(Moco) domain(Garattinietal.,2003;Mahro2011). The containing domainandan85 melanogaster specificities andexpressionprofilesofthefourAOXenzymesin identified. Inthisstudywecharacterizedtheactivities, involvedandthecorrespondingmutationshavenotyetbeen characterized byundetectableAOXenzymaticactivity. However, the We demonstratethatthe AOX2 detectable onlyduringmetamorphosisandcharacterizea encoded .Furthermore,weshowthatAOX2activityis shift event,whichremovesthemolybdenumcofactordomainof andAOX4activityisnotdetectableinourassays. via LaMasa19,20157Milan,Italy. Biology, IRCCS-Istituto diRicerche Farmacologiche MarioNegriIRCCS, University of Potsdam, 14476Potsdam, Germany.University of identified an11-bp deletioninthe consequence ofastructuralalterationtheAOX1 © 2014.PublishedbyTheCompanyofBiologistsLtd|JournalExperimentalBiology(2014)217,2201-2211 doi:10.1242/jeb.102129 Received 7January 2014;Accepted20March2014 1 a 20 al., 2012).Thestructureofeachmonomertheseenzymesincludes subunits, withdifferences in the substratebindingpocket(Coelhoet butterfly-shaped XORenzymes,consistingoftwoidentical150 mouse AOX3wasshowntobehighlyidenticalthehomodimeric and xanthineintouricacid(Enrothetal.,2000).Thestructure of the catabolismofpurines,metabolizinghypoxanthineintoxanthine xanthine (XOR)enzyme,whichservesakeyrole in molybdo-flavoenzymes togetherwiththestructurallyrelated Aldehyde oxidases(AOX;EC1.2.3.1)belongtothefamilyof melanogaster KEY WORDS:Aldehydeoxidase,Molybdoenzymes, mutant allelesinthisgeneregion, distinct lineages. Thefunctionalroleandthesubstratespecificityof independent duplicationeventsinthevertebrateandinvertebrate Phylogenetic analysisshowedthattheAOXgeneclusterevolvedvia aldehyde oxidases( In thegenomeofDrosophilamelanogaster Zvonimir Marelja specificities different geneexpression patternsandenzymesubstrate The fouraldehydeoxidasesof RESEARCH ARTICLE *Authors for correspondence ([email protected];*Authors for correspondence [email protected]) CP 07360Mexico City, Mexico. Neurociencias, CentrodeInvestigación ydeEstudiosAvanzados, Av. IPN2508, -1 Fanis Missirlis INTRODUCTION ABSTRACT Institute of Biochemistry and Biology, Department of MolecularEnzymology, andBiology, Biochemistry of Department Institute of kDa N-terminal2x[2Fe-2S]domain,a40 insertion inthisdevelopmentalgenethatdisruptsitsactivity. . We showthatthe , Geneduplication,Substratespecificities 3, AOX enzymesisunknown.Two loss-of-function * andSilkeLeimkühler AOX1– ( 1 Aldox-1 , MiriamDambowsky Aldox-1 4 3

Departamento deFisiología, Biofísicay Departamento kDa C-terminalmolybdenumcofactor n1 ) wereidentifiedonchromosome3. ) areassociatedwithaphenotype Po n1 low pyridoxaloxidase Po lpo phenotype mapstotheAOX3 lpo -associated phenotypeisthe allele, resultinginaframe- 2 Laboratory of Molecular of Laboratory , fourgenescodingfor

1, 1 kDa centralFAD- , MarcoBolis * Drosophila ( gene. We Po lpo Minos- ) and

kDa D. Drosophila melanogaster 2 , MarinaL.Georgiou metabolic pathways(Kamlehet al.,2008;Kamlehet2009). of thetryptophan,arginine, pyrimidineandglycerophospholipid hypoxanthine anduraterevealed unsuspectedchangesineach AOXs) mutantsconfirmedthe biochemicalchangesinxanthine, suppressing MocosulfurationandinactivatingbothXOR and and Rajagopalan, 1982;Bray, 1988; Hille,1996). required forthecatalyticactivityofenzymes(Wahl and Moco ofeukaryoticAOXscontainsanequatorialsulfurligand mammalian AOXsareunknown. physiological roleandtheendogenoussubstratesofhuman than XOR(Pauff etal.,2009;Coelho2012).The 2012). Nevertheless,AOXsexhibitabroadersubstratespecificity common reactionmechanisms(Schumannetal.,2009;Coelho and thehighstructuralsimilaritybetweenAOXsXORsuggest presence ofsomehighlyconservedresiduesinthecatalyticcenters Terao, 2011; Mahroetal.,2011; GarattiniandTerao, 2012).The relevance (Schumannetal.,2009;Pryde2010;Garattiniand and aza-oxo-heterocyclesofpharmacologicaltoxicological specificity, catalyzingtheoxidationofvarioustypesaldehydes 2008; Garattinietal.,2009;Kurosaki2013). AOX3 non-functional pseudogenes,representingthevestigesofmouse contains onesingleandfunctional ( marsupials containthelargest numberof Rodríguez-Trelles etal.,2003;Garattini 2008).Rodentsand ancestor (Forrestetal.,1956;NashandHenderson,1982; arose fromaseriesofduplicationeventscommon Metabolic profilingofthe et al.,1992;Vorbach etal.,2003;Glantzounis2005). pigments occurringintheabsenceofantioxidanturate(Hilliker mutant arisesfromtheprogressiveoxidationanddegradationof eye Forrest, 1980).Thissuggeststhatthebrownisheyecolorof (yellow brown)toxanthommatin(dulldark(Phillips and chromogenic oxidationoftheeyepigmentdihydroxanthommatin the redeyepigmentdrosopterin(brightred),andincreased reddish-brown eyecolourbecauseoftheloweredconcentrations of Drosophila melanogasterry them hypersensitivetooxidativestress(Hillikeretal.,1992). urate (Morita,1958;GlassmanandMitchell,1959),whichmakes mutants accumulatexanthineandhypoxanthinearedevoidof (Chovnick etal.,1976;Keith1987).Loss-of-function to controlAOX enzymaticactivity(Collinsand Glassman,1969; AOX1, Two loss-of-functionmutantalleles, Most animalandplantgenomescontain In Mammalian AOXsarecharacterizedbyabroadsubstrate aldehyde oxidase-1 Drosophila melanogaster,therosy and AOX3, AOX3L1 AOX4 orthologs (Garattinietal.,2003;Garattini 1 , EnricoGarattini and ( Aldox-1 AOX3L1), whereasthehumangenome Drosophila ry mutants arecharacterizedbyadull n1 ), havebeenpreviouslydescribed AOX1 low pyridoxaloxidase ( ry) locusencodesXOR and gene (hAOX1)andtwo AOX AOX 2 , maroon-like have gene clustersthat functional genes ( ( Po 2201 ma-l, XOR lpo ry ry )

The Journal of Experimental Biology indicated. Theinsertion pointsof CG18516 melanogaster the activityofencodedenzymeisundetectablein provide evidencethat AOX3 Df(3R)Exel7326 2202 Fig. Sequencing ofthe the dataavailable,itwasproposedthat Dickinson, 1970;DickinsonandGaughan,1981).Onthebasisof RESEARCH ARTICLE four genes,CG18522, predicted addition, itremainstobeestablishedwhetherallofthefour genes correspondingto al., 2000;Garattiniet2008).Atpresent,theidentitiesof four two distinctandunidentified the productof The The RESULTS (RNAi). We clonedtheclassical activities hadbeendisruptedbymutationorRNA interference extracts fromavarietyof and Williamson, 1976;Cypheretal.,1982). physiological relevance(Stanulovi , thelevelsofpyridoxalareconsideredtobeparticular physiological significanceofthe associated withthe during metamorphosisandthattwofurtherAOXactivitiesare experiments suggestingthattheAOX2activityisonlypresent substrate ofthe form oftheactivevitaminB6metabolite,seemstobeaspecific ascertained. With respecttothis,pyridoxal,thedephosphorylated In thisstudyweusedanativein-gelactivityassaytoanalyze O xanthineoxidoreductase 2,4,5-trimethoxybenzaldehyde RNA interference pyridoxaloxidase XOR pyridoxal-5-phosphate TMB 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazoliumbromide RNAi PO molybdenumcofactor 2,4-dihydroxybenzaldehyde PLP aldehydeoxidase MTT Moco DHB AOX List of abbreviations List of

