The Role of Latero-Frontal Cirri in Particle Capture by the Gills of Mytilus edulis

9 Pages·1999·4.1 MB·English

by  H Silverman

Reference: Biol. Bull. 197: 368-376. (December 1999)
The Role of Latero-Frontal Cirri in Particle Capture
by the Gills of Mytilus edulis
HAROLD SILVERMAN1 JOHN W. LYNN, PETER G. BENINGER*. AND THOMAS H. DIETZ
,
Department ofBiological Sciences, Louisiana State University, Baton Rouge, Louisiana 70803; and
* Departement de Biohgie, Faculte des Sciences, Universite de Nantes, 2 rue de la Houssiniere,
44322 Nantes Cedex, France
Abstract. In this study we examined the mechanism of capture-transport-selection-ingestion. However, the actual
particle capture in Mytilus edulis, using radioactive-label mechanism(s) of particle capture remains difficult to deter-
clearance studies, progressive fixation, and scanning elec- mine.
tron microscopy to visualize in detail the cirri and their A number of components are important in understanding
range of motion. Confocal laser scanning microscopy was the mechanism of particle capture. Fluid mechanics has
used to observe the interaction of cirri with 1 /xm fluores- been used to describe both the delivery of particles to the
cent latex particles on live strips of control and serotonin- gill filaments and their movement on the gill (J0rgensen,
treated isolated gill tissue. The gills of M. edulis possess 1983; Nielsen etai, 1993; Ward etai. 1998a; Riisgard and
large, complex latero-frontal cirri composed of 18-26 pairs Larsen, 2000). The potential interaction of particles with a
of cilia. Particles that were intercepted by the cirri were structure on the gill would be an equally important compo-
transferred to the watercurrent on the frontal surface ofthe nent to understanding the mechanisms leading to particle
filament where they were propelled toward the ventral par- capture events. Generally, suspension feeding bivalves can
bedmrtilxieocoplmuvceooimksvngeearegdtdodoa4ttvn.hoEee9ds.1cfm0bhCrleeolrcn3egitaaMac~mrlh'aeisnsaeducfrrreiocyfxtoaesoltcdtinieuisidnfsnoi,rufeeaosttmhmfhdeliee5enlmxfa~Coeit'ndel.sraspomte-oWerafsnahrwtitoe.taenitntdCoeanlrlttehhtacbaehiratarattrgMinhilp.cislanestrgetosdwiptuamepulleredil-ed-ys cRctRolaioeipsss1teg0u,iar2nre1dpt9,uph8nae8r,1)t9r.iw7aci8nlT;teghhseTeomeovonmfeseratWcihafsnaehkwnwoieiwdlstieomnasrtgneadunnessfgDfeeaiodvcfoiifted/onsxst,miczcae1lp(s9etM8au0f2rrhr;aeloneSmcpneparbr1toeu0ifr"nc'gglpeasraa/tunniradd-tn
ies demonstrated that removal ofE. coli from the seawater the extremes of the size range may differ, because the
bathing medium was reduced 90% to 0.5 ml g~' dry tissue behavior in fluid would change depending on particle den-
min"1 when 10~3 M serotonin was present. These data sity and geometry.
demonstrated that for small particles (< 2 ;um) in the near To observe the actual particle capture event, one needs to
field, movement ofcirri was essential forsuccessful capture be able to resolve both the particle and the cellular or-
either by direct contact or with water acting as a hydrome- ganelles that are involved with the capture. To date, Riis-
chanical coupler. gard and his colleagues have used isolated preparations and
constructed half filament models to analyze particle move-
Introduction ments closetothe filaments ofMvtilusedulis (Nielsen etai,
1993; Riisgardetai, 1996). Theirobservations indicate that
Considerable progress in understanding particle process- particles can be directed to the frontal surface of gill fila-
ing mechanisms in suspension-feeding bivalves has been ments by direct interception of particles by latero-frontal
made in recent years (Beninger et ai, 1993, 1997; J0r- cirri. They haveextended these observations by determining
gensen, 1990; Ward et ai, 1998b). Investigations have the path as particles approach the gill filaments, in vivo,
focused on various components of the feeding sequence: usinga side-mounted microscopic preparation (Riisgard and
Larsen, 2000).
Received 3 June 1999; accepted 5 October 1999. Silverman etai. ( 1996, 2000) and Beningeretai. (1997)
1 Towhom correspondence should be addressed. E-mail: [email protected] have examined gill strips using confocal laser scanning
368
CIRRI AND PARTICLE CAPTURE 369
microscopy (CLSM). The disadvantages of this system are were maintained in the laboratory for several months in
thatthe observationsaremadeongill strips that are isolated: a4rt-if6icCial seavvater (Chambers and de Armendi. 1979) at
normal fluid flow is altered; and normal neural orhormonal and fed algae weekly until used.
