The compass depressors of Paracentrotus lividus (Echinodermata, Echinoida): ultrastructural and mechanical aspects of their variable tensility and contractility

August 9, 2017 | Autor: Iain Wilkie | Categoria: Zoology, Acetylcholine, Soft Tissue, Thin Layer
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Zoomorphology (1992) 112:143-153

Zoomorphology 9 Springer-Verlag1992

The compass depressors of Paracentrotus lividus (Echinodermata, Echinoida): ultrastructnral and mechanical aspects of their variable tensility and contractility I.C. Wilkie 1, M . D . Candia Carnevali 2, and F. Bonasoro 2 1 Department of Biological Sciences, Glasgow Polytechnic, 70 Cowcaddens Road, Glasgow G4 0BA, Scotland, UK 2 Dipartimento di Biologia "Luigi Gorini", Universitfi degli Studi di Milano, Via Celoria 26, 1-20133 Milano, Italy Received February 12, 1992

Summary. The compass depressors are bands of soft tissue which connect the compass ossicles of the echinoid lantern to the inner edge of the test. They are essentially ligaments with on one side a thin layer of muscle cells. The ligamentous c o m p o n e n t consists mainly of a parallel array of collagen fibrils with interspersed 12 nm microfibrils. The most notable cellular constituents are granulecontaining cell bodies and their processes which resemble the juxtaligamental cells that have been found in all echinoderm mutable collagenous tissues and which m a y control the tensility of these tissues. The muscle cells occupy about 8% of the total cross-sectional area of the compass depressor and are located in a richly innervated pseudostratified myoepithelium. When subjected to constant low loads in creep tests the compass depressor stretches to a fixed length beyond which there is no further extension. The length at this creep limit coincides with the m a x i m u m length to which the compass depressor is stretched by natural movements of the intact lantern. Stress-strain tests show that treatment with 1 m M acetylcholine or 100 m M K + ions can increase reversibly the stiffness of the compass depressor to an extent that cannot be due to contraction of the myoepithelium, suggesting that the mechanical properties o f the ligament are under physiological control. Tension-length data on the myoepithelium suggest that it generates a m a x i m u m active tension when the compass depressor is stretched to the creep limit. The implications of these results for the function of the compass depressors are discussed.

et al. 1988; Candia Carnevali et al. 1991) and certain aspects of its mechanical behaviour have been explored (Candia Carnevali et al. 1988; Andrietti et al. 1990), little attention has been paid to those components of the lantern that form the compass system. The compass system consists of: (1) five compass ossicles which are located on the aboral (upper) surface of the lantern, (2) five compass elevator muscles which interconnect transversely the compasses, (3) ten ligaments linking the compasses to the underlying rotular ossicles, and (4) ten compass depressors - straps of soft tissue which extend from the distal lobes of the compasses to the interambulacral processes of the perignathic girdle (inner edge of the test) at its junction with the flexible peristomial m e m b r a n e (Fig. 1). The compasses

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A. Introduction

I

All regular sea-urchins possess a complex masticatory apparatus known as Aristotle's lantern. Although much is k n o w n of the functional m o r p h o l o g y of this structure (e.g. Mfirkel 1979; Mfirkel etal. 1990; Lanzavecchia Correspondence to:

I.C. Wilkie

2 mm I

ed

pr

Fig. 1. Diagrammatic representation of the lantern of P a r a c e n t r o t u s lividus. The peripharyngeal coelomic membranes are omitted. Only the soft tissue components of the right side have been included; on the left side the insertion facets of the muscles are indicated by stippling, c d compass depressor; ce compass elevator; co compass; pm peristomial membrane; pr protractor muscle; re retractor muscle; ro rotula. A r r o w indicates one of the pair of ligaments that link the compass to the rotula

