Antennal uridine diphosphate(UDP)-glycosyltransferases in a pest insect:diversity and putative function in odorant andxenobiotics clearance

F. Bozzolan, D. Siaussat, A. Maria, N. Durand,M.-A. Pottier, T. Chertemps and M. Maïbèche-Coisne

Département d’Ecologie Sensorielle, Institut d’Ecologieet des Sciences de l’Environnement de Paris, UniversitéPierre et Marie Curie, Paris, France

Abstract

Uridine diphosphate UDP-glycosyltransferases(UGTs) are detoxification enzymes widely distributedwithin living organisms. They are involved in thebiotransformation of various lipophilic endogenouscompounds and xenobiotics, including odorants.Several UGTs have been reported in the olfactoryorgans of mammals and involved in olfactory pro-cessing and detoxification within the olfactorymucosa but, in insects, this enzyme family is stillpoorly studied. Despite recent transcriptomic analy-ses, the diversity of antennal UGTs in insects has notbeen investigated. To date, only three UGT cDNAshave been shown to be expressed in insect olfactoryorgans. In the present study, we report the identifica-tion of eleven putative UGTs expressed in the anten-nae of the model pest insect Spodoptera littoralis.Phylogenetic analysis revealed that these UGTsbelong to five different families, highlighting theirstructural diversity. In addition, two genes, UGT40R3and UGT46A6, were either specifically expressed oroverexpressed in the antennae, suggesting specificroles in this sensory organ. Exposure of male mothsto the sex pheromone and to a plant odorant differen-tially downregulated the transcription levels of these

two genes, revealing for the first time the regulation ofinsect UGTs by odorant exposure. Moreover, the spe-cific antennal gene UGT46A6 was upregulated byinsecticide topical application on antennae, suggest-ing its role in the protection of the olfactory organtowards xenobiotics. This work highlights the struc-tural and functional diversity of UGTs within thishighly specialized tissue.

Keywords: UDP-glycosyltransferases, olfaction, de-toxification, insect.

Abbreviations: CCE, carboxylesterases; GST, gluta-thione S-transferases; P450, cytochromes P450; UGT,UDP-glycosyltransferase.

Introduction

Uridine diphosphate (UDP)-glycosyltransferases (UGTs)catalyse the addition of glycosyl groups from activatednucleotide sugar to a variety of small hydrophobic mol-ecules (aglycones), resulting in more hydrophilic com-pounds, which can thus be efficiently excreted. Glycosideconjugation is therefore an important metabolic pathwayfor the biotransformation of a wide range of lipophilicendogenous and exogenous compounds. Members ofthis superfamily are found in all living organisms,animals, plants, bacteria and virus, suggesting an ancientorigin (Burchell & Coughtrie, 1989; Mackenzie et al.,1997).

Mammalian UDP-glucuronosyltransferases are themost studied. They use UDP-glucuronic acid as donor forcatalysing the glucuronidation of various endogenoussubstrates, such as steroids or vitamins, or of variousexogenous compounds, such as drugs, pollutants,odorants and dietary compounds. The major site ofglucuronidation is the liver, but other organs, including thebrain and olfactory tissues also exhibit UGT activity(Heydel et al., 2010). Compared with other detoxification

Correspondence: Dr Martine Maïbèche-Coisne. Institut d’Ecologie etdes Sciences de l’Environnement de Paris, Département d’EcologieSensorielle, 7 Quai St-Bernard, BC 1211, F-75005 Paris, France. Tel.:+ 33 144276592; fax: + 33 144276509; e-mail: martine.maibeche@snv.jussieu.fr

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InsectMolecular

Biology

Insect Molecular Biology (2014) doi: 10.1111/imb.12100

© 2014 The Royal Entomological Society 1

enzymes, such as cytochrome P450s (P450s), gluta-thione S-transferases (GSTs) or carboxylesterases(CCEs), UGTs have been less studied in insects (Despréset al., 2007). Insect UGTs typically use UDP-glucose asa donor rather than UDP-glucuronic acid (Ahmad &Hopkins, 1993b; Luque & O’Reilly, 2002; Ahn et al., 2012).UGT activities have been investigated in the fruit flyDrosophila melanogaster (Luque & O’Reilly, 2002), thegrasshopper Melanoplus sanguinipes, the cockroachPeriplaneta americana, two Tenebrio species and inseveral moths species (Ahmad & Hopkins, 1993a; Luqueet al., 2002; Ahn et al., 2011). These activities were mainlyfound in larvae and directed towards various ingestedplant allelochemicals (Ahn et al., 2011, 2012). A recentphylogenetic analysis including 310 putative UGT genesidentified from genomic/transcriptomic databases hasshed a new light on the diversification of this multigenefamily in insects (Ahn et al., 2012); however, only twoUGTs were characterized both at the molecular and func-tional level in insects: a phenol-UGT from Bombyx mori,which exhibited a wide substrate specificity towardsallelochemicals (Huang et al., 2008), and an UGTfrom Zygaena filipendulae, which has been involved incyanogenic defence compound biosynthesis (Jensenet al., 2011). Insect UGTs were also suspected to playa role in resistance mechanisms towards insecticides,including dichlorodiphenyltrichloroethane (DDT; Pedraet al., 2004), carbamates (Silva et al., 2012), neoni-cotinoids (Yang et al., 2013) and pyrethroids (Vontaset al., 2005). All these data suggest that insect UGTs mayplay important roles in the detoxification and biotrans-formation of exogenous and endogenous compounds, asalready shown in mammals.

The role of UGTs in vertebrate olfaction was demon-strated a long time ago (Lazard et al., 1991 and re-viewed in Heydel et al., 2010). UGT2A1, which is highlyexpressed in rat olfactory epithelium (Heydel et al., 2001;Zhang et al., 2005), conjugates odorants (Lazard et al.,1991). As the glucuronidated products did not elicit anolfactory response, it was proposed that this reactionparticipates in the termination of the olfactory signal.In insects, a variety of enzymes has been proposedto play a role in odorant/pheromone termination withinthe antennae (Jacquin-Joly & Maïbèche-Coisne, 2009).In particular, esterases, P450s and GSTs have beeninvolved in odorant degradation (Vogt et al., 1985;Rogers et al., 1999; Wojtasek & Leal, 1999; Ishida &Leal, 2005; Durand et al., 2011) and detection(Maïbèche-Coisne et al., 2004). Concerning UGTs,studies are limited to reports of their expression in anten-nae of three species, including D. melanogaster (Wanget al., 1999), B. mori (Huang et al., 2008) and Man-duca sexta (Robertson et al., 1999), suggesting a role inolfactory processing.

Using the moth Spodoptera littoralis, a worldwide pestof cotton and vegetable crops as a model, we identified 11putative antennal UGTs by analysis of an expressedsequence tag (EST) collection from male antennae(Jacquin-Joly et al., 2007; Legeai et al., 2011). Phylo-genetic analysis revealed an unsuspected structural, andpotentially functional, diversity of this gene family withinthis tissue. Expression patterns were studied using quali-tative and quantitative PCR. Two transcripts restricted tothe olfactory or to the chemosensory tissues of adultmales were studied in detail. Both were overexpressed inadult male antennae. Moreover, their transcription wasdifferentially regulated after exposure of the males toeither the sex pheromone, a plant odorant or a pyrethroidinsecticide. Interestingly, the transcription of the antennal-specific UGT was clearly upregulated by deltamethrinexposure, strongly suggesting a role in insecticide detoxi-fication within the olfactory organ. Together, these datareveal the structural and functional diversity of UGTs thatmay be expressed in the olfactory organ of an insectspecies.

