Rev. bras. farmacogn. vol.27 número4

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RevistaBrasileiradeFarmacognosia27(2017)529–532

w ww . e l s e v i e r . c o m / l o c a t e / b j p

Short

communication

Aflatoxins

produced

by

Aspergillus

nomius

ASR3,

a

pathogen

isolated

from

the

leaf-cutter

ant

Atta

sexdens

rubropilosa

Eduardo

Afonso

da

Silva-Junior

a

_,

_Camila

_Raquel

_Paludo

a

_,

_Lohan

_Valadares

b

_,

Norberto

Peporine

Lopes

c

_,

_Fábio

_Santos

_do

_Nascimento

b

_,

_Mônica

_Tallarico

_Pupo

a,∗

a_Departamento_de_Ciências_{Farmacêuticas,}_Faculdade_de_Ciências_{Farmacêuticas}_de_Ribeirão_Preto,_Universidade_de_São_Paulo,_Ribeirão_Preto,_SP,_Brazil b_Departamento_de_Biologia,_Faculdade_de_Filosofia,_Ciências_e_Letras_de_Ribeirão_Preto,_Universidade_de_São_Paulo,_Ribeirão_Preto,_SP,_Brazil

c_Núcleo_de_Pesquisa_em_Produtos_Naturais_e_Sintéticos,_Faculdade_de_Ciências_{Farmacêuticas}_de_Ribeirão_Preto,_Universidade_de_São_Paulo,_Ribeirão_Preto,_SP,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received21February2017 Accepted15May2017 Availableonline7June2017

Keywords:

Aflatoxins

Aspergillusnomius

Leaf-cutterants Massspectrometry Chemicalecology

a

b

s

t

r

a

c

t

Aspergillusspp.causeeconomicimpactsduetoaflatoxinsproduction.Althoughthetoxicityofaflatoxins isalreadyknown,littleinformationabouttheirecologicalrolesisavailable.Hereweinvestigatedthe compoundsproducedbyAspergillusnomiusASR3directlyfromadeadleaf-cutterqueenantAttasexdens rubropilosaandthefungalaxenicculture.Chemicalanalyseswerecarriedoutbyhigh-resolutionmass spectrometryandtandemmassspectrometrytechniques.AflatoxinsB1andG1weredetectedinboth theaxeniccultureandinthedeadleaf-cutterqueenant.Thepresenceofthesemycotoxinsinthedead leaf-cutterqueenantsuggeststhatthesecompoundscanberelatedtotheinsectpathogenicityofA. nomiusagainstA.sexdensrubropilosa.

Introductoryremarks

Fungus-farmingantscultivateasymbiontfungusastheir pri-maryfoodsource.Agroupoffungus-farmingantsstartedtofeed thesymbiontfunguswithplantmaterial inabout8–12million yearsagoandtheyareknownasleaf-cutterants(SchultzandBrady, 2008).Leaf-cutterantsofthegeneraAttaandAcromyrmex culti-vatethefungusLeucoagaricusgongylophorus(Lichtetal.,2014)and areconsideredasdominantherbivoresoftheNew World trop-ics (Schultz and Brady,2008).Anadult colonyof Attaspp. can havemillionsofants,whichcollectlargeamountsofplant mate-rialtofeedtheirmutualisticfungus(HölldoblerandWilson,1990). Leaf-cutterantsareimportantfortheecosystemequilibrium in temperateareasofAmericaandcanalsocauseagriculturallosses whentheycollectplantmaterialfromcrops(Weber,1966).

Anewleaf-cutter antcolonystartswitha queenantthat is fecundatedduringthenuptialflight.Mostofthequeenantsare pre-datedandasmallnumbersucceedinstartinganewcolony(Weber, 1966).Leaf-cutter antsareassociated withantibiotic-producing bacteria to protect their colonies (Santos et al., 2004; Haeder etal.,2009;Seipkeetal.,2011;Schoenianetal.,2011). Fungus-farming ants possess evolvedglands to feedsymbiont bacteria

∗ Correspondingauthor.

