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InsectBiochemistry

andMolecularBiology

Insect Biochemistry and Molecular Biology 37 (2007) 702–712

The sialotranscriptome of the blood-sucking bug Triatoma brasiliensis(Hemiptera, Triatominae)

Adriana Santosa, Jose Marcos C. Ribeirob, Michael J. Lehanec, Nelder Figueiredo Gontijoa,Artur Botelho Velosoa, Mauricio R.V. Sant’Annac, Ricardo Nascimento Araujoa,

Edmundo C. Grisardd, Marcos Horacio Pereiraa,�

aLaboratorio de Fisiologia de Insetos Hematofagos, Departamento de Parasitologia/ICB, Universidade Federal de Minas Gerais,

Caixa postal 486, 31270-901, Belo Horizonte, MG, BrazilbSection of Vector Biology, Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health,

12735 Twinbrook Parkway, Room 2E32D, Rockville, MD 20852, USAcLiverpool School of Tropical Medicine, Pembrok Place, Liverpool L3 5QA, UK

dLaboratorio de Protozoologia, MIP, CCB, Universidade Federal de Santa Catarina, Caixa postal 476, 88040-900, Florianopolis, SC, Brazil

Received 9 February 2007; received in revised form 27 March 2007; accepted 1 April 2007

Abstract

Triatoma brasiliensis is the most important autochthon vector of Trypanosoma cruzi in Brazil, where it is widely distributed in the

semiarid areas of the Northeast. In order to advance the knowledge of the salivary biomolecules of Triatominae, a salivary gland cDNA

library of T. brasiliensis was mass sequenced and analyzed. Polypeptides were sequenced by HPLC/Edman degradation experiments.

Then 1712 cDNA sequences were obtained and grouped in 786 clusters. The housekeeping category had 24.4% and 17.8% of the clusters

and sequences, respectively. The putatively secreted category contained 47.1% of the clusters and 68.2% of the sequences. Finally, 28.5%

of the clusters, containing 14% of all sequences, were classified as unknown. The sialoma of T. brasiliensis showed a high amount and

great variety of different lipocalins (93.8% of secreted proteins). Remarkably, a great number of serine proteases that were not observed

in previous blood-sucking sialotranscriptomes were found. Nine Kazal peptides were identified, among them one with high homology to

the tabanid vasodilator vasotab, suggesting that the Triatoma vasodilator could be a Kazal protein.

r 2007 Elsevier Ltd. All rights reserved.

Keywords: Saliva; Transcriptome; Hematophagy; Salivary proteins; Triatoma brasiliensis

1. Introduction

Triatoma brasiliensis is the most important autochtho-nous vector of Trypanosoma cruzi in Brazil, where it iswidely distributed in the semiarid areas of the Northeast(Costa et al., 2003; Dias et al., 2000; Silveira et al., 1984).This species is able to colonize houses and peridomiciliaryareas, and is also widely distributed in sylvatic habitatswhere it is mainly found among rock piles associated withvarious species of vertebrates. This increases its importanceas a vector, because it can re-colonize domestic habitats

after it has been eliminated through insecticide spraying(Alencar, 1987; Diotaiuti et al., 2000).Blood feeders have evolved a wide set of pharmacolo-

gically active molecules to counteract host defense systems(haemostasis, inflammation, immune response) in thefeeding site (Andrade et al., 2005; Ribeiro and Francischet-ti, 2003). Several biomolecules have already been describedin triatomine bug saliva, including anticoagulants(Hellmann and Hawkins, 1964, 1965; Pereira et al., 1996;Ribeiro et al., 1998), vasodilators (Ribeiro et al., 1990,1993; Ribeiro and Nussenzveig, 1993), antihistamines(Ribeiro and Walker, 1994), sialidase (Amino et al.,1998), a sodium channel blocker (Dan et al., 1999),immunosuppressors (Kalvachova et al., 1999), a poreformer (Amino et al., 2002), a complement system inhibitor

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0965-1748/$ - see front matter r 2007 Elsevier Ltd. All rights reserved.

doi:10.1016/j.ibmb.2007.04.004

�Corresponding author. Tel.:+5531 3499 2835, fax: +5531 3499 2970.

