Progress in Understanding Harmful Algal Blooms: Paradigm Shifts and New Technologies for

36 Pages·2012·2.08 MB·English

MA04CH07-Anderson ARI 3November2011 14:18
Progress in Understanding
Harmful Algal Blooms:
Paradigm Shifts and New
orgonly. Technologies for Research,
s.e
ws
vieal u Monitoring, and Management
en
nualrperso
ww.an2. For Donald M. Anderson,1 Allan D. Cembella,2
w1
m 9/ and Gustaaf M. Hallegraeff3
wnloaded frobrary on 10/0 012BA25ilof4rl3eod;geyWmDaeeiglp:eandreatrnmdIenenrstts,[e-mail]rnaepRhiecseInarsctihtu,t2i7o5n7,0WBoroemdseHrhoalvee,nM,Gasesramchaunsye;tts
oLi email:[email protected]
176. Dathers A3Iunssttriatulitae;efomraMil:[email protected],UniversityofTasmania,Hobart,Tasmania7001,
3-m
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2.D Annu.Rev.Mar.Sci.2012.4:143–76 Keywords
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Sci. 2FLO FSeirpsttepmubbelirsh1e9d,2o0n1l1ineasaReviewinAdvanceon redtide,shellfishtoxicity,fishkills,ecogenomics,climatechange
ne. OF TheAnnualReviewofMarineScienceisonlineat Abstract
Annu. Rev. Mariby UNIVERSITY mT1CA0lohal.rp1irsi1yingar4ehri6.gtat/ihsncatlnrnee(cid:2)nu’cssuaedlrr2revo0eveiv1:-di2mewbaysr.ionAreng-n1u2a0l3R08ev-0ie8w1s1.21 TaloTelfgdhhmaeitslo,apbniuhlnyobeoltinigumcehrwsnhte,e(nHtaiesletcAdhlheB,nsasctdo)oiileuhnonraggtiviisetfiemosca,aslalfiinngisddnnhciearfirrpeecigpeaaurssno,eltadaatpcnoohadrvreyaeesdrcafiottogthsermyensrttpesiehsoamesinfta,trifsmacenhwwpdiaatdchntehtdcsreaefmddgroeeaasmvrn.deaTlghoteohapmr,imsmeehe.nfgnaut.stl.,
1941-1405/12/0115-143$20.00 ourinterpretationofthephytoplanktonspeciesconcept(strainvariation),the
dogmaoftheirapparentcosmopolitanism,theroleofbacteriaandzooplank-
tongrazinginHABs,andourapproachestoinvestigatingtheecologicaland
geneticbasisfortheproductionoftoxinsandallelochemicals.Increasingly,
eutrophicationandclimatechangeareviewedandmanagedasmultifactorial
environmentalstressorsthatwillfurtherchallengemanagersofcoastalre-
sourcesandthoseresponsibleforprotectinghumanhealth.Herewereview
HABsciencewithaneyetowardnewconceptsandapproaches,emphasizing,
wherepossible,theunexpectedyetpromisingnewdirectionsthatresearch
hastakeninthisdiversefield.
143
MA04CH07-Anderson ARI 3November2011 14:18
INTRODUCTION
Virtuallyeverycoastalregionoftheworldisaffectedbyharmfulalgalblooms(HABs)commonly
calledredtides(Figure1).Sincethelattertermerroneouslyincludesmanybloomsthatdiscolor
thewaterbutcausenoharm,andalsoexcludesbloomsofhighlytoxiccellsthatcauseproblems
atlow(andessentiallyinvisible)cellconcentrations,scientistspreferthetermHAB.
HABsaremostcommonincoastalmarineecosystems,buttheymayalsoaffecttheopenocean
as well as brackish or freshwater ecosystems. Most HAB phenomena are caused by blooms of
microscopicalgaeorphytoplankton,includingcertaincyanobacteria(blue-greenalgae),although
the term also applies to harmful blooms of macroalgae (seaweeds). HAB events are typically
associatedwithrapidproliferationand/orhighbiomassaccumulationoftoxicorotherwisenoxious
microalgaeattheseasurfaceorinthewatercolumn,butevenlowcellnumbersofhighlytoxic
planktonicspeciesoraccumulationsofcellsonbenthicsubstratesmaycauseproblems.
