SESSION SUMMARY
PHARMACOLOGY
SessionChairperson
BettyTwarog
TuftsUniversity Medford, Massachusetts
Co-Chairman
Edward Gilfil lan
BigelowLaboratory for OceanSciences McKown Point WestBoothbay Harbor, Maine 7his sess«in served to bring t II. I:.ffects<>I Toxins on Organisms Mat 335 m M. H, Evans Agricultural Research Cc!u ttct l instt ut te ofAnima! Physio logy, Babraham,Cambridge, England. 337 ABSTI AC T Outbreaksntparalytic sht'llfish poisoning alongthe 1'acific coast ofNorth Ameticaappearto be eau~ed bysaxitoxin derived from ~nnvauiai careiiellu Saxitoxincausesparalysis inman and mammals bya specific anddirect action «nnerve and skeletal muscle, inwhich it prevents initiation andpropagation ofaction potentials, byblocking thesodium channels ofthe excitable cell membrane.Theprobably caust.of death isthe failure ofthe movements of respiration.resultingfrom this peripheral paralys~s. Theredoes notappear to beany siy nit icant paralysis of the central nervous system, except in experimentalanimalswhich have hadsaxitoxin administered directlyinto the cerebrospinalParalysisassticiatedfluid, withblooms IN1 ROD UCTION paralyticshellfish poiscining isa well recognized clinical condition, Halstead inloti& ! listed85 separate outbreaks, between theyears 1689 and 1962, with a totalestimate «1over 959 victims and more than 222 deaths. A morerecent estimatebyI'rakash etat. in 1971 9! putsthe world-wide total at about 1600 t ast.1 h»signs and symptoms thatvictims present toa clinicianhave been des LA8ORATORYlNVESTK ATIONS Followinga major outbreak ofparalytic shellfish poisoning inEngland atthe endof May 1968 2! an attempt was made toextract saxitoxin from some of thepoisonous mussels, Mytilus edah's !, lt wasfound that when a crude extractwas injected into mice, following theassay technique developed by McFarren 3! fromSommer k Meyer 5! the dose-survival timecurve was significantlydifferent from that obtained when pure saxitoxin wasinjected. At aboutthe same time it becameclear that these mussels hadacquired theirpoison from a bloomof G. tamarerrsis 8,20!, so this finding gave supportto an earliersuggestion by Schantt 2! that the poisonof C, tarriarertsismight be differentfrom saxitoxin. Attemptswere then made to purifythe crude extracts. The first attempt madeuse of the ion-exchange technique used successfully bySchantz ntzete aL 3! inpur>fying saxitoxin. However, only 18%%uo of thetotal toxicity present in the crudeextract was recovered by meansof theirtechnique. About 45,o of the toxicityran off thecolumn during the preliminary wash with buffer, having failedto bind to the resin.It waslater partiallypurified by gelfiltration in columnsofSephadex G,10 and G.25. All therest of the original toxic material wasirrecoverably lost, and losses of themajor toxic fraction were high throughoutthesubsequent stages of purification. The specific toxicity of this materialwas eventually raised to about 270 rn.u. /rng. Because atleast 90% of theimpurity was found to be NaCl, it wasdecided that this was sufficiently pureto be used inpreliminary pharmacological tests.The other poison that didbind to theresin was quite easily purified by followingthe preliminary stagesof the process for saxitoxin 3!, reachingabout 1,550 rn.u./mg !. Pure saxitoxin assaysat about 5,500 m.u. /mg. Neuropharmrtco!ogicalactionsof saxitoxin and the G. tamarensrspoisons. Thematerial which formed 18% of thetotal mussel toxicity, and which behavedlike saxitoxinduring the stagesof purification,was pharma- cologicallyindistinguishable frompure saxitoxin. Itblocked the conduction of nerveimpulses infilaments from the rat cauda equina and in desheathed frog sciaticnerve. In eachcase the degree ofblock was dose dependent, and equal to theblocking effect of a comparableconcentration ofsaxitoxin, expressed as m.u./1. Figure 1 showsanexample ofthe compound action potential becoming reducedby a!: saxitoxinat 20 pg/l and b!: the saxitoxin-likepoison A e C Figure1,Compound action potentials recorded from a nerve,showing the effects of shellfishtoxins. Each photograph shows 4 recordings; top- control, second - after 3 min,in poison, third - afterwashing for 3 min.fourth - afterwashing far 15 min.The 3 photographsshowthe actiom of: {A! saxitoxin 20kg l., B!minor saxitoxin-likecomponent from Mytilus edulis 100 m,u./l., C! majorunknown components100m,u, /l. Eachtrace has a 1m V calibrationpulseon the left and thetiming pips at the bottom of thephotographs show 1 mSecintervals. at100 m.u. CONCl USIONS The laboratory experimentsdescribed above confirm the suggestionmade by Schantzin 1960 Z2! that shellfishtoxicity derived from G, tarnarertsisis not due to the presenceof saxitoxin, but to some other compoundwith similar neuropharrnac«logical effects. It is true that somematerial indistinguishable frownsaxitoxin was extractedfrom the rnusselsresponsible for the 1968 outbreakin Fngland,but it accountedfor lessthan 20% of the totaltoxicity. Most of the toxicitywas due to anotherpoison, chemically distinct from saxitoxin,with similarthough not identicalpharmacological effects. One cannotyet say whether this unknown poison causes the nausea, vomiting and othergastro-intestinal effects that arecommonly seen when paralytic shellfish poisoningfollows a bloomof G. tamarerrsis.It is equallypossible for these effectsto bedue to someother irritant produced by thisorganism but not by C. catenelfa. Kaoin 19' 9! stressedthat one of the dangersassociated with bloomsof G catertel4was due to theheat stability of saxitoxin.Cooking poisonous Pacific coastshellfish lessens their toxicity only slightly. Both the poisons extracted fromthe poisonous Englishmussels werefound tobe heat-stable atpH 1, but neverthelesstherewereunexplained andheavy losses af the major unknown componentstable,ingeneral, throughout than thesaxitoxinstages is.of purification.Various accountsIt maynt thereforeoutbreaks bealongless Atlanticcoastshave suggested thatthe toxicity ofthese shellfish islowered considerablybycooking 8, 14, 12!. A reductionoftoxicity by cooking has beenthought toexplain theabsence offatalities duringthe 1968 outbreak in England,whenthe raw mussels hadvery high levels aftaxicity, Nevertheless, manyfatalities haveoccurred duringother Atlantic coastoutbreaks, andthe aver'allfatality rates are not noticeably different between thePacific and A tl antic areas. Whendeath occurs, itis almost certainly duetorespiratory paralysiscaused bythe action ofthese poisons onthe respiratory musclesand their peripheral motorinnervation. Thepoisons seemunable topass thraugh thebload-brain barrier,sothe respiratory centersin the CNS are not affected, Therefore, administrationofcentral stimulants andanaleptics willnot be beneficial, Allauthorities agreethat artificial ventilation isthe measure mostlikely to preventdeath ofa badlypoisoned victim.However, itseems thatvery few caseshave been treated inthis way, perhaps because insuch grave cases the respirationfailsbefore skilled medical assistance canbe abtained, Wider publictraining in such 'first aid' measures asmouth-to-mauth artificial ventilationmight enable victims tobe kept alive long enough ta receive professionalmedical care. REFERENCES 1. BOND,R.M, and l. C.MEDCOF. 19$8, Epidemic shellfish poisoning in NewBrunswick, 1957. Canad. Med, Ass. J., 79, 19-24. 