**2. Brief history of crustacean heart physiology**

Cardiac nerves—comprising accelerator nerves (CA) and inhibitory nerves (CI)—govern the activity of the heart in an involuntary manner in both model animals and humans. CA and CI are cardiovascular components of the ANS that transmit psychological information to the heart. Alterations in the activity of these neurons can modulate momentary heart rate and contractile force in a reflex manner in crustaceans as shown in Figures 1–3. Because the degree of stress can be measured by monitoring heart rate variability, the heart can be called a reflection of the neural activity of an animal.

However, the cardiac regulatory function of the human ANS is not clearly understood because of the difficulty associated with recording ANS activity. Nevertheless, I have successfully recorded ANS activity in crustaceans. Both CA and CI are active when the heart is beating. This is evident in *in situ* recordings of nerve activity in hermit crabs in the 1970s (Figure 1, unpublished data). In decapod crustaceans, only one pair of nerves, termed the cardiac or regulator nerves, innervate the heart bilaterally. The crustacean system therefore appears simpler than the human cardiac nervous system. However, appearances can be deceiving: the fundamental mechanisms of the acceleration/inhibition functions are similar in humans and crustaceans, and the ANS controls the cardiac region in both animals. It is evident in Figure 1 that three different impulses varying in size indicate discharging: small (not marked), medium (shown by triangles), and large (not marked) impulses. A significant feature of Figure 1 is that the crab's heartbeat stopped completely during the period when the large-sized impulses were active, which is shown by a train discharge at a high frequency of approximately 50 or 60 Hz. Based on these observations, I was able to identify the neurons (in this case, the large-sized impulses) that transmit inhibitory commands. CI and CA always function in balance with each other. Figure 1 shows that the ANS dynamically controls the heart.

Healthy human hearts do not stop beating; only the rate of the heartbeat changes. However, crustacean hearts can cease beating for a period of time (Figure 2). A key finding in my crustacean study was that specimens showed a pattern of intermittent heartbeat cessation under healthy, normal, non-stressed conditions. The cessation was induced by an intermittent burst discharge of CI as mentioned above (Figure 1). An important observation was that the presence of a human caused interruptions in this intermittency. In other words, stress from an approaching human caused an alteration in ANS function: i.e., the intermittent pattern changed to a continuous pattern [2] (Figure 3).

This intermittency was actually documented in the 1970s by Canadian crustacean heart researchers, J. L. Wilkens and B. McMahon, though they did not mention the role played by stress. (They found a strong relationship between cardiac and respiratory control.) The physiology of the crustacean ANS has been studied throughout the 20th century by Carlson (1900s), Alexandrowicz (1930s), Maynard (1950s, 1960s), Young [3], Field and Larimer [4], and Yazawa and Kuwasawa [5]. However, these authors did not provide information on the relationship between heartbeat and behavior of crustaceans.

**2. Brief history of crustacean heart physiology**

reflection of the neural activity of an animal.

360 Advances in Bioengineering

changed to a continuous pattern [2] (Figure 3).

relationship between heartbeat and behavior of crustaceans.

Cardiac nerves—comprising accelerator nerves (CA) and inhibitory nerves (CI)—govern the activity of the heart in an involuntary manner in both model animals and humans. CA and CI are cardiovascular components of the ANS that transmit psychological information to the heart. Alterations in the activity of these neurons can modulate momentary heart rate and contractile force in a reflex manner in crustaceans as shown in Figures 1–3. Because the degree of stress can be measured by monitoring heart rate variability, the heart can be called a

However, the cardiac regulatory function of the human ANS is not clearly understood because of the difficulty associated with recording ANS activity. Nevertheless, I have successfully recorded ANS activity in crustaceans. Both CA and CI are active when the heart is beating. This is evident in *in situ* recordings of nerve activity in hermit crabs in the 1970s (Figure 1, unpublished data). In decapod crustaceans, only one pair of nerves, termed the cardiac or regulator nerves, innervate the heart bilaterally. The crustacean system therefore appears simpler than the human cardiac nervous system. However, appearances can be deceiving: the fundamental mechanisms of the acceleration/inhibition functions are similar in humans and crustaceans, and the ANS controls the cardiac region in both animals. It is evident in Figure 1 that three different impulses varying in size indicate discharging: small (not marked), medium (shown by triangles), and large (not marked) impulses. A significant feature of Figure 1 is that the crab's heartbeat stopped completely during the period when the large-sized impulses were active, which is shown by a train discharge at a high frequency of approximately 50 or 60 Hz. Based on these observations, I was able to identify the neurons (in this case, the large-sized impulses) that transmit inhibitory commands. CI and CA always function in

balance with each other. Figure 1 shows that the ANS dynamically controls the heart.

