Detection of carboxylesterase and esterase activity in culturable gut bacterial flora isolated from diamondback moth, Plutella xylostella (Linnaeus), from India and its possible role in indoxacarb degradation

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h tt p : / / w w w . b j m i c r o b i o l . c o m . b r /

Environmental

Microbiology

Detection

of

carboxylesterase

and

esterase

activity

in

culturable

gut

bacterial

ﬂora

isolated

from

diamondback

moth,

Plutella

xylostella

(Linnaeus),

from

India

and

its

possible

role

in

indoxacarb

degradation

Shanivarsanthe

Leelesh

Ramya,

Thiruvengadam

Venkatesan

∗

,

Kottilingam

Srinivasa

Murthy,

Sushil

Kumar

Jalali,

Abraham

Verghese

ICAR-NationalBureauofAgriculturalInsectResources,Hebbal,Bangalore,Karnataka,India

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received24June2014 Accepted8September2015 Availableonline2March2016 AssociateEditor:ValeriaMaiade Oliveira Keywords: Plutellaxylostella Bacteria 16SrRNA Phylogeny Carboxylesterase Indoxacarb

a

b

s

t

r

a

c

t

Diamondbackmoth(DBM),Plutellaxylostella(Linnaeus),isanotoriouspestofbrassicacrops worldwideandisresistanttoallgroupsofinsecticides.Theinsectsystemharborsdiverse groupsofmicrobiota,whichinturnhelpsinenzymaticdegradationofxenobiotic-like insec-ticides. The presentstudy aimed todetermine thediversity ofgutmicroflorain DBM, quantifyesteraseactivityandelucidatetheirpossibleroleindegradationofindoxacarb. Wescreened11geographicpopulationsofDBMinIndiaandanalyzedthemforbacterial diversity.Theculturablegutbacterialfloraunderwentmolecularcharacterizationwith16S rRNA.Weobtained25bacterialisolatesfromlarvae(n=13)andadults(n=12)ofDBM.In larvalgutisolates,gammaproteobacteriawasthemostabundant(76%),followedbybacilli (15.4%).Molecularcharacterizationplacedadultgutbacterialstrainsintothreemajorclasses basedonabundance:gammaproteobacteria(66%),bacilli(16.7%)andflavobacteria(16.7%). Esteraseactivityfrom19gutbacterialisolatesrangedfrom0.072to2.32␮mol/min/mg pro-tein.Esterasebandswereobservedin15bacterialstrainsandthebandingpatterndiffered inBacilluscereus–KC985225andPantoeaagglomerans–KC985229.Thebandswere character-izedascarboxylesterasewithprofenofosusedasaninhibitor.Minimalmediastudyshowed thatB.cereusdegradedindoxacarbupto20%,soitcoulduseindoxacarbformetabolism andgrowth.Furthermore,esteraseactivitywasgreaterwithminimalmediathancontrol media:1.87versus0.26␮mol/min/mgprotein.Apartfromtheinsectesterases,bacterial carboxylesterasemayaidinthedegradationofinsecticidesinDBM.

(http://creativecommons.org/licenses/by-nc-nd/4.0/).

∗ _{Corresponding}_author.

E-mail:tvenkat12@gmail.com(T.Venkatesan).

http://dx.doi.org/10.1016/j.bjm.2016.01.012

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Introduction

Diamondbackmoth(DBM),Plutella xylostella(Linnaeus), isa seriousdestructivepestofcruciferouscropsworldwide.1_The costassociatedwithitsmanagementisestimatedtobe4–5 USbillionperyear.2,3_In_India,_DBM_was_identiﬁed_as_a_major pest,withreported lossesofup to50%.4 _DBM_has_become resistanttoeach newclassofinsecticidebecauseof inten-siveandrepeateduse ofinsecticides. Insecticideresistance andabsenceofnaturalenemiesarebelievedtoexplaintheP. xylostellapeststatusworldwide.3,5 _Insects_gain_resistance_to insecticidesbyincreasingproductionofmetabolicenzymes byesterases,glutathione-S-transferasesandP450-dependent monooxygenasesaswellasnon-metabolicmechanisms. Fur-thermore,anincreasednumberofbacteriaintheinsectgut mayproducedetoxifyingenzymessuchascarboxylesterase andhelpinthedetoxiﬁcationofthexenobioticcompounds. DBMpopulationswerefoundtoharbormanygutbacteria.6

