Parasitol Res (2015) 114:4503–4511 DOI 10.1007/s00436-015-4694-6

ORIGINAL PAPER

Genes encoding defensins of important Chagas disease vectors used for phylogenetic studies Catarina Andréa Chaves de Araújo 1,4 & Ana Carolina Bastos Lima 1 & Ana Maria Jansen 2 & Cleber Galvão 4 & José Jurberg 4 & Jane Costa 1 & Patricia Azambuja 3 & Peter Josef Waniek 3

Received: 18 May 2015 / Accepted: 24 August 2015 / Published online: 4 September 2015 # Springer-Verlag Berlin Heidelberg 2015

Abstract Insects possess both cellular and humoral immune responses. The latter makes them capable to recognize and control invading pathogens after synthesis of a variety of small proteins, also known as antimicrobial peptides. Defensins, cysteine-rich cationic peptides with major activity against Gram-positive bacteria, are one ubiquitous class of antimicrobial peptides, widely distributed in different animal and plant taxa. Regarding triatomines in each of the so far analyzed species, various defensin gene isoforms have been identified. In the present study, these genes were sequenced and used as a molecular marker for phylogenetic analysis. Considering the vectors of Chagas disease the authors are reporting for the first time the presence of these genes in Triatoma sordida (Stål, 1859), Rhodnius nasutus (Stål, 1859), and Panstrongylus megistus (Burmeister, 1835). Members of the Triatoma brasiliensis species complex were included into the study to verify the genetic variability within these taxa. Mainly in their mature peptide, the deduced

Electronic supplementary material The online version of this article (doi:10.1007/s00436-015-4694-6) contains supplementary material, which is available to authorized users. * Peter Josef Waniek [email protected] 1

Laboratório de Biodiversidade Entomológica, Instituto Oswaldo Cruz – FIOCRUZ, Rio de Janeiro, Brazil

2

Laboratório de Biologia de Tripanosomatídeos, Instituto Oswaldo Cruz – FIOCRUZ, Rio de Janeiro, Brazil

3

Laboratório de Bioquímica e Fisiologia de Insetos, Instituto Oswaldo Cruz – FIOCRUZ, Rio de Janeiro, Brazil

4

Laboratório Nacional e Internacional de Referência em Taxonomia de Triatomíneos, Instituto Oswaldo Cruz – FIOCRUZ, Av. Brasil 4365, 21045-900 Rio de Janeiro, Brazil

defensin amino acid sequences were highly conserved. In the dendrogram based on defensin encoding nucleotide, sequences the Triatoma Def3/4 genes were separated from the rest. In the dendrogram based on deduced amino acid sequences the Triatoma Def2/3/4 together with Rhodnius DefA/B pre-propeptides were separated from the rest. In the sub-branches of both the DNA and amino acid dendrograms, the genus Triatoma was separated from the genus Rhodnius as well as from P. megistus. Keywords Defensin . Triatominae . Antimicrobial peptides . Phylogenetic analysis . Chagas disease

Introduction Antimicrobial peptides (AMPs) are usually composed of less than 100 amino acid residues participating in the humoral immune response of nearly all life forms and are considered as an ancient host defense mechanism to control their natural microbiota flora and/or combat pathogen infections (Ganz 2003; Thomma et al. 2003; Bulet et al. 2004; Thevessin et al. 2007). Insects are the major animal group and some species are medically important because of their ability to transmit diseases (Lehane 2005). One of the reasons for the insects’ evolutionary success is their highly developed immune system, which recognizes and eliminates invading microorganisms (Lehane 2005). Insects possess both innate cellular and humoral defense strategies of which the latter comprise the production of many different antimicrobial molecules, including defensins (Hoffmann 1995, 1997). Defensins are a group of small, cationic AMPs and have been found in not only Paneth cells, mammalian epithelial cells, macrophages, and neutrophils of vertebrates, but also plants, fungi, and invertebrates where the cysteine-stabilized

