. This is why we paired the physical response with a suite of physiological, cellular and hormonal stress metrics. Importantly, and regardless of the varying efficacy levels discussed below, none of the treatments were lethal and all sea cucumbers recovered fully from short-term exposures.

Ethanol showed the least potential as an immobilizing agent since the physical response of treated individuals was heightened relative to the controls. Treatment with clove oil and MgCl₂ induced a lessened behavioural response compared to controls but did not result in complete limpness. This could imply that longer immersion times or greater concentrations of these anesthetics may be required to achieve the full effectiveness, although high concentrations could also be lethal in echinoderms¹⁸. MS-222 stood out of the lot, as all individuals exposed to it were rendered unresponsive almost immediately, implying that this anesthetic is highly potent and effective as a means of immobilizing sea cucumbers.

Respiration rates represent a non-invasive, visually observable physiological biomarker of stress in sea cucumbers^22,29. The response of a relaxed sea cucumber would be a reduction in respiration rate, the adverse response would be a faster respiration rate, as shown in Gianasi et al.²⁹ after injection of trace markers in the aquapharyngeal bulb of C. frondosa. Unsurprisingly, the ethanol-treated individuals showed a higher respiration rate than the controls, both at the beginning and the end of the treatment period, confirming that ethanol was not an effective relaxing agent and suggesting that sea cucumbers may have been trying to flush it out of their systems by increasing water exchanges. Individuals exposed to MgCl₂ decreased their respiration rate upon contact and over time. Similarly, with clove oil, respiration rates at the beginning and the end of the exposure were both lower than those of control individuals, and seemingly decreased over time during the exposition period. MS-222 once again stood out, as the exposed individuals completely closed their cloaca upon entering the anesthetic bath, with only a few individuals displaying one last opening. It is possible that muscles (including those that open and close the cloaca) could have been paralyzed upon submersion. Accordingly, in fishes, this chemical was demonstrated to elicit rapid anesthesia in about 2 min³⁰ and to completely abolish muscle movement³¹.

Coelomocytes are versatile cells in echinoderms, where their main functions relate to the immune system^22,32. If an anesthetic is triggering an immune response, it most likely means that the individual is still capable of perception. In fact, spikes in coelomocytes have been shown to occur as a response to non-physical threats, including nearby predators (scent only) and injured conspecific (alarm signals) in C. frondosa⁹. Here, during the 20-min exposure trials, the sea cucumbers treated with ethanol showed an increase in coelomocytes relative to the controls, suggesting that cellular markers mirrored the adverse behavioural and physiological responses discussed earlier for this chemical, while providing further support that this metric reflects stress. Ethanol could be perceived as a foreign substance or threatening chemical cue when entering the respiratory tree. This contrasts with ethanol producing general anesthesia in mammals³³. At least in sea cucumbers, its application has none of the desired effects of an anesthetic and its usage should be avoided. The cellular response to clove oil did not differ much between treated and control individuals, suggesting that while it did not render the sea cucumbers limp under the concentration and duration tested, it did not trigger any perceptible cellular stress response either. Similarly, treatment with MgCl₂ elicited lower total coelomocyte densities, which suggests it was either relaxing the individuals despite not immobilizing them (see above), or just not affecting them at all, perhaps because it is naturally found at low concentrations in seawater²⁴. In sea cucumbers treated with MS-222, there was a lower mean density of total coelomocytes compared to controls, and while it was not statistically significant, it aligns with the depressed or unaffected behavioural and physiological markers to support MS-222 as a candidate for invertebrate anesthesia.

When examined in detail, phagocytes were consistently the most abundant of all coelomocyte types, explaining the variations in total abundance, as previously described in other studies that exposed C. frondosa to various stressors^9,21. As the present trials were short (20 min), phagocytes dominated, consistent with the assumption that they lead the initial cellular response. The other coelomocytes like morula and fusiform cells, and eventually hemocytes, appear later in the response, i.e. after 60–90 min (see Ref.⁹). Despite their potential importance in the longer term, their low recorded abundance in the present study precludes any finer analysis or definitive conclusion.

