Summary. 1. The gastropod mollusk Pleurobranchaea performs at least two distinct cyclic behaviours using its buccal musculature, namely ingestion of food and.
Journal of Comparative Physiology, A
J Comp Physiol (1981) 145:277-287
9 Springer-Verlag 1981
Motor Program Switching in Pleurobranchaea I. Behavioural and Electromyographic Study of Ingestion and Egestion in Intact Specimens Roger P. Croll and W.J. Davis The Thimann Laboratories and The Long Marine Laboratories, University of California at Santa Cruz, Santa Cruz, California 95064, USA Accepted July 29, 1981
Summary. 1. The gastropod mollusk Pleurobranchaea performs at least two distinct cyclic behaviours using its buccal musculature, namely ingestion of food and egestion of unpalatable objects. The movements and motor programs underlying each of these behaviours have been characterized by cinematography and electromyography in intact specimens. A third buccal movement of unknown behavioural significance, is also described. 2. Both ingestion and egestion entail cyclic protraction and retraction of the radula within the buccal mass at slightly different cycle frequencies (Table 1). Cinematography has revealed three principal differences in the respective movements (Figs. 1, 2): 1) during egestion, the radula protraction phase of the movement cycle (the functional power stroke) is proportionately longer in duration than protraction during ingestion (the functional return stroke); 2) during the retraction phase of ingestion the radula is medially folded and rolls inward, while during the retraction phase of egestion the radula is flattened and does not roll inward; 3) during ingestion the jaws close near the end of retraction, while during egestion the jaws close near the beginning of retraction. 3. The functional morphology of the buccal musculature was studied anatomically (Fig. 3) and physiologically (Table 2). Attention was focussed on seven muscles, two radula retractors, two protractors, a buccal constrictor, jaw closer and lip retractor. 4. Electromyograms (EMG's) from five of these muscles were made during ingestion, egestion and the third, undefined rhythm. Ingestion and egestion are characterized by reliable differences in EMG activity which are consistent with observed differences in the movements (Figs. 4-6, Tables 3 and 4). The EMG differences during egestion include: 1) a relative increase in protractor muscle discharge (duration and intensity); 2) reduction or elimination of discharge in the buccal constrictor, a muscle which contributes
to radular folding; 3) increase in the activity of lip retractor and jaw closer muscles; 4) a phase advance of the jaw closer muscle in the retraction phase of the cycle. The respective motor programs for the different behaviours are summarized in bar diagrams (Fig. 7). 5. The results are consistent with the hypothesis that ingestion and egestion employ the same basic central nervous oscillator and motor neurons, with distinctions between these behaviours caused by shifts in the balance of activity in different motor pools.
Introduction Everyday experience illustrates that many motor systems are multifunctional, i.e., they are used to perform more than one behaviourally distinct task. Thus the tongue is used in speaking and eating; leg muscles for walking and jumping, etc. This phenomenon of 'metastable coordination' (Ayers and Davis 1977) has been analyzed from the viewpoint of the associated motor programs in several motor systems. One of the first such studies involved thoracic muscles of locusts which can cause movements of both the legs and the wings. Pairs of muscles that operate as antagonists during flight act as synergists during walking (Wilson 1962). Similarly, limb muscles in lobsters that are synergic during forward walking become antagonistic during backward walking (Ayers and Davis 1977; Ayers and Clarac 1978). Slight phase shifts of the activity of two muscles in an otherwise stable motor program underlie a transition from ingestion to egestion movements in Limulus (Wyse and Dwyer 1973). In mammals, the two forelimbs operate in phase during some forms of locomotion (galloping and jumping) but in anti-phase during others (walking, trotting and swimming; Miller et al. 1975a, b).
