Paralytic Shellfish Toxins and Cyanotoxins in the Mediterranean

24 Pages·2017·2.2 MB·English

microorganisms
Article
Paralytic Shellfish Toxins and Cyanotoxins in the
Mediterranean: New Data from Sardinia and
Sicily (Italy)
AntonellaLugliè1 ID,MariaGraziaGiacobbe2,ElenaRiccardi3,MilenaBruno4,
SilviaPigozzi3,MariaAntoniettaMariani1,CeciliaTeodoraSatta1,5,* ID,DanielaStacca1,
AnnaMariaBazzoni6 ID,TizianaCaddeo1,PasqualinaFarina1,BachisioMarioPadedda1,
SilviaPulina1,7,NicolaSechi1andAnnaMilandri3
1 DipartimentodiArchitettura,DesigneUrbanistica,UniversityofSassari,ViaPiandanna4,07100Sassari,
Italy;[email protected](A.L.);[email protected](M.A.M.);[email protected](D.S.);[email protected](T.C.);
[email protected](P.F.);[email protected](B.M.P.);[email protected](S.P.);[email protected](N.S.)
2 Istitutoperl’AmbienteMarinoCostiero,CNR,SpianataS.Raineri86,98122Messina,Italy;
[email protected]
3 FondazioneCentroRicercheMarine,NationalReferenceLaboratoryforMarineBiotoxins,
VialeA.Vespucci2,47042Cesenatico(FC),Italy;[email protected](E.R.);
[email protected](S.P.);[email protected](A.M.)
4 EnvironmentalQualityandFishFarming,EnvironmentandPrimaryPrevention,IstitutoSuperiorediSanità,
V.leReginaElena299,00161Rome,Italy;[email protected]
5 AgenziaRegionaleperlaRicercainAgricoltura(AGRIS),ServizioIttico,S.S.Sassari-FertiliaKm18,600,
Bonassai,07040Olmedo,Italy
6 DipartimentodiIspezionedegliAlimenti,IstitutoZooprofilatticoSperimentaledellaSardegnaG.Pegreffi,
ViaDucadegliAbruzzi8,07100Sassari,Italy;[email protected]
7 DepartmentofLifeandEnvironmentalSciences,UniversityofCagliari,ViaFiorelli1,09126Cagliari,Italy
* Correspondence:[email protected];Tel.:[phone]
Received:8August2017;Accepted:13November2017;Published:16November2017
Abstract: Harmful algal blooms represent a severe issue worldwide. They affect ecosystem
functionsandrelatedservicesandgoods,withconsequencesonhumanhealthandsocio-economic
activities. Thisstudyreportsnewdataonparalyticshellfishtoxins(PSTs)fromSardiniaandSicily
(Italy),thelargestMediterraneanislandswheretoxicevents,mainlycausedbyAlexandriumspecies
(Dinophyceae),havebeenascertainedinmusselfarmssincethe2000s. ThetoxicityoftheA.minutum,
A.tamarenseandA.pacificumstrains,establishedfromtheisolationofvegetativecellsandresting
cysts,wasdeterminedbyhighperformanceliquidchromatography(HPLC).Theanalysesindicated
the highest toxicity for A. pacificum strains (total PSTs up to 17.811 fmol cell−1). The PSTs were
also assessed in a strain of A. tamarense. The results encourage further investigation to increase
the knowledge of toxic species still debated in the Mediterranean. This study also reports new
data on microcystins (MCs) and β-N-methylamino-L-alanine (BMAA) from a Sardinian artificial
lake (Lake Bidighinzu). The presence of MCs and BMAA was assessed in natural samples and
incellculturesbyenzyme-linkedimmunosorbentassay(ELISA).BMAApositiveswerefoundin
all the analysed samples with a maximum of 17.84 µg L−1. The obtained results added further
informationoncyanotoxinsinMediterraneanreservoirs,particularlyBMAA,whichhavenotyet
beenthoroughlyinvestigated.
Keywords: paralyticshellfishtoxins;microcystins;BMAA;Alexandrium;artificiallakes;Mediterranean
Microorganisms2017,5,72;doi:10.3390/microorganisms5040072 www.mdpi.com/journal/microorganisms
Microorganisms2017,5,72 2of24
1. Introduction
Harmfulalgalblooms(HABs)arerecognizedasoneofthemajorthreatsinaquaticecosystems
worldwide[1–4]. HABsaffectecosystemfunctioning(e.g.,biodiversity,energyflowandfoodchains)
and, consequently, they negatively impact functions and related goods and services (e.g., food
production,recreationalandculturalactivities),leadingtoeconomicconsequences[5–7].
AmajornegativeaspectofHABsistheproductionoftoxiccompoundswhichimpactonhuman
health [8,9], both directly (e.g., during work and recreational outdoor activities) and indirectly
(e.g., ingesting contaminated seafood). In this regards, dinoflagellates and cyanobacteria are the
mostimportantrepresentativesofharmfulclassesinmarineandfreshwaterecosystems,respectively.
