Comparison of two domoic acid-producing diatoms: a review

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Hydrobiologia269/270: 213-224, 1993. H. vanDam(ed.),TwelfthInternationalDiatom Symposium.

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@ 1993 Kluwer Academic Publishers. Printed in Belgium.

Comparisonof two domoic acid-producing diatoms: a review M.C. Villac I, D.L. Roelke1,T.A. Villareal2 & G.A. Fryxelll 1Departmentof Oceanography, TexasA&M University,CollegeStation, TX 77843-3146, USA; 2EnvironmentalSciences,BostonHarbor Campus,Universityof Massachusetts, Boston.MA 02125-3393, USA

Key words:Pseudo nitzschiapungensf. multiseries,Pseudo nitzschiaaustralis,domoic acid, neurotoxin, amnesicshellfishpoisoning

;

Abstract In the past five years,awarenessof domoic acid has increasedfrom localized problems in Canadato outbreaksalong both North American coasts. The phycotoxin domoic acid causesAmnesic Shellfish Poisoning (ASP) in humans and can be fatal. The known speciesof phytoplankton responsiblefor production of domoic acid include somepennatediatom speciesof the genusNitzschia,sensulatu, which form steppedchains typical of the Pseudonitzschia. Thesediatoms are widely distributed, but their life historiesand populationdynamicsarepoorly understood.This reviewaddresseshistoriesof occurrences, morphology,geographicaldistributions, seasonalpatterns, growth requirements,domoic acid production, and trophic interactions,with emphasison a comparisonof Pseudonitzschia pungensf. multiseries (Hasle) Hasle and Pseudonitzschia australisFrenguelli.Through continuedresearchit will becomepossible to provide guidelinesfor regulatory agenciesthat protect both the consumer and the seafoodindustry.

Introduction The genusPseudonitzschia was describ~dby H. Peragallo(Peragallo& Peragallo,1897-1908)and typified by Pseudonitzschia seriata(Cleve)H. Peragallo(= Nitzschiaseriata)(Fryxell et al., 1991b). Thereis somedebatewhetherto considerPseudonitzschiaa separategenus (e.g. Hustedt, 1930; Frenguelli,1939;Hasle, 1993)or a sectionof the large, diverse genusNitzschia(e.g. Hasle, 1965; Takano & Kuroki, 1977; Ricard, 1987).Nevertheless,the group is uniform in its marine planktonic habitat, eccentriccanal raphe systems,and elongatevalves with a growth habit of clonal chains formed by the connectionsfrom overlap-

ping tips of cells (refer to discussionbelow). On the other hand, when one examinestheir occurrences,it becomesevident that severalmembers of Pseudonitzschia and closely related Nitzschia presentdiversegeographicaldistribution and distinct types of ecologicalrelationships: N. granii Hasle and N. norvegicaHasle are found in Phaeocystisgelatinouscolonieson cold northern waters (Hasle, 1964);N. interruptestriata?Simonsenon Thalassiosira threadsin gelatinouscolonieson the Gulf Streamwarm corerings(Fryxell et ai., 1984); N. lecointel[VanHeurck on Thalassiosiragravida Cleve in gelatinouscolonies at the Antarctic ice edge during the austral spring bloom (Fryxell, 1989); and Pseudonitzschiaamericana (Hasle)

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~ 214 Fryxell (= N. americana)in Chaetoceros setaeon the coastal Gulf of Mexico (Fryxell et al., 1990). Other Pseudonitzschiaspecies have become known for their toxin production (Table 1). Pseudonitzschiapungens f. multiseries (Hasle) Hasle was the cause of Amnesic Shellfish Poisoning (ASP) in Canada in 1987 (Bates et al., 1988, 1989; Subba Rao et al., 1988). This first was thought to be an isolated occurrence,since severalrelatedspeciesof diatoms testednegative for domoic acid production in the laboratory (Bates et al., 1989; Fryxell et al., 1990; Martin etal., 1990; Fryxell etal., 1991b; Kang etal., 1993).However, several additional Pseudonitzschia specieshave now been implicated in toxic events from field observations or cultures (Table 1). Of particular interest was the occurrence of domoic acid on the west coast of the U.S.A. in 1991,concurrent with large Pseudonitzschia aUj'tralis Frenguellipopulations.This event was evident as pelicansdied with their stomachsfull of anchoviesthat had been eating the P. auj'tralis (Work et al., 1993).In view of the danger from domoic acid on the North American coasts and possibleproblems on similar coasts around the world (seebelow),we find it essentialto compare the known occurrences,morphology,growth conditions, and toxin production particularly of P. pungensf. multiseriesand P. australis,which are now known from field obserVationsand culture studies. Although these two taxa may not be uniquein productionof this neurotoxin,theyhave beenintensivelystudied,and it is our objectiveto

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help pinpoint researchareasof vital importance in order to formulate future predictive tools. It is important that researchprovidesthe background for regulationsto protect consumersand fisheries around the world.

