lable at ScienceDirect

Insect Biochemistry and Molecular Biology 40 (2010) 506e515

Contents lists avai

Insect Biochemistry and Molecular Biology

journal homepage: www.elsevier .com/locate/ ibmb

Study of the aminopeptidase N gene family in the lepidopterans Ostrinianubilalis (Hübner) and Bombyx mori (L.): Sequences, mapping and expression

Cristina M. Crava a, Yolanda Bel a, Siu Fai Lee b, Barbara Manachini c, David G. Heckel d, Baltasar Escriche a,*

aDepartment of Genetics, University of Valencia, Dr. Moliner 50, Burjassot 46100, Valencia, SpainbDepartment of Genetics, Bio21 Institute, 30 Flemington Road, Parkville, VIC 3010, AustraliacDepartment of Animal Biology, University of Palermo, via Archirafi 18, Palermo 90123, ItalydDepartment of Entomology, Max Planck Institute for Chemical Ecology, Hans Knöll Strasse 8, Jena D-07745, Germany

a r t i c l e i n f o

Article history:Received 29 January 2010Received in revised form16 April 2010Accepted 16 April 2010

Keywords:Quantitative PCRMidgut APNPuromycin-sensitive aminopeptidaseBt toxin-binding proteinsLarval development expression

* Corresponding author. Tel.: þ34 96 3543401; faxE-mail address: Baltasar.escriche@uv.es (B. Escrich

0965-1748/$ e see front matter � 2010 Elsevier Ltd.doi:10.1016/j.ibmb.2010.04.010

a b s t r a c t

Aminopeptidases N (APNs) are a class of ectoenzymes present in lepidopteran larvae midguts, involvedin the Bacillus thuringiensis (Bt) toxins mode of action. In the present work, seven aminopeptidases havebeen cloned from the midgut of Ostrinia nubilalis, the major Lepidopteran corn pest in the temperateclimates. Six sequences were identified as APNs because of the presence of the HEXXH(X)18E and GAMENmotifs, as well as the signal peptide and the GPI-anchor sequences. The remaining sequence did notcontain the two cellular targeting signals, indicating it belonged to the puromycin-sensitive amino-peptidase (PSA) family. An in silico analysis allowed us to find orthologous sequences in Bombyx mori. Aphylogenetic study of lepidopteran aminopeptidase sequences resulted in their clustering into nineclasses. Linkage analysis revealed that the onapn genes as well as all bmapn genes clustered in a singlelinkage group. O. nubilalis aminopeptidases were expressed in all larval instars. In 5th instar larvaetissues, apns transcripts were found mainly in midguts while apn8 was also highly expressed in Mal-pighian tubules, and psa showed an ubiquitous expression pattern in O. nubilalis and B. mori. Thesequence homology and gene organization of apns suggest a single origin from an ancestral lepidopteranapn gene.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Aminopeptidases N (APNs) are a class of endoproteases thatcleave the N-terminus of the polypeptides to release single aminoacids (Piggot and Ellar, 2007). They are members of the zinc-dependant metalloprotease M1 type, that needs the divalent cationzinc to activate a molecule of water, and belong to a subfamilynamed gluzincins (Albiston et al., 2004). These ectoenzymes arelocated in the brush border membranes of the alimentary tract ina variety of different organisms, including the midgut of Lepidop-tera larvae. In these kinds of insects, a glycosyl-phosphatidylino-sitol (GPI) anchor is used for their attachment to the membrane(Garczynski and Adang, 1995). APNs are involved in several func-tions in a wide range of organisms, but in the lepidopteran larvalmidgut they play an important role in protein digestion, co-oper-ating with the carboxypeptidases in the digestion of the peptidesproduced by the action of the digestive endopeptidases (Terra andFerreira, 1994).

: þ34 96 3543029.e).

All rights reserved.

Lepidopteran APNs are widely studied not only because of theirrole in digestion but also for their involvement in the binding to theBacillus thuringiensis (Bt) crystal insecticidal toxins, also called Crytoxins (Knight et al., 1994; Bravo et al., 2007). These toxins act in themidgut of the lepidopteran larvae and their binding to one or morereceptors located in the brush border membrane is the key step ofthe mechanism of toxicity (Ferré and Van Rie, 2002). Cadherin-likeand alkaline phosphatase proteins together with different isoformsof APNs have been shown to interact with different types of Crytoxins (Piggot and Ellar, 2007; Jurat-Fuentes and Adang, 2004;Flanagan et al., 2005). Indeed, the gut transgenic expression ofthe APN1 from Manduca sexta generated susceptibility to Cry1Actoxin in tolerant Drosophila melanogaster larvae (Gill and Ellar,2002) and the reduction of the expression of an APN of Spo-doptera litura with RNAi resulted in a reduced susceptibility of thelarvae to Cry1C (Rajagopal et al., 2002). These direct evidences ofthe participation of APNs in the Bt mode of action have beencomplemented by reports about association between APN alter-ations and resistance to Bt toxins: Herrero et al. (2005) describedthe lack of APN1 expression in a Cry1Ca resistant Spodoptera exiguastrain and Zhang et al. (2009) showed a deletion mutation in theapn1 gene of a Cry1Ac resistant Helicoverpa armigera strain.

C.M. Crava et al. / Insect Biochemistry and Molecular Biology 40 (2010) 506e515 507

To date, different isoforms from more than 20 lepidopteranspecies have been cloned and characterized (Piggot and Ellar,2007), but the number of apn genes for each single species is stilluncertain. Angelucci et al. (2008) reported seven different apncDNA sequences in H. armigera, as well as the orthologous genes inthe Bombyx mori genome suggesting the occurrence of at leastseven classes of APNs in Lepidoptera. So far, the phylogenic analysishas shown that the APNs within a particular species cluster intodifferent clades, which unfortunately are given different names bydifferent authors (Herrero et al., 2005; Angelucci et al., 2008). Withrespect to the involvement of these aminopeptidases in the Bttoxins mode of action, there is evidence of interaction only for APNsbelonging to five of the classes (Piggot and Ellar, 2007). In general,all the reported lepidopteran APN sequences have been obtainedfrom midgut cDNA analysis.

In the present study, aminopeptidases of the polyphagousLepidoptera Ostrinia nubilalis (European corn borer) and the lepi-dopteran model B. mori have been studied. O. nubilalis is the majorinsect pest of the corn crops in Europe and in United States, andCry1 toxins are widely used for its control, primarily the activatedCry1Ab expressed in transgenic corn (Koziel et al., 1993). Alterna-tively, B. mori is a domesticated lepidopteran species susceptible toCry1Aa toxin. Its use in comparative studies is favored by theavailability of genetic stocks, and its commercial interest hasprovided many studies of B. mori during the past century. Indeed,there are large databases and its genome is fully sequenced (Duanet al., 2009; Shimomura et al., 2009).

Currently, more than 100 isoforms of lepidopteran APNs havebeen reported in databases, but despite the economical importanceof O. nubilalis, no complete sequence of any apn has been cloned. Inthe present work, the full-length cDNA sequences of sevenaminopeptidases expressed in the midgut of the European cornborer larvae are reported, as well as their linkage relations and theirexpression during the larval development and in different larvaltissues. The results obtained are further in silico analyzed andcompared with data of the aminopeptidase sequences andexpression levels obtained from B. mori database mining.