E-mail address: marcoshp@icb.ufmg.br (M.H. Pereira).

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(Cavalcante et al., 2003) and inhibitors of plateletaggregation induced by collagen (Noeske-Jungblut et al.,1994; Ribeiro and Garcia, 1981), ADP (Ribeiro andGarcia, 1980; Sarkis et al., 1986), arachidonic acid (Ribeiroand Sarkis, 1982), thrombin (Francischetti et al., 2000;Noeske-Jungblut et al., 1995) or PAF (Golodne et al.,2003). Few of these activities have been reported fromT. brasiliensis. Sant’Anna et al. (2002), using the suppres-sion subtractive hybridization (SSH) technique, identifiedsix full-length differentially expressed cDNAs fromT. brasiliensis. Among them, three had their activitiesinferred by similarity with other sequences in GenBank,but the others had no obvious orthologues.

Until now, only the sialome of Rhodnius prolixus hasbeen published (Ribeiro et al., 2004), but the evolutionarydiversity among triatomines suggests that extrapolatingfindings from one group to another should be undertakenwith caution. So we can see that there are 138 species in theTriatominae placed in six tribes (Triatomini, Rhodniini,Cavernicoloni, Bolboderini, Alberproseniini, Linschostei-ni) and 19 genera (Galvao et al., 2003). Schofield (1988)suggested that the Triatominae had different origin fromReduviidae predators that converged to haematophagyhabit independently. Several studies showed differencesbetween Rhodniini and Triatomini tribes (Bargues et al.,2000; Catala, 1997; Dujardin et al., 1999; Garcia andPowell, 1998; Jurberg, 1996; Marcilla et al., 2001; Stothardet al., 1998). Marked differences are also seen betweenRhodnius and Triatoma saliva, in that Triatoma lacksnitrophorins, and their apyrase, anticoagulant and vasodi-lator activities show distinct mechanisms of action (Ribeiroet al., 1998). So a comparative study of a sialome from theTriatomini will be very informative and permit interestingcomparisons with that from the Rhodniini alreadypublished. Thus, in this study a salivary gland cDNAlibrary of Triatoma brasiliensis was mass sequenced andanalyzed, and polypeptides were sequenced by HPLC/Edman degradation experiments.

2. Materials and methods

2.1. Insect rearing

T. brasiliensis were captured in Simplıcio Mendes, Piauı(Northeastern region of Brazil) and reared in the insectaryof the Centro de Pesquisas Rene Rachou—Fiocruz MG,maintained at 2872 1C and 65710% relative humidity.They were kept in cages containing vertical strips of coarsefilter paper and fed weekly on chickens.

2.2. Salivary gland cDNA library construction

T. brasiliensis salivary gland mRNA was isolated from 50salivary gland pairs from starved adult insects using theDynabeads mRNAs DIRECTTM kit (DYNAL, GreatNeck, NY). The PCR-based cDNA library was constructedusing a SMART cDNA library construction kit (BD-

Clontech, Palo Alto, CA), according to the manufacturer’sinstructions. The obtained library was plated by infecting log-phase XL-1 Blue cells (Stratagene, La Jolla, CA, USA). Thetiter of the cDNA library was 0.492� 106 pfu/ml, with arecombination efficiency of 87%.