144 are c o n n e c t e d to delicate c o e l o m i c m e m b r a n e s t h a t delim i t p e r i p h a r y n g e a l cavities s u r r o u n d i n g v a r i o u s c o m p o nents o f the l a n t e r n a n d it has been suggested t h a t the c o m p a s s system serves either as a r e s p i r a t o r y p u m p , w h i c h assists the delivery o f o x y g e n to the l a n t e r n m u s cles ( N i c h o l s 1969), o r as a p r e s s u r e - c o m p e n s a t i n g device to stabilise p r e s s u r e w i t h i n the rigid test as the l a n t e r n is p r o t r a c t e d a n d r e t r a c t e d d u r i n g feeding activity ( S m i t h 1984). T h e c o m p a s s e l e v a t o r muscles are so-called because their c o n t r a c t i o n results in the u p w a r d s r o t a t i o n o f the c o m p a s s e s u p o n the r o t u l a e , a n d it has u s u a l l y been a s s u m e d t h a t the c o m p a s s d e p r e s s o r s are s i m p l y muscles w h o s e a c t i o n a n t a g o n i s e s t h a t o f the elevators (e.g. I t y m a n 1955; N i c h o l s 1969; S m i t h 1984). H o w e v e r , C h a d wick (1900) referred to t h e m as " l i g a m e n t s " a n d in an u l t r a s t r u c t u r a l i n v e s t i g a t i o n S a i t a (1969) n o t e d that, a p a r t f r o m a s m a l l p r o p o r t i o n o f m u s c l e cells, they c o n sist m a i n l y o f c o l l a g e n fibres. O n the basis o f f u r t h e r u l t r a s t r u c t u r a l o b s e r v a t i o n s , L a n z a v e c c h i a et al. (1988) c o n j e c t u r e d t h a t the c o l l a g e n o u s c o m p o n e n t m i g h t be a m u t a b l e c o l l a g e n o u s tissue ( M C T ) , i.e. t h a t its m e c h a n ical p r o p e r t i e s c a n be a d j u s t e d r a p i d l y u n d e r p h y s i o l o g i cal c o n t r o l . Such v a r i a b l e tensility has been d e m o n s t r a t e d in m a n y e c h i n o d e r m c o l l a g e n o u s structures i n c l u d i n g seau r c h i n spine l i g a m e n t s ( H i d a k a a n d T a k a h a s h i 1983; M o t o k a w a 1983; D i a b a n d G i l l y 1984; M o r a l e s et al. 1989) a n d p e r i s t o m i a l m e m b r a n e ( C a n d i a C a r n e v a l i et al. 1990). In these l o c a t i o n s , stiffening o f the collagen o u s tissue m a y serve to lock, respectively, the spines a n d l a n t e r n in a p a r t i c u l a r p o s i t i o n , while their softening a l l o w s these structures to be m o v e d by c o n v e n t i o n a l muscles. M C T s can thus f u n c t i o n as passive m e c h a n o effectors (for reviews see M o t o k a w a 1984; W i l k i e 1984; W i l k i e a n d E m s o n 1988; C a n d i a C a r n e v a l i a n d W i l k i e 1992). This p a p e r focuses o n the m e c h a n i c a l b e h a v i o u r a n d r e l a t e d aspects o f the m o r p h o l o g y o f the c o m p a s s dep r e s s o r s o f the c a m a r o d o n t s e a - u r c h i n P a r a c e n t r o t u s lividus ( L a m a r c k , 1816). T h e u l t r a s t r u c t u r e o f their ligam e n t o u s a n d m u s c u l a r c o m p o n e n t s is d e s c r i b e d a n d inf o r m a t i o n is p r o v i d e d o n b o t h their passive m e c h a n i c a l p r o p e r t i e s a n d their c o n t r a c t i l e activity. E v i d e n c e is presented w h i c h c o n f i r m s p r e v i o u s suspicions t h a t the stiffness o f the c o l l a g e n o u s c o m p o n e n t is u n d e r direct nervous control.

B. Materials and methods Specimens of P. lividus were collected by SCUBA diver from the Ligurian coast of Italy and kept in tanks of aerated sea-water at 16~ C in the University of Milan. Transmission electron microscopy. Material was fixed at 4~ C for 2 h with either 2% glutaraldehyde alone, or a 2% glutaraldehyde4% paraformaldehyde mixture, in 0.1 M cacodylate buffer. After an overnight wash in buffer, the specimens were postfixed for 2 h with 1% osmic acid in 0.1 M cacodylate buffer, prestained with 1% uranyl acetate in 25% ethanol for 2 h, and then dehydrated in a graded ethanol series and embedded in an Epon-Araldite mixture. Sections were cut with an LKB V Ultrotome using a diamond

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Fig. 2a-c. Apparatus used for the investigation of a the creep behaviour of single compass depressors, and the stress-strain behaviour of b single compass depressors and c complete compass depressor sets. Not to scale, ar aluminium rod; cd compass depressor; cl clamp; gl glass hook; ht half-test; imt isometric transducer; itt isotonic transducer; la lantern; ma manipulator; w weight