Results and discussion

Identification of 11 putative UGT genes andnomenclature

A total of 108 ESTs were first identified, leading afterassembly to 11 partial contigs encoding partial UGTsequences. Complete coding regions were thenisolated using 3′/5′rapid amplification of cDNA ends(RACE)-PCR, leading to 11 ORFs encoding putativeUGTs (Table 1). Sequences were named according tothe recommendation of the UGT nomenclature com-mittee (http://www.flinders.edu.au/medicine/sites/clinical-pharmacology/ugt-homepage.cfm).

The structural features of the putative UGTs fromS. littoralis are highlighted in Fig. 1. The N-terminal sub-strate binding domain is highly variable whereas theC-terminal sugar-donor binding domain is more con-served, as described for other insect UGTs (Ahn et al.,2012). The two predicted sugar donor binding sites(Fig. 1) are also conserved as compared with other UGTsfrom mammals or insects (Ahn et al., 2012). All thesequences displayed the typical UGT motif signature(Mackenzie et al., 1997). The two catalytic residuesdeduced from human UGT2B7 modelling (H35 and D151)were located in the N-terminal substrate binding site,as expected (Miley et al., 2007). Several putative N-glycosylation sites were found within each sequence(Table 1), as determined by searching the Prositedatabase (http://prosite.expasy.org/), suggesting thatS. littoralis UGTs might be glycosylated, as reported forUGT37a1 from D. melanogaster (Luque & O’Reilly, 2002).Signal peptides were predicted for all the sequences

2 F. Bozzolan et al.

© 2014 The Royal Entomological Society

(SIGNALP 3.0; (Bendtsen et al., 2004)) and could mediatethe integration of the protein precursors into the endo-plasmic reticulum (ER) compartment, whereas the hydro-phobic transmembrane domain found at the C-terminalend could retain the mature proteins in the ER membrane.All these data suggested that S. littoralis UGTs were prob-ably active proteins, anchored in the ER membrane.

Phylogenetic analysis

The 11 S. littoralis UGTs were included in a phylogeneticanalysis (Fig. 2), together with the UGTs recently identi-fied in Helicoverpa armigera and B. mori through genomeanalysis (Ahn et al., 2012). Assignment to family and sub-family on the basis of amino acid identity was supportedby the phylogenetic analysis. Among the 14 UGT familiesidentified to date in Lepidoptera (Ahn et al., 2012),S. littoralis antennal UGTs distribute into five families.Most of them (eight sequences) clustered within UGT33and UGT40 families, the two largest lepidopteran UGTfamilies. Orthologous pairs with H. armigera UGTs werefound except for one gene, UGT33B13. Orthologoussequences for this UGT could not be identified, even afteradding several UGT sequences from other Lepidopteraspecies in the analysis (not shown). Two sequences,UGT40R2 and UGT40R3 were very similar: theirC-terminal parts were nearly identical, whereas theirN-terminal substrate binding domains were more variable.This suggested that they might be produced by alternativesplicing of the same gene, according to different substratespecificities, as shown for several B. mori and H. armigeraUGTs (Ahn et al., 2012).

The number of UGT genes identified from insectgenomes ranges between 12 in the bee Apis mellifera and58 in the aphid Acyrthosiphon pisum. In Lepidoptera, this

number ranges between 42 and 45 in H. armigera andB. mori, respectively. We could thus presume that theantennae of S. littoralis expressed around one quarter ofthe UGT repertoire of this lepidopteran species. Thisstructural diversity supports a functional diversification ofantennal UGTs in insects and a high potential of UGT-dependent metabolism within this highly specializedtissue.

Tissue-related and temporal expressions of S. littoralisantennal UGTs

Tissue-related expressions of the 11 putative UGTs weredetermined by reverse-transcriptase (RT)-PCR in order toidentify the antennal-specific or antennal-enriched genesthat may be more likely to be involved in olfactory func-tions in male adult antennae. Indeed, such restrictedexpression is a useful criterion that has already beenhelpful in identifying specific olfactory genes such asolfactory receptors and odorant-binding proteins invarious species (Benton et al., 2007). Expression was firstcompared among the male antenna, thorax and abdomen(Table 1). Most of the UGTs showed a wide range ofexpression through the body, except for two sequences,UGT40R3 and SlUGT46A6, which were only amplified inantennae.

To define the expression patterns of UGT40R3 andSlUGT46A6 in adult males, various non-olfactory tissueswere then tested (Fig. 3A). UGT46A6 expression wasrestricted to the antennae, whereas UGT40R3 was alsoexpressed in the proboscis and slightly in the legs. Asthese two appendages bear gustatory hairs, this sug-gested that UGT40R3 expression was restricted tochemosensory organs. Only two other insect UGTshave been reported to be expressed in the antennae,UGT33D8 from B. mori (Huang et al., 2008) and

Table 1. Summary of Spodoptera littoralis antennal uridine diphosphate-glycosyltransferase sequences and tissue distribution, as analysed byreverse-transcriptase PCR on male tissues

NameGenBankAccession

Length(aa)

Closest relatedsequence and identity

Tissue

Sequenceinformation ORFcompleted by

N-glycosylationpredicted sitesAnt Tho Abd

Numberof ESTs

UGT33B13 KF777109 515 HaUGT33B5 (70%) x x x 39 – 68, 187, 466UGT33F4 KF777110 517 HaUGT33F3 (70%) x x x 2 5′RACE 69, 191, 469UGT33J2 KF777111 514 HaUGT33J1 (75%) x x x 4 – 64, 184, 416, 463UGT33T2 KF777112 516 HaUGT33T1 (60%) x x x 3 – 69, 191, 418, 470UGT40U1 KF777113 520 HaUGT40D1 (57%) x x – 5 – 111, 124, 245, 338UGT40L2 KF777114 522 HaUGT40L1 (71%) x x x 11 PCR 66, 71, 248UGT40R3 KF777115 520 HaUGT40R1 (63%) x – – 8 – 123, 244UGT40R2 KF777116 524 HaUGT40R1 (63%) x – x 5 PCR 123, 244UGT41D2 KF777117 520 HaUGT41D1 (67%) x x x 14 3′RACE 102, 236UGT42B4 KF777118 516 HaUGT42B2 (82%) x x x 14 PCR 53, 456UGT46A6 KF777119 526 HaUGT46A3 (84%) x – – 3 – 64, 246, 292

UGT, uridine diphosphate-glycosyltransferase; RACE, rapid amplification of cDNA ends; ORF, open reading frame; Ant, antenna; Tho, thorax; Abd,abdomen; Ha, Helicoverpa armigera; EST, expressed sequence tag.

