One of the most remarkable adaptations to survive attacks from predators is to detach an appendage—a process known as autotomy. This occurs in a variety of animals, including lizards (tail), crabs (legs), and starfish (arms). There has been extensive investigation of the evolution, ecology, and biomechanical impact of autotomy, but little is known about neural mechanisms controlling autotomy in animals. However, evidence for the existence of a peptide that acts as an autotomy-promoting factor in starfish has been reported. While investigating in vivo effects of a sulfakinin/cholecystokinin-type neuropeptide (ArSK/CCK1) in the starfish Asterias rubens, we observed that this peptide triggered arm autotomy in some animals. Furthermore, when injection of ArSK/CCK1 was combined with mechanical clamping of an arm, autotomy of the clamped arm occurred in 85% of animals tested, with 46% also autotomizing one or more other arms. In contrast, no autotomy was observed in clamped animals that were injected with water (control). To examine the physiological relevance of these findings, we analyzed expression of ArSK/CCK1 in the autotomy plane, a specialized region at the base of the arms in A.rubens. In accordance with its in vivo effects, nerve fibers expressing ArSK/CCK1 were revealed in the tourniquet muscle, a band of muscle that mediates constriction of the arm during and after autotomy. We conclude that ArSK/CCK1 acts as an autotomy-promoting factor in starfish and as such it is the first neuropeptide to be identified as a regulator of autotomy in animals.
Advances in transcriptome/genome sequencing have facilitated discovery of a huge variety of neuropeptides in the starfish A.rubens. We recently reported the identification of two neuropeptides in A.rubens derived from the same precursor protein that are related to sulfakinin (SK)-type neuropeptides in insects and cholecystokinin (CCK)-type neuropeptides in vertebrates and that we therefore refer to as ArSK/CCK1 and ArSK/CCK2. SK/CCK-type neuropeptides act as inhibitory regulators of feeding behavior in insects and vertebrates and, accordingly, we discovered that ArSK/CCK1 and ArSK/CCK2 trigger cardiac stomach contraction and retraction and inhibit feeding behavior in A.rubens. However, when testing the in vivo pharmacological effects of ArSK/CCK1 we have also observed that it induces arm autotomy in some animals. Thirty-three animals were injected with 10 μL of 10−3 M ArSK/CCK1 and in five of these animals, arm autotomy was observed following injection of the neuropeptide. In contrast, arm autotomy was not observed in animals injected with 10 μL of 10−3 M ArSK/CCK2 (n= 25) or in control animals injected with 10 μL of water (n= 54). Among starfish injected with ArSK/CCK1, in two animals autotomy of one arm was observed, in one animal autotomy of three arms was observed, and in two animals autotomy of four arms was observed. Examples of the latter are illustrated and described henceforth. Within a 10 min period after injection of ArSK/CCK1, constriction of the body wall was observed at the junction of the arms with the central disc region and at a position consistent with that of the tourniquet muscle in the autotomy plane. Following this, arm autotomy began to occur, with breakage of the body wall first occurring on both sides of the ambulacrum and with autotomizing arms being twisted into an abnormal position. Eventually the body wall of the autotomizing arms became completely separated from the central disc and the only structures connecting those arms with the central disc were the pyloric ducts that link the pyloric stomach in the central disc with the pyloric caeca in the arms. Finally, the pyloric ducts of the autotomizing arms broke and only one arm remained attached to the central disc.
Sulfakinin/cholecystokinin-type precursor protein (ArSK/CCKP) has an N-terminal signal peptide (blue) and two SK/CCK-type neuropeptide sequences (red), which are bounded by putative dibasic or tetrabasic cleavage sites (green). The C-terminal glycine of each neuropeptide sequence is shown in orange to indicate that this residue is a potential substrate for post-translational conversion to an amide group. Use of mass spectrometry to determine mature structures of the neuropeptides ArSK/CCK1 and ArSK/CCK2 derived from ArSK/CCKP reveals three post-translational modifications. In both peptides, the C terminus is amidated and the tyrosine residue is sulfated. Furthermore, an N-terminal glutamine residue is converted to pyroglutamate in ArSK/CCK1. Sequence and structural data are from Semmens et al. and Tinoco et al.
(A) A control animal that was injected with 10 μL of distilled water; no autotomy was observed during a 5 h period of monitoring after injection or when examined 24 h after injection in this animal or in 53 other animals injected with water.