E-mail:[email protected](M.T.Pupo).

hostedontheirexoskeletonand,inturn,bacteriaproduce antimi-crobialcompounds(Currieetal.,2006).Ant-symbiontbacteriaare consideredapromisingsourceofantibiotics,thenaturalproducts dentigerumycinandselvamicinarenovelantifungal agents pro-ducedbyant-symbiontbacteria(Ohetal.,2009;VanArnametal., 2016).

Fungalpathogens alsoplaya roleinthis multilateral symbi-oticsystem.Thewell-knownspecializedfungalgardenpathogen

Escovopsisspp.candestroycoloniesoffungus-farmingants(Currie etal.,1999).OtherfungifromthegeneraAspergillus,Fusarium,and

Trichoderma,canalsoparasiteleaf-cutter antscolonies(Poulsen etal.,2006;Pagnoccaetal.,2012).Aspergillusnomius,forinstance, producesinsecticidalnaturalproductssuchasnominineand asper-nomine(Gloeretal.,1989;Staubetal.,1992).Themaingoalofthis workwastoidentifythemajorsecondarymetabolitesproducedby

A.nomiusASR3afteritsproliferationinanAttasexdensrubropilosa

queenant.

Materialsandmethods

Fungalspores werecollectedfromthebodyof anA.sexdens rubropilosaqueeninfestedbyagreenish-yellowfungusandwere inoculatedonPetriplatescontainingPDAmedium.Theplateswere incubatedat30◦Cuntilfungalgrowthandsporulation.Sporesof

theisolatedfunguswerepreservedat−80◦C incryotubes con-taining a20%glycerolaqueoussolution.ThefungalstrainASR3

http://dx.doi.org/10.1016/j.bjp.2017.05.001

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530 E.A.Silva-Junioretal./RevistaBrasileiradeFarmacognosia27(2017)529–532

Fig.1.AttasexdensrubropilosaqueeninfestedbyAspergillusnomius(A),andAspergillusnomiusASR3isolatedfromtheant(B).

Intens. x105

2.0

1.5

1.0

0.5

0.0

Intens. x105

5

4

3

2

1

0

217.0671 229.0680

289.0892 259.0795

301.0916

313.0711 335.0530

351.0454

335.0532

351.0451

369.0556

413.2656 463.2981

377.3197 393.3142 419.2741

441.3085 463.3007

+MS, 5.0 min #297

+MS, 3.3 min #194 485.3410

[B1+Na]+

[G1+Na]+

[B1+Na]+

200 250 300 350 400 450 500 m/z

Fig.2.ESI-HRMSspectraofethylacetateextractsofA.sexdensqueen(A)andA.nomiusASR3cultivatedinPDAmedium(B).Theredarrowsindicatetheaflatoxinsions.

wasidentifiedbysequencingtheITS(internaltranscribedspacer) regionusingtheprimersITS-1(5′TCCGTAGGTGAACCTGCGG3′)and

ITS-4(5′TCCTCCGCTTATTGATATGC3′).Thespecies(GenBank

acces-sion number KY986877) was identified by comparison (BLAST search)withsequencesdepositedatNCBI.

SporesofASR3wereinoculatedon80mmPetridishes contain-ing25mlofPDAmedium.Thefungalcultureandthecontrol(PDA only)wereincubatedfor14daysat30◦C.Afterthisperiod,the

cul-turemediumofeachplatewascollectedandextractedwith60ml ofethylacetate.Theinfestedqueenantwasalsoextractedwith ethylacetate.Finally,theextractswerefilteredandevaporatedto dryness.

Thecrude extractsandauthenticSigma–Aldrichstandardsof aflatoxinsB1andG1solutionsinmethanol/water(1:1v/v)were analyzedbydirectinfusionintotheelectrospray(ESI)sourceof themicrOTOF Q II-ESI-TOF mass spectrometer(Bruker Dalton-ics).Formic acid was added in the samples beforethe ESI-MS acquisitionstoimprovetheprotonation.Thedataacquisitionwas performedat thepositive ion-monitoringmodeand theMS/MS spectrawereobtainedbycollisionoftheselectedionswith nitro-gengas.Asolutionoftrifluoroaceticacid(TFA-Na+)at10mgml−1

wasusedforinternalcalibration,thecapillaryvoltagewassetfor 3.5kVandthedryingtemperaturewas180◦C.