Histories of occurrences The first outbreak of domoic acid occurred in eastern Prince Edward Island, Canada, in late fall-early winter of 1987. The symptoms of the intoxication to humans were vomiting and diarrhea, followed in some casesby confusion, disorientation, and memory loss, which led to the name" Amnesic Shellfish Poisoning". Three peopledied, and somevictims sufferedfrom apparently chronic short-term memory loss (Teitelbaum et al., 1990). The detailed chronology of this ASP event(Addison & Stewart, 1989;Todd, 1990)and the epidemiologyof the poisoning(Perl et al., 1990)have been describedelsewhere.The sourceof domoic acid was traced to the diatom P. pungensf. multiseriej',and the vector to humans was the blue-mussel M)'tiluj' edulis Linnaeus (Bateset al., 1988,1989;SubbaRao et al., 1988). Despite the recurrenceof domoic acid in Prince Edward Island during autumnal blooms of P. pungensf. multiseriesfor the following threeyears, ASP caseswere preventedby a monitoring program for early warning of increasingdomoic acid levels(Bateset al., 1989;Smith et al., 1990a). The Canadian ASP eventsprompted officials to monitor the northeastcoast of the U. S.A. for

TableI. Pseudonitzschia speciesreportedto producedomoic acid. Species

Location

Reference

P. altstralisFrenguelli (=N. pseudoseriata)

Monterey Bay. U.S.A.

Buck et al. (1992); Garrison etal. (1992)

P. delicatissima (Cleve)Heidenin Heiden& Kolbe ( = ,v. actydrophila)

PrinceEdward Island, Canada

Smith etal, (1991)

P. (Hasle) Hasle ( =pseudodelicatissima N, delicatula)

Bay of Fundy, Canada .

Martin (1990); Haya etet ai,al.(1991)

p, pungen.~ f. multiseries(Hasle) Hasle (= .V.pungensf. multiseries)

Prince Edward Island, Canada GalvestonBay, U.S.A.

Bird etltl. (1988); Fryxell et al. (199Ia)

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~.'.' ..

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215 domoic acid. At present,althoughan outbreakof the toxin has not been reported, severalorganisms have tested positive for domoic acid. Cultures of P. pungensf. multiseries,isolated from MassachusettsBay,haveproducedthe toxin (Villareal et al., in review). Domoic acid was also found in sea scallops from Eastport, Maine (Shumway,1989),andin bay scallops(Argopectan irradians Lamarck) and blue-musselsharvested from Nantucket Shoalsand GeorgesBank areas, Massachusetts(Nassif & Timperi, 1991).Except for one specificsite at Eastport, domoic acid was found in concentrationsless than 20 ,Ltgg -1, the regulatoryguidelinefor human consumptionestablishedduring the first eventby the Health and WelfareMinistry of Canadafrom estimationsof amount ingestedby those hospitalized. In Galveston Bay, Gulf of Mexico, the population of P. pungensf. multiserieshas beenmonitored sinceFebruary1989.It is alsopresentin an archivedcollection from 1974,which showsthat it has not beenrecentlyintroduced, and that it is well adaptedto survivalin this area(Fryxell et al., 1991a).It occursat low densitiesthroughoutthe )lear (increasingduring the colder months), and producesdomoic acid in culture (Fryxell et al., 1991a;Reap, 1991;Dickey etal., 1992). The domoicacid outbreakthat occurredon the west coast of the U.S.A. during the (all of 1991 was tracedto the diatom P. australis(Buck et al., 1992;Garrison et al., 1992).The die-off of pelicans(Pelecanus occidentalisRidgway)and cormorants (Phalacrocora."\" [Brandt]) in . penicillatus . . Monterey Bay, CalIfornia, which had fed on con. d h . tammate anc ovles(Engrau/.rs mardax G.Irar,d) I . (F . 11992Wk I gaveearywarnmg ntzeta., ; or eta., 1993).Toxic levelsof domoic acid weredetected in razor clams(Siliquapatula Dixon) and Dungeness crabs (Cancer magister Dana) harvested along the coasts of California, Oregon, and Washington,leadingto the closureof commercial

egon),and the mouth of the Columbia River (Villac et al., in review).Only a dozen casesof illness in humansassociatedwith shellfishconsumption were reported, with mild gastrointestinal symptoms and one complaint of memory deficit (Anonymous, 1991). As for the other diatoms that may produce domoic acid, Pseudonitzschia pseudodelicatissima (Hasle) Hasle, Pseudonitzschiadelicatissima (Cleve)Heidenin Heiden & Kolbe, and Amphora coffeaeformisAgardh, the information in the literature is sparce.During August-October 1988, domoic acid was detected in blue-musselsand clams (Mya arenariaLinnaeus) from the Bay of Fundy, New Brunswick,Canada;in this case,the toxin sourcewas traced to the dominant diatom in the plankton at the time, P. pseudodelicatissima Hasle (Martin et al., 1990;Haya et al., 1991).No casesof ASP were reported due to an intensive monitoring effort and timely closures (Gilgan et al., 1990).Both P. delicatissima(Smith el al., 1991)and A. coffeaeformis (MarandCl. et al., 1990) producedtrace to small amountsof domoic acid in culturesof clonesisolatedfrom the Prince Edward Island area(respectively,5 x 10-3 pg and 0.2 pg of domoic acid per cell).

