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Cinefilms of particle capture by an induced local change of beat of lateral cilia of a bryozoan
J. Exp. Mar. Biol. Ecol., 1982, Vol. 62,
pp. 225-236
225
Elsevier Biomedical Press
CINEFILMS
OF PARTICLE
CAPTURE BY
AN INDUCED LOCAL CHANGE OF BEAT OF LATERAL CILIA OF A BRYOZOAN
RICHARD R. STRATHMANN Zoology Department and Friday Harbor Laboratories, University of Washington, Friday Harbor, WA 98250, U.S.A.
Abstract: A local disruption of the metachronal wave always accompanies capture of algal cells by tentacles of Fhutrellidra hispida (Fabricius). Beat changes for % 0.2 s over k 100 pm of the ciliated band during capture of a 10-w particle. The halted parcel of water is therefore larger than the particle of food but much smaller than the flow that continues past the tentacles elsewhere. These events are consistent with the hypothesis that an induced local reversal of beat concentrates particles for those suspension feeders that retain particles upstream from a band of simple cilia (adults or larvae of bryozoans, brachiopods, phoronids, hemichordates, and echinoderms). These events are not explained by other hypotheses that have been advanced for concentration of particles by these suspension feeders. Aerosol fdtration models of direct interception are not applicable to this type of ciliary suspension feeder because retention depends on the magnitude of a stimulus and response to it. The stimulus will not be the same function of diameter of the food particle, and response is unlikely below a threshold stimulus.
INTRODUCTION
Suspension feeders in the Bryozoa, Phoronida, Brachiopoda, Hemichordata, and Echinodermata retain particles upstream from a band of simple cilia (Strathmann, 1978). Similarities in the size of cilia and in the motion of cilia and particles suggest that the mechanism of retaining particles upstream from the ciliary band is the same in all five phyla. Investigators have differed in their interpretation of the mechanism of capture of particles and the physical processes involved in concentrating particles from suspension. Bullivant (1968) and Gilmour (1978,1979,198 1) suggested an impingement mechanism. I have argued that an impingement mechanism is implausible at such low Reynolds numbers (Strathmann, 1971) and have suggested that food particles induce cilia on a small region of the ciliary band to reverse the effective stroke for a short period. Films of echinoplutei support this hypothesis (Strathmann et al., 1972), but these films were taken in the plane of the effective stroke. The films of echinoplutei, therefore, show the reversed beat of cilia but do not show the extent of the ciliary band affected. The bryozoan Flustrellidra hispida (Fabricius) was selected for this study. Because the zooids are sessile, they were easily oriented with a tentacle in the plane of focus and stationary. Because they live on intertidal algae moved by the waves, the zooids remain extended or soon reextend when particles are added or the light intensity changed. 0022-0981/82/0000-0000/%02.75 0 1982 Elsevier Biomedical Press
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RICHARD
R. STRATHMANN
The films demonstrate that a local change of beat induced by a particle is associated with retention of a particle, as in the echinoplutei filmed previously. The extent of the band affected in the local change of beat is measured for the first time. The films also provide a qu~titative description of other aspects of the capture and transport of particles. Aerosol models of filtration do not include this mechanism of particle capture and cannot predict a relation between size of particle and intensity of capture. Retention depends on the magnitude of a stimulus and response to it, so retention is only indirectly related to the diameter of elements of the filter (cilia) and of particles. Also, there is no evidence that particles adhere to elements of the filter.
METHODS
The camera is a high speed 16-mm cinecamera (Redlake Locam). The films are at w 100 frames/s and 200 frames/s. A timing light places a spot on the margin of the film every 0.01 s, and the spacing of these spots gives more exact intervals between frames for calculations of rates. Kodak 4 x negative film provides a rather grainy picture but requires less light than slower films. The pictures at higher magn&ation are taken with optics for dserential interference contrast and a 10 x objective. Viewing during filming is through a Wild cinetube rather than through the camera. The microscope is equipped with a Cloney cooling stage (Cloney et al., 1970). The animals were usually at 18 to 19 *C during filming, which is above ambient sea-water temperatures and aquarium temperatures at Plymouth, England in February. A faulty cooling module prevented cooling to lower temperatures during filming. Portions of colonies were held in a chamber > 1 mm deep until polypides emerged. Small flagellates of the species C~co~~~ae~~carterue (Braarud and Fagerland) Braarud, Dunaliella tertiolecta Butcher, and Isochrysis galhana Parke were added. by pipet in various mixtures. It is not possible to identify the flagellates from the ftis. Most cells are 5 to 10 pm in diameter. Some cells of C~eo~phaera carterae are as large as 14 pm. Upon addition of algae the polypides usually first withdraw, then emerge and capture many particles, then remain extended and produce currents but capture few particles. The films therefore record greatly varying frequencies of capture of passing particles. Drawings are either from prints (Figs. 2, 3B,C) or from projected images reflected by a mirror at 45” onto a piece of paper (Fig. 3A,D,E,F). RESULTS
Some particles pass a tentacle without noticeable delay and are not captured Other particles pass close to a tentacle, stop, and then move back toward the frontal side of the tentacle (Figs. 1, 2, 3A). After a brief delay a captured particle moves proximally along the frontal side of the tentacle (Fig. 4). During its journey toward the base of a tentacle a particle frequently moves toward the side of the tentacle. Occasionally a
Fig. 1. Capture and transport of a particle on a tentacle of Flusnellidra hispidu: the tentacle is in the plane of the page; the lateral cilia beat at a right angle to the page; numbers at the upper right of each picture are frame numbers from a film with 0.0103 s between frames; the particle is to the left of the short dark line; at 0 and 1 the particle, moving toward the plane of focus, approaches the tentacle, and the metachronal wave of beat of the lateral cilia is clearly visible; the metachronal wave is altered near the particle at 2, and the disruption of beat spreads at 3 and 4, while the particle moves toward the frontal surface of the tentacle; from 4 to 12 the particle is transported proximally along the frontal surface; at 10, 12, and 14 a new disruption ofmetachrony on the other side accompanies movement of the particle away from that side; at 16 and 18 an inward flick of the tentacle carries it out of focus.