(B–D) Test animal (no. 3) after injection of ArSK/CCK1. By ∼10 min after injection of ArSK/CCK1, indentation of the body wall at the junctions of the arms with the central disc region can be seen at a position that corresponds with the location of the tourniquet muscle (B; arrowheads). The first signs of autotomy were observed at ∼30 min after injection, with one of the arms twisted and partially detached from the central disc region (C; white arrowhead). By 2 h after injection, four of the five arms were twisted and partially detached from the central disc (D; black asterisks) and only one arm remained untwisted and completely attached to the central disc (D; white asterisk). By 2 h and 40 min after injection of ArSK/CCK1, four of the five arms were completely autotomized and a single arm remained attached to the central disc (image not shown).
(E and F) Test animal (no. 4) after injection of ArSK/CCK1; by 1 h and 40 min after injection of ArSK/CCK1, autotomy of four arms is ongoing, with the black arrowheads in (E)labeling regions of the body wall on either side of the ambulacrum where breakage has occurred. By 2 h and 15 min after injection of ArSK/CCK1, autotomy of three arms is completed (F; white arrowhead showing an autotomized arm), one arm remains attached to the central disc only by the pyloric duct, which links the pyloric stomach in the disc with pyloric caeca in the arm, (F; black arrowhead), and only one arm remains completely attached to the central disc (F; white asterisks).
To facilitate investigation of a potential physiological role of ArSK/CCK1 as a regulator of autotomy in A.rubens, we performed experiments to identify mechanical stimuli that trigger arm autotomy in this species. Our aim was to use a method that mimics the pressure exerted on arms when starfish are attacked by predators; for example, when the arm of a starfish is clamped in the beak of a seagull. To accomplish this, a Mohr tubing clip was used to clamp one arm and then the animal was suspended by this clip, but with its other arms in contact with seawater contained in a glass bowl below. Arms were clamped with the Mohr tubing clip in one of three positions: (1), proximal (P)to the junction of the arm with the central disc, coinciding with the position of the autotomy plane; (2), mid-way (M)along the length of an arm; or (3), distal (D)from the central disc and proximal to the arm tip. In animals with arm clamping proximal to the central disc region, autotomy of the clamped arm was observed in 80% of animals tested (16 out of 20). In contrast, when the arm was clamped mid-way along its length or proximal to the arm tip, autotomy of the clamped arm was observed in only 10% (2 out of 20) and 4.2% (1 out of 24) of animals tested, respectively. The time taken for arm autotomy to occur was also measured and this ranged from as little as 103 s in an animal that was clamped proximal to the central disc to as much as 1,333 s in one starfish that exhibited arm autotomy with clamping proximal to the arm tip. The mean time taken for arm autotomy was 303.4 s (n= 16) and 118.5 s (n= 2) in starfish clamped proximal to the central disc or mid-way along the length of an arm, respectively. Following autotomy, we also examined the precise location where autotomy had occurred in animals and found that it occurred at a position corresponding to the junction between the second and third, third and fourth, or fourth and fifth ambulacral segments, consistent with previously reported findings for autotomy in this species.
(A) One arm of each starfish tested was clamped using a Mohr tubing clip (white arrowhead) and a retort stand clamp (black arrowhead) was used to support the Mohr tubing clip so that the starfish was suspended over a glass bowl containing seawater. The clip was positioned proximal to the disc (n= 20), mid-way along the length of the arm (n= 20; as shown in this photograph), or at the arm-tip distal from the disc (n= 24).
(B) Clamping of an arm proximal (P)to the central disc triggers autotomy in 80% of animals tested, whereas clamping mid-way (M)along the length of an arm or at the arm-tip distal (D)from the central disc only triggers autotomy in 10% and 4.2% of animals, respectively.
(C) The mean time taken for autotomy of the clamped arm to occur was 303.4 s (n= 16), 118.5 s (n= 2), and 1,333 s (n= 1) for animals clamped in the P, M, and D positions, respectively.
(D) Proportion of animals in which arm autotomy occurred between the second and third(2–3), third and fourth (3–4), and fourth and fifth (4–5) ambulacral segments of the clamped arm.
(E) When starfish injected with 10 μL water (control) are clamped M along the length of an arm (Water+ M), no arm autotomy is observed. However, when starfish injected with ArSK/CCK1 (10 μL 10−3 M) are clamped M along the length of an arm (ArSK/CCK1+ M), autotomy of the clamped arm occurs in 84.6% of animals.
(F) Clamped animals injected with ArSK/CCK1 (ArSK/CCK1+ M) autotomized one (54.5%), two (9.1%), three (18.2%), or four arms (18.2%).
(G) The mean time taken for autotomy of the clamped arm to occur was 440.8 s in ArSK/CCK1-injected animals (ArSK/CCK1+ M).
(H) Proportions of ArSK/CCK1-injected animals (ArSK/CCK1+ M) in which arm autotomy occurred between the first and second (1–2), second and third (2–3), and third and fourth (3–4) ambulacral segments of the clamped arm.
(I) When starfish injected with 10 μL water (control)are clamped M along the length of