2. Materials and methods

2.1. Insects

O. nubilalis individuals used for cDNA cloning were taken froma laboratory colony founded from a field sample collected in theLombardia area and maintained in the University of Milan (Italy).Larvae used for the expression studies were obtained from theInstitut National de Recherche Agronomique (INRA, Montpellier,France). Both colonies were reared at the University of Valencia. O.nubilalis insects from the area of Bonn (Germany), Germany wereused to generate the linkage mapping families. Bonn-E wascollected from mugwort (Artemisia vulgaris) in 2000 and Bonn-Ncollected from maize (Zea mays) in 2002, by C. Saeglitz of RWTHUniversity, Aachen, Germany. The insects had been propagated inthe lab for 24 and 13 generations respectively, when the crossescommenced in October 2003. Rearing procedures were modifiedfrom Wyniger (1974). The diet consisted of crouched corn, wheatgerm and yeast, to which a vitamin mixture, antibiotics, andpreserving agents were added, and solidified with agar. Afterhatching, larvae were kept at 25 �C at a photoperiod of 16:8 (L:D) hand 70% RH in a climate chamber. Pupae were moved to plasticboxes provided with filter paper. Emerging adults were transferredto mating cages and maintained at 22 �C at the same photoperiodand humidity as larvae, and provided with 10% honey in water.Cages were misted daily with water to increase humidity. Crosseswere conducted at the RWTH University. The initial single-pair

cross involved a virgin Bonn-E female and a virgin Bonn-N male(grandparents). Egg masses were collected daily for five consecu-tive days. The mating pair was then snap frozen in liquid nitrogen.F1 larvae were reared to adulthood. To generate a female infor-mative mapping family (F305), a virgin hybrid F1 female wasbackcrossed to a freshly emerged Bonn-E male; their progeny (F2)were reared to adulthood, sexed and snap frozen at �50 �C untilDNA isolation.

2.2. Screening for apn sequence fragments

Genomic DNA (gDNA) was isolated from adult moths with headsand wings removed, using three extractions with phenol (equili-brated with TE buffer, pH 8.0) and one with chloroform. Theaqueous fraction was treated with 10 mg of RNAse at 37 �C for 1 h.DNA was precipitated overnight in two volumes of ethanol in thepresence of 0.2 M NaCl, and re-dissolved in TE buffer, pH 8.

Degenerate primers (Table 1 in Supplementary material) wereused to amplify products from isolated O. nubilalis gDNA (10 ng)with Accu-Taq polymerase (Sigma, St. Louis, MO, USA) by touch-down PCR using the following cycling conditions: initial denatur-ation of 5 min at 96 �C, followed by 30 cycles of 30 s at 95 �C, 30 s at60 �C (annealing temperature reduced by 0.5 �C per cycle) and1min at 72 �C, followed by another 30 cycles of 30 s at 95 �C, 30 s at45 �C, and 1 min at 72 �C. PCR products were separated on a 1%agarose gel. DNA fragments were purified, mass-cloned into pGem-T Easy vector (Promega, Madison, WI, USA), heat transformed intoJM109 competent cells and sequenced.

2.3. apn isoform cDNA cloning

Midguts were dissected from fifth instar larvae and washed infresh cold MET buffer (300 mM mannitol, 17 mM TriseHCl pH 7.5,5 mM EGTA). Samples were quickly homogenized with a pestle ina microtube containing TriPure reagent (Roche, Mannheim,Germany) and total RNA was prepared following the manufac-turer’s protocol. Total RNAwas resuspended in free-nuclease water.The A260/A280 ratios of the preparations were checked for purity,and RNA was quantified by measuring A260.

Information to prepare primers (Table 1 in Supplementarymaterial) to amplify cDNA fragments was obtained from thescreening previously described (2.2 section), or from partialsequences previously reported in NCBI GenBank (http://www.ncbi.nlm.nih.gov/) as in the case of onapn3a and onapn4 (EF103944 andEF103945 respectively). The total midgut RNAwas retrotranscribedusing the Super Script� II Reverse Transcriptase kit (Invitrogen,Carlsbad, CA, USA) following the manufacturer’s instructions andwith 0.5e1 mg of total RNA. PCR reactions were carried out withNetzyme� polymerase (Molecular Netline Bioproducts, Valencia,Spain) using a Mastercycler personal (Eppendorf, Hamburg,Germany) under the following conditions: 94 �C for 3min, 30 cyclesof 94 �C for 30 s, 55 �C for 30 s and 72 �C for 1 min, followed bya elongation step of 72 �C for 7 min. Amplified products were gel-purified and cloned into pGEM-T Easy vector (Promega) withligation overnight at 4 �C, and the resulting constructs weretransformed into competent heat-shock Escherichia coli DB3.1 cellsor electro-competent E. coli DH10B cells. Plasmids were purifiedwith High Pure Plasmid Isolation Kit from Roche and sequenced atIBMCP (Instituto de Biologia Molecular y Celular de Plantas,Valencia, Spain).

The full-length cDNAs sequences were obtained with SMART�RACE cDNA Amplification Kit (Clontech, Mountain View, CA, USA)following the manufacturer’s protocol, using RNA from a pool ofthree larval midguts. Primers based on the internal gene sequencespreviously amplified and sequenced were used to design primers

C.M. Crava et al. / Insect Biochemistry and Molecular Biology 40 (2010) 506e515508

for the RACE-PCR reactions (Table 1 in Supplementary material).Amplified products were gel-purified, cloned and sequenced.

2.4. Sequencing and bioinformatics analysis

The plasmids were sequenced usingM13 primers appropriate tothe cloning vector. Contigs were assembled with the SeqMansoftware (DNAstar, Madison, WI) to obtain full-length cDNAsequences. The sequences were translated into putative proteinsusing the EditSeq software (DNAstar). Structural domains of theputative proteins were analyzed with the Prosite program availableat the URL http://www.expasy.ch/prosite/. The presence of thesignal peptide and the GPI-anchor sequences was screened withthe SignalP program (http://www.cbs.dtu.dk/services/SignalP/)(Bendtsen et al., 2004) and the GPI-SOM program (http://gpi.unibe.ch/), respectively.

The homology of the protein sequences was analyzed usingClustal X software program (Thompson et al., 1997). All completelepidopteran APN protein sequences reported in GenBank andsequences with high homology to aminopeptidases annotated inthe assembled genome of B. mori (http://silkdb.org) were obtained.Single representative sequence of each APN group for each insectspecies was randomly picked from redundant sequences (identityhigher than 90%). A multiple alignment analysis was performedwith all selected and O. nubilalis APN sequences. The phylogenetictree was constructed with neighbor-joining method with 1000bootstrap resamplings with the Clustal X program. The clusteringnaming has been performed following mostly Herrero et al. (2005).Data about tissue-specific expression of the B. mori aminopepti-dases were obtained in silico from the B. mori Microarray Database(http://silkworm.swu.edu.cn/microarray, Xia et al., 2007).

2.5. Linkage mapping

AFLP analysis was conducted at CESAR, University of Melbourne,Australia, as outlined in Vos et al. (1995) with minor modifications(Lee, 2006). 500 ng of gDNA was digested with EcoRI and Tru1I;double-stranded Eco and Mse adapters were ligated to the restric-tion fragments with T4 ligase; the ligationmixtures were incubatedovernight at 4 �C and diluted 10 times with TE pH 8. First-stageamplifications employed the EcoþA and MseþC primers, and werethen diluted 10-fold for selective amplifications with three selec-tive bases on each of the IRD-700-labelled Eco and unlabelled Mseprimers. Selective amplified products were resolved by electro-phoresis in 6% polyacrylamide gels (SequaGel-6, National Diag-nostic, Atlanta, GE, USA) using the LiCor Global IR2 System (LiCorBiotechnology, Lincoln, NE, USA). Grandparents, parents andoffspring of Family 305 (2.1 section) were run on the same gel. AFLPgel imageswere saved in TIFF format (16 bits). AFLP polymorphismswere scored by hand from the LiCor printouts (on A3 papers).Presence (scored as 1) and absence (scored as 0) of polymorphicAFLP bands were entered into a Microsoft Excel spreadsheet forpreliminary linkage analysis. Thirty autosomes and the sex chro-mosome were identified as sets of nonrecombining AFLPs, sincethere is no crossing-over within chromosomes in femaleLepidoptera.

