Experimental Parasitology 137 (2014) 25–34

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Experimental Parasitology

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

A family of serine protease inhibitors (serpins) in the cattle tickRhipicephalus (Boophilus) microplus

0014-4894/$ - see front matter � 2013 Elsevier Inc. All rights reserved.http://dx.doi.org/10.1016/j.exppara.2013.12.001

⇑ Corresponding author at: Centro de Biotecnologia, Universidade Federal do RioGrande do Sul, Avenida Bento Gonçalves 9500, Prédio 43421, Porto Alegre 91501-970, RS, Brazil. Fax: +55 (51) 33087309.

E-mail address: itabajara.vaz@ufrgs.br (I. da Silva Vaz Jr.).

Lucas Tirloni a, Adriana Seixas a,b, Albert Mulenga c, Itabajara da Silva Vaz Jr. a,d,⇑, Carlos Termignoni a,e

a Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Avenida Bento Gonçalves 9500, Prédio 43421, Porto Alegre 91501-970, RS, Brazilb Departamento de Ciências Básicas da Saúde, Universidade Federal de Ciências da Saúde de Porto Alegre, Rua Sarmento Leite 245, Porto Alegre 90050-170, RS, Brazilc Department of Entomology, Texas A&M University, Minnie Heep Center, College Station, TX, USAd Faculdade de Veterinária, Universidade Federal do Rio Grande do Sul, Avenida Bento Gonçalves 9090, Porto Alegre 91540-000, RS, Brazile Departamento de Bioquímica, Universidade Federal do Rio Grande do Sul, Rua Ramiro Barcelos 2600, Porto Alegre 90035-000, RS, Brazil

h i g h l i g h t s

� Eighteen full-length serpin sequenceswere found in Rhipicephalus(Boophilus) microplus.� R. microplus serpins are conserved

across other tick serpins.� Genes are differentially expressed in

analysed tissues.� Tick serpins possesses a serpin

conserved tertiary structure.

g r a p h i c a l a b s t r a c t

a r t i c l e i n f o

Article history:Received 17 June 2013Received in revised form 29 November 2013Accepted 3 December 2013Available online 11 December 2013

Keywords:SerpinTickInhibitorPhysiologyBlood-feeding

a b s t r a c t

Proteins belonging to the serine protease inhibitor (serpin) superfamily play essential roles in manyorganisms. In arthropods these proteins are involved in innate immune system, morphogenesis anddevelopment. In mammals serpins regulate pathways that are essential to life such as blood coagulation,fibrinolysis, inflammation and complement activation, some of which are considered the host’s first lineof defense to hematophagous and/or blood dueling parasites. Thus, it is hypothesized that ticks use ser-pins to evade host defense, facilitating parasitism. This study describes eighteen full-length cDNAsequences encoding serpins identified in Rhipicephalus (Boophilus) microplus, here named RmS 1–18 (R.microplus serpin). Spatial and temporal transcriptional profiling demonstrated that R. microplus serpinsare transcribed during feeding, suggesting their participation in tick physiology regulation. We speculatethat the majority of R. microplus serpins are conserved in other ticks, as indicated by phylogeny analysis.Over half of the 18 RmSs are putatively functional in the extracellular environment, as indicated by puta-tive signal peptides on 11 of 18 serpins. Comparative modeling and structural-based alignment revealedthat R. microplus serpins in this study retain the consensus secondary of typical serpins. This descriptivestudy enlarges the knowledge on the molecular biology of R. microplus, an important tick species.

� 2013 Elsevier Inc. All rights reserved.

1. Introduction industry worldwide due to its spoliation action and its role of

The tick Rhipicephalus (Boophilus) microplus is a hematophagousectoparasite that infests bovines, with significant impact on cattle

vector of the etiologic agents of babesiosis and anaplasmosis (Grisiet al., 2002; Jonsson, 2006). Most tick control strategies to datehave resorted to the use of acaricides. Although these chemicalsoffer effective protection in the short term, they nevertheless failto offer a more permanent solution to tick infestations, due to seri-ous limitations such as selection of acaricide-resistant tick popula-tions, not to mention environment and food chain contamination

26 L. Tirloni et al. / Experimental Parasitology 137 (2014) 25–34

hazards (Guerrero et al., 2012). To solve this problem, considerableresearch efforts have been devoted to develop alternative tick con-trol methods (Parizi et al., 2012; Seixas et al., 2012). In this sense,one of the approaches to select targets consists of the identificationof tick proteins that play key roles in tick survival, such as acquisi-tion and digestion of blood meal and modulation of host immuneresponse (Mulenga et al., 2001). The purpose of this study is to en-large the knowledge on the tick serine protease inhibitors in R.microplus allowing to explore the role(s) of these proteins in tickphysiology.

Serpins (serine protease inhibitors) are a superfamily of pro-teins containing between 350 and 500 amino acid residues withmolecular weights ranging from 40 to 60 kDa (Gettins, 2002).Although intracellular serpins have been described, most serpinsoperate in the extracellular environment (Silverman et al., 2001;Gettins, 2002). Over 500 members of the serpin superfamily havebeen identified in animals, plants, bacteria, archaea and viruses(Irving et al., 2000). In mammals, most serpins play crucial rolesin the control of endopeptidases that mediate important pathwayssuch as blood coagulation, fibrinolysis, inflammation and comple-ment activation (Silverman et al., 2001; Rau et al., 2007). Asendopeptidases and serpins play pivotal roles in mammalianhomeostasis maintenance, it has been hypothesized that ticks uti-lize serpins to disrupt the host homeostatic balance in order tofacilitate parasitism (Mulenga et al., 2001). As in mammals, serpinsregulate a similarly wide range of physiological responses inarthropods. In some arthropods these inhibitors are involved inthe regulation of endogenous endopeptidases, protection againstmicrobial endopeptidases, hemolymph coagulation, phenoloxidaseactivation, regulation of the Toll pathway as well as reproductionand development (Gulley et al., 2013).