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RICHARD R. STRATHMANN
particle is lost into the outgoing current between the tentacles, but usually the motion toward the side of the tentacle is halted, and the particle is returned to the frontal side of the tentacle and then con~ues pro~~y (Figs. 3A, 4). During the initial capture of a particle the beat of the lateral cilia changes over a short length of tentacle near the site of capture. This alteration of beat usually is limited to the side of the tentacle approached by the particle. The zone of disruption of ciliary beat often moves proximally with the transported particle, but a disrupted metachrony does not accompany a particle the entire distance it is transported along a tentacle. Short zones of altered beats appear when a particle veers to the side, and, as in the initial capture, usually only the side toward which the particle is moving is affected. Particles seldom move far before the tentacle tlicks inwards (Figs. 1, 3D,E,F, 4, 5). The tentacle Ilick appears to aid transport toward the mouth (see below). 0 set L
1
: a.
i
Fig. 2. Diagram of particle capture based on first five frames of Fig. 1: the particle is shown as a stippled spot.
In this study the band of lateral cilia is in the plane of focus and the direction of the ef%ctive stroke is appro~~ateIy p~n~~~~to the plane of focus. Though the direction of the effective stroke cannot be directly observed, a disruption of the metachronal wave indicates a change in beat (Figs. 1, 2,4), and a reversal of the effective stroke during the alteration of beat can be inferred from the changed direction of motion of the particle. The alteration of beat coincides with the time and place that a particle arrives and thus appears to be induced by the particle. The alteration of beat is local. The hypothesis that particles are captured and conc~~a~~ by an induced local reversal of beat is supported by these films. Particles often pass without a disruption of beat, and sometimes metachrony is disrupted when no particle is visible. One can reject the null hypothesis that coincidence of particies and alteration of beat is random by a quantitative analysis of a ftim. For one test I followed a continuous sequence of 17.4 s (which does not include the time the tentacle was out of focus during inward flicks). Ten particles passed the tentacle without an alteration of beat and were in view from 0.05 to 0.11 s (mean 0.09 s). The
UPSTREAM CAF’TURE BY CILIARY BANDS
229
passage of five other particles was accompanied by a disruption of metachrony and these were in view for 0.15 to 0.35 s (mean 0.22 s). There is no overlap in the lengths of time that particles are in view in the two cases, so the di&rence is si~~c~t at P < 0.001 (Wilcoxon’s two-sample test). For a second test I selected a long interval between flicks (14.8 s) and compared (1) the total time that particles are in view (1.17 s), (2) the total time metachrony is disrupted (0.97 s), and (3) the total time both occur together (0.50 s). If appearance of particles and disruption of metachrony is independent, the expected co-occurrence as a fraction of the total interval is 0.079 times 0.065, which is 0.0052. The observed fraction of time for co-occurrence is 0.50/14.8, which is 0.034. The observed co-occurrence is about seven times that expected if the
Fig. 3. A, capture and transport of a particle on a tentacle: complete circles show position of the particle every 0,026 s; dashed circles show additional positions; the figure is not a direct frontal view ofthe tentacle, and the particle path is not parallel to the plane of focus; B,C, tentacle flick from same frames as Fig. 5; B, frames 0 to 15; C, frames 15 to 40; D,E,F, capture of a particle and transport by a tentacle flick; circles show position of the particle every 0.026 s; the tentacle positions in E and F correspond to the first three particle pa&ions in the inward flick and the retnrn; the last position in D is repeated as the first in E; the last in E is repeated as the Srst in F; the paths of the particle and the tentacle are not paraBel to the plane of focus; the long scale line is for A, D, E, F; the short scale line for B, C.
RICHARD R. STRATHMANN
230
events were independent. With lower concentration of particles or a more actively feeding animal one would find an even greater diB%rence between expected and observed co-occurrence. (This second test ignores the spatial coincidence of particles and disrupted metac~ony.) Finally, in all fxlmed captures in which both cilia and particles could be viewed in the same frames, a disruption of metachrony accompanied retention of the particle. A quantitative analysis confirms one’s impression from the films: retention of particles is associated with an alteration of beat. Usually > 100 pm of the ciliary band is affected when a particie is retained, though the length of band over which metachrony is disrupted varies. Table I summarizes data
Length of baud with disrupted metachrony during retention of a food particle: intervals are from the last frame with no visible disruption. Time (s) Mean length (pm) Range in lengths n
0.01 60 30-90 9
0.02 80 45-115 4
0.03 or 0.04 105 60-14s 6
from several events well in focus. In these data the interval between frames is 0.0103 s. Some of the variation arises from variation in the time disruption began relative to the times pictures were taken, but in all the sequences analyzed, the greatest spread in disruption occurred in the first 0.01 s. Extension of the area atTected proceeded at a slower rate in subsequent intervals. Spread from 0.03 to 0.04 s was about half that in the initial 0 to 0.01 s. At intervals > 0.04 s the dis~ption sometimes extended to z 150 pm of band but not to greater lengths in connection with a single retention. The total time for metachrony to recover is 0.22 s (range 0.14 to 0.3 1 s, n = 6). The time for a part of the band disrupted within the first 0.01 s to recover metachrony is 0.16 s (range 0.14 to 0.22 s, n = 9). The latter values are lower than the time for complete recovery because the disruption often moves along the tentacle with the particle. Particles are transported proximally along the tentacles at w lmm per s (mean 1.1 mm/s, range 0.75 to 1.55, n = 6). The rate of transport along the tentacle varies because particles are delayed when they wander to the side of the tentacle and are recaptured (Fig. JA). This occurs about every 75 pm (range 55 to 110 pm, n = 6). Captured particles are seldom transported far before the tentacle flicks inward (Figs. 1, 3D,E,F, 4). Figs. 3B,C and 5 show the form of a flick in the plane of focus; no particle is visible in this sequence. In all the flicks analyzed the inward stroke is faster than the return. Amplitude varies greatly with a range of arcs of at least 45’ to 100”. The time to complete a tlick also varies greatly. The inward movement takes 0.035 to 0.100 s (mean 0.055 s, n = 14). The movement takes 3 0.1 to 0.3 s, although very slow movement at the end of the return can extend these times. In two of the filmed flicks the capture of particles is clearly visible, though the flick is not in the plane of focus.
