ARTICLE IN PRESS

Journal of Insect Physiology 53 (2007) 1112–1124

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Identification of midgut microvillar proteins from Tenebrio molitor andSpodoptera frugiperda by cDNA library screenings with antibodies

A.H.P. Ferreiraa,1, P.T. Cristofolettia,1, D.M. Lorenzinic,2, L.O. Guerraa, P.B. Paivab,M.R.S. Brionesb, W.R. Terraa, C. Ferreiraa,�

aDepartamento de Bioquımica, Instituto de Quımica, Universidade de Sao Paulo, C.P. 26077, Sao Paulo 05513-970, BrasilbDepartamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de Sao Paulo, Rua Botucatu 862,

04023-062 Sao Paulo, BrasilcDepartamento de Parasitologia, Instituto de Ciencias Biomedicas, Universidade de Sao Paulo, Av. Lineu Prestes 1374, 05508-900 Sao Paulo, Brasil

Received 2 April 2007; received in revised form 29 May 2007; accepted 5 June 2007

Abstract

The objective of this study was to identify midgut microvillar proteins in insects appearing earlier (Coleoptera) and later (Lepidoptera)

in evolution. For this, cytoskeleton-free midgut microvillar membrane from Spodoptera frugiperda (Lepidoptera) and Tenebrio molitor

(Coleoptera) were used to raise antibodies. These were used for screening midgut cDNA expression libraries. Positive clones were

sequenced, assembled and searched for similarities with gene/protein databases. The predicted midgut microvillar proteins from

T. molitor were: cockroach allergens (unknown function), peritrophins (peritrophic membrane proteins), digestive enzymes

(aminopeptidase, a-mannosidase) and unknown proteins. Predicted S. frugiperda midgut proteins may be grouped into six classes: (a)

proteins involved in protection of midgut (thioredoxin peroxidase, aldehyde dehydrogenase, serpin and juvenile hormone epoxide

hydrolase); (b) digestive enzymes (astacin, transporter-like amylase, aminopeptidase, and carboxypeptidase); (c) peritrophins; (d)

proteins associated with microapocrine secretion (gelsolin, annexin); (e) membrane-tightly bound-cytoskeleton proteins (fimbrin,

calmodulin) and (f) unidentified proteins. The novel approach is compared with others and microvillar function is discussed in the light

of the predicted proteins.

r 2007 Elsevier Ltd. All rights reserved.

Keywords: Microvillar membrane; Sequencing; Antibody screening; EST; Coleoptera; Lepidoptera; Digestion

1. Introduction

The insect midgut cell microvillus is homologous to thatdescribed in vertebrates and reviewed by Bement andMooseker (1996). Thus, a bundle of parallel actin fila-ments cross-linked by actin-bundling proteins like fimbrinand villin forms the core of a microvillus. Lateral sidearms (composed of myosin I and calmodulin) connect thesides of the actin bundle to the overlying plasmamembrane.

e front matter r 2007 Elsevier Ltd. All rights reserved.

sphys.2007.06.007

ing author. Tel.: +55 11 3091 2180; fax: +55 11 3091 2186.

ess: clfterra@iq.usp.br (C. Ferreira).

ution of these authors is equal.

ress: Centro de Biotecnologia, Universidade Federal do

Sul, Caixa Postal 15005, Porto Alegre 91501-970, Brasil.

Insect midgut microvilli were isolated for the first time byFerreira and Terra (1980) from an insect midgut having asingle cell type (midgut caeca from an early divergingDiptera) using a differential calcium (magnesium) precipita-tion technique (Schmitz et al., 1973) developed for mammals.A few months later, Hanozet et al. (1980) used the sametechnique to isolate microvilli from the columnar (principal)cell of the midgut of lepidopteran larvae. Nevertheless, thelack of electron microscopy monitoring could not rule outthat the preparation was contaminated with membranes ofgoblet cells, the other cells forming the midgut of lepidopter-ans. Later on, Cioffi and Wolfsberger (1983) fractionatedlepidopteran midgut cells with an ultrasound technique andwere able to isolate both microvilli from columnar cells andthose from goblet cells. Santos et al. (1986) compared severalprocedures to prepare microvilli from lepidopteran midgut

ARTICLE IN PRESSA.H.P. Ferreira et al. / Journal of Insect Physiology 53 (2007) 1112–1124 1113

cells with electron microscopy monitoring. After this paper,and complementary data from Eisen et al. (1989), differentialprecipitation, mainly as modified by Wolfersberger et al.(1987), became the method of choice to prepare microvillifrom columnar cells of lepidopteran midguts. In addition toearly diverging Diptera and Lepidoptera, differential pre-cipitation has also been used to isolate microvilli frommidgut cells from other insect taxa, such as Dictyoptera(Parenti et al., 1986), Coleoptera (Ferreira et al., 1990;Reuveni et al., 1993) and late diverging Diptera (Lemos andTerra, 1992).

Microvilli preparations have been used to show thatmicrovillar integral digestive enzymes vary among differenttaxa. Most frequently they are: aminopeptidase, alkalinephosphatase, carboxypeptidase, dipeptidase, and a- gluco-sidase (Terra and Ferreira, 1994). Microvilli preparationshave also been used to reveal amino acid symporters (Terraet al., 2006). Ion transporters (Pullikuth et al., 2003) andaquaporins (Le Caherec et al., 1997) have been found ininsect apical cell membranes and are also supposed to bemicrovillar proteins. In addition to those studies, the searchfor Bt toxin receptors has led to the identification of severallepidopteran midgut microvillar proteins like aminopepti-dases (Knight et al., 1994) and cadherins (Vadlamudi et al.,1995). More recently, proteomic analysis of lepidopteranmidgut microvillar preparations found previously de-scribed digestive enzymes and identified several otherproteins like ABC transporters, V-ATPAse, actin, cadher-ins, afadin, etc. (McNall and Adang, 2003; Krishna-moorthy et al., 2007).

Microvilli prepared by differential precipitation methodsare free from contaminants from other cells (like gobletcells in the case of lepidopterans), but still contain most ofthe microvillus skeleton (see micrographs accompanyingthe above cited papers). Thus, a protein identified inmicrovilli preparations to be assigned to the microvillarmembrane must have its occurrence in the cytoskeletonruled out. This trouble may be avoided by preparingmicrovillar membranes free from cytoskeleton.