Amongdinoflagellates,themajorityoftheknownAlexandriumspeciesaretoxic[10]. Thetoxic
compounds produced belong to saxitoxins, spirolides, goniodomins and gymnodimines, but the
most important in terms of impact on humans are saxitoxins [11–13]. Saxitoxin and its analogues
(paralyticshellfishtoxins, PSTs)areneurotoxicalkaloidsmainlyresponsibleforparalyticshellfish
poisoning(PSP),oneofthemostseriousbio-intoxications[14,15]. PSPoutbreakshaveharmfuleffects
onhumanhealthaswellasimportanteconomicimplicationsforaquacultureandshellfishfarms,the
impairmentoftourismandrecreationalactivities,alterationofmarinetrophicstructureandthedeath
ofmarinemammals,fishesandseabirds[11]. HumanscancontractPSPbyingestingseafood,mainly
shellfishthathaveaccumulatedPSTsintheirtissues. ThememberstatesoftheEuropeanUnionmust
banmusselharvestingifPSTsinshellfishexceedthelimitof800µgSTXeqkg−1 inanyediblepart
(2004/853/EC).
Since the beginning of 2000, eight closures and marketing blocks of farming areas (closure
perioduptotwenty-sevendaysintheGulfofOlbiain2003,[16])haveoccurredalongthecoastof
Sardinia,thesecondlargestMediterraneanisland(westernMediterraneanSea,Italy),makingthis
regionthemostaffectedbyPSPinItaly[16–18]. Themainorganismsresponsiblefortheseevents
wereAlexandriumpacificumLitaker(exA.catenella(WhedonandKofoid),Balech),andA.minutum
Halim [16,19]. These two species bloomed singularly or together in mixed blooms when mussel
contaminationsweredetected. Morerecently(2011),mixedbloomsofA.minutumandA.pacificum
were also observed along the Sicilian coasts (Syracuse Bay), in addition to the recurrent, seasonal
blooms of A. minutum already recorded since the beginning of the 2000s [20]. However, only two
casesofshellfishfarmclosuresinSicilyareknown,inwinter2007andautumn2008,(unpublished
dataoftheRF-IZI-2008-1139874project),duetolocaldifficultiesinestablishingaregularcontrolplan
formussels.
Cyanobacteria are known to produce harmful major hepato- and neuro-toxins (microcystins,
anatoxins, cylindrospermopsins, saxitoxins, β-N-methyl-amino-L-alanine), and a lot of minor
toxins (e.g., anabaenopeptilides, anabaenopeptins, cyanopeptolins, aeruginosins, microginins,
microviridins, nostophycins)[21–24], withvariousenzymeinhibitionrolesincellularmetabolism.
CyanobacteriacanalsoproducePSTs[25];furthermore,themajorityofthemappearsinvolvedinthe
β-N-methylamino-L-alanine(BMAA)production[26],apotent,notyetwell-investigatedneurotoxin.
OriginallydiscoveredincyanobacterialspeciessymbiontsofCycadaceae,butthendetectedin95%
ofcyanobacteria[27,28],BMAA,likeitsisomerDABA(2,4-diaminobutyricaciddihydrochloride),is
anon-proteicaminoacidassociatedwithneurodegenerativesyndromes,particularlyamyotrophic
lateralsclerosisandAlzheimer’sdisease. BMAAmaybemisincorporatedbynervecellsinplaceof
serineinproteins,leadingtoamisfoldingoftheproteinstructureandgivingrisetoneurodegeneration
andthedeathofbrainmotorneurons. Thismechanismofbio-incorporationwassuggestedearly[29].
Particular attention has to be paid to BMAA co-presence with microcystins (MCs) in
environmental matrices, due to the ability of the latter to generate oxidative stress to DNA and
neurologicaldamageinthehippocampus,withamnesiceffects[30]. MCsareafamilyofmorethan
90potenteptapeptidehepatotoxins[21],andarethemostfrequentfreshwatercyanotoxinsrecognized
worldwide[31],includingintheMediterraneanregion[32,33].
Microorganisms2017,5,72 3of24
Oneofthemainconcernsconnectedtocyanotoxinsisinregardtotheirpresenceinwaterfor
drinking[34,35]. About90%ofthedrinkingwaterinSardiniaisderivedfromartificiallakes,which
aremainlyeutrophicanddominatedbycyanobacteria. Cyanotoxinshavebeendetectedbothinwaters
andscumsinSardinianartificiallakessincethe1980s[33],andastrongpositiverelationshipbetween
MCsandeutrophicationwasassessedmorerecently[36].
ThisstudyreportsnewdataonPSTprofiles,cellmorphologyandthegeneticsofAlexandrium
strains(AM.icmrooirngauntisumms 2,01A7, .5,p 7a2 cificum,A.tamarense)obtainedfromseveralsitesalongtheS3a orfd 24i nianand
Siciliancoasts. Further,thestudyincludesthefirstdataonBMAAconcentrationsinadditiontoMCs
are mainly eutrophic and dominated by cyanobacteria. Cyanotoxins have been detected both in
data in nwatauterras lansadm scpumless infr SoamrdianiaSna radrtiifniciiaanl laakretsi fisicnicael thlaek 1e98(0Ls a[3k3e], Banidd iag shtrionnzgu p)o,switihveo sreelawtioantsehrisp are used
fordrinkbinetgw.eBenM MACAs aandn deuMtroCphsicwateiorne walasso asasnesasleyds medorein reccyenatnlyo [b36a]c. terialstrainsobtainedfromsamples
This study reports new data on PST profiles, cell morphology and the genetics of Alexandrium
collectedinthesamelake. Theaimofthisstudywastogivenovelinformationonthetoxicityofboth
strains (A. minutum, A. pacificum, A. tamarense) obtained from several sites along the Sardinian and
dinoflagellatesandcyanobacteria,tocontributetotheknowledgeofthetoxicityandbiodiversityof
Sicilian coasts. Further, the study includes the first data on BMAA concentrations in addition to MCs
thesetwodatotax iinc ngartouruapl ssaimnptlhese frMomed ai Stearrdrainniaena nartriefigciiaol nla.ke (Lake Bidighinzu), whose waters are used for
drinking. BMAA and MCs were also analysed in cyanobacterial strains obtained from samples
2. Matericaollsleactnedd iMn theet hsaomdes lake. The aim of this study was to give novel information on the toxicity of both
dinoflagellates and cyanobacteria, to contribute to the knowledge of the toxicity and biodiversity of
