Table Of ContentReference:Bio/. Bull. 193: 107-115.(October. 1997)
Chemosensory Activation of an Antennular
Grooming Behavior in the Spiny Lobster, Panulirus
argus, Is Tuned Narrowly to L-Glutamate
JOHN C. BARBATO AND PETER C. DANIEL*
DepartmentofBiology, Hofstra University. Hempstead, New York 11549
Abstract. Antennular grooming behavior (AGB) is a staticadherencetotheantennularcuticle,L-Gluisasensi-
stereotypedbehaviorincrustaceansinwhichthefirstpair tiveindicatoroffoulingbyfood-associatedchemicalsand
ofantennae, the majorolfactory organs, are clasped and thus an appropriate compound to stimulate antennular
wiped repetitively by the third maxillipeds, which also grooming.
serve as feeding appendages. AGB apparently functions
to clear away accumulating debris on or between the Introduction
antennular aesthetascs (olfactory sensilla). The purpose
ofthis research was to determine whether AGB can be Odorantaccessandremovalfromreceptorsareequally
activated by chemicals commonly found in food odors. important for effective chemical detection. To facilitate
Lobsterswerepresented, viaheadsetorhandheldpipette, these two processes, animals have evolved short- and
with 27 chemicals found in theirfood. One chemical, L- long-term mechanisms that include elimination of stag-
glutamate,evokedveryhighfrequenciesofwiping. Most nant boundary layers, alteration and internalization of
chemicals tested were not stimulatory and only a few chemostimulants,andgroomingofchemosensoryorgans.
were weakly stimulatory (adenosine-5'-monophosphate. Thesemechanismsmaintainthesensitivityofthereceptor
glycine,D-glutamate).Thisissurprisingbecauseprevious cell and. inthecase ofgrooming, the structural integrity
studieshaveshownthatotherbehaviors(antennularflick, ofthe chemosensory organ.
search)canbeevokedbyamuchbroaderarrayofchemi- Crustaceans are good models in which to investigate
calsfoundinfoododorants. Onthebasisoftheseresults, chemoreceptionbecausetheyrespondtochemostimulants
we propose that chemosensory neurons that specifically with highly stereotypical and quantifiable behaviors.
detect L-Glu activate AGB through a recently described Chemical cues regulate many components ofcrustacean
non-olfactorypathway. Furthermore,weproposethatthe behaviorincludingfoodrecognition(Hazlett, 1968;Carr,
roleofL-GluinevokingAGBisbasedonitselectrostatic 1978; Carr, 1982; Schembri, 1981; Fine-Levy et al.,
properties. Because it has a high probability ofelectro- 1988), courtship behavior (Atema and Engstrom, 1971;
Dunham, 1978; Atemaetal., 1979; Gleeson, 1980), and
predator avoidance (Mackie and Grant, 1974).
Received 18February 1997;accepted20June 1997. Electrophysiological recordings of receptor neurons
caelpaA*ntbAiobunrretenhv(eoiDura-rto&tionosnLws-(:hAOloaRam)nN,tsce)Lno-.nraurralegdaisernpnioongnserdioe(nonLecm--eAi5rn'gsg-)h,mobouLenl-hodaapsvbhpieoaosrraapdgh(diarAnteGeesBs()(eL,dA-.AMsoPnl))f,,acLtD-o-arsy&parrLe--- a((enD.deg.r,bbyDeehaarnvbdiyoAraatnledmasAt.cuhd1ei9,e8s21;9e8mF4pi;lnoeAy-niLdneevgrysoaebntlaaalt.ni,don1A9c8th8ee;c,hDna1in9qi8ue5el)s
tate(L-Asp),betaine(Bet),L-cysteine(L-Cys),D-&L-glutamate(D-& and Derby, 1991) have shown that antennules are the
L-Glu),glycine(Gly),L-histidine(L-His),L-hydroxyproline(L-Hyp),L- primary organs for detecting odors. Therefore, mecha-
isoleucine(L-Ile),L-lactate(L-Lac),L-leucine(L-Leu),L-lysine(L-Lys), nisms that enhance odorant access to and removal from
tLSae-trme)e,t(hLNi-MosnDuicAnc)ei.na(tLLe--Mpe(htLe)-n,Syulaca)ml,manotiannuireiun(meL-c(PhThlaeou)r),i,dLe-Lp-(rtNohlrHie4noCen1i)(n,Le-NP-(rmLo-e)Tt,hhryL)l-,-seDLr--iavnsaeplai(rnL-e- ttihaelpteorircercuespttaocreaennvbierhoanvmieonr.toSfutchheamnetcehnanunliessmsareinecslsuedne-
(L-Val),artificialseawater(ASW). antennular flicking (Snow, 1973; Gleeson et al., 1993),
107
108 J C. BARBATO AND P.C. DANIEL
biochemicalremovalofstimuli(Gleesonetul., 1987;Carr nules are favorable substrates for microbes and detritus.