2.3. Sequencing of T. brasiliensis cDNA library

T. brasiliensis salivary gland cDNA library was plated toapproximately 100 plaques per plate (80mm Petri dish).The plaques were randomly picked and transferred to1.5ml centrifuge microtubes containing 100 ml of distilledwater. Five microliters of the phage sample were used astemplate for a PCR to amplify random cDNAs. Theprimers TriplEx2-F (50-CTC CGA GAT CTG GAC GAGC-30) positioned upstream of the cDNA of interest (50 end),and TriplEx2-R (50-TAA TAC GAC TCA CTA TAGGGC-30) positioned downstream of the cDNA of interest(30 end) were used for the PCR (94 1C/4min followed by 35cycles of 94 1C/1min, 52.5 1C/1min and 72 1C/1.1min, anda final extension of 72 1C/7min) carried out with the PhtTaq DNA polymerase system (Phoneutria, Belo Horizonte,MG, Brazil). Amplified products were visualized by 1.0%agarose gel electrophoresis and cleaned up using the GFXPCR DNA and Gel Band Purification Kit (GE/AmershamBiosciences, Buckinghamshire, UK) or the Wizards SVGel and PCR Clean-Up System kit (Promega, Madison,WI, USA). Four microliters of the cleaned PCR productwas used as a template for a cycle-sequencing reactionusing the DYEnamic ET dye terminator cycle sequencingkit (GE/Amersham Biosciences). The primer Seq. Clon-tech-F (50-CTC GGG AAG CGC GCC ATT GTG TTGGT-30) was used for sequencing. Conditions were 94 1C/1min,and 35 cycles of 94 1C/30 s, 51 1C/25 s and 60 1C/4min.After cycle-sequencing the samples, a post-reaction clean-up step, consisted of isopropanol precipitation followedby 70% ethanol wash, was performed. After the super-natant removal, each pellet was dissolved in 6 mL ofMegaBACE loading solution and sequenced on a Mega-BACETM 1000 sequencing instrument (GE/AmershamBiosciences).

2.4. Chromatography

Approximately 20 ml of saliva from starved adult insectswere chromatographed according Ribeiro et al. (2004).Briefly, experiments used 0.24ml bed volume columns ofstrong cation (Mono-S) and strong anion (Mono-Q) ionexchangers obtained from Amersham Biosciences (Piscat-away, NJ). To elute the proteins of interest, the ion-exchange columns were submitted to gradients of NaCl(0–1M). For the cation exchange column, the buffer usedwas 50mM sodium acetate at pH 5.0 and for the anionexchange, 50mM Tris-Cl at pH 8.0. Fractions of interesthad 40 ml removed and diluted with an equal volume of20% methanol containing 0.4% tri-fluoroacetic acid (TFA)and were applied to a ProSorb cartridge (Perkin Elmer,

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Foster City, CA) previously treated with 10 ml of methanol.After absorption of the solution through the polyvinyli-dene difluoride (PVDF) membrane, the cartridge waswashed three times with the same volume of 10% methanolcontaining 0.1% TFA.

2.5. Bioinformatics

Bioinformatics procedures were as by Francischetti et al.(2002) and Valenzuela et al. (2002), except that the clusteringof the cDNA sequences was accomplished using the CAP3program (Huang, 1992) after initial clustering of the databasefollowing a BLASTN (Altschul et al., 1997) of the databaseagainst itself and reading the output to join those sequencesthat had at least 95 identical residues in a window of 100residues. Accession numbers for the National Center forBiology Information (NCBI) databases are given asgi|XXXX, where XXXX is the accession number. Signalpeptide predictions were done with the SignalP program(Nielsen et al., 1997). Transmembrane helices were predictedwith the TMHMM program (Sonnhammer et al., 1998).Sequence alignments and phylogenetic tree analysis used theClustalW package (Thompson et al., 1994). Phylogenetictrees were constructed by the neighbor-joining method(Saitou and Nei, 1987). Boot strapping of phylogenetic treeswas done with the Clustal package for 1000 trials.Phylogenetic trees and dendograms were formatted withTreeView (Page, 1996) using the ClustalW output. Theelectronic version of the complete tables in Microsoft Excelformat with hyperlinks to web-based databases and toBLAST results is available at http://www.ncbi.nlm.nih.gov/projects/omes/T_brasiliensis_sialome.

2.6. Supplemental material

Supplemental Tables S1 and S2 and figures are hyperlinkedthroughout the paper to the NCBI pages, http://www.ncbi.nlm.nih.gov/projects/omes/T_brasiliensis_sialome/Sup_tab1/TB-Sup-table1.xls and http://www.ncbi.nlm.nih.gov/projects/omes/T_brasiliensis_sialome/Sup_tab2/TB-Sup-table2.xls, respectively.

