w w w. s b f g n o s i a . o r g . b r / r e v i s t a
Original
Article
Amphidinolide
P
from
the
Brazilian
octocoral
Stragulum
bicolor
Thiciana
S.
Sousa
a,1,
Genoveffa
Nuzzo
b,1,
Maria
C.M.
Torres
a,
Norberto
P.
Lopes
c,
Adele
Cutignano
b,
Paula
C.
Jimenez
d,
Evelyne
A.
Santos
a,
Bruno
A.
Gomes
a,
Angela
Sardo
b,
Otilia
D.L.
Pessoa
a,
Leticia
V.
Costa-Lotufo
e,
Angelo
Fontana
a,∗aDepartamentodeQuímicaOrgânicaeInorgânica,UniversidadeFederaldoCeará,Fortaleza,CE,Brazil
bCNR,IstitutodiChimicaBiomolecolare,Bio-OrganicChemistryUnit,Pozzuoli,Naples,Italy
cNúcleodePesquisaemProdutosNaturaiseSintéticos,FaculdadedeCiênciasFarmacêuticasdeRibeirãoPreto,UniversidadedeSãoPaulo,RibeirãoPreto,SP,Brazil dDepartamentodeCiênciasdoMar,UniversidadeFederaldeSãoPaulo,Santos,SP,Brazil
eDepartamentodeFarmacologia,InstitutodeCiênciasBiomédicas,UniversidadedeSãoPaulo,SãoPaulo,SP,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory: Received15June2015 Accepted17August2015 Availableonline9October2015
Keywords: Symbiont
Marinenaturalproduct Chromatography
Nuclearmagneticresonance Marineecology
a
b
s
t
r
a
c
t
Dinoflagellatesareanimportantsourceofuniquebioactivesecondarymetabolites.Symbioticspecies, commonlynamedzooxanthellae,transfermostoftheirphotosyntheticallyfixedcarbontotheirhost. Themutualisticrelationshipprovidestheorganicmetabolitesusedforenergyproductionbutthereare veryfewreportsoftheroleofthedinoflagellatesintheproductionofsecondarymetabolitesinthe symbioticassociation.Coralsandotherrelatedcnidariansarethemostwell-knownanimalscontaining symbioticdinoflagellates.InthepresentpaperwedescribetheisolationofamphidinolideP(1)fromthe octocoralStragulumbicoloranditsprey,thenudibranchMarionialimceana,collectedoffthecoastsof Fortaleza(Ceará,Brazil).Thecoralextractsalsocontained3-O-methylderivative(2)ofamphidinolide P,togetherwithminorcompoundsstillunderinvestigation.Amphidinolideshavebeensofarreported onlyinlaboratoryculturesofAmphidiniumsp.,thuscompounds1and2representsthefirstidentification ofthesepolyketidesininvertebrates.Thefindingprovesthepossibilitytoisolateamphidinolidesfroma naturalsymbiosis,enablingfurtherbiologicalandbiotechnologicalstudies.
©2015SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Allrightsreserved.
Introduction
Manymarineinvertebrateshostsymbioticdinoflagellates, com-monly referred to as zooxanthellae, which provide nutritional requirementsin the form of photosynthetic products and also facilitatetheassimilationofdissolvedinorganicnitrogen(Hallock, 2001;Kitaetal.,2010).Dinoflagellatesareunicellulareukaryotes whichexhibita greatdiversity offormandlifestyle.Free living species are an important source of natural products, including marinetoxinsandbioactivecompounds(Wang,2008;Bluntetal., 2012),whereassymbioticmicroalgaehavebeenoftensuggested toberesponsiblefortheproductionofsecondarymetabolitesin marineinvertebrates,especiallycnidariansandporifera(Kobayashi and Ishibashi, 1993; Kobayashi and Kubota, 2010; Mydlarz etal.,2003).Symbioticcommunityofdinoflagellatescomprisesa
∗ Correspondingauthor.
E-mail:afontana@icb.cnr.it(A.Fontana).