2.1. Dinoflthaegseel ltawtoe stoxic groups in the Mediterranean region.
2.1.1. Sam2.p Mliantgeriaanl danCd eMlleCthuodltsu res
In th2e.1.c Doinnotfelaxgtelolafteps rogrammes for the monitoring and management of HABs (Projects MiPA
5C8—Fisheries and Aquaculture, Strategy EVK3-CT-2001-00046, SEED GOCE-CT-2005-00387, PO
2.1.1. Sampling and Cell Cultures
SardegnaFSE2007-2013-L.R.7/2007,regionalplanofsurveillance,vigilanceandsanitarycontrolof
In the context of programmes for the monitoring and management of HABs (Projects MiPA
production and marketing of molluscs of Sardinia), water sampling was carried out in the period
5C8—Fisheries and Aquaculture, Strategy EVK3-CT-2001-00046, SEED GOCE-CT-2005-00387, PO
from 2001 to 2013 in the Olbia harbour (Gulf of Olbia, Tyrrhenian Sea, Sardinia, Italy; GO) and in
Sardegna FSE 2007-2013-L.R.7/2007, regional plan of surveillance, vigilance and sanitary control of
theSyracpursoeduhcatirobno aunrd (mSyarrkaectiunsge obf amyo,lIluosncsia onf Scaoradsint,iaS),i cwilayt,erI tsaalmyp;lSinYg) w(Faisg cuarrreie1d) .ouSta irnd tihnei apeirsioadm ongthe
mostrelefvraomnt 2s0h0e1 ltlofi 2s0h13p irno tdhue cOelrbsiai hnaIrtbaoluyr w(Giuthlf o9f7 O53lbitao, nTsyryrh−e1n,ioann eSetah, iSradrdoinfiwa, hItaiclyh; GdOer)i avneds ifnr othme theGulf
ofOlbia.SSyhraeclulsfies hharpbrooudr (uScytriaocunsein baSyi,c Iiolnyiarne caocahset,s Si6c2ily5, tItoanlys; SyY−) 1(F[i3g7u]r.e 1S)u. Srafradcieni(a− is0 a.m30onmg )thwe matoesrt samples
relevant shellfish producers in Italy with 9753 tons y−1, one third of which derives from the Gulf of
werecollectedat2–4samplingpointsateachsite. Duringsomeoccasions,verticalnetsamples(mesh
Olbia. Shellfish production in Sicily reaches 625 tons y−1 [37]. Surface (−0.30 m) water samples were
10µm),fromabout−2mtothesurface,andahighernumberofstationsweresampled. Theseextra
collected at 2–4 sampling points at each site. During some occasions, vertical net samples (mesh 10
sampleswµmer)e, flrionmk eadbotuot s−p2e mci atol stihteu sautirofancse, ianntdh ae hsiagmhepr lninugmsbietre osf( est.agt.i,oPnsS Twser>e 8sa0m0pµlgedS. TTXheesqe kexgt−ra1 )and/or
thespecisaalmnpeleesd swoerfe tlhinekreeds teoa rscpheceiarls s(ietu.gat.i,otnhse ino bthjee cstaimvepslinogf ssipteesc i(efi.gc.,p PrSoTjes c>t s8)0.0S µugr fSaTcXeewq kagte−1r) samples
and/or the special needs of the researchers (e.g., the objectives of specific projects). Surface water
werealsocollectedinthePortoTorresharbour(GulfofAsinara,Sardinia,Italy;PT)atonesampling
samples were also collected in the Porto Torres harbour (Gulf of Asinara, Sardinia, Italy; PT) at one
pointalonganannualcyclein2002. Asinglesedimentsamplingwascarriedoutbyascubadiver
sampling point along an annual cycle in 2002. A single sediment sampling was carried out by a
usingcylisncudbraic dailvperl ausstinicg ccoyrlienrdsri(c2a0l pclmastlioc ncogrewrsi t(h20a cmdi alomnge tweirtho fa 5dicamme)tienr oMf 5a ycm2)0 i1n0 M,iany t2h0e10G, iunl fthoef Alghero
(SardiniaGnuSlef aof, ASalgrhdeirnoi (aS,aIrtdainlyia;nG SAea),. Sardinia, Italy; GA).

Figure1.FLigoucraet 1io. Lnoocaftitohne osf ttuhde ystuadrye aaraena dansda smampplilinngg ssiitteess. .(L(aLkaek BeidBigidhiingzhui =n zBuD;= GBulDf o;fG Oulblifao =f GOOl;b ia=GO;
Porto Torres harbour = PT; Gulf of Alghero = GA; Syracuse bay = SY).
PortoTorresharbour=PT;GulfofAlghero=GA;Syracusebay=SY).
Microorganisms2017,5,72 4of24
Twelve cell cultures of Alexandrium species were obtained from the above sites, eleven by
the isolation of vegetative cells from water samples, and one by the germination of resting cysts
from sediment samples (Table 1). Cells and cysts were isolated with glass micropipettes or a
micromanipulatorNarishigeandthentransferredintotissueculturemultiplatesfilledwithL1,F20or
F2medium,withouttheadditionofsilicate.Culturesweregrownat(17–20)±1◦Cwithaphotoperiod
of12:12light:darkcycleandirradianceof100µmolphotonsm−2s−1.
