Cardiovascular physiology of decapod crustaceans: from scientific inquiry to practical applications Free

The cardiovascular system of crustaceans, like the majority of invertebrate circulatory systems, is classified as open, meaning that the circulating fluid is not completely enclosed within vessels. However, this classification covers a broad array of systems, from simple open spaces or sinuses between the tissues as in the Cirripedia, through the tubular hearts of isopods, to the globular heart and complex series of arteries and capillary-like vessels of decapods (McGaw and Reiber, 2015). Despite a plethora of work on the cardiac physiology of crabs and lobsters carried out between the 1960s and 1990s, the system was, until recently, still viewed as fairly rudimentary when compared with vertebrate cardiovascular systems. However, this view is changing as a result of a number of substantive articles published in the last two decades. We now know that the crustacean cardiovascular system is more complex than first thought, with control mechanisms akin to some of the simple vertebrate closed systems (Wilkens, 1999; McMahon, 2001; Reiber and McGaw, 2009; McGaw and Reiber, 2015). It has been proposed that, at least in physiological terms, the decapod cardiovascular system may now be classified as one that is partially closed, rather than open (Reiber and McGaw, 2009).

Changes in cardiac parameters can be used as a proxy for metabolic rate, stress levels and physiological responses to environmental and biotic variables (DeFur and Mangum, 1979; Handy and Depledge 1999; Wikelski and Cooke 2006; Green 2011). As such, they change in response to exercise, feeding, hypoxia, emersion, low salinity and temperature in a variety of decapod species. A comprehensive analysis of these responses in crustaceans is discussed in other works (McGaw and Reiber, 2015; McGaw and Whiteley, 2023). Instead of reiterating that information, this Review concentrates on more recent, though somewhat disparate areas of investigation that were not extensively explored in previous review articles. These encompass a re-evaluation of the anatomical function of the heart and an exploration of how heart rate and cardiac pausing (brief stoppage of the heart) can be used to interpret reactions of crustaceans to environmental stressors. This Review concludes by investigating practical applications of heart rate monitoring in decapod crustaceans.

The heart of the decapod crustacean is described as a single-chambered ventricle suspended within the pericardial sinus (see Glossary) by elastic ligaments (Maynard, 1960). The heart is neurogenic (see Glossary); signals that stimulate its contraction originate from cardiac pacemaker cells situated in the cardiac ganglion, which synapses onto the dorsal surface of the heart (Wiersma and Novitski, 1942; Maynard, 1960; McMahon and Wilkens, 1983). Seven arteries originate from the decapod crustacean heart (Fig. 1). These arteries branch into smaller arteries and capillary-like vessels that divide within the tissues, where the exchange of gas, nutrients and waste occurs (Maynard, 1960). Once haemolymph has bathed the tissues, it drains into a network of interstitial lacunae, which are distributed throughout the tissue (McLaughlin, 1983; McGaw and Reiber, 2002, 2015; McGaw, 2005). Haemolymph from the lacunae drains into larger irregular spaces, called sinuses, all of which eventually drain into the large ventral thoracic sinus. Subsequently, haemolymph is reoxygenated as it flows through the branchiocardiac veins of the gills. It is thought that movement of blood through the gills is facilitated by contraction of the skeletal muscles, the scaphognathite pump (see Glossary) and one-way valves in the gill vessels (Taylor and Taylor, 1986, 1992). From the gills, oxygenated haemolymph enters the pericardial sinus, from which it fills the ventricle through a series of paired ostia (see Glossary).

Decapod crustaceans can partition haemolymph flow through the different arterial systems, sending blood to more metabolically active tissues. However, unlike mammals, the majority of crustaceans do not have layers of smooth muscle in their arterial wall to aid in selective tissue perfusion (Shadwick et al., 1990). Instead, haemolymph flow is thought to be regulated by muscular valves at the base of each major artery (Kuramoto and Ebara, 1984; Kihara and Kuwasawa, 1984; Tsukamoto et al., 1992; Kuramoto et al., 1995; Wilkens and Kuramoto, 1998). These valves are innervated (Alexandrowicz, 1932), and various neurohormonal modulators can cause them to contract or relax, leading to the redistribution of arterial haemolymph flow (Alexandrowicz, 1932; Kuramoto and Ebara, 1984, 1991; Hill and Kuwasawa, 1992). Unlike the major arteries, there is no evidence of valves in the smaller arteries (McGaw and Reiber, 2015). Partitioning of haemolymph flow within these smaller vessels may be aided by changes in their resistance, which is regulated by neurotransmitters and neurohormones. Small changes in the lumen diameter brought about by these modulators can cause large changes in the resistance of the vessel (Wilkens et al., 1997a,b; Wilkens and Taylor, 2003).