1Theseauthorshaveequallycontributedtothiswork.
heterogeneousgroupofmanystrainsandspeciesthatinpartcan beisolated(AndersenandKawachi,2005).Althoughsomeofthese isolateshaveproventobeabletosynthesizesecondarymetabolites inlabculture(Kelleretal.,1987),thesymbioticassociationimposes abilateralexchangebetweenhostandsymbionts,whichleadsto productionofmetabolitesthatarenotgenerallyformedbyeither organismseparately(Trench,1993).Thus,synthesisofsecondary metabolitesbyzooxanthellaeinhospiteremainsanopenquestion.
Amphidiniumisa widespreadgenusof dinoflagellates,found
in temperate and tropical marine waters, in both free-living benthicandendosymbioticstates.Amphidiniumspeciesareoften amongstthemostabundantdinoflagellatesinbenthicecosystems, and a few species are knownas a prolific sourceof secondary metabolites. Symbiotic strains of thegenus have been isolated from the Okinawan flatworm Amphiscolops sp. (Kobayashi and Tsuda,2004).Alongseveralgenerations,laboratoryculturesofthis dinoflagellatehaveproducedanumberofbioactivemacrolactones designatedasamphidinolides(Kobayashi,2008).Thefamily–to datealmost40members–showsavarietyofbackboneskeletons and ring size from 12 to 29 atoms. Furthermore, due to their
http://dx.doi.org/10.1016/j.bjp.2015.08.010
diversefunctionality, stereochemical complexity and promising cytotoxicity activity, amphidinolides have been also subject of manychemicalsyntheses(KobayashiandKubota,2010).
AspartofaBilateralProgramonidentificationandbiosynthesis ofbioactivemetabolitesfromBrazilianmarineorganisms,wehave recentlystudiedthechemicalcompositionandcytotoxicactivityof theextractoftheoctocoralStragulumbicolor(Octocorallia: Clavu-lariidae)(BumbeeranddaRocha,2012).Theaimofthisworkisto describetheidentificationofamphidinolideP(1)(Ishibashietal., 1995)anditsderivative3-O-methylamphidinolideP(2)fromthe invertebrateandfromitspredator,thenudibranchMarionia lim-ceana.
O
O O
14
3 19 5 3
1
18 7 8
9
14 11 12
13 15
16 17 21 20
22
O O
O O O
OH OMe
1
2
Materialsandmethods
General
OpticalrotationsweremeasuredonaJascoP-2000 Polarime-ter.UVspectrawereacquiredonaJascoV-650spectrophotometer. High resolution electrospray ionization mass spectra (HRES-IMS)wereacquiredonaQ-ExactiveHybridQuadrupole-Orbitrap Mass Spectrometer(Thermo Scientific) or AmaZonSL (Bruker®
–Billa Rica,USA)instrument. 1H(600MHz)and 13C(150MHz)
NMR spectra were performed on a Bruker Avance DRX 600 equippedwitha cryoprobe.Chemicalshifts valuesarereported in ppm (␦) and referenced to internal signals of residual protons (C6D6 1H ␦ 7.15, 13C 128.0ppm). Solid phase
extrac-tion was carried out with polystyrene–divinyl benzene resin (CHROMABOND® HR-X). Silica gel chromatography was
per-formedusingprecoatedMerckF254platesandMerckKieselgel60 powder.