Fig. 4. Capture and transport of a particle on a tentacle ofFhtrellidra hispidawith 0.0103 s between frames and frame numbers at upper right: the orientation of tentacle and beat are as in Fig. 1; the particle (at left of dark line) approaches at 0; metachrony of the lateral cilia is disrupted at 1; disruption spreads at 2; by 12 the particle has moved proximally; transport continues along the tentacle through 24 with disruption of metachrony behind the particle; note that the particle has moved toward the side opposite capture in 12 and 14, and this is followed by disruption of metachrony on that side and return of the particle by 16: at 28 the tentacle flicks inward and out of focus.
232
RICHARD R. STRATHMANN
In one case (Fig. 3D,E,F) the transport proximally along the tentacle as a result of the flick is 90 pm at 1.0 mm/s. In the other case the transport proximally as a result of the flick is 215 pm at 1.4 mm/s. The transport proximally in these Ilicks is about as fast as in transport along the tentacle (see above). The particle is not simply entering a current which will meet a tentacle at a more proximal position. A particle moves inward with the tentacle and away from it on the inward flick, then returns to the tentacle at a more proximal point as the tentacle returns to its original position (Fig. 3D,E,F). The events in a flick are more complex than diagrammed by Strathmann (1973). I earlier suggested that a flick may speed transport toward the mouth, but the two cases analyzed here do not support this hypothesis. The filmed sequences may be biased,
Fig. 5. Tentacle flick parallel to the plane of focus; interval between frames is 0.00515 s; frame numbers are at upper right; the flick is just beginning at 0, is at the innermost position at 15 (0.077 s) and has not yet fully returned at 30 and 40 (0.16 s and 0.21 s).
UPSTREAM
CAPTURE
BY CILIARY
BANDS
233
however, because in the two sequences the captures were not near the tips of the tentacles. More complex sequences of events can occur. Sometimes a particle is first detained on one tentacle and then, during a local alteration of beat on the first tentacle, it is transferred to a neighboring tentacle.
DISCUSSION
The lateral cilia alter beat at the time and place that a particle is detained and moved toward the frontal surface of a tentacle. The direction of motion of captured particles suggests a reversal of the effective stroke of the lateral cilia. The extent of the ciliated band affected (about 100 pm) suggests that water currents are affected in a region much larger than the captured food particle. The films thus support the hypothesis that food particles are captured and retained by a local reversal of beat induced by the particle. A parcel of water much larger than the particle is detained with the particle, but this parcel of water is but a small fraction of the volume flowing past the tentacles elsewhere during the 0.2 s of altered beat. The films support my earlier interpretation of particle capture by lophophorates, echinoderms, and hemichordates (Strathmann, 1973, 1975; Strathmann & Bonar, 1976). Previous films of plutei in the plane of the effective stroke show a reversal of ciliary beat during capture of a particle (Strathmann et al., 1972). The present films in the plane of the ciliated band of bryozoans show the extent of the band affected. The form of the tentacle flick is shown more accurately in these films than in my report in 1973, and the distances which particles move during capture (or during recapture while in transport) are measured more accurately from the films. High speed microcinephotography offers obvious advantages but also imposes some restrictions. Though the chambers holding the bryozoan were several mm thick, the chamber walls could affect current velocities past some of the zooids observed, and intact bryozoan colonies could not be observed. Observations at lower magnification in larger chambers show interactions among zooids and alternate methods of particle capture that are also important for feeding (Winston, 1978). Also, laterofrontal cilia are reported from diverse bryozoans (Winston, 1978). The laterofrontal cilia are usually motionless but could affect particle capture as sensors or filters. In a scanning electron micrograph of a tentacle of Membruniporu sp., the laterofrontal cilia are spaced about as widely as the metachronal wave length (Strathmann, 1973). In some cyphonautes larvae of bryozoa, the laterofrontal cilia can act as a sieve (Strathmann, unpubl. obs.). Their function is not yet demonstrated and I do not know whether laterofrontal cilia play any role in feeding by the zooids of Fiusrrellidru hispidu. The films contradict many of Gihnour’s (1978,1979,1981) conclusions about feeding by lophophorates and hemichordates. Substantial parts of his accounts of ciliary feeding are certainly wrong. There is as yet no evidence that momentum carries particles
‘34
RICHARD
R. STRATHMANN
across flow lines to an extent significant for the feeding of these animals. Several calculations have demonstrated the implausibility of an impingement mechanism for these animals (Strathmann, 1971; Rubenstein & Koehl, 1977). Even neutrally buoyant particles could cross flow lines in a gradient of velocities. Velocity gradients generated by bands of cilia may be steep enough to have such an effect on algal cells. It is not known whether movement across flow lines is important in capture or transport of particles by ciliary feeders. Jorgensen’s (1981) model is not supported by the cited reference (Cox & Hsu, 1977) and is too vague to be tested by measurements of current velocities and sizes of particles captured. The importance of velocity gradients for capture and retention of particles by bryozoa or other ciliary suspension feeders cannot yet be assessed. Rubenstein & Koehl(1977) applied aerosol models of filtration to suspension feeding animals in an attempt to determine which physical processes contribute to the concentration of particles. They distinguish inertial impaction, direct interception, motile particle deposition, and gravitational sedimentation as mechanisms of capture of particles smaller than the pore size of the filter. For a given size of cilium the intensity of capture by direct interception increases as a function of the diameter of the particle. The models predict that echinoderm larvae will capture particles by direct interception. The prediction also applies to those lophophorates and hemichordates which feed by the same mechanism as the echinoderm larvae. The aerosol models of fdtration are for filters which sit passively in a current, however. In upstream retention of particles by ciliary bands of echinoderms, hemichordates, and lophophorates, the filter generates the current by its own motion and also alters its motion in response to particles. Because the cilia alter beat when they capture particles, the aerosol model of filtration is an inadequate basis for predicting either the mechanism of particle capture or the effect of particle size on intensity of capture. Instead the intensity of capture depends on the stimulus which causes the cilia to alter beat. The cilia respond to a great variety of particles, so a mechanical stimulus is likely to be sufficient for capture. If deformation of either a cilium or the cell bearing the cilium is the stimulus, then