cues are modified or missing. However, the advantages ot
this system include magnification, resolution, tunable depth Confocal laser scanning microscopy
offocus, andpreselection ofagill strip where all cilia are in In larger specimens (> 30 mm anterior-posterior shell
motion as confirmed by observationbefore adding particles. length), the posterior extremity of the gill was used for
The near-fieldeventsofparticle interactionwithcirri are not CLSM preparations, because the demibranchs are thinnest
altered by isolation procedures. The only bivalve investi- and shortest in this region, while still presenting the typical
gated to date using this technique is Dreissenapolymorpha, gill architecture. This preparation facilitatedthe observation
a species with a homorhabdic eulamellibranch gill type of4-5 mm wide strips of gill extending from the gill arch
(Silverman et <//., 1996). In this freshwater bivalve, the to the ventral particle groove. Strips from the posterior
latero-frontal cirri direct particles from the water onto the extremity of each demibranch were removed using micro-
frontal surface ofthe gill filaments, where the particles were surgical instruments, and placed in isosmotic (1060 mosm)
transportedtothe ventral particle grooveby the frontal cilia. seawateron a microscope slide lined with Nitex screen (125
M\tilus ednlis. a marine bivalve species often used for a /urn mesh). This screen allowed cilia-generated water cur-
variety of particle capture studies, has a homorhabdic rili- rents to flow on both sides ofthe demibranch. and mechan-
bparratnicchlegigllrotoypvee. I(tAtaklisnosh.as19la3t7e:ro-Ofwreonnt,al c1i9r7ri4:anOdwaevnentarnadl cicoavlerdsilsitpurabbaonvceetohfetphreepgairllatwioanswaivtohidsieldicboynesvuapcpourutminlgubtrhie-
McCrae, 1976). The organization and structure of latero- cant posts at each corner. For larger animals with thicker
f(rMoonotrale.cir1ri97i1n:MO.weednu,lis19h7a4v:eObweeennexatnednsiMvceClryaede,scr1i9b76e;d giimlelsn,sw<e a3l0sommob.setrhveegdilsliwngalse siusfofliactieedntfliylasmmeanltls.anFdorthsipnect-o
Jones et at.. 1990). The cirri are large, complex structures allow any region to be used. We observed the gills of more
composed of two ciliary plates: each plate consists of be- than 40 specimens (range of shell length = 18-81 mm)
tween 18 and 26 fused cilia that are hinged at the base
during this study.
(Owen, 1974). The tips of the cilia are free and oriented at The preparations were observed using the CLSM tech-
an angle, suggesting a "paddle" or "filtering" structure nique previously described (Silverman et al.. 1996). The
(Riisgard et al, 1996). Capture and retention studies have were examined with a Noran Instruments confocal
shown that M. edulis can intercept particles down to 1 ^m gsiylslstem attached to a Nikon Optiphot microscope with a
with retention efficiencies of approximately 50% (M0hlen- 40X fluor lens (NA 1.3). The laser wavelength was set at
berg and Riisgard, 1978). Using a video microscope to 529 nm with an FITC barrier filter in the return image path.
observe single isolated gill filaments, Riisgard etal., ( 1996) Reflected channel images were acquired (laser scan and
showedthatthe latero-frontalcirri ofM. edulis can intercept image digitization < 1 /us/frame) and sequentially stored at
relatively large (7 ju.m) particles. However, the exact se- 120 frames s~' (~ 8 ms between frames). Images were
quence ofparticle-cirrus interaction was not readily resolv- captured and analyzed using the Odyssey InterVision (No-
able. ran) software on a Silicon Graphics Indy computer. Images
In the present study we examined small particle capture were not altered except to adjust the contrast.
by live gill strips using confocal microscopy and high-speed Fluorescent beads of 0.7 ju,m or 1.0 jnm diameter were
video recording. This approach enabled us to document in added to the preparations to track particle motion in cilia-
detail particle-cirrus interactions in the marine bivalve. aenerated water currents and particle transport on the cili-
Mytilus edulis. We reported some of the preliminary data ated epithelia. Gill preparations were oriented such that
recently in a brief commentary (Silverman et al., 2000). beads could be added under the coverslip in the dorsal
resjion of the strip and subsequently moved from dorsal to
Material and Methods ventral. Particle velocities were determined after observing
Animals the movement of individual fluorescent beads over known
distances and times.
Observations were made on Mytilus edulis individuals
collected at low tide in Chamcook Harbour (Passama- Scanning electron microscopy
quoddy Bay, Bay of Fundy, Canada) and on animals pur-
chased from the Marine Biological Laboratory (Woods Toexamine the spatial relationships ofcirri and cilia, gill
Hole. Massachusetts). The specimens were flown on moist tissue was prepared for scanning electron microscopy using
isicaenpaacSktsatteoUtnhieveLrisfietyS.ciSepnecceismeMniscrforsocmopCyanFaadcialiwteyraetsLtoourie-d acilpiraogatreaslslivsetagfeisxatoifonthetierchbneiaqtuecycthlaets.alSlmoawleldmtuhsesealrsreswteroef
for 1-2 days at 4C in seawater-soaked towels prior to placed in containers with enough seawater (~ 20-30 ml) to
dissection and observation. Specimens from Woods Hole cover the shells. They were left undisturbed until their
370 SILVERMAN ET AL.
siphons were clearly visible. Drops of 2% OsO4 (1-2 mil
were slowly added near the edge of the container; the
specimen was left intact for 20 min. The posterior adductor
muscle was then cut and the animal exposed to fixative for
another 40 min. In a few cases, the adductor muscle was
carefully cut before OsO4, was added, with total exposure to
osmium in the bath being 30 min. The mussels were then
rinsed in seawater and fixed for an hour in 2% glutaralde-
hyde in seawater. After fixation, the gill was removed,
dehydrated in a graded ethanol series, critical-point dried,
and sputter-coated with gold-palladium. Specimens were
mounted on stubs and visualized with a Cambridge Scan-
ning Electron Microscope. Some gills were removed and
photographed before and after fixation, and again after
critical-point drying to monitor tissue shrinkage.