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R.P. Croll and W.J. Davis: Motor Program Switching in Pleurobranchaea. I
T h u s different m o t o r p r o g r a m s i n v o l v i n g the same muscles are selected a c c o r d i n g to the b e h a v i o u r a l needs o f the o rg a n i s m . A l t h o u g h m a n y m o t o r systems are m u l t i f u n c t i o n al, little is k n o w n a b o u t the cellular m e c h a n i s m s t h a t underlie switching f r o m o n e m o t o r p r o g r a m to another. W e h a v e b e g u n to address this q u e s t i o n w i t h the present study o f a m u l t i f u n c t i o n a l m o t o r system in a g a s t r o p o d m o l l u s k , Pleurobranchaea, t h a t is a m e n a b l e to n e u r o p h y s i o l o g i c a l analysis on the cellular level. It has been s h o w n t h a t several g a s t r o p o d species exhibit b o t h egestive a n d ingestive b e h a v i o u r using their buccal m u s c u l a t u r e (Aplysia, K u p f e r m a n n 1974; Tritonia, A u d e s i r k a n d A u d e s i r k 1979; Pleurobranchaea, M c C l e l l a n 1978, 1979). In the present w o r k we h a v e studied the k i n e m a t i c s o f egestion a n d ingestion in Pleurobranchaea using v id e o analysis a n d d e v e l o p e d the m e a n s to r e c o r d the c o r r e s p o n d i n g m o t o r p r o g r a m s f r o m the p a r t i c i p a t i n g buccal muscles. This w o r k in t u r n p r o v i d e s essential b a c k g r o u n d d a t a for a n a l y z i n g this instance o f m o t o r p r o g r a m switching in the r e d u c e d p r e p a r a t i o n . A n a b s t r a c t o f this w o r k has been p u b l is h e d (Crotl a n d D a v i s 1980). Subs eq u en t w o r k is a i m e d at d e t e r m i n i n g the cellular m e c h a n i s m s by w h i c h the switch f r o m one b e h a v i o u r to the o t h e r is a c c o m p l i s h e d .
Materials and Methods Specimens of Pleurobranchaea caliJbrnica 100-450 ml in volume were purchased from Dr. Rim Fay of Pacific Biomarine, Venice, CA. Animals were maintained in fresh, running seawater at ambient temperatures (14 17 ~ in the Long Marine Laboratories at U.C.S.C.
Cinematographic Analysis. Twelve specimens were photographed at 60 frames/s using a Panasonic television camera (model WV 1350) and a video tape recorder (model NV 8030). Ingestion behaviour was elicited by presenting specimens with strips of fresh, raw squid, pieces of mussel, a liquefied homogenate of fresh squid, or a solution of hydrolysed vegetable protein concentrate consisting of 10% Dr. Bronner's Balanced Protein Seasoning (All-One-GodFaith, Inc., Escondido, CA) in tapwater. Egestion was elicited by injecting 5-10% ethanol or a 10% solution of detergent (Alconox) in sea water through a fine tube inserted through the mouth into the buccal cavity. The recorded behaviours were viewed on a Panasonic video monitor (model WV 5300) using stop-frame or slow motion (1/9 or 1/18 normal speed). A third type of buccal movement of unknown behavioural significance occurred either in response to food stimuli or spontaneously in otherwise quiescent animals.
Anatomy. Observations on buccal mass musculature were made on freshly dissected specimens. Muscles were numbered according to previous nomenclature (Davis and Mpitsos I971; Davis et al. 1973; Lee and Ligeois 1974).
