Identification and Activity-Dependent Labeling of Peripheral Sensory Structures on a Spionid Polychaete

2004·6 MB·

by  Sara M Lindsay

Reference:Biol.Bull.206:65-77.(April20041
2004MarineBiologicalLaboratory
Identification and Activity-Dependent Labeling of
Peripheral Sensory Structures on a Spionid Polychaete
SARA M. LINDSAY*. TIMOTHY J. RIORDAN. JR.. AND D. FOREST
SchoolofMarine Sciences, 575J MurrayHall, UniversityofMaine, Orono, Maine 04469
Abstract. In marine sedimentary habitats, chemorecep- Introduction
tion is thought to coordinate feeding in many deposit-
feedinginvertebratessuchaspolychaetes.snails,andclams. In marine sedimentary habitats, deposit-feeding inverte-
Relativelylittleisknown,however,aboutthechemosensory brates such as polychaetes, bivalves, gastropods, crusta-
structures and mechanism ofsignal transduction in deposit ceans,holothurians,andhemichordatesare abundantmem-
feeders.Usingelectronmicroscopy,confocallaserscanning bers of the tnacrofauna. These organisms ingest large
microscopy(CLSM),andimmunohistochemistry,weinves- volumes of sediment with typically low food value, often
tigatedthestructureandfunctionofputativechemosensory processingoneormorebodyweightsofsedimenteachday
cells on the feeding appendages of a deposit-feeding (reviewedinLopezandLevinton, 1987). They alsodisturb
polychaete species. Dipolydora quadrilobata. Tufts ofpu- thesedimentastheyburroworbuildtubes.Asbioturbators.
tative sensory cilia were distributed over the prostomium depositfeedershaveaprofoundinfluenceonthebiological,
and feeding palps and typically occurred next to pores. chemical, geological, and even physical properties oftheir
dmEmuixilcauarmrmoisncacaelnotldpisyopnbarelenopvefseaattwlhheeedrtsehemeuslriptemoigircileiaoslrn.i,saTtdehwidiestpchlsealelyntsisrnoawgrniystamhnicesalasdlibjsouanncoedfneatlpnercgcoelstatrnooo--nf lht11aar99byi89ib23teu;;atttia.AollSn.lh,Deue(rl1pe,l.9og,9s.10,i9;t29N04Gif)0rt,e1teo)srd.seoesrudmsesiaremandefainfanmtendecdtmnitScRstreeeoigdrcbeinhiobmaaecerlrhndgettc,m,oitmsr11mat99unr89sny11pi;;otr(iPtSMleuaasacrnnhit(dnaeFenildaenlinkdisd.-,-
apical mitochondria and relatively short ciliary rootlets. Mayer, 1994), nutrient cycling (Widbom and Frithsen,
eStxaaimniinnegdwibtyhCaLnStiMs,eruamndagraeivnesatleadceatxyolnaateldprao-ctuebsusleisnfwraosm (1M99a5y;erCehtriasl.t.en1s9e9n6;eWtesalt..on20ct00a)l.,, 2a0n0d0).fatSeedoifmepnotllduitsatnutrs-
putative sensorytuftson thepalpsurfacetopalpnerves,as bance by deposit feeders mediates competitive interactions
well as many free nerve endings. Activity-dependent cell (e.g.. Rhoads and Young. 1970; Woodin. 1976; Wilson,
labeling experiments were used to test the sensitivity of 1981; Brenchley, 1981). and influences animal distribution
putativesensorycellsonthepalpstoanaminoacidmixture patternsanddispersal (Wilson, 1981; Gunther, 1992; Brey.
thatelicited feeding inpreviousbehavioral experiments. In 1991),aswellasrecruitment(Williams, 1980;Posey, 1986:
static exposures, the number of lateral and abfrontal cells Luckenbach, 1987; Mines et al.. 1989; Olafsson. 1989:
labeled in response to the amino acid mixture was signifi- Flach, 1992). In addition, deposit feeders can influence
cantly greater than in the controls. Ultrastructural. posi- recruitmentdirectlyby ingesting larvaeandjuveniles(Wil-
tional, and now physiological evidence strongly suggests son. 1980; Tamaki. 1985; Miliekovsky. 1974: Elmgren et
that spionid feeding palps are equipped with sensory cells, nl.. 1986; Albertsson and Leonardsson. 2001).
at least some ofwhich function as chemoreceptors. Ratesofsedimentmobilizationbydepositfeedersdepend
on food supply. In the last twodecades, behavioral, physi-
ological,andmathematical modelingapproacheshavebeen
applied to the question of what makes some sediments
better food than others (e.i;.. review by Jumars, 1993).
Received 17September2002;accepted4February2004. Largely absent from this body of research, however, are
*To whom correspondence should be addressed. E-mail: slmdsay(s> studies that investigate the cues that initiate ingestion and
rnaiue.edu modulate feeding rates in deposit feeders. Jumars (1993)
66 S. M. LINDSAY ETAL
suggested several stimuli that could operate to regulate they may also be involved in osmoregulation (Fewou and
ingestion rate: smell, taste, distension ofthegut, and inter- Dhainaut-Courtois. 1995). Nuchal organs typically are
nal detection ofthe absorbed products in body fluids. The pairedepidermal structures found on the dorsal side ofthe
physiologicalandmolecularmechanismsfordetectingthese prostomiumorperistomium (/.<., the anteriorpresegmental
stimuli remain poorly understood for mostdepositfeeders. region). Somespionidpolychaetes(notDipolydorat/iuidri-
Chemoreceptionisimplicatedinthecoordinationoffeed- lohatii) alsohave metameric nuchal organs on theirbodies
ingbyavarietyofdepositfeeders.Forexample,freshfecal (Jelsing, 2002). Cephalic nuchal organs are typically com-
material depresses feeding rate in the snail Hvdrohiti tnin- posed ofciliated supporting cells, bipolar primary sensory
cata (Forbes and Lopez. 1986) and the spionid polychaete cells with cilia, unmodified epidermal cells, and retractor
Pseudopolydorakempijaponica(MillerandJumars, 1986). musclecellsinthosespeciesinwhichnuchalorganscanbe
Phagostimulants are implicated as well. When given a retracted (reviewed by Purschke, 1997).