Bacterial clearance
Clearance of "S-labeled Excherichia coli from the bath-
ing medium was quantified with an adaptation of the etlnFliigsutrreea1t.ed Stcoaanlnlionwgefilxeacttiroonnomficcriorrgiradpuhrionfggitlhlefibleaamtenctysclfer.oCmirAr/Vi///fIlIe.xV
mmeatnhoetdaolf,Ri1i9s9g5a,rd19(9179)8.8)Muasssedelsscr(i<bed30prmevmioushsellyl(lSeinlgvtehr)- dfuilraimnegnt.thCeiirrripaorweeirntshteroekxetemnodevdinpgosiotvieorn(tahrerofwrhoentaadl)isnurtfhaeceint(efr)fiolfametnhte
were placed in individual test tubes containing 20 ml aer- space,and intheflexedposition(c)withtheirfreetipsbentoverthefrontal
ated artificial seawater (5C) with or without 10~3 M sero- surface. Adjacent cirri on a filament were stopped at different stages ofa
tonin for 15 min. Radiolabeled bacteria were added to each heat cycle (arrow). The inset ofa higher magnification indicates that the
treeciliarytipsassociatedwiththetwociliaryplatesofacirrusangleaway
tube after the mussels opened their siphons, and after 1 min fromthebodyofthecirrustogivea 'V likeappearance(*). Bar = 25 ;u,m;
equilibration, four samples (100 /Ltl) were collected at 10- inset bar = ? /uni-
min intervals. The reduction ofbathing medium radioactiv-
ity followed first order kinetics, and the clearance was
expressed as ml g~' dry tissue min"'. Dataareexpressed as rate ofcapture ofeach image (< 1 /x.s) minimized blurring
the mean one standard error with the number ofanimals due to the movement ofthe cirrus (Fig. 2E). The cirral beat
in parentheses. averaged 7.9 0.4 Hz (n = 6).
Results Cirral interaction with particles
Cimil structure Particles interacted with moving cirri in several different
ways but usually were (i) swept onto the frontal surface of
Progressive fixation stopped the cirri and cilia in various the filaments and entrained into the frontal water current
stages of their beat cycle. The cirri position varied from (Fig. 3). Fluorescent particles (1 /urn) entering the interfila-
being arched over the frontal surface of a gill filament to ment space were captured within the 2.2-2.8 /urn wide "V"
extending into the interfilument space (Fig. 1). Adjacent of the free ciliary tips of the cirrus and moved toward the
cirri on a filament were arrested during fixation in a stag- frontal surface as the cirrus beat from the extended into the
gered pattern, such that neighboring cirri were not in the flexed position. In most cases, particles interacting with a
same position relative to the frontal surface (Fig. 1). The cirrus were moved directly into the frontal current and
CLSM time series micrographs (Figs. 3-6) are of filaments transported at 307 36 /am s l, n = 4 (Figs. 3, 4) toward
in the same orientation as in Figure 1. Confocal images of the ventral particle groove.
a cirrusbeatcycle indicated that during the powerstroke the Three additional types ofparticle interaction with a beat-
cirrus bent over the frontal surface into a flexed position ingcirrus were observed, (ii) During flexion ofthecirrus the
(Fig. 2D). On recovery it returned to an extended position particle may be deposited at the edge of the frontal surface
perpendicular to the apical surface of the latero-frontal without being immediately transported. Subsequent cirral
cirrus cell (Fig. 2A, I). The body of the cirrus beat as a movement positioned the particle into the frontal surface
single unit; a complete beat required the cirral body to pivot current (Fig. 4). (iii) Some particles remained between the
at its base in addition to moving through the ciliary wave- cirral plates and moved with the cirrus for multiple beat
form (Fig. 2). The cirrus moved out of the plane of focus cycles (Fig. 5). These particles were finally incorporated
during the flexion, but as the free ciliary tips were moving into the frontal flow after several cirral beat cycles. The
out ofview they were clearly resolvable (Fig. 2E). The high particles that moved with the cirral extension away from the
CIRRI AND PARTICLE CAPTURE 371
Figure 2. A series ofconfocal video images (120 frames s ') ofa single cirrus from an in vitro gill strip
ofM\tilus etlulix. The time between each frame shown in this series is 25 ms. The focal plane ofthe video is
slightly offthe perpendicularto the dorso-ventral axisofthe filament. The parallel bright lines flank the lateral
surface ofthe filament. Thus, the beat ofthe cirral tip is toward the frontal surface ofthe filament that lies to
the right and into the background, at a right angle to the visible lateral surface ofthe filament. The resolution
ofindividual ciliary tips (arrowhead) on one side ofthe cirrus (one cirral plate) is clear. The bending (power
stroke) in A-D moves theplane oftheciliary tips toward the frontal surface ofthe filament. Images in frames
E-Irepresenttherecoverystroke.Thetotaltimetakenforthisbeatcyclewas 200ms(beatfrequency ~ 5Hz).