Resultsanddiscussion

ThefungusASR3wasisolatedfromaqueenoftheleaf-cutterant

A.sexdensrubropilosa,whichhaddiedafterthenuptialflightand itsbodywasinfestedbyagreenish-yellowfungus(Fig.1).The iso-latedfunguswasidentifiedasA.nomiusbyaBLASTsearchoftheITS sequencefromASR3strain,whichrevealed99%ofidentitymatch withITSsequenceforA.nomiusMW16(GenBankaccession num-berKF312149.1).Thisspecieshasbeendescribedaspathogenicfor socialinsectssuchasleaf-cuttingants(Poulsenetal.,2006)and ter-mites(Chouvencetal.,2012);howeverthecompoundsinvolvedin thepathogenesisremainunderexplored.

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E.A.Silva-Junioretal./RevistaBrasileiradeFarmacognosia27(2017)529–532 531

Table1

CompoundsproducedbyAspergillusnomiusidentifiedusingESI-HRMSandESI-MS/MS.

Compound Axenicfungalculture Queenant Fragmentions(m/z)a

Molecularformula [M+H]+_(error_(ppm)) _Molecular_formula _[M+H]+_(error_(ppm))

AflatoxinB1 C17H12O6 313.0712(1.6) C17H12O6 313.0711(1.3) 298,285,270,257,241

AflatoxinG1 C17H12O7 329.0661(1.5) C17H12O7 329.0658(0.6) 311,283,243,215 a_Fragment_ions_are_common_for_aflatoxins_produced_by_A._nomius_ASR3_and_authentic_standards._Protonated_aflatoxins_ions_were_observed_after_the_addition_of_formic

acidinthesamples.

preparation.Themajorionsatm/z335.0530andm/z351.0454were presentinbothextracts,butabsentinPDAcontrolmediumextract (Fig.2).Thehigh-resolutionmassanalysissuggestedtheiridentities asaflatoxinB1(1)(C17H13O6+,error1.6ppm)andaflatoxinG1(2) (C17H13O7+,error1.5ppm;Table1).Thefragmentationspatterns atESI-MS/MSfor theseionswereconsistentwiththeliterature datafor1and2(Cavaliereetal.,2007;Sirhanetal.,2013;Tóth

etal.,2013)andmatchedwithauthenticstandards(Fig.S2-S5). ThesefragmentationsinvolvelossesofCO,CO2,H2O,andmethyl radicals, which are commoneliminationsfor lactones and aro-maticmethoxylgroup(Crottietal.,2009;Vessecchietal.,2011; Demarqueetal.,2016)

. AflatoxinsaremycotoxinsgenerallyproducedbyAspergillusspp. thathavebeendescribedastoxictohumans,animalsandinsects (MatsumuraandKnight,1967;Kirk,1971;GacemandHadj-Khelil, 2016).ThecarcinogeniceffectsofaflatoxinsB1andG1aredueto theCYP450activationofthedoublebondintheringAtoanactive epoxide,whichcovalentlybindstotheDNA(Niuetal.,2008). How-ever,despitetheknowledgeabouttheproductionandbiological activityofaflatoxins,littleisknownabouttheecologicalfunctions ofthesenaturalproducts.Ithasbeenshownthatsomeinsectssuch astheearwormHelicoverpazeaareabletodetoxifyaflatoxins(Niu etal.,2009), whileaflatoxinsimpairtheproperdevelopmentof otherinsects (Matsumuraand Knight,1967;Gunstetal.,1982). Thehightoxicityofaflatoxinsandthedetectioninthedead leaf-cutterqueenant,suggestthatthesecompoundsmaybeinvolved withthepathogenicityofA.nomiusASR3fortheleaf-cutterantsA. sexdensrubropilosa.