Healthy human hearts do not stop beating; only the rate of the heartbeat changes. However, crustacean hearts can cease beating for a period of time (Figure 2). A key finding in my crustacean study was that specimens showed a pattern of intermittent heartbeat cessation under healthy, normal, non-stressed conditions. The cessation was induced by an intermittent burst discharge of CI as mentioned above (Figure 1). An important observation was that the presence of a human caused interruptions in this intermittency. In other words, stress from an approaching human caused an alteration in ANS function: i.e., the intermittent pattern

This intermittency was actually documented in the 1970s by Canadian crustacean heart researchers, J. L. Wilkens and B. McMahon, though they did not mention the role played by stress. (They found a strong relationship between cardiac and respiratory control.) The physiology of the crustacean ANS has been studied throughout the 20th century by Carlson (1900s), Alexandrowicz (1930s), Maynard (1950s, 1960s), Young [3], Field and Larimer [4], and Yazawa and Kuwasawa [5]. However, these authors did not provide information on the

**Figure 3.** Intermittent heartbeat cessation of a Japanese spiny lobster (*Panulirus japonicus*) was interrupted by an ap‐ proaching human shown in an EKG recording from a live specimen.

**Figure 1.** Simultaneous recording of heartbeat (EKG) and cardiac nerve activity (ANS) of the hermit crab (*Dardanus crassimanus*). Asterisks (\*) indicate heartbeats.

**Figure 2.** Intermittent cessation of heartbeat shown in an EKG recording from a live saw tooth Gazami crab (*Scyll serra‐ ta*) specimen.

In summary, I recorded impulses of the cardiac nerves of crustacean hearts. I observed that CI discharged a burst of impulses at a high-rate, ~60 Hz, and concomitantly, CA momentarily ceased their impulse discharge [1]. During the CI burst period, the heart rate disappeared or decreased significantly, although a rapid restitution of heartbeats occurred within one second. These brief cessations of heartbeats occurred intermittently and somewhat regularly during "stress-free" periods, for example, when the animal was hiding in a shelter. In contrast, the stress-free behavior was not exhibited if the lobsters or crabs were approached by humans. This response can be likened to the way cicadas stop singing when humans are in close proximity. These findings suggest that crustaceans are incredible specimens because their stress can be detected by electrocardiograms.

Crustacean and human hearts strongly resemble each other in structure and function. It is known that homologous genes (e.g., Nkx2-5, the NK2 homeobox gene) function to form the developing heart of all animals: [6]. In crustaceans and humans, both CA and CI connect with the cardiac pacemaker cells, but CA further proceed to the ventricular muscles beyond the pacemaker cells. Why do CA control the entire heart? The answer is that CA–muscle connections can implement direct modulation of contractile force, whereas CI merely sup‐ press rhythm [5]. The resemblance between crustacean and human indicates that some knowledge obtained from crustacean should be applicable to human. See Recent report by Fossat et al.

(1) P. Fossat et al. "Anxiety-like behavior in crayfish is controlled by serotonin." Science 344, 1293–1297 (2014).

(2) P. FOSSAT et al. "Anxiety-like behavior in crayfish is controlled by serotonin." Society for Neuroscience, Poster 655.18/UU43 - Motivation and Emotions: Rodent Anxiety Models Tue, Nov 18, 1:00 - 5:00 PM 2014 Washington DC.

(3) OUTSIDE JEB. The Journal of Experimental Biology (2014) 217, 3389-3391

I studied electrocardiograms (EKG) of both animal models and humans and used modified Detrended Fluctuation Analysis (mDFA) to calculate the scaling exponent (SI) (originally, Peng et al., [7]). In this article, I show that the SI numerically is capable of distinguishing between healthy and unhealthy hearts, and between "stressed" and "relaxed" hearts. Further, I propose the mDFA to be a viable potential method for health/stress checking if incorporated into a device that can quantify stress through EKGs.