knife, stained with aqueous uranyl acetate and lead citrate, and observed in a Jeo1100 SX electron microscope. Mechanical testing. The mechanical properties of both single compass depressors and complete compass depressor sets were investigated. The extension of single compass depressors under constant load was recorded by removing the top half of the test to expose the lantern, and detaching a compass depressor by breaking off the abaxial lobe of the compass to which it was attached. The half-test was rigidly clamped and the detached end of the compass depressor was linked by a heart-clip and silver chain to one end of the lever of an isotonic transducer from the other end of which was suspended a small weight (Fig. 2a). To investigate the relationship between compass depressor tension and length, single compass depressors were excised with attached fragments of compass and test, and were linked by heartclips to a clamped glass hook and to an isometric transducer which was fixed to the moving section of a manipulator. The manipulator allowed the vertical position of the transducer and therefore the length of the preparation to be adjusted by known amounts (Fig. 2b). Complete compass depressor sets were also prepared by removing the aboral half of the test, transecting the retractor, protractor and compass elevator muscles, and cutting round the whole peristomial membrane, leaving the lantern connected to the oral half-test by only the ten intact compass depressors. The half-test was clamped rigidly and the lantern was linked to an isometric transducer (fixed to a manipulator as above) via an aluminium rod inserted through the pharynx and between the teeth of the sea-urchin (Fig. 2c). In the tension-length tests single compass depressors and complete compass depressor sets were subjected to stepwise increases in length (expressed below as "strain": increase in length as a fraction of the initial length). To obtain tension-length data on the contractile component, complete compass depressor sets were stimulated with 1 mM acetylcholine firstly at zero strain (i.e. length at which passive tension is just zero) and then at each subsequent increment in strain 20-30 s after the peak passive tension. At each position the acetylcholine was washed out after the active tension became maximal (Fig. 3). In order to compare the behaviour of

145

ACh Tension(g)

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Fig. 3. Diagrammatic representation of an oscillograph recording to show the experimental protocol used to investigate the contractile behaviour of compass depressor sets. See text for further de-

tails. A Ch acetylcholine; A T active tension; PT passive tension

Fig. 4. Diagrammatic transverse section of the compass depressor. li ligament; m myoepithelium; I peripharyngeal coelomic channel of the compass depressor; 2 peripharyngeal coelomic space between the compass depressor and the underlying protractor muscle; 3 perivisceral coelom. Arrows indicate mesothelia

the compass depressors with that of conventional muscles, this technique was also applied to complete protractor muscle sets. In the case of compass depressor sets, strain was calculated indirectly from the vertical displacement of the lantern using a relationship developed from the model of Andrietti et al. (1990). Compass depressor cross-sectional area was estimated from a relationship between this parameter and test diameter which was derived from transverse histological sections of compass depressors from animals of different sizes. All experiments were performed in air at room temperature (ca. 25~ C).

C. Results L Ultrastructure

The general structure and anatomical relations of the compass depressors have been described by Lanzavecch.ia et al. (1988). They are essentially collagenous ligaments enclosed by mesothelia (coelomic epithelia) which delimit the peripharyngeal coelomic compartments and which, on the inner (adaxial) 1 surface of the compass depressors take the form of a myoepithelium (Fig. 4). 1. Ligament. The ligamentous portion consists mainly of densely packed collagen fibrils with periodicity ca. 1 The myoepithelium was mistakenly reported as occurring on the outer (abaxial) surface by Wilkie et al. (1992).

60 nm (Figs. 5, 1 l) which are mostly parallel to the longitudinal axis of the compass depressor, except towards the compass insertions where bundles of fibrils diverge to form an almost orthogonal fibre network. Also common are hollow microfibrils, ca. 12 nm in diameter, which sometimes have a beaded appearance (periodicity 30 4 0 n m ) and which form cylindrical clusters (i.e. fibres) running usually parallel to the longitudinal axis of the compass depressor. Towards the insertions these microfibrils are organised into both fibres and a loose meshwork separating adjacent bundles of collagen fibrils (Figs. 5, 6, 11). The ligament also contains many cellular elements, the most prominent of which are bipolar granule-containing cells whose processes extend longitudinally through the ligament. The perikarya of these cells are particularly abundant near the test insertion of the ligament and contain a nucleus, Golgi body and concentric layers of rough endoplasmic reticulum which often surround a central core of electron-dense granules (Figs. 8 10). Such granules are also present in the cell processes and have variable electron density and circular or oval profiles (greatest dimensions 280 and 370 nm respectively). Other cells containing envacuolated collagen fibrils and many other vacuoles of heterogeneous appearance may be phagocytic fibroclasts (Fig. 7). Also c o m m o n are large cell processes with finely granular, electronlucent cytoplasm which resemble axons. Although there are close contacts between the latter and granule-containing elements (Fig. l l), there is as yet no evidence for the presence of synaptic junctions. 2. Myoepithelium. The inner surface of the compass depressor is furrowed longitudinally and covered by a pseudostratified myoepithelium which apparently consists of a single layer of mesothelial cells (peritoneocytes) and one to a few underlying layers of myocytes (Fig. 12). The monociliated peritoneocytes touch the basal lamina by narrow processes (Figs. 12, 14) and are linked to each other at apical junctional complexes consisting of apical zonulae and septate junctions (see Rieger and Lombardi 1987). The myocytes occupy 8_+ 2% of the total cross-sectional area of the compass depressor. Within their contractile processes can be seen thin and thick myofilaments arranged parallel to the longitudinal axis of the compass depressor, Z-bodies, mitochondria and subsarcolemmal cisternae. The cells contact the basal lamina at hemidesmosomes and each other at spot desmosomes (Fig. 14). Profiles of axons are abundant within the myoepithelium. Tracts of possibly aminergic varicose axons containing dense-core granules 130 170 nm in diameter are located against the basal lamina, between the myocytes, or immediately below the peritoneocytes (Fig. 13). Less abundant are large granule-containing processes indistinguishable from those within the ligament, and axonlike processes with sparse, electron-lucent cytoplasm. Along its length the myoepithelium is attached to the ligament at contacts between the filamentous material of the basal lamina and the microfibrillar meshwork