The individuals treated with ethanol, clove oil and MS-222 showed a mean cortisol level that was comparable to that of their control groups. This was surprising for ethanol, given the adverse behavioural, physiological and cellular responses and considering the known toxicity of this chemical in some invertebrates³⁴. It is possible that ethanol interfered with the normal hormonal response in C. frondosa. Inversely, while the behavioural, physiological and cellular results suggest that MgCl₂ was not a stressful anesthetic agent for sea cucumbers, the cortisol levels were higher than that of controls, questioning whether C. frondosa was completely desensitized by this anesthetic agent. As cortisol is not as well studied in invertebrates as in vertebrates, we have yet to unravel the full meaning of this response, but propose that MgCl₂ should be used with caution, as its innocuity remains ambiguous.

The depth of anesthesia generated by the three most promising chemicals (leaving out ethanol, which had already been dismissed based on the initial results; see above) was further tested during direct exposure to a predator (the sea star Solaster endeca). In line with earlier results, the most efficient anesthetic against the stress of imminent predation was MS-222, where treated individuals did not react perceptibly. In small mammals, the efficacy of anesthetic agents is measured with various metrics, including respiratory efforts and muscle tone decreases³⁵, which are somewhat comparable to the physical reaction and respiration rates measured here in C. frondosa. These markers, along with the cellular and hormonal markers, suggest unresponsiveness in MS-222-treated sea cucumbers, even in the face of imminent peril or while being preyed upon. The two other chemicals tested (clove oil and MgCl₂) gave unconvincing results under the same predatory threat, based on the behavioural response and on somewhat inconsistent cellular and hormonal markers. As expected, the control sea cucumbers exposed to their predators showed the highest mean cortisol levels. The group anesthetized with MgCl₂ showed the second highest hormonal stress response, even though their cellular response was weak. Inversely, sea cucumbers treated with clove oil before exposure to the predator exhibited a measurable increase in coelomocytes but no cortisol spike. Only the sea cucumbers that had been treated with MS-222 showed consistently lower mean hormonal and cellular responses relative to controls under predatory threat. Support for MS-222 as an invertebrate anesthetic, admittedly based on variable doses and measures of efficacy, has so far been ambiguous, with positive results obtained for horseshoe crabs, many cnidarians and echinoderms, a few molluscs, and generally not with decapod crustaceans¹³. This overview, combined with the present findings, suggests that it may predominately work through immersion in soft-bodied, unshelled aquatic taxa.

It is important to acknowledge that our understanding of the perception and expression of stress and pain in holothuroid echinoderms and other invertebrate taxa is far from complete, since we barely grasp their peculiar anatomies and nervous systems. It was only recently discovered that echinoderms are mostly head-like animals, calling for a re-evaluation of the evolutionary events that led to their pentameric adult body plan³⁶. Identifying the metrics necessary to assess the mode of action and true efficacy of anesthetics will remain a challenge, and their applicability may not be universal. It is likely that studies similar to the present one will have to be conducted across a diversity of taxa. Moreover, in the many aquatic taxa with permeable body walls and circulatory systems, the use of anesthetic agents may not always be appropriate, e.g. where chemicals could interfere with metabolites or cells under study. Finally, beyond ethical considerations in echinoderms and other invertebrates³⁷, there remains important debates around the very definition of sentience and the need for legislating the welfare of invertebrate taxa⁷.

Conclusions

Combining all biomarkers (behavioural, physiological, cellular, and hormonal responses; reaction to a predator; recovery) ultimately provided a baseline of information regarding the efficacy of each of the anesthetic agents tested (Table 5). Ethanol, a known depressant in mammals²³ and common anesthetic for molluscs¹, was the most stress-inducing chemical for sea cucumbers across all of the metrics, indicating its use should minimally be reassessed and certainly avoided in echinoderms. MgCl₂ emerged as a promising anesthetic since it reduced the physical response and respiration rate, and elicited a mild cellular stress response, although it triggered elevated cortisol levels that need clarification and could suggest that sea cucumbers were not totally insensible. Overall, this chemical might only be effective at a higher concentration than the one used here, which might make anesthesia and euthanasia difficult to untangle¹⁸. Combined results for clove oil revealed that it might have merit after making some alterations to the protocol, as it reduced the behavioural and physiological responses, and kept the cellular and hormonal responses at baseline levels, however, these results and those of the predation trials suggest that one or many of the following may be required: an increase in concentration, longer exposure time, or combination of this muscle relaxant with another chemical. Ultimately, MS-222 clearly was the most effective in the present settings. Its use in holothuroids mirrored its prior use in fish. It completely immobilized the individuals, it blocked their cloacal movements (respiration rate), and it kept the coelomocytes and cortisol level at low levels, even in a life-threatening situation. Here, the higher concentration of MS-222 from Applegate et al.¹⁴ was used (0.8 g L^-1); however, lower concentrations would be worth exploring in holothuroids because it might be more effective, both biologically and economically. Overall, this study provided insight into invertebrate anesthesia, including baseline results that could be used to develop new or refined protocols. Additional work is needed to reach definitive conclusions regarding the mode of action, efficacy and broad applicability of these (and other) anesthetic agents in echinoderms and other invertebrate taxa.