Electromyographic Recording. Chronically implanted electrodes were made from 25 cm lengths of Teflon coated silver wire (O.D. = 0.007", Medwire Corp., Mt. Vernon, NY). An overhand knot was made 3 cm from one end and drawn tight. The insulation
on the knot was scraped off under a dissecting microscope. To implant these electrodes, a specimen was slowly cooled to 4 ~ After 0.5 h at 4 ~ it was injected with 15% of its volume with 2 x isotonic MgC12, and then submerged in a container of 1 • isotonic MgCIz at 4 ~ Specimens were typically paralyzed and flaccid after 0.5 h. The animal was then placed dorsal side up in a dissecting tray and covered with cold (4 ~ sea water. The proboscis was everted by applying gentle pressure on the body wall. Pinning a section of rubber tubing across the animal immediately posterior to the rhinophore maintained the proboscis in the everted position and minimized blood loss during subsequent surgery. The muscles of the buccal mass could be visualized directly through the thin skin of the everted proboscis. A small incision (< 1 cm long) was made directly over the muscle(s) from which recordings were desired. The knotted end of the electrode wire was then looped around the muscle bundle and tied off with a square knot so that the deinsulated portion of the wire was held firmly against the muscle without constricting the tissue unduely. The short end segment of wire was trimmed adjacent to the knot. Up to four electrodes were so implanted on different muscles in single specimens. The placement of each electrode was encoded by a series of knots near the free end opposite from the muscle, After implanting the electrodes the wound was closed with 2-3 sutures and the animal was placed in a large aquarium of sea water at 14~ for a three-day recovery period. Fourteen of 15 operated specimens survived for successful recording sessions. Approximately 75% of implanted electrodes exhibited sufficient signal-to-noise ratios to yield useful data. Muscle potentials were recorded differentially against silver electrodes in the bath. Electrode wires were led to preamplifiers and then to an eight-channel Gould-Brush chart recorder for making permanent records. The direction of movement of strips of squid was usually observed as an index of ingestion or egestion, and coded directly on the chart paper adjacent to electromyograms using an electronic event indicator or by hand.
Results Th e d a t a to be presented here are o r g a n i z e d in three sections, c o r r e s p o n d i n g to the c i n e m a t o g r a p h i c analysis, f u n c t i o n a l m o r p h o l o g y o f the buccal mass, a n d e l e c t r o m y o g r a p h i c analysis in intact, b e h a v i n g specimens.
Cinematographic Analyses B o t h ingestion an d egestion b e h a v i o u r s in Pleurobranchaea entail repeated, al t er n at e p r o t a c t i o n a n d retraction o f the r a d u l a with respect to the j a w s (Fig. 1 A). T h e c o m p l e t e ingestive cycle (i.e., a single representative bite o f a r h y t h m i c sequence) o c c u p y i n g a p p r o x i m a t e l y 2 s at 0.1 s intervals, is s h o w n in Fig. 1 B. T h e sequence in this case was elicited by liquefied squid h o m o g e n a t e . Th e b eg i n n i n g o f the sequence is arbitrarily chosen as slightly before the b e g i n n i n g o f j a w opening. W i t h i n 0.3 s o f o p e n i n g o f the j a w s the internal r a d u l a was visible t h r o u g h the m o u t h . P r o t r a c t i o n o f the r a d u l a c o n t i n u e d until 0.7 s into the sequence, at w h i ch p o i n t the f o r m a t i o n o f a deep m e d i a l fold in the r a d u l a was visible. R e t r a c t i o n o f the r a d u l a
R.P. Croll and W.J. Davis: Motor Program Switching in Pleurobranehaea. I
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9
Fig. 1A-C. Stop-frame analysis of ingestion and egestion. A shows an artist's rendition of the frontal view of an animal at the point of maximal radular protraction during ingestion. B is a sequential series of tracing from a video monitor during a complete ingestion cycle. Numbers at the lower left of each animal signify tenths of seconds of elapsed time. C is a similar series of tracings during a complete egestion cycle
began at this point, and was accompanied by deepening of this center fold, accomplished by medial movement of the more lateral radular regions. This movement is not clearly detectable in the stop-frame analysis of Fig. 1 but is plainly seen in videotapes played at normal speed. Such inward medial movement of the radula occurred continuously until 1.5 s of elapsed time had passed. During active feeding the food object was positioned in the center of this fold, held in place by rows of tiny teeth that project obliquely backward from the radular surface into the buccal cavity. The mouth remained open until after the radula was retracted out of view (Fig. 1 B, 1.5 s frame). The jaw began to close shortly after, and was fully closed within 0.2-0.3 s. The mouth then remained closed in this sequence for an additional 0.2 s, at which time the cycle began to repeat. A complete cycle of buccal movement during an egestive sequence is shown in Fig. 1 C. The cycle is again considered to begin just before jaw opening, in order to facilitate comparison with the ingestive sequence of Fig. 1 B. As in the case of the ingestive
