Effects of Ca2+ and catecholamines on swimming cilia of the trochophore larva of the polychaete Spirobranchus giganteus (Pallas)

J. Exp. Mar. Bial. Ecol., 1986, Vol. 95, pp. 245-255

245

Elsevier

JEM 628

EFFECTS OF CA’+ AND CATECHOLAMINES ON SWIMMING CILIA OF THE TROCHOPHORE LARVA OF THE POLYCHAETE SPZROBRANCHUS GZGANTEUS (Pallas)

JOAN R. MARSDEN

and HAROUT HASSESSIAN

McGill University, Montreal, Quebec, Canada, and the Bellairs Research Institute, Barbados

(Received 28 May 1985; revision received 19 August 1985; accepted 15 November 1985) Abstratt: Trochophore larvae of the tropical serpulid S~~ob~a~chus giganteus (Pallas) swim by means of prototrochal and metatrochal rings ofcilia. A system of developing neurites carrying vesicles of several kinds is located on the inner surfaces of both prototrochal and metatrochal cells. The swimming cilia arrest on exposure to EDTA, Ba(OH),, lanthanum chloride, trifluoperazine and Ca*+-free sea water, i.e. under conditions that interfere with the supply of external Ca ‘+. Swimming cilia are also arrested by the bblocker alprenolol, an effect ameliorated by the a, agonist phenylephrine or the @agonist isoproterenol. We conclude that there is a Ca’+-dependent, catecholaminer~c excitation of the swimming cilia of the S. giganteus trochophore larva, involving fi receptors and probably neurally mediated. Other cilia on the larval body are insensitive to the agents affecting the activity of swimming cilia. Key words: calcium; catecholamines; swimming cilia; trochophore; polychaete; Spirobranchus giganteus

The planktotrophic trochophore larva of serpulid polychaetes such as Spirobranchus giganteus (Pallas) swims by the coordinated beating of prototrochal and metatrochal cilia. The larva bears an apical organ sensitive to mechanical and chemical stimulation, as well as a single eyespot, and summing direction has been shown to be influenced by visible light (Young & Chia, 1982; Marsden, 1984). In the larva of S. gigunteus there is a system of developing neurites comprising a small apical nemopile, a nerve beneath the prototroch and a suboral complex which includes a metatrochal nerve and others associated with mouth and pharynx. The morphology of these neurites, as reported in this study, supports the view that there is nervous transmission to cells bearing swimm~g cilia. Details of a similar system have been described for the trochophore of S. polycerus (La&i, 1984). The pharmacology of the larval nervous system is unknown, Studies on three-setiger larvae of species without a free trochophore stage (Marsden & Lacalli, 1978; Bell, 1980; Bhup & Marsden, 1982) indicate a cholinergic excitation of muscarinic receptors on segmental muscle and a serotonergic stimulation of mucus secretion but provide no evidence, morphological or pharmacological, for nervous control of swimming cilia. This paper reports on the effects of experimental exposure of trochophore larvae of 0022-0981/86/$03.50 0 1986 Elsevier Science Publishers B.V. (Biomedical Division)

JOAN R. MARSDEN

246

AND HAROUT

HASSESSIAN

S. giganteus to agents known to influence catecholamine transmission and Ca’ + levels. The impact of these drugs has been estimated in terms of their effect on swimming and examined in the context of the control of the action of trochal cilia.

MATERIALS

AND

METHODS

Observations on living larvae were conducted at the Bellairs Research Institute, Barbados. Larval cultures were made from spontaneous spawnings of adults collected on the nearby reef and removed from their tubes in the laboratory. Culture dishes were kept in the water table at 25 *C on a 12 : 12 light : dark regime. All larvae used in this study were 24 to 48-h trochophores in which posterior elongation and segment formation had not yet begun. MORPHOLOGY

Larvae were prepared for electron microscopy by an initial fixation for 1 h in 1 ml 70% glut~~dehyde mixed with 28 ml of 0.2 M cacodylate buffer, 26 ml distilled water and 1.3 g of NaCI. They were then rinsed twice in 0.2 M cacodylate buffer, post fixed for 1 h in 1 ml of 4% 0~0, mixed with 3 ml of 2.5 % sodium bicarbonate and dehydrated in ethyl alcohol. Embedding in Epon was preceded by a three-stage impregnation: 1 h in 50 :50 100% ethy1 alcohol and Epon, 1 h in 100% Epon, and overnight in fresh Epon. Microscopy was carried out in the Biology Department, McGill University. Silver-gold sections were cut with a diamond knife and stained with hot uranyl acetate and lead citrate. Philips 200 and 410 electron microscopes were used. EXPOSURE Larval