2. EVANS,M. H. 1965,Cause of death in experimentalparalytic shellfish poisoning PSP!. Brit. J. exp.Pathol 46, 24S-253. 3. EVANS,M, H. 1969a.Spinal reflexes in catafter intravenous saxitaxin and tetrodotoxin.Toxicon, 7, 131-138. 4. EVANS,M. H, 1969b.Differences between the effectsof saxitorin paralyticshellfish poison! and tetrodotaxin onthe frog neuromuscular junction. Brit. J. Pharmac 35, 426-436. EVANS,M. H. 1970,Two toxins from a poisonoussample of mussels Mytilus edulis.Brit. J. Pharmac.,40, 847-865, CEMMILL, J. S. and W. G. MANDERSON.1960. Neurotoxic mussel poisoning.Lancet, 2, 307-309. 1fAI c1 AI 1!It 5'i'tactic I' MI ! I H, k I- . ff %$4MI:.R,arid V, SCHOENHOLZ,19Z8. Mussel [i«l~oolr'igI fire'vent Meif . Z. 305-394. NI I I!I I R A ft ]04< paralyticshellfish poisoning and Goriyaufrr> rprriiriru~ri I I-ish Res Bcj. Canad.. 7. 490-$04. 18 I'FR41I-.WAN'lV ISS8 A fatal caseof poisoningby mussels.with rrmark»«ri the an't«iniif the poison;illustrated by two slightercases i~ c uriingat the same time, lancet, 2, 568. tu f'RAkA4II A, I C.' MEfX.OF and h, D. TENNANT. ],971.Paralytic shellfishI~»seining in Eastern Canada, Fisheries Res. Bd. Canad, Bull, 177. b" pp R 13[NbN, i R . I M [!istrihution4< ionvuii/pi tuniayq nci[ ebour inthe ivesterri North Sea in April, May arid liine 3. [ ![F[-, R. 14 Twoj<, cases olpoisoning b!mussets - onetata[ l,ancet. », S 'HANTZ,E l.[oeO. [3iochernical studiesonparalytic ihellti~h[ oi~ons. Ann.N. Y. Acad.Sci.. ut>, t343- 55, 73 SCHANTZ.E.l.. l. D,MGl.[l, lV. STAN ER, l. SHAVE[F [. ['. [3QV,'[7EN,l. M,[,'t'N W, R, S. WYLER. [3,RIF .E[..and H,SOMMER. [957.['aralytic shellfish poison Vl.A procedureforthe isolationandpurification ofthe poison from toxic clam and tnussel tissue.J, Amer.chem. Soc,9, 5Z30-5235, 24.SEVEN. M.J. ]958. Mussel poisoning. Ann,int. Mad 48, 84l-897. 25.SQ[vlMER, H.and K. F. METER. 1437, Paralytic shell-fish poisoning, Arch, ['ath<>l,24, 5'-598 Zo.TENNANT, R. D., I. NAUHERT,andH. E. CORBE[[1955. An outbreakofparalytic shellfish poisoning. Canad, Med, Ass. l, .2, 436-439. Z7.Vv'ATE RFIELD, C. J. andM. W, EVANS.1472. A method for distinguishingtetrodotoxin [romsaxitoxin. bycomparing theirrelative stabilitieswben heated inacid solution. Experientia, 'H.o7 j-e71. CARMOVASCULARACTIONS OF SAXITOXIN C. Y. Kao Departmentof I'harmacnIogy StateUniversity ot Nev York Doe:nstateMedicaI Center Brookjyn, New' York ABSTRAC I In whole antrnals, saxitoxin causes a marked fall in the arterial blood pressure which is frequentlv followed by a compensatory pressor response of moderate deyree. The hypotensive action is due to a lowering of the peripheral resistance and not to anv selective action of saxitoxin on the central vasomotor rnechanisrn or on the heart, The peripheral action is due to a combination of direct relaxant effect on the vascular smooth muscle at Iow doses, and of a release of vasomotor tone in higher doses which blocked the adrenergic vasoconstrictor nerves. The late compensatory pressor phase is due to released c;ttecholamines ln clinical casesof' paralytic shellfish poisoning, the initial hvpotensivephase is almost neverseen, but the late pressorphase may be a presenting sign. INTRODUCTION Saxitoxin, like tetrodotoxin, is among the most deadly poisons known, the rninirnal lethal dose determined in mice being about 8 gg/kg body weight. Assttn»ng homogeneous distribui.ion throughout body water, the lethal concentration is Frobably about IDnM. Although the fundamental acticn of saxitoxin is a highly selectiveblockage of an increasein sodium permeability in many excitablemembranes, interfering with the generationof actionpotentials l, there are important actions on the cardiovascttlarsystem in whole animals. For more references, see 1, 2 and 3!. Site of hypotertsiue actiort Except in very low doses less than 'l pg 'kg intravenously!, the cardiovascular actions of saxitoxin are manifested as a marked fall in the arterial blood pressure Fig. 1!. Although there was some old information l00STXt.opg/kg 100 sec Figurel. Effect of saxitoxtn STX! on systemicarterial pressure. Cat, V, 3,8 tcg, The systemicarterial pressurewas originally 138/98mm Hg, visible at the left end of the record. STX injection ts indicatedby arrow. Note that the pressurebegan to fall within 40 seconds,and reachedits lowest at 62140 rnm Hg in 200 sec. Note that the pressurereturned to pretoxinlevel in about8 mni., and wasfollowed by a phase of pressorresponse from ref. lob 348 i~hichsuggested th,itthe hypotension wasdue to some action ofsaxitoxin a ratl-.eraninipure extract ofcontaminated musselsand clams! ancent y asoiliot~'Icent'efs oi'ret., see I ancl 2 I,recent expel irnerlts using head-ban centra cios i liert usion technique showed thatthe primary site far the hypotensivt o actionscould ni>t be in any central structures. Briefly, theexperiment: inv it,li i-liiiinsut of' !ieri}flu,riffuasocfi1atiou Tounderst mrlthe mechanism nfthe hypotensive action,experiments using a techniqueotregional perfusion wa necessary. Inthis technique, a vascular bedwith relativelv homogeneous pharmacological responses wasisolated troutthe circulation ot the rest ot the body. In the cat and in thedog, the vasculatureotskelet;il muscles waschosen; inthe cat, a wholehind leg, ipprrpriatelyprepared, was used ib, 10!,and in thedog, the gracilis muscle wasused i'8!. The ctrculatinn to the muscle bed was then provided via a constantvolume pump tram either the animal's own body, or from a separate donoranimal, Since the flaw into the perfused muscular bedis constant, cliangin sthe peripheral vascular resistance became manifested aschanges in thepressure required to perfuse the vasculature. Asin thehead-body cross-perfusionstudies, the nervous innervation between the perfused regions and the rest of the body was lett intact. Whensaxitaxin was injected intravenously intothe body of an animal, the systeinicarterial blood pressure fell rapidly, but. in theperfused region, the immeJiate response tothe hypotension inthe body was a riseinblood pressure IFig. l. In otherwords, instead of a fallin peripheralresistance, there was actuallyan increasein peripheral resistance in the perfused region, a vasoconstriction.If the circulation to theperfused region was obtained from thesame anima, then this pressor response wasfollowed bya depressor response.If the circulation to the perfused region was supplied by a donor animalwhose blood was not poisoned bysaxitoxin, then the pressor response in theperfusion pressure remained, and only gradually subsided coincident ~'1'h .1pt«gr ii figure2. l:tte<1<|ti< 3cQ Thefull hy p< tcnstic action ofsaxitoxin hi fecause itis dose-dependent. Theevent~c xln,desowcver. 'l'L lsl s 8Itt tlvmore h conhplex reachedarevalid when dosci otsaxitoxin n s1 esciii i x'c1 at icivrand the conclll!iorl >g'hcn theJose was increased toabove oxinabout equi t.ci ian I kto 1 ~h 5pg ~k,g werr useif perfusedtnusc.ularvasculature were asetollows: a ou .. 