choice,thecommondeposit-feedingpolychaeteStreblospio Other presumed chemosensory structures have been de-
benedicti fed preferentially on organically enriched sedi- scribed from polychaetes, including epidermal papillae of
ments rather than on unaltered sediments (Kihslinger and thedeposit-feedinglugwormArenicohinuirinti(Jouincttil..
Woodin,2000).Feedingrateinadeposit-feedinghemichor- 1985).compoundsensoryorgansontheprostomialcirriand
date was strongly correlated with sediment chlorophyll n palpsofNereisdiversicolor(Dorsett and Hyde. 1969). and
concentrations (Karrh and Miller. 1994). Robertson et <//. the parapodial cirri ofnereidid polychaetes (Boilly-Marer.
(1980) found that fiddler crabs, Ucu pii^ilutor, fed selec- 1972). InPlan-nereisdinner!ii. thereceptorsoftheparapo-
tively in diatom-enriched patches of sediment and could dial cirri function in perception of sexual pheromones
resolve patches at the millimeter scale by probing with (Boilly-Marer. 1968. 1974). Peripheral sensory structures
sensorysetaeontheirlegs. Fiddlercrabshavealsobeenthe have also been observed on the feeding palps, prostomia.
focus ofseveral studies that explicitly tested the effects of and peristomia of several spionid polychaete species
chemicalcompoundsondepositfeeders.Avarietyofamino (Dauer. 1984. 1987. 1991. 1997: Worsaee, 2001). With the
acids, peptides, and sugars appear to stimulate feeding exception of the nereidid pheromone receptors, studies
(Robertson et ai, 1981; Rittschof and Buswell, 1989; demonstrating functions of these putative polychaete che-
Weissburg and Zimmer-Faust, 1991; Weissburg. 1993). moreceptorsarelargelyabsent.Thegoalsofthisstudywere
Similarly, Ferner and Jumars (1999) found that dissolved to(1)describethedistribution and ultrastructureofperiph-
cues (amino acids and complex mixtures) influenced the eral sensory cells of the spionid polychaete Dipolydont
cpafeoemnleitdynlicoynhgaeaectxbietedhesDanivdpaieunodldryitshohuifigsraasrewqsvoueraridaknlrfiltlusooepnbicaosenthiaodfw(eRepidotoilhnryagtdcahanipenatarenttsdhi.ecLliWesn-pedbisooaunrynie,dd- 2bitqou0uerr0alae2dels)rd.irbcleeyoslblpausotsnaris,enesgsapnoaidnnnd(iiD2n.)mgmauqstunosaoicdghhrnieisamltoiofbcucaanhtlceatmciiuo(necRasaillotrrhdaoaatlpenperltaooicnatidchtheeLdsietbnhesadtthsraaulvyca-.--
2002).
Although chemical mediation of deposit feeding seems
likely, relatively little is known about the chemosensory Materials and Methods
fseterduecrtsu.resOannedemxeccehpatinoinsmisoftisdidglnearl tcrraabnss.ducWteiiosnsbinurdgepoasnidt Collection andmaintenance ofanimals
colleagues have identified gender-specific differences in Individuals of Dipolydoru t/iuulrilohtitit (Jacobi 1883)
tiddlercrab feeding behaviorin response tochemical cues, were sieved (0.5 mm) out of cores collected from the
and these are linked to differences in the number and mudflats of Lowe's Cove at the University of Maine's
sensitivity of chemoreceptor neurons (Weissburg, 1993. DarlingMarineLaboratory(Walpole. ME.USA)onseveral
1999; WeisshurgandDerby. 1995; Weissburgi-tai. 1996). days in SeptemberandOctoberof2000. and March. April,
Recent work (Weissburg. 2001) suggests the adenylate- and May of 2001. Animals and natural sediments were
cyclase-cAMPsecond messengercascade mediates inhibi- transported to the University of Maine in Orono and
tionofchemosensory neurons, andthatgender-specificdif- maintainedinaquaria insideanenvironmentalchamber(14
ferences inthispathwaycontributetothephysiologicaland C:10 C, 12 h light:dark cycle). Individual worms that
behavioral differences in fiddlercrabchemosensitivity. measured 10-20 mm in length and showed no signs of
Among the polychaetes, nuchal organs are presumed to gametogenesis. loss of segments, or other bodily damage
he involved in Chemoreception basedon histological. ultra- were used in the experiments.
structural, and positional criteria (Storch and Schlotzer-
Schrehardt. 19X8; Purschke. 1997). Several authors have Electron microscopy
speculated that nuchal organs may be involved in food
selection (Rullier. 1951; Rhode. 1940; No/ais </til.. 1997) For scanning electron microscopy (SEM). intact worms
or reproduction (Schlot/er-Schrehaidt. 19X7). although were relaxed in chilled 37'i magnesium chloride, fixed in
SPIONID PERIPHERAL SENSORY CELLS 67
3% glutaraldehyde in 0.1 M phosphate buffer with 10% bolicallyactivecells(Dwyerel<//.. 1980:PiccoandMenini.