In recent decades, numerous investigations have aimed to understand the metabolic roles of associated microorganisms,7,8 _mainly _bacteria, _in _gut _microbiota.9–12 However, despite the growing awareness of the key roles microbesplayinmetabolism,vitaminsynthesis,pheromone production,pathogenpreventionandpesticidedegradation,13 thesymbiotic relationshipwithinthe gutisfar frombeing fullyunderstood.Microscopyexaminationofthecontentof thealimentarycanalofinsectsrevealsavarietyofmicroﬂora ranging from bacteria, yeast, molds and protozoa, but the interactionwithhostinsectsrangesfrommutualistic symbio-sistopathogenesis.Insectswithpoororrestrictednutrient dietlikelyhaveprimaryendosymbionts,andnoneofthese bacterialendosymbiontshavebeencultured.14

Degradationof insecticides usually combinesa number ofprocesses, includingmicrobial degradationandchemical hydrolysis,which is alsoaffected bysomephysiochemical propertiessuchaspH,organiccarbonandmoisturecontent.15 However,degradation istheprimarymechanismof insecti-cidebreakdown and detoxiﬁcationininsect systems; thus, microbesmayhaveamajorroleininsecticideresistance.A signiﬁcantnumberofmicrobialcarboxylesteraseshavebeen discovered,16_but_the_role_of_bacterial_{carboxylesterase}_from insectsinthedegradationofinsecticidesislittleknown.

WeaimedtodeterminewhethergutmicroﬂoraofDBM lar-vaeplayaroleininsecticidedegradation.Wecharacterized andanalyzedthediversityofgutbacteriaoflarvaeandadults ofDBMpopulationsbybiochemical and culture-dependent 16SrRNA gene-basedmolecularmethods. Furthermore,we detectedgeneralesteraseandcarboxylesteraseactivityof bac-terialstrains byspectrophotometry and native PAGEassay, respectively,toexaminethepossibleroleoflarvalgutbacteria ininsecticidedegradation.

Materials

and

methods

CollectionofDBMpopulations

Larvaeandadultswerecollectedfromdifferentgeographical locationsofIndia(Table1S)andrearedonmustard(Brassica

juncia L.)seedlings in plasticcups (5.5cm×6.9cm×4.5cm) containingmoistenedvermiculate.Theindividualcupswere placedinplasticcages(24cm×24cm×24cm)foradult emer-gence.

Culture-dependentisolationofbacterialﬂoraandbacterial growth

Before dissection, 4th-instar larvae and 1-week-old DBM adultsweresurface-sterilizedinsodiumhypochlorite(0.1%) and ethanol(70%) for5s toremove the adhering contam-inants,especiallyexternal microﬂora.17 _The_entire_gut _was removedunderasepticconditionsfromsterilizedlarvaeand adults.Guthomogenates(100␮L)wereplatedonsterile LB-agar media (10gtryptone, 5g yeast extract, 10gNaCl and 15gagar)inthreereplicatesandincubatedat30◦Cfor48h. Thecolonieswereselectedbymorphologicalcharacteristics (shape, color, and elevation) from the three replicates and byGramstaining,thenwerestreakedontoLBagarplatesto obtainpureculture,whichwasinoculatedonLBbroth(10g tryptone,5gyeastextract,10gNaCl)andincubatedat30◦C for48–72hformaximumgrowth.

Screeningandidentiﬁcationbasedonbiochemicaland morphologicalcharacteristics

Preliminaryidentiﬁcationofbacterialisolateswasbasedon biochemicalanalysis,morphologiccharacteristicsandGram staining.AllisolatesunderwentVoges–Proskauerandmethyl redtesting,withredindicatingpositivestainingandyellowor brownnegativestaining.

DNAextraction,PCRampliﬁcationand16SrRNAgene sequenceanalysis

Culturable bacterial genomic DNA was isolated by SDS-lysis18 _and _the _quantity _of _DNA _was _checked _with _1% agarose gel. The 16S rRNA gene was amplified with the 16S primer (27F: AGAGTTTGATCMTGGCTCAG and 1492R: CGGTTACCTTGTTACGACTT).ThePCRprotocolwasaninitial denaturationat95◦Cfor1minfollowedby30cyclesof95◦C (1min),annealing55◦C(1min)and72◦C(2min)andafinal extensionstepof72◦Cfor2min(C1000_Thermal_cycler)._The amplifiedPCRproductwasverifiedwith1.5%agarosegel.The PCRproductwaspurifiedbyuseoftheQuaginPCRpurification kit.Directsequencingofthe16SrRNAgeneforallbacterial iso-lateswasperformedatEurofinsanalyticalservices(Bangalore, India).