6: yy, g1 t ~e re~ponies h h in the hvpotension,therewasa fall inthe perfus ows:onpfoincic ent wit t esystemic peripheialresistance, ora vasodilatory crresponseusion pressureA lat in icatintt a ta in attributabletothe direct action of saxitoxin onthe vase'utature.a cr cc prrisorw,issimilarrcsponic. to incases withIow doses, Thefirst depressor responsew ducasto blockadeofsympathetic vasomotornerves which provided thenot'mal vasomotortone.The proof forthis conclusion isthat vase@ onstrictor responsesinthe muscular vasculaturenorma! lvelicited bselectrical stimulatictn ofthe sympathetic nervesweremarkedly reduced.or abolished, aftersuch saxitoxintreatment. Therefore,a morecomplete pictureofthe hypotensive actionofsaxitoxin isthat inlow doses thereisa direct vascQilatory actionon thevascular smoothmuscle, and inhigh doses thereisan additional releasecif vasomotortone.Other evidence whichpermitted theconclusion ofa direct vasodilatoryresponseisthat the effect ispresent evenafter the'vascular smoothmusclehas been blocked completely withalpha- and beta-adrenergic blockingagents phento!amine andpropranolol! «ndwith a cholinergic blockingagent atropine!. Byanalogy withthelack ofeffect oflow doses «f tetrodotoxinonthe release of norepinephrine fromthesplenic nerve t,it may beassumed thatlow doses ofsaxitoxin whichcaused reflexvasoconstriction didnot interfere with release ofnorepinephrine. Differencesbetu.cert thecurdiovascalur uitioris ofsu~ttcxi»i uniftetr<>roti! tiki Thebiological actionsof saxitoxin arevery similar tothose oftetriidotoxtn, eventhough theyare different chemically. A fewminor clilferences betveen thecardiovascular actionsofthese twotoxins arepresent, ancemay explain someofthe seemingly basicdifferences inthe clinical symptomatiil~itcy incasts of paralyticshellfish poisoning dueto saxitoxin!andof trtriidontish poisoning duetotetrodotoxin! 0!.ln very low doses lc»s than ! pg kgh saxitoxincanproduce rieuromuscular paralysiswithout appreciable hypoten- sion !. Kithtetrodotoxin, effectson these two systems are~nsep~rable occurringtogether, andin parallel degrees. withall doses. Saxitoxin isslightly lesspotent than tetrodotoxin inits hypotensive effects,causing lesscif a fall in bloodpressure, andthe effect isshorter lasting. Saxitoxin hasa greater selectivityforadrenergic nervesthan cholinergic nervesas compared with tetrodotoxin.Thisselectivity ismanifested bythe more frequent occurrence of nerveelicitedvasodilatory response in the skeletal muscle vascular bed. a responsewhich isknown tobe mediated bysome postganglionic cholinergic syznpatheticnerves, Lastly, there is greater tendency, in the case of saxitoxin hypotension,fora late phase ofpressor response toappear. This late pressor responseiscaused by catecholamine released either from theadrenal medulla >r l« , ll; lrrrr» rr Pt! A puss!blv<,}}nickel relevance <>f this last observatirin is that whereas,in cases »f tetrud»n po!siining,hyp<>tendaifthe syrnptornato- logy,in paralyticshellfish poisoning no caseof hypotensionhas appeared in theclinical literature !, My guessis that in paralyticshellfish poisoning the < >ncentrati<>n REFERENCES 1. EVANS. M. H., Io72 T trr> lotpire rttr>tirrrrr 'rcr«l>r6, 2 KA !, 3', lore. T KA !, . Y. Io72. Pharmacologyof tetrorlotozrrrand saxitoxin. Fed. I'r kAt,!, C. >, and l. R. MCCUI.I,.OUGH. 1973. Flectroplrysr'ologic'ai pr<>j>r'rtl 's>l spl .'1!I tlr'rve irr relation to norepirrephrineOverf lot !. J, I'harm<}< >I.exp. Therap. 185: 49-59, kA '}. '!'., and A. NI'SHIYAMA. 1965, Actions of sax}toxin on I ! Allan HancockFoundation Universityof 'Southern California LosAngeles, California ABSTRACT 1hedin«tlagellate Gyntrto<]ittr'urtr f>re;e which is the maj re Igof e Figure Z, Endplatepotential» inFriig nerve-»artiirius musclepreparation soakedin toxin tractionTI at4 TUm] in addition tnZ.5 x f0+d tubocurarinetoehniinate mechanicalresponses tiiindirect stimulation. Oneevoked EPPis imp Figure3. Continuationof Figure2, endof fir»tburst z r! art oo 8' f igiure 4 1!ivt yr.rm vumrnarv ii ipoirt,rrirrrui f:pp~ prriducod m troy, sartiirius nrrvi-rnirirlrfirvparatriin ih preach P MOLE. F SERtt4F 4FTWNL ' ~F f U/ML ~V~/V. 2&0 hM 40 IPrt>! figure!r. l ACKNOWLEDGEMENT Appreciationis expressedto thehard work anddevotion of JaneSpitzer to thisproject. Support for the researchhas been through contracts from the Office of Naval Researchand the USC Sea Grant Program.Grateful acknowledgementsfor cultures are made to Dr. S, M. Rayand to Dr. D. F. Martin, 36] Figuree<.Squ d ax< Figure'. Squidax 362 Figure8..>qu. Figure9 Squid axon. Spike which induces smaJJdamped osciJJations only.Toxin 4 FTU inJ. REFERENCES BEIN, S, ], 1955. Red tide bacterial »tudie», Univ. ot 4'liami Marine Lab. Speci~I Serv. Bull IC: 1-4 2 COLLIER, ALBERT. 1958,Some biceanographi 3. CUMMINS, I, M., and A. A. STFVEINS.;O70. Investigations Gyr»» 4, DAVIS. C . C. Io48,Gyi»»ok»i><»< brevis sp. nov., a cause<~f discoI 5. INC>LE,ROBERT W. 'lo54. Irritant gasesassociated with red tide. Univ. ot 'Miansi I,ah. Special Servic'eBull. o: 1-4 6. JENSON-HOLM, I I. I I. LAUSEN, K. M ILTHERS and KNUD O. MOI.I.ER,1959. I!etermination of the cholinesteraseactivity in blood and 7. MCFAI REN,E. F., H. TANABE,F, J. SILVA, W. B. WILSON, J, E, CAMPBELL,and K. H. LEWIS, 1965. The occurrenceof a ciguatera-likepoison in oysters,clams and G, brevecultures, Toxicon 3: 11-123. 8. MARTIN, D. F. and A. B, CHATTERJFE.1970. Some chemical and physicalproperties of two toxins from the red tide organism Gym»oui»incanbreve, U. S. FishWildl. Serv. Fish. Bull 68: 433-443. 9. RAY,S, M, andW, B.WILSON, 1957, Effects of unialgaland bacteria freecultures of Gyr»»odi»i«rr 10. RAY, S. M.,D. V. ALDRICH, andG. VREVE,1965. Induction of shellfish poisoningin ch~cks,Science 148: 1748-1749. 11. SASVER,l. l., M. IKAWA, F. THURGERG,and M. AI AIN. 1972. Physiol<>gicaland chemicalstudies on Gym»ok»imambreve toxin, Toxicon 0: 163-172. 5A4 Vl:R.I. 1. 1<73.C«mparative stu Iles»nalgal toxm r tnar n , pharmqc~>gn<»y.I'. II?. X'Iartin and G.XI I'a '>lia.Puh.Acad I'ress,,'mi sew York, pp. 12> 17i 13 Sl'IFC ELSTEIN, i%1. 5'., 7.. PASTEIan , iB. C. ABIEC!TT.1<73 I'urificati»nand hi«l >gicalactivity of Gy>»n > firn:!>r ', n>t >mini. I «xic»n 11; 85-o3. STAI Edward S, Ci! fillan Bigelowl.aboratory For Ocean Sciences WestBoothbay Harbor, Maine 04575 Sherry A. Hanson BigelowLaboratory for Ocean Sciences WestBoothbay Harbor, Maine 04S75 ABSTRACT Paralyticshelltish poisoning toxin can afl'ect marine invertebrates in varietyof ways,It appearsthat the roost striking effec.ts were caused by the toxineffects on the animals nerves, ln Filter Feeding spec ies filtiation rates mav begreatly depressed by PSPtoxi~, JNTRODUCTIQ Y, Oneof the problems indescribing theeffects ofparalytic shellfish poisoning PSJ'!toxin on marine invertebrates isthat there is very little published work thatbears directly onthe subject. Perhaps thehest way to approach thesubject isto consider the factors such as selective feeding, varying filtration rates and susceptibilityofnerves toPSP toxin which affect the toxicitv of filter-feeding organismsexposed to a toxicdinoflagellate. Thequestions of which species of i~vertebratesbecome toxic, as welt as which species of dinoflagellates are toxic,is covered exhaustively ina number ofrecent reviews 9,14, 15!. EFFECTSOF SEI.ECTIVF.FEEDING lt appearstobe a generalfeature ofPSP testing that different spec.ies of animalsexposed toroughly similar amounts of'dinoflagellates willfrequently accumulatequite different amounts oftoxin, Once explanation forthis is that whilesome species may feed on toxic dinoflagellates, othersmay find them unpalatable.Bothof the above situations have been reported. Forexample, Buley ! observedthatthe mussel Mytitis caIiforriircnus fedselectively on