et ul., 1989), and antennular grooming (Bauer, 1989). Ifthismaterialisnotremoved,chitinivorousmicroorgan-
Antennularflicking,whichoccursatfrequenciesof0.5 isms can damage the exoskeleton and the presence of
to 2Hz in Punulirusargus(Schmitt and Ache, 1979). is otherfoulingorganismscancauserespiratoryandsensory
defined asthe verticaldeflectionofthelateralfilamentto impairment. When a shrimp, Heptacarpus pictus, was
apositionnearlycontactingthemedialfilament(Zimmer- experimentally prevented from grooming the antennules,
Faustetai, 1984). Antennular flicking reduces the stag- extensive structural damage of the aesthetascs occurred
nant boundary layers of seawater created by the dense (Bauer. 1977).Therefore,anythingthatenhancesthelevel
tufts of aesthetascs (Snow, 1973; Gleeson et ai, 1993) ofmicrobialfoulingisdetrimentaltothestructuralinteg-
which are the sensilla housing the dendritic processes of rity of the antennule and, presumably, to its functional
hundreds ofolfactory receptorneurons (ORNs) (Griinert role as achemoreceptororgan.
and Ache, 1988). This removal mechanism uses move- Althoughfoulingoftheantennulesoccurscontinually,
mentsoftheantennulestosplayaparttheaesthetaschairs, feedingmayresultinparticularlyhighratesofaccumula-
allowingwatertorushinanddisplacethewatercollected tionofdebris. Byprovidinganutrient-richsubstrate,this
duringthepreviousflick.Flickingisanalogoustoaverte- debris facilitates microbial colonization. Results ofstud-
brate sniff (Schmitt and Ache, 1979) because sniffing ies ofseveral crustacean species show that AGB can be
flushes air from the upper nasal cavity, the location of elicitedinresponsetocompoundstypicallyfoundinfood
theolfactoryepithelium. InP. argus, flickingisactivated (Snow, 1973; Zimmer-Faust et ai, 1984). In this paper,
inadose-dependentmannerbyabroadrangeofchemical we examine which chemicals found in the food of P.
