Proceedings of the Fifth International Echinoderm Conference / Galway / 24-29 September 1984 Ultrastructural changes in the autotomy tissues of Eupentacta quinquesemita (Selenka) (Echinodermata: Holothuroidea) during evisceration MARIA BYRNE Smithsonian Marine Station at Link Port, Fort Pierce, Fla., USA ABSTRACT: Evisceration in the dendrochirote holothurian Eupentacta quinquesemita (Selenka) is associated with the sudden breakdown of three autotomy tissues. These tissues were examined before, during and after evisceration to investigate the presence of autotomy related specializations and to elucidate the morphological events associated with autotomy. The tissues are characterized by a preponderance of connective tissue and also contain muscle and nerve. Axon-like processes containing large electron-dense vesicles (LDVs) are found in the connective tissue and in association with muscle cells. These processes are similar to the neurosecretory-like processes described for other echinoderm autotomy tissues. Autotomy is part of the general phenomenon of variable tensility in echinoderm connective tissues and involves a change in the connective tissue matrix. During autotomy, the matrix loses its structural integrity causing collagen fibril disarray and disorgani- zation of associated cells. Some axon membrane and basal lamina disruption occurs, but the vesicles contained in axons and the LDVs appear to remain in tact. The vesicles con- tained in locally distributed axons do not appear to be the source of agents that effect connective tissue breakdown; an alternate source is discussed. 1 INTRODUCTION The autotomy of body parts through a sud- den reduction in the tensility of connec- tive tissue structures is characteristic of the Phylum Echinodermata (Emson & Wilkie 1980) and appears to be unique to the group. Echinoderm autotomy differs from autotomy in other invertebrates where body parts are cast off through rupture of muscle tissue specializations (McVean 1975). The sudden breakdown of connective tissue during autotomy is part of the general phenomenon of variable tensility of echi- noderm connective tissues (Motokawa 1984; Wilkie 1984). Another aspect of the phe- nomenon is the reversible stiffening/sof- tening changes demonstrated by echinoderm catch ligaments. The changes associated with variable tensility have attracted attention because they occur in an extracellular tis- sue and are considered by many workers to be under neutral control (Jordan 1914,1919; Serra-von Buddenbrockl963; talkie 1978,1983, 1984; Holland & Grimmer 1981a,b; Motokawa 1981,1982a,1984; Byrne 1982; Hilgers & Splechtna 1982; flidaka & Takahashi 1983). Autotomy is usually associated with anat- omical specializations that facilitate ejection of body parts (McVean 1975). This aspect of variable tensility has re- ceived relatively little attention, al- though there are several ultrastructural studies of echinoderm catch ligaments (Holland & Grimmer 1981b; Smith et al 1981; Motokawa 1982b; Hidaka & Takahashi 1983; Wilkie 1983). Thus far, the fine structure of ophiuroid and crinoid arm autotomy tis- sues has been described (Wilkie 1979; Holland & Grimmer 1981a). The presence of axon-like processes, filled with large electron-dense vesicles (LDVs), appears to be characteristic of echinoderm connective tissues (Wilkie, 1979,1984; Holland & Grimmer 1981a,b; Smith et al 1981; Byrne 1982; Hilgers i Splechtna 1982; Motokawa 1982b,1984; Hidaka & Takahashi 1983). The LDVs resemble neu- rosecretory vesicles found in firmly estab- lished neurosecretory neurons (Maddrell & Nordmann 1979) and, based on their morphol- ogy, LDVs have been suggested to be in- volved in the control of variable tensility (Wilkie 1979, 1984; Holland & Grimmer 1981 a,b; Smith et al 1981; Motokawa 1982b, 1984; Hidaka & Takahashi 1983). Holothurian evisceration results in au- totomy of the digestive tract and tentacu- lar crown (Kille 1935; Smith & Greenberg 1973; Byrne 1982), In the dendrochirote 413 holothurian Eupentacta quinquesemita (Selenka) evisceration is associated with the irreversible breakdown of three struc- tures, (1) the introvert (the anterior ex- tensible portion of the body wall), (2) the tendon (P-L tendon) that connects the pha- ryngeal retractor muscle (PRM) to the lon- gitudinal