.Genomic locationofthe 1. AOX AOX are conservedinmost / AOX4 Drosophila AOX genes evolved bygeneduplication events genes presenton extracts usingawidevarietyofsubstrates. Deficiencies: Genes: Minos insertions: Cytogenetic map: and Df(3R)BSC515 are located on the right arm of the third (3R). Gene and inter-gene sizes are shown in bp and the number of exons per geneis are locatedontherightarmofthird chromosome(3R).Geneandinter-genesizesareshowninbp thenumberofexonsper Po D. melanogaster encoding enzymepyridoxaloxidase(PO).In AOX3 CG18519, AOX4 Drosophila AOX1 Po genes arefunctionallyactive.Finally, the gene. We showthat D. melanogaster Drosophila Mi{ET1}CG18519 rspiamelanogasterAOX Drosophila AOX is evolutionarilymorerecentandthat lpo 5163 bp 4 exons . Theprimersused tomapthe rosy 87D Drosophila AOX and Po CG6045 Drosophila genes belongingtotheclusterof ć genome ledtotheannotationof lpo andChaykin,1971;Browder Aldox-1 allele andshowedthatPOis Po chromosome 3(Adamset and lpo . b 6 p41b 1234bp 481bp 768bp 2.5 Mbp gene. We alsoreport MB05465 and . Finally, we n1 CG18516, codingfor strains, whereAOX AOX1, Df(3R)Exel6174 are unknown.In genes needstobe Aldox-1 and Mi{y CG18522/AOX1/PO 4749 bp 6 exons Po AOX2 n1 lpo +mDint2 genes. lpo map to mutation bysequencing areshownasarrows. AOX }CG6045 and The genes D. MI02285 originated fromanancient phylogenetic reconstructionsuggeststhat genomic sequencingdatacoveringatotalof12distinctlineages.Our and correspondingproteinproductsinDrosophila phylogenetic analysisonthepredictedstructuresof primers usedformolecularanalysis. deletion mutants(deficiencies)thatwereusedinthisstudyandPCR cluster. Fig. 1992). Thismappingisconsistentwiththelocationof 88F-89A, 0.08 (Garattini etal.,2008): same nomenclaturegiventothefourgenesbyGarattinietal. characteristics oftheAOXenzymes.Inthisstudy, weadoptedthe an equivalentnumberofpredictedproteinshavingallthestructural Po Chovnick etal.,1990).Usingappropriatecytogeneticmarkers,the XOR/ry are clusteredonchromosome3R,2.5 (CG6045) andAOX4 duplication gaveriseto AOX1, Drosophila yakuba into resulted intheappearanceof giving risetovertebrate with asingle AOX3. material Fig. in complement ofthepredictedmolybdo-flavoenzymespresent among downstreamlineages.Theduplicationledtotheextant also supplementarymaterialFig. be theorthologsof duplication, whichledtotheoriginof D Drosophila grimshawi,twodistinctduplicationsgivingriseto of themostancientcomplement characterized byfourdistinct subsequent evolutionaryhistoryof AOX1, virilis CG18519/AOX2 Mi{ET1}CG18519 and To reconstructtheevolutionaryhistory, weperformeda A majorevolutionaryeventisrepresented byathird lpo 88F7-89A Dr 4546 bp ry/ 5 exons AOX2. A furtherduplicationeventinvolving are shownandalso thebreakpointsfor osophila simulans, and Df(3R)BSC515 XOR, CG18522 is endowedbyaspecies-specific AOX3-D2 Drosophila willistoni AOX2 AOX2, Intron 2 locus (BridgesandBrehme,1944;Keithetal.,1987; Aldox-1 The JournalofExperimentalBiology(2014)doi:10.1242/jeb.102129

1 alsoshowsthetransposoninsertionsandgenomic XOR

and S1). InDrosophila ananassae,afurther MB05465 cM apartfromeachother(NelsonandSzauter, AOX3 are evident(Fig. n1 AOX3 Df(3R)Exel7326 and threeactive / and D. melanogasterAOX1, AOX1, CG18519 CG6045/AOX3 Mi{y alleles weremappedtothecytogeneticregion C156.Fg illustratesthatthesegenes 1 (CG18516). Fig. Aldox-1 4524 bp 7 exons AOX1 +mDint2 s (Fig. 2). Itislikelythatthisduplication 2). AOXs (Fig. and Drosophila erecta , whichconsistsofXOR AOX3-D3 (Fig. Drosophila sechellia Exon 3 }CG6045 n1 XOR and AOX1, which,subsequently, duplicated AOX3 AOX4 (CG18522), 2, supplementarymaterialFig.

S1). Thesespeciesarerepresentative Drosophila mojavensis 2, supplementarymaterialFig. duplication, distinctfromtheone MI02285 / AOX2, CG6045 (Fig. 2, supplementarymaterial duplications (Fig. AOX genes (Fig. AOXs intheDrosophilidaeis AOX3-D

CG18516/AOX4 Mbp downstreamfromthe AOXs inDrosophila. The Df(3R)Exel6174 genes thatarepredictedto 4144 bp AOX2 7 exons AOX4 3R AOX2 Drosophila , in additionto (CG18519), 2, supplementary using theavailable AOX2 / D. melanogaster, and isconserved AOX3 and

2). Drosophila , are endowed and gave riseto AOX AOX3 AOX

AOX2- S1). In AOX1 AOX3 AOX3 AOX3 genes XOR, gene

(see S1).

The Journal of Experimental Biology RESEARCH ARTICLE doublet (bandsa 3A).Thetoptwobandsconstituteanincompleteresolved (Fig. With benzaldehydeasasubstrate,four bandsweredetected thiazolyl bluetetrazoliumbromide(MTT)astheelectronacceptor. determined byanin-gelstainingassayusingvarioussubstrates and Proteins wereseparatedbynativePAGE andAOXactivitywas code foractiveproteins,weperformedexperimentsonflyextracts. To evaluatewhetherthe Fig. aldehyde-catalyzing enzymesinadultwild-typeflies four substratesreveals of different thepresence useof The acetaldehyde asasubstrate(Fig. pyridoxal, whileband results showthatbanda products oftheactivitydistinctAOXisoenzymes.Inaddition, the acetaldehyde andhypoxanthineindicatethatthefourbandsare the Drosophila pseudobscura predicted inthe whereas in the lacking distinctgenomicregions ofthechromosome3Raffecting products, weevaluatedthecomplement ofbandsinthreeflystrains incubation withhypoxanthine. acetaldehyde. Band lineages, four AOX7 AOX6 XDHs To relateeachofthebandstoactivity AOX S1). Finally, onedeletionandthree AOX (Fig.

and I

gene cluster(Fig. AOX3 XDH nsect

Vertebrate XD genes. OnlyAOX1

Df(3R)Exel7326[AOX2-4]

