article: Triatomines are hematophagous insects that act as vectors of Chagas disease. Rhodnius neglectus is one of these kissing bugs found, contributing to the transmission of this American trypanosomiasis. The saliva of hematophagous arthropods contains bioactive molecules responsible for counteracting host haemostatic, inflammatory, and immune responses. Next generation sequencing and mass spectrometry-based protein identification were performed to investigate the content of triatomine R. neglectus saliva. We deposited 4,230 coding DNA sequences (CDS) in GenBank. A set of 636 CDS of proteins of putative secretory nature was extracted from the assembled reads, 73 of them confirmed by proteomic analysis. The sialome of R. neglectus was characterized and serine protease transcripts detected. The presence of ubiquitous protein families was revealed, including lipocalins, serine protease inhibitors, and antigen-5. Metalloproteases, disintegrins, and odorant binding protein families were less abundant. The data presented improve our understanding of hematophagous arthropod sialomes, and aid in understanding hematophagy and the complex interplay among vectors and their vertebrate hosts.
  Blood-sucking triatomines (Hemiptera: Reduviidae) feed exclusively on blood in all life stages. They obtain their blood meal from venules or arterioles of their vertebrate hosts. The steps during feeding include piercing of the host skin, followed by a probing period, and finally engorgement [1]. In support of this habit, these arthropods have evolved effective mechanisms to counteract host responses, such as haemostasis, inflammation and immunological reactions. While biting, their salivary glands (SG) release potent pharmacological substances, including vasodilator, anti-inflammatory, antiplatelet, anticlotting and immunomodulatory molecules, to enable the arthropod to obtain a successful blood-meal [2,3]. These bioactive salivary components represent a promising source of molecules with therapeutic potential for treating circulatory disorders [4,5]. In the 1990s, multinational control programs against Chagas disease led to a significant reduction of acute cases in many endemic regions of Latin America, mainly through a reduction of domestic vectors [6]. However, factors such as the wide geographical distribution of triatomine species and the availability of different infection reservoirs remain multifactorial obstacles in the control of the disease. Nowadays, there is constant concern regarding the sporadically or progressive (re) invasion and (re) colonization of human dwellings by wild secondary vectors [7,8]. Rhodnius neglectus is found in the Brazilian Savanna (Cerrado) in association with different wild palms, playing an important role in the sylvatic maintenance of T. cruzi and Trypanosoma rangeli [9–11]. In nature, R. neglectus feeds mainly on birds and much less on rodents, and rarely on opossum [12]. This species is able to act as a secondary vector, being observed in both intra and peridomestic environments in five Brazilian states [13–17], a possible result of deforestation and wild ecotope invasion. These anthropogenic environmental changes favor vector dispersion, bridging sylvatic/domestic cycles of the disease. Sialome studies (from the Greek sialo = saliva) have been developed for many species of bloodsucking insects, which are frequently vectors of human and animal diseases. Sanger automated sequencing technology has been used to investigate the salivary transcriptome for almost two decades. However, Next Generation Sequencing (NGS) is capable of providing much more sequence data in a single run, with a higher resolution than that from the Sanger technique, allowing for deeper analysis of the transcripts. One important application of NGS is RNA sequencing (RNA-seq), used to describe transcriptomes of cells and tissues. Deep sequencing increases the possibilities of finding new biological molecules in the saliva of bloodsucking insects, offering a new array of substances to be further investigated and functionally characterized. The aim of this report is to catalog the transcripts of R. neglectus SGs with probable function in hematophagy using RNAseq and mass spectrometry. This strategy was used to describe the bioactive molecules in triatomine saliva and improve our understanding on the dynamics of the blood-feeding process, vector-host interaction and disease transmission. The data is available at the National Center for Biotechnology Information (NCBI) and can be used in different scientific research projects.