body wall muscle (LBWM), and (3) the intestine-cloacal junction* The fine structure of the intact P-L tendon has pre- viously been reported (Byrne 1982) and will be described here briefly. The ultrastruc- ture of the introvert and intestine-cloacal junction is also described* Particular emphasis is placed on the changes that oc- cur in the three structures during eviscer- ation and for evidence of neural involve- ment in the connective tissue change. Fig. 1. One micrometer cross section of the epidermis (E) and superficial dermis (S). The ossicles (0) are surrounded by collagen fibrils (CF). Scale=l .Oum. Fig. 2. Cross section of the dense connec- tive tissue (D) and the loose connective tissue (L). M, muscle fibres; Arrow, DCT- LCT interface. Scale=1.0vim. Fig. 3. Cross section of the 'transparent' LCT (L). M, muscle fibres MC, morula cells; P, peritoneum. Scale=1.0um, Fig. 4. LDV-filled process and muscle fibre bundle (M) in the OCT. BL, basal lamina; CF, collagen fibrils. Scale=2.0,jm. 2 MATERIALS AND METHODS Specimens of E. quinquesemita were collec- ted using S.C.U.B.A. near Victoria, Brit- ish Columbia. Evisceration was induced in freshly-collected specimens by squeezing the body transversely with forceps. For transmission electron microscopy, intact and eviscerating specimens were injected with 3% glutaraldehyde in 0.2M cacodylate buffer (pH 7.4). The autotomy tissues were dissected and placed in fresh fixative for one hour at room temperature. The tis- sues were then rinsed in the same buffer, post-fixed in 1% 0s04 in cacodylate for one hour at 4?C, decalcified in ascorbic acid (Dietrich & Fontaine, 1975), dehydrated, and embedded in Epon 812. Thick sections were stained with Richardson's stain (Richardson et al 1960) for light micro- scopic examination. Thin sections were stained with uranyl acetate and lead cit- rate and viewed with a Philips EM 300. 3 RESULTS 3.1 Structure of intact introvert The introvert is predominantly dermal con- nective tissue with the cuticle and epider- mis on the outside and the musculature, nerve plexus and peritoneum on the coelomic side (Figs. 1-3). The dermal connective tissue has three layers, the superficial dermis, a dense connective tissue (DCT) layer and a loose connective tissue layer (LCT). The superficial dermis contains ossicles, cross striated collagen fibrils and connective tissue matrix (Fig. 1), The DCT contains abundant collagen fibrils varying in diameter from 20-160nm associ- ated with an interfibrillar matrix, Fig. 4, Fig. 5. LDV-filled process in LCT (L). The LCT is electron-lucent and contains a few unstriated fibrils (UF). BL, basal lamina. Scale-l.Oum. Fig. 6. Elongate LDV-filled process in autotomizing introvert near the DCT-LCT interface. CF, collagen fibrils; 0, DCT; L, LCT. Scale=2..0um. Fig. 7. LDV-filled processes in the gut connective tissue contain axial micro- tubules (MT) and occasional mitochondria (MI). CF, collagen fibrils. Scale=0.5ym. Fig. 8. One micrometer cross section of the intestine adjacent to the intestine- cloacal junction. B, brown body; C, con- nective tissue; EP, intestinal epithelium; G, gut lumen; P, peritoneum. Scale-O.lmm. Fig. 9. Cross section of a PRM bundle sur- rounded by P-L tendon collagen fibrils (CF). LDV-filled processes and axons (A) are found with the muscle fibres (M). BL, basal lamina, NT, neurotubules. Scale= l.Oym. Fig. 10. Cross section of autotomized P-L tendon. An intact LDV-filled process is alongside a dispersed PRM bundle. The muscle fibres (M) lack thick filaments, and the contractile elements disperse (asterisk). The collagen fibrils (CF) are in disarray, and the sarcolemma (5) has lifted away,. An axon-like profile (A) has an irregular membrane profile and contains few vesicles. BL, basal lamina. Scale= 2.0um? 414 * .