Zoomorphology 96, 3343 (1980) Zoomorphology © by Springer-Verlag 1980

Autotomy in a Polychaete: Abscission Zone at the Base of the Tentacular Crown of Sabella penicillus

Bill Kennedy I and Harald Kryvi 2 1 Bodega Marine Laboratory, Bodega Bay, CA 94923, USA 2 Institute of Anatomy, University of Bergen, Arstadvollen, N-5000 Bergen, Norway

Summary. Under various circumstances the tentacular crown of some sabellid polychaetes becomes detached from the body. Separation occurs always at a preestablished zone of abscission at the base of the crown. We used electron microscopy .to study the abscission zone of Sabella penicillus, both in specimens whose crown was intact and in those whose crown had separat- ed.

The abscission zone is within the intermediate layer, between the crown skeleton and the body wall musculature, and only structures supported by the crown skeleton separate from the animal 's body. Abscission involves a rupture of the paramyosin muscle cells which form bridges connecting extensions f rom the epimysium of the body wall musculature and from the cartilage matrix of the crown. After abscission the anterior and posterior ends of the cells remain in place on the crown and body respectively.

Sabella penicillus appears able to control the loss of its tentacular crown, so this abscission is a kind of autotomy. Under some circumstances autotomy of the crown may permit escape or confer some surgical benefit to the animal. Using standard histology we found the same anatomical provision for crown abscission in a variety of sabellids. We conclude that differences in their capacities to autotomize the crown have a behavioral/physiological basis rather than an anatomical one.

A. Introduction

Polychaetes in the family Sabellidae have a tentacular crown, comprising a number of pinnately branched radioles surrounding the mouth at the anterior end of the body. The crown is held outside the opening of the animal 's tube, where it creates a current in the surrounding seawater and removes from it particulate matter and dissolved oxygen.

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34 B. Kennedy and H, Kryvi

The tentacular crown of sabellids is supported by an internal skeleton. Some earlier workers declared that the structure bore no resemblance to cartilage (see Nicol 1931), but Kryvi (1977) has shown that histochemical and ul trastruc- rural evidence indicates that the crown skeleton of Sabella penicillus (Linn6) is a cartilage fully deserving of the term.

The cart i laginous skeleton is jo ined at its base to the longi tudina l body wall muscula ture in an in termediate layer, which Kryvi (1975) has described in detail. In the in termediate layer, posteriorly project ing extensions of the cartilage matr ix of the crown skeleton interdigitate with anteriorly projecting extensions of the ep imysium of the longi tudinal body wall muscles. The epimysial extensions and the cartilage matrix extensions are no t in contact with one another , but rather are connected by short pa ramyos in muscle cells.

We observed while ma in ta in ing Sabella penicillus in aquar ia that when the animals are roughly handled, the tentacular crown often falls away f rom the body. In part icular , a gentle pull on the crown or a lateral pressure at the head end of the animal causes a separat ion of the crown from the body. The separat ion happens suddenly and with very little loss of blood, and appears always to occur at a point very near the in termediate layer where the crown skeleton jo ins the body wall musculature .

Here we present the results of an electron microscopical invest igat ion into the zone of abscission of the ten tacular crown of Sabella penicillus and in to the roles played by various parts of the intermediate layer in the abscission process.

B. Materials and Methods

We removed Sabella penicitlus in their tubes from the walls of seawater reservoirs at the Bergen Aquarium. Other sabeltids were collected in the vicinity of Bodega Bay, Sonoma County, California: Myxicola infundibulurn (Myxicolinae) from harbor mooring floats, Chone tnollis (Fabricinae) from harbor flats, and Chone ecaudata (Fabricinae) from intertidal rock. We either held the animals on aquarium tables for further examination, or brought them immediately to the laboratory for treatment for electron microscopy or standard histology. We urged Sabella penicillus from their tubes by inserting a blunt probe into the posterior end of each tube and running it carefully through to the other end.

Specimens for standard histology were fixed in seawater Bouin's, dehydrated in graded ethanols, embedded in polyester wax 90/10 (Steedman 1960), and cut into 5-tam or 7-1~m sections on a rotary microtome, using razor blades. Wax sections were stained with paraldehyde-fuchsin and a modification of Halmi's trichrome counterstain (Clark 1955).