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α-helical and β-sheet fold is used (Dimarcq et al. 1998; Ganz 2003; Zhu 2008; Carvalho Ade and Gomes 2011). This AMP type belongs to the innate immunity system of insects, with high activity against Gram-positive bacteria and was identified in almost all insect orders (Bulet et al. 1992, 1999, 2004; Lamberty et al. 1999; Dassanayake et al. 2007). Insect defensins are synthesized mainly in the fat body (organ analogous to the liver in higher animals) and midgut tissue and are released into the hemolymph and intestinal lumen, respectively (Hetru et al. 1998; Nayduch et al. 2013). Chagas disease or American trypanosomiasis was primarily considered a zoonosis that occurs in wild animals only and which was disseminated by the triatomine vectors; later, man entered the transmission of the disease because of expansion of civilization into new undeveloped regions (Jurberg and Galvão 2006; Lima and Sarquis 2008; Araújo et al. 2009; Coura 2013). Considering triatomines, which are vectors of Trypanosoma cruzi (Kinetoplastida, Trypanosomatidae), the etiologic agent of Chagas disease, the identification and characterization of genes encoding defensin have so far been reported in three species. In Rhodnius prolixus (Stål, 1859), three defensin encoding genes and their transcript abundances after bacterial induction have been described (Lopez et al. 2003). In Triatoma brasiliensis Neiva, 1911, the messenger RNA (mRNA) encoding four different defensin isoforms has been detected in various organs of this triatomine species (Araújo et al. 2006; Waniek et al. 2009). In two independent studies, defensin transcripts were identified in the salivary glands of Triatoma infestans (Klug, 1834) (Assumpção et al. 2008; Schwarz et al. 2014). In a recent study which analyzed midgut transcriptome of R. prolixus, eight defensin encoding sequences has been identified (Ribeiro et al. 2014). Except one, all identified transcripts have been assigned to the previously characterized defensin genes A–C. Previous studies involving defensin and triatomines have demonstrated the relation of this gene to the blood ingestion, regulation of symbiont, and parasite development in the intestinal tract and hemocytes of these insects (Araújo et al. 2006; Boulanger et al. 2006; Waniek et al. 2009, 2011). Although molecular markers such as Cytochrom b encoding gene (Cytb) and internal transcribed spacer (ITS) have extensively been used for phylogenetic analyses the phylogenetic position of triatomines among the Reduviidae is still controversial and new approaches to clarify the origin of triatomines are needed (Hypsa et al. 2002; Weirauch 2008; Schofield and Galvão 2009; Weirauch and Munro 2009; Blandón-Naranjo et al. 2010; Quisberth et al. 2011; Bargues et al. 2014). In the present study, genes encoding defensins were used as molecular markers to evaluate the phylogenetic relation of some epidemiologically important triatomine species and taxa from the Triatoma brasiliensis species complex. Phylogenetic studies involving insect defensin genes showed the existence of two defensin lineages, whereas one is

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associated to the Lepidoptera, while the second one is related to the Hemiptera (Heteroptera), Coleoptera, Diptera, and Hymenoptera (Dassanayake et al. 2007). Since defensins are closely related to the insect’s microbiota, their analysis might contribute in a better understanding of triatomine evolution and in the T. cruzi-vector interactions.

Material and methods Origin of insects Fifth instar nymphs of Triatoma sordida, R. prolixus, Rhodnius nasutus (Stål, 1859), Panstrongylus megistus and two members of the T. brasiliensis complex (Triatoma sherlocki Papa, Jurberg, Carcavallo, Cerqueira & Barata, 2002; Triatoma juazeirensis Costa & Felix, 2007) were provided by Laboratório Nacional e Internacional de Referência em Taxonomia de Triatomíneos, FIOCRUZ, Rio de Janeiro. The other two members of the T. brasiliensis complex (Triatoma brasiliensis macromelasoma Galvão, 1956 and Triatoma melanica Neiva & Lent, 1941) were obtained from Laboratório de Biodiversidade Entomológica – FIOCRUZ. In addition, specimen of T. b. brasiliensis (Neiva, 1911) from Caico, Rio Grande do Norte (Brazil) were used to verify whether or not there are genetically variances in different populations of this triatomine species, since the already known defensin genes were characterized from T. brasiliensis from another locality (Araújo et al. 2006; Waniek et al. 2009). Preparation of tissues For sequence identification, the enlarged anterior midgut (stomach) wall and fat body tissue of the different triatomine species 7 days after a blood meal were dissected. Tissues of ten insects were pooled and stored at −80 °C until use. RNA extraction and reverse transcription Total RNA was isolated using the SV Total RNA Isolation System (Promega, Madison, USA), following the manufacturer’s protocol. Reverse transcription was carried out as described previously (Araújo et al. 2006). Nucleic acid concentrations were determined using a Bio Photometer (Eppendorf, Hamburg, Germany). To verify the complementary DNA (cDNA) quality, the gene-encoding β-actin was amplified as an internal control as described previously (Waniek et al. 2009). Primers, PCR, and sequencing Oligonucleotide primers and PCR conditions were carried out according to Araújo et al. (2006) and Waniek et al. (2009) for