stimuli detected by the antennules (Daniel and Derby, argus stimulate the release of AGB. Compounds found
1991). infood,includingaminoacids,nucleotides,organicacids
The aesthetascs of P. argus contain membrane-bound and ammonium (Carrand Derby, 1986) were tested. Re-
enzymes and transporters that serve to eliminate specific sults ofbehavioral assays revealed that only one amino
chemical stimuli including nucleotides and amino acids acid, L-glutamate (L-Glu), evoked AGB with great fre-
(Trapido-Rosenthal etal. 1988; Carr etal.. 1989). Ecto- quency. Even analoguesofL-Glu (D-glutamate, L-aspar-
nucleotidases catalyze the dephosphorylation of adenine tate, N-methyl-D-aspartate) were not excitatory. On the
nucleotides,creatinganodorantdifferentfromtheonethat basisoftheseresults,weproposethatchemosensoryneu-
initiallyenteredthereceptorenvironment.Theendproduct rons that specifically detect L-Glu activate AGB through
of dephosphorylation, adenosine, a molecule for which a recently described non-olfactory pathway (Schmidt et
there is little receptorsensitivity, is internalized by a spe- ai, 1992; Schmidt and Ache. 1996). Furthermore, we
cific uptake system (Trapido-Rosenthal et ai. 1987). In proposethatL-Gluevokes AGB because the high proba-
additiontotheadenosineuptakesystem,anuptakesystem bility ofelectrostatic adherence to the antennularcuticle
fortaurine has been identified (Gleeson etai, 1987) and makes it a sensitive indicatoroffouling by food-associ-
uptake systems for other amino acids, including L-gluta- atedchemicals.
mate, also exist (Trapido-Rosenthal et ai, 1988; Carr et
ul., 1989). Prolonged stimulationofORNs limitsthetem- Materials and Methods
poralandspatialresolutionof"patchy"olfactoryenviron- Sourceandmaintenance oflobsters
bmyentthses(eAtbeimocaheetmialc.a,l19r8e9m)o.vaQluimcekcehlainmiisnamtsioansowfeloldoarsanbtys Spiny lobsters (55 to 70mm carapace length) were
antennular flicking enhances receptorsensitivity. obtainedfromtheFloridaKeysRegionalMarineLabora-
The least studied mechanism for facilitating odorant tory in Long Key. Florida, and maintained in separate
removal and maintaining receptor accessibility is anten- 80-1 aquaria (one lobster/aquarium). Aquaria were lined
nulargroomingbehavior(AGB). AlthoughAGB iscom- with crushed coral; filled with aerated, recirculating In-
mon in many crustaceans, very little is known about stantOcean(specificgravity, 1.021-1.023);andequipped
which stimuli activate it (Bauer, 1989). This behavior withgravel-bottomfiltersystems.Lobsterswerefedscal-
consistsofanantennulardeflectiondownward,permitting loporshrimpdaily adlibitum and the uneaten food was
tbhyetlhaetetrhailrdanmdaxmileldiipaeldsan(tpeaninrueldaarpfpielnadmaegntesstoonbeeitghrerasspiedde rtheemoavmebdienafttetrem1phe.raTthuereliwgahst:dmaarikntcayicnleedwbaestw1e2e:n122a5n-d
of the mouth) and pulled repeatedly through the setal 27C. Red light (25W, ceramic-coated light bulbs) was
combsofthe maxillipeds. The resulting action facilitates provided during the darkcycle.
removal ofmaterial that has accumulated on orbetween
ChemicalStimuli
the aesthetascs (Snow, 1973; Bauer, 1989). Fouling is a
recurring problem forcrustaceans because both the exo- Thefollowingcompounds wereusedasstimuli: adeno-
skeletonandspecializedstructuressuchasgillsandanten- sine-5'-monophosphate (AMP), D- & L-alanine (D- & L-
CHEMOSENSORY ACTIVATION OF GROOMING 109
Ala), L-arginine(L-Arg), L-asparagine(L-Asn), i.-aspartate attached by hook and loop fasteners to the rostrum. A
(L-Asp), betaine (Bet), D- & L-glutamate (D- & L-Glu), bentglassrod(5-mmdiameter) wasgluedtotherostrum
glycine (Gly). L-histidine (L-His), L-hydroxyproline (L- attachment with cyanoacrylate quick-bonding nontoxic
Hyp), L-isoleucine (L-Ile). L-lactate (L-Lac), L-leucine (L- glue.Thisprovidedsupportforthetubingthrough which
Leu), L-lysine (L-Lys), L-methionine (L-Met), ammonium the stimulus was introduced (1-mm inner diameter,