Specimens for scanning electron microscopy (SEM) were fixed in 3% glutaraldehyde in phos- phate buffer made isotonic to sea water by addition of NaC1 (Millonig 1962), and later rinsed in buffer, post-fixed in 1% OsO4-buffer, rinsed again, dehydrated in graded ethanols, and transferred to amyt acetate for critical point drying in liquid CO~. Specimens were affixed to stubs with silver cement, and thermal-coated under high vacuum with gold-palladium. We examined the specimens in a Philips PSEM-500 scanning electron miscoscope.

For semithin sections and for transmission electron microscopy (TEM), small pieces of tissue were fixed and dehydrated as for SEM, embedded in Epon, and sectioned on a LKB III Ultratome, using glass knives. Semithin (0.5-1.0 ~tm) sections were stained in toluidine blue; ultrathin sections were contrasted with uranyl acetate and lead citrate and examined with a Siemens Elmiskop I.

The tentacular crown of Sabella penicillus separates from the body under a variety of circum- stances (see below). For microscopical study of the abscission zone after separation, we held the

Abscission Zone in Sabella Tentacle Crown 35

specimen's body in one hand and the crown in the other and gently pulled until the crown was released, and then immediately plunged the pieces into the fixative.

C. Results

In a specimen o f Sabella penicillus whose tentacular crown is intact, the interme- diate layer between the crown skeleton and the body wall musculature has the a r rangement o f features shown in Figs. t and 2. Using transmission electron microscopy, Kryvi (1975) found that the short, densely stained cells (p) which fo rm bridges connect ing epimysial extensions (eve) with cartilage matrix exten- sions (cme) contain thick and thin filaments, and he concluded that these are muscle cells. Where these cells contact the extensions, much o f their surface is covered with hemidesmosome-l ike structures, into which the thin filaments appear to be inserted. The thick filaments are cross-striated in the manner o f pa ramyos in muscle filaments. The cartilage matr ix and the epimysium consist largely o f collagen-like fibrils (Kryvi 1975, t977), loosely packed and haphaza rd in orientation. Near the intermediate layer these fibrils are ar ranged in compac t bundles f rom which fibrils extend into the finest branches o f the extensions.

When separation o f the tentacular crown occurs in Sabella penicillus, only those anterior structures supported by the crown skeleton part f rom the body

Fig. 1. Semithin longitudinal section through the intermediate layer, light micrograph. The vacuolated cells (cc) of the cartilaginous skeleton of the crown at upper left, longitudinal body wall musculature (Ira) extends beyond the lower right, Both the cartilage matrix (cm) and the epimysium (ep) send extensions (arrows) into the intermediate layer, where they are joined by paramyosin muscle cells. Toluidine blue, x 550. Scale = 10 gm

Fig. 2. Higher magnification: Interdigitating extensions of the cartilage matrix (cme) and the epimy- sium (epe) are joined by paramyosin muscle cells (p). Nuclei of some fibroblast-like cells are visible among the extensions. Toluidine blue. × 1,400, Scale= 10 gm

36 B. Kennedy and H. Kryvi

Fig, 3. The posterior end of an abscised tentacular crown showing the exposed surface of the base, scanning electron micrograph. Orientation: dorsal side downward in the figure; the pinnules of the crown are out of view beyond the upper edge of the photograph. The undisturbed crown epidermis (ep) is neatly torn (arrows) at the abscission zone. The abscission surface is covered by extensions of the cartilage matrix on both lateral (ab) and transverse (tb) bars of the crown skeleton base. x 135. Scale= 100 ~tm

Fig. 4. The anterior end of the body of an animal whose tentacular crown has been pulled away, scanning electron micrograph. The exposed surface of the abscission zone (as) is face-on; orientation is dorsal side downward. The abscised crown shown in Fig. 6 belongs to this animal. Dorsal and ventral collar folds (dJ] vJ) appear at the borders of the photograph. An arrow indicates the approximate position of the section which produced Figs. 5 and 7. x 135. Scale= 100 lam

Abscission Zone in Sabella Tentacle Crown 37

(Fig. 3): the crown base, radioles, and pinnules; and the 'palps' . The body of the animal retains the remaining anteriorly situated structures, including (Fig. 4) the mouth and associated lateral lips, and the ventral sacs and parallel folds.