2.1.2. MorphologicalAnalysis
Themorphologyofaliveandfixedcells(Lugol’ssolution,2%finalconcentration)fromcultures
wasanalysedusinginvertedZeissmicroscopes(Axiovert10,25,35and200;CarlZeiss,Oberkochen,
Germany)at400×and1000×magnifications,equippedwithdigitalcameras(SpotFlex,Spotimaging,
SterlingHeights,Michigan,USAandZeissAxiocam,CarlZeiss,Oberkochen,Germany).Platepatterns
werestudiedfollowingBalech(1995)[38]afterstainingwithCalcofluorwhite(FluorescentBrightener
28,Sigma,Steinheim,Germany)andobservationunderUVepifluorescence(filterset487902,Zeiss
filters,CarlZeiss,Oberkochen,Germany)[39]. Cellsizesweredeterminedusingcalibratedeyepieces
andtheAxioVision3.1software(CarlZeissVision,GmbH,München-Hallbergmoos,Germany).
2.1.3. ToxinAnalysis
PSTanalysiswasconductedonculturesintheexponentialgrowthphase,withinthreemonths
fromtheisolationofvegetativecells. Exactvolumesandcellcountspercultureweredulyrecorded
forfurthercalculations. PSTanalysiswasconductedbyhighperformanceliquidchromatography
(HPLC) with pre-column oxidation and fluorescence detection (FLD) [40] for the Alexandrium
strains CNR–ACATSRA4, UNISS3, UNISS4, CNR–AMID6, CNR–AMISY1 and UNISS5, and with
HPLC–FLDpost-columnoxidation[41–43]fortheAlexandriumstrainsCNR–ACATS2,CNR–ACATC2,
CNR–AMIA2PT,CNR–AMIA4,CNR–AMIA5,andCNR–ATA6PT.
Aliquots of the cultures of the Alexandrium strains CNR–ACATSRA4, UNISS3, UNISS4,
CNR–AMID6, CNR–AMISY1 and UNISS5 were centrifuged at 4500 rpm to obtain a concentrated
algalpelletthatwassubsequentlyextractedusingamaximumof5mLaceticacid0.1M.Theacidic
pelletwassonicated(Ultrasonic®LiquidProcessorModelXL2020,HeatSystemsInc.,NewYork,NY,
USA)for30mininpulsemode,tobreakthecells,andthencentrifugedagainat4500rpmfor10min.
Aliquotsof1mLofthesupernatantwerefurthercleanedupusingC18SPEcartridges(Supelclean
LC18,500mg/3mL,Supelco,Bellefonte,PA,USA)andeventually10-foldconcentratedbyevaporation
under stream of nitrogen using mild heating. Analysis was performed using both peroxide and
periodateoxidationsteps. ThemethodallowedthisstudytoquantifyindividualPSTs,exceptforthe
epimeric pairs (for example GTX1 and 4), which form identical oxidation products and cannot be
separated[44].
ToxinswerequantifiedagainstlinearcalibrationsofallcurrentlyavailablePSTcertifiedreference
standards. No ion exchange fractionation was undertaken prior to the quantification of the
N-hydroxylated toxins, such as GTX1 and 4. The limit of quantification (LOQ) of the analytical
methodforeachtoxin(STX,GTX1and4,GTX2and3,GTX5,C1/2,neoSTX,dcSTX,dcGTX2and3)
wasintherangeof0.02–0.15pmolinjectedonthecolumn. Thelimitofdetection(LOD)wasassumed
tobeone-thirdofthelimitofquantification.
AliquotsoftheculturesofAlexandriumstrainsCNR–ACATS2,CNR–ACATC2,CNR–AMIA2PT,
CNR–AMIA4, CNR–AMIA5, and CNR–ATA6PT were sonicated with 0.1 M acetic acid. After
centrifugation, the supernatants were treated for SPE clean-up according to [45] and eluates were
injectedintoaHPLCsystem. TheLCdeterminationofPSTswascarriedoutusingion-pairelution
withsodiumhexanesulfonateandsodiumheptanesulfonate,andpost-columnoxidationwithperiodic
acidandfluorescencedetectionbasedon[41–43]. Toxinswerequantifiedusingthereferencestandards
availableatthetime.
Microorganisms2017,5,72 5of24
Table1.ListofAlexandriumisolatesconsideredinthisstudyandreferencefortoxins,morphologyandgeneticdata.