* itf LDV K m fe ' 10 m 415 6,11,12), Many of the collagen fibrils have unstriated fibrils (10-14nm in diame- ter) attached. Bundles of muscle cells surrounded by a basal lamina are scattered through the DCT (Figs. 4,11). Axons are present in the DCT in association with muscle fibres and also surrounded by con- nective tissue. The axons are enshe.athed in a basal lamina and contain neurotubules, occasional mitochondria and two types of vesicles, clear vesicles (70-80nm) and dense-core vesicles (90-140nm). The DCT also contains axon-like processes (Figs. 4, 5) filled with large, electron-dense ves- icles (LDVs) that vary in shape from round (150-300nm) through ellipsoidal to sausage- shaped (180-550nm X 125-220nm). Axial microtubules and occasional mitochondria are present in the processes (Fig. 7). These LDV-filled processes are found beside muscle bundles along with axons and are scattered throughout the connective tissue (Figs. 4,6). The LCT is an amorphous electron-lucent layer of connective tissue matrix with occasional collagen fibrils (4O-60nm in diameter) and more common, smal] diameter (7-12nm), unstriated fibrils (Figs. 3,5,6) Muscle fibres, morula cells, axons and LDV- filled processes are also present in the LCT. diameter (4-10nm) unstriated fibrils (Fig. 7)? A diffuse layer of collagen fibrils (20-4Onm in diameter) is found along the basal lamina near the gut musculature (Fig. 15). Axons are rarely found in the intestinal connective tissue but LDV-filled processes are encountered, especially near the basal lamina (Fig. 7). Morula cells and brown bodies are common in the connec- tive tissue (Figs. 8,15). 3.4 Structure of autotomizing introvert During evisceration the introvert changes from a solid, opaque structure to one that is soft and translucent. Introvert thick- ness decreases as it distends, with the viscera and coelomic fluid propelled for- Figs. 11-16. Similar fields of intact and automized tissues. Fig. 11, Intact introvert DCT. The muscle fibres (M) are contracted and sarcolemroal extensions (S) lie within basal laminar (BL) folds. CF, collagen fibrils. Scale= 2.0um. 3.2 Structure of intact P-L tendon The P-L tendon is comprised of a collage- nous connective tissue layer surrounding a central muscle region containing the tapered ends of PRM cells and is overlain by peritoneum (Figs. 9,13). Connective tissue ramifies throughout the interior of the tendon so the PRM fibres are completely embedded in connective tissue. The tendon contains abundant small diameter collagen fibrils (30-4Onm) and unstriated fibrils (10-15nm) in an interfibrillar matrix. Axons and LDV containing processes similar to those described for the introvert are present in the tendon in association with muscle and connective tissue (Fig. 9). 3.3 Structure of intact intestine cloacal junction The intestine cloacal junction is the posterior end of the intestine where it joins with the cloaca. It contains a thick connective tissue layer with the gut musculature and peritoneum on the coelomic side and the epithelium on the luminal side (Figs. 8,15), The connective tissue is largely matrix and contains small Fig. 12. Autotomizing DCT. The muscle fibres (M) are extended. The sarcolemmal extensions and basal laminar folds have straightened out and the basal lamina (BL) is disrupted (arrows). CF, collagen fibrils. Scale=2.0ym. Fig. 13. Intact P-L tendon. The perito- neum (P) overlies the relatively compact collagenous connective tissue of the tendon. CF, collagen fibrils; M, muscle; MV, microvilli. Scale=3.0pm. Fig. 14. Automized P-L tendon. The peritoneum (P) is disrupted (arrow), and the collagen fibrils (CF) are dispersed.. M, muscle; MV, microvilli. Scale=2.0ym. Fig, 15. Intact intestine-cloacal junction. The peritoneum (P), nerve plexus (NP) and muscle (M) layers cover the intestinal con- nective tissue. Collagen fibrils (CF) are particularly abundant along the basal lamina (BL), B, brown body; MC, morula cell. Scale=5.0Wm, Fig. 16. Autotomized intestine-cloacal junction. The peritoneum (P), nerve plexus and muscle layer are disrupted (arrow), and the collagen fibrils (CF) are dispersed. Scale-5.0ym. 416 417 ward by body wall muscle contraction. As the introvert autotomizes, the superficial dermis and epithelium delaminate from the underlying DCT, and the ossicles embedded within it become visible to the eye. Changes are evident when similar fields of normal and autotomizing DCT are compared (Figs. 11,12). The muscle fibres extend as the introvert distends, and the basal lamina surrounding the muscle fibres is disrupted. Structural examination reveals that the collagen fibrils remain intact and that they appear to slide across and away from each other. This suggests that changes occur in the interfibrilar matrix. Axons and LDV-filled processes also appear to extend, with some damage to axon mem- branes and the basal lamina, but their vesicular contents appear intact (Fig. 6). The peritoneal cells, muscle fibres and nerve plexus axons dissociate into the coelom, and the LCT is infiltrated by coelomic fluid. Collagen fibrils and un- striated fibrils in the LCT also remain intact, but the matrix loses its structural integrity and the cells embedded in it become disorganized. LDV-filled processes in the LCT and among the dissociated plexus axons retain their vesicles during autotomy. 