the intensity of capture might be in proportion to the change in drag produced by the particle. It is not clear how this drag will scale with particle size. The following discussion is speculative and does not indicate the probable complexity of motion as a particle passes the lateral cilia, but it does point out some of the problems to be considered. Though the flow of water around individual cilia in their effective strokes is not known for any animals with upstream retention, one can assume a velocity gradient between cilia in their effective strokes. The larger, compound cilia of veliger larvae of molluscs move up to two times as fast as particles passing within the length of the cilia (Strathmann & Leise, 1979). The veliger’s compound cilia are longer, wider, and probably faster than the simple cilia of F. hispida and other animals with upstream retention, and the velocity gradients next to the veligers’ cilia in their effective strokes may differ from those of F. hispida; but the lateral cilia of F. hkpida are probably moving faster in their effective strokes than the water midway between cilia. Cilia may therefore
UPSTREAMCAPTUREBYCILIARYBANDS
235
overtake or pass particles. First, consider a sphere passing the tentacle midway between two cilia in their effective strokes. The larger the sphere the larger the surface which will be passed by a cilium and the drag on the cilium will increase with the surface which it is passing. In addition, for a larger sphere the surface will be closer to the cilium because the radius is larger and this will also increase the drag. If a near approach is sufficient to trigger a reversal of beat, then probability of a reversal and thus a capture may increase not as the function of diameter in the aerosol filtration model of direct interception but some function of diameter related to drag. This could be quite a different function of size. (If a cilium overtakes a particle directly from behind it will also experience a change in drag before it contacts the particle.) Secondly, if the cilium actually pushes on the particle, the added drag may not be in proportion to the particle’s diameter, as in Stokes’ law, because there is a velocity gradient to which other cilia and the nearby surface of the tentacle contribute, and this gradient changes as the metachronal wave progresses along the tentacle. The change in drag may be a different function of the particle’s diameter in this case also. Thirdly, there could be a threshold, below which the stimulus would be insufficient to trigger a reversal of beat and capture. The change in beat occurs or it does not. It is likely that particles below some size will not trigger a change in beat. Because the magnitude of the stimulus depends on the position of the particle relative to cilia as it passes the band, there may not be an abrupt threshold, but there is likely to be a minimum size sufficient to trigger a reversal at any of the possible paths through the band. This threshold has not yet been demonstrated, and it could be small. Nevertheless, any threshold is a departure from the aerosol filtration model. Fourthly, particles may move across flow lines in the velocity gradient between cilia, with uncertain effects. Copepods and nauplii may respond to individual particles while filtering, so aerosol models of filtration may have limited application to these animals, just as for upstream ciliary suspension feeders. For most suspension feeders the mechanism of capture is unknown and properties of food particles are poorly known. For most suspension feeders, aerosol models of filtration or other models that take account of inertial and viscous forces are effective in rejecting implausible interpretations of feeding mechanisms but less powerful in identifying the mechanism of capture or predicting the intensity of capture of different foods. LaBarbera (1978) makes a similar point about intensity of capture by passive filters: surface charges on particles affect intensity of capture, probably by affecting adhesion.
ACKNOWLEDGEMENTS
The John Simon Guggenheim Foundation and NSF Grant OCE 8008310 supported this study. I filmed the animals at the Marine Biological Association laboratory at Plymouth, England and ‘analyzed the films at the Friday Harbor Laboratories. I was helped and advised by J. C. Green, J. E. Green, J. B. Gilpin-Brown, R. G. Harris, J. E.
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RICHARD R. STRATHMANN
Woollard, and other scientists and staff at Plymouth and Friday Harbor. An anonymous reviewer mad6 helpful comments.
REFERENCES BULLIVANT,J. S., 1968. The method of feeding of lophophorates (Bryozoa, Phoronida, Brachiopoda). N.Z. J. Mar. Freshwater Res., Vol. 2, pp. 135-146. CLONEY, R.A., J. SCHAADT& J.V. DU~DEN, 1970. Thermoelectric cooling stage for the compound microscope. Actu Zooiogka, Vol. 5 1, pp. 95-98. Cox, R.G. % S.K. Hsu, 1977. The lateral migration of solid particles in laminar flow near a plane. Int. .I. Muli$hase Flow, Vol. 3, pp. 201-222. GILMOUR, T.H.J., 1978. Cihation and function of the food-collecting and waste-rejecting organs of lophophorates. Can. J. Zool., Vol. 56, pp. 2142-2155. GILMOUR,T. H. J., 1979. Feeding in pterobranch hemichordates and the evolution of gill slits. Can. J. Zool., Vol. 57, pp. 1136-l 142. GILMOUR,T. H. J., 1981. Food collecting and waste-rejecting mechanisms in Glottidiapyramidata and the persistence of lingulacean inarticulate brachiopods in the fossil record. Can. J. Zool., Vol. 59, pp. 1539-1547. JBRGENSEN,C.B., 1981. A hydromechanical principle for particle retention in Mytilus edulis and other ciliary suspension feeders. Mar. Eiol., Vol. 61, pp. 277-282. LABARBERA,M., 1978. Particle capture by a Pacific brittle star: experimental test of the aerosol suspension feeding model. Science, Vol. 201, pp. 1147-l 149. RUBENSTEIN,D. I. & M. A. R. KOEHL, 1977. The mechanisms of filter feeding: some theoretical considerations. Am. Nat., Vol. 111, pp. 981-994. STRATHMANN,R.R., 1971. The feeding behavior of planktotrophic echinoderm larvae: mechanisms, regulation, and rates of suspension-feeding. J. Exp. Mar. Biol. Ecol., Vol. 6, pp. 109-160. STRATHMANN, R.R., 1973. Function of lateral cilia in suspension feeding of lophophorates (Brachiopoda, Phoronida, Ectoprocta). Mar. Biol., Vol. 23, pp. 129-136. STRATHMANN,R. R., 1975. Larval feeding in echinoderms. Am. Zool., Vol. 15, pp. 717-730. STRATHMANN,R. R. & D. BONAR, 1976. Ciliary feeding of tornaria larvae of Ptychodera fravu (Hemichordata : Enteropneusta). Mnr. Biol., Vol. 34, pp. 317-324. STRATHMANN,R.R., T.L. JAHN & J. R.C. FONSECA, 1972. Suspension feeding by marine invertebrate larvae: clearance of particles from suspension by ciliated bands of a rotifer, pluteus, and trochophore. Biol. Bull. (Woods Hole, Muss.), Vol. 142, pp. 505-519. STRATHMANN, R. R. & E. LEISE, 1979. On feeding mechanisms and clearance rates of molluscan veligers. Biol. Bull. (Woods Hole, Mass.), Vol. 157, pp. 524-535. WINSTON, J.E., 1978. Polypide morphology and feeding behavior in marine ectoprocts. Bull. Mar. Sci., Vol. 28, pp. l-31.
pp. 225-236
225
Elsevier Biomedical Press
CINEFILMS
OF PARTICLE
CAPTURE BY
AN INDUCED LOCAL CHANGE OF BEAT OF LATERAL CILIA OF A BRYOZOAN
RICHARD R. STRATHMANN Zoology Department and Friday Harbor Laboratories, University of Washington, Friday Harbor, WA 98250, U.S.A.