dinoflagellatesevenwhen they only accounted for some2% of the phytoplanktoniccommunity. Dupuy and Sparks ! repor t that the Pacific oyster,Crassostrecr gigas,does not readily accept thedinoflagelJate Goriyaulax a>ashirigtonensisasfood. Other than these reports there appear tobe no other studiesin the Jiterature. Selective feeding needs further research, EFFECTSOF VARYINGFJLTRATION RATES Anotherpossible source of thevariations in toxicity between different speciesof filter-feeding animals would be a differencein filtration rates for specificspecies. Comparative dataof this sort is difficult to find. One such set ofdata is available fora populationofmussels, Mytilus edulis, from Casco Bay,Maine, and a populationof soft shell clams, Mya arenaria from Ogunquit.During July and August 1973 100 mg M. edutis from Casco Bay filteredanaverage of1875 ml/h; during this same period 100 rng M. areriaria fromOgunquit filtered anaverage of1100 ml/h Gilfillan, unpublished data!. Overthis period mussels ofcomparable sizefiltered approximately 1.63times asmuch water as clams. If one assumes equal efficiencies offiltration, then rnusselsshould gain toxin at 1.6 times the rate seen inclams. This being the 4 0 F 0 I ME ~ PDPIOP4 550Pst t 55 Fh,t R*t 5 EE5 DDODht 'PSJ Pd PJ 0 0 5 55P 515 I 5 P55 545 ~ 00 111 I 'IO 1 ~ 5I 92 P1I 455 POS 54 141J JDJ JJIJ IDS I ~ M 5 5 P 1 5 5541ME555555555 I 1 case,if the ratesof toxin excretionare not markedlydifferent For the two species,mussels shouldbe about twice as toxic as clams of comparablesize for thesame exposure. Qn thebasis of datacollected in I972mussels appear to detoxifymore quickly than clams, yet duringa risein PSPlevel, mussels are alwaysvery considerably more toxic than clams: usually by a factorcloser to IO than2 JohnHurst, Dept. MarineResources, West Boothbay Harbor, Maine,pers, comm.!, Certainly such a situationexisted in Ogunquitin early Septemberof this year whenM, erlrrliswere more than 6 timesas toxic as lVl. arerrariaof comparableweight. Figs,1 and2!. EFFECTSOF SENSITIVITY OF NERVESTO PSP TOXIN Investigationscarried out by Twarog,et al. 6!, suggestthat the relative toxicitiesattained by a groupoF filter-feeding mollusks may be morenearly a resultof diFferingsensitivities of eachof the species'nerves to saxitoxinr'STX! thananything else. Without exception, those species which Twarog, et al. haveshown to bemore resistant to STXaccumulate toxin to a greaterextent in anygiven area than those which are less resistant, The one exception is thecase of Mercenariarrrercerraria, thequahog, whose nerves are considerably more resistant to STX than those of Mya arerraria, the soft shell clam. In Massachusettsduring the 1972 bloom of Corryarrlaxtarnarerrsisiquahogs from Ipswich did not accumulatenearly as much toxin as did soft shell clams from nearbyareas. Whether this situationresulted from selectivefeeding by the quahogsor froma patchydistribution of G, tamarerrsiscan only beanswered b~further work. Thename Cortyaulax excavata has been suggested for this speciesbut not officially adopted. 369 RECENT EXPERIMENTAL RESULTS 4'orkcarried out withM, arena@'aand M. edulisfrom Ogunquit, Maine duringthe September 1974 bloom of Gorjyau]axtamarensis may shed some lighton the relation of the resistance of a speciesnerves to STXto the species' physiology. Mya arrnaria, soft shell clams and Mytifus edulis, blue musse]s, were collected fromthe Oqunquit River, Maine. Clams were collected on 4, 5, 19September and 9 October;mussels were collected on S Septemberand 9 October. 370 Table I Temperaturesalwhich filtration rates were determined foranimals from Ogunquit. Date T oC! 4-5 September 15.0 9 September 14.3 10 September 11.5 Filtrationrateswere determined foras wide a variety ofsizes ofanimal as possibleoneach date. Filtration rateswere determined inful] strength sea water 0 o/oot using methods described inGilfillan !.Temperatures at whichthefi]tration rateswere determined areshown in Table 1.PSP content wasdetermined forthree size ranges withineach species, ThePSP assays were carriedoutaccording tothe methods described inPrakash, etaI, 4!. Results ofPSP assays Foreach sizerange areshown inFigures 1and 2. Results are showninFigures 1and 2 as plots offiltration rateagainst dryweight. Straight linesshown onthe plots arelinear regressions offiltraiton rateonthe cornrnon logarithmofdry weight. In normal clams artd mussels thisre]ation approximatesa straight line. Pointstonote onFigure 1are that on4-5 September thereappears tobe very littie relation between filtration rateand dry weight, Theregression is non-significant r=0.1046, t =0.6896!. Itshou]d alsobe noted thaton 4 -5 Septemberallanimals filtered lowvolumes ofwater. Nine animals didnot filteratall; five ofthese werelarge animals weighingmore than1000 mg.Only 1 outof50 animals filtered morethan 400 m]/h. These arevery ]ow fi]tration ratesforthe time ofyear. Inlate August 1973100 rng clams fromOgunquit Riverfiltered aboutb times asmuch asinSeptember 1974.PSPcontent didnot appear to vary with size OnSeptember 19a highly significant regression r=0.613, t=3,33! of filtrationrateon log dry weight wasobtained. Controldata from 1973 are availableonlyfor small animals weighing about100 mg. For animals ofthis sizefiltration ratesobtained on19 September appearednormal. By 19 SeptemberPSPcontent hadfallen toabout 20%of the values observed on4 5- September, On9 Octobera non-significant regressionwas obtained r=0.361, t =1,397!,Filtration ratesobserved forsmall anima]s 00rng! arecomparab]e withthose observed theprevious fall 973! inOctober, Theseanima]s filtered lesswater onthe whole thanthose collected on19 September. Theprimary reasonforthis isthat by 9 Octoberthewater temperature haddropped to 11.5oC Table 1!and theclams werepreparing toenter their winter period of dormancy,Whateffect these physiological changeshave on the relation betweenfi]tration rateand dry weight isunknown. By9 October theanimals wereessentially clean of PSP. Figure 2 shcws results c>btainedwith Mvtilus edrdis on 5 September and 9 October. On 5 September a non-significant regression of filtration rate on log dry weight was obtained r=0.265, t =-0.91!; larger animals ! 600 mg! tended to filter less water per unit dry weight than smaller animals. Looking at the plot shown in Figure 2 it appears that tiltration approximates a linear function ot log body weight up to a weight of about F00 mg. This trend appears to be reflected in the distribution of PSP in these animals, Animals weighing600 mg or more tend to have abo~t half the PSPcontent of smaller animals.Compared with controlmussels from CascoBay in September1973 small mussels' 00 mg! f'iltration rate was reduced by ra. 50 .o, On 9 October the PSP content of the musselswas < 58 and they appeared in all respectshealthy and normal. A highly significant regressionof filtration rate on log dry weight was obtained r=0,783, t=4.18!. Smaller animals filtered about the same amount of water as on 5 September, larger animals ! 