The arrow identifies the midpoint ofthe cirrus. Bar = 4 /j,m.
frontal surface suggest that there was intimate contact, ei- used to construct Figures 3-6 are archived in the electronic
ther directly or within a few tenths of a /xm. from a cirrus databaseofTheBiologicalBulletin atwww.mbl.edu/litml/BB/
(Fig. 5). (iv) Finally, on rare occasions the particle did not VIDEO/BB.video.html.
reappear, indicating it became dislodged from the cirrus in
the interfilament space (not shown). Serotonin effects on cirral position and clearance of
Particle entrainment in the frontal surface current ofthe gill Escherichia coli
wasobservedbothin association with mucusrafts(Beningeret
at., 1993, 1997) and in the absence ofany visible mucus rafts Progressive fixation (as described above) revealed that
(Figs. 3, 4). Indeed, we have observed filaments where two the cirri in the serotonin-treated animals were arrested in the
particles were being transported, but at different velocities. flexed position, occluding most ofthe frontal surfaces ofthe
One moving particle can overtake another particle on the gill filament (Fig. 7). The amount offrontal surface covered
frontal surface(Fig.6). Sucheventsindicate independenttrans- by the cirri is somewhat exaggerated in this figure because
port ofthe two particles along the frontal surface, and would the fixation caused 44 3% (;; = 4) shrinkage. From our
suggestthattheywere notmoving inasingle mucusraft,ornot CLSM observations in some sectionsofthe gill, both frontMal
at the same height above the epithelium. Quick-time videos and lateral ciliacontinued to beat in the presence of 10
372 SILVERMAN ET AL
To assess particle capture in the absence ofmovement by
latero-frontal cirri, cirral motion was arrested by adding
M
10 serotonin to the seawater bathing medium (Jor-
gensen, 1983; Ward el ai. 1998a). Clearance (ml g ' dry
tissue min ') of 35S-labeled E. coti by M. edulis in 5C
seawater was 4.92 0.43 (n = 3) for controls (130 12
=
mg dry tissue) compared to 0.51 0.18 (n 3) for treated
animals (142 26 mg dry tissue). Clearance of bacteria
from control mussels displayed first order kinetics with 21
nun required to remove 5Q7c of the label (Fig. 8). The
serotonin-treated mussels also displayed first order kinetics,
but experienced an 89.6% reduction in the rate of removal
of bacteria from the seawater.
Discussion
Figure3. A confocal microscopy time series or the frontal surface of
an in vitn> gill filament from Myiilus ednlis. Dorsal is at the top ofeach The movement ofan individual cirrus on a living strip of
Tsiuhmcaecgecesirsriaivnebdefavrteanimtneraailnsd3t3oouwmtasro.dfCtithrhereapllbatointpetsooamfr.ethTveihseiibmlategimeasea'nedVlatpshsteerud'cVtb'useretaswree(eatnrhreoewtasic)ph.s cMyotnifloictasli'ltalusleirs gsiclalncnainngbemiocbrsoesrcvoepdyat(ChiLgShM)r.esoTlhuetiobneautsicnyg-
ofthecirriasthey flexoverthefrontal surfaceoneachsideofthefilament. cle is similar to that described for the cirri of Drcissena
One cirrus directed a I-/LUTI fluorescent latex particle onto the edge ofthe IHilyniorplui (Silverman et ai, 1996). In the flexed position
filamentfromtheright interhlamentspace(A:brightspotattopright). The the tips ofthe cirri are located overthe frontal surface ofthe
pdaorwtinclethewafsronetnatlrasiunrefdacein(tBh)e. Tfrhoentaplartwiactleerwcausrrternatnsapnodrtemdovonedthveenftrroanltlayl gill filament, whereas in the extended position they project
surface ofthe filament (C and D) moving 20 ju.nl in 100 ms by frame D. into the interfilament space at the level ofthe latero-frontal
Bar = 10 |um. cells from which they originate.
Confocal microscopy permits resolution of individual
cirri and. depending on reflectance and light scattering,
serotonin. This was also evident in some areas of gill that includes their individual ciliary tips. The lateral (x-y plane)
were fixed forscanning microscopy. This motion is inferred resolution (about 0.2 /xrn) of CLSM, the enhanced rate of
in the wave-like pattern formed by the tips of lateral cilia sequential image acquisition (~ 8 ms/frame), and high
(Fig. 7). Although the fixation was not instantaneous, the speed of capture of individual images (< I /xs) enabled
positions of the lateral cilia tips are suggestive of a syn- observation ofthe interaction of l-/xm particles with latero-
chronized or metachronal motion (Fig. 7). frontal cirri. Although particles were seen to be directed
Figure4. A cnnlocul microscopy time seriesofan in run*MyiiltiscJnli.\ gill filament. A l-/j,m fluorescent
particle (brightspot underthe letterA) interacted with acirrus and was moved toward, but notonto,the frontal
surface ofthe filament (A-C). On the nextcontact with acirrus, the particle was moved intothefrontal surface
watercurrent and transported rapidly (D-F). The reference line marks the initial location ofthe particle in all
ol the images. The elapsed time ol the series was 125 ms. Bar = 10 /urn
CIRRI AND PARTICLE CAPTURE 373
Figure5. Aconfocalmicroscopy timeseriesofaninvitroMytiluseiliilisgilltilumenl.Afluorescentparticle
movedrapidly down thecenterofthe filament (A-F) in 125 nis. A secondparticle wasassociatedwithacirrus
(A; upperleft) whoseciliary tipswere partiallyoutoftheplaneoffocus. Asthecirruscontinuedto move hack
toward the extended position in the left interfilament space, the particle was drawn back with the cirrus (B-C)
until it moved out ofthe plane offocus in D. It reappeared in E-Fas thecirrus moved into its flexed position
=
over the frontal surface ofthe filament. Bar 10 fim.