Abstract: A local disruption of the metachronal wave always accompanies capture of algal cells by tentacles of Fhutrellidra hispida (Fabricius). Beat changes for % 0.2 s over k 100 pm of the ciliated band during capture of a 10-w particle. The halted parcel of water is therefore larger than the particle of food but much smaller than the flow that continues past the tentacles elsewhere. These events are consistent with the hypothesis that an induced local reversal of beat concentrates particles for those suspension feeders that retain particles upstream from a band of simple cilia (adults or larvae of bryozoans, brachiopods, phoronids, hemichordates, and echinoderms). These events are not explained by other hypotheses that have been advanced for concentration of particles by these suspension feeders. Aerosol fdtration models of direct interception are not applicable to this type of ciliary suspension feeder because retention depends on the magnitude of a stimulus and response to it. The stimulus will not be the same function of diameter of the food particle, and response is unlikely below a threshold stimulus.
INTRODUCTION
Suspension feeders in the Bryozoa, Phoronida, Brachiopoda, Hemichordata, and Echinodermata retain particles upstream from a band of simple cilia (Strathmann, 1978). Similarities in the size of cilia and in the motion of cilia and particles suggest that the mechanism of retaining particles upstream from the ciliary band is the same in all five phyla. Investigators have differed in their interpretation of the mechanism of capture of particles and the physical processes involved in concentrating particles from suspension. Bullivant (1968) and Gilmour (1978,1979,198 1) suggested an impingement mechanism. I have argued that an impingement mechanism is implausible at such low Reynolds numbers (Strathmann, 1971) and have suggested that food particles induce cilia on a small region of the ciliary band to reverse the effective stroke for a short period. Films of echinoplutei support this hypothesis (Strathmann et al., 1972), but these films were taken in the plane of the effective stroke. The films of echinoplutei, therefore, show the reversed beat of cilia but do not show the extent of the ciliary band affected. The bryozoan Flustrellidra hispida (Fabricius) was selected for this study. Because the zooids are sessile, they were easily oriented with a tentacle in the plane of focus and stationary. Because they live on intertidal algae moved by the waves, the zooids remain extended or soon reextend when particles are added or the light intensity changed. 0022-0981/82/0000-0000/%02.75 0 1982 Elsevier Biomedical Press
726
RICHARD
R. STRATHMANN
The films demonstrate that a local change of beat induced by a particle is associated with retention of a particle, as in the echinoplutei filmed previously. The extent of the band affected in the local change of beat is measured for the first time. The films also provide a qu~titative description of other aspects of the capture and transport of particles. Aerosol models of filtration do not include this mechanism of particle capture and cannot predict a relation between size of particle and intensity of capture. Retention depends on the magnitude of a stimulus and response to it, so retention is only indirectly related to the diameter of elements of the filter (cilia) and of particles. Also, there is no evidence that particles adhere to elements of the filter.
METHODS
The camera is a high speed 16-mm cinecamera (Redlake Locam). The films are at w 100 frames/s and 200 frames/s. A timing light places a spot on the margin of the film every 0.01 s, and the spacing of these spots gives more exact intervals between frames for calculations of rates. Kodak 4 x negative film provides a rather grainy picture but requires less light than slower films. The pictures at higher magn&ation are taken with optics for dserential interference contrast and a 10 x objective. Viewing during filming is through a Wild cinetube rather than through the camera. The microscope is equipped with a Cloney cooling stage (Cloney et al., 1970). The animals were usually at 18 to 19 *C during filming, which is above ambient sea-water temperatures and aquarium temperatures at Plymouth, England in February. A faulty cooling module prevented cooling to lower temperatures during filming. Portions of colonies were held in a chamber > 1 mm deep until polypides emerged. Small flagellates of the species C~co~~~ae~~carterue (Braarud and Fagerland) Braarud, Dunaliella tertiolecta Butcher, and Isochrysis galhana Parke were added. by pipet in various mixtures. It is not possible to identify the flagellates from the ftis. Most cells are 5 to 10 pm in diameter. Some cells of C~eo~phaera carterae are as large as 14 pm. Upon addition of algae the polypides usually first withdraw, then emerge and capture many particles, then remain extended and produce currents but capture few particles. The films therefore record greatly varying frequencies of capture of passing particles. Drawings are either from prints (Figs. 2, 3B,C) or from projected images reflected by a mirror at 45” onto a piece of paper (Fig. 3A,D,E,F). RESULTS
Some particles pass a tentacle without noticeable delay and are not captured Other particles pass close to a tentacle, stop, and then move back toward the frontal side of the tentacle (Figs. 1, 2, 3A). After a brief delay a captured particle moves proximally along the frontal side of the tentacle (Fig. 4). During its journey toward the base of a tentacle a particle frequently moves toward the side of the tentacle. Occasionally a
Fig. 1. Capture and transport of a particle on a tentacle of Flusnellidra hispidu: the tentacle is in the plane of the page; the lateral cilia beat at a right angle to the page; numbers at the upper right of each picture are frame numbers from a film with 0.0103 s between frames; the particle is to the left of the short dark line; at 0 and 1 the particle, moving toward the plane of focus, approaches the tentacle, and the metachronal wave of beat of the lateral cilia is clearly visible; the metachronal wave is altered near the particle at 2, and the disruption of beat spreads at 3 and 4, while the particle moves toward the frontal surface of the tentacle; from 4 to 12 the particle is transported proximally along the frontal surface; at 10, 12, and 14 a new disruption ofmetachrony on the other side accompanies movement of the particle away from that side; at 16 and 18 an inward flick of the tentacle carries it out of focus.