Antennal UDP-glycosyltransferases in a pest insect 3

© 2014 The Royal Entomological Society

10 20 30 40 50 60 70 80 90 100 | | | |* | | | | | | UGT33B13 ---MSTGVTLFLLSVSIAYNDAAKILALFPVPSISHQVVFRPYTQELARRGHQVTVITPDPAFPKGDVPANLTEIDVHDFSYEMW----------KSFYE UGT33F4 --MRNFIYIILCLSTFASHDEAAKILAVFPIPSISHQVVFRPLTHELAKRGHEVTVITTDPAFPKGTAPANLTEIDVHDISYDLWN---------KIFMA UGT33J2 ------MQILLCIALQVIFADAARILAVFPVPSFSHQITFRPITMELIKRGHEVVVMTPDPMFKKGETPANLTEIDVHDISYQAW----------QVFME UGT33T2 --MSVLWCIVSAYILVVSPIQAARILFYIPTPSISHQVVFRPLAQTLASRGHDVTIITADPAFPKGKSPANLTEIDVHDMSYEMWQ---------EEIMK UGT40U1 --MERIQTFWLALSVLLVCAEASKVLVVFPLPSRSHANLGDGIVRHLLNAGHEVTYITPFV---YKNAPPNLRTIDVSSNFDVWP--------AHLITIK UGT40L2 --MKYKIVTSIFLLSLLVSSEALRILVCYPMTSKSHSILGHGIVNRLLEAGHEVVHITSFP---NGKVLPNLTEVNVSSIAEVFTKDVDG---VEQFKLK UGT40R3 ---MALAICLFF-LLLSSSCEAYKALVVFGMPATSHSNLGRGVVRNLLKDGHEVTFITPIP---IKDPPPNLHQIDVSSNFELLP--------LDLMKIE UGT40R2 ---MALAILLFLGLLLSSSCEAYKALVVFGMPSTSHFHLGNGVVRNLLRDGHEVTYITPIE---YKNPPPNLRQIDVSSNFDVLP--------TYQINLK UGT41D2 -----MKLSILLLLGVVAHCHGYHIITMFPMPSHSHNQLSKGIVDALLQGGHTVTWVTPFPGK-ADKNNPKLKLIDISSVLSHVS----------NIDMT UGT42B4 MKPSVLIATILSLLVLTDYAYSLNILGIFPYQGKSHFIVFRVYLRELAKRGHNVTVISHFP---EQDPPANYHDISLAG-STEAIEGVGP----FQTSYL UGT46A6 ----MRTLAILLVAIFAVNVQSARILGLFPHTGKSHQMVFDPLLRTLAERGHDVTVVSFFP---IKNPPANYTIVSLEGLAAQGVETIDLSYFDSQNKLL UGT2B7 MSVKWTSVILLIQLSFCFSSGNCG-KVLVWAAEYSHWMNIKTILDELIQRGHEVTVLASSASILFDPNNSSALKIEIYPTSLTKTELENFIMQQIKRWSD UGT2A1 ----MLKNILLWSLQLSLLGMSLGGNVLIWPMEGSHWLNVKIIIDELLRKEHNVTVLVASGALFITPSVSPSLTFEIYPVPFGKEKIESVIKDFVLTWLE 110 120 130 140 150 160 170 180 190 200 | | | | *| | | | | | UGT33B13 VTSTGDN--DLVLQMTLAFTIIVDIVEMQLK--LDAVQKILK--EEKFDLLLLEACVKPALVLSHVY---KVPVIQVSSFGPMNFNIETIG-SAWHPLLY UGT33F4 STKGNKD--DVVMVMSAIMDALVAIVDAQLK--DDKVQNLIRDKSKQFDLLLLEACVRPALVYSHIY---KAPVIQISSLGAALDNYANVG-ASTHPFLY UGT33J2 AAKGKKE--DLITQMRVGSEMLIDLVDLQFQ--TKEVKEIIKG-KQHFDLLLVEACVRPALAFSHVY---KVPLIQISSFGAMFDNYAVIG-APIHPFLY UGT33T2 ETRGETG--DLKAQLAPVMRTLYMVFDKQLQ--DEQIQTLINDKNKQFDLVILEGVLPPLLVFSHLY---KAPAILMSSFGGVYGHYESVG-APTHPLLY UGT40U1 SIIEDP---DAFANMNMMAFLVTTIMNHTYE--NEAVAALLNDSKEHFDAVIVEWIFNEAIGGIATI--FDCPLIWMSSVEVHWKLLSLID-QPSNPAYS UGT40L2 NLIGKG----NFGDSALFMYYVYIIHRNFLE--EPSVVKLFSDPKEKFDAVVLEWFFTEMNAGIPAL--FNCPLIWVCSTEPHWQSMRVMD-GITNPAYT UGT40R3 RFLGPNS--MPALPRFFVKMMMMNLVSKTME--HENVQKLLNDTIAHFDVVIVEWMFTSLSAGYATI--FDCPLIWLIPVEVNSMTIGLVD-AVPHPAYS UGT40R2 HLMEAP---KPSGHRNFVKLMLINLVMKTLE--HENVQRLLNDTNEHFDVVIVEHMMSDLSASYATI--FDCPLIWVSPVEVNALSIGLID-VLPNPAYT UGT41D2 DQSHNN------AGISFIKEFAANMTLEMLN--TPAVKEALIKG--NFDAVVTENFFCDFLAGIPAV--LQVPWIQLAATQLHPDIEAQID-EVRSVPTI UGT42B4 VLLGVS---------LFLAHTGVQNCDTMLE--NDRVQSLIKEKP-KFDVIVVEQFNSDCALGVAHK--FDAPVVGIMSHILMPYHYQRLG-IPYNPAYV UGT46A6 NTLGIEKVIKQILDFQPLADMATGICSNIVD--FVPLSNAMKKS---YDVILVENFNSDCMLGLMHVHGLKAPYISLSSSAMMQWSADRIG-VNDNPSYV UGT2B7 --LPKDTFWLYFSQVQEIMSIFGDITRKFCKDVVSNKKFMKKVQESRFDVIFADAIFPCSELLAELFN---IPFVYSLSFSPGYTFEKHSGGFIFPPSYV UGT2A1 NRPSPSTIWTFYKEMAKVIEEFHLVSRGICDGVLKNEKLMTKLQRGKFEVLLSDPVFPCGDIVALKLG---IPFIYSLRFSPASTVEKHCGKVPFPPSYV 210 220 230 240 250 260 270 280 290 300 | | | | | | | | | | UGT33B13 PSILTKRSLNLSKWEKLEA--------VWNFYRIEKILKGIENSENEMAKRLFGPDVPSISELNNNVHMLFLNAHPIWDGNRPVPPSVIYMGGLHQKPQ- UGT33F4 PAVTRQRLHNLTLIEKIKE--------LYYDFMINYMYGKTMVKENEMLRTHFR-DVPPVTELNNNVDMLFLNVHPIFEGIRPVPPSVVYMGGLHQKPN- UGT33J2 PNTINQRIYNLTMYEKLYE--------LYNNYMMDQLTVDMTVEENKMLKKHFGPDVPEITELQNRVEMLFLNVNPIWEGIRPVPPSVIHLGGLPQKPH- UGT33T2 PLNIRQKLGNLTLWDKLRE--------LFLYYAVENLWYSFYDLGDSILKKNFGPDVPNVWGLMDNIDMLFLNIHPMFEGIRPVPPNAIYLHGLHQKPT- UGT40U1 VDMTSSNQPPLSFTERVSELWTQIQISILSYFIFDKMQDETYQKYVVPAITKRGRDAPSFYEMKYNASLILANAYVSTAIPQTMPQSHKYIGGYHVDEVV UGT40L2 LDIFTHNKLPLNFWQRAEGLWKVVKKAVQ-VLILNQFEKWSYYSIYPEIAAKRGVTMPSYEEAVYNGSFMLINAHPSIGGAIKLPQNSANIAGYHIDK-V UGT40R3 TDPLSSYLPPFSFLERATEIWTRLQESVLGFLYYESKDAANYERIVVPQVQKRGRQAPPLSEVQYNASLVLGNSHVSMGLPLSLPQNYKPVGGYHIEEEV UGT40R2 TDTMALYTAPFTFLERLEELWMRISDSYNDYMVYEPTEEAEYQRLIVPQLQKRGRQVPPYSEVRYNATLVLGNSHVSTGIPLGFPQNYKSMGGYHIEEEV UGT41D2 PLMFNPSGVPMTFASRAINAMIFSMMTMYRWTGR-AATIKQYEDIFTPIAAARGVSLPPFDDAFHNVSITFVNSHESLTPAFTTPPNVISIAGYHMDENI UGT42B4 PFHFLEGGTKPTLYQRVER-------TIFDCYFRTLFEYYTQRNNQNSLAKYFDD-IPPLDDLARNMKFLLLYHNFVLTGSRLFPSNVIEIGSFHVGDA- UGT46A6 