y. The HAB designation is a societal concept rather than a scientific definition—blooms are
orgonl consideredtofittheHABcriterioniftheycauseinjurytohumanhealthorsocioeconomicinterests,
s.e
eviewnal us ocarutsoeciollmnepsosnaenndtsdoefaatqhuoaftificsehc,osseyasbteirmdss.,SmoammemHaAlsB,asnpdecoiethsearremtoarxiingeenliifcea,nodftpernovdiuacteobxlionotmrasntshfeart
nualrperso throughthefoodweb.Humanconsumersofseafoodcontaminatedbythesetoxinsmayalsobe
w.anFor poisoned,sufferingacutetoxicsymptomsandevenfatalitiesinextremecases.Furthertoxicthreats
w2. tohumanhealthareposedbytoxicaerosolsandwaterbornecompoundsthatcauserespiratory
w1
m 9/ andskinirritationwhenreleasedfromtoxiccells.
wnloaded frobrary on 10/0 [e.gC.,erretaacintivHeAoBxygspenecsiepsecciaens(dRirOecSt)l,yproellyeuansesactuormatpeodufnadttsythacaitdsar(eP,UstFriAcstl)y,mspuecaiklaigneg],bnuottctaonxibnes
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176. Dathers
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Figure1
NoctilucascintillansredtideatapristinetourismresortofftheeastcoastofTasmania,southeastAustralia,
believedtorepresentarecentclimate-drivenrangeextensionfromSydneycoastalwaters.(Source:Erin
Watson,UniversityofTasmania.)
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144 Anderson Cembella Hallegraeff
MA04CH07-Anderson ARI 3November2011 14:18
injuriousandevenlethaltofinfish,especiallywhenheldcaptiveinaquacultureoperations.Non-
toxicHABscausedamagetoecosystems,fisheryresources,andrecreationalfacilities,oftendue
tothehighbiomassofaccumulatedalgae,whichcancreatenoxiousscumsandfoam,shadeother
phytoplanktonandseagrassbeds,andcausefaunalmortalitiesviadecayandoxygendepletion.
ThediversityofHABspeciesandtheirimpactspresentsasignificantchallengetothoserespon-
sibleformanagementofcoastalresourcesandtheprotectionofpublichealth.HABsarecomplex
oceanographicphenomenathatrequiremultidisciplinarystudyandmethodologiesrangingfrom
molecular and cell biology to large-scale field surveys, numerical modeling, and remote sens-
ing.Ourknowledgebaseandabilitytounderstandandmanagethesephenomenahaveexpanded
greatlyinthepastseveraldecades.Consequently,therearemanyexcellentbooksandpapersthat
reviewthisprogress(e.g.,Hallegraeff1993,Andersonetal.1998,Hallegraeffetal.2003,Grane´li
&Turner2006).Thepurposeofthiscontributionisnottoduplicatetheseefforts,butratherto
highlightnewdiscoveries,technologicaladvances,andparadigmshiftsinourviewofHABs.
y.
orgonl
s.e
ws
vieal u TRENDS
en
nualrperso Fcoournmtraiensyrayneaprrso,gHraAmBsrinesdeeaprecnhdwenatslyc,ownidtuhcfteewdtiankirneglaativgeloibsoallapteiorsnp.eIcntidviev.idGuiavlernetsheaerdchiveerrssiotyr
w.anFor ofHABsandtheirimpacts,itwasdifficulttoperceivetrendsinincidence—regionally,nationally,
ww12. orglobally.ThefirstsynthesestoprovideaglobalperspectiveonHABtrendswerebyAnderson
m 9/
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176. Doathers Li cimatpiornov,eimdpcroomvemdudneicteactitoionnaomfoHnAgBssciaenndtisthtse,irintcorxeianssedduaeqtuoacbueltttuerrechoepmeriactaiolninsstinrucmoeansttaaltiwonataenrsd,
3-m andstimulationofHABsasaresultofculturaleutrophicationorperhapsevenclimatechange.
4S
4:1A - Morethan20yearslater,thereisageneralscientificconsensusthatglobally,thenumberoftoxic
012.RID blooms,theresultingeconomiclosses,thetypesofresourcesaffected,andthenumberoftoxins
Sci. 2FLO aanndd tdoisxaicgrsepeemcieenstrseaproisrete,dhohwaveevearl,loinvcerretahseedreoavseorntshfeopratshtefeewxpdanecsiaodnesin(Fpigarutriceu2la)r. Arerggiuomnse,notrs
ne. OF whethertherehasbeenanyincreaseatallorevenadecreaseinotherareas.Itisfartooeasyto
MariTY extrapolatefromoneregiontoanotherwithoutadequatescientificjustification,ortoinferatrend
v. RSI withoutsufficientlong-termdata.Withthebenefitofhindsightwecanhighlightregionalcases
nu. ReNIVE whereexpansionhasoccurredandprobablemechanismscanbeidentified.OneexampleofaHAB
nU expansioncausedbystormsorothernaturaleventsisintheGulfofMaine,UnitedStates,where
Ay
b in1972amajortropicalstormstimulatedanddispersedalargeAlexandriumredtidethroughout
theregion.Thisledtothedepositionofdormantcystsinwatersthathadvirtuallynohistoryof
paralyticshellfishpoisoning(PSP),butthathavehadannualoutbreaksnearlyeveryyearthereafter
(Anderson1998).Anexampleofshipballastwater–mediatedrangeexpansionistheintroduction
of the toxic dinoflagellate Gymnodinium catenatum into Tasmania in the mid-1970s (see review
of molecular, plankton, and cyst evidence in Bolch & de Salas 2007), from where it has since
dispersedtomainlandAustralia.