  R. neglectus triatomines originating from insects collected in 1982 at Itambaracá, in Paraná State, Brazil, were reared in the insectarium at the University of Brasília (Brazil). They were kept at 27±1°C, a relative humidity of 70–75%, under a 12 h/12 h light/dark cycle. The blood source of these insects was Gallus gallus domesticus. The SGs of 5th instar nymphs and adults were dissected at 5,12, and 24 days post blood meal in cold Trizol reagent (Invitrogen, Carlsbad, CA, USA). A pool of thirty SG pairs was stored at -80°C prior to RNA extraction. Total RNA was extracted following the Trizol manufacturer’s instructions. RNA integrity and concentration were checked by lab-on-chip analysis using an Agilent 2100 Bioanalyzer (Agilent Technologies, USA). A RNA sample was sent to the Federal District High-Performance Genome Center (DF, Brazil) for Illumina cDNA library construction and next generation sequencing. A Library was prepared with standard protocols using TruSeq RNA kit, v2 (Illumina, San Diego, CA). To generate paired-end reads of 300 nucleotides in length, the sequencing of cDNA libraries was performed on an Illumina MiSeq sequencer (Illumina, USA). One lane of the MiSeq machine was used for sequencing this and another library, distinguished by bar coding. The RNA-seq sequencing generated a total of 12,049,305 reads. The nominal length of the sequences was 301 nt. Following trimming of low quality bases (quality 20 or lower), the average length was 248. 07, the median was 301 and L50 was 296 nt. Sequences smaller than 25 nt or with average quality < 20 were rejected. Bioinformatic analyses were conducted as previously described [18]. As there was no reference genome to map, the strategy was to perform a de novo assembly with Abyss [19] and Soapdenovo Trans [20] assemblers using different kmer (k) values (from 20 to 90). The resulting assemblies were joined by an iterative BLAST and cap3 assembler [21]. Sequence contamination between bar-coded libraries were identified and removed when their sequence identities were over 98%. Coding sequences (CDS) were extracted based on the existence of a signal peptide and on similarities to other known proteins [22]. Coding and protein sequences were mapped into a hyperlinked Excel spreadsheet. Reads were mapped into contigs using blastn [23] with a word size of 25, masking homonucleotide decamers and allowing mapping to up to five different CDS if the BLAST results had the same scores. Mapping of the reads was also included in the Excel spreadsheet. CDS were automatically annotated a program written by JMCR that searched a vocabulary of nearly 250 words for matches various databases, including Swissprot, Gene Ontology, KOG, PFAM, and SMART, and a subset of the non-redundant protein database containing proteins from vertebrates (NCBI). Further manual annotation was done as required. Alignment analysis were done with Bioedit software [24] after sequence alignment performed using ClustalW [25]. Phylogenetic analysis and statistical neighbor-joining bootstrap tests of the phylogenies were done with Mega package [26]. The sequences used in alignments with R. neglectus CDS were obtained from the non-redundant protein database of the NCBI and are represented by six letters followed by the NCBI GI number. The letters derive from the first three letters of the genus and the first three letters of the species name. The raw reads were deposited at the Sequence Read Archive (SRA) in NCBI under bioproject PRJNA292130. A total of 4,230 coding sequences were deposited in DDBJ/EMBL/GenBank through the Transcriptome Shotgun Annotation portal under the accession GDKW00000000. The SGs were dissected from 5th instar nymphs and adults at 5,12 and 24 days post blood meal and carefully punctured at 4°C. Following centrifugation (16. 000 × g, 15 min, 4°C), the soluble protein fraction from fifteen pairs of SG homogenates was ethanol/acetone precipitated. Resuspended proteins were consecutively alkylated, reduced, digested by trypsin, and subjected to LC-MS/MS analysis as previously described [27]. Briefly, the tryptic peptides were loaded onto a 2 cm fused silica trap column (150 μm inner diameter) packed in-house with reverse phase capillary column ReproSil-Pur C18-AQ 5 μm resin (Dr. Maisch GmbH, Germany) and separated using a DIONEX 3000 nanoUPLC system coupled to an LTQ-Orbitrap Elite mass spectrometer (Thermo Scientific, Waltham, USA). MS1 spectra were recorded in the Orbitrap mass analyzer with 120,000 resolution. After ion fragmentation, MS/MS spectra of the 15 most intense ions were acquired. Raw files were generated and used for protein identification using Proteome Discoverer v. 1. 3 (Thermo Scientific, Waltham, USA) with in-house SequestHT algorithm for R. neglectus SG transcriptome and human keratins, BSA and porcin trypsin. The false discovery rate was less than 1%, with peptide rank of 1 and at least 2 peptides per protein.