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the Triatoma species. Defensin encoding cDNAs of Rhodnius were amplified using the oligonucleotide primers pairs RpDefAF 5′-GAATACTCCACTCAACCGCAAC-3′/ RpDefAR 5′-TCTAAGGAGAATGTTCACTTCA-3′, RpDefBF 5′-CAGTACCTAGGATATTCCACTCAAC-3′/ RpDefBR 5′-AGAAATTCTCCAAATACACTTCA-3′, and RpDefCF 5′-CAGTACAGTCCTAATACCTAGCC-3′/ RpDefCR 5′-GCGTAAATGACTTGAATATTGAGT-3′ based on the sequences described previously and at the same conditions as mentioned earlier (Lopez et al. 2003). The amplified products were visualized by electrophoresis, using 1 % agarose gels containing ethidium bromide. Amplicons were purified using the Wizard SV Gel and PCR Purifications System (Promega). The obtained PCR products were sequenced at least three times at each direction by Plataforma Genômica de Sequenciamento de DNA/PDTIS-FIOCRUZ, IOC, Rio de Janeiro, using the respective defensin forward and reverse primers. Panstrongylus megistus DefA encoding gene (PmDefA) was amplified using the primers (Def1 T. brasiliensis) as described above. P. megistus DefB encoding gene (PmDefB) was identified using the 5′- and 3′-RACE (rapid amplification of cDNA ends) Kits (Invitrogen, Carlsbad, CA, USA) (Waniek et al. 2005; Moreira et al. 2014). For the amplification of the 3′-end the primers Def-deg-F and Abridged Universal Amplification Primer were used (Waniek et al. 2009). For the subsequent amplification of the 5′-end the reverse primers PmGSP1 (5′-ACCATTTCACTTCCTA-3′), PmGSP2 (5′-CATTCACCGCCACGATTACCCAGA-3′) and PmGSP3 (5′-GTGCGGCACAGGCTGAATGGT-3′) with the respective forward primers supplied in the 5′-RACE kit were used. The obtained products were cloned, sequenced and assembled as described previously (Waniek et al. 2009). Sequence identity and phylogenetic analyses Identity was assessed using BLAST X at the web servers of the National Center for Biotechnology Information (http:// www.ncbi.nlm.nih.gov/) (Altschul et al. 1990). The deduced amino acid sequences were aligned using ClustalW version 1. 83 (Thompson et al. 1994), and slight corrections were carried out visually. Predicted signal-peptide cleavage sites were calculated using SignalP, version 3.0 (Bendtsen et al. 2004). In a first step of the tree reconstruction (based on maximum likelihood), the defensin alignment was analyzed with jModelTest 2.1.2 (Darriba et al. 2012) and ProtTest 2.4 (Abascal et al. 2005), respectively, to determine an appropriate models of nucleotides and amino acid substitutions. Comparing the available substitution models jModelTest supported JC (Jukes and Cantor 1969) and ProTest supported the use of the JTT+G (Jones et al. 1992), in which G indicates the use of a gamma distribution to describe the rate heterogeneity. Phylogenetic analysis of the cDNA and deduced amino acid

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sequences of the pre-prodefensins were also conducted by the neighbor-joining (NJ) method using Kimura-2 (Kimura 1980) and Dayhoff (Schwarz and Dayhoff 1979) models with Gamma distribution. For the construction of the defensin phylogenetic dendrograms, MEGA version 6.06 was used (Tamura et al. 2013). Three outgroup sequences of two taxa were added to the data set the heteropteran Pyrrhocoris apterus (GenBank accession no. JX560432) and two of the homopteran Nilaparvata lugens (GenBank accession nos. KC355195 and KC355196) and the trees were calculated based on the cDNA and deduced amino acid sequences obtained in the present study. For comparison of the obtained results with defensin sequences previously described, the DefA, DefB, and DefC sequences of R. prolixus (Lopez et al. 2003), T. brasiliensis Def1, Def2, Def3, and Def4 (Araújo et al. 2006; Waniek et al. 2009) and two defensins identified in T. infestans (TiDefA, TiDef) were used (Assumpção et al. 2008; DQ391188). Another not characterized R. prolixus gene (GAHY01001257) was included in the present study (Ribeiro et al. 2014). For maximum likelihood and neighbor-joining analyses, 500 bootstrap repetitions were carried out.