Similar to haemolymph redistribution, cardiac function can also be modulated by both neural and hormonal control mechanisms. Neural control involves the cardioregulatory nerves, which include both inhibitory and acceleratory fibres. These nerves act directly on the cardiac ganglion to influence heart rate and stroke volume (reviewed in Saver and Wilkens, 1998; McMahon, 2001; McGaw and Reiber, 2015; Muscato et al., 2022). In addition to acting on the cardioregulatory nerves, a number of peptide and amine neurohormones act directly on the cardioarterial valves, the artery walls, the central nervous system or the cardiac muscle itself (Wilkens et al., 1996; Saver et al., 1998; McMahon, 2001; Muscato et al., 2022; Dickinson and Powell, 2023).

In order to understand the evolution of thinking in this field, it is important to consider the methods used to investigate crustacean cardiovascular function in the past, and how new biomedical innovations are being used today. The direct measurement of heart rate in decapod crustaceans can be accomplished using techniques such as impedance conversion, pulsed-Doppler probes and infrared transmitters (DeFur and Mangum, 1979; Depledge and Andersen, 1990; Airriess et al., 1994; Handy and Depledge, 1999; McGaw et al., 1994, 2018; Stillman, 2004; Tepolt and Somero, 2014). These techniques are relatively straightforward to set up, and this ease of use probably accounts for why heart rate (rather than stroke volume or cardiac output) is the most commonly used indicator of cardiac function in decapod crustaceans. In contrast, accurate measurement of stroke volume and cardiac output is more difficult to perform; thus, these parameters are often calculated rather than measured directly. For example, cardiac output can be calculated using the thermodilution principle. Here, catheters implanted in arteries allow for the measurement of downstream temperature changes following the injection of a bolus of cold saline. The temperature drop is measured and a thermodilution curve is generated: the area under the curve is inversely proportional to cardiac output (Nanchal and Taylor, 2008). To determine stroke volume, pulsed Doppler probes can be implanted on all arteries exiting the heart and the summation of these flows gives a value for stroke volume (Airriess et al., 1994; DeWachter and McMahon, 1996). Alternatively, in the heart of transparent species, such as grass shrimp (Palaemonetes pugio), stroke volume can be measured using high-speed videography (Spicer, 2001, 2006; Harper and Reiber, 2004; Guadagnoli and Reiber, 2005; Göpel and Wirkner, 2018). Playback of the video allows the ventricular cross-sectional area during diastole and systole to be traced. The change in two-dimensional ventricular area during a cardiac cycle can then be converted to volume by modelling the ventricle as a prolate spheroid or trapezoidal shape (Guadagnoli et al., 2007). Sonomicrometry can also be used to estimate stroke volume by measuring dimensional changes in semi-isolated decapod crustacean hearts (Rose et al., 2001). By placing several sonomicrometry crystals on either side of semi-isolated heart preparations, the sonomicrometer can calculate the linear distance based on the time it takes for a sound wave emitted from one crystal to reach the second crystal (Rose et al., 2001). The advantages of sonomicrometry are that it allows for direct measurement of dimensional changes of the heart, and these changes are presumed to be equivalent to stroke volume (Rose et al., 2001). However, recent work by Maus et al. (2019a,b, 2021) brings this presumption into question, because the inner chamber of the heart does not follow the outer dimensions during cardiac contraction; in fact, the inner chamber is more complex in shape (Fig. 2). These findings are important, and their implications are discussed below.

Although the ‘single-chambered ventricle’ has typically been regarded as rather simple, knowledge regarding some of its anatomical complexities is not new. As far back as 1921, Baumann showed that, anatomically, three dimensional bands of muscle cross the inner surface of the heart, dividing the lumen into sub-chambers. The presence of these muscle bands was noted by Maynard (1960), who stated that ‘the effect of such cardiac divisions on blood flow is not known’. Over 50 years later, Wirkner and colleagues used microcomputed tomography to section casts of the cardiovascular system of pagurid and lithodid crabs; they also found asymmetrical muscle strands crossing the lumen of the heart, dividing it into ‘cavities’ (Keiler et al., 2013, 2015a,b; Wirkner et al., 2013).

Recently, Maus et al. (2019a) used magnetic resource imaging to investigate heart contraction and haemolymph flow in intact brown crabs (Cancer pagurus). They confirmed earlier work (Baumann, 1921; Maynard, 1960) showing that the single ventricle is subdivided into cavities by folds of muscle (Fig. 3). The largest sub-structure connects the paired ostia, extending to the posterior and sternal arteries; three smaller cavities occur at the base of the paired hepatic and anterolateral arteries and the anterior aorta (Maus et al., 2019a,b, 2021; Fig. 3). These authors note that the myocardial folds ‘may assist in the distribution of haemolymph, in conjunction with arterial resistance and cardiac valves, but this remains to be verified’ (Maus et al., 2019b). This represents an exciting new hypothesis that could potentially change the way we view regional haemolymph perfusion in decapod crustaceans. As discussed above, until now, it was assumed that differential haemolymph flow was controlled by the cardioarterial valves at the base of each artery (Alexandrowicz, 1932; Kuramoto and Ebara, 1984, 1991; Hill and Kuwasawa, 1992). However, this knowledge on valve function is based on the use of semi-isolated heart preparations, and, to our knowledge, the function of the cardiac valves in vivo has not been investigated. This could have important connotations, because cardiac responses to neurohormone modulators can differ in in vivo whole-animal preparations (McGaw et al., 1995; Powell et al., 2023). If these hypotheses that the heart is indeed functionally chambered are proved correct, it will change our understanding of cardiovascular dynamics of invertebrate open circulatory systems.