  The assembly of R. neglectus SG transcriptome enabled the extraction of 5,705 CDS. These CDS mapped a total of over 11 million reads. Following automated and manual annotation, the CDS were classified into putative secreted, housekeeping, unknown, transposable element, and viral product. The CDS of the housekeeping class comprised the largest class (Table 1). They were further characterized into 24 subclasses, according to their possible function, summarized in Table 2. The secreted class was organized in subclasses that include previously known gene families present in hematophagous saliva, such as lipocalin, nitrophorin, antigen-5, as well as gene families not commonly reported in triatomine saliva, such as serine protease and disintegrin (Table 3). The following section describes the putative secreted proteins present in R. neglectus sialome, highlighting the remarkable finding of serine proteases in this group. Lipocalins comprised one of the most abundant groups of transcripts, with 16. 29% of putatively secreted reads. These include a large group of extracellular proteins that usually bind to small hydrophobic molecules, cell surface receptors or other proteins. The members of this family have little similarity in peptide sequence, however share a conserved three-dimensional structure, comprised of a single eight-stranded antiparallel β-barrel [28]. In blood-sucking insect and tick saliva the lipocalins are abundantly expressed, but not in Diptera or fleas. In ticks, their function is associated with binding to histamine and serotonin [29]. Triabin and nitrophorin, the two major groups found here, are discussed below. The CAP superfamily members [Cysteine-Rich Secretory Proteins (CRISPS), Antigen 5 (Ag5), and Pathogenesis-Related 1 (Pr-1) ] are found in a wide range of organisms, most often as secreted proteins [38]. Ag5, present in the venom of wasps and ants, are considered potent allergens to mammals [39,40]. This superfamily can also block smooth muscle contraction when present in snake venom [41] and act in the defense response in plants [42]. They have been described in the saliva of some hematophagous, including mosquitoes [43,44] and sand flies [45]. Among triatomines, Ag5 genes have been reported in the sialotranscriptomes of R. prolixus [46], T. infestans [47], D. maxima [48], T. matogrossensis [49] and Triatoma rubida [50]. Their functions in blood-feeder saliva remained unexplored for a long time, but a recent report revealed salivary Ag5 of D. maxima and T. infestans as Cu+2-dependent antioxidant enzymes that inhibit neutrophil oxidative burst and platelet aggregation induced by collagen [51]. The sialotranscriptome analysis revealed eight CDS related to the Ag5 family. The alignment of R. neglectus Ag5 with other triatomine Ag5 sequences showed some conserved motifs (S1 Fig). Phylogenetic analysis offers support for the formation of clades I and II comprising triatomine and Diptera sequences, respectively (Fig 3). For blood-feeders, targeting components of the coagulation cascade is essential to attenuate the haemostatic response of their hosts. All enzymes participating in this cascade are serine proteases associated with complement activation [52,53]. The R. neglectus sialotranscriptome exhibited a variety of transcripts coding for proteins with serine protease inhibitory function, comprising 14 CDS and 3,164 reads. Based on their Pfam signature, kazal, pacifastin and serpin families were extracted. The odorant-binding protein (OBP) family is a chemosensory protein ubiquitous in insects commonly associated with solubilizers and carriers of odorants and pheromones. Although associated with chemosensory organs, in recent times this family has been related to other roles such as hydrophobic chemical transportation [102]. The OBPs are characterized by a variable amino acid sequence, but conserve a pattern of six conserved cysteines residues paired to form three disulfide bridges [103]. The folding is a typical six α-helices assembled in a compact and stable structure [104,105]. Eleven CDS containing protein sequences related to OBPs were recognized in our transcriptome analysis, all possessing signal peptide prediction. The conserved cysteine residues of R. neglectus CDS (Fig 10) were seen during alignment. Phylogenetic analysis with good bootstrap support shows Clade I containing most R. neglectus sequences grouped with R. prolixus OBP. However, RN_3440 was grouped in Clade III, suggesting this is a more distant OBP (S4 Fig). R. neglectus saliva content was tryptic digested and subjected to mass spectrometry to validate the analysis of the transcripts possibly associated with secreted products. Among the 73 identified secreted