PLVSSEFTSQMTFLQRLEN-------TILNVYYKTWFRYAIQMKEKAIIEKRFGRRIPDLQEIAKNVSMMLVNTFHSLNGVRPLLPGVVEVGGMHLDHSR UGT2B7 PVVMSELTDQMTFMERVKN--------MIYVLYFDFWFEIFDMKKWDQFYSEVLGRPTTLSETMGKADVWLIRNSWNFQFPYPLLPNVDFVGGLHCKPA- UGT2A1 PAILSELTDQMSFADRVRN--------FISYRMQDYMFETL-WKQWDSYYSKALGRPTTLCETMGKAEIWLMRTYWDFEFPRPYLPNFEFVGGLHCKPA- 310 320 330 340 350 360 370 380 390 400 | | a | | | | | a a | |bb a| UGT33B13 KEIPAD-LKKYLDSSKNGVVYISFGTNVQPSLLPQDRVQMMAKVFSQLPYDVLWKWDKDELPG-RSKNIRISKWLPQSDLLRHPKIKLFITQGGLQSTDE UGT33F4 KPLPEE-LKSYMDSSKNGVIYISFGTNVDPTQLPADRIEVLVKTLSQLPYDVLWKWNGDVLPG-RTDNIRIAKWLPQQDLLHHPKLKLFITQAGLQSTDE UGT33J2 KELPQD-LKTYLDSSKNGVIYISFGTNVKPSLLPPEKIQTLVKAFSKMPYDVLWKWDKDELPG-RTANIRISKWLPQSDLLKHPKIKLFVTQGGLQSTDE UGT33T2 KDLPQD-LKSYLDSSKHGVIYVSFGTNVDPALLPPEKIQILVRVFSQLPYDVLWKWSKDELPG-RSPNIKIAKWFPQPDLLKHPNVKLFITQGGLQSTDE UGT40U1 KPLLED--KKLIESSKDGVIYFSLGSNLKSKDLPEEIRVSLLKMFGTLKQTVLWKFEAN-MTD-LPPNVHILEWAPQQAILSHPKLAVFITHGGLLSTIE UGT40L2 KPLPKD-LQKIMDEAKNGVIYFSMGSIVQSDGMSEQMQKSILNMFSKYKQTVIWKFESD-MKDNIPSNVHLVKWAPQQSILAHPNLKLFITHGGQLSTSE UGT40R3 KPLPED-LEKIMMNSKNGVIYFSMGSNLKSKDWPEEIKRDLLKLFGELKQTVLWKFEEE-LPN-VPKNVHILKWAPQPSILAHPKCVLFITHGGLLSTTE UGT40R2 KPLPED-LEKIMMNSKNGVIYFSMGSNLKSKDWPEDIKRDLLKLFGELKQTVIWKFEEE-LPN-VPKNVHILKWAPQPSILAHPKCVLFITHGGLLSTTE UGT41D2 PPLPKD-LQELLDGSPKGVIYFSMGSVLRSSALKPHTCAALLKLFASLPYTVLWKFEEP-LKD-LPPNVHVRPWMPQPSILAHPNVKVFITHGGQLSSLE UGT42B4 KPLTGD-LKKFVDEAEHGVIYISFGSVVRASTMPADKVQDVLNVMKKLPQRFIWKWEDKTLLVDKN-KLYTNNWLPQVDILAHPKTLAFLSHAGNGGTTE UGT46A6 KPIPHY-IERFLNESEHGVVLFSWGSLIKTASIPKYKEEIIVNALSKLKQRVIWKYENSNEEGTLTGNILKVKWIPQYELLQHEKVIAFIAHGGLLGMTE UGT2B7 KPLPKEMEDFVQSSGENGVVVFSLGSMVSN--MTEERANVIASALAQIPQKVLWRFDGN-KPDTLGLNTRLYKWIPQNDLLGHPKTRAFITHGGANGIYE UGT2A1 KPLPKEMEEFVQTSGEHGVVVFSLGSMVKN--LTEEKANLIASALAQIPQKVLWRYKGK-IPATLGSNTRLFDWIPQNDLLGHPKTRAFITHGGTNGIYE 410 420 430 440 450 460 470 480 490 500 | cc | | | | | | | | | UGT33B13 AITAGVPLIGFPMLGDQWYNVEKYVHHGIGLQLDLLSTNEVEFKNAINTVVNDERYRQNVHKLRSVMFDQPQPPLERAVWWTEHVLRHGGARHLRGPAAN UGT33F4 AISAGVPLIAIPMFGDQWYNSEKYEYFKIGKKLFMERLTVEEFTNAIKTVINDDSYRKNIVKLRSVMRDESETPLERAVWWTEHVLRHGGAKHLRGPAAN UGT33J2 AITAGVPLVGVPMLGDQWYNVEKYVHHGIGVKLELISLTEETFSTAVNTVIGNESYRKNIQKLRTLMNDQPMKPLDRGIWWIEHVLRHGGAKHLRSPVAN UGT33T2 AITAGVPLVGMPMLGDQYFNVERYVCHGFGIRMDMETLTEEKLKHAINATIEDNSYRRNMERFRTLINDSPQTPLERAVWWTEYLLRHGDAKHLRSPSAN UGT40U1 SVHFGIPIIGIPVLADQHMNIKKAVRNGFALKVDLSYTMADQLKKAIIEVTSNSKYAQKAKELSFIHHDRPVKPGVELVHWVNHVINTHGAPHLRSPALH UGT40L2 AIHYGIPLVGIPVMADQVLNMISVENKGFGIKVTLSEDMIPELNAAIKKVLTDDAYRKKAKEISALFHDRVMTPGAAVPYWIEYVVRTRGAPHLRSPALD UGT40R3 TIHFGVPTIAIPVFGDQFINVKKSVARGFTLQVDLSYKLAADLKVAIEEMLSNPKYRQRVKELSYIYHDRPVKPGAELRHWVQHVVNTRGASHLRSPALQ UGT40R2 TIHYGVPTIAIPVFGDQFINVKKAVARGYALEVKLSHSIAAELKVAIQEMLNNPKYRQRVKELSYIYHDRPVKPGAELRHWVQHVVNTRGAPHLRSPALQ UGT41D2 AVRAGVPLLAVPVFGDQPANAERARRSGYALKVDFSPDMVPELEVALKEILNNDQYSKRVKYLSKVFTNRPTTPKKLINHYVELAIETKGALHLRSTSLQ UGT42B4 AIHYGVPMVAMPIVGDQPANAAAVEESGLGVQLQVRDLNEENLLNAFKKVS-DPKFRERVKEVSKAWHDRPLSPMDTAIYWTTEFAAKHPNFTFRTAAAD UGT46A6 AISAGKPMLIVPFYGDQMVNGAAATTIGLGKAISYADMSEKSLLEGLQSVL-SPEMRMSARRASKIWQDRIADPLDTAVYWVERVIRWGHQDPLHSTSKD UGT2B7 AIYHGIPMVGIPLFADQPDNIAHMKARGAAVRVDFNTMSSTDLLNALKRVINDPSYKENVMKLSRIQHDQPVKPLDRAVFWIEFVMRHKGAKHLRVAAHD UGT2A1 AIYHGIPMVGVPMFADQPDNIAHMKAKGAAVEVNMNTMTSADLLSAVRAVINEPFYKENAMRLSRIHHDQPVKPLDRAVFWIEFVMRHKGAKHLRVAAHD 510 520 530 540 550 | | | | | UGT33B13 ISWADYLELELVLIVLAVIIGFSIVLSLIAHYLWKFVKIQIKYR-KLKET------ UGT33F4 MSWAEYLELELVFTLLLGVITAIAVLYVILRFLNKMVSANTVKKSKRS-------- UGT33J2 ISWAEYLEFELVAIVLSALLAALAVAIAVVYSLYGFVTRNYIIRAKVKSS------ UGT33T2 ISWRQYLELDLVLILLSVLLTAIAVTVIVLRLVYRSIRSYLMVTLR---------- UGT40U1 VPFYQKMYLDLAAVLIILFLAGRLLLKKAYAAVFSKS--------KSNKKKTH--- UGT40L2 VPLYQKLYLDLAAFIAVVEIVLKKVVKYLRKREVIKRR-------RAKNL------ UGT40R3 VPLYQRLYLDLVAFLSVAFIVLYMLIKKLYSRVRSKK--------IVNNKKRN--- UGT40R2 VPLYQRLYLDLAALLLVVILVLKLLLKNLYHRIRPKKT-------NVNIKKKDKKN UGT41D2 YRWYERWMLDVVLVLLVTLASILALLVFAAKKVINKVTGKKVYTEKSSKKKRN--- UGT42B4 VPFYQYINLDVAAVLITIFVLNIVVLRIVINKCRSKKK-----VVTKTEKKKAKRA UGT46A6 MGFIEYNLLDVAAVILLSFVFLILVLRIVLNQILRLFG-----AGTSKKEKLH--- UGT2B7 LTWFQYHSLDVIGFLLVCVATVIFIVTKCCLFCFWKFA------RKAKKGKND--- UGT2A1 LSWFQYHSLDVIGFLLACMASAILLVIKCCLFVFQKIG------KTXKKNKRD--- Transmembrane domain cytoplasmic tail