AOX5, respectively. Inturn,

. AOX1 . 2, supplementarymaterialFig. h.s

Nematoda XDHs Nematoda s Vertebrate AOXs Invertebrate AOXs is deletedwhile

Drosophila obscura

AOX2 and Hs AOXs AOX3 d b AOX5 ). Theenzyme-specificsubstratespyridoxal,

corresponds toXOR,asitisstainedafter Bacteria XDHs AOX6 AOX4 (XDH Ancestral duplicationevent2) AOX b is stainedexclusivelyinthepresenceof AOX1

(XDH Ancestral duplicationevent1) 1). Plant AOXs AOX7 is stainedonlyinthepresenceof and is deletedin Df(3R)BSC515[AOX1-4] Plant XDHs genes predictedin Drosophila AOXs AOX1

Fungii XDH 3A). Bandc Drosophila persimilis.Inthese group, beforethebranchingof , and AOX2– Drosophila XDHs AOX5 Df(3R)Exel6174[AOX1], AOX4

S1). AOX is detectableonlywith 4 are deletedwithout are duplicatedinto is duplicatedinto D. melanogaster duplications are 100 AOX1– 63 100 removes all 36 100 78 74 75 94 4 100 D. yakubaXDH 100 gene 100 D. ananassaeXDH D. mojavensisXDH D. sechelliaXDH D. erectaXDH D. melanogasterXDH 95 D. persimilisXDH D. pseudoobscuraXDH 84 D. virilisXDH 100 89 100 D. grimshawiXDH D. willistoniXDH 62 100 97 100 100 98 96 100 100 100 100 100 100 100 85 contrary towhatisobserved in the fastest migratingband were screenedwith20different substrates(Fig. (Dickinson andGaughan,1981).Flyextractsofthesegenotypes the twob material Fig. extracts. Incontrast,the Deficiency ( affecting used. IntheDf(3R)Exel7326[AOX2-4]/ry previously showntobespecific forPO(Cypheretal.,1982),was codes fortheclassicalPOenzyme.Insteadofpyridoxal,TMB, AOX substrates (DickinsonandGaughan,1981). Finally, ourresults confirms previousstudiesthatindicate thatXORcanutilizecertain extracts, bandsb 1 Po These resultsconfirmthatdifferent highlights onlythetopthree (Fig. distinguishing thevariousAOXbands.Similartobenzaldehyde trimethoxybenzaldehyde (TMB)arethemosteffective in dihydroxybenzaldehyde (DHB),4-nitrobenzaldehydeand2,4,5- Out ofthe20substratesconsidered,salicylaldehyde,2,4- four AOX/XORbandsobservedinwild-typeflies(seeFig. used ascontrolsinourexperiments,theyshowthesame detectable in supplementary materialFig. characterized bydifferent substratespecificities(Fig. 100 100 100 D. mojavensisAOX3 n1 99 100 100 No bandsstainablewiththeAOXsubstratesconsidered are D. melanogasterAOX3 D. sechelliaAOX3 D. erectaAOX3 D. yakubaAOX3 D. ananassae lpo D. virilisAOX3 D. willistoniAOX3 100 D. pseudoobscuraAOX5 D. persimilisAOX5 D. mojavensisAOX1 D. virilisAOX1 64 D. virilisAOX3-D mutant extractsonlylackband D. erectaAOX1 D. yakubaAOX1 D. grimshawiAOX3 D. willistoni D. ananassaeAOX1 D. melanogasterAOX1 D. sechelliaAOX1 , Aldox-1 D. pseudoobscuraAOX1 100 D. persimilisAOX1 3A), salicylaldehydeshowsfourbands(Fig. D. grimshawiAOX1 D. persimilisAOX7 D. pseudoobscuraAOX7 100 D. grimshawiAOX3-D2 D. persimilisAOX6 D. pseudoobscuraAOX6 D. ananassaeAOX3-D3 D. persimilisAOX4 D. pseudoobscuraAOX4 100 D. simulansAOX1 D. erectaAOX4 D. yakubaAOX4 D. melanogasterAOX4 D. sechelliaAOX4 D. grimshawiAOX2 D. mojavensisAOX2 D. virilisAOX2 AOX1. We analyzedthetrans-heterozygotesbetween D. grimshawiAOX2-D D. willistoniAOX2 D. pseudoobscuraAOX2 D. ananassaeAOX2 D. sechelliaAOX2 D. yakubaAOX2 D. persimilisAOX2 and D. erectaAOX2 AOX3 AOX1 D. melanogasterAOX2 The JournalofExperimentalBiology(2014)doi:10.1242/jeb.102129 Df) chromosomesandthetriplemutantrecombinant

Df(3R)BSC515[AOX1-4]/ry S2). Extractsofthehomozygote n1 c that removesalldetectableAOXandXORactivity bands, andTMBonlythetop and c d are notdetectable.Inthesame mutant,the Df(3R)Exel6174[AOX1]/ry is visiblewhensalicylaldehyde isused,

S2). a– distinct colors. AOXs duplicationsareindicatedwith shown. primitive branchesoftherootedtreeare data. Thebootstrapscoresforthemost was deducedfromgenomesequencing AOX andXORproteinswhosestructure phylogenetic treeconsistsofallthe protein sequences.Theunrooted reconstructed (right) wasgeneratedfromthe lineages. Therootedphylogenetictree representative ofdifferent evolutionary eukaryotic AOX/XORthatare publicly availableprokaryoticand Drosophila was generatedfromcombining The unrootedphylogenetictree(left) and XORproteinsofDrosophilidae. Fig. c bands, 4-nitrobenzaldeyhdeonly a

, suggestingthatthe .PhylogenetictreesoftheAOX 2. ry D. melanogaster Drosophila mutant. Thisobservation 2 2 protein sequenceswith , Po , Po Drosophila lpo lpo

white 3B, supplementary , Aldox-1 XOR andtheseven , Aldox-1 a band (Fig. 2 , Po AOX andXOR mutant were

3B). DHB AOX1 AOXs are lpo n1 n1 , Aldox- mutant mutant 2203

gene 3A). 3B).

3B, ry 2 ,

The Journal of Experimental Biology from thecontrolflies Aldox-1 corresponding recombinant proteinwasexpressed andpurifiedin wild-type ubiquitous 2204 Df(3R)Exel6174[AOX1]/ry analyzed theAOXactivityprofilesindeletionmutant To validateandextendthe findingsofthesubstratescreen,wefurther (Fig. might implythatthesetwobandsaregeneratedbythesameprotein show thatbandsb RESEARCH ARTICLE The bandisabsentinextractsof ry and mutantflies(Fig. acetaldehyde asasubstratearepresentintheextractsofallcontrol AOX1 for isresponsible POactivity the acetaldehyde-dependentbands annihilatestheTMB-dependent band specific RNAidrivenbytheubiquitous act-Gal4 missing inDf(3R)Exel6174[AOX1]/ry a c b c b a d c b a B d c b a A 506 To furtherconfirmthatAOX1isindeedtheenzymaticactivity 3, supplementarymaterialFig. show aspecificAOX1bandfollowingincubationwithTMB.