onto the frontal surface during the power stroke ofa cirrus, surface of the filaments even when no mucus is visible or
we could not determine whether the interaction was direct onto visible mucus rafts that are observed to be moving
or ifwater acted as a mechanical couplerbetween the cirrus along the frontal surface (Beninger el at, 1997). In both
and particle (Riisgard ct til.. 1996). However, the close cases, transport of panicles along the frontal surface aver-
interaction (herein defined as within a few tenths of /urn) of aged about 307 ^im & l, and was similar to previously
the beating cirrus with the particle is responsible for the reported rates (Jones et at. 1990; Ward et at, 1991. 1993;
movement of many particles to the frontal surface of the Nielsen et at. 1993).
filament where particles subsequently move in the frontal
water flow. Under the experimental conditions described
here for M. edulis. the particles can be moved to the frontal
Figure 7. A scanning electron micrograph of two filaments from a
Mytiluseilnlis gill treated with 10 'Mserotonin.Thespacebetweenthese
fixedfilaments\\aslargerthanthatobservedinFigure I (20/im.versusthe
15 jj.m). Visible in the enlarged interfilament space are the tips ot the
underlying lateral cilia (Ic). whoseorigins fromtwoadjacent filaments are
Figure 6. A confocal microscopy time series (25 ms between each easily discernible. The arrows indicate areas where the lateral cilia were
image)ofan HI vitroMytilusediilis gill filament showing thattransportof fixed in the \arious phases ofbeat associated with the metachronal wave
particlesalong a filament can occurat differentrates. In these imagestwo form unset, lower magnification). The cirri (c) in these preparations are
particlesmovedalongthefrontalsurfaceofthefilament.Thelargerparticle stopped over the frontal surface of the filament, occluding much of the
(arrowhead) moved at 750 jum s~'. while the velocity of the smaller frontal surface. Each cirrus is composed of pairs (arrowheads) of cirral
particle (arrow) was 350 /j.m s '. Bar = 10 jim. plates containing IS-26 cilia. Bar = 10 /im. inset bar = 20 /j.m.
374 SILVERMAN ET AL
12.5 capture process, it does not provide the information under
the normal water flow conditions expected in vivo. Ward
12.2- and his colleagues have providedendoscopic videos (1998a,
b) that permit observation ofdye streams and the motion of
E 12.0- relatively large particles (7 jam) as they move within the
EQ. pallial cavity toward the gill of a living M. edulis. Ward et
73 11.8- ul. ( 1998a) indicate that particles approach the gill at a low
angle (less than 30) and are either captured by the filament
1 1.5- or bounce until they are subsequently captured. Observing
these endoscopic videos, it appears that there is a non-
1 1.2 random location of the particles interacting at or near the
10 20 30 edge of the filament. These near-field interactions probably
time (min) represent interception by the cirri, rather than particle cap-
Figure8. Time-dependent removal ofEscherichiacolifrom5C sea- ture by the frontal water flow (Ward et ul.. 1998a). The
water by Mytilin ctlnlis controls (open square) or treated with 10"' M endoscopic observations are valuable to describe flows and
serotonin (filled square). Each point represents the mean SEM for 3 gross movements ofparticle approach to the filaments. The
eaaqnnuidamtatirleosant(efdoS:rEtYMhe=scmoan-ltl0re.orl00tm3huasXnse+tlhse1w2sa.ys3m:7b6Y.olrs=a=re00..n09o03t83.vXis+ibl1e2)..3T65h.erre=gr0e.s9s9i4o.n tvhaerioabbsleervraetsioolnustitohnataanrde nreelcaetsisvaerlyy tlooiwdemnatigfnyifpirceactiisoenmelcimhi-t
anism!s) of interaction between filament organelles and
individual particles. Thus, it is not possible to determine the
The role of these ciliary organelles in particle capture distances separating particles from the structures on the gill
associated with suspension feeding has been controversial. filament. Despite the particular limitations ofboth the con-
Early workers including Atkins (1937) and Oral (1967) focal and the endoscopic observational approaches, the data
suggested that cirri were important as "mechanical" filters obtained from each appear to be complementary and sug-
or traps that moved particles onto the frontal surface ofthe gest the importance of both cirral interaction (near-field)
gill filament for subsequent transport. Support for this view and water flow (far-field) forparticle capture in this species.