Samplecollection
Stragulumbicolorwascollectedontheundersideofbeachrocks intheintertidalzonealong Capongabeach(04◦02′ S38◦11′ W)
located61kmeastfromFortaleza,Ceará,Brazil.Specimen identi-ficationwascarriedoutbyProf.TitoMonteirodaCruzLotufoat UniversityofSãoPaulo,andavouchersample(ColBIOTL1364e ColBIOTL1365)weredepositedatthe“Prof.EdmundoF.Nonato” collectionintheOceanographicInstituteoftheUniversityofSão Paulo(Brazil).FirstsamplingwascarriedinAugust2013,andthe octocoralwascollectedalong withitspredator,thenudibranch
Marionialimceana.ThemolluscwasidentifedbythebiologistFelipe deVasconcelosSilvaoftheFederalUniversityofCeará(Fortaleza, Brasil).Colonieswerewashedwithsterileseawaterand keptin iceuntilMeOH extractionin thelaboratory. InApril 2014,new sampleswerecollectedaimedtoisolationofassociatedmicroalgae byusingtheprotocoldescribedbyAndersenandKawachi(2005). Thecoloniesweretransportedtothelaboratoryinsterileseawater andkeptinice.Afteravigorouslyagitation,thesupernatantwas collectedinaPetridish,anddinoflagellateswereisolatedwitha micropipetteunderinvertedmicroscopy(200×).Eachsamplewas
transferredtoa24multiwellplatecontainingKmedium(Keller
etal.,1987)andmaintainedat23◦Cunderaphotoperiodof12h
(light):12h(dark).
Cytotoxicactivity
Cytotoxicitywasevaluatedagainsttheadenocarcinomacolon cancer cell line HCT-116 (ATCC CCL-247TM). Cells were
main-tainedinRPMI1640mediumsupplementedwith10%fetalbovine serum(v/v),2mMglutamine,100U/mlpenicillin,100g/ml strep-tomycin at 37◦C under a 5% CO2 atmosphere. Extracts and
fractions were tested at concentrations ranging from 0.001 to 50g/ml, while compounds (1 and 2)were tested at concen-trations ranging from 0.001 to 25M during 72h. The effect on cell proliferation was evaluated in vitro using the MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazoliumbromide] assay,asdescribedbyMosmann(1983).Doxorubicinwasusedas positive control.IC50 (the concentrationthatinhibitsgrowthin
50%)valueswerecalculated,alongwiththerespective95%CI (con-fidenceinterval),bynon-linearregressionusingGraphPadPrism 5.0.
Extractionandisolationofmetabolites
TheoctocoralcoloniesweresuccessivelyextractedwithMeOH (3×100ml).Thisdryextract(250mg)wasresuspendedin1.5ml
waterandfractionatedon2.5gHR-Xresinbyelutionwith90ml H2O,60mlCH3CN/H2O7:3,45mlCH3CNand45mlCH2Cl2/CH3OH
9:1(Cutignanoetal.,2015).TheactiveCH3CNfraction(11.2mg)
waschromatographedonsilicagelwithagradientofdiethylether inpetroleumethertogivetwelvefractions(A–L).FractionFandH containedcompound2(0.1mg)and1(0.5mg)respectively.LC–MS andNMR analysesoffractionJ,KandLrevealedminoranalogs whoseinvestigationiscurrentlyinprogress.
AmphidinolideP(1):colorlessoil;[␣]25
D+12.8(c0.04,MeOH);
UV(MeOH)max(ε)225(26,452),231(28,101),239(18,095)nm;
HRESIMS[M+Na]+m/z397.1982(calcdforC
22H30O5Na397.1991).
3-O-Methyl-amphidinolideP(2):colorlessoil;UV(MeOH)max
(ε) 225 (16,452), 231 (18,101), 239 (12,095) nm; 1H and 13C
NMRdataseeTable2;HRESIMS[M+Na]+m/z411.2149(calcdfor
C23H32O5Na411.2147).
Massspectrometryanalysis(ESI-MS/MS)
Comparativeanalysisofnudibranchextractsandpure1was performedonAmaZonSL(Bruker®–BillaRica,USA)instruments.
Nitrogenwasusedasanebulizing(10psi)anddryinggas(5l/min, 180◦C)whilethecapillaryhighvoltagewassetto3.5kVandtheend
plateat500V.Sampleswereinfused(5l/min)bysyringepump (Bruker®–BillaRica,USA).