Linkage mapping of aminopeptidases was performed onwhole-genome-amplified DNAusing GenomiPhi DNA amplification kit (GEHealthcare, Buckinghamshire, UK) of 20 backcross progeny, parentsand grandparents of Family 305. The genes analyzed were onapn1,onapn2, onapn3b and onapn8. The primers used for each amplifi-cation (Table 1 in Supplementary material) produced ampliconswhich included one (onapn2 and onapn8) or two introns (in thecase of onapn1 and onapn3b genes). The gDNA amplification reac-tions were performed using the Expand High Fidelity PCR System

(Roche) under the following conditions: 94 �C for 2 min,10 cycles of94 �C for 15 s, annealing at 55 �C for 30 s, extension at 72 �C for3 min, followed by 20 cycles of 94 �C for 15 s, annealing at 55 �C for30 s, and extension at 72 �C for 3 min increasing 5 s for every cycle,and ending with a final extension step of 72 �C for 5 min. To assessthe identity of the different onapn amplicons, some of the PCR thereaction products were band purified using the High Pure PCRProduct Purification Kit (Roche) and sequenced. Intron size poly-morphismswere found only in the case of onapn2. For the other apngenes it was necessary to perform RFLP analyses. For this purpose,onapn1 amplicons were treated with ApaI restriction enzyme,onapn3b amplicons were digested with EcoRI, and onapn8 PCRproducts were treated with HinfI. Fragment size polymorphismpatterns in the 20 progeny were compared with previously-deter-mined AFLP scores in Family 305 for assignment of the genes tolinkage groups.

2.6. Developmental expression analysis

Total RNA was purified from at least three samples of 1 and 7days old single whole larvae and from single midguts of 14, 21 and28 days old larvae (corresponding to each one of the 5 larvalinstars), using TriPure reagent (Roche). Internal segments of theapn mRNA transcripts were amplified by RT-PCR, as describedabove (2.3 section), employing primers specific to each gene (Table1 in Supplementary material). A portion of a housekeeping genecoding for the O. nubilalis ribosomal protein subunit S3 (rps3,DQ988989) was RT-PCR amplified as a positive control and waterwas used as negative control. Each amplification was performed intriplicate. In all samples, the negative controls did not showamplification products.

Amplifications of gDNA with the same primer pairs, resulted inreaction products with different sizes from the ones obtained afterRT-PCR, due to a presence of introns. Therefore, amplicon sizeswere used to test of the absence of contaminant gDNA in thesamples. Amplified fragments from RNA or gDNA were sequencedto verify that correspond to the expected sequences.

2.7. Tissue expression localization using quantitative reverse-transcription PCR (qRT-PCR)

Three RNA samples were prepared from midgut, Malpighiantubules, fat body and carcass of fifth instar larvae. Larvae weredissected in MET buffer, isolated tissues were immediately snapfrozen in liquid nitrogen and total RNA was purified with TriPurereagent (Roche). Total RNA (2 mg) from each sample was treatedwith 2 ml of DNase I (Invitrogen) for 15 min at 25 �C to removecontaminant gDNA. DNase I was deactivated with 1 ml of EDTA25 mM and heating to 65 �C for 10 min. RNA (1.5 mg) was thenretrotranscribed into cDNA using the Super Script� II ReverseTranscriptase kit (Invitrogen), according to the protocol providedby the manufacturer. Contamination of gDNA in the RNA prepara-tions was checked by using total RNA as template in qRT-PCRreactions with aminopeptidase specific primers (Table 1 inSupplementary material). The primers were designed withPrimer3 software (http://frodo.wi.mit.edu/) to amplify productsless than 100 bp. ABI Prism 7000 Sequence Detection System(Applied Biosystems, Carlsbad, CA, USA) was used for qRT-PCRreactions with 12.5 mg of Power SYBR� Green PCR Master Mix(Applied Biosystems), 4.5 ml of water, 3 ml of primers mix (2.5 mMeach) and 5 ml of cDNA diluted 1:7 (final volume 25 ml). Thefollowing conditions were employed: 1 cycle at 50 �C for 2 min, 1cycle at 95 �C for 10 min, 40 cycles at 95 �C for 15 s and 60 �C for1 min. Dissociation curves analysis of the amplification productswere performed at the end of each PCR reaction with a dissociation

C.M. Crava et al. / Insect Biochemistry and Molecular Biology 40 (2010) 506e515 509

stage of 1 cycle at 60 �C to confirm that only the corresponding PCRproduct was amplified and detected. The housekeeping gene rps3was used as internal control to normalize RNA levels between thesamples and water was used for negative controls. The negativecontrols showed no amplification products. Each reaction wasperformed in triplicate. Data analysis of qRT-PCR was performedwith the SDS software V1.2. (Applied Biosystems). To maintainconsistency, the baseline was set automatically by the software.Differences in the CT (cycle threshold) between each aminopepti-dase gene and the rps3, (DCT), were calculated to normalize thedifferences in the amount of total nucleic acid added to cDNAreaction mixture. For the genes with expression in other tissuesthan midgut, the relative expression levels were calculated andtested for significance with the REST� software (Pfaffl et al., 2002),employing the midgut as calibrator. Differences were consideredsignificant when P < 0.05.

3. Results

3.1. Cloning of aminopeptidase isoforms from O. nubilalis

PCRwith degenerate primers designed from two conserved APNmotifs (AFPCYDEP and AGAMENWG) yielded amplicons from gDNAcontaining partial sequences of about 200 bp of onapn2, onapn3b,onapn8, each containing a single intron, and onpsa with intronabsent. An additional partial sequence of onapn1 was obtained asa second product of a RACE-PCR 30 reaction performed to amplifyonapn3b. These five partial sequences were used to design gene-specific primers for amplification of the entire coding sequences.The work was completed by incorporating the sequences of thereported EST of onapn3a and onapn4 (Coates et al., 2008).

Seven full-length aminopeptidase cDNAswere cloned from totalRNA isolated from midgut of fifth instar larvae of O. nubilalis.According to the results of the phylogenetic analysis the sevengenes were named onapn1 (EU878375), onapn2 (EU878376),onapn3a (FJ896130), onapn3b (FJ492806), onapn4 (FJ694793),onapn8 (FJ647796), and onpsa (FJ492807) and the relativesequences consisted of 3160, 3622, 3461, 3545, 3054, 3129 and2812 nucleotides, respectively. Analysis of cDNA sequences showed50-UTR sequences range from 7 to 109 bp, open reading frames(ORFs) of about 3 kb and 30-UTR sequences from 168 to 748 bplength. The onpsa sequence was an exception, since exhibiteda truncated ORF of 1668 bp and a 30-UTR of 1035 bp.