3.5 Structure of autotomized P-L tendon During autotomy, the P-L tendon changes from a relatively compact collagenous tis- sue, to one comprised of disorganized fi- brils. Similar fields of normal and auto- tomized tendon show the increase in inter- fibril distance and fibril disarray result- ing from autotomy (Figs. 13,14). After autotomy, the peritoneum is disrupted, and portions dissociate from the basal lamina. Although the collagen fibrils remain in- tact, it appears that the associated matrix undergoes a structural change. This re- sults in fibril disorganization and a marked increase in the interfibril distance. The PRM muscle bundles are disrupted, and the contractile filament organization is lost with a disappearance of the thick filaments (Figs. 10,14). Sarcolemmal dis- ruption is evident, and eventually the muscle cell contents are dispersed. Axons and LDV-filled processes also disperse and show membrane irregularity, but for the most part retain their vesicles. Some axon-like profiles devoid of vesicles may represent axons which have lost vesicles during autotomy (Fig. 10). The LDV-filled processes remain surprisingly intact (Fig. 10), although some vesicular loss through mechanical damage might be expected. 3.6 Structure of autotomized intestine- cloacal junction As for the other autotomy tissues, the intestinal peritoneum, muscle layer and nerve plexus dissociate during autotomy, and the underlying connective tissue is infiltrated with coelomic fluid (compare Figs. 15,16). The collagen fibrils are disorganized, but remain intact. Some axons in the dissociated nerve plexus and connective tissue have an irregular con- tour, and their basal lamina is disrupted. Vesicles are present in most axons. How- ever, some axons completely surrounded by coelomic fluid contain few or no vesicles, suggesting that the vesicles may have been lost during autotomy. 4 DISCUSSION Previous examination of echinoderm catch and autotomy tissues revealed that they are collagenous connective tissues (Takahashi 1967; Wilkie 1979; Holland & Grimmer 1981a, b; Smith et al 1981; Motokawa 1982b; Hidaka & Takahashi 1983) and the ultrastructure of the P-L tendon and introvert dense connec- tive tissue is similar. The structure of the introvert loose connective tissue and the intestine differ by having a high ma- trix content relative to fibril content. The collagen fibrils and unstriated fibrils of the autotomy tissues are characteristi- cally of small diameter and are similar to those observed in other echinoderm connec- tive tissues (Junquelra et al 1980; Holland & Grimmer 1981a,b; Smith et al 1981; Motokawa 1982b; Hidaka & Takahashi 1983). Unlike ophiuroid and crinoid autotomy structures that are comprised entirely of connective tissue (Wilkie 1979; Holland & Grimmer 1981a), the autotomy tissues of E. quinquesemita also contain muscle cells. Axons and LDV-containing processes are present in the autotomy structures of E. quinquesemita. They are surrounded by connective tissue as well as in association with muscle cells and the nerve plexus. The axons and their vesicular contents appear to be typical of echinoderm neurons. The clear vesicles may be cholinergic, while the dense-core vesicles may be mono- aminergic, as suggested elsewhere (Prosser & Mackie 1980; Byrne 1982; Pentreath & Cobb 1982). The LDV-filled processes resemble axons in containing axial micro- tubules and vesicles, and in their associ- ation with muscle fibres. Based on their morphology, the LDV-filled processes of E. quinquesemita are considered to be axons. 