Results Analyses of the cDNA sequences As a control of the cDNA quality, the gene encoding β-actin was used. After the PCR with β-actin oligonucleotides, a band of about 300 bp—corresponding to the expected length of this PCR product—was generated for all nucleotide samples, which were used in the present study. After amplification purification and sequencing, 681 defensin encoding sequences were obtained. Because a part of the sequences was truncated, for the present analyses, 483 full sequences (including 5′- and 3′-RACE) were used. Considering the Def1 gene of Triatoma and DefA, DefB, and DefC of Rhodnius, all sequences had a coding region (including stop codon) of 285 bp, showed the ATG start codon and terminated with the TGA stop codon (Fig. 1). The Def3 and Def4 cDNA sequences from the genus Triatoma presented a coding region of 282 bp and the same stop codons as the above-mentioned Def1 gene. The coding region of both P. megistus defensin encoding genes (PmDefA and PmDefB) had a length of 273 bp and terminated with the TGA stop codon. The data showed a high similarity between T. brasiliensis Def1 and R. prolixus/R. nasutus DefC and T. brasiliensis Def3/4 and R. prolixus/R. nasutus DefA/B, respectively. The Def1 sequence characterized in T. b. brasiliensis originated from Caico, Rio Grande do Norte was identical to the Def1 sequence previously characterized from T. brasiliensis. When comparing the similarity of the Triatominae cDNA sequences encoding defensin, the lowest

4506 RnDefC RpDefC RnDefB RpDefB RnDefA RpDefA TmeDef3a1 TshDef3b TmeDef4a TjDef4a2 TbDef2 TbDef3a TshDef3a TiDefA TshDef13 TsoDef1 TiDef TmaDef14 PmDefA PmDefB Rp GAHY01001257 Papt NlDefA NlDefB

Parasitol Res (2015) 114:4503–4511 MKCILSLFTLFLVATLAY------SYPAE-WNYQHQLDDAQWE-PAGEITEEHLS---RMKRATCDLLSFTSKWFTPNHAGCAAHCIFLGNRGGRCVGTVCHCRK MKCILSLFTLFLVATLVY------SYPAE-WNSQHQLDDAQWE-PAGELTEEHLS---RMKRATCDLLSLTSKWFTPNHAGCAAHCIFLGNRGGRCVGTVCHCRK MKCILSLVTLFLVAVLVH------SHPAE-WNTQQELDDALWE-PAGEVTEEHVA---RLKRATCDLLSFSSKWVTPNHAGCAAHCLLRGNRGGQCKGTVCHCRK MKCILSLVTLFLVAVLVH------SHPAE-WNTQQELDDALWE-PAGEVTEEHVA---RLKRATCDLLSFSSKWVTPNHAGCAAHCLLRGNRGGHCKGTICHCRK MKCILSLVTLFLVAVLVH------THPAE-WNTQQELDDALWE-PAGEVTEEHVA---RLKRATCDLFSFQSKWVTPNHAACAAHCLLRGNRGGQCKGTICHCRK MKCILSLVTLFLVAVLVH------SHPAE-WNTHQQLDDALWE-PAGEVTEEHVA---RLKRATCDLFSFRSKWVTPNHAACAAHCLLRGNRGGRCKGTICHCRK MKCALSLVTLFLVVALAY------SHPAE-W-TQQQLDEALWE-PAGEVTEEHVA---RLKRATCDLFSFQSKWVTPNHAACAAHCLLRGNRGGQCKGTICHCRK MKCALSLVTLFLVVALAY------SHPAE-W-TEQQLDEALWE-PAGEVTEEHVA---RLKRATCDLFSFQSKWVTPNHAACAAHCLLRGNRGGQCKGTICHCRK MKCALSLVTLFLVVALAY------SHPAE-C-TQQQLDEALWE-PAGEVTEEHVA---RLKRATCDLFSFQSKWVTPNHAACAAHCLLRGNRGGQCKGTICHCRK MKCALSLVTLFLVVALAY------SHPAE-W-TQQQLDEALWE-PAGEVTEEHVT---RLKRATCDLFSFQSKWVTPNHAACAAHCLLRGNRGGQCKGTICHCRK MKCALSLVTLFLVAALAY------SYPAD-L-AQQPLDETEWEQPAGEVTEEHVT---RLKRATCDLFSLQSKWVTPNHAACAAHCLLRGNRGGQCKGTICHCRK MKCALSLVTLFLVAALAY------SHPAE-W-TEQQLDEDIWE-PAGEVTEEHVA---RLKRATCDLFSFQSQWVTPNHAACAAHCLLRGNRGGECKGTICHCRK MKCALSLVTLFLVAALAY------SHPAE-W-TEQQLDEDIWE-QAGEVTEEHVA---RLKRATCDLFSFQSQWVTPNHAACAAHCLLRGNRGGECKGTICHCRK MKCALSLVTLLLVAALAY------SHPAE-W-TQQQLDEALWE-PAGEVTEEHVA---RLKRATCDLFSFQSQWVTPNHAACAAHCLLRGNRGGECKGTICHCRK MKCALSLVTLFLVAALAY------SYPAD-L-AQQPLDETQWEQPAGEITEEHAA---RLKRATCDLFSFESKWFTPNHAACAAHCILLGNRGGHCVGTVCHCRK MKCALSLVTLFLVAALAY------SYPAD-L-AEQPLDETQWEQPAGEITEEHAA---RLKRATCDLFSFSSKWFTPNHAACAAHCILLGNRGGHCVGTVCHCRK MKCILSLVTLFLVAALAY------SYPAD-L-AQQPLDEAQWEQPAGEITEEHAA---RLKRATCDLFSFSSKWFTPNHAACAAHCILLGNRGGHCVGTVCHCRK MKCALSLVTLFLVAALAY------SYPAD-L-AQQPLDETEWEQPAGEITEEHGA---RLKRATCDLFSFESKWFTPNHAACAAHCILLGNRGGHCVGTVCHCRK MKCALCLVTLFLLATVAY------SFPAVEL-EQQELDDAIWT-PA----EENGA---RVKRATCDLFSFESKWFTPNHAACAAHCIFLGNRGGHCVGTVCHCRK MKCALCLVTLFLVAALAY------SYPA-EW-VHQQLDDTIWKQ--G--TQQNGV---RVKRATCDLFSFESQWFTPNHSACAAHCIILGNRGGECKGTICHCRK MKCILSLVTLFLVAAMVY------SYPAE-LILLQQLDDARWE-PAGEITEEHLA---RMKRATCDLLSFQSQWFTPNHAACAAHCLFLGYKGGNCQGTICHCRN MKFVV-LFIFTVVVAMAS------AHPYIPVDEDADVPDAIPE-------EYHSL---RVKRATCDVLSFSSKWFTPNHSACAIHCIAKGYKGGSCKKAICHCRR MNSSMTAVLLLVASVMALYVVHVNSLPTG-MPVEDDLVLTGDELPAAVGRESAVATGSRAKRATCDLFSFETQWVTPNHAACAAHCIVLGKKGGYCSNTICYCRN MHSSITAVLLLVASVMALYVVHVNSLPTG-MPVEDDLVLTEDELPAAVGRESAVATGSRAKRATCDLLSFNSKWVTPNHAGCAAHCLIRGKKGGSCKNAICYCRN *: : . : : .:. : * : : * ******::*: ::*.****:.** **: * :** * ::*:**.