Phylogeneticsanalysis

TheNCBIdatabase(http://www.ncbi.nlm.nih.gov)wasBLAST searched for the 16S rRNA gene sequences, which were used to construct a phylogenetic tree by the character-basedmaximum-likelihoodmethodbasedontheTamura–Nei model19 _with_MEGA6_and_{1000-iteration}_{bootstrapping.}_The phylogenetictreewasvisualizedbyusingTreeView.Gamma distributionparametersandsubstitutionrateswereusedin phylogeneticanalysis.

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Quantitativeanalysisofesteraseandproteinestimation

Bacterialculturesweretransferred(1mL)toEppendorftubes andcellsuspensionswerecentrifugedat8000rpmfor10min (4◦C).Pelletswerere-suspendedin100mMpotassium phos-phatebuffer(pH7.5)andDNaseandcentrifugedat8000rpm for 15min (4◦C). Esterase activity was determined spec-trophotometricallyat450nm.20_An_amount_of₉₀₀_␮L_reaction mixture[␣-naphthylacetate+fastblueRR (4-benzoylamino-2,5-dimethoxyaniline)]and4mLpotassiumphosphatebuffer was added to each 100␮L of enzyme extract. The initial spectrophotometricreadingwasat450nm.Themixturewas incubatedatroomtemperature(RT)for10minandabsorbance wasrecordedat450nm.Astandardcurvewaspreparedwith anidenticalprocedurewithaknownamountof␣-napthol. Therate of hydrolysis was expressed asmicromoles of ␣-naptholproducedperminuteatRTandthespeciﬁcactivity was expressed as micromoles of ␣-napthol produced per minutepermilligram proteinatRT.Theprotein concentra-tionofthesamplewasdeterminedbytheLowrymethod21 withbovineserumalbuminusedasastandard.

Quantiﬁcationofesteraseinbacterialﬂoraanddetection ofcarboxylesterase

Thecarboxylesteraseactivitypatternin25bacterialisolates wasdetermined in8%polyacrylamidegel(pH8.8)bya Bio-radminigelelectrophoresissystem.22 _Esterase_activity_was estimated in 20min at RT. Profenofos was used as a car-boxylesteraseinhibitorfordetection.23_Native_PAGE_gel_with theenzymesamplewasincubatedinthePAGEbuffer contain-ing100␮Linhibitorsolutionfor30minatRTandthecontrol wasno treatment. Gelwas observedafterstainingwith ␣-naphthylacetate(0.4%)andFastBlueBsalt(0.1%)in40mM sodiumphosphatebuffer(pH7.4)andcomparedwiththe con-trolforcarboxylesterasedetection.

Screeningofindoxacarb(92.02%purity)degrading bacteria(BayerCropSciences,Mumbai,India)

Previouslyidentiﬁedlarvalgutbacterialstrainswerescreened fortheirabilitytodegradeindoxacarb.Thebacterialculture wasplatedonagarmediacontaining5mLindoxacarb(25,50 and100ppm).Thecoloniesabletogrowontheagarplatewith indoxacarbwereselectedandstreakedonanLBagarplateto obtainpureculture.Thisprocedurewasrepeatedthreetimes toensurethepurityoftheisolatedcolonies.Thepureculture wasinoculatedinLBbrothandincubatedat30◦Cfor48–72h formaximumgrowth.

IndoxacarbdegradationbyB.cereusandgas chromatography–massspectroscopy(GC–MS)

Aflask(250mL) wassupplementedwithindoxacarb asthe onlycarbonsource.Intotal,50mLminimalmediaand1mL inoculumweretransferredtotheflaskandthoroughly homog-enized.Thefinalconcentrationofindoxacarb(5mL)was25,50 and100ppm.Minimalmediawithinoculumandwith indox-acarbwithoutinoculumwasacontrol.ThepHofallmediawas adjustedto7.0andtheculturewasincubatedat37◦Cwithout

Table1–Culturablelarvalgutbacteriaisolatedfrom diamondbackmoth(DBM)populations.

Sampleno. Isolate Bacteria Accessionno.

1 PX-1 Enterococcusmundtii KC985227 2 PX-2 Bacilluscereus KC985225 3 PX-3 Enterobacterasburiae KC410777 4 PX-3 Leclerciaadecarboxylata KC410778 5 PX-3 Enterobacteriaceaebacterium KC410779 6 PX-4 Enterobactersp. KF468763 7 PX-5 Pseudomonassp. KF597541 8 PX-6 Bacilluscereus KC139357 9 PX-7 Pseudomonassp. KF485084 10 PX-8 Enterococcusmundtii KC985226 11 PX-9 Enterobactersp. KF485082 12 PX-10 Enterococcusmundtii KF597539 13 PX-11 Enterobactersp. KC441058

contactwiththeambientatmosphereandkeptinashakerat 120rpmfor6days.Cellgrowthwasmeasured spectrophoto-metricallybymeasuringabsorbanceat660nm.Growthrate wasestimatedbytheslopeofthelinerepresentingthe lin-ear ﬁtofthe increase inabsorbance over time,during the exponentialphaseofculturegrowth.