sucrose, post-fixed in 1% osmium tetroxide inO.I A7phos- 1993).Theseanalogsenteractive neurons through nonspe-
phate buffer, and dehydrated in an ethanol series. Samples cificcationchannelsactivatedandopenedbythebindingof
were thencritical-point-dried with liquidCO:, mounted on a ligand with its receptor protein. Sequestration of the
stubs, and coated with gold palladium in a Conductavac I analogs in these cells allows for the activity-dependent
(SpeeVac. Inc.) sputter coaler. Samples were viewed with labeling ofindividual receptorneurons.
an AMRay AMR1000A scanning electron microscope op- For example, the guanidinium analog, l-amino-4-gua-
eratingat5 kV. Negativeswerescannedat800dpi withan nidobutane (= agmatine), has been shown to enter into
HP ScanJet 7400C flatbed scanner equipped with an HP receptor neurons through such open cation channels (Yo-
ScanJet XPA attachment, and the images were saved as shikami, 1981).Whencoupledwithknownstimulatorycues
TIFFfiles. in solution and perfused over olfactory organs, agmatine
For transmission electron microscopy (TEM), worms accumulates in activated odorant receptor neurons (Michel
were relaxed in chilled 37% magnesium chloride, fixed in ftnI.. 1999:Steulletettil., 2000).Cellsstimulatedbyacue
p3o%st-gfliuxteadrailndeh1y%deoisnmpihuomsphtaettreoxbiudfefeirnw0i.t1h M10%phsouscprhoastee, aacgcmuamtuilnaeteIgaGgmaanttiinbeodayndfoclalnowbeed ibdyentsiifliveedr uisnitenngsiafnicaatnitoin-
buffer, dehydrated in an acetone series, and embedded in labeling (Marc. 1995, 1999a. b). We presented agmatine
Spurr's resin. Ultrathin sections were collected onto slot plus a mixture of amino acids known to elicit behavioral
garciedtsa,te.thSeanmpstlaeisnewderweithvi0ew.e5d%wlietahdaciPthrialtleipasndCM2%10utrraannysl- rbieistpuoninsebsottho ifnldoiwv-itdhuraolugshpeacnidmesntsatiocfeDxippeorliymdenotrsa.c/uadrilo-
missionelectron microscopeoperatingat 80kV. Negatives
wscearnenesrcaenqnueidppated80w0itdhpiawHiPthSacnanHJePtSXcPanAJetat7ta4c0h0mCenftl,atabnedd sFelaowwatterrial(s.ASIWnd,iviindumalM:wo4r2m3sNwaeCrle.i9mmKeCr1.sed13inCaaCrtUif,ici2a3l
the images were saved asTIFFfiles. MgCU, 26 MgSO4 (Cavanaugh, 1975) pH adjusted to 7.2)
insideasmall coverslipperfusionchamber(WarnerInstru-
Confocal laserscanning microscopy ments,Model#RC21B).Odorantstimuliwereaddedtothe
thFtAehyaSredtWnWnehao,tofrimiiomxnswnesaodlCar.rwromtyeviBssrfeatierclawnitlraieilegrmSlheoeatsrxaeeseaa,otdMwaaa4MiktrneDeid)arCn.e3f(io7nArMA%fiSat5xWeM.sr,hoglMtCuiah3tnKrr2ieoi%aenscnob,eol1lfu0oEpt-cn4inHktio%ieinnrn7gpff.roo4rris.irsonm5elsaFsuelmostdiriIentoni-,yn-n Tc6Awr0ouhaS-nbesrtbWnrelAobrlypSslteyeuWrrgdbfiriunbanasgvygineiodstsanytnctodofhfplneecuaneoiedodcm,cdkatoinnreaviadanf5nldo-tvtlsoedfss.tp;lthuoieFlfwmllsupuueleirisidradtfefesufvlsosleoliorwuo(wyt0nwi.fo56acrn0shoscamtsmmwubterfheornserreeds6t'yh)o0hrenrilwomndeauigrngneie.dhsn
cact6AmAFoh-olfcoSne1tnbutne1jsuWsarpBumeie-griacn1aainii)ntnaffn,siedni(gnetdcBadiawlS-Ip0gaAagpsio.s)cenlGa5e.ri)tt%iedAyeTdidlsahlSiaTnuloeotrtWtuvfeiietedpt-deArrdmoaniSonnim1u-1-gdW:atsX:h1ru1et0yibr00uni10(salnc0aesinwui0benctnibosoita,u0nbah(tt.donwSe5addido0%1yrg'.r8ymf05m(aoTh.%1s,r)ra"5.inw%tt5TASSoietrbpn.bri)hboete,odXcoLivnow-mmieiuioom1snitXneu0esho-nn0;(csItS/FslOeiAcIeowgOdlSTnremoWauCroani.lm-ne:en osmaA(trAfuliteeGlSofarnmfnnetBbWoaiasptynvt,eeempear,eel(nilnmurodeAtsotedc,Ghsd.rtarieBernedAlolm)uonaiaSn=ixxliiVceWntnaCdAeu5l-A.rGlwb6eySwyaB)vos..aoWaprcfllpmtiWSae(asnitcocrevm)iio.far.imnnutuntgmestrlFodseaotioudlhlpar,wceilioieinnomnvnredcecw.seihlri=nun(tatvdgaIh4hnaeeelddfmtnwvrhweeoMeg2ioserl0mzr6myee(mmc0sraWemi-s)carn(mhMres-fia)noe2onnredfd0ari5sgnpi2tmrnImiC0oanAimfl)stumrintSilenrfnaesuMWote-.-hor
gsaloLcaiseasitcesasl,iTdeBCsiSrinmSiFPnl2guhocaormno,fmoocAuaLnl)t.l-aGPsre(erSpsoacuraatnthineoirnnnsgBwmieiorctereocishcmnoaoplgeoe.gdyNweAigst--h p(1h01a%tmeipnabrupafrffieoorrrmtwaoiltfdihexhiny1gd0.e%.Whs2ou%lcreogswleuotr(awmr/asvl)dw,eehrpyeHdepl7ia.n2c)e0d.f2irnoMmfixpoahvtoeisrv--e
ativecontrolstotestforthespecificityofprimaryantibodies night to several days.