600 mg! filtered more water than on 5 September,all this notwithstanding that the temperatureon 9 October was nearly 5 C cooler, Small 00 mgl musselswere filtering at about the rate observedin Casco Bay, 17 October 1973. Detailed comparisonof thesesets ot data is complicated by both the lack of control data for all size rangesand by the over-riding influence of autumnal cooling. However, it seems clear that both the clams and the mussels were adverselyaffected by intoxicationwith I'Sl' toxin. The clams appearedto be more severelyaffected at lower PSP levels than the mussels.This is in keeping with the findings of Twarog, et al, 6! who found that Mya arerraria nerves were completelyblocked by a concentration of STX tv o orders of magnitudelower than that which had no effect on Mytilris edulis nerves. On 4 5 September M. arerraria in the flats were observed to be very sluggishand to behaveas though they were partially paralyzed.Similarly apparently paralyzedsoft shellclams were reported from both WesternMaine and Massachusettsin 1972.Apparently no suchparalysis of soft shell clams has ever been reportedfrom EasternMaine and Atlantic Canada, Prakash et Q l. 4! ! . Neither McFarrenet al. 2!, Quayle 5!, nor Halstead 9j make any mention of detrimentaleffects of PSP toxin on the affected animals. The only referencein the literatureto deleteriouseffects of PSP toxin appearsto have risenfrom the 196Sbloom of G. tarnarensisoff thecoast of England 0, 1!. Both morbidity and mortality of shellfish were associatedwith the 1968 bloom. Figure3 showsa plot of filtrationrate v dry weight for Mya arenariafrom Black'sHarbor, New Brunswick,These animals were collectedon 25 July 1974; filtration rateswere determinedon 28 July 1974, On 25 July these animalswere assayedat 1400yg PSP/100g. shellfishmeat, yet when their filtration rates were determinedthey appearedhealthy i. e. a significant regressionof filtration rate on log dry weight was obtained r=0.693, t =-2.88!.Aocontrol data for non-toxic clamsfrom this location isavailable. buttliese clams filtered much mere water per unit time than clams of e quivalentweightand half the toxicity fromOgunquit, Theydid not appear sluggishasdid the Ogunquit clamshaving halfthe toxicity, Theabove results posea lotmore questions thanthey answer. Forexarnpl, areCanadian Aibaarenaria moreresistant toPSP because theyhave been chronicallyexposed toit? These results clearly indicate thatmuch Further researchisneeded toclarify therelation between PSPand the physiology oF filter feeding mnlluscs, WRIMErllHIfD ml,rh 2 500 3000 BIBLIOGRAPHY:1 ADAMS, J. A, D. D, SEATON, J B. BUCHANAN, and M. R. I.ONGBOTTOM. 1968. Biological observations associated with the toxic phytcplankton bloom otf the east coast <'< 2. BOURNE,N. 1965.Paralytic shellfishpoison in seascallops Placo!><' 3, BI!LEY, J. M. 1936.Consumption ot diatorns and dino!lagellatesby the mussel. Hull, Scripts Irrst. OcearIogr, U«iu, L«lif. Te 4, CADDY, J. F. and R, A. CHANDLER 1968, Accumulation of paralytic shellfish poison by the rough whelk H«ccini«iran«neat«rn L,! Proc. N 5. COULSON, J. C G, R. POTTS, I. R. DEANS, and S. M. FRASER, 1968. Mortality nf shags and other sea birds causedby paralytic shellfish poison. Nat«re 220 162!;23-24. 6. DuPUY, J. L. and A. K. SPARKS. 1967. Gonya«lax washingtonensis, its relationship to Mytilus catiforr 7. GILFILLAN, E, S. 1973, Effects of seawater extracts of crude oil an carbon budgets in two speciesof mussels.Proc, Joint. Conf. on preventionand control of oil spills. March 13-15, 1973, Washington,D. C. American Petroleum Institute. pp 691-695. 8, GOGGINS, P. L, 1961. Paralytic shellfishpoison. ln Proceedir 9. IIALSTEAD, B. W. ed.! 1965. Poisonousand venomousmarine animals. Voh I. U,S. Gov't Printing Office. pp. i-xxxv, 1-994. 10, INGHAM, H. R., J. MASON, and E. P. C. WOOD. 1968. Distribution of toxin in molluscan shellfishfollowing the occurrenceof musseltoxicity in north-eastEngland. Nature 220 162! 25-27. 11. LOOSANOFF, V. L. 1949. On the food selectivityof oysters. Science 110:122, INot all referencesare cited in thetext; those which are not are goodsources of general information. 374 12.McFARREN, E,F., M. L, SCHAFER, J.E. CAhfPBELL, K.H, I EWIS, E. T,JENSON, andE. J, SCHANTZ 1956; Public health significance of paralyticshellfish poison: A review ofliterature andunpublished research.Proc, Mat. Shellfish. Assn. 47:114-141. 13.NEEDLER, A.B. 1949. Paralytic shellfish poisoning andGonyaulax tamarensis.Jour. Fish. Res, Bd. Canada. 7490-904, 14.PRAKASH, A.,J. C, MEDCOF andA. D. TENNANT. 1971.Paralytic shellfishpoisoning ineastern Canada. Fish.Res. Bd, Can, Bull. 177, pp, i-vii,i, 1-87. 15.QUALE, D.B. 1969. Paralytic shellfish poisoning inBritish Columbia Fish.Res, Bd, Can. Bull. 168, pp. i-x, 1-68. lb. TWAROG,B.M., T. HIDAKA, and H. YAMAGUCHI. 1972.Resistance to tetrodotoxinand saxitoxin in nervesof bivalvemolluscs, Toxicon; 10273-278. PHYLOGENETICGRADATION OF RESISTANCE TO TETRODOTOXIN AND SAXITOXIN IN PUFFERFISHESAND RELATEDFISHES Alan D. Grinnell Departmentof Biology UCLA Los Angeles, California Thepotent nerve poisons, tetrodotoxin TTX! and saxitoxin STX! have beenshown to be extremely powerful, specific blockers ofthe early inward Na'current of action potentials inexcitable membranes, Studies of thekinetics of thisblocking action by Hille ! andby Cuervoand Adelman ! have revealedthat it isa simpleadsorption phenomenon, following the l.angmuir adsorptionisotherm, Other types of ionicconductance channels are not affected,eg. the Ca"channels of'many invertebrate tissues !, orare very little affected,eg, the depolarizing channels ofmechanosensory terminals 1, 12!, chemosensorysynaptic membranes !, and theNa~ - conductancechannel of vertebratecardiac action potentials !. Most interestingis theobservation thatNa' spikesin theanimals that produce TTX, pufferfishesand certain newts,are resistantto TTX but can be blockedby STX 9, 8, 5!, Themolecular basis for this resistance is not known, but might provide importantinsight into the mechanism of actionof TTX and of the channel itself.Further interest isadded by theobservation that normally sensitive Na-channelsin mammalian muscle can become TTX andSTX resistant as ACh-sensitivityspreads across the surface ofa denervatedordamaged muscle fiber 3, 6h Thishas been interpreted asa conversionof some channels from sensitive to insensitive form, Thusthere are several questions posed by the known instances of TTX and STXinsensitivity that we feltwere worth pursuing. For example, is the resistanceall-or-none, or gradedfor any given channel. If graded,can this gradationbecorrelated with toxin concentrations inan animal's tissues, and is thisphylogenetically governed7 ls the resistance due to differencesin toxin binding,orin its effect onconductance through a given channel after binding7 DoesTTX interferecompetitively with STX binding7 Can the resistance be attributedin any given instanceto the appearanceof a new kind of conductancechannel, eg,to Ca~ ~7. These and other questions were addressed, at leastin part, during a recentAlpha Helix Expedition to the Great Barrier Reef,where TTX and STX resistance were examined in several species of pufferfishesandtheir relatives eg. porcupine fish, boxfish, triggerfish!. This workhas been reported indetail elsewhere 0, 2!. I3riefiy summarized, our findings were as follows: Theaction potentials of muscle fibers in thepufferfishes examined were insensitiveto3 x10 5 M TTX,the highest concentration tried. These action potentialsv ere found to be Na-dependent,with no apparentCa+ ~ -component.On theother hand, TTX blockedspikes by 50%in three unrelatedcontrol species at a concentrationof 3 to 5 x 108 M. Pufferfish relativeswere less sensitive than were control fish, but neverthelesswere susceptibletothe toxin. 