TO DRUGS behaviour

was

observed

in

drugs

at

concentrations

ranging

from

10-3-10-6 M (l-0.001 mM), a series similar to those found to be effective with both polychaete (Marsden & Lacalli, 1978) and echinoderm (Burke, 1983a) larvae. If swimming was altered at a concentration of 5 x 10F4 M or less the effect was then explored in a more finely graded series. All effective levels of concentration were then tested twice. Experiments were carried out on l-ml samples of fluid taken from near the surface of cultures 24-48 h old, thus avoiding abnormal larvae which tend to remain near the bottom. The number of larvae/ml varied from ~20 to 100. A 1O-2 M stock solution of each drug in sea water was mixed with sea water and culture fluid to produce the desired final concentrations. When two agents were used they were mixed before the addition of culture fluid. Observations, for periods of 1 min to 2 h were carried out on glass spot plates viewed under a dissecting microscope. Larvae were then sieved through plankton netting and washed into fresh sea water where they were watched for recovery. Drugs used were noradrenaline, isoproterenol, L-phenylephrine, adrenaline, dopam-

CaZ+ ,

CATECHOLAMINES AND TROCHOPHORESWIMMING

247

ine, octopamine, and the antagonists alprenolol and yohimbine. &OHDA, lanthanum chloride, trifluoperazine (TFP), EDTA and Ba(OH), were also tested. All drugs were from Sigma Chemical Co. Ca ’ + -free sea water was made up according to Horstadius (1973). RESULTS MORPHOLOGY The chief components of the nervous system of the trochophore of S. gigunteus are a sub-apical neuropile, nerves at the base of the prototroch and mctatroch and others associated with pharyngeal and oral muscles. Prototrochal and metatrochal neurites contain small numbers of dense, oval granules (maximum size 60 x 90 nm) as well as clusters of small (34-40 nm in diameter), semi-lucent vesicles (Figs. 1,2,4, and 5), and occasional dense cored vesicles (Fig. 2). The prototroch consists of three to four tiers of heavily ciliated cells with the nerve lying on the medial surface of the second tier (Figs. 2 and 4). The number of neurites making up the nerve varies, being greatest in the ventral region where small vesicles frequently occur in synaptic-like clusters closely associated with the adjacent trochal cell membrane (Fig. 3). The metatrochal nerve is smaller and consists of two neurites in a groove at the base of a two-tiered ring of ciliated cells (Fig. 1). APPLICATIONOF DRUGS Solutions at 10 - 3 M and 10 - 4 M are well outside the range of concentration used in ne~o~~smitter studies in which the reactive membrane comes into direct contact with the applied solution. In studies on whole larvae, however, it seems reasonable to assume that active agents are taken up through the cuticle and reach the relevant internal receptors at a concentration considerably lower than that in the external medium. Indirect evidence to this effect can be seen in Burke’s (1983a) study on the induction of metamorphosis by dopamine in whole larvae and excised larval arms of Dendraster excentrikus. The effect of added chemical agents was assessed in terms of changes in swimming

ability. Very little normal variation in swimming behaviour was encountered, and in most cases a given group of larvae could be readily assessed as swimming normally, abnormally, or not at all. Cessation of swimming is a consequence of the arrest of prototrochal cilia in a horizontal or apically directed position and metatrochal cilia in a posteriorly directed position (Fig. 6). During arrest individual cilia (particularly those of the prototroch) flicker occasionally but there is no effective locomotion. Larvae with arrested cilia sink, anal side up. The small cilia in the food groove and those in the gut continued to beat when the swimmin g cilia were arrested. Indeed, the gut cilia were so persistently active that they were used as a criterion for judging the larva to be alive. Adrenaline, noradrenaline, dopamine, octopamine, yohimbine and isoproterenol at

JOAN

Fig , 1. Cr~~sc~tion

R. MARSDEN

ofmetatroch,

35ooO x

AND

HAROUT

: metatrochal

HASSESSIhN

nerve fasmwi); cm, circular muscle; m. metal

chal cell.