There are several methods to separate the microvillarmembrane from the enclosed cytoskeleton of mammalmidgut cells (Critchley et al., 1975; Carlsen et al., 1983;Hopfer et al., 1983; Riendeau et al., 1986). These methodswere tested in insects and a successful procedure wasdeveloped for cytoskeleton removal from insect midgutmicrovilli, resulting in purified membranes with negligibleamounts of cytoskeleton and with a small contaminationby basolateral membranes (Coleoptera and Diptera, seeJordao et al., 1995; Lepidoptera, Capella et al., 1997). As inthe case of mammals, the purified microvillar membranesof insects have a 1.5–3-fold enrichment in marker enzymesover the microvilli preparation. Also similar to mammals(Proulx, 1991), the insect microvillar membrane density isproportional to the protein–lipid mass ratio (Jordao et al.,1995; Capella et al., 1997).

The physiology of midgut microvillar membrane mayvary with position along the midgut and among insect taxa,

including differences in terminal digestion, chemicaldefenses, ion homeostasis, signaling, and secretory me-chanisms. The midgut microvillar proteins are expected toreflect this complexity. Insect midgut microvillar mem-brane densities vary widely, with insects appearing later inevolution (more-derived insects) having denser membranes(and hence a higher protein content) than insects appearingearlier in evolution (less-derived insects, Terra et al., 2006).Although a higher protein content does not necessarilymean a richer variety of proteins, there is evidencesupporting this. Sodium dodecyl sulfate–polyacrylamidegel electrophoresis (SDS–PAGE) of midgut microvillarproteins of a coleopteran (membrane density 1.08–1.10)resolves fewer clearly visible bands than in the case of alepidopteran or a dipteran (membrane density 1.14–1.16,Jordao et al., 1995; Capella et al., 1997). If it is indeed truethat more-derived insects have a greater variety ofmicrovillar membrane proteins than less-derived insects,the midgut cell surface would appear to play a moresophisticated role in more-derived than in less-derivedinsects. The identification of microvillar proteins is, thus, astep forward in understanding microvillar function.This study describes the immunoscreening of Tenebrio

molitor and Spodoptera frugiperda expression midgutcDNA libraries with antibodies raised against microvillarproteins. Sequences obtained here were used in conjunctionwith previously published data to identify microvillarfunctions. This novel approach complements others.

2. Material and methods

2.1. Animals

Stock cultures of T. molitor were maintained undernatural photoregime conditions on wheat bran at 24–26 1Cand 70–75% r.h. Larvae of both sexes (each weighingabout 0.12 g), having midguts full of food, were used.

S. frugiperda (Lepidoptera: Noctuidae) were laboratoryreared according to Parra (1986). The larvae wereindividually contained in glass vials with a diet based onkidney bean (Phaseolus vulgaris), wheat germ, yeast andagar and were maintained under a natural photoregime(summer, 14L: 10D; winter, 10L: 14D) at 25 1C. Adultswere fed a 10% honey solution. Fifth (last) instar larvae ofboth sexes were used in the experiments.Larvae of S. frugiperda and T. molitor were immobilized

by placing them on ice. Their guts were dissected in cold125mM NaCl for S. frugiperda and 342mM NaCl forT. molitor, and the midgut tissue was pulled apart. Midguttissue, after being rinsed thoroughly with saline, was storedat �70 1C until use.

2.2. Chemicals

The Wizard Plasmid Miniprep System and thepGEM-T Easy Vector plasmid kits were purchased fromPromega (Madison, WI); the DNA gel extraction kit was

ARTICLE IN PRESSA.H.P. Ferreira et al. / Journal of Insect Physiology 53 (2007) 1112–11241114

from QIAGEN Inc. (Santa Clarita, CA); agar, agarose andoligonucleotides were from Invitrogen (Carlsbad, CA) anddNTPs, modification and restriction enzymes from NewEngland Biolabs (Ipswich, MA). All other chemicals werepurchased from Merck (Darmstadt, Germany) or Aldrich-Sigma (USA) unless otherwise stated.

2.3. Purification of microvillar membrane proteins from

midgut

Microvilli were isolated from midgut tissue with aprocedure derived from that of Schmitz et al. (1973). Thetissue was homogenized with Potter–Elvehjem homogeni-zer. The homogenization buffer used was 50mM mannitolin 2mM Tris-HCl buffer, pH 7.1 and the homogenate waspassed through 45-mm-pore nylon net. The filtrate wasmade of 10mM CaCl2 (T. molitor) or 12mM MgCl2(S. frugiperda). After 15min with periodic stirring, thesuspension was centrifuged. The following sequentialfractions were collected: P1, pellet obtained by centrifugingat 2800g (T. molitor) or 2300g (S. frugiperda) for 10min; S1(supernatant) and P2 (pellet) resulting after centrifugationat 15,500g, (T. molitor) or 12,000g (S. frugiperda) for15min; P2 was resuspended in homogenization mediumwith the aid of a Potter–Elvehjem homogenizer in onevolume of a cold 10mM CaCl2 solution (T. molitor) or12mM MgCl2 (S. frugiperda). After centrifugation, thefollowing fractions were obtained: P3, pellet at 2800g

(T. molitor) or 2300g (S. frugiperda) for 15min; P4(microvilli) and S2, pellet and supernatant at 15,500g

(T. molitor) or 12,000g (S. frugiperda), for 15min. Allcentrifugations were carried out at 4 1C. The preparationswere maintained at �20 1C until used.

Purification of microvillar membranes, which consists infreeing the microvillar membranes from the enclosedcytoskeletal elements, was performed as described byJordao et al. (1995) and Capella et al. (1997). The fractionP4 described above was suspended in a fresh solution of1M Tris-HCl buffer, pH 7.0. After placing for 1 h on icewith periodic stirring, the sample was diluted to 50mMTris with cold distilled water and centrifuged (4 1C) at19,000g (S. frugiperda) or 25,000g (T. molitor) for 30min.The resulting pellet (purified microvillar membranes, P5)was suspended in 17mM Tris buffer, pH 7.0, containing10mM NaCl.

Aminopeptidase and g-glutamyl-transferase activitieswere used as microvillar membrane markers (Ferreiraet al., 1990; Jordao et al., 1995; Capella et al., 1997).

2.4. Preparation of soluble midgut contents and peritrophic

membrane from T. molitor

The peritrophic membrane and its contents werehomogenized in double distilled water with the aid of aPotter–Elvehjem homogenizer and centrifuged at 25,000g

for 30min at 4 1C. The resulting supernatant was used assource of soluble luminal contents. The pellet contains the

insoluble fraction of ingested food as well as the peritr-ophic membrane that corresponds to a gel-like materialseen at the top of the pellet. This gel-like material wascollected with a spatula and resuspended in 60% glyceroland centrifuged at 10,000g for 15min at 4 1C. Thesupernatant was diluted in water to a final 10% glyceroland centrifuged at 25,000g for 30min at 4 1C. The resultingpellet was resuspended in double distilled water and labeledperitrophic membrane preparation.