50% blocking concentrations werein the range of1 to 5 x 10~ M. Theshellfish toxin, saxitoxin STX!, which blocks electrical activityin thesame manner as TTX, was also tried. Pufferfish muscle fibers wereinsensitive to STX, but other fishes showed action potential block at approximately the same concentrationsas with TTX. Pufferfish supramedullarycells SMC! had Na spikes which were not blocked by TTX or 378 STX,in contrast tounrelated fishor the related triggerfish. Bothpufferfish and triggerfishSMCs showed prolonged Ca+- dependentaction potentials in the absenceof external Na~. Thevariable degree ofresistance taTTX shows a phylogenetic dependence, andreflects differences inthe ability ofthe toxin to bindto a receptorsite ratherthan inthe degree ofTTX effectiveness followingbinding. Theability of TTXat sufficientconcentration tocompletely block aetio~ potentials in pufferfishrelatives indicates thatthere are not some sensitive channels, some insensitiveones.The fact that adaptations imparting resistance toTTX also provideimmunity toSTX indicates thatthe two toxins bind in similar fashion tothe same receptor, and are similarly atfected bycharges in the membrane environment, REFERENCES 1. CUERVO,L.A. andLV. J. ADELMAN. 1970. Equilibrium andkinetic propertiesof the interaction between tetrodotoxin and the excitable membraneofthe squid giant axon. J. Gen Physiol, 55: 309-335. 2. EATON,D., A. D. GRINNELLand Y, KIDOKORO,1975, TTX-re- sistanceandCa spikes inpufferfish suprarnedullary cells in press!. 3, FURUKAWA,T., T. SASAOKA,and Y. HOSOYA.1959, Effects of tetrodotoxinonthe neuromuscular junction Jap. J. Physiol. 9:143-152. 4. HAGIWARA,S.and S NAKAJIMA.1966. Differences in Na andCa + spikesas examined by application of tetrodotoxin, procaine, and manganeseions. J. Gen. Physiok49: 793-806. S. HAGIWARA,S. and K. TAKAHASHI,19&7. Resting and spike potentialsof skeletalmuscle fibers of saltwater elasrnobranch and teleostfish, J. Physiol. 190:499-517, 6. HARRIS,J B. and S. THESLEFF. 1971. Studies on tetrodotoxin resistant actionpotentials in denervated skeletal muscle. Acta. Physiol. Scand. 83; 382-388. 7. HILLE,B. 1968. Pharmacological modifications ofthe sodium channels of frognerve. J. Gen. Physiol 51: 199-219. 379 S. KAO.C. Y. 196b. Tetrodotoxin. saxitoxin, andtheir significance inthe studyofexcitation phenomena. Vharrnacol. Rev,18: 997-1049. KAO,C, Y. and F. A. FUHRMAN, 1967.Differentiation ofthe actions of tetrod »»»II RESISTANCETO PARALYTICSHELLFISH TOXINS IN BIVALVE MOLLUSCS BettyM. Twarogand Hiroshi Yamaguchi Departmentof Biology Tufts University Medford, Massachusetts02155 381 ABSTRACT Nervesof Mytiluseduiis, L. and . an certainother bivalves are resistantan toot the blockingeffects of saxitoxin STX!, , an activeprinciple of paralytic sh llf' h oxin TTX!, a toxinderived from pufferfish. It ha b shown that sp ec iesresistant to STX accumulate level feris - ] . t' as h een toxinoxin dangerous a to man. Resistance toSTX and TTXs o parais a yticro sert ellfish individual nervefib ibers, and d is not due to a protectivesheath around thefibers. Sodiumdeficiency reduces andblocks theaction potential, sothe resistance oes not dependon developmentof a non-sodiumsspi ike- e-generating mec anism. Physiological,biochemical, ecologica!, andbehavioral problems with respectto toxinresistance and accumulation will be discussed. Saxitoxin STX!; Mode of actionand structural refationshi s ip too te t ro d oto~iri TTX! . Saxitoxin STX!, a powerfulneurotoxin, isa major constituent ofparalytic shellfishtoxin. It wasfirst isolated from the Pacific butter clam, Snxidornus nuttalfi Conrad! and later identified in Gonyaulaxcatenella bySchantz and associates 6! G. tarnarensiscontains STX, and in addition,an unidentified toxin perhapschemically related to STX Evans,7! Narahashi personal communication!has calledthis unidentified toxin Gonyariiaxtarnarensis toxin GTTX!. The mode of action of STX and GTTX are similar to that of tetrodotoxin TTX!, the activeprinciple of the toxin of pufferfish Sphaeroides!and the Ca!ifornianewt Tarichatorosa: Both STX and TTX block conductionof thenerve impulse by reversiblyinterfering with the early voltage-dependent increasein sodiumion conductancewhich generatesthe nerve action potential, 3!. Conductionblock leads to paralysis.This paralysis superficially resemblescurare poisoning but is fundamentally different since curare selectivelyblocks neurornuscularand synaptic transmission and does not affect conduction in nerve fibres. STX and TTX do not interfere with synaptic transmission. The molecular structure of STX has been deducedby Won' and associates 0!. Further information on the chemical structure of STX and GTTX is presentedin this symposiumby Schantz. Comparative studies of sensitivityta STX and TTX in mollusc nerve. In view of the fact that somemolluscs are exposedto large amountsof paralytictoxin and nevertheless survive, it wasof interestto analyzethe effects of the toxin on their neuromuscularsys terns. It hadbeen found previously that TTX doesnot block the action potential in theanterior byssus retractor muscle of Mytilus edulis Linnaeus!;and it had been suggestedthat the spike-generatingmechanism in this muscle depends on increasedconductance tocilrium rather th.in sodium ions t lit,Siii sequent~ei iien eviirnieevil n ittpporteu tr t~ suggestion 19! It wasalso cibserved thatnerve fibered tnAfynlti ~ere restitartt toTTX lt wastherefore ofinterest tofind that incontrast tiimuscle thespike-generating mechanismisdependent onsodium tons.The possibilttv thatthe neural sheath mayprotect thenerve fibers was excluded inexperiments whichshowesl that "naked"nerve fibers, wtth neural sheath stripped off,are as insensitive toTTX as normallysheathed fibers. Since the put terfish, which secretes and accumulatesTTX,has nerve fibers which are resistant tothe toxin, itseemed possiblethatTTX resistance irtJVlytilus nerve might be related tothe ahilitv Mytr'lustofeed on G. catene0a andCi. tutrturrnsrs andto accumulate large quantitiesofparalytic shellfish toxin, containing STX, which resembles TTXin structureand mode of action. The sensitivity toSTX was tested and indeed. Mytilusnerve was resistant. Since Kao and Fuhrman 2! hadfc und that the pufferfishisselectively immune toTTX but sensitive toSTX. ttwas surprising to find Mytilus resistantto both. Presumablya bivalve mollusc would be exposed only to STX under normal conditions.Does cross resistance to STX and TTX developin molluscs.in contrastto the pufferfish, on the basis of similar chemical structure of STX and TTX7Alternatively, isMytilus resistant toz toxicprinciple very similar to TTXwhich remain» unidentified in paralyticshellfish toxin that is, GTIX Narahashiand Evans in these proceedings!. ln order to answer some ot these questions,fTX andSTX sensitivity were compared ineight species ofbivalve molluscs.These data are discussed along with preliminary observations on toxinaccumulation and behaviorin someot thesespec tes during the September,]972 outbreak of paralyticshellfish pi»soning in lvlassachusetts A contparisoitaf sensitivityfo STX and TTX in f ti alt e mi>llus~~ Themarine species tested were Mytilus eifulis Linnacust, the bay musselor ediblemussel: Modiolus