and recreational fisheries in those shores (Horner

4-5 ,Ltm.Coarse striae, 10-13 in 10 ,Ltm; striae

& Postel,1993;Wood& Shapiro,1992).Withthe

and fibulae in approximatelyequal numbers,

,

exceptionof the bloom in Monterey Bay, P. australis was found only at low densitiesin some coastal sites that were investigatedat the time, such as Newport (Los Angeles),Coos Bay, (Or-

sometimesdisplacedin relation to each other. Electron microscope:Interstriae membraneperforated by three or four, seldom two, rows of closely spacedporoids, 4-6 in 1 ,Ltm.

1

Morphology and geographicaldistributions The morphologiesof both P. pungensf. mu/riseries and P. auslralishave been studied in detail by Hasle (1965) and are summarizedbelow: Pseudomtzsc . hra . pungens f . muItrserres .. (H asIe) H I F. 1 d 2 (H I 1965. 14- 15. I ., as e, IgS an as e, . p. , p . -. I 6 fi 4. I 7 figs 1 an_,p.,gs d~.,. I 5 fi 10- 12.,p.,g.,p., fi 9-11) gs , . Light microscope:Frustules in stepped chains formed by overlappingof the cell ends.Valve and girdle views with a linear-lanceolateshape.Central nodulus absent. Length 68-140 ,Ltm,width

: i

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Fi",!.\" J--', Figs 1 :1nd 2, P,elldll//itz.\"('hiapt/llc~en".f. mll/tilerie.~: clone TV-14 isolated from Massachusetts Bay (Fig, I). :1nd clone F-307 i~olat.:d from Galveston Bay (Fig. 2). Figs 3. 4. 5. Pselldlmitzschiaall.l/ra/i.\":.:Ionc ORI-5 i~olated from the mouth of the Columbia River (Fig, 3). ~pecimen from net haul from Monterey Bay (Fig. 4) :1nd from net haullrom Newport B.:ach. Los Angeles (Fig. 5), Scales tor Figs la. 2a. 3.4. and 5a = 10 11m.Scales for Figs lb. 2bc. and 5bc = 111m.

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217 Pseudonitzschiaaustralis Frenguelli, Figs 3-5 (Frenguelli,1939;Hasle, 1965:p. 11; pl. 4, figs 3 and 4; pl. 5, figs 1-6; pl. 6, fig. 1). L.h . . stepped cams. h . Ig t mIcroscope:FrustuIes m db I . f th II d V I l' lorme y over appmg 0 e ce en s. aves I. I I . h .ddl ( b mear to anceo ate, wIt mI e part a out oneth O d) I II I .d II d I. h I Ir more or ess para e SI es; ce en s s Ig t Y d d d . F I I. rostrate an roun e at tIp. rustu es mear to . dl h . . dl . L h spm e-s apem gtr e vIew. engt 75- 144J1m, width 6.5-8 J1m.Canal raphe eccentric,central . .. . nodulus absent. FIbulae and strIae m apprOXI.

matelyequalnumbers,12-18 m 10J1m.Trans-

" . verse strIae of the mtercalary bands strongly SII . .fi d d h I I d h ICI e an somew at more c osey space t an

th f hit ose 0 t e va ves.

Electron microscope:Interstriae membraneperforatedby two rows of fairly largeporoids at each side of a striae,4-5 in 1 J1m. As already mentioned,Pseudonitzschia was first describedas a genusby H. Peragallo(Peragallo & Peragallo,1897-1908),and later considereda sectionof thegenusNitzschia(Hasle, 1965).Since the genusPseudonitzschia has beenreconsidered (Hasle, 1993),we have already updated the nomenclaturefor these species.Most of the literature on this subject,however,refer to someof the diatoms in questionas Nitzschia.

with the valveendsdistinguishesP. australisfrom P. pungens(Hasle, 1965; 1972). Distributional patterns add to the understanding of the existenceof different species, . and are basedon thosefew studIesthat presentmforma. . tIon at the ultrastructure level. There IS now an . .. mcreased need for more detaIled taxonomIC work . . . ~

on the Pseudomt.;.schla spp., due to theIr cosmo.. polttan dIstrIbutIon and potentIal as toxm prod UTcehrs... .. e ongmaI descnptlon 0f P . pungensf . muItl-. . (H asIe, 1965)was made on materIa senes . I lrom ~ five Iocatlons: . . (0 sI0tiJord, t he N 0rth A t IantIc N Ch k B U SA ) h S h orway; esapeae ay, ..., t e out A IantIc . (A t Ianti.da, U ruguay,. Quequen,A rgentina), and the North Pacific (Oregon,U.S.A.). In addition to thoserecords,P. pungensf. multiseries has beenfound in Prince Edward Island, eastern Canada; off Brook Point, Vancouver Island, western Canada; Massachusetts Bay, eastern U.S.A.; Gulf Stream warm-core rings; Puget Sound,Washington,and Monterey Bay, California, western U.S.A.; Galveston Bay, Texas, northwestGulfof Mexico; Jinhae Bay, off southeastern South Korea; and Ofunato Bay, Japan (T akano & Kuroki, 1977; Kaczmarska et al., 1986;Bateset al.. 1989;Forbes& Denman,1991; Fryxell et al., 1990;Buck et al., 1992:Horner & Postel, 1993;Villareal et al., in review).These10-