Within the field of vertebrate cardiovascular physiology, an increasing body of literature shows that the time interval between individual heart beats is highly variable (reviewed in Ernst, 2014). In mammals, this represents the influence of the autonomic nervous system on the underlying cardiac rhythm generated by the pacemaker cells (von Borrell et al., 2007). This natural heart rate variation is characteristic of a healthy heart, whereas steady inter-beat frequency is usually associated with some type of cardiac impairment (Ernst, 2014; Ibbini et al., 2022). This is similar for decapod crustaceans: resting, unstressed crabs exhibit intermittent heart activity, whereas heart rate variation is more consistent in stressed and/or immobilized animals (Yazawa and Katsuyama, 2001; Yazawa, 2015; McGaw and Nancollas, 2021). Deciphering heart rate variation in mammals usually requires longer-term recording (24 h) and specific algorithms to be applied to the data (von Borell et al., 2007). However, heart rate variation is more obvious and accentuated in crustaceans because they can completely stop their heart for extended periods (this is known as ‘acardia’). This phenomenon distinguishes crustaceans from vertebrates and many invertebrate taxa; however, we still do not fully understand its purpose (McGaw and Nancollas, 2021). In this section, we consider the variance in resting heart rate and discuss the patterns and possible function of cardiac pauses.

Resting heart rate varies considerably among individual decapods of the same species and is independent of mass or time of year (Yazawa et al., 2005; Kushinsky et al., 2019; McGaw and Nancollas, 2021). In resting, unstressed crabs, the heart rate can be quite variable from minute to minute (Fig. 4), which is likely the natural pattern (Cumberlidge and Uglow, 1977; McGaw and Nancollas, 2018, 2021). For example, in green crabs (Carcinus maenas), heart rate varies between 15 and 150 beats min⁻¹ at 11–15°C (Cumberlidge and Uglow, 1977; Depledge, 1978; McGaw and Nancollas, 2021).

As might be expected, stressed crabs have higher heart rates than unstressed animals, but their heart rate is also more stable and continuous (Cumberlidge and Uglow, 1977; McGaw and Nancollas, 2018, 2021). ‘Immobilized’ C. maenas strapped to a plastic grate have a more consistent heart rate compared with those maintained in a small box (Fig. 4) where they are not restrained but movement is restricted (McGaw and Nancollas, 2018). The immobilized crabs can still move their legs, and this will increase heart rate and haemolymph flow (McGaw and McMahon, 1996); it is therefore likely that the steady heart rate in immobilized individuals results from stress, and not from the loss of locomotor activity (Yazawa and Katsuyama, 2001; Yazawa, 2015; McGaw and Nancollas, 2018).

When compared with decapods in their natural habitat, individuals that are kept in the laboratory for long periods or are in poor condition tend to have lower heart rates (Depledge, 1985; Aagaard, 1996). This is likely due to continuous sensory input experienced by an animal as a result of the constant changing of multiple environmental parameters in the natural environment (Aagaard, 1996; Styrishave et al., 2003).

In decapod crustaceans, brief pauses in heart rate lasting 1–5 s can be provoked by touch, vibrations, visual disturbance or simply a person entering the room where the animal is housed (McMahon and Wilkens, 1977; Hermitte and Maldonado, 2006; Yang et al., 2013; Canero and Hermitte, 2014; Yazawa, 2015). These pauses are also sometimes associated with ventilatory reversals (see Glossary; McMahon, 1999; McGaw, 2004). Because both of these (cessations of heart rate) tend to be spontaneous and instantaneous, it suggests direct control of the heart by neurons of the cardiac ganglion (Yazawa and Kuwasawa, 1992; Yang et al., 2013).

Extended periods of acardia (>10 s) primarily occur in unstressed, resting animals (Yazawa and Katsuyama, 2001; McGaw, 2004; Kushinsky et al., 2019; McGaw and Nancollas, 2018, 2021). These cardiac pauses rarely exceed 400 s, and are commonly between 10 and 90 s in duration, interspersed with occasional single heart beats (Kushinsky et al., 2019; McGaw and Nancollas, 2021; Fig. 5). Variation in heart rate in crustaceans is, at least in part, linked to these bouts of acardia, because the heart rate gradually slows and then increases as the animal enters and exits periods of cardiac pausing, respectively (Kushinsky et al., 2019; McGaw and Nancollas, 2021). Immediately after a period of acardia, the heart rate is elevated; but it slowly declines during the interpause period (Figs 5 and 6). Because these changes in heart rate are gradual, they are likely to be mediated by neurohormonal input either on the cardiac ganglion or directly on the cardiac muscle, rather than by a purely neural control mechanism, which would be expected to cause more rapid reductions in heart rate (McMahon, 1999; Wilkens, 1999; McGaw and Reiber, 2015; Dickinson and Powell, 2023). In support of this theory, in lobsters, regular periods of acardia of 10–15 min are associated with release of the neurotransmitter GABA and its subsequent uptake (Yazawa and Katsuyama, 2001).