The inferred protein sequences (Fig. 1) ranged from 925 to 1017amino acids long, except the OnPSA protein that comprised 555amino acids. In silico analysis of the putative proteins identifiedAPN common motifs in homologous positions: a signal peptide inthe N-terminus, a GPI-anchor sequence in the C-terminus, the zinc-binding motif HEXXH(X)18E, and the GAMEN gluzincin amino-peptidase sequence (Albiston et al., 2004). Moreover, eachsequence exhibited several possible N-glycosylation sites (data notshown). The signal peptide and GPI-anchor motifs were absent inthe OnPSA protein. The lack of both signal sequences in the onpsagene suggested that was not a typical lepidopteran APN (Piggot andEllar, 2007) and could be classified by sequence similarity asa puromycin-sensitive aminopeptidase (PSA). Alignment with theB. mori (BGIBMGA011674) and D. melanogaster (NP728618) PSAproteins evidenced the lack of both signals in this class of insectaminopeptidases, although the protein of 555 amino acidsexpressed in O. nubilalis could be a truncated form since bothorthologous PSAs had an average length of 900 amino acids.

The percentage of identity among the APN proteins expressed inthe O. nubilalis midgut ranged from 74% to 25% (Table 2 inSupplementary material). The OnPSA sequence exhibited thelowest identity with the other OnAPNs (from 15 to 18%).

3.2. In silico analysis of homologous sequences toOnAPNs and OnPSA in B. mori

The annotated genome of B. mori was screened with the BLASTtool implemented in the Silkworm Genome Database for searchingorthologous sequences to the O. nubilalis aminopeptidasesdescribed in the present study. Sequence with an E-value close to0 and a score higher than 990 was found for each case, and itwas named according to the correspondent O. nubilalis amino-peptidase. The sequences BGIBMGA008017, BGIBMGA008060 andBGIBMGA011674 were named as bmapn2, bmapn4 and bmpsa. Thebmapn8 gene sequence was identified within the sequenceBGIBMGA008063. This sequence of 44.7 kb included not one buttwo aminopeptidase coding regions: the B. mori sequence orthol-ogous to the onapn8 and another aminopeptidase orthologous toH. armigera apn7 (EU328180). In the same way, the bmapn1 and thebmapn3 were identified on a single sequence of 24.2 kb(BGIBMGA008059). Although two onapn3 isoforms were cloned,only one sequence for the apn3was found in the genome of B. mori.The BmAPN3 protein was more similar to OnAPN3a, showing anidentity percentage of 64% while with OnAPN3b identity decreasedto 60%. Both identity values are in the range found between theother predicted individual O. nubilalis aminopeptidase sequencesand their orthologues in B. mori (about 60%).

Identity percentages among the O. nubilalis orthologousaminopeptidase proteins from B. mori varied from 24 to 40%, withthe exception of BmPSA that showed the lower amino acid identitywith the BmAPNs (from 20 to 23%) (Table 2 in Supplementarymaterial).

The five O. nubilalis orthologous apn genes found in the genomeof B. mori clustered all together in the scaffold with accessionnumber nscaf2889, located on the chromosome 9. The sequencesthat coded for the other APNs were found in the same scaffold,including the sequences for bmapn5 (BGIBMGA008062), bmapn6(BGIBMGA008061), and bmapn7 (BGIBMGA008063), according toour phylogenetic analysis. In the section of the scaffold thatcomprises these eight apns, all of them were transcribed in thesame DNA strand, except bmapn2 that was on the complementarystrand. The bmapn1 was the first, followed by bmapn3, bmapn4,bmapn6, bmapn5, bmapn8, bmapn7, and bmapn2, in this order. Thebmpsa sequence was found in the scaffold nscaf3031, located onchromosome 11, and no other aminopeptidase sequences weredetected in the same scaffold.

The B. mori microarray database on the SilkDB website, basedon early fifth instar larvae RNA, was used to screen the expres-sion levels of the O. nubilalis orthologous genes (Table 1). Nineprobes were reported to be related to bmapns or bmpsa, anda tenth probe (sw17741), classified as aminopeptidase but notdirectly linked with the orthologous aminopeptidase genes ofB. mori, was identified as bmapn3. Each probe was alignedwith the corresponding aminopeptidase coding region to confirmits identity. The bmapn2, bmapn3 and bmapn8 transcriptswere recognized for more than one probe. The sequencesBGIBMGA008059 and BGIBMGA008063 included both two apngenes (bmapn1 and bmapn3, and bmapn7 and bmapn8, respec-tively) and our analysis showed that the probe sw03042 wasrelated to bmapn1, the probe sw18255 was identified as bmapn3and the probes sw06103 and sw02792 as bmapn8. The micro-array data showed that the highest expression level of thebmapns occurred in the midgut, while in Malpighian tubules onlybmapn1 and bmapn8 were highly expressed and bmapn3 was juston the threshold of expression discrimination. Similarly, bmapn1expression levels were around the threshold in head and in theintegument. The bmpsa transcripts were widely found in all theanalyzed tissues.

Fig. 1. Clustal X alignment of aminopeptidase putative protein sequences from O. nubilalis. GAMEN and HEXXH(X)18E zinc-binding site motif are framed. Signal peptides and GPIanchor are underlined. Black shades indicate 100% of identity among amino acid sequences; grey ones indicate 80% identity while light-grey ones indicate 60% identity.

C.M. Crava et al. / Insect Biochemistry and Molecular Biology 40 (2010) 506e515510

Table 1Signal intensity units (S.I.U.) obtained frommicroarray analysis, using RNA from different B. mori tissues. Data extrapolated from Xia et al. (2007). Tissues with the S.I.U. higherthan 400 are highlighted in bold.

Probe ID CorrespondingB. mori gene

Midgut Malpighian tubules Fat body Gonads Silk glands Hemocyte Integument Head

sw03042 bmapn1 99934 (�9254) 1422 (�270) 167 (�50) 161 (�28) 82 (�16) 286 (�100) 598 (�110) 654 (�60)sw05220 bmapn2 2763 (�252) 66 (�15) 28 (�19) 18 (�14) 36 (�14) 101 (�73) 45 (�18) 52 (�12)sw04261 bmapn2 809 (�88) 18 (�19) �11 (�15) 5 (�7) 19 (�3) 14 (�7) �2 (�20) 22 (�14)sw10041 bmapn2 30274 (�2139) 271 (�66) 266 (�58) 42 (�30) 110 (�17) 69 (�22) 204 (�37) 396 (�80)sw17741 bmapn3 33099 (�3405) 318 (�55) 25 (�25) 32 (�17) 16 (�6) 77 (�34) 55 (�42) 3 (�26)sw18255 bmapn3 147035 (�10,534) 629 (�121) 42 (�17) 36 (�23) 64 (�12) 213 (�119) 83 (�24) 187 (�52)sw19250 bmapn4 57729 (�5841) 317 (�94) 89 (�54) 10 (�17) 18 (�4) 77 (�43) 120 (�26) 126 (�36)sw06103 bmapn8 6352 (�389) 2088 (�226) 3 (�8) 18 (�11) 20 (�4) 5 (�7) 13 (�12) 15 (�8)sw02792 bmapn8 711 (�126) 435 (�50) 54 (�16) 39 (�6) 115 (�16) 59 (�13) 65 (�37) 47 (�7)sw09454 bmpsa 1339 (�70) 1613 (�212) 758 (�40) 2510 (�253) 1296 (�75) 1653 (�158) 1588 (�129) 1991 (�159)

C.M. Crava et al. / Insect Biochemistry and Molecular Biology 40 (2010) 506e515 511

3.3. Phylogenetic analysis of the sequences

A phylogenetic tree (Fig. 2) was generated with lepidopterancomplete aminopeptidase protein sequences deposited in GenBankand Silkworm Genome Database, and the nearly-completesequences from S. litura fat-body APN (ABN04204) and Plutellaxylostella APN3 (AAF01259). Nine phylogenetic clusters of lepi-dopteran aminopeptidases were identified, adding four additionalclusters to those reported by Herrero et al. (2005). Each lepidop-teran species showed no more than one sequence for each cluster,with the exception of O. nubilalis for which two genes belonging to

Fig. 2. Phylogenetic tree derived from Clustal X alignment of complete Lepidoptera aminopewith S. litura APN8 (ABN04204) and P. xylostella APN3 (AAF01259) sequences, which werHelicoverpa armigera, Hp: Helicoverpa punctigera, Hv: Heliothis virescens, On: Ostrinia nubilalisSpodoptera exigua, Sl: Spodoptera litura, Ep: Epiphas postvittiana, Tn: Trichoplusia ni, Ld: Lymvalues are indicated for the principal nodes.