Table 5. Summary of stress biomarkers, i.e. behavioural, physiological (respiration rate), cellular (coelomocytes), hormonal (cortisol) and recovery (survival) results exhibited by individuals of Cucumaria frondosa after exposure to all four anesthetic agents for 20 min.

Metric	Ethanol	MgCl2	Clove oil	MS-222
Physical response	Control < Treatment	Control > Treatment	Control > Treatment	Control > Treatment
Respiration rates	Control < Treatment	Control > Treatment	Control > Treatment	Control > Treatment
Coelomocyte densities	Control < Treatment	Control > Treatment	Control ≈ Treatment	Control > Treatment
Cortisol levels	Control ≈ Treatment	Control < Treatment	Control > Treatment	Control ≈ Treatment
Coelomocyte densities (predator present)	Not tested	Control > Treatment	Control < Treatment	Control > Treatment
Cortisol levels (predator present)	Not tested	Control ≈ Treatment	Control > Treatment	Control >  > Treatment
Recovery (survival post-experiment)	Survived	Survived	Survived	Survived

Note that trends may not all be statistically significant (see Tables 1, 2, 4 for statistical analyses).

Materials and methods

Experimental design

Sea cucumbers (Cucumaria frondosa) were hand collected by divers in Tors Cove (Avalon Peninsula, Newfoundland, eastern Canada) at ~ 10 m depth and were acclimated to flow-through conditions in the laboratory (Ocean Sciences Centre, Memorial University) over several weeks. The individuals (n ~ 60) were kept in large tanks (500 L) with ambient unfiltered seawater flow (75–250 L h⁻¹) that provided natural seston as food, along with natural temperature and salinity fluctuations. The light intensity was kept between 32 and 58 lx to mimic field conditions, as described for C. frondosa in Gianasi et al.²⁹.

For the experimental trials, individuals with a mean body-wall wet weight of ~ 106 g (measured after the trials) were placed individually into a separate experimental vessel (5 L) filled with either only seawater (control) or seawater with the treatment anesthetic (see below for details). The vessels were set in a water table continuously supplied with running ambient seawater, so that experimental temperatures followed those in the field and fluctuated around 8.4 °C over the study period. A total of six individuals were used in each trial (n = 3 for control and n = 3 for each treatment condition). These trials were performed twice for each chemical agent, such that 6 individuals per treatment were analysed, with the same number of paired controls. Experiments occurred at the same time each day, one at a time, beginning at 9:00 and finishing before 16:00, alternating between control and treatment trials, and were all completed within a few weeks, in order to avoid potential biases from diurnal and seasonal cycles in C. frondosa³⁸.

Experimental procedures

Exposure to anesthetic agents

The purpose of this experiment was to assess whether the anesthetic agents performed as expected (rendered the subjects unresponsive) and concomitantly caused any responses consistent with stress. Common concentrations and protocols outlined in the literature for each chemical were used (see below). Anesthetic stock solutions, where necessary, were prepared on the eve or morning of the corresponding trials and refrigerated until use. The solution of ethanol (5%) was made by diluting 100% ethanol with seawater to the appropriate proportion and stirring¹. To make the clove oil solution, it was first diluted in a 1:9 ratio in 100% ethanol (clove oil is poorly soluble in water) and proportion of this stock solution was added to seawater to the desired concentration (0.125 ml L^-1) and then stirred as per Lewbart et al.¹. To make the solution of MS-222, the powder was added to seawater up to the desired concentration (0.8 g L^-1) and stirred¹⁴. For MgCl_2, a 1 M stock solution was made with filtered seawater (100 µm mesh size) and mixed at a 1:1 ratio with seawater to reach the desired concentration of 7.5%, then stirred^1,39. Immediately after the vessels were prepared (control with seawater and treatments with seawater + anesthetic), the sea cucumbers were added, and the trial was initiated. We used an exposure time of 20 min for all chemicals, which acts as a suitable average of the immersion times in the current literature¹.