Non-stressed decapods can exhibit periods of acardia that are remarkably uniform in both the length of the bout of acardia and the interpause period (Cumberlidge and Uglow, 1977; Kushinsky et al., 2019; McGaw and Nancollas, 2021; Fig. 6). The rhythmicity of these events is controlled by central pattern generators in the cardiac ganglion and modulated by neurohormones (Muscato et al., 2022; Dickinson and Powell, 2023; Powell et al., 2023), changes in pH and associated ion levels (Haley et al., 2018), or by extrinsic input (from another neural system) to the cardiac pacemaker neurons (Powell et al., 2023).

Changes in heart rate and ventilation are closely coupled, such that when heart rate ceases, there is a concurrent cessation in ventilation of the branchial chambers (see Glossary; Wilkens et al., 1974; McMahon, 2001; McGaw, 2004). As such, haemolymph flow through the branchial veins and irrigation of the gill chambers stops. Although these events interrupt oxygen supply and delivery, the effects on downstream processes are likely not that pronounced. The overall haemolymph volume of decapod crustaceans is approximately 25–30% of their body mass (McMahon and Wilkens, 1977; Airriess and McMahon, 1994); this carries enough oxygen for up to 5–6 min of acardia (Johansen et al., 1970). Indeed, McMahon and Wilkens (1972, 1977) report that during periods of ventilatory and cardiac cessation, the haemolymph oxygen tension remains stable for 5–6 min before dropping sharply. This correlates with time periods of acardiac events for a number of decapod species, where periods of acardia lasting more than 400 s (i.e. 6 min 40 s) are rare (Kushinsky et al., 2019; McGaw and Nancollas, 2021). This would suggest that the timing of acardia and restarting of heart rate are dependent on the levels of oxygen, CO₂ and H⁺ in the haemolymph (McMahon and Wilkens, 1977).

The exact function of longer cardiac pauses, which are distinct from the short-term (<5 s) startle response (McMahon and Wilkens, 1972, 1975, 1977; Canero and Hermitte, 2014; Yazawa, 2015), is still unclear. The most common explanation is that they act as an energy conservation mechanism when metabolic demands are low (McMahon and Wilkens, 1977; McDonald et al., 1977; Burnett and Bridges, 1981): the fact that they only occur in inactive crabs corroborates this theory (McGaw and Nancollas, 2018, 2021). That being said, cardiac pauses are rarely observed during hypoxic exposure when one would expect an animal to attempt to reduce its energy needs – instead a consistent bradycardia (see Glossary) is observed (McGaw and Nancollas, 2018). It is also suggested that these cardiac pauses are an example of invertebrate ‘sleep’, but this too has not been proven (Yazawa and Katsuyama, 2001). Determining the functional significance of these periods of acardia clearly warrants further investigation.

Although heart rate has been and continues to be used extensively in crustaceans as an indicator of stress or metabolic work, one must be cautious against relying too heavily on heart rate alone as a proxy for these states. In crustaceans, stroke volume can vary independently of heart rate to influence the overall cardiac output (Airriess and McMahon, 1994; McGaw and McMahon, 1996). This may be especially pertinent given the fact that the heart is partitioned internally by bands of muscle (Maus et al., 2019a,b, 2021; Fig. 3) and may act as a chambered structure (R.A.E. and I.J.M., unpublished observation). In addition, because periods of acardia appear to be an essential part of the cardiac repertoire, taking into account the length and frequency of these events may be just as important as monitoring heart rate, stroke volume and/or cardiac output when investigating the responses of decapod crustaceans to environmental stressors.

Within decapod crustaceans, cardiac responses to similar environmental challenges are quite disparate, even within a single species (McGaw and Whiteley, 2023). This could be due to the nature of the experimental set-up (Cumberlidge and Uglow, 1977; McGaw and Nancollas, 2018), lack of settling time in the apparatus prior to recording (Taylor, 1976; Butler et al., 1978; Wilson et al., 2021) or even unforeseen laboratory elements, such as vibrations from water pumps and electrical motors, which can disturb an animal (Florey and Kriebel, 1974; Burnovicz et al., 2009). Until recently, the nature of the equipment used to record heart rate meant that all experiments had to be carried out in the laboratory, and the animals were tethered or their movement restricted. Sound scientific practice also dictated that animals were acclimated to static conditions beforehand, and the effect of one environmental parameter was usually determined in isolation. In reality, environmental parameters rarely remain stable, and changes in heart rate that are evident in the lab can be accentuated, masked or absent altogether in the natural environment where animals are exposed to multiple stimuli (Styrishave et al., 2003). Therefore, in order to achieve more biological relevance in our understanding of crustacean cardiac function, in situ measurements of heart function would be informative.