Class 3 have been cloned. Midgut O. nubilalis aminopeptidasescloned in the present work and their B. mori homologues wereassigned to Classes 1e4, 8 and PSA.

Classes 1e4 were the most represented in terms of numbers ofgenes reported in different species, with nine or more lepidopteranspecies included in each one. Class 5 was composed of only threesequences, while Classes 6 and 7 were formed by isoforms from B.mori and H. armigera. Regarding O. nubilalis, in the present work noorthologous isoforms belonging to Classes 5e7 have been detected,although they could be present in the genome. The determinationof Class 8 was supported by the cloning of the OnAPN8 mRNA that

ptidase protein sequences reported in GenBank and in Silkworm Genome Database, ande almost entire. Accession numbers of each amino acid sequence are indicated. Ha:, Of: Ostrinia furnacalis, Px: Plutella xylostella, Bm: Bombyx mori, Ms: Manduca sexta, Se:antria dispar, Aj: Achea janata, Pi: Plodia interpunctella, Cs: Chilo suppressalis. Bootstrap

C.M. Crava et al. / Insect Biochemistry and Molecular Biology 40 (2010) 506e515512

clustered together with S. litura fat-body APN and the BmAPN8defined in this paper.

3.4. Mapping the O. nubilalis aminopeptidase genes

PCR amplicons from onapn1, onapn2, onapn3b and onapn8 werescored for allelic polymorphisms for linkage group assignment inFamily 305. Amplicons from onapn2 were 500 or 535 bp, reflectingan allelic intron size polymorphism; both parents were heterozy-gous 500/535. The amplicon from onapn1 was 1030 bp; whendigested with ApaI one allele was uncut and the other produceda band of 970 bp (the smaller restriction fragment of expected size60 bp was not visible on the gel), and both parents were hetero-zygous 1030/970. The amplicon from onapn3b was 850 bp; whendigested with EcoRI one allele was uncut and the other producedbands of size 475 and 375; both parents were heterozygous 850/(475 þ 375). For these three loci, progeny occurred in the expected1:2:1 ratio (homozygote:heterozygote:homozygote). The maternalsegregation pattern could be deduced for the homozygousoffspring of both classes, enabling assignment of all three loci toAFLP Linkage Group 8.

The amplicon from onapn8 was 920 bp; when digested withXhoI one allele was uncut and the other produced bands of size 720and 200; only the father was heterozygous so a maternal segre-gation pattern could not be deduced. However the paternalsegregation pattern matched the pattern of onapn3 for 19 of 20progeny, enabling assignment of onapn8 to AFLP Linkage Group 8also. Ribosomal protein genes had been mapped in Family 305 toserve as anchor loci, and RpL24 was also found to map to AFLPLinkage Group 8.

3.5. Aminopeptidase genes expression during larval development

RT-PCR analysis of the aminopeptidase gene transcripts duringlarval development showed expression of all the studied genesthroughout the entire larval cycle (Fig. 3). The amplification prod-ucts showed the expected sizes of 399, 286, 391, 473, 421, 590, and

Fig. 3. RT-PCR expression analysis of O. nubilalis aminopeptidases during larvaldevelopment. Larval ages were 1 day (1d), 7 days (7d), 14 days (14d), 21 days (21d) and28 days (28d). Expression of the rps3 housekeeping gene is shown as a control.

560 bp for onapn1, onapn2, onapn3a, onapn3b, onapn4 onapn8 andonpsa, respectively. Sequencing of the amplified fragmentsconfirmed their identity. Amplification products were detectedfrom the first to the 28th day of life of the larvae. The amplificationof a 343 bp-length fragment of the control rps3 gene was obtainedin all samples.

3.6. Differential tissue expression

Midgut, Malpighian tubules, fat body and carcass larval tissueswere selected to be studied for expression of the aminopeptidasegenes by qRT-PCR (Fig. 4), using the housekeeping gene rps3 asinternal control. Dissociation curve analysis indicated singlesequence amplification for all genes. Results suggested the occur-rence of mRNA transcripts of each aminopeptidase gene describedin this work, in the midgut. The onapn4, the onapn8 and onapn3awere also expressed in Malpighian tubules, and the latter was alsodetected in the fat body. Expression of onpsa was found in all theanalyzed tissues. In this case, onpsa was expressed at the samelevels in midgut and in Malpighian tubules, while in the fat bodythe expression decreased to a third and in the carcass to one fifth.The onapn3a and onapn4 had a very low expression level in theMalpighian tubules relative to the midgut (about 700 and 400times, respectively), while the onapn8 was expressed in both theMalpighian tubules and the midgut, without a statistically signifi-cant difference. Expression of onapn3a detected in fat body wasmore than 2000 times lower than in the midgut.

4. Discussion

Seven cDNA sequences coding for aminopeptidases expressed inthemidgut of O. nubilalis have been cloned and sequenced and theirorthologous sequences have been identified in the genome of B.mori. Availability of the complete annotated genome of B. mori wasa useful tool for in silico identification of the Lepidoptera homolo-gous genes, despite numerous errors in the automatic annotation ofORFs. For example in the present study, the bmapn1 and the bmapn3were identified in a single predicted sequence (BGIBMGA008059),as well as the bmapn7 and bmapn8 that were annotated together aspart of the same single sequence (BGIBMGA008063). Indeed, Beland Escriche (2006) reported inconsistencies in the B. mori cad-herin annotated genomic sequence.

A database search for O. nubilalis aminopeptidases in the Gen-Bank produced four apn partial sequences, three of them reportedpreviously by Coates et al. (2008) (onapn1, onapn3a and onapn4)

Fig. 4. O. nubilalis aminopeptidase gene expression levels relative to the rps3 house-keeping gene in different larval tissues, measured by qRT-PCR. Vertical bars repre-sented the means � S.E. (N ¼ 3). Mal. Tub., Malpighian tubules.

C.M. Crava et al. / Insect Biochemistry and Molecular Biology 40 (2010) 506e515 513

and an additional one (onapn2, ABL01482). Moreover, a BLASTsearch performed in the GenBank EST database provided 32sequences derived from an O. nubilalismidgut EST library (Khajuriaet al., 2009). Screening of these EST sequences with the amino-peptidases described in this work found homologies for all classesof apns, but no psa homologous EST sequence was detected.

In the present study, whole aminopeptidase protein sequenceshave been used for phylogenetic analyses and the nomenclatureproposed by Herrero et al. (2005) was followed. The most recentanalysis on lepidopteran APNs (Angelucci et al., 2008) identified upto seven clusters, however the additional sequences obtained in thepresent work from O. nubilalis, with homologues identified in B.mori too, showed the occurrence of two additional ones. These newAPN8 and PSA classes are also supported by homologous partialsequences (PxAPN-G, EF579957 and PxAPN-E, EF579959) reportedin a P. xylostella larval midgut cDNA library (Baxter et al., 2008).