Important residues in donor binding regions

a nucleotide interacting residues b phosphate interacting residues c glucoside interacting residues

Figure 1. Multiple alignment of the 11 Spodoptera littoralis uridine diphosphate-glycosyltransferases (UGTs) with human UGT2B7 and rat UGT2A1.Predicted signal peptides in the N-terminal are underlined. The UGT signature motif is boxed. The transmembrane domains based on UGT2A1 in theC-terminal half and cytoplasmic tails are in white boxes under alignment. Important catalytic residues (H41 and D139) are indicated by asterisks abovethe alignment. Grey bars under the sequences indicate the two donor binding regions and several residues interacting with the sugar donor are indicatedby letters (a, b, c) above the alignment.

4 F. Bozzolan et al.

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UGT35B1 from D. melanogaster (Wang et al., 1999), butthese two UGTs were also expressed in other tissues.The restricted pattern of UGT46A6 and UGT40R3 sug-gests that these genes might be specifically involved inchemoperception or in maintaining chemosensory organhomeostasis.

Sex expression levels of these two genes (Fig. 3B)during development (Fig. 3C) were studied more precisely

by quantitative RT-PCR. Both genes showed more pro-nounced expression in male antennae: 4.4-fold more forUGT40R3 and 2.4-fold more for UGT46A6 as comparedwith the female antennae. Moreover, temporal expressionduring post-embryonic development revealed that bothUGTs were predominantly expressed during adult life, withbarely detectable expression at the end of the pupalstage, followed by a rapid rise in transcript number

0.1Color ranges:

UGT48

UGT48

UGT41

UGT39

UGT47

UGT46

UGT43

UGT44

UGT42

UGT50

UGT34

UGT33

SlUGT40R3

SlUGT40R2

HaUGT40R1

BmUGT40K1BmUGT40G1

BmUGT40G2

SlUGT40U1

HaUGT40D1

HaUGT40D2

BmUGT40P1

HaUGT40M1BmUGT40N1

BmUGT40S1

HaUGT40Q1BmUGT40A1

BmUGT40H1

BmUGT40B1

BmUGT40B3

BmUGT40B2P

BmUGT40B4

HaUGT40F1

HaUGT40F2

HaUGT40L1

SlUGT40L2

HaUGT48A1

BmUGT48C1

BmUG

T41A

2Bm

UGT4

1A3

BmU

GT4

1A1

HaUG

T41D

1Sl

UGT4

1D2

HaUGT4

1B1

HaUGT4

1B2

HaUGT41

B3

HaUGT39B2

BmUGT39B1BmUGT39C1

HaUGT47A2

BmUGT47A1

HaUGT46A3

SlUG

T46A6

HaUGT46A4

BmUGT46A1

BmUG

T46A2HaUGT46B1BmUGT46C2

HaU

GT43A1

BmU

GT43B1

HaU

GT44A2

BmU

GT44A1 H

aUG

T42B

2

SlU

GT4

2B4

Bm

UG

T42B1

HaU

GT4

2C1

BmU

GT4

2A1

BmU

GT4

2A2

HaUGT50A2

BmUG

T50A1

HaUGT34A3

BmUG

T34A2

BmU

GT33R

1

BmU

GT33R

2

BmU

GT340C

1

BmU

GT340C

2

BmU

GT33Q

1

Bm

UG

T33N1B

mU

GT3

3K1Bm

UGT3

3D6

BmUG

T33D

7

BmUG

T33D

3Bm

UG

T33D

8Bm

UG

T33D

2

BmU

GT3

3D1

BmU

GT3

3D5

BmU

GT3

3D4

HaUGT33F3SlUGT33F4

HaUGT33

F1

HaUGT33M1HaUGT33T1SlUGT33T2

HaUGT3

3F2

HaUGT3

3J1

SlUGT3

3J2

SlUGT33B13

HaUGT33B9HaUGT33B11

HaUGT33B8

HaUGT33B4

HaUGT33B7

HaUGT33B12

HaUGT33B1

HaUGT33B5

HaUGT33B2

HaUGT33B3

Figure 2. Phylogenetic tree of Spodoptera littoralis (SlUGT), Helicoverpa armigera (HarUGT) and Bombyx mori (BmUGT) uridinediphosphate-glycosyltransferases (UGTs). Three partial sequences from B. mori and H. armigera (HarUGT43, HarUGT28 and BmUGT009787P) have notbeen included. The consensus phylogenetic tree was built using the neighbour-joining method, with a total of 1000 bootstrap replications. Branchescorresponding to partitions reproduced in < 70% bootstrap replicates were collapsed.

Antennal UDP-glycosyltransferases in a pest insect 5

© 2014 The Royal Entomological Society

after adult emergence. UGT40R3 expression level washowever clearly lower compared with that of UGT46A6during all developmental stages.

The expression profiles of these two UGTs, with asexual dimorphism and a late onset during development,are similar to those observed for several olfactory genes,such as odorant-binding proteins or odorant-degradingenzymes (Ishida & Leal, 2005; Durand et al., 2011) andare concomitant with the electrophysiological responsive-ness of males to odorants, and particularly to pheromonecomponents (Györgyi et al., 1988; Vogt et al., 1989). Thissuggests a more specific role in olfactory processes forthese UGTs.

UGT46A6 localization within antennae

The cellular localization of UGT46A6 transcripts in maleantennae was characterized by in situ hybridization(Fig. 4). In S. littoralis antennae, olfactory sensilla are dis-played on the ventral side and scales of the dorsal side(Ljungberg et al., 1993). UGT46A6 signals were restrictedto the sensilla side of the antennae, with no labelling on

the scale side. Labelling was located at the base of theolfactory sensilla, in cells that could correspond to theaccessory cells or the olfactory receptor neurons. In situhybridization with a UGT40R3 probe was however unsuc-cessful, probably because of its low expression level withregard to the method sensitivity.

Nevertheless, to our knowledge, this is the first report ofan UGT expression restricted to the olfactory sensillarcells of an insect. This suggests that the correspondingenzyme could act on substrate molecules entering thesensilla through its cuticular pores, including odorants orxenobiotics.

Effect of odorant and insecticide exposure on UGT40R3and UGT46A6 expression levels

To test whether UGT40R3 and UGT46A6 expressioncould be modulated by odorant exposure, males wereeither exposed to Z9E11-14:Ac, the main female sexpheromone component attractive to males, or to Z3-6:Ac,a green leaf volatile used as a chemical cue by both sexesto locate the host plant (Durand et al., 2010a). After

UGT40R3 UGT46A6 0.00

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

Nor

mal

ized

exp

ress

ion

rpL13

UGT40R3

UGT46A6

A B

0,00

0,01

0,02

0,03

0,04

0,05

0,06

Larvae D-5 D-3 D-1 D+1 D+2 D+3 D+4 D+5

UGT40R3 UGT46A6

Nor

mal

ized

exp

ress

ion

C

a a a

b, c

b

b

c c

a A A A A B B

C C B,C

*

*

0.06

0.05

0.04

0.03

0.02

0.01

Figure 3. (A) Analysis of UGT40R3 and UGT46A6 expression throughout different body parts of male adults by reverse-transcriptase PCR.(B) Comparison of normalized expression levels of UGT40R3 and UGT46A6 in male and female antennae after quantitative PCR analysis. Asterisksindicate statistical differences (Student’s t test, P < 0.01). (C) Analysis of UGT40R3 and UGT46A6 expression in larval antennae and male antennae byquantitative PCR during insect life. Expression levels were normalized to that of rpL13. Data are given as the mean ± SD. Different letters (in capital forUGT40R3) indicate statistical differences (ANOVA, Tukey’s post hoc test, P < 0.05).

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Z3-6:Ac exposure, UGT40R3 and UGT46A6 transcriptswere both significantly decreased (4.6- and 2.2-fold,respectively). Only UGT40R3 transcripts decreased 2.6-fold after pheromone exposure (Fig. 5).

The median lethal dose (LD50) value for deltamethrinwas 7.6 ng ± 2.3 in males after topical application onantennae. Interestingly, UGT46A6 increased 2.2-foldwhen the highest of the two sublethal doses was used(1/10 of the LD50), whereas no variation was observed withthe lower dose (1/100of the LD50).

Both UGTs exhibited different transcriptional regulationafter odorant or insecticide exposure. UGT40R3 wasdownregulated by both odorants but the insecticide didnot affect its transcription, whereas UGT46A6 was re-gulated in opposite ways by the green leaf volatileand the insecticide. Induction of xenobiotic-metabolizingenzymes by xenobiotics or plant allelochemicals fromthe diet (Feyereisen, 1999; Després et al., 2007) or byvolatiles (Durand et al., 2010a; Pottier et al., 2012) hasbeen documented in insects, but, to our knowledge,never for antennal UGTs. More globally, little informationis available on the roles of UGTs in the detoxificationof allelochemicals: metabolism of plant compounds byUGTs has been reported in M. sexta larvae (Ahmad &Hopkins, 1993b) and an UGT capable of degradingflavonoids and coumarins in vitro has been identified inB. mori (Huang et al., 2008). In the absence of a knownfunction for the antennal UGTs identified in the presentstudy, it is difficult to speculate on the physiological sig-nificance of the downregulation observed after odorantexposure. Little is known regarding UGT downregulation,

A Ventral face

(3)

(2)

(4)

(1)

Dorsal face

B Trichoidsensilla

Figure 4. Expression of UGT46A6 after in situ hybridization in longitudinal sections of Spodoptera littoralis male antennae. (A) View of a segmentshowing the disposition of the olfactory epithelium (1), the antennal nerve (2), the antennal lumen (3) and the ventral cuticle (4). Arrows show the trichoidolfactory sensilla. (B) Higher magnification of the olfactory epithelium, showing labelled cells. Scale: 50 μm in A; 5 μm in B.