n1 Benzaldehyde white flies (Fig. AOX1 D. melanogasterAOX1

silencing withaspecificRNAi(Fig. Df(3R)Exel6174[AOX1]/ry and

4A). Incontrast,thebands Pyridoxal Df(3R)Exel6174[AOX1]/TM6B,Tb

c 4A). Similarly, silencingof have thesamesubstratespecificity, which

2 Df(3R)Exel7326[AOX2-4]/ry , Po

Acetaldehyde lpo Df(3R)Exel6174[AOX1]/ry , Aldox-1 b

and Df(3R)BSC515[AOX1-4]/ry2 S2). , Po gene wasclonedandthe 2 lpo , Po c

a Hypoxanthine

, whileitleavesunaffected , Aldox-1 (Fig. n1 Fg 4 (Fig. lpo ry

b 5

, Aldox-1 06 2 4B). , Po n1 and

promoter inwhite lpo A) andfollowing 2,4,5-Trimethoxybenzaldehyde (TMB) 4-Nitrobenzaldehyde 2,4-Dihydroxybenzaldehyde Salicylaldehyde

c , Aldox-1 AOX1 observed with

4B). Extracts 2 1 n1 , Po , white n1

flies, the lpo 2 , Po with a , Aldox-1 and lpo

n1 , from extracts obtainedforthe acetaldehyde (Fig. stained stronglywithbenzaldehyde/TMBandmoreweakly with unequivocally demonstratethatthe Minos Df(3R)Exel7326[AOX2-4]/ry Acetylaldehyde-dependent bands recombinant AOX1andwhole--extractband extracts usingin-gelstaininganalysis(Fig. migration behaviorandsubstratespecificitytothatinthewhole-fly Escherichia coli domain of which resultsinaframe-shiftmutationthateliminatestheMoco classical POenzyme,whichisinactivatedinthe for theAOXactivityassociated withbands other thanAOX1 confirming thatthetwobands must betheproductsof Df(3R)Exel7326[AOX2-4]/Mi-AOX3 likely toencodetwo variants protein AOX3 ry , the isoform affected bythe affected , theisoform insertion mutantofthe 2 , Po AOX1 The JournalofExperimentalBiology(2014)doi:10.1242/jeb.102129 lpo , Aldox-1 . RecombinantAOX1wasusedtocomparethe Fg 5A).To identifythe (Fig. (supplementary materialFig. 2,4,5-trimethoxybenzaldehyde (TMB)wereused. dihydroxybenzaldehyde, 4-nitrobenzaldehydeand used. Assubstrates,salicylaldehyde,2,4- Df(3R)BSC515[AOX1-4] Df(3R)Exel6174[AOX1] procedure wasappliedtothetrans-heterozygotes hypoxanthine banddwasdetected.(B)Thesame acetaldehyde bandsbandcweredetected,with detected, withpyridoxalonlybandawas acceptors: withbenzaldehydethebandsa–dwere bromide andphenazinemethosulfateaselectron four different substrateswiththiazolylbluetetrazolium extracts wereanalyzedinnativegelsthepresenceof AOXs. Fig. controls, white Df(3R)Exel7326[AOX2-4]

4C). Cloningandsequencingofthe

.Substratespecificitiesofthe 3. (A) TheAOXandXORactivitiesin n1 white flies demonstratedan11-bp deletion, AOX3 2 , Po and rosy , ry gene (Mi-AOX3;Fig. lpo 506 b Aldox-1 , Aldox-1 counterparts show noAOX / 506 AOX1 ry / ry / 2 ry and parentalstrains,the and , Po ( 2 ry , Po 2 , Po 506 AOX lpo b lpo ) mutantextractswere gene codesforthe n1 lpo , Aldox-1 a

c 4C). Asexpected, , Aldox-1 and

Po , Aldox-1 S3). Theseresults co-migrate andare mutant allele,is n1 gene responsible are absentin lpo .melanogaster D. c mutant flies, , weusedthe mutant allele. n1 white n1 AOX1 AOX , n1 . As 1). Unlike and fly genes gene

The Journal of Experimental Biology RESEARCH ARTICLE due totwoseparable proteinproductsoriginating fromthe down. Theresultsareconsistent withtheideathatbands flies, demonstratingthespecificity ofthe comparable in associated withtheAOX1band afterincubationwithTMBis disappearance ofband causes astrongreductionin the levelsofband silencing ofthe band uponincubationwithacetaldehyde(Fig. C B A A A A A A A A A A A XDH OX1 OX3 OX1 OX1 OX3 OX1 OX3 OX1 OX3 OX1

Wild-type white white lysate AOX3 Df(3R)Exel7326[AOX2-4]/Mi-AOX3 act>AOX1RNAi

Purified AOX1 gene withaspecificRNAiinadult Df(3R)Exel6174, [AOX1]/ry UAS-AOX1-RNAi c TMB Acetaldehyde Coomassie Benzaldehyde (Fig.

act-Gal4 5C). Theintensityofthesignal Df(3R)Exel6174 [AOX1]/TM6B, Tb

white

AOX3 2 , Po

ry lpo ma-l 2 , Po , Aldox-1 RNAi geneknock- 5B). Furthermore, F1 lpo , Aldox-1 ry 5 n1 and parental 0 6 b

b ry white 5 n1 Acetaldehyde TMB

and 06 and the AOX3 c Acetaldehyde TMB flies are are accessible onFlyBase(http://flybase.org) andprovide The dataproducedbythemodENCODE andFlyAtlasprojects translational processing. the productsofalternativesplicingeventsorresult post- gene. Atpresent,itisunclearwhetherthetwoformsofAOX3 are generally correlated generally active AOX mRNAs are iso-enzymesandcorresponding thecatalytically expression of Tissue- andstage-specific total proteins. acetaldehyde assubstrates.Coomassiestainwasusedto extracts (40 (3 μg) wascomparedwithendogenousactivitiesfrom parentals act-Gal4 ( (A) Extractsfrommaleandfemale Fig. Po activity (seealsoFig. visualized withacetaldehyde,whichcorrespondtotheAOX3 trimethoxybenzaldehyde (TMB),whereastwobandswere activity. ForAOX1noactivitywas detectable with2,4,5- 1 AOX1-RNAi were: white acetaldehyde werenotalteredincomparisontothecontrolswhich whereas thetwobandsthatappearinpresenceof n1 lpo The JournalofExperimentalBiology(2014)doi:10.1242/jeb.102129 were usedascontrols.(B)UbiquitoussilencingofAOX1with

.DetectionofthePOactivityAOX1 4. , Aldox-1 act>AOX1RNAi Df(3R)Exel6174[AOX1]/TM6B,Tb , maroon-like μg totalprotein)usingbenzaldehyde,TMBand and w n1 adult flieswereanalyzedforAOX1andAOX3 1118

5). Thegenotypes ; act-Gal4 F1 ) abolishesTMB-dependentactivity, ( ma-l / F1 CyO ), ry Df(3R)Exel6174[AOX1]/ry ( 506 act-Gal4 white and theparentals 1 and ry , ry ). (C)PurifiedAOX1 506 gene . 2 , Po and the white lpo , Aldox- UAS- fly 2 2205 ,

The Journal of Experimental Biology complementary peaksofexpression relativeto mRNA isalsomeasurablethroughoutdevelopment,with embryonic stages,allthelarval stagesandduringadulthood. expressed throughoutdevelopment, withpeaksinthelate 2206 older pupaeandinadultflies. the earlyembryonicstages,in secondandthirdinstarlarvae,in AOX2 al., 2013).Comparingtheexpressionpatternsofvarious in theD.melanogaster expression profilesofalmostalltheopenreadingframesidentified comprehensive informationaboutthetissue-andstage-specific RESEARCH ARTICLE expression in10- to16-h-oldembryosandin thirdinstarlarvae, A C B A A A A A A OX3 OX1 OX3 OX1 OX3 OX1 and

Wild-type

AOX3 white white

act>AOX3RNAi i.6A),itisevidentthat mRNAs (Fig.