146

147 separating adjacent collagen fibril bundles. The myocytes penetrate the ossicles at the insertions of the compass depressor into the compass and test, and here the ultrastructure is similar to that of other sea-urchin skeletomuscular junctions (see Stauber and M/irkel 1988). Immediately below the myocyte sarcolemma there is a continous electron-dense layer which is penetrated by thin myofilaments. The basal lamina is extended to form loops round which are knitted bundles of collagen fibrils which themselves wrap round the calcite bars of the skeletal stereom (Figs. 15, 16). The muscle cells thus appear to form mechanically stable unions both with the skeleton and along their area of contact with the ligament.

II. Mechanical properties 1. Constant load experiments. Under a low load (i.e. 0.3 g, representing tensile stress 4.5 4.9 kPa) single compass depressors stretched rapidly at first. However, the extension rate decelerated until after 1-5 min a constant length was reached (Fig. 17a). Compass depressors could sustain such low loads without further extension for at least 24 minutes. The significance of this creep limit was revealed during the preparation of lanterns for physiological investigation. The length of the compass depressors of exposed, but still intact, lanterns was measured firstly when the compasses were fully depressed and then after the addition of a few drops of 0.56 M KC1 which caused contraction of the compass elevators and other lantern muscles. The maximum length to which the compass depressors were stretched by the resultant elevation of the compasses and lateral tilting of the lantern was found to be consistent within individuals and in most cases corresponded exactly to the length at which extension stopped in subsequent creep tests (Table 1). Removal of the load from single compass depressors that had reached the creep limit resulted in both instantaneous elastic recoil and longer term, time-dependent elastic recovery (Fig. 17b). Since these responses persisted during anaesthesia, it is unlikely that they were caused by contractile activity of the compass depressor myoepithelium. In the course of the creep tests a number of compass depressors were subjected to stepwise increases in load

Figs. 5--11. Transmission electron micrographs (TEM) of the ligamentous component of the compass depressor Fig. 5. Longitudinal section showing collagen fibrils on the left and a bundle of microfibrils on the right Fig. 6. Transverse section showing two bundles of microfibrils, some of which appear to be hollow (fine arrow), and collagen fibrils

(double arrowhead) Fig. 7. Possible fibroblast or fibroclast containing envacuolated collagen fibrils (arrow) Fig. 8. Granule-containing cell body. Note rough endoplasmic reticulum (arrow)

until failure occurred. Assuming that mesothelia and muscle layers make no significant contribution to the resistance to imposed loads, the mean tensile strength of the compass depressor ligament was estimated to be 135.8+ (SD) 125.0 kPa (n=13). Stimulation of compass depressors at the creep limit with sea-water containing 100 m M K + ions resulted in a slowly developing active contraction (Fig. 17 c).

2. Stress-strain experiments. Figure 18 a shows three tension-strain curves obtained from a single compass depressor and two compass depressor sets. Since we calculated that muscle ceils occupy only about 8% of the cross-sectional area of the compass depressor, and in order to permit comparison with other collagenous structures, it was assumed that muscle and mesothelial components make no significant contribution to the passive resistance of the compass depressors to imposed stretch, and tension was calculated as the measured force (g) per unit cross-sectional area o f collagen. The elastic stiffness (i.e. maximum slope of the tension-strain curve) was 1.43 MPa for the single compass depressor and 1.41 MPa and 2.20 MPa respectively for the two compass depressor sets. The resistance of the compass depressors to imposed strain could be increased by treatment with sea-water containing either a high potassium ion concentration or 1 m M acetylcholine. Figure 18b shows the reversible effect of 100 m M K + on the passive tension of a single compass depressor stretched repeatedly to a strain of 0.67. Assuming muscle fibres occupy 8% of the crosssectional area of the compass depressor, if isometric contraction of these fibres had been entirely responsible for this increase in passive tension, they would have had to generate an active tension of 2 MPa. However, since the strongest tension recorded previously in any muscle is about 1.4 MPa (anterior byssus retractor muscle of bivalve molluscs: Watabe and Hartshorne 1990), it is much more likely that the increase in compass depressor passive tension depended at least partly on an intrinsic stiffening of the collagenous component. The effect of 1 m M ACh on the tension-strain curve of a compass depressor set is shown in Fig. 18 c. Before ACh treatment the elastic stiffness was 1.88 MPa; during treatment it was 3.37 MPa. To be entirely responsible for the ACh-induced increase in passive tension observed at strain 0.2, the muscle fibres would have had to generate an active tension of 4 MPa. It is more credible that ACh induced a change in the collagenous component of the compass depressors. The contractile behaviour o f a compass depressor set is compared in Fig. 18 d with that of a protractor muscle set (tested by the same method) from another animal of similar size. From this it can be seen that the maximum tension generated by the compass depressors was about half that of the protractor muscles. However, if tension is calculated as force per unit cross-sectional area of muscle alone, it appears that the maximum protractor tension was 0.08 MPa and the maximum compass depressor muscle tension was 1.27 MPa. Other compass depressor sets generated high tensions (the next highest