0

0,5

1

1,5

2

2,5

Naive

Exposed

Z9E11-14:Ac

Z3-6:Ac

DeltamethrinLD50 1/100

DeltamethrinLD50 1/100

UG

T40R

3

UG

T46A

6

UG

T40R

3

UG

T46A

6

UG

T40R

3

UG

T46A

6

UG

T40R

3

UG

T46A

6 *

* *

*

Nor

mal

ized

exp

ress

ion

fold

2.5

1.5

0.5

Figure 5. Transcript levels variations of UGT40R3 and UGT46A6 inantennae after exposure of males to odorants and after topicalapplication of deltamethrin, as measured by quantitative PCR analysis.Expression levels were normalized to those of rpL13. N ≥ 3 for eachcondition. Data are given as normalized expression fold variations ascompared with controls. Asterisks indicate statistical differences(Student’s t-test, P < 0.01).

Antennal UDP-glycosyltransferases in a pest insect 7

© 2014 The Royal Entomological Society

even in mammals. Downregulation of UGT transcriptionduring infection or inflammatory responses has beenobserved in mice and it has been proposed that thiscould reduce glucuronidation and contribute to alterationof drug efficacy (Richardson et al., 2006). Downregula-tion of antennal UGT transcription by odorant moleculesmight reduce the amount of the corresponding enzymesand thus the glycosylation processes mediated by theseUGTs.

Little information is available on UGT regulation ininsecticide resistant strains or after insecticide exposurein insects. Several UGTs are constitutively overexpressedin strains of D. melanogaster (Pedra et al., 2004), Myzuspersicae (Silva et al., 2012) or Bemisia tabaci (Yang et al.,2013) resistant to DDT, carbamates and neonicotinoids,respectively. In the case of pyrethroids, one study reportsUGT induction after permethrin exposure in Anophelesgambiae (Vontas et al., 2005). To our knowledge, induc-tion of an UGT by deltamethrin was never reportedbefore, especially in such specialized organs as theantennae. Dose-dependent specific UGT46A6 inductionafter insecticide topical application suggests a role of thisenzyme in deltamethrin detoxification at the antennallevel.

Spodoptera littoralis antennae were known to expressvarious CCEs (Durand et al., 2010b) and P450s (Pottieret al., 2012), two families of detoxification enzymesinvolved in deltamethrin detoxification and resistancein various insects, including the genus Spodoptera(Oakeshott et al., 2005). These enzymes are involved inthe first phase of xenobiotic detoxification by adding afunctional group to the molecule (functionalization step),leading to a metabolite that could be conjugated by phase-two enzymes (conjugation step), such as GSTs or UGTs(Guéguen et al., 2006).

As the olfactory receptor neurons are exposed to theairborne volatiles, including potentially toxic ones, wecould presume that some antennal enzymes belonging tovarious families have evolved to sequentially detoxifyinsecticides within the antennae, participating in the pres-ervation of the sensillar function, whereas some othershave evolved towards an odorant clearance function.

S. littoralis is a highly polyphagous pest species whichhas to deal with a wide range of allelochemicals fromplants. This species is also known for its high ability todevelop resistance to various insecticides. As olfactionplays a vital role in the adult reproductive behaviours ofthis nocturnal moth, keeping antennal functioning iscrucial. Recruitment of different detoxification enzymes,including UGTs, for the detoxification and clearance ofvarious volatile allelochemicals, xenobiotics and odorantswithin the olfactory organs may participate in the optimi-zation of the antennal function in various ecologicalcontexts.

Experimental procedures

Chemicals

(Z,E)-9,11-tetradecadienyl acetate (Z9E11-14:Ac) was synthe-sized in the laboratory (courtesy of Dr M. Lettere, >97% purity,CAS 50767-79-8). (Z)-3-hexenyl acetate (Z3-6:Ac) was from Lan-caster Synthesis (Alpha Aesar, Ward Hill, MA, USA; 99% purity,CAS 3681-71-8). Deltamethrin (Pestanal®, CAS Number 52918-63-5) was from Sigma-Aldrich (St.Louis, MO, USA).

Animals, exposure to odorants, insecticide and tissue collection

Insects were reared on semi-artificial diet at 24 °C, 60–70% rela-tive humidity, and under a 16 h light:8 h dark photoperiod asdescribed in Durand et al. (2010b). Sexes were separated atpupal stage. Antennae from last instar larvae, 3-day-old male andfemale adults, from male pupae and various tissues from 3-day-old males were dissected and stored at −80 °C until use.

Odorant exposure experiments were conducted as describedin Durand et al. (2010a). Twenty 1-day-old males were set for 48hinto hermetically sealed boxes containing either 1 μg of Z3-6:Ac,a green leaf volatile from host plant, or Z9E11-14:Ac, the mainsex pheromone component, loaded onto a filter paper. Controlanimals were kept in the same conditions except that the filterpaper was only loaded with solvent (hexane). Antennae werethen dissected for quantitative PCR analysis.

Insecticide exposure was performed using a brief topical appli-cation of 2-day-old male antennae with a solution of deltamethrindiluted in hexane. A 1-μl droplet of each dilution was applied onthe distal part of each antenna, avoiding any contact with thehead. The LD50 was estimated 24 h after the exposure of males todeltamethrin serial dilutions (n = 30 for each dilution). The LD50

was computed using Probit analysis. Statistics were evaluatedusing an α-risk of 0.05. Two sublethal doses corresponding to1/100 and 1/10 of the LD50 were then used for antennal applica-tions. Control insects were treated with hexane only. Antennaewere dissected 24 h after treatment.

For in situ hybridization, 3-day-old male antennae were fixedfor 6 h at 4 °C in 4% PFA-3% Tween 20, rinsed twice inphosphate-buffered saline (PBS; 0.85% NaCl, 1.4 mM KH2PO4,8 mM Na2HPO4, pH 7.1; Sigma-Aldrich) and then cryoprotectedin PBS-18% sucrose at 4 °C.

RNA isolation and cDNA synthesis

Total RNAs were extracted with TRIzol Reagent (Invitrogen,Carlsbad, CA, USA), quantified with a spectrophotometer(BioPhotometer; Eppendorf, Hamburg, Germany) then treatedwith DNase I (Roche, Basel, Switzerland) according to the man-ufacturer’s instructions. For PCR, single-stranded cDNAs weresynthesized from total RNAs (5 μg) from various tissues asdescribed in Durand et al. (2010a). For 5′ and 3′-RACE PCR,antennal cDNAs were synthesized from 1 μg of male antennalRNA at 42 °C for 1.5 h using the SMART™ RACE cDNA Ampli-fication Kit (Clontech, Mountain View, CA, USA) with 200 U ofSuperscript II, 3′-cDNA synthesis (CDS)-primer and SMART IIoligonucleotide.