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particle is lost into the outgoing current between the tentacles, but usually the motion toward the side of the tentacle is halted, and the particle is returned to the frontal side of the tentacle and then con~ues pro~~y (Figs. 3A, 4). During the initial capture of a particle the beat of the lateral cilia changes over a short length of tentacle near the site of capture. This alteration of beat usually is limited to the side of the tentacle approached by the particle. The zone of disruption of ciliary beat often moves proximally with the transported particle, but a disrupted metachrony does not accompany a particle the entire distance it is transported along a tentacle. Short zones of altered beats appear when a particle veers to the side, and, as in the initial capture, usually only the side toward which the particle is moving is affected. Particles seldom move far before the tentacle tlicks inwards (Figs. 1, 3D,E,F, 4, 5). The tentacle Ilick appears to aid transport toward the mouth (see below). 0 set L
1
: a.
i
Fig. 2. Diagram of particle capture based on first five frames of Fig. 1: the particle is shown as a stippled spot.
In this study the band of lateral cilia is in the plane of focus and the direction of the ef%ctive stroke is appro~~ateIy p~n~~~~to the plane of focus. Though the direction of the effective stroke cannot be directly observed, a disruption of the metachronal wave indicates a change in beat (Figs. 1, 2,4), and a reversal of the effective stroke during the alteration of beat can be inferred from the changed direction of motion of the particle. The alteration of beat coincides with the time and place that a particle arrives and thus appears to be induced by the particle. The alteration of beat is local. The hypothesis that particles are captured and conc~~a~~ by an induced local reversal of beat is supported by these films. Particles often pass without a disruption of beat, and sometimes metachrony is disrupted when no particle is visible. One can reject the null hypothesis that coincidence of particies and alteration of beat is random by a quantitative analysis of a ftim. For one test I followed a continuous sequence of 17.4 s (which does not include the time the tentacle was out of focus during inward flicks). Ten particles passed the tentacle without an alteration of beat and were in view from 0.05 to 0.11 s (mean 0.09 s). The
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passage of five other particles was accompanied by a disruption of metachrony and these were in view for 0.15 to 0.35 s (mean 0.22 s). There is no overlap in the lengths of time that particles are in view in the two cases, so the di&rence is si~~c~t at P < 0.001 (Wilcoxon’s two-sample test). For a second test I selected a long interval between flicks (14.8 s) and compared (1) the total time that particles are in view (1.17 s), (2) the total time metachrony is disrupted (0.97 s), and (3) the total time both occur together (0.50 s). If appearance of particles and disruption of metachrony is independent, the expected co-occurrence as a fraction of the total interval is 0.079 times 0.065, which is 0.0052. The observed fraction of time for co-occurrence is 0.50/14.8, which is 0.034. The observed co-occurrence is about seven times that expected if the
Fig. 3. A, capture and transport of a particle on a tentacle: complete circles show position of the particle every 0,026 s; dashed circles show additional positions; the figure is not a direct frontal view ofthe tentacle, and the particle path is not parallel to the plane of focus; B,C, tentacle flick from same frames as Fig. 5; B, frames 0 to 15; C, frames 15 to 40; D,E,F, capture of a particle and transport by a tentacle flick; circles show position of the particle every 0.026 s; the tentacle positions in E and F correspond to the first three particle pa&ions in the inward flick and the retnrn; the last position in D is repeated as the first in E; the last in E is repeated as the Srst in F; the paths of the particle and the tentacle are not paraBel to the plane of focus; the long scale line is for A, D, E, F; the short scale line for B, C.
RICHARD R. STRATHMANN
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events were independent. With lower concentration of particles or a more actively feeding animal one would find an even greater diB%rence between expected and observed co-occurrence. (This second test ignores the spatial coincidence of particles and disrupted metac~ony.) Finally, in all fxlmed captures in which both cilia and particles could be viewed in the same frames, a disruption of metachrony accompanied retention of the particle. A quantitative analysis confirms one’s impression from the films: retention of particles is associated with an alteration of beat. Usually > 100 pm of the ciliary band is affected when a particie is retained, though the length of band over which metachrony is disrupted varies. Table I summarizes data
Length of baud with disrupted metachrony during retention of a food particle: intervals are from the last frame with no visible disruption. Time (s) Mean length (pm) Range in lengths n
0.01 60 30-90 9
0.02 80 45-115 4
0.03 or 0.04 105 60-14s 6
from several events well in focus. In these data the interval between frames is 0.0103 s. Some of the variation arises from variation in the time disruption began relative to the times pictures were taken, but in all the sequences analyzed, the greatest spread in disruption occurred in the first 0.01 s. Extension of the area atTected proceeded at a slower rate in subsequent intervals. Spread from 0.03 to 0.04 s was about half that in the initial 0 to 0.01 s. At intervals > 0.04 s the dis~ption sometimes extended to z 150 pm of band but not to greater lengths in connection with a single retention. The total time for metachrony to recover is 0.22 s (range 0.14 to 0.3 1 s, n = 6). The time for a part of the band disrupted within the first 0.01 s to recover metachrony is 0.16 s (range 0.14 to 0.22 s, n = 9). The latter values are lower than the time for complete recovery because the disruption often moves along the tentacle with the particle. Particles are transported proximally along the tentacles at w lmm per s (mean 1.1 mm/s, range 0.75 to 1.55, n = 6). The rate of transport along the tentacle varies because particles are delayed when they wander to the side of the tentacle and are recaptured (Fig. JA). This occurs about every 75 pm (range 55 to 110 pm, n = 6). Captured particles are seldom transported far before the tentacle flicks inward (Figs. 1, 3D,E,F, 4). Figs. 3B,C and 5 show the form of a flick in the plane of focus; no particle is visible in this sequence. In all the flicks analyzed the inward stroke is faster than the return. Amplitude varies greatly with a range of arcs of at least 45’ to 100”. The time to complete a tlick also varies greatly. The inward movement takes 0.035 to 0.100 s (mean 0.055 s, n = 14). The movement takes 3 0.1 to 0.3 s, although very slow movement at the end of the return can extend these times. In two of the filmed flicks the capture of particles is clearly visible, though the flick is not in the plane of focus.