Fig. 2. Cross-section Fig :.3. Synaptic-like

of protatroch,

clusters

35000 x

ofvesicleson

: protntrochalnwve (arrows): p, prototrochal

the ventral side of the body (arrows): nerve; b, in the mttatrochal nerve.

~11,

a, in the pmtotroc

Cal’,

CATECHOLAMINES

AND TROCHOPHORE

SWIMMING

Fig. 4. Cross-section

of metatrochal

nerve, 60480 x

: m, metatrochal

Fig. 5. Cross-section

of prototrochal

nerve, 20 160 x

: n, neurite; p, prototrochal

Fig. 8. Swollen membranes

of metatroch~

cilia (arrows) 6450x.

after a 4-h exposure

249

cell; n, neurite. cell.

to 1O-6 M 6-OHDA,

250

JOAN R. MARSDEN AND HAROUT HASSESSIAN

concentrations of 5 x 10m4 M to lo- ’ M evoked no change in the swimming behaviour of the larvae. Alprenolol, a P-blocker, had a strongly inhibitory effect on swimming at concentrations of 10 - 3 M, 5 x 10 - 4 M and 10 - 4 M (Fig. 7). At lower concentrations, 6-8 x 10e5 M, swimming continued for 50-60 min. At 5 x 10e5 M larvae behaved as they do in fresh sea water, swimming normally for over 2 h. Effects in the 10e4 M to 8 x lo- 5 M range were reversible in fresh sea water. The effect of alprenolol could be modified by simultaneous addition of the /? agonist isoproterenol or the /3 agonist phenylephrine (Table I). Trifluoperazine (TFP), at all concentrations used (10 - 3 to 10 - 6 M), evoked an initial, transitory slowing of both prototrochal and metatrochal cilia. Recovery from this

Fig. 6. Diagram oftrochophore larva with arrested swimming cilia: a, apical tuft; an, anus; av, anal vesicle; mc, metatrochal cilia; o, oesophagus; pc, prototrochal cilia; st, stomach.

CONCENTRATION

(mM)

Fig. 7. Swimming responses to alprenolol and trifluoperazine.

Ca’+ , CATECHOLAMINES

AND TROCHOPHORE

SWIMMING

2’1

effect occurred in all but the highest concen~ations in 1-2 min. Trochophores in 10eW3M and 10e4 M TFP stopped swimming in 2-10 min; those in 1O-5-1O-6 M continued to swim as long as did controls in normal sea water. In the intermediate 10 -4-10- ’ M range swimming lasted from 30 min to 2 h (Fig. 7). TABLE I

Effect of alprenolol and catecholamine agonists on swimming time. Agent(s)

Concentration

Time to cessation of swimming (min)

Alprenolol Aiprenoloi + isoproterenof Alprenolol + I-phenyiephrine

7 x 10m4M Both at 7 x IOm4M Both at 7 x 10e4 M

4.5 (* 2.5) 21 (*I) 22.5 ( + 0.5)

A three-degree polynomial regression was calculated for both the alprenolol and TFP data (time to cessation of swimming against concentration of drug). The fit to the expected curve was good with significance levels of 0.000008 and 0.00036, respectively. In lop6 M 6-hydroxydop~ne (6-OHDA) larvae swam for as long as one h but swimming was slow and erratic. Prototrochal cilia remained active and normal in appearance but those on the metatroch became ragged in outline and largely immobile. In lo- 5 M 6-OHDA swimming was inte~ttent and very erratic. After 5 min most larvae had stopped and the cilia of both metatroch and prototroch were ragged, shortened or entirely lost, although the cilia of the food groove, the gut and the apical tuft remained unaffected. In 10 - 4 M 6-OHDA larvae stopped swimming immediately; prototrochal and metatrochal cilia were held stiffly at first, but within 1 or 2 min they became ragged and then dissolved. Other cilia on the body remained normal. Ultrastructural examination of larvae exposed to 10 -6 M 6-OHDA for 4 h revealed some swelling and membrane damage in the trochal cilia (Fig. 8). In Ca2 + -free sea water trochophores became either instantly immobile or adopted a jerky, circling mode of locomotion. Immobilized larvae settled on the bottom and within 10 min began to move again, swimming in an irregular, spasmodic fashion. Normal swimming was not resumed when affected larvae were transferred to fresh sea water. Control animals in complete artificial sea water swam normally. Ba(OH), at 10 4 M and 10 - ’ M caused an immediate arrest of swimming cilia and a sustained muscular con~a~tion. When placed in fresh sea water larvae did not recover. EDTA at 10m6 M had no effect on larval locomotion. At 10e5 M swimming was jerky although sustained for up to 2 h and in a 10m4 M solution larvae stopped swimming within 10 min. Lanthanum chloride had a similar effect on swimming and, in addition, appeared to stimulate mucus secretion. In a lo- 4 M solution larvae stopped swimming in 5 to 10 mm; in a lo- 5 M solution they swam for up to 20 min by which time most larvae were stuck to the bottom of the dish by a mucus-like material. In a