The typical features present in classical lepidopteranaminopeptidases were reviewed by Piggot and Ellar (2007). Thecharacteristic motif essential for their enzymatic activity, the zinc-binding motif HEXXH(X)18E (Hooper, 1994) and the GAMENsequence have been found in all the O. nubilalis and B. moriaminopeptidases described in the present study. Six out of sevenputative protein sequences (OnAPN1, OnAPN2, OnAPN3a,OnAPN3b, OnAPN4 and OnAPN8) were similar in size and includeda potential signal peptide at the N-terminal, a GPI-anchor signalsequence at the C-terminal, and showed a high similarity with thereported aminopeptidases N from Lepidoptera. A threonine-richsequence downstream of the C-terminal GPI signal sequence wasdescribed by Piggot and Ellar (2007) as a feature for Classes 1 and 3,and also was found in the APN2 of H. armigera (Angelucci et al.,2008). Our study confirmed this observation in the O. nubilalisand B. mori APN1 and APN3 sequences, but not in the APN2sequence.

The signal peptide motif and the GPI-anchor signal were absentfrom the OnPSA cDNA sequence. The putative protein was trun-cated and composed of 555 amino acids due to a premature stopcodon in the ORF, in contrast to its orthologue in B. mori where theprotein had 865 amino acids and to the other OnAPNs composed byan average of 950 amino acids. Although this truncated proteinshowed the typical APN structural motif, it is possible that it isa non-functional protein. In D. melanogaster, mutant flies witha deletion within the psa locus were viable, with normal body sizeand structure and also produced viable offspring (Schulz et al.,2001). Homology analysis with tBLASTx program showed that theOnPSA protein had higher similarity to the aminopeptidase fromthe puromycin-sensitive family than to the APNs. Therefore, iden-tity analyses revealed that the protein sequence was more related(about 40% identity) to the D. melanogaster PSA (AF327435) or tothe human PSA (NM006310) than to the lepidopteran APNs (about15% identity), even considering that these two proteins werecomposed of around 900 amino acids. The puromycin-sensitiveaminopeptidases are described as a family of mainly cytoplasmicproteins involved in a large number of different cellular processes(Tobler et al., 1997; Thompson et al., 1999).

Pairwise identity percentages between the APNs of O. nubilalisvaried from 25 to 39% and from 23 to 40% for the BmAPNs, exceptfor BmAPN7 which the identity decreased to 13%. The amino acididentity increased within the classes ranging from 50% (whenphylogenetically distant species are compared) to 95% (for the samegenus species). As differing from other species of Lepidoptera, inO. nubilalis two isoforms of apn3 have been found and cloned.The amino acid identity between these two isoforms (74%) and thehigher identity of OnAPN3a (96%) versus OnAPN3b (74%) to theOstrinia furnacalis APN3, suggest that the sequences are not allelesof the same locus but rather they are transcribed from different loci.

A homology search with the Blast tool on the Silkworm GenomicDatabase using both apn3 sequences detected only a single genebelonging to the APN class 3. Probably, a duplication event occurredspecifically in O. nubilalis for APN3 (or maybe in other phyloge-netically related species). In fact, Baxter et al. (2008) evidencedpossible gene duplication when they reported in P. xylostella twodifferent partial sequences with similarity to Class 4. Also, the sameauthors reported an EST clone from P. xylostella with homology toClass 3 but consistently different from the APN3 previously repor-ted by other group (Nakanishi et al., 2002). The similarity insequence amongst the aminopeptidases belonging to each one ofthe clustered groups is higher than within the aminopeptidases ofeach one of the species. This seems to suggest that APNs are derivedfrom multiple gene duplications as has been also proposed byChang et al. (1999) and Angelucci et al. (2008) that claimedaminopeptidases in Lepidoptera derived from gene duplicationevents in ancestral lepidopteran. The possible APN duplicationevents are supported by the structural genome grouping in tandemof the aminopeptidase genes. In fact, the apn genes are clustered ina region of almost 150 kb on chromosome 9 of B. mori, and syntenywas observed among the O. nubilalis apns mapped in the presentwork. Also, data reported from P. xylostella (Baxter et al., 2008)indicate that aminopeptidases are located in the same linkagegroup. Chromosome 9 of B. mori also contains RpL24, thus linkagebetween this anchor locus and the APN cluster is conserved inB. mori and O. nubilalis. The PSA is the most phylogenetically distantprotein of the studied lepidopteran aminopeptidase family, andthe position of psa gene in a different linkage group from the otheraminopeptidases in B. mori (chromosome 11) and P. xylostella(Baxter et al., 2008), suggests a chromosomal translocation duringthe evolution from the lepidopteran ancestor.

The mRNA transcripts for the aminopeptidases expressed in themidgut of O. nubilalis and studied in this work were detectedthroughout the whole larval developmental cycle. In Epiphyaspostvittana, the expression of the aminopeptidases of classes from 1to 5 was detected in all larval stages, and, in lower proportion, inthe eggs, pupae and adults (Simpson et al., 2008). Similarly,H. armigera apns from Classes 1 to 4were expressed at similar levelsin all larval stages (Angelucci et al., 2008) and the same results weredescribed for Helicoverpa punctigera apns from Classes 1 to 3,although northern blot analysis revealed a developmental regula-tion, principally for apn Class 1 (Emmerling et al., 2001). The APNsexpression throughout the whole larval cycle is probably relatedwith the need of nearly constant eating to store energy andmetabolites for the growth and the metamorphosis.

Expression of lepidopteran aminopeptidases has been generallyobserved in midgut tissues (Piggot and Ellar, 2007). Similarly, thefirst O. nubilalis apn EST sequences were cloned from a midgutlibrary (Coates et al., 2008). The midgut expression of all testedaminopeptidases in O. nubilalis and B. mori is confirmed from ouranalysis of the mRNA transcripts levels. However, the midgut wasnot the only tissue where aminopeptidases were expressed. Forexample, onapn8 was expressed in Malpighian tubules at the samelevels than in the midgut, and in this tissue, a low expression ofonapn3a and onapn4 was also found. The expression of onapn3a infat body was minor. In the carcass, a lack of expression of any of thestudied apns was observed. Only onpsa showed a general expres-sion in all analyzed tissues. Expression profiles of bmapns obtainedfrommicroarrays data (Xia et al., 2007) agreed with the expressionpatterns observed for O. nubilalis. All the bmapns homologous tothose described in O. nubilalis were strongly expressed in midgut,and bmapn8 was also expressed in Malpighian tubules. Expressionof bmapn2, bmapn3 and bmapn4 genes seemed to be midgutspecific, while bmapn1 transcripts were detected also in theMalpighian tubules at consistent levels. On the other hand, the

C.M. Crava et al. / Insect Biochemistry and Molecular Biology 40 (2010) 506e515514

widespread expression of bmpsa was also confirmed. Consistentwith these observations, in Trichoplusia ni larvae apn1 and apn4expression was clearly detected in Malpighian tubules at lowerlevels than in the midgut and no aminopeptidase activity wasdemonstrated in fat body, carcass and salivary glands (Wang et al.,2005). On the contrary, the expression of the S. litura Class 8 apnwas described as specific for fat body (Budatha et al., 2007). InE. postvittana, the expression of apns belonging to groups from 1 to5 was detected in all the tested tissues (midgut, hindgut, fat body,Malpighian tubules and carcass) although the highest expressionwas found in themidgut for all of them (Simpson et al., 2008). Asidefrom the high expression levels detected in the lepidopteranmidgut, where APNs could play an important digestive role, itseems that the specific tissue expression in the larva vary fromspecies to species. Differential gut expression of aminopeptidasesfrom H. armigera has been demonstrated in larvae fed withdifferent diets (Chougule et al., 2005). As Malpighian tubulescollaborate with the larval gut in the adsorption of nutritivesubstances (Ogutcu et al., 2005), probably the differential expres-sion of APNs from different clusters in the lepidopteran speciescould be linked to the components of the diets. The apn expressionin midgut and Malpighian tubules could resemble their prevalentexpression in the corresponding mammal tissues which are theintestine (where APNs act as digestive enzymes) and the kidney(where APNs play a role activating or inactivating hormones)(Vlahovic and Stefanovic, 1998). The expression of the psa in everytissue analyzed in O. nubilalis and B. mori, and the absence of thesignal peptide and the GPI-anchor signal, suggested for this proteina function different from that of the rest of midgut aminopepti-dases. Indeed, the human PSA is similarly ubiquitously expressedand its function has been associated to the cell cycle-regulatingproteolysis amongst other processes (Tobler et al., 1997; Thompsonet al., 1999).