chloride (NH4C1), N-methyl-D-aspartate (NMDA), L-phe- attachedtotherodwithcableties)andallowedtestsolu-
nylalanine (L-Phe), L-proline (L-Pro), L-serine (L-Ser), L- tions to be injected at aconstant distance in the vicinity
succinate (L-Suc), taurine (Tau), L-threonine (L-Thr) and oftheantennules. Aperistalticpumpwas usedtodeliver
L-valine (L-Val). Stock solutions (lOmM. pH 8.1) ofall allsolutions.Todesensitizetheanimalstothemechanical
the stimuli were prepared in artificial seawater (ASW) stimulus,tankwaterwaspumpedcontinuously,viaplastic
(Cavanaugh, 1964). L-cysteine (L-Cys) was prepared on flexibletubing,fromthetankthroughthestimulus-intro-
the day oftesting to prevent precipitation from freezing; ductiontubing.Eachtestsolution(5-mlina 10-mlplastic
allotherswerestoredat -70Cuntilused.Withtheexcep- syringe) was injected into the tubing through a two-way
tion ofD-Ala, D-Glu (a stereoisomerofGlu). NH4C1. and stopcockvalve.Theflowrateofthepumpwasmaintained
NMDA (an agonist of a subclass of glutamate receptors at 10-ml min~'. In subsequent trials, stimuli were pre-
(Schoepp, 1994)),thesecompoundswereidentifiedbyCarr sentedviaahandheld5-ml pipette.Thetipofthe pipette
and Derby (1986) ascomponents ofprey extracts. On the was gently placed in the vicinity ofthe antennules. This
ddialyutoefdtetsotienigt,hearpp5romprMiatoers0t.o5cmksMoluwtiitohnsAwSeWr.ethawedand omfetAhGodBafppreoamretdhetoloeblsictietra(cFoing.sis1t).enAtlllygtrrieaaltserwemraegnviitduedoe-
taped, beginning0.25min beforeeach stimuluswaspre-
Experimentaldesign sented and continuing forup to 2min afterward.
The 26 compounds listed above were each assayed at
0.5mM. To ensure that the lobsters would not become Data analysis
desensitized, we presented these chemicals in five sepa- The magnitudes of the AGB responses in all experi-
rate trials (maximum of 10 chemicals pertrial including ments were determined from videotapes. Pre-stimulus
ASW & L-Glu) so that no experiment lasted longerthan wipe rates were determined by counting the number of
4h. We subsequently tested 10 ofthese compounds (in wipesthatoccurredinthe0.25-min periodbefore stimu-
three separate trials) at 5.0mM. In previous trials, these lus presentation. Post-stimulus wipe rates were deter-
10 chemicals had elicited either a highly significant re- mined by recording the number of wipes that occurred
sponse (i.e., one that was significantly greater than the for 1 min after stimulus presentation. Wipe rates were
response toward ASW) orthe response was greaterthan thereforedefinedasthepost-stimulusresponserateminus
theresponsetowardASWforamajorityoflobsters. The the pre-stimulus response rate. The three wipe rates
exception was NMDA, which was assayed at both con- (wipes-min ') counted for each stimulus per trial were
centrations even though it failed to meet the criteria. Fi- averaged and reported as the mean wipe rate.
nally,aseriesofconcentrationsofL-Glu(0.01,0.05,0.08, Inmostcases,datadidnotmeettheassumptionsneces-
and 0.1 mM) and three othercompounds (AMP, Gly, D- saryforparametricstatisticaltests;therefore,nonparamet-
Gluat0.5, 1.0, 5.0and 10.0mM) shown toelicit signifi- rictestswereused.Friedman'srepeatedmeasurestestson
cant responses at 5.0mM were tested. Each ofthe four rankswasusedtocompareresponsestochemicalstimuli
ccloumdpeodunAdSsWwaassaasscaoynetdrolinsatismeuplaursateantdria0l..5AlmlMtriLal-sGliun,- c(aSnitgmdaisftfaetr,enJcaensdewleSrceiefntoiufnidc,).pWaihrewriesestcaotmipstaircialsloynssigwneifrie-
which preliminary experiments showed tobe very effec- performedusingtheStudent-Newman-Keuls(S-N-K)test
tive in evoking AGB, as a response standard. adapted forranked data. Forthe concentration seriesex-
periments, least-squares regression analyses were per-
Presentation ofstimuli formedonlog-transformedconcentrationsofeachstimu-
Experimentaltrialswereblindinthattheexperimenter lus vs. wipe rates standardized to the L-Glu (0.5 mM)
did notknowtheorderin whichthechemicals were pre- response.