Advances in, and miniaturization of, heart rate data loggers have reached a point where these tools can now be used in situ on relatively small, free-moving aquatic animals (McGaw et al., 2018; Steell et al., 2020; Brijs et al., 2021; Gutzler and Watson, 2022). At the time of writing, heart rate data loggers have not been widely used in crustaceans, but the initial findings are interesting. For example, a comparison between the slipper lobster (Scyllarides aequinoctialis) and the Caribbean spiny lobster (Panulirus argus) shows that heart rate of these two species is entrained by different environmental variables in the field. The slipper lobster exhibits an elevated heart rate during nocturnal high tides (McGaw et al., 2018; Fig. 7A); certainly, many crustaceans are more active at night than during daytime high tides (Naylor, 1985). In contrast, spiny lobster heart rate does not change on a tidal or diel basis (McGaw et al., 2018; Steell et al., 2020). Instead, an increase in heart rate of 15–20 beats min⁻¹ correlates with an increase in water temperature in this species (Fig. 7B). This is not unexpected, as heart rate increases in line with temperature for most crustaceans (reviewed in McGaw and Reiber, 2015); however, in this case, the change in heart rate is associated with an increase in water temperature of just 2–3°C (McGaw et al., 2018). This is noteworthy because, in many research articles, the authors state that they used a ‘stable’ water temperature of X±1°C: these data would suggest that a 2°C range in water temperature is not stable with respect to heart rate. Over a slightly greater temperature change of 5°C (11–16°C change), Pacific spiny lobsters (Panulirus interruptus) fitted with heart rate data loggers exhibit an even more pronounced increase in heart rate, of approximately 50 beats min⁻¹ (Csik, 2020).

In stable environmental conditions (e.g. temperature, salinity, oxygen), rapid or large-scale increases in cardiac parameters are usually associated with an increase in locomotor activity (DeWachter and McMahon, 1996; Burnovicz et al., 2009; McGaw and Nancollas, 2021). However, an increase in heart rate is not observed in free-moving P. argus, suggesting that there is a dissociation between heart rate and activity (Steell et al., 2020). Heart rate does increase by approximately 20% in free-moving American lobsters (Homarus americanus); however, it eventually plateaus, despite a further increase in walking speed, again showing a dissociation between heart rate and activity (Gutzler and Watson, 2022). One limitation of miniaturized data loggers is that they only record heart rate; it could be that an increase in stroke volume (independent of rate) – as occurs during walking in Dungeness crabs, Cancer magister – supplies the increased metabolic demand (DeWachter and McMahon, 1996).

Although the data discussed above are novel, the relatively high cost of data loggers and the fact that one has to retrieve the animal and logger in order to download data will mean, at least in the short term, that most experiments monitoring heart rate will still be carried out in the laboratory. In addition, the comparatively short battery life and limited onboard memory of miniaturized data loggers means that data have to be recorded intermittently, missing short-term changes in heart rate (McGaw and Nancollas, 2021; Gutzler and Watson, 2022). Finally, because one cannot measure all the variables that a free-moving crustacean will experience, it could be very difficult to actually determine what may be influencing changes in heart rate.

Historically, research on the cardiovascular responses of decapod crustaceans has largely been directed towards increasing our scientific knowledge of physiological control mechanisms. As such, it has sometimes been difficult to describe, in layman's terms, how this basic or pure scientific research may actually be of benefit to humanity. However, this does appear to be changing; in the last two decades, physiological measures have been used to determine stress responses and disease progression in aquacultured crustaceans. In addition, the general public are becoming more concerned about animal welfare within aquaculture operations, as well as during the subsequent handling and dispatching of crustaceans that are destined for the table. Below, we discuss how our understanding of cardiac function is being applied.

Most of the research on the cardiovascular responses of decapods has concentrated on that of large commercially fished species, or smaller intertidal animals that are readily available and easily maintained in the lab. In the past three decades, a ‘Blue Transformation’ has taken place, whereby seafood produced by aquaculture now constitutes a greater percentage than that landed in capture fisheries (FAO, 2022). During this time, a >1200% increase in aquaculture of crustaceans has occurred (FAO, 2022; Kültz, 2022). This has been paralleled by an increased research emphasis on aquacultured species (e.g. shrimp/prawn, crayfish, swimming crab). Despite this upswing, much of this research still focuses on how changes in temperature (Chung et al., 2012; Ren et al., 2021; Duan et al., 2022), oxygen (Zheng et al., 2022) and salinity (Chen et al., 2016) affect cardiac and metabolic processes and, ultimately, growth and survival. However, changes in heart rate are starting to be used in a number of more novel applications, such as in monitoring disease and stress in aquacultured animals.