Df(3R)Exel7326[AOX2-4]/ry genome (Celnikeretal.,2009;Robinson Df(3R)Exel7326[AOX2-4]/Mi-AOX3 UAS-AOX3-RNAi

act-Gal4 Df[AOX2-4]/TM6B, Tb Df(3R)Exel7326[AOX2-4]/TM6B, Tb AOX2

white

2

shows lowlevelsof , Po ry Mi-AOX3/TM3, Sb, Ser 2 lpo ma-l , Po , Aldox-1 AOX1, i.e.during lpo F1 1 , Aldox-1

ry n1 5 0 ry 6 ry 5 n1 5 06 AOX1 0 6 Acetaldehyde TMB AOX1, 1 AOX3 Acetaldehyde TMB Acetaldehyde TMB is pupae. while large amountsofthe transcript areexpressedin2-day-old seen inearlyembryos, whitepre-pupaeandpupae (2to3daysold). activity aredetectedinlarvaeand adultfliesbutlowactivitiesare AOX bands.Consistentwiththe mRNA data,highlevelsofAOX1 cinnamaldehyde wasusedasacommon substratetovisualizeallthe acetaldehyde wasusedfor thedetectionofAOX3and developmental stages(Fig. analyzed theprofilesofAOX activityinextractsofdifferent samples (FlyAtlasdataset). measurable levelsofthetranscriptarepresentinadult head To confirmsomeofthemRNA dataattheproteinlevel,we AOX4 parentals activity (seealsoFig. top bandwasvisualizedwithTMB,whichcorrespondstotheAOX1 AOX3, noactivitywasdetectablewithacetaldehyde,whereasthe Aldox-1 visualized withTMB.Ascontrols,theextractsof detectable withacetaldehyde,whereastheAOX1bandwas analyzed forAOX1andAOX3activity. ForAOX3,noactivitywas female from maleandfemale Fig. act-Gal4 white presence ofTMBwasnotreduced.Ascontrols,theextracts dependent activity, whereastheAOX1 bandthatappearsinthe with act-Gal4( AOX3/ the parentals 1 n1 The JournalofExperimentalBiology(2014)doi:10.1242/jeb.102129 adult flieswereanalyzedforAOX1andAOX3activity. For

.Identificationofthe 5. , ma-l TM3, Sb,Ser Df(3R)Exel7326[AOX2-4]/Mi-AOX3 n1 is undetectablethroughoutdevelopment,while / CyO Df(3R)Exel7326[AOX2-4]/TM6B, Tb were usedascontrols.(B)Extractsfrommaleand F1 , ry Df(3R)Exel7326[AOX2-4]/TM6B ( act>AOX3RNAi act-Gal4 506 and theparental were used.(C)UbiquitoussilencingofAOX3

4). Thegenotypes

Df(3R)Exel7326[AOX2-4]/ry 6B). AOX1wasassayedwith TMB, ) wereused. AOX3-dependent activities. ) stronglyreducestheacetaldehyde- UAS-AOX3-RNAi white adult flieswere , ry , 1 Tb and ry white 506 1 2 and Mi- , Po and the 2 , ry and w , Po lpo 506 , Aldox- Extracts lpo and 1118 , ;

The Journal of Experimental Biology RESEARCH ARTICLE Fig. the substratesTMB andacetaldehyde. flyheadswereanalyzed with old pupaeandinadult malesofindicatedgenotypes. (D) AOXactivitiesinextractsofdecapitated flies(abdomenandthorax) and white and cinnamaldehyde(allAOXs XOR).Theenzymeactivitypatternsareshownatdifferentembryo totheadultfly for developmentalstagesfromtheearly acetaldehyde(forAOX3) 10–25, moderate;25–50,moderately high;50–100,>100,veryhigh.(B)Nativeactivitygelswerestained withTMB(forAOX1), bars), AOX2

.Developmental expressionpatternof 6. (left gel)and (black bars)and C B A AOX3 AOX1 AOX2 AOX3 AOX1 AOX2 AOX3 AOX1

XDH XDH Embryo 0–2 h Mi-AOX2 Expression level scale 100 120 130 Embryo 2–4 h 110 10 20 30 40 50 80 90 0 Embryo 4–6 h Embryo 0–6 h (right gel)strains.Notetheappearance oftheAOX2activityin

AOX3 white Embryo 6–8 h Embryo 8–10 h 1st instar larva Embryo 10–12 h Pupae 2days

(gray bars)throughouttheflydevelopmental stages(www.flybase.org). Expression levelscale:0–3,verylow;5–10, Mi-AOX2 2nd instar larva Embryo 12–14 h Embryo 14–16 h ry 3rd instar larva 2 Embryo 16–18 h , Po white AOX1, AOX2 lpo White prepupae new Embryo 18–20 h white , Aldox-1 Embryo 20–22 h Pupae 2 days post WPP Embryo 22–24 h Mi-AOX2 n1 Pupae 4 days post WPP 3rd instar larva (L3) 12 h

and AOX3. 1st instar larva 2nd instar larva ry Adult male Adult female 2 , Po Adult male lpo Cinnamaldehyde L3 puffstage 1–2 , Aldox-1 White prepupae (WPP) new

(A) TheplotrepresentsthemodENCODE mRNA expressionlevelsfor L3 puffstage 3–6

L3 puffstage 7–9 n1 Embryo 0–6 h

1st instar larva Pupae 2 days post WPP

2nd instar larva Pupae 3 days post WPP WPP 12 h white Pupae 4 days post WPP

Mi-AOX2 WPP 24 h D AOX3 AOX1 3rd instar larva The JournalofExperimentalBiology(2014)doi:10.1242/jeb.102129 pupae. (C)ComparisonofAOX2activity inextractsof2-day- White prepupae new Adult female 1 day Adult female 5 days Pupae 2 days post WPP Whole Adult female 30 days

Pupae 4 days post WPP

Adult male 1 day Head Adult female Adult male 5 days Adult male 30 days Adult male Abdomen/thorax Cinnamaldehyde Acetaldehyde TMB Acetaldehyde TMB AOX1 (white 2207

The Journal of Experimental Biology their expressionprofilesduringthedevelopmentofinsect.Inthis of theseproteinsindifferent tissues,theirsubstratespecificitiesand number ofquestionsregardingthephysiologicalsignificanceall characterization ofAOXactivitieswasincompleteandraisesa Garattini etal.,2008).Thisfindingindicatesthattheinitial right armofthethirdchromosome(Fig. Predicted pseudogenes arecrossedthrough.AOXorthologs aredrawninthesamecolor. event. Curved blackarrowsindicateaduplication Drosophila (B) Schematicrepresentationofthe inferredevolutionaryhistoryoftheAOXgenes.Eachpanelindicatesa different andcorresponding AOXcomplement ( sequencing of (Warner etal.,1980;Warner andFinnerty, 1981).However, genome from XOR(Dickinson,1970;DickinsonandGaughan,1981), permitted theidentificationofonlytwoAOXenzymesseparable Szauter, 1992).However, theavailablein-gelstainingassays Dickinson, 1970;DickinsonandGaughan,1981;Nelson chromosomal region88F-89A (Collins andGlassman,1969; mRNAs andcorrespondingcatalyticallyactiveAOXiso-. between thetissue-andstage-specificexpressionofAOX together, thesedataindicatethatthereisageneralcorrelation lower intheheadandstrongerabdomen/thorax.Taken samples (Fig. demonstrate noenrichmentinAOX4activityatthelevelofhead whole body, headsandthorax/abdomen ofwhite compared theAOXenzymaticprofileinextractsobtainedfrom into thesynthesisofcatalyticallyactiveenzyme.To thispurpose,we head oftheadultfly, assuggested bytheFlyAtlasdata,translates genes. 2208 Fig. Po activity wereidentifiedusingthetwoloss-of-functionmutantalleles In previousstudies,twogeneticlociaffecting pupae andadultflies,asexpectedfromthe In contrast,AOX3activityishighinearlyembryos,larvae,older RESEARCH ARTICLE Aldox-1 DISCUSSION Aldox-1 additional bandisabsentinthe2-day-oldpupaeof and viable the pupaestagesofcontrol AOX1– We evaluatedwhethertheexpressionof Cinnamaldehyde revealsanadditionalband,whichisobservedin lpo