Fig. 4. Capture and transport of a particle on a tentacle ofFhtrellidra hispidawith 0.0103 s between frames and frame numbers at upper right: the orientation of tentacle and beat are as in Fig. 1; the particle (at left of dark line) approaches at 0; metachrony of the lateral cilia is disrupted at 1; disruption spreads at 2; by 12 the particle has moved proximally; transport continues along the tentacle through 24 with disruption of metachrony behind the particle; note that the particle has moved toward the side opposite capture in 12 and 14, and this is followed by disruption of metachrony on that side and return of the particle by 16: at 28 the tentacle flicks inward and out of focus.
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In one case (Fig. 3D,E,F) the transport proximally along the tentacle as a result of the flick is 90 pm at 1.0 mm/s. In the other case the transport proximally as a result of the flick is 215 pm at 1.4 mm/s. The transport proximally in these Ilicks is about as fast as in transport along the tentacle (see above). The particle is not simply entering a current which will meet a tentacle at a more proximal position. A particle moves inward with the tentacle and away from it on the inward flick, then returns to the tentacle at a more proximal point as the tentacle returns to its original position (Fig. 3D,E,F). The events in a flick are more complex than diagrammed by Strathmann (1973). I earlier suggested that a flick may speed transport toward the mouth, but the two cases analyzed here do not support this hypothesis. The filmed sequences may be biased,
Fig. 5. Tentacle flick parallel to the plane of focus; interval between frames is 0.00515 s; frame numbers are at upper right; the flick is just beginning at 0, is at the innermost position at 15 (0.077 s) and has not yet fully returned at 30 and 40 (0.16 s and 0.21 s).
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however, because in the two sequences the captures were not near the tips of the tentacles. More complex sequences of events can occur. Sometimes a particle is first detained on one tentacle and then, during a local alteration of beat on the first tentacle, it is transferred to a neighboring tentacle.
DISCUSSION
The lateral cilia alter beat at the time and place that a particle is detained and moved toward the frontal surface of a tentacle. The direction of motion of captured particles suggests a reversal of the effective stroke of the lateral cilia. The extent of the ciliated band affected (about 100 pm) suggests that water currents are affected in a region much larger than the captured food particle. The films thus support the hypothesis that food particles are captured and retained by a local reversal of beat induced by the particle. A parcel of water much larger than the particle is detained with the particle, but this parcel of water is but a small fraction of the volume flowing past the tentacles elsewhere during the 0.2 s of altered beat. The films support my earlier interpretation of particle capture by lophophorates, echinoderms, and hemichordates (Strathmann, 1973, 1975; Strathmann & Bonar, 1976). Previous films of plutei in the plane of the effective stroke show a reversal of ciliary beat during capture of a particle (Strathmann et al., 1972). The present films in the plane of the ciliated band of bryozoans show the extent of the band affected. The form of the tentacle flick is shown more accurately in these films than in my report in 1973, and the distances which particles move during capture (or during recapture while in transport) are measured more accurately from the films. High speed microcinephotography offers obvious advantages but also imposes some restrictions. Though the chambers holding the bryozoan were several mm thick, the chamber walls could affect current velocities past some of the zooids observed, and intact bryozoan colonies could not be observed. Observations at lower magnification in larger chambers show interactions among zooids and alternate methods of particle capture that are also important for feeding (Winston, 1978). Also, laterofrontal cilia are reported from diverse bryozoans (Winston, 1978). The laterofrontal cilia are usually motionless but could affect particle capture as sensors or filters. In a scanning electron micrograph of a tentacle of Membruniporu sp., the laterofrontal cilia are spaced about as widely as the metachronal wave length (Strathmann, 1973). In some cyphonautes larvae of bryozoa, the laterofrontal cilia can act as a sieve (Strathmann, unpubl. obs.). Their function is not yet demonstrated and I do not know whether laterofrontal cilia play any role in feeding by the zooids of Fiusrrellidru hispidu. The films contradict many of Gihnour’s (1978,1979,1981) conclusions about feeding by lophophorates and hemichordates. Substantial parts of his accounts of ciliary feeding are certainly wrong. There is as yet no evidence that momentum carries particles
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across flow lines to an extent significant for the feeding of these animals. Several calculations have demonstrated the implausibility of an impingement mechanism for these animals (Strathmann, 1971; Rubenstein & Koehl, 1977). Even neutrally buoyant particles could cross flow lines in a gradient of velocities. Velocity gradients generated by bands of cilia may be steep enough to have such an effect on algal cells. It is not known whether movement across flow lines is important in capture or transport of particles by ciliary feeders. Jorgensen’s (1981) model is not supported by the cited reference (Cox & Hsu, 1977) and is too vague to be tested by measurements of current velocities and sizes of particles captured. The importance of velocity gradients for capture and retention of particles by bryozoa or other ciliary suspension feeders cannot yet be assessed. Rubenstein & Koehl(1977) applied aerosol models of filtration to suspension feeding animals in an attempt to determine which physical processes contribute to the concentration of particles. They distinguish inertial impaction, direct interception, motile particle deposition, and gravitational sedimentation as mechanisms of capture of particles smaller than the pore size of the filter. For a given size of cilium the intensity of capture by direct interception increases as a function of the diameter of the particle. The models predict that echinoderm larvae will capture particles by direct interception. The prediction also applies to those lophophorates and hemichordates which feed by the same mechanism as the echinoderm larvae. The aerosol models of fdtration are for filters which sit passively in a current, however. In upstream retention of particles by ciliary bands of echinoderms, hemichordates, and lophophorates, the filter generates the current by its own motion and also alters its motion in response to particles. Because the cilia alter beat when they capture particles, the aerosol model of filtration is an inadequate basis for predicting either the mechanism of particle capture or the effect of particle size on intensity of capture. Instead the intensity of capture depends on the stimulus which causes the cilia to alter beat. The cilia respond to a great