148

Figs. 9, 10. Longitudinal and transverse sections of granule-containing cel! bodies showing concentric layers or rough endoplasmic reticulum surrounding aggregations of electron-dense granules and other organelles

Fig. 11. Transverse section including close contacts between axonlike structures (asterisks') and granule-containing cell processes. Note also bundles of extracellular microfibrils (arrows)

149 was 0.81 MPa), but in another nine protractor sets the range was 0.07 0.19 MPa (mean_+SD: 0.15_+ 0.04 MPa), and so it appears that a feature of the compass depressor myocytes may be their capacity to generate particularly high active tensions. Figure 18d also highlights the different passive properties of compass depressors and protractors, the much greater stiffness of the former being attributable to their collagenous component. D. Discussion

mental cells. Since somatostatin or somatostatin immunoreactivity has been found in the central and peripheral nervous systems of vertebrates (Grimmelikhuijzen et al. 1987) and in the insect central nervous system (N/issel 1987), this result provides independent evidence for the neuronal nature of these cells. Our finding of close contacts between juxtaligamental elements and axon-like processes in the compass depressor ligament suggests a possible link with the motor nervous system, although without unequivocal evidence for synaptic junctions such as those occurring between ophiuroid hyponeural axons and juxtaligamental cells (Cobb 1985) this must remain speculative.

L Ultrastructural aspects

The compass depressor ligament consists predominantly of a parallel array of typical collagen fibrils. However, although sparser in distribution, the 12 nm microfibrils are also present in significant numbers, in the form of loose networks and fibre-like aggregates. Microfibrils of identical appearance form a distinct sheet in the peristomial membrane of P. lividus (Candia Carnevali et al. 1990) and similar 10 20 nm filaments have been described in many other echinoderm collagenous tissues (see e.g. Hidaka and Takahashi 1983), sometimes being identified as "elastic" or "elastic-like" fibres (e.g. Bachmann and Goldschmid 1978; Walker 1979). They bear a close resemblance to the fibrillin-containing microfibrils associated with amorphous vertebrate elastin which are about 10 nm in diameter, often appear to be hollow and can show a periodicity of around 50 nm (Keene etal. 1991). Since ultrastructural similarities do not prove biochemical or functional affinity, and since the mechanical role of the vertebrate microfibrils has not been established (Oxlund et al. 1988), it would be premature to assume that the echinoderm microfibrils represent elastic elements. It is notable, however, that Shadwick et al. (1990) found the presumptive elastic tissue of crustacean arteries to be composed only of 25 nm microfibrils. The most striking cellular constituents of the compass depressor ligament are the electron-dense, granule-containing cells and their processes. In their general cytology these resemble the neurosecretory-like cells which have been found in all echinoderm mutable collagenous tissues (MCTs) investigated so far and which have been called "juxtaligamental cells" (Wilkie 1979). However, they show two peculiarities: (1) their fusiform, bipolar shape is unusual, most juxtaligamental perikarya having a single process, and (2) there did not appear to be different populations of cells distinguishable by granule size and/or shape, as have been found in the majority of MCTs (see reviews by Wilkie 1984; Wilkie and Emson 1988). It has been hypothesised that juxtaligamental cells are modified neurons which bring about directly changes in the tensile properties of MCTs, perhaps by controlling the extracellular Ca 2§ ion concentration (Wilkie 1979, 1992). Using immunohistochemical methods, Welsch et al. (1989) have demonstrated the presence of somatostatin (or at least material having the same antigenic determinants as somatostatin) in certain crinoid juxtaliga-