werepreparedbytreatingspecimensasdescribed,butomit-
tleiansgtth1e01woArbm.sRewpeeraeteudseidnctuobatetsitonfsorwererperopdeurcfiobirlmietdy,.andat Nm10oMmflAtwGABS(sWtiantiiAncs)SitdWreiaa(lscs.omnaItlrnlodli,pveintdrui=aldi6swhow,roamrnmsdsw)1e0orrmel4i0omfmmeeMirtsheAerdG4Bi0n
plusthe mixture ofaminoacids inASW(treatment,n = 6
Activity-dependentcell labeling worms)wasslowlyaddedbypipette.Wormswereexposed
labWeleaacdtaipvatteeddthoelfmacettohroydnuesuerdonbsyiMniczheeblrafeitsthil.a(n1d99s9p)intyo troelatthievetlryeaqtumieenstcsenftorin60thmeindi,shdeusr,inigmmwehriscehdtiinmefrtehsehyAwSerWe
dloibnsiteurms.anTahliosgsmettoheondteerxpilnotiotssttihmeulabaitleidtynoefurcoantsionaincdgmueatnai-- tfohrem5 imnianftroeerzeemro(v-e20resCid)uaflorA1GBm,inanpdriroreltaoxefidxibnyg.pWlhacoilneg
68 S. M. LINDSAY ETAL.
gwlourtmarsalwdeerheydpelaince0d.2inMfipxhaotsipveha(t1e%bupfafrearfwoirtmhal1d0eh%ydseu,cr2os%e pprreosceenstseisnttyhpeicsalalmyewpelraenenaost tchoepllaanbaerledincemlolrbeodtyh;anthoensee
(w/v), pH 7.2) from overnight to several days. adjacentsection. Second,the locationsoflabeledcells ina
single section were compared to labeled cells in adjacent
Tissueprocessing, immunolabeling, and visualization sections toavoid counting the samecell twice.
Labeled cells were grouped by type according to their
Fixed worms were rinsed in a phosphate buffer (PB: location within the sections (Fig. 1). Abfrontal cells were
1.76 g NaH2PO4 H:O + 7.67 g Na:HPO4 in 1 1 of locatedbehindastraight linedrawnacrossthesectionsand
deionized water), dehydrated through a graded series of tangent to the back ofthe food groove. Lateral cells were
absoluteethanol andacetone,embeddedinEpon 812resin, foundbetweenthefoodgrooveandtheabfrontalcilia.Cells
cured, and sectioned using a microtome and glass knives. immediately adjacent to the frontal ciliated food groove
An average of 156 sections were processed for each indi- werecalled laterofrontalcells. Andcellslocatedwithin the
vidual. Semi-thick sections (2 jum) were placed in 7-mm frontal ciliated food groove were called frontal cells. Be-
wellsofaTeflon-coatedspotslide(ErieScientific),deplas- causedifferent numbersofsectionswereanalyzedforeach
ticized in a 1:5 v/v solution ofmature sodium ethoxide in worm, we calculated the number of labeled cells per 100
anhydrous ethanol. and subsequently washed in three /j.m palp length, based on the total number of sections
changes of anhydrous ethanol. The slides were rinsed in analyzed perindividual. The resulting datawere compared
deionized water, air-dried, and then incubated overnight in among treatments using a nonparametric Kruskal-Wallis
a 1:100dilution ofapolyclonal rabbit anti-AGB IgG anti- analysis of variance (SAS version 9, SAS Institute, Inc.).
body (Signature Immunologies and courtesy of R. Marc. Data from flow experiments and static experiments were
tUhneinverrsiintsyedoifnUPtBa.h wSacshohoeldoifn M\e7dticgionaet).seTrhuem silnidPesBwpelrues anaTloyzeesdtismeaptaerattehley.total number of each cell type in any
0.05%thimerosal(1%GSPBT)for 10min.andincubatedin lengthofpalp, serial sections madefromthepalps (middle
aant1i:b5o0ddyilfuotrio6n0omfina.1A-fntemrgaolfdinaclonPjBugwaatesdhaanntdi-araibrb-idtryiInggG. pstoaritni.onT)oloufidfeounrewboluremsstawienrseacsitdaiicnecdelwlitpahrtast(oi.leu.i,dnenueclbeluuse)
labeled cells were visualized using silver intensification and allows the different types ofcells to be identified and
(Marc, 1999a, b). The silver nitrate solution for silver in- counted. Individualcellsweretracedthroughthesectionsto
tohienfyndss6riooflimquctluaiitonionoofnnAedwe(aii1nso1n14pi5rzmeempgdlarcoweiaftdtrdeiberci)ya.ocnmiiiIdzxeim+dnlgw3ao4ttf2ehrrse)ioe,nlgaustnosildooudnt1iioumBnmls:(co0if5.tr51amt%lge taaivbsoosiuudted5woa%usblosefe-ctcthoieuonnatevidenrgfa.ogreOenlaecanhvgetohrfagotefh,etshaeebwopuoatrlmp1ss2,1ofr/euDpn.rieiso/efmnuptlairlnipg-
aqueoussilvernitrate. Followingadipin5% acetic acidto lobaia.
stop the intensification reaction, the slides were washed in
deionized water for 10 min. air-dried, and mounted in
Results
Permount (Fisher) for viewing on a light microscope.