Authors’contributions

LCAVandFSNcontributedwiththecollectionandidentification oftheleaf-cutterqueenant.EASJandCRPcontributedwiththe lab-oratorywork,dataanalysisanddraftedthepaper.NPLcontributed withmassspectrometrydataacquisitionandanalysis.MTP super-visedthelaboratoryworkandcontributedwithcriticalreadingof themanuscript.Alltheauthorshavereadthefinalmanuscriptand approvedthesubmission.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

The authors thank São Paulo Research Foundation (FAPESP grants 2012/24204-1, 2012/22487-6, 2013/50954-0 and

2013/04092-7), CAPES and CNPq for funding and research support.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,atdoi:10.1016/j.bjp.2017.05.001.

References

Cavaliere,C.,Foglia,P.,Guarino,C.,Nazzari,M.,Samperi,R.,Laganà,A.,2007. Deter-mination of aflatoxins in olive oil by liquid chromatography–tandem mass spectrometry. Anal. Chim. Acta 596, 141–148.

Chouvenc,T.,Efstathion,C.A.,Elliott,M.L.,Su,N.Y.,2012.Resource competition between two fungal parasites in subterranean termites. Naturwissenschaften 99, 949–958.

Crotti,A.E.M.,Bronze-Uhle,E.S.,Nascimento,P.G.B.D.,Donate,P.M.,Galembeck,S.E., Vessecchi,R.,Lopes,N.P.,2009.Gas-phase fragmentation of ␥-lactone

deriva-tives by electrospray ionization tandem mass spectrometry. J. Mass Spectrom. 44,1733–1741.

Currie,C.R.,Mueller,U.G.,Malloch,D.,1999.Theagriculturalpathologyofantfungus gardens.Proc.Natl.Acad.Sci.U.S.A.96,7998–8002.

Currie,C.R.,Poulsen,M.,Mendenhall,J.,Boomsma,J.J.,Billen,J.,2006.Coevolved cryptsandexocrineglandssupportmutualisticbacteriainfungus-growingants. Science311,81–83.

Demarque,D.P.,Crotti,A.E.M.,Vessecchi,R.,Lopes,J.L.C.,Lopes,N.P.,2016. Fragmen-tationreactionsusingelectrosprayionizationmassspectrometry:animportant toolforthestructuralelucidationandcharacterizationofsyntheticandnatural products.Nat.Prod.Rep.33,432–455.

Gacem,M.A.,Hadj-Khelil,A.O.E.,2016.Toxicology,biosynthesis,bio-controlof afla-toxinandnewmethodsofdetection.AsianPac.J.Trop.Biomed.6,808–814.

Gloer,J.B.,Rinderknecht,B.L.,Wicklow,D.T.,Dowd,P.F.,1989.Nominine:anew insecticidalindolediterpenefromthesclerotiaofAspergillusnomius.J.Org. Chem. 54, 2530–2532.

Gunst,K.,Chinnici,J.P.,Llewellyn,G.C.,1982.Effects of aflatoxin B1, aflatoxin B2, aflatoxin G1, and sterigmatocystin on viability, rates of development, and body length in two strains of Drosophilamelanogaster(Diptera). J. Invertebr. Pathol. 39, 388–394.

Haeder,S.,Wirth,R.,Herz,H.,Spiteller,D.,2009.Candicidin-producing Streptomyces support leaf-cutting ants to protect their fungus garden against the pathogenic fungus Escovopsis. Proc. Natl. Acad. Sci. U. S. A. 106, 4742–4746.

Hölldobler,B.,Wilson,E.O.,1990.The Ants. Belknap Press, Cambridge.

Kirk,H.D.,1971.EffectofaflatoxinB1ondevelopmentofDrosophilamelanogaster (Diptera).J.Invertebr.Pathol.18,313–315.

Licht,H.H.,Boomsma,J.J.,Tunlid,A.,2014.Symbioticadaptationsinthefungal cul-tivarofleaf-cuttingants.Nat.Commun.5,5675.

Matsumura,F.,Knight,S.G.,1967.Toxicityandchemosterilizingactivityofaflatoxin againstinsects.J.Econ.Entomol.60,871–872.