is gained from the differential particle capture in bivalve These data also are complementary to those obtained by
species with cirri of different sizes (Owen and McCrae, Riisgard and his colleagues (1996, 2000) demonstrating
1976; M0hlenberg and Riisgard, 1978; J0rgensen et ai, cirral interaction with particles in various isolated prepara-
1984). McHenery and Birkbeck ( 1985) suggested that Esch- tions and also in young undisturbed mussels.
erichia coli were captured more effectively by marine bi- Although most reports indicate that isolated M. edulis
M
valves with larger cirri than by those with small or no gills must be stimulated by 10 7 serotonin to maintain
latero-frontal cirri. A similarrelationship betweencirral size ciliary activities, we have found that sections of isolated
and particlecapture has been reported for several freshwater gills commonly have beating cilia. We selected preparations
bivalves (Silverman ft cil.. 1995. 1997; Tankersley. 1996). where latero-frontal cirri, and frontal and lateral cilia were
The observations of particle/cirral interactions described all beating. The beat frequency of the cirri (8 Hz) and the
here extend the observations of Riisgard et a/.. (1996). patterns of motion were similar to the 4-9 Hz observed by
These earlier observations were based on traditional light Oral (1967) in young mussels in vivo. He noted that intact
microscopic lenses immersed in water (theoretical maxi- M. edulis displayed a diverse repertoire of ciliary activity,
mum resolution approximately 0.5 jam) to observe particle including spontaneous arrest. While Oral (1967) highlights
interaction with cirri in isolated M. cdMulis gills. Their iso- abeatcycle that places adjacent cirri offsetby Vi beat cycle,
lated gill preparation contained 10 7 serotonin to stim- he points out that there are many other relationships be-
ulate ciliary activity and were positioned to maintain the tween adjacent cirri that would not be detected with the
normal interfilament gap (40 jiim) at the level of the lateral methods used. The data presented in this study also are
ciliated cells. The greater resolution of CLSM provides consistent with the recent in vivo observations of Riisgard
more structural detail, and the high image capture speed (< and Larsen (2000) and with the modeling done by Riisgard
1 jas) and recording speed (120 frames s~') allow better and colleagues based on isolated filament preparations
visualization of particle interaction with the cirri. The con- (Nielsen et ul.. 1993: Riisgard et a!., 1996). Indeed, the
focal images indicate that the cirrus was responsible for the observations made here are consistent with the calculations
movement of 1 jam particles onto the frontal surface ofthe of Silvester and Sleigh (1984) who suggest that latero-
gill filament. The similarity ofthe results between these two frontal cirri can act as "sieves." although perhaps more
studies, where different methods were used, supports the water moves around ratherthan through the cirrus (Riisgard
accuracy of information gained from both studies. et ul.. 1996).
While observation of cirral/particle interactions with the The finding of an 83% reduction in particle capture in
CLSM provides evidence for (he role of cirri in the particle Mvtilus edulis after serotonin is used to block cirral move-
CIRRI AND PARTICLE CAPTURE 375
merit (Ward et til., 1998a) also is consistent with the previ- contact with the plates. The amount of water that might be
ous results of Jorgensen (1983), who showed that cirral moving with the cirri is small according to the model of
movement was critical to particle capture. The reduction in Riisgard etul. ( 1996). In addition, their model indicates that
particle capture efficiency afterserotonin administration has particles can make contact with the cirral plate. The mech-
been ascribed to a breakdown in cirral-generated water anismforparticlecapture isclose interaction withthecirrus,
currents (Ward et ai, 1998a). However, the data of the and at low Reynolds numbers, hydromechanical and me-
present study provide an alternate explanation. The obser- chanical are essentially the same, and consistent with the
vations of the present study confirm that the cirri oftreated confocal observations made in this study.
specimens are arrested in the flexed position (Jorgensen, Both the eulamellibranch. Dreissenapolymorpha and the
1975, 1983). eliminating direct cirri particle interaction filibranchMytilusedulis specieshave homorhabdic gills and
while the frontal and lateral cilia continue to beat. Further- complex cirri, and capture small particles in the near-field
more, the positions of the arrested cirri physically block when cirral movements cause particles to be deflected onto
particles from approaching the frontal surface following the frontal surface. The cirri stop the particles and then
serotonin treatment. Bacterial clearance was reduced 90% transfer them to the frontal flow (Riisgard et ai. 1996;
under these conditions. On occasion we have noted that Beninger et ai. 1997; Riisgard and Larsen. 2000; this
cirral beating may be spontaneously arrested. Oral (1967) study). The environment ofthe transfer is non-steady state.
also noted that ciliary organelles on /;; vivo mussel gills low Reynolds number (Griinbaum et ai. 1998). Predicting
would spontaneously become motionless. Fluorescent par- exact particle movements without actually observing them
ticles moving in the frontal water current were observed to is difficult (Shimeta and Jumars, 1991). Far-field observa-
"fall" occasionally or be deflected from the frontal surface tions are possible using endoscopy that situate particle cap-
(Nielsen et ai, 1993; unpub. obs. in D. polymorpha). Thus, ture in intact specimens nearthe latero-frontal cirri (Wardet
CLSM
cirral activity is important for both particle capture and. ul.. 1998a). Near-field interactions are visible with
perhaps, keeping particles entrained in the frontal water (Silverman et ai. 1996; this study). These observations,
current. together with microscopic observations on single filaments
In addition to the cirral arrest, serotonin also caused gill or isolated gills (Nielsen et ai. 1993; Riisgard etai. 1996),
musculature to relax in preparations (J0rgensen, 1983; Me- and studies using in vivo preparations (Oral, 1967; Riisgard
dler and Silverman. 1997). which can increase the interfila- and Larsen, 2000) all demonstrate the importance of cirri
ment distance. In modeling particle-capture mechanisms, and their direct interaction with particles during the capture
the dimensions of gill structures (ostial size, intertilament process.