The consensus serpin structure contains three b-sheets (sA, sBand sC) and eight or nine a-helices (hA–hI), and possesses a reac-tive center loop (RCL). RCL is a solvent exposed flexible stretch of21 amino acid residues positioned between b-sheets sA and sC,which acts as a pseudo-substrate of the specific target endopepti-dase (Gettins, 2002). RCL structure extends from residue P17 toresidue P04, in the Schechter and Berger (1967) notation, and aregenerally recognized by a consensus pattern P17½E�—P16½E=K=R�—P15½G�—P14½T=S�—P13½X�—P12—9½A=G=S�—P8—1½X�—P01—P04½X�. RCL con-tains the peptide bond between residues P1 and P01, which iscleaved by the target endopeptidase, where the P1 residue in RCLis critical to define the specificity of a serpin for a particular endo-peptidase (Hopkins et al., 1993; Hopkins and Stone, 1995; Irvinget al., 2000). After cleavage, serpin covalently traps and distortsthe target endopeptidase. Therefore, serpins are classified as ‘‘sui-cide’’ or ‘‘single use’’ inhibitors (Huntington et al., 2000). Basedon consensus serpin amino acid motifs and secondary structures,a number of serpin-encoding nucleotide sequences have beenidentified in various tick species, including Ixodes ricinus (Leboulleet al., 2002a), Haemaphysalis longicornis (Sugino et al., 2003; Imam-ura et al., 2005), Rhipicephalus appendiculatus (Mulenga et al.,2003), Amblyomma americanum (Mulenga et al., 2007), Amblyommacajennense (Batista et al., 2008), Dermacentor variabilis (Andersonet al., 2008), Ixodes scapularis (Mulenga et al., 2009), Amblyommavariegatum (Ribeiro et al., 2011), Amblyomma maculatum (Karimet al., 2011), Antricola delacruzi (Ribeiro et al., 2012), Rhipicephaluspulchellus (Tan et al., unpublished) and Rhipicephalus haemaphysa-loides (Yu et al., 2013). Only one serpin-encoding gene has been re-ported in R. microplus (Kaewhom et al., 2007).

A limited number of studies have demonstrated that recombi-nant tick serpins have anticoagulant and immunosuppressiveactivities (Leboulle et al., 2002b; Imamura et al., 2005; Prevotet al., 2006, 2009; Mulenga et al., 2013). Moreover, mortality andreduced feeding efficiency have been reported when H. longicornis(Sugino et al., 2003), R. appendiculatus (Imamura et al., 2006, 2008),

I. ricinus (Prevot et al., 2007) or R. microplus (Jittapalapong et al.,2010) were fed on hosts immunized with tick recombinant serpins.These observations point to the importance of these inhibitors intick physiology, tick-host interaction and also as a target for tickcontrol.

This study reports the identification and characterization of 18serpin-encoding sequences in the cattle tick, which were named R.microplus serpin (RmS). cDNA and amino acid sequences were usedto characterize the serpin gene family and to examine serpinexpression profile among different tissues and developmentalstages of the cattle tick.

2. Materials and methods

2.1. Identification of R. microplus serpins

Source sequences encoding R. microplus serpins were foundusing BLAST (Basic Local Alignment and Search Tool) with theBLASTP, BLASTN and BLASTX algorithms (Altschul et al., 1990)against Bmi Gene Index (BmiGI) available at DFCI (http://comp-bio.dfci.harvard.edu/tgi/) (Guerrero et al., 2005; Wang et al.,2007; Saldivar et al., 2008; Bellgard et al., 2012), and a R. microplustranscriptome database (RmINCT-EM) created by our researchgroup using Illumina sequencing (unpublished). Twenty-six ser-pins from other ticks and seven serpins from mammals, all se-quences deposited in GenBank (Table S1), were used as queriesagainst these databases. About 52 expressed sequence tags (ESTs)were found, and sequences with >95% nucleotide identity wereused to assemble complete coding sequences using the contigassembly program (CAP) (Huang, 1992).

To validate the accuracy of sequence encoding serpin identifica-tion, assembled sequences were inspected for the presence of start-codon and stop-codon, amino acid length (350–450 amino acids oftranslated protein sequences), and the presence of two amino acidsmotifs described as conserved in known serpins: NAVYFKG andDVNEEG (Miura et al., 1995; Han et al., 2000). To confirm the iden-tity of the assembled sequences, the deduced amino acid sequenceswere scanned against GenBank using the BLASTP homology searchprogram against the non-redundant protein sequence database andagainst amino acid motif in ScanProsite (Sigrist et al., 2010).

2.2. Bioinformatic analyses

Analyses of deduced amino acid sequences were carried outusing the GeneDoc software (Nicholas et al., 1997) and BioEdit 7.1(Hall, 1999). Sequence alignment was performed using the Musclealgorithm (Edgar, 2004) in the MEGA5.1 program (Tamura et al.,2011). The presence of secretion signal sequence was predictedusing the SignalP 4.0 server (Petersen et al., 2011). Putative N-glyco-sylation sites were found using the NetNGly1.0 server (Gupta et al.,unpublished, http://cbs.dtu.dk/services/NetNGlyc/). Theoreticalmolecular weight and isoelectric points of the mature serpin pro-teins were calculated using the Compute pI/Mw tool via the ExPASywebsite (http://web.expasy.org/compute_pi/pi_tool-Ref.html).

2.3. Biological material

Ticks were obtained from a R. microplus colony (Porto Alegrestrain) maintained at the Faculdade de Veterinária of UniversidadeFederal do Rio Grande do Sul, Brazil. Ticks were reared on calves,which were brought from a naturally tick-free area and maintainedin individual boxes. Calves were infested with 10-day-old larvae.After 21 days, partially engorged females (P) were manually re-moved from the cattle and fully engorged females (F) droppedfrom calves were collected and dissected to obtain of the tissues.

L. Tirloni et al. / Experimental Parasitology 137 (2014) 25–34 27

2.4. Tick dissection and RNA extraction

Ticks were rinsed with 70% ethanol. The dorsal surface was dis-sected with a scalpel blade. Salivary glands (SG), midguts (MG) andovaries (OV) were separated with fine-tipped forceps. The remain-ing material was named carcass (CA), representing the tickremnant after removal of these tissues. All tissues were dissectedwithin 2 h and washed with phosphate-buffered solution (PBS) inan RNase free environment. The dissected tissues were rapidlyremoved and placed in a tube containing TRIzol� reagent (Invitro-gen, USA). Total RNA was extracted according to the manufac-turer’s recommendations. The RNA samples were resuspended indiethylpyrocarbonate (DEPC)-treated water. RNA concentrationand purity were determined using a spectrophotometer (Pharma-cia Biotech, Sweden).