Fig. 1 Alignment of Hemiptera defensin pre-propeptides. Identical and similar amino acid residues between the defensins are indicated below the alignment sequences by asterisks and dots, respectively. The first amino acid residue of propeptide and mature peptide are in bold and italic. Cysteine amino acid residues forming the disulfide bridges are bold and

gray shaded. The abbreviations are composed as follows: the first two/ three letters are the species name followed by the gene. Pre-prodefensins marked by superscript numbers include the following identical sequences: 1TshDef4; 2TjDef4, TmaDef4, TbDef4; 3TmDef1, 4TjDef1, TbDef1, TjDef1a

similarity of 66.6 % was found between PmDef3 and R. prolixus DefC and the highest similarity of 99.6 % between several Triatoma Def1 and Def3/4 cDNA sequences. All cDNA percentage similarities are listed in the supplemental file 1. The sequences were submitted to the GenBank and received the respective accession numbers: KF056935KF056936, KF056938-KF056989, KP893619.

Some deduced defensin amino acid sequences were identical; among others, the Def1 sequences obtained from T. juazeirensis and T. b. macromelasoma with that of T. brasiliensis (Fig. 1). The lowest similarity on the amino acid level of 62.1 % was found between PmDef3 and RnDefB and the highest of 97.8 % between RpDefB and RnDefB and several Triatoma Def1 and Def4 pre-prodefensins. A detailed list of amino acid similarities is shown in the supplemental file 1.

Structure of the deduced peptides The obtained cDNAs encoded triatomine pre-prodefensins of 94 (Def1, DefA, DefB, and DefC); 93 (Def3/4); and 90 (PmDefA/B) amino acid residues. All analyzed mature peptides contained six cysteines, a specific defensin characteristic to form three disulfide bridges (Fig. 1). At the amino terminal end, all Triatoma Def1/2, Rhodnius DefC and PmDefB deduced amino acid sequences had a predicted signal-peptide with a cleavage site between Ser19 and Tyr20 whereas in Triatoma Def3/4 and Rhodnius DefA/B this cleavage site was found between Ser19 and His20 (Fig. 1). In RnDefA and PmDefA the signal peptide cleavage sites were located between Thr19/His20 and Ser19/Phe20, respectively. The only difference in the number of amino acids in triatomine defensins was found in the length of activation peptides of 28 amino acid residues in both Panstrongylus defensins, 31 in Def3/Def4 of Triatoma, and 32 in Def1/Def2 of Triatoma, and all Rhodnius sequences. The Lys/Arg-Ala/Thr (KR-AT) activation peptide cleavage site is a typical characteristic in the defensin structure and occurred in all analyzed triatomine species as well as in the deduced defensin amino acid sequences of the hemipteran outgroup species.