II. Mechanical aspects

Under a low load the compass depressor stretches to a certain length beyond which there is no further extension. Although more work is required to identify the microarchitectural correlates of this creep limit, it seems clear that it is not dependent on strong intermolecular linkages. The tensile strength of the compass depressor ligament was estimated to be about 0.14 MPa; that of mammalian tendon, which, like the compass depressor ligament, consists mainly of a parallel array of collagen fibrils, is around 10 MPa (Wainwright et al. 1976). Assuming that echinoid and mammalian collagen fibrils have roughly the same tensile properties (which appears to be the case from the work of Hidaka and Takahashi 1983), this therefore indicates that in the compass depressor ligament cohesion between these fibrils cannot involve stable covalent bonds, as in mammalian tendon. This was confirmed by the results of the stress-strain tests illustrated in Fig. 18 a, in which the elastic stiffness of the compass depressor was found to be 1.412.20 MPa, well within the range of values published for echinoderm MCTs (see Wilkie 1984), but much lower than the elastic stiffness of mammalian tendon which is around 1 GPa (Wainwright et al. 1976). It was found that exposure of the compass depressors to either a high K § concentration or to acetylcholine resulted in a reversible increase in stiffness that was too great to be due to active tension development by the myoepithelium. This is compatible with the view of Lanzavecchia et al. (1988) that the compass depressor ligament is composed of MCT whose passive stiffness is under direct nervous control. A similar response to K § ions and acetylcholine has been demonstrated in other sea-urchin mutable collagenous structures: the peristomial membrane (Candia Carnevali et al. 1990 and unpublished results) and the ligaments of the spine-test joint (Takahashi 1967; Hidaka and Takahashi 1983; Motokawa 1983). On the basis of our preliminary results we surmise that the compass depressor ligament may be innervated by a cholinergic pathway activation of which, e.g. by exogenous acetylcholine or the depolarising effect of K § ions, leads to an increase in the stability of the linkages responsible for interfibrillar cohesion and so inhibits slippage between the collagen fibrils. The relationship between compass depressor strain

151 Table 1. Compass depressor (CD) length measured in four intact lanterns when compasses were fully depressed and during K-stimulation, compared with length at creep limit under constant load. All lengths in mm

f a

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No.

CD length when compasses fully depressed

MaximumCD length in K-stimulated lantern

CD length at creep limit

1 2 3 4

11 9 11 12

14 13 13 13

14 12 13 13

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Fig. 17 a-e. Tracings of oscillograph recordings illustrating the mechanical behaviour of the compass depressors under constant load. a Under a low load (0.3 g) the compass depressor stretches to a creep limit beyond which there is no further extension, b Unloading (U) of a compass depressor at the creep limit results in both an instantaneous shortening (the full extent of which was not recorded in this case) and slower elastic recovery: the longer the duration of the unloading period, the shorter the !ength at which the extension rate begins to decelerate on subsequent reloading (L). e Addition of sea-water containing 100 mM K + irons (K) causes active contraction. W wash in normal sea-water. In all cases vertical bar = 1 mm and horizontal bar = 1 min

and active tension (Fig. 18 d) conforms to a general pattern observed in the protractors and in most other muscles: as the starting length increases the force of contraction increases, up to a certain length (corresponding to the maximum length taken up by the muscle in the intact body) beyond which (sometimes after a plateau phase) the active tension declines. The compass depressors differed from the protractors in showing considerable strain (and a marked rise in passive tension) before any active tension was detected. For the compass depressors the maximum force of contraction was recorded at a strain of around 20%. In the results shown in Table 1, compass depressor length at the creep limit was on average 25% greater than when the compasses were fully depressed.

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Figs. 12-16. TEMs of the myoepithelium of the compass depressor

Fig. 12. General view showing relationship with the ligamentous component (10. Note my0cytes (my), peritoneocytes (pe) and basal lamina (double arrowheads) Fig. 13. Adluminal side of the myoepithelium with a bundle of varicose axons (arrow) located between the peritoneocytes and myocytes Fig. 14. Basal side of the myoepithelium. Note that a myocyte (arrow) and the process of a peritoneocyte (double arrowhead) both contact the basal lamina by means of hemidesmosomes Fig. 15. Longitudinal section of a myocyte (my) at its insertion into the test. Note the bundles of microfilaments (arrow) diverging from the thickened basal lamina (b/) Fig. 16. Myocyte (my) at its test insertion, with collagen fibrils (co) knitted round tendinous loops (arrow) emanating from the basal lamina

If these results are representative then the myoepithelium may operate most effectively when the compass depressor has been fully stretched to the creep limit.

I~I. Implications for the function of the compass depressors The echinoid compass system is linked physically, and probably functionally, to the coelomic membranes that form the cavities of the peripharyngeal coelom. Whether these coelomic cavities have mainly a respiratory or pressure-compensating function, and the exact contribution of the compass system to this function, have still to be established (compare the views of Nichols 1969 and Smith 1984). It is our opinion that such an elaborate anatomical system cannot be involved simply in regulating pressure within the test and that the conventional view that it is first and foremost a respiratory ventilator is correct. According to this view, elevation and depression of the compasses stirs up fluid in the peripharyngeal coelomic spaces and enhances the delivery to the lantern muscles of oxygen which has perhaps diffused from the perivisceral coelom. However, the compass system has the capacity for more sophisticated behaviour than this, as is described below. We have shown that, although possessing a myoepithelium which is responsible for the contractility that was previously assumed to be their only function, the compass depressors are primarily collagenous ligaments with the capacity to undergo rapid changes in their mechanical properties. Acting as conventional ligaments they may serve to limit aboral elevation of the compasses to a position which avoids overstretching the delicate peripharyngeal coelomic membrane and at which the myoepithelium can exert its maximum active tension. The exact role of the myoepithelium needs t o be investigated. While its contraction n o doubt assists gravity and passive elastic recoil of the ligaments to lower the compasses, it may be involved in subtler activities. We have observed in intact lanterns rotational oscillations of the whole compass system around its vertical axis, with the compasses set at a fixed level above the rotulae. These movements may have been generated by rhythmical contractions of the myoepithelium in reciprocal sets of compass depressors. The functional significance of the variable tensility