2.5. Preparation of soluble midgut contents and peritrophic

membrane from S. frugiperda

The peritrophic membrane was isolated from midgutcontents by dissection with a fine forceps and rinsed with125mM NaCl solution to remove food particles. Themembrane was then homogenized with the aid of an Omni-Mixer (Sorvall, USA) at 5000 rpm for 3 cycles of 30 s with30 s pause, followed by centrifugation at 25,000g for 30minat 4 1C. The supernatant was discarded and the pellet wasdispersed in double distillated water by ultrasound in aBranson 250 sonifier. The preparation was centrifugedagain at 2500g for 5min at 4 1C to remove nondispersedmaterial and the supernatant was used as a source ofperitrophic membranes.Soluble midgut contents of S. frugiperda were prepared

as described for T. molitor.

2.6. Protein quantification and hydrolase assays

Protein was quantified according to Smith et al. (1985),as modified by Morton and Evans (1992), using chickenovalbumin as a standard.Aminopeptidase activity was determined with 1mM

leucine p-nitroanilide (LeupNA) as substrate in 100mMTris HCl, pH 7.5, whereas g-glutamyl-transferase wasassayed with 1mM L-g-glutamyl-p-nitroanilide in 50mMTris HCl buffer, pH 8.8 containing 10mM glycyl-glycine.In both methods, the appearance of p-nitroaniline wasfollowed according to Erlanger et al. (1961). Incubationswere carried out at 30 1C for at least four different timeperiods, and initial rates of hydrolysis were calculated. Allassays were performed under conditions so that productformation was proportional to enzyme concentration andto time. Controls without enzyme and others withoutsubstrate were included. One unit of enzyme (U) is definedas the amount that hydrolyses 1 mmol of substrate per min.

2.7. Preparation of microvillar protein antisera

The pre-immune blood of all the rabbits used to raiseantibodies was nonreactive against proteins of insectmidgut and Escherichia coli XL1-Blue. Antibodies wereraised as follows. Two milliliters of a microvillar proteinpreparation were dispersed with an equal volume ofFreund’s complete adjuvant. This suspension (containing4mg of the purified microvillar membrane proteins) was

ARTICLE IN PRESSA.H.P. Ferreira et al. / Journal of Insect Physiology 53 (2007) 1112–1124 1115

then injected into the inguinal nodes of a rabbit. After 4weeks, another injection of microvillar proteins (with2.9mg for T. molitor and 4.2mg for S. frugiperda) wasadministered, but now with Freund’s incomplete adjuvant.The protein mass of the injected samples was calculated sothat at least 15 mg (T. molitor) or 50 mg (S. frugiperda) ofthe less-represented bands resolved by SDS–PAGE wereadministered. After 7 days the rabbit was bled. The bloodwas left standing for 1 h at 37 1C and overnight at 4 1C,before being centrifuged at 3000g for 10min at 4 1C. Thesupernatant was added to a suitable solution to become50% saturated in (NH4)2SO4, pH 6.8. After standingovernight at room temperature (25 1C), the resultingsuspension was centrifuged at 5000g for 15min at 4 1C.The pellet was resuspended in 0.1M NaCl and dialyzedovernight against 1000 volumes of 100mM NaCl at 4 1C.The resulting antiserum was stored at �20 1C. Antibodyproduction and specificity was checked on Western blotsafter SDS–PAGE.

2.8. SDS–PAGE and Western blotting

SDS–PAGE of samples was carried out in 12% (w/v)polyacrylamide gels containing 0.1% (w/v) SDS, on adiscontinuous pH system (Laemmli, 1970), using BioRad(USA) Mini-Protein II equipment. Lyophilized sampleswere dissolved in sample buffer containing 60mMTris–HCl, pH 6.8, 2.5% (w/v) SDS, 0.36mM 2-mercap-toethanol, 10% glycerol and 0.05% (w/v) bromophenolblue and heated for 2min at 95 1C in a water bath beforebeing loaded onto the gels. Electrophoresis was carried outat 200V until the front marker (bromophenol blue)reached the bottom of the gel. The gel was stained with asolution of 0.1% (w/v) Comassie Blue R in 10% acetic acidand 40% methanol for 30min. Destaining was achievedwith several washes in a solution containing 40% methanoland 10% acetic acid. The masses of individual bandsresolved after SDS–PAGE of the samples were evaluatedby comparison with SDS–PAGE controls having differentamounts of egg albumin. Molecular-weight markers used:lysozyme (14.4 kDa), soybean trypsin inhibitor (21.5 kDa),carbonic anhydrase (31 kDa), chicken ovalbumin (45 kDa),bovine serum albumin (66 kDa) and phosphorylase b(97.4 kDa).

Western blotting was performed as follows. AfterSDS–PAGE, the proteins were electrophoretically trans-ferred onto a nitrocellulose membrane filter (pore size0.45mm; BioRad, USA, Towbin et al., 1979). The transferefficiency was evaluated by observing the pre-stainedmolecular weight markers (BioRad or Sigma, USA). Thefilters were first reacted (after a blocking step) with theantiserum diluted 1000-fold in TBS (Tris-buffered solution:50mM Tris–HCl buffer, pH 7.4, with 0.15M NaCl)containing 0.05% Tween 20 (TBS-T) for 2 h at roomtemperature (25 1C). After extensive washing with TBS-T,the filters were reacted with anti-rabbit IgG coupled withperoxidase (Sigma, USA) diluted 1:1000 in TBS-T for 2 h

at room temperature. After washing extensively with thesame buffer, the strips were incubated with 0.08%4-chloro-1-naphthol in TBS containing 0.1% hydrogenperoxide until gray bands appeared where antigens wererecognized. The reagent was prepared before use by theaddition of one volume of 0.5% chloro-naphthol inmethanol to five volumes of TBS with hydrogen peroxide.Pre-immune serum was used in control experiments toshow that antisera were specific.