The culture was extracted twice with anequal volume ofethylacetateasanextracting reagent.Themixturewas centrifuged at 8000rpm for 10min and ethylacetate with residualindoxacarbwasfiltered.Thefiltratewasevaporated to drynessand re-dissolved in50␮Lhigh-performance liq-uid chromatography-gradedichloromethaneforanalysisby GC–MS.Theoperatingconditionswereinjectortemperature 245◦C,detectortemperature280◦Candcarrierflow2mL/min. Minimal media and indoxacarb without bacteria and with inoculumandwithoutindoxacarbwerecontrols.

Quantiﬁcationofesteraseduringindoxacarbdegradation

byB.cereus

Thetestsample(minimalmedia+indoxacarb+B.cereus)and controlsample(minimalmedia+B.cereus)wereassayedfor esterase activity.Thetestsamplewascentrifuged topellet outthecells(8000rpm,4◦C,and15min).Esteraseactivitywas determinedasdescribedabove.

Results

Biochemicalcharacteristics,morphologicalcharacteristics

andGramstainingpropertiesoflarvalandadultgut

bacterialisolatesofDBM

Analysisofmorphologicalcharacteristicsrevealedthesame typeofbacterialcoloniesinallthreereplicates.Therefore,a singlecolonywasselectedtomaintainapureculture.Among the13larvalisolates,9wereGram-negativerods,2were Gram-positiverodsandtheremaining2wereGram-positivecocci. Among12 adultisolates,9were Gram-negativerodsand3 wereGram-positiverods.Among25bacterialstrains,21had negative resultson methyl red testing,and 4 had positive results.Voges–Proskauertestingwaspositivefor12isolates andnegativefor13(Tables2Sand3S).Forfurtherconﬁrmation

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Table2–CulturableadultgutbacteriaisolatedfromDBM populations.

Sampleno. Isolates Bacteria Accessionno. 1 PX-1 Chryseobacteriumsp. KF312303 2 PX-2 Chryseobacteriumsp. KC985228 3 PX-3 Enterobactersp. KC512248 4 PX-4 Enterobactersp. KF468764 5 PX-5 Enterococcusmundtii KC512247 6 PX-6 Bacilluscereus KC139358 7 PX-6 Weissellaconfusa KC139359 8 PX-7 Pseudomonassp. KF597543 9 PX-8 Pantoeaagglomerans KC985229 10 PX-9 Morganellamorganii KF468765 11 PX-10 Pseudomonassp. KF597542 12 PX-11 Pseudomonasaeruginosa KF485083

andidentiﬁcationofisolates,molecularcharacterizationwas performed.

Diversityanalysisofculturablebacteria

Intotal,13and12bacterialfromlarvaeisolates(Table1)and adults(Table2),respectively,wereidentiﬁedandsequenced. 16S rRNA sequencing placed these 13 larval bacterial iso-latesintotwomajorclasses–gammaproteobacteria(76%)and

bacilli (15.4%)andthe 12adultbacterialisolatesinto three major classes basedon abundance– gammaproteobacteria (66%),bacilli(16.7%)andﬂavobacteria(16.7%).

Molecularcharacterizationandphylogenyanalysis

Thephylogenyanalysisof16SrRNAgenefromlarval(Fig.1) and adult(Fig.2)bacterialisolatesshowed twoclades. 16S rRNA gene sequencingwas usedto determinebroad taxo-nomicgroupings;thepercentagedistributionisinFig.3.For larvalgutisolates,thephylogenetictreeshowedtwoclades with94%bootstrapvalue:clade1,Enterococcus,Bacillus, Pseu-domonasandEnterobacter,andclade2,Enterobacterasburiaeand Enterobactersp.Furthermore,theﬁrstcladewasdividedinto twobranches:branch1,EnterococcusmundtiiandB.cereus,and branch2,PseudomonasandEnterobacter.Inthesecondbranch, Pseudomonasshowed98%similarity,Enterobactersp.and Entero-bacteriaceaebacteriumwerecloselyrelated,andthetreewas rootedwithCrenarchaeota.