Itisalsoclearthatsomeaspectsoftheapparentexpansionareduetoheightenedandmore
effectivescientificandregulatoryattentiontotheproblem.Ineffect,wearebetterdefiningthe
boundariesofaproblemwhosescalewehadnotfullyunderstoodorappreciated.Aprominent
exampleinthisregardisassociatedwiththeamnesicshellfishpoisoning(ASP)syndrome,which
causedmorethanahundredcasesofhumanillnessandseveraldeathsin1987afterconsumption
www.annualreviews.org • ProgressinHABScienceandManagement 145
MA04CH07-Anderson ARI 3November2011 14:18
a
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b
Figure2
Distributionofeventsupto(a)1970and(b)2009,respectively,whereparalyticshellfishpoisoningtoxins
weredetectedinshellfishorfish.(Source:U.S.NationalOfficeforHarmfulAlgalBlooms.)
ofmusselsfromAtlanticCanada(Batesetal.1989).ThecausativeagentofASPwaspromptly
identifiedastheneurotoxindomoicacid,producedbyseveralspeciesofmarinediatoms.Following
thisdiscovery,novelanalyticalmethodswerequicklyintroducedintotheregulatorystructureand
usedforscientificresearch.Afewyearslater,theunusualmortalityofseabirdsinCalifornialed
totheidentificationofdomoicacidasthecause,withthatdiscoveryattributeddirectlytoreports
fromthe1987Canadianevent(Worketal.1993).ThesubsequentdocumentationofASPtoxins
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146 Anderson Cembella Hallegraeff
MA04CH07-Anderson ARI 3November2011 14:18
yearafteryearalongmuchoftheWestCoastoftheUnitedStates(Andersonetal.2008)leaves
no doubt that the toxin had been present in those waters for many years but had never been
properlyidentified.Twenty-fiveyearsaftertheAtlanticCanadianpoisoningevent,ASPisnowa
recognizedpublichealththreatinmanycountries.
A number of researchers have investigated potential linkages between HAB expansion and
eutrophication(e.g.,Smayda1989,Andersonetal.2002,Glibertetal.2008).Asdiscussedbelow,
in some instances, the linkage is clear and unequivocal, whereas in others, it is subtle or even
nonexistent(Andersonetal.2008).
KEYDEVELOPMENTSINHARMFULALGALBLOOM
SCIENCEANDTECHNOLOGY
The significant public health, tourism, fishery, and ecosystem impacts from HABs have led to
y.
orgonl majorresearchprogramsworldwidethatseektounderstandthesecomplexphenomena.Progress
s.e hasbeenrapidinmanyareas,andnewapproachesandtechnologiesforresearchandmanagement
ws
vieal u arenowavailable.
en
nualrperso
w.anFor GeneticsandTaxonomy
ww12. HABs are often nearly monospecific events. Correctly assessing the precise taxonomic identity
m 9/
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176. Doathers Li athrgeugeotlhdasttfaonrdtahredsfaokretorafdstitaiboinliatlysopfencoiemsecnlacslsaitfiucraet,ioconn,saenrdvaiftigveenmetoircpdhioffleorgeinccaelstrcaaitnsnmotubsterleimnkaeind
3-m todifferencesinmorphology,wecannotspeakofdifferentspecies.Otherscontendthatgenetic
4S
4:1A - differencesaresufficienttoseparatespecies,evenwhentheseassignmentsdifferfromthosethat
012.RID would be made on the basis of morphology. Increasingly, nuclear or plastid DNA sequences
Sci. 2FLO imnocropmhobsipneactiioesn. with lipid, pigment, and toxin biochemistry are now used to redefine existing
ne. OF PhylogeneticreconstructionofrelationshipsamongHABtaxaaremostoftenpredicatedupon
MariTY sequenceanalysisofoneorafewgenes,typicallyincludingtheribosomalDNA(rDNA),inter-
v. RSI nal transcribed spacer (ITS), or cytochrome c oxidase subunit 1 (cox1) genes. Ribotyping and
nu. ReNIVE application of conservative single-gene markers such as rDNA have been valuable in resolving
nU taxonomicandphylogeneticissuesamongHABspecies,butrevealingcrypticdiversityatthein-
Ay
b traspecificandpopulationlevelsrequiresfinerdiscrimination.Forexample,molecularsequencing
ofstrainsinculturerevealedthatthewidelyreportedcosmopolitancoastaldiatomSkeletonema
costatum includes five distinct morphotaxa, at least some of which have discrete regional distri-
butions and distinct ecophysiological characteristics (Kooistra et al. 2008). The dinoflagellate
generaGymnodiniumandGyrodiniumwereinitiallyseparatedmorphologicallyonthebasisofgir-
dledisplacement;withsupportfromanewmolecularphylogeny,thesegenerawereredefinedon
thebasisoftheshapeoftheapicalgroove,allowingforthecreationofthefamilyKareniaceae
forthefucoxanthin-containing,fish-killingdinoflagellategeneraKarenia(10+spp.nowknown),
Karlodinium (8 spp.), and Takayama (6 spp.) (Daugbjerg et al. 2000, de Salas et al. 2008). This
combinationofimprovementsinmorphotaxonomycoupledwiththedevelopmentofmolecular
probes (see below) also led to the new recognition that what were thought to be monospecific
KareniabrevisredtidesinFloridaareinfactoftenmultispeciesblooms(Steidingeretal.2008).