Reference
YearandSourceof
Species Strain GeographicOrigin CodeSite PSPToxicity
Culture Toxins Morphology Genetic
A.pacificum CNR-ACATS2 GulfofOlbia,Tyrrhenian 2002,vegetativecell GO yes † † *
A.pacificum CNR-ACATC2 GulfofOlbia,Tyrrhenian 2002,vegetativecell GO yes † † *
A.pacificum CNR-ACATSRA4 Syracuseharbour,Ionian 2012,vegetativecell SY yes † † **
A.minutum UNISS3 GulfofOlbia,Tyrrhenian 2012,vegetativecell GO yes †,§ †,§ †,§
A.minutum UNISS4 GulfofOlbia,Tyrrhenian 2012,vegetativecell GO yes †,§ †,§ †,§
A.minutum CNR-AMIA2PT PortoTorresharbour,AsinaraGulf 2002,vegetativecell PT yes † † *
A.minutum CNR-AMID6 Syracuseharbour,Ionian 2012,vegetativecell SY no † n.d. **
A.minutum CNR-AMISY1 Syracuseharbour,Ionian 2012,vegetativecell SY no † n.d. **
A.minutum CNR-AMIA4 Syracuseharbour,Ionian 2001,vegetativecell SY yes † # #
A.minutum CNR-AMIA5 Syracuseharbour,Ionian 2001,vegetativecell SY yes † # #
A.tamarense CNR-ATA6PT PortoTorresharbour,AsinaraGulf 2002,vegetativecell PT yes † † ##
A.tamarense UNISS5 GulfofAlghero,Sardinian 2010,restingcyst GA no †,§ †,§ †,§
†Thisstudy,§[46]Stacca,2013,*[47]Pennaetal.2008,**[48]Pennaetal.2015,#[20]Vilaetal.2005,##[49]Pennaetal.2007,n.d.notdescribed.
Microorganisms2017,5,72 6of24
2.1.4. BiomolecularAnalysis
ThegenomicDNAofA.tamarenseUNISS5wasextractedfromapproximately10–20mLofculture
inlogarithmicgrowthphase. Cellswereharvestedbycentrifugationat3000rpmfor15min. Thepellet
wastransferredtoa2mLEppendorftubeandcentrifugedagainat10,000rpmfor5min.Totalgenomic
DNAwasextractedfromtheresultingpelletusingaDNeasyPlantKit(Qiagen,Valencia,CA,USA),
accordingtothemanufacturer’sinstructions. TheextractedDNAwasimmediatelyfrozenat−80◦C.
A.minutumUNISS3andUNISS4wereanalysedapplyingthesingle-cellPCRmethod[50]. Individual
cellswereisolatedunderaninvertedmicroscope(ZeissAxiovert25,CarlZeiss,Oberkochen,Germany)
withaglasspipetteandtransferredto200µLPCRtubescontaining5µLoflysissolution(0.005%
SDSand400ngµL−1ProteinaseK).Tubeswerebrieflycentrifugedtoensurethatthecellswereatthe
bottom. Tubeswerestoredat−20◦C.Lysisinvolvedthethawingoftubesandthefreezingat−80◦C
foratleast10min,incubationat60◦Cfor30minandfinallyat95◦Cfor10min. Thelysateswere
usedimmediately,orstoredat−80◦CuntilusedforthePCR.
The primers D1R and D2C [51] were used to amplify the large subunit LSU rRNA gene and
theprimersITSF01andITS4[52,53]wereusedtoamplifythe5.8SrDNAandinternaltranscribed
spacers(ITS1andITS2regions). ThePCRsforA.tamarenseUNISS5werecarriedoutin25µLreactions
containing1µLofDNAextract,0.625µLofeachprimer(10µM),1.5µLofdNTPs(200µMofeach),
1.5µLofMgCl (25mM),2.5µLof10×PCRbuffer,and0.125µLofTaqDNApolymerase(Qiagen).
2
ThePCRsforA.minutumUNISS3andUNISS4werecarriedoutin50µLreactionscontaining1µLof
DNAextract,4µLofeachprimer(10µM),1µLofdNTPs(200µMofeach),1µLofMgCl (25mM),
2
5µLof10×PCRbufferand0.25µLofTaqDNApolymerase(Qiagen). PCRcyclesincludedoneinitial
stepat95◦Cfor5minfollowedby40cyclesat95◦Cfor20s,55◦C(forprimersD1RandD2C)or
53 ◦C (for primers ITSF01 and ITS4) for 30 s and at 72 ◦C for 1 min, followed by a final extension
at72◦Cfor10min. AliquotsofthePCRproductswereelectrophoresedin1.2%agarosegels. The
remainingproductwasstoredat4◦Cuntilsequenced.
The sequences obtained for UNISS3, UNISS4 and UNISS5 were aligned with those obtained
from GenBank using the MAFFT v.6 program) [54] under FFT–NS–i (slow; iterative refinement
method). Phylogenetic relationships were done with the maximum-likelihood (ML) method and
the GTRGAMMA evolution model on Randomized Axelerated Maximum Likelihood (RAxML) v.
7.0.4 [55]. Repeated runs on distinct starting trees were carried out to select the tree with the best
topology (the one with the greatest likelihood of 1000 alternative trees). Bootstrap ML analysis
was done with 1000 pseudo-replicates and the consensus tree was computed with Mr. Bayes [56].
AllanalysesweredonethroughthefreelyavailableUniversityofOsloBioportal[57].
2.2. Cyanobacteria
2.2.1. Sampling
SamplingwasconductedmonthlyfromApril2014toMarch2015atasinglestationclosetothe
deepestpart(depth: 25m)inLakeBidighinzu(North-WesternSardinia; Figure1). Watersamples
werecollectedusingaNiskinbottlealongaverticalprofilefromthesurface(−0.5m)andatdepths
of−1,−2.5,−5,−7.5and−10m. Fromeachdepth,asample(100mL)wasfixedinthefieldusing
Lugol’ssolutionforcyanobacteriacountsandspeciesidentificationandanothersample(1000mL)
was collected for quantitative toxin analysis. Samples for toxin analysis were stored at −20 ◦C
untilprocessing.