2.9. Midgut cDNA library construction and screening

Total RNA was extracted from midgut tissue ofT. molitor and S. frugiperda larvae with Trizol followingthe instructions of the manufacturer, Invitrogen, which arebased on Chomczynski and Sacchi (1987), and sent toStratagene (La Jolla, CA), in order to construct a cDNAlibrary. At Stratagene the mRNAs were isolated, dividedinto two equal samples and used in cDNA synthesis with apoly-T and a random primer. Finally, the two cDNA poolswere mixed (1:1) and nondirectionally inserted in the vectorl ZAPII. The library titer was 1.5� 1010 pfuml�1. Screen-ing used antibodies raised against microvillar membraneproteins in rabbits, following the library manufacturerprotocol (picoBlueTM immunoscreening kit, Stratagene)instructions in nitrocellulose membranes. Phages wereplated at low density on an E. coli lawn, to allow individualcollection of positive phage plaques. The inserts of clonedcDNA were excised from the phages and inserted intopBluescript plasmids (following Stratagene cDNA libraryprotocol) and checked for the presence of insert using PCRreaction with primer M13 forward (50 CCC AGT CACGAC GTT GTA AAA CG 30) and M13 reverse (50 AGCGGA TAA CAA TTT CAC AC- A GG 30) at standardconditions for the TAQ DNA Polymerase (Invitrogen),except for annealing temperature at 50 1C for 45 s. The 50

end of amplified PCR product was sequenced (‘‘ABI3100’’, Applied Biosystems) performed with the DNA kitBig Dye Terminator Cycle sequencing (Applied Biosys-tems). All clones were sequenced once using a T3 primer.Random sequencing of cDNA library was used as a controlof its quality.

2.10. Sequence assembly

The electropherograms of the sequenced clones wereautomatically processed for base calling and low-qualitytrimming using Phred set to minimum quality 10 (non-default parameters: –trim_alt –trim_cutoff 0.09). Vectorsequence trimming was done by Crossmatch (nondefaultparameters: -minmatch 10 –minscore 20) and contaminantsequences were identified by BlastN set to e-value cutoff1e�30 (nondefault parameters: -e 1e�30) against adatabase of possible contaminants (ribosomal RNA,E. coli genome, mitochondria and plasmid sequences, allfrom GenBank). Sequences containing more than 200 bpafter trimming (for quality and remaining vector

ARTICLE IN PRESS

Fig. 1. Specificity of antibodies raised against microvillar membrane from

T. molitor (A) and S. frugiperda (B). (MW) Molecular weight markers; (1)

Coomassie-stained SDS–PAGE of microvillar proteins (40mg); (2–5)

Western blots stained with antibodies raised against the microvillar

membrane preparation: (2) 10 mg of midgut tissue protein; (3) 10mg of

soluble midgut luminal protein; (4) 10mg of peritrophic membrane

proteins; (5) 10 mg of microvillar membrane proteins.

A.H.P. Ferreira et al. / Journal of Insect Physiology 53 (2007) 1112–11241116

sequences) and not identified as contaminants wereconsidered high-quality sequences.

High-quality sequences were assembled by sequencesimilarity using CAP3 (http://seg.cs.iastate.edu; Huang andMadan, 1999) set to minimum overlap 40 and minimumidentity 95% (nondefault parameters: -o 40 –p 95).

Assembled sequences with two or more reads and inpositive frame were considered to correspond to micro-villar proteins.

The extent to which the genes coding for midgutmicrovillar proteins had been identified at each sequencingstep was evaluated by the novelty rate, defined as the ratiobetween the number of bases in new sequences and thetotal number of sequenced bases.

2.11. Functional annotation and sequence analysis

Assembled sequences were searched for similarity(BlastX, e-value cutoff 1e�6, Interpro) against publicdatabases (nr, Swissprot, TREmbl, Interpro) and the bestmatches identified. Gene Ontology entries associated tosimilar sequences from Interpro, Swissprot and TREmblwere used for the automatic functional annotation of theassembled sequences originating from at least twosequenced clones.

Special features in sequences were predicted with the aidof the following softwares: (a) glycosyl phosphatidylinositol (GPI) anchors, DGPI (http://129.194.185.165/dgpi/index_en.html); (b) chitin-binding domains, Prosite(http://ca.expasy.org/prosite/; Hulo et al., 2006); (c) trans-membrane helices and topology of proteins, HMMTOP(http://www.enzim.hu/hmmtop/html/submit.html; Tusnadyand Simon, 1998) and HMMTOP 2.0 version (Tusnady andSimon, 2001); (d) tree reconstruction using boot-strap algorithm (http://atgc.lirmm.fr/phyml, Guindonet al., 2005).

3. Results

3.1. Preparation of antibodies against midgut microvillar

proteins from T. molitor and S. frugiperda

The isolation of midgut microvillar membranes fromT. molitor and S. frugiperda followed the two-stepprocedure previously described (Jordao et al., 1995;Capella et al., 1997). We found similar enrichments andyields, both regarding T. molitor and S. frugiperda samples,when aminopeptidase activity (and also g-glutamyl trans-ferase activity in the case of S. frugiperda) was used as amarker enzyme. Thus, T. molitor microvillar membranepreparation was enriched 22-fold with a 23% recovery(Jordao et al., 1995 found 23-fold and 23% recovery). Thefigures for S. frugiperda were: enrichment, 7.8-fold;recovery, 19% (according to Capella et al., 1997: 8.0-foldand 18% recovery).

The midgut microvillar membranes were injected intorabbits in amounts such that the less-concentrated bands

seen in Coomassie-stained SDS–PAGE had at least 50 mg(S. frugiperda) or 15 mg (T. molitor). The resulting antiserawere quite specific and seem to recognize most majormicrovillar proteins, a few peritrophic membrane proteinsand almost no protein from the midgut contents, both inT. molitor (Fig. 1(A)) and S. frugiperda (Fig. 1(B)).

3.2. Identification of midgut microvillar proteins from

T. molitor and S. frugiperda

The midgut microvillar protein antisera were used toscreen cDNA expression libraries of T. molitor and S.

frugiperda midguts. The expected result was that clonesrecognized by the antibodies should correspond toexpressed microvillar membrane proteins.Positive clones generated ESTs that, after trimming and

quality estimates were used for comparative analyses(Table 1). The extent of library screening was followedby the novelty rate. The novelty rate decreased fast,showing that a few expressed sequences correspond to mostof the antibody-reacting microvillar proteins (Fig. 2).Library screening was discontinued when nearly allsequences corresponding to antibody-reacting microvillarproteins had been identified. In other words, library

ARTICLE IN PRESSA.H.P. Ferreira et al. / Journal of Insect Physiology 53 (2007) 1112–1124 1117

screening was discontinued when the novelty ratebecomes approximately constant. This happened whenthe novelty rate becomes close to zero (T. molitor) orapproximately 0.15 (S. frugiperda). ESTs in a positiveframe were clusterized using the CAP3 program and theresulting contigs were blasted against GenBank. Singlets,with a single exception, were not considered in the analysisand were discarded. The ESTs from T. molitor weredeposited in GenBank with access numbers fromEG358387 to EG358894 and EG358896. Those obtainedfrom S. frugiperda were assigned access numbers fromEG358897 to EG359147 and from EG359149 toEG359150.

The assembled sequences were then annotated accordingto Gene Ontology (Fig. 3). The most represented categoryin both insects is related to chitin binding, followed bypeptidases, glycosidases and oxyreductases.