sented. Each stimulus was administered to six lobsters,
except where noted in Results, in triplicate tests. Two Results
methods of stimulus presentation were employed: auto- Responses to compounds at0.5mM
matedandhandheldpipette. Forthe firsttwotrialsusing
stimuliat0.5mM,presentationofstimuliwasautomated Ofthe compoundspresentedat0.5mM, L-Gluwasby
viaaheadsetapparatusasdescribedpreviously inDaniel farthemosteffectivecompoundatelicitingAGB.Intrials
and Derby (1988). A 13-mm diameter acrylic rod was #l-#4,reportedinFigure 1,L-Gluproducedsignificantly
10 J. C. BARBATO AND P.C DANIEL
-
Z(J
v 15 -
C
CHEMOSENSORY ACTIVATION OF GROOMING 11
higherwipe rates than all otherstimuli tested at0.5mM significant, the 50% maximal responses based on visual
and ASW (Fig 1; Friedman repeated measures ANOVA; inspection were at least 10mM.
trial #1. x' = 29.7, = 9, P = 0.0001; trial #2. r
34.6. = 9, P = 0.0001; trial #3, \2 = 24.4, n = 6, P Discussion
= 0.0002; trial#4. x~ = 23.1, n = 6, P = 0.006; S-N-K AGB
pairwise comparisons test for ranked data, P < 0.05). specificity to L-gliitamate: implicationsfor
Two compounds, Gly and L-Met (trial #3), elicited re- sen.soiy-motorintegration
sponses that were significantly greater than ASW (S-N- Our results show that, unlike other behaviors studied
K pairwise comparisons testforranked data, P < 0.05). in P. argus (Fine-Levy et al.. 1988. 1989; Daniel and
Neither the stereoisomer of L-Glu. D-Glu, nor the ana- Derby. 1988;Fine-Levyand Derby, 1991, 1992; Lynnet
logue, L-Asp(sidechainhas oneless carbon), elicited re- al.. 1994), AGB is elicited almost exclusively by one
sponsesgreaterthanthosetoward ASWin trial#3 (S-N- chemical, the L-enantiomer of glutamate (L-Glu). L-Glu
IKnpaadidriwtiisone,coNmMpaDriAsodnisdtnesottfeolricritanskiegdnidfaitcaa.ntPre>sp0o.n0s5e)s. twhaasnawtelreeastAM1P00,tiGmleys, maonrdeD-efGfleuc,tivteheinneexlti-cbietsitngsAinGglBe
(trial #5, \~ = 4.91, H = 3, P = 0.194). However, none stimuli. Furthermore, theD-enantiomerofL-Glu (D-Glu).
of the compounds tested in this trial, including L-Glu, astructural analogueofL-Glu (L-Asp), andan agonistof
were significantly different from ASW. This is probably a major class of glutamate receptors (NMDA). either
because the small sample size used in this experiment failed to activate AGB (L-Asp, NMDA) or were only
resultedinaverylow-powertest. However,allthree lob- weaklyeffectiveathighconcentrations(D-Glu).Wepro-
ostrerNsMteDstAedarte0s.p5onmdMe.dmuch moretoL-Gluthan to ASW paosspeectihfaitceclliacsistaotfiocnhoefmoAsGenBsorreyquinreeusrosnesnsnoarryroiwnpluyttfurnoemd
to L-Glu.
