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,∗aDepartamentodeCiênciasFarmacêuticas,FaculdadedeCiênciasFarmacêuticasdeRibeirãoPreto,UniversidadedeSãoPaulo,RibeirãoPreto,SP,Brazil bDepartamentodeBiologia,FaculdadedeFilosofia,CiênciaseLetrasdeRibeirãoPreto,UniversidadedeSãoPaulo,RibeirãoPreto,SP,Brazil
cNúcleodePesquisaemProdutosNaturaiseSintéticos,FaculdadedeCiênciasFarmacêuticasdeRibeirãoPreto,UniversidadedeSãoPaulo,RibeirãoPreto,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.
©2017SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Thisisanopen accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
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
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]+
[G1+Na]+
[B1+Na]+
200 250 300 350 400 450 500 m/z
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.
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)) Molecularformula [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 aFragmentionsarecommonforaflatoxinsproducedbyA.nomiusASR3andauthenticstandards.Protonatedaflatoxinsionswereobservedaftertheadditionofformic
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.
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