Table 1

ESTs corresponding to proteins reacting with midgut microvillar protein

antibodies from T.molitor and S. frugiperda

T.molitor S. frugiperda

Total ESTs 332 592

High-quality ESTs 247 493

Clustering in frame 177 419

Unmounted 70 74

Total discarded ESTs 85 99

Incorrected frame 6 4

Mitochondrial sequences 9 –

Ribosomal sequences 15 20

Vector sequences 5 9

Low qualitya 45 49

Low complexity – 12

Short sequencesb 5 5

Average sequence length after trimming: 421.3 (T.molitor) and 503.3

(S. frugiperda).aSequence shorter than 200bp with Phred value above 10.bSequences shorter than 200 bp after vector and low quality trimming.

1.2

1

0.8

0.6

0.4

0.2

0

0 50 100 150 200

Sequenced clone

Novelty R

ate

Fig. 2. Changes in the novelty rate accompanying the immunoscreening of t

corresponds to the ratio of the number of new bases with the number of tota

4. Discussion

4.1. Extent to which microvillar proteins were identified

There are two major approaches to identify proteinsexpressed in a tissue: transcriptome and proteome. In thecase of a tissue organelle, like the microvilli, thetranscriptomics approach cannot be used because it is notpossible to isolate a group of mRNAs (and hence toprepare a cDNA library) that correspond only to micro-villar proteins. Massive random sequencing of midguttissue cDNA libraries is not an alternative procedure.There is no way to recognize, among the ESTs, thoserelated with microvillar proteins, except for the few onesthat are homologous to proteins found only in midgutmicrovillar membranes.The proteomics approach is therefore the method of

choice and its use has resulted in several findings (e.g.McNall and Adang, 2003; Krishnamoorthy et al., 2007).This method, nevertheless, is limited by the occasionalfailure of protein bands to yield useful mass spectra and bythe quality of peptide mass fingerprints obtained when thesequences of the specific organism under study are notabundant in the data bases (frequent among insects)(Krishnamoorthy et al., 2007).In this paper, a novel approach is described to identify

microvillar proteins. The method consists in using micro-villar proteins to generate antibodies that were employed toscreen an expression cDNA library. The advantages of themethod over the proteomic approach are: (a) the sequencesof the cloned genes that correspond to microvillar proteinspermit identification by similarity searches in data banks,even if sequences of the specific (or a close related)organism under study are lacking; (b) the cloning approachallows complete gene sequences to be obtained, which maybe used in functional studies regarding the role of theproteins. This is the case even where the sequences have nomatch in the data banks, or where they match proteins withunknown functions.

Sequenced clone

Novelty R

ate

0 100 200 300 400 500

1.2

1

0.8

0.6

0.4

0.2

0

he cDNA libraries of T. molitor (A) and S. frugiperda (B). Novelty rate

l sequenced bases.

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Table 2

Clusters coding for midgut microvillar membrane proteins from Tenebrio molitor

Cluster

number

No. seqs. in

cluster

Best NR protein match E value E value against

Beetle baseaPredicted protein Putative

function

1 17 AAD28248.19mucin-like protein MUC1

[Aedes]

5.0e�12 5e�013 Peritrophin Peritrophic

membrane

2 7 AAL05409.19peritrophin [Aedes aegypti] 4.0e�18 1e�032 Peritrophin Peritrophic

membrane

3 6 AAM94156.19mucin-like peritrophin [Aedes

aegypti]

7.0e�11 5e�007 Peritrophin Peritrophic

membrane

4 3 XP_308952.19ENSANGP00000013237

[Anopheles]

1.0e�14 2e�007 Peritrophin Peritrophic

membrane

5 4 NP_609408.19CG6206-PA [Drosophila] 7.0e�62 0.33 a-Mannosidase Digestion

6 2 AAP940459membrane alanyl minopeptidase

[Tenebrio]

2e�92 No hits found Aminopeptidase Digestion

7 6 AAB82404.199Cr-PII [Periplaneta americana] 9.0e�14 2e�037 Cockroach allergen Unknown

8 50 AAD13530.29major allergen Bla g 1.0101

[Blattella]

5.0e�31 1e�046 Cockroach allergen Unknown

9 48 AAD13530.29major allergen Bla g 1.0101

[Blattella]

1.0e�22 2e�034 Cockroach allergen Unknown

10 27 AAD13531.19major allergen Bla g 1.02

[Blattella]

2.0e�70 1e�086 Cockroach allergen Unknown

11 2 XP_319154.19ENSANGP00000011976

[Anopheles]

5.0e�61 0.24 Unknown Unknown

12 3 No similar protein described 0.45 Unknown Unknown

13 2 No similar protein described 2e�087 Unknown Unknown

ESTs obtained by immunoscreening a T. molitor midgut expression cDNA library were clusterized using CAP3. Clusters obtained with more than 2 clones

sequenced were blasted against Genbank and the best Blast hit and their corresponding E values were collected.aBlast against Tribolium ESTs in Beetle Base Databank at /http://www.bioinformatics.ksu.edu/BeetleBase/S.

0

2

4

6

8

10

12

14

O-g

lyco

syl h

yd

rola

se

oxid

ore

du

cta

se

pe

ptid

ase

actin

bin

din

g ch

itin

bin

din

g

ca

lciu

m-d

ep

en

de

nt

ph

osp

ho

lipid

bin

din

g

ca

lciu

m io

n b

ind

ing

se

rin

e p

rote

ase

in

hib

ito

r un

kn

ow

n

Nu

mb

er o

f C

lus

ters

Fig. 3. Midgut cDNA clusters corresponding to antibody-reacting

microvillar proteins grouped by Gene Ontology. Figures are the number

of clusters grouped in GO categories of Molecular Function Ontology.

Only the most represented categories are presented. Empty histograms

correspond to S. frugiperda and solid histograms to T. molitor.

A.H.P. Ferreira et al. / Journal of Insect Physiology 53 (2007) 1112–11241118

The accuracy of the novel method is based on theassumptions that (a) the preparation used to raiseantibodies contains only microvillar membranes; (b) theantibodies used were able to detect most clones expressingmicrovillar proteins and (c) the cDNA libraries used wererepresentative of the midgut proteins, including those ofmicrovilli. The three assumptions will be discussed below.