movements, the radula was first visible 0.3 s after jaw opening. The protraction phase of the egestive cycle, however, continued until 1.4 s into the sequence, as compared with 0.7 s during ingestion. In other words, the radula protraction phase was twice as long during egestion as during ingestion. Additional distinctions from the ingestive movements were evident during radular retraction. Whereas the retraction during ingestion was characterized by the deepening center fold in the radula, this movement was absent in the retraction phase of egestion. Instead the radula was held in a flattened configuration, without the lateral-to-medial and inward movement of rows of radular teeth. These features are not easily detectable in stop frame analysis shown in Fig. 1 but are clearly seen in videotape played at normal speed and by direct observation. In addition, during egestion the jaws began to close almost coincidentally with the beginning of retraction (Fig. 1 C, 1.5 s), rather than near the end of retraction as occurred during the ingestion (Fig. 1 B, 1.4 s). As in the case of ingestion, the egestion sequence was ter-
280
R.P. Croll and W.J. Davis: Motor Program Switching in Pleurobranchaea. I
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minated with a 0.2 s period of complete jaw closure (approximately 2.1-2.2 s in Fig. 1 C). The difference between ingestion and egestion with regard to protraction and retraction times is seen more clearly in a bar diagram of the corresponding movements. The sequences shown in Fig. 1 were replayed at slow motion in order to clearly determine the end of protraction. Figure 2 illustrates that the protraction duty cycle increases during egestion, i.e., the protraction phase is longer and the retraction phase is shorter during egestion than during ingestion. To assess the reliability of this observation, videotaped sequences of ingestion and egestion similar to those portrayed qualitatively in Figs. 1 and 2 were analyzed in 7 animals. This was accomplished by comparing the second to last ingestion cycle immediately prior to injection o f ethanol or detergent into the buccal cavity with the second subsequent egestion cycle in the same specimen. The time from mouth opening to end of protraction was measured and divided by the total time the jaws were open. This furnishes a measure of the dominance of protraction within this portion of the cycle. For ingestion sequences the mean ratio was 0.40 (range 0.30~).50, s.d. =0.08). Thus in no case during ingestion did protraction occupy more of the time that the mouth was open than did retraction. For egestion sequences from the same 7 specimens, the mean ratio was 0.68 (range 0.55-0.80, s.d.=0.10). Thus during egestion, protraction always occupied more of the mouth open time than did retraction. The mean ratios for the two behaviours were significantly different (matched pair t-test, P 0.4). In the course of these cinematographic studies we also observed a third behaviour or set of behaviours involving rhythmic buccal movements. The most characteristic feature of this third behaviour(s) was weak, relatively slow rhythmic opening and closing of the jaws in the absence of eversion of the proboscis. Rhythmic movements of the internal buccal mass were sometimes evident from movements o f the overlying body wall, although the radula never protracted far enough to be seen through the open mouth. Such a behaviour pattern occasionally occurred spontaneously in unrestrained specimens, but was also occasionally elicited by applying liquefied food stimuli to the oral veil region. In particular, specimens with high feeding thresholds often exhibited this behaviour pattern prior to initiating ingestion. The frequency of these movements was slow compared with both ingestion and egestion; in five sequences elicited in three specimens by application of vegetable protein solution, the mean frequency was 0.10 Hz (range 0.05-0.15 Hz, s.d.=0.041). The behavioural signifi-
R.P. Croll and W.J. Davis: Motor Program Switchingin Pleurobranchaea. I cance of this movement pattern is not known. A fourth behaviour was also sometimes observed in association with egestion, namely the expulsion of a large quantity of a viscous fluid from the buccal cavity following injection of a small amount of the noxious stimulus. This action, which we term regurgitation, did not appear to alter the ongoing egestion movements but simply accompanied them, and thus presumably involved internal musculature which was inaccessible to video analysis in intact animals. It seems likely that the regurgitation involves reverse peristalsis of the esophagus and/or contraction of gut musculature without altering the basic buccal sequence of egestion.