www.annualreviews.org • ProgressinHABScienceandManagement 147
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1
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176. Dathers Figure3
3-m MolecularbiogeographyofthedinoflagellateAlexandriumtamarense/catenellaspeciescomplexbasedonlarge-subunit(LSU)ribosomal
4S (cid:2) (cid:3) (cid:4) (cid:5) (cid:6)
2.4:1DA - RasNsisAtesdeqduisepnecressa.l.B(lMacokdaerlreodwasft(erS,cho,lainndetal).1in9d9i5c,atReupizutSaetibvaestniaa´ntuertaladl.is2p0e0r5s.a)l,whereasgrayarrows( and )suggesthuman-
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Sci. 2FLO meaLnoogkeinnegtiaclailklyedisooelastnedo.tMneocrepsshaorsilpyemcieesandegseignnetaitciaolnlys,idtheenrteicfoalr,ea,ncdanlosookminegtimdieffserbeentofdoliemsinteodt
ne. OF useforecologicalpurposes.ThedinoflagellateAlexandriumtamarenseisknowntoexistastoxic
MariTY and nontoxic strains, bioluminescent and nonbioluminescent populations, and cold-water and
v. RSI warm-waterforms.Furthermore,rRNAsequencesofisolatesoftheA.tamarensespeciescomplex
nu. ReNIVE clustered more logically on the basis of geographic origin than morphotaxonomy (Scholin &
nU Anderson 1994, Scholin et al. 1995; Figure 3). Morphologically indistinguishable populations
Ay
b thuscanhidecrypticgenotypes,someofwhichareconsistentlytoxicwhereasothers(European
andTasmanianribotypes,nowtermedGroupsIIIandV;Lillyetal.2007)aremostlynontoxic.
Insuchcasesitiscriticalthatmolecularprobesusedinshellfishandphytoplanktonmonitoring
programs discriminate between ribotypes rather than simply morphospecies. On the basis of
rRNA studies the appearance of the temperate Asian (Group IV) ribotype of A. tamarense in
1983intheMediterraneancouldonlybeexplainedbyhuman-assistedintroduction(Lillyetal.
2002). However, when Masseret et al. (2009) examined these same strains using hypervariable
microsatellitemarkersthisrevealedrelationshipsthatwerenotapparentfromrRNAsequences
studiesonthesamegroup.Mediterraneanpopulationswereshowntobeadistinctlineage,and
thereforeotheroriginsmustnowbeexplored.
Thesameprinciplesapplytotheciguatera-causingbenthicdinoflagellategenusGambierdiscus.
Until recently, only a single species of G. toxicus was recognized even though a >100-fold
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148 Anderson Cembella Hallegraeff
MA04CH07-Anderson ARI 3November2011 14:18
variation in toxicity and variations in ciguatoxin profiles have long been known. Molecular
sequencing in combination with a reexamination of morphotaxonomy allowed discrimination
of12species,ofwhich5areendemictotheAtlantic(includingtheCaribbean/WestIndiesand
GulfofMexico),5areendemictothetropicalPacific,and2(G.carpenteriandG.caribaeus)are
circumtropicallydistributed.ThedifferencesinGambierdiscusspeciescompositionintheAtlantic
and Pacific correlate with structural differences in the ciguatoxins accumulated in Atlantic and
Pacificfish(Litakeretal.2009)andwithdifferencesinthesymptomsofpoisonedindividuals.