variety of particles, so a mechanical stimulus is likely to be sufficient for capture. If deformation of either a cilium or the cell bearing the cilium is the stimulus, then the intensity of capture might be in proportion to the change in drag produced by the particle. It is not clear how this drag will scale with particle size. The following discussion is speculative and does not indicate the probable complexity of motion as a particle passes the lateral cilia, but it does point out some of the problems to be considered. Though the flow of water around individual cilia in their effective strokes is not known for any animals with upstream retention, one can assume a velocity gradient between cilia in their effective strokes. The larger, compound cilia of veliger larvae of molluscs move up to two times as fast as particles passing within the length of the cilia (Strathmann & Leise, 1979). The veliger’s compound cilia are longer, wider, and probably faster than the simple cilia of F. hispida and other animals with upstream retention, and the velocity gradients next to the veligers’ cilia in their effective strokes may differ from those of F. hispida; but the lateral cilia of F. hkpida are probably moving faster in their effective strokes than the water midway between cilia. Cilia may therefore
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overtake or pass particles. First, consider a sphere passing the tentacle midway between two cilia in their effective strokes. The larger the sphere the larger the surface which will be passed by a cilium and the drag on the cilium will increase with the surface which it is passing. In addition, for a larger sphere the surface will be closer to the cilium because the radius is larger and this will also increase the drag. If a near approach is sufficient to trigger a reversal of beat, then probability of a reversal and thus a capture may increase not as the function of diameter in the aerosol filtration model of direct interception but some function of diameter related to drag. This could be quite a different function of size. (If a cilium overtakes a particle directly from behind it will also experience a change in drag before it contacts the particle.) Secondly, if the cilium actually pushes on the particle, the added drag may not be in proportion to the particle’s diameter, as in Stokes’ law, because there is a velocity gradient to which other cilia and the nearby surface of the tentacle contribute, and this gradient changes as the metachronal wave progresses along the tentacle. The change in drag may be a different function of the particle’s diameter in this case also. Thirdly, there could be a threshold, below which the stimulus would be insufficient to trigger a reversal of beat and capture. The change in beat occurs or it does not. It is likely that particles below some size will not trigger a change in beat. Because the magnitude of the stimulus depends on the position of the particle relative to cilia as it passes the band, there may not be an abrupt threshold, but there is likely to be a minimum size sufficient to trigger a reversal at any of the possible paths through the band. This threshold has not yet been demonstrated, and it could be small. Nevertheless, any threshold is a departure from the aerosol filtration model. Fourthly, particles may move across flow lines in the velocity gradient between cilia, with uncertain effects. Copepods and nauplii may respond to individual particles while filtering, so aerosol models of filtration may have limited application to these animals, just as for upstream ciliary suspension feeders. For most suspension feeders the mechanism of capture is unknown and properties of food particles are poorly known. For most suspension feeders, aerosol models of filtration or other models that take account of inertial and viscous forces are effective in rejecting implausible interpretations of feeding mechanisms but less powerful in identifying the mechanism of capture or predicting the intensity of capture of different foods. LaBarbera (1978) makes a similar point about intensity of capture by passive filters: surface charges on particles affect intensity of capture, probably by affecting adhesion.
ACKNOWLEDGEMENTS
The John Simon Guggenheim Foundation and NSF Grant OCE 8008310 supported this study. I filmed the animals at the Marine Biological Association laboratory at Plymouth, England and ‘analyzed the films at the Friday Harbor Laboratories. I was helped and advised by J. C. Green, J. E. Green, J. B. Gilpin-Brown, R. G. Harris, J. E.
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Woollard, and other scientists and staff at Plymouth and Friday Harbor. An anonymous reviewer mad6 helpful comments.
REFERENCES BULLIVANT,J. S., 1968. The method of feeding of lophophorates (Bryozoa, Phoronida, Brachiopoda). N.Z. J. Mar. Freshwater Res., Vol. 2, pp. 135-146. CLONEY, R.A., J. SCHAADT& J.V. DU~DEN, 1970. Thermoelectric cooling stage for the compound microscope. Actu Zooiogka, Vol. 5 1, pp. 95-98. Cox, R.G. % S.K. Hsu, 1977. The lateral migration of solid particles in laminar flow near a plane. Int. .I. Muli$hase Flow, Vol. 3, pp. 201-222. GILMOUR, T.H.J., 1978. Cihation and function of the food-collecting and waste-rejecting organs of lophophorates. Can. J. Zool., Vol. 56, pp. 2142-2155. GILMOUR,T. H. J., 1979. Feeding in pterobranch hemichordates and the evolution of gill slits. Can. J. Zool., Vol. 57, pp. 1136-l 142. GILMOUR,T. H. J., 1981. Food collecting and waste-rejecting mechanisms in Glottidiapyramidata and the persistence of lingulacean inarticulate brachiopods in the fossil record. Can. J. Zool., Vol. 59, pp. 1539-1547. JBRGENSEN,C.B., 1981. A hydromechanical principle for particle retention in Mytilus edulis and other ciliary suspension feeders. Mar. Eiol., Vol. 61, pp. 277-282. LABARBERA,M., 1978. Particle capture by a Pacific brittle star: experimental test of the aerosol suspension feeding model. Science, Vol. 201, pp. 1147-l 149. RUBENSTEIN,D. I. & M. A. R. KOEHL, 1977. The mechanisms of filter feeding: some theoretical considerations. Am. Nat., Vol. 111, pp. 981-994. STRATHMANN,R.R., 1971. The feeding behavior of planktotrophic echinoderm larvae: mechanisms, regulation, and rates of suspension-feeding. J. Exp. Mar. Biol. Ecol., Vol. 6, pp. 109-160. STRATHMANN, R.R., 1973. Function of lateral cilia in suspension feeding of lophophorates (Brachiopoda, Phoronida, Ectoprocta). Mar. Biol., Vol. 23, pp. 129-136. STRATHMANN,R. R., 1975. Larval feeding in echinoderms. Am. Zool., Vol. 15, pp. 717-730. STRATHMANN,R. R. & D. BONAR, 1976. Ciliary feeding of tornaria larvae of Ptychodera fravu (Hemichordata : Enteropneusta). Mnr. Biol., Vol. 34, pp. 317-324. STRATHMANN,R.R., T.L. JAHN & J. R.C. FONSECA, 1972. Suspension feeding by marine invertebrate larvae: clearance of particles from suspension by ciliated bands of a rotifer, pluteus, and trochophore. Biol. Bull. (Woods Hole, Muss.), Vol. 142, pp. 505-519. STRATHMANN, R. R. & E. LEISE, 1979. On feeding mechanisms and clearance rates of molluscan veligers. Biol. Bull. (Woods Hole, Mass.), Vol. 157, pp. 524-535. WINSTON, J.E., 1978. Polypide morphology and feeding behavior in marine ectoprocts. Bull. Mar. Sci., Vol. 28, pp. l-31.