Distribution ofperipheralsensory cells
digitization andanalysis
JavIemlaingeJsEo1f2tHhMeVsevcitdieonoscwaemreeracampotuunrteedddiognitaalnlyOlusyimnpgusa cirrSic,a\ncnnixniigeWloercstaroane,mi2c0r0o1s)codpiystrreivbeuatlededotnufttsheofpcaillipas(/a'.n'.d,
fBrXa6m0e glirgahbtbemricrboosacrodpe(.ScVioindeoLGsig3n)alsinweareDelplassOepdtiptloexa ppurtoasttiovmeiisiemnsoofryDifci>loila\dwnerrueqmfiodurnidloobnataall(Fisgu.rf2a)c.esTuofftsthoef
GX110 computer. The images were analyzed using the prostomium (Fig. 2B) and were typically adjacent to pores
Scion ImagePC software. Beta ver. 4.02 (Scion Corpora- (Fig. 2B). The ciliaofthe prostomial tufts were nonmotile
tion, Frederick, MD). Cells labeled with agmatine were and relatively short; they numbered about 13-20 cilia per
identified byquantifying the pixel intensity inside acell ol tuft (Fig. 2C). On the palps, we observed motile cilia
interest inthedigiti/ed imagesandcomparing ittothepixel arra\ed in a row immediately adjacent to each side ofthe
intensity of an unlaheled region adjacent to the cell d'.v,. foodgroove (laterofrontal, Fig. 3A, B), and tufts ofmotile
Micheletal.. 1999).Themeanandstandarddeviationofthe cilia arrayed in a second lateral row and on the abfrontal
pixel intensity inside cells ofinterest wasused to calculate surface (Fig. 3A, B). Laterofrontal, lateral, and abfrontal
a95% confidence interval; cells werecounted as labeled if ciliaoccurredwithsimilarfrequencyalongthelengthofthe
the lower limit of this interval was higher than the upper palp (Fig. 3C). The putative sensory tufts of cilia on the
limit ofthe unlabeled region. Two strategies were used to abfrontal surface seemed regularly dispersed; and. interest-
avoid double-counting of labeled cells. First, cells were ingly, we observed fewer adjacent pores than on the pros-
counted only if the distal processes and apical cilia were tomium.Theciliaoftheabfrontalsurfaceswerelongerthan
SPIONID PERIPHERAL SENSORY CELLS 69
Singlecross-section(2jam)
throughthepalp
,
B
AbfrontalCell
Location
Lateral
Cell
Location
Latero-
FrontalCell
Location
FrontalCiliaand FrontalCellLocation
Figure 1. (A) Gross palp morphology and section placement for cell labeling studies in Dipohdora
quadrilobata.(B)Celltypelocationwithinsections.Cellsalongthefoodgrooveandwithinthefrontalciliawere
designatedfrontalcells.Cellsimmediatelyadjacenttothefrontalciliaweredesignatedlaterofrontalcells.Cells
behindalinedrawnacrossthebackendofthefoodgrooveweredesignatedabfrontalcells.Cellsinfrontofthat
lineandlateraltothefrontalsurfaceweredesignatedlateralcells.
thoseoftheprostomium,andtherewerefewerciliapertuft the muscle layers below, and cell bodies often projected
(7-14) (Fig. 3C. inset). laterally (Fig. 5). Ciliary rootlets ofboth palp and prosto-
mial cells, whenobserved,wereshortrelativetothe length
Season'cell ultrastructure ofthecellbody(Fig.5A).Allciliawerereadilydistinguish-
Palp and prostomial sensory cells shared similar ultra- able from the mic9roXvi2llarcuticle (Figs. 4, 5) anddisplayed
structural characteristics. All were multiciliated and con- a characteristic + 2 microtubular arrangement in
tained many apical mitochondria(Figs. 4, 5). Laterofrontal cross-section.
cells on the palp were typically adjacent to glandularcells
(Fig. 4) associated with pores on the surface. Gland cells Confocal laserscanning microscopy
contained either globules of very electron-dense material
(Fig. 4C) orlessdense material that appeared tobe mucus Theantibodyforacetylateda-tubulintargetsnerveaxons
(not shown). Abfrontal cells ofthe palp were restricted by and cilia axoneme microtubules. Cilia ofthe food groove.