2.5. Semi-quantitative RT-PCR

To get an insight into tissue distribution profiles of RmS se-quences, gene specific primers for each RmS sequence (ORF) wereused (Table S2) to investigate the mRNA transcription patterns oftissues mentioned above. OligodT primed cDNA was synthesizedfrom 5 lg of total RNA using the SuperScript III kit (Invitrogen,USA). Five hundred nanogram of resulting cDNA was used as tem-plate for RT-PCR using Taq DNA Polimerase (Ludwig Biotecnologia,Brazil). The reactions were performed according to the followingsteps: 5 min at 94 �C followed by 35 cycles of 30 s at 94 �C, 30 sat 55 �C, and 1 min and 30 s at 72 �C, with a final elongation at72 �C for 5 min. PCR products were electrophoresed on a 0.8% aga-rose gel and visualized by staining with ethidium bromide. Tick ac-tin primers were used as positive control (da Silva Vaz et al., 2005).

2.6. cDNA cloning of R. microplus serpins

To confirm assembled sequences, RmS-1, RmS-3, RmS-6, RmS-7,RmS-9, RmS-13, RmS-15, RmS-17 and RmS-18 were cloned andsequenced. The amplifications were carried out by RT-PCR using500 ng of resulting cDNA (see above) and Elongase� Enzyme(Invitrogen, USA). Reactions were performed as cycles: 5 min at94 �C followed by 35 cycles of 30 s at 55 �C and 1 min and 30 s at68 �C, with a final elongation at 68 �C for 5 min. Amplified ampli-cons were purified using GeneClean� II Kit (MP Biomedicals,USA) according to the manufacturer’s instructions. Purified ampli-cons were cloned into pGEM-Teasy (Promega, USA) according tothe manufacturer’s protocol. Cloning was confirmed by automatedsequencing.

2.7. Phylogenetic analysis

Multiple amino acids sequences alignments of RmS and othertick serpins retrieved from GenBank (Accession Nos. in Fig. 4) werecreated using the Muscle algorithm (Edgar, 2004) in the Mega5.1program (Tamura et al., 2011). The phylogenetic analyses wereperformed according to the neighbor-joining method (Kumaret al., 2008). Gapped positions were treated by complete deletion.Poisson correction was used as a substitution model to determinepairwise distances. Confidence was determined using bootstrapvalues at 1000 replicates.

2.8. Structure-based alignment and molecular modeling

To get an insight into the RmS structure, selected sequences(RmS-3, RmS-6, RmS-9, RmS-13, RmS-15 and RmS-16) weresubjected to structural based alignment using STRAP (StructureBased Alignment Program) (Gille and Frömmel, 2001) and theMuscle algorithm (Edgar, 2004). The secondary structures were

superimposed on the structurally aligned sequences using the hu-man a1-antitrypsin tertiary structure (PDB 1HP7) as template (Kimet al., 2001). The alignment sequences were subsequently viewedusing the GeneDoc software (Nicholas et al., 1997).

These mature putative secreted RmS sequences were subjectedto molecular modeling using the SWISS-MODEL workspace in theautomated mode (Arnold et al., 2006). The best 3D structure tem-plates were predicted using the alignment of our sequences againststructures of Protein Data Bank (PDB) proteins using BLASTP(Altschul et al., 1990). QMEAN4 was the method used to estimatemodel reliability and predict quality (Benkert et al., 2009). Illustra-tions of the 3D structures were generated using PyMol (DeLano,2002).

3. Results

3.1. Identification of R. microplus serpins (RmS) encoding sequences

3.1.1. Sequence analysisEighteen full-length sequences encoding putative serpins were

successfully identified through BLAST, probing R. microplus ESTdatabases with available tick and mammalian serpin sequences(Table 1). These sequences were named RmS 1–18, and were se-lected for detailed studies.

3.1.2. Serpin feature of RmSPredicted amino acid sequences showed the presence of the

two consensus sequences found in serpins: N-[AT]-[VIM]-[YLH]-F-[KRT]-[GS] and [DERQ]-[VL]-[NDS]-E-[EVDKQ]-G, correspondingto the conserved serpin amino acid motifs NAVYFKG and DVNEEG(Fig. S1). All RmS predicted polypeptides showed high similarity toseveral known tick serpins available in GenBank (Table 2). Scan-ning for amino acid sequence patterns on the ScanProsite demon-strated that most deduced proteins contain the serpin signaturePS00284 ([LIVMFY]-[G]-[LIVMFYAC]-[DNQ]-[RKHQS]-[PST]-F-[LIV-MFY]-[LIVMFYC]-X-[LIVMFAH]), except for RmS-5, RmS-13 andRmS-14.

3.1.3. RmS polypeptide propertiesDeduced RmS amino acid sequences are in accordance with typ-

ical serpin size, ranging from 375 to 410 amino acid residues(Fig. S1) with molecular weight and theoretical isoelectric pointof the mature putative proteins ranging from 41 to 44.8 kDa and5.35 to 9.08, respectively (Table 2). All RmS were predicted to haveone to four potential N-glycosylation sites (N-X-[T/S]), except forRmS-13 and RmS-18, which do not possess any such sites.Scanning for signal peptides revealed that 11 out of 18 deducedproteins possess leader sequence, indicating that they are poten-tially secreted serpins (Table 2).

3.1.4. RmS reactive center loop (RCL)Most RmS have the consensus sequence typical of the RCL hinge

region, a characteristic common to all inhibitory serpinsP17½E�—P16½E=K=R�—P15½G�—P14½T=S�—P13½X�—P12—9½A=G=S�—P8—1½X�ð

—P01—P04½X�Þ, with some variations among them. RmS-12 has thelowest number of conserved residues in the hinge region (G-L-F-L residues in the P12–9 position). Of the 18 serpin sequences,17 showed conservation of the threonine side chain at the P8 po-sition, consistent with inhibitory serpin characteristics (Gettins,2002). A small serine side chain residue replaced the threonineside chain at P8 only in RmS-7. Although the putative P1 residues(which determines primary inhibitor specificity) are variable(Fig. 1), 44.4% (8/18) are basic (arginine or lysine) residues.

Table 1Accession Nos. of RmS sequences.