space, filament size, cirral size), rate of ciliary beat, and
ambient hydrostatic pressure are all critical components
(Silvester and Sleigh, 1984; Famme etai. 1986; Griinbaum Acknowledgments
ft ul.. 1998). Riisgard and Larsen (2000) point out that the
calculations based on /';; vitro gill dimensions must at least This project was supported by Louisiana State University
match intact clearance values, microscopic measurements Sea Grant Program NA46RG0096 R/ZMM-5. We thank
on intact tissues, and particle transport rates, if such calcu- Julie Cherry, Ron Bouchard, Paul Bruce, and Chris Thi-
lations are to accurately predict mechanism. Many ofthese bodaux for technical assistance. This research was aided by
components are neitherfixed nornecessarily uniform across the M. D. Socolofsky Microscopy Facility. Additional video
a gill at any particular time (Medler and Silverman. 1997; images may be viewed at http://www.biology.lsu.edu/re-
Medler f/ /., 1999). search.
Wecannotdetermine whetherthe particle-delivery mech-
anism involves direct particle contact with the cirri. How-
ever, at theobserved proximity ofthe particle with the cirrus Literature Cited
(< 1 ju,m), the question of 'hydromechanical' versus 'me- Atkins, D. 1937. On the ciliary mechanisms and interrelationships of
chanical' becomes irrelevant, as any intervening water is lamellibranchs. II. Sortingdeviceson the gills. Q. J. Microsc. Set. 79:
essentially a mechanical coupler. Evidence for this reason- 339-373.
ing comes from particles that were not successfully moved Benifnugnectri,onP.aGn.d,mSu.cSotc-y.t|eeand,isYt.riPboutuisosnaritn,PalnacdopJ.ecEt.enWamragde.ll1a9n9i3c.usGainldl
to the frontal surface on the first cirral interaction. Many of M\tilus eilulis (Mollusca: Bivalvia): the role of mucus in particle
these particles were drawn back with the cirrus as it moved transport. Mm: /-..,>/. Prog. Ser. 98: 275-282.
toward the extended position (Figs. 4. 5). These particles Beninger, P. G., J. VV. Lynn, T. H. Dietz, and H. Silverman. 1997.
appeared to reside within the space (2.2-2.8 /urn) between Mucociliarytransportinlivingtissue:thetwo-layermodelconfirmedin
tThheefpraeretitcilpes(o1f^tihme itnwdoiacimleitaerry)pcloatuelsdmbaekpihnygsicuaplltyhewecidrgruesd. Chaptmhobeteenmrtusis,asleEla.nIMd.y..raicaltnmidv.aJte.idoudnleipsAortLem.netBniiadolli..ofB1ue9lg7l.g9.s1o9f3M:teh4me-b7sr.eaanuercphoitne.ntLiyatlc,claicitiinoins
between the plates, moving within the water being drawn niricxatits. Exp. Cell Rex. 122: 203-218.
back by the cirrus during its extension, or by cohesive Dral, A. D. G. 1967. The movements ofthe latero-frontal cilia and the
376 SILVERMAN ET AL.
mechanismofparticleretention inthe mussel(MytilusediilisL.).Neth. techniquesforstudyingparticlecapture in filter-feedingbivalves. Lim-
J. Sea Res. 3: 391-422. nol. Oceanogr. (In press).
Famine. P.. H. V. Riisgard, and C. B. Jorgensen. 1986. On direct Riisgard.H.U.,P.S.Larsen,andN.F.Nielsen. 1996. Particlecapture
measurementofpumping rates in the musselMytilusediilis. Mar. Biol. in the mussel Mytilusediilis: the role oflatero-frontal cirri. Mar. Biol.
92: 323-327. 127: 259-266.
1,1iinli.inm. D.. D. Eyre, and A. Fogelson. 1998. Functional geometry Shimeta.J..and P.A.Jumars. 1991. Physicalmechanismsandratesof
ofciliated tentaculararrays in active suspension feeders. /. .vp. Biol. particle capture by suspension-feeders. Oceanogr. Mar. Biol. Annn.
201: 2575-2589. Rc\: 29: 191-257.
Jones. H. D.. O. G. Richards, and S. Hutchinson. 1990. The role of Silverman, H., E. C. Achberger, J. W. Lynn, and T. H. Dielz. 1995.
ctenidial abfrontal cilia in water pumping in Mytilus ediilis L. J. E.\p. Filtration and utilization oflaboratory-cultured bacteria by Dreissena
Mar. Biol. Ecol. 143: 15-26. polymorpha, Corhicula fiuminea, and Caninciilina te.\asensis. Biol.
Jorgensen, C. B. 1975. On gill function in the musselMytilusediilis L. Bull. 189: 308-319.