70 S. M. LINDSAY ETAL.
sensory tufts to the palp nerves (Fig. 6B). There are also
manynerveswithapparentfreeendingsonthepalpsurface
(i.e., not associated with ciliated cells). A more detailed
mwfif^^^^
&fSJNP
?TS v-;syXj V c"**S v J*r.'-."^ '^s^L^^sr-'.,-
i*.i? /" -.it.^t.*i '>*/!fldBMl3Ht^Hp' ~'^ jd^^H^^l
Figure2. Peripheral sensoiy cellson iheprostomiimiofl)i/>i>lydoru
i/utulrilohiitu. (A) Anterior prolilc. shiming pulps (pa) and piosionmini
(pro);scale 10(1/.nil Inselslum'sentireworm.(HiCiliatedstrueluresarc
ilislnhuteil on theentiresurfaceoftheprostoniium (anowsi; palps wcic
ieino\cd. leavingascaripsi; scale 1(10/mi. (Cl I'oiesipo)arefound i^A.- '?>'" .o
ud|accnllothesensory sliueluresontheprosiominm;scale = 10/Mill Inset 3. Peripheralsensoiy cellsonIhepalpofDi/'olyilni'iii/utulri-
showsthesensoryciliaofthepiostomium; insetscale - 2.5juin. lohulii. (A)Lateralviewofthemiddleportionofafeedingpalp,showing
fsrconnstoarl\fcoiloida(gsrllooovnet(hIe'gll.alcliaalleraonlidonalhallrocnillailas(n11r1t.acaensdoflutllhsepoaflpn;onscmaolleil=e
lateral and abl'ronlul surfaces of palp, as well as (he main so/mi. (Hi Middleportionofthe feedingpalpshowinglaterolrontal(II)
cpeanltpranlernveervaonudssesvyesrtaelmsmsahlolweredpalapeentyelravleesdprao-jteucbtuilnigntroeatch-e cssiehlniosawoiatndvjgalctuehlneltsltoiousidiih.geisfocroaol\nectalilgflo.IoId)lagl/erjroMoo.lvieo(nCl(iafgl)H.(i1sa1l1saclwiellipalo,raatsinodlnatseeornfasllofyreyeddi(isltnifrgtisbpuisatlle)dp;
tivity(Fig.6A).Ourinitialobservationsclearlyshow axons scale = 10/jiii. Inset palpsensoiy tuftsappeartohesimilarlothoseon
that project from the cilia of putative lateral and ahfrontal thepiosioiiiiuni;insetscale = I jim.
SPIONID PERIPHERAL SENSORY CELLS 71
Figure 4. Ultrastructure ofDipolydora quadrilobata palp laterofrontal cells. (A) Semi-oblique section
throughpalpfoodgrooveshowingdensefieldoffrontalcilia(fc)andadjacentlaterofrontalcell(If);scale=2
/jm.(B)Samelaterofrontalcellshowingnumerouscilia(c)andmanyapicalmitochondria(mt);scale=2fj,m;
thebasalportionofthecellprojectsoutoftheplaneofsection.(C)Anothersemi-obliquesectionofthepalp
revealsthelaterofrontalcellandadjacentglandularcell(gc)withpore(po);scale = 2/j,m.
examination ofthe innervation ofthese peripheral sensory pared to controls in all cell types pooled (Kruskal-Wallis,
cells, as well as their reactivity to anti-serotonin and anti- X2 = 9.88,d.f. = 1,P = 0.002, n = 36). Weobservedthe
FMRFamide, is in progress. greatest amount of labeling in lateral and abfrontal cells
(Fig. 8B).
Activity-dependentlabeling Based on counts from the serial sections stained with
toluidine blue, frontal cells and laterofrontal cells occurat
Four types of putative sensory cells were labeled by aboutthesamefrequency,averaging27frontalcellsand28
eafprrxmoopincotnesaoslisnaecgceslitledhsxetm(iepFxinagtdl.uiprsn7eg):o.tfTfhDrhr.oeonustqgaeulha,fdtorhluaeirtleecorpeboliafldtreoatrnytmptaioelss,atgalolmalattethhrieaanlvs,eeuracafnenalddcleuatlboha-erf cc2leea.llt5lle5ssroompfcermcronutr1fa0ollr0ecsDe/s.lulrfsnrqeupoqaefudrerpnia1tll0lopy0b.,ajtGaaatim,vaeoacnfnopdmaanblapisa.nsveLuedamrtiaeagnrvegaelrpnaaaoglnepdchrlaaaebtnnfeggroeotfnhst1aoilnf1
tshuerfpaaclepo(fFigt.he7),pamlap.nyFrwointthalvisainbdlelcaitliearoefxrtonetnadlincgelflrsomwetrhee daipsptrroixbiutmiaotnelaylo6n8g8thferolnetnagltchelolfs,th7e14pallpa,tewroefrcoanntatlhceenllesx,paencdt
generally found as groups of several cells close to one atotalof280lateralandabfrontalcellstooccuronasingle
oafnotthheesreacteltlhsewpeerriephlearbyeloedf.thIenfcooontdragsrto,ovlea,bealnedd olaftteernalmoasndt pinalspt.atBiacsteridalosnontlhye,owcecucrornesnecrevaotfivlaebleyliensgtitmhaattewtehatob1s4er%veodf
abfrontal cells were always found in isolation. the total number of lateral and abfrontal cells, 47c of the
The occurrence oflabeledcells in the flow-through per- total number of laterofrontal cells, and 3% of the total
fusionexperimentwasquite variable(Fig. 8A). Poolingall number of frontal cells were labeled by agmatine in the
c(eclule)tyopnesc,eltlhelraebelwiansg (nKorussikganlif-iWcaalnltisefffect=o0f.7a4m,idn.of.a=cid1s, presence ofamino acids.