Microvillar membranes were isolated using proceduresthat result in apparently contaminant-free preparations, asshown using several marker enzymes and extensive electronmicroscopy monitoring (Jordao et al., 1995; Capella et al.,1997). Preparation enrichments and yields obtained in thiswork were the same in the case of both T. molitor andS. frugiperda and provide evidence that our preparationsare virtually free from contaminants. This is also supportedby the finding of clones expressing enzymes known to beassociated with the microvillar membranes, like amino-peptidase in both insects (Cristofoletti and Terra, 1999;Ferreira et al., 1994), but not those expressing solubleenzymes like maltase, even though this enzyme is foundassociated with midgut cell glycocalyx, as is the case inS. frugiperda (Ferreira et al., 1994).The extent to which the antibodies allowed the identi-

fication of the microvillar proteins has no direct answer.The raised antibodies apparently recognize all or at leastmost microvillar proteins in each species as evaluated byWestern-blots, provided that immunoreactive-carbohy-drate epitopes are not important (see Fig. 1). Also, cDNAexpression libraries were screened with antibodies until therate of appearance of new sequences relative to sequencedclones had declined almost to zero for T. molitor or to avery small value in the case of S. frugiperda (see Fig. 2). Asa consequence, it is possible that the vast majority of clonesexpressing antibody-reacting proteins were identified and

ARTICLE IN PRESSA.H.P. Ferreira et al. / Journal of Insect Physiology 53 (2007) 1112–1124 1119

sequenced. Nevertheless, it is impossible to disregard thefollowing possible major sources of errors: (a) undetectedproteins because of lack of reacting antibodies caused byextremely low amounts of antigens or because they wereinsufficiently immunogenic; (b) contaminants detectedbecause they are highly immunogenic; (c) nonmicrovillarproteins detected because they share epitopes or wereaccidentally associated with microvillar proteins and (d)failure of inserted-cDNA-phage expression.

It should be stressed here that no claim is made that anearly complete identification of microvillar proteins ofboth insects were attained. McNall and Adang (2003), asan example, visualized (silver staining) about 45 proteinson 1D gels and about 450 proteins in 2D gels oflepidopteran microvilli preparations. Although their pre-parations were contaminated with cytoskeleton and lateralmembrane proteins (see Introduction), the actual numberof proteins in microvillar membranes should on this basisfar exceed the number seen in Fig. 1 (Coomassie Bluestaining), although there are no data to provide a preciseestimate. The predicted proteins represent, thus, onlymajor microvillar proteins that were present in sufficientamounts to generate antibodies. In spite of this limitations,the antibody library screening approach used in this studyhas led to the prediction of 27 proteins (Table 3), whereasthe best proteomic study to date (Krishnamoorthy et al.,2007) predicted only 13 lepidopteran midgut microvillarproteins.

With the necessary caution it is possible now to discussthe proteins found in the midgut microvillar membranes ofT. molitor and S. frugiperda.

4.2. Midgut microvillar proteins from T.molitor

This is the first systematic study of midgut microvillarproteins in coleopterans, after the search for the majordigestive enzymes (reviewed in Terra et al., 2006). Fourgroups of predicted microvillar proteins were recognized inT. molitor midguts: cockroach allergens, peritrophins,hydrolases (aminopeptidase and a-mannosidase), andproteins with unknown function (Table 2).

Cockroach allergens are proteins with tandem aminoacid repeats that were first described in cockroaches(Pomes et al., 1998; Yang et al., 2000), and which are animportant cause of asthma. Their functions in the insect areunknown. T. molitor has four closely related clusters ofcockroach allergens that may correspond to the sameprotein. They have no predicted GPI-anchor, in contrastwith the very similar GPI-anchored protein from Aedes

aegypti (Shao et al., 2005).The four clusters that best match peritrophins or mucin-

like proteins are probably all peritrophins, because theyhave predicted chitin-binding type-2 domains. Accordingto Tellam et al. (1999), peritrophins are proteins thatinterlock chitin fibers forming the peritrophic membrane,an anatomical structure that separates food from themidgut cells. Peritrophins may have mucin domains in

addition to chitin-binding domains. The four clustersprobably correspond to different proteins. How peritro-phins are immobilized on the midgut cell surface (withoutanchors) and concur in peritrophic membrane formationare discussed below for S. frugiperda.The aminopeptidase cluster corresponds to the micro-

villar enzyme previously isolated and characterized(Cristofoletti and Terra, 1999, 2000). Cloning and sequen-cing this enzyme revealed that it has a predictedGPI-anchor (GenBank accession number AY332269).The a-mannosidase cluster has a sequence similar to

mammalian lysosomal a-mannosidase and may correspondto the enzyme found in midgut cell membranes (unpub-lished results). The other clusters correspond to proteinswith unknown function, although one of them matchesXP_319154.1, a protein that putatively has four transmem-brane domains also found in a Tribolium EST (cluster 13).

4.3. Midgut microvillar proteins from S. frugiperda

Previous attempts to identify midgut microvillar proteinsin lepidopterans (other than major digestive enzymesreviewed in Terra et al., 2006) used proteomics. A totalof 13 proteins were predicted, including previouslyidentified aminopeptidases, ABC transporters, V-ATPase,cytoskeleton proteins and proteins without a clear functionin microvilli (McNall and Adang, 2003; Krishnamoorthyet al., 2007).In this paper, a total of 27 proteins were predicted. The

predicted S. frugiperda proteins may be grouped into 5classes: midgut protection, peritrophic membrane forma-tion, hydrolases, cytoskeleton organization and secretion,and unknown function (Table 3).Four predicted proteins are related to protection of

midgut: thioredoxin peroxidase, aldehyde dehydrogenase,serpin, and juvenile hormone (JH) epoxide hydrolase.Thioredoxin peroxidase best matched with one enzyme ofBombyx mori (Lee et al., 2005) that plays a protective roleagainst oxidative stress caused by temperature and viralinfection. In the case of S. frugiperda, the role of themicrovillar thioredoxin peroxidase is probably to detoxifyH2O2 produced by plant allelochemicals ingested by thelarvae (Terra and Ferreira, 2005). The enzyme is probablyinserted in the microvillar membrane by a putativemembrane-spanning segment.The predicted aldehyde dehydrogenase probably detoxi-

fies aldehydes present in diet or originating after the actionof insect b-glucosidases on toxic b-glucosides (see details inTerra and Ferreira, 2005).In insects, serpins have been described as involved in

immune responses in plasma and in tissues (Tong andKanost, 2005). In Anopheles gambiae midgut, expression ofserpin SRPN6 is induced by E. coli and malaria parasites(Abraham et al., 2005). In S. frugiperda midgut, thepredicted serpin with unknown anchor probably has asimilar role, protecting the midgut from microorganisminfection. Alternatively, it could protect the surface of the

ARTICLE IN PRESS

Table 3

Clusters coding for microvillar membrane proteins from Spodoptera frugiperda

Cluster

Number

No. seqs. in

cluster

Best NR protein match E value E value

against

Butterfly Basea

Predicted protein Putative

function

1 42 XP_319075.1|ENSANGP00000013314

[Anopheles]