418 The LDVs are similar to the neurosecretory- tissues, LDV-prcrc esses are found only with like vesicles in other echinoderm connec- connective tissue (Wilkie 1979; Holland & tive tissues that are typically large, Grimmer 1981a); perhaps the LDV-processes electron-dense and of variable shape. The of E. quinquesemita are not homologous to LDV-containing processes appear character- those of ophiuroid and crinoid autotomy istic of echinoderm connective tissues and tissues, are thought to play a role in connective Coelomic fluid factors that alter connec- tissue variable tensility (Wilkie 1979, tive tissue tensility have been isolated 1984; Holland & Grimmer 1981a,b; Hilgers & from several echinoderms (Motokawa 1981, Splechtna 1982; Motokawa 1982b,1984; 1982a,c). There is evidence for the pres- Hidaka & Takahashi 1983). ence of an autotomy-inducing factor in the The sudden breakdown of the autotomy tis- eviscerated coelomic fluid of E. quinque- sues during evisceration involves a change semita (Byrne 1983), as found in another in the connective tissue matrix, as occurs dendrochirote holothurian, Sclerodactyla in arm autotomy in ophiuroids and crinoids briareus (Smith & Greenberg 1973). The (Wilkie 1979; Holland & Grimmer 1981a). peritoneal disruption, followed by coelomic During autotomy the fibrillar elements of fluid infiltration into the connective tis- the autotomy tissues remain intact, but sues during evisceration, may be important they disperse as the matrix loses its to the autotomy process of E. quinquesemita. structural integrity. Although it is dif- The three autotomy tissues are partially or ficult to describe change in the electron- completely bathed in coelomic fluid and in luscent matrix, the fibril disarray sug- this respect, differ from the arm autotomy gests that the matrix has undergone a phy- tissues of ophiuroids and crinoids. The sical and chemical change, perhaps a sol- autotomy tissues of these groups are not gel-like change. associated with a large coelomic fluid In the autotomy tissues of ophiuroids medium and so ophiuroid and crinoid arm and crinoids, the LDV-filled processes autotomy may depend on locally distributed appear to be the only potential source of cells. In contrast, cells involved in autotomy-inducing agents that can be de- holothurian evisceration may be located at tected microscopically within the connec- some distance from the autotomy tissues tive tissue (Wilkie 1979; Holland & Grimmer and effect the change through the medium 1981a). The contents of the LDVs have been of the coelomic fluid. suggested to be proteolytic enzymes or Introvert and P-L tendon autotomy was chelating agents that when secreted, effect mimicked in vitro by altering the ionic connective tissue change (Wilkie 1979; composition of test solutions (Byrne in Holland & Grimmer 1981a). Axon profiles press). The results suggest that the mech- without vesicles and LDV exocytosis ob- anism of autotomy involves a change in the served in the autotomized crinoid syzygy ionic interations within the proteoglycan have been suggested to be part of a mas- matrix. How this is brought about is not sive vesicle exocytosis that plays a role known. Although echinoderm autotomy ap- in effecting autotomy (Holland & Grimmer Pears t0 be neurally controlled, the mor- 1981a). However, it is not clear whether phological basis for neural mediation has these profiles are a cause or a result of not been established, autotomy. During evisceration in E? quinquesemita, the LDVs appear to remain intact. The disruption of axon membranes and basal T thank Professor &.R. Fontaine for his lamina observed in autotomized tissues may enthusiastic supervision of my research, be mechanical rupture occurring during Dr- K-J- Eckelbarger and Mr= J>E. Miller autotomy. Swollen axons devoid of vesicles read the manuscript. The work was sup- were observed adjacent to intact vesiculat- ported by a university of Victoria Fellow- ed axons and are likely to be a result ship and this report is Smithsonian Marine rather than a cause of autotomy. Eviscer- station contribution no. 134. ation and autotomy in E. quinquesemita appear to be neurally controlled (Byrne 1983) but, based on morphological evidence, REFERENCES the vesicles contained in locally distrib- uted neurons do not appear to be the source Byrne, M. 1982. Functional morphology of of agents that effect connective tissue a holothurian autotomy plane and its breakdown. 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