Metrics recorded

Two organismal metrics were used; one was the physical response to a stimulus to test for immobilization, measured at the end of the exposure; the other was the respiration rate (cloacal opening and closing) used as a proxy of physiological stress level, measured during the exposure. In addition, a cellular metric of coelomocyte counts, and a hormonal metric of cortisol levels were also measured following terminal sampling of the sea cucumbers. All metrics are described below.

Physical response (body contraction)

The physical stimulation was used to test for behavioural response (mostly along the body wall) and was measured at the end of the exposure in both the treatment and control individuals. This metric provided information on the general efficacy of the procedure at rendering the subject unresponsive. The individuals were poked in the middle of the body (bivium side) with a metal probe three times (~ 1 s) with a force of ~ 3.5 N. The response to this stimulus was scored in a non-blind fashion on a scale of 0 to 3; 0 being unresponsive, 1 being characterised by slow body wall contractions (lasting > 3 s), 2 being fast body wall contractions (lasting < 3 s), and 3 representing a rapid immediate contraction.

Physiological response (respiration rate)

The respiration rate, identified as a proxy of stress level^22,38, was measured during the exposure. It is defined as the opening and closing of the cloaca (sometimes referred to as the anus), to pump seawater in and out of the respiratory tree where oxygenation occurs⁴⁰. Each time the cloaca opened and closed counted as one respiration. For each sea cucumber (control and treatment), counts were taken over two periods of 5 min, once at the beginning of the procedure (0–5 min) and once at the end (15–20 min).

Cellular response (coelomocytes)

Cellular metrics using counts of coelomocyte were assessed in treated individuals and controls. At the end of the trial (see above), a small longitudinal opening (~ 1 cm) was made with a scalpel near the posterior end of the body, between rows of tube feet. Following a protocol developed by Caulier et al.²¹ and Jobson et al.²², the intact Polian vesicle was rapidly removed and isolated and its fluid poured into a petri dish, transferred to 15 ml vials, and the total fluid amount noted. Subsamples for cortisol analysis (see below) were placed into 1.5 ml centrifuge tubes and stored at − 80 °C. All organs were then removed, and the eviscerated body weight (which included muscle bands and aquapharyngeal bulb) was recorded.

Coelomocyte analysis began immediately after collection due to their tendency to agglomerate together, as mentioned by Caulier et al.²¹. Vials were constantly swirled to keep the cells free and suspended. Aliquots of fluid were placed into a hemocytometer (Neubauer) and random grids were photographs (eight images per individual) under the microscope (Nikon Eclipse 80i) at 200 × magnification. Cells were counted and ascribed to one of three coelomocyte types: phagocytes, morula, and fusiform^21,41.

Hormonal response (cortisol)

Fluid from the Polian vesicle (minimum of 2 ml) was obtained from the terminally sampled sea cucumbers during the analysis of coelomocytes (described above), placed in Eppendorf tubes and frozen undiluted at − 80 °C within 10 min of collection. To prepare samples for processing, the fluid was thawed, and the pH lowered to 1.5–2.0 using 0.5 M HCl. In order to extract cortisol from the fluid, samples were washed once with 4 ml of methylene chloride following the procedure provided for a competitive cortisol ELISA assay (Cayman Chemical—Item 500360). The remainder of methylene chloride was evaporated out of the washed sample using a nitrogen stream and reconstituted with 250 µl of ELISA buffer. Preparation of assay-specific reagents was done following standard ELISA kit protocol. Before plating, each sample of extracted cortisol was centrifuged at 4000 rpm for 5 min. The incubation and development of the plate was completed as per the instructions of the Cayman ELISA manual. As suggested for the 96 well plate, each sample, the 9-point standard curve, blank, total activity, non-specific binding and maximum binding wells were all run in duplicate. Before reading, the plate was shaken mechanically by the microplate reader (Molecular Devices SpectraMax® M5) for 3 s. Using a microplate reader and SoftMax® Pro v7.1 software, the plates were read using a wavelength of 420 nm. Data was analysed using an Excel program from Cayman Chemical specifically designed for this ELISA kit and publicly available (https://www.caymanchem.com/analysisTools/elisa).