Phylogenetic analysis Maximum likelihood analyses showed very low bootstrap support and partly inconsistent branching (supplemental files 2 and 3). Neighbor-joining trees showed higher bootstrap values and were more conclusive. The NJ tree constructed based on the cDNA sequences showed the presence of three main branches (Fig. 2). In the first branch, the genes encoding defensins of Rhodnius (DefA, B, C, and Rp GAHY01001257) and Def1/Def2 of the genus Triatoma were grouped as well as both Panstrongylus defensin encoding genes (PmDefA, PmDefB). In the first sub-branch all Triatoma Def1 genes, besides the T. brasiliensis species complex. also T. sordida and T. infestans were separated from the Panstrongylus encoding genes with high bootstrap support. In this clade, highly variable bootstrap values ranged from 35 to 99 % (Fig. 2). In the second sub-branch, Rhodnius DefC and GAHY01001257 were separated from DefA and DefB with low bootstrap support. The T. brasiliensis Def2 gene was in an intermediate position separated by low bootstrap support from the defensins of the genera Triatoma and Panstrongylus.

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4507 87 TmaDef1, TjDef1, TbDef1 65 97 99

TjDef1a TshDef1, TmeDef1

35

TsoDef1 TiDef PmDefA

51

PmDefB

92

TbDef2 48

99 83

RnDefC RpDefC

R. prolixus GAHY01001257 RnDefA

78

58

RpDefA RnDefB

78

99

97

RpDefB

99 TbDef3a 95 52

TshDef3a TiDefA

TshDef3b 55

61

TjDef4 TjDef4a, TmaDef4, TbDef4

61 TshDef4 52 TmeDef3a, TmeDef4 64 TmeDef4a

Papt NlDefA 100

NlDefB

0.1

Fig. 2 Neighbor-joining dendrogram of Triatominae defensin encoding cDNA sequences. One sequence of Pyrrhocoris apterus and two of Nilaparvata lugens were used as outgroup. Branch labels show the percentages of bootstrap samples supporting that branch

In the second main branch, Triatoma Def3 and Def4 genes were separated by low bootstrap support (Fig. 2). Def3a of T. melanica (TmeDef3a) was located in the Def4 branch whereas TiDefA was located in the Def3 branch. Also in this clade, strongly variable bootstrap values (52–99 %) were observed (Fig. 2). However, inside of the T. brasiliensis species complex, the defensin gene analysis could not separate these insect species and subspecies efficiently. A similar effect was observed in the branch of T. brasiliensis Def1genes. In the third branch clustered the hemipteran outgroup species (Fig. 2). The second NJ phylogenetic tree was constructed based on the deduced defensin amino acid sequences (Fig. 3). After the analysis, two main branches with high bootstrap support containing triatomine sequences could be identified; the first sub-branch contained the Triatoma Def2/3/4 and Rhodnius DefA/B sequences, respectively, where all Triatoma Def3/4 sequences clustered together with TbDef2 and a low bootstrap support. The bootstrap support ranged in the Triatoma clade from 21 to 94 %. In the second clade, grouped DefA and DefB of both Rhodnius species, with a bootstrap support ranging from 47 to 89 %. In both clades, the respective defensins forms 3/4 and A/B could not be separated. Rhodnius DefC sequences localized in the second branch grouped together with R. prolixus GAHY01001257, Triatoma Def1 and both Panstrongylus defensins. The three Rhodnius defensins grouped together separated from the sequences of Panstrongylus and Triatoma with a low bootstrap value 34 %.

The Triatoma sub-branch presented two different clades in which TiDef was separated from the T. brasiliensis species complex sequences with a bootstrap support of 73 %. Both P. megistus defensins were separated from all the Triatoma defensin amino acid sequences by low bootstrap support (Fig. 3). The outgroup defensins of P. apterus (Papt) and N. lugens (NlDefA, NlDefB) were separated from the triatomines.