3. Results and discussion

3.1. cDNA library characteristics

To obtain an insight into the salivary transcriptome ofT. brasiliensis, we randomly sequenced 1712 cDNA clones

from a salivary gland cDNA library from this insect andassembled a clusterized database (Supplemental Table S1),yielding 786 clusters of related sequences, 666 of whichcontained only a single EST. The consensus sequence ofeach cluster is designated either a contig (deriving from twoor more sequences) or a singleton (deriving from a singlesequence). In this paper, for simplicity sake, we will use thedenomination contig to address sequences deriving bothfrom consensus sequences and from singletons.Using the BLAST package of programs (Altschul et al.,

1997), we compared the sequence of each cluster in thedatabase with the non-redundant protein and nucleotidesets of the NCBI and the gene ontology database(Ashburner et al., 2000; Hvidsten et al., 2001; Lewiset al., 2000). The translated sequences were also screenedwith RPSBLAST for protein motifs of the combined set ofPfam (Bateman et al., 2000) and SMART (Schultz et al.,2000) databases (also known as the Conserved DomainsDatabase—CDD). Finally, we submitted all translatedsequences (starting with a Met) to the SignalP server(Nielsen et al., 1997) to detect the presence of signalpeptides indicative of secretion. The EST assembly,BLAST, and signal peptide results were piped into anExcel spreadsheet for manual annotation. Three categoriesof expressed genes derived from the manual annotation ofthe contigs (Table 1). The housekeeping (H) category had24.4% and 17.8% of the clusters and sequences, respec-tively, and an average of 1.6 sequences per cluster. Incontrast, the putatively secreted (S) category contained47.1% of the clusters and 68.2% of the sequences, with anaverage number of 3.2 sequences per cluster. Similar resultswere observed in other transcriptome analysis of salivaryglands in Aedes aegypti, Anopheles gambiae, Ixodes

scapularis and R. prolixus (Francischetti et al., 2002;Ribeiro et al., 2004; Valenzuela et al., 2002). Finally,28.5% of the clusters, containing 14% of all sequences,were classified as unknown (U) because no assignment fortheir putative function could be made; most of theseconsisted of singletons.

3.2. Housekeeping (H) genes

The 192 gene clusters (comprising 305 EST) attributed toH genes expressed in the salivary glands of T. brasiliensis

were further characterized into 17 subgroups according tofunction (Table 2). Two of the largest sets were associatedwith protein synthesis machinery (117 EST in 53 clusters)and with energy metabolism (38 EST in 30 clusters). This is

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Table 1

Types of transcripts found in Triatoma brasiliensis salivary glands

Types of transcripts Clusters Sequences Sequences/cluster % Cluster % Sequence

Secreted 370 1168 3.2 47.1 68.2

Housekeeping 192 305 1.6 24.4 17.8

Unknown 224 239 1.1 28.5 14.0

Total 786 1712

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consistent with an organ specialized in secreting polypep-tides. Also, the 40 EST (37 clusters), representingconserved proteins of unknown function and indicated as‘‘Conserved’’ in Table 2, are presumably associated withcellular metabolism. This group also includes a homologueof the Rhodnius prolixus salivary protein MYS3 precursor(Ribeiro et al., 2004). Twelve clusters code for productsassociated with protein modification function, includingtwo glutathione S-transferase proteins and a glutathioneS-transferase-like protein and, also, two chaperones. Ninetransporters/storage protein genes were also identified,including three coding for energy production and conver-sion, two for inorganic ion transport and metabolism, oneinvolved with intracellular trafficking, secretion, andvesicular transport, and another two coding for carbohy-drate or lipid transport and metabolism. Additionalinformation on each of the 192 gene clusters is availableonline (Supplemental Table S1).

3.3. Possibly secreted proteins and peptides

Supplemental Table S1 indicates the presence of severalgene families previously described in the salivary glands ofR. prolixus (Ribeiro et al., 2004) and T. infestans (NCBI).Remarkably, of the 370 clusters of transcripts possiblyassociated with secretory products, 341 code for proteins ofthe lipocalin family (Flower et al., 2000). A summary ofthese transcripts organized by their abundance and proteinfamily is shown in Table 3.