In Lepidoptera, midgut membrane APNs have been widelystudied because they have been proposed as toxin-binding proteinsinvolved in the mode of action of the Bt toxins (Bravo et al., 2007)and at least one protein from the groups one to five was reported tointeract with Cry toxins (Piggot and Ellar, 2007). A BmAPN1 func-tional analysis suggested a role in the irreversible binding of theCry1Ab toxin to the brush border membrane vesicles (Ibiza-Palacios et al., 2008). In addition, ligand blot analyses have shownthat at least three putative APN proteins can bind Cry1A toxins in O.nubilalis (Hua et al., 2001).

Our work provides the characterization of seven midgut specificaminopeptidases in O. nubilalis and B. mori and their expressionpattern. The phylogenetic analysis has revealed that aminopepti-dases in the Lepidopteran larvae midguts cluster in at least ninegroups. Furthermore, linkage studies of the onapn genes and theanalysis of the orthologous genes present in the genome of B. moriprovided strong evidence for gene clustering of Lepidopteranaminopeptidases, probably deriving from multiple gene duplica-tions. The APN identifications will facilitate further studies tocharacterize the Bt toxin-APN binding interactions, providing awayto understand the development of the resistance to B. thuringiensis,which is crucial to design appropriate strategies for the pestresistance management.

Acknowledgements

The present project was funded by EU Fifth Framework Project“ProBenBt” (QLK3-CT-2002-01969). Additional support wasprovided by the Generalitat Valenciana (GVARVIV2007-090 andACOMP/2009/313), by the Spanish Ministerio de Educación yCiencia (AGL2006-11914), by European FEDER funds, by CESAR,a Special Research Centre of the Australian Research Council, and by

the Max-Planck-Gesellschaft. M.C. Crava was funded by a fellow-ship V Segles from the University of Valencia (UV-BVS-07-2083),Y. Bel was financed by the Spanish Ministerio de Educación yCiencia (AGL2006-11914), and S.F. Lee was supported by theUniversity of Melbourne and the Max-Planck-Gelsellschaft. Thanksto Prof. Dr. Ingolf Schuphan and Dr. Christiane Saeglitz of RWTHUniversity, Aachen, for support of the mapping project, and to Dr.Heiko Vogel and Domenica Schnabelrauch of MPI Chemical Ecologyfor EST sequencing.

Appendix. Supplementary material

Supplementary data associated with this article can be found inthe online version at doi:10.1016/j.ibmb.2010.04.010.

References

Albiston, A.L., Ye, S., Chai, S.Y., 2004. Membrane bound members of the M1 familymore than aminopeptidases. Protein Pept. Lett. 11, 491e500.

Angelucci, C., Barrett-Wilt, G.A., Hunt, D.F., Akhurst, E.J., East, P.D., Gordon, K.H.,Campbell, P.M., 2008. Diversity of aminopeptidases, derived from four lepi-dopteran gene duplications, and polycalins expressed in the midgut of Heli-coverpa armigera: identification of proteins binding the d-endotoxin, Cry1Ac ofBacillus thuringiensis. Insect Biochem. Mol. Biol. 38, 685e696.

Baxter, S.W., Zhao, J.Z., Shelton, A.M., Vogel, H., Heckel, D.G., 2008. Genetic mappingof Bt-toxin binding proteins in a Cry1A-toxin resistant strain of diamondbackmoth Plutella xylostella. Insect Biochem. Mol. Biol. 38, 125e135.

Bel, Y., Escriche, B., 2006. Common genomic structure for the Lepidoptera cadherin-like genes. Gene 381, 71e80.

Bendtsen, J.D., Nielsen, H., von Heijne, G., Brunak, S., 2004. Improved prediction ofsignal peptides SignalP 3.0. J. Mol. Biol. 340, 783e795.

Bravo, A., Gill, S.S., Soberon, M., 2007. Mode of action of Bacillus thuringiensis Cryand Cyt toxins and their potential for insect control. Toxicon 49, 423e435.

Budatha, M., Meur, G., Dutta-Gupta, A., 2007. A novel aminopeptidase in the fatbody of the moth Achaea janata as a receptor for Bacillus thuringiensis Cry toxinsand its comparison with midgut aminopeptidase. Biochem. J. 405, 287e297.

Chang, W.X., Gahan, L.J., Tabashnik, B.E., Heckel, D.G., 1999. A new aminopeptidasefrom diamondback moth provides evidence for a gene duplication event inLepidoptera. Insect Mol. Biol. 8, 171e177.

Chougule, N.P., Giri, A.P., Mohini, N.S., Gupta, V.S., 2005. Gene expression patterns ofHelicoverpa armigera gut proteases. Insect Biochem. Mol. Biol. 35, 355e367.

Coates, B.S., Sumerford, D.V., Hellmich, R.L., Lewis, L.C., 2008. Mining an Ostrinianubilalis midgut expressed sequence tag (EST) library for candidate genes andsingle nucleotide polymorphisms (SNPs). Insect Mol. Biol. 17, 607e620.

Duan, J., Li, R., Cheng, D., Fan, W., Zha, X., Cheng, T., Wu, Y., Wang, J., Mita, K.,Xiang, Z., Xia, Q., 2009. SilkDB v2.0 a platform for silkworm (Bombyx mori)genome biology. Nucleic Acids Res. 38, D453eD456.

Emmerling, M., Chandler, D., Sandeman, M., 2001. Molecular cloning of three cDNAsencoding aminopeptidases from the midgut if Helicoverpa punctigera, theAustralian native budworm. Insect Biochem. Mol. Biol. 31, 899e907.

Ferré, J., Van Rie, J., 2002. Biochemistry and genetics of insect resistance to Bacillusthuringiensis. Annu. Rev. Entomol. 47, 501e533.

Flanagan, R.D., Cao-Guo, Y., Mathis, J.P., Meyer, T.E., Shi, X.M., Siquiera, H.A.A.,Siegfried, B.D., 2005. Identification, cloning and expression of a Cry1Ab cad-herin receptor of European corn borer Ostrinia nubilalis (Hübner) (Lepidoptera:Crambidae). Insect Biochem. Mol. Biol. 35, 33e40.

Garczynski, S.F., Adang, M.J., 1995. Bacillus thuringiensis CrylA(c) d-endotoxinbinding APN in the Manduca sexta midgut has a glycosyl-phosphatidylinositolanchor. Insect Biochem. Mol. Biol. 25, 409e415.

Gill, M., Ellar, D.J., 2002. Transgenic Drosophila reveals a functional in vivo receptorfor the Bacillus thuringiensis toxin Cry1Ac1. Insect Mol. Biol. 11, 619e625.