2.2.2. CyanobacterialAbundanceandSpeciesCompositioninNaturalSamples
Phytoplankton samples were analysed using Utermöhl’s technique [58]. Cyanobacteria cell
density was determined microscopically from subsamples (5–10 mL) of the fixed samples, using
an inverted microscope (Zeiss, Axiovert 10, Carl Zeiss, Oberkochen, Germany) at 200× and 400×
magnifications,basedoncellcountsfromanappropriatenumberoffields. Specieswereidentified
Microorganisms2017,5,72 7of24
fromaliveandfixedsamplesaccordingtotheavailabletaxonomicguides[59–63]. Cyanobacteriadata
weretransformed(weightedmean)torepresentintegratedsamplesinthephoticzone(Zeu=2.5times
theSecchidiskdepth;[64]).
2.2.3. CellCultures
OnestrainofMicrocystisaeruginosa(UNISS12)andoneofDolichospermumflos-aquae(UNISS13)
wereobtainedbyisolationofvegetativecells(respectively,onecolonyandonetrichome)fromwater
samplescollectedinLakeBidighinzu.
Specimenswereisolatedwithglassmicropipettesandtransferredintotissueculturemultiplates
filledwithBG11medium. Culturesweregrownat21±1◦Cwithaphotoperiodof16:8light:dark
cycleandirradianceof50–70µmolphotonsm−2s−1.
2.2.4. ToxinDetermination
Enzyme-linked immunosorbent assay (ELISA) detected BMAA, MCs and nodularins (NOD)
innaturalwatersamplesandcultures. ThesampleswereanalysedusingELISAkitsfromAbraxis
(Microcystins ADDA–ELISA Microtiter Plate for MCs and BMAA kit), after a slow freeze–thaw
cycle followed by ultrasonic treatment (ELMA S 10, Elmasonic, Singen, Germany), according to
themanufacturer’sprotocols. Despiteinitialcriticismsofitsreliability[65],theAbraxisBMAAkit
waspositivelyvalidatedinrecentstudies[66]. TheabsorbancewasreadusingaMiniphotometer
(model 6+, Metertech Inc., Taipei, Taiwan). The results were expressed, respectively, as µg L−1 of
MC–LRequivalents,asindicatedbythemanufacturer,andwerereportedinthetextasMCs,andas
µgL−1forBMAA.SampleswereconsideredpositivewhentheMCsandBMAAconcentrationswere
higherthanthelowestdetectionlimit(LDL;0.10µgL−1and4µgL−1,respectively). Duringthisstudy,
toxinanalyseswerecarriedoutonthewatersamplescollectedatthetwodepthsofcyanobacteria
abundancemaximaineachdateofsampling,andreportedasaveragedata.
3. Results
3.1. Dinoflagellates
3.1.1. MorphologyofAlexandriumStrains
A.minutumculturesexhibitedsinglecellswithaventralporeinthe1(cid:48) plate. UNISS3andUNISS4
showedthesameplatepatternwiththe1(cid:48)platetruncatedanteriorlyandposteriorly,theanteriorsulcal
platewideandthe2(cid:48)(cid:48)(cid:48)(cid:48) plateoftypeB(transversalaxislongerthanthelongitudinalaxis)(Figure2).
Cellsizesrangedfrom20µmto26.3µminlength(mean: 22.3;n=40),andfrom17.5µmto25µm
inwidth(mean: 20.6;n=40). A.minutumCNR–AMIA2PTcellswereslightlysmallerthantheother
specimens. The size range was 16.9 µm to 26.5 µm (mean = 21 µm; n = 20) and 15 µm to 19.8 µm
(mean=17.8µm;n=20),respectivelyforlengthandwidth.
A.tamarenseUNISS5culturewasderivedfromthegerminationofagreyish,ellipsoidalcyst(50µm
longand28µmwide),withagranularcontentandayellowaccumulationbody. Thearcheopylewas
ovalandextendedforahalf-lengthofthecyst(Figure3). A.tamarenseUNISS5andCNR–ATA6PT
cellsweremainlysingleorinchainsoftwoandhadaventralporeinthe1(cid:48) plate. Cellsofbothstrains
sharedthesameplatepattern,characterizedbyalarge6(cid:48)(cid:48) plateand1(cid:48) plateindirectcontactwithPo
(Figure3). ThecellsizeofUNISS5rangedfrom22.5µmto32.5µm(mean=27.7µm;n=20)inlength
andfrom22.5µmto33.8µm(mean=28.7µm;n=20)inwidth. Also,thecellsizeofCNR–ATA6PT
wasverysimilar,withameanlengthandwidthof27.8µmand28.3µm,respectively.
Microorganisms 2017, 5, 72 7 of 24
from alive and fixed samples according to the available taxonomic guides [59–63]. Cyanobacteria
data were transformed (weighted mean) to represent integrated samples in the photic zone (Zeu =
2.5 times the Secchi disk depth; [64]).
2.2.3. Cell Cultures
One strain of Microcystis aeruginosa (UNISS12) and one of Dolichospermum flos-aquae (UNISS13)
were obtained by isolation of vegetative cells (respectively, one colony and one trichome) from
water samples collected in Lake Bidighinzu.
Specimens were isolated with glass micropipettes and transferred into tissue culture
multiplates filled with BG11 medium. Cultures were grown at 21 ± 1 °C with a photoperiod of 16:8
light:dark cycle and irradiance of 50–70 µmol photons m−2 s−1.