Becauseoftheapparentcontinuityoftheworld’soceans,similarhydrologicalenvironmentsin
differentoceanstendtohavemorphologicallysimilarphytoplanktonassemblages(referredtoas
latitudinalcosmopolitanism;Taylor&Pollingher1987),andmanyscientistshavelongclaimed
thatmarineprotistshavehadampleevolutionarytimetoreachandinhabitallsuitableenviron-
ments. Molecular taxonomy is, however, increasingly contradicting the dogma of widespread
cosmopolitanismofmicroalgae.Genetic,reproductive,andmorphologicalvariationin193global
y.
orgonl strainsofthecosmopolitanmarinediatomPseudo-nitzschiapungensallowedforthediscrimination
s.e ofthreeITScladeswithdifferentgeographicdistributions.CladeIIwasrestrictedtothenortheast
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vieal u Pacific and Clade III originated from widely separated areas (Vietnam, China, and Mexico);
en
nualrperso o20n0ly8)C.lade I was recovered in all global locations in temperate coastal waters (Casteleyn et al.
w.anFor Thetoolsofmolecularbiologyhaveenabledincreasinglyfinescalesofgeneticdiscrimination
ww12. withinandamongspecies,rangingfromtherelativelycoarseinter-andintraspecificresolution
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176. Doathers Li pstoapbulelapthioennsootyfpAiclecxhaanrdarcituemrs,asnudchthaesathpepaprreondtulcatcikonofofcotorxreinlastaionndawlliethlohchigehmlyicvaalrs.iaSbimleiblaurtlyc,loNnaagllayi
3-m etal.(2009)usedmicrosatellitestoelucidatereasonsfortheexpansionofCochlodiniumpolykrikoides
4S
4:1A - redtidesinJapanandKorea,beingabletoresolvelarge-scalegenetictransferfromwesttoeast
012.RID viatheTushimaWarmCurrentaswellasfrequentapparenthuman-assisteddispersalwithinthe
Sci. 2FLO SsiegtnoifiIcnalnancedoSfetah.eTsehcishahraascteenrgisetnicdseirnedthneecwonHteAxtBofcopnocpeupltastioonnatnhdebeclooolomgidceavlealnodpmeveonltu.tionary
ne. OF
MariTY
v. RSI EcogenomicsandGeneExpressionandFunction
nu. ReNIVE Therecentdevelopmentandapplicationofadvancedtechnologiesfromthegenericallydefined
nU “-omics”sciences(genomics,transcriptomics,proteomics,metabolomics)coupledwithbioinfor-
Ay
b maticsplatformshasalreadyprovideddeepandoftenrevolutionaryshiftsinunderstandingthe
ecology and evolution of HAB species and bloom dynamics. Although the field remains in its
infancy, noteworthy contributions to HAB research have already been made in addressing the
followingfourquestions:(a)WhatarethephylogeneticrelationshipsamongHABtaxathatac-
countfortheirpatternsofevolution?(b)Whatarethebiosyntheticgenesandmetabolicpathways
involvedinbiosynthesisoftoxinsandotherallelochemicals?(c)Whatarethepatternsofdiver-
sityatthepopulationandspecieslevelwithinandamongnaturalblooms?And(d)whatarethe
mechanismsofgeneexpressionandregulationwithincellsofHABtaxaandthatgoverninter-
and intraspecific responses to putative competitors and/or grazers? The ecogenomic approach
to HAB research provides the opportunity to quantify functions and interactions of organisms
(cellstopopulations)atanecosystemlevel—relevanttocausesandconsequencesofHABs—by
determiningtherelationshipstoecologicalandevolutionaryprocesses.
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Conventionalgenomicsisoftenbaseduponthesequencingandannotationofwholegenomes,
withsubsequentbioinformaticfocusonthestructureandfunctionofkeygroupsofgenes.This
approachhasbeensuccessfullyappliedtoelucidatethecomparativestructureandfunctionofgene
clustersforthetoxinanalogsofsaxitoxinsandmicrocystinsoftoxigenicHAB-formingcyanobacte-
ria,suchasRaphidiopsisbrookiiandCylindrospermopsisraciborskii(Stuckenetal.2010),withgenome
sizes of <4 Mb. The much larger size of the nuclear genomes of eukaryotic microalgae has
severely restricted the whole-genome sequencing approach—there is only one published fully
sequencedandannotatedclosedgenomeforaHABspecies,thatofthebrowntidespeciesAureo-
coccusanophagefferens(Gobleretal.2011).ThispaucityofwholegenomesforHABtaxaisbeing
addressed,albeitnotsystematically.Adraftgenome(300Mb)hasbeengeneratedforthetoxigenic
diatomPseudo-nitzschiamultiseries(Armbrust2009),whichproducestheneurotoxindomoicacid,
andtheexpectedpartialannotationisexpectedtoyieldinsightsintopathwaysoftoxigenesisand
ironacquisitionrelevanttobloomdynamics.Nevertheless,mosttoxigenicHABtaxabelongto
y.