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of the compass depressor ligaments is also unclear. It is possible that when the compass system is stationary "stiffening" of the compass depressors locks the compass ossicles in the fully lowered position, and that when the compass system is active the compass depressors switch to a compliant state thus facilitating displacement of the compasses by muscular activity. Andrietti et al. (/990) also suggested that in their stiffened state the compass depressors might help to stabilise the vertical attitude of the lantern when the lantern muscles (protractors and retractors) are inactive. It is obvious that much work is required to clarify both the overall physiological function of the compass system and the contribution of the compass depressors and other components to this function. Acknowledgements. This research received financial support from the Consiglio Nazionale delle Ricerche, Rome, and the Royal Society of London. We are grateful to Dr Elisa Lucca for technical assistance and to Professor F. Andrietti for valuable discussion.

References Andrietti F, Candia Carnevali MD, Wilkie IC, Lanzavecchia G, Melone G, Celentano FC (1990) Mechanical analysis of the sea-urchin lantern: the overall system in Paracentrotus lividus. J Zool Lond 220:345 366 Bachmann S, Goldschmid A (1978) Fine structure of the axial complex of Sphaerechinus granularis (Lain.) (Echinodermata: Echinoidea). Cell Tissue Res 193:10%123

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iour of the compass depressors. a Tension-strain curves obtained from a single compass depressor and two compass depressor sets. Tension was calculated as force per unit cross-sectional area of collagen. The initial length was that at which passive tension just ceased to register, b Effect of seawater containing 100 m M K + ions (K) on the stiffness of a single compass depressor. At the intervals shown, the preparation was subjected to a strain of 0.67 for ca. 1 min then returned to its initial length; values represent passive tension at strain 0.67. e Tension-strain curve of a compass depressor set before, during and after treatment with I mM acetylcholine, d Contractile behaviour of a compass depressor set (CD) compared with that of a protractor muscle set (PR) from an animal of similar size (test diameters 45 mm and 47 mm respectively). The initial length in both cases was that at which the passive tension just ceased to register. A T active tension; PT passive tension

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Candia Carnevali MD, Wilkie IC (1992) Gli straordinari tessuti connettivi degli echinodermi 286 : 58-70 Candia Carnevali MD, Andrietti F, Lanzavecchia G, Melone G, Celentano FC (1988) Aristotle's lantern in the regular sea-urchin Paracentrotus lividus. II. Biochemical approach to the interpretation of movement. In : Burke RD, Mladenov PV, Lambert P, Parsley RL (eds) Echinoderm biology. Balkema, Rotterdam, pp 663 672 Candia Carnevali MD, Bonasoro F, Andrietti F, Melone G, Wilkie IC (1990) Functional morphology of the peristomial membrane of regular sea-urchins: general organization and mechanical properties in Paracentrotus lividus. In: De Ridder C, Dubois P, LaHaye M, Jangoux M (eds) Echinoderm research. Balkema, Rotterdam, pp 207-216 Candia Carnevali MD, Bonasoro F, Melone G (1991) Microstructural and mechanical design in the lantern ossicles of the regular sea-urchin Paracentrotus lividus: a scanning electron microscope study. Boll Zool 58 : 1-42 Chadwick HC (1900) Echinus. Liverpool Marine Biology Committee Memoirs, Number 3 Cobb JLS (1985) The motor innervation of the oral plate ligament in the brittlestar Ophiura ophiura (L.). Cell Tissue Res 242:685688 Diab M, Gilly WF (1984) Mechanical properties and control of non-muscular catch in spine ligaments of the sea urchin, Strongylocentrotusfranciscanus. J Exp Biol 1 ] 1 : 155 170 Grimmelikhuijzen CJP, Graft D, Groeger A, McFarlane ID (1987) Neuropeptides in invertebrates. In: Ali MA (ed) Nervous systems in invertebrates. Plenum Press, New York, pp 105-132 Hidaka M, Takahashi K (1983) Fine structure and mechanicai properties of the catch apparatus of the sea urchin spine, a collagenous connective tissue with muscle-like holding capacity. J Exp Biol 103:1-14 Hyman LH (1955) The Invertebrates, vol IV. Echinodermata. McGraw-Hill, New York