proteins groups, 48 were from the lipocalin family, including triabin, pallidipin and nitrophorin proteins, reaffirming their abundance (Table 4). Other soluble proteins, predicted as being secreted by these arthropods, were: secreted metalloprotease, antigen-5, serpin and trypsin-like protease, each with at least one observation. It is intriguing that only one serine protease was detected by proteomic analysis, regardless the high number of transcripts reads assigned to this subclass of putative secreted proteins (Table 3). There are several possible explanations for this observation. First, the proteins are expressed in a such a small amount not detectable by our proteomic approach; second, the proteins are not secreted; third and most likely, these proteins present in SGs are expressed upon specific physiological conditions, such as during stimulation of salivation (feeding). In addition, it is also possible that those enzymes have both intracellular and extracellular functions as many other proteases do. A comparative blastp analysis was employed to address the similarity of the SGs proteins from R. neglectus compared to R. prolixus. The two species do not show high evolutionary divergence, presenting at least 80% identity in analyzed sequences (Table 5), suggesting both species share a common ancestral lineage. As described before, this high degree of protein similarity was also seen with R. brethesi and R. robustus in the Amazon rainforest [106]. Identity among sequences was greater in housekeeping class members, showing that these proteins have a lower evolutionary rate than those of the secreted class. This indicates that antihaemostatic proteins evolve faster after divergence. Different molecular mechanisms may be responsible for the variation between these closely related Rhodnius species, expanding their biological diversity patterns. The particularity of each species could be related to their different habitats, including different prey and abiotic factors. Hematophagy evolved independently at least six times in approximately 15,000 species allowing for adaptation to an existing complex host haemostatic system [5,107]. Thus, many salivary molecules target different pathways for the insect to achieve a successful blood meal. Here, we described R. neglectus sialome in all its complexity to expand our knowledge of the salivary proteins from hematophagous triatomine bugs. R. neglectus is considered of secondary importance in the transmission of T. cruzi, causative agent of Chagas disease. The analysis of salivary secretory products of R. neglectus that might be involved in vector-host interactions share similarity with other triatomine species, which can also be infected by and transmit the protozoan. It is possible that the expression of putative trypsin-like serine proteases in the SGs of R. neglectus correlates with blood sources of this species of triatomine. Their role and that of other secreted class, hypothetical and conserved secreted proteins, in hematophagy should be analyzed in future works, and we accentuate that sialome study is still an open field for new discoveries.
summary: Chagas disease is caused by the Trypanosoma cruzi protozoan, which is transmitted to vertebrates through the feces of infected triatomines during blood sucking. The vascular injury caused by the bite triggers mechanisms capable of preventing the association with hosts, such as immune response, inflammation and haemostasis. However, hematophagous insects are able to counteract these defenses through a complex repertoire of salivary molecules that have specific targets in the host. Our results show that R. neglectus salivary glands express different protein gene families, possessing multi-functional features directly related to different anti-haemostatic activities. For instance, lipocalins are proteins possessing anti-coagulant and vasodilator functions. Saliva contents have evolved to adapt to blood-feeding habit, ensuring the maintenance of blood flow, the success of the meal, and transmission of diseases.
keywords: sequencing techniques
  invertebrates
  medicine and health sciences
  body fluids
  enzymes
  metalloproteases
  enzymology
  saliva
  animals
  serine proteases
  genome analysis
  odorant binding proteins
  sequence motif analysis
  molecular biology techniques
  research and analysis methods
  sequence analysis
  sequence alignment
  proteins
  molecular biology
  insects
  arthropoda
  biochemistry
  anatomy
  physiology
  transcriptome analysis
  genetics
  biology and life sciences
  proteases
  genomics
  computational biology
  organisms
title: A Deep Insight into the Sialome of Rhodnius neglectus, a Vector of Chagas Disease