2.2.4. Toxin Determination
Enzyme-linked immunosorbent assay (ELISA) detected BMAA, MCs and nodularins (NOD) in
natural water samples and cultures. The samples were analysed using ELISA kits from Abraxis
(Microcystins ADDA–ELISA Microtiter Plate for MCs and BMAA kit), after a slow freeze–thaw cycle
followed by ultrasonic treatment (ELMA S 10, Elmasonic, Singen, Germany), according to the
manufacturer’s protocols. Despite initial criticisms of its reliability [65], the Abraxis BMAA kit was
positively validated in recent studies [66]. The absorbance was read using a Mini photometer (model
6+, Metertech Inc., Taipei, Taiwan). The results were expressed, respectively, as µg L−1 of MC–LR
equivalents, as indicated by the manufacturer, and were reported in the text as MCs, and as µg L−1
for BMAA. Samples were considered positive when the MCs and BMAA concentrations were higher
than the lowest detection limit (LDL; 0.10 µg L−1 and 4 µg L−1, respectively). During this study, toxin
analyses were carried out on the water samples collected at the two depths of cyanobacteria
abundance maxima in each date of sampling, and reported as average data.
3. Results
3.1. Dinoflagellates
3.1.1. Morphology of Alexandrium Strains
A. minutum cultures exhibited single cells with a ventral pore in the 1′ plate. UNISS3 and
UNISS4 showed the same plate pattern with the 1′ plate truncated anteriorly and posteriorly, the
anterior sulcal plate wide and the 2′′′′ plate of type B (transversal axis longer than the longitudinal
axis) (Figure 2). Cell sizes ranged from 20 µm to 26.3 µm in length (mean: 22.3; n = 40), and from 17.5
µm to 25 µm in width (mean: 20.6; n = 40). A. minutum CNR–AMIA2PT cells were slightly smaller
Mthiacrnoo trghaen iosmthse20r1 s7p,5e,c7i2mens. The size range was 16.9 µm to 26.5 µm (mean = 21 µm; n = 20) and 185 oµfm24
to 19.8 µm (mean = 17.8 µm; n = 20), respectively for length and width.
Microorganisms 2017, 5, 72 8 of 24
A. tamarense UNISS5 culture was derived from the germination of a greyish, ellipsoidal cyst (50
µm long and 28 µm wide), with a granular content and a yellow accumulation body. The archeopyle
was oval and extended for a half-length of the cyst (Figure 3). A. tamarense UNISS5 and

CNR–ATA6PT cells were mainly single or in chains of two and had a ventral pore in the 1′ plate.
CellsF oigfu broe t2h. Lsitgrhaitn ms icsrhoagrreadp htsh oef sAalmexean pdlraiutem pmainttuetrunm, ccehlalsr a(UctNerISizSe3d a nbdy U aN lIaSrSg4e s 6tr′a′ ipnlsa),t est aainnded 1 w′ pitlha te in
Figure2.LightmicrographsofAlexandriumminutumcells(UNISS3andUNISS4strains),stainedwith
direcCt acloconftlaucotr wanitdh o Pbose (rFveigdu ursei n3g) . UTVh efl ucoelrle sscieznec oe.f (Ua,bN)I aSpSi5c arla anngde vde fnrtoraml v2ie2w.5s µshmo wtoin 3g2 t.h5 eµ vmen (tmrale an =
CalcofluorandobservedusingUVfluorescence.(a,b)apicalandventralviewsshowingtheventral
27.7 pµpoomrree; (n(vv p=p) )2i ni0n)t htihene 1l (cid:48)e1pn′ lpgalttaehta ean andndtdh f etrhowem idw e2id2ae.n5 ta eµnrimtoerr itsoour l 3csa3ul.l8pc alµal tmpel( as(tame) ;e((saca)n)a; =n(c t2a) 8pa.in7cta aµlpvmiice;aw ln v s=hie o2ww0 )is nhigno twwhienid2g(cid:48)t (cid:48)ht(cid:48)h(cid:48). peA l2alt′s′e′o′. , the
cell spizlaet eo. fS CcaNleR b–aArsT 1A0 µ6PmT. was very similar, with a mean length and width of 27.8 µm and 28.3 µm,
Scalebars10µm.
respectively.

Figure 3. Light micrographs of Alexandrium tamarense cyst and cells (UNISS5 strain), stained with
Figure3. LightmicrographsofAlexandriumtamarensecystandcells(UNISS5strain),stainedwith
Calcofluor and observed using UV fluorescence. (a) empty cyst showing the shape of archeopyle;
CalcofluorandobservedusingUVfluorescence. (a)emptycystshowingtheshapeofarcheopyle;
((bb,,cc)) vveennttrraall vviieewwss sshhoowwiinngg tthhee vveennttrraall ppoorree ((vvpp)) iinn tthhee 11(cid:48)′ ppllaattee,, tthhee wwiiddee 66(cid:48)′(cid:48)′ ppllaattee aanndd tthhee ccoonnnneeccttiioonn
bbeettwweeeenn tthhee 11(cid:48)′ ppllaattee aanndd aappiiccaall ppoorree ppllaattee ((PPoo)).. SSccaallee bbaarrss 1100 µµmm..
Specimens of A. pacificum (CNR–ACATS2 and CNR–ACATC2) from cultures exhibited the
SpecimensofA.pacificum(CNR–ACATS2andCNR–ACATC2)fromculturesexhibitedthesame
same cell size and pattern of thecal plates of those described from the field in the Gulf of Olbia [67].
cell size and pattern of thecal plates of those described from the field in the Gulf of Olbia [67].