6.0e�46 0.0e+00 Aldehyde

dehydrogenase

Protection

2 25 AAR15420.1| thiol peroxiredoxin [Bombyx

mori]

2e�105 0.0e+00 Thioredoxin

peroxidase

Protection

3 2 AF242200_19serpin-2 [Bombyx mori] 5.0e�38 1.0e�126 Serpin Protection

4 1 AAQ87024.1| juvenile hormone epoxide

hydrolase [B]

1e�84 0.0e+00 JH epoxide

hydrolase

Protection

5 105 EAL24878.1|GA21261-PA [Drosophila

pseudoobscura]

0.0e+00 0.0e+00 Transporter-like

amylase

Digestion

6 47 AAL48097.19RE72980p [Drosophila

melanogaster]

5.0e�96 0.0e+00 Transporter-like

amylase

Digestion

7 41 NP_610384.19CG8690-PA [Drosophila

melanogaster]

3.0e�63 0.0e+00 Transporter-like

amylase

Digestion

8 19 AAP44964.1| midgut class 1 aminopeptidase

N [Spod]

0.0e+00 0.0e+00 Aminopeptidase Digestion

9 8 AAQ24379.1| midgut aminopeptidase N2

[Helicove]

0.0e+00 0.0e+00 Aminopeptidase Digestion

10 4 BAA32476.19aminopeptidase N [Manduca

sexta]

8.0e�78 0.0e+00 Aminopeptidase Digestion

11 2 AAP37951.19midgut aminopeptidase N2

[Helicoverpa]

2.0e�42 0.0e+00 Aminopeptidase Digestion

12 2 XP_317868.19ENSANGP00000020016

[Anopheles]

5.0e�28 0.0e+00 Carboxypeptidase A Digestion

13 2 XP_318552.19ENSANGP00000019641

[Anopheles]

2.0e�23 0.0e+00 Astacin Digestion

14 9 AAS89976.1| peritrophin membrane protein

1 [Spodo]

3e�81 0.0e+00 Peritrophin Peritrophic

membrane

15 9 AAP33177.19peritrophin 1 [Mamestra

configurata]

0.0e+00 0.0e+00 Peritrophin Peritrophic

membrane

16 4 AAP33177.19peritrophin 1 [Mamestra

configurata]

4.0e�29 0.0e+00 Peritrophin Peritrophic

membrane

17 2 AAP33177.19peritrophin 1 [Mamestra

configurata]

2.0e�64 1.0e�140 Peritrophin Peritrophic

membrane

18 33 XP_317951.19ENSANGP00000020539

[Anopheles]

1.0e�87 0.0e+00 Gelsolin Enzyme

secretion

19 8 BAB16697.19annexin [Bombyx mori] 7.0e�77 0.0e+00 Annexin Enzyme

secretion

20 3 XP_309749.19ENSANGP00000012700

[Anopheles]

3.0e�62 0.0e+00 Calmodulin Cytoskeleton

signalling

21 2 XP_309626.19ENSANGP00000011155

[Anopheles]

0.0e+00 5e�49 Fimbrin Cytoskeleton

22 2 BAD93613.1| protein disulfide-isomerase

like pro

1.0e�90 0.0e+00 Disulfide isomerase Unknown

23 25 NP_727068.19CG32751-PA [Drosophila

melanogaster]

5.0e�27 0.0e+00 Vanin-like protein Unknown

24 11 No similar protein described 0.0e+00 Unknown Unknown

25 4 No similar protein described No hits found Unknown Unknown

26 4 No similar protein described 4e�27 Unknown Unknown

27 2 No similar protein described 1.0e�172 Unknown Unknown

ESTs obtained by immunoscreening a S. frugiperda midgut cDNA expression library were clusterized using CAP3. Clusters obtained with more than 2

clones sequenced were blasted against Genbank and the best Blast hit and their corresponding E values were collected.aBlast against Lepidoptera ESTs in Butterfly BaseDatabank at /http://heliconius.cap.ed.ac.uk/butterfly/db/S.

A.H.P. Ferreira et al. / Journal of Insect Physiology 53 (2007) 1112–11241120

midgut cells from the action of luminal serine proteinases.In mammalian midgut cells, that protection is conferred bymucus proteins, which are not present in insects.

A possible function of the microvillar JH epoxidehydrolase might be to deactivate plant JH analogues.Many of these analogues are not epoxides, like the

historical juvabione (Slama and Williams, 1966), but thosethat are epoxides may be affected by JH epoxide hydrolase.It is not known how this protein is associated with themicrovillar membrane.The predicted S. frugiperda peritrophins (see definition

and other details above under T. molitor) are those that

ARTICLE IN PRESS

Amino acid transporter

Salivary amylase

Pancreatic amylase

S_frugiperda cluster 1

S_frugiperda amylase

Organic anion transporter

100

100

100

Fig. 4. Cladogram of peptide sequences of chosen proteins from

the amylase family deposited in GenBank. Amino acid transporter

(neutral) (AAB26524), organic ion transporter (CAB77184), S. frugiperda

amylase (AAO13754), salivary amylase (human) (P04745) and pancreatic

amylase (human) (P04746). The cladogram was prepared with ClustalX

(Thompson et al., 1997) and PHYML Online (Guindon et al., 2005).

A.H.P. Ferreira et al. / Journal of Insect Physiology 53 (2007) 1112–1124 1121

have been previously described (Bolognesi et al., 2001,peritrophin-33k, AA58997.1), and three others. Peritro-phin-33k does not have any kind of anchor to be inserted inthe microvillar membrane and is secreted as a solubleprotein by a microapocrine mechanism from the anteriormidgut cells of S. frugiperda. The other peritrophins aredifferent but are homologous to peritrophin-33k and arealso supposed to have no anchor. After secretion,molecules of peritrophin-33k are seen associated withmidgut cell microvilli and this was thought to correspondto proteins entrapped in the glycocalyx (Bolognesi et al.,2001). Glycocalyx entrapping was also described formaltase and cellobiase (Ferreira et al., 1994) inS. frugiperda midgut cells. However, no sequences pre-dicted as corresponding to either maltase or cellobiase werefound in the present immunoscreening of the cDNAexpression library. It is possible, however, that the failureto find those sequences resulted from maltase andcellobiase not being as immunogenic as peritrophins.Nevertheless, we consider that it is more probable thatthe recovery of clones encoding cloned peritrophinsresulted from their being more strongly bound to theglycocalyx than maltase and cellobiase, which are set freeon freezing and thawing. If this is true, the chitin-bindingdomain of peritrophins may bind to the N-acetylglucosa-mine residues of the carbohydrate chains of microvillarglycoproteins that form the glycocalyx. As an alternativehypothesis, peritrophins may associate with the microvillarsurface because they bind to nascent chitin fibers duringtheir synthesis by chitin synthase. Both views are supportedby the facts: (a) chitin molecules are synthesized outside themidgut cells under the action of microvillar-bound chitinsynthase (Zimoch and Merzendorf, 2002) and (b) theperitrophic membrane is formed among midgut microvilliin lepidopteran larvae (Harper and Hopkins, 1997).