Thephylogenetictreeforadultsshowedtwoclades:clade 1, Pseudomonas, Enterococcus, Weissella, Pantoea, Enterobacter, Morganella and Bacillus, and clade 2, Chryseobacterium. The ﬁrstcladewasfurtherdividedintotwobranches;intheﬁrst branch,Pseudomonassp.andP.aeruginosawererelatedtoeach

Enterococcus mundtii NBRC 12367 (AB680277) Enterococcus mundtii qz963 (AB761305) Enterococcus mundtii PX-E.C (KC985226) *

Enterobacter sp PX-L-B.E.c (KF468763) * Enterobacter sp. Hauzhou07 (JX220913) Enterobacter sp. PXE-1 (KC441058)*

Enterobacter asburiae m-4 (KF757336) Enterobacter asburiae PXE (KC410777) *

Enterobacter sp PX-L-S.E.sp (KF485082) * Enterobacter sp BSP6 (KF360068). 0.1 91 85 99 87 82 75 94 98 85 88 85 82 92 Crenarchaeota CQ786528 Enterobacteriaceae bacterium PXS (KC410779) * Enterobacteriaceae bacterium B28 (FJ628394) Bacillus cereus PXD (KC139357) *

Bacillus cereus PX-B.c.Or (KC985225) * Pseudomonas sp PX-L-Y.P.sp (KF485084) * Pseudomonas sp PX-L-P.strt (KF597541) * Pseudomonas sp ME-1 (KF975434) Pseudomonas sp SNPO614 (KF851246) Bacillus cereus CSB17 (KC999982) Bacillus cereus JKR62 (HQ705975) Bacillus cereus JP44SK43 (JX155760)

Enterococcus mundtii PX-L-Em (KF597539) * Enterococcus mundtii PX_Ec.Mt.Pun (KC985227) * Enterococcus mundtii APU IK-3 (AB898301)

Enterococcus mundtii APU IK-12 (AB898308)

Fig.1–Maximum-likelihoodtreededucedfrompartialsequencesof16SrRNAgeneoflarvalgutbacterialisolatesofDBM. Entriesfromthisworkarerepresentedwithastar(*),accessionnumbersandphylotypes.Entriesfromthepublicdatabase arerepresentedbyagenericnameandaccessionnumbers.Crenarchaeotawasusedasanoutgroup.

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Pseudomonas sp PX-A-P.st (KF597542)* Pseudomonas sp ME-1 (KF975434)

Pseudomonas sp EGY-WPhB6 (KF576651) Pseudomonas sp PX-A-P.p (KF597543)*

Pseudomonas aeruginosa PX-A-D.P.a (KF485083)* Pseudomonas aeruginosa BM6 (JX14417)

Bacilllus cereus PXDK-2 (KC139358)* Bacilllus cereus 3170O2 (KF598976)

Morganella morganii PX-A.S.M.m (KF468765)*

Morganella morganii 3D4A (JF710989) Pantoea agglomerans PX-Pt.ag.jor (KC985229)* Pantoea agglomerans WAB1951 (AM184290) Enterobacter sp PX-A-E.cl (KC512248)* Enterobacter sp R9 (KF843699) Enterobacter sp Px-A-Mi (KF468764)*

Enterobacter sp AL (KF747680) Enterobacter sp yingde07 (JX220911)

Enterococcus mundtii PX-A-Ec.m (KC512247)* Enterococcus mundtii APUIK-47(AB898343) Weissella confusa PXDK-3 (KC139359)*

Weissella confusa PUFSTMId055 (KR780676.1)

Chryseobacterium sp PX-A-PUJ-Cry.sp (KF312303)* Chryseobacterium sp K2 (KM192326) Chryseobacterium sp PX-Cry.bt.sp.Ori (KC985228)* Chryseobacterium sp JAS14 (KF313552) Crenarchaeota SCGC ASPO01000004 98 89 63 64 79 68 40 34 70 60 92 2720 99 98 8 81 81 0.5

Fig.2–Maximum-likelihoodtreeconstructedforpartial16SrRNAgeneofadultgutbacterialisolatesofDBM.Entriesfrom thisworkarerepresentedwithastar(*),accessionnumbersandphylotypes.Entriesfromthepublicdatabaseare

representedbyagenericnameandaccessionnumbers.Crenarchaeotawasusedasanoutgroup.

otherwith99%similarity.Enterobactersp.wasclosetoPantoea agglomerans.ThetreewasrootedwithCrenarchaeota. Entero-bacter,Enterococcus, Pseudomonasand Bacilluswere the most commoninbothlarvalandadultisolates.

0 20 Flavobacteria 40 60 80 100 120 140 160 Gammaproteobacteria Bacilli

Adult Larvae

Fig.3–Percentagedistributionofadultandlarvalgut isolatesofDBM.Percentagedistributioncalculatedonthe basisofrelativeabundance.

Esteraseanalysis

In total, 19 of 25 strains showed esterase activity, which ranged from 0.072 to 2.32␮mol/min/mg protein (Fig. 4). B. cereus (KC985225) showed high esterase activity. Esterase activity was observed in 15 of 25 bacterial strains: E. mundtii–KC985227,KF597539,KC512247,KC985226;B.cereus – KC985225, KC139358; E. asburiae – KC410777, Enterobacter sp. – KF485082; Enterobacter sp.– KC512248; E. cancerogenus – KF468763; P. agglomerans – KC985229; Pseudomonas sp. – KF597541,KF597542;Morganellamorganii–KF468765and Chry-seobacteriumsp.–KC985228.Theesterasebandpatternsofthe strainsandthefrequenciesofeachesterasebandareinFig.5. Fouresterasebandsweredetected,namelyEst-1,Est-2,Est-3 andEst-4,wasaccordingtotheirrelativemobilityinthegel. Est-2andEst-4werecommonamongall15strains,whereas Est-3wasobservedinP.agglomerans–KC985229andEst-1inB. cereus–KC985225.Thebandswerescatteredthroughoutthree zonesofthegel:fast-moving(Est-1),medium-moving(Est-2 and Est-3)andslow-moving(Est-4).Allhydrolyzedthe sub-strate␣-naphthylacetateandwereclassiﬁedas␣-esterases.

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0 0.5

Enterococcus mundtii (KF597539) Enterobacter asburiae (KC410777) Enterobacter sp. (KF485082)

Pantoea agglomerans (KC985229)Pseudomonas stutzeri (KF597541)Pseudomonas stutzeri (KF597542)Morganella morganii (KF468765)Chryseobacterium sp (KC985228) Bacillus cereus (KC139357)_{Enterobacter sp (KC441058)}

Enterococcus mundtii (KC985227) Enterobacter cloacae (KC512248)

Enterobacter cancerogenus (KF468763) Enterococcus mundtii (KC512247)Enterococcus mundtii (KC985226)

Bacillus cereus (KC985225)Bacillus cereus (KC139358)

1 1.5 2 2.5 Esterase activity µ moles/min/mg protein Bacterial isolates

Fig.4–SpeciﬁcactivityofbacterialesteraseisolatedfromlarvalandadultgutofDBM.

1 2 3 4 5 6 7 8 9 10 11 12 EST-4 EST-3 EST-2 EST-1 13 14 15

Fig.5–EsteraseisozymepatternfrombacterialisolatesofDBM.

Detectionofcarboxylesterase

The bands inhibited by profenofos were classiﬁed as car-boxylesterases.Incubationofthegelinprofenofoscompletely inhibitedEst-2inallbacterialstrains,Est-4inallstrainsexcept P.agglomerans–KC985229andEst-1inB.cereus–KC985225, whereasEst-3andEst-4werenotinhibitedbyprofenofosinP. agglomerans–KC985229.Accordingtothisclassiﬁcation, Est-2,Est-3andEst-4intheremainingstrainswerefoundtobe carboxylesterase.

Screeningofindoxacarb-degradingbacteria

Of13larvalgutbacterialisolates,onlyB.cereus–KC985225 was able togrow in the agar medium containing100ppm indoxacarb.B.cereus grownon minimalmedia lacking car-bon and supplementedwith indoxacarb showed enhanced growthrate,at660nm,whereasB.cereusgrownonminimal mediawithoutindoxacarb showedretardedgrowth (Fig. 6), whichconﬁrmsthatgrowthwaslimitedbydepletionof indox-acarbasanavailablecarbon source.Themaximumgrowth wasobservedinmediumsupplementedwith100ppm indox-acarb. 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0 24 48 72 96 120 Ab so rbance at 66 0n m Incubation time (h) 25 50 100 C

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Quantiﬁcationofesteraseactivityduringindoxacarb degradationbyB.cereus

The esterase activity in the test sample (minimal media+indoxacarb+B. cereus) was 1.87, 0.46 and 0.35␮mol/min/mg protein for 100, 50 and 25ppm respec-tively,andthesameinthecontrol(minimalmedia+B.cereus) was0.26␮mol/min/mgprotein(Fig.7).

GC–MSanalysis

The sample with B. cereus+indoxacarb+minimal media showed a single peak at retention time 22.9min in the chromatogram,and MSanalysisdetermined thatit had an identicalspectrumofindoxacarb(Fig.8).Thesamespectrum ofindoxacarbwasobservedatretentiontime22.86minwhen analyzingthecontrolsamplewithoutB.cereus(Fig.9),butthe totalareaoftheindoxacarb(Fig.10)peakwasreducedinthe

0 0.5 1 1.5 2 Indoxacarb (100 ppm) Indoxacarb (50ppm) Indoxacarb concentration (ppm) Esterase activity µ moles/min/mg protein Indoxacarb (25 ppm) Control

Fig.7–QuantiﬁcationofesterasesfromBacilluscereusin minimalmediawithindoxacarb.

2.8e+07 TIC: 30-1-14-STD.D\data.ms Abundance AbundanceTime--> 59.0 150.0 218.0 264.0 177.0 114.0 88.0 31.0 m/z--> 240.0 289.0 321.1 343.0 366.0 392.0 424.1 468.1 499.1 549.2 527.1 4.798 7.787 11.643 22.863 2.6e+07 2.4e+07 2.2e+07 1.8e+07 1.6e+07 1.4e+07 1.2e+07 1e+07 8000000 6000000 4000000 2000000 1000000 900 000 800 000 700 000 600 000 500 000 400 000 300 000 200 000 100 000 20 40 60 80 100120140160180200220240260280300320340360380400420440460480500520540560 Indoxacarb 0 6.00 8.00 10.00 12.00 14.00

Scan 2896 (22.826 min) 30-1-14-STD.D\data.ms

16.00 18.00 20.00 22.00 24.00 26.00 28.00 2e+07

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Abundance 22.901 300 250 200 150 100 50 7000 6500 5500 4500 3500 2500 1500 500 0 20 6000 5000 4000 3000 2000 1000 0 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 40 60 80 100 120 140 160180 200220240260 280300 320340 360380400 420 440460 480500520540 Indoxacarb m/z--> Abundance Time--> 207.0 32.0 59.0 95.9 150.0 122.8 176.9 235.0 281.0 320.9 355.1 429.0 401.0 475.0 527.0

Scan 2906 (22.891 min) 25-1-14-4B.D\data.ms lon 527.00 (526.70 to 527.00): 25-1-14-4B.D\data.ms

Fig.9–ChromatogramandmassspectrumofindoxacarbdegradationbyB.cereusintestsample.

O Cl O O O F F F O O O N N N

Fig.10–Structureofindoxacarb.

testsample.B.cereusshowedpartialdegradationofindoxacarb (20%).

Discussion

Inthepresentstudy,weisolatedvariousbacteriaassociated withdifferentﬁeldpopulationsofDBM,whichledtothe iden-tiﬁcationof13culturableisolatesfromlarvaegutand12from adultgut.WefoundE.mundtii,B.cereus,E.asburiae,Enterobacter sp.,Pseudomonassp.,P.aeruginosaandBacillussp.asthemajor bacterialisolatesfromlarvalandadultgut.Bacillussp., Pseu-domonassp.hasbeenisolatedfromthegutoftheDiscladispa armigera(Olivier).24ThePseudomonassp.andStenotrophomonas

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sp.were reportedinthe larval gutofDBM.6 _Presence_of_P. aeruginosa,Pseudomonassp.andEnterococcussp.wasreported inthelarvalgutofH.armigera.25_Therefore,_Bacillus_sp., Psue-domonasandEnterobacteroccurcommonlyindifferentgroups ofinsects.

Atthegenuslevel, Enterobacter(38.46%)and Pseudomonas (25%)dominatedinlarvalandadultDBMgut,respectively.The adultgutshowedmorediversebacterialcommunitiesas com-paredwithlarvae.Thegutduringthelarvaetoadulttransition isbelievedtoundergoasterilizationprocessandadultsrecruit newmicrobiota.26_B._cereus_was_exclusively_retained_by_both larvaeandadultsinthePX-6strain.Thedifferencesinmidgut bacterialcompositionbetweenlarvaeandadultswere large andcouldbeduetodifferentfoodhabitsandenvironmental conditions.Here,thelarvalgutbacterialassociationsaremore importantbecausetheyinvolvedetoxiﬁcationofinsecticide andmayconferresistancetoDBM.

BacteriaassociatedwithLepidopteraarelittleknown,and studies related togut microbiota suggest that microorgan-ismsplayaroleinhostdigestion,provideessentialnutrients andhelpindetoxiﬁcationofpesticides.27_{Microorganisms}_are knowntometabolize severalinsecticides. Insecticides with aliphatic,aromaticand heterocycliccompounds were com-pletelymineralizedbyvariousmicrobes.Catabolismisatype ofdegradation whereby an organic chemical iscompletely degraded, and the energy or nutrient gained used for the cellgrowthandco-metabolismisanothermethodof degra-dationwithnonetbeneﬁttotheorganism.28 _Three_classes ofbacterialenzymesareknowntodegradeorganophosphate compounds,namely,phosphotriesterases(EC3.1.8.1),methyl parathion hydrolases (MPH, EC 3.1.8.1) and organophos-phorus acidanhydrolases (EC3.1.8.2).29 _Our_study _revealed esteraseinthegutbacteriaofDBMlarvae.Theroleofinsect gut bacterial esterases in degradation of insecticides has not been studied extensively; however, the role played by soilbacterialesterases ininsecticide degradation hasbeen reported.30

Native PAGE is animportant technique in the study of bacterialesterases.Thecharacteristicsofeachesterase isoen-zyme can be determined by the use of specific inhibitors. Carboxylesterases (EC 3.1.1.1) from the thermophillic bac-terium Alicyclobacillus tengchongensis completely degraded malathion.31_In_our_study,_esterases_(EC_3.1.1)_from_B._cereus degradedindoxacarbupto20%.Fromthepreliminary char-acterization of insect esterases from DBM, a high level of resistancewasfoundassociatedwithslow-movingesterases, whicharemoreimportantinresistancemonitoringthan fast-movingesterases.32_In_the_present_study,_among₂₅_bacterial strainstested,15showedesterasebands,whereas19showed esteraseactivitywhenquantifiedspectrophotometrically.All 15 strains showed slow-moving esterase and were charac-terizedascarboxylesterases,exceptinonestrain.Therefore, thepresenceofslow-movingcarboxylesteraseinthe bacte-rialstrainsmayplayaroleininsecticidedetoxificationand mayconferresistancetoDBM.Studiesoftheuseof micro-bialcarboxylesteraseforinsecticidedegradationarelimited. Indoxacarbisoneofthemostcommonlyusedinsecticides belongingtotheoxadiazinegroupworldwideanduntilnow, welackpublishedinformationon microbialdegradation of indoxacarb. Our study reveals that bacteria (B. cereus) in

DBM could utilizeindoxacarb for metabolism and growth. GC–MSanalysisoftheextractsoftheminimalmediastudy withB.cereusrevealeditsindoxacarb-degradingcapability.B. cereuswasalsofoundtodegradefenvalerate,cypermethrin, andchlorpyrifosinsoil.30,33,34_In_the_present_study,_B._cereus mayhavedegradedindoxacarbbyenhancedesterase activ-ity. Quantificationofesteraseintheminimalmediaclearly showed the role of esterase in degradation ofindoxacarb. From the resultsof this study,we cannotpredict whether this hydrolysis resultsincomplete mineralization or if the resultingmetaboliteremainsinthemedium.Bacteriaisolated fromtheinsectgutexposedtoinsecticidesgenerateenzymes, therebyresultinginanewmetabolicpathwayofinsecticide degradation.Our studyrevealed the role ofesterase inthe partialdegradationofindoxacarb.Wehaveinsufficient evi-dencetostatethesymbioticroleofthesebacteriawithDBM. Thepresentresultsopenanavenuefortheidentificationof novelenzymecarboxylesteraseanditsroleinthedegradation ofindoxacarb.Furthermore,intensiveworkonunderstanding thepathwaysandaminoacidsinvolvedmayleadtothe syn-thesisofspecificinsecticideswithanewmodeofactionfor themanagementofDBM.

Conclusions

Weisolated13and12bacterialisolatesfromlarvalandadult gutofDBM,respectively.Thegutbacterialassociationdiffered betweenadultsandlarvae.Inlarvalgutisolates, gammapro-teobacteriawasthemostabundant(76%),followedbyBacilli (15.4%).Esterasebandswereincreasedin15bacterialstrains. B.cereusdegradedindoxacarbupto20%,whichshowedits abil-itytoutilizeindoxacarbformetabolismandgrowth.Besides theinsectesterases,bacterialcarboxylesterasemayaidinthe degradationofinsecticidesinDBM.

Conﬂicts

of

interest

Theauthorsdeclarenoconﬂictsofinterest.

Acknowledgement

ThisisapartofPh.D.workoftheﬁrstauthor.

Appendix

A. Supplementary

data

Supplementarydataassociatedwiththisarticlecanbefound, intheonlineversion,atdoi:10.1016/j.bjm.2016.01.012.

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