Additionally, the isolates from the Sicily culture (CNR–ACATSRA4) displayed similar
Additionally,theisolatesfromtheSicilyculture(CNR–ACATSRA4)displayedsimilarmorphological
morphological traits with a size range of 27.7–36.6 µm for length and 28.5–37.4 µm for width.
traitswithasizerangeof27.7–36.6µmforlengthand28.5–37.4µmforwidth.
3.1.2. Toxins of Alexandrium Strains
3.1.2. ToxinsofAlexandriumStrains
Strains of A. minutum from Sardinian sites (CNR–AMIA2PT, UNISS3 and UNISS4) and two
StrainsofA.minutumfromSardiniansites(CNR–AMIA2PT,UNISS3andUNISS4)andtwostrains
strains (CNR–AMIA4 and CNR–AMIA5) from SY were found to be toxic (Table 2). These strains
(CNR–AMIA4andCNR–AMIA5)fromSYwerefoundtobetoxic(Table2). Thesestrainsrevealed
revealed higher concentrations of GTX1 and 4 than GTX2 and 3 (Figure 4). CNR–AMIA2PT was the
higher concentrations of GTX1 and 4 than GTX2 and 3 (Figure 4). CNR–AMIA2PT was the most
most toxic strain, followed by the CNR–AMIA4 and CNR–AMIA5 strains, and UNISS3 and UNISS4
toxicstrain,followedbytheCNR–AMIA4andCNR–AMIA5strains,andUNISS3andUNISS4strains
strains (Table 2).
(Table2).
A. tamarense CNR–ATA6PT showed moderate toxicity, especially due to GTX5 and GTX1 and 4
A.tamarenseCNR–ATA6PTshowedmoderatetoxicity,especiallyduetoGTX5andGTX1and4
(Table 2, Figure 4). No toxicity (<LOD) was found for A. tamarense UNISS5 (Table 2).
(Table2,Figure4). Notoxicity(<LOD)wasfoundforA.tamarenseUNISS5(Table2).
AA.. ppaacciifificcuumm CCNNRR––AACCAATTSS22 aanndd CCNNRR––AACCAATTCC22 sshhoowweedda at toottaallt otoxxicicitityyu uppt oto1 177.8.81111f mfmoollc ceelll−l−11,,
which was markedly higher than the total toxicity found for A. pacificum CNR–ACATSRA4 (Table 2).
whichwasmarkedlyhigherthanthetotaltoxicityfoundforA.pacificumCNR–ACATSRA4(Table2).
GTX1 and 4 and GTX5 were the most abundant toxins for A. pacificum CNR–ACATS2 and
GTX1 and 4 and GTX5 were the most abundant toxins for A. pacificum CNR–ACATS2 and
CNR–ACATC2 (Table 2, Figure 4). STX and GTX5 values were notably higher for CNR–ACATS2
CNR–ACATC2(Table2,Figure4). STXandGTX5valueswerenotablyhigherforCNR–ACATS2than
than CNR–ACATC2. Rather, GTX2 and 3, GTX1 and 4 and NeoSTX were higher for CNR–ACATC2
CNR–ACATC2. Rather,GTX2and3,GTX1and4andNeoSTXwerehigherforCNR–ACATC2than
than CNR–ACATS2. GTX1 and 4 and C1/2 prevailed in the toxin profile of A. pacificum
CNR–ACATS2. GTX1and4andC1/2prevailedinthetoxinprofileofA.pacificumCNR–ACATSRA4
CNR–ACATSRA4 (Table 2; Figure 4).
(Table2;Figure4).
Microorganisms2017,5,72 9of24
Table2.ListofAlexandriumisolatesanalysedinthisstudyandrelativecontentinPSTs.
Toxins(fmolcell−1)
Strain Code Site
STX GTX5 GTX2,3 GTX1,4 C1/2 dcGTX2,3 dcSTX NeoSTX Total
A.pacificum CNR-ACATS2 GO 0.229 11.808 0.579 5.129 — — — 0.066 17.811
A.pacificum CNR-ACATC2 GO 0.048 4.280 0.622 6.159 — — — 0.505 11.614
A.pacificum CNR-ACATSRA4* SY 0.008 0.022 0.045 0.057 0.082 <LOD 0.005 <LOD 0.219
A.minutum UNISS3* GO <LOQ <LOD 0.004 0.013 <LOD <LOQ <LOD <LOD 0.017
A.minutum UNISS4* GO <LOQ <LOQ 0.001 0.018 <LOD <LOQ <LOD <LOD 0.020
A.minutum CNR-AMIA2PT GPT <LOD <LOD 0.012 0.308 — — — <LOD 0.320
A.minutum CNR-AMID6* SY <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD
A.minutum CNR-AMISY1* SY <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD
A.minutum CNR-AMIA4 SY <LOD <LOD 0.008 0.100 — — — <LOD 0.108
A.minutum CNR-AMIA5 SY <LOD <LOD 0.007 0.096 — — — <LOD 0.103
A.tamarense CNR-ATA6PT GPT <LOD 0.044 0.007 0.048 — — — <LOD 0.099
A.tamarense UNISS5* GA <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD
LOD=limitofdetection,LOQ=limitofquantification,—=toxinsnotanalysed,*concentrated1:10times.
MMicriocroorogragnainsmisms 2s021071,7 5,,5 7,27 2 10of249 of 24

Figure4.PercentagecompositionofPSTtoxinsintheanalysedstrains.a=Alexandriumminutumstrains;b=A.tamarensestrain;c=A.pacificumstrains.
Figure 4. Percentage composition of PST toxins in the analysed strains. a = Alexandrium minutum strains; b = A. tamarense strain; c = A. pacificum strains.