3.4. Preliminary characterization of the salivary

transcriptome and proteome of T. brasiliensis

Several clusters of sequences coding for housekeepingand putative secreted polypeptides, indicated in Supple-mental Table S1, are abundant and complete enough to

extract consensus sequences of novel cDNA. Additionally,primer extension studies on several clones allowed us toobtain full- or near full-length sequences of products ofinterest. A total of 41 novel sequences, 36 of which code forputative secreted proteins, are grouped in SupplementalTable S2.To obtain information on the most abundant proteins in

the salivary glands of T. brasiliensis, 20 ml of saliva werecollected from starved adult insects to perform chromato-graphic experiments. The ion-exchange chromatography isbetter suited than SDS-PAGE for this purpose becausemost T. brasiliensis salivary proteins are lipocalins with amolecular mass of 18–22 kDa. SDS-PAGE leads to poorseparation of such proteins. Peaks of interest were collectedand submitted to Edman degradation. These results are

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Table 2

Functional classification of the housekeeping genes expressed in Triatoma brasiliensis salivary glands

Types of transcripts Clusters Sequences Sequences/cluster % Cluster % Sequence

Protein synthesis machinery 53 117 2.2 27.6 38.4

Conserved 37 40 1.1 19.3 13.1

Metabolism, energy 30 38 1.3 15.6 12.5

Protein modification 12 29 2.4 6.3 9.5

Transport/storage 9 18 2.0 4.7 5.9

Signal transduction 8 11 1.4 4.2 3.6

Cytoskeletal 9 10 1.1 4.7 3.3

Metabolism, oxidant 5 9 1.8 2.6 3.0

Transcription machinery 7 7 1.0 3.6 2.3

Protein export machinery 5 6 1.2 2.6 2.0

Metabolism, nucleotides 2 4 2.0 1.0 1.3

Proteasome machinery 3 4 1.3 1.6 1.3

Transcription factors 3 3 1.0 1.6 1.0

Metabolism, lipid 3 3 1.0 1.6 1.0

Nuclear regulation 2 2 1.0 1.0 0.7

Metabolism, amino acid 2 2 1.0 1.0 0.7

Metabolism, carbohydrate 2 2 1.0 1.0 0.7

Total 192 305

Table 3

Classification of transcripts coding for putative secreted proteins in

Triatoma brasiliensis salivary glands

Types of transcripts Clusters Sequences %

Sequences

Lipocalins 341 1096 93.8

Kazal domain containing 9 26 2.2

Secreted serine protease 5 29 2.5

Others proteins 5 5 0.4

Similar to Rhodnius binding

protein

2 2 0.2

Inositol phosphatase 2 4 0.3

Similar to Rhodnius MYS

protein

1 1 0.1

Sulfatase—secreted? 1 1 0.1

Similar to Culicoides protein 1 1 0.1

Similar to major epididymal

secreted protein

1 1 0.1

50-nucleotide/apyrase 1 1 0.1

Antigen 5 protein 1 1 0.1

Total 370 1168

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summarized in Fig. 1. Descriptions of the proteinsidentified are provided below.

3.4.1. Putative salivary-secreted proteins for which a

function is known or presumed

From 370 clusters of transcripts possibly associated withsecretory products, 341 (93.8%) are lipocalins. This familycontains salivary proteins from triatomine insects and ticksthat counteract vertebrate host haemostasis events such ascoagulation, vasoconstriction and platelet aggregation(Ribeiro, 1995). All the results obtained in Edman degra-dation experiments were from proteins of this group(Supplemental Table S1).

Lipocalins. Among the lipocalin family, 218 sequenceshad similarity to Triabins from other triatomines. Theseinclude: (i) Triabin, a serine-protease inhibitor that forms anon-covalent complex with thrombin (Noeske-Jungblutet al., 1995); (ii) Pallidipin, an anticollagen that preventsplatelet aggregation (Noeske-Jungblut et al., 1994); (iii)procalin, the major allergen of T. protracta saliva (Paddocket al., 2001); (iv) several triabin-like sequences and (v)others.