252

JOAN R. MARSDEN

AND HAROUT

HASSESSIAN

10 - 6 M solution secretion was obvious in 10 min although larvae appeared to continue to struggle to swim for up to 30 min.

DISCUSSION

The swimming cilia of the trochophore of Spirobrunchus gikunteus can be arrested by exposure to TFP, Ba(OH),, Ca 2+-free sea water, EDTA and l~~~urn chloride. EDTA is a Ca’+ chelating agent, lanthanum chloride an inhibitor of Ca2 + flux and TFP an agent which, in micromolar concentrations, will reduce the availability of Ca2 + , by interrupting processes independent of (Sand et al., 1983) or mediated by (Levin & Weiss, 1976; Landry et al., 198 1; Otter et af., 1984) calmodulin. TFP is known to effect an inhibition of a Ba2 + -induced, Ca’ + -dependent ciliary reversal in Paramecium (Rauh et al., 1980). A similar inhibition of a Ca2 + -dependent reversal of the lateral cilia on the mussel gill has been interpreted as a binding of TFP (at concen~ations in the 20-45 FM range) to a Ca2+ -cahnodulin complex that forms when Ca2+ is present at levels of 10’. 6-10 - 5 M, rendering the complex unavailable to sensors controlling the active sliding of ciliary microtubule proteins (Reed et al., 1982). A non-specific inhibition of gill PDE can be effected by TFP but requires concentrations in the 100 PM range. TFP also acts as an anticholinergic agent, possibly by inhibiting vesicular exocytosis (Clapham & Neher, 1984) or otherwise disturbing the linkage between receptor stirnulation and Ca2 + channel activation (Wada et al., 1983). In addition it is known to act, at nanomolar levels, as a neurileptic, blocking dopaminergic transmission in the mammalian nervous system (Seeman, 1980). It is possible, therefore, that the TFPinduced ciliary arrest in Spirobrunchus giganteus is related to the interruption of a dopamine-generated excitation. However, since the dop~ine-blocking effect requires only very low concentrations, our observed decline in effectiveness of TFP in a 10 -4 M as compared with a 10 - s M solution is more consistent with a role in control of Ca2 + av~ability. In Paramecium Ba*+ is believed to reverse ciliary beating by depolarization of the ciliary membrane following entry via Ca2 + channels (Ling 8c Kung, 1980; Forte et al., 1981). The results of exposure of Spirobranchus gzganteus larvae to Ba(OH), and TFP may, therefore, be the consequence, respectively, of an entry of Ba2 + through Ca2 + channels and a binding of TFP to the Ca2 + -calrnodulin complex in the cilium, both events leading to a depletion of available internal Ca2+. It must be emphasized, however, that the internal concentration of Ca 2 + in the trochal cell is unknown in this study on whole larvae whereas information on ciliated protozoa and mussel gill comes from work with both intact cells and demernbranated preparations. It remains possible that the Ca” effect observed with larvae is a membrane-related event but the cellpenetrating quality of TFP, its effectiveness on larvae in micromolar concentrations and the understanding that cahnodulin is internal and not associated with the ciliary membrane (Stephens, 1983; Stommel, 1984) argue against this interpretation. The