As Hasle (1972) pointed out, although the

cations reflect that this toxic species is found in

uniquetype of colony-formationand shapeof the frustulereadilyidentify a diatom as a Pseudonitzschia,differentiationat the specificlevelrequiresa detailed examinationof the outline and the fine structureof the valve.P. pungensf. multiseriescan be easilymistakenfor its nominate form P. pungens f. pungens(Grunow in Cleve & Moller) Hasle, which has been shown to be narrower (74-142 J1mlong and 3-4.5 J1mwide), to have 9-15 striae in 10 J1m,and to have 2 rows of interstriae poroids (Hasle, 1965; Fryxell et al., 1990).P. australisand P. seriataalso show great similarity.The shapeof the valve,mainly the rostrate ends,and the distribution of interstriaeporoids distinguishesP. australis from P. seriata, whosevalveis alsomore asymmetricand has3-5 rows of poroids, 7-8 in 1 J1m;the width together

the Atlantic waters of North America, Europe, and South America, and in the Pacific waters of North America and northeastAsia (seedistribution map in Fryxell et al., 1990: 178, Fig. 5). P. pungensis characterizedas mostly confined to' coastal waters (no distinction betweenforms is referred to here), and seemsto extend further north in the easternthan in the westernparts of the Atlantic, which may correspond to current systems(Hasle, 1972). Pseudonitzschia australiswas initially reported only in samplesfrom the southern hemisphere (coastalwatersof Chile, Peru,Argentina, Southwest Africa, and inshore and off-shorewatersof New Zealand); it was observed at various seasons and in differentyears, suggestingthat it is a regularcomponentof the phytoplankton in those

"

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i '.~.~4"\

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218 areas(Hasle, 1972).At that time, Haslealsonoted two questionablerecords from the west coast of the U.S.A. P. australisis now known to be found at severalsitesalongthe westcoastof the U.S.A., from San Diego, California, to the mouth of the Columbia River (Villac et al., in review), at the Strait of Juan de Fuca and Puget Sound, Washington (Homer & Postel, 1993), and to have bloomedin MontereyBay in the fall of 1991(Buck et al., 1992).Avaria & Munoz (1987) have also found P. australisin Chilean coastal waters at high concentrationsduring non-EI Nino years. Such findings raise concern about climatic fluctuationsaffectingoceanographicconditions,leading to interannualvariations in the distribution of this toxic diatom.

Seasonalpatternsand hydrography Sincethe ASP eventin Canada,the autecologyof P. pungensf. mu/tiserieshas beenintensivelystudied both in the field and in cultures. Although it hasbeenreportedto dominate at colder temperatures (autumn in Oslofjord, late autumn-early winter in Prince Edward Island, and winter in GalvestonBay), it is able to survive up to 30 cC (Fryxell et al., 1990).The bloom of 1987in Prince Edward Island extendedca. 10km seawardfrom the mouth of the Cardigan River; it lasted for about three months (mid-October to earlyJanuary),reachingthe highestdensity (ca. 1.5 x 107cellsl-l)by 11December(Bateset al., 1989). P. pungenscan be found in the areafrom August through March. In early August f. pungensis dominant with no detectabledomoic acid, while by mid-Octoberf. mu/tiseriesbecomesdominant with the appearanceof domoic acid in phytoplankton samples and mussels (Smith et al., 1990a). The 1987 P. pungensf. mu/tiseriesbloom in Canada took place after an unusually long dry spell in late summer,followed by a severerainstorm in early September(Bird & Wright, 1989). Smith et al. (1990b)found a relationshipbetween pulsesof nitrate availability and P. pungenspeaks that were attributed mostly to freshwaterrunoff