Diseases such as white spot virus syndrome (WSVS) are a problem in high-density shrimp-farming operations, leading to mortality rates of 90–100% (Walker et al., 2012). WSVS presents as white spots on the body in heavily infected Kuruma shrimp (Marsupenaeus japonicas), and is accompanied by reduced swimming and lethargy (Cheng et al., 2021). However, at lower viral loads, this disease is not visually obvious. Physiological responses investigated during the progression of WSVS infection show that heart rate increases during the early infection stages, but starts to decrease as more cells become infected and break down (Cheng et al., 2021). Importantly, there is a decrease in heart rate variation as the viral load increases, because it affects the cardiac autonomic nervous inputs. These changes in cardiac function could provide a mechanism of early detection before the disease is visually apparent (Cheng et al., 2021).

The transport and marketing of live crustaceans is increasing rapidly in line with the demand for high-quality seafood. As such, methods that reduce stress and minimize product loss during transport have received significant attention. It has been postulated that if heart rate can be lowered, this would reduce oxygen use and energy expenditure during transport, and measurements of heart rate and its variability have therefore been used as a way of investigating best practice in animal transport. For example, temperature stress during shipping can affect the quality and post-harvest survival of mud crabs (Scylla spp.). As expected, the heart rate of mud crabs increases with temperature between 10 and 38°C (Tsai et al., 2019). A lower heart rate at lower temperatures might be expected to reduce transport stress, as the crabs would have a lower metabolic rate (Tsai et al., 2019). However, using analysis of heart rate variation, a temperature of 14–18°C was determined to be the optimal range to reduce stress during transport (Tsai et al., 2019).

Another example of the use of heart rate to investigate animal welfare during transport is provided by studies on AQUI-S and adenosine. AQUI-S is a commercially produced aquatic anaesthetic that has been investigated as a means to reduce stress during transport in the southern rock lobster (Jasus edwardsii; Robertson et al., 2018). When AQUI-S is administered into the water at increasing concentrations, heart rate increases with dosage between 10 and 90 ppm, with a noticeable drop thereafter. At around 200 ppm, heart rate stabilizes at 44 beats min⁻¹ and remains at consistently low levels (Robertson et al., 2018). However, this effect is only sustained while the lobster is stored in water with AQUI-S. When the lobster is moved to clean water or air, the heart rate increases, although to lower levels than those observed for untreated lobsters. Heart rate is regained within an hour of emersion in clean water, as AQUI-S is rapidly metabolized, meaning it is only suitable for short-term transport. Willis et al. (2022) followed up on this work using the freshwater prawn Macrobrachium rosenbergii. They found that adenosine, which is associated with the innate torpor reaction in hibernating species (Bundgaard et al., 2020), injected at a concentration of 2.5 nmol l⁻¹, reduces heart rate for 2–3 h. As with AQUI-S, this would be appropriate to reduce stress during short-term transport, but further research is needed to identify agents suitable for longer-term transport (Willis et al., 2022).

Changes in heart rate occur in sick crustaceans and may indicate that death is imminent (Yazawa et al., 2005). For example, in coconut crabs (Birgus latro) and Mokuzu crabs (Eriocheir japonicus), there is a change in the interval between similar points of an electrocardiogram (EKG) trace in sick crabs prior to their death. In healthy crabs, the interval between similar points on the EKG is close to one. However, in the 24 h preceding death, this interval fluctuates, and it drops below one in the 12 h prior to the animal expiring (Yazawa et al., 2005). This change in interval rate on the EKG trace results from changes in neural or hormonal control systems or fluctuating ion levels in sick animals; ultimately, it could be used to predict loss of stock (Yazawa et al., 2005). The analysis of heart rate variation even suggests that lobsters may experience stressful episodes that are akin to vertebrate emotions (Yazawa, 2015, 2017). Of course, the idea of using heart rate variation to predict death and identify ‘emotions’ is an example of early work that needs more substantiation. However, such ideas lead nicely into the next section, which discusses a more recent focus on the welfare of crustaceans.

The use of vertebrates and cephalopods in scientific research typically requires animal care protocols to be submitted prior to experimentation, to ensure that animals are treated in an ethical and humane manner (Elwood, 2021). In recent years, the welfare of decapod crustaceans has gained recognition, with several review articles emerging as a result (e.g. Sneddon et al., 2014; Diggles, 2019; Elwood, 2021). These articles all suggest the need for increased protection for large crustaceans (Elwood and Adams, 2015; De Morri and Normando, 2019; Passantino et al., 2021; Crump et al., 2022). Guided by an independent review (Birch et al., 2021), the UK government have recently legally recognized decapod crustaceans as sentient beings [Animal Welfare (Sentience) Act 2022]; this will have significant implications for animal welfare protocols. This designation will probably also be implemented in other countries throughout Europe, with ramifications for the use of decapod crustaceans in scientific research. Thus, it will be important to understand best practices to reduce stress or suffering when carrying out experiments on decapod crustaceans (Carder, 2017; De Morri and Normando, 2019; Passantino et al., 2021).