Niu,G.,Siegel,J.,Schuler,M.A.,Berenbaum,M.R.,2009.Comparativetoxicityof mycotoxinstonavelorangeworm(Amyeloistransitella)andcornearworm( Heli-coverpazea).J.Chem.Ecol.35,951–957.

Niu,G., Wen, Z., Rupasinghe,S.G., Ren, S.Z.,Berenbaum, M.R., Schuler, M.A., 2008.AflatoxinB1detoxificationbyCYP321A1inHelicoverpazea.Arch.Insect Biochem.Physiol.69,32–45.

Oh,D.C.,Poulsen,M.,Currie,C.R.,Clardy,J.,2009.Dentigerumycin:abacterial medi-ator of an ant-fungus symbiosis. Nat. Chem. Biol. 5, 391–393.

Pagnocca, F.C.,Masiulionis, V.E., Rodrigues, A., 2012. Specialized fungal para-sites and opportunistic fungi in gardens of attine ants. Psyche (Stuttg),

http://dx.doi.org/10.1155/2012/905109.

Poulsen,M.,Hughes,W.O.H.,Boomsma,J.J.,2006.Differential resistance and the importance of antibiotic production in Acromyrmexechinatiorleaf-cutting ant castes towards the entomopathogenic fungus Aspergillusnomius. Insect. Soc. 53, 349–355.

Santos,A.V.,Dillon,R.J.,Dillon,V.M.,Reynolds,S.E.,Samuels,R.I.,2004.Occurrence oftheantibioticproducingbacteriumBurkholderiasp.incoloniesofthe leaf-cuttingantAttasexdensrubropilosa.FEMSMicrobiol.Lett.239,319–323.

(4)

532 E.A.Silva-Junioretal./RevistaBrasileiradeFarmacognosia27(2017)529–532

Schultz,T.R.,Brady,S.G.,2008.Majorevolutionarytransitionsinantagriculture. Proc.Natl.Acad.Sci.U.S.A.105,5435–5440.

Seipke,R.F.,Barke,J.,Brearley,C.,Hill,L.,Yu,D.W.,Goss,R.J.M.,Hutchings,M.I., 2011.AsingleStreptomycessymbiontmakesmultipleantifungalstosupport thefungusfarmingantAcromyrmexoctospinosus.PLoSONE6,1–8.

Sirhan,A.Y.,Tan,G.H.,Wong,R.C.S.,2013.Determinationofaflatoxinsinfoodusing liquidchromatographycoupledwithelectrosprayionizationquadrupoletime offlightmassspectrometry(LC-ESI-QTOF-MS/MS).FoodControl31,35–44.

Staub,G.M.,Gloer,J.B.,Wicklow,D.T.,Dowd,P.F.,1992.Aspernomine:acytotoxic antiinsectanmetabolitewithanovelringsystemfromthesclerotiaofAspergillus nomius.J.Am.Chem.Soc.114,1015–1017.

Tóth,K.,Nagy,L.,Mándi,A.,Kuki,Á.,Mézes,M.,Zsuga,M.,Kéki,S.,2013. Collision-induceddissociationofaflatoxins.RapidCommun.MassSpectrom.27,553–559.

VanArnam,E.B.,Ruzzini,A.C.,Sit,C.S.,Horn,H.,Pinto-Tomás,A.A.,Currie,C.R.,Clardy, J.,2016.Selvamicin,anatypicalantifungalpolyenefromtwoalternativegenomic contexts.Proc.Natl.Acad.Sci.U.S.A.113,12940–12945.

Vessecchi,R.,Zocolo,G.J.,Gouvea,D.R.,Huebner,F.,Cramer,B.,Marchi,M.R.R., Humpf,H.U.,Lopes,N.P.,2011.Re-examinationoftheanionderivativesof isoflavonesbyradicalfragmentationinnegativeelectrosprayionizationtandem massspectrometry:experimentalandcomputationalstudies.RapidCommun. MassSpectrom.25,2020–2026.