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Fig. 3. Morphology of the buccal mass as seen in dorsal aspect. Progression from left to right represents sequential stages of dorsal dissection. E esophagus; R radula; S radular sac; T lateral tooth. For descriptivenames of numbered muscles see Table 2. Anterior is toward the bottom
Functional Morphology of the Buccal Mass Pleurobranchaea typically feeds by cyclical eversion of a proboscis, consisting of an outer sheath and an internal buccal mass. The buccal mass in turn contains the radula, paired lateral support structures (the lateral teeth), and a complicated set of muscles. The muscular anatomy of the buccal mass has been described by Davis and Mpitsos (1971), Davis et al. (1973), Lee and Liegeois (1974). The present observations confirm previous ones but are accompanied here by new functional data which provide independent insights into the role of certain key muscles. Figure 3 is a diagrammatic representation of the buccal mass in the resting position (approximately half way between the points of maximum possible protraction and retraction of the radula) as seen in dorsal aspect. The sheet-like radula (R) and radular sac (S) move inside the buccal mass anteriad and posteriad with respect to the enclosing musculature. Muscles 1 and 3 originate posteriorly on the lateral wall of the buccal mass, and insert medially on the back of the radula. Both of these muscles are innervated primarily by the second nerve root of the buccal ganglion, although muscle 3 also receives innervation from the third buccal root (Davis et al. 1973; and Croll, unpublished data). In the present study we severed these nerves from the buccal ganglion and stimulated them electrically, which resulted in contraction of muscles 1 and 3 and retraction of the radula. Direct electrical stimulation of the respective muscles individually yielded similar movements. These data support the hypothesis that muscles 1 and 3 normally play the role of radula retractors (ventral and dorsal, respectively). Muscles 2 and 4 insert posteriorly on the radular sac. Muscle 2 originates anteriorly at the inner surface of the rigid lateral tooth which is in turn embedded within surrounding musculature. Muscle 4 originates anteriorly in the lining of the buccal cavity which
is itself attached to lateral musculature. Muscles 2 and 4 are both innervated by the first root of the buccal ganglion (Davis et al. 1973). In the present work we severed root 1 from the buccal ganglion and stimulated it electrically, which caused contraction of muscles 2 and 4 and protraction of the radula. Direct electrical stimulation of the same muscles individually caused similar movements. These data support the hypothesis that muscles 2 and 4 represent radula protractors (lateral and medial, respectively). Muscle 5 is the most conspicuous single muscle in the buccal mass, comprising a flattened sheet of tissue that completely encircles the anterior two thirds of the buccal mass. Stimulation of buccal root 3 causes muscle 5 to contract, contributing to medial infolding of the radula similar to that seen during the retraction phase of feeding. Localized electrical stimulation of muscle 5 has a similar effect. These data collectively indicate that muscle 5 is a buccal constrictor. Two additional muscles have been examined in the present work, namely muscle 8 and muscle 27. Muscle 8 is attached posteriorly to the external portion of the buccal mass and inserts on the inner surface of the lips. Since both attachments are mobile the terms 'origin' and 'insertion' are meaningless in the present context. Contraction of muscle 8, induced by direct electrical stimulation of the muscle, has the dual effect of retracting the lip near the insertion and pointing the buccal mass toward the contralateral side. The muscle may thus play a bifunctional role. Muscle 27 is a circumferential muscle that encircles the mouth region. Direct electrical stimulation of the muscle causes the jaws to clamp closed, suggesting that this muscle is a jaw closer. The functions of the various buccal muscles shown in Fig. 3 are summarized in Table 2 together with the evidence on which the functional assignment is based.
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R.P. Croll and W.J. Davis: Motor Program Switching in Pleurobranchaea. I
Table 2. Tabular summary of buccal muscles shown in Fig. 3. The first column shows the number used to identify the muscle. The second column is the functional name of the muscle. Columns 3 5 indicate the kind of evidence used to arrive at the muscle's function, including stimulation of buccal nerve roots (column 3), movement caused by direct electrical stimulation of the muscle (column 4) and recording of electromyograms during behaviour in intact specimens (column 5) Muscle no.