Serpin IDa TIGRb NCBIc

RmS-1 TC24850 KC990100RmS-2 CV442791, CV442792 KC990101RmS-3 TC16894 KC990102RmS-4 TC22306, CV440429, EST894342 KC990103RmS-5 TC16400, EST904431 KC990104RmS-6 TC15941 KC990105RmS-7 TC15217 KC990106RmS-8 – KC990107RmS-9 – KC990108RmS-10 – KC990109RmS-11 – KC990110RmS-12 TC16456, TC20312, TC15274 KC990111RmS-13 TC17980 KC990112RmS-14 TC23827 KC990113RmS-15 – KC990114RmS-16 TC15356 KC990115RmS-17 – KC990116RmS-18 TC18048 KC990117

a Sequences encoding R. microplus serpins were named RmS, in an acronymrepresenting R. microplus serpin.

b Nucleotide sequences retrieved from the DFCI Boophilus microplus gene index –TIGR used to assemble complete RmS coding sequences.

c GenBank Access No. of RmS sequences characterized in this article.

28 L. Tirloni et al. / Experimental Parasitology 137 (2014) 25–34

3.2. Expression pattern analysis

Semi-quantitative RT-PCR analysis showed that 16 out of 18RmS mRNA were transcribed (Fig. 2). RmS-1 and RmS-2 weretranscribed in all analyzed tissues, with predominant expressionobserved in ovary (POV and FOV), and lower transcription detectedin FCA. RmS-3 transcripts were found in most of the analyzedtissues, except POV, FOV and FMG, and its transcription is up-reg-ulated in PSG and FSG. RmS-4 and RmS-10 were specific to bothovary stages (POV and FOV). Apparently, RmS-6 and RmS-17 wereuniformly expressed in all tissues. RmS-7 was transcribed only inpartially engorged female tissues, except in CA and OV, where aweak transcription was observed in fully engorged females. Exceptfor PCA and FCA, all tissues transcribed RmS-8. RmS-9 was tran-scribed in all tissues, but was more abundant in fully engorged

Table 2Polypeptide features of R. microplus serpins (RmS) sequences.

ID SPa Putative functionb N-gly sitesc

RmS-1 � Inhibitory 2RmS-2 � Inhibitory 1RmS-3 + Inhibitory 2RmS-4 + Inhibitory 3RmS-5 + Inhibitory 1RmS-6 + Inhibitory 2RmS-7 + Inhibitory 1RmS-8 � Inhibitory 4RmS-9 + Inhibitory 1RmS-10 + Inhibitory 3RmS-11 � Inhibitory 2RmS-12 � Non-inhibitory 1RmS-13 + Inhibitory 0RmS-14 � Inhibitory 4RmS-15 + Inhibitory 2RmS-16 + Inhibitory 3RmS-17 + Inhibitory 3RmS-18 � Inhibitory 0

a Signal peptide (SP) was predicted using the SignalP 4.0 server, symbols (+) and (�)b Prediction to inhibitory or non-inhibitory function was based on the RCL consensusc Putative N-glycosylation sites were predicted using the NetNGlyc 1.0 server, basedd Molecular weight (MW) and theoretical isoelectric point (pI) were calculated usinge The best matches identities were obtained using BLASTP against the non-redundant p

(R. pulchellus serpin), RaS (R. appendiculatus serpin), Lospin (Amblyomma americanum se

female MG and SG. RmS-11 and RmS-12 had a weak transcription,solely in FOV and POV, respectively. RmS-13 was transcribed in alltissues, except in PMG and FMG. RmS-14 was transcribed in all tis-sues, except FCA. RmS-15 and RmS-16 had a nearly similar tran-scriptional profile, with RmS-16 not transcribed in PSG. RmS-18was transcribed in most tissues analyzed, except in PSG. RmS-5and RmS-14 were not transcribed in the tissues analyzed.

3.3. Phylogenetic analysis

Amino acid sequences of all RmS and serpin sequences of othertick species (RpS – R. pulchellus serpin, RaS – R. appendiculatus ser-pin, lospin – A. americanum serpin and IsS – I. scapularis serpin)were used to analyze the phylogenetic relationship among tick ser-pins. The analysis showed the presence of two major groups of tickserpins. Group A comprises potentially secreted serpins formed byclusters A–G. Group B comprises the potentially intracellular ser-pins, formed by the clusters H–K. The 11 phylogenetic clustersare supported by bootstrap values of 58% for cluster E, 95% for clus-ter J, 99% for cluster H and 100% for cluster A, B, C, D, F, G, I and K(Fig. 3).

Group A: RmS-17 was grouped with RpS-2 in cluster A. Theseserpins show a K residue at the P1 position in RCL. Serpins of thiscluster are closely related with RaS-4, lospin-8, lospin-9, lospin-10, AmS-5 and AmS-7. In cluster B, RmS-15 was grouped withRpS-6, and both are related with AmS-3 and AmS-9. Cluster B isformed by serpins sharing 100% identity in RCL, with an R at theP1 position. Cluster C is formed by RmS-6, RpS-3 and AmS-6. All se-quences in this cluster share have R residue at the P1 position inRCL. In cluster D, RmS-9 was grouped with RpS-5, sharing a se-quence identity of 90% and has a K at RCL the P1 position. In clusterF, RmS-5 was grouped with RhS-1, which is related with lospin-1,lospin-2 and lospin-3. Serpins in this cluster have a K residue at theP1 position in RCL, and only lospin-2 and lospin-3 are potentiallyintracellular serpins. In cluster G, RmS-4 and RmS-10 grouped inthe same branch. The expression profile of these sequences issimilar; both were predominantly transcribed in ovaries. RmS-3grouped in another branch with RaS-3, which are related withHlS-2 and lospin-12. RmS-3 possesses close identity with RaS-3(93%), HlS-2 (70%) and lospin-12 (71%), all sequences display

MW (kDa)d pId Best match (% identity)e

42.5 6.36 RpS-4 JAA54313.1 (89)42.7 5.65 RpS-9 JAA54312.1 (91)41.5 5.35 RaS-3 AAK61377.1 (93)42.0 5.81 Lospin-4 ABS87356.1 (64)42.0 9.07 RhS-1 AFX65224.1 (87)41.9 5.72 RpS-3 JAA54308.1 (90)42.3 5.74 RpS-14 JAA63258.1 (98)42.9 5.95 RpS-13 JAA54311.1 (88)41 8.73 RpS-5 JAA54309.1 (90)41.9 5.67 Lospin-4 ABS87356.1 (64)42.6 6.05 RpS-1 JAA54314.1 (90)44.8 5.59 IsS-2 XP_002401187.1 (59)43.5 5.90 RpS-7 JAA54167.1 (95)41.5 6.27 RpS-15 JAA54312.1 (88)42.6 7.98 RpS-6 JAA54307.1 (93)41.9 9.08 Lospin-8 ABS87360.1 (44)41.5 5.75 RpS-2 JAA54310.1 (91)41.1 5.88 RpS-16 JAA63611.1 (64)

represent presence and absence of signal peptide sequence, respectively.sequence (Hopkins et al., 1993).

on the putative N-glycosylation site sequence N-X-T/S.the Compute pI/Mw tool via the ExPASy website.rotein database in GenBank. The numbers represents the GenBank Accession No. RpSrpin) and IsS (Ixodes scapularis serpin).