orgonl thedinoflagellates,agroupthathasproventobeparticularlyintractablewithrespecttogenomic
s.e analysis.Thedinoflagellatenucleargenomeistypicallylarge—often>250Gb,andinthecaseof
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vieal u Alexandrium,morethan40timesthesizeofthehumangenome!Thissizeposesanimpedimentto
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nualrperso w(Rhooclhe-eg4e5n4o)mpeyrsoeqseuqeunecnincgin,gevteenchwniotlhogthye.Fimurptlheemrecnotmatpiolenxoitfiemsaasnsdivpeleycuplairaarlilteielshoigfht-htehrdoinugohflpagu-t
w.anFor ellategenome—permanentlycondensedchromatin,generallackofhistones,frequentbase-pair
ww12. substitution,highG-Cbase-pairratio—compoundthegenomesequencingproblem.
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176. Doathers Li theSdoinmoefloagfethlleatceosm,hpalveexirteiecsenintlhyebreenentiandtdhreesgseendovmiaetroafnesuckriaprtyoomtiiccHs,oArBgsepneeceiexsp,raensdsioinnpparrotfiicluinlagr.
3-m The transcriptome comprises all of the messenger RNA (mRNA) transcripts expressed within
4S
4:1A - a given time frame, and therefore can provide dynamic insights into shifts in gene expression
012.RID relevanttomechanismssuchastoxinbiosynthesisorbloomgrowthkinetics.SeveralHABtaxa
Sci. 2FLO hsuabvseebqeueennrceecoefnatltyrasunbscjercibteeddtcooamnpalleymsiseonftaerxypDreNsseAd(sceDqNueAn)cesetqaugesn(EceS.TAsl)t,heoaucghhotfhwehEicShTisaanashlyosrist
ne. OF isrestrictedbythefactthatonlyafractionofthetranscribedgenomeissurveyed,andthenonly
MariTY whenthecorrespondingmRNAisexpressed,thisapproachhasbeeneffectivelyusedtoidentify
v. RSI genetranscripts,discovernewgenes,anddeterminesequencehomologyforvariousHABtaxa.
nu. ReNIVE ESTdatabasesarenowextensiveandrepresentativeofawidediversityoforganisms,including
nU the HAB species Alexandrium fundyense (Hackett et al. 2005), A. catenella (Uribe et al. 2008),
Ay
b A. ostenfeldii (Jaeckisch et al. 2008), A. minutum (Yang et al. 2010), Karenia brevis (Lidie et al.
2005),Chrysochromulinapolylepis(Johnetal.2010),andPrymnesiumparvum(LaClaire2006),with
moreinthepipelineawaitingfurtherannotationandverification.
AnESTlibraryconstructedforthedinoflagellateA.minutum(Yangetal.2010)combinedwith
theapplicationofanoligonucleotidemicroarrayuncoveredthepresenceof192genesthatwere
differentiallyexpressedbetweentoxicandnontoxicstrainsofthisspecies(Figure4).Neverthe-
less,althoughpotentialcandidategenesweredetectedforpossibleinvolvementingrowthregu-
lationand/ortoxinbiosynthesis,noconfirmedhitsforthePSPtoxinbiosyntheticgenesfoundin
cyanobacteriaweredetected.AmajoradvanceinthisregardwasreportedbyHackettetal.(2011),
whoassembledcomprehensivetranscriptomedatasetsforseveralsaxitoxin-producingdinoflag-
ellatesandarelatednontoxicspecies.Theyidentified265putativehomologsof14cyanobacterial
saxitoxinsynthesisgenes,includingallofthegenesdirectlyinvolvedintoxinsynthesis,asdescribed
· ·
150 Anderson Cembella Hallegraeff
MA04CH07-Anderson ARI 3November2011 14:18
Microarray design
Designed using Agilent eArray platform
60mer oligonucleotides ensure high-binding, organism-specific stringency
Contig from overlapping ESTs

ESTs = raw sequences
y. >Alexandrium-C-a-01a02.m13f 1026 letters
orgonl TTTTTTTTTTTTTTTTTTTTTTTTCAACGGCGCAAATGTCCCCGCTCAAGCTATGTGGTCCAGCGACCGAGCCGCTCGCGTCTGCAG