following the rains. The waters of the Cardigan estuaryundergoexchangeswith the adjacentoffshore area (eastern Northumberland Strait that separates Prince Edward Island from Nova Scotia) through a combination of forces,including tides,winds, and freshwaterrunoff; mid-depth temperaturevaluesfor the mouth of the Cardigan River (1982-87) vary seasonallyfrom 5-20 °C and salinity varies from 20 to 30 (Drinkwater & Petri, 1988). Pseudonitzschiapseudodelicatissimawas the dominant speciesduring the summer period of 1987-89 in the western Bay of Fundy, east Canada (Wildish et al., 1990),and is presentin the southwesternpart of the Bay year round (Martin et al., 1990).More than 105cells1- 1were requiredto contaminateshellfish.P. pungensalso occurred during 1987, but it was not reported whetherit was the toxic f. mu/tiseriesor the nontoxic f. pungen.\'. The Bay of Fundy region is the sourceof a southwardflowing plume (Townsend et al.. 1987)that may seedmore southerlyregions. Such events remove toxic blooms from control solelyby local speciessuccessionalforces(sensu Smayda.1980),and introduce a probabilistic element dominated by large scale advective features. Suchmechanismshave beenshownto initiate the introduction of toxic dinoflagellate speciesinto the southernGulf of Maine, U. S.A. (Franks & Anderson, 1992). Both P. pungensf. mu/tiseriesand P. pseudodelicatissima arefound in MassachusettsBay (adjacentto the southernGulf of Maine), and the former has produceddomoic acid in culture (Villareal et al., in review). Seasonalitybetween the toxic and non-toxic forms of P. pungensis also found in Galveston Bay, Texas (Fryxell et al., 1991a; Dickey et al., 1992).P. pungensf. pungensis more abundantin May-December,while f. mu/tiseriesincreaseswith the advent of cold fronts, and peaks in the winter and spring. Nevertheless, oysters, the dominantfisheryin GalvestonBay,that werecollected from a shallow reef in the area during the springof 1991testednegativefor domoic acid (D. L. Roelke, unpublished data). The cold fronts that reach Galveston Bay also often result in heavyrainfall that replacesmarine plankton with

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219 more estuarine forms. Galveston Bay fisheries may be somewhatprotected from domoic acid becauseevenwhen f. multiseriesshows a higher relative abundance,blooms have not been reported. The environmentalsettingfor the bloom of P. australishas its own peculiaritiesdue to the seasonalhydrographiccyclefound in Monterey Bay, located at the edge of the California Current. September-Octoberis an intermediatestagebetween the upwelling and the Davidson Current periods;thus it may presentwell-stratifiedwaters with surgesof upwelling, followed by offshore surface water contribution and deeper colder watermixingin November(Bolin & Abbot, 1963). The autecologyof P. australisin Monterey Bay hasbeenstudiedfor 1989-91(Buck et al., 1992), and includesbiotic and abiotic data on the ASP eventitself. Peaksof P. australisoccurredduring the autumnof those years,but it was also abundant in the spring of 1990and summerof 1991. At thetime of the 1991bloom, MontereyBay area was completingits annualdry season;the waters were moderatelystratified, the surfacetemperature was 13-14 cC, salinity was 30-33, and nutrients were relativelydepleted.Maximum abundancein surfacewaters (6.7 x 105cells 1- 1) was found in November 1991. The abundanceof Nitzschiaspp. in Monterey Bay reported sincethe early 1950'sseemsto indicatethat P. australismay havebeena common inhabitant of thesewaters, and could also be related to the high mortality of pelicansin the central California coast during the autumnsof 1971, 1976, and 1981 (Buck et al., 1992). Moreover, samples of a Pseudonitzschiaspp.-dominated bloom from November 1977have been recently reexaminedand the dominant specieswas identified as P. australis,andnot P. seriataas initially reported(Garrison et al., 1992).Blooms of Nitzschia dating back to at least 1922-23 have also beenfound in somenarrow bays in the region of Friday Harbor, Washington (Phifer, 1933); the diatom was then identified as P. seriataand observedin concentrations exceeding 2.5 x 106cells 1- 1.Although most historical identificationscannot be verified in the electron microscope,it is

clearthat Pseudonitzschia speciesarewide spread, often dominant diatoms, which might represent potential ASP risks in many areas around the world. Consideringthat the sourceof domoic acid on the westcoastwas solelyfrom P. australis(which may not be the case- seediscussionbelow), the large geographical extension of contaminated shellfishfound posesa questionaboutthe dynamics of the bloom. It is not known whether there were severalsimultaneousblooms, or one bloom that was somehow displaced along the coast. Data on local phytoplankton population dynamics is required for a better understandingof this aspect. Unfortunately, researchhas been dominated by process-orientedstudies such as pigmentand primary productivity, and little is known about the speciescomposition and its variations in relation to environmentalvariables for those shores(Wood & Shapiro, 1992).