ID P17 P16 P15 P14 P13 P12 P11 P10 P9 P8 P7 P6 P5 P4 P3 P2 P1 P1'P2' P3' P4'RmS-1 E E G T E A A A A T A V M M A A C C L S SRmS-2 E E G T E A A A A T A V T L M T Y C A R IRmS-3 E E G T I A T A V T G L G F V P L S A H YRmS-4 E E G T I A A A V T G L F V M P S S S L YRmS-5 E Q G T V A A A V T A I R V S V K S G K SRmS-6 E E G T E A A A V T G V I G V N R I G I ERmS-7 E K G T E A V A L S S G I V R H S K T P GRmS-8 E E G T E A A A A T A V V M G F G C S A NRmS-9 E E G S E A D S A T L L R I S G K A A E ERmS-10 E E G T V A A A V T G L F V R P T A P L PRmS-11 E E G T E A A A A T A V M M V A C C M S SRmS-12 E D G V E G L F L T P L I M M C Y A G V SRmS-13 E V G T R A V A A T Q A Q F V S K S L V HRmS-14 E E G T E A A A A T G V T M M T Y C A R MRmS-15 E E G S E A A A V T G F V I Q L R T A A FRmS-16 E E G S E A A A V T G V T I N T R T T T GRmS-17 E E G S E A A G A T A V I F Y T K S A V VRmS-18 E E G T E A A A A T G M V A M A R C A S M

scissile bond

P17[E]-P16[E/K/R]-P15[G]-P14[T/S]-P13[X]-P12-9[A/G/S]-P8-1[X]-P1’-P4[X]

Fig. 1. Sequence alignment of 18 R. microplus serpin putative reactive center loops (RCL). The residues in RCL are numbered according to the nomenclature developed bySchechter and Berger (1967), where residues on the amino-terminal side of the scissile bond P1—P01

� �are noted as primed and those on the carboxyl-terminal side are

primed. Residues were marked in gray in accordance with the consensus sequence of RCL. The boxed residues representing the scissile-bond and bold-faced residues werepredicted P1.

L. Tirloni et al. / Experimental Parasitology 137 (2014) 25–34 29

similar P1 residue in RCL, a L for RmS-3, RaS-3, HlS-2 and an I forlospin-12. Curiously, serpins of this cluster have poly-proline(PPPP) after the RCL sequence, a feature absent in most serpins.

Group B: In cluster H, RmS-18 grouped with AmS-1, the aminoacid sequences have 61% identity and a R residue at P1 in RCL. Incluster I, RmS-8 was grouped with RpS-13, sharing a sequenceidentity of 87% and having a residue G at RCL P1 position. In clusterJ, RmS-1 appears closely related to RaS-1 and RpS-4, sharing a se-quence identity of 82% and 89%, respectively. In another branch,RmS-11 was grouped with RpS-1, sharing a sequence identity of89%. The serpins from this cluster share 95% identity in RCL andhave C at the P1 position. In cluster K, RmS-2 is closely related toRpS-9, RhS-2, and RaS-2. These serpins share a Y residue at theP1 RCL position.

RmS-12, RmS-14 and RmS-16 are distantly related to any othertick serpin, they segregated alone in separated branches. However,RmS-16 is related with mammalian serpins. When we used bovineantithrombin as outlier, they segregated in the same branch (datenot shown).

3.4. Serpin conserved structure of RmS

A structure-based sequence alignment of some putative se-creted proteins transcribed in salivary gland (RmS-3, RmS-6,RmS-9, RmS-13, RmS-15 and RmS-16) revealed that all have threeb-sheet (sA, sB and sC) and nine a-helices (from hA to hI), present-ing a typical serpin secondary structure (Fig. 4). In order to confirmthese features, the tertiary structures of RmS proteins were mod-eled using the SwissModel Server (Fig. S2). For RmS-3, the modelwas generated from amino acid 5 to 382 using heparin cofactor II(1JMJ) as template (28% identity). This model has a QMEAN4 scoreof 0.612 and Z-scores of �2.543. The RmS-6 model was generatedfrom amino acid 2 to 373 and plasminogen activator inhibitor 1(1DB2) was used as template (26% identity). This model had aQMEAN4 score of 0.541 and Z-score of �3.748. The RmS-9 model

was generated from amino acid 2 to 375 using the human anti-thrombin (1ATH) as template (25% identity), with a QMEAN4 scoreof 0.524 and a Z-score of �3.966. RmS-13 was generated from ami-no acid 23 to 389 using native plasminogen activator inhibitor 1(1DB2) as template (29% identity) and had a QMEAN4 score of0.546 and a Z-score of �3.696. RmS-15 was generated from aminoacid 14 to 382 using the native activated protein C inhibitor (2OL2)as template (30% identity) and had a QMEAN4 score of 0.585 and aZ-score of �3.051. RmS-16 was generated from amino acid 7 to 380using the antithrombin complexed with factor IXa and a pentasac-charide (3KCG) as template (34% identity) and had a QMEAN4score of 0.582 and a Z-score of �3.021.

All RmSs comparative models showed a three-dimensionalstructure typical of native inhibitory serpins with a RCL completelyexposed (Fig. S2). Furthermore, comparing 219 serpins sequences,Irving et al. (2000) identified 51-core amino acid residues as beingassociated to the inhibitory action of serpins. Adapting this analy-sis aligning all 18 tick RmS sequences with the human a1-antitryp-sin (1HP7) revealed that the 51-core residues that underpin theefficient functioning of an inhibitory serpin are 68–90% conserved(Fig. S1).