Discussion Comparing the genes encoding defensin (Def1) from T. brasiliensis (Araújo et al. 2006; Waniek et al. 2009), T. infestans DefA (Assumpção et al. 2008) and Def (DQ391188), with DefA, B, and C from R. prolixus, all open reading frames presented 285 bp in size, in contrast to T. brasiliensis Def3 and Def4 and both Panstrongylus defensin genes which present 282 and 270 bp in size, respectively. When the obtained defensin sequences were compared with the non-blood sucking hemipterans P. apterus and N. lugens the defensin encoding sequence of P. apterus was shorter, presenting 267 bp while that of N. lugens had 315 bp, all ending with the same stop codons as the sequences identified in the triatomine species. All the obtained defensin sequences presented an 19 amino acid residue signal peptide, in contrast to 18 and 17 amino acid residues identified in P. apterus and N. lugens (DefA and DefB) sequences, respectively. In addition, all Hemipteran defensins analyzed in the present study

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Parasitol Res (2015) 114:4503–4511 1 29 TmeDef3a

33

TmeDef4a

47

TjDef4a2 TshDef3b

21

TiDefA 26

TbDef3a

57

94 TshDef3a 50

TbDef2 RpDefA RnDefA

86

RnDefB

47

85

89

RpDefB 99 RnDefC

38

RpDefC R. prolixus GAHY01001257

34

PmDefA

35

PmDefB TiDef

12

TsoDef1

73 48 41

TshDef13 TmaDef14 NlDefA 100

NlDefB Papt

0.1

Fig. 3 Neighbor-joining dendrogram of deduced Triatominae defensin amino acid sequences. Sequences of Pyrrhocoris apterus and Nilaparvata lugens were used as outgroup. Branch labels show the percentages of bootstrap samples supporting that branch

including the outgroup species showed a 43 amino acid mature peptide in which 21 amino acid residues were identical, emphasizing the high conservation of these AMPs in Hemiptera. The present data showed most differences found in the insect defensins are localized in the pre-pro region, while the mature peptide primary structure is highly conserved (Araújo et al. 2006; Waniek et al. 2009), this applies to the Hemiptera, in a comparison of Triatominae with bugs from other orders like P. apterus and N. lugens. All activation peptide sequences of the genus Triatoma Def1/2, R. prolixus DefA-C, GAHY01001257 and T. infestans Def presented 32 amino acid residues, differing from Triatoma Def3 and Def4 sequences and DefA from T. infestans which presented 31 amino acid residues (Araújo et al. 2006; Assumpção et al. 2008; Waniek et al. 2009; Ribeiro et al. 2014). Activation peptides of both P. megistus defensins had a length 28 amino acid residues. The insect species used as outgroup (P. apterus and N. lugens) presented 27 and 44 amino acid residues, respectively. The similarity of the pre-prodefensin considering the genus Triatoma Def1 was 78–100 % inside of this genus, when compared to Rhodnius DefC it ranged between 75 and 79 %. Comparing the obtained defensin sequences with the outgroup insects, the identity percentage decreased to 42.7 % (P. apterus) and 43.2 and 40.3 % (DefA and DefB of N. lugens), respectively. Consequently, the obtained results show the defensins of triatomines are more similar among them when compared to more phylogenetic distant Hemipteran insects.

Many questions about the origin and geographic distribution of the Triatominae subfamily are still unsolved. Various studies related to the phylogeny of Chagas disease vectors have widely used molecular markers such as Cytb and ITS genes (Monteiro et al. 2000; Hypsa et al. 2002; Tartarotti and Ceron 2005; de Paula et al. 2007; Weirauch and Munro 2009; Quisberth et al. 2011) since these markers seem to well separate the clades related to the different triatomine species subpopulations (Yao et al. 2010). Considering Cytb as a molecular marker, the population structure of the genus Triatoma has been well defined, mainly the species and subspecies of the T. brasiliensis complex (Monteiro et al. 2004; Pfeiler et al. 2006). ITS is another molecular marker extensively explored for phylogenetic studies regarding the evolution questions and phylogeny of triatomines (Marcilla et al. 2001, 2002; Tartarotti and Ceron 2005; Quisberth et al. 2011). Even though Cytb and ITS have extensively been used, new markers are needed for better evaluation of the origin and position of triatomines. Recent studies have demonstrated genes encoding defensins can be used as molecular markers in different organisms (Seufi et al. 2011; Chen et al. 2012; d’Alençon et al. 2013). In the present work using genes encoding defensins, considering the phylogenetic trees constructed based on the cDNA and amino acid sequences, both showed the genera Triatoma and Panstrongylus located in a branch separated from the genus Rhodnius. These results corroborates the polyphyletic origin hypothesis that the triatomine species from the tribes Triatomini and Rhodniini originated from various ancestrals (Tartarotti and Ceron 2005;