Recovery post anesthesia

In order to confirm that the anesthetizing procedure was not permanently damaging or lethal, trials used the same protocols as described earlier, except the subjects were not terminally analysed at the end of the 20-min exposure to the anesthetics. Instead, both the control individuals and those exposed to the anesthetic were returned individually to recovery vessels (5 L beakers with flowing seawater under conditions stated above). The two organismal metrics (physical response and respiration rate, described above) were assessed immediately post transfer and post exposure (0 min and 15 min) and then after 24 h and 1 wk.

Predation trials

In order to further test the efficiency of the anesthetic as a sensory inhibitor, the main predator of C. frondosa, the sea star Solaster endeca was used, since it is well known to induce strong behavioural, cellular and hormonal responses^9,29,42,43. The predation trials consisted of control and treatment trials. Control individuals were exposed to seawater for 20 min, then exposed to direct contact with S. endeca for 1 h. Treatment individuals were exposed to the anesthetic for 20 min (as described above), then exposed to direct contact with S. endeca for 1 h. Ethanol was excluded from these trials since preliminary results showed it could already be dismissed as an anesthetic.

During the exposure to S. endeca, respiration rate was recorded over a 5-min interval at the beginning of the 1-h trial (0 min) and before the end (55 min), following the procedures described earlier. Additional scores were assigned for stages of active buoyancy adjustment (ABA). ABA can be described as the increase in movement and in water-to-flesh ratio in a sea cucumber and is typically used to escape threats like a predator or sub-optimal environmental conditions⁴⁴. The stages of ABA were scored on a scale of 0–3 based on Hamel et al.⁴⁴, where 0 = no signs of ABA, 1 = slightly bloated + podia extended; 2 = slightly bloated + podia retracted; 3 = full ABA display, i.e. podia retracted, fully bloated, contractions/fast movement. At the completion of the monitoring period, individuals were terminally sampled, and fluid collected to determine the cellular and hormonal metrics as per the methods used in the original anesthetic trials (see above).

Data analysis

Data are provided as mean ± standard deviation. All statistical analyses were performed in JASP and used α < 0.05 for significance. Assumptions of normality (Shapiro–Wilk for t-tests, Q-Q plot of residuals for ANOVA) and equal variance (Levene’s test) were explored to confirm the use of parametric tests. If either of the assumptions failed, the data was log transformed and tested again. If this failed to correct data distributions, a non-parametric test was conducted. Only the qualitative behavioural scores (response to poking and ABA) were not statistically tested.

A two-way analysis of variance (ANOVA) was first performed to test the differences between the respiration rates of control and treatment groups at beginning and end of the trial. Because of a significant interaction between treatment and time within the clove oil trial, independent one-way ANOVA tests had to be conducted for each of these factors. A two-way ANOVA was used in the same way to test differences of respiration rates across the 1 h predation trials.

The coelomocyte density for control and treatment groups of each chemical were compared with a t-test. The data for MgCl₂ and MS-222 had to be log transformed due to failure of the normality test. Once transformed, both data sets met the assumptions. Similarly, t-tests were performed for comparison between control and treatment groups for phagocyte density. The coelomocyte densities for the predation trials were tested with a one-way ANOVA followed by pairwise post-hoc analyses (Bonferroni method) to compare each treatment to the pooled controls (controls were pooled when not statistically different).

Any cortisol values that read below the detection limit were considered unreliable and assigned the minimum value within the detection limit. There were no readings above the detection limit. Cortisol levels obtained following the 20-min anesthesia trials and the predatory trials were log transformed and the effect of treatment tested with a one-way ANOVA.

Acknowledgements

We thank the members of the Mercier Lab for their continued support, Javier Santander and Ian Fleming for allowing us to use their stocks of MS-222, and Andrea Leyte for providing help during the experiments. This research was partly supported by a grant from the Natural Sciences and Engineering Research Council of Canada (NSERC) to AM.

Author contributions

JC: conceptualization; methodology; investigation; formal analysis; data curation; visualization; writing—original draft. SJ: conceptualization; methodology; investigation; data curation; visualization; writing–review and editing. JFH: conceptualization; methodology; validation; supervision; resources, writing–review and editing. AM: conceptualization; methodology; validation; funding acquisition; project administration; supervision; resources, writing–review and editing.

Data availability

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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