4. Discussion

Analysis of transcriptomic and proteomic data has led to thediscovery of large families of serpins in multicellular organisms(Irving et al., 2000; Silverman et al., 2001; Rawlings et al., 2004)including ticks (Leboulle et al., 2002a; Sugino et al., 2003; Imamuraet al., 2005; Mulenga et al., 2003, 2007, 2009; Ribeiro et al., 2011,2012). In ticks, these data provide evidence that these arthropodsencode a large and variable family of serpins. The analysis ofI. scapularis genome data showed the presence of 45 putative ser-pins genes (Mulenga et al., 2009). Moreover, transcriptomic andproteomic analysis revealed seventeen serpin coding sequencesin A. americanum (Mulenga et al., 2007), four in R. appendiculatus

Fig. 2. The transcription profile of RmSs was analyzed using cDNA synthesized fromtotal RNA extracted from partially engorged (P) and fully engorged (F) R. microplusfemales salivary gland (SG), midgut (MG), ovaries (OV) and carcass (CA, represent-ing the tick remnant after removal of the tissues mentioned). Tick actin was used asinternal RT-PCR control. The primers used in the RT-PCR analysis are listed onTable S2.

30 L. Tirloni et al. / Experimental Parasitology 137 (2014) 25–34

(Mulenga et al., 2003), two in H. longicornis (Sugino et al., 2003;Imamura et al., 2005), thirty-two in A. maculatum (Karim et al.,2011), six in I. ricinus (Leboulle et al., 2002a; Chmelar et al.,2011), four in A. variegatum (Nene et al., 2002; Ribeiro et al.,2011), nine in A. cajennense (Batista et al., 2008) and one in A. del-acruzi (Ribeiro et al., 2012) and R. microplus (Kaewhom et al., 2007).

The analyses of two R. microplus-transcript databases (RmINCT-EM and BmiGI) allowed the identification of 18 full-length ORFswith similarity to tick serpin encoding sequences. Sequence analy-ses revealed that all 18 RmS sequences have the best-match to ser-pins from R. pulchellus (Tan et al., unpublished), R. appendicullatus(Mulenga et al., 2003), A. americanum (Mulenga et al., 2007),I. scapularis (Mulenga et al., 2009) or A. maculatum (Karim et al.,2011). Eleven of the 18 RmS are putative secreted proteins, sincethey have a signal peptide sequence. A recent study showed thattick-resistant bovine sera recognize recombinant RmS-3, suggest-ing that the native serpin is secreted into the bite site (Rodri-guez-Valle et al., 2012). The presence of this serpin in salivacould be responsible for the anticoagulant properties of tick saliva(Horn et al., 2000). Similarly to other blood-sucking arthropods,ticks need to avoid host defense mechanisms (hemostasis andimmunity) systems, which are regulated by serpins (Ribeiro,

1987; Gettins, 2002; Rau et al., 2007). Therefore, serpin presencein tick saliva suggests that these proteins might contribute to mod-ulate host hemostasis and immunity after tick feeding (Leboulleet al., 2002a; Chalaire et al., 2011; Chmelar et al., 2011; Mulengaet al., 2013).

The numbers of secreted and non-secreted serpins observed inR. microplus have been shown to be similar, since the 11 out of 18RmS have a signal peptide. This proportion differs from otherarthropods, where the majority of known serpins exist as an extra-cellular form (Reichhart, 2005; Mulenga et al., 2007, 2009). How-ever, serpins without a classical secretory signal might eitherfunction in the intracellular compartment, controlling endogenousproteolytic pathways, or are exported by non-canonical secretorypathways (Silverman et al., 2001). Consistent with the fact thatalmost all known serpins are glycosylated, 16 out of 18 RmS haveN-glycosylation sites, a general feature of serpins and are impor-tant for biological activity (Rau et al., 2007).

The gene expression profile analysis provides an insight into thebiological relationship of serpins concerning tick feeding biology.Our RT-PCR results revealed that RmSs are largely transcribed intick tissues, though at different transcription levels, suggesting thatthey regulate different pathways in tick physiology. Transcriptionof some putative secreted RmS in SG and MG (RmS-3, RmS-6,RmS-9, RmS-13, RmS-15, RmS-16 and RmS-17) suggests that theyplay a role in hematophagy, allowing the blood meal uptake andmaintaining blood in a fluid state at the feeding site and afteringestion. The detection of transcripts in internal organs impliesthat RmSs also play a role regulating other biological systems.Indeed, most arthropod serpins are involved in innate immune re-sponse regulation (Gulley et al., 2013). Proteolytic cascades take acentral role in many immune reactions, amplifying signals in sev-eral defense mechanisms after pathogen invasion (Silverman andManiatis, 2001). Many of these cascades are regulated by serpins,including the hemolymph coagulation cascade (Iwanaga et al.,1998), the activation of the Toll pathway (Reichhart, 2005) andpro-phenoloxidase (proPO) activation for melanization (Cereniuset al., 2008). Also, serpins have been found in insect male accessoryglands, indicating that they play a role in reproduction (Colemanet al., 1995), and that they are essential for dorsal–ventral axis for-mation and wing expansion (Charron et al., 2008).

The absence of RmS-5 and RmS-14 transcripts in all analyzedtissues is not surprising, because these sequences are present inthe BmiGI database but not in the RmINCT-Rm database. This isa consequence of the differences between these two databases,since the BmiGI database was constructed using a mixture ofdevelopment stages submitted to different environmental condi-tions, and RmINCT-EM was constructed using ovary, synglanglion,salivary glands, fat body and embryo. Also BmiGI (Guerrero et al.,2005; Wang et al., 2007; Saldivar et al., 2008; Bellgard et al.,2012) was carried out with an R. microplus population geographi-cally distant from Porto Alegre strain used in this work.

Phylogenetic analyses revealed the presence of two majorgroups, predominantly divided by intracellular and extracellularserpins. Most of RmS grouped with paralogs or orthologs from R.pulchellus, R. appendiculatus and R. haemaphysaloides. Tick serpinsgrouped in the same cluster in the phylogenetic tree, have similarRCL structures, mainly considering the P1 residue, which is deter-minant to serpin inhibitory specificity. This phylogenetic analysisreinforces the idea that ticks possess paralogous and orthologousserpins that are involved in regulation of similar pathways indifferent tick species. This is consistent with observed data ofAnopheles gambiae serpins, where serpins that grouped in the samecluster have similar peptidase target, acting in the same pathwayin different insects (Suwanchaichinda and Kanost, 2009). Also,the presence of orthologs serpins if capable of triggering protectiveimmune response against heterologous tick challenges can be

Fig. 3. Neighbor-joining analysis of RmS and other tick serpins amino acid sequences. The alignment of retrieved sequences was created using Muscle (Edgar, 2004) in theMega 5.0 program. Except for RmS sequences, all other sequences were retrieved from GenBank (Accession No. in parenthesis).