Abscission exposes the posterior face of the crown skeleton (Fig. 3), present- ing a clear view of the lateral bars (ab) and transverse bar (tb) of the skeleton base. The epidermis (ep) of the crown tears cleanly (arrows), forming a line describing the perimeter of the exposed surface.

Similarly, abscission exposes the anterior face of the body wall. Muscular contraction near the abscission site distorts the surface (Fig. 4), and if put back together the two complementary parts would match only approximately. On each side of the body in an animal whose crown is intact, an extension of the coelom and a large blood vessel pass across the abscission zone and into the tentacular crown. During abscission muscular contraction closes these spaces as they rupture (Fig. 4, arrow), and reduces loss of blood and coelomic fluid.

The relation of the abscission zone to the intermediate layer is revealed in sections. After abscission extensions of the epimysium (epe, Figs. 5 and 7) and of the cartilage matrix (cme, Figs. 6 and 8) remain on the body and the crown, respectively. Other features of the epimysium and the crown skeleton also appear as in animals whose crown is intact. Some of the fibroblast-like interstitial cells appear to have ruptured, while others may be undamaged.

After abscission, the distal parts of the epimysial extensions and the cartilage matrix extensions (cme, Fig. 9) are left covered by flocculent masses (mr) of filaments. These are the remains of the paramyosin muscle cells. Thin myofila- ments remain inserted on hemidesmosome-like structures which surround the ends of the epimysial extensions (epe, Fig. 10) and the cartilage matrix extensions (cme, Fig. 11). Thick filaments are left among the thin filaments.

During abscission, or immediately afterward, the wound closes at the anterior end of the animal's body (Figs. 4, 5 and 7). Maintained in an aquarium, the animal soon effects a healing of the abscission surface and then begins to regenerate the tentacular crown (Fig. 12). Regeneration appears to proceed from two areas, each situated near the point where the tentacular blood vessels passed through the original intermediate layer into the original tentacular crown.

D. Discussion

Abscission of the tentacular crown of Sabella penicillus principally involves a severing of the bridges connecting epimysial extensions of the body wall musculature and cartilage matrix extensions of the crown skeleton. This skeleto- muscular disjunction is accompanied by a destruction of some fibroblast-like cells associated with the epimysium and the perichondrium, by a clean tearing of the adjacent epidermis, and by muscular contraction near the wound site which limits loss of body fluids.

This skeletomuscular disjunction is a rupture of short paramyosin muscle cells. We cannot determine from our material whether myofilaments simply

38 B. Kennedy and H. Kryvi

. . . . . . . . . . . . .

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Fig. 5. The abscission zone of an animal whose tentacular crown has been pulled away. Light micrograph. The longitudinal body wall musculature (lm) has contracted, drawing the torn edges (e) of epidermis together and closing the wound. Some debris (d) has accumulated at the wound site, but very little fluid has leaked to the outside. Toluidine blue. × 560. Scale= 10/am

Fig. 6. A light micrograph, at the abscission zone of the tentacular crown which was removed from the animal shown in Fig, 4. The vacuolated cells (co) and cartilage matrix (cm) of the crown skeleton appear as in animals having the crown intact. Extensions (cme) of the matrix, exposed by the abscission, prqiect from the surface among fibroblast-like cells. Totuidine blue. x 560. Scale = 10 ~tm

Abscission Zone in Sabella Tentacle Crown 39

Fig. 10. Part of an epimysial extension (epe) in the abscission zone of an animal whose tentacular crown has been pulled away. Thick and thin myofilaments (my) remain in place about the end of the epimysial extension. Transmission electron micrograph. × 15,000. Scale = 1 ~tm

Fig. 11. Part of a cartilage matrix extension (cme) in the abscission zone on a crown which has been pulled away from the animal. Thick and thin myofilaments (my) remain in place about the cartilage matrix extension. Hemidesmosome-like figures appear where myofilaments are attached. Transmission electron micrograph, x 15,000. Scale = 1 lam