Ophelia 13: 187-232. Silverman, H.,J. W. Lynn, and T. H. Dietz. 1996. Particlecapture by
Jorgensen, C. B. 1983. Fluidmechanical aspectsofsuspension feeding. the gills of Dreissena polymorpha: structure and function of latero-
Mar. Ecol. Prog. Ser. 11: 89-103. frontal cirri. Biol. Bull. 191: 42-54.
Jorgensen, C. B. 1990. Bivalve FilterFeeding: Hydrodynamics, Bioen- Silverman, H., S. J. Nichols, J. S. Cherry, E. Achberger, J. VV. Lynn.
ergetics. PhvsiologvandEcology. OlsenandOlsenPress,Fredensborg, and T. H. Dietz. 1997. Clearance oflaboratory-cultured bacteriaby
Denmark.
freshwaterbivalves:differencesbetweenlenticandloticunionids.Can.
Jorgensen,C.B.,T.Kiorboe.F.Mohlenberg,andH.U.Riisgard. 1984. J. Zool. 75: 1857-1866.
Ciliary and mucus-net filler feeding, with special reference to fluid Silverman, H..J. VV. Lynn, and T. H. Dietz. 2000. In vitro studies of
McHmeencehrayni.cJa.lGc.h.araanctderTi.stHic.s.BiMrakrb.ecEkc.ol.198P5r.og.USpetr.ak1e5:an2d83p-r2o9ce2s.singof particle capture and transport in suspension-feeding bivalves. Liinnol.
cultured microorganisms by bivalves. J. Ev/>. Mar. Biol. Ecol. 90: SilvOescteearn.ogNr..R(.I,naprnedss)M.. A. Sleigh. 1984. Hydrodynamic aspects of
MedlsB1iei4cvr5a.-gli1vlS6il.a3,).m,aunsadcnldHe.sreSisinplozvneesbrermaatnom.utsr1sa9en9ls7sm.i,ttDerFreusinscitsnieovninatarlop.oolrIygnmavnoeinrzepabhtrai.on(BMiooofll.liunts1rc1ia6n::- Sprupoannrgt,tihcMele.,fceaaepndtidunrgVe.obRfyotsMheye.timl1uu9ss8.8s.eJl. MDIarnrefl.iusesBnieconela.opfAosfslooyocmd.osrUipzhKeaa.6n4d:Oef8co5oo9/do-qg8ui7aa9n.t7i7t:y
526-532.
200-212.
Medler. S., C. C. Thompson, T. H. Dietz, and H. Silverman. 1999. Tankersley, R.A. 1996. Multipurposegills: effectoflarvalbroodingon
Ionic effects on intrinsic gill muscles in the freshwater bivalve. Dre- the feeding physiology offreshwaterunionid mussels. Invertebr. Biol.
issenapolymorpha. Conip. Biocheni. Ph\\iol. A. 122: 163172. 115: 243-255.
Mohlenberg, F., and H. U. Riisgard. 1978. Efficiency of particle re- Ten Winkel, E. H.,and C. Davids. 1982. Food selection by Dreissena
tention in 13 species of suspension feeding bivalves. Ophelia 17: polymorpha Pallas (Mollusca: Bivalvia). Freshwater Biol. 12: 553-
239-246. 558.
Moore,H.J. 1971. Thestructureofthelatero-frontalcirrionthegillsof Ward, J. E., P. G. Beninger, B. A. MacDonald, and R. J. Thompson.
certain lamellibranch molluscs and their role in suspension feeding. 1991. Direct observations of feeding structures and mechanisms in
Mar. Biol. 11: 23-27. bivalve molluscs using endoscopic examination and videoimage anal-
Nielsen. N. F., P. S. Larsen, H. U. Riisgard. C. B. Jorgensen. 1993. ysis. Mai: Biol. Ill: 287-291.
Fluid motion and particle retention in the gill ofMytilus ediilis: video Ward, J. E., B. A. MacDonald. R. J. Thompson, and P. G. Beninger.
recordings and numerical modelling. Mar. Biol. 116: 61-71. 1993. Mechanisms of suspension feeding in bivalves: resolution of
Owen. G. 1974. Studies on the gill of Mytilus ediilis: the eu-latero- current controversies by means ofendoscopy. Liinnol. Oceanogr. 38:
frontal cirri. Proc. R. Soc. Loiul. B. 187: 83-91. 265-272.
Owen,G.,andJ.M.McCrae. 1976. Furtherstudiesonthelatero-frontal Ward. J. E., L. P. Sanford. R. I. E. Newell, and B. A. MacDonald.
tracts ofbivalves. Proc. R. Soc. Lund. B. 194: 527-544. I998a. An newexplanationofparticlecapture in suspension-feeding
Riisgard. H. U. 1988. Efficiency ofparticle retention and filtration rate bivalve molluscs. Liinnol. Oceanogr. 43: 741-752.
in 6speciesofNortheast American bivalves. Mar. Ecol. Prog. Ser. 45: Ward, J. E.. J. S. Levinton, S. E. Shumway, and T. Cucci. 1998b.
217-223. Particle sorting in bivalves: in vivodetermination ofthe pallial organs
Riisgard, H. U., and P. S. Larsen. 2000. A comment on experimental ofselection. Mar. Biol. 131: 283-292.