Four predicted aminopeptidase clusters with putativeGPI-anchors and one predicted carboxypeptidase withunknown anchor correspond to enzymes previouslydescribed as microvillar enzymes in S. frugiperda midgut(Ferreira et al., 1994). As the sequences are incomplete, it isnot possible to relate them to the four classes described foraminopeptidases (Wang et al., 2005), or even to be surewhether the clusters obtained correspond (or not) to thesame aminopeptidase.

Trypsin was previously described as transiently asso-ciated with midgut microvillar membranes (Ferreira et al.,1994; Jordao et al., 1999). In spite of that, no clone oftrypsin was obtained in the present paper, which may resultfrom this enzyme being insufficiently immunogenic orpresent in amounts that were too small.

Three closely related clusters (5–7) corresponding tocomplete sequences are identified as a-amylases if the Blastis done against all sequences deposited in the GenBank. Ifthe Blast is done against only mammalian sequences,however, the best hit is to the neutral amino acidtransporter. This noncatalytical protein is related to thea-amylase family from the structural and evolutionary

points of view (Janecek, 2002). A sequence tree of cluster 5,neutral amino acid transporter, organic ion transportersand amylases shows that cluster 5 is more similar to aminoacid transporter than to amylases (Fig. 4). A closeinspection of the cluster 5 sequence reveals that the 3amylase catalytic residues and the 2 strictly conservedresidues involved in Ca2+ binding (D’Amico et al., 2000)are present. As amylase-related transporters do not havethe catalytic proton donor and at least one of the strictlyconserved Ca2+-binding residues (D’Amico et al., 2000),clusters 5–7 can be identified as microvillar transporter-likea-amylases having a putative GPI anchor at their C-terminal, as has been found for another S. frugiperda

amylase (access no. GI AA013754, Da Lage et al., 2002)having a typical sequence. Part of the amylase activitydetermined to be present in S. frugiperda midgut micro-villar membranes (Ferreira et al., 1994) may depend onthese transporter-like amylases. The same might not betrue for the amylase (undergoing microapocrine secretion)that was immunocytolocalized with a heterologous(T. molitor) amylase antiserum (Bolognesi et al., 2001),which is not expected to recognize the microvillartransporter-like amylases with their peculiar sequences,although some epitope-sharing recognition cannot be ruledout. The relationship among S. frugiperda amylases andtheir secretion/microvillar-insertion mechanisms deservereinvestigation in the light of this new data.The best hit for cluster 13 is metallopeptidase. Never-

theless, cluster 13 is incomplete, has an unknown anchorand possesses the zinc-binding motif HEXXHXXGXXHthat is characteristic of astacins (Stocker et al., 1995).

ARTICLE IN PRESSA.H.P. Ferreira et al. / Journal of Insect Physiology 53 (2007) 1112–11241122

The astacins are found throughout the animal kingdomand include the meprins, which are anchored to the surfaceof epithelial cell, where they are thought to processbiologically active peptides (Bond and Beynon, 1995). Itis tempting to speculate that the role of the midgutmicrovillar S. frugiperda astacin-like enzyme might be toinactivate signaling peptides, such as those that putativelyinduce digestive enzyme secretion. This subject obviouslydemands further research.

The predicted proteins calmodulin, fimbrin, annexin,and gelsolin are not anchored. They might be recovered inthe microvillar membrane preparations because they mayassociate with membranes or with cystoskeleton elementsfound contaminating the preparation. Calmodulin is aCa2+-binding protein that is part (with myosin I) of thelateral arm that connects the actin bundle to the overlyingplasma membrane in midgut microvilli (Bement andMooseker, 1996). Furthermore, calmodulin may alsoparticipate in numerous regulatory processes (Tong et al.,2006), putatively including those in S. frugiperda midgutmicrovilli (see below). Annexin is a family of structurallyrelated proteins with Ca2+-dependent phospholipid-bind-ing properties that may promote membrane fusions(Rescher and Gerke, 2004). Gelsolin-like proteins areactivated by high levels of cytosolic Ca2+ and severe actinfilaments by insinuating between actin units (McGoughet al., 2003).

Calmodulin, annexin and gelsolin putatively interplay inthe microapocrine secretory process of digestive enzymesdescribed in S. frugiperda midgut (Ferreira et al., 1994;Jordao et al., 1999; Bolognesi et al., 2001) as follows.Calmodulin may control the Ca2+ concentration inside themicrovilli in response to some signaling process, thusaffecting the activity of gelsolin and annexin. The move-ment of secretory vesicles through the microvilli might behelped by the actin-filament severing activity of gelsolin,whereas secretory vesicle fusion with microvillar membranemay be promoted by annexin.

The predicted vanin-like protein and disulfide isomerasehave unknown anchors and their function has not beenestablished. The functions of clusters 24–27 (with nosimilar protein described) are also unknown, despite thefact that all of them (except cluster 25) are homologous tosequences in the butterfly base databank.

4.4. Midgut microvillar proteins in Coleoptera and

Lepidoptera: comparative aspects

The results described in this paper support a hypothesisregarding the reason why the lepidopteran midgut micro-villar membranes are denser and show a greater variety ofproteins than those of coleopterans. There are a host ofmicrovillar proteins in lepidopterans that assist the larvaein dealing quickly with huge amounts of food derived froma variety of plants. There are proteins involved incounteracting plant chemical defenses, in protecting themidgut surface against the larval serine proteinases, and in

promoting peculiar secretory mechanisms. In addition tothose proteins, there are still others that lepidopteransshare with coleopterans, namely those forming theperitrophic membrane, a few digestive enzymes and thosenot found among predicted proteins like receptors and ionand organic compound transporters. There are coleopter-ans attacking a variety of plants, but they usually deal withtheir food more slowly than lepidopterans. Perhapsbecause of that, their digestive physiology strategy seemsto rely less on midgut microvillar proteins than in the caseof lepidopterans.

Acknowledgments

This work was supported by the Brazilian researchagencies FAPESP, and CNPq (PRONEX program). AHPFerreira and PT Cristofoletti are post-doc fellows ofFAPESP, LO Guerra is a graduate fellow of FAPESP.MRS Briones received an international research grant fromthe Howard Hughes Medical Institute, WR Terra and CFerreira are staff members of their respective departmentsand research fellows of CNPq.

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