Typically, behavioural responses such as increased activity, righting reflexes or tail flipping/antennal flicking are used to indicate distress in crustaceans (Stoner, 2012). Levels of crustacean hyperglycaemic hormone and haemolymph glucose and lactate are also effective indicators (reviewed in Albalat et al., 2022; Wuertz et al., 2023). Heart rate has been used for decades in physiological research as a bioindicator of responses to environmental perturbations or as a proxy for metabolic rate (reviewed in McGaw and Reiber, 2015; McGaw and Whiteley, 2023). Heart rate is also being incorporated as a stress indicator in crustacean welfare for testing the efficacy of anaesthetics and for assessing effective stunning or euthanasia methods (Wuertz et al., 2023), as we discuss below. At present, heart rate is classified as a moderate indicator of pain and stress in decapods (Diggles et al., 2024). Despite the new legislation and the increasing number of articles discussing crustacean welfare, Diggles et al. (2024) state that we still need to maintain scepticism until we have more scientific validation of different methods to assess pain and sentience in decapod crustaceans.

Anaesthesia in decapod crustaceans, as mentioned above, has primarily been used to reduce stress (and thus improve survival) during live transport (e.g. Robertson et al., 2018; Pozhoth and Jeffs, 2022; Rotllant et al., 2023). However, given the change in status for decapods, its use for general handling and surgical procedures will likely increase substantially in the coming years (de Souza-Valente, 2022; Rotllant et al., 2023; Spoors et al., 2023). In the past decade, work in this area has incorporated heart rate as a physiological measurement to complement behavioural indicators to assess the efficacy of various anaesthetics; however, this might not always be appropriate, as discussed below.

Eugenol, an active ingredient in clove oil, is used extensively as a fish anaesthetic (Martins et al., 2019; Aydin and Barbas, 2020). Intravenous injection of eugenol into blue crab (Callinectes sapidus), red swamp crayfish (Procambarus clarkii) and whiteleg shrimp (Litopenaeus vannamei) at concentrations of 200 ppm and above results in lethargy and lack of proprioception, lasting for up to 30 min (Wycoff et al., 2018). The authors hypothesized that because the heart is neurogenic, eugenol should bind to the cardiac ganglion, causing a decrease or cessation of heart rate (Wycoff et al., 2018). However, the heart rate of C. sapidus and P. clarkii is unchanged after administration of eugenol (Wycoff et al., 2018). Litopenaeus vannamei does exhibit a 35% drop in heart rate at the highest injection concentrations (400 ppm), but this decline in L. vannamei is probably an artifact due to an abnormally high pre-treatment heart rate of 200–250 beats min⁻¹ (Wycoff et al., 2018). The heart rate of C. sapidus does decline by 35% following intravascular injection of the drug alfaxalone (100 mg kg⁻¹), but recovers after 10 min (Minter et al., 2013). These authors suggest that this ‘bradycardia’ may also be an experimental artifact associated with a pre-injection tachycardia (see Glossary) caused by handling stress. As with eugenol, sensory and behavioural inactivity are observed for over an hour following injection, so despite the lack of a cardiac response, eugenol and alfaxalone are acting as efficient anaesthetics, indicating that analysis of heart rate might not be the best way to assess the suitability of anaesthetics in crustaceans (Minter et al., 2013; Wycoff et al., 2018).

Intravascular injection of ketamine, propofol, tiletamine–zolazepam and xylacine into C. sapidus causes a bradycardia during the injection process, but heart rate is regained within 5–10 min after treatment (Quesada et al., 2011). Because behavioural responsiveness also resumes within 5–10 min, these are deemed to be unsuitable anaesthetics. The fact these substances do cause prolonged anaesthesia in other decapod species (Oswald, 1977; Gardner, 1997) suggests that responses are species specific, and this will need to be tested for in the future (Quesada et al., 2011; Willis et al., 2022). In each of these studies, the authors emphasize a decrease in heart rate as an indicator of effective anaesthesia (Quesada et al., 2011; Minter et al., 2013; Wycoff et al., 2018). However, a decline in heart rate does not necessarily have to occur for a substance to be an effective anaesthetic (Cooke et al., 2004; Cotter and Rodnick, 2006; Kübra, 2022). Indeed, if a substantial decrease in heart rate occurred, it would indicate the deepest level of anaesthesia with severe depression of nerve impulses, from which crustaceans typically do not recover (de Souza Valente, 2022).

In most Western nations, farmed animals destined for human consumption have to be stunned prior to slaughter (reviewed in Terlouw et al., 2008). There is increasing pressure to find appropriate methods for stunning or rapid euthanasia of crustaceans that are destined for the table (Elwood, 2021). The inability to detect a viable heart beat can be used as an indicator of unresponsiveness and/or death (Weineck et al., 2018), potentially making it a more useful measurement in this regard than in assessments of anaesthesia. A number of different treatments have been used to render crustaceans ‘unresponsive’ prior to slaughtering and/or cooking, and these are discussed below.