~,

Growth requirementsand domoic 'acid production Domoic acid is a water soluble,heat stableamino acid compoundthat disruptsnormal neurochemical transmission in the brain by binding to glutamatereceptors,so that the continuousstimulation leads to the destruction of the neurons (Bird et al., 1988;Wright et al., 1989).Beforethe ASP eventin Canada,the only reported sources of domoic acid were seaweedsof the family Rhodomelaceae: Chondria armata Okamura (Takemoto & Daigo, 1958),C. baileyana(Montagne) Harvey (Edelstein et al., 1974), and AIsidium corallinumC. Agardh (Impellizzeri et al., 1975). Most data about P. pungens f. multiseries growth requirementscomefrom batch culture experimentsrelated to domoic acid production. It requiresthreeconditions to producedomoic acid in culture: (1) cessationof cell division, (2) availability of nitrate or other nitrogen sourceduring stationary phase, and (3) the presenceof light (Bateset al., 1991a).Suchfindingsapplyto clones isolatedfrom the widely separatedsitesof Prince

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220 Edward Island and GalvestonBay (Reap, 1991). It was later shown that, although exponentially growing cells in the presenceof nitrate did not producedomoic acid, cells using440 ,urnammonium did (Bates et ai., 1991b).Axenic cultures producethe neurotoxin,but xeniconesyield more domoic acid and the cultures last severalweeks longer (Douglas& Bates, 1992).A transmission electronmicroscopestudy of P. pungensf. muitiseriesstructureshas found no evidencefor the presenceof viruses,ectosymbionts,or endosymbionts (MacPheeet ai., 1992).A possiblerole of microorganismsin the process of domoic acid production awaits further investigations. Lewis et ai. (1993) have shown that growth rates of P. pungensf. muitiseriesisolated from Canadian waters increasedfrom 0.25 divisions d- 1 (5 "C) to a maximum of 0.65 divisions d- 1

P. pungensf. muitiseries,domoic acid production started during the exponentialphase,reachinga maximum value of 37 pg cell- 1 during the stationary phase;however,domoic acid during the exponentialphasecould be attributed to the initial inoculum, as the authors suggest.P. austraiis clones isolated from Monterey Bay, Coos Bay and the mouth of the Columbia River have also tested positive for domoic acid (Villac et ai., in review). As researchprogresses,it is expectedthat different clones will produce different amounts of the toxin, and that the environmental requirementsfor domoic acid production will also differ dependingon the speciesbeingtested,the growth stage,and whetherthe test is done with batch or continuouscultures. Studieson the physiologyof the Pselldonit=schia spp. will add to the under-

(20 ° C), declining slightly at 25 "C; the rate of

standing of the dynamics of toxin production and

domoic acid production increased from 0.01 to 0.51 pgcell-1 d-1 from 5 to 25 :C, respectively. Other Canadian clones tested at 10 °C showed a

degree of differentiation among taxa (Pan et ai., 1991,1993; Hargraves etai., 1993).

variable production of domoic acid among cultures(0.3-18 pgcell- 1;Bateset ai., 1989).Clones isolatedfrom Galveston Bay and testedat 20 ° C presenteda growth rate of 0.5-0.7 divisions d - 1

Trophic interactions

and domoic acid valuesrangingfrom 0.31-19.67 pg cell- 1 (Reap, 1991); those cuitures grew best

tensified over a 3-month period. Bates et ai.

at salinity 25. Bates etai. (199la) have demonstrated that low irradiance levels (45 ,uE m - 2 s - I) slowed the division rate and therefore de-

domoic acid (Maeda etai., 1984) could have inhibited grazing by planktonic crustacea. On the other hand, in feeding and uptake experiments

layed the attainment of stationary phase; however, once the plateau phase was reached, domoic acid production was comparable to conditions under higher irradiance (145 ,uE m - 2 S- 1). Cul-

with zooplankton, Windust & Wright (1991) have found domoic acid to be moderately toxic to some small size copepods and that they can function as a vector. Allelopathic effects might have also al-

tures promptly ceased to produce domoic acid during the dark period of the light:dark cycle; its production also stopped in the absence of nitrogen during the stationary phase, but resumed after nitrate was added to the medium (Bates et ai., 1991a).

lowed the toxic speciesto maintain its dominance over other phytoplankton organisms (Bird & Wright, 1989). Blue mussels were the principal organism studied during the toxic outbreaks at Prince Edward Island. Domoic acid concentrations reached as

(

growth rate of 0.64-0.71 divisions d - I, and a

Data on growth requirementsof P. allstraiisare

sparce.Two clonesisolated from Monterey Bay (Garrison et ai., 1992)grown at 15 °C, 22, 32,uE m - 2 S- I, and 12:12 light:dark cycle, showed a growthrateof 0.8and0.82divisionsd ~ 1.Unlike

It is not known why the P. pllngensf. Inllitiseries bloom in Prince Edward Island persistedand in(1989) suggest that the insecticidal property of

high as 900 ,ugg -

1

of whole body mussel(Addi-

son & Stewart, 1989).Becausedomoic acid is a water soluble compound, it was thought that it would not accumulatein tissues.This belief was reinforcedwhen it was shown that the blue mus-