Five contigs of T. brasilensis sialome were foundmatching the Nitrophorins. All of them matched theNitrophorin 3B from R. prolixus with identity values thatranged from 31% to 42%. The findings are of interest,because Nitrophorins are hemeproteins found in the saliva

of blood-feeding insects that carry nitric oxide—typical ofRhodnius species. These proteins do not reveal amino acidssequence similar to lipocalins but their predicted crystalstructure is typical of this large family of proteins(Andersen et al., 1997, 1998). Saliva of the blood-suckingbug R. prolixus contains four homologous nitrophorins,named NP1 to NP4, according to their relative abundancein the glands. Combined they represent near 50% of thesalivary protein and confer a deep cherry color to thegland. However, heme-containing proteins have not beendescribed in the saliva of Triatoma species, nor they appearto exist in large concentrations, as the Triatoma salivaryglands are colorless.Lipocalins are remarkably diverse at the amino acid

sequence level, often falling below 20% identity betweenmembers, yet have highly conserved structures (Flower etal., 2000). Functional genomic, proteomic, and functionalstudies have been performed to probe the role of salivarylipocalins in blood-feeding arthropods. In the course ofthese investigations, anticoagulant, antiplatelet, anti-in-flammatory, and vasodilatory molecules have been de-scribed (Andersen et al., 2005). The high number andvariety of lipocalins found in T. brasiliensis has beenreported previously in the saliva of R. prolixus and ticks.Ticks and kissing bugs evolved salivary lipocalins that actas efficient scavengers of biogenic amines, that is of strongadaptive value in the convergent evolution of arthropods tohematophagy (Calvo et al., 2006). As in R. prolixus, theproliferation of lipocalin genes from Triatoma species hasprobably occurred via gene duplication and subsequentdivergence (Ribeiro et al., 2004).A phylogenetic tree containing all published salivary

lipocalins from triatomines including those described inthis work reveals the large divergence of this group ofproteins, indicated by the weak bootstrap support formany of its clades (Fig. 2). In particular, the tree clearlyshows divergence between Rhodnius and Triatoma proteins,and, within Triatoma, it shows several instances of cladeswith strong bootstrap support containing solely membersof one species (although clades with members derivingfrom more than one Triatoma species also exist). Some ofthese closely related proteins from the same speciesproteins could be alleles, but when the amino acid sequencediverge by more than 10% they are most probably derivedfrom gene duplication events. The existence of mono-specific clades is indicative of gene duplication events thatoccurred after the ancestral Triatoma originated thepresent day Triatoma species, supporting the idea thatgene duplication events must have been important in theevolution to blood feeding, as proposed before for ticks(Mans and Neitz, 2004).

Kazal peptides. Nine clusters of sequences displayed theKazal domain signature. The similarity of the contigs withother Kazal inhibitors is shown in Fig. 3A. Among them,TBQ-contig 259 codes for a protein 53% identical toinfestin, the intestinal thrombin inhibitor from T. infestans

(Lovato et al., 2006), coherent to the neighbor-joining tree

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Fig. 1. (A) Strong cation exchange and (B) strong anion exchange

chromatography of 20 ml of saliva from T. brasiliensis starving adult

insects. The bars with number represent the fractions submitted to Edman

degradation to obtain amino terminal sequence information.

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Fig. 2. Phylogenetic tree of lipocalin sequences among triatomine species with bootstrap concordance higher than 85%, obtained by the Neighbour

Joining method. The bar shows 20% divergence at the amino acid level. The numbers in the tree branches indicate the bootstrap value from 10,000

interactions. Red braches—lipocalins from R. prolixus; Green branches—lipocalins from only one Triatoma species; Blue branches—lipocalins from other

Triatoma species; Black branches—lipocalins from R. prolixus and Triatoma species. T. brasiliensis sequences are indicated by TB. Other sequences are

indicated by the first three letter of the genus followed by the first two letters of the species name followed by the GenBank accession number.