Fig. 7. Part of the section shown in Fig. 5, at higher magnification. Extensions (epe) of the epimysium appear as in an animal whose crown is intact. Toluidine blue. x 1,400. Scale = 10 ~m

Fig. 8. Higher magnification of an abscised crown. Densely stained anchoring structures pass f rom the extensions (epe) into the cartilage matrix, as in the intact crown. A fibroblast-like perichondrial cell (n) appears in the section. Toluidine blue. x 1,400. Scale = l0 p.m

Fig. 9. The surface exposed at the base of the tentacular crown by abscission, scanning electron micrograph. Extensions (cme) of the cartilage matrix rise from the surface; myofilaments of ruptured paramyosin muscle cells form a flocculent mass (mJ) covering the distal parts of the extensions. × 4,250. Scale = 10/.tm

40 B. Kennedy and H. Kryvi

Fig. 12. The anterior end of an animal with a partially regenerated tentacular crown. Scanning electron micrograph. Orientation is ventral side downward. The base (b) of the regenerated crown and parts of some tentacular filaments (J) are visible. This is the result of three weeks' regeneration, and the crown is still much smaller than the original, x 320. Scale= 10 ~tm

break or whether they slide apart, but at least some of the thin filaments remain fixed at the ends of the muscle cells after abscission is complete. I f disjunction is effected by sliding of myofilaments, then the animal may be capable of controlling abscission. If, on the other hand, myofilaments are merely snapping under tension, then the abscission zone represents a plane of least strength in the myoskeletal system.

Abscission under the animal 's control is a kind of autotomy (Federicq 1883). McVean (1975) recognizes autotomy as the discarding of a body part by an animal, performed reliably and easily whenever the context is appropriate, asso- ciated with physiological and anatomical specializations which reduce the extent of injury, and followed often by regeneration of the lost part. Auto tomy is usually discussed in relation to the context in which it occurs, and different categories of autotomy reflect their contexts.

Fauvel (1927) thought that the ease with which the tentacular crown of Sabella is detached by autotomy and later regenerated accounted for the extreme variability in size of the crowns on animals brought in from the field. Nicol (1931) wrote (p. 558) that 'specimens of Sabella are fairly frequently collected without, or with regenerating, branchial crowns ... Under aquarium conditions it is often found that worms throw off the entire branchial crown and retire into their tubes, ' and then regenerate the crown. All the Sabella penicillus

Abscission Zone in Sabella Tentacle Crown 41

we collected from the protected environment at the Bergen Aquarium possessed fully grown crowns. No apparently spontaneous autotomy occurred in any of the dozens of specimens we kept in aquaria over periods as long as several months.

Our routine technique for inducing abscission does not provide a suitable context in which to discuss autotomy. We kept some animals in their tubes in aquaria and simulated a fish predator by slowly approaching an expanded crown with forceps in hand and then quickly grasping it. Sometimes the animal escaped entirely by rapid withdrawal into the tube. When the crown was caught with the forceps, the animals always autotomized it, usually following a brief struggle, as it retreated into the tube. Viewed in this context, abscission of the tentacular crown by Sabellapenicillus might be termed an 'escape autotomy' .

Nicol (1931) suggested that loss of the crown might also be a normal reaction to wear or damage to its parts. We have no evidence of such a 'surgical auto- tomy ' by Sabella penicillus.

Some sabellid polychaetes autotomize the tentacular crown more readily than others. Sander (1976) used a technique like ours to remove crowns from Sabellastarte rnagnifica (Sabellinae), and commented that crown loss in that species often accompanies the stress of collection. But in Dales's (1961) hands Schizobranchus affinis (Sabellinae) did not autotomize, and crown removal in this species resulted in considerable loss of blood. Berrrill (1931) mentioned that Sabella penicillus (as Sabella pavonina) autotomizes the crown readily, but Branchiomma vesiculosum (Sabellinae) dos not. Branchiomma has better devel- oped photoreceptors in the crown than Sabella has, and Berrill supposed that Branchiomma might depend for escape upon a better developed ' shadow reflex' instead of autotomy. Wells (1951, 1952) observed autotomy in Sabella penicillus (as Sabella pavonina) but not in Myxicola infundibulum (Myxicolinae). He found behavioral differences between these two species which relate to differences in their respiratory physiology. Sabella penicillus irrigates its tube, and acquires dissolved oxygen across the body wall as well as through the tentacular crown. Myxicola infundibulum does not irrigate its tube, and depends upon the tentacular crown and the anteriormost part of the body for gas exchange. Moreover, Myxicola infundibulum has a much better developed startle reflex than has Sabella, but apparently is capable of anterior regeneration only if damage is limited to the crown itself, whereas Sabella regenerates even from body frag- ments.