Resultsanddiscussion
Stragulum bicolor belongs toa recently described genusand
speciesofoctocoralthatcommonlyoccursalongtheshallowwaters oftheBraziliancoasts(BumbeeranddaRocha,2012).Thespecies hasattractedmuchattentionforitssuspectedinvasivenaturebut sofarthereisnoreportofsecondarymetabolitesfromthis organ-ism.Theoctocoralisalsothemaincomponentofthedietofthe newspeciesofnudibranchMarionialimceana(Nudibranchia: Tri-toniidae)(Silvaetal.,2013).Bothinvertebrateswerecollectedin theintertidalzoneofthebeachrocksonthecoastofCapongaBeach (Cascavel, Ceará,Brazil)in August2013.Thecnidarianand two specimensofnudibranchswereextractedbyMeOH.Theorganic extractofS.bicolorwasfurtherfractionatedusinganovel proto-colofsolidphase extraction(SPE)basedonpolystyrene-divinyl benzenecolumns(CHROMABOND®
Table1
Cytotoxicactivityofextracts,SPE-HRXfractions(#1–#4)andpurecompounds1
and2fromtheoctocoralStragulumbicolorandofthemethanolextractofthe nudi-branchMarionialimceana.ValuesareexpressedasIC50ing/mlagainstthecolon
adenocarcinomacelllineHCT-116.
Sample IC50
CI95%(g/ml)
S.bicolor(methanolextract) 0.505 0.24–1.07 S.bicolor(Fraction#1) >50 S.bicolor(Fraction#2) >50 S.bicolor(Fraction#3) 12.44 S.bicolor(Fraction#4) >50 AmphidinolideP(1) >25a
3-O-Methyl-amphidinolideP(2) 12.15a
6.98–21.17
M.limceana(methanolextract) 47.20
3.46–643.8
Doxorubicin 0.02a
0.02–0.03
aThesevaluesareexpressedinMconcentration.
ExtractsandfractionsofS.bicolorwerescreenedforcytotoxicity usingtheMTTassayonadenocarcinomacelllineHCT-116and rel-evantcytotoxicity(>80%inhibition)wasfoundonlyinthefraction elutedbyCH3CN(Table1).Thismaterialwasfurtherfractionated
onsilicagelcolumnwithanelutiongradientofdiethyletherin petroleumethertogiveamphidinolideP(1,0.5mg),its3-O-methyl derivative(2,0.1mg)andanumberofminorcompoundsthatare stillunderinvestigation.
Compound 1 was identified by NMR analysis and compari-son of theresulting NMR and MS datawith those reported in theliterature(Ishibashietal.,1995;Trostetal.,2005)whereas, despitetheclearstructuralrelationship,theassignmentof com-pound 2 required an accurate spectroscopic study. In fact, the
1HNMRspectrumofcompound2wasratherdifferentfromthat
of 1 mainlyfor what concernedthe presenceof a methyl sin-gletat3.20ppmandthedistributionoftheallylicprotonsaround 2.5ppmandmethylsignalsbetween0.84 and0.93ppm(Fig.1). However,2D-NMRexperimentsallowedanunambiguous charac-terizationofallspinsystemsaccordingtothedepictedstructure (Table2).
RecollectionofS.bicolorfromdifferentsitesoffCearácoastin April2014confirmedthepresenceofamphidinolideP(1)inthe tis-sueofthecnidarian.Furthermore,fullscanESI-MSanalysiscarried outbydirectinfusionofthemethanolextractofthenudibranchM. limceanacollectedupontheoctocoralshowedamajorionspecies atm/z397.2(C22H30NaO5+)(Fig.2).TheMS/MSfragmentation
pat-ternofthisionwassuperimposabletosodiatedamphidinolideP(1) thatgavetwoseriesoffragmentsarisingbyeitherneutrallossof water(m/z379.2)orsequentiallossofCO2,waterandketene(m/z
353.2,335.2and311.2,respectively)(seeSupportingInformation) accordingtothemechanismpreviouslydescribedforthe polyke-tidestetronasinandmycolactone(Fonsecaetal.,2004;Hongetal., 2003).