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221 sel rapidly depuratedover a 72 hour-period, and that only 1-2% of the domoic acid residedin the tissues(Novaczek et al., 1992). The west coasteventis complicatedin that the toxin was found in high concentrationsin both anchovy and razor clam tissues. Despite the characteristicof domoic acid as a water soluble compound, tissues were affected and acted as accumulatingagentsof the toxin. Contaminated razor clams were still being reported six months after the outbreakwas detectedin Monterey Bay (Horner & Postel,1993;Wood & Shapiro, 1992), and it is not known whetherthis is a consequence of multiple sourcesof domoic acid or of a very long depurationrate. Different accumulationand depurationratesof domoic acid by different shellfish have alreadybeen suggestedby Gilgan et al. (1990).The possibility that more than one toxic diatom (or someother domoic acid source)might be involvedmust be considered.Not only P. australis, but also P. pungensf. multiserieshas been reportedfor those shores.At present,only nearsurfacewater phytoplankton abundancedata are available, and its composition and variations throughout the water column could certainly clarify someaspects.Possibletrophic interactions among toxic phytoplankton, shellfish, crabs, fishes,and birds on the west coast denotesquite a complexissue.As routine testinghasneverbeen done,nothing is known about backgroundlevels or fluctuations of domoic acid levels along the Pacific coast of North America (Anonymous, 1991). A very short food chain is involved in ASP events,when one considers that people can be affectedby eatingshellfishthat are contaminated with toxic phytoplankton. Although 20 Jig g- 1 has been establishedas the regulatoryguideline for human consumption, effects of chronic low level ingestionof domoic acid are unknown. Future considerations A toxic algal bloom eventis both a public health hazard and an economicthreat. It damagesthe shellfish industry, disrupts international trade, and discourages expaQsion into aquaculture

(Shumway, 1989).'Blooms of different speciesof phytoplanktonpossessuniqueenvironmentalto1erances and preferencesduring developmental phases(Taylor, 1990).Thus, there is much to be done in order to understandthe causes,predict the occurrences,and mitigatethe effectsof harmful algal blooms. There is a real need for managementactivities to interact with the scientific community. At present,monitoring the phytoplankton is a powerful tool to forewarn of potentially harmful conditions and detectnew speciesthat may pose a hazard. Nevertheless,the occurrenceof P. pungensf. multiseriesand P. pseudodelicatissima in the samecontiguouswater masseson the eastcoast, togetherwith the widespreadAS P phenomenon on the west coast, indicatesthat the examination of local dynamics must be augmentedby an understandingof large-scalecirculation and transport patterns to provide a picture of processes responsiblefor toxic bloom formation. The first challenge, in studying a harmful bloom, is to determinethe causativeorganism(s). Light and electron microscopycan so far distinguish these taxa. Molecular biology and immunologicalmethodsareunderway,and will provide new tools for rapid assayof toxic species(Bates et al., in review).Furthermore,theymay shedlight on important issuesin taxonomy, evolution, and population biology (Anderson, 1992). The physiological,chemical, and toxicological mechanismsunderlyingtoxin production and retention are an open and promising field of research.The biosyntheticpathway of domoic acid is also of greatimportance,sinceother secondary metabolitesderived from marine organismshave served as chemical markers through successive trophic levelsand/or have already becomevaluable tools in biomedicine (Laycock et al., 1989; Scheuer, 1990). Most medications are toxic in some concentrations,and a naturally occurring neurotoxin may have biomedical applicationsor even provide insight into neural pathways of speechand learning. International trade in seafood may foster the shipment of domoic acid across international boundaries; therefore, regulatory issuesfor do-

-

,

.

.

'

I

222 moic acid extend from local shellfish management .. f to the level of 1~~ernatl~nalcommerce. One.o us (GAF, 1990) vIsited Pnnce Edward Island m the autumn and was struck with how rapidly the industry

had

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eastern PnnceEdwardIsland.Canada. Can.J. Fish.aquat. Sci.46: 1203-1215. Bates,S. S. A. S. W. de Freitas,R. Pocklington, M. A. Quilliam,!. C. Smi~h& J. Wo.rms.19.?la.~ontrolson dom?lc.acl.d production~ythedla~om .~It=.\"chla pungel~s f. lIIultl.\"ene.\"m culture: nutnents

and Irradlance.

Can. J. Fish.

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on Harmful Sci. 1799: 5.

Bates. S. S..~C. Leger. B. A. Keafer & D. M. Anderson, revie\v.

for Exploratory Research aCE Faculty Development Award, assac

w. Gilgan et al., 1989. Pennate diatom ,Vit=schia pungens as the primary source ofdomoic acid, a toxin in she!lfish from

ceedings of the Second Canadian \\"orkshop Marine Algae. Can. Tech. Rep. Fish. aquatic

Fryxell

Institute

1 S .

atlona

Chile. J.