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analysis that demonstrated higher similarity to triatomineintestinal inhibitors (Fig. 3B). Predicted amino acidsequence from TBQ-contig 54 (near full-length sequence)was similar to the vasodilator from horse flies (Hybomitra

bimaculata, Diptera, Tabanidae), named vasotab precursor(Takac et al., 2006). Vasotab is a member of the Kazal-typeprotease inhibitor family. Physiologic tests with vasotabdemonstrated positive inotropism in isolated rat hearts,vasodilatation of coronary and peripheral vessels, and Na,K-ATPase inhibition. Accordingly, we can speculate thatthe Triatoma’s vasodilator could be a Kazal peptide. Theremaining six Kazal peptides showed homology to putativesequences of different species but with expected high blastvalues (40.01).

Secreted serine proteases. Five contigs had similarity toserine proteases. Among them, one (TBQ-contig 692)showed 39% identity to a trypsin-like serine protease fromZebrafish (gi|66911393). The four remaining matchedtrypsin-like salivary precursors previously identified in theplant-feeding bug Lygus lineolaris (Zhu et al., 2003),

showing identity to the amino acid level ranging from 31%to 67%. The most abundant contig (TBQ-40), with 24sequences, codes for a protein 41% identical (BLAST Evalue 3E -37) to the L. lineolaris trypsin-like precursor(LlSgP4). The ClustalW alignment from T. brasiliensis andL. lineolaris trypsin-like sequences is shown in Fig. 4. Lygus

bugs ingest plant liquids by inserting their suckingmouthparts into plant tissues where extra-oral digestionis facilitated by the secretion of digestive enzymes from thesalivary glands (Cohen, 1998). Proteolytic activity has beendetected in salivary glands from many mirids, includingL. rugulipennis (Laurema et al., 1985) and Creontiades

dilutus (Colebatch et al., 2001). In triatomines, a trypsin-like activity was described for the T. infestans triapsin(Amino et al., 2001). The protein was identified as aninactive precursor and a second trypsin-like protein may beresponsible for the triapsin activation upon saliva release.Therefore, the trypsin-like sequences found in theT. brasiliensis sialome could be related to saliva processingupon the release on the host skin. Although they showed

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Fig. 3. (A) ClustalW alignment of predicted sequences from seven contigs that showed homology to kazal peptides with other kazal inhibitors—vasotab

from Hybomitra bimaculata (gi94730670), INF2R from T. infestans (gi14211145), dipetalin from Dipetalogaster maximus (gi21465882), rhodniin1 from R.

prolixus (gi1827578). Residues identical in more than four sequences are boxed and gaps are indicated by dashes. Amino acids that define the kazal family

protease inhibitor domain are indicated below the sequences. (B) NJ phylogenetic tree showing the sequence distance between members of the group. The

two most divergent contigs (TBQ-531 and 155) were excluded from the analysis.

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similarity to the typical trypsins with digestive functionthat are capable of cleaving a variety of proteins, thesalivary trypsin-like from haematophagous Hemipteramight be very specific in relation to their substrates. Theseproteases could participate in the processing of salivaryproteins or act in specific targets from the host, once typicaltrypsins could elicit itch in the skin (Thomsen et al., 2002)and increase the possibility of the bug to be perceived bythe host.

Apyrase. Apyrases are enzymes ubiquitously found inthe salivary glands of blood-feeding insects and ticks(Valenzuela et al., 2003). These enzymes, belonging todifferent protein families, degrade the neutrophil-activatingsubstance ATP and the platelet-aggregating nucleotideADP to AMP, presumably facilitating blood feeding.T. infestans apyrase is a member of the 50-nucleotidasefamily (Champagne et al., 1995) and was reported as a setof five different molecular weight proteins (Faudry et al.,2004). The contigs showed 43% similarity to an Aedes

aegypti apyrase. The fact that only one contig with onlyone EST was found suggests that the level of expression inT. brasiliensis is low, and indeed T. infestans apyraseactivity has been recorded to be about 10-fold higher thanthat in T. brasiliensis (Ribeiro et al., 1998).