.Dynamic evolutionaryhistoryofthe 7. A melanogaster and n1 n1 4 ) located~2.5 species are indicated. The various AOX genes are represented in boxes; colors are associated with their corresponding tree branches shownin A. species areindicated. ThevariousAOXgenesarerepresented inboxes;colorsareassociated withtheircorrespondingtree branches mutant strains(Fig. locus impairstheexpressionofboth Aldox-1 Minos-AOX2 willistoni obscura hawaiian 6D). Interestingly, weobserved thatAOX1activityis D. melanogaster repleta virilis n1 , whicharelocatedincloseproximityonthe

D. grimshawi D. virilis D. mojavensis D. willistoni D. persimilis D. pseudoobscura D. ananassae D. erecta D. yakuba D. melanogaster D. sechellia D. simulans Mbp downstreamfromthe insertion mutant(

6C). Theobservationsuggeststhatthe white predicts aclusteroffour flies butnotinthehomozygous Drosophila

Mi-AOX2; Fig. 1) (Adamsetal.,2000; AOX3 B D. melanogaster AOX4 AOX3 flies. Theresults mRNA profile. rosy AOX geneclusters. mRNA inthe D. ananassae locus onthe AOX .vrlsD ilsoimjvni D.grimshawi D.willistoni/mojavensis D. virilis and ry 6B). This 2 , Po AOX2 genes AOX lpo , (A) Thecoloredtreerepresentsthe inferred are questionsthatremainopen. of thisfamilygenesin Kurosaki etal.,2013).Thereconstructionoftheevolutionaryhistory pseudogenization andgenedeletionevents(Garattinietal.,2008; the andtheRNAimethodology. staining method,newtransposon-inducedalleles,deficiency genetic andbiochemicaltools,includinganoptimizedin-gelactivity AOX4 (Fig. establish thatthe disrupted intheclassical gene encodesforPO(Fig. Drosophila study wereconstructthephylogenesisof the distinct fromtheonegivingrisetovertebrate originated fromanancientduplicationof genes, AOX3 complement of active Garattini etal.,2008),theDrosophilidaegenomesincludeuptosix one functional counterparts. Incontrasttothehumangenome,whichcontainsonly AOX4 during different developmentalstagesoftheinsect.Incontrast, AOX2 species analyzedarecharacterizedbythepresenceof lacks 7).With theexceptionof (Fig. melanogaster AOX4 any oftheorgan anddevelopmentalstagesconsidered.Whether AOX4 in (Hawaiian was duplicatedinto Previous phylogeneticstudieshaveindicatedthattheevolutionof D. melanogaster,whosegenomeispredictedtocontainanactive Drosophila AOX AOX 6), whilenoenzymaticactivitycouldbeassociatedwiththe AOX3, buthasacquired has aspecializedfunctionin is notpresentinall loci, isobservedin gene. To reachtheseconclusions,weusedacombinationof gene, nocatalyticallyactiveAOX4enzymewasdetectedin AOX and AOX1, D. erecta/sechellia/yakuba/ genes istheresultofaseriesgeneduplication, melanogaster/simulans Dr The JournalofExperimentalBiology(2014)doi:10.1242/jeb.102129 AOX3 (Figs genes. Theminimalandprobablythemostancient osophila AOX AOX2 species group,and AOX 2, 7).Inaddition,wedemonstratethattheAOX1 AOX2 genes. OutofthefourD.melanogasterAOX gene (Berger etal.,1995;Garattini2003; AOX3-D genes arenotdirectlyrelatedtothevertebrate Precursor and genes, consistingofthe species group)andAOX3-D3 gene isexpressedduringmetamorphosis D. virilis, Drosophila AOX3 Aldox-1 Drosophila 4), andthattheAOX3activityis ( D. virilis AOX5, D. obscura are expressedinanactiveform D. melanogaster n1 D. mojavensis Drosophila species (Fig. mutant (Fig. D. ananassae), D. pseudoobscura/persimilis AOX6 indicates that species group),AOX4 XOR. Thisduplicationis species group,which AOXs, suggestingthat phylogeny. and AOX1, and 7). Furthermore,

AOX 5). Finally, we and whyAOX3 ( AOX7, allthe D. ananassae D. willistoni AOXs have AOX2 AOX3-D2 genes in AOX1, and ( D. )

The Journal of Experimental Biology unknown. Furtherobservationsofthe absent inthe white flies suggestthatthismutantalleleaffects also RESEARCH ARTICLE as substrate.Incontrast, than 2%ofthewild-typeAOXactivitywhenacetaldehydeisused Moreover, consistentwithaformerstudyonthe AOX1 AOX enzymes in physiological tasksinvivo recognize auniquesetofsubstrates andcarryoutdifferent (Fig. is detectablewithcinnamaldehyde butnotwithbenzaldehyde Gaughan, 1981;Cypheretal., 1982). Interestingly, AOX2activity heterocycles (Fig. substrate specificitiesforanumberofaldehydes,andaza- oxo- intheorganism. pyridoxal kinasefortheregulationofhomeostaticlevels PLP by theactionofAOX1,representingacompetitivereactionto the (Toney, 2005).ExcessofvitaminB6inthefoodmight becountered cofactor formanyenzymesinvolvedinaminoacidmetabolism pyridoxal kinaseintopyridoxal-5-phosphate(PLP),whichacts asa with pyridoxamineandpyridoxine.Pyridoxalisconvertedby the Pyridoxal isoneofthethreenaturalformsvitamineB6, along Malphigian tubules(DickinsonandGaughan,1981)(FlyAtlas). amounts oftheenzymeAOX1aremeasurableinmidgut and excreted bythegutsystem(Forrestetal.,1961),asabundant B6 frompyridoxalto4-pyridoxicacid,whichthenmightbe hypothesize thatAOX1playsaroleinthedecompositionofvitamin TMB (BrowderandWilliamson, 1976;Cypheretal.,1982).We enzyme capableofoxidizingpyridoxalorthebenzaldehydederivate is specificallyoxidizedbyAOX3.Incontrast,AOX1theonly purified recombinantAOX1(Fig. oxidation isdetectableonlyinthepresenceofhighamounts We confirmtheseobservations,demonstratingthatacetaldehyde wild-type flies(DickinsonandGaughan,1981;Cypheretal.,1982). with levelsofAOXactivitythatareclosetothosedeterminedin compounds indicatethat (Dickinson andSullivan,1975).Theresultsobtainedwiththese pyridoxal hasbeenreportedtobeoxidizedbyflyextractsaswell molecules knowntobeoxidizedbymammalianAOXs,although the minorbandtothoseobservedwouldbeexpected. this case,however, greaterinter-sample variationsintheintensityof possible thatthefasterbandisadegradationproductofAOX3.In specificities (Fig. enzymatically activebandscharacterizedbyidenticalsubstrate from theAOX3 suggest thattwoAOX3isoformswithdistinctsizesaregenerated acids fromintheNterminus(Celnikeretal.,2009).Therefore,we them (NCBIaccessionAY094721) hasadeletionof226amino support thepresenceoftwoalternative band forms.ThecDNA sequencesavailableinmodENCODE activities existedasmodifiedminorbandandunmodifiedmajor (Dickinson andGaughan,1981)suggestedthattwo supplementary materialFig. migrating bandforAOX3(indicatedas (Dickinson andGaughan,1981),wealsodetectedamajorminor the homozygote have different substratespecificities.Itwaspreviouslyshownthat In thecaseofAOX1andAOX3,weconfirmoverlapping The 20substratesusedinthisstudywereselectedamongthe While wewereabletoshowthatthe 6). Thesedatasupporttheidea thatdifferent AOXisoforms pupae areendowedwithanadditionalAOXband,whichis gene, themolecularnatureof ry 2 , Po gene, whichisconsistentwiththeexistenceoftwo Aldox-1

3, supplementarymaterialFig.