P = 0.39, n = 27). There was a non-significant trend Discussion
towardgreaterlabelinginthecontrolsforlaterofrontal and
frontal cells (e.g., Kruskal-Wallis test of the difference The diversity of chemosensory organs among marine
betweenthe numberoffrontalcellslabeledinthepresence organisms is great, from crustacean aesthetasc sensilla to
ofaminoacidscomparedtocontrols:x2 = 2.7655,d.f. = 1, molluscan osphradium, rhinophores. and oral tentacles, to
P = 0.09, n = 9). Relativelyfewercells were labeled in polychaete nuchal organs (Laverack, 1968; Emery, 1992).
thestaticexperiments(Fig. 8B,noteaxisscale).Incontrast Despite this diversity, the transduction of externally de-
to the flow experiment, however, addition ofamino acids tected signals to the brain follows a similar pathway, be-
significantly increased cell labeling with agmatine com- ginning with the activation of a chemoreceptor cell that
72 S. M. LINDSAY ETAL.
leadstothecentral nervoussystem.Thestructuralcommon
denominator for all chemosensory cells is the presence of
ciliary or microvillarextensions into theenvironment. The
peripheralcellsofthespionidpalpsandprostomiumclearly
meet this requirement (Figs. 2, 3).
Dauer (1984, 1987. 1991, 1997) and Worsaee (2001)
observednonmotileciliaonpalpsandprostomiaofseveral
spionid species, including Dipolydora quadrilobata, and
classifiedthemassensoryonthebasisofscanningelectron
microscopy data and positional criteria. Our initial TEM
observations (Figs. 4, 5) support these classifications, and
suggest that these peripheral sensory cells have features
similar to the caudal chemoreceptors that Jouin el ai.
(1985) described in Arenicola marina namely an abun-
dance of apical mitochondria and short ciliary rootlets.
Confocal laser scanning microscopy furtherreveals axonal
processes to palp nerves from the lateral and abfrontal
sensory tufts (Fig. 6), as well as many free nerve endings
projectingtothelateral andabfrontal surfacesofthepalps.
Assigning function tocells based solely on morphologi-
cal criteria can be difficult, however. For example, it ap-
pears that no single morphological character defines che-
moreceptor cilia: on the crustacean aesthetasc, olfactory
ciliacan have "significant" ciliary rootlets and motile cilia
(Griinert and Ache, 1988). Weusedthe activity-dependent
labelingexperimentstoexplorethepossiblefunctionofthe
palpsensorycells.Althoughwedesignedtheexperimentto
assayforcellactivityinthepresenceorabsenceofchemical
cues (aminoacids), it is important to note that this method
of labeling active sensory cells does not discriminate be-
tweentypesofreceptors. It simply identifiescellsthathave
accumulated agmatine, regardless of the stimulus source.
Thus,thelabeledcellsmayincludeavarietyofsensorycell
types. The initial stimulation of a mechanoreceptor (i.e..
stretching orbendingofthe cell membraneoraprotruding
cilium) in at least one invertebrate (the crayfish) opens a
stretch-activatedionchannelthatappearstobepermeableto
divalent cations (Edwards et ai. 1981). Chemoreceptor
transductionproceedssimilarly,withthebindingofaligand
tothe receptorin the membranecausing the activation and
opening ofionchannels. In bothcases, suchcation-perme-
able channels should be permeable toagmatine, andthere-
fore both mechanoreceptors and chemoreceptors could be
labeled using this technique.
Spionid palps are probably equipped with mechanosen-
.. 5. Ultrastructure ofDipolyJuni iimulriluhiitii palp abfrontal
cellandprostomialsensorycells.(A)Constrainedbyamusclelayerbelow.
Ihis ahtrontal palp sensory cell projects laterally. Cilia (c) are readily
distinguished fromthemicrovilli(mv)ofthecuticle;ciliaryrootlets(cr)
appearrelativelyshort;scale=0.5/J.m.(B)Prostomialsensorycellshave
asimilarstructure, withcilia(c)projectingthroughthemicrovillar(mv)
cuticle,numerousmitochondria(nit),andabilobednucleus(nu);scale=
_ /urn. (C) Highermagnification ofthe samecell, showingapical mito-
chondria(nit);scale = 0.7/im.
SPIONID PERIPHERAL SENSORY CELLS 73
*,
Figure 6. Fluorescence of anti-acetylated a-tubulin in Dipol\dora qiiadrilnhata palps examined with
confocallaserscanningmicroscopyreveals(A)palpnerves(pn),andciliaofthefrontalfoodgroove(scalebar
40juin)aswellas(B)ciliaoftheabfrontalputativesensorytufts(st),theiraxons(ax),andtheirproximityto
thepalpnerves(pn);scale = 25/AMI).
sory cells. Many spionids (including D. i/uculrilobatd; T. cells. There was virtually no labeling in the agmatine con-
Riordan, pers. obs.)switchfromdepositfeedingtosuspen- trols. These results suggest that the lateral and abfrontal
sionfeedinginthepresenceofhigherflowrates(Taghonet cellsarechemosensory,althoughitispossiblethatthecells
al., 1980; Dauer et al. 1981). This switch is probably were stimulated by a response triggered elsewhere on the
mediated by mechanosensory detection of flow rates. In body. We believe that body movement was an unlikely
addition, when suspension feeding, spionids collect sus- stimulus, however, because the worms werequiescentdur-
pendedparticlesthatdirectlyinterceptthepalps.Cilialining ing the static trials.