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Martínez et al. 2006; Tartarotti et al. 2006). However, on both the nucleic acid and amino acid level both defensins of Pan stron gy lus —whi ch also b elongs to the tribe Triatomini—grouped with Triatoma. It also could be observed the gene encoding defensin separated the genes into two (Def1 and Def3/Def4) different branches, though it was not possible to separate the closely related taxa from the T. brasiliensis complex within the branches of the respective orthologous genes. In addition, Def3a of T. melanica (TmeDef3a) was located in the Def4 branch and possibly corresponds to Def4 gene identified in the other Triatoma species. Within genera, similarity was very high and some orthologous defensin encoding genes and deduced defensin amino acid sequences were identical even if they are from different species of the genus Triatoma. These problems might be explained by the fact that the sequences are relatively short (~300 bp) and that the defensin encoding genes are highly conserved. These results also showed DefA gene from T. infestans is probably orthologous to the Def3 gene from T. brasiliensis, since both genes are in the same branch, while the Def gene also obtained from T. infestans can be associated to the Def1 from T. brasiliensis (Araújo et al. 2006; Assumpção et al. 2008; Waniek et al. 2009). R. prolixus GAHY01001257 and both Panstrongylus defensin genes grouped in the DefC/Def1 branch and it is probable that they are orthologues. Regarding evolution, the control of some pathogens mediated by AMPs has been displayed as a high efficient mechanism of immune defense, which can be found even in most primitive life organisms but also in humans and particularly considering humans, AMPs such as defensin have a highly conserved molecule structure (Wiesner and Vilcinskas 2010). Despite the high conservation of triatomine defensin sequences, the present results showed a clear separation between the Triatoma Def1 and Def3/Def4 branches, in both trees constructed based on nucleic acid and amino acid defensin sequences. This fact was already observed by other authors, when Def1 gene was isolated and characterized from the stomach and intestine of T. brasiliensis (Araújo et al. 2006), while Def3 and Def4 genes were identified predominantly in the fat body of the same insect species (Waniek et al. 2009). Triatomines from the T. brasiliensis complex are morphologically different and, e.g., show variable isoenzyme patterns (Costa et al. 1997). A striking feature in the analysis of defensin encoding genes of the present study was the fact that Def1 and Def4 could be amplified from all Triatoma species used in the present study whereas Def3 was only found in T. b. brasiliensis and T. sherlocki. The absence of Def3 in T. juazeirensis, T. melanica, and T. b. macromelasoma cannot be justified by differing sequences and thus reduced primer annealing because the members of the T. brasiliensis species complex are closely related and the target sequences highly conserved. Poor cDNA quality also can be excluded because the same template produced clear PCR amplification products

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for Def1 and Def4 as well as ß-actin. In addition, to take the different gene expression sites into account, cDNAs synthesized from total RNA of the midgut and fat body were used. Another more likely possibility for this phenomenon might be that Def3 and Def4 are alleles and Def3 occurs in heterozygous (Def3-Def4) insects while in homozygous insects (Def4Def4) only Def4 is detectable. A differing presence of defensin genes might affect the T. cruzi establishment and thus the vector competence within the T. brasiliensis complex. If Def3 and Def4 play a role in the intestinal humoral immune response, the respective peptides might influence the T. cruzi population and/or the microbiota in the digestive tract. Kitani et al. (2009) have shown a dose-dependent negative effect of synthetic defensin fragments on different Trypanosoma species in axenic cultures. Even if the intestinal defensins do not affect T. cruzi directly, the modification of gut microbiota, concomitant infections, and also many factors involved in the immune system of the vector might influence the development of this parasite and other intestinal parasites like Trypanosoma rangeli (Mello et al. 1996; Kollien et al. 1998; Kollien and Schaub 2000; Azambuja et al. 2005; Castro et al. 2007; Araújo et al. 2007, 2008; Garcia et al. 2010; Vieira et al. 2014, 2015). As it was shown previously, bacteria are able to influence the T. cruzi development in the intestine of R. prolixus as well as the presence of different T. cruzi strains sharing the same intestinal tract of a bug (Azambuja et al. 2004; Araújo et al. 2014). A variation in the presence of the AMP defensin might strongly modify the gut microbiota and thus influence indirectly the T. cruzi establishment. Acknowledgments The authors thank Fundação de Amparo à Pesquisa no Estado do Rio de Janeiro-FAPERJ (E-26/100.456/2007; E-26/ 110.403/2011), Conselho Nacional de Desenvolvimento Científico e Tecnológico-CNPq (PDJ: 151187/2009-6). CNPq – Ministério da Saúde/Secretaria de Vigilância Sanitária (SVS). Authors also thank Pesquisador Visitante FIOCRUZ/CNPq (158817/2010-9). CACA is a Post-doctor fellow by Capes/PNPD in Biodiversidade e Saúde-IOC/ FIOCRUZ Research Fellow and PJW is a PDS FAPERJ Fellow (E-26/ 200.117/2015).

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