Identification and cloning of SlUGTs

Putative UGT sequences were identified from a S. littoralis maleantennal EST library (Jacquin-Joly et al., 2007; Legeai et al.,

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© 2014 The Royal Entomological Society

2011) by local TBLASTN analysis with the BioEdit SequenceAlignment Editor software (http://www.mbio.ncsu.edu/bioedit/bioedit.html) and using the sequences of rat olfactory UGT2A1and of UGT33D8 from B. mori (Huang et al., 2008) as queries.ESTs were assembled and for five sequences, the missingregions of the corresponding contigs were obtained by 5′- and/or3′-RACE-PCR and/or PCR. For 5′-RACE, we used 2.5 μl of5′-RACE-ready cDNA with specific reverse primers anduniversal primer mix as the anchor primer. 3′-RACE amplifica-tions were carried out with UPM as reverse primer and specificforward primers. The PCR products were purified (Nucleospin®Extract II; Macherey-Nagel, Düren, Germany) and clonedinto pCR®II-TOPO® plasmid (Invitrogen). After isolation(Nucleospin® Plasmid; Macherey-Nagel), recombinant plasmidswere sequenced (GATC Biotech, Marseille, France) and the over-lapping sequences assembled to obtain full-length cDNAs. Thecomplete ORFs were then amplified from antennal cDNA with ahigh-fidelity Taq DNA polymerase (Roche) and sequenced toconfirm that the sequences were not chimeric. All the primersused are reported in Table 2.

Nomenclature and phylogenetic analysis

According to the UGT nomenclature Committee, UGT familiesand subfamilies are defined at 40% and 60% amino acid identity,respectively (Mackenzie et al., 1997). Names were assigned tothe S. littoralis sequences on this basis. The UGT predictedprotein sequences from H. armigera and B. mori were extractedfrom the National Center for Biotechnology Information (http://

www.ncbi.nlm.nih.gov/) and analysed with the S. littoralis UGTsusing CLUSTALW alignment through MEGA5 software (http://www.megasoftware.net; Tamura et al., 2011). This alignment wasused to build a consensus phylogenetic tree using the neighbour-joining method. Pairwise and multiple alignments were performedwith a gap opening penalty at 10 and the gap extension penaltyleft at 0.2. A total of 1000 bootstrap replications were used.Branches corresponding to partitions reproduced in <70% boot-strap replicates were collapsed.

Tissue-related expression analysis by reverse-transcriptasePCR and quantitative PCR

Expression of the UGT genes was first qualitatively studied in theantenna, thorax and abdomen of males by RT-PCR. Primer pairswere chosen in the less conserved regions of the varioussequences in order to avoid cross amplification and are shown inTable 2, together with PCR conditions. A ribosomal control generpL13 (Maïbèche-Coisne et al., 2004) was used to check thequality of the cDNAs. PCR was performed on 100 ng of cDNAs.PCR products were loaded on 1.5% agarose gels and visualizedusing Gel RED (VWR; Biotium, Hayward, CA, USA) fluorescence.For two genes that exhibited an expression restricted to theantenna additional tissues were then tested to refine their respec-tive distribution; quantitative PCR was also conducted to com-pare their expression between male and female antennae andduring development. Amplification by quantitative PCR was per-formed as described in detail in Durand et al. (2010b) using theLightCycler® 480 Real-Time PCR System (Roche). Each

Table 2. List of the primers used for reverse-transcriptase PCR, rapid amplification of cDNA ends PCR or quantitative PCR, indicating their sequencesand annealing temperatures

Primer Purpose Sequence 5′-3′ Temp (°C)

UGT 33B13 do RT-PCR CTGAGAGAACACTTTGGCCATCAT 67UGT 33B13 up RT-PCR AGCAGTGTGGAACTTTTACAGAATAGAAAA 66UGT 33F4 5R RT-PCR GGGCGTTCACGATGATCTCTT 63.9UGT 33F4 do RT PCR CGTCCAGGTAGAACATCACCATTC 68UGT 33F4 up RT PCR GGCAGTGACAAGACAACGTCTGC 70UGT 33J2 do RT-PCR TTTGAGAAGGCTTTGACAAGCGTCT 70UGT 33J2 up RT-PCR AATTGTATAACAATTACATGATGGATCAAT 63UGT 33T2 do RT-PCR GGCAACTGAGAGAACACTCGTACTAAT 66UGT 33T2 up RT-PCR TTCCTTTATTATGCAGTAGAAAATCTTTGG 66UGT 40U1 do RT-PCR TTCAATAGGCTCTCGGATTTCTT 64UGT 40U1 up RT-PCR CAGTAGACATGACGTCGTCCAATC 67UGT 40 L2 do RT-PCR TCACACGATCATGGAACAGAGCT 68UGT 40 L2 up RT-PCR CCCACAACAAGCTGCCTTTGA 69UGT 40R3 do RT-PCR-qPCR ACAGATCAACTTGCAGAGTAAAGCC 66UGT 40R3 up RT-PCR TGTCATCGTACCTGCCTCCCT 68UGT 40R3qrev qPCR CTTGCCGACCTCTCTTTTGGACTT 66UGT 40R2 do RT-PCR GAGTGAGATAGCTTAACTTCCAAAGCA 66UGT 40R2 up RT-PCR GTTGTCAATTGGATTGATCGATGT 66UGT 41D2 do RT-PCR CCAGCCAAGAAGTCACAGAAGAAG 67UGT 41D2 up RT-PCR ACAACCAGCTCTCCAAGG 60UGT 41D2 3R 3′RACE PCR CCCCCATAGCCGCAGCGAGA 71.8UGT 42B4 do RT PCR TTCTGTCCAGTATATAGCGGTATCCAT 65UGT 42B4 up RT PCR CAGACAAAGTGCAGGACGTACTAAA 65UGT 46A6 up RT-PCR AAGTTCTGAGTTCACATCCCAGATG 66UGT46A6 do RT-PCR CGTTCACGATGATCTCTTCTTTGTAC 66UGT 46A6 5R 5′RACE PCR CGGCGAAGATCGCGACGAGTAGTA 70.3UGT 46A6 qdo qPCR, ISH CGTGGTTCCGATACGCGATACA 67,1UGT 46A6 qrev qPCR, ISH ACACCAGGCAATAGAGGACGAACA 66,1

RT, reverse transcriptase; qPCR, quantitative PCR; RACE, rapid amplification of cDNA ends; ISH, in situ hybridization.

Antennal UDP-glycosyltransferases in a pest insect 9

© 2014 The Royal Entomological Society

reaction was run in technical triplicate with three independentbiological replicates. Data were analysed with LightCycler 480®Software (Roche). The crossing-point values were first deter-mined for the reference genes with a run formed by the fivefolddilution series, the measuring points and three negative controls.The normalized UGT expression was calculated with Q-GENE

software (Simon, 2003) using rpL13 as reference. This gene hasbeen already demonstrated as the best reference gene in theseconditions (Durand et al., 2010b).

In situ hybridization

A 398 bp cDNA fragment was amplified by PCR using two specificprimers (Table 2) and used as template for in vitro transcription togenerate digoxigenin-labelled RNA sense and antisense probes.Antennae from 3-day-old male moths were embedded in Tissue-Tek mediumTM compound (CellPath, Newtown Powys, UK).Cryosections (7 μm) were set in cell culture insert (GreinerBio-one, Monroe, NC, USA). Hybridization was conducted asdescribed previously (Durand et al., 2010a). Pictures wereacquired (Olympus BX61 microscope, IMAGEPRO software) anddigitalized using Adobe Photoshop® 7.0 (Adobe, USA).

Acknowledgements

We thank M. Solvar for her help in histology and insectrearing and A. Haines for the nomenclature of S. littoralisUGTs.

This work was supported by University Pierre & MarieCurie (Emergence Project).

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