Evidently Sabella penicillus discards the crown at comparatively low cost. The intact crown may supply little oxygen to the body (Wells 1952); and, although the gathering of particulate matter ceases when the crown is lost, the organs that manipulate particles (Nicol 1931) remain in place on the body. Tube construction and feeding resume as soon as the first regenerating tentacular filaments appear. A predator that succeeds in getting only the crown of Sabella penicillus deprives its prey of very little.

Differences among sabellids in their capacity to autotomize probably have little anatomical basis. We were unable to induce autotomy of the crown in Myxicola infundibulum (Myxicolinae), while Chone ecaudata (Fabricinae) autoto- mized as readily as Sabella penicillus under similar conditions, and Chone mollis

42 B. Kennedy and H. Kryvi

autotomized, but much less readily. In one figure, Bonar (1972) shows Chone mollis with the crown removed, but he does not mention autotomy. These species all have an intermediate layer which is structurally indistinguishable in wax sections from the abscission zone of Sabella penicillus.

Although sabellid polychaetes appear to exercise some control over autotomy of the crown, their zone of abscission seems to be the point of least strength in the skeletomuscular system. The crown of Sabella penicillus parts from the body at the abscission zone even in specimens which have been killed by various means.

The abscission zone of Sabella penicillus interests us from the physiological viewpoint. We suppose that the epidermis and interstitial cells offer little resis- tance to separation, and autotomy follows as a matter of course when the paramyosin muscle cells cease to function. In other instances of skeletomuscular breakage which have come to our attention (e. g., Takahashi 1967; Eylers, 1967), the failure is at some point other than within myofilament systems. Kryvi (1975) found nerves containing dense-cored vesicles terminating on the paramyo- sin muscle cells. An electrophysiological or pharmacological study of this system might reveal how the failure of the paramyosin muscle cells is brought about. We suggest that failure occurs when, upon receipt at the neuromuscular junctions of a command to autotomize, the myofilaments slide freely past one another; then the paramyosin muscle cells are forced to extreme hyperextension by con- traction of nearby body wall muscles, by expansion of interstitial cells surround- ing the paramyosin muscle cells, or simply by traction of the crown away from the body.

A capacity for autotomy is widespread among polychaetes. In species from several families reproductive stolons part from stocks at sexual maturity. Body parts are cast off under a variety of circumstances: the operculum or the whole crown of Serpulidae; gill filaments and tentacles or 'palps' in Cirratulidae, Acrocirridae, Flabelligeridae, Spionidae, etc.; and scales in Aphroditacea, to name only a few examples In all these the parts separate at abscission zones, anatomically characterized in a variety of ways, and abscission occurs with little loss of body fluids and may be followed by regeneration (Kennedy, unpub- lished).

Acknowledgements. Most of this investigation was conducted at the Institute of Anatomy, University of Bergen, during a one-year visit by BK to Norway. Expenses of the visit were defrayed in large part by the King Olav V Fellowship (1976-1977), and we gratefully acknowledge the interest shown by the American-Scandinavian Foundation, administrators of the Fellowship. We appreciate the generosity of the directors of the Institute, W. Harkmark and P.R. Flood and the hospitality and technical assistance of the staff. Part of the work was done at the Bodega Marine Laboratory, and we thank Cadet Hand, director of the Laboratory, and the Department of Zoology, University of California at Berkeley, for space and supplies for BK during 1978-1979. We thank Ms. V.G. Campen for assistance in preparation of the manuscript, and Ms. K. Weltzin for typing.

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Received February 25, 1980