Ca’+ , CATECHOLAMINES

AND TROCHOPHORE

SWIMMING

153

slowing and cessation of swimming on exposure to EDTA, lanthanum chloride and Ca2 +-free sea water support the view that action by the trochal cilia is dependent on Ca2+ uptake from the external medium. The beating of the trochal cilia on the trochophore of S. giganteus may, therefore, depend on an inflow of Ca’ + , an event associated with membrane excitation, Ciliary activity, in most examples studied, takes place when the cell membrane is in resting condition. Excitation of the membrane may result in reversal of the direction of the power stroke as in ~uru~eciu~ (N~toh & Kaneko, 1973) and ~ikopleura (Gait & Mackie, 1971), in ciliary arrest as in the mussel gill (Walter & Satir, 1978), Core&a (Mackie et al., 1974) and gastropod veliger larvae (Mackie et al., 1976), or in an alteration of the frequency of the beat as in the gill of Mytilus (Saimi et al., 1983). On the other hand, cilia of mammalian trachea and cultured oviduct cells (Lee et al., 1976), the abfrontal cilia on the ~ytiiu~ gill (Stommel, 1984) and the lateral compound cilia of the protozoan Sty~onich~a(de Peyer & Machemer, 1982) are quiescent when the membrane is in a resting state and require Ca2+ for activation. In the case of the mammalian cells and the abfrontal cilia, ciliary arrest can be obtained with micromolar levels of TFP (Verdugo et al., 1983; Stommel, 1984). Our observations suggest that the trochai cilia of S~jru~ranchu~gigante~ may operate in a similar way. The response of S. g~gante~ larvae to alprenolol, isoproterenol and phenylephrine is consistent with a catecholaminergic excitation of the trochal cilia. The consequence of application of 6-OHDA is, however, less clear. 6-OHDA, a selective agent for the destruction of adrenergic neurons, is believed to act in a manner related to its rapid autoxidation and the consequent production of neurotoxic substances such as quinones and superoxide radicals and/or to the inhibition of tyosine hydroxylase by lack of oxygen (Javoy, 1975). The swimming cilia of trochophore larvae appear to be more sensitive than other parts of the body to deteriorating environmental conditions; in covered well slides they have been observed to suffer fragmentation or to be cast off in ribbons. It may be, therefore, that they are particularly reactive to oxygen deprivation and respond to the oxygen-scaven~ng property of 6-OHDA by ciliary histolysis. The absence in the larva of cells packed with dense cored vesicles eliminates the possibility of demonstrating a 6-OHDA anti-adrenergic effect at the morphological level but does not preclude the possibility of a catecholaminergic excitation of swimming cilia. Dense cored vesicles are present in small numbers in neurites in the trochal region along with clusters of semi-lucent vesicles, a situation similar to that described for the velar nerve of gastropod larvae (Mackie et al., 1976). It is likely, therefore, that the trochal neurites of S. giganteus carry more than one transmitter and that one of these is a catecholamine, a possibility supported by studies indicating that more than one transmitter may be secreted by one neuron (Chan-Palay et al., 1982) or indeed by one granule (Pelletier et al., 1981). The aminergic transmitter most frequently suggested as involved in ciliary control is serotonin (Aiello, 1974; Jorgensen, 1975; Mackie et al., 1976), although there is some less positive evidence for a role for dopamine in the control of cilia on the lamellibranch gill (Paparo & Aiello, 1970; Paparo & Finch, 1972). Catecholamines have been found

254

JOAN R. MARSDEN AND HAROUT

HASSESSIAN

in both echinoderm (Burke, 1983a,b) and polychaete (Marsden & Lacalli, 1978) larvae but there is no evidence relating them to the control of ciliary activity. The trochophore of S. gigunteus carries other cilia: in the gut, the food groove and the apical tuft, that do not respond in the same way as the trochal cilia, indicating physiological differences between the control of cilia serving purposes such as feeding and perception and those responsible for swimming. Analogous differences have been demonstrated for two sets of compound cilia in Stylcmichia (Dietmer, 1984), the paired flagella of C~lu~y~u~#na~ (Kamiya & Whitm~, 1984) and the abfrontal, lateral and laterofrontal cilia on the Mytilus gill (Stommel, 1984). In the latter case possible relationships between Ca*+ sensitivity and levels or kinds of calmodulin are suggested.

ACKNOWLEDGEMENTS

This research was supported by a National Science and Engineering Research Council Operating Grant to Joan Marsden. The authors are grateful to R. Lamarche, Dr. M. Neuwirth, and Dr. W. Hunte for facilities at the Bellairs Research Institute.

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