Herrero, S., Gechev, T., Bakker, P.L., Moar, W.J., de Maagd, R.A., 2005. Bacillus thur-ingiensis Cry1Ca-resistant Spodoptera exigua lacks expression of one of fouraminopeptidases N genes. BMC Genomics 6, 96.

Hooper, N.M., 1994. Families of zinc metalloproteases. FEBS Lett. 354, 1e6.Hua, G., Masson, L., Jurat-Fuentes, J.L., Schwab, G., Adang, M.J., 2001. Binding

analysis of Bacillus thuringiensis Cry d-endotoxins using brush bordermembrane vesicles of Ostrinia nubilalis. Appl. Environ. Microbiol. 67, 872e879.

Ibiza-Palacios, M.S., Ferré, J., Higurashi, S., Miyamoto, K., Sato, R., Escriche, B., 2008.Selective inhibition of binding of Bacillus thuringiensis Cry1Ab toxin to cad-herin-like and aminopeptidase proteins in brush-border membranes anddissociated epithelial cells from Bombyx mori. Biochem. J. 409, 215e221.

Jurat-Fuentes, J.L., Adang, M.J., 2004. Characterization of a Cry1Ac-receptor alkalinephosphatase in susceptible and resistant Heliothis virescens larvae. Eur. J. Bio-chem. 271, 3127e3135.

Khajuria, C., Zhu, Y.C., Chen, M.S., Buschman, L.L., Higgins, R.A., Yao, J., Crespo, A.L.B.,Siegfried, B.D., Muthukrishnan, S., Zhu, K.Y., 2009. Expressed sequence tagsfrom larval gut of the European corn borer (Ostrinia nubilalis): exploringcandidate genes potentially involved in Bacillus thuringiensis toxicity andresistance. BMC Genomics 10, 286.

C.M. Crava et al. / Insect Biochemistry and Molecular Biology 40 (2010) 506e515 515

Knight, P.J., Crickmore, N., Ellar, D.J., 1994. The receptor for Bacillus thuringiensisCrylA(c) delta-endotoxin in the brush border membrane of the lepidopteranManduca sexta is aminopeptidase N. Mol. Microbiol. 11, 429e436.

Koziel, M.G., Beland, G.L., Bowman, C., Carozzi, N.B., Crenshaw, R., Crossland, L.,Dawson, J., Desai, N., Hill, M., Kadwell, S., Launis, K., Lewis, K., Maddox, D.,McPherson, K., Meghji, M.R., Merlin, E., Rhodes, R., Warren, G.W., Wright, M.,Evola, S.V., 1993. Field performance of elite transgenic maize plants expressing aninsecticidal protein derived fromBacillus thuringiensis. Biotechnology 11,194e200.

Lee, Siu Fai, 2006. Genome mapping in Lepidoptera. PhD Thesis, Department ofGenetics, University of Melbourne, Melbourne, Australia.

Nakanishi, K., Yaoi, K., Nagino, Y., Hara, H., KitamiAtsumi, S., Miura, N., Sato, R., 2002.Aminopeptidase N isoforms from the midgut of Bombyx mori and Plutellaxylostella e their classification and the factors that determine their bindingspecificity to Bacillus thuringiensis Cry1A toxin. FEBS Lett. 9, 215e220.

Ogutcu, A., Suludere, Z., Uzunhisarcikli, M., Kalender, Y., 2005. Effects of Bacillusthuringiensis on Malpighian tubule cells of Thaumetopoea pityocampa (Lepi-doptera: Thaumetopoeidae) larvae. Folia Biol. (Krakow) 53, 7e11.

Pfaffl, M.W., Horgan, G.W., Demple, L., 2002. Relative expression software tool(REST�) for group-wise comparison and statistical analysis of relative expres-sion results in real-time PCR. Nucleic Acids Res. 30, e36.

Piggot, C.R., Ellar, D.J., 2007. Role of receptors in Bacillus thuringiensis crystal toxinactivity. Microbiol. Mol. Biol. Rev. 71, 255e281.

Rajagopal, R., Sivakumar, S., Agrawal, N., Malhotra, P.M., Bhatnagar, R.K., 2002.Silencing of midgut aminopeptidase N of Spodoptera litura by double strandedRNA establishes its role as Bacillus thuringiensis receptor. J. Biol. Chem. 277,46849e46851.

Schulz, C., Perezgasga, L., Fuller, M.T., 2001. Genetic analysis of dPsa, the Drosophilaorthologous of puromycin-sensitive aminopeptidase, suggests redundancy ofaminopeptidases. Dev. Genes Evol. 211, 581e588.

Simpson, R.M., Poulton, J., Markwick, N.P., 2008. Expression levels of aminopepti-dase-N genes in the lightbrown apple moth, Epiphyas postvittana. Insect Sci. 15,505e512.

Shimomura, M., Minami, H., Suetsugu, Y., Ohyanagi, H., Satoh, C., Antonio, B.,Nagamura, Y., Kadono-Okuda, K., Kajiwara, H., Sezutsu, H., Nagaraju, J.,Goldsmith, M.R., Xia, Q., Yamamoto, K., Mita, K., 2009. KAIKObase: an integratedsilkworm genome database and data mining tools. BMC Genomics 10, 486.

Terra, W.R., Ferreira, C., 1994. Insect digestive enzymes: properties compartmen-talization and function. Comp. Biochem. Phys. 109, 1e62.

Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G., 1997. TheCLUSTAL_X windows interface: flexible strategies for multiple sequence align-ment aided by quality analysis tools. Nucleic Acids Res. 25, 4876e4882.

Thompson, M.W., Tobler, A., Fontana, A., Hersh, L.B., 1999. Cloning and analysis ofthe gene for the human puromycin-sensitive aminopeptidase. Biochem. Bio-phys. Res. Commun. 258, 234e240.

Tobler, A.R., Constam, D.B., Schmitt-Graff, A., Malipiero, U., Schlapbach, R.,Fontana, A., 1997. Cloning the human puromycin-sensitive aminopeptidase andevidence for expression in neurons. J. Neurochem. 68, 889e897.

Vlahovic, P., Stefanovic, V., 1998. Kidney ectopeptidases. Structure, functions andclinical significance. Pathol. Biol. 46, 779e786.

Vos, P., Hogers, R., Bleeker, M., Reijans, M., van de Lee, T., Hornes, M., Frijters, A.,Pot, J., Peleman, J., Kuiper, M., Zabeu, M., 1995. AFLP: a new technique for DNAfingerprinting. Nucleic Acids Res. 23, 4407e4414.

Wang, P., Zhang, X., Zhang, J., 2005. Molecular characterization of four midgutaminopeptidase N isozymes from the cabbage looper, Trichoplusia ni. InsectBiochem. Mol. Biol. 35, 611e620.

Wyniger, R., 1974. Insektenzucht. Methoden der Zucht und Haltung von Insektenund Milben im Laboratorium. Verlag Eugen Ulmer, Stuttgart, Germany.

Xia, Q., Cheng, D., Duan, J., Wang, G., Cheng, T., Zha, X., Liu, C., Zhao, P., Dai, F.,Zhang, Z., He, N., Zhang, L., Xiang, Z., 2007. Microarray-based gene expressionprofiles in multiple tissues of the domesticated silkworm, Bombyx mori.Genome Biol. 8, R162.

Zhang, S., Cheng, H., Gao, Y., Wang, G., Liang, G., Wu, K., 2009. Mutation of anaminopeptidase N gene is associated with Helicoverpa armigera resistance toBacillus thuringiensis Cry1Ac toxin. Insect Biochem. Mol. Biol. 39, 421e429.