153 Keene DR, Maddox BK, Kuo H, Sakai LY, Glanville RW (1991) Extraction of extendable beaded structures and their identification as fibrillin-containingextracellular matrix microfibrils. J Histochem Cytochem 39:441-449 Lanzavecchia G, Candia Carnevali MD, Melone G, Celentano FC, Andrietti F (1988) Aristotle's lantern in the regular sea urchin Paracentrotus lividus I. Functional morphology and significance of bones, muscles and ligaments. In: Burke RD, Mladenov PV, Lambert P, Parsley RL (eds) Echinoderm biology. Balkema, Rotterdam, pp 649-662 M/irkel K (1979) Structure and growth of the cidaroid socket-joint lantern of Aristotle compared to the hinge-joint lanterns of non-cidaroid regular echinoids (Echinodermata, Echinoidea). Zoomorphology 94:1 32 M/irkel K, R6ser U, Stauber M (1990) The interpyramidal muscle of Aristotle's lantern: its myoepithelial structure and growth (Echinodermata, Echinoida). Zoomorphology 109:251-262 Morales M, Del Castillo J, Smith DS (1989) Acetylcholine sensitivity of the spine-test articular capsule of the sea-urchin Eucidaris tribuloides. Comp Biochem Physiol 94C : 547 554 Motokawa T (1983) Mechanical properties and structure of the spine-joint central ligament of the sea-urchin, Diadema setosurn (Echinodermata, Echinoidea). J Zool Lond 201:223 235 Motokawa T (1984) Connective tissue catch in echinoderms. Biol Rev 59:255-270 Nfissel DR (1987) Neuroactive substances in the insect CNS. in: All MA (ed) Nervous systems in invertebrates. Plenum Press, New York, pp 171-212 Nichols D (1969) Echinoderms. Hutchinson, London Oxlund H, Manschot A, Viidik A (1988) The role of elastin in the mechanical properties of skin. J Biomechanics 21:213 218 Rieger RM, Lombardi J (1987) Ultrastructure of coelomic lining in echinoderm podia: significance for concepts in the evolution of muscle and peritoneal cells. Zoomorphology 107:191 208 Saita A (1969) La morfologia ultrastrutturale dei muscoli della "Lanterna di Aristotele" di alcuni echinoidi. Istituto Lombardo (Rend Sc) B 103:297 313 Shadwick RE, Pollock CM, Stricker SA (1990) Structure and biomechanical properties of crustacean blood vessles. Physiol Zool 63:90-101

Smith AB (1984) Echinoid Palaeobiology. Allen and Unwin, London Stauber M, M/irkel K (1988) Comparative morphology of muscleskeleton attachments in the Echinodermata. Zoomorphology 108:137-148 Takahashi K (1967) The catch apparatus of the sea-urchin spine. II. Responses to stimuli. J Fac Sci Univ Tokyo, Sect 4, 11:121130 Wainwright SA, Biggs WD, Currey JD, Gosline JM (1976) Mechanical design in organisms. Edward Arnold, London Walker CW (1979) Ultrastructure of the somatic portion of the gonads in asteroids, with emphasis on flagellated-collar cells and nutrient transport. J Morphol 162 : 127-162 Watabe S, Hartshorne DJ (1990) Paramyosin and the catch mechanism. Comp Biochem Physiol 96B:639-646 Welsch U, Heinzeller T, Cobb JLS (1989) Histochemical and finestructural observations on the nervous tissue of Antedon bifida and Decametra spec. (Echinodermata:Crinoidea). Biomed Res 10 (Supplement 3): 145 154 Wilkie IC (1979) The juxtaligamental cells of Ophiocomina nigra (Abildgaard) (Echinodermata:Ophiuroidea) and their possible role in mechano-effector function of collagenous tissue. Cell Tissue Res 197:515 530 Wilkie IC (1984) Variable tensility in echinoderm collagenous tissues : a review. Mar Behav Physiol 11 : 1-34 Wilkie IC (1992) Variable tensility of the oral arm plate ligaments of the brittlestar Ophiura ophiura L. (Echinodermata : Ophiuroidea). J Zool Lond (in press) Wilkie IC, Emson RH (1988) Mutable collagenous tissues and their significance for echinoderm palaeontology and phylogeny. In: Paul CRC, Smith AB (eds) Echinoderm phylogeny and evolutionary biology, Clarendon Press, Oxford, pp 311-330 Wilkie IC, Candia Carnevali MD, Bonasoro F (1992) Structure and mechanical behaviour of the compass depressors of Paracentrotus lividus (Lamarck) (Echinodermata:Echinoidea). In: Canicattl C, Scalera-Liaci L (eds) Echinoderm research 1991. Balkema, Rotterdam, pp 99-105

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