Low-temperature exposure is an effective method to incapacitate tropical and temperate crustacean species. When P. clarkii and L. vannamei are immersed in an ice slurry, the heart beat becomes undetectable within 5 min and the animals remain unresponsive to external stimuli during this period (Piana et al., 2018; Weineck et al., 2018). Whether the animal is still responsive can be checked by a tap on the cephalothorax. If the animal is still responsive, this tap results in a rapid, short-term increase in heart rate in an animal with no measurable heart beat or, if the heart is still beating (even slowly), it will cause an immediate cessation of heart rate for a few seconds. The fact that P. clarkii and L. vannamei are unresponsive during cold exposure, but heart rate recovers after 5 min when animals are put back into warmer water, indicates that this is an effective stunning rather than killing method (Weineck et al., 2018). In other studies, chilling cold-water crustaceans (American lobster, H. americanus; European crayfish, Astacus astacus) to 0°C depresses or halts cardiac function; however, because the heart still responds to sensory stimulation during this chilling period, this is likely to be an unsuitable method for rendering cold-water crustaceans unresponsive (Fregin and Bickmeyer, 2016).

Heating is also used to incapacitate crustaceans, and cardiac function can be used to assess the efficacy of this method. Adams et al. (2019) used the breakdown of a regular heart beat and its subsequent cessation to determine how different rates of warming affect P. clarkii. During slow warming (1°C min⁻¹), heart rate becomes sporadic upon reaching 40°C and ceases at 44°C; animals do not recover when returned to 15°C. With rapid heating (12°C min⁻¹), disruption of heart rate occurs at a mean of 51°C, with a measurable beat still persisting in some individual animals at 60°C. When crayfish are placed directly into hot water (>80°C) it still takes several minutes for the heart to flat-line, representing the time taken for the body temperature to equilibrate with that of the water. However, when an animal is placed directly into boiling water (100°C), heart rate becomes irregular after 5 s, ceasing completely a few seconds later. In all cases, it was determined that when heart rate becomes irregular, the crayfish are no longer responsive and are essentially dead. The recommendation is that immediate introduction into boiling water shortens the noxious event as opposed to slow heating (Adams et al., 2019).

Although live boiling would appear to be a fast and effective method to kill crustaceans, it has been outlawed in a number of countries (New Zealand, Norway, Switzerland and Tasmania, Australia) (EFSA Panel on Animal Health and Welfare, 2005; European Food Safety Panel, 2005; Passantino et al., 2021). Here, stunning by electrocution is recommended; custom machines such as Crustastun^® (Studham Technologies, Luton, UK) are designed to administer a lethal electric shock prior to cooking (Neil et al., 2022). When an electrical shock (120 V) is applied to P. clarkii, C. sapidus and L. vannamei for 10 s, heart rate becomes erratic and does not fully regain its prior function, suggesting damage to the heart tissue itself (Fig. 8; Weineck et al., 2018). Despite this, the majority of animals do regain motor function and start moving within 10 min, showing that this method is effective in stunning an animal prior to dispatching it (Roth and Øines, 2010; Roth and Grimsbo, 2016; Weineck et al., 2018).

Chemical methods of euthanasia include injection of substances into the circulatory system. Injection of KCl into the leg sinus of H. americanus causes heart rate to stop after <1 min (Battison et al., 2000); a similar response occurs following direct injection into the pericardial sac of C. sapidus (Mones et al., 2023). Ivermectin acts as an effective agent for C. sapidus and, like KCl, causes a cessation of heart rate and activity after <1 min (Mones et al., 2023). Both lobsters and crabs injected with KCl and ivermectin never regain a viable heartbeat. Although lidocaine HCl can stop the heart of C. sapidus, it is not considered an effective euthanasia agent because 20% of crabs treated with lidocaine HCl can be resuscitated (Mones et al., 2023). Because KCl and ivermectin have to be injected at a relatively high dose directly into the animal, it is not clear whether this method would be of use for animals destined for human consumption (Schnick, 2006; Mones et al., 2023).

Most of the articles on whole-animal cardiovascular physiology were published between the 1960s and 1990s and, as mentioned above, this literature shows disparities in cardiovascular responses to similar environmental challenges, even within a single species. Given the subsequent technological advances for recording physiological parameters, we suggest that there is an increasing need to re-analyse some of the classical whole-animal physiological responses. This could be carried out using new analytical techniques, such as magnetic resonance imaging, ultrasound echocardiogram and mini-computerized axial tomography scans, which can give a much more detailed assessment of the internal mechanics of in vivo hearts (Pereira and Pizzi, 2012; Landschoff et al., 2018; Maus et al., 2019a,b). Further advances in data loggers will allow us to record cardiac responses of smaller free-ranging animals in the field over longer time periods (Brijs et al., 2021; Steell et al., 2020; Gutzler and Watson, 2022). This will enable us to determine whether physiological responses recorded during ‘controlled’ laboratory experiments are similar to those in the natural environment where decapod crustaceans are simultaneously exposed to multiple biotic and physicochemical variables (Turko et al., 2023). These advances, coupled with a focus on sentience and pain in crustaceans, from both advocacy groups and scientists, will undoubtedly drive the direction of research in the field of crustacean physiology in the future (Diggles et al., 2024).