Geophys. Res. 92.: 14369-14382. . Bates,S.S.,C.J.Blrd,R.K.Boyd,A.S.W.deFreltas,M. Falk et al., 1988. Investigations on the source of domoic

aquat. Sci. 48: 1136-1144. Bates. S. S., L. Bourque, P. Cormier, C. Leger. J. C. Smith & J. Worn1S, 1991b. Ammonium or nitrate: is there an influence on the growth and domoic acid production by the diatom Nit=.\"chia pun,s:en.\"?In D. C. Gordon Jr. (ed.). Pro-

Acknowledgements Support Ih N

off northern

EI

acid responsible for the outbreak of amnesic shellfish poisoning (ASP) in eastern Prince Ed\~.ard Island. Atlantic Res. Lab. Tec. Rep. NRCC 29086: )9 pp. Bates, S. S.. C. J. Bird, A. S. W. de Freitas, R. Foxall, M.

pro-

to minimize the concentration domoic acid, e.g., excising the

.

egon Health Division, Portland. 40: 1-2. Avaria, S. & P. Mui'loz, 1987. Effects of the 1982-1983 Nii'lo on the marine phytoplankton

thereby provided a safe markets and protected

e same

cessing procedures of the water soluble h . hI t . t .

Th

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darkness t t 11 Th e na ura y. ese

in

harvesting procedures product for international t

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tOXIC

dissipate

d

0

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adapted

Anderson,D. H., 1992.The Fifth International Conference on Toxic MarinePhytoplankton: a personalperspective. HarmfulAlgaeNews62:6-7. Anonymous, 1991.Domoicacidfoundin OregonandWashingtonshellfish.Communicable Diseases Summary,Or-

Discrimi;ation

between

in

domoic-acid-producing

and non-toxic forms of the diatom Pseud'lIIit=.\"chia pungens using immunofluorescence. Mar. Ecol. Prog. Ser. Bird, C. J.. R. K. Bovd. D. Bre\ver. C. A. Cr;ft. A. S. W. de Freitas el ul., 1988. Identification of domoic acid as the ~oxin agent resp?nsible for the P.E.I. co~taminated mussel Incident. AtlantIc Res. Lab. Tec. Rep. )6: 86 pp. Bird, C. J. & J. L. C. Wright, 1989. The shellfish toxin domoic

support for T. A. Villareal. We thank K. R. Buck and F. P. Chavez, Monterey Bay Aquarium Reh I t .t t L Sh . 0 I t .t t f sear~ n~ I u e, . apIrO, regon ns I U e 0 Manne BIology, and W. E. Keene, Oregon Health Division, for operational support in field and lab research on t hewes t coas.t L . A . C.t! I uentes and

acid.WorldAquacult;;re 20:40-.:11. Boli~,R. L. & D. P. Abbot, 1963.Studieson the marine climate and phytoplankton of the central coastal area of California, 1954-1960.Cal. Coop. Oceanic Inves. Rep.9:23-.:15. Buck,K. .~., L. Uttal-Cooke, C. H..Pilskaln,D. L. Roelke, M.C.villac,G.A.Fryxell,L.A.Clfuentes&F.P.Chavez, 1992.Autoecologv of P.\"eudonit=.\"chia au.\"trali.\" Frenguelli. a

J. Manhart, Texas A&M University, and R. A. Horner, University of Washington, participated . f 1d . . J 1. N .f M h

suspecteddomoi~.acid producer, fromMontereyB~v.Cali.cornia.Mar. Ecol.Prog.Ser.84: 293-302.

muse

u

ISCUSSlons.

Department

u Ie

assl,

assac

usetts

of Health, and R. S. Becka, Texas

A&M University, provided assistance we appre. t CIa e.

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Villareal, T. A.. D. L. Roelke & G. A. Ff)'xell. in revie\v. Occurrenceof the toxic diatoms Nit=chiapungen.\' f. multiseriesand Nit=schiapseudodelicatissima in Massachusetts Bay. Massachusetts,U.S.A.A.. Mar. Environ. Res. Wildish, D. J.. J. L. Martin. A. J. Wildish & M. Ringuett. 1990. Environmental monitoring of the Bay of Fundy salmonidmaricultureindustryduring 1988-89.Can.Tech. Rep. Fish. aquat.Sci. 1760:123pp. \\indust. A. & J. L. C. Wright. 1991.The effectof domoic acid and Nil=schiapungenson zooplankton:toxicity, feeding and uptake. In D. C. Gordon Jr. (ed.), Proceedingsof theSecondCanadianWorkshopon HarmfulMarineAlgae. Can. Tech. Rep. Fish. aquatic Sci. 1799:29. Wood. A. M, & L. Shapiro. 1992.Domoic acid workshop. Final report-OregonInstitute of Marine Biology: 53 pp. Work, T. M., A. M. Beale,L. Fritz, M. A. Quilliam,M. Silver, K. Buck & J. L. C. Wright, 1993.Domoic acid intoxication of brown pelicans(Pelecanus occidentalis) in California. In T. J. Smayda& Y. Shimizu(eds),Proceedings of the Fifth InternationalConferenceon Toxic Marine Phytoplankton. Elsevier,New York: 643-649. Wright. J. L. C., R. K. Boyd. A. S. W. de Freitas,M. Falk, R. A. Foxall et al., 1989.Identificationof domoic acid, a neuroexcitatoryamino acid, in toxic musselsfrom eastern Prince Edward Island. Can. J. Chern.67: 481-490.

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