Likeothermembersofthefamily(KobayashiandKubota,2010), amphidinolideP(1)hasbeenneverreportedfromothersources exceptforthelaboratoryculturesofthedinoflagellateAmphidinium
sp.Thus,thepresenceofamphidinolideP(1)intheoctocoralisthe firstreportofthisclassofmacrolidesininvertebratesand,tothe bestofourknowledge,inafieldsample.S.bicolorhostsarichand apparentlyuncultivablecommunityofdinoflagellates.Analysisof theinhospitegroupofzooxanthellaeofS.bicolorcollectedinApril 2014revealedamajorpresenceofasingletypeofdinoflagellate cellthatwasisolatedandmaintainedforseveralweeksinmultiwell platesbyusingdifferentculturemedia(k,f/2andsupplementedES). Unfortunatelythecells,whoseidentityisstillunderdetermination,
Table2
1Hand13CNMRspectroscopicdataofamphidinolideP(1)and3-O-methyl-amphidinolideP(2).
1a,b 2a
No. ␦H(multi,JinHz) ␦C ␦H(multi,JinHz) ␦C
1 172.3 168.7
2 2.27,d(12.0)
2.36,d(12.0)
44.7 2.44,d(12.5)
2.57,d(12.5)
44.9
3 99.0 100.7
4 1.94,m 45.1 1.98,m 47.7
5 – 143.5 – 143.8
6 2.09,dd(12.0,12.0)
2.51,dd(2.5,12.0)
38.9 2.11,dd(12.0,12.6)
2.51,dd(2.5,12.6)
39.1
7 3.46,m 73.3 3.13,ddd(2.5,3.0,
8.0)
74.1
8 2.61,dd(2.0,8.0) 62.3 2.71,dd(2.0,8.0) 63.2
9 2.47,dd(2.0,8.0) 57.4 2.57c 56.7
10 2.17,dd(8.0,14,0)
2.64,d(14.0)
36.2 2.19,dd(9.0,14,0)
2.74,d(14.0)
36.0
11 142.7 140.8
12 6.19,d(15.0) 133.4 6.33,d(15.0) 134.8
13 5.59,dd(8.0,15.0) 128.8 5.57,dd(8.0,15.0) 129.1
14 5.29,t(8.0) 78.1 5.33,t(8.0) 78.1
15 2.42,m 45.0 2.47,m 44.6
16 – 146.5 – 146.8
17 4.87,brs
4.88,brs
111.9 4.87,brs
4.90,brs
111.7
18 0.91,d(7.0) 11.4 0.84,d(7.0) 10.7
19 4.76,s
4.81,s
109.5 4.69,s
4.77,s
108.6
20 4.80,s
4.93,s
117.6 4.86,s
4.91,s
119.0
21 0.90,d(7.0) 16.0 0.93,d(7.0) 16.2
22 1.66,s 19.4 1.71,s 19.1
OMe – – 3.20,s 49.5
aDataweremeasuredinC
6D6at600MHz.
6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm
Fig.1.1HNMRspectra(C
6D6,600MHz)ofcompounds1(top)and2(bottom).
remainedintheirnon-motileformandanyattempttogrowthem turnedouttobeunsuccessfulsofar.Itisworthnotingthatdespite thecommonbeliefthatmanysecondarymetabolitesisolatedfrom invertebratesmaybeproducedbysymbioticzooxanthellae,only theoriginoftheterpeneglycosidespseudopterosinsfromthesoft coralPseudopterogorgiaelisabethaehasbeenrigorouslytracedback
toasymbioticdinoflagellateofthegenusSymbiodinium(Mydlarz etal.,2003;Boehnleinetal.,2005).
Amphidinolidesarepotentanticanceragentsandsomeofthem exhibitedcytotoxicitytocancercelllinesinsub-nanomolarrange (KobayashiandTsuda,2004).Althoughpreviousstudiesindicated amoderate cytotoxicityfor amphidinolideP(1)(Kobayashiand
100 200 300 400 m/z
0.0 0.2 0.4
0.00
250 300 350 400 m/z
0.25 0.50 0.75 1.00 1.25
335.2
311.2 379.2
OH
(1)
O O
O O
m/z 311.2
m/z 379.2
m/z 353.2
M+ Na+
397.24 353.2
0.6 0.8 1.0 Intens. x107
Intens. x106
Tsuda,2004; Kobayashi,2008), theproductof S.bicolordidnot showanyactivityagainstadenocarninomacoloncancerHCT-116 cells,whilecompound2wasonlymoderatelyactive,presentingan IC50of12.2M.