ws.se CCGAATCCAAGGAAAATAGAGGCTGCAACAAAAAAAGGAGGCCAAACAGGGGCCATCACTACGTACCTGCGGCGGCTCA
evienal u CAGAGGTCCCGGGCACGGCCCGACTCCGAGCAACCCCGACAACATAGAGCGCGGGGACGAGCTTATACAACGGCCGCGGGTCTCTGCCGCCAGATTGCCATACAGATACATAGTCCCCAGTGGAGAGGTGCCCGGGTTAATCCCGCCCATCCAAAAAAAC
nualrperso CTTGCGGACCGGAGGACGGCATACGTCTCGCGCACCATACGGAAGTCGACAGGCGGAGGGGAGTCGCAATGCACTTCGGCCCCCTCTAGACAACGCCTGCCGGGCGCGCTCGGGACGTCCTACGAGGACTTGCTACTGGGGTGGGGAAGCGTTTTCGGC
www.an12. For CGGGGGGGACCGAGCCCCGACCCTCTCCCGTGGCGCCCCTGTTCTGCCGGGTGCGAAAGGACCGCCGCTTACGTGTGCAAACAGCATTGGCTCCGCGCGTGTGTGGGGGTGCACAGCCCCTATTCTCTCCCGTCTGTATGGCGCAAAATTTCACACATAAGCGTCCCGACAGGAGTGCGGGTGCGCGCGGGCACAATCCGGGTTCGAACGGTGGCTCCGGAGGTGTCGCTGAGTTGGGCTTGCATGGCAGGAGCGGTTTCCCCCCA
m 9/ AGTCGTCGCGGAACGTCTTGACGACCCACTTGGCCCTTTTTTGGGAAAGGGGGGCCCCCCTTTTTTCCCGGGTTTCCCCCCGGGGGGGGGCCCCCCCCCTTTCCCCCCACTTAATGGGGCAGAAGGGGAAC
wnloaded frobrary on 10/0 TTGTTCCCATCGCGCGCATTGTCGGCCTCAATGGGTAAGGACCCTTATTTGGCTATAATCCCGCTTAGGGGCCTCCACGCTGCCCTACGTCGTATCAATTCCCTGTCGGCCCGCACCACCTACATCCGACGATTTTTTCCGCCGCGAACGGGCGGCTCGTCGGCCGCCGTGGTGTCGTCGGGCGGCTCCCTCAACAACCGACGAGTAGTAGACCGAGCGCGGTTATTAACGCGACGGCGGGAGGGAAGGGTGGTGGGAAAACAGCC CTATGCTTCCCCTGTTGGTGTGCGTTCGGCCAATGGCCCTGGT
oLi
176. Dathers
3-m
4S
4:1A -
2.D
01RI
Sci. 2FLO FOilgiguorneu4cleotidemicroarrayforstudiesofgeneexpressioninthetoxigenicdinoflagellateAlexandriumminutum.Themicroarraywas
ne. OF derivedfromthetranscriptomicanalysisofthecorrespondingcomplementaryDNA(cDNA)andsequencingof>15,000expressed
MariTY sequencetags(ESTs)(Yangetal.2010).Geneexpressionpatternscanbedeterminedfromautomatedscanningofthecolorpattern
v. RSI generatedviahybridizationandfluorescencelabeling.(AdaptedafterimagesuppliedbyI.Yang,AlfredWegenerInstituteforPolarand
nu. ReNIVE MarineResearch.)
nU
Aby by Kellmann et al. (2008). Putative homologs of four proteins grouped closely in phylogenies
withcyanobacteriaandarelikelythefunctionalhomologsofsxtA,sxtG,andsxtBindinoflagel-
lates.However,thephylogeniesdonotsupportthetransferofthesegenesdirectlybetweentoxic
cyanobacteriaanddinoflagellates.Thesaxitoxinsynthesispathwaywaslikelyassembledindepen-
dently in the distantly related cyanobacteria and dinoflagellates. The independent evolution of
thesaxitoxinbiosyntheticgenesinthesetwoecologicallydistinctgroupsoforganismssuggests
thatthistoxinmayconferabenefittoproducersthatwedonotyetfullyunderstand.
In dinoflagellate species producing polyketide toxins, including spirolides (Jaeckisch et al.
2008)andbrevetoxins(Monroe&VanDolah2008),searchstrategiesbasedonESTorcDNA
librariesweresuccessfulinidentifyingarangeofPKSsfromhighsequenceconservationinseveral
PKSdomains.Yetitremainsunclearwhichofthesegenesareresponsibleforproductionofthe
correspondingtoxins.
www.annualreviews.org • ProgressinHABScienceandManagement 151
MA04CH07-Anderson ARI 3November2011 14:18
Sequence analysis based upon a normalized cDNA library of the fish-killing haptophyte
C.polylepis(Johnetal.2010)revealedseveralgenesputativelyrelatedtotoxinsynthesisand13
putative PKS-related gene sequences. Semiquantitative reverse-transcription polymerase chain
reaction(RT-PCR)followingexpressionofPKSgenesoverthephotocycleofsynchronizedcul-
turesyieldedthefirstexpressionofPKSgenesinatoxichaptophyte.