dernissus Dillwynh the ribbedmussel; My~ arenartti Linnaeus!,the softshellclam, steamer;Merrenaria rriercenaria Linnaeus!, the quahogor cherrystoneclam; Placopectenrrtagellarttcus Gmelinf. the sea scallop;Pectert irradiarrs Lamarck!, the bay scallop, and Criissostreiavi rginicv Gme!in!, the edibleoyster. Thesewere collectedeither at Nahant or Woods Hole, Massachusetts.The freshwaterclam, Elliptio complanata Lightfoot} was obtained from the ConnecticutValley Supply House in Massachusetts. The experimental method has been describedelsewhere t]Hi. Effectsof STX arrd TTX ort conductionof the rtertieirnpul~e Figure1 showsrecords of compoundaction potentials in desheathedvisceral connectivesof Nfytilusand Pbcopectert,two specieshighly resistant to STX. After ten minutesin 10 g perml STX, theaction potential is normal.No block was observedeven after thirty minutes, RES lSTANT SPEC1ES PLACOPFCTEN MAGELLAN lCUS SEA SCALLOP! MVT1LUS EGULtS 4IUSSEL! CONTROL STX 10~ 9 m lOMINS! Figure1. The effect ofSTX on compound action potentials innerves ofresistant species. Notethat action potentials in10 gper ml STX do not differ from the control, 5EN5lTJVE5PE :lEQ CeaSSOSTaeav tt0 HKA OYSTER! ST X WASH CONTROL STX 0st!HS! 'I0+9 m 10-Tgtml 0 M NS! !M! N! Figure2. The dfect ofSTX on compound actionpotentials innerves ofa sensitivespecies Notethat block is completeat 10 ~ g perml. RELATI VELY 10 ~ 5 MINS! l NSEhlSITIVE SPECiES llo-5 I MY A ARE NAR A ' 5;EAMERC LAN' 'O-5 l OM NS! PECTEN!RRAD ANS AY SCaLLOP! ' O 4 MERCE NAR!A l '. MERCENARI A QUAHOG! CONTROL ST!l 9 ml! STxlqrml PARTtAL FULL RECOYERY 3LOCK 3LOCK Figore3.The effect of STXon compound action potentials in nerves of relatively insensitivespecies. Records of partialblock are shown,at concentrations indicated,Full block occurred at the concentrations indicated in parenthesis: at 10 tn Mya, 1Cr inPecferr, and above 10 g perml in Mercenariac Figure2 displaysresults obtained with thehighly sensitive Crassostrea. The actior potential is almostcompletely blocked at 10, andfully blocked at 10 g per ml STX, Similarresults were obtainedwith Hltptio, The remainingspecies resisted full blockby STX untilconcentrations of 10 ~ g perml or morewere applied. Fig, 3 showsrecords obtained with Mercetmria, Pecten,and Mya, whichare all consideredrelatively insensitive, although not f'ully resistantto STX. Sirrtilar experimentswere performed with TTX. In all cases,it is apparent that the blocking effect is reversed after washing off STX or TTX. Sinceit seemedpossible that resistancein somespecies might be dueto limitationof TTX or STXdiffusion by a protectivecovering around the nerve fibers,all theexperiments were done using nerve fibers from which the neural sheathhad been removed. In someexperiments, resistance was studied before andafter removal of theneural sheath. No significantdifference was noted. Thus,resistance to TTX andSTX is a propertyof thenerve fibers, not the sheath. ERR Table l Blockof actionpotential by saxitoxin STX! Species 1O~ 1O-'7 1O~ 10~ 104 g/ml I Mytilus edulis Placopectenrrragellarticus Mercerrarfa rrrercenarfa Modiolus derrrissus Pecten irrad arts Mya arerraria Crassostreao rgirtica Hl t>ti o corrrplurrata Thespecies arelisted in orderof increasing sensitivtty toSTX. i = Full Block. lf theaction potential ofthe resistant species isnot generated byan increase inconductance tosodium ions, then resistance toTTX or STX could easily be explained,since these substances specifically block the early sodium current 3!.The desheathed nerves ofall resistant and sensitive species were exposed tosodium deficient andsodium free solutions, lnevery case sodium deficiency reducedand bltrcked the action potential within minutes. Relativebloclcirrg poterrcies of STX and TTX. Tablesl and 2indicate theconcentrations ofSTX and TTX required fully to blockthe compound action potential in various species. Resistance to STX roughlyparallel» TTX resistance, Myt lusand Pfacopectert are resistant to bothtoxins, Crassostrea andElliptio are highly sensitive. Among the species relativelyinsensitive toSTX, the correlation isless striking: STXresistance, MercerIar >Modiolas a >Pecterr >Mya TTX resistance,Mya» Modiolus> Mercerraria7>Pecten. With respectto TTX,Mya shouldbe classed as resistantand Pecten as sensi tive. Table 2 Blockof action potential bytetrodatoxin TTX! Species 10-8 107 10-6 1P-5 g err!l!, Mytilus edu!is P1aco pect err rnage11anicus Mya arenaria Alodiotus dernt'ssus Mercertarra rrtercenarta Pecten irradians Crassostreavirgittica Eltiptiocon!ptanata Thespecies arelisted in order of increasing sensitivity toTTX, + = Ful!Block. Table 3 WholeBody Accumulation ofParalytic Shellfish Toxin Spenes STX content Maximum Location Jigpef S7X g 100 g per m} Afytilus ednlis York Harbor, Me. edible mussel! 10,092 10+ MerrirnackRiver es tuary 7. 392 7 x 1O-5 Essex, !v}ass. 7,200 7x 10 S Eastham. Mass. 0 to "high" 0 Mercenarra mercenaria Eastham, Ma ss, Quahog, cherrystonec}am! Pecten irrad>ans Eastham, Mass. bay sca}}op! 2. 040 2 x 10"S JV}yaa renaria York Harbor, Me, steamer.htt!eneck clam! l. 726 2 x ~ " vterrimackRiver estuary 9. 600 10< ' ' Hampton Beach. N, H. 6,000 6 x 10 5 "Essex, Mass, 3,500 35xlOS Crassostreauirginica Eastharn,Mass. 0 0 oy stet'! Great Bay, N. H. 56 6x 7 ' Quarantine}eve}-. 80!tg per 100 g ' ' Manyh4ya were "weak"; siphons were f}accid. Effectsofaccumulation ofparalytic shellfish toxin on the molluscs themselves, Duringthe Red Tide of September,1972, along the Northeast coast of the UnitedStates, some observations were recorded on therelationship between wholebody accumulation of paralytic shellfish toxin and behavioral symptomsin the molluscs !. Table3 summarizestheobservations. The level of toxinis calculatedas rnicrogramsofSTX per 100 g ofwet tissue pg per 100 g!, on the basis of measurementsof mouse-toxicity of extracts of wholemolluscs, It is probably thattoxins other than STX are being detected, causing an overestimate of STX. Themaximum concentrations of STX in thewhole volume of softparts are calculatedfrom the data, on the assumptionthat the toxinis uniformly distributedthroughout the body of the mollusc.This assumptionis convenient,butquestionable. Since a singlemeasurement inPecten! showed thatthe concentration of toxin in musclewas about one-tenth of thewhole anima!concentration, it is probablethat the actual concentration in muscle and in nerveis lessthan the concentrationin the wholeorganism. Resistanceto STXand TTX as relatedto paralyticshellfish poisoning. Speciesshown in thepresent study to haveSTX and TTX-resistant spike mechanismshave all beenimplicated in outbreaksof paralyticshellfish poisoningin humans. Halstead S! and Bourne ! indicatedthat species implicatedin massoutbreaks of paralyticshellfish poisoning involving fatalitiesinclude M. edulis,Mya arenaria, and Placopecten rnagellanicus. Of these,Mytilus and Placopecten are resistant to STXand TTX; Mya is relativelyinsensitive toSTX and resistant to TTX.!n contrast Crassostrea, an ediblespecies highly sensitive to both toxins, has never been implicated in paralytic shellfish poisoning. We suggestedearlier S! that speciesresistant to STX and TTX can accumulatethese toxins without harmwhile sensitivespecies cannot without 'themselvesbeing poisoned unless the toxin can be prevented from reaching the nervoussystem in