L. Tirloni et al. / Experimental Parasitology 137 (2014) 25–34 31

useful in the development of a universal anti-tick vaccine (Pariziet al., 2012).

The residues and structures involved in inhibitory activity ofserpin families have been identified and the conservation patternsare clearly correlated to serpin inhibitory mechanism (Irving et al.,2000). Analysis of the tick serpin genes showed high conservation

among primary, secondary and tertiary structures, suggesting thatRmS-3, RmS-6, RmS-9, RmS-13, RmS-15 and RmS-16 are functionalinhibitory serpins. The P1 residue in RCL is critical to serpininhibitory specificity. Important mammalian serpins such as a1-antiplasmin, antithrombin, protein C inhibitor and C1 inhibitorhave a basic amino acid residue (R or K) at P1 (Rau et al., 2007).

HHHHHHHHH1HP7 : -----------------EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEF : 33 RmS-3 : ------------------MVAKFLFLASALAVAHCETDDSTLLARAHNQF : 32 RmS-6 : --------------MKPLVAVATLLALSCLQLSLCQTEQEHKLVAANNQF : 36 RmS-9 : -----------------MKIGRFVFLFWTCVMHMCSAND----ATGSTNL : 29 RmS-13 : MQVTWLFLCVVAPLGVQGGLIDSIRERKAPKAWRRSQETISDVVNSNNGL : 50 RmS-15 : ---------------MYRLLCLVTTVAVITMATEPEPEDRWKLALANNYL : 35 RmS-16 : ----------------MQFTVVMVIILMGAIHAVAPASDALKVTVANNAF : 34 * hA s6B hB hC HHHHHHHHHHHHH EEEEHHHHHHHHHHHHHH HHHHHHHHHHHH1HP7 : AFSLYR-QLAHQSN--STNIFFSPVSIATAFAMLSLGTKGDTHDEILEGL : 80 RmS-3 : AVNLLK-ELATENP--SSNVFFSPTSIAAAFGMAYLGARGGSESELNSVF : 79 RmS-6 : AIHLLK-VLPSLPN---ENVFFSPYSLSTALGMVFLGARGDTLEELSQGL : 82 RmS-9 : ALGLFQ-QLSTDTS---DNVLFSPFGVSVLLAILSAATKGDTSKEIVSAF : 75 RmS-13 : GISMLK-ALG------DQNVLLSPISVNIVLHMVLIGARGRTAYQMSAQM : 93 RmS-15 : GLNLLK-ALPSNAN---TNVFFSPFSVSAALGMAYIGARGDTLEQLTLNF : 81 RmS-16 : AFELLP-LMSTNQL---DNVFYSPYSVTTALAMVHAGADALTLRELHEAL : 80 * ** * * * * * hD s2A HHHHHHHHHHHHHHHHHH EEEEEEEEEEE1HP7 : NFNLTEIPE-AQIHEGFQELLHTLNQPDSQLQLTTGNGLFLSEGL--KLV : 127 RmS-3 : GHTDVGLTDRSRLLTAYKNLLELSAS--PNVTLDVANMVLAQDRF--PIS : 125 RmS-6 : GYKALSLSE-SDVREAFALQNNRLQAHARQAGLDVANSAAVQQGL--DVI : 129 RmS-9 : GSDTAIAAVKQDIESAFKEMATGAPSAVRKSPILLGNLLAVKKGLKSKLL : 125 RmS-13 : GNPSSS------LMSELLHKMSSPDGNSRKVALDHASAVLIQEGA--SFN : 135 RmS-15 : GYTAEELNE-QKILALFKEQLEAARDLPHEYTLDIANAAVAQEGY--GVL : 128 RmS-16 : KYNLVGLAE-DKVVSAHADFNRHLLG-PSNSTLEVANAAVLDQRL--NAL : 126

hE s1A hF HHHHHHHHHH EEEEE HHHHHHHHHHHHHHHH1HP7 : DKFLEDVKKLYHSEAFTVNF--GDTEEAKKQINDYVEKGTQGKIVDLVKE : 175 RmS-3 : DSYKQQLREIFDADLRSANF-VEDGPRVAAEVNAWVREKTRGKISGILPE : 174 RmS-6 : DTYYDALNRTFNAHVFNVDF-QGNGQQAVDTINEWVKQATHNKIDKLFHE : 178 RmS-9 : PQFLNFLKDGGPFSTLVDEL----DENLASRSNKWVSNQTKGQITKILDD : 171 RmS-13 : ASYYREINRLFDASLATVQFGEGRGSDVVKEVNEWASRKTQGRIPQFLEE : 185 RmS-15 : PNYTEALVSAFGAEYLEADF-SKKGQEAIDNINKWVREKTHGKIRSLFDM : 177 RmS-16 : SSYLNALKNGFAAELLKADF-SGDERATLNAINSWVSQKTQQKISKLFDE : 175 * * ** * * * * s3A hG s4C s3C EEEEEEEEEE HHHHEEEEEEE EEEEEEEEE1HP7 : --LDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKR : 223 RmS-3 : G-QPLDIVLFILNAVYFKGTWVTKFDAHRTINKPFLNLGTTEVSKPAMHL : 223 RmS-6 : P-LETNTRLVLMNAILFKGFWERQFDPSHTTKHVFYNGGIQGTPVDTMFL : 227 RmS-9 : DHTEDDARMLLLNAIHFKGEWKEKFEKQLAFKGTFKNYDGTDTPTTFMFK : 221 RmS-13 : V-PEETTKMLVLNAMYFKSDWKTQFDPEFTDKRVFRNEDGTTANVPIMFV : 234 RmS-15 : P-PDISTRLILLNAVYFKGTWLYEFNKLKTKPRSFYNGGVTKVLIPMMKM : 226 RmS-16 : P-LPSFTKLVLLNAIYFKGTWLSKFDQSKTSRAPFYTADGRSTSVDTMQG : 224 * * * *** * * * * * **