ThedevelopmentandapplicationofcDNAandoligonucleotidemicroarrays—well-established
technologiesinbiomedicalandenvironmentalstudies—provideaquantumleapforwardforHAB
research.Patternsofgeneexpressionandregulationcanbescreenedsimultaneouslyusingthou-
sandsofgeneprobesspottedonthe“labonachip.”Wecannowsurveygenesrelatedtogrowth
dynamics, toxigenesis, interspecific interactions, and ecophysiology, to name but a few of the
processesunderstudy.Inanovelstudyofpredator-preyinteractionsandpossibledefensemech-
anisms,anEST-basedoligonucleotidemicroarrayforthedinoflagellateA.minutumwasusedto
demonstrateashiftingeneexpressionof14genesinresponsetoacopepodgrazer,concomitant
y.
orgonl withanincreaseindinoflagellatecelltoxincontent(Yangetal.2010).
s.e Transcriptomic analysis is, however, subject to a critical limitation, as this type of analysis
ws
vieal u applies only to expression at the transcriptional level. Many key genes may be posttranslation-
en
nualrperso aplolysttmraondsilfiaetido,ntahlemreobdyifiacffaetciotinngofthbeiosstyrnutchtuetriecagnednefupnrcotdiounctosfmparyotbeeinas.coInntfraicbtu,tainhgigfahctdoergrineethoef
w.anFor failureofmicroarrayanalysistoconfirmexpressionoftoxin-specificgenesinAlexandrium(Yang
ww12. etal.2010).
m 9/
wnloaded frobrary on 10/0 cafiopnmpgAleipnerlpdaelrmyitnsoeitns”Ht—obAfyBcttawhnteaoxa-apdd.ridmoErteeaesrnoslsimietohrene—parlicsogosteumeleopemolreiefscitpanrnogosaptlthtyhrosaeirsnessswltiarsatu.siocBdtnyuearptleheminsadonmeddneifittfhcuuoanptdcio,otniCnognhbeaunnoteferhtatathasielo.nn(eo2not0tf0ior5afe)t“ecppnlarrooibmtteeeeeiinnnd
176. Doathers Li tnoonbteoaxbiclesttroairnessoolfvAe.semvienruatlugmro.uTphsisofappproroteaicnhshasastabxeoennocmomicpmleamrkeenrtse,ddiosrcrsiumpienrasteidnegdtobxyieclveecrtrsou-s
3-m sprayionization(ESI)andmatrix-assistedlaserdesorption/ionization(MALDI)ofwholeproteins
4S
4:1A - orpeptidedigestsfollowedbydenovosequencingorpeptidemassfingerprintingbytandemmass
012.RID spectrometry(e.g.,Chanetal.2006).Proteomicshasprovidedvaluableinformationontheeffects
Sci. 2FLO a2n0d09m),oadnedaacntiaolnysoisfothfethdeinpolaflsatgidelplartoetemoemtaebooflittheeyeFslsoortiodxairneodntiadheudminaonfllaivgeerllcaetellKli.nber(eYviosudnegteectteadl.
ne. OF anelectrontransferprotein(plastocyanin)inheritedfromgreenalgaethatmaycontributetoeco-
MariTY logicalsuccessinironutilization(Nosenkoetal.2006).Nevertheless,muchpromiseremainstobe
v. RSI fulfilledintheapplicationofproteomicstostudiesofHABecophysiologyandbloomdynamics.
nu. ReNIVE Thestudyoftheprofileoflow-molecular-weightmetaboliteswithincellshasyettobeapplied
nU toHABresearchinaconcertedfashion.Therearenoapparentmajortechnologicalimpediments,
Ay
b and the metabolomics approach has already been successfully applied to the dinoflagellate en-
dosymbiontSymbiodinium(reviewedinGordon&Leggatt2010).AmongHABspecies,metabolic
fingerprintingofK.brevisextractsbymassspectrometrywasnotsuccessfulinidentifyingspecific
compoundsresponsibleforallelopathy(Princeetal.2008).Thisfrontierfieldislikelytobemore
fruitfulwhenmorecomprehensivespectrallibrariesbecomeavailableandasstructural-functional
relationshipsofthemetabolitesarebetterdefined.
DiversityandInteractionofPhycotoxinsandAllelochemicals
MuchoftheattentiondirectedtowardsHABs—bysocietyingeneral,socioeconomicinterests,
regulatoryagencies,andthescientificcommunity—hasbeenfocusedonthefactthatmanyHAB
taxa produce potent toxins with ecosystem and human health consequences. Regulatory and
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152 Anderson Cembella Hallegraeff