Inconclusion,wehaveidentifiedamphidinolideP(1)andits derivative3-O-methylamphidinolideP(2)intheoctocoralS.bicolor
evenifthelatterproductisprobablyanartifactthatarises dur-ingextractionwithMeOH.Amphidinolideshavebeenrecognized aspotentanticanceragents(KobayashiandTsuda,2004)but com-pounds1and2didnotshowsignificantcytotoxicity.Sincethepure compoundswereless activethanrawfractionsoftheoctocoral (Table1),itisconceivablethatotherminoramphidinolidescould bepresentinthetissueoftheinvertebrate.Thisbringsintodebate thesynthesisofsecondarymetabolitesbyS.bicolorandthe uncul-tivablestrainofaputativeAmphidiniumsymbiont.Infact,despite theprimaryecologicalrelevanceoftheinteractionbetween cnidar-iansanddinoflagellatealgae(Davyetal.,2012),theisolationof1is thefirstandsofaronlyevidenceaboutproductionofapolyketide byasymbioticassociation.ItisworthnotingthatamphidinolideP (1)hasbeenrecoveredfromthecoraltissuesinamountfarhigher thanthatreportedfromculturesofAmphidiniumisolate(0.25%w/w intheoctocoralextractfor1and0.005%w/wintheextractofthe dinoflagellatepellet)(Ishibashietal.,1995).Thisapparentincrease intheproductionofthesecondarymetabolitesisinagreementwith thesymbioticoriginoftheproductsinceitiswelldocumentedthat thehostsplaysanimportantroleinregulatingthetypeandthe quantityofmetabolitesproducedbythesymbiont(Trench,1993; GordonandLeggat,2010).Furthermore,occurrenceof amphidi-nolidesinanaturalassemblageopenstotheintriguingpossibility tofindcompoundsdifferentfromthosesofarreportedfrompure culturesofthedinoflagellate,aswellasraisesaquestionaboutthe ecologicalorphysiologicalroleofthesecompounds.Tothisregards, thepresenceofamphidinolideP(1)intheextractofthenudibranch provesthedietarydependenceofthetwoanimalsandprovidesthe firstevidenceoftransferofthisclassofsecondarymetabolitesalong thetrophicmarinelevels.Thisfindingalsosuggeststhatthese com-poundsmaynothaveinthecoraladefensiveroleagainstselective predators.
Authorscontributions
GN,TSSandMCMTcarriedoutpurificationandstructure eluci-dation;EAS,BAGandODLPcontributedincollectionandextraction oftheanimals;ACcontributedintheNMRandMSinvestigations; NPLcontributedinthemassspectrometryanalysisand fragmenta-tionstudiesoncrudesamples;ASisolatedthesymbiont;PCS,EAS andLVCSperformedthebiologicalassays.AFdraftedthepaper. AFand LVCL developedtheprojectand interpretedtheresults. Alltheauthorshavereadthefinalmanuscriptandapprovedthe submission.
Conflictsofinterest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgements
Fundingwas provided by Bilateral Project CNR-CNPq (Italy-Brazil) “Characterization and Biosynthetic Origin of Bioactive NaturalProductsinMarineInvertebratesandAssociated Microor-ganismsfromtheNortheasternBrazilianCoasts”.Mrs.Dominique
Melck of Servizio NMR at ICB-NMR and Mr. Maurizio Zampa of Servizio Spettrometria di Massa at ICB-CNR were gratefully acknowledgedforrecordingspectra.LVCLandAFaregratefulto Prof.TitoMonteirodaCruzLotufoandFelipedeVasconcelosSilva forthetaxonomicidentification.
AppendixA. Supplementarydata
Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,atdoi:10.1016/j.bjp.2015.08.010.
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