Muscle name Evidence for functional role and abbreviation Buccal Direct Electromyonerve muscle grams stimulation stimulation (r = root)
1
Ventral radula retractor Lateral radula protractor Dorsal radula retractor Medial radula protractor Buccal constrictor Dorsal lip retractor Circumferential jaw closer
2 3 4 5 8 27
Yes (r.2)
Yes
No
Yes (r. 1)
Yes
No
Yes (r.2,3)
Yes
Yes
Yes (r.1)
Yes
Yes
Yes (r.3)
Yes
Yes
No
Yes
Yes
No
Yes
Yes
Electromyograms (EMG's) During Behaviour of Intact Specimens Electrical recordings from selected buccal muscles shown in Table 2 and Fig. 3 were obtained from several (14) specimens. Muscles representative of each functional group were selected on the basis of accessibility for electrode implantation. Thus muscle 4 was selected to represent activity patterns for radular protraction, and muscle 3 was selected to represent the activity patterns for the radular retraction. Recordings were also obtained from muscles 5, 8 and 27. EMG's were recorded under three general behavioural conditions: 1) ingestion or egestion of a strip of squid; 2) ingestion or egestion movements induced by application of liquefied food externally or noxious liquid internally, respectively; and 3) the relatively feeble, rhythmic buccal movements characterized in the cinematographic analysis as a third but unspecified behaviour or set of behaviours. The first of these three conditions allowed unambiguous distinction between ingestion and egestion based on the direction of squid movement. The second condition allowed better simultaneous visualization of the buccal apparatus, which was not obscured by food within the buccal cavity. Figure 4 shows typical electromyograms from an intact, behaving Pleurobranchaea under the first of
the above conditions (squid strip present). A sequence of egestive movements (beginning in the middle of the record and indicated by downward arrows), induced by squirting noxious fluid into the buccal mass, is bracketed by ingestive movements (indicated by upward arrows). During ingestion, muscle 3 (dorsal radula retractor) and 5 (buccal constrictor) are vigorously active in simultaneous bursts of muscle potentials, alternating with bursts in muscle 4 (medial radula protractor). The squid strip moved inward during the bursts in muscles 3 and 5, and was stationary with respect to the mouth during muscle 4 activity. During the egestion sequence the relative electrical activities in these three muscles changed dramatically in comparison with ingestion. Activity in muscle 5 was suppressed, in this instance fully, as was cyclic activity in muscle 3. In contrast, the muscle potentials recorded from muscle 4 increased in amplitude. In addition, during egestion the proportion of each cycle occupied by the muscle 4 burst increased by approximately 50% in comparison with ingestion movements. The changes in activity patterns of these buccal muscles were seen also under the second EMG recording condition, i.e., during feeding movements induced by liquefied rather than solid food. No significant differences were observed when food was absent from the buccal cavity. As demonstrated by the above cinematographic analysis, ingestion and egestion can be distinguished also on the basis of the timing of mouth closure relative to retraction. In order to establish an electromyographic correlate to this criterion, several animals were implanted with electrodes in both the dorsal radula retractor (muscle 3) and the circumferential jaw closer (muscle 27). Figure 5 shows a series of such recordings from a single specimen during ingestive movements induced by hydrolyzed vegetable protein solution (part A), during egestive movements induced by injection of 10% detergent (part B), and during weak opening and Closing of the jaws in the absence of full eversion of the proboscis elicited in this instance by vegetable protein solution (part C). During the ingestive sequence, activity in muscle 3 begins about 0.5 s prior to the burst in muscle 27 (Fig. 5A). When egestive movements were induced in the same specimen, the intensity of muscle activity in muscle 3 declined, while the activity of muscle 27 increased substantially. In addition the bursts on these two muscles began nearly simultaneously during egestive movements. Of 13 ingestive cycles selected on the basis of a high signal-to-noise ratio from four specimens, muscle 3 fired prior to muscle 27 in all cases. Of 18 egestive cycles selected on the same basis from 5 specimens, muscle 3 in no case fired prior to muscle 27. Muscle 27 fired first in 8/18 cases, while
R.P. Croll and W.J. Davis: Motor Program Switching in Pleurobranchaea. I
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