Inositol phosphatase. Two contigs produced similarity toinositol phosphatases from R. prolixus. The inositolpolyphosphate 5-phosphatase (IPP) enzymes act on bothsoluble inositol phosphate and phosphoinositide substratesand are involved in many cellular processes related tosignal transduction, secretion, and cytoskeletal structure.In R. prolixus, it was previously thought to be responsiblefor the salivary apyrase activity, but later identified with noapyrase activity (Ribeiro et al., 2004). R. prolixus inositolpolyphosphate 5-phosphatase exists as an isolated IPPdomain, which is secreted into the saliva of this blood-feeding insect. It shows selectivity for soluble and lipidsubstrates having a 1,4,5-trisphosphate substitution patternwhile only poorly hydrolyzing substrates containing a D3

phosphate (Andersen and Ribeiro, 2006). The role ofsalivary Inositol phosphatases for triatomine bugs remainsunclear.

Salivary protein MYS2 precursor. One contig showedsimilarity to the R. prolixus MYS-2. The MYS proteinshave no homology to sequences with known functions.Two MYS proteins were identified in the T. brasiliensis

sialome, MYS-2 and MYS-3. Each of these showed highhomology to R. prolixus, probably secreted MYS protein,but while MYS-2 was classified as a secreted protein, theMYS-3 was classified as housekeeping. The R. prolixus

sialome also identified a third member of the family, theMYS-1, which was also predicted as probably secreted andshowed no homology to the ESTs sequenced here (Ribeiroet al., 2004).

Antigen-5-like protein precursor. One cDNA contigindicated similarity to the antigen-5- protein family fromR. prolixus (Ribeiro et al., 2004). These are a widespreadextracellular family of proteins ubiquitously found inanimals and plants with mostly unknown functions(Schreiber et al., 1997; Valenzuela et al., 2003). Closelyrelated proteins from this family have been reported in thesalivary glands of Hymenoptera and Diptera, such as sandflies (Charlab et al., 1999), tsetse (Li et al., 2001),mosquitoes (Francischetti et al., 2002; Valenzuela et al.,2002) and Culicoides sonorensis (Campbell et al., 2005).

Other proteins. Other contigs were identified withhomology to proteins deposited in the GenBank. Theyare a contigs with similarity to sulfatase, similar toC. sonorensis protein, similar to major epididymal secretedprotein and two contigs with similarity to heme- bindingproteins from R. prolixus.

3.5. Concluding remarks

The sialoma of T. brasiliensis showed a high number andvariety of lipocalins. A similar situation has been reportedpreviously in the saliva of R. prolixus and ticks (Ribeiro

ARTICLE IN PRESS

Fig. 4. ClustalW alignment of predicted sequences from four contigs that showed similarity to trypsin-like precursors with five trypsin-like sequences from

L. lineolaris (Zhu et al., 2003). Amino acid gaps are indicated by dashes. Identical or highly similar residues among sequences are highlighted with a black

or gray background. Functionally important residues are boxed. The most divergent contig (TBQ-692) was excluded from the analysis.

A. Santos et al. / Insect Biochemistry and Molecular Biology 37 (2007) 702–712 709

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et al., 2004). Remarkably, the sialoma showed the presenceof a great number of serine proteases that were notobserved in previous sialotranscriptomes. Although mos-quitoes have a chymotrypsin-like enzyme, the number oftranscripts is low. Ticks have a metalloprotease that breaksfibrin into fibrinogen, but it is a completely different familyof proteins.

The presence of the Kazal peptide with high homologyto the tabanid vasodilator is also interesting, suggestingthat the Triatoma vasodilator could be a Kazal protein.

Acknowledgements

We thank Fabrıcio Rodrigues dos Santos and PaulaLara Ruiz from the Laboratorio de Biodiversidade eEvoluc- ao Molecular (ICB/UFMG), and Cristiane QuimelliSnoeijer and Carlos Rodrigo Bueno from the Laboratoriode Protozoologia, (MIP—CCB/ UFSC) for helping in themass sequencing reactions. This work was supported by theWellcome Trust Fundation, CNPq, CAPES, Fapemig,ECLAT and the Intramural Research Program of theNational Institute of Allergy and Infectious Diseases.

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