anactive form!. This idea is supportedby thefield data, whichshow that Mytilusaccumulates the highest levels of toxin of all species studied,yet showsno overtlyabnormal symptoms whatsoever; whereas, whensamples of Myashowed levels of toxinabout 2000 pg per 100g, many clamswere obviously abnormal, with siphonsflaccid. That the Mya were indeeddisplaying paralytic symptoms due to STX is supportedby observationsthat theclams recovered within hourswhen restored to toxin-free seawater.Rapid complete reversal would be expectedafter nerve block by STX,but not if theclams suffered from a moregeneral metabolic poison. In no casedid theamount of toxinaccumulated by thesensitive Crassostrea exceed 5bpg per100 g eventhough it wassurely exposed to theRed Tide. It is interestingthat Mercenaria, although resistant, also did not accumulate toxin; perhapsit escapedexposure to Gonyaulax, 3BB Mechanisms of Resistanceto STX arid TTX. A. The physiologicalbasis of resistance, ln the speciesstudied, resistance is not conferredby an impermeableneural sheath, nor does it dependon abandoninga sodium mechanismfor the generationof theaction potential. One mechanism could be specific resistance of the sodiumchannel to the toxins. Kao and Fuhrrnan 2! found that the pufterfishand the newt haveswerves which are highly resistantto TTX but sensitiveto STX. In our observations,on thecontrary, the resistanceof several speciesto TTX seemsto parallelresistance to STX, There couldbe a cross-resistanceto the closely related rnolecules of STX andTTX in molluscs. Resistanceto GTTX, thethird toxin, has not yet beenstudied, Further research may revealthat the sodiumchannels of the nervesof resistantspecies are associatedwith a substanceor structurethat impedesaccess and prevents binding of the large toxin rnolecules.Alternatively, the molecular characteristicsof the channelsmay be specificallyaltered to preclude interaction with STX or TTX. Thislast question can best be approachedby biochemicalstudies of the site s!of attachmentof thetoxins to the nervemembrane. Methods for doing sohave recently been cteveloped. Isolation, from speciessensitive to TTX and STX, of protein and/or lipoproteinfractions of nerve membranesthat specificallybind thesetoxins has recently been described by Hendersonand Wang 9!; Benzerand Raftery, , 3!; Barnolaet al, ! and Henderson,et a1. 0!. Also Benzerand Raftery , 3!; have studiedthe susceptibilityof the TTX-bindingcomponent ofgarfish olfactory nerve membrane to a numberof enzymes.Their data suggest that there exists some phospholipid component, aswell asprotein, in thespecific binding site of TTX andpresumably STX! to the sodium channelsof garfish olfactory nerve. ln somespecies toxins may be stored in sucha wayas to reducethe levels of toxin in the circulation,thus protecting the nervoussystem from exposure. Sucha mechanismhas been described by Priceand Lee 4,1S! in Saxidomus, wherethe toxin is sequesteredin pigmented granules in thesiphon, Norris and Chew in thisconference have cited another example of this mechanism,the accumulationof toxinselectively in thedigestive gland of theliest Coastrazor clam,The musculartissues contain no toxin, and thusthe clam is safeto eat once cleanedof the digestivegland, B. Geneticor acquiredresistances Possiblyspecies which are periodically exposed to dinoflagellateblooms have evolved mecharusmspermitting them to exploit the toxic organismsas food.It wouldbe interesting toknow if resistanceto TI X andSTX is acquired on exposureto the toxinsor is inherited.It seemslikely that exposureto din»flagellateblooms means exposure to STX,not TTX; many of thespecies studied never encounter TTX in their natural habitat. However exposure to a TTX-likesubstance is not ruledout, GTTXas reported by Narahashiin this conferencemay be identicalto the neurotoxindetected by Evans ! in poisonousMyti4s. The neurotoxin described by Evans isnot identical with TTX,and blocks action potentials in Tarichatorosa, a speciesresistant to TTX but sensitiveto STX.Isolation and identification of thissubstance may clarify theproblem. lt wouldbe of interestto studyNiytifus from an areawhich is knowntobe free of RedTides, Unless the resistance is a geneticcharacteristic of thespecies, one would expect to find manysensitive individuals. In a previous study I8! my associatesand I found nu correlationbetween systematic relationshipand resistance to STX or TTX. Some sensitivity to TTXwas noted amongindividual Nfytifus from Maine in contrastto thosefrom California and Massachusetts,but no data were found in the literature on relative exposureto Ra rid .. Ecol Amongthe sensitivespecies, ecological isolation, that is, lack of exposureto theset«xins. would permit survival, Gorryaulaxis found only in a marine habitat. The fresh water speciesare very sensitiveto STX and TTX. During bloomsthe algaAphariiromenoit f los-aquae,in freshwater habitats was found by ]ackim 8rGentile l ]! to releasea toxinsimilar to STX. However,we found no rep»rtsconcerning whether or not fresh water molluscsdie during these algal blooms. and no toxicity data are availablesince these molluscs are not considered edible. Crassastrra in very sensitive to STX and TTX, and is surely exposedto blooms of Gnrtyaufax. lt is probable that a behavioral responseprotects this species. Crassostrra gigas has been shown by Dupuy k Sparks ! to cease filtering when exposed to Goriyaulax. The oyster shuts its valves tightly and d«esn»t resumefiltering until the water is clear. However, after prolonged exposure the oyster filters minimally. lf Mytitus and Crassostrea are both exposedto the toxic dinof'lagellates,Myti4s accumulatesmore than four times as inuch toxin as does Crassostrea,Protective behavioral responsestnay well be «und in other sensitive marine species. Dunng th» 1972Red Tide in Massachusetts,Mercenaria was never found to accumulate tcixin, even in heavily infested areas i,e. localities where high t»xin I vels were found in Mytifus and Myaj, Since Mercenaria is resistant, it could theoreticallyaccumulate toxin without harm to itself. lt is possiblethat Merce«ariais protectedfrom exposureby its habitat, whichis deeperthan that of Mya and'or by its feedinghabits; unlike Mya and Pectert,it may not be a surfaceteeder or it may reject detritus. This interestingproblem deserves further exploration. An important observationwas madeduring the 1972Red Tide emphasizing that not on!y habitat but behavior feeding habits! determinetoxin accumulation;A number ofspecies col'lected ata depth of35 feet contained ntorethan 1000wg per100 g toxin.Since Gonuaylux isnot known topene trite tothis depth, it seems possible thatthese species filtered detritus towhich th» toxinwas absorbed. Deepwater species which do not filter detritus cnuld not be exposed. CONCLUSION An investigationof resistance to STX and TTX in bivalvemolluscs has contributecttoour understanding of accumulation ofparalytic shellfish toxin duringRed Tides. Continued studies of the neurophysiology. ecology, and behaviorofmolluscs and other marine organisms during exposure toRed Tide shouldprove to beof considerablepractical value and theoretical interest. ACKNOWLEDGEMENT The investigationwas supported by U. S. NationalInstitutes of Health Grants AM 11996 and NS 10554. I amindebted to Dr. E.J. 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