Responses to compounds at5mM Accordingtoelectrophysiological studies.ORNsinP.
mM argusarenarrowlytunedtospecificchemicalstimuliand
ResponsestoL-Gluat0.5 weresignificantlyhigher can be classified by best-compound. "Best" cells have
than responses totest stimuli when presented at 5.0mM been identified for AMP, ATP, Cys. Bet, Glu, NH4C1,
(Fig. 2; Friedman'srepeatedmeasuresANOVA; trial#1, andTau(DerbyandAche, 1984;Carretal., 1986;Derby
X2 = 43.1, n = 9. P = 0.0001; trial #2, x2 = 24.7, n = etal., 1991;Danieletal., 1994).Similartuningcharacter-
6, P = 0.0002; trial #3, x2 = 10.4. n = 6, P = 0.0055; isticshavebeenidentifiedforolfactoryandnon-olfactory
S-N-K pairwise comparisons test for ranked data, P < chemosensitive neurons distributed on second antennae,
0.05). Six compounds, L-Asn, L-Cys, AMP. and Tau in antennules.maxillipeds,andlegsofHomarusamericanus
trial #1, Gly and D-Glu in trial #2, evoked wipe rates (Johnsonetal., 1985;Tierneyetal.. 1988;Corottoetal.,
significantlygreaterthanthoseforASW(S-N-Kpairwise 1992; Voigt etal., 1997). Biochemical receptor-binding
comparisons test for ranked data, P < 0.05). NMDA assaysofantennulesofP. argushaveidentified indepen-
produced no significant response at 5.0mM (trial #3, S- dentolfactory receptorsites forAMP,Tau (Olsonetal.,
N-Kpairwisecomparisonstestforrankeddata,P>0.05). 1992; Olson and Derby, 1995; Sung et al., 1996), both
stereoisomers of Ala (Michel et al.. 1993), and L-Glu
Responses to concentration series (Burgess etal., 1994).
Compounds that weakly evoke AGB may do so by
The magnitude ofAGB towards L-Glu increased as a activatingL-Glu-bestneurons. Electrophysiological stud-
linear function of the log of its concentration (Fig. 3. ies ofORNs showed that responses to next-best stimuli
least-squares regression, F = 86.6, n = 30, P < 0.001. were generally 100-fold less than to the best compound
r = 0.76, standardized wipes-min"' = 107.7 + (Daniel etal., 1994). Hence the amino acids Gly and D-
(53.1 log[concentration]) and were at least 100times Glu, and the nucleotide, AMP, are possibly "next-best"
moreeffectivethan anyoftheotherchemicalsacrossthe stimulantsthatweaklyactivateAGBviaL-Glu-bestcells.
rangeofconcentrationstested. OnlytheAMPconcentra- However, the response spectra of AGB and of ORNs
NMDA
tion series yielded a significant regression (least-squares sensitivetoL-Gluarenotentirelyconsistent. and
regression, F = 22.6, n = 26, P < 0.0001, r = 0.55, L-Cysserveaspartialagonistsandantagonistsforchemo-
standardized wipes-min~' = 7.26 + (39.5 log[concen- receptors presumed to be ORNs sensitive to glutamate
tration]). From the linear regression equations, the con- (Burgess and Derby. 1995). Since AGB was elicited by
centrations needed to achieve 25% and 50% maximal L-GlubutnotNMDAorL-Cysevenat5mM,itispossible
responses were 0.03 and 0.08mM, respectively, for L- that non-olfactory neurons mediate AGB.
Glu.and3.0and 14mM,respectively,forAMP.Although Howmightsuchaspecificstimulusleadtoactivationof
thelinearregressionequationsforGlyandD-Gluwerenot AGB? There appearto be two antennularchemosensory
112 J. C. BARBATO AND P.C. DANIEL
35
-i
30 -
CHEMOSENSORY ACTIVATION OF GROOMING 13
fo
114 J C. BARBATO AND P.C. DANIEL
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