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;luglie@uniss.it(A.L.);marianim@uniss.it(M.A.M.);dstacca@uniss.it(D.S.);tcaddeo@uniss.it(T.C.); pasqui81@hotmail.it(P.F.);bmpadedda@uniss.it(B.M.P.);pulinasi@uniss.it(S.P.);sechi@uniss.it(N.S.) 2 Istitutoperl’AmbienteMarinoCostiero,CNR,SpianataS.Raineri86,98122Messina,Italy; mariagrazia.giacobbe@iamc.cnr.it 3 FondazioneCentroRicercheMarine,NationalReferenceLaboratoryforMarineBiotoxins, VialeA.Vespucci2,47042Cesenatico(FC),Italy;elena.riccardi@centroricerchemarine.it(E.R.); silvia.pigozzi@centroricerchemarine.it(S.P.);anna.milandri@centroricerchemarine.it(A.M.) 4 EnvironmentalQualityandFishFarming,EnvironmentandPrimaryPrevention,IstitutoSuperiorediSanità, V.leReginaElena299,00161Rome,Italy;milena.bruno@iss.it 5 AgenziaRegionaleperlaRicercainAgricoltura(AGRIS),ServizioIttico,S.S.Sassari-FertiliaKm18,600, Bonassai,07040Olmedo,Italy 6 DipartimentodiIspezionedegliAlimenti,IstitutoZooprofilatticoSperimentaledellaSardegnaG.Pegreffi, ViaDucadegliAbruzzi8,07100Sassari,Italy;bazzoni.annamaria@tiscali.it 7 DepartmentofLifeandEnvironmentalSciences,UniversityofCagliari,ViaFiorelli1,09126Cagliari,Italy * Correspondence:ctsatta@uniss.it;Tel.:+39-079-213-042 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 (

Paralytic Shellfish Toxins and Cyanotoxins in the Mediterranean

Similar Paralytic Shellfish Toxins and Cyanotoxins in the Mediterranean