theedgeofthe frontal foodgroove (i.e., laterofrontal cilia) Ifweacceptthatthelateralandabfrontalciliatedtuftsare
and inside the food groove (i.e., frontal cilia) move such chemosensory. then the question arises whether the cells
particlestowardthe mouth(Dauer. 1984. 1985, 1987). and functionasdistanceorcontactchemoreceptors.Whenspio-
may be mechanosensitive. For example, the laterofrontal nidpolychaetesaredepositfeeding,theytypicallyprobethe
cwetsmmivhueehleugcciTersgahhh,neaaeswtnonthficeoeieocsxnnaotpegctlnrenaaeearsncnsinotdtmmsoerieepttdymcniouothwlfbmnsaaauyiantrndwkiodc,oaeetsniiePnocsnaacanousrnsrnsoaeyfdpraopauersyrccnefiottddaionhceennecltdfdoalolspburudaepspeillaiaindirpfooctnfnenigessopcceotloitnnefnaisennbotxaag(ohrpctDueltyucatii,elvucclaiieelatttlrbyell,e.lrsypaa.ootbfis1erHss9looooi8inlnb5ntwall)gat-ye.;le eDtmaswtoc.eaeidtancdlie(ot.smr,/nuebtpaa1anornd9ctrodrt8nbi1seiltusn;ohrucgebLfgu,aiaaestncreawedswdshiawia(eminsyRnetc,inrhmopetpiretarndrhsseisaeues.nmrdedfanoiaalbstacstncee.aotdd.l)ri,vPLpwneruioioesnrtnfvitdheeinisxeogoaipdunysaist,trnhloetgeyfn2,i,ta0ct,b0lhwife2enewr)-cop.eblsnaoutlhuBcdaponeiolsdwncngse(caaudlDurmsuasfeitdueadhneeciaodre-at
and frontal cells in the flow-through controls (Fig. 8A) is thatthepalpsmediatedtheresponse.Inaddition.Fernerand
intriguing, and the possibility that these cells function as Jumars (1999) reported repeated "pore-water-sniffing" be-
mechanoreceptors should be further examined using elec- haviorby Boccardiaproboscidea in which the palps were
trophysiological approaches. It is also possible that the raised intothe watercolumn, sankpassivelyontothe sedi-
increased labeling in controls was due to the presence of ment, and then quickly returned to an upright position.
agmatine, whichitselfisapotentchemicalcueinzebrafish Again, it was the abfrontal palp surface that contacted the
(Michel ettil.. 1999). Inthe static trials, significantly more sediment. Given such behaviors, we speculate thatthe ab-
cells were labeled in the presence of amino acids and frontal cells act as contact chemoreceptors. Yet spionids
agmatinethaninagmatinealone(Fig. 8B);increased label- alsousetheirpalpsto"monitor"thewatercolumn(Daueret
ing was especially pronounced in the lateral and abfrontal til.. 1981; Ferner and Jumars, 1999), and the lateral and
74 S. M. LINDSAY ETAL
Labeled Labeled
laterofrontalcells lateralcell
Labeled IV . \
frontalcell *
.
,Vv
*'
ftn 25|Jtn B
Labeled
Y'#'" - abfrontalcell
, :..- ' ,
"Unlabeled
abfrontalcell
;*
D
Figure7. LabelingofDipolydnniqiuulrilobatapalpcellsbyagmatineinthepresenceandabsenceofthe
aminoacid mixture. (A) Semi-thin section frompalpexposedto agmatine + aminoacids showing labeled
laterofrontalandfrontalcells;scale=25ju,m.Insetshowsthesamecellsathighermagnification;scale=5/xm.
(IBns)etSesmhio-twhsinthseecstaimonefcrelolmaptalhpigehxeprosmaegdnitofiacgamtaiotni;nesc+aleam=ino5a;cuimd.s(sCh)owSiemnig-tlahbienlesdecltaitoenralfrceolml;psaclapleex=po2s5efdj.ntio.
agmatine + aminoacidsshowinglabeledabfrontalcell;scale = 25/xm.Insetshowsthesamecellathigher
magnification; scale = 5 /xm. (D) Semi-thin section from palp exposed to agmatine without amino acids
(control),showingunlabeledahfrontulcellanditsciliaprojectingfromthecuticle;scale=25fxm.Insetshows
thesamecellathighermagnification;scale = 5/xm.
abfrontal sensorycells mightalsofunction asdistanceche- (i.e., lackingcontact, this paper)andadsorbed (i.e., contact
moreceptors. necessary. Riordan and Lindsay. 2002) cues. The initial
Among invertebrates, preliminary studies suggest that ultrastructuralinformationrevealsnosignificantdifferences
gustatory("taste."orcontactchemosensitive)andolfactory among the groups of palp sensory cells (laterofrontal, lat-
("smell,"ordistancechemosensitive)sensoryneuronsshow eral,andabfrontal).Characteri/ationoftheinnervationpat-
aconsiderable amount ofstructural similarity (Dionne and ternsandimmunoreactivitytoserotoninandFMRFamideis
Dubin, 1994). For example, two populations of sensory ongoing.
cells are distributed on the sensory tentacles of the nudi-
branch Plicstilla sihoi>(ie. The intraepithelial sensory cells Conclusion
(contactorshort-distancechemoreceptors)andsubepithelial
sensory cells (olfaction) have distinct spatial distributions, Previousresearchestablishedthatselectionofparticlesof
axonal organizations, and sensitivity to chemical cues, yet certain sizes by spionid palps can he influenced by mucus
haveverysimilarelectrophysiologicalandpharmacological adhesion strength and particle "stickiness" (Jumars et /..
characteristics (Boudko ct til.. 1997, 1999). Our experi- 19X2: Taghon. 19X2: Dauer. 1985). Qian and Chia (1997)
mentswithD. c/UMirilo/xitiidemonstrateihat the lateraland havespeculatedaboutapossiblesensoryrolethepalpsmay
abfrontalsensorycellsofthepalpsrecogni/ebothdissolved play in selective feeding, and putative sensory structures