s1B s2B s3B hH EEEEEEEEEE EEEEEEEE EEEEEEEEE HHHHHHHHHHH1HP7 : LGMFNIQHCKKLSSWVLLMKYLGNATA-IFFLPDEGK--LQHLENELTHD : 270 RmS-3 : RARFPYARVEPLHASALEIPYEGDRFTMVVLLPDNATG-LPAVRNGLSLA : 272 RmS-6 : RHTTRRGFSVELQSKVLELPYRDSDYSMVIVLPEERDG-ADAVKQVLTID : 276 RmS-9 : RRHFEYALDATLKTHVISLPYKDVKARFVLLLPLNRAG-AKALAGLLTAS : 270 RmS-13 : IETFDFSHDDELNVDALRVPYADNQYSMILMLPRSRQSPLSSVVQGLTAA : 284 RmS-15 : KSTLNHTFDAMLNADVVDLPYVGKDIAMTILLPSERNG-IEHLKSALTTH : 275 RmS-16 : VVKAGYAYVRNLAATMLELPYNGLDYSMILLVPQNGSS-VEVLKRQLNGT : 273 * ** s3C s4A hI HHHHHH EEE EEEEEEEEEEEEEEHHHHHHHHHHHHHHH1HP7 : IITKFLENEDR-RSASLHLPKLSITGTYDLKSVLGQLGITKVFS-NGADL : 318 RmS-3 : ALEDVGSRLSF-RDVILQLPKFDMSLSYGLVPAMKAIKLNSVFG-GSADF : 320 RmS-6 : KLNIAVGSLTS-GPVAISLPKFKIDKLNPLKSNLTFLGLRKMFG-SEADL : 324 RmS-9 : EWKRLLGTLRN-TSVALSMPKFELSKKYNLMKSLEYLGIKKIFT-DKADL : 318 RmS-13 : KLNEIIGEMEP-EQVELSLPKMSVQKMLKLRAPLEKLGLRVPFT-DAADF : 332 RmS-15 : NLNKAVARMYP-KDMKVRLPKFKLDNKYTLKPTLEALGITKIFS-ADADL : 323 RmS-16 : AIAAARHRLRE-SKIKLHLPRFKLEQKYDLKEVLPKLGIKRIFNAGEADL : 322 ** * * * * * s5A RCL EEEEEE ------------------------- E1HP7 : SGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGA-----MFLEAIPMSIPPE : 363 RmS-3 : SGISEAVPLVISDVLHKAAVEVNEEGTIATAVTG-LGFVPLSAHYNPPPP : 369 RmS-6 : SGITGDRRLYVSDVLQRAVVEVNEEGTEAAAVTGVIGVNRIGI-----EP : 369 RmS-9 : SGMIGTGDMFVSAIEQVSKLRLDEEGSEADSATLLRISGKAAEEGE---- : 364 RmS-13 : KGISKAEDLRLNEILHKAALDVDEVGTRAVAATQAQFVSKSLVHFK---- : 378 RmS-15 : SGISGAKNLYVSDVLHKAVLEVNEEGSEAAAVTGFVIQLRTAAFVTPPPL : 373 RmS-16 : SQINGEKSLFVDRAVHKAVVEVNEEGSEAAAVTGVTINTRTTTGPS---- : 368 * * * * * s1C s4B s5B EEE EEEEEEE EEEEEEE1HP7 : VKF--NKPFVFLMIDQNTKSPLFMGKVVNPTQK : 394 RmS-3 : IEFTVDHPFIFYIRDRSTNRVLFIGEVNTL--- : 399 RmS-6 : FLFTADHPFLFFIRDRKTNEIFFAGQVNVL--- : 399 RmS-9 : -SVVADHPFIFAVTAGRRGDILFMGQVNKLPAT : 396 RmS-13 : -QFTVDRPFLALIRHEPTGAILFIAQVLSMQS- : 409 RmS-15 : PKVYVDHPFIFLIRNVHTNTIMFLGEVNAL--- : 403 RmS-16 : -EIHVDRPFLFYITNKRIGNILFVGQVNKID-- : 398 ** ** * *

Fig. 4. Structure-based sequence alignment was constructed by comparison withhuman a1-antitrypsin (PDB 1HP7) as modeling template, against putative secretedRmS (RmS-3, 6, 9, 13, 15 and 16), using STRAP (Structure-based AlignmentProgram). Secondary structures assigned based on the 1HP7 tertiary structure, arelabeled as ‘‘H’’ for a-helix and ‘‘E’’ for b-strand. Helices are labeled from ‘‘hA’’ to ‘‘hI’’and b-strands that constitute b-sheet A–C are labeled as ‘‘sA’’, ‘‘sB’’ and ‘‘sC’’,respectively. The residues that correspond to the 51 core residues (Irving et al.,2000) are indicated by an asterisk (*). The high conserved residues are labeled inblack and low in gray.

32 L. Tirloni et al. / Experimental Parasitology 137 (2014) 25–34

They participate in the regulation of blood coagulation, inflamma-tion and complement activation inhibiting enzymes with a primaryspecificity to substrates having R or K at the P1 position, and theyare important in mammalian host defenses against blood suckingparasites (Ribeiro, 1987; Francischetti et al., 2009). For example,serpin-1B from Manduca sexta has A at P1 (residue 343) and is anelastase inhibitor. If this P1-A residue is changed by site-directedmutagenesis by a P1-K residue (A343K), the inhibitory activityagainst elastase is lost, whereupon this serpin inhibits trypsin,plasmin and thrombin (Li et al., 1999). The Ixodes ricinus immuno-suppressor (Iris) is a serpin with M at P1 (position 340), and it is aspecific elastase inhibitor (Leboulle et al., 2002b), on the otherhand, mutant M340R does not inhibit elastase, but it is a powerfulinhibitor of factor Xa and thrombin (Prevot et al., 2006). Eight RmShave a basic residue at the P1 position in RCL, and except for RmS-18, all are putative secreted serpins. In four of them (RmS-9, RmS-13, RmS-15 and RmS-16), transcription is higher in the salivarygland of fully engorged than in partially engorged females, point-ing to a role that modulates one or more of the host defense mech-anisms. However, additional biochemical and biological studies arerequired to determine the specific function of each serpin.

Although different tick ESTs encoding serpin fragments arepresent in GenBank and BmiGI databases, only a few members ofthe R. microplus serpin protein family have been characterized. Inthis sense, in this report we described an in silico characterizationof various members of the R. microplus serpin family, as a first stepto broaden our understanding about how these proteins contributeto the tick physiological processes. Better understanding of theseinhibitors biological functions could be a helpful tool for the devel-opment of new tick control methods.

Acknowledgments

This work was supported by Grants from CNPq-InstitutoNacional de Ciência e Tecnologia de Entomologia Molecular, FINEP,CAPES, CNPq, FAPERJ and FAPERGS (Brazil). We dedicate this paperto the memory of Alexandre A. Peixoto.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.exppara.2013.12.001.

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