Aldox-1 3, supplementarymaterialFig. lpo , Aldox-1 D. melanogaster Po n1 . Inpreviousstudiesitwasshown thatthe n1 extracts (containingAOX1)haveless lpo and n1 extracts (containingAOX3)present

S2). DickinsonandGaughan and Po

4C). Inflyextractsacetaldehyde lpo Mi-AOX2 mutants havetissue-specific ry AOX3 2 AOX1, AOX2andAOX3 , Po Aldox-1 Po lpo lpo mutant flies(Fig. AOX2, as2-day-old

transcripts. Oneof S2) (Dickinsonand , Aldox-1 allele mapstothe b n1 Aldox and Aldox-1

S2). Itisalso allele remains c -dependent n1 in Fig. n1 mutant locus

6). 3, hypothesize thatthe band correspondingtoAOX4cannotbeidentified,leadingus FlyAtlas). InD.melanogaster cinnamaldehyde. trimethoxybenzaldehyde, valeraldehyde,vanillin,xanthineand trans- pyridoxal-HCl, phenylacetaldehyde,salicylaldehyde, 2,4,5- naphtalenedicarbaldehyde, 4-nitrobenzaldehyde,phenanthridine,phthalazin, insect’s development.We showedthatthe complementary expressionpatternsduringthedifferent stagesofthe AOX2 andAOX3in rodents, dogsandinsects. primates, taxawhoseolfactorysystemislessdevelopedrelativeto ortholog isafunctionallyinactivepseudogeneinhumansand 2006; Kurosakietal.,2013).ItisremarkablethattheAOX3l1 that areoftenaromaticaldehydes(Kurosakietal.,2004;Terao etal., and mayfunctioninthedegradationofodorantspheromones rodents anddogs,asAOX3l1isrestrictedtotheolfactorymucosa the mammalianAOX3l1enzymemayserveasimilarfunctionin pheromone degradation.Inthisrespect,itisinterestingtonotethat proposed thatantennae-specificAOXsmayplayarolein present inmaleinsects.Basedontheseobservations,ithasbeen antennae ofbothsexes,althoughlarger amountsoftheenzymeare al., 2007).Inthesestudies,AOXactivityhasbeendetectedinthe al., 1989;Rybczynski etal.,1990;Merlin2005;Pelletier in theantennaeofseveralmothspeciesareavailable(Rybczynski et methodology. Reportsdemonstrating thepresenceofAOXenzymes antennae, precludingdeterminationoftheenzymewithour synthesis isrestrictedtosub-structuresofthehead,suchas propose thatAOX4iseithernotexpressedinanactiveformorits to findahead-specificAOX4havebeeninconclusive.Thus,we available inFlyAtlas(Robinsonetal.,2013).However, ourattempts the headofadultflies,assuggestedbymRNA expressiondata expressed inthehead(Fig. Malpighian tubules.IncontrasttoAOX3,AOX1isweakly two enzymesaresynthesizedpredominantlyinthemidgutand tissue distributionsofAOX1andAOX3areoverlappingthe (Dickinson andGaughan,1981;FlyAtlas).Itwasshownthatthe the modENCODEandFlyAtlasdatapresentedinFlyBase and overlappingexpressionpatterns,whichisalsoconsistentwith Specifically, fromtheVienna All fliesusedinthisstudywereretrievedfrompublicstockcenters. 2-hydroxypyrimidine, hypoxanthine, dihydroxybenzaldehyde, 3-ethoxy-4-hydroxybenzaldehyde,heptaldehyde, Reachim: benzaldehyde,acetaldehyde,allopurinol,butyraldehyde, 2,4- AOX substrateswereobtainedfromFluka,Sigma-Aldrich,Merck and al., 2007),weusedsecondchromosomeinsertionfor unresolved. AOXs indifferent species,tissuesanddevelopmentalstagesremain However, theactualphysiologicalfunctionsof continuous evolutionarychangeinotherspeciesof Drosophila Chemicals MATERIALS ANDMETHODS disrupting Bloomington was usedasdescribedpreviously (Missirlisetal.,2003)(Table induce theRNAi,aubiquitous an insertionontheXchromosome for were herereferredtoas the followingcharacteristics: (1) addition, threedeficiencychromosomes wereobtainedfromBDSCthathad In conclusion,wewereabletodistinguishtheactivitiesofAOX1, AOX2 The JournalofExperimentalBiology(2014)doi:10.1242/jeb.102129 cultures Drosophila and AOX3, respectively(Metaxakisetal.,2005): thestocks AOX4 Stock Center(BDSC),weused Mi-AOX2 D. melanogaster Drosophila w gene exertsatissue-specificfunctionin

*, 6D) (DickinsonandGaughan,1981; whole-body extracts,azymographic Actin5C-Gal4/CyO and Df(3R)Exel6174[AOX1] Mi-AOX3 RNAi Center(VDRC)(Dietzlet UAS-AOX3-RNAi N ′ -methylnicotinamide, 2,3- AOX and toconfirmtheir gene clusterisunder (Table UAS-AOX1-RNAi ( act-Gal4) driverline Minos

1, Fig. Drosophila. Tbe1).To (Table Drosophila insertions

uncovers 1). From 1A). In 2209 and

The Journal of Experimental Biology genes (Table out at4°C. remove additionalcelldebris.Thehomogenizationprocedurewascarried and asecondcentrifugationstepwasperformedat17,000 affecting Po ma-l ry Df(3R)BSC515[AOX1-4] Df(3R)Exel7326[AOX2-4] Df(3R)Exel6174[AOX1] Mi-AOX3 Mi-AOX2 10 by aSuperose12sizeexclusionchromatographycolumn(GEHealthcare). nitrilotriacetic acid(Ni-NTA) resin(QIAGEN,Valencia, CA,USA)and recombinant proteinwaspurifiedbyaffinity chromatographyusingnickel- visible asbandin thenativegel. reaction, thereducing agentprecipitatesintoapurple-black color, makingit (MTT) and0.1 additionally containing100 2210 stained forAOXactivityusing5 extracts wereseparatedby6%native PAGE andthegelsweresubsequently colorimetric in-gelactivityassay. Here,40 To detecttheactivityofAOX enzymesinflyextracts,weperformeda collected ina1.5 instar larvae,20 For eachsample,10flies,abdomens,50heads,200embryos,100first AOX1 The cDNA cloneLD37006containingthecDNA of specifically b a UAS-AOX1-RNAi Stock name N-terminal His resulting plasmidwasdesignatedpZM171andexpressesAOX1withan NdeI-SalI sitesoftheexpressionvectorpTrcHis (Temple etal.,2000). The TGAGCTTGAACTG GCTG wereused,whichallowedcloningintothe AATTACAATC AACG andthereverseprimerGTCGACCTAATResource Center, andtheforwardprimerCATATGGCTGGAAG - - Project (BDGP)GoldcDNA collectionofthe act-Gal4 coli adult fly, larvaeorpupae(except:2 and Pelletpestlesbluepolypropylenesticks,wasused.A volumeof8 grinder (Sigma),composedofabattery-drivenPelletpestlescordlessmotor at Table 0.4 homozygous viablechromosome 1969) andAldox-1 RESEARCH ARTICLE performed for30 inhibitor cocktail(Roche)wasaddedtothefliesandhomogenization was (Gutiérrez etal.,2013)wereusedascontrols. BDRC (Table Detection of in-gelAOX extracts fly Detection of activityfrom extracts fly of Production Cloning andpurification of UAS-AOX3-RNAi per head;5 Identity (ID)numbersoftheusedstockfromcorrespondingpubliccenters. Stocks wereretrievedfrompublicstockcenters:Vienna 506 − lpo min, thesupernatantwastransferredintoanew1.5 mmol 80°C untilfurtherusage.Forthepreparationofflyextracts,amicro F1 TP1000 cellsandHis Aldox-1 1. Drosophilamelanogaster ( CG18522) wasobtainedfromtheBerkeley AOX1, and(3)(Df(3R)BSC515[AOX1-4] l − μl perabdomen)of0.1 1 AOX1, (2)Df(3R)Exel7326[AOX2-4]

n1 1, Fig. 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazoliumbromide 1). Wild-type TAN3 (SadraieandMissirlis,2011) andwhite 6 -tag fusion.TheplasmidpZM171wastransformedinto

mmol

s instarlarvae,10thirdlarvaeorpupaewere ml Eppendorftube,shock-frozeninliquidN

s. Thecrudeextractwascentrifugedat14,000 n1 1A). Theclassicmutations (Dickinson, 1970)wereusedfromthecompound,

l − 1 μmol phenazine methosulfate(PMS). Upon AOX 6 -AOX1 washeterologouslyexpressed.The Drosophila l

mmol ry − 1 μl perembryo;4 2 EDTA, 1

, Po mol lines usedinthisstudy

w w w y,w*; Mi{y w P{KK105948}VIE-260B Genotype w* ry ma-l ry P{GD16750} l lpo − l 1118 1118 1118 1118 2 506 1 − , Actin5C-Gal4/CyO , Aldox-1 μg totalproteinofthewholefly 1 Po of aAOXsubstrate(seeabove), F1 ;Df(3R)BSC515/TM6C,Sb ;Df(3R)Exel7326/TM6B,Tb ;Df(3R)Exel6174,P{w ; Mi{ET1}CG18519 Tris-HCl (pH AOX1 mmol lpo Po Aldox-1 +mDint2 uncovers lpo

l n1 Drosophila v48191 − (Collins andGlassman, uncovers allfour μl perfirstlarvae;3 1 , Sb Drosophila DTT and2×protease }CG6045 Drosophila n1

ml Eppendorftube sbd-2 D. melanogaster Sb 8.0) lysisbuffer AOX2-4

g sbd-2 obtained from for 10 ) MB05465 2 MI02285 Genomics +mC and stored Genome RNAi Center(VDRC)orBloomington without }Exel6174/TM6B,Tb min to μl per /TM3,Sb 1

g 1 ,cu AOX for μl 1 E. 1 ,Ser given (i.e.AOX4duplicationinboth duplication eventwasdetectedinmorethanonelineage,anewname ananassae added tothe‘AOX’ speciesthatwaslikelytohaveoriginatedit(i.e. duplications weredetectedinnomorethanonespecies,the‘-D’ suffix was unknown AOXisoenzymes,thefollowingcriteriawereadopted:if maintaining theopenreadingframe.To assignnamestonewpreviously reading frame;and(3)thelackofarecognizableintron/exonjunction identifiable exonsresultingindisruptionoftheexpecteddownstreamopen STOP codonlocatedincodingsequence;(2)theabsenceofoneormore considered aspseudogenesbythefollowingcriteria:(1)presenceofone may betranscribed,butdonotencodeacompleteprotein. coding foranactiveAOXprotein(activegenes)frompseudogenes,which phase ofexon/intronjunctions.A majorproblemwassortingofgenes account conservationofthecodingexonsintermssequence,length,and necessary. Reconstructionofthe AOX3 andAOX4proteinsoranevolutionarilycloserAOX,when polypeptides withsimilaritytothe Using thetBlastn grant OD010949-10. Drosophila Volkes (toZ.M.).A clone/plasmidforAOX1cloningwasobtainedfromthe (EMBO) [grantnumberASTF226-2011] and theStudienstiftungdesdeutschen [grant number179835toF.M.]; bytheEuropean MolecularBiologyOrganisation number LE1171/8-1 toS.L.];theConsejo NacionaldeCienciayTecnología project This workwassupportedbytheDeutsche Forschungsgemeinschaft(DFG)[grant the experimentsandanalyzeddata.Z.M.,E.G.,F.M. andS.L.wrotethepaper. Z.M., F.M. andS.L.designedtheresearch.Z.M.,M.D.,M.L.G.M.B.conducted The authorsdeclarenocompetingfinancialinterests. Phylogenetic calculations Funding Author contributions Competing interests material Table well aseukaryoticAOXandXORproteinsequences(seesupplementary generated throughBioNJ,wasbasedonpubliclyavailableprokaryotic as comprehensive phylogenetictreespanningfrombacteriatovertebrates, bionj/) (Gascuel,1997)and100bootstrapreplicateswerecalculated. A through thedistance-basedmethodBioNJ(http://www.atgc-montpellier.fr/ the available Workbench (http://www.clcbio.com/). Thephylogenetictreecontainingall (http://www.ebi.ac.uk/Tools/msa/muscle/) andvisualizedwithCLCMain annotated. MultiplesequencealignmentswerepreparedusingMuscle loci andchromosomalposition(ifavailable)ofthepredictedgeneswere sequences, seesupplementarymaterialTable the correspondingproteinproductswasperformed(12taxaand59 was namedAOX5). To determinetheevolutionofAOXgenes,aphylogeneticanalysis 1 1 Genomic Resource Center, supportedbyNational InstitutesofHealth AOX3 andD.ananassae The JournalofExperimentalBiology(2014)doi:10.1242/jeb.102129 Drosophila

S2) (Kurosakietal.,2013). algorithm, wesearchedforexonicsequencescoding Drosophila Source BDSC BDSC BDSC BDSC BDSC BDSC BDSC BDSC VDRC BDSC VDRC AOX andXORproteinstructureswasgenerated a AOX Stock Center(BDSC). D. melanogaster D. persimilis genes wasperformed,takinginto AOX3-D); ifthesameAOX

S1). Exonicstructure,genomic 5125 180 225 25019 7980 7653 33191 25450 48191 4414 Stock IDnumber 100330 and XOR, AOX1,AOX2, D. pseudoobscura AOX b genes were D.

The Journal of Experimental Biology Dickinson, ikno,W. J. Dickinson, M., Terao, M.J., Ramos, A.T., Carvalho, J., Trincão, M., Mahro, C., Coelho, ikno,W .adGuhn S. Gaughan, and W. J. Dickinson, aatn,E n ea,M. Terao, and E. Garattini, M. Terao, and E. Garattini, yhr .J,Tdso .L,Cutih,J .adKmrn A.K. Kumaran, and J.B. Courtright, J.L., Tedesco, J.J., Cypher, E. Glassman, and J.F. Collins, B. Osmond, and M. McCarron, W., Gelbart, A., Chovnick, hvik . lr,S . utn .L,J n eue A.G. Reaume, and Jr F. L., Dutton, S.H., Clark, A., Chovnick, Karpen, S., Henikoff, K.C., Gunsalus, M.B., Gerstein, L.A., Dillon, S.E., Celniker, K.S. Brehme, and C.B. Bridges, Brown, J.E., Repine, R.M., Wright, G., Harta, K.P., Clancy, E., Mezey, R., Berger, RESEARCH ARTICLE http://jeb.biologists.org/lookup/suppl/doi:10.1242/jeb.102129/-/DC1 Supplementary materialavailableonlineat